Application of methyl esterification substituted acid lycium ruthenicum polysaccharide extract in prevention and treatment of diabetic nephropathy

Abstract

The invention discloses application of a methyl esterification substituted acid lycium ruthenicum polysaccharide extract in prevention and treatment of diabetic nephropathy, and belongs to the field of biological medicine. Compared with lycium barbarum polysaccharide LBPS which is not substituted by methyl esterification, the methyl esterified lycium barbarum polysaccharide extract provided by the invention has higher activity, and intervenes different links of DKD, including inhibition of proliferation of mesangial cells, oxidative damage, inflammation and the like. The comprehensive effect is particularly important for complicated and multi-factor diseases such as diabetic nephropathy, the methyl esterification substituted acidic LRPS can radically relieve the damage of high glucose to the kidney by reconstructing the steady state of mesangial cells, and a new strategy is provided for the treatment of diabetic nephropathy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and relates to application of a methyl-esterified substituted acidic black wolfberry polysaccharide extract in prevention and treatment of diabetic nephropathy. BACKGROUND

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease caused by diabetes. According to incomplete statistics, about 40% of diabetic patients can develop DKD. The latest research shows that DKD has replaced primary glomerular disease to become the primary cause of chronic kidney disease (CKD) in China, and is also the main cause of end-stage renal disease (ESRD). Therefore, early intervention of DKD has become the focus of attention in the field of kidney disease. Glomerular mesangial cells (GMC) are intrinsic cells of the kidney, which are distributed in the glomerular capillary and form a functional unit with adjacent podocytes and glomerular endothelial cells, and regulate glomerular filtration by performing functions such as contraction, phagocytosis and maintenance of normal matrix metabolism. In the process of DKD, high blood sugar increases the advanced glycation end products (AGE), which causes glomerular and renal function damage. AGE is formed by catalytic and non-enzymatic reaction of amino acids derived from proteins, fats and nucleic acids and reducing sugars, and mesangial cells can uptake and degrade AGE when synthesizing and secreting matrix components. The increase of AGE concentration promotes the overexpression of TGF-β1 and CTGF, stimulates the excessive synthesis of collagen matrix components and GBM thickening, causes mesangial proliferation and glomerular sclerosis, and leads to loss of glomerular function.

[0003] In the treatment of diabetic nephropathy, inhibiting the excessive proliferation of mesangial cells is one of the key strategies to delay the progression of the disease. Traditional drugs such as angiotensin-converting enzyme inhibitors have certain effects, but are often accompanied by adverse reactions. As a natural active ingredient, plant polysaccharides have the characteristics of multi-target and low toxicity, and can effectively block the high glucose-induced proliferation of mesangial cells through multiple mechanisms, providing a new intervention strategy for the prevention and treatment of diabetic nephropathy and showing broad prospects. Ganoderma lucidum polysaccharides can reduce the excessive glycogen deposition of diabetic mice, alleviate the excessive glucose metabolism in the glomerular region, and effectively inhibit the abnormal proliferation of mesangial cells induced by high glucose. Cistanche polysaccharides can activate the TGFβ / Smad signal, down-regulate the expression of type IV collagen and fibronectin, and improve the accumulation of renal lipids and glomerular fibrosis in the kidney tissue of diabetic mice. Xiyu crude polysaccharides can inhibit the proliferation of human glomerular mesangial cells induced by high glucose, the expression of inflammatory factors and fibrosis, and the inhibition tendency is concentration-dependent.

[0004] Black wolfberry (Lycium ruthenicum Murr), a traditional and precious Chinese medicinal material, primarily grows in areas such as the Qinghai-Tibet Plateau. Modern research indicates that black wolfberry polysaccharides (LRPS) are one of the most important active ingredients in black wolfberry, possessing a complex chemical structure and diverse biological activities. Existing literature reports that extracted LRPS have a wide molecular weight distribution, ranging from 17 to 2650 kDa, and are composed of a heteropolysaccharide composed of three or more monosaccharides, such as L-rhamnose (Rha), L-arabinose (Ara), D-glucose (Glc), D-galactose (Gal), D-galacturonic acid (GalA), and D-glucuronic acid (GlcA).

[0005] There are already literature reports that Lycium barbarum polysaccharides have an alleviating effect on diabetic nephropathy, but how to obtain a polysaccharide with better effects and more comprehensive functions to alleviate diabetic nephropathy has become a difficulty and hot topic in research. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of an acidic black wolfberry polysaccharide extract with methyl ester substitution, which can effectively prevent and treat diabetic nephropathy and effectively inhibit high glucose-induced mesangial cell proliferation and inflammatory changes.

[0007] By improving the extraction method, the technicians of this study obtained a methyl ester-substituted acidic black wolfberry polysaccharide extract LRPS with a unique structural composition:

[0008] ① It is a mixed polysaccharide composed of three single polysaccharides with molecular weights of 1692kD, 57kD and 7.9kD respectively;

[0009] ②The monosaccharide composition is D-mannose (Man) (9.329%), D-glucosamine (GlcN) (0.698%), L-rhamnose (Rha) (4.542%), D-glucuronic acid (GlcA) (6.424%), D-galacturonic acid (GalA) (6.424%), D-glucose (Glc) (8.322%), D-galactose (Gal) (8.322%), D-xylose (Xyl) (2.658%) and L-arabinose (Ara) (24.621%);

[0010] LRPS infrared analysis of the methylated black wolfberry polysaccharide extract indicated that the polysaccharide sample was an acidic polysaccharide with methylation. Methylated polysaccharides are polysaccharides in which the carboxyl groups of the uronic acid units are substituted with methyl groups. They are widely found in the pectin component of plant cell walls. Methylation significantly alters the physicochemical properties of polysaccharides. With increasing methylation, the lipophilicity of the polysaccharide increases, intra- and intermolecular electrostatic repulsions decrease, and the molecular conformation becomes more compact. These changes directly affect the solubility, viscosity, and gelation behavior of the polysaccharide. The activity of the black wolfberry polysaccharide extract obtained in this study is comparable to that reported in the literature. Its novel polysaccharide structure offers unique advantages in the treatment of diabetic nephropathy. Lycium barbarum polysaccharide (LBPS) reported in previous literature and patents does not have methylation. The acidic black wolfberry polysaccharide with methylation obtained in this study exhibits higher activity than its unmethylated counterpart and intervenes in various aspects of diabetic nephropathy, including inhibiting mesangial cell proliferation, oxidative damage, and inflammation. This combined effect is particularly important for complex and multifactorial diseases such as diabetic nephropathy. Methylated acidic LRPS can fundamentally reduce the damage of high sugar to the kidneys by reestablishing the homeostasis of mesangial cells, providing a new strategy for the treatment of diabetic nephropathy.

[0011] In order to achieve the above object, the present invention adopts the following technical solutions:

[0012] The invention provides a methyl ester-substituted black wolfberry polysaccharide extract.

[0013] In one embodiment of the present invention, the methyl-esterified substituted Lycium ruthenicum polysaccharide extract is prepared according to the following method:

[0014] (1) After grinding the black wolfberry powder, adding boiling water, stirring, ultrasonicating, and centrifuging, taking the supernatant, extracting the filter residue again, and combining the two supernatants; preferably, adding ultrapure boiling water at a material-liquid ratio of 10 to 20 mL per gram of black wolfberry powder;

[0015] Preferably, the ultrasonic power is 900-1000W, 40-45°C;

[0016] Preferably, the centrifugation condition is 10000-15000 rpm for 15-25 minutes;

[0017] Preferably, the conditions for the second extraction are the same as those for the first extraction;

[0018] (2) concentrating the supernatant obtained in step (1) to 1 / 3 to 1 / 2 of the original volume to obtain a crude extract of Lycium ruthenicum;

[0019] Preferably, the concentration is carried out in a rotary evaporator at 50°C;

[0020] (3) adding 8% to 12% (w / v) trichloroacetic acid solution to the crude extract of Lycium ruthenicum Murr obtained in step (2), standing for 3 to 4 hours, centrifuging, taking the supernatant, and repeatedly extracting the precipitate three times according to the above method, and combining the obtained supernatant;

[0021] Preferably, the w / v is g / 10ml;

[0022] Preferably, the centrifuging condition is centrifuging at 4 to 5°C and 8000 to 9000 rpm for 15 to 20 min.

[0023] (4) adding 3 to 4 times volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, standing for 8 to 12 hours (preferably standing for 12 hours in a 4°C refrigerator), centrifuging the solution after standing, discarding the supernatant, washing the filter residue, dissolving, and using a dialysis bag with a molecular weight cut-off of 2.5 to 3 kDa to dialyze for 48 to 72 hours, and freeze-drying to obtain the methyl ester-substituted Lycium ruthenicum Murr polysaccharide extract;

[0024] Preferably, the centrifuging condition is centrifuging at 4 to 5°C and 8000 to 9000 rpm for 15 to 20 min.

[0025] In an embodiment of the present application, the Lycium ruthenicum Murr polysaccharide extract is a mixed polysaccharide composed of three single polysaccharides with molecular weights of 1692 kD, 57 kD and 7.9 kD, respectively.

[0026] In an embodiment of the present application, the monosaccharide composition of the Lycium ruthenicum Murr polysaccharide extract is D-mannose (Man) (9.329%), D-glucosamine (GlcN) (0.698%), L-rhamnose (Rha) (4.542%), D-glucuronic acid (GlcA) (6.424%), D-galacturonic acid (GalA) (6.424%), D-glucose (Glc) (8.322%), D-galactose (Gal) (8.322%), D-xylose (Xyl) (2.658%) and L-arabinose (Ara) (24.621%).

[0027] In an embodiment of the present application, the Lycium ruthenicum Murr polysaccharide extract is an acidic polysaccharide with methyl ester substitution.

[0028] The present application provides the use of the above-mentioned methyl ester-substituted Lycium ruthenicum Murr polysaccharide extract in the preparation of a medicament for treating diabetic nephropathy.

[0029] In one embodiment of the present invention, the black wolfberry polysaccharide extract has the following characteristics at the cellular level: inhibiting high glucose-induced mesangial cell proliferation; inhibiting high glucose-induced inflammatory response of mesangial cells; inhibiting high glucose-induced oxidative damage to mesangial cells; and is more active than non-methylated wolfberry polysaccharide (LBPS, Shanghai Yuanye Biotechnology Co., Ltd.).

[0030] In one embodiment of the present invention, the black wolfberry polysaccharide extract has the following characteristics at the animal level: significantly improving renal function-related indicators (urine protein); improving glomerular hypertrophy, mesangial area widening and glomerular basement membrane thickening (pathology).

[0031] The present invention also provides a use of a methyl-esterified substituted Lycium ruthenicum polysaccharide extract in the preparation of a product for preventing, improving and / or treating diabetic nephropathy.

[0032] In one embodiment of the present invention, the prevention, improvement and / or treatment of diabetic nephropathy includes at least one of the following:

[0033] (a) Inhibits high glucose-induced mesangial cell proliferation, inflammatory response, and oxidative damage;

[0034] (b) Improve abnormal weight gain caused by diabetes;

[0035] (c) improving individual renal function-related indicators;

[0036] (d) Improve the degree of glomerular hypertrophy, mesangial area widening and glomerular basement membrane thickening in individuals.

[0037] In one embodiment of the present invention, the product includes but is not limited to one or more of medicines, food, feed, and feed additives.

[0038] In one embodiment of the present invention, the dosage form of the drug is a liquid preparation or a solid preparation.

[0039] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

[0040] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0041] In one embodiment of the present invention, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0042] In one embodiment of the present application, the food product comprises a health food product, a special medical purpose food product, a functional food product, or a pet food product;

[0043] In one embodiment of the present application, the food product comprises a grain product, a starch product, a vegetable product, a fruit product, a meat product, a poultry product, an egg product, a dairy product.

[0044] In one embodiment of the present application, the methyl ester substituted Lycium ruthenicum polysaccharide extract is prepared according to the following method:

[0045] (1) After the Lycium ruthenicum powder is crushed, boiling water is added, and after stirring, ultrasonic treatment, and centrifugation, the supernatant is taken, the filter residue is extracted again, and the two supernatants are combined; preferably, 10-20 mL of the feed liquid ratio of ultrapure boiling water is added per gram of Lycium ruthenicum powder;

[0046] Preferably, the ultrasonic power is 900-1000 W, and the temperature is 40-45°C;

[0047] Preferably, the centrifugation conditions are 10000-15000 rpm for 15-25 minutes;

[0048] Preferably, the re-extraction conditions are the same as the first extraction;

[0049] (2) The supernatant obtained in step (1) is concentrated to 1 / 3-1 / 2 of the original volume to obtain a Lycium ruthenicum crude extract;

[0050] Preferably, the concentration is performed in a rotary evaporator at 50°C;

[0051] (3) 8%-12% (w / v) trichloroacetic acid solution is added to the Lycium ruthenicum crude extract obtained in step (2), and after standing for 3-4 hours, centrifugation is performed, the supernatant is taken, and the precipitate is repeatedly extracted three times according to the above method, and the obtained supernatants are combined;

[0052] Preferably, the w / v is g / 10 ml;

[0053] Preferably, the centrifugation conditions are 8000-9000 rpm for 15-20 minutes at 4-5°C;

[0054] (4) 3-4 times the volume of anhydrous ethanol is added to the supernatant obtained in step (3) for alcohol precipitation, and after standing for 8-12 hours (preferably standing in a 4°C refrigerator for 12 hours), the solution after standing is centrifuged, the supernatant is discarded, the filter residue is washed, dissolved, and dialyzed using a dialysis bag with a molecular weight cut-off of 2.5-3 kDa for 48-72 hours, and then freeze-dried to obtain the methyl ester substituted Lycium ruthenicum polysaccharide extract;

[0055] Preferably, the centrifugation is performed at 4-5°C and 8000-9000 rpm for 15-20 min.

[0056] The present invention also provides a medicine for preventing, improving and / or treating diabetic nephropathy, wherein the medicine comprises a methyl-esterified substituted Lycium ruthenicum polysaccharide extract.

[0057] In one embodiment of the present invention, the dosage form of the drug is a liquid preparation or a solid preparation.

[0058] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

[0059] In one embodiment of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0060] In one embodiment of the present invention, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

[0061] In one embodiment of the present invention, the methyl-esterified substituted Lycium ruthenicum polysaccharide extract is prepared according to the following method:

[0062] (1) After crushing the black wolfberry powder, adding boiling water, stirring, ultrasonicating, and centrifuging, taking the supernatant, extracting the filter residue again, and combining the two supernatants; preferably, adding ultrapure boiling water at a material-liquid ratio of 10 to 20 mL per gram of black wolfberry powder;

[0063] Preferably, the ultrasonic power is 900-1000W, 40-45°C;

[0064] Preferably, the centrifugation condition is 10000-15000 rpm for 15-25 minutes;

[0065] Preferably, the conditions for the second extraction are the same as those for the first extraction;

[0066] (2) concentrating the supernatant obtained in step (1) to 1 / 3 to 1 / 2 of the original volume to obtain a crude extract of Lycium ruthenicum;

[0067] Preferably, the concentration is carried out in a rotary evaporator at 50°C;

[0068] (3) adding 8% to 12% (w / v) trichloroacetic acid solution to the crude extract of Lycium barbarum obtained in step (2), letting it stand for 3 to 4 hours, centrifuging, taking the supernatant, and repeatedly extracting the precipitate three times according to the above method, and combining the obtained supernatants;

[0069] Preferably, the w / v is g / 10ml;

[0070] Preferably, the centrifugation is carried out at 4-5°C and 8000-9000 rpm for 15-20 min;

[0071] (4) adding 3 to 4 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, standing for 8 to 12 hours (preferably standing in a 4°C refrigerator for 12 hours), centrifuging the solution after standing, discarding the supernatant, washing the filter residue, dissolving it, and dialyzing it using a dialysis bag with a molecular weight cutoff of 2.5 to 3 kDa for 48 to 72 hours, and freeze-drying to obtain a methylated ester-substituted black fruit wolfberry polysaccharide extract;

[0072] Preferably, the centrifugation is performed at 4-5°C and 8000-9000 rpm for 15-20 min.

[0073] The present invention also provides the use of the methyl-esterified substituted Lycium ruthenicum polysaccharide extract in the preparation of a medicament for preventing, improving and / or treating diabetic nephropathy. It should be understood that the above general description and the detailed description below are merely exemplary and explanatory and do not limit the application of the present invention.

[0074] Beneficial effects

[0075] The present invention provides a novel acidic black wolfberry polysaccharide extract with methyl ester substitution, which has a more significant effect on preventing diabetic nephropathy and delaying disease progression than non-methyl esterified wolfberry polysaccharides.

[0076] (1) Ultrasound-assisted water extraction and alcohol precipitation were used to extract black wolfberry polysaccharide to obtain a new acidic polysaccharide with methyl ester substitution;

[0077] (2) The extract of black wolfberry polysaccharide (methyl esterification) has an inhibitory effect on the proliferation of mesangial cells under high glucose and is more effective than the wolfberry polysaccharide (unmethyl esterification);

[0078] (3) The extract of black wolfberry polysaccharide (methyl ester substitution) has an inhibitory effect on oxidative damage of mesangial cells under high glucose and is more effective than wolfberry polysaccharide (non-methyl ester substitution);

[0079] (4) Black wolfberry polysaccharide extract (methyl ester substitution) has an inhibitory effect on the inflammatory response of mesangial cells under high glucose and is more effective than wolfberry polysaccharide (unmethyl ester substitution);

[0080] (5) Black wolfberry polysaccharide extract (methyl ester substitution) can significantly improve the abnormal weight gain caused by diabetes after 8 weeks of intervention;

[0081] (5) After 8 weeks of intervention with black wolfberry polysaccharide extract (methyl ester substitution), the 24-hour urine microalbumin level in DKD mice was significantly reduced;

[0082] (6) After 8 weeks of intervention with black wolfberry polysaccharide extract (methyl ester substitution), the pathological changes of DKD mice, such as glomerular hypertrophy, mesangial area widening, glomerular basement membrane thickening, and renal interstitial fibrosis, were reversed to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 : Polysaccharide composition and molecular weight of black wolfberry polysaccharide extract.

[0084] Figure 2 : Infrared spectrum of black wolfberry polysaccharide extract.

[0085] Figure 3 : Inhibitory effects of black wolfberry polysaccharide extract (methyl esterification) and wolfberry polysaccharide (non-methyl esterification) on mesangial cell proliferation under high glucose.

[0086] Figure 4 : Inhibitory effects of black wolfberry polysaccharide extract (methyl esterification) and wolfberry polysaccharide (non-methyl esterification) on oxidative damage of mesangial cells under high glucose.

[0087] Figure 5 : Inhibitory effects of black wolfberry polysaccharide extract (methyl esterification) and wolfberry polysaccharide (non-methyl esterification) on inflammatory changes of mesangial cells under high glucose.

[0088] Figure 6 : Improvement effect of black wolfberry polysaccharide extract (methyl ester substitution) on diabetic symptoms in db / db mice.

[0089] Figure 7 : Effects of black wolfberry polysaccharide extract (methyl ester substitution) on renal function in DKD mice.

[0090] Figure 8 : Effects of black wolfberry polysaccharide extract (methyl ester substitution) on renal pathology in DKD mice. DETAILED DESCRIPTION

[0091] The sources of the raw materials and reagents involved in the following examples are as follows:

[0092] Black wolfberry (from Qinghai Province) was purchased from Zaokang Wolfberry Co., Ltd. Bleomycin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. SPF-grade db / db and db / m mice (20 ± 2 g, 4 weeks old) were purchased from Weitong Lihua Animal Research Center (Beijing, China); mouse glomerular mesangial cells SV40-Mes13 were purchased from Suzhou Haixing Biotechnology Co., Ltd. DMEM / F12 medium was purchased from Nanjing Shenghang Biotechnology Co., Ltd. Fetal bovine serum was purchased from Thermo Fisher Scientific (China) Co., Ltd. Xylene, ethanol, and paraffin were purchased from Sinopharm Chemical Reagent Co., Ltd. Phosphate-buffered saline and trypsin were purchased from Wuhan Shangen Biotechnology Co., Ltd. Glucose was purchased from Sigma. MTT assay kits and H&E, PAS, and Masson staining kits were purchased from Beijing Solebao Biotechnology Co., Ltd. Hydrogen peroxide (H2O2) kit, reactive oxygen species (ROS) kit, mouse interleukin-1β (IL-1β) kit, and mouse tumor necrosis factor (TNF-α) kit were purchased from Biyuntian Biotechnology Co., Ltd.

[0093] Lycium barbarum polysaccharides were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0094] The detection methods involved in the following embodiments are as follows:

[0095] Calculation method of cell viability:

[0096] Dehydrogenases within the mitochondria of living cells reduce water-soluble tetrazolium salts to water-insoluble blue-purple formazan crystals (MTT). The amount of formazan produced is proportional to the number of living cells and their metabolic activity. The number of living cells can be estimated by measuring the absorbance (OD) of the solution containing dissolved formazan.

[0097] Reactive oxygen species detection:

[0098] The intracellular reactive oxygen species detection kit utilizes the fluorescent probe DCFH-DA for reactive oxygen species detection. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by esterases within the cell to produce DCFH. DCFH cannot penetrate the cell membrane, making it easy for the probe to be loaded into the cell. Intracellular reactive oxygen species can oxidize the non-fluorescent DCFH to produce fluorescent DCF. Measuring the fluorescence of DCF provides an indication of the intracellular reactive oxygen species level.

[0099] Hydrogen peroxide detection:

[0100] The hydrogen peroxide detection kit oxidizes divalent iron ions with H2O2 to produce trivalent iron ions, which then react with xylenolorange in a specific solution to form a purple product, thereby determining the H2O2 concentration.

[0101] Example 1: Preparation and structural identification of methyl-esterified substituted Lycium ruthenicum polysaccharide extract

[0102] 1. Preparation of methyl-esterified substituted Lycium ruthenicum polysaccharide extract

[0103] (1) A suitable amount of Lycium ruthenicum powder was ground into powder and passed through an 80-mesh sieve. Ultra-pure boiling water (100°C) was added at a ratio of 10 mL / g, and the mixture was stirred uniformly and extracted under ultrasonic conditions at 900 W and 40°C for 60 min. The mixture was then centrifuged at 10,000 rpm for 15 min to obtain a supernatant and a precipitate.

[0104] (2) The precipitate obtained in step (1) was added with ultra-pure boiling water at a ratio of 10 mL / g, and the mixture was stirred uniformly and extracted under ultrasonic conditions at 900 W and 40°C for 60 min. The mixture was then centrifuged at 10,000 rpm for 15 min, and the supernatant was taken and the precipitate was discarded.

[0105] (3) The supernatants obtained in steps (1) and (2) were combined and concentrated to 1 / 3 of the original volume in a rotary evaporator at 50°C to obtain a Lycium ruthenicum crude extract.

[0106] (4) A 10% (10 g / 100 ml) trichloroacetic acid solution was added to the Lycium ruthenicum crude extract obtained in step (3) to remove proteins. After standing for 4 h, the mixture was centrifuged at 8000 rpm and 4°C for 15 min, and the supernatant was taken. The precipitate was repeatedly extracted three times according to the above method, and the obtained supernatants were combined.

[0107] (5) The supernatant obtained in step (4) was treated with 4 volumes of anhydrous ethanol for alcohol precipitation, and the solution was placed in a 4°C refrigerator for 12 h. After centrifugation at 8000 rpm and 4°C for 10 min, the supernatant was discarded, and the filter residue was washed with anhydrous ethanol three times. The residue was re-dissolved in purified water and dialyzed for 48 h using a dialysis bag with a molecular weight cut-off of 3 kDa. After freeze-drying, the LRPS sample was obtained.

[0108] 2. Characterization of methyl-esterified substituted Lycium ruthenicum polysaccharide extract Figures 1-2 )

[0109] (1) The molecular weight and distribution of the polysaccharide were determined by high-performance gel permeation chromatography (HPGPC).

[0110] ① Establishment of molecular weight calibration curve: Accurately weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards), add 0.05M NaCl solution to prepare 5 mg / ml dextran standard solution, filter with 0.22μm microporous filter membrane for later use, adopt HPGPC method, use high performance gel permeation chromatography tandem columns for detection, use the logarithm of the relative molecular mass of the standard as the ordinate, and use the retention time of the corresponding chromatographic peak as the abscissa for linear regression to obtain the calibration curve.

[0111] ② Sample Solution Preparation: Accurately weigh 5 mg of LRPS sample and add 1 ml of 0.05 M NaCl solution to the sample to prepare a 5 mg / ml test sample solution. Centrifuge at 8000 rpm for 10 min. Filter the supernatant through a 0.22 μm microporous filter membrane. Transfer the sample to a 2 ml injection vial for later use. Chromatographic Method: HPGPC was used, with detection performed using high-performance gel permeation chromatography (HPLC) columns in series. Instrument: Waters HPLC; Detector: Waters differential detector; Column: Two polymer-based aqueous SEC (GFC) columns (8 × 300 mm) connected in series; Mobile phase: 0.05 M NaCl solution; Flow rate: 0.50 ml / min; Column temperature: 40°C; Injection volume: 30 μl.

[0112] The experimental results showed that it was a mixed polysaccharide composed of three single polysaccharides with molecular weights of 1692kD, 57kD and 7.9kD respectively.

[0113] Table 1: Molecular weight results

[0114]

[0115] Note: The peak near 42.2 min is the mobile phase salt peak. The peak after the mobile phase salt peak may contain small molecules such as oligosaccharides.

[0116] (2) Determine the monosaccharide composition of black wolfberry polysaccharide.

[0117] Weigh 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose, dissolve, and dilute to 10 ml in a volumetric flask to prepare a standard stock solution. Dilute each solution to the following gradient dilutions, filter through a 0.22 μm microporous filter, and transfer to a sample vial.

[0118] Table 2: Monosaccharide standard mixture gradient concentration information

[0119]

[0120] ① Preparation of sample solution

[0121] Solid sample extraction: Take a clean chromatographic flask, accurately weigh 5 mg (±0.05 mg) of polysaccharide sample, add 1 mL of 2M TFA acid solution, heat at 121°C for 2 hours. Blow nitrogen, dry. Add 3 mL of methanol, dry again, repeat the methanol washing 2-3 times. Add 1 mL of sterile water to dissolve, transfer into a chromatographic flask for testing.

[0122] ② PMP derivatization: Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolysate in a stoppered eppendorf tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution, 0.5 mL of 0.5 mol / L PMP methanol solution, vortex mix, then react in a 70°C water bath for 1 hour. After the reaction is completed, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add chloroform 1 mL and vortex extract 3 times to remove excess PMP. After discarding the chloroform layer, take 0.3 mL and add water to 1 mL.

[0123] The experimental results show that the monosaccharide composition is D-mannose (Man) (9.329%), D-glucosamine (GlcN) (0.698%), L-rhamnose (Rha) (4.542%), D-glucuronic acid (GlcA) (6.424%), D-galacturonic acid (GalA) (6.424%), D-glucose (Glc) (8.322%), D-galactose (Gal) (8.322%), D-xylose (Xyl) (2.658%), and L-arabinose (Ara) (24.621%).

[0124] (3) Infrared spectrum determination of polysaccharide

[0125] Weigh 1-2 mg of each dried polysaccharide sample in a mortar, add 200 mg of KBr powder respectively, grind uniformly, press the tablet, use Fourier transform micro infrared spectrometer to scan the sample, wavelength range 4000-400 cm -1 , record the infrared spectrum.

[0126] The results show that the infrared spectrum shows the typical characteristics of polysaccharide substances. As can be seen from the figure, a wide and strong absorption peak appears at 3420 cm -1 , which is the stretching vibration of O-H of sugar, a smaller absorption peak appears at 2933 cm -1 , which is the stretching vibration of methyl or methylene C-H, the peak between 1400-1200 cm -1 should be the C-H variable angle vibration of sugar. From the above, it can be determined that the sample is a polysaccharide substance.

[0127] At 1739cm -1 The absorption peak at 1632 cm-1 can be attributed to the symmetrical stretching vibration absorption peak of C=O of the esterified carboxyl group in galacturonic acid, while the absorption peak at 1632 cm-1 can be attributed to the symmetrical stretching vibration absorption peak of C=O of the esterified carboxyl group in galacturonic acid. -1 The peak at ( ) is the characteristic absorption peak of the asymmetric stretching vibration of the free carboxyl group COO- in galacturonic acid, indicating that the polysaccharide sample has methyl ester substitution.

[0128] The above infrared test results show that the polysaccharide sample is an acidic polysaccharide with methyl ester substitution.

[0129] Example 2: Improvement of the Methylated Black Lycium Barbarum Polysaccharide Extract on Mesangial Cell Proliferation, Oxidative Damage and Inflammation Under High Glucose

[0130] 1. Cell proliferation assay

[0131] The MTT method was used to detect the effect of the methylated black wolfberry polysaccharide extract prepared in Example 1 on the proliferation of mesangial cells (SV40-Mes13 cells) and compare its activity with that of non-methylated black wolfberry polysaccharide.

[0132] The experiment is divided into two parts:

[0133] (1) Cytotoxicity test of black wolfberry polysaccharide and determination of drug concentration

[0134] Blank group Control, black wolfberry polysaccharide 10μg / mL group, black wolfberry polysaccharide 25μg / mL group, black wolfberry polysaccharide 50μg / mL group;

[0135] The experimental steps are as follows:

[0136] 1) 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured. 100 μL of the corresponding drug was then added. After drug addition, the cells were incubated in a 37°C cell culture incubator for 24 h. No drug was added to the blank group.

[0137] 2) Add 10 μL of MTT solution to each well and incubate at 37°C in a cell culture incubator for 4 h;

[0138] 3) Add 100 μL of Formazan solution to each well, mix well, and incubate at 37°C in a cell culture incubator for 3-4 hours. Observe under a conventional optical microscope until the purple formazan crystals have completely dissolved.

[0139] 4) Measure the absorbance at 570 nm.

[0140] The results of toxicity experiments showed that ( Figure 3 A), the methyl ester-substituted black wolfberry polysaccharide extract LRPS alone has no effect on cell proliferation activity.

[0141] (2) Comparison of the effects of methylated and non-methylated black wolfberry polysaccharide extracts on mesangial cell proliferation in polysaccharide systems

[0142] Blank group Control, glucose group HG (the added drug was 25 mM glucose), methyl-esterified black wolfberry polysaccharide group HG+LRPS (the added drug was 25 mM glucose + 25 μg / mL methyl-esterified black wolfberry polysaccharide prepared in Example 1), and wolfberry polysaccharide group HG+LBPS (the added drug was 25 mM glucose + 25 μg / mL wolfberry polysaccharide);

[0143] The experimental steps are as follows:

[0144] 1) 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured with 100 μL of the corresponding drug. After drug addition, the cells were incubated in a 37°C cell culture incubator for 24 h. No drug was added to the blank group.

[0145] 2) Add 10 μL of MTT solution to each well and incubate at 37°C in a cell culture incubator for 4 h;

[0146] 3) Add 100 μL of Formazan solution to each well, mix well, and incubate at 37°C in a cell culture incubator for 3-4 hours. Observe under a conventional optical microscope until the purple formazan crystals have completely dissolved.

[0147] 4) Measure the absorbance at 570 nm.

[0148] The results showed that ( Figure 3 B):

[0149] The survival rates of SV40-Mes13 cells cultured for 24 hours in the four groups of control, HG (Glucose 25mM), HG+LRPS (25μg / mL), and HG+LBPS (25μg / mL) were 99.78%, 129.69%, 105.15% and 115.15%, respectively. It can be seen that after pre-culture with the methylated black wolfberry polysaccharide extract LRPS, the proliferation of SV40-Mes13 cells was inhibited with statistical significance and the activity was significantly better than that of wolfberry polysaccharide LBPS, indicating that the cell proliferation of SV40-Mes13 cells induced by high glucose can be effectively reversed by LRPS (##P<0.01).

[0150] 2. Detection of antioxidant activity

[0151] The detection kit was used to detect the effect of black wolfberry polysaccharide extract on ROS and H2O2 levels in mesangial cells (SV40-Mes13 cells) under high glucose.

[0152] (1) Hydrogen peroxide detection

[0153] The experiment was divided into four groups: blank group Control, glucose group HG (the added drug was 25 mM glucose), Lycium ruthenicum polysaccharide group HG+LRPS (the added drug was 25 mM glucose+25 μg / mL of the methylated substituted Lycium ruthenicum polysaccharide prepared in Example 1), Lycium barbarum polysaccharide group HG+LBPS (the added drug was 25 mM glucose+25 μg / mL of Lycium barbarum polysaccharide);

[0154] Blank group Control, glucose group HG: 100 μL / 5000 cells (SV40-Mes13 cells) were added in each well of a 96-well plate for culture and given 100 μL of corresponding drug stimulation, and after the addition of the drug, the cells were incubated at 37°C in a cell incubator for 24 h, wherein no drug was added in the blank group;

[0155] Lycium ruthenicum polysaccharide group HG+LRPS, Lycium barbarum polysaccharide group HG+LBPS: 100 μL / 5000 cells (SV40-Mes13 cells) were added in each well of a 96-well plate for culture and given 100 μL of methylated substituted Lycium ruthenicum polysaccharide or Lycium barbarum polysaccharide, and after the addition of the drug, the cells were incubated at 37°C in a cell incubator for 24 h, and then high glucose HG was added for induction at 37°C for 24 h;

[0156] The experimental steps after induction were as follows:

[0157] ① The supernatant sample of cells (SV40-Mes13 cells) was centrifuged at 100-500 g for 5 min; the number of pre-coated plate strips required for one experiment was calculated and determined, and the required plate strips were taken out and placed in a 96-well frame;

[0158] ② Standard samples were prepared for each experiment, and a standard curve was drawn, and a background correction hole, i.e., a blank hole, was set.

[0159] ③ The sample or different concentrations of standard samples were added to the corresponding holes at 100 μL / hole, respectively.

[0160] ④ 100 μL of hydrogen peroxide detection reagent was added to each hole.

[0161] ⑤ Gently shake or knock to mix, and place at room temperature for 30 minutes. Then immediately measure A560.

[0162] ⑥ The concentration of hydrogen peroxide in the sample was calculated according to the standard curve.

[0163] (2) Active oxygen detection

[0164] The experiment was divided into four groups: a blank group (Control), a glucose group (HG) (the added drug was 25 mM glucose), a black wolfberry polysaccharide group (HG+LRPS) (the added drug was 25 mM glucose + 25 μg / mL methylated black wolfberry polysaccharide prepared in Example 1), and a wolfberry polysaccharide group (HG+LBPS) (the added drug was 25 mM glucose + 25 μg / mL wolfberry polysaccharide).

[0165] Blank group (Control), glucose group (HG): 100 μL / 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured, followed by 100 μL of the corresponding drug stimulation. After drug addition, the cells were incubated in a cell culture incubator at 37°C for 24 h. No drug was added to the blank group.

[0166] Black wolfberry polysaccharide group HG+LRPS, wolfberry polysaccharide group HG+LBPS: 100 μL / 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured and given 100 μL of methylated black wolfberry polysaccharide or wolfberry polysaccharide. After adding the drug, the cells were incubated at 37°C for 24 hours in a cell culture incubator, and then high-glucose HG was added and induced at 37°C for 24 hours.

[0167] The experimental steps after induction are as follows:

[0168] ① Dilute CM-H2DCFDA with PBS solution to a final concentration of 5 μM;

[0169] ② Remove the cell culture medium, add 1 ml of diluted CM-H2DCFDA, and incubate in a 37°C cell culture incubator for 30 minutes;

[0170] ③ Wash the cells three times with PBS solution to fully remove CM-H2DCFDA that has not entered the cells;

[0171] ④ Stimulate cells with ROS positive control for 30 minutes

[0172] ⑤Use 495nm excitation wavelength and 530nm emission wavelength to detect the intensity of fluorescence before and after stimulation at each time point.

[0173] The results show that ( Figure 4 ):

[0174] The ROS and H2O2 produced by SV40-Mes13 cells in the glucose HG group were significantly higher than those in the Control group, and the difference was statistically significant (**P<0.01).

[0175] In the black wolfberry polysaccharide group HG+LRPS, methylated black wolfberry polysaccharide LRPS was first added to the cells and cultured for 24 hours (37°C), and then high sugar was added and induced at 37°C for 24 hours. The production of cellular ROS and H2O2 decreased, which was statistically significant and the activity was significantly better than LBPS, indicating that the oxidative stress of SV40-Mes13 cells induced by high sugar can be effectively reversed by LRPS (##P<0.01).

[0176] 3. Detection of inflammatory factors

[0177] The ELISA method was used to detect the effect of black wolfberry polysaccharide on the secretion of cytokines IL-1β and TNF-α by mesangial cells (SV40-Mes13 cells) under high glucose.

[0178] Blank group Control, glucose group HG (the added drug was 25 mM glucose), black wolfberry polysaccharide group HG+LRPS (the added drug was 25 mM glucose + 25 μg / mL methyl-esterified black wolfberry polysaccharide prepared in Example 1), and wolfberry polysaccharide group HG+LBPS (the added drug was 25 mM glucose + 25 μg / mL wolfberry polysaccharide);

[0179] Blank group (Control), glucose group (HG): 100 μL / 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured, followed by 100 μL of the corresponding drug stimulation. After drug addition, the cells were incubated in a cell culture incubator at 37°C for 24 h. No drug was added to the blank group.

[0180] Black wolfberry polysaccharide group HG+LRPS, wolfberry polysaccharide group HG+LBPS: 100 μL / 5000 cells (SV40-Mes13 cells) were added to each well of a 96-well plate and cultured and given 100 μL of methylated black wolfberry polysaccharide or wolfberry polysaccharide. After adding the drug, the cells were incubated at 37°C for 24 hours in a cell culture incubator, and then high-glucose HG was added and induced at 37°C for 24 hours.

[0181] The experimental steps after induction are as follows:

[0182] ① Centrifuge the cell supernatant sample at 100-500g for 5 minutes; calculate and determine the number of pre-coated plates required for one experiment, take out the required plates and place them in the 96-well frame;

[0183] ② Each experiment requires the preparation of standard products and the drawing of a standard curve, while setting up a background comparison well, i.e. a blank well;

[0184] ③ Add 100 μL / well of samples or standards of different concentrations to the corresponding wells, seal the wells with transparent sealing film, and incubate at room temperature for 120 min; wash the plate 5 times, and pat dry on thick absorbent paper for the last time;

[0185] ④ Add 100 μL / well of biotinylated antibody, seal the reaction wells with transparent sealing film, incubate at room temperature for 60 min, wash the plate 5 times, and pat dry on thick absorbent paper for the last time;

[0186] ⑤ Add 100 μL / well of horseradish peroxidase-labeled streptavidin. Seal the wells with white sealing film and incubate at room temperature in the dark for 20 minutes. Wash the plate five times, patting dry on thick absorbent paper for the final wash.

[0187] ⑥Add 100 μL / well of TMB solution, seal the reaction wells with white sealing film, and incubate at room temperature in the dark for 15-20 minutes until the standard shows a very significant color change; add 50 μL / well of stop solution, mix well and immediately measure the A450 value.

[0188] The results show that ( Figure 5 ):

[0189] The production of IL-1β and TNF-α inflammatory factors by SV40-Mes13 cells in the glucose HG group was significantly higher than that in the control group, and the difference was statistically significant (**P<0.01).

[0190] In the black wolfberry polysaccharide group (HG+LRPS), pre-incubation with methyl-esterified LRPS for 24 hours followed by high glucose induction for 24 hours reduced IL-1β and TNF-α production in cells, with statistical significance and significantly superior activity to that of the wolfberry polysaccharide LBPS. These results indicate that LRPS can effectively reverse the inflammatory response of SV40-Mes13 cells under high glucose conditions (##P<0.01).

[0191] Example 3: Effect of Black Wolfberry Polysaccharide Extract on the Improvement of Diabetes Symptoms

[0192] Male db / db mice (4 weeks old) and db / m mice (4 weeks old) were purchased from the Weitonglihua Animal Research Center (Beijing, China). All animals were housed under standard conditions (room temperature 22°C, humidity 60%, light / dark cycle) for 2 weeks and received a standard pellet diet and adequate drinking water.

[0193] Six db / m mice were selected as the normal control group (CT), and 12 db / db mice were numbered according to their fasting weight. Six mice were randomly selected as the DKD (diabetic retinopathy) group, and six mice were selected as the black wolfberry polysaccharide extract treatment group (50 mg / kg gavage / day, 8 weeks).

[0194] The specific method is:

[0195] Adaptive feeding for two weeks, normal control group and DKD group (model group) normal feeding for 8 weeks, black nightshade polysaccharide extract intervention group in addition to normal feeding, 50mg LRPS / kg mice per day, a total of 8 weeks of intragastric administration, once a week to measure the body weight and blood glucose.

[0196] Experimental results( Figure 6 ) :

[0197] Diabetic mice all appeared symptoms of diabetes (high blood sugar and weight gain), while LRPS can inhibit the development of weight gain symptoms after intragastric administration, and prevent the progression of diabetes by controlling weight and other symptoms of diabetes.

[0198] Example 4: Improvement of methylated substituted black nightshade polysaccharide extract on kidney damage in DKD mice

[0199] Male db / db mice (db / db mice, 4 weeks old) and db / m mice (db / m mice, 4 weeks old) were purchased from Vivotec Laboratory Animal Research Center (Beijing, China). All experimental animals were adaptively fed for 2 weeks under standard conditions (room temperature 22℃, humidity 60%, light / dark cycle), and standard pellets and sufficient drinking water were ingested. All mice were weighed before the experiment, and the blood glucose level of db / db mice was measured using a blood glucose meter (Roche). db / db mice with fasting blood glucose > 300mg / dl were considered diabetic.

[0200] Select 6 db / m mice as the normal control group (CT), and 12 db / db mice according to the fasting body weight number, randomly select 6 as the DKD group (model group), and 6 as the methylated substituted black nightshade polysaccharide extract intervention group (50mg LRPS / kg intragastric administration / day, 8 weeks).

[0201] Specific method:

[0202] Adaptive feeding for two weeks, normal control group and DKD group (model group) normal feeding for 8 weeks; among them, the black nightshade polysaccharide extract intervention group in addition to normal feeding, 50mg LRPS / kg mice per day, a total of 8 weeks of intragastric administration.

[0203] Measure the body weight and blood glucose once a week, and sacrifice after 8 weeks, collect blood and urine and kidney organs for subsequent experiments.

[0204] Experimental results( Figures 7-8 ) :

[0205] (1) Improvement of black nightshade polysaccharide extract on kidney function in DKD mice:

[0206] The blood urea nitrogen (BUN), serum creatinine (CR) and 24h urine microalbumin levels of the mice in the three groups were determined by using biochemical kits (Nanjing Jiancheng Biological Engineering Institute) after the last administration.

[0207] The results showed that:

[0208] The blood urea nitrogen (BUN), serum creatinine (CR) and 24h urine microalbumin levels of the mice in the blank group (normal control group (CT)) were 6.15 mmol / L, 15.13 μg / 24h and 45.49 μmol / L, respectively.

[0209] The blood urea nitrogen (BUN), serum creatinine (CR) and 24h urine microalbumin levels of the mice in the DKD group (model group) were 15.62 mmol / L, 80.39 μg / 24h and 321.11 μmol / L, respectively.

[0210] The blood urea nitrogen (BUN), serum creatinine (CR) and 24h urine microalbumin levels of the mice in the black nightshade polysaccharide extract intervention group were 12.07 mmol / L, 68.54 μg / 24h and 275.47 μmol / L, respectively.

[0211] It can be seen that the 24h urine microalbumin level of the mice in the DKD group was significantly higher than that in the control group (**P<0.01), and the black nightshade polysaccharide extract intervention for 8 weeks significantly reduced the 24h urine microalbumin level of the DKD mice (##P<0.01), but slightly reduced the serum creatinine and urea nitrogen levels (##P<0.05).

[0212] (2) Improvement of black nightshade polysaccharide extract on kidney pathology of DKD mice:

[0213] The kidney tissues of the three groups of mice were taken after being sacrificed at the end of the last administration, and HE staining, glycogen (PAS) staining and Masson staining were performed to evaluate the improvement of black nightshade polysaccharide extract on kidney pathology of DKD mice.

[0214] HE staining showed that compared with the kidney tissues of normal mice, the glomerular volume of the mice in the DKD group was significantly increased, the mesangial area was significantly widened, the mesangial cells were hyperplastic, the mesangial matrix was aggregated, and the glomerular basement membrane was diffusely thickened. After 8 weeks of intervention of black nightshade polysaccharide extract, the glomerular hypertrophy, mesangial area widening and glomerular basement membrane thickening were improved. PAS staining showed that there was no obvious mesangial expansion in the glomeruli of the mice in the CT group, the glomerular mesangial expansion, mesangial matrix aggregation and mesangial cell hyperplasia in the DKD group were significantly inhibited after 8 weeks of intervention of black nightshade polysaccharide extract.

[0215] Masson staining showed that there was no significant renal interstitial fibrosis in DKD group and Lycium ruthenicum polysaccharide extract intervention group.

[0216] Although the present application has been disclosed in preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Application of a methyl-esterified substituted black wolfberry polysaccharide extract in the preparation of a product for preventing, improving and / or treating diabetic nephropathy.

2. The use according to claim 1, characterized in that The prevention, improvement and / or treatment of diabetic nephropathy includes at least one of the following functions: (a) Inhibits high glucose-induced mesangial cell proliferation, inflammatory response, and oxidative damage; (b) Improve abnormal weight gain caused by diabetes; (c) improving individual renal function-related indicators; (d) Improve the degree of glomerular hypertrophy, mesangial area widening and glomerular basement membrane thickening in individuals.

3. The use according to claim 1 or 2, characterized in that The products include but are not limited to one or more of medicines, foods, feeds, and feed additives.

4. The use according to claim 3, characterized in that The dosage form of the drug is a liquid preparation or a solid preparation. Optionally, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids. Preferably, the drug further includes pharmaceutically acceptable excipients. Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants. Preferably, the food includes health food, food for special medical purposes, functional food or pet food; Preferably, the food includes grain products, starch products, vegetable products, fruit products, meat products, poultry products, egg products, and dairy products.

5. The use according to any one of claims 1 to 4, characterized in that: The methyl ester-substituted black wolfberry polysaccharide extract is prepared according to the following method: (1) After grinding the black wolfberry powder, adding boiling water, stirring, ultrasonicating, and centrifuging, taking the supernatant, extracting the residue again, and combining the two supernatants; (2) concentrating the supernatant obtained in step (1) to 1 / 3 to 1 / 2 of the original volume to obtain a crude extract of Lycium ruthenicum; (3) adding 8% to 12% (w / v) trichloroacetic acid solution to the crude extract of Lycium barbarum obtained in step (2), letting it stand for 3 to 4 hours, centrifuging, taking the supernatant, and repeatedly extracting the precipitate three times according to the above method, and combining the obtained supernatants; (4) adding 3 to 4 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, standing for 8 to 12 hours, centrifuging the solution after standing, discarding the supernatant, washing the filter residue, dissolving it, and dialyzing it using a dialysis bag with a molecular weight cutoff of 2.5 to 3 kDa for 48 to 72 hours, and freeze-drying to obtain a methylated substituted black fruit wolfberry polysaccharide extract.

6. A drug for preventing, improving and / or treating diabetic nephropathy, characterized in that: The medicine contains methyl ester-substituted black wolfberry polysaccharide extract.

7. The drug according to claim 6, characterized in that The dosage form of the drug is a liquid preparation or a solid preparation. Optionally, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids.

8. The drug according to claim 6 or 7, characterized in that The drug also includes pharmaceutically acceptable excipients; preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants.

9. The drug according to any one of claims 6 to 8, characterized in that The methyl ester-substituted black wolfberry polysaccharide extract is prepared according to the following method: (1) After grinding the black wolfberry powder, adding boiling water, stirring, ultrasonicating, and centrifuging, taking the supernatant, extracting the residue again, and combining the two supernatants; (2) concentrating the supernatant obtained in step (1) to 1 / 3 to 1 / 2 of the original volume to obtain a crude extract of Lycium ruthenicum; (3) adding 8% to 12% (w / v) trichloroacetic acid solution to the crude extract of Lycium barbarum obtained in step (2), letting it stand for 3 to 4 hours, centrifuging, taking the supernatant, and repeatedly extracting the precipitate three times according to the above method, and combining the obtained supernatants; (4) adding 3 to 4 times the volume of anhydrous ethanol to the supernatant obtained in step (3) for alcohol precipitation, standing for 8 to 12 hours, centrifuging the solution after standing, discarding the supernatant, washing the filter residue, dissolving it, and dialyzing it using a dialysis bag with a molecular weight cutoff of 2.5 to 3 kDa for 48 to 72 hours, and freeze-drying to obtain a methylated substituted black fruit wolfberry polysaccharide extract.

10. Use of a methyl-esterified substituted Lycium ruthenicum polysaccharide extract in the preparation of a drug for preventing, improving and / or treating diabetic nephropathy.

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