Plebopus portentosus polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses phlebopus portentosus polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of polysaccharide extraction. The phlebopus portentosus polysaccharide (PP-0A) comprises galactose, fucose, glucose, mannose and xylose, and the molar ratios of the galactose, the fucose, the glucose, the mannose and the xylose are 62.26%, 16.96%, 15.23%, 4.68% and 0.87% respectively. According to the invention, the structural characteristics of PPP-0A are measured by using methylation, a scanning electron microscope, thermogravimetric analysis, nuclear magnetic resonance, an atomic force microscope and other technologies, and the composition and properties of PPP-0A and the inhibition effect of PPP-0A on the activity of alpha-glucosidase and alpha-amylase are analyzed. In addition, the hypoglycemic effect of the PPP-0A is researched by using an in-vitro HepG2 cell model induced by hyperglycemia and high insulin level. The invention provides a theoretical basis for clarification of the relationship between the hypoglycemic activity of the PPP-0A and the hypoglycemic effect of the PPP-0A on type 2 diabetes mellitus mice.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extraction, in particular to a Boletus edulis polysaccharide and a preparation method and application thereof. Background Art

[0002] In recent decades, diabetes mellitus (DM) has become a major health problem of global concern, and its incidence is closely related to modern dietary habits. Diabetes is a chronic metabolic disease characterized by imbalanced glucose and lipid metabolism, leading to chronic hyperglycemia, impaired insulin action, and pancreatic islet cell damage. Currently, about 537 million adults worldwide suffer from diabetes, of which about 90% suffer from type 2 diabetes (T2DM). Hyperglycemia can lead to long-term complications, including macrovascular and microvascular damage, cardiovascular disease, and retinopathy, and increase the risk of cardiovascular disease (CVD). The key to effectively controlling diabetes is to control blood glucose levels through a combination of dietary adjustment, physical exercise, and drug therapy. The basic strategies for controlling diabetes include regular monitoring of blood glucose, adherence to a healthy diet plan, maintaining moderate exercise, and using drugs when necessary. A variety of glucose-lowering drugs have been developed clinically for different mechanisms of action, such as α-glucosidase inhibitors and oral biguanides. However, long-term use of these drugs may cause serious adverse reactions. Therefore, there is an urgent need to find natural products with significant glucose-lowering effects, low toxicity, and low side effects for the prevention and treatment of T2DM.

[0003] Polysaccharides are macromolecular compounds composed of multiple monosaccharide molecules connected by glycosidic bonds. In addition to glucose, these compounds may also contain other types of monosaccharides, such as galactose, rhamnose, xylose, fucose, and arabinose. In recent years, dietary polysaccharides extracted from mushrooms have received increasing attention due to their various pharmacological activities and low toxicity. Studies have shown that fungal polysaccharides have anti-inflammatory, anti-tumor, immunomodulatory, antioxidant, and hypoglycemic properties. Among these biological activities, the potential of polysaccharides in controlling diabetes is particularly noteworthy.

[0004] Phlebopus portentosus (Berk. and Broome) Boedijin is a famous edible wild mushroom widely distributed in tropical areas of China, especially Yunnan, Guangxi and Hainan. It is also popular in northern Thailand. The fruiting body of the fungus is large, tastes good, and is rich in a variety of functional compounds, including polysaccharides, proteins, fibers, fats, amino acids, and essential minerals for the human body. Kamchantat et al. obtained a polysaccharide-protein complex of Phlebopus portentosus by water extraction and ethanol precipitation, and the obtained polysaccharide fragment PPC-P11 has antioxidant, antitumor and hypoglycemic effects. In previous studies, the inventors also preliminarily proved that the crude polysaccharide extracted from Phlebopus portentosus and several polysaccharide components (PPP-0, PPP-1, PPP-2) all have an inhibitory effect on α-glucosidase. However, the specific relationship between the structure of the component PPP-0A obtained by further purification from the PPP-0 component and its hypoglycemic activity is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a dark brown veined boletus polysaccharide and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The dark brown veined boletus polysaccharide PPP-0A separated by the present invention has a significant inhibitory effect on the activity of digestive enzymes, especially α-glucosidase and α-amylase, and also has the function of lowering blood sugar.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a dark brown stalked boletus polysaccharide. The dark brown stalked boletus polysaccharide comprises galactose, fucose, glucose, mannose and xylose, and the molar ratios thereof are 62.26%, 16.96%, 15.23%, 4.68% and 0.87% respectively.

[0008] Preferably, the structure of the Boletus edulis polysaccharide is as follows:

[0009]

[0010] The present invention also provides the use of the dark brown veined boletus polysaccharide in any of the following items:

[0011] (1) Application in the preparation of drugs for treating diabetes;

[0012] (2) Application in the preparation of α-glucosidase and / or α-amylase activity inhibitors;

[0013] (3) Application in the preparation of drugs for improving insulin resistance in HepG2 cells;

[0014] (4) Application in the preparation of drugs for lowering blood sugar.

[0015] The invention also provides a medicine for treating diabetes, comprising the dark brown vein boletus polysaccharide.

[0016] The present invention also provides an alpha-glucosidase and / or alpha-amylase activity inhibitor, comprising the boletus edulis polysaccharide.

[0017] The invention also provides a medicine for improving insulin resistance of HepG2 cells, comprising the dark brown stalked boletus polysaccharide.

[0018] The present invention also provides a blood sugar lowering medicine, characterized in that it comprises the Boletus edulis polysaccharide.

[0019] The present invention also provides a method for preparing the dark brown veined boletus polysaccharide, comprising the following steps:

[0020] Mixing and leaching the fruiting bodies of Boletus edulis with ethanol, discarding the supernatant, adding water to extract the precipitate, concentrating the extract, adding ethanol to collect the precipitate and freeze-drying it to obtain a freeze-dried powder; eluting the freeze-dried powder with HPD-100 macroporous adsorption resin to obtain crude polysaccharide;

[0021] The crude polysaccharide is eluted through a DEAE-52 cellulose chromatographic column, fractions are collected, and then the fractions are further purified using Sephacryl S-400HR to obtain the dark brown veined boletus polysaccharide.

[0022] Preferably, the mass volume ratio of the dark brown veined boletus polysaccharide to ethanol is 1:4, and the extraction time is 20-30 minutes;

[0023] The mass volume ratio of the dark brown veined boletus fruiting body to the water is 1:20, and the extraction conditions are: extraction at 80° C. for 2-3 times, each time for 50-100 minutes.

[0024] Preferably, the elution condition of Sephacryl S-400HR is: elution with distilled water at 1 mL / min.

[0025] The present invention discloses the following technical effects:

[0026] The present invention isolates a novel neutral polysaccharide (PPP-0A) from the fruiting body of Boletus edulis. The chemical structure of PPP-0A is characterized by ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, circular dichroism, scanning electron microscopy and atomic force microscopy. The research results show that PPP-0A is mainly composed of mannose, glucose, galactose and fucose. Methylation and nuclear magnetic resonance spectroscopy analysis showed that the glycosidic bonds of PPP-0A were composed of →6)-α-D-Galp-(1→, →2,6)-α-D-Galp-(1→ and →3)-α-D-Glcp-(1→. In addition, within the experimental concentration range, PPP-0A could effectively inhibit the activities of α-glucosidase and α-amylase. The in vitro anti-diabetic effect of PPP-0A was evaluated using IR-HepG2 cells, and the results showed that PPP-0A could increase the glucose consumption of IR-HepG2 cells, showing an in vitro hypoglycemic effect. The present invention provides a theoretical basis for clarifying the relationship between the hypoglycemic activity of Boletus edulis polysaccharide and its hypoglycemic effect on type 2 diabetic mice, and reveals the potential of PPP-0A in treating diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is a flow chart of extraction, separation and purification of Boletus edulis polysaccharide;

[0029] Figure 2 Characteristic analysis of Boletus edulis polysaccharide; A: DEAE-52 column elution curve of Boletus edulis polysaccharide; B: Sephacryl S-400HR column chromatography elution curve of Boletus edulis polysaccharide; C: Thermal analysis of PPP-0A; D: Molecular configuration analysis of PPP-0A; E: Molecular weight analysis of PPP-0A;

[0030] Figure 3 Structural analysis of PPP-0A; A: The monosaccharide composition of PPP-0A was determined by HPAEC; B: UV-visible spectrum of PPP-0A; C: Fourier transform infrared spectrum of PPP-0A; D: The glycosyl linkage type of PPP-0A was analyzed by methylation and GC-MS;

[0031] Figure 4 For PPP-0A part in D2O 1 HNMR spectrum;

[0032] Figure 5 For PPP-0A part in D2O 13 C NMR spectrum;

[0033] Figure 6 is the HSQC spectrum of PPP-0A;

[0034] Figure 7 is the COSY spectrum of PPP-0A;

[0035] Figure 8 is the HMBC spectrum of PPP-0A;

[0036] Fig. 9 is the NOESY spectrum of PPP-0A;

[0037] Fig.10 The morphological characteristics of PPP-0A; AC: SEM image, D: AFM image, E: CD spectrum image, F: XRD image;

[0038] Fig.11 is the change of maximum absorption wavelength of PPP-0A and Congo red in NaOH solution with different concentrations;

[0039] Fig.12 The results of the inhibitory activity and inhibition kinetics evaluation of PPP-0A on α-glucosidase and α-amylase; AB: the inhibitory effects of PPP-0A on α-glucosidase and α-amylase activities, respectively; CD: the reversibility of the inhibitory effects of PPP-0A on α-glucosidase and α-amylase, respectively; EF: Lineweaver-Burk plot of the inhibition types of PPP-0A on α-glucosidase and α-amylase;

[0040] Fig.13 Results of cell viability assay; A: PPP-0A cytotoxicity test results, B: stability of IR-HepG2 cell model, C-D: effects of different concentrations of insulin and different time on HepG2 cell activity and glucose consumption;

[0041] Fig.14 Effects of PPP-0A on glucose consumption (A), glycogen synthesis (B), HK (C) and PK (D) activities of IR-HepG2 cells. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1

[0045] 1. Experimental methods

[0046] 1.1 Extraction, separation and purification of polysaccharides

[0047] The flow chart of polysaccharide extraction, separation and purification is shown in Figure 1 , the specific operations are as follows:

[0048] The dried fruiting bodies (200 g) of Boletus edulis were crushed and fine powder was screened out using a 60-mesh (0.3 mm) sieve. Degrease with 95% ethanol at a ratio of 1:4 (m / v) for 30 min. Then, the precipitate was mixed with distilled water at a ratio of 1:20 (mass volume ratio of powder to distilled water) (m / v) and extracted three times at 80°C for 80 min each time. The extracts were combined and concentrated to 1 / 4 of the original volume at 60°C under reduced pressure using a rotary evaporator. 95% ethanol with a volume of 4 times the concentrate was slowly added and allowed to stand overnight, and the precipitate was collected and freeze-dried. Finally, the polysaccharide was redissolved, and a macroporous resin (HPD-100) with a liquid volume of 50% was added and shaken at a temperature of 75°C for 6.3 h. The decolorized eluate was then adsorbed with 12% polyamide powder for 4 h, eluted, the eluate was collected, and freeze-dried to obtain crude polysaccharide (C-PPP).

[0049] 1g C-PPP was dissolved in distilled water and loaded into a balanced DEAE-52 cellulose column. It was eluted with distilled water and NaCl solutions of 0.1, 0.2, 0.4, 0.8 and 1.0 M at a flow rate of 1 mL / min. The total sugar content of each collected fraction was analyzed by phenol-sulfuric acid method. PPP-0 with the highest polysaccharide content was further purified by Sephacryl S-400HR to obtain PPP-0A, and the elution condition was: eluted with distilled water at a flow rate of 1 mL / min. In the present invention, the PPP-0A component was selected for structural characterization and subsequent cell experiments.

[0050] 1.2 Structural characteristics of PPP-0A

[0051] Chemical composition analysis

[0052] The polysaccharide content of PPP-0A was quantified by the phenol-sulfuric acid method using D-glucose as the standard. In addition, the content of reducing sugar was determined by the 3,5-dinitrosalicylic acid colorimetric method.

[0053] 1.2.2. Molecular weight determination

[0054] The homogeneity and molecular weight of PPP-0A were determined using the SEC-MALLS-RI method. The molecular weight (Mw) and polydispersity index (Mw / Mn) of PPP-0A in a 0.1 M NaNO3 aqueous solution containing 0.02% NaN3 were determined using a DAWN HELEOS-II laser photometer. The laser photometer was equipped with two tandem chromatographic columns (300×8 mm, Shodex OH-pak SB-803 and 804; ShowaDenko KK, Tokyo, Japan). The chromatographic conditions were a column temperature of 45°C, an eluent flow rate of 0.6 mL / min, and an injection volume of 100 μL. A calibration curve was drawn using dextran standards.

[0055] 1.2.3. Monosaccharide composition analysis

[0056] The monosaccharide composition of PPP-0A was analyzed using high performance anion exchange chromatography (HPAEC). In a sealed test tube, 5 mg of PPP-0A was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 121 ° C for 2 h. After hydrolysis, the sample was blown dry under nitrogen. Methanol was then added for washing, and then blown dry with nitrogen again, and the methanol wash was repeated 2 to 3 times. The resulting residue was redissolved in deionized water, filtered through a 0.22 μm microporous filter membrane, and then analyzed using a Thermo ICS 5000+ ion chromatography system (Thermo Fisher Scientific, USA). The system is equipped with Dionex TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatography column. The retention time of monosaccharides was determined using a mixed monosaccharide standard solution.

[0057] 1.2.4 Thermogravimetric analysis (TG)

[0058] The thermal stability of PPP-0A was evaluated using a TGA / DSC3+ thermogravimetric analyzer (Mettler Toledo, CHE). The analysis was performed in the temperature range of 20-800°C with a linear heating rate of 10°C / min. The nitrogen flow rate in the sample chamber was maintained at 70 mL / min.

[0059] 1.2.5. Specific rotation determination

[0060] The optical rotation of the polysaccharide samples was determined using a SGW-531 digital fully automatic polarimeter (Shanghai Inisa Physical Optical Instrument Co., Ltd., Shanghai, China). First, the PPP-0A sample (100 ± 0.10 mg) was dissolved in 10 mL of distilled water and then transferred to a polarizing tube. The optical rotation of the sample was directly measured using an automatic polarimeter at a temperature of 25 ± 0.1 °C. To ensure the accuracy of the results, the experiment was repeated six times. Subsequently, the specific optical rotation was calculated using the following formula:

[0061]

[0062] α, optical rotation; t, detection temperature; λ, light source wavelength (λ=589 nm); L, polarization tube length; C, PPP-0A solution concentration (g / mL).

[0063] 1.2.6. UV-Vis and Fourier Transform Infrared Spectroscopy Analysis

[0064] A 1 mg / mL PPP-0A polysaccharide solution was analyzed using a UV-Vis spectrophotometer (JASCO, Japan) at a wavelength range of 200-400 nm. 2 mg of PPP-0A sample was mixed with 200 mg of dry KBr powder, fully ground and pressed into tablets. Then, a Fourier transform infrared spectrometer (Thermo Scientific, USA) was used at 4000-400 cm -1 Scan within the range.

[0065] 1.2.7. Methylation analysis

[0066] The dried sample (3 mg) of PPP-0A was dissolved in 500 μL of dimethyl sulfoxide (DMSO). The obtained polysaccharide solution was then mixed with 1 mL of distilled water and 500 μL of dichloromethane (CH2Cl2), centrifuged to discard the aqueous phase, and washed with water several times. The methylated polysaccharide was hydrolyzed with 2 M TFA at 120 °C for 90 min, then reduced with NaBD4, acetylated with acetic anhydride, and finally analyzed using a GC-MS system (Agilent Technologies, USA) equipped with an Agilent BPX70 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The temperature program was set as follows: the initial temperature was 140 °C for 2 min, then increased to 230 °C, and the flow rate was 3 °C / min for 3 min. The scanning mode was SCAN, and the mass-to-charge ratio (m / z) range was 50 to 350.

[0067] 1.2.8. Nuclear magnetic resonance (NMR) analysis

[0068] Dissolve the dried PPP-0A in 0.5 mL of D2O to a final concentration of 40 mg / mL and place it in an NMR tube. 1D and 2D NMR analysis, including 1 H-NMR, 13 C-NMR, COSY, NOESY, HSQC and HMBC were measured at 25 °C using a Bruker AVANCE NEO 500M spectrometer system (Bruker, Germany).

[0069] 1.2.9. Morphological characteristics of PPP-0A

[0070] (1) Scanning electron microscopy (SEM) analysis

[0071] The surface morphology of PPP-0A was observed on a TESCAN MIRALMS scanning electron microscope. The sample was fixed on the scanning electron microscope stage and coated with gold powder. The scanning electron microscope observation was performed at an accelerating voltage of 15 kV.

[0072] (2) Atomic force microscopy (AFM) analysis

[0073] The polysaccharide sample (PPP-0A) was dissolved in ultrapure water at a concentration of 10 μg / mL. Subsequently, 5 μL of the sample solution was deposited on a clean mica sheet and air-dried at room temperature for 2 h before atomic force microscopy observation.

[0074] (3) Congo red test

[0075] PPP-0A (1.5 mg / mL) was thoroughly mixed with 80 μM Congo red solution. Subsequently, the maximum absorbance was measured in the wavelength range of 400-700 nm using a UV / visible spectrophotometer (V650 spectrophotometer, JASCO, Japan).

[0076] (4) Circular dichroism analysis (CD)

[0077] PPP-0A polysaccharide 1 mg was accurately weighed, dissolved in ultrapure water, and then centrifuged at 12000 r / min for 5 min. The resulting supernatant was then transferred to a JASCO J-1500 circular dichroism spectrometer (JASCO, Japan) and CD spectra were scanned at a speed of 20 nm / min.

[0078] (5) X-ray diffraction analysis (XRD)

[0079] XRD analysis was performed using Rigaku Ultima IV (Japan) to evaluate the crystallinity of purified PPP-0A. The evaluation was performed in the range of 0-80° (2θ), the radiation used was Cu-Kα generated at 40 kV and 40 mA, and the scanning speed was 4° per minute.

[0080] 1.3. In vitro hypoglycemic activity

[0081] 1.3.1.α-Glucosidase activity inhibition test

[0082] 40 μL of PPP-0A solution (concentrations were 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 mg / mL, respectively) was mixed with 40 μL of α-glucosidase solution (0.5 U / mL) and pre-incubated at 37°C for 15 min. After the pre-incubation, 20 μL of 2.5 mmol / L p-nitrophenyl-α-D-pyranogalactoside (PNPG) solution was added as a substrate for reaction. After the reaction was carried out at 37°C for 15 min, 50 μL of 1 mol / L Na2CO3 solution was immediately added to terminate the reaction. Finally, the absorbance was measured at 405 nm using an ELISA reader. Acarbose was used as a positive control, and the α-glucosidase inhibition rate was calculated according to the following formula:

[0083] α-glucosidase inhibition rate (%) = [1-(A1-A2) / A0] × 100%

[0084] Wherein A1 represents the absorbance of the mixture reaction solution; A2 represents the absorbance of the sample reaction solution without the enzyme system; A0 represents the absorbance of the sample reaction solution without the sample.

[0085] 1.3.2. α-Glucosidase inhibition kinetics

[0086] PPP-0A (concentrations of 0, 2, and 4 mg / mL) was incubated with different amounts of α-glucosidase (30, 60, 90, and 120 μL) for 15 min, and then a 2.5 mM solution of p-nitrophenyl-β-D-pyranogalactoside (PNPG) was added. Subsequent steps were performed according to the standard α-glucosidase inhibition assay protocol. The reversibility of the inhibition was evaluated with the enzyme amount as the horizontal axis and the activity as the vertical axis.

[0087] PPP-0A was selected as an inhibitor to evaluate the enzymatic reaction rate of α-glucosidase under different substrate concentrations of PNPG (2.5, 2, 1.5, 1, and 0.5 mM) while keeping the concentration of α-glucosidase constant. Subsequently, regression analysis was performed using the Lineweaver-Burk method, comparing 1 / [V] on the vertical axis with 1 / [S] on the horizontal axis. The analysis yielded K m and V max The values ​​of PPP-0A were calculated and the reversible inhibition types of α-glucosidase activity by different concentrations of PPP-0A were determined.

[0088] V max and K mAccording to formula (1) and (2), we can calculate:

[0089]

[0090] 1.3.3. α-Amylase activity inhibition test

[0091] 100 μL of sample solution (concentrations were 0.125, 0.25, 0.5, 1, 2, 4, 8 and 16 mg / mL, respectively) was thoroughly mixed with an equal volume of α-amylase (0.1 U / mL) and incubated at 37°C for 5 min. Subsequently, 50 μL of 1% starch solution (mass fraction) was added and mixed thoroughly. After reacting for 5 min, 100 μL of DNS solution was added and placed in a boiling water bath for 10 min to terminate the reaction. After the solution was cooled to room temperature, the absorbance was measured at a wavelength of 540 nm. The α-amylase inhibition rate was calculated using the following formula:

[0092] α-Amylase inhibition rate (%) = [1-(A1-A0) / (A2-A3)] × 100%

[0093] Wherein, A1 is the absorbance of the test sample in the hypotonic solution; A0 is the absorbance of the drug without enzyme solution; A2 is the absorbance of the control group; A3 is the absorbance of the control group without enzyme solution.

[0094] 1.3.4. α-Amylase inhibition kinetics

[0095] 50 μL of different concentrations of PPP-0A (0, 2 and 4 mg / mL) were mixed with an equal volume of α-amylase (0.4, 0.2, 0.1, 0.05 and 0.025 U / mL) and the reaction was continued for 5 min. After the reaction was completed, a solution containing 1% starch was added and the reaction was continued for another 10 min. The subsequent steps were carried out according to the α-amylase activity inhibition test protocol outlined in 1.3.3. above.

[0096] PPP-0A (50 μL, concentrations of 0, 2, and 4 mg / mL) was incubated with α-amylase (0.1 U / mL, 50 μL) for 5 min, and then different concentrations of substrate (0.5-1.5%, 50 μL) were added. The reaction was carried out under the conditions of 1.3.3. The Lineweaver-Burk double reciprocal plot was used to analyze the α-amylase inhibition kinetics.

[0097] 1.3.5. Cell culture and viability assay

[0098] After recovery, the cells were transferred to a T25 culture flask containing 10% DMEM low-glucose medium and cultured at 37°C and 5% CO2. After the cells adhered to the surface of the culture flask, they were digested with trypsin. The cells were passaged at a ratio of 1:3 every three days. Cells in the logarithmic growth phase were used for subsequent experiments.

[0099] HepG2 cells were plated at 5 × 10 3 The density of the unit was inoculated into a 96-well plate and cultured for 24 hours. Then, different concentrations of PPP-0A (0.4, 0.8, 1.6, 3.2 and 6.4 mg / mL) of DMEM were added and the cells were allowed to continue to be cultured for 24 hours. After the treatment period, the old culture medium was removed and 100 μL DMEM and 10 μL CCK-8 solution were added to each well. The resulting mixture was cultured at 37 ° C for 1 hour, and then the absorbance was measured at a wavelength of 450 nm using an ELISA reader.

[0100] 1.3.6. Establishment of HepG2 cell insulin resistance model

[0101] Cells were plated at 10 per well 4 The cells were seeded at a density of 10 μg / mL in a 96-well plate and cultured in low-glucose complete medium for 24 h. After cell adhesion, the original medium was removed. To establish an insulin resistance cell model, the initial insulin concentration tested was 10 μg / mL. -5 , 10 -6 , 10 -7 , 10 -8 , and 10 -9 M, duration was 24 h. Subsequently, glucose consumption was measured at 24, 48, 60 and 72 h.

[0102] Effect of PPP-0A on glucose consumption in IR-HepG2 cells

[0103] According to 1.3.6, the HepG2 model was established in a 96-well plate. The experimental groups included: a normal control (NC) group, consisting of HepG2 cells treated with DMEM medium; a positive control group, consisting of IR-HepG2 cells treated with 0.8 mg / mL metformin; a model group, consisting of IR-HepG2 cells treated with DMEM medium; and an experimental group, consisting of IR-HepG2 cells treated with 0.8, 1.6, and 3.2 mg / mL metformin. Each group included three replicate wells. After 24 h of culture, glucose consumption was measured using a glucose kit.

[0104] 1.3.8. Determination of glycogen content

[0105] According to 1.3.6, the HepG2 model was established in a 6-well plate. The experimental grouping was carried out as described in section 1.3.7. After 24 h, the old culture medium was removed and cell lysis buffer was added to lyse the cells. The intracellular glycogen level was measured using a glycogen detection kit according to the manufacturer's instructions.

[0106] 1.3.9. Determination of intracellular hexokinase and pyruvate kinase

[0107] Treat and culture the cells according to 1.3.8. After 24 h of culture, trypsinize the cells and collect them in a centrifuge tube. After centrifugation, discard the supernatant. The activities of hexokinase (HK) and pyruvate kinase (PK) were determined using a hexokinase assay kit and a pyruvate kinase assay kit, respectively. In addition, the protein concentration was determined using a BCA protein assay kit, and the results were expressed as U / mg protein.

[0108] Statistical analysis

[0109] Data were processed using GraphPad version 9.5, and all graphs were generated using Origin 2021 (Origin Lab). Values ​​are presented as mean ± standard error of the mean (SEM). Variance tests were performed using ANOVA followed by multiple comparison tests. Statistical significance was defined as *p<0.05 and **p<0.01.

[0110] 2. Results and Analysis

[0111] 2.1 Isolation and purification of polysaccharides

[0112] After decolorization, deproteinization and ethanol precipitation, the yield of crude polysaccharide of Boletus edulis was 2.07±1.56%. Figure 2 A), four different polysaccharide fractions were obtained: PPP-0, PPP-1, PPP-2 and PPP-3. The polysaccharide fraction eluted with distilled water was determined to be the main component, among which PPP-0 had the highest polysaccharide content with a yield of 29.3±9.05%. Subsequently, the main component PPP-0A was further purified by Sephacryl S-400HR column chromatography to obtain a symmetrical sharp peak ( Figure 2 B), indicating that this is a homogeneous polysaccharide. The present invention further studied the structure and hypoglycemic activity of PPP-0A.

[0113] 2.2.Structural characteristics of PPP-0A

[0114] 2.2.1. Analysis of physical and chemical properties

[0115] The total sugar content of PPP-0A was determined to be 90.3% by the phenol-sulfuric acid method. The reducing sugar content of PPP-0A was 0.87% by the DNS method. In addition, the specific rotation of PPP-0A in distilled water was = +103°, indicating that PPP-0A is dominated by α-type glycosidic bonds.

[0116] The thermal stability of PPP-0A was determined by thermogravimetric analysis (TG). Figure 2 As shown in Figure C, the overall thermogravimetric curve of PPP-0A is divided into three different stages. In the temperature range of 80-290°C, the mass of PPP-0A decreased by 8.99%, which may be due to the loss of bound water or free water. Between 290-450°C, the mass dropped sharply, and the total weight decreased by 65.99%. This stage is related to the thermal decomposition of the polysaccharide chain, resulting in gas production or carbonization, which includes carboxyl decarboxylation reactions and the breaking of chemical bonds in the sugar chain. The third stage occurs between 455-500°C and may involve the cleavage of some stable structures, resulting in a weight loss rate of 23.92%. After exceeding 500°C, the mass loss rate tends to stabilize, and the final residual mass produced is about 1.1% of the original mass. These findings indicate that PPP-0A has significant thermal stability, which is also confirmed by TG analysis.

[0117] 2.2.2. Molecular weight and chain conformation

[0118] The homogeneity and molecular weight of PPP-0A were determined using size exclusion chromatography-multi-angle laser light scattering and refractive index detection (SEC-MALLS-RI). Figure 2 As shown in Figure E, the results show that the RI curve presents a single, symmetrical peak, indicating that the PPP-0A fraction is uniform. Figure 2 It can also be seen in Figure E that both the laser light scattering (LS) and refractive index (RI) detectors detected a single peak, indicating that PPP-0A had no obvious aggregation in aqueous solution. According to the retention time of SEC-MALLS-RI, the weight average molecular weight (Mw) of PPP-0A was 173.768 kDa, the number average molecular weight (Mn) was 14.54 kDa, and the peak molecular weight (Mp) was 14.49 kDa. Generally speaking, when the slopes of the conformational graph are 0.2-0.4, 0.5-0.6, and 1.0, respectively, they reflect spherical polymers, flexible coil polymers, and rigid rod polymers, respectively. After measurement, the slope of the conformational graph of PPP-0A is 0.55±0.11, indicating that the molecular configuration of PPP-0A has the characteristics of a flexible coil polymer, see Figure 2 Middle D.

[0119] 2.2.3. Monosaccharide composition analysis

[0120] The monosaccharide composition of PPP-0A was determined by HPAEC. Figure 3 As shown in Figure A, PPP-0A was identified to contain galactose (Gal), fucose (Fuc), glucose (Glc), mannose (Man) and xylose (Xyl), with molar ratios of 62.26%, 16.96%, 15.23%, 4.68% and 0.87%, respectively. These results indicate that PPP-0A is a heteropolysaccharide, mainly composed of galactose. Previous research reports indicated that crude polysaccharides of Boletus edulis consisted of mannose, glucose, galactose and fucose, with molar ratios of 4.4%, 22.8%, 60.2% and 12.6%, respectively. These two are obviously different heteropolysaccharides.

[0121] 2.2.4. UV-visible spectroscopy and Fourier transform infrared spectroscopy analysis

[0122] The UV-visible spectrum of PPP-0A is shown in Figure 3 As shown in B. There are no obvious absorption peaks at 260 nm and 280 nm, indicating that nucleic acids and proteins have been effectively removed from PPP-0A.

[0123] Fourier transform infrared spectroscopy elucidates the characteristic functional groups of polysaccharides. Figure 3 As shown in C, at 3390cm -1 and 2930cm -1 The broad and strong peaks at 1650 cm-1 are attributed to the OH and CH stretching vibrations of the polysaccharide. -1 and 1402cm -1 The absorption peaks at 1152cm correspond to C=O stretching vibration and CH bending vibration. -1 The absorption peak at 1080cm is attributed to the COC stretching vibration. -1 and 1030cm -1 The peak at 820 cm indicated that the backbone pattern of PPP-0A contained a pyranose ring. -1 and 570cm -1 The characteristic absorption band of α-glycosidic bond indicates the presence of α-glycosidic bond in PPP-0A. This observation is consistent with the conformation obtained by specific rotation.

[0124] Methylation analysis

[0125] like Figure 3As shown in D, the glycosyl linkage types of PPP-0A were analyzed by methylation and GC-MS. The methylation analysis results (Table 1) showed that PPP-0A contained 13 different glycosidic bonds, including t-Fucp, t-Manp, t-Glcp, t-Galp, 4-Fucp, 3-Glcp, 2-Galp, 6-Glcp, 4-Glcp, 6-Galp, 3,6-Glcp, 2,6-Manp and 2,6-Galp. Among them, Gal is the most important sugar residue, involving four glycosidic bonds: t-Galp, 2-Galp, 6-Galp and 2,6-Galp. It was preliminarily inferred that the main chain of PPP-0A is composed of the following sugar residues: →6)-α-D-Galp-(1→, →2,6)-α-D-Galp-(1→ and →3-α-D-Glcp-(1→. Methylation analysis showed that the molar ratio of the four main monosaccharides in PPP-0A (Gal:Fuc:Glc:Man=62.33:14.67:19.16:3.85) was basically consistent with the monosaccharide composition results.

[0126] Table 1 Methylation analysis of PPP-0A

[0127]

[0128] 2.2.6. Nuclear magnetic resonance analysis

[0129] To further explain the structure of PPP-0A, the inventors performed nuclear magnetic resonance spectroscopy analysis on the polysaccharide, including one-dimensional and two-dimensional nuclear magnetic resonance. 1 H NMR spectroscopy ( Figure 4 ), the sample signal is mainly located in the range of δ3.0-5.5, and multiple coupling signal peaks are observed in the region of δ4.3-5.4. This observation indicates that the sample contains a variety of sugar residues. The corresponding chemical shifts of the heterotopic hydrogen are recorded at δ4.89, 4.97, 4.98 and 5.28, respectively. 1 The heterotopic proton signals (δ4.89-5.28) in the H spectrum indicate that these residues are connected by α-glycosidic bonds, which is consistent with the results of Fourier transform infrared spectroscopy. In contrast, the non-homologous hydrogen signals are mainly concentrated in the δ3.1-4.2 region. Due to the obvious overlap between the individual signals, it is necessary to combine COSY and HSQC spectra to determine the H2-H6 chemical shifts of each sugar residue separately. In addition, 13 The isomeric carbon signals at δ 97.81, 97.81, 101.45 and 99.57 in the C spectrum ( Figure 5 ) corresponds to C-1 of the four sugar residues in PPP-0A. Figure 6) were combined with the reference data, and the cross-peak assignments of proton signals / isomeric carbon signals (H1 / C1) were as follows: δ 4.89 / 97.81, 4.97 / 97.81, 4.98 / 101.45, and 5.28 / 99.57, and were designated as residues A, B, C, and D, respectively.

[0130] The isomeric signals of residue A at δ4.89 / 97.81 (H1 / C1) indicated that it was probably an α-galactosyl residue. The other proton signals (H2-H6) of residue A were classified according to the cross peaks shown in the COSY spectrum ( Figure 7 ). The signals H2 to H6 of residue A are δ3.73, 3.90, 3.86, 3.75, and (3.80, 3.58), respectively. The correlations between proton and carbon signals, such as H2 / C2 (δ3.73 / 68.74), H3 / C3 (δ3.90 / 69.45), H4 / C4 (δ3.86 / 68.21), H5 / C5 (δ3.75 / 68.64), and H6 / C6 (δ3.80, 3.58 / 66.42), were determined from the HSQC spectrum ( Figure 6 ). Therefore, the C2-C6 signals of residue A were assigned as δ97.81, 68.74, 69.45, 68.21, 68.64 and 66.42. It is worth noting that the chemical shifts of C1 and C6 shifted to the lower area, indicating that residue A was substituted at the O1 and O6 positions of the sugar ring. Therefore, combined with the methylation analysis and literature reports, residue A was determined to be →6)-α-D-Galp-(1→). By the same method, the present invention inferred the types of other residues, and as a result, residues B, C and D were determined to be α-L-Fucp-(1→, →2,6)-α-D-Galp-(1→ and →3)-α-D-Glcp-(1→, respectively. Following similar steps, the assignment of all sugar residue shifts was completed. Table 2 lists the residues AD 1 H and 13 The chemical shift of C.

[0131] Table 2 1 H and 13 Chemical shift of PPP-0A resonance in CNMR spectra

[0132]

[0133] The present invention combines HMBC and NOESY spectra to analyze the sugar residues in PPP-0A. 13 C and 1 H chemical shift to clarify its structure and connection mode. Figure 8 and Fig. 9As shown in the figure, five cross peaks related to the H1 signal of residue A were observed, located at δ4.89 / 66.42, 4.89 / 67.17, 4.89 / 3.8, 4.89 / 3.58 and 4.89 / 4.12, respectively. Among them, the cross peak at δ4.89 / 66.42 indicates that H1 of residue A is correlated with C6 of residue A. The peaks at δ4.89 / 3.8 and δ4.89 / 3.58 correspond to the H1 / H6 signals of residue A. In addition, the cross peak at δ4.89 / 4.12 shows that there is a strong correlation between H1 of residue A and H6 of residue C. The HMBC spectrum also shows overlapping peaks between other residues, with H1 of residue B corresponding to C2 of residue C, and H1 of residue C corresponding to C6 of residue C. As shown in the figure Figure 4 As shown in Figure 5F, NOESY correlations between residues were observed between H1 of residue B and H2 of residue C, between H1 of residue C and H6 of residue C, between H1 of residue C and H3 of residue D, and between H1 of residue D and H6 of residue A. The relevant assignments are shown in Table 3.

[0134] Table 3 HMBC and NOESY related attributes of PPP-0A

[0135]

[0136] Based on the analysis of one-dimensional and two-dimensional NMR data and the methylation results, it was inferred that the polysaccharide was mainly composed of →6)-α-D-Galp-(1→, →2,6)-α-D-Galp-(1→ and →3)-α-D-Glcp-(1→. These sugar residues are connected to each other to form the main chain, while the side chain is composed of α-L-Fucp-(1→, which is connected to the O-2 position of the →2,6)-α-D-Galp-(1→ sugar residue. Therefore, the structure of PPP-0A is deduced as shown below.

[0137]

[0138] 2.3. Morphological characteristics of PPP-0A

[0139] SEM analysis

[0140] The molecular morphology of the polysaccharide can be observed using a scanning electron microscope (SEM). Scanning electron microscope images of PPP-0A at ×30kx, ×50kx, and ×100kx magnifications ( Fig.10 AC) shows disordered lamellar structures and broken branching structures. At lower magnification ( Fig.10 AB), PPP-0A appears to have a fragment-like shape; however, the bottom flake of PPP-0A has a smooth and intact surface, while in the high-magnification image ( Fig.10The branching structure in the upper part is fragmented. This unique structure reflects the complexity of PPP-0A.

[0141] 2.3.2. Atomic force microscopy analysis

[0142] Atomic force microscopy images of PPP-0A are shown in Figure 2. Fig.10 As shown in D. The results showed that PPP-0A had an island structure with separated wheel blocks, indicating that the sugar chain molecules were aggregated together. In addition, Fig.10 The atomic force microscopy image in center D shows that PPP-0A is rod-shaped with a diameter between 15 and 30 nm, while the diameter of a single polysaccharide molecule chain is usually between 0.1 and 1.0 nm. These findings suggest that the structure of PPP-0A is branched and entangled, which is consistent with the results obtained from SEM analysis.

[0143] CD spectrometry

[0144] like Fig.10 As shown in Figure E, the negative Cotton effect at 200 nm can be attributed to the n→π* transition of the carboxyl group. In addition, the negative effect observed in the range of 220-228 nm indicates that PPP-0A exhibits an ordered helical structure in aqueous solution. These findings are consistent with the results obtained from Congo red analysis.

[0145] XRD analysis

[0146] The crystal structure characteristics of PPP-0A were further analyzed by X-ray diffraction (XRD) pattern. Fig.10 F. The results show that PPP-0A exhibits a broad diffraction peak at 2θ≈20°, which indicates that PPP-0A has a typical amorphous structure.

[0147] 2.3.5. Triple helix structure analysis

[0148] like Fig.11 As shown in the figure, in a weak alkaline solution, the maximum absorption peak wavelength of the complex formed by PPP-0A and Congo red showed a significant red shift compared to the Congo red control sample. In addition, with the increase of sodium hydroxide concentration, the maximum absorption peak wavelength also showed irregular changes. These results show that PPP-0A maintains a triple helical structure. When the sodium hydroxide concentration gradually increases to 0.5M, the change in the maximum absorption wavelength is very small, which indicates that the triple helical structure of PPP-0A polysaccharide remains intact in an alkaline environment.

[0149] 2.4. In vitro hypoglycemic activity

[0150] 2.4.1. Evaluation of α-glucosidase inhibitory activity and inhibition kinetics

[0151] Fig.12 Figure A shows the inhibitory effects of PPP-0A and acarbose at different concentrations (0.125-16 mg / mL) on α-glucosidase. The results showed that the inhibitory effects of PPP-0A and acarbose on α-glucosidase were concentration-dependent. It is worth noting that the maximum inhibition rate of PPP-0A was 77.9%, slightly lower than the 98.2% of the positive control acarbose. The results showed that the IC value of PPP-0A on the inhibitory activity of α-glucosidase was 1.34. 50 The value was 3.9 mg / mL. Nevertheless, PPP-0A is still expected to be further developed into a potential anti-diabetic drug.

[0152] The present invention studies the reversibility and type of α-glucosidase inhibition to reveal the inhibitory mechanism of PPP-0A on the enzyme. According to the characteristics of the inhibitor-enzyme binding process, the inhibition can be divided into irreversible and reversible. Fig.12 Figure C shows the inhibition kinetic curve of PPP-0A on α-glucosidase at different mass concentrations (0, 2 and 4 mg / mL). After adding PPP-0A to the enzyme reaction system, three regression lines with zero intercept were generated. The slopes of these regression lines are inversely proportional to the mass concentration of PPP-0A, indicating that the inhibition type of PPP-0A on α-glucosidase is reversible. In addition, changes in kinetic parameters can also help to further understand the type of inhibition. While keeping the concentration of α-glucosidase unchanged, the mass concentrations of PPP-0A and substrate (PNPG) were changed, and the reaction rate (V) of the enzyme inhibition system was measured. The Lineweaver-Burk method was used to plot a double reciprocal plot of [V] versus [S]. As shown Fig.12 As shown in Figure E, there is a linear relationship between 1 / [V] and 1 / [S]. The Lineweaver-Burk curve of PPP-0A intersects in the third quadrant. As the mass concentration of PPP-0A increases, K m and V max These results indicate that the inhibition type of PPP-0A on α-glucosidase can be determined as a combination of uncompetitive inhibition and noncompetitive inhibition.

[0153] 2.4.2 Evaluation of α-amylase inhibitory activity and inhibition kinetics

[0154] The inhibitory effects of PPP-0A and acarbose on amylase are shown in Fig.12 In B. Within the experimental concentration range of 0.125-16 mg / mL, both samples showed inhibitory effects on α-glucosidase. The inhibitory effect of PPP-0A was dose-dependent. Within the concentration range of 2-8 mg / mL, the inhibition rate increased significantly and then gradually stabilized with the increase in concentration. The IC of PPP-0A was calculated.50 The value was 10.4 mg / mL, indicating that its inhibitory effect on α-amylase was weaker than that on α-glucosidase (IC 50 =3.9 mg / mL). In addition, the inhibition kinetics of α-amylase inhibition was studied, and it was found that there was a linear relationship between enzyme concentration and reaction rate. As the concentration of PPP-0A increased, the slope gradually decreased, indicating that PPP-0A reduced the catalytic efficiency of the enzyme and the inhibition process was reversible (see Fig.12 D). In the reaction system, when the enzyme concentration remains constant, the reaction rates of PNPG at concentrations of 0.5%, 0.75%, 1%, 1.25% and 1.5% are measured. Fig.12 As shown in Figure F, there is a significant linear relationship between 1 / [V] and 1 / [S], ​​and all straight lines intersect at one point in the third quadrant. According to the kinetic equation, when the concentration of PPP-0A is 0, 2, and 4 mg / mL, the measured V m The values ​​were 0.0082, 0.0053 and 0.003 respectively (Table 4). m The values ​​were 5.27, 3.67 and 2.45, respectively (Table 4). These results show that as the inhibitor concentration increases, the reaction rate (V m ) and Michaels constant (K m ) were all reduced, which is consistent with the relevant characteristics of mixed inhibition. Therefore, the inhibition type of PPP-0A on α-amylase can be classified as reversible mixed inhibition. This result is consistent with the kinetic results of PPP-0A inhibiting α-glucosidase.

[0155] Table 4 Kinetic parameters of α-glucosidase and α-amylase

[0156]

[0157] Cell viability assay

[0158] The cytotoxicity of the purified polysaccharide fraction PPP-0A extracted from Boletus edulis was evaluated using the CCK-8 assay in order to select the appropriate concentration of the polysaccharide solution for subsequent experiments. Fig.13 Figure A shows the toxicity of different concentrations of PPP-0A to HepG2 cells. Fig.13As shown in Figure A, when the concentration of PPP-0A was between 0.4 and 6.4 mg / mL, the cell viability remained above 95% after 24 hours of treatment. This indicates that within this concentration range and treatment duration, PPP-0A had no significant toxicity or side effects on HepG2 cells (p<0.05). However, at a concentration of 6.4 mg / mL, although no obvious cytotoxicity was observed, cell viability decreased. Therefore, subsequent experiments were conducted in the concentration range of 0.8 to 3.2 mg / mL to study the effect of polysaccharides on improving insulin resistance in HepG2 cells, and the treatment duration was also 24 hours.

[0159] 2.4.4. Establishment of HepG2 cell insulin resistance model

[0160] Fig.13 Figures C and D describe the effects of different insulin concentrations and treatment durations on HepG2 cell viability and glucose consumption. Fig.13 As shown in C, with the increase of insulin concentration (10 -9 , 10 -8 , 10 -7 , 10 -6 and 10 -5 M) and the extension of exposure time (24, 36, 48 and 60 h), the cell survival rate gradually decreased. Long-term use of high-concentration insulin has an inhibitory effect on cell growth. It is worth noting that when the insulin concentration is 10 -6 M, within the first 48 h, the cell viability was not significantly different from that of the control group, with a survival rate of more than 93%. However, after 48 h, the cell activity was significantly inhibited (p < 0.01). -5 The survival rates of cells treated with M at 24h, 36h, 48h and 60h were also significantly different from those of the control group (p<0.05 or p<0.01).

[0161] Insulin concentration and duration of treatment significantly affect glucose consumption in HepG2 cells, a key factor in assessing the development of insulin resistance. Fig.13 As shown in Figure D, compared with the control group, the glucose consumption of HepG2 cells increased after 48 h of exposure to different concentrations of insulin. In addition, as the insulin concentration increased, the glucose consumption gradually decreased. -5 and 10 -6 M, glucose consumption was the lowest, and the difference was extremely significant compared with the control group. -5 and 10 -6M) had lower glucose consumption than the control group, with inhibition rates of 9.6% and 8.9%, respectively. The results of CCK-8 cell activity assay also confirmed these findings. -6 HepG2 cells treated with M insulin for 48 h were considered to be in the optimal state for cell modeling.

[0162] 2.4.5. Stability study of insulin resistance cell model

[0163] After the model is successfully established using the optimal insulin concentration and duration as described in 2.4.4, cells from the control and experimental groups are simultaneously cultured in normal medium without insulin to assess the duration of insulin resistance in the model. Fig.13 As shown in B, during the 48h and 72h culture process, the glucose consumption of the experimental group cells was significantly different from that of the control group.

[0164] Effects of PPP-0A on glucose consumption and glycogen synthesis in IR-HepG2 cells

[0165] The effect of PPP-0A on glucose metabolism in IR-HepG2 cells under high glucose and high fat conditions was evaluated by glucose oxidase assay. -6 M insulin model was cultured for 48 h. According to the toxicity of the drug, IR-HepG2 cells were treated with PPP-0A at 0.8-3.2 mg / mL for 24 h. The experiment included a normal group, a model group, and a positive control group. Fig.14 As shown in A, at an insulin concentration of 10 -6 Under the induction of M, the glucose consumption of the model group was significantly different from that of the normal group (p<0.05), indicating that the insulin resistance model was successfully established and can be used for subsequent experiments. Compared with the model group, the glucose consumption of the positive control group (metformin hydrochloride) and the PPP-0A treatment group increased, indicating that PPP-0A can effectively promote the glucose consumption of IR-HepG2 cells. It is worth noting that the glucose consumption of the 3.2mg / mL PPP-0A treatment group reached (13.19±0.92mM). In addition, insulin resistance is closely related to liver glycogen synthesis. As Fig.14 As shown in Figure B, the glycogen content in the PPP-0A group increased in a dose-dependent manner compared with the model group. When the concentration was 3.2 mg / mL, the glycogen content in the PPP-0A group was 1.33 times that of the model group. This result suggests that PPP-0A can promote glycogen synthesis and thus reduce blood sugar levels.

[0166] 2.4.7.HK and PK activities

[0167] In order to evaluate the regulatory effect of PPP-0A on these two key liver enzymes in the process of glucose metabolism, the present invention treated the IR-HepG2 cell model with various concentrations of PPP-0A to determine its regulatory effect on key liver glucose production enzymes. Fig.14 As shown in C and D, the HK and PK activities in the PPP-0A-treated group were significantly increased compared with the model group. At a concentration of 3.2 mg / mL, the HK and PK activities were close to those of the positive control group treated with metformin (4 mM). Taken together, these findings suggest that PPP-0A can enhance glucose metabolism by increasing HK and PK activities, thereby promoting glycogen synthesis and glycolysis. Therefore, PPP-0A has the potential to reduce blood glucose levels.

[0168] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A dark brown veined boletus polysaccharide, characterized in that: The dark brown veined boletus polysaccharide comprises galactose, fucose, glucose, mannose and xylose, and the molar ratios thereof are 62.26%, 16.96%, 15.23%, 4.68% and 0.87% respectively.

2. The dark brown veined boletus polysaccharide according to claim 1, characterized in that The structure of the dark brown veined boletus polysaccharide is shown below:

3. Use of the dark brown veined boletus polysaccharide according to claim 1 or 2 in any of the following items: (1) Application in the preparation of drugs for treating diabetes; (2) Application in the preparation of α-glucosidase and / or α-amylase activity inhibitors; (3) Application in the preparation of drugs for improving insulin resistance in HepG2 cells; (4) Application in the preparation of drugs for lowering blood sugar.

4. A drug for treating diabetes, characterized in that: The invention comprises the Suillus edulis polysaccharide as claimed in claim 1 or 2.

5. An α-glucosidase and / or α-amylase activity inhibitor, characterized in that: The invention comprises the Suillus edulis polysaccharide as claimed in claim 1 or 2.

6. A drug for improving insulin resistance in HepG2 cells, characterized in that: The invention comprises the Suillus edulis polysaccharide as claimed in claim 1 or 2.

7. A drug for lowering blood sugar, characterized in that: The invention comprises the Suillus edulis polysaccharide as claimed in claim 1 or 2.

8. The method for preparing Boletus edulis polysaccharide according to claim 1 or 2, characterized in that: The following steps are involved: Mixing and leaching the fruiting bodies of Boletus edulis with ethanol, discarding the supernatant, adding water to extract the precipitate, concentrating the extract, adding ethanol to collect the precipitate and freeze-drying it to obtain a freeze-dried powder; eluting the freeze-dried powder with HPD-100 macroporous adsorption resin to obtain crude polysaccharide; The crude polysaccharide is eluted through a DEAE-52 cellulose chromatographic column, fractions are collected, and then the fractions are further purified using Sephacryl S-400HR to obtain the dark brown veined boletus polysaccharide.

9. The preparation method according to claim 8, characterized in that: The mass volume ratio of the dark brown veined boletus polysaccharide to ethanol is 1:4, and the extraction time is 20-30 minutes; The mass volume ratio of the dark brown veined boletus fruiting body to the water is 1:20, and the extraction conditions are: extraction at 80° C. for 2-3 times, each time for 50-100 minutes.

10. The preparation method according to claim 8, characterized in that: The elution condition of Sephacryl S-400HR is: elution with distilled water at 1 mL / min.

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