Enzyme extraction method polygonum cuspidatum homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and relates to polygonum cuspidatum homogeneous polysaccharide and a preparation method and application thereof.The polygonum cuspidatum homogeneous polysaccharide is mainly composed of galacturonic acid, rhamnose, galactose, glucuronic acid, glucose, mannose, xylose and glucuronide amine according to the molar ratio of 41.967: 24.624: 17.951: 5.427: 4.59: 3.501: 1.236: 0.358, and the weight-average molecular weight of the polygonum cuspidatum homogeneous polysaccharide is 26.57 kDa. The polygonum cuspidatum homogeneous polysaccharide is fixed in component, high in yield, good in hypoglycemic effect and good in safety, and a basis is provided for development of innovative hypoglycemic drugs.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a homogeneous polysaccharide of Polygonum cuspidatum, its preparation method and application. Background Technology

[0002] Polygonum cuspidatum Sieb. et Zucc., commonly known as Japanese knotweed, is a plant belonging to the genus Polygonum in the family Polygonaceae. It has a bitter and sour taste and is slightly cold in nature. It enters the liver and gallbladder meridians and is widely used in traditional Chinese medicine, possessing the effects of soothing the liver and gallbladder, clearing heat and detoxifying, promoting blood circulation and removing blood stasis, relieving pain and eliminating phlegm. Polygonum cuspidatum and its active ingredients have various pharmacological effects. Anthraquinones such as emodin have antibacterial effects; resveratrol can suppress cough, lower blood pressure, dilate coronary vessels, increase coronary blood flow, and mildly enhance cardiac contractility, as well as having some lipid-lowering effects; in addition, Polygonum cuspidatum also has anti-liver damage, anti-shock, anti-asthmatic, antioxidant, sedative, and microcirculation-improving effects.

[0003] Diabetes mellitus (DM) is a chronic systemic metabolic disease characterized by hyperglycemia, caused by insulin secretion deficiency and / or insulin resistance. With a significant increase in morbidity and mortality, the prevention and treatment of type 2 diabetes has become a critical challenge urgently needing to be addressed. In recent years, traditional Chinese medicine (TCM) has become a research hotspot due to its effectiveness, multi-target effects, and low toxicity. Increasing research has found that TCM polysaccharides, due to their hypoglycemic effects, have great potential in the treatment of diabetes, demonstrating good clinical efficacy, increasing cure rates, and reducing adverse reactions.

[0004] Currently, research on the preparation and pharmacological activity of Polygonum cuspidatum polysaccharides is limited, and the composition of Polygonum cuspidatum polysaccharides obtained by different extraction methods varies significantly. Existing Polygonum cuspidatum polysaccharide preparation processes have low extraction rates, restricting their application in practical production. Various polysaccharide extraction methods exist, but enzymatic extraction offers significant advantages over other methods, including mild reaction conditions, less product degradation, high extraction efficiency, low cost, environmental friendliness and energy conservation, and optimization of effective components. In particular, auxiliary extraction methods combining enzymatic extraction with other methods can improve extraction efficiency and reduce extraction time. Therefore, developing a method for preparing Polygonum cuspidatum polysaccharides with a fixed composition and high yield would greatly promote the industrialization of Polygonum cuspidatum and expand its application scope. Summary of the Invention

[0005] This invention addresses the technical problem of limited application of Polygonum cuspidatum polysaccharide by providing a homogeneous Polygonum cuspidatum polysaccharide, its preparation method, and its application. The prepared homogeneous Polygonum cuspidatum polysaccharide has a fixed composition and a high yield, exhibits hypoglycemic effect, and has good safety, providing an important basis for developing novel T2DM treatment regimens.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a homogeneous polysaccharide of Polygonum cuspidatum, which is mainly composed of galacturonic acid, rhamnose, galactose, glucuronic acid, glucose, mannose, xylose, and glucuronide in a molar ratio of 41.967:24.624:17.951:5.427:4.59:3.501:1.236:0.358, and has a weight-average molecular weight of 2.657 kDa. Its structural unit has the following structural formula:

[0008] .

[0009] Secondly, the present invention provides a method for preparing the above-mentioned homogeneous polysaccharide from Polygonum cuspidatum, comprising the following steps:

[0010] 1) After air-drying Polygonum cuspidatum at 50 ℃ for 48 h, grind it into powder using a grinder. Mix the air-dried Polygonum cuspidatum powder, compound enzyme preparation and water and add it to the Chinese medicine extractor. Under certain temperature conditions, each sample is extracted twice. After centrifugation at 500 r / min, the supernatant from the two extractions is combined and concentrated in a vacuum rotary evaporator at 45 ℃.

[0011] 2) The concentrated Polygonum cuspidatum polysaccharide solution was subjected to alcohol precipitation to obtain crude polysaccharide precipitated with alcohol;

[0012] 3) The crude polysaccharide precipitated with alcohol was deproteinized using the Sevag method to obtain total polysaccharide of Polygonum cuspidatum;

[0013] 4) The total polysaccharides of Polygonum cuspidatum were separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharides of Polygonum cuspidatum.

[0014] Preferably, the compound enzyme preparation consists of 500 U / mg pectinase, 50 U / mg cellulase and 800 U / mg papain.

[0015] Preferably, the extraction time in step 1) is 180 min.

[0016] Preferably, the extraction temperature in step 1) is 40~60℃.

[0017] Preferably, in step 2), the alcohol precipitation uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25 °C, and the precipitation time is 12-24 h.

[0018] Preferably, in step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:1.

[0019] Preferably, the ion exchange chromatography column in step 4) is a DEAE-52 cellulose column, the flow rate of the ion exchange column chromatography is 50 mL / h, and the gradient elution program is: 0–50 min water, 20–40 min 0.1 mol / L NaCl, 30–40 min 0.3 mol / L NaCl; the packing material of the gel column is Sephadex G-200.

[0020] Thirdly, the present invention also provides the application of the above-mentioned Polygonum cuspidatum homogeneous polysaccharide in the preparation of drugs for treating type 2 diabetes.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention relates to a novel homogeneous polysaccharide compound, Polygonum cuspidatum (PCPs), which has a fixed composition and a high yield. It exhibits hypoglycemic effects and good safety, providing an important basis for developing novel T2DM treatment regimens and for the quality control and standardized production of Polygonum cuspidatum homogeneous polysaccharide. Attached Figure Description

[0023] Figure 1 The hydrogen spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0024] Figure 2 This is the carbon spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0025] Figure 3 The HH-COSY spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0026] Figure 4 The HSQC spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0027] Figure 5 The NOESY spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0028] Figure 6 The HNBC map of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0029] Figure 7 DEPT-135 NMR spectrum of homogeneous polysaccharide PCPs from Polygonum cuspidatum.

[0030] Figure 8Polygonum cuspidatum polysaccharide PCPs can alleviate HFD / STZ-induced T2DM in C57BL / 6 mice, including: (A) experimental flowchart; (B) FBG and OGTT content; (C) OGTT-AUC, FINS concentration, HOMA-IR, GLP-1, LEPH and ADP; (D) HDL-C, LDL-C, TG and TC content levels and mRNA expression levels of SCD1, ACL-Y, ACC2, FASN, and SREBP-1C; (E) SQLE, MVK, LSS, LDLR, HMGCR, and SREBP-2 mRNA expression levels; (F) pathological images of liver and kidney.

[0031] Figure 9 Polygonum cuspidatum polysaccharide PCPs can protect against colonic disorders induced by HFD / STZ in T2DM mice, including: (A) changes in body weight; (B) DAI score; (C) intestinal tissue photographs; (D) colon length; (E) MPO level; (F) FITC content; (G) intestinal pathological effects; (H) levels of IL-6, IL-22, IL-10, TNF-α, and IL-1β; (I) mRNA expression levels of Atf4, Xbp1s, Muc5ac, and Perk; (J) mRNA expression levels of B3gnt6, C1galt1, St3gal3, and Fut8; and (K) immunohistochemical images of MUC2 mice. Detailed Implementation

[0032] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0033] The compound enzyme preparation used in the following examples consists of 500 U / mg pectinase, 50 U / mg cellulase and 800 U / mg papain, purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0034] Example 1: Extraction of homogeneous polysaccharide PCPs from Polygonum cuspidatum

[0035] (1) Extraction of total polysaccharides from Polygonum cuspidatum

[0036] Polygonum cuspidatum (from Ji'an City, Jiangxi Province) was air-dried at 50 ℃ for 48 h, then ground into powder. 500 g of the dried Polygonum cuspidatum powder, 20 g of compound enzyme preparation, and 1000 mL of water were mixed and added to a traditional Chinese medicine extractor. Extraction was performed at 45 ℃ for 180 min, with each sample extracted twice. After centrifugation at 500 r / min, the supernatants from both extractions were combined and concentrated in a vacuum rotary evaporator at 45 ℃. The concentrated solution was precipitated with four volumes of anhydrous ethanol and then allowed to stand at a constant temperature of 4 ℃ for 24 h. The precipitate was then collected by centrifugation to obtain crude polysaccharide precipitated with alcohol.

[0037] The crude polysaccharide was precipitated with alcohol, and then extracted with 1 / 5 volume of chloroform-n-butanol (4:1 volume ratio) using the Sevag method. The mixture was shaken for 20 min, transferred to a separatory funnel, allowed to stand, and the supernatant was collected and centrifuged for 1 min to remove residual protein precipitate. This process was repeated 3-5 times to obtain a crude polysaccharide solution of Polygonum cuspidatum after protein removal. The solution was concentrated under reduced pressure and dried, yielding a crude polysaccharide yield of 6.9%.

[0038] (2) Isolation and purification of total polysaccharides from Polygonum cuspidatum

[0039] A DEAE-52 cellulose column was used, connected to a fraction collector and a peristaltic pump. Total polysaccharides from Polygonum cuspidatum were eluted sequentially with distilled water, 0.1 mol / L NaCl, and 0.3 mol / L NaCl at a flow rate of 50 mL / h for 40 min, 30 min, and 35 min, respectively. The eluent was collected in 10 mL tubes and labeled. The absorbance was measured at 490 nm using the phenol-sulfuric acid method, and a scatter plot was plotted. 0–20 tubes of eluent (eluent containing 0–0.2 mol / L NaCl) were collected, concentrated, dialyzed through a 3500 Da dialysis bag, and freeze-dried to obtain PCPs-A, PCPs-B, and PCPs-C with yields of 18.3%, 6.9%, and 14.8%, respectively.

[0040] After further purification using a Sephadex G-200 column, the combined solutions were concentrated using a rotary evaporator and freeze-dried to obtain the component named PCPs, with a final yield of 68.2%.

[0041] Example 2: Structural Identification of Homogeneous Polysaccharide PCPs from Polygonum cuspidatum

[0042] (1) According to the established experimental protocol, the carbon content (MC) of PCPs was analyzed using the HPAEC system equipped with a pulsed current detector. The molecular weight (MW) was determined by the HPSEC system (MALLS-VIS-R) with multiple detectors, and its weight-average molecular weight was 2.657 kDa.

[0043] (2) Monosaccharide profiles were evaluated by preparing aqueous standards of 10 monosaccharides (rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride) at concentrations of 5 mg and 10 mg / mL. These solutions were diluted, filtered (0.22 μm), and stored for analysis. Subsequently, 5 mg of HP was hydrolyzed in 2 M trifluoroacetic acid (TFA) at 121 °C for 2 hours, dried under nitrogen, washed with methanol, and resuspended in 5 mL of sterile water. HP was then transferred to HPLC vials. For derivatization, 0.2 mL of each standard or hydrolysis product was mixed with 0.2 mL of 0.5 M sodium hydroxide and 0.5 mL of 0.5 M PMP in methanol, heated at 70 °C for 1 hour, and then neutralized with 0.2 mL of 0.5 M hydrochloric acid. PMP was extracted with chloroform. The volume was brought to 1 mL with 0.3 mL of sample. Chromatographic analysis was performed on a Thermo U3000 C18 column using a 17:83 (v / v) mixture of acetonitrile and phosphate buffer as the mobile phase at a flow rate of 0.8 mL / min. The column temperature was maintained at 30 °C, the injection volume was 10 μL, and the detection wavelength was 250 nm.

[0044] The results showed that the homogeneous polysaccharide of Polygonum cuspidatum was mainly composed of galacturonic acid, rhamnose, galactose, glucuronic acid, glucose, mannose, xylose, and glucuronide in a molar ratio of 41.967:24.624:17.951:5.427:4.59:3.501:1.236:0.358.

[0045] (3) Reconstitute the lyophilized sample with 0.5 mL of heavy water (D2O). Measure the one-dimensional NMR (1D NMR), carbon-13 NMR (13C NMR), DEPT-135, and two-dimensional NMR spectra (COSY, HMBC, HSQC, and NOESY) by nuclear magnetic resonance spectroscopy. Calibration parameters: HDO hydrogen signal at δH = 4.70 ppm, TMS carbon signal at δC = 0.00 ppm.

[0046] The methylation analysis results of PCPs are summarized in Table 1. The results indicate that PCPs are a complex polysaccharide structure containing at least nine monosaccharide types and 24 different glycosidic bond linkage modes. Five major linkage modes account for 74.65% of the total sugar residues, specifically: →4)-galactose pA-(1→ (37.34%), →2)-rhamnose-(1→ (15.22%), t)-galactose pA-(1→ (8.41%), t)-galactose pA-(1→ (6.60%), and →2,4)-rhamnose-(1→ (7.05%), with their branching structures mainly located at the C-2 site. Minor sugar residues may be distributed in the main chain or side chain regions.

[0047] Table 1 Analysis of methylation and linkage patterns of PCPs

[0048]

[0049] Table 2 1 H and 13 C signal assignments

[0050]

[0051] A series of structural characterizations were also carried out in this invention, including: methylation experiments to analyze its sugar residues and nuclear magnetic resonance spectrum analysis (see Table 2) to determine the glycosidic bond linkage mode. Figure 1 For the 1 1H nuclear magnetic resonance spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 2 For the 13 13C nuclear magnetic resonance spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 3 For the HH-COSY spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 4 For the HSQC spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 5 For the NOESY spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 6 For the HMBC spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Figure 7 For the DEPT-135 nuclear magnetic resonance spectrum of the uniform polysaccharide PCPs from Polygonum cuspidatum. Through various analysis methods, the structural formula of the structural unit of the uniform polysaccharide PCPs from Polygonum cuspidatum is as follows:

[0052] .

[0053] Example 3 Therapeutic effect of the uniform polysaccharide from Polygonum cuspidatum on T2DM mice

[0054] 3.1 Effects of PCPs on symptoms, intestinal barrier (IB) and intestinal microbiota in T2DM mice

[0055] 3.1.1 Animals

[0056] In this example, 50 male C57BL / 6 mice aged 7-8 weeks (body weight 20-22 g) were used, provided by Beijing Biohit Biotechnology Co., Ltd. (License number: SCXK (Beijing) 2019-0010). All experimental animals were housed in a standard environment (temperature 22±1 °C, relative humidity 50±5%) and maintained a 12-hour light / dark cycle. All experimental protocols have been approved by the Laboratory Animal Management Committee of Henan University of Chinese Medicine (Approval number: DWLL202402406).

[0057] 3.1.2 Animal experiments

[0058] After an acclimatization period, the mice were randomly divided into two groups. The normal control group (NC) (n=10) received a standard diet, while the T2DM group was fed a high-fat diet (60% fat) throughout the experiment. This high-fat diet was provided by Nutrition Animal Feed High Technology Co., Ltd. (China) and contained TBHQ (0.067 g / kg), choline tartrate (3 g / kg), L-cysteine ​​(3 g / kg), a vitamin mixture (14 g / kg), a mineral mixture (68 g / kg), cellulose (69 g / kg), lard (301 g / kg), soybean oil 179 (33 g / kg), sucrose (89 g / kg), maltodextrin (225 g / kg), and casein (195 g / kg). After six weeks of dietary intervention, all animals were given free access to water after a 12-hour fast. Mice in the diabetic group received two intraperitoneal injections of STZ (dissolved in 0.1 mol / L citrate buffer at pH 4.5, with a concentration of 60 mg / kg) within 72 hours, while mice in the non-diabetic group received an equal volume of citrate buffer. After a 2-week observation period, fasting blood glucose (FBG) levels were measured using a Sinok glucometer; animals with FBG levels ≥11.1 mmol / L were classified as T2DM models.

[0059] These diabetic mice were randomly divided into two groups: a PCPs treatment group and a model control group (MC group, n=10 each). Based on previous studies, the PCPs group mice were administered PCPs (200 mg / kg / day) via oral gavage daily, while the NC and MC groups received an equal volume of PBS via gavage daily for 6 weeks. Food and water intake were monitored weekly, and fasting blood glucose (FBG) was measured at weeks 8, 9, 11, 13, and 14. Fecal samples were collected at the end of the week. The entire experimental period lasted 14 weeks. After the experiment, mice were anesthetized with sodium pentobarbital, and blood was drawn from the orbital sinus. The pancreas, kidneys, liver, and colon were then immediately removed for further analysis.

[0060] 3.1.3 Serum-related marker analysis

[0061] Serum samples were collected and centrifuged at 2000 r / min for 10 minutes to detect lipopolysaccharide (LPS), insulin-like growth factor-1 (GLP-1), leptin (LEP), adiponectin (ADP), alanine aminotransferase (ALT), fasting serum insulin (FINS), creatinine (Cr), aspartate aminotransferase (AST), blood urea nitrogen (BUN), triglycerides (TG), and total cholesterol. The detection methods followed the instructions of each kit. All kits were purchased from Beijing Solarbio Science & Technology Co., Ltd. The formula for calculating the insulin resistance index (HOMA-IR) is: HOMA-IR = (fasting blood glucose × fasting serum insulin) / 22.5.

[0062] 3.1.4 Oral glucose tolerance test (OGGT)

[0063] Six weeks after administration of PCPs, an oral glucose tolerance test (OGTT) was performed according to the method described in previous literature. Briefly, each mouse was orally administered a glucose solution (2 g / kg), and blood glucose concentrations were measured at 120, 90, 60, 30, and 0 minutes after gavage.

[0064] 3.1.5 Histopathological Analysis

[0065] Tissue sections (5 μm thick) prepared from fixed pancreatic, kidney, liver, and colon samples undergo trimming, washing, dehydration, clearing, and sectioning. The samples are then dewaxed, rehydrated, stained with hematoxylin and eosin (H&E), and mounted with neutral resin. The stained sections are then observed and imaged under a microscope.

[0066] 3.1.6 16S rDNA Sequencing

[0067] Bacterial DNA was extracted from fecal samples using a DNA kit. DNA purity and concentration were assessed using a NanoDrop 2000 spectrophotometer, and DNA integrity was verified by 1% agarose gel electrophoresis. Subsequently, the V3-V4 region of the 16S rRNA gene was amplified using primers 806R (5′-GGACTACHVGGGTWTCTAAT-3′) and 338F (5′-ACTCCTACGGGAGGCAGCAG-3′). After verification by 2% agarose gel electrophoresis, the PCR products were purified using an AxyPrep DNA gel recovery kit, quantified using the Quanti Fluor™-ST blue fluorescence system, and finally sequenced on an Illumina MiSeq PE 300 sequencing platform.

[0068] 3.2 Effects of fecal microbiota transplantation (FMT) on IB and symptoms in T2DM mice

[0069] To investigate the causal relationship between the gut microbiota and the PCPs-mediated effects, a fecal microbiota transplantation (FMT) experiment was conducted. Fecal samples collected from mice in the NC and PCPs-treated groups (as described in Section 3.1.2) at weeks 13 and 14 were stored at -80 °C. These samples were resuspended in sterile saline (100 mg / mL) under sterile low-temperature conditions and centrifuged (4 °C, 1000 r / min, 1 min) to prepare fecal microbiota suspensions. Type 2 diabetic mice selected according to the criteria in Section 3.1.2 were randomly assigned to three groups (n=10 per group): the FMT-NC group, the MC group, and the FMT-PCPs group. The FMT-NC group received fecal suspension (2 mL / kg) from mice in the NC group, the MC group received PBS (2 mL / kg), and the FMT-PCPs group received fecal suspension (2 mL / kg) from mice in the PCPs group. Gavage was performed once daily for four weeks. Food and water intake were recorded weekly throughout the intervention period, and fasting blood glucose (FBG) was measured at weeks 1, 2, 3, and 4. Mice were euthanized at the end of the study, and blood, pancreas, and colon samples were collected for subsequent analysis. Serum biomarker detection (HOMA-IR, LPS, FINS, Cr, BUN, AST, ALT, TG, 241, and TC), inflammatory cytokines (TNF-α, IL-6, and IL-1β), tight junction proteins, and TLR4 / NF-κB pathway detection all followed the procedures described in Section 3.1.

[0070] 3.5 Experimental Results

[0071] 3.5.1 PCPs alleviate T2DM in HFD / STZ-triggered C57BL / 6 mice

[0072] Adopting such Figure 8 The experimental design scheme shown in Figure A is as follows. Figure 8 As shown in Figure B, compared with the control group (NC), the fasting blood glucose (FBG) level in the MC group remained elevated throughout the experiment (P<0.05). Figure 8 The results showed that the values ​​in the PCPs group were significantly lower than those in the MC group (P<0.05). Furthermore, compared with the MC group, the PCPs group also had significantly lower fasting insulin (FINS) levels and a higher HOMA-IR index. Figure 8 E and 8F; P<0.05). PCPs not only significantly reduced serum leptin (LEP) levels, but also increased serum adenosine diphosphate (ADP) and leptin gene 1 (GLP-1) levels. Figure 8 C; P<0.05). These results indicate that PCPs can effectively regulate glucose metabolism and hormone levels in T2DM mice.

[0073] like Figure 8 As shown in Figure D, compared to the MC group, PCPs treatment significantly reduced serum triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) levels, while low-density lipoprotein cholesterol (LDL-C) showed no significant change. These results indicate that PCPs can alleviate hyperlipidemia but cannot directly improve the hyperglycemic state in mice exposed to a high-fat diet. Furthermore, PCPs significantly reduced liver triglyceride and total cholesterol levels induced by a high-fat diet / STZ. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), key indicators of liver injury, were significantly elevated in the MC group compared to the NC group; however, after PCPs administration, the levels of both enzymes decreased. Simultaneously, blood urea nitrogen (BUN) and creatinine (Cr) levels, indicators of renal function, were significantly higher in the MC group than in the NC group (P<0.05), suggesting renal impairment in T2DM mice. PCPs supplementation significantly inhibited the increase of these indicators, indicating its protective effect on renal function.

[0074] In terms of cholesterol metabolism, PCPs can reduce the abnormal mRNA levels of genes such as SREBP-2, LDLR, LSS, MVK, and SQLE in the liver of mice fed a high-fat diet, but HMGCR expression is not affected. Figure 8 E). Histological analysis of the kidney showed ( Figure 8 In the NC group, glomerular structure remained intact and renal function was normal; the MC group, however, exhibited characteristics of renal injury, including glomerular swelling, enlarged intercapsular space, and blurred glomerular boundaries. PCPs intervention significantly improved these pathological changes. Overall, these results indicate that PCPs can effectively alleviate high-fat diet / STZ-induced renal injury.

[0075] 3.5.2 PCPs protect colonic IB in HFD / STZ-triggered T2DM mice

[0076] Mice in the MC group with type 2 diabetes induced by a high-fat diet / streptozotocin (HFD / STZ) exhibited symptoms such as rectal bleeding, diarrhea, and weight loss compared to the NC group. In contrast, mice treated with PCPs showed reduced diarrhea and faster weight recovery. Figure 9 A and 9B). PCPs also significantly prevented colonic shortening in these mice (A and 9B). Figure 9 C and 9D). Myeloperoxidase (MPO) activity, a marker of colonic tissue inflammation, was elevated in the MC group, but significantly decreased after PCP treatment. Figure 9 E).

[0077] The results showed that the FITC-glucan content in the blood of mice in the MC group was significantly increased, while that in the PCPs group was significantly decreased. Figure 9 F).

[0078] Compared with the MC group, PCP supplementation significantly increased the protein expression levels and mRNA expression of closure protein and ZO-1. Figure 9 G). Furthermore, although the levels of pro-inflammatory cytokines (such as IL-6, TNF-α, and IL-1β) and anti-inflammatory cytokines (such as IL-1 and IL-10) were elevated in the MC group, they all decreased significantly after PCPs treatment. Figure 9 H).

[0079] Studies have found that PCPs significantly altered the expression of endoplasmic reticulum stress genes such as Atf4, Perk, and Xbp1s. Figure 9 I), while simultaneously enhancing the expression levels of the core type 3 O-glycan gene B3gnt6, the core type 1 O-glycan gene C1galt1, and the sialyl transferase gene St3gal3. Figure 9 J).

[0080] High-fat diet / streptozotocin (HFD / STZ) induced type 2 diabetes (T2DM) mice showed reduced mucus layer thickness and decreased goblet cell count in the proximal colon, while PCP intervention restored these indicators. Figure 9 K). Compared with the MC group, PCPs treatment significantly increased the expression level of MUC2 protein and the number of goblet cells, thereby enhancing the thickness of the mucus layer.

[0081] The results in summary indicate that PCPs can strengthen the intestinal mucus barrier and improve colonic integrity in high-fat diet / streptozotocin-induced type 2 diabetic mice.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A uniform polysaccharide of Polygonum cuspidatum, characterized in that, The uniform polygon of the giant knotweed is mainly composed of galacturonic acid, rhamnose, galactose, glucuronic acid, glucose, mannose, xylose and glucosamine in a molar ratio of 41.967:24.624:17.951:5.427:4.59:3.501:1.236:0.358, and has a weight average molecular weight of 26.57 kDa, and the structural unit has the following structural formula: 。 2. The method of preparing the homogeneous polysaccharide of the Polygonum cuspidatum according to claim 1, characterized in that, The method comprises the following steps: 1) After the giant knotweed is air-dried at 50 DEG C for 48 h, the air-dried giant knotweed is ground into powder by a grinding machine, and the air-dried giant knotweed powder, a composite enzyme preparation and water are mixed and then added into a traditional Chinese medicine extractor, the extraction temperature is 180 min, each sample is extracted twice, and after centrifugation at 500 r / min, the supernatants of the two times of extraction are combined and concentrated in a 45 DEG C vacuum rotary evaporator; 2) The concentrated giant knotweed polysaccharide solution is subjected to alcohol precipitation to obtain alcohol-precipitated crude polysaccharide; 3) The alcohol-precipitated crude polysaccharide is subjected to protein removal by a Sevag method to obtain total giant knotweed polysaccharide; 4) The total giant knotweed polysaccharide is sequentially subjected to ion exchange chromatography and gel column chromatography for separation and purification, and then concentrated and freeze-dried to obtain the uniform giant knotweed polysaccharide.

3. The preparation method of the uniform polysaccharide of the Reynoutria japonica according to claim 2, characterized in that, The composite enzyme preparation is composed of 500 U / mg of pectinase, 50 U / mg of cellulase and 800 U / mg of papain.

4. The preparation method of the uniform polysaccharide of the Reynoutria japonica according to claim 2, characterized in that, In step 1), the extraction time is 180 min.

5. The preparation method of the uniform polysaccharide of the Reynoutria japonica according to claim 2, characterized by, In step 2), the alcohol precipitation is performed by using 80-95% ethanol, the alcohol precipitation temperature is 4-25 DEG C, and the time is 12-24 h.

6. The preparation method of the uniform polysaccharide of the Reynoutria japonica according to claim 2, characterized in that, In step 3), the Sevag method is used for protein removal, and a mixed solution of chloroform and n-butanol is used for extraction, and the volume ratio of chloroform to n-butanol is 3-5:

1.

7. The preparation method of the uniform polysaccharide of the Reynoutria japonica according to claim 2, characterized in that, In step 4), the ion exchange chromatography column is a DEAE-52 cellulose column, the flow rate of the ion exchange column chromatography is 50 mL / h, the gradient elution program is 0-50 min of water, 20-40 min of 0.1 mol / L NaCl and 30-40 min of 0.3 mol / L NaCl, and the filler of the gel column is Sephadex G-200.

8. The uniform giant knotweed polysaccharide of claim 1 is used for preparing a drug for treating type 2 diabetes.

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