Tibetan medicine scindapsus aureus flower polysaccharide A1 with lipid-lowering function as well as preparation method and application thereof

Abstract

The invention relates to the field of natural medicines, and particularly discloses Tibetan medicine scindapsus aureus flower polysaccharide A1 with a lipid-lowering function as well as a preparation method and application thereof. The Tibetan medicine scindapsus aureus flower polysaccharide A1 is prepared from the following components in mol percent: 55 to 60 percent of arabinose, 15 to 20 percent of galactose, 5 to 10 percent of galacturonic acid, 5 to 10 percent of rhamnose and 3 to 5 percent of glucose. The Tibetan medicine scindapsus aureus flower polysaccharide A1 is extracted and separated from Tibetan medicine scindapsus aureus flowers. The method comprises the following steps: S1, extracting and preparing Tibetan medicine scindapsus aureus flower total polysaccharide by adopting a water extraction and alcohol precipitation method; s2, separating and purifying the scindapsus aureus flower total polysaccharide by adopting an anion exchange chromatography technology to obtain EGP-A, and further separating and purifying the EGP-A by adopting a gel column chromatography technology to obtain EGP-A1. Through research and implementation of the invention, the EGP-A1 has good lipid-lowering activity, can play a role in protecting blood fat and cardiovascular health, and has a good medicinal prospect in the aspect of preparing lipid-lowering medicines.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and particularly relates to a Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function, a preparation method and an application thereof. Background Art

[0002] Hypertriglyceridemia is currently considered to be one of the important risk factors for cardiovascular events, mainly referring to excessively high levels of total cholesterol, triglycerides and low-density lipoprotein or too low levels of high-density lipoprotein in serum. Long-term lipid imbalance leads to increased burden on the cardiovascular system. The human body obtains lipids through two pathways: autonomous synthesis by the liver and absorption of dietary lipids by the small intestine. Dietary intake is the main source of lipids in the human body, and more than 95% of lipids come from dietary fat. In the small intestine, the ingested lipids are emulsified by bile acids and hydrolyzed by digestive enzymes into monoglycerides and free fatty acids. These digested lipids pass through the intestinal mucosa through passive diffusion or protein-mediated transport mechanisms. Chylomicrons are resynthesized in intestinal cells and enter the body circulation. The content of triglyceride-rich lipoproteins in postprandial plasma continues to increase and returns to normal after 6-8 hours, but due to the eating habit of three meals a day, the body is in a non-fasting state most of the time, which causes the circulatory system to be exposed to a high triglyceride environment for a long time, greatly increasing cardiovascular risk. Therefore, postprandial hyperlipidemia requires strict treatment. Many clinical studies have shown that the increased mortality of patients with coronary heart disease is closely related to high levels of TG. People at high risk of cardiovascular diseases such as obesity and diabetes are also often accompanied by high TG blood.

[0003] The TG-lowering drugs currently used in clinical practice are fibrates and niacin drugs. The target of fibrates is peroxisome proliferators activated receptor α (PPAR-α), which can regulate lipid metabolism in the body. After PPAR-α is activated, fatty acid oxidation is enhanced and TG levels are reduced. The fibrates currently on the market include clofibrate, bezafibrate, fenofibrate, etc. Nicotinic acid drugs belong to the B vitamins and can show lipid-lowering effects when used in large doses. However, due to the side effects of niacin drugs such as skin flushing and gastrointestinal tract, their application is also limited.

[0004] The Tibetan medicine green radish flower is the dried flower buds of the plant of the genus Thymelaeaceae, which is used in many Tibetan secret recipes. It is listed as a second-level protected variety of rare Tibetan medicinal materials and is also known as one of the "Eighteen Treasures of Qinghai-Tibet". According to the "Tibetan Medical Health Illustrations", green radish flowers are mainly used to treat diabetes, coronary heart disease, hypertension, hyperlipidemia, etc.

[0005] Current research mainly focuses on the hypoglycemic mechanism, anti-tumor and immune regulation, and hypolipidemic effects of green radish flowers, as well as the recent discovery that green radish flower extracts reduce the blood lipid level of mice fed a high-fat diet, inhibit atherosclerosis, and protect myocardial infarction. However, the above studies are all based on the total extract of green radish flowers, and it is not clear which component plays a related role. For example: Invention patent publication number CN 105267058 A discloses a green radish flower polysaccharide and its application, that is, the total polysaccharide content in the green radish flower polysaccharide is 5-15%. Prepared by the following method: crush the green radish flower, add water to extract, filter, remove the solvent from the filtrate and concentrate, add an organic solvent to precipitate, filter, wash the filter residue with an organic solvent, and dry to obtain green radish flower polysaccharide. The green radish flower polysaccharide has good moisturizing and antioxidant effects, and can be used in cosmetics, health foods and medicine. The invention patent only discloses the total polysaccharide in the Tibetan medicine green radish flower, and does not separate and extract the green radish flower polysaccharide components and study the active ingredients.

[0006] The composition of the total polysaccharide of the Tibetan medicine green radish flower is complex and huge, which brings great difficulties and challenges to its development and utilization. Therefore, it is of great significance to separate the active polysaccharide with a single structure for the development of green radish flower polysaccharide. Green radish flower polysaccharide (EGP) is one of the important active substances in the Tibetan medicine green radish flower, which has pharmacological activities such as anti-tumor, hypoglycemic and immunosuppressive. There is no research report on the lipid-lowering effect of green radish flower polysaccharide. Summary of the invention

[0007] The technical problems to be solved by the present invention are: in view of the existing problems in the research on the Tibetan medicine green radish flower in the prior art: first, the separation of polysaccharides is relatively rough, and the types and structural characteristics of the polysaccharides in the green radish flower have not been clarified; second, the discussion on the anti-hyperlipidemia and immune enhancement functions of the total polysaccharides of the green radish flower has not been implemented to the specific effective ingredients; third, there is a lack of research on the mechanism of action of the total polysaccharides of the green radish flower in the lipid-lowering effect, and other defects and deficiencies, and provide a Tibetan medicine green radish flower polysaccharide A1 with lipid-lowering function and a preparation method and application.

[0008] The present invention aims to clarify that the main component of green radish flower polysaccharide that reduces intestinal epithelial cell lipid absorption and improves hyperlipidemia is the A1 component and the mechanism behind it. Based on the existing technology of preparing total polysaccharides by water extraction and alcohol precipitation, the polysaccharide components in the green radish flower total polysaccharide are further separated and purified by anion exchange chromatography technology and gel column chromatography technology to obtain the green radish flower polysaccharide A1 component; and GC-MS, gel chromatography, Fourier transform infrared spectroscopy, methylation, one-dimensional and two-dimensional nuclear magnetic resonance technology are used to perform structural analysis on the green radish flower polysaccharide A1 component, and its structural composition is predicted; a monolayer intestinal epithelial cell absorption model is constructed using human colon cancer Caco-2 cells, oleic acid is used to simulate dietary long-chain fatty acids, and the inhibitory effect of the green radish flower polysaccharide A1 component on intestinal epithelial cell lipid absorption is observed, and the main mechanism of green radish flower polysaccharide A1 inhibiting intestinal lipid absorption is explored from the point of lipid-promoting internalization of CD36 on the surface of intestinal epithelial cell membrane. The main innovation of the present invention is that chromatography technology is used to separate and purify the components in the green radish flower polysaccharide, and the structure of the green radish flower polysaccharide A1 component is analyzed and predicted for the first time by combining spectroscopy, GC-MS, gel chromatography, methylation modification and nuclear magnetic resonance technologies, clarifying that the green radish flower polysaccharide A1 component has the effect of reducing intestinal dietary lipid absorption.

[0009] The present invention adopts the following technical scheme to achieve the purpose of the invention.

[0010] Firstly, the present invention extracts Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function from the Tibetan medicine scindapsus flower.

[0011] The Tibetan medicine green radish flower polysaccharide A1 contains, by mol%, 55-60% arabinose (Ara), 15-20% galactose (Gal), 5-10% galacturonic acid (Gal-UA also called GalA), 5-10% rhamnose (Rha), and 3-5% glucose (Glc).

[0012] The structural formula of EGP-A1 is as follows:

[0013] Secondly, the present invention also provides a method for preparing Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function, comprising the following steps: S1 uses water extraction and alcohol precipitation method to extract and prepare total polysaccharides from the Tibetan medicine Scindapsus flower; S2 uses anion exchange chromatography technology to separate and purify the total polysaccharide of the Tibetan medicine Epimedium flower to obtain EGP-A, and uses gel column chromatography technology to further separate and purify EGP-A to obtain EGP-A1.

[0014] Furthermore, the water extraction and alcohol precipitation method described in step S1 is used to extract and prepare the total polysaccharides of Tibetan medicine green radish flower, specifically: take the Tibetan medicine green radish flower, add pure water to reflux and extract, concentrate the obtained extract, add ethanol, stir and stand, filter out the ethanol solution, collect the precipitate, dry and dissolve it in water, add Savege reagent to remove protein, collect the supernatant and concentrate and freeze-dry to obtain the total polysaccharides of Tibetan medicine green radish flower.

[0015] Preferably, the Tibetan medicine green radish flower is added with pure water for reflux extraction as follows: take the Tibetan medicine green radish flower and add pure water according to the solid-liquid ratio of 1:10-30, reflux and extract at 90-100℃ for 2-4 times, 3-5 h / time, and combine the extracts; the concentration is carried out by reduced pressure vacuum concentration; the drying is carried out by reduced pressure vacuum drying; the addition of ethanol, stirring and standing is as follows: add 5-15 times the volume of anhydrous ethanol, stir well and stand at 2-6℃ for 12-36h; the volume ratio of Savege reagent is chloroform: n-butanol = 4:1.

[0016] Furthermore, the anion exchange chromatography technique described in step S2 is used to separate and purify the total polysaccharides of the Tibetan medicine Epimedium flower to obtain EGP-A, which includes the steps of separating the polysaccharides with an ion exchange column, drawing an elution curve of the ion exchange column, and dialysis desalting.

[0017] Furthermore, the ion exchange column is used to separate polysaccharides, specifically: weigh the total polysaccharides of the Tibetan medicine green radish flower obtained in step S1, add pure water to fully dissolve it, slowly add it to the anion exchange fiber column, adsorb it overnight, and then use ultrapure water and sodium chloride solutions of different concentrations for gradient elution.

[0018] Preferably, the added pure water is added in a ratio of 80-120 times that of the Tibetan medicine scindapsus flower polysaccharide; the anion exchange fiber column is DEAE-52; and the different concentrations of sodium chloride solution are 0.1 mol / L, 0.3 mol / L and 0.5 mol / L.

[0019] Furthermore, the drawing of the ion exchange column elution curve is specifically as follows: collecting the eluate, determining the absorbance of each tube of eluate by the phenol-sulfuric acid method, taking the tube number as the X-axis and the absorbance value as the Y-axis, and drawing the anion exchange cellulose chromatography column elution curve of the Tibetan medicine green radish flower polysaccharide.

[0020] Furthermore, the dialysis desalination is specifically as follows: collecting the main elution peaks at each concentration, concentrating under reduced pressure, pouring the concentrated solution into a dialysis bag, placing it in ultrapure water for magnetic stirring dialysis; collecting the dialyzed solution, freeze-drying to obtain each elution component, and the component obtained from the ultrapure water eluate is named EGP-A.

[0021] Preferably, the dialysis bag is MD44; the dialysis time is 36-72 h, ultrapure water is replaced every 6-10 h, and repeated 4-8 times.

[0022] Furthermore, gel column chromatography technology is used to further separate and purify EGP-A to obtain EGP-A1, specifically: weigh EGP-A, add ultrapure water to fully dissolve, absorb the EGP-A supernatant solution after rotary centrifugation, slowly add it to dextran gel G-200, elute with deionized water, use phenol-sulfuric acid method to detect the whole process, collect the main elution peak, and perform segmented separation after detection to obtain EGP-A1; Preferably, the ultrapure water is added in an amount of 800-1200 times that of EGP-A; and the rotary centrifugation is performed at 4000-6000 rpm for 10-20 min.

[0023] The EGP-A1 obtained by the above method steps contains, by mol%, 55-60% arabinose (Ara), 15-20% galactose (Gal), 5-10% galacturonic acid (Gal-UA), 5-10% rhamnose (Rha), and 3-5% glucose (Glc).

[0024] In addition, the present invention also provides the use of the Tibetan medicine scindapsus flower polysaccharide A1 component in the preparation of lipid-lowering drugs.

[0025] Furthermore, the Tibetan medicine green radish flower polysaccharide A1 has a significant lipid-lowering function; the Tibetan medicine green radish flower polysaccharide A1 contains, by mol%, 55-60% arabinose (Ara), 15-20% galactose (Gal), 5-10% galacturonic acid (GalA), 5-10% rhamnose (Rha), and 3-5% glucose (Glc).

[0026] Furthermore, the lipid-lowering drug has dosage forms including tablets, pills, capsules, granules, injections, oral liquids, mixtures and other common dosage forms, or is formulated with Tibetan medicine scindapsus flower polysaccharide A1 to produce a synergistic drug.

[0027] Beneficial effects: (1) The present invention uses GC-MS, gel chromatography, Fourier transform infrared spectroscopy, methylation, one-dimensional and two-dimensional nuclear magnetic resonance technology to clarify the monosaccharide composition of the Tibetan medicine green radish flower polysaccharide A1 component and illustrate the structural characteristics of the Tibetan medicine green radish flower polysaccharide A1. The Tibetan medicine green radish flower polysaccharide A1 component has clear ingredients and strong pharmacological effects.

[0028] (2) Through the research and implementation of the present invention, it is clarified that the Tibetan medicine green radish flower polysaccharide A1 component has good lipid-lowering activity, can play a protective role on blood lipids and cardiovascular health, and has good medicinal prospects in the preparation of lipid-lowering drugs.

[0029] (3) The present invention obtains total polysaccharides from the flowers of the Tibetan medicinal herb Epimedium through hot water extraction, ethanol precipitation, and protein removal by the Savege method, and separates and purifies the total polysaccharides from the flowers of the Tibetan medicinal herb Epimedium by ion exchange column chromatography to obtain Tibetan medicinal herb Epimedium polysaccharide A (EGP-A), and further separates and purifies EGP-A by gel column chromatography to obtain EGP-A1. The preparation process is reasonable and simple, and can be easily industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The structural formula of the Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1); Figure 2 This is the extraction flow chart of the Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1); Figure 3 This is the elution curve of the total polysaccharide of the Tibetan medicine Scindapsus flower on the DEAE-52 anion exchange chromatography column; Figure 4 This is the elution curve of the Tibetan medicine green radish flower polysaccharide A on the Sephadex G200 exchange chromatography column; Figure 5 Ion chromatograms of monosaccharide standards (upper figure) and EGP-A1 (lower figure); Figure 6 This is the infrared spectrum analysis chart of EGP-A1; Figure 7 This is the UV spectrum analysis chart of EGP-A1; Figure 8 This is the total ion current of PMAAs, the methylation product of EGP-A1; Fig. 9 is the nuclear magnetic resonance spectrum of EGP-A1; Fig.10 This is a diagram of EGP-A1 inhibiting the accumulation of lipids induced by oleic acid in Caco-2 cells. Among them: A is BODIPY and Oil Red O staining after incubation of Caco-2 cells with 250μM OA or EGP-A1 for 24 hours; B and C are the statistics of the average fluorescence intensity of BODIPY and the average Oil Red area in the cells; D is the quantitative result of intracellular TG, and the intracellular triglyceride level is analyzed by comparing the triglyceride content per microgram of total protein in the cell lysate of each group; Fig.11 Figure 1 is an analysis of the anti-lipid activity of EGP-A1. A is the Oil Red O staining result of HEGP2 cells incubated with 100 μM OA and 200 μM PA for 24 h with 1, 10, and 100 μg / mL EGP-A1; B is the quantitative result of intracellular TG; C is the fluorescence quantitative qPCR result of RAW264.7 cells treated with 50 μg / mL Ox-LDL or EGP-A1 for 24 h. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0032] Example 1: Preparation of Tibetan medicine Scindapsus scindapsus flower total polysaccharide The total polysaccharide of the Tibetan medicine green radish flower was prepared by water extraction and alcohol precipitation. Take 1.5 kg of the Tibetan medicine green radish flower, add 30 L of pure water according to the solid-liquid ratio of 1:20, reflux and extract at 95 ° C for 3 times, 4 h / time, combine the extracts, reduce pressure and vacuum concentration, add 9 times the volume of anhydrous ethanol, stir well and let stand at 4 ° C for 24 hours. Filter out the ethanol solution, collect the precipitate, and dry it under reduced pressure. The dried precipitate was dissolved in water and the Savege reagent (chloroform: n-butanol = 4:1) was added to remove the protein. The supernatant was collected and concentrated and freeze-dried to obtain the total polysaccharide of the Tibetan medicine green radish flower.

[0033] 1.5 kg of Tibetan medicine green radish flower was extracted by the above-mentioned water extraction and alcohol precipitation method to obtain 207.6 g of total polysaccharides from Tibetan medicine green radish flower, with an extraction yield of 13.82%.

[0034] Example 2: Obtaining Tibetan Medicine Epimedium Flower Polysaccharide A1 Component (EGP-A1) Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1) was obtained from the total polysaccharides of Epimedium flower by separation and purification.

[0035] The extraction process flow chart of the Tibetan medicine green radish flower polysaccharide A1 component is as follows Figure 2 shown.

[0036] (1) Separation of polysaccharides using ion exchange column: Weigh 5 g of the obtained Tibetan medicine green radish flower polysaccharide, add 500 mL of pure water to fully dissolve it, and slowly add 10 mL of it into a DEAE-52 anion exchange fiber column (1.8×50 cm). After adsorption overnight, ultrapure water and different concentrations of sodium chloride solution (0.1 mol / L, 0.3 mol / L and 0.5 mol / L) were used for gradient elution at a flow rate of 1.2 mL / min.

[0037] (2) Draw the elution curve of the ion exchange column: collect the eluent (6 mL / tube, collect 40 tubes of each eluent) (tubes 0-40 are ultrapure water eluents, tubes 41-80 are 0.1 mol / L sodium chloride solution eluents, tubes 81-120 are 0.3 mol / L sodium chloride solution eluents, and tubes 121-160 are 0.5 mol / L sodium chloride solution eluents). Use the phenol-sulfuric acid method to determine the absorbance of each tube of eluent, and draw the anion exchange cellulose chromatography column elution curve of green radish flower polysaccharide with the tube number as the X-axis and the absorbance as the Y-axis.

[0038] (3) Dialysis desalination: collect the main elution peaks at each concentration, concentrate under reduced pressure, pour the concentrated solution into an MD44 dialysis bag, and dialyze in ultrapure water with magnetic stirring for 48 hours, replace the ultrapure water once every 8 hours, and repeat 6 times. Collect the dialyzed solution, freeze-dry to obtain the elution components, and obtain four components: EGP-A (obtained from the ultrapure water eluate), EGP-B (obtained from the 0.1 mol / L sodium chloride solution eluate), EGP-C (obtained from the 0.3 mol / L sodium chloride solution eluate), and EGP-D (obtained from the 0.5 mol / L sodium chloride solution eluate).

[0039] Figure 3 This is the elution curve of the total polysaccharide of Tibetan medicine Epimedium flower on DEAE-52 anion exchange chromatography column.

[0040] After elution with the above-mentioned pure water and NaCl solutions of various concentrations, a total of four elution peaks were obtained, named EGP-A, EGP-B, EGP-C, and EGP-D. The elution peaks were collected, concentrated, dialyzed for desalination, and freeze-dried to obtain different components of 2.98 g, 1.427 g, 2.20 g, and 0.682 g, with yields of 29.80%, 14.27%, 20.20%, and 6.82%, respectively; among them, EGP-A eluted from pure water had the highest yield and was the main component of the Tibetan medicine green radish flower polysaccharide.

[0041] (4) Gel column chromatography separation: Weigh 1 g of EGP-A component and add 1000 mL of ultrapure water to fully dissolve it. Centrifuge at 5000 rpm for 15 min at room temperature. Pipette 7 mL of EGP-A supernatant solution and slowly add it to dextran gel G-200 (1.6×60 cm) along the inner wall of the chromatography column. Elute with deionized water at a flow rate of 0.7 mL / min. Use the phenol-sulfuric acid method for full detection, collect the main elution peaks for detection, and then perform segmented separation to obtain EGP-A1 and EGP-A2.

[0042] Figure 4 This is the elution curve of Tibetan medicine Epimedium flower polysaccharide A (EGP-A) on the Sephadex G200 exchange chromatography column.

[0043] Tibetan medicine epimedium flower polysaccharide A (EGP-A) was separated by the above-mentioned gel "molecular sieve" to obtain two main elution peaks, named EGP-A1 and EGP-A2 respectively. The elution peaks were collected and concentrated under reduced pressure and freeze-dried to obtain the separated and purified Tibetan medicine epimedium flower polysaccharide EGP-A1.

[0044] Example 3: Purity Identification and Molecular Weight Determination of Tibetan Medicine Epimedium Flower Polysaccharide A1 Component (EGP-A1) The purity and molecular weight of the Tibetan medicine Epimedium scindapsus flower polysaccharide A1 (EGP-A1) were determined by high performance gel permeation chromatography (HPGPC).

[0045] HPGPC chromatographic conditions: TSKgel G3000PWXL gel column (7.8×300mm, 7μm); column temperature 40℃; mobile phase 0.1mol / L NaNO3 solution, flow rate 0.6mL / min; injection volume 10μL; HPLC system equipped with RID-20A differential refractometer detector.

[0046] Standard curve drawing: Accurately weigh dextran standards of different molecular weights (D0, D1, D2, D3, D4, D5, D6 and D7) and dissolve them in ultrapure water to prepare a 10 mg / mL solution. After injection, record the retention time of each standard solution, and finally draw a standard curve with the logarithm of the retention time and the standard average molecular weight (lgMw).

[0047] Sample determination: Accurately weigh 20 mg of EGP-A1 component and dissolve it in 2 mL of ultrapure water to prepare a 10 mg / mL polysaccharide solution. After injection testing, the molecular weight of the homogeneous polysaccharide is finally calculated based on the standard curve. The sample determination results are shown in Table 1 below: Table 1 Sample measurement results

[0048] The results in Table 1 above show that the molecular weight of Tibetan medicine green radish flower polysaccharide A1 is 23.42 KDa.

[0049] Example 5: Composition Analysis of Tibetan Medicine Epimedium Flower Polysaccharide A1 Component (EGP-A1) Weigh 2 mg of Tibetan medicine green radish flower polysaccharide A1 component (EGP-A1) and add it to the sample bottle. Add 1 mL of 2M trifluoroacetic acid solution to the bottle, seal it, and use 121°C and oil bath for 2 hours to fully hydrolyze the Tibetan medicine green radish flower polysaccharide A1. Blow dry the liquid in the bottle with nitrogen, add methanol to wash, blow dry again, repeat 3 times to remove the residual trifluoroacetic acid, and finally, add 20 mL of ultrapure water to fully dissolve it.

[0050] EGP-A1 composition analysis was performed using Thermo ICS 5000 + The EGP-A1 component was analyzed and detected by an ion chromatography system (ICS 5000+, ThermoFisher Scientific, USA) with an electrochemical detector.

[0051] Chromatographic conditions: Dionex™ CarboPac™ PA20 (150*3.0 mm, 10μm) liquid chromatography column; mobile phase: A (pure water), B (0.1M sodium hydroxide) and C (0.1M sodium hydroxide, 0.2M sodium acetate), 5μL injection volume, flow rate 0.5mL / min; column temperature was set to 30℃.

[0052] Gradient elution: elution time 60 min, initial conditions Phase A / B / C (95:5:0, V / V), 26 min Phase A / B / C (85:5:10, V / V), 42 min Phase A / B / C (85:5:10, V / V), 42.1 min Phase A / B / C (60:0:40, V / V), 52 min Phase A / B / C (60:40:0, V / V), 52.1 min Phase A / B / C (95:5:0, V / V), 60 min return to initial conditions.

[0053] Figure 5 The ion chromatograms of monosaccharide standards (upper figure) and EGP-A1 (lower figure).

[0054] By comparing the retention time with the standard, and then substituting it into the standard curve for quantification, the experiment was repeated three times and the results are shown in Table 2: EGP-A1 is mainly composed of 8 monosaccharides, namely rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (Gal-UA) and glucuronic acid (Glc-UA), which indicates that EGP-A1 is a heteropolysaccharide composed of 8 monosaccharides. The average molar ratio of the monosaccharide components of Rha: Ara: Gal: Glc: Xyl: Man: Gal-UA: Glc-UA is 8.26: 58.61: 17.14: 4.34: 1.63: 0.71: 8.76: 0.55, of which arabinose accounts for as high as 58.6%, and the molar ratio of galacturonic acid in EGP-A1 is 8.76%, which indicates that EGP-A1 is an acidic polysaccharide.

[0055] Table 2 Molar ratio (mol%) of monosaccharide composition of EGP-A1

[0056] Note: mol% represents the total content of monosaccharides Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru, Rib, Gal-UA, Gul-UA, Glc-UA and Man-UA.

[0057] Example 6: Infrared spectroscopy analysis of Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1) EGP-A1 and potassium bromide were dried to constant weight. 200 mg of potassium bromide was weighed on a precision balance and fully ground in an agate mortar. Transparent potassium bromide flakes with a diameter of 13 mm were prepared by mechanical tableting. The wavelength of the Fourier transform infrared spectrometer was 4000-400 cm -1 Scan within the range, set the measured value as a reference, weigh 1 mg of dried EGP-A1 and 200 mg of dried potassium bromide, grind and mix thoroughly, and then press to form a transparent sheet. Spectrometer with a wavelength of 4000-400 cm -1 The infrared spectrum of EGP-A1 was measured by scanning within the range, and the measured data was imported into Origin drawing software to draw the infrared spectrum of EGP-A1.

[0058] Figure 6 This is the infrared spectrum analysis chart of EGP-A1.

[0059] Fourier transform infrared spectroscopy is one of the effective methods to identify characteristic functional groups in polysaccharide structure research. The results of Fourier transform infrared spectroscopy detection of characteristic functional groups in EGP-A1 are shown in Table 3 and Figure 6 Shown Table 3 Special absorption peaks of functional groups in infrared spectrum of EGP-A1

[0060] from Figure 6 From Table 3, we can see that: 3370cm -1 The strong absorption peak near 2931.5cm is generated by the antisymmetric stretching vibration of the hydroxyl group (-OH) with the highest content in the sugar chain. -1 The absorption peak at 1737.66 cm is the antisymmetric stretching vibration peak of methylene (-CH2-). -1 and 1252.65cm -1 The weaker peak at 1622.95 cm is attributed to the stretching vibration of the carboxyl group (C=O), which indicates that EGP-A1 may contain a certain amount of uronic acid. -1 The strong absorption peak near the 1200-1000 cm also represents the stretching vibration of the carboxyl group (C=O), indicating that the sample may contain functional groups such as carboxyl (COOH). This result is consistent with the monosaccharide composition analysis, proving that EGP-A1 is an acidic polysaccharide. -1 The wavelength range is the unique characteristic absorption region of polysaccharides, and EGP-A1 appears at 1037.42 cm -1 A group of absorption peaks near 810 and 896 cm -1 The appearance of weak absorption peaks at , indicated the existence of α- and β-configuration glycosidic bonds in EGP-A1.

[0061] Example 7: UV-spectral analysis of Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1) A certain amount of EGP-A1 was weighed and dissolved in pure water to prepare a 1 mg / mL polysaccharide solution. The baseline was leveled with pure water. The UV scanner was set to a scanning range of 190-400 nm, and the polysaccharide solution was scanned to obtain the UV scanning spectrum of the Tibetan medicine green radish flower polysaccharide A1.

[0062] Figure 7 This is the UV spectrum analysis chart of EGP-A1.

[0063] from Figure 7 It can be seen that 260nm is the maximum ultraviolet absorption area of ​​nucleic acid, and 280nm is the maximum ultraviolet absorption area of ​​protein. No obvious absorption peak was observed in the EGP-A1 solution by scanning at 260nm and 280nm wavelengths, indicating that no obvious protein and nucleic acid residues were found in EGP-A1. EGP-A1 has a high purity and meets the basic requirements of nuclear magnetic resonance and methylation analysis.

[0064] Example 8: Methylation and GC-MS analysis of Tibetan medicine Epimedium flower polysaccharide A1 component (EGP-A1) The polysaccharide methylation experiment was processed according to the acidic polysaccharide methylation sample: (1) Weigh EGP-A1 (about 5 mg), add 1 mL of water to dissolve it, then pipette 1 mL of the prepared 100 mg / mL 1-cyclohexyl-2-morpholineethylcarbodiimide methyl p-toluenesulfonate, mix well, and react for 2 h; (2) Add 1 mL of 2 M imidazole and mix thoroughly. Then, transfer 1.5 mL of the mixture into new sample bottles. Add 1 mL of 30 mg / mL sodium borohydride solution to one bottle and 1 mL of 30 mg / mL sodium borodeuteride solution to the other bottle. Mix thoroughly and react for 3 h. (3) Add glacial acetic acid to the sample bottle, mix well, and let stand until the reaction stops. Collect the sample, dialyze it completely, and freeze-dry it; (4) Add 500 μL of anhydrous DMSO to the frozen sample, vortex thoroughly to dissolve, add 1 mg of sodium hydroxide powder to the system, and incubate at room temperature for 30 min; (5) Add 50 μL of iodomethane solution and react for 1 h; (6) Mix water and dichloromethane in a ratio of 1:2 and pipette 3 mL into a sample bottle, vortex thoroughly to mix, centrifuge, and retain the lower organic phase. After washing the organic phase three times, pipette the organic phase and blow dry with nitrogen; (7) Repeat the imidazole, sodium borohydride and sodium borodeuteride experimental system in the second step. After reacting for 3 h, add 100 μL 2 M trifluoroacetic acid and heat at 121 °C for 90 min to fully hydrolyze the polysaccharide; (8) After the reaction, evaporate the liquid, take 50 μL of 2M ammonia water and 1M sodium borodeuteride solution, add them in sequence, mix well, and let them react for 2.5 h; (9) Add 20 μL of glacial acetic acid, mix well, and let stand until the reaction stops. Collect the sample and blow dry with nitrogen, wash with methanol, blow dry again, and repeat twice; (10) Add 250 μL of acetic anhydride to the sample bottle, vortex thoroughly to mix, and incubate at 100°C in an oil bath for 2.5 h. After returning to room temperature, add 1 mL of water to the sample, mix well, and let stand for 10 min. (11) Add 500 μL of dichloromethane, mix thoroughly, extract the sample, collect the organic phase after centrifugation, extract it repeatedly with dichloromethane, combine the organic phases, concentrate and evaporate to dryness, add 500 μL of dichloromethane to dissolve, and then detect on GC-MS.

[0065] In this experiment, GC-MS technology was used to analyze the samples, and the autosampler model was G4567A. The chromatographic system used was Agilent7890A (Agilent Technologies, USA), and the chromatographic column was BPX70 (30m × 0.25mm ×0.25µm, SGE, Australia). The injection volume was 1μL, high-purity helium was loaded, and the split ratio was set to 10:1; the column oven was 140℃ for 2min, and the temperature program was set at 3℃ / min to 230℃ and maintained for 3min. The mass spectrometry system used was Agilent 5977B (AgilentTechnologies, USA), equipped with an electron impact ion source (EI) and a MassHunter workstation. The analytes were detected in full scan (SCAN) mode using an electron impact ion source (EI), and the mass scan range (m / z): 50-350.

[0066] Methylation is an effective method to predict the connection sites of polysaccharide residues. This method can be used to obtain the relative proportions of monosaccharides with different connection modes in the polysaccharide system. The modified products of EGP-A1 monosaccharide components were obtained by methylation, acid hydrolysis, reduction and acetylation of EGP-A1. The products were detected by GC-MS based on the retention time and characteristic fragments ( Figure 8 ) for structural identification. Figure 8 This is the total ion current diagram of PMAAs, the methylation product of EGP-A1, where the upper diagram is H substitution and the lower diagram is D substitution.

[0067] Table 4 EGP-A1 methylation analysis results

[0068] Comparison with the CCRC database showed that the PMAAs sugar residues of EGP-A1 (Table 4) consisted of t-Ara(f), 2-Rha(p), 3-Ara(f), 5-Ara(f), 2,3-Rha(p), 2,4-Rha(p), 3-Gal(p), 4-Gal(p), 4-Gal(p)-UA, 2,5-Ara(f), 3,4-Gal(p) and 3,6-Glc(p), with molar ratios of 34.92%, 5.60%, 1.03%, 20.23%, 1.70%, 4.09%, 1.40%, 15.06%, 8.56%, 2.31%, 2.28% and 2.7%, respectively. The above results were basically consistent with the results of monosaccharide composition analysis. In addition, among the residues with a molar ratio exceeding 5%, t-Ara(f) is the only terminal residual sugar, and its ratio with non-terminal sugars is close to 1:2, followed by 5-Ara(f) residual sugar, which accounts for about 20%. These two are speculated to constitute the main part of the EGP-A1 side chain in the form of terminal group and side chain connection, respectively; the residual sugars except arabinose are all hexoses, mainly 4-Gal(p), 4-Gal(p)-UA and 2-Rha(p), 2,4-Rha(p), which are speculated to constitute the linear connection of the main chain.

[0069] Example 9: Nuclear Magnetic Resonance Analysis of Tibetan Medicine Epidendrum Scindapsus Flower Polysaccharide A1 Component (EGP-A1) The structure of EGP-A1 was analyzed by nuclear magnetic resonance spectroscopy.

[0070] Weigh 20 mg of EGP-A1 and place it in a sample bottle. Add sufficient deuterated aqueous solution to dissolve it, freeze-dry it, and repeat 3 times to fully replace the deuterium in the heavy water. After 0.5 mL of deuterated water is taken to fully dissolve the sample, transfer it to a 5 mm NMR tube, and scan the polysaccharide sample in a BrukerAVANCE III HD 600 MHz NMR scanner to obtain a one-dimensional NMR ( 1 H NMR, 13 C NMR), two-dimensional nuclear magnetic resonance (COSY (HH), HSQC (HC), HMBC (HC), NOESY (HH)). The nuclear magnetic resonance scanning results were analyzed by MestRenova.

[0071] Fig. 9 This is the nuclear magnetic resonance spectrum of EGP-A1.

[0072] In order to clarify the main glycosidic bond connection mode in EGP-A1, the nuclear magnetic resonance spectroscopy was used to 1 H NMR, 13 EGP-A1 was analyzed by C NMR, (CH) HSQC, (HH) COSY, HMBC and NOESY. 1 H NMR and13 The spectral information of C NMR was used to assign the anomeric hydrogen and carbon in EGP-A1. 1 In the anomeric hydrogen (4.3-5.5 ppm) region of H NMR, multiple groups of anomeric hydrogen signals were found ( Fig. 9 A in the figure), the main peak positions are 5.24, 5.17, 5.10, 5.07, 5.03, 5.01, and 4.39 ppm. The number of anomeric hydrogens represents the number of residues in the polysaccharide, thus proving that EGP-A1 is composed of multiple residual sugars, which is consistent with the methylation results. In the 3.0-4.2 ppm range of the hydrogen spectrum, there are many signals and the proton signals outside the anomeric hydrogens of the residual sugar ring are in line with the general characteristics of the hydrogen spectrum of polysaccharides. Three main peaks at 1.24, 1.23, and 1.16 ppm were identified near 1.20 ppm. The peaks at this position correspond to the H6 signals of 2-Rha(p), 2,4-Rha(p), and 2,3-Rha(p) in the methylation experiment.

[0073] By identifying the one-dimensional carbon NMR spectrum 13 C NMR ( Fig. 9 In the anomeric region (90-110 ppm) of the carbon spectrum in B), three groups of anomeric carbon signals were found, located at 107.43, 107.08, and 106.88 ppm respectively; the range of 55-85 ppm is the peak information area of ​​the carbon outside the polysaccharide residual sugar excluding the anomeric carbon, the signal near 61.06 ppm is attributed to the residual sugar ring exomethylene -CH2-secondary carbon, and the multiple signals at 74-85 ppm represent the carbon information of various furanose residual sugars C2-C4 or pyranose residual sugars C2-C5. It may be due to this 13 The C NMR spectrum signal is weak, and no obvious uronic acid carbonyl carbon signal is found in the range of 170-210 ppm. However, the carbonyl position of the acidic residual sugar can be determined from the two-dimensional spectrum.

[0074] Combined with two-dimensional NMR spectra, the structure of the polysaccharide was further analyzed. 13 C NMR and (CH) HSQC NMR information ( Fig. 9C in the figure), five groups of H1 / C1 related weak signals were identified in the anomeric region, which were 5.17 / 106.38, 5.10 / 106.88, 5.07 / 107.08, 5.01 / 107.43, and 4.40 / 102.95 ppm respectively. Combined with the methylation results and the integrated intensity of the anomeric hydrogen in the hydrogen spectrum, the five major residual sugars with a proportion greater than 5% were identified, among which 5.17 / 106.38 were H1 / C1 signals of α→t)-Ara-(1→, 5.10 / 106.88 were H1 / C1 signals of α→4)-GalA-(1→, 5.07 / 107.08 were H1 / C1 signals of α→4)-Gal-(1→, 5.01 / 107.43 were H1 / C1 signals of α→5)-Ara-(1→, and 4.40 / 102.95 were H1 / C1 signals of β→2)-Rha-(1→.

[0075] Based on the inferred 5.01 ppm attributed to the anomeric H1 in the residual sugar α→5)-Ara-(1→, combined with the COSY two-dimensional spectrum that identifies hydrogen-hydrogen correlation ( Fig. 9 D) can find a strongly correlated signal of 4.05 ppm, confirming the positions of H1 and H2 are 5.01 and 4.05 ppm; combined with the HSQC spectrum, it is confirmed that the signal of H2 / C2 is 4.05 / 81.24; according to the COSY information, the signal related to H2 and H3 is 4.05 / 3.99, confirming the hydrogen spectrum position of C3 is 3.99 ppm, and combined with HSQC to find 3.99 / 82.20, confirming the position of C3 is 82.20 ppm, and the position information of H4 / C4 and H5 / C5 is determined to be 4.24 / 84.01 and 3.75 (3.86) / 66.07 by the same method. According to this method, other main positions of residual sugars are found and the same signal positions are assigned, forming the following Table 5: Table 5 EGP-A1 13 C and 1 H NMR chemical shift

[0076] NMR 2D spectrum HMBC( Fig. 9The E in the figure has a greater effect on determining the hydrogen-carbon correlation of 2-3 chemical bonds. In the analysis of polysaccharides, it has a certain effect on determining the connection of the main glycosidic bonds, and then it has a great significance for determining the main structure of the polysaccharide. Through the HMBC spectrum, the correlation signal of 5.17 / 66.07 was found in the EGP-A1 α→t)-Ara-(1→ residual sugar anomeric carbon. Combined with the assigned residual sugar, it was found that the α→t)-Ara-(1→ anomeric hydrogen was correlated with the C5 of α→5)-Ara-(1→, confirming the existence of a glycosidic bond connecting α→t)-Ara-(1→ residual sugar and α→5)-Ara-(1→ residual sugar 1→5, and the two constituted the main terminal sugar glycosidic bond in EGP-A1; the correlation signal of 5.01 / 66.07 confirmed the existence of the same residual sugar 1 in the residual sugar α→5)-Ara-(1→ →5 connected glycosidic bond. In addition, the related signals of α→4)-GalA-(1→ related anomeric carbon were assigned, and the related signals at 5.10 / 84.01 were found, so it was speculated that α→4)-GalA-(1→ and β→2)-Rha-(1→ had a 1→2 glycosidic bond connection; the overall signal of HMBC judged that EGP-A1 may be composed of galactose and rhamnose as the main chain part, and arabinose as the branch part. The site of branch formation in the main chain is speculated to be the C3 position of residual sugar 2,3-Rha, the C4 position of 3,4-Rha, the C3 position of 3,4-Gal, etc.

[0077] By NOESY ( Fig. 9 F) The spectrum in Figure 5 verifies the existence of glycosidic bonds. The signal of 5.07 / 4.23 confirms that α→4)-Gal-(1→ is connected to α→4)-GalA-(1→ by a 1→4 glycosidic bond; the signal of 5.10 / 4.22 confirms that the main residual sugar α→4)-GalA-(1→ is connected to β→2)-Rha-(1→ by a 1→2 glycosidic bond; the signal of 5.17 / 3.75 in the anomeric part speculates that the residual sugar α→t)-Ara-(1→ is connected to the residual sugar α→5)-Ara-(1→ by a 1→5 glycosidic bond; and the signal of 5.01 / 3.75 in the anomeric part speculates that the residual sugar is connected to the same residual sugar α→5)-Ara-(1→ by a 1→5 glycosidic bond.

[0078] Therefore, combined with the above test results and analysis, the main structure diagram of the green radish flower polysaccharide A1 component (EGP-A1) is obtained as follows Figure 1 shown.

[0079] Example 10: Tibetan medicine Epimedium polysaccharide A1 fraction (EGP-A1) inhibits lipid accumulation in Caco-2 cells treated with oleic acid (1) Construction of Caco-2 monolayer cell model

[0080] On the 3rd, 6th, 9th, 12th, 15th, 18th and 21st days after inoculation, three wells of cells were taken for each concentration to measure the TEER value, and the dynamic formation process of tight junctions of cell monolayers with three different inoculation concentrations and their differences were observed. TEER value = (R measurement well - R blank well) × A, R measurement well is the resistance value measured by the Transwell culture dish with Caco-2 cells grown, R blank well is the resistance value of the Transwell culture dish without inoculation of cells and only with the same volume of culture medium, A is the effective membrane area (0.33cm 2 ).

[0081] (2) Verification of Caco-2 cell monolayer integrity Caco-2 cell monolayer integrity was assessed by FITC-labeled dextran. A standard curve of FITC-labeled dextran was prepared using PBS at 0.005, 0.01, 0.025, 0.05, 0.1, and 0.25 μg / mL. FITC-dextran standard solution. The fluorescence intensity was measured by a microplate reader (excitation wavelength 427 nm, emission wavelength 536 nm). The standard curve was drawn by linear regression with fluorescence intensity as the ordinate and concentration (μg / mL) as the abscissa. Caco-2 cells were cultured on a 24-well Transwell plate. After the cell monolayer grew for 21 days, 3 wells of cells were taken and the culture medium was discarded. The AP and BL sides were washed twice with preheated PBS, and the PBS was discarded. 0.4 mL of FITC-dextran (20 μg / mL) was added to the AP side, and 0.6 mL of PBS was added to the BL side. The cells were cultured in a 37°C, 5% CO2 incubator for 1 hour. 100 μL of samples were taken from the BL pool at 30 and 60 min, and the same volume of blank PBS was added. The fluorescence intensity was detected by a microplate reader under the conditions of excitation wavelength 427 nm and emission wavelength 536 nm. The concentration of FITC-dextran in the transport solution on the BL side and Papp were calculated according to the standard curve and regression equation to verify the integrity of the Caco-2 cell monolayer. Papp = (dQ / dt) / (AC0), where dQ / dt is the amount of FITC dextran transported per unit time (μg / s), A is the effective membrane area (0.33 cm 2 ), C0 is the initial concentration (20 μg / mL).

[0082] (3) Triglyceride extraction The chloroform-methanol system was used to extract lipids in the cells and the lower chamber culture medium, that is, the cells and the lower chamber culture medium on the Transwell surface were collected, and 2 ml of chloroform: methanol: water (2:1:1) was added thereto, and centrifuged at 8000 rpm, 4 degrees for 10 min; the lower organic phase was aspirated; 50 ul of formic acid and 1 ml of a mixture (chloroform: methanol: water) were added to the remaining liquid, vortexed, centrifuged at 8000 rpm, 4 degrees for 10 min, and the lower organic phase was aspirated; the lower organic phases aspirated twice were mixed and dried; 100 ul of a mixture (chloroform: methanol 2:1) was added for re-dissolution, and the amount of triglycerides contained was detected using the TG kit of Nanjing Jiancheng Biotechnology Co., Ltd.

[0083] (4) Oil Red O staining / BODIPY staining After Caco-2 cells were treated with 250 μM oleic acid or different polysaccharide components, the original culture medium was discarded and washed with PBS; 4% paraformaldehyde solution was added for fixation at room temperature for 15 min, washed with PBS, and the cells were infiltrated with isopropanol for 30 s. Then, 0.3% Oil Red O / 1 μg / mL BODIPY dye solution was added for staining in the dark for 15 min. After staining, the dye solution was discarded, and isopropanol or acidic PBS was added to wash away the residual Oil Red or residual BODIPY dye. The average area of ​​Oil Red O and BODIPY in the cells was observed under a microscope.

[0084] (5) Cell surface biotinylation Caco-2 cells were plated in 6-well plates and treated with 250 μM oleic acid or 50 μg / mL polysaccharide A for 4, 2, 1, 0.5, 0.25, and 0 h, and then washed with pre-cooled PBS to remove the residual culture medium. Biotin solution (0.5 mg / mL) was added to the cells and incubated on ice for 30 min. The cells were then lysed after washing away the residual biotin with acidic PBS, and cell membrane proteins were extracted using streptavidin-avidin receptor magnetic beads. The effect of A on the internalization of CD36 in Caco-2 cells was detected by SDS-PAGE gel electrophoresis.

[0085] (6) Cell immunofluorescence The same cell administration method as in "(6) Cell surface biotinylation" was used to intervene the Caco-2 cells plated on 24-well plates. After the intervention, the original culture medium was discarded, and 4% paraformaldehyde solution was added to fix the cells for 15 minutes. The blocking solution (PBS solution containing 5% goat serum and 0.3% BSA) was added and blocked at room temperature for 1 hour. CD36 (1:1000) and CAV-1 (1:2000) were incubated overnight, and the primary antibody was discarded and the secondary antibody was incubated at room temperature for 1 hour. The residual primary antibody was washed and the secondary antibody was incubated in the dark for 1 hour. After the incubation, the cells were washed and the nuclei were stained with DAPI. The images were collected under a microscope and the expression level of CD36 on the membrane was counted.

[0086] Fig.10 The figure shows that EGP-A1 inhibits the accumulation of lipids in Caco-2 cells induced by oleic acid. A shows BODIPY and Oil Red O staining after incubation of Caco-2 cells with 250 μM OA or EGP-A1 for 24 h; B and C show the statistics of the average fluorescence intensity of BODIPY and the average Oil Red area in the cells; D shows the quantitative results of intracellular TG, and the intracellular triglyceride level is analyzed by comparing the triglyceride content per microgram of total protein in the cell lysate of each group.

[0087] BODIPY and Oil Red O staining showed that lipid accumulation in the 250 μM OA-treated group increased significantly compared with the control group. EGP-A1 significantly reduced lipid accumulation in the cells compared with the OA-treated group. At the same time, the content of total triglyceride (TG) in the cells was detected. Fig.10 (D) Consistent with the staining results, the TG level in the 250 μM OA-treated group was significantly increased compared with the control group. Compared with the OA-treated group, EGP-A1 significantly reduced the TG content in the cells.

[0088] To evaluate the inhibitory effect of EGP-A1 on lipid accumulation in Caco-2 cells after oleic acid treatment, we first selected 1 / 10 / 100 μg / mL EGP-A1, which did not affect the viability of Caco-2 cells, and co-incubated Caco-2 cells with 250 μM oleic acid (OA) for 24 h ( Fig.11 ). Fig.11 Figure 1 is an analysis of the anti-lipid activity of EGP-A1. A is the Oil Red O staining result of HEGP2 cells incubated with 100 μM OA and 200 μM PA with 1, 10, and 100 μg / mL EGP-A1 for 24 hours; B is the quantitative result of intracellular TG; C is the fluorescence quantitative qPCR result of RAW264.7 cells treated with 50 μg / mL Ox-LDL or EGP-A1 for 24 hours.

[0089] To verify the lipid-lowering effect of EGP-A1, HEGP2 hepatocytes treated with palmitic acid (PA) and RAW264.7 cells treated with OX-LDL were co-incubated with EGP-A1 and OA ( Fig.11 A in Fig.11 B in Fig.11C), Oil Red O staining and intracellular TG content detection results showed that compared with the control group, the OA+PA treatment group significantly increased the lipid level in HEGP2 cells. Compared with the OA+PA group, the EGP-A1 group could significantly reduce the accumulation of lipids in HEGP2 cells. Fluorescence quantitative qPCR results showed that it could reduce the expression of lipid efflux gene ABCG1 in RAW264.7 cells, and reduce the expression of CD36 and SRA1 related to lipid absorption. The above results suggest that EGP-A1 has the effect of reducing cellular lipid accumulation.

[0090] The above experimental results show that EGP-A1 has good lipid-lowering activity, can protect blood lipids and cardiovascular health, and has good medicinal prospects in the preparation of lipid-lowering drugs.

[0091] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention are all within the scope of the present invention.

Claims

1. Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function, characterized by: The Tibetan medicine green radish flower polysaccharide A1 comprises arabinose, galactose, galacturonic acid, rhamnose and glucose components.

2. The Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 1, characterized in that: The structural formula of the Tibetan medicine green radish flower polysaccharide A1 is as follows: 。 3. The Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 1, characterized in that: The Tibetan medicine green radish flower polysaccharide A1 contains, in mol%, 55-60% arabinose, 15-20% galactose, 5-10% galacturonic acid, 5-10% rhamnose and 3-5% glucose.

4. A method for preparing the Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function, characterized in that: The following steps are involved: S1 uses water extraction and alcohol precipitation method to extract and prepare the total polysaccharide of the Tibetan medicine Scindapsus flower; S2 uses anion exchange chromatography technology to separate and purify the total polysaccharide of the Tibetan medicine Epimedium flower to obtain EGP-A, and uses gel column chromatography technology to further separate and purify EGP-A to obtain EGP-A1.

5. The method for preparing the Tibetan medicine scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 4, characterized in that: The water extraction and alcohol precipitation method described in step S1 is used to extract and prepare the total polysaccharides of the Tibetan medicine green radish flower, specifically: take the Tibetan medicine green radish flower, add pure water to reflux and extract, concentrate the obtained extract, add ethanol, stir and stand, filter out the ethanol solution, collect the precipitate, dry it and dissolve it in water, then add Savege reagent to remove the protein, collect the supernatant and concentrate and freeze-dry it to obtain the total polysaccharides of the Tibetan medicine green radish flower.

6. The method for preparing the Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 4, characterized in that: The separation and purification of the total polysaccharide of the Tibetan medicine scindapsus flower to obtain EGP-A described in step S2 includes the steps of separating the polysaccharide with an ion exchange column, drawing an elution curve of the ion exchange column, and dialysis desalination.

7. The method for preparing the Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 4, characterized in that: The gel column chromatography technique described in step S2 is used to further separate and purify EGP-A to obtain EGP-A1, specifically: weigh EGP-A, add ultrapure water to fully dissolve it, absorb the EGP-A supernatant solution after rotary centrifugation, slowly add it to dextran gel G-200, elute with deionized water, use phenol-sulfuric acid method to detect the whole process, collect the main elution peak, and perform segmented separation after detection to obtain EGP-A1.

8. The method for preparing the Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 5, characterized in that: The Tibetan medicine green radish flower is added with pure water for reflux extraction as follows: take the Tibetan medicine green radish flower and add pure water according to the solid-liquid ratio of 1:10-30, reflux and extract at 90-100°C for 2-4 times, 3-5 h / time, and combine the extracts; the concentration is reduced pressure vacuum concentration; the drying is reduced pressure vacuum drying; the addition of ethanol, stirring and standing is as follows: add 5-15 times the volume of anhydrous ethanol, stir thoroughly and stand at 2-6°C for 12-36h; the volume ratio of the Savege reagent is chloroform: n-butanol = 4:

1.

9. The method for preparing the Tibetan medicine Scindapsus scindapsus flower polysaccharide A1 with lipid-lowering function according to claim 6, characterized in that: The ion exchange column polysaccharide separation method comprises the following steps: weighing the total polysaccharide of the Tibetan medicine green radish flower obtained in step S1, adding pure water to fully dissolve it, slowly adding it to the anion exchange fiber column, adsorbing it overnight, and gradient eluting it with ultrapure water and sodium chloride solutions of different concentrations; the drawing of the ion exchange column elution curve comprises the following steps: collecting the eluate, determining the absorbance of each tube of the eluate by the phenol-sulfuric acid method, drawing the anion exchange cellulose chromatography column elution curve of the green radish flower polysaccharide with the tube number as the X-axis and the absorbance as the Y-axis; the dialysis desalination method comprises the following steps: collecting the main elution peaks at each concentration, concentrating it under reduced pressure, pouring the concentrated solution into a dialysis bag, placing it in ultrapure water for magnetic stirring dialysis, collecting the dialyzed solution, and freeze-drying it to obtain the elution components, and the component obtained from the ultrapure water eluate is named EGP-A.

10. Application of Tibetan medicine green radish flower polysaccharide A1 with lipid-lowering function, characterized in that: The Tibetan medicine scindapsus flower polysaccharide A1 component is used to prepare lipid-lowering drugs.

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