A new grifola frondosa homogeneous polysaccharide gfp-z and its simple and rapid preparation method and use

Abstract

This invention discloses a novel homogeneous polysaccharide GFP-Z from Grifola frondosa, along with its simple and rapid preparation method and applications. This homogeneous Grifola frondosa polysaccharide GFP-Z differs from previously reported Grifola frondosa polysaccharides. It is composed of α(1,4)-D-Glc, contains branches linked by α-1,6 glycosidic bonds, has a branching degree of approximately 1 / 10, a molecular weight greater than 1000 kDa, and consists solely of glucose, containing no protein. This invention provides a novel Grifola frondosa polysaccharide. The preparation method of this invention eliminates the need for column chromatography, yielding homogeneous GFP-Z, and offers advantages such as simplicity, speed, and low cost. The hypoglycemic effect of the homogeneous Grifola frondosa polysaccharide GFP-Z provided by this invention is significantly superior to that of the positive control drug metformin. Therefore, this invention lays the foundation for developing new drugs for the treatment of diabetes and actively promotes the research and development of active ingredients in natural drugs for diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a novel homogeneous polysaccharide GFP-Z from Grifola frondosa, its simple and rapid preparation method, and its applications. Background Technology

[0002] Diabetes is a serious chronic disease caused by the body's inability to produce enough insulin or to use the insulin effectively, leading to elevated blood sugar levels. This causes damage to many organs, resulting in disability and life-threatening complications. According to data released by the International Diabetes Federation (IDF) in 2021, there are 537 million adults aged 20-79 (10.5% of the global population) with diabetes, of which type 2 diabetes accounts for more than 90% of all diabetes cases worldwide. The total number of people with diabetes globally is projected to increase to 643 million (11.3% of the global population) by 2030 and to 783 million (12.2% of the global population) by 2045. Diabetes is an unprecedented pandemic, one of the fastest-growing global health emergencies of the 21st century, and it is spiraling out of control.

[0003] The pathogenesis of diabetes is not fully understood, and it is difficult to cure. Clinically, the main treatment for diabetes is long-term use of hypoglycemic drugs to control and alleviate the condition. Currently, the drugs used to treat type 2 diabetes are mainly chemical or biochemical drugs, including alpha-glucosidase inhibitors, insulin secretagogues, insulin sensitizers, DPP4 inhibitors, SGLT2 inhibitors, GLP-1 receptor agonists, insulin and its analogues, etc. However, they all have their own significant side effects, such as gastrointestinal discomfort, hypoglycemia, weight gain, sodium and water retention, heart failure, nasopharyngitis, skin edema and peeling, severe arthritis, acute pancreatitis, bladder cancer, congestive heart failure, and many other adverse reactions. This serious situation compels the search for safer and more effective hypoglycemic active substances. Finding natural, safe, and effective new active ingredients from nature has always been a key focus of new drug development.

[0004] Currently, there are various methods for the extraction, separation, and purification of Grifola frondosa polysaccharides. The main extraction methods include cold water soaking, hot water extraction, ultrasound-assisted extraction, subcritical water extraction (SWE), enzymatic extraction, and high-pressure extraction. The polysaccharides obtained by these methods differ significantly in molecular structure, chain conformation, and biological activity. Each extraction method has its own advantages and disadvantages. After extraction, the extract needs to be processed through deproteinization, ultrafiltration, dialysis, and freeze-drying to obtain crude polysaccharides. To accurately assess its structural characteristics and biological activity, the crude polysaccharides need further separation and purification. Generally, the crude polysaccharides are first deproteinized and decolorized, then separated and purified using column chromatography, mainly including anion exchange chromatography, filtration chromatography, affinity chromatography, and molecular sieve chromatography. Finally, the polysaccharides are concentrated, dialyzed, and freeze-dried to obtain relatively pure polysaccharides.

[0005] Although Grifola frondosa and its active ingredients have been found to have significant hypoglycemic effects, the diverse and complex extraction processes significantly impact the structure and pharmacological activity of the products. Existing separation and purification methods are not only time-consuming, labor-intensive, and costly, but also fail to yield pure active polysaccharides, instead producing mixtures of various components (polysaccharides, proteins, small molecules, etc.). Furthermore, the chemical structure of polysaccharides is inherently complex. As a result, the composition, structure, and mechanism of action of most of Grifola frondosa's hypoglycemic active substances remain unclear, directly hindering their development into new drugs for treating diabetes. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a novel homogeneous polysaccharide GFP-Z from Grifola frondosa, along with its simple and rapid preparation method and applications. The method of this invention enables the rapid preparation of a novel homogeneous polysaccharide GFP-Z from Grifola frondosa, which exhibits significant hypoglycemic activity and can markedly improve the symptoms of type 2 diabetes.

[0007] The first objective of this invention is to provide a novel homogeneous polysaccharide GFP-Z from Grifola frondosa, which is composed of α(1,4)-D-Glc and contains branches linked by α-1,6 glycosidic bonds, with a branching degree of approximately 1 / 10. Its chemical structure is shown below:

[0008]

[0009] The appearance and texture of the lyophilized Grifola frondosa homogeneous polysaccharide GFP-Z are as follows: white, loose and porous, with a soft, cotton-like texture. HPLC analysis shows a single peak with a weight-average molecular weight of approximately 1765 kDa and a peak molecular weight of approximately 1354 kDa, classifying it as a macromolecular polysaccharide with a molecular weight greater than 1000 kDa. The sugar content of homogeneous polysaccharide GFP-Z is 98.5% ± 1%, consisting only of glucose (Glu 100%), and contains no protein, suggesting it is a protein-free dextran. DEAE ion-exchange column chromatography confirmed that it was almost completely eluted in the 0.1 M NaCl region. Structural analysis revealed the presence of α-glycosidic bonds, specifically α-1,4 and α-1,6 glycosidic bonds. Based on these parameters, no similar Grifola frondosa polysaccharides were found in comparison with existing Grifola frondosa polysaccharides; therefore, the Grifola frondosa homogeneous polysaccharide GFP-Z of this invention is a novel Grifola frondosa polysaccharide.

[0010] The second objective of this invention is to provide a simple and rapid method for preparing a new homogeneous polysaccharide GFP-Z from Grifola frondosa that has a hypoglycemic effect, replacing the traditional column chromatography method for obtaining homogeneous polysaccharides. This method specifically includes the following steps:

[0011] (1) The fruiting bodies of the Grifola frondosa were crushed, extracted with hot water, filtered, and the residue was extracted with hot water again, filtered, and the filtrates were combined. Then centrifuged, the supernatant was filtered with filter paper and concentrated. After the concentrated water extract was placed at room temperature, ethanol was added to it, and the mixture was stirred slowly and then stirred until it was evenly mixed. After centrifugation, the precipitate was resuspended with purified water, and the ethanol was removed by rotary evaporation. The resuspended liquid was labeled GF42.

[0012] (2) Add purified water to GF42, stir and centrifuge, filter the supernatant, add ethanol to the filtrate, stir slowly and then centrifuge, collect the precipitate, reconstitute the precipitate with purified water, freeze dry, and obtain Grifola frondosa homogeneous polysaccharide GFP-Z.

[0013] Preferably, in step (1), the volume ratio of the concentrated water extract to ethanol is 1.5:(1-1.25).

[0014] Preferably, in step (2), the volume ratio of the filtrate to ethanol is 6:(4-5).

[0015] Preferably, the concentration of the ethanol is 42% by volume.

[0016] Preferably, in step (2), the filtration is performed using a 0.22 μm aqueous microporous membrane.

[0017] A third objective of this invention is to provide the application of Grifola frondosa homogeneous polysaccharide GFP-Z in the preparation of drugs for treating diabetes, particularly in the preparation of drugs for treating type 2 diabetes.

[0018] A fourth objective of this invention is to provide a medicament for treating diabetes, wherein the medicament comprises Grifola frondosa homogeneous polysaccharide GFP-Z as an active ingredient, wherein the diabetes is type 2 diabetes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) This invention provides a novel homogeneous polysaccharide GFP-Z from Grifola frondosa. This homogeneous polysaccharide GFP-Z is composed of α(1,4)-D-Glc and has branches linked by α-1,6 glycosidic bonds. The degree of branching is about 1 / 10, and the molecular weight is greater than 1000kDa. It is a protein-free glucan, which is different from previously reported Grifola frondosa polysaccharides. It is a novel Grifola frondosa polysaccharide, and therefore this invention expands the types of Grifola frondosa polysaccharides.

[0021] (2) The present invention also provides a simple and rapid method for preparing a new homogeneous polysaccharide GFP-Z from Grifola frondosa. The method of the present invention does not require the use of column chromatography (DEAE ion column or molecular sieve column chromatography, etc.) to obtain homogeneous polysaccharide GFP-Z, and has the advantages of being simple, rapid and low cost.

[0022] (3) The hypoglycemic effect of the homogeneous polysaccharide GFP-Z provided by the present invention is significantly better than that of the positive drug metformin. Therefore, the present invention lays the foundation for the development of new drugs for the treatment of diabetes and actively promotes the research and development of active ingredients of natural drugs for diabetes. Attached Figure Description

[0023] Figure 1 Calibration curve for GPC (glucan) standard.

[0024] Figure 2 This is an HPLC analysis chromatogram of the homogeneous polysaccharide GFP-Z from Grifola frondosa.

[0025] Figure 3 The appearance of Grifola frondosa homogeneous polysaccharide GFP-Z.

[0026] Figure 4 This is a liquid chromatography chromatogram of PMP-derived monosaccharide mixed standards. PMP: 1-Phenylon-3-methyl-5-pyrazolone, Man: mannose, Rib: ribose, Rha: rhamnose, GlcA: glucuronic acid, GalA: galacturonic acid, Glc: glucose, Gal: galactose, Xly: xylose, Ara: arabinose, Fuc: fucose.

[0027] Figure 5 Liquid chromatography diagram showing the monosaccharide composition analysis of Grifola frondosa homogeneous polysaccharide GFP-Z.

[0028] Figure 6 Infrared (IR) spectral analysis of GFP-Z, a homogeneous polysaccharide from Grifola frondosa.

[0029] Figure 7 For Grifola frondosa homogeneous polysaccharide GFP-Z 1 H-NMR spectrum.

[0030] Figure 8 For Grifola frondosa homogeneous polysaccharide GFP-Z 13 C-NMR spectrum.

[0031] Figure 9 For Grifola frondosa homogeneous polysaccharide GFP-Z 1 HNMR (315K) spectrum.

[0032] Figure 10 For Grifola frondosa homogeneous polysaccharide GFP-Z 1 H- 13 C HSQC spectrum.

[0033] Figure 11 For Grifola frondosa homogeneous polysaccharide GFP-Z 1 H- 1 H COSY spectrum.

[0034] Figure 12 For Grifola frondosa homogeneous polysaccharide GFP-Z 1 H- 1 H ROSY spectrum.

[0035] Figure 13 This is a chemical structural fragment of the homogeneous polysaccharide GFP-Z from Grifola frondosa.

[0036] Figure 14 Fasting blood glucose levels in mice of each group after 30 days. NC: normal control, DC: model control, PC: positive control, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001.

[0037] Figure 15 The values ​​represent the water intake / body weight of mice in each group over 30 days. NC: normal control, DC: model control, PC: positive control.

[0038] Figure 16 The values ​​represent the feed / body weight of mice in each group over 30 days. NC: normal control, DC: model control, PC: positive control. Detailed Implementation

[0039] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0040] Example 1

[0041] Preparation of homogeneous polysaccharide GFP-Z from Grifola frondosa:

[0042] Take 50g of *Grifola frondosa* fruiting bodies, crush them, add 0.6L of pure water, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, add another 0.35L of pure water to the filter residue, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, combine the two filtrates, centrifuge at 4000r / min for 10min, filter the supernatant through filter paper to obtain the water extract. Concentrate the water extract by rotary evaporation at 52℃, and make up to a certain volume with a graduated cylinder to obtain the concentrated water extract. After the concentrated water extract is placed at room temperature, add anhydrous ethanol at approximately 200mL / min, stirring slowly and quickly until the final ethanol concentration is 45% (v / v). After mixing evenly, cover with plastic wrap and place in a refrigerator at 4℃ for 1h, centrifuge at 7000r / min for 15min, add purified water to the precipitate for resuspending, remove residual ethanol by rotary evaporation, label the resuspended solution GF45, and it is grayish-white. Further purification of GF45: Dilute GF45 with an appropriate amount of pure water, centrifuge at 8000 r / min for 30 min, filter with a 0.22 μm aqueous microporous membrane to remove poorly soluble substances, bring the filtrate to a certain volume, add anhydrous ethanol to make the final concentration 45% (v / v), stir slowly and quickly, centrifuge at 8000 r / min for 5 min, collect the precipitate (the further purification step of GF45 can be repeated once as needed), the precipitate is white, redissolve the precipitate with an appropriate amount of purified water, freeze dry, and that is the GFP-Z component.

[0043] Example 2

[0044] Preparation of homogeneous polysaccharide GFP-Z from Grifola frondosa:

[0045] Take 300g of *Grifola frondosa* fruiting bodies, crush them, add 3.5L of pure water, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, add another 2L of pure water to the filter residue, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, combine the two filtrates, centrifuge at 4000r / min for 10min, filter the supernatant through filter paper to obtain the water extract. Concentrate the water extract by rotary evaporation at 52℃, and bring it to a certain volume with a graduated cylinder to obtain the concentrated water extract. After the concentrated water extract has been placed at room temperature, add anhydrous ethanol at approximately 200mL / min, stirring slowly and quickly until the final ethanol concentration is 40% (v / v). After mixing evenly, cover with plastic wrap and place in a refrigerator at 4℃ for 1h, centrifuge at 7000r / min for 15min, add purified water to the precipitate for resuspending, remove residual ethanol by rotary evaporation, label the resuspended solution GF40, and it should be grayish-white. Further purification of GF40: Dilute GF40 with an appropriate amount of pure water, centrifuge at 8000 r / min for 30 min, filter with a 0.22 μm aqueous microporous membrane to remove poorly soluble substances, bring the filtrate to a certain volume, add anhydrous ethanol to make the final concentration 40% (v / v), stir slowly and quickly, centrifuge at 8000 r / min for 5 min, collect the precipitate (the further purification step of GF40 can be repeated once as needed), the precipitate is white, redissolve the precipitate with an appropriate amount of purified water, freeze dry, and that is the GFP-Z component.

[0046] Example 3

[0047] Preparation of homogeneous polysaccharide GFP-Z from Grifola frondosa:

[0048] Take 300g of *Grifola frondosa* fruiting bodies, crush them, add 3.5L of pure water, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, add another 2L of pure water to the filter residue, boil at 90℃ for 75min, filter through a 0.125mm metal sieve, combine the two filtrates, centrifuge at 4000r / min for 10min, filter the supernatant through filter paper to obtain the water extract. Concentrate the water extract by rotary evaporation at 52℃, and bring it to a certain volume with a graduated cylinder to obtain the concentrated water extract. After the concentrated water extract has been placed at room temperature, add anhydrous ethanol at approximately 200mL / min, stirring slowly and quickly until the final ethanol concentration is 42% (v / v). After mixing evenly, cover with plastic wrap and place in a refrigerator at 4℃ for 1h, centrifuge at 7000r / min for 15min, add purified water to the precipitate for resuspending, remove residual ethanol by rotary evaporation, label the resuspended solution GF42, and it is grayish-white. Further purification of GF42: Dilute GF42 with an appropriate amount of pure water, centrifuge at 8000 rpm for 30 min, filter through a 0.22 μm aqueous microporous membrane to remove poorly soluble substances, bring the filtrate to a certain volume, add anhydrous ethanol to a final concentration of 42% (v / v), stir slowly and quickly, centrifuge at 8000 rpm for 5 min, collect the precipitate (the further purification step of GF42 can be repeated once if necessary), the precipitate is white, redissolve the precipitate with an appropriate amount of purified water, and lyophilize to obtain the GFP-Z fraction. Subsequent experiments will use this preparation process to provide compounds.

[0049] Example 4

[0050] Purity and molecular weight determination of Grifola frondosa homogeneous polysaccharide GFP-Z, and purity verification by DEAE ion exchange column chromatography:

[0051] 1. Chromatographic conditions:

[0052] An Agilent 1260 Infinity II liquid chromatograph was used; the column was a TSKgel G5000PW. XL (7.8mm ID

[0053] (×30cm, 10μm) + TSKgel G3000PW XL (7.8mm ID×30cm, 7μm) tandem analysis; column temperature 35℃; mobile phase 0.01M NaCl solution; flow rate 0.28mL / min; isothermal 35℃; analysis time 120min; differential refractive index detector; injection volume 20μL.

[0054] 2. Establishment of GPC calibration curve:

[0055] Two mg of GPC standards with molecular weights of 1 kDa, 5 kDa, 12 kDa, 25 kDa, 50 kDa, 270 kDa, 670 kDa, and 1194 kDa were weighed and dissolved in 200 μL of 0.01 M NaCl solution. The solutions were filtered through a 0.22 μm filter and analyzed under the chromatographic conditions described above. Data are summarized in Table 1. A GPC calibration curve was plotted with molecular weight of the GPC standards on the ordinate and retention time on the abscissa, and the curve equation was fitted. The calibration curve is shown below. Figure 1 The fitted curve equation is:

[0056] Y = -0.000355166x 3 +0.0618383x 2 -3.67089x+78.7202.

[0057] Table 1. Molecular weight and retention time of GPC standards

[0058]

[0059] 3. Determination of purity and molecular weight of Grifola frondosa homogeneous polysaccharide GFP-Z:

[0060] Dissolve 2 mg of GFP-Z lyophilized powder in 0.01 M NaCl solution, filter through a 0.22 μm filter membrane, and analyze under the above chromatographic conditions. The GFP-Z peak eluted at approximately 46.043 min, with a solvent peak eluting at around 84 min. The chromatogram is shown below. Figure 2 Corresponding to the established GPC standard curve, the weight-average molecular weight of GFP-Z was calculated to be approximately 1765 kDa, the peak molecular weight was approximately 1354 kDa, and the dispersibility was 1.7.

[0061] 4. Verification of the purity of Grifola frondosa homogeneous polysaccharide GFP-Z by DEAE ion exchange column chromatography:

[0062] 0.5 g of GFP-Z lyophilized powder was dissolved in an appropriate amount of pure water and loaded onto a DEAE ion exchange column equilibrated with pure water. The column was eluted sequentially with pure water, 0.1 M NaCl, 0.5 M NaCl, and 2 M NaCl. The eluent from each segment was collected, dialyzed to remove salt for 2 days, concentrated, freeze-dried, and weighed. The results showed that GFP-Z was almost completely eluted (>95%) in the 0.1 M NaCl segment.

[0063] Example 5

[0064] Physicochemical properties determination of Grifola frondosa homogeneous polysaccharide GFP-Z:

[0065] 1. Appearance and texture of GFP-Z:

[0066] GFP-Z lyophilized powder appears as follows: white, loose and porous, with a soft, cotton-like texture. Figure 3 ).

[0067] 2. Polysaccharide and protein content of GFP-Z:

[0068] This experiment used the phenol-sulfuric acid method to determine the polysaccharide content, following the "People's Republic of China Agricultural Industry Standard NY / T 1676-2008 Determination of Crude Polysaccharide Content in Edible Fungi". Eleven gradient concentration standard solutions (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 mg / mL) were prepared using standard dextran. A standard curve was established with each concentration gradient as the x-axis and the product absorbance as the y-axis. The fitted linear equation was y = 6.4896x + 0.072, R0. 2 =0.996. GFP-Z was configured with solubility within two linear ranges, with three replicates for each concentration. The sugar concentration of GFP-Z was calculated by substituting the absorbance values ​​into the equation, and the sugar content was calculated and averaged. The calculation results showed that the sugar content of GFP-Z was 98.5% ± 1%.

[0069] This experiment used the BCA method to determine protein content. Eight gradient standard protein solutions (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL) were prepared using fetal bovine serum albumin. A standard curve was constructed with each concentration gradient as the x-axis and the product absorbance as the y-axis. The fitted linear equation was y = 1.2185x + 0.1611, R0. 2 =0.999. GFP-Z was configured with two concentrations within a linear range, with three replicates for each concentration. The GFP-Z protein concentration was calculated using the absorbance values, and the protein content was calculated and averaged. The results showed that the GFP-Z protein content was 0. Therefore, it is inferred from this data that GFP-Z does not contain protein.

[0070] 3. Monosaccharide composition of GFP-Z:

[0071] This experiment used PMP-HPLC pre-column derivatization to analyze the monosaccharide composition. (PMP: 1-phenyl-3-methyl-5-pyrazolone)

[0072] Prepare standard solutions for each of the 10 monosaccharide standards (D-Arabinose, D-Mannose, D-Glucose, D-Galactose, D-Xylose, D-Ribose, L-Rhamnose, D-Glucuronic acid, D-Galacturonic acid, and D-Fucose). Then, take 100 μL of each monosaccharide standard solution to form a mixed standard solution.

[0073] Sample acid hydrolysis: Take 2 mg of GFP-Z into a colorimetric tube, add 1 mL of ultrapure water to dissolve it, add 1 mL of 4 M trifluoroacetic acid (TFA), seal the tube, and heat it in a 100 °C water bath for 4 h. After the reaction is complete, add methanol and evaporate it by rotary evaporation. Repeat this process several times until TFA is completely removed. The hydrolysis product is then redissolved in 200 μL of ultrapure water.

[0074] PMP Derivatization: 150 μL of the GFP-Z hydrolyzed sample solution, 150 μL of the mixed standard solution, and 150 μL of each monosaccharide standard were added to separate 2 mL EP tubes. Then, 150 μL of 0.6 M NaOH solution was added, and the mixture was stirred. Next, 300 μL of 0.5 M PMP methanol solution was added, and the mixture was shaken and incubated in a 70 °C water bath for 100 min. After the reaction, 300 μL of 0.3 M HCl was added for neutralization, followed by 100 μL of ultrapure water and 1 mL of chloroform extraction. The mixture was centrifuged at 2000 rpm for 7 min, and the upper aqueous phase was collected. This extraction was repeated three times. The upper aqueous phase was filtered through a 0.22 μm microporous membrane and then analyzed by HPLC.

[0075] HPLC analysis: The mobile phase was prepared as follows (0.34 L acetonitrile + 1.66 L phosphate buffer). The column was YMC-Pack ODS-AM (5 μm, 250 mm × 4.6 mm). The column temperature was 25℃. The UV detection wavelength was 254 nm. The flow rate was 1 mL / min. The injection volume was 10 μL. The elution procedure is shown in Table 2 below.

[0076] Table 2. Elution Procedure

[0077]

[0078] Figure 4 and Figure 5 The results showed that the homogeneous polysaccharide GFP-Z consisted only of glucose (Glu 100%). It was inferred that it was a protein-free dextran.

[0079] 4. Infrared spectrum (IR) of GFP-Z:

[0080] Take approximately 2 mg of Grifola frondosa homogeneous polysaccharide GFP-Z lyophilized powder, mix it with dried KBr, grind it into a fine powder, compress it into tablets, and then press it into tablets at 400-4000 cm⁻¹. -1 Infrared scanning was performed on the interval, and the absorption peaks in the scan pattern were interpreted.

[0081] Figure 6 The results showed that 3387cm -1 The absorption peak at 2920 cm⁻¹ exhibits a broad and strong peak, which is due to the stretching vibration of OH, directly related to the stretching frequency caused by the large number of hydroxyl (OH) groups in GFP-Z; -1 1650cm -1 The absorption peak at 1200-1500 cm⁻¹ is due to the stretching vibration of CH; -1 The absorption peak at 1000-1200 cm⁻¹ is due to the in-plane bending vibrations of CH and OH. -1 The absorption peak at 930 cm⁻¹ is due to carbohydrate ring vibration; -1 The absorption peak at 850 cm⁻¹ indicates the presence of an α-glycosidic bond; -1 The absorption peak at 575 cm⁻¹ indicates the possible presence of a β-glycosidic bond; -1 The absorption peaks at that location are a result of the pyranose ring skeleton. The results indicate that the IR spectrum of GFP-Z contains characteristic absorption peaks of polysaccharides, including α-glycosidic bonds and possibly β-glycosidic bonds.

[0082] 5. GC-MS analysis of GFP-Z methylation:

[0083] To determine the linkage type between glycoside residues, a methylation-hydrolysis-reduction-acetylation reaction was performed, and the products were analyzed by GC-MS. First, GFP-Z was methylated with CH3I under anhydrous, alkaline conditions to methylate the free hydroxyl groups; infrared spectroscopy confirmed the disappearance of the hydroxyl absorption peak. The methylated GFP-Z polysaccharide was hydrolyzed by heating with 4M trifluoroacetic acid (TFA) at 100°C for 4 h, thereby exposing the hydroxyl groups at the linkage sites. Pure water and sodium borohydride were added to the hydrolyzed methylated polysaccharide, and the reaction was allowed to proceed overnight at room temperature. The next day, 20% glacial acetic acid was added to terminate the reaction, reducing the hydrolysate, opening the sugar ring, and reducing the terminal groups to hydroxyl groups. After drying, acetic anhydride was added and reacted at 100°C for 2 h to acetylate the exposed hydroxyl groups, yielding partially methylated adipol acetates (PMAAs). GC-MS analysis was used to determine the structure of the PMAAs in the reaction product and to identify the linkage type in GFP-Z.

[0084] GC-MS conditions: RXI-5SIL MS column 30m×0.25mm×0.25μm; temperature program conditions: initial temperature 120℃, increase to 250℃ / min at 3℃ / min; hold for 5min; injection port temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1mL / min.

[0085] Table 3. GC-MS analysis results of methylated derivatives of GFP-Z

[0086]

[0087] 2,3,4,6-Me4-Glc: 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol;

[0088] 2,3,6-Me3-Glc: 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol;

[0089] 2,3-Me2-Glc: 1,4,5,6-tetra-O-acetyl-2,3,-di-O-methyl-D-glucitol.

[0090] GC-MS data revealed the presence of three sugar alcohols in the methylated derivatives of GFP-Z: 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol, and 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl-D-glucitol. The integral area ratio of the three sugar alcohols was approximately 1:7.5:0.95. Based on these data, it is inferred that the neutral sugar GFP-Z contains (1,4) and (1,6) glycosidic bonds. The precise structure of GFP-Z will be further elucidated using spectroscopic methods.

[0091] 6. Nuclear magnetic resonance (NMR) of GFP-Z: 1 H-NMR and 13 C-NMR

[0092] from 1 H-NMR spectrum ( Figure 7 In the sample, the broad peak at δ(ppm) 6.02 was identified as an active H signal of -OH, and the peak at δ(ppm) 5.40 was identified as an α-glycosidic bond terminal H, possibly an α-1,4 glycosidic bond terminal H; combined with... 1 H-NMR and1 H- 1 H ROSY spectrum ( Figure 12 The H signal at δ(ppm) 4.80 may be a β-glycosidic bond or an α-glycosidic bond. This signal is present in... 1 The peaks overlap with the solvent peaks in the H-NMR spectrum. 1 H-NMR (315K) proves this. Figure 9 ); 1 The signal in the δ (ppm) range of 3.30-4.30 in the H-NMR spectrum was determined to be the H signal of H2-H6 in the sugar ring.

[0093] from 13 C NMR spectrum ( Figure 8 In the spectrum, δ(ppm) 99.81 was identified as the terminal C signal of an α-glycosidic bond. No terminal C signal of a β-glycosidic bond was found in the spectrum. Therefore, the H signal at δ(ppm) 4.80 was identified as the terminal H of an α-glycosidic bond, and possibly the terminal H of an α-1,6 glycosidic bond. 13 In the C NMR spectrum, the signal in the range of δ(ppm) 65-85 is the C signal of sugar ring C2-C5; the signal at δ(ppm) 60.52 is the C signal of -CH2OH, i.e., the C6 signal.

[0094] Combination 1 H- 13 C HSQC and 1 H- 1 H COSY spectrum ( Figure 10 , Figure 11 The glycosyl groups can be clearly identified as four groups of carbon and hydrogen signals, namely A, B, C, and D (E signal is not obvious), as shown in the GFP-Z chemical structure fragment diagram. Figure 13 According to glycosylation NMR 1 H and 13 The C signal indicates that positions 4 and 6 of glycosyl group C are connected to other glycosyl groups. Based on existing information, it is determined that the other glycosyl groups are connected to glycosyl group C via α-1,4 and α-1,6 glycosidic bonds, respectively. Similarly, it is determined that the other glycosyl groups are connected to glycosyl groups B and D via α-1,4 glycosidic bonds. 1 H- 1 The correlation signal at δ (ppm) 4.80 (D1) in the H ROSY spectrum indicates that glycosyl D and glycosyl C are linked at position 6. 1 H- 1 The correlation signal at δ (ppm) 5.40 in the H ROSY spectrum corroborates the previous conclusion of an α-1,4 glycosidic bond. These corroborating signals suggest that GFP-Z should be linked to AB via an α-1,4 glycosidic bond. x1 -CB x2 The main chain is formed by branches AB linked by α-1,4 glycosidic bonds. x3-D is linked to the 6-position of the glycosyl C via an α-1,6 glycosidic bond. Combined with GC-MS data on methylation of GFP-Z and 1 The HNMR spectrum indicates that the branching degree of GFP-Z is approximately 1 / 10.

[0095] Based on the monosaccharide composition analysis, infrared (IR) spectrum, methylation GC-MS analysis, and NMR spectrum data of GFP-Z, it is inferred that the homogeneous polysaccharide GFP-Z is composed of α(1,4)-D-Glc, with branched chains connected by α-1,6 glycosidic bonds, and a branching degree of approximately 1 / 10, as shown in the chemical structure fragment diagram of GFP-Z. Figure 13 ).

[0096] Example 6

[0097] Blood sugar lowering effect experiment:

[0098] This experiment used male BKS-db / db mice (6-7 weeks old) and male BKS-m littermate negative mice (6-7 weeks old), both fed a maintenance diet. After one week of acclimatization, fasting blood glucose was monitored. BKS-db / db mice with blood glucose levels in the range of 10-20 mmol / L were randomly assigned to three groups (model control, positive control, and GFP-Z treatment group, n=7) for subsequent experiments. BKS-m mice served as normal controls (n=7). Metformin was administered at a dose of 200 mg / kg / day for the positive control, and GFP-Z was administered at a dose of 60 mg / kg / day via intraperitoneal injection for 30 consecutive days. The normal control and model control groups received an equal volume of saline intraperitoneally. Fasting blood glucose levels were monitored every 10 days. Specifically, after fasting for 5 hours, blood glucose was measured using a blood glucose meter (ACCU-CHEK Performa Roche Advanced Glucose Meter) within 30 minutes. Water intake and feed weight were recorded during the experiment.

[0099] Fasting blood glucose results showed that on day 20, the blood glucose levels in the GFP-Z treatment group and the model group began to differ significantly (P < 0.001), and the effect remained significant on day 30 (P < 0.001). Furthermore, the hypoglycemic effect of GFP-Z was significantly superior to that of the positive control drug metformin (P < 0.05). Figure 14 ).

[0100] The water intake / body weight ratio results showed that the water intake / body weight ratio of the model control group gradually increased over time; the water intake / body weight ratios of both the metformin positive control group and the GFP-Z administration group were significantly lower than those of the model control group. Temporally, there was no significant difference between the two groups from 0 to 10 days, but as time increased, the water intake / body weight ratio of the metformin positive control group gradually increased, while the water intake / body weight ratio of the GFP-Z administration group remained at a low level or even showed a decreasing trend. Overall, there was a significant correlation between the water intake / body weight ratio and blood glucose levels in each group of mice, a result consistent with the symptoms of type 2 diabetes. Figure 15 ).

[0101] The feed-to-weight ratio results showed that the feed-to-weight ratio of the model control group mice remained at a high level over time; the feed-to-weight ratios of the positive control group (metformin) and the GFP-Z group were lower than those of the model control group and showed a decreasing trend. Comparatively, the decrease in the GFP-Z group was significantly greater than that of the metformin group. At 10-20 days, the feed-to-weight ratio of the GFP-Z group was already lower than that of normal mice, presumably because the db / db mice weighed more than twice as much as normal mice. Overall, there was a significant correlation between the feed-to-weight ratio and blood glucose levels in each group of mice, a result consistent with the symptoms of type 2 diabetes. Figure 16 ).

[0102] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Grifola frondosa homogeneous polysaccharide GFP-Z, characterized in that, The homogeneous polysaccharide GFP-Z from Grifola frondosa is composed of α(1,4)-D-Glc and contains side chains linked by α-1,6 glycosidic bonds. Its chemical structure fragment is shown below: ； The weight-average molecular weight of the homogeneous polysaccharide GFP-Z from Grifola frondosa is 1765 kDa, and the degree of branching is 1 / 10.

2. A simple and rapid method for preparing the homogeneous polysaccharide GFP-Z from Grifola frondosa as described in claim 1, characterized in that, Includes the following steps: (1) Crush the fruiting bodies of the maitake mushroom, extract with hot water, filter, extract the residue with hot water again, filter, and combine the filtrates; Then centrifuge, filter the supernatant with filter paper and concentrate; after the concentrated water extract is placed at room temperature, add ethanol, stir slowly and quickly, mix evenly, let stand, centrifuge, resuspend the precipitate with purified water, remove ethanol by rotary evaporation, and label the resuspended liquid as GF42. (2) Add purified water to GF42, stir and centrifuge, filter the supernatant, add ethanol to the filtrate, stir slowly and then centrifuge, collect the precipitate, reconstitute the precipitate with purified water, freeze dry, and obtain Grifola frondosa homogeneous polysaccharide GFP-Z. In step (1), the volume ratio of the concentrated water extract to ethanol is 1.5:(1~1.25); In step (2), the volume ratio of the filtrate to ethanol is 6:(4~5).

3. The preparation method according to claim 2, characterized in that, In step (2), the filtration is performed using a 0.22 μm aqueous microporous membrane.

4. The use of the homogeneous polysaccharide GFP-Z from Grifola frondosa as described in claim 1 in the preparation of a drug for treating diabetes.

5. The application according to claim 4, characterized in that, The diabetes mentioned is type 2 diabetes.

6. A drug for treating diabetes, characterized in that, The drug contains the homogeneous polysaccharide GFP-Z of Grifola frondosa as described in claim 1 as the active ingredient.

7. The drug according to claim 6, characterized in that, The diabetes mentioned is type 2 diabetes.

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