WO2002000234A1 - A pharmaceutical preparation of chinese herbs for preventing and treating cardiovascular and cerebrovascular disease and the use thereof - Google Patents

A pharmaceutical preparation of chinese herbs for preventing and treating cardiovascular and cerebrovascular disease and the use thereof Download PDF

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Publication number
WO2002000234A1
WO2002000234A1 PCT/CN2001/001061 CN0101061W WO0200234A1 WO 2002000234 A1 WO2002000234 A1 WO 2002000234A1 CN 0101061 W CN0101061 W CN 0101061W WO 0200234 A1 WO0200234 A1 WO 0200234A1
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Prior art keywords
extract
ginkgo
ginkgo biloba
astragalus
ethanol
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PCT/CN2001/001061
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French (fr)
Chinese (zh)
Inventor
Xinan Zhao
Yanban Pan
Maofang Wu
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Xinan Zhao
Yanban Pan
Maofang Wu
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Priority to AU2001277446A priority Critical patent/AU2001277446A1/en
Publication of WO2002000234A1 publication Critical patent/WO2002000234A1/en

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    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • 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
    • A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the invention relates to a traditional Chinese medicine preparation, in particular to a traditional Chinese medicine preparation with functions of preventing and treating heart and brain blood vessels, delaying aging and regulating human immune function.
  • the present invention also relates to a Chinese medicinal preparation containing ginkgo biloba leaf extract, astragalus extract and spirulina and its preparation method.
  • Patent application No. 96117700 in Chinese patent literature describes a ginkgo biloba capsule, which is based on ginkgo biloba leaf extract and blue-green algae to promote blood circulation.
  • the ingredients for removing blood stasis are added with salvia powder.
  • the invention concept strengthens the idea of promoting blood circulation and removing blood stasis.
  • Ginkgo biloba extract has the characteristics of absorbing free radicals, which can remove or reduce peroxyl free radicals, and inhibit cell membrane lipid peroxidation reactions; increase the SOD activity of red blood cells, reduce the production of peroxidized lipids, and maintain the vessel wall Health, enhance the ability of red blood cells to deform, reduce blood viscosity, prevent thrombosis and anti-platelet aggregation; expand blood vessels, enhance cardio-cerebral blood flow, improve and promote cardio-blood and fine blood circulation throughout the body; promote brain cell metabolism and improve brain function. Therefore, it is used for cardiovascular, cerebrovascular and peripheral vascular diseases, preventing atherosclerosis, and preventing and treating dementia.
  • Ginkgo biloba extract has a protective effect on cardiac dysfunction caused by myocardial ischemia, and has a certain improvement on cellular edema and ion disturbances caused by cerebral anemia; cerebral vasospasm has been significantly relieved.
  • Astragalus is the essential medicine of Chinese medicine. It has the function of strengthening qi and solid surface, and is used for qi deficiency and fatigue, internal heat and thirst. Pharmacological experiments show that astragalus can enhance the immune function of the body. It can enhance the learning and memory of mice, and can promote the generation, development and development of various cells. The maturation process promotes hematopoietic function of the bone marrow.
  • Spirulina is rich in essential nutrients for the human body.
  • the protein content is three times that of meat, up to 60-70%, of which 8 kinds of human essential amino acids account for 40%, and chlorophyll is 10 times that of ordinary vegetables and fruits.
  • Microbiotin, minerals, trace elements and unique biologically active substances constitute a comprehensive nutrition, and can regulate the physiological balance of the human body and gastrointestinal functions.
  • the purpose of the present invention is to provide a new medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
  • the present inventors have found through research that a pharmaceutical preparation composed of ginkgo biloba extract, astragalus extract and spirulina has excellent effects in preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating the immune function of the human body.
  • the first aspect of the present invention relates to a medicinal preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
  • the preparation is composed of ginkgo biloba extract, astragalus extract and spirulina.
  • Another aspect of the present invention relates to the use of a combination of ginkgo biloba extract, astragalus extract and spirulina for the preparation of a medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
  • Another aspect of the present invention relates to a method for preparing a pharmaceutical preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function, which comprises mixing Ginkgo biloba extract, astragalus extract and spirulina together.
  • the invention relates to a pharmaceutical preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function, which is composed of ginkgo biloba extract, astragalus extract and spirulina.
  • the pharmaceutical preparation of the present invention is composed of 8-24 ginkgo biloba extract, 12-36 astragalus extract and 30-90 spirulina.
  • the pharmaceutical preparation of the present invention is composed of 10 ginkgo biloba extract, 30 astragalus extract and 60 spirulina in parts by weight.
  • the pharmaceutical preparation of the present invention is composed of 16 ginkgo biloba extract, 24 astragalus extract and 50 spirulina in parts by weight.
  • Another aspect of the present invention relates to the use of ginkgo biloba extract, astragalus extract and spirulina in the preparation of a medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
  • 10 ginkgo biloba extract, 30 astragalus extract and 60 spirulina are preferably used by weight.
  • 16 ginkgo biloba extract, 24 astragalus extract and 50 spirulina are preferably used by weight.
  • Another aspect of the present invention relates to a method for preparing the pharmaceutical preparation of the present invention, which comprises mixing together ginkgo biloba extract, astragalus extract and spirulina.
  • ginkgo extract used in the present invention is prepared as follows:
  • ginkgo biloba After ginkgo biloba is dried and crushed, it is extracted with 40-80% ethanol at 40-70 ° C under reflux for 2-4 times. After the ethanol is distilled off, the extract is adsorbed with a macroporous adsorption resin (D-101 type, etc.). It was eluted with 90% ethanol, the eluent was recovered with ethanol, and the residue was dried to obtain ginkgo biloba extract.
  • a macroporous adsorption resin D-101 type, etc.
  • the obtained ginkgo extract contains no less than 24% ginkgo flavonoids and no less than ginkgo lactones
  • the ginkgo extract used therein can also be prepared as follows:
  • Dry and crush ginkgo biloba (pass through 20 mesh sieve), use water containing 1-2% antioxidant 10 to 15 times the amount of solution, boil and extract three times, each time 1-2 hours; combine the three water extracts, pass through the polyamide column, the ratio of the column height to the width is 4-15; wash with three times the amount of water, and then use Elute with 60-90% ethanol, concentrate, extract twice with n-hexane, and concentrate to dry to obtain the total flavonoids of ginkgo.
  • the aqueous solution of the antioxidant is a 0.5% sodium metabisulfite solution; the ratio of the height and width of the polyamide column is 4; and the ethanol concentration is 70% when the column is eluted.
  • Astragalus slices were boiled and extracted three times with 15-30 times water for 0.5-2 hours each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous resin (D-101, etc.), and washed with 40-90% ethanol. The ethanol was removed from the eluent, and the residue was dried to obtain astragalus extract.
  • macroporous resin D-101, etc.
  • the astragalus extract contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
  • the spirulina used in the present invention is an edible standard (see National Standard GB / T16919-1977)
  • the pharmaceutical preparations of the present invention are preferably used in the form of oral preparations such as tablets, capsules, and the like.
  • oral preparations such as tablets, capsules, and the like.
  • capsule preparations trials of 100 patients with cardiovascular and cerebrovascular diseases have obvious effects and a wide range of indications. The efficiency is 99%.
  • 100 clinical trials have shown that in the treatment of cardiovascular and cerebrovascular diseases, it has exact effects on dizziness, chest tightness, palpitations and limb numbness caused by insufficient cardiovascular blood supply. It is effective in improving myocardial ischemia, arrhythmia, and hyperlipidemia. It has certain curative effects on asthma, allergies, diabetes, beauty, etc., especially for improving gastrointestinal weakness and hair loss symptoms.
  • Ginkgo biloba leaves were dried and pulverized, and then extracted with 60% ethanol at 60 ° C for three times. After the ethanol was distilled off, the extract was adsorbed with a macroporous resin such as D101, and eluted with 70% ethanol. The eluent was used to recover ethanol. The residue was dried to obtain ginkgo biloba extract.
  • the extract contains no less than 24% ginkgo flavonoids and no less than 6% ginkgo lactones.
  • Astragalus slices were boiled and extracted three times with 20 times water for 1 hour each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous adsorption resin (D-101, etc.), eluted with 60% ethanol, and the eluent was recovered. The ethanol and residue were dried to obtain Huang's extract.
  • macroporous adsorption resin D-101, etc.
  • This product contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
  • 'Ginkgo biloba leaves are dried, crushed, and refluxed for three times under 70% ethanol.
  • the extract is distilled off with ethanol and adsorbed with a macroporous adsorption resin (D-101 type, etc.). It is eluted with 60% ethanol, and the eluent is used to recover ethanol. The residue was dried to obtain ginkgo biloba extract.
  • the extract contains no less than 24% ginkgo flavones and no less than 6% ginkgo lactones.
  • Astragalus slices were boiled and extracted three times with 25 times water for 1.5 hours each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous adsorption resin (D-101, etc.), eluted with 70% ethanol, and the eluent was recovered. Ethanol and residue were dried to obtain astragalus extract.
  • macroporous adsorption resin D-101, etc.
  • the extract contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
  • Test samples Capsules prepared in Example 1 or 2 of the present invention.
  • Animal grouping and gavage Animals are retrieved, and after 3 days of basic feed feeding, they are divided into 2.5g / kg group, 5.0g / kg group, and 10g / kg group according to the principle of random grouping (equivalent to the adult day recommended by the factory) Drink 5 times, 10 times, and 20 times the recommended dosage (30g / 60kg) and the control group.
  • the experimental group was administered with the corresponding dose of the capsules of the present invention, and the control group was administered with distilled water once a day for 25 consecutive hours. day.
  • SRBC Sheep red blood cells
  • cRBC chicken red blood cells
  • oxidized coenzyme I product of Sigma
  • 511 type microplate analyzer Shanghai third Analytical Instrument Factory.
  • Gavage was continued for 20 consecutive days, and each mouse was injected with 2% SRBC 0.2ml for intraperitoneal immunization. After continued gavage for 4 days, the eyeballs were removed and blood was collected in a centrifuge tube, left for 1 hour, and peeled off.
  • mice were injected with 20% chicken red blood cell suspension lml. After 30 minutes, it was killed by cervical dislocation and fixed in mice. Cut the skin of the abdominal wall in the middle of the plate, inject 2ml of normal saline through the abdominal cavity, rotate the mouse plate for 1 minute, aspirate 1ml of peritoneal washing solution, drop it on 2 slides, place in a wet box at 37 ° C for 30 minutes, and take it out in normal saline.
  • the animals were sacrificed by cervical dislocation, and the spleen was removed and crushed. After passing through a 200-mesh sieve, they were washed 3 times with Hank's solution, and 5 ⁇ 10 6 cells / ml cell suspension was prepared with complete 1640 culture solution. Take 300 ⁇ 1 of the cell suspension of each mouse and place it in a 96-well culture plate, 100 ⁇ per well, and add 100 ⁇ target cells (YAC-1 cells, 1> ⁇ 10 5 cells / ml) to each well. Release wells (target cells 100 ⁇ l + culture medium 100 ⁇ l) and maximum release wells (target cells 100 ⁇ l + 1% NP-40 100 ⁇ l) were each 3 wells, cultured at 37 ° C.
  • the animals were sacrificed by cervical dislocation, and the spleen and thymus were removed.
  • the weights of the animal, spleen and cysts were weighed out on the analytical balance, and the organ / body weight ratio was calculated according to the formula. Value (expressed as mg / 10g) and statistically processed.
  • Example 1 Effects of the capsules of Example 1 or 2 of the present invention on delayed allergic reaction in mice: Compared with the control group, the mean toe swelling degree of the 2.5 g / kg dose group is significantly different (P ⁇ 0.05), Compared with the control group, the average toe swelling degree of the 5.0g / kg group and the 10g / kg dose group has a highly significant difference (PO.01). (Table 1 )
  • Control group 12 121.67 ⁇ 8.45
  • Example 1 or 1 of the invention Effect of Example 1 or 1 of the invention on the activity of mouse NK cells:
  • mice (BALB / C) were dosed with 2.5 g / kg, 5.0 g / kg, and 10 g / kg of Ganoderma lucidum (infusion form) for 25 days. In the delayed allergy test, each dose group was sufficient. The degree of toe swelling is significantly different from that of the control group. In the determination of serum hemolysin, the 5.0g / kg group and the 10g / kg dose have a highly significant difference in volume number compared with the control group (PO.01). ; In the mouse peritoneal macrophage phagocytosis test, compared with the control group, the phagocytic index of the 10g / kg group had a highly significant difference (P ⁇ 0.01).
  • the doses of 5.0g / kg and 10g / kg can increase the serum antihemolysin volume and the level of delayed allergies in mice; the dose of 2.5g / kg can increase the levels of delayed allergies in mice; the dose of 10g / kg can Increase mouse phagocytosis index, according to the evaluation basis (3.1.3. It can be determined that the capsule of Example 1 or 2 of the present invention has an immunomodulatory effect.
  • Experimental example 2
  • the recommended amount is 0.75g / day, that is, 0.0125 / kg.bw (based on 60k body weight), and the dose is increased by 5 times, 10 times, and 20 times, which are 0.0625g / kg.bw., 0.175g / kg. bw, 0.05g / kg.bw.
  • UV spectrophotometer UV spectrophotometer, fluorescence spectrophotometer, refrigerated high-speed centrifuge, constant temperature and boiling water bath vortex, tissue homogenizer, etc.
  • Reagents thiobarbituric acid, tetraethoxypropane, stilbungstic acid, n-butanol, hydroxylamine hydrochloride, xanthine, menaline, p-aminobenzylsulfonic acid, sodium azide, phosphate, etc., MAO-B Assay kit (purchased from Ilicon Biotechnology Co., Ltd.) 1.5 Experimental method:
  • Blood, brain, and liver tissue homogenates were prepared: weighed with ether, weighed by celiac artery, and 5% EDTA-Na anticoagulated glutathione peroxidase ( GSH-Px) Lipid Peroxide (MDA) and Superoxide Dismutase (SOD).
  • GSH-Px EDTA-Na anticoagulated glutathione peroxidase
  • MDA Lipid Peroxide
  • SOD Superoxide Dismutase
  • the MDA content in blood was measured on the same day by TBA fluorescence method.
  • 75 ⁇ 1 of the aforementioned anticoagulant was added to 1.5 ml of physiological saline at 2000 r / min, centrifuged for 10 minutes, 0.5 ml of the supernatant was added, the required reagents were added, and the supernatant was shaken, centrifuged and discarded.
  • TBA fluorescence intensity (while using 10nmol / ml tetraethoxypropane as a standard) Curve), the measured fluorescence intensity can be checked on the standard curve for the corresponding MDA content.
  • the TBA fluorescence method was used to determine the MDA content in the tissues. The liver tissue was slowly frozen from the cryogenic water tank on the day of the measurement, and 10 times cold physiological saline was added according to the weight of the tissue. Repeat 3 times. The homogenate was centrifuged at 4000 ° C / min for 30 minutes at 4 ° C, and 0.1 ml of the supernatant was taken. The other steps were the same as the determination of MDA in blood.
  • DTNB method was used to measure the activity of GSH-Px in blood on the same day. Take 10 ⁇ of the above anticoagulant and add it to 990 ⁇ 1 double-distilled water, shake it thoroughly to make it all hemolyzed, take 0.4ml, add For the required reagents, perform an enzymatic reaction in a 37 ° C water bath for 3 minutes, stop the reaction, centrifuge, and develop the color. Read the OD value at a wavelength of 422nm, and calculate the number of enzyme activity units according to the formula.
  • the activity of GSH-Px in the tissues was measured.
  • the liver tissue was slowly frozen from the low-temperature refrigerator on the day of measurement, and 10 times cold physiological saline was added according to the weight of the tissue.
  • the homogenizer was homogenized at 20,000 r / min for 10 seconds with a 15-second interval and repeated 3 times. . Centrifuge at 10000 r / min for 10 minutes and take the supernatant for measurement (the procedure is the same as that for blood measurement).
  • the hydroxylamine method was used to measure the SOD activity on the day.
  • the anticoagulant was centrifuged at 3000 r / min for 10 minutes. Take 30 ⁇ 1 of plasma, add the required reagents, mix well, and place the reagents in a 37 ° C water bath for 40 minutes. Stop the reaction by adding reagents. After 10 minutes of color development, measure the OD value at 550nm, and calculate the number of SOD activity units based on the OD value.
  • the brain tissue was slowly frozen from the low-temperature water tank, and 5 times cold physiological saline was added according to the weight of the tissue, and the slurry was hooked in an ice bath at 20,000 r / min for 10 seconds, with an interval of 15 seconds, and repeated 3 times.
  • the homogenate was centrifuged at 4 ° C l0000r / min for 20 minutes. After the supernatant was diluted 5 times, 0.2 ml was taken, and the required reagents were added.
  • the reaction was stopped in a constant temperature water bath for 1 hour.
  • the OD was measured at 242 nm. The value calculates the number of MAO-B vitality units.
  • the capsule of the present invention is orally administered for 90 days, and the low and medium dose can obviously increase the blood glutathione peroxidase (GSH -Px) activity, the difference is significant (P ⁇ 0.01, P ⁇ 0.05).
  • Lipid peroxide (MDA) levels and superoxide dismutase (SOD) activity in blood of each dose group were not statistically different from those of the aging control group (P> 0.05).
  • Table 8 Effects of the capsules of Example 1 or 2 of the present invention on the activity of MAO-B in brains of rats, the content of MDA in liver tissues, and the activity of GSH-P
  • the capsules of the present invention were orally administered for 90 days, and each dose can make monoamine oxidase (MAO) in rat brain tissue -B) Vigor was significantly reduced and the difference was significant (PO.05, P ⁇ 0.01), and the difference was also significant compared with the young control group (P ⁇ 0.05, P ⁇ 0.01).
  • MAO monoamine oxidase
  • GSH-Px glutathione peroxidase
  • MDA lipid peroxides
  • Rats of Example 1 or 2 of the present invention were orally administered for 90 days. Compared with aging control rats, each dose can significantly reduce the activity of monoamine oxidase (MAO-B) in rat brain tissues. There are also significant differences in ratios; low and medium doses can significantly increase glutathione peroxidase (GASH-Px) activity in rat liver tissues and blood; high doses can cause lipid peroxide (MDA) levels in liver tissues obviously decased.
  • MAO-B monoamine oxidase
  • GASH-Px glutathione peroxidase
  • MDA lipid peroxide
  • Example 1 Effects of the capsules of Example 1 or 2 of the present invention on lipid peroxides (MDA) in rat blood Effects of levels and superoxide dismutase (SOD) activity in rat blood. Based on the results of the fruit fly survival test being positive, according to the criteria for determining the function of delaying aging in the "Functional Evaluation Procedures and Test Methods for Health Foods", it is believed that the capsules of Example 1 or 2 of the present invention have a delaying aging effect.
  • MDA lipid peroxides
  • SOD superoxide dismutase
  • Example 1 or 2 of the present invention There were 100 patients with cardiovascular and cerebrovascular diseases, including 51 male patients and 49 female patients, aged 13-80 years. Take the capsules of Example 1 or 2 of the present invention to the above patients 3 times a day, 2 capsules each time, and the administration time is 1 to 4 months. The results are shown in Tables 9-13. From the results in Tables 9-13, it can be seen that the capsules of Example 1 or 2 of the present invention have good effects on various symptoms related to cardiovascular and cerebrovascular diseases, and the effective rate is 99%.

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Abstract

the present invention discloses a pharmaceutical preparation of Chinese herbs for preventing and treating cardiovascular and cerebrovascular disease. The preparation may be formulated by using 8-24 weight parts of extract from Ginkgo biloba leaf, 12-36 weight parts of extract from Astragalus membranaceus, 30-90 weight parts of Spirulina as raw material.

Description

一种防治心脑血管疾病的中药制剂及其制备方法 发明领域  Traditional Chinese medicine preparation for preventing and treating cardiovascular and cerebrovascular diseases and preparation method thereof
本发明涉及一种中药制剂， 特别是涉及一种具有防治心脑血 管、 延緩衰老及调节人体免疫功能的中药制剂。 具体讲， 本发明 还涉及含银杏叶提取物， 黄芪提取物及螺旋藻的中药制剂及其制 备方法。  The invention relates to a traditional Chinese medicine preparation, in particular to a traditional Chinese medicine preparation with functions of preventing and treating heart and brain blood vessels, delaying aging and regulating human immune function. Specifically, the present invention also relates to a Chinese medicinal preparation containing ginkgo biloba leaf extract, astragalus extract and spirulina and its preparation method.
背景技术 Background technique
在治疗^、脑血管疾病方面的药物， 国内外文献中均有过大量 的报道， 纯中药治疗心脑血管疾病的药物也较多， 如银杏叶制剂 络欣通、 丹参滴丸等。 此类药物多是单味药制剂， 疗效单一； 而 中国专利文献中的第 96117700 号专利申请记载了一种银杏参胶 嚢， 该药物在银杏叶提取物， 蓝绿藻的基础上加了活血化瘀的成 份， 即加入了丹参粉。 其发明构思上强化了活血化瘀的思想。  In the treatment of cerebrovascular diseases, a large number of reports have been reported in the literature at home and abroad, and pure Chinese medicines are also used to treat cardiovascular and cerebrovascular diseases, such as Ginkgo biloba preparation Luoxintong and Danshen dripping pills. Most of these drugs are single-drug preparations with a single curative effect. Patent application No. 96117700 in Chinese patent literature describes a ginkgo biloba capsule, which is based on ginkgo biloba leaf extract and blue-green algae to promote blood circulation. The ingredients for removing blood stasis are added with salvia powder. The invention concept strengthens the idea of promoting blood circulation and removing blood stasis.
现代药理研究还表明：银杏叶提取物具有吸收自由基的特性， 可清除或減少过氧自由基， 抑制细胞膜脂质过氧化反应； 提高红 细胞 SOD活性， 降低过氧化脂质的产生， 维护血管壁的健康， 增 强红细胞变形能力， 降低血液粘度， 防止血栓形成和抗血小板凝 聚； 扩张血管， 增强心脑血流量， 改善和促进心血及全身微细血 液循环； 促进脑细胞代谢、 改善脑功能。 因而用于心脑血管及外 周血管疾病、 预防动脉粥样硬化， 防治老年痴呆。 动物实验证明， 银杏叶提取物对心肌缺血引起的心功能紊乱有保护作用， 对脑缺 血引起的细胞水肿和离子紊乱均有一定的改善； 脑血管痉挛有明 显緩解。  Modern pharmacological research also shows that: Ginkgo biloba extract has the characteristics of absorbing free radicals, which can remove or reduce peroxyl free radicals, and inhibit cell membrane lipid peroxidation reactions; increase the SOD activity of red blood cells, reduce the production of peroxidized lipids, and maintain the vessel wall Health, enhance the ability of red blood cells to deform, reduce blood viscosity, prevent thrombosis and anti-platelet aggregation; expand blood vessels, enhance cardio-cerebral blood flow, improve and promote cardio-blood and fine blood circulation throughout the body; promote brain cell metabolism and improve brain function. Therefore, it is used for cardiovascular, cerebrovascular and peripheral vascular diseases, preventing atherosclerosis, and preventing and treating dementia. Animal experiments have shown that Ginkgo biloba extract has a protective effect on cardiac dysfunction caused by myocardial ischemia, and has a certain improvement on cellular edema and ion disturbances caused by cerebral anemia; cerebral vasospasm has been significantly relieved.
黄芪是中医补气之要药。 具有益气固表的功能， 用于气虚乏 力， 内热消渴等症。 药理实验表明黄芪有增强机体免疫功能的作 用。 有增强小鼠学习记忆的作用， 能促进各类细胞生成、 发育及 成熟过程， 促进骨髓的造血功能。 Astragalus is the essential medicine of Chinese medicine. It has the function of strengthening qi and solid surface, and is used for qi deficiency and fatigue, internal heat and thirst. Pharmacological experiments show that astragalus can enhance the immune function of the body. It can enhance the learning and memory of mice, and can promote the generation, development and development of various cells. The maturation process promotes hematopoietic function of the bone marrow.
螺旋藻含有丰富的人体必须的营养成份， 蛋白质的含量是肉 类的三倍， 高达 60 - 70 % , 其中 8种人体必须氨基酸占 40 % , 叶 绿素是普通蔬茱和水果的 10倍， 丰富的微生素、 矿物质、 微量元 素及特有的生物活性物质构成了全面营养， 并能调节人体生理机 能平衡和肠胃功能。  Spirulina is rich in essential nutrients for the human body. The protein content is three times that of meat, up to 60-70%, of which 8 kinds of human essential amino acids account for 40%, and chlorophyll is 10 times that of ordinary vegetables and fruits. Microbiotin, minerals, trace elements and unique biologically active substances constitute a comprehensive nutrition, and can regulate the physiological balance of the human body and gastrointestinal functions.
发明目的 Object of the invention
本发明目的在于提供一种新的用于防治心脑血管疾病、 延緩 衰老及调节人体免疫功能的药物。  The purpose of the present invention is to provide a new medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
发明简述 Brief description of the invention
本发明人经研究现发现由银杏叶提取物， 黄芪提取物及螺旋 藻组成的药物制剂在防治心脑血管疾病， 延緩衰老及调节人体免 疫功能方面置示出优良效果。  The present inventors have found through research that a pharmaceutical preparation composed of ginkgo biloba extract, astragalus extract and spirulina has excellent effects in preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating the immune function of the human body.
因此， 本发明第一方面涉及一种用于防治心脑血管疾病、 延 緩衰老及调节人体免疫功能的药物制剂，该制剂由银杏叶提取物， 黄芪提取物及螺旋藻组成。  Therefore, the first aspect of the present invention relates to a medicinal preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function. The preparation is composed of ginkgo biloba extract, astragalus extract and spirulina.
本发明再一方面涉及银杏提取物、 黄芪提取物和螺旋藻结合 用于制备用于防治心脑血管疾病、 延緩衰老及调节人体免疫功能 的药物中用途。  Another aspect of the present invention relates to the use of a combination of ginkgo biloba extract, astragalus extract and spirulina for the preparation of a medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
本发明再一方面涉及制备一种用于防治心脑血管疾病、 延緩 衰老及调节人体免疫功能的药物制剂的方法， 其包括将银杏叶提 取物、 黄芪提取物及螺旋藻混合在一起。  Another aspect of the present invention relates to a method for preparing a pharmaceutical preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function, which comprises mixing Ginkgo biloba extract, astragalus extract and spirulina together.
发明详迷 Invention details
本发明涉及一种用于防治心脑血管疾病， 延緩衰老及调节人 体免疫功能的药物制剂， 其是由银杏叶提取物， 黄芪提取物及螺 旋藻组成。  The invention relates to a pharmaceutical preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function, which is composed of ginkgo biloba extract, astragalus extract and spirulina.
根据本发明的一个优选实施方案， 本发明药物制剂按重量份 计是由 8- 24银杏叶提取物、 12- 36黄芪提取物及 30- 90螺旋 藻组成。 According to a preferred embodiment of the present invention, the pharmaceutical preparation of the present invention The meter is composed of 8-24 ginkgo biloba extract, 12-36 astragalus extract and 30-90 spirulina.
根据本发明的一个优选实施方案， 本发明药物制剂按重量份 计是由 10银杏叶提取物、 30黄芪提取物及 60螺旋藻组成。  According to a preferred embodiment of the present invention, the pharmaceutical preparation of the present invention is composed of 10 ginkgo biloba extract, 30 astragalus extract and 60 spirulina in parts by weight.
根据本发明的一个优选实施方案， 本发明药物制剂按重量份 计是由 16银杏叶提取物、 24黄芪提取物及 50螺旋藻组成。  According to a preferred embodiment of the present invention, the pharmaceutical preparation of the present invention is composed of 16 ginkgo biloba extract, 24 astragalus extract and 50 spirulina in parts by weight.
本发明再一方面涉及银杏叶提取物、 黄芪提取物及螺旋藻用 于制备用于防治心脑血管疾病、 延緩衰老及调节人体免疫功能的 药物中用途。  Another aspect of the present invention relates to the use of ginkgo biloba extract, astragalus extract and spirulina in the preparation of a medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
根据本发明的用途，优选按重量计使用 8- 24银杏叶提取物、 12 - 36黄芪提取物及 30 - 90螺旋藻。  According to the use of the present invention, it is preferable to use 8-24 ginkgo biloba extract, 12-36 astragalus extract and 30-90 spirulina by weight.
根据本发明的用途， 优选按重量计使用 10银杏叶提取物、 30 黄芪提取物及 60螺旋藻。  According to the use of the present invention, 10 ginkgo biloba extract, 30 astragalus extract and 60 spirulina are preferably used by weight.
根据本发明的用途， 优选按重量计使用 16银杏叶提取物、 24 黄芪提取物及 50螺旋藻。  According to the use of the present invention, 16 ginkgo biloba extract, 24 astragalus extract and 50 spirulina are preferably used by weight.
本发明再一方面涉及制备本发明药物制剂的方法， 其包括将 银杏叶提取物、 黄芪提取物及螺旋藻混合在一起。  Another aspect of the present invention relates to a method for preparing the pharmaceutical preparation of the present invention, which comprises mixing together ginkgo biloba extract, astragalus extract and spirulina.
根据本发明的制备方法， 其中本发明中所用的银杏提取物是 如下制备的：  According to the preparation method of the present invention, wherein the ginkgo extract used in the present invention is prepared as follows:
银杏叶经烘干、 粉碎， 用 40- 80%乙醇于 40- 70°C回流提取 2-4 次， 提取液蒸去乙醇后用大孔吸附树脂（D- 101 型等） 吸 附， 用 50- 90%乙醇洗脱、 洗脱液回收乙醇， 残留液干燥， 得银 杏叶提取物。  After ginkgo biloba is dried and crushed, it is extracted with 40-80% ethanol at 40-70 ° C under reflux for 2-4 times. After the ethanol is distilled off, the extract is adsorbed with a macroporous adsorption resin (D-101 type, etc.). It was eluted with 90% ethanol, the eluent was recovered with ethanol, and the residue was dried to obtain ginkgo biloba extract.
所得银杏提取物含银杏黄酮不低于 24% , 含银杏内脂不低于 The obtained ginkgo extract contains no less than 24% ginkgo flavonoids and no less than ginkgo lactones
6%。 6%.
根据本发明， 其中所用银杏提取物还可如下制备：  According to the present invention, the ginkgo extract used therein can also be prepared as follows:
将银杏烘干、 粉碎（过 20 目筛） ， 用含 1-2%抗氧剂的水 溶液 10- 15倍量， 煮沸提取三次， 每次 1-2小时； 合并三次水 提液， 过聚酰胺柱， 柱高与宽的比值为 4-15; 用三倍量的水洗 涤， 再用 60- 90%乙醇洗脱， 浓缩， 用正己烷萃取二次， 浓缩干 燥得银杏总黄酮。 Dry and crush ginkgo biloba (pass through 20 mesh sieve), use water containing 1-2% antioxidant 10 to 15 times the amount of solution, boil and extract three times, each time 1-2 hours; combine the three water extracts, pass through the polyamide column, the ratio of the column height to the width is 4-15; wash with three times the amount of water, and then use Elute with 60-90% ethanol, concentrate, extract twice with n-hexane, and concentrate to dry to obtain the total flavonoids of ginkgo.
根据本发明所述银杏液提取物的制备方法， 其中所迷抗氧剂 水溶液为 0.5%焦亚硫酸钠水溶液； 聚酰胺柱高与宽的比例为 4; 过柱洗脱时乙醇浓度为 70%。  According to the method for preparing a ginkgo biloba extract, the aqueous solution of the antioxidant is a 0.5% sodium metabisulfite solution; the ratio of the height and width of the polyamide column is 4; and the ethanol concentration is 70% when the column is eluted.
根据本发明的制备方法， 其中本发明所用黄芪提取物是如下 制备的：  According to the preparation method of the present invention, wherein the astragalus extract used in the present invention is prepared as follows:
黄芪切片， 用 15- 30倍水煮沸提取三次， 每次 0.5- 2小时， 三次煎液合并、 滤过， 滤液用大孔吸附树脂（D- 101型等）吸附， 用 40- 90%乙醇洗脱、 洗脱液回收乙醇、 残留液干燥， 得黄芪提 取物。  Astragalus slices were boiled and extracted three times with 15-30 times water for 0.5-2 hours each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous resin (D-101, etc.), and washed with 40-90% ethanol. The ethanol was removed from the eluent, and the residue was dried to obtain astragalus extract.
所得黄芪提取物含黄芪甲甙（C44H6804) 不低于 0.1% The astragalus extract contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
根据本发明， 本发明所用的螺旋藻为食用标准 （见国家标准 GB/T16919 - 1977 )  According to the present invention, the spirulina used in the present invention is an edible standard (see National Standard GB / T16919-1977)
根据本发明， 本发明的药物制剂优选以口服制剂如片剂、 胶 嚢等形式使用， 其中以胶嚢制剂为例， 试用 100例心脑血管疾病 患者， 效果明显， 适应症范围广， 总有效率为 99%。 经临床试用 100 例证明， 在治疗心脑血管疾病中， 对心血管供血不足引起的 头晕、 胸闷、 心悸、 肢体麻木等有确切疗效。 在改善心肌缺血、 心律失常、 高血脂症等方面效果明显。 对于气喘、 过敏、 糖尿病、 美容等有一定疗效， 特别是对肠胃虛弱、 脱发症状的改善效果明 置。  According to the present invention, the pharmaceutical preparations of the present invention are preferably used in the form of oral preparations such as tablets, capsules, and the like. Taking capsule preparations as an example, trials of 100 patients with cardiovascular and cerebrovascular diseases have obvious effects and a wide range of indications. The efficiency is 99%. 100 clinical trials have shown that in the treatment of cardiovascular and cerebrovascular diseases, it has exact effects on dizziness, chest tightness, palpitations and limb numbness caused by insufficient cardiovascular blood supply. It is effective in improving myocardial ischemia, arrhythmia, and hyperlipidemia. It has certain curative effects on asthma, allergies, diabetes, beauty, etc., especially for improving gastrointestinal weakness and hair loss symptoms.
下面的实施例或实验用于进一步说明本发明， 但其不意味着 对本发明的任何限制。  The following examples or experiments are used to further illustrate the present invention, but they are not meant to limit the present invention in any way.
实施例 1: 1.1 银杏叶提取物的制备 Example 1: 1.1 Preparation of Ginkgo biloba extract
银杏叶经烘干、 粉碎， 用 60%乙醇于 60°C下回流提取 3次， 提取液蒸去乙醇后用 D101型等大孔吸附树脂吸附， 用 70%乙醇 洗脱、 洗脱液回收乙醇， 残留液干燥， 得银杏叶提取物。  Ginkgo biloba leaves were dried and pulverized, and then extracted with 60% ethanol at 60 ° C for three times. After the ethanol was distilled off, the extract was adsorbed with a macroporous resin such as D101, and eluted with 70% ethanol. The eluent was used to recover ethanol. The residue was dried to obtain ginkgo biloba extract.
该提取物含银杏黄酮不低于 24% , 含银杏内脂不低于 6%。 The extract contains no less than 24% ginkgo flavonoids and no less than 6% ginkgo lactones.
1.2 黄芪提取物的制备 1.2 Preparation of Astragalus Extract
黄芪切片， 用 20倍水煮沸提取三次， 每次 1小时， 三次煎液 合并， 滤过， 滤液用大孔吸附树脂（D- 101 型等） 吸附， 用 60 %乙醇洗脱、 洗脱液回收乙醇、 残留液干燥， 得黄氏提取物。  Astragalus slices were boiled and extracted three times with 20 times water for 1 hour each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous adsorption resin (D-101, etc.), eluted with 60% ethanol, and the eluent was recovered. The ethanol and residue were dried to obtain Huang's extract.
本品含黄芪甲甙（C44H6804) 不低于 0.1% This product contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
1.3 制剂的制备  1.3 Preparation of preparation
取银杏叶提取物 10kg,黄芪提取物 30kg,螺旋藻食用标准（国 家标准 GB/T16919- 1977) 螺旋藻 60kg， 混匀， 制粒、 装胶嚢， 0.25g/粒。  Take 10kg of ginkgo biloba extract, 30kg of astragalus extract, Spirulina edible standard (national standard GB / T16919-1977), 60kg of spirulina, mix well, granulate, fill with capsules, 0.25g / capsule.
实施例 2:  Example 2:
2.1 银杏叶提取物  2.1 Ginkgo biloba extract
'银杏叶经烘干、 粉碎， 70%乙醇 下回流提取 3次， 提取 液蒸去乙醇后用大孔吸附树脂（D- 101 型等） 吸附， 用 60%乙 醇洗脱、 洗脱液回收乙醇， 残留液干燥， 得银杏叶提取物。  'Ginkgo biloba leaves are dried, crushed, and refluxed for three times under 70% ethanol. The extract is distilled off with ethanol and adsorbed with a macroporous adsorption resin (D-101 type, etc.). It is eluted with 60% ethanol, and the eluent is used to recover ethanol. The residue was dried to obtain ginkgo biloba extract.
该提取物含银杏黄酮不低于 24%, 含银杏内脂不低于 6%。 The extract contains no less than 24% ginkgo flavones and no less than 6% ginkgo lactones.
2.2 黄芪提取物的制备 2.2 Preparation of Astragalus Extract
黄芪切片， 用 25倍水煮沸提取三次， 每次 1.5小时， 三次煎 液合并、 滤过， 滤液用大孔吸附树脂（D- 101 型等） 吸附， 用 70%乙醇洗脱、 洗脱液回收乙醇、 残留液干燥， 得黄芪提取物。  Astragalus slices were boiled and extracted three times with 25 times water for 1.5 hours each time. The three decoctions were combined and filtered. The filtrate was adsorbed with macroporous adsorption resin (D-101, etc.), eluted with 70% ethanol, and the eluent was recovered. Ethanol and residue were dried to obtain astragalus extract.
该提取物含黄芪甲甙（C44H6804) 不低于 0.1% The extract contains astragaloside IV (C 44 H 68 0 4 ) not less than 0.1%
2.3 制剂的制备  2.3 Preparation of preparation
取银杏叶提取物 16kg, 黄芪提取物 24kg, 食用标准（国家标 准 GB/T61919 - 1977 )螺旋藻 50kg, 混匀， 制粒、 装胶嚢， 0.25g/ 粒。 Take 16kg of ginkgo biloba extract, 24kg of astragalus extract, edible standard (national standard) Quasi-GB / T61919-1977) Spirulina 50kg, mixed, granulated, filled with gelatin, 0.25g / capsule.
实验例 1  Experimental example 1
本发明药物制剂对动物免疫***的调节  Regulation of the pharmaceutical preparation of the present invention on animal immune system
一、 材料与方法 I. Materials and methods
1 材料  1 Material
1.1 受试样品： 本发明实施例 1或 2制备的胶嚢。  1.1 Test samples: Capsules prepared in Example 1 or 2 of the present invention.
1.2 动物： 雌性 BALB/C小鼠， 体重 18 - 22克， 华西医科大 学实验动物中心提供（合格证号： 川实动管质第 64号） 。  1.2 Animals: Female BALB / C mice, weighing 18 to 22 grams, provided by the Experimental Animal Center of West China Medical University (Certificate No .: Chuanshi Dynamic Tuberculosis No. 64).
1.3 动物分組及灌胃： 动物领回， 基础饲料喂饲 3 天后， 按 随机分組原则分为 2.5g/kg组、 5.0g/kg组、 10g/kg组〔分别相当 于厂方推荐的成人日饮用建议剂量（30g/60kg ) 的 5倍， 10倍和 20倍〕及对照组， 实验组以相应剂量的上述本发明胶嚢灌胃， 对 照组以蒸馏水灌胃， 每天一次， 连续灌胃 25天。  1.3 Animal grouping and gavage: Animals are retrieved, and after 3 days of basic feed feeding, they are divided into 2.5g / kg group, 5.0g / kg group, and 10g / kg group according to the principle of random grouping (equivalent to the adult day recommended by the factory) Drink 5 times, 10 times, and 20 times the recommended dosage (30g / 60kg) and the control group. The experimental group was administered with the corresponding dose of the capsules of the present invention, and the control group was administered with distilled water once a day for 25 consecutive hours. day.
1.4 主要试剂和仪器 绵羊红细胞（SRBC ) ： 采自健康綿羊 颈静脉； 鸡红细胞（cRBC ) : 采自健康公鸡心脏血； 氧化型辅酶 I: Sigma公司产品； 511型酶标分析仪： 上海第三分析仪器厂。  1.4 Main reagents and instruments Sheep red blood cells (SRBC): collected from jugular vein of healthy sheep; chicken red blood cells (cRBC): collected from healthy rooster heart blood; oxidized coenzyme I: product of Sigma; 511 type microplate analyzer: Shanghai third Analytical Instrument Factory.
2 方法  2 methods
2.1 迟发性***反应（足趾增厚法）  2.1 Delayed allergies (toe thickening method)
连续灌胃 20天后，用 2 % ( v/v )SRBC腹腔免疫，每只 0.2ml, 免疫 4天后， 测量左右足趾部厚度， 测定 3次平均值； 然后在测 量部位皮下注射 20 % ( v/v ) SRBC, 每只 2μ1, 注射后于 24小时 测量左后足趾部厚度， 测定 3次取平均值。 计算攻击前后足趾厚 度的差值， 以差值表示小鼠的 DTH的程度， 并进行统计学处理。  After continuous gastric administration for 20 days, 2% (v / v) SRBC was used for intraperitoneal immunization, 0.2ml each. After 4 days of immunization, the thickness of the left and right toes was measured, and the average value was measured 3 times. Then, 20% (v / v) SRBC, each 2 μ1, the thickness of the left hind toe was measured 24 hours after the injection, and the average was measured 3 times. The difference between toe thickness before and after the challenge was calculated, and the degree of DTH in mice was expressed as the difference, and statistical processing was performed.
2.2 血清溶血素测定（血凝法）  2.2 Determination of serum hemolysin (hemagglutination method)
连续灌胃 20天， 每只小鼠腹腔注射 2 % SRBC 0.2ml免疫， 继续灌胃 4天后， 摘除眼球取血于离心管内， 放置 1小时， 剥离， 2000rpm离心 10分钟， 收集血清， 用生理盐水将血清进行倍比稀 释， 每份稀释 14 孔， 将不同稀释度的血清置于血凝板中， 每孔 ΙΟΟμΙ, 再加入 0.5 % SRBC悬液 ΙΟΟμΙ, 混匀， 置湿盒 37°C 3小时， 观察结果， 记录每孔的凝集程度， 按公式计算抗体积数（抗体积 ¾ = Si + 2S2 + 3S3 + …… nSn ) , 并进行统计学处理。 Gavage was continued for 20 consecutive days, and each mouse was injected with 2% SRBC 0.2ml for intraperitoneal immunization. After continued gavage for 4 days, the eyeballs were removed and blood was collected in a centrifuge tube, left for 1 hour, and peeled off. Centrifuge at 2000 rpm for 10 minutes, collect the serum, dilute the serum with physiological saline, dilute 14 wells each, put the serum of different dilutions on the blood coagulation plate, 100 μl per well, and add 0.5% SRBC suspension 100 μl, Mix well, place in a humidified box at 37 ° C for 3 hours, observe the results, record the degree of aggregation of each well, and calculate the anti-volume number (anti-volume ¾ = Si + 2S 2 + 3S 3 + ...... nS n ) according to the formula, and make statistics Learn to handle.
2.3 小鼠腹腔巨噬细胞吞噬鸡红细胞试验（半体内法） 连续灌胃 25夭后， 于每鼠腹腔注射 20 %鸡红细胞悬液 lml, 30分钟后， 颈推脱臼处死， 将其固定在鼠板上， 正中剪开腹壁皮 肤， 经腹腔注入生理盐水 2ml, 转动鼠板 1分钟， 吸出腹腔洗液 lml, 平均滴于 2张载玻片上， 置湿盒 37°C 30分钟， 取出在生理 盐水中漂洗， 晾干， 固定， 4 % Giemsa PBS染色 3分钟， 蒸镏水 漂洗晾干， 镜检。 按公式计算吞噬百分率及吞噬指数， 进行统计 学处理。  2.3 Test of peritoneal macrophages phagocytosing chicken red blood cells in mice (semi-in vivo method) After 25 g of continuous intragastric administration, each mouse was injected with 20% chicken red blood cell suspension lml. After 30 minutes, it was killed by cervical dislocation and fixed in mice. Cut the skin of the abdominal wall in the middle of the plate, inject 2ml of normal saline through the abdominal cavity, rotate the mouse plate for 1 minute, aspirate 1ml of peritoneal washing solution, drop it on 2 slides, place in a wet box at 37 ° C for 30 minutes, and take it out in normal saline. Medium rinsing, drying, fixing, staining with 4% Giemsa PBS for 3 minutes, rinsing with steamed water, drying, and microscopy. Calculate the percentage of phagocytosis and phagocytosis index according to the formula for statistical processing.
2.4 NK细胞活性测定  2.4 NK cell activity determination
连续灌胃 25天后， 颈推脱臼处死动物， 取出脾脏， 街碎， 过 200 目筛网后， 用 Hank's液洗 3次， 用完全 1640培养液配成 5 X 106个 /ml细胞悬液。将各只鼠的细胞悬液取 300μ1分置于 96孔 培养板中， 每孔 ΙΟΟμΙ, 每孔加靶细胞 ( YAC - 1 细胞， 1 ><105 个 /ml ) ΙΟΟμΙ, 同时做靶细胞自然释放孔（靶细胞 ΙΟΟμΙ +培养液 ΙΟΟμΙ )及最大释放孔（靶细胞 ΙΟΟμΙ + 1 % ΝΡ - 40 ΙΟΟμΙ )各 3孔， 37°C 5 % C02培养 4小时， 取出 1500rpm离心 5分钟。 将各孔上 清液 ΙΟΟμΙ置于另一培养板中， 每孔再加入 ΙΟΟμΙ基质液， 100分 钟后加 lmol/L HC 130μ1终止反应， 在 490nm处测定 O.D.值， 按 公式计算 NK细胞活性率， 并进行统计学处理。 After 25 consecutive days of gavage, the animals were sacrificed by cervical dislocation, and the spleen was removed and crushed. After passing through a 200-mesh sieve, they were washed 3 times with Hank's solution, and 5 × 10 6 cells / ml cell suspension was prepared with complete 1640 culture solution. Take 300μ1 of the cell suspension of each mouse and place it in a 96-well culture plate, 100μΙ per well, and add 100μΙ target cells (YAC-1 cells, 1><10 5 cells / ml) to each well. Release wells (target cells 100 μl + culture medium 100 μl) and maximum release wells (target cells 100 μl + 1% NP-40 100 μl) were each 3 wells, cultured at 37 ° C. 5% CO 2 for 4 hours, and centrifuged at 1500 rpm for 5 minutes. 100 μl of the supernatant of each well was placed in another culture plate, and 100 μl of matrix solution was added to each well. 100 minutes later, 1 mol / L HC 130 μ1 was added to stop the reaction. The OD value was measured at 490 nm, and the NK cell activity rate was calculated according to the formula. Statistical processing.
2.5 脏器 /体重比值测定  2.5 Organ / weight ratio determination
连续灌胃 25天后， 颈推脱臼处死动物， 取出脾脏、 胸腺， 分 析天平分别称出动物、 脾脏、 胞腺重量， 按公式计算脏器 /体重比 值（以 mg/10g表示） ， 并进行统计学处理。 After 25 consecutive days of gavage, the animals were sacrificed by cervical dislocation, and the spleen and thymus were removed. The weights of the animal, spleen and cysts were weighed out on the analytical balance, and the organ / body weight ratio was calculated according to the formula. Value (expressed as mg / 10g) and statistically processed.
二、 结果 Second, the results
1 本发明实施例 1或 2胶嚢对小鼠迟发性***反应的影响： 2.5 g/kg剂量组与对照组相比，其足趾肿胀度均数有显著性差 异（ P<0.05 ) , 5.0g/kg組和 10g/kg剂量组与对照组相比， 其足趾 肿胀度均数有高度显著性差异（ PO.01 ) 。 （表 1 )  1 Effects of the capsules of Example 1 or 2 of the present invention on delayed allergic reaction in mice: Compared with the control group, the mean toe swelling degree of the 2.5 g / kg dose group is significantly different (P <0.05), Compared with the control group, the average toe swelling degree of the 5.0g / kg group and the 10g / kg dose group has a highly significant difference (PO.01). (Table 1 )
表 1 本发明胶嚢对小鼠返发性***反应影响 組别 例数（只） 足趾肿胀度（mm ) ( z±S ) 对照组 12 0.31±0.08 Table 1 Effects of capsules of the present invention on retrograde allergies in mice Group Number of cases (only) Toe swelling (mm) (z ± S) Control group 12 0.31 ± 0.08
2.5g/k 组 13 0.40±0.10ΛΛ 2.5g / k group 13 0.40 ± 0.10 ΛΛ
5.0g/kg组 12 0.56±0.11Λ 5.0g / kg group 12 0.56 ± 0.11 Λ
1 Og/kg组 12 0.48±0.08*  1 Og / kg group 12 0.48 ± 0.08 *
*与对照组相比 ( Ρ<0.01 ) **与对照組相比（ΡΟ.05 )  * Compared with the control group (P <0.01) ** Compared with the control group (P05.05)
2 本发明实施例 1或 2胶嚢对小鼠血清溶血素产生的影响： 2.5g/kg 剂量组与对照組相比， 其抗体积数无显著性差异 ( P>0.05 ) , 5.0g/k 组和 10g/kg剂量組和对照组相比， 其抗体积 数有高度显著性差异（ PO.01 ) 。 （表 2 ) 表 2 本发明胶嚢对小鼠血清溶血素抗体积数的影响 2 Effect of capsules of Example 1 or 2 of the present invention on serum hemolysin production in mice: Compared with the control group, the 2.5 g / kg dose group had no significant difference in anti-volume numbers (P> 0.05), 5.0 g / k Compared with the 10 g / kg dose group and the control group, the anti-volume numbers have a highly significant difference (PO.01). (Table 2) Table 2 Effects of capsules of the present invention on serum hemolysin anti-volume numbers in mice
組别 例数（只） 抗体积数（ 士 S ) 对照组 12 121.67±8.45 Group Number of cases (only) Anti-volume number (± S) Control group 12 121.67 ± 8.45
2.5g/kg组 13 127.15±13.212.5g / kg group 13 127.15 ± 13.21
5.0g/k 組 12 141.25±8.00Λ lOg/kg组 12 144.08±13.28* 5.0g / k group 12 141.25 ± 8.00 Λ lOg / kg group 12 144.08 ± 13.28 *
*与对照組相比（P<0.01 )  * Compared with the control group (P <0.01)
3. 本发明实施例 1 或 2胶嚢对小鼠巨噬细胞吞噬功能的影 响： 3. Effect of the capsules of Example 1 or 2 of the present invention on phagocytosis of mouse macrophages:
2.5g/kg组， 5.0g/kg組和 10g/kg各剂量组与对照组相比， 其 吞噬百分率均无显著性差异（P>0.05 )； 2.5g/kg组， 5.0g/kg组与 对照组相比， 其吞噬指数无显著性差异（P>0.05 ) ， 10g/kg组与 对照組相比， 其吞噬指数有高度显著性差异（P<0.01 ) 。 （表 3 ) 表 3 本发明实施例 1或 2胶嚢对小鼠巨噬细胞吞噬功能的影响 组别 例数（只） 吞噬率（％ ) 吞噬指数（ 士 S )  There was no significant difference in the percentage of phagocytosis between the 2.5g / kg group, 5.0g / kg group and 10g / kg group compared with the control group (P> 0.05); 2.5g / kg group, 5.0g / kg group and Compared with the control group, the phagocytic index was not significantly different (P> 0.05). Compared with the control group, the 10g / kg group had a highly significant phagocytic index (P <0.01). (Table 3) Table 3 Effects of the capsules of Example 1 or 2 of the present invention on the phagocytic function of mouse macrophages Group Number of cases (only) Phagocytic rate (%) Phagocytic index (± S)
( ±S )  (± S)
对照组 11 31·45±5·47 0.82±0.15 Control group 11 31 · 45 ± 5 · 47 0.82 ± 0.15
2.5g/kg組 12 33.67士 9.82 0.93±0.252.5g / kg group 12 33.67 ± 9.82 0.93 ± 0.25
5.0g/k 组 13 33.85士 7.47 0.91士 0.235.0g / k group 13 33.85 ± 7.47 0.91 ± 0.23
10g/k 组 12 38.42±6.13 1.16±0.26Λ 10g / k group 12 38.42 ± 6.13 1.16 ± 0.26 Λ
*与对照组相比（Ρ<0.01 )  * Compared with the control group (P <0.01)
4 本发明实施例 1或 1胶嚢对小鼠 ΝΚ细胞活性的影响： 4 Effect of Example 1 or 1 of the invention on the activity of mouse NK cells:
2.5g/kg, 5.0g/kg和 10g/kg剂量組与对照组相比， 其 NK细 胞活性率均无显箸性差异（P>0.05 ) 。 （表 4 )  Compared with the control group, the 2.5 g / kg, 5.0 g / kg and 10 g / kg dose groups had no significant difference in NK cell activity rate (P> 0.05). (Table 4 )
表 4 本发明胶; ft对小鼠 NK细胞活性的影响  Table 4 Effects of the gel of the invention; ft on mouse NK cell activity
组别 例数（只） 足趾肿胀度（mm ) ( ±S ) 对照组 11 55.98±13.54  Group Number of cases (only) Toe swelling (mm) (± S) Control group 11 55.98 ± 13.54
2.5g/k 組 12 56.72±12.54  2.5g / k group 12 56.72 ± 12.54
5.0g/k 组 13 48.94±13.36  5.0g / k group 13 48.94 ± 13.36
10g/kg组 12 50.62±14.00 5 本发明实施例 1或 2胶嚢对小鼠脏器 /体重比值的影响： 各剂量组与对照组相比， 其脾脏 /体重比值、胸腺 /体重比值无 显著性差异（ P>0.05 ) 。 （表 5 ) 10g / kg group 12 50.62 ± 14.00 5 Effect of the capsule of Example 1 or 2 of the present invention on the organ / body weight ratio of mice: Compared with the control group, each dose group had no significant difference in spleen / body weight ratio and thymus / body weight ratio (P> 0.05). (table 5 )
表 5 本发明胶; 对小鼠脏器 /体重比值的影响 组别 例数（只） 脾^/体重比值 胸^/体重比值 Table 5 Effects of the gel of the present invention on the organ / weight ratio of mice Group Number of cases (only) Spleen ^ / weight ratio Chest ^ / weight ratio
( mg/10g ) ( x±S ) ( mg/lOg ) (： ±S ) 对照组 11 69.93±5.37 16.12±2.72 (mg / 10g) (x ± S) (mg / lOg) (: ± S) Control group 11 69.93 ± 5.37 16.12 ± 2.72
2.5g/k 组 12 64.76±5.92 16.08±1.932.5g / k group 12 64.76 ± 5.92 16.08 ± 1.93
5.0g/kg组 13 65.63±6.20 15.60±1.725.0g / kg group 13 65.63 ± 6.20 15.60 ± 1.72
10g/k 组 12 64.47士 7. 80 15.66±2.29 小结 10g / k group 12 64.47 ± 7. 80 15.66 ± 2.29 Summary
分别经口给予小鼠（BALB/C ) 2.5g/kg、 5.0g/kg, 和 10g/kg 剂量的佰路灵芝（冲剂型） 25天， 在迟发性***反应试验中， 各 剂量组足趾肿胀度与对照组相比均有显著性差异； 在血清溶血素 测定中， 5.0g/kg组和 10g/kg剂量与对照組相比， 其 体积数有 高度显著性差异（PO.01 ) ； 在小鼠腹腔巨噬细胞吞噬试验中， 10g/kg组与对照组相比，其吞噬指数有高度显著性差异（ P<0.01 )。 因此， 5.0g/kg、 10g/kg剂量能提高小鼠血清溶血素抗体积数和迟 发性***反应水平； 2.5g/kg 剂量能提高小鼠迟发性***反应水 平； 10g/kg剂量能提高小鼠吞噬指数， 据评价依据（ 3.1.3结果判 定） 可判定本发明实施例 1或 2胶嚢具有免疫调节作用。 实验例 2 Mice (BALB / C) were dosed with 2.5 g / kg, 5.0 g / kg, and 10 g / kg of Ganoderma lucidum (infusion form) for 25 days. In the delayed allergy test, each dose group was sufficient. The degree of toe swelling is significantly different from that of the control group. In the determination of serum hemolysin, the 5.0g / kg group and the 10g / kg dose have a highly significant difference in volume number compared with the control group (PO.01). ; In the mouse peritoneal macrophage phagocytosis test, compared with the control group, the phagocytic index of the 10g / kg group had a highly significant difference (P <0.01). Therefore, the doses of 5.0g / kg and 10g / kg can increase the serum antihemolysin volume and the level of delayed allergies in mice; the dose of 2.5g / kg can increase the levels of delayed allergies in mice; the dose of 10g / kg can Increase mouse phagocytosis index, according to the evaluation basis (3.1.3. It can be determined that the capsule of Example 1 or 2 of the present invention has an immunomodulatory effect. Experimental example 2
本发明药物制剂对动物的延緩衰老实验  Experiment of delaying aging of the pharmaceutical preparation of the present invention on animals
1. 材料与方法：  1. Materials and methods:
1.1 受试样品： 本发明实施例 1或 2的胶嚢  1.1 Test sample: Capsules of Example 1 or 2 of the present invention
1.2实验动物：  1.2 Experimental animals:
选 18月龄雌性 Wistar大鼠 36只， （购自中国医学科学院实 验动物研究所繁育场， 合格证书： 医动字第 01 - 3008号）购入后 在动物房适应一周， 禁食 12小时称体重， 随机分为四组， 每组 9 只， 分别是对照組（体重 295g ±29.7 ) , 低剂量实验组（体重 295g ±17.1 ) 0.038g/kg.bw, 中剂量组 ( 299g ±29.7 ) 0.075g/kg.bw和 高剂量实验組（ 293 ±19.1 ) 0.15g/kg.bw。 另购三月龄雌性 Wistar 大鼠 9只作为青年对照实验组（225g .77 ) 。  Thirty-six female Wistar rats aged 18 months were selected (purchased from the Experimental Animal Breeding Institute of the Chinese Academy of Medical Sciences, certificate of qualification: Yidongzi No. 01-3008). Body weight was randomly divided into four groups of 9 animals each, which were the control group (body weight 295g ± 29.7), the low-dose experimental group (body weight 295g ± 17.1) 0.038g / kg.bw, and the medium-dose group (299g ± 29.7) 0.075. g / kg.bw and high-dose experimental group (293 ± 19.1) 0.15g / kg.bw. Nine three-month-old female Wistar rats were purchased as a young control experimental group (225g.77).
1.3 剂量选择： 1.3 Dosage selection:
人推荐量为 0.75g/曰, 即 0.0125/kg.b.w (以 60k 体重计 ) , 以此剂量上浮 5 倍、 10 倍、 20 倍， 分别为 0.0625g/kg.bw.、 0.175g/kg.bw、 0.05g/kg.bw。  The recommended amount is 0.75g / day, that is, 0.0125 / kg.bw (based on 60k body weight), and the dose is increased by 5 times, 10 times, and 20 times, which are 0.0625g / kg.bw., 0.175g / kg. bw, 0.05g / kg.bw.
1.4. 试剂与仪器： 1.4. Reagents and instruments:
仪器： 紫外分光光度计、 荧光分光光度计、 冷冻高速离心机、 恒温及煮沸水浴涡、 组织匀浆器等。  Apparatus: UV spectrophotometer, fluorescence spectrophotometer, refrigerated high-speed centrifuge, constant temperature and boiling water bath vortex, tissue homogenizer, etc.
试剂： 硫代巴比妥酸、 四乙氧基丙烷、 碑钨酸、 正丁醇、 盐 酸羟胺、 黄嘌呤、 甲萘胺、 对氨基笨磺酸、 叠氮钠、 磷酸盐等， MAO - B测定试剂盒（购自伊力康生物技术有限公司 ) 1.5 实验方法： Reagents: thiobarbituric acid, tetraethoxypropane, stilbungstic acid, n-butanol, hydroxylamine hydrochloride, xanthine, menaline, p-aminobenzylsulfonic acid, sodium azide, phosphate, etc., MAO-B Assay kit (purchased from Ilicon Biotechnology Co., Ltd.) 1.5 Experimental method:
1.5.1抗氧化实验：  1.5.1 Anti-oxidation experiment:
连续灌胃 90天， 实验结束处死动物， 制备血液、 脑、 肝组织 匀浆标本： ***麻醉称体重、 腹腔动脉取血， 5 % EDTA - Na 抗 凝待测谷胱甘肽过氧化物酶（GSH - Px ) 脂质过氧化物 （MDA ) 和超氧化物歧化酶（SOD ) 。 取肝脏用冷生理盐水洗两次， 在水 浴上用滤纸吸干后称肝总重，用病理刀片切下 0.5克左右（三份）， 称重后分别放入冷冻管中立即投入液氮中速冻后， 存低温水箱保 存待测 GSH - Px和 MDA。 取全脑称重后放入冷冻管中立即投入 液氮中速冻后存低温冰箱待测单氨氧化酶。  The animals were sacrificed for 90 consecutive days, and the animals were sacrificed at the end of the experiment. Blood, brain, and liver tissue homogenates were prepared: weighed with ether, weighed by celiac artery, and 5% EDTA-Na anticoagulated glutathione peroxidase ( GSH-Px) Lipid Peroxide (MDA) and Superoxide Dismutase (SOD). Take the liver and wash it twice with cold physiological saline, blot it with filter paper on a water bath, and weigh the total weight of the liver. Cut out about 0.5 grams (three portions) with a pathological blade, weigh them, place them in a freezing tube, and immediately put them into liquid nitrogen. After quick freezing, store the GSH-Px and MDA to be tested in a cryogenic water tank. Take the whole brain, weigh it, put it into a freezing tube, and immediately put it into liquid nitrogen, and then store it in a low-temperature freezer for testing.
1.5.1.1脂质过氧化物（MDA ) 的测定： 1.5.1.1 Determination of Lipid Peroxide (MDA):
用 TBA荧光法当日测定血中 MDA含量，取前述抗凝血 75μ1 加入 1.5ml生理盐水 2000r/分钟， 离心 10分钟， 取上清 0.5ml, 加所需试剂， 摇匀、 离心、 弃上清液， 沉淀加水、 再加 TBA试剂， 沸水浴 75分钟， 流水冷却至室温， 加正丁醇 5ml， 震荡萃取， 离 心， 取上清测定荧光强度（同时用 10nmol/ml四乙氧基丙烷做一 标准曲线） ， 测得的荧光强度在标准曲线上可查得相应的 MDA 含量。 用 TBA荧光法测定组织中 MDA含量， 测定当日从低温水 箱取出肝组织緩冻， 根据组织重量加入 10倍的冷生理盐水， 超速 匀浆器 20，000r/分钟匀浆 10秒， 间歇 15秒， 重复 3次。 匀浆于 4 °C 4000r/分钟离心 30分钟， 取上清 0.1ml, 其它步骤同血中 MDA 测定。  The MDA content in blood was measured on the same day by TBA fluorescence method. 75 μ1 of the aforementioned anticoagulant was added to 1.5 ml of physiological saline at 2000 r / min, centrifuged for 10 minutes, 0.5 ml of the supernatant was added, the required reagents were added, and the supernatant was shaken, centrifuged and discarded. Add precipitation, add TBA reagent, add boiling water bath for 75 minutes, cool the running water to room temperature, add 5ml of n-butanol, shake extract, centrifuge, take the supernatant to determine the fluorescence intensity (while using 10nmol / ml tetraethoxypropane as a standard) Curve), the measured fluorescence intensity can be checked on the standard curve for the corresponding MDA content. The TBA fluorescence method was used to determine the MDA content in the tissues. The liver tissue was slowly frozen from the cryogenic water tank on the day of the measurement, and 10 times cold physiological saline was added according to the weight of the tissue. Repeat 3 times. The homogenate was centrifuged at 4000 ° C / min for 30 minutes at 4 ° C, and 0.1 ml of the supernatant was taken. The other steps were the same as the determination of MDA in blood.
1.5.1.2谷胱甘肽过氧化物酶（ GASH - Px ) 的测定： 1.5.1.2 Determination of glutathione peroxidase (GASH-Px):
DTNB法当日测血中 GSH - Px活力， 取 ΙΟμΙ上述抗凝血加 入到 990μ1双蒸水中， 充分震荡， 使之全部溶血， 取 0.4ml, 加入 所需试剂， 37°C水浴中进行酶促反应 3分钟， 终止反应， 离心， 显色， 于 422nm波长读 OD值， 按照公式计算出酶活力单位数。 DTNB method was used to measure the activity of GSH-Px in blood on the same day. Take 10μΙ of the above anticoagulant and add it to 990μ1 double-distilled water, shake it thoroughly to make it all hemolyzed, take 0.4ml, add For the required reagents, perform an enzymatic reaction in a 37 ° C water bath for 3 minutes, stop the reaction, centrifuge, and develop the color. Read the OD value at a wavelength of 422nm, and calculate the number of enzyme activity units according to the formula.
组织中 GSH - Px 活力， 测定当日从低温冰箱取出肝组织緩 冻， 根据组织重量加入 10倍的冷生理盐水， 超速勾浆器 20，000r/ 分钟匀浆 10秒， 间歇 15秒， 重复 3次。 勾浆以 10000r/分钟离心 10分钟， 取上清液测定（步骤同血测定） 。  The activity of GSH-Px in the tissues was measured. The liver tissue was slowly frozen from the low-temperature refrigerator on the day of measurement, and 10 times cold physiological saline was added according to the weight of the tissue. The homogenizer was homogenized at 20,000 r / min for 10 seconds with a 15-second interval and repeated 3 times. . Centrifuge at 10000 r / min for 10 minutes and take the supernatant for measurement (the procedure is the same as that for blood measurement).
1.5.1.3超氧化物歧化酶（ SOD ) ： 1.5.1.3 Superoxide dismutase (SOD):
羟胺法当日测 SOD 活性， 将前述抗凝血 3000r/分钟， 离心 10分钟。 取血浆 30μ1, 加所需试剂， 混匀， 于 37°C水浴 40分钟， 加试剂终止反应， 显色 10分钟后， 于 550nm处测定 OD值， 根 据 OD值计算出 SOD活力单位数。  The hydroxylamine method was used to measure the SOD activity on the day. The anticoagulant was centrifuged at 3000 r / min for 10 minutes. Take 30μ1 of plasma, add the required reagents, mix well, and place the reagents in a 37 ° C water bath for 40 minutes. Stop the reaction by adding reagents. After 10 minutes of color development, measure the OD value at 550nm, and calculate the number of SOD activity units based on the OD value.
1.5.1.4盐酸苄胺荧光法测 MAO一 B活性， 1.5.1.4 Measurement of MAO-B activity by benzylamine hydrochloride fluorescence method,
测定当日从低温水箱取出脑组织緩冻， 根据组织重量加入 5 倍的冷生理盐水， 在冰浴中 20，000r/分钟勾浆 10秒， 间歇 15秒， 重复 3次。 匀浆于 4°C l0000r/分钟离心 20分钟， 取上清 5倍稀释 后， 取 0.2ml, 加所需试剂， 恒温水浴 1小时， 加试剂终止反 应， 于 242nm波长处测 OD值， 根据 OD值计算出 MAO - B活 力单位数。  On the day of the measurement, the brain tissue was slowly frozen from the low-temperature water tank, and 5 times cold physiological saline was added according to the weight of the tissue, and the slurry was hooked in an ice bath at 20,000 r / min for 10 seconds, with an interval of 15 seconds, and repeated 3 times. The homogenate was centrifuged at 4 ° C l0000r / min for 20 minutes. After the supernatant was diluted 5 times, 0.2 ml was taken, and the required reagents were added. The reaction was stopped in a constant temperature water bath for 1 hour. The OD was measured at 242 nm. The value calculates the number of MAO-B vitality units.
1.6 实验数据用 DBASEIII软件建立数据库， 用 SYSTAT软 件分析处理。 1.6 The experimental data was set up with DBASEIII software and analyzed and processed with SYSTAT software.
2. 结果： 见表 6 - 8 2. Results: see Table 6-8
表 6 各组大鼠的初始体重、 中期体重、 结束体重  Table 6 Initial weight, intermediate weight, and end weight of each group of rats
初始体重 中期体重 结束体重 组别 动物数 体重 动物数 体重 动物数 体重Initial weight mid weight end weight Number of animals in group Weight of animals
(只） ( g ) (只） ( g ) (只） ( g ) 青年对照组 9 225±6.77 9 243±10.5 9 256±14.6 老年对照组 9 295±41.3 9 306±36.6 9 312±27.7 低剂量組 9 295±17.1 9 298±18.3 9 288±17.4 中剂量組 9 299±29.7 9 294±18.0 9 295±24.1 高剂量组 9 293±19.1 9 293±37.3 9 304±21.7 表 1结果显示， 各剂量組大鼠体重与老龄对照组相比无统计 学差异 ( P>0.05 ) 。 (Only) (g) (only) (g) (only) (g) Youth control group 9 225 ± 6.77 9 243 ± 10.5 9 256 ± 14.6 Aged control group 9 295 ± 41.3 9 306 ± 36.6 9 312 ± 27.7 Low dose Group 9 295 ± 17.1 9 298 ± 18.3 9 288 ± 17.4 Medium dose group 9 299 ± 29.7 9 294 ± 18.0 9 295 ± 24.1 High dose group 9 293 ± 19.1 9 293 ± 37.3 9 304 ± 21.7 Table 1 shows the results for each dose There was no statistical difference in body weight between the rats in the group and the aging control group (P> 0.05).
表 7 本发明实施例 1或 2胶嚢对大鼠血中 SOD、 GSH - PX 活力及 MDA含量的影响  Table 7 Effects of capsules of Example 1 or 2 of the present invention on SOD, GSH-PX activity and MDA content in rat blood
組别 n SOD GSH - Ρχ MDA g/kg.bw NU/ml血 U/ml血 nmol/ml血 Group n SOD GSH-Ρχ MDA g / kg.bw NU / ml blood U / ml blood nmol / ml blood
0(青年对照） 9 151.4±5·47 28.02±2.07 2.78±0.880 (youth control) 9 151.4 ± 5 · 47 28.02 ± 2.07 2.78 ± 0.88
0(老龄对照） 9 149.9±3.62 24.08±2.70# 2.67±0.720 (aged control) 9 149.9 ± 3.62 24.08 ± 2.70 # 2.67 ± 0.72
0.038 9 152.1±4.33 27.13±2.21** 2.58±0.780.038 9 152.1 ± 4.33 27.13 ± 2.21 ** 2.58 ± 0.78
0.075 9 149.5±2.62 26.69±2.40* 2·88±0·540.075 9 149.5 ± 2.62 26.69 ± 2.40 * 2.88 ± 0.54
0.150 9 150.7±2.38 25.84±2.85 2.64士 0.65 与老龄对照组比较 *P<0.05 **P<0.01 0.150 9 150.7 ± 2.38 25.84 ± 2.85 2.64 ± 0.65 compared with the aging control group * P <0.05 ** P <0.01
与青年对照组比较 #P<0.05 由表 7可见，与老龄对照组相比，经口给予本发明胶嚢 90天， 低， 中剂量能明显升高血中谷胱甘肽过氧化物酶（GSH - Px ) 活 力， 差异有显著性（P<0.01、 P<0.05 ) 。 各剂量组血中脂质过氧 化物（MDA )水平和超氧化物歧化酶（SOD ) 活力与老龄对照组 比较无统计学差异（P>0.05 ) 。 表 8 本发明实施例 1或 2胶嚢对大鼠脑中 MAO - B活力和 肝组织中 MDA含量及 GSH - P 活力的影响 Compared with the young control group #P <0.05 It can be seen from Table 7 that compared with the aging control group, the capsule of the present invention is orally administered for 90 days, and the low and medium dose can obviously increase the blood glutathione peroxidase (GSH -Px) activity, the difference is significant (P <0.01, P <0.05). Lipid peroxide (MDA) levels and superoxide dismutase (SOD) activity in blood of each dose group were not statistically different from those of the aging control group (P> 0.05). Table 8 Effects of the capsules of Example 1 or 2 of the present invention on the activity of MAO-B in brains of rats, the content of MDA in liver tissues, and the activity of GSH-P
組别 n 脑中 MAO -活力 脑中 MDA含量 肝組织中 GHS - g/kg.bw nmol苄醛 /mg組织 nmol/mg組织 PxU/mg蛋白 Group n MAO in brain-MDA content in brain GHS-g / kg in liver tissues bw nmol benzaldehyde / mg tissue nmol / mg tissue PxU / mg protein
0 (青年对照） 9 2.32±0.53 0.047±0.005 256.3±25.120 (youth control) 9 2.32 ± 0.53 0.047 ± 0.005 256.3 ± 25.12
0 (老龄对照 ) 9 2.24±0.43 0.052±0.013 157.8±38.0靈 0 (Age control) 9 2.24 ± 0.43 0.052 ± 0.013 157.8 ± 38.0 spirit
0.038 9 1.89±0.20*# 0.049±0.004 182.6±20.07* 0.038 9 1.89 ± 0.20 * # 0.049 ± 0.004 182.6 ± 20.07 *
0.075 9 1.72±0.45*# 0.048±0.004 181.3±17.43*0.075 9 1.72 ± 0.45 * # 0.048 ± 0.004 181.3 ± 17.43 *
0.150 9 1.59±0.32**## 0.043±0.004*# 171.5±25.39 与老龄对照組相比 AP<0.05 **Ρ<0·01 0.150 9 1.59 ± 0.32 ** ## 0.043 ± 0.004 * # 171.5 ± 25.39 A P <0.05 ** P <0.01
与青年对照組相比 #P<0.05 ##P<0.01 由表 8可知，与老龄对照组相比，经口给予本发明胶嚢 90天， 各剂量均能使大鼠脑组织中单胺氧化酶（MAO - B ) 活力明显降 低， 差异显著（PO.05, Ρ<0.01 ) , 与青年对照组相比差异也有 明显性（P<0.05, P<0.01 ) 。 低、 中剂量能使肝組织中谷胱甘肽 过氧化物酶（GSH - Px ) 活力明显升高， 差异显箸（P<0.05 ) ； 高剂量能使肝组织中脂质过氧化物（MDA )水平明显降低， 差异 有显著性（ P<0.05 ) 。 Compared with the young control group #P <0.05 ## P <0.01 As can be seen from Table 8, compared with the aging control group, the capsules of the present invention were orally administered for 90 days, and each dose can make monoamine oxidase (MAO) in rat brain tissue -B) Vigor was significantly reduced and the difference was significant (PO.05, P <0.01), and the difference was also significant compared with the young control group (P <0.05, P <0.01). Low and medium doses can significantly increase the activity of glutathione peroxidase (GSH-Px) in liver tissues, with a significant difference (P <0.05); high doses can make lipid peroxides (MDA) in liver tissues The level was significantly reduced, and the difference was significant (P <0.05).
3 结论： 3 Conclusions:
经口给予大鼠本发明实施例 1或 2胶嚢 90天，与老龄对照組 大鼠相比， 各剂量能明显降低大鼠脑组织中单胺氧化酶（ MAO - B )活力， 并与青年对照组相比差异也有显著性； 低、 中剂量能明 显升高大鼠肝组织和血中谷胱甘肽过氧化物酶（GASH - Px ) 活 力； 高剂量能使肝組织中脂质过氧化物（MDA ) 水平明显降低。  Rats of Example 1 or 2 of the present invention were orally administered for 90 days. Compared with aging control rats, each dose can significantly reduce the activity of monoamine oxidase (MAO-B) in rat brain tissues. There are also significant differences in ratios; low and medium doses can significantly increase glutathione peroxidase (GASH-Px) activity in rat liver tissues and blood; high doses can cause lipid peroxide (MDA) levels in liver tissues obviously decased.
本发明实施例 1或 2胶嚢对大鼠血中脂质过氧化物（MDA ) 水平和大鼠血中超氧化物歧化酶（SOD ) 活性的影响。 结合果蝇 生存实验结果为阳性， 依据《保健食品功能学评价程序和检验方 法》 中延緩衰老作用判定的标准， 认为本发明实施例 1或 2胶嚢 有延緩衰老作用。 实施例 3 Effects of the capsules of Example 1 or 2 of the present invention on lipid peroxides (MDA) in rat blood Effects of levels and superoxide dismutase (SOD) activity in rat blood. Based on the results of the fruit fly survival test being positive, according to the criteria for determining the function of delaying aging in the "Functional Evaluation Procedures and Test Methods for Health Foods", it is believed that the capsules of Example 1 or 2 of the present invention have a delaying aging effect. Example 3
本发明药物制剂对心脑血管疾病的作用。  Effect of the pharmaceutical preparation of the present invention on cardiovascular and cerebrovascular diseases.
心脑血管疾病患者 100例， 其中男性患者 51例， 女性患者 49例， 年龄 13 - 80岁。 给上述患者服用本发明实施例 1或 2胶 嚢， 每天 3次， 每次 2粒， 给药时间 1 - 4个月。 结果见表 9 - 13。 从表 9 - 13中结果可看到本发明实施例 1或 2胶嚢对与心脑血管 疾病有关的各种症状均有良好疗效， 且有效率为 99 % 。  There were 100 patients with cardiovascular and cerebrovascular diseases, including 51 male patients and 49 female patients, aged 13-80 years. Take the capsules of Example 1 or 2 of the present invention to the above patients 3 times a day, 2 capsules each time, and the administration time is 1 to 4 months. The results are shown in Tables 9-13. From the results in Tables 9-13, it can be seen that the capsules of Example 1 or 2 of the present invention have good effects on various symptoms related to cardiovascular and cerebrovascular diseases, and the effective rate is 99%.
表 9实施例 1或 2胶嚢对与心脑血管疾病有关症状的疗效 肌肉酸痛 10 0 5 5  Table 9 Efficacy of capsules of Example 1 or 2 on symptoms related to cardiovascular and cerebrovascular diseases Muscle soreness 10 0 5 5
面唇紫暗 7 0 7 0  Face and lips purple and dark 7 0 7 0
心悸 34 0 18 16 胸闷 31 0 12 19 心前隐痛 11 0 6 5  Palpitations 34 0 18 16 Chest tightness 31 0 12 19 Pain in front of the heart 11 0 6 5
失眠 50 2 18 30 抑郁 7 0 7 0  Insomnia 50 2 18 30 Depression 7 0 7 0
脱发 28 1 10 17  Hair loss 28 1 10 17
而 15 0 9 6  And 15 0 9 6
过敏 4 0 3 1  Allergies 4 0 3 1
糖尿病 4 0 3 1  Diabetes 4 0 3 1
肠胃虛弱 35 0 9 26  Gastrointestinal weakness 35 0 9 26
其他 19 0 6 13 合计 343 4 (1% ) 157 ( 46% ) 180 ( 53% ) 表 10 实施例 1或 2胶嚢对心脑血管疾病症状的改善状况 Other 19 0 6 13 Total 343 4 (1%) 157 (46%) 180 (53%) Table 10 Example 1 or 2 improvement of cardio-cerebral-vascular disease symptoms
Figure imgf000018_0001
表 12 给实施例 1或 2胶嚢前后心电图改善情况
Figure imgf000018_0001
Table 12 ECG improvement before and after the capsules of Example 1 or 2
Figure imgf000018_0002
表 13 实施例 1或 2胶嚢对心脑血管疾病的改善
Figure imgf000018_0002
Table 13 Cardio-cerebral-vascular disease improvement in Example 1 or 2
病种分类 给药后  Classification of diseases
例次 显效 有效 无效 冠心病 11 0 11 0 高血压 14 5 9 0 风心病 3 0 3 0 高脂血症 12 7 5 0 肺心病 0 0 2 0 甲心病 2 0 2 0 心律失常 5 1 4 0 动脉硬化 5 0 5 0 肠胃虚弱 35 26 9 0 糖尿病 4 1 2 0 抑郁症 7 0 7 0 脱发 28 17 10 1 Cases markedly effective and ineffective coronary heart disease 11 0 11 0 hypertension 14 5 9 0 wind heart disease 3 0 3 0 Hyperlipidemia 12 7 5 0 Pulmonary heart disease 0 0 2 0 Coronary heart disease 2 0 2 0 Arrhythmia 5 1 4 0 Arteriosclerosis 5 0 5 0 Gastrointestinal weakness 35 26 9 0 Diabetes 4 1 2 0 Depression 7 0 7 0 Hair loss 28 17 10 1

Claims

权 利 要 求 Rights request
1. 一种防治心脑血管疾病、 延緩衰老及调节人体免疫功能的 药物制剂， 其按重量份计是由下述成分組成： 1. A pharmaceutical preparation for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function, which is composed of the following components in parts by weight:
银杏叶提取物为 8-24, Ginkgo biloba extract is 8-24,
黄芪提取物为 12-36, Astragalus extract is 12-36,
螺旋藻为 30 - 90。 Spirulina is 30-90.
2. 权利要求 1所述的药物制剂， 其中按重量份计  2. The pharmaceutical preparation according to claim 1, wherein the part is by weight
银杏叶提取物为 10， Ginkgo biloba extract is 10,
黄芪提取物为 30, Astragalus extract is 30,
螺旋藻为 60。 Spirulina is 60.
3. 权利要求 1所述的药物制剂， 其中按重量份计  3. The pharmaceutical preparation according to claim 1, wherein in terms of parts by weight
银杏叶提取物为 16, Ginkgo biloba extract is 16,
黄芪提取物为 24, Astragalus extract is 24,
螺旋藻为 50。 Spirulina is 50.
4. 权利要求 1-3任一要求的药物制剂的制备方法， 其包括将 银杏叶提取物， 黄芪提取物及螺旋藻混合在一起。  4. A method for preparing a pharmaceutical preparation according to any one of claims 1-3, comprising mixing together ginkgo biloba extract, astragalus extract and spirulina.
5. 权利要求 4所述的方法， 其中银杏叶提取物如下制备： 将银杏叶烘干、 粉碎， 用乙醇（40- 80% ) 回流提取（40- 70°C ) 2-4 次， 提取液蒸去乙醇后用大孔吸附树脂（D- 101 型等） 吸 附， 用乙醇（50- 90% ) 洗脱、 洗脱液回收乙醇， 残留液干燥， 得银杏叶提取物；  5. The method according to claim 4, wherein the ginkgo biloba extract is prepared as follows: ginkgo biloba leaves are dried, pulverized, and extracted with ethanol (40-80%) under reflux (40-70 ° C) for 2-4 times, and the extract solution After evaporating the ethanol, it was adsorbed with a macroporous adsorption resin (type D-101, etc.), eluted with ethanol (50-90%), and the eluent was recovered to recover ethanol, and the residue was dried to obtain a ginkgo biloba extract;
该银杏叶提取物含银杏黄酮不低于 24% ,含银杏内脂不低于 6%。The ginkgo leaf extract contains no less than 24% ginkgo flavonoids and no less than 6% ginkgo lactones.
6. 权利要求 4所述的方法， 其中银杏叶提取物如下制备： 将银杏叶烘干、 粉碎（过 20 目筛） ， 用含 1-2%抗氧剂的水溶 液 10- 15倍量， 煮沸提取三次， 每次 1-2小时； 合并三次水提 液， 过聚酰胺柱， 柱高与宽的比值为 4- 15; 用三倍量的水洗涤， 再用 60- 90%乙醇洗脱， 浓缩， 用正己烷萃取二次， 浓缩干燥得 银杏总黄酮。 6. The method according to claim 4, wherein the ginkgo biloba extract is prepared as follows: ginkgo biloba leaves are dried, pulverized (passed through a 20-mesh sieve), and boiled with 10-15 times the amount of an aqueous solution containing 1-2% antioxidant. Extract three times, 1-2 hours each time; combine three water extracts, pass through a polyamide column, the ratio of the column height to the width is 4- 15; wash with three times the amount of water, Then it was eluted with 60-90% ethanol, concentrated, extracted twice with n-hexane, and concentrated and dried to obtain the total flavonoids of ginkgo.
7. 权利要求 6所迷的方法， 其中所述的抗氧剂水溶液为 0.5% 的焦亚硫酸钠溶液； 聚酰胺柱高与宽的比例为 4; 过柱洗脱时乙 醇浓度为 70%。  7. The method of claim 6, wherein the aqueous solution of the antioxidant is a 0.5% sodium metabisulfite solution; the ratio of the height of the polyamide column to the width of the column is 4; and the concentration of ethanol when the column is eluted is 70%.
8. 权利要求 4的方法， 其中黄芪提取物如下制备： 黄芪切片， 用 15 -30倍水煮沸提取三次， 每次 0.5- 2小时， 三次煎液合并、 滤过， 滤液用大孔吸附树脂 （D- 101 型等） 吸附， 用乙醇（40 -90% ) 洗脱、 洗脱液回收乙醇、 残留液干燥， 得黄芪提取物； 该黄芪提取物含黄芪甲甙（C44H6804) 不.低于 0.1%。 8. The method of claim 4, wherein the astragalus extract is prepared as follows: Astragalus slices are boiled and extracted three times with 15-30 times water for 0.5-2 hours each time. The three decoctions are combined and filtered, and the filtrate is adsorbed with a macroporous resin ( D-101 type, etc.) adsorption, eluting with ethanol (40 -90%), recovering the eluent to ethanol, and drying the residue to obtain astragalus extract; the astragalus extract contains astragaloside IV (C 44 H 68 0 4 ) No. Less than 0.1%.
9. 权刮要求 1-3任一要求的制剂， 其中所述银杏提取物含银 杏黄酮不低于 24%, 含银杏内酯不低于 6%, 黄芪提取物含黄芪 甲甙不低于 0.1%。  9. The preparation of any one of claims 1-3, wherein the ginkgo extract contains no less than 24% ginkgo flavonoids, no less than 6% of ginkgolides, and no less than 0.1 of astragaloside in astragalus extracts %.
10. 银杏提取物， 黄芪提取物和螺旋藻结合在制备用于防治心 脑血管疾病、 延緩衰老及调节人体免疫功能的药物中用途。  10. Use of ginkgo biloba extract, astragalus extract and spirulina in the preparation of a medicament for preventing and treating cardiovascular and cerebrovascular diseases, delaying aging and regulating human immune function.
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* Cited by examiner, † Cited by third party
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MD2516C2 (en) * 2004-04-07 2005-03-31 Валериу РУДИК Anti-inflammatory, antiseptic and antimycotic medicament in the form of gel
MD2671G2 (en) * 2004-08-10 2005-08-31 Валериу РУДИК Antiviral and antiherpetic medicine in the form of gel
CN108125995A (en) * 2018-01-26 2018-06-08 扬州工业职业技术学院 A kind of ginko leaves flavone extract and its application
CN108125995B (en) * 2018-01-26 2021-04-16 扬州工业职业技术学院 Ginkgo leaf flavone extract and application thereof
CN110338326A (en) * 2019-07-10 2019-10-18 中慈保健品科技开发有限公司 Assist in the treatment of the soybean peptide compounding solid beverage and its preparation method and application of cardiovascular and cerebrovascular

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