WO2023174205A1 - Pharmaceutical formulation and use thereof - Google Patents

Pharmaceutical formulation and use thereof Download PDF

Info

Publication number
WO2023174205A1
WO2023174205A1 PCT/CN2023/081052 CN2023081052W WO2023174205A1 WO 2023174205 A1 WO2023174205 A1 WO 2023174205A1 CN 2023081052 W CN2023081052 W CN 2023081052W WO 2023174205 A1 WO2023174205 A1 WO 2023174205A1
Authority
WO
WIPO (PCT)
Prior art keywords
quercetin
marshmallow
group
extract
glucuronide
Prior art date
Application number
PCT/CN2023/081052
Other languages
French (fr)
Chinese (zh)
Inventor
唐海涛
王丹丹
葛海涛
王殿广
彭小兰
梁慧
王富江
Original Assignee
苏中药业集团股份有限公司
江苏苏中药业研究院有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/CN2022/080818 external-priority patent/WO2023173268A1/en
Priority claimed from CN202210251763.8A external-priority patent/CN116785305A/en
Priority claimed from CN202210990942.3A external-priority patent/CN117582459A/en
Application filed by 苏中药业集团股份有限公司, 江苏苏中药业研究院有限公司 filed Critical 苏中药业集团股份有限公司
Publication of WO2023174205A1 publication Critical patent/WO2023174205A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents

Definitions

  • the invention belongs to the field of pharmaceuticals, and specifically relates to a marshmallow flower flavonoid pharmaceutical preparation and its application.
  • Medicinal hollyhock flower is the dried corolla of the Malvaceae plant Abelmoschus manihot (L.) Medic. Its taste is sweet and cold, and it returns to the kidney and bladder meridian. It has the effects of clearing away dampness and heat, reducing swelling and detoxifying, and is used for carbuncle, swollen poison, water-fire scald. This medicine was first recorded in "Jiayou's Materia Medica”. "Jiayou's Materia Medica” records: marshmallow flowers are used to treat malignant sores that have pus that have not healed for a long time. "Compendium of Materia Medica” says: The flowers of hollyhock are sweet, cold, slippery, and non-toxic. They can be used to treat malignant sores that have pus and water that have not bled for a long time. They can be cured immediately after applying it for a long time. It is an important medicine for treating sores.
  • Flavonoids are one of the main components of marshmallow flowers, and they are also one of its pharmacologically active ingredients. Huangkui capsules have been on the market for many years, and their main active ingredient is marshmallow flower extract. Research has confirmed that the flavonoids in marshmallow flower extract not only have a significant therapeutic effect on ulcers, but also have a significant protective effect on damage to the heart, brain, tissue, etc. caused by ischemia. In addition, many literature reports have shown that the total flavonoids of marshmallow flowers It may have good effects in anti-inflammatory, antipyretic and analgesic, protecting heart and brain from ischemic damage, lowering blood sugar, and anti-viral.
  • patent CN201210082553.7 discloses a total flavonoid extract of marshmallow flowers, containing gossipin-3'-glucoside, quercetin-3'-glucoside and isoquercetin in a weight ratio of (11-16 ): (2.5-6): (4-6.5) flavonoids can be used to prepare and treat nephritis diseases.
  • Patent CN200610097615.6 discloses a total flavonoid extract of marshmallow flowers, with a total flavonoid content of 50-90% by weight.
  • the flavonoid components contained in it are: quercetin-3-acaiglycoside 1.0-5.0%, golden silk peach glycoside 8-24.0%, isoquercetin 7.0-20.0%, quercetin-3'-glucoside 5.0-15.0%, gossyrin-3'-glucoside 3.0-10.0%, myricetin 0.5-5.0% , cottonseed 0.5-5.0%, quercetin 2.0-8.0%, and several other flavonoid components.
  • the total flavonoid extract of marshmallow flowers can be used to prepare drugs for treating nephritis.
  • the flavonoid components are qualitatively and quantitatively clear, and the curative effect is reliable. .
  • bioflavonoids have various therapeutic effects, there are many types of flavonoids, which can currently be divided into seven different subtypes, and different flavonoid compositions will lead to different therapeutic effects.
  • bioflavonoids have been reported in the literature to be effective in preventing or treating diabetic retinopathy
  • compounds with five hydroxyl groups, such as quercetin have negative effects on ocular blood flow
  • flavonoids are dehydrogenated to flavanones
  • Significant improvements in ocular blood flow were obtained.
  • compounds that increase blood flow also lead to a significant increase in retinal functional recovery after ischemic injury. How to develop such compounds to effectively treat eye diseases such as macular degeneration, visual fatigue and cataracts and even improve the effect on diabetic retinopathy is an important research task.
  • CNV Choroidal neovascularization
  • AMD age-related macular degeneration
  • AMD central exudative chorioretinitis
  • macular degeneration caused by high myopia.
  • CNV secondary to AMD is the most common and has become One of the main causes of irreversible visual impairment in the elderly. Insufficient blood flow is also a factor affecting visual fatigue. Enhancing eye blood flow or acting on eye smooth muscles can help restore visual fatigue.
  • Glucose reabsorption in the proximal renal tubule is mediated by the sodium-glucose cotransporter (SGLT) 1 and SGLT2 transporters, of which approximately 90% of glucose reabsorption is mediated by SGLT2 and the remaining 10% is mediated by SGLT1.
  • SGLT2 inhibitors inhibit renal tubular glucose reabsorption by selectively binding to the SGLT2 receptor, thereby lowering the renal glucose threshold, increasing urinary glucose excretion, and achieving significant blood sugar lowering purposes.
  • SGLT2 inhibitors have a renal protective effect on non-diabetic chronic kidney disease (CKD).
  • SGLT2 inhibitors can reduce the expression of collagen and fibronectin by reducing TGF ⁇ -1, PAI1, STAT 1, MMP 7 and inhibiting the AGEs-RAGE axis, thereby reducing the accumulation of extracellular matrix and alleviating the progression of DN renal fibrosis.
  • SGLT2 inhibitors can also reduce uric acid (SUA) in patients with diabetes.
  • SGLT2 inhibitors empagliflozin
  • serum uric acid levels significantly reduced serum uric acid levels.
  • SGLT2 inhibitors can be beneficial to the prevention and treatment of heart failure by reducing plasma volume, reducing before and after load, improving myocardial energy metabolism, and improving myocardial remodeling.
  • EGFRIs Epidermal growth factor receptor antagonists
  • rash mainly acne/acne-like rash
  • Acne/acne-like rash not only affects the patient's quality of life, but may also prevent normal treatment, seriously affecting the efficacy of tumor treatment.
  • Acne/acneiform rash limits the application of EGFRIs to a certain extent.
  • Western medicine often uses antibiotics, external hormones and other methods to treat the disease, but the effect is not very obvious.
  • Skin wounds are a common clinical symptom, and conventional skin wound treatments include skin grafting and artificial substitute coverage.
  • Skin ulcers are common clinical diseases and frequently-occurring diseases. They refer to local skin tissue defects caused by various reasons. The exposed wounds are prone to infection, especially chronic skin ulcers that cannot heal for a long time or are prone to recurrence. Treatment is difficult and seriously affects the patient's quality of life. It also brings a certain social burden. Clinically, there is an urgent need for the research and development of more safe, effective, and economical treatments and drugs.
  • Inflammatory bowel disease is a special chronic intestinal inflammatory disease, including ulcerative colitis and Crohn's disease. Clinical patients will present with repeated abdominal pain, diarrhea, and mucus and bloody stools. Most patients have repeated attacks and protracted recovery. On the one hand, it seriously affects the patient's quality of life; on the other hand, it can increase the risk of colorectal cancer. The longer the course of the disease, the higher the degree of inflammation, and the greater the possibility of cancer; UC can increase the risk of cancer by about 2.4 times; the course of the patient's disease The cancer rates at 10, 20 and 30 years were 2%, 8% and 18% respectively.
  • IBD-related colorectal cancer has the characteristics of younger age, disseminated and multifocal lesions, and its pathology and pathogenesis are different from sporadic CRC; "Inflammatory bowel disease-dysplasia- "Colorectal cancer” is an important pathway for the occurrence of CRC.
  • Colorectal cancer is the most common malignant tumor of the digestive tract, with an incidence rate second only to gastric cancer and esophageal cancer. The incidence rate of colorectal cancer in my country is currently on the rise. The domestic incidence rate is approximately 4.8%, far exceeding the international level of 2%.
  • Enteric-coated preparations refer to preparations that do not release or release almost no drug in the stomach within a specified period of time, but enter the intestine and can release most or all of the drug in a certain part of the intestine. Making enteric-coated preparations can reduce the irritation of the drug to the stomach, improve the bioavailability of the drug, and allow the drug to be more completely absorbed in the intestines. There are currently no enteric-coated preparations containing marshmallow flower extract.
  • marshmallow flower reported in the literature include ethanol reflux extraction, ultrasonic extraction, and room temperature soaking extraction.
  • the components of marshmallow flower extract are complex, the content of active ingredients varies greatly, the extraction transfer rate is low, the extraction energy consumption is high, and the amount of solvent is large. . Therefore, it is necessary to explore a marshmallow flower therapeutic drug and its extraction process that can jointly treat kidney disease, especially diabetic nephropathy, and simultaneously treat multiple diseases such as eye diseases.
  • the present invention provides an effective part of marshmallow flower flavonoids, an extract, a composition, a preparation process, an application, a pharmaceutical preparation and its application.
  • the present invention prepares a high-purity marshmallow flower flavonoid effective part, extract, composition, and pharmaceutical preparation through an extraction coupling resin method, and finds that it has better therapeutic activity for kidney diseases, such as diabetic nephropathy, contrast-induced kidney injury, or erythema.
  • kidney diseases such as diabetic nephropathy, contrast-induced kidney injury, or erythema.
  • Lupus nephritis it also has the effect of treating eye diseases, it also has the effect of anti-fibrosis, the effect of preventing and treating ulcer diseases, the effect of preventing and treating inflammation, the effect of preventing and treating skin diseases, and the effect of promoting wound healing.
  • the marshmallow flower flavonoid pharmaceutical preparation of the present invention has lower side effects and provides a better therapeutic drug for patients.
  • the present invention provides a high-purity effective part, extract and composition of flavonoids from marshmallow flowers.
  • the present invention provides an effective part of flavonoids from marshmallow flowers, including the following mass ratio of flavonoid components: gossyrin-8-O- ⁇ -D-glucuronide: hypericin: isoquercetin.
  • the mass ratio of dermatin:myricetin:quercetin-3'-O-glucoside is: 10:3.0-20:4.0-18:1.9-6.0:6.0-20.
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is: 10 : 4.0-17: 4.50-16: 1.9-5.5: 7.0-16; preferably: 10: 6.0-15: 5.0-13: 2.0-5.0: 8.0-14; further, containing quercetin, wherein gossydertin -8-O- ⁇ -D-glucuronide: quercetin is 10:1.0-10.0, preferably 10:1.0-6.0, preferably 10:1.5-5.0; further, it contains rutin, wherein cotton Cortin-8-O- ⁇ -D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably 10:0.1-0.3; further, it contains quercetin- 3-O-Acaiglycoside, wherein Gossipin-8-O- ⁇ -D-glucuronide: Quer
  • the present invention provides an effective part of flavonoids from marshmallow flowers, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin:
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide:hyperin is: 0.8-1.2:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8 -1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.09-1.6:0.2-2.0:0.05-1.2:0.5-3.0:0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.12-0.7:0.8-1.6:0.2-0.5:0.85-1.7:1.0-1.4.
  • the mass ratio value of the isoquercetin is 1.2 or 1.0.
  • the effective part of the flavonoids of the marshmallow flower further contains rutin, and the mass ratio of isoquercitrin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05 , preferably 1:0.007-0.04, preferably 1:0.009-0.04, or preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, it contains quercetin-3-O-acaxiglucoside, The mass ratio of quercetin and quercetin-3-O-sophoroside is 10:0.1-2.5, 10:0.1-0.9, preferably 10:0.15-0.5.
  • the effective part of the flavonoids of the marshmallow flower contains quercetin, quercetin-3'-O-glucoside, myricetin, and gossyderm-8-O- ⁇ -D-glucuronide.
  • the total content of isoquercetin and hyperoside is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%; further, quercetin-3'-O-glucose
  • the glycoside content is 12.1-25%, further the quercetin-3'-O-glucoside content is 12.6-23% or 13-20%, 13-20%, or 14-25%; or further , the content of quercetin is higher than 0.7%, further higher than 1.0%, further 1.0%-10%, further 1.5%-5%; or further, cottonseed-8-O- ⁇ -D-glucuronide is 8.5-30%, further 12%-23%.
  • the effective part contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O- ⁇ -D-glucuronide, and isoquercetin.
  • the total content of rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82%, or 69-90%; further, quercetin
  • the content of quercetin-3'-O-glucoside is 12.1-25%, and further, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%.
  • the present invention provides a plant extract containing flavonoid components, including the following mass content components: hyperoside 10-25%, isoquercitrin 8-19%, gossipin-8- O- ⁇ -D-glucuronide 5-30%, quercetin-3'-O-glucuronide 12.1-25%; further hyperoside 12-23%, isoquercetin 10-17% , gossipin-8-O- ⁇ -D-glucuronide 8.5-25%, quercetin-3'-O-glucuronide 12.6-23%; further hyperoside 13-22%, isosin Quercetin 11-17%, Gossipin-8-O- ⁇ -D-glucuronide 12-21%, Quercetin-3'-O-glucuronide 13-20%.
  • the extract also contains quercetin 2-10%, preferably quercetin 2.5-9%, preferably quercetin 3-8.5%, further 1.5%-5%; further, it contains bayberry. 2-11% of myricetin, preferably 3-7% of myricetin, preferably 3-5% of myricetin, and further, 0.08-2.5% of quercetin-3-O-sophoraside, preferably 0.1-1.5%, further 0.1 -0.9%, and further 0.1-0.5%; further, the plant is a flower of marshmallow okra, which can be a whole flower or a corolla, preferably a marshmallow flower.
  • the present invention provides a marshmallow flower extract, containing flavonoid components, including the following mass content components: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossipin-8 -O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-O-glucuronide 14-25%, quercetin 1.6-4.9 %; furthermore, hyperoside 11-22%, isoquercetin 12.0-17%, gossyrin-8-O- ⁇ -D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-O-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%.
  • mass content components including the following mass content components: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossipin-8 -O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0-4.9%
  • the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, it contains quercetin- 3-O-Asophoroside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, still further 0.1-0.5%.
  • the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82% or 69-90%. .
  • the present invention provides a composition, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-
  • the mass ratio of 8-O- ⁇ -D-glucuronide:hyperoside is: 1:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6; further 1:0.1- 1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8- 1.4; further to 1: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3 or
  • the composition also includes rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably It is 1:0.007-0.04, preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, the content of quercetin-3'-O-glucoside in the flavonoids is 12.1-25%, Furthermore, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%, and further, it contains 0.08-2.5% of quercetin-3-O-acaxiglucoside, preferably 0.1-1.5 %, further 0.1-0.9%, further 0.1-0.5%.
  • the composition contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O- ⁇ -D-glucuronide, and isoquercetin.
  • the total content of rutin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%.
  • the present invention provides a pharmaceutical composition, which contains the above-mentioned effective part, the above-mentioned marshmallow extract or the above-mentioned composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, diluents, and wetting agents.
  • the lubricant does not contain magnesium stearate.
  • the dosage forms of the pharmaceutical composition include injections, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures, suspensions, and dispersible tablets.
  • the present invention provides a pharmaceutical composition containing the following components by weight: hyperoside 3.0-20.0, isoquercetin 4.0-18.0, myricetin 1.9-6.0, quercetin-3'- O-glucoside 6.0-20, gossyrin-8-O- ⁇ -D-glucuronide 8.0-12.0; further, the pharmaceutical composition contains the following components by weight: hyperoside 4.0-17.0, isoquercetin 4.5-16.0, myricetin 1.9-5.5, quercetin-3'-O-glucoside 7.0-16.0, gossipin-8-O- ⁇ -D-glucuronide 8.0 -12.0;
  • the pharmaceutical composition contains the following components by weight: hyperoside 6.0-15.0, isoquercetin 5.0-13.0, myricetin 2.0-5.0, quercetin-3'-O-glucose Glycoside 8.0-14.0, gossyrin-8-O- ⁇ -D-glucuronide 8.0-12.0; the further pharmaceutical composition contains the following components by weight: hyperoside 8.0-15.0, Isoquercetin 6.0-12.5, myricetin 2.0-4.0, quercetin-3'-O-glucuronide 9.0-13.0, gossyrin-8-O- ⁇ -D-glucuronide 8.0-12.0; follow Furthermore, the pharmaceutical composition contains the following components by weight: hyperoside 10-15.0, isoquercetin 7.0-12.0, myricetin 2.0-3.0, quercetin-3'-O- Glucoside 9.0-12.0, gossyrin-8-O- ⁇ -D-glucuronide 8.0-12.0.
  • the pharmaceutical composition further contains quercetin, wherein the weight ratio of gossydisin-8-O- ⁇ -D-glucuronide to quercetin is 9-11:1.0-10.0, preferably 10 : 1.0-6.0, more preferably 10: 1.5-5.0, further preferably 10: 1.0-10.0.
  • the pharmaceutical composition further contains quercetin-3-O-acaiglycoside, wherein gossipin-8-O- ⁇ -D-glucuronide and quercetin-3-O-acaiglycoside
  • the weight ratio is 9-11:0.05-2.5, 10:0.1-0.9, preferably 10:0.15-0.5, and further preferably 10:0.05-2.5.
  • the content of the component as a pharmaceutical active ingredient is more than 60%, further more than 65%, further more more than 75%, preferably the cottonseed-8-O- ⁇ -D-
  • the weight of glucuronide is 9-11 parts. Preferably it is 10 parts.
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier.
  • the present invention provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O- ⁇ -D-glucuronide:hyperoside: The mass ratio of isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:4.0-18:1.9-6.0:6.0-20.
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 4.0-17: 4.50-16: 1.9-5.5: 7.0-16; preferably 10: 6.0-15.6: 5.0-13: 2.0-5.0: 8.0-14, or 10: 6.0-15: 5.0-13: 2.0-5.0 : 8.0-14; further, containing quercetin, wherein the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide and quercetin is 10:1.0-10.0, preferably 10:1.0- 6.0, preferably 10:1-5.6, preferably 10:1.5-5.0; further, it contains rutin, wherein the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide to rutin is 10: 0.05-0.6, preferably 10: 0.1-0.5, preferably 10: 0.1-0.45, or 10:
  • the present invention also provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O- ⁇ -D-glucuronide: hyperoside. : Isoquercetin: Myricetin: Quercetin-3'-O-glucoside: The mass ratio of quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, Or 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99.
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, preferably 10:0.1 -0.3; further, it contains quercetin-3-O-a Sophora glycoside, in which the mass ratio of Gossipin-8-O- ⁇ -D-glucuronide to Quercetin-3-O-Asophoroside It is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.5.
  • the present invention provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:
  • the mass ratio of isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, or 10: 6-15: 5-13: 1.0-1.8: 4.0-5.9: 0.6-0.99.
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, preferably 10:0.1 -0.3; further, it contains quercetin-3-O-a Sophora glycoside, in which the mass ratio of Gossipin-8-O- ⁇ -D-glucuronide to Quercetin-3-O-Asophoroside It is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.5.
  • the present invention provides an application of an effective part of a flavonoid in the preparation of a drug for treating and/or preventing ulcerative diseases.
  • the effective part of the flavonoid includes the following mass ratio of flavonoid components: isoquercitrin : Quercetin: Quercetin-3'-O-glucoside: Myricetin: Gossyrin-8-O- ⁇ -D-glucuronide: The mass ratio of hyperoside is: 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8 -1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.09-1.6:0.2-2.0:0.05-1.2:0.5-3.0:0.25-3.6.
  • the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8
  • the mass ratio of -O- ⁇ -D-glucuronide:hyperoside is: 0.8-1.2:0.12-0.7:0.8-1.6:0.2-0.5:0.85-1.7:1.0-1.4.
  • the mass ratio value of the isoquercetin is 1.2 or 1.0.
  • the effective part of the flavonoids of the marshmallow flower further contains rutin
  • the mass ratio of isoquercitrin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05 , preferably 1:0.007-0.04, preferably 1:0.008-0.04, or preferably 1:0.009-0.04; further, it contains quercetin-3-O-acaxiglucoside, isoquercetin and quercetin-
  • the mass ratio of 3-O-sophoroside is 10:0.01-0.25, 10:0.01-0.09, preferably 1:0.015-0.065, preferably 10:0.015-0.05.
  • the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis.
  • the flavonoid extract or effective part of the marshmallow flower contains quercetin, quercetin-3'-O-glucoside, myricetin, and gossydertin-8-O.
  • the total content of ⁇ -D-glucuronide, isoquercetin and hyperoside is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%; further, quercetin Cortin-3'-O-glucoside content is 12.1-25%, preferably 12.6-25%, preferably 13-25%, preferably 14-25%, or, 12.6-23%, or 13-20% ; Further, the content of quercetin is more than 0.7%, preferably more than 1.0%, preferably 1.0-12%, preferably 1.5-12%, or 1.0%-10%, or 1.5%-5%; further Preferably, gossydisin-8-O- ⁇ -D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%
  • the extract or effective part contains quercetin, quercetin-3'-O-glucoside, myricetin, gossyderm-8-O- ⁇ -D-
  • the total content of glucuronide, isoquercetin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82% or 69- 90%; further, the quercetin-3'-O-glucoside content is 12.1-25%, preferably 12.6-23%, preferably 13-20%.
  • the present invention provides an extract of marshmallow flowers, containing flavonoid components.
  • the extract includes components with the following mass contents: hyperoside 8-30%, isoquercetin 10-24%, Gossyrin-8-O- ⁇ -D-glucuronide 8.5-34%, myricetin 3.0-4.9% or 5.1-7.0%, quercetin-3'-O-glucuronide 14-25%, quercetin Cortin 1.6-12%; or hyperoside 8-26%, isoquercetin 12.0-19.8%, gossyrin-8-O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0 -4.9% or 5.1-6.0%, quercetin-3'-O-glucoside 14-25%, quercetin 1.6-4.9%; or hyperoside 11-22%, isoquercetin 12.0-17 %, gossyrin-8-O- ⁇ -D-glucuronide 12-23%, myricetin 1.6-4.
  • the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.95%, further 0.09-0.95%, further 0.1-0.95%, or 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, containing quercetin-3-O-acaxiglucoside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, and further 0.1-0.8 %, and further 0.1-0.5%.
  • the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, preferably 65-90%, preferably 69-82%.
  • the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis.
  • the present invention provides a composition comprising the following mass ratio of flavonoid components: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin -8-O- ⁇ -D-glucuronide: Hypericum
  • the mass ratio of glycosides is: 1:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6; further: 1:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5- 3.0; further 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; Further is 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further is 1: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3 or 1.2
  • the composition also includes rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably It is 1:0.007-0.04, preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, the content of quercetin-3'-O-glucoside in the flavonoids is 12.1-25%, Furthermore, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%, and further, it contains 0.08-2.5% of quercetin-3-O-acaxiglucoside, preferably 0.1-1.5 %, further 0.1-0.9%, further 0.1-0.5%.
  • the composition contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O- ⁇ -D-glucuronide, and isoquercetin.
  • the total content of rutin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%.
  • the present invention provides a pharmaceutical composition, which contains the above-mentioned effective part, the above-mentioned marshmallow extract or the above-mentioned composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, diluents, and wetting agents.
  • the lubricant does not contain magnesium stearate.
  • the dosage forms of the pharmaceutical composition include injections, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures, suspensions, and dispersible tablets.
  • the present invention provides methods for preparing the effective parts, extracts and compositions of flavonoids in each technical solution of the above-mentioned first main aspect.
  • the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract.
  • the method includes the following steps: (1) Extract marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) The extract is concentrated and extracted to obtain an extract; (3) the extract is freed of solvent and eluted with a macroporous resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is percolation.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution
  • the extraction agent used for extraction in step (2) It is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is level 1-5
  • the macroporous resin model described in step (3) is D101, HPD100 or AB-8.
  • step (2) further includes subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
  • the elution process of the macroporous resin described in step (3) is: the diameter-to-height ratio of the macroporous resin is 1:4-1:9, the concentration of the loading solution is 0.10-0.30g crude drug/mL, and the loading solution The volume is 4-12BV, load the sample at a flow rate of 1-4BV/h, use 4-8 or 0.5-8BV pure water and 1-5BV of 3-15% ethanol to remove impurities at a flow rate of 0.5-4BV/h, use 2- 8BV 50-80% ethanol is used for elution at a flow rate of 1-5BV/h.
  • the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract.
  • the method includes the following steps: (1) The flowers or medicinal parts of marshmallow are treated with ethanol. Extract with alcohol to prepare an extract; (2) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (3) The supernatant is eluted with polyamide resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; The preferred extraction method is percolation.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution;
  • the water bath temperature described in step (2) is 50-25%. 70°C, water bath time is 30-90min;
  • the diameter-to-height ratio of the resin described in step (3) is 1:4-1:9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, and the volume of the sample solution is 4- 12BV, elute with 4-8BV pure water and 4-8BV 60-95% ethanol.
  • the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract.
  • the preparation method includes the following steps: (1) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Extraction The pH value of the liquid is adjusted to 2.0-3.0, refrigerated, filtered to collect the precipitate, and dissolved in water; (3) the dissolved liquid is extracted to obtain an extraction liquid; (4) the extraction liquid is eluted with polyamide resin after removing the solvent to obtain marshmallow flowers.
  • the effective fraction or extract of flavonoids; the preferred extraction method is reflux.
  • the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution;
  • the pH regulator described in step (2) is hydrochloric acid , sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid;
  • the extraction agent used for extraction in step (3) is n-butanol, petroleum ether or ethyl acetate, and the extraction method is continuous countercurrent extraction, so
  • the material-to-liquid ratio of the extraction is 0.8-4:1, and the extraction stages of the extraction are level 1-5;
  • the diameter-to-height ratio of the resin in step (4) is 1:4-1:9, and the loading liquid
  • the concentration is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4-8BV 60-95% ethanol are used for elution.
  • the present invention provides the application of the effective parts, extracts and compositions in each of the technical solutions of the above-mentioned first and second main aspects.
  • the application is application in the preparation of drugs for kidney diseases, or application in the preparation of drugs for eye diseases, or preparation of lupus nephritis, or kidney damage caused by contrast agents, or pulmonary fibrosis, or heart failure, or reducing uric acid, or
  • the kidney disease is diabetic nephropathy, or diabetic nephropathy or nephritis accompanied by renal fibrosis, lupus erythematosus nephritis, or kidney damage caused by contrast agents; preferably, the kidney disease is diabetic nephropathy or nephritis accompanied by renal fibrosis.
  • the eye disease is preferably macular degeneration, visual fatigue or cataracts, diabetic retinopathy, age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia;
  • the Fibrosis can be selected from pulmonary fibrosis, and further is specific pulmonary fibrosis; preferably, the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis;
  • the inflammatory bowel disease is ulcerative colitis and Crohn's disease; preferably, the skin disease is any one of rash and acne; preferably, the wound includes but is not limited to burns Burn wounds, residual small wounds, donor site wounds, chronic ulcer wounds, and fresh and refractory skin wounds; preferably, the chronic ulcer wounds include but are not limited to diabetic, vascular, and radiation ulcers.
  • the pharmaceutical composition of the present invention when combined according to a specific ratio, has a good effect in treating diabetic nephropathy, has few side effects, and also has the effect of treating pulmonary fibrosis.
  • the present invention provides a pharmaceutical preparation containing the effective parts, extracts, compositions and preparation methods and applications described in the technical solutions of the above-mentioned first and second main aspects.
  • the effective parts and extracts contained in the pharmaceutical preparation invention are called extracts, and the effective parts and extracts prepared by extracting marshmallow flowers are called marshmallow flower extracts.
  • the present invention provides a pharmaceutical preparation, which contains marshmallow flower extract and pharmaceutically acceptable auxiliary materials.
  • the marshmallow flower extract contains marshmallow flower extract and pharmaceutically acceptable auxiliary materials.
  • flavonoids in the following mass ratio: gossyrin-8-O- ⁇ -D-glucuronide: hyperoside: isoquercetin
  • the mass ratio of dermatin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 2.0-25: 3.0-20: 1.0-7.0: 4.0-23: 0.6-12.0;
  • the flavonoid component contains the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O- ⁇ -D-glucuronic acid Glycosides: Hyperin is 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6;
  • flavonoids with the following mass content: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossyrin-8-O- ⁇ -D-glucuronide 5-30%, Myricetin 1.6-11%, quercetin-3'-O-glucoside 12.1-25%, quercetin 1.4-10%; further, hyperoside 8-26%, isoquercetin 12.0-19.8 %, gossyrin-8-O- ⁇ -D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-O-glucuronide 14-25% , Quercetin 1.6-4.9%; here refers to the proportion of flavonoids such as hyperoside in marshmallow flower extract;
  • the preparation is a non-aqueous liquid preparation
  • the mass content of the pharmaceutically acceptable excipients in the preparation is 0.5%-99.5%.
  • the marshmallow flower extract contains flavonoid components in the following mass ratio:
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperin:isoquercetin:myricetin:quercetin-3'-O-glucuronide:quercetin is 10 : 3.0-20: 4.0-18: 1.9-6.0: 6.0-20: 1.0-10.0, or 10: 6-15: 5-13: 1.0-2.9: 4.0-9.5: 0.6-6.0, or 10: 6-15 :5-13:1.0-1.8:4.0-5.9:0.6-0.99;
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 4.0-17: 4.50-16: 1.9-5.5: 7.0-16;
  • the mass ratio of gossyrin-8-O- ⁇ -D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 6.0-15:5.0-13:2.0-5.0:8.0-14;
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide: quercetin is 10:1.0-6.0, preferably 10:1.2-6, further preferably 10:1.5-5.0;
  • the mass ratio of gossydisin-8-O- ⁇ -D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, and further preferably 10:0.1-0.3;
  • quercetin-3-O-acaiglycoside contains quercetin-3-O-acaiglycoside, and the ratio of gossyrin-8-O- ⁇ -D-glucuronide: quercetin-3-O-acaiglycoside is 10:0.1- 2.5, preferably 10:0.1-0.9, further preferably 10:0.15-0.5;
  • gossyrin-8-O- ⁇ -D-glucuronide hypericin: isoquercetin: myricetin: quercetin-3'-O-glucuronide: quercetin
  • the mass ratio is 10:6-16:5-13:1.0-4.5:4.0-12:0.6-6.0, or 10:15.1-16:5-13:3.0-4.5:9.6-12:0.6-6.0;
  • the mass ratio of the cottonseed-8-O- ⁇ -D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably is 10: 0.1-0.3;
  • the mass ratio of the cottonseed-8-O- ⁇ -D-glucuronide:quercetin-3-O-acaiglycoside is: 10: 0.1-2.5, preferably 10: 0.1-0.9, further preferably 10: 0.15-0.5.
  • the marshmallow flower extract contains flavonoid components in the following mass ratio:
  • Isoquercetin Quercetin: Quercetin-3'-O-glucoside: Myricetin: Gossyrin-8-O- ⁇ -D-glucuronide: Hypericin 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 0.8-1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 0.8- 1.2: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8-1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3; Or 0.8-1.2: 0.09-1.6: 0.2-
  • the mass ratio value of the isoquercetin is 1.2 or 1.0;
  • isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O- ⁇ -D-glucuronide: hyperoside is 1 : 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16 -1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7 :0.8-1.6:0.2-0.5:0.8-1.8:0.8-1.4; further 1:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3; or 1.2:0.05-1.6:0.2 -4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1.2: 0.1-1.2:
  • the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, further preferably 1:0.006-0.05, even more preferably 1:0.007 -0.04, further preferably 1:0.008-0.03, most preferably 1:0.009-0.03;
  • quercetin-3-O-acaxiglucoside contains quercetin-3-O-acaxiglucoside, and the mass ratio of the isoquercetin and quercetin-3-O-acaxiglucoside is 10:0.1-2.5, preferably 10:0.1-0.9, More preferably, it is 10:0.15-0.5.
  • the marshmallow flower extract contains flavonoid components with the following mass content: hyperin 10-25%, isoquercetin 8-19%, gossipin-8-O- ⁇ -D -Glucuronide 5-30%, quercetin-3'-O-glucoside 12.1-25%;
  • quercetin-3'-O-glucoside content is 12.6-23% or 13-20%;
  • hyperoside 12-23% isoquercetin 10-17%, gossyrin-8-O- ⁇ -D-glucuronide 8.5-25%, quercetin-3'-O - Glucoside 12.6-23%;
  • hyperoside 13-22% isoquercetin 11-17%, gossyrin-8-O- ⁇ -D-glucuronide 12-21%, quercetin-3'-O - Glucoside 13-20%;
  • quercetin preferably 2.5-9% quercetin, further preferably 3-8.5% quercetin, even more preferably 1.5%-5% quercetin;
  • myricetin preferably 3-7% myricetin, further preferably 3-5% myricetin
  • quercetin-3-O-aphoretin glycoside contains 0.08-2.5% of quercetin-3-O-aphoretin glycoside, preferably 0.1-1.5% of quercetin-3-O-aphoretin glycoside, and further preferably 0.1-0.9% of quercetin-3-O-aphoretin glycoside. %, and further preferably quercetin-3-O-acaxiglucoside 0.1-0.5%;
  • hyperoside 11-22% isoquercetin 12.0-17%, gossipin-8-O- ⁇ -D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1 -9.0%, quercetin-3'-O-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%;
  • rutin preferably 0.01-1.0% rutin, preferably 0.05-0.8% rutin, further preferably 0.09-0.8% rutin, even more preferably 0.1-0.6% rutin.
  • the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, more preferably 65-85% or 66-84%, even more preferably 69-82% % or 69-90%;
  • quercetin contains quercetin, quercetin-3'-O-glucoside, myricetin, gossyrin-8-O- ⁇ -D-glucuronide, isoquercitrin and hyperoside.
  • the total content is more than 55%, preferably more than 60%, more preferably 65-85%, even more preferably 69-82%;
  • quercetin-3'-O-glucoside content is 12.1-25%
  • the preferred quercetin-3'-O-glucoside content is 12.6-23%, 13-20%, 13-20%, Or 14-25%;
  • the content of quercetin is higher than 0.7%, preferably higher than 1.0%, further preferably 1.0%-10%, More preferably, 1.5%-5%;
  • gossydisin-8-O- ⁇ -D-glucuronide is 8.5-30%, preferably 12%-23%.
  • the marshmallow flower extract is prepared by the following steps: (1) extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) extracting the extract after concentration to prepare Extract; (3) remove the solvent from the extract and elute with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
  • the amount of ethanol used is 10-25 times that of marshmallow flowers or marshmallow medicinal parts, and the ethanol is a 60-95% ethanol solution; in step (2), the
  • the extraction agent used for the extraction is n-butanol, petroleum ether or ethyl acetate.
  • the extraction method is continuous countercurrent extraction.
  • the material-liquid ratio of the extraction is 0.8-4:1.
  • the extraction stages of the extraction are Level 1-5; in step (3), the macroporous resin model is D101, HPD100 or AB-8; in step (22), the water bath temperature is 50-70°C, and the water bath time is 30-90 minutes; In step (33), the diameter-to-height ratio of the resin is 1:4-9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4- Elute with 8BV in 60-95% ethanol.
  • step (2) includes subjecting the extraction liquid to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
  • the elution process of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:4-9, the concentration of the sample solution is 0.10-0.30g crude drug/mL, and the volume of the sample solution is 4- 12BV, load the sample at a flow rate of 1-4BV/h, use 4-8BV pure water and 1-5BV 3-15% ethanol at a flow rate of 0.5-4BV/h to remove impurities, use 2-8BV 50-80% ethanol at a flow rate of 1 -5BV/h flow rate for elution.
  • the present invention also provides a pharmaceutical preparation of marshmallow flower extract, which contains pharmaceutically acceptable auxiliary materials.
  • the preparation is a non-aqueous liquid preparation; the pharmaceutically acceptable auxiliary materials are in the preparation.
  • the content is 0.5%-99.5%;
  • the extract is prepared by the following steps: (1) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Concentrating the extract and extracting to prepare the extract liquid; (3) remove the solvent from the extraction liquid and then elute with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
  • the amount of ethanol used is 10-25 times that of marshmallow flowers or marshmallow medicinal parts, and the ethanol is a 60-95% ethanol solution; in step (2), the
  • the extraction agent used for the extraction is n-butanol, petroleum ether or ethyl acetate.
  • the extraction method is continuous countercurrent extraction.
  • the material-liquid ratio of the extraction is 0.8-4:1.
  • the extraction stages of the extraction are Level 1-5; in step (3), the macroporous resin model is D101, HPD100 or AB-8; in step (22), the water bath temperature is 50-70°C, and the water bath time is 30-90 minutes; In step (33), the diameter-to-height ratio of the resin is 1:4-9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4- Elute with 8BV in 60-95% ethanol.
  • step (2) includes subjecting the extraction liquid to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
  • the elution process of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:4-9, the concentration of the sample solution is 0.10-0.30g crude drug/mL, and the volume of the sample solution is 4- 12BV, load the sample at a flow rate of 1-4BV/h, use 4-8BV pure water and 1-5BV 3-15% ethanol at a flow rate of 0.5-4BV/h to remove impurities, use 2-8BV 50-80% ethanol at a flow rate of 1 -5BV/h flow rate for elution.
  • the preparation method of the marshmallow flower extract in the pharmaceutical preparation described in the first aspect includes The steps are as follows: (101) Extract marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (102) Adjust the pH value of the extract to 2.0-3.0, refrigerate, filter to collect the precipitate, and add water to dissolve; (103) The dissolved solution is Extraction is performed to obtain an extraction liquid; (104) the solvent is removed from the extraction liquid and then eluted with polyamide resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is reflux.
  • the amount of ethanol used in step (101) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution;
  • the pH adjustment in step (102) The agent is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid;
  • the extraction agent used for extraction in step (103) is n-butanol, petroleum ether or ethyl acetate, and the extraction method is continuous countercurrent Extraction, the material-to-liquid ratio of the extraction is 0.8-4:1, the extraction level of the extraction is level 1-5; the diameter-to-height ratio of the resin in step (104) is 1:4-9, and the sample is loaded
  • the liquid concentration is 0.10-0.60g crude drug/mL, the sample liquid volume is 4-12BV, and 4-8BV pure water and 4-8BV 60-95% ethanol are used for elution.
  • the non-aqueous liquid preparation is a solid preparation or a semi-solid preparation; further selected from the group consisting of suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures.
  • the pharmaceutically acceptable excipients include solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, Any one, any two combinations, any three combinations, or multiple combinations of diluents, wetting agents, adhesives or film-forming agents;
  • the auxiliary materials are selected from lactose, polyethylene glycol, Cross-linked polyvinylpyrrolidone, magnesium lauryl sulfate, hydroxypropyl cellulose, talc, calcium sulfate, microcrystalline cellulose, low-substituted hydroxypropyl methylcellulose, croscarmellose sodium, magnesium stearate Any one or any two combinations or any three combinations, or multiple combinations;
  • the filler and disintegrant are respectively 1%-80% of the weight of the preparation, and further 3%-78% , further 5%-75%, further 10%-70%, 15%-75%, 15%-65%,
  • the mass content of the pharmaceutically acceptable excipients in the preparation is 2%-95%; further 5%-95%; further 10%-90%; further 15 %-85%; further 20%-80%, 25%-75%, 30%-70%, 40%-60%, 50%-80%, 15%-90%; further 20%-85% , 4%-50%.
  • the present invention also provides the use of the above-mentioned pharmaceutical preparation in the preparation of drugs for kidney diseases, or the preparation of drugs for eye diseases, or the preparation of lupus erythematosus nephritis, or kidney damage caused by contrast agents, or pulmonary fibrosis, or Application in heart failure, or in the preparation of drugs that lower uric acid, or in the preparation of anti-fibrotic drugs, or in the preparation of drugs for the treatment and/or prevention of ulcerative diseases, or in the preparation of products for the prevention and treatment of inflammatory bowel disease, Or the application in the preparation of medicines for treating and/or preventing skin diseases, or the application in the preparation of medicines for promoting wound healing; preferably, the kidney disease is diabetic nephropathy, or diabetic nephropathy accompanied by renal fibrosis or nephritis, lupus erythematosus Nephritis, or kidney damage caused by contrast agents; preferably, the kidney disease is diabetic ne
  • the peak area of hyperoside reference substance/concentration of hyperoside reference substance*correction factor* is calculated as the peak area of quercetin-3-O-acaciaglycoside*constant volume/weighed sample amount (after deducting water), and the content is not calculated. Higher than 2.0%, further not higher than 0.9%, preferably 0.1%-0.7%.
  • the total content of various flavonoids tested was above 70%, further above 75%, and further between 80% and 90%.
  • the % ethanol mentioned in the present invention is a volume percentage unless otherwise specified.
  • the effective parts or extracts of flavonoids from marshmallow flowers according to the present invention and their preparations have the effect of treating various nephritis, such as diabetic nephropathy, contrast agent kidney injury or lupus erythematosus nephritis, and also have the effect of treating eye diseases, In particular, various diseases related to the suppression of blood flow and suppression of ocular blood vessels, such as age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia. In particular, the hollyhock flower of the present invention The effective parts or extracts of flavonoids also have the effect of treating pulmonary fibrosis.
  • various nephritis such as diabetic nephropathy, contrast agent kidney injury or lupus erythematosus nephritis
  • eye diseases In particular, various diseases related to the suppression of blood flow and suppression of ocular blood vessels, such as age-related macular degeneration, central exud
  • the preparation method of the present invention has simple technology, short production cycle, mild processing conditions, low energy consumption and good separation effect of 7 kinds of flavonoids from marshmallow flowers.
  • the raw materials and reagents used come from a wide range of sources, are low in cost, and are easy to achieve industrialized large-scale production.
  • the effective parts or extracts and preparations of flavonoids from marshmallow flowers provided by the present invention have the effect of promoting wound healing, and have obvious effects of promoting wound healing and treating diabetic ulcers and have good safety.
  • the present invention provides an enteric-coated preparation containing marshmallow flower extract.
  • the preparation has the characteristics of good stability and fast dissolution rate of the product. It is almost not released in the stomach, can be released quickly in the intestinal environment, and can alleviate the symptoms. Drugs stimulate the stomach and improve the bioavailability of the drug, allowing the drug to be more completely absorbed in the intestines. At the same time, it has obvious therapeutic effect on ulcerative colitis and provides a brand-new preparation for the clinical treatment of ulcerative colitis.
  • Figure 1 shows the dissolution process of 5-ASA and ZHT.
  • Figure 2 shows the effect of ZHT on DSS-induced colon atrophy
  • A Pictures of colon appearance of mice in each group
  • Figure 3 shows the effect of ZHT on the pathological changes of colon tissue induced by DSS;
  • Figure 4 shows the pathological pictures of each group in the oral ulcer drug efficacy experiment.
  • Figure 5 is a flow chart of acne model preparation, drug administration, and material collection.
  • Figure 6 is a flow chart of atopic dermatitis model construction and drug administration.
  • Figure 7 shows the effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model.
  • Figure 8 shows the effects of ZHT and HKY on the pathological changes in the skin of the DNFB-induced atopic dermatitis model.
  • Figure 9 is a representative graph of the skin wound area of each group of rats at different time points; (A) a representative graph of the skin wound area of each group of rats at different time points, (B) a graph of the skin wound healing curve of each group of rats. *P ⁇ 0.05, **P ⁇ 0.01vs.Model.
  • Figure 10 shows the results of HE staining of rat skin wound tissue and the quantitative graph of epidermal thickness;
  • A The graph of HE staining result of rat wound and edge tissue,
  • B Quantification of epidermal thickness of rat skin wound in each group. **P ⁇ 0.01, vs.Model; ##, P ⁇ 0.01, vs.Control.
  • Figure 11 shows the results of Masson staining of rat skin wound tissue and the quantitative map of collagen deposition;
  • A the result of Masson staining of rat wound and edge tissue,
  • B the quantitative map of collagen deposition in skin wounds of rats in each group.
  • N 6, *P ⁇ 0.05, **P ⁇ 0.01vs.Model; ##P ⁇ 0.01, vs.Control.
  • Figure 12 shows the changes in CD31 and VEGF mRNA levels in rat skin wound tissue;
  • A Changes in CD31 mRNA levels in rat skin tissue and rat skin wound tissue after 9 days of treatment,
  • B VEGF mRNA levels in rat skin tissue and rat skin wound tissue after 9 days of treatment Variety.
  • Figure 13 shows the results of CD31 immunofluorescence staining of rat skin wound tissue and the fluorescence intensity quantification chart;
  • A CD31 immunofluorescence staining of rat skin tissue after 9 days of treatment, the scale bar is 250 ⁇ m;
  • marshmallow flowers in the following examples are dried marshmallow corollas. Unless otherwise specified, they are from the same batch of marshmallow flowers.
  • the contents of seven components of the flavonoid effective parts of the marshmallow flowers prepared in each example were measured. These 7 ingredients refer to: hyperoside, rutin, isoquercetin, gossyrin-8-O- ⁇ -D-glucuronide, myricetin, quercetin-3'-O-glucose Glycosides, quercetin.
  • the detection method adopts the UPLC method reported in "Determination of 7 Components in Hollyhock Flowers by One Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis, Issue 12, 2013, 2082-2087) to determine the content of the components.
  • the material-to-liquid ratio of extraction (that is, extraction agent:extraction liquid) refers to the volume ratio.
  • Huangkui capsules described in the following examples are all Huangkui capsules produced and sold by Suzhong Pharmaceutical Group Co., Ltd. unless otherwise specified.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, and the loading liquid (water-soluble (liquid) concentration is 0.15g crude drug/mL, the sample liquid volume is 7BV, the sample is loaded at a flow rate of 2BV/h, 6BV pure water and 3BV 5% ethanol are used to remove impurities successively at a flow rate of 2BV/h, and 4BV 60% ethanol is used to remove impurities. Elute at a flow rate of 2BV/h to obtain an eluate. The eluate is then recovered under reduced pressure at 50°C to ethanol to obtain marshmallow flower flavonoid extract. The test content is adjusted according to the content measurement results through the addition method. The content of the seven ingredients is adjusted. The content of each component is as shown in the table below, and the total solid content of the effective parts of marshmallow flower flavonoids is 2.23g, and the total of the seven components is 1769mg, accounting for 79.32% of the total solid content.
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 2.5:1, the injection speed of the extraction is 80mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 4, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, and the sample solution is 2BV/ h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate, and then use the eluate at 60 After recovering ethanol under reduced pressure at °C, obtain the flavonoid extract of the marshmallow flower. Test the content.
  • Example 2-1 According to the method of the above-mentioned Example 2-1, the marshmallow flowers were enlarged 10 times, and two batches of flavonoid effective parts of the marshmallow flowers were prepared separately to obtain the content measurement data of the samples in Table 2-2. Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
  • 2-A1 contains 0.24% quercetin-3-O-acaxiglucoside based on peak area.
  • the proportion of each component is shown in Table 2-3 below.
  • Example 2-A1 Get the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and follow the steps of Example 2-A1 Mix the formula according to the proportion (replace rutin with quercetin 3-O-acaxiglucoside, adjust the proportion to 0.02), and prepare the total flavonoid composition as follows.
  • Example 2-A1 Get the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and follow the steps of Example 2-A1
  • the formula was mixed according to the proportion (replacing rutin with quercetin 3-O-acaxiglucoside, and the proportion was adjusted to 0.05) to prepare a total flavonoid composition.
  • the material-to-liquid ratio of the extraction i.e., extraction agent:extraction liquid
  • the injection speed of the extraction is 120mL/min
  • the speed of the extraction machine is 60Hz
  • the extraction level of the extraction is 4.
  • the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.15g crude drug/mL, and the volume of the sample solution is For 7BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluate, concentrate, and incubate at 60 Dry under reduced pressure at °C to obtain total solids, containing rutin 0.22%, hyperin 20.58%, isoquercetin 17.81, hibifolin 16.07%, myricetin 5.15%, quercetin-3'-O- Glucoside is 17.37%, quercetin is 1.77%, and the peak area of quercetin-3-O-acacia glycoside is converted to 0.43%.
  • the mass ratio of each component is shown in Table 2
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 3:1, the injection speed of the extraction is 100mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 5, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:8, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 8BV, and the sample solution is 2BV/ Load the sample at a flow rate of h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 3BV/h to obtain the eluate, and then use the eluate at 60°C After recovering ethanol under reduced pressure, obtain the flavonoid extract of marshmallow flowers.
  • the test content is based on the content measurement results.
  • Example 3-1 According to the preparation method of Example 3-1, 500g of marshmallow flowers were used for repeated experiments, and two batches of content data were obtained as shown in Table 3-2 below. Quercetin-3-O-Acacia glycoside content is 0.30-0.80%. Among them, A represents “each ingredient/total solids (%)" and B represents “each ingredient/isoquercitrin”.
  • the material-liquid ratio of the extraction (that is, the extraction agent :extraction liquid) is 3:1, the injection speed of extraction is 120mL/min, the speed of extraction machine is 60Hz, and the extraction level of extraction is 5.
  • the extraction liquid is processed D101 macroporous resin treatment
  • the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 7BV, and the sample is loaded at a flow rate of 2BV/h , use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 2BV/h to obtain an eluate, and then use the eluate to recover ethanol under reduced pressure at 60°C.
  • the marshmallow flower extract was obtained, and the content was tested. According to the content measurement results, the contents of the seven components were adjusted by the addition method so that the contents of each component were as shown in the table below, and 2.05g of the total solid content of the flavonoids effective parts of the marshmallow flower was obtained.
  • the resin treatment process is: the resin diameter-to-height ratio is 1:7, and the sample solution concentration is 0.15-0.5g crude drug/mL, the volume of the sample solution is 8BV, elute with 5BV pure water and 5BV 80% ethanol to obtain the eluate, concentrate, dry under reduced pressure at 60°C, test the content, and add Method, adjust the contents of the 7 components so that the contents of each component are as shown in the table below, and the total solids of the effective parts of the flavonoids of the marshmallow flowers are obtained, and the content of quercetin-3-O-acacia glycoside is 0.39%.
  • the extraction liquid is processed D101 macroporous resin treatment
  • the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.2g crude drug/mL, the volume of the sample solution is 6BV, and the sample is loaded at a flow rate of 2BV/h , use 1BV pure water and 4BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 3BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, and recover the ethanol under reduced pressure at 60°C to obtain
  • the content of the marshmallow flower extract was tested. According to the content measurement results, the contents of the seven ingredients were adjusted by the addition method to obtain 2.86g of the total solid content of the flavonoids effective parts of the marshmallow flower.
  • Extract the marshmallow flowers with 15 times of 70% ethanol under reflux Remove the ethanol from the extract to 0.5-1.0g crude drug/ml, add 10% hydrochloric acid solution to adjust the pH to 2.0-3.0, refrigerate, filter, wash, and take the precipitate. Add water to dissolve, and use ethyl acetate for continuous countercurrent extraction.
  • the material-to-liquid ratio of the extraction i.e., extraction agent:extraction liquid
  • the extraction level of the extraction is 4.
  • the extraction liquid is recovered under reduced pressure at 50°C to recover the solvent. Then, add water to dilute it, and process it with polyamide resin.
  • the resin treatment process is: the resin diameter-to-height ratio is 1:6, the concentration of the sample solution is 0.15-0.5g crude drug/mL, the volume of the sample solution is 6BV, and 4BV pure water is used. Elute with 4BV 70% ethanol to obtain the eluate, concentrate, and dry under reduced pressure at 60°C to obtain. The content of quercetin-3-O-acaxiglucoside is 0.47%.
  • the extracted material-to-liquid ratio (that is, the extraction agent :extraction liquid) is 2:1, the injection speed of extraction is 100mL/min, the speed of extraction machine is 40Hz, and the extraction level of extraction is 4.
  • the extraction liquid is processed D101 macroporous resin treatment
  • the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.3g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 2BV/h , use 3BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 70% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, test the content and adjust 7 types by adding 7 specific ingredients
  • the content of ingredients is as shown in the table below.
  • the eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain marshmallow flower extract.
  • the content is tested. According to the content measurement results, the content of the seven ingredients is adjusted by the addition method. , the addition amount does not exceed 10% of the total weight, so that the content of each component is as shown in the table below, and 3.16g of the total solid content of the effective parts of marshmallow flower flavonoids is obtained.
  • macroporous resin The diameter-to-height ratio is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, the sample is loaded at a flow rate of 2BV/h, and 6BV pure water and 3BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. , use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain an eluate. The eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain 3.56g of total solids of marshmallow flowers.
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 2.5:1, the injection speed of the extraction is 80mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 4, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/ml, the volume of the sample solution is 6BV, and the sample solution is 2BV/ h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate, test the content, and then Rutin and hyperoside were added to the eluent so that the contents of each component are as shown in the table below, and then ethanol was recovered under reduced pressure at 60°C to obtain 4.28g of total solids of marshmallow flowers.
  • the extracted material-to-liquid ratio is ( That is, the extraction agent:extracting liquid) is 2.5:1, and the extraction stage of the extraction is 4. After the extracting liquid is recovered under reduced pressure at 40°C, it is treated with D101 macroporous resin.
  • the processing process of macroporous resin is:
  • the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 1.5BV/h.
  • Example 2-B 30kg of the marshmallow flower mixture was crushed to coarse powder, and extracted by percolation with 18 times of 70% ethanol to obtain a marshmallow flower extract; the ethanol was removed from the marshmallow flower extract, diluted with water, and continuously counter-currently used with ethyl acetate.
  • the material-to-liquid ratio of extraction that is, extraction agent:extraction liquid
  • the injection rate of extraction is 80mL/min
  • the extraction speed is 80mL/min.
  • the extraction machine speed is 40Hz
  • the extraction level is 4.
  • the processing technology of the macroporous resin is: the diameter of the macroporous resin is high The ratio is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 5BV, the sample is loaded at a flow rate of 1.5BV/h, and 3BV pure water and 4BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. Elute with 5BV 60% ethanol at a flow rate of 1.5BV/h to obtain the eluate. The eluate is recovered under reduced pressure at 65°C to recover the ethanol to obtain the total solids of marshmallow flowers. Dry in vacuum, crush, and detect 7 kinds of flavonoids. Content (%), the results are as follows, in which the content of quercetin-3-O-acaxiglucoside is 0.20-0.50%.
  • Preparation process Prepare the effective part according to any method of Examples 1-7, add water for injection, stir and dissolve, add to 100L, adjust the pH to 7, filter, fill, and sterilize to obtain eye drops.
  • the pH adjuster used is sodium hydroxide or/and hydrochloric acid.
  • Mode of administration intragastric administration.
  • STZ sodium citrate solution was injected intraperitoneally at a dose of 100 mg/kg for 7 consecutive days.
  • the blood glucose level of the mice was measured in the tail vein, and the diabetes model was established based on blood glucose greater than 16.7 mmol/L. If blood sugar is greater than 13.8mmol/L, urine protein and renal function abnormalities appear, it indicates that the modeling is successful.
  • Experimental groups normal group, model group, positive drug group (dapagliflozin tablets, 45.5 mg/kg), sample group prepared by the method of Example 2-1 (62.4 mg/kg) and sample group prepared by the method of Example 6 (62.4 mg/kg), and the sample group (62.4 mg/kg) was prepared by the method of Example 8, with a total of 6 groups, 15 animals in each group. After 4 weeks of administration, urine protein and urinary creatinine were detected, and daily food intake and mouse status were calculated. The test results are shown in Table 12-1 below.
  • Each group of Examples has a reducing effect on urinary protein, among which the sample group of Example 2-1 can significantly reduce urinary protein and urinary protein/urinary creatinine (ACR).
  • ACR urinary protein/urinary creatinine
  • mice Animal models: db/db mice, 16 weeks old, C57BL/6 mice, 16 weeks old.
  • Mode of administration intragastric administration.
  • mice C57BL/6 mice were divided into normal group, db/db mice were divided into model group, positive drug group (dapagliflozin tablets, 45.5mg/kg), sample group prepared by the method of Example 3-1 ( 62.4 mg/kg) and the sample group (62.4 mg/kg) prepared by the method of Example 9-1, the sample group (62.4 mg/kg) prepared by the method of Example 2-A3, and the sample group (62.4 mg/kg) of Example 7-1 ), 15 mice in each group. After 4 weeks of administration, urine protein, urinary creatinine and daily food intake were measured, and the status of the mice was observed. The test results are shown in Table 12-2 below.
  • ACR urinary protein/urinary creatinine
  • Example 13 Effects of flavonoids on ocular blood flow and choroidal neovascularization
  • mice rats, body weight 150-180g, SPF grade, half male and half male.
  • Mode of administration intragastric administration.
  • Drug grouping and dosage normal group, model group, positive control drug group (vertibofen 1.35 mg/kg), Example 2-1 sample group (69.9 mg/kg), Example 3-1 sample group ( 87.36mg/kg), Example 7-2 sample group (87.36mg/kg), Example 9-1 sample group (87.36mg/kg), Example 9-2 sample group (87.36 mg/kg), 15 rats in each group.
  • the choroidal blood flow inhibition rate and choroidal angiogenesis area of the eye were measured. The test results are shown in Table 13 below.
  • Example 2-1 sample group, Example 3-1 sample group, Example 9-1 sample group, Example 9- Sample group 2 can significantly reduce choroidal angiogenesis.
  • Example 4-1, Example 4-2, Example 5-1, and Example 5-2 sample experiments also have the same characteristics as Example 2-1, Example 3-1, and Example 9-1 in this example.
  • the above-mentioned aspects of Embodiment 9-2 basically have the same functions or effects.
  • mice SD rats, body weight 200-250g, SPF grade, half male and half male.
  • the modeling success criteria are based on the diagnostic criteria for acute kidney injury recommended by the 2012 KDIGOAKI clinical guidelines (people who meet one of the following conditions can be diagnosed): 1 Serum creatinine rises by more than 26.5umol/L (0.3mg/dl) within 48 hours; 2 Serum creatinine increases by more than 50% of the baseline - confirmed or presumed to occur within 7 days; 3 Urine output decreases ⁇ 0.5ml/(kg*h) and lasts for more than 6 hours. Among them, in animal models, an increase in serum creatinine exceeding 50% of the baseline is often used as the diagnostic criterion for acute kidney injury, that is, the modeling success criterion.
  • Drug grouping and dosage normal group, model group, positive control drug group (irbesartan 50 mg/kg/d), Example 2-1 sample group (69.9 mg/kg), Example 3-1 sample group (87.36mg/kg), Example 7 sample group (87.36mg/kg), Example 9-1 sample group (87.36mg/kg), Example 9-2 sample group (87.36mg/kg, according to Example 9 -2 prepared solid material), 15 animals per group, and 4 weeks after administration, the choroidal blood flow inhibition rate and choroidal angiogenesis area of the eye were measured. The test results are shown in Table 14 below.
  • the serum creatinine and serum urea nitrogen of rats with contrast agent renal injury in the model group were significantly higher than those of each administration group; Example 9-1 sample group, Example 2-A1 sample group, Example 3.1 sample, Example 8 sample and Examples All 6 samples reduced serum creatinine and serum urea nitrogen to varying degrees.
  • mice Twenty-six MRL/lpr male mice (lupus nephritis mice) were selected and one group was observed. The body weight was 18-22g and they were 13 weeks old. They were purchased from Nanjing Junke Bioengineering Co., Ltd. and 24 male C57BL/6 mice were selected. Only as normal group, body weight 18-22g, 13 weeks old, purchased from Wuhan Hualianke Biotechnology Co., Ltd.
  • Drug grouping and dosage normal group, MRL/lpr mouse model group, positive control drug group (dexamethasone 1 mg/kg), Example 2-B sample group (69.9 mg/kg), Example 3-1 Sample group (87.36mg/kg), Example 7-1 sample group (87.36mg/kg), Example 5-2 sample group (87.36mg/kg), Example 8 sample group (87.36mg/kg) each group 15 rats were administered intragastrically for 4 weeks.
  • Example 5-2 sample group and Example 2-B sample Group Example 3-1 sample, Example 8 sample and Example 7-1 sample can all improve urinary protein, serum antinuclear antibody levels, and IgG and C3 deposition in renal tissue, and Example 2-B The sample, Example 3-1 sample, and Example 5-2 sample have better effects.
  • mice rats, body weight 150-180g, SPF grade, half male and half male.
  • Mode of administration intragastric administration.
  • Drug grouping and dosage normal group, Huangkui capsule group (extract 8g/kg), Example 2-A1 sample group (5g/kg), Example 9-2 sample group (8g/kg), each group 30 animals, once a day for 12 weeks, the dosage is calculated based on the extract.
  • Embodiment 2-A3, Embodiment 3-1, Embodiment 4-1, Embodiment 5-2, and Embodiment 9-1 have been verified to be equivalent to Embodiment 2-A1 and Embodiment 9-2 in this embodiment.
  • the above-mentioned safety functions or effects are basically the same.
  • Example 17 Effect of effective fractions of total flavonoids on idiopathic pulmonary fibrosis
  • Preparation of experimental sample 1 Take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, as shown in the table 17-1 Feeding ratio, weigh each active ingredient, mix, and prepare a pharmaceutical composition.
  • mice Kunming mice, half male and half female, weighing 18-22 grams, clean grade.
  • Animal grouping randomly divided into normal group, model group, control drug group (rosiglitazone 5mg/kg), Example 2-A3 sample group (70mg/kg), Example 9-2 sample group (70mg/kg) kg), Huangkui capsule sample group (180mg/kg based on extract), experimental sample group 1 (62.4mg/kg), 10 mice in each group.
  • mice were adaptively raised for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01ml/g). The patients were placed in a supine fixed position, routine disinfection was performed, a midline cervical incision was made, and the trachea was exposed by blunt dissection. The model group, control drug group, and treatment group were punctured and slowly injected bleomycin (5 mg/kg) into the tracheal cartilage ring space, and the normal control group was injected Equal volume of normal saline was injected. Immediately after injecting the drug, the mice were rotated upright for 3-5 minutes to allow the drug solution to be evenly distributed in the lungs on both sides. The skin was then sutured and the sutures were disinfected. After they woke up, they were sent to a clean observation room for feeding.
  • bleomycin 5 mg/kg
  • Administration will begin on the second day after modeling.
  • the administration group will be administered intragastrically according to the above dosage, once a day.
  • the normal group and the model group will be administered the same volume of distilled water 10ml/kg in the same way for 28 consecutive days. .
  • Staining of pathological sections of lung tissue After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis is divided into four grades.
  • the experimental results are shown in Table 17-2 below.
  • the experimental results showed that the degree of alveolitis and pulmonary fibrosis in the lung tissue of mice in the model group was significantly aggravated, showing typical pulmonary fibrosis parenchymal lesions. After 28 days of administration, the lung coefficient of mice in each dose group decreased significantly. The degree of alveolitis and pulmonary fibrosis in the lung tissue is significantly reduced.
  • the effective part of the total flavonoids of the present invention can significantly reduce the degree of alveolitis and pulmonary fibrosis in mice with pulmonary fibrosis, has a protective effect on the lungs of mice with pulmonary fibrosis, and can slow down the damage to the lung tissue caused by fibrosis.
  • Example 1 Example 2-1, Example 2-A2, Example 3.2-1, and Example 5-2 also have the same characteristics as Example 2-A3 and Example 9-2 in this example. Fibrosis basically has the same effect or effect.
  • Sodium-glucose contransporter2 (SLC5A2, also known as SGLT2) is a gene encoding a sodium-glucose cotransporter and can reabsorb more than 90% of the glucose filtered by the glomerulus. SLC5A2 is only expressed at the brush border within the epithelial cell membrane of the proximal tubule of the kidney and is considered a marker of the proximal tubule. The main function is to recover filtered sodium and glucose. In the kidney tissue of patients with diabetes and diabetic nephropathy, inhibiting the expression of SLC5A2 protein can play a role in excreting sodium and reducing blood sugar, thus protecting the kidney tissue of patients with diabetes and diabetic nephropathy.
  • Mode of administration intragastric administration.
  • mice were divided into 3 groups when UACR>200mg/g, diabetic nephropathy group (DKD), Huangkui capsule group (DKD+HK, 0.84g/kg/d) and total flavonoids group (DKD+HT, Example 2-A2 sample, 0.0755g/kg/d), Huangkui capsule group and total flavonoids group were administered for four weeks (28d).
  • DKD diabetic nephropathy group
  • DKD+HK Huangkui capsule group
  • DKD+HT total flavonoids group
  • Example 2-A2 sample 0.0755g/kg/d
  • Huangkui capsule group and total flavonoids group were administered for four weeks (28d).
  • the Enzyme-linked immunosorbent assay (ELISA) method is used to detect trace albumin (UACR) in the urine of db/db mice
  • ELISA Enzyme-linked immunosorbent assay
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • IHC ImmunoHistoChemistry
  • DKD body weight before grouping of the HK group (Huangkui capsule group) and HT group (total flavonoids group)
  • HT total flavonoids group
  • the UACR value of the HT group was significantly lower, and there was a significant difference.
  • Huangkui capsules and total flavonoids can effectively reduce microalbuminuria in db/db mice.
  • Huangkui capsules and total flavonoids can inhibit the expression of SLC5A2 protein on the cell membrane of the proximal convoluted tubule, reduce the expression of SLC5A2 protein in the kidneys of DKD mice, and effectively inhibit the activity of SLC5A2 protein, thereby effectively reducing the reabsorption of glucose in the kidneys of DKD mice.
  • Example 3-1 Referring to the preparation method of the above-mentioned Example 3-1, repeated experiments were carried out with 5000g of marshmallow flowers, and two batches of content (unit: %) data were obtained as shown in the table below. Quercetin-3-O-Acacia glycoside content is 0.30-0.80%.
  • mice were kept in an SPF-level animal room throughout the entire process, with a light-to-dark time ratio of 1:1, room temperature controlled at 23 ⁇ 2°C, and humidity controlled at 55%. The mice could eat and drink freely; the bedding was changed every three days. , to ensure that the mice are in a dry and clean environment.
  • mice After 5 days of adaptive rearing, the mice were randomly divided into 6 groups, with two cages in each group and 4 mice in each cage. They are the blank control group (Control), the ambrosia total flavonoid extract control group (150mg/kg/day ZHT), the model group (DSS), and the model + 5-aminosalicylic acid group (DSS + 200mg/kg/day 5 -ASA), model+ZHT group (DSS+75mg/kg/day ZHT).
  • Control the ambrosia total flavonoid extract control group
  • DSS model group
  • DSS + 5-aminosalicylic acid group DSS + 200mg/kg/day 5 -ASA
  • model+ZHT group DSS+75mg/kg/day ZHT
  • mice ulcerative colitis model was induced by free drinking of 3% DSS for 7 consecutive days.
  • Each mouse cage (4 mice) was replaced with 3% DSS solution (80mL) every 2 days to ensure that the DSS solution was effective; blank
  • the mice in the control group drank pure water freely, and each cage (4 mice) was changed every 2 days.
  • Different doses of ZHT or the positive therapeutic drug 5-ASA were administered intragastrically from 9:00 to 10:00 every day at a dose of 0.1mL/10g b.w., and the blank group and model group were given the same dose of blank carrier solution (containing 2% DMSO, 2 % Tween 80 and 0.5% CMC-Na) for 7 consecutive days.
  • blank carrier solution containing 2% DMSO, 2 % Tween 80 and 0.5% CMC-Na
  • DAI disease activity index
  • mice The body weight of mice was recorded before daily administration, and feces were collected for fecal occult blood test.
  • the DAI score is used to score the severity of the disease based on three indicators: weight loss, fecal viscosity and fecal occult blood. Each indicator corresponds to 0-4 points.
  • the scoring scale Accurate as Table 20-2.
  • Fecal occult blood test Take a small amount of feces and apply it in the middle of the glass slide (the glass slide has been pre-fired to reduce color errors), add 3 drops of 10/L methaminophenol sulfate acetic acid solution and 3 drops of 3% hydrogen peroxide solution , mix well, observe the results immediately, and rate according to Table 20-3.
  • mice in each group were fasted for 24 hours and sacrificed by cervical dislocation. Cut open the mouse's abdominal cavity to expose the mouse's colon. Take all the mouse's colon tissue, measure and record its length, place it in newly prepared pre-cooled physiological saline to flush the intestinal contents, and cut out 1cm of the colon near the rectum. The tissue was fixed overnight in 4% paraformaldehyde for preparation of paraffin sections and subsequent pathological examination. The remaining tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The blood samples were allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 15 min. The serum was collected and quickly stored at -80°C for subsequent testing.
  • MPO myeloperoxidase
  • MPO is a functional marker and activation marker of neutrophils. Under pH 6.0 conditions, using H 2 O 2 and 3,3′-dimethoxybenzidine hydrochloride as substrates, MPO can catalyze the formation of substrates. For orange-yellow product, the amount of product produced was determined by colorimetry at 460 nm, thereby calculating the MPO content.
  • Sample preprocessing Accurately weigh the tissue weight, add homogenization medium at a weight-to-volume ratio of 1:9, grind continuously for 1 minute in a tissue grinder at a frequency of 60Hz, repeat 5 times, until no precipitation is visible to the naked eye, and prepare 10% tissue homogenate;
  • the colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in conventional paraffin. Take serial transverse sections (10 ⁇ m) of the embedded colon tissue, and then perform H&E staining.
  • the H&E staining steps are as follows:
  • Dewaxing Dewaxing with xylene I for 10 minutes, dewaxing with xylene II for 5 minutes;
  • Ethanol soaking soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
  • Anti-blue Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
  • Eosin staining stain for 5 minutes, then wash with tap water for 30 seconds;
  • Ethanol dehydration Use different contents of ethanol for dehydration in sequence. The order is as follows: 85% ethanol dehydration for 20 seconds, 90% ethanol dehydration for 30 seconds, 95% ethanol I for 1 minute, 95% ethanol II for 1 minute, and absolute ethanol I for 2 minutes. , dehydrate with absolute ethanol II for 2 minutes;
  • Pathological examination The degree of colon damage is scored based on pathological indicators such as the degree of colon tissue lesions, inflammatory cell infiltration, and crypt cell damage. The specific scoring standards are shown in Table 20-5.
  • a 260 /A 280 should be between 1.8-2.0.
  • Use enzyme-free water to adjust the RNA concentration to 1 ⁇ g add 3 ⁇ L of 5 ⁇ gDNA wiper Mix and heat at 42°C for 2 min.
  • After removing the genomic DNA add 5 ⁇ L of 4 ⁇ qRT Super MixII to reach a 20 ⁇ L system, and perform a 20 ⁇ L system at 25°C-2min, 50 °C-30min, 85°C-5min reverse transcription reaction, cDNA is stored at -80°C for later use.
  • the contents of TNF- ⁇ , IL-1 ⁇ and IL-6 in colon tissue were detected.
  • the DAI score is calculated by adding the three indicator values in Table 20-2 and taking the average. As shown in Table 20-7, the DAI score of the mice in the DSS model group increased significantly from the 3rd day. Compared with the Control group, the difference was statistically significant (P ⁇ 0.01). On the 6th day of the experiment, 3 mice had diarrhea. There were 7 diarrhea cases in 7 days; the DSS+ZHT (75mg/kg/day) group’s DAI score increased more slowly. On days 4, 5, 6, and 7, the difference in DAI score was statistically significant compared with the DSS model group ( P ⁇ 0.01), no mice developed diarrhea during the experiment; the positive therapeutic drug 5-ASA (200mg/kg/day) alleviated the symptoms of DSS-induced colitis and reduced the DAI score.
  • 5-ASA 200mg/kg/day
  • the colon length of the Control group was 7.34 ⁇ 0.08cm, and the colon of mice in the DSS model group shrunk to 4.85 ⁇ 0.18cm. Compared with the Control group, the difference was significant (P ⁇ 0.01); ZHT ( The colon length (7.43 ⁇ 0.13cm) of the 150mg/kg/day) group was close to that of the Control group.
  • the MPO content in the colon tissue of the Control group was 0.30 ⁇ 0.04U/g, and the MPO content in the colon tissue of the mice in the DSS model group increased to 1.04 ⁇ 0.10U/g.
  • the difference was statistically significant (P ⁇ 0.01) ; 75mg/kg/day ZHT has an inhibitory effect on the increase in MPO content in the colon induced by DSS.
  • the inhibition rate of 75mg/kg/day ZHT is 92.8 ⁇ 19.4%, and the difference is statistically significant (P ⁇ 0.01); Positive therapeutic drugs 5 -ASA (200mg/kg/day) significantly inhibited the DSS-induced increase in MPO content in the colon, with an inhibition rate of 88.7 ⁇ 9.9% (P ⁇ 0.01).
  • This example uses a mouse model of ulcerative colitis induced by DSS, and uses 5-aminosalicylic acid as a positive control drug to examine the therapeutic effect of 75 mg/kg/day ambrosia total flavonoids in the treatment of colitis.
  • ZHT 75 mg /kg/day
  • mice significantly protected mice from DSS-induced weight loss, diarrhea, hematochezia and other colitis symptoms; significantly improved DSS-induced colon atrophy, crypt structure damage, tissue inflammatory infiltration and other histopathological changes; reduced DSS-induced
  • the tissue MPO levels increased and the expression of inflammatory factors showed a good therapeutic effect on colitis, which was equivalent to the effect of the positive therapeutic drug 5-aminosalicylic acid.
  • ZHT has certain curative effects on colitis.
  • the effect at 75mg/kg/day is equivalent to that of the positive drug 5-ASA, and its safety is good.
  • Example 1 It has been verified that the samples of Example 1, Example 2-A3, Example 3.2-2, Example 5-2, and Example 19-2 also have the same effect on colitis as described in Example 19-1 in this example. or effect.
  • SPF male SD rats were randomly divided into groups, with 9-11 rats in each group. They are blank group (normal group), model group, marshmallow flower ethanol extract group, marshmallow flower water extract group, marshmallow flower total flavonoid extract group, and watermelon frost group. All rats were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg). A glass tube with an inner diameter of 4 mm at the lower end was taken and a small cotton ball was placed at the lower end. For the rats in the model group and each administration group, drop 90% phenol solution into the glass tube until the cotton ball is just soaked, and place the lower end flatly on the buccal membrane about 1 mm from the left and right corners of the mouth of the rat for 30 seconds. It can be seen that there is a white lesion about 4-5mm in diameter in this area. In the blank group, double distilled water was used instead of phenol for the same treatment.
  • each drug into a suspension with double-distilled water, and apply it evenly on the oral mucosal wound after modeling, 4 times a day.
  • the dosage of each drug is as follows: watermelon cream, 20 mg/wound/day; marshmallow flower ethanol extract 10 mg/wound/day; marshmallow flower aqueous extract 8.75 mg/wound/day, marshmallow flower total flavonoid extract 1.05 mg/wound/day.
  • Administration was continued for 5 days, and the ulcer area and ulcer healing were observed before and after administration. On the 6th day, the ulcer surface was photographed after being anesthetized with chloral hydrate (400 mg/kg). After the rats were sacrificed by cervical dislocation, the bilateral buccal membrane tissues of the rats were removed.
  • Data are expressed as mean ⁇ SD.
  • the significant difference between the model group and the normal group (blank control group) was tested with Student's-t test; the significant difference between the model group and the other drug groups was analyzed with One-way ANOVA test, and further with Newman- Keuls multiple comparison test is used to test the significant difference between two groups.
  • the oral mucosa surface of the rats in the blank group was smooth, uniform in color, and free of blood spots. After phenol stimulation, the local mucosa quickly turns white, and edema can be seen after 2 hours. Inflammatory exudation, bleeding, and local ulcer formation can be seen within 1 to 5 days, and a yellow-white pseudomembrane appears on the surface and falls off. On the 6th day, except for the blank group, all rats in each group could see sunken wounds; 50% of the rats in the model group had obvious dilation of blood vessels in the wounds, and 1 rat had yellow pus spots. The results are shown in Table 21-1 (each rat There are two wounds in the mouth).
  • Scoring standard for the degree of lesions 0.5-4 points according to the severity of the lesions from mild to severe.
  • the scoring standards are: 0.5 points for mild or very mild lesions; 1 point for mild lesions; 2 points for moderate lesions; Severe lesions were scored as 3 points; very severe lesions were scored as 4 points; no lesions were scored as 0 points.
  • the ulcer length is measured with a "microscope side micro-ruler" under a 40x light microscope (eyepiece 4 ⁇ , objective lens 10 ⁇ ). The length of the ulcer is scored as 1 point if it is ⁇ 5mm/40X, and 2 points if it is >5mm/40X or 5mm/40X. . Add all scores by category to calculate the average lesion score of each group of animals. The higher the score, the more severe the disease.
  • the pathological changes in each group are shown in Table 21-2, and the pathological pictures are shown in Figure 4.
  • the short black arrow indicates skin necrosis and shedding to form ulcers (without epidermal coverage)
  • the long black arrow indicates inflammatory cells infiltrating into the dermis
  • the green arrow indicates The blood vessels are dilated and congested
  • the green arrow indicates fibroblasts
  • the green star indicates the thin layer of stratified squamous epithelium on the skin surface, with mild surface keratinization.
  • Example 22-1 Pharmacodynamic evaluation of total flavonoids from ambrosia in the treatment of acne
  • ZHT total flavonoids
  • ZHT-1 Total flavonoids of Z. annua
  • ZHT-2 Total flavonoids of ambrette
  • Table 22-1 ZHT Cream Prescription.
  • Aqueous phase Weigh the prescribed amount of ethanol, add ZHT, stir to dissolve, add Tween 80, sodium lauryl sulfate, glycerin, isopropyl myristate, methylparaben, azone, and purify into the beaker. water and stir until evenly dispersed.
  • Oil phase Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petroleum jelly into a beaker, heat and dissolve in a water bath at 80°C until it becomes liquid.
  • Cultivation method of P.acnes Add 4mL of RCM culture medium and 1mL of P.acnes bacterial liquid into a sterile test tube and mix evenly by pipetting. Wrap the sterile container with a sealing film and put it vertically into an anaerobic container containing an anaerobic gas-generating bag. placed in an oxygen culture bag, placed in an electric constant-temperature incubator at 37°C, and cultured once every 2 days.
  • Preparation method of P.acnes injection ready for use. Use physiological saline to dilute the concentration of the bacterial solution to 6 ⁇ 10 7 CFU/mL to obtain P.acnes injection (the McFarland turbidity of the bacterial liquid is approximately 2 McFarland. After diluting 10 times with normal saline, 6 ⁇ 10 7 CFU is obtained. /mL concentration of bacterial suspension, measured OD 600nm between 0.63-0.65.)
  • the rat acne model establishment and drug efficacy evaluation method are shown in Figure 5-1.
  • the acne model was established using 100% oleic acid combined with P.acnes. On the 8th day, treatment with ZHT cream, a therapeutic drug, was started, and 2.5% oxygen was used. Benzoyl/1% clindamycin (BPO) hydrogel was used as a positive control.
  • BPO clindamycin
  • the acne model was constructed using the right auricle of the rat as a carrier, and the left The ears were not treated. 100% oleic acid was applied to the inner side of the right ear of the rats once at 9 am every day, 0.5 mL each time.
  • the blank group was applied with the same amount of normal saline; oleic acid was applied to the inner side of the right ear of the rats for 1 hour every other day.
  • the prepared P.acnes injection was injected into the inner side of the ear, 50 ⁇ L each time; the blank group was injected with physiological saline.
  • the rats were regrouped to ensure that the severity of the model in each group of rats was basically the same. They were divided into blank control group (Veh), model group (Model), model + benzoyl peroxide/ Clindamycin group (Model+BPO), model+1%ZHT-1 group (Model+1%ZHT-1), model+1%ZHT-2 group (Model+1%ZHT-2), female in each group There are one cage each for rats and male rats, with three rats in each cage. Starting from the afternoon of the 8th day after grouping, 50 mg of the drug to be tested was applied to the ears every afternoon.
  • the right auricle of the rat was photographed and recorded, and symptoms were scored.
  • the severity of the disease was evaluated from the four indicators of redness, swelling, desquamation, papules, and cysts. Rating, each indicator corresponds to 0-4 points, and the scores of the four indicators are added to obtain the total score.
  • the scoring standard is: 0 points, no symptoms; 1 point, mild; 2 points, moderate; 3 points, severe; 4 points, extremely severe.
  • the rats were sacrificed on the 19th day, and the right auricle of the rats was completely cut off. 1/4 of the ear tissue was fixed in 4% paraformaldehyde overnight for paraffin section preparation and pathological examination. The remaining ear tissue was quick-frozen in liquid nitrogen. Afterwards, it was stored at -80°C for subsequent ELISA and qPCR detection.
  • the rat ear tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and then stained with H&E.
  • the pathological changes of the rat acne model were evaluated according to histological criteria.
  • the inner surface of the right auricle of the rats in the Veh group was always smooth and flat, without symptoms such as redness, swelling, desquamation, papules, and cysts, and the thickness of the right auricle remained basically unchanged.
  • the right auricle of the rats in the Model group showed obvious symptoms of redness, swelling, and desquamation on the 7th day of modeling, and the symptoms were redness, swelling, and desquamation.
  • the symptoms continued to worsen as the modeling progressed, and the surface of the right auricle was uneven and accompanied by cysts in the later stages;
  • the thickness of the right auricle of the rats increased significantly after modeling, and starting from the 6th day, it increased significantly compared with the Veh group , the difference was statistically significant (P ⁇ 0.01) (Table 22-3);
  • the skin lesion symptom score showed that the degree of skin lesions gradually worsened after modeling. Starting from the 3rd day, there was a significant difference compared with the Veh group (P ⁇ 0.01) (Table 22-4).
  • the thickness of the epidermal layer was measured to be 12.56 ⁇ 2.39 ⁇ m.
  • the boundaries between hair follicles, epidermis, and dermis were clearly visible, and inflammatory cell infiltration was occasionally seen in the dermis;
  • the thickness of the epidermal layer of the rat ear tissue in the Model group was 171.73 ⁇ 17.18 ⁇ m, which was significantly increased compared with the Veh group (P ⁇ 0.01).
  • the epidermis and cuticle were excessively thickened, inflammatory cells could be gathered in the cuticle, hair follicles were expanded, and the hair follicle mouth,
  • the infundibulum was filled with keratinized material, the keratin plugs were severely blocked and enlarged into a pot shape, the sebaceous glands were atrophied, and the dermis was edematous with a large amount of inflammatory infiltration.
  • the pathological score was 3.83 ⁇ 0.17 points, which was significant compared with the Veh group. difference (P ⁇ 0.01).
  • the thickness of the epidermal layer of the rat ear tissue in the Model+BPO group was 104.90 ⁇ 7.35 ⁇ m, which was significantly lower than that of the Model group (P ⁇ 0.01); the stratum corneum was slightly thickened, there were less keratinized substances at the hair follicle opening and infundibulum, and the sebaceous glands were normal in shape. , there was no edema in the dermis, and the degree of inflammatory cell infiltration was reduced compared with the Model group.
  • the pathological score was 2.67 ⁇ 0.33 points, which was significantly lower than the Model group (P ⁇ 0.01).
  • the thickness of the epidermal layer of rat ear tissue in the Model+1% ZHT-2 group and the Model+1.0% ZHT-1 group were 133.36 ⁇ 13.17 ⁇ m and 128.08 ⁇ 8.83 ⁇ m respectively. 1.0% ZHT-1 can significantly reduce the epidermal growth caused by modeling. Thick (P ⁇ 0.05).
  • 1% ZHT-2 and 1.0% ZHT-1 can improve the excessive thickening of the epidermis and stratum corneum, reduce dermal edema and reduce inflammatory cell infiltration in a dose-dependent manner.
  • the pathological scores of each group were 3.33 ⁇ 0.33 points, 3.33 ⁇ 0.33 points.
  • the mRNA expression of Mcp-1 in the rat ear tissue of the Model group increased to 3.11 ⁇ 0.30 times that of the Veh group, (P ⁇ 0.01); compared with the Model group, applying BPO hydrogel could significantly improve the Mcp-1 induced by modeling.
  • the mRNA expression of Mcp-1 was up-regulated, and the improvement rate was 70.9 ⁇ 14.9% (P ⁇ 0.01vs.Model); applying 1% ZHT-1 cream can improve the up-regulation of Mcp-1 mRNA expression induced by modeling, and the improvement rate was 25.5 ⁇ 16.0%, but the difference was not statistically significant (P>0.05 vs. Model); while applying 1% ZHT-2 cream had an inhibitory effect on the up-regulation of Mcp-1 mRNA expression induced by modeling.
  • the mRNA expression of Il-1 ⁇ in the rat ear tissue of the Model group increased to 14.23 ⁇ 2.24 times that of the Veh group, and the difference was statistically significant (P ⁇ 0.01); compared with the Model group, applying BPO hydrogel can improve modeling.
  • the induced Il-1 ⁇ mRNA expression was up-regulated, and the improvement rate was 76.6 ⁇ 6.9% (P ⁇ 0.05vs.Model); applying 1% ZHT-2 and 1% ZHT-1 cream can improve the modeling-induced Il-1 ⁇
  • the mRNA expression was up-regulated, and the improvement rates were 18.5 ⁇ 15.3% (P>0.05vs.Model) and 58.4 ⁇ 6.5% (P ⁇ 0.01vs.Model) respectively.
  • the mRNA expression of Il-6 in the rat ear tissue of the Model group was significantly increased to 4.62 ⁇ 0.50 times that of the Veh group, and the difference was statistically significant (P ⁇ 0.01); apply BPO hydrogel, 1% ZHT-2, 1% ZHT-1 cream reduces the up-regulation of Il-6 mRNA expression induced by modeling, and the improvement rates are 60.0 ⁇ 13.5% (P ⁇ 0.05vs.Model), 46.2 ⁇ 5.4% (P ⁇ 0.05vs.Model), and 61.1 ⁇ 20.3% (P ⁇ 0.05vs.Model).
  • the mRNA expression of Tnf- ⁇ in the rat ear tissue of the Model group was significantly increased to 3.98 ⁇ 0.52 times that of the Veh group (P ⁇ 0.01); applying BPO hydrogel can improve the up-regulation of Tnf- ⁇ mRNA expression induced by modeling and improve The rate was 71.2 ⁇ 10.0% (P ⁇ 0.05vs.Model); applying 1% ZHT-2 and 1% ZHT-1 cream could inhibit the up-regulation of Tnf- ⁇ mRNA expression induced by modeling, and the improvement rates were 18.2 ⁇ 12.9 respectively. % (P>0.05vs.Model), 58.3 ⁇ 15.0% (P ⁇ 0.05vs.Model).
  • the protein content of IL-1 ⁇ in the ear tissue of the Veh group was 175.2 ⁇ 29.5pg/mL, and the protein content of IL-1 ⁇ in the ear tissue of the rats in the Model group was 270.1 ⁇ 18.9pg/mL, which increased to 1.54 ⁇ 0.11 times that of the Veh group ( P ⁇ 0.05); the protein content of IL-1 ⁇ in rat ears after administration of BPO, 1% ZHT-2 and 1% ZHT-1 was 224.0 ⁇ 20.0pg/mL, 194.4 ⁇ 20.5pg/mL, 179.7 ⁇ 25.8pg/ mL, respectively improved the IL-1 ⁇ induced by modeling by 48.5 ⁇ 21.1% (P>0.05vs.Model), 79.8 ⁇ 21.6% (P ⁇ 0.05)vs.Model and 95.2 ⁇ 27.2% (P ⁇ 0.05vs.Model). Increase in protein content.
  • the protein content of IL-6 in the ear tissue of the Veh group was 93.7 ⁇ 20.4pg/mL, and the protein content of IL-6 in the ear tissue of the rats in the Model group was 266.4 ⁇ 15.2pg/mL, which increased to 2.84 ⁇ 0.16 times that of the Veh group.
  • the difference was extremely significant (P ⁇ 0.01); after administration of BPO hydrogel, the protein content of IL-6 in rat ear tissue was 189.0 ⁇ 10.8pg/mL, inhibiting 44.8 ⁇ 6.3% (P ⁇ 0.01vs.Model). Model-induced increase in IL-6 protein content in rat ear tissue.
  • the protein content of IL-6 in the ear tissue of the rats in the groups given 1% ZHT-2 and 1% ZHT-1 was 237.2 ⁇ 21.4pg/mL and 235.2 ⁇ 25.6pg/mL respectively, and each group improved by 16.9 ⁇ 12.4% respectively.
  • P>0.05vs.Model 18.1 ⁇ 14.8%
  • P>0.05vs.Model Model-induced increase in IL-6 protein content in rat ear tissue.
  • the TNF- ⁇ protein content in the ear tissue of the Veh group was 103.7 ⁇ 23.7pg/mL, and the Tnf- ⁇ protein content in the ear tissue of the Model group was 423.2 ⁇ 37.0pg/mL, which increased to 4.08 ⁇ 0.58 times that of the Veh group.
  • the difference is extremely significant (P ⁇ 0.01); the protein content of TNF- ⁇ in rat ear tissue after administration of BPO, 1% ZHT-2, and 1% ZHT-1 was 286.2 ⁇ 46.1pg/mL and 224.9 ⁇ 49.1pg/mL respectively.
  • Example 4-1 and Example 19-2 have been verified to have functions or effects that are basically consistent with the above-mentioned acne effects of Example 19-1 and Example 6 in this example.
  • Example 22-2 Pharmacodynamic evaluation of total flavonoids from ambrosia in the treatment of atopic dermatitis
  • mice SPF grade Balb/c mice were purchased from the Comparative Medicine Center of Yangzhou University [Experimental Animal Production License Number: SCXK (Su) 2017-0007], 7-8 weeks old, weighing 18-20 g. The mice were kept in an environment with a room temperature of 23 ⁇ 2°C, a humidity of 55%, and a light to dark time ratio of 1:1. The mice were allowed to eat and drink freely.
  • mice After adaptive feeding for 5 days, 80 clean-grade Balb/c mice were randomly divided into 10 groups, namely the blank control group (Sham), the DNFB model group (DNFB), the positive drug mometasone furoate group (Mome), and the yellow group.
  • ZHT total flavonoids
  • ZHT total flavonoids
  • HKY HKY cream 0.5%
  • 100 mg/kg SZHT oral administration group 100 mg/kg SZHT oral administration group.
  • fluorobenzene (DNFB) fluorobenzene (DNFB), in the 4th, 6th, 8.
  • Aqueous phase Weigh the prescribed amount of ethanol, add ZHT/HKY, stir to dissolve, and add Tween 80, sodium lauryl sulfate, glycerin, isopropyl myristate, methylparaben, and azone into the beaker in sequence. , purified water, stir until evenly dispersed.
  • Oil phase Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petroleum jelly into a beaker, heat and dissolve in a water bath at 80°C until it becomes liquid.
  • AD Score Atopic dermatitis severity score
  • the back skin of each mouse was photographed and recorded, and the severity of dermatitis appearance was evaluated through four indicators: 1 erythema/hemorrhage, 2 dryness, 3 exudation/scab, and 4 edema. Each indicator is scored from 0 to 3, and the scores of the four indicators are added together to obtain the total score.
  • the AD Score scoring criteria are as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms.
  • Paraffin sections were routinely dewaxed to water, and the sections were placed in hematoxylin dye, stained for 5 minutes, and rinsed with tap water. The sections were then placed in hydrochloric acid ethanol, differentiated for 30 seconds, soaked in tap water for 15 minutes, and then placed in eosin dye and stained for 2 minutes. Routinely dehydrate, clear, and mount with neutral resin. Use a digital pathology slide scanner to take pictures and count the thickness of the epidermal layer.
  • GraphPad Prism 6.0 software was used for statistical analysis and graphing of data. All data are expressed as mean ⁇ standard error (mean ⁇ SEM). Dermatitis scores were analyzed using two-way ANOVA, other data were analyzed using one-way ANOVA, and Dunnett's multiple test was used to analyze the significance of differences between groups. When P ⁇ 0.05, the differences between groups were significant and statistically significant.
  • the skin acanthosis of the % ZHT group was improved, and the epidermal layer thickness was 100.59 ⁇ 10.95 ⁇ m.
  • Example 3.2-1, Example 4-2, and Example 9-2 also have functions or effects that are basically consistent with the above-mentioned aspects of the total flavonoids of ambrosia in this example.
  • Model group (Model): Apply a blank cream without total flavonoids from ambrosia;
  • Positive medicine group Apply Deshi cleansing water colloid
  • Low-dose group (Low-0.5%): apply 0.5% total flavonoids cream of ambrette;
  • Middle-dose group (Middle-1%): apply 1% ambrette total flavonoids cream;
  • High-dose group (High-2%): apply 2% ambrette total flavonoids cream;
  • STZ solution configuration 1 Configuration of citric acid buffer: add 2.1g of citric acid to 100mL of distilled water to prepare liquid A; add 2.94g of sodium citrate to 100mL of distilled water to prepare liquid B. 2 When using, mix A and B in proportion and adjust the pH to 4.2-4.5, which is the citric acid buffer solution for STZ. 3 Dissolve STZ in citrate buffer at a concentration of 1% before injection. Protect from light and refrigerate at 4°C for later use.
  • Preparation of 4% chloral hydrate Weigh 4g of chloral hydrate, add appropriate amount of physiological saline to dissolve, and adjust the volume to 100mL. Protect from light and refrigerate at 4°C for later use.
  • STZ solution and citrate buffer were injected into the left lower abdomen of rats according to their fasting body weight, and the injection was completed within 10 minutes. All rats were fasted for 6 hours before administration, and blood was collected from the tail vein 72 hours after injection to measure fasting blood glucose. Diabetes models were successfully constructed if the fasting blood glucose value was ⁇ 16.7 mmol/L and there was no significant decrease in body weight. If the blood sugar of some rats is between 5-8mmol/L, then inject STZ (35mg/kg) again; if the blood sugar of some rats is between 8-10mmol/L, then inject STZ (25mg/kg) again; if the blood sugar of some rats is between 10-16mmol /L, then inject STZ (20mg/kg).
  • the rats were anesthetized by intraperitoneal injection of 4% chloral hydrate according to body weight (2 mg/kg). After the rats were completely anesthetized, the backs of the rats were shaved with an electric shaver. After shaving, Use a medical cotton swab dipped in iodophor and apply it continuously on the back of the rat 4-5 times for disinfection. Use a sterile 8-mm Biopsy Punch to print a circular area with a diameter of 8 mm in the middle of the rat's back. Use sterile forceps to pick up the skin in the middle of the circular area and use sterile iris scissors to follow the outline of the Biopsy Punch.
  • the skin is removed with a circular impression to create a circular, full-thickness skin resection open wound with a diameter of 8mm, which reaches deep into the muscles and avoids large arterial and venous blood vessels.
  • iodophor continuously to the wound 4-5 times, and apply appropriate pressure to fully stop bleeding.
  • Rats in the blank control group were only injected with an equal dose of chloral hydrate according to their body weight, and their backs were shaved without establishing a full-thickness skin excision open wound.
  • Rats that died during modeling were eliminated, and diabetic rats that were successfully modeled, awake and in good condition were randomly divided into groups according to the random number table method, namely: model group (smeared with blank cream), positive drug group (smeared with Deshi Water colloid), low-dose group (smear 0.5% total flavonoids cream of Scutellaria baicalensis), medium-dose group (smear 1% cream of total flavonoids of Scutellaria sibiricum), high-dose group (smear 2% cream of total flavonoids of Scutellaria sibiricum).
  • Drug treatment was given immediately after the full-thickness skin excision wound model was established, and photos were taken and recorded on 0d (before application and administration), 3d, 7d, and 9d after treatment, and the wound area was measured using ImageJ software.
  • the fasting blood glucose measured by tail vein blood sampling is 16.7-33.3mmol/L;
  • the skin tissue was attached to the filter paper, fixed vertically in 4% paraformaldehyde solution for 48 hours, and then embedded in conventional paraffin. Perform continuous full-thickness skin sections (5 ⁇ m) on the embedded skin tissue, place 2 sections on each slide, and then perform HE staining.
  • the specific steps are as follows:
  • Ethanol soaking soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
  • Anti-blue Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
  • Eosin staining stain for 5 minutes, then wash with tap water for 30 seconds;
  • Ethanol dehydration Use different contents of ethanol to dehydrate in sequence. The sequence is as follows: 85% ethanol dehydration. 20s, 90% ethanol dehydration for 30s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, absolute ethanol I for 2 min, absolute ethanol II for 2 min;
  • Skin tissue was taken, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and made into skin paraffin sections. Put the paraffin slices into a 60°C oven and bake them for 20 minutes, then soak them in xylene I for 15 minutes, xylene II for 15 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and 95% alcohol for 5 minutes. Soak in 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then rinse with distilled water and PBS for 5 minutes respectively. Place the sections in a staining vat filled with antigen retrieval buffer, and perform antigen heat retrieval in a pressure cooker.
  • VEGF vascular endothelial growth factor
  • FIG. 9A is a representative diagram of the skin wound area of rats in each group at different time points.
  • fibrin clots (“scabs") appeared in the skin wounds of each group, and a small amount of exudate existed. , indicating that there is an inflammatory reaction in the wound at this time; on the third day after administration, the wound scabs in each administration group began to harden, and the scabs became dry. The dry scabs can play an antibacterial role, thus having an antibacterial effect on the wounds.
  • the wound area in the model group was 99.48 ⁇ 8.76% of the original wound area, and the rats in the Deshijie hydrocolloid group Compared with the administration group and the model group, the wound area began to shrink.
  • the wound area was 64.52 ⁇ 7.04% of the initial wound area.
  • the wound area of the 0.5%, 1%, and 2% Huangkui total flavonoid cream groups was the initial wound area respectively. 106.80 ⁇ 9.37%, 91.41 ⁇ 5.51%, 98.38 ⁇ 8.74% ( Figure 9B).
  • Figure 9B On the 7th day after administration, the hard scabs in each group began to fall off.
  • the wound area in the model group was 47.62 ⁇ 10.68% of the initial wound area.
  • the wound area in the 0.5%, 1%, and 2% Huangkui total flavonoid cream groups was divided into 47.62 ⁇ 10.68% of the initial wound area. 49.65 ⁇ 8.79%, 27.38 ⁇ 6.61%, 36.54 ⁇ 5.72% of the area.
  • the 1% cream group promoted wound healing the most, while the wound area of the Deshijie hydrocolloid group was 17.86 ⁇ 4.68% of the initial wound area, and its effect on promoting wound healing was better than that of the 1% Huangkui total flavonoids cream group.
  • the wound area in the model group was 12.05 ⁇ 4.03% of the original wound area; the wound area in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 15.98 ⁇ 5.63% and 10.04% of the initial wound area, respectively. ⁇ 5.64%, 10.60 ⁇ 3.14%; the wound area of the Deshijie water colloid group was 8.03 ⁇ 4.70% of the initial wound area.
  • the wound healing in the positive medicine Deshijie hydrocolloid group was obvious, with significant differences compared with the model on 3 days and 7 days.
  • the 1% Huangkui total flavonoids cream group showed basic recovery after treatment.
  • HE staining is one of the commonly used paraffin section staining methods. It uses hematoxylin, eosin and other dyes as dyes, so it is also called hematoxylin-eosin staining. Hematoxylin stain is a basic dye that stains cell nuclei purple-blue, eosin stains components of the extracellular matrix and cytoplasm pink, and other structures appear in different hues and combinations of these colors.
  • the overall color pattern of the stained tissue shows the overall layout and distribution of cells and provides an overall overview of the structure of the tissue sample. It is possible to observe the number and distribution of fibroblasts, capillaries, inflammatory cells, etc. during the wound healing process.
  • the results of tissue section microscopy showed that the skin tissue of rats in the normal group showed a complete keratinocyte layer, skin appendages, collagen fibers cross-linked and arranged into parallel bundles, and a small amount of fibroblasts and inflammatory cells.
  • the skin tissue of the rats in the model group was loosely arranged, and it was seen that keratinocytes began to proliferate, but have not yet migrated to the center of the wound.
  • the overall skin structure was disordered, the new epidermis was thinner, there was less new granulation tissue, and there were inflammatory cells.
  • fibroblasts migrated to the wound, among which the number of inflammatory cells was relatively large, indicating that the rats in the model group were mainly in the first stage of wound healing: the inflammatory phase.
  • the keratinocyte layer in the wound of different concentrations of total flavonoids cream group and Deshijie hydrocolloid group has proliferated and migrated to the center of the wound.
  • the collagen fibers in the granulation tissue under the keratinocyte layer are closely arranged and more visible. Fibroblasts grew, new capillaries were scattered and visible, the new epidermis was thicker, and there were relatively few inflammatory cells, indicating that the skin wounds of rats in each treatment group had entered the wound healing process. Stages 2-3 of synthesis: proliferation and remodeling.
  • Figure 10B is a quantitative statistical diagram of the epidermal thickness of rats.
  • the epidermal thickness of the blank control group was 26.83 ⁇ 2.65 ⁇ m.
  • the epidermal thickness of the model group increased significantly to 87.72 ⁇ 1.95 ⁇ m (P ⁇ 0.01vs. control group).
  • Dejie Wet was given to
  • the epidermal thickness of the hydrocolloid group was 112.70 ⁇ 7.58 ⁇ m, and the epidermal thickness of the 0.5% total flavonoids cream group, 1% total flavonoids cream group, and 2% total flavonoids cream group were 99.97 ⁇ 3.06 ⁇ m and 121.00 ⁇ m, respectively. ⁇ 2.35 ⁇ m, 121.10 ⁇ 5.64 ⁇ m.
  • Masson staining is a commonly used staining method in histology. It can dye collagen fibers blue and keratin and muscle fibers red, and is used to evaluate the formation of collagen fibers in wounds.
  • Figure 11A shows the collagen deposition in each group after 9 days of treatment. The darker the color, the greater the amount of collagen deposition. It can be intuitively seen from the figure that the model group has lighter blue staining, and the Deshiji hydrocolloid group and different concentrations The blue dyeing in the total flavonoids cream group of Huang Kui was deepened to varying degrees. Among them, the blue area of the 1% Total Flavonoids Cream group was larger, darker and more evenly distributed than in the other groups.
  • Figure 11B is a quantitative statistical diagram of the collagen area percentage. The collagen area percentage in the blank control group was 36.02 ⁇ 2.95%. The collagen area percentage in the model group dropped significantly to 14.74 ⁇ 1.64% (P ⁇ 0.01vs. control group).
  • the collagen area percentage of the colloid group was 23.24 ⁇ 1.83%, and the collagen area percentages of the 0.5% total flavonoid cream group, 1% total flavonoid cream group, and 2% total flavonoid cream group were 19.33 ⁇ 2.46%, respectively. 27.45 ⁇ 1.74%, 22.76 ⁇ 2.48%.
  • the difference analysis results showed that there was a significant difference between the Deshijie hydrocolloid group and the model group (P ⁇ 0.05).
  • P ⁇ 0.05 In the different concentrations of total flavonoid cream smear and administration groups, 1 The collagen content in the % ambrosia total flavonoids cream group was the highest, which was significantly different from the model group (P ⁇ 0.01).
  • vascular markers CD31 and pro-angiogenic factor (VEGF) were used to determine the changes in the vascular markers CD31 and VEGF mRNA on the 9th day after administration.
  • Figure 12A shows that the expression of Cd 31mRNA in the modeling group decreased significantly, from 0.22 ⁇ 0.03 to 0.10 ⁇ 0.01 (P ⁇ 0.01).
  • the expression of CD31mRNA increased to varying degrees after administration.
  • the expression of CD31mRNA in the Dejie wet hydrocolloid group was 0.17 ⁇ 0.02, and the expression of CD31mRNA in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 0.10 ⁇ 0.01, respectively. 0.21 ⁇ 0.02, 0.12 ⁇ 0.02.
  • the application of 1% ambrosia total flavonoid cream enhanced the expression of CD31mRNA most significantly, with a statistical difference (P ⁇ 0.01). There was no statistical difference in the other groups.
  • Figure 12B shows that the expression of Vegf mRNA decreased after modeling, from 0.25 ⁇ 0.07 to 0.14 ⁇ 0.04.
  • the expression of VEGF mRNA increased to varying degrees after administration.
  • the expression of VEGF mRNA in the Dejie wet hydrocolloid group The expression of VEGF mRNA in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 0.10 ⁇ 0.02, 0.21 ⁇ 0.04, and 0.14 ⁇ 0.04 respectively, but compared with the model group, there was no See significant differences.
  • CD31 is a typical marker of vascular endothelial cells, which can be used to measure angiogenesis.
  • anti-CD31 antibodies to immunohistochemically stain tissue sections, the distribution, density and content of CD31 in new tissues can be obtained very intuitively and objectively. Then analyze whether the microcirculation is successfully established. Using the average fluorescence intensity as an indicator, multiple fields of view from different sections were selected to take pictures to evaluate the angiogenesis in different groups.
  • DAPI blue
  • anti-CD31 antibody red
  • CD31 vascular endothelial cells
  • the number of new blood vessels in the wound of the model group was small, and CD31 showed weak positive expression.
  • the relative fluorescence intensity was 41.63 ⁇ 7.46% of that of the control group (P ⁇ 0.05).
  • the expression of CD31 in the Dejie wet hydrocolloid group was higher than that of the model group, and the relative fluorescence intensity was 41.63 ⁇ 7.46% of that of the control group. 73.75 ⁇ 18.13%, but there is no statistical difference.
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelium and blood vessel formation
  • the VEGF protein level in the model group decreased compared with the blank group, and increased to varying degrees after administration.
  • the VEGF protein level in the model group was 44.93 ⁇ 3.69pg/ml
  • the VEGF level in the Dejie wet hydrocolloid group was 44.93 ⁇ 3.69pg/ml.
  • the protein level was 114.30 ⁇ 13.11pg/ml
  • the VEGF protein levels in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups were 77.74 ⁇ 5.90pg/ml, 98.14 ⁇ 11.53pg/ml, and 99.29 ⁇ 17.35pg/ml respectively. .
  • the VEGF protein expression level in the 0.5% ambrosia total flavonoid cream group increased compared with the model group, but there was no statistical difference.
  • an STZ-induced diabetes model was used to construct a rat full-thickness skin excision model using sterile 8-mm Biopsy Punch.
  • Dejie wet hydrocolloid was used as the positive control drug
  • blank cream was used as the blank control drug in the model group to investigate Huang Kui. Therapeutic effect of total flavonoids on diabetic ulcer rats.
  • total flavonoids from ambrosia can accelerate wound re-epithelialization, promote collagen fiber deposition, increase the expression of VEGF protein levels, and promote the healing of full-thickness skin wounds in diabetic rats.
  • the total flavonoids of ambrosia can also promote the formation of new blood vessels by increasing the expression of CD31, thereby accelerating the healing process of diabetic ulcer wounds.
  • the total flavonoids of ambrosia have certain effects in treating diabetic ulcers.
  • Example 2-A2, Example 4-2, Example 9-2, Example 19-1, and Example 19-2 also have functions that are basically consistent with the above-mentioned aspects of the total flavonoids 1 of ambrosia in this example. or effect.
  • Dry mixing, granulation, and whole granulation Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
  • enteric coating solution Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
  • Coating The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
  • the processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, the concentration of the sample solution (aqueous solution) is 0.15g crude drug/mL, and the sample is loaded The liquid volume is 7BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol at a flow rate of 2BV/h to remove impurities, and use 4BV 60% ethanol at a flow rate of 2BV/h to elute to obtain the eluate. Then, the eluate was recovered under reduced pressure at 50°C to recover ethanol to obtain marshmallow flower flavonoid extract. The tested content was based on the content measurement results and the content of 7 components was adjusted by the addition method to obtain marshmallow flower extract. The 7 components accounted for the total The solid content ratio is 83%.
  • enteric coating solution Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
  • Coating The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
  • Dry mixing, granulation, and whole granulation Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
  • enteric coating solution Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
  • Coating The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
  • Dry mixing, granulation, and whole granulation Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
  • enteric coating solution Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 180mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
  • Dry mixing, granulation, and whole granulation Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
  • enteric coating solution Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 210mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
  • Coating The plain tablets are coated with enteric coating, and the weight is increased by 3% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
  • Total mixing and tableting Add the prescribed amount of magnesium stearate and mix in the mixing machine for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
  • enteric coating solution Take the prescribed amount of enteric film coating pre-suspension enteric hydroxypropyl methylcellulose 320 mg and slowly add it to 800 mL of purified water under stirring. Stir for 15-30 minutes and then wait for 80 seconds. Mesh sieve, spare;
  • Coating The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
  • Total mixing and tableting Add the prescribed amount of magnesium stearate and mix in the mixing machine for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
  • enteric coating solution Take the prescribed amount of enteric film coating pre-suspension enteric hydroxypropyl methylcellulose 240 mg and slowly add it to 800 mL of purified water under stirring. Stir for 15-30 minutes and then wait for 80 seconds. Mesh sieve, spare;
  • enteric-coated tablets prepared in Examples 24-1 to 24-5 and Comparative Examples 24-1 to 24-2 were sequentially dissolved in artificial gastric juice (pH 1.2 hydrochloric acid solution) for 2 hours; artificial intestinal juice (pH 6.8 phosphate buffer) Dissolution was conducted for 1 hour; artificial colon fluid (phosphate buffer solution with pH 7.8) was dissolved for 3 hours, and the dissolution was investigated under the conditions.
  • artificial gastric juice pH 1.2 hydrochloric acid solution
  • intestinal juice pH 6.8 phosphate buffer
  • Dissolution was conducted for 1 hour
  • artificial colon fluid phosphate buffer solution with pH 7.8 was dissolved for 3 hours, and the dissolution was investigated under the conditions.
  • the specific test method first add 900mL of hydrochloric acid solution (pH 1.2) into the dissolution cup and examine the release amount of the enteric preparation within 2 hours; then discard the acid solution and immediately add phosphate buffer with a temperature of 37°C ⁇ 0.5°C solution (pH 6.8), and measure the release amount of the enteric preparation in the buffer; then discard the buffer solution, and immediately add phosphate buffer (pH 7.8) with a temperature of 37°C ⁇ 0.5°C, and measure the enteric preparation. Release in buffer.
  • the release test is to detect the total flavonoids contained in each tablet, including hyperoside, rutin, isoquercetin, gossipin-8-O- ⁇ -D-glucuronide, myricetin, and quercetin.
  • the total amount of 7 ingredients including glucoside-3'-O-glucoside and quercetin.
  • the acidic medium with pH 1.2 simulates the gastric acid environment
  • the medium with pH 6.8 simulates the intestinal environment
  • the medium with pH 7.8 simulates the colon environment.
  • the dissolution rate of the examples does not exceed 10%, while the dissolution rate of the comparative examples reaches more than 20%.
  • Examples 24-1 to 24.5 have a fast dissolution rate and no time lag effect.
  • the dissolution rate reaches more than 90% at 210 minutes (4th hour), indicating excellent performance.
  • the dissolution rate of Examples 24-1 to 24.3 reached more than 95% in 210 minutes.
  • the stability of the enteric-coated tablets prepared in Examples 24-1 to 24-3 of the present invention was investigated.
  • the examination conditions were high temperature (40°C), high humidity (RH75% ⁇ 5%), and light experiment (4500lx ⁇ 500lx), respectively. Leave it for 10 days under the above inspection conditions, and take samples to measure its release on days 0, 5, and 10.
  • the measurement method is UV spectrophotometry.
  • Release degree 1 refers to the release degree in simulated artificial intestinal fluid (pH 6.8 phosphate buffer solution); release degree 2 refers to the release degree in simulated artificial colon fluid (pH 7.2-7.8 phosphate buffer solution).
  • the release test is to detect the total flavonoids contained in each tablet, including hyperoside, rutin, isoquercetin, gossipin-8-O- ⁇ -D-glucuronide, myricetin, and quercetin.
  • the total amount of 7 ingredients including glucoside-3'-O-glucoside and quercetin.
  • the result data shows that the content of the example is relatively stable when this product is placed under high temperature (40°C), high humidity (RH75% ⁇ 5%), and light experiment (4500lx ⁇ 500lx) for 10 days. And in the simulated artificial intestinal fluid environment, there is almost no release, and the release rate is relatively stable in the artificial colon fluid environment.
  • Ulcerative colitis model rats were selected to conduct pharmacodynamic evaluation of the medicine of the present invention.
  • the experimental rats were kept in an SPF-level animal room throughout the entire process, with a light-to-dark time ratio of 1:1, room temperature controlled at 23 ⁇ 2°C, and humidity controlled at 55%.
  • the rats could eat and drink freely; the bedding was changed every three days. , to ensure that the rats are in a dry and clean environment.
  • 3% DSS solution preparation Weigh 3g of DSS powder and fully dissolve it in 100mL of pure water, and the result is a 3% DSS solution.
  • the rats were randomly divided into 5 groups, with two cages in each group and 4 rats in each cage. They are the blank control group (Control), the model group (DSS), the model + 5-aminosalicylic acid group (DSS + 140mg/kg/day 5-ASA), and the model + HK1 group (DSS + 280mg/kg/day HK1 ), model+HK2 group (DSS+52.5mg/kg/day HK2).
  • the dosage is calculated based on the active ingredient.
  • the rat ulcerative colitis model was induced by drinking 3% DSS freely for 7 consecutive days.
  • Each rat (4 rats) in each cage was replaced with 3% DSS solution (80mL) every 2 days to ensure that the DSS solution was effective; blank
  • the rats in the control group drank pure water freely, and each cage (4 rats) was changed every 2 days.
  • the rats were orally administered with corresponding doses in custom-made enteric-coated capsules at 9:00-10:00 am every day.
  • HK1, HK2 and the positive therapeutic drug 5-ASA were administered.
  • the blank group and the model group were given the same dose.
  • DAI disease activity index
  • the body weight of rats was recorded before daily administration, and feces were collected for fecal occult blood test.
  • the DAI score is used to score the severity of the disease based on three indicators: weight loss, fecal viscosity and fecal occult blood. Each indicator corresponds to 0-4 points.
  • the scoring standards are as shown in Table 24-5.
  • Fecal occult blood test Take a small amount of feces and apply it in the middle of the glass slide (the glass slide has been pre-fired to reduce color errors), add 3 drops of 10/L methaminophenol sulfate acetic acid solution and 3 drops of 3% hydrogen peroxide solution , mix well, observe the results immediately, and rate according to Table 24-11.
  • rats in each group were fasted for 24 hours and sacrificed by cervical dislocation. Cut open the rat's abdominal cavity to expose the rat's colon. Take all the rat's colon tissue, measure and record its length, place it in newly prepared pre-cooled physiological saline to flush the intestinal contents, and cut out 1cm of the colon near the rectum. The tissue was fixed overnight in 4% paraformaldehyde for preparation of paraffin sections and subsequent pathological examination. The remaining tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The blood samples were allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 15 min. The serum was collected and quickly stored at -80°C for subsequent testing.
  • the colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in conventional paraffin. Take serial transverse sections (10 ⁇ m) of the embedded colon tissue, and then perform H&E staining.
  • the H&E staining steps are as follows:
  • Dewaxing Dewaxing with xylene I for 10 minutes, dewaxing with xylene II for 5 minutes;
  • Ethanol soaking soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
  • Anti-blue Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
  • Eosin staining stain for 5 minutes, then wash with tap water for 30 seconds;
  • Ethanol dehydration Use different contents of ethanol for dehydration in sequence. The sequence is as follows: 85% ethanol dehydration for 20s, 90% ethanol dehydration for 30s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, and absolute ethanol I for dehydration. Water for 2 minutes, dehydrate with absolute ethanol II for 2 minutes;
  • Pathological examination The degree of colon damage is scored based on pathological indicators such as the degree of colon tissue lesions, inflammatory cell infiltration, and crypt cell damage. The specific scoring standards are shown in Table 24-13.
  • the DAI score is calculated by adding the three indicator values in Table 24-13 and taking the average. As shown in Table 24-14, the DAI score of the rats in the DSS model group increased significantly from the 3rd day. Compared with the Control group, the difference was statistically significant (P ⁇ 0.01). On the 6th day of the experiment, 3 rats had diarrhea, and on the 6th day of the experiment, 3 rats had diarrhea. There were 7 diarrhea cases in 7 days; the DAI score of the DSS+HK1 (280mg/kg/day) and DSS+HK2 (52.5mg/kg/day) groups showed a relatively gentle growth trend. On days 4, 5, 6, and 7, the DAI scores were the same as Compared with the DSS model group, the difference was statistically significant (P ⁇ 0.01).
  • the colon length of the Control group was 17.05 ⁇ 0.04cm, and the colon of rats in the DSS model group shrunk to 14.32 ⁇ 0.15cm.
  • the difference was significant (P ⁇ 0.01); 280 mg/kg/day HK1, 52.5mg/kg/day HK2 significantly improved colon atrophy induced by DSS, and the difference between the DSS+HK2 (52.5mg/kg/day) group and the DSS model group was statistically significant (P ⁇ 0.05);
  • the positive drug 5-ASA 140mg/kg/day
  • the difference was statistically significant (P ⁇ 0.05) compared with the Control group.
  • This example uses a DSS-induced rat ulcerative colitis model, and uses 5-aminosalicylic acid as a positive control drug to examine the therapeutic effect of enteric-coated capsules containing marshmallow flower extract in treating colitis.
  • the results show that HK1 (280mg /kg/day) and HK2 (52.5mg/kg/day) groups significantly protected DSS-induced colitis symptoms such as weight loss, diarrhea, and hematochezia in rats; among them, the effect of HK2 (52.5mg/kg/day) was better than other groups good.
  • Significantly improves histopathological changes such as DSS-induced colon atrophy, crypt structural damage, and tissue inflammatory infiltration.
  • the enteric-coated capsules containing marshmallow flower extract have certain curative effects on colitis.
  • the effect at 52.5 mg/kg/day is equivalent to that of the positive drug 5-ASA, and the safety is good.
  • Example 25 Experiment on pharmaceutical composition and its effect on idiopathic pulmonary fibrosis
  • mice Animal models: db/db mice, 16 weeks old, C57BL/6 mice, 16 weeks old.
  • mice C57BL/6 mice were divided into normal group, db/db mice were divided into model group, positive drug group (dapagliflozin tablets, 45.5mg/kg), and Example 25-1 pharmaceutical composition group ( 62.4mg/kg), 15 mice in each group. After 4 weeks of administration, urine protein, urinary creatinine and daily food intake were detected, and the status of the mice was observed. The test results are shown in the table below.
  • Example 25-1 can reduce urinary protein and urinary protein/urinary creatinine (ACR). Only 1 animal developed diarrhea/abdominal bloating, with low side effects.
  • ACR urinary protein and urinary protein/urinary creatinine
  • Example 25-4 Experiment on idiopathic pulmonary fibrosis
  • mice 60 Kunming mice, half male and half female, weighing 18-22 grams, clean grade.
  • Animal grouping randomly divided into 5 groups, namely normal group, model group, control drug group (rosiglitazone 5 mg/kg), Example 25-1 pharmaceutical composition group (70 mg/kg), Example 25-2 drug Composition group (70 mg/kg), Huangkui capsule sample group (180 mg/kg based on extract), Example 25-2 pharmaceutical composition group (70 mg/kg), 10 mice in each group.
  • mice were adaptively raised for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01ml/g). The patients were placed in a supine fixed position, routine disinfection was performed, a midline cervical incision was made, and the trachea was exposed by blunt dissection. The model group, control drug group, and treatment group were punctured and slowly injected bleomycin (5 mg/kg) into the tracheal cartilage ring space, and the normal control group was injected Equal volume of normal saline was injected. Immediately after injecting the drug, the mice were rotated upright for 3-5 minutes to allow the drug solution to be evenly distributed in the lungs on both sides. The skin was then sutured and the sutures were disinfected. After they woke up, they were sent to a clean observation room for feeding.
  • bleomycin 5 mg/kg
  • Administration will begin on the second day after modeling.
  • the administration group will be administered according to the above dosage, once a day.
  • the normal group and The model group was given the same volume of distilled water 10ml/kg in the same way for 28 consecutive days.
  • Staining of pathological sections of lung tissue After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis is divided into four grades.
  • Crush the marshmallow flower extract of Example 2-A4 take 400g, add the following weight portions of 20g hydroxypropyl cellulose, 15g talc, 20g microcrystalline cellulose, and 20g calcium sulfate, mix well, and pass through an 80-mesh sieve. Add an appropriate amount of absolute ethanol to make a soft material, then use a 20-mesh sieve to make granules, dry, whole granules, and put into capsules.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Pulmonology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Alternative & Traditional Medicine (AREA)

Abstract

The present invention relates to a pharmaceutical formulation and use thereof, belonging to the field of pharmacy. The pharmaceutical formulation has significant effects in preventing and treating renal diseases, eye diseases, ulcerative diseases, inflammatory bowel diseases and skin diseases, resists fibrosis, promotes wound healing, treats diabetic ulcers, and has good safety.

Description

一种药物制剂及其应用A pharmaceutical preparation and its application 技术领域Technical field
本发明属于制药领域，具体涉及一种黄蜀葵花黄酮类药物制剂及其应用。The invention belongs to the field of pharmaceuticals, and specifically relates to a marshmallow flower flavonoid pharmaceutical preparation and its application.
背景技术Background technique
药用黄蜀葵花为锦葵科植物黄蜀葵Abelmoschus manihot(L.)Medic的干燥花冠，其味甘、寒，归肾、膀胱经；具有清利湿热，消肿解毒功效，用于痈疽肿毒，水火烫伤。该药始载于《嘉佑本草》，《嘉佑本草》记载：黄蜀葵花，主诸恶疮脓水久不瘥者，作末敷。《本草纲目》曰：黄蜀葵花，其花气味甘、寒、滑、无毒，治诸恶疮脓水久不瘥者，作末敷之即愈，为疮家要药。Medicinal hollyhock flower is the dried corolla of the Malvaceae plant Abelmoschus manihot (L.) Medic. Its taste is sweet and cold, and it returns to the kidney and bladder meridian. It has the effects of clearing away dampness and heat, reducing swelling and detoxifying, and is used for carbuncle, swollen poison, water-fire scald. This medicine was first recorded in "Jiayou's Materia Medica". "Jiayou's Materia Medica" records: marshmallow flowers are used to treat malignant sores that have pus that have not healed for a long time. "Compendium of Materia Medica" says: The flowers of hollyhock are sweet, cold, slippery, and non-toxic. They can be used to treat malignant sores that have pus and water that have not bled for a long time. They can be cured immediately after applying it for a long time. It is an important medicine for treating sores.
黄酮类化合物是黄蜀葵花的主要成分之一，同时也是其药理活性成分之一，黄葵胶囊已上市多年，其主要活性成分为黄蜀葵花提取物。研究证实黄蜀葵花提取物中的黄酮类成分不仅对疮疡有明显的治疗作用，对缺血导致心、脑、组织等损伤也具有明显的保护作用，此外，众多文献报道表明，黄蜀葵花总黄酮在抗炎、解热镇痛、保护心脑缺血损伤、降血糖、抗病毒等方面可能具有较好效果。例如，专利CN201210082553.7公开了一种黄蜀葵花的总黄酮提取物，含有棉皮素-3'-葡萄糖苷、槲皮素-3'-葡萄糖苷和异槲皮苷重量比为(11-16)∶(2.5-6)∶(4-6.5)的黄酮，可用于制备治疗肾炎疾病。专利CN200610097615.6则公开了一种黄蜀葵花总黄酮提取物，按重量百分比总黄酮含量为50-90％，其所含黄酮成分为：槲皮素-3-洋槐糖苷1.0-5.0％、金丝桃苷8-24.0％、异槲皮苷7.0-20.0％、槲皮素-3'-葡萄糖苷5.0-15.0％、棉皮素-3'-葡萄糖苷3.0-10.0％、杨梅素0.5-5.0％、棉皮素0.5-5.0％、槲皮素2.0-8.0％，以及其它数种黄酮类成分，其所述黄蜀葵花总黄酮提取物可用于制备治疗肾炎药物，黄酮成分定性和定量明确，疗效可靠。Flavonoids are one of the main components of marshmallow flowers, and they are also one of its pharmacologically active ingredients. Huangkui capsules have been on the market for many years, and their main active ingredient is marshmallow flower extract. Research has confirmed that the flavonoids in marshmallow flower extract not only have a significant therapeutic effect on ulcers, but also have a significant protective effect on damage to the heart, brain, tissue, etc. caused by ischemia. In addition, many literature reports have shown that the total flavonoids of marshmallow flowers It may have good effects in anti-inflammatory, antipyretic and analgesic, protecting heart and brain from ischemic damage, lowering blood sugar, and anti-viral. For example, patent CN201210082553.7 discloses a total flavonoid extract of marshmallow flowers, containing gossipin-3'-glucoside, quercetin-3'-glucoside and isoquercetin in a weight ratio of (11-16 ): (2.5-6): (4-6.5) flavonoids can be used to prepare and treat nephritis diseases. Patent CN200610097615.6 discloses a total flavonoid extract of marshmallow flowers, with a total flavonoid content of 50-90% by weight. The flavonoid components contained in it are: quercetin-3-acaiglycoside 1.0-5.0%, golden silk peach glycoside 8-24.0%, isoquercetin 7.0-20.0%, quercetin-3'-glucoside 5.0-15.0%, gossyrin-3'-glucoside 3.0-10.0%, myricetin 0.5-5.0% , cottonseed 0.5-5.0%, quercetin 2.0-8.0%, and several other flavonoid components. The total flavonoid extract of marshmallow flowers can be used to prepare drugs for treating nephritis. The flavonoid components are qualitatively and quantitatively clear, and the curative effect is reliable. .
尽管生物类黄酮具有各种治疗作用，但是，黄酮种类众多，目前主要可以分为7种不同的亚型，且不同的黄酮成分组成将导致不同的治疗作用。例如，虽然文献报道生物类黄酮可有效预防或治疗糖尿病视网膜病变，但具有五个羟基的化合物，如槲皮素对眼部血流量有负面影响，而当黄酮脱氢为黄烷酮时，则获得了眼部血流量的显着改善。此外，增加血流量的化合物还导致缺血性损伤后视网膜功能恢复的显着增加。如何开发此类化合物有效用于治疗眼部疾病如黄斑变性、视觉疲劳和白内障甚至提升对糖尿病视网膜病变的效果，是研究的重要任务。Although bioflavonoids have various therapeutic effects, there are many types of flavonoids, which can currently be divided into seven different subtypes, and different flavonoid compositions will lead to different therapeutic effects. For example, although bioflavonoids have been reported in the literature to be effective in preventing or treating diabetic retinopathy, compounds with five hydroxyl groups, such as quercetin, have negative effects on ocular blood flow, and when flavonoids are dehydrogenated to flavanones, Significant improvements in ocular blood flow were obtained. Furthermore, compounds that increase blood flow also lead to a significant increase in retinal functional recovery after ischemic injury. How to develop such compounds to effectively treat eye diseases such as macular degeneration, visual fatigue and cataracts and even improve the effect on diabetic retinopathy is an important research task.
脉络膜新生血管(CNV)见于许多眼底疾病，如年龄相关性黄斑变性(AMD)、中心性渗出性脉络膜视网膜炎以及高度近视引起的黄斑病变等，其中以AMD继发的CNV最常见，已成为老年人不可逆性视功能损害的主要原因之一。血流量不足也是视觉疲劳的一个影响因素，增强眼部血流量，或者作用于眼部平滑肌，有助于恢复视觉疲劳。Choroidal neovascularization (CNV) is seen in many fundus diseases, such as age-related macular degeneration (AMD), central exudative chorioretinitis, and macular degeneration caused by high myopia. Among them, CNV secondary to AMD is the most common and has become One of the main causes of irreversible visual impairment in the elderly. Insufficient blood flow is also a factor affecting visual fatigue. Enhancing eye blood flow or acting on eye smooth muscles can help restore visual fatigue.
近端肾小管重吸收葡萄糖是由钠-葡萄糖协同转运蛋白(SGLT)1和SGLT2转运体介导，其中约有90％的葡萄糖重吸收由SGLT2介导，其余10％由SGLT1介导。SGLT2抑制剂通过选择性结合SGLT2受体，抑制肾小管葡萄糖的重吸收，可以降低肾糖阈，增加尿糖***，达到明显的降血糖目的。近年来临床研究发现SGLT2抑制剂对非糖尿病导致的慢性肾脏病(CKD)具有肾脏保护作用。SGLT2抑制剂能通过降低TGFβ-1,PAI1,STAT 1,MMP 7以及抑制AGEs-RAGE轴，减少胶原蛋白、纤连蛋白的表达，从而减少细胞外基质积累，缓解DN肾脏纤维化进展。SGLT2抑制剂除了调节管球反馈、减少蛋白尿进程、抗炎、抗纤维化的作用外，还能降低糖尿病患者体内尿酸(SUA)，例如，相比对照组，SGLT2抑制剂(恩格列净、卡格列净、达格列净、托格列 净、鲁格列净、依格列净)显著降低血清尿酸水平。SGLT2抑制剂通过减少血浆容量，降低前后负荷、改善心肌能量代谢、改善心肌重构可有益于心力衰竭的防治。Glucose reabsorption in the proximal renal tubule is mediated by the sodium-glucose cotransporter (SGLT) 1 and SGLT2 transporters, of which approximately 90% of glucose reabsorption is mediated by SGLT2 and the remaining 10% is mediated by SGLT1. SGLT2 inhibitors inhibit renal tubular glucose reabsorption by selectively binding to the SGLT2 receptor, thereby lowering the renal glucose threshold, increasing urinary glucose excretion, and achieving significant blood sugar lowering purposes. In recent years, clinical studies have found that SGLT2 inhibitors have a renal protective effect on non-diabetic chronic kidney disease (CKD). SGLT2 inhibitors can reduce the expression of collagen and fibronectin by reducing TGFβ-1, PAI1, STAT 1, MMP 7 and inhibiting the AGEs-RAGE axis, thereby reducing the accumulation of extracellular matrix and alleviating the progression of DN renal fibrosis. In addition to regulating tubuloglomerular feedback, reducing the progression of proteinuria, and having anti-inflammatory and anti-fibrotic effects, SGLT2 inhibitors can also reduce uric acid (SUA) in patients with diabetes. For example, compared with the control group, SGLT2 inhibitors (empagliflozin) , canagliflozin, dapagliflozin, togliflozin net, lupagliflozin, empagliflozin) significantly reduced serum uric acid levels. SGLT2 inhibitors can be beneficial to the prevention and treatment of heart failure by reducing plasma volume, reducing before and after load, improving myocardial energy metabolism, and improving myocardial remodeling.
表皮生长因子受体拮抗剂(epidermal growth factor receptor inhibitors，EGFRIs)类药物是近年来临床应用最为广泛的抗肿瘤药，此类药以其突出的临床疗效而备受医生及患者的青睐。但其不良反应表现多样，皮疹(主要表现为痤疮/痤疮样皮疹)是其中最为常见的。痤疮/痤疮样皮疹不但影响患者的生活质量，而且可能导致治疗无法正常进行，严重影响肿瘤的治疗效果。痤疮/痤疮样皮疹在一定程度上限制了EGFRIs类药物的应用。西医常应用抗生素、外用激素等方法治疗，效果不甚明显。Epidermal growth factor receptor antagonists (EGFRIs) are the most widely used anti-tumor drugs in clinical practice in recent years. These drugs are favored by doctors and patients for their outstanding clinical efficacy. However, its adverse reactions are diverse, and rash (mainly acne/acne-like rash) is the most common one. Acne/acne-like rash not only affects the patient's quality of life, but may also prevent normal treatment, seriously affecting the efficacy of tumor treatment. Acne/acneiform rash limits the application of EGFRIs to a certain extent. Western medicine often uses antibiotics, external hormones and other methods to treat the disease, but the effect is not very obvious.
皮肤伤口是一种常见的临床症状，常规的皮肤创伤治疗包括植皮和人工替代物覆盖。皮肤溃疡是临床的常见病、多发病，指各种原因引起的局部皮肤组织缺损，创口暴露容易感染，尤其是慢性皮肤溃疡长期不能愈合或易复发，治疗比较棘手，严重影响患者的生活质量，并带来一定的社会负担，临床上亟需更多安全、有效、经济的治疗方式及药物的研究开发。Skin wounds are a common clinical symptom, and conventional skin wound treatments include skin grafting and artificial substitute coverage. Skin ulcers are common clinical diseases and frequently-occurring diseases. They refer to local skin tissue defects caused by various reasons. The exposed wounds are prone to infection, especially chronic skin ulcers that cannot heal for a long time or are prone to recurrence. Treatment is difficult and seriously affects the patient's quality of life. It also brings a certain social burden. Clinically, there is an urgent need for the research and development of more safe, effective, and economical treatments and drugs.
流行病学调查研究显示，我国的糖尿病发病率呈逐年上升趋势，该趋势与生活模式的改变和社会老龄化问题密切相关，并且糖尿病患者中有15％最终会出现长期不愈的皮肤溃疡。如何促进糖尿病溃疡创面快速愈合，降低致残率，保护肢体功能，是当今医学领域研究的重要课题和热点。可通过在实验性糖尿病大鼠背部人工造成的无菌创面建立糖尿病状态下慢性难愈合创面动物模型，以此开展促进伤口愈合的药物的研究。Epidemiological surveys show that the incidence of diabetes in my country is increasing year by year. This trend is closely related to changes in lifestyle and the aging of society, and 15% of diabetic patients will eventually develop long-term unhealed skin ulcers. How to promote rapid healing of diabetic ulcer wounds, reduce disability rates, and protect limb functions is an important topic and hot topic in today's medical field. An animal model of chronic refractory wounds under diabetes can be established by artificially creating sterile wounds on the backs of experimental diabetic rats, so as to conduct research on drugs that promote wound healing.
炎性肠病是一种特殊的慢性肠道炎症性疾病，包括溃疡性结肠炎和克罗恩病，临床患者会表现为反复的腹痛、腹泻、粘液血便，多数患者反复发作，迁延不愈，一方面它严重影响病人生活质量，另一方面它能使结直肠癌变风险增加，病程越长炎性程度越高癌变的可能性越大；其中UC能使癌变的风险增加约2.4倍；患者病程10年、20年和30年的癌变率分别为2％、8％和18％。同时，IBD相关的结直肠癌(colorectalcancer,CRC)具有年轻化、呈散播性和多灶性病变的特点，其病理学及发病机制均不同于散发性CRC；“炎性肠病-异常增生-结直肠癌”是CRC发生的重要途径。结直肠癌是最常见的消化道恶性肿瘤，发生率仅次于胃癌和食道癌，目前我国结直肠癌的发病率呈上升趋势，国内发病的上升速度约为4.8％远远超过国际水平2％，因此炎性肠病的治疗及结直肠癌的防治受到国内外学者的普遍关注。中国专利CN201310645683.1公开了黄蜀葵花及其提取物在制备防治炎性肠病药物中的应用。实验结果表明，该发明提供的黄蜀葵花及其黄蜀葵花提取物能够明显抑制TNBS诱导的慢性结肠炎小鼠炎性肠病，并能显著降低长期给予TNBS引起的模型小鼠升高的死亡率、并能显著降低结肠重量/长度比值；病理检查结果表明，能显著降低TNBS诱导所产生的肠道炎症，限制病变严重程度，减轻隐窝损害，能明显减轻TNBS诱导的炎性肠病，有望开发成为新的防治炎性肠病的药物。肠溶制剂系指在规定的时间内在胃中不释放或是几乎不释放药物，而进入肠中，在肠的某部位能大部分或全部释放药物的制剂。制成肠溶制剂可以减轻药物对胃部的刺激，也可以提高药物的生物利用度，可以让药物更完全的在肠道中吸收。目前尚未有含有黄蜀葵花提取物的肠溶制剂的出现。Inflammatory bowel disease is a special chronic intestinal inflammatory disease, including ulcerative colitis and Crohn's disease. Clinical patients will present with repeated abdominal pain, diarrhea, and mucus and bloody stools. Most patients have repeated attacks and protracted recovery. On the one hand, it seriously affects the patient's quality of life; on the other hand, it can increase the risk of colorectal cancer. The longer the course of the disease, the higher the degree of inflammation, and the greater the possibility of cancer; UC can increase the risk of cancer by about 2.4 times; the course of the patient's disease The cancer rates at 10, 20 and 30 years were 2%, 8% and 18% respectively. At the same time, IBD-related colorectal cancer (CRC) has the characteristics of younger age, disseminated and multifocal lesions, and its pathology and pathogenesis are different from sporadic CRC; "Inflammatory bowel disease-dysplasia- "Colorectal cancer" is an important pathway for the occurrence of CRC. Colorectal cancer is the most common malignant tumor of the digestive tract, with an incidence rate second only to gastric cancer and esophageal cancer. The incidence rate of colorectal cancer in my country is currently on the rise. The domestic incidence rate is approximately 4.8%, far exceeding the international level of 2%. , therefore the treatment of inflammatory bowel disease and the prevention and treatment of colorectal cancer have received widespread attention from scholars at home and abroad. Chinese patent CN201310645683.1 discloses the application of marshmallow flowers and their extracts in the preparation of drugs for preventing and treating inflammatory bowel disease. Experimental results show that the marshmallow flowers and marshmallow flower extracts provided by the invention can significantly inhibit inflammatory bowel disease in mice with chronic colitis induced by TNBS, and can significantly reduce the increased mortality of model mice caused by long-term administration of TNBS. It can significantly reduce the colon weight/length ratio; pathological examination results show that it can significantly reduce intestinal inflammation induced by TNBS, limit the severity of lesions, reduce crypt damage, and significantly reduce inflammatory bowel disease induced by TNBS. It is expected to be developed Become a new drug to prevent and treat inflammatory bowel disease. Enteric-coated preparations refer to preparations that do not release or release almost no drug in the stomach within a specified period of time, but enter the intestine and can release most or all of the drug in a certain part of the intestine. Making enteric-coated preparations can reduce the irritation of the drug to the stomach, improve the bioavailability of the drug, and allow the drug to be more completely absorbed in the intestines. There are currently no enteric-coated preparations containing marshmallow flower extract.
文献报道的黄蜀葵花提取方法有乙醇回流提取、超声提取、常温浸泡提取，但黄蜀葵花提取物的成分复杂，有效成分含量变化较大，且提取转移率偏低，提取能耗高，溶剂用量大。因此，有必要探索一种可以联合治疗肾病特别是糖尿病肾病以及同时可以治疗眼部疾病等多种疾病的黄蜀葵花治疗药物及其提取工艺。The extraction methods of marshmallow flowers reported in the literature include ethanol reflux extraction, ultrasonic extraction, and room temperature soaking extraction. However, the components of marshmallow flower extract are complex, the content of active ingredients varies greatly, the extraction transfer rate is low, the extraction energy consumption is high, and the amount of solvent is large. . Therefore, it is necessary to explore a marshmallow flower therapeutic drug and its extraction process that can jointly treat kidney disease, especially diabetic nephropathy, and simultaneously treat multiple diseases such as eye diseases.
申请人统计黄葵胶囊的不良反应病例发现，最近某一年度共收到关于黄葵胶囊不 良反应194例，按照药物不良反应表现统计，出现次数超过10例次的有恶心、瘙痒、皮疹、呕吐、腹泻、上腹部胀满不适等，均为已知不良反应。最近出现新的ADR表现有头晕、头痛、食欲下降等。另外，在已收到的严重不良反应中，有1例涉及到肝细胞损害，有可能是黄葵胶囊损害肝细胞的信号。因此，开发一种新的黄蜀葵花黄酮提取物以降低副作用且用于治疗疾病例如溃疡的药物具有临床意义。The applicant counted the adverse reaction cases of Huangkui Capsules and found that in a recent year, a total of There were 194 cases of adverse reactions. According to the statistics of adverse drug reactions, nausea, itching, rash, vomiting, diarrhea, upper abdominal distension and discomfort, etc. that occurred more than 10 times were all known adverse reactions. New ADR manifestations that have recently appeared include dizziness, headache, and loss of appetite. In addition, among the serious adverse reactions that have been received, one case involved liver cell damage, which may be a signal of Huangkui capsule damaging liver cells. Therefore, it is of clinical significance to develop a new flavonoid extract of marshmallow flowers to reduce side effects and be used as a drug to treat diseases such as ulcers.
发明内容Contents of the invention
针对上述不足，本发明提供了一种黄蜀葵花黄酮类有效部位、提取物、组合物，制备工艺，应用，以及药物制剂及其应用。In view of the above shortcomings, the present invention provides an effective part of marshmallow flower flavonoids, an extract, a composition, a preparation process, an application, a pharmaceutical preparation and its application.
本发明通过萃取耦合树脂法制备得到了一种高***蜀葵花黄酮类有效部位、提取物、组合物、药物制剂，发现其具有更好的肾病治疗活性，例如糖尿病肾病、造影剂肾损伤或红斑狼疮肾炎，其还具有治疗眼部疾病的作用，其还具有抗纤维化的作用，防治溃疡性疾病的作用，防治炎症的作用，防治皮肤疾病的作用，以及促进伤口愈合的作用。同时本发明所述的黄蜀葵花黄酮类药物制剂具有较低的副作用，为患者提供了一种更好的治疗药物。The present invention prepares a high-purity marshmallow flower flavonoid effective part, extract, composition, and pharmaceutical preparation through an extraction coupling resin method, and finds that it has better therapeutic activity for kidney diseases, such as diabetic nephropathy, contrast-induced kidney injury, or erythema. Lupus nephritis, it also has the effect of treating eye diseases, it also has the effect of anti-fibrosis, the effect of preventing and treating ulcer diseases, the effect of preventing and treating inflammation, the effect of preventing and treating skin diseases, and the effect of promoting wound healing. At the same time, the marshmallow flower flavonoid pharmaceutical preparation of the present invention has lower side effects and provides a better therapeutic drug for patients.
为解决上述技术问题，本发明采用的技术方案如下：In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
第一主要方面，本发明提供了一种高***蜀葵花黄酮类有效部位、提取物、组合物。In the first main aspect, the present invention provides a high-purity effective part, extract and composition of flavonoids from marshmallow flowers.
第一方面，本发明提供了一种黄蜀葵花黄酮类有效部位，包括如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为：10：3.0-20：4.0-18：1.9-6.0：6.0-20。In a first aspect, the present invention provides an effective part of flavonoids from marshmallow flowers, including the following mass ratio of flavonoid components: gossyrin-8-O-β-D-glucuronide: hypericin: isoquercetin. The mass ratio of dermatin:myricetin:quercetin-3'-O-glucoside is: 10:3.0-20:4.0-18:1.9-6.0:6.0-20.
具体地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为：10：4.0-17：4.50-16：1.9-5.5：7.0-16；优选为：10：6.0-15：5.0-13：2.0-5.0：8.0-14；进一步地，含有槲皮素，其中棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素为10：1.0-10.0，优选为10：1.0-6.0，优选为10：1.5-5.0；更进一步地，含有芦丁，其中棉皮素-8-O-β-D-葡萄糖醛酸苷：芦丁为10：0.05-0.6，优选为10：0.1-0.4,进一步优选为10：0.1-0.3；再进一步，含有槲皮素-3-O-洋槐糖苷，其中棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素-3-O-洋槐糖苷为10：0.1-2.5，10：0.1-0.9，优选为10：0.15-0.5。Specifically, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is: 10 : 4.0-17: 4.50-16: 1.9-5.5: 7.0-16; preferably: 10: 6.0-15: 5.0-13: 2.0-5.0: 8.0-14; further, containing quercetin, wherein gossydertin -8-O-β-D-glucuronide: quercetin is 10:1.0-10.0, preferably 10:1.0-6.0, preferably 10:1.5-5.0; further, it contains rutin, wherein cotton Cortin-8-O-β-D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably 10:0.1-0.3; further, it contains quercetin- 3-O-Acaiglycoside, wherein Gossipin-8-O-β-D-glucuronide: Quercetin-3-O-Acaiglycoside is 10:0.1-2.5, 10:0.1-0.9, preferably 10:0.15-0.5.
第二方面，本发明提供了一种黄蜀葵花黄酮类有效部位，包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6。In a second aspect, the present invention provides an effective part of flavonoids from marshmallow flowers, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: The mass ratio of gossydisin-8-O-β-D-glucuronide:hyperin is: 0.8-1.2:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为0.8-1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为0.8-1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为0.8-1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为0.8-1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8 -1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.09-1.6：0.2-2.0：0.05-1.2：0.5-3.0：0.25-3.6。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.09-1.6:0.2-2.0:0.05-1.2:0.5-3.0:0.25-3.6.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.12-0.7：0.8-1.6：0.2-0.5：0.85-1.7：1.0-1.4。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.12-0.7:0.8-1.6:0.2-0.5:0.85-1.7:1.0-1.4.
进一步具体地，所述异槲皮苷的质量比数值为1.2或1.0。 Further specifically, the mass ratio value of the isoquercetin is 1.2 or 1.0.
进一步具体地，所述的黄蜀葵花黄酮类有效部位进一步含有芦丁，异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，优选为1：0.006-0.05，优选为1：0.007-0.04，优选为1：0.009-0.04，或者优选为1：0.008-0.03，优选为1：0.009-0.03；进一步地，含有槲皮素-3-O-洋槐糖苷，异槲皮苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，10：0.1-0.9，优选为10：0.15-0.5。More specifically, the effective part of the flavonoids of the marshmallow flower further contains rutin, and the mass ratio of isoquercitrin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05 , preferably 1:0.007-0.04, preferably 1:0.009-0.04, or preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, it contains quercetin-3-O-acaxiglucoside, The mass ratio of quercetin and quercetin-3-O-sophoroside is 10:0.1-2.5, 10:0.1-0.9, preferably 10:0.15-0.5.
进一步具体地，所述的黄蜀葵花黄酮类有效部位含有的槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷及金丝桃苷的总含量为55％以上，优选60％以上，优选为65-85％，优选为69-82％；进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，更进一步地槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％、13-20％，或者为14-25％；或者进一步地，槲皮素的含量高于0.7％，进一步地高于1.0％，进一步地为1.0％-10％，进一步地为1.5％-5％；或者更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷为8.5-30％，进一步地为12％-23％。Further specifically, the effective part of the flavonoids of the marshmallow flower contains quercetin, quercetin-3'-O-glucoside, myricetin, and gossyderm-8-O-β-D-glucuronide. , the total content of isoquercetin and hyperoside is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%; further, quercetin-3'-O-glucose The glycoside content is 12.1-25%, further the quercetin-3'-O-glucoside content is 12.6-23% or 13-20%, 13-20%, or 14-25%; or further , the content of quercetin is higher than 0.7%, further higher than 1.0%, further 1.0%-10%, further 1.5%-5%; or further, cottonseed-8-O-β -D-glucuronide is 8.5-30%, further 12%-23%.
进一步具体地，所述的有效部位含有槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷、芦丁及金丝桃苷的总含量为55％以上，优选60％以上，优选65-85％或66-84％，优选为69-82％,或69-90％；进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，更进一步地槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％。More specifically, the effective part contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O-β-D-glucuronide, and isoquercetin. The total content of rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82%, or 69-90%; further, quercetin The content of quercetin-3'-O-glucoside is 12.1-25%, and further, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%.
第三方面，本发明提供了一种植物提取物，含有黄酮类成分，包括如下质量含量的成分：金丝桃苷10-25％、异槲皮苷8-19％、棉皮素-8-O-β-D-葡萄糖醛酸苷5-30％、槲皮素-3'-O-葡萄糖苷12.1-25％；进一步地金丝桃苷12-23％、异槲皮苷10-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-25％、槲皮素-3'-O-葡萄糖苷12.6-23％；进一步地金丝桃苷13-22％、异槲皮苷11-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-21％、槲皮素-3'-O-葡萄糖苷13-20％。In a third aspect, the present invention provides a plant extract containing flavonoid components, including the following mass content components: hyperoside 10-25%, isoquercitrin 8-19%, gossipin-8- O-β-D-glucuronide 5-30%, quercetin-3'-O-glucuronide 12.1-25%; further hyperoside 12-23%, isoquercetin 10-17% , gossipin-8-O-β-D-glucuronide 8.5-25%, quercetin-3'-O-glucuronide 12.6-23%; further hyperoside 13-22%, isosin Quercetin 11-17%, Gossipin-8-O-β-D-glucuronide 12-21%, Quercetin-3'-O-glucuronide 13-20%.
具体地，所述的提取物还含有槲皮素2-10％，优选槲皮素2.5-9％，优选槲皮素3-8.5％，进一步为1.5％-5％；更进一步地，含有杨梅素2-11％，优选杨梅素3-7％，优选杨梅素3-5％，进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选0.1-1.5％，进一步地0.1-0.9％，再进一步地0.1-0.5％；进一步地，所述植物为黄蜀葵秋葵的花，可以为全花，或花冠，优选为黄蜀葵花。Specifically, the extract also contains quercetin 2-10%, preferably quercetin 2.5-9%, preferably quercetin 3-8.5%, further 1.5%-5%; further, it contains bayberry. 2-11% of myricetin, preferably 3-7% of myricetin, preferably 3-5% of myricetin, and further, 0.08-2.5% of quercetin-3-O-sophoraside, preferably 0.1-1.5%, further 0.1 -0.9%, and further 0.1-0.5%; further, the plant is a flower of marshmallow okra, which can be a whole flower or a corolla, preferably a marshmallow flower.
第四方面，本发明提供了一种黄蜀葵花提取物，含有黄酮类成分，包括如下质量含量的成分：金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-30％、杨梅素3.0-4.9％或5.1-6.0％、槲皮素-3'-O-葡萄糖苷14-25％、槲皮素1.6-4.9％；进一步地金丝桃苷11-22％、异槲皮苷12.0-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-23％、杨梅素1.6-4.9％或5.1-9.0％、槲皮素-3'-O-葡萄糖苷13-22％、槲皮素1.4-8％或1.4-7.8％。In the fourth aspect, the present invention provides a marshmallow flower extract, containing flavonoid components, including the following mass content components: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossipin-8 -O-β-D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-O-glucuronide 14-25%, quercetin 1.6-4.9 %; furthermore, hyperoside 11-22%, isoquercetin 12.0-17%, gossyrin-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-O-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%.
具体地，所述的提取物还包含质量含量为0.01-1.0％的芦丁，进一步为0.05-0.8％，进一步为0.09-0.8％，进一步为0.1-0.6％；进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选0.1-1.5％，进一步地0.1-0.9％，再进一步地0.1-0.5％。Specifically, the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, it contains quercetin- 3-O-Asophoroside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, still further 0.1-0.5%.
具体地，所述的黄蜀葵花提取物中黄酮类成分的质量含量为55％以上，优选60％以上，优选为65-85％或66-84％，优选为69-82％或69-90％。Specifically, the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82% or 69-90%. .
第五方面，本发明提供了一种组合物，包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：1：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5： 0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3或者为1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3。In a fifth aspect, the present invention provides a composition, including flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin- The mass ratio of 8-O-β-D-glucuronide:hyperoside is: 1:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6; further 1:0.1- 1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8- 1.4; further to 1: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3 or 1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further For 1: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further for 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further for 1 : 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 1: 0.2 -0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3.
具体地，所述的组合物还包括芦丁，所述的异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，优选为1：0.006-0.05，优选为1：0.007-0.04，优选为1：0.008-0.03，优选为1：0.009-0.03；进一步地，槲皮素-3'-O-葡萄糖苷在黄酮类成分中的含量为12.1-25％，更进一步地槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％，进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选0.1-1.5％，进一步地0.1-0.9％，再进一步地0.1-0.5％。Specifically, the composition also includes rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably It is 1:0.007-0.04, preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, the content of quercetin-3'-O-glucoside in the flavonoids is 12.1-25%, Furthermore, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%, and further, it contains 0.08-2.5% of quercetin-3-O-acaxiglucoside, preferably 0.1-1.5 %, further 0.1-0.9%, further 0.1-0.5%.
进一步具体地，所述的组合物含有槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷、芦丁及金丝桃苷的总含量为55％以上，优选60％以上，优选65-85％，优选为69-82％。Further specifically, the composition contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O-β-D-glucuronide, and isoquercetin. The total content of rutin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%.
第六方面，本发明提供了一种药物组合物，所述的药物组合物包含上述有效部位、上述黄蜀葵提取物或上述组合物，所述的药物组合物还包括药学上可接受的载体。In a sixth aspect, the present invention provides a pharmaceutical composition, which contains the above-mentioned effective part, the above-mentioned marshmallow extract or the above-mentioned composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
具体地，所述药学上可接受的载体包括溶剂、乳化剂、崩解剂、填充剂、增溶剂、抗氧剂、pH调节剂、渗透压调节剂、抑菌剂、稀释剂、润湿剂、粘合剂和/或成膜剂，所述的润滑剂不包含硬脂酸镁。Specifically, the pharmaceutically acceptable carrier includes solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, diluents, and wetting agents. , adhesive and/or film-forming agent, the lubricant does not contain magnesium stearate.
具体地，所述的药物组合物的剂型包括注射剂、片剂、栓剂、软膏剂、凝胶剂、丸剂、片剂、颗粒剂、胶囊剂或合剂、混悬剂、分散片。Specifically, the dosage forms of the pharmaceutical composition include injections, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures, suspensions, and dispersible tablets.
第七方面，本发明提供一种药物组合物，含有下列重量份的组分：金丝桃苷3.0-20.0，异槲皮苷4.0-18.0，杨梅素1.9-6.0，槲皮素-3'-O-葡萄糖苷6.0-20，棉皮素-8-O-β-D-葡萄糖醛酸苷8.0-12.0；进一步地，所述的药物组合物，含有下列重量份的组分：金丝桃苷4.0-17.0，异槲皮苷4.5-16.0，杨梅素1.9-5.5，槲皮素-3'-O-葡萄糖苷7.0-16.0，棉皮素-8-O-β-D-葡萄糖醛酸苷8.0-12.0；In the seventh aspect, the present invention provides a pharmaceutical composition containing the following components by weight: hyperoside 3.0-20.0, isoquercetin 4.0-18.0, myricetin 1.9-6.0, quercetin-3'- O-glucoside 6.0-20, gossyrin-8-O-β-D-glucuronide 8.0-12.0; further, the pharmaceutical composition contains the following components by weight: hyperoside 4.0-17.0, isoquercetin 4.5-16.0, myricetin 1.9-5.5, quercetin-3'-O-glucoside 7.0-16.0, gossipin-8-O-β-D-glucuronide 8.0 -12.0;
进一步地，所述的药物组合物，含有下列重量份的组分：金丝桃苷6.0-15.0，异槲皮苷5.0-13.0，杨梅素2.0-5.0，槲皮素-3'-O-葡萄糖苷8.0-14.0，棉皮素-8-O-β-D-葡萄糖醛酸苷8.0-12.0；进一步地所述的药物组合物，含有下列重量份的组分：金丝桃苷8.0-15.0，异槲皮苷6.0-12.5，杨梅素2.0-4.0，槲皮素-3'-O-葡萄糖苷9.0-13.0，棉皮素-8-O-β-D-葡萄糖醛酸苷8.0-12.0；跟更进一步地，所述的药物组合物，含有下列重量份的组分：金丝桃苷10-15.0，异槲皮苷7.0-12.0，杨梅素2.0-3.0，槲皮素-3'-O-葡萄糖苷9.0-12.0，棉皮素-8-O-β-D-葡萄糖醛酸苷8.0-12.0。Further, the pharmaceutical composition contains the following components by weight: hyperoside 6.0-15.0, isoquercetin 5.0-13.0, myricetin 2.0-5.0, quercetin-3'-O-glucose Glycoside 8.0-14.0, gossyrin-8-O-β-D-glucuronide 8.0-12.0; the further pharmaceutical composition contains the following components by weight: hyperoside 8.0-15.0, Isoquercetin 6.0-12.5, myricetin 2.0-4.0, quercetin-3'-O-glucuronide 9.0-13.0, gossyrin-8-O-β-D-glucuronide 8.0-12.0; follow Furthermore, the pharmaceutical composition contains the following components by weight: hyperoside 10-15.0, isoquercetin 7.0-12.0, myricetin 2.0-3.0, quercetin-3'-O- Glucoside 9.0-12.0, gossyrin-8-O-β-D-glucuronide 8.0-12.0.
所述的药物组合物，进一步地含有槲皮素，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素的重量比为9-11：1.0-10.0，优选为10：1.0-6.0，进一步优选为10：1.5-5.0，进一步优选为10：1.0-10.0。The pharmaceutical composition further contains quercetin, wherein the weight ratio of gossydisin-8-O-β-D-glucuronide to quercetin is 9-11:1.0-10.0, preferably 10 : 1.0-6.0, more preferably 10: 1.5-5.0, further preferably 10: 1.0-10.0.
所述的药物组合物，进一步地含有槲皮素-3-O-洋槐糖苷，其中，棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素-3-O-洋槐糖苷的重量比为9-11：0.05-2.5，10：0.1-0.9，优选为10：0.15-0.5，进一步优选为10：0.05-2.5。The pharmaceutical composition further contains quercetin-3-O-acaiglycoside, wherein gossipin-8-O-β-D-glucuronide and quercetin-3-O-acaiglycoside The weight ratio is 9-11:0.05-2.5, 10:0.1-0.9, preferably 10:0.15-0.5, and further preferably 10:0.05-2.5.
所述的药物组合物，所述组分作为药物活性成分其含量在60％以上，进一步为65％以上，更进一步为75％以上，优选所述棉皮素-8-O-β-D-葡萄糖醛酸苷的重份为9-11份， 优选为10份。In the pharmaceutical composition, the content of the component as a pharmaceutical active ingredient is more than 60%, further more than 65%, further more more than 75%, preferably the cottonseed-8-O-β-D- The weight of glucuronide is 9-11 parts. Preferably it is 10 parts.
所述的药物组合物，进一步的含有药学上可接受的载体。The pharmaceutical composition further contains a pharmaceutically acceptable carrier.
第八方面，本发明提供了一种黄蜀葵花提取物，所述提取物包括如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：3.0-20：4.0-18：1.9-6.0：6.0-20。In an eighth aspect, the present invention provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O-β-D-glucuronide:hyperoside: The mass ratio of isoquercetin:myricetin:quercetin-3'-O-glucoside is 10:3.0-20:4.0-18:1.9-6.0:6.0-20.
具体地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：4.0-17：4.50-16：1.9-5.5：7.0-16；优选为10：6.0-15.6：5.0-13：2.0-5.0：8.0-14，或10：6.0-15：5.0-13：2.0-5.0：8.0-14；进一步地，含有槲皮素，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素的质量比为10：1.0-10.0，优选为10：1.0-6.0，优选为10：1-5.6，优选为10：1.5-5.0；更进一步地，含有芦丁，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.5，优选为10：0.1-0.45，或10：0.1-0.3；再进一步地，含有槲皮素-3-O-洋槐糖苷，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素-3-O-洋槐糖苷为10：0.1-2.5，优选为10：0.1-0.9，优选为10：0.15-0.5。Specifically, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 4.0-17: 4.50-16: 1.9-5.5: 7.0-16; preferably 10: 6.0-15.6: 5.0-13: 2.0-5.0: 8.0-14, or 10: 6.0-15: 5.0-13: 2.0-5.0 : 8.0-14; further, containing quercetin, wherein the mass ratio of gossyrin-8-O-β-D-glucuronide and quercetin is 10:1.0-10.0, preferably 10:1.0- 6.0, preferably 10:1-5.6, preferably 10:1.5-5.0; further, it contains rutin, wherein the mass ratio of gossydisin-8-O-β-D-glucuronide to rutin is 10: 0.05-0.6, preferably 10: 0.1-0.5, preferably 10: 0.1-0.45, or 10: 0.1-0.3; further, it contains quercetin-3-O-acaxiglucoside, wherein gossyderin- The ratio of 8-O-β-D-glucuronide and quercetin-3-O-acaxiglucoside is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.5.
第九方面，本发明还提供了一种黄蜀葵花提取物，所述提取物包括如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：6-15：5-13：1.0-2.9：4.0-9.5：0.6-6.0，或10：6-15：5-13：1.0-1.8：4.0-5.9：0.6-0.99。In a ninth aspect, the present invention also provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O-β-D-glucuronide: hyperoside. : Isoquercetin: Myricetin: Quercetin-3'-O-glucoside: The mass ratio of quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, Or 10:6-15:5-13:1.0-1.8:4.0-5.9:0.6-0.99.
进一步地，含有芦丁，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，优选为10：0.1-0.3；更进一步地，含有槲皮素-3-O-洋槐糖苷，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，优选为10：0.15-0.5。Further, it contains rutin, wherein the mass ratio of gossydisin-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, preferably 10:0.1 -0.3; further, it contains quercetin-3-O-a Sophora glycoside, in which the mass ratio of Gossipin-8-O-β-D-glucuronide to Quercetin-3-O-Asophoroside It is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.5.
第十方面，本发明提供了一种黄蜀葵花提取物，所述提取物包括如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：6-15：5-13：1.0-2.9：4.0-9.5：0.6-6.0，或10：6-15：5-13：1.0-1.8：4.0-5.9：0.6-0.99。In a tenth aspect, the present invention provides a marshmallow flower extract, which includes flavonoid components in the following mass ratio: gossyrin-8-O-β-D-glucuronide:hyperoside: The mass ratio of isoquercetin:myricetin:quercetin-3'-O-glucoside:quercetin is 10:6-15:5-13:1.0-2.9:4.0-9.5:0.6-6.0, or 10: 6-15: 5-13: 1.0-1.8: 4.0-5.9: 0.6-0.99.
进一步地，含有芦丁，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，优选为10：0.1-0.3；更进一步地，含有槲皮素-3-O-洋槐糖苷，其中棉皮素-8-O-β-D-葡萄糖醛酸苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，优选为10：0.15-0.5。Further, it contains rutin, wherein the mass ratio of gossydisin-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, preferably 10:0.1 -0.3; further, it contains quercetin-3-O-a Sophora glycoside, in which the mass ratio of Gossipin-8-O-β-D-glucuronide to Quercetin-3-O-Asophoroside It is 10:0.1-2.5, preferably 10:0.1-0.9, preferably 10:0.15-0.5.
第十一方面，本发明提供了一种黄酮类有效部位在制备治疗和/或预防溃疡性疾病的药物中的应用，所述黄酮类有效部包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6。In an eleventh aspect, the present invention provides an application of an effective part of a flavonoid in the preparation of a drug for treating and/or preventing ulcerative diseases. The effective part of the flavonoid includes the following mass ratio of flavonoid components: isoquercitrin : Quercetin: Quercetin-3'-O-glucoside: Myricetin: Gossyrin-8-O-β-D-glucuronide: The mass ratio of hyperoside is: 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为0.8-1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为0.8-1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为0.8-1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5-3.0; further 0.8-1.2 : 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8 -1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3。 Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.09-1.6：0.2-2.0：0.05-1.2：0.5-3.0：0.25-3.6。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.09-1.6:0.2-2.0:0.05-1.2:0.5-3.0:0.25-3.6.
具体地，所述的黄蜀葵花黄酮类有效部位包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷的质量比为：0.8-1.2：0.12-0.7：0.8-1.6：0.2-0.5：0.85-1.7：1.0-1.4。Specifically, the effective part of the flavonoids of the marshmallow flower includes the flavonoid components in the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin-8 The mass ratio of -O-β-D-glucuronide:hyperoside is: 0.8-1.2:0.12-0.7:0.8-1.6:0.2-0.5:0.85-1.7:1.0-1.4.
进一步具体地，所述异槲皮苷的质量比数值为1.2或1.0。Further specifically, the mass ratio value of the isoquercetin is 1.2 or 1.0.
进一步具体地，所述的黄蜀葵花黄酮类有效部位进一步含有芦丁，异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，优选为1：0.006-0.05，优选为1：0.007-0.04，优选为1：0.008-0.04，或者优选为1：0.009-0.04；进一步地，含有槲皮素-3-O-洋槐糖苷，异槲皮苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.01-0.25，10：0.01-0.09，优选为1：0.015-0.065，优选为10：0.015-0.05。More specifically, the effective part of the flavonoids of the marshmallow flower further contains rutin, and the mass ratio of isoquercitrin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05 , preferably 1:0.007-0.04, preferably 1:0.008-0.04, or preferably 1:0.009-0.04; further, it contains quercetin-3-O-acaxiglucoside, isoquercetin and quercetin- The mass ratio of 3-O-sophoroside is 10:0.01-0.25, 10:0.01-0.09, preferably 1:0.015-0.065, preferably 10:0.015-0.05.
其中，所述溃疡性疾病为溃疡性肠炎、溃疡性胃炎、溃疡性咽喉炎和溃疡性口腔炎中的任意一种。Wherein, the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis.
上述第八-第十一方面中，所述的黄蜀葵花黄酮类提取物或有效部位含有的槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷及金丝桃苷的总含量为55％以上，优选60％以上，优选为65-85％，优选为69-82％；进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，优选为12.6-25％，优选为13-25％，优选为14-25％，或，12.6-23％，或13-20％；进一步地，槲皮素的含量为0.7％以上，优选为1.0％以上，优选为1.0-12％，优选为1.5-12％，或1.0％-10％，或1.5％-5％；更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷为8.5-34％，优选为12-34％，或8.5-30％，或12％-23％。In the above-mentioned eighth to eleventh aspects, the flavonoid extract or effective part of the marshmallow flower contains quercetin, quercetin-3'-O-glucoside, myricetin, and gossydertin-8-O. -The total content of β-D-glucuronide, isoquercetin and hyperoside is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%; further, quercetin Cortin-3'-O-glucoside content is 12.1-25%, preferably 12.6-25%, preferably 13-25%, preferably 14-25%, or, 12.6-23%, or 13-20% ; Further, the content of quercetin is more than 0.7%, preferably more than 1.0%, preferably 1.0-12%, preferably 1.5-12%, or 1.0%-10%, or 1.5%-5%; further Preferably, gossydisin-8-O-β-D-glucuronide is 8.5-34%, preferably 12-34%, or 8.5-30%, or 12%-23%.
上述第八-第十一方面中，所述的提取物或有效部位含有槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷、芦丁及金丝桃苷的总含量为55％以上，优选60％以上，优选65-85％或66-84％，优选为69-82％或69-90％；进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，优选为12.6-23％，优选为13-20％。In the above-mentioned eighth to eleventh aspects, the extract or effective part contains quercetin, quercetin-3'-O-glucoside, myricetin, gossyderm-8-O-β-D- The total content of glucuronide, isoquercetin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85% or 66-84%, preferably 69-82% or 69- 90%; further, the quercetin-3'-O-glucoside content is 12.1-25%, preferably 12.6-23%, preferably 13-20%.
第十二方面，本发明提供了一种黄蜀葵花提取物，含有黄酮类成分，所述提取物包括如下质量含量的成分：金丝桃苷8-30％、异槲皮苷10-24％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-34％、杨梅素3.0-4.9％或5.1-7.0％、槲皮素-3'-O-葡萄糖苷14-25％、槲皮素1.6-12％；或者金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-30％、杨梅素3.0-4.9％或5.1-6.0％、槲皮素-3'-O-葡萄糖苷14-25％、槲皮素1.6-4.9％；或者金丝桃苷11-22％、异槲皮苷12.0-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-23％、杨梅素1.6-4.9％或5.1-9.0％、槲皮素-3'-O-葡萄糖苷13-22％、槲皮素1.4-8％或1.4-7.8％。In a twelfth aspect, the present invention provides an extract of marshmallow flowers, containing flavonoid components. The extract includes components with the following mass contents: hyperoside 8-30%, isoquercetin 10-24%, Gossyrin-8-O-β-D-glucuronide 8.5-34%, myricetin 3.0-4.9% or 5.1-7.0%, quercetin-3'-O-glucuronide 14-25%, quercetin Cortin 1.6-12%; or hyperoside 8-26%, isoquercetin 12.0-19.8%, gossyrin-8-O-β-D-glucuronide 8.5-30%, myricetin 3.0 -4.9% or 5.1-6.0%, quercetin-3'-O-glucoside 14-25%, quercetin 1.6-4.9%; or hyperoside 11-22%, isoquercetin 12.0-17 %, gossyrin-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1-9.0%, quercetin-3'-O-glucuronide 13-22% , Quercetin 1.4-8% or 1.4-7.8%.
具体地，所述的提取物还包含质量含量为0.01-1.0％的芦丁，进一步为0.05-0.95％，进一步为0.09-0.95％，进一步为0.1-0.95％，或0.05-0.8％，进一步为0.09-0.8％，进一步为0.1-0.6％；进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选0.1-1.5％，进一步地0.1-0.9％，再进一步地0.1-0.8％，再进一步地0.1-0.5％。Specifically, the extract also contains rutin with a mass content of 0.01-1.0%, further 0.05-0.95%, further 0.09-0.95%, further 0.1-0.95%, or 0.05-0.8%, further 0.09-0.8%, further 0.1-0.6%; further, containing quercetin-3-O-acaxiglucoside 0.08-2.5%, preferably 0.1-1.5%, further 0.1-0.9%, and further 0.1-0.8 %, and further 0.1-0.5%.
具体地，所述的黄蜀葵花提取物中黄酮类成分的质量含量为55％以上，优选60％以上，优选为65-90％，优选为69-82％。Specifically, the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, preferably 65-90%, preferably 69-82%.
其中，所述溃疡性疾病为溃疡性肠炎、溃疡性胃炎、溃疡性咽喉炎和溃疡性口腔炎中的任意一种。Wherein, the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis.
第十三方面，本发明提供了一种组合物，包括如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃 苷的质量比为：1：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3或者为1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3。In a thirteenth aspect, the present invention provides a composition comprising the following mass ratio of flavonoid components: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossydisin -8-O-β-D-glucuronide: Hypericum The mass ratio of glycosides is: 1:0.05-1.6:0.2-4.6:0.05-1.2:0.2-3.5:0.25-3.6; further: 1:0.1-1.2:0.4-3.1:0.10-0.9:0.4-3.1:0.5- 3.0; further 1: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; Further is 1: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further is 1: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3 or 1.2 : 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16 -1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7 :0.8-1.6:0.2-0.5:0.8-1.8:0.8-1.4; further 1:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3.
具体地，所述的组合物还包括芦丁，所述的异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，优选为1：0.006-0.05，优选为1：0.007-0.04，优选为1：0.008-0.03，优选为1：0.009-0.03；进一步地，槲皮素-3'-O-葡萄糖苷在黄酮类成分中的含量为12.1-25％，更进一步地槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％，进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选0.1-1.5％，进一步地0.1-0.9％，再进一步地0.1-0.5％。Specifically, the composition also includes rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, preferably 1:0.006-0.05, preferably It is 1:0.007-0.04, preferably 1:0.008-0.03, preferably 1:0.009-0.03; further, the content of quercetin-3'-O-glucoside in the flavonoids is 12.1-25%, Furthermore, the content of quercetin-3'-O-glucoside is 12.6-23% or 13-20%, and further, it contains 0.08-2.5% of quercetin-3-O-acaxiglucoside, preferably 0.1-1.5 %, further 0.1-0.9%, further 0.1-0.5%.
进一步具体地，所述的组合物含有槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷、芦丁及金丝桃苷的总含量为55％以上，优选60％以上，优选65-85％，优选为69-82％。Further specifically, the composition contains quercetin, quercetin-3'-O-glucoside, myricetin, gossydisin-8-O-β-D-glucuronide, and isoquercetin. The total content of rutin, rutin and hyperin is more than 55%, preferably more than 60%, preferably 65-85%, preferably 69-82%.
第十四方面，本发明提供了一种药物组合物，所述的药物组合物包含上述有效部位、上述黄蜀葵提取物或上述组合物，所述的药物组合物还包括药学上可接受的载体。In a fourteenth aspect, the present invention provides a pharmaceutical composition, which contains the above-mentioned effective part, the above-mentioned marshmallow extract or the above-mentioned composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
具体地，所述药学上可接受的载体包括溶剂、乳化剂、崩解剂、填充剂、增溶剂、抗氧剂、pH调节剂、渗透压调节剂、抑菌剂、稀释剂、润湿剂、粘合剂和/或成膜剂，所述的润滑剂不包含硬脂酸镁。Specifically, the pharmaceutically acceptable carrier includes solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, diluents, and wetting agents. , adhesive and/or film-forming agent, the lubricant does not contain magnesium stearate.
具体地，所述的药物组合物的剂型包括注射剂、片剂、栓剂、软膏剂、凝胶剂、丸剂、片剂、颗粒剂、胶囊剂或合剂、混悬剂、分散片。Specifically, the dosage forms of the pharmaceutical composition include injections, tablets, suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures, suspensions, and dispersible tablets.
第二主要方面，本发明提供了上述第一主要方面的各个技术方案中，所述黄酮类有效部位、提取物、组合物的制备方法。In the second main aspect, the present invention provides methods for preparing the effective parts, extracts and compositions of flavonoids in each technical solution of the above-mentioned first main aspect.
第一方面，本发明提供了上述有效部位或上述黄蜀葵提取物的制备方法，所述的方法包括以下步骤：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液浓缩后经萃取，制得萃取液；(3)萃取液去除溶剂后经大孔树脂洗脱，制得黄蜀葵花黄酮类有效部位或提取物；优选提取方法为渗漉。In a first aspect, the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract. The method includes the following steps: (1) Extract marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) The extract is concentrated and extracted to obtain an extract; (3) the extract is freed of solvent and eluted with a macroporous resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is percolation.
具体地，步骤(1)所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)所述的萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(3)所述的大孔树脂型号为D101、HPD100或AB-8。Specifically, the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution; the extraction agent used for extraction in step (2) It is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, the material-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is level 1-5; The macroporous resin model described in step (3) is D101, HPD100 or AB-8.
具体地，步骤(2)进一步包括在萃取之前，对提取液经活性炭吸附、醇沉或酸沉处理。Specifically, step (2) further includes subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
具体地，步骤(3)所述的大孔树脂的洗脱工艺为：大孔树脂径高比为1：4-1：9，上样液浓度为0.10-0.30g生药/mL，上样液体积为4-12BV，以1-4BV/h流速上样，用4-8或0.5-8BV纯水和1-5BV的3-15％乙醇以0.5-4BV/h流速进行除杂，用2-8BV 50-80％乙醇以1-5BV/h流速进行洗脱。Specifically, the elution process of the macroporous resin described in step (3) is: the diameter-to-height ratio of the macroporous resin is 1:4-1:9, the concentration of the loading solution is 0.10-0.30g crude drug/mL, and the loading solution The volume is 4-12BV, load the sample at a flow rate of 1-4BV/h, use 4-8 or 0.5-8BV pure water and 1-5BV of 3-15% ethanol to remove impurities at a flow rate of 0.5-4BV/h, use 2- 8BV 50-80% ethanol is used for elution at a flow rate of 1-5BV/h.
第二方面，本发明提供了上述有效部位或上述黄蜀葵提取物的制备方法，所述的方法包括以下步骤：其制备方法包括如下步骤：(1)黄蜀葵花或黄蜀葵药用部位用乙 醇提取，制得提取液；(2)提取液加入澄清剂，水浴处理，过滤去上清；(3)上清液经聚酰胺树脂洗脱，制得黄蜀葵花黄酮类有效部位或提取物；优选提取方法为渗漉。In a second aspect, the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract. The method includes the following steps: The preparation method includes the following steps: (1) The flowers or medicinal parts of marshmallow are treated with ethanol. Extract with alcohol to prepare an extract; (2) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (3) The supernatant is eluted with polyamide resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; The preferred extraction method is percolation.
具体地，步骤(1)所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)所述的水浴温度为50-70℃，水浴时间为30-90min；步骤(3)所述的树脂径高比为1：4-1：9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。Specifically, the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution; the water bath temperature described in step (2) is 50-25%. 70℃, water bath time is 30-90min; the diameter-to-height ratio of the resin described in step (3) is 1:4-1:9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, and the volume of the sample solution is 4- 12BV, elute with 4-8BV pure water and 4-8BV 60-95% ethanol.
第三方面，本发明提供了上述有效部位或上述黄蜀葵提取物的制备方法，其制备方法包括如下步骤：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液调节pH值为2.0-3.0，冷藏，过滤取沉淀，加水溶解；(3)溶解液经萃取，制得萃取液；(4)萃取液去除溶剂后经聚酰胺树脂洗脱，制得黄蜀葵花黄酮类有效部位或提取物；优选提取方法为回流。In a third aspect, the present invention provides a method for preparing the above-mentioned effective part or the above-mentioned marshmallow extract. The preparation method includes the following steps: (1) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Extraction The pH value of the liquid is adjusted to 2.0-3.0, refrigerated, filtered to collect the precipitate, and dissolved in water; (3) the dissolved liquid is extracted to obtain an extraction liquid; (4) the extraction liquid is eluted with polyamide resin after removing the solvent to obtain marshmallow flowers. The effective fraction or extract of flavonoids; the preferred extraction method is reflux.
具体地，步骤(1)所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)所述的pH调节剂为盐酸、硫酸、醋酸、磷酸、枸橼酸、酒石酸或马来酸；步骤(3)的萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(4)所述的树脂径高比为1：4-1：9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。Specifically, the amount of ethanol used in step (1) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution; the pH regulator described in step (2) is hydrochloric acid , sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid; the extraction agent used for extraction in step (3) is n-butanol, petroleum ether or ethyl acetate, and the extraction method is continuous countercurrent extraction, so The material-to-liquid ratio of the extraction is 0.8-4:1, and the extraction stages of the extraction are level 1-5; the diameter-to-height ratio of the resin in step (4) is 1:4-1:9, and the loading liquid The concentration is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4-8BV 60-95% ethanol are used for elution.
第三主要方面，本发明提供了上述第一、第二主要方面的各个技术方案中，所述有效部位、提取物、组合物的应用。In the third main aspect, the present invention provides the application of the effective parts, extracts and compositions in each of the technical solutions of the above-mentioned first and second main aspects.
所述应用为在制备肾病药物中的应用，或制备眼部疾病药物中的应用，或制备红斑狼疮肾炎，或者造影剂导致的肾损伤、或者肺纤维化、或者心力衰竭、或者降低尿酸、或者SGLT2抑制剂的药物中的应用，或制备抗纤维化药物中的应用，或制备治疗和/或预防溃疡性疾病的药物中的应用，或制备防治炎症性肠病产品中的应用，或制备治疗和/或预防皮肤疾病的药物中的应用，或制备促进伤口愈合的药物中的应用。The application is application in the preparation of drugs for kidney diseases, or application in the preparation of drugs for eye diseases, or preparation of lupus nephritis, or kidney damage caused by contrast agents, or pulmonary fibrosis, or heart failure, or reducing uric acid, or The application of SGLT2 inhibitors in medicines, or the application in the preparation of anti-fibrotic medicines, or the application in the preparation of medicines for the treatment and/or prevention of ulcerative diseases, or the application in the preparation of products for the prevention and treatment of inflammatory bowel disease, or the preparation of treatment and/or application in medicines for preventing skin diseases, or application in preparing medicines for promoting wound healing.
优选地，所述肾病为糖尿病肾病，或者伴有肾纤维化的糖尿病肾病或肾炎、红斑狼疮肾炎，或者造影剂导致的肾损伤；优选所述的肾病为伴有肾纤维化的糖尿病肾病或肾炎；优选地，所述眼部疾病优选为黄斑变性、视觉疲劳或白内障、糖尿病视网膜病变、年龄相关性黄斑变性、中心性渗出性脉络膜视网膜炎、高度近视引起的黄斑病变；优选地，所述纤维化可选自肺纤维化，进一步的为特异性肺纤维化；优选地，所述溃疡性疾病为溃疡性肠炎、溃疡性胃炎、溃疡性咽喉炎和溃疡性口腔炎中的任意一种；优选地，所述的炎性肠病为溃疡性结肠炎和克罗恩病；优选地，所述皮肤疾病为皮疹、痤疮中的任意一种；优选地，所述伤口包括但不限于烧烫灼伤创面、残余小创面、供皮区创面、慢性溃疡创面和新鲜及难愈性皮肤创面；优选地，所述慢性溃疡创面包括但不限于糖尿病性、血管性、放射性溃疡。Preferably, the kidney disease is diabetic nephropathy, or diabetic nephropathy or nephritis accompanied by renal fibrosis, lupus erythematosus nephritis, or kidney damage caused by contrast agents; preferably, the kidney disease is diabetic nephropathy or nephritis accompanied by renal fibrosis. ; Preferably, the eye disease is preferably macular degeneration, visual fatigue or cataracts, diabetic retinopathy, age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia; Preferably, the Fibrosis can be selected from pulmonary fibrosis, and further is specific pulmonary fibrosis; preferably, the ulcer disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis; Preferably, the inflammatory bowel disease is ulcerative colitis and Crohn's disease; preferably, the skin disease is any one of rash and acne; preferably, the wound includes but is not limited to burns Burn wounds, residual small wounds, donor site wounds, chronic ulcer wounds, and fresh and refractory skin wounds; preferably, the chronic ulcer wounds include but are not limited to diabetic, vascular, and radiation ulcers.
本发明所述的药物组合物，按照特定比例进行组合具有良好的治疗糖尿病肾病的效果，副作用小，还具有治疗肺纤维化作用。The pharmaceutical composition of the present invention, when combined according to a specific ratio, has a good effect in treating diabetic nephropathy, has few side effects, and also has the effect of treating pulmonary fibrosis.
第四主要方面，本发明提供了一种药物制剂，含有上述的第一、第二主要方面的各个技术方案中所述的有效部位、提取物、组合物及其制备方法与应用。In the fourth main aspect, the present invention provides a pharmaceutical preparation containing the effective parts, extracts, compositions and preparation methods and applications described in the technical solutions of the above-mentioned first and second main aspects.
含有的有效部位、提取物在本药物制剂发明中称为提取物，由黄蜀葵花提取制备得到有效部位、提取物则称为黄蜀葵花提取物。The effective parts and extracts contained in the pharmaceutical preparation invention are called extracts, and the effective parts and extracts prepared by extracting marshmallow flowers are called marshmallow flower extracts.
第一方面，本发明提供了一种药物制剂，含有黄蜀葵花提取物及药学上可接受的辅料，所述黄蜀葵花提取物，In a first aspect, the present invention provides a pharmaceutical preparation, which contains marshmallow flower extract and pharmaceutically acceptable auxiliary materials. The marshmallow flower extract,
含有如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲 皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：2.0-25：3.0-20：1.0-7.0：4.0-23：0.6-12.0；Contains flavonoids in the following mass ratio: gossyrin-8-O-β-D-glucuronide: hyperoside: isoquercetin The mass ratio of dermatin: myricetin: quercetin-3'-O-glucoside: quercetin is 10: 2.0-25: 3.0-20: 1.0-7.0: 4.0-23: 0.6-12.0;
或者，含有如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷为0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；Alternatively, the flavonoid component contains the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O-β-D-glucuronic acid Glycosides: Hyperin is 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6;
或者，含有如下质量含量的黄酮类成分：金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷5-30％、杨梅素1.6-11％、槲皮素-3'-O-葡萄糖苷12.1-25％、槲皮素1.4-10％；进一步地，金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-30％、杨梅素3.0-4.9％或5.1-6.0％、槲皮素-3'-O-葡萄糖苷14-25％、槲皮素1.6-4.9％；这里指金丝桃苷等黄酮类在黄蜀葵花提取物中的占比；Alternatively, it contains flavonoids with the following mass content: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossyrin-8-O-β-D-glucuronide 5-30%, Myricetin 1.6-11%, quercetin-3'-O-glucoside 12.1-25%, quercetin 1.4-10%; further, hyperoside 8-26%, isoquercetin 12.0-19.8 %, gossyrin-8-O-β-D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-O-glucuronide 14-25% , Quercetin 1.6-4.9%; here refers to the proportion of flavonoids such as hyperoside in marshmallow flower extract;
所述制剂为非水性液体的制剂；The preparation is a non-aqueous liquid preparation;
所述药学上的可接受的辅料在制剂中的质量含量为0.5％-99.5％。The mass content of the pharmaceutically acceptable excipients in the preparation is 0.5%-99.5%.
在一些实施例中，所述黄蜀葵花提取物含有如下质量比的黄酮类成分：In some embodiments, the marshmallow flower extract contains flavonoid components in the following mass ratio:
棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：3.0-20：4.0-18：1.9-6.0：6.0-20：1.0-10.0，或10：6-15：5-13：1.0-2.9：4.0-9.5：0.6-6.0，或10：6-15：5-13：1.0-1.8：4.0-5.9：0.6-0.99；The mass ratio of gossyrin-8-O-β-D-glucuronide:hyperin:isoquercetin:myricetin:quercetin-3'-O-glucuronide:quercetin is 10 : 3.0-20: 4.0-18: 1.9-6.0: 6.0-20: 1.0-10.0, or 10: 6-15: 5-13: 1.0-2.9: 4.0-9.5: 0.6-6.0, or 10: 6-15 :5-13:1.0-1.8:4.0-5.9:0.6-0.99;
进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：4.0-17：4.50-16：1.9-5.5：7.0-16；Further, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 4.0-17: 4.50-16: 1.9-5.5: 7.0-16;
进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：6.0-15：5.0-13：2.0-5.0：8.0-14；Further, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 6.0-15:5.0-13:2.0-5.0:8.0-14;
进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素的质量比为10：1.0-6.0，优选为10：1.2-6，进一步优选为10：1.5-5.0；Further, the mass ratio of gossydisin-8-O-β-D-glucuronide: quercetin is 10:1.0-6.0, preferably 10:1.2-6, further preferably 10:1.5-5.0;
更进一步地，含有芦丁，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，进一步优选为10：0.1-0.3；Furthermore, it contains rutin, and the mass ratio of gossydisin-8-O-β-D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, and further preferably 10:0.1-0.3;
更进一步地，含有槲皮素-3-O-洋槐糖苷，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素-3-O-洋槐糖苷为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5；Furthermore, it contains quercetin-3-O-acaiglycoside, and the ratio of gossyrin-8-O-β-D-glucuronide: quercetin-3-O-acaiglycoside is 10:0.1- 2.5, preferably 10:0.1-0.9, further preferably 10:0.15-0.5;
再更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：6-16：5-13：1.0-4.5：4.0-12：0.6-6.0，或10：15.1-16：5-13：3.0-4.5：9.6-12：0.6-6.0；Furthermore, gossyrin-8-O-β-D-glucuronide: hypericin: isoquercetin: myricetin: quercetin-3'-O-glucuronide: quercetin The mass ratio is 10:6-16:5-13:1.0-4.5:4.0-12:0.6-6.0, or 10:15.1-16:5-13:3.0-4.5:9.6-12:0.6-6.0;
再更进一步地，含有芦丁，所述棉皮素-8-O-β-D-葡萄糖醛酸苷与芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，进一步优选为10：0.1-0.3；Furthermore, it contains rutin, and the mass ratio of the cottonseed-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably is 10: 0.1-0.3;
再更进一步地，含有槲皮素-3-O-洋槐糖苷，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5。Further still, it contains quercetin-3-O-acaiglycoside, and the mass ratio of the cottonseed-8-O-β-D-glucuronide:quercetin-3-O-acaiglycoside is: 10: 0.1-2.5, preferably 10: 0.1-0.9, further preferably 10: 0.15-0.5.
在一些实施例中，所述黄蜀葵花提取物含有如下质量比的黄酮类成分：In some embodiments, the marshmallow flower extract contains flavonoid components in the following mass ratio:
异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷为0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为0.8-1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为0.8-1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为0.8-1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为0.8-1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为0.8-1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；或者为0.8-1.2：0.09-1.6：0.2-2.0：0.05-1.2：0.5-3.0：0.25-3.6；或者为0.8-1.2：0.12-0.7：0.8-1.6：0.2-0.5：0.85-1.7：1.0-1.4； Isoquercetin: Quercetin: Quercetin-3'-O-glucoside: Myricetin: Gossyrin-8-O-β-D-glucuronide: Hypericin 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 0.8-1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 0.8- 1.2: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 0.8-1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3; Or 0.8-1.2: 0.09-1.6: 0.2-2.0: 0.05-1.2: 0.5-3.0: 0.25-3.6; Or 0.8-1.2: 0.12-0.7: 0.8-1.6: 0.2-0.5: 0.85-1.7: 1.0- 1.4;
进一步地，所述异槲皮苷的质量比数值为1.2或1.0；Further, the mass ratio value of the isoquercetin is 1.2 or 1.0;
进一步地，异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷为1：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；或者为1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；Further, isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O-β-D-glucuronide: hyperoside is 1 : 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16 -1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7 :0.8-1.6:0.2-0.5:0.8-1.8:0.8-1.4; further 1:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3; or 1.2:0.05-1.6:0.2 -4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1.2: 0.16-1.0: 0.6-2.4 : 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1.2: 0.2-0.7: 0.8-1.6: 0.2 -0.5: 0.8-1.8: 0.8-1.4; further 1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3;
进一步地，含有芦丁，所述异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，进一步优选为1：0.006-0.05，更进一步优选为1：0.007-0.04，再更进一步优选为1：0.008-0.03，最优选为1：0.009-0.03；Further, it contains rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, further preferably 1:0.006-0.05, even more preferably 1:0.007 -0.04, further preferably 1:0.008-0.03, most preferably 1:0.009-0.03;
进一步地，含有槲皮素-3-O-洋槐糖苷，所述异槲皮苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5。Further, it contains quercetin-3-O-acaxiglucoside, and the mass ratio of the isoquercetin and quercetin-3-O-acaxiglucoside is 10:0.1-2.5, preferably 10:0.1-0.9, More preferably, it is 10:0.15-0.5.
在一些实施例中，所述黄蜀葵花提取物含有如下质量含量的黄酮类成分：金丝桃苷10-25％、异槲皮苷8-19％、棉皮素-8-O-β-D-葡萄糖醛酸苷5-30％、槲皮素-3'-O-葡萄糖苷12.1-25％；In some embodiments, the marshmallow flower extract contains flavonoid components with the following mass content: hyperin 10-25%, isoquercetin 8-19%, gossipin-8-O-β-D -Glucuronide 5-30%, quercetin-3'-O-glucoside 12.1-25%;
进一步地，槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％；Further, the quercetin-3'-O-glucoside content is 12.6-23% or 13-20%;
进一步地，金丝桃苷12-23％、异槲皮苷10-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-25％、槲皮素-3'-O-葡萄糖苷12.6-23％；Further, hyperoside 12-23%, isoquercetin 10-17%, gossyrin-8-O-β-D-glucuronide 8.5-25%, quercetin-3'-O - Glucoside 12.6-23%;
进一步地，金丝桃苷13-22％、异槲皮苷11-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-21％、槲皮素-3'-O-葡萄糖苷13-20％；Further, hyperoside 13-22%, isoquercetin 11-17%, gossyrin-8-O-β-D-glucuronide 12-21%, quercetin-3'-O - Glucoside 13-20%;
进一步地，还含有槲皮素2-10％，优选槲皮素2.5-9％，进一步优选槲皮素3-8.5％，更进一步优选槲皮素1.5％-5％；Further, it also contains 2-10% quercetin, preferably 2.5-9% quercetin, further preferably 3-8.5% quercetin, even more preferably 1.5%-5% quercetin;
进一步地，含有杨梅素2-11％，优选杨梅素3-7％，进一步优选杨梅素3-5％，Further, it contains 2-11% myricetin, preferably 3-7% myricetin, further preferably 3-5% myricetin,
进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选槲皮素-3-O-洋槐糖苷0.1-1.5％，进一步优选槲皮素-3-O-洋槐糖苷0.1-0.9％，再进一步优选槲皮素-3-O-洋槐糖苷0.1-0.5％；Further, it contains 0.08-2.5% of quercetin-3-O-aphoretin glycoside, preferably 0.1-1.5% of quercetin-3-O-aphoretin glycoside, and further preferably 0.1-0.9% of quercetin-3-O-aphoretin glycoside. %, and further preferably quercetin-3-O-acaxiglucoside 0.1-0.5%;
进一步地，金丝桃苷11-22％、异槲皮苷12.0-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-23％、杨梅素1.6-4.9％或5.1-9.0％、槲皮素-3'-O-葡萄糖苷13-22％、槲皮素1.4-8％或1.4-7.8％；Further, hyperoside 11-22%, isoquercetin 12.0-17%, gossipin-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1 -9.0%, quercetin-3'-O-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%;
进一步地，含有芦丁0.01-1.0％，优选芦丁0.05-0.8％，进一步优选芦丁0.09-0.8％，更进一步优选芦丁0.1-0.6％。Further, it contains 0.01-1.0% rutin, preferably 0.05-0.8% rutin, further preferably 0.09-0.8% rutin, even more preferably 0.1-0.6% rutin.
在一些实施例中，所述黄蜀葵花提取物中黄酮类成分的质量含量为55％以上，优选为60％以上，进一步优选为65-85％或66-84％，更进一步优选为69-82％或69-90％；In some embodiments, the mass content of flavonoids in the marshmallow flower extract is more than 55%, preferably more than 60%, more preferably 65-85% or 66-84%, even more preferably 69-82% % or 69-90%;
进一步地，含有的槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷及金丝桃苷的总含量为55％以上，优选60％以上，进一步优选为65-85％，更进一步优选为69-82％；Further, it contains quercetin, quercetin-3'-O-glucoside, myricetin, gossyrin-8-O-β-D-glucuronide, isoquercitrin and hyperoside. The total content is more than 55%, preferably more than 60%, more preferably 65-85%, even more preferably 69-82%;
进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，优选槲皮素-3'-O-葡萄糖苷含量为12.6-23％、13-20％、13-20％，或者为14-25％；Further, the quercetin-3'-O-glucoside content is 12.1-25%, and the preferred quercetin-3'-O-glucoside content is 12.6-23%, 13-20%, 13-20%, Or 14-25%;
或者进一步地，槲皮素的含量高于0.7％，优选高于1.0％，进一步优选1.0％-10％， 更进一步优选1.5％-5％；Or further, the content of quercetin is higher than 0.7%, preferably higher than 1.0%, further preferably 1.0%-10%, More preferably, 1.5%-5%;
或者更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷为8.5-30％，优选12％-23％。Or further, gossydisin-8-O-β-D-glucuronide is 8.5-30%, preferably 12%-23%.
在一些实施例中，所述的黄蜀葵花提取物由如下步骤制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液浓缩后经萃取，制得萃取液；(3)萃取液去除溶剂后经大孔树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉；In some embodiments, the marshmallow flower extract is prepared by the following steps: (1) extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) extracting the extract after concentration to prepare Extract; (3) remove the solvent from the extract and elute with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
或者，由如下方法制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(22)提取液加入澄清剂，水浴处理，过滤去上清；(33)上清液经聚酰胺树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉。Alternatively, it is prepared by the following method: (1) Extract the flowers or medicinal parts of Hollyhock with ethanol to prepare an extract; (22) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (33) The supernatant After elution with polyamide resin, the marshmallow flower extract is obtained; the preferred extraction method is percolation.
在一些实施例中，步骤(1)中，所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)中，所述萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(3)中，所述大孔树脂型号为D101、HPD100或AB-8；步骤(22)中，所述水浴温度为50-70℃，水浴时间为30-90min；步骤(33)中，所述树脂径高比为1：4-9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。In some embodiments, in step (1), the amount of ethanol used is 10-25 times that of marshmallow flowers or marshmallow medicinal parts, and the ethanol is a 60-95% ethanol solution; in step (2), the The extraction agent used for the extraction is n-butanol, petroleum ether or ethyl acetate. The extraction method is continuous countercurrent extraction. The material-liquid ratio of the extraction is 0.8-4:1. The extraction stages of the extraction are Level 1-5; in step (3), the macroporous resin model is D101, HPD100 or AB-8; in step (22), the water bath temperature is 50-70°C, and the water bath time is 30-90 minutes; In step (33), the diameter-to-height ratio of the resin is 1:4-9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4- Elute with 8BV in 60-95% ethanol.
在一些实施例中，步骤(2)包括在萃取之前，对提取液经活性炭吸附、醇沉或酸沉处理。In some embodiments, step (2) includes subjecting the extraction liquid to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
在一些实施例中，所述大孔树脂的洗脱工艺为：大孔树脂径高比为1：4-9，上样液浓度为0.10-0.30g生药/mL，上样液体积为4-12BV，以1-4BV/h流速上样，用4-8BV纯水和1-5BV的3-15％乙醇以0.5-4BV/h流速进行除杂，用2-8BV 50-80％乙醇以1-5BV/h流速进行洗脱。In some embodiments, the elution process of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:4-9, the concentration of the sample solution is 0.10-0.30g crude drug/mL, and the volume of the sample solution is 4- 12BV, load the sample at a flow rate of 1-4BV/h, use 4-8BV pure water and 1-5BV 3-15% ethanol at a flow rate of 0.5-4BV/h to remove impurities, use 2-8BV 50-80% ethanol at a flow rate of 1 -5BV/h flow rate for elution.
第二方面，本发明还提供了一种黄蜀葵花提取物的药物制剂，含有药学上可接受的辅料，所述制剂为非水性液体的制剂；所述药学上的可接受的辅料在制剂中的含量为0.5％-99.5％；所述提取物由如下步骤制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液浓缩后经萃取，制得萃取液；(3)萃取液去除溶剂后经大孔树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉；In a second aspect, the present invention also provides a pharmaceutical preparation of marshmallow flower extract, which contains pharmaceutically acceptable auxiliary materials. The preparation is a non-aqueous liquid preparation; the pharmaceutically acceptable auxiliary materials are in the preparation. The content is 0.5%-99.5%; the extract is prepared by the following steps: (1) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Concentrating the extract and extracting to prepare the extract liquid; (3) remove the solvent from the extraction liquid and then elute with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
或者，由如下方法制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(22)提取液加入澄清剂，水浴处理，过滤去上清；(33)上清液经聚酰胺树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉。Alternatively, it is prepared by the following method: (1) Extract the flowers or medicinal parts of Hollyhock with ethanol to prepare an extract; (22) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (33) The supernatant After elution with polyamide resin, the marshmallow flower extract is obtained; the preferred extraction method is percolation.
在一些实施例中，步骤(1)中，所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)中，所述萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(3)中，所述大孔树脂型号为D101、HPD100或AB-8；步骤(22)中，所述水浴温度为50-70℃，水浴时间为30-90min；步骤(33)中，所述树脂径高比为1：4-9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。In some embodiments, in step (1), the amount of ethanol used is 10-25 times that of marshmallow flowers or marshmallow medicinal parts, and the ethanol is a 60-95% ethanol solution; in step (2), the The extraction agent used for the extraction is n-butanol, petroleum ether or ethyl acetate. The extraction method is continuous countercurrent extraction. The material-liquid ratio of the extraction is 0.8-4:1. The extraction stages of the extraction are Level 1-5; in step (3), the macroporous resin model is D101, HPD100 or AB-8; in step (22), the water bath temperature is 50-70°C, and the water bath time is 30-90 minutes; In step (33), the diameter-to-height ratio of the resin is 1:4-9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and 4-8BV pure water and 4- Elute with 8BV in 60-95% ethanol.
在一些实施例中，步骤(2)包括在萃取之前，对提取液经活性炭吸附、醇沉或酸沉处理。In some embodiments, step (2) includes subjecting the extraction liquid to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
在一些实施例中，所述大孔树脂的洗脱工艺为：大孔树脂径高比为1：4-9，上样液浓度为0.10-0.30g生药/mL，上样液体积为4-12BV，以1-4BV/h流速上样，用4-8BV纯水和1-5BV的3-15％乙醇以0.5-4BV/h流速进行除杂，用2-8BV 50-80％乙醇以1-5BV/h流速进行洗脱。In some embodiments, the elution process of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:4-9, the concentration of the sample solution is 0.10-0.30g crude drug/mL, and the volume of the sample solution is 4- 12BV, load the sample at a flow rate of 1-4BV/h, use 4-8BV pure water and 1-5BV 3-15% ethanol at a flow rate of 0.5-4BV/h to remove impurities, use 2-8BV 50-80% ethanol at a flow rate of 1 -5BV/h flow rate for elution.
第三方面，上述第一方面所述的药物制剂中所述黄蜀葵花提取物的制备方法包括 如下步骤：(101)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(102)提取液调节pH值为2.0-3.0，冷藏，过滤取沉淀，加水溶解；(103)溶解液经萃取，制得萃取液；(104)萃取液去除溶剂后经聚酰胺树脂洗脱，制得黄蜀葵花黄酮类有效部位或提取物；优选提取方法为回流。In a third aspect, the preparation method of the marshmallow flower extract in the pharmaceutical preparation described in the first aspect includes The steps are as follows: (101) Extract marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (102) Adjust the pH value of the extract to 2.0-3.0, refrigerate, filter to collect the precipitate, and add water to dissolve; (103) The dissolved solution is Extraction is performed to obtain an extraction liquid; (104) the solvent is removed from the extraction liquid and then eluted with polyamide resin to obtain the effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is reflux.
在一些实施例中，步骤(101)所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(102)所述的pH调节剂为盐酸、硫酸、醋酸、磷酸、枸橼酸、酒石酸或马来酸；步骤(103)的萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(104)所述的树脂径高比为1：4-9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。In some embodiments, the amount of ethanol used in step (101) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution; the pH adjustment in step (102) The agent is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid; the extraction agent used for extraction in step (103) is n-butanol, petroleum ether or ethyl acetate, and the extraction method is continuous countercurrent Extraction, the material-to-liquid ratio of the extraction is 0.8-4:1, the extraction level of the extraction is level 1-5; the diameter-to-height ratio of the resin in step (104) is 1:4-9, and the sample is loaded The liquid concentration is 0.10-0.60g crude drug/mL, the sample liquid volume is 4-12BV, and 4-8BV pure water and 4-8BV 60-95% ethanol are used for elution.
在上述第一～第三方面，所述非水性液体的制剂为固体制剂或半固体制剂；进一步选自栓剂、软膏剂、凝胶剂、丸剂、片剂、颗粒剂、胶囊剂或合剂。In the above first to third aspects, the non-aqueous liquid preparation is a solid preparation or a semi-solid preparation; further selected from the group consisting of suppositories, ointments, gels, pills, tablets, granules, capsules or mixtures.
在上述第一～第三方面，所述药学上可接受的辅料包括溶剂、乳化剂、崩解剂、填充剂、增溶剂、抗氧剂、pH调节剂、渗透压调节剂、抑菌剂、稀释剂、润湿剂、粘合剂或成膜剂中的任一种或任两种组合或任三种组合、或多种组合；进一步地，所述辅料选自乳糖、聚乙二醇、交联聚乙烯吡咯烷酮、月桂醇硫酸镁、羟丙基纤维素、滑石粉、硫酸钙、微晶纤维素、低取代羟丙甲基纤维素、交联羧甲基纤维素钠、硬脂酸镁中的任一种或任两种组合或任三种组合、或多种组合；进一步地，所述填充剂和崩解剂分别为制剂重量的1％-80％，进一步为3％-78％，进一步为5％-75％，进一步为10％-70％、15％-75％、15％-65％、20％-70％、20％-60％、25％-65％、25％-55％、30％-60％、30％-50％、35％-55％，进一步为1％-30％、25％-55％、50％-80％。In the above first to third aspects, the pharmaceutically acceptable excipients include solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, bacteriostatic agents, Any one, any two combinations, any three combinations, or multiple combinations of diluents, wetting agents, adhesives or film-forming agents; further, the auxiliary materials are selected from lactose, polyethylene glycol, Cross-linked polyvinylpyrrolidone, magnesium lauryl sulfate, hydroxypropyl cellulose, talc, calcium sulfate, microcrystalline cellulose, low-substituted hydroxypropyl methylcellulose, croscarmellose sodium, magnesium stearate Any one or any two combinations or any three combinations, or multiple combinations; further, the filler and disintegrant are respectively 1%-80% of the weight of the preparation, and further 3%-78% , further 5%-75%, further 10%-70%, 15%-75%, 15%-65%, 20%-70%, 20%-60%, 25%-65%, 25%- 55%, 30%-60%, 30%-50%, 35%-55%, further 1%-30%, 25%-55%, 50%-80%.
在上述第一～第三方面，所述药学上的可接受的辅料在制剂中的质量含量为2％-95％；进一步为5％-95％；进一步为10％-90％；进一步为15％-85％；进一步为20％-80％、25％-75％、30％-70％、40％-60％、50％-80％、15％-90％；进一步为20％-85％、4％-50％。In the above first to third aspects, the mass content of the pharmaceutically acceptable excipients in the preparation is 2%-95%; further 5%-95%; further 10%-90%; further 15 %-85%; further 20%-80%, 25%-75%, 30%-70%, 40%-60%, 50%-80%, 15%-90%; further 20%-85% , 4%-50%.
第四方面，本发明还提供了上述药物制剂在制备肾病药物中的应用，或制备眼部疾病药物中的应用，或制备红斑狼疮肾炎，或者造影剂导致的肾损伤、或者肺纤维化、或者心力衰竭、或者降低尿酸的药物中的应用，或制备抗纤维化药物中的应用，或制备治疗和/或预防溃疡性疾病的药物中的应用，或制备防治炎症性肠病产品中的应用，或制备治疗和/或预防皮肤疾病的药物中的应用，或制备促进伤口愈合的药物中的应用；优选地，所述肾病为糖尿病肾病，或者伴有肾纤维化的糖尿病肾病或肾炎、红斑狼疮肾炎，或者造影剂导致的肾损伤；优选所述的肾病为伴有肾纤维化的糖尿病肾病或肾炎；优选地，所述眼部疾病优选为黄斑变性、视觉疲劳或白内障、糖尿病视网膜病变、年龄相关性黄斑变性、中心性渗出性脉络膜视网膜炎、高度近视引起的黄斑病变；优选地，所述纤维化可选自肺纤维化，进一步的为特异性肺纤维化；优选地，所述溃疡性疾病为溃疡性肠炎、溃疡性胃炎、溃疡性咽喉炎和溃疡性口腔炎中的任意一种；优选地，所述的炎性肠病为溃疡性结肠炎和克罗恩病；优选地，所述皮肤疾病为皮疹、痤疮中的任意一种；优选地，所述伤口包括但不限于烧烫灼伤创面、残余小创面、供皮区创面、慢性溃疡创面和新鲜及难愈性皮肤创面；优选地，所述慢性溃疡创面包括但不限于糖尿病性、血管性、放射性溃疡。In a fourth aspect, the present invention also provides the use of the above-mentioned pharmaceutical preparation in the preparation of drugs for kidney diseases, or the preparation of drugs for eye diseases, or the preparation of lupus erythematosus nephritis, or kidney damage caused by contrast agents, or pulmonary fibrosis, or Application in heart failure, or in the preparation of drugs that lower uric acid, or in the preparation of anti-fibrotic drugs, or in the preparation of drugs for the treatment and/or prevention of ulcerative diseases, or in the preparation of products for the prevention and treatment of inflammatory bowel disease, Or the application in the preparation of medicines for treating and/or preventing skin diseases, or the application in the preparation of medicines for promoting wound healing; preferably, the kidney disease is diabetic nephropathy, or diabetic nephropathy accompanied by renal fibrosis or nephritis, lupus erythematosus Nephritis, or kidney damage caused by contrast agents; preferably, the kidney disease is diabetic nephropathy or nephritis accompanied by renal fibrosis; preferably, the eye disease is macular degeneration, visual fatigue or cataracts, diabetic retinopathy, age Related macular degeneration, central exudative chorioretinitis, macular degeneration caused by high myopia; preferably, the fibrosis can be selected from pulmonary fibrosis, and further is specific pulmonary fibrosis; preferably, the ulcer The disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis; preferably, the inflammatory bowel disease is ulcerative colitis and Crohn's disease; preferably, The skin disease is any one of rash and acne; preferably, the wounds include but are not limited to burn wounds, residual small wounds, donor site wounds, chronic ulcer wounds and fresh and difficult-to-heal skin wounds; Preferably, the chronic ulcer wounds include but are not limited to diabetic, vascular, and radiation ulcers.
本发明所述的黄蜀葵花黄酮类有效部位，除检测金丝桃苷、芦丁、异槲皮苷、棉皮素-8-O-β-D-葡萄糖醛酸苷(hibifolin)、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素这7 种成分以外，还在检测过程中，同时对本发明实施例制备得到的固形物样品进行了槲皮素-3-O-洋槐糖苷含量进行峰面积按比例进行含量折算，含量折算计算方式为＝金丝桃苷对照品峰面积/金丝桃苷对照品浓度*校正因子*了槲皮素-3-O-洋槐糖苷峰面积*定容体积/称样量(折水后)，含量计其不高于2.0％，进一步地不高于0.9％，优选为0.1％-0.7％。检测的多种黄酮总成分含量在70％以上，进一步地在75％以上，更进一步地在80％-90％。In addition to detecting hyperoside, rutin, isoquercitrin, gossipolin-8-O-β-D-glucuronide (hibifolin), the effective parts of flavonoids in marshmallow flowers of the present invention, Quercetin-3'-O-glucoside, quercetin 7 In addition to the above ingredients, during the detection process, the quercetin-3-O-acacia glycoside content of the solid samples prepared in the embodiments of the present invention was also measured and the peak area was proportionally converted to the content. The content conversion calculation method was =gold The peak area of hyperoside reference substance/concentration of hyperoside reference substance*correction factor*is calculated as the peak area of quercetin-3-O-acaciaglycoside*constant volume/weighed sample amount (after deducting water), and the content is not calculated. Higher than 2.0%, further not higher than 0.9%, preferably 0.1%-0.7%. The total content of various flavonoids tested was above 70%, further above 75%, and further between 80% and 90%.
本发明中提及的％乙醇无特殊说明皆为体积百分比。The % ethanol mentioned in the present invention is a volume percentage unless otherwise specified.
有益效果：Beneficial effects:
(1)本发明所述的黄蜀葵花黄酮类有效部位或提取物及其制剂具有治疗多种肾炎的作用，例如糖尿病肾病、造影剂肾损伤或红斑狼疮肾炎，还具有治疗眼部疾病的作用，尤其是与抑制血流量和抑制眼血管相关的各种疾病，例如年龄相关性黄斑变性、中心性渗出性脉络膜视网膜炎以及高度近视引起的黄斑病变等，特别地，本发明所述的黄蜀葵花黄酮类有效部位或提取物还具有治疗肺纤维化的作用。(1) The effective parts or extracts of flavonoids from marshmallow flowers according to the present invention and their preparations have the effect of treating various nephritis, such as diabetic nephropathy, contrast agent kidney injury or lupus erythematosus nephritis, and also have the effect of treating eye diseases, In particular, various diseases related to the suppression of blood flow and suppression of ocular blood vessels, such as age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia. In particular, the hollyhock flower of the present invention The effective parts or extracts of flavonoids also have the effect of treating pulmonary fibrosis.
(2)本发明所述的制备方法，工艺简单，生产周期短，处理条件温和，能耗低且黄蜀葵花7种黄酮类成分离效果好。所用的原料、试剂来源广泛，成本低，容易实现工业化大规模生产。(2) The preparation method of the present invention has simple technology, short production cycle, mild processing conditions, low energy consumption and good separation effect of 7 kinds of flavonoids from marshmallow flowers. The raw materials and reagents used come from a wide range of sources, are low in cost, and are easy to achieve industrialized large-scale production.
(3)本发明所述的黄蜀葵花黄酮类有效部位或提取物或组合物及其制剂具有较低的副作用，为患者提供了一种更好的治疗手段。(3) The effective parts or extracts or compositions of flavonoids from marshmallow flowers according to the present invention and their preparations have lower side effects and provide patients with a better treatment method.
(4)本发明提供的黄蜀葵花黄酮类有效部位或提取物及其制剂对皮肤疾病，例如皮炎、痤疮具有很好的预防和治疗作用。(4) The effective parts or extracts of flavonoids from marshmallow flowers provided by the present invention and their preparations have good preventive and therapeutic effects on skin diseases, such as dermatitis and acne.
(5)本发明所提供的黄蜀葵花黄酮类有效部位或提取物或组合物及其制剂可以在更低的用量下有效治疗皮肤疾病。(5) The effective parts or extracts or compositions of flavonoids from marshmallow flowers provided by the present invention and their preparations can effectively treat skin diseases at lower dosages.
(6)本发明提供的黄蜀葵花黄酮类有效部位或提取物及制剂具有促进伤口愈合的作用，具有明显的促进伤口愈合、治疗糖尿病溃疡作用及较好的安全性。(6) The effective parts or extracts and preparations of flavonoids from marshmallow flowers provided by the present invention have the effect of promoting wound healing, and have obvious effects of promoting wound healing and treating diabetic ulcers and have good safety.
(7)本发明所提供的黄蜀葵花黄酮类有效部位或提取物或组合物及其制剂可以在更低的用量下有效治疗口腔溃疡药效。(7) The effective parts or extracts or compositions of flavonoids from marshmallow flowers provided by the present invention and their preparations can effectively treat oral ulcers at lower dosages.
(8)本发明提供了一种含有黄蜀葵花提取物的肠溶制剂，该制剂具有稳定性较好，产品溶出速度快等特点，在胃中几乎不释放、肠道环境能迅速释放，可以减轻药物对胃部的刺激，提高药物的生物利用度，可以让药物更完全的在肠道中吸收。同时其对溃疡性结肠炎具有明显治疗效果，为临床治疗溃疡性结肠炎提供了一种全新的制剂。(8) The present invention provides an enteric-coated preparation containing marshmallow flower extract. The preparation has the characteristics of good stability and fast dissolution rate of the product. It is almost not released in the stomach, can be released quickly in the intestinal environment, and can alleviate the symptoms. Drugs stimulate the stomach and improve the bioavailability of the drug, allowing the drug to be more completely absorbed in the intestines. At the same time, it has obvious therapeutic effect on ulcerative colitis and provides a brand-new preparation for the clinical treatment of ulcerative colitis.
附图说明Description of the drawings
图1为5-ASA及ZHT溶解流程。Figure 1 shows the dissolution process of 5-ASA and ZHT.
图2为ZHT对DSS诱导的结肠萎缩的影响；(A)各组小鼠结肠外观图片，(B)各组小鼠结肠长度统计图，数据以平均值±标准误差表示。N＝8.**,P＜0.01,vs.Control；#,P＜0.05,vs.DSS，使用单因素方差及Dunnett’s多重比较检验进行分析。Figure 2 shows the effect of ZHT on DSS-induced colon atrophy; (A) Pictures of colon appearance of mice in each group, (B) Statistical diagram of colon length of mice in each group. Data are expressed as mean ± standard error. N＝8.**, P＜0.01, vs.Control; # , P＜0.05, vs.DSS, analyzed using one-way variance and Dunnett's multiple comparison test.
图3为ZHT对DSS诱导的结肠组织病理改变的影响；(A)各组小鼠结肠组织H&E染色图片，5倍物镜放大下，Scale bar＝500μm；20倍物镜放大下，Scale bar＝100μm，(B)各组小鼠结肠组织炎症评分统计图。数据以平均值±标准误差表示。N＝7.**,P＜0.01,vs.Control；##,P＜0.01,vs.DSS，使用单因素方差及Dunnett’s多重比较检验进行分析。Figure 3 shows the effect of ZHT on the pathological changes of colon tissue induced by DSS; (A) H&E staining pictures of colon tissue of mice in each group, under 5x objective lens magnification, Scale bar = 500 μm; under 20x objective lens magnification, Scale bar = 100 μm, (B) Statistical chart of colon tissue inflammation scores of mice in each group. Data are expressed as mean ± standard error. N＝7.**, P＜0.01, vs.Control; ## , P＜0.01, vs.DSS, analyzed using one-way variance and Dunnett's multiple comparison test.
图4为口腔溃疡药效实验中各组病理图。Figure 4 shows the pathological pictures of each group in the oral ulcer drug efficacy experiment.
图5为痤疮模型制备、给药、取材流程图。 Figure 5 is a flow chart of acne model preparation, drug administration, and material collection.
图6为特应性皮炎模型构建和给药流程图。Figure 6 is a flow chart of atopic dermatitis model construction and drug administration.
图7为ZHT和HKY对DNFB诱导的特应性皮炎模型外观的影响。Figure 7 shows the effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model.
图8为ZHT和HKY对DNFB诱导的特应性皮炎模型皮肤病理变化的影响，其中，各组小鼠皮肤组织HE染色代表图，蓝色线条指示表皮层厚度，Scale bar＝100μm。Figure 8 shows the effects of ZHT and HKY on the pathological changes in the skin of the DNFB-induced atopic dermatitis model. Among them, the representative images of HE staining of mouse skin tissue in each group, the blue line indicates the thickness of the epidermal layer, Scale bar = 100 μm.
图9为不同时间点各组大鼠皮肤伤口面积代表性图；(A)不同时间各组大鼠皮肤伤口面积代表性图，(B)各组大鼠皮肤伤口愈合曲线图。*P<0.05，**P<0.01vs.Model。Figure 9 is a representative graph of the skin wound area of each group of rats at different time points; (A) a representative graph of the skin wound area of each group of rats at different time points, (B) a graph of the skin wound healing curve of each group of rats. *P<0.05, **P<0.01vs.Model.
图10为大鼠皮肤伤口组织HE染色结果图及表皮厚度定量图；(A)大鼠伤口及边缘组织HE染色结果图，(B)各组大鼠皮肤伤口表皮厚度定量。**P<0.01，vs.Model；##,P<0.01,vs.Control。Figure 10 shows the results of HE staining of rat skin wound tissue and the quantitative graph of epidermal thickness; (A) The graph of HE staining result of rat wound and edge tissue, (B) Quantification of epidermal thickness of rat skin wound in each group. **P<0.01, vs.Model; ##, P<0.01, vs.Control.
图11为大鼠皮肤伤口组织Masson染色结果图及胶原沉积定量图；(A)大鼠伤口及边缘组织Masson染色结果图，(B)各组大鼠皮肤伤口胶原沉积定量图。N＝6，*P＜0.05，**P＜0.01vs.Model；##P<0.01,vs.Control。Figure 11 shows the results of Masson staining of rat skin wound tissue and the quantitative map of collagen deposition; (A) the result of Masson staining of rat wound and edge tissue, (B) the quantitative map of collagen deposition in skin wounds of rats in each group. N＝6, *P＜0.05, **P＜0.01vs.Model; ##P＜0.01, vs.Control.
图12为大鼠皮肤伤口组织CD31和VEGF mRNA水平变化；(A)治疗9天后大鼠皮肤组织鼠皮肤伤口组织CD31mRNA水平变化，(B)治疗9天后大鼠皮肤组织鼠皮肤伤口组织VEGF mRNA水平变化。Figure 12 shows the changes in CD31 and VEGF mRNA levels in rat skin wound tissue; (A) Changes in CD31 mRNA levels in rat skin tissue and rat skin wound tissue after 9 days of treatment, (B) VEGF mRNA levels in rat skin tissue and rat skin wound tissue after 9 days of treatment Variety.
图13为大鼠皮肤伤口组织CD31免疫荧光染色结果图及荧光强度定量图；(A)治疗9天后大鼠皮肤组织CD31免疫荧光染色，比例尺为250μm；(B)CD31的荧光强度定量图，N＝6，#P<0.05vs.control。Figure 13 shows the results of CD31 immunofluorescence staining of rat skin wound tissue and the fluorescence intensity quantification chart; (A) CD31 immunofluorescence staining of rat skin tissue after 9 days of treatment, the scale bar is 250 μm; (B) The fluorescence intensity quantification chart of CD31, N =6, #P<0.05vs.control.
图14为大鼠皮肤伤口组织VEGF蛋白水平变化；造模9天后大鼠皮肤组织鼠皮肤伤口组织VEGF蛋白水平变化，N＝6，**P<0.05vs model。Figure 14 shows the changes in VEGF protein levels in rat skin wound tissue; changes in VEGF protein levels in rat skin tissue after 9 days of modeling, N=6, **P<0.05 vs model.
具体实施方式Detailed ways
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The present invention will be further described in detail below with reference to specific examples. The following examples are not used to limit the present invention, but are only used to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following examples are generally in accordance with conventional conditions. Unless otherwise specified, the experimental methods used in the examples are generally in accordance with conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all Available commercially.
以下实施例的黄蜀葵花为黄蜀葵干燥花冠，如无特别说，为同一批次黄蜀葵花。The marshmallow flowers in the following examples are dried marshmallow corollas. Unless otherwise specified, they are from the same batch of marshmallow flowers.
对各实施例制备得到的黄蜀葵花黄酮类有效部位进行7种成分的含量的测定。这7种成分是指：金丝桃苷、芦丁、异槲皮苷、棉皮素-8-O-β-D-葡萄糖醛酸苷、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素。有效部位样品制备过程中，还对有效部位干燥前的溶液进行了各成分含量的测试，干燥前后样品含量有一定的变化。检测方法采用《一测多评法测定黄蜀葵花中7个成分》(《药物分析杂志》，2013年第12期，2082-2087)中报道的UPLC法测定其中成分的含量。The contents of seven components of the flavonoid effective parts of the marshmallow flowers prepared in each example were measured. These 7 ingredients refer to: hyperoside, rutin, isoquercetin, gossyrin-8-O-β-D-glucuronide, myricetin, quercetin-3'-O-glucose Glycosides, quercetin. During the preparation process of the effective part sample, the content of each component was also tested on the solution of the effective part before drying. There was a certain change in the sample content before and after drying. The detection method adopts the UPLC method reported in "Determination of 7 Components in Hollyhock Flowers by One Test and Multiple Evaluation Method" (Journal of Pharmaceutical Analysis, Issue 12, 2013, 2082-2087) to determine the content of the components.
所述的萃取的料液比(也即萃取剂:提取液)是指体积比。The material-to-liquid ratio of extraction (that is, extraction agent:extraction liquid) refers to the volume ratio.
以下实施例中所述黄葵胶囊若无特殊说明均为苏中药业集团股份有限公司生产销售的黄葵胶囊。Huangkui capsules described in the following examples are all Huangkui capsules produced and sold by Suzhong Pharmaceutical Group Co., Ltd. unless otherwise specified.
实施例1黄蜀葵花黄酮类有效部位的制备Example 1 Preparation of effective parts of flavonoids from marshmallow flowers
将100g黄蜀葵花粉碎至粗粉，用15倍的60％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，经活性炭吸附后，用正丁醇连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为1.5:1，萃取的进样速度为40mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为2级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液(水溶 液)浓度为0.15g生药/mL，上样液体积为7BV，以2BV/h流速上样，用6BV纯水和3BV 5％乙醇以2BV/h流速先后进行除杂，用4BV 60％乙醇以2BV/h流速进行洗脱得到洗脱液，然后将该洗脱液在50℃下减压回收乙醇后得到黄蜀葵花黄酮提取物，测试含量根据含量测定结果通过加入法，调整7种成分含量，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物2.23g，7种成分合计1769mg，占总固形物比例为79.32％。Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 15 times of 60% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, adsorb it with activated carbon, and use n-butanol for continuous countercurrent extraction. The material-to-liquid ratio (that is, extraction agent:extraction liquid) is 1.5:1, the injection speed of the extraction is 40mL/min, the extraction machine speed of the extraction is 40Hz, the extraction level of the extraction is 2, and the extraction liquid is placed at 40 After recovering the solvent under reduced pressure at ℃, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, and the loading liquid (water-soluble (liquid) concentration is 0.15g crude drug/mL, the sample liquid volume is 7BV, the sample is loaded at a flow rate of 2BV/h, 6BV pure water and 3BV 5% ethanol are used to remove impurities successively at a flow rate of 2BV/h, and 4BV 60% ethanol is used to remove impurities. Elute at a flow rate of 2BV/h to obtain an eluate. The eluate is then recovered under reduced pressure at 50°C to ethanol to obtain marshmallow flower flavonoid extract. The test content is adjusted according to the content measurement results through the addition method. The content of the seven ingredients is adjusted. The content of each component is as shown in the table below, and the total solid content of the effective parts of marshmallow flower flavonoids is 2.23g, and the total of the seven components is 1769mg, accounting for 79.32% of the total solid content.
表1样品的含量测定数据
Table 1 Content measurement data of samples
实施例2黄蜀葵花黄酮类有效部位的制备Example 2 Preparation of effective parts of flavonoids from marshmallow flowers
实施例2-1Example 2-1
将100g黄蜀葵花粉碎至粗粉，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2.5:1，萃取的进样速度为80mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为4级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:5，上样液浓度为0.15g生药/mL，上样液体积为6BV，以2BV/h流速上样，用6BV纯水和3BV 5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以1.5BV/h流速进行洗脱得到洗脱液，然后将该洗脱液在60℃下减压回收乙醇后得到黄蜀葵花黄酮提取物，测试含量，根据含量测定结果通过加入法，调整7种成分含量，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物2.99。槲皮素-3-O-洋槐糖苷含量为0.28％。Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 70% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, and use ethyl acetate for continuous countercurrent extraction. The extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 2.5:1, the injection speed of the extraction is 80mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 4, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, and the sample solution is 2BV/ h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate, and then use the eluate at 60 After recovering ethanol under reduced pressure at ℃, obtain the flavonoid extract of the marshmallow flower. Test the content. According to the content measurement results, adjust the content of the 7 components through the addition method so that the content of each component is as shown in the table below, and obtain the total effective part of the flavonoids of the marshmallow flower. Solids 2.99. The content of quercetin-3-O-acaiglycoside is 0.28%.
表2-1样品的含量测定数据

Table 2-1 Content measurement data of samples

实施例2-A黄蜀葵花黄酮类有效部位的制备Example 2-A Preparation of effective parts of flavonoids from marshmallow flowers
按照上述实施例2-1方法，黄蜀葵花放大10倍，另行制备2批黄蜀葵花黄酮类有效部位，获得表2-2样品的含量测定数据的样品。槲皮素-3-O-洋槐糖苷含量为0.20-0.50％。According to the method of the above-mentioned Example 2-1, the marshmallow flowers were enlarged 10 times, and two batches of flavonoid effective parts of the marshmallow flowers were prepared separately to obtain the content measurement data of the samples in Table 2-2. Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
表2-2样品的含量测定数据
Table 2-2 Content measurement data of samples
其中，2-A1含有按峰面积折算的槲皮素-3-O-洋槐糖苷含量含量为0.24％，各成分的比例关系如下表2-3所示。Among them, 2-A1 contains 0.24% quercetin-3-O-acaxiglucoside based on peak area. The proportion of each component is shown in Table 2-3 below.
表2-3样品各组分之间的含量比例数据
Table 2-3 Content ratio data between each component of the sample
实施例2-A3Example 2-A3
取金丝桃苷、异槲皮苷、hibifolin、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素、槲皮素3-O-洋槐糖苷原料，按照实施例2-A1的比例进行组方混合(以槲皮素3-O-洋槐糖苷替换芦丁，比例调整为0.02)，如下，制备得到总黄酮组合物。Get the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and follow the steps of Example 2-A1 Mix the formula according to the proportion (replace rutin with quercetin 3-O-acaxiglucoside, adjust the proportion to 0.02), and prepare the total flavonoid composition as follows.
表2-4
Table 2-4
实施例2-A4Example 2-A4
取金丝桃苷、异槲皮苷、hibifolin、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素、槲皮素3-O-洋槐糖苷原料，按照实施例2-A1的比例进行组方混合(以槲皮素3-O-洋槐糖苷替换芦丁，比例调整为0.05)，制备得到总黄酮组合物。Get the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and follow the steps of Example 2-A1 The formula was mixed according to the proportion (replacing rutin with quercetin 3-O-acaxiglucoside, and the proportion was adjusted to 0.05) to prepare a total flavonoid composition.
实施例2-BExample 2-B
为制得质量符合均一性要求的黄蜀葵花有效部位，根据多批实施例2-1的提取物各成分含量结果，研究药材与提取物种所述成分的含量关系，将3批来源不同、成分差异较大的黄蜀葵花按软件计算方式进行混合，取混合后的黄蜀葵花2000g用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液，去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为3:1，萃取的进样速度为120mL/min，萃取的萃取机转速为60Hz，萃取的萃取级数为4级，将萃取液在50℃下减压回收溶剂后， 加水稀释，离心，取上清液经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液浓度为0.15g生药/mL，上样液体积为7BV，以2BV/h流速上样，用6BV纯水和3BV5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以2BV/h流速进行洗脱得到洗脱液，浓缩，在60℃下减压干燥，即得总固形物，含芦丁0.22％，金丝桃苷20.58％，异槲皮苷17.81，hibifolin16.07％，杨梅素5.15％，槲皮素-3'-O-葡萄糖苷17.37％，槲皮素1.77％，含槲皮素-3-O-洋槐糖苷峰面积折算为0.43％。各组分之间的质量比例如下表2-5所示。In order to prepare the effective parts of marshmallow flowers whose quality meets the requirements of uniformity, based on the content results of each component of the extract in multiple batches of Example 2-1, the content relationship between the medicinal materials and the components of the extracted species was studied, and three batches of different sources and different components were studied. The larger marshmallow flowers are mixed according to the software calculation method. 2000g of the mixed marshmallow flowers are extracted by percolation with 18 times of 70% ethanol to obtain the marshmallow flower extract. Remove the ethanol, dilute with water, and continuously extract with ethyl acetate in countercurrent. The material-to-liquid ratio of the extraction (i.e., extraction agent:extraction liquid) is 3:1, the injection speed of the extraction is 120mL/min, the speed of the extraction machine is 60Hz, and the extraction level of the extraction is 4. Put the extraction liquid in After recovering the solvent under reduced pressure at 50°C, Add water to dilute, centrifuge, take the supernatant and process it with D101 macroporous resin. The processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.15g crude drug/mL, and the volume of the sample solution is For 7BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluate, concentrate, and incubate at 60 Dry under reduced pressure at ℃ to obtain total solids, containing rutin 0.22%, hyperin 20.58%, isoquercetin 17.81, hibifolin 16.07%, myricetin 5.15%, quercetin-3'-O- Glucoside is 17.37%, quercetin is 1.77%, and the peak area of quercetin-3-O-acacia glycoside is converted to 0.43%. The mass ratio of each component is shown in Table 2-5 below.
表2-5样品各组分之间的含量比例数据
Table 2-5 Content ratio data between each component of the sample
实施例3黄蜀葵花黄酮类有效部位的制备Example 3 Preparation of effective parts of flavonoids from marshmallow flowers
实施例3-1Example 3-1
将100g黄蜀葵花粉碎至粗粉，用18倍的60％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为3:1，萃取的进样速度为100mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为5级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:8，上样液浓度为0.15g生药/mL，上样液体积为8BV，以2BV/h流速上样，用6BV纯水和3BV 5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以3BV/h流速进行洗脱得到洗脱液，然后将该洗脱液在60℃下减压回收乙醇后得到黄蜀葵花黄酮提取物，测试含量根据含量测定结果通过加入法，调整7种成分含量，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物3.18g。槲皮素-3-O-洋槐糖苷含量0.20-0.50％。Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 60% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, and use ethyl acetate for continuous countercurrent extraction. The extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 3:1, the injection speed of the extraction is 100mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 5, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:8, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 8BV, and the sample solution is 2BV/ Load the sample at a flow rate of h, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 3BV/h to obtain the eluate, and then use the eluate at 60°C After recovering ethanol under reduced pressure, obtain the flavonoid extract of marshmallow flowers. The test content is based on the content measurement results. Adjust the contents of the 7 components by the addition method so that the contents of each component are as shown in the table below. The total solids of the effective parts of flavonoids of marshmallow flowers are obtained. 3.18g. Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
表3-1样品的含量测定数据
Table 3-1 Content measurement data of samples
实施例3-2 Example 3-2
按照实施例3-1的制备方法，黄蜀葵花药材500g进行重复实验，得到2批含量数据如下表3-2所示。槲皮素-3-O-洋槐糖苷含量0.30-0.80％。其中A代表“各成分/总固形物(％)”，B代表“各成分/异槲皮苷”。According to the preparation method of Example 3-1, 500g of marshmallow flowers were used for repeated experiments, and two batches of content data were obtained as shown in Table 3-2 below. Quercetin-3-O-Acacia glycoside content is 0.30-0.80%. Among them, A represents "each ingredient/total solids (%)" and B represents "each ingredient/isoquercitrin".
表3-2
Table 3-2
实施例4黄蜀葵花黄酮类有效部位的制备Example 4 Preparation of effective parts of flavonoids from marshmallow flowers
实施例4-1Example 4-1
将100g黄蜀葵花，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用正丁醇连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为3:1，萃取的进样速度为120mL/min，萃取的萃取机转速为60Hz，萃取的萃取级数为5级，将萃取液在50℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液浓度为0.15g生药/mL，上样液体积为7BV，以2BV/h流速上样，用6BV纯水和3BV5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以2BV/h流速进行洗脱得到洗脱液，然后将该洗脱液在60℃下减压回收乙醇后得到黄蜀葵花提取物，测试含量，根据含量测定结果通过加入法，调整7种成分含量，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物2.05g。100g of marshmallow flowers were percolated and extracted with 18 times of 70% ethanol to obtain marshmallow flower extract; the ethanol was removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with n-butanol. The material-liquid ratio of the extraction (that is, the extraction agent :extraction liquid) is 3:1, the injection speed of extraction is 120mL/min, the speed of extraction machine is 60Hz, and the extraction level of extraction is 5. After recovering the solvent under reduced pressure at 50℃, the extraction liquid is processed D101 macroporous resin treatment, the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 7BV, and the sample is loaded at a flow rate of 2BV/h , use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 2BV/h to obtain an eluate, and then use the eluate to recover ethanol under reduced pressure at 60°C. Afterwards, the marshmallow flower extract was obtained, and the content was tested. According to the content measurement results, the contents of the seven components were adjusted by the addition method so that the contents of each component were as shown in the table below, and 2.05g of the total solid content of the flavonoids effective parts of the marshmallow flower was obtained.
表4-1样品的含量测定数据
Table 4-1 Content measurement data of samples
实施例4-2 Example 4-2
将黄蜀葵花，用18倍的70％乙醇渗漉提取，提取液去除乙醇至0.5-1.0g生药/ml，加入量ZTC1+1-II澄清剂分别为4％B组分和2％A组分，水浴温度60℃，水浴保温时间60min，离心，滤过，稀释，经聚酰胺树脂(80-100目)处理，树脂的处理工艺为：树脂径高比为1:7，上样液浓度为0.15-0.5g生药/mL，上样液体积为8BV，用5BV纯水和5BV 80％乙醇洗脱得到洗脱液，浓缩，在60℃下减压干燥，测试含量，根据含量测定结果通过加入法，调整7种成分含量，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物即得，含槲皮素-3-O-洋槐糖苷含量为0.39％。Extract the marshmallow flowers by percolation with 18 times of 70% ethanol. Remove the ethanol from the extract to 0.5-1.0g crude drug/ml. Add the ZTC1+1-II clarifying agent at 4% component B and 2% component A respectively. , water bath temperature is 60°C, water bath holding time is 60 minutes, centrifuge, filter, dilute, and treated with polyamide resin (80-100 mesh). The resin treatment process is: the resin diameter-to-height ratio is 1:7, and the sample solution concentration is 0.15-0.5g crude drug/mL, the volume of the sample solution is 8BV, elute with 5BV pure water and 5BV 80% ethanol to obtain the eluate, concentrate, dry under reduced pressure at 60°C, test the content, and add Method, adjust the contents of the 7 components so that the contents of each component are as shown in the table below, and the total solids of the effective parts of the flavonoids of the marshmallow flowers are obtained, and the content of quercetin-3-O-acacia glycoside is 0.39%.
表4-2
Table 4-2
实施例5黄蜀葵花黄酮类有效部位的制备Example 5 Preparation of effective parts of flavonoids from marshmallow flowers
实施例5-1Example 5-1
将100g黄蜀葵花，用15倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用正丁醇连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2.5:1，萃取的进样速度为80mL/min，萃取的萃取机转速为50Hz，萃取的萃取级数为4级，将萃取液在50℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液浓度为0.2g生药/mL，上样液体积为6BV，以2BV/h流速上样，用1BV纯水和4BV5％乙醇以2BV/h流速进行除杂，用3BV 60％乙醇以2BV/h流速进行洗脱得到洗脱液，将洗脱液在60℃下减压回收乙醇后得到黄蜀葵花提取物，测试含量，根据含量测定结果通过加入法，调整7种成分含量，制得黄蜀葵花黄酮类有效部位总固形物2.86g。100g of marshmallow flowers were percolated and extracted with 15 times of 70% ethanol to obtain marshmallow flower extract; ethanol was removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with n-butanol. The material-to-liquid ratio of the extraction (that is, the extraction agent :extraction liquid) is 2.5:1, the injection speed of extraction is 80mL/min, the speed of extraction machine is 50Hz, and the extraction level of extraction is 4. After recovering the solvent under reduced pressure at 50℃, the extraction liquid is processed D101 macroporous resin treatment, the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.2g crude drug/mL, the volume of the sample solution is 6BV, and the sample is loaded at a flow rate of 2BV/h , use 1BV pure water and 4BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 3BV 60% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, and recover the ethanol under reduced pressure at 60°C to obtain The content of the marshmallow flower extract was tested. According to the content measurement results, the contents of the seven ingredients were adjusted by the addition method to obtain 2.86g of the total solid content of the flavonoids effective parts of the marshmallow flower.
实施例5-2Example 5-2
将黄蜀葵花，用15倍的70％乙醇回流提取，提取液去除乙醇至0.5-1.0g生药/ml，加入10％盐酸溶液调节pH值为2.0-3.0，冷藏，滤过，洗涤，取沉淀，加水溶解，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为3:1，萃取的萃取级数为4级，将萃取液在50℃下减压回收溶剂后，加水稀释，经聚酰胺树脂处理，树脂的处理工艺为：树脂径高比为1:6，上样液浓度为0.15-0.5g生药/mL，上样液体积为6BV，用4BV纯水和4BV 70％乙醇洗脱得到洗脱液，浓缩，在60℃下减压干燥，即得。槲皮素-3-O-洋槐糖苷含量为0.47％。Extract the marshmallow flowers with 15 times of 70% ethanol under reflux. Remove the ethanol from the extract to 0.5-1.0g crude drug/ml, add 10% hydrochloric acid solution to adjust the pH to 2.0-3.0, refrigerate, filter, wash, and take the precipitate. Add water to dissolve, and use ethyl acetate for continuous countercurrent extraction. The material-to-liquid ratio of the extraction (i.e., extraction agent:extraction liquid) is 3:1. The extraction level of the extraction is 4. The extraction liquid is recovered under reduced pressure at 50°C to recover the solvent. Then, add water to dilute it, and process it with polyamide resin. The resin treatment process is: the resin diameter-to-height ratio is 1:6, the concentration of the sample solution is 0.15-0.5g crude drug/mL, the volume of the sample solution is 6BV, and 4BV pure water is used. Elute with 4BV 70% ethanol to obtain the eluate, concentrate, and dry under reduced pressure at 60°C to obtain. The content of quercetin-3-O-acaxiglucoside is 0.47%.
表5

table 5

实施例6黄蜀葵花黄酮类有效部位的制备Example 6 Preparation of effective parts of flavonoids from marshmallow flowers
将100g黄蜀葵花，用16倍的60％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2:1，萃取的进样速度为100mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为4级，将萃取液在50℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液浓度为0.3g生药/mL，上样液体积为3BV，以2BV/h流速上样，用3BV纯水和3BV 5％乙醇以2BV/h流速进行除杂，用4BV 70％乙醇以2BV/h流速进行洗脱得到洗脱液，测试含量并通过加入7种具体成分，调整7种成分含量，使得其各成含量如下表所示，然后将该洗脱液在60℃下减压回收乙醇后得到黄蜀葵花提取物，测试含量，根据含量测定结果通过加入法，调整7种成分含量，加入量不超过总重的10％，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物3.16g。100g of marshmallow flowers were percolated and extracted with 16 times of 60% ethanol to obtain marshmallow flower extract; ethanol was removed from the marshmallow flower extract, diluted with water, and continuously countercurrent extracted with ethyl acetate. The extracted material-to-liquid ratio (that is, the extraction agent :extraction liquid) is 2:1, the injection speed of extraction is 100mL/min, the speed of extraction machine is 40Hz, and the extraction level of extraction is 4. After recovering the solvent under reduced pressure at 50℃, the extraction liquid is processed D101 macroporous resin treatment, the processing technology of macroporous resin is: the diameter-to-height ratio of macroporous resin is 1:6, the concentration of the sample solution is 0.3g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 2BV/h , use 3BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 70% ethanol to elute at a flow rate of 2BV/h to obtain the eluent, test the content and adjust 7 types by adding 7 specific ingredients The content of ingredients is as shown in the table below. Then the eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain marshmallow flower extract. The content is tested. According to the content measurement results, the content of the seven ingredients is adjusted by the addition method. , the addition amount does not exceed 10% of the total weight, so that the content of each component is as shown in the table below, and 3.16g of the total solid content of the effective parts of marshmallow flower flavonoids is obtained.
表6样品的含量测定数据
Table 6 Content measurement data of samples
实施例7黄蜀葵花黄酮类有效部位的制备(对比例)Example 7 Preparation of effective parts of flavonoids from marshmallow flowers (comparative example)
实施例7-1Example 7-1
将500g黄蜀葵花粉碎至粗粉，用20倍的70％乙醇渗漉提取，得黄蜀葵花提取液；减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:4，上样液浓度为0.15g生药/mL，上样液体积为6BV，以2BV/h流速上样，用7BV纯水和4BV5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以1.5BV/h流速进行 洗脱得到洗脱液，将洗脱液在60℃下减压回收乙醇后得到黄蜀葵花总固形物16.12g，其各成分含量如下表所示。Crush 500g of marshmallow flowers into coarse powder, and extract by percolation with 20 times of 70% ethanol to obtain marshmallow flower extract. After recovering the solvent under reduced pressure, it is treated with D101 macroporous resin. The processing technology of macroporous resin is: macroporous resin The diameter-to-height ratio is 1:4, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, the sample is loaded at a flow rate of 2BV/h, and 7BV pure water and 4BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. , using 4BV 60% ethanol at a flow rate of 1.5BV/h The eluate was obtained by elution. The eluate was recovered under reduced pressure at 60°C to recover ethanol, and 16.12g of total solids of marshmallow flowers were obtained. The contents of each component are as shown in the table below.
表7-1样品的含量测定数据
Table 7-1 Content measurement data of samples
实施例7-2Example 7-2
将100g黄蜀葵花粉碎至粗粉，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:5，上样液浓度为0.15g生药/mL，上样液体积为6BV，以2BV/h流速上样，用6BV纯水和3BV5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以1.5BV/h流速进行洗脱得到洗脱液，将洗脱液在60℃下减压回收乙醇后得到黄蜀葵花总固形物3.56g。Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 70% ethanol to obtain marshmallow flower extract. After recovering the solvent under reduced pressure, it is treated with D101 macroporous resin. The processing technology of macroporous resin is: macroporous resin The diameter-to-height ratio is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 6BV, the sample is loaded at a flow rate of 2BV/h, and 6BV pure water and 3BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. , use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain an eluate. The eluate is recovered under reduced pressure at 60°C to recover ethanol to obtain 3.56g of total solids of marshmallow flowers.
表7-2样品的含量测定数据
Table 7-2 Content measurement data of samples
实施例8黄蜀葵花黄酮类有效部位的制备(对比例)Example 8 Preparation of effective parts of flavonoids from marshmallow flowers (comparative example)
将100g黄蜀葵花粉碎至粗粉，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2.5:1，萃取的进样速度为80mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为4级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:5，上样液浓度为0.15g生药/ml，上样液体积为6BV，以2BV/h流速上样，用6BV纯水和3BV5％乙醇以2BV/h流速进行除杂，用4BV 60％乙醇以1.5BV/h流速进行洗脱得到洗脱液，测试含量，然后 在洗脱液中加入芦丁，金丝桃苷，使得其各成含量如下表所示，然后在60℃下减压回收乙醇后得到黄蜀葵花总固形物4.28g。Crush 100g of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 70% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, and use ethyl acetate for continuous countercurrent extraction. The extracted material-to-liquid ratio is ( That is, the extraction agent:extraction liquid) is 2.5:1, the injection speed of the extraction is 80mL/min, the speed of the extraction machine is 40Hz, the extraction level of the extraction is 4, and the extraction liquid is recovered under reduced pressure at 40°C. After solvent, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/ml, the volume of the sample solution is 6BV, and the sample solution is 2BV/ h flow rate to load the sample, use 6BV pure water and 3BV 5% ethanol to remove impurities at a flow rate of 2BV/h, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain the eluate, test the content, and then Rutin and hyperoside were added to the eluent so that the contents of each component are as shown in the table below, and then ethanol was recovered under reduced pressure at 60°C to obtain 4.28g of total solids of marshmallow flowers.
表8样品的含量测定数据
Table 8 Content measurement data of samples
实施例9Example 9
实施例9-1黄蜀葵花黄酮类有效部位的制备Example 9-1 Preparation of effective parts of flavonoids from marshmallow flowers
将25kg黄蜀葵花粉碎至粗粉，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2.5:1，萃取的萃取级数为4级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:5，上样液浓度为0.15g生药/mL，上样液体积为3BV，以1.5BV/h流速上样，用6BV纯水和3BV 10％乙醇以2BV/h流速进行除杂，用4BV60％乙醇以1.5BV/h流速进行洗脱得到洗脱液，将该洗脱液在60℃下减压回收乙醇后得到黄蜀葵花总固形物通过加入7种具体成分，调整7种成分(加入成分含量不超过总重的20％)，使得其各成含量如下表所示，制得黄蜀葵花黄酮类有效部位总固形物。槲皮素-3-O-洋槐糖苷含量0.20-0.50％。Crush 25kg of marshmallow flowers to coarse powder, and extract by percolation with 18 times of 70% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, and use ethyl acetate for continuous countercurrent extraction. The extracted material-to-liquid ratio is ( That is, the extraction agent:extracting liquid) is 2.5:1, and the extraction stage of the extraction is 4. After the extracting liquid is recovered under reduced pressure at 40°C, it is treated with D101 macroporous resin. The processing process of macroporous resin is: The diameter-to-height ratio of the macroporous resin is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 3BV, and the sample is loaded at a flow rate of 1.5BV/h. Use 6BV pure water and 3BV 10% ethanol at a flow rate of 2BV/ h flow rate to remove impurities, use 4BV 60% ethanol to elute at a flow rate of 1.5BV/h to obtain an eluent, and recover the ethanol under reduced pressure at 60°C to obtain the total solids of marshmallow flowers by adding 7 specific components. , adjust the 7 ingredients (the content of the added ingredients does not exceed 20% of the total weight) so that the content of each component is as shown in the table below, and the total solid content of the effective parts of the flavonoids in the marshmallow flower is obtained. Quercetin-3-O-Acacia glycoside content is 0.20-0.50%.
表9-1样品的含量测定数据
Table 9-1 Content measurement data of samples
实施例9-2黄蜀葵花黄酮类有效部位的制备Example 9-2 Preparation of effective parts of flavonoids from marshmallow flowers
参考实施例2-B，将30kg黄蜀葵花混合物粉碎至粗粉，用18倍的70％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，用乙酸乙酯连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为2.5:1，萃取的进样速度为80mL/min，萃 取的萃取机转速为40Hz，萃取的萃取级数为4级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:5，上样液浓度为0.15g生药/mL，上样液体积为5BV，以1.5BV/h流速上样，用3BV纯水和4BV5％乙醇以2BV/h流速进行除杂，用5BV 60％乙醇以1.5BV/h流速进行洗脱得到洗脱液，将-洗脱液在65℃下减压回收乙醇后得到黄蜀葵花总固形物，真空干燥，粉碎，检测7种成分黄酮含量(％)，结果如下，其中槲皮素-3-O-洋槐糖苷含量0.20-0.50％。Referring to Example 2-B, 30kg of the marshmallow flower mixture was crushed to coarse powder, and extracted by percolation with 18 times of 70% ethanol to obtain a marshmallow flower extract; the ethanol was removed from the marshmallow flower extract, diluted with water, and continuously counter-currently used with ethyl acetate. Extraction, the material-to-liquid ratio of extraction (that is, extraction agent:extraction liquid) is 2.5:1, the injection rate of extraction is 80mL/min, and the extraction speed is 80mL/min. The extraction machine speed is 40Hz, and the extraction level is 4. After the extraction liquid is decompressed at 40°C to recover the solvent, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter of the macroporous resin is high The ratio is 1:5, the concentration of the sample solution is 0.15g crude drug/mL, the volume of the sample solution is 5BV, the sample is loaded at a flow rate of 1.5BV/h, and 3BV pure water and 4BV 5% ethanol are used to remove impurities at a flow rate of 2BV/h. Elute with 5BV 60% ethanol at a flow rate of 1.5BV/h to obtain the eluate. The eluate is recovered under reduced pressure at 65°C to recover the ethanol to obtain the total solids of marshmallow flowers. Dry in vacuum, crush, and detect 7 kinds of flavonoids. Content (%), the results are as follows, in which the content of quercetin-3-O-acaxiglucoside is 0.20-0.50%.
表9-2样品的含量测定数据
Table 9-2 Content measurement data of samples
实施例11黄蜀葵花黄酮类有效部位的滴眼液的制备Example 11 Preparation of Eye Drops from the Effective Parts of Althea Flower Flavonoids
制备过程：按实施例1-7任一种方法制备得到的有效部位，加注射用水搅拌溶解，加至100L，并调节pH至7，过滤、灌装，灭菌，即得滴眼液。所用pH调节剂为氢氧化钠或/和盐酸。Preparation process: Prepare the effective part according to any method of Examples 1-7, add water for injection, stir and dissolve, add to 100L, adjust the pH to 7, filter, fill, and sterilize to obtain eye drops. The pH adjuster used is sodium hydroxide or/and hydrochloric acid.
实施例12糖肾模型药理实验Example 12 Pharmacological Experiment on Glucose Kidney Model
实施例12-1Example 12-1
动物模型：链脲佐菌素(STZ)+高脂饲料糖肾模型，C57BL/6小鼠，20±10g，SPF级，雄性。Animal model: Streptozotocin (STZ) + high-fat diet glucose renal model, C57BL/6 mice, 20±10g, SPF grade, male.
给药方式：灌胃给药。Mode of administration: intragastric administration.
实验造模：实验小鼠适应性饲养3天后，喂养高脂高糖饲料(实验室自制，配方：猪油：蔗糖：蛋黄：基础饲料＝18：20：3：59)，连续8周，然后开始按100mg/kg剂量腹腔注射STZ柠檬酸钠溶液，连续7天，第8天尾静脉检测小鼠血糖水平，以血糖大于16.7mmol/L为糖尿病模型成模标准。以血糖大于13.8mmol/L，出现尿蛋白、肾功能异常，说明建模成功。Experimental modeling: After the experimental mice were adaptively raised for 3 days, they were fed high-fat and high-sugar feed (made in the laboratory, formula: lard: sucrose: egg yolk: basic feed = 18:20:3:59) for 8 consecutive weeks, and then STZ sodium citrate solution was injected intraperitoneally at a dose of 100 mg/kg for 7 consecutive days. On the 8th day, the blood glucose level of the mice was measured in the tail vein, and the diabetes model was established based on blood glucose greater than 16.7 mmol/L. If blood sugar is greater than 13.8mmol/L, urine protein and renal function abnormalities appear, it indicates that the modeling is successful.
实验分组：正常组，模型组，阳性药组(达格列净片，45.5mg/kg)，实施例2-1方法制备样品组(62.4mg/kg)和实施例6方法制备样品组(62.4mg/kg)、实施例8方法制备样品组(62.4mg/kg)，总共6组，每组15只，给药4周后检测尿蛋白及尿肌酐及计算每日摄食量及小鼠状态。检测结果如下表12-1所示。Experimental groups: normal group, model group, positive drug group (dapagliflozin tablets, 45.5 mg/kg), sample group prepared by the method of Example 2-1 (62.4 mg/kg) and sample group prepared by the method of Example 6 (62.4 mg/kg), and the sample group (62.4 mg/kg) was prepared by the method of Example 8, with a total of 6 groups, 15 animals in each group. After 4 weeks of administration, urine protein and urinary creatinine were detected, and daily food intake and mouse status were calculated. The test results are shown in Table 12-1 below.
表12-1

Table 12-1

注:*与正常对照组比较，P＜0.05；#与模型对照组比较，P＜0.05。Note: *Compared with the normal control group, P＜0.05; #Compared with the model control group, P＜0.05.
实施例各组对尿蛋白均有降低作用，其中实施例2-1样品组可显著降低尿蛋白和尿蛋白/尿肌酐(ACR)。小鼠状态观察结果显示，实施例2-1样品组腹泻、腹胀小鼠明显少于其他各组。Each group of Examples has a reducing effect on urinary protein, among which the sample group of Example 2-1 can significantly reduce urinary protein and urinary protein/urinary creatinine (ACR). The observation results of the mouse status showed that the number of mice with diarrhea and abdominal distension in the sample group of Example 2-1 was significantly less than that of the other groups.
实施例12-2Example 12-2
动物模型：db/db小鼠，16周龄，C57BL/6小鼠，16周龄。Animal models: db/db mice, 16 weeks old, C57BL/6 mice, 16 weeks old.
给药方式：灌胃给药。Mode of administration: intragastric administration.
实验造模：db/db小鼠饲养17周后，检测尿蛋白，出现尿蛋白、肾功能异常，则模型成功，用于后期实验。Experimental modeling: After 17 weeks of raising db/db mice, urine protein is detected. If urine protein and renal function abnormalities appear, the model is successful and can be used for later experiments.
实验分组：C57BL/6小鼠分为正常组，将db/db小鼠分为模型组，阳性药组(达格列净片，45.5mg/kg)，实施例3-1方法制备样品组(62.4mg/kg)和实施例9-1方法制备样品组(62.4mg/kg)、实施例2-A3方法制备样品组(62.4mg/kg)，实施例7-1样品组(62.4mg/kg)，每组15只，给药4周后检测尿蛋白及尿肌酐及每日摄食量，并观察小鼠状态。检测结果如下表12-2所示。Experimental grouping: C57BL/6 mice were divided into normal group, db/db mice were divided into model group, positive drug group (dapagliflozin tablets, 45.5mg/kg), sample group prepared by the method of Example 3-1 ( 62.4 mg/kg) and the sample group (62.4 mg/kg) prepared by the method of Example 9-1, the sample group (62.4 mg/kg) prepared by the method of Example 2-A3, and the sample group (62.4 mg/kg) of Example 7-1 ), 15 mice in each group. After 4 weeks of administration, urine protein, urinary creatinine and daily food intake were measured, and the status of the mice was observed. The test results are shown in Table 12-2 below.
表12-2
Table 12-2
注:*与正常对照组比较，P＜0.05；#与模型对照组比较，P＜0.05。Note: *Compared with the normal control group, P＜0.05; #Compared with the model control group, P＜0.05.
实施例各组对尿蛋白均有降低作用，各样品组可降低尿蛋白和尿蛋白/尿肌酐(ACR)。小鼠状态观察结果显示，实施例3-1和实施例9-1样品组、实施例2-A3样品组腹泻、腹胀小鼠明显少于其他各组。Example: Each group has a reducing effect on urinary protein, and each sample group can reduce urinary protein and urinary protein/urinary creatinine (ACR). The observation results of the mouse status showed that there were significantly fewer mice with diarrhea and abdominal distension in the sample groups of Example 3-1 and Example 9-1, and the sample group of Example 2-A3 than in the other groups.
实施例13黄酮对眼部血流量和脉络膜新生血管的作用Example 13 Effects of flavonoids on ocular blood flow and choroidal neovascularization
实验动物：大鼠，体质量150-180g，SPF级，雌雄各半。Experimental animals: rats, body weight 150-180g, SPF grade, half male and half male.
给药方式：灌胃给药。Mode of administration: intragastric administration.
实验造模：选择健康雄性Brown-Norway大鼠，***肌肉注射(50mg/kg)麻醉动物，用0.5％托吡卡胺和新福林混合散瞳剂散瞳，采用氪黄激光(美国科以人公司)(波长568nm，参数：光斑直径100μm，曝光时间0.1s，功率150-200mW)，在120D专用前置镜下，距视盘2-4盘径，围绕视盘在视网膜大血管间光凝8个点，看到Bruch膜被击破后产生小气泡为合格光凝点，有视网膜、脉络膜或玻璃体出血者剔除。Experimental modeling: Select healthy male Brown-Norway rats, anesthetize the animals with intramuscular injection of ketamine (50 mg/kg), use a mixed mydriatic agent of 0.5% tropicamide and phenylephrine to dilate the pupils, and use krypton yellow laser (American Science and Technology Company) (wavelength 568nm, parameters: spot diameter 100μm, exposure time 0.1s, power 150-200mW), under the 120D special front mirror, 2-4 disk diameters away from the optic disc, photocoagulate 8 between the large blood vessels of the retina around the optic disc If you see small bubbles produced after Bruch's membrane is broken, it is a qualified photocoagulation point. Those with retinal, choroidal or vitreous hemorrhage will be excluded.
药物分组及给药剂量：正常组，模型组，阳性对照药组(维替搏芬1.35mg/kg)，实施例2-1样品组(69.9mg/kg)，实施例3-1样品组(87.36mg/kg)，实施例7-2样品组(87.36mg/kg)、实施例9-1样品组(87.36mg/kg)，实施例9-2样品组 (87.36mg/kg)每组15只，给药4周后检测眼部脉络膜血流抑制率及脉络膜血管新生面积。检测结果如下表13所示。Drug grouping and dosage: normal group, model group, positive control drug group (vertibofen 1.35 mg/kg), Example 2-1 sample group (69.9 mg/kg), Example 3-1 sample group ( 87.36mg/kg), Example 7-2 sample group (87.36mg/kg), Example 9-1 sample group (87.36mg/kg), Example 9-2 sample group (87.36 mg/kg), 15 rats in each group. After 4 weeks of administration, the choroidal blood flow inhibition rate and choroidal angiogenesis area of the eye were measured. The test results are shown in Table 13 below.
表13
Table 13
注:*与正常对照组比较，P＜0.01；#与模型对照组比较，P＜0.05。Note: *Compared with the normal control group, P＜0.01; #Compared with the model control group, P＜0.05.
结论：各实施例样品对脉络膜血流抑制率及脉络膜血管新生面积均有抑制作用，实施例2-1样品组、实施例3-1样品组、实施例9-1样品组、实施例9-2样品组能够显著降低脉络膜血管新生。Conclusion: The samples of each example have inhibitory effects on the choroidal blood flow inhibition rate and choroidal angiogenesis area. Example 2-1 sample group, Example 3-1 sample group, Example 9-1 sample group, Example 9- Sample group 2 can significantly reduce choroidal angiogenesis.
实施例4-1，实施例4-2，实施例5-1，实施例5-2样品实验也具备与本实施例中的实施例2-1，实施例3-1，实施例9-1，实施例9-2上述各方面基本一致的作用或效果。Example 4-1, Example 4-2, Example 5-1, and Example 5-2 sample experiments also have the same characteristics as Example 2-1, Example 3-1, and Example 9-1 in this example. , the above-mentioned aspects of Embodiment 9-2 basically have the same functions or effects.
实施例14黄酮对造影剂肾损伤的作用Example 14 Effect of flavonoids on contrast-induced renal injury
实验动物：SD大鼠，体质量200-250g，SPF级，雌雄各半。Experimental animals: SD rats, body weight 200-250g, SPF grade, half male and half male.
给药方式：灌胃给药Mode of administration: intragastric administration
实验造模：选择健康雄性SD大鼠，首先，肌内注射给予呋塞米(10ml/kg)，之后每隔15分钟尾静脉注射***素合成抑制剂(吲哚美辛，10mg/kg，溶于二甲基亚砜，1.25％NAHCO3溶液稀释)及一氧化氮合酶抑制剂(N-硝基-L-精氨酸甲酯基，L-NAME，10mg/kg，溶于0.9％NaCl溶液)和76％泛影葡胺(10ml/kg)。Experimental modeling: Healthy male SD rats were selected. First, furosemide (10 ml/kg) was administered intramuscularly, and then a prostaglandin synthesis inhibitor (indomethacin, 10 mg/kg, dissolved) was injected into the tail vein every 15 minutes. diluted in dimethyl sulfoxide, 1.25% NAHCO 3 solution) and nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester, L-NAME, 10 mg/kg, dissolved in 0.9% NaCl solution) and 76% diatrizoate meglumine (10ml/kg).
建模成功标准根据2012年KDIGOAKI临床指南推荐的急性肾损伤诊断标准(符合以下情况之一者即可被诊断)：①48小时内血清肌酐升高超过26.5umol/L(0.3mg/dl)；②血清肌酐升高超过基线50％—确认或推测7天内发生；③尿量减少＜0.5ml/(kg*h)，且持续6小时以上。其中，在动物模型中常使用血清肌酐升高超过基线50％作为急性肾损伤的诊断标准，即建模成功标准。The modeling success criteria are based on the diagnostic criteria for acute kidney injury recommended by the 2012 KDIGOAKI clinical guidelines (people who meet one of the following conditions can be diagnosed): ① Serum creatinine rises by more than 26.5umol/L (0.3mg/dl) within 48 hours; ② Serum creatinine increases by more than 50% of the baseline - confirmed or presumed to occur within 7 days; ③ Urine output decreases <0.5ml/(kg*h) and lasts for more than 6 hours. Among them, in animal models, an increase in serum creatinine exceeding 50% of the baseline is often used as the diagnostic criterion for acute kidney injury, that is, the modeling success criterion.
药物分组及给药剂量：正常组、模型组，阳性对照药组(厄贝沙坦50mg/kg/d)、实施例2-1样品组(69.9mg/kg)，实施例3-1样品组(87.36mg/kg)，实施例7样品组(87.36mg/kg)、实施例9-1样品组(87.36mg/kg)，实施例9-2样品组(87.36mg/kg，按照实施例9-2制备得到的固形物)每组15只，给药4周后检测眼部脉络膜血流抑制率及脉络膜血管新生面积。检测结果如下表14所示。Drug grouping and dosage: normal group, model group, positive control drug group (irbesartan 50 mg/kg/d), Example 2-1 sample group (69.9 mg/kg), Example 3-1 sample group (87.36mg/kg), Example 7 sample group (87.36mg/kg), Example 9-1 sample group (87.36mg/kg), Example 9-2 sample group (87.36mg/kg, according to Example 9 -2 prepared solid material), 15 animals per group, and 4 weeks after administration, the choroidal blood flow inhibition rate and choroidal angiogenesis area of the eye were measured. The test results are shown in Table 14 below.
表14

Table 14

注:*与正常组比较，P＜0.01；#与模型组比较，P＜0.05Note: *Compared with the normal group, P＜0.01; #Compared with the model group, P＜0.05
结论：in conclusion:
模型组造影剂肾损伤大鼠血清肌酐、血清尿素氮显著高于各给药组；实施例9-1样品组、实施例2-A1样品组、实施例3.1样品、实施例8样品和实施例6样品均对血清肌酐、血清尿素氮均不同程度降低。The serum creatinine and serum urea nitrogen of rats with contrast agent renal injury in the model group were significantly higher than those of each administration group; Example 9-1 sample group, Example 2-A1 sample group, Example 3.1 sample, Example 8 sample and Examples All 6 samples reduced serum creatinine and serum urea nitrogen to varying degrees.
实施例15黄酮对红斑狼疮肾炎的作用Example 15 Effect of flavonoids on lupus erythematosus nephritis
选择MRL/lpr雄性小鼠(红斑狼疮肾炎小鼠)26只，观察1组，体质量为18-22g，13周龄，购于南京君科生物工程有限公司，取雄性C57BL/6小鼠24只作为正常组，体质量18-22g，13周龄，购于武汉华联科生物技术有限公司。Twenty-six MRL/lpr male mice (lupus nephritis mice) were selected and one group was observed. The body weight was 18-22g and they were 13 weeks old. They were purchased from Nanjing Junke Bioengineering Co., Ltd. and 24 male C57BL/6 mice were selected. Only as normal group, body weight 18-22g, 13 weeks old, purchased from Wuhan Hualianke Biotechnology Co., Ltd.
药物分组及给药剂量：正常组、MRL/lpr小鼠模型组，阳性对照药组(***1mg/kg)、实施例2-B样品组(69.9mg/kg)，实施例3-1样品组(87.36mg/kg)，实施例7-1样品组(87.36mg/kg)、实施例5-2样品组(87.36mg/kg)，实施例8样品组(87.36mg/kg)每组15只，灌胃给药4周。Drug grouping and dosage: normal group, MRL/lpr mouse model group, positive control drug group (dexamethasone 1 mg/kg), Example 2-B sample group (69.9 mg/kg), Example 3-1 Sample group (87.36mg/kg), Example 7-1 sample group (87.36mg/kg), Example 5-2 sample group (87.36mg/kg), Example 8 sample group (87.36mg/kg) each group 15 rats were administered intragastrically for 4 weeks.
实验指标：给药结束后，接取24小时尿液，用于检测尿蛋白，尿液接取后，所有小鼠摘眼球取血，离心获得血清，用于抗核抗体检测，同时，取出肾脏，速置于液氮中速冻，转移到负八十冰箱保存，用于肾脏免疫检测。检测结果如下表15所示。Experimental indicators: After the administration, 24-hour urine was collected for detection of urinary protein. After the urine was collected, the eyes of all mice were removed to collect blood, and centrifuged to obtain serum for anti-nuclear antibody detection. At the same time, the kidneys were removed. , quickly frozen in liquid nitrogen, transferred to a minus 80 refrigerator for storage, and used for renal immune testing. The test results are shown in Table 15 below.
表15
Table 15
注:*与正常组比较，P＜0.01；#与模型组比较，P＜0.05。Note: *Compared with the normal group, P＜0.01; #Compared with the model group, P＜0.05.
结论：模型组红斑狼疮肾炎小鼠尿蛋白、血清抗核抗体水平、肾组织内IgG和C3沉积量均明显高于各给药组；而实施例5-2样品组、实施例2-B样品组、实施例3-1样品、实施例8样品和实施例7-1样品均对尿蛋白、血清抗核抗体水平、肾组织内IgG和C3沉积能够起到改善作用，且实施例2-B样品、实施例3-1样品、实施例5-2样品效果较优。Conclusion: The urinary protein, serum antinuclear antibody levels, and IgG and C3 deposition in renal tissue of mice with lupus erythematosus nephritis in the model group were significantly higher than those of each administration group; while the Example 5-2 sample group and Example 2-B sample Group, Example 3-1 sample, Example 8 sample and Example 7-1 sample can all improve urinary protein, serum antinuclear antibody levels, and IgG and C3 deposition in renal tissue, and Example 2-B The sample, Example 3-1 sample, and Example 5-2 sample have better effects.
实施例16黄酮毒理作用 Example 16 Toxicological effects of flavonoids
实验动物：大鼠，体质量150-180g，SPF级，雌雄各半。Experimental animals: rats, body weight 150-180g, SPF grade, half male and half male.
给药方式：灌胃给药。Mode of administration: intragastric administration.
药物分组及给药剂量：正常组、黄葵胶囊组(提取物8g/kg)，实施例2-A1样品组(5g/kg)，实施例9-2样品组(8g/kg)，每组30只，每天1次，连续12周，给药剂量按提取物计算。Drug grouping and dosage: normal group, Huangkui capsule group (extract 8g/kg), Example 2-A1 sample group (5g/kg), Example 9-2 sample group (8g/kg), each group 30 animals, once a day for 12 weeks, the dosage is calculated based on the extract.
实验结果：给药结束后，较正常组相比，黄葵胶囊组肾脏重量、脏体和脏脑系数升高，病理显示，黄葵胶囊组有中度肾小管肥大，而实施例9-2样品组和实施例2-A1组，病理检测未出现肾小管肥大，检测结果如下表16所示。Experimental results: After the administration, compared with the normal group, the kidney weight, viscera and viscera-brain coefficients of the Huangkui capsule group increased. Pathology showed that the Huangkui capsule group had moderate renal tubular hypertrophy, while Example 9-2 In the sample group and the Example 2-A1 group, no tubular hypertrophy was found in pathological examination, and the detection results are shown in Table 16 below.
表16
Table 16
注:与正常组相比，*P<0.05。Note: Compared with the normal group, *P<0.05.
实施例2-A3，实施例3-1，实施例4-1，实施例5-2，实施例9-1经验证也具备与本实施例中的实施例2-A1，实施例9-2上述安全性基本一致的作用或效果。Embodiment 2-A3, Embodiment 3-1, Embodiment 4-1, Embodiment 5-2, and Embodiment 9-1 have been verified to be equivalent to Embodiment 2-A1 and Embodiment 9-2 in this embodiment. The above-mentioned safety functions or effects are basically the same.
实施例17总黄酮有效部位对特发性肺纤维化的作用Example 17 Effect of effective fractions of total flavonoids on idiopathic pulmonary fibrosis
实验样品1制备：取金丝桃苷、异槲皮苷、hibifolin、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素、槲皮素3-O-洋槐糖苷原料，按表17-1投料比例，称取各活性成分，混合，制备得到药物组合物。Preparation of experimental sample 1: Take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, as shown in the table 17-1 Feeding ratio, weigh each active ingredient, mix, and prepare a pharmaceutical composition.
表17-1
Table 17-1
实验动物：昆明种小鼠，雌雄各半，体重18-22克，清洁级。Experimental animals: Kunming mice, half male and half female, weighing 18-22 grams, clean grade.
动物分组：随机分组，分为正常组、模型组、对照药组(罗格列酮5mg/kg)、实施例2-A3样品组(70mg/kg)、实施例9-2样品组(70mg/kg)、黄葵胶囊样品组(以提取物计180mg/kg)，实验样品1组(62.4mg/kg)，每组小鼠10只。Animal grouping: randomly divided into normal group, model group, control drug group (rosiglitazone 5mg/kg), Example 2-A3 sample group (70mg/kg), Example 9-2 sample group (70mg/kg) kg), Huangkui capsule sample group (180mg/kg based on extract), experimental sample group 1 (62.4mg/kg), 10 mice in each group.
实验造模：实验小鼠适应性饲养3天后，用4％的水合氯醛(0.01ml/g)腹腔注射麻醉。取仰卧固定位，常规消毒，行颈正中切口，钝性分离暴露气管，模型组，对照药组、治疗组于气管软骨环间隙穿刺缓慢注入博莱霉素(5mg/kg)，正常对照组注入等体积生理盐水，注药后立即将小鼠直立旋转3-5min，使药液均匀分布于两侧肺内，然后缝合皮肤，并在缝合处消毒，待清醒后送清洁级观察室饲养。Experimental modeling: After the experimental mice were adaptively raised for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01ml/g). The patients were placed in a supine fixed position, routine disinfection was performed, a midline cervical incision was made, and the trachea was exposed by blunt dissection. The model group, control drug group, and treatment group were punctured and slowly injected bleomycin (5 mg/kg) into the tracheal cartilage ring space, and the normal control group was injected Equal volume of normal saline was injected. Immediately after injecting the drug, the mice were rotated upright for 3-5 minutes to allow the drug solution to be evenly distributed in the lungs on both sides. The skin was then sutured and the sutures were disinfected. After they woke up, they were sent to a clean observation room for feeding.
给药方式：造模第二天开始给药，给药组按上述剂量灌胃给药，每天一次，正常组和模型组按同样的方法给予同等体积的蒸馏水10ml/kg，连续给药28天。Method of administration: Administration will begin on the second day after modeling. The administration group will be administered intragastrically according to the above dosage, once a day. The normal group and the model group will be administered the same volume of distilled water 10ml/kg in the same way for 28 consecutive days. .
肺组织病理切片染色：给药结束后，取右肺，按常规病理学方法进行固定、包埋、切片、HE染色。将肺泡炎和肺纤维化的程度分为四级。Staining of pathological sections of lung tissue: After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis is divided into four grades.
实验结果如下表17-2所示，实验结果表明，模型组小鼠肺组织肺泡炎和肺纤维化程度显著加重，呈典型的肺纤维化实质病变。给药28天，各剂量组小鼠肺系数明显降 低，肺组织肺泡炎及肺纤维化程度明显减轻。本发明总黄酮有效部位可明显降低肺纤维化小鼠的肺泡炎和肺纤维化程度，对肺纤维化小鼠的肺部具有保护作用，可以减缓由纤维化对方肺部组织造成的损伤。The experimental results are shown in Table 17-2 below. The experimental results showed that the degree of alveolitis and pulmonary fibrosis in the lung tissue of mice in the model group was significantly aggravated, showing typical pulmonary fibrosis parenchymal lesions. After 28 days of administration, the lung coefficient of mice in each dose group decreased significantly. The degree of alveolitis and pulmonary fibrosis in the lung tissue is significantly reduced. The effective part of the total flavonoids of the present invention can significantly reduce the degree of alveolitis and pulmonary fibrosis in mice with pulmonary fibrosis, has a protective effect on the lungs of mice with pulmonary fibrosis, and can slow down the damage to the lung tissue caused by fibrosis.
表17-2
Table 17-2
注:*与正常组比较，P＜0.01；#与模型组比较，P＜0.05。Note: *Compared with the normal group, P＜0.01; #Compared with the model group, P＜0.05.
经验证实施例1，实施例2-1，实施例2-A2，实施例3.2-1，实施例5-2，也具备与本实施例中的实施例2-A3，实施例9-2上述纤维化基本一致的作用或效果。It has been verified that Example 1, Example 2-1, Example 2-A2, Example 3.2-1, and Example 5-2 also have the same characteristics as Example 2-A3 and Example 9-2 in this example. Fibrosis basically has the same effect or effect.
实施例18 SGLT2作用机制研究Example 18 Study on the mechanism of action of SGLT2
Sodium-glucose contransporter2(SLC5A2，也称SGLT2)是一个编码基因，该基因编码钠-葡萄糖共转运体，且可以重吸收肾小球滤过的大约90％以上的葡萄糖。SLC5A2只在肾脏近端小管上皮细胞膜内刷状缘表达，被认为是近端小管的标志物。主要功能为回收滤过的钠和葡萄糖。在糖尿病及糖尿病肾病患者肾脏组织中，抑制SLC5A2蛋白的表达能够起到排钠、排水降糖的作用，从而能够保护糖尿病和糖尿病患者肾脏组织。Sodium-glucose contransporter2 (SLC5A2, also known as SGLT2) is a gene encoding a sodium-glucose cotransporter and can reabsorb more than 90% of the glucose filtered by the glomerulus. SLC5A2 is only expressed at the brush border within the epithelial cell membrane of the proximal tubule of the kidney and is considered a marker of the proximal tubule. The main function is to recover filtered sodium and glucose. In the kidney tissue of patients with diabetes and diabetic nephropathy, inhibiting the expression of SLC5A2 protein can play a role in excreting sodium and reducing blood sugar, thus protecting the kidney tissue of patients with diabetes and diabetic nephropathy.
动物模型：db/db小鼠，18周龄。Animal model: db/db mice, 18 weeks old.
给药方式：灌胃给药。Mode of administration: intragastric administration.
实验分组：db/db小鼠UACR>200mg/g时分为3组，糖尿病肾病组(DKD)、黄葵胶囊组(DKD+HK，0.84g/kg/d)和总黄酮组(DKD+HT，实施例2-A2样品，0.0755g/kg/d)，黄葵胶囊组和总黄酮组给药四周(28d)。Enzyme-linked immunosorbent assay(ELISA)方法测db/db小鼠尿液中微量白蛋白(UACR)，Reverse Transcription Polymerase Chain Reaction(RT-PCR)方法检测SLC5A2mRNA表达水平，ImmunoHistoChemistry(IHC)的方法检测SLC5A2蛋白表达水平。Experimental grouping: db/db mice were divided into 3 groups when UACR>200mg/g, diabetic nephropathy group (DKD), Huangkui capsule group (DKD+HK, 0.84g/kg/d) and total flavonoids group (DKD+HT, Example 2-A2 sample, 0.0755g/kg/d), Huangkui capsule group and total flavonoids group were administered for four weeks (28d). The Enzyme-linked immunosorbent assay (ELISA) method is used to detect trace albumin (UACR) in the urine of db/db mice, the Reverse Transcription Polymerase Chain Reaction (RT-PCR) method is used to detect the expression level of SLC5A2 mRNA, and the ImmunoHistoChemistry (IHC) method is used to detect the SLC5A2 protein. The expression level.
实验结果：Experimental results:
与DKD组相比，HK组(黄葵胶囊组)、HT组(总黄酮组)分组前体重DKD(N＝5)，HK(N＝5)，HT(N＝6)、给药第一周体重和第四周体重DKD(N＝5)，HK(N＝5)，HT(N＝6)无显著性差异。给药第一周血糖和第四周血糖DKD(N＝5)，HK(N＝5)，HT(N＝6)无显著性差异。给药第一周UACR值DKD(N＝5)，HK(N＝5)，HT(N＝6)，无显著性差异。给药第四周HT组与DKD相比，*P<0.05，UACR值明显降低，有显著性差异。给药第四周肾脏均值DKD(N＝5)，HK(N＝5)，HT(N＝6)；无显著性差异。给药第四周肾脏指数DKD(N＝5)，HK(N＝5)，HT(N＝6)；*P<0.05，肾脏指数明显增加，有显著性差异。给药第四周DKD组部分肾小球呈分叶状，基底膜明显增厚，肾小球毛细血管受压明显，系膜区系膜细胞增生及系膜基质明显扩增；HK组肾小球基底膜增厚明显改善，肾小球毛细血管完好，系膜细胞和系膜基质增生明显改善；HT组肾小球基底膜明显变 薄，肾小球无增生且肾小球毛细血管间质无增生，系膜细胞和系膜基质增生无扩增。给药第四周IHC-P量化结果表明与DKD组(N＝5)相比，黄葵HK组(N＝5)、HT组(N＝6)SLC5A2蛋白表达水平明显降低。给药第四周RT-PCR结果验证免疫组化结果表明，与DKD组(N＝5)相比，黄葵HK组(N＝5)、HT组(N＝6)SLC5A2mRNA表达水平明显降低。给药第四周与DKD组(N＝5)相比，黄葵HK组(N＝5)、HT组(N＝6)SLC5A2IHC-P量化表达量与肾小球数量的比值明显降低，与IHC-P量化结果相一致。Compared with the DKD group, the body weight before grouping of the HK group (Huangkui capsule group) and HT group (total flavonoids group) was DKD (N=5), HK (N=5), HT (N=6), and the first dose There was no significant difference between weekly body weight and fourth week body weight in DKD (N=5), HK (N=5), and HT (N=6). There was no significant difference between blood glucose in the first week of administration and blood glucose in DKD (N=5), HK (N=5) and HT (N=6) in the fourth week. There was no significant difference in the UACR values of DKD (N=5), HK (N=5), and HT (N=6) in the first week of administration. Compared with DKD in the fourth week of administration, *P<0.05, the UACR value of the HT group was significantly lower, and there was a significant difference. In the fourth week of administration, the mean renal values were DKD (N=5), HK (N=5), and HT (N=6); there was no significant difference. In the fourth week of administration, the renal index was DKD (N=5), HK (N=5), HT (N=6); *P<0.05, the renal index increased significantly, and there was a significant difference. In the fourth week of administration, some glomeruli in the DKD group were lobulated, with significantly thickened basement membrane, obvious glomerular capillary compression, proliferation of mesangial cells in the mesangial area, and significant expansion of the mesangial matrix; glomeruli in the HK group The thickening of the glomerular basement membrane was significantly improved, the glomerular capillaries were intact, and the proliferation of mesangial cells and mesangial matrix was significantly improved; the glomerular basement membrane of the HT group was significantly changed. Thin, there is no proliferation of glomeruli, no proliferation of glomerular capillary interstitium, and no expansion of mesangial cells and mesangial matrix. The IHC-P quantification results in the fourth week of administration showed that compared with the DKD group (N=5), the SLC5A2 protein expression levels in the Huangkui HK group (N=5) and HT group (N=6) were significantly reduced. The immunohistochemical results verified by RT-PCR results in the fourth week of administration showed that compared with the DKD group (N=5), the expression levels of SLC5A2 mRNA in the Huangkui HK group (N=5) and HT group (N=6) were significantly reduced. Compared with the DKD group (N=5) in the fourth week of administration, the ratio of SLC5A2IHC-P quantified expression to the number of glomeruli in the Huangkui HK group (N=5) and HT group (N=6) was significantly lower. The IHC-P quantification results were consistent.
因此，黄葵胶囊和总黄酮能够有效降低db/db小鼠的微量白蛋白尿。黄葵胶囊和总黄酮能够抑制近曲小管细胞膜上SLC5A2蛋白的表达，能够降低DKD小鼠肾脏SLC5A2蛋白的表达，能够有效抑制SLC5A2蛋白的活性，从而有效减少DKD小鼠肾脏对葡萄糖的重吸收。Therefore, Huangkui capsules and total flavonoids can effectively reduce microalbuminuria in db/db mice. Huangkui capsules and total flavonoids can inhibit the expression of SLC5A2 protein on the cell membrane of the proximal convoluted tubule, reduce the expression of SLC5A2 protein in the kidneys of DKD mice, and effectively inhibit the activity of SLC5A2 protein, thereby effectively reducing the reabsorption of glucose in the kidneys of DKD mice.
实施例19黄蜀葵花总黄酮提取物的制备Example 19 Preparation of total flavonoid extract of marshmallow flowers
参照上述实施例3-1的制备方法，黄蜀葵花5000g进行重复实验，得到2批含量(单位：％)数据如下表所示。槲皮素-3-O-洋槐糖苷含量0.30-0.80％。Referring to the preparation method of the above-mentioned Example 3-1, repeated experiments were carried out with 5000g of marshmallow flowers, and two batches of content (unit: %) data were obtained as shown in the table below. Quercetin-3-O-Acacia glycoside content is 0.30-0.80%.
表19
Table 19
实施例20溃疡性结肠炎动物实验Example 20 Animal Experiment on Ulcerative Colitis
1.实验材料1. Experimental materials
1.1实验动物1.1 Experimental animals
SPF级雄性C57BL/6J小鼠，6-8周龄，体重22-25g，由扬州大学比较医学中心提供。SPF grade male C57BL/6J mice, 6-8 weeks old, weighing 22-25g, were provided by the Comparative Medicine Center of Yangzhou University.
1.2试剂及药品1.2 Reagents and drugs
表20-1

Table 20-1

2.实验方法2. Experimental methods
2.1动物饲养和管理2.1 Animal feeding and management
实验小鼠全程饲养于SPF级动物房，光照与黑暗时间比为1:1，室温控制在23±2℃，湿度控制在55％，小鼠能够自由进食和饮水；每三天更换一次垫料，以保证小鼠处于干燥清洁的环境中。Experimental mice were kept in an SPF-level animal room throughout the entire process, with a light-to-dark time ratio of 1:1, room temperature controlled at 23±2°C, and humidity controlled at 55%. The mice could eat and drink freely; the bedding was changed every three days. , to ensure that the mice are in a dry and clean environment.
2.2药物配制2.2 Drug Preparation
所有药物都按图1步骤进行溶解：①：于给药前一天配置0.5％CMC-Na：于100mL70℃纯水中缓慢加入0.5g CMC-Na粉末，搅拌均匀，于室温静置过夜。次日，配置药物溶液前超声，直至得到均质的0.5％CMC-Na溶液。②：分别称取12.5mg，37.5mg，75mg ZHT和100mg 5-ASA，分别加入0.1mL DMSO配置成DMSO母液。③：向②中各母液分别加入0.1mL Tween-80。④：向③中各溶液加入4.8mL 0.5％CMC-Na溶液。分别配制成2.5mg/mL，7.5mg/mL和15mg/mL的ZHT混悬液及20mg/mL的5-ASA混悬液。All drugs are dissolved according to the steps in Figure 1: ①: Prepare 0.5% CMC-Na one day before administration: slowly add 0.5g CMC-Na powder to 100 mL 70°C pure water, stir evenly, and let stand at room temperature overnight. The next day, ultrasonicate before preparing the drug solution until a homogeneous 0.5% CMC-Na solution is obtained. ②: Weigh 12.5mg, 37.5mg, 75mg ZHT and 100mg 5-ASA respectively, and add 0.1mL DMSO respectively to prepare DMSO mother solution. ③: Add 0.1mL Tween-80 to each mother liquor in ②. ④: Add 4.8mL of 0.5% CMC-Na solution to each solution in ③. Prepare respectively 2.5mg/mL, 7.5mg/mL and 15mg/mL ZHT suspension and 20mg/mL 5-ASA suspension.
3％DSS溶液配置方法：称取3g DSS粉末充分溶解于100mL纯水中，所得即为3％DSS溶液。Preparation method of 3% DSS solution: Weigh 3g of DSS powder and fully dissolve it in 100mL of pure water. The result is 3% DSS solution.
2.3DSS诱导小鼠溃疡性结肠炎(Ulcer colitis,UC)模型的制备2. Preparation of 3DSS-induced ulcerative colitis (UC) model in mice
适应性饲养5天后，将小鼠随机分组，6组，每组两笼，每笼4只。分别为空白对照组(Control)，黄葵总黄酮提取物对照组(150mg/kg/day ZHT)，模型组(DSS)，模型+5-氨基水杨酸组(DSS+200mg/kg/day 5-ASA)，模型+ZHT组(DSS+75mg/kg/day ZHT)。After 5 days of adaptive rearing, the mice were randomly divided into 6 groups, with two cages in each group and 4 mice in each cage. They are the blank control group (Control), the ambrosia total flavonoid extract control group (150mg/kg/day ZHT), the model group (DSS), and the model + 5-aminosalicylic acid group (DSS + 200mg/kg/day 5 -ASA), model+ZHT group (DSS+75mg/kg/day ZHT).
如下方法所示，小鼠溃疡性结肠炎模型由连续7天自由饮用3％DSS诱发，每笼小鼠(4只)每2天更换一次3％DSS溶液(80mL)以确保DSS溶液有效；空白对照组小鼠自由饮用纯水，每笼小鼠(4只)每2天更换一次。As shown in the following method, the mouse ulcerative colitis model was induced by free drinking of 3% DSS for 7 consecutive days. Each mouse cage (4 mice) was replaced with 3% DSS solution (80mL) every 2 days to ensure that the DSS solution was effective; blank The mice in the control group drank pure water freely, and each cage (4 mice) was changed every 2 days.
每日上午9：00-10：00以0.1mL/10g b.w.剂量灌胃给予不同剂量ZHT或阳性治疗药物5-ASA，空白组和模型组给予同等剂量的空白载体溶液(含2％DMSO、2％吐温80以及0.5％CMC-Na)，连续7天。每天给药前，进行疾病活跃指数(Disease activity index,DAI)评分；第8天，对小鼠实施安乐死，采集血液及结肠组织。Different doses of ZHT or the positive therapeutic drug 5-ASA were administered intragastrically from 9:00 to 10:00 every day at a dose of 0.1mL/10g b.w., and the blank group and model group were given the same dose of blank carrier solution (containing 2% DMSO, 2 % Tween 80 and 0.5% CMC-Na) for 7 consecutive days. Before daily administration, the disease activity index (DAI) score was performed; on the 8th day, the mice were euthanized, and blood and colon tissue were collected.
2.4粪便粘稠度/粪便隐血测试及DAI评分2.4 Fecal viscosity/fecal occult blood test and DAI score
于每日给药前记录小鼠体重，采集粪便进行粪便隐血测试。从体重下降、粪便粘稠度和粪便潜血三个指标对疾病严重程度进行DAI评分，每个指标对应0-4分，评分标 准如表20-2。粪便隐血测试：取少量粪便涂布于玻片中间(玻片预先经火灼烧，以减少颜色误差)，滴加10/L硫酸甲氨基酚乙酸溶液3滴及3％过氧化氢溶液3滴，混匀，立即观察结果，并按照表20-3进行评分。The body weight of mice was recorded before daily administration, and feces were collected for fecal occult blood test. The DAI score is used to score the severity of the disease based on three indicators: weight loss, fecal viscosity and fecal occult blood. Each indicator corresponds to 0-4 points. The scoring scale Accurate as Table 20-2. Fecal occult blood test: Take a small amount of feces and apply it in the middle of the glass slide (the glass slide has been pre-fired to reduce color errors), add 3 drops of 10/L methaminophenol sulfate acetic acid solution and 3 drops of 3% hydrogen peroxide solution , mix well, observe the results immediately, and rate according to Table 20-3.
表20-2 DAI评分细则
Table 20-2 DAI scoring rules
表20-3粪便隐血测试评分表
Table 20-3 Fecal occult blood test score sheet
2.5标本采集2.5 Specimen collection
各组小鼠末次给药后，禁食24h，脱颈椎处死。剪开小鼠腹腔，暴露出小鼠的结肠，取小鼠的全部结肠组织测量并记录其长度，置于新配制的预冷生理盐水中进行肠内容物冲洗，截取1cm近直肠段结肠，于4％多聚甲醛中固定过夜，用于石蜡切片制备及随后的病理学检查，剩余组织取中段液氮速冻后，保存于-80℃，以供后续实验使用。血液样品于室温条件下静置2-3h，然后以3500rpm离心15min，取血清并迅速保存于-80℃，用于后续检测。After the last administration, mice in each group were fasted for 24 hours and sacrificed by cervical dislocation. Cut open the mouse's abdominal cavity to expose the mouse's colon. Take all the mouse's colon tissue, measure and record its length, place it in newly prepared pre-cooled physiological saline to flush the intestinal contents, and cut out 1cm of the colon near the rectum. The tissue was fixed overnight in 4% paraformaldehyde for preparation of paraffin sections and subsequent pathological examination. The remaining tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The blood samples were allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 15 min. The serum was collected and quickly stored at -80°C for subsequent testing.
2.6结肠组织中髓过氧化物酶(Myeloperoxidase，MPO)含量测定2.6 Determination of myeloperoxidase (MPO) content in colon tissue
MPO是中性粒细胞的功能标志和激活标志，在pH 6.0的条件下，以H2O2及3,3′-二甲氧基联苯胺盐酸盐为底物，MPO能够催化底物生成桔黄色产物，在460nm处通过比色法测定产物的生成量，从而计算出MPO含量。MPO is a functional marker and activation marker of neutrophils. Under pH 6.0 conditions, using H 2 O 2 and 3,3′-dimethoxybenzidine hydrochloride as substrates, MPO can catalyze the formation of substrates. For orange-yellow product, the amount of product produced was determined by colorimetry at 460 nm, thereby calculating the MPO content.
具体测定步骤如下：The specific measurement steps are as follows:
1)样本前处理：精确称取组织重量，按1:9的重量体积比加匀浆介质，于组织研磨机中以60Hz的频率持续研磨1min，重复5次，直至无肉眼可见沉淀，制备成10％的组织匀浆；1) Sample preprocessing: Accurately weigh the tissue weight, add homogenization medium at a weight-to-volume ratio of 1:9, grind continuously for 1 minute in a tissue grinder at a frequency of 60Hz, repeat 5 times, until no precipitation is visible to the naked eye, and prepare 10% tissue homogenate;
2)配制反应体系：待测样本均分别取0.09mL 10％组织匀浆或血清样本至2个新的2mL EP管(对照管及测定管)，并按表20-4加入试剂盒内相应试剂进行反应；2) Prepare reaction system: For each sample to be tested, take 0.09mL of 10% tissue homogenate or serum sample into two new 2mL EP tubes (control tube and measurement tube), and add the corresponding reagents in the kit according to Table 20-4 to react;
3)测定吸光度：取150μL反应产物于96孔板，在460nm处，酶标仪测定吸光度；3) Measure the absorbance: Take 150 μL of the reaction product in a 96-well plate, and measure the absorbance at 460 nm with a microplate reader;
4)按如下公式计算MPO含量：
4) Calculate the MPO content according to the following formula:
其中，W为样本取样量；对于组织匀浆，W(g)＝匀浆液浓度×取样体积(mL)。Among them, W is the sample sampling volume; for tissue homogenate, W (g) = homogenate concentration × sampling volume (mL).
表20-4 MPO含量测定操作表

Table 20-4 MPO content determination operation table

2.7 H&E染色2.7 H&E staining
结肠于4％多聚甲醛固定24h后，进行常规石蜡包埋。对包埋好的结肠组织进行连续横向切片(10μm)，然后进行H&E染色，H&E染色步骤如下：The colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in conventional paraffin. Take serial transverse sections (10 μm) of the embedded colon tissue, and then perform H&E staining. The H&E staining steps are as follows:
1)脱蜡：二甲苯I脱蜡10min，二甲苯II脱蜡5min；1) Dewaxing: Dewaxing with xylene I for 10 minutes, dewaxing with xylene II for 5 minutes;
2)乙醇浸泡：用无水乙醇浸泡2次，然后依次用95％、90％、85％的乙醇浸泡1次，每次浸泡1min，最后用水洗涤2min；2) Ethanol soaking: soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
3)染色：用苏木精进行染色，染色5min，然后用自来水洗涤1min；3) Staining: Stain with hematoxylin for 5 minutes, then wash with tap water for 1 minute;
4)分化：用1％盐酸酒精分化20s，然后用自来水洗涤1min；4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;
5)反蓝：用1％稀氨水反蓝30s，然后用自来水洗涤1min；5) Anti-blue: Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
6)伊红染色：染色5min，然后用自来水洗涤30s；6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;
7)乙醇脱水：依次用不同含量的乙醇进行脱水处理，顺序如下：85％乙醇脱水20s，90％乙醇脱水30s，95％乙醇I脱水1min，95％乙醇II脱水1min，无水乙醇I脱水2min，无水乙醇II脱水2min；7) Ethanol dehydration: Use different contents of ethanol for dehydration in sequence. The order is as follows: 85% ethanol dehydration for 20 seconds, 90% ethanol dehydration for 30 seconds, 95% ethanol I for 1 minute, 95% ethanol II for 1 minute, and absolute ethanol I for 2 minutes. , dehydrate with absolute ethanol II for 2 minutes;
8)透明：二甲苯I处理2min，二甲苯II处理2min，二甲苯III处理2min；8) Transparency: xylene I treatment for 2 minutes, xylene II treatment for 2 minutes, xylene III treatment for 2 minutes;
9)最后用中性树胶封片，用数字病理切片扫描仪(NanoZoomer 2.0RS)对切片扫描成像；9) Finally, seal the slides with neutral gum, and use a digital pathology slide scanner (NanoZoomer 2.0RS) to scan and image the slides;
10)病理学检查：根据结肠组织病变程度、炎症细胞浸润、隐窝细胞损伤等病理学指标对结肠损伤程度进行评分，具体评分标准如表20-5所示。10) Pathological examination: The degree of colon damage is scored based on pathological indicators such as the degree of colon tissue lesions, inflammatory cell infiltration, and crypt cell damage. The specific scoring standards are shown in Table 20-5.
表20-5炎症评分表

Table 20-5 Inflammation score sheet

2.8实时荧光定量PCR2.8 Real-time fluorescence quantitative PCR
将结肠组织置于研磨管中，加入研磨珠和400μL Trizol试剂，于组织研磨机中以60Hz的频率持续研磨1min，重复5次，直至无肉眼可见沉淀。收集上清，12,000rpm离心10min后转移上清至新的1.5mL无酶管。按氯仿-异丙醇萃取过夜后离心，弃上清，75％乙醇洗去附着在RNA上的离子，晾干后加入DEPC水溶解，吸取2μL用于Nano-100微量分光光度计定量，检测时A260/A280应处于在1.8-2.0之间。使用无酶水调整RNA浓度至1μg，加入5×gDNA wiper Mix 3μL后42℃加热2min，去除基因组DNA后，再加入4×qRT Super MixⅡ5μL，达成20μL体系，于PCR仪器进行25℃-2min、50℃-30min、85℃-5min的逆转录反应，cDNA于-80℃保存备用。Place the colon tissue into a grinding tube, add grinding beads and 400 μL Trizol reagent, and grind continuously in a tissue grinder at a frequency of 60 Hz for 1 min, repeating 5 times until no precipitation is visible to the naked eye. Collect the supernatant, centrifuge at 12,000 rpm for 10 min, and then transfer the supernatant to a new 1.5 mL enzyme-free tube. Extract with chloroform-isopropyl alcohol overnight and centrifuge. Discard the supernatant. Wash away the ions attached to the RNA with 75% ethanol. After drying, add DEPC water to dissolve. Pipette 2 μL for quantification with Nano-100 Micro Spectrophotometer. During detection A 260 /A 280 should be between 1.8-2.0. Use enzyme-free water to adjust the RNA concentration to 1 μg, add 3 μL of 5×gDNA wiper Mix and heat at 42°C for 2 min. After removing the genomic DNA, add 5 μL of 4×qRT Super MixⅡ to reach a 20 μL system, and perform a 20 μL system at 25°C-2min, 50 ℃-30min, 85℃-5min reverse transcription reaction, cDNA is stored at -80℃ for later use.
按照1μL cDNA+10μLGreen+0.8μL Primers+8.2μL ddH2O的体系进行实时荧光定量PCR反应，采用Comparative Ct(2-ΔΔCt)方法计算相对mRNA表达，各基因的表达量以GAPDH的表达量进行校正。所用引物见表20-6。According to 1μL cDNA+10μL The system of Green+0.8μL Primers+8.2μL ddH 2 O was used for real-time fluorescence quantitative PCR reaction, and the relative mRNA expression was calculated using the Comparative Ct (2 -ΔΔCt ) method. The expression level of each gene was corrected by the expression level of GAPDH. The primers used are shown in Table 20-6.
表20-6 PCR所用引物序列
Table 20-6 Primer sequences used in PCR
2.8 TNF-α，IL-1β和IL-6含量的测定2.8 Determination of TNF-α, IL-1β and IL-6 contents
依据ELISA试剂盒的操作流程，检测结肠组织中TNF-α，IL-1β和IL-6的含量。According to the operating procedure of the ELISA kit, the contents of TNF-α, IL-1β and IL-6 in colon tissue were detected.
2.9统计学分析2.9 Statistical analysis
运用GraphPad Prism(Ver 6.0)进行统计学分析，所有数据采用平均值±标准误(Mean±SEM)表示，体重变化和DAI评分变化采用Two-way ANOVA分析，其他数据采用One-way ANOVA分析，利用Dunnett’s多重检验分析组间差异的显著性，当P＜0.05时，组间差异具有统计学意义。GraphPad Prism (Ver 6.0) was used for statistical analysis. All data were expressed as mean ± SEM. Weight changes and DAI score changes were analyzed by Two-way ANOVA. Other data were analyzed by One-way ANOVA. Using Dunnett's multiple test analyzed the significance of differences between groups. When P<0.05, the differences between groups were statistically significant.
3.实验结果3.Experimental results
3.1不同剂量黄葵总黄酮对小鼠DAI评分的影响3.1 Effects of different doses of total flavonoids from ambrosia on the DAI score of mice
DAI的评分，为按表20-2的3项指标值进行加和后取平均值计算。如表20-7所示，DSS模型组小鼠DAI评分从第3天开始显著升高，与Control组相比，差异具有统计学意义(P<0.01)，实验第6天腹泻3只，第7天腹泻7只；DSS+ZHT(75mg/kg/day)组DAI评分增长趋势较为平缓，第4，5，6，7天，其DAI评分与DSS模型组相比，差异具有统计学意义(P<0.01)，实验过程中无小鼠出现腹泻；阳性治疗药物5-ASA(200mg/kg/day)缓解DSS诱导的结肠炎症状，降低DAI评分，从第4天开始，与DSS模型组相比，差异显著(P<0.01)，实验过程中无小鼠出现腹泻。实验过程中，ZHT150mg/kg/day正常组与control组无显著差异，对小鼠体重无影响，第6天1只小鼠出现腹泻症状。The DAI score is calculated by adding the three indicator values in Table 20-2 and taking the average. As shown in Table 20-7, the DAI score of the mice in the DSS model group increased significantly from the 3rd day. Compared with the Control group, the difference was statistically significant (P<0.01). On the 6th day of the experiment, 3 mice had diarrhea. There were 7 diarrhea cases in 7 days; the DSS+ZHT (75mg/kg/day) group’s DAI score increased more slowly. On days 4, 5, 6, and 7, the difference in DAI score was statistically significant compared with the DSS model group ( P<0.01), no mice developed diarrhea during the experiment; the positive therapeutic drug 5-ASA (200mg/kg/day) alleviated the symptoms of DSS-induced colitis and reduced the DAI score. Starting from the 4th day, it was comparable to the DSS model group. Ratio, the difference was significant (P<0.01), and no mice had diarrhea during the experiment. During the experiment, there was no significant difference between the ZHT150mg/kg/day normal group and the control group, and it had no effect on the weight of the mice. On the 6th day, 1 mouse developed diarrhea symptoms.
表20-7各组小鼠DAI评分
Table 20-7 DAI scores of mice in each group
注:数据以平均值±标准误表示。N＝8.*,P＜0.05,**,P＜0.01,vs.Control；#,P＜0.05,##,P＜0.01,vs.DSS，使用双因素方差及Dunnett’s多重比较检验进行分析。Note: Data are expressed as mean ± standard error. N＝8.*,P＜0.05,**,P＜0.01,vs.Control; # ,P＜0.05, ## ,P＜0.01,vs.DSS, analyzed using two-factor variance and Dunnett's multiple comparison test.
3.2不同剂量黄葵总黄酮对小鼠结肠长度的影响3.2 Effects of different doses of total flavonoids from ambrosia on the colon length of mice
如图2和表20-8所示，Control组结肠长度为7.34±0.08cm，DSS模型组小鼠结肠萎缩至4.85±0.18cm，与Control组相比，差异显著(P<0.01)；ZHT(150mg/kg/day)组结肠长度(7.43±0.13cm)与Control组接近。75mg/kg/day ZHT显著改善了由DSS诱导的结肠萎缩，其中DSS+ZHT(75mg/kg/day)组与DSS模型组相比，差异具有统计学意义(P<0.05)；阳性药物5-ASA(200mg/kg/day)也改善了由DSS诱导的结肠萎缩，与Control组相比，差异具有统计学意义(P<0.05)。As shown in Figure 2 and Table 20-8, the colon length of the Control group was 7.34±0.08cm, and the colon of mice in the DSS model group shrunk to 4.85±0.18cm. Compared with the Control group, the difference was significant (P<0.01); ZHT ( The colon length (7.43±0.13cm) of the 150mg/kg/day) group was close to that of the Control group. 75mg/kg/day ZHT significantly improved colon atrophy induced by DSS, and the difference between the DSS+ZHT (75mg/kg/day) group and the DSS model group was statistically significant (P<0.05); positive drugs 5- ASA (200mg/kg/day) also improved colon atrophy induced by DSS, and the difference was statistically significant (P<0.05) compared with the Control group.
表20-8各组小鼠结肠长度统计表
Table 20-8 Statistical table of colon length of mice in each group
注:数据以平均值±标准误差表示。N＝8.**,P＜0.01,vs.Control；#,P＜0.05,vs.DSS,使用单因素方差及Dunnett’s多重比较检验进行分析。Note: Data are expressed as mean ± standard error. N＝8.**, P＜0.01, vs.Control; #, P＜0.05, vs.DSS, analyzed using one-way variance and Dunnett’s multiple comparison test.
3.3不同剂量黄葵总黄酮对小鼠结肠组织中髓过氧化物酶(MPO)含量的影响3.3 Effects of different doses of ambrosia total flavonoids on myeloperoxidase (MPO) content in mouse colon tissue
Control组结肠组织中MPO含量为0.30±0.04U/g，DSS模型组小鼠结肠组织中MPO含量上升至1.04±0.10U/g，与Control组相比，差异具有统计学意义(P<0.01)；75mg/kg/day ZHT对DSS诱导的结肠中MPO含量上升有抑制作用，75mg/kg/day ZHT的抑制率为92.8±19.4％，差异具有统计学意义(P<0.01)；阳性治疗药物5-ASA(200mg/kg/day)显著抑制了DSS诱导的结肠中MPO含量上升，抑制率为88.7±9.9％(P<0.01)。The MPO content in the colon tissue of the Control group was 0.30±0.04U/g, and the MPO content in the colon tissue of the mice in the DSS model group increased to 1.04±0.10U/g. Compared with the Control group, the difference was statistically significant (P<0.01) ; 75mg/kg/day ZHT has an inhibitory effect on the increase in MPO content in the colon induced by DSS. The inhibition rate of 75mg/kg/day ZHT is 92.8±19.4%, and the difference is statistically significant (P<0.01); Positive therapeutic drugs 5 -ASA (200mg/kg/day) significantly inhibited the DSS-induced increase in MPO content in the colon, with an inhibition rate of 88.7±9.9% (P<0.01).
3.4不同剂量黄葵总黄酮对小鼠结肠组织病理改变的影响3.4 Effects of different doses of ambrosia total flavonoids on pathological changes in colon tissue of mice
实验结果显示(图3)，Control组和ZHT(150mg/kg/day)组小鼠结肠组织结构正常，鲜有炎性细胞浸润，隐窝细胞及表皮细胞完好。DSS模型组小鼠结肠组织中炎性细胞明显增多，隐窝细胞与表皮细胞全部缺失，病变范围达80％以上，与Control组相比，病理评分具有显著性差异(P<0.01)；DSS+ZHT(75mg/kg/day)组结肠组织显著改善，与DSS模型组相比，DSS+ZHT(75mg/kg/day)组的病理评分差异极显著(P<0.01)。阳性治疗药物5-ASA(200mg/kg/day)显著改善DSS诱导的结肠组织病理学变化，与DSS模型组相比，病理评分具有显著性差异(P<0.01)。The experimental results showed (Figure 3) that the colon tissue structure of mice in the Control group and ZHT (150 mg/kg/day) group was normal, with little inflammatory cell infiltration, and crypt cells and epidermal cells intact. There was a significant increase in inflammatory cells in the colon tissue of mice in the DSS model group, all crypt cells and epidermal cells were lost, and the lesion range reached more than 80%. Compared with the Control group, the pathological scores were significantly different (P<0.01); DSS+ The colon tissue of the ZHT (75mg/kg/day) group was significantly improved. Compared with the DSS model group, the pathological score of the DSS+ZHT (75mg/kg/day) group was significantly different (P<0.01). The positive therapeutic drug 5-ASA (200 mg/kg/day) significantly improved the histopathological changes of the colon induced by DSS. Compared with the DSS model group, the pathological scores were significantly different (P<0.01).
3.5黄葵总黄酮对DSS诱导的IL-1β，IL-2，IL-4，IL-6，TNF-α和IFN-γmRNA表达水平的影响3.5 Effect of total flavonoids of ambrosia on DSS-induced IL-1β, IL-2, IL-4, IL-6, TNF-α and IFN-γ mRNA expression levels
实验结果显示，DSS模型组小鼠结肠组织中IL-1β的mRNA表达升高至Control组 的7.36±0.79倍(P<0.01)；单独给予150mg/kg/day ZHT使小鼠结肠组织中IL-1β的mRNA表达升高至Control组的2.21±0.24倍，但与Control组相比差异不显著(P>0.05)。75mg/kg/day ZHT可抑制DSS诱导的IL-1βmRNA表达上调，抑制率为81.9±5.0％(P<0.01)；阳性治疗药物5-ASA(200mg/kg/day)抑制DSS诱导的IL-1βmRNA表达，抑制率为91.6±3.6％(P<0.01)。Experimental results showed that the mRNA expression of IL-1β in the colon tissue of mice in the DSS model group increased to that in the Control group. 7.36±0.79 times (P<0.01); giving 150mg/kg/day ZHT alone increased the mRNA expression of IL-1β in the colon tissue of mice to 2.21±0.24 times that of the Control group, but there was no difference compared with the Control group. Significant (P>0.05). 75mg/kg/day ZHT can inhibit the up-regulation of IL-1βmRNA expression induced by DSS, with an inhibition rate of 81.9±5.0% (P<0.01); the positive therapeutic drug 5-ASA (200mg/kg/day) inhibits the DSS-induced IL-1βmRNA expression. Expression, the inhibition rate was 91.6±3.6% (P<0.01).
实验结果显示，DSS模型组小鼠结肠组织中IL-2的mRNA表达显著升高至Control组的6.65±0.56倍(P<0.01)；单独给予ZHT(150mg/kg/day)使小鼠结肠组织中IL-2的mRNA表达升高至Control组的1.74±0.15倍，但差异无统计学意义(P>0.05)。75mg/kg/day ZHT抑制了78.7±4.5％由DSS诱导的IL-2mRNA表达上升，与DSS模型组相比，差异极显著(P<0.01)；阳性治疗药物5-ASA(200mg/kg/day)显著抑制由DSS诱导的结肠组织中IL-2的mRNA表达上升，抑制率为94.2±2.8％(P<0.01)。Experimental results showed that the mRNA expression of IL-2 in the colon tissue of mice in the DSS model group was significantly increased to 6.65±0.56 times that of the Control group (P<0.01); ZHT (150 mg/kg/day) alone increased the expression of IL-2 in the colon tissue of mice. The mRNA expression of IL-2 in the control group increased to 1.74±0.15 times that of the Control group, but the difference was not statistically significant (P>0.05). 75mg/kg/day ZHT inhibited the increase in IL-2 mRNA expression induced by DSS by 78.7±4.5%. Compared with the DSS model group, the difference was extremely significant (P<0.01); the positive therapeutic drug 5-ASA (200mg/kg/day ) significantly inhibited the increase in IL-2 mRNA expression in colon tissue induced by DSS, with an inhibition rate of 94.2±2.8% (P<0.01).
实验结果显示，DSS模型组小鼠结肠组织中IL-4的mRNA表达显著升高至Control组的5.78±1.22倍(P<0.01)；单独给予150mg/kg/day ZHT组小鼠结肠组织中IL-4的mRNA表达为Control组的1.07±0.16倍，与Control组相比，差异不显著(P>0.05)。75mg/kg/day ZHT抑制DSS诱导的IL-4mRNA表达上调，抑制率为78.65±7.18％，与DSS模型组相比，差异显著(P<0.01)；阳性治疗药物5-ASA(200mg/kg/day)显著抑制DSS诱导的IL-4转录上调，抑制率为69.15±6.17％(P<0.01)。Experimental results showed that the mRNA expression of IL-4 in the colon tissue of mice in the DSS model group was significantly increased to 5.78±1.22 times that of the Control group (P<0.01); IL-4 in the colon tissue of mice in the ZHT group given 150 mg/kg/day alone The mRNA expression of -4 was 1.07±0.16 times that of the Control group. Compared with the Control group, the difference was not significant (P>0.05). 75mg/kg/day ZHT inhibited the up-regulation of IL-4mRNA expression induced by DSS, with an inhibition rate of 78.65±7.18%. Compared with the DSS model group, the difference was significant (P<0.01); the positive therapeutic drug 5-ASA (200mg/kg/ day) significantly inhibited the up-regulation of IL-4 transcription induced by DSS, with an inhibition rate of 69.15±6.17% (P<0.01).
实验结果显示，DSS模型组小鼠结肠组织中IL-6的mRNA表达显著升高至Control组的13.53±2.34倍(P<0.01)；ZHT(150mg/kg/day)组与Control组相比，小鼠结肠组织中IL-6的mRNA表达无显著变化。75mg/kg/day ZHT抑制了79.4±6.5％由DSS诱导的IL-6转录上调，与DSS模型组相比，差异极显著(P<0.01)；；阳性治疗药物5-ASA(200mg/kg/day)抑制DSS诱导的IL-6转录上调，抑制率为91.5±6.2％(P<0.01)。Experimental results showed that the mRNA expression of IL-6 in the colon tissue of mice in the DSS model group was significantly increased to 13.53±2.34 times that of the Control group (P<0.01); compared with the Control group, the ZHT (150 mg/kg/day) group There was no significant change in IL-6 mRNA expression in mouse colon tissue. 75mg/kg/day ZHT inhibited 79.4±6.5% of the up-regulation of IL-6 transcription induced by DSS. Compared with the DSS model group, the difference was extremely significant (P<0.01); the positive therapeutic drug 5-ASA (200mg/kg/ day) inhibited the up-regulation of IL-6 transcription induced by DSS, and the inhibition rate was 91.5±6.2% (P<0.01).
实验结果显示，DSS模型组小鼠结肠组织中TNF-α的mRNA表达显著升高至Control组的7.82±1.68倍(P<0.01)；150mg/kg/day ZHT使小鼠结肠组织中TNF-α的mRNA表达升高至Control组的3.29±1.09倍，但差异无统计学意义(P>0.05)。75mg/kg/day ZHT抑制了88.5±7.1％由DSS诱导的TNF-α转录上调，，DSS+ZHT(75mg/kg/day)组与DSS模型组相比，差异显著(P<0.01)。阳性治疗药物5-ASA(200mg/kg/day)组抑制DSS诱导的TNF-α转录上调，抑制率为87.4±7.2％(P<0.01)。Experimental results showed that the mRNA expression of TNF-α in the colon tissue of mice in the DSS model group was significantly increased to 7.82±1.68 times that of the Control group (P<0.01); 150 mg/kg/day ZHT increased the level of TNF-α in the colon tissue of mice. The mRNA expression of the control group increased to 3.29±1.09 times, but the difference was not statistically significant (P>0.05). 75mg/kg/day ZHT inhibited the up-regulation of TNF-α transcription induced by DSS by 88.5±7.1%. Compared with the DSS model group, the difference between the DSS+ZHT (75mg/kg/day) group and the DSS model group was significant (P<0.01). The positive therapeutic drug 5-ASA (200 mg/kg/day) group inhibited the up-regulation of TNF-α transcription induced by DSS, and the inhibition rate was 87.4±7.2% (P<0.01).
实验结果显示，DSS模型组小鼠结肠组织中IFN-γ的mRNA表达显著升高至Control组的5.60±0.50倍(P<0.01)；ZHT(150mg/kg/day)组与Control组相比，小鼠结肠组织中的IFN-γmRNA表达无显著改变。75mg/kg/day ZHT抑制了74.2±6.0％由DSS诱导的IFN-γ转录上调，DSS+ZHT(75mg/kg/day)组与DSS模型组相比，差异极显著(P<0.01)。阳性治疗药物5-ASA(200mg/kg/day)抑制DSS诱导的IFN-γ转录上调，抑制率为83.0±4.2％(P<0.01)。Experimental results showed that the mRNA expression of IFN-γ in the colon tissue of mice in the DSS model group was significantly increased to 5.60±0.50 times that of the Control group (P<0.01); compared with the Control group, the ZHT (150 mg/kg/day) group There was no significant change in IFN-γmRNA expression in mouse colon tissue. 75mg/kg/day ZHT inhibited the up-regulation of IFN-γ transcription induced by DSS by 74.2±6.0%. Compared with the DSS model group, the difference between the DSS+ZHT (75mg/kg/day) group and the DSS model group was extremely significant (P<0.01). The positive therapeutic drug 5-ASA (200mg/kg/day) inhibited the DSS-induced upregulation of IFN-γ transcription, with an inhibition rate of 83.0±4.2% (P<0.01).
4.实验结论和讨论4. Experimental conclusion and discussion
本实施例采用DSS诱导的小鼠溃疡性结肠炎模型，以5-氨基水杨酸为阳性对照药物，考察75mg/kg/day黄葵总黄酮治疗结肠炎的治疗效果，结果表明，ZHT(75mg/kg/day)组明显保护DSS诱导的小鼠体重降低、腹泻、便血等结肠炎症状；显著改善DSS诱导的结肠萎缩、隐窝结构损伤及组织炎性浸润等组织病理学变化；降低DSS诱导的组织MPO水平上升及炎症因子表达，表现出对结肠炎较好的治疗效果，与阳性治疗药物5-氨基水杨酸的效果相当。This example uses a mouse model of ulcerative colitis induced by DSS, and uses 5-aminosalicylic acid as a positive control drug to examine the therapeutic effect of 75 mg/kg/day ambrosia total flavonoids in the treatment of colitis. The results show that ZHT (75 mg /kg/day) group significantly protected mice from DSS-induced weight loss, diarrhea, hematochezia and other colitis symptoms; significantly improved DSS-induced colon atrophy, crypt structure damage, tissue inflammatory infiltration and other histopathological changes; reduced DSS-induced The tissue MPO levels increased and the expression of inflammatory factors showed a good therapeutic effect on colitis, which was equivalent to the effect of the positive therapeutic drug 5-aminosalicylic acid.
综上所述，ZHT对结肠炎具有一定的疗效，在75mg/kg/day的效果与阳性药5-ASA相当，安全性较好。 In summary, ZHT has certain curative effects on colitis. The effect at 75mg/kg/day is equivalent to that of the positive drug 5-ASA, and its safety is good.
经验证实施例1，实施例2-A3，实施例3.2-2，实施例5-2，实施例19-2样品也具备与本实施例中的实施例19-1上述结肠炎基本一致的作用或效果。It has been verified that the samples of Example 1, Example 2-A3, Example 3.2-2, Example 5-2, and Example 19-2 also have the same effect on colitis as described in Example 19-1 in this example. or effect.
实施例21口腔溃疡药效实验Example 21 Oral ulcer drug efficacy experiment
1实验材料1Experimental materials
1.1动物：SPF级雄性SD大鼠，250-300g，购于上海杰思捷实验动物有限公司。1.1 Animals: SPF grade male SD rats, 250-300g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.
1.2药品：桂林西瓜霜，桂林三金药业股份有限公司；黄蜀葵花乙醇提物(干粉，黄蜀葵花95％乙醇回流提取3次浓缩干燥制备得到)；黄蜀葵花水提物(稠膏，黄蜀葵花水回流提取三次浓缩干燥制备得到)，黄蜀葵花总黄酮提取物(实施例19制备得到，批号19-2)。1.2 Drugs: Guilin Watermelon Frost, Guilin Sanjin Pharmaceutical Co., Ltd.; Ethanol extract of marshmallow flowers (dry powder, prepared by refluxing and extracting 95% ethanol 3 times of marshmallow flowers and concentrating and drying them); Water extract of marshmallow flowers (thick paste, marshmallow flowers) Prepared by water reflux extraction three times, concentration and drying), total flavonoid extract of marshmallow flowers (prepared in Example 19, batch number 19-2).
1.3试剂：苯酚(分析纯)，南京化学试剂有限公司；水合氯醛(分析纯)，国药集团化学试剂有限公司。1.3 Reagents: phenol (analytical grade), Nanjing Chemical Reagent Co., Ltd.; chloral hydrate (analytical grade), Sinopharm Chemical Reagent Co., Ltd.
2实验方法2Experimental methods
2.1造模：将SPF雄性SD大鼠随机分组，每组9-11只。分别为空白组(正常组)，模型组，黄蜀葵花醇提物组，黄蜀葵花水提物组，黄蜀葵花总黄酮提取物组，西瓜霜组。全部大鼠腹腔注射水合氯醛(400mg/kg)麻醉，取一根下端内径为4mm的玻璃管，将小棉球放置于其下端。对于模型组和各给药组大鼠，向玻璃管内滴加90％苯酚溶液至刚浸透小棉球，下端分别平置于大鼠左、右两侧口角约1mm处颊膜上黏膜接触30s，可见该区域有直径约4-5mm的白色损害。空白组则以双蒸水代替苯酚作相同处理。2.1 Modeling: SPF male SD rats were randomly divided into groups, with 9-11 rats in each group. They are blank group (normal group), model group, marshmallow flower ethanol extract group, marshmallow flower water extract group, marshmallow flower total flavonoid extract group, and watermelon frost group. All rats were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg). A glass tube with an inner diameter of 4 mm at the lower end was taken and a small cotton ball was placed at the lower end. For the rats in the model group and each administration group, drop 90% phenol solution into the glass tube until the cotton ball is just soaked, and place the lower end flatly on the buccal membrane about 1 mm from the left and right corners of the mouth of the rat for 30 seconds. It can be seen that there is a white lesion about 4-5mm in diameter in this area. In the blank group, double distilled water was used instead of phenol for the same treatment.
2.2给药：将各药以双蒸水配制成混悬液，于造模后均匀涂沫在口腔黏膜创面，每天4次。各药剂量如下：西瓜霜，20mg/创面/天；黄蜀葵花醇提物10mg/创面/天；黄蜀葵花水提物8.75mg/创面/天，黄蜀葵花总黄酮提取物1.05mg/创面/天。连续给药5天，给药前后观察溃疡面积以及溃疡愈合情况。第6天以水合氯醛(400mg/kg)麻醉后进行溃疡面拍照，在大鼠脱颈椎处死后，取下大鼠双侧侧颊膜组织，每组随机挑选7-9个样本浸泡于4％甲醛(生理盐水配制)固定，常规取材，脱水，石蜡包埋。切片经HE染色，光学显微镜观察。剩余颊膜组织冻于-70℃冰箱备用，待测其他炎症指标。2.2 Administration: Prepare each drug into a suspension with double-distilled water, and apply it evenly on the oral mucosal wound after modeling, 4 times a day. The dosage of each drug is as follows: watermelon cream, 20 mg/wound/day; marshmallow flower ethanol extract 10 mg/wound/day; marshmallow flower aqueous extract 8.75 mg/wound/day, marshmallow flower total flavonoid extract 1.05 mg/wound/day. Administration was continued for 5 days, and the ulcer area and ulcer healing were observed before and after administration. On the 6th day, the ulcer surface was photographed after being anesthetized with chloral hydrate (400 mg/kg). After the rats were sacrificed by cervical dislocation, the bilateral buccal membrane tissues of the rats were removed. 7-9 samples from each group were randomly selected and soaked in 4 % formaldehyde (prepared with physiological saline), fixed, routinely collected, dehydrated, and embedded in paraffin. Sections were stained with HE and observed under a light microscope. The remaining buccal membrane tissue was frozen in a -70°C refrigerator for later use, to be tested for other inflammatory indicators.
2.3数据统计：数据以平均值±SD表示。模型组与正常组(空白对照组)之间的显著性差异以Student’s-t test进行检验；模型组与其余给药组之间以One-way ANOVA test进行显著性差异分析，并进一步以Newman-Keuls multiple comparison test进行两组间显著性差异检验。2.3 Data statistics: Data are expressed as mean ± SD. The significant difference between the model group and the normal group (blank control group) was tested with Student's-t test; the significant difference between the model group and the other drug groups was analyzed with One-way ANOVA test, and further with Newman- Keuls multiple comparison test is used to test the significant difference between two groups.
3结果3 results
3.1肉眼观察及评判3.1 Visual observation and evaluation
空白组大鼠口腔黏膜表面光滑，色泽均匀，无血点血斑。苯酚刺激后，局部黏膜快速发白，2h后可见水肿，1-5天内可见炎性渗出，出血、局部形成溃疡创面，表面出现黄白色假膜覆盖并脱落。第6天，除空白组外，各组大鼠均可见创面凹陷；50％模型组大鼠创面血管扩张较明显，有1只大鼠可见黄色脓点，结果见表21-1(每个老鼠口腔有两个创面)。The oral mucosa surface of the rats in the blank group was smooth, uniform in color, and free of blood spots. After phenol stimulation, the local mucosa quickly turns white, and edema can be seen after 2 hours. Inflammatory exudation, bleeding, and local ulcer formation can be seen within 1 to 5 days, and a yellow-white pseudomembrane appears on the surface and falls off. On the 6th day, except for the blank group, all rats in each group could see sunken wounds; 50% of the rats in the model group had obvious dilation of blood vessels in the wounds, and 1 rat had yellow pus spots. The results are shown in Table 21-1 (each rat There are two wounds in the mouth).
表21-1大鼠苯酚口腔颊膜溃疡程度统计

Table 21-1 Statistics on the degree of oral and buccal ulcers in rats with phenol

0：无溃疡，口腔黏膜正常；0: No ulcer, normal oral mucosa;
Ⅰ：有溃疡凹，无明显出血或血管扩张、无明显肿胀、无明显假膜；Ⅰ: There is ulcer pit, no obvious bleeding or dilation of blood vessels, no obvious swelling, and no obvious pseudomembrane;
Ⅱ：有溃疡凹，有血点但较少；Ⅱ: There are ulcer pits and blood spots but less;
Ⅲ：有溃疡凹，有血斑且较明显，有轻微肿胀。III: There are ulcer pits, obvious blood spots, and slight swelling.
3.2病理改变3.2 Pathological changes
光学显微镜检查下列项目：(1)黏膜上皮细胞有无变性、坏死，糜烂、溃疡，有无炎细胞浸润；(2)黏膜下组织有无充血水肿、炎细胞浸润。The following items were examined under an optical microscope: (1) Whether the mucosal epithelial cells are degenerated, necrotic, erosion, ulcer, and whether there is inflammatory cell infiltration; (2) whether the submucosal tissue is congestion, edema, and inflammatory cell infiltration.
病变程度评分标准：按病变由轻到重的程度分别记为0.5-4分，积分标准为：轻微或极轻度病变记为0.5分；轻度病变记1分；中度病变记2分；重度病变记3分；极重度病变记为4分；无病变记为0分。溃疡长度用“显微镜用侧微尺”在40倍光镜下(目镜4×，物镜10×)测量溃疡的长度＜5mm/40X记为1分，＞5mm/40X或5mm/40X记为2分。类加所有分数，计算出每组动物病变均分分值越高，提示病变程度越重。Scoring standard for the degree of lesions: 0.5-4 points according to the severity of the lesions from mild to severe. The scoring standards are: 0.5 points for mild or very mild lesions; 1 point for mild lesions; 2 points for moderate lesions; Severe lesions were scored as 3 points; very severe lesions were scored as 4 points; no lesions were scored as 0 points. The ulcer length is measured with a "microscope side micro-ruler" under a 40x light microscope (eyepiece 4×, objective lens 10×). The length of the ulcer is scored as 1 point if it is <5mm/40X, and 2 points if it is >5mm/40X or 5mm/40X. . Add all scores by category to calculate the average lesion score of each group of animals. The higher the score, the more severe the disease.
各组病理改变统计结果见表21-2，病理图片见图4，图4b中，黑短箭头示皮肤坏死脱落形成溃疡(无表皮覆盖)，黑长箭示真皮浸润的炎细胞，绿箭示血管扩张充血，绿箭示成纤维细胞，绿星示皮肤表被的薄层复层鱗状上皮，表面轻度角化。The statistical results of pathological changes in each group are shown in Table 21-2, and the pathological pictures are shown in Figure 4. In Figure 4b, the short black arrow indicates skin necrosis and shedding to form ulcers (without epidermal coverage), the long black arrow indicates inflammatory cells infiltrating into the dermis, and the green arrow indicates The blood vessels are dilated and congested, the green arrow indicates fibroblasts, and the green star indicates the thin layer of stratified squamous epithelium on the skin surface, with mild surface keratinization.
表21-2大鼠口腔黏膜病理改变评分结果
Table 21-2 Score results of pathological changes in rat oral mucosa
注:##P＜0.01vs空白组；*P＜0.05,**P＜0.01vs模型组。Note: ## P＜0.01vs blank group; *P＜0.05, **P＜0.01vs model group.
实施例22黄葵总黄酮治疗皮肤病的药效学评价Example 22 Pharmacodynamic evaluation of total flavonoids from ambrosia in treating skin diseases
实施例22-1黄葵总黄酮治疗痤疮的药效学评价Example 22-1 Pharmacodynamic evaluation of total flavonoids from ambrosia in the treatment of acne
1.实验目的：1. Experimental purpose:
考察涂抹给药黄葵总黄酮(ZHT)对100％油酸联合痤疮丙酸杆菌(Propionibacterium acnes,P.acnes)诱导的痤疮的治疗效果，并与阳性药过氧苯甲酰/克林霉素的疗效进行比较。To investigate the therapeutic effect of smear-administered total flavonoids (ZHT) on acne induced by 100% oleic acid combined with Propionibacterium acnes (P. acnes), and compare it with the positive drug benzoyl peroxide/clindamycin Compare the efficacy.
2.实验材料 2. Experimental materials
2.1.实验动物2.1. Experimental animals
SPF级SD大鼠，雌雄各半，分笼饲养，6-8周龄，体重140-150g，由南京医科大学医药实验动物中心提供(实验动物生产许可证号：SCXK(苏)2021-0011)。SPF grade SD rats, half male and half female, raised in separate cages, 6-8 weeks old, weighing 140-150g, provided by the Medical Experimental Animal Center of Nanjing Medical University (Experimental Animal Production License Number: SCXK (Su) 2021-0011) .
试剂及药品Reagents and medicines
黄葵总黄酮(ZHT-1)，按照实施例19方法制备，也称为“黄葵总黄酮提取物”，批号19-1。黄葵总黄酮(ZHT-2)，按照实施例6方法制备。Total flavonoids of Z. annua (ZHT-1) was prepared according to the method of Example 19. It is also called "Z. annuus total flavonoids extract", batch number 19-1. Total flavonoids of ambrette (ZHT-2) was prepared according to the method of Example 6.
2.3.实验用药的配制2.3. Preparation of experimental drugs
ZHT乳膏配制方法：1％的黄葵总黄酮乳膏按照如表22-1处方配置。Preparation method of ZHT cream: 1% total flavonoid cream of ambrette is prepared according to the prescription in Table 22-1.
表22-1：ZHT乳膏配置处方。
Table 22-1: ZHT Cream Prescription.
水相：称取处方量乙醇，加入ZHT，搅拌溶解，依次向烧杯内加入吐温80、十二烷基硫酸钠、甘油、肉豆蔻酸异丙酯、尼泊金甲酯、氮酮、纯化水，搅拌至分散均匀。Aqueous phase: Weigh the prescribed amount of ethanol, add ZHT, stir to dissolve, add Tween 80, sodium lauryl sulfate, glycerin, isopropyl myristate, methylparaben, azone, and purify into the beaker. water and stir until evenly dispersed.
油相：称取处方量单硬脂酸甘油酯、棕榈酸、白凡士林于烧杯中，于80℃水浴下加热溶解至液态。Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petroleum jelly into a beaker, heat and dissolve in a water bath at 80°C until it becomes liquid.
将水相缓慢加入油相中，边加边进行搅拌，水相全部加入后，从水浴锅内取出，于室温下搅拌直至冷却成乳膏。Slowly add the water phase into the oil phase, stirring while adding. After all the water phase has been added, remove from the water bath and stir at room temperature until it cools into a cream.
强化梭菌培养基(RCM)的配制方法：称取强化梭菌培养基粉末19.0g于500mL锥形瓶中，加500mL蒸馏水加热搅拌，分装，121℃高压灭菌15分钟，以备使用。Preparation method of enhanced Clostridial medium (RCM): Weigh 19.0g of enhanced Clostridial medium powder into a 500mL Erlenmeyer flask, add 500mL distilled water, heat and stir, aliquot, and autoclave at 121°C for 15 minutes before use.
P.acnes的培养方法：无菌试管中加入4mL的RCM培养基和1mL的P.acnes菌液并吹打混匀，包上无菌容器封口膜后竖直放入含有厌氧产气袋的厌氧培养袋中，置于37℃电热恒温培养箱中培养，每2天传代1次。Cultivation method of P.acnes: Add 4mL of RCM culture medium and 1mL of P.acnes bacterial liquid into a sterile test tube and mix evenly by pipetting. Wrap the sterile container with a sealing film and put it vertically into an anaerobic container containing an anaerobic gas-generating bag. placed in an oxygen culture bag, placed in an electric constant-temperature incubator at 37°C, and cultured once every 2 days.
P.acnes注射液的配制方法：现用现配。生理盐水将菌液浓度稀释至6×107CFU/mL，即得P.acnes注射液(菌液的麦氏浊度约为2McFarland，用生理盐水稀释10倍后，即得6×107CFU/mL浓度的细菌混悬液，测其OD600nm在0.63-0.65之间。)Preparation method of P.acnes injection: ready for use. Use physiological saline to dilute the concentration of the bacterial solution to 6×10 7 CFU/mL to obtain P.acnes injection (the McFarland turbidity of the bacterial liquid is approximately 2 McFarland. After diluting 10 times with normal saline, 6×10 7 CFU is obtained. /mL concentration of bacterial suspension, measured OD 600nm between 0.63-0.65.)
2.3大鼠痤疮模型的构建及治疗2.3 Construction and treatment of rat acne model
大鼠痤疮模型建立和药物疗效评价方法如图5-1所示，利用100％油酸联合P.acnes建立痤疮模型，在第8天开始涂抹治疗药物ZHT乳膏治疗，并以过2.5％氧苯甲酰/1％克林霉素(BPO)水凝胶为阳性对照。The rat acne model establishment and drug efficacy evaluation method are shown in Figure 5-1. The acne model was established using 100% oleic acid combined with P.acnes. On the 8th day, treatment with ZHT cream, a therapeutic drug, was started, and 2.5% oxygen was used. Benzoyl/1% clindamycin (BPO) hydrogel was used as a positive control.
将大鼠，雌雄各半，每组6只，分笼饲养，适应性饲养一周后，对空白组以外的大鼠用100％油酸联合P.acnes诱导痤疮模型。以大鼠右耳廓为载体构建痤疮模型，左 耳不做处理，每日上午九点在大鼠右耳内侧涂抹100％油酸1次，每次0.5mL，空白组涂抹等量生理盐水；每隔一日涂抹油酸1h后于大鼠右耳内侧注射制备好的P.acnes注射液，每次50μL；空白组注射生理盐水。Rats, half male and half female, 6 rats per group, were raised in separate cages. After one week of adaptive rearing, rats other than the blank group were treated with 100% oleic acid combined with P. acnes to induce an acne model. The acne model was constructed using the right auricle of the rat as a carrier, and the left The ears were not treated. 100% oleic acid was applied to the inner side of the right ear of the rats once at 9 am every day, 0.5 mL each time. The blank group was applied with the same amount of normal saline; oleic acid was applied to the inner side of the right ear of the rats for 1 hour every other day. The prepared P.acnes injection was injected into the inner side of the ear, 50 μL each time; the blank group was injected with physiological saline.
在造模第8天，对大鼠进行重新分组，以保证每组大鼠的模型严重程度基本相同，分为空白对照组(Veh)，模型组(Model)，模型+过氧苯甲酰/克林霉素组(Model+BPO)，模型+1％ZHT-1组(Model+1％ZHT-1)，模型+1％ZHT-2组(Model+1％ZHT-2)，每组雌鼠和雄鼠各一笼，每笼各三只。分组后于第8天下午开始，每天下午在耳部涂抹50mg待测药物。On the 8th day of modeling, the rats were regrouped to ensure that the severity of the model in each group of rats was basically the same. They were divided into blank control group (Veh), model group (Model), model + benzoyl peroxide/ Clindamycin group (Model+BPO), model+1%ZHT-1 group (Model+1%ZHT-1), model+1%ZHT-2 group (Model+1%ZHT-2), female in each group There are one cage each for rats and male rats, with three rats in each cage. Starting from the afternoon of the 8th day after grouping, 50 mg of the drug to be tested was applied to the ears every afternoon.
2.4耳廓肿胀测量及症状评分2.4 Auricle swelling measurement and symptom scoring
于第0、3、6、9、12、15、18日上午造模前用游标卡尺测量大鼠右耳廓厚度，每只鼠选取3个不同的测量位点测量，取其平均值，以保证测量的准确性与客观性，并绘制大鼠右耳廓厚度随时间变化的曲线图。Before modeling on the morning of days 0, 3, 6, 9, 12, 15, and 18, use a vernier caliper to measure the thickness of the right auricle of the rat. Select 3 different measurement points for each rat and take the average value to ensure The accuracy and objectivity of the measurement were measured, and the thickness of the right auricle of the rat was plotted as a function of time.
于第1、3、7、11、15、17日上午造模前对大鼠右耳廓进行拍照记录及症状评分，分别从红肿、脱屑、丘疹、囊肿4个指标对疾病的严重程度进行评分，每个指标对应0-4分，将4个指标的得分相加得到总分。评分标准下：0分，无症状；1分，轻度；2分，中度；3分，重度；4分，极重度。Before modeling on the morning of days 1, 3, 7, 11, 15, and 17, the right auricle of the rat was photographed and recorded, and symptoms were scored. The severity of the disease was evaluated from the four indicators of redness, swelling, desquamation, papules, and cysts. Rating, each indicator corresponds to 0-4 points, and the scores of the four indicators are added to obtain the total score. The scoring standard is: 0 points, no symptoms; 1 point, mild; 2 points, moderate; 3 points, severe; 4 points, extremely severe.
2.5标本采集2.5 Specimen collection
大鼠于第19天处死，完整剪下大鼠右耳廓，取1/4耳组织于4％多聚甲醛中固定过夜，用于石蜡切片制备及病理学检查，剩余耳组织经液氮速冻后，保存于-80℃，以供后续ELISA及qPCR检测使用。The rats were sacrificed on the 19th day, and the right auricle of the rats was completely cut off. 1/4 of the ear tissue was fixed in 4% paraformaldehyde overnight for paraffin section preparation and pathological examination. The remaining ear tissue was quick-frozen in liquid nitrogen. Afterwards, it was stored at -80°C for subsequent ELISA and qPCR detection.
2.6 H&E染色及病理学检查2.6 H&E staining and pathological examination
大鼠耳组织经4％多聚甲醛固定24h后，用石蜡包埋后进行切片，然后进行H&E染色，依据组织学判定标准评价大鼠痤疮模型的病理变化情况。The rat ear tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and then stained with H&E. The pathological changes of the rat acne model were evaluated according to histological criteria.
大量炎性细胞浸润等表现，具体评分标准如表22-2所示。A large number of inflammatory cell infiltration and other symptoms, the specific scoring criteria are shown in Table 22-2.
表22-2痤疮病理组织学评分标准表
Table 22-2 Acne histopathological scoring standard table
2.8大鼠耳组织IL-1β、IL-6、TNF-α和MCP-1的蛋白含量测定2.8 Determination of protein content of IL-1β, IL-6, TNF-α and MCP-1 in rat ear tissue
依据ELISA试剂盒厂家建议的操作流程，检测各个耳组织中IL-1β、IL-6、TNF-α和MCP-1的含量。According to the operating procedures recommended by the ELISA kit manufacturer, the contents of IL-1β, IL-6, TNF-α and MCP-1 in each ear tissue were detected.
3.实验结果3.Experimental results
3.1 ZHT对痤疮大鼠耳廓外观及厚度的影响3.1 Effect of ZHT on the appearance and thickness of auricles in rats with acne
在整个实验过程中，Veh组大鼠右耳廓内侧面始终光滑平整，无红肿、脱屑、丘疹、囊肿等症状，其右耳廓厚度基本保持不变。During the entire experiment, the inner surface of the right auricle of the rats in the Veh group was always smooth and flat, without symptoms such as redness, swelling, desquamation, papules, and cysts, and the thickness of the right auricle remained basically unchanged.
Model组大鼠右耳廓在造模第7天出现明显的红肿、脱屑症状，且红肿、脱屑症 状随造模的进行持续加重，且后期右耳廓表面凹凸不平并伴有囊肿出现；造模后大鼠的右耳廓厚度显著增加，从第6天开始，与Veh组相比显著升高，差异具有统计学意义(P<0.01)(表22-3)；皮损症状评分显示，造模后皮损程度逐渐加重，从第3天开始，与Veh组相比具有显著性差异(P<0.01)(表22-4)。The right auricle of the rats in the Model group showed obvious symptoms of redness, swelling, and desquamation on the 7th day of modeling, and the symptoms were redness, swelling, and desquamation. The symptoms continued to worsen as the modeling progressed, and the surface of the right auricle was uneven and accompanied by cysts in the later stages; the thickness of the right auricle of the rats increased significantly after modeling, and starting from the 6th day, it increased significantly compared with the Veh group , the difference was statistically significant (P<0.01) (Table 22-3); the skin lesion symptom score showed that the degree of skin lesions gradually worsened after modeling. Starting from the 3rd day, there was a significant difference compared with the Veh group (P <0.01) (Table 22-4).
涂抹BPO水凝胶改善了造模引起的红肿、脱屑及囊肿的出现，Model+BPO组大鼠右耳廓表面也较为光滑平整，给予BPO水凝胶治疗后逐渐延缓造模引起的耳朵增厚，在第18天Model+BPO组大鼠右耳廓厚度与Model组相比有显著降低(P<0.01)(表22-3)；皮损症状评分显示，涂抹BPO水凝胶对造模引起的痤疮样症状有一定治疗作用，从第11天开始，与Model组相比具有显著性差异(P<0.01)(表22-4)。Applying BPO hydrogel improved the redness, desquamation and cysts caused by modeling. The surface of the right auricle of rats in the Model+BPO group was also relatively smooth and flat. After treatment with BPO hydrogel, the ear growth caused by modeling was gradually delayed. On the 18th day, the thickness of the right auricle of the rats in the Model+BPO group was significantly reduced compared with the Model group (P<0.01) (Table 22-3); the skin lesion symptom score showed that applying BPO hydrogel had a negative impact on the modeling The acne-like symptoms caused have a certain therapeutic effect. Starting from the 11th day, there was a significant difference (P<0.01) compared with the Model group (Table 22-4).
涂抹1％ZHT-1、1％ZHT-2乳膏后在第12天均能够明显降低造模引起的耳朵增厚，与Model组相比具有显著性差异(P<0.01)(表22-3)；涂抹1％ZHT-2在第15天改善了造模引起的脱屑，与Model组相比皮损症状评分显著降低(P<0.01)，但无法进一步改善造模引起的红肿和丘疹，因此在其他时间点与Model组相比皮损症状评分无显著性差异(P>0.05)。涂抹1.0％ZHT-1缓解了造模引起的红肿、脱屑和丘疹等痤疮样症状，在第11、15、17天，Model+1.0％ZHT组与Model组相比症状评分显著降低(P<0.01)(表22-4)。Applying 1% ZHT-1 and 1% ZHT-2 cream can significantly reduce ear thickening caused by modeling on the 12th day, with a significant difference compared with the Model group (P<0.01) (Table 22-3 ); Applying 1% ZHT-2 improved the desquamation caused by modeling on the 15th day, and the skin lesion symptom score was significantly reduced compared with the Model group (P<0.01), but it could not further improve the redness, swelling and papules caused by modeling. Therefore, there was no significant difference in skin lesion symptom scores compared with the Model group at other time points (P>0.05). Applying 1.0% ZHT-1 alleviated acne-like symptoms such as redness, scaling, and papules caused by modeling. On days 11, 15, and 17, the symptom scores of the Model+1.0% ZHT group were significantly lower than those of the Model group (P< 0.01) (Table 22-4).
表22-3各组大鼠右耳廓厚度变化情况(单位：mm)
Table 22-3 Changes in the thickness of the right auricle of rats in each group (unit: mm)
数据以平均值±标准误差表示。N＝6.**,P＜0.01,vs.Veh；##,P＜0.01,vs.Model,使用双因素方差及Dunnett’s多重比较检验进行分析。Data are expressed as mean ± standard error. N＝6.**, P＜0.01, vs.Veh; ## , P＜0.01, vs.Model, analyzed using two-factor variance and Dunnett's multiple comparison test.
表22-4各组大鼠右耳廓皮损症状评分
Table 22-4 Symptom scores of skin lesions on the right auricle of rats in each group
3.2 ZHT对痤疮大鼠耳朵病理形态的影响3.2 Effect of ZHT on the pathological morphology of the ears of acne rats
Veh组大鼠耳组织未见明显异常，测量其表皮层厚度为12.56±2.39μm，毛囊、表皮、真皮层界线清晰可见，真皮层内偶见炎症细胞浸润；There were no obvious abnormalities in the ear tissue of rats in the Veh group. The thickness of the epidermal layer was measured to be 12.56±2.39 μm. The boundaries between hair follicles, epidermis, and dermis were clearly visible, and inflammatory cell infiltration was occasionally seen in the dermis;
Model组大鼠耳组织表皮层厚度为171.73±17.18μm，较Veh组显著增加(P<0.01)，表皮层和角质层过度增厚，角质层中可见炎性细胞聚集，毛囊扩张，毛囊口、漏斗部被角化物质填充，角栓堵塞较重、扩大呈壶状，皮脂腺萎缩，真皮层水肿并伴有大量炎性浸润，其病理评分为3.83±0.17分，与Veh组相比具有显著性差异(P<0.01)。 The thickness of the epidermal layer of the rat ear tissue in the Model group was 171.73±17.18 μm, which was significantly increased compared with the Veh group (P<0.01). The epidermis and cuticle were excessively thickened, inflammatory cells could be gathered in the cuticle, hair follicles were expanded, and the hair follicle mouth, The infundibulum was filled with keratinized material, the keratin plugs were severely blocked and enlarged into a pot shape, the sebaceous glands were atrophied, and the dermis was edematous with a large amount of inflammatory infiltration. The pathological score was 3.83±0.17 points, which was significant compared with the Veh group. difference (P<0.01).
Model+BPO组大鼠耳组织表皮层厚度为104.90±7.35μm，与Model组相比显著降低(P<0.01)；角质层略增厚，毛囊口、漏斗部角化物质较少，皮脂腺形态正常，真皮层未见水肿，炎性细胞浸润程度较Model组减轻，其病理评分为2.67±0.33分，与Model组相比显著降低(P<0.01)。The thickness of the epidermal layer of the rat ear tissue in the Model+BPO group was 104.90±7.35μm, which was significantly lower than that of the Model group (P<0.01); the stratum corneum was slightly thickened, there were less keratinized substances at the hair follicle opening and infundibulum, and the sebaceous glands were normal in shape. , there was no edema in the dermis, and the degree of inflammatory cell infiltration was reduced compared with the Model group. The pathological score was 2.67±0.33 points, which was significantly lower than the Model group (P<0.01).
Model+1％ZHT-2组、Model+1.0％ZHT-1组大鼠耳组织表皮层厚度分别为133.36±13.17μm、128.08±8.83μm，1.0％ZHT-1能够显著降低造模引起的表皮增厚(P<0.05)。The thickness of the epidermal layer of rat ear tissue in the Model+1% ZHT-2 group and the Model+1.0% ZHT-1 group were 133.36±13.17μm and 128.08±8.83μm respectively. 1.0% ZHT-1 can significantly reduce the epidermal growth caused by modeling. Thick (P<0.05).
涂抹1％ZHT-2、1.0％ZHT-1能够改善表皮层和角质层的过度增厚，减轻真皮层水肿并减少炎性细胞浸润，具有一定的剂量依赖性，各组病理评分分别为3.33±0.33分、3.33±0.33分。Applying 1% ZHT-2 and 1.0% ZHT-1 can improve the excessive thickening of the epidermis and stratum corneum, reduce dermal edema and reduce inflammatory cell infiltration in a dose-dependent manner. The pathological scores of each group were 3.33± 0.33 points, 3.33±0.33 points.
3.3 ZHT对痤疮大鼠耳组织Mcp-1、Il-1β、Il-6和Tnf-α的mRNA表达水平的影响3.3 Effect of ZHT on the mRNA expression levels of Mcp-1, Il-1β, Il-6 and Tnf-α in ear tissue of acne rats
Model组大鼠耳组织中Mcp-1的mRNA表达升高至Veh组的3.11±0.30倍，(P<0.01)；与Model组相比，涂抹BPO水凝胶可显著改善造模诱导的Mcp-1的mRNA表达上调，改善率为70.9±14.9％(P<0.01vs.Model)；涂抹1％ZHT-1乳膏，可改善造模诱导的Mcp-1的mRNA表达上调，改善率为25.5±16.0％，但差异无统计学意义(P>0.05vs.Model)；而涂抹1％ZHT-2乳膏对造模诱导的Mcp-1的mRNA表达上调有抑制作用。The mRNA expression of Mcp-1 in the rat ear tissue of the Model group increased to 3.11±0.30 times that of the Veh group, (P<0.01); compared with the Model group, applying BPO hydrogel could significantly improve the Mcp-1 induced by modeling. The mRNA expression of Mcp-1 was up-regulated, and the improvement rate was 70.9±14.9% (P<0.01vs.Model); applying 1% ZHT-1 cream can improve the up-regulation of Mcp-1 mRNA expression induced by modeling, and the improvement rate was 25.5± 16.0%, but the difference was not statistically significant (P>0.05 vs. Model); while applying 1% ZHT-2 cream had an inhibitory effect on the up-regulation of Mcp-1 mRNA expression induced by modeling.
Model组大鼠耳组织中Il-1β的mRNA表达升高至Veh组的14.23±2.24倍，差异具有统计学意义(P<0.01)；与Model组相比，涂抹BPO水凝胶可改善造模诱导的Il-1β的mRNA表达上调，改善率为76.6±6.9％(P<0.05vs.Model)；涂抹1％ZHT-2、1％ZHT-1乳膏均可改善造模诱导的Il-1β的mRNA表达上调，改善率分别为18.5±15.3％(P>0.05vs.Model)、58.4±6.5％(P<0.01vs.Model)。The mRNA expression of Il-1β in the rat ear tissue of the Model group increased to 14.23±2.24 times that of the Veh group, and the difference was statistically significant (P<0.01); compared with the Model group, applying BPO hydrogel can improve modeling. The induced Il-1β mRNA expression was up-regulated, and the improvement rate was 76.6±6.9% (P<0.05vs.Model); applying 1% ZHT-2 and 1% ZHT-1 cream can improve the modeling-induced Il-1β The mRNA expression was up-regulated, and the improvement rates were 18.5±15.3% (P>0.05vs.Model) and 58.4±6.5% (P<0.01vs.Model) respectively.
Model组大鼠耳组织中Il-6的mRNA表达显著升高至Veh组的4.62±0.50倍，差异具有统计学意义(P<0.01)；涂抹BPO水凝胶、1％ZHT-2、1％ZHT-1乳膏降低造模诱导的Il-6的mRNA表达上调，改善率分别为60.0±13.5％(P<0.05vs.Model)、46.2±5.4％(P<0.05vs.Model)、61.1±20.3％(P<0.05vs.Model)。The mRNA expression of Il-6 in the rat ear tissue of the Model group was significantly increased to 4.62±0.50 times that of the Veh group, and the difference was statistically significant (P<0.01); apply BPO hydrogel, 1% ZHT-2, 1% ZHT-1 cream reduces the up-regulation of Il-6 mRNA expression induced by modeling, and the improvement rates are 60.0±13.5% (P<0.05vs.Model), 46.2±5.4% (P<0.05vs.Model), and 61.1± 20.3% (P<0.05vs.Model).
Model组大鼠耳组织中Tnf-α的mRNA表达显著升高至Veh组的3.98±0.52倍(P<0.01)；涂抹BPO水凝胶可改善造模诱导的Tnf-α的mRNA表达上调，改善率为71.2±10.0％(P<0.05vs.Model)；涂抹1％ZHT-2、1％ZHT-1乳膏可抑制造模诱导的Tnf-α的mRNA表达上调，改善率分别为18.2±12.9％(P>0.05vs.Model)、58.3±15.0％(P<0.05vs.Model)。The mRNA expression of Tnf-α in the rat ear tissue of the Model group was significantly increased to 3.98±0.52 times that of the Veh group (P<0.01); applying BPO hydrogel can improve the up-regulation of Tnf-α mRNA expression induced by modeling and improve The rate was 71.2±10.0% (P<0.05vs.Model); applying 1% ZHT-2 and 1% ZHT-1 cream could inhibit the up-regulation of Tnf-α mRNA expression induced by modeling, and the improvement rates were 18.2±12.9 respectively. % (P>0.05vs.Model), 58.3±15.0% (P<0.05vs.Model).
3.5 ZHT对痤疮大鼠耳组织IL-1β、IL-6、TNF-α和MCP-1的含量的影响3.5 Effect of ZHT on the contents of IL-1β, IL-6, TNF-α and MCP-1 in ear tissue of acne rats
Veh组耳组织中IL-1β蛋白含量为175.2±29.5pg/mL，Model组大鼠耳组织中IL-1β的蛋白含量为270.1±18.9pg/mL，升高至Veh组的1.54±0.11倍(P<0.05)；给予BPO、1％ZHT-2和1％ZHT-1后大鼠耳中IL-1β的蛋白含量为224.0±20.0pg/mL、194.4±20.5pg/mL、179.7±25.8pg/mL，分别改善了48.5±21.1％(P>0.05vs.Model)、79.8±21.6％(P<0.05)vs.Model和95.2±27.2％(P<0.05vs.Model)造模诱导的IL-1β蛋白含量的增加。The protein content of IL-1β in the ear tissue of the Veh group was 175.2±29.5pg/mL, and the protein content of IL-1β in the ear tissue of the rats in the Model group was 270.1±18.9pg/mL, which increased to 1.54±0.11 times that of the Veh group ( P<0.05); the protein content of IL-1β in rat ears after administration of BPO, 1% ZHT-2 and 1% ZHT-1 was 224.0±20.0pg/mL, 194.4±20.5pg/mL, 179.7±25.8pg/ mL, respectively improved the IL-1β induced by modeling by 48.5±21.1% (P>0.05vs.Model), 79.8±21.6% (P<0.05)vs.Model and 95.2±27.2% (P<0.05vs.Model). Increase in protein content.
Veh组耳组织中IL-6蛋白含量为93.7±20.4pg/mL，Model组大鼠耳组织中IL-6的蛋白含量为266.4±15.2pg/mL，升高至Veh组的2.84±0.16倍，差异极显著(P<0.01)；给予BPO水凝胶后，大鼠耳组织中IL-6的蛋白含量为189.0±10.8pg/mL，抑制了44.8±6.3％(P<0.01vs.Model)造模诱导的大鼠耳组织中IL-6蛋白含量的增加。 给予1％ZHT-2、1％ZHT-1后组大鼠耳组织中IL-6的蛋白含量分别为237.2±21.4pg/mL、235.2±25.6pg/mL，各组分别改善了16.9±12.4％(P>0.05vs.Model)、18.1±14.8％(P>0.05vs.Model)造模诱导的大鼠耳组织中IL-6蛋白含量的增加。The protein content of IL-6 in the ear tissue of the Veh group was 93.7±20.4pg/mL, and the protein content of IL-6 in the ear tissue of the rats in the Model group was 266.4±15.2pg/mL, which increased to 2.84±0.16 times that of the Veh group. The difference was extremely significant (P<0.01); after administration of BPO hydrogel, the protein content of IL-6 in rat ear tissue was 189.0±10.8pg/mL, inhibiting 44.8±6.3% (P<0.01vs.Model). Model-induced increase in IL-6 protein content in rat ear tissue. The protein content of IL-6 in the ear tissue of the rats in the groups given 1% ZHT-2 and 1% ZHT-1 was 237.2±21.4pg/mL and 235.2±25.6pg/mL respectively, and each group improved by 16.9±12.4% respectively. (P>0.05vs.Model), 18.1±14.8% (P>0.05vs.Model) Model-induced increase in IL-6 protein content in rat ear tissue.
Veh组耳组织中TNF-α蛋白含量为103.7±23.7pg/mL，Model组大鼠耳组织中Tnf-α的蛋白含量为423.2±37.0pg/mL，升高至Veh组的4.08±0.58倍，差异极显著(P<0.01)；给予BPO、1％ZHT-2、1％ZHT-1后大鼠耳组织中TNF-α的蛋白含量分别为286.2±46.1pg/mL、224.9±49.1pg/mL、221.5±60.3pg/mL，与Model组相比，分别改善了62.1±15.4％(P<0.01vs.Model)、42.9±14.4％(P<0.05vs.Model)、63.1±18.9％(P<0.01vs.Model)造模诱导的大鼠耳组织中TNF-α蛋白含量的增加。The TNF-α protein content in the ear tissue of the Veh group was 103.7±23.7pg/mL, and the Tnf-α protein content in the ear tissue of the Model group was 423.2±37.0pg/mL, which increased to 4.08±0.58 times that of the Veh group. The difference is extremely significant (P<0.01); the protein content of TNF-α in rat ear tissue after administration of BPO, 1% ZHT-2, and 1% ZHT-1 was 286.2±46.1pg/mL and 224.9±49.1pg/mL respectively. , 221.5±60.3pg/mL, compared with the Model group, they were improved by 62.1±15.4% (P<0.01vs.Model), 42.9±14.4% (P<0.05vs.Model), and 63.1±18.9% (P< 0.01 vs. Model) model-induced increase in TNF-α protein content in rat ear tissue.
实施例4-1，实施例19-2样品经验证也具备与本实施例中的实施例19-1和实施例6上述各方面痤疮作用基本一致的作用或效果。The samples of Example 4-1 and Example 19-2 have been verified to have functions or effects that are basically consistent with the above-mentioned acne effects of Example 19-1 and Example 6 in this example.
实施例22-2黄葵总黄酮治疗特应性皮炎的药效学评价Example 22-2 Pharmacodynamic evaluation of total flavonoids from ambrosia in the treatment of atopic dermatitis
1.研究内容及目的：1. Research content and purpose:
考察不同剂量的黄葵总黄酮(ZHT-1，如实施例22-1)和黄葵HKY对DNFB诱导的特应性皮炎(Atopic dermatitis,AD)的治疗效果，并与阳性药糠酸莫米松(Mometasone,Mome)比较，以确定ZHT和HKY对AD的治疗效果。Examine the therapeutic effects of different doses of total flavonoids of Z. annua (ZHT-1, such as Example 22-1) and Z. alba. HKY on DNFB-induced atopic dermatitis (Atopic dermatitis, AD), and compare them with the positive drug mometasone furoate. (Mometasone, Mome) comparison to determine the therapeutic effect of ZHT and HKY on AD.
2.实验材料2. Experimental materials
2.1实验动物2.1 Experimental animals
SPF级Balb/c小鼠，购自扬州大学比较医学中心[实验动物生产许可证号：SCXK(苏)2017-0007]，7-8周龄，体重18-20g。饲养于室温23±2℃、湿度55％、光照与黑暗时间比为1∶1的环境中，小鼠自由进食和饮水。SPF grade Balb/c mice were purchased from the Comparative Medicine Center of Yangzhou University [Experimental Animal Production License Number: SCXK (Su) 2017-0007], 7-8 weeks old, weighing 18-20 g. The mice were kept in an environment with a room temperature of 23±2°C, a humidity of 55%, and a light to dark time ratio of 1:1. The mice were allowed to eat and drink freely.
2.2药物与主要试剂
2.2 Drugs and main reagents
2.3实验仪器
2.3 Experimental instruments
3.实验方法3. Experimental methods
3.1动物分组及特应性皮炎模型构建3.1 Animal grouping and atopic dermatitis model construction
将80只清洁级Balb/c小鼠适应性饲养5天后，随机分成10组，分别为空白对照组(Sham)、DNFB造模组(DNFB)、阳性药糠酸莫米松组(Mome)、黄葵总黄酮(ZHT)乳膏0.5％组、黄葵HKY(HKY)乳膏0.5％组以及100mg/kg SZHT灌胃给药组。松香石蜡脱毛，每只小鼠在第1天上午在背部涂抹50μL浓度为1.0％[W/V，溶剂为丙酮橄榄油混合物(丙酮：橄榄油＝3:1)]的2，4-二硝基氟苯(DNFB)，在第4、6、 8、10天上午涂抹给予0.5％(W/V)DNFB，每次50μL，对照组涂抹等量的溶剂对照(丙酮:橄榄油＝3:1)。动物实验遵循实验动物饲养管理与使用指南，符合动物伦理学规范。特应性皮炎模型构建图6所示。After adaptive feeding for 5 days, 80 clean-grade Balb/c mice were randomly divided into 10 groups, namely the blank control group (Sham), the DNFB model group (DNFB), the positive drug mometasone furoate group (Mome), and the yellow group. The total flavonoids (ZHT) cream 0.5% group, the HKY (HKY) cream 0.5% group and the 100 mg/kg SZHT oral administration group. For rosin paraffin hair removal, each mouse applied 50 μL of 2,4-dinitrine with a concentration of 1.0% [W/V, the solvent was an acetone-olive oil mixture (acetone:olive oil = 3:1)] on the back on the morning of the first day. fluorobenzene (DNFB), in the 4th, 6th, 8. On the morning of day 10, apply 0.5% (W/V) DNFB, 50 μL each time, and apply the same amount of solvent control (acetone:olive oil=3:1) to the control group. Animal experiments follow the guidelines for the care, management and use of experimental animals and comply with animal ethics standards. The construction of atopic dermatitis model is shown in Figure 6.
3.2给药方式及给药剂量3.2 Administration method and dosage
自第5天开始，每日下午用电子分析天平准确称取100mg不同剂量的ZHT乳膏及HKY乳膏，均匀涂抹于背部造模区域，对照组给予100mg空白乳膏；100mg/kg SZHT组采用灌胃给药。具体造模、给药流程如图6所示。Starting from the 5th day, use an electronic analytical balance to accurately weigh 100 mg of different doses of ZHT cream and HKY cream every afternoon, and apply it evenly on the back modeling area. The control group is given 100 mg of blank cream; the 100 mg/kg SZHT group is given Administer by gavage. The specific modeling and drug administration process are shown in Figure 6.
3.2.1 ZHT乳膏及HKY乳膏配制3.2.1 Preparation of ZHT cream and HKY cream
ZHT乳膏及HKY乳膏处方见表22-3-1。The prescriptions of ZHT cream and HKY cream are shown in Table 22-3-1.
表22-3-1黄葵总黄酮乳膏及黄葵HKY处方
Table 22-3-1 Huangkui total flavonoid cream and Huangkui HKY prescription
乳膏配制过程：Cream preparation process:
水相：称取处方量乙醇，加入ZHT/HKY，搅拌溶解，依次向烧杯内加入吐温80、十二烷基硫酸钠、甘油、肉豆蔻酸异丙酯、尼泊金甲酯、氮酮、纯化水，搅拌至分散均匀。Aqueous phase: Weigh the prescribed amount of ethanol, add ZHT/HKY, stir to dissolve, and add Tween 80, sodium lauryl sulfate, glycerin, isopropyl myristate, methylparaben, and azone into the beaker in sequence. , purified water, stir until evenly dispersed.
油相：称取处方量单硬脂酸甘油酯、棕榈酸、白凡士林于烧杯中，于80℃水浴下加热溶解至液态。Oil phase: Weigh the prescribed amount of glyceryl monostearate, palmitic acid, and white petroleum jelly into a beaker, heat and dissolve in a water bath at 80°C until it becomes liquid.
将水相缓慢加入油相中，边加边进行搅拌，水相全部加入后，从水浴锅内取出，于室温下搅拌直至冷却成乳膏。Slowly add the water phase into the oil phase, stirring while adding. After all the water phase has been added, remove from the water bath and stir at room temperature until it cools into a cream.
3.2.2 SZHT混悬液配制3.2.2 Preparation of SZHT suspension
0.5％CMC-Na的配制：在给药前一天，于100mL 70℃纯水中缓慢加入0.5g CMC-Na粉末，搅拌均匀，于室温静置过夜。次日，准确称取50mg ZHT，加入0.1mL DMSO配置成DMSO母液。向ZHT母液中加入0.1mL Tween-80及9.8mL超声至均质的0.5％CMC-Na溶液，得到5mg/mL的ZHT混悬液。Preparation of 0.5% CMC-Na: One day before administration, slowly add 0.5g CMC-Na powder to 100mL 70℃ pure water, stir evenly, and let it stand at room temperature overnight. The next day, accurately weigh 50mg ZHT and add 0.1mL DMSO to prepare DMSO mother solution. Add 0.1 mL of Tween-80 and 9.8 mL of sonicated 0.5% CMC-Na solution to the ZHT mother solution to obtain a 5 mg/mL ZHT suspension.
3.3特应性皮炎严重程度评分(AD Score)3.3 Atopic dermatitis severity score (AD Score)
于造模第4、7、9、11天，对每只小鼠背部皮肤进行拍照记录，通过①红斑/出血②干燥③渗出/结痂④水肿4项指标评价皮炎外观严重程度。各项指标以0～3分进行记分，将4个指标的得分相加得到总分。AD Score评分标准如下：0，无症状；1，症状轻微；2，中度症状；3，严重症状。On the 4th, 7th, 9th, and 11th days of modeling, the back skin of each mouse was photographed and recorded, and the severity of dermatitis appearance was evaluated through four indicators: ① erythema/hemorrhage, ② dryness, ③ exudation/scab, and ④ edema. Each indicator is scored from 0 to 3, and the scores of the four indicators are added together to obtain the total score. The AD Score scoring criteria are as follows: 0, no symptoms; 1, mild symptoms; 2, moderate symptoms; 3, severe symptoms.
3.5 HE染色 3.5 HE staining
石蜡切片常规脱蜡至水，将切片置于苏木素染料中，染色5分钟后，自来水冲洗。再将切片置于盐酸乙醇中，分化30秒后，置于自来水中浸泡15分钟，再置于伊红染料中，染色2分钟。常规脱水，透明，用中性树脂封片。使用数字病理切片扫描仪拍照，并统计表皮层厚度。Paraffin sections were routinely dewaxed to water, and the sections were placed in hematoxylin dye, stained for 5 minutes, and rinsed with tap water. The sections were then placed in hydrochloric acid ethanol, differentiated for 30 seconds, soaked in tap water for 15 minutes, and then placed in eosin dye and stained for 2 minutes. Routinely dehydrate, clear, and mount with neutral resin. Use a digital pathology slide scanner to take pictures and count the thickness of the epidermal layer.
3.10统计学分析3.10 Statistical analysis
采用GraphPad Prism 6.0软件进行数据的统计分析及制图，所有数据以平均值±标准误(mean±SEM)表示。皮炎评分采用two-way ANOVA分析，其他数据采用one-way ANOVA分析，利用Dunnett's多重检验分析组间差异的显著性,当P<0.05时，组间差异显著，具有统计学意义。GraphPad Prism 6.0 software was used for statistical analysis and graphing of data. All data are expressed as mean ± standard error (mean ± SEM). Dermatitis scores were analyzed using two-way ANOVA, other data were analyzed using one-way ANOVA, and Dunnett's multiple test was used to analyze the significance of differences between groups. When P<0.05, the differences between groups were significant and statistically significant.
4.实验结果4.Experimental results
4.1 ZHT、HKY对DNFB诱导的特应性皮炎模型外观的影响4.1 Effects of ZHT and HKY on the appearance of DNFB-induced atopic dermatitis model
如图7所示，在整个实验过程中，空白对照组(Sham)背部皮肤始终光滑平整，没有出现渗出结痂，红斑出血等症状。经过DNFB反复刺激，从第4天开始，DNFB组小鼠的结痂，红斑和水肿等症状明显，其皮炎评分(AD Score)相较于Sham组差异明显(P＜0.01，N＝8)，说明AD模型建立成功；阳性药物糠酸莫米松(Mome)能够逐步改善由DNFB引起的AD样症状，在第11天的皮炎评分中Mome组与DNFB组存在显著差异(P＜0.05，N＝8)；涂抹0.5％ZHT能够较明显地缓解AD样症状，在第11天的皮炎评分中0.5％ZHT组与DNFB组的差异极显著(P＜0.01，N＝8)；灌胃给予100mg/kg SZHT没有表现出对AD样症状的缓解作用(P＞0.05，N＝8)；0.5％HKY在一定程度上能起到减轻AD样症状的作用，但0.5％HKY组与DNFB组相比，皮炎评分不存在显著性差异(P＞0.05，N＝8)。As shown in Figure 7, throughout the entire experiment, the skin on the back of the blank control group (Sham) was always smooth and flat, with no symptoms such as exudation, scabs, erythema, and bleeding. After repeated stimulation by DNFB, starting from the 4th day, the mice in the DNFB group showed obvious symptoms such as scabs, erythema and edema, and their dermatitis score (AD Score) was significantly different from that of the Sham group (P<0.01, N=8). This shows that the AD model was successfully established; the positive drug mometasone furoate (Mome) can gradually improve AD-like symptoms caused by DNFB. There was a significant difference in the dermatitis score on the 11th day between the Mometas group and the DNFB group (P<0.05, N=8 ); applying 0.5% ZHT can significantly alleviate AD-like symptoms. In the dermatitis score on the 11th day, the difference between the 0.5% ZHT group and the DNFB group was extremely significant (P<0.01, N=8); 100 mg/kg was given by gavage SZHT did not show any alleviation effect on AD-like symptoms (P>0.05, N=8); 0.5% HKY can alleviate AD-like symptoms to a certain extent, but compared with the DNFB group, the 0.5% HKY group had less dermatitis There was no significant difference in scores (P>0.05, N=8).
4.3 ZHT和HKY对DNFB诱导的特应性皮炎模型皮肤病理变化的影响。4.3 Effects of ZHT and HKY on skin pathological changes in DNFB-induced atopic dermatitis model.
各组皮肤组织切片HE染色结果如图8所示，Sham组背部皮肤组织无明显改变，皮肤各层结构完整，真皮与表皮交界界限清楚，无明显炎症细胞浸润，表皮厚度(蓝色线条所示)为37.18±8.93μm；DNFB组皮肤组织出现明显的角化过度和棘皮层肥厚，表皮厚度为134.28±22.46μm，相较于Sham组有明显增加(P＜0.01，N＝6)，符合特应性皮炎皮肤组织病理的表征；在涂抹Mome治疗后，可明显观察到表皮增厚程度减轻，表皮层厚度为46.09±14.29μm(P＜0.01，N＝6，与DNFB组相比)；0.5％ZHT组的皮肤棘皮层肥厚有所改善，表皮层厚度为100.59±10.95μm，与DNFB组相比，0.5％ZHT降低了34.7±5.0％的增厚，具有显著性差异(P＜0.05，N＝6)，这与皮炎评分结果一致；然而0.5％HKY对DNFB诱导的AD样病理变化没有显著的改善作用，其表皮厚度也与DNFB组没有显著性差异(P＞0.05，N＝6)。The results of HE staining of skin tissue sections in each group are shown in Figure 8. There was no obvious change in the skin tissue on the back of the Sham group. The structure of each layer of the skin was intact. The boundary between the dermis and the epidermis was clear. There was no obvious inflammatory cell infiltration. The thickness of the epidermis (shown by the blue line) ) was 37.18±8.93μm; the skin tissue of the DNFB group showed obvious hyperkeratosis and acanthosis hypertrophy, and the epidermal thickness was 134.28±22.46μm, which was significantly increased compared with the Sham group (P<0.01, N=6), in line with the characteristics Characterization of skin histopathology of atopic dermatitis; after applying Mome treatment, the degree of epidermal thickening can be obviously reduced, and the thickness of the epidermal layer is 46.09±14.29μm (P<0.01, N=6, compared with the DNFB group); 0.5 The skin acanthosis of the % ZHT group was improved, and the epidermal layer thickness was 100.59±10.95 μm. Compared with the DNFB group, 0.5% ZHT reduced the thickness by 34.7±5.0%, with a significant difference (P＜0.05, N =6), which is consistent with the dermatitis score results; however, 0.5% HKY has no significant improvement effect on AD-like pathological changes induced by DNFB, and its epidermal thickness is also not significantly different from that of the DNFB group (P>0.05, N=6).
实施例3.2-1，实施例4-2，实施例9-2，也具备与本实施例中的黄葵总黄酮上述各方面基本一致的作用或效果。Example 3.2-1, Example 4-2, and Example 9-2 also have functions or effects that are basically consistent with the above-mentioned aspects of the total flavonoids of ambrosia in this example.
实施例23黄葵总黄酮促进伤口愈合的药效学评价Example 23 Pharmacodynamic evaluation of total flavonoids from ambrosia in promoting wound healing
1.实验材料1. Experimental materials
1.1实验动物1.1 Experimental animals
SPF级SD大鼠，6-8周龄，150-200g。SPF grade SD rat, 6-8 weeks old, 150-200g.
1.2试剂和药品1.2 Reagents and drugs
表23-1

Table 23-1

2.实验方法2. Experimental methods
2.1动物饲养和管理2.1 Animal feeding and management
SD大鼠(购自南京医科大学，许可证号：SCXK(苏)2021-0001)全程饲养于SPF级动物房，光照与黑暗时间比为1:1，室温控制在23±2℃，湿度控制在55％，大鼠自由进食和饮水。高糖高脂饲料饲养阶段每三天更换一次垫料，注射STZ建立糖尿病模型阶段，每天更换一次垫料，建立皮肤全层切除模型后改为每半天更换一次垫料，以保证大鼠处于干燥清洁的环境中。所有动物实验方案均获得中国药科大学实验动物管理和使用委员会的批准(许可证号：SYXK(苏)2018-0019)。SD rats (purchased from Nanjing Medical University, license number: SCXK (Su) 2021-0001) were kept in an SPF-level animal room throughout the whole process. The light to dark time ratio was 1:1, the room temperature was controlled at 23±2°C, and the humidity was controlled. At 55%, rats had free access to food and water. During the high-sugar and high-fat feed feeding stage, the bedding was changed every three days. During the diabetes model stage when STZ was injected, the bedding was changed once a day. After the full-thickness skin resection model was established, the bedding was changed every half a day to ensure that the rats were dry. in a clean environment. All animal experiment protocols were approved by the Experimental Animal Care and Use Committee of China Pharmaceutical University (license number: SYXK (Su) 2018-0019).
2.2给药方式和给药剂量2.2 Administration method and dosage
电子分析天平分别称取100mg的空白乳膏、0.5％、1％、2％黄葵总黄酮乳膏、德湿洁水胶体，均匀涂抹在溃疡伤口表面，每日两次。组别及各组涂抹药物如下所示：Use an electronic analytical balance to weigh 100 mg of blank cream, 0.5%, 1%, and 2% total flavonoid cream of Huang Kui, and Deshijie hydrocolloid, and apply them evenly on the surface of the ulcer wound twice a day. The groups and the drugs applied in each group are as follows:
空白对照组(Control)：不作处理Blank control group (Control): no treatment
模型组(Model)：涂抹不含黄葵总黄酮的空白乳膏剂；Model group (Model): Apply a blank cream without total flavonoids from ambrosia;
阳性药组(Positive)：涂抹德湿洁水胶体；Positive medicine group (Positive): Apply Deshi cleansing water colloid;
低剂量组(Low-0.5％)：涂抹0.5％黄葵总黄酮乳膏剂；Low-dose group (Low-0.5%): apply 0.5% total flavonoids cream of ambrette;
中剂量组(Middle-1％)：涂抹1％黄葵总黄酮乳膏剂；Middle-dose group (Middle-1%): apply 1% ambrette total flavonoids cream;
高剂量组(High-2％)：涂抹2％黄葵总黄酮乳膏剂；High-dose group (High-2%): apply 2% ambrette total flavonoids cream;
2.3试剂配制2.3 Reagent preparation
STZ溶液配置：①柠檬酸缓冲液的配置：蒸馏水100mL中加入柠檬酸2.1g配成A液；蒸馏水100mL加入柠檬酸钠2.94g配成B液。②使用时将A、B按比例混合，调节pH至4.2-4.5，即是配置STZ的柠檬酸缓冲液。③注射前用柠檬酸缓冲液以1％的浓度溶解STZ。避光、4℃冰箱冷藏备用。STZ solution configuration: ① Configuration of citric acid buffer: add 2.1g of citric acid to 100mL of distilled water to prepare liquid A; add 2.94g of sodium citrate to 100mL of distilled water to prepare liquid B. ② When using, mix A and B in proportion and adjust the pH to 4.2-4.5, which is the citric acid buffer solution for STZ. ③ Dissolve STZ in citrate buffer at a concentration of 1% before injection. Protect from light and refrigerate at 4°C for later use.
4％水合氯醛配制：称取4g水合氯醛，加适量生理盐水溶解，定容至100mL。避光、4℃冰箱冷藏备用。Preparation of 4% chloral hydrate: Weigh 4g of chloral hydrate, add appropriate amount of physiological saline to dissolve, and adjust the volume to 100mL. Protect from light and refrigerate at 4°C for later use.
不同浓度黄葵总黄酮乳膏配制：0.5％、1％和2％的黄葵总黄酮乳膏按照如实施例22表22-1处方配置，不同浓度的按水补足。Preparation of total flavonoid creams of different concentrations: 0.5%, 1% and 2% total flavonoid creams of ambrette are prepared according to the prescription in Table 22-1 of Example 22, and the different concentrations are supplemented with water.
2.4糖尿病溃疡大鼠模型的建立2.4 Establishment of diabetic ulcer rat model
2.4.1STZ法诱导SD大鼠建立糖尿病动物模型2.4.1STZ method induced SD rats to establish diabetic animal model
适应性饲养一周后，将70只健康雄性SD大鼠(SPF级，4-6周龄，体重150-200g)，随机分笼，每笼5只大鼠，给予高糖高脂饲料喂养，作为糖尿病模型组。另选10只正常大鼠，每笼5只，给予普通饲料喂养，作为空白对照组。对60只SD大鼠分别 测量空腹血糖(Fasting Blood Glucose，FBG)和体重。喂养四周后，对糖尿病模型组大鼠一次性按体重腹腔注射链脲佐菌素(35mg/kg)，空白对照组一次性按体重注入等量的柠檬酸缓冲液。STZ溶液及柠檬酸缓冲液按空腹体重于大鼠左下腹注射，在10min内注射完毕。所有大鼠给药前均禁食水6h，注射72h后进行尾静脉采血，测量空腹血糖，空腹血糖值≥16.7mmol/L、体重无明显降低者为糖尿病模型构建成功。部分大鼠血糖位于5-8mmol/L，则再次注射STZ(35mg/kg)；部分大鼠血糖位于8-10mmol/L，则再次注射STZ(25mg/kg)；部分大鼠血糖位于10-16mmol/L，则注射STZ(20mg/kg)。注射72h后进行尾静脉采血测量空腹血糖，空腹血糖值≥16.7mmol/L，认定为糖尿病模型构建成功，若空腹血糖＜16.7mmol/L，则按体重再次补注STZ，直至空腹血糖值≥16.7mmol/L，认定为糖尿病模型制备成功。本次实验注射STZ诱导SD大鼠建立糖尿病动物模型耗时12天，全部成模后给予高糖高脂饲料喂养一周，使其稳定成模。After one week of adaptive feeding, 70 healthy male SD rats (SPF grade, 4-6 weeks old, weight 150-200g) were randomly divided into cages, with 5 rats in each cage, and fed with high-sugar and high-fat feed. Diabetes model group. Another 10 normal rats were selected, 5 in each cage, and fed with ordinary feed as a blank control group. For 60 SD rats, respectively Measure fasting blood glucose (FBG) and body weight. After four weeks of feeding, the rats in the diabetic model group were intraperitoneally injected with streptozotocin (35 mg/kg) based on body weight at one time, and the rats in the blank control group were injected with the same amount of citrate buffer based on body weight at one time. STZ solution and citrate buffer were injected into the left lower abdomen of rats according to their fasting body weight, and the injection was completed within 10 minutes. All rats were fasted for 6 hours before administration, and blood was collected from the tail vein 72 hours after injection to measure fasting blood glucose. Diabetes models were successfully constructed if the fasting blood glucose value was ≥16.7 mmol/L and there was no significant decrease in body weight. If the blood sugar of some rats is between 5-8mmol/L, then inject STZ (35mg/kg) again; if the blood sugar of some rats is between 8-10mmol/L, then inject STZ (25mg/kg) again; if the blood sugar of some rats is between 10-16mmol /L, then inject STZ (20mg/kg). 72 hours after injection, blood was collected from the tail vein to measure fasting blood glucose. If the fasting blood glucose value is ≥16.7mmol/L, the diabetes model is deemed to have been successfully constructed. If the fasting blood glucose is <16.7mmol/L, STZ will be re-injected according to body weight until the fasting blood glucose value is ≥16.7 mmol/L, it was deemed that the diabetes model was successfully prepared. In this experiment, it took 12 days to establish a diabetic animal model in SD rats induced by STZ injection. After all the models were formed, they were fed with high-sugar and high-fat feed for one week to stabilize the models.
2.4.2糖尿病大鼠全层皮肤切除伤口模型的建立2.4.2 Establishment of full-thickness skin excision wound model in diabetic rats
糖尿病大鼠稳定成模后，将大鼠按体重(2mg/kg)腹腔注射4％水合氯醛麻醉，待大鼠完全麻醉后，用电动剃毛器对大鼠背部剃毛，剃毛后，使用医用棉签蘸上碘伏连续涂抹大鼠背部4-5次进行消毒。采用无菌8-mm Biopsy Punch在大鼠背部中间位置印出一个直径8mm的圆形区域，使用无菌镊子将圆形区域中间的皮肤夹起，用无菌虹膜剪沿着Biopsy Punch勾勒出的圆形印子将皮肤切除，制造一个直径为8mm的圆形全层皮肤切除开放创面，深达肌肉，避开动、静脉大血管。随后立即用碘伏连续涂抹伤口4-5次，并给予适当的按压，充分止血。空白对照组大鼠仅按体重注射等剂量的水合氯醛，并在背部剃毛，不建立全层皮肤切除开放创面。剔除造模时死亡的大鼠，将造模成功，苏醒且状态良好的糖尿病大鼠按照随机数字表法进行随机分组，分别是：模型组(涂抹空白乳膏)、阳性药组(涂抹德湿洁水胶体)、低剂量组(涂抹黄葵总黄酮0.5％乳膏剂)、中剂量组(涂抹黄葵总黄酮1％乳膏剂)、高剂量组(涂抹黄葵总黄酮2％乳膏剂)。全层皮肤切除伤口模型建立以后立即给予药物治疗，并于治疗后第0d(涂抹给药前)、3d、7d、9d拍照记录，利用ImageJ软件测量伤口面积。After the diabetic rats were stabilized and established as models, the rats were anesthetized by intraperitoneal injection of 4% chloral hydrate according to body weight (2 mg/kg). After the rats were completely anesthetized, the backs of the rats were shaved with an electric shaver. After shaving, Use a medical cotton swab dipped in iodophor and apply it continuously on the back of the rat 4-5 times for disinfection. Use a sterile 8-mm Biopsy Punch to print a circular area with a diameter of 8 mm in the middle of the rat's back. Use sterile forceps to pick up the skin in the middle of the circular area and use sterile iris scissors to follow the outline of the Biopsy Punch. The skin is removed with a circular impression to create a circular, full-thickness skin resection open wound with a diameter of 8mm, which reaches deep into the muscles and avoids large arterial and venous blood vessels. Immediately afterwards, apply iodophor continuously to the wound 4-5 times, and apply appropriate pressure to fully stop bleeding. Rats in the blank control group were only injected with an equal dose of chloral hydrate according to their body weight, and their backs were shaved without establishing a full-thickness skin excision open wound. Rats that died during modeling were eliminated, and diabetic rats that were successfully modeled, awake and in good condition were randomly divided into groups according to the random number table method, namely: model group (smeared with blank cream), positive drug group (smeared with Deshi Water colloid), low-dose group (smear 0.5% total flavonoids cream of Scutellaria baicalensis), medium-dose group (smear 1% cream of total flavonoids of Scutellaria sibiricum), high-dose group (smear 2% cream of total flavonoids of Scutellaria sibiricum). Drug treatment was given immediately after the full-thickness skin excision wound model was established, and photos were taken and recorded on 0d (before application and administration), 3d, 7d, and 9d after treatment, and the wound area was measured using ImageJ software.
2.5型糖尿病溃疡造模的整体评价Overall evaluation of type 2.5 diabetic ulcer modeling
1.一般症状的观察，即是否有多饮、多食、多尿的变化，并且观察是否有精神状态差，毛色枯黄、脱毛，活动明显减少现象；1. Observation of general symptoms, that is, whether there are changes in polydipsia, polyphagia, and polyuria, and whether there are poor mental status, yellowish coat color, hair loss, and significant reduction in activity;
2.尾静脉采血测得空腹血糖在16.7-33.3mmol/L；2. The fasting blood glucose measured by tail vein blood sampling is 16.7-33.3mmol/L;
3.全层皮肤缺损的典型外观表现：溃疡、红肿，以溃疡面积为指标进行统计，计算公式如下：伤口面积(％)＝未愈合创口面积/原始创面面积×100％。3. Typical appearance of full-thickness skin defects: ulcers, redness and swelling. The ulcer area is used as an indicator for statistics. The calculation formula is as follows: wound area (%) = unhealed wound area/original wound area × 100%.
2.6 HE染色2.6 HE staining
将皮肤组织贴附在滤纸片上，于4％多聚甲醛溶液中垂直固定48h，进行常规石蜡包埋。对包埋好的皮肤组织进行连续皮肤全层切片(5μm)，每个载玻片放置2张切片，然后进行HE染色，具体步骤如下：The skin tissue was attached to the filter paper, fixed vertically in 4% paraformaldehyde solution for 48 hours, and then embedded in conventional paraffin. Perform continuous full-thickness skin sections (5 μm) on the embedded skin tissue, place 2 sections on each slide, and then perform HE staining. The specific steps are as follows:
1)脱蜡：二甲苯Ⅰ脱蜡10min，二甲苯Ⅱ脱蜡5min；1) Dewaxing: xylene I dewaxing for 10 minutes, xylene II dewaxing for 5 minutes;
2)乙醇浸泡：用无水乙醇浸泡2次，然后依次用95％、90％、85％的乙醇浸泡1次，每次浸泡1min，最后用水洗涤2min；2) Ethanol soaking: soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
3)染色：用苏木精进行染色，染色5min，然后用自来水洗涤1min；3) Staining: Stain with hematoxylin for 5 minutes, then wash with tap water for 1 minute;
4)分化：用1％盐酸酒精分化20s，然后用自来水洗涤1min；4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;
5)反蓝：用1％稀氨水反蓝30s，然后用自来水洗涤1min；5) Anti-blue: Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
6)伊红染色：染色5min，然后用自来水洗涤30s；6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;
7)乙醇脱水：依次用不同含量的乙醇进行脱水处理，顺序如下：85％乙醇脱水 20s，90％乙醇脱水30s，95％乙醇I脱水1min，95％乙醇II脱水1min，无水乙醇I脱水2min，无水乙醇II脱水2min；7) Ethanol dehydration: Use different contents of ethanol to dehydrate in sequence. The sequence is as follows: 85% ethanol dehydration. 20s, 90% ethanol dehydration for 30s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, absolute ethanol I for 2 min, absolute ethanol II for 2 min;
8)透明：二甲苯I处理2min，二甲苯II处理2min，二甲苯III处理2min；8) Transparency: xylene I treatment for 2 minutes, xylene II treatment for 2 minutes, xylene III treatment for 2 minutes;
9)最后用中性树胶封片，用数字病理切片扫描仪(NanoZoomer 2.0RS)对切片扫描成像。9) Finally, seal the slides with neutral gum, and use a digital pathology slide scanner (NanoZoomer 2.0RS) to scan and image the slides.
2.7 Masson染色2.7 Masson staining
将石蜡切片脱蜡至水，用weigert氏苏木溶液染处理10min，1％盐酸乙醇分化1min，蒸馏水冲洗25min，丽春红酸性品红液染色10min，1％磷钼酸水溶液分化5min，2％亮绿染液染5min，1％冰醋酸水溶液浸洗1min。随后分别用95％乙醇、无水乙醇脱水，二甲苯中透明，中性树胶封片。用数字病理切片扫描仪(NanoZoomer 2.0RS)对切片扫描成像。Dewax the paraffin sections to water, stain with Weigert's hematoxylin solution for 10 min, differentiate with 1% hydrochloric acid ethanol for 1 min, rinse with distilled water for 25 min, stain with Ponceau acid fuchsin solution for 10 min, differentiate with 1% phosphomolybdic acid aqueous solution for 5 min, 2% Dye with bright green dye for 5 minutes and soak with 1% glacial acetic acid aqueous solution for 1 minute. Then, they were dehydrated with 95% ethanol and absolute ethanol, made transparent in xylene, and sealed with neutral gum. The slides were scanned and imaged using a digital pathology slide scanner (NanoZoomer 2.0RS).
2.8血管标志物CD31的免疫荧光2.8 Immunofluorescence of vascular marker CD31
取皮肤组织，4％多聚甲醛过夜固定，行石蜡包埋，制成皮肤石蜡切片。将石蜡切片放入60℃烘箱中，烤片20min，然后依次放入二甲苯Ⅰ浸泡15min、二甲苯Ⅱ浸泡15min、无水乙醇Ⅰ浸泡5min、无水乙醇Ⅱ浸泡5min、95％酒精浸泡5min、85％酒精浸泡5min、75％酒精浸泡5min，再用蒸馏水和PBS分别润洗5min。将切片置于盛满抗原修复缓冲液的染色缸中，于高压锅中进行抗原热修复。流水冲洗高压锅，冷却至室温后，甩去多余的液体，滴加5％BSA，室温封闭1h。接着，甩去封闭液，滴加稀释的一抗(1:400)，并将切片置于湿盒中4℃过夜孵育。次日，甩去一抗，用PBS润洗切片3次，每次5min，随后滴加稀释的荧光二抗(1:1000)，室温避光孵育1h后，甩去二抗，用PBS中润洗3次，每次5min。甩干切片后，滴加含Hoechst的抗荧光淬灭剂，于荧光显微镜下观察并拍照。Skin tissue was taken, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and made into skin paraffin sections. Put the paraffin slices into a 60°C oven and bake them for 20 minutes, then soak them in xylene I for 15 minutes, xylene II for 15 minutes, absolute ethanol I for 5 minutes, absolute ethanol II for 5 minutes, and 95% alcohol for 5 minutes. Soak in 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then rinse with distilled water and PBS for 5 minutes respectively. Place the sections in a staining vat filled with antigen retrieval buffer, and perform antigen heat retrieval in a pressure cooker. Rinse the pressure cooker with running water, cool to room temperature, shake off excess liquid, add 5% BSA dropwise, and seal at room temperature for 1 hour. Then, shake off the blocking solution, add diluted primary antibody (1:400) dropwise, and place the sections in a humidified box for overnight incubation at 4°C. The next day, remove the primary antibody and rinse the sections with PBS three times for 5 minutes each time. Then add diluted fluorescent secondary antibody (1:1000) dropwise. After incubation at room temperature in the dark for 1 hour, remove the secondary antibody and moisten with PBS. Wash 3 times, 5 minutes each time. After drying the sections, anti-fluorescence quenching agent containing Hoechst was added dropwise, and the sections were observed and photographed under a fluorescence microscope.
2.9实时荧光定量PCR2.9 Real-time fluorescence quantitative PCR
将皮肤组织置于研磨管中，加入研磨珠和800μL Trizol试剂，于组织研磨机中以60Hz的频率持续研磨1min，重复5次，直至无肉眼可见沉淀。收集上清，12,000rpm离心10min后转移上清至新的1.5mL无酶管。按氯仿-异丙醇萃取过夜后离心，弃上清，75％乙醇洗去附着在RNA上的离子，晾干后加入DEPC水溶解，吸取2μL用于Nano-100微量分光光度计定量，检测时A260/A280应处于在1.8-2.0之间。使用无酶水调整RNA浓度至1μg，加入5×gDNA wiper Mix 3μL后42℃加热2min，去除基因组DNA后，再加入4×qRT Super MixⅡ5μL，达成20μL体系，于PCR仪器进行25℃-2min、50℃-30min、85℃-5min的逆转录反应，cDNA于-80℃保存备用。Place the skin tissue into a grinding tube, add grinding beads and 800 μL Trizol reagent, and grind continuously in a tissue grinder at a frequency of 60 Hz for 1 min, repeat 5 times, until no precipitation is visible to the naked eye. Collect the supernatant, centrifuge at 12,000 rpm for 10 min, and then transfer the supernatant to a new 1.5 mL enzyme-free tube. Extract with chloroform-isopropyl alcohol overnight and centrifuge. Discard the supernatant. Wash away the ions attached to the RNA with 75% ethanol. After drying, add DEPC water to dissolve. Pipette 2 μL for quantification with Nano-100 Micro Spectrophotometer. During detection A260/A280 should be between 1.8-2.0. Use enzyme-free water to adjust the RNA concentration to 1μg, add 3μL of 5×gDNA wiper Mix and heat it at 42°C for 2 minutes. After removing the genomic DNA, add 5μL of 4×qRT Super MixⅡ to reach a 20μL system. Run the PCR instrument at 25℃-2min, 50 ℃-30min, 85℃-5min reverse transcription reaction, cDNA is stored at -80℃ for later use.
按照1μL cDNA+10μLGreen+0.8μL Primers+8.2μL ddH2O的体系进行实时荧光定量PCR反应，采用2-ΔCt方法计算mRNA表达，各基因的表达量以GAPDH的表达量进行校正。所用引物见表23-2。According to 1μL cDNA+10μL The system of Green+0.8μL Primers+8.2μL ddH 2 O was used for real-time fluorescence quantitative PCR reaction. The 2 -ΔCt method was used to calculate the mRNA expression. The expression level of each gene was corrected by the expression level of GAPDH. The primers used are shown in Table 23-2.
表23-2 PCR所用引物序列
Table 23-2 Primer sequences used in PCR
2.10血管内皮生长因子(VEGF)含量的测定2.10 Determination of vascular endothelial growth factor (VEGF) content
依据ELISA试剂盒的操作流程，检测皮肤组织中VEGF的含量。Detect the VEGF content in skin tissue according to the operating procedure of the ELISA kit.
2.11统计学分析2.11 Statistical analysis
运用GraphPad Prism(Ver 6.0)进行统计学分析，所有数据采用平均值±标准误 (Mean±SEM)表示，伤口外观愈合率数据采用Two-way ANOVA分析，其他数据采用One-way ANOVA分析，利用Dunnett’s多重检验分析组间差异的显著性，当P＜0.05时，组间差异具有统计学意义。Statistical analysis was performed using GraphPad Prism (Ver 6.0), and all data were expressed as mean ± standard error. (Mean±SEM) means that the wound appearance healing rate data were analyzed by Two-way ANOVA, and other data were analyzed by One-way ANOVA. Dunnett's multiple test was used to analyze the significance of differences between groups. When P<0.05, the difference between groups has Statistical significance.
3.实验结果3.Experimental results
3.1不同浓度黄葵总黄酮乳膏对大鼠皮肤伤口面积的影响3.1 Effects of different concentrations of ambrosia total flavonoid cream on skin wound area in rats
为了考察不同浓度黄葵总黄酮乳膏促进伤口愈合的作用，构建了糖尿病大鼠全层皮肤创伤模型，观察各组大鼠伤口的愈合情况，分别在第0、3、7、9天涂抹给药前进行拍照，记录伤口变化情况，计算伤口面积，并进行统计学分析。In order to investigate the effect of different concentrations of total flavonoid cream on wound healing, a full-thickness skin wound model of diabetic rats was constructed. The wound healing of rats in each group was observed. The cream was applied on days 0, 3, 7, and 9 respectively. Take photos before medication, record the changes in the wound, calculate the wound area, and conduct statistical analysis.
图9A是不同时间点各组大鼠皮肤伤口面积代表性图，给药前(致伤后24h)，各组皮肤伤口处均有纤维蛋白凝块(“痂”)出现，存在少量渗出物，说明此时伤口处存在炎症反应；给药后第三天，各给药组伤口结痂开始***，痂皮变得干燥，干燥的痂皮能够起到抗菌的作用，从而对伤口起到一定的保护作用，并为角质形成细胞的增殖和迁移提供了临时的细胞外基质，促进伤口再上皮化；模型组伤口面积为原始伤口面积的99.48±8.76％，德湿洁水胶体组大鼠创面与给药组和模型组相比，创伤面积开始缩小，伤口面积为初始伤口面积的64.52±7.04％，0.5％、1％、2％黄葵总黄酮乳膏剂组伤口面积分别为初始伤口面积的106.80±9.37％、91.41±5.51％、98.38±8.74％(图9B)。给药后第7天，各组硬痂开始脱落，模型组伤口面积为初始伤口面积的47.62±10.68％，0.5％、1％、2％黄葵总黄酮乳膏剂组伤口面积分为其初始伤口面积的49.65±8.79％，27.38±6.61％，36.54±5.72％。其中以1％乳膏剂组促进伤口愈合最为明显，而德湿洁水胶体组伤口面积为初始伤口面积的17.86±4.68％，其促进伤口愈合的作用优于1％黄葵总黄酮乳膏剂组。给药后第九天，模型组伤口面积为原始伤口面积的12.05±4.03％；0.5％、1％、2％黄葵总黄酮乳膏剂组伤口面积分别为初始伤口面积的15.98±5.63％、10.04±5.64％、10.60±3.14％；德湿洁水胶体组伤口面积为初始伤口面积的8.03±4.70％。总体而言，阳性药德湿洁水胶体组伤口愈合明显，在3天和7天与模型相比都具有显著性差异，1％黄葵总黄酮乳膏剂组呈现治疗基本痊愈。Figure 9A is a representative diagram of the skin wound area of rats in each group at different time points. Before administration (24 hours after injury), fibrin clots ("scabs") appeared in the skin wounds of each group, and a small amount of exudate existed. , indicating that there is an inflammatory reaction in the wound at this time; on the third day after administration, the wound scabs in each administration group began to harden, and the scabs became dry. The dry scabs can play an antibacterial role, thus having an antibacterial effect on the wounds. It has a certain protective effect and provides a temporary extracellular matrix for the proliferation and migration of keratinocytes to promote wound re-epithelialization; the wound area in the model group was 99.48±8.76% of the original wound area, and the rats in the Deshijie hydrocolloid group Compared with the administration group and the model group, the wound area began to shrink. The wound area was 64.52±7.04% of the initial wound area. The wound area of the 0.5%, 1%, and 2% Huangkui total flavonoid cream groups was the initial wound area respectively. 106.80±9.37%, 91.41±5.51%, 98.38±8.74% (Figure 9B). On the 7th day after administration, the hard scabs in each group began to fall off. The wound area in the model group was 47.62±10.68% of the initial wound area. The wound area in the 0.5%, 1%, and 2% Huangkui total flavonoid cream groups was divided into 47.62±10.68% of the initial wound area. 49.65±8.79%, 27.38±6.61%, 36.54±5.72% of the area. Among them, the 1% cream group promoted wound healing the most, while the wound area of the Deshijie hydrocolloid group was 17.86±4.68% of the initial wound area, and its effect on promoting wound healing was better than that of the 1% Huangkui total flavonoids cream group. On the ninth day after administration, the wound area in the model group was 12.05±4.03% of the original wound area; the wound area in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 15.98±5.63% and 10.04% of the initial wound area, respectively. ±5.64%, 10.60±3.14%; the wound area of the Deshijie water colloid group was 8.03±4.70% of the initial wound area. Overall, the wound healing in the positive medicine Deshijie hydrocolloid group was obvious, with significant differences compared with the model on 3 days and 7 days. The 1% Huangkui total flavonoids cream group showed basic recovery after treatment.
3.2HE染色结果分析3.2 Analysis of HE staining results
为了进一步评价不同浓度黄葵总黄酮乳膏促进糖尿病伤口愈合的效果，我们造模9天后的大鼠皮肤组织进行了HE染色，以观察组织微观层面的改变。HE染色法是常用的石蜡切片染色方法之一，它以苏木精、依红等为染料进行染色，故也叫苏木精-伊红染色法(hematoxylin-eosin staining)。苏木精染液是碱性染料，可将细胞核染成紫蓝色，伊红则将细胞外基质和细胞质中的成分染成粉红色，其它结构则呈现出不同的色调和这些颜色的组合。此外，染色后组织的整体颜色模式显示了细胞的总体布局和分布，并提供了组织样本结构的总体概况，可以观察创面愈合过程中成纤维细胞、毛细血管数量及分布、炎症细胞等情况。In order to further evaluate the effect of different concentrations of ambrosia total flavonoid cream on promoting diabetic wound healing, we performed HE staining on rat skin tissue 9 days after the model was established to observe microscopic changes in the tissue. HE staining is one of the commonly used paraffin section staining methods. It uses hematoxylin, eosin and other dyes as dyes, so it is also called hematoxylin-eosin staining. Hematoxylin stain is a basic dye that stains cell nuclei purple-blue, eosin stains components of the extracellular matrix and cytoplasm pink, and other structures appear in different hues and combinations of these colors. In addition, the overall color pattern of the stained tissue shows the overall layout and distribution of cells and provides an overall overview of the structure of the tissue sample. It is possible to observe the number and distribution of fibroblasts, capillaries, inflammatory cells, etc. during the wound healing process.
如图10A所示，组织切片镜检结果表明，正常组大鼠皮肤组织可见完整的角质形成细胞层，皮肤附属器，胶原纤维交联排列成平行束，有少量的成纤维细胞和炎性细胞，对比之下，模型组大鼠皮肤组织排列松散，可见角质形成细胞开始增殖，但仍未迁移至伤口中心，整体皮肤结构紊乱，新生表皮较菲薄，新生的肉芽组织较少，有炎性细胞以及成纤维细胞向伤口处迁移，其中炎性细胞的数目相对较多，表明模型组大鼠主要处于伤口愈合的第1阶段：炎症期。而不同浓度黄葵总黄酮乳膏剂组和德湿洁水胶体组伤口处的角质形成细胞层已经增殖、迁移至伤口中心，角质形成细胞层下的肉芽组织中的胶原纤维排列紧密，可见较多的成纤维细胞生长，新生毛细血管散在可见，新生表皮较厚，炎性细胞相对较少，表明各给药组大鼠皮肤伤口已经进入伤口愈 合的第2-3阶段：增殖和重塑期。图10B是大鼠表皮厚度的定量统计图，空白对照组表皮厚度为26.83±2.65μm，模型组表皮厚度显著增加，为87.72±1.95μm(P<0.01vs.对照组)，给与德洁湿水胶体组表皮厚度为112.70±7.58μm，0.5％黄葵总黄酮乳膏剂组、1％黄葵总黄酮乳膏剂组、2％黄葵总黄酮乳膏剂组表皮厚度分别为99.97±3.06μm、121.00±2.35μm、121.10±5.64μm。结果显示1％黄葵总黄酮乳膏剂组、2％黄葵总黄酮乳膏剂组、德湿洁水胶体组大鼠表皮厚度相对于模型组组大鼠显著增高，表明其伤口处再上皮化程度高于模型组、可有效促进大鼠全层皮肤伤口愈合。As shown in Figure 10A, the results of tissue section microscopy showed that the skin tissue of rats in the normal group showed a complete keratinocyte layer, skin appendages, collagen fibers cross-linked and arranged into parallel bundles, and a small amount of fibroblasts and inflammatory cells. , In contrast, the skin tissue of the rats in the model group was loosely arranged, and it was seen that keratinocytes began to proliferate, but have not yet migrated to the center of the wound. The overall skin structure was disordered, the new epidermis was thinner, there was less new granulation tissue, and there were inflammatory cells. And fibroblasts migrated to the wound, among which the number of inflammatory cells was relatively large, indicating that the rats in the model group were mainly in the first stage of wound healing: the inflammatory phase. However, the keratinocyte layer in the wound of different concentrations of total flavonoids cream group and Deshijie hydrocolloid group has proliferated and migrated to the center of the wound. The collagen fibers in the granulation tissue under the keratinocyte layer are closely arranged and more visible. Fibroblasts grew, new capillaries were scattered and visible, the new epidermis was thicker, and there were relatively few inflammatory cells, indicating that the skin wounds of rats in each treatment group had entered the wound healing process. Stages 2-3 of synthesis: proliferation and remodeling. Figure 10B is a quantitative statistical diagram of the epidermal thickness of rats. The epidermal thickness of the blank control group was 26.83±2.65 μm. The epidermal thickness of the model group increased significantly to 87.72±1.95 μm (P<0.01vs. control group). Dejie Wet was given to The epidermal thickness of the hydrocolloid group was 112.70±7.58 μm, and the epidermal thickness of the 0.5% total flavonoids cream group, 1% total flavonoids cream group, and 2% total flavonoids cream group were 99.97±3.06 μm and 121.00 μm, respectively. ±2.35μm, 121.10±5.64μm. The results showed that the epidermal thickness of rats in the 1% total flavonoids cream group, 2% total flavonoids cream group, and Deshijie hydrocolloid group was significantly higher than that of the rats in the model group, indicating the degree of re-epithelialization of the wound. Higher than that of the model group, it can effectively promote full-thickness skin wound healing in rats.
3.3 Masson染色结果分析3.3 Analysis of Masson staining results
Masson染色是组织学中常用的染色方法，可将胶原纤维染成蓝色，角蛋白和肌肉纤维染成红色，用于评价创面胶原纤维形成情况。Masson staining is a commonly used staining method in histology. It can dye collagen fibers blue and keratin and muscle fibers red, and is used to evaluate the formation of collagen fibers in wounds.
图11A可以看到各组治疗9天后的胶原沉积情况，颜色越深代表胶原沉积的量越多，从图中可以直观地看出模型组蓝染较浅，德湿洁水胶体组和不同浓度黄葵总黄酮乳膏剂组蓝染有不同程度的加深，其中1％黄葵总黄酮乳膏剂组较其他组蓝色部分面积更大，颜色更深，分布更为均匀。图11B是胶原面积百分比的量化统计图，空白对照组胶原面积百分比为36.02±2.95％，模型组胶原面积百分比显著下降，为14.74±1.64％(P<0.01vs.对照组)，德洁湿水胶体组胶原面积百分比为23.24±1.83％，0.5％黄葵总黄酮乳膏剂组、1％黄葵总黄酮乳膏剂组、2％黄葵总黄酮乳膏剂组胶原面积百分比分别为19.33±2.46％、27.45±1.74％、22.76±2.48％，差异性分析结果表明德湿洁水胶体组与模型组相比有显著差异(P＜0.05)，不同浓度黄葵总黄酮乳膏剂涂抹给药组中，1％黄葵总黄酮乳膏剂组的胶原含量最多，与模型组相比具有显著差异(P＜0.01)。Figure 11A shows the collagen deposition in each group after 9 days of treatment. The darker the color, the greater the amount of collagen deposition. It can be intuitively seen from the figure that the model group has lighter blue staining, and the Deshiji hydrocolloid group and different concentrations The blue dyeing in the total flavonoids cream group of Huang Kui was deepened to varying degrees. Among them, the blue area of the 1% Total Flavonoids Cream group was larger, darker and more evenly distributed than in the other groups. Figure 11B is a quantitative statistical diagram of the collagen area percentage. The collagen area percentage in the blank control group was 36.02±2.95%. The collagen area percentage in the model group dropped significantly to 14.74±1.64% (P<0.01vs. control group). Dejie Wet Water The collagen area percentage of the colloid group was 23.24±1.83%, and the collagen area percentages of the 0.5% total flavonoid cream group, 1% total flavonoid cream group, and 2% total flavonoid cream group were 19.33±2.46%, respectively. 27.45±1.74%, 22.76±2.48%. The difference analysis results showed that there was a significant difference between the Deshijie hydrocolloid group and the model group (P<0.05). In the different concentrations of total flavonoid cream smear and administration groups, 1 The collagen content in the % ambrosia total flavonoids cream group was the highest, which was significantly different from the model group (P<0.01).
3.4 CD31和VEGF mRNA水平的变化3.4 Changes in CD31 and VEGF mRNA levels
在正常状态下，血管处于稳定状态，且得到充分灌注，为组织输送充足的营养物质和氧气。一旦组织发生损伤，局部缺血缺氧，伤口部位会产生大量促血管生成因子，从而促进微血管的生长。为了确定血管标志物CD31和促血管生成因子(VEGF)在基因水平上的变化，在给药后第9天进行RT-qPCR检测CD31和VEGF mRNA的表达。Under normal conditions, blood vessels are in a stable state and fully perfused, delivering sufficient nutrients and oxygen to tissues. Once tissue damage occurs and local ischemia and hypoxia occur, a large amount of pro-angiogenic factors will be produced at the wound site, thereby promoting the growth of microvessels. In order to determine the changes in the vascular markers CD31 and pro-angiogenic factor (VEGF) at the gene level, RT-qPCR was performed to detect the expression of CD31 and VEGF mRNA on the 9th day after administration.
图12A表明造模组Cd 31mRNA的表达显著下降，从0.22±0.03下降至0.10±0.01(P<0.01)。给药后CD31mRNA的表达存在不同程度的增加，德洁湿水胶体组CD31mRNA的表达为0.17±0.02，0.5％、1％、2％黄葵总黄酮乳膏剂组CD31mRNA的表达分别为0.10±0.01、0.21±0.02、0.12±0.02，其中1％黄葵总黄酮乳膏剂涂抹给药增强CD31mRNA的表达最为显著，具统计学差异(P<0.01)，其余各组均无统计学差异。图12B表明造模后Vegf mRNA的表达下降，从0.25±0.07下降至0.14±0.04，给药后VEGF mRNA的表达存在不同程度的增加，与模型组相比，德洁湿水胶体组VEGF mRNA的表达为0.23±0.08，0.5％、1％、2％黄葵总黄酮乳膏剂组VEGF mRNA的表达分别为0.10±0.02、0.21±0.04、0.14±0.04，但给药组和模型组相比，未见显著性差异。Figure 12A shows that the expression of Cd 31mRNA in the modeling group decreased significantly, from 0.22±0.03 to 0.10±0.01 (P<0.01). The expression of CD31mRNA increased to varying degrees after administration. The expression of CD31mRNA in the Dejie wet hydrocolloid group was 0.17±0.02, and the expression of CD31mRNA in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 0.10±0.01, respectively. 0.21±0.02, 0.12±0.02. Among them, the application of 1% ambrosia total flavonoid cream enhanced the expression of CD31mRNA most significantly, with a statistical difference (P<0.01). There was no statistical difference in the other groups. Figure 12B shows that the expression of Vegf mRNA decreased after modeling, from 0.25±0.07 to 0.14±0.04. The expression of VEGF mRNA increased to varying degrees after administration. Compared with the model group, the expression of VEGF mRNA in the Dejie wet hydrocolloid group The expression of VEGF mRNA in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups was 0.10±0.02, 0.21±0.04, and 0.14±0.04 respectively, but compared with the model group, there was no See significant differences.
3.5皮肤组织免疫荧光染色结果分析3.5 Analysis of immunofluorescence staining results of skin tissue
CD31是血管内皮细胞的典型标志物，可以用来衡量血管生成的情况，利用抗CD31抗体对组织切片进行免疫组化染色，可以非常直观且客观地得到新生组织内CD31的分布、密度和含量，继而分析其微循环建立成功与否。以平均荧光强度为指标，选取不同切片的多个视野进行拍照，以评价不同组的血管生成情况。CD31 is a typical marker of vascular endothelial cells, which can be used to measure angiogenesis. Using anti-CD31 antibodies to immunohistochemically stain tissue sections, the distribution, density and content of CD31 in new tissues can be obtained very intuitively and objectively. Then analyze whether the microcirculation is successfully established. Using the average fluorescence intensity as an indicator, multiple fields of view from different sections were selected to take pictures to evaluate the angiogenesis in different groups.
如图13A所示，用DAPI(蓝)进行细胞核的定位，抗CD31抗体(红)定位血管内皮细胞，可得到CD31在组织中的明确分布。模型组创面新生血管数量少，CD31呈弱阳性表达，相对荧光强度为对照组的41.63±7.46％(P<0.05)，德洁湿水胶体组CD31表达高于模型组，相对荧光强度为对照组的73.75±18.13％，但无统计学差异。不同浓 度黄葵总黄酮乳膏剂组CD31表达均高于模型组，0.5％、1％、2％黄葵总黄酮乳膏剂组相对荧光强度分别为对照组的49.49±13.50％、70.92±9.53％、47.69±8.99％，但均未显示统计学差异。As shown in Figure 13A, DAPI (blue) is used to localize cell nuclei, and anti-CD31 antibody (red) is used to localize vascular endothelial cells, and a clear distribution of CD31 in tissues can be obtained. The number of new blood vessels in the wound of the model group was small, and CD31 showed weak positive expression. The relative fluorescence intensity was 41.63±7.46% of that of the control group (P<0.05). The expression of CD31 in the Dejie wet hydrocolloid group was higher than that of the model group, and the relative fluorescence intensity was 41.63±7.46% of that of the control group. 73.75±18.13%, but there is no statistical difference. Different concentrations The expression of CD31 in the total flavonoids cream group was higher than that in the model group. The relative fluorescence intensities of the 0.5%, 1%, and 2% total flavonoid cream groups were 49.49±13.50%, 70.92±9.53%, and 47.69% of those in the control group, respectively. ±8.99%, but none showed statistical differences.
3.6血管内皮生长因子(VEGF)蛋白水平的变化3.6 Changes in vascular endothelial growth factor (VEGF) protein levels
糖尿病创面难以愈合的重要原因是高血糖以及持续的炎症反应导致的微循环灌注不足。因此，其愈合过程中最关键的一步是新生血管的重建，微循环的有效建立使修复过程更加高效，加速毛细血管间营养物质的交换，以及激活的白细胞的迁徙。VEGF属于生长因子的一个亚家族，即血小板衍生生长因子家族，是参与血管形成和血管生成的重要信号蛋白，能为血管内皮的迀移及血管形成提供基质，促进溃疡创面的新生毛细血管增生，刺激肉芽组织的生长，从而促进创面的愈合。An important reason why diabetic wounds are difficult to heal is insufficient microcirculatory perfusion caused by hyperglycemia and sustained inflammatory response. Therefore, the most critical step in the healing process is the reconstruction of new blood vessels. The effective establishment of microcirculation makes the repair process more efficient, accelerates the exchange of nutrients between capillaries, and the migration of activated leukocytes. VEGF belongs to a subfamily of growth factors, namely the platelet-derived growth factor family. It is an important signaling protein involved in blood vessel formation and angiogenesis. It can provide a matrix for the migration of vascular endothelium and blood vessel formation, and promote the proliferation of new capillaries in ulcer wounds. Stimulates the growth of granulation tissue, thereby promoting wound healing.
如图14所示，模型组VEGF蛋白水平与空白组相比有所下降，在给药后存在不同程度的增加，模型组VEGF蛋白水平为44.93±3.69pg/ml，德洁湿水胶体组VEGF蛋白水平为114.30±13.11pg/ml，0.5％、1％、2％黄葵总黄酮乳膏剂组VEGF蛋白水平分别为77.74±5.90pg/ml、98.14±11.53pg/ml、99.29±17.35pg/ml。数据表明，各给药组以德洁湿水胶体组增加VEGF蛋白水平表达最为显著(P<0.01)，1％和2％黄葵总黄酮乳膏剂组的效应弱于德洁湿水胶体，但也均能够显著增加皮肤组织VEGF蛋白水平(P<0.01)，而0.5％黄葵总黄酮乳膏剂组VEGF蛋白表达水平与模型组相比有所增加，但无统计学差异。As shown in Figure 14, the VEGF protein level in the model group decreased compared with the blank group, and increased to varying degrees after administration. The VEGF protein level in the model group was 44.93±3.69pg/ml, and the VEGF level in the Dejie wet hydrocolloid group was 44.93±3.69pg/ml. The protein level was 114.30±13.11pg/ml, and the VEGF protein levels in the 0.5%, 1%, and 2% ambrosia total flavonoid cream groups were 77.74±5.90pg/ml, 98.14±11.53pg/ml, and 99.29±17.35pg/ml respectively. . The data showed that among the various administration groups, the Dejie Wet Hydrocolloid group increased the expression of VEGF protein levels most significantly (P<0.01). The effects of the 1% and 2% Huangkui total flavonoid cream groups were weaker than those of Dejie Wet Hydrocolloid, but They were also able to significantly increase the VEGF protein level in skin tissue (P<0.01). The VEGF protein expression level in the 0.5% ambrosia total flavonoid cream group increased compared with the model group, but there was no statistical difference.
4.实验结论4.Experimental conclusion
本实验使用STZ诱导糖尿病模型，利用无菌8-mm Biopsy Punch构建大鼠全层皮肤切除模型，以德洁湿水胶体为阳性对照药物，以空白乳膏为模型组空白对照药物，考察黄葵总黄酮对糖尿病溃疡大鼠的治疗作用。In this experiment, an STZ-induced diabetes model was used to construct a rat full-thickness skin excision model using sterile 8-mm Biopsy Punch. Dejie wet hydrocolloid was used as the positive control drug, and blank cream was used as the blank control drug in the model group to investigate Huang Kui. Therapeutic effect of total flavonoids on diabetic ulcer rats.
结果表明，黄葵总黄酮可加快伤口再上皮化，促进胶原纤维沉积，提高VEGF蛋白水平的表达增加，促进糖尿病大鼠全层皮肤伤口的愈合。此外，黄葵总黄酮还可以通过增加CD31的表达促进新生血管的生成，进而加快糖尿病溃疡伤口愈合进程。综上所述，黄葵总黄酮具有一定的治疗糖尿病溃疡的作用。The results showed that total flavonoids from ambrosia can accelerate wound re-epithelialization, promote collagen fiber deposition, increase the expression of VEGF protein levels, and promote the healing of full-thickness skin wounds in diabetic rats. In addition, the total flavonoids of ambrosia can also promote the formation of new blood vessels by increasing the expression of CD31, thereby accelerating the healing process of diabetic ulcer wounds. In summary, the total flavonoids of ambrosia have certain effects in treating diabetic ulcers.
实施例2-A2，实施例4-2，实施例9-2，实施例19-1，实施例19-2实验样品也具备与本实施例中黄葵总黄酮1上述各方面基本一致的作用或效果。The experimental samples of Example 2-A2, Example 4-2, Example 9-2, Example 19-1, and Example 19-2 also have functions that are basically consistent with the above-mentioned aspects of the total flavonoids 1 of ambrosia in this example. or effect.
实施例24黄蜀葵花提取物肠溶制剂Example 24 Enteric-coated preparation of marshmallow flower extract
实施例24-1一种黄蜀葵花提取物肠溶制剂的制备Example 24-1 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的制备1. Preparation of marshmallow flower extract
取黄蜀葵花1kg，加18倍量的乙醇，加热回流1小时，滤过，滤液减压回收乙醇，浓缩，调节pH至6.0，静置，除去上层的油层，真空干燥，粉碎，得到黄蜀葵花提取物，测得黄蜀葵花总黄酮7种成分的总含量为9％。Take 1kg of marshmallow flowers, add 18 times the amount of ethanol, heat and reflux for 1 hour, filter, recover the filtrate under reduced pressure to recover the ethanol, concentrate, adjust the pH to 6.0, let it stand, remove the upper oil layer, vacuum dry, and pulverize to obtain marshmallow flower extract The total content of 7 total flavonoids in marshmallow flowers was measured to be 9%.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-1

Table 24-1

3.肠溶片或肠溶胶囊的制备3. Preparation of enteric-coated tablets or enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，乳糖、低取代羟丙纤维素、聚乙二醇、月桂醇硫酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; lactose, low-substituted hydroxypropylcellulose, polyethylene glycol, and magnesium lauryl sulfate are crushed and passed through a 60-mesh sieve for later use; prepare an 85% ethanol solution for later use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的乳糖、低取代羟丙纤维素、聚乙二醇放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
3)总混、压片：加入处方量的月桂醇硫酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium lauryl sulfate, put it in the mixing machine and mix for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型欧巴代200mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
5)包衣：素片包肠溶衣，增重4％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
实施例24-2一种黄蜀葵花提取物肠溶制剂的制备Example 24-2 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的制备1. Preparation of marshmallow flower extract
将1kg黄蜀葵花粉碎至粗粉，用15倍的60％乙醇渗漉提取，得黄蜀葵花提取液；黄蜀葵花提取液去除乙醇，水稀释，经活性炭吸附后，用正丁醇连续逆流萃取，萃取的料液比(也即萃取剂:提取液)为1.5:1，萃取的进样速度为40mL/min，萃取的萃取机转速为40Hz，萃取的萃取级数为2级，将萃取液在40℃下减压回收溶剂后，经D101大孔树脂处理，大孔树脂的处理工艺为：大孔树脂径高比为1:6，上样液(水溶液)浓度为0.15g生药/mL，上样液体积为7BV，以2BV/h流速上样，用6BV纯水和3BV 5％乙醇以2BV/h流速先后进行除杂，用4BV 60％乙醇以2BV/h流速进行洗脱得到洗脱液，然后将该洗脱液在50℃下减压回收乙醇后得到黄蜀葵花黄酮提取物，测试含量根据含量测定结果通过加入法，调整7种成分含量，制得黄蜀葵花提取物，7种成分占总固形物比例为83％。Crush 1kg of marshmallow flowers to coarse powder, and extract by percolation with 15 times of 60% ethanol to obtain marshmallow flower extract; remove the ethanol from the marshmallow flower extract, dilute it with water, adsorb it with activated carbon, and use n-butanol for continuous countercurrent extraction. The material-to-liquid ratio (that is, extraction agent:extraction liquid) is 1.5:1, the injection speed of the extraction is 40mL/min, the extraction machine speed of the extraction is 40Hz, the extraction level of the extraction is 2, and the extraction liquid is placed at 40 After recovering the solvent under reduced pressure at ℃, it is treated with D101 macroporous resin. The processing technology of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:6, the concentration of the sample solution (aqueous solution) is 0.15g crude drug/mL, and the sample is loaded The liquid volume is 7BV, load the sample at a flow rate of 2BV/h, use 6BV pure water and 3BV 5% ethanol at a flow rate of 2BV/h to remove impurities, and use 4BV 60% ethanol at a flow rate of 2BV/h to elute to obtain the eluate. Then, the eluate was recovered under reduced pressure at 50°C to recover ethanol to obtain marshmallow flower flavonoid extract. The tested content was based on the content measurement results and the content of 7 components was adjusted by the addition method to obtain marshmallow flower extract. The 7 components accounted for the total The solid content ratio is 83%.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-2
Table 24-2
3.肠溶片、肠溶胶囊制备方法3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，乳糖、低取代羟丙纤维素、聚乙二醇、月桂醇硫酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; lactose, low-substituted hydroxypropylcellulose, polyethylene glycol, and magnesium lauryl sulfate are crushed and passed through a 60-mesh sieve for later use; prepare an 85% ethanol solution for later use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的乳糖、低取代羟 丙纤维素、聚乙二醇放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulating, and whole graining: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose and low substituted hydroxyl. Put cellulose propylene glycol and polyethylene glycol into a wet mixing granule machine and dry mix for 5-10 minutes, then add an appropriate amount of 85% ethanol solution to make wet granules; pass the wet granules through a 20-mesh screen and then dry them at an electric constant temperature Dry in a blast drying oven to make the moisture content of the intermediate particles less than or equal to 3%; pass the dry particles through a 20-mesh screen to make them whole;
3)总混、压片：加入处方量的月桂醇硫酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium lauryl sulfate, put it in the mixing machine and mix for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型欧巴代200mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
5)包衣：素片包肠溶衣，增重4％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
实施例24-3一种黄蜀葵花提取物肠溶制剂的制备Example 24-3 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的1. Marshmallow flower extract
将黄蜀葵花1kg加80％乙醇适量，提取1次，提取2小时，合并提取液，滤过，滤液浓缩成60℃时相对密度为1.13～1.16的浓缩液，加适量水水沉24h，抽滤，浓缩至相应体积，通过大孔吸附树脂柱，采用醇水梯度洗脱，水洗脱3个柱体积，10％及30％乙醇各洗脱3个柱体积，70％乙醇洗脱1个柱体积。洗脱液浓缩、干燥、粉碎后得到黄蜀葵花总黄酮提取物，其总黄酮含量为65％。Add an appropriate amount of 80% ethanol to 1kg of marshmallow flowers, extract once, extract for 2 hours, combine the extracts, filter, and concentrate the filtrate into a concentrated solution with a relative density of 1.13 to 1.16 at 60°C. Add an appropriate amount of water and let it settle for 24 hours, and filter with suction. , concentrate to the corresponding volume, pass through the macroporous adsorption resin column, use alcohol-water gradient elution, water elutes 3 column volumes, 10% and 30% ethanol each elutes 3 column volumes, 70% ethanol elutes 1 column volume. The eluate was concentrated, dried and pulverized to obtain a total flavonoid extract of marshmallow flowers, with a total flavonoid content of 65%.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-3
Table 24-3
3.肠溶片、肠溶胶囊制备方法3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，乳糖、低取代羟丙纤维素、聚乙二醇、月桂醇硫酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; lactose, low-substituted hydroxypropylcellulose, polyethylene glycol, and magnesium lauryl sulfate are crushed and passed through a 60-mesh sieve for later use; prepare an 85% ethanol solution for later use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的乳糖、低取代羟丙纤维素、聚乙二醇放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
3)总混、压片：加入处方量的月桂醇硫酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium lauryl sulfate, put it in the mixing machine and mix for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型欧巴代200mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 200mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
5)包衣：素片包肠溶衣，增重4％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
实施例24-4一种黄蜀葵花提取物肠溶制剂的制备 Example 24-4 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的制备1. Preparation of marshmallow flower extract
如实施例24-1制备得到的黄蜀葵花提取物。The marshmallow flower extract prepared in Example 24-1.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-4
Table 24-4
3.肠溶片、肠溶胶囊制备方法3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，乳糖、低取代羟丙纤维素、聚乙二醇、月桂醇硫酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; lactose, low-substituted hydroxypropylcellulose, polyethylene glycol, and magnesium lauryl sulfate are crushed and passed through a 60-mesh sieve for later use; prepare an 85% ethanol solution for later use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的乳糖、低取代羟丙纤维素、聚乙二醇放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
3)总混、压片：加入处方量的月桂醇硫酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium lauryl sulfate, put it in the mixing machine and mix for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型欧巴代180mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 180mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
5)包衣：素片包肠溶衣，增重5％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 5% to obtain enteric-coated tablets; or 2) the medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
实施例24-5一种黄蜀葵花提取物肠溶制剂的制备Example 24-5 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵葵花提取物的制备1. Preparation of Hollyhock Sunflower Extract
如实施例24-1制备得到的黄蜀葵花提取物。The marshmallow flower extract prepared in Example 24-1.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-5
Table 24-5
3.肠溶片、肠溶胶囊制备方法 3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，乳糖、低取代羟丙纤维素、聚乙二醇、月桂醇硫酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; lactose, low-substituted hydroxypropylcellulose, polyethylene glycol, and magnesium lauryl sulfate are crushed and passed through a 60-mesh sieve for later use; prepare an 85% ethanol solution for later use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的乳糖、低取代羟丙纤维素、聚乙二醇放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of lactose, low-substituted hydroxypropylcellulose, and polyethylene glycol, and put them into a wet mixing granulator to dry mix for 5-10 minutes. Minutes, add an appropriate amount of 85% ethanol solution to prepare wet granules; pass the wet granules through a 20-mesh screen and then dry them in an electric constant-temperature blast drying oven to make the moisture content of the intermediate granules less than or equal to 3%; dry granules pass through a 20-mesh screen. Mesh screen for whole grains;
3)总混、压片：加入处方量的月桂醇硫酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium lauryl sulfate, put it in the mixing machine and mix for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型欧巴代210mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film-coated pre-suspension enteric Opadry 210mg and slowly add it to 800mL of purified water under stirring. Stir for 15-30 minutes and then pass through an 80-mesh sieve. spare;
5)包衣：素片包肠溶衣，增重3％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 3% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
对比例24-1一种黄蜀葵花提取物肠溶制剂的制备Comparative Example 24-1 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的制备1. Preparation of marshmallow flower extract
如实施例24-1制备得到的黄蜀葵花提取物。The marshmallow flower extract prepared in Example 24-1.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-6
Table 24-6
3.肠溶片、肠溶胶囊制备方法3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，微晶纤维素、交联聚乙烯吡咯烷酮、羧甲基纤维素钠、硬脂酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; microcrystalline cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethylcellulose, and magnesium stearate are crushed and passed through a 60-mesh sieve; prepare 85% ethanol. solution, ready for use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的微晶纤维素、交联聚乙烯吡咯烷酮、羧甲基纤维素钠放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, and sodium carboxymethylcellulose and put them into a wet mixing granulator. Mix for 5-10 minutes, then add an appropriate amount of 85% ethanol solution to prepare wet particles; pass the wet particles through a 20-mesh screen and dry them in an electric constant-temperature blast drying oven to make the moisture of the intermediate particles less than or equal to 3%; dry The particles are passed through a 20-mesh sieve to make them whole;
3)总混、压片：加入处方量的硬脂酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium stearate and mix in the mixing machine for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型羟丙甲基纤维素320mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film coating pre-suspension enteric hydroxypropyl methylcellulose 320 mg and slowly add it to 800 mL of purified water under stirring. Stir for 15-30 minutes and then wait for 80 seconds. Mesh sieve, spare;
5)包衣：素片包肠溶衣，增重4％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 4% to obtain enteric-coated tablets; or 2) The medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
对比例24-2一种黄蜀葵花提取物肠溶制剂的制备Comparative Example 24-2 Preparation of an enteric preparation of marshmallow flower extract
1.黄蜀葵花提取物的制备 1. Preparation of marshmallow flower extract
如实施例24-1方法制备得到。Prepared by the method of Example 24-1.
2.处方组成由下表所示2. The composition of the prescription is shown in the table below
表24-7
Table 24-7
3.肠溶片、肠溶胶囊制备方法3. Preparation method of enteric-coated tablets and enteric-coated capsules
1)过筛：黄蜀葵花提取物粉碎过八十目筛，微晶纤维素、交联聚乙烯吡咯烷酮、羧甲基纤维素钠、硬脂酸镁粉碎过六十目筛备用；配制85％乙醇溶液，备用；1) Sieve: marshmallow flower extract is crushed and passed through an 80-mesh sieve; microcrystalline cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethylcellulose, and magnesium stearate are crushed and passed through a 60-mesh sieve; prepare 85% ethanol. solution, ready for use;
2)干混、制粒、整粒：取处方量的黄蜀葵花提取物，和处方量的微晶纤维素、交联聚乙烯吡咯烷酮、羧甲基纤维素钠放入湿法混合颗粒机中干混5-10分钟，在加入适量的85％乙醇溶液制成湿颗粒；将湿颗粒过20目筛网湿整理后于电热恒温鼓风干燥箱干燥，使中间体颗粒水分小于等于3％；干颗粒过二十目筛网整粒；2) Dry mixing, granulation, and whole granulation: Take the prescribed amount of marshmallow flower extract, and the prescribed amount of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, and sodium carboxymethylcellulose and put them into a wet mixing granulator. Mix for 5-10 minutes, then add an appropriate amount of 85% ethanol solution to prepare wet particles; pass the wet particles through a 20-mesh screen and dry them in an electric constant-temperature blast drying oven to make the moisture of the intermediate particles less than or equal to 3%; dry The particles are passed through a 20-mesh sieve to make them whole;
3)总混、压片：加入处方量的硬脂酸镁，置总混机中混合10-15分钟；测定颗粒含量，计算片重，压片，制得素片；3) Total mixing and tableting: Add the prescribed amount of magnesium stearate and mix in the mixing machine for 10-15 minutes; measure the particle content, calculate the tablet weight, and compress the tablets to prepare plain tablets;
4)配制肠溶衣液：取处方量肠溶型薄膜包衣预混悬剂肠溶型羟丙甲基纤维素240mg在搅拌下缓慢加入到纯化水800mL中，搅拌15-30分钟后过80目筛，备用；4) Prepare enteric coating solution: Take the prescribed amount of enteric film coating pre-suspension enteric hydroxypropyl methylcellulose 240 mg and slowly add it to 800 mL of purified water under stirring. Stir for 15-30 minutes and then wait for 80 seconds. Mesh sieve, spare;
5)包衣：素片包肠溶衣，增重5％，得肠溶片；或者2)中黄蜀葵花颗粒整粒、抛光，装入肠溶胶囊壳中，制得肠溶胶囊。5) Coating: The plain tablets are coated with enteric coating, and the weight is increased by 5% to obtain enteric-coated tablets; or 2) the medium-sized marshmallow flower particles are whole, polished, and put into enteric-coated capsule shells to prepare enteric-coated capsules.
实施例24-6产品溶出曲线测试(pH1.2-pH7.8)Example 24-6 Product Dissolution Curve Test (pH1.2-pH7.8)
实施例24-1～24-5及对比例24-1～24-2制成的肠溶片依次在人工胃液(pH1.2盐酸溶液)溶出2小时；人工肠液(pH6.8磷酸缓冲液)溶出1小时；人工结肠液(pH7.8的磷酸缓冲液)溶出3小时，条件下的溶出度考察。The enteric-coated tablets prepared in Examples 24-1 to 24-5 and Comparative Examples 24-1 to 24-2 were sequentially dissolved in artificial gastric juice (pH 1.2 hydrochloric acid solution) for 2 hours; artificial intestinal juice (pH 6.8 phosphate buffer) Dissolution was conducted for 1 hour; artificial colon fluid (phosphate buffer solution with pH 7.8) was dissolved for 3 hours, and the dissolution was investigated under the conditions.
测试具体方法：首先在溶出杯中加入盐酸溶液(pH1.2)900mL，考察肠溶制剂在2小时内的释放量；然后弃去酸液，立即加入温度为37℃±0.5℃的磷酸盐缓冲液(pH6.8)，测定肠溶制剂在缓冲液中的释放量；然后弃去缓冲液液，立即加入温度为37℃±0.5℃的磷酸盐缓冲液(pH7.8)，测定肠溶制剂在缓冲液中的释放量。The specific test method: first add 900mL of hydrochloric acid solution (pH 1.2) into the dissolution cup and examine the release amount of the enteric preparation within 2 hours; then discard the acid solution and immediately add phosphate buffer with a temperature of 37℃±0.5℃ solution (pH 6.8), and measure the release amount of the enteric preparation in the buffer; then discard the buffer solution, and immediately add phosphate buffer (pH 7.8) with a temperature of 37°C ± 0.5°C, and measure the enteric preparation. Release in buffer.
释放度的检测是检测每片中含黄葵总黄酮以金丝桃苷、芦丁、异槲皮苷、棉皮素-8-O-β-D-葡萄糖醛酸苷、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素7种成分总量计。The release test is to detect the total flavonoids contained in each tablet, including hyperoside, rutin, isoquercetin, gossipin-8-O-β-D-glucuronide, myricetin, and quercetin. The total amount of 7 ingredients including glucoside-3'-O-glucoside and quercetin.
表24-8

Table 24-8

在上述实验中，pH1.2的酸性介质是模拟胃酸环境，pH6.8介质是模拟肠道环境，pH7.8介质是模拟结肠环境。In the above experiment, the acidic medium with pH 1.2 simulates the gastric acid environment, the medium with pH 6.8 simulates the intestinal environment, and the medium with pH 7.8 simulates the colon environment.
通过表24-8中数据可以看出，在pH1.2的酸性介质中，实施例24-1～24.5在120min时溶出度为小于1％，对比例1和2分别在120min时溶出度达到为4％。It can be seen from the data in Table 24-8 that in the acidic medium of pH 1.2, the dissolution of Examples 24-1 to 24.5 at 120 min is less than 1%, and the dissolution of Comparative Examples 1 and 2 at 120 min respectively reaches 4%.
在pH6.8的介质中，实施例溶出度不超过10％，而对比例溶出度达到20％以上。In a medium with pH 6.8, the dissolution rate of the examples does not exceed 10%, while the dissolution rate of the comparative examples reaches more than 20%.
在pH7.8的介质中，实施例24-1～24.5溶出速度快，不存在时滞效应，210min(第4h)时溶出度达到90％以上，性能优越。且实施例24-1～24.3在210min溶出度达到95％以上。In a medium with pH 7.8, Examples 24-1 to 24.5 have a fast dissolution rate and no time lag effect. The dissolution rate reaches more than 90% at 210 minutes (4th hour), indicating excellent performance. And the dissolution rate of Examples 24-1 to 24.3 reached more than 95% in 210 minutes.
实施例24-7稳定性试验Example 24-7 Stability test
对本发明实施例24-1～24-3制成的肠溶片进行稳定性考察，考察条件为高温(40℃)、高湿(RH75％±5％)、光照实验(4500lx±500lx)，分别在以上考察条件下放置10天，在0、5、10天进行取样测定其释放度，测定方法为紫外分光光度法。释放度1是指模拟人工肠液(pH6.8磷酸盐缓冲溶液)中的释放度；释放度2是指模拟人工结肠液(pH7.2-7.8磷酸盐缓冲溶液)中的释放度。The stability of the enteric-coated tablets prepared in Examples 24-1 to 24-3 of the present invention was investigated. The examination conditions were high temperature (40°C), high humidity (RH75%±5%), and light experiment (4500lx±500lx), respectively. Leave it for 10 days under the above inspection conditions, and take samples to measure its release on days 0, 5, and 10. The measurement method is UV spectrophotometry. Release degree 1 refers to the release degree in simulated artificial intestinal fluid (pH 6.8 phosphate buffer solution); release degree 2 refers to the release degree in simulated artificial colon fluid (pH 7.2-7.8 phosphate buffer solution).
释放度的检测是检测每片中含黄葵总黄酮以金丝桃苷、芦丁、异槲皮苷、棉皮素-8-O-β-D-葡萄糖醛酸苷、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素7种成分总量计。The release test is to detect the total flavonoids contained in each tablet, including hyperoside, rutin, isoquercetin, gossipin-8-O-β-D-glucuronide, myricetin, and quercetin. The total amount of 7 ingredients including glucoside-3'-O-glucoside and quercetin.
表24-9

Table 24-9

结果数据表明，本品在高温(40℃)、高湿(RH75％±5％)、光照实验(4500lx±500lx)放置10天下，实施例的含量比较稳定。并且在模拟人工肠液环境下，几乎不释放，在人工结肠液环境中释放度较稳定。The result data shows that the content of the example is relatively stable when this product is placed under high temperature (40°C), high humidity (RH75%±5%), and light experiment (4500lx±500lx) for 10 days. And in the simulated artificial intestinal fluid environment, there is almost no release, and the release rate is relatively stable in the artificial colon fluid environment.
实施例24-8溃疡性结肠炎动物实验Example 24-8 Animal Experiment on Ulcerative Colitis
选用溃疡性结肠炎模型大鼠进行本发明药物的药效学评价。Ulcerative colitis model rats were selected to conduct pharmacodynamic evaluation of the medicine of the present invention.
1.1实验动物1.1 Experimental animals
健康雄性SD大鼠，SPF级，体重180～200g。Healthy male SD rats, SPF grade, weight 180~200g.
1.2试剂及药品1.2 Reagents and drugs
表24-10
Table 24-10
2.实验方法2. Experimental methods
2.1动物饲养和管理2.1 Animal feeding and management
实验大鼠全程饲养于SPF级动物房，光照与黑暗时间比为1:1，室温控制在23±2℃，湿度控制在55％，大鼠能够自由进食和饮水；每三天更换一次垫料，以保证大鼠处于干燥清洁的环境中。The experimental rats were kept in an SPF-level animal room throughout the entire process, with a light-to-dark time ratio of 1:1, room temperature controlled at 23±2°C, and humidity controlled at 55%. The rats could eat and drink freely; the bedding was changed every three days. , to ensure that the rats are in a dry and clean environment.
2.2 DSS诱导大鼠溃疡性结肠炎(Ulcer colitis,UC)模型的制备2.2 Preparation of DSS-induced ulcerative colitis (UC) model in rats
3％DSS溶液配置：称取3g DSS粉末充分溶解于100mL纯水中，所得即为3％DSS溶液。3% DSS solution preparation: Weigh 3g of DSS powder and fully dissolve it in 100mL of pure water, and the result is a 3% DSS solution.
适应性饲养5天后，将大鼠随机分组，5组，每组两笼，每笼4只。分别为空白对照组(Control)，模型组(DSS)，模型+5-氨基水杨酸组(DSS+140mg/kg/day 5-ASA)，模型+HK1组(DSS+280mg/kg/day HK1)、模型+HK2组(DSS+52.5mg/kg/day HK2)。给药剂量以活性成分计算。After adaptive feeding for 5 days, the rats were randomly divided into 5 groups, with two cages in each group and 4 rats in each cage. They are the blank control group (Control), the model group (DSS), the model + 5-aminosalicylic acid group (DSS + 140mg/kg/day 5-ASA), and the model + HK1 group (DSS + 280mg/kg/day HK1 ), model+HK2 group (DSS+52.5mg/kg/day HK2). The dosage is calculated based on the active ingredient.
如下方法所示，大鼠溃疡性结肠炎模型由连续7天自由饮用3％DSS诱发，每笼大鼠(4只)每2天更换一次3％DSS溶液(80mL)以确保DSS溶液有效；空白对照组大鼠自由饮用纯水，每笼大鼠(4只)每2天更换一次。 As shown in the following method, the rat ulcerative colitis model was induced by drinking 3% DSS freely for 7 consecutive days. Each rat (4 rats) in each cage was replaced with 3% DSS solution (80mL) every 2 days to ensure that the DSS solution was effective; blank The rats in the control group drank pure water freely, and each cage (4 rats) was changed every 2 days.
每日上午9：00-10：00以相对应剂量装于定制大鼠肠溶型胶囊经口灌胃给药，给予HK1、HK2和阳性治疗药物5-ASA，空白组和模型组给予同等剂量的空白载体溶液The rats were orally administered with corresponding doses in custom-made enteric-coated capsules at 9:00-10:00 am every day. HK1, HK2 and the positive therapeutic drug 5-ASA were administered. The blank group and the model group were given the same dose. blank carrier solution
(含2％DMSO、2％吐温80以及0.5％CMC-Na)，连续7天。每天给药前，进行疾病活跃指数(Disease activity index,DAI)评分；第8天，对大鼠实施安乐死，采集血液及结肠组织。(Containing 2% DMSO, 2% Tween 80 and 0.5% CMC-Na) for 7 consecutive days. Before daily administration, the disease activity index (DAI) score was performed; on the 8th day, the rats were euthanized, and blood and colon tissue were collected.
2.3粪便粘稠度/粪便隐血测试及DAI评分2.3 Fecal viscosity/fecal occult blood test and DAI score
于每日给药前记录大鼠体重，采集粪便进行粪便隐血测试。从体重下降、粪便粘稠度和粪便潜血三个指标对疾病严重程度进行DAI评分，每个指标对应0-4分，评分标准如表24-5。粪便隐血测试：取少量粪便涂布于玻片中间(玻片预先经火灼烧，以减少颜色误差)，滴加10/L硫酸甲氨基酚乙酸溶液3滴及3％过氧化氢溶液3滴，混匀，立即观察结果，并按照表24-11进行评分。The body weight of rats was recorded before daily administration, and feces were collected for fecal occult blood test. The DAI score is used to score the severity of the disease based on three indicators: weight loss, fecal viscosity and fecal occult blood. Each indicator corresponds to 0-4 points. The scoring standards are as shown in Table 24-5. Fecal occult blood test: Take a small amount of feces and apply it in the middle of the glass slide (the glass slide has been pre-fired to reduce color errors), add 3 drops of 10/L methaminophenol sulfate acetic acid solution and 3 drops of 3% hydrogen peroxide solution , mix well, observe the results immediately, and rate according to Table 24-11.
表24-11 DAI评分细则
Table 24-11 DAI scoring rules
表24-12粪便隐血测试评分表
Table 24-12 Fecal occult blood test score sheet
2.4标本采集2.4 Specimen collection
各组大鼠末次给药后，禁食24h，脱颈椎处死。剪开大鼠腹腔，暴露出大鼠的结肠，取大鼠的全部结肠组织测量并记录其长度，置于新配制的预冷生理盐水中进行肠内容物冲洗，截取1cm近直肠段结肠，于4％多聚甲醛中固定过夜，用于石蜡切片制备及随后的病理学检查，剩余组织取中段液氮速冻后，保存于-80℃，以供后续实验使用。血液样品于室温条件下静置2-3h，然后以3500rpm离心15min，取血清并迅速保存于-80℃，用于后续检测。After the last administration, rats in each group were fasted for 24 hours and sacrificed by cervical dislocation. Cut open the rat's abdominal cavity to expose the rat's colon. Take all the rat's colon tissue, measure and record its length, place it in newly prepared pre-cooled physiological saline to flush the intestinal contents, and cut out 1cm of the colon near the rectum. The tissue was fixed overnight in 4% paraformaldehyde for preparation of paraffin sections and subsequent pathological examination. The remaining tissue was quickly frozen in liquid nitrogen and stored at -80°C for subsequent experiments. The blood samples were allowed to stand at room temperature for 2-3 hours, and then centrifuged at 3500 rpm for 15 min. The serum was collected and quickly stored at -80°C for subsequent testing.
2.5 H&E染色2.5 H&E dyeing
结肠于4％多聚甲醛固定24h后，进行常规石蜡包埋。对包埋好的结肠组织进行连续横向切片(10μm)，然后进行H&E染色，H&E染色步骤如下：The colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in conventional paraffin. Take serial transverse sections (10 μm) of the embedded colon tissue, and then perform H&E staining. The H&E staining steps are as follows:
1)脱蜡：二甲苯I脱蜡10min，二甲苯II脱蜡5min；1) Dewaxing: Dewaxing with xylene I for 10 minutes, dewaxing with xylene II for 5 minutes;
2)乙醇浸泡：用无水乙醇浸泡2次，然后依次用95％、90％、85％的乙醇浸泡1次，每次浸泡1min，最后用水洗涤2min；2) Ethanol soaking: soak twice in absolute ethanol, then soak once in 95%, 90%, and 85% ethanol for 1 minute each time, and finally wash with water for 2 minutes;
3)染色：用苏木精进行染色，染色5min，然后用自来水洗涤1min；3) Staining: Stain with hematoxylin for 5 minutes, then wash with tap water for 1 minute;
4)分化：用1％盐酸酒精分化20s，然后用自来水洗涤1min；4) Differentiation: Differentiate with 1% hydrochloric acid alcohol for 20 seconds, then wash with tap water for 1 minute;
5)反蓝：用1％稀氨水反蓝30s，然后用自来水洗涤1min；5) Anti-blue: Use 1% dilute ammonia to reverse blue for 30 seconds, then wash with tap water for 1 minute;
6)伊红染色：染色5min，然后用自来水洗涤30s；6) Eosin staining: stain for 5 minutes, then wash with tap water for 30 seconds;
7)乙醇脱水：依次用不同含量的乙醇进行脱水处理，顺序如下：85％乙醇脱水20s，90％乙醇脱水30s，95％乙醇I脱水1min，95％乙醇II脱水1min，无水乙醇I脱 水2min，无水乙醇II脱水2min；7) Ethanol dehydration: Use different contents of ethanol for dehydration in sequence. The sequence is as follows: 85% ethanol dehydration for 20s, 90% ethanol dehydration for 30s, 95% ethanol I for 1 min, 95% ethanol II for 1 min, and absolute ethanol I for dehydration. Water for 2 minutes, dehydrate with absolute ethanol II for 2 minutes;
8)透明：二甲苯I处理2min，二甲苯II处理2min，二甲苯III处理2min；8) Transparency: xylene I treatment for 2 minutes, xylene II treatment for 2 minutes, xylene III treatment for 2 minutes;
9)最后用中性树胶封片，用数字病理切片扫描仪(NanoZoomer 2.0 RS)对切片扫描成像；9) Finally, seal the slides with neutral gum, and use a digital pathology slide scanner (NanoZoomer 2.0 RS) to scan and image the slides;
10)病理学检查：根据结肠组织病变程度、炎症细胞浸润、隐窝细胞损伤等病理学指标对结肠损伤程度进行评分，具体评分标准如表24-13所示。10) Pathological examination: The degree of colon damage is scored based on pathological indicators such as the degree of colon tissue lesions, inflammatory cell infiltration, and crypt cell damage. The specific scoring standards are shown in Table 24-13.
表24-13炎症评分表
Table 24-13 Inflammation score sheet
2.6统计学分析2.6 Statistical analysis
运用GraphPad Prism(Ver 6.0)进行统计学分析，所有数据采用平均值±标准误(Mean±SEM)表示，体重变化和DAI评分变化采用Two-way ANOVA分析，其他数据采用One-way ANOVA分析，利用Dunnett’s多重检验分析组间差异的显著性，当P＜0.05时，组间差异具有统计学意义。GraphPad Prism (Ver 6.0) was used for statistical analysis. All data were expressed as mean ± SEM. Weight changes and DAI score changes were analyzed by Two-way ANOVA. Other data were analyzed by One-way ANOVA. Using Dunnett's multiple test analyzed the significance of differences between groups. When P<0.05, the differences between groups were statistically significant.
3.实验结果3.Experimental results
3.1黄蜀葵花提取物肠溶胶囊对大鼠DAI评分的影响3.1 Effect of marshmallow flower extract enteric-coated capsules on DAI scores in rats
DAI的评分，为按表24-13的3项指标值进行加和后取平均值计算。如表24-14所示，DSS模型组大鼠DAI评分从第3天开始显著升高，与Control组相比，差异具有统计学意义(P<0.01)，实验第6天腹泻3只，第7天腹泻7只；DSS+HK1(280mg/kg/day)、DSS+HK2(52.5mg/kg/day)组DAI评分增长趋势较为平缓，第4，5，6，7天，其DAI评分与DSS模型组相比，差异具有统计学意义(P<0.01)，实验过程中，DSS+HK1(280mg/kg/day)组第6天腹泻1只，第7天腹泻2只；DSS+HK2(52.5mg/kg/day)组无大鼠出现腹泻；阳性治疗药物5-ASA(140mg/kg/day)缓解DSS诱导的结肠炎症状，降低DAI评分，从第4天开始，与DSS模型组相比，差异显著(P<0.01)，实验过程中无大鼠出现腹泻。The DAI score is calculated by adding the three indicator values in Table 24-13 and taking the average. As shown in Table 24-14, the DAI score of the rats in the DSS model group increased significantly from the 3rd day. Compared with the Control group, the difference was statistically significant (P<0.01). On the 6th day of the experiment, 3 rats had diarrhea, and on the 6th day of the experiment, 3 rats had diarrhea. There were 7 diarrhea cases in 7 days; the DAI score of the DSS+HK1 (280mg/kg/day) and DSS+HK2 (52.5mg/kg/day) groups showed a relatively gentle growth trend. On days 4, 5, 6, and 7, the DAI scores were the same as Compared with the DSS model group, the difference was statistically significant (P<0.01). During the experiment, one animal in the DSS+HK1 (280mg/kg/day) group had diarrhea on the 6th day and 2 had diarrhea on the 7th day; DSS+HK2 ( No rats in the 52.5 mg/kg/day) group had diarrhea; the positive therapeutic drug 5-ASA (140 mg/kg/day) alleviated the symptoms of DSS-induced colitis and reduced the DAI score. Starting from the 4th day, it was comparable to the DSS model group. Ratio, the difference was significant (P<0.01), and no rats had diarrhea during the experiment.
表24-14各组大鼠DAI评分
Table 24-14 DAI scores of rats in each group
注:数据以平均值±标准误表示。N＝8.*,P＜0.05,**,P＜0.01,vs.Control；#,P＜0.05,##,P＜0.01,vs.DSS，使用双因素方差及Dunnett’s多重比较检验进行分析。Note: Data are expressed as mean ± standard error. N＝8.*,P＜0.05,**,P＜0.01,vs.Control; # ,P＜0.05, ## ,P＜0.01,vs.DSS, analyzed using two-factor variance and Dunnett's multiple comparison test.
3.2黄蜀葵花提取物对大鼠结肠长度的影响3.2 Effect of marshmallow flower extract on colon length in rats
如表24-15所示，Control组结肠长度为17.05±0.04cm，DSS模型组大鼠结肠萎缩至14.32±0.15cm，与Control组相比，差异显著(P<0.01)；280mg/kg/day HK1、52.5mg/kg/day HK2显著改善了由DSS诱导的结肠萎缩，其中DSS+HK2(52.5mg/kg/day)组与DSS模型组相比，差异具有统计学意义(P<0.05)；阳性药物5-ASA(140mg/kg/day)也改善了由DSS诱导的结肠萎缩，与Control组相比，差异具有统计学意义(P<0.05)。As shown in Table 24-15, the colon length of the Control group was 17.05±0.04cm, and the colon of rats in the DSS model group shrunk to 14.32±0.15cm. Compared with the Control group, the difference was significant (P<0.01); 280 mg/kg/day HK1, 52.5mg/kg/day HK2 significantly improved colon atrophy induced by DSS, and the difference between the DSS+HK2 (52.5mg/kg/day) group and the DSS model group was statistically significant (P<0.05); The positive drug 5-ASA (140mg/kg/day) also improved the colon atrophy induced by DSS, and the difference was statistically significant (P<0.05) compared with the Control group.
表24-15各组大鼠结肠长度统计表
Table 24-15 Statistical table of colon length of rats in each group
注:数据以平均值±标准误差表示。N＝8.**,P＜0.01,vs.Control；#,P＜0.05,vs.DSS,使用单因素方差及Dunnett’s多重比较检验进行分析。Note: Data are expressed as mean ± standard error. N＝8.**, P＜0.01, vs.Control; #, P＜0.05, vs.DSS, analyzed using one-way variance and Dunnett’s multiple comparison test.
4.实验结论和讨论4. Experimental conclusion and discussion
本实施例采用DSS诱导的大鼠溃疡性结肠炎模型，以5-氨基水杨酸为阳性对照药物，考察含有黄蜀葵花提取物的肠溶胶囊治疗结肠炎的治疗效果，结果表明，HK1(280mg/kg/day)、HK2(52.5mg/kg/day)组明显保护DSS诱导的大鼠体重降低、腹泻、便血等结肠炎症状；其中HK2(52.5mg/kg/day)效果相比于其他更好。显著改善DSS诱导的结肠萎缩、隐窝结构损伤及组织炎性浸润等组织病理学变化。This example uses a DSS-induced rat ulcerative colitis model, and uses 5-aminosalicylic acid as a positive control drug to examine the therapeutic effect of enteric-coated capsules containing marshmallow flower extract in treating colitis. The results show that HK1 (280mg /kg/day) and HK2 (52.5mg/kg/day) groups significantly protected DSS-induced colitis symptoms such as weight loss, diarrhea, and hematochezia in rats; among them, the effect of HK2 (52.5mg/kg/day) was better than other groups good. Significantly improves histopathological changes such as DSS-induced colon atrophy, crypt structural damage, and tissue inflammatory infiltration.
综上所述，含黄蜀葵花提取物的肠溶胶囊对结肠炎具有一定的疗效，在52.5mg/kg/day的效果与阳性药5-ASA相当，安全性较好。In summary, the enteric-coated capsules containing marshmallow flower extract have certain curative effects on colitis. The effect at 52.5 mg/kg/day is equivalent to that of the positive drug 5-ASA, and the safety is good.
实施例25药物组合物及其在特发性肺纤维化作用的实验Example 25 Experiment on pharmaceutical composition and its effect on idiopathic pulmonary fibrosis
实施例25-1药物组合物的制备Example 25-1 Preparation of pharmaceutical composition
取金丝桃苷、异槲皮苷、hibifolin、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素、槲皮素3-O-洋槐糖苷原料，按下表投料比例，称取各活性成分，混合，制备得到药物组合物。

Take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and add the proportions as shown in the table below. Take each active ingredient and mix it to prepare a pharmaceutical composition.

实施例25-2药物组合物的制备Example 25-2 Preparation of pharmaceutical composition
按下表投料比例，称取各活性成分，混合，制备得到药物组合物。
According to the feeding ratio in the table below, weigh each active ingredient and mix to prepare a pharmaceutical composition.
取金丝桃苷、异槲皮苷、hibifolin、杨梅素、槲皮素-3'-O-葡萄糖苷、槲皮素、槲皮素3-O-洋槐糖苷原料，按下表投料比例，称取各活性成分，混合，制备得到药物组合物。Take the raw materials of hyperoside, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside, quercetin, and quercetin 3-O-acaxiglucoside, and add the proportions as shown in the table below. Take each active ingredient and mix it to prepare a pharmaceutical composition.
实施例25-3糖尿病肾病实验Example 25-3 Diabetic nephropathy experiment
动物模型：db/db小鼠，16周龄，C57BL/6小鼠，16周龄。Animal models: db/db mice, 16 weeks old, C57BL/6 mice, 16 weeks old.
给药方式：灌胃给药Mode of administration: intragastric administration
实验造模：db/db小鼠饲养17周后，检测尿蛋白，出现尿蛋白、肾功能异常，则模型成功，用于后期实验。Experimental modeling: After 17 weeks of raising db/db mice, urine protein is detected. If urine protein and renal function abnormalities appear, the model is successful and can be used for later experiments.
实验分组：C57BL/6小鼠分为正常组，将db/db小鼠分为模型组，阳性药组(达格列净片，45.5mg/kg)，实施例25-1药物组合物组(62.4mg/kg)，每组15只，给药4周后检测尿蛋白及尿肌酐及每日摄食量，并观察小鼠状态。检测结果如下表所示。Experimental grouping: C57BL/6 mice were divided into normal group, db/db mice were divided into model group, positive drug group (dapagliflozin tablets, 45.5mg/kg), and Example 25-1 pharmaceutical composition group ( 62.4mg/kg), 15 mice in each group. After 4 weeks of administration, urine protein, urinary creatinine and daily food intake were detected, and the status of the mice was observed. The test results are shown in the table below.
表25-1
Table 25-1
注:*与正常对照组比较，P＜0.05；#与模型对照组比较，P＜0.05；Note: *Compared with the normal control group, P＜0.05; #Compared with the model control group, P＜0.05;
结果显示，实施例25-1的药物组合物组可降低尿蛋白和尿蛋白/尿肌酐(ACR)。仅1个动物出现腹泻/腹胀情形，具有较低的副作用反应。而在同期研究的不同黄酮成分的黄蜀葵花提取物的研究中发现，由与实施例25-1不同比例黄酮成分组成的黄蜀葵花提取物，动物呈现不同的副作用，含有本发明比例的黄酮成分的黄蜀葵花提取物具有较低副作用，比剂量更大的黄葵胶囊还显示出更低的副作用。The results show that the pharmaceutical composition group of Example 25-1 can reduce urinary protein and urinary protein/urinary creatinine (ACR). Only 1 animal developed diarrhea/abdominal bloating, with low side effects. In the study of marshmallow flower extracts with different flavonoid components studied at the same time, it was found that the marshmallow flower extracts composed of flavonoid components in different proportions as in Example 25-1 showed different side effects in animals, and those containing the flavonoid components of the present invention showed different side effects. Marshmallow flower extract has lower side effects and has been shown to have lower side effects than higher dose marshmallow capsules.
实施例25-4特发性肺纤维化作用实验Example 25-4 Experiment on idiopathic pulmonary fibrosis
实验动物：昆明种小鼠60只，雌雄各半，体重18-22克，清洁级。Experimental animals: 60 Kunming mice, half male and half female, weighing 18-22 grams, clean grade.
动物分组：随机分为5组，即正常组、模型组、对照药组(罗格列酮5mg/kg)、实施例25-1药物组合物组(70mg/kg)、实施例25-2药物组合物组(70mg/kg)、黄葵胶囊样品组(以提取物计180mg/kg)，实施例25-2药物组合物组(70mg/kg)、每组小鼠10只。Animal grouping: randomly divided into 5 groups, namely normal group, model group, control drug group (rosiglitazone 5 mg/kg), Example 25-1 pharmaceutical composition group (70 mg/kg), Example 25-2 drug Composition group (70 mg/kg), Huangkui capsule sample group (180 mg/kg based on extract), Example 25-2 pharmaceutical composition group (70 mg/kg), 10 mice in each group.
实验造模：实验小鼠适应性饲养3天后，用4％的水合氯醛(0.01ml/g)腹腔注射麻醉。取仰卧固定位，常规消毒，行颈正中切口，钝性分离暴露气管，模型组，对照药组、治疗组于气管软骨环间隙穿刺缓慢注入博莱霉素(5mg/kg)，正常对照组注入等体积生理盐水，注药后立即将小鼠直立旋转3-5min，使药液均匀分布于两侧肺内，然后缝合皮肤，并在缝合处消毒，待清醒后送清洁级观察室饲养。Experimental modeling: After the experimental mice were adaptively raised for 3 days, they were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.01ml/g). The patients were placed in a supine fixed position, routine disinfection was performed, a midline cervical incision was made, and the trachea was exposed by blunt dissection. The model group, control drug group, and treatment group were punctured and slowly injected bleomycin (5 mg/kg) into the tracheal cartilage ring space, and the normal control group was injected Equal volume of normal saline was injected. Immediately after injecting the drug, the mice were rotated upright for 3-5 minutes to allow the drug solution to be evenly distributed in the lungs on both sides. The skin was then sutured and the sutures were disinfected. After they woke up, they were sent to a clean observation room for feeding.
给药方式：造模第二天开始给药，给药组按上述剂量给药，每天一次，正常组和 模型组按同样的方法给予同等体积的蒸馏水10ml/kg，连续给药28天。Method of administration: Administration will begin on the second day after modeling. The administration group will be administered according to the above dosage, once a day. The normal group and The model group was given the same volume of distilled water 10ml/kg in the same way for 28 consecutive days.
肺组织病理切片染色：给药结束后，取右肺，按常规病理学方法进行固定、包埋、切片、HE染色。将肺泡炎和肺纤维化的程度分为四级。Staining of pathological sections of lung tissue: After administration, the right lung was removed and fixed, embedded, sectioned, and stained with HE according to conventional pathological methods. The degree of alveolitis and pulmonary fibrosis is divided into four grades.
实验结果：
Experimental results:
注:*与正常组比较，P＜0.01；#与模型组比较，P＜0.05Note: *Compared with the normal group, P＜0.01; #Compared with the model group, P＜0.05
实验结果表明，模型组小鼠肺组织肺泡炎和肺纤维化程度显著加重，呈典型的肺纤维化实质病变。给药28天，各剂量组小鼠肺系数明显降低，肺组织肺泡炎及肺纤维化程度明显减轻。总黄酮有效部位可明显降低肺纤维化小鼠的肺泡炎和肺纤维化程度，对肺纤维化小鼠的肺部具有保护作用，可以减缓由纤维化对方肺部组织造成的损伤。Experimental results showed that the degree of alveolitis and pulmonary fibrosis in the lung tissue of mice in the model group was significantly aggravated, showing typical pulmonary fibrosis parenchymal lesions. After 28 days of administration, the lung coefficient of mice in each dose group was significantly reduced, and the degree of alveolitis and pulmonary fibrosis in the lung tissue was significantly reduced. The effective fraction of total flavonoids can significantly reduce the degree of alveolitis and pulmonary fibrosis in mice with pulmonary fibrosis, has a protective effect on the lungs of mice with pulmonary fibrosis, and can slow down the damage to the lung tissue caused by fibrosis.
实施例26制剂的制备Example 26 Preparation of Formulation
将实施例2-A4的黄蜀葵花提取物粉碎后取400g，加入下述重量份的羟丙基纤维素20g、滑石粉15g、微晶纤维素20g、硫酸钙20g混匀，过80目筛，加入适量的无水乙醇，制软材，再用20目筛制颗粒，干燥，整粒，装胶囊。Crush the marshmallow flower extract of Example 2-A4, take 400g, add the following weight portions of 20g hydroxypropyl cellulose, 15g talc, 20g microcrystalline cellulose, and 20g calcium sulfate, mix well, and pass through an 80-mesh sieve. Add an appropriate amount of absolute ethanol to make a soft material, then use a 20-mesh sieve to make granules, dry, whole granules, and put into capsules.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。 The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (16)

  1. 一种药物制剂，含有黄蜀葵花提取物及药学上可接受的辅料，其特征在于，所述黄蜀葵花提取物，A pharmaceutical preparation containing marshmallow flower extract and pharmaceutically acceptable auxiliary materials, characterized in that the marshmallow flower extract,
    含有如下质量比的黄酮类成分：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：2.0-25：3.0-20：1.0-7.0：4.0-23：0.6-12.0；Contains flavonoids in the following mass ratio: gossyrin-8-O-β-D-glucuronide: hyperoside: isoquercetin: myricetin: quercetin-3'-O-glucuronide : The mass ratio of quercetin is 10:2.0-25:3.0-20:1.0-7.0:4.0-23:0.6-12.0;
    或者，含有如下质量比的黄酮类成分：异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷为0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；Alternatively, the flavonoid component contains the following mass ratio: isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O-β-D-glucuronic acid Glycosides: Hyperin is 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6;
    或者，含有如下质量含量的黄酮类成分：金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷5-30％、杨梅素1.6-11％、槲皮素-3'-O-葡萄糖苷12.1-25％、槲皮素1.4-10％；进一步地，金丝桃苷8-26％、异槲皮苷12.0-19.8％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-30％、杨梅素3.0-4.9％或5.1-6.0％、槲皮素-3'-O-葡萄糖苷14-25％、槲皮素1.6-4.9％；Alternatively, it contains flavonoids with the following mass content: hyperoside 8-26%, isoquercetin 12.0-19.8%, gossyrin-8-O-β-D-glucuronide 5-30%, Myricetin 1.6-11%, quercetin-3'-O-glucoside 12.1-25%, quercetin 1.4-10%; further, hyperoside 8-26%, isoquercetin 12.0-19.8 %, gossyrin-8-O-β-D-glucuronide 8.5-30%, myricetin 3.0-4.9% or 5.1-6.0%, quercetin-3'-O-glucuronide 14-25% , Quercetin 1.6-4.9%;
    所述制剂为非水性液体的制剂；The preparation is a non-aqueous liquid preparation;
    所述药学上的可接受的辅料在制剂中的质量含量为0.5％-99.5％。The mass content of the pharmaceutically acceptable excipients in the preparation is 0.5%-99.5%.
  2. 根据权利要求1所述的药物制剂，其特征在于，所述黄蜀葵花提取物含有如下质量比的黄酮类成分：The pharmaceutical preparation according to claim 1, wherein the marshmallow flower extract contains flavonoid components in the following mass ratio:
    棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：3.0-20：4.0-18：1.9-6.0：6.0-20：1.0-10.0，或10：6-15：5-13：1.0-2.9：4.0-9.5：0.6-6.0，或10：6-15：5-13：1.0-1.8：4.0-5.9：0.6-0.99；The mass ratio of gossyrin-8-O-β-D-glucuronide:hyperin:isoquercetin:myricetin:quercetin-3'-O-glucuronide:quercetin is 10 : 3.0-20: 4.0-18: 1.9-6.0: 6.0-20: 1.0-10.0, or 10: 6-15: 5-13: 1.0-2.9: 4.0-9.5: 0.6-6.0, or 10: 6-15 :5-13:1.0-1.8:4.0-5.9:0.6-0.99;
    进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：4.0-17：4.50-16：1.9-5.5：7.0-16；Further, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 4.0-17: 4.50-16: 1.9-5.5: 7.0-16;
    进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷的质量比为10：6.0-15：5.0-13：2.0-5.0：8.0-14；Further, the mass ratio of gossyrin-8-O-β-D-glucuronide:hyperoside:isoquercitrin:myricetin:quercetin-3'-O-glucuronide is 10: 6.0-15:5.0-13:2.0-5.0:8.0-14;
    进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素的质量比为10：1.0-6.0，优选为10：1.2-6，进一步优选为10：1.5-5.0；Further, the mass ratio of gossydisin-8-O-β-D-glucuronide: quercetin is 10:1.0-6.0, preferably 10:1.2-6, further preferably 10:1.5-5.0;
    更进一步地，含有芦丁，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，进一步优选为10：0.1-0.3；Furthermore, it contains rutin, and the mass ratio of gossydisin-8-O-β-D-glucuronide:rutin is 10:0.05-0.6, preferably 10:0.1-0.4, and further preferably 10:0.1-0.3;
    更进一步地，含有槲皮素-3-O-洋槐糖苷，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素-3-O-洋槐糖苷为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5；Furthermore, it contains quercetin-3-O-acaiglycoside, and the ratio of gossyrin-8-O-β-D-glucuronide: quercetin-3-O-acaiglycoside is 10:0.1- 2.5, preferably 10:0.1-0.9, further preferably 10:0.15-0.5;
    再更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷：异槲皮苷：杨梅素：槲皮素-3'-O-葡萄糖苷：槲皮素的质量比为10：6-16：5-13：1.0-4.5：4.0-12：0.6-6.0，或10：15.1-16：5-13：3.0-4.5：9.6-12：0.6-6.0；Furthermore, gossyrin-8-O-β-D-glucuronide: hypericin: isoquercetin: myricetin: quercetin-3'-O-glucuronide: quercetin The mass ratio is 10:6-16:5-13:1.0-4.5:4.0-12:0.6-6.0, or 10:15.1-16:5-13:3.0-4.5:9.6-12:0.6-6.0;
    再更进一步地，含有芦丁，所述棉皮素-8-O-β-D-葡萄糖醛酸苷与芦丁的质量比为10：0.05-0.6，优选为10：0.1-0.4，进一步优选为10：0.1-0.3；Furthermore, it contains rutin, and the mass ratio of the cottonseed-8-O-β-D-glucuronide to rutin is 10:0.05-0.6, preferably 10:0.1-0.4, further preferably is 10: 0.1-0.3;
    再更进一步地，含有槲皮素-3-O-洋槐糖苷，所述棉皮素-8-O-β-D-葡萄糖醛酸苷：槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5。Further still, it contains quercetin-3-O-acaiglycoside, and the mass ratio of the cottonseed-8-O-β-D-glucuronide:quercetin-3-O-acaiglycoside is: 10: 0.1-2.5, preferably 10: 0.1-0.9, further preferably 10: 0.15-0.5.
  3. 根据权利要求1所述的药物制剂，其特征在于，所述黄蜀葵花提取物含有如下质量比的黄酮类成分：The pharmaceutical preparation according to claim 1, wherein the marshmallow flower extract contains flavonoid components in the following mass ratio:
    异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖 醛酸苷：金丝桃苷为0.8-1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为0.8-1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为0.8-1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为0.8-1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为0.8-1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为0.8-1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；或者为0.8-1.2：0.09-1.6：0.2-2.0：0.05-1.2：0.5-3.0：0.25-3.6；或者为0.8-1.2：0.12-0.7：0.8-1.6：0.2-0.5：0.85-1.7：1.0-1.4；Isoquercetin: Quercetin: Quercetin-3'-O-glucoside: Myricetin: Gossipin-8-O-β-D-glucoside Aldehyde glycosides: Hypericin is 0.8-1.2: 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 0.8-1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9 : 0.4-3.1: 0.5-3.0; further 0.8-1.2: 0.16-1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 0.8-1.2: 0.2-0.8: 0.8-2.1: 0.18 -0.6: 0.8-2.0: 0.7-2.0; further 0.8-1.2: 0.2-0.7: 0.8-1.6: 0.2-0.5: 0.8-1.8: 0.8-1.4; further 0.8-1.2: 0.2-0.6: 1.0-1.2 : 0.2-0.4: 0.9-1.7: 1.0-1.3; or 0.8-1.2: 0.09-1.6: 0.2-2.0: 0.05-1.2: 0.5-3.0: 0.25-3.6; or 0.8-1.2: 0.12-0.7: 0.8 -1.6: 0.2-0.5: 0.85-1.7: 1.0-1.4;
    进一步地，所述异槲皮苷的质量比数值为1.2或1.0；Further, the mass ratio value of the isoquercetin is 1.2 or 1.0;
    进一步地，异槲皮苷：槲皮素：槲皮素-3'-O-葡萄糖苷：杨梅素：棉皮素-8-O-β-D-葡萄糖醛酸苷：金丝桃苷为1：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；或者为1.2：0.05-1.6：0.2-4.6：0.05-1.2：0.2-3.5：0.25-3.6；进一步为1.2：0.1-1.2：0.4-3.1：0.10-0.9：0.4-3.1：0.5-3.0；进一步为1.2：0.16-1.0：0.6-2.4：0.14-0.75：0.6-2.5：0.6-2.4；进一步为1.2：0.2-0.8：0.8-2.1：0.18-0.6：0.8-2.0：0.7-2.0；进一步为1.2：0.2-0.7：0.8-1.6：0.2-0.5：0.8-1.8：0.8-1.4；进一步为1.2：0.2-0.6：1.0-1.2：0.2-0.4：0.9-1.7：1.0-1.3；Further, isoquercetin: quercetin: quercetin-3'-O-glucoside: myricetin: gossyrin-8-O-β-D-glucuronide: hyperoside is 1 : 0.05-1.6: 0.2-4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1: 0.16 -1.0: 0.6-2.4: 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1: 0.2-0.7 :0.8-1.6:0.2-0.5:0.8-1.8:0.8-1.4; further 1:0.2-0.6:1.0-1.2:0.2-0.4:0.9-1.7:1.0-1.3; or 1.2:0.05-1.6:0.2 -4.6: 0.05-1.2: 0.2-3.5: 0.25-3.6; further 1.2: 0.1-1.2: 0.4-3.1: 0.10-0.9: 0.4-3.1: 0.5-3.0; further 1.2: 0.16-1.0: 0.6-2.4 : 0.14-0.75: 0.6-2.5: 0.6-2.4; further 1.2: 0.2-0.8: 0.8-2.1: 0.18-0.6: 0.8-2.0: 0.7-2.0; further 1.2: 0.2-0.7: 0.8-1.6: 0.2 -0.5: 0.8-1.8: 0.8-1.4; further 1.2: 0.2-0.6: 1.0-1.2: 0.2-0.4: 0.9-1.7: 1.0-1.3;
    进一步地，含有芦丁，所述异槲皮苷与芦丁的质量比为1：0.001-0.08，优选为1：0.005-0.06，进一步优选为1：0.006-0.05，更进一步优选为1：0.007-0.04，再更进一步优选为1：0.008-0.03，最优选为1：0.009-0.03；Further, it contains rutin, and the mass ratio of isoquercetin to rutin is 1:0.001-0.08, preferably 1:0.005-0.06, further preferably 1:0.006-0.05, even more preferably 1:0.007 -0.04, further preferably 1:0.008-0.03, most preferably 1:0.009-0.03;
    进一步地，含有槲皮素-3-O-洋槐糖苷，所述异槲皮苷与槲皮素-3-O-洋槐糖苷的质量比为10：0.1-2.5，优选为10：0.1-0.9，进一步优选为10：0.15-0.5。Further, it contains quercetin-3-O-acaxiglucoside, and the mass ratio of the isoquercetin and quercetin-3-O-acaxiglucoside is 10:0.1-2.5, preferably 10:0.1-0.9, More preferably, it is 10:0.15-0.5.
  4. 根据权利要求1所述的药物制剂，其特征在于，所述黄蜀葵花提取物含有如下质量含量的黄酮类成分：金丝桃苷10-25％、异槲皮苷8-19％、棉皮素-8-O-β-D-葡萄糖醛酸苷5-30％、槲皮素-3'-O-葡萄糖苷12.1-25％；The pharmaceutical preparation according to claim 1, characterized in that the marshmallow flower extract contains flavonoids with the following mass content: hyperoside 10-25%, isoquercetin 8-19%, gossipin -8-O-β-D-glucuronide 5-30%, quercetin-3'-O-glucuronide 12.1-25%;
    进一步地，槲皮素-3'-O-葡萄糖苷含量为12.6-23％或者为13-20％；Further, the quercetin-3'-O-glucoside content is 12.6-23% or 13-20%;
    进一步地，金丝桃苷12-23％、异槲皮苷10-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷8.5-25％、槲皮素-3'-O-葡萄糖苷12.6-23％；Further, hyperoside 12-23%, isoquercetin 10-17%, gossyrin-8-O-β-D-glucuronide 8.5-25%, quercetin-3'-O - Glucoside 12.6-23%;
    进一步地，金丝桃苷13-22％、异槲皮苷11-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-21％、槲皮素-3'-O-葡萄糖苷13-20％；Further, hyperoside 13-22%, isoquercetin 11-17%, gossyrin-8-O-β-D-glucuronide 12-21%, quercetin-3'-O - Glucoside 13-20%;
    进一步地，还含有槲皮素2-10％，优选槲皮素2.5-9％，进一步优选槲皮素3-8.5％，更进一步优选槲皮素1.5％-5％；Further, it also contains 2-10% quercetin, preferably 2.5-9% quercetin, further preferably 3-8.5% quercetin, even more preferably 1.5%-5% quercetin;
    进一步地，含有杨梅素2-11％，优选杨梅素3-7％，进一步优选杨梅素3-5％，Further, it contains 2-11% myricetin, preferably 3-7% myricetin, further preferably 3-5% myricetin,
    进一步地，含有槲皮素-3-O-洋槐糖苷0.08-2.5％，优选槲皮素-3-O-洋槐糖苷0.1-1.5％，进一步优选槲皮素-3-O-洋槐糖苷0.1-0.9％，再进一步优选槲皮素-3-O-洋槐糖苷0.1-0.5％；Further, it contains 0.08-2.5% of quercetin-3-O-aphoretin glycoside, preferably 0.1-1.5% of quercetin-3-O-aphoretin glycoside, and further preferably 0.1-0.9% of quercetin-3-O-aphoretin glycoside. %, and further preferably quercetin-3-O-acaxiglucoside 0.1-0.5%;
    进一步地，金丝桃苷11-22％、异槲皮苷12.0-17％、棉皮素-8-O-β-D-葡萄糖醛酸苷12-23％、杨梅素1.6-4.9％或5.1-9.0％、槲皮素-3'-O-葡萄糖苷13-22％、槲皮素1.4-8％或1.4-7.8％；Further, hyperoside 11-22%, isoquercetin 12.0-17%, gossipin-8-O-β-D-glucuronide 12-23%, myricetin 1.6-4.9% or 5.1 -9.0%, quercetin-3'-O-glucoside 13-22%, quercetin 1.4-8% or 1.4-7.8%;
    进一步地，含有芦丁0.01-1.0％，优选芦丁0.05-0.8％，进一步优选芦丁0.09-0.8％，更进一步优选芦丁0.1-0.6％。Further, it contains 0.01-1.0% rutin, preferably 0.05-0.8% rutin, further preferably 0.09-0.8% rutin, even more preferably 0.1-0.6% rutin.
  5. 根据权利要求1-4任一项所述的药物制剂，其特征在于，所述黄蜀葵花提取物中黄 酮类成分的质量含量为55％以上，优选为60％以上，进一步优选为65-85％或66-84％，更进一步优选为69-82％或69-90％；The pharmaceutical preparation according to any one of claims 1 to 4, wherein the yellow marshmallow flower extract contains The mass content of ketone components is more than 55%, preferably more than 60%, more preferably 65-85% or 66-84%, even more preferably 69-82% or 69-90%;
    进一步地，含有的槲皮素、槲皮素-3'-O-葡萄糖苷、杨梅素、棉皮素-8-O-β-D-葡萄糖醛酸苷、异槲皮苷及金丝桃苷的总含量为55％以上，优选60％以上，进一步优选为65-85％，更进一步优选为69-82％；Further, it contains quercetin, quercetin-3'-O-glucoside, myricetin, gossyrin-8-O-β-D-glucuronide, isoquercitrin and hyperoside. The total content is more than 55%, preferably more than 60%, more preferably 65-85%, even more preferably 69-82%;
    进一步地，槲皮素-3'-O-葡萄糖苷含量为12.1-25％，优选槲皮素-3'-O-葡萄糖苷含量为12.6-23％、13-20％、13-20％，或者为14-25％；Further, the quercetin-3'-O-glucoside content is 12.1-25%, and the preferred quercetin-3'-O-glucoside content is 12.6-23%, 13-20%, 13-20%, Or 14-25%;
    或者进一步地，槲皮素的含量高于0.7％，优选高于1.0％，进一步优选1.0％-10％，更进一步优选1.5％-5％；Or further, the content of quercetin is higher than 0.7%, preferably higher than 1.0%, further preferably 1.0%-10%, even more preferably 1.5%-5%;
    或者更进一步地，棉皮素-8-O-β-D-葡萄糖醛酸苷为8.5-30％，优选12％-23％。Or further, gossydisin-8-O-β-D-glucuronide is 8.5-30%, preferably 12%-23%.
  6. 根据权利要求1-5任一项所述的药物制剂，其特征在于，所述的黄蜀葵花提取物由如下步骤制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液浓缩后经萃取，制得萃取液；(3)萃取液去除溶剂后经大孔树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉；The pharmaceutical preparation according to any one of claims 1 to 5, characterized in that the marshmallow flower extract is prepared by the following steps: (1) extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract ; (2) The extract liquid is concentrated and then extracted to obtain an extract liquid; (3) The solvent is removed from the extract liquid and then eluted with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
    或者，由如下方法制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(22)提取液加入澄清剂，水浴处理，过滤去上清；(33)上清液经聚酰胺树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉。Alternatively, it is prepared by the following method: (1) Extract the flowers or medicinal parts of Hollyhock with ethanol to prepare an extract; (22) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (33) The supernatant After elution with polyamide resin, the marshmallow flower extract is obtained; the preferred extraction method is percolation.
  7. 一种黄蜀葵花提取物的药物制剂，含有药学上可接受的辅料，其特征在于，所述制剂为非水性液体的制剂；所述药学上的可接受的辅料在制剂中的含量为0.5％-99.5％；所述提取物由如下步骤制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(2)提取液浓缩后经萃取，制得萃取液；(3)萃取液去除溶剂后经大孔树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉；A pharmaceutical preparation of marshmallow flower extract, containing pharmaceutically acceptable auxiliary materials, characterized in that the preparation is a non-aqueous liquid preparation; the content of the pharmaceutically acceptable auxiliary materials in the preparation is 0.5% - 99.5%; the extract is prepared by the following steps: (1) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract; (2) Concentrating the extract and then extracting to prepare an extract; (3) After removing the solvent from the extract, it is eluted with macroporous resin to obtain marshmallow flower extract; the preferred extraction method is percolation;
    或者，由如下方法制备得到：(1)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(22)提取液加入澄清剂，水浴处理，过滤去上清；(33)上清液经聚酰胺树脂洗脱，制得黄蜀葵花提取物；优选提取方法为渗漉。Alternatively, it is prepared by the following method: (1) Extract the flowers or medicinal parts of Hollyhock with ethanol to prepare an extract; (22) Add a clarifying agent to the extract, treat it in a water bath, and filter to remove the supernatant; (33) The supernatant After elution with polyamide resin, the marshmallow flower extract is obtained; the preferred extraction method is percolation.
  8. 根据权利要求6-7任一项所述的药物制剂，其特征在于，步骤(1)中，所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(2)中，所述萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(3)中，所述大孔树脂型号为D101、HPD100或AB-8；步骤(22)中，所述水浴温度为50-70℃，水浴时间为30-90min；步骤(33)中，所述树脂径高比为1：4-9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。The pharmaceutical preparation according to any one of claims 6-7, characterized in that in step (1), the amount of ethanol used is 10-25 times that of marshmallow flowers or marshmallow medicinal parts, and the ethanol is 60-25 times that of marshmallow flowers or medicinal parts of marshmallow. 95% ethanol solution; in step (2), the extraction agent used for extraction is n-butanol, petroleum ether or ethyl acetate, the extraction method is continuous countercurrent extraction, and the solid-liquid ratio of the extraction is 0.8 -4:1, the extraction level of the extraction is level 1-5; in step (3), the macroporous resin model is D101, HPD100 or AB-8; in step (22), the water bath temperature The temperature is 50-70°C, and the water bath time is 30-90min; in step (33), the diameter-to-height ratio of the resin is 1:4-9, the concentration of the loading solution is 0.10-0.60g crude drug/mL, and the volume of the loading solution is 4-12BV, elute with 4-8BV pure water and 4-8BV 60-95% ethanol.
  9. 根据权利要求6-7任一项所述的药物制剂，其特征在于，步骤(2)包括在萃取之前，对提取液经活性炭吸附、醇沉或酸沉处理。The pharmaceutical preparation according to any one of claims 6 to 7, wherein step (2) includes subjecting the extract to activated carbon adsorption, alcohol precipitation or acid precipitation before extraction.
  10. 根据权利要求6-7任一项所述的药物制剂，其特征在于，所述大孔树脂的洗脱工艺为：大孔树脂径高比为1：4-9，上样液浓度为0.10-0.30g生药/mL，上样液体积为4-12BV，以1-4BV/h流速上样，用4-8BV纯水和1-5BV的3-15％乙醇以0.5-4BV/h流速进行除杂，用2-8BV 50-80％乙醇以1-5BV/h流速进行洗脱。The pharmaceutical preparation according to any one of claims 6-7, characterized in that the elution process of the macroporous resin is: the diameter-to-height ratio of the macroporous resin is 1:4-9, and the concentration of the sample solution is 0.10- 0.30g crude drug/mL, the sample liquid volume is 4-12BV, load the sample at a flow rate of 1-4BV/h, and remove with 4-8BV pure water and 1-5BV of 3-15% ethanol at a flow rate of 0.5-4BV/h. For impurities, use 2-8BV 50-80% ethanol to elute at a flow rate of 1-5BV/h.
  11. 根据权利要求1-5任一项所述的药物制剂，其特征在于，所述黄蜀葵花提取物的制备方法包括如下步骤：(101)黄蜀葵花或黄蜀葵药用部位用乙醇提取，制得提取液；(102)提取液调节pH值为2.0-3.0，冷藏，过滤取沉淀，加水溶解；(103)溶解液经萃取，制得萃取液；(104)萃取液去除溶剂后经聚酰胺树脂洗脱，制得 黄蜀葵花黄酮类有效部位或提取物；优选提取方法为回流。The pharmaceutical preparation according to any one of claims 1 to 5, characterized in that the preparation method of the marshmallow flower extract includes the following steps: (101) Extracting marshmallow flowers or medicinal parts of marshmallow with ethanol to prepare an extract liquid ; (102) Adjust the pH value of the extract to 2.0-3.0, refrigerate, filter to collect the precipitate, and add water to dissolve; (103) The dissolved liquid is extracted to obtain an extract; (104) The extract is eluted with polyamide resin after removing the solvent ,be made of Effective parts or extracts of flavonoids from marshmallow flowers; the preferred extraction method is reflux.
  12. 根据权利要求11所述的药物制剂，其特征在于，步骤(101)所述乙醇的用量为黄蜀葵花或黄蜀葵药用部位的10-25倍，所述乙醇为60-95％的乙醇溶液；步骤(102)所述的pH调节剂为盐酸、硫酸、醋酸、磷酸、枸橼酸、酒石酸或马来酸；步骤(103)的萃取用的萃取剂为正丁醇、石油醚或乙酸乙酯，所述萃取的方法为连续逆流萃取，所述萃取的料液比为0.8-4：1，所述萃取的萃取级数为1级-5级；步骤(104)所述的树脂径高比为1：4-9，上样液浓度为0.10-0.60g生药/mL，上样液体积为4-12BV，用4-8BV纯水和4-8BV的60-95％乙醇进行洗脱。The pharmaceutical preparation according to claim 11, wherein the amount of ethanol used in step (101) is 10-25 times that of marshmallow flowers or medicinal parts of marshmallow, and the ethanol is a 60-95% ethanol solution; step The pH regulator described in (102) is hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid or maleic acid; the extraction agent used for extraction in step (103) is n-butanol, petroleum ether or ethyl acetate, The extraction method is continuous countercurrent extraction, the material-to-liquid ratio of the extraction is 0.8-4:1, and the extraction level of the extraction is level 1-5; the diameter-to-height ratio of the resin in step (104) is 1:4-9, the concentration of the sample solution is 0.10-0.60g crude drug/mL, the volume of the sample solution is 4-12BV, and elution is carried out with 4-8BV pure water and 4-8BV 60-95% ethanol.
  13. 根据权利要求1-12任一项所述的药物制剂，其特征在于，所述非水性液体的制剂为固体制剂或半固体制剂；进一步选自栓剂、软膏剂、凝胶剂、丸剂、片剂、颗粒剂、胶囊剂或合剂。The pharmaceutical preparation according to any one of claims 1 to 12, wherein the non-aqueous liquid preparation is a solid preparation or a semi-solid preparation; further selected from the group consisting of suppositories, ointments, gels, pills, and tablets. , granules, capsules or mixtures.
  14. 根据权利要求1-12任一项所述的药物制剂，其特征在于，所述药学上可接受的辅料包括溶剂、乳化剂、崩解剂、填充剂、增溶剂、抗氧剂、pH调节剂、渗透压调节剂、抑菌剂、稀释剂、润湿剂、粘合剂或成膜剂中的任一种或任两种组合或任三种组合、或多种组合；进一步地，所述辅料选自乳糖、聚乙二醇、交联聚乙烯吡咯烷酮、月桂醇硫酸镁、羟丙基纤维素、滑石粉、硫酸钙、微晶纤维素、低取代羟丙甲基纤维素、交联羧甲基纤维素钠、硬脂酸镁中的任一种或任两种组合或任三种组合、或多种组合；进一步地，所述填充剂和崩解剂分别为制剂重量的1％-80％，进一步为3％-78％，进一步为5％-75％，进一步为10％-70％、15％-75％、15％-65％、The pharmaceutical preparation according to any one of claims 1 to 12, wherein the pharmaceutically acceptable excipients include solvents, emulsifiers, disintegrants, fillers, solubilizers, antioxidants, and pH adjusters. , any one or any two combinations or any three combinations, or multiple combinations of osmotic pressure regulators, bacteriostatic agents, diluents, wetting agents, adhesives or film-forming agents; further, the Excipients are selected from lactose, polyethylene glycol, cross-linked polyvinylpyrrolidone, magnesium lauryl sulfate, hydroxypropyl cellulose, talc, calcium sulfate, microcrystalline cellulose, low-substituted hydroxypropylmethyl cellulose, cross-linked carboxylic acid Any one, any two combinations, any three combinations, or multiple combinations of sodium methylcellulose and magnesium stearate; further, the filler and disintegrant are respectively 1%-1% of the weight of the preparation. 80%, further 3%-78%, further 5%-75%, further 10%-70%, 15%-75%, 15%-65%,
    20％-70％、20％-60％、25％-65％、25％-55％、30％-60％、30％-50％、35％-55％，进一步为1％-30％、25％-55％、50％-80％。20%-70%, 20%-60%, 25%-65%, 25%-55%, 30%-60%, 30%-50%, 35%-55%, further 1%-30%, 25%-55%, 50%-80%.
  15. 根据权利要求1-12任一项所述的药物制剂，其特征在于，所述药学上的可接受的辅料在制剂中的质量含量为2％-95％；进一步为5％-95％；进一步为10％-90％；进一步为15％-85％；进一步为20％-80％、25％-75％、30％-70％、40％-60％、50％-80％、15％-90％；进一步为20％-85％、4％-50％。The pharmaceutical preparation according to any one of claims 1 to 12, characterized in that the mass content of the pharmaceutically acceptable excipients in the preparation is 2%-95%; further, 5%-95%; further 10%-90%; further 15%-85%; further 20%-80%, 25%-75%, 30%-70%, 40%-60%, 50%-80%, 15%- 90%; further 20%-85%, 4%-50%.
  16. 权利要求1-15任一项所述的药物制剂在制备肾病药物中的应用，或制备眼部疾病药物中的应用，或制备红斑狼疮肾炎，或者造影剂导致的肾损伤、或者肺纤维化、或者心力衰竭、或者降低尿酸的药物中的应用，或制备抗纤维化药物中的应用，或制备治疗和/或预防溃疡性疾病的药物中的应用，或制备防治炎症性肠病产品中的应用，或制备治疗和/或预防皮肤疾病的药物中的应用，或制备促进伤口愈合的药物中的应用；优选地，所述肾病为糖尿病肾病，或者伴有肾纤维化的糖尿病肾病或肾炎、红斑狼疮肾炎，或者造影剂导致的肾损伤；优选所述的肾病为伴有肾纤维化的糖尿病肾病或肾炎；优选地，所述眼部疾病优选为黄斑变性、视觉疲劳或白内障、糖尿病视网膜病变、年龄相关性黄斑变性、中心性渗出性脉络膜视网膜炎、高度近视引起的黄斑病变；优选地，所述纤维化可选自肺纤维化，进一步的为特异性肺纤维化；优选地，所述溃疡性疾病为溃疡性肠炎、溃疡性胃炎、溃疡性咽喉炎和溃疡性口腔炎中的任意一种；优选地，所述的炎性肠病为溃疡性结肠炎和克罗恩病；优选地，所述皮肤疾病为皮疹、痤疮中的任意一种；优选地，所述伤口包括但不限于烧烫灼伤创面、残余小创面、供皮区创面、慢性溃疡创面和新鲜及难愈性皮肤创面；优选地，所述慢性溃疡创面包括但不限于糖尿病性、血管性、放射性溃疡。 The application of the pharmaceutical preparation according to any one of claims 1 to 15 in the preparation of drugs for kidney diseases, or the preparation of drugs for eye diseases, or the preparation of lupus erythematosus nephritis, or kidney damage caused by contrast agents, or pulmonary fibrosis, Or the application in heart failure, or the application of drugs that lower uric acid, or the application in the preparation of anti-fibrosis drugs, or the application in the preparation of drugs for the treatment and/or prevention of ulcerative diseases, or the application in the preparation of products for the prevention and treatment of inflammatory bowel disease. , or application in the preparation of medicines for treating and/or preventing skin diseases, or application in the preparation of medicines for promoting wound healing; preferably, the kidney disease is diabetic nephropathy, or diabetic nephropathy accompanied by renal fibrosis or nephritis, erythema Lupus nephritis, or kidney damage caused by contrast agents; preferably, the kidney disease is diabetic nephropathy or nephritis accompanied by renal fibrosis; preferably, the eye disease is preferably macular degeneration, visual fatigue or cataracts, diabetic retinopathy, Age-related macular degeneration, central exudative chorioretinitis, and macular degeneration caused by high myopia; preferably, the fibrosis can be selected from pulmonary fibrosis, and further is specific pulmonary fibrosis; preferably, the fibrosis can be The ulcerative disease is any one of ulcerative enteritis, ulcerative gastritis, ulcerative pharyngitis and ulcerative stomatitis; preferably, the inflammatory bowel disease is ulcerative colitis and Crohn's disease; preferably , the skin disease is any one of rash and acne; preferably, the wounds include but are not limited to burn wounds, residual small wounds, donor site wounds, chronic ulcer wounds and fresh and difficult-to-heal skin wounds ; Preferably, the chronic ulcer wounds include but are not limited to diabetic, vascular, and radiation ulcers.
PCT/CN2023/081052 2022-03-15 2023-03-13 Pharmaceutical formulation and use thereof WO2023174205A1 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CNPCT/CN2022/080818 2022-03-15
PCT/CN2022/080818 WO2023173268A1 (en) 2022-03-15 2022-03-15 Effective parts of flavonoids from flos abelmoschus manihot, and preparation method therefor and use thereof
CN202210251763.8 2022-03-15
CN202210251763.8A CN116785305A (en) 2022-03-15 2022-03-15 Pharmaceutical composition and application thereof
CN202210990942.3 2022-08-18
CN202210990942.3A CN117582459A (en) 2022-08-18 2022-08-18 Application of flos Abelmoschi Manihot extract in preparing medicine for treating and/or preventing ulcer diseases

Publications (1)

Publication Number Publication Date
WO2023174205A1 true WO2023174205A1 (en) 2023-09-21

Family

ID=88022205

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/081052 WO2023174205A1 (en) 2022-03-15 2023-03-13 Pharmaceutical formulation and use thereof

Country Status (1)

Country Link
WO (1) WO2023174205A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1994337A (en) * 2006-11-11 2007-07-11 周亚球 Total flavone extract of maniod eibish, its preparation and application
CN103610712A (en) * 2013-12-03 2014-03-05 江苏省中医院 Application of sunset abelmoschus flowers and extract thereof to preparation of inflammatory bowel disease prevention and treatment medicament
CN111920836A (en) * 2019-06-13 2020-11-13 江苏苏中药业集团股份有限公司 Application of abelmoschus manihot extract in preparation of medicine for treating fibrosis
CN112870236A (en) * 2019-11-29 2021-06-01 江苏苏中药业集团股份有限公司 Flavone effective part of abelmoschus manihot and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1994337A (en) * 2006-11-11 2007-07-11 周亚球 Total flavone extract of maniod eibish, its preparation and application
CN103610712A (en) * 2013-12-03 2014-03-05 江苏省中医院 Application of sunset abelmoschus flowers and extract thereof to preparation of inflammatory bowel disease prevention and treatment medicament
CN111920836A (en) * 2019-06-13 2020-11-13 江苏苏中药业集团股份有限公司 Application of abelmoschus manihot extract in preparation of medicine for treating fibrosis
CN112870236A (en) * 2019-11-29 2021-06-01 江苏苏中药业集团股份有限公司 Flavone effective part of abelmoschus manihot and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
XU YU, SUN XINYI, ZHU BOYU, LOU YAN, ZHAO YUE: "Research Progress of Hollyhock Flower in the Treatment of Diabetic Retinopathy", MODERN JOURNAL OF INTEGRATED TRADITIONAL CHINESE AND WESTERN MEDICINE, vol. 30, no. 6, 20 February 2021 (2021-02-20), pages 678 - 680, XP093091796 *
XUE-MEI WANG, GAO SU-LIAN, LIU MING: "The application of abelmoschus manihot in cosmatics-anti-acne beauty toner", JOURNAL OF ANHUI UNIVERSITY(NATURAL SCIENCES), vol. 27, no. 1, 28 March 2003 (2003-03-28), pages 79 - 83, XP093091797 *

Similar Documents

Publication Publication Date Title
AU2007307427B2 (en) Composition for treating atopic dermatitis comprising extracts of Bamboo and Scutellaria
RU2759382C2 (en) Drug for injection based on saponin b4 pulsatilla
CN108904685A (en) Purposes of the fritillaria total alkaloids extract in the drug or health care product of preparation prevention and treatment Hashimoto thyroiditis
CN102652778A (en) Medicinal composition
CN101856418B (en) Pharmaceutical preparation for preventing nephritis and preparation method thereof
WO2023174205A1 (en) Pharmaceutical formulation and use thereof
CN112691114B (en) Application of momordica polysaccharide in preparation of medicine for treating ulcerative colitis and pharmaceutical preparation of momordica polysaccharide
CN114869952A (en) Application of cortex lycii radicis extract in preparation of product for repairing reproductive system injury
CN111494489B (en) Gel sustained-release agent for treating otitis externa and preparation method thereof
CN112076249B (en) Application of perilla leaf extract in preparing medicament for treating inflammatory bowel disease
WO2023173268A1 (en) Effective parts of flavonoids from flos abelmoschus manihot, and preparation method therefor and use thereof
CN103083289A (en) Application of curcumin in preparation of medicine for treating psoriasis
CN111407783B (en) Application of penthorum chinense pursh in preparation of medicine for treating high proteinuria
CN110279754A (en) A kind of Chinese medicinal granule that treating suppurative dermatosis, preparation method and applications
CN117582459A (en) Application of flos Abelmoschi Manihot extract in preparing medicine for treating and/or preventing ulcer diseases
CN115252631B (en) Application of pseudo-ginseng extract in preparation of medicine for treating diabetic nephropathy
CN107281209B (en) A kind of pharmaceutical composition of anti-gastric-ulcer and preparation method thereof and purposes
CN103566276B (en) A kind of Tibetan medicinal composition being used for the treatment of gastropathy and preparation method thereof
CN111643498B (en) Pharmaceutical composition for treating kidney stone and application thereof
CN110721193B (en) Application of cynomorium songaricum total polysaccharide in preparation of medicine for treating asthma
CN116688000B (en) Application of ramulus mori total alkaloids in preparation of medicines for treating polycystic ovary syndrome (PCOS)
CN102258557A (en) Medicament for treating prostatic hyperplasia
TWI661832B (en) Use of hsian-tsao (mesona procumbens hemsl.) extracts for preparing a pharmaceutical composition for promoting wound healing
CN116920019A (en) Application of haw extract in preparing medicine for treating constipation
CN117618498A (en) External therapeutic medicine for wounds

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23769708

Country of ref document: EP

Kind code of ref document: A1