Sea cucumber polysaccharide and application thereof in preparation of products for treating diabetic nephropathy

Abstract

The invention belongs to the technical field of ocean, and particularly relates to sea cucumber polysaccharide and application thereof in preparation of products for treating diabetic nephropathy. The invention discloses a novel holothuria leucospilota PS, which is a sulfated polysaccharide with a fucosylated glycosaminoglycan (FGs)-like structure, and the main chain structure of the novel holothuria leucospilota PS is-> 3)-beta-D-Galp NAc-(1-> 4)-beta-D-Glcp UA-(1->. Results show that after the H.leucospilota PS is intervened, the contents of SCr, BUN and UA in serum of a DKD rat can be reduced, and kidney injury and fibrosis of the DKD rat are improved. The compound can be used for developing functional foods or medicines for treating diabetic nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of marine technology, and specifically relates to sea cucumber polysaccharide and application thereof in preparing a product for treating diabetic nephropathy. Background Art

[0002] Diabetes mellitus (DM) is a complex chronic metabolic disease that is prone to occur when insulin is insufficient or the produced insulin cannot be effectively utilized. The disease can lead to many complications such as cardiovascular disease, renal failure and retinopathy, which seriously affect the quality of life of the patients and may even be life-threatening. DM mainly includes type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), of which the latter accounts for about 90% of the total incidence. Generally, patients with chronic kidney disease (CKD) and DM (about 30% of T1DM and 40% of T2DM) may suffer from true diabetic nephropathy (DKD), which may lead to death. DKD is one of the microvascular complications of DM, which is prone to cause changes in the normal function and structure of the kidneys, including increased urinary albumin excretion, thickening of the glomerular basement membrane and increased chyloprotein, tubulointerstitial fibrosis and, eventually developing into end-stage renal disease.

[0003] Oral hypoglycemic drugs and insulin injections are the main treatments for DM at this stage. However, long-term use of these drugs can cause side effects such as hypoglycemia, gastrointestinal discomfort and insulin resistance, so DM patients are in urgent need of effective, safe and reasonably priced drugs. Tropical marine polysaccharides refer to polysaccharide compounds extracted from tropical marine organisms (such as seaweed and marine animals). These polysaccharides contain sulfate groups and have multiple biological activities such as antioxidant, lipid-lowering, and blood sugar-lowering. In addition, because marine biopolysaccharides have good biocompatibility and no toxic side effects to human cells, they have good utilization and development prospects in medicine and nutrition. However, there are still deficiencies in the resource development of tropical marine polysaccharides. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a sea cucumber polysaccharide.

[0005] The purpose of the second aspect of the present invention is to provide a method for preparing the sea cucumber polysaccharide of the first aspect of the present invention.

[0006] The purpose of the third aspect of the present invention is to provide the use of the sea cucumber polysaccharide of the first aspect of the present invention in the preparation of a product for treating diabetic nephropathy.

[0007] The fourth aspect of the present invention aims to provide a polysaccharide composition.

[0008] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is: The first aspect of the present invention aims to provide a sea cucumber polysaccharide, which is isolated from sea cucumber and named as sea cucumber polysaccharide ( Holothuria leucospilota polysaccharide, H. leucospilota PS).

[0009] In some embodiments of the present invention, the sea cucumber polysaccharide comprises a polysaccharide represented by the structure of formula 1): Formula 1); Wherein, the R group is selected from H, SO3 - One of; R1 group is selected from Fucp, H, SO3 - One of; n is 75-157, and p is the abbreviation of pyranose, which represents a hexavalent sugar.

[0010] In some embodiments of the present invention, the sea cucumber polysaccharide is composed of the following monosaccharides: D-glucuronic acid (GlcpUA), N-acetylgalactosamine (GalpNAc) and fucose (Fucp); the molar ratio of D-glucuronic acid (GlcpUA), N-acetylgalactosamine (GalpNAc) and fucose (Fucp) is: (0.2~0.6):1:(2.0~3.0).

[0011] In some embodiments of the present invention, the sea cucumber polysaccharide is a sulfated polysaccharide having a fucosylated glycosaminoglycan (FGs)-like structure, and its main chain structure is →3)-β-D-Galp NAc-(1→4)-β-D-Glcp UA-(1→.

[0012] In some embodiments of the present invention, the sea cucumber polysaccharide has a substitution group, wherein the R group substitution occurs at the 4-O and 6-O positions of GalpNAc, and the R group is selected from H, SO3 - The Fucp side chain is located at the C3 site of Glcp UA, wherein the 2C and 3C sites of Fucp have replaceable R groups, and the R groups are selected from H, SO3 - The 4C position of Fucp has a replaceable R1 group, and the R1 group is selected from Fucp, H, SO3 - One of them.

[0013] In some embodiments of the present invention, the weight average molecular weight of the polysaccharide of formula 1) is 9×10 4 ~1.1×10 5 Da; preferably, 1.05×10 5 ~1.08×10 5 Da.

[0014] In some embodiments of the present invention, the number average molecular weight of the polysaccharide of formula 1) is 0.5×10 5 ~0.8×10 5 Da; preferably, 0.6×10 5 ~0.7×10 5 Da.

[0015] In some embodiments of the present invention, the Z average molecular weight of the polysaccharide of formula 1) is 5×10 5 ~7×10 5 Da; preferably, 6×10 5 ~6.5×10 5 Da.

[0016] The second aspect of the present invention provides a method for preparing the sea cucumber polysaccharide according to the first aspect of the present invention, comprising the following steps: 1) Defatting and enzymatic hydrolysis of sea cucumber; 2) Hexadecylpyridinium chloride (CPC) precipitation; 3) Alcohol precipitation; 4) Activated carbon decolorization; 5) Deproteinization; 6) Dialysis desalination; 7) Chromatographic purification.

[0017] In some embodiments of the present invention, the degreasing is performed using an organic reagent, and the organic reagent includes at least one of acetone, petroleum ether, and chloroform.

[0018] In some embodiments of the present invention, the enzyme used for enzymatic hydrolysis includes at least one of papain, alkaline protease, and trypsin.

[0019] In some embodiments of the present invention, the temperature of the enzymolysis is 50-70° C.; the time of the enzymolysis is 22-26 h; and the amount of enzyme used in the enzymolysis is 8000-11000 (U / g).

[0020] In some embodiments of the present invention, the concentration of CPC used for precipitation is 0.08-0.12 g / mL.

[0021] In some embodiments of the present invention, the reagent used for the alcohol precipitation is selected from at least one of ethanol, isopropanol and butanol.

[0022] In some embodiments of the present invention, the deproteinization method is selected from Sevage reagent or trichloroacetic acid.

[0023] In some embodiments of the present invention, the chromatography purification includes ion chromatography purification and molecular sieve chromatography purification.

[0024] In some embodiments of the present invention, the ion chromatography purification is performed using a DEAE Sepharose fast flow chromatography column, and other conventional ion chromatography columns in the art can also achieve corresponding effects.

[0025] In some embodiments of the present invention, the molecular sieve chromatography purification is performed using a Sephadex G-100 chromatography column, and other conventional molecular sieve chromatography columns in the art can also achieve corresponding effects.

[0026] The third aspect of the present invention provides the use of the sea cucumber polysaccharide described in the first aspect of the present invention in the preparation of a product for treating diabetic nephropathy.

[0027] In some embodiments of the present invention, the product comprises a food or a medicine.

[0028] In some embodiments of the present invention, the dosage form of the food or medicine includes at least one of powder, tablet, capsule, sustained-release agent, and granule.

[0029] In some embodiments of the present invention, in a rat model, the sea cucumber polysaccharide H. leucospilota PS dose is 100-200 mg / kg.

[0030] The fourth aspect of the present invention provides a sea cucumber polysaccharide composition, comprising sea cucumber polysaccharide H. leucospilota PS and pharmaceutically acceptable excipients.

[0031] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a flavoring agent, a binder, and a filler.

[0032] In some embodiments of the present invention, the sea cucumber polysaccharide composition includes sea cucumber polysaccharide H. leucospilota PS, gelatin, erythritol, coconut sugar and modified starch.

[0033] In some embodiments of the present invention, the sea cucumber polysaccharide H. leucospilota The mass ratio of PS, gelatin, erythritol, coconut sugar and modified starch is 2-3:2-3:2-3:5-6:2-3.

[0034] The beneficial effects of the present invention are: The present invention discovered a new type of sea cucumber polysaccharide H. leucospilota PS is a sulfated polysaccharide with a fucosylated glycosaminoglycan (FGs)-like structure, and its main chain structure is →3)-β-D-Galp NAc-(1→4)-β-D-GlcpUA-(1→.

[0035] The present invention uses a Goto-Kakizaki (GK) rat model of diabetic nephropathy induced by diet, and clarifies the anti-diabetic nephropathy effect of H.leucospilota PS with a specific structure on model rats from the aspects of renal pathology, serum indicators, and changes in renal transcriptional genes. The results show that H.leucospilota PS can reduce the levels of SCr, BUN, and UA in the serum of DKD rats after intervention, and improve renal damage and fibrosis in DKD rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the process flow chart of sea cucumber polysaccharide extraction.

[0037] Figure 2 Analysis of monosaccharide composition of sea cucumber polysaccharides by HPAEC-PAD (A) and HPLC (B).

[0038] Figure 3 This is the HPSEC chromatogram of sea cucumber polysaccharide.

[0039] Figure 4 This is the infrared spectrum of sea cucumber polysaccharide.

[0040] Figure 5 This is the NMR spectrum of sea cucumber polysaccharide.

[0041] Figure 6 This is the COSY spectrum of sea cucumber polysaccharide.

[0042] Figure 7 This is the TOCSY spectrum of sea cucumber polysaccharide.

[0043] Figure 8 This is the HSQC spectrum of sea cucumber polysaccharide.

[0044] Fig. 9 This is the NOESY spectrum of sea cucumber polysaccharides.

[0045] Fig.10 This is the HMBC spectrum of sea cucumber polysaccharides.

[0046] Fig.11 This is a schematic diagram of the structure of sea cucumber polysaccharides.

[0047] Fig.12 These are the structural formulas of three sea cucumber polysaccharides.

[0048] Fig.13 Serum SCr (A), BUN (B) and UA levels (C) in rats with diabetic nephropathy.

[0049] Fig.14 H&E staining of kidney tissue in rats with diabetic nephropathy (bar=100 μm).

[0050] Fig.15 Sirius red staining of kidney tissue in rats with diabetic nephropathy (magnification: 400×). DETAILED DESCRIPTION

[0051] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0052] Example 1 Sea Cucumber Polysaccharide H. leucospilota PS separation and purification The invention provides a method for extracting sea cucumber body wall polysaccharides. The sea cucumber is selected from sea cucumbers of the genus Tripterygium wilfordii. Sea cucumbers of the genus Tripterygium wilfordii are one of the most common edible sea cucumbers in the South China Sea of ​​my country and have medicinal and edible values.

[0053] The specific implementation method of the present invention is as follows: soak the dried sea cucumber body wall for 20 to 24 hours, remove the internal organs and body cavity membrane, mince it with a meat grinder, and freeze-dry it into powder. The freeze-drying parameters of the freeze-drying powder are -60 to -25°C, vacuum degree 1-3 bar, and 10-14 hours. Soak in acetone for defatting, and the mass ratio of sodium acetate, papain, EDTA, cysteine ​​and water is 3.28:0.26:0.58:0.24. Stir and centrifuge, take the supernatant, add cetylpyridinium chloride (Cetyl-Pyridinium Chloride, CPC) to the supernatant at a ratio of 10 (W / V)%, centrifuge after standing at room temperature, and redissolve the precipitate in NaCl: ethanol (100:15V / V) solution. Add anhydrous ethanol to precipitate, stand for 10 to 12 hours, and then centrifuge. Add 0.5%-0.75% activated carbon to the supernatant for decolorization, and stir and decolorize at 40°C for 60 minutes. Trifluoroacetic acid was used to remove protein, and the solution was dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 8000 Da; and freeze-dried (temperature -60~-25℃, vacuum degree 1-3 bar) for 10-14 h to obtain crude sea cucumber polysaccharides.

[0054] The graded column chromatography of the present invention specifically uses a DEAE-Sepharose Fast Flow column for graded separation, and then eluted and purified by a Sephadex G-100 column. After assembling the chromatography column, fill it with fully degassed DEAE-FF filler and balance it with 0.1 M sodium acetate buffer. Weigh 200 mg of the sample, add 10 mL of buffer to dissolve it, pass the prepared polysaccharide solution through a 0.45 μm filter membrane, and add it to the chromatography column. The eluent uses a 0.1 M sodium acetate solution containing 1.5 M, set the flow rate to 1 mL / min, 5 min / tube, collect tubes 61-68, and lyophilize to obtain HLP-1. Further purification using Sephadex G-100: weigh 100 mg of HLP-1 lyophilized powder, add 5 mL of 0.1 M sodium acetate buffer containing 2 M NaCl, fully dissolve it, pass it through a 0.45 μm filter membrane, and set the flow rate to 0.5 mL / min. The main peak (tubes 19-30) was collected, and 5 mL was collected in each tube. After reduced pressure concentration, the solution was dialyzed for 48 h and freeze-dried to obtain the purified sample of sea cucumber polysaccharide.

[0055] Example 2 Sea Cucumber Polysaccharide H. leucospilota Structural identification of PS 1) Analysis of monosaccharide composition by HPLC The monosaccharide composition was determined by high performance liquid chromatography (HPLC) with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. 6 mg of the polysaccharide prepared in Example 1 was dissolved in 3 mL of trifluoroacetic acid (4 M) to prepare a 2 mg / mL mixed solution, which was transferred to an ampoule tube, sealed, and hydrolyzed at 120 °C for 4 h. After the reaction was completed, it was cooled to room temperature, 100 μL of the hydrolyzate was added with an equal volume of methanol, and dried with nitrogen to remove trifluoroacetic acid. The above operation was repeated 3 times until the sample tube had a constant weight. The dried sample was dissolved in 100 μL of ultrapure water, and an equal volume of 0.6 M NaOH solution was added and mixed evenly, and then an equal volume of 0.5 M PMP-methanol solution was added for pre-column derivatization reaction, and the reaction was carried out in a 70 °C water bath for 1.5 h. After the reaction was finished and cooled to room temperature, 100 μL of the reactant was taken out and neutralized with 50 μL of hydrochloric acid (0.3 M), 1 mL of chloroform and 900 μL of water were added, the chloroform layer was discarded after vigorous shaking and centrifugation for 5 min, and 1 mL of chloroform was added to the aqueous layer. The extraction was repeated 3 times to obtain a derivatized polysaccharide solution. Finally, the derivatized polysaccharide solution of the upper layer (aqueous layer) was filtered using a 0.22 μm water filter membrane and then detected by high performance liquid chromatography. The monosaccharide standard was prepared as a 1 mg / mL solution and the polysaccharide hydrolyzate was derivatized and labeled at the same time. The monosaccharide standards are glucosamine, glucuronic acid, aminogalactose, galactose and fucose.

[0056] The detection conditions of liquid chromatography are shown in Table 1.

[0057] Table 1 Liquid chromatography operating parameters

[0058] 2) Analysis of monosaccharide composition by ion chromatography The derivatized polysaccharide solution in the upper layer (aqueous phase) obtained in step 1) was passed through a 0.22 μm water filter membrane and injected. Different concentrations of monosaccharide standard solutions and mixed standard solutions were prepared, and the samples were analyzed using the HPACE-PAD method. The mixed standard solution was prepared with the same concentration of glucosamine, glucuronic acid, aminogalactose, galactose and fucose. The five monosaccharide standard samples were prepared into 5.0 mg / mL stock solutions, and 1.0 mL was drawn from each of the five monosaccharide standard sample stock solutions, mixed to prepare 0.5 mg / mL mixed standard solutions, and the mixed standard solutions were diluted to the required concentration gradient, such as 0.02, 0.04, 0.06, 0.08, 0.1 mg / mL mixed standard solutions. Equipped with a Carbo Pac TMPA 20 (4 mm × 250 mm) ion chromatography column, set the flow rate to 0.5 mL / min, and the mobile phase A was UP water, expressed in percentage.

[0059] Table 2 Monosaccharide gradient elution program

[0060] Due to the limitations of high performance anion exchange chromatography (HPAEC) and the separation effect of the PA-20 column, the peak times of galactosamine and rhamnose are very close ( Figure 2 In the structural analysis, HPSEC and PMP-HPLC with Agilent C18 column were used to measure the monosaccharide composition. Each peak was integrated and quantified according to the HPLC peak position and time ( Figure 2 In B), it can be seen that the monosaccharide composition of HLP-Ⅰ is mainly D-glucuronic acid (GlcpUA), N-acetylgalactosamine (GalpNAc) and fucose (Fucp), with a molar ratio of 0.45:1.00:2.40.

[0061] 3) Determination of homogeneity and molecular weight High-performance size exclusion chromatography-multi-angle laser light scattering (HPSEC-MALLS) measurements were performed using an Agilent 1200 series LC system (Agilent Technologies, Palo Alto, USA) at 25 °C. The mobile phase was 0.1 M NaNO3 in water with a flow rate of 0.6 mL / min and the specific refractive index increment (dn / dc) was set to 0.145 mL / g. The molecular weight was calculated by the Zimm method of static light scattering according to the basic light scattering equation.

[0062] H. leucospilota The weight average molecular weight (Mw), number average molecular weight (Mn) and Z average molecular weight (Mz) of PS were measured to be 1.062×10 5 Da, 0.652×10 5 Da and 6.232×10 5 Da. The narrow and symmetrical peaks on the HPSEC-RI chromatogram ( Figure 3 ) and a relatively low polydispersity index value (1.63, PDI = Mw / Mn) indicated that H. leucospilota PS has higher purity and better uniformity.

[0063] 4) Infrared spectroscopy analysis of molecular structure The sample and KBr mixture (1:100, w / w) were compressed into thin sheets and transferred to an FT-IR spectrometer (Nicolet6700, Thermo Fisher Scientific Technology Co., Ltd, USA). -1 FT-IR spectra were recorded in the range of 100 nm and 64 scans.

[0064] 5) Analysis of molecular structure by nuclear magnetic resonance spectroscopy Deuterated polysaccharides (50 mg) were dissolved in 0.6 mL D2O and subjected to 1D and 2D NMR experiments. All experiments were performed on a Bruker AVANCE spectrometer (Bruker BioSpin, Rheinstetten, Germany). 1H and 13C NMR spectra were acquired at 298.2 K using 600.58 and 151.01 MHz proton frequencies and processed with MestReNova 14.0 software. 2D NMR experiments, including 1H-1H correlation spectroscopy (1H-1H COSY), 1H-13C heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), total correlation spectroscopy (TOCSY), and NOESY were performed for structural analysis.

[0065] like Figure 4 As shown, 3478.53 cm -1 The strong absorption band at 2929.85cm represents the stretching vibration of OH in the sugar residue. -1 The band nearby is related to the stretching vibration of CH in the sugar ring. -1 and 1650 cm -1 The peak at 1253.12 cm is attributed to the -COOH bending vibration of the carboxylic acid product and the bending and stretching vibration of CO in glucuronic acid and N-acetylgalactosamine. -1 The signal at 1050.93 cm is attributed to the asymmetric stretching vibration of the sulfate group (O=S=O), which is a typical feature of sulfated polysaccharides. -1 The absorption peak is in the fingerprint region of polysaccharides (400-1300 cm -1 , which can be used to distinguish small differences in the structure of different compounds) can identify specific chemical groups in HLP-Ⅰ, which are generated by COC stretching vibration. 962.47, 850.71 and 686.36 cm -1 The asymmetric absorption peak of H. leucospilota There are abundant sulfate groups in PS. 820 cm -1 - 850 cm -1 The nearby bands have obvious differences, which may be related to the different substitution positions of sulfate groups in the polysaccharide chain structure. -1 The characteristic peak of COS stretching vibration indicates that the C-4 position of the fucose residue contains an axial sulfate group.

[0066] like Figure 5As shown, the chemical shift of the anomeric proton signal region in the 1H NMR spectrum is 5.0-5.8 ppm, indicating the presence of α-sulfated fucose residues and H. leucospilota The sulfate group substitution types in PS are Fucp2,4S, Fucp4S, and Fucp0S. The signal ranges of the methyl protons (-CH3) of Fucp and GalpNAc are in the 1.1-1.4 ppm and 1.8-2.1 ppm regions, and the signal integration results in an area ratio of 1.05:1.00. The 13C NMR spectrum further illustrates the H. leucospilota The carbon skeleton of PS polysaccharide is composed of L-Fucp, D-GalpNAc and D-GlcpUA, and D-GalpNAc and D-GlcpUA are in the β-anomeric carbon configuration ( Figure 6 ).

[0067] According to H. leucospilota HSQC spectral data of PS ( Figure 7 ), the positions of the H-1 signals belonging to the H and I units are 5.62 ppm and 5.32 ppm, respectively. Using 1H-1H COSY ( Figure 8 ) spectrum to further analyze other signals of the resonance system. The H-2 (4.39 ppm) and H-4 (4.58 ppm) signals are in the low field position, which verifies the presence of sulfate groups at the C-2 and C-4 positions in the H unit. The H-3 (3.91 ppm) and H-4 (4.14 ppm) signals confirm the sulfation of the I unit at O-3 and O-4. Fig. 9 As shown, signals A, B, and C are from (H-1, C-1), (H-2, C-2), (H-3, C-3), (H-4, C-4), (H-5, C-5), and (H-6, C-6) of GalpNAc, GalpNAc4S6S, and GalpNAc4S. Signals D, E, and F are from (H-1, C-1), (H-2, C-2), (H-3, C-3), (H-4, C-4), and (H-5, C-5) of GlcpUA, GlcpUA 2S, and GlcpUA2S3S. The chemical shifts of H-4 and H-6 in GalpNAc4S6S are downfield compared to GalpNAc, indicating that its O-4 and O-6 positions are sulfated. Based on the related peaks in HMBC and NOESY, the residue sequence of the glycosidic bond was clarified. Fig.10 The two clear signals at 4.39 / 3.86 ppm and 4.64 / 4.43 ppm (denoted as D1 / A3, B3, C3 and H1, B1, C1 / D1, E1, F1, respectively) indicated that GlcpUA was linked to the C-3 position of GalN, which was associated with the C-3 position of GalpNAc. Fig.11The cross peaks of H1(D) / H3(E), H1(G,I) / H3(E), and H1(G,I) / H3(F) interactions confirmed the replacement of fucosyl residue at O-3 by GlcpUA.

[0068] H. leucospilota The H / C chemical shift data of PS are shown in Table 3. Based on the above structural analysis, H. leucospilota PS contains three main monosaccharides (GlcpUA, GalpNAc and Fucp), and its main chain structure is similar to that of some sea cucumber body wall FGs. Therefore, it is speculated that H. leucospilota PS is a sulfated glycosaminoglycan with a FGs-like structure, and sulfate substitution occurs at the O-4 and O-6 positions of GalpNAc. In addition, H. leucospilota PS has fucose branches with different sulfation patterns (Fucp2, 4S, Fucp3, 4S, and Fucp0S).

[0069] Table 3 H leucospilota P.S. 1 H and 13 Assignment of C NMR spectra

[0070] Based on the above data, H. leucospilota PS includes the following three different polysaccharide structures: Formula 2); Formula 3); Formula 4).

[0071] Combining infrared and nuclear magnetic data, it is predicted that H. leucospilota The structural diagram of PS is shown in Fig.11 shown.

[0072] Example 3 Sea Cucumber Polysaccharide H. leucospilota Study on the activity of PS on diabetic nephropathy 1) Animal experiment design GK rats (Goto-Kakizaki rats) are a commonly used animal model of spontaneous type 2 diabetes. Male GK rats were used as DKD model rats, and 30 rats were randomly divided into three groups: diabetic nephropathy group (DKD); H. leucospilota PS low-dose group (HLP-L, 100 mg / kg HLP); H. leucospilotaPS high-dose group (HLP-H, 200 mg / kg HLP). Wistar rats (n=10) were used as the normal group (NC). The male Goto-Kakizaki rats and male Wistar rats were provided by Tianqin Biotechnology Co., Ltd. (Changsha, China) with the number SCXK (Xiang) 2014-0011. After one week of adaptation to the experimental conditions, all rats were gavaged once a day at 10 mL / kg body weight for 4 weeks. The water and food intake of the rats on the same day were recorded. After fasting for 10 h, all rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. Blood samples were collected from the orbits and centrifuged at 3500g at 4°C for 10 min. Serum was collected for further analysis. All animals were killed by cervical vertebrae, and kidney tissues were immediately removed from the samples and stored at -80°C for later use.

[0073] 2) Serum index detection The urea nitrogen, uric acid and creatinine contents in rat serum were determined according to the kit instructions. The urea nitrogen test kit (BC1530-50T / 24S) was purchased from Beijing Solebow Technology Co., Ltd.; the creatinine content determination kit (C486296-1kit) and the uric acid test kit (U486299-1kit) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0074] 3) Renal tissue pathology observation Kidney tissue was taken, washed with saline to remove blood, and fixed in 10% formalin for one week. After paraffin embedding, dewaxing and dehydration of sections, staining (hematoxylin for nuclei, eosin for cytoplasm), dehydration and sealing, optical microscopy was used for histopathological observation, and images were collected for analysis.

[0075] 4) Sirius red staining of kidney tissue Prepare paraffin sections of kidney tissue with a thickness of about 5 μm, dewax to remove moisture, take out after immersion in Sirius red staining solution for 60 minutes, remove the surface staining solution, take out after immersion in Mayer hematoxylin staining solution for 5-10 minutes, rinse with running water, dehydrate to make it transparent in a conventional way, seal with neutral gum, and observe and analyze.

[0076] 5) Experimental results BUN and SCr are indicators of glomerular filtration capacity. High levels of serum BUN, SCr, UA, etc. are one of the manifestations of DKD. Fig.13 As shown in the results, the contents of each index in the DKD group were significantly higher than those in the NC group. Among them, the BUN, Scr, and UA in the high-dose group were significantly reduced by 1.11mmolL / L, 15.44μmol / L, and 80.00 μmol / L, respectively. The results suggest that H. leucospilota PS can improve the glomerular filtration capacity and repair proximal tubular epithelial cells.

[0077] Histopathological results such as Fig.14 As shown in the figure, the glomeruli and tubules of mice in the NC group were intact, while the glomeruli of rats in the DKD group were atrophic, the glomerular cystoids were severely hyperplastic, the tubular endothelial cells were detached, the basement membrane was exposed, the inflammatory cells infiltrated in the cytoplasm, and the interstitial edema. After HLP intervention, the above symptoms were significantly alleviated. Compared with the DKD group, the renal structure was more intact, and the changes in renal tubular morphology were similar to those in the NC group. This shows that HLP can reduce renal damage in diabetic rats and has a certain protective effect.

[0078] Renal fibrosis in T2DM rats is obviously one of the early signs of DKD. In this example, the area of ​​collagen fibers was determined by Sirius red staining; Fig.15 As shown in the figure, the collagen deposition area in the HLP group was significantly reduced, and the HLP-H group had a better relief effect. The synthesis of type IV collagen, fibronectin, and laminin in renal interstitial cells, tubular epithelial cells, endothelial cells, and other interstitial cells increased, accelerating the accumulation of extracellular matrix (ECM), thus forming renal interstitial fibrosis.

[0079] Example 4 A sea cucumber polysaccharide microgel enteric-coated sustained-release granule A sea cucumber polysaccharide microcapsule enteric-coated sustained-release granule, the formula of which is 10g of sea cucumber polysaccharide, 10g of gelatin, 10g of erythritol, 30g of coconut sugar and 15g of modified starch, and the microcapsule enteric-coated sustained-release granules are obtained by spray drying and packaging to obtain the sea cucumber polysaccharide microcapsule enteric-coated sustained-release granules. The enteric-coated granules have a good effect on improving diabetic nephropathy.

[0080] Example 5 A sea cucumber polysaccharide microgel enteric-coated sustained-release granule A sea cucumber polysaccharide microcapsule enteric-coated sustained-release granule, the formula of which is 15g of sea cucumber polysaccharide, 15g of gelatin, 15g of erythritol, 25g of coconut sugar and 10g of modified starch, and the microcapsule enteric-coated sustained-release granules are obtained by spray drying and packaging to obtain the sea cucumber polysaccharide microcapsule enteric-coated sustained-release granules. The enteric-coated granules have a good effect on improving diabetic nephropathy.

[0081] Example 6 A sea cucumber polysaccharide microgel enteric-coated sustained-release granule A sea cucumber polysaccharide microcapsule enteric-coated sustained-release granule, the formula of which is 12.5 g of sea cucumber polysaccharide, 12.5 g of gelatin, 12.5 g of erythritol, 30 g of coconut sugar and 10 g of modified starch, and the microcapsule enteric-coated sustained-release granules are obtained by spray drying and packaging to obtain the sea cucumber polysaccharide microcapsule enteric-coated sustained-release granules. The enteric-coated granules have a good effect on improving diabetic nephropathy.

Claims

1. A sea cucumber polysaccharide, characterized in that: The polysaccharide comprises a polysaccharide shown in formula 1): Formula 1); Wherein, the R group is selected from H, SO3 - One of; R1 group is selected from Fucp, H, SO3 - One of; n is 75-157.

2. The sea cucumber polysaccharide according to claim 1, characterized in that: The polysaccharide is composed of the following monosaccharides: D-glucuronic acid, N-acetylgalactosamine and fucose; The molar ratio of D-glucuronic acid, N-acetylgalactosamine and fucose is: (0.2-0.6):1:(2.0-3.0).

3. The sea cucumber polysaccharide according to claim 2, characterized in that: The weight average molecular weight of the polysaccharide of formula 1) is 9×10 4 ~1.1×10 5 Da.

4. The method for preparing the polysaccharide according to any one of claims 1 to 3, comprising the following steps: 1) Defatting and enzymatic hydrolysis of sea cucumber; 2) Hexadecylpyridinium chloride (CPC) precipitation; 3) Alcohol precipitation; 4) Decolorization; 5) Deproteinization; 6) Dialysis desalination; 7) Chromatographic purification.

5. The preparation method according to claim 4, characterized in that: The enzyme used for the enzymolysis includes at least one of papain, alkaline protease and trypsin.

6. The preparation method according to claim 5, characterized in that: The chromatography purification includes ion chromatography purification and molecular sieve chromatography purification.

7. A sea cucumber polysaccharide composition, characterized in that: The sea cucumber polysaccharide composition comprises the polysaccharide according to any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

8. The polysaccharide composition according to claim 7, characterized in that: The pharmaceutically acceptable excipients include at least one of a flavoring agent, a binder, and a filler.

9. Use of the sea cucumber polysaccharide according to any one of claims 1 to 3 and / or the sea cucumber polysaccharide composition according to any one of claims 7 to 8 in the preparation of a product for treating diabetic nephropathy.

10. The use according to claim 9, characterized in that: The dosage form of the product includes one of powder, tablet, capsule, sustained-release agent and granule.

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