Effective part of pulse-activating decoction as well as extraction and purification method and application of effective part

Abstract

The invention discloses an effective part of a pulse-activating decoction as well as an extraction and purification method and application thereof, and the extraction and purification method comprises the following steps: taking red ginseng, radix ophiopogonis and schisandra chinensis as raw materials, adding water for extraction, and freeze-drying to obtain freeze-dried powder; dissolving the freeze-dried powder with distilled water, loading the dissolved powder on a macroporous adsorption resin column, carrying out gradient elution with water and a 20% ethanol aqueous solution in sequence, and separating fractions Fr1-Fr8; the chromatographic characteristics of each fraction are analyzed by using a C18 chromatographic column through a liquid chromatograph, the fractions with the same chromatographic characteristics are combined, the fractions Fr1-Fr4 are combined into a component yf-1, the fraction Fr5 is combined into a component yf-2, and the component yf-2 is the effective part of the pulse-activating decoction. According to the effective part disclosed by the invention, toxic parts are removed, so that H9c2 cell injury caused by adriamycin can be more effectively and stably protected with lower toxicity, and the application of the component yf-2 in preparation of medicines for treating myocardial cell injury is prompted.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to an effective part of Shengmai Yin and an extraction and purification method and application thereof. BACKGROUND

[0002] Shengmai Yin is from Medical Enlightenment, which is composed of red ginseng, ophiopogon and schisandra, has the effects of benefiting qi and nourishing yin, restoring pulse and preventing collapse, and is clinically used for treating acute myocardial infarction, cardiogenic shock, toxic shock, hemorrhagic shock, and coronary heart disease, endocrine disorders and other diseases caused by qi and yin deficiency. 〔1〕 The prior art mostly adopts whole formula extraction or rough separation, and the effective components and toxic components are not clear.

[0003] Wang P, Zhao Z, Wang D, et al. Progress in Chemical Composition, Pharmacological Action and Clinical Application of Shengmai Yin. Journal of Tianjin University of Traditional Chinese Medicine, 2024, 43(6):565-570 SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art, and provide a low-toxicity effective part of Shengmai Yin.

[0005] The second purpose of the present application is to provide an extraction and purification method of the effective part of Shengmai Yin.

[0006] The third purpose of the present application is to provide an application of the effective part of Shengmai Yin in the preparation of a drug for treating myocardial cell damage.

[0007] The technical scheme of the present application is summarized as follows:

[0008] An extraction and purification method of an effective part of Shengmai Yin, comprising the following steps:

[0009] 1) Take red ginseng, ophiopogon and schisandra as raw materials in a mass ratio of 1:2:1, add water for extraction, freeze-dry to obtain a freeze-dried powder;

[0010] 2) Take 10g of the freeze-dried powder obtained in step 1) according to the proportion, dissolve in 30ml of distilled water, load a macroporous adsorption resin column with a model D101 of 300g, sequentially elute with 1200ml of water and 1200ml of an ethanol aqueous solution with a volume concentration of 20%, and collect each 300ml of effluent as a fraction from the start of loading, and collect fractions Fr1-Fr8; analyze the chromatographic characteristics of each fraction by using a C18 chromatographic column through a liquid chromatograph, combine fractions with the same chromatographic characteristics, and combine fractions Fr1-Fr4 as component yf-1 and fraction Fr5 as component yf-2, wherein the component yf-2 is an effective part of Shengmai Yin.

[0011] The above method extracts and purifies an effective part of Shengmai Yin.

[0012] Use of the effective part of Shengmai Drink in the preparation of a drug for treating myocardial cell injury.

[0013] Advantages of the present application:

[0014] The effective part yf-2 of Shengmai Drink is obtained by the component optimization strategy of "leaving effect and removing toxicity", using fingerprint technology and modern new separation technology. The effective part can more effectively, more stably and less toxicly protect H9c2 cell injury caused by doxorubicin, indicating that the effective part yf-2 of Shengmai Drink is used for preparing a drug for treating myocardial cell injury. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Effect of yf-1-yf-12 and original Shengmai Drink (referred to as original prescription) with a drug dose of 10 μg / mL on the survival rate of H9c2 cells induced by 0.1 μM doxorubicin.

[0016] Figure 2 Effect of yf-1-yf-12 and original Shengmai Drink with a drug dose of 50 μg / mL on the survival rate of H9c2 cells induced by 0.1 μM doxorubicin.

[0017] Figure 3 Effect of yf-11 with different drug doses on the survival rate of H9c2 cells induced by 0.1 μM doxorubicin.

[0018] Figure 4 Total ion flow chart of yf-2 in positive ion mode.

[0019] Figure 5 Total ion flow chart of yf-2 in negative ion mode. DETAILED DESCRIPTION

[0020] The present application will be further described below in combination with specific examples. The examples of the present application are only a part of the examples of the present application, but not all the examples. Based on the examples in the present application, all the other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0021] Example 1

[0022] The extraction and purification method of the effective part of Shengmai Drink comprises the following steps:

[0023] 1) Red ginseng, Ophiopogon japonicus and Schisandra chinensis (2020 and 2025 edition of Chinese Pharmacopoeia Shengmai Yin) were taken as raw materials in a mass ratio of 1:2:1, 10 times the mass of distilled water was added to soak for 2 hours, heated to reflux for 40 min, cooled to room temperature, filtered, recovered the filtrate, and the residue was extracted with 8 times the mass of distilled water, and the extraction was repeated twice for 20 min, the filtrate was combined, concentrated under reduced pressure, and freeze-dried to obtain a freeze-dried powder (i.e. the freeze-dried powder of the original Shengmai Yin, abbreviated as the original prescription in Figure 1 and Figure 2

[0024] 2) 10 g of the freeze-dried powder obtained in step 1) was dissolved in 30 ml of distilled water, and a macroporous adsorption resin column with a model D101 of 300 g was loaded, and then 1200 mL of water, 1200 mL of 20% (volume concentration) ethanol aqueous solution, 1200 mL of 50% (volume concentration) ethanol aqueous solution, 1200 mL of 75% (volume concentration) ethanol aqueous solution and 1200 mL of anhydrous ethanol were used for gradient elution, and from the start of loading, every 300 mL of effluent was collected as a fraction to obtain fractions Fr1-Fr20.

[0025] The chromatographic characteristics of each fraction were analyzed by a liquid chromatograph using a C18 chromatographic column, and the fractions with the same chromatographic characteristics were combined.

[0026] Fractions Fr1-Fr4 were combined as component yf-1, fraction Fr5 as component yf-2, fraction Fr6 as component yf-3, fraction Fr7 as component yf-4, fraction Fr8 as component yf-5, fractions Fr9-Fr10 as component yf-6, fractions Fr11-Fr12 as component yf-7, fraction Fr13 as component yf-8, fraction Fr14 as component yf-9, fractions Fr15-Fr16 as component yf-10, fractions Fr17-Fr19 as component yf-11 and fraction Fr20 as component yf-12.

[0027] The component yf-2 is an effective part of Shengmai Yin of the application, and the compound identification results are shown in Figures 4-5 and Table 1.

[0028] Table 1 Identification of compounds in yf-2 sample

[0029] Example 2

[0030] Activity test of component yf-2 on inhibition of H9c2 myocardial cell injury.

[0031] 1. Test cell line

[0032] ​Highly differentiated rat myocardial cell line H9c2 (hereinafter referred to as H9c2 cells) was purchased from the Cell Resource Center of Shanghai Life Science Institute, Chinese Academy of Sciences.

[0033] 2. Experimental materials

[0034] DMEM medium, Gibco company; Hyclone fetal bovine serum, Hyclone company; trypsin, Biosharp company; penicillin-streptomycin mixture (double antibiotic), Wuhan Saivier Biotechnology Co., Ltd.; doxorubicin, Sigma-Aldrich company; 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), Sigma-Aldrich company; Dimetylsulfoxide (DMSO), Solabio company;

[0035] Shengpian Yin freeze-dried powder, in Figure 1 and Figure 2 , referred to as the original prescription.

[0036] yf-2 freeze-dried powder (component yf-2 freeze-dried), in Figure 1 and Figure 2 , referred to as 2.

[0037] 3. Instruments and equipment

[0038] Carbon dioxide incubator (Shanghai Lishen Co., Ltd.), biological safety cabinet (China Haier Co., Ltd.), inverted microscope (Japan Nikon Co.), Milli-Q ultrapure water machine (America Merck Millipore Co.), multifunctional enzyme label instrument (Shanghai Gene Co., Ltd.).

[0039] 4. Experimental method

[0040] (1) Cell culture

[0041] H9c2 cells were cultured in normal culture medium (DMEM medium containing 10% fetal bovine serum + 1% double antibiotic) in a 37℃, 5% CO2 saturated humidity incubator, and the medium was changed every 2 days. The logarithmic growth phase cells were used for experiment.

[0042] (2) Doxorubicin injury protection experiment

[0043] Cells were inoculated in 96-well plates, and the following groups were set (n = 6 for each group):

[0044] Blank control group: normal culture medium;

[0045] Model group: normal culture medium + 0.1 μM doxorubicin;

[0046] yf-2 low dose group: normal medium + 0.1 μΜ doxorubicin + 10 μg / mL yf-2 lyophilized powder;

[0047] yf-2 high dose group: normal medium + 0.1 μΜ doxorubicin + 50 μg / mL yf-2 lyophilized powder;

[0048] original recipe low dose group: normal medium + 0.1 μΜ doxorubicin + 10 μg / mL Shengpiyin lyophilized powder;

[0049] original recipe high dose group: normal medium + 0.1 μΜ doxorubicin + 50 μg / mL Shengpiyin lyophilized powder;

[0050] MTT method for detecting cell survival rate: H9c2 cells were seeded in 96-well plates at a density of 1 x 10 4 cells / well, and cultured for 24 h to adhere, then the fresh normal medium containing different concentrations of yf-2 lyophilized powder (10, 50 μg / mL) and Shengpiyin lyophilized powder (10, 50 μg / mL) was replaced according to the grouping, 6 replicate wells were set in each group, and after 3 h of culture, doxorubicin (final concentration 0.1 μΜ) was added, and the same amount of normal medium was added to the blank control group. After 24 h of continuous culture, 20 μL of MTT (5 mg / mL) was added to each well, and incubated for 4 h; the supernatant was discarded, 150 μL of DMSO was added, and shaken for 10 min; the absorbance (OD value) at 570 nm was detected by a microplate reader.

[0051] Cell survival rate (%) = (OD 实验组 / OD 空白对照组 ) x 100%.

[0052] (3) Experimental results

[0053] Compared with the blank control group (cell viability 100%), the model group significantly (p<0.05) induced myocardial cell damage (cell survival rate 59.33%) ( Figures 1-2 ).

[0054] 10 μg / mL of Shengpiyin lyophilized powder (original recipe) and component yf-2 (abbreviated as 2 in the figure) can significantly (p<0.05) protect this damage (cell survival rates are 72.02% and 70.14%, respectively) ( Figure 1 ).

[0055] 50 μg / mL of Shengpiyin lyophilized powder (original recipe) and component yf-2 can significantly (p<0.05) protect this damage (cell survival rates are 60.63% and 65.92%, respectively), and component yf-2 is better ( Figure 2 ).

[0056] Compared with the original recipe, the component yf-2 can more effectively protect adriamycin-induced H9c2 cell damage, suggesting that the component yf-2 can be used as an anti-cardiac injury drug.

[0057] The component yf-2 is a core active substance for treating heart injury by Shengpi Yin, and has better anti-cardiac injury activity.

[0058] Example 3

[0059] Activity test of the component yf-11 on aggravating H9c2 myocardial cell damage.

[0060] 1. Test cell line

[0061] The H9c2 myocardial cell line of high differentiation was purchased from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences.

[0062] 2. Experimental materials

[0063] DMEM medium, Gibco Company; Hyclone fetal bovine serum, Hyclone Company; trypsin, Biosharp Company; adriamycin, Sigma-Aldrich Company; 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), Sigma-Aldrich Company; Dimetylsulfoxide (DMSO), Solabio Company;

[0064] Shengpi Yin freeze-dried powder, in Figure 1 and Figure 2 , is referred to as the original recipe.

[0065] yf-11 freeze-dried powder (component yf-11 freeze-dried). In Figure 1 and Figure 2 , it is referred to as 11.

[0066] 3. Instruments and equipment

[0067] Carbon dioxide incubator (Shanghai Lishen Instrument Co., Ltd.), biological safety cabinet (Haier Co., Ltd.), inverted microscope (Nikon, Japan), Milli-Q ultrapure water machine (Merck Millipore, USA), multifunctional enzyme marker (Shanghai Gene Co., Ltd.).

[0068] 4. Experimental method

[0069] (1) Cell culture

[0070] H9c2 cells were subcultured in normal medium (DMEM medium containing 10% fetal bovine serum + 1% double-antibiotic) in a 37°C, 5% CO2 saturated humidity incubator, and the medium was changed every 2 days. The logarithmic growth phase cells were used for experiments.

[0071] (2) Toxicity evaluation of component yf-11

[0072] The cells were inoculated in a 96-well plate, and the following groups were set (n = 6 for each group):

[0073] Blank control group: normal medium;

[0074] Model group: normal medium + 0.1 μM adriamycin;

[0075] yf-11 dose 1 group: normal medium + 0.1 μM adriamycin + 50 μg / mL yf-11 lyophilized powder (see Figure 2 );

[0076] yf-11 dose 2 group: normal medium + 0.1 μM adriamycin + 10 μM yf-11 lyophilized powder (see Figure 3 );

[0077] yf-11 dose 3 group: normal medium + 0.1 μM adriamycin + 25 μM yf-11 lyophilized powder (see Figure 3 );

[0078] yf-11 dose 4 group: normal medium + 0.1 μM adriamycin + 50 μM yf-11 lyophilized powder (see Figure 3 );

[0079] yf-11 dose 5 group: normal medium + 0.1 μM adriamycin + 100 μM yf-11 lyophilized powder (see Figure 3 );

[0080] MTT method for detecting cell survival rate: same as in Example 2.

[0081] (3) Experimental results

[0082] Compared with the blank control group (cell survival rate 100%), the model group significantly (p < 0.05) induced myocardial cell damage (cell survival rate 59.33%), and 50 μg / ml of component yf-11 significantly (p < 0.05) aggravated the damage (cell survival rate 10.85%) Figure 2 .

[0083] At the same time, 10, 25, 50, and 100 μM of yf-11 lyophilized powder pre-administration (3 hours) significantly (p < 0.05) aggravated the damage, and the toxic gradient dose relationship was obvious Figure 3 .

[0084] The results show that component yf-11 has damaging effect on myocardial cells, and is a toxic substance for treating heart damage by Shengpi Drink.

[0085] Compared with the original formula, component yf-11 can more significantly aggravate the damage of H9c2 cells induced by adriamycin, and there is a significant dose-dependent relationship, which suggests that the weakly toxic effective part yf-2 obtained by separating component yf-11 is a safer drug for treating heart damage.

Claims

1. A method for extracting and purifying the effective components of Shengmai Yin (a traditional Chinese medicine formula), characterized by: Includes the following steps: 1) Take red ginseng, ophiopogon japonicus and schisandra chinensis in a mass ratio of 1:2:1 as raw materials, add water to extract, freeze dry to obtain freeze-dried powder; 2) Take 10g of the lyophilized powder obtained in step 1) according to the proportion, dissolve it in 30ml of distilled water, and load it onto a 300g macroporous adsorption resin column of model D101. Elute sequentially with 1200mL of water and 1200mL of 20% ethanol aqueous solution. From the start of sample loading, collect every 300mL of eluent as a fraction, and divide the fractions Fr1-Fr8 into fractions Fr1-Fr8. Analyze the chromatographic characteristics of each fraction using a C18 column in liquid chromatography, and combine fractions with the same chromatographic characteristics. Combine fractions Fr1-Fr4 into component yf-1 and fraction Fr5 into component yf-2. Component yf-2 is the effective part of a Shengmai Yin (a traditional Chinese medicine formula).

2. The effective component of Shengmai Yin extracted and purified by the method of claim 1.

3. The use of the effective component of Shengmai Yin according to claim 2 in the preparation of a drug for treating myocardial cell damage.

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