A polysaccharide from Pulveroboletus ravenelii and its preparation method and application

Abstract

The present invention discloses a polyporus badius polysaccharide and its preparation method and application, belonging to the technical field of polysaccharide extraction. The polyporus badius polysaccharide (PPP-0A) includes galactose, fucose, glucose, mannose and xylose, and the molar ratios are 62.26%, 16.96%, 15.23%, 4.68% and 0.87% respectively. The present invention uses techniques such as methylation, scanning electron microscopy, thermogravimetric analysis, nuclear magnetic resonance and atomic force microscopy to determine the structural characteristics of PPP-0A, analyze its composition and properties, and its inhibitory effects on the activities of α-glucosidase and α-amylase. In addition, an in vitro HepG2 cell model induced by high blood glucose and high insulin levels was used to study the hypoglycemic effect of PPP-0A. The present invention provides a theoretical basis for clarifying the relationship between the hypoglycemic activity of PPP-0A and its hypoglycemic effect on type 2 diabetic mice.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide extraction, and particularly to a polysaccharide from Phlebopus portentosus and its preparation method and application. Background Art

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

[0003] Polysaccharides are macromolecular compounds formed by the linkage of multiple monosaccharide molecules through 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 well-known edible wild mushroom that is widely distributed in the tropical regions of China, especially in Yunnan, Guangxi, and Hainan. It is also popular in northern Thailand. The fruiting body of this mushroom is relatively large, with a good taste, and is rich in various functional compounds, including polysaccharides, proteins, fibers, fats, amino acids, and essential minerals for the human body. Kamchantat et al. obtained a polysaccharide-protein complex from Phlebopus portentosus using water extraction and ethanol precipitation methods, and the resulting polysaccharide fragment PPC-P11 has antioxidant, anti-tumor, and hypoglycemic effects. In previous studies, the inventors also preliminarily demonstrated that the crude polysaccharide extracted from Phlebopus portentosus and several polysaccharide components (PPP-0, PPP-1, PPP-2) all have inhibitory effects on α-glucosidase. However, the specific relationship between the structure of the further purified component PPP-0A from the PPP-0 fraction and its hypoglycemic activity remains unclear. Summary of the Invention

[0005] The object of the present invention is to provide a polysaccharide from Phlebopus portentosus, its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The polysaccharide PPP-0A from Phlebopus portentosus isolated in the present invention has significant inhibitory effects on the activities 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 present invention provides a polysaccharide from Phlebopus portentosus, which comprises galactose, fucose, glucose, mannose, and xylose, and the molar ratios are 62.26%, 16.96%, 15.23%, 4.68%, and 0.87% respectively.

[0008] Preferably, the structure of the polysaccharide from Phlebopus portentosus is as follows:

[0009]

[0010] The present invention also provides the application of the polysaccharide from Phlebopus portentosus in any one of the following:

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

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

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

[0014] (4) Use in the preparation of antidiabetic drugs.

[0015] The present invention also provides a drug for treating diabetes, comprising the polysaccharide of Phlebopus portentosus.

[0016] The present invention also provides an inhibitor of α-glucosidase and / or α-amylase activity, comprising the polysaccharide of Phlebopus portentosus.

[0017] The present invention also provides a drug for improving insulin resistance in HepG2 cells, comprising the polysaccharide of Phlebopus portentosus.

[0018] The present invention also provides an antidiabetic drug, characterized by comprising the polysaccharide of Phlebopus portentosus.

[0019] The present invention also provides a method for preparing the polysaccharide of Phlebopus portentosus, comprising the following steps:

[0020] Mix the fruiting bodies of Phlebopus portentosus with ethanol for extraction, discard the supernatant, add water to the precipitate for extraction, concentrate the extract, add ethanol to collect the precipitate and freeze-dry to obtain a freeze-dried powder; elute the freeze-dried powder with HPD-100 macroporous adsorption resin to obtain a crude polysaccharide;

[0021] Elute the crude polysaccharide with a DEAE-52 cellulose chromatography column, collect the fractions, and further purify the fractions with Sephacryl S-400HR to obtain the polysaccharide of Phlebopus portentosus.

[0022] Preferably, the mass-volume ratio of the polysaccharide of Phlebopus portentosus to ethanol is 1:4, and the extraction time is 20 - 30 min;

[0023] The mass-volume ratio of the fruiting bodies of Phlebopus portentosus to the water is 1:20, and the extraction conditions are: extraction at 80 °C for 2 - 3 times, 50 - 100 min each time.

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

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

[0026] A novel neutral polysaccharide (PPP-0A) was isolated from the fruiting bodies of Boletus obscure-venulosus. The chemical structure of PPP-0A was characterized by ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, scanning electron microscopy and atomic force microscopy techniques. The results showed that PPP-0A was 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 hypoglycemic effect in vitro. This invention provides a theoretical basis for clarifying the relationship between the hypoglycemic activity of Boletus obscure-venulosus 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0028] Figure 1 Flow chart for the extraction, isolation and purification of Boletus obscure-venulosus polysaccharide;

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

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

[0031] Figure 4 For part of PPP-0A in D 2 O1 HNMR spectrum;

[0032] Figure 5 For the PPP-0A part in D 2 O 13 C NMR spectrum;

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

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

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

[0036] Figure 9 NOESY spectrum of PPP-0A;

[0037] Figure 10 Morphological characteristics of PPP-0A; A-C: SEM images, D: AFM image, E: CD spectral image, F: XRD image;

[0038] Figure 11 Wavelength changes of the maximum absorption of PPP-0A and Congo red in NaOH solutions with different concentrations;

[0039] Figure 12 Evaluation results of the inhibitory activity and inhibitory kinetics of PPP-0A against α-glucosidase and α-amylase; A-B: Inhibitory effects of PPP-0A on the activities of α-glucosidase and α-amylase, respectively; C-D: Reversibility of the inhibitory effects of PPP-0A on α-glucosidase and α-amylase, respectively; E-F: Lineweaver-Burk plots of the inhibitory types of PPP-0A against α-glucosidase and α-amylase;

[0040] Figure 13 Results of cell viability assay; A: Results of the cytotoxicity test of PPP-0A, B: Stability of the IR-HepG2 cell model, C-D: Effects of insulin at different concentrations and different times on the activities and glucose consumption of HepG2 cells, respectively;

[0041] Figure 14 Effects of PPP-0A on glucose consumption (A), hepatic glycogen synthesis (B), HK (C) and PK (D) activities in IR-HepG2 cells. Detailed implementation method

[0042] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all 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 for the extraction, separation, and purification of polysaccharides is shown in Figure 1 , and the specific operations are as follows:

[0048] The dried fruiting bodies of Boletellus obscure-venulosus (200 g) were crushed, and the fine powder was sieved out using a 60-mesh (0.3 mm) sieve. Defatting was carried out 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-to-volume ratio of powder to distilled water) (m / v), and extracted three times at 80 °C for 80 min each time. The extraction solutions were combined and concentrated under reduced pressure to 1 / 4 of the original volume using a rotary evaporator at 60 °C. 95% ethanol with a volume 4 times that of the concentrated solution was slowly added and left to stand overnight. The precipitate was collected and freeze-dried. Finally, the polysaccharide was redissolved, and macroporous resin (HPD-100) accounting for 50% of the liquid volume was added, and the mixture was shaken at 75 °C for 6.3 h. Then, the decolorized eluate was adsorbed with 12% polyamide powder for 4 h, eluted, the eluate was collected, and freeze-dried to obtain crude polysaccharide (C-PPP).

[0049] 1 g of C-PPP was dissolved in distilled water and loaded onto a pre-equilibrated DEAE-52 cellulose chromatography column. Elution was carried out successively with distilled water and NaCl solutions with concentrations 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 using the phenol-sulfuric acid method. The polysaccharide PPP-0 with the highest content was further purified using Sephacryl S-400HR to obtain PPP-0A, and the elution conditions were: elution with distilled water at a flow rate of 1 mL / min. In the present invention, the PPP-0A fraction was selected for structural characterization and subsequent cell experiments.

[0050] 1.2 Structural characteristics of PPP-0A

[0051] 1.2.1 Chemical composition analysis

[0052] Using D-glucose as the standard, the polysaccharide content of PPP-0A was quantified by the phenol-sulfuric acid method. 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 SEC-MALLS-RI method. The molecular weight (Mw) and polydispersity index (Mw / Mn) of PPP-0A in 0.1 M NaNO 3 aqueous solution containing 0.02% NaN 3 were measured using a DAWN HELEOS-II laser photometer. The laser photometer was equipped with two chromatographic columns in series (300×8 mm, Shodex OH-pak SB-803 and 804; Showa Denko K.K., 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 dried under nitrogen. Subsequently, methanol was added for washing, and then dried again with nitrogen. The methanol washing was repeated 2 to 3 times. The obtained residue was redissolved in deionized water, filtered through a 0.22 μm microporous membrane, and then analyzed using a Thermo ICS 5000+ ion chromatography system (Thermo Fisher Scientific, USA). The system was equipped with a Dionex TM CarboPac TM PA20 (150×3.0 mm, 10 μm) liquid chromatography column. The retention times of monosaccharides were 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 carried out in the temperature range of 20 - 800 °C with a linear heating rate of 10 °C / min. The nitrogen gas flow rate in the sample chamber was maintained at 70 mL / min.

[0059] 1.2.5. Specific rotation determination

[0060] The specific rotation of the polysaccharide sample was determined using an SGW-531 digital automatic polarimeter (Shanghai Inessa Physical Optics 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 polarimeter tube. At a temperature of 25 ± 0.1 °C, the specific rotation of the sample was directly measured using an automatic polarimeter. To ensure the accuracy of the results, the experiment was repeated six times. Subsequently, the specific rotation was calculated using the following formula:

[0061]

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

[0063] 1.2.6. UV-Vis spectroscopy and Fourier transform infrared spectroscopy analysis

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

[0065] 1.2.7. Methylation analysis

[0066] The dried sample of PPP-0A (3 mg) was dissolved in 500 μL of dimethyl sulfoxide (DMSO). Then the resulting polysaccharide solution was mixed with 1 mL of distilled water and 500 μL of dichloromethane (CH 2 Cl 2 ), centrifuged to discard the aqueous phase, and washed several times with water. The methylated polysaccharide was hydrolyzed with 2 M TFA at 120 °C for 90 min, then reduced with NaBD 4 , acetylated with acetic anhydride, and finally analyzed using a GC-MS system (Agilent Technologies, USA) equipped with an Agilent BPX70 chromatographic 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 it was heated to 230 °C at a rate of 3 °C / min for 3 min. The scan 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 D 2 O to a final concentration of 40 mg / mL, and then placed into an NMR tube. One-dimensional and two-dimensional 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 a Rigaku Ultima IV (Japan) to evaluate the crystallinity of purified PPP-0A. The evaluation was carried out in the range of 0 - 80° (2θ), and the radiation used was Cu-Kα generated at 40 kV and 40 mA, with a scanning speed of 4° per minute.

[0080] 1.3. In vitro hypoglycemic activity

[0081] 1.3.1. α-Glucosidase activity inhibition assay

[0082] Forty microliters of PPP-0A solutions (at concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 mg / mL) were 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-galactopyranoside (PNPG) solution was added as a substrate for the reaction. After the reaction was carried out at 37 °C for 15 min, 50 μL of 1 mol / L Na 2 CO 3 solution was immediately added to terminate the reaction. Finally, the absorbance was measured at 405 nm using a microplate 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 - (A 1 - A 2 ) / A 0 × 100%

[0084] where A 1 represents the absorbance of the mixture reaction solution; A 2 represents the absorbance of the sample reaction solution without the enzyme system; A 0 represents the absorbance of the sample reaction solution without the sample.

[0085] 1.3.2. α-Glucosidase inhibition kinetics

[0086] PPP-0A (at 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 2.5 mM p-nitrophenyl-β-D-galactopyranoside (PNPG) solution was added. The subsequent steps were carried out 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] With the concentration of α-glucosidase remaining constant, PPP-0A was selected as the inhibitor to evaluate the enzymatic reaction rate of α-glucosidase at different substrate concentrations of p-nitrophenyl-α-D-glucopyranoside (PNPG) (2.5, 2, 1.5, 1, and 0.5 mM). 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 the values of K m and V max , and determined the type of reversible inhibition of α-glucosidase activity by different concentrations of PPP-0A.

[0088] V max and K m were calculated according to formulas (1) and (2):

[0089]

[0090] 1.3.3. α-Amylase Activity Inhibition Test

[0091] 100 μL of sample solutions with concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 mg / mL were 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 well. After reacting for 5 min, 100 μL of DNS solution was added and the mixture was placed in a boiling water bath for 10 min to terminate the reaction. After the solution 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 - (A 1 - A 0 ) / (A 2 - A 3 )] × 100%

[0093] where A 1 is the absorbance of the test sample in the hypotonic solution; A 0 is the absorbance of the drug in the enzyme-free solution; A 2 is the absorbance of the control group; A 3 is the absorbance of the control group in the enzyme-free solution.

[0094] 1.3.4. α-Amylase Inhibition Kinetics

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

[0096] Incubate PPP-0A (50 μL, at concentrations of 0, 2, and 4 mg / mL) with α-amylase (0.1 U / mL, 50 μL) for 5 min, and then add substrates at different concentrations (0.5 - 1.5%, 50 μL). The reaction is carried out under the conditions of 1.3.3. Analyze the α-amylase inhibition kinetics using the Lineweaver-Burk double reciprocal plot.

[0097] 1.3.5. Cell culture and viability assay

[0098] After resuscitation, transfer the cells to a T25 culture flask containing 10% DMEM low-glucose medium and culture at 37 °C and 5% CO 2 . After the cells adhere to the surface of the culture flask, digest them with trypsin. Passage the cells at a ratio of 1:3 every three days. Cells in the logarithmic growth phase are used for subsequent experiments.

[0099] Seed HepG2 cells into a 96-well plate at a density of 5 × 10 3 per well and culture for 24 h. Then, add DMEM containing different concentrations of PPP-0A (0.4, 0.8, 1.6, 3.2, and 6.4 mg / mL) and let the cells continue to culture for 24 h. After the treatment period, remove the old medium, add 100 μL of DMEM and 10 μL of CCK-8 solution to each well. Incubate the resulting mixture at 37 °C for 1 h, and then measure the absorbance at a wavelength of 450 nm using a microplate reader.

[0100] 1.3.6. Establish a HepG2 cell insulin resistance model

[0101] Seed the cells into a 96-well plate at a density of 10 4 per well and culture in low-glucose complete medium for 24 h. After the cells adhere, remove the original medium. To establish an insulin-resistant cell model, the initially tested insulin concentrations are 10 -5 , 10 -6 , 10 -7 , 10 -8 , and 10 -9 M, for a duration of 24 h. Subsequently, measure the glucose consumption at 24, 48, 60, and 72 h respectively.

[0102] 1.3.7. Effect of PPP-0A on Glucose Consumption in IR-HepG2 Cells

[0103] According to 1.3.6, a 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 culturing for 24 h, the glucose consumption was measured using a glucose assay kit.

[0104] 1.3.8. Determination of Glycogen Content

[0105] According to 1.3.6, a 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 medium was removed, and cell lysate was added to lyse the cells. The intracellular glycogen level was measured using a glycogen assay kit according to the manufacturer's instructions.

[0106] 1.3.9. Determination of Intracellular Hexokinase and Pyruvate Kinase

[0107] The cells were treated and cultured according to 1.3.8. After culturing for 24 h, the cells were digested with trypsin and collected into a centrifuge tube. After centrifugation, the supernatant was discarded. 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 measured using a BCA protein assay kit, and the results were expressed as U / mg protein.

[0108] 1.4. Statistical Analysis

[0109] The data were processed using GraphPad version 9.5, and all graphs were generated using Origin 2021 (Origin Lab). The values were expressed as the mean ± standard error of the mean (SEM). Analysis of variance was used for variance testing, followed by multiple comparison testing. 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 polysaccharides from Boletus obscure-venosus was 2.07 ± 1.56%. After separation using a DEAE-52 cellulose column( Figure 2In 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 symmetric sharp peak ( Figure 2 In 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. Physicochemical property analysis

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

[0116] The thermal stability of PPP-0A was determined by thermogravimetric analysis (TG). As Figure 2 shown in 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 decreased sharply, and the total weight decreased by 65.99%. This stage is related to the thermal decomposition of the polysaccharide chain, resulting in gas generation or carbonization, which includes decarboxylation of carboxyl groups and cleavage of chemical bonds in the sugar chain. The third stage occurred between 455 - 500 °C, which 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 tended to be stable, and the final residual mass produced was approximately 1.1% of the original mass. These findings indicate that PPP-0A has significant thermal stability, and TG analysis also proves this.

[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). As Figure 2 shown in E, the results showed that the RI curve presented a single, symmetric peak, indicating that the PPP-0A fraction was homogeneous. In addition, from Figure 2It can also be seen from Figure E that both the laser light scattering (LS) and refractive index (RI) detectors detected a single peak, indicating that PPP-0A did not show 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, when the slope of the conformation diagram is 0.2 - 0.4, 0.5 - 0.6, and 1.0 respectively, it reflects spherical polymers, flexible coil polymers, and rigid rod-like polymers. After measurement, the slope of the conformation diagram of PPP-0A was 0.55 ± 0.11, indicating that the molecular configuration of PPP-0A had the characteristics of flexible coil polymers, as shown in Figure 2 Figure D.

[0119] 2.2.3. Monosaccharide composition analysis

[0120] The monosaccharide composition of PPP-0A was determined by HPAEC. As Figure 3 shown in Figure A, it was identified that PPP-0A contained galactose (Gal), fucose (Fuc), glucose (Glc), mannose (Man), and xylose (Xyl), and their molar ratios were 62.26%, 16.96%, 15.23%, 4.68%, and 0.87% respectively. These results indicated that PPP-0A was a heteropolysaccharide mainly composed of galactose. Previous research reports pointed out that the crude polysaccharide from Boletus obscure-venosus consisted of mannose, glucose, galactose, and fucose, and their molar ratios were 4.4%, 22.8%, 60.2%, and 12.6% respectively. These two were obviously different heteropolysaccharides.

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

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

[0123] Fourier transform infrared spectroscopy elucidated the characteristic functional groups of polysaccharides. As Figure 3 shown in Figure C, the broad and strong peaks at 3390 cm -1 and 2930 cm -1 were attributed to the O-H and C-H stretching vibrations of polysaccharides. The absorption peaks at 1650 cm -1 and 1402 cm -1 corresponded to the C=O stretching vibration and C-H bending vibration respectively. The absorption peak at 1152 cm -1 was attributed to the C-O-C stretching vibration. The absorption peaks at 1080 cm -1 and 1030 cm-1 The peak at indicates that the backbone pattern of PPP-0A contains a pyranose ring. 820 cm -1 and the characteristic absorption bands at 570 cm -1 indicate the presence of an α-glycosidic bond in PPP-0A. This observation is consistent with the conformation obtained from the specific rotation results.

[0124] 2.2.5. Methylation Analysis

[0125] As shown in D of Figure 3 , the glycosyl linkage types of PPP-0A were analyzed by methylation and GC-MS. The results of methylation analysis (Table 1) show that PPP-0A contains 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 abundant sugar residue, involving four glycosidic bonds: t-Galp, 2-Galp, 6-Galp, and 2,6-Galp. It is 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→. As determined by methylation analysis, the molar ratio of the four main monosaccharides in PPP-0A (Gal:Fuc:Glc:Man = 62.33:14.67:19.16:3.85) is 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. In the 1 1H NMR spectrum ( Figure 4 ), the sample signals are mainly in the range of δ 3.0 - 5.5, and multiple coupled signal peaks are observed in the region of δ 4.3 - 5.4. This observation indicates that the sample contains multiple sugar residues. The corresponding chemical shifts of the anomeric hydrogens are recorded at δ 4.89, 4.97, 4.98, and 5.28, respectively. 1The anomeric proton signals (δ 4.89 - 5.28) in the ¹H spectrum indicate that these residues are linked by α-glycosidic bonds, which is consistent with the results of Fourier transform infrared spectroscopy studies. In contrast, the non-anomeric hydrogen signals are mainly concentrated in the region of δ 3.1 - 4.2. Due to the significant overlap between individual signals, it is necessary to combine the COSY and HSQC spectra to determine the H2 - H6 chemical shifts of each sugar residue separately. In addition, 13 the isomeric carbon signals ( Figure 5 ) at δ 97.81, 97.81, 101.45, and 99.57 in the ¹³C spectrum correspond to the C-1 of the four sugar residues in PPP-0A. By combining the HSQC spectrum ( Figure 6 ) with reference data, the cross-peak assignments of the proton signals / isomeric carbon signals (H1 / C1) are as follows: δ 4.89 / 97.81, 4.97 / 97.81, 4.98 / 101.45, and 5.28 / 99.57, and are designated as residues A, B, C, and D, respectively.

[0130] The isomeric signal of residue A at δ 4.89 / 97.81 (H1 / C1) indicates that it may be an α-galactosyl residue. The other proton signals (H2 - H6) of residue A are classified according to the cross-peaks shown in the COSY spectrum ( Figure 7 ). The H2 to H6 signals of residue A are δ 3.73, 3.90, 3.86, 3.75, and (3.80, 3.58), respectively. The correlations between proton and carbon signals, such as H2 / C2 (δ 3.73 / 68.74), H3 / C3 (δ 3.90 / 69.45), H4 / C4 (δ 3.86 / 68.21), H5 / C5 (δ 3.75 / 68.64), and H6 / C6 (δ 3.80, 3.58 / 66.42), are determined by the HSQC spectrum ( Figure 6 ). Therefore, the C2 - C6 signals of residue A are designated as δ 97.81, 68.74, 69.45, 68.21, 68.64, and 66.42. It is worth noting that the chemical shifts of C1 and C6 shift to a lower region, indicating that residue A is substituted at the O1 and O6 positions of the sugar ring. Therefore, combining methylation analysis and literature reports, residue A is determined to be →6)-α-D-Galp-(1→). By the same method, the types of other residues were deduced in the present invention, and the results determined residues B, C, and D to be α-L-Fucp-(1→, →2,6)-α-D-Galp-(1→, and →3)-α-D-Glcp-(1→, respectively. Following similar steps, the assignments of all sugar residue shifts were completed. Table 2 lists the 1 ¹H and 13 ¹³C chemical shifts of residues A - D.

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

[0132]

[0133] The present invention combines HMBC and NOESY spectra to analyze each sugar residue in PPP-0A 13 C and 1 the chemical shifts of H to clarify its structure and linkage mode. In the HMBC and NOESY spectra (as shown in Figure 8 and Figure 9 ), 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 related to 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 a strong correlation between H1 of residue A and H6 of residue C. The HMBC spectrum also shows overlapping peaks between other residues. H1 of residue B corresponds to C2 of residue C, and H1 of residue C corresponds to C6 of residue C. As shown in Figure 4 F, 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 correlation assignments of PPP-0A

[0135]

[0136] Based on the one-dimensional and two-dimensional nuclear magnetic resonance data analysis and methylation results, it is inferred that the polysaccharide is mainly composed of →6)-α-D-Galp-(1→, →2,6)-α-D-Galp-(1→, and →3)-α-D-Glcp-(1→. These sugar residues are interconnected 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 follows.

[0137]

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

[0139] 2.3.1. SEM analysis

[0140] The molecular morphological characteristics of polysaccharides can be observed using a scanning electron microscope (SEM). The scanning electron microscope images of PPP-0A at magnifications of ×30kx, ×50kx, and ×100kx ( Figure 10 in A-C) show a disordered stacked sheet structure and a fragmented branched structure. At lower magnifications ( Figure 10 in A-B), PPP-0A appears to have a fragmented shape; however, the bottom sheet surface of PPP-0A is smooth and intact, while in the high-magnification image ( Figure 10 in C), the upper branched structure is fragmented. This unique structure reflects the complexity of PPP-0A.

[0141] 2.3.2. Atomic force microscopy analysis

[0142] The atomic force microscopy image of PPP-0A is shown in Figure 10 in D. The results show that PPP-0A has an island-like structure with separated wheel blocks, indicating that the sugar chain molecules aggregate together. In addition, Figure 10 the atomic force microscopy image in D shows that PPP-0A is rod-shaped with a diameter between 15 and 30 nm, while the diameter of a single-chain polysaccharide molecular chain is usually between 0.1 and 1.0 nm. These findings indicate that the structure of PPP-0A is mutually branched and entangled, which is consistent with the results obtained from SEM analysis.

[0143] 2.3.3. CD spectroscopy determination

[0144] As shown in Figure 10 in 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 has an ordered helical structure in aqueous solution. These findings are consistent with the results obtained from Congo red analysis.

[0145] 2.3.4. XRD analysis

[0146] The crystal structure characteristics of PPP-0A were further analyzed by X-ray diffraction (XRD) patterns. The X-ray diffraction pattern of PPP-0A is shown in Figure 10 in F. The results show that PPP-0A shows a broad diffraction peak at 2θ≈20°, indicating that PPP-0A has a typical amorphous structure.

[0147] 2.3.5. Triple helix structure analysis

[0148] As shown in Figure 11As shown, in a weak base 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, as the concentration of sodium hydroxide increased, the maximum absorption peak wavelength also showed irregular changes. These results indicate that PPP-0A maintains a triple helix structure. When the sodium hydroxide concentration gradually increased to 0.5 M, the change in the maximum absorption wavelength was very small, indicating that the triple helix structure of the PPP-0A polysaccharide remained intact in the alkaline environment.

[0149] 2.4. In vitro hypoglycemic activity

[0150] 2.4.1. α-Glucosidase inhibitory activity and inhibition kinetics evaluation

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

[0152] This invention studied the reversibility and type of α-glucosidase inhibitory effect to reveal the inhibitory mechanism of PPP-0A on this enzyme. According to the characteristics of the binding process between the inhibitor and the enzyme, the inhibitory effect can be divided into irreversible and reversible types. Figure 12 Figure C shows the inhibition kinetic curves of PPP-0A at different mass concentrations (0, 2, and 4 mg / mL) on α-glucosidase. After adding PPP-0A to the enzyme reaction system, three regression lines with zero intercepts were generated. The slopes of these regression lines were inversely proportional to the mass concentration of PPP-0A, indicating that the inhibitory type of PPP-0A on α-glucosidase is reversible. In addition, the change in kinetic parameters also helps to further understand the inhibitory type. With the concentration of α-glucosidase remaining constant, the mass concentrations of PPP-0A and the substrate (PNPG) were changed, and the reaction rate (V) of the enzyme inhibition system was measured. A double-reciprocal plot of [V] vs. [S] was plotted using the Lineweaver-Burk method. As Figure 12 shown in Figure E, a linear relationship exists 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 maxGradually decreased (Table 3). These results indicate that the inhibition type of PPP-0A on α-glucosidase can be determined as a combination of uncompetitive inhibition and non-competitive inhibition.

[0153] 2.4.2 α-Amylase inhibition activity and inhibition kinetics evaluation

[0154] The inhibitory effects of PPP-0A and acarbose on amylase are shown in Figure 12 Figure B. In 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. In the concentration range of 2 - 8 mg / mL, the inhibition rate increased significantly and then gradually stabilized with the increase in concentration. The calculated IC 50 value of PPP-0A 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 was also studied, and a linear relationship was found between the enzyme concentration and the 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 Figure 12 Figure D). In the reaction system, when the enzyme concentration remained constant, the reaction rates of pNPG at concentrations of 0.5%, 0.75%, 1%, 1.25%, and 1.5% were measured respectively. As shown in Figure 12 Figure F, a significant linear relationship was presented between 1 / [V] and 1 / [S], and all the straight lines intersected at a point in the third quadrant. According to the kinetic equation, when the concentrations of PPP-0A were 0, 2, and 4 mg / mL respectively, the measured V m values were 0.0082, 0.0053, and 0.003 (Table 4). Similarly, the recorded K m values were 5.27, 3.67, and 2.45 (Table 4). These results indicate that with the increase in the inhibitor concentration, both the reaction rate (V m ) and the Michaelis constant (K m ) decreased, which was in line 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 was consistent with the kinetic results of PPP-0A inhibiting α-glucosidase.

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

[0156]

[0157] 2.4.3. Cell viability assay

[0158] The cytotoxicity of the purified polysaccharide component PPP-0A extracted from Boletellus obscure- brunneus was evaluated using the CCK-8 assay to select an appropriate concentration of the polysaccharide solution for subsequent experiments. Figure 13 Panel A in [Figure] shows the toxicity of different concentrations of PPP-0A to HepG2 cells. As Figure 13 shown in Panel A, when the concentration of PPP-0A was between 0.4 and 6.4 mg / mL and the cells were treated for 24 h, the cell viability remained above 95%. This indicates that within this concentration range and treatment duration, PPP-0A has no obvious 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, the cell viability decreased. Therefore, subsequent experiments were carried out in the concentration range of 0.8 to 3.2 mg / mL to study the effect of the polysaccharide on improving insulin resistance in HepG2 cells, and the treatment duration was also 24 h.

[0159] 2.4.4. Establishment of an insulin resistance model in HepG2 cells

[0160] Figure 13 Panels C and D in [Figure] describe the effects of different concentrations of insulin and different treatment durations on the viability and glucose consumption of HepG2 cells. As Figure 13 shown in Panel C, as the insulin concentration (10 -9 , 10 -8 , 10 -7 , 10 -6 and 10 -5 M) increased and the exposure time (24, 36, 48, and 60 h) prolonged, the cell viability gradually decreased. Prolonged use of high concentrations of insulin inhibited cell growth. It should be noted that when the insulin concentration was 10 -6 M, within the first 48 h, the cell viability was not significantly different from that of the control group, with a viability exceeding 93%. However, after 48 h, the cell activity was significantly inhibited (p < 0.01). In addition, the viabilities of cells treated with 10 -5 M at 24 h, 36 h, 48 h, and 60 h were also significantly different from those of the control group (p < 0.05 or p < 0.01).

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

[0162] 2.4.5. Study on the stability of insulin resistance cell model

[0163] After successfully establishing the model using the optimal insulin concentration and duration described in 2.4.4, the cells of the control group and the experimental group were cultured simultaneously in normal medium without insulin to evaluate the duration of insulin resistance in the model. As Figure 13 shown in B, during the 48 h and 72 h of culture, there were significant differences in the glucose consumption of the experimental group cells compared with the control group.

[0164] 2.4.6. Effects of PPP-0A on glucose consumption and glycogen synthesis in IR-HepG2 cells

[0165] Under the induction of high glucose and high fat, the effects of PPP-0A on glucose metabolism in IR-HepG2 cells were evaluated by the glucose oxidase method. HepG2 cells were cultured in a high-glucose environment with an insulin model at a concentration of 10 -6 M for 48 h. According to the administration toxicity, IR-HepG2 cells were treated with 0.8 - 3.2 mg / mL PPP-0A for 24 h. The experiment included a normal group, a model group, and a positive control group. As Figure 14 shown in A, under the induction of an insulin concentration of 10 -6 M, there were significant differences in the glucose consumption of the model group compared with the normal group (p < 0.05), indicating that the insulin resistance model was successfully established and could 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 both increased, indicating that PPP-0A could effectively promote the glucose consumption of IR-HepG2 cells. It should be noted that the glucose consumption of the 3.2 mg / mL PPP-0A treatment group reached (13.19 ± 0.92 mM). In addition, insulin resistance is closely related to hepatic glycogen synthesis. As Figure 14 shown in B, compared with the model group, the increase in glycogen content in the PPP-0A group was dose-dependent. 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 indicates that PPP-0A can promote glycogen synthesis, thereby reducing blood glucose levels.

[0166] 2.4.7. HK and PK Activities

[0167] To evaluate the regulatory effect of PPP-0A on these two key hepatic enzymes during glucose metabolism, the present invention treated the IR-HepG2 cell model with various concentrations of PPP-0A to determine its regulatory effect on key hepatic gluconeogenic enzymes. As Figure 14 shown in C and D, compared with the model group, the activities of HK and PK in the PPP-0A treatment group were significantly increased. At a concentration of 3.2 mg / mL, the activities of HK and PK were close to those of the positive control group treated with metformin (4 mM). In summary, these research results indicate that PPP-0A can enhance glucose metabolism by increasing the activities of HK and PK, thereby promoting glycogen synthesis and glycolysis. Therefore, PPP-0A has the potential to lower blood glucose levels.

[0168] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention 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 are 62.26%, 16.96%, 15.23%, 4.68% and 0.87% respectively; The structure of the dark brown veined boletus polysaccharide is shown below: The preparation method of the dark brown veined boletus polysaccharide comprises the following steps: Mix and extract the fruiting body of Boletus edulis with ethanol, discard the supernatant, add water to the precipitate for extraction, concentrate the extract, add ethanol to collect the precipitate and freeze-dry it to obtain a freeze-dried powder; elute the freeze-dried powder with HPD-100 macroporous adsorption resin, adsorb the decolorized eluate with 12% polyamide powder for 4 hours, elute, collect the eluate, and freeze-dry it to obtain crude polysaccharide; The crude polysaccharide is eluted through a DEAE-52 cellulose chromatographic column, and fractions are collected by eluting with distilled water and NaCl solutions with concentrations of 0.1, 0.2, 0.4, 0.8 and 1.0 M at a flow rate of 1 mL / min, and the fraction with the highest polysaccharide content is further purified by Sephacryl S-400HR to obtain dark brown veined boletus polysaccharide; 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: 80° C. for 2-3 times, each time for 50-100 minutes; The elution condition of the Sephacryl S-400HR is: elution with distilled water at 1 mL / min.

2. Use of the dark brown veined boletus polysaccharide according to claim 1 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.

3. An α-glucosidase and / or α-amylase activity inhibitor, characterized in that: The invention comprises the dark brown veined boletus polysaccharide as claimed in claim 1.

4. A drug for improving insulin resistance in HepG2 cells, characterized in that: The invention comprises the dark brown veined boletus polysaccharide as claimed in claim 1.

5. A drug for lowering blood sugar, characterized in that: The invention comprises the dark brown veined boletus polysaccharide as claimed in claim 1.

6. The method for preparing the polysaccharide of Boletus edulis according to claim 1, characterized in that: The following steps are involved: Mix and extract the fruiting body of Boletus edulis with ethanol, discard the supernatant, add water to the precipitate for extraction, concentrate the extract, add ethanol to collect the precipitate and freeze-dry it to obtain a freeze-dried powder; elute the freeze-dried powder with HPD-100 macroporous adsorption resin, adsorb the decolorized eluate with 12% polyamide powder for 4 hours, elute, collect the eluate, and freeze-dry it to obtain crude polysaccharide; The crude polysaccharide is eluted through a DEAE-52 cellulose chromatographic column, and fractions are collected by eluting with distilled water and NaCl solutions with concentrations of 0.1, 0.2, 0.4, 0.8 and 1.0 M at a flow rate of 1 mL / min, and the fraction with the highest polysaccharide content is further purified by Sephacryl S-400HR to obtain dark brown veined boletus polysaccharide; 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: 80° C. for 2-3 times, each time for 50-100 minutes; The elution condition of the Sephacryl S-400HR is: elution with distilled water at 1 mL / min.

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