American ginseng composition for reducing blood fat and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to an American ginseng composition for reducing blood fat and application thereof. The oligopeptide AQFTHWKLH with biological activity is extracted through enzymolysis of American ginseng with neutral protease and papain, and the oligopeptide can effectively reduce the levels of serum total cholesterol, triglyceride and low-density lipoprotein cholesterol and improve the level of high-density lipoprotein cholesterol and also has the function of enhancing an antioxidant system. The invention also relates to a composition containing the oligopeptide and an application of the oligopeptide in preparation of drugs for reducing blood fat. Experiments prove that the composition has a remarkable blood fat reducing effect and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly, to a American ginseng composition for reducing blood lipid and its application. Background Art

[0002] Hyperlipidemia is a common metabolic disease, mainly manifested as elevated levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol in the blood, and has become an important risk factor for cardiovascular diseases such as atherosclerosis and coronary heart disease. Although existing lipid-lowering drugs such as statins and fibrates have good efficacy, long-term use may cause adverse reactions such as muscle damage and abnormal liver function. Therefore, there is an urgent need to develop new lipid-lowering active substances with high safety and natural sources.

[0003] American ginseng (Panax quinquefolium L.), as a traditional Chinese medicine, has various pharmacological activities, but the active ingredients and mechanisms directly applied to reduce blood lipid have not been clarified. In recent years, bioactive short peptides have received extensive attention due to their good bioavailability and specific functions. Based on this background, the present invention extracts a novel short peptide from American ginseng by proteolytic enzyme hydrolysis and confirms its significant blood lipid-lowering effect. Summary of the Invention

[0004] The purpose of the present invention is to provide an active short peptide AQFTHWKLH extracted from American ginseng and its composition, as well as the application of the composition in the preparation of lipid-lowering drugs or health foods, so as to solve the problem of lack of natural and safe lipid-lowering substances in the prior art.

[0005] The present invention provides an American ginseng composition for reducing blood lipid; In some embodiments, the composition contains the short peptide AQFTHWKLH obtained by proteolytic enzyme hydrolysis of American ginseng.

[0006] In some embodiments, the composition further contains a pharmaceutically acceptable carrier or excipient.

[0007] In some embodiments, the carrier is selected from normal saline, mannitol, glucose or a mixture thereof.

[0008] The present invention provides the use of the American ginseng composition in the preparation of a drug for treating or preventing diseases related to abnormal blood lipid metabolism.

[0009] The present invention provides the use of the American ginseng composition in the preparation of a drug for improving the levels of serum total cholesterol, triglycerides and low-density lipoprotein cholesterol.

[0010] For the first time, a biologically active short peptide AQFTHWKLH was extracted from American ginseng by enzymatic hydrolysis with neutral protease and papain, and the composition containing this short peptide has a significant effect on reducing blood lipid, showing broad application prospects. Description of the Drawings

[0011] Figure 1 Structural diagram of short peptide AQFTHWKLH.

[0012] Figure 2 Analysis of the mass spectrometry results of short peptide AQFTHWKLH.

[0013] Figure 3 Detection of the cell viability by short peptide AQFTHWKLH.

[0014] Figure 4 Detection of intracellular lipid droplets by Oil Red O staining. Detailed Description of the Invention

[0015] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0016] Example 1 Preparation of Active Short Peptide from American Ginseng Take 100 g of dry powder of American ginseng, add it to 1000 mL of deionized water, stir evenly and then carry out hydration treatment, and leave it standing at 4 °C for 12 hours. Adjust the pH of the hydrated American ginseng suspension to 7.5, add neutral protease (100 U / mg) and papain (800 U / mg) in a ratio of 1:1, and the enzyme addition amount is 2% of the mass of the dry powder of American ginseng. Carry out enzymatic hydrolysis reaction in a water bath at 50 °C for 6 hours. After the reaction is completed, terminate the enzymatic hydrolysis by heating at 95 °C for 10 minutes, cool and then centrifuge (8000 rpm, 10 minutes), and take the supernatant. The supernatant is separated by ultrafiltration, and the filtrate with a molecular weight less than 3 kDa is collected. The collected filtrate with a molecular weight less than 3 kDa is analyzed by liquid chromatography-mass spectrometry. The mobile phases used are A (0.1% formic acid aqueous solution) and B (0.1% formic acid-acetonitrile aqueous solution). From 0 to 50 min, the linear gradient of solution B is from 4% to 50%; from 50 to 54 min, the linear gradient of solution B is from 50% to 100%; from 54 to 60 min, solution B is maintained at 100%. Subsequently, the corresponding amino acid sequence is obtained by searching the corresponding database with the software MaxQuant, and its mass spectrometry results are as Figure 1 shown. At the same time, PEP-FOLD is used as a de novo short peptide structure prediction tool based on the structural alphabet (SA), and its structural diagram is as Figure 2 shown.

[0017] The results showed that the molecular weight of the short peptide AQFTHWKLH (SEQ ID NO:1) was 1.17 kDa, composed of 9 amino acids, with a purity of 96.32%. The quality of the polypeptide met the experimental requirements, and it had good water solubility, conforming to the characteristics of bioactive peptides.

[0018] Example 2 Preparation of a composition containing active short peptides from American ginseng Take 50 mg of the prepared short peptide AQFTHWKLH and dissolve it in 5 mL of sterile physiological saline. Add an appropriate amount of mannitol (5% w / v) as a stabilizer and adjust the pH to 7.0. After filtering the solution through a sterile filter membrane (0.22 μm), it was dispensed into sterile vials, with each vial containing 5 mg of short peptide AQFTHWKLH, and stored frozen at -20°C for later use.

[0019] Example 3 Experiment on the lipid-lowering activity of a composition containing active short peptides from American ginseng Experimental animals and grouping: SPF-grade C57BL / 6J mice, male, weighing 20 - 25 g, were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., production license number: SCXK (Jiangsu) 2023 - 0016; animal batch number: 20231001 - BL6 - M - 20 - 25G.

[0020] After one week of adaptive feeding of the experimental animals, they were randomly divided into the following four groups, with 10 mice in each group: Normal control group (NC group): Fed with normal diet and gavaged with physiological saline; High-fat model group (MC group): Fed with high-fat diet and gavaged with physiological saline; Low-dose short peptide group (LD group): Fed with high-fat diet and gavaged with the composition containing active short peptides from American ginseng prepared in Example 2 at a dose of 5 mg / kg; High-dose short peptide group (HD group): Fed with high-fat diet and gavaged with the composition containing active short peptides from American ginseng prepared in Example 2 at a dose of 15 mg / kg; Positive control group (PC group): Fed with high-fat diet and gavaged with atorvastatin at a dose of 5 mg / kg.

[0021] Modeling and treatment: Mice in the MC, LD, HD, and PC groups were fed with a high-fat diet (containing 45% fat) to establish a model for 8 weeks. During this period, they were treated according to the group every day, and the NC group was fed with normal diet.

[0022] Index detection: At the end of the 8th week of the experiment, after the mice were fasted for 12 hours, blood was taken from the orbital cavity, serum was separated, and blood lipid-related indexes were detected, including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), SOD and MDA concentrations. Among them, total cholesterol (TC) was detected by the CHOD-PAP method (cholesterol oxidase-peroxidase method) at a detection wavelength of 500 - 550 nm; triglyceride (TG) was detected by the GPO-PAP method (glycerol phosphate oxidase-peroxidase method) at a detection wavelength of 546 nm; LDL-C was detected by the homogeneous method at a detection wavelength of 600 nm; HDL-C was detected by the homogeneous method at a detection wavelength of 700 nm; SOD was detected by the xanthine oxidase method at a detection wavelength of 450 nm; MDA was detected by the TBA method at a detection wavelength of 532 nm; determination was carried out using a Hitachi 7170A automatic biochemical analyzer, as shown in Table 1.

[0023] Table 1 Index detection of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in mice of each group Note: * indicates significant difference compared with the MC group (p < 0.05), ** indicates extremely significant difference (p < 0.01); a indicates significant difference compared with the NC group (p < 0.05), b indicates extremely significant difference (p < 0.01); The results in Table 1 show that compared with the NC group, the serum TC, TG, and LDL-C levels of the mice in the MC group increased significantly, and the HDL-C level decreased significantly, indicating that the high blood lipid mouse model was successfully constructed; compared with the MC group, the serum TC, TG, and LDL-C levels of the mice in the LD group, HD group, and PC group decreased significantly (p < 0.05 or p < 0.01), and the decrease in the HD group was more significant than that in the PC group (p < 0.01); at the same time, the HDL-C level of the mice in the HD group was significantly higher than that in the MC group (p < 0.05), and the effect was slightly better than that in the PC group; that is, the American ginseng active short peptide AQFTHWKLH can effectively reduce the blood lipid level of high-fat-fed mice and has potential blood lipid-lowering activity.

[0024] Table 2 Antioxidant capacity indexes of mice in each group

[0025] Note: * indicates significant difference compared with the MC group (p < 0.05), ** indicates extremely significant difference (p < 0.01); a indicates significant difference compared with the NC group (p < 0.05), b indicates extremely significant difference (p < 0.01); As can be seen from the data in Table 2, compared with the NC group, the content of MDA in the serum of mice in the MC group increased significantly (p < 0.05), the activity of SOD increased, but there was no significant difference; compared with the MC group, the activity of SOD in the serum of mice in the LD group, HD group and PC group increased extremely significantly (p < 0.05), and the content of MDA decreased extremely significantly (p < 0.01); compared with the PC group, the increase in SOD in the serum of mice in the LD group and HD group was more significant, and the decrease in the content of MDA was more significant; this indicates that the active short peptide AQFTHWKLH of American ginseng may enhance the antioxidant system of hyperlipidemic mice by increasing the level of SOD and decreasing the level of MDA.

[0026] Example 4 Cell Experiment on the Hypolipidemic Activity of the Active Short Peptide AQFTHWKLH of American Ginseng In the cell incubator, human hepatoma cells HepG2 (ATCC HB-8065) were cultured in a complete medium (DMEM high-glucose medium + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin); culture environment: 37 °C, 5% CO2, saturated humidity; then the HepG2 cells were seeded in a 96-well plate (5 × 10³ cells / well) and treated according to the grouping after adhesion.

[0027] Blank control group: After the cells were seeded and adhered for 24 hours, they were replaced with fresh normal medium (without any inducer or drug) and continued to be cultured for 24 hours to synchronize the treatment time of other groups; Model group: Oxidized low-density lipoprotein (ox-LDL) was prepared at 50 μg / mL (dissolved in serum-free DMEM); after the cells adhered, the original medium was aspirated, and serum-free medium containing 50 μg / mL ox-LDL was added (to avoid interference from serum proteins); continued to be cultured for 24 hours; Low-dose group: 25 μM of the active short peptide AQFTHWKLH of American ginseng was dissolved in PBS with pH 7.4; after the cells adhered, the original medium was aspirated; Pretreatment: Serum-free medium containing 25 μM of the short peptide was added and incubated for 2 hours; ox-LDL (50 μg / mL) was added and continued to be cultured for 24 hours. High-dose group: 100 μM of the active short peptide AQFTHWKLH of American ginseng was dissolved in PBS with pH 7.4; after the cells adhered, the original medium was aspirated; Pretreatment: Serum-free medium containing 100 μM of the short peptide was added and incubated for 2 hours; ox-LDL (50 μg / mL) was added and continued to be cultured for 24 hours.

[0028] Positive control group: Atorvastatin was dissolved in DMSO at 10 μM to ensure that the final concentration of DMSO < 0.1% was non-toxic to the cells; after the cells adhered, the original medium was aspirated; Pretreatment: Serum-free medium containing 10 μM of atorvastatin was added and incubated for 2 hours; ox-LDL (50 μg / mL) was added and continued to be cultured for 24 hours.

[0029] Cell viability detection (MTT method): MTT solution (5 mg / mL) was added to the cells in each group and incubated for 4 hours. After dissolving formazan, the OD570 nm was measured, and the cell survival rate was calculated. See Figure 3 .

[0030] Figure 3 The results showed that the cell survival rate in the model group decreased significantly (p < 0.01 vs. the blank control group), indicating that treatment with 50 μg / mL ox-LDL for 24 hours could induce lipotoxic damage in HepG2 cells, resulting in reduced metabolic activity; the cell survival rate in the high-dose group was close to the normal level (96.8%, p < 0.01 vs. the model group), suggesting that the high-concentration short peptide had stronger cytoprotective effects and no cytotoxicity (no significant difference from the normal group, p > 0.05); atorvastatin (positive control group) restored the survival rate to 86.5%, verifying the reliability of the experimental system and indicating that the protective effect of the high-dose short peptide group was better than that of the traditional drug.

[0031] Detection of intracellular lipid droplets by Oil Red O staining: After fixing the cells in each group, Oil Red O staining (0.5% isopropanol solution) was performed, and the lipid droplet area was observed under a microscope and quantitatively analyzed by ImageJ. See Figure 4 .

[0032] Figure 4 The results showed that the proportion of the lipid droplet area in the normal group was only 2.1%, indicating that HepG2 cells not treated with ox-LDL had no obvious lipid accumulation under normal metabolic conditions; the model group showed that after induction with ox-LDL, the lipid droplet area increased significantly (p < 0.01 vs. the blank control group), indicating that a lipid accumulation model was successfully constructed, simulating the pathological state under a high-fat environment. The positive control group showed that after intervention with atorvastatin (10 μM), the lipid droplet area decreased to 15.3% (p < 0.01 vs. the model group), verifying that the drug effectively reduced lipid accumulation by inhibiting cholesterol synthesis. The lipid droplet area in the high-dose group further decreased significantly (p < 0.05 vs. the model group), even better than the positive control group, suggesting that the short peptide was superior to the traditional drug atorvastatin at high concentrations and might play a lipid-lowering role through multiple pathways such as regulating lipid uptake, decomposition, and storage.

[0033] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A blood lipid lowering American ginseng composition, characterized in that: The composition comprises the short peptide AQFTHWKLH obtained by enzymatic hydrolysis of American ginseng by protease.

2. The American ginseng composition according to claim 1, characterized in that The composition further comprises a pharmaceutically acceptable carrier or excipient.

3. The composition according to claim 2, wherein the carrier is selected from physiological saline, mannitol, glucose or a mixture thereof.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for treating or preventing diseases related to dyslipidemia.

5. Use of the composition according to any one of claims 1 to 3 in the preparation of a medicament for improving serum total cholesterol, triglyceride and low-density lipoprotein cholesterol levels.

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