WO2022228095A1 - Application of prismatomeris connata y. z. ruan root extract in preparation of medicine for treating pulmonary fibrosis - Google Patents

Application of prismatomeris connata y. z. ruan root extract in preparation of medicine for treating pulmonary fibrosis Download PDF

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WO2022228095A1
WO2022228095A1 PCT/CN2022/086065 CN2022086065W WO2022228095A1 WO 2022228095 A1 WO2022228095 A1 WO 2022228095A1 CN 2022086065 W CN2022086065 W CN 2022086065W WO 2022228095 A1 WO2022228095 A1 WO 2022228095A1
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nanshan
flower root
extract
pulmonary fibrosis
anthraquinone
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PCT/CN2022/086065
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French (fr)
Chinese (zh)
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靳洪涛
张金兰
胡广
生宁
李思铮
李梦林
江海燕
王冬梅
王喆
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中国医学科学院药物研究所
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    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4418—Non condensed pyridines; Hydrogenated derivatives thereof having a carbocyclic group directly attached to the heterocyclic ring, e.g. cyproheptadine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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)
    • A61K36/74—Rubiaceae (Madder family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of medicine, and in particular relates to the application of extraction parts and compounds obtained by extracting the root of the traditional Chinese medicine Nanshan flower in preventing and treating pulmonary fibrosis diseases.
  • Pulmonary fibrosis is a type of disease in which diffuse exudation, infiltration and fibrosis of the pulmonary interstitium are the main lesions. Its main symptoms include dyspnea, shortness of breath, dry cough, and wheezing. die.
  • idiopathic interstitial pneumonia IIP
  • Idiopathic pulmonary fibrosis IPF
  • Idiopathic pulmonary fibrosis IPF is the most common type of disease with pulmonary fibrosis as the main manifestation in idiopathic interstitial pneumonia. Sexual lung disease.
  • Pirfenidone was first used in Japan for the treatment of IPF in 2008, and in 2014, pirfenidone and nintedanib were approved by the US FDA for the specific treatment of IPF.
  • neither of these two drugs can reverse or prevent the progression of IPF, and they are expensive, placing a greater burden on patients and their families.
  • Pulmonary fibrosis can be divided into primary pulmonary disease and partial manifestations of systemic disease.
  • the types of pulmonary fibrosis as primary lung diseases include: radiation fibrosis (ie, fibrosis caused by radiation), fibrosis as a result of damaging inflammatory lung lesions (such as sclerosing tuberculosis, honeycomb lung, Small cystic pulmonary fibrosis), fibrosis as the result of allergic reaction, drug-induced interstitial fibrosis, fibrosis as the end-stage manifestation of interstitial inflammation, etc.; (2) pulmonary fibrosis is the result of systemic disease Some manifestations include: granulomatous disease (such as sarcoidosis, amyloidosis, nevus hamartoma, xanthoma), collagen disease and rheumatic disease (such as scleroderma, lupus erythematosus, nodular disease) periarteritis, dermatomyositis,
  • Nanshan flower is also known as dog bone wood. It is the root of Prismatomeris connata Y.Z. Ruan, a Rubiaceae plant, and is one of the characteristic medicinal materials in Guangxi. There are strong roots and bones, dampness and yellowing, blood stasis and regeneration, cooling blood and hemostasis.
  • Nanshan flower root extract can inhibit liver fibrosis by inducing apoptosis of rat astrocytes.
  • scopolide can protect the liver of hepatic fibrosis rats, positively regulates the expression of cytokines related to hepatic fibrosis, It can reduce the level of extracellular matrix in liver, improve the degree of hepatic fibrosis in rats, and has anti-hepatic fibrosis effect.
  • the prior art Journal of Beijing University of Traditional Chinese Medicine, Vol. 32, No. 1, Pages 42-44 discloses that the alcohol extract of Nanshan flower root can significantly reduce the proliferation and deposition of collagen fibers in the liver of rats, and reduce the area of fibrotic tissue. It has certain development value in the treatment of liver fibrosis.
  • liver fibrosis is mostly fibrosis caused by bacterial and viral infection, toxin damage, and abnormal metabolism. It is currently believed that liver fibrosis is a reversible process. Pulmonary fibrosis is considered to be an irreversible process, and the lungs are directly connected to the outside world. The causes of pulmonary fibrosis are more complicated, and the etiology of most patients is unknown. From the prior art, many drugs have the effect of preventing and treating liver fibrosis, but the drugs that clearly have the effect of preventing and treating pulmonary fibrosis are very rare.
  • pirfenidone and nintedanib are known to be For pulmonary fibrosis, that is to say, drugs used to prevent and treat liver fibrosis are generally not used to prevent and treat pulmonary fibrosis.
  • Guangxi Yingkang Pharmaceutical Co., Ltd. has developed a tablet prepared from Nanshan flower for the treatment of silicosis, and its indication is "treatment of silicosis”.
  • liquid, filtered, the filtrate was decompressed to recover ethanol, concentrated to a thick paste, stirred three times with 10 times the amount of warm water, allowed to stand, filtered, the filtrate was concentrated under reduced pressure, vacuum-dried, pulverized, added with appropriate amount of auxiliary materials, made into granules, Compressed into 1,000 tablets, coated with sugar, and obtained.
  • silicosis is known to be a lung disease caused by inhalation of silica or silica and its crystalline forms such as quartz and other less common forms such as cristobalite and phosphoquartz, and its rapidly progressive manifestations are similar to interstitial pneumonia. performance, and pulmonary fibrosis is not the same.
  • the main active ingredient of the above-mentioned tablet is a polymer formed by a variety of metal elements mainly composed of organoaluminum, and the mechanism of action is that aluminum forms insoluble aluminum silicate on the surface of silicon dioxide, which makes it difficult to dissolve.
  • the preparation method of the polysaccharide is as follows: after crushing 10 kg of decoction pieces, extracting with ultrasonic heating water, concentrating under reduced pressure, and precipitating with 80% ethanol to obtain 905 g of crude polysaccharide. After the crude polysaccharide was redissolved in distilled water, the protein was removed by the Savage method, passed through the filter membrane U under high pressure, and then concentrated through the ultrafiltration membrane A to remove low molecular weight components, and the concentrate was dried to obtain 560 g of polysaccharide.
  • the above content suggests that polysaccharide components are the active components of Nanshan flower root in preventing and treating pulmonary fibrosis.
  • the present invention is proposed in order to find an effective part having a definite effect on prevention and treatment of pulmonary fibrosis from Nanshan flower root, and to provide its pharmaceutical use.
  • the purpose of the present invention is to provide a new pharmaceutical use of the Nanshan flower root extract, that is, the use of the Nanshan flower root extract in the preparation of a medicine for treating pulmonary fibrosis.
  • the anti-pulmonary fibrosis effective parts of the extract are discovered for the first time, and the preparation method is simple, the effective parts are clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is obviously better than the prior art, and can be combined with the existing technology.
  • Anti-pulmonary fibrosis chemicals have the advantages of synergistic effect.
  • effective part refers to an extract composed of one or several types of components extracted from a single plant with relatively clear components, which can also be called effective components or effective intermediates ("Quality of New Chinese Medicines"). Research Technical Guiding Principles (for Trial Implementation, January 2021). "Anthraquinones, Anthraquinone Glycosides and Iridoids Effective Parts” means that the compounds of anthraquinones, anthraquinone glycosides and iridoids in the extract are the constituents of the extract. Although the extract may also contain other components derived from the same plant, these other components are usually not chemical components that can effectively play a therapeutic role in the extract. Because its content is lower than the threshold for exerting a therapeutically effective effect or itself does not have a desired therapeutically effective effect, etc.
  • the object of the present invention is to provide a kind of preparation method of the above-mentioned effective parts of Nanshan flower root.
  • the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of anthraquinones, anthraquinone glycosides and iridoids active ingredients of Radix Nanshanii.
  • the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of scopolide and methylisorubidin-1-methyl ether.
  • the object of the present invention is to provide a pharmaceutical composition for the treatment of pulmonary fibrosis, which combines the above-mentioned Nanshan flower root extract with anti-pulmonary fibrosis chemical drug pirfenidone or nintedanib, and has Obvious synergistic effect.
  • Nanshan flower root extract in preparing a medicine for preventing and treating pulmonary fibrosis, characterized in that the Nanshan flower root extract is anthraquinones, anthraquinone glycosides and iridoids effective parts.
  • the active ingredients of anthraquinones, anthraquinone glycosides and iridoids account for more than 60% of the content of the extract, more preferably more than 70%, and most preferably 100% by weight.
  • the anthraquinone active ingredients include 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylisorubicin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy anthraquinone -3-Hydroxymethyl anthraquinone, desmethyl oxal, methyl isoalizarin-1-methyl ether, ox aldehyde, 1,3-dihydroxy-2-methoxymethyl anthraquinone, 3-hydroxy -1-Methoxy-2-hydroxymethylanthraquinone, 1,3-dihydroxy-2-ethoxymethylanthraquinone, 1-hydroxy-2,3-dimethoxy-7-methylanthraquinone , 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5,6 -Dimethoxy-2-methyl
  • the anthraquinone glycoside active ingredients include one or more of lucidin 3-O- ⁇ -primeveroside, damnacanthol 3-O- ⁇ -primeveroside, and rubiadin 3-O- ⁇ -primerveroside.
  • the iridoid active ingredients include one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, and asperulosidic acid.
  • the Nanshan flower root extract contains at least 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin Alizarin and scopolactone.
  • the Nanshan flower root extract contains at least scopolactone and methylisorubidin-1-methyl ether.
  • the combination of scopolactone and methylisorubidin-1-methyl ether produces a synergistic effect, and its effect is obviously better than that of scopolide and methylisorubidin-1-methyl ether.
  • the present invention also provides the use of scopolide in preparing a medicine for preventing and treating pulmonary fibrosis.
  • the present invention also provides the use of methylisorubicin-1-methyl ether in preparing a medicine for preventing and treating pulmonary fibrosis.
  • the extract of the present invention has a comprehensive prevention and treatment effect on pulmonary fibrosis, can improve pulmonary fibrosis caused by a variety of different factors, can improve lung function, and reduce collagen deposition in lung tissue and fibroblast proliferation and activation. , can inhibit lung tissue TGF- ⁇ and TNF- ⁇ and inhibit pulmonary fibrosis.
  • the ethyl acetate extract of Nanshan flower root can reduce the elevated expression of COL1A1, COL3A1 and ⁇ -SMA proteins caused by pulmonary fibrosis in mice. Its action pathway is not through the formation of insoluble aluminum silicate on the surface of silica, which makes it lose its toxic effect on macrophages.
  • the pulmonary fibrosis is not caused by silicosis.
  • the present invention also provides the preparation method of the Nanshan flower root extract, namely extracting Nanshan flower root with ethanol, then using preparative chromatography to remove impurities, and refining and purifying to obtain anthraquinones, anthraquinone glycosides and iridoids
  • the active ingredient is obtained by extracting Nanshan flower root with ethyl acetate solvent; or after extracting Nanshan flower root with solvent, concentrating the extract, and then extracting with ethyl acetate.
  • the solvent is one or a combination of two or more of water, methanol, ethanol, propanol, butanol and the like.
  • the Nanshan flower root is extracted with petroleum ether first, the extract is discarded, then extracted with ethyl acetate, and concentrated under reduced pressure to obtain the ethyl acetate extract.
  • the preparation method can also be as follows: extracting Nanshan flower root with water, then extracting with 95% ethanol, combining water and ethanol extracts, concentrating the extract under reduced pressure, extracting with ethyl acetate and then rotary evaporation to remove the solvent obtained from Nanshan Flower root extract.
  • the preparation method can also be as follows: 95% ethanol reflux extraction of Nanshan flower root three times, the extract is concentrated under reduced pressure, the impurities are removed by preparative chromatography, and the anthraquinones, anthraquinone glycosides and iridoids are obtained by refining and purification
  • the chromatographic method can be one or more of macroporous resin method, molecular sieve chromatography, silica gel adsorption chromatography, polyamide adsorption chromatography, etc. Preferred is silica gel column chromatography, eluting with a gradient of water and acetonitrile.
  • the preparation method can also be: the Nanshan flower root extract obtained by refluxing extraction with 95% ethanol for three times, ethyl acetate extraction, and rotary evaporation to remove the solvent.
  • the present invention also provides a pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that it is composed of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids in Nanshan flower root.
  • a pharmaceutical composition for treating pulmonary fibrosis which is characterized in that it is composed of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids in Nanshan flower root.
  • the weight ratio of the above three effective extraction parts is 1-10:1-10:1-5.
  • the pharmaceutical composition for treating pulmonary fibrosis is composed of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methyl Composed of isolarin and scopolactone.
  • the weight ratio of the above four compounds is 1-10:1-10:1-10:1-10.
  • the pharmaceutical composition for treating pulmonary fibrosis is composed of scopolactone and methylisorubicin-1-methyl ether.
  • the weight ratio of the above two compounds is 1-10:1-10.
  • the present invention also preferably provides a pharmaceutical composition for the treatment of pulmonary fibrosis, which is characterized in that it is composed of the above-mentioned Nanshan flower root extract and an anti-pulmonary fibrosis chemical, and the anti-pulmonary fibrosis chemical is pyridine Nintedone or nintedanib.
  • the combination of the Nanshan flower root extract and the chemical drug against pulmonary fibrosis has a synergistic effect, and its effect is obviously better than the simple superposition of the respective effects of the Nanshan flower root extract and the chemical drug for preventing and treating pulmonary fibrosis.
  • the weight ratio of Nanshan flower root extract and anti-pulmonary fibrosis chemical is 2-20:1-10.
  • the medicine for preventing and treating pulmonary fibrosis according to the present invention can prevent and/or treat pulmonary fibrosis, and the medicine is composed of active ingredients and pharmaceutically acceptable excipients, and the active ingredients are Nanshan flower root extract, scopolamine Any one of lactone, methylisorubicin-1-methyl ether, and the pharmaceutical composition.
  • the content of the active ingredient is 0.1-99.9% (w/w), and the content of pharmaceutically acceptable excipients is 0.1-99.9% (w/w), and the total is 100% .
  • the medicament of the present invention can be in any pharmaceutically acceptable dosage form, and these dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, buccal preparations, granules, granules, Pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, aerosols, drops, patches.
  • excipients can be used for the medicine of the present invention, and specific examples of excipients include: fillers, diluents, carriers, excipients, disintegrants, binders, lubricants, flavoring agents, surfactants, colorants , coating agent, propellant, stabilizer, etc.
  • a suitable pharmaceutically acceptable carrier can be optionally added when preparing a medicament, and the pharmaceutically acceptable carrier is selected from: starch, sucrose, lactose, mannitol, mannitol, sorbitol, sodium metabisulfite, hydrogen sulfite Sodium, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, Acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, silicon derivatives, cellulose and its derivatives, alginate, gelatin , polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbon
  • the effective anti-pulmonary fibrosis part of the extract is discovered for the first time, the preparation method is simple, the effective part is clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is comprehensive. It is obviously superior to the prior art, wherein the two specific compounds have a synergistic effect against pulmonary fibrosis, and the extract can also have the advantages of synergistic effect with anti-pulmonary fibrosis chemical drugs.
  • Figure 1 Effects of different extraction parts of Nanshan flower root on lung function in mice with bleomycin-induced pulmonary fibrosis.
  • Figure 2 The effect of different extraction parts of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.
  • Fig. 3 Effects of different extraction parts of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
  • Figure 4 Effects of different preparation techniques of Nanshan flower root on lung function of mice with bleomycin-induced pulmonary fibrosis.
  • Figure 5 Effects of different preparation processes of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.
  • Figure 6 Effects of different preparation techniques of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
  • Figure 7 Effects of different preparation techniques of Nanshan flower root on the expression of TGF- ⁇ and TNF- ⁇ in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
  • Figure 8 Effects of different preparation techniques of Nanshan flower root on the expression of COL1A1, COL3A1 and ⁇ -SMA proteins in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
  • Fig. 9 Effects of scopolactone and methylisorubicin-1-methyl ether on the expression of ⁇ -SMA protein after TGF- ⁇ 1-induced fibroblast activation.
  • Figure 10 The effect of the pharmaceutical combination of the present invention on the expression of ⁇ -SMA protein after TGF- ⁇ 1-induced fibroblast activation.
  • Nanshan flower root extract Take 1 kg of Nanshan flower root, extract 3 times with 8 times the amount of 95% ethanol, combine the extracts, concentrate under reduced pressure, extract 3 times with 100 ml of ethyl acetate, combine the extracts, concentrate under reduced pressure to remove the solvent, and then dry to obtain the obtained 3 g of Nanshan flower root extract was detected by chromatography, and the content of anthraquinones, anthraquinone glycosides and iridoids accounted for 64% of the total extract.
  • Nanshan flower root Take 1kg of Nanshan flower root, extract twice with 8 times the amount of water, and then extract twice with 5 times the amount of 95% ethanol, combine the water and ethanol extracts, concentrate the extract under reduced pressure, extract with 200ml of petroleum ether, and discard the extract , and then extracted 3 times with 100ml of ethyl acetate, combined the extracts, concentrated under reduced pressure to remove the solvent and then dried.
  • the obtained Nanshan flower root extract 8g was detected by chromatography, among which anthraquinones, anthraquinone glycosides and cycloalkenyl ethers The content of terpenoid active ingredients accounted for 71% of the total extract.
  • Nanshan flower root Take 1kg of Nanshan flower root, pulverize it to a fineness of about 50 mesh, first extract it once with an appropriate amount of petroleum ether, discard the petroleum ether extract, and then extract three times with 5 times the amount of ethyl acetate, combine the extracts, and concentrate and dry under reduced pressure. 11 g of the ethyl acetate extract was obtained, and the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounted for 79% of the total amount of the extracts through chromatographic detection.
  • the adopted chromatographic column is a C18 column, and the mobile phase is the mobile phase A phase: Water (0.1% formic acid, 1 mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1 mmol/L ammonium acetate), gradient elution conditions: 0-10min, 20%B; 10-60min, 20- 100% B, flow rate: 0.3 mL/min, column temperature: 30°C.
  • the three active components of anthraquinones, anthraquinone glycosides and iridoids were collected in sections, and concentrated and dried under reduced pressure to obtain the purified three active components of anthraquinones, anthraquinone glycosides and iridoids. part.
  • the above three kinds of effective parts can also be mixed evenly to obtain the Nanshan flower root extract. After chromatographic detection, the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounts for 100% of the total amount of the extract. %.
  • Example 1 Take the Nanshan flower root extract of Example 1 and pirfenidone, and mix them in a weight ratio of 10:1.
  • Nanshan flower root extract of Example 1 Take the Nanshan flower root extract of Example 1 and nintedanib, and mix them in a weight ratio of 8:1.
  • Nanshan flower root extract in Example 2 Take 10 parts by weight of Nanshan flower root extract in Example 2, add 12 parts by weight of lactose and 2 parts by weight of microcrystalline cellulose, dry granulation, sieve, granulate, put into capsules, and obtain capsules.
  • Example 6 Take the pharmaceutical composition of Example 6, add appropriate amount of surfactant, propellant and stabilizer to prepare an inhalation spray.
  • SPF pathogen-free
  • mice About 80 SPF grade male C57BL/6N mice were selected, 10 were used as blank control group, and the rest were treated with tracheal instillation of bleomycin to cause pulmonary fibrosis.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital 80 mg/kg, and the upper incisors of the mice were fixed on the tracheal instillation console.
  • a trocar was inserted into the mouse trachea, connected to an HRH-MAG4 pulmonary liquid quantitative nebulizer, and quickly pushed into 50 ⁇ L of sterile PBS-dissolved bleomycin solution at a dose of 0.04 U per mouse.
  • mice After administration, the mice were kept upright and swirled to make the solution evenly distributed in the lungs, and water and feed were given to the mice after they recovered, and the pulmonary fibrosis mouse model was established. In the blank control group, other operations were the same except that sterile PBS solution was administered to the trachea.
  • the animals in the surviving modeling group were randomly divided into model group, nintedanib positive drug group, Huanggen tablet group, Nanshan flower root ethyl acetate extract group, and Nanshan flower root n-butanol extract group. , Nanshan flower root water extract group.
  • mice in each group were given the corresponding drug intervention by gavage every day for 14 days.
  • the blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution,
  • the administration dose was 50 mg/kg; the Huanggen tablet group was administered with Huanggen tablet suspension, the administration dose was 200 mg/kg; the Nanshan flower root extract administration group was given Nanshan flower root ethyl acetate extract, Nanshan flower root extract Butanol extract, Nanshan flower root water extract suspension, dosage 100mg/kg.
  • mice The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis. Each lobe of the right lung was taken and rinsed in normal saline, and then the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content in lung tissue.
  • HEP hydroxyproline
  • the observation by the cage includes but is not limited to the following indicators: the death of the mice, whether there are wet rales, nasal sprays, arched back, erect hair, etc., as well as changes in limb activity and behavior. Record the occurrence time, extent and duration, etc. The body weight of the mice before and after the modeling, and before the mice were sacrificed, were recorded.
  • mice On the 14th day of administration, the mice were fully anesthetized by intraperitoneal injection of pentobarbital sodium 90 mg/kg within 24 hours after the last administration, and the mice were placed in the supine position on the dissection board.
  • the skin of the mouse neck was wiped with alcohol, the skin was cut open, the neck muscles were bluntly separated, the trachea was exposed, a small incision was made transversely under the thyroid cartilage, a tracheal cannula was inserted and ligated with sutures.
  • tracheal intubation and Flexivent instrument detect mouse total inspiratory volume (IC) by Deep Inflation module, detect respiratory system resistance (Rrs), compliance (Crs), elasticity (Ers) by SnapShot-150 module, Quick Prime-3 The module detects the main airway resistance (Rn), tissue damping (G) and tissue elasticity (H) and other indicators, and PVs-P draws the P-V loop of mouse breathing.
  • IC total inspiratory volume
  • RVs respiratory system resistance
  • Crs compliance
  • Ers elasticity
  • SnapShot-150 module Quick Prime-3
  • the module detects the main airway resistance (Rn), tissue damping (G) and tissue elasticity (H) and other indicators, and PVs-P draws the P-V loop of mouse breathing.
  • mice The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 ⁇ m and baked at 60°C for 1 h.
  • the Nanjing Jiancheng Hydroxyproline Detection Kit was used to detect the content of hydroxyproline in the right lung tissue of mice according to the instructions.
  • the specific steps are as follows: (1) Sample hydrolysis: Accurately weigh 20-40 mg of tissue wet weight into a 15 mL centrifuge tube, Add 1 mL of the hydrolyzate and mix well. Cover with a boiling water bath for 10 minutes, shake well, and continue the boiling water bath for 10 minutes to fully hydrolyze. (2) Adjust pH: add 10 ⁇ L of indicator after cooling down the centrifuge tube, shake well, and adjust the pH to about 6.0-6.8. Add double distilled water to 10mL and mix well.
  • Sample detection It is divided into blank tube, standard product tube, and measuring tube, except that 1 mL of double-distilled water is added to the blank tube, 1 mL of standard application solution with a concentration of 5 ⁇ g/mL is added to the standard product tube, and 1 mL of the sample to be tested is added to the sample tube. , the operation is the same.
  • mice The body weight of the mice before and after modeling was recorded, and the results are shown in Table 1: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the body weight of the nintedanib and Nanshan flower root ethyl acetate extract groups improved. effect.
  • the results of respiratory function are shown in Table 2 and Figure 1: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the ethyl acetate extract of Nanshan flower root can effectively improve the respiratory system of the mice The total system capacity and respiratory system compliance were better than those of the Huanggen tablet group, and the results were statistically different. It shows that the ethyl acetate extract of Nanshan flower root can improve the respiratory function of mice.
  • mice (unit g, compared with blank group, # p ⁇ 0.05, ## p ⁇ 0.01, compared with model group, *p ⁇ 0.05)
  • Nanshan flower root The effect of different extraction parts of Nanshan flower root on the content of hydroxyproline in mouse lung tissue.
  • Hydroxyproline accounts for 13.4% in collagen, a very small amount in elastin, and does not exist in other proteins.
  • pulmonary fibrosis the main component that increases is collagen fibers, and the content of hydroxyproline in lung tissue can be converted into the content of lung collagen to reflect the degree of pulmonary fibrosis.
  • Table 3 and Figure 3 The hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p ⁇ 0.01).
  • the ethyl acetate extract of Nanshan flower root showed statistical difference (p ⁇ 0.01).
  • the results showed that the ethyl acetate extract of Nanshan flower root could reduce the collagen deposition and pulmonary fibrosis in mice induced by bleomycin.
  • SPF pathogen-free
  • mice About 80 SPF grade male C57BL/6N mice were selected, 10 were used as blank control group, and the rest were treated with tracheal instillation of bleomycin to cause pulmonary fibrosis.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital 80 mg/kg, and the upper incisors of the mice were fixed on the tracheal instillation console.
  • a trocar was inserted into the mouse trachea, connected to an HRH-MAG4 pulmonary liquid quantitative nebulizer, and quickly pushed into 50 ⁇ L of sterile PBS-dissolved bleomycin solution at a dose of 0.04 U per mouse.
  • mice After administration, the mice were kept upright and swirled to make the solution evenly distributed in the lungs, and water and feed were given to the mice after they recovered, and the pulmonary fibrosis mouse model was established. In the blank control group, other operations were the same except that sterile PBS solution was administered to the trachea.
  • the body weight of the surviving model animals After 14 days, according to the body weight of the surviving model animals, they were randomly divided into the model group, the nintedanib positive drug group, the embodiment 1 group, the embodiment 2 group, the embodiment 3 group, and the embodiment 4 group, with 10 animals in each group. .
  • mice in each group were given the corresponding drug intervention by gavage every day for 14 days.
  • the blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution, The administration dose was 50 mg/kg;
  • Example 1-Example 4 groups were respectively administered the Nanshan flower root extract suspension prepared with 0.5% CMC-Na aqueous solution, and the administration dose was 100 mg/kg.
  • mice The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis.
  • the lobes of the right lung were taken and rinsed in normal saline, and the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content, TGF- ⁇ and TNF- ⁇ content in lung tissue. and Western Blot to detect the expression of pulmonary fibrosis-related proteins.
  • HEP hydroxyproline
  • mice The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 ⁇ m and baked at 60°C. After dewaxing with xylene, hydration with gradient ethanol, antigen retrieval (0.01M citrate buffer, pH 6.0, high pressure repair for 2 min); blocking endogenous catalase with 3% hydrogen peroxide, 5% BSA at room temperature closed.
  • Sections were incubated with ⁇ -SMA primary antibody overnight at 4°C. After the sections were washed with PBS buffer, the corresponding secondary antibodies were incubated at room temperature for 1 h. Freshly prepared DAB chromogenic reagent was added dropwise, hematoxylin counterstained, hydrochloric acid alcohol differentiation, dehydration and drying with gradient ethanol, and xylene was transparent. Staining was quantitatively analyzed using ImageJ v1.50C image software. Five different fields of view were randomly selected for each section, and the relative area of positive expression was analyzed.
  • Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of PBS buffer after weighing, and use a homogenizer to fully homogenize. Centrifuge at 3500rpm for 20min, and collect the supernatant for ELISA test. The supernatant was collected and the contents of TNF- ⁇ and TGF- ⁇ in the lung tissue were detected according to the instructions of the rat TNF- ⁇ and TGF- ⁇ kit of Jiangsu Jingmei Biotechnology Co., Ltd. The total protein concentration of tissue was detected by BCA protein quantification kit, and the results were expressed as the ratio of the content of TNF- ⁇ or TGF- ⁇ to total protein.
  • Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of RIPA lysis buffer containing 1% protease inhibitor and 1% protein phosphatase inhibitor after weighing, grind and lyse on ice for 45 min. The lysate was centrifuged at 12,000 rpm at 4°C for 20 min, and the supernatant was taken, and the protein concentration was measured using the BCA protein assay kit.
  • 50 ⁇ g protein sample was diluted with 5 ⁇ loading buffer, heated at 100°C for 5 minutes, electrophoresed in SDS-PAGE gel, then transferred to PVDF membrane, blocked with 5% nonfat milk powder for 1h, and primary antibody at 4°C After incubation overnight, the membrane was washed three times with TBST buffer and incubated with the corresponding secondary antibody for 1 h at room temperature. After washing, the membrane was developed with ECL chemiluminescence solution. After recording by chemiluminometer exposure, bands were analyzed using ImageJ.
  • the experimental results are expressed as mean ⁇ standard deviation Statistical software SPSS 20 was used for analysis, Student's t test was used to compare the model group with the blank group, One Way ANOVA was used between each administration group and the model group, and the homogeneity of variance was tested by Homogeneity. Least significant test (LSD) test; Tamhane's T2 test was used if the variance was unequal. The difference was considered significant at p ⁇ 0.05.
  • mice The body weight, mortality and respiratory function of mice were recorded after modeling and after treatment with Nanshan flower root extract.
  • the body weight results are shown in Table 4: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the Nanshan flower root extracts with different preparation methods recovered to varying degrees after treatment, and the mortality rate of the mice in the administration group was lower than the model group.
  • the results of respiratory function are shown in Table 5 and Figure 4: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the examples 1-4 groups can effectively improve the total respiratory system of the mice. Volume and respiratory compliance, the results were statistically different. It is shown that the groups of Examples 1-4 can improve the respiratory function of mice.
  • Nanshan flower root extracts The effects of different preparation methods of Nanshan flower root extracts on the degree of bleomycin-induced pulmonary fibrosis in mice.
  • Masson staining (Fig. 5) showed that in the blank control group, there was only positive staining of blue collagen around the bronchial wall and blood vessel wall, and the morphological structure was basically normal.
  • the mice in the model group developed areas of pulmonary fibrosis, and collagen fibers proliferated and thickened.
  • the administration groups had different degrees of improvement, among which the positive drug nintedanib group and the Example 4 group had the best improvement effect.
  • the percentage of positive area is shown in the bar graph in Figure 2.
  • Nanshan flower root extract The effect of different preparation methods of Nanshan flower root extract on the content of hydroxyproline in mouse lung tissue.
  • the results are shown in Table 6 and Figure 6.
  • the hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p ⁇ 0.01).
  • the hydroxyproline content in the lung tissue of the Nanshan flower root administration groups with different concentrations decreased in a dose-dependent manner, and there was a statistical difference between each example group and the model group (p ⁇ 0.01).
  • the results show that Nanshan flower root extract can reduce the collagen deposition and pulmonary fibrosis in mice caused by bleomycin, among which Example 4 has the best effect.
  • TGF- ⁇ is the most important regulator of tissue fibrosis caused by dysregulation of extracellular matrix synthesis and deposition, and is associated with various organ fibrosis, sclerosis, and atherosclerosis.
  • TNF- ⁇ is a cytotoxic factor that plays a key role in the pathogenesis of pulmonary fibrosis. Therefore, we detected TGF- ⁇ and TNF- ⁇ content in mouse lung tissue by ELISA. The results are shown in Table 7 and Figure 7. The contents of TGF- ⁇ and TNF- ⁇ in the lung tissue of the mice in the model group increased, and there was a statistical difference; the administration group decreased the contents of TGF- ⁇ and TNF- ⁇ in the lung tissue. The results showed that the corresponding extracts of Nanshan flower root may inhibit the progression of pulmonary fibrosis by affecting the expression of TGF- ⁇ and TNF- ⁇ .
  • fibroblasts are activated to release a large amount of extracellular matrix.
  • the main components of extracellular matrix are COL1A1 and COL3A1, and ⁇ -SMA is the main marker of fibroblast activation.
  • COL1A1 and COL3A1 proteins are expressed in mouse lung tissue by Western Blot.
  • ⁇ -SMA is the main marker of fibroblast activation.
  • Nanshan flower root extract can reduce the elevated expression of COL1A1, COL3A1 and ⁇ -SMA proteins caused by pulmonary fibrosis in mice, and the effect of Example 4 is the most significant.
  • Fibroblasts do not express ⁇ -actin ( ⁇ -SMA) in the unactivated state, after being activated by TGF- ⁇ 1, fibroblasts are activated to myofibroblasts and stop proliferating, express ⁇ -SMA and release extracellular matrix related proteins.
  • ⁇ -SMA ⁇ -actin
  • Human lung fibroblast cell line HPF cells were purchased from Shanghai Enzyme Link Biotechnology Co., Ltd., human recombinant TGF- ⁇ 1 was purchased from Peprotech Company, and scopolide and methylisorubidin-1-methyl ether were purchased from Beijing Biterenkang Biotechnology Co., Ltd. Pharmaceutical Technology Co., Ltd.
  • the HPF cells were spread on a 6-well plate, and the medium was discarded after growing to about 70%.
  • the negative control group was added with 1 mL of serum-free medium (containing 0.1% DMSO), and the modeling group and the administration group were added with 1 mL containing 10ng/ml.
  • TGF- ⁇ 1 serum-free medium, scopolactone or methylisorubicin-1-methyl ether were added to the administration group to make the final concentration reach 5 ⁇ g/mL, and the culture plate was shaken horizontally to mix the liquid. Place in a 37 °C, 5% CO2 incubator for 48 h.
  • the experimental materials and experimental methods were the same as those in Experimental Example 3, and the administration groups were the nintedanib group and the Examples 1, 5, 6, 7, 8, and 9 groups, respectively.
  • the administration dose of nintedanib group, Example 8 and Example 9 group was 2 ⁇ M, and the administration dose of the other example groups was 5 ⁇ g/mL.
  • Example 1 is the ethyl acetate extract of Nanshan flower root;
  • Example 5 drug combination is anthraquinones, anthraquinone glycosides and iridoids are mixed in a weight ratio of 8:1:1;
  • Example 6 drug combination For 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin and scopolactone according to 1:5: Mixing in a weight ratio of 5:1: the drug combination in Example 7 is scopolactone and alizarin-1-methyl ether, which are mixed in a weight ratio of 1:1; the drug combination in Example 8 is Nanshan in Example 1 The flower root extract and pirfenidone were mixed in a weight ratio of 10:1; the drug combination in Example 9 was that the Nanshan flower root extract and nintedanib of Example 1 were mixed in a weight ratio of 8:1.
  • the instructions combine the Nanshan flower root extract with chemical drugs such as pirfenidone or nintedanib, which has a synergistic effect.
  • the chromatographic column is Waters BEH C18 (2.1*100mm, 1.7 ⁇ m), mobile phase A phase: water (0.1% formic acid, 1mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1mmol/L ammonium acetate) , gradient elution conditions: 0-5min, 20%B; 5-30min, 20-100%B, flow rate: 0.3mL/min, column temperature: 30°C.
  • Adopt the negative ion acquisition mode of primary high resolution (HRMS) and secondary high resolution (HRMS/MS) mass spectrometry drying gas temperature: 280 °C, drying gas flow rate: 11L/min, sheath gas temperature: 325 °C, sheath gas flow rate: 12L /min, scanning range of primary mass spectrometry: 150-1200 m/z, scanning range of secondary mass spectrometry: 50-1200 m/z, secondary collision voltage: 10/20/30/40/50V.
  • first-order high-resolution mass spectrometry (HRMS) data were collected.
  • the HRMS data was compared with the self-built database to identify the traditional Chinese medicine components in the extract of Nanshan flower root, and the secondary high-resolution mass spectrometry (HRMS/MS) data of the traditional Chinese medicine component in the Nanshan flower root extract was further collected under different collision voltages.
  • Thirty traditional Chinese medicine components in Nanshan flower root extract were identified, including 23 anthraquinones, 4 iridoids and 3 anthraquinone glycosides, and some compounds were isomer compounds. The basic information of each component identified is shown in the table below.
  • the Nanshan flower root extract of the present invention can improve pulmonary fibrosis caused by different factors, can improve lung function, reduce collagen deposition in lung tissue and fibroblast proliferation and activation, and can inhibit lung tissue TGF- ⁇ and TNF- ⁇ . ⁇ inhibits pulmonary fibrosis.
  • the extracting parts of Nanshan flower root in the present invention are anthraquinones, iridoids and anthraquinone glycosides. Two of the specific compounds, scopolactone and methylisorubidin-1-methyl ether, clearly inhibit the activation of fibroblasts.
  • the Nanshan flower root extract of the present invention has a definite anti-pulmonary fibrosis effect, and scopolactone and methylisorubidin-1-methyl ether have proved the anti-pulmonary fibrosis potential at the cellular level, and their combination has a synergistic increase effect.
  • chemical drugs such as pirfenidone or nintedanib for anti-pulmonary fibrosis, it has a synergistic effect.
  • the Nanshan flower root extract and its constituent components of the present invention can be used to prepare medicines against pulmonary fibrosis.

Abstract

A use of Prismatomeris connata Y. Z. Ruan root extract in the preparation of medicine for treating pulmonary fibrosis. The extract has effective parts of anthraquinones, anthraquinone glycosides and iridoids. The extract can be used alone or in combination with a chemical agent capable of resisting pulmonary fibrosis, and is prepared into a pharmaceutical composition for treating pulmonary fibrosis.

Description

南山花根提取物在肺纤维化治疗的应用Application of Nanshan Flower Root Extract in the Treatment of Pulmonary Fibrosis 技术领域technical field
本发明属于医药领域，具体涉及从中药南山花根提取得到的提取部位和化合物在防治肺纤维化疾病中的应用。The invention belongs to the field of medicine, and in particular relates to the application of extraction parts and compounds obtained by extracting the root of the traditional Chinese medicine Nanshan flower in preventing and treating pulmonary fibrosis diseases.
背景技术Background technique
肺纤维化(PF)是肺间质弥漫性渗出、浸润和纤维化为主要病变的一类疾病，其主要症状包括呼吸困难、气短、干咳、喘憋现象，严重时会导致患者呼吸衰竭而死亡。绝大部分肺纤维化病人病因不明(特发性)，这组疾病称为特发性间质性肺炎(IIP)，是间质性肺病中一大类。而特发性间质性肺炎中最常见的以肺纤维化病变为主要表现形式的疾病类型为特发性肺纤维化(IPF)，是一种能导致肺功能进行性丧失的严重的间质性肺疾病。目前对其发病机制仍不太清楚，在全球范围内，仅有两个专门用于治疗特发性肺纤维化的药物上市。2008年吡非尼酮(Pirfenidone)首先在日本用于IPF治疗，2014年吡非尼酮和尼达尼布(Nintedanib)被美国FDA批准用于特异性地治疗IPF。但是，这两种药物也不能逆转或阻止IPF的病情发展，且价格昂贵，给患者及家庭带来较大的负担。Pulmonary fibrosis (PF) is a type of disease in which diffuse exudation, infiltration and fibrosis of the pulmonary interstitium are the main lesions. Its main symptoms include dyspnea, shortness of breath, dry cough, and wheezing. die. The vast majority of patients with pulmonary fibrosis are of unknown etiology (idiopathic). This group of diseases is called idiopathic interstitial pneumonia (IIP), which is a large category of interstitial lung diseases. Idiopathic pulmonary fibrosis (IPF) is the most common type of disease with pulmonary fibrosis as the main manifestation in idiopathic interstitial pneumonia. Sexual lung disease. At present, its pathogenesis is still unclear, and only two drugs are marketed specifically for the treatment of idiopathic pulmonary fibrosis worldwide. Pirfenidone was first used in Japan for the treatment of IPF in 2008, and in 2014, pirfenidone and nintedanib were approved by the US FDA for the specific treatment of IPF. However, neither of these two drugs can reverse or prevent the progression of IPF, and they are expensive, placing a greater burden on patients and their families.
肺纤维化可以分为原发性肺疾病和全身疾病的部分表现。(1)肺纤维化作为原发性肺疾病的类型有：放射纤维化(即由放射性导致的纤维化)、纤维化为损伤性炎性肺病变的结果(如硬化性肺结核、蜂窝状肺、小囊状肺纤维化)、纤维化为过敏反应性反应的结果、由于药物引起的间质性纤维化、纤维化作为间质性炎症的末期表现等；(2)肺纤维化为全身疾病的部分表现有：肉芽肿病(如结节病、淀粉样变、斑痣性错构瘤病、黄瘤病)、胶原性疾病及风湿性疾病(如硬皮症、红斑性狼疮、结节性动脉周围炎、皮肌炎、类风湿性关节炎)等。Pulmonary fibrosis can be divided into primary pulmonary disease and partial manifestations of systemic disease. (1) The types of pulmonary fibrosis as primary lung diseases include: radiation fibrosis (ie, fibrosis caused by radiation), fibrosis as a result of damaging inflammatory lung lesions (such as sclerosing tuberculosis, honeycomb lung, Small cystic pulmonary fibrosis), fibrosis as the result of allergic reaction, drug-induced interstitial fibrosis, fibrosis as the end-stage manifestation of interstitial inflammation, etc.; (2) pulmonary fibrosis is the result of systemic disease Some manifestations include: granulomatous disease (such as sarcoidosis, amyloidosis, nevus hamartoma, xanthoma), collagen disease and rheumatic disease (such as scleroderma, lupus erythematosus, nodular disease) periarteritis, dermatomyositis, rheumatoid arthritis), etc.
近年来，我国医学工作者用传统中药中发现治疗和延缓肺纤维化的有效药物取得了***成果，从天然来源寻找治疗肺纤维化的药物被认为是研发此类药物的有效途径。In recent years, Chinese medical workers have achieved fruitful results in discovering effective drugs for treating and delaying pulmonary fibrosis in traditional Chinese medicine. Finding drugs for the treatment of pulmonary fibrosis from natural sources is considered an effective way to develop such drugs.
南山花又名狗骨木。为茜草科植物南山花[Prismatomeris connata Y.Z.Ruan]的根，是广西特色药材之一。有强根壮骨，利湿退黄，祛瘀生新，凉血止血之功。Nanshan flower is also known as dog bone wood. It is the root of Prismatomeris connata Y.Z. Ruan, a Rubiaceae plant, and is one of the characteristic medicinal materials in Guangxi. There are strong roots and bones, dampness and yellowing, blood stasis and regeneration, cooling blood and hemostasis.
近年来，有学者发现南山花根提取物能够通过诱导大鼠星形胶质细胞凋亡，从而抑制肝纤维化的作用。但未见南山花根提取物或南山花根来源得到的化合物用于抗肺纤维化活性和用途的报道。现有技术(中国新药杂志，2016年第25卷和第24期，第2868-2869页)中公开了南山花根乙醇提取物的乙酸乙酯萃取部位和水部位可能为抗肝纤维化的活性部位，从中可以分离纯化得到豆甾-4-烯-3-酮、去甲虎刺醛等几种化合物，但是没有证据表明分离纯化鉴定的化合物是活性成分，而且其用途为抗肝纤维化的。现有技术(中国比较医学杂志，第30卷第2期，第9-14页)中公开了东莨菪内酯能保护肝纤维化大鼠肝，正向调控与肝纤维相关细胞因子的表达，降低肝细胞外基质的水平，改善大鼠肝纤维化病变程度，具有抗肝纤维化作用。现 有技术(北京中医药大学学报，第32卷第1期，第42-44页)公开了南山花根醇提物可明显减轻大鼠肝脏内胶原纤维增生沉积，减少纤维化组织面积，其在肝纤维化治疗方面有一定的开发价值。但是，上述用途为抗肝纤维化，不同器官的纤维化发生机制各有不同。临床上肝纤维化多为细菌病毒感染、毒素损害、代谢异常等导致的纤维化，目前认为肝纤维化是一个可逆的过程。而肺纤维化被认为是不可逆过程，肺脏与外界直接相通，肺纤维化的诱因更为复杂，且绝大部分病人的病因不明。从现有技术看，很多药物都有防治肝纤维化的作用，但是明确具有防治肺纤维化作用的药物非常少见，如上所述，截止目前，仅仅已知吡非尼酮和尼达尼布被用于肺纤维化，也就是说，防治肝纤维化的药物通常并不能用于防治肺纤维化。In recent years, some scholars have found that Nanshan flower root extract can inhibit liver fibrosis by inducing apoptosis of rat astrocytes. However, there is no report on the anti-pulmonary fibrosis activity and use of Nanshan flower root extract or compounds derived from Nanshan flower root. In the prior art (Chinese Journal of New Drugs, Vol. 25 and No. 24 in 2016, pages 2868-2869), it was disclosed that the ethyl acetate extraction site and the water site of the ethanolic extract of Nanshan flower root may be active against liver fibrosis Several compounds such as stigma-4-en-3-one and norsine can be obtained by separation and purification, but there is no evidence that the compounds identified by separation and purification are active ingredients, and their use is anti-hepatic fibrosis. . The prior art (Chinese Journal of Comparative Medicine, Vol. 30, Issue 2, Pages 9-14) discloses that scopolide can protect the liver of hepatic fibrosis rats, positively regulates the expression of cytokines related to hepatic fibrosis, It can reduce the level of extracellular matrix in liver, improve the degree of hepatic fibrosis in rats, and has anti-hepatic fibrosis effect. The prior art (Journal of Beijing University of Traditional Chinese Medicine, Vol. 32, No. 1, Pages 42-44) discloses that the alcohol extract of Nanshan flower root can significantly reduce the proliferation and deposition of collagen fibers in the liver of rats, and reduce the area of fibrotic tissue. It has certain development value in the treatment of liver fibrosis. However, the above-mentioned uses are anti-hepatic fibrosis, and the mechanisms of fibrosis in different organs are different. Clinically, liver fibrosis is mostly fibrosis caused by bacterial and viral infection, toxin damage, and abnormal metabolism. It is currently believed that liver fibrosis is a reversible process. Pulmonary fibrosis is considered to be an irreversible process, and the lungs are directly connected to the outside world. The causes of pulmonary fibrosis are more complicated, and the etiology of most patients is unknown. From the prior art, many drugs have the effect of preventing and treating liver fibrosis, but the drugs that clearly have the effect of preventing and treating pulmonary fibrosis are very rare. As mentioned above, so far, only pirfenidone and nintedanib are known to be For pulmonary fibrosis, that is to say, drugs used to prevent and treat liver fibrosis are generally not used to prevent and treat pulmonary fibrosis.
广西盈康药业有限责任公司已开发治疗矽肺的用南山花制备的片剂，其适应症为“治疗矽肺”，其制备方法为用60％乙醇加热回流提取三次，每次2小时，合并提取液，滤过，滤液减压回收乙醇，浓缩至稠膏状，用10倍量温水分三次搅匀，静置，滤过，滤液减压浓缩，真空干燥，粉碎，加入适量辅料，制颗粒，压制成1000片，包糖衣，即得。但是，已知矽肺是由于吸入二氧化硅或者硅石及其晶体形式如石英和其他比较少见的形式如方石英和磷石英而引起的肺疾病，其急进型的表现与间质性肺炎有类似的表现，与肺纤维化并不等同。其次，现有技术中认为上述片剂的主要作用成分为以有机铝为主的多种金属元素形成的聚合物，作用机理为铝在二氧化硅表面形成难溶性的硅酸铝，使之对巨噬细胞丧失毒性作用，从而起拮抗石英细胞毒效应(参见李钰等主编，《矿山粉尘及职业危害防控技术》，冶金工业出版社，2017年10月，第211页)。但是，发明人出人意料地发现，在防治肺纤维化过程中的有效成分与治疗矽肺中的有效成分并不相同，并通过大量研究和实验找到了优选地防治肺纤维化有效成分。Guangxi Yingkang Pharmaceutical Co., Ltd. has developed a tablet prepared from Nanshan flower for the treatment of silicosis, and its indication is "treatment of silicosis". liquid, filtered, the filtrate was decompressed to recover ethanol, concentrated to a thick paste, stirred three times with 10 times the amount of warm water, allowed to stand, filtered, the filtrate was concentrated under reduced pressure, vacuum-dried, pulverized, added with appropriate amount of auxiliary materials, made into granules, Compressed into 1,000 tablets, coated with sugar, and obtained. However, silicosis is known to be a lung disease caused by inhalation of silica or silica and its crystalline forms such as quartz and other less common forms such as cristobalite and phosphoquartz, and its rapidly progressive manifestations are similar to interstitial pneumonia. performance, and pulmonary fibrosis is not the same. Secondly, it is considered in the prior art that the main active ingredient of the above-mentioned tablet is a polymer formed by a variety of metal elements mainly composed of organoaluminum, and the mechanism of action is that aluminum forms insoluble aluminum silicate on the surface of silicon dioxide, which makes it difficult to dissolve. Macrophages lose their toxic effect, thus antagonizing the cytotoxic effect of quartz (see Li Yu et al., Editor-in-Chief, "Mine Dust and Occupational Hazard Prevention and Control Technology", Metallurgical Industry Press, October 2017, p. 211). However, the inventors unexpectedly found that the active ingredients in the prevention and treatment of pulmonary fibrosis are not the same as those in the treatment of silicosis, and through a large number of studies and experiments, they found the preferred active ingredients for prevention and treatment of pulmonary fibrosis.
现有技术(中草药，第51卷第4期，第1031-1036页)公开了来自南山花根的多糖可以通过抑制炎症细胞浸润降低炎症水平，下调肺组织中的α-SMA、Vimentin表达水平，上调E-cadherin表达水平，提示黄根多糖能够有效的改善免疫平衡，抑制炎症反应和EMT的发展，从而改善矽肺组织纤维化程度，对矽肺纤维化有较好的治疗作用。其多糖的制备方法为饮片10kg粉碎后，经超声波加热水提取，减压浓缩，80％乙醇沉淀，得粗多糖905g。粗多糖复溶于蒸馏水后，用Savage法除去蛋白质，高压通过滤膜U，再通过超滤膜A浓缩并除去低相对分子质量组分，浓缩物干燥得560g多糖。上述内容启示多糖类成分是南山花根防治肺纤维化的活性成分。但是，上述实验中并未进行南山花根中的多糖与非多糖成分的有效性对比，发明人经过深入研究后发现，多糖类成分并非南山花根防治肺纤维化的主要有效成分。The prior art (Chinese Herbal Medicine, Vol. 51, No. 4, pp. 1031-1036) discloses that polysaccharides from Nanshan flower root can reduce the level of inflammation by inhibiting the infiltration of inflammatory cells, and down-regulate the expression levels of α-SMA and Vimentin in lung tissue, Up-regulation of the expression level of E-cadherin suggests that the polysaccharide of yellow root can effectively improve the immune balance, inhibit the development of inflammatory response and EMT, thereby improving the degree of fibrosis in silicosis tissue, and has a good therapeutic effect on silicosis fibrosis. The preparation method of the polysaccharide is as follows: after crushing 10 kg of decoction pieces, extracting with ultrasonic heating water, concentrating under reduced pressure, and precipitating with 80% ethanol to obtain 905 g of crude polysaccharide. After the crude polysaccharide was redissolved in distilled water, the protein was removed by the Savage method, passed through the filter membrane U under high pressure, and then concentrated through the ultrafiltration membrane A to remove low molecular weight components, and the concentrate was dried to obtain 560 g of polysaccharide. The above content suggests that polysaccharide components are the active components of Nanshan flower root in preventing and treating pulmonary fibrosis. However, the above experiments did not compare the effectiveness of polysaccharide and non-polysaccharide components in Nanshan flower root. After in-depth research, the inventor found that polysaccharide components are not the main effective components of Nanshan flower root for preventing and treating pulmonary fibrosis.
为了从南山花根中找到对防治肺纤维化具有明确作用的有效部位，并提供其制药用途，提出了本发明。The present invention is proposed in order to find an effective part having a definite effect on prevention and treatment of pulmonary fibrosis from Nanshan flower root, and to provide its pharmaceutical use.
发明内容SUMMARY OF THE INVENTION
本发明目的是提供一种南山花根提取物的新的制药用途，即南山花根提取物在制备治疗肺纤维化的药物中的用途。所述提取物的抗肺纤维化有效部位是首次发现的，具有制备方法简单，有效部位明确，活性成分清楚，治疗肺纤维化作用全面，治疗肺纤维化效果明显优于 现有技术，可以与抗肺纤维化的化学药产生协同增效作用等优点。The purpose of the present invention is to provide a new pharmaceutical use of the Nanshan flower root extract, that is, the use of the Nanshan flower root extract in the preparation of a medicine for treating pulmonary fibrosis. The anti-pulmonary fibrosis effective parts of the extract are discovered for the first time, and the preparation method is simple, the effective parts are clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is obviously better than the prior art, and can be combined with the existing technology. Anti-pulmonary fibrosis chemicals have the advantages of synergistic effect.
在本发明中，“有效部位”是指从单一种植物中提取的一类或数类成份组成的成分相对明确的提取物，也可以称为有效组分或者是有效中间体(《中药新药质量研究技术指导原则(试行)，2021年1月》)。其中“蒽醌类、蒽醌糖苷类和环烯醚萜类有效部位”是指在提取物中蒽醌类成分、蒽醌糖苷类成分和环烯醚萜类成分的化合物是提取物中组成结构上占主要的和发挥主要治疗作用的化学成分，提取物中虽然还可能含有来源于同一种植物的其他成分，但这些其他成分在提取物中通常不是能够有效发挥治疗作用的化学成分，这是由于其含量低于发挥治疗有效作用的阈值或者其本身就不具有所期望的治疗有效作用等。In the present invention, "effective part" refers to an extract composed of one or several types of components extracted from a single plant with relatively clear components, which can also be called effective components or effective intermediates ("Quality of New Chinese Medicines"). Research Technical Guiding Principles (for Trial Implementation, January 2021). "Anthraquinones, Anthraquinone Glycosides and Iridoids Effective Parts" means that the compounds of anthraquinones, anthraquinone glycosides and iridoids in the extract are the constituents of the extract. Although the extract may also contain other components derived from the same plant, these other components are usually not chemical components that can effectively play a therapeutic role in the extract. Because its content is lower than the threshold for exerting a therapeutically effective effect or itself does not have a desired therapeutically effective effect, etc.
更进一步地，本发明的目的是提供一种上述南山花根有效部位的制备方法。Further, the object of the present invention is to provide a kind of preparation method of the above-mentioned effective parts of Nanshan flower root.
更进一步地，本发明的目的是提供一种治疗肺纤维化的药物组合物，其由南山花根的蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分组成。Further, the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of anthraquinones, anthraquinone glycosides and iridoids active ingredients of Radix Nanshanii.
更进一步地，本发明的目的是提供一种治疗肺纤维化的药物组合物，其由东莨菪内酯和甲基异茜草素-1-甲醚组成。Further, the object of the present invention is to provide a pharmaceutical composition for treating pulmonary fibrosis, which is composed of scopolide and methylisorubidin-1-methyl ether.
更进一步地，本发明的目的是提供一种治疗肺纤维化的药物组合物，其将上述南山花根提取物与抗肺纤维化的化学药吡非尼酮或尼达尼布联合使用，具有明显的协同增效作用。Further, the object of the present invention is to provide a pharmaceutical composition for the treatment of pulmonary fibrosis, which combines the above-mentioned Nanshan flower root extract with anti-pulmonary fibrosis chemical drug pirfenidone or nintedanib, and has Obvious synergistic effect.
为了实现上述目的，本发明提供了如下具体地技术方案：In order to achieve the above object, the present invention provides the following specific technical solutions:
一种南山花根提取物在制备防治肺纤维化的药物中的用途，其特征在于：所述南山花根提取物是蒽醌类、蒽醌糖苷类和环烯醚萜类有效部位。优选地，上述提取物中按重量计，蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分含量占提取物含量的60％以上，更优选地70％以上，最优选100％。A use of Nanshan flower root extract in preparing a medicine for preventing and treating pulmonary fibrosis, characterized in that the Nanshan flower root extract is anthraquinones, anthraquinone glycosides and iridoids effective parts. Preferably, in the above extract, the active ingredients of anthraquinones, anthraquinone glycosides and iridoids account for more than 60% of the content of the extract, more preferably more than 70%, and most preferably 100% by weight.
优选地，所述蒽醌类有效成分包括2-甲基蒽醌、1-羟基-2-甲基蒽醌、甲基异茜草素、2-羟基-3-甲氧基蒽醌、2-羟基-3-羟甲基蒽醌、去甲基虎刺醛、甲基异茜草素-1-甲醚、虎刺醛、1,3-二羟基-2-甲氧甲基蒽醌、3-羟基-1-甲氧基-2-羟甲基蒽醌、1,3-二羟基-2-乙氧甲基蒽醌、1-羟基-2,3-二甲氧基-7-甲基蒽醌、7-羟基-1,2-二甲氧基-6-甲基蒽醌、1,3,8-三羟基-7-甲氧基-2-甲基蒽醌、3-羟基-5,6-二甲氧基-2-甲基-1,2,3,4-四氢蒽醌、1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、1,3-二羟基-6-甲氧基-2-甲氧甲基蒽醌、东莨菪内酯、3,5-二羟基-6,7-二甲氧基-2-甲基-1,2,3,4-四氢蒽醌、3-羟基-1,5,6-三甲氧基-2-甲基蒽醌、2-羟基-4,6,7-三甲氧基-3-甲基蒽醌、4-羟基-1,2,3-三甲氧基-7-羟甲基蒽醌、1,3-二羟基-5,6-二甲氧基-2-甲氧基甲基蒽醌的一种或多种。Preferably, the anthraquinone active ingredients include 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylisorubicin, 2-hydroxy-3-methoxyanthraquinone, 2-hydroxy anthraquinone -3-Hydroxymethyl anthraquinone, desmethyl oxal, methyl isoalizarin-1-methyl ether, ox aldehyde, 1,3-dihydroxy-2-methoxymethyl anthraquinone, 3-hydroxy -1-Methoxy-2-hydroxymethylanthraquinone, 1,3-dihydroxy-2-ethoxymethylanthraquinone, 1-hydroxy-2,3-dimethoxy-7-methylanthraquinone , 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methylanthraquinone, 3-hydroxy-5,6 -Dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, 1,3 -Dihydroxy-6-methoxy-2-methoxymethylanthraquinone, scopolactone, 3,5-dihydroxy-6,7-dimethoxy-2-methyl-1,2,3 , 4-tetrahydroanthraquinone, 3-hydroxy-1,5,6-trimethoxy-2-methylanthraquinone, 2-hydroxy-4,6,7-trimethoxy-3-methylanthraquinone, A kind of 4-hydroxy-1,2,3-trimethoxy-7-hydroxymethylanthraquinone and 1,3-dihydroxy-5,6-dimethoxy-2-methoxymethylanthraquinone or more.
优选地，所述蒽醌糖苷类有效成分包括lucidin 3-O-β-primeveroside、damnacanthol 3-O-β-primeveroside、rubiadin 3-O-β-primerveroside的一种或多种。Preferably, the anthraquinone glycoside active ingredients include one or more of lucidin 3-O-β-primeveroside, damnacanthol 3-O-β-primeveroside, and rubiadin 3-O-β-primerveroside.
优选地，所述环烯醚萜类有效成分包括Prismatomerin、deacetylasperuloside、deacetylasperulosidic acid、asperulosidic acid的一种或多种。Preferably, the iridoid active ingredients include one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, and asperulosidic acid.
优选地，所述南山花根提取物中至少含有1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、甲基异 茜草素-1-甲醚、甲基异茜草素和东莨菪内酯。Preferably, the Nanshan flower root extract contains at least 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin Alizarin and scopolactone.
更优选地，所述南山花根提取物中至少含有东莨菪内酯和甲基异茜草素-1-甲醚。其中通过东莨菪内酯和甲基异茜草素-1-甲醚的搭配组合，产生了协同增效作用，其效果明显好于东莨菪内酯和甲基异茜草素-1-甲醚各自防治肺纤维化效果的简单叠加。More preferably, the Nanshan flower root extract contains at least scopolactone and methylisorubidin-1-methyl ether. Among them, the combination of scopolactone and methylisorubidin-1-methyl ether produces a synergistic effect, and its effect is obviously better than that of scopolide and methylisorubidin-1-methyl ether. Simple superposition of pulmonary fibrosis effects.
更进一步地，本发明还提供了一种东莨菪内酯在制备防治肺纤维化的药物中的用途。Further, the present invention also provides the use of scopolide in preparing a medicine for preventing and treating pulmonary fibrosis.
更进一步地，本发明还提供了一种甲基异茜草素-1-甲醚在制备防治肺纤维化的药物中的用途。Further, the present invention also provides the use of methylisorubicin-1-methyl ether in preparing a medicine for preventing and treating pulmonary fibrosis.
更优选地，本发明的所述提取物具有全面的防治肺纤维化作用，可以改善多种不同因素导致的肺纤维化，能够改善肺功能，降低肺组织的胶原沉积和成纤维细胞增殖和活化，能够抑制肺组织TGF-β和TNF-α而抑制肺纤维化。南山花根乙酸乙酯提取物可以降低小鼠肺纤维化导致的COL1A1、COL3A1和α-SMA蛋白表达升高。其作用途径不是通过在二氧化硅表面形成难溶性的硅酸铝，使之对巨噬细胞丧失毒性作用。More preferably, the extract of the present invention has a comprehensive prevention and treatment effect on pulmonary fibrosis, can improve pulmonary fibrosis caused by a variety of different factors, can improve lung function, and reduce collagen deposition in lung tissue and fibroblast proliferation and activation. , can inhibit lung tissue TGF-β and TNF-α and inhibit pulmonary fibrosis. The ethyl acetate extract of Nanshan flower root can reduce the elevated expression of COL1A1, COL3A1 and α-SMA proteins caused by pulmonary fibrosis in mice. Its action pathway is not through the formation of insoluble aluminum silicate on the surface of silica, which makes it lose its toxic effect on macrophages.
更进一步地，所述肺纤维化不是由矽肺引起的。Still further, the pulmonary fibrosis is not caused by silicosis.
本发明还提供了所述南山花根提取物的制备方法，即取南山花根用乙醇提取，再使用制备色谱法去除杂质，精制纯化得到蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分；或者取南山花根用乙酸乙酯溶剂提取得到；或者取南山花根用溶剂提取后，浓缩提取液，再使用乙酸乙酯萃取得到。优选地，所述的溶剂为水、甲醇、乙醇、丙醇、丁醇等的一种或二种以上的组合。The present invention also provides the preparation method of the Nanshan flower root extract, namely extracting Nanshan flower root with ethanol, then using preparative chromatography to remove impurities, and refining and purifying to obtain anthraquinones, anthraquinone glycosides and iridoids The active ingredient is obtained by extracting Nanshan flower root with ethyl acetate solvent; or after extracting Nanshan flower root with solvent, concentrating the extract, and then extracting with ethyl acetate. Preferably, the solvent is one or a combination of two or more of water, methanol, ethanol, propanol, butanol and the like.
优选地，取南山花根先用石油醚提取，弃去提取液，再用乙酸乙酯提取，减压浓缩得到乙酸乙酯提取物。Preferably, the Nanshan flower root is extracted with petroleum ether first, the extract is discarded, then extracted with ethyl acetate, and concentrated under reduced pressure to obtain the ethyl acetate extract.
优选地，所述制备方法还可以为：南山花根用水提取，再使用95％乙醇提取，合并水和乙醇提取液，提取液减压浓缩，使用乙酸乙酯萃取后旋蒸去除溶剂获得的南山花根提取物。Preferably, the preparation method can also be as follows: extracting Nanshan flower root with water, then extracting with 95% ethanol, combining water and ethanol extracts, concentrating the extract under reduced pressure, extracting with ethyl acetate and then rotary evaporation to remove the solvent obtained from Nanshan Flower root extract.
优选地，所述制备方法还可以为：南山花根95％乙醇回流提取三次，提取液减压浓缩，采用制备色谱法去除杂质，精制纯化得到蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分，所述色谱法可以为大孔树脂法、分子筛色谱法、以及硅胶吸附色谱法、聚酰胺吸附色谱法等的一种或多种。优选为硅胶柱色谱法，采用水和乙腈梯度洗脱。Preferably, the preparation method can also be as follows: 95% ethanol reflux extraction of Nanshan flower root three times, the extract is concentrated under reduced pressure, the impurities are removed by preparative chromatography, and the anthraquinones, anthraquinone glycosides and iridoids are obtained by refining and purification The chromatographic method can be one or more of macroporous resin method, molecular sieve chromatography, silica gel adsorption chromatography, polyamide adsorption chromatography, etc. Preferred is silica gel column chromatography, eluting with a gradient of water and acetonitrile.
可选地，所述制备方法还可以为：南山花根95％乙醇回流提取三次，乙酸乙酯萃取后旋蒸去除溶剂获得的南山花根提取物。Optionally, the preparation method can also be: the Nanshan flower root extract obtained by refluxing extraction with 95% ethanol for three times, ethyl acetate extraction, and rotary evaporation to remove the solvent.
本发明还提供一种治疗肺纤维化的药物组合物，其特征在于：由南山花根中的蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分组成。优选地，上述三种有效提取部位的重量配比为1-10：1-10：1-5。The present invention also provides a pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that it is composed of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids in Nanshan flower root. Preferably, the weight ratio of the above three effective extraction parts is 1-10:1-10:1-5.
进一步地，所述治疗肺纤维化的药物组合物由1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、甲基异茜草素-1-甲醚、甲基异茜草素和东莨菪内酯组成。优选地，上述四种化合物的重量配比为1-10：1-10：1-10：1-10。Further, the pharmaceutical composition for treating pulmonary fibrosis is composed of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methyl Composed of isolarin and scopolactone. Preferably, the weight ratio of the above four compounds is 1-10:1-10:1-10:1-10.
进一步地，所述治疗肺纤维化的药物组合物由东莨菪内酯和甲基异茜草素-1-甲醚组成。优选地，上述两种化合物的重量配比为1-10：1-10。Further, the pharmaceutical composition for treating pulmonary fibrosis is composed of scopolactone and methylisorubicin-1-methyl ether. Preferably, the weight ratio of the above two compounds is 1-10:1-10.
本发明还优选地提供一种治疗肺纤维化的药物组合物，其特征在于：由上述南山花根提取物和抗肺纤维化的化学药组成，所述抗肺纤维化的化学药为吡非尼酮或尼达尼布。南山花根提取物和抗肺纤维化的化学药组合具有协同增效作用，其效果明显好于所述南山花根提取物和所述化学药各自防治肺纤维化效果的简单叠加。优选地，南山花根提取物和抗肺纤维化的化学药的重量配比为2-20：1-10。The present invention also preferably provides a pharmaceutical composition for the treatment of pulmonary fibrosis, which is characterized in that it is composed of the above-mentioned Nanshan flower root extract and an anti-pulmonary fibrosis chemical, and the anti-pulmonary fibrosis chemical is pyridine Nintedone or nintedanib. The combination of the Nanshan flower root extract and the chemical drug against pulmonary fibrosis has a synergistic effect, and its effect is obviously better than the simple superposition of the respective effects of the Nanshan flower root extract and the chemical drug for preventing and treating pulmonary fibrosis. Preferably, the weight ratio of Nanshan flower root extract and anti-pulmonary fibrosis chemical is 2-20:1-10.
本发明所述防治肺纤维化的药物，其可以预防和/或治疗肺纤维化，所述药物中由活性成分和药学上可接受辅料组成，所述活性成分为南山花根提取物、东莨菪内酯、甲基异茜草素-1-甲醚、所述药物组合物的任一种。The medicine for preventing and treating pulmonary fibrosis according to the present invention can prevent and/or treat pulmonary fibrosis, and the medicine is composed of active ingredients and pharmaceutically acceptable excipients, and the active ingredients are Nanshan flower root extract, scopolamine Any one of lactone, methylisorubicin-1-methyl ether, and the pharmaceutical composition.
在所述的药物中，所述活性成分的含量占比为0.1-99.9％(w/w)，药学上可接受辅料的含量占比为0.1-99.9％(w/w)，合计为100％。In the medicine, the content of the active ingredient is 0.1-99.9% (w/w), and the content of pharmaceutically acceptable excipients is 0.1-99.9% (w/w), and the total is 100% .
本发明的药物可以是任何可药用的剂型，这些剂型包括：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、口服液、***剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、气雾剂、滴剂、贴剂。The medicament of the present invention can be in any pharmaceutically acceptable dosage form, and these dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, oral liquids, buccal preparations, granules, granules, Pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, aerosols, drops, patches.
本发明的药物可以使用适宜的药物辅料，具体的辅料实例包括：填充剂、稀释剂、载体、赋形剂、崩解剂、粘合剂、润滑剂、矫味剂、表面活性剂、着色剂、包衣剂、抛射剂、稳定剂等。Appropriate pharmaceutical excipients can be used for the medicine of the present invention, and specific examples of excipients include: fillers, diluents, carriers, excipients, disintegrants, binders, lubricants, flavoring agents, surfactants, colorants , coating agent, propellant, stabilizer, etc.
在制备成药剂时可选择性的加入适合的药物可接受的载体，所述药物可接受的载体选自：淀粉、蔗糖、乳糖、甘露糖醇、甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、吐温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。A suitable pharmaceutically acceptable carrier can be optionally added when preparing a medicament, and the pharmaceutically acceptable carrier is selected from: starch, sucrose, lactose, mannitol, mannitol, sorbitol, sodium metabisulfite, hydrogen sulfite Sodium, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or their aqueous solutions, hydrochloric acid, Acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, silicon derivatives, cellulose and its derivatives, alginate, gelatin , polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, stearin Calcium acid, magnesium stearate, etc.
本发明的优点和有益效果为：所述提取物的抗肺纤维化有效部位是首次发现的，具有制备方法简单，有效部位明确，活性成分清楚，治疗肺纤维化作用全面，治疗肺纤维化效果明显优于现有技术，其中两种具体的化合物之间具有抗肺纤维化的协同增效作用，所述提取物还可以与抗肺纤维化的化学药产生协同增效作用等优点。The advantages and beneficial effects of the present invention are as follows: the effective anti-pulmonary fibrosis part of the extract is discovered for the first time, the preparation method is simple, the effective part is clear, the active ingredients are clear, the effect of treating pulmonary fibrosis is comprehensive, and the effect of treating pulmonary fibrosis is comprehensive. It is obviously superior to the prior art, wherein the two specific compounds have a synergistic effect against pulmonary fibrosis, and the extract can also have the advantages of synergistic effect with anti-pulmonary fibrosis chemical drugs.
附图说明Description of drawings
图1南山花根不同提取部位对博来霉素诱导肺纤维化小鼠肺功能的影响。Figure 1 Effects of different extraction parts of Nanshan flower root on lung function in mice with bleomycin-induced pulmonary fibrosis.
图2南山花根不同提取部位对博来霉素诱导肺纤维化小鼠纤维化程度的影响。Figure 2 The effect of different extraction parts of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.
图3南山花根不同提取部位对博来霉素诱导肺纤维化小鼠肺组织羟脯氨酸含量的影响。Fig. 3 Effects of different extraction parts of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
图4南山花根不同制备工艺对博来霉素诱导肺纤维化小鼠肺功能的影响。Figure 4 Effects of different preparation techniques of Nanshan flower root on lung function of mice with bleomycin-induced pulmonary fibrosis.
图5南山花根不同制备工艺对博来霉素诱导肺纤维化小鼠纤维化程度的影响。Figure 5. Effects of different preparation processes of Nanshan flower root on the degree of fibrosis in mice with bleomycin-induced pulmonary fibrosis.
图6南山花根不同制备工艺对博来霉素诱导肺纤维化小鼠肺组织羟脯氨酸含量的影响。Figure 6. Effects of different preparation techniques of Nanshan flower root on the content of hydroxyproline in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
图7南山花根不同制备工艺对博来霉素诱导肺纤维化小鼠肺组织TGF-β和TNF-α表达的影响。Figure 7 Effects of different preparation techniques of Nanshan flower root on the expression of TGF-β and TNF-α in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
图8南山花根不同制备工艺对博来霉素诱导肺纤维化小鼠肺组织COL1A1、COL3A1、α-SMA蛋白表达的影响。Figure 8 Effects of different preparation techniques of Nanshan flower root on the expression of COL1A1, COL3A1 and α-SMA proteins in lung tissue of mice with bleomycin-induced pulmonary fibrosis.
图9东莨菪内酯和甲基异茜草素-1-甲醚对TGF-β1诱导的成纤维细胞激活后α-SMA蛋白表达的影响。Fig. 9 Effects of scopolactone and methylisorubicin-1-methyl ether on the expression of α-SMA protein after TGF-β1-induced fibroblast activation.
图10本发明药物组合对TGF-β1诱导的成纤维细胞激活后α-SMA蛋白表达的影响。Figure 10 The effect of the pharmaceutical combination of the present invention on the expression of α-SMA protein after TGF-β1-induced fibroblast activation.
具体实施方式Detailed ways
实施例1南山花根提取物的制备Example 1 Preparation of Nanshan Flower Root Extract
取南山花根1kg，用8倍量95％乙醇回流提取3次，合并提取液后减压浓缩，再使用100ml乙酸乙酯萃取3次，合并萃取液，减压浓缩去除溶剂后干燥，获得的南山花根提取物3g，经色谱法检测，其中蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分含量占提取物总量的64％。Take 1 kg of Nanshan flower root, extract 3 times with 8 times the amount of 95% ethanol, combine the extracts, concentrate under reduced pressure, extract 3 times with 100 ml of ethyl acetate, combine the extracts, concentrate under reduced pressure to remove the solvent, and then dry to obtain the obtained 3 g of Nanshan flower root extract was detected by chromatography, and the content of anthraquinones, anthraquinone glycosides and iridoids accounted for 64% of the total extract.
实施例2南山花根提取物的制备Example 2 Preparation of Nanshan Flower Root Extract
取南山花根1kg，用8倍量水提取2次，再使用5倍量95％乙醇提取2次，合并水和乙醇提取液，提取液减压浓缩，使用200ml石油醚萃取，弃去萃取液，再使用100ml乙酸乙酯萃取3次，合并萃取液，减压浓缩去除溶剂后干燥，获得的南山花根提取物8g，经色谱法检测，其中蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分含量占提取物总量的71％。Take 1kg of Nanshan flower root, extract twice with 8 times the amount of water, and then extract twice with 5 times the amount of 95% ethanol, combine the water and ethanol extracts, concentrate the extract under reduced pressure, extract with 200ml of petroleum ether, and discard the extract , and then extracted 3 times with 100ml of ethyl acetate, combined the extracts, concentrated under reduced pressure to remove the solvent and then dried. The obtained Nanshan flower root extract 8g was detected by chromatography, among which anthraquinones, anthraquinone glycosides and cycloalkenyl ethers The content of terpenoid active ingredients accounted for 71% of the total extract.
实施例3南山花根提取物的制备Example 3 Preparation of Nanshan Flower Root Extract
取南山花根1kg，粉碎细度为约50目，先用适量石油醚提取1次，弃去石油醚提取液，再用5倍量乙酸乙酯提取3次，合并提取液，减压浓缩干燥得到乙酸乙酯提取物11g，经色谱法检测，其中蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分含量占提取物总量的79％。Take 1kg of Nanshan flower root, pulverize it to a fineness of about 50 mesh, first extract it once with an appropriate amount of petroleum ether, discard the petroleum ether extract, and then extract three times with 5 times the amount of ethyl acetate, combine the extracts, and concentrate and dry under reduced pressure. 11 g of the ethyl acetate extract was obtained, and the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounted for 79% of the total amount of the extracts through chromatographic detection.
实施例4南山花根提取物的制备Example 4 Preparation of Nanshan Flower Root Extract
取南山花根1kg，用8倍量95％乙醇回流提取3次，提取液合并，减压浓缩，采用制备色谱柱去除杂质，所采用的色谱柱为C18柱，流动相为流动相A相：水(0.1％甲酸，1mmol/L乙酸铵)，流动相B相：乙腈(0.1％甲酸，1mmol/L乙酸铵)，梯度洗脱条件：0-10min，20％B；10-60min，20-100％B，流速：0.3mL/min，柱温：30℃。分别分段收集蒽醌类、蒽醌糖苷类 和环烯醚萜类三种有效成分部位段，减压浓缩干燥得到纯化的蒽醌类、蒽醌糖苷类和环烯醚萜类三种有效成分部位。也可以再将上述三种有效部位混合均匀，即得到南山花根提取物，经色谱法检测，其中蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分含量占提取物总量的100％。Take 1kg of Nanshan flower root, extract 3 times with 8 times the amount of 95% ethanol, the extracts are combined, concentrated under reduced pressure, and a preparative chromatographic column is used to remove impurities. The adopted chromatographic column is a C18 column, and the mobile phase is the mobile phase A phase: Water (0.1% formic acid, 1 mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1 mmol/L ammonium acetate), gradient elution conditions: 0-10min, 20%B; 10-60min, 20- 100% B, flow rate: 0.3 mL/min, column temperature: 30°C. The three active components of anthraquinones, anthraquinone glycosides and iridoids were collected in sections, and concentrated and dried under reduced pressure to obtain the purified three active components of anthraquinones, anthraquinone glycosides and iridoids. part. The above three kinds of effective parts can also be mixed evenly to obtain the Nanshan flower root extract. After chromatographic detection, the content of the active ingredients of anthraquinones, anthraquinone glycosides and iridoids accounts for 100% of the total amount of the extract. %.
实施例5药物组合物的制备The preparation of embodiment 5 pharmaceutical composition
取实施例4中制备蒽醌类、蒽醌糖苷类和环烯醚萜类三种有效部位，分别按照重量8：1：1的重量比混合。Take three effective parts of anthraquinones, anthraquinone glycosides and iridoids prepared in Example 4, and mix them according to the weight ratio of 8:1:1 respectively.
实施例6药物组合物的制备The preparation of embodiment 6 pharmaceutical composition
取1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、甲基异茜草素-1-甲醚、甲基异茜草素和东莨菪内酯，按照1：5：5：1的重量比混合。Take 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin and scopolactone in a ratio of 1:5 : 5:1 weight ratio mixing.
实施例7药物组合物的制备The preparation of embodiment 7 pharmaceutical composition
取东莨菪内酯和甲基异茜草素-1-甲醚,按照1：1的重量比混合。Take scopolactone and methylisorubicin-1-methyl ether and mix them according to the weight ratio of 1:1.
实施例8药物组合物的制备The preparation of embodiment 8 pharmaceutical composition
取实施例1的南山花根提取物和吡非尼酮，按照10：1的重量比混合。Take the Nanshan flower root extract of Example 1 and pirfenidone, and mix them in a weight ratio of 10:1.
实施例9药物组合物的制备The preparation of embodiment 9 pharmaceutical composition
取实施例1的南山花根提取物和尼达尼布，按照8：1的重量比混合。Take the Nanshan flower root extract of Example 1 and nintedanib, and mix them in a weight ratio of 8:1.
实施例10胶囊剂的制备Example 10 Preparation of capsules
取实施例2的南山花根提取物10重量份，加入12重量份乳糖和2重量份的微晶纤维素，干法制粒，过筛，整粒，装入胶囊，得到胶囊剂。Take 10 parts by weight of Nanshan flower root extract in Example 2, add 12 parts by weight of lactose and 2 parts by weight of microcrystalline cellulose, dry granulation, sieve, granulate, put into capsules, and obtain capsules.
实施例11吸入喷雾剂的制备Example 11 Preparation of Inhalation Spray
取实施例6的药物组合物，加入适量表面活性剂、抛射剂和稳定剂，制备为吸入喷雾剂。Take the pharmaceutical composition of Example 6, add appropriate amount of surfactant, propellant and stabilizer to prepare an inhalation spray.
实验例1：南山花根不同提取部位对博来霉素致小鼠肺纤维化的治疗作用Experimental Example 1: Therapeutic effect of different extraction parts of Nanshan flower root on bleomycin-induced pulmonary fibrosis in mice
1.实验材料1. Experimental materials
1.1实验动物1.1 Experimental animals
无特定病原菌(Specific pathogen-free,SPF)级C57BL/6N小鼠(雄性，体重18-22g)，购自北京维通利华实验动物技术有限公司，生产许可证号：SCXK-(京)2016-0011，发证机关：北京市科学技术委员会，合格证号：11400700300671。Specific pathogen-free (SPF) grade C57BL/6N mice (male, body weight 18-22g), purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK-(Beijing) 2016 -0011, issuing authority: Beijing Municipal Science and Technology Commission, certificate number: 11400700300671.
1.2药物与主要试剂1.2 Drugs and main reagents
Figure PCTCN2022086065-appb-000001
Figure PCTCN2022086065-appb-000001
1.3实验主要仪器1.3 The main instruments of the experiment
Figure PCTCN2022086065-appb-000002
Figure PCTCN2022086065-appb-000002
2.实验方法2. Experimental method
2.1造模及分组2.1 Modeling and grouping
取SPF级雄性C57BL/6N小鼠约80只，10只作为空白对照组，其余进行气管滴注博来霉素造成肺纤维化。腹腔注射戊巴比妥钠80mg/kg麻醉小鼠，将小鼠上门齿固定在气管滴注操作台上，LED灯光透过颈部皮肤，自口腔向内观察小鼠气管口。将套管针***小鼠气管，连接HRH-MAG4肺部液体定量雾化器快速推入50μL无菌PBS溶解的博来霉素溶液，造模剂量0.04U/只。给药后保持直立轻旋小鼠使溶液在肺部分布均匀，待其苏醒后给予水和饲料，肺纤维化小鼠模型建立完成。空白对照组除气管给药无菌PBS溶液外，其他操作相同。About 80 SPF grade male C57BL/6N mice were selected, 10 were used as blank control group, and the rest were treated with tracheal instillation of bleomycin to cause pulmonary fibrosis. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital 80 mg/kg, and the upper incisors of the mice were fixed on the tracheal instillation console. A trocar was inserted into the mouse trachea, connected to an HRH-MAG4 pulmonary liquid quantitative nebulizer, and quickly pushed into 50 μL of sterile PBS-dissolved bleomycin solution at a dose of 0.04 U per mouse. After administration, the mice were kept upright and swirled to make the solution evenly distributed in the lungs, and water and feed were given to the mice after they recovered, and the pulmonary fibrosis mouse model was established. In the blank control group, other operations were the same except that sterile PBS solution was administered to the trachea.
14天后，根据存活的造模组动物的体重随机分组至模型组、尼达尼布阳性药组、黄根片组、南山花根乙酸乙酯提取物组、南山花根正丁醇提取物组、南山花根水提物组。After 14 days, the animals in the surviving modeling group were randomly divided into model group, nintedanib positive drug group, Huanggen tablet group, Nanshan flower root ethyl acetate extract group, and Nanshan flower root n-butanol extract group. , Nanshan flower root water extract group.
2.2给药方法2.2 Method of administration
模型建立14天后，每日灌胃给予各组小鼠相应的药物干预14天，空白组和模型组给药0.5％CMC-Na水溶液；尼达尼布阳性药组给药尼达尼布水溶液，给药剂量50mg/kg；黄根片组给药黄根片混悬液，给药剂量200mg/kg；南山花根提取物给药组分别给予南山花根乙酸乙酯提取物、南山花根正丁醇提取物、南山花根水提取物混悬液，给药剂量100mg/kg。14 days after the establishment of the model, the mice in each group were given the corresponding drug intervention by gavage every day for 14 days. The blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution, The administration dose was 50 mg/kg; the Huanggen tablet group was administered with Huanggen tablet suspension, the administration dose was 200 mg/kg; the Nanshan flower root extract administration group was given Nanshan flower root ethyl acetate extract, Nanshan flower root extract Butanol extract, Nanshan flower root water extract suspension, dosage 100mg/kg.
2.3观察指标2.3 Observation indicators
自造模后每周记录小鼠体重，每天记录小鼠死亡情况。给药14天后小鼠腹腔注射戊巴比妥钠90mg/kg充分麻醉，暴露气管，连接Flexivent检测小鼠肺功能。打开小鼠胸腔，心脏灌流冲去肺脏血液后，取出肺脏，小鼠肺左大叶固定于4％多聚甲醛溶液中，常规石蜡包埋，用于后续Masson染色和免疫组织化学观察肺组织病理改变以及肺纤维化程度。取右肺各叶在生 理盐水中充分漂洗后，滤纸吸干表面水分，于-80℃冰箱保存，用于后续肺组织的羟脯氨酸(HYP)含量检测。The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis. Each lobe of the right lung was taken and rinsed in normal saline, and then the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content in lung tissue.
2.3.1小鼠体重及笼旁观察2.3.1 Mice body weight and observation around the cage
笼旁观察包括但不限于如下指标：小鼠死亡情况，是否有湿罗音、喷鼻、弓背、竖毛等，以及四肢活动和行为方式的变化。记录出现时间、程度和持续时间等。记录造模前、造模后每周、小鼠处死前体重。The observation by the cage includes but is not limited to the following indicators: the death of the mice, whether there are wet rales, nasal sprays, arched back, erect hair, etc., as well as changes in limb activity and behavior. Record the occurrence time, extent and duration, etc. The body weight of the mice before and after the modeling, and before the mice were sacrificed, were recorded.
2.3.2呼吸功能检测2.3.2 Respiratory function test
给药14天，最后一次给药后24小时内腹腔注射戊巴比妥钠90mg/kg充分麻醉，仰卧位置于小鼠解剖板上。酒精擦净小鼠颈部皮肤，剪开皮肤，钝性分离颈部肌肉，暴露气管，于甲状软骨下横切一小口，***气管插管并用缝合线结扎。连接气管插管和Flexivent仪器，通过Deep Inflation模块检测小鼠吸气总容积(IC)，SnapShot-150模块检测呼吸***阻力(Rrs)、顺应性(Crs)、弹性(Ers)，Quick Prime-3模块检测主气道阻力(Rn)、组织阻尼(G)和组织弹性(H)等指标，PVs-P绘制小鼠呼吸P-V loop。On the 14th day of administration, the mice were fully anesthetized by intraperitoneal injection of pentobarbital sodium 90 mg/kg within 24 hours after the last administration, and the mice were placed in the supine position on the dissection board. The skin of the mouse neck was wiped with alcohol, the skin was cut open, the neck muscles were bluntly separated, the trachea was exposed, a small incision was made transversely under the thyroid cartilage, a tracheal cannula was inserted and ligated with sutures. Connect tracheal intubation and Flexivent instrument, detect mouse total inspiratory volume (IC) by Deep Inflation module, detect respiratory system resistance (Rrs), compliance (Crs), elasticity (Ers) by SnapShot-150 module, Quick Prime-3 The module detects the main airway resistance (Rn), tissue damping (G) and tissue elasticity (H) and other indicators, and PVs-P draws the P-V loop of mouse breathing.
2.3.3Masson染色2.3.3 Masson staining
取小鼠左肺于4％多聚甲醛固定，用于制备病理切片。经过至少72小时固定组织，取组织经水冲洗固定液，酒精梯度脱水、二甲苯透明组织后包埋。组织包埋后，采用半自动轮转式切片机进行切片，切片厚度为4μm，60℃烤片1h。使用南京建成快速Masson染色染色，具体步骤为：(1)石蜡切片脱蜡入水：二甲苯10min两次、100％乙醇、100％乙醇、95％乙醇、75％乙醇、30％乙醇各两min、温蒸馏水漂洗60s两次。(2)核染色：Regaud氏苏木精染色60s，0.1％乙酸洗液漂洗30s。(3)浆染色：丽春红染液染色60s，0.1％乙酸洗液漂洗30s。(4)分化：1％磷钼酸水溶液分化6-8min，镜下观察至纤维部分为淡粉色，弃去分色液，不需水洗。(5)胶原复染：用苯胺蓝水溶液复染5min，倒丢，用无水乙醇冲洗干净。(6)封片：吹干后用中性树脂封片。(7)镜检：胶原纤维、粘液、软骨呈蓝色；胞浆、肌肉、纤维素、神经胶质呈红色；胞核蓝紫色。染色采用ImageJ v1.50C图像软件进行定量分析。每张切片随机选取5个不同视野，呈现蓝色的胶原蛋白区域为阳性，以阳性面积与整个视野组织总面积的比值作为肺脏纤维化指数。The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 μm and baked at 60°C for 1 h. Using Nanjing Jiancheng rapid Masson staining, the specific steps are: (1) Paraffin sections are dewaxed and put into water: xylene for 10 min twice, 100% ethanol, 100% ethanol, 95% ethanol, 75% ethanol, 30% ethanol for two minutes each, Rinse twice with warm distilled water for 60 s. (2) Nuclear staining: Regaud's hematoxylin staining for 60s, 0.1% acetic acid washing solution for 30s. (3) Pulp dyeing: Ponceau red dyeing solution for 60s, 0.1% acetic acid washing solution for 30s rinsing. (4) Differentiation: 1% phosphomolybdic acid aqueous solution was differentiated for 6-8 minutes, and the fiber part was light pink when observed under a microscope, and the color separation solution was discarded without washing. (5) Collagen counterstaining: counterstaining with aniline blue aqueous solution for 5 min, discarded, and rinsed with absolute ethanol. (6) Sealing: After drying, seal the film with neutral resin. (7) Microscopic examination: collagen fibers, mucus, and cartilage are blue; cytoplasm, muscle, cellulose, and glia are red; nucleus is blue-purple. Staining was quantitatively analyzed using ImageJ v1.50C image software. Five different fields of view were randomly selected for each section, and the blue collagen area was positive, and the ratio of the positive area to the total area of the entire field of view was used as the pulmonary fibrosis index.
2.3.4羟脯氨酸(HYP)检测2.3.4 Hydroxyproline (HYP) detection
使用南京建成羟脯氨酸检测试剂盒，依说明书检测小鼠右肺组织羟脯氨酸含量，具体步骤如下：(1)样品水解：精确称取组织湿重20-40mg于15mL离心管中，加入水解液1mL，混匀。加盖沸水浴10min，摇匀，继续沸水浴10min使水解充分。(2)调节pH：待离心管流水冷却后加入10μL指示剂，摇匀，调节pH在6.0-6.8左右。加入双蒸水至10mL，混匀。取3ml稀释后的水解液加入约30mg活性炭，使溶液澄清无色，混匀，3500rpm离心10min，小心取上清作检测。(3)样品检测：分为空白管、标准品管、测定管，除空白管加入1mL双蒸水，标准品管加入1mL浓度为5μg/mL的标准应用液，样品管加入1mL待测样品外，操作相同。加 入0.5mL试剂一(氯胺T溶液和柠檬酸缓冲液)，混匀静置10min；加入0.5mL试剂二(高氯酸溶液)，混匀静置10min；加入0.5mL试剂三(二甲基氨基苯甲醛溶液)，混匀，60℃水浴15min，冷却后，3500rpm离心10min。(4)检测吸光度：取上清检测550nm波长的吸光度，测定各管吸光度值。根据计算公式计算组织中羟脯氨酸的含量。The Nanjing Jiancheng Hydroxyproline Detection Kit was used to detect the content of hydroxyproline in the right lung tissue of mice according to the instructions. The specific steps are as follows: (1) Sample hydrolysis: Accurately weigh 20-40 mg of tissue wet weight into a 15 mL centrifuge tube, Add 1 mL of the hydrolyzate and mix well. Cover with a boiling water bath for 10 minutes, shake well, and continue the boiling water bath for 10 minutes to fully hydrolyze. (2) Adjust pH: add 10 μL of indicator after cooling down the centrifuge tube, shake well, and adjust the pH to about 6.0-6.8. Add double distilled water to 10mL and mix well. Take 3ml of the diluted hydrolyzate and add about 30mg activated carbon to make the solution clear and colorless, mix well, centrifuge at 3500rpm for 10min, and carefully take the supernatant for detection. (3) Sample detection: It is divided into blank tube, standard product tube, and measuring tube, except that 1 mL of double-distilled water is added to the blank tube, 1 mL of standard application solution with a concentration of 5 μg/mL is added to the standard product tube, and 1 mL of the sample to be tested is added to the sample tube. , the operation is the same. Add 0.5mL reagent one (chloramine T solution and citric acid buffer), mix well and let stand for 10min; add 0.5mL reagent two (perchloric acid solution), mix well and let stand for 10min; add 0.5mL reagent three (dimethyl Aminobenzaldehyde solution), mix well, water bath at 60°C for 15min, after cooling, centrifuge at 3500rpm for 10min. (4) Detection of absorbance: Take the supernatant to detect the absorbance at a wavelength of 550 nm, and measure the absorbance value of each tube. The content of hydroxyproline in the tissue was calculated according to the calculation formula.
3.实验结果3. Experimental results
3.1南山花根不同提取部位对小鼠呼吸功能的影响。3.1 The effect of different extraction parts of Nanshan flower root on the respiratory function of mice.
记录小鼠在造模前后的体重，结果如表1所示：与空白对照组相比，模型组的小鼠体重大幅下降，尼达尼布和南山花根乙酸乙酯提取物组有体重改善作用。呼吸功能结果如表2和图1所示：与空白对照组相比，模型组的小鼠呼吸***总容量下降，呼吸***顺应性下降；南山花根乙酸乙酯提取物能够有效提高小鼠呼吸***总容量和呼吸***顺应性，且效果优于黄根片组，结果有统计学差异。表明南山花根乙酸乙酯提取物能够改善小鼠的呼吸功能。The body weight of the mice before and after modeling was recorded, and the results are shown in Table 1: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the body weight of the nintedanib and Nanshan flower root ethyl acetate extract groups improved. effect. The results of respiratory function are shown in Table 2 and Figure 1: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the ethyl acetate extract of Nanshan flower root can effectively improve the respiratory system of the mice The total system capacity and respiratory system compliance were better than those of the Huanggen tablet group, and the results were statistically different. It shows that the ethyl acetate extract of Nanshan flower root can improve the respiratory function of mice.
Figure PCTCN2022086065-appb-000003
Figure PCTCN2022086065-appb-000003
表1小鼠体重记录(单位g，与空白组比较， #p<0.05， ##p<0.01，与模型组比较，*p<0.05) Table 1 Records of body weight of mice (unit g, compared with blank group, # p<0.05, ## p<0.01, compared with model group, *p<0.05)
Figure PCTCN2022086065-appb-000004
Figure PCTCN2022086065-appb-000004
表2小鼠呼吸***功能(与空白组比较， ##p<0.01，与模型组比较，*p<0.05,**p<0.01) Table 2. Mouse respiratory system function (compared with blank group, ## p<0.01, compared with model group, *p<0.05, **p<0.01)
3.2南山花根不同提取部位对博来霉素致小鼠肺纤维化程度影响。3.2 The effect of different extraction parts of Nanshan flower root on the degree of pulmonary fibrosis in mice induced by bleomycin.
Masson染色(图2)显示，空白对照组仅在支气管壁、血管壁周围有蓝色胶原蛋白的阳性 染色，形态结构基本正常。模型组小鼠出现肺纤维化区域，胶原纤维组织增生、变厚。阳性药尼达尼布和南山花根乙酸乙酯提取物有较好的改善作用。阳性面积占比见图2柱状图。Masson staining (Fig. 2) showed that in the blank control group, there was only positive staining of blue collagen in the bronchial wall and around the blood vessel wall, and the morphological structure was basically normal. The mice in the model group developed areas of pulmonary fibrosis, and collagen fibers proliferated and thickened. The positive drug nintedanib and ethyl acetate extract of Nanshan flower root had better improvement effect. The percentage of positive area is shown in the bar graph in Figure 2.
3.3南山花根不同提取部位对小鼠肺组织羟脯氨酸含量的影响。3.3 The effect of different extraction parts of Nanshan flower root on the content of hydroxyproline in mouse lung tissue.
羟脯氨酸在胶原蛋白中占13.4％，在弹性蛋白中占极少量，其它蛋白中均不存在。肺纤维化时，主要增加的成分为胶原纤维，测定肺组织羟脯氨酸的含量，可换算成肺脏胶原蛋白的含量，以反映肺纤维化程度。结果如表3和图3所示，模型组小鼠肺脏的羟脯氨酸含量增加，有统计学差异(p<0.01)。南山花根乙酸乙酯提取物与模型组相比均出现统计学差异(p<0.01)。结果表明南山花根乙酸乙酯提取物可以降低博来霉素导致的小鼠肺部胶原沉积和肺纤维化程度。Hydroxyproline accounts for 13.4% in collagen, a very small amount in elastin, and does not exist in other proteins. In pulmonary fibrosis, the main component that increases is collagen fibers, and the content of hydroxyproline in lung tissue can be converted into the content of lung collagen to reflect the degree of pulmonary fibrosis. The results are shown in Table 3 and Figure 3. The hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p<0.01). Compared with the model group, the ethyl acetate extract of Nanshan flower root showed statistical difference (p<0.01). The results showed that the ethyl acetate extract of Nanshan flower root could reduce the collagen deposition and pulmonary fibrosis in mice induced by bleomycin.
Figure PCTCN2022086065-appb-000005
Figure PCTCN2022086065-appb-000005
表3小鼠肺组织羟脯氨酸含量(与空白组比较， ##p<0.01，与模型组比较，**p<0.01) Table 3 Content of hydroxyproline in lung tissue of mice (compared with blank group, ## p<0.01, compared with model group, **p<0.01)
实验例2：南山花根不同制备方法的提取物对博来霉素致小鼠肺纤维化的治疗作用Experimental Example 2: Therapeutic effect of extracts of Nanshan flower root with different preparation methods on bleomycin-induced pulmonary fibrosis in mice
1.实验材料1. Experimental materials
1.1实验动物1.1 Experimental animals
无特定病原菌(Specific pathogen-free,SPF)级C57BL/6N小鼠(雄性，体重18-22g)，购自北京维通利华实验动物技术有限公司，生产许可证号：SCXK-(京)2016-0011，发证机关：北京市科学技术委员会，合格证号：11400700300671。Specific pathogen-free (SPF) grade C57BL/6N mice (male, body weight 18-22g), purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK-(Beijing) 2016 -0011, issuing authority: Beijing Municipal Science and Technology Commission, certificate number: 11400700300671.
1.2药物与主要试剂1.2 Drugs and main reagents
Figure PCTCN2022086065-appb-000006
Figure PCTCN2022086065-appb-000006
Figure PCTCN2022086065-appb-000007
Figure PCTCN2022086065-appb-000007
1.3实验主要仪器1.3 The main instruments of the experiment
Figure PCTCN2022086065-appb-000008
Figure PCTCN2022086065-appb-000008
2.实验方法2. Experimental method
2.1造模及分组2.1 Modeling and grouping
取SPF级雄性C57BL/6N小鼠约80只，10只作为空白对照组，其余进行气管滴注博来霉素造成肺纤维化。腹腔注射戊巴比妥钠80mg/kg麻醉小鼠，将小鼠上门齿固定在气管滴注操作台上，LED灯光透过颈部皮肤，自口腔向内观察小鼠气管口。将套管针***小鼠气管，连接HRH-MAG4肺部液体定量雾化器快速推入50μL无菌PBS溶解的博来霉素溶液，造模剂量0.04U/只。给药后保持直立轻旋小鼠使溶液在肺部分布均匀，待其苏醒后给予水和饲料，肺纤维化小鼠模型建立完成。空白对照组除气管给药无菌PBS溶液外，其他操作相同。About 80 SPF grade male C57BL/6N mice were selected, 10 were used as blank control group, and the rest were treated with tracheal instillation of bleomycin to cause pulmonary fibrosis. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital 80 mg/kg, and the upper incisors of the mice were fixed on the tracheal instillation console. A trocar was inserted into the mouse trachea, connected to an HRH-MAG4 pulmonary liquid quantitative nebulizer, and quickly pushed into 50 μL of sterile PBS-dissolved bleomycin solution at a dose of 0.04 U per mouse. After administration, the mice were kept upright and swirled to make the solution evenly distributed in the lungs, and water and feed were given to the mice after they recovered, and the pulmonary fibrosis mouse model was established. In the blank control group, other operations were the same except that sterile PBS solution was administered to the trachea.
14天后，根据存活的造模组动物的体重随机分组至模型组、尼达尼布阳性药组、实施例1组、实施例2组、实施例3组、实施例4组，每组10只。After 14 days, according to the body weight of the surviving model animals, they were randomly divided into the model group, the nintedanib positive drug group, the embodiment 1 group, the embodiment 2 group, the embodiment 3 group, and the embodiment 4 group, with 10 animals in each group. .
2.2给药方法2.2 Method of administration
模型建立14天后，每日灌胃给予各组小鼠相应的药物干预14天，空白组和模型组给药0.5％CMC-Na水溶液；尼达尼布阳性药组给药尼达尼布水溶液，给药剂量50mg/kg；实施例1-实施例4组分别给药0.5％CMC-Na水溶液配制的南山花根提取物混悬液，给药剂量100mg/kg。14 days after the establishment of the model, the mice in each group were given the corresponding drug intervention by gavage every day for 14 days. The blank group and model group were given 0.5% CMC-Na aqueous solution; the nintedanib positive drug group was given nintedanib aqueous solution, The administration dose was 50 mg/kg; Example 1-Example 4 groups were respectively administered the Nanshan flower root extract suspension prepared with 0.5% CMC-Na aqueous solution, and the administration dose was 100 mg/kg.
2.3观察指标2.3 Observation indicators
自造模后每周记录小鼠体重，每天记录小鼠死亡情况。给药14天后小鼠腹腔注射戊巴比妥钠90mg/kg充分麻醉，暴露气管，连接Flexivent检测小鼠肺功能。打开小鼠胸腔，心脏灌流冲去肺脏血液后，取出肺脏，小鼠肺左大叶固定于4％多聚甲醛溶液中，常规石蜡包埋，用于后续Masson染色和免疫组织化学观察肺组织病理改变以及肺纤维化程度。取右肺各叶在生理盐水中充分漂洗后，滤纸吸干表面水分，于-80℃冰箱保存，用于后续肺组织的羟脯氨酸(HYP)含量检测、TGF-β和TNF-α含量的检测、Western Blot检测肺纤维化相关蛋白的表达。The body weight of mice was recorded every week after modeling, and the death of mice was recorded every day. After 14 days of administration, the mice were fully anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg/kg, the trachea was exposed, and Flexivent was connected to detect the lung function of the mice. The thoracic cavity of the mice was opened, and the lungs were removed by cardiac perfusion. The left lobe of the mouse lungs was fixed in 4% paraformaldehyde solution and embedded in conventional paraffin for subsequent Masson staining and immunohistochemical observation of lung histopathology. changes and the degree of pulmonary fibrosis. The lobes of the right lung were taken and rinsed in normal saline, and the surface water was absorbed by filter paper and stored in a -80°C refrigerator for subsequent detection of hydroxyproline (HYP) content, TGF-β and TNF-α content in lung tissue. and Western Blot to detect the expression of pulmonary fibrosis-related proteins.
2.3.1小鼠体重及笼旁观察2.3.1 Mice body weight and observation around the cage
同实验例1。Same as experimental example 1.
2.3.2呼吸功能检测2.3.2 Respiratory function test
同实验例1。Same as experimental example 1.
2.3.3Masson染色2.3.3 Masson staining
同实验例1。Same as experimental example 1.
2.3.4羟脯氨酸(HYP)检测2.3.4 Hydroxyproline (HYP) detection
同实验例1。Same as experimental example 1.
2.3.5免疫组织化学(IHC)染色2.3.5 Immunohistochemical (IHC) staining
取小鼠左肺于4％多聚甲醛固定，用于制备病理切片。经过至少72小时固定组织，取组织经水冲洗固定液，酒精梯度脱水、二甲苯透明组织后包埋。组织包埋后，采用半自动轮转式切片机进行切片，切片厚度为4μm，60℃烤片。经二甲苯脱蜡、梯度乙醇水化、抗原修复(0.01M柠檬酸盐缓冲液，pH6.0，高压修复2min)；用3％过氧化氢封闭内源性过氧化氢酶，5％BSA室温封闭。切片用α-SMA一抗4℃孵育过夜。切片用PBS缓冲液冲洗后，室温下孵育相应二抗1h。滴加新鲜配制的DAB显色剂，苏木精复染，盐酸酒精分化，梯度乙醇脱水干燥，二甲苯透明，封片后显微镜下观察棕褐色为阳性表达。染色采用ImageJ v1.50C图像软件定量分析。每张切片随机选取5个不同视野，分析阳性表达的相对面积。The left lungs of mice were fixed in 4% paraformaldehyde for preparation of pathological sections. After at least 72 hours of fixing the tissue, the tissue was washed with water, the fixative solution, dehydrated with alcohol gradient, and the tissue was cleared with xylene before embedding. After the tissue was embedded, it was sliced with a semi-automatic rotary microtome with a slice thickness of 4 μm and baked at 60°C. After dewaxing with xylene, hydration with gradient ethanol, antigen retrieval (0.01M citrate buffer, pH 6.0, high pressure repair for 2 min); blocking endogenous catalase with 3% hydrogen peroxide, 5% BSA at room temperature closed. Sections were incubated with α-SMA primary antibody overnight at 4°C. After the sections were washed with PBS buffer, the corresponding secondary antibodies were incubated at room temperature for 1 h. Freshly prepared DAB chromogenic reagent was added dropwise, hematoxylin counterstained, hydrochloric acid alcohol differentiation, dehydration and drying with gradient ethanol, and xylene was transparent. Staining was quantitatively analyzed using ImageJ v1.50C image software. Five different fields of view were randomly selected for each section, and the relative area of positive expression was analyzed.
2.3.6酶联免疫吸附实验(ELISA)检测2.3.6 Enzyme-linked immunosorbent assay (ELISA) detection
收集每组小鼠肺组织样品，组织样本在-80℃冻存直至检测。取一小块肺组织，称重后加入一定量PBS缓冲液，使用匀浆器充分匀浆。3500rpm离心20min，收集上清用于ELISA检验。取上清按照江苏晶美生物科技有限公司大鼠TNF-α和TGF-β试剂盒说明书方法，检测肺组织中TNF-α和TGF-β的含量。使用BCA蛋白定量试剂盒检测组织总蛋白浓度，结果用TNF-α或TGF-β的含量与总蛋白的比值表示。Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of PBS buffer after weighing, and use a homogenizer to fully homogenize. Centrifuge at 3500rpm for 20min, and collect the supernatant for ELISA test. The supernatant was collected and the contents of TNF-α and TGF-β in the lung tissue were detected according to the instructions of the rat TNF-α and TGF-β kit of Jiangsu Jingmei Biotechnology Co., Ltd. The total protein concentration of tissue was detected by BCA protein quantification kit, and the results were expressed as the ratio of the content of TNF-α or TGF-β to total protein.
2.3.7Western blot检测相关蛋白的表达2.3.7 Western blot to detect the expression of related proteins
收集每组小鼠肺组织样品，组织样本在-80℃冻存直至检测。取一小块肺组织，称重后加入一定量含1％蛋白酶抑制剂和1％蛋白磷酸酶抑制剂的RIPA裂解液，研磨后冰上裂解45min。裂解液于4℃12000rpm离心20min后取上清，使用BCA蛋白质测定试剂盒测量蛋白质浓度。50微克蛋白样品用5×上样缓冲液稀释，并100℃加热5分钟后，在SDS-PAGE胶中电泳，随后转移到PVDF膜上，用5％脱脂奶粉封闭1h，4℃条件下一抗孵育过夜，TBST缓冲液洗膜3次后室温下孵育相应二抗1h，洗膜后用ECL化学发光液显影。经化学发光仪曝光记录后，使用ImageJ分析条带。Lung tissue samples were collected from each group of mice, and the tissue samples were frozen at -80°C until assayed. Take a small piece of lung tissue, add a certain amount of RIPA lysis buffer containing 1% protease inhibitor and 1% protein phosphatase inhibitor after weighing, grind and lyse on ice for 45 min. The lysate was centrifuged at 12,000 rpm at 4°C for 20 min, and the supernatant was taken, and the protein concentration was measured using the BCA protein assay kit. 50 μg protein sample was diluted with 5× loading buffer, heated at 100°C for 5 minutes, electrophoresed in SDS-PAGE gel, then transferred to PVDF membrane, blocked with 5% nonfat milk powder for 1h, and primary antibody at 4°C After incubation overnight, the membrane was washed three times with TBST buffer and incubated with the corresponding secondary antibody for 1 h at room temperature. After washing, the membrane was developed with ECL chemiluminescence solution. After recording by chemiluminometer exposure, bands were analyzed using ImageJ.
2.4统计方法2.4 Statistical methods
实验结果以均数±标准差
Figure PCTCN2022086065-appb-000009
表示，使用统计软件SPSS 20进行分析，模型组与空白组比较采用Student's t检验，给药各组与模型组采用单因素方差分析(One Way ANOVA)，Homogeneity检验方差齐性，若方差齐时采用最小显著法(LSD)检验；若方差不齐时采用Tamhane's T2检验。以p<0.05为差异有显著性。 The experimental results are expressed as mean ± standard deviation
Figure PCTCN2022086065-appb-000009
Statistical software SPSS 20 was used for analysis, Student's t test was used to compare the model group with the blank group, One Way ANOVA was used between each administration group and the model group, and the homogeneity of variance was tested by Homogeneity. Least significant test (LSD) test; Tamhane's T2 test was used if the variance was unequal. The difference was considered significant at p<0.05.
3.实验结果3. Experimental results
3.1南山花根不同制备方法的提取物对小鼠呼吸功能的影响。3.1 The effects of different preparation methods of Nanshan flower root extracts on respiratory function of mice.
记录小鼠在造模后和给予南山花根提取物治疗后的体重、死亡率和呼吸功能。体重结果 如表4所示：与空白对照组相比，模型组的小鼠体重大幅下降，不同制备方法的南山花根提取物治疗后出现不同程度的恢复，且给药组小鼠的死亡率较模型组较低。呼吸功能结果如表5和图4所示：与空白对照组相比，模型组的小鼠呼吸***总容量下降，呼吸***顺应性下降；实施例1-4组能够有效提高小鼠呼吸***总容量和呼吸***顺应性，结果有统计学差异。表明实施例1-4组能够改善小鼠的呼吸功能。The body weight, mortality and respiratory function of mice were recorded after modeling and after treatment with Nanshan flower root extract. The body weight results are shown in Table 4: compared with the blank control group, the body weight of the mice in the model group decreased significantly, and the Nanshan flower root extracts with different preparation methods recovered to varying degrees after treatment, and the mortality rate of the mice in the administration group was lower than the model group. The results of respiratory function are shown in Table 5 and Figure 4: compared with the blank control group, the total volume of the respiratory system of the mice in the model group decreased, and the compliance of the respiratory system decreased; the examples 1-4 groups can effectively improve the total respiratory system of the mice. Volume and respiratory compliance, the results were statistically different. It is shown that the groups of Examples 1-4 can improve the respiratory function of mice.
Figure PCTCN2022086065-appb-000010
Figure PCTCN2022086065-appb-000010
表4小鼠体重记录(单位g)和最终死亡数(与空白组比较， #p<0.05) Table 4. Recording of body weight of mice (unit g) and the final number of deaths (compared with blank group, # p<0.05)
Figure PCTCN2022086065-appb-000011
Figure PCTCN2022086065-appb-000011
表5小鼠呼吸***功能(与空白组比较， ##p<0.01，与模型组比较，*p<0.05,**p<0.01) Table 5. Mouse respiratory system function (compared with blank group, ## p<0.01, compared with model group, *p<0.05, **p<0.01)
3.2南山花根不同制备方法的提取物对博来霉素致小鼠肺纤维化程度影响。3.2 The effects of different preparation methods of Nanshan flower root extracts on the degree of bleomycin-induced pulmonary fibrosis in mice.
Masson染色(图5)显示，空白对照组仅在支气管壁、血管壁周围有蓝色胶原蛋白的阳性染色，形态结构基本正常。模型组小鼠出现肺纤维化区域，胶原纤维组织增生、变厚。给药各组有不同程度的改善，其中阳性药尼达尼布组和实施例4组有最好的改善作用。阳性面积占比见图2柱状图。Masson staining (Fig. 5) showed that in the blank control group, there was only positive staining of blue collagen around the bronchial wall and blood vessel wall, and the morphological structure was basically normal. The mice in the model group developed areas of pulmonary fibrosis, and collagen fibers proliferated and thickened. The administration groups had different degrees of improvement, among which the positive drug nintedanib group and the Example 4 group had the best improvement effect. The percentage of positive area is shown in the bar graph in Figure 2.
3.3南山花根不同制备方法的提取物对小鼠肺组织羟脯氨酸含量的影响。3.3 The effect of different preparation methods of Nanshan flower root extract on the content of hydroxyproline in mouse lung tissue.
结果如表6和图6所示，模型组小鼠肺脏的羟脯氨酸含量增加，有统计学差异(p<0.01)。南山花根不同浓度给药组的肺组织羟脯氨酸含量程剂量依赖性下降，各实施例组与模型组相 比均出现统计学差异(p<0.01)。结果表明南山花根提取物可以降低博来霉素导致的小鼠肺部胶原沉积和肺纤维化程度，其中实施例4组效果最佳。The results are shown in Table 6 and Figure 6. The hydroxyproline content in the lungs of the mice in the model group increased, and there was a statistical difference (p<0.01). The hydroxyproline content in the lung tissue of the Nanshan flower root administration groups with different concentrations decreased in a dose-dependent manner, and there was a statistical difference between each example group and the model group (p<0.01). The results show that Nanshan flower root extract can reduce the collagen deposition and pulmonary fibrosis in mice caused by bleomycin, among which Example 4 has the best effect.
Figure PCTCN2022086065-appb-000012
Figure PCTCN2022086065-appb-000012
表6小鼠肺组织羟脯氨酸含量(与空白组比较，##p<0.01，与模型组比较，**p<0.01)Table 6. The content of hydroxyproline in the lung tissue of mice (compared with the blank group, ##p<0.01, compared with the model group, **p<0.01)
3.4南山花根不同制备方法的提取物对小鼠肺组织TGF-β和TNF-α表达的影响。3.4 The effects of different preparation methods of Nanshan flower root extracts on the expression of TGF-β and TNF-α in mouse lung tissue.
TGF-β是细胞外基质合成与沉积失调导致组织纤维化最重要的调节因子，多种器官纤维化、硬化、动脉粥样硬化都与之相关。TNF-α是一种细胞毒因子,在肺纤维化的发病机制中起着关键作用。因此，我们通过ELISA检测小鼠肺组织中的TGF-β和TNF-α含量。结果如表7和图7所示，模型组小鼠肺组织中TGF-β和TNF-α含量上升，有统计学差异；给药组降低了肺组织中TGF-β和TNF-α的含量。结果显示南山花根相应提取物可能通过影响TGF-β和TNF-α的表达抑制肺纤维化的进展。TGF-β is the most important regulator of tissue fibrosis caused by dysregulation of extracellular matrix synthesis and deposition, and is associated with various organ fibrosis, sclerosis, and atherosclerosis. TNF-α is a cytotoxic factor that plays a key role in the pathogenesis of pulmonary fibrosis. Therefore, we detected TGF-β and TNF-α content in mouse lung tissue by ELISA. The results are shown in Table 7 and Figure 7. The contents of TGF-β and TNF-α in the lung tissue of the mice in the model group increased, and there was a statistical difference; the administration group decreased the contents of TGF-β and TNF-α in the lung tissue. The results showed that the corresponding extracts of Nanshan flower root may inhibit the progression of pulmonary fibrosis by affecting the expression of TGF-β and TNF-α.
Figure PCTCN2022086065-appb-000013
Figure PCTCN2022086065-appb-000013
表7小鼠肺组织TGF-β和TNF-α含量(与空白组比较，##p<0.01，与模型组比较，*p<0.05,**p<0.01)Table 7 Contents of TGF-β and TNF-α in lung tissue of mice (compared with blank group, ##p<0.01, compared with model group, *p<0.05, **p<0.01)
3.5南山花根不同制备方法的提取物对小鼠肺组织COL1A1、COL3A1和α-SMA蛋白表达的影响。3.5 The effects of different preparation methods of Nanshan flower root extracts on the expression of COL1A1, COL3A1 and α-SMA proteins in mouse lung tissue.
肺纤维化发生后，成纤维细胞被激活释放大量细胞外基质，细胞外基质的主要组成成分有COL1A1、COL3A1，而α-SMA是成纤维细胞激活的主要标志。我们通过Western Blot检验小鼠肺组织中COL1A1、COL3A1蛋白表达情况。同时，我们还通过免疫组织化学检测α-SMA在小鼠肺组织的分布情况。结果(表8和图8)显示，博来霉素刺激后小鼠肺组织I型胶原和III 型胶原释放增加，α-SMA表达增加且广泛分布，与空白组相比有统计学差异(p<0.01)。南山花根不同制备方法的提取物治疗后有明显的改善作用，与模型组相比有统计学差异(p<0.01)。结果显示南山花根提取物可以降低小鼠肺纤维化导致的COL1A1、COL3A1和α-SMA蛋白表达升高，实施例4的效果最显著。After the occurrence of pulmonary fibrosis, fibroblasts are activated to release a large amount of extracellular matrix. The main components of extracellular matrix are COL1A1 and COL3A1, and α-SMA is the main marker of fibroblast activation. We examined the expression of COL1A1 and COL3A1 proteins in mouse lung tissue by Western Blot. At the same time, we also detected the distribution of α-SMA in mouse lung tissue by immunohistochemistry. The results (Table 8 and Figure 8) showed that after stimulation with bleomycin, the release of type I collagen and type III collagen in the lung tissue of mice increased, and the expression of α-SMA was increased and widely distributed, which was statistically different from the blank group (p <0.01). The extracts of Nanshan flower root with different preparation methods have obvious improvement effect after treatment, and there is a statistical difference compared with the model group (p<0.01). The results show that Nanshan flower root extract can reduce the elevated expression of COL1A1, COL3A1 and α-SMA proteins caused by pulmonary fibrosis in mice, and the effect of Example 4 is the most significant.
Figure PCTCN2022086065-appb-000014
Figure PCTCN2022086065-appb-000014
表8南山花根不同制备方法提取物对博来霉素诱导的小鼠肺组织蛋白表达影响Table 8 Effects of different preparation methods of Nanshan flower root extract on bleomycin-induced mouse lung tissue protein expression
实验例3：南山花根提取物中的有效化合物抑制成纤维细胞激活Experimental Example 3: Effective Compounds in Nanshan Flower Root Extract Inhibit Fibroblast Activation
1.实验背景1. Experimental background
成纤维细胞在未激活状态下不表达α肌动蛋白(α-SMA)，在受TGF-β1激活后，成纤维细胞激活为肌成纤维细胞并停止增殖，表达α-SMA并释放细胞外基质相关蛋白。我们从文献和实验得到，南山花根蒽醌提取部位含有东莨菪内酯和甲基异茜草素-1-甲醚两种已知化合物，我们设计成纤维细胞激活实验并给予南山花根提取物中含有的化合物治疗，检验东莨菪内酯和甲基异茜草素-1-甲醚的抗成纤维细胞激活活性。Fibroblasts do not express α-actin (α-SMA) in the unactivated state, after being activated by TGF-β1, fibroblasts are activated to myofibroblasts and stop proliferating, express α-SMA and release extracellular matrix related proteins. We obtained from literature and experiments that the extract of Nanshan flower root anthraquinone contains two known compounds, scopolactone and methylisorubicin-1-methyl ether, we designed a fibroblast activation experiment and gave Nanshan flower root extract The anti-fibroblast activating activity of scopolactone and methylisorubicin-1-methyl ether was examined in the treatment of the compounds contained in the study.
2.实验材料2. Experimental materials
人肺成纤维细胞系HPF细胞购自上海酶联生物科技有限公司，人重组TGF-β1购自Peprotech公司，东莨菪内酯和甲基异茜草素-1-甲醚购自北京倍特仁康生物医药科技有限公司。Human lung fibroblast cell line HPF cells were purchased from Shanghai Enzyme Link Biotechnology Co., Ltd., human recombinant TGF-β1 was purchased from Peprotech Company, and scopolide and methylisorubidin-1-methyl ether were purchased from Beijing Biterenkang Biotechnology Co., Ltd. Pharmaceutical Technology Co., Ltd.
3.实验方法3. Experimental method
将HPF细胞铺于6孔板，待生长至约70％，弃去培养基，阴性对照组加入1mL无血清培养基(含0.1％DMSO)，造模组和给药组加入1mL含10ng/ml TGF-β1的无血清培养基，给药组中分别加入东莨菪内酯或甲基异茜草素-1-甲醚，使终浓度达到5μg/mL，水平旋摇培养板使药液混匀。置于37℃，5％CO 2培养箱中孵育48小时。随后弃去培养基，加入RIPA裂解液100μL裂解细胞(含1％蛋白酶，磷酸酶抑制剂)，使用刮刀收集细胞，13000rpm，4℃，离心10min。取上清，并转移至新的EP管中，进行Western blot实验检测α-SMA蛋白表达情况。 The HPF cells were spread on a 6-well plate, and the medium was discarded after growing to about 70%. The negative control group was added with 1 mL of serum-free medium (containing 0.1% DMSO), and the modeling group and the administration group were added with 1 mL containing 10ng/ml. TGF-β1 serum-free medium, scopolactone or methylisorubicin-1-methyl ether were added to the administration group to make the final concentration reach 5 μg/mL, and the culture plate was shaken horizontally to mix the liquid. Place in a 37 °C, 5% CO2 incubator for 48 h. Then, the medium was discarded, 100 μL of RIPA lysis solution was added to lyse the cells (containing 1% protease, phosphatase inhibitor), and the cells were collected using a scraper, centrifuged at 13000 rpm, 4° C. for 10 min. The supernatant was taken and transferred to a new EP tube for Western blot to detect the expression of α-SMA protein.
4.实验结果4. Experimental results
结果如表9和图9所示，在TGF-β1诱导后，成纤维细胞表达α-SMA增多。而东莨菪内 酯和甲基异茜草素-1-甲醚均有抑制α-SMA表达增多的作用，有抑制成纤维细胞激活的效果，结果有统计学差异(p<0.01)。The results are shown in Table 9 and Figure 9. After induction of TGF-β1, fibroblasts express increased α-SMA. However, both scopolactone and methylisorubidin-1-methyl ether inhibited the increased expression of α-SMA and inhibited the activation of fibroblasts, and the results were statistically different (p<0.01).
Figure PCTCN2022086065-appb-000015
Figure PCTCN2022086065-appb-000015
表9东莨菪内酯和甲基异茜草素-1-甲醚对成纤维细胞抑制活性Table 9 Fibroblast inhibitory activity of scopolactone and methylisorubicin-1-methyl ether
实验例4：本发明药物组合物抑制成纤维细胞激活Experimental Example 4: The pharmaceutical composition of the present invention inhibits the activation of fibroblasts
实验材料与实验方法与实验例3相同，给药组分别为尼达尼布组，实施例1、5、6、7、8、9组。尼达尼布组、实施例8和实施例9组给药剂量为2μM，其余各实例组给药剂量为5μg/mL。实施例1为南山花根乙酸乙酯提取物；实施例5药物组合为蒽醌类、蒽醌糖苷类和环烯醚萜类按照重量8：1：1的重量比混合；实施例6药物组合为1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、甲基异茜草素-1-甲醚、甲基异茜草素和东莨菪内酯按照1：5：5：1的重量比混合：实施例7药物组合为东莨菪内酯和甲基异茜草素-1-甲醚,按照1：1的重量比混合；实施例8药物组合为实施例1的南山花根提取物和吡非尼酮按照10：1的重量比混合；实施例9药物组合为实施例1的南山花根提取物和尼达尼布按照8：1的重量比混合。The experimental materials and experimental methods were the same as those in Experimental Example 3, and the administration groups were the nintedanib group and the Examples 1, 5, 6, 7, 8, and 9 groups, respectively. The administration dose of nintedanib group, Example 8 and Example 9 group was 2 μM, and the administration dose of the other example groups was 5 μg/mL. Example 1 is the ethyl acetate extract of Nanshan flower root; Example 5 drug combination is anthraquinones, anthraquinone glycosides and iridoids are mixed in a weight ratio of 8:1:1; Example 6 drug combination For 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, methylisorubicin and scopolactone according to 1:5: Mixing in a weight ratio of 5:1: the drug combination in Example 7 is scopolactone and alizarin-1-methyl ether, which are mixed in a weight ratio of 1:1; the drug combination in Example 8 is Nanshan in Example 1 The flower root extract and pirfenidone were mixed in a weight ratio of 10:1; the drug combination in Example 9 was that the Nanshan flower root extract and nintedanib of Example 1 were mixed in a weight ratio of 8:1.
实验结果Experimental results
结果如表10和图10所示，与实验例3中表9的数据比较可知，在相同剂量的情况下，东莨菪内酯组的数值为5.10，甲基异茜草素-1-甲醚组为4.22，将东莨菪内酯和甲基异茜草素-1-甲醚组合后的实施例7为3.10，可见实施例7组结果明显优于东莨菪内酯、甲基异茜草素-1-甲醚单独使用的结果，即东莨菪内酯和甲基异茜草素-1-甲醚组合后具有协同增效作用。另外，通过比较尼达尼布组、实施例1组和实施例9的结果可知，在与尼达尼布组相同剂量和比实施例1组剂量更低的情况下，实施例9组有明显更优的技术效果，说明书将南山花根提取物与吡菲尼酮或尼达尼布等化学药组合后，具有协同增效作用。The results are shown in Table 10 and Figure 10. Compared with the data in Table 9 in Experimental Example 3, it can be seen that under the same dose, the value of the scopolactone group is 5.10, and the methylisorubidin-1-methyl ether group It is 4.22, and the Example 7 after combining scopolactone and methylisorubicin-1-methyl ether is 3.10. It can be seen that the results of Example 7 group are obviously better than those of scopolactone and methylisorubidin-1-methyl ether. The result of methyl ether alone, that is, the combination of scopolactone and methylisorubicin-1-methyl ether has a synergistic effect. In addition, by comparing the results of the nintedanib group, the example 1 group and the example 9, it can be seen that in the case of the same dose as the nintedanib group and a lower dose than the example 1 group, the example 9 group has obvious Better technical effect, the instructions combine the Nanshan flower root extract with chemical drugs such as pirfenidone or nintedanib, which has a synergistic effect.
Figure PCTCN2022086065-appb-000016
Figure PCTCN2022086065-appb-000016
表10不同药物组合对成纤维细胞抑制活性Table 10 Inhibitory activity of different drug combinations on fibroblasts
实验例5：基于液质联用技术的黄根提取物中药成分分析Experimental Example 5: Analysis of Chinese medicinal components in Huang root extract based on LC/MS technology
1.色谱和质谱条件1. Chromatographic and Mass Spectrometry Conditions
安捷伦1290超高效液相色谱联用安捷伦6550三重四极杆飞行时间质谱。色谱柱为Waters  BEH C18(2.1*100mm，1.7μm)，流动相A相：水(0.1％甲酸，1mmol/L乙酸铵)，流动相B相：乙腈(0.1％甲酸，1mmol/L乙酸铵)，梯度洗脱条件：0-5min，20％B；5-30min，20-100％B，流速：0.3mL/min，柱温：30℃。Agilent 1290 UPLC coupled to Agilent 6550 Triple Quadrupole Time-of-Flight Mass Spectrometer. The chromatographic column is Waters BEH C18 (2.1*100mm, 1.7μm), mobile phase A phase: water (0.1% formic acid, 1mmol/L ammonium acetate), mobile phase B phase: acetonitrile (0.1% formic acid, 1mmol/L ammonium acetate) , gradient elution conditions: 0-5min, 20%B; 5-30min, 20-100%B, flow rate: 0.3mL/min, column temperature: 30°C.
采用一级高分辨(HRMS)和二级高分辨(HRMS/MS)质谱负离子采集模式，干燥气温度：280℃，干燥气流速：11L/min，鞘气温度：325℃，鞘气流速：12L/min，一级质谱扫描范围：150-1200m/z，二级质谱扫描范围：50-1200m/z，二级碰撞电压：10/20/30/40/50V。Adopt the negative ion acquisition mode of primary high resolution (HRMS) and secondary high resolution (HRMS/MS) mass spectrometry, drying gas temperature: 280 °C, drying gas flow rate: 11L/min, sheath gas temperature: 325 °C, sheath gas flow rate: 12L /min, scanning range of primary mass spectrometry: 150-1200 m/z, scanning range of secondary mass spectrometry: 50-1200 m/z, secondary collision voltage: 10/20/30/40/50V.
2.实验结果2. Experimental results
基于液相色谱高分辨质谱技术识别和鉴定南山花根提取物中的中药成分Identification and identification of traditional Chinese medicine components in Nanshan flower root extract based on liquid chromatography and high-resolution mass spectrometry
基于已优化的色谱质谱分析条件，采集一级高分辨质谱(HRMS)数据。将HRMS数据与自建数据库进行比对，识别南山花根提取物中的中药成分，进一步在不同碰撞电压下采集南山花根提取物中药成分的二级高分辨质谱(HRMS/MS)数据，鉴定了南山花根提取物中30个中药成分，包括23个蒽醌类成分、4个环烯醚萜类成分和3个蒽醌糖苷类成分，部分化合物为同分异构体化合物。鉴定的各成分基本信息见下表。Based on optimized chromatographic mass spectrometry conditions, first-order high-resolution mass spectrometry (HRMS) data were collected. The HRMS data was compared with the self-built database to identify the traditional Chinese medicine components in the extract of Nanshan flower root, and the secondary high-resolution mass spectrometry (HRMS/MS) data of the traditional Chinese medicine component in the Nanshan flower root extract was further collected under different collision voltages. Thirty traditional Chinese medicine components in Nanshan flower root extract were identified, including 23 anthraquinones, 4 iridoids and 3 anthraquinone glycosides, and some compounds were isomer compounds. The basic information of each component identified is shown in the table below.
Figure PCTCN2022086065-appb-000017
Figure PCTCN2022086065-appb-000017
表11基于液相色谱高分辨质谱鉴定的黄根提取物中药成分Table 11 Identification of Chinese medicinal components of Huang Root Extract based on liquid chromatography high-resolution mass spectrometry
实验例结论：本发明南山花根提取物可以改善不同因素导致的肺纤维化，能够改善肺功能，降低肺组织的胶原沉积和成纤维细胞增殖和活化，能够抑制肺组织TGF-β和TNF-α而抑制肺纤维化。本发明南山花根提取部位为蒽醌类、环烯醚萜类和蒽醌糖苷类。其中的两个具体化合物东莨菪内酯和甲基异茜草素-1-甲醚，明确具有抑制成纤维细胞激活的作用。本发明南山花根提取物具有确切的抗肺纤维化作用，东莨菪内酯和甲基异茜草素-1-甲醚从细胞水平上证明了有抗肺纤维化潜力，其组合后具有协同增效作用。同时，本发明的南山花根提取物与抗肺纤维化的化学药吡菲尼酮或尼达尼布等化学药组合后，具有协同增效作用。综上，本发明南山花根提取物及其组成成分，可用于制备抗肺脏纤维化的药物。Experimental example conclusion: The Nanshan flower root extract of the present invention can improve pulmonary fibrosis caused by different factors, can improve lung function, reduce collagen deposition in lung tissue and fibroblast proliferation and activation, and can inhibit lung tissue TGF-β and TNF-α. α inhibits pulmonary fibrosis. The extracting parts of Nanshan flower root in the present invention are anthraquinones, iridoids and anthraquinone glycosides. Two of the specific compounds, scopolactone and methylisorubidin-1-methyl ether, clearly inhibit the activation of fibroblasts. The Nanshan flower root extract of the present invention has a definite anti-pulmonary fibrosis effect, and scopolactone and methylisorubidin-1-methyl ether have proved the anti-pulmonary fibrosis potential at the cellular level, and their combination has a synergistic increase effect. At the same time, after the Nanshan flower root extract of the present invention is combined with chemical drugs such as pirfenidone or nintedanib for anti-pulmonary fibrosis, it has a synergistic effect. In conclusion, the Nanshan flower root extract and its constituent components of the present invention can be used to prepare medicines against pulmonary fibrosis.

Claims (16)

  1. 一种南山花根提取物在制备治疗肺纤维化的药物中的用途，其特征在于：所述南山花根提取物是蒽醌类、蒽醌糖苷类和环烯醚萜类有效部位；所述南山花根提取物的制备方法为取南山花根用乙醇提取，再使用制备色谱法去除杂质，精制纯化得到蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分；或者取南山花根用乙酸乙酯提取得到；或者取南山花根用溶剂提取后，浓缩提取液，再使用乙酸乙酯萃取得到。A use of Nanshan flower root extract in preparing medicine for treating pulmonary fibrosis, characterized in that: the Nanshan flower root extract is anthraquinones, anthraquinone glycosides and iridoids effective parts; The preparation method of Nanshan flower root extract is to take Nanshan flower root and extract it with ethanol, then use preparative chromatography to remove impurities, and refine and purify to obtain anthraquinones, anthraquinone glycosides and iridoids active components; It can be obtained by extracting with ethyl acetate; or after extracting Nanshan flower root with solvent, concentrating the extract, and then extracting with ethyl acetate.
  2. 根据权利要求1所述的用途，其特征在于：所述南山花根提取物的制备方法为，南山花根95％乙醇回流提取三次，提取液减压浓缩，采用制备色谱法去除杂质，精制纯化得到蒽醌类、蒽醌糖苷类和环烯醚萜类有效成分，所述色谱法为大孔树脂法、分子筛色谱法、以及硅胶柱色谱法、聚酰胺柱色谱法的一种或多种。The use according to claim 1, characterized in that: the preparation method of the Nanshan flower root extract is: 95% ethanol reflux extraction of Nanshan flower root three times, the extract is concentrated under reduced pressure, the impurities are removed by preparative chromatography, and the purification is carried out. The active ingredients of anthraquinones, anthraquinone glycosides and iridoids are obtained, and the chromatography is one or more of macroporous resin method, molecular sieve chromatography, silica gel column chromatography and polyamide column chromatography.
  3. 根据权利要求2所述的用途，其特征在于：所述色谱法为硅胶柱色谱法，采用水和乙腈梯度洗脱。The use according to claim 2, wherein the chromatography is silica gel column chromatography, and gradient elution with water and acetonitrile is adopted.
  4. 根据权利要求1所述的用途，其特征在于：所述南山花根提取物的制备方法为，取南山花根先用石油醚提取，弃去提取液，再用乙酸乙酯提取，减压浓缩得到乙酸乙酯提取物。Use according to claim 1, is characterized in that: the preparation method of described Nanshan flower root extract is: take Nanshan flower root and extract with petroleum ether first, discard the extract, then extract with ethyl acetate, and concentrate under reduced pressure An ethyl acetate extract was obtained.
  5. 根据权利要求1所述的用途，其特征在于：所述南山花根提取物的制备方法为，取南山花根用溶剂提取后，浓缩提取液，再使用乙酸乙酯萃取得到，所述的溶剂为水、甲醇、乙醇、丙醇、丁醇等的一种或二种以上的组合。The use according to claim 1, wherein: the preparation method of the Nanshan flower root extract is: after taking Nanshan flower root and extracting it with a solvent, concentrating the extract, and then extracting it with ethyl acetate to obtain the solvent. It is one or a combination of two or more of water, methanol, ethanol, propanol, butanol, etc.
  6. 根据权利要求5所述的用途，其特征在于：所述南山花根提取物的制备方法为，南山花根用水提取，再使用95％乙醇提取，合并水和乙醇提取液，提取液减压浓缩，使用乙酸乙酯萃取后旋蒸去除溶剂获得的南山花根提取物。The use according to claim 5, characterized in that: the preparation method of the Nanshan flower root extract is as follows: Nanshan flower root is extracted with water, then extracted with 95% ethanol, combined with water and ethanol extract, and the extract is concentrated under reduced pressure , using ethyl acetate extraction and rotary evaporation to remove the solvent of Nanshan flower root extract.
  7. 根据权利要求5所述的用途，其特征在于：所述南山花根提取物的制备方法为，南山花根95％乙醇回流提取三次，乙酸乙酯萃取后旋蒸去除溶剂获得的南山花根提取物。The use according to claim 5, characterized in that: the preparation method of the Nanshan flower root extract is: the Nanshan flower root is extracted three times by refluxing with 95% ethanol, and the Nanshan flower root is extracted by rotary evaporation to remove the solvent after ethyl acetate extraction. thing.
  8. 根据权利要求1所述的用途，其特征在于：所述肺纤维化不是由矽肺引起的。The use according to claim 1, wherein the pulmonary fibrosis is not caused by silicosis.
  9. 根据权利要求2所述的用途，其特征在于：所述治疗肺纤维化是改善肺功能，降低肺组织的胶原沉积和成纤维细胞增殖和活化，抑制肺组织TGF-β和TNF-α而抑制肺纤维化。The use according to claim 2, characterized in that: the treatment of pulmonary fibrosis is to improve lung function, reduce collagen deposition in lung tissue and fibroblast proliferation and activation, and inhibit lung tissue TGF-β and TNF-α. Pulmonary Fibrosis.
  10. 根据权利要求1所述的用途，其特征在于：所述蒽醌类有效成分包括2-甲基蒽醌、1-羟基-2-甲基蒽醌、甲基异茜草素、2-羟基-3-甲氧基蒽醌、2-羟基-3-羟甲基蒽醌、去甲基虎刺醛、甲基异茜草素-1-甲醚、虎刺醛、1,3-二羟基-2-甲氧甲基蒽醌、3-羟基-1-甲氧基-2-羟甲基蒽醌、1,3-二羟基-2-乙氧甲基蒽醌、1-羟基-2,3-二甲氧基-7-甲基蒽醌、7-羟基-1,2-二甲氧基-6-甲基蒽醌、1,3,8-三羟基-7-甲氧基-2-甲基蒽醌、3-羟基-5,6-二甲氧基-2-甲基-1,2,3,4-四氢蒽醌、1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、1,3-二羟基-6-甲氧基-2-甲氧甲基蒽醌、东莨菪内酯、3,5-二羟基-6,7-二甲氧基-2-甲基-1,2,3,4-四氢蒽醌、3-羟基-1,5,6-三甲氧基-2-甲基蒽醌、2-羟基-4,6,7-三甲氧基-3-甲基蒽醌、4-羟基-1,2,3-三甲氧基-7-羟甲基蒽醌、1,3-二羟基-5,6-二甲氧基-2- 甲氧基甲基蒽醌的一种或多种；The use according to claim 1, wherein the anthraquinone active ingredients include 2-methylanthraquinone, 1-hydroxy-2-methylanthraquinone, methylisorubicin, 2-hydroxy-3 -Methoxyanthraquinone, 2-Hydroxy-3-hydroxymethylanthraquinone, Demethyl succinylaldehyde, Methylisorubicin-1-methyl ether, stigma aldehyde, 1,3-dihydroxy-2- Methoxymethyl anthraquinone, 3-hydroxy-1-methoxy-2-hydroxymethyl anthraquinone, 1,3-dihydroxy-2-ethoxymethyl anthraquinone, 1-hydroxy-2,3-dihydroxymethyl anthraquinone Methoxy-7-methylanthraquinone, 7-hydroxy-1,2-dimethoxy-6-methylanthraquinone, 1,3,8-trihydroxy-7-methoxy-2-methyl Anthraquinone, 3-hydroxy-5,6-dimethoxy-2-methyl-1,2,3,4-tetrahydroanthraquinone, 1,3-dihydroxy-5,6-dimethoxy- 2-methylanthraquinone, 1,3-dihydroxy-6-methoxy-2-methoxymethylanthraquinone, scopolactone, 3,5-dihydroxy-6,7-dimethoxy- 2-Methyl-1,2,3,4-tetrahydroanthraquinone, 3-hydroxy-1,5,6-trimethoxy-2-methylanthraquinone, 2-hydroxy-4,6,7-trimethyl Oxy-3-methylanthraquinone, 4-hydroxy-1,2,3-trimethoxy-7-hydroxymethylanthraquinone, 1,3-dihydroxy-5,6-dimethoxy-2- one or more of methoxymethylanthraquinone;
    所述蒽醌糖苷类有效成分包括lucidin 3-O-β-primeveroside、damnacanthol 3-O-β-primeveroside、rubiadin 3-O-β-primerveroside的一种或多种；The anthraquinone glycoside active ingredients include one or more of lucidin 3-O-β-primeveroside, damnacanthol 3-O-β-primeveroside, and rubiadin 3-O-β-primerveroside;
    所述环烯醚萜类有效成分包括Prismatomerin、deacetylasperuloside、deacetylasperulosidic acid、asperulosidic acid的一种或多种。The iridoid active ingredients include one or more of Prismatomerin, deacetylasperuloside, deacetylasperulosidic acid, and asperulosidic acid.
  11. 一种南山花根提取物在制备治疗肺纤维化的药物中的用途，其特征在于：所述南山花根提取物是莨菪内酯、甲基异茜草素-1-甲醚的一种或两种。A use of Nanshan flower root extract in preparing medicine for treating pulmonary fibrosis, characterized in that: the Nanshan flower root extract is one or both of scopolactone and methylisorubidin-1-methyl ether. kind.
  12. 一种治疗肺纤维化的药物组合物，其特征在于：由南山花根中的蒽醌类、蒽醌糖苷类和环烯醚萜类有效提取部位组成，上述三种有效提取部位的重量配比为1-10：1-10：1-5。A medicinal composition for the treatment of pulmonary fibrosis, characterized in that: it is composed of the effective extraction parts of anthraquinones, anthraquinone glycosides and iridoids in the root of Nanshan flower, and the weight proportions of the above three effective extraction parts are 1-10:1-10:1-5.
  13. 一种治疗肺纤维化的药物组合物，其特征在于：由1,3-二羟基-5,6-二甲氧基-2-甲基蒽醌、甲基异茜草素-1-甲醚、甲基异茜草素和东莨菪内酯组成，上述四种化合物的重量配比为1-10：1-10：1-10：1-10。A pharmaceutical composition for treating pulmonary fibrosis, characterized in that it is composed of 1,3-dihydroxy-5,6-dimethoxy-2-methylanthraquinone, methylisorubicin-1-methyl ether, It is composed of methylisorubicin and scopolactone, and the weight ratio of the above four compounds is 1-10:1-10:1-10:1-10.
  14. 一种治疗肺纤维化的药物组合物，其特征在于：由东莨菪内酯和甲基异茜草素-1-甲醚组成，上述两种化合物的重量配比为1-10：1-10。A pharmaceutical composition for treating pulmonary fibrosis, which is characterized in that: it is composed of scopolactone and methylisorubicin-1-methyl ether, and the weight ratio of the two compounds is 1-10:1-10.
  15. 一种治疗肺纤维化的药物组合物，其特征在于：由南山花根提取物和抗肺纤维化的化学药组成，所述抗肺纤维化的化学药为吡非尼酮或尼达尼布，南山花根提取物和抗肺纤维化的化学药的重量配比为2-20：1-10。A pharmaceutical composition for the treatment of pulmonary fibrosis, characterized in that: it is composed of an extract of Nanshan flower root and an anti-pulmonary fibrosis chemical, wherein the anti-pulmonary fibrosis chemical is pirfenidone or nintedanib , the weight ratio of Nanshan flower root extract and anti-pulmonary fibrosis chemical is 2-20:1-10.
  16. 权利要求12-15任一项所述的组合物在制备治疗肺纤维化的药物中的用途。Use of the composition of any one of claims 12-15 in the preparation of a medicament for treating pulmonary fibrosis.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101972236A (en) * 2010-10-13 2011-02-16 北京诚创康韵医药科技有限公司 Sustained release preparation containing pirfenidone
CN104922247A (en) * 2015-06-08 2015-09-23 广西壮族自治区民族医药研究院 Extraction method and application of ethyl acetate extract of Prismatomeris tetrandra
CN107019697A (en) * 2016-02-02 2017-08-08 瑞阳(苏州)生物科技有限公司 The pharmaceutical composition and its application of prevention or treatment fibrotic disease
CN112972459A (en) * 2021-03-04 2021-06-18 广西国际壮医医院 Application of radix astragali monomer in liver protection

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106389596B (en) * 2016-11-10 2020-05-29 广西中医药大学 Medicine for treating pulmonary fibrosis
CN107669625A (en) * 2017-09-29 2018-02-09 昆明医科大学 Anti-fibrosis drug, preparation method, application and animal model constructing method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101972236A (en) * 2010-10-13 2011-02-16 北京诚创康韵医药科技有限公司 Sustained release preparation containing pirfenidone
CN104922247A (en) * 2015-06-08 2015-09-23 广西壮族自治区民族医药研究院 Extraction method and application of ethyl acetate extract of Prismatomeris tetrandra
CN107019697A (en) * 2016-02-02 2017-08-08 瑞阳(苏州)生物科技有限公司 The pharmaceutical composition and its application of prevention or treatment fibrotic disease
CN112972459A (en) * 2021-03-04 2021-06-18 广西国际壮医医院 Application of radix astragali monomer in liver protection

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LU QING-LAN;HUANG SHI-WEN;WEI XUE-PING;ZHOU HAI-DONG;LIU GUI-CHENG;QIN HUI-YING;YAO HONG-FEI: "Anti-Fibrosis Effects of Polysaccharide of Prismatomeris Tetrandra on Silicosis Rats", CHINESE TRADITIONAL AND HERBAL DRUGS, vol. 51, no. 4, 28 February 2020 (2020-02-28), pages 1031 - 1036, XP055981866, ISSN: 0253-2670, DOI: 10.7501/j.issn.0253-2670.2020.04.029 *
MOHR EDUARDA TALITA BRAMORSKI, DOS SANTOS NASCIMENTO MARCUS VINICIUS PEREIRA, DA ROSA JÚLIA SALVAN, VIEIRA GUILHERME NICÁCIO, KRET: "Evidence That the Anti-Inflammatory Effect of Rubiadin-1-methyl Ether Has an Immunomodulatory Context", MEDIATORS OF INFLAMMATION, vol. 2019, 3 November 2019 (2019-11-03), GB , pages 1 - 12, XP055981862, ISSN: 0962-9351, DOI: 10.1155/2019/6474168 *
NAM HYANG; KIM MOON-MOO: "Scopoletin has a potential activity for anti-aging via autophagy in human lung fibroblasts", PHYTOMEDICINE, vol. 22, no. 3, 4 February 2015 (2015-02-04), AMSTERDAM, NL , pages 362 - 368, XP029582878, ISSN: 0944-7113, DOI: 10.1016/j.phymed.2015.01.004 *
YANG ZENG-YAN, ZHAO XIANG-PEI;WEI JIN-YU;ZHANG QING-QING;BAI CHUN-HUI;TANG JIN: "Effect of Prismatomeris Connata Y.Z.Ruan. Extract on the Proliferation, Apoptosis and Extracellular Matrix in Rat Hepatic Stellate Cell", LISHIZHEN MEDICINE AND MATERIA MEDICA RESEARCH, vol. 25, no. 3, 20 March 2014 (2014-03-20), CN , pages 519 - 521, XP055981871, ISSN: 1008-0805, DOI: 10.3969/j.issn.1008-0805.2014.03.003 *
ZHAO SA-NA;WANG CHUN-XIANG;ZHANG XIAO-PING;CHEN TAO: "Anthraquinones from Prismatomeris connata", CHINESE TRADITIONAL PATENT MEDICINE, vol. 38, no. 10, 18 January 2016 (2016-01-18), pages 2200 - 2203, XP055981875, ISSN: 1001-1528, DOI: 10.3969/j.issn.1001-1528.2016.10.022 *

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