Traditional Chinese medicine composition for treating apoplexy and preparation method thereof

Abstract

The invention provides a medicine composition for treating apoplexy and a preparation method thereof. The traditional Chinese medicine composition is prepared from astragalus membranaceus, ligusticum wallichii, angelica sinensis, safflower carthamus, peach kernels, red peony roots, elecampane, rhizoma acori graminei, parasitic loranthus, leeches, earthworms and acanthopanax. According to the traditional Chinese medicine composition, an original preparation process is improved, and the traditional Chinese medicines are prepared respectively according to the characteristics of the medicinal materials, so that anticoagulant and antithrombotic active ingredients in the medicinal materials are increased, a relatively remarkable treatment effect is achieved in clinical treatment, and the blood viscosity condition of a stroke patient is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine preparation, and relates to a traditional Chinese medicine composition for treating stroke and a preparation method thereof. Background Art

[0002] Stroke is a disease of qi and blood in the brain meridians. This summarizes the root causes, pathogenesis, and location of the disease. The disorder of qi and blood is a pathological change in which the qi and blood of the human body lose their mutual coordination, which is one of the main pathogeneses of stroke. The qi and blood in the human body are mutually root and used physiologically: qi is yang, and blood is yin; qi mainly warms and nourishes, and blood mainly moistens and nourishes. When qi moves, blood moves; when blood arrives, qi arrives. Pathologically, they damage and lose each other: qi disease affects blood, and blood disease affects qi. When qi declines, blood declines; when blood declines, qi declines; when qi stagnates, blood stasis occurs, and when blood stasis occurs, qi stagnates. When qi rebels upward, qi sinks with blood; qi escapes with blood, and blood perishes with qi. Therefore, "Huangdi Neijing" says: "When qi and blood are not in harmony, all diseases will occur and change." The basic pathogenesis of stroke belongs to the category of blood stasis syndrome in traditional Chinese medicine, and is often closely related to qi stagnation, qi deficiency, qi rebellion, qi sinking, blood deficiency, blood overflow, blood heat, yin deficiency, yang deficiency, etc. in clinical practice. Stroke is a disease syndrome mainly characterized by sudden fainting, unconsciousness, hemiplegia, deviation of the mouth and eyes, speech disorders, and numbness of the body. In mild cases, there may be no fainting, and only symptoms such as hemiplegia and deviation of the mouth and eyes are seen. According to the clinical manifestation characteristics of stroke, acute cerebrovascular diseases in Western medicine are similar to it, including ischemic stroke and hemorrhagic stroke.

[0003] Longshengzhi Capsule is one of the core products of Shaanxi Buchang Pharmaceutical. It has the effects of replenishing qi and activating blood circulation, removing stasis and dredging collaterals. It is used for patients with recovery stage of arteriosclerotic cerebral infarction who are diagnosed as having apoplexy of qi deficiency and blood stasis type in traditional Chinese medicine differentiation of symptoms and signs, showing symptoms such as hemiplegia, unilateral numbness, deviation of the mouth and eyes, speech impairment, etc. The applicant has applied for patents on the corresponding formula, preparation method, detection method, new uses, etc. of this product. The specific application number 02114551.2 patent discloses the formula of Longshengzhi Capsule, including the formula and dosage ratio of twelve traditional Chinese medicines such as Astragalus membranaceus, Hirudo, Ligusticum chuanxiong, Angelica sinensis, Carthamus tinctorius, Prunus persica, Paeonia lactiflora, Aucklandia lappa, Acorus tatarinowii, Pheretima, Taxillus chinensis, Acanthopanax senticosus, etc. This traditional Chinese medicine composition has the effects of replenishing qi and activating blood circulation, removing stasis and dredging collaterals. The patent with application number 200510043066.X discloses the preparation method of Longshengzhi Capsule, and its technological steps include: pulverizing the dry extracts of Hirudo, Pheretima, and Acanthopanax senticosus into fine powder; decocting 9 kinds of medicinal materials such as Astragalus membranaceus twice with water, and mixing them evenly with the powders of Pheretima, Hirudo, and Acanthopanax senticosus to obtain the product. The patent with application number 201210228897.4 discloses a preparation method of a Longshengzhi oral preparation. Among them, 6 kinds of medicinal materials such as Astragalus membranaceus, Ligusticum chuanxiong, Angelica sinensis, Carthamus tinctorius, Prunus persica, and Aucklandia lappa are extracted by heating under reflux with ethanol. The medicinal residues are first extracted with water with pH 3 - 5, then with water with pH 5.5 - 7.5, and finally with water with pH 8.0 - 9.0, and then combined and precipitated with ethanol; after the rough pulverization of Hirudo and Pheretima, they are percolated with 50% - 70% ethanol, the percolation liquid is collected, the ethanol solutions are combined, dried and pulverized into dry extract powder, and the extract powder of Acanthopanax senticosus is added to obtain the product. However, the products prepared by the above preparation methods have poor curative effects in clinical use and also have the disadvantage of low enrichment of active ingredients.

[0004] The above existing patented technologies also have the following problems. For example, the volatile oil components in the formula are treated separately, and with the loss of volatile components, it will also lead to uncertain contents of the components constituting the effective substances in the finally obtained Longshengzhi Capsule, having problems of low accuracy and unstable quality, which affect the actual curative effect of the drug. And the existing technologies have low extraction efficiency for animal and plant medicinal materials and fail to increase the contents of anticoagulant and antithrombotic active ingredients. Therefore, the existing technologies still need to be improved. Summary of the Invention

[0005] The purpose of the present invention is to provide a traditional Chinese medicine composition for treating stroke and its preparation method to solve the deficiencies in the prior art.

[0006] Another object of the present invention is to improve the preparation process of the 12 herbs in Longshengzhi Capsule, including: extracting volatile oils from Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii by steam distillation method; enzymatically extracting Hirudo and Pheretima, which improves the anticoagulant and antithrombotic active ingredients in the herbs; and adding cellulase and pectinase to the extracts of Astragalus membranaceus, Carthamus tinctorius, Prunus persica, Paeonia lactiflora, Taxillus chinensis, and Acanthopanax senticosus to further enrich the content of the effective substances in the above traditional Chinese medicines and improve the therapeutic effect on stroke and cardio-cerebral thrombosis diseases.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A traditional Chinese medicine composition for treating stroke, which is made from the following raw materials in parts by weight: 153 - 226 parts of Astragalus membranaceus, 48 - 71 parts of Ligusticum chuanxiong, 48 - 71 parts of Angelica sinensis, 56 - 83 parts of Carthamus tinctorius, 72 - 107 parts of Prunus persica, 56 - 83 parts of Paeonia lactiflora, 52 - 77 parts of Aucklandia lappa, 52 - 77 parts of Acorus tatarinowii, 52 - 77 parts of Taxillus chinensis, 69 - 101 parts of Hirudo, 40 - 59 parts of Pheretima, and 16 - 24 parts of Acanthopanax senticosus.

[0009] Further, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 174 - 205 parts of Astragalus membranaceus, 55 - 65 parts of Ligusticum chuanxiong, 55 - 65 parts of Angelica sinensis, 64 - 75 parts of Carthamus tinctorius, 82 - 97 parts of Prunus persica, 64 - 75 parts of Paeonia lactiflora, 59 - 70 parts of Aucklandia lappa, 59 - 70 parts of Acorus tatarinowii, 59 - 70 parts of Taxillus chinensis, 78 - 92 parts of Hirudo, 46 - 54 parts of Pheretima, and 18 - 22 parts of Acanthopanax senticosus.

[0010] Even further, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 190 parts of Astragalus membranaceus, 60 parts of Ligusticum chuanxiong, 60 parts of Angelica sinensis, 70 parts of Carthamus tinctorius, 90 parts of Prunus persica, 70 parts of Paeonia lactiflora, 65 parts of Aucklandia lappa, 65 parts of Acorus tatarinowii, 65 parts of Taxillus chinensis, 85 parts of Hirudo, 50 parts of Pheretima, and 20 parts of Acanthopanax senticosus.

[0011] In the formula of Longshengzhi, Astragalus membranaceus and Hirudo are the monarch drugs, Prunus persica, Carthamus tinctorius, Paeonia lactiflora, Ligusticum chuanxiong, and Angelica sinensis are the ministerial drugs, and Acanthopanax senticosus, Taxillus chinensis, Acorus tatarinowii, Pheretima, and Aucklandia lappa are the adjuvant drugs.

[0012] Astragalus membranaceus, sweet in taste, warm in nature, attributing to the lung, spleen, liver and kidney meridians. It has the effects of invigorating qi and lifting yang, strengthening the exterior resistance and consolidating the superficies, inducing diuresis and expelling toxins, and promoting granulation and wound healing. It is mainly used to treat symptoms such as qi deficiency and weakness, anorexia and loose stools, middle qi sinking, chronic diarrhea and rectal prolapse, hematochezia and metrorrhagia, spontaneous sweating due to superficies deficiency, difficult ulceration of carbuncles and sores, non-healing of long-term ulcers, blood deficiency and sallow complexion, internal heat and polydipsia, etc. Astragalus mainly contains triterpenoid saponin components: astragaloside I, II, III, IV, among which the one with the best biological activity is astragaloside IV, namely astragaloside A. Some studies have shown that astragaloside A can play a protective role in cardiovascular diseases by inhibiting inflammatory reactions, inhibiting cardiomyocyte apoptosis, improving myocardial ischemia-reperfusion injury, regulating lipid metabolism, promoting cardiac angiogenesis, inhibiting myocardial fibrosis, and improving myocardial hypertrophy.

[0013] Hirudo, salty and bitter in taste, cold in nature, slightly poisonous, attributing to the liver meridian. Its effects are promoting blood circulation to remove blood stasis and eliminating masses, and it is clinically used for symptoms such as blood stasis and amenorrhea, mass in the abdomen, hemiplegia due to stroke, and injuries from falls. Hirudo mainly contains proteins, including 17 kinds of amino acids, and the hydrolyzed amino acid content reaches 49.4%, including 8 kinds of essential amino acids for the human body, among which the content of glutamic acid is as high as 8.3%; it also contains 14 kinds of trace elements such as Zn, Mn, Fe, Co, Cr, Se, Mo, Ni, etc. The active components isolated from different species of Hirudo are different, which can be roughly divided into two categories: one is the components that directly act on the coagulation system, including thrombin inhibitors and some other substances related to blood coagulation, such as hirudin, philippinensis hirudin, haemadipsa sylvestris hirudin, etc.; the second category is other protease inhibitors and other active components, such as fibrinolysin, decorsin, etc. Hirudin is the main effective medicinal component of Hirudo, which is an acidic single-chain polypeptide composed of 65 amino acid residues and without glycosylation. Hirudin has strong anticoagulant, thrombolytic and lipid-lowering effects.

[0014] Peach kernel, bitter and sweet in taste, flat in nature, attributing to the heart, liver and large intestine meridians. Its effects are promoting blood circulation to remove blood stasis, loosening the bowels and relieving constipation, and relieving cough and asthma. Modern research has found that peach kernel mainly contains: amygdalin, triglyceride, as well as carbohydrates, proteins and amino acids, volatile oils, vitamins, etc. Modern research shows that amygdalin has the effect of anti-atherosclerosis.

[0015] Safflower, pungent in taste, warm in nature, attributing to the heart and liver meridians, has the effects of promoting blood circulation to dredge the meridians and dissipating stasis to relieve pain, and mainly contains compounds such as flavonoids, alkaloids, polyynes, lignans, organic acids and steroids, etc. Among them, the flavonoid component safflower yellow pigment is its main effective component. The main effective components of safflower on the cardiovascular system are safflower yellow pigment and safflower quinone glycoside and other effective components.

[0016] Paeonia lactiflora Pall. has a bitter taste and a slightly cold nature, and it pertains to the Liver Meridian. It is good at promoting qi stagnation in blood, and has the effects of activating blood circulation to dredge the meridian, dissipating stasis and relieving pain. It mainly contains monoterpene glycoside components: paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, albiflorin, paeoniflorigenone, paeonoside, etc.; it also contains paeonol and other alcohols and phenols. Modern research shows that paeoniflorin can play a role in protecting the heart by resisting myocardial cell oxidation, inhibiting myocardial cell apoptosis, inhibiting myocardial inflammation, regulating autophagy, and regulating signal transduction pathways such as MAPK and PI3K / Akt.

[0017] Chuanxiong Rhizoma has a pungent taste and a warm nature, and it pertains to the Liver, Gallbladder and Pericardium Meridians. It has the effects of promoting blood circulation and qi movement, dispelling wind and relieving pain. It contains phthalide derivatives (ligustilide, chuanxiong lactone, chuanxiong phenol, etc.), bisphthalide derivative alkaloids (tetramethylpyrazine, choline, etc.), organic acids (ferulic acid, folic acid, vanillic acid, protocatechuic acid, etc.), vanillin and other chemical components. Modern research shows that phthalide derivatives are important active components of Chuanxiong Rhizoma, which are closely related to the effects of promoting blood circulation to remove blood stasis, promoting qi movement, dispelling wind, dampness and relieving pain of Chuanxiong Rhizoma. Ligustilide has the functions of improving microcirculation, dilating blood vessels and resisting myocardial ischemia.

[0018] Angelica sinensis (Oliv.) Diels has a sweet and pungent taste and a warm nature, and it pertains to the Liver, Heart and Spleen Meridians. It has the effects of enriching blood and promoting blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relaxing bowel. Angelica volatile oil is one of the main active components of Angelica sinensis (Oliv.) Diels. Ligustilide, n-butylidenephthalide, phenol, p-cresol, m-ethylphenol, thymol, allo-ocimene, β-ocimene-X, palmitic acid, phthalic anhydride, etc. in Angelica volatile oil all belong to phthalide compounds. The components of Angelica volatile oil have the function of stabilizing the myocardial cell membrane, maintaining the normal functions of lysosomes and mitochondria, enhancing the anti-hypoxia ability of myocardial cells, and protecting myocardial cells from ischemia-induced damage.

[0019] Acanthopanax senticosus (Rupr. et Maxim.) Harms has a bitter and sweet taste and a warm nature, and it pertains to the Liver, Spleen and Lung Meridians. Its effects are to replenish qi and strengthen the spleen, tonify the kidney and soothe the nerves. Acanthopanax senticosus (Rupr. et Maxim.) Harms glycoside components can improve myocardial ischemia and the local damage caused by it, and can also enhance microcirculation, improve blood supply to the brain tissue, and relieve the clinical symptoms brought by cerebral ischemia.

[0020] Taxillus chinensis (DC.) Danser has a bitter and sweet taste and a neutral nature, and it pertains to the Liver and Kidney Meridians. Its effects are to dispel wind-dampness, tonify the liver and kidney, and strengthen the bones and muscles. The chemical components of Taxillus chinensis (DC.) Danser mainly contain flavonoid components: taxilluside, quercetin, hyperoside, quercitrin, etc.

[0021] Acorus tatarinowii has pungent and bitter tastes, is warm in nature, and belongs to the heart and stomach meridians. Its effects are to open orifices and resolve phlegm, awaken the mind and improve intelligence, and promote digestion by removing dampness. The main components in Acorus tatarinowii are volatile components, including β-asarone, α-asarone, γ-asarone, cis-methylisoeugenol, methyleugenol, khellinone, etc. Modern research shows that Acorus tatarinowii has the effect of nourishing the myocardium. β-asarone in Acorus tatarinowii can significantly reduce the blood lipid level in atherosclerotic rats; it can improve the blood rheology in hyperlipidemic rats; it can reduce the endothelin level in myocardial ischemia rats, increase the content of nitric oxide, and reduce the degree of myocardial tissue damage and necrosis rate.

[0022] Pheretima aspergillum has a salty taste, is cold in nature, and belongs to the liver, spleen, and bladder meridians. Its effects are to clear heat and calm convulsions, dredge collaterals, relieve asthma, and induce diuresis. Pheretima aspergillum mainly contains components such as amino acids, nucleotides, and trace elements. Pheretima aspergillum also contains a variety of enzymes, among which fibrinolysin, lumbrokinase, and lumbrocollegenase are the main active components for anticoagulation and antithrombosis.

[0023] Aucklandia lappa has pungent and bitter tastes and is warm. It belongs to the spleen, stomach, gallbladder, and large intestine meridians. Its effects are to promote qi circulation to relieve pain, strengthen the spleen, promote digestion, and relieve stagnation. Aucklandia lappa mainly contains volatile oil chemical components such as dehydrocostus lactone, longifolene, β-costol, costunolide, and cis-farnesol. Modern research shows that dehydrocostus lactone has the effects of dilating blood vessels and improving microcirculation.

[0024] The present invention also provides a preparation method of the above traditional Chinese medicine composition, and the preparation method includes the following steps:

[0025] (1) Take the Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii in the above weight ratio, extract the volatile oil with water to obtain the mixed volatile oil and medicinal residues;

[0026] (2) Encapsulate the mixed volatile oil with cyclodextrin to obtain the volatile oil inclusion compound, and obtain the first powder after drying;

[0027] (3) Take the Hirudo and Pheretima aspergillum in the above weight ratio, add a compound enzyme preparation and water for extraction to obtain an enzymatic hydrolysate, filter, and obtain the second powder after drying;

[0028] (4) Take Astragalus membranaceus, Carthamus tinctorius, Prunus persica, Paeonia lactiflora, Taxillus chinensis, and Acanthopanax senticosus, mix them evenly with the medicinal residues in step (1), add water for soaking, enzymatically hydrolyze with a cellulase and pectinase buffer solution, after centrifuging the enzymatic hydrolysate, take the supernatant, filter and reserve it, add ethanol to the remaining precipitate after centrifugation and stir, centrifuge to take the supernatant, combine the supernatants, and obtain the third powder after drying;

[0029] (5) Mix the first powder, the second powder, and the third powder evenly to obtain the traditional Chinese medicine composition.

[0030] Furthermore, the preparation method of the traditional Chinese medicine composition includes the following steps:

[0031] (1) Grinding the Chuanxiong rhizome, Chinese angelica root, costus root and Acorus calamus into 40-60 mesh, extracting volatile oil by steam distillation to obtain mixed volatile oil and medicinal residues;

[0032] (2) The volatile oil prepared in step (1) is included in the cyclodextrin, a 40%-60% ethanol solution is added, ultrasonic treatment is performed, and centrifugal drying is performed to obtain a volatile oil inclusion compound, which is then vacuum dried at 40° C. to obtain a powder, thereby obtaining the first powder;

[0033] (3) Take leech and earthworm powder, add 4-5 times the total weight of the medicinal materials in water, then add a composite enzyme preparation with an enzyme-substrate ratio of 5-6%, and perform enzymolysis at pH 2.0 and temperature 45-50°C for 1-2 hours to obtain an enzymatic solution. Adjust the pH of the enzymatic solution to 6.0-8.0, and continue enzymolysis at temperature 50-60°C for 1-2 hours. Heat the enzymatic solution to 85°C for inactivation for 10 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the solution pH to 7.0, filter, and use a ceramic filter membrane to intercept components with a molecular weight of less than 3000 Da in the enzymatic solution. Collect the non-permeated liquid and freeze-dry to obtain a second powder;

[0034] (4) Astragalus, safflower, peach kernel, red peony root, mistletoe, and Acanthopanax senticosus are ground into 40-60 mesh, mixed with the dregs of step (1), added with 8-12 times the amount of water, soaked for 0.5 hours, added with a cellulase and pectinase buffer solution with an enzyme-substrate ratio of 3-4% at 45-50°C, and hydrolyzed for 2-3 hours. The hydrolyzed solution is heated to 85°C for inactivation for 15 minutes, cooled to room temperature, centrifuged, and the supernatant is taken. The pH value of the solution is adjusted to 7.0 and set aside; 4-5 times the amount of 60%-80% ethanol is added to the remaining precipitate after centrifugation, stirred evenly, and allowed to stand for 12-24 hours. The supernatant is taken by centrifugation and the ethanol is recovered under reduced pressure; the supernatants are combined and spray-dried to obtain a third powder;

[0035] (5) The first powder, the second powder and the third powder are mixed evenly to obtain a Chinese medicine composition.

[0036] The purpose of crushing the medicinal materials into 40-60 mesh is to increase the specific surface area of the medicinal materials by 3-5 times and shorten the solvent penetration path.

[0037] The steam distillation method in step (1) is specifically to add 10-15 times the total weight of the medicinal materials in water, distill for 2-3 hours, and the distillation process parameters are: steam pressure 0.07-0.08Mpa, distillate temperature 40-50°C.

[0038] Step (2) The ultrasonic treatment is specifically carried out under the conditions of a temperature of 30 - 50 °C for 0.5 - 1 hour with a power of 200 - 300 W. The volatile oil is included with cyclodextrin to obtain a volatile oil inclusion compound; the cyclodextrin is one of α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin, and γ-cyclodextrin (γ-CD), preferably hydroxypropyl-β-cyclodextrin. Hydroxypropyl-β-cyclodextrin can better precipitate oily substances with the mixed volatile oil and improve the stability of the volatile oil.

[0039] The compound enzyme preparation in step (3) is composed of acidic protease, neutral protease, and alkaline protease with a mass ratio of 3:2:5. The acidic protease is pepsin, the neutral protease is papain or subtilisin, and the alkaline protease is trypsin.

[0040] The cellulase and pectinase buffer solution in step (4) is composed of cellulase, pectinase, and citrate-phosphate buffer solution with a mass ratio of 10:1:1000. The use of a citrate-phosphate buffer system can improve the activity of the compound enzyme and enhance the enzymatic hydrolysis effect on traditional Chinese medicinal materials.

[0041] The present invention also provides a pharmaceutical preparation for treating stroke. The pharmaceutical preparation includes the above-mentioned traditional Chinese medicine composition and pharmaceutically acceptable excipients. The dosage form of the pharmaceutical preparation is not limited, but preferably an oral dosage form, such as soft capsules, hard capsules, pills, tablets, granules, powders, etc., and preferably capsules. The pharmaceutically acceptable excipients include but are not limited to starch, dextrin, sucrose, microcrystalline cellulose, lactose, polyethylene glycol 4000, polyethylene glycol 6000, vegetable oil, beeswax, etc.

[0042] The present invention provides the use of the above-mentioned traditional Chinese medicine composition for preparing a drug for treating stroke.

[0043] The technical solution provided by the present invention has the following beneficial technical effects:

[0044] First, Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii all contain volatile oil components. In the prior art, water extraction and alcohol extraction are mostly used for Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii. However, in the above extraction processes, the physicochemical properties of the volatile oil components change during high-temperature and long-time decoction, resulting in isomerization reactions such as oxidation, hydrolysis, and degradation, leading to the loss of their effective substance components. In contrast, the present invention optimizes the parameter system of the water vapor distillation method. By adjusting the steam pressure gradient (0.07 - 0.08 MPa) and the distillate temperature control range (40 - 50 °C), the average contents of ligustilide, dihydrocostus lactone, and β-asarone, which have effects on the cardiovascular system, are increased by more than 20%.

[0045] Second, in the prior art, after pulverizing insect medicinal materials, alcohol extraction is often carried out. This method cannot extract protein polypeptide components in animal medicinal materials well. In the present invention, leeches and earthworms are treated separately, and a composite enzyme is added for enzymatic hydrolysis. Two-stage hydrolysis is carried out under the conditions of pH 2.0 - 8.0 and 45 - 60 °C for a total duration of 2 - 4 hours. Through the synergistic effect of enzyme cleavage sites, macromolecular proteins (molecular weight > 10 kDa) are efficiently converted into soluble polypeptide fragments. Then, components with a molecular weight less than 3000 Da in the enzymatic hydrolysate are intercepted by a ceramic filter membrane, and polypeptides with a relative molecular weight less than 3000 Da of earthworms and leeches are obtained. The composite enzyme enzymatic hydrolysis process adopted in the present invention is simple, can effectively separate polypeptide components from leech and earthworm tissues, greatly improves the resource utilization rate, reduces the cost, and the obtained polypeptide powders of earthworms and leeches and other extract powders are made into oral preparations, which can further improve the therapeutic effect of the oral preparation on stroke patients

[0046] Third, there are many disadvantages in the prior art for the extraction process of the effective components of traditional astragalus membranaceus, safflower, peach kernel, and red peony root. For example, water decoction and ethanol reflux extraction have low extraction efficiency, easily inactivate thermosensitive components, and serious component loss. The present invention adopts a continuous extraction technology that combines cellulose enzyme, pectinase, and citrate-phosphate buffer extraction and alcohol extraction, which improves the dissolution of active components such as astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin

[0047] Fourth, the traditional Chinese medicine composition provided by the present invention can effectively improve the blood viscosity of stroke patients, promote cerebral blood circulation, improve the symptoms of stroke patients, and the therapeutic effect is better than that of the marketed products Longshengzhi Capsule and Tongxinluo Capsule Specific Embodiments

[0048] In order to make the purpose and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention

[0049] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. existing in the prior art

[0050] Example 1

[0051] (1) Crush 600 g of Chuanxiong, 600 g of Angelica sinensis, 650 g of Aucklandia lappa, and 650 g of Acorus tatarinowii to 40 mesh, and extract the volatile oil by steam distillation to obtain a mixed volatile oil and medicinal residues; the steam distillation method is specifically adding 10 times the total weight of the medicinal materials of water, with a distillation time of 2 hours, and the distillation process parameters are: steam pressure 0.07 Mpa, distillate temperature 40 °C;

[0052] (2) Incorporate the volatile oil obtained in the previous step with 5 times the amount of hydroxypropyl-β-cyclodextrin, add a 40% ethanol solution, perform ultrasonic treatment, and centrifuge and dry to obtain a volatile oil inclusion complex, and vacuum dry at 40 °C to a powder to obtain the first powder; the ultrasonic treatment is specifically: ultrasonic treatment for 0.5 hours at a temperature of 40 °C and a power of 200 W;

[0053] (3) Take 850 g of Hirudo powder and 500 g of Pheretima aspergillum powder, mix them evenly, add 4 times the total weight of the medicinal materials of water, then add a composite enzyme preparation with an enzyme-substrate ratio of 5%, and carry out enzymatic hydrolysis at pH 2.0 and a temperature of 45 °C for 1 hour to obtain an enzymatic hydrolysate. Adjust the pH of the enzymatic hydrolysate to 6.0 and continue enzymatic hydrolysis at a temperature of 50 °C for 1 hour. Heat the enzymatic hydrolysate to 85 °C for inactivation for 10 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, filter, and use a ceramic filter membrane to intercept the components with a molecular weight less than 3000 Da in the enzymatic hydrolysate, and freeze-dry to obtain the second powder; the composite enzyme preparation is composed of pepsin, papain, and trypsin with a mass ratio of 3:2:5.

[0054] (4) Take 1900 g of Astragalus membranaceus, 700 g of Carthamus tinctorius, 900 g of Prunus persica, 700 g of Paeonia lactiflora, 650 g of Taxillus chinensis, and 200 g of Acanthopanax senticosus, crush them to 40 mesh, mix them with the medicinal residues in step (1), add 8 times the amount of water, soak for 0.5 hours, add a cellulase and pectinase buffer solution with an enzyme-substrate ratio of 3% and carry out enzymatic hydrolysis at 45 °C for 2 hours. Heat the enzymatic hydrolysate to 85 °C for inactivation for 15 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, and set aside; add 4 times the amount of 60% ethanol to the remaining centrifuged precipitate, stir evenly, stand for 12 hours, centrifuge and take the supernatant, and recover ethanol under reduced pressure; combine the supernatants and spray-dry to obtain the third powder;

[0055] (5) Mix the first powder, the second powder, and the third powder evenly to obtain the traditional Chinese medicine composition.

[0056] Example 2

[0057] (1) Crush 550 g of Chuanxiong, 550 g of Angelica sinensis, 590 g of Aucklandia lappa, and 590 g of Acorus tatarinowii to 50 mesh, and extract the volatile oil by steam distillation to obtain a mixed volatile oil and medicinal residues; the steam distillation method is specifically adding 15 times the total weight of the medicinal materials of water, with a distillation time of 2 hours, and the distillation process parameters are: steam pressure 0.07 Mpa, distillate temperature 45 °C;

[0058] (2) The volatile oil described in the previous step is included with 5 times the amount of hydroxypropyl-β-cyclodextrin, 50% ethanol solution is added, ultrasonic treatment is carried out, centrifuged and dried to obtain the volatile oil inclusion complex, and vacuum dried at 40 °C to powder to obtain the first powder; The ultrasonic treatment is specifically carried out under the condition of a temperature of 40 °C for 0.5 hours with a power of 240 W;

[0059] (3) Take 780 g of Hirudo powder and 460 g of Pheretima powder, mix them evenly, add 4 times the amount of water based on the total weight of the medicinal materials, then add a complex enzyme preparation with an enzyme-substrate ratio of 6%, and carry out enzymatic hydrolysis at pH 2.0 and a temperature of 48 °C for 1 hour to obtain an enzymatic hydrolysate. Adjust the pH of the enzymatic hydrolysate to 7.0 and continue enzymatic hydrolysis at a temperature of 55 °C for 1 hour. Heat the enzymatic hydrolysate to 85 °C to inactivate the enzyme for 10 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, filter, and use a ceramic filter membrane to intercept the components with a molecular weight less than 3000 Da in the enzymatic hydrolysate, and freeze-dry to obtain the second powder; The complex enzyme preparation is composed of pepsin, papain, and trypsin with a mass ratio of 3:2:5;[[ID=^6]]

[0060] (4) Take 1740 g of Astragalus membranaceus, 640 g of Carthamus tinctorius, 820 g of Prunus persica, 640 g of Paeonia lactiflora, 590 g of Taxillus chinensis, and 180 g of Acanthopanax senticosus, crush them to 50 meshes, mix them with the medicinal residues in step (1), add 10 times the amount of water, soak for 0.5 hours, add a cellulase and pectinase buffer solution with an enzyme-substrate ratio of 4%, and carry out enzymatic hydrolysis at a temperature of 48 °C for 2 hours. Heat the enzymatic hydrolysate to 85 °C to inactivate the enzyme for 15 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, and set aside; Add 4 times the amount of 70% ethanol to the remaining precipitate after centrifugation, stir evenly, stand for 18 hours, centrifuge to take the supernatant, and recover ethanol under reduced pressure; Combine the supernatants and spray dry to obtain the third powder;

[0061] (5) Mix the first powder, the second powder and the third powder evenly to obtain the traditional Chinese medicine composition.

[0062] Example 3

[0063] (1) Crush 650 g of Ligusticum chuanxiong, 650 g of Angelica sinensis, 700 g of Aucklandia lappa, and 700 g of Acorus tatarinowii to 60 meshes, and extract the volatile oil by steam distillation to obtain a mixed volatile oil and medicinal residues; The steam distillation method is specifically to add 15 times the amount of water based on the total weight of the medicinal materials, with a distillation time of 3 hours, and the distillation process parameters are: steam pressure 0.08 Mpa, distillate temperature 50 °C;

[0064] (2) The volatile oil described in the previous step is included with 5 times the amount of hydroxypropyl-β-cyclodextrin, 60% ethanol solution is added, ultrasonic treatment is carried out, centrifuged and dried to obtain the volatile oil inclusion complex, and vacuum dried at 40 °C to powder to obtain the first powder; The ultrasonic treatment is specifically carried out under the condition of a temperature of 50 °C for 1 hour with a power of 300 W;

[0065] (3) Take 920 g of leech powder and 540 g of earthworm powder, mix them evenly, add 5 times the total weight of the medicinal materials in water, then add a compound enzyme preparation with an enzyme-substrate ratio of 6%, and carry out enzymatic hydrolysis at pH 2.0 and a temperature of 50 °C for 2 hours to obtain an enzymatic hydrolysate. Adjust the pH of the enzymatic hydrolysate to 8.0 and continue enzymatic hydrolysis at a temperature of 60 °C for 2 hours. Heat the enzymatic hydrolysate to 85 °C for inactivation for 10 minutes, cool it to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, filter, and use a ceramic filter membrane to intercept the components with a molecular weight less than 3000 Da in the enzymatic hydrolysate, and then carry out freeze-drying to obtain the second powder; the compound enzyme preparation is composed of pepsin, subtilisin, and trypsin with a mass ratio of 3:2:5;

[0066] (4) Take 2050 g of astragalus membranaceus, 750 g of safflower, 970 g of peach kernel, 750 g of red peony root, 700 g of taxillus chinensis, and 220 g of acanthopanax senticosus, pulverize them to 60 mesh, mix them with the medicinal residues in step (1), add 12 times the amount of water, soak for 0.5 hour, add a cellulase and pectinase buffer solution with an enzyme-substrate ratio of 3%, and carry out enzymatic hydrolysis at 50 °C for 3 hours. Heat the enzymatic hydrolysate to 85 °C for inactivation for 15 minutes, cool it to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, and set aside; add 5 times the amount of 80% ethanol to the remaining centrifuged precipitate, stir evenly, let it stand for 24 hours, centrifuge to take the supernatant, and recover ethanol under reduced pressure; combine the supernatants and carry out spray drying to obtain the third powder;

[0067] (5) Mix the first powder, the second powder, and the third powder evenly to obtain the traditional Chinese medicine composition.

[0068] Example 4

[0069] Preparation method of granules: Take 500 g of the traditional Chinese medicine composition prepared by the method of Example 1, add 800 g of soluble dextrin, 300 g of lactose, and 2 g of steviol glycoside, mix them evenly, granulate with 75% ethanol, dry, pack, and package to obtain the product.

[0070] Example 5

[0071] Preparation method of hard capsules: Take 260 g of the traditional Chinese medicine composition prepared by the method of Example 2 and fill it into hard capsules to obtain the product.

[0072] Example 6

[0073] Preparation method of tablets: Take 400 g of the traditional Chinese medicine composition prepared by the method of Example 3, add 40 g of sodium carboxymethyl starch and 50 g of lactose, granulate with 70% ethanol, dry, screen the granules, add 5 g of magnesium stearate and 5 g of microcrystalline silica, mix them evenly, press into 2000 tablets, and coat them with a film coating to obtain the product.

[0074] Example 7

[0075] Preparation method of pellets: Take 270 g of the traditional Chinese medicine composition prepared by the method of Example 4, make pellets with 60% ethanol, polish, and dry to obtain the pellets.

[0076] Experimental Example 1

[0077] To study the extraction effect of volatile components of traditional Chinese medicine composition by steam distillation under different distillation conditions, choose ligustilide in Ligusticum chuanxiong Hort. and Angelica sinensis (Oliv.) Diels, dehydrocostus lactone in Aucklandia lappa Decne., and β-asarone in Acorus tatarinowii Schott as target components. According to the preparation method recorded in step (1), add 600 g of Ligusticum chuanxiong Hort., 600 g of Angelica sinensis (Oliv.) Diels, 650 g of Aucklandia lappa Decne., and 650 g of Acorus tatarinowii Schott, and carry out the preparation under different steam pressures and distillate temperatures, and detect the content of the target components after preparation.

[0078] Determination of the contents of ligustilide and dehydrocostus lactone by high performance liquid chromatography (HPLC). The chromatographic conditions are as follows: Hypersil ODS2 column (200 mm×4.6 mm, 5 μm), acetonitrile-water (80:20), flow rate: 1 ml / min, column temperature: 35 °C, detection wavelength: 350 nm (ligustilide), 225 nm (dehydrocostus lactone).

[0079] Preparation of mixed reference substance solution: Weigh ligustilide and dehydrocostus lactone as reference substances respectively, dissolve and dilute with water to prepare a mixed reference substance solution.

[0080] Preparation of test solution: Weigh 0.5 g of the first powder prepared in Example 1, add 10 mL of water to dissolve and prepare, ultrasonicate (power: 250 W, frequency 40 kHz) for 30 min, shake well, filter, and take the subsequent filtrate to obtain the test solution. Record the peak area, and calculate the contents of ligustilide and dehydrocostus lactone by the external standard method.

[0081] Determination of the content of β-asarone by ultraviolet spectrophotometry at 302 nm. Instrument and equipment: TU-1810 type ultraviolet-visible spectrophotometer.

[0082] Preparation of reference substance solution: Accurately weigh β-asarone reference substance, dissolve it with methanol, respectively pipette 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mL of this reference substance solution into a 10 mL volumetric flask, and make up the volume to the mark with methanol. Preparation of test solution: Weigh 0.5 g of the first powder prepared in Example 1, add 10 mL of methanol to dissolve and prepare, weigh accurately, ultrasonicate (power: 250 W, frequency 40 kHz) for 30 min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution. The experimental results are shown in Table 1.

[0083] Table 1 Detection results of three volatile components

[0084]

[0085]

[0086] Comparative Example 1

[0087] The raw material ratio composition and steps of Comparative Example 1 are basically the same as those of step (1) of Example 1, except that after the medicinal materials are pulverized, they are soaked in 5 times the amount of water by weight of the medicinal materials for 30 minutes, and then steam distilled under normal pressure to obtain the distillate. The distillate is included with 5 times the amount of hydroxypropyl-β-cyclodextrin and dried into powder. Using the detection method in Experimental Example 1 above, the content of ligustilide was measured to be 0.42 mg / g, the content of dehydrocostus lactone was 2.25 mg / g, and the content of β-asarone was 6.28 mg / g.

[0088] Comparative Example 2

[0089] The raw materials and steps of Comparative Example 2 are basically the same as those of step (1) of Example 1, except that after the medicinal materials are pulverized, the powders of Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii are extracted by heating under reflux with 60% ethanol twice, each time for 1 hour, filtered, the ethanol solution is reserved, the solvent is removed by reduced pressure distillation, and 5 times the amount of hydroxypropyl-β-cyclodextrin is used and dried into powder. Using the detection method in Experimental Example 1 above, the content of ligustilide was measured to be 0.61 mg / g, the content of dehydrocostus lactone was 2.73 mg / g, and the content of β-asarone was 6.81 mg / g.

[0090] In the traditional medicinal material extraction process, although the steam distillation method can promote the dissolution of active ingredients through soaking, it is easy to cause the degradation of thermosensitive components under normal pressure operation conditions (100 °C). Although the ethanol reflux extraction method uses a low-boiling solvent (ethanol boiling point 78 °C) to reduce the risk of thermal denaturation, long-term heating will cause the destruction of terpene structural components such as ligustilide, dehydrocostus lactone, and β-asarone, resulting in a general decrease in the yield of the target product. In addition to ligustilide, fatty acids and fatty acid esters are also extracted by the ethanol reflux extraction method, which further affects the yield of the target product.

[0091] The preparation method provided by the present invention can significantly improve the extraction efficiency of thermally unstable components by optimizing the pressure-temperature coordination parameters of the distillation system. When the steam pressure is controlled in the range of 0.07 - 0.08 MPa and the distillate temperature is at 40 - 50 °C, the enrichment concentration of key active ingredients shows a significant increase: the ligustilide can reach above 0.80 mg / g (about 30% higher than the ethanol reflux extraction process), the dehydrocostus lactone can reach above 3.50 mg / g (about 28% higher than the ethanol reflux extraction process), and the β-asarone can also stably reach above 8.30 mg / g (about 21% higher than the ethanol reflux extraction process), as shown in Table 1.

[0092] Experimental Example 2

[0093] To further study the extraction effects of leech and earthworm proteins under different preparation processes.

[0094] Set up experimental group A, experimental group B, experimental group C, experimental group D, experimental group E, and example 1 group. Each group took 850 g of leech powder and 500 g of earthworm powder.

[0095] Experimental group A: The leech and earthworm medicinal materials were not treated.

[0096] Experimental group B: The leech and earthworm medicinal materials were percolated with 50%-70% ethanol, the percolate was collected, the ethanol was recovered, concentrated, dried, and pulverized into dry extract powder.

[0097] Experimental group C: Add 5 times the total weight of water of the leech and earthworm medicinal materials, then add pepsin with an enzyme-substrate ratio of 5%, and carry out enzymatic hydrolysis at pH 2.0 and a temperature of 45 °C for 2 hours to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was heated to 85 °C to inactivate for 10 minutes, cooled to room temperature, centrifuged, the supernatant was taken, the pH value of the solution was adjusted to 7.0, filtered, and the components with a molecular weight less than 3000 Da in the enzymatic hydrolysate were intercepted by a ceramic filter membrane, and freeze-dried to obtain.

[0098] Experimental group D: Take the leech and earthworm medicinal materials, add subtilisin with an enzyme-substrate ratio of 5%, and carry out enzymatic hydrolysis at pH 6.0 and a temperature of 55 °C for 2 hours. The other steps are the same as the preparation method of experimental group C.

[0099] Experimental group E: Take the leech and earthworm medicinal materials, add trypsin with an enzyme-substrate ratio of 5%, and carry out enzymatic hydrolysis at pH 8.0 and a temperature of 55 °C for 2 hours. The other steps are the same as those of experimental group C.

[0100] Example 1 group: Take the leech and earthworm medicinal materials and prepare them according to the method in step (3) of Example 1 to obtain.

[0101] The BCA method was used to measure the protein concentration

[0102] Principle: The protein concentration range measured by the BCA method can reach 25-500 g / mL, and the sensitivity is relatively high. Its principle is based on the fact that protein molecules reduce Cu 2+ to Cu + , and BCA specifically combines with Cu + to form a purple complex, which has the maximum light absorption value at 562 nm and is proportional to the protein concentration. Using the protein reference solution as the standard curve, the protein concentration can be measured according to the absorption value.

[0103] Dissolve bovine serum albumin (BSA) standard in PBS buffer to prepare a protein standard solution with a concentration of 0.5 mg / ml. Dilute the standard solution with PBS to 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml, where 0 mg / ml is the blank control. Take the samples of experimental groups A - E and Example 1 group, dissolve them in water, centrifuge and take the supernatant, and dilute to the same protein concentration as the control solution. Determine the concentration according to the following method:

[0104] Add 0, 1, 2, 4, 8, 12, 16, 20 μl of the protein standard solution into a 96 - well plate respectively, and add PBS to make the volume of each well up to 20 μl. The concentrations of the protein standard in each well are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / ml respectively. Add an appropriate volume of the sample and make corresponding dilutions according to the expected protein concentration in the sample. Add 200 μl of BCA working solution (100 ml of copper - BCA solution, add 2 ml of 4% copper sulfate solution and mix to get the working solution, and the working solution can be stored at room temperature for 24 hours), then incubate at 37 °C for 30 minutes. Read A562 with an enzyme - linked immunosorbent assay (ELISA) reader, draw a standard curve, and calculate the linear regression equation. Substitute the A562 value of the sample into the linear regression equation and multiply by the dilution factor to obtain the protein concentration in the sample.

[0105] Table 2 Analysis of the results of extracting proteins from leeches and earthworms by several methods

[0106]

[0107] The protein concentration (8.56 mg / mL) and protein percentage (11.85%) of the Example 1 group (extracted by composite enzyme) are the highest, significantly higher than those of other single - enzyme treatment groups (groups C / D / E). This indicates that the composite enzyme may, through the synergistic action of multiple enzymes, more efficiently decompose macromolecular proteins into polypeptide substances, and improve the extraction rate and purity of the target product. The protein concentration (3.46 mg / mL) and protein percentage (5.66%) of the experimental group B (treated with ethanol) are the lowest, even lower than those of the untreated experimental group A (8.29%), indicating that ethanol may damage the protein structure or dissolve non - target components (such as lipids, polysaccharides), resulting in protein loss. In contrast, the enzymatic hydrolysis method (groups C / D / E) significantly improves the extraction efficiency of the target product by specifically cleaving protein peptide bonds.

[0108] Experimental Example 3

[0109] To further study the extraction effect of the preparation process on the anticoagulant and anti-atherosclerotic active ingredients, baicalin in Scutellaria baicalensis, safflower yellow pigment A in Carthamus tinctorius, amygdalin in Prunus persica, and paeoniflorin in Paeonia lactiflora were selected as the target detection components. The preparation process of the present invention was compared with several other different preparation processes, and the contents of the target components after preparation were detected respectively. Experimental group F, experimental group G, experimental group H, and example 1 group were set up, and 1900 g of Astragalus membranaceus, 700 g of Carthamus tinctorius, 900 g of Prunus persica, and 700 g of Paeonia lactiflora were taken in each group.

[0110] Samples of experimental group F: Prepared according to the process described in the application number 201210228897.4. Weigh the powders of Astragalus membranaceus, Carthamus tinctorius, and Prunus persica, add ethanol with a weight ratio of 6 times and a concentration of 50% to the medicinal materials, heat and extract 2 times, 1 hour each time, filter, and keep the ethanol solution for use; Mix the medicinal residues with Paeonia lactiflora, first add water with a weight ratio of 6 times and a pH value of 3.0 to soak for 30 min, then heat and reflux for 1 hour, filter, and keep the filtrate for use; Second, add water with a weight ratio of 5 times and a pH value of 5.5, heat and reflux for 1 hour, filter, and keep the filtrate for use; Third, add water with a weight ratio of 5 times and a pH value of 8.0, heat and reflux for 1 hour, filter, combine the filtrates of the above three acid water extractions, add ethanol to make the alcohol content reach 50%, stand for 24 h, filter, and keep the ethanol solution for use.

[0111] Samples of experimental group G: Prepared according to the process described in the application number 200510043066.X. Weigh the Astragalus membranaceus, Carthamus tinctorius, Prunus persica, and Paeonia lactiflora, decoct with water 2 times, 2 hours for the first time and 1.5 hours for the second time, filter, and combine the decoction.

[0112] Samples of experimental group H: Take Astragalus membranaceus, Carthamus tinctorius, Prunus persica, and Paeonia lactiflora, add 8 times the amount of water, soak for 0.5 hour, add cellulase with an enzyme-substrate ratio of 3% and enzymolyze at 45 °C for 2 hours, heat the enzymolysis solution to 85 °C to inactivate for 15 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, remove the solvent, and spray dry to obtain.

[0113] Samples of example 1 group: Take Astragalus membranaceus, Carthamus tinctorius, Prunus persica, and Paeonia lactiflora, add 8 times the amount of water, soak for 0.5 hour, add cellulase and pectinase buffer with an enzyme-substrate ratio of 3% and enzymolyze at 45 °C for 2 hours, heat the enzymolysis solution to 85 °C to inactivate for 15 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, and reserve; Add 4 times the amount of ethanol with a concentration of 60% to the remaining centrifuged precipitate, stir evenly, stand for 12 hours, centrifuge and take the supernatant, recover ethanol under reduced pressure; Combine the supernatants, remove the solvent, and spray dry to obtain.

[0114] Determination of astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin by HPLC. The chromatographic conditions are as follows: Agilent XDB-C18 chromatographic column (150 mm × 4.6 mm, 5 μm), mobile phase: methanol - acetonitrile - 0.15% phosphoric acid aqueous solution (26:2:72), flow rate: 1 ml / min, column temperature 30°C, detection wavelengths: 203 nm (astragaloside IV), 337 nm (hydroxysafflor yellow A), 217 nm (amygdalin), 230 nm (paeoniflorin).

[0115] Preparation of the mixed reference substance solution: Weigh astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin as reference substances respectively, dissolve and dilute them with methanol to prepare a mixed reference substance solution.

[0116] Preparation of the test solution: Take 0.5 g of the samples from experimental groups F - H and Example 1 group, add 10 mL of methanol to dissolve and prepare. Weigh accurately, sonicate (power: 250 W, frequency 40 kHz) for 30 min, cool to room temperature, weigh accurately again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0117] Record the peak areas and calculate the contents of astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin by the external standard method.

[0118] Table 3 Detection results of the contents of astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin

[0119]

[0120] In the comparative test of the above preparation methods, the extraction efficiency of the Example 1 group for astragaloside IV, hydroxysafflor yellow A, amygdalin, and paeoniflorin is the highest, significantly higher than that of the extraction with single cellulase (group H). This indicates that the combination of cellulase and pectinase buffer can improve the dissolution of the above active ingredients. In experimental group F (semi-bionic, ethanol extraction method) and experimental group G (water extraction), there is long-term heating, resulting in the loss of active ingredients and less dissolution amount.

[0121] In order to illustrate the application effect of the traditional Chinese medicine composition provided by the present invention in the prevention and treatment of qi deficiency and blood stasis type stroke diseases, capsules with a loading specification of 0.4 g / capsule were prepared according to the method of Example 1 for clinical trials.

[0122] Clinical trials

[0123] General information

[0124] From January 2022 to January 2023, 93 stroke patients with qi deficiency and blood stasis type admitted to Xi'an Buchang Traditional Chinese Medicine Heart and Brain Disease Hospital were selected as the research objects. Inclusion criteria: (1) Meeting the diagnostic criteria in the "Diagnostic and Efficacy Evaluation Criteria for Stroke", qi deficiency and blood stasis type; (2) Age > 18 years old; (3) Having signs and symptoms of focal brain damage; (4) Signing the informed consent form of the patient. Exclusion criteria: (1) Complicated with other organic diseases; (2) Complicated with malignant tumors; (3) Mental system diseases; (4) Patients with drug allergies; (5) Coagulation dysfunction.

[0125] After admission, the patients were randomly divided into an observation group, control group 1, and control group 2 according to a ratio of 1:1:1, with 31 cases in each group. In the observation group, there were 17 males and 14 females; the age was 42 - 75 years old, with an average of (63.6 ± 5.4) years old, and the course of disease was 26 - 92 days, with an average of (48.7 ± 7.2) days; in control group 1, there were 22 males and 9 females, the age was 46 - 73 years old, with an average of (61.7 ± 4.2) years old, and the course of disease was 25 - 88 days, with an average of (46.9 ± 8.1) days; in control group 2, there were 19 males and 12 females, the age was 45 - 77 years old, with an average of (64.8 ± 3.9) years old, and the course of disease was 26 - 89 days, with an average of (47.5 ± 7.8) days. There was no statistically significant difference in the general data of the 3 groups of clinical patients (P > 0.05), and they were comparable.

[0126] Research methods

[0127] After the patients were admitted to the hospital, they were given routine treatments, including blood pressure lowering, blood sugar lowering, electrolyte supplementation, and nutritional support. On this basis, the observation group was given capsules prepared according to the method of Example 1, with a loading of 0.4 g per capsule, 5 capsules each time, 3 times a day, for a course of 4 weeks; control group 1 was given Tongxinluo Capsules on the basis of routine treatment, with the national drug approval number Z19980015, 0.26 g per capsule, 4 capsules each time, 3 times a day, for a course of 4 weeks; control group 2 was given Longshengzhi Capsules on the basis of routine treatment, with the national drug approval number Z20010059, the specification is 0.4 g per capsule, 5 capsules each time, 3 times a day, for a course of 4 weeks;

[0128] Observation indicators

[0129] Efficacy judgment criteria: Refer to the "Diagnosis and Efficacy Evaluation Criteria for Stroke" formulated by the Emergency Collaboration Group for Encephalopathy of the State Administration of Traditional Chinese Medicine. According to the patient's consciousness (0 - 9 points), language (0 - 6 points), facial paralysis (0 - 2 points), eye symptoms (2 - 4 points), upper limb paralysis (0 - 6 points), finger paralysis (0 - 5 points), lower limb paralysis (0 - 6 points), toe paralysis (0 - 5 points), and other syndromes (abnormal pupil 7 points, convulsion 7 points, hematemesis and hematochezia 8 points, involuntary defecation and urination 8 points, eyes closed and mouth open 8 points, snoring and weak breathing 9 points, faint pulse 9 points, cold hands and feet 9 points) for scoring. The total score is 0 - 53 points. The higher the score, the more severe the stroke symptoms. The sum of each score is the syndrome score classification standard. The percentage conversion formula for the pre-treatment score and post-treatment score of the disease type is: [(pre-treatment score - post-treatment score) / pre-treatment score] × 100%, expressed as a percentage. Basic recovery: ≥81%, significant progress: ≥56%, <81%; progress: ≥11%, <56%; no change: <11% or negative value. Total effective rate = (number of cases with basic recovery + number of cases with significant progress + number of cases with progress) / number of cases included in the group × 100%

[0130] Hemorheology indexes: Collect fasting venous blood from patients. The levels of plasma viscosity, whole blood high shear viscosity, fibrinogen, and whole blood low shear viscosity are detected using an automatic hemorheology analyzer.

[0131] Table 4 Comparison of the effective rates of three groups of patients

[0132]

[0133] After treatment, Longshengzhi Capsule and Tongxinluo Capsule both showed good therapeutic effects on stroke patients with qi deficiency and blood stasis. In comparison, the traditional Chinese medicine composition provided by the present invention has a better therapeutic effect.

[0134] Table 5 Comparison of hemorheology indexes of three groups of patients

[0135]

[0136] Note: Compared with before treatment, #P < 0.05; compared with control group 1 after treatment, *P < 0.05; compared with control group 2 after treatment, △P < 0.05

[0137] Both Longshengzhi Capsule and Tongxinluo Capsule can improve the blood viscosity of stroke patients with qi deficiency and blood stasis. After treatment, the whole blood low shear viscosity, whole blood high shear viscosity, plasma viscosity, and fibrinogen of the patients decreased to varying degrees. Compared with Tongxinluo Capsule and Longshengzhi Capsule, the traditional Chinese medicine composition provided by the present invention has a better improvement effect on the blood viscosity of stroke patients.

Claims

1. A traditional Chinese medicine composition for treating stroke, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 153 - 226 parts of Astragalus membranaceus, 48 - 71 parts of Ligusticum chuanxiong, 48 - 71 parts of Angelica sinensis, 56 - 83 parts of Carthamus tinctorius, 72 - 107 parts of Prunus persica, 56 - 83 parts of Paeonia lactiflora, 52 - 77 parts of Aucklandia lappa, 52 - 77 parts of Acorus tatarinowii, 52 - 77 parts of Taxillus chinensis, 69 - 101 parts of Hirudo nipponia, 40 - 59 parts of Pheretima aspergillum, 16 - 24 parts of Eleutherococcus senticosus.

2. The traditional Chinese medicine composition according to claim 1, wherein It is prepared from the following raw materials in parts by weight: 174 - 205 parts of Astragalus membranaceus, 55 - 65 parts of Ligusticum chuanxiong, 55 - 65 parts of Angelica sinensis, 64 - 75 parts of Carthamus tinctorius, 82 - 97 parts of Prunus persica, 64 - 75 parts of Paeonia lactiflora, 59 - 70 parts of Aucklandia lappa, 59 - 70 parts of Acorus tatarinowii, 59 - 70 parts of Taxillus chinensis, 78 - 92 parts of Hirudo nipponia, 46 - 54 parts of Pheretima aspergillum, 18 - 22 parts of Eleutherococcus senticosus.

3. The traditional Chinese medicine composition according to claim 1, wherein It is prepared from the following raw materials in parts by weight: 190 parts of Astragalus membranaceus, 60 parts of Ligusticum chuanxiong, 60 parts of Angelica sinensis, 70 parts of Carthamus tinctorius, 90 parts of Prunus persica, 70 parts of Paeonia lactiflora, 65 parts of Aucklandia lappa, 65 parts of Acorus tatarinowii, 65 parts of Taxillus chinensis, 85 parts of Hirudo nipponia, 50 parts of Pheretima aspergillum, 20 parts of Eleutherococcus senticosus.

4. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that The preparation method comprises the following steps: (1) Take Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii in the above weight ratio, extract volatile oil with water to obtain mixed volatile oil and medicinal residues; (2) Encapsulate the mixed volatile oil with cyclodextrin to obtain a volatile oil inclusion compound, and dry it to obtain the first powder; (3) Take Hirudo nipponia and Pheretima aspergillum in the above weight ratio, add a compound enzyme preparation and water for extraction to obtain an enzymolysis solution, filter it, and dry it to obtain the second powder; (4) Take Astragalus membranaceus, Carthamus tinctorius, Prunus persica, Paeonia lactiflora, Taxillus chinensis, and Eleutherococcus senticosus, mix them evenly with the medicinal residues in step (1), add water for soaking, enzymolyze with a cellulase and pectinase buffer solution. After centrifuging the enzymolysis solution, take the supernatant, filter it for standby. Add ethanol to the remaining precipitate after centrifugation, stir, centrifuge to take the supernatant, combine the supernatants, and dry to obtain the third powder; (5) Mix the first powder, the second powder, and the third powder evenly to obtain the traditional Chinese medicine composition.

5. The preparation method of the traditional Chinese medicine composition according to any one of claims 1-3, characterized in that, The preparation method comprises the following steps: (1) Crush Ligusticum chuanxiong, Angelica sinensis, Aucklandia lappa, and Acorus tatarinowii to 40 - 60 mesh, and extract volatile oil by steam distillation to obtain mixed volatile oil and medicinal residues; (2) Encapsulate the volatile oil in step (1) with cyclodextrin, add a 40% - 60% ethanol solution, perform ultrasonic treatment, centrifuge and dry it to prepare a volatile oil inclusion compound, and vacuum dry it at 40°C to powder to obtain the first powder; (3) Take the powders of Hirudo nipponia and Pheretima aspergillum, add 4 - 5 times the amount of water based on the total weight of the medicinal materials, then add a compound enzyme preparation with an enzyme - substrate ratio of 5 - 6%, enzymolyze at pH 2.0 and a temperature of 45 - 50°C for 1 - 2 hours to obtain an enzymolysis solution. Adjust the pH of the enzymolysis solution to 6.0 - 8.0, continue to enzymolyze at a temperature of 50 - 60°C for 1 - 2 hours. Heat the enzymolysis solution to 85°C for inactivation for 10 minutes, cool it to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, filter, and use a ceramic filter membrane to intercept the components with a molecular weight less than 3000Da in the enzymolysis solution. Collect the non - permeated solution and freeze - dry it to obtain the second powder; (4) Take Astragalus membranaceus, Carthamus tinctorius, Prunus persica, Paeonia lactiflora, Taxillus chinensis, and Eleutherococcus senticosus, pulverize them to 40 - 60 mesh, mix them with the medicinal residues from step (1), add 8 - 12 times the amount of water, soak for 0.5 hour, add a cellulase and pectinase buffer solution with an enzyme - substrate ratio of 3 - 4%, enzymatically hydrolyze at 45 - 50 °C for 2 - 3 hours, heat the enzymatic hydrolysate to 85 °C to inactivate the enzymes for 15 minutes, cool to room temperature, centrifuge, take the supernatant, adjust the pH value of the solution to 7.0, and set aside; add 4 - 5 times the amount of ethanol with a concentration of 60% - 80% to the remaining precipitate after centrifugation, stir evenly, let stand for 12 - 24 hours, centrifuge to take the supernatant, and recover ethanol under reduced pressure; combine the supernatants and spray - dry to obtain the third powder; (5) Mix the first powder, the second powder, and the third powder evenly to obtain the traditional Chinese medicine composition.

6. The preparation method of the traditional Chinese medicine composition according to claim 4, wherein, (2) The specific method of steam distillation in step (1) is to add 10 - 15 times the amount of water based on the total weight of the medicinal materials, distill for 2 - 3 hours, and the distillation process parameters are: steam pressure 0.07 - 0.08 Mpa, distillate temperature 40 - 50 °C.

7. The preparation method of the traditional Chinese medicine composition according to claim 4, wherein, (3) The specific ultrasonic treatment in step (2) is carried out at a temperature of 30 - 50 °C, with an ultrasonic time of 0.5 - 1 hour and an ultrasonic power of 200 - 300 W.

8. The preparation method of the traditional Chinese medicine composition according to claim 4, wherein (4) The composite enzyme preparation in step (3) is composed of acidic protease, neutral protease, and alkaline protease with a mass ratio of 3:2:

5.

9. The preparation method of the traditional Chinese medicine composition according to claim 4, wherein, (5) The cellulase and pectinase buffer solution in step (4) is composed of cellulase, pectinase, and citrate - phosphate buffer solution with a mass ratio of 10:1:1000. (6) Use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating stroke.

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