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

Abstract

The method comprises the following steps: taking red ginseng, radix ophiopogonis and schisandra chinensis, carrying out water extraction and freeze drying to obtain freeze-dried powder, taking the freeze-dried powder, adding distilled water to dissolve the freeze-dried powder, adding the dissolved freeze-dried powder to a macroporous adsorption resin column, carrying out gradient elution with water, a 20% ethanol aqueous solution, a 50% ethanol aqueous solution and a 75% ethanol aqueous solution in sequence, and carrying out vacuum concentration to obtain the effective part of the pulse-activating decoction. From the beginning of sample loading, collecting every 300mL of effluent as a fraction, and collecting the fractions Fr1-Fr16; the chromatographic characteristics are analyzed, fractions with the same chromatographic characteristics are combined, the fractions Fr15-Fr16 are combined into a component yf-10, and the component yf-10 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 protected in a more effective, more stable and lower-toxicity manner, and the application of the component yf-10 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 Shengpi Yin and an extraction and purification method and application thereof. BACKGROUND

[0002] Shengpi 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 astringing, and is commonly used in the treatment of 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 adopts whole formula extraction or rough separation, and the effective components and toxic components are not clear.

[0003] Wang Ping, Zhao Zhiwen, Wang Dinding, et al. Progress of Chemical Composition, Pharmacological Action and Clinical Application of Shengpi 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 an effective part of Shengpi Yin with low toxicity.

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

[0006] The third purpose of the present application is to provide an application of the effective part of Shengpi 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] The extraction and purification method of the effective part of Shengpi Yin comprises the following steps:

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

[0010] 2) according to the proportion, 10 g of the freeze-dried powder obtained in step 1) is dissolved in 30 ml of distilled water, and is loaded onto a macroporous adsorption resin column with a model D101, and is eluted with 1200 ml of water, 1200 ml of an ethanol aqueous solution with a volume concentration of 20%, 1200 ml of an ethanol aqueous solution with a volume concentration of 50%, and 1200 ml of an ethanol aqueous solution with a volume concentration of 75%, respectively, and from the start of loading, every 300 ml of effluent is collected as a fraction to obtain fractions Fr1-Fr16; the chromatographic characteristics of each fraction are analyzed by using a C18 chromatographic column through a liquid chromatograph, and the fractions with the same chromatographic characteristics are combined, and the fractions Fr1-Fr4 are combined as component yf-1; the fraction Fr5 is component yf-2; the fraction Fr6 is component yf-3; the fraction Fr7 is component yf-4; the fraction Fr8 is component yf-5; the fractions Fr9-Fr10 are combined as component yf-6; the fractions Fr11-Fr12 are combined as component yf-7; the fraction Fr13 is component yf-8; the fraction Fr14 is component yf-9; and the fractions Fr15-Fr16 are combined as component yf-10; and the component yf-10 is the effective part of Shengmai Drink.

[0011] The effective part of Shengmai Drink extracted and purified by the above method.

[0012] The effective part of Shengmai Drink is used for preparing a drug for treating myocardial cell injury.

[0013] Advantages of the present application:

[0014] The present application obtains the component yf-10 which is an effective part of Shengmai Drink through the component optimization strategy of 'leaving efficacy and removing toxicity', and uses the fingerprint technology and modern new separation technology, and the effective part of the present application can more effectively, more stably and more lowly protect the H9c2 cell injury caused by adriamycin by removing the toxic part, which indicates that the component yf-10 of the present application is used for preparing a drug for treating myocardial cell injury. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0017] Figure 3 Effects of yf-10 with different drug dosages on the survival rate of H9c2 cells induced by 0.1 μM adriamycin.

[0018] Figure 4 Effect of yf-11 at different dosages on H9c2 cell survival rate induced by 0.1 μM adriamycin.

[0019] Figure 5 Total ion current chromatogram of yf-10 in positive ion mode.

[0020] Figure 6 Total ion current chromatogram of yf-10 in negative ion mode. DETAILED DESCRIPTION

[0021] The application will be further described below in conjunction with specific examples. The examples of the application are only a part of the examples of the application, rather than all the examples. Based on the examples in the application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the application.

[0022] Example 1

[0023] An extraction and purification method of an effective part of Shengpi Yin, comprising the following steps:

[0024] 1) Take red ginseng, ophiopogon and schisandra (2020 and 2025 edition of Chinese Pharmacopoeia Shengpi Yin) as raw materials in a mass ratio of 1:2:1, soak in 10 times the mass of distilled water for 2 hours, heat reflux for 40 min, cool to room temperature, filter, recover the filtrate, and then repeat the extraction with 8 times the mass of distilled water for 20 min for 2 more times, combine the 3 times of filtrate, reduce pressure and concentrate, freeze-dry to obtain a freeze-dried powder (i.e. the freeze-dried powder of the original prescription Shengpi Yin, referred to as the original prescription in the following Figure 1 and Figure 2

[0025] 2) Take 10 g of the freeze-dried powder obtained in step 1), dissolve in 30 ml of distilled water, and load a macroporous adsorption resin column with a model D101 of 300 g, and elute with 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, respectively, and collect each 300 mL of effluent as a fraction from the start of loading, to obtain fractions Fr1-Fr20; analyze the chromatographic characteristics of each fraction by a liquid chromatograph using a C18 chromatographic column, and combine the fractions with the same chromatographic characteristics; combine fractions Fr1-Fr4 as component yf-1; fraction Fr5 as component yf-2; and fraction Fr6 as component yf-3;

[0026] ​Flow fraction Fr7 is component yf-4; flow fraction Fr8 is component yf-5; flow fractions Fr9-Fr10 are combined into component yf-6; flow fractions Fr11-Fr12 are combined into component yf-7; flow fraction Fr13 is component yf-8; flow fraction Fr14 is component yf-9; flow fractions Fr15-Fr16 are combined into component yf-10; flow fractions Fr17-Fr19 are combined into component yf-11 and flow fraction Fr20 is component yf-12.

[0027] The component yf-10 is the effective part of the Shengmai Decoction of the present invention, and the compound identification results are as follows: Figures 5-6 As shown in Table 1.

[0028] Table 1. Information on compounds identified in YF-10 samples.

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] Example 2

[0036] Activity test of component yf-10 in inhibiting H9c2 cardiomyocyte damage.

[0037] 1. Test cell lines

[0038] The highly differentiated rat cardiomyocyte line H9c2 (hereinafter referred to as H9c2 cells) was purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0039] 2. Experimental Materials

[0040] DMEM medium, Gibco; Hyclone fetal bovine serum, Hyclone; trypsin, Biosharp; penicillin-streptomycin mixture (double antibiotic), Wuhan Saiwei Biotechnology Co., Ltd.; doxorubicin, Sigma-Aldrich; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Sigma-Aldrich; Dimetylsulfoxide (DMSO), Solarbio.

[0041] Shengmaiyin freeze-dried powder, in Figure 1 and Figure 2 is abbreviated as the original formula.

[0042] yf-10 freeze-dried powder (obtained by freeze-drying component yf-10), in Figure 1 and Figure 2 is abbreviated as 10.

[0043] 3. Instrument and equipment

[0044] Carbon dioxide incubator (Shanghai Lishen Co., Ltd.), biological safety cabinet (China Haier Co., Ltd.), inverted microscope (Nikon Co., Ltd., Japan), Milli-Q ultrapure water integrated machine (Merck Millipore Co., Ltd., USA), multifunctional microplate reader (Shanghai Gene Co., Ltd.).

[0045] 4. Experimental method

[0046] (1) Cell culture

[0047] H9c2 cells were cultured in normal medium (DMEM medium containing 10% fetal bovine serum + 1% double antibody) in a 37 °C, 5% CO2 saturated humidity incubator, and the medium was changed every 2 days. Cells in the logarithmic growth phase were used for experiments.

[0048] [[ID=二十九]](2) Adriamycin injury protection experiment

[0049] Cells were seeded in 96-well plates and divided into the following groups (n = 6 for each group): <00,00127>Blank control group: normal medium;

[0051] Model group: normal medium + 0.1 μM adriamycin;

[0052] yf-10 low-dose group: normal medium + 0.1 μM adriamycin + 10 μg / mL yf-10 freeze-dried powder (see Figure 1 );

[0053] yf-10 high-dose group: normal medium + 0.1 μM adriamycin + 50 μg / mL yf-10 freeze-dried powder (see Figure 2 );

[0054] yf-10 dose 1 group: normal medium + 0.1 μM adriamycin + 10 μM yf-10 freeze-dried powder (see Figure 3 );

[0055] yf-10 dose 2 group: normal medium + 0.1 μM adriamycin + 25 μM yf-10 freeze-dried powder (see Figure 3 );

[0056] YF-10 dosage group 3: normal culture medium + 0.1 μM doxorubicin + 50 μM YF-10 lyophilized powder (see...) Figure 3 );

[0057] YF-10 dosage group 4: normal culture medium + 0.1 μM doxorubicin + 75 μM YF-10 lyophilized powder (see...) Figure 3 );

[0058] YF-10 dosage group 5: normal culture medium + 0.1 μM doxorubicin + 100 μM YF-10 lyophilized powder (see...) Figure 3 );

[0059] Low-dose group of the original formula: normal culture medium + 0.1 μM doxorubicin + 10 μg / mL Shengmai Yin lyophilized powder (see...) Figure 1 );

[0060] High-dose group of the original formula: normal culture medium + 0.1 μM doxorubicin + 50 μg / mL Shengmai Yin lyophilized powder (see original formula) Figure 2 );

[0061] MTT assay for cell viability: H9c2 cells were inoculated at 1×10⁻⁶ 4 Cells were seeded at a density of 1 / well in 96-well plates. After 24 hours of culture and adherence, the medium was replaced with fresh normal medium containing different concentrations of YF-10 lyophilized powder (10, 50 μg / mL) and Shengmai Yin lyophilized powder (10, 50 μg / mL) according to the groups. Each group had 6 replicates. After 3 hours of culture, doxorubicin (final concentration 0.1 μM) was added, while the blank control group was added with an equal volume of normal medium. After another 24 hours of culture, 20 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added. The plates were shaken for 10 minutes. The absorbance (OD value) at 570 nm was measured using a microplate reader.

[0062] Cell viability (%) = (OD) 实验组 / OD 空白对照组 )×100%.

[0063] (3) Experimental Results

[0064] Compared with the blank control group (100% cell viability), the model group significantly (p<0.05) induced cardiomyocyte damage (cell survival rate 59.33%). Figures 1-2 ).

[0065] 10 μg / mL of Shengmai Yin lyophilized powder (original formula) and component yf-10 (abbreviated as 10 in the figure) significantly (p<0.05) protected against this damage (cell viability was 72.02% and 71.89%, respectively), with component yf-10 showing more stable effects. Figure 1 );

[0066] Lyophilized Shengmaiyin powder (original formula) at 50 μg / mL and component yf-10 can significantly (p < 0.05) protect this kind of damage (the cell survival rates are 60.63% and 65.15% respectively), and the effect of component yf-10 is better ( Figure 2 ).

[0067] Meanwhile, pre-administering yf-10 lyophilized powder at 10, 25, and 50 μM (for 3 hours) can all significantly (p < 0.05) protect against damage, and the gradient dose relationship is obvious ( Figure 3 ).

[0068] Compared with the original formula, component yf-10 of the present invention can more effectively protect H9c2 cells from adriamycin-induced damage, suggesting that component yf-10 can be used as a drug for anti-cardiac injury.

[0069] Component yf-10 is the core active substance of Shengmaiyin in treating cardiac injury and has better anti-cardiac injury activity.

[0070] Example 3

[0071] Activity test of component yf-11 in aggravating H9c2 cardiomyocyte injury.

[0072] 1. Test cell line

[0073] Highly differentiated rat cardiomyocyte strain H9c2, purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0074] 2. Experimental materials

[0075] DMEM medium, Gibco; Hyclone fetal bovine serum, Hyclone; Trypsin, Biosharp; Adriamycin, Sigma-Aldrich; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Sigma-Aldrich; Dimetylsulfoxide (DMSO), Solarbio;

[0076] Lyophilized Shengmaiyin powder, in Figure 1 and Figure 2 is abbreviated as the original formula.

[0077] yf-11 lyophilized powder (abbreviated as 11 in the figure).

[0078] 3. Instrument equipment

[0079] Carbon dioxide constant temperature incubator (Shanghai Lishen Co., Ltd.), biosafety cabinet (China Haier Co., Ltd.), inverted microscope (Japan Nikon Corporation), Milli-Q ultrapure water system (Merck Millipore Corporation, USA), multi-functional microplate reader (Shanghai Gene Co., Ltd.).

[0080] 4. Experimental Methods

[0081] (1) Cell Culture

[0082] H9c2 cells were passaged in normal culture medium (DMEM medium containing 10% fetal bovine serum + 1% penicillin and antibiotics) at 37°C and 5% CO2 saturated humidity incubator, with the medium changed every 2 days. Cells in the logarithmic growth phase were used for experiments.

[0083] (2) Toxicity evaluation of component YF-11

[0084] Cells were seeded in 96-well plates and divided into the following groups (n=6 per group):

[0085] Blank control group: normal culture medium;

[0086] Model group: normal culture medium + 0.1 μM doxorubicin;

[0087] YF-11 Dosage Group 1: Normal culture medium + 0.1 μM doxorubicin + 50 μg / mL YF-11 lyophilized powder (see...) Figure 2 );

[0088] YF-11 dosage group 2: normal culture medium + 0.1 μM doxorubicin + 10 μM YF-11 lyophilized powder (see...) Figure 4 );

[0089] YF-11 dosage group 3: normal culture medium + 0.1 μM doxorubicin + 25 μM YF-11 lyophilized powder (see...) Figure 4 );

[0090] YF-11 dosage group 4: normal culture medium + 0.1 μM doxorubicin + 50 μM YF-11 lyophilized powder (see...) Figure 4 );

[0091] YF-11 dosage group 5: normal culture medium + 0.1 μM doxorubicin + 100 μM YF-11 lyophilized powder (see...) Figure 4 );

[0092] MTT assay for cell viability: Same as in Example 2.

[0093] (3) Experimental Results

[0094] Compared with the blank control group (cell viability 100%), the model group significantly (p<0.05) induced cardiomyocyte damage (cell viability 59.33%), and 50 μg / ml of component YF-11 significantly (p<0.05) aggravated this damage (cell viability 10.85%). Figure 2 ).

[0095] Meanwhile, pre-administration of lyophilized component YF-11 at concentrations of 10, 25, 50, and 100 μM (3 hours) significantly aggravated (p<0.05) the damage, and the toxicity gradient dose relationship was obvious. Figure 4 ).

[0096] This indicates that component YF-11 has a damaging effect on cardiomyocytes and is a toxic substance in Shengmai Yin for treating heart damage.

[0097] Compared to the original formulation, component yf-11 significantly aggravated doxorubicin-induced H9c2 cell damage, exhibiting a significant dose-dependent relationship. This suggests that yf-10, the weakly toxic effective fraction obtained by isolating component yf-11, could be a more effective and safer drug for treating cardiac damage.

Claims

1. A method for extracting and purifying the effective components of Shengmai Yin, 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, 1200mL of 20% (v / v) ethanol aqueous solution, 1200mL of 50% (v / v) ethanol aqueous solution, and 1200mL of 75% (v / v) ethanol aqueous solution. From the start of sample loading, collect every 300mL of eluent as a fraction to obtain fractions Fr1-Fr16. Analyze the chromatographic characteristics of each fraction using a C18 column in liquid chromatography. Fractions with the same chromatographic characteristics were combined, and fractions Fr1-Fr4 were combined into component yf-1; fraction Fr5 into component yf-2; fraction Fr6 into component yf-3; fraction Fr7 into component yf-4; fraction Fr8 into component yf-5; fractions Fr9-Fr10 into component yf-6; fractions Fr11-Fr12 into component yf-7; fraction Fr13 into component yf-8; fraction Fr14 into component yf-9; and fractions Fr15-Fr16 into component yf-10. Component yf-10 is the effective component of Shengmai Yin.

2. The method of claim 1 for extracting and purifying the effective components of Shengmai Yin.

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

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