WO2016107540A1 - 一种预防或治疗心脑血管疾病或痴呆症的中药组合物及其制备方法和用途 - Google Patents

一种预防或治疗心脑血管疾病或痴呆症的中药组合物及其制备方法和用途 Download PDF

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WO2016107540A1
WO2016107540A1 PCT/CN2015/099381 CN2015099381W WO2016107540A1 WO 2016107540 A1 WO2016107540 A1 WO 2016107540A1 CN 2015099381 W CN2015099381 W CN 2015099381W WO 2016107540 A1 WO2016107540 A1 WO 2016107540A1
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chinese medicine
group
medicine composition
traditional chinese
ginseng
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PCT/CN2015/099381
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English (en)
French (fr)
Inventor
张志伟
李志刚
张纲
关秀伟
逯蕴
高伟博
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神威药业集团有限公司
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Priority to AU2015373702A priority Critical patent/AU2015373702B2/en
Priority to CA2971080A priority patent/CA2971080C/en
Priority to KR1020177021241A priority patent/KR102005265B1/ko
Priority to US15/540,831 priority patent/US20170354700A1/en
Priority to JP2017535818A priority patent/JP6548734B2/ja
Priority to DE112015005869.9T priority patent/DE112015005869T5/de
Priority to RU2017121638A priority patent/RU2674264C1/ru
Publication of WO2016107540A1 publication Critical patent/WO2016107540A1/zh
Priority to US16/506,426 priority patent/US10967023B2/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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • 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
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the invention relates to a traditional Chinese medicine composition for preventing or treating cardiovascular and cerebrovascular diseases or dementia, and discloses a preparation method and use of the traditional Chinese medicine composition.
  • Cardiovascular and cerebrovascular diseases are the most important diseases that threaten human life and health. They are characterized by high morbidity, high mortality, high disability, and high recurrence rate. According to statistics in recent years, the annual death of cardiovascular and cerebrovascular diseases in China accounts for about half of the causes of death. The prevalence of cardiovascular and cerebrovascular diseases is closely related to the development of society and the improvement of living standards. Excessive psychological stress, unreasonable diet, lack of exercise, excessive smoking and drinking, and obesity are all causes of cardiovascular and cerebrovascular diseases. Cardiovascular and cerebrovascular diseases include coronary heart disease, angina pectoris, myocardial infarction, blood stasis, ischemic encephalopathy, cerebral thrombosis, hypertension, hyperlipidemia, etc.
  • cardiovascular and cerebrovascular diseases seriously endanger human health, and it is extremely important for its early prevention and timely treatment.
  • Dementia is an acquired, persistent intellectual impairment syndrome caused by organic brain lesions. Dementia occurring in the elderly is mainly divided into: A. primary degenerative dementia, namely Alzheimer's disease (AD); B. Vascular dementia (VaD); C. mixed dementia (AD combined with VaD); D. Other dementia (Pick disease, Louis inclusion body dementia). AD and VaD are the two most important types of dementia.
  • A. primary degenerative dementia namely Alzheimer's disease (AD); B. Vascular dementia (VaD); C. mixed dementia (AD combined with VaD); D. Other dementia (Pick disease, Louis inclusion body dementia).
  • AD and VaD are the two most important types of dementia.
  • Vascular dementia is associated with one or repeated strokes and hypertension, hyperlipidemia, diabetes, smoking and drinking, etc., manifested as memory, computational power, orientation and judgmental disorders, may have affective disorders and behavioral abnormalities, and loss of life ability in the late stage.
  • Recent studies have noted that the cerebral blood flow and glucose metabolism rate of the frontal lobe, temporal lobe, especially the thalamus and basal ganglia of VaD patients are significantly lower than other sites, revealing that VaD may be disconnected from the cortical and subcortical structures. That is, the brain is in contact with incompleteness; cerebrovascular resistance, blood viscosity, and neurohumoral factors all have direct or indirect effects on cerebral blood flow.
  • cerebral ischemic injury is a cascade process, and in each link will induce different molecular networks of neuronal degeneration damage: after cerebral infarction, cerebral blood flow disruption and reperfusion, resulting in brain tissue energy depletion, nerve
  • the elevation of the transmitter, especially the excitatory amino acid causes a further decrease in blood flow and releases calcium ions from the intracellular calcium pool, triggering a large number of enzymes to trigger a signal cascade, and some enzymes lead to oxygen free radical production.
  • Ginseng is warm, sweet and bitter, functional to nourish the kidney and nourish the heart, benefit Qi and increase wisdom; Ginkgo biloba leaves are flat, bitter taste, phlegm, and good blood circulation.
  • the saffron is flat and sweet, and it is good for blood circulation and phlegm.
  • ginseng contains a variety of saponins that can significantly improve cerebral ischemia-reperfusion injury and learning and memory impairment in a variety of experimental animals, can promote the learning and memory ability of normal animals, and increase the synaptic pair Quality intake, enhance the role of nerve growth factor;
  • Ginkgo biloba extract can inhibit cell membrane lipid peroxidation, can expand blood vessels, increase blood flow, reduce blood viscosity, inhibit thrombosis and anti-platelet aggregation, improve brain metabolism, protect nerves Cytosine;
  • safflorin has the effect of inhibiting the influx of extracellular calcium ions and the release of calcium ions on the endoplasmic reticulum, anti-oxidation, anti-hypertension, atherosclerosis, cerebral edema, etc., and can also increase blood oxygen in mammals.
  • it has also been found to have anti-ethanol-induced memory and learning disabilities.
  • the invention patent ZL02131435.7 discloses a traditional Chinese medicine composition containing a therapeutically effective amount of ginseng, ginkgo leaf and saffron for treating ischemic cerebrovascular disease, vascular dementia, senile dementia disease and the like. Based on this patent, the applicant developed the “Suoluotong Capsule” (the original trade name is “Wei Brain Kang”), which is currently in clinical trials.
  • the invention patent WO2007118363A1 discloses a similar traditional Chinese medicine composition for treating ischemic cerebrovascular disease and senile dementia, which is composed of ginseng, ginkgo leaf, saffron and soybean.
  • the inventors have systematically studied the above-mentioned patented technology and found that when a specific composition and content ratio are used, a better therapeutic effect can be obtained.
  • the inventors conducted a systematic experimental study on the effects of the components of the compositions referred to in the invention patents ZL02131435.7 and WO2007118363A1 in the prevention or treatment of cardiovascular and cerebrovascular diseases and dementia, and found that soybeans play a role in the composition. The effect is not obvious.
  • the pharmacological effects of the compositions of ginseng, ginkgo biloba and saffron can be achieved only by using the medicinal ingredients of ginseng, ginkgo biloba and saffron.
  • compositions made of ginseng, ginkgo biloba and saffron with a specific weight ratio have better effects in treating or preventing cardiovascular and cerebrovascular diseases and dementia, especially in the treatment of ischemia. It is more effective in the treatment of cerebrovascular disease, coronary heart disease, angina pectoris and senile dementia, especially vascular dementia.
  • the dosage of the composition composed of ginseng, ginkgo biloba and saffron in the medicine can be further reduced, which not only saves energy and reduces cost, but further reduces the possibility of long-term use of the composition. side effect.
  • the pharmaceutical raw material of the traditional Chinese medicine composition is the following pharmaceutical ingredients:
  • the pharmaceutical raw material of the traditional Chinese medicine composition is a pharmaceutical flavor of the following parts by weight:
  • the pharmaceutical raw material of the traditional Chinese medicine composition is a pharmaceutical flavor of the following weight ratio:
  • the pharmaceutical raw material from which the traditional Chinese medicine composition is prepared is the following pharmaceutical ingredients:
  • the invention also provides a preparation method of the above traditional Chinese medicine composition.
  • the traditional Chinese medicine composition of the present invention can be prepared by various methods.
  • the three traditional Chinese medicines of ginseng, ginkgo biloba, and saffron are weighed according to the above ratio, mixed, pulverized, or pulverized and mixed.
  • the three traditional Chinese medicines of ginseng, ginkgo biloba and saffron are weighed according to the above ratio, and are mixed and extracted.
  • the preparation method of the traditional Chinese medicine composition comprises the steps of: preparing the ginseng extract by the steps of weighing the three traditional Chinese medicines of ginseng, ginkgo biloba and saffron according to the above ratio; a step of preparing a ginkgo biloba extract, a step of preparing a saffron extract, and a step of mixing the above ginseng extract, ginkgo biloba extract and saffron extract.
  • the main component obtained is ginseng total saponin; in the step of preparing the ginkgo biloba extract, the main component obtained is ginkgo biloba total flavonoids and total lactone; In the step of the saffron extract, the main component obtained is saffron total glycosides.
  • the above three extracts can be prepared by the existing method for extracting the above components, and the extraction and preparation methods of the components are relatively mature.
  • total ginsenosides, total flavonoids of ginkgo biloba and total lactone can be extracted by the method described in the pharmacopoeia.
  • the method for extracting the saffron total glycosides can be extracted and prepared by the method described in the Chinese Journal of Modern Applied Pharmacy (March 28, 2008, vol. 28, pp. 729-731).
  • the method for preparing the ginseng extract is: pulverizing the ginseng into a powder, refluxing the ethanol twice, filtering, and recovering the solvent under reduced pressure to a relative density of 1.12 to 1.14 at 70 ° C. About 2-6 times the amount of crude drug is mixed well, cold, and the macroporous resin is adsorbed on the supernatant. The drug-loaded resin is washed with distilled water, then eluted with ethanol, and the alcohol eluate is collected and concentrated to dryness to obtain ginseng extract. Things.
  • the method for preparing the ginkgo biloba extract is: ginkgo biloba leaf powder and ethanol immersion, filtration, filter residue and ethanol immersion, filtration, combining two leach filtrates, and concentrating under reduced pressure.
  • the temperature is 70 ° C
  • the relative density is 1.12 ⁇ 1.14
  • the relative density was 1.02 to 1.04 when concentrated to 70 ° C.
  • the ethyl acetate: n-butanol was extracted, and the extracts were combined, and the solvent was evaporated under reduced pressure to obtain a ginkgo leaf extract.
  • the method for preparing the saffron extract is: the western red peanut medicine is added to the ethanol cold dip, filtered, and the dregs are further added with ethanol cold soaking, filtered, and the two leaching filtrates are combined.
  • relative density when concentrated to 70 ° C under reduced pressure 1.12 ⁇ 1.14 adding water, adsorbing the resin on the macroporous, the drug-loading resin is first washed with distilled water, then washed with ethanol, and the ethanol eluent is concentrated to 70 ° C, the relative density is 1.02 to 1.04, and concentrated to dryness to obtain western red. Flower extract.
  • the present invention also provides a traditional Chinese medicine preparation for preventing or treating cardiovascular and cerebrovascular diseases or dementia, which preparation is prepared from the above traditional Chinese medicine composition, and at least one pharmaceutically acceptable auxiliary material.
  • the formulation may be in a solid dosage form or a semi-solid dosage form, a liquid dosage form or a gaseous dosage form.
  • the solid or semisolid dosage form is selected from the group consisting of a tablet, a pill, an ointment, a dandruff, a powder, a granule, a suppository, a powder, an emulsion, a chewable, and a capsule.
  • the liquid preparation is selected from any one of an oral liquid, a suspension, a syrup, an injection, a medicinal liquor, and an elixir.
  • the gas formulation is an aerosol or inhaler.
  • the present invention also provides the use of the above traditional Chinese medicine composition or preparation for the preparation of a medicament for preventing or treating cardiovascular or cerebrovascular diseases or dementia.
  • the cardiovascular and cerebrovascular diseases are at least one selected from the group consisting of ischemic cerebrovascular diseases, coronary heart disease, and angina pectoris.
  • the dementia is selected from the group consisting of senile dementia, especially vascular dementia.
  • the traditional Chinese medicine composition of the present invention has the following technical effects:
  • the traditional Chinese medicine composition and preparation of the present invention have a better therapeutic effect in preventing or treating cardiovascular and cerebrovascular diseases or dementia.
  • the traditional Chinese medicine composition of the present invention is used in a lower amount in medicine, which is advantageous for cost saving and energy saving.
  • the traditional Chinese medicine composition of the invention has small toxic and side effects, good safety performance, can be taken for a long time, and has good medicinal prospects.
  • the invention is further illustrated by the following specific examples, comparative examples and related test examples, which are intended to illustrate the invention and not to limit the scope of the invention.
  • the experimental methods in the following examples and test examples which do not specify the specific experimental conditions are carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
  • the ginseng extract, the ginkgo leaf extract and the saffron extract are respectively extracted, and the three extracts are mixed, and further prepared into granules and capsules according to a conventional method. Agent, injection.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the ginseng extract and ginkgo biloba extract were extracted and prepared according to the methods described in the Chinese Pharmacopoeia (2010 Edition), pp. 367-368 and 392-393, according to the Chinese Modern Applied Pharmacy (August, 2011, Vol. 28).
  • the method described in the eighth paragraph 729-731) extracts the saffron extract, mixes the three extracts, and further prepares granules, capsules, and injections according to a conventional method.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • the extract mixture and formulation were prepared in accordance with the method described in Example 1 of the present invention.
  • composition of the composition and the preparation method described in Example 1 of Patent WO2007/118363 were prepared as an extract mixture and a corresponding capsule.
  • the extract mixture and capsules were prepared according to the preparation method of Example 1 of the patent WO2007/118363 according to the composition of the composition described in Example 2 of the patent WO2007/118363.
  • Test Example 1 Protective effect on experimental rat ischemic myocardium
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition are prepared into three kinds of extract mixtures), and the test is prepared by using distilled water to form 0.15 g/mL (according to the combination of traditional Chinese medicines) The original dose of the substance is calculated).
  • Reagents urethane, isoproterenol hydrochloride, sodium chloride injection, CK, CK-MB kit.
  • Instrument physiological recorder BL-420 physiological signal acquisition system, centrifuge, semi-automatic biochemical analyzer.
  • the rats in each group were first examined by electrocardiogram, and the ST segment, abnormal T wave and arrhythmia were discarded.
  • the rats were randomly divided into blank control and model group, 10 rats in each group were given daily.
  • the blank control group was administered with physiological saline, and the traditional Chinese medicine composition of the present invention (Example 1, Example 2, Example 5, Example 6, Example 9, Example 11 was administered at a dose of 3 g/kg/d, and the dose used was According to the original dose of ginseng, ginkgo biloba, and saffron), the comparison group (in which, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, and comparative example 6 were 3 g/kg/ d dose, the dosage was calculated according to the original dose of ginseng, ginkgo biloba and saffron; the proportion of 7 groups and the proportion of 8 were administered at a dose of 3g/kg/d, and the dosage used was ginseng, ginkgo biloba, and west.
  • One of the following conditions is judged to be myocardial ischemia positive: 1) ST segment level is downward or upward ⁇ 0.1 mv; 2) T wave is higher than 1/2 of the same lead R wave; 3) T wave is high ST segment shift. Negative judgment criteria: 1) S-T segment oblique offset, or horizontal offset ⁇ 0.1mv; 2) T wave low level or bidirectional inverted.
  • the ST segment elevation of the electrocardiogram of the model group was ⁇ 0.1mv as a marker for successful modeling.
  • the experimental data were expressed as mean ⁇ standard deviation and statistically processed by t test.
  • the traditional Chinese medicine composition of the present invention has a very significant difference; compared with the model group, the creatine kinase CK and the creatine kinase isoenzyme CK-MB can be significantly reduced after 6 hours of modeling; compared with the comparative group.
  • the composition of the invention is more effective. It is indicated that the traditional Chinese medicine composition of the invention has better protective effect on acute myocardial ischemia injury induced by isoproterenol. See Table 2.
  • Myocardial ischemia refers to a decrease in blood perfusion of the heart, resulting in a decrease in oxygen supply to the heart, abnormal myocardial energy metabolism, and a pathological condition that does not support the normal functioning of the heart.
  • Coronary heart disease is the most common and most common cause of myocardial ischemia.
  • the traditional Chinese medicine composition of the present invention has a significant decrease in the displacement of the J point at different time points in the acute myocardial ischemia induced by isoproterenol (P ⁇ 0.05-0.01), and the serum CK and CK.
  • -MB has a significant reducing effect, indicating that the traditional Chinese medicine composition of the present invention has a significant protective effect on myocardial ischemia, and the effect thereof is better than that of the comparative Chinese medicine composition.
  • Kunming mice weighing 18-22 g, male and female, were provided by the Viton Lihua Animal Experimental Center.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.2 g/mL with distilled water (by composition) The original dose is calculated).
  • TC Total cholesterol
  • TG Triglyceride
  • Kunming mice were randomly divided into normal control group (16), high fat model group (16), composition group of the present invention (10 in each group), and comparison group (10 in each group). Each group is male and female.
  • the normal control group was fed with normal feed, and the other groups were fed with high fat diet (formulation: 77.5% basic feed, 2% cholesterol, 10% lard, 10% egg yolk powder, 0.5% sodium deoxycholate).
  • Mice were weighed once a week for 5 weeks. Five weeks later, three males and three females in the normal control group and the high-fat model group were randomly selected, and blood was taken from the eyeballs to measure serum TC and TG.
  • mice can be used for formal experiments. Subsequently, each group of mice was intragastrically administered in the same dosage as described in Table 4 (wherein the Chinese medicine composition of the present invention (Example 1, Example 2, Example 5, Example 6, Example 9, Example 11) , according to the dose of 4g / kg / d, the dosage used is calculated according to the original dose of ginseng, ginkgo biloba, saffron), the comparative group (wherein, comparative example 1, comparative example 2, comparative example 3, comparative example 4 Comparative Example 5 and Comparative Example 6 were administered at a dose of 4 g/kg/d.
  • Table 4 wherein, comparative example 1, comparative example 2, comparative example 3, comparative example 4 Comparative Example 5 and Comparative Example 6 were administered at a dose of 4 g/kg/d.
  • the dosages were calculated according to the original dose of ginseng, ginkgo biloba and saffron; the comparison group 7 and the comparative 8 were dosed at 3 g/kg/d. For administration, the dosage is calculated based on the original dose of ginseng, ginkgo biloba, saffron, and soybean)).
  • Each group of drugs was made up of a suspension of 1% sodium carboxymethylcellulose. After 3 weeks of administration, the animals in each group were fasted for 12 hours, weighed, blood was taken from the eyeball, blood samples were centrifuged at 3000 rpm for 10 min, serum was separated, and TC and TG were measured according to the instructions of total cholesterol and triglyceride kit. The data obtained were statistically analyzed using SPSS10.0 statistical software. Said.
  • the weight of each group of mice was about 18-20g.
  • the body weight of each group varied. It can be seen that the body weight changes of the high-fat model group, the composition group of the present invention, and the comparative group were significantly increased compared with the normal control group, and after 3 weeks of administration, the body weight of the model group rose to the highest, suggesting high fat.
  • the maintenance of the model group, the traditional Chinese medicine composition group and the comparative Chinese medicine composition group of the present invention can significantly reduce the body weight of the high fat model group animals (P ⁇ 0.05).
  • the Chinese medicine composition group of the present invention has the best effect of reducing body weight. See Table 3.
  • Hyperlipidemia refers to elevated levels of cholesterol or triglycerides in the plasma. Hyperlipidemia is mainly associated with cholesterol, and elevated cholesterol is one of the most important risk factors for increased mortality of cardiovascular and cerebrovascular diseases such as coronary heart disease. Abnormal blood lipids, especially elevated cholesterol levels, are prone to "blood thickening", depositing on the walls of blood vessels, gradually forming small plaques (which we often call “atherosclerosis”), these "plaques” increase, increase Large, gradually block the blood vessels, slowing the blood flow, and the blood flow is interrupted in severe cases; if this happens in the heart, it will cause coronary heart disease; in the brain, there will be cardiovascular and cerebrovascular diseases such as stroke.
  • the traditional Chinese medicine composition of the invention reduces the serum TC and TG of the hyperlipidemic mice, and has significant difference (**P ⁇ 0.01) compared with the model group, suggesting that the traditional Chinese medicine composition of the invention is superior to the treatment and prevention of cardiovascular and cerebrovascular diseases. Comparative Chinese medicine composition.
  • Kunming mice weighing 18-22 g, male, were provided by the Viton Lihua Animal Experimental Center.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.2 g/mL with distilled water (by composition) The original dose is calculated).
  • Reagents alloxan, insulin radioimmunoassay kit.
  • mice After 1 week of adaptive feeding in male Kunming mice, 10 rats were randomly selected as normal controls. The other mice were fasted (without water) for 24 hours, and then alloxan was freshly formulated into 2% solution with physiological saline to 200 mg/kg. The dose was intraperitoneal injection. After 72 hours, blood was taken from the tail to measure blood glucose. The mice with blood glucose above 11.1 mmol/L were used as diabetic model mice and included in the experiment.
  • Diabetes model mice were randomly divided into groups of 10 each.
  • the blood glucose levels of each group were similar, among which 1 group was the model control group, 6 groups were the composition group of the invention, and 8 groups were the comparison group.
  • Each group of mice was intragastrically administered with the corresponding drug (the dose was the same as in Test Example 2), and the normal control group and the model control group were given corresponding amounts of physiological saline, and the body weight was adjusted regularly to adjust the administration amount for 21 consecutive days.
  • mice were administered glucose at a dose of 2.5 g/kg and blood glucose was measured at 0 min, 30 min, 60 min, and 120 min. The mice were bled on the 22nd day, and the serum was separated by centrifugation at 3000 rpm for 10 min at 4 ° C, and serum insulin levels were measured according to the kit instructions.
  • the oral glucose tolerance test showed that the traditional Chinese medicine composition of the present invention can significantly improve the glucose tolerance of the experimental mice (P ⁇ 0.01), and the comparison group can significantly improve the glucose tolerance of the experimental mice (P ⁇ 0.05). .
  • the results suggest that the hypoglycemic effect of the traditional Chinese medicine composition of the present invention is superior to that of the comparative group. See Table 5.
  • the serum insulin level of the model group was significantly decreased, indicating that the function of mouse islet ⁇ cells was destroyed after intraperitoneal injection of alloxan, and the modeling was successful; compared with the model control group, the traditional Chinese medicine combination of the present invention was compared with the model control group.
  • the serum insulin levels in the group and the comparison group were increased to different extents, wherein the Chinese medicine composition group of the present invention had a very significant difference (P ⁇ 0.01), while the comparative groups 1, 2, 4, 7, and 8 had significant differences.
  • Sexual differences (P ⁇ 0.05) the other comparison groups of serum insulin increased but not statistically significant, suggesting that the composition of the present invention is superior to the comparative group in improving serum insulin in alloxan-induced diabetic mice.
  • the results are shown in Table 6.
  • cardiovascular and cerebrovascular diseases With the improvement of people's living standards and environmental factors, more and more out of the cardiovascular and cerebrovascular patients Abnormalities in glucose metabolism, patients with cardiovascular and cerebrovascular diseases often have poor prognosis if they are accompanied by abnormal glucose metabolism. Diabetic patients with myocardial infarction have a risk of recurrent MI in the future of more than 40%. In addition, abnormal blood glucose can also cause endothelial dysfunction, decreased aortic elasticity, left ventricular hypertrophy, carotid atherosclerotic plaque, microalbuminuria and other pathological processes, eventually leading to atherosclerosis.
  • the traditional Chinese medicine composition of the invention can significantly reduce the blood glucose of the mouse model of alloxan hyperglycemia and improve the serum insulin of the alloxan-induced diabetic mice, indicating that the traditional Chinese medicine composition of the invention has a good preventive and therapeutic effect on cardiovascular and cerebrovascular diseases.
  • SD rats weighing 180.0-220.0 g, were used by both male and female. They were provided by the Viton Lihua Animal Experimental Center and were housed in the animal room (control temperature) for one week before the animal experiment.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, prepared according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.15 g/mL with distilled water (by composition) The original dose is calculated).
  • Aspirin white tablets, 50mg/tablet, supplied by Shanghai Branch of China Pharmaceutical Corporation.
  • Sodium pentobarbital white powder, supplied by Shanghai Chemical Reagent Co., Ltd. of China Pharmaceutical (Group), formulated with 0.8% aqueous solution in physiological saline, and used.
  • Heparin sodium white powder, 125u/mg, supplied by Shanghai Chemical Reagent Co., Ltd., China Pharmaceutical (Group), and prepared with 0.1% solution of normal saline.
  • Sodium citrate Provided by the First Pharmaceutical Factory of China Pharmaceutical Industry Corporation, the normal saline is formulated into a 3.8% solution for use.
  • ADP Sigma production, pH 7.4 phosphate buffer was prepared into a 200 ⁇ mol / L concentration solution for use.
  • MK4/HC platelet counter manufactured by Baker Instrument, USA
  • Labor aggregometer-153 dual-platelet aggregation instrument manufactured by Labor GmbH, Hanburg, Germany
  • Rats were randomly divided into groups, 15 rats in each group, namely, blank control group and aspirin group (1% CMC-Na solution was ground to prepare 0.88 mg/mL suspension, according to 0.0044 g/kg/d), and the traditional Chinese medicine composition group of the present invention. (6 groups, the same dosage as Test Example 1), and a comparative group (8 groups, the same dosage as Test Example 1).
  • the blank control group was given the same volume of normal saline. It was administered once a day for 7 consecutive days.
  • the middle plastic tube 4 in the middle plastic tube, one end of which is exposed to the plastic tube 0.5cm to fix the position, and the length of the thread in the tube is 6.5cm, and the heparin sodium solution is filled with polyethylene.
  • one end of the plastic tube was inserted into the left external jugular vein, and the other end of the plastic tube was inserted into the right common carotid artery, and the blood flow was immediately opened. After the blood flow was opened for 20 minutes, the blood flow was immediately interrupted and the middle wire was quickly taken out, and the weight of the wire was weighed, which is the total weight. The total weight minus the weight of the wire is the wet weight of the thrombus. Calculate the thrombus inhibition rate according to the following formula:
  • Inhibition rate (control group thrombus wet weight - experimental group thrombus wet weight) / control group thrombus wet weight ⁇ 100%. The wet weight of thrombus in each group was statistically compared.
  • Platelet aggregation inhibition test After 1 hour of administration on the 7th day of each group of rats, the rats were anesthetized by the above method, the common carotid artery was intubated, and the blood was anticoagulated with 3.8% sodium citrate at a ratio of 1:9. The heart was transferred/separated for 5 minutes, and the upper plasma was platelet-rich plasma (PRP). The remaining part was again centrifuged at 3500 rpm for 15 minutes. The supernatant was platelet-poor plasma (PPP), and the platelet counter was used to count PRP platelets. PPP was used to adjust the platelet count of PRP to 3 ⁇ 105/mm2 about.
  • PRP platelet-rich plasma
  • Aggregation inhibition rate (%) (blank control group aggregation rate - experimental group aggregation rate) / blank control group aggregation rate ⁇ 100%.
  • each of the drug-administered groups had a significant inhibitory effect on thrombus formation, and the thrombus wet weight of the traditional Chinese medicine composition group, the comparative example 2, and the Chinese medicine composition group of the present invention was very significant compared with the control group.
  • Sexual differences P ⁇ 0.01
  • there were significant differences in the wet weight of thrombus in the Chinese medicine composition group of Comparative Examples 1, 3, 4, 5, 6, and 8 P ⁇ 0.05
  • the effect of thrombosis was better than that of the proportional group.
  • Table 7 The results are shown in Table 7.
  • each of the administration groups had a significant inhibitory effect on ADP-induced rat platelet aggregation compared with the blank control group.
  • the inhibition rate of platelet aggregation (**P ⁇ 0.01 compared with the control group) of the traditional Chinese medicine composition group of the present invention exceeds the comparative group (*P ⁇ 0.05 compared with the control), suggesting that the traditional Chinese medicine composition of the present invention is anti-platelet.
  • the effect of aggregation is better than the comparison group.
  • Thrombosis is the accumulation of some components in the blood, forming a mass, affecting the flow of blood.
  • the main cause of thrombosis is the change of blood components, the damage of vascular endothelium and the change of blood flow velocity.
  • Thrombosis can block the coronary arteries, causing a sharp decrease or interruption of blood flow, causing severe and long-lasting acute ischemia in the corresponding myocardium, eventually leading to ischemic necrosis of the myocardium; in addition, a certain part of the cerebral blood vessels spontaneously forms a thrombus, resulting in The blood vessels in the brain are blocked and the blood flow is not smooth, forming a "cerebral thrombosis".
  • the traditional Chinese medicine composition of the invention has better effect of reducing thrombus size, inhibiting thrombus formation and lowering platelet aggregation rate than the traditional Chinese medicine composition, suggesting that the traditional Chinese medicine composition of the invention has better treatment and prevention of cardiovascular and cerebrovascular diseases. The role.
  • ICR mice 18-22 g, half male and half female. Provided by Beijing Weitong Lihua Experimental Animal Technology Development Co., Ltd.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition are prepared into a corresponding extract mixture by using distilled water to prepare 0.2 g/mL (according to the composition) The original dose is calculated).
  • Haberin (Shishanjia Tablet), 50 ⁇ g/table, produced by Henan Zhulin Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, using distilled water to prepare 4 ⁇ g/mL; Danakan tablets (tanakan), Ginkgo biloba standard extraction (Egb761) 40mg / tablet, produced by France Beaufort-Epson Pharmaceutical Industry Co., Ltd., prepared with distilled water to prepare 1.5mg / mL; scopolamine hydrobromide, 0.6mg / mL, produced by Shanghai Hefeng Pharmaceutical Co., Ltd.
  • TT-2 mouse platform automatic control instrument produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences.
  • Animals were male and female, randomly divided into: blank control group, model group, Haberin 0.08 mg/kg group, Danacon 30 mg/kg group, and Chinese medicine composition group (6 groups, the same dosage as test case 2) , Comparative group (8 groups, same as the dose used in Test Example 2).
  • mice were intragastrically administered daily at a dose of 20 mL/kg, and the blank control group and the model group were given distilled water once daily for 15 consecutive days.
  • the control group mice were intraperitoneally injected with physiological saline at a volume of 10 mL/kg, and the other groups were injected with scopolamine (6 mg/kg).
  • the mice were placed in the automatic control system of the platform for 3 minutes, then energized, and the mice were subjected to electric shock stimulation for 5 times. When the mice were shocked, they jumped onto the platform, escaped the electric shock, and trained for 5 minutes to obtain memory.
  • mice Sixty minutes after the administration on the 15th day, each group of animals was placed in a platform automatic control instrument to measure the number of times (the number of errors) and the occurrence time (latency period) of the mice subjected to electric shock from the jumping platform. Results were statistically processed (t-test). See Table 9.
  • ICR mice 25-32 g, half male and half female. Provided by Beijing Weitong Lihua Experimental Animal Technology Development Co., Ltd.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.2 g/mL with distilled water (by composition) The original dose is calculated).
  • Haberin (Shishanjia Tablet), 50 ⁇ g/table, produced by Henan Zhulin Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, using distilled water to prepare 4 ⁇ g/mL; Danakan tablets (tanakan), Ginkgo biloba standard extraction (Egb761) 40mg / tablet, France Bofu - Yipusheng Pharmaceutical Industry Co., Ltd., using distilled water to prepare 1.5mg / mL; reserpine injection, 1mg / mL, Shanghai Medical University Hongqi Pharmaceutical Factory, test time The solution was made up to 0.05 mg/mL with physiological saline.
  • TT-2 mouse platform automatic control instrument produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences
  • test group was the same as Test Example 5.
  • mice in the blank control group were given subcutaneous injection of normal saline in the volume of 10 mL/kg, and the other groups were subcutaneously injected with reserpine hydrochloride (0.5 mg/kg).
  • the mice were placed in the automatic control system of the platform for 3 min, then energized, and the mice were subjected to electric shock stimulation for 5 times. When the mice were shocked, they jumped onto the platform, escaped the electric shock, and trained for 5 minutes to obtain memory.
  • Sixty minutes after the 15th day of administration each group of animals was placed in a platform automatic control instrument to measure the number of errors and latency of the mice within 5 minutes. Results were statistically processed (t-test).
  • the number of errors in the blank control group was significantly reduced within 5 min (P ⁇ 0.05), and the latency was significantly prolonged (P ⁇ 0.05), indicating successful modeling.
  • the number of errors in the mice of the traditional Chinese medicine composition group was significantly reduced within 5 min (P ⁇ 0.05), and the latency was significantly prolonged (P ⁇ 0.05).
  • the number of errors in the control drug Haberin group and Danakang group was significantly reduced within 5 min (P ⁇ 0.05), and the latency was prolonged, but it was not statistically significant.
  • the number of errors in the Chinese medicine composition group of the first to fourth and seventh groups was significantly reduced within 5 min (P ⁇ 0.05), and the incubation period was prolonged, but it was not statistically significant. In the other comparison groups, the number of errors within 5 min decreased, and the incubation period prolonged, but it was not statistically significant. See Table 10.
  • the traditional Chinese medicine composition of the present invention has an effect of improving the acquired memory disorder of the animal, suggesting that it may be related to the mechanism of action of the traditional Chinese medicine composition of the present invention to increase the content of catechol amino neurotransmitters, and the effect is better than that of the comparative group.
  • Test Example 7 Effect of sodium nitrite on mice with acquired acquired memory impairment in mice
  • ICR mice 18-22 g, half male and half female. Provided by Beijing Weitong Lihua Experimental Animal Technology Development Co., Ltd.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.2 g/mL with distilled water (by composition) The original dose is calculated).
  • Haberin (Shishanjia Tablet), 50 ⁇ g/table, produced by Henan Zhulin Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, using distilled water to prepare 4 ⁇ g/mL; Danakan tablets (tanakan), Ginkgo biloba standard extraction (Egb761) 40mg / tablet, produced by France Beaufort-Epson Pharmaceutical Industry Co., Ltd., prepared with distilled water to prepare 1.5mg / mL; sodium nitrite, Beijing Yili Fine Chemicals Co., Ltd. production. At the time of the test, 12 mg/mL was prepared with physiological saline.
  • TT-2 mouse platform automatic control instrument produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences
  • test group was the same as Test Example 5.
  • mice were intragastrically administered daily at a dose of 20 mL/kg, and the blank control group and the model group were given distilled water once daily for 15 consecutive days.
  • the mice were placed in a platform automatic control instrument for 5 min, and the mice were subjected to electric shock stimulation for 5 times.
  • the mice in the control group were injected subcutaneously with normal saline in a volume of 10 mL/kg, and the other groups were injected with sodium nitrite (120 mg/kg).
  • ty minutes after the 15th day of administration each group of animals was placed in a platform automatic control instrument to measure the number of errors and latency of the mice within 5 minutes. Results were statistically processed (t-test).
  • the number of errors in the blank control group was significantly reduced within 5 min (P ⁇ 0.05), and the latency was significantly prolonged (P ⁇ 0.01), indicating successful modeling.
  • the number of errors in the Chinese herbal medicine composition group, the control drug Haberin and the Danakang group were significantly decreased within 5 min (P ⁇ 0.05), and the latency was significantly prolonged (P ⁇ 0.01-0.05).
  • the number of errors in the Chinese medicine composition group was significantly decreased within 5 min (P ⁇ 0.05), and the latency was significantly prolonged (P ⁇ 0.05).
  • the error number of the Chinese medicine composition group in the comparison group 2 was significantly reduced within 5 min (P ⁇ 0.05).
  • the incubation period has an extended trend The significance of accounting.
  • the number of errors in other comparison groups decreased, and the incubation period prolonged, which was not statistically significant.
  • the results are shown in Table 11.
  • composition of the present invention has an effect of improving the consolidation memory disorder of the animal, suggesting that the traditional Chinese medicine composition of the present invention may improve learning and memory by improving brain circulation and improving ischemia and hypoxia of the brain tissue, and the effect is better than that of the comparative group.
  • Test Example 8 Effect of VaD on permanent bilateral ligation of bilateral common carotid arteries in rats
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition are prepared into a corresponding extract mixture, and the test is formulated into 0.3 g/mL with distilled water (by composition) The original dose is calculated).
  • Haberin (Shishan A), 50 ⁇ g/table, produced by Henan Zhonglin Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, using distilled water to prepare 6 ⁇ g/mL; Danakan tablets (tanakan), Ginkgo biloba standard extraction (Egb761) 40 mg/tablet, produced by Beaufort-Epson Pharmaceutical Industries, France, and formulated into 2 mg/mL with distilled water.
  • High performance liquid phase acetylcholine chloride (AchCl), disodium hydrogen phosphate (Na 2 HPO 4 ), tetramethylammonium chloride (TMACl), sodium octyl sulfonate (OSA), sodium thiosulfate (Na 2 S 2 O 5 ), ethylenediaminetetraacetic acid tetrasodium salt (EDTA), all HPLC grade, produced by Sigma, USA; phosphoric acid (H 3 PO 4 , 85%), perchloric acid (HClO 4 ) are HPLC Grade, produced by Fisher Scientific, USA; MB reagent, produced by ESA, USA.
  • Biochemical assay acetylcholinesterase (TCHE) kit; superoxide dismutase (SOD) kit, malondialdehyde (MDA) kit; protein quantification (biuret method) kit; all built by Nanjing Bioengineering Provided by the Institute; neuropeptide Y (NPY) kit, provided by the PLA General Hospital of Science and Technology Development Center; beta-endorphin ( ⁇ -EP) kit, provided by the Naval Radiology Technology Center.
  • TCHE acetylcholinesterase
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • protein quantification protein quantification
  • MORRIS water maze produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences. FT-630G ⁇ -counter, produced by Beijing Nuclear Equipment Factory. Spectrophotometer, UV-120-02, produced by Shimadzu Corporation, Japan.
  • 16-channel Coularray Coulomb array electrochemical high performance liquid chromatography and chromatography workstation 582 pump, 5600A electrochemical detector, 542 autosampler, 5040 solid microporous electrode (platinum electrode, solid palladium electrode), CH150 column temperature Box, pre-column immobilized enzyme reactor, post-column immobilized enzyme reactor, all products of American ESA company; column (C18150 ⁇ 3mm ID) American ESA company product; ultracentrifuge 55P-72, Japan HITACHI company product; low temperature Refrigerator, French JOUAN products; syringe microporous membrane filter: water system (0.22 ⁇ m), Tianjin Tengda filter plant products.
  • Mobile phase Na 2 HPO 4 (100 mmol / L), TMACl (0.5 mmol / L), OSA (2.0 mmo / L), MB reagent (0.005% v / v), re-distilled deionized water to volume, 85% H 3 PO4 adjusted pH value of 8.00, 0.22 ⁇ m water system membrane filtration.
  • ESA582 binary pump system ESA ACH-3 column (150 ⁇ 3mm 5 ⁇ m ID), ESA pre-column immobilized enzyme reactor (ESA ACH-SPR, 3cm), 0.35mL / min flow rate, 35 ° C column temperature.
  • ESA ACH-SPR ESA pre-column immobilized enzyme reactor
  • Detection conditions 5600A electrochemical detector; 5040 solid microporous electrode (platinum working electrode, solid palladium reference electrode) potential: +300 mV.
  • Rats were chloral hydrate anesthesia (350 mg/kg), a midline incision was made in the neck, bilateral common carotid arteries were isolated and ligated (surgical group was only ligated without ligation of the common carotid artery), sutured and returned to the cage, and penicillin was infected for 4 days.
  • the MORRIS water maze swimming test was conducted, and the rats with the tendency of learning and memory disorder were selected by the duration as the index, randomly grouped: model group; Haberin 60 ⁇ g/kg group; Danakang 20 mg/kg group; the present invention
  • the Chinese medicine composition group and the comparative Chinese medicine composition group (the dose is the same as in Test Example 1).
  • the drug was administered once daily (10 mL/kg) for 2 months.
  • the sham operation group and the model group were given an equal volume of distilled water.
  • the duration of the MORRIS water maze was measured 2 months and 3 months after the modeling (1 month and 2 months after administration).
  • the abdominal aorta was bled, and the brain was quickly taken, and the left hemisphere liquid was solidified by nitrogen.
  • 0.15 M HCLO 4 homogenate 100 mg brain weight plus 1 ml was centrifuged at 14000 ⁇ g for 4 min at 4° C., the supernatant was taken, filtered through a 0.22 ⁇ m filter, and 10 ⁇ l was injected into the autosampler to determine the ACh content by HPLC; Four groups of formalin were fixed in the right hemisphere.
  • Dehydration, embedding, sectioning and HE staining were performed for pathological examination.
  • Six brain tissues were homogenized to determine AchE, SOD activity and MDA content (colorimetric method).
  • Plasma measures neuropeptide Y, ⁇ -EP and other indicators (radiopheresis). Results were statistically processed (t-test).
  • Table 15 shows the effect of plasma NPY and ⁇ -EP on VaD rats
  • Comparative Example 4 the pyramidal cells in the hippocampus of the Chinese medicine composition group were loosely arranged, the cells were small, some were deeply stained, blurred, and the boundaries were unclear, and the individual cells shrunk and were small triangles or clumps.
  • the pyramidal cells in the hippocampus of the Chinese medicine composition group were well arranged, the levels were visible, the boundary between the pyramidal cells was unclear, the nuclear membrane was diminished, the nucleolus was small, and it was denatured and partially disappeared.
  • Chronic cerebral ischemia is a common pathological process in the development of various diseases such as vascular dementia (VaD), Alzheimer's disease (AD) and subcortical arteriosclerotic encephalopathy (Binswanger disease).
  • VaD vascular dementia
  • AD Alzheimer's disease
  • Binswanger disease subcortical arteriosclerotic encephalopathy
  • Cerebral ischemia and hypoxia may cause mitochondrial oxidative metabolism dysfunctional, leading to excessive generation of reactive oxygen species, including O -2, HO -2, OH - , H 2 O 2 and the like.
  • reactive oxygen species can react with proteins, lipids, nucleic acid molecules, etc. in neurons and destroy their molecular structure, causing superoxide changes, which can further cause damage to brain cell structure and function, and generate excessive peroxidation products such as C.
  • Dialdehyde malondialdehyde, MDA
  • Neurons in the hippocampus, prefrontal cortex, temporal lobe, and cerebral cortex are very sensitive to ischemia, and these regions are brain regions closely related to learning and memory. Damage to neuronal function and structure in these brain regions after cerebral ischemia can cause neurotransmitter disorders associated with learning and memory, causing a decline in learning and memory, and even dementia.
  • the 2VO-induced VaD model in this experiment confirmed the above biochemical, pathological and behavioral changes, and observed that the traditional Chinese medicine composition group can significantly inhibit AchE activity, increase the ACh content in the brain of model animals, and improve learning and memory disorders; It can also significantly increase SOD activity in the brain and scavenge oxygen free radicals to protect brain tissue. It is shown that the traditional Chinese medicine composition group of the present invention has a significant therapeutic effect on VaD, and the effect is better than that of the comparative Chinese medicine composition.
  • the content of neuropeptide Y in plasma of patients with VaD decreased, and the content of ⁇ -EP increased.
  • the composition group can significantly increase plasma neuropeptide Y content and reduce plasma ⁇ -EP content.
  • Test Example 9 Effect of ischemia-reperfusion combined with subcutaneous injection of D-galactose on learning and memory impairment in mice
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.2 g/mL with distilled water (by composition) The original dose is calculated).
  • Haberin (Shishanjia Tablet), 50 ⁇ g/table, produced by Henan Zhulin Zhongsheng Pharmaceutical Co., Ltd. Yuzhong Pharmaceutical Factory, using distilled water to prepare 4 ⁇ g/mL; Danakan tablets (tanakan), Ginkgo biloba standard extraction (Egb761) 40mg/ The film was produced by Beaufort-Epson Pharmaceutical Industries, France, and was formulated into 1.5 mg/mL with distilled water.
  • D-galactose produced by Shanghai Hengxin Chemical Reagent Co., Ltd.; acetylcholinesterase (TCHE) kit; superoxide dismutase (SOD) kit; malondialdehyde (MDA) kit; glutathione peroxide (GSH) -Px) kit; nitric oxide (NO) kit; protein quantification (biuret method) kit; all provided by Nanjing Jiancheng Bioengineering Center.
  • TCHE acetylcholinesterase
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • GSH glutathione peroxide
  • NO nitric oxide
  • protein quantification protein quantification
  • MORRIS water maze produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences.
  • Spectrophotometer UV-120-02 produced by Shimadzu Corporation, Japan.
  • mice were randomly divided into two groups: sham operation group, composite model group, D-galactose group, ischemia reperfusion group, Haberin group, Danacon group, and Chinese medicine composition group (6 groups, with The doses used in Test Example 2 were the same), and the comparative group (8 groups, which was the same as the dose used in Test Example 2).
  • Anaesthesia with chloral hydrate, neck incision, ischemia reperfusion surgery separation of bilateral common carotid arteries, arterial clip clamping for 20 minutes, loosening for 20 minutes, then clamping for 20 minutes and then loosening, while cutting the rat tail, bleeding 0.5mL, formed repeated cerebral ischemia and reperfusion injury, the wound was sutured and returned to the cage. 7 days after surgery, D-galactose (100mg/kg, 10mL/kg) was injected subcutaneously in the back, and administered by intragastric administration (20mL/kg) once daily for 8 weeks. In the sham operation group, only the common carotid artery was not clipped.
  • mice After 8 weeks of administration, the mice were trained in the MORRIS water maze for 3 days, twice a day, and the learning and memory behavior was measured on the 4th day. The brain was decapitated on the 5th day, and 10% brain homogenate was prepared in physiological saline to measure the brain tissue. AchE, SOD, GSH-Px activity, MDA, NO content and other biochemical indicators (colorimetric method). Results were statistically processed (t test)
  • the duration of swimming and the length of swimming path in the MORRIS water maze after ischemia-reperfusion combined with subcutaneous D-galactose were significantly different (P ⁇ 0.01).
  • Search strategy Usually marginal or random. There was no significant difference in swimming duration and length of swimming path between the sham-operated group and the model group in the ischemia-reperfusion group. The search strategy was significantly different from the composite model group (P ⁇ 0.05); There was no significant difference in swimming duration, swimming path length and search strategy between the subcutaneous D-galactose group and the composite model group. It indicated that the subcutaneous injection of D-galactose and ischemia-reperfusion injury could form mice.
  • the activity of SOD and GSH-Px in the brain of mice (complex model group) after ischemia-reperfusion combined with subcutaneous injection of D-galactose decreased, and the content of MDA and NO increased, which was significantly different from the sham operation group. P ⁇ 0.05 ⁇ 0.01); the activity of SOD and GSH-Px in the brain tissue of D-galactose group decreased, and the content of MDA and NO increased, which was significantly different from the sham operation group (both P ⁇ 0.05-0.01).
  • the SOD, GSH-Px activity and NO content in the brain tissue of the ischemia-reperfusion group were significantly lower than those in the sham-operated group (P ⁇ 0.05).
  • the MDA content was not significantly different from that in the sham-operated group.
  • the SOD, GSH-Px activity, MDA and NO content of the control drug Haberin group did not change significantly; the activity of SOD and GSH-Px in the Danakang group was significantly enhanced, and the contents of MDA and NO were significantly decreased ( P ⁇ 0.05 ⁇ 0.01); the activity of SOD and GSH-Px in the traditional Chinese medicine composition group was significantly enhanced, and the contents of MDA and NO were significantly decreased (P ⁇ 0.05-0.01); the mice in the Chinese medicine composition group of Comparative Examples 1 ⁇ 3 and 7 were compared.
  • the SOD activity of the tissue was significantly enhanced, the NO content was significantly decreased (P ⁇ 0.05), and the GSH-Px activity and MDA content were not significantly changed.
  • the NO content of the 4 ⁇ 6 and 8 groups was significantly lower (P ⁇ 0.05), SOD, There was no significant change in GSH-Px activity and MDA content. The results are shown in Table 17.
  • the AchE activity in the brain of mice (complex model group) after ischemia-reperfusion combined with subcutaneous injection of D-galactose was significantly increased, which was significantly different from the sham operation group (P ⁇ 0.01); D-half was injected subcutaneously.
  • the activity of AchE in the brain tissue of the lactose group and the simple ischemia-reperfusion group was significantly higher than that of the sham operation group (P ⁇ 0.05), and there was no significant difference compared with the composite model group.
  • the AchE activity of the control drug Haberin group was significantly decreased (P ⁇ 0.05); the AchE activity of the Danacon group was not significantly changed; the traditional Chinese medicine composition group, the comparative example 1 ⁇ 3, 7 Chinese medicine composition The AchE activity of the group was significantly decreased (P ⁇ 0.05); the AchE activity of the Chinese medicine composition group of Comparative Examples 4-6, 8 did not change significantly.
  • the results are shown in Table 18.
  • MDA oxygen free radicals
  • NO is an extremely important messenger in the body and plays a dual role in the process of cerebral ischemia. On the one hand, it can reduce the infarct area, increase the blood supply to the cortex, and on the other hand, it can synergize with the oxygen free radicals produced by ischemia, causing nerve cell damage.
  • Monitoring the levels of SOD, GSH-Px, MDA and NO in brain tissue is of great significance for the study of brain ischemia-reperfusion injury.
  • the D-galactose subacute aging model was established by continuously injecting large doses of D-galactose into the mice, causing disorder of glucose metabolism in the animals.
  • D-galactose can produce oxygen radicals such as superoxide anions under the catalysis of galactose oxidase.
  • Oxygen free radicals are highly active and can attack unsaturated fatty acids, proteins, enzymes and DNA in the nucleus of membrane phospholipids; free radicals can also cause lipid peroxidation to form lipid peroxide (LPO), LPO MDA is formed under acidic conditions after precipitation with protein.
  • LPO lipid peroxide
  • MDA is an active cross-linking agent, which can rapidly form fluorochrome with phosphatidylethanolamine and then combine with proteins, peptides and lipids to form lamellar lipofuscin (LF).
  • Continuous injection with D-galactose can produce a variety of biochemical changes, such as increased MDA and LF levels in brain tissue, SOD in the brain, GSH-Px in the blood, and decreased CAT activity in red blood cells.
  • the changes in natural aging are consistent, indicating that D-galactose can cause aging of cells and organisms.
  • the activities of SOD, GSH-Px and CAT indirectly reflect the body's ability to scavenge oxygen free radicals; while the levels of MDA and LF indirectly reflect the severity of the cells being attacked by free radicals.
  • mice cerebral ischemia-reperfusion injury combined with subcutaneous injection of D-galactose resulted in a significant increase in AchE activity after brain injury, resulting in cognitive impairment and a significant decrease in spatial learning ability.
  • the activities of SOD and GSH-Px decreased significantly, and the contents of MDA and NO increased significantly.
  • the activity of AchE in the brain tissue of the mice decreased, the activities of SOD and GSH-Px were significantly enhanced, the contents of MDA and NO were significantly decreased, and the spatial learning ability was significantly enhanced, indicating that the traditional Chinese medicine composition was resistant to ischemia.
  • the active oxygen production and lipid peroxidation caused by perfusion have an inhibitory effect, thereby preventing irreversible nerve cell damage, thereby improving learning and memory ability, and the pharmacological effect of the traditional Chinese medicine composition of the present invention is better than that of the comparative Chinese medicine composition.
  • the traditional Chinese medicine composition of the present invention and the comparative Chinese medicine composition (self-made, prepared according to the methods of the examples and the comparative examples of the present invention are prepared into corresponding extract mixtures), and the test is formulated into 0.15 g/mL with distilled water (by composition) The original dose is calculated).
  • Danakan tablets (tanakan), Ginkgo biloba leaf standardized extract (Egb761) 40mg/tablet, produced by Beaufort-Epson Pharmaceutical Industries, France, formulated with distilled water to form 2.0mg/mL.
  • Alginate microsphere vascular embolization agent KMG
  • KMG Alginate microsphere vascular embolization agent
  • SS somatostatin
  • NPY neuropeptide Y
  • CGRP Calcitonin Gene Related Peptide
  • CGRP Calcitonin Gene Related Peptide
  • ET Endothelin
  • OSA octyl sulfonate Sodium
  • OSA is HPLC grade, all of which are products of Sigma, USA
  • Ultracentrifuge HITACHI 55P-72, Japan
  • 16-channel Coularray Coulomb array electrochemical high performance liquid chromatography and chromatography workstation 580 pump, 5600A electrochemical detector, 542 autosampler, US ESA product; column (C1815 ⁇ 4.6mm 5 ⁇ m), the US WATERS product.
  • Low temperature refrigerator French JOUAN product
  • syringe microporous membrane filter water system (0.22 ⁇ m)
  • Tianjin Tengda Filter Factory products MORRIS water maze, produced by the Institute of Materia Medica, Chinese Academy of Medical Sciences.
  • FT-630G ⁇ -counter produced by Beijing Nuclear Equipment Factory.
  • the mobile phase consisted of sodium dihydrogen phosphate 90 mmol/L, citric acid 50 mmol/L, sodium octyl sulfonate 1.7 mmol/L, and ethylenediaminetetraacetic acid tetrasodium (EDTA) 0.05 ⁇ mol/L.
  • the above solution was filtered through a 0.2 um aqueous membrane. Acetonitrile (10%) was added to the filtrate.
  • the flow rate of the mobile phase was 0.6 mL/min, the working electrode was 1:-150 mV, the working electrode was 2:450 mV, the working electrode was 3:500 mV, and the working electrode was 4:550 mV.
  • 0.1 mol/L perchloric acid standard was used as a stock solution (100 ⁇ g/mL) and stored in a -70 ° C refrigerator.
  • the stock solution was diluted to 10 ⁇ g/mL as a diluent.
  • the dilution is formulated into a series of concentrations as a standard solution.
  • 0.15 mol/L perchloric acid (containing 0.04% sodium metabisulfite and 0.04% EDTA) was used as the working fluid.
  • the external carotid artery was ligated, the common carotid artery was clamped by the artery, and 0.1 mL of KMG was injected from the external carotid artery into the internal carotid artery with a syringe.
  • the artery clamp was loosened, the external carotid artery was ligated, the wound was sutured, and the cage was returned.
  • the sham operation group only separated the common carotid artery, internal carotid artery and external carotid artery, and did not inject microspheres. Penicillin sodium for injection was anti-infected for four days. The behavior of the animals was observed on the next day of surgery. The behavioral changes of the former limbs were judged as the model was established (otherwise discarded), and they were divided into model group, Danakang 20 mg/kg group, and the traditional Chinese medicine composition group (6). The group was the same as the dose used in Test Example 1 and the comparative group (8 groups, which were the same as those used in Test Example 1).
  • the intragastric administration was started 10 days after the operation, and the sham operation group and the model group were given an equal volume of distilled water once a day for 90 days. Animals were tested for duration and path length of the MORRIS water maze search platform 40 and 100 days after modeling (30 and 90 days after drug administration). The abdominal aorta was drawn by laparoscopic chloral hydrate for 60 minutes after the last administration. Condensation, separation of plasma, and rapid decapitation of the brain, separation of cortex, hippocampus, striatum, weighing, liquid nitrogen solidification, -70 ° C low temperature refrigerator.
  • the microspheres After the injection of the biological microspheres, the microspheres enter the blood vessels of the brain with the blood flow, and multiple cerebral infarctions of different sizes can be formed in 24 hours, and the parts are mainly cortex, hippocampus and striatum. After 100 days, the liquefaction necrosis in the infarct area of the model group showed a cavity, the brain tissue was obviously atrophied, the hippocampus became smaller, the cortex became thinner, and the weight was significantly reduced. There was a significant difference between the sham operation group and the model group (P ⁇ 0.05-0.01).
  • the weight of the cerebral cortex of the Chinese medicine composition group and the comparative Chinese medicine composition group of the present invention increased, but there was no statistical significance, and the weight of the striatum and hippocampus of the traditional Chinese medicine composition group increased significantly (P ⁇ 0.05); the weight of the cerebral cortex, striatum and hippocampus of the control group of Danacon and the comparative Chinese medicine composition group increased, but there was no statistical significance. See Table 19.
  • the duration of the sham operation model group in the water maze was significantly shortened (P ⁇ 0.05); 30 days after the administration, the duration of the water maze was significantly shortened in the Chinese medicine composition group and the model group (P ⁇ 0.05), no significant changes were observed in other groups; after 90 days of administration, the duration of the water maze was significantly shortened in the Chinese medicine composition group and the model group (P ⁇ 0.05-0.01). The proportions 1 to 3, 7 and the model group were compared. The duration of the water maze was significantly shortened (P ⁇ 0.05), There were no significant changes in its group. See Table 20.
  • the plasma NPY content of the sham-operated group was significantly increased, the ET content was significantly decreased (P ⁇ 0.01), and the SS and CGRP levels were increased but not statistically significant; 100 days after modeling (90 days after drug), compared with the model group, the NPY and SS contents of the traditional Chinese medicine composition group were significantly increased (P ⁇ 0.01), the ET content was significantly decreased (P ⁇ 0.05), and the CGRP content was not significantly changed; The contents of NPY and SS in the Chinese medicine composition group were significantly increased (P ⁇ 0.05-0.01), the ET content was decreased, and the CGRP content was not significantly changed.
  • the brain mechanism of learning and memory mainly includes neurophysiological mechanism and neurochemical mechanism.
  • the neurophysiological mechanism focuses on the localization of memory in the brain and its accompanying bioelectrical activity.
  • the neurobiochemical mechanism mainly describes neurotransmitters, neuropeptides and large organisms. The role of molecules in learning and memory and their relationship.
  • Neuropeptides are endogenous active substances that are found in neural tissues and participate in the function of the nervous system. They are a special kind of information substance. It is characterized by low content, high activity and wide effect It is ubiquitous and complex, and regulates a variety of physiological functions in the body, such as pain, sleep, emotion, learning and memory, and even the differentiation and development of the nervous system itself are regulated by neuropeptides.
  • Somatostatin is a widely distributed neuroactive peptide in the nervous system, in which the cortex and hippocampus of the hypothalamus and brain are the highest, which is related to the regulation of cognitive function.
  • the SS in the brain has a positive regulatory effect on learning and memory.
  • SS regulates the pathways of learning and memory.
  • One is the direct pathway, which causes the increase of calcium influx by exciting the SS nerve, and the second is possible through other neurological properties.
  • SS-energy neurons are closely related to cholinergic neurons and have synaptic connections, which provides evidence for their functional association: SS may promote acetylcholine release and affect learning and memory functions.
  • Endothelin-1 (ET) is a vasoactive peptide that has been deeply studied in recent years.
  • NPY Neuropeptide Y
  • NPY neuropeptide Y2 receptor
  • NPY neuropeptide Y2 receptor
  • LTP long-term potentiation
  • the test used a method of injecting biological microspheres into the internal carotid artery to cause a multi-infarct dementia (MID) model in rats.
  • the results showed that the neurological behavior disorder occurred in the model group after 24 hours, and multiple infarctions were formed.
  • the cerebral tissue liquefaction necrosis occurred in the 10 days of infarction.
  • the cortical thinning occurred in 100 days, the hippocampus was atrophied, and the weight of cortex, striatum and hippocampus was significantly reduced.
  • Memory impairment, cortex and hippocampus 5-HT, 5-HIAA content were significantly reduced, DA content decreased but no significant difference (P ⁇ 0.1), plasma NPY content decreased significantly, ET content increased significantly, SS and CGRP have Reduce the trend.
  • the rats of the traditional Chinese medicine composition group of the present invention were significantly shortened in the MORRIS water maze, and the learning and memory ability was significantly improved;
  • the hippocampus weight increased significantly (P ⁇ 0.05).
  • the contents of 5-HT, 5-HIAA, DA, striatum 5-HT, 5-HIAA and hippocampus NE, DA and 5-HT were significantly increased. High (P ⁇ 0.05 ⁇ 0.01), plasma NPY and SS content increased significantly, and ET content decreased significantly.
  • the traditional Chinese medicine composition of the invention can increase the neurotransmitter content associated with learning and memory in the brain of MID rats, thereby having a therapeutic effect on vascular dementia.
  • the effect of the traditional Chinese medicine composition of the present invention is better than that of the comparative Chinese medicine composition group, and is statistically significant.

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Abstract

一种预防或治疗心脑血管疾病和/或痴呆症的中药组合物及其制备方法和用途，所述组合物由下列重量份的药物原料制成：人参1、银杏叶0.8-1.5、西红花0.018-0.030。

Description

一种预防或治疗心脑血管疾病或痴呆症的中药组合物及其制备方法和用途 技术领域
本发明涉及一种用于预防或治疗心脑血管疾病或痴呆症的中药组合物，并公开了所述中药组合物的制备方法和用途。
背景技术
心脑血管疾病是威胁人类生命和健康的最主要的疾病，具有高发病率、高死亡率、高致残率、高复发率的特点。根据近年来的资料统计，我国每年心脑血管疾病死亡者占死亡病因的半数左右。心脑血管疾病的流行，与社会的发展和生活水平的提高紧密相关，心理压力过大、饮食结构不合理、缺乏运动、过量抽烟饮酒、肥胖等都是心脑血管疾病发病的诱因。心脑血管疾病包括冠心病、心绞痛、心肌梗塞、癖血型肺心病、缺血性脑病、脑血栓、高血压、高血脂等，这些疾病的主要诱发原因是动脉硬化造成管腔狭窄、管道阻塞，从而导致心脑供血不足，引起头沉、头晕、头痛、胸闷等症状，严重者可导致脑中风及心肌梗塞的发生，心脑缺血后影响能量代谢，继发乳酸堆积、钙超载、自由基损伤等多种变化。因此对心脑血管疾病严重危害着人类的健康，对其早期的预防、及时治疗有着极其重要的意义。
痴呆是由于大脑器质性病变引起的一种获得性、持续性智力损害综合征。发生于老年人的痴呆主要分为：A.原发性退行性痴呆，即阿尔茨海默病(Alzheimer’s disease，简称AD)；B.血管性痴呆(Vascular dementia，VaD)；C.混合型痴呆(AD合并VaD)；D.其他痴呆(匹克病、路易包涵体痴呆)。老年痴呆中AD和VaD是两大最主要的类型。
血管性痴呆与一次或反复中风以及高血压、高血脂、糖尿病、抽烟饮酒等有关，表现为记忆力、计算力、定向力及判断力障碍，可有情感障碍及行为异常，晚期丧失生活能力。
研究认为脑梗塞是否引起痴呆主要与梗塞灶的大小、多少及部位有关。调查表明，梗死灶体积大于50mL可以合并痴呆，大于100mL则经常合并痴呆；也有调查发现，VaD患者中大面积梗死占11.2％，小面积梗死灶占88.8％，多发病灶占97.6％，提示病灶体积小，也可发生痴呆，尤其是梗死灶数目越多，痴呆发生率越高。也有研究认为,脑梗死的部位是导致痴呆的关键性因素；多数报道指出，CT上发现有脑室旁白质病变改变者，痴呆发生率显著增高。Pavics研究患者的脑血流量变化，发现血管性痴呆患者平均半球血流量明显降低，梗死区域的灌注量比正常组织明显降低，完全不灌注区很少见；采用认知量表与脑血流对比研究发现，平均半球血流量与痴呆程度呈正相关，提示VaD血流量的降低与智能活动有非常密切的关系。近年来的研究注意到，VaD患者的额叶、颞叶，尤其是丘脑、基底节等部位的脑血流及糖代谢率较其他部位显著下降，揭示VaD可能与皮质、皮质下结构的联系中断，即大脑神经机能联系不全有关；脑血管阻力、血液粘滞性以及神经体液因素都会对脑血流量保持恒定有直接或间接影响。
研究表明，脑缺血损伤是级联反应过程，而在每一个环节会诱导对神经元变性损伤的不同分子网络：脑梗塞发生后，脑血流量中断和再灌注，造成脑组织能量耗竭，神经递质尤其是兴奋性氨基酸的升高又造成血流量进一步下降，并使钙离子内流或从胞内的钙库释放，引发大量的酶引发信号级联反应，某些酶导致氧自由基产生，它本身也作为第二信使， 损害细胞蛋白质、糖、脂肪酸等，进一步造成梗塞周围去极化，使梗塞区向半暗区扩展；自由基和其它信使激活炎性细胞因子和酶，导致小胶质细胞被激活产生炎症反应，使血管通透性增加，神经元骨架破坏，而继发性损伤最终导致痴呆。研究表明，缺血后神经递质表现为先升后降，包括乙酰胆碱、儿茶酚胺、神经肽等神经递质与认知能力下降密切相关。它们共同参与VaD的病理生理过程，并可能成为反映VaD病情严重程度的重要指标。
近年来，中药在治疗心脑血管疾病、老年性痴呆尤其是血管性痴呆方面已经显示出了独特的优越性，中药疗效确切、持久、不良反应少，尤其以远期疗效满意而广泛应用于临床。大量的研究结果和临床实践证明，中药以其耐受性较好、毒副作用小等特点显示出其具有独特的优势，适合患者长期服用。
人参性温，味甘微苦，功能补肾养心，益气增智；银杏叶性平，味苦、涩，功擅活血化瘀。西红花性平，味甘，善活血化瘀，散瘀开结。现代药理研究结果也证实，人参所含多种皂苷对多种试验动物脑缺血再灌注损伤及学习记忆障碍有明显的改善作用，可促进正常动物的学习记忆能力，有增加突触体对递质的摄取，增强神经生长因子的作用；银杏叶提取物可抑制细胞膜脂质过氧化反应，能扩张血管、增加血流量、降低血液粘度、抑制血栓形成和抗血小板聚集、改善脑代谢、保护神经细胞；西红花苷具有抑制胞外钙离子的内流及内质网上钙离子的释放，抗氧化，抗高血压、动脉粥样硬化、脑水肿等作用，还能提高哺乳动物血流氧分压，近年来还发现其有抗乙醇所致记忆和学习障碍的作用。
发明专利ZL02131435.7公开了一种含有治疗有效量的人参、银杏叶和西红花的中药组合物，用于治疗缺血性脑血管疾病和血管性痴呆、老年性痴呆疾病等。申请人以该专利为基础研制了“塞络通胶囊”(其最初商品名是“维脑康”)，目前正处于临床试验阶段。
发明专利WO2007118363A1公开了一种类似的用于治疗缺血性脑血管病和老年痴呆的中药组合物，由人参、银杏叶、西红花、大豆组成。
发明人在对上述专利技术进行系统研究时发现，当采用特定的成分组成和含量配比时，能够取得更好的疗效。
发明内容
发明人对发明专利ZL02131435.7和WO2007118363A1中涉及的组合物的组分在预防或治疗心脑血管疾病和痴呆症中的作用进行了系统的实验性研究，发现大豆在组合物中的起到的作用并不明显。通过组方优化研究，发现完全可以仅用人参、银杏叶、西红花三种中药成分达到甚至超过WO2007118363A1的组合物的药理药效。
另外，发明人还发现，采用特定重量份配比的人参、银杏叶和西红花制成的组合物具有更优的治疗或预防心脑血管疾病和痴呆症的作用，尤其是在治疗缺血性脑血管疾病、冠心病、心绞痛和老年性痴呆，尤其是血管性痴呆疾病方面治疗效果更佳。根据该研究，可以进一步降低人参、银杏叶和西红花组成的组合物在药物中的使用剂量，不仅能起到节约能源、降低成本的作用，更进一步降低了该组合物长期服用可能产生的副作用。
因此，本发明的目的在于提供一种效果更好的、用于预防或治疗心脑血管疾病或痴呆症的中药组合物及其制备方法和用途。
本发明的目的是通过如下技术方案实现的：
一种用于预防或治疗心脑血管疾病或痴呆症的中药组合物，制成所述中药组合物的药 物原料为下列重量份配比的药味：
人参1份、银杏叶0.8-1.5份、西红花0.018-0.030份。
优选地，制成所述中药组合物的药物原料为下列重量份配比的药味：
人参1份、银杏叶1份、西红花0.018-0.030份。
更优选地，制成所述中药组合物的药物原料为下列重量份配比的药味：
人参1份、银杏叶0.9-1.2份、西红花0.020-0.025份。
再优选地，制成所述中药组合物的药物原料为下列重量份配比的药味：
人参1份、银杏叶1份、西红花0.020-0.025份。
最优选地，制成所述中药组合物的药物原料为下列重量份配比的药味：
人参1份、银杏叶1份、西红花0.022份。
本发明还提供上述中药组合物的制备方法。
本发明的中药组合物可以采用多种方法制备得到。
在本发明的一种实施方式中，按上述配比称取人参、银杏叶、西红花三种中药，混合后粉碎，或粉碎后混合得到。
在本发明另一种实施方式中，按上述配比称取人参、银杏叶、西红花三种中药，混合后提取制备而成。
在本发明一种优选的实施方式中，所述中药组合物的制备方法包括如下步骤：按上述配比称取人参、银杏叶、西红花三种中药的步骤，制备人参提取物的步骤，制备银杏叶提取物的步骤，制备西红花提取物的步骤，以及将上述人参提取物、银杏叶提取物和西红花提取物混合的步骤。
优选地，所述制备人参提取物的步骤中，得到的主要成分为人参总皂苷；所述制备银杏叶提取物的步骤中，得到的主要成分为银杏叶总黄酮和总内酯；所述制备西红花提取物的步骤中，得到的主要成分为西红花总苷。
上述三种提取物可采用现有的提取上述成分的方法进行制备，所述成分的提取制备方法已较为成熟，例如人参总皂苷、银杏叶总黄酮和总内酯可采用药典中记载的方法提取制备，所述西红花总苷的提取方法可采用文献《中国现代应用药学》(2011年8月第28卷第8期729-731页)记载的方法提取制备。
在本发明的一种实施方式中，所述制备人参提取物的方法为：将人参粉碎成粉末，乙醇回流提取2次，过滤，滤液减压回收溶剂至70℃时相对密度1.12～1.14，加入相当生药2-6倍量的水搅匀，冷沉，上清液上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗脱，收集醇洗脱液浓缩至干，得到人参提取物。
在本发明的一种实施方式中，所述制备银杏叶提取物的方法为：银杏叶粗粉加乙醇温浸，过滤，滤渣再加乙醇温浸，过滤，合并两次浸滤液，减压浓缩至70℃时相对密度为1.12～1.14，加入生药2-6倍量的水，充分搅匀，冷沉，滤过，滤过液上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗脱，收集乙醇洗脱液，浓缩至70℃时相对密度为1.02～1.04，乙酸乙酯：正丁醇萃取，合并萃取液，减压回收溶剂，得到银杏叶提取物。
在本发明的一种实施方式中，所述制备西红花提取物的方法为：西红花生药加入乙醇冷浸，滤过，药渣再加入乙醇冷浸，滤过，合并两次浸滤液，减压浓缩至70℃时相对密度 为1.12～1.14，加入水，上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗，将乙醇洗脱液浓缩至70℃时相对密度1.02～1.04，浓缩至干，得到西红花提取物。
本发明还提供一种用于预防或治疗心脑血管疾病或痴呆症的中药制剂，所述制剂由上述中药组合物，和至少一种药学上可接受的辅料制成。
所述制剂可以为固体剂型或半固体剂型、液体剂型或气体剂型。
所述的固体或半固体剂型选自片剂、丸剂、膏剂、丹剂、散剂、颗粒剂、栓剂、粉剂、乳剂、咀嚼剂和胶囊剂中的任一种。
所述液体制剂选自口服液、混悬液、糖浆、注射液、药酒和酊剂中的任一种。
所述的气体制剂为气雾剂或吸入剂。
本发明还提供上述中药组合物或制剂在制备预防或治疗心脑血管疾病或痴呆症的药物中的应用。
优选地，所述心脑血管疾病选自缺血性脑血管疾病、冠心病或心绞痛中的至少一种。
优选地，所述痴呆症选自老年性痴呆，尤其是血管性痴呆。
与现有技术相比，本发明的中药组合物具有如下技术效果：
(1)本发明的中药组合物及制剂在预防或治疗心脑血管疾病或痴呆症中具有更好的疗效。
(2)本发明中药组合物在药品中的使用量更低，有利于节约成本，节约能源。
(3)本发明中药组合物的毒副作用小、安全性能好、可以长期服用，具有良好的药用前景。
具体实施方式
下述结合具体实施例、对比例和相关试验例，进一步阐述本发明，但这些实施例和试验例仅限于说明本发明而不是用于限制本发明的范围。下列实施例和试验例中未注明具体实验条件的实验方法，是按照常规条件，或按照厂商所建议的条件实施的。
第一部分：本发明中药组合物及其制剂的制备
实施例1
人参       1份
银杏叶     0.8份
西红花     0.018份
按发明专利ZL02131435.7实施例1公开的方法，分别提取得到人参提取物、银杏叶提取物、西红花提取物，将三种提取物混合，进一步按常规的方法分别制备成颗粒剂、胶囊剂、注射剂。
实施例2
人参       1份
银杏叶     1.5份
西红花     0.030份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例3
人参       1份
银杏叶      1份
西红花      0.018份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例4
人参        1份
银杏叶      1份
西红花      0.030份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例5
人参        1份
银杏叶      0.9份
西红花      0.020份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例6
人参        1份
银杏叶      1.2份
西红花      0.025份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例7
人参        1份
银杏叶      1份
西红花      0.20份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例8
人参        1份
银杏叶      1份
西红花      0.025份
按本发明实施例1所述方法制备提取物混合物和制剂。
实施例9
人参        1份
银杏叶      1份
西红花      0.022份
分别参照中国药典(2010年版 一部)367～368页和392～393页下记载的方法提取制备人参提取物和银杏叶提取物，按照文献《中国现代应用药学》(2011年8月第28卷第8期729-731页)记载的方法提取制备西红花提取物，将三种提取物混合，进一步按常规的方法分别制备成颗粒剂、胶囊剂、注射剂。
实施例10
人参        1份
银杏叶      0.8份
西红花      0.030份
按本发明实施例9所述方法制备提取物混合物和制剂。
实施例11
人参        1份
银杏叶      1.5份
西红花      0.018份
按本发明实施例9所述方法制备提取物混合物和制剂。
实施例12
人参        1份
银杏叶      0.9份
西红花      0.025份
按本发明实施例9所述方法制备提取物混合物和制剂。实施例13
人参        1份
银杏叶      1.2份
西红花      0.02份
按本发明实施例9所述方法制备提取物混合物和制剂。
第二部分：对比例中药组合物及其制剂的制备
对比例1
人参        1份
银杏叶      1份
西红花      0.1份
按本发明实施例1所述方法制备提取物混合物和制剂。
对比例2
人参        1份
银杏叶      1份
西红花      0.015份
按本发明实施例1所述方法制备提取物混合物和制剂。
对比例3
人参        1份
银杏叶      1.5份
西红花      0.4份
按本发明实施例1所述方法制备提取物混合物和制剂。
对比例4
人参        1份
银杏叶      1.9份
西红花      0.05份
按本发明实施例9所述方法制备提取物混合物和制剂。
对比例5
按发明专利ZL02131435.7实施例1所述的组方及制备方法制备成提取物混合物和相应的制剂。
对比例6
按发明专利ZL02131435.7实施例2所述的组方及制备方法制备成提取物混合物和相应的制剂。
对比例7
按专利WO2007/118363实施例1所述组合物处方及制备方法制备成提取物混合物和相应胶囊剂。
对比例8
按专利WO2007/118363实施例2所述组合物处方，按专利WO2007/118363实施例1制备方法制备提取物混合物和胶囊剂。
第三部分 药效学实验
试验例1 对实验性大鼠缺血心肌的保护作用
实验材料
健康雄性Wastar大鼠，体重180～220g，清洁级，由维通利华动物实验中心提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成三种提取物混合物)，试验时用蒸馏水配成0.15g/mL(按中药组合物的原药量计算)。
试剂：乌拉坦、盐酸异丙肾上腺素、氯化钠注射液、CK、CK-MB试剂盒。
仪器：生理记录仪BL-420生理信号采集系统、离心机、半自动生化分析仪。
试验方法
a)分组及模型制备：
试验用药前各组大鼠先做心电图检查，弃去S-T段、T波有异常改变者和心率失常者，将大鼠随机分为空白对照和造模组，每组10只，每天分别灌胃：空白对照组给予生理盐水、本发明中药组合物(实施例1、实施例2、实施例5、实施例6、实施例9、实施例11，按3g/kg/d剂量给药，所用剂量按人参、银杏叶、西红花的原药量计算)、对比例组(其中，对比例1、对比例2、对比例3、对比例4、对比例5、对比例6按3g/kg/d剂量给药，所用剂量按人参、银杏叶、西红花的原药量计算；对比例7组、对比例8按3g/kg/d剂量给药，其中所用剂量按人参、银杏叶、西红花、大豆的原药量计算)，连续给药10天，于第10天灌药后30min，按照如下方法造模：将各组大鼠用20％乌拉坦5ml/kg腹腔注射浅麻醉后，背位固定，皮下多点注射盐酸异丙肾上腺素，正常对照组给予等容量的生理盐水，连接心电图机，走纸速度50cm/s，标准电压10mm/mv，记录成膜后心电图变化。导联心电图，观察ST段的改变。具备以下条件之一者判断为心肌缺血阳性：1)S-T段水平向下或向上偏移≥0.1mv；2)T波高耸超过同导联R波的1/2；3)T波高耸伴S-T段移位。阴性判断标准：1)S-T段斜形偏移，或水平偏移<0.1mv；2)T波低平或双向倒置。以模型组大鼠心电图ST段抬高≥0.1mv作为造模成功的标志。
b)实验方法：
连续给药10天，于末次灌药后30min造模，造模前、造模后即刻、5min、10min、20min、30min时用生理记录仪导联心电图，并在6h后采血，离心取血清检测CK、CK-MB。
c)统计分析：
实验数据用均数±标准差表示，以t检验作统计学处理。
3结果
a)对J点位移的影响
空白组与模型组比较有非常显著性差异(P<0.01)；与模型组比较，本发明中药组合物组在不同时间对J点位移有显著下降作用(P<0.05～0.01)，且本发明中药组合物的作用好于各对比例组。见表1。
表1本发明中药组合物对大鼠J点位移的影响(
Figure PCTCN2015099381-appb-000001
n＝10)
Figure PCTCN2015099381-appb-000002
注：与模型组相比*P<0.05；**P<0.01
b)对异丙肾上腺素所致急性心肌缺血大鼠血清CK、CK-MB的影响
与空白组比较，本发明中药组合物有非常显著性差异；与模型组比较，造模6h后，可使肌酸激酶CK、肌酸激酶同功酶CK-MB明显减少；与对比例组比较，本发明组合物的效果更好。表明本发明中药组合物对异丙肾上腺素致大鼠急性心肌缺血损伤具有更好的保护作用。见表2。
表2对异丙肾上腺素所致急性心肌缺血大鼠血清CK、CK-MB的影响(
Figure PCTCN2015099381-appb-000003
n＝10)
Figure PCTCN2015099381-appb-000004
Figure PCTCN2015099381-appb-000005
注：与模型组相比*P<0.05；**P<0.01
小结：心肌缺血是指心脏的血液灌注减少，导致心脏的供氧减少，心肌能量代谢不正常，不能支持心脏正常工作的一种病理状态。冠心病是引起心肌缺血最主要、最常见的病因。本发明中药组合物与对比例中药组合物相比，对异丙肾上腺素所致急性心肌缺血大鼠不同时间对J点位移有显著下降作用(P<0.05～0.01)，对血清CK、CK-MB具有明显的降低作用，说明本发明中药组合物对心肌缺血具有明显的保护作用，且其效果好于对比例中药组合物。
试验例2 降血脂实验
实验材料
昆明种小鼠，体重18～22g，雌雄各半，由维通利华动物实验中心提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
试剂：总胆固醇(TC)测试试剂盒；甘油三脂(TG)测定试剂盒。
仪器：B-260型恒温水浴锅；TDL 80-2B型低速离心机；THER-MO LABSYSTEM MK3型酶标仪。
试验方法
取昆明种小鼠，按体重随机分为正常对照组(16只)、高脂模型组(16只)、本发明组合物组(每组10只)、对比例组(每组10只)，每组雌雄各半。正常对照组喂普通饲料，其余各组饲喂高脂饲料(配方为：77.5％基础饲料，2％胆固醇，10％猪油，10％蛋黄粉，0.5％去氧胆酸钠)。小鼠每周称体重一次，喂养5周。5周后，随机取正常对照组和高脂模型组雌雄动物各3只，摘取眼球取血，测定血清TC、TG。正常对照组的血清TC、TG均低于高脂模型组，数据差异显著，提示小鼠的高脂血症模型已经形成，该批小鼠可用于正式试验。随后给各组小鼠按表4所述剂量等体积灌胃给药(其中，本发明中药组合物(实施例 1、实施例2、实施例5、实施例6、实施例9、实施例11，按4g/kg/d剂量给药，所用剂量按人参、银杏叶、西红花的原药量计算)、对比例组(其中，对比例1、对比例2、对比例3、对比例4、对比例5、对比例6按4g/kg/d剂量给药，所用剂量按人参、银杏叶、西红花的原药量计算；对比例7组、对比例8按3g/kg/d剂量给药，所用剂量按人参、银杏叶、西红花、大豆的原药量计算))。各组药物均用1％羧甲基纤维素钠制成混悬液。给药3周后，各组动物均禁食12h，称重，摘取眼球取血，血液样品3000rpm离心10min，分离血清，按照总胆固醇和甘油三酯试剂盒说明书操作，测定TC、TG。所得数据用SPSS10.0统计软件进行统计，数据以
Figure PCTCN2015099381-appb-000006
表示。
结果
a)各组药物对高血脂症小鼠体重增长的影响
实验开始时各组小鼠体重均在18-20g左右，经过5周的高脂饲料造模，各组小鼠的体重变化不一。可以看出：高脂模型组、本发明组合物组、对比例组的体重变化与正常对照组相比，均有明显增大，给药3周后，模型组体重升至最高，提示高脂模型组的保持，本发明中药组合物组和对比例中药组合物组均能明显降低高脂模型组动物的体重(P<0.05)。且本发明中药组合物组降低体重的效果最好。见表3。
表3本发明中药组合物对小鼠体重影响变化表(g)
Figure PCTCN2015099381-appb-000007
注：与模型组相比*P<0.05
b)各组药物对高血脂症小鼠血清TC、TG的影响结果
给药3周后，测定各组小鼠血清的总胆固醇(TC)和甘油三酯(TG)。结果见表4。
表4本发明中药组合物对小鼠血脂水平的影响(
Figure PCTCN2015099381-appb-000008
n＝10)
Figure PCTCN2015099381-appb-000009
注：与模型组相比*P<0.05；**P<0.01
由表4可以看出，高脂模型组的TC、TG水平均比正常对照组明显升高，表明小鼠高脂血症模型造模成功。与模型组相比，本发明中药组合物组和对比例组均可明显降低小鼠的TC、TG水平，且本发明中药组合物组效果优于对比例组，表明本发明组合物组降血脂作用好于对比例组。
小结：高脂血症(hyperlipidemia)是指血浆中胆固醇或甘油三酯水平升高。高血脂主要与胆固醇相关，胆固醇升高是冠心病等心脑血管疾病死亡率上升的最重要的危险因素之一。血脂异常，尤其是胆固醇浓度升高，容易造成“血稠”，在血管壁上沉积，逐渐形成小斑块(就是我们常说的“动脉粥样硬化”)，这些“斑块”增多、增大，逐渐堵塞血管，使血流变慢，严重时血流被中断；这种情况如果发生在心脏，就引起冠心病；发生在脑，就会出现脑中风等心脑血管疾病。本发明中药组合物降低高血脂症小鼠血清TC、TG与模型组相比具有显著性差异(**P<0.01)，提示本发明中药组合物在治疗和预防心脑血管疾病方面效果优于对比例中药组合物。
试验例3 降血糖实验
实验材料
昆明种小鼠，体重18～22g，雄性，由维通利华动物实验中心提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
试剂：四氧嘧啶、胰岛素放射免疫分析药盒。
仪器：血糖/血酮仪及配套试纸、电热恒温水浴锅、电子天平、低速冷冻离心机、放射免疫计数器。
试验方法
糖尿病模型小鼠的制备：
雄性昆明小鼠适应性饲养1周后，随机抽取10只作为正常对照，其余小鼠禁食(不禁水)24小时后，将四氧嘧啶用生理盐水新鲜配成2％溶液，以200mg/kg剂量腹腔注射，72小时后剪尾取血检测血糖，以血糖在11.1mmol/L以上的小鼠为糖尿病模型小鼠，纳入实验。
b)分组及给药：
取糖尿病模型小鼠随机分组，每组10只。各组血糖值相近，其中1组为模型对照组，6组为本发明组合物组、8组为对比例组。各组小鼠均灌胃相应药物(剂量与试验例2相同)，正常对照组和模型对照组给予相应量的生理盐水，定期称量体重调整给药量，连续21天。
c)指标检测：
实验期间分别于0d、7d、14d、21d剪尾取血检测血糖。口服糖耐量实验在灌胃结束检测血糖后，将小鼠以2.5g/kg剂量灌服葡萄糖，并检测0min、30min、60min、120min血糖。小鼠于第22天眼眶取血，4℃,3000rpm离心10min分离血清，按照试剂盒说明书检测血清胰岛素水平。
实验数据均用SPSS软件进行统计学处理，各项指标结果以均数±标准差表示，组间比较采用t检验。
结果
a)对四氧嘧啶高血糖模型小鼠血糖的影响
小鼠腹腔注射四氧嘧啶后，血糖显著升高，给药7d、14d模型组小鼠血糖很高，与正常组比较具有显著性差异，说明四氧嘧啶致糖尿病模型成功，并在试验期间持续高血糖。给药7d后各给药组小鼠血糖开始下降，给药14d后，本发明组合物组小鼠血糖下降明显，与模型组比较有显著性差异。灌胃21d后本发明组合物比较有非常显著性差异(P<0.01)，对比例组有显著性差异(P<0.05)。结果提示本发明组合物具有较好的降血糖作用，且本发明组合物的降血糖效果优于对比例组。
口服糖耐实验表明，本发明中药组合物能使实验小鼠糖耐受量有非常明显改善(P<0.01)，对比例组能使实验小鼠糖耐受量有明显改善(P<0.05)。结果提示本发明中药组合物的降血糖效果优于对比例组。见表5。
表5本发明中药组合物对四氧嘧啶高血糖模型小鼠血糖的影响(
Figure PCTCN2015099381-appb-000010
n＝10)
Figure PCTCN2015099381-appb-000011
Figure PCTCN2015099381-appb-000012
注：与模型组比较，*P<0.05；**P<0.01
b)对四氧嘧啶性糖尿病小鼠血清胰岛素的影响
与正常对照对相比较，模型组小鼠的血清胰岛素水平明显降低，说明腹腔注射四氧嘧啶后，小鼠胰岛β细胞的功能受到破坏，造模成功；与模型对照组比较，本发明中药组合物组与对比例组血清胰岛素水平均有不同程度的升高，其中本发明中药组合物组具有非常显著性差异(P<0.01)，而对比例1、2、4、7、8组具有显著性差异(P<0.05)，其他对比例组血清胰岛素的升高但无统计学意义，提示本发明组合物组对提升四氧嘧啶性糖尿病小鼠血清胰岛素的效果优于对比例组。结果见表6。
表6本发明中药组合物对四氧嘧啶性糖尿病小鼠血清胰岛素的影响(
Figure PCTCN2015099381-appb-000013
n＝10)
Figure PCTCN2015099381-appb-000014
注：与模型组比较，*P<0.05；**P<0.01
小结：随着人们生活水平的提高和环境因素的影响，在心脑血管患者中越来越多地出 现糖代谢方面的异常，心脑血管疾病患者若同时伴有糖代谢异常，常常预后不良。患过心肌梗死的糖尿病患者未来再发心梗的危险超过40％。另外，血糖异常还可引起内皮功能障碍、大动脉弹性降低、左室肥厚、颈动脉粥样斑块、微量白蛋白尿等一系列病理过程，最终导致动脉粥样硬化。本发明中药组合物可以显著降低四氧嘧啶高血糖模型小鼠血糖，提高四氧嘧啶性糖尿病小鼠血清胰岛素，说明本发明中药组合物对心脑血管疾病具有很好的预防和治疗作用。
试验例4 抗血栓形成实验
1.实验材料
SD大鼠，体重180.0～220.0g，雌雄兼用，由维通利华动物实验中心提供，动物实验前在动物房内(控制温度)饲养适应一周。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.15g/mL(按组合物的原药量计算)。
阿司匹林：白色片剂，50mg/片，中国医药公司上海分公司提供。戊巴比妥钠：白色粉末，中国医药(集团)上海化学试剂公司提供，用生理盐水配制成0.8％水溶液，备用。肝素钠：白色粉末，125u/mg，中国医药(集团)上海化学试剂公司提供，生理盐水配制成0.1％的溶液备用。枸橼酸钠：中国医药工业公司西南制药一厂提供，生理盐水配制成3.8％溶液备用。ADP：Sigma公司生产，pH 7.4磷酸缓冲液配制成200μmol/L浓度溶液备用。
MK4/HC血小板计数仪(美国Baker Instru-ments公司生产)，Labor aggregometer-153型双道血小板聚集仪(德国Labor GmbHHanburg公司生产)
2.试验方法
将大鼠随机分组，每组15只，即空白对照组、阿司匹林组(1％CMC-Na溶液研磨配成0.88mg/mL悬混液，按0.0044g/kg/d)、本发明中药组合物组(6组，剂量与试验例1相同)、对比例组(8组，剂量与试验例1相同)。空白对照组给同体积生理盐水。每天给药一次，连续7天。
体内血栓形成实验：各组大鼠第7天给药后1h，参照动-静脉旁路血栓形成法【张钧田.现代药理学实验方法.北京：北京医科大学出版社，1998；1216-1217】，用0.8％的戊巴比妥钠40mg/kg剂量腹腔注射麻醉，分离右侧颈总动脉和左侧颈外静脉。取三段聚乙烯塑料管，中间一段内径为2mm，长约8.5cm，两端的两段塑料管内径为1.5mm，各长10cm。在中段塑料管内放入一根长约7cm预先称重的4号手术丝线，其中一端露出塑料管0.5cm以固定位置，并保持在管中的丝线长度为6.5cm，将肝素钠溶液充满聚乙烯塑料管内，将塑料管的一端插入左侧颈外静脉后，将塑料管的另一端插入右侧颈总动脉，随即立刻开放血流。血流开放20分钟后，立即中断血流并迅速取出中段的丝线，称取丝线的重量，即为总重量。总重量减去丝线的重量即为血栓湿重。按下列公式计算血栓抑制率：
抑制率＝(对照组血栓湿重-实验组血栓湿重)/对照组血栓湿重×100％。将各组血栓湿重进行统计学比较。
血小板聚集抑制试验：在各组大鼠第7天给药1h后，用上述方法麻醉大鼠，颈总动脉插管放血，血液用3.8％枸橼酸钠按1:9比例抗凝，以500转/分离心5分钟，取上部血浆即为富血小板血浆(PRP)，余下部分再次以3500转/分离心15分钟，上清液为贫血小板血浆(PPP)，用血小板计数仪计数PRP血小板，用PPP调节PRP的血小板计数至3×105个/mm2 左右。样品管37℃保温3分钟后加入ADP 5μL(终浓度为5μmol/L)，搅拌子转速500r/min，记录加入ADP后的记录笔移动的行距，按下式计算聚集率：
聚集率(％)＝加入ADP的行程×100％/0～100％的行程
计算出各实验组的平均聚集率和标准差，并按下式计算各实验组的血小板聚集抑制率：
聚集抑制率(％)＝(空白对照组聚集率-实验组聚集率)/空白对照组聚集率×100％。
3.结果
a)对实验性血栓形成的影响
各给药组与空白对照组相比，均对血栓形成有明显的抑制作用，其中与对照组比较本发明中药组合物组、对比例2、7中药组合物组大鼠血栓湿重具有非常显著性差异(P<0.01)，对比例1、3、4、5、6、8中药组合物组大鼠血栓湿重具有显著性差异(P<0.05)，提示本发明中药组合物组对大鼠血栓形成的影响好于对比例组。结果见表7。
表7本发明中药组合物对大鼠血栓形成的影响(
Figure PCTCN2015099381-appb-000015
n＝15)
Figure PCTCN2015099381-appb-000016
注：与对照组比较，*P<0.05；**P<0.01
(2)对血小板聚集性的影响
实验过程中，各组动物所取血样的血小板数均在3×105个/mm2左右，无显著性差异。实验结果见表8。
表8本发明中药组合物对大鼠血小板聚集率的影响(
Figure PCTCN2015099381-appb-000017
n＝15)
Figure PCTCN2015099381-appb-000018
Figure PCTCN2015099381-appb-000019
注：与对照组相比*P<0.05；**P<0.01
由上表可知，各给药组与空白对照组相比，对ADP诱导的大鼠血小板聚集均有明显的抑制作用。其中本发明中药组合物组对血小板聚集抑制率(与对照组相比，**P<0.01)超过对比例组(与对照相比，*P<0.05)，提示本发明中药组合物在抗血小板聚集方面效果优于对比例组。
小结：血栓是血液里的一些成分发生聚集，形成团块，影响了血液的流动，出现血栓的原因主要是血液成分的改变、血管内皮受损和血流流动速度的改变。血栓可以阻塞冠状动脉血管，致使血流急剧减少或中断，使相应的心肌出现严重而持久的急性缺血，最终导致心肌的缺血性坏死；另外，脑血管的某个部位自发形成血栓，造成脑部血管堵塞、血液流通不畅，形成“脑血栓”，脑血栓形成后，血栓脱落引起血管堵塞，形成脑梗。本发明中药组合物与对比例中药组合物相比，具有更好的降低血栓大小，抑制血栓形成、降低血小板凝聚率的作用，提示本发明中药组合物具有更好的治疗和预防心脑血管疾病的作用。
试验例5 对氢溴酸东莨菪碱致获得性记忆障碍模型小鼠的影响
1.实验材料
ICR种小鼠，18-22g，雌雄各半。由北京维通利华实验动物技术发展有限公司提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
哈伯因(石杉甲片)，50μg/片，由河南竹林众生制药股份有限公司豫中制药厂生产，试验时用蒸馏水配成4μg/mL；达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成1.5mg/mL；氢溴酸东莨菪碱，0.6mg/mL，上海禾丰制药有限公司生产。
TT-2型小鼠跳台程序自动控制仪：中国医学科学院药物研究所生产。
2.试验方法
动物雌雄各半，随机分组：空白对照组，模型组，哈伯因0.08mg/kg组，达纳康30mg/kg组，本发明中药组合物组(6组，与试验例2所用剂量相同)，对比例组(8组，与试验例2所用剂量相同)。
动物每日按20mL/kg体积灌胃给药，空白对照组及模型组动物给予蒸馏水，每日一次，连续15日。第14天给药后50min按10mL/kg体积给予空白对照组小鼠腹腔注射生理盐水，其余各组动物均注射东莨菪碱(6mg/kg)。10min后将小鼠放入跳台程序自动控制仪内适应3min，然后通电，使小鼠遭受电击刺激5次，小鼠遭到电击时跳上跳台，逃避电击，训练5min，使获得记忆。第15日给药后60min，将各组动物放入跳台程序自动控制仪中，测定小鼠离开跳台遭受电击的次数(错误次数)及发生时间(潜伏期)。结果进行统计学处理(t检验)。见表9。
表9本发明组合物对东莨菪碱所致小鼠获得性记忆障碍的影响(
Figure PCTCN2015099381-appb-000020
n＝12)
Figure PCTCN2015099381-appb-000021
注：与模型组相比*P<0.05；**P<0.01
结果：与模型组比较，空白对照组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.01)，说明造模成功。与模型组比较，哈伯因组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.01)；本发明中药组合物组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.01)；达纳康组和对比例1～4、7、8中药组合物组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.05)，对比例5、6中药组合物组错误次数有减少趋势、潜伏期有延长趋势，无统计学意义。
结论：训练前给予小鼠M-受体阻断剂东莨菪碱，可造成脑内乙酰胆碱含量降低，形成获得性记忆缺损。本试验在此化学损伤基础上进行跳台试验，以小鼠5min内错误次数、潜伏期为指标，观察本发明组合物及对比例组合物对该模型的影响。结果表明本发明中药组合物明显改善东莨菪碱所致小鼠获得性记忆障碍，效果好于对比例组，提示本发明中药组合物可能有增加脑内乙酰胆碱含量的作用，且作用强于对比例组。
试验例6 对利血平所致小鼠获得性记忆障碍模型小鼠的影响
1.实验材料
ICR种小鼠，25-32g，雌雄各半。由北京维通利华实验动物技术发展有限公司提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
哈伯因(石杉甲片)，50μg/片，由河南竹林众生制药股份有限公司豫中制药厂生产，试验时用蒸馏水配成4μg/mL；达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成1.5mg/mL；利血平注射液，1mg/mL，上海医科大学红旗制药厂，试验时用生理盐水配成0.05mg/mL。
TT-2型小鼠跳台程序自动控制仪：中国医学科学院药物研究所生产
2.试验方法
试验分组同试验例5。
动物每日按20mL/kg体积灌胃给药，空白对照组及模型组动物给予蒸馏水，每日一次，连续15日。第14天给药后即刻按10mL/kg体积给予空白对照组小鼠背颈部皮下注射生理盐水，其余各组动物均背颈部皮下注射盐酸利血平(0.5mg/kg)。注射后60min将小鼠放入跳台程序自动控制仪内适应3min，然后通电，使小鼠遭受电击刺激5次，小鼠遭到电击时跳上跳台，逃避电击，训练5min，使获得记忆。第15日给药后60min，将各组动物放入跳台程序自动控制仪中，测定5min内小鼠的错误次数及潜伏期。结果进行统计学处理(t检验)。
3.结果
与模型组比较，空白对照组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.05)，说明造模成功。与模型组比较，本发明中药组合物组小鼠5min内错误次数均明显减少(P<0.05)，潜伏期均明显延长(P<0.05)。对照药哈伯因组、达纳康组小鼠5min内错误次数明显减少(P<0.05)，潜伏期有延长趋势，但无统计学意义。对比例1～4、7中药组合物组小鼠5min内错误次数明显减少(P<0.05)，潜伏期有延长趋势，但无统计学意义。其他对比例组小鼠5min内错误次数有减少趋势，潜伏期有延长趋势，但无统计学意义。见表10。
表10本发明组合物对利血平所致小鼠获得性记忆障碍的影响(
Figure PCTCN2015099381-appb-000022
n＝12)
Figure PCTCN2015099381-appb-000023
Figure PCTCN2015099381-appb-000024
注：与模型组相比*P<0.05；**P<0.01
结论：研究证实，利血平可使脑内单胺类神经递质耗竭，损害学习和记忆过程，训练前给予利血平可造成小鼠记忆获得或保持缺损。本试验以5min内跳台错误发生的次数及错误发生的潜伏期为指标，观察本发明中药组合物及对比例组对该模型的影响。灌胃给予本发明中药组合物及对比例组合物后，二指标均获不同程度改善，且本发明中药组合物组的改善程度明显好于对比例组。表明本发明中药组合物有改善动物获得性记忆障碍的作用，提示可能与本发明中药组合物能够增加儿茶酚氨类神经递质含量的作用机制有关，且作用好于对比例组。
试验例7 对亚硝酸钠所致小鼠获得性巩固性记忆障碍模型小鼠的影响
1.实验材料
ICR种小鼠，18-22g，雌雄各半。由北京维通利华实验动物技术发展有限公司提供。
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
哈伯因(石杉甲片)，50μg/片，由河南竹林众生制药股份有限公司豫中制药厂生产，试验时用蒸馏水配成4μg/mL；达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成1.5mg/mL；亚硝酸钠，北京益利精细化学品有限公司生产。试验时用生理盐水配成12mg/mL。
TT-2型小鼠跳台程序自动控制仪：中国医学科学院药物研究所生产
2.试验方法
试验分组同试验例5。
动物每日按20mL/kg体积灌胃给药，空白对照组及模型组动物给予蒸馏水，每日一次，连续15日。第14天给药后60min将小鼠放入跳台程序自动控制仪内训练5min，并使小鼠遭受电击刺激5次。训练结束后，立即按10mL/kg体积给予对照组小鼠背颈部皮下注射生理盐水，其余各组动物均注射亚硝酸钠(120mg/kg)。第15日给药后60min，将各组动物放入跳台程序自动控制仪中，测定5min内小鼠的错误次数及潜伏期。结果进行统计学处理(t检验)。
3.结果
与模型组比较，空白对照组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.01)，说明造模成功。与模型组比较，本发明中药组合物组、对照药哈伯因和达纳康组小鼠5min内错误次数均明显减少(P<0.05)，潜伏期均明显延长(P<0.01～0.05)。对比例7中药组合物组小鼠5min内错误次数明显减少(P<0.05)，潜伏期明显延长(P<0.05)，对比例2中药组合物组小鼠5min内错误次数明显减少(P<0.05)，潜伏期有延长趋势无统 计学意义。其他对比例组错误次数有减少趋势、潜伏期有延长趋势，无统计学意义。结果见表11。
表11本发明组合物对亚硝酸钠所致小鼠巩固性记忆障碍的影响(
Figure PCTCN2015099381-appb-000025
n＝12)
Figure PCTCN2015099381-appb-000026
注：与模型组相比*P<0.05；**P<0.01
研究证实，亚硝酸钠可使血红蛋白变性，使脑组织缺血缺氧，损害学习和记忆过程，训练后立即给予亚硝酸钠可造成小鼠记忆巩固或保持缺损。本试验在此化学损伤基础上，以5min内跳台错误次数、潜伏期为指标，观察本发明组合物及对比例组合物对该模型的影响。灌胃给予发明组合物及对比例组合物后，二指标有不同程度改善，且本发明组合物组的改善程度好于对比例组。表明本发明组合物有改善动物巩固性记忆障碍的作用，提示本发明中药组合物可能通过改善脑部循环、改善脑组织缺血缺氧从而改善学习记忆，且该作用好于对比例组。
试验例8 对大鼠双侧颈总动脉永久性结扎所致VaD的影响
1.实验材料
(1)动物：SD大鼠，250～270g体重，雄性。由北京维通利华实验动物技术发展有限公司提供。
(2)药物：
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.3g/mL(按组合物的原药量计算)。
哈伯因(石杉甲片)，50μg/片，由河南竹林众生制药股份有限公司豫中制药厂生产，试验时用蒸馏水配成6μg/mL；达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成2mg/mL。
(3)试剂：
高效液相：氯化乙酰胆碱(AchCl)、磷酸氢二钠(Na2HPO4)、氯化四甲基氯化铵(TMACl)、辛烷基磺酸钠(OSA)、硫代硫酸钠(Na2S2O5)、乙二胺四乙酸四钠盐(EDTA)、均为HPLC级，美国Sigma公司生产；磷酸(H3PO4，85％)、高氯酸(HClO4)均为HPLC级，美国Fisher Scientific公司生产；MB试剂，美国ESA公司生产。
生化测定：乙酰胆碱酯酶(TCHE)试剂盒；超氧化物歧化酶(SOD)试剂盒，；丙二醛(MDA)试剂盒；蛋白定量(双缩脲法)试剂盒；均由南京建成生物工程研究所提供；神经肽Y(NPY)试剂盒，由解放军总医院科技开发中心放免所提供；β－内啡肽(β－EP)试剂盒，由海军放免技术中心提供。
(4)仪器：
MORRIS水迷宫，中国医学科学院药物研究所生产。FT-630Gγ-计数仪，北京核设备厂生产。分光光度计，UV-120-02，日本岛津公司生产。16通道Coularray库仑阵列电化学高效液相色谱仪及色谱工作站，582泵，5600A电化学检测器，542自动进样器，5040型固态微孔电极(铂电极、固态钯电极)、CH150型柱温箱、柱前固定化酶反应器、柱后固定化酶反应器，均为美国ESA公司产品；柱子(C18150×3mm I.D.)美国ESA公司产品；超速离心机55P-72，日本HITACHI公司产品；低温冰箱，法国JOUAN公司产品；针筒式微孔滤膜过滤器：水系(0.22μm)，天津腾达过滤器厂产品。
(5)乙酰胆碱(ACh)含量测定条件：
流动相：Na2HPO4(100mmol/L)、TMACl(0.5mmol/L)、OSA(2.0mmo/L)、MB试剂(0.005％v/v)，重蒸去离子水定容，85％H3PO4调PH值为8.00，0.22μm水系膜过滤。
色谱条件：ESA582型二元泵体系、ESA ACH-3色谱柱(150×3mm 5μm I.D.)、ESA柱前固定化酶反应器(ESA ACH-SPR，3cm)、0.35mL/min流速、35℃柱温。
检测条件：5600A电化学检测器；5040型固态微孔电极(铂工作电极、固态钯参比电极)电势：+300mV。
2.试验方法
假手术组、模型组、哈伯因组、达纳康组、本发明中药组合物组(6组，与试验例1所用剂量相同)，对比例组(8组，与试验例1所用剂量相同)
大鼠水合氯醛麻醉(350mg/kg)，颈部正中切口，分离双侧颈总动脉并结扎(假手术组只分离不结扎颈总动脉)，缝合后回笼饲养，青霉素抗感染4天。1个月后进行MORRIS水迷宫游泳试验，以持续时间为指标筛选出现学习记忆障碍趋势的大鼠，随机分组：模型组；哈伯因60μg/kg组；达纳康20mg/kg组；本发明中药组合物组及对比例中药组合物组(剂量与试验例1相同)。每日灌胃给药一次(10mL/kg)，连续2个月。假手术组和模型组灌胃等体积蒸馏水。于造模后2个月、3个月(给药后1个月、2个月)分别测定MORRIS水迷宫的持续时间。末次测定后腹主动脉抽血，迅速取脑，左半脑液氮固化。冰浴中以0.15M HCLO4匀浆(100mg脑重加1ml)14000×g 4℃离心20min，取上清液，0.22μm滤膜过滤，自动进样器进样10μl，HPLC测定ACh的含量；右半脑每组4只福尔马林固定，经脱水、包埋、切片、HE染色，进行病理学检查，6只脑组织匀浆，测定AchE、SOD活性及MDA含量(比色法)；血浆测定神经肽Y、β-EP等指标(放免法)。结果进行统计学处理(t检验)。
3.结果
3.1对大鼠水迷宫持续时间的影响
大鼠双侧颈总动脉结扎后1个月，虽然出现了学习记忆障碍的趋势，但与假手术组比较尚无明显差异。结扎2个月、3个月后，与模型组比较，大鼠在MORRIS水迷宫的持续时间假手术组显著短于模型组(P<0.01)，表明造模成功。给药2个月至3个月后，本发明中药组合物组大鼠学习记忆能力均有明显提高，持续时间与模型组比较明显缩短(P<0.01)；对照药哈伯因组及对比例2、3、6、7中药组合物组也有相同作用(P<0.05～0.01)；给药后3个月，对照药达纳康组大鼠学习记忆能力有提高趋势，无统计学意义。结果见表12。
表12对VaD大鼠MORRIS水迷宫持续时间的影响
Figure PCTCN2015099381-appb-000027
Figure PCTCN2015099381-appb-000028
注：与模型组相比*P<0.05；**P<0.01
3.2对大鼠脑内AchE活性及ACh含量的影响
模型组大鼠双侧颈总动脉结扎后3个月，脑内AchE活性升高、ACh含量降低，假手术组与模型组比较有显著性差异(P<0.01)；给药2个月后，本发明中药组合物组大鼠的脑内AchE活性均明显降低(P<0.05)，大鼠全脑ACh含量均明显升高(P<0.01)，对照药哈伯因及对比例7中药组合物组亦有相同作用(P<0.05～0.01)，其他对比例中药组合物组与达纳康组AchE活性未见明显改变，ACh含量明显升高(P<0.05)。结果见表13。
表13对VaD大鼠脑内AchE活性及ACh含量的影响
Figure PCTCN2015099381-appb-000029
Figure PCTCN2015099381-appb-000030
Figure PCTCN2015099381-appb-000031
注：与模型组相比*P<0.05；**P<0.01
3.3对脑组织SOD活性、MDA含量的影响
模型组大鼠双侧颈总动脉结扎后3个月，脑内SOD活性明显降低，假手术组与模型组比较有显著性差异(P<0.01)，MDA含量未见明显改变；与模型组比较，本发明中药组合物组SOD活性明显升高(P<0.05)，其余各组SOD活性未见明显改变。与模型组比较，各组大鼠脑内MDA含量未见明显改变。结果见表14。
表14对VaD大鼠脑内SOD活性及MDA含量的影响
Figure PCTCN2015099381-appb-000032
Figure PCTCN2015099381-appb-000033
Figure PCTCN2015099381-appb-000034
注：与模型组相比*P<0.05；**P<0.01
3.4对大鼠血浆NPY、β-EP的影响
大鼠双侧颈总动脉结扎后3个月，血浆NPY含量明显降低，β-EP的含量明显升高，假手术组与模型组比较均有显著性差异(P<0.01)；给药2个月后，本发明中药组合物组、对比例2、7中药组合物组大鼠的NPY含量明显升高，各剂量组β-EP显著降低(均P<0.01)，其他对比例中药组合物组大鼠的NPY含量明显升高，各剂量组β-EP显著降低(均P<0.05)，对照药哈伯因及达纳康作用均不明显。结果见表15
表15对VaD大鼠血浆NPY、β-EP含量的影响
Figure PCTCN2015099381-appb-000035
Figure PCTCN2015099381-appb-000036
注：与模型组相比*P<0.05；**P<0.01
3.5病理学检查
病理学检查表明：假手术组海马区锥体细胞层排列尚可排列规则，结构紧密，层次丰富，细胞核膜饱满、圆滑。模型组海马区大部分锥体细胞部分区域排列稀疏，细胞核膜可见皱缩，核仁较小，部分锥体细胞结构不清、深染，呈三角形或团块状，周边可见空隙，核仁消失。哈伯因组海马区锥体细胞排列略有疏松，大部分细胞核膜可见，少许核仁略小，部分消失。达纳康组和对比例1～3、7中药组合物组海马区大部分锥体细胞排列较为整齐，细胞大小尚可，核膜清楚，部分核仁可见。本发明中药组合物组海马区大部分锥体细胞排列有序，细胞间结构紧密，核膜较饱满，核仁尚可，散在区域可见皱缩、深染的锥体细胞。对比例4中药组合物组海马区锥体细胞排列较为疏松，细胞较小，部分深染、模糊，界限不清，个别皱缩，呈小三角形或团块状。对比例5～6、8中药组合物组海马区锥体细胞排列尚可，层次可见，锥体细胞间界限不清，核膜淡化，核仁较小，呈变性状态，部分消失。
小结：
慢性脑缺血是血管性痴呆(VaD)、Alzheimer病(AD)及皮质下动脉硬化性脑病(Binswanger病)等多种疾病发展过程中的一个共同病理过程。研究证实，大鼠双侧颈总动脉结扎(2VO)后血流可持续下降，3周后为慢性期，1个月仍低于正常组；皮质、海马等参与学习记忆的神经元萎缩、变性、脱失呈进行性加重；2VO后3个月皮质和海马M型乙酰胆碱受体结合力下降；学习记忆等行为学出现明显障碍。
许多研究表明，脑缺血后可引起细胞内Ca2+超载、兴奋性毒性和炎性反应等，从而进一步导致神经元功能和结构损害。脑缺血缺氧还可引起线粒体氧化代谢功能出现障碍，导致生成超量的活性氧自由基，包括O-2,HO-2,OH-,H2O2等。这些活性氧自由基可与神经元内的蛋白质、脂质、核酸分子等反应并破坏其分子结构，发生超氧变化，进一步可引起脑细胞结构和功能破坏，并生成过量的过氧化产物如丙二醛(malondialdehyde,MDA)。海马、前额叶、颞叶、大脑皮层的神经元对缺血十分敏感，而这些区域是与学习记忆能力密切相关的脑区。脑缺血后这些脑区神经元功能和结构的损害会造成与学习记忆相关的神经递质紊乱，引起学习记忆能力的下降，甚至痴呆。
本试验2VO致VaD模型证实了上述生化、病理学及行为学变化过程，并观察到本发明中药组合物组可明显抑制AchE活性，升高模型动物脑内ACh含量，改善学习记忆障碍；该药还可明显增加脑内SOD活性，清除氧自由基保护脑组织。表明本发明中药组合物组对VaD有明显的治疗作用，且效果好于对比例中药组合物。
据报道，VaD病人血浆中神经肽Y含量降低，β-EP含量升高，申请人测定了2VO致VaD模型动物的神经肽Y及β-EP含量，与报道相一致，并观察到本发明中药组合物组可明显升高血浆神经肽Y含量、降低血浆β－EP含量。
病理学检查表明：模型组海马区部分区域排列稀疏，细胞核膜可见皱缩，核仁较小，部分锥体细胞结构不清、深染，呈三角形或团块状，周边可见空隙，核仁消失。本发明中药组合物组海马区大部分锥体细胞排列有序，细胞间结构紧密，核膜较饱满，病变程度明显减轻。
试验例9 缺血再灌注合并皮下注射D-半乳糖所致小鼠学习记忆障碍的影响
1.实验材料
(1)动物：ICR种小鼠，25-32g，雌雄各半。由北京维通利华实验动物技术发展有限公司提供。
(2)药物：
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.2g/mL(按组合物的原药量计算)。
哈伯因(石杉甲片)，50μg/片，由河南竹林众生制药股份有限公司豫中制药厂生产，试验时用蒸馏水配成4μg/mL；达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/ 片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成1.5mg/mL。D-半乳糖，上海恒信化学试剂有限公司生产；乙酰胆碱酯酶(TCHE)试剂盒；超氧化物歧化酶(SOD)试剂盒；丙二醛(MDA)试剂盒；谷光甘肽过氧化物(GSH-Px)试剂盒；一氧化氮(NO)试剂盒；蛋白定量(双缩脲法)试剂盒；均由南京建成生物工程中心提供。
(3)仪器：
MORRIS水迷宫，中国医学科学院药物研究所生产。分光光度计UV-120-02，日本岛津公司生产。
2.试验方法
小鼠随机分组，雌雄各半：假手术组、复合模型组、D-半乳糖组、缺血再灌组、哈伯因组、达纳康组、本发明中药组合物组(6组，与试验例2所用剂量相同)，对比例组(8组，与试验例2所用剂量相同)。
水合氯醛麻醉，颈部切口，施缺血再灌手术：分离双侧颈总动脉，动脉夹夹闭20分钟，松开20分钟，再夹闭20分钟后松开，同时切断鼠尾，放血0.5mL，形成反复脑缺血再灌注损伤，伤口缝合后回笼饲养。术后7天背部皮下注射D-半乳糖(100mg/kg，10mL/kg)，同时灌胃给药(20mL/kg)，每日一次，连续8周；假手术组仅分离颈总动脉不夹闭，背部皮下同体积注射生理盐水；D-半乳糖组仅分离颈总动脉不夹闭，背部皮下注射D-半乳糖(100mg/kg，10mL/kg)；缺血再灌组施缺血再灌手术，背部皮下同体积注射生理盐水。假手术组、复合模型组、D-半乳糖组均灌胃等体积蒸馏水。
给药8周后小鼠在MORRIS水迷宫中训练3天，每天2次，第4天测定学习记忆行为，第5天断头取脑，生理盐水制成10％脑匀浆，测定脑组织中AchE、SOD、GSH-Px活力，MDA、NO含量等生化指标(比色法)。结果进行统计学处理(t检验)
3.结果
3.1对小鼠MORRIS水迷宫学习记忆行为的影响
缺血再灌注合并皮下注射D-半乳糖后8周的小鼠在MORRIS水迷宫中的游泳持续时间、游泳路径长度延长，与假手术组比较均有显著性差异(P<0.01)；搜索策略多为边缘式或随机式。单纯缺血再灌组在水迷宫中的游泳持续时间、游泳路径长度与假手术组及模型组比较均未见明显差异，搜索策略与复合模型组比较有显著性差异(P<0.05)；单纯皮下注射D-半乳糖组在水迷宫中游泳持续时间、游泳路径长度及搜索策略与复合模型组比较均无明显差异；表明单纯皮下注射D-半乳糖及缺血再灌注损伤可形成小鼠学习记忆障碍，但程度不及合并处理显著；与复合模型组比较，对照药哈伯因组的游泳路径长度均显著缩短，搜索策略明显改进(P<0.05～0.01)；达纳康组的游泳持续时间及路径长度显著缩短(P<0.05～0.01)，搜索策略未见明显改进；本发明中药组合物组小鼠的游泳持续时间、游泳路径长度均显著缩短(P<0.05～0.01)，搜索策略明显改进(P<0.01)；对比例组2、3、7、8小鼠的游泳持续时间、游泳路径长度均显著缩短(P<0.05～0.01)，搜索策略明显改进(P<0.05～0.01)；对比例组1、4、5、6小鼠的游泳持续时间、游泳路径长度均显著缩短(P<0.05)，搜索策略无明显改进。结果见表16。
表16对小鼠MORRIS水迷宫学习记忆行为的影响(
Figure PCTCN2015099381-appb-000037
n＝12)
Figure PCTCN2015099381-appb-000038
Figure PCTCN2015099381-appb-000039
注：①搜索策略：直线式1分；趋向式2分；边缘式3分；随机式4分；②与假手术组比较#P<0.05，##P<0.01；③与模型组比较*P<0.05，**P<0.01。
3.2对脑组织SOD、GSH-Px活性及MDA、NO含量的影响
缺血再灌注合并皮下注射D-半乳糖后8周的小鼠(复合模型组)脑组织SOD、GSH-Px活性下降，MDA、NO含量升高，与假手术组比较均有显著性差异(P<0.05～0.01)；单纯皮下注射D-半乳糖组脑组织SOD、GSH-Px活性下降，MDA、NO含量升高，与假手术组比较有明显差异(均P<0.05～0.01)，与复合模型组比较无明显变化；单纯缺血再灌注组脑组织SOD、GSH-Px活性及NO含量与假手术组比较明显降低(P<0.05)，MDA含量与假手术组比较无明显变化。与复合模型组比较，对照药哈伯因组的SOD、GSH-Px活性及MDA、NO含量未见明显改变；达纳康组的SOD、GSH-Px活性明显增强，MDA、NO含量明显降低(P<0.05～0.01)；本发明中药组合物组SOD、GSH-Px活性明显增强，MDA、NO含量明显降低(P<0.05～0.01)；对比例1～3、7中药组合物组小鼠脑组织SOD活性明显增强，NO含量明显降低(均P<0.05)，GSH-Px活性及MDA含量未见明显改变；对比例4～6、8组NO含量明显降低(均P<0.05)，SOD、GSH-Px活性及MDA含量未见明显改变。结果见表17。
表17对脑组织SOD、GSH-Px活性及MDA、NO含量的影响(
Figure PCTCN2015099381-appb-000040
n＝12)
Figure PCTCN2015099381-appb-000041
Figure PCTCN2015099381-appb-000042
注：与假手术组比较#P<0.05，##P<0.01；与复合模型组比较*P<0.05，**P<0.01
3.3对脑组织AchE活性的影响
缺血再灌注合并皮下注射D-半乳糖后8周的小鼠(复合模型组)脑组织AchE活性明显增强，与假手术组比较有显著性差异(P<0.01)；单纯皮下注射D-半乳糖组和单纯缺血再灌注组脑组织AchE活性增加，与假手术组比较有明显差异(P<0.05)，与复合模型组比较无明显差异。与复合模型组比较，对照药哈伯因组的AchE活性明显下降(P<0.05)；达纳康组AchE活性无明显改变；本发明中药组合物组、对比例1～3、7中药组合物组AchE活性明显降低(P<0.05)；对比例4～6、8中药组合物组AchE活性未见明显改变。结果见表18。
表18对脑组织AchE活性的影响(
Figure PCTCN2015099381-appb-000043
n＝10)
Figure PCTCN2015099381-appb-000044
Figure PCTCN2015099381-appb-000045
注：与假手术组比较#P<0.05，##P<0.01；与复合模型组比较*P<0.05，**P<0.01
小结：
脑缺血后再灌注损伤的确切机制目前尚不清楚，多数学者认为与氧自由基增多诱发的级联反应有关。正常情况下氧自由基的形成量极少，寿命也短，故不构成对机体的危害，其中内源性的氧自由基清除剂起到了很重要的作用。谷胱甘肽过氧化物酶(GSH-Px)是一种重要的催化氢氧自由基分解的酶；SOD是内源性氧自由基清除剂的代表，其活力在一定程度上反映了内源性氧自由基的清除活力；膜脂质降解产生的主要代谢产物MDA是常用来反映脂质过氧化的一个指标，NO是机体内极其重要的信使，在脑缺血的过程中有双重作用，一方面可以缩小梗死的面积，增加皮质供血，另一方面可与缺血产生的氧自由基协同作用，造成神经细胞损伤。监测脑组织SOD、GSH-Px、MDA、NO水平对研究脑组织缺血再灌注损伤有重要意义。
D-半乳糖亚急性衰老模型是利用给小鼠连续皮下注射大剂量D-半乳糖，造成动物糖代谢紊乱而建立的。D-半乳糖在半乳糖氧化酶催化下，可产生超氧阴离子等氧自由基。氧自由基具有高度活性，可以攻击膜磷脂中的不饱和脂肪酸、蛋白质、酶及胞核内的DNA等；自由基还可使脂质发生过氧化作用而形成过氧化脂质(LPO)，LPO随蛋白沉淀后在酸性条件下生成MDA，MDA是活泼的交联剂，可迅速与磷脂酰乙醇胺生成荧光色素，然后与蛋白质、肽类、脂类结合形成板层状脂褐质(LF)。用D-半乳糖连续注射，小鼠可产生多项生化指标的变化，如脑组织MDA、LF含量的升高；脑中SOD、血中GSH-Px、红细胞中CAT活力的降低，这些变化和自然衰老的变化相一致，说明D-半乳糖可引起细胞和机体的衰老。SOD、GSH-Px、CAT活性间接反映机体清除氧自由基的能力；而MDA、LF水平高低间接反映细胞受自由基攻击的严重程度。
本试验通过小鼠脑缺血再灌注损伤合并皮下注射D-半乳糖，造成脑损伤后AchE活性明显增强，从而出现认知障碍，空间学习能力明显下降；试验还观察到复合模型小鼠脑组织SOD、GSH-Px活性明显下降，MDA、NO含量明显升高。灌胃给予本发明中药组合物后小鼠脑组织AchE活性下降，SOD、GSH-Px活性明显增强，MDA、NO含量明显下降，空间学习能力明显增强，表明表明本发中药组合物对缺血再灌注造成的活性氧生成及脂质过氧化有抑制作用，从而阻止不可逆性神经细胞损伤，进而改善学习记忆能力，且本发明中药组合物的药效好于对比例中药组合物的药效。
试验例10 对多发梗塞性痴呆(MID)模型大鼠的影响
1.实验材料
(1)动物：SD大鼠，雄性，260～280g体重。由北京维通利华实验动物技术发展有限公司提供。
(2)药物：
本发明中药组合物及对比例中药组合物(自制，按本发明各实施例及对比例所述的方法制备成相应的提取物混合物)，试验时用蒸馏水配成0.15g/mL(按组合物的原药量计算)。
达纳康片(tanakan)，银杏叶标准化萃取物(Egb761)40mg/片，法国博福－益普生制药工业公司生产，试验时用蒸馏水配成2.0mg/mL。海藻酸钠微球血管栓塞剂(KMG)，规格：100～200μm，北京圣医耀科技发展有限责任公司；生长抑素(SS)放免试剂盒，神经肽Y(NPY)，均由海军放免技术中心提供；降钙素基因相关肽(CGRP)放免试剂盒，北京福瑞生物工程公司提供，内皮素(ET)，由北京北免东雅生物技术研究所提供。去甲肾上腺素(NE)、多巴胺(DA)、3，4－二羟基苯乙酸(DOPAC)、高香草酸(HVA)、5-羟色胺(5-HT)、5-羟吲哚乙酸(5-HIAA)、磷酸二氢钠(NaH2PO4)、柠檬酸、硫代硫酸钠(Na2S2O5)、乙二胺四乙酸四钠盐(EDTA)均为AR级；辛烷基磺酸钠(OSA)为HPLC级，均为美国Sigma公司产品；乙腈(ACN)、高氯酸(HClO4)均为HPLC级，美国Fisher Scientific公司产品。
(3)仪器：
超速离心机(日本HITACHI 55P-72)；16通道Coularray库仑阵列电化学高效液相色谱仪及色谱工作站，580泵，5600A电化学检测器，542自动进样器，美国ESA公司产品；柱子(C1815×4.6mm 5μm)，美国WATERS公司产品。低温冰箱，法国JOUAN公司产品；针筒式微孔滤膜过滤器：水系(0.22μm)，天津腾达过滤器厂产品。MORRIS水迷宫，中国医学科学院药物研究所生产。FT-630Gγ-计数仪，北京核设备厂生产。
(4)色谱条件：
流动相组成：磷酸二氢钠90mmol/L，柠檬酸50mmol/L，辛烷基磺酸钠1.7mmol/L，乙二胺四乙酸四钠(EDTA)0.05μmol/L。上述溶液以0.2um水系膜过滤。滤过液中加入乙腈(10％)。流动相的流速为0.6mL/min，工作电极1：-150mV，工作电极2：450mV，工作电极3：500mV，工作电极4：550mV。
标准液的配制：以0.1mol/L的高氯酸配标准品作为储备液(100μg/mL)，存放于-70℃冰箱中。将储备液稀释至10μg/mL作为稀释液。将稀释液配成系列浓度作为标准液。用0.15mol/L的高氯酸(含0.04％焦亚硫酸钠和0.04％EDTA)为工作液。
2.实验方法
大鼠水合氯醛腹腔麻醉(350mg/kg)，颈部备毛、消毒，正中切口，分离颈总动脉、颈内动脉及颈外动脉。结扎颈外动脉，动脉夹夹闭颈总动脉，以注射器从颈外动脉向颈内动脉注入0.1mL KMG，松开动脉夹，结扎颈外动脉，缝合伤口，回笼饲养。假手术组只分离颈总动脉、颈内动脉及颈外动脉，不注射微球。注射用青霉素钠抗感染四天。手术次日观察动物行为表现，以前肢有行为学改变者判定为模型成立(否则弃去不用)，并将其分为模型组，达纳康20mg/kg组，本发明中药组合物组(6组，与试验例1所用剂量相同)，对比例组(8组，与试验例1所用剂量相同)。
手术10天后开始灌胃给药，假手术组和模型组均灌胃等体积蒸馏水，每日一次，连续90天。动物于造模后40、100天(药后30、90天)测定大鼠MORRIS水迷宫寻找平台的持续时间和路径长度，末次给药后60min经水合氯醛腹腔麻醉腹主动脉抽血，抗凝、分离血浆，并迅速断头取出大脑，分离皮层、海马、纹状体，称重，液氮固化，-70℃低温冰箱保存。每100mg脑组织加1mL冰冷的工作液，在冰浴中用电动匀浆器匀浆15s(11000转/min)，0-4℃14000rpm离心20min，提取上清液，用0.22μm滤膜和注射器式的过滤器过滤， 滤液分装、保存于-20℃冰箱中待测。自动进样器每次进样10μl，HPLC测定5-HT、5-HIAA、NE、DA等单胺类神经递质的含量(HPLC-ECD法)。血浆测定ET、NPY、SS、CGRP(放免法)。结果进行统计学处理(t检验)。
3.结果
3.1对大脑皮层、纹状体、海马重量的影响
注射生物微球后，微球随血流进入脑部血管，24h可形成大小不一的多发性脑梗塞，其部位主要为皮层、海马、纹状体。100天后，模型组梗塞区液化坏死出现空洞，脑组织明显萎缩，海马变小，皮层变薄，重量明显减轻，假手术组与模型组比较有显著性差异(P<0.05～0.01)。与模型组比较，本发明中药组合物组及对比例中药组合物组大鼠大脑皮层重量有所增加，但无统计学意义，本发明中药组合物组纹状体、海马重量明显增加(P<0.05)；对照药达纳康及对比例中药组合物组大鼠大脑皮层、纹状体、海马重量均有所增加，但无统计学意义。见表19。
表19.对MID大鼠大脑皮层、纹状体、海马重量的影响(
Figure PCTCN2015099381-appb-000046
n＝10)
Figure PCTCN2015099381-appb-000047
注：与模型组相比*P<0.05；**P<0.01
3.2对MORRIS水迷宫持续时间的影响
与模型组比较，假手术模型组在水迷宫中的持续时间比较明显缩短(P<0.05)；给药后30天，本发明中药组合物组与模型组比较水迷宫持续时间明显缩短(P<0.05)，其它组未见明显改变；给药90天后，本发明中药组合物组与模型组比较水迷宫持续时间均明显缩短(P<0.05～0.01)对比例1～3、7组与模型组比较水迷宫持续时间均明显缩短(P<0.05)，其 它组未见明显改变。见表20。
表20对MID大鼠MORRIS水迷宫持续时间的影响
Figure PCTCN2015099381-appb-000048
Figure PCTCN2015099381-appb-000049
注：与模型组相比*P<0.05；**P<0.01
3.3对血浆ET、NPY、SS、CGRP含量的影响
在造模后100天后与模型组比较，假手术组动物血浆NPY含量明显升高，ET含量明显降低(均P<0.01)，SS及CGRP含量升高但无统计学意义；造模后100天(药后90天)，与模型组比较，本发明中药组合物组NPY及SS含量显著升高(P<0.01)，ET含量明显降低(P<0.05)，CGRP含量未见明显改变；对比例1～3、7中药组合物组NPY及SS含量显著升高(P<0.05～0.01)，ET含量有降低趋势，CGRP含量未见明显改变；对比例4～6、8中药组合物组NPY及SS含量有升高趋势，ET含量有降低趋势，CGRP含量未见明显改变；对照药达纳康组NPY及SS含量明显升高(P<0.05)，ET含量显著降低(P<0.01)，CGRP含量未见明显改变。见表21。
表21对血浆ET、NPY、SS、CGRP含量的影响(
Figure PCTCN2015099381-appb-000050
n＝10)
Figure PCTCN2015099381-appb-000051
Figure PCTCN2015099381-appb-000052
注：与模型组相比*P<0.05；**P<0.01
3.4对脑内单胺类神经递质的影响
(1)对皮层单胺类神经递质的影响
造模后100天，与模型组比较，假手术组大鼠皮层5-HT、5-HIAA含量明显升高(P<0.05)，NE、DA含量升高，但无统计学意义；本发明中药组合物组5-HT含量明显升高(P<0.01)，DA、5-HIAA含量均明显升高(P<0.05)；对比例1～3、7中药组合物组DA、5-HT含量均明显升高(P<0.05)；对比例4～6、8中药组合物组5-HT含量有所升高，但无统计学差异；对照药达纳康组5-HT、5-HIAA含量明显升高(P<0.01)，其它指标均未见明显改变。见表22。
表22对MID大鼠皮层单胺类神经递质水平的影响(
Figure PCTCN2015099381-appb-000053
n＝10)
Figure PCTCN2015099381-appb-000054
Figure PCTCN2015099381-appb-000055
注：与模型组相比*P<0.05；**P<0.01
(2)对纹状体单胺类神经递质的影响
造模后100天，与模型组比较，假手术组大鼠纹状体5-HT、5-HIAA含量明显升高(P<0.05)，说明造模成功；造模后100天(给药后90天)，与模型组比较，本发明实施例5、9组5-HT含量明显升高(P<0.05)，本发明实施例1，2，6，11组5-HIAA含量明显升高(P<0.05)；对比例中药组合物组和对照药达纳康组5-HT、5-HIAA含量未见明显改变。见表23。
表23对MID大鼠纹状体单胺类神经递质水平的影响(
Figure PCTCN2015099381-appb-000056
n＝10)
Figure PCTCN2015099381-appb-000057
注：与模型组相比*P<0.05；**P<0.01
(3)对海马单胺类神经递质的影响
造模后100天(给药后90天)，与模型组比较，假手术组组大鼠海马DA、5-HT、5-HIAA含量明显升高(P<0.05～0.01)，NE含量升高，但无统计学意义；本发明中药组合物组NE、DA、5-HT含量均明显升高(P<0.05～0.01)；对比例中药组合物组5-HT含量明显升高(P<0.05)；对照药达纳康组各项指标均无明显改变。见表24。
表24对MID大鼠海马单胺类神经递质水平的影响(
Figure PCTCN2015099381-appb-000058
n＝10)
Figure PCTCN2015099381-appb-000059
注：与模型组相比*P<0.05；**P<0.01
小结
研究认为脑梗塞是否引起痴呆主要与梗塞灶的大小、多少及部位有关。调查表明，梗死灶体积大于50mL可以合并痴呆，大于100mL则经常合并痴呆；也有调查发现，VaD患者中大面积梗死占11.2％，小面积梗死灶占88.8％，多发病灶占97.6％，提示病灶体积小，也可发生痴呆，尤其是梗死灶数目越多，痴呆发生率越高。也有研究认为，脑梗死的部位是导致痴呆的关键性因素；多数报道指出，CT上发现有脑室旁白质病变改变者，痴呆发生率显著增高。因而基于多发梗塞性痴呆(MID)模型进行相关研究对判断药物治疗VaD的有效性有指导作用。
学习和记忆的脑机制主要包括神经生理机制和神经生化机制，神经生理机制着重探讨记忆在脑中的定位及其伴随的生物电活动规律，神经生化机制主要阐述神经递质、神经肽及生物大分子对学习和记忆的作用及其关系。神经肽是泛指存在于神经组织并参与神经系统功能作用的内源性活性物质，是一类特殊的信息物质。特点是含量低、活性高、作用广 泛而又复杂，在体内调节多种多样的生理功能，如痛觉、睡眠、情绪、学习与记忆乃至神经系统本身的分化和发育都受神经肽的调节。生长抑素(somatostatin,SS)是神经系统中广泛分布的神经活性肽，其中以下丘脑和大脑的皮层和海马含量最高，这种分布与其对认知功能的调控有关。脑中的SS对学习和记忆具有积极的调控作用，SS调控学习和记忆的途径，一是直接途径，通过兴奋SS能神经，引起钙离子内流增加，二是可能通过其他神经道质来实现。SS能神经元和胆碱能神经元相距很近，有突触联系，这为其功能联系提供了证据：SS可能促进乙酰胆碱释放从而影响学习和记忆功能。内皮素(endothelin-1，ET)是近年来深入研究的一种血管活性肽，也是一种神经介质，具有强烈的收缩血管特性，广泛分布于机体各个器官,除能引起血管收缩外，尚能对神经元和胶质细胞造成直接的损害，在出血性、缺血性脑血管病患者血浆ET浓度明显升高，ET水平与脑梗死灶的大小呈显著正相关。ET又作用于神经细胞，使神经细胞钙超载，产生自由基，进一步加重脑损害。神经肽Y(neuropeptide Y,NPY)是由36个氨基酸构成的一种活性多肽，在中枢神经系统中主要分布于大脑皮层、海马、丘脑、下丘脑及脑干，以海马浓度最高，而海马是调节学习和记忆的关键部位。NPY原在神经细胞胞体内合成后，运送至神经末梢并储存在囊泡中，与去甲肾上腺素、肾上腺素、γ-氨基丁酸、生长抑素等经典神经递质共存。NPY不但与长时记忆存在密切联系，而且对短时记忆也有一定的影响。NPY主要通过其受体发挥生物学作用，目前研究认为，海马中Y2受体的mRNA表达最为丰富，该受体对学习记忆的调节可能是长时程传递增强(long-term potentiation，LTP)机制。由于上述神经肽与学习记忆的相关性以及它们与胆碱能神经递质以及肾上腺素能神经递质的相关性，我们考察了MID模型大鼠的血浆神经肽和皮层、纹状体、海马的单胺类神经递质水平以及药物的作用。
试验以生物微球注入颈内动脉的方法，造成大鼠多发梗塞性痴呆(MID)模型。结果表明，模型组动物24h后出现神经行为障碍，形成多发梗塞灶，10天梗塞部位脑组织液化坏死，100天皮质变薄、海马萎缩，皮层、纹状体、海马重量均明显减轻，出现学习记忆障碍，皮层及海马5-HT、5-HIAA含量均明显降低，DA含量有降低趋势但无明显差异(P<0.1)，血浆中NPY含量明显降低，ET含量明显升高，SS及CGRP有降低趋势。
灌胃给予本发明中药组合物30～90天后(造模40～100天)，与模型组比较，本发明中药组合物组大鼠在MORRIS水迷宫中持续时间明显缩短，学习记忆能力明显提高；给药90天后海马重量明显增加(P<0.05)，皮层5-HT、5-HIAA、DA含量、纹状体5-HT、5-HIAA含量及海马NE、DA、5-HT含量均明显升高(P<0.05～0.01)，血浆NPY和SS含量明显升高，ET含量明显降低。提示本发明中药组合物可升高MID大鼠脑内与学习记忆相关的神经递质含量，从而对血管性痴呆有治疗作用。另外，从对比试验数据可知本发明中药组合物的效果好于对比例中药组合物组，且具有统计学意义。

Claims (10)

  1. 一种用于预防或治疗心脑血管疾病和/或痴呆症的中药组合物，制成所述中药组合物的药物原料为下列重量份配比的药味：
    人参1份、银杏叶0.8-1.5份、西红花0.018-0.030份。
  2. 根据权利要求1所述的中药组合物，其特征在于，制成所述中药组合物的药物原料为下列重量份配比的药味：
    人参1份、银杏叶1份、西红花0.018-0.030份。
  3. 根据权利要求2所述的中药组合物，其特征在于，制成所述中药组合物的药物原料为下列重量份配比的药味：
    人参1份、银杏叶0.9-1.2份、西红花0.020-0.025份。
  4. 根据权利要求3所述的中药组合物，其特征在于，制成所述中药组合物的药物原料为下列重量份配比的药味：
    人参1份、银杏叶1份、西红花0.020-0.025份。
  5. 根据权利要求4所述的中药组合物，其特征在于，制成所述中药组合物的药物原料为下列重量份配比的药味：
    人参1份、银杏叶1份、西红花0.022份。
  6. 根据权利要求1-5任一项所述的中药组合物的制备方法，包括：称取人参、银杏叶、西红花的步骤，制备人参提取物的步骤，制备银杏叶提取物的步骤，制备西红花提取物的步骤，以及将上述人参提取物、银杏叶提取物和西红花提取物混合的步骤。。
  7. 根据权利要求6所述的方法，其特征在于，所述制备人参提取物的步骤中得到的主要成分为人参总皂苷，所述制备银杏叶提取物的步骤中得到的主要成分为银杏叶总黄酮和总内酯，所述制备西红花提取物的步骤中得到的主要成分为西红花总苷。
  8. 根据权利要求7所述的方法，其特征在于，所述制备人参提取物的步骤为：将人参粉碎成粉末，乙醇回流提取2次，过滤，滤液减压回收溶剂至70℃时相对密度1.12～1.14，加入相当生药2-6倍量的水搅匀，冷沉，上清液上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗脱，收集醇洗脱液浓缩至干，得到人参提取物；
    所述制备银杏叶提取物的步骤为：银杏叶粗粉加乙醇温浸，过滤，滤渣再加乙醇温浸，过滤，合并两次浸滤液，减压浓缩至70℃时相对密度为1.12～1.14，加入生药2-6倍量的水，充分搅匀，冷沉，滤过，滤过液上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗脱，收集乙醇洗脱液，浓缩至70℃时相对密度为1.02～1.04，乙酸乙酯：正丁醇萃取，合并萃取液，减压回收溶剂，得到银杏叶提取物；
    所述制备西红花提取物的步骤为：西红花生药加入乙醇冷浸，滤过，药渣再加入乙醇冷浸，滤过，合并两次浸滤液，减压浓缩至70℃时相对密度为1.12～1.14，加入水，上大孔吸附树脂，载药树脂先以蒸馏水冲洗，再以乙醇洗，将乙醇洗脱液浓缩至70℃时相对密度1.02～1.04，浓缩至干，得到西红花提取物。
  9. 一种用于预防或治疗心脑血管疾病和/或痴呆症的中药制剂，所述制剂由根据权利要求1-5任一项所述的中药组合物或根据权利要求6-8任一项所述的方法制备得到的中药组合 物，和至少一种药学上可接受的辅料制成。
  10. 根据权利要求1-5任一项所述的中药组合物、权利要求6-8任一项所述方法制备得到的中药组合物、或权利要求9所述的中药制剂在制备用于预防或治疗心脑血管疾病和/或痴呆症的药物中的用途；优选地，所述心脑血管疾病选自缺血性脑血管疾病、冠心病或心绞痛中的至少一种；优选地，所述痴呆症为老年性痴呆，尤其是血管性痴呆。
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US10967023B2 (en) 2021-04-06
RU2674264C1 (ru) 2018-12-06
AU2015373702B2 (en) 2018-08-09
CN104587087A (zh) 2015-05-06
KR102005265B1 (ko) 2019-07-30
JP2018505155A (ja) 2018-02-22
AU2015373702A1 (en) 2017-07-13

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