Application of cichorium glandulosum polysaccharide in preparation of medicine for basic characterization and relieving type 2 diabetes mellitus

Abstract

The invention provides an application of cichorium glandulosum polysaccharide in preparation, basic characterization and type 2 diabetes mellitus relieving medicines, and belongs to the technical field of medicines. Animal experiment results show that after the cichorium glandulosum polysaccharide is applied to type 2 diabetic mice, serum biochemical indexes can be adjusted, the blood sugar level can be reduced, organ injuries can be protected or repaired, and PI3K / AKT / mTOR signal channel related protein expression in the main glycometabolism target organ liver can be adjusted. Therefore, the prepared cichorium glandulosum polysaccharide can be used as a medicine for relieving type 2 diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of inulin from Cichorium glandulosum Boiss. in the preparation, basic characterization and drugs for alleviating type 2 diabetes. Background Art

[0002] Diabetes is a chronic metabolic disease, usually characterized by hyperglycemia, and is closely related to insulin secretion and disorders of protein, carbohydrate and fat metabolism. Due to changes in work and eating habits, more and more people are suffering from diabetes. The latest statistics of the International Diabetes Federation predict that by 204, the number of patients worldwide will reach 642 million, more than 90% of which are type 2 diabetes (T2DM). Diabetes not only seriously affects the quality of life of patients, but also brings a significant economic burden to the country. In addition, the mortality rate of diabetic patients is relatively high, and they are prone to develop various complications. The current treatment methods can not only prevent the occurrence and development of the disease, but also bring inevitable adverse reactions, such as hypoglycemia, constipation and other problems. Therefore, it is urgent to find drugs with low toxicity and good hypoglycemic effect.

[0003] Cichorium glandulosum Boiss. is widely distributed in Xinjiang and belongs to a traditional Chinese medicine with both medicinal and edible properties. It tastes slightly bitter and salty, is cool in nature, and belongs to the liver, gallbladder and stomach meridians. It has the effects of clearing heat and detoxifying, clearing the liver and gallbladder, strengthening the stomach and promoting digestion, diuresis and detumescence. In recent years, Cichorium glandulosum Boiss. has been developed into Chinese patent medicines with significant curative effects, such as Hugan Buzure Granules, Compound Muniziqi Granules, Qingre Kasen Granules, Yanxiao Dina'er Syrup, etc., and has been widely used in clinical treatment of various liver and gall diseases. Modern research shows that Cichorium glandulosum Boiss. is rich in active ingredients such as polysaccharides, flavonoids and terpenoids, and has significant pharmacological activities such as lipid-lowering, hypoglycemic, liver-protecting and antibacterial.

[0004] Plant-based treatment methods have attracted many researchers to explore various natural sources to find new compounds for treating diseases. It has been found that many chemical components in plants, such as polysaccharides, polyphenols and alkaloids, have good hypoglycemic biological activities. Polysaccharides are natural dietary fibers, which can prevent and treat diabetes with little side effects, and have gradually become a research hotspot. Polysaccharides are high molecular weight biopolymers formed by the linkage of more than ten monosaccharides through glycosidic bonds, and their molecular weights are generally between tens of thousands and millions. At present, many studies have shown that polysaccharides can regulate diabetes through different mechanisms. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of inulin from Cichorium glandulosum Boiss. in the preparation, basic characterization and drugs for alleviating type 2 diabetes.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] Application of inulin polysaccharide in the preparation, basic characterization and drugs for alleviating type 2 diabetes

[0008] Preferably, the extraction method of the inulin polysaccharide includes: after inulin is soaked with ethanol, extracted by enzyme method, precipitated with alcohol, decolorized and deproteinized, inulin polysaccharide is obtained; the ethanol concentration is 90-98%.

[0009] Preferably, the enzyme extraction is carried out with constant temperature oscillation for 1-3 h, the extraction temperature is 40-60 °C, the solid-liquid ratio is 1:(30-50) g / mL, and the amount of cellulase is 1-3%.

[0010] Preferably, the alcohol precipitation includes fully mixing the concentrated extract with ethanol and standing at 0-4 °C for 12-24 h; the ethanol concentration in the mixed solution is 70-80%.

[0011] Preferably, the decolorization and deproteinization adopt the macroporous resin adsorption method, and the macroporous resin is of AB-8 type.

[0012] Preferably, the inulin polysaccharide can reduce the phenomenon of high blood sugar in type 2 diabetes.

[0013] Preferably, the inulin polysaccharide can improve the serum biochemical indexes and organ damage of type 2 diabetes.

[0014] Preferably, the inulin polysaccharide can alleviate type 2 diabetes through the PI3K / AKT / mTOR signaling pathway.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides an application of inulin polysaccharide in the preparation, basic characterization and drugs for alleviating type 2 diabetes. The results of animal experiments show that after the inulin polysaccharide of the present invention is used in type 2 diabetic mice, it can regulate serum biochemical indexes, reduce blood sugar levels, protect or repair organ damage, and regulate the expression of proteins related to the PI3K / AKT / mTOR signaling pathway in the main sugar metabolism target organ liver. Therefore, the inulin polysaccharide prepared by the present invention can be used as a drug for alleviating type 2 diabetes. Description of the Drawings

[0017] Figure 1 is the color contrast before and after decolorization of macroporous resin;

[0018] Figure 2 is the ultraviolet scanning chart of inulin polysaccharide;

[0019] Figure 3 is the Fourier transform infrared spectrum chart of inulin polysaccharide;

[0020] Figure 4 is the molecular weight spectrum chart of inulin polysaccharide;

[0021] Figure 5 is the analysis of monosaccharide composition of Cichorium glandulosum polysaccharide; Figure A is the mixed standard map of 11 monosaccharides; Figure B is the monosaccharide spectrogram of Cichorium glandulosum polysaccharide;

[0022] Figure 6 is the scanning electron microscope image of Cichorium glandulosum polysaccharide; Figure A is the electron microscope image of Cichorium glandulosum polysaccharide at 500 times magnification; Figure B is the electron microscope image of Cichorium glandulosum polysaccharide at 5000 times magnification;

[0023] Figure 7 is the graph of the effect of Cichorium glandulosum polysaccharide on blood glucose in type 2 diabetic mice; Figure A is the change in OGTT of the normal group and the model group after 7 weeks of high-sugar and high-fat diet; Figure B is the change in fasting blood glucose of mice in each group during treatment; Figure C is the change in OGTT of mice in each group at the third week of treatment; Figure D is the area under the curve of OGTT of mice in each group at the third week of treatment; Figure E is the change in ITT of mice in each group at the third week of treatment; Figure F is the area under the curve of ITT of mice in each group at the third week of treatment; Figure G is the content of glycosylated serum protein in mice in each group; Figure H is the content of liver glycogen in mice in each group;

[0024] Figure 8 is the graph of the effect of Cichorium glandulosum polysaccharide on serum lipids and liver function in type 2 diabetic mice; Figure A is the content of TG in mice in each group; Figure B is the content of TC in mice in each group; Figure C is the content of HDL-C in mice in each group; Figure D is the content of LDL-C in mice in each group; Figure E is the content of ALT in mice in each group; Figure F is the content of AST in mice in each group;

[0025] Figure 9 is the graph of the effect of Cichorium glandulosum polysaccharide on serum insulin and insulin resistance index in type 2 diabetic mice; Figure A is the content of serum insulin in mice in each group; Figure B is the insulin resistance index of mice in each group;

[0026] Figure 10 is the graph of the effect of Cichorium glandulosum polysaccharide on oxidative stress in type 2 diabetic mice; Figure A is the content of MDA in mice in each group; Figure B is the content of GSH in mice in each group; Figure C is the content of CAT in mice in each group; Figure D is the content of T-SOD in mice in each group;

[0027] Figure 11 is the graph of the effect of Cichorium glandulosum polysaccharide on organ index in type 2 diabetic mice; Figure A is the liver index of mice in each group; Figure B is the kidney index of mice in each group;

[0028] Figure 12 is the graph of the effect of Cichorium glandulosum polysaccharide on liver tissue morphology in type 2 diabetic mice; Figure A is the liver morphology of mice in each group; Figure B is the HE staining map (×200, ×400); Figure C is the ORO staining map (×200, ×400); Figure D is the PAS staining map (×200, ×400);

[0029] Figure 13 Effect diagram of inulin polysaccharide on pancreatic tissue of type 2 diabetic mice (×200, ×400);

[0030] Figure 14 Effect diagram of inulin polysaccharide on colonic tissue morphology and related protein expression in type 2 diabetic mice; Figure A is the diagram of colonic morphology; Figure B is the HE staining of pancreas (×200, ×400); Figure C is the statistics of colonic length; Figure D is the immunoblotting of ZO-1 and Occludin proteins; Figure E is the statistics of Occludin protein expression; Figure F is the statistics of ZO-1 protein expression;

[0031] Figure 15 Effect diagram of inulin polysaccharide on protein expression of PI3K / AKT / mTOR signaling pathway in liver tissue of type 2 diabetic mice; Figure A is the immunoblotting of P-mTOR, mTOR, P-PI3K, PI3K, P-AKT, and AKT proteins; Figure B is the expression ratio of P-AKT / AKT; Figure C is the expression ratio of P-PI3K / PI3K; Figure D is the expression ratio of P-mTOR / mTOR;

[0032] Note: Figure 7-15 Data are expressed as mean ± SD. Compared with the control group, the difference was statistically significant (P<0.05); compared with the control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; Detailed implementation manners

[0033] The present invention provides the application of inulin polysaccharide in the preparation, basic characterization, and alleviation of drugs for type 2 diabetes.

[0034] In the present invention, the preparation method of the inulin polysaccharide preferably includes:

[0035] After Cichorium glandulosum Boiss. et Huet was soaked in ethanol, extracted by enzymatic method, precipitated with alcohol, decolorized and defatted, Cichorium glandulosum Boiss. et Huet polysaccharide was obtained. The ethanol concentration was 90-98%, more preferably 95%. The enzymatic extraction was carried out by constant temperature shaking for 1-3 h, preferably 2 h, the extraction temperature was 40-60 °C, preferably 50 °C, the solid-liquid ratio was 1:(30-50) g / mL, preferably 1:30 g / mL, and the amount of cellulase was 1-3%, more preferably 2%. The alcohol precipitation included fully mixing the concentrated extract with ethanol, standing at 0-4 °C for 12-24 h, more preferably standing at 4 °C for 24 h, and repeating the operation 3 times. The ethanol concentration in the mixed solution was 70-80%, preferably 75%. The decolorization and defatting were carried out by macroporous resin adsorption method. The macroporous resin was AB-8 type, more preferably AB-8 macroporous resin chromatography column method. In the present invention, after repeated soaking with 95% ethanol, a large amount of fat, pigment, small molecular lipid-soluble compounds, etc. could be removed. Under the conditions of the enzymatic extraction, more polysaccharides could be obtained. Under the conditions of the alcohol precipitation method, most of the polysaccharides could be precipitated, and most of the pigments and small molecular substances could be removed by repeated operation. The decolorization and defatting by AB-8 macroporous resin method could change the polysaccharide from dark brown to light yellow and remove a large amount of protein, which was suitable for industrial production application.

[0036] In the present invention, the Cichorium glandulosum Boiss. et Huet polysaccharide could relieve the symptoms of hyperglycemia in type 2 diabetes; the Cichorium glandulosum Boiss. et Huet polysaccharide could reduce the phenomenon of high blood sugar in type 2 diabetes; the Cichorium glandulosum Boiss. et Huet polysaccharide could improve the serum biochemical indexes and organ damage in type 2 diabetes; the Cichorium glandulosum Boiss. et Huet polysaccharide could relieve type 2 diabetes through the PI3K / AKT / mTOR signaling pathway. After the Cichorium glandulosum Boiss. et Huet polysaccharide was used in type 2 diabetic mice, it could regulate the serum biochemical indexes, reduce the blood sugar level, protect or repair the organ damage, and regulate the expression of proteins related to the PI3K / AKT / mTOR signaling pathway in the liver, the main target organ of sugar metabolism. The experimental results showed that the Cichorium glandulosum Boiss. et Huet polysaccharide of the present invention had potential medicinal value in the treatment of type 2 diabetes.

[0037] Except that Cichorium glandulosum Boiss. et Huet involved in the present invention was purchased from Hotan County, Xinjiang Uygur Autonomous Region and identified by Professor Qin Dongmei of the College of Pharmacy, Shihezi University as the underground root part of Cichorium glandulosum Boiss. et Huet (Cichorium glandulosum Boiss.et Huet, CG), a plant of the genus Cichorium in the Compositae family, there were no special limitations for the other raw materials, and commercially available products in the art could be used.

[0038] The present invention will be described in detail below with reference to the accompanying drawings. The description of this part is only exemplary and explanatory, and should not have any limiting effect on the protection scope of the present invention. In addition, those skilled in the art can make corresponding combinations of the features in the examples and different examples in this document according to the description of this document.

[0039] Example 1

[0040] 1.1 Preparation of Cichorium glandulosum polysaccharide

[0041] An appropriate amount of CG roots was soaked in 95% ethanol and repeatedly soaked until the ethanol solution became colorless. The residue was dried until there was no alcohol smell, pulverized, and sieved through a 24-mesh sieve to obtain CG root residue powder. 2% cellulase was added to the CG residue powder, and extraction was carried out by constant temperature shaking at 50 °C, with a solid-liquid ratio of 1:30. After extraction, it was concentrated to 1 / 10 of the original solution, centrifuged at 4500 r / min for 10 minutes, the precipitate was discarded, the supernatant was collected, 95% ethanol with a concentration about 3 times that of the concentrated solution was added to about 70%, and it was left at 4 °C for 24 hours. The supernatant was discarded, 95% ethanol was added again to about 70%, and the operation was repeated two or three times. Then it was centrifuged at 5000 r / min for 10 minutes, the supernatant was discarded, the precipitate was collected, volatilized until there was no alcohol smell, and freeze-dried to obtain a powder for standby. The powder was dissolved in an appropriate amount of pure water, added to a pretreated AB-8 macroporous resin column, slowly absorbed, eluted at a flow rate of 1 mL / min, detected by the phenol-sulfuric acid method, and collected until it was colorless. The eluate was collected, concentrated, and freeze-dried to obtain the crudely purified CG polysaccharide (CGP) with preliminary impurity removal.

[0042] 1.2 Basic characterization of Cichorium glandulosum polysaccharide

[0043] 1.2.1 Ultraviolet-visible spectrophotometer (UV-Vis)

[0044] A 2 mg / mL CGP solution was prepared, and the absorption wavelength in the range of 250 - 600 nm was scanned with a UV spectrophotometer. If absorption peaks appeared at 260 nm and 280 nm, it indicated the presence of substances such as proteins and nucleic acids.

[0045] 1.2.2 Fourier transform infrared spectroscopy (FT-IR)

[0046] Fourier transform infrared spectroscopy was used to determine the characteristic absorption peaks of CGP in the wavelength range of 4000 - 500 cm -1

[0047] 1.2.3 Molecular weight determination

[0048] A gel chromatography differential system was used. According to the properties of the compound, a gel exclusion chromatography column with an appropriate molecular weight range was selected. The column temperature was 40 °C; the injection volume was 100 μL; the mobile phase was 0.1 M aqueous sodium nitrate solution; the flow rate was 1.0 mL / min; the elution gradient: isocratic for 40 min; the dn / dc value was 0.138 mL / g, and the standard was dextran solutions with different molecular weights to determine the molecular weight of CGP.

[0049] 1.2.4 Monosaccharide composition analysis

[0050] ​Add 11 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galactose, glucuronic acid) into ampoules respectively. Subsequently, hydrolyze them with 2 mL of 3M trans-fatty acid at 120 °C for 3 hours. The generated acid hydrolysis solution is evaporated in nitrogen, and then 5 mL of water is added for rotation to prepare the standard stock solution. The absolute quantification method is used to evaluate the quantity of each monosaccharide, and the molar ratio is determined according to the molar mass of the monosaccharide. A similar treatment is also performed on 5 mg of CGP. Centrifuge at 12,000 r / min for 5 minutes and conduct ion chromatography analysis.

[0051] 1.2.5 Scanning Electron Microscope (SEM)

[0052] Use a scanning electron microscope to observe the molecular morphology of CGP, and use an ion sputtering sprayer to uniformly deposit a thin gold coating on the conductive adhesive for observation at 500× and 5000× conditions respectively.

[0053] Example 2

[0054] Animal experiments

[0055] 2.1 Animal experiment model establishment

[0056] 70 male Kunming mice are adaptively fed for 2 weeks. Randomly select 10 of them as the blank group and give them normal feed, and the remaining mice are used as the experimental group and fed with a high-sugar and high-fat diet. At the 7th week, an oral glucose tolerance test (OGTT) is performed on each group of mice. After fasting the experimental group mice at the 8th week, inject STZ (dose: 100 mg / kg) intraperitoneally, and the blank group is injected with an equal amount of citrate buffer solution for 3 days. One week later, if the fasting blood glucose of the experimental group mice exceeds 11.1 mmol / L, it is regarded as successful model establishment. For the mice with failed model establishment, a one-time supplementary injection of STZ (dose: 120 mg / kg) is given until the model is successfully established.

[0057] 2.2 Animal experiment grouping and drug administration

[0058] The mice were re-divided into 6 groups with six mice in each group. The specific grouping was as follows: blank group (Ctrl), model group (Mod), positive control group (Met), low-dose group of crude polysaccharide from Cichorium glandulosum Boiss. et Huet (L-CGP), medium-dose group of crude polysaccharide from Cichorium glandulosum Boiss. et Huet (M-CGP), and high-dose group of crude polysaccharide from Cichorium glandulosum Boiss. et Huet (H-CGP). Among them, the Ctrl group and the Mod group were gavaged with normal saline, the Met group was given metformin hydrochloride (250 mg / kg·bw), and the L-CGP, M-CGP, and H-CGP groups were respectively given crude polysaccharide from Cichorium glandulosum Boiss. et Huet at 300 mg / kg·bw, 600 mg / kg·bw, and 1200 mg / kg·bw (the dosing doses were determined according to the preliminary pre-tests). Each group was dosed according to a body weight of 0.1 mL / 10 g. Gavage was performed once a day, and the changes in fasting blood glucose of the mice in each group were recorded. Administration was continued for four weeks. An oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT) were performed in the third week, and dissection was carried out in the fourth week.

[0059] 2.3 Sample collection

[0060] After blood was collected from the mouse eyeballs, the mouse was sacrificed by cervical dislocation. The blood was placed in a refrigerator at 4°C for static preservation. After dissection, the liver, colon, and pancreas were quickly removed. One part was stored at room temperature in 4% paraformaldehyde, and the other part was wrapped with tin foil, labeled, and frozen in a -80°C refrigerator. After the dissection was completed, the blood was balanced. The serum was collected and stored in a -20°C refrigerator after centrifugation at 3500 r / min for 15 min using a centrifuge.

[0061] 2.4 Determination of fasting blood glucose

[0062] After the mice were fasted for 12 h (prepare 20% glucose solution to prevent death from hypoglycemia), the tail tips of the mice were wiped with an alcohol cotton ball and scissors. The tail tip of the mouse was slightly cut with scissors, and the mouse was gently pressed from the root of the tail to the tip. After discarding the first drop of blood, a SanNuo blood glucose meter was used to insert the blood glucose paper for measurement, and the reading was recorded after waiting for 5 seconds.

[0063] 2.5 Oral glucose tolerance test

[0064] After the mice were fasted for 12 h (prepare an insulin solution at 2 U / mL (refrigerated) to prevent death from hyperglycemia), the mice were first weighed and their fasting blood glucose values were measured. Then, a 20% glucose aqueous solution was gavaged (at a body mass of 0.1 mL / 10 g), and the blood glucose values at 30, 60, 90, and 120 min after gavage were recorded. A blood glucose change curve was plotted, and the area under the blood glucose curve (Area under the curve, AUC) of each group was calculated.

[0065] Oral glucose tolerance AUC (h·mmol / L) = 0.5 × [(A + B) × 0.5 + (B + C) × 0.5 + (C + D) × 0.5 + (D + E)]

[0066] The blood glucose values at 0, 0.5, 1.0, 1.5, and 2.0 h are A, B, C, D, and E respectively.

[0067] 2.6 Insulin tolerance test

[0068] After fasting the mice for 12 h (prepare 20% glucose solution to prevent death from hypoglycemia), weigh the mice and measure their fasting blood glucose values first. Then inject an insulin solution at 2 U / mL intraperitoneally, record the blood glucose values at 30, 60, 90, and 120 min, plot the blood glucose change curve, and calculate the area under the blood glucose curve (AUC) for each group.

[0069] Insulin tolerance AUC (h·mmol / L) = 0.5 × [(A + B) × 0.5 + (B + C) × 0.5 + (C + D) + (D + E)]

[0070] The blood glucose values at 0, 0.5, 1.0, 1.5, and 2.0 h are A, B, C, D, and E respectively.

[0071] 2.7 Determination of biochemical indicators

[0072] Determine glycated serum protein (GSP), liver glycogen, insulin and insulin resistance index, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and malondialdehyde (MDA) according to the kit instructions.

[0073] 2.8 Determination of organ index

[0074] Weigh the excised liver and kidney and record, and calculate the organ index for each organ in combination with the body weight.

[0075] Organ index (%) = organ weight (g) / body weight (g) × 100%

[0076] 2.9 Histopathological examination

[0077] After trimming the liver, pancreas, and colon tissues preserved in 4% paraformaldehyde to be flat, dehydrate them in a gradient of alcohol, treat them with xylene and then infiltrate with wax, embed, section, stain with hematoxylin and eosin, and mount the slides for microscopic examination of H&E staining. The liver tissue sections are processed by frozen section and then stained with oil red O for microscopic examination of oil red O staining. The liver tissue sections are stained with periodic acid, Schiff, and hematoxylin for microscopic examination of glycogen staining.

[0078] 2.10 Detection of related expressions in liver and colon tissues by Western Blot

[0079] According to the operation method of the BCA kit, extract the protein from liver and colon tissues, measure the protein concentration, and after normalization, boil for 10 min to obtain the protein samples of each group. Prepare the PAGE gel according to the instruction manual of the gel preparation kit, add the protein samples of each group and the Marker indicating the protein position in turn. After electrophoresis at 250 V for 25 min, transfer the gel to the PVDF membrane at 600 mA for 1.5 h. Block for 35 min, wash off the blocking solution, cut according to the molecular weight of the target protein, put the band containing the target protein into the pre-diluted primary antibody box, incubate overnight. The next day, wash off the residual primary antibody, incubate with the secondary antibody for 1 h, and then wash off the residual secondary antibody to enter the exposure chamber for exposure to observe the protein expression.

[0080] 2.11 Data processing

[0081] Use Image J software to process the gray value of the Western Blot experimental results. Use GraphPad Prism 8.0.2 for statistical analysis and drawing. The data are expressed in the form of mean ± standard deviation (X±SD). For two independent sample data, t-test is used; for comparison between groups, analysis of variance is used, and statistical significance is considered when p<0.05.

[0082] 3 Results and analysis

[0083] As Figure 1 shown, after treatment with AB-8 macroporous resin, the inulin polysaccharide changed from the original dark brown to light yellow, indicating that this method has a good decolorization effect; from Figure 2 the ultraviolet scan, it can be seen that the absorption peaks at 260 nm and 280 nm are not obvious, indicating the presence of a small amount of protein or nucleic acid; Figure 3 shows the absorption peak characteristics of polysaccharides in the Fourier transform infrared spectrum in the range of 4000 - 500 cm -1 . The broad band near 3401 cm -1 is the O-H stretching vibration; the weak band near 2932 cm -1 is the C-H stretching vibration; 1652 cm -1 is the bending vibration of -OH; 1415 cm -1 is the C-H bending vibration; 1325 cm -1 may be the C-H bending vibration; the C-O-C bond appears at 1245 cm -1 ; the absorption band in the range of 1148 - 1047 cm -1 can be classified as the stretching and bending vibrations of C-O-C and C-O-H, indicating the presence of a pyranose ring; the characteristic absorption band at 910 cm -1 indicates the presence of a pyranoside configuration. Figure 4The molecular weight determination results showed that there were at least three polysaccharides with different molecular weights in CGP, which were 624769 Da, 12926 Da, and 3880 Da respectively, and the polydispersities were 1.247160, 3.029179, and 1014.021088 respectively; Figure 5 It can be known that CGP is composed of fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, galacturonic acid, and glucuronic acid, and the molar ratios are 0.012, 0.071, 0.071, 0.178, 0.423, 0.025, 0.046, 0.112, and 0.063 respectively; Figure 6 Scanning electron microscopy showed that CGP was mainly in the form of lamellae, with a rough surface and irregular shape, possibly due to the lack of purification and the presence of impurities.

[0084] Such as Figure 7 A, an oral glucose tolerance test was performed on the mice in the model group and the blank group. Compared with the control group mice, the blood glucose of the model group mice increased faster and recovered more slowly, indicating the phenomenon of insulin resistance. At this time, it may be easier to establish the model by administering the modeling drug STZ; Figure 7 B, compared with the Mod group, the drug administration groups all showed varying degrees of blood glucose reduction during the drug administration period. Among them, the Met group and the H-CGP group had the most significant blood glucose regulation effects on type 2 diabetic mice (p<0.001), and the blood glucose reduction levels of the two groups were relatively close; the oral glucose tolerance and insulin tolerance tests were as Figure 7 C-F After treatment with CGP, it may be able to relieve the impaired glucose tolerance and insulin resistance of diabetic mice, even better than metformin hydrochloride; as shown in Figures G and H, CGP can reduce GSP and promote glycogen synthesis.

[0085] Such as Figure 8 As shown, compared with the Mod group, each treatment group could reduce the levels of TG, TC, LDL-C, ALT, and AST, and increase the level of HDL-C. The treatment level of the CGP group was related to the dose. It is worth mentioning that the treatment effect of the H-CGP group was similar to that of the Met group. CGP can improve the blood lipid and liver function related indicators and relieve type 2 diabetes.

[0086] Such as Figure 9 It can be known that the insulin level of the Mod group was significantly higher than that of the Ctrl group (p<0.001), while the insulin level of the treatment group was significantly lower than that of the Mod group (p<0.001). Further observation of the insulin resistance index found that the insulin resistance in the Mod group was obvious, while that in the treatment group decreased significantly, indicating that CGP can relieve the insulin resistance phenomenon of type 2 diabetes.

[0087] Such as Figure 10A - D. Compared with the Ctrl group, the serum MDA and CAT levels of the Mod group mice were significantly increased (P < 0.001), while the GSH and T - SOD levels were significantly decreased (P < 0.001). Compared with the Mod group, all treatment groups could reduce the MDA and CAT levels and increase the GSH and T - SOD levels, and the CGP group showed a dose - dependent effect. Among them, the improvement effects of the Met group and the H - CGP group were relatively close. CGP can relieve type 2 diabetes by regulating the in - vivo oxidative stress level.

[0088] As Figure 11 A, B. Compared with the Mod group, the mice in each treatment group showed a decrease in liver and kidney indices, and the decreases in the Met group and the H - CGP group were the most significant (p < 0.001). This indicates that the enlargement of the liver and kidneys in diabetic mice may be related to inflammation, and CGP can improve this phenomenon.

[0089] As Figure 12 A - D. To more intuitively understand the effect of CGP on the liver, H&E, Oil Red O and PAS staining analyses were performed on the liver. From the appearance of the liver, the liver of the Ctrl group mice was dark red with a complete and smooth surface, while the liver of the Mod group mice was light yellow with some depressions on the surface. Compared with the Mod group, each treatment group gradually alleviated this symptom. The HE staining results showed that there were no obvious fat vacuoles in the liver of the Ctrl group mice, and the hepatic lobules were arranged neatly and densely; in the liver of the Mod group mice, there were a large number of inflammatory infiltrations, severe fatty vacuolization, disordered cell arrangement and unclear structure. Compared with the Mod group, all treatment groups had improvements, especially the Met group and the H - CGP group had the most significant improvements. The Oil Red O staining showed that there was a large amount of fat accumulation in the Mod group, while the treatment groups could reduce the fat accumulation, and there was a certain dose - dependence. The PAS staining results showed that there were a large number of purple - red granules in the liver of the Ctrl group mice, indicating the presence of glycogen in the liver, while there were fewer purple - red granules in the liver of the Mod group mice, indicating a lower glycogen content. Compared with the Mod group, the hepatic glycogen content in all CG groups increased, indicating that administering CGP can improve the hepatic glycogen synthesis ability and relieve type 2 diabetes.

[0090] As Figure 13 , By observing the HE staining of the pancreas, the islet cell lines of the Ctrl group mice were clear, while the pancreatic cells of the Mod group mice were severely damaged, with blurred boundaries, a large number of inflammatory infiltrations and cell swelling, and the pancreatic injury of the treatment group mice could be gradually relieved.

[0091] As Figure 14 A - F. By observing and measuring the colon, it was found that CGP could extend the colon length; by detecting the expression of HE in the colon and the intestinal barrier function - related proteins Occludin and ZO - 1, it was speculated that CGP might protect or repair the intestinal barrier.

[0092] As Figure 15 shown in A-D, by detecting the expression of proteins related to the PI3K / AKT / mTOR signaling pathway, each treatment group showed the effects of reducing the expression of P-mTOR and promoting the phosphorylation of PI3K and AKT, and these effects showed a dose-dependent manner. Among them, the H-CGP group had better effects than the Met group in inhibiting P-mTOR protein and promoting PI3K phosphorylation. The research results indicate that CGP may regulate type 2 diabetes through the PI3K / AKT / mTOR signaling pathway.

[0093] In summary, chicory polysaccharide has a certain alleviating and repairing effect on STZ-induced type 2 diabetes, improves the hyperglycemia symptoms caused by type 2 diabetes, regulates blood lipids, liver function and oxidative stress levels, repairs or protects organ damage, and may improve type 2 diabetes by regulating the protein expression of the PI3K / AKT / mTOR signaling pathway.

[0094] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of inulin polysaccharide in the preparation, basic characterization and drugs for relieving type 2 diabetes, characterized in that, The preparation method of Cichorium glandulosum polysaccharide includes: after Cichorium glandulosum is soaked with ethanol, extracted by enzyme method, precipitated with alcohol, decolorized and deproteinized, Cichorium glandulosum polysaccharide is obtained; The Cichorium glandulosum is purchased from Hotan County, Xinjiang Uygur Autonomous Region, and is identified by Professor Qin Dongmei of the School of Pharmacy, Shihezi University as the underground root part of Cichorium glandulosum Boiss.et Huet, CG, a plant of the genus Cichorium in the Compositae family; The ethanol concentration is 90-98%; The enzyme extraction is carried out with constant temperature shaking for 1-3 h, the extraction temperature is 40-60 °C, the solid-liquid ratio is 1:(30-50) g / mL, and the amount of cellulase is 1-3%; The alcohol precipitation includes fully mixing the concentrated extract with ethanol and standing at 0-4 °C for 12-24 h; the ethanol concentration in the mixed solution is 70-80%; The decolorization and deproteinization adopt the macroporous resin adsorption method, and the macroporous resin is of the AB-8 type; The Cichorium glandulosum polysaccharide can reduce the phenomenon of high blood sugar in type 2 diabetes; The Cichorium glandulosum polysaccharide can improve the serum biochemical indexes and organ damage of type 2 diabetes; The Cichorium glandulosum polysaccharide can relieve type 2 diabetes through the PI3K / AKT / mTOR signaling pathway.

2. The application according to claim 1, characterized in that The Cichorium glandulosum polysaccharide can relieve type 2 diabetes.

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