A pharmaceutical composition for treating coronary heart disease and a preparation method thereof

Abstract

The application provides a pharmaceutical composition for treating coronary heart disease, which is prepared from raw materials in the following weight ratio: 60-80 parts of Fructus Choerospondiatis, 15-22 parts of Salvia miltiorrhiza, 5-8 parts of clove, 0.5-6 parts of borneol and 0-8 parts of Radix Ophiopogonis. The pharmaceutical composition has the advantages of simple prescription, stable and reliable preparation process, remarkable efficacy and low toxicity and side effects.

Description

Technical Field

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

[0002] Coronary atherosclerotic heart disease, abbreviated as coronary heart disease (CHD), is a heart disease caused by coronary atherosclerosis that leads to stenosis, spasm or obstruction of the lumen, resulting in myocardial ischemia, hypoxia or necrosis. Affected by various risk factors such as genetics, metabolism, and lifestyle, it is the leading cause of death worldwide. It is reported that the prevalence of CHD in people over 60 years old in China is 27.8%, and it shows an increasing trend year by year. CHD is seriously threatening the physical health of Chinese residents. Traditional Chinese medicine believes that CHD belongs to the category of "chest impediment", and blood stasis syndrome is one of the main traditional Chinese medicine syndrome types of CHD. The method of promoting blood circulation to remove blood stasis is an important treatment method for CHD. Modern medicine believes that the main symptom of CHD is chest pain in front of the heart, and the nature of the pain is mostly colic and stuffy pain, and stabbing pain is relatively rare. In traditional Chinese medicine textbooks such as "Diagnostics of Traditional Chinese Medicine" and "Internal Medicine of Traditional Chinese Medicine", stabbing pain is mostly the main manifestation of blood stasis syndrome of CHD (chest impediment). Clinically, the western medicine treatment of angina pectoris is mainly class II β-adrenergic receptor blockers, calcium channel blockers (CCBs), and vasodilator drugs. Although it has achieved good clinical efficacy, patients have a high dependence on drugs, 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 impediment", and clinical syndrome differentiation and treatment can significantly improve symptoms and prognosis. The treatment of CHD with traditional Chinese medicine has obvious advantages, and it is significantly better than western medicine treatment in improving clinical symptoms, prognosis, etc.

[0003] The pharmaceutical composition referred to in the present invention is Guanxin Shutong Capsule, which is composed of the monarch drug Guangzao and Danshen, the ministerial drugs Dingxiang and Tianzhuhuang, and the adjuvant and guiding drug Bingpian. In the formula, Guangzao has the functions of promoting qi and activating blood circulation, nourishing the heart and calming the mind, resisting myocardial ischemia, and protecting heart function, etc., and is used for qi stagnation and blood stasis, chest pain, palpitations and shortness of breath, and restlessness of mind; Danshen is recorded in "Compendium of Materia Medica": "Activating blood circulation, dredging the pericardium collaterals, treating hernia pain", and has the effects of promoting blood circulation to remove blood stasis, nourishing blood, and calming the mind, etc., to assist Guangzao in promoting blood circulation to remove blood stasis and clearing the heart to relieve pain; Dingxiang has the effects of warming the middle and descending adverse qi, tonifying the kidney and assisting yang, and is used for diseases of the "life gate" (aorta), heart diseases, etc.; Bingpian has the effects of resuscitating the patient from unconsciousness, clearing heat and relieving pain, and is used for febrile disease with unconsciousness, convulsions, chest pain due to chest impediment, etc.; Tianzhuhuang has the effects of clearing heat and resolving phlegm, cooling the heart and calming fright. Guanxin Shutong Capsule is used for chest impediment with blood stasis in the heart meridian syndrome, and is clinically used to treat symptoms such as chest pain, chest tightness, palpitation, shortness of breath, etc. caused by CHD and angina pectoris. The main treatment functions are promoting blood circulation to remove blood stasis, dredging meridians and collaterals, promoting qi and relieving pain.

[0004] The applicant has filed multiple patent applications for this product, specifically involving the following related information: 02114549.0 (Dosage and formulation ratio of traditional Chinese medicine), 200510041670.9 (Preparation process), 200510041695.9 (Drip pills), 200510041884.6 (Pharmaceutical composition), 200510124510.0 (Quality control method for capsules), 201010500159.1 (Application in drugs for diabetes and impaired glucose tolerance), and 201210124018.3 (Preparation process). (Improved), 201210123918.6 (Oral Preparations), 201210124063.9 (Improved Oral Liquids), 201210124064.3 (Improved Soft Capsules), 201410003476.0 (Fingerprint Spectrum Determination Method), 201410148569.2 (Application in Drugs for Treating Myocardial Ischemia and Protecting the Cardiac), 201510870868.1 (Extract Drug Compositions), 201610005020.7 (Multiple Content Detection Methods), 202211404731.3

[0005] (Identification method of biomarkers for anti-myocardial ischemia), 202410092635.2 (Coronary heart disease relief extract).

[0006] However, in the early clinical and basic research conducted on this drug, we found that the formulation and dosage ratio of this traditional Chinese medicine composition have not yet reached the optimal level. Therefore, it is particularly important to develop a traditional Chinese medicine formula with good efficacy and few toxic side effects. Summary of the Invention

[0007] This invention provides a pharmaceutical composition for treating coronary heart disease and its preparation method. The pharmaceutical composition is simple to formulate and has a stable and reliable preparation process, and has the advantages of significant efficacy and low toxicity.

[0008] The formulation and dosage ratio of this invention were derived by the inventors of this application through extensive practical research and experience, and it was found that the dosage of each component within the following weight range can present better efficacy.

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

[0010] The pharmaceutical composition for treating coronary heart disease described in this invention is made from the following raw materials in the indicated weight ratios: 60-80 parts of jujube, 15-22 parts of salvia miltiorrhiza, 5-8 parts of clove, 0.5-6 parts of borneol, and 0-8 parts of bamboo shavings.

[0011] Preferably, the pharmaceutical composition is made from the following raw materials in the indicated weight ratios: 61.8 parts of jujube, 20.7 parts of salvia miltiorrhiza, 6 parts of clove, 5.5 parts of borneol, and 6 parts of bamboo shavings.

[0012] As a further preferred embodiment of the present invention, the pharmaceutical composition is made from the following raw materials in the indicated weight ratios: 75.4 parts of jujube, 16.6 parts of salvia miltiorrhiza, 7 parts of clove, and 1 part of borneol.

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

[0014] Take jujubes and grind them into fine powder, then divide them into two equal portions for later use. Grind bamboo shavings into fine powder for later use. Grind the remaining jujubes into the coarsest powder, use 70% ethanol as a solvent for percolation, collect the percolate, recover the ethanol and concentrate it to a thick paste with a relative density of 1.30-1.35 (50℃). Add one portion of the jujube fine powder, mix well, dry, and grind into fine powder for later use. The danshen extract was prepared three times. For the first extraction, the extract was heated under reflux with ethanol for 1.5 hours, filtered, and the ethanol was recovered from the filtrate. The extract was then concentrated to a thick paste with a relative density of 1.30–1.35 and set aside. For the second extraction, the extract was heated under reflux with 50% ethanol for 1.5 hours, filtered, and the filtrate was set aside. For the third extraction, the extract was decocted with water for 2 hours, filtered, and the filtrate was combined with the filtrate from the second extraction. The ethanol was recovered, and the extract was concentrated to a thick paste with a relative density of 1.30–1.35. This paste was then combined with the thick paste from the first extraction, mixed thoroughly, and concentrated to a thick paste with a relative density of 1.30–1.35. Another portion of finely powdered jujube was added, mixed well, dried, and pulverized into a fine powder. The volatile oil of cloves was extracted using steam distillation. The volatile oil was evenly sprayed into finely powdered bamboo shavings, mixed thoroughly, and sealed. Borneol was mixed with the remaining finely powdered bamboo shavings, ground finely, and then mixed with the above-mentioned fine powders. The mixture was then filled into capsules to obtain the final product.

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

[0016] Take coarse jujube powder (3 / 4 the amount of the prescribed jujube), moisten it evenly with 70% ethanol, seal and let it stand for 60 minutes, put it into a percolator, soak it in 70% ethanol for 48 hours, then slowly percolate at a rate of 2-3 mL / min, collect the percolate, recover the ethanol, concentrate it under reduced pressure to a relative density of 1.30-1.35, then add fine jujube powder (1 / 8 the amount of the prescribed jujube), mix well, dry, and pulverize; take danshen for extraction 3 times, the first time add 95% ethanol and heat under reflux for 1.5 hours, filter, recover the ethanol from the filtrate, and concentrate it to a thick paste with a relative density of 1.30-1.35, for later use; the second time heat under reflux for 1.5 hours with 50% ethanol, filter, and reserve the filtrate; the third time add water and decoct for 2 hours, filter... The filtrate was combined with the filtrate from the second extraction, the ethanol was recovered, and the mixture was concentrated to a thick paste with a relative density of 1.30–1.35. This paste was then combined with the thick paste from the first extraction, mixed, and concentrated to a thick paste with a relative density of 1.30–1.35. One part of fine jujube powder (1 / 8 of the amount of jujube in the prescription) was added, mixed well, dried, and pulverized into a fine powder. The volatile oil of clove was extracted by steam distillation. The amount of volatile oil added to the 60g clove in the prescription was fixed at 9.5mL. The volatile oil was evenly sprayed into the fine powder of bamboo shavings (1 / 2 of the amount of bamboo shavings in the prescription), mixed well, and sealed (1 / 2 of the amount of bamboo shavings in the prescription). Borneol was mixed with the fine powder of bamboo shavings, ground into a fine powder, and mixed with the above fine powders to obtain the mixture of the effective components 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 bamboo shavings with water or alcohol solvent to obtain a mixture of the active ingredients of jujube extract, salvia miltiorrhiza extract, clove extract, borneol extract, and bamboo shavings extract.

[0018] The above-mentioned Chinese herbal extracts can be added to pharmaceutical excipients to make pills, granules, tablets or capsules.

[0019] Droplets: The total mixture of the obtained Coronary Heart Relief Drug Composition is mixed with an appropriate amount of one or two excipients such as matrix PEG4000, PEG6000, stearic acid, or poloxamer, and then hot-melted, stirred and mixed evenly, and then dripped to make droplets.

[0020] Granules: The total mixture of the obtained coronary heart disease relief drug composition is mixed with an appropriate amount of one or two excipients such as dextrin, sucrose, lactose, mannitol, etc., and then wet-granulated, dried, and sieved to make granules.

[0021] Tablets: The obtained mixture of the coronary heart disease relief drug composition is mixed with an appropriate amount of corn starch, hydroxypropyl methylcellulose, magnesium stearate, dextrin and other one or two excipients, granulated, mixed and compressed into tablets;

[0022] Capsules: The obtained mixture of Guanxin Shutong drug composition is mixed with one or two of the following excipients: mannitol, lactose, microcrystalline cellulose, starch, magnesium stearate, talc, dextrin, etc., mixed evenly, granulated, and filled into capsules to make hard capsules.

[0023] Solution of the pharmaceutical composition of the present invention:

[0024] Explanation: In this formula, jujube is selected to invigorate qi and blood circulation, and to clear obstructed meridians. Combined with danshen, it can remove blood stasis, promote new blood production, invigorate blood, and relieve pain. The combined effect of these two herbs in invigorating qi and blood circulation is even more pronounced, making them the principal herbs. Clove is then added, which is pungent and warm, warming the middle jiao, dispelling cold, and promoting blood circulation. Bamboo shavings are used as the assistant herbs, resolving phlegm, clearing heat, and calming the nerves. These assist the principal herbs in warming and unblocking the blood vessels, thus eliminating blood stasis and obstruction. Borneol is used as the guiding herb to open the orifices and awaken the mind, guiding the medicine upwards to the head and eyes, thus ensuring smooth blood flow to the heart and brain, and relieving symptoms such as chest tightness and dark tongue.

[0025] In the early stages of this invention, four Q-Markers for the Coronary Heart Disease Relief Formula were screened: protocatechuic acid, cryptotanshinone, eugenol, and borneol. Based on the prescription of Guanxin Shutong Capsules, a uniform design method was used, with the prescription compatibility ratio as the influencing factor. Fifteen Guanxin Shutong formulas with different prescription compatibility ratios were prepared from the same batch of medicinal materials / processed slices. Since jujubes from different producing areas grow at the same latitude and longitude, the content of their main components does not fluctuate much, so using jujubes from different producing areas as an influencing factor is not appropriate. Salvia miltiorrhiza is widely distributed due to its extensive planting area, and the quality of Salvia miltiorrhiza from different producing areas varies, resulting in large fluctuations in the content of its main components. Cloves are mostly produced abroad, and there are few producing areas in my country, so using different producing areas as a variation factor provides limited reference. Borneol is mostly artificially synthesized. Bambusa textilis is less distributed. Therefore, Salvia miltiorrhiza from different producing areas was tested to prepare samples with differences in the quality of Guanxin Shutong. Based on the Guanxin Shutong prescription, 15 formulas with different Salvia miltiorrhiza from different producing areas conforming to the prescription compatibility ratio of Guanxin Shutong were prepared. A total of 30 samples with differences in Guanxin Shutong that met the research characteristics were prepared, including qualified and unqualified samples tested according to pharmacopoeia standards.

[0026] Beneficial effects of the present invention

[0027] (1) In the research on the improvement of the formulation process of the Guanxin Shutong product, the inventors of this application, guided by traditional Chinese medicine theory, obtained the following two optimal formulation ratios, achieving the best efficacy. The optimal formulation ratio is: 61.8 parts of jujube, 20.7 parts of salvia miltiorrhiza, 6 parts of clove, 5.5 parts of borneol, and 6 parts of bamboo shavings. Surprisingly, based on the original formulation, by reducing the amount of the assistant herb bamboo shavings and changing the dosage ratio of jujube, salvia miltiorrhiza, clove, and borneol, and after repeated research and experimentation, another optimal formulation ratio was obtained: 75.4 parts of jujube, 16.6 parts of salvia miltiorrhiza, 7 parts of clove, and 1 part of borneol.

[0028] (2) Through improvements in the preparation process, this invention obtains more refined and optimal preparation process parameters, resulting in a protocatechuic acid content ≥0.372mg / g, eugenol content ≥0.415mg / g, cryptotanshinone content ≥0.468mg / g, and borneol content ≥13.443mg / g in the Guanxin Shutong sample. This also makes the content of effective components in the extract of the drug composition of this invention higher and relatively uniform and stable.

[0029] (3) Based on the CCK-8 method, the GXST differential sample safety drug dosage screening showed that the Guanxin Shutong drug composition of the present invention was non-toxic to H9C2 cells, which also shows that the drug composition of the present invention has the advantages of low toxicity and high safety.

[0030] (4) Compared with the original formula of Guanxin Shutong, the drug composition of the present invention (Guanxin Shutong formula) has a significant protective effect on H9C2 cells. The GXST differential samples in groups 11, 15, 17, 18, 19, 21, 22, 23, 24, 27 and 29 also have a significant protective effect on H9C2 cells, which further indicates that the drug composition of the present invention has a protective effect on cardiomyocytes. Attached Figure Description

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

[0032] Figure 2 - The effect of differentially expressed Guanxin Shutong samples on the H9C2 cell H / R model; among which, experiments 11, 15, 17, 18, 19, 21, 22, 23, 24, 27, and 29 were the differentially expressed Guanxin Shutong samples of groups 11, 15, 17, 18, 19, 21, 22, 23, 24, 27, and 29, 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 Implementation

[0034] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0035] Example 1: Preparation of Differentiated Samples of the Drug Composition Coronary Heart Disease Relief of the Present Invention

[0036] 1. Experimental Materials and Instruments

[0037] 1.1 Reagents and Tests

[0038] Guangzao coarse powder (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231101), Guangzao fine powder (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231106), Danshen (Shaanxi Buchang Pharmaceutical Co., Ltd., batch number: 20231107), Dingxiang (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 Danshen (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040311), Henan Danshen (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040315), Sichuan Danshen (Shaanxi Kangshengtang Pharmaceutical Co., Ltd., batch number: 2024040313), Shaanxi Danshen (Shaanxi Tasly Herbal Medicine Technology Co., Ltd. GAP base, 24030102), Shandong Danshen (Heze Danhong Pharmaceutical Danshen GAP base, 20230301), 95% ethanol (Shaanxi Baoji Alcohol Factory).

[0039] 1.2 Experimental Apparatus

[0040] Electric thermostatic water bath: Shanghai Zhulan Instrument Technology Co., Ltd., HH-21-6; Forced air drying oven: Shanghai Zhulan Instrument Technology Co., Ltd., PHG-9075A; High-speed multi-functional 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 Scheme

[0043] 2.1.1 Preparation of differentiated samples with different compatibility ratios within the same batch

[0044] In the original formula of Guanxin Shutong Capsules, the proportions of jujube, danshen, clove, borneol, and bamboo shavings were 57%, 29%, 7%, 3.5%, and 3.5%, respectively. In a uniform design, the proportions of jujube ranged from 25.6% to 87.9%, danshen from 0% to 58%, clove from 0% to 14%, and borneol and bamboo shavings from 0% to 7%. Based on the principle that the number of levels in a uniform design should be greater than twice the number of factors, the five herbs were set as five factors. A five-factor, 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 shows the uniform design table for U5(515) obtained using the DPS data processing system.

[0046]

[0047] Table 2. Medicinal herb dosage for samples with differences in Guanxin Shutong capsules.

[0048]

[0049]

[0050] 2.1.2 Preparation of differentiated samples of Salvia miltiorrhiza from different origins and with different preparation processes

[0051] The experiment involved processing Danshen slices from different origins, including Shaanxi, Anhui, Shandong, Sichuan, and Henan. Considering the significant differences in efficacy due to variations in processing techniques, the experiment included alterations to the preparation process of Danshen slices, specifically, alcohol extraction of Danshen slices from different origins at 0, 1, and 2 times. The experimental prescriptions are shown in Table 3 below.

[0052] Table 3. Preparation of Differential Samples of Guanxin Shutong 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: Crude jujube powder (3 / 4 of the prescribed amount of jujube) was evenly moistened with 70% ethanol, sealed and left to stand for 60 minutes, then placed in a percolator, soaked in 70% ethanol for 48 hours, and then slowly percolated at a rate of 2-3 mL / min until completion. The percolate was collected, the ethanol was recovered, and the mixture was concentrated to a relative density of 1.15-1.20 (50℃), and then concentrated under reduced pressure to a relative density of... Heat the extract to 1.30–1.35 (50℃), then add finely powdered jujube (1 / 8 the amount of jujube in the prescription), mix well, dry, and pulverize. Extract the danshen three times. For the first extraction, add 95% ethanol and heat under reflux for 1.5 hours, filter, recover the ethanol from the filtrate, and concentrate to a thick paste with a relative density of 1.30–1.35 for later use. For the second extraction, heat under reflux for 1.5 hours with 50% ethanol, filter, and reserve the filtrate. For the third extraction, add water and decoct for 2 hours, filter, combine the filtrate with the filtrate from the second extraction, and recover the saturation. Ethanol was concentrated to a thick paste with a relative density of 1.30–1.35 (55–60℃), combined with the thick paste from the first extraction, mixed well, and concentrated to a thick paste with a relative density of 1.30–1.35 (55–60℃). One part of finely powdered jujube (1 / 8 of the amount of jujube in the prescription) was added, mixed well, dried, and pulverized into a fine powder. Cloves were steam distilled to extract volatile oil. Cloves contain 15–20% volatile oil, and the content of volatile oil in the medicinal material is not less than the specified content in Part I of the 2020 Chinese Pharmacopoeia. 16%, therefore, to ensure product quality stability, Buchang Pharmaceutical Co., Ltd. fixed the amount of volatile oil added to the 60g clove in the prescription to 9.5mL, and evenly sprayed the volatile oil into the fine powder of Tianzhuhuang (half the amount of Tianzhuhuang in the prescription), mixed well, and sealed (the prescription specifies half the amount of Tianzhuhuang) (the prescription specifies half the amount of Tianzhuhuang (15g) to absorb 9.5mL of clove oil (approximately 1.6g:1mL)); borneol was mixed with the fine powder of Tianzhuhuang, ground finely, and mixed evenly with the above-mentioned fine powders. In summary, a total of 30 batches of differentiated samples of Guanxin Shutong were obtained.

[0056] 2.3 Establishment of the Quality Information Dataset for the Pharmaceutical Compositions of the Present Invention

[0057] The quality of traditional Chinese medicine (TCM) is the foundation of clinical efficacy, and differences in TCM quality are a key factor affecting changes in efficacy. Exploring the precise quantitative correlation between TCM quality information and biological effects is a crucial scientific issue that needs to be addressed in TCM quality evaluation and the development of Q-Markers. It is also a critical scientific and technological problem that urgently needs to be solved in the development of TCM quality evaluation standards, moving from simply determining whether a product is qualified to evaluating high-quality products. Our research group has previously screened four Q-Markers in the Guanxin Shutong formula through extensive experiments: protocatechuic acid from jujube, cryptotanshinone from danshen, eugenol from clove, and borneol from borneol. The 2020 edition of the Chinese Pharmacopoeia for Guanxin Shutong Capsules uses two methods for content determination: gas chromatography (GC) and high-performance liquid chromatography (HPLC). GC uses borneol, isoborneol, and eugenol as indicator components, while HPLC uses tanshinone IIA and salvianolic acid B as indicator components. This method provides relatively comprehensive quality control, but it has the following two problems: 1. There are many chromatographic conditions (GC has one detection condition, HPLC has two), which makes content determination inconvenient; 2. The principal ingredient, jujube, has the effects of promoting blood circulation, nourishing the heart, and calming the mind, which is crucial for the efficacy of Guanxin Shutong Capsules. Furthermore, the amount of jujube (480g) accounts for approximately 57% of the total prescription amount (840g), a significant proportion, but the content determination of jujube as an indicator component is not included.

[0058] Borneol is a natural or synthetic substance with the effects of clearing the mind, relieving fever, and alleviating pain. Natural borneol is mainly produced in Sumatra and the Nanyang Islands of Indonesia, while synthetic borneol is produced in Guangzhou, Nanjing, Zhuzhou, Tianjin, and other places. It is used for febrile delirium, convulsions, stroke with phlegm, sudden syncope due to qi stagnation, coma due to sudden illness, red eyes, mouth sores, sore throat, and ear discharge. Borneol is the main active ingredient of borneol and has a significant analgesic effect. This invention establishes a gas chromatography method for determining the content of borneol in borneol, providing a data foundation for the establishment of a quality information dataset.

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

[0060] This invention also establishes a multi-index determination method to simultaneously determine the content of three components: protocatechuic acid, cryptotanshinone, and eugenol. While determining the content of Q-Marker, it aims to solve the problems mentioned above, such as the need for multiple chromatographic conditions and the absence of the main medicinal component of jujube in the pharmacopoeia.

[0061] 2.3.2 Materials and Instruments

[0062] 2.3.2.1 Experimental Apparatus

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

[0064] 2.3.2.2 Reference Standards and Drugs

[0065] Borneol: Batch No. 110881-201508, Purity: 96.8%, China National Institutes for Food and Drug Control; Guanxin Shutong Capsules: Batch No. 240201, Shaanxi Buchang Pharmaceutical Co., Ltd.; S1-S30 Guanxin Shutong Differentiated Samples, Self-made, Protocatechuic Acid, Batch No. 110809-202207, Purity: 99.99%, China National Institutes for Food and Drug Control; Cryptotanshinone: 110852-201807, Purity: 99.0%, China National Institutes for Food and Drug Control; Eugenol: Batch No. 110725-202318, Purity: 99.99%, China National Institutes for Food and Drug Control.

[0066] 2.3.2.3 Reagents

[0067] Ethyl acetate: Batch No.: 210329, Luoyang Haohua Chemical Reagent Co., Ltd.; Methanol: Chromatographic alcohol, F24O5K2O3, Fisher Reagent Company, USA; Formic acid: Batch No.: 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: flame ionization detector, temperature: 250℃; injector temperature: 220℃; split ratio: 2:1; column temperature: 80℃, held for 5 min, then increased to 150℃ at a rate of 5℃ / min, held for 5 min, then increased to 220℃ at a rate of 25℃ / min, held for 5 min; injection volume: 2μL. Under these conditions, borneol and adjacent peaks achieved good separation, with theoretical plate numbers 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 standard, place it in a 5 mL volumetric flask, and dilute to the mark with ethyl acetate to prepare a solution of 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 standard, place it in a 5 mL volumetric flask, and dilute to the mark with ethyl acetate to prepare a solution of 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 standard, place it in a 5 mL volumetric flask, and dilute to the mark with ethyl acetate to prepare a solution of 3,5932 mg / mL. -1 The solution was used as a linear solution reference stock solution.

[0076] Accuracy reference solution: Take 207.13 mg of borneol reference standard, place it in a 200 mL volumetric flask, and dilute to the mark with ethyl acetate to prepare a solution of 1.00251 mg / mL. -1 The solution was used as an accuracy control solution.

[0077] 2.3.3.2.2 Preparation of the test solution

[0078] Take 0.5g of each of the S1-S30 coronary artery shunt differential samples, accurately weigh them, accurately add 20mL of ethyl acetate, sonicate (power 180W, frequency 42kHz) for 30min, let it cool to room temperature, weigh it again, make up the weight loss with ethyl acetate, shake well, filter through a microporous membrane (0.22μm), and take the filtrate.

[0079] 2.3.4 Methodological Validation

[0080] 2.3.4.1 System Adaptability

[0081] Reference solution 1 was injected 5 times, reference solution 2 was injected 2 times, and the system confirmation solution (reference solution 1) was injected 1 time. The peak areas were recorded. The RSD of the precision of reference solution 1 was 0.28%, indicating good instrument precision. The F value of the two references was 100.06%, and the average theoretical plate number was 10846, indicating that the system suitability meets the pharmacopoeia requirements.

[0082] 2.3.4.2 Repeatability Test

[0083] Six samples of Guanxin Shutong S30 were taken and prepared according to the test solution method in section 2.2.2. The chromatographic conditions in section 2.1 were followed, the peak area was recorded, and the content was calculated. The RSD was 0.046%, indicating that the method has good repeatability.

[0084] 2.3.4.3 Stability Test

[0085] 2.3.4.3.1 Stability of the reference solution

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

[0087] 2.3.4.3.2 Stability of the test solution

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

[0089] 2.3.4.4 Limit of Quantitation and Limit of Detection

[0090] The reference solution was gradually diluted to a concentration of 0.0794 mg / mL, which was then used as the limit of quantitation solution. Five consecutive injections were performed, and the mean signal-to-noise ratio was 15.82, with an RSD of 1.81 for the peak area, indicating that the limit of quantitation met the pharmacopoeia standard. The reference solution was diluted to a concentration of 0.03974 mg / mL, which was then used as the limit of detection solution. Two consecutive injections were performed, and the signal-to-noise ratio was greater than 8.1 in both cases.

[0091] 2.3.4.5 Examination of Linear Relationships

[0092] Accurately measure 200 μL, 500 μL, 1000 μL, 1500 μL, and 2000 μL of the linear stock solution into 2 mL volumetric flasks, dilute to the mark with ethyl acetate to prepare linear solutions with concentrations of 0.3593, 0.8983, 1.7966, 2.6949, and 3.5932, respectively. Inject these solutions into the gas chromatograph under the chromatographic conditions described in section 2.3.3.1, and record the peak areas. Plot a standard curve with concentration (X) on the x-axis and peak area (Y) on the y-axis (results are shown in Table 4). R 2 The value was 0.9992, indicating a good linear relationship.

[0093] Table 4. Regression Equation, Correlation Coefficient, and Linear Range

[0094]

[0095] 2.3.4.6 Accuracy Test

[0096] Accurately weigh 0.25 g of the test sample, add the accuracy reference solution, and prepare the test sample solution according to the preparation method in section 2.2.2. Prepare 6 parallel samples, inject them into the gas chromatograph under the chromatographic conditions in section 2.3.3.1, record the peak area, and calculate the recovery rate. The results are 93.19%-99.32%, with an average of 97.41% and an RSD of 2.29. The experimental results show that the recovery meets the pharmacopoeia requirements.

[0097] 2.3.4.7 Recovery Test

[0098] Six portions of a sample with known content (batch number: 240201), each approximately 0.25 g, were accurately weighed and added to the specified amount of borneol reference solution as described in section 2.3.3.2.2. The test solutions were prepared according to the above method, and the chromatographic conditions were followed as described in section 2.3.3.1. The peak areas were recorded, and the results are shown in Table 5. The results showed that the average recovery rate of borneol was 97.41%, and the RSD was 2.29%.

[0099] Table 5. Recovery rate of borneol sample

[0100]

[0101]

[0102] 2.3.4.8 Determination of content in differentiated samples

[0103] The content of borneol in the differential samples of S1-S30 coronary artery shutong was determined. An appropriate amount of sample was accurately weighed and the test solution was prepared according to the method in section 2.2.2. Two samples were prepared for each batch of differential samples. Two injections were performed on each sample. The chromatographic conditions in section 2.1 were followed and the peak area was recorded. The average content of borneol in each batch of differential samples was calculated. The results for each batch are shown in Table 6.

[0104] Table 6. Borneol content (mg / g) in differentially expressed samples of Guanxin Shutong.

[0105]

[0106] This chapter establishes a GC method for determining the borneol content in differentially expressed samples of Guanxin Shutong (a traditional Chinese medicine). The results show good linearity, with an average recovery rate of 97.41%, indicating that the method is accurate, sensitive, and reproducible. Ethyl acetate has good solubility for borneol, and other interfering components in the sample dissolve in relatively little, so it was chosen as the extraction solvent. The preparation methods of the test solution were investigated, including reflux extraction, overnight cold soaking, and ultrasonic extraction. The peak areas were recorded and the contents were calculated according to the chromatographic conditions described in section 2.1. The results show that ultrasonic extraction yields a higher borneol content. Furthermore, ultrasonic extraction (power 250 Hz, frequency 50 kHz) for 10, 30, 45, and 60 min was also investigated. The test solutions were prepared and the peak areas were recorded and the contents were calculated according to the chromatographic conditions described in section 2.1. The results indicate that ultrasonic extraction for 30 min resulted in near-complete extraction of borneol.

[0107] 2.3.5 HPLC Experimental Methods and Results

[0108] 2.3.5.1 Chromatographic conditions

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

[0110] 2.3.5.2 Solution Preparation

[0111] 2.3.5.2.1 Preparation of reference solution

[0112] Accurately weigh appropriate amounts of the reference standards protocatechuic acid, cryptotanshinone, and eugenol, and add methanol to prepare a mixed reference 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 the test solution

[0114] Accurately weigh 0.5g of Guanxin Shutong capsules and 0.5g of S1-S30 differential samples, place them in a stoppered conical flask, and accurately add 10mL of methanol; stopper tightly, weigh, sonicate (power: 250W, frequency: 40kHz) for 30min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[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 and inject it six times consecutively under the chromatographic conditions described in section 2.1. Record the chromatograms 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 indicate that the instrument has good precision.

[0118] 2.3.5.2.3.2 Stability Test

[0119] The test solution of Guanxin Shutong capsules was injected and analyzed at 0h, 6h, 12h, 24h, and 48h under the chromatographic conditions described in section 2.1. The calculated RSDs of the peak areas of protocatechuic acid, eugenol, and cryptotanshinone were 0.39%, 0.42%, and 0.46%, respectively, indicating that the test solution had good stability within 48h. The reference solution was also injected and analyzed at 0h, 6h, 12h, 24h, and 48h under the chromatographic conditions described in section 2.1. The calculated RSDs of the peak areas of protocatechuic acid, eugenol, and cryptotanshinone were 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 Guanxin Shutong capsules and prepare 6 test solutions in parallel according to the method in section 2.2. Determine the results under the chromatographic conditions in section 2.1. The RSDs of the peak areas of protocatechuic acid, eugenol, and cryptotanshinone were calculated to be 0.39%, 0.4%, and 0.37%, respectively, indicating that the method has good repeatability.

[0122] 2.3.5.2.3.4 Examination of Linear Relationships

[0123] Accurately pipette the mixed reference solution, dilute it with methanol at different ratios, and then inject it for determination. Plot a standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate, calculate the regression equation, and the results are shown in Table 7.

[0124] Table 7 Results of the linear relationship examination

[0125]

[0126] 2.3.5.2.3.5 Recovery Test

[0127] Accurately weigh 0.5 g of the contents of Guanxin Shutong capsules with known component content, add reference standards at a ratio of 100% of component content, prepare the test solution according to the method in section 2.2, and determine the chromatographic conditions in section 2.1. Calculate the average recovery rate and RSD of protocatechuic acid, eugenol, and cryptotanshinone. The results are shown in Table 8.

[0128] Table 8 Results of the spiking recovery test of three components in Guanxin Shutong Capsules

[0129]

[0130]

[0131] 2.3.5.2.3.6 Determination of content in differentiated samples

[0132] The content of different samples of Guanxin Shutong from S1 to S30 was determined. An appropriate amount of sample was accurately weighed and the test solution was prepared according to the method in section 2.2.2. Two injections were performed on each sample. The chromatographic conditions in section 2.1 were followed and the peak area was recorded. The average content of protocatechuic acid, eugenol and cryptotanshinone in each batch of different samples was calculated. The results for each batch are shown in Table 9.

[0133] Table 9. Content (mg / g) of protocatechuic acid, eugenol, and cryptotanshinone in differentiated samples of Guanxin Shutong.

[0134]

[0135] This invention establishes an HPLC method for determining the contents of protocatechuic acid, eugenol, and cryptotanshinone in differentially expressed samples of coronary artery disease relieving agents. The results show good linearity, with an average recovery rate of 98.56% for protocatechuic acid, 100.91% for eugenol, and 104.9353% for cryptotanshinone, indicating that the method is accurate, sensitive, and reproducible. Methanol exhibits good solubility for protocatechuic acid, eugenol, and cryptotanshinone, and exhibits low dissolution of other interfering components in the samples; therefore, it was chosen as the extraction solvent. The preparation methods of the test solution were studied in the experiment. Reflux extraction, overnight cold soaking extraction, and ultrasonic extraction were investigated. The chromatographic conditions under section 2.1 were followed, peak areas were recorded, and contents were calculated. The results showed that ultrasonic extraction yielded the highest content among the three Q-Markers. Simultaneously, ultrasonic extraction (power 250 Hz, frequency 50 kHz) for 10, 30, 45, and 60 min was also investigated. The test solutions were prepared and the chromatographic conditions under section 2.1 were followed, peak areas were recorded, and contents were calculated. This indicated that ultrasonic extraction for 30 min resulted in near-complete extraction of the three Q-Markers.

[0136] Example 2: Pharmacodynamic analysis of the differentiated samples of Guanxinshtong at the in vitro level

[0137] In the early stage, our research group has carried out a preliminary study on the biomarkers of Guanxinshtong against myocardial cell ischemia based on the TMT quantitative proteomics technology, and found 43 differential proteins. Based on this, in this chapter, the pharmacodynamics of 30 batches of differentiated samples will be verified at the cellular level to lay a foundation for the later work.

[0138] 1. Experimental materials and instruments

[0139] 1.1 Drugs and reagents <00003z16>Differentiated samples of Guanxinshtong S1 - S30: self - made, Guanxinshtong Capsules, Shaanxi Buchang Pharmaceutical Co., Ltd., batch numbers: 240201 / 240202 / 240203 / 240204 / 240205. PBS buffer: Biosharp, 24149299; Special medium for H9C2 cells: Wuhan Punosai Life Science and Technology Co., Ltd., WHAA24A023; DMEM basal medium: Gibco, 6124332; Cell Counting kit - 8, Proteintech, 20010859; Serum - free cell cryopreservation solution, Biosharp, 23163960; 0.25% trypsin solution: Wuhan Punosai Life Science and Technology Co., Ltd., WHAB24C191; Dimethyl sulfoxide, MP Biomedicals, USA, Y181205; FBS, Gibco, 2281654CP.

[0141] 1.2 Main instruments

[0142] -80°C low - temperature refrigerator, Qingdao Haier Special Electric Appliance Co., Ltd., DW - 86L338J; Carbon dioxide incubator:

[0143] Thermo 300264168; Clean bench: Suzhou Antai Air Technology Co., Ltd., AZ2019071523; Vertical pressure steam sterilizer: Shanghai Shen'an Medical Instrument Factory, LDZX - 75KBS; Microplate reader: MD, USA, Spectra[[ID=z4]] M2; Rapid Mixer: Changzhou Guohua Electric Co., Ltd., Changzhou Guohua SK-1; Electric Thermostatic Water Bath: Beijing Kewei Yongxing Instrument Co., Ltd., HH-2A; Tri-Gas Incubator: Wuxi Puhe Biomedical Technology Co., Ltd., PH-1-A; Flying Pigeon Centrifuge: Shanghai Anting Scientific Instrument Technology Factory, TDL-80-2B; Inverted Microscope: Shanghai Optical Instrument Factory No. 1, XSP-SG; Handheld Cell Counter: Scepter™ 3.0, EMD Millipore Corporation 2107600217; Cell Counting Sensor: EMD Millipore Corporation 60-0067; High-Power Data Ultrasonic Cleaner: Kunshan Shumei Ultrasonic Instruments, KQ-800KDE; Electronic Balance: Sartorius Scientific Instruments (Beijing) Co., Ltd., Germany, SQP-SECURA225D-1CN.

[0144] 1.3 Experimental Cells

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

[0146] 2 Experimental Methods

[0147] 2.1 Preparation of experimental reagents

[0148] Differential samples S1-S30 of Guanxin Shutong were taken on a sterile operating table. The powder was yellowish-brown and partially insoluble in water. 200 mg of Guanxin Shutong powder was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain a drug solution of 200 mg / mL. The drug solution was sealed with sealing film, quickly mixed with a rapid mixer, and then placed in a high-power ultrasonic cleaner. After ultrasonic dissolution for 30 min at room temperature, it was placed at -20℃ for later use. In the formal experiment, the drug solution was taken out and used with the corresponding cell culture medium to prepare different concentrations of the drug.

[0149] 2.2 H9C2 cell culture and drug administration

[0150] 2.2.1 Cell resuscitation and medium change

[0151] The water bath was heated to 37°C. The H9C2 cell cryopreservation tubes were removed from liquid nitrogen and rapidly thawed within 1 minute by shaking in the water bath. The surface of the tubes was sterilized with 75% ethanol solution and placed on a purification rack. The cell cryopreservation solution was then transferred to 10 mL centrifuge tubes, balanced, and centrifuged at 1800 rpm. -1Centrifuge for 10 min, remove the cells, discard the supernatant, add 1 mL of complete culture medium, mix well by pipetting, and transfer the cells to a 25 cm² culture flask. Add 4 mL of H9C2-specific culture medium and incubate at 37°C with 5% CO2. After 24 h, remove the cells for observation to ensure they are in good condition. Change the medium: aspirate the waste liquid, wash twice with 2 mL of L-PBS, and add 5 mL of complete culture medium to continue culturing.

[0152] 2.2.2 Cell passage and cryopreservation

[0153] Remove the cells from the incubator and observe them under a microscope. When the H9C2 cell adhesion area reaches 80-90%, cell passage can be performed. Remove the original culture medium, wash with 2 mL of PBS to remove dead cells and residual culture medium, repeat the process once, then add 1 mL of 0.25% trypsin and incubate the cells at 37°C for 3 min. Observe under a microscope whether the cells have become rounded and detached. Then, add 3 mL of H9C2-specific culture medium to stop the digestion. Then, gently pipette the cells that have not completely detached from the culture flask surface, transfer the cell suspension to a 10 mL EP tube, and centrifuge for 10 min at 1800 rpm. -1 Then remove the supernatant. Passaging: Add 2 mL of H9C2 special culture medium to suspend the cells. Take two 25 cm2 culture flasks, add the suspension to the flasks, and mix well by pipetting. Then add 4 mL of fresh complete culture medium and mix the cells evenly. Observe under a microscope whether the cells are evenly distributed. Then place the cells in a cell culture incubator (37℃, 5% CO2) for culture. Cryopreservation: Add 2 mL of serum-free cell cryopreservation solution, mix the cells evenly by pipetting, transfer them to cell cryopreservation tubes, perform gradient cryopreservation, and finally store them in a liquid nitrogen tank.

[0154] 2.2.3 CCK-8 assay for the toxicity of differentially expressed Guanxin Shutong samples to H9C2 cells

[0155] The CCK-8 assay was used to detect the cytotoxicity of the differentially differentiated Guanxin Shutong samples S1-S30 to H9C2 cells. The specific experimental steps are as follows:

[0156] (1) Select cells in good growth condition, discard the culture medium, wash gently twice with PBS buffer, and then 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 it in a 37°C, 5% CO2 cell culture incubator. Observe under a microscope for 3 min to see if the cells become round and begin to detach. Then add 3 mL of H9C2 special culture medium to stop the digestion.

[0158] (3) Gently pipette the cells that have not yet completely detached from the culture flask surface, transfer the cell suspension to a 10 mL centrifuge tube, and centrifuge for 10 min at 1800 r·min. -1 Then discard the supernatant;

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

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

[0161] (6) The Control group was given incomplete culture medium DMEM, and each drug concentration group was given the corresponding drug concentration.

[0162] (7) After 24 hours, discard the liquids from each group and add 10% CCK-8 detection solution (diluted with DMEM) in the dark;

[0163] (8) Incubate at 37℃ and 5% CO2 in a cell culture incubator for 2 hours, and detect the absorbance of each well at 450nm wavelength using an ELISA reader.

[0164]

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

[0166] 2.2.4 Cell plating and cell model establishment

[0167] Remove H9C2 cells in the logarithmic growth phase from the incubator, remove the culture medium, add 2 mL of PBS, wash away residual culture medium and dead cells, repeat the process once, then add 1 mL of 0.25% trypsin and incubate at 37°C for 3 min. Remove the culture flask and observe under a microscope whether the cells have become rounded and begun to detach. Then add 3 mL of H9C2-specific culture medium to stop the digestion. Gently pipette the cells that have not yet completely detached from the culture flask surface, transfer the cell suspension to a 10 mL centrifuge tube, and centrifuge for 10 min at 1800 rpm. -1 After discarding the supernatant, add 1 mL of complete culture medium, and gently pipette the cells to mix them thoroughly. Turn on the handheld cell counter, and once the equipment is ready, insert the cell counting sensor and immerse the sensor probe into the well-mixed cell suspension. Wait for the device to display "Remove sensor from sample" before taking the reading. Seed 5 × 10⁵ cells per well at a volume of 100 μL. 3Cells were cultured in 96-well plates using a figure-eight shaking method to ensure even distribution of the cell suspension. The plates were then placed in a cell culture incubator (37℃, 5% CO2) for 24 hours. The supernatant was discarded, and serum-free DMEM was added. For each drug concentration group, the corresponding drug concentration was added. The experiment was divided into a normal control group, a model group, and a drug-treated group. Based on the previous CCK-8 cytotoxicity dose screening results, the drug dosages for each group in the S1-S30 differential drug-treated groups were set as shown in Table 10. The normal control group plates were cultured in a cell culture incubator (37℃, 5% CO2), and the model group plates were cultured in a tri-gas incubator (37℃, 5% CO2, 2% O2, 93% N2) for 16 hours. Serum was added to the normal and model groups, and the plates were placed in a normal incubator for 2 hours. The supernatant was discarded, and 10% CCK8 was added for incubation for 2 hours. The OD values ​​were then measured.

[0168] Table 10. Dosage settings for GXST differentiated dosing groups

[0169]

[0170]

[0171] 2.2.5 Comparison of efficacy with the control group drug of this invention (Guanxin Shutong Capsules)

[0172] To compare the efficacy of the preferred differentiated samples of this invention with that of commercially available Guanxin Shutong capsules, we performed pharmacodynamic analysis on the differentiated samples and also analyzed the efficacy of the contents of commercially available Guanxin Shutong capsules. Following the procedure in "2.2.4 Cell Plating and Cell Model Establishment," the results were obtained and are detailed in the appendix to the instruction manual. Figure 1 As shown.

[0173] 3 Results and Discussion

[0174] 3.1 Screening of safe drug dosage for GXST differential samples based on CCK-8 assay

[0175] The effects of different concentrations of GXST samples (S1-S30) on H9C2 cell viability were detected using the CCK-8 assay. Groups S1, S2, S3, S4, S5, S8, S12, S13, S14, S16, S18, S19, S23, S24, S25, S26, S27, S28, S29, and S30 showed no toxicity to H9C2 cells at 50 μg / mL. Groups S6, S7, S9, S10, S11, S15, S17, S20, S21, and S22 showed no toxicity to H9C2 cells at 100 μg / mL.

[0176] 3.2 Effects of GXST differential samples on H / R damage in H9C2 cells

[0177] The Guanxin Shutong formula has a significant protective effect against H9C2 cells. Differentially tested GXST samples (groups 11, 15, 17, 18, 19, 21, 22, 23, 24, 27, and 29) also showed significant protective effects against H9C2 cells. (See the instruction manual appendix.) Figure 2 As shown.

Claims

1. A pharmaceutical composition for treating coronary heart disease, characterized in that, The pharmaceutical composition is made of raw medicinal materials in the following weight ratio: 61.8 parts of Kuangzao, 20.7 parts of Danshen, 6 parts of Dingxiang, 5.5 parts of Bingpian and 6 parts of Tianzhu Huang.

2. A pharmaceutical composition for treating coronary heart disease, characterized by comprising the compound of claim 1. The pharmaceutical composition is made of raw medicinal materials in the following weight ratio: 75.4 parts of Kuangzao, 16.6 parts of Danshen, 7 parts of Dingxiang and 1 part of Bingpian.

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