Edible and herbal tea polysaccharide, preparation method thereof and application of edible and herbal tea polysaccharide in preparation of medicine for treating diabetes mellitus

Abstract

The invention discloses edible and herbal tea polysaccharide, a preparation method thereof and application of the edible and herbal tea polysaccharide in preparation of a medicine for treating diabetes mellitus. The edible and herbal tea polysaccharide consists of arabinose, galactose and glucose in a molar ratio of 4: 5: 10, and the molecular weight of the edible and herbal tea polysaccharide is 637.5 kD. The research finds that the edible and herbal tea polysaccharide can effectively improve the glucose tolerance and serum TG, TC, HDL-C and LDL-C levels of alloxan mice; the conditions of intestinal villus edema and shedding of an alloxan mouse can be effectively relieved, and the expression level of Occludin, Claudin-1, ZO-1, TNF-alpha, IL-1beta, IL-6, MPO and NF-kappa B pathway related proteins in colon tissues of the alloxan mouse can be effectively recovered; the herbal tea polysaccharide has a good curative effect on diabetes mellitus and enteritis complicated by diabetes mellitus, and a new treatment approach is provided for diabetes mellitus and enteritis complicated by diabetes mellitus.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an edible herbal tea polysaccharide, a preparation method thereof, and an application thereof in preparing a drug for treating diabetes. Background Art

[0002] Diabetes is the most common chronic disease in the world, which can cause a series of complications, such as insulin resistance, cardiovascular disease, retinopathy, intestinal barrier structure and function damage, etc., which seriously endanger human health and quality of life. Among them, intestinal barrier structure and function damage is one of the most common complications of diabetes. Intestinal barrier dysfunction can promote the absorption of pathogens and toxins from the lumen, induce intestinal inflammation and worsening of hyperglycemia. These pathological factors can worsen the prognosis of diabetes.

[0003] For diabetes and its complications, choosing Chinese medicine to lower blood sugar is a good choice. Chinese medicine lowers blood sugar and has the characteristics of multi-pathway, multi-channel, and multi-target. It has a mild and lasting blood sugar lowering effect, small toxic side effects, and can effectively delay the occurrence and development of complications. Therefore, finding efficient, non-toxic, and new blood sugar lowering drugs from natural medicines has become a research direction for diabetes treatment.

[0004] Edible herbal tea is a commonly used medicinal material of the She ethnic group. It is also a local specialty medicinal material that is commonly used for both medicine and food. It has the effects of dispelling wind and relieving exterior symptoms, clearing away heat and detoxifying, regulating qi and strengthening the spleen, and has a very good medicinal effect on digestive system diseases. The main chemical components of edible herbal tea include volatile oils, flavonoids, alkaloids, etc. At present, the research on edible herbal tea mainly focuses on total flavonoids and total alkaloids, etc., and there are few reports on the research of polysaccharides in edible herbal tea. Summary of the invention

[0005] The invention aims to provide an edible herbal tea polysaccharide and a preparation method thereof and application thereof in the preparation of a diabetes treatment drug, thereby providing a new and effective way for the treatment of diabetes.

[0006] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] The invention firstly provides an edible herbal tea polysaccharide, which is composed of arabinose, galactose and glucose in a molar ratio of 4:5:10 and has a molecular weight of 637.5 kD.

[0008] After further analysis, it was found that the structural formula of the herbal tea polysaccharide is as follows:

[0009]

[0010] Among them, A is Araf-(1→, B is →5)-Araf-(1→, C is →3,5)-Araf-(1→, G is

[0011] →4)-Galp-(1→, E is →3,6)-Galp-(1→, D is Galp-(1→, F is →4,6)-Galp-(1→, M is →3)-Galp-(1→, H is →4)-Glcp-(1→, I is →3)-Glcp-(1→.

[0012] The present invention also provides a method for preparing the above-mentioned edible herbal tea polysaccharide, which comprises the following steps:

[0013] (1) crushing the edible herbal tea, soaking it in ultrapure water and refluxing it to obtain an extract;

[0014] Preferably, the w / v ratio of the herbal tea to ultrapure water is 1:10.

[0015] (2) concentrating the extract and then precipitating it with anhydrous ethanol, and drying the precipitate to obtain a first-grade edible herbal tea crude polysaccharide;

[0016] (3) using the Seveag method to remove protein from the crude edible herbal tea polysaccharide, and then using anhydrous ethanol to precipitate to obtain secondary crude edible herbal tea polysaccharide;

[0017] (4) The secondary edible herbal tea polysaccharide is purified by DEAE-52 fiber column chromatography and Sephadex G-100 gel column chromatography to obtain the edible herbal tea polysaccharide.

[0018] The present invention has found that the edible herbal tea polysaccharide can not only effectively improve the glucose tolerance, serum TG, TC, HDL-C and LDL-C levels of alloxan mice, but also effectively reduce the edema and shedding of intestinal villi of alloxan mice, and effectively restore the expression levels of Occludin, Claudin-1, ZO-1, TNF-α, IL-1β, IL-6, MPO and NF-κB pathway related proteins in the colon tissue of alloxan mice, indicating that the edible herbal tea polysaccharide has a good therapeutic effect on diabetes and diabetic enteritis.

[0019] Therefore, the present invention also provides the use of the above-mentioned edible herbal tea polysaccharide in the preparation of diabetes treatment drugs and diabetes-related enteritis treatment drugs; and a diabetes treatment drug and a diabetes-related enteritis treatment drug, the active ingredients of which at least include the above-mentioned edible herbal tea polysaccharide.

[0020] Compared with the prior art, the technical effects of the present invention are embodied in:

[0021] The present invention separates and identifies edible herbal tea polysaccharides from edible herbal tea for the first time, and finds for the first time that the edible herbal tea polysaccharides can not only effectively improve the glucose tolerance, serum TG, TC, HDL-C and LDL-C levels of alloxan mice, but also effectively reduce the edema and shedding of intestinal villi of alloxan mice, and effectively restore the expression levels of Occludin, Claudin-1, ZO-1, TNF-α, IL-1β, IL-6, MPO and NF-κB pathway related proteins in the colon tissue of alloxan mice; indicating that the edible herbal tea polysaccharides have good therapeutic effects on diabetes and diabetic enteritis, and provide a new treatment approach for diabetes and diabetic enteritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the glucose standard curve;

[0023] Wherein, Concentration means concentration, Absrobance at 490nm means absorbance at 490nm;

[0024] Figure 2 The DEAE-52 elution curve of edible herbal tea polysaccharide;

[0025] Wherein, OD represents absorbance, the same below;

[0026] Figure 3 This is the Sephadex G-100 elution curve of edible herbal tea polysaccharide;

[0027] Figure 4 Calibration curve for polysaccharides in edible herbal tea;

[0028] Wherein, time / min represents the retention time (of the standard) / minute, and log Mw represents the logarithm of the molecular weight (of the standard);

[0029] Figure 5 This is the ion chromatogram of the mixed standard 16 sugars;

[0030] Figure 6 HNMR spectrum of edible herbal tea polysaccharide;

[0031] Figure 7 CNMR spectrum of edible herbal tea polysaccharide;

[0032] Figure 8 HSQC spectrum of edible herbal tea polysaccharides;

[0033] Fig. 9 HMBC spectrum of edible herbal tea polysaccharides;

[0034] Fig.10 COSY spectrum of edible herbal tea polysaccharides;

[0035] Fig.11 Effects of herbal tea polysaccharides on body weight in alloxan diabetic mice (n=5, );

[0036] Wherein, Time (week) indicates medication time (week), Bodyweight (g) indicates body weight (g), control indicates blank control group, model indicates model group, PCSA indicates herbal tea polysaccharide group, METH indicates metformin hydrochloride group; the same below; ## indicates comparison with control, p < 0.01, * indicates comparison with model, p < 0.05, ** indicates comparison with model, p < 0.01;

[0037] Fig.12 Effects of herbal tea polysaccharides on glucose tolerance in alloxan-treated mice (n=5, );

[0038] Wherein, Time(h) represents time, Glucose represents glucose;

[0039] Fig.13 Effects of herbal tea polysaccharides on the area under the glucose tolerance curve in alloxan-treated mice (n=5, );

[0040] Among them, ALX represents alloxan, AUC represents area under the curve; ** represents comparison with the model, p < 0.01;

[0041] Fig.14 Effects of herbal tea polysaccharides on serum TG in mice fed with alloxan (n=5, );

[0042] Fig.15 Effects of herbal tea polysaccharides on serum TC in mice fed with alloxan (n=5, );

[0043] Fig.16 Effects of herbal tea polysaccharides on serum HDL-C in mice fed with alloxan (n=5, );

[0044] Fig.17 Effects of herbal tea polysaccharides on serum LDL-C in mice fed with alloxan (n=5, );

[0045] Figures 14 to 17 In the table, * represents comparison with the model, p < 0.05, ** represents comparison with the model, p < 0.01;

[0046] Fig.18 HE staining images of intestinal tissues of mice in each group;

[0047] Fig.19 Occludin antibody co-staining images of colon tissues of mice in each group;

[0048] Fig. 20 Effects of herbal tea polysaccharides on the level of occludin in colon tissue of mice fed with alloxan (n=3,

[0049] );

[0050] Among them, Related expression of Occludin indicates the relative expression level of Occludin protein, * represents comparison with the model, p < 0.05; the same below;

[0051] Fig.21 The co-staining images of the colon tissues of mice in each group were obtained by using Claudin-1 antibody;

[0052] Fig. 22 Effects of herbal tea polysaccharides on the level of Claudin-1 in colon tissue of mice fed with alloxan (n=3,

[0053] );

[0054] Fig.23 The co-staining images of the colon tissues of mice in each group were obtained by using ZO-1 antibody;

[0055] Fig.24 Effects of herbal tea polysaccharides on the level of ZO-1 in colon tissue of mice fed with alloxan (n=3, );

[0056] Fig.25 Effect of herbal tea polysaccharides on the mRNA level of occludin in the colon tissue of alloxan-treated mice (n=3, );

[0057] Fig.26 Effects of herbal tea polysaccharides on the mRNA level of Claudin-1 in the colon tissue of alloxan-treated mice (n=3, );

[0058] Fig. 27 Effects of herbal tea polysaccharides on the mRNA level of ZO-1 in the colon tissue of mice irradiated with alloxan (n=3, );

[0059] Fig.28 The TNF-α antibody co-staining images of colon tissues of mice in each group;

[0060] Fig.29Effects of herbal tea polysaccharides on TNF-α levels in colon tissue of mice fed with alloxan (n=3, );

[0061] Fig.30 The IL-1β antibody co-staining images of the colon tissues of mice in each group;

[0062] Fig.31 Effects of herbal tea polysaccharides on IL-1β levels in colon tissue of mice fed with alloxan (n=3, );

[0063] Fig.32 The IL-6 antibody co-staining images of the colon tissues of mice in each group;

[0064] Fig.33 Effects of herbal tea polysaccharides on IL-6 levels in colon tissue of mice fed with alloxan (n=3, );

[0065] Fig.34 Effect of herbal tea polysaccharides on the mRNA level of TNF-α in the colon tissue of alloxan-treated mice (n=3, );

[0066] Fig.35 Effects of herbal tea polysaccharides on the mRNA level of IL-1β in the colon tissue of alloxan-treated mice (n=3, );

[0067] Fig.36 Effect of herbal tea polysaccharides on the mRNA level of IL-6 in the colon tissue of alloxan-treated mice (n=3, );

[0068] Fig.37 Effects of herbal tea polysaccharides on the level of MPO in the colon tissue of mice fed with alloxan (n=3, );

[0069] Fig.38 The electrophoresis diagrams of NF-κB pathway-related proteins in the colon tissues of mice in each group;

[0070] Fig.39 Effects of herbal tea polysaccharides on the level of NF-κB pathway-related protein p65 in colon tissue of alloxan mice (n=3, );

[0071] Fig.40 Effects of herbal tea polysaccharides on the level of p-IκB, a protein associated with the NF-κB pathway, in the colon tissue of mice fed alloxan (n=3, ). DETAILED DESCRIPTION

[0072] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0073] In this example, data are reported as mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism 8 (GraphPad software Inc., USA). The effects of hypertension and herbal medicine as grouping variables were analyzed by t-test. In all analyses, two-tailed p < 0.05 was considered statistically significant.

[0074] Example 1 Extraction and structural identification of edible herbal tea polysaccharides

[0075] 1. Preparation of crude polysaccharides for edible herbal tea

[0076] The edible herbal tea medicinal materials were crushed, passed through a 60-mesh sieve, soaked in ultrapure water for 30 minutes, the solid-liquid ratio of the medicinal materials to the ultrapure water was 1:10 (w / v), reflux extraction was performed twice, each time for 1 hour, the extracts were combined, concentrated under reduced pressure, and precipitated with 4 times the volume of anhydrous ethanol, allowed to stand in a refrigerator at 4°C overnight, and filtered; the precipitate was washed with anhydrous ethanol, and vacuum dried to obtain crude edible herbal tea polysaccharides.

[0077] 2. Determination of sugar content of crude polysaccharides in herbal tea

[0078] (1) Drawing of standard curve

[0079] Prepare 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL glucose standards, respectively. Take 0.4 mL of each concentration of glucose standard in a test tube, add 0.2 mL of 5% phenol solution and 2 mL of concentrated sulfuric acid, mix thoroughly, and measure the absorbance at 490 nm (maximum absorption wavelength); draw a standard curve with the concentration of glucose standard as the horizontal axis and the absorbance as the vertical axis. Figure 1 The standard curve equation is shown.

[0080] (2) Determination of sugar content of crude polysaccharides in herbal tea

[0081] A crude polysaccharide solution of edible herbal tea with a concentration of 0.1 mg / mL was prepared, and the absorbance was measured according to the standard curve method. According to the standard curve equation, the sugar content of the crude polysaccharide of edible herbal tea was calculated to be 53.07%.

[0082] 3. Separation and purification of herbal tea polysaccharides

[0083] (1) Seveag method for protein removal

[0084] The protein in the crude polysaccharide of edible herbal tea was removed by Seveag method (chloroform: n-butanol 4:1), and the extract after protein removal was precipitated with anhydrous ethanol, and the supernatant was discarded after standing and stratification; a small amount of distilled water was added to the precipitate to dissolve the precipitate into a polysaccharide solution; the precipitation step was repeated 3 times to ensure that there was no Sevage reagent in the solution.

[0085] (2) Purification of polysaccharides by DEAE-52 fiber column chromatography

[0086] 1) Activation and column loading of DEAE-52 filler: Soak the DEAE-52 filler in distilled water for 3 hours to remove impurities; then soak it in 0.1 mol / L NaOH solution for 1 hour, and wash it with distilled water until the pH value is neutral; then soak it in HCl solution of the same concentration for 1 hour, and wash it with water until the pH value is neutral; then soak it in NaOH solution of the same concentration for 1 hour, and wash it with water until the pH value is neutral; finally, load it into a column with a specification of The flow rate was controlled at 1.5 mL / min in the chromatography column using a constant flow pump.

[0087] 2) DEAE-52 fiber column chromatography separation: Take an appropriate amount of edible herbal tea crude polysaccharide and dissolve it in 10mL ultrapure water. Centrifuge (3500r / min) for 10min, take the supernatant and load it on DEAE-52 column, elute with ultrapure water, collect the eluate, and collect the eluate for 5min / tube. The eluate of each tube is detected by phenol-sulfuric acid method, and the absorbance value is measured at 490nm. The elution time is used as the horizontal axis and the absorbance is used as the vertical axis to draw the elution curve (such as Figure 2 ), the collected eluate was concentrated and dialyzed for 24 h using a dialysis bag with a cutoff of 3500 KDa. The dialysate was concentrated and lyophilized for the next purification step.

[0088] (3) Purification of polysaccharides by Sephadex G-100 gel column chromatography

[0089] 1) Activation and column packing of Sephadex G-100 filler

[0090] Weigh an appropriate amount of Sephadex G-100 filler and soak it in 20 times the volume of ultrapure water for 24 hours to allow it to fully absorb water and swell. Pour off the unsinkable material on the upper layer and then fill it into a container with a specification of The flow rate was controlled at 0.3 mL / min in the chromatography column using a constant flow pump.

[0091] 2)Sephadex G-100 purification

[0092] Weigh an appropriate amount of the freeze-dried sample separated by DEAE-52, dissolve it in ultrapure water, filter it through a 0.45 μm microporous filter membrane, load it onto a Sephadex G-100 column, elute it with ultrapure water, collect the eluate, and the collection time is 15 min / tube. The eluate in each tube is detected by the phenol-sulfuric acid method, and the absorbance value is measured at 490 nm. The elution curve is drawn with the elution time as the horizontal axis and the absorbance as the vertical axis, as shown in FIG. Figure 3 shown.

[0093] 4. Analysis of the structure and characteristics of herbal tea polysaccharides

[0094] (1) Determination of relative molecular weight

[0095] High performance size exclusion chromatography (HPSEC) was used to determine the molecular weight of edible herbal tea polysaccharides. Different relative molecular weight dextran reference substances were used as reference, the retention time or elution volume of the standard substance was used as the horizontal axis, and the logarithm of the molecular weight of the standard substance was used as the vertical axis to draw a calibration curve, and the calibration curve equation (such as Figure 4 ). Dissolve the edible herbal tea polysaccharide in ultrapure water to prepare a 1.0 mg / mL sample solution, measure its retention time, and calculate the molecular weight of each edible herbal tea polysaccharide according to the calibration curve equation. Ultrapure water is used as the mobile phase, the injection volume is 10 μL, the flow rate is 0.6 mL / min, and the column temperature is 30°C.

[0096] The retention time of the edible herbal tea polysaccharide in this example is 7.793 min, and the molecular weight of the edible herbal tea polysaccharide is calculated to be 637.5 kD according to the calibration curve equation.

[0097] (2) Determination of monosaccharide composition

[0098] Weigh 5 mg of sample accurately and place it in an ampoule, add 2 ml of 3M TFA, and hydrolyze at 120°C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry with nitrogen, add 5 ml of water and vortex to mix, pipette 50 μL and add 950 μL of deionized water, centrifuge at 12000 rpm for 5 min; take the supernatant for ion chromatography analysis, and the analysis conditions are as follows:

[0099] Chromatographic column: Dionex CarbopacTM PA20 (3*150mm);

[0100] Mobile phase: A: H2O; B: 15mM NaOH; C: 15mMNaOH&100mM NaOAC;

[0101] Flow rate: 0.3ml / min;

[0102] Injection volume: 5 μL;

[0103] Column temperature: 30°C;

[0104] Detector: Electrochemical detector.

[0105] In this embodiment, fucose, galactosamine hydrochloride, rhamnose, arabinose, glucosamine hydrochloride, galactose, glucose, N-acetyl-D-glucosamine, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid are used to prepare a mixed standard. The ion chromatogram of the mixed standard 16 sugars is as follows: Figure 5 The results of monosaccharide analysis are shown in Table 1.

[0106] Table 1 Monosaccharide analysis results

[0107]

[0108]

[0109] It can be seen that the monosaccharides of the edible herbal tea polysaccharide of this embodiment are composed of arabinose, galactose and glucose, and the molar ratio is 4:5:10.

[0110] (3) NMR analysis

[0111] Take an appropriate amount of edible herbal tea polysaccharide and dissolve it in D2O. Then use nuclear magnetic resonance spectrometer to measure the 1 H-NMR spectrum (such as Figure 6 )and 1 C-NMR spectrum (such as Figure 7 ), HSQC spectrum, HMBC spectrum, COSY spectrum are as follows Figure 8 , Fig. 9 and Fig.10 .

[0112] (4) Methylation assay

[0113] After methylation, hydrolysis and acetylation, the edible herbal tea polysaccharide samples were measured by GC-MS and compared with the standard mass spectrum library. Specifically including:

[0114] First, weigh the edible herbal tea polysaccharide sample (2-3 mg) and place it in a glass reaction bottle, add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution; react in a magnetic stirring water bath at 30°C for 60 minutes; finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction.

[0115] Then, take the methylated edible herbal tea polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) to hydrolyze for 90 minutes, and evaporate to dryness on a rotary evaporator; add 2 ml of double distilled water to the residue, reduce it with 60 mg of sodium borohydride for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry it in an oven at 101°C, then add 1 ml of acetic anhydride to acetylate at 100°C for 1 hour, and cool it. Then add 3 mL of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4-5 times to remove excess acetic anhydride.

[0116] Then, the acetylated product was dissolved with 3 mL of CH2Cl2 and transferred to a separatory funnel. After adding a small amount of distilled water and shaking sufficiently, the upper aqueous solution was removed, and this process was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, fixed to 10 mL, and placed in a liquid phase vial.

[0117] Finally, the acetylation product samples were determined using a Shimadzu GCMS-QP 2010 gas chromatograph-mass spectrometer. The GC-MS conditions were: RXI-5SILMS column 30m*0.25mm*0.25μm; the programmed temperature conditions were: starting temperature 120℃, heating to 250℃ / min at 3℃ / min; maintaining for 5min; the inlet temperature was 250℃, the detector temperature was 250℃ / min, the carrier gas was helium, and the flow rate was 1mL / min.

[0118] The analysis results are shown in Table 2.

[0119] Table 2 Analysis of results of polysaccharide methylated sugar alcohol acetyl ester (PMAA)

[0120]

[0121]

[0122] (5) Prediction of the structure of herbal tea polysaccharides

[0123] Table 3. Polysaccharides in herbal tea 1 H and 13 C NMR chemical shift assignment (δ unit: ppm)

[0124]

[0125] According to the analysis results of Table 2 and Table 3, the structural formula of edible herbal tea polysaccharide is predicted as follows:

[0126]

[0127] Example 2 Pharmacological Analysis of Edible Herbal Tea Polysaccharides

[0128] (1) Experimental animals

[0129] Twenty C57BL / 6 male mice were raised in the Animal Experiment Center of Zhejiang Institute of Traditional Chinese Medicine, with a body weight of 20±3g. The temperature was 25℃ and the humidity was 60±5%. Subsequent experiments were conducted after 7 days of adaptive feeding.

[0130] (2) Establishment and grouping of T1DM mouse model

[0131] After the adaptive feeding, 5 mice were randomly selected as the blank control group, and the initial blood glucose values ​​of all mice were measured. The other 15 mice were intraperitoneally injected with 180 mg / kg of alloxan to establish a T1DM mouse model. 72 hours after the intraperitoneal injection of alloxan, the mice were fasted for 8 hours (fasting but not water), and tail vein blood was collected for FBG (fasting blood glucose) measurement (see Table 4).

[0132] Table 4 Effects of alloxan on mouse FBG

[0133]

[0134] The standard for successful modeling is that FBG is higher than 11.1 mmol / L. As can be seen from Table 4, the FBG of mice in the model group was 12.97 mmol / L, indicating that the modeling was successful.

[0135] The mice with successful modeling were randomly divided into 3 groups, 5 in each group, namely the model group, the herbal tea polysaccharide group, and the metformin hydrochloride group. Among them, the herbal tea polysaccharide group was gavaged with 50 mg / kg / d of herbal tea polysaccharide; the metformin hydrochloride group was gavaged with 120 mg / kg / d of metformin hydrochloride. The blank group and the model group were gavaged with an equal amount of normal saline; the body weight of all mice was recorded every week; after 4 weeks, the mice were anesthetized and blood was collected from their orbits, serum was separated, and various biochemical indicators were measured.

[0136] (3) Determination of various indicators of diabetic mice

[0137] 1) Weight

[0138] Body weight was measured by weighing method, and the results are shown in Fig.11 .

[0139] Depend on Fig.11 It can be seen that compared with the normal group of mice, the body weight of the model group decreased significantly since the first week of administration (p < 0.01); compared with the model group of mice, the body weight of the herbal tea polysaccharide group and the metformin hydrochloride group increased significantly (p < 0.05), and the herbal tea polysaccharide group was very close to the normal group of mice; indicating that herbal tea polysaccharides can significantly inhibit weight loss in diabetic mice.

[0140] 2) Glucose tolerance test (OGTT)

[0141] After the last administration, the mice were fasted overnight and gavage concentration was 2.0 g / kg glucose. The blood glucose levels of mice were measured and recorded at 0, 30, 60, and 120 min after gavage. The calculation formula used was as follows: AUC = 0.5 × 0 min blood glucose + 30 min blood glucose + 1.5 × 60 min blood glucose + 120 min blood glucose.

[0142] Test results such as Fig.12 and Fig.13 As shown. It can be seen that compared with the blank group mice, the glucose tolerance of the model group was significantly reduced; but after being treated with herbal tea polysaccharides or metformin hydrochloride, the glucose tolerance of mice in each group showed a recovery trend (see Fig.12 ). At the same time, compared with the blank group mice, the area under the oral glucose tolerance curve (AUC) of the model group mice increased significantly; while compared with the model group mice, the AUC values ​​of the herbal tea polysaccharide group and the metformin hydrochloride group mice were significantly reduced (see Fig.13 ), which had no significant difference compared with the blank group mice.

[0143] (4) Measurement of serum biochemical parameters

[0144] Blood was collected from the mouse orbits and centrifuged at 3500 rpm for 15 min at 4°C; each sample was stored at 4°C for biochemical analysis. ELISA was used to determine the levels of TG, TC, HDL-C, and LDL-C in mouse serum according to the procedures described in the commercial kit; the results are shown in Fig.14 , Fig.15 , Fig.16 and Fig.17 shown.

[0145] Depend on Figures 14 to 17 It can be seen that the TC, TG, HDL-C and LDL-C levels of the model group mice were significantly different from those of the normal group (P < 0.01), but the levels of TC, TG and LDL-C were significantly improved after treatment with herbal tea polysaccharides (P < 0.05, p < 0.01); compared with the model group, the HDL-C level in the herbal tea polysaccharides group showed a certain upward trend, but the difference was not statistically significant (P>0.05).

[0146] (5) Histopathological analysis

[0147] Colon tissues from each mouse were removed and fixed in 10% neutral buffered formalin for 24 h. Tissue samples were dehydrated and embedded in paraffin. Each tissue wax block was sectioned (5 μm) over its entire length, and the sections were stained with hematoxylin and eosin (H&E).

[0148] Fig.18The H&E staining results of the tissues show color images captured under an optical microscope at 400 times magnification. It can be seen that the intestinal mucosal epithelial cell layer of the blank group mice was intact, the intestinal villi were clearly visible, and no obvious pathological damage was observed; while the intestinal mucosal structure of the model group mice was chaotic, the villi fell off in pieces, and there was exposure of the lamina propria, necrosis, and inflammatory cell infiltration; the edema and shedding of the intestinal villi of the mice in the low-dose herbal tea group and the metformin hydrochloride group were significantly reduced.

[0149] (6) Immunohistological analysis

[0150] 1) Effects of herbal tea polysaccharides on the genes and proteins of occludin, claudin-1, and ZO-1 in the colon tissue of alloxan-treated mice

[0151] Part of the colon tissue samples were fixed with 4% paraformaldehyde at room temperature for 24 h and then embedded in paraffin. Each block was sectioned (3 μm) over its entire length, then co-stained with ZO-1, Claudin-1, and Occludin antibodies, and images were captured under a fluorescence microscope equipped with a charge-coupled device camera (Olympus); and the expression levels of ZO-1, Claudin-1, and Occludin genes and proteins were analyzed.

[0152] like Fig.19 and Fig. 20 , Fig.21 and Fig. 22 , Fig.23 and Fig.24 As shown in the results, compared with the control group, the levels of Occludin, Claudin-1 and ZO-1 proteins in the colon tissue of the model group were significantly decreased (P < 0.05); compared with the model group, the expressions of Occludin, Claudin-1 and ZO-1 proteins in the colon tissue of the mice in the herbal tea polysaccharide group and the metformin hydrochloride group were increased (P < 0.05).

[0153] like Fig.25 , Fig.26 and Fig. 27 As shown, the analysis results at the gene level were consistent with those at the protein level.

[0154] 2) Effects of herbal tea polysaccharides on TNF-α, IL-1β, and IL-6 genes and proteins in colon tissue of alloxan-treated mice

[0155] The analysis method is the same as above.

[0156] like Fig.28 and Fig.29 , Fig.30 and Fig.31 , Fig.32 and Fig.33As shown in the results, compared with those in the control group, the levels of TNF-α, IL-1β, and IL-6 proteins in the colon tissue of the model group were significantly increased (P < 0.05); compared with the model group, the expressions of TNF-α, IL-1β, and IL-6 proteins in the colon tissue of the mice in the herbal tea polysaccharide group and the metformin hydrochloride group were significantly decreased (P < 0.05).

[0157] like Fig.34 , Fig.35 and Fig.36 As shown, the analysis results at the gene level were consistent with those at the protein level.

[0158] 3) Effect of herbal tea polysaccharides on MPO in colon tissue of alloxan-treated mice

[0159] like Fig.37 As shown, compared with the control group, the MPO protein content in the colon tissue of the model group was significantly increased (P < 0.05); compared with the model group, the MPO protein expression in the colon tissue of the mice in the herbal tea polysaccharide group and the metformin hydrochloride group was significantly decreased (P < 0.05).

[0160] 4) Effects of herbal tea polysaccharides on NF-κB pathway-related proteins in colon tissue of alloxan-treated mice

[0161] like Fig.38 , Fig.39 and Fig.40 As shown in the results, compared with the control group, the levels of p65 and p-IκB proteins in the colon tissue of the model group were significantly increased (P < 0.01, P < 0.05); compared with the model group, the expressions of p65 and p-IκB proteins in the colon tissue of the mice in the herbal tea polysaccharide group and the metformin hydrochloride group were decreased (P < 0.01, P < 0.05).

Claims

1. A herbal tea polysaccharide, characterized in that: It is composed of arabinose, galactose and glucose in a molar ratio of 4:5:10, with a molecular weight of 637.5kD.

2. The edible herbal tea polysaccharide according to claim 1, characterized in that The structural formula is as follows: Among them, A is Araf-(1→, B is →5)-Araf-(1→, C is →3,5)-Araf-(1→, G is →4)-Galp-(1→, E is →3,6)-Galp-(1→, D is Galp-(1→, F is →4,6)-Galp-(1→, M is →3)-Galp-(1→, H is →4)-Glcp-(1→, I is →3)-Glcp-(1→.

3. The method for preparing the edible herbal tea polysaccharide according to claim 1 or 2, characterized in that: The following steps are involved: (1) crushing the edible herbal tea, soaking it in ultrapure water and refluxing it to obtain an extract; (2) concentrating the extract and then precipitating it with anhydrous ethanol, and drying the precipitate to obtain a first-grade edible herbal tea crude polysaccharide; (3) using the Seveag method to remove protein from the crude edible herbal tea polysaccharide, and then using anhydrous ethanol to precipitate to obtain secondary crude edible herbal tea polysaccharide; (4) The secondary edible herbal tea polysaccharide is purified by DEAE-52 fiber column chromatography and Sephadex G-100 gel column chromatography in sequence to obtain the edible herbal tea polysaccharide.

4. The preparation method according to claim 3, characterized in that: In step (1), the material-liquid ratio (w / v) of the edible herbal tea and ultrapure water is 1:

10.

5. Use of the edible herbal tea polysaccharide according to claim 1 or 2 in the preparation of a drug for treating diabetes.

6. Use of the edible herbal tea polysaccharide according to claim 1 or 2 in the preparation of a drug for treating diabetes mellitus complicated with enteritis.

7. A drug for treating diabetes, characterized in that: The effective ingredients at least include the edible herbal tea polysaccharide as claimed in claim 1 or 2.

8. A drug for treating diabetes-related enteritis, characterized in that: The effective ingredients at least include the edible herbal tea polysaccharide as claimed in claim 1 or 2.

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