Preparation of a traditional Chinese medicine component and its application in treating hyperlipidemia

Abstract

The present invention discloses the preparation of a Chinese medicine component and its application in treating hyperlipidemia. Hyperlipidemia is extremely harmful to the human body and is a risk factor for coronary heart disease, stroke, sudden cardiac death, myocardial infarction, etc. At present, the most widely used and relatively effective western medicines in the treatment guidelines for hyperlipidemia are statins, fibrates and nicotinic acid, but western medicines have many adverse reactions, such as liver damage, gastrointestinal reactions, etc. The present invention prepares a Chinese medicine component, which has obvious lipid-lowering activity and has the prospect of being developed into a lipid-lowering and hyperlipidemia treatment drug.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and specifically relates to the preparation of a traditional Chinese medicine component and its application in treating hyperlipidemia. Background Art

[0002] Hyperlipidemia refers to a systemic lipid metabolism disorder characterized by elevated low-density lipoprotein cholesterol, triglycerides, and total cholesterol and low high-density lipoprotein cholesterol in the blood. With the improvement of people's living standards and changes in lifestyle, the number of people with hyperlipidemia is increasing, and the age of onset is also earlier. Hyperlipidemia is extremely harmful to the human body and is a risk factor for coronary heart disease, stroke, sudden cardiac death, myocardial infarction, etc. At present, the most widely used and relatively effective Western medicines in the treatment guidelines for hyperlipidemia are statins, fibrates, and niacin, but Western medicines have many adverse reactions, such as liver damage and gastrointestinal reactions. Traditional Chinese medicine has a long history, and Chinese medicine has unique advantages in treating hyperlipidemia and preventing cardiovascular and cerebrovascular diseases. It can effectively prevent the occurrence of atherosclerosis, and has mild efficacy and few adverse reactions, which are favored by more and more patients.

[0003] White mustard seeds are the dried mature seeds of Sinapis alba L. of the Cruciferae family. As a traditional Chinese medicinal material, it has the effects of warming the lungs, dispelling phlegm and promoting qi, dispersing nodules, unblocking meridians and relieving pain. In modern pharmacological studies, white mustard seeds and related extracts have the effects of treating pneumonia, asthma and cough in children, and have an inhibitory effect on liver cancer cells and cervical cancer.

[0004] White mustard polysaccharide is a polysaccharide compound extracted from white mustard. Due to the differences in the primary structure composition and higher-order spatial structure such as the secondary structure of the polysaccharide, the polysaccharide composition is complex and the biological activity varies greatly. Although it is difficult to find the truly active part of the polysaccharide, it is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of a Chinese medicinal ingredient and its application in treating hyperlipidemia. The Chinese medicinal ingredient is obtained by fractionating and refining white mustard polysaccharide.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A Chinese medicine ingredient is a polysaccharide, which is prepared by the following method:

[0008] Take an appropriate amount of white mustard, soak and defat it with petroleum ether, then heat it with water for extraction, concentrate the extract under reduced pressure, add anhydrous ethanol to adjust the ethanol volume fraction to 90%, let it stand at low temperature overnight, collect the precipitate by centrifugation, re-dissolve it in water and freeze-dry it to obtain white mustard crude polysaccharide; take an appropriate amount of white mustard crude polysaccharide, add an appropriate amount of water to prepare a polysaccharide solution, centrifuge to remove insoluble impurities, add anhydrous ethanol to the solution to make the ethanol volume fraction reach 30%, let it stand at low temperature overnight, centrifuge, collect the supernatant, add anhydrous ethanol to make the ethanol volume fraction reach 60%, let it stand at low temperature overnight, centrifuge, collect the supernatant, add anhydrous ethanol to the supernatant to make the ethanol volume fraction reach 90%, let it stand at low temperature overnight, centrifuge to collect the precipitate, re-dissolve it in water and freeze-dry it to obtain.

[0009] Preferably, the solid-liquid ratio of water heating extraction is 1kg:5L.

[0010] Preferably, the water heating extraction temperature is 90°C.

[0011] More preferably, the water is heated for extraction twice, each time for 4 hours.

[0012] Preferably, the centrifugation condition is 4000 r / min for 10 minutes.

[0013] Preferably, standing overnight at low temperature refers to standing overnight at 4°C.

[0014] Use of any of the above-mentioned Chinese medicinal ingredients in preparing lipid-lowering drugs.

[0015] Use of any of the above-mentioned Chinese medicinal ingredients for preparing a drug for treating hyperlipidemia.

[0016] Beneficial effects:

[0017] The present invention separates a Chinese medicinal ingredient from white mustard seeds, which is a 90% ethanol graded polysaccharide prepared by a method of ethanol graded alcohol precipitation (30%, 60%, 90%). Activity research results show that the 90% ethanol graded polysaccharide provided by the present invention has obvious lipid-lowering activity, while other alcohol-precipitated graded polysaccharides (30%, 60%) have no obvious lipid-lowering activity. Therefore, the Chinese medicinal ingredient provided by the present invention has the prospect of being developed into a lipid-lowering and hyperlipidemia treatment drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The statistical results of the weight of mice in each group (n=10); Note: Compared with the control group at the same time, * indicates a significant difference, P<0.05; Compared with the model group at the same time, # indicates a significant difference, P<0.05;

[0019] Figure 2: The liver weight of mice in each group was measured (n=10); Note: Compared with the control group, * indicates a significant difference, P<0.05; Compared with the model group, # indicates a significant difference, P<0.05;

[0020] Figure 3 The results of Oil Red O staining of the liver of mice in each group (n=10); Note: Compared with the control group, * indicates a significant difference, P<0.05; Compared with the model group, # indicates a significant difference, P<0.05;

[0021] Figure 4 The results of epididymal fat weight measurement of mice in each group (n=10); Note: Compared with the control group, * indicates a significant difference, P<0.05; Compared with the model group, # indicates a significant difference, P<0.05;

[0022] Figure 5 The results of H&E staining of epididymal fat of mice in each group (n=10); Note: Compared with the control group, * indicates a significant difference, P<0.05; compared with the model group, # indicates a significant difference, P<0.05. DETAILED DESCRIPTION

[0023] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0024] 1. Experimental Materials

[0025] 1. Materials and reagents

[0026] White mustard seeds were purchased from Beijing Tongrentang Co., Ltd. (origin: Anhui). They were identified as dried mature seeds of the cruciferous plant white mustard Sinapis alba L. by Associate Professor Wan Jinyi of Beijing University of Chinese Medicine. The voucher samples were stored in Beijing University of Chinese Medicine. High-fat feed (composition: 77.5% basal feed, 2% cholesterol, 10% egg yolk powder, 10% lard, 0.5% sodium cholate) was purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Serum total cholesterol (TC) detection kit, triglycerides (TG) detection kit, high-density lipoprotein cholesterol (HDL-C) detection kit, low-density lipoprotein cholesterol (LDL-C) detection kit were purchased from Nanjing Jiancheng Bioengineering Institute. Oil Red O staining kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Other reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0027] 2. Experimental Animals

[0028] 60 healthy C57BL / 6 male mice, 6-8 weeks old, weighing 20-25g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. They were kept in the Experimental Animal Center of the Institute of Traditional Chinese Medicine of Beijing University of Chinese Medicine in an SPF environment. The temperature of the breeding environment was always maintained at 20-25°C, the relative humidity was controlled at 50%-60%, and the light and dark were alternated every day for 12 hours. After one week of pre-adaptation, the mice were randomly divided into the following 6 groups: control group, model group, white mustard polysaccharide 30% alcohol precipitation fraction (BJZP-30%) group, white mustard polysaccharide 60% alcohol precipitation fraction (BJZP-60%) group, white mustard polysaccharide 90% alcohol precipitation fraction (BJZP-90%) group and simvastatin group (positive drug).

[0029] 2. Experimental Methods

[0030] 1. Preparation of white mustard polysaccharide

[0031] Take 2000g of white mustard seeds, crush them, soak and defatted them with petroleum ether at room temperature for 2 times, 2 days each time, recover the petroleum ether, and evaporate the residue until there is no ether smell. Use water (according to the solid-liquid ratio of 1:5, kg:L) as the extraction solvent, heat and extract at 90℃ for 2 times, 4 hours each time, combine the filtrate and concentrate under reduced pressure to obtain a concentrate, then add anhydrous ethanol to adjust the final ethanol concentration to 90%, and let it stand at 4℃ overnight. Centrifuge at 4000r / min for 10 minutes, collect the precipitate, re-dissolve in water and freeze-dry to obtain white mustard seed crude polysaccharide.

[0032] The crude polysaccharide of white mustard seeds is added with water to prepare a polysaccharide solution with a mass fraction of 10%, and the solution is centrifuged at 4000r / min for 10 minutes to remove insoluble impurities. Anhydrous ethanol is added to the solution to make the ethanol volume fraction reach 30%, and the solution is precipitated overnight in a refrigerator at 4°C. The polysaccharide is centrifuged at 4000r / min for 10 minutes to obtain a 30% alcohol precipitation component (BJZP-30%); a certain amount of anhydrous ethanol is added to the supernatant to make the ethanol volume fraction reach 60%, and the solution is precipitated overnight (4°C), and the polysaccharide is centrifuged at 4000r / min for 10 minutes to obtain a 60% alcohol precipitation component (BJZP-60%); in the same way, a 90% alcohol precipitation component (BJZP-90%) is obtained. BJZP-30%, BJZP-60%, and BJZP-90% are respectively reconstituted with water and freeze-dried for standby use.

[0033] 2. Hyperlipidemic mouse model

[0034] Sixty 6-8 week old C57BL / 6J mice were randomly divided into the following 6 groups after 1 week of adaptive feeding:

[0035] There were 10 rats in each group: control group (oral administration of normal saline), model group (oral administration of normal saline), BJZP-30% group (oral administration of 200 mg / kg BJZP-30%), BJZP-60% group (oral administration of 200 mg / kg BJZP-60%), BJZP-90% group (oral administration of 200 mg / kg BJZP-90%) and simvastatin group (oral administration of 50 mg / kg simvastatin).

[0036] Except for the control group that ate regular feed, the other groups ate high-fat feed freely every day to establish an obesity model. Each drug-treated group was continuously gavaged with the corresponding dose of 0.2mL / 10g for 30 days, once a day. The weight of the mice was recorded daily during the experiment. The weight of the model group mice was 20% higher than that of the control group mice as a sign of successful modeling. After the experiment, orbital blood was taken from each group of mice, centrifuged at 4000r / min for 10 minutes, and the upper serum was taken and stored in a -80℃ refrigerator. The mice in each group were immediately killed, the mice were fixed on the dissection table, the abdominal cavity was opened, the liver and epididymal fat were cut and weighed, and stored in a -80℃ refrigerator.

[0037] 3. Determination of serum lipid indexes in mice

[0038] Mouse serum was obtained, and the levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in mouse serum were detected according to the experimental steps in the kit instructions.

[0039] 4. Oil Red O staining of mouse liver

[0040] Mouse liver tissue was obtained and fixed in 4% paraformaldehyde for 24 hours. After gradient alcohol dehydration, it was embedded in paraffin and sliced. The sections of each group were stained with Oil Red O staining kit, and the experimental steps were carried out according to the instructions of the kit. The fat staining of mouse liver was observed and analyzed under a microscope.

[0041] 5. H&E staining of mouse epididymal fat

[0042] Mouse epididymal fat tissue was obtained and fixed in 4% paraformaldehyde for 24 hours. After gradient alcohol dehydration, it was embedded in paraffin and sliced. It was stained with hematoxylin and eosin, dehydrated and sealed. The staining of mouse epididymal fat was observed and analyzed under a microscope.

[0043] 6. Data processing

[0044] The experimental results all obeyed the normal distribution, and the data were expressed as mean ± standard deviation. SPSS20.0 statistical software was used for one-way analysis of variance. P < 0.05 was considered to be statistically significant.

[0045] 3. Experimental Results

[0046] 1. Effect of white mustard polysaccharide on the body weight of obese mice

[0047] In order to investigate the effect of white mustard polysaccharide on the body weight of obese mice, the body weight of each group of mice was recorded. Figure 1 The results showed that from the 15th day of modeling, the body weight of the mice in the model group was significantly increased compared with that in the control group (P<0.05), and then the weight difference between the model group and the control group gradually widened; compared with the model group, the body weight of the mice in the BJZP-90% group was significantly reduced from the 25th day of administration (P<0.05); compared with the model group, the body weight of the mice in the simvastatin group was significantly reduced on the 30th day of administration (P<0.05); and there was no significant difference in the body weight of the mice in the BJZP-30% group and the BJZP-60% group compared with the model group in the same period of time.

[0048] 2. Effects of white mustard polysaccharide on serum lipid indexes in obese mice

[0049] Table 1 lists the results of the determination of serum lipid indexes of mice in each group. Compared with the control group, the serum TC, TG, and LDL-C contents of mice in the model group were significantly increased (P<0.05), and the HDL-C content was significantly decreased (P<0.05), showing a hyperlipidemia state; compared with the model group, the serum TC, TG, and LDL-C contents of mice in the BJZP-90% group and the simvastatin group were significantly decreased (P<0.05), and the HDL-C content was significantly increased (P<0.05); the serum TC, TG, LDL-C, and HDL-C contents of mice in the BJZP-30% group were almost the same as those in the model group, and the blood lipid indexes of mice in the BJZP-60% group were slightly better than those in the model group, but there was no significant difference. The above results show that BJZP-90% has a significant lipid-lowering effect, while BJZP-30% and BJZP-60% have no significant lipid-lowering effect.

[0050] Therefore, other lipid-lowering experiments were continued using BJZP-90% as the test polysaccharide.

[0051] Table 1 Results of serum lipid index testing in each group of mice (n=10, mM)

[0052]

[0053] 3. Effects of white mustard polysaccharide on liver weight and adipose tissue content in obese mice

[0054] Based on the above research, the effect of BJZP-90% on the liver of obese mice was investigated. The liver weights of mice in the control group, model group, BJZP-90% group and simvastatin group were measured. Figure 2As shown, compared with the control group, the liver weight of the mice in the model group increased significantly (P<0.05), and compared with the model group, the liver weight of the mice in the BJZP-90% group and the simvastatin group decreased significantly (P<0.05), indicating that BJZP-90% can significantly reduce the liver weight of obese mice. In addition, the results of liver oil red O staining and quantitative analysis showed that compared with the control group, the liver cells of the mice in the model group had a large number of scattered lipid droplets; compared with the model group, the scattered lipid droplets between the liver cells of the mice in the BJZP-90% group and the simvastatin group were significantly reduced ( Figure 3 ), indicating that BJZP-90% can significantly reduce the liver fat content in obese mice.

[0055] 4. Effect of white mustard polysaccharide on epididymal fat weight and adipose tissue content in obese mice

[0056] The epididymal fat of mice is distributed more centrally, and its structure is intact and easy to obtain. The epididymal fat weighing results of mice in the control group, model group, BJZP-90% group and simvastatin group are shown in the figure. Figure 4 As shown in the figure, compared with the control group, the epididymal fat weight of mice in the model group increased significantly (P<0.05), and compared with the model group, the epididymal fat weight of mice in the BJZP-90% group and simvastatin group decreased significantly (P<0.05), indicating that BJZP-90% can significantly reduce the epididymal fat weight of obese mice. Figure 5 As shown, at the same magnification, the size of epididymal adipocytes in the model group mice was significantly increased compared with that in the control group mice; compared with the model group, the epididymal adipocytes in the BJZP-90% group and the simvastatin group mice were uniform in size and smaller in size, indicating that BJZP-90% can significantly reduce the epididymal fat content in obese mice.

[0057] The above experimental results show that the 90% ethanol fractionated polysaccharide of white mustard has obvious lipid-lowering activity, while other alcohol-precipitated fractionated polysaccharides (30%, 60%) have no obvious lipid-lowering activity.

[0058] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A Chinese medicinal ingredient, which is a polysaccharide, characterized in that: Prepared by the following method: Take an appropriate amount of white mustard, soak and defat it with petroleum ether, then heat it with water for extraction, concentrate the extract under reduced pressure, add anhydrous ethanol to adjust the ethanol volume fraction to 90%, let it stand at low temperature overnight, collect the precipitate by centrifugation, re-dissolve it in water and freeze-dry it to obtain white mustard crude polysaccharide; take an appropriate amount of white mustard crude polysaccharide, add an appropriate amount of water to prepare a polysaccharide solution with a mass fraction of 10%, centrifuge to remove insoluble impurities, add anhydrous ethanol to the solution to make the ethanol volume fraction reach 30%, let it stand at low temperature overnight, centrifuge, collect the supernatant, add anhydrous ethanol to make the ethanol volume fraction reach 60%, let it stand at low temperature overnight, centrifuge, collect the supernatant, add anhydrous ethanol to the supernatant to make the ethanol volume fraction reach 90%, let it stand at low temperature overnight, collect the precipitate by centrifugation, re-dissolve it in water and freeze-dry it to obtain; wherein: the solid-liquid ratio of water heating extraction is 1kg:5L; the temperature of water heating extraction is 90℃; water heating extraction is performed twice, each time for 4 hours; standing at low temperature overnight means standing at 4℃ overnight.

2. The Chinese medicinal ingredient according to claim 1, characterized in that: The centrifugation condition was 4000 r / min for 10 minutes.

3. Use of the Chinese medicinal ingredients according to claim 1 or 2 for preparing lipid-lowering drugs.

4. Use of the Chinese medicinal ingredient according to claim 1 or 2 for preparing a drug for treating hyperlipidemia.

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