Application of ligustroside J and composition thereof in preparation of medicine for treating hyperlipidemia and reducing weight

Abstract

The invention discloses a novel medical application of ligustroside J, and particularly provides an application of ligustroside J in preparation of drugs for treating hyperlipidemia and obesity. A large number of experiments prove that the ligustroside J can effectively reduce the content of triglyceride and total cholesterol in a hepatocyte lipid accumulation model, and can be applied to preparation of the medicine for treating hyperlipidemia and obesity.

Description

Technical Field

[0001] The present invention relates to a new medical use of Ligupurpuroside J, specifically to the use of Ligupurpuroside J in the preparation of drugs for treating hyperlipidemia and losing weight, and belongs to the field of medical uses of Ligupurpuroside J. Background Art

[0002] Hyperlipidemia, also known as dyslipidemia or lipid metabolism disorder, is a common metabolic disease. Its main characteristics are abnormal elevation of lipid contents such as triglyceride (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in the blood or too low content of high density lipoprotein cholesterol (HDL-C). Hyperlipidemia is an important risk factor for promoting the formation of atherosclerosis, and atherosclerosis is the main pathological basis for cardiovascular and cerebrovascular diseases. With the improvement of living standards and the change of lifestyle, hyperlipidemia has become a common clinical disease at present, and its incidence rate increases year by year. Therefore, studying the prevention and treatment measures of hyperlipidemia and finding effective therapeutic drugs are research fields that are highly valued by domestic and foreign research scholars.

[0003] Obesity refers to a certain degree of obvious overweight and too thick fat layer, which is a state caused by excessive accumulation of body fat, especially triglyceride, and is a chronic disease caused by the imbalance between energy intake and consumption. With the improvement of living standards, people's diet structure and lifestyle have changed greatly, resulting in an increasing number of obese people in society. Obesity is often accompanied by abnormal glucose and lipid metabolism, which not only affects people's appearance, but also increases the incidence of diseases such as diabetes, hypertension, coronary heart disease, and hyperlipidemia, and has become a global public health problem. The treatment of obesity has always been a hot topic at home and abroad, and drug treatment is one of the commonly used means for treating obesity at present.

[0004] Through a large number of previous experiments, we found that Ligupurpuroside J has the effect of reducing cholesterol and triglyceride. So far, there is no literature report on the pharmacological effects of Ligupurpuroside J in treating hyperlipidemia and losing weight. Summary of the Invention

[0005] The purpose of the present invention is to apply Ligupurpuroside J and its compositions to the preparation of drugs for treating hyperlipidemia or losing weight.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] Evaluation of the lipid-lowering and weight-loss effects of Ligustroside J - Through a large number of experiments, the present invention found that Ligustroside J can effectively reduce the contents of cholesterol and triglyceride in the in vitro lipid accumulation model of HepG2 cells, and its effect on triglyceride is particularly significant. Therefore, Ligustroside J can be prepared into a drug for treating hyperlipidemia and losing weight.

[0008] Another technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating hyperlipidemia and losing weight, which is prepared by combining an effective amount of Ligustroside J with a pharmaceutically acceptable carrier, excipient or diluent. According to different administration methods, the pharmaceutical composition of the present invention may contain 0.1% - 99% by weight of Ligustroside J. Among them, after adding various excipients and pharmaceutically acceptable carriers, excipients or diluents required for preparing different dosage forms to Ligustroside J, it can be prepared into any suitable clinical preparation by conventional preparation methods, such as oral preparations (tablets, oral liquids, granules, capsules, soft capsules or dripping pills), etc.; among them, the excipients may be antioxidant complexing agents, fillers, matrix materials, etc.; the pharmaceutically acceptable carriers are one or more of xylitol, mannitol, lactose, fructose, dextran, glucose, polyvinylpyrrolidone, low molecular dextran, sodium chloride, calcium gluconate or calcium phosphate. Description of the Drawings

[0009] Figure 1 Chemical structure of Ligustroside J

[0010] Figure 2 Regression curve of the dose-effect relationship of Ligustroside J in reducing TG content

[0011] Figure 3 Regression curve of the dose-effect relationship of Ligustroside J in reducing TC content Detailed Embodiments

[0012] The present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention can be modified or replaced, but these modifications and replacements all fall within the protection scope of the present invention.

[0013] Experimental Example 1

[0014] Preparation of Ligustroside J

[0015] The old leaves of Ligustrum robustum (Roxb.) Bl. were extracted with 95% ethanol, and the extract was obtained by reduced pressure recovery. The extract was suspended and distributed twice with 10 times deionized water, and the obtained aqueous solution was dried to obtain a water-soluble fraction. The water-soluble fraction was loaded onto D101 macroporous resin and eluted with ethanol of different concentrations. The fraction eluted with 60% ethanol was separated and purified by normal-phase silica gel, Sephadex LH-20 gel column chromatography and preparative liquid chromatography to obtain a monomeric crystal, which was determined to be ligupurpuroside J by 1H-NMR and 13C-NMR data.

[0016] Experimental Example 2

[0017] Evaluation of the lipid-lowering activity of ligupurpuroside J using an in vitro HepG2 cell lipid accumulation model

[0018] 1. Preparation of reagents and drugs

[0019] (1) Preparation of oleic acid modeling solution: 0.056 g potassium hydroxide, 10 ml PBS, 0.1 g BSA, vortex and mix well, then add 31.6 μL oleic acid to make a 10 mmol·L-1 stock solution, filter with a 0.22 μm filter membrane and aliquot, store at -20 °C, and dilute to 100 μmol·L-1 with culture medium when using, and prepare fresh.

[0020] (2) Cholesterol modeling solution: Accurately weigh 20 mg cholesterol, dissolve it in 1 ml absolute ethanol, and store at -20 °C. Make a 20 mg·mL-1 stock solution, filter with a 0.22 μm filter membrane and aliquot, and dilute to 10 μg·mL-1 with culture medium when using, and prepare fresh.

[0021] (3) 25-Hydroxycholesterol modeling solution: Accurately weigh 2 mg 25-hydroxycholesterol, dissolve it in 1 ml absolute ethanol, and store at -20 °C. Make a -1 stock solution, filter with a 0.22 μm filter membrane and aliquot, and dilute to 1 μg·mL -1 when using, and prepare fresh.

[0022] (4) Ligupurpuroside J was dissolved in an appropriate amount of pure water to prepare a stock solution with a concentration of 10 mmol·L -1 , aliquoted, stored at -20 °C, and diluted with culture medium when using, and prepared fresh.

[0023] (5) Fenofibrate: Accurately weigh 3.608 mg fenofibrate, dissolve it in 1 ml DMSO to prepare a stock solution with a concentration of 10 mmol·L -1 , aliquoted, stored at -20 °C, and diluted with culture medium when using, and prepared fresh.

[0024] 2. Experimental method

[0025] (1) Cell culture and passage

[0026] Human hepatocarcinoma HepG 2 cells were inoculated in high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. They were cultured in an incubator at 37°C and 5% CO 2 2. When observed under a microscope, when the cell confluence rate in the culture flask reached approximately 90%, the original culture medium was discarded, and the cells were washed 2 - 3 times with autoclaved PBS. After discarding the PBS, 3 ml of 0.25% trypsin was added to digest the cells, and they were placed in an incubator at 37°C and 5% CO 2 2 - 3 minutes. The cells were observed under a microscope. When the cells changed from an irregular adherent state to round and separated from each other, indicating complete digestion, the trypsin was discarded, and 3.5 ml of complete culture medium containing 10% inactivated fetal bovine serum was added to terminate the digestion of trypsin. The culture medium was aspirated with a bent pipette and gently pipetted repeatedly to make the cells detach from the flask wall into a cell suspension. The cell suspension was collected in a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. The original culture medium in the centrifuge tube was discarded, and the cells were collected. 2 ml of high-glucose DMEM culture medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin was used to resuspend the cells into a uniform single-cell suspension. The cells were passaged at a ratio of 1:2 - 1:4, an appropriate volume of cell culture medium was added to each flask, the cells were mixed evenly, and they were continued to be cultured in an incubator at 37°C and 5% CO 2 2, and the culture flask opening was slightly suspended.

[0027] (2) Determination of intracellular triglyceride (TG) content

[0028] Take HepG cells in the logarithmic growth phase with good status 2 , wash the cells 2 - 3 times with PBS. After digestion with 0.25% trypsin, discard the trypsin, add fresh culture medium to terminate the digestion, blow down the cells into a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the original culture medium, add fresh culture medium, resuspend the cells into a single-cell suspension, and inoculate them at 1×10 6 / ml into a 24-well plate, with 0.5 mL of cell suspension in each well, and culture for 24 hours until the cells are completely adherent. Use incomplete culture medium containing 100 μmol·L -1 oleic acid (OA) to stimulate the cells to establish a lipid accumulation model. At the same time, the blank control group was given incomplete culture medium, the positive drug administration group was given 10 μmol·L -1 fenofibrate, and the drug administration groups were given 6.25, 12.5, 25, 50, 100 μmol·L -1 lignanside J from Ligustrum purpurascens, at 37°C and 5% CO 2Incubate in an incubator for 24 h. Discard the culture medium, wash 2 - 3 times with PBS, discard the PBS, add 100 μl of cell lysate to each well, mix well and let stand at room temperature for 10 min. Transfer an appropriate amount of supernatant to a 1.5 mL centrifuge tube, heat at 70 °C for 10 min, centrifuge at 2000 rpm at room temperature for 5 min, take the supernatant and mix well, and measure the TG content in the sample using a kit.

[0029] (3) Determination of total cholesterol (TC) content in cells

[0030] Take HepG cells in the logarithmic growth phase with good condition 2 cells, wash the cells 2 - 3 times with PBS, digest with 0.25% trypsin, discard the trypsin, add fresh culture medium to terminate digestion, blow down the cells into a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the original culture medium, add fresh culture medium, blow into a single cell suspension, inoculate at 1×10 6 / ml into a 24 - well plate, add 0.5 mL of cell suspension to each well, and culture for 24 hours until the cells are completely adherent. Then discard the original medium, replace it with 1% BSA culture, add 10 μg·mL -1 cholesterol and 1 μg·mL -1 25 - hydroxycholesterol and act for 24 h. At the same time, the blank control group is given incomplete culture medium, the positive drug administration group is given 10 μmol·L -1 fenofibrate, and the administration groups are respectively given 6.25, 12.5, 25, 50, 100 μmol·L -1 lignan J from Ligustrum purpurascens, and incubate in an incubator at 37 °C with 5% CO 2 for 24 h. Discard the culture medium, wash 2 - 3 times with PBS, discard the PBS, add 100 μl of cell lysate to each well, mix well and let stand at room temperature for 10 min. Transfer an appropriate amount of supernatant to a 1.5 mL centrifuge tube, heat at 70 °C for 10 min, centrifuge at 2000 rpm at room temperature for 5 min, take the supernatant and mix well, and measure the TC content in the sample using a kit.

[0031] 3. Experimental results

[0032] As shown in Table 1, 24 h after modeling, compared with the blank group, the TG and TC contents in the cells of the model group were significantly increased, and the difference was extremely significant. The positive drug fenofibrate (10 μmol·L -1 ) could significantly reduce the TG content and TC content in the HepG 2 cell lipid accumulation model. The dose - effect relationship of lignan J from Ligustrum purpurascens in reducing the TG and TC contents in the HepG 2 cell lipid accumulation model was obvious. Lignan J from Ligustrum purpurascens at 25 - 100 μmol·L -1 could significantly reduce the TG and TC contents in the HepG 2TG content in the cell lipid accumulation model, 50 - 100 μmol·L -1 Ligustroside J can significantly reduce the TC content in the HepG2 cell lipid accumulation model. According to its dose-effect relationship regression curve graph ( Figure 1-2 ), the median effective concentration (EC 50 ) of ligustroside J for reducing triglyceride and total cholesterol is 22.8 μmol·L -1 and 38.4 μmol·L -1 respectively.

[0033] Table 1. Lipid-lowering effect of ligustroside J (LP-J) on the HepG2 cell lipid accumulation model (X±SD) **P<0.01, ***P<0.001 vs. blank group; ▲ P<0.05, ▲▲ P<0.01 vs. model group

[0034]

Claims

1. Use of Ligustrum lucidum glycoside J and its composition in the preparation of drugs for treating hyperlipidemia and losing weight.

2. The use according to claim 1, characterized in that: Adenophora glycoside J can be extracted and separated from plants; it can also be synthesized by chemical synthesis.

3. A pharmaceutical composition for treating hyperlipidemia and losing weight according to claim 1, characterized in that The drug is composed of an effective amount of adenophora glycoside J and a pharmaceutically acceptable carrier. Adenophora glycoside J can be processed into capsules (soft capsules), granules (dry suspension mixtures), tablets (dispersible tablets, effervescent tablets, chewable tablets, orodisintegrating tablets), solutions (syrups), pills (concentrated pills, dripping pills, micropills), etc.

4. Claims 1-3 describe the lipid-lowering and weight-loss activities of the compound adenophora glycoside J and its composition, and their use in the preparation of clinical drugs for the treatment of hyperlipidemia and weight loss.

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