A composite plant extract for promoting vascular regeneration and repair, and its preparation method and application

Abstract

The present invention relates to the technical field of composite plant extracts, and in particular to a composite plant extract for promoting blood vessel regeneration and repair, and a preparation method and application thereof, wherein the composite plant extract is made of the following components in parts by weight: 6 to 12 parts of Cyathula officinalis, 3 to 8 parts of Ganoderma lucidum, 14 to 20 parts of Salvia miltiorrhiza, 5 to 10 parts of Panax notoginseng, 12 to 18 parts of Carthamus tinctorius, 8 to 14 parts of Astragalus, 40 to 50 parts of Leonurus japonicus and 1 to 5 parts of Bupleurum chinense. The composite plant extract prepared by the Chinese medicine compatibility principle can be effectively applied to blood vessel regeneration and repair, is safer and has no side effects, and has a good therapeutic effect on cardiovascular and cerebrovascular diseases such as atherosclerosis.

Description

Technical Field

[0001] The invention relates to the technical field of composite plant extracts, in particular to a composite plant extract for promoting blood vessel regeneration and repair, and a preparation method and application thereof. Background Art

[0002] The vascular system is like the "lifeline" of the human body, shouldering the critical mission of delivering oxygen and nutrients to tissues throughout the body and taking away metabolic waste. However, under the impact of various pathogenic factors, blood vessels are very susceptible to damage, and this condition is closely related to many difficult diseases. For example, the onset of myocardial infarction often results from the sudden blockage of the coronary artery, which causes the myocardial blood supply to be interrupted instantly, and large areas of myocardium necrotize due to ischemia and hypoxia; diabetic foot is a serious complication caused by diabetes. Long-term high blood sugar causes lesions, stenosis, or even occlusion of blood vessels in the lower limbs, which in turn causes ulceration and infection of foot tissues, making healing difficult, causing great pain to patients, seriously affecting their quality of life, and may even lead to amputation.

[0003] At present, there are many shortcomings in the clinical treatment methods used to promote vascular regeneration and repair. Although traditional chemical synthetic drugs can intervene in the process of vascular regeneration to a certain extent, they are often accompanied by many disadvantages. This type of drug has poor targeting, and when trying to stimulate vascular repair, it is easy to have unexpected effects on other healthy tissues and organs, causing side effects such as gastrointestinal discomfort, liver and kidney damage, etc. Moreover, some patients are intolerant to chemical drugs due to individual differences, making it difficult to continue treatment.

[0004] In comparison, extracts from natural plants have gradually become the focus of medical research due to their unique advantages. Plant extracts come from nature. In the long history of medical practice, many plants have been proven to have excellent medicinal value, high safety, and good human acceptance. More importantly, the composition of plant extracts is complex and diverse, and multiple active ingredients coexist. They work together and are likely to produce synergistic effects, providing a new, gentler and more effective solution to the problem of vascular regeneration and repair. This is the traditional application field of traditional Chinese medicine. The use of natural plant Chinese medicinal materials has higher safety performance when acting on lesions. However, the current research on plant extracts to promote vascular regeneration and repair is not deep and systematic enough, and there is still a lot of potential to be tapped, and more groundbreaking exploration and innovation are urgently needed.

[0005] Endothelial progenitor cells (EPCs) are the precursor cells of endothelial cells (ECs). The signal molecules produced by tissue ischemia and hypoxia can promote the mobilization of bone marrow EPCs to the peripheral circulation, which has a positive significance for the repair and angiogenesis of vascular endothelial damage. Endothelial damage is the basis for the onset of cardiovascular and cerebrovascular diseases such as atherosclerosis. In recent years, EPCs have become a hot topic in the study of cardiovascular and cerebrovascular diseases. A large number of studies have reported that when the vascular endothelium is damaged, the number of endothelial progenitor cells in the peripheral blood is significantly lower than that of normal people, accompanied by a decrease in the levels of stromal cell-derived factor 1 and interleukin 8, and the levels of stromal cell-derived factor 1 and interleukin 8 in the peripheral blood have recovered with the progression of the disease. It has long been confirmed that endothelial progenitor cells have paracrine functions, and can release active cytokines such as vascular endothelial growth factor, basic fibroblast growth factor, granulocyte-macrophage colony-stimulating factor, stromal cell-derived factor 1, granulocyte colony-stimulating factor and interleukin 8. These cytokines can not only mobilize endothelial progenitor cells from the bone marrow into the peripheral blood, but also promote endothelial progenitor cell proliferation, differentiation, chemotaxis and homing. How to apply Chinese medicinal materials to this research field and obtain safer and side-effect-free drugs for the treatment of cardiovascular and cerebrovascular diseases is the ultimate goal of our research. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a composite plant extract for promoting vascular regeneration and repair, and a preparation method and application thereof. The composite plant extract prepared using the principles of Chinese medicine compatibility can be effectively applied to vascular regeneration and repair, is safer and has no side effects, and has a good therapeutic effect on cardiovascular and cerebrovascular diseases such as atherosclerosis.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A composite plant extract for promoting blood vessel regeneration and repair is prepared from the following components in parts by weight: 6-12 parts of Cyathula officinalis, 3-8 parts of Ganoderma lucidum, 14-20 parts of Salvia miltiorrhiza, 5-10 parts of Panax notoginseng, 12-18 parts of Carthamus tinctorius, 8-14 parts of Astragalus membranaceus, 40-50 parts of Leonurus japonicus and 1-5 parts of Bupleurum chinense.

[0009] Preferably, the composite plant extract is made of the following components in parts by weight: 8-10 parts of Cyathula officinalis, 4-6 parts of Ganoderma lucidum, 17-19 parts of Salvia miltiorrhiza, 7-9 parts of Panax notoginseng, 14-16 parts of Carthamus tinctorius, 10-12 parts of Astragalus, 43-47 parts of Leonurus japonicus and 2-4 parts of Bupleurum chinense.

[0010] Preferably, the composite plant extract is made of the following components in parts by weight: 9 parts of Cyathula officinalis, 5 parts of Ganoderma lucidum, 18 parts of Salvia miltiorrhiza, 8 parts of Panax notoginseng, 15 parts of Carthamus tinctorius, 11 parts of Astragalus membranaceus, 45 parts of Leonurus japonicus and 3 parts of Bupleurum chinense.

[0011] A method for preparing a composite plant extract for promoting vascular regeneration and repair, characterized in that it comprises the following steps:

[0012] S1, weighing the medicinal materials of the formula;

[0013] S2, mixing and crushing the Chinese cyperus rotundus, Panax notoginseng, safflower and Leonurus japonicus into powder, adding Ganoderma lucidum powder to the mixture to obtain a mixed coarse powder A, adding 3.5 times the volume of drinking water to the mixed coarse powder A to soak for 4 to 8 hours, adding complex enzyme A to perform enzymolysis, then adding 75% ethanol to perform hot reflux extraction twice, each time for 1.5 hours, recovering the ethanol and drying to obtain a mixed extract A;

[0014] S3, the decoction pieces of Danshen and Bupleurum are crushed into a mixed coarse powder B, and the compound enzyme B is added for enzymolysis, and then the mixture is extracted by adding water and hot reflux twice, each time for 1.5 hours, and concentrated to a density of 1.10-1.15, and ethanol is added to an alcohol content of 70%, and the mixture is allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B;

[0015] S4, fully mixing the mixed extract A obtained in step S2 and the mixed extract B obtained in step S3 using a stirring mixer to obtain a composite plant extract.

[0016] Preferably, the complex enzyme A in step S2 is cellulase, glycosidase and papain in a mass ratio of 5:8:6.

[0017] Preferably, the dosage ratio of the complex enzyme A to the mixed coarse powder B in step S2 is 1:28, the extraction temperature is 43-46° C., the enzymolysis time is 90 min, and the enzymolysis pH is 5.7.

[0018] Preferably, the complex enzyme B in step S3 is cellulase and β-glucosidase in a mass ratio of 3:1.

[0019] Preferably, the dosage ratio of the complex enzyme B to the mixed coarse powder A in step S3 is 1:32, the extraction temperature is 50-54° C., the enzymolysis time is 130 min, and the enzymolysis pH is 4.2.

[0020] According to the use of a composite plant extract for promoting vascular regeneration and repair as described in any one of the above items, the composite plant extract is used to prepare drugs for treating atherosclerosis and cardiovascular and cerebrovascular diseases.

[0021] Preferably, the composite plant extract is used in the preparation of a drug for promoting vascular regeneration and repair.

[0022] Beneficial effects of the present invention:

[0023] 1. From the perspective of traditional Chinese medicine, the present invention uses Sichuan Cyathula and Ganoderma Lucidum as the main medicine of the whole prescription, mainly targeting the disease of blood stasis and obstruction, and improves the symptoms of heart depression and insufficient heart qi by promoting blood circulation and combining qi-tonifying and tranquilizing the mind, thereby improving the microcirculation of the human body, dilating blood vessels, and reducing blood viscosity to play the effect of promoting blood circulation and removing blood stasis, thereby preventing or treating cardiovascular and cerebrovascular diseases; then, Danshen, Panax Notoginseng and Carthamus Tinctorius are used to regulate the qi mechanism, replenish the middle and lower qi, and promote qi and blood circulation, and are altogether the ministerial medicines, which assist in enhancing the efficacy of Sichuan Cyathula and Ganoderma Lucidum; Astragalus and Leonurus japonicus are adjuvants, which promote the production of body fluids and promote blood circulation, replenish qi and raise yang; and then, Bupleurum chinense is used as the guiding medicine to dredge the meridians, lead the medicine into the pericardium meridian, and assist the whole prescription in regulating body functions. The whole prescription works together to promote blood circulation and remove blood stasis, replenish qi and nourish the heart, and relieve stasis and resolve stagnation, and is used to treat cardiovascular and cerebrovascular diseases such as atherosclerosis.

[0024] 2. According to modern medical theory, the active ingredient of Sichuan Cyathula is cyperasterone, a natural EGFR inhibitor, which exhibits anti-proliferative effects by inducing cell apoptosis and cell cycle arrest, and has the effects of promoting blood circulation and improving the symptoms of atherosclerotic diseases. The triterpenes of Ganoderma lucidum in Ganoderma lucidum have the effects of promoting blood circulation, improving metabolism, and regulating blood lipids, while adenine nucleosides can inhibit platelet aggregation, prevent thrombosis, and have anti-inflammatory, analgesic, and sedative effects. Combined with Ganoderma lucidum polysaccharides, it can enhance the body's immunity and assist in lowering blood sugar. Assisted by salvianolic acid B in Salvia miltiorrhiza, it can effectively help improve the bone marrow mobilization efficiency of endothelial progenitor cells, and can also promote the adhesion of endothelial progenitor cells to the collagen surface, which is conducive to the homing of endothelial progenitor cells. Assisted by the effects of other drugs, it acts on cardiovascular and cerebrovascular diseases such as atherosclerosis.

[0025] 3. The medicinal materials used in the formula of the present invention are simple, but can be prepared by the simplest and most convenient extraction method. After a large number of experiments, the original medicinal materials of the formula were finally divided into two groups for enzymatic extraction. The experiment obtained the optimal extraction conditions. Compared with the extraction and mixing of single medicinal materials separately, the preparation efficiency is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the percentage of endothelial progenitor cells in the surgical group and the sham-operated group of the present invention (Note: Compared with the sham-operated group, a P<0.05);

[0027] Figure 2 is the percentage of endothelial progenitor cells occupying nucleated cells in the surgical group of the present invention (Note: Compared with the control subgroup, aP<0.05); among them, control subgroup (A1), Example 1 subgroup (A2), Example 2 subgroup (A3), Example 3 subgroup (A4), Comparative Example 1 subgroup (A5), Comparative Example 2 subgroup (A6), Comparative Example 3 subgroup (A7) and vascular endothelial growth factor subgroup (A8);

[0028] Figure 3 is the percentage of endothelial progenitor cells occupying nucleated cells in the sham operation group of the present invention (Note: compared with the control subgroup, aP<0.05; compared with the subgroup of Example 1, b P<0.05); among them, control subgroup (B1), Example 1 subgroup (B2), Example 2 subgroup (B3), Example 3 subgroup (B4), Comparative Example 1 subgroup (B5), Comparative Example 2 subgroup (B6), Comparative Example 3 subgroup (B7) and vascular endothelial growth factor subgroup (B8);

[0029] Figure 4 The results of identification of endothelial progenitor cells in the peripheral blood of mice of the present invention;

[0030] Figure 5 The endothelial progenitor cell counts in the low-power field of view of the control group of the present invention and the group 1 of Example 1. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments and drawings. The contents mentioned in the implementation modes are not intended to limit the present invention.

[0032] Sichuan Achyranthes bidentata tastes sweet and slightly bitter, and is neutral in nature. It enters the liver and kidney meridians. Its effects are to remove blood stasis and dredge menstruation, open up joints, and act as a diuretic and relieve stranguria. Sichuan Achyranthes bidentata has a strong blood-breaking ability, so pregnant women and those with heavy menstruation should not take it.

[0033] Lingzhi is sweet and neutral in nature. It enters the heart, lung, liver and kidney meridians and has the functions of replenishing qi and calming the mind, relieving cough and relieving asthma.

[0034] Salvia miltiorrhiza is bitter and slightly cold in nature. It enters the heart and liver meridians. It has the functions of promoting blood circulation and removing blood stasis, relieving menstruation and relieving pain, clearing the heart and eliminating restlessness, cooling blood and eliminating carbuncle.

[0035] Panax notoginseng tastes sweet, slightly bitter, and warm in nature. It enters the liver and stomach meridians. It has the effects of dispersing blood stasis and stopping bleeding, reducing swelling and relieving pain. Panax notoginseng is warm in nature and can promote blood circulation, so pregnant women should take it with caution. It should not be used alone by those with blood heat or yin deficiency with fire.

[0036] Safflower is pungent and warm in nature and enters the heart and liver meridians. It has the effects of promoting blood circulation, relieving blood stasis and relieving pain.

[0037] Astragalus has the effects of tonifying Qi and raising Yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, promoting fluid and nourishing blood, relieving stagnation and relieving numbness, expelling toxins and discharging pus, and astringing sores and regenerating tissues. Astragalus is sweet, slightly warm, and can nourish and stop sweating, and it can easily help fire and restrain evil. Therefore, it should not be taken by people with symptoms such as excessive exterior evil, stagnation of Qi and dampness, stagnant food, the initial stage of carbuncle or severe heat and toxicity after ulceration, as well as those with Yin deficiency and Yang hyperactivity.

[0038] Motherwort has the functions of promoting blood circulation and regulating menstruation, promoting diuresis and reducing swelling, clearing away heat and detoxifying.

[0039] Bupleurum has a pungent, bitter and slightly cold nature. It enters the liver, gallbladder and lung meridians. It has the effects of dispersing and reducing fever, soothing the liver and relieving depression, and raising Yang Qi.

[0040] Example 1

[0041] Weigh according to the formula: 9g of Cyathula, 5g of Ganoderma lucidum, 18g of Salvia miltiorrhiza, 8g of Panax notoginseng, 15g of Carthamus tinctorius, 11g of Astragalus, 45g of Leonurus japonicus and 3g of Bupleurum chinense.

[0042] A mixture of Cyathula officinalis, Panax notoginseng, safflower and Leonurus japonicus is crushed into powder, and then Ganoderma lucidum powder is added to obtain a mixed coarse powder A. The mixed coarse powder A is added into 3.5 times the volume of drinking water and soaked for 6 hours. A composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43-46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; 75% ethanol is then added for hot reflux extraction twice, each time for 1.5 hours, and the ethanol is recovered and dried to obtain a mixed extract A.

[0043] The decoction pieces of salvia miltiorrhiza and bupleurum are crushed into a mixed coarse powder B, and a composite enzyme B (cellulase and β-glucosidase in a mass ratio of 3:1) is added for enzymolysis, wherein the dosage ratio of the composite enzyme B to the mixed coarse powder B is 1:32, the extraction temperature is 50-54°C, the enzymolysis time is 130min, and the enzymolysis pH is 4.2; then, water is added for hot reflux extraction twice, each time for 1.5h, concentrated to a density of 1.10-1.15, ethanol is added to an alcohol content of 70%, allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B.

[0044] The mixed extract A and the mixed extract B are fully mixed with a stirring mixer to obtain a composite plant extract.

[0045] Example 2

[0046] Weigh according to the formula: 6g of Cyathula, 8g of Ganoderma lucidum, 14g of Salvia miltiorrhiza, 10g of Panax notoginseng, 12g of Carthamus tinctorius, 14g of Astragalus membranaceus, 40g of Leonurus japonicus and 5g of Bupleurum chinense.

[0047] A mixture of Cyathula officinalis, Panax notoginseng, safflower and Leonurus japonicus is crushed into powder, and then Ganoderma lucidum powder is added to obtain a mixed coarse powder A. The mixed coarse powder A is added into 3.5 times the volume of drinking water and soaked for 6 hours. A composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43-46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; 75% ethanol is then added for hot reflux extraction twice, each time for 1.5 hours, and the ethanol is recovered and dried to obtain a mixed extract A.

[0048] The decoction pieces of salvia miltiorrhiza and bupleurum are crushed into a mixed coarse powder B, and a composite enzyme B (cellulase and β-glucosidase in a mass ratio of 3:1) is added for enzymolysis, wherein the dosage ratio of the composite enzyme B to the mixed coarse powder B is 1:32, the extraction temperature is 50-54°C, the enzymolysis time is 130min, and the enzymolysis pH is 4.2; then, water is added for hot reflux extraction twice, each time for 1.5h, concentrated to a density of 1.10-1.15, ethanol is added to an alcohol content of 70%, allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B.

[0049] The mixed extract A and the mixed extract B are fully mixed with a stirring mixer to obtain a composite plant extract.

[0050] Example 3

[0051] Weigh according to the formula: 12g of Cyathula, 3g of Ganoderma lucidum, 20g of Salvia miltiorrhiza, 5g of Panax notoginseng, 18g of Carthamus tinctorius, 10g of Astragalus, 47g of Leonurus japonicus and 1g of Bupleurum chinense.

[0052] A mixture of Cyathula officinalis, Panax notoginseng, safflower and Leonurus japonicus is crushed into powder, and then Ganoderma lucidum powder is added to obtain a mixed coarse powder A. The mixed coarse powder A is added into 3.5 times the volume of drinking water and soaked for 6 hours. A composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43-46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; 75% ethanol is then added for hot reflux extraction twice, each time for 1.5 hours, and the ethanol is recovered and dried to obtain a mixed extract A.

[0053] The decoction pieces of salvia miltiorrhiza and bupleurum are crushed into a mixed coarse powder B, and a composite enzyme B (cellulase and β-glucosidase in a mass ratio of 3:1) is added for enzymolysis, wherein the dosage ratio of the composite enzyme B to the mixed coarse powder B is 1:32, the extraction temperature is 50-54°C, the enzymolysis time is 130min, and the enzymolysis pH is 4.2; then, water is added for hot reflux extraction twice, each time for 1.5h, concentrated to a density of 1.10-1.15, ethanol is added to an alcohol content of 70%, allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B.

[0054] The mixed extract A and the mixed extract B are fully mixed with a stirring mixer to obtain a composite plant extract.

[0055] Comparative Example 1

[0056] Weigh according to the recipe: 9g of Cyathula capitata and 5g of Ganoderma lucidum.

[0057] The Cyathula officinalis is crushed into powder and then added with Ganoderma lucidum powder to obtain a mixed coarse powder A. The mixed coarse powder A is added with 3.5 times the volume of drinking water and soaked for 6 hours. A composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43-46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; 75% ethanol is then added for hot reflux extraction twice, each time for 1.5 hours, and the ethanol is recovered and dried to obtain a mixed extract A.

[0058] Comparative Example 2

[0059] Weigh according to the formula: 9g of Cyathula, 18g of Salvia miltiorrhiza, 8g of Panax notoginseng, 15g of Carthamus tinctorius, 11g of Astragalus membranaceus, 45g of Leonurus japonicus and 3g of Bupleurum chinense.

[0060] The herbs of Cyathula officinalis, Panax notoginseng, safflower and Leonurus japonicus are mixed and crushed into powders to obtain a mixed coarse powder A, the mixed coarse powder A is added into 3.5 times the volume of drinking water and soaked for 4 to 8 hours, and a composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43 to 46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; then 75% ethanol is added for hot reflux extraction twice, each time for 1.5 hours, and the mixed extract A is obtained after the ethanol is recovered and dried.

[0061] The decoction pieces of salvia miltiorrhiza and bupleurum are crushed into a mixed coarse powder B, and a composite enzyme B (cellulase and β-glucosidase in a mass ratio of 3:1) is added for enzymolysis, wherein the dosage ratio of the composite enzyme B to the mixed coarse powder B is 1:32, the extraction temperature is 50-54°C, the enzymolysis time is 130min, and the enzymolysis pH is 4.2; then, water is added for hot reflux extraction twice, each time for 1.5h, concentrated to a density of 1.10-1.15, ethanol is added to an alcohol content of 70%, allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B.

[0062] The mixed extract A and the mixed extract B are fully mixed with a stirring mixer to obtain a composite plant extract.

[0063] Comparative Example 3

[0064] Weigh according to the formula: 9g of Cyathula, 5g of Ganoderma lucidum, 18g of Salvia miltiorrhiza, 8g of Panax notoginseng, 15g of Carthamus tinctorius, 11g of Astragalus, and 45g of Leonurus japonicus.

[0065] A mixture of Cyathula officinalis, Panax notoginseng, safflower and Leonurus japonicus is crushed into powder, and then Ganoderma lucidum powder is added to obtain a mixed coarse powder A. The mixed coarse powder A is added into 3.5 times the volume of drinking water and soaked for 6 hours. A composite enzyme A (cellulase, glycosidase and papain in a mass ratio of 5:8:6) is added for enzymolysis, wherein the dosage ratio of the composite enzyme A to the mixed coarse powder A is 1:28, the extraction temperature is 43-46°C, the enzymolysis time is 90 minutes, and the enzymolysis pH is 5.7; 75% ethanol is then added for hot reflux extraction twice, each time for 1.5 hours, and the ethanol is recovered and dried to obtain a mixed extract A.

[0066] The salvia miltiorrhiza medicinal material slices are crushed into coarse powder B, and complex enzyme B (cellulase and β-glucosidase in a mass ratio of 3:1) is added for enzymolysis, wherein the dosage ratio of complex enzyme B to coarse powder B is 1:32, the extraction temperature is 50-54°C, the enzymolysis time is 130min, and the enzymolysis pH is 4.2; then, water is added for hot reflux extraction twice, each time for 1.5h, concentrated to a density of 1.10-1.15, ethanol is added to an alcohol content of 70%, allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B.

[0067] The mixed extract A and the mixed extract B are fully mixed with a stirring mixer to obtain a composite plant extract.

[0068] Test Case

[0069] 1. Materials

[0070] 1.1 Experimental Animals 85 C57BL / 6 mice, SPF grade, 8 weeks old, weighing 19-22 g, of either sex, were purchased from the Experimental Animal Center of Nanjing Medical University.

[0071] 1.2 Experimental reagents and instruments Example 1 composite plant extract, Example 2 composite plant extract, Example 3 composite plant extract, Comparative Example 1 composite plant extract, Comparative Example 2 composite plant extract, Comparative Example 3 composite plant extract; fetal bovine serum was purchased from Hyclone, USA; M199 culture medium, D-Hank's solution and PBS were purchased from GE, USA; vascular endothelial growth factor, basic fibroblast growth factor, photocross-linked collagen and pancreatic enzyme were purchased from Sigma-Aldrich, USA; vascular endothelial growth factor, stromal cell-derived factor 1 and interleukin 8 enzyme-linked immunosorbent assay kits were purchased from ebios, USA The cells were collected from 100 samples of 400 μg / ml ELISA kit, and 100 μg / ml ELISA kit for 30 min. The kit was purchased from 5 μg / ml ELISA kit of 5 μg / ml. The kit was from 4 μg / ml ELISA kit of 5 μg / ml. Science Company; Percoll separation solution was purchased from GE Company, USA; mouse VEGFR-2-APC, CD31-FITC, CD34-PE, CD133-APC and CD45-FITC were purchased from BD Company, USA; culture flasks and cell culture plates were purchased from Corning Company, USA; inverted phase contrast microscope CX51 was purchased from Olympus Company, Japan; cell flow chamber experimental device system was purchased from Flexcell Company, USA; low-speed centrifuge was purchased from Hunan Hercy Instrument Equipment Co., Ltd.; FACSCalibur flow cytometer was purchased from BD Company, USA; CO2 incubator was purchased from Thermo Company, USA.

[0072] 1.3 Experimental methods

[0073] 1.3.1 Experimental Grouping and Methods of Endothelial Progenitor Cell Bone Marrow Mobilization

[0074] A total of 80 8-week-old C57BL / 6 mice were selected and randomly divided into two groups: surgery group and sham operation group, 40 mice in each group. After the mice in the surgery group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital 10 mg / kg, the skin of the left neck was incised to expose the left carotid sheath, and the common carotid artery, internal carotid artery and external carotid artery were bluntly separated. The internal carotid artery and common carotid artery were clamped with a vascular clamp, and the iron wire was inserted into the common carotid artery through the external carotid artery to injure the common carotid artery for 3 minutes. The external carotid artery was ligated with No. 0 surgical thread. Note that the external carotid artery must be ligated at the insertion point during the ligation process to prevent bleeding. The mice in the sham operation group only underwent carotid skin incision and separation and external carotid artery ligation without vascular endothelial injury. The surgical group C57BL / 6 mice that had been injured by the wire were randomly divided into 8 subgroups, namely the control subgroup (A1), the Example 1 subgroup (A2), the Example 2 subgroup (A3), the Example 3 subgroup (A4), the Comparative Example 1 subgroup (A5), the Comparative Example 2 subgroup (A6), the Comparative Example 3 subgroup (A7) and the vascular endothelial growth factor subgroup (A8), with 5 mice in each group. The control subgroup mice were injected with 5% glucose injection via the tail vein at a dose of 10 mL / kg; the Example 1 subgroup mice were injected with 5% glucose dissolved at a mass concentration of 100 g / L Example 1 subgroup, the dose was 10 mL / kg, the Example 2 subgroup mice were injected with 5% glucose dissolved at a mass concentration of 100 g / L Example 2 subgroup, the dose was 10 mL / kg, the Example 3 subgroup mice were injected with 5% glucose dissolved at a mass concentration of 100 g / L Example 3 subgroup, the dose was 10 mL / kg; the Comparative Example 1 subgroup mice were injected with The mice in the subgroup of Comparative Example 2 were injected with 5% glucose dissolved at a mass concentration of 100 g / L and a dose of 10 mL / kg; the mice in the subgroup of Comparative Example 3 were injected with 5% glucose dissolved at a mass concentration of 100 g / L and a dose of 10 mL / kg through the tail vein; the mice in the subgroup of Comparative Example 3 were injected with 5% glucose dissolved at a mass concentration of 100 g / L and a dose of 10 mL / kg through the tail vein; the mice in the vascular endothelial growth factor subgroup were injected with vascular endothelial growth factor at a concentration of 10 μmol / L through the tail vein, and the dose was 10 mL / kg. After 7 days of continuous administration, 0.5 mL of blood from each group of mice was collected by heart blood sampling, diluted with an equal volume of D-Hank's solution, added to 1.5 times the volume of Percoll separation solution with a relative density of 1.090, centrifuged at 2000 r / min for 30 minutes, and the middle layer cells were taken, and then washed again with D-Hank's solution.Resuspend with 50 μL M199 culture medium, add 2 μL CD34-PE flow antibody, wash twice with PBS after 20 min, add 5 μL VEGFR-2-APC flow antibody, wash twice with PBS after 20 min, discard the culture medium after centrifugation, add 150 μL PBS to resuspend the cells, and detect and calculate the percentage of endothelial progenitor cells by flow cytometry. Similarly, the sham-operated C57BL / 6 mice were randomly divided into 6 subgroups, namely, control subgroup (B1), Example 1 subgroup (B2), Example 2 subgroup (B3), Example 3 subgroup (B4), Comparative Example 1 subgroup (B5), Comparative Example 2 subgroup (B6), Comparative Example 3 subgroup (B7) and vascular endothelial growth factor subgroup (B8), 5 mice in each group, and the administration method was consistent with the surgical group.

[0075] 1.3.2 Isolation and identification of endothelial progenitor cells from mouse peripheral blood

[0076] Five C57BL / 6 mice were taken. After blood was collected from the heart, 1 mL of blood was taken from each mouse and added to an equal volume of Percoll separation solution. The blood was placed in a 50 mL centrifuge tube and centrifuged at 1500 r / min for 20 min. The nucleated cells at the interface of the liquid surface were aspirated, diluted with PBS, and centrifuged at 1500 r / min for 5 min. The supernatant was discarded, and the cells after centrifugation were cultured in M199 medium containing 10% fetal bovine serum, 10 μg / L vascular endothelial growth factor, and 3 μg / L basic fibroblast growth factor for 24 h. After that, the non-adherent cells were removed, and the adherent cells were cultured for 7 days. After cell digestion, centrifuge at 1500r / min for 5min, discard the supernatant, wash twice with PBS, resuspend the cells to prepare a single cell suspension and adjust the cell concentration to 1×109L-1, take 200μL of cell suspension, add 5μL of CD34-PE, CD133-APC and CD45-FITC flow cytometry antibodies respectively, incubate at room temperature in the dark for 20min, and detect the expression of CD34, CD133 and CD45 by flow cytometry.

[0077] 1.3.3 Flow chamber experiment

[0078] 100 mg of photo-crosslinked collagen was fully mixed with 10 mg of the composite plant extract (No. 1) of Example 1, and then 2 mL of distilled water was added to fully dissolve the mixture. The mixture was covered on a glass slide and placed under an ultraviolet lamp for crosslinking for 4 hours.

[0079] At the same time, 100 mg of photo-cross-linked collagen was dissolved in 2 mL of distilled water as a control group, covered on a glass slide and placed under ultraviolet light for cross-linking for 4 hours.

[0080] After 7 days of isolation and culture, the EPCs were digested with trypsin, and the floating dead cells were washed with PBS to prepare a cell suspension with a cell concentration of 1×108L-1. The suspension containing EPCs was allowed to flow over a collagen-coated glass slide at 0.01mL / min. The time it took for the EPCs to flow over the same distance and the number of EPCs that finally adhered to the glass slide were detected by an inverted microscope.

[0081] 1.3.4 Levels of stromal cell-derived factor-1 and interleukin-8 in the serum of mice in each group

[0082] 0.5 mL of blood from each group of mice was anticoagulated and centrifuged at 1500 r / min for 20 min before collecting 10 μL of serum. The levels of stromal cell-derived factor-1 and interleukin-8 in the serum of each group of mice were detected by enzyme-linked immunosorbent assay according to the instructions of the kit.

[0083] 1.4 Main outcome measures

[0084] ① Flow cytometry was used to detect the expression of CD34, CD133 and CD45 in endothelial progenitor cells. ② Flow chamber experiment was used to detect the time it took for endothelial progenitor cells to flow through the same distance of collagen-coated glass slides and the number of endothelial progenitor cells that finally adhered to the glass slides. ③ ELISA was used to detect the secretion levels of stromal cell-derived factor-1 and interleukin-8 in mouse serum.

[0085] 1.5 Statistical analysis

[0086] SPSS16.5 statistical software was used. One-way analysis of variance was used for comparison among multiple groups, and SNK test (q test) was used for pairwise comparison among groups. P < 0.05 was considered statistically significant.

[0087] 2Results

[0088] 2.1 Results of Endothelial Progenitor Cell Bone Marrow Mobilization Experiment

[0089] After the carotid artery injury experiment, the percentage of endothelial progenitor cells in the surgery group increased significantly compared with the sham surgery group by marking CD34-PE and VEGFR-2-APC double positive as endothelial progenitor cells. Figure 1 ).

[0090] In the surgical group, the percentage of endothelial progenitor cells occupying nuclei in the Example 1-3 subgroup and the vascular endothelial growth factor subgroup increased significantly compared with the control subgroup, and the difference was significant ( Figure 2 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 1 did not increase significantly, and the difference was significant ( Figure 2 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 2 did not increase significantly, and the difference was significant ( Figure 2 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 3 increased, and the difference was significant ( Figure 2 ).

[0091] In the sham operation group, the percentage of endothelial progenitor cells occupying nuclei in the vascular endothelial growth factor subgroup increased significantly compared with the subgroups of Examples 1-3 and the control subgroup, and the difference was significant ( Figure 3 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 1 did not increase significantly, and the difference was significant ( Figure 3 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 2 did not increase significantly, and the difference was significant ( Figure 3 ); Compared with the control subgroup, the percentage of endothelial progenitor cells occupying nuclear cells in the subgroup of Example 3 increased, and the difference was significant ( Figure 3 ).

[0092] 2.2 Identification results of endothelial progenitor cells in mouse peripheral blood

[0093] Figure 4 The results of the flow cytometry staining with CD34-PE, CD133-APC and CD45 antibodies are shown. The percentage of positive samples for the three antigen markers CD34, CD133 and CD45 is (88.19±5.16)％ .

[0094] 2.3 Flow chamber experiments

[0095] After the slide was removed, the cells were observed and counted under a phase contrast microscope ×40 eyepiece. The results showed that the counting results of Example 1 group were significantly different from those of the control group (P<0.05). Figure 5 .

[0096] 2.4 The composite plant extracts in each group promoted the levels of stromal cell-derived factor-1 and interleukin-8 in paracrine secretion of endothelial progenitor cells

[0097] In the surgical group, the levels of stromal cell-derived factor-1 and interleukin-8 in the serum of the mice in the subgroups of Examples 1-3 and the vascular endothelial growth factor subgroup were significantly different from those in the control subgroup (P<0.05), as shown in Table 1.

[0098] Table 1 Levels of stromal cell-derived factor-1 and interleukin-8 in the serum of mice in the surgical group after 7 days of drug administration ( n=5,ng / L)

[0099] Group Stromal cell-derived factor-1 Interleukin-8 Control subgroup 11.2±1.3 21.5±2.1 Example 1 Subgroup <![CDATA[19.9±1.6 a ]]> <![CDATA[44.4±3.1 a ]]> Example 2 Subgroup <![CDATA[18.3±1.8 a ]]> <![CDATA[42.3±1.8 a ]]> Example 3 Subgroup <![CDATA[17.4±0.9 a ]]> <![CDATA[41.9±3.7 a ]]> Comparative Example 1 Subgroup <![CDATA[13.6±3.1 a ]]> <![CDATA[32.7±2.4 a ]]> Comparative Example 2 Subgroup <![CDATA[12.7±2.49 a ]]> <![CDATA[26.6±0.7 a ]]> Comparative Example 3 Subgroup <![CDATA[17.3±1.7 a ]]> <![CDATA[36.4±1.5 a ]]> VEGF subgroup <![CDATA[20.1±2.9 a ]]> <![CDATA[44.1±2.3 a ]]>

[0100] Note: Compared with the control subgroup, a P<0.05.

[0101] In the sham operation group, the levels of stromal cell-derived factor-1 and interleukin-8 in the serum of mice in the vascular endothelial growth factor subgroup were significantly different from those in the subgroups of Examples 1-3 and the control subgroup (P<0.05), as shown in Table 2.

[0102] Table 2 Levels of stromal cell-derived factor-1 and interleukin-8 in the serum of mice in the sham operation group after 7 days of drug administration ( n=5,ng / L)

[0103] Group Stromal cell-derived factor-1 Interleukin-8 Control subgroup 2.1±0.3 3.3±0.4 Example 1 Subgroup <![CDATA[16.9±0.4 ab ]]> <![CDATA[33.1±1.9 ab ]]> Example 2 Subgroup <![CDATA[14.7±0.8 ab ]]> <![CDATA[27.1±2.7 ab <!-- 8 -->]]> Example 3 Subgroup <![CDATA[15.4±1.6 ab ]]> <![CDATA[24.1±3.6 ab ]]> Comparative Example 1 Subgroup <![CDATA[12.8±0.7 ab ]]> <![CDATA[19.1±1.3 ab ]]> Comparative Example 2 Subgroup <![CDATA[9.3±1.4 ab ]]> <![CDATA[8.2±2.6 ab ]]> Comparative Example 3 Subgroup <![CDATA[14.1±1.7 ab ]]> <![CDATA[22.1±3.2 ab ]]> VEGF subgroup <![CDATA[16.7±1.2 ab ]]> <![CDATA[34.9±2.1 ab ]]>

[0104] Note: Compared with the control subgroup, a P<0.05; compared with the subgroup in Example 1, b P<0.05.

[0105] All technical features in this embodiment can be modified in appearance according to actual needs.

[0106] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious replacement without departing from the concept of the present technical solution is within the protection scope of the present invention.

Claims

1. A composite plant extract for promoting vascular regeneration and repair, characterized in that: The composite plant extract is prepared from the following components in parts by weight: 6-12 parts of Cyathula officinalis, 3-8 parts of Ganoderma lucidum, 14-20 parts of Salvia miltiorrhiza, 5-10 parts of Panax notoginseng, 12-18 parts of Carthamus tinctorius, 8-14 parts of Astragalus, 40-50 parts of Leonurus japonicus and 1-5 parts of Bupleurum chinense.

2. The composite plant extract for promoting angiogenesis and repair according to claim 1, characterized in that: The composite plant extract is prepared from the following components in parts by weight: 8-10 parts of Cyathula officinalis, 4-6 parts of Ganoderma lucidum, 17-19 parts of Salvia miltiorrhiza, 7-9 parts of Panax notoginseng, 14-16 parts of Carthamus tinctorius, 10-12 parts of Astragalus, 43-47 parts of Leonurus japonicus and 2-4 parts of Bupleurum chinense.

3. A composite plant extract for promoting angiogenesis and repair according to claim 2, characterized in that: The composite plant extract is prepared from the following components in parts by weight: 9 parts of Cyathula officinalis, 5 parts of Ganoderma lucidum, 18 parts of Salvia miltiorrhiza, 8 parts of Panax notoginseng, 15 parts of Carthamus tinctorius, 11 parts of Astragalus, 45 parts of Leonurus japonicus and 3 parts of Bupleurum chinense.

4. The method for preparing a composite plant extract for promoting vascular regeneration and repair according to claim 1, characterized in that: The following steps are involved: S1, weighing the medicinal materials of the formula amount; S2, mixing and crushing the Chinese cyperus rotundus, Panax notoginseng, safflower and Leonurus japonicus into powder, adding Ganoderma lucidum powder to the mixture to obtain a mixed coarse powder A, adding 3.5 times the volume of drinking water to the mixed coarse powder A to soak for 4 to 8 hours, adding complex enzyme A to perform enzymolysis, then adding 75% ethanol to perform hot reflux extraction twice, each time for 1.5 hours, recovering the ethanol and drying to obtain a mixed extract A; S3, the decoction pieces of Danshen and Bupleurum are crushed into a mixed coarse powder B, and the compound enzyme B is added for enzymolysis, and then the mixture is extracted by adding water and hot reflux twice, each time for 1.5 hours, and concentrated to a density of 1.10-1.15, and ethanol is added to an alcohol content of 70%, and the mixture is allowed to stand for 16 hours, and concentrated and dried to obtain a mixed extract B; S4, fully mixing the mixed extract A obtained in step S2 and the mixed extract B obtained in step S3 using a stirring mixer to obtain a composite plant extract.

5. The method for preparing a composite plant extract for promoting vascular regeneration and repair according to claim 4, characterized in that: The complex enzyme A described in step S2 is cellulase, glycosidase and papain in a mass ratio of 5:8:

6.

6. The method for preparing a composite plant extract for promoting vascular regeneration and repair according to claim 4, characterized in that: The dosage ratio of the complex enzyme A to the mixed coarse powder A in step S2 is 1:28, the extraction temperature is 43-46° C., the enzymolysis time is 90 min, and the enzymolysis pH is 5.

7.

7. The method for preparing a composite plant extract for promoting vascular regeneration and repair according to claim 4, characterized in that: The complex enzyme B in step S3 is cellulase and β-glucosidase in a mass ratio of 3:

1.

8. The method for preparing a composite plant extract for promoting vascular regeneration and repair according to claim 4, characterized in that: The dosage ratio of the complex enzyme B to the mixed coarse powder B in step S3 is 1:32, the extraction temperature is 50-54° C., the enzymolysis time is 130 min, and the enzymolysis pH is 4.

2.

9. The use of a composite plant extract for promoting vascular regeneration and repair according to any one of claims 1 to 3, characterized in that: The composite plant extract is used for preparing medicines for treating atherosclerosis and cardiovascular and cerebrovascular diseases.

10. The use of a composite plant extract for promoting vascular regeneration and repair according to claim 9, characterized in that: The composite plant extract is used in preparing a medicine for promoting blood vessel regeneration and repair.

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