CN113350396B - Preparation method of traditional Chinese medicine composition for resisting myocardial ischemia - Google Patents

Abstract

The invention relates to a preparation method of a traditional Chinese medicine composition for resisting myocardial ischemia, which comprises the following steps: (1) weighing the following raw materials: weighing the following raw materials in parts by weight: 15 parts of fructus choerospondiatis, 10 parts of radix salviae miltiorrhizae, 10 parts of sandalwood, 10 parts of myrobalan and 10 parts of kapok; (2) water extraction: adding water into the raw materials, and performing hot reflux extraction; filtering, and collecting the water extractive solution; (3) alcohol precipitation: adding ethanol solution into the water extract, precipitating, filtering, collecting filtrate, concentrating to remove ethanol or solvent to obtain water solution of the Chinese medicinal composition for resisting myocardial ischemia. The traditional Chinese medicine composition has the advantages of simple preparation method, wide raw material source and low cost, and has the effects of resisting acute myocardial ischemia of rats caused by ISO and treating coronary heart disease.

Description

A kind of preparation method of anti-myocardial ischemia traditional Chinese medicine composition

technical field

The invention relates to the field of traditional Chinese medicines, in particular to a preparation method of a new medicinal composition for treating myocardial ischemia.

Background technique

Myocardial ischemia is a pathological state in which the body's supply of blood flow to the heart is reduced, resulting in reduced oxygen supply to the heart, disturbance of myocardial metabolism, and the inability of the heart to function normally. Ischemic heart disease, including angina pectoris, myocardial infarction, coronary heart disease, etc., has attracted worldwide attention because of its high morbidity and high mortality. The results of the Global Burden of Disease (GBD) study show that in 2017, there were more than 10 million new cases of ischemic heart disease, 120 million existing cases, and more than 8 million deaths worldwide, making it the leading cause of death in the global population. . With the improvement of the economic level of Chinese residents and changes in their lifestyles, the morbidity and mortality rates of ischemic heart disease in my country have been increasing. The second cause of death from cerebrovascular disease.

Existing treatments for ischemic heart disease mostly use chemical drugs, such as nitrates, statins hypolipidemic drugs, anti-platelet preparations, etc. Currently, there is no Chinese medicine with definite curative effect for clinical treatment.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the defects existing in the prior art, to seek a preparation method of a traditional Chinese medicine prescription with definite curative effect on myocardial ischemia, and to broaden the drug resources for ischemic heart disease.

In order to achieve the above object, the present invention provides a preparation method of an anti-myocardial ischemia traditional Chinese medicine composition, comprising the following steps:

(1) Weigh the raw materials

The raw materials are weighed by the following parts by weight: 15 parts of jujube, 10 parts of Salvia miltiorrhiza, 10 parts of sandalwood, 10 parts of myrobalan, 10 parts of kapok;

(2) water extraction

The raw materials are added to water, and extracted under heat reflux; filtered, and the water extract is taken for later use;

(3) Alcohol precipitation

After concentrating the aqueous extract, adding ethanol solution, precipitation, filtration, taking the filtrate, concentrating to remove ethanol in the solvent or removing the solvent, the aqueous solution of the anti-myocardial ischemia traditional Chinese medicine composition or the anti-myocardial ischemia traditional Chinese medicine composition is obtained.

Further, in step (2), the number of times of hot reflux extraction is 3 times, the amount of water added each time is 12 times the total weight of the raw materials, and the time for each hot reflux extraction is 1.5h.

The specific steps of alcohol precipitation in step (3) are: concentrating the water extract to a relative density of 1.1-1.2, adding ethanol until the ethanol content in the solution is 85%, precipitation, and taking the filtrate; drying to obtain the anti-myocardial ischemia traditional Chinese medicine combination.

Compared with the prior art, the present invention has the following advantages:

1. The pharmaceutical composition of the present invention is derived from natural pharmaceutical ingredients, and is effective for biochemical indicators such as glutathione peroxidase activity, superoxide dismutase content and malondialdehyde content and myocardial necrosis in the blood and myocardial tissue of acute myocardial ischemia rats. The area has the effect of improving, and has the effect of resisting ISO-induced acute myocardial ischemia in rats and treating coronary heart disease.

2. The preparation method of the traditional Chinese medicine composition of the present invention is simple, the source of raw materials is wide, and the cost is low.

Description of drawings

Fig. 1 is the HPLC chromatogram for standard curve preparation;

In the figure, A- reference substance, B- test solution, C- negative control solution;

Figure 2 is a comparison diagram of the histopathological sections of rat myocardial tissue under different treatments.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments.

Example 1

Screening example of preparation technology of traditional Chinese medicine composition:

1. Extraction process

1) Weigh the raw materials

Take the following raw materials by weight according to the prescription amount: 15 parts of jujube, 10 parts of Salvia miltiorrhiza, 10 parts of sandalwood, 10 parts of myrobalan, 10 parts of kapok;

(2) water extraction

The raw materials are added to water, and extracted under heat reflux; filtered, and the water extract is taken for later use;

(3) Alcohol precipitation

Concentrating the extract to a relative density of 1.1-1.2, adding ethanol to a certain concentration, precipitation, taking the filtrate, and concentrating to remove ethanol in the solvent to obtain the SXK aqueous solution of the anti-myocardial ischemia traditional Chinese medicine composition.

2. Optimization of extraction process

1 Instruments and materials

1.1 Instruments High performance liquid chromatograph (Dinex Ultiate 3000, American Dian Instruments Co., Ltd.), electric heating blast drying oven (GZX-9070MBE, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), ultrasonic cleaner (SB-5200DT, Ningbo New Art Ultrasonic Equipment Co., Ltd.), rotary evaporator (RE-52AA, Shanghai Yarong Biochemical Instrument Factory), digital display electric heating constant temperature water bath (XMTD-204, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory). Electronic analytical balance (CPA225D, Sartorius Scientific Instruments Co., Ltd.).

1.2 Materials Salvia miltiorrhiza (batch number 1601002, Hebei Quantai Pharmaceutical Co., Ltd.), tanshinone IIA reference substance (China Institute for Food and Drug Control, batch number: 110766-201721), methanol (analytical grade, Nanjing Chemical Reagent Factory); acetonitrile (chromatographic alcohol, DikmaPure).

2 Methods and results

2.1 Determination of Tanshinone IIA

2.1.1 Chromatographic conditions and system suitability test: Diamonsil C18 column (250mm×4.6mm, 5μm) was used; acetonitrile (A)-phosphoric acid water (B) was used as the mobile phase, and gradient elution was carried out according to the setting conditions in Table 1 ; The column temperature is 20℃; the detection wavelength is 270nm; the theoretical plate number should not be less than 60000 according to the tanshinone IIA peak.

Table 1 Mobile phase setting conditions

2.1.2 Preparation of reference solution Precisely weigh 5.0012 mg of tanshinone IIA, put it in a 5 mL volumetric flask, dissolve it with methanol and dilute to the mark, and shake well. Precisely pipette 1 mL of the above-prepared reference solution, put it in a 50 mL volumetric flask, add methanol to dilute to the mark, shake well, and use it as the tanshinone IIA reference solution with a concentration of 20.0048 μg·mL-1.

2.1.3 Preparation of the test solution Take the prepared SXK aqueous solution, dilute to 100mL, and shake well to get it.

2.1.4 Preparation of Negative Control Solution: Weigh the whole prescription medicinal materials lacking Salvia miltiorrhiza, prepare a sample without Salvia miltiorrhiza according to the same process, set the volume to 100mL, and shake well.

2.1.5 Specificity test The above reference solution, test solution and negative control solution were filtered through a 0.45 μm microporous membrane, and 10 μL was injected into each. The results are shown in Figure 1: In the chromatogram of the test product, there are chromatographic peaks at the same retention time as the chromatogram of the tanshinone IIA reference substance, which can be separated from other components; There are no interfering peaks at the corresponding positions of the chromatographic peaks, indicating that tanshinone IIA has specificity under the above chromatographic conditions: .

2.1.6 Determination method Precisely draw 10 μL of the reference solution and the test solution, respectively, and inject them into a high-performance liquid chromatograph for measurement.

2.1.7 Preparation of standard curve: Dilute the reference solution with methanol by 2, 4, 6, 8, and 10 times, accurately pipette 10 μL into the high performance liquid chromatograph, and measure according to the chromatographic conditions under “2.1.1”. Taking the chromatographic peak area as the ordinate and the content of tanshinone IIA as the abscissa, using Excel software to draw the standard curve, the regression equation is: Y=69.66X+0.2946, (R ² =0.9998).

2.1.8 Precision test: Take 10 μL of the reference solution under “2.1.2” continuously, inject 6 injections respectively, record the peak area of each injection, the RSD of the peak area of tanshinone IIA is 1.86%, indicating that the precision of the instrument is good .

2.1.9 Stability test: Take the prepared SXK aqueous solution, prepare it according to the method under "2.1.2", inject samples at 2, 4, 6, 8 and 10 hours respectively, and measure according to the chromatographic conditions under "2.1.1" The content of tanshinone IIA, calculated RSD was 1.13%, indicating that the tanshinone IIA in the SXK aqueous solution was stable for at least 10h.

2.2 Orthogonal experimental design: The yield and paste yield of tanshinone IIA in the extract are used as the investigation indicators, and the concentration of alcohol precipitation in step (3), the extraction times in step (1), the amount of water added, and the extraction time are selected according to 4 L ₉ (3 ⁴ ) orthogonal experimental design was carried out for the factor 3 level, and the factor level table (see Table 2).

Table 2 Extraction factor level table of traditional Chinese medicine composition orthogonal test of the present invention

2.2.1 Extraction method: Weigh the whole prescription medicinal materials according to the prescribed amount, a total of 55g, and take 9 parts respectively. An orthogonal design test L ₉ (3 ⁴ ) was used, and four factors were selected: A concentration of alcohol precipitation, B extraction times, C water addition, and D extraction time, as shown in Table 2. According to the orthogonal test scheme, decoction and filtration were carried out in water. 75-85% ethanol solution was added to the filtrate, and after standing for precipitation, filtration was performed, and the filtrate was concentrated to 100 ml to obtain 9 sample solutions for orthogonal tests.

2.2.2 Determination of the yield of paste: Precisely measure 50 mL of the sample solution under each condition in item "2.2.1", place it in an evaporating dish that has been dried to constant weight, evaporate to dryness in a water bath, and dry under reduced pressure at 80°C to constant weight , Precisely weigh the quality, and calculate the yield of the paste.

2.2.3 Evaluation indicators and result analysis The orthogonal test results are combined with visual analysis and variance analysis. The results are shown in Table 3 and Table 4.

Table 3 Intuitive analysis diagram of traditional Chinese medicine composition water extraction and alcohol precipitation orthogonal test of the present invention

Table 4 Results of ANOVA for Comprehensive Score

According to the results of visual analysis and variance analysis, the range of comprehensive scores shows that the primary and secondary effects of each factor are B>D>C>A, that is, the extraction times have the greatest impact on the content of tanshinone IIA, followed by extraction time, water amount, and alcohol precipitation concentration. , the optimal combination of water extraction and alcohol precipitation is A ₃ B ₃ C ₃ D ₂ . That is, 12 times of water was used, and the mixture was extracted under heat reflux for 3 times for 1.5 hours each time. After precipitation with 85% ethanol, the filtrate was collected.

2.2.4 Confirmatory experiment: Weigh 3 parts of medicinal materials according to the prescription ratio, each 55g, carry out 3 times of confirmation experiment according to the best water extraction and alcohol precipitation process, and determine the content of tanshinone IIA and the yield of ointment. The results showed that the content of Tanshinone IIA in the preferred process had the highest comprehensive score of dry extract yield, and the RSD was 2.21%. The results show that the optimized process is stable and feasible.

Example 2

Product Preparation Example

1) Weigh the raw materials

Take the following raw materials by weight according to the prescription amount: 15 parts of jujube, 10 parts of Salvia miltiorrhiza, 10 parts of sandalwood, 10 parts of myrobalan, 10 parts of kapok;

(2) water extraction

Add 12 times by weight of water to the raw material, extract under reflux for 3 times, 1.5h each time; filter, combine the extracts, and set aside;

(3) Alcohol precipitation

After combining the extracts, concentrate the extracts to a relative density of 1.1-1.2, add ethanol until the ethanol content in the solution is 85%, precipitate, and take the filtrate to obtain the water extract of the traditional Chinese medicine composition of the present invention.

Example 3

Application Example

1 material

1.1 Animal health 50 SD rats, half male and half male, weighing 200-250 g. Provided by the GLP Center of Heilongjiang University of Traditional Chinese Medicine, license number SCXK-(Beijing) 2009-0001.

1.2 Instruments KY2000 semi-automatic biochemical analyzer (Shimadzu Corporation, Japan); 756PC Spectrum UV spectrophotometer (Shanghai Spectrum Instrument Co., Ltd.); BL-420F biological function experimental system.

1.3 Reagents GSH-Px test kit (batch number 20180626), SOD kit (batch number 20181105), and MDA kit (batch number 20171214) were purchased from Nanjing Jiancheng Bioengineering Institute.

1.4 Drugs Isoproterenol Hydrochloride (Shanghai Hefeng Pharmaceutical Co., Ltd.); Di'ao Xinxuekang Capsule (Chengdu Di'ao Group Co., Ltd.); The aqueous solution of the Chinese medicine composition SXK of the present invention prepared in Example 2.

2 methods

2.1 Establishment of isoproterenol (Iso)-induced acute myocardial ischemia model: The rats were randomly divided into blank group, model group, SXK high, medium and low dose groups and positive control drug (Diao Xinxuekang Capsule) group. The aqueous solution of SXK is converted by body surface area according to the normal dosage, and the dosage is respectively high, middle and low dosage groups: 4mL (L (after the preparation of the traditional Chinese medicine composition in the above example 1 is dried, the concentration is 0.4g with water). /ml, 0.2g/ml, 0.1g/ml of high, medium and low concentrations as high, medium and low dose groups, with 1ml/100g for intragastric administration). Positive control group: 4mL (equivalent to Di'ao Xinxuekang 30 mg·kg ^-1 ). The blank group and the model group were given distilled water of the corresponding volume, and all rats were given continuous gavage for 15 days, once/day. Effect of oxidative stress and oxidative stress levels, Gu Yuanyuan, Yu Ying, Zhou Zhongguang, etc., Shizhen Chinese Medicine Chinese Medicine, 2019, 30(10): 2370-2372) and pre-test, starting from the 12th day, except for the blank group, the rest of the All groups were intraperitoneally injected with isoproterenol at a dose of 5 mg·kg ^-1 , once a day for 3 consecutive days.

2.2 Detection indicators and methods

2.2.1 Electrocardiogram detection: After the last gavage, the rats were anesthetized with 10% chloral hydrate (3ml·kg ^-1 ) by intraperitoneal injection. After the anesthesia was completed, they were supine and fixed on the operating table, and the needle electrode was placed on one side. The rats were inserted under the skin of the limbs, and the other side was connected to the BL-420F biological function experimental system, the standard lead II electrocardiogram was recorded, the changes of the electrocardiogram before and after the modeling were observed, and the voltage value of the J point and the changes of the T wave and the height of the T wave were compared before and after the modeling. The measurement takes the PR segment as the baseline, and 5 continuous waveforms are measured and the average value is taken for statistical analysis.

2.2.2 Determination of GSH-Px, SOD and MDA in serum: the rat abdominal aorta was intubated to take blood, a part of plasma was taken to separate serum, and the contents of GSH-Px, SOD and MDA in serum were determined according to the instructions of the kit.

2.2.3 Preparation of myocardial tissue pathological sections: The apical tissue was taken out, fixed in 10% formaldehyde, stained with HE, and the pathological changes of the specimens in each group were observed under a light microscope.

2.3 Statistical processing: SPSS 13.0 software was used, the data were represented by x±s, the comparison between the two groups was performed by t test, and P<0.05 was considered statistically significant.

3 results

3.1 Effect on the electrocardiogram of rats with acute myocardial ischemia: after injection of Iso, as shown in Table 5, compared with the control group, the T wave of the model group increased and the J point moved down, indicating myocardial ischemia; compared with the model group , SXK high dose can significantly improve the J point and T wave changes (p<0.05).

Table 5 Effects of T wave height and J point height after injection of isoproterenol (x±s, n=10)

Compared with blank group ^▲ P<0.05, ^▲▲ P<0.01; compared with model group ^■ P<0.05, ^■■ P<0.01.

3.2 Effects on GSH-Px, SOD and MDA in serum: After injection of Iso, the activities of GSH-Px and SOD in serum of rats were significantly decreased, and the content of MDA was significantly increased. Compared with the model group, the activities of GSH-Px and SOD in the serum of the rats in the three dose groups of SXK were increased to a certain extent, and the content of MDA was decreased. <0.05), can significantly reduce the content of MDA in rat serum (p<0.05). See Table 6.

Table 6 Effects of serum GSH-Px, MDA and SOD activities in myocardial ischemia rats (x±s, n=10)

Compared with blank group ^▲ P<0.05, ^▲▲ P<0.01; compared with model group ^■ P<0.05, ^■■ P<0.01.

3.3 Myocardial tissue pathological section The myocardial cells in the blank group were neatly arranged, without obvious degeneration and necrosis, and there was no inflammatory cell infiltration in the interstitial cells. Compared with the blank control group, the myocardial tissue of the model group had obvious ischemic damage, mainly showing myocardial fiber disorder, interstitial edema and degeneration, and myocardial cells showed obvious pathological changes such as vacuolar degeneration, uneven cytoplasmic coloration, and pyknosis. The morphological and structural changes showed that intraperitoneal injection of isoproterenol could cause acute myocardial ischemia injury; compared with the model group, the myocardial necrosis area was significantly reduced in each administration group; It is shown that SXK can alleviate the degeneration and necrosis of cardiomyocytes, and the results are shown in Figure 2.

The etiology of ischemic heart disease (coronary heart disease) is closely related to the production of oxygen free radicals in the heart. Myocardial hypoxia-ischemia induces oxidative stress, and a large number of neutrophils accumulate in the ischemic area, accelerating the generation of oxygen free radicals. The accumulation of oxygen free radicals causes lipid peroxidation in membranes, causing structural abnormalities and functional damage of cell membranes, thereby aggravating myocardial tissue damage. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) are endogenous enzyme scavengers that can counteract the oxidative damage of free radicals, and malondialdehyde (MDA) is a stable The aldehyde products of lipid peroxidation are widely used to reflect the level of oxidative stress. The traditional Chinese medicine composition SXK high-dose group prepared by the invention has strong antioxidant capacity, can improve the activities of GSH-Px and SOD in myocardial tissue, can inhibit the process of lipid peroxidation, and reduce the activity of lipid peroxidation MDA , reduce the damage of oxygen free radicals to the myocardium, protect ischemic myocardium, and treat ischemic heart disease.

In the research process of the present invention, it was observed that after oral administration of SXK in rats, myocardial tissue ischemia caused by Iso can be effectively inhibited, the height of T wave and J point is significantly changed, the activities of GSH-Px and SOD in serum are increased, and the content of MDA is significantly decreased, suggesting that SXK can protect cardiomyocytes and improve the stability of cell membranes. From the pathological sections, it can be seen that after taking SXK, a large number of inflammatory cells infiltrated in the area of myocardial degeneration and necrosis, and myocardial fibers were disordered and broken. Experiments show that the traditional Chinese medicine composition SXK prepared by the present invention can effectively inhibit myocardial tissue ischemia caused by isoproterenol (Iso), protect myocardial cells from damage, and thus play a role in treating coronary heart disease.

Claims (3)

1. The preparation method of the traditional Chinese medicine composition for resisting myocardial ischemia is characterized by comprising the following steps:

(1) weighing raw materials

Weighing the following raw materials in parts by weight: 15 parts of fructus choerospondiatis, 10 parts of radix salviae miltiorrhizae, 10 parts of sandalwood, 10 parts of myrobalan and 10 parts of kapok;

(2) water extraction

Adding water into the raw materials, and performing hot reflux extraction; filtering, and collecting the water extractive solution;

(3) alcohol precipitation

Concentrating the water extract, adding ethanol solution, precipitating, filtering, collecting filtrate, concentrating, and removing ethanol or solvent to obtain the final product.

2. The preparation method according to claim 1, wherein the alcohol precipitation in the step (3) comprises the following specific steps: concentrating the water extract to relative density of 1.1-1.2, adding ethanol until ethanol content is 85%, precipitating, and collecting filtrate; drying to obtain the traditional Chinese medicine composition for resisting myocardial ischemia.

3. The preparation method according to claim 2, wherein the number of hot reflux extractions in step (2) is 3, each time water is added in an amount 12 times the total weight of the raw materials, and each time the hot reflux extraction time is 1.5 h.

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