Pharmaceutical composition for treating coronary heart disease and preparation method thereof

Abstract

The invention provides a medicine composition for treating coronary heart disease. The medicine composition is prepared from the following raw material medicines in parts by weight: 60-80 parts of fructus choerospondiatis, 15-22 parts of radix salviae miltiorrhizae, 5-8 parts of clove, 0.5-6 parts of borneol and 0-8 parts of tabasheer. The pharmaceutical composition is simple and convenient in formula compatibility and stable and reliable in preparation process, and has the advantages of remarkable efficacy and low toxic and side effects.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and in particular relates to a pharmaceutical composition for treating coronary heart disease and a preparation method thereof. Background Art

[0002] Coronary atherosclerotic heart disease (CHD) is a heart disease caused by coronary atherosclerosis causing lumen stenosis, spasm or obstruction, leading to myocardial ischemia, hypoxia or necrosis. It is affected by multiple risk factors such as genetics, metabolism, and lifestyle, and is the leading cause of death worldwide. It is reported that the prevalence of coronary heart disease in people over 60 years old in my country is 27.8%, and it is showing an upward trend year by year. CHD is seriously threatening the health of Chinese residents. Traditional Chinese medicine believes that CHD belongs to the category of "chest pain", blood stasis syndrome is one of the main syndrome types of CHD in traditional Chinese medicine, and blood circulation and stasis treatment is an important treatment for CHD. Modern medicine believes that CHD is mainly characterized by pain in the precordial area, and the nature of the pain is mostly colic and dull pain, while stabbing pain is relatively rare. In traditional Chinese medicine textbooks such as "Chinese Medicine Diagnosis" and "Chinese Medicine Internal Medicine", stabbing pain is often regarded as the main manifestation of CHD (chest pain) blood stasis syndrome. Clinically, Western medicine treatment for angina pectoris mainly uses class II beta-adrenergic receptor blockers, calcium channel blockers (CCBs), and vasodilators. Although it has achieved good clinical results, patients are highly dependent on drugs, and the condition is prone to recurrence, and there are many related adverse reactions. These problems seriously affect the treatment and prognosis of patients. Traditional Chinese medicine has unique insights into the etiology and pathogenesis of "chest pain", and clinical syndrome differentiation and treatment can significantly improve symptoms and prognosis. Traditional Chinese medicine has obvious advantages in treating CHD, and it is significantly superior to Western medicine in improving clinical symptoms and prognosis.

[0003] The product referred to in the pharmaceutical composition of the present invention is Guanxin Shutong Capsule, which is composed of five flavors of main medicines, namely, Radix Achyranthis Bidentatae and Salvia miltiorrhizae, auxiliary medicines, namely, Clove and Radix Glechomae Dahuricae, and adjuvant medicine, namely, Borneol. In the prescription, Radix Achyranthis Bidentatae has the effects of promoting qi and blood circulation, nourishing the heart and tranquilizing the mind, resisting myocardial ischemia, protecting cardiac function, and the like, and is used for qi stagnation and blood stasis, chest wall pain, palpitations, shortness of breath, and restlessness; Salvia miltiorrhizae is recorded in Compendium of Materia Medica as "promoting blood circulation, unblocking pericardium, and treating hernia pain", and has the effects of promoting blood circulation and removing blood stasis, nourishing blood, and tranquilizing the mind, so as to help Radix Achyranthis Bidentatae promote blood circulation and remove blood stasis, clear the heart and relieve pain; Clove has the effects of warming the middle and descending adverse, tonifying the kidney and assisting yang, and is used for "lifeline" (aorta) disease, heart disease, and the like; Borneol has the effects of invigorating the mind and refreshing the mind, clearing away heat and relieving pain, and is used for symptoms such as coma, convulsion, chest obstruction and heart pain caused by febrile diseases; Radix Glechomae Dahuricae Dahuricae has the effects of clearing heat and removing phlegm, cooling the heart and calming convulsion. Guanxinshutong Capsule is used for chest obstruction and blood stasis in the heart. It is clinically used to treat chest pain, chest tightness, palpitation, shortness of breath and other symptoms caused by CHD and angina pectoris. Its main functions are to promote blood circulation and remove blood stasis, promote blood circulation and activate collaterals, and relieve pain.

[0004] The applicant has previously applied for multiple patent applications for the product, including: 02114549.0 (dosage ratio of Chinese patent medicine formula), 200510041670.9 (preparation process), 200510041695.9 (pills), 200510041884.6 (pharmaceutical composition), 200510124510.0 (quality control method for capsules), 201010500159.1 (application in diabetes and impaired glucose tolerance drugs), 201210124018.3 (preparation process), 201210124018.4 (preparation process), 201210124018.5 (preparation process), 201210124018.6 (preparation process), 201210124018.7 (preparation process), 201210124018.8 (preparation process), 201210124510.0 (quality control method for capsules), 201010500159.1 (application in diabetes and impaired glucose tolerance drugs), 201210124018.3 (preparation process), 201210124018.6 (preparation process), 201210124510.0 (quality control method for capsules), 201010500159.1 (application in diabetes and impaired glucose tolerance drugs), 201210124018.3 (preparation process), 201210124018.3 (preparation process), 201210124018.6 (preparation process), 200510041884.6 (pharmaceutical composition), 20 Improvement), 201210123918.6 (oral preparation), 201210124063.9 (improvement of oral liquid), 201210124064.3 (improvement of soft capsule), 201410003476.0 (fingerprint determination method), 201410148569.2 (application in the treatment of myocardial ischemia and protection of the heart), 201510870868.1 (extract pharmaceutical composition), 201610005020.7 (one-test-multiple-evaluation content detection method), 202211404731.3

[0005] (Method for identifying biomarkers against myocardial ischemia), 202410092635.2 (Guanxinshutong extract).

[0006] However, in a large number of clinical uses and basic research conducted in the early stages of this drug, we found that the dosage ratio of the Chinese medicine combination has not yet reached the optimal level. Therefore, it is particularly important to develop a Chinese medicine prescription with good efficacy and few toxic and side effects. Summary of the invention

[0007] The invention provides a pharmaceutical composition for treating coronary heart disease and a preparation method thereof. The pharmaceutical composition has simple formulation and compatibility, stable and reliable preparation process, and has the advantages of significant efficacy and low toxic and side effects.

[0008] The dosage ratio of the formula of the present invention is the work of the inventor of the present application. Through a large amount of practical research experience, it is concluded that the dosage of each component within the following weight range can exhibit better efficacy.

[0009] The technical solution of the patent application of this invention is:

[0010] The pharmaceutical composition for treating coronary heart disease of the present invention is prepared from raw materials in the following weight ratio: 60-80 parts of Fructus Axillae, 15-22 parts of Salvia miltiorrhiza, 5-8 parts of Cloves, 0.5-6 parts of Borneol and 0-8 parts of Radix Glehniae.

[0011] Preferably, the pharmaceutical composition is made of raw materials in the following weight ratio: 61.8 parts of Fructus Choerospondiae, 20.7 parts of Salvia miltiorrhiza, 6 parts of Cloves, 5.5 parts of Borneol, and 6 parts of Radix dasyphyllae.

[0012] As a further preferred embodiment of the present invention, the pharmaceutical composition is made of raw materials in the following weight ratio: 75.4 parts of Choerospondias axillaris, 16.6 parts of Salvia miltiorrhiza, 7 parts of Cloves, and 1 part of Borneol.

[0013] The preparation method of the pharmaceutical composition is:

[0014] Grind the jujube into fine powder and divide it into two parts for later use; grind the rhizoma damicornis into fine powder and set aside; grind the remaining jujube into the coarsest powder, use 70% ethanol as solvent for percolation, collect the percolation liquid, recover the ethanol and concentrate it to a thick paste with a relative density of 1.30-1.35 (50°C), add one part of the jujube fine powder, mix well, dry, grind into fine powder and set aside. Extract Danshen three times. The first time, heat and reflux with ethanol for 1.5 hours, filter, recover ethanol from the filtrate, and concentrate to a thick paste with a relative density of 1.30-1.35 for use; the second time, heat and reflux with 50% ethanol for 1.5 hours, filter, and the filtrate is used for use; the third time, add water and boil for 2 hours, filter, combine the filtrate with the filtrate of the second extraction, recover ethanol, and concentrate to a thick paste with a relative density of 1.30-1.35, combine with the thick paste of the first extraction, mix, concentrate to a thick paste with a relative density of 1.30-1.35, add another portion of Guangzao fine powder, mix well, dry, and crush into fine powder. Extract volatile oil from cloves by steam distillation, spray the volatile oil evenly into the Tianzhuhuang fine powder, mix well, and seal; mix borneol with the remaining Tianzhuhuang fine powder, grind it into fine powder, mix it with the above-mentioned fine powders, and put it into capsules to obtain it.

[0015] As a further preferred embodiment of the present invention, the preparation method of the pharmaceutical composition is:

[0016] Take the coarse powder of Ziziphus jujuba (3 / 4 of the amount of the prescription Ziziphus jujuba), moisten it evenly with 70% ethanol, place it in a sealed container for 60 minutes, put it into a percolator, add 70% ethanol and soak it for 48 hours, then slowly percolate at a speed of 2-3 mL / min, collect the percolation liquid, recover the ethanol, and concentrate it under reduced pressure to a relative density of 1.30-1.35, then add the fine powder of Ziziphus jujuba (1 / 8 of the prescription Ziziphus jujuba), mix well, dry, and crush; extract Danshen 3 times, add 95% ethanol for the first time, heat and reflux for 1.5 hours, filter, recover ethanol from the filtrate, and concentrate it to a thick paste with a relative density of 1.30-1.35 for use; heat and reflux with 50% ethanol for the second time for 1.5 hours, filter, and set aside the filtrate; add water and boil for 2 hours for the third time, filter The filtrate is combined with the second extraction filtrate, ethanol is recovered, concentrated to a thick paste with a relative density of 1.30-1.35, combined with the thick paste extracted for the first time, mixed, concentrated to a thick paste with a relative density of 1.30-1.35, added with a portion of jujube strychnos nucifera powder (1 / 8 of the amount of jujube strychnos nucifera in the prescription), mixed, dried, and crushed into fine powder; volatile oil is extracted from cloves by steam distillation, and the amount of volatile oil added to 60g of cloves in the prescription is fixed at 9.5mL, and the volatile oil is evenly sprayed into the dahlia fine powder (1 / 2 of the amount of dahlia in the prescription), mixed, and sealed (1 / 2 of the amount of dahlia in the prescription); borneol is mixed with the dahlia fine powder, ground, and mixed with the above-mentioned fine powders to obtain a mixture of active ingredients of the pharmaceutical composition of the present invention.

[0017] The pharmaceutical composition of the present invention can also be prepared by extracting jujube, salvia miltiorrhiza, clove, borneol and jasmine with water or alcohol solvent to prepare a mixture of the active substance components of jujube extract, salvia miltiorrhiza extract, clove extract, borneol extract and jasmine extract.

[0018] The above Chinese herbal medicine extract can be added with pharmaceutical excipients to be made into pills, granules, tablets or capsules.

[0019] Dropping pills: the total mixture of the obtained Guanxinshutong pharmaceutical composition and an appropriate amount of one or two auxiliary materials such as matrix PEG4000, PEG6000, stearic acid, or poloxamer are hot-melted, stirred and mixed evenly, and then dripped to make dropping pills;

[0020] Granules: The total mixture of the Guanxinshutong pharmaceutical composition obtained is mixed evenly with an appropriate amount of dextrin, sucrose, lactose, mannitol, or one or two of the auxiliary materials, wet granulated, dried, sieved, and made into granules;

[0021] Tablets: The obtained Guanxinshutong pharmaceutical composition mixture is mixed evenly with an appropriate amount of one or two auxiliary materials such as corn starch, hydroxypropyl methylcellulose, magnesium stearate, dextrin, etc., granulated, mixed, and pressed into tablets;

[0022] Capsules: Add one or two of the appropriate auxiliary materials such as mannitol, lactose, microcrystalline cellulose, starch, magnesium stearate, talc, dextrin, etc. to the obtained Guanxinshutong pharmaceutical composition mixture, mix evenly, granulate, and encapsulate to make hard capsules.

[0023] The prescription of the pharmaceutical composition of the present invention is as follows:

[0024] Explanation of the prescription: Choosing Fructus Atractylodes in the prescription of the present invention can invigorate qi and activate blood circulation, dredge the blood vessels blocked by blood stasis, and adding Salvia miltiorrhiza can remove blood stasis, promote new blood circulation, and relieve pain. The two medicines are used together to invigorate qi and activate blood circulation, and they are the main medicine. It is supplemented with cloves, which are pungent and warm, warm the middle and dispel cold, and promote blood circulation. Rhizoma Cibotii is the assistant medicine, which can resolve phlegm, clear heat and calm the nerves. By helping the main medicine to warm and dredge the blood vessels, blood stasis and numbness can be eliminated. The auxiliary medicine is borneol to open the mind and refresh the mind, which can lead the medicine to go up to the head and eyes, so that the heart and brain blood vessels are unblocked, and the chest tightness and dark tongue symptoms are eliminated.

[0025] In the early stage of the present invention, four Q-Markers of Guanxinshutong prescription have been screened out, which are protocatechuic acid, cryptotanshinone, eugenol and borneol. Based on the prescription of Guanxinshutong Capsules, the uniform design method was adopted, and the prescription compatibility ratio was taken as the influencing factor. 15 groups of Guanxinshutong prescriptions with different prescription compatibility ratios were prepared from the same batch of medicinal materials / decoction pieces; because the Fructus Achyranthis Bidentatae from different origins grows at the same longitude and latitude, the content of the main components does not fluctuate much, and it is still inappropriate to use Fructus Achyranthis Bidentatae from different origins as the influencing factor; Salvia miltiorrhizae is widely planted and distributed, and the quality of Salvia miltiorrhizae from different origins is different, so the content of the main components fluctuates greatly; cloves are mostly produced abroad, and there are fewer production sites in my country, so there is less reference content for taking different origins as the variation factor; borneol is mostly artificially synthesized borneol; and Rhizoma Cibotii is less distributed; therefore, Salvia miltiorrhizae from different origins were selected for testing and then samples with different quality differences in Guanxinshutong were prepared. Based on the prescription of Guanxinshutong, 15 groups of prescriptions of Salvia miltiorrhizae from different origins that met the prescription compatibility ratio of Guanxinshutong were prepared, and a total of 30 groups of Guanxinshutong difference samples that met the research characteristics were prepared, including qualified samples and unqualified samples tested according to the pharmacopoeia standards.

[0026] Beneficial effects of the present invention

[0027] ⑴. In the research on the improvement of the formula process of Guanxinshutong product, the inventor of this application, guided by the theory of traditional Chinese medicine, obtained the following two groups of optimal formulas and obtained the best drug efficacy. The optimal formula ratio is: 61.8 parts of jujube, 20.7 parts of salvia, 6 parts of cloves, 5.5 parts of borneol, and 6 parts of tianzhuhuang. It was surprisingly found that on the basis of the original formula, the auxiliary drug tianzhuhuang was reduced, and the dosage ratio of jujube, salvia, cloves, and borneol was changed. After repeated research and experimental summary, another optimal formula ratio was obtained: 75.4 parts of jujube, 16.6 parts of salvia, 7 parts of cloves, and 1 part of borneol.

[0028] ⑵, the present invention improves the preparation process to obtain more refined and optimal preparation process parameters, which obtains the protocatechuic acid content in the Guanxinshutong sample ≥ 0.372 mg / g, eugenol content ≥ 0.415 mg / g, cryptotanshinone content (≥ 0.468 mg / g, borneol content ≥ 13.443 mg / g, which also makes the content of the active ingredient in the extract of the pharmaceutical composition of the present invention higher and relatively uniform and stable.

[0029] (3) Based on the GXST differential sample safety drug dose screening by CCK-8 method, the Guanxinshutong pharmaceutical composition of the present invention showed no toxicity to H9C2 cells, which also shows that the pharmaceutical composition of the present invention has the advantages of small toxic and side effects and high safety.

[0030] (4) Compared with the original formula of Guanxinshutong, the pharmaceutical composition of the present invention (Guanxinshutong formula) has a significant protective effect on H9C2 cells. GXST differentiated sample group 11, group 15, group 17, group 18, group 19, group 21, group 22, group 23, group 24, group 27, and group 29 also have a significant protective effect on H9C2 cells, which further indicates that the pharmaceutical composition of the present invention has a protective effect on cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 -The effect of the control group drugs on the H9C2 cell H / R model, where GXST capsule represents Guanxin Shutong capsule;

[0032] Figure 2 -The effects of differentiated samples of Guanxinshutong on the H9C2 cell H / R model; among them, experiments 11, 15, 17, 18, 19, 21, 22, 23, 24, 27, and 29 are the 11th, 15th, 17th, 18th, 19th, 21st, 22nd, 23rd, 24th, 27th, and 29th groups of differentiated samples of Guanxinshutong, respectively.

[0033] Note: Compared with the blank group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001. DETAILED DESCRIPTION

[0034] Unless otherwise defined, the technical terms or scientific terms used in the patent application specification and claims of the present invention shall have the common meanings understood by persons having ordinary skills in the field to which the present invention belongs.

[0035] Example 1 Preparation of samples of Guanxinshutong with different quality for the pharmaceutical composition of the present invention

[0036] 1 Experimental materials and instruments

[0037] 1.1 Drugs and Reagents

[0038] The coarsest powder of jujube (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231101), fine powder of jujube (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231106), salvia miltiorrhiza (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231107), clove (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231101), borneol (synthetic borneol) (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 2305002), Tianzhuhuang fine powder (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20230607), Anhui Salvia miltiorrhiza (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040311), Henan Salvia miltiorrhiza (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040315), Sichuan Salvia miltiorrhiza (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040313), Shaanxi Salvia miltiorrhiza (Shaanxi Tasly Botanical Medicine Technology Co., Ltd. GAP base, 24030102), Shandong Salvia miltiorrhiza (Heze Danhong Pharmaceutical Salvia miltiorrhiza GAP base, 20230301), 95% ethanol (Shaanxi Baoji Alcohol Factory).

[0039] 1.2 Experimental instruments

[0040] Electric constant temperature water bath: Shanghai Zhulan Instrument Technology Co., Ltd., HH-21-6; blast drying oven: Shanghai Zhulan Instrument Technology Co., Ltd., PHG-9075A; high-speed multifunctional pulverizer: Yongkang Yunda Machinery Equipment Factory, JR-100; rotary evaporator: Shanghai Yarong Biochemical Instrument Factory, RE-52AA.

[0041] 2 Experimental methods

[0042] 2.1 Uniform design experimental plan

[0043] 2.1.1 Preparation of differentiated samples with different proportions in the same batch

[0044] In the original formula of Guanxinshutong Capsule, the proportions of Fructus jujubae, Salvia miltiorrhizae, Clove, Borneol, and Rhizoma dahliae were 57%, 29%, 7%, 3.5%, and 3.5%, respectively. In uniform design, Fructus jujubae varied uniformly between 25.6% and 87.9%, Salvia miltiorrhizae varied uniformly between 0% and 58%, Clove varied uniformly between 0% and 14%, and Borneol and Rhizoma dahliae varied uniformly between 0% and 7%. According to the principle that the number of uniform design levels should be greater than twice the number of factors, the five Chinese medicines were set as 5 factors, and the five-factor and fifteen-level uniform design method was selected in the DPS data processing system, and the uniform design scheme was based on the U5 (515) table.

[0045] Table 1 U5 (515) uniform design table obtained using DPS data processing system

[0046]

[0047] Table 2 Medicinal material dosage of different samples of Guanxinshutong Capsules

[0048]

[0049]

[0050] 2.1.2 Differentiated sample preparation of Salvia miltiorrhiza from different origins and different preparation processes

[0051] Experimental design: Considering the significant difference in the efficacy of the experimental prescription process, this experiment designed a change in the preparation process of Danshen slices, that is, Danshen slices from different origins were extracted 0 times, 1 time, and 2 times. The experimental prescription is shown in Table 3 below.

[0052] Table 3 Preparation of different samples of Guanxinshutong capsules

[0053]

[0054] 2.2 Preparation method of differentiated samples

[0055] According to the standard process flow of Guanxin Shutong Capsules in the 2020 edition of the Pharmacopoeia of the People's Republic of China, the medicinal materials were added according to Table 3: the coarse powder of Fructus jujubae (3 / 4 of the amount of Fructus jujubae in the prescription) was evenly moistened with 70% ethanol, sealed and placed for 60 minutes, placed in a percolator, and soaked in 70% ethanol for 48 hours. The percolation was completed slowly at a speed of 2-3 mL / min, the percolation liquid was collected, the ethanol was recovered, and it was concentrated to a relative density of 1.15-1.20 (50°C), and then concentrated to a relative density under reduced pressure. The temperature was 1.30~1.35 (50℃), and then fine powder of Fructus jujuba (1 / 8 of the amount of Fructus jujuba in the prescription) was added, mixed evenly, dried, and crushed; Danshen was extracted 3 times, and 95% ethanol was added for the first time, heated and refluxed for 1.5h, filtered, and the filtrate was recovered and concentrated to a thick paste with a relative density of 1.30~1.35 for later use; 50% ethanol was added for the second time, heated and refluxed for 1.5h, filtered, and the filtrate was used for later use; water was added for the third time, boiled for 2h, filtered, and the filtrate was combined with the filtrate of the second extraction, and recovered Ethanol, and concentrated to a thick paste with a relative density of 1.30-1.35 (55-60°C), combined with the thick paste extracted for the first time, mixed, concentrated to a thick paste with a relative density of 1.30-1.35 (55-60°C), added a portion of Guangzao fine powder (1 / 8 of the Guangzao in the prescription), mixed, dried, and crushed into fine powder; cloves are used to extract volatile oil by steam distillation. Cloves contain 15-20% volatile oil. The Chinese Pharmacopoeia 2020 Class 1 stipulates that the content of medicinal materials shall not be less than 16%. Therefore, in order to ensure the stability of product quality, Buchang Pharmaceutical Co., Ltd. fixed the amount of volatile oil added to 60g of cloves in the prescription to 9.5mL, sprayed the volatile oil evenly into the fine powder of dahlia (1 / 2 of the amount of dahlia in the prescription), mixed it evenly, and sealed it (the prescription stipulates 1 / 2 of dahlia) (the prescription stipulates that 1 / 2 of dahlia (15g) absorbs 9.5mL of clove oil (about 1.6g: 1mL)); borneol is mixed with the fine powder of dahlia, ground into powder, and mixed evenly with the above fine powders. In summary, a total of 30 batches of differentiated samples of Guanxinshutong were obtained.

[0056] 2.3 Establishment of the quality information data set of the pharmaceutical composition of the present invention

[0057] The quality of traditional Chinese medicine is the basis of clinical efficacy. The difference in the quality of traditional Chinese medicine is a key factor affecting the change in efficacy. How to explore the precise quantitative correlation between the quality information of traditional Chinese medicine and the biological effect is a key scientific problem to be solved in the quality evaluation of traditional Chinese medicine and the development of Q-Marker. It is also a key scientific and technological problem that urgently needs to be solved in the development of the quality evaluation standard of traditional Chinese medicine from whether the product is qualified or not to the high-quality development of high-quality products. Our research group has screened out 4 Q-Markers in Guanxinshutong Recipe through a large number of experiments in the early stage, namely protocatechuic acid in Choerospondias australis, cryptotanshinone in Salvia miltiorrhiza, eugenol in cloves, and borneol in borneol. The content determination method of Guanxinshutong Capsules in the 2020 edition of the Chinese Pharmacopoeia is divided into gas chromatography and high-performance liquid chromatography. Gas chromatography uses borneol, isoborneol, and eugenol as indicator components, and high-performance liquid chromatography uses tanshinone ⅡA and tanshinonic acid B as indicator components. The quality control is relatively comprehensive, but there are two problems: 1. There are many chromatographic conditions (1 detection condition for gas chromatography and 2 detection conditions for high-performance liquid chromatography), which brings inconvenience to content detection; 2. The main medicine Guangzao has the effects of promoting qi and blood circulation, nourishing the heart and calming the mind, which is crucial to the therapeutic effect of Guanxinshutong Capsules, and the amount of Guangzao (480g) accounts for about 57% of the total prescription amount (840g), which accounts for a large proportion, but the indicator components of the main medicine Guangzao are not included in the content determination.

[0058] Borneol is a natural or synthetic substance with the effects of refreshing the mind, clearing away heat and relieving pain. Natural borneol is mainly produced in Sumatra and Nanyang Islands of Indonesia, and synthetic borneol is produced in Guangzhou, Nanjing, Zhuzhou, Tianjin and other places. It is used for fever coma, convulsions, stroke phlegm, qi stagnation, coma, red eyes, mouth sores, sore throat, pus in the ear canal, etc. Borneol is the main active ingredient of borneol and has obvious analgesic effect. The present invention establishes a gas chromatography method for determining the content of borneol in borneol, which provides a data basis for the establishment of a quality information data set.

[0059] 2.3.1 Method for determining the content of multiple indexes in the pharmaceutical composition of the present invention

[0060] The present invention also establishes a multi-index determination method to simultaneously determine the contents of three components, namely, protocatechuic acid, cryptotanshinone and eugenol. While determining the Q-Marker content, it is intended to solve the problems mentioned above, such as the large number of chromatographic conditions and the lack of the main drug, Fructus Atractylodes multiflorus, in the pharmacopoeia content determination.

[0061] 2.3.2 Materials and instruments

[0062] 2.3.2.1 Experimental instruments

[0063] Gas chromatograph: Agilent, USA, GC7820A; electronic balance: Sartorius Scientific Instruments (Beijing) Co., Ltd., Germany, SQP-SECURA225D-1CN; ultrasonic cleaner: Kunshan Shumei Ultrasonic Instrument Co., Ltd., KQ-800KED; UPT series reverse osmosis ultrapure water machine: Merck Chemical Technology (Shanghai) Co., Ltd., UPT-I10T; liquid chromatograph: Shimadzu, Japan, LC-2030C.

[0064] 2.3.2.2 Reference substances and drugs

[0065] Camphor: batch number, 110881-201508, purity: 96.8%, China Food and Drug Inspection Institutes; Guanxinshutong Capsules: batch number: 240201, Shaanxi Buchang Pharmaceutical Co., Ltd.; S1-S30 Guanxinshutong differentiated samples, homemade, protocatechuic acid, batch number: 110809-202207, purity: 99.99%, China Food and Drug Inspection Institutes; Cryptotanshinone: 110852-201807, purity: 99.0%, China Food and Drug Inspection Institutes; Eugenol: batch number, 110725-202318, purity: 99.99%, China Food and Drug Inspection Institutes.

[0066] 2.3.2.3 Reagents

[0067] Ethyl acetate: batch number: 210329, Luoyang Haohua Chemical Reagent Co., Ltd.; methanol: chromatographic alcohol, F24O5K203, Fisher Reagent Company, USA; formic acid: batch number: 10010118, Sinopharm Chemical Reagent Co., Ltd.

[0068] 2.3.3 GC gas chromatography experimental methods and results

[0069] 2.3.3.1 Chromatographic conditions

[0070] Chromatographic column: HP-5 quartz capillary column (30m×0.32mm, 0.25μm); carrier gas nitrogen; flow rate 20mL·min-1; detector is hydrogen flame ionization detector, temperature 250℃; injector temperature 220℃; split ratio 2:1; column temperature is 80℃, maintained for 5min, then increased to 150℃ at a rate of 5℃ per minute, maintained for 5 minutes, and then increased to 220℃ at a rate of 25 degrees Celsius per minute, maintained for 5 minutes; injection volume: 2μL. Under these conditions, borneol and adjacent peaks can be well separated, and the number of theoretical plates is not less than 2000.

[0071] 2.3.3.2 Solution preparation

[0072] 2.3.3.2.1 Preparation of reference solution

[0073] Reference solution 1: Accurately weigh 8.20 mg of borneol reference substance, place in a 5 mL volumetric flask, add ethyl acetate to dilute to the mark to make 1.58752 mg mL -1 The solution was used as reference solution 1.

[0074] Reference solution 2: Accurately weigh 8.01 mg of borneol reference substance, place in a 5 mL volumetric flask, and dilute to scale with ethyl acetate to make 1.550736 mg mL -1 The solution was used as reference solution 2.

[0075] Linear stock solution: Accurately weigh 18.56 mg of borneol reference substance, place in a 5 mL volumetric flask, add ethyl acetate to dilute to the mark, and make 3,5932 mg mL -1 The solution was used as the linear solution reference stock solution.

[0076] Accuracy reference solution: Take 207.13 mg of borneol reference substance and place it in a 200 mL volumetric flask. Add ethyl acetate to dilute to the mark to make 1.00251 mg mL -1 of solution as the accuracy reference solution.

[0077] 2.3.3.2.2 Preparation of test solution

[0078] Take 0.5 g of differentiated samples of S1-S30 Guanxinshutong respectively, accurately weigh them, accurately add 20 mL of ethyl acetate, ultrasonically treat (power 180 W, frequency 42 kHz) for 30 min, let it cool to room temperature, weigh it again, make up the lost weight with ethyl acetate, shake it well, filter it through a microporous filter membrane (0.22 μm), and take the filtrate to obtain the product.

[0079] 2.3.4 Methodological validation

[0080] 2.3.4.1 System adaptability

[0081] Take reference solution 1 5 times, reference solution 2 2 times, and the system reconfirmation solution (reference solution 1) once, record the peak area, and the precision RSD of reference solution 1 is 0.28%, indicating that the instrument has good precision. The F value of the two references is 100.06%, and the average number of theoretical plates is 10846, indicating that the system adaptability meets the requirements of the pharmacopoeia.

[0082] 2.3.4.2 Repeatability test

[0083] Take 6 samples of Guanxinshutong S30 group, prepare them according to the test solution method in 2.2.2, operate according to the chromatographic conditions in 2.1, record the peak area, calculate the content, and its RSD is 0.046%, indicating that this method has good repeatability.

[0084] 2.3.4.3 Stability test

[0085] 2.3.4.3.1 Stability of reference solution

[0086] Take the reference solution and operate according to the above chromatographic conditions. Record the peak areas at 0h, 6h, 10h, 30h, 40h and 48h respectively. The RSD is 1.33%, indicating that the reference solution has good stability within 48h.

[0087] 2.3.4.3.2 Stability of test solution

[0088] Take the test solution and operate according to the above chromatographic conditions. Record the peak areas at 0h, 5h, 24h, 34h, and 48h, respectively. The RSD is 1.496%, indicating that the test solution has good stability within 48h.

[0089] 2.3.4.4 Limit of quantification and detection limit

[0090] The reference solution was gradually diluted to a concentration of 0.0794 mg / mL as the quantitative limit solution, and 5 injections were made continuously. The average signal-to-noise ratio was 15.82, and the RSD of the peak area was 1.81, indicating that the quantitative limit met the pharmacopoeia standard. The reference solution was diluted to a concentration of 0.03974 mg / mL as the detection limit solution, and 2 injections were made continuously. The signal-to-noise ratio was greater than 8.1.

[0091] 2.3.4.5 Investigation of linear relationship

[0092] Accurately take 200μL, 500μL, 1000μL, 1500μL, and 2000μL of the linear stock solution and place them in a 2mL volumetric flask, dilute to the mark with ethyl acetate, and prepare linear solutions with concentrations of 0.3593, 0.8983, 1.7966, 2.6949, and 3.5932. Inject into the gas chromatograph according to the chromatographic conditions under 2.3.3.1, and record the peak area. Draw a standard curve with concentration (X) as the horizontal axis and peak area (Y) as the vertical axis (see Table 4 for the results). 2 It is 0.9992, and the results show that the linear relationship is good.

[0093] Table 4 Regression equation, correlation coefficient, linear range

[0094]

[0095] 2.3.4.6 Accuracy test

[0096] Take 0.25g of the test sample, weigh it accurately, add the accuracy reference solution, prepare it according to the test sample solution preparation method under 2.2.2, prepare 6 parallel portions, inject it into the gas chromatograph according to the chromatographic conditions under 2.3.3.1, record the peak area, and calculate the recovery rate. The result is 93.19%-99.32%, the average is 97.41%, and the RSD is 2.29. The experimental results show that the sample recovery meets the requirements of the pharmacopoeia.

[0097] 2.3.4.7 Sample recovery test

[0098] Take 6 portions of samples with known content (batch number: 240201), each about 0.25g, accurately weighed, and added a certain amount of borneol reference solution in item 2.3.3.2.2. Prepare the test solution according to the above method, and operate according to the chromatographic conditions in item 2.3.3.1, record the peak area, and calculate the results in Table 5. The results show that the average recovery rate of borneol is 97.41% and the RSD is 2.29%.

[0099] Table 5 borneol sample recovery

[0100]

[0101]

[0102] 2.3.4.8 Differential sample content determination

[0103] The differentiated samples of S1-S30 Guanxinshutong were assayed. An appropriate amount of samples were accurately weighed and the test solution was prepared according to the method in 2.2.2. Two samples were prepared for each batch of differentiated samples. Each sample was injected twice continuously. The chromatographic conditions in 2.1 were followed and the peak areas were recorded. The mean borneol content of each batch of differentiated samples was calculated. The results for each batch of samples are shown in Table 6.

[0104] Table 6 Content of borneol in differentiated samples of Guanxinshutong (mg / g)

[0105]

[0106] This chapter established a GC method for determining the content of borneol in differentiated samples of Guanxinshutong. The results showed a good linear relationship and the average recovery rate of borneol was 97.41%, indicating that the method was accurate, sensitive and reproducible. Ethyl acetate has good solubility for borneol and less dissolution of other interfering components in the sample, so it was selected as the extraction solvent. In the experiment, the preparation method of the test solution was studied, and the extraction methods of reflux method, cold soaking overnight method and ultrasonic method were investigated. The chromatographic conditions under 2 and 1 were operated, the peak area was recorded, and the content was calculated. The results showed that the ultrasonic extraction of borneol had a higher content; at the same time, the ultrasonic extraction (power 250HZ, frequency 50kHz) was investigated for 10, 30, 45, and 60min, and the test solution was prepared. The chromatographic conditions under 2 and 1 were operated, the peak area was recorded, and the content was calculated. It showed that the ultrasonic extraction of 30min was basically complete for borneol.

[0107] 2.3.5 HPLC experimental methods and results

[0108] 2.3.5.1 Chromatographic conditions

[0109] Using UltimateAQ-C 18 (4.6×250mm, mm) chromatographic column; methanol (B)-0.2% formic acid water (A) as mobile phase, gradient elution (0-15min, 5%-28% B; 15-20min, 28%-36%; 20-22min, 36%-38%; 22-42min, 38%-58%; 42-55min, 58%-75%; 55-65min, 75%-90%; 65-70min, 90%-5%; 70-80min, 5%); detection wavelengths of 280nm (eugenol) and 254nm (protocatechuic acid, cryptotanshinone); flow rate of 1.0mL min -1 ; Column temperature was 35°C; Injection volume was 10 μL.

[0110] 2.3.5.2 Solution preparation

[0111] 2.3.5.2.1 Preparation of reference solution

[0112] Accurately weigh appropriate amounts of reference substances protocatechuic acid, cryptotanshinone, and eugenol, and add methanol to prepare a mixed reference substance solution containing 1.64 mg of protocatechuic acid, 3.9 mg of cryptotanshinone, and 4 mg of eugenol per 1 mL.

[0113] 2.3.5.2.2 Preparation of test solution

[0114] Accurately weigh 0.5 g of Guanxin Shutong Capsules and 0.5 g of S1-S30 differentiated samples, place them in a stoppered conical flask, and accurately add 10 mL of methanol; seal the flask, weigh the weight, 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 filtrate to obtain.

[0115] 2.3.5.2.3 Methodological validation

[0116] 2.3.5.2.3.1 Precision test

[0117] Accurately pipette an appropriate amount of the mixed reference solution, inject it continuously for 6 times according to the chromatographic conditions in 2.1, record the chromatogram, and calculate the RSDs of the peak areas of protocatechuic acid, eugenol, and cryptotanshinone to be 0.66%, 0.65%, and 0.65%, respectively. The results show that the instrument has good precision.

[0118] 2.3.5.2.3.2 Stability test

[0119] The test solution of Guanxinshutong Capsules was taken and sampled and determined at 0h, 6h, 12h, 24h and 48h according to the chromatographic conditions under 2.1. The RSDs of the peak areas of protocatechuic acid, eugenol and cryptotanshinone were calculated to be 0.39%, 0.42% and 0.46%, respectively, indicating that the test solution had good stability within 48h. The reference solution was taken and sampled and determined at 0h, 6h, 12h, 24h and 48h according to the chromatographic conditions under 2.1. The RSDs of the peak areas of protocatechuic acid, eugenol and cryptotanshinone were calculated to be 0.20%, 0.29% and 0.24%, respectively, indicating that the reference solution had good stability within 48h.

[0120] 2.3.5.2.3.3 Repeatability test

[0121] Take an appropriate amount of the contents of Guanxinshutong Capsules and prepare 6 test solutions in parallel according to the method in 2.2. Determine according to the chromatographic conditions in 2.1, and calculate the RSDs of the peak areas of protocatechuic acid, eugenol, and cryptotanshinone to be 0.39%, 0.4%, and 0.37%, respectively, indicating that the method has good repeatability.

[0122] 2.3.5.2.3.4 Investigation of linear relationship

[0123] Accurately pipette the mixed reference solution, dilute it with methanol at different proportions and then inject it for measurement. Draw the standard curve with the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis, and calculate the regression equation. The results are shown in Table 7.

[0124] Table 7 Linear relationship investigation results

[0125]

[0126] 2.3.5.2.3.5 Sample recovery test

[0127] Take 0.5 g of the test sample of Guanxinshutong Capsules with known ingredient content, weigh it accurately, add the reference substance at a ratio of 100% of the ingredient content, prepare the test sample solution according to the method under 2.2, and determine it according to the chromatographic conditions under 2.1. Calculate the average sample recovery rate and RSD of protocatechuic acid, eugenol and cryptotanshinone. The results are shown in Table 8.

[0128] Table 8 The recovery test results of the three components in Guanxinshutong Capsules

[0129]

[0130]

[0131] 2.3.5.2.3.6 Differential sample content determination

[0132] The differentiated samples of S1-S30 Guanxinshutong were assayed. An appropriate amount of samples were accurately weighed and the test solution was prepared according to the method in 2.2.2. Each sample was injected twice continuously. The operation was carried out according to the chromatographic conditions in 2.1. The peak areas were recorded and the mean contents of protocatechuic acid, eugenol and cryptotanshinone in each batch of differentiated samples were calculated. The results for each batch of samples are shown in Table 9.

[0133] Table 9 Contents of protocatechuic acid, eugenol and cryptotanshinone in differentiated samples of Guanxinshutong (mg / g)

[0134]

[0135] The present invention establishes an HPLC method for determining the contents of protocatechuic acid, eugenol and cryptotanshinone in differentiated samples of Guanxinshutong, and the results show good linearity, an average recovery rate of protocatechuic acid of 98.56%, an average recovery rate of eugenol of 100.91, and an average recovery rate of cryptotanshinone of 104.9353, indicating that the method is accurate, sensitive and reproducible. Methanol has good solubility for protocatechuic acid, eugenol and cryptotanshinone, and other interfering components in the sample are less dissolved, so it is selected as the extraction solvent. In the experiment, the preparation method of the test solution was studied, and three extraction methods, namely reflux method, cold soaking overnight method and ultrasonic method, were investigated. The operation was carried out according to the chromatographic conditions under items 2 and 1, the peak area was recorded, and the content was calculated. The results showed that the ultrasonic extraction content of the above three Q-Markers was higher. At the same time, ultrasonic extraction (power 250HZ, frequency 50kHz) for 10, 30, 45, and 60 minutes was investigated to prepare the test solution. The operation was carried out according to the chromatographic conditions under items 2 and 1, the peak area was recorded, and the content was calculated. It was shown that the above three Q-Markers were basically completely extracted after 30 minutes of ultrasonic extraction.

[0136] Example 2 Analysis of the efficacy of differentiated samples of Guanxinshutong based on in vitro levels

[0137] Our research group has previously conducted preliminary research on biomarkers of Guanxinshutong against myocardial ischemia based on TMT quantitative proteomics technology and discovered 43 differentially expressed proteins. Based on this, this chapter will verify the efficacy of 30 batches of differentiated samples at the cellular level, laying the foundation for subsequent work.

[0138] 1. Experimental Materials and Instruments

[0139] 1.1 Drugs and Reagents

[0140] Differentiation samples of Guanxinshutong S1-S30: homemade, Guanxinshutong capsule Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 240201 / 240202 / 240203 / 240204 / 240205. PBS buffer: Biosharp, 24149299; H9C2 cell-specific culture medium: Wuhan Punuo Life Science Technology Co., Ltd., WHAA24A023; DMEM basal culture medium: Gibco, 6124332CellCounting kit-8, Proteintech, 20010859; serum-free cell freezing solution Biosharp, 23163960; 0.25% trypsin solution: Wuhan Punuo Life Science Technology Co., Ltd., WHAB24C191; dimethyl sulfoxide, American MPBiomedicals Y181205; FBS, Gibco, 2281654CP.

[0141] 1.2 Main instruments

[0142] -80℃ low temperature refrigerator, Qingdao Haier Special Electric Appliance Co., Ltd., DW-86L338J; CO2 incubator:

[0143] Thermo 300264168; Clean bench: Suzhou Antai Air Technology Co., Ltd., AZ2019071523; Vertical pressure steam sterilizer: Shanghai Shen'an Medical Equipment Factory, LDZX-75KBS; Microplate reader: MD, USA, Spectra M2; fast mixer: Changzhou Guohua Electric Co., Ltd. Changzhou Guohua SK-1; electric constant temperature water bath; Beijing Kewei Yongxing Instrument Co., Ltd., HH-2A; three-gas incubator: Wuxi Puhe Biomedical Technology Co., Ltd. PH-1-A; Fei Ge brand centrifuge: Shanghai Anting Scientific Instrument Technology Factory, TDL-80-2B; inverted microscope: Shanghai Optical Instrument Factory No. 1, XSP-SG; handheld cell counter: ScepterTM3.0, EMD Millipore Corporation2107600217; cell counting sensor: EMD Millipore Corporation 60-0067; high-power data ultrasonic cleaner: Kunshan Shumei Ultrasonic Instrument, KQ-800KDE; electronic balance: Sartorius Scientific Instrument (Beijing) Co., Ltd., Germany, SQP-SECURA225D-1CN.

[0144] 1.3 Experimental cells

[0145] Rat cardiomyocytes (H9C2) were purchased from Wuhan Pronocell Life Science Co., Ltd.

[0146] 2 Experimental methods

[0147] 2.1 Preparation of experimental drugs

[0148] Take differentiated samples of Guanxinshutong S1-S30 on a sterile operating table. The drug powder is yellow-brown and partially insoluble in water. Take 200 mg of Guanxinshutong powder and dissolve it in 1 m dimethyl sulfoxide (DMSO) to obtain a 200 mg / mL drug solution. The drug solution is sealed with a sealing film, quickly mixed with a fast mixer, and placed in a high-power data ultrasonic cleaner. After ultrasonic dissolution at room temperature for 30 minutes, it is placed at -20°C for use. In the formal experiment, the drug solution is taken out and different concentrations of drugs are prepared using the corresponding cell culture medium.

[0149] 2.2H9C2 cell culture and drug administration

[0150] 2.2.1 Cell recovery and medium replacement

[0151] Heat the water bath to 37°C, take out the H9C2 cell cryopreservation tube from the liquid nitrogen, shake it quickly in the water bath to make it melt quickly within 1 minute; disinfect its surface with 75% ethanol solution, place it on the purification rack, and then transfer the cell cryopreservation solution to a 10mL centrifuge tube, balance it and put it in the centrifuge at 1800r·min -1Centrifuge for 10 minutes, remove and discard the supernatant, add 1mL of complete culture medium, blow evenly, transfer the cells to a 25cm2 culture flask, then add 4mL of H9C2 special culture medium, and place the culture flask in a 37℃, 5% CO2 incubator for culture. After 24 hours, remove the cells for observation to ensure that the cells are in good condition. Change the medium: aspirate the waste liquid, add 2mL of D-PBS to rinse twice, and add 5mL of complete culture medium to continue culture.

[0152] 2.2.2 Cell passaging and cryopreservation

[0153] Take the cells out of the incubator and observe under a microscope. When the H9C2 cell adhesion area reaches 80-90%, the cells can be passaged. Remove the original culture medium, add 2mL PBS to wash, remove dead cells and residual culture medium, repeat the operation once, add 1mL 0.25% trypsin, put the cells into a 37℃ incubator for digestion for 3min, observe under a microscope whether the cells become round and fall off, and then directly add 3mL H9C2 special culture medium to terminate digestion. Then use a pipette to gently blow the cells that have not completely fallen off the surface of the culture bottle, transfer the cell suspension to a 10mL EP tube, centrifuge for 10min, and rotate at 1800r·min -1 , and then remove the supernatant. Passaging: add 2mL H9C2 special culture medium to suspend the cells, take 2 25cm2 culture flasks and add the suspension into the flasks, blow evenly, then add 4mL fresh complete culture medium, mix the cells evenly, observe whether the cells are evenly distributed under a microscope, and then place the cells in a cell culture incubator (37°C, 5% CO2) for culture; Cryopreservation: add 2mL serum-free cell freezing solution to blow evenly on the cells, transfer them to cell cryopreservation tubes, perform gradient freezing, and finally store them in a liquid nitrogen tank.

[0154] 2.2.3 Detection of toxicity of differentiated Guanxinshutong samples on H9C2 cells by CCK-8 method

[0155] The CCK-8 method was used to detect the toxicity of Guanxinshutong differentiated samples S1-S30 to H9C2 cells. The specific experimental steps are as follows:

[0156] (1) Select cells with good growth status, discard the culture medium, gently wash twice with PBS buffer, and discard the liquid;

[0157] (2) Add 1 mL of 0.25% trypsin to the culture flask, shake gently to allow the trypsin to fully contact the cells, and then place in a 37°C, 5% CO2 cell culture incubator for 3 min of digestion. Observe under a microscope to see if the cells become round and begin to fall off. Then add 3 mL of H9C2 special culture medium to terminate digestion.

[0158] (3) Use a pipette to gently blow away the cells that have not completely fallen off the surface of the culture flask, transfer the cell suspension to a 10 mL centrifuge tube, and centrifuge for 10 min at 1800 r / min. -1 , and then discard the supernatant;

[0159] (4) Resuspend the cell pellet and evenly seed it in a 96-well plate at 5000 cells / well;

[0160] (5) After the cells adhered and grew for 24 h, the 96-well plate was washed twice with PBS buffer to remove residual serum;

[0161] (6) The control group was added with incomplete medium DMEM, and each drug concentration group was added with drugs of corresponding concentrations;

[0162] (7) After 24 h, the liquid in each group was discarded and 10% CCK-8 detection solution (diluted in DMEM) was added in a dark place;

[0163] (8) Incubate in a cell culture incubator at 37°C and 5% CO2 for 2 h, and measure the absorbance of each well at a wavelength of 450 nm using an enzyme reader;

[0164]

[0165] (9) The obtained data were analyzed using Graph Pad Prism 9.0.0.

[0166] 2.2.4 Cell plating and cell model establishment

[0167] Take out the H9C2 cells in the logarithmic growth phase from the incubator, remove the culture medium, add 2mL PBS, wash away the residual culture medium and dead cells, repeat the operation once, add 1mL 0.25% trypsin, and digest at 37℃ for 3min. Take out the culture flask and observe under a microscope whether the cell morphology becomes round and begins to fall off, then add 3mL H9C2 special culture medium to terminate the digestion. Then gently blow the cells that have not completely fallen off the surface of the culture flask, transfer the cell suspension to a 10mL centrifuge tube, centrifuge for 10min, and rotate at 1800r·min -1 , discard the supernatant, add 1mL of complete culture medium, blow the cells evenly, turn on the handheld cell counter, insert the cell counting sensor when the device is ready, and insert the sensor probe into the evenly blown cell suspension. Wait for the device to display the words "Move the sensor out of the sample to be tested" and wait for the reading. Inoculate 5×10 cells per well at a volume of 100μL. 3The cells were placed in a 96-well plate, and the 96-well plate was gently shaken by the "eight-shaped shaking method" to make the cell suspension evenly distributed. The 96-well plate was placed in a cell culture incubator (37°C, 5% CO2) for culture. The culture was continued for 24 hours, the supernatant was discarded, serum-free DMEM and the corresponding concentration of drugs were added to each drug concentration group. The experiment was divided into a normal group, a model group and a drug administration group. According to the results of the previous CCK-8 cytotoxicity dose screening, the drug doses of each group of S1-S30 differentiated drug administration groups were set as shown in Table 10. The normal control group plate was placed in a cell culture incubator (37°C, 5% CO2) for culture, and the model plate was placed in a three-gas incubator (37°C, 5% CO2, 2% O2, 93% N2) for 16 hours. After adding serum to the normal group and the model group, they were placed in a normal incubator for 2 hours, the supernatant was removed and discarded, 10% CCK8 was added and incubated for 2 hours, and the OD value was measured.

[0168] Table 10 Dose setting of GXST differentiated administration group

[0169]

[0170]

[0171] 2.2.5 Comparison of efficacy with the control group drug (Guanxinshutong Capsules) of the present invention

[0172] In order to compare the efficacy of the preferred differentiated samples of the present invention with that of the commercially available Guanxinshutong capsules, we analyzed the efficacy of the contents of the commercially available Guanxinshutong capsules while conducting the pharmacodynamic analysis of the differentiated samples, and referred to the method steps of "2.2.4 Cell plating and cell model establishment" to determine the results. See the attached manual for the results. Figure 1 shown.

[0173] 3 Results and discussion

[0174] 3.1 GXST Differentiated Sample Safety Drug Dose Screening Based on CCK-8 Method

[0175] The CCK-8 method was used to detect the effects of different concentrations of S1-S30GXST differentiated samples on the activity of H9C2 cells. Group S1, group S2, group S3, group S4, group S5, group S8, group S12, group S13, group S14, group S16, group S18, group S19, group S23, group S24, group S25, group S26, group S27, group S28, group S29, and group S30 showed no toxicity to H9C2 cells at 50 μg / mL, and group S6, group S7, group S9, group S10, group S11, group S15, group S17, group S20, group S21, and group S22 showed no toxicity to H9C2 cells at 100 μg / mL.

[0176] 3.2 Effects of GXST-differentiated samples on H9C2 cell H / R injury

[0177] Guanxinshutong prescription has obvious protective effect on H9C2 cells. GXST differentiated samples 11, 15, 17, 18, 19, 21, 22, 23, 24, 27 and 29 also have obvious protective effect on H9C2 cells. Figure 2 shown.

Claims

1. A pharmaceutical composition for treating coronary heart disease, characterized in that: The pharmaceutical composition is prepared from raw materials in the following weight ratio: 60-80 parts of Choerospondias axillaris, 15-22 parts of Salvia miltiorrhiza, 5-8 parts of Cloves, 0.5-6 parts of Borneol, and 0-8 parts of Radix dasyphyllae.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is prepared from raw materials in the following weight ratio: 61.8 parts of Fructus Achyranthis Bidentatae, 20.7 parts of Salvia miltiorrhizae, 6 parts of Cloves, 5.5 parts of Borneol, and 6 parts of Radix Glehniae.

3. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is prepared from raw materials in the following weight ratio: 75.4 parts of Choerospondias axillaris, 16.6 parts of Salvia miltiorrhiza, 7 parts of Cloves, and 1 part of Borneol.

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