Zinc-modified cordyceps militaris polysaccharide, and preparation method and application thereof

Abstract

This invention discloses a zinc-modified Cordyceps militaris polysaccharide, wherein the zinc-modified Cordyceps militaris polysaccharide is a Cordyceps militaris polysaccharide-zinc complex (CMP-Zn), wherein the Cordyceps militaris polysaccharide (CMP) is in the form of α-D-pyranoyl glucan (α-D-Glc). p The polysaccharide with α-D-glucan as its main chain has a weight-average molecular weight of 9.6-9.7 kDa and a Glc content ≥92% in its monosaccharide composition; the CMP-Zn complex has a weight-average molecular weight of 1.5-1.6 kDa and a zinc content of 3.8-4.2% (w / w). The application of zinc-modified Cordyceps militaris polysaccharide in the preparation of products with antioxidant, hypoglycemic, or high-glucose-induced intestinal flora imbalance functions is disclosed. This invention achieves structural analysis, green zinc modification, and significant activity enhancement of Cordyceps militaris α-D-glucan, providing a new strategy for the high-value utilization of α-configuration edible fungal polysaccharides.

Description

Technical Field

[0001] This invention relates to the field of natural polysaccharide structure modification and functional food preparation technology, and particularly to a zinc-modified Cordyceps militaris polysaccharide and its preparation method and application. Background Technology

[0002] Diabetes has become the most burdensome chronic non-communicable disease globally, with direct medical expenditures and indirect social costs ranking first among all chronic diseases. Most diabetic patients are diagnosed with non-insulin-dependent diabetes mellitus, or type 2 diabetes, characterized by hyperglycemia and dyslipidemia caused by defects in insulin secretion and action. Considering the accompanying side effects and adverse reactions of chemically synthesized drugs such as acarbose and biguanides, edible and medicinal resources have become potential candidates for novel natural compounds with hypoglycemic and lipid-lowering activities. Multiple studies have reported that some plant polysaccharides exhibit hypoglycemic activity. These plant polysaccharides include those derived from mulberry bark, bamboo shoots, shellfish, wolfberry, and moringa.

[0003] Cordyceps militaris polysaccharide ( Cordyceps militaris polysaccharides CMP possesses various biological activities, including antioxidant, immune-enhancing, and anti-tumor effects, but natural CMP is primarily composed of α-D-Glc... p As the main chain, its antioxidant and hypoglycemic activities are significantly weaker than those of β-configuration polysaccharides, severely limiting the activity of α-D-Glc. p Applications of α-D-Glc are limited to complex organic reactions such as sulfation and carboxymethylation, which suffer from drawbacks such as cumbersome steps, residual organic solvents, and configuration destruction. Metal complexation, as a green, simple, and scalable modification strategy, has not yet been applied to α-D-Glc. p System report in type CMP.

[0004] Based on this, the present invention aims to construct a green, one-step, scalable ultrasonic hydrothermal complexation process for Zn²⁺. + Cordyceps militaris α-D-Glc p Highly active CMP-Zn complexes were obtained through complexation. Using an in vitro-in vivo-multi-omics strategy, the antioxidant, hypoglycemic, and gut microbiota remodeling effects were systematically evaluated, providing a promising technical route for the metallization modification of α-configuration polysaccharides and promoting the high-value application of Cordyceps militaris polysaccharides in functional foods and as adjuvant hypoglycemic inhibitors. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned problems. In a first aspect, it provides a zinc-modified Cordyceps militaris polysaccharide, wherein the zinc-modified Cordyceps militaris polysaccharide is a Cordyceps militaris polysaccharide-zinc complex (CMP-Zn), wherein the Cordyceps militaris polysaccharide (CMP) is in the form of α-D-pyranoyl glucan (α-D-Glc). pThe polysaccharide with the main chain structure of Glc has a weight-average molecular weight of 9.6-9.7 kDa and a Glc content of ≥92% in the monosaccharide composition; the CMP-Zn complex has a weight-average molecular weight of 1.5-1.6 kDa and a zinc content of 3.8-4.2% (w / w).

[0006] Furthermore, the glycosidic bond type of the CMP includes: →4)-Glc p -(1→、→3,4)-Glc p -(1→、→4,6)-Glc p -(1→、→3)-Glc p -(1→and Glc p -(1→, where →4)-Glc p -(1→ is the main chain skeleton.)

[0007] Secondly, a method for preparing any of the zinc-modified Cordyceps militaris polysaccharides as described in the first aspect is provided, comprising the following steps: S1: Dissolve Cordyceps militaris polysaccharide (CMP) and trisodium citrate in water at a mass ratio of 0.5:0.6, and pre-stir at 45-55℃ to form a transparent solution; S2: Slowly add ZnCl2 solution dropwise to the solution obtained in step S1, with the mass ratio of CMP to ZnCl2 being 0.5:1.0, and adjust the pH to 5.0-5.5 with NaOH; S3: The mixture obtained in step S2 was subjected to hydrothermal reaction at 50°C for 2.5 hours under ultrasonic power of 300 W and frequency of 40 kHz. S4: The reaction solution obtained in step S3 is subjected to ethanol precipitation, centrifugation, rotary evaporation to remove alcohol and freeze drying to obtain the CMP-Zn complex.

[0008] Furthermore, in step S1, the pre-stirring temperature is 50°C and the time is 10 min; in step S2, the ZnCl2 solution concentration is 100 mg / mL and the dropping rate is slow; in step S3, sonication and stirring are performed simultaneously.

[0009] Thirdly, the present invention also provides the application of zinc-modified Cordyceps militaris polysaccharide as described in the first aspect or zinc-modified Cordyceps militaris polysaccharide prepared by the method described in the second aspect in the preparation of products with antioxidant, hypoglycemic, or high-sugar-induced intestinal flora imbalance functions.

[0010] Furthermore, the antioxidant function is manifested in CMP-Zn increasing the scavenging rate of DPPH free radicals and ABTS. + Free radical scavenging rate and total reducing power.

[0011] Furthermore, the blood sugar lowering function is achieved through one or more of the following pathways: (1) In vitro inhibition of α-amylase and α-glucosidase activities, with CMP-Zn inhibiting α-amylase by ≥75% at a concentration of 0.8 mg / mL; and inhibiting α-glucosidase by 63% at high concentrations. (2) It lowers blood glucose levels in zebrafish with high glucose levels in vivo, with a hypoglycemic effect of 52% at a concentration of 20 μg / mL; (3) Upregulation of insulin receptor gene Insr and the gene of silence information regulator 1 Sirt1 Expression of lactate dehydrogenase A gene is downregulated. Ldha Hypoxia-inducible factor 1 subunit A gene Hif1a and pyruvate dehydrogenase kinase subtype 2 gene Pdk2 The expression of [something] promotes glucose oxidation and reduces lactic acid accumulation.

[0012] Furthermore, the restoration of the function of high-sugar-induced intestinal flora imbalance is manifested in: restoring the Shannon index of the intestinal flora to more than 85% of the normal level, and reducing the Proteobacteria phylum ( Proteobacteria The abundance of Bacteroidetes decreased by at least 11.72%, making the abundance of Bacteroidetes (Bacteroidetes) Bacteroidetes The abundance of Firmicutes increased by at least 99.35%, making Firmicutes ( Firmicutes ) and Actinobacteria ( Actinobacteriota They rebounded by 35.54% and 59.94% respectively.

[0013] Fourthly, the present invention also provides the application of zinc-modified Cordyceps militaris polysaccharide as described in the first aspect or zinc-modified Cordyceps militaris polysaccharide prepared by the method described in the second aspect in lowering blood sugar.

[0014] The present invention has the following beneficial effects: 1. This invention provides a structurally well-defined α-D-pyranoyl glucan (CMP). The invention obtains homogeneous CMP (Mw 9669 Da, glucose content 92%) through a modified hydrothermal method combined with fractional alcohol precipitation. Methylation and NMR analysis confirm that the main chain is →4)-α-D-Glc. p -(1→ is a typical α-configuration polysaccharide, providing a standard sample for structure-activity relationship studies.

[0015] 2. This invention establishes a green and simple zinc modification process: using an ultrasonic-assisted hydrothermal method, CMP-Zn complex (Zn content 3.8-4.2%) is prepared in one step under mild conditions (50℃, pH 5.0-5.5). Zn forms Zn-O / Zn-OH with hydroxyl groups without destroying the α-configuration. The process is green and can be scaled up.

[0016] 3. This invention can significantly improve antioxidant activity, increasing the DPPH scavenging rate of CMP-Zn from 16% to over 30% (an increase of >80%), ABTS + The clearance rate and total reducing power were significantly better than those of CMP (P<0.05).

[0017] 4. Significantly enhanced hypoglycemic activity and revealed a new mechanism: In vitro, CMP-Zn at 0.8 mg / mL inhibited α-amylase by >75%, and at high concentrations, it inhibited α-glucosidase by 63%; in a high-glucose zebrafish model, its hypoglycemic efficacy was 52% (comparable to metformin). Mechanistically, CMP-Zn upregulates... Insr / Sirt1 , lower Ldha / Hif1a / Pdk2 It promotes glucose oxidation and reduces lactic acid accumulation.

[0018] 5. Effectively reshapes the gut microbiota: CMP-Zn restored the Shannon index of the gut microbiota in high-sugar zebrafish to more than 85%, reduced the Proteobacteria phylum by 11.72%, and increased the Bacteroidetes, Firmicutes, and Actinobacteria phyla by 99.35%, 35.54%, and 59.94%, respectively, while downregulating oxidative stress-related functional genes.

[0019] 6. High safety and broad application prospects: The MTC level in the zebrafish model reaches 20 μg / mL with no toxic side effects. It can be used as an ingredient in functional foods, health foods, or as a precursor for adjuvant hypoglycemic drugs, showing great potential for industrialization.

[0020] In summary, this invention has achieved structural analysis, green zinc modification, and significant activity enhancement of Cordyceps militaris α-D-glucan, providing a new strategy for the high-value utilization of α-configuration edible fungi polysaccharides. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0022] Figure 1 : CMP monosaccharide composition ion chromatogram, where A is the standard chromatogram and B is the sample chromatogram; Figure 2 CMP molecular weight gel chromatogram; Figure 3 Microstructure of CMP at 40nm (A) and 2nm (B); Figure 4 CMP ultraviolet spectroscopy analysis (A) and infrared spectroscopy analysis (B) Figure 5CMP thermogravimetric analysis; Figure 6 GS-MS total ion chromatogram of PMAAs belonging to CMP; Figure 7 Mass spectra of various PMAAs belonging to CMP; Figure 8 CMP 1 H(A) and 13 C NMR (B) spectrum, two-dimensional COSY (C) and HSQC (D) spectra; Figure 9 Scanning electron microscope (SEM) images and EDS scan images (E) of CMP (A, C) and CMP-Zn (B, D) at different magnifications; Figure 10 : CMP and CMP-Zn UV (A) and IR spectral (B) scans; Figure 11 XRD patterns of CMP and CMP-Zn; Figure 12 Thermogravimetric curves of CMP(A) and CMP-Zn(B); Figure 13 XPS full spectra of CMP (A) and CMP-Zn (B), Zn 2p spectrum of high-resolution CMP-Zn (C), and C 1s (D) and (E), O 1s (F) and (G) spectra of high-resolution CMP and CMP-Zn; Figure 14 DPPH radical scavenging activity (A), ABTS+ scavenging activity (B) and total reducing power (C) of CMP and CMP-Zn; Figure 15 Inhibition rates of CMP and CMP-Zn on α-amylase (a, b, and c represent significant differences within groups; A, B, and C represent significant differences between groups) Figure 16 Inhibition rates of CMP and CMP-Zn on α-glucosidase (a, b, and c represent significant differences within groups; A, B, and C represent significant differences between groups) Figure 17 Glucose levels in zebrafish after CMP and CMP-Zn treatment; Figure 18 CMP and CMP-Zn affect insulin receptor sirtuin 1 ( Sirt1 (A), ( Insr (B) Lactate dehydrogenase A ( Ldha (C) Inhibit hypoxia-inducible factor 1 subunit A ( Hif1a (D) and pyruvate dehydrogenase kinase subtype 2 ( Pdk2 (E) The effect of expression level; Figure 19 The effects of each group on the Chao1 (A) index of gut microbiota and the number of observed (B) observations; Figure 20 The effects of each group on the Simpson (A) and Shannon (B) indices of gut microbiota; Figure 21 Venn diagrams of different sample groups; Figure 22 The relative abundance of each group of samples at the phylum (A) and genus (B) levels; Figure 23 Zebrafish gut microbiota PICRUSt2 functional annotation cluster heatmap (top 35). Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0024] Example 1: Purification, characterization and structural analysis of Cordyceps militaris polysaccharides 1.1 Experimental Materials Cordyceps militaris fruiting bodies were purchased from RT-Mart supermarket on Chunyang Road, Chengyang District, Qingdao City, Shandong Province.

[0025] 1.2 Experimental Methods 1.2.1 Extraction and purification of polysaccharides The fruiting bodies of *Cordyceps militaris* were dried and pulverized into powder (100 g). This powder was mixed with anhydrous ethanol (1:8, v / v) for 12 h to remove ethanol-soluble impurities such as fats and polyphenols. After filtration, the residue was collected and dried in a fume hood to evaporate the organic solvent. The pretreated sample was extracted with distilled water at a 1:50 material-to-liquid ratio at 70°C for 1 h with continuous stirring, followed by centrifugation (4800 rpm, 10 min). The supernatant was collected, and the precipitate was retained. This extraction was repeated twice, and the supernatants were combined. The sample was concentrated using a rotary evaporator at 55°C. Anhydrous ethanol was then slowly added to the concentrated sample while stirring to induce alcohol precipitation, resulting in a final ethanol concentration of 80%. The precipitate was then incubated overnight at 4°C. The precipitate was then collected by centrifugation (4800 rpm, 10 min), redissolved in ultrapure water, and deproteinized using the Sevag method (CHCl3 / BuOH = 4:1, v / v). The sample was dialyzed at 8000 Da for 72 h and then lyophilized to obtain crude *Cordyceps militaris* polysaccharide.

[0026] The polysaccharide purification method employed fractional alcohol precipitation. 15 g of crude polysaccharide was weighed and dissolved in 500 mL of water to obtain a homogeneous solution. Anhydrous ethanol was added to the CMP to a final concentration of 20%, and precipitation was carried out for 12 h. After precipitation, the solution was centrifuged at 10,000 rpm and 4 °C for 10 min, and the supernatant was retained. Excess organic matter was removed from the supernatant by rotary evaporation. This process was repeated to achieve a final concentration of 40%. The precipitate was discarded by centrifugation, and the supernatant was used to repeat the above steps to obtain a system with a final concentration of 80%. The precipitate of the final 80% system was washed twice with acetone and diethyl ether. Subsequently, it was dissolved in ultrapure water, and organic matter was removed by rotary evaporation. The solution was then lyophilized to obtain purified CMP.

[0027] 1.2.2 Ultraviolet Spectral Scan The ultraviolet absorption of polysaccharides and zinc polysaccharides was analyzed using ultraviolet spectroscopy. The test samples were prepared as 2 mg / mL aqueous solutions, and the full wavelength range of 190–600 nm was scanned using a UV-Vis spectrophotometer.

[0028] 1.2.3 Chemical Composition Analysis (1) Determination of total sugar content The total sugar content of CMP was determined using the phenol-sulfuric acid method. Polysaccharides hydrolyze into monosaccharides under concentrated sulfuric acid, which then rapidly dehydrate to form uronic acid derivatives. These derivatives react with phenol to form an orange-red compound with a maximum absorption peak at 490 nm. A glucose standard curve was plotted using a colorimetric method, and the polysaccharide concentration was calculated accordingly.

[0029] The specific steps are as follows: Take 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 100 μg / mL standard glucose solution into test tubes, and add distilled water to a final volume of 1 mL. Add 1 mL of 5% phenol to each tube, then quickly add 5 mL of concentrated sulfuric acid. Let stand for 10 min, vortex, and incubate in a 30℃ water bath for 20 min. Measure the absorbance at a wavelength of 490 nm (three parallel sets). Plot a standard curve with the absorbance value on the ordinate and the glucose concentration in the corresponding test tube on the abscissa.

[0030] Sample determination: First, prepare the sample to a concentration of approximately 100 μg / mL. Take 1 mL of the sample, 1 mL of phenol, and 5 mL of concentrated sulfuric acid. After the reaction, measure the absorbance and calculate the total sugar content based on the standard curve.

[0031] (2) Protein content determination The protein content of CMP was determined using the Coomassie Brilliant Blue method.

[0032] The specific steps are briefly described as follows: Take 0.0, 0.1, 0.2, 0.4, 0.6, and 0.8 mL of 0.1 mg / mL standard bovine serum albumin solution into test tubes, and add distilled water to a final volume of 1.0 mL. Add 5.0 mL of Coomassie Brilliant Blue G-250 reagent to each tube and vortex to mix. After 2 min, measure the absorbance at 595 nm (using 0.0 mL of protein standard solution as a blank control; three groups are parallel). Perform a linear regression of the absorbance value (Y) against the protein standard solution concentration (X, μg / mL) to obtain a standard curve.

[0033] Sample determination: Accurately transfer 1 mL of polysaccharide solution with a concentration of approximately 1 mg / mL into a test tube, add 5 mL of Coomassie Brilliant Blue, mix well, let stand at room temperature for 3 min, measure the absorbance, substitute it into the standard curve equation, and calculate the protein content.

[0034] (3) Determination of total phenol content The total phenol content was determined using the Folin-Ciocalteu method.

[0035] Construction of the standard curve: Pipette 0.0, 0.05, 0.10, 0.15, 0.20, and 0.25 mL of 100 μg / mL gallic acid standard solution into test tubes, and add distilled water to a final volume of 0.5 mL. Add 1 mL of 10-fold diluted Folin-Ciocalteu working solution, incubate at 30°C in the dark for 5 min, then add 2 mL of 10% sodium carbonate solution and react at 30°C in the dark for 1 h. Measure the absorbance at 760 nm (three parallel sets) and plot the gallic acid standard curve.

[0036] Sample determination: Accurately transfer 0.5 mL of a 1 mg / mL polysaccharide solution into a test tube and measure the absorbance using the same method as for the standard curve. Substitute the measured absorbance of the sample into the standard curve to determine the polyphenol content. The polyphenol content of the sample is expressed as gallic acid equivalents.

[0037] 1.2.4 Polysaccharide molecular weight determination and homogeneity determination The molecular weight of the samples was determined using the HPGPC-MALLS / RID system.

[0038] A Shodex SB-806 HQ gel column was used with a mobile phase of 0.1 mol / L NaNO3 solution. The sample (3.0 mg / mL) was injected into the elution column at a flow rate of 0.5 mL / min, with an injection volume of 200 μL. The column temperature was 40 °C. The refractive index increment remained at 0.145 mL / g. Furthermore, HPLC was performed on an LC-20A system equipped with a RID-10A refractive index detector to determine the weight-average molecular weight of the sample.

[0039] 1.2.5 Determination of polysaccharide and monosaccharide composition and proportion The monosaccharide composition was determined using the HPAEC-PAD method.

[0040] Accurately weigh 10 mg of the sample and dissolve it in 2 mL of 2.0 mol / L trifluoroacetic acid (TFA). Hydrolyze the solution at 80℃ for 5 h. After hydrolysis, add deionized water and remove the remaining TFA by rotary evaporation (60℃). Prepare a 1 mg / mL monosaccharide mixed standard solution (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid).

[0041] Clarification was performed using Supelclean™ ENVI-18 SPE tubes (500 mL / 6 mL) and a 0.22 μm aqueous filter. Samples were separated using a Dionex™ AminoPac™ PA10 IC column (Dionex, 3 × 250 mm) with 0.20 mol / L NaOH solution as the mobile phase at a flow rate of 0.25 mL / min at 30 °C. Monosaccharide composition was analyzed using a Thermo Fisher ICS-5000+ ion chromatograph.

[0042] 1.2.6 Microstructure Analysis The surface morphology of the sample under test was observed using a scanning electron microscope.

[0043] Take an appropriate amount of sample powder and sprinkle it evenly on the carbon conductive tape. Use a bulb syringe to blow away any unattached samples. After gold sputtering, vacuum the sample tray. Observe and photograph the surface morphology of the sample at 200 and 1000 magnifications under an accelerating voltage of 6.0 kV.

[0044] 1.2.7 Infrared Spectroscopy Scanning Infrared spectroscopy was performed using the potassium bromide pellet method.

[0045] A small amount of dried CMP was thoroughly mixed with KBr and pressed into thin sheets. To analyze the functional groups of the polysaccharide samples, FT-IR of the CMP was measured on a Nicolet 5700 spectrometer (Thermo Co., Madison, USA) in the range of 4000–400 cm⁻¹. 1. Resolution is 4 cm 1. The number of scans was 64.

[0046] 1.2.8 Thermogravimetric Analysis Accurately weigh 3 mg of sample and determine the thermal stability of the sample using a thermogravimetric analyzer with N2 (99.99%) as the protective gas. Measurement conditions: detection temperature 50~600℃, heating rate 10℃ / min; flow rate 30 mL / min.

[0047] 1.2.9 Methylation Analysis Weigh 2 mg of CMP into a glass reaction flask, add 1 mL of anhydrous DMSO, quickly add methylation reagent A, seal the flask, dissolve the methylation reagent under sonication, and then add methylation reagent B. React in a magnetically stirred water bath at 30°C for 60 min. Finally, add 2 mL of ultrapure water to the mixture to terminate the methylation reaction.

[0048] The methylated polysaccharide was hydrolyzed in 1 mL of 2 M trifluoroacetic acid (TFA) for 90 min, and then evaporated to dryness using a rotary evaporator. The residues were reduced with 2 mL of double-distilled water and 60 mg of sodium borohydride for 8 h, neutralized with glacial acetic acid, rotary evaporated, and dried in an oven at 101 °C. Then, 1 mL of acetic anhydride was added for acetylation, and the reaction was carried out at 100 °C for 1 h, followed by cooling. Then, 3 mL of toluene was added, and the mixture was concentrated under reduced pressure and evaporated to dryness. This process was repeated 4-5 times to remove excess acetic anhydride.

[0049] The acetylated product was dissolved in 3 mL of CH₂Cl₂ and transferred to a separatory funnel. A small amount of distilled water was added, and the mixture was shaken thoroughly. The supernatant was removed, and this process was repeated four times. The CH₂Cl₂ layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid chromatography vial. Analysis of the acetylated product sample was performed using a Thermo Scientific 1300-7000 gas chromatograph-mass spectrometer. GC-MS conditions: HP-INNOWAX column 30 m*0.32 mm*0.25 μm; temperature program conditions: initial temperature 140℃, increased to 230℃ at 1℃ / min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1 mL / min.

[0050] 1.2.10 1D and 2D nuclear magnetic resonance analysis (NMR) A 30 mg sample of CMP components was dissolved in 1 mL of D2O, placed in an NMR tube, and collected and recorded on an NMR spectrometer. 1 H NMR and 13 Chemical shifts of C NMR and acquisition of two-dimensional NMR data. 1 H- 1 H-related spectra (COSY) and 1 H- 13 C(HSQC) spectral chemical shift, expressed in ppm.

[0051] 1.2.11 Data Statistics and Analysis Each treatment consisted of three independent biological replicates. All data are expressed as mean ± standard deviation. Data differences were analyzed using SPSS 20.0 (SPSS Inc., USA), Duncan's multiple range test, and p < 0.05. Figures were plotted using Origin 2021.

[0052] 1.3 Experimental Results and Analysis 1.3.1 CMP Chemical Composition Analysis The maximum content of crude CMP extracted by hydrothermal method was 100 mg / g. After purification, the total sugar, protein, and total phenol content of CMP were determined. The results are shown in Table 1. The purified polysaccharide had lower protein and total phenol content, while the total sugar content reached 89.93 ± 0.23%. This indicates that the CMP purification effect was good.

[0053] Table 1. Composition of CMP monosaccharides

[0054] 1.3.2 Composition and Proportion of CMP Monosaccharides like Figure 1 As shown in Table 2, after monosaccharide component analysis of CMP, the main monosaccharide components were obtained and their proportions were summarized. The specific experimental results are shown in Table 2. The main components of CMP are Glc (92%), Man (3.4%), Gal (2.8%), and Ara, etc. Therefore, CMP purified by hydrothermal method is a neutral sugar mainly composed of Glc, but the purified CMP is specifically α-D-Glc. por β-D-Glc p It's not yet certain; more precise structural analysis is needed to prove that CMP is based on α-D-Glc p Primarily polysaccharides.

[0055] Table 2. Composition of CMP monosaccharides

[0056] 1.3.3 Determination of CMP Molecular Weight like Figure 2 The diagram shows the CMP distribution uniformity curve and molecular weight. Experimental results indicate that the molecular weight distribution curve has only one single peak, without any extraneous peaks, indicating good purification effect. The molecular weight is approximately 9669 Da, which differs from the molecular weight of polysaccharides extracted by other existing preparation methods. This may be due to temperature settings; some studies have indicated that the temperature in hydrothermal methods affects molecular weight. Furthermore, changes in molecular weight may lead to changes in biological function. Although some studies suggest that lower molecular weight reduces activity, others indicate that lower molecular weight may enhance activities such as hypoglycemic activity. Therefore, although the CMP purified in this invention has a lower molecular weight, this may actually promote the development of biological functional activities.

[0057] 1.3.4 CMP Microstructure Analysis like Figure 3 As shown in Figures A and B, CMP exhibits a smooth, sheet-like structure under a scanning electron microscope. The structure is loosely and irregularly arranged, lacking a network structure. It is speculated that the irregular morphology of CMP may be related to the type of glycosidic bonds.

[0058] 1.3.5 CMP Infrared and Ultraviolet Spectroscopy Detection like Figure 4 As shown in Figure -A, there is no absorption peak in the 260 nm-280 nm range, indicating that CMP contains no proteins or nucleic acids. Figure 4 As shown in -B, at 3339 cm 1 The strong absorption band nearby belongs to the OH stretching vibration, 2929 cm. 1 The nearby weak peak value belongs to CH stretching vibration. 1631 cm 1 and 1412 cm 1 The signals on the left and right are related to -C=O and CO stretching, respectively. In CMP, 768 cm 1 The absorption peak at 1024 cm⁻¹ is a characteristic absorption of the stretching vibration of the pyran ring, indicating that the sugar ring in the sugar chain is a pyran ring. 1 and 936 cm 1 The absorption peak at this point indicates both asymmetric and symmetric stretching vibrations of the D-glucopyranose ring COC skeleton, suggesting that this component may be D-Glc. p In addition, 859 cm 1 The absorption peak at that location represents the CH vibration of the α-terminal group of the D-glucopyranose ring. Therefore, the infrared spectroscopy results preliminarily indicate that the CMP extracted in this invention is α-D-Glc p It is a polysaccharide with a structure that does not contain β-glycosidic bonds.

[0059] 1.3.6 CMP Thermogravimetric Analysis like Figure 5 As shown, the TG and DTG curves of CMP were obtained by thermogravimetric analysis. The TG curve characterizes the thermogravimetric loss rate of CMP, showing two distinct mass losses. The first mass loss is due to water evaporation; the polysaccharide loses 6.9% of its mass in the range of 38.4℃ to 107.3℃, and the endothermic peak at 77.3℃ is due to the vaporization of adsorbed water in CMP. The second mass loss occurs in the range of 107.3℃ to 1016.2℃, with a mass loss rate of 82.1%, indicating that CMP undergoes thermal decomposition and chemical bond breaking in this temperature range. Furthermore, the second stage of mass loss is extremely rapid, with complete loss occurring around 360℃. The DTG curve shows that CMP reaches its maximum decomposition rate at 285.5℃. This is consistent with most natural polysaccharides, although some reports suggest that β-1,3-D-Glc... p It exhibits strong thermal stability, but the natural compound structure is prone to decomposition. This may be because at lower molecular weights, a triple helix structure is not formed, making the structure easily decomposed.

[0060] 1.3.7 CMP methylation analysis Since the monosaccharide composition of purified CMP does not contain uronic acid and is a neutral polysaccharide, methylation analysis can be performed directly. The methylated CMP sample was then subjected to acid hydrolysis, reduction, and acetylation to obtain partially methylated sugar alcohol acetate derivatives (PMAAs). These were then analyzed by GS-MS to obtain the total ion chromatogram of CMP PMAAs. Figure 6 ) and the corresponding mass spectra of each peak ( Figure 7 ).

[0061] Since the monosaccharide composition of purified CMP does not contain uronic acid and is a neutral polysaccharide, methylation analysis can be performed directly. The methylated CMP sample was then subjected to acid hydrolysis, reduction, and acetylation to obtain partially methylated sugar alcohol acetate derivatives (PMAAs). These were then analyzed by GS-MS to obtain the total ion chromatogram of CMP PMAAs. Figure 6) and the corresponding mass spectra of each peak ( Figure 7 ).

[0062] The mass spectra corresponding to the PMAAs peaks were compared with the standard spectral library, and relevant literature was consulted to determine the type of methylated sugar residues. At the same time, the relative molar ratio of each methylated sugar residue was calculated based on the peak area of ​​the chromatographic peaks. Finally, the CMP glycosidic bond type and the corresponding molar ratio were obtained. The specific statistics are shown in Table 3.

[0063] CMP contains nine major glycosidic bonds. Glucose residues have been identified as having five glycosidic bond forms, primarily →4)-Glc. p -(1→,small amount→3,4)-Glc p -(1→,→4,6)-Glc p -(1→,→3)-Glc p -(1→and Glc p -(1→, the total molar ratio in CMP is 0.961. The mannose residues are →2)-Manp-(1→ and →2,4)-Manp-(1→, the total molar ratio in CMP is 0.026. Among the three monosaccharide residues, galactose residues have the lowest proportion, being →4-Gal p -(1→and Gal p -(1→, the total molar ratio in CMP is 0.012. Meanwhile, →4)-Glc p -(1→ is the most abundant bond type in CMP. Therefore, 1→4 linked dextran may form the backbone of CMP.

[0064] Table 3 Major Glycosidic Bond Types in CMP

[0065] 1.3.8 CMP 1D and 2D NMR Analysis CMP 1 H NMR spectrum as shown Figure 8 As shown in -A, the D2O signal is at 4.79 ppm. The spectral peaks were deconvolved using the GSD function in MestReNova software. Three signals were observed in the isoproton region, which were then classified as α-D-Glc based on monosaccharide percentage and methylation. p (5.38 ppm), α-D-Gal p (5.22 ppm) and α-D-Manp (5.18 ppm). No signal was detected in the 4.3–4.8 ppm range, indicating the absence of β-glycosidic bonds in CMP. More signals were found in the aprotic region, closely spaced, suggesting they will be assigned to a two-dimensional spectrum.

[0066] 1 H-1 H-correlation spectroscopy (COSY) is a common analytical technique that reflects the correlation between adjacent hydrogen atoms. From isomeric hydrogen atoms, the relationships between all hydrogen atoms are determined through the coupling relationships between adjacent hydrogen atoms. For example... Figure 8 The relevant signal of dextran can be clearly observed in the COSY spectra of -C,CMP. However, the coupling data of hydrogen atoms in the other two monosaccharides are hardly reflected. The corresponding hydrogen atoms of α-D-Glcp can be easily assigned to 1 1H NMR and COSY spectra: H1: 5.38 ppm, H2: 3.57 ppm, H3: 3.74 ppm, H4: 3.60 ppm, H5: 3.85 ppm and H6: 3.76 ppm.

[0067] 13 C NMR spectra such as Figure 8 As shown in -B, a signal of 101.96 ppm was assigned to α-D-Glc. p Furthermore, the absence of signal after 103 ppm further confirms that CMP does not contain β-glycosidic bonds, but only α-glycosidic bonds. According to 1 H- 13 C(HSQC) spectrum ( Figure 8 -D), see reference 1 1H NMR spectroscopy: most signals can be found in the nonproton region (α-D-Glc). p The residues were distributed as follows: C2: 69.39 ppm, C3: 74.59 ppm, C4: 77.16 ppm, C5: 69.32 ppm, C6: 63.29 ppm.

[0068] Based on the above analysis, combined with infrared spectroscopy and monosaccharide composition, it can be concluded that the purified CMP does not have a β-configuration and is in the form of α-D-Glc p Primarily, and most likely α-1,4-D-Glc p It forms the basic framework.

[0069] In summary, this embodiment established a modified hydrothermal method and successfully purified CMP. The structure of CMP was identified using infrared spectroscopy and nuclear magnetic resonance (NMR). Ultraviolet spectroscopy showed that CMP did not contain proteins, indicating good purification efficiency. The purified CMP had a molecular weight of 9669 Da, with Glc as the predominant monosaccharide. SEM showed that CMP exhibited a sheet-like structure. Infrared spectroscopy revealed the presence of α-glycosidic bonds and a pyranose ring in CMP. 1D NMR analysis showed that CMP did not contain β-glycosidic bonds, with α-glycosidic bonds being the predominant component. Furthermore, methylation and NMR analyses suggested that the backbone of CMP is α-1,4-D-Glc. pThis chapter establishes an improved hydrothermal method for the successful purification of α-D-Glc. p The primary CMP.

[0070] Example 2: Modification and optimization of biological functions of α-D-pyranoyl glucan from Cordyceps militaris This embodiment involves complexing CMP and Zn under ultrasound-assisted hydrothermal conditions. The structure of CMP-Zn is characterized by infrared and X-ray energy dispersive spectroscopy (EDS) to explore its complexation mechanism. Antioxidant and hypoglycemic functions are also investigated. Zebrafish is used as a biological model to study hypoglycemic-related genes (…). sirt1 , insr , ldha , hif1a , pdk2 This study investigates the expression levels of CMP and intestinal 16S rRNA to elucidate the mechanisms underlying CMP's hypoglycemic function. A simple chemical modification method will be developed to enhance the traditional biological functions of CMP and refine its underlying mechanisms of action.

[0071] 2.1 Experimental Materials CMP was derived from α-D-Glc purified in Example 1. p Structural polysaccharides.

[0072] 2.2 Experimental Methods 2.2.1 Preparation of Zinc-Modified Polysaccharides Dissolve 0.5 g CMP and 0.6 g trisodium citrate in 120 mL of distilled water and stir at 50 °C for 10 min. Then slowly add 10 mL ZnCl2 (100 mg / mL, pH=3 HCl solution). Adjust the pH to 5-5.5 with NaOH solution (1.0 mol / L). After sonication for 50 min, stir at 50 °C for 2.5 h. After the reaction is complete, centrifuge at 3000 rpm for 5 min to remove undissolved substances. Add 4 times (v / v) of anhydrous ethanol to form a white precipitate, and keep it at 4 °C overnight. Centrifuge at 2500 g for 5 min, dissolve the precipitate in distilled water, centrifuge at 2500 rpm for 5 min, and rotary evaporate the supernatant to dryness of the ethanol. Freeze-dry to obtain the CMP-Zn complex.

[0073] 2.2.2 X-ray diffraction scanning (X-RD) The X-ray diffractometer is equipped with a Cu target ceramic X-ray tube, with a tube voltage of 40 kV, a tube current of 40 mA, a minimum step size of 0.001°, a maximum output of 18 kW, and an absolute accuracy of 2. The divergence slit is 0.76 mm, the scanning range is 5~80°, and the scanning rate is 5° / min.

[0074] 2.2.3 X-ray energy dispersive spectroscopy (XPS) A Kratos AXIS ULTRA DLD X-ray photoelectron spectrometer (XPS) was used, with a monochromatic X-ray source Al Kα as the excitation source and an excitation voltage of 1486.6 eV. The binding energy was calibrated using C1s at 284.8 eV.

[0075] 2.2.4 In vitro antioxidant activity assay (1) The DPPH radical scavenging activity was detected by the method described by Braca et al. (2001).

[0076] (2) The total reducing power was determined using the method of Yuan et al. (2005).

[0077] (3) ABTS + Free radical scavenging activity was detected using a method reported by Chen et al. (2019).

[0078] 2.2.5 In vitro hypoglycemic activity assay The inhibitory effects of CMP and CMP-Zn on α-glucosidase and α-amylase activities were investigated to determine their in vitro hypoglycemic effects.

[0079] Take five test tubes and add 100 μL of CMP and CMP-Zn solutions of different concentrations (1.0, 1.2, and 1.6 mg / mL), respectively. Then add 100 μL of α-glucosidase and incubate at 37°C. Next, add PNPG solution to each CMP and CMP-Zn sample and incubate for 20 min. Finally, add sodium carbonate solution to terminate the reaction and measure the absorbance (A) at 450 nm. Similarly, replace the polysaccharide with acarbose (concentration reduced by 10 times) as a positive control group, keeping other steps unchanged, and calculate the inhibition rate according to the following formula.

[0080]

[0081] In the formula: A 样品 : Absorbance values ​​of α-glucosidase + CMP and CMP-Zn solutions + PNPG solution + Na2CO3 solution; A 对照1 Absorbance values ​​of CMP and CMP-Zn solutions + PNPG solution + Na2CO3 solution; A 对照2 Absorbance of α-glucosidase + PNPG solution + Na2CO3 solution.

[0082] Take 5 test tubes and add 0.4 mL of α-amylase solution to each tube. Add 0.2 mL of CMP and CMP-Zn solutions of different concentrations (1.0, 1.2, 1.6 mg / mL) and mix for 10 min. Add 0.3 mL of starch solution and mix. Finally, add 2.0 mL of DNS reagent to terminate the experiment and react at 100℃ for 15 min. Measure the absorbance (A) at 540 nm. Use acarbose (0.01, 0.02, 0.04, 0.08, 0.16 mg / mL) instead of CMP and CMP-Zn as a positive control, keeping other steps unchanged, and calculate the inhibition rate according to the following formula.

[0083]

[0084] In the formula: A 样品 : α-Amylase + CMP and CMP-Zn solution + starch solution + DNS reagent absorbance; A 对照 : Absorbance values ​​of CMP and CMP-Zn solutions + starch solution + DNS reagent; A 空白 : Absorbance of α-amylase + starch solution + DNS reagent.

[0085] 2.2.6 In vivo hypoglycemic activity assay (1) Determination of maximum tolerated concentration (MTC) Wild-type AB strain zebrafish, 5 days post-fertilization, were used to determine the maximum detectable concentration (MTC) of CMP and CMP-Zn in lowering blood glucose and to evaluate their efficacy. A total of 630 zebrafish were randomly selected and placed in seven six-well plates, with three replicates per group. Twelve groups were experimental groups, containing different concentrations (5, 10, 20, 62.5, 125, and 250 μg / mL) of CMP and CMP-Zn solutions. Normal control and model control groups were also included. Except for the normal control group, all groups were fed a high-sugar diet to establish a high-sugar zebrafish model. The zebrafish were treated at 28℃ for 48 h, and the MTC of CMP and CMP-Zn on the zebrafish was determined.

[0086] (2) Study on hypoglycemic efficacy Forty-five zebrafish were randomly selected and placed in five six-well plates: a normal control group, a model control group, a positive control group (methylbenzyl peroxide), and a polysaccharide sample group (CMP and CMP-Zn), with three replicates per group. Except for the normal control group, all groups were fed a high-sugar diet to establish a high-sugar zebrafish model. The zebrafish were treated at 28°C for 48 h, and glucose levels were analyzed using a glucose assay kit.

[0087] 2.2.7 Expression detection of key genes for lowering blood glucose Total RNA was extracted from zebrafish using an animal tissue RNA extraction kit. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. Quantitative qPCR was performed using a qPCR kit on a Real-Time PCR system, with a total reaction volume of 20 μL. SlActin was selected as the internal control gene; the primers used in this study are listed in Table 4. Relative target gene expression levels were measured using a 22... ΔΔ CT method calculation.

[0088] Table 4 Primer design for the genes being tested

[0089] 2.2.8 Analysis of 16S rRNA in gut microbiota 16S rRNA gene sequencing analysis was performed on the control group (CK), high glucose treatment group (HG), positive control group (HG+MTF), polysaccharide treatment group (CMP), and polysaccharide zinc treatment group (CMP-Zn). Gut microbiota analysis was conducted using the Novaseq sequencing platform. DNA extraction and detection, PCR amplification, product purification, and library construction were performed. After sequencing the paired ends to obtain raw PE reads (Raw Tags), these were assembled and filtered to obtain effective tag sequences (Nochime). The effective data (Clean data) was denoised using DADA2 (DADA2 was used by default) to obtain the final ASVs. For each ASV, species annotation was performed on the representative sequence to obtain corresponding species information and species-based abundance distribution. Simultaneously, abundance, alpha diversity calculations, Venn diagrams, and petal diagrams were performed on the ASVs to obtain information on species richness and evenness within the samples, as well as shared and unique ASVs among different samples or groups. After amplicon annotation, differentially expressed species communities between groups were identified through relative abundance analysis at different levels. LEfSe was then used to analyze the significance of these differences, revealing the impact and variations of different treatments on gut microbiota species and community structure. The amplicon annotation results can also be correlated with relevant functional databases, and PICRUST2 software was used to perform functional prediction analysis on the microbial communities in the ecological samples.

[0090] Other experimental methods and data statistical analysis methods are the same as in Example 1.

[0091] 2.3 Experimental Results and Analysis 2.3.1 Chemical composition and structural characterization of CMP-Zn The chemical composition of the Zn-modified CMP-Zn complex was analyzed and compared with that of CMP. The monosaccharide composition and molecular weight were determined, as shown in Table 5. CMP-Zn mainly contains Glc, Gal, and Man, and the main components remained unchanged, although Glc accounted for approximately 78.3% of CMP-Zn. Molecular weight analysis showed that the molecular weight of CMP-Zn decreased to 1548 Da. Chemical composition analysis results showed that the sugar content of CMP-Zn was 74.07 ± 2.71%, with lower protein and total phenolic content, similar to unmodified CMP.

[0092] Table 5. Composition, molecular weight, and chemical composition analysis of CMP and CMP-Zn monosaccharides.

[0093] like Figure 9 As shown, the morphology of CMP-Zn was analyzed by SEM and compared with that of CMP. Figure 9 As can be seen from A and B, after Zn complexation, the thickness becomes thinner and the edges become more pronounced. Under high magnification, the surface of CMP-Zn is relatively rough, with obvious wrinkles. Figure 9 -D). But for α-D-Glc p The overall morphology of the CMP structure was not significantly affected. After EDS energy dispersive spectroscopy and fluorescence identification of C, O, and Zn elements, as shown... Figure 9 -E indicates that the Zn element was detected, signifying successful Zn complexation.

[0094] like Figure 10 As shown, the modified CMP did not exhibit a significant absorption peak between 260 nm and 280 nm. Combined with the physicochemical results, the CMP-Zn sample contained very little protein. Figure 10 -A). Fourier transform infrared spectroscopy (FTIR) can reflect the structural characteristics of modified polysaccharide samples. For example... Figure 10 As shown in -B, 2929 cm 1 The absorption peak at 3365 cm⁻¹ disappears in CMP-Zn, possibly because zinc binding reduces the intensity of CH₄. Meanwhile, the absorption peak at 3365 cm⁻¹ in CMP-Zn... 1 The peak at 1631 cm⁻¹ indicates that the -OH group may have coordinated with Zn. (CMP-Zn, 1631 cm⁻¹) 1 The signal at that location has redshifted to 1558 cm. 1 At this location, it is evident that Zn complexation influences the tensile vibrations of C=O. Furthermore, at 1254 cm⁻¹... 1The strong peak at this point may be due to the influence of polysaccharide and Zn complexation on the COC tensile vibration. In CMP-Zn, there is a peak at 922 cm⁻¹. 1 851 cm 1 and 768 cm 1 The three peaks indicate that complexation did not alter the α-configuration of the polysaccharide. Meanwhile, 922 cm⁻¹ 1 The enhanced absorption peak at 600-500 cm⁻¹ is attributed to the Zn-OH vibration. In CMP-Zn, the peak is at 600-500 cm⁻¹. 1 There are differences between CMP and CMP-Zn; the peak value in CMP-Zn is 522 cm⁻¹. 1 Moved to 552 cm 1 This may be attributed to the vibration of Zn-O. In summary, Zn may interact with α-D-Glc... p The hydroxyl groups in CMP underwent coordination reactions, while the complexation did not alter the main components of CMP. like Figure 11 As shown, the XRD results of the two samples indicate that neither complex has high crystallinity and no sharp crystallization peaks. Polysaccharides are typically composed of hundreds or even thousands of monosaccharides linked by glycosidic bonds with complex chemical structures. After complexation with Zn, the amorphous properties of the polysaccharide remain unchanged.

[0095] like Figure 12 As shown, the DTG curve shows the temperature at which CMP-Zn exhibits the maximum mass loss rate, and the TG curve shows the change in the percentage of mass loss of CMP-Zn over the decomposition temperature range.

[0096] Comparison with CMP revealed that the thermal decomposition of the modified polysaccharide sample in the TG curves involved two stages: loss of moisture and volatiles within the 28-150℃ range. The mass loss rate of the CMP-Zn complex was 7.7%, while that of CMP was 6.9%, indicating that the free water content of CMP remained almost unchanged after modification. The decarboxylation and thermal decomposition rates of the polysaccharide structure were 82.1% for CMP and 56.3% for CMP and CMP-Zn complexes, respectively. The lower mass loss rate of CMP-Zn indicates that CMP contains more hydrogen-bound water.

[0097] Significant differences exist between the CMP and CMP-Zn complexes in the DTG curves. The first endothermic peak of the CMP-Zn complex is similar to that of CMP, but the maximum decomposition rate of the CMP-Zn complex is 354.1℃, while that of CMP is only 285.5℃. This indicates that CMP-Zn has better thermal stability than CMP. The highest mass loss rate for CMP-Zn occurs at 779.4℃, lower than that of CMP, and this loss does not decrease further with increasing temperature. In contrast, CMP exhibits thermal decomposition around 850℃, suggesting that the carbon chains in CMP are more easily pyrolyzed than those in CMP-Zn. The large difference in thermal properties between CMP and CMP-Zn complexes is likely due to the introduction of zinc to replace the hydrogen atoms in the hydroxyl groups in CMP.

[0098] like Figure 13 As shown, the high-resolution narrow-scan XPS spectra of carbon, oxygen, and zinc peaks of CMP and CMP-Zn complexes are displayed. Figure 13 -A and B). Narrow scan of Zn 2p in CMP-Zn ( Figure 13 -C) is a function that can be deconvolved into Zn 2p 3 / 2 Bimodal (1022.4, 1021.8 eV) and Zn 2p 1 / 2 (1044.7, 1045.4 eV), these are bonds related to Zn-OH and Zn-O, which is consistent with the results in the infrared.

[0099] like Figure 13 As shown in -D, the observed high-resolution narrow scan of the carbon peak C1s deconvolves into three carbon components in CMP, and also deconvolves into three carbon components in CMP-Zn. Figure 13 -E). The carbon peak at a binding energy of 284.8 eV is due to the presence of C-C bonds. In CMP, the peaks at binding energies of 286.3 and 287.9 ​​eV are the absorption peaks of CO and C=O, respectively. In CMP-Zn, the peaks at binding energies of 286.3 and 288.5 eV are the absorption peaks of CO and C=O, respectively. Simultaneously, O1s (E) bonds of both CMP and CMP-Zn are present. Figure 13 The peak widths of Zn-F and G increase after complexation with Zn, possibly due to the formation of Zn-O. The intensities of C=O and COC increase after complexation, consistent with infrared results.

[0100] Furthermore, the decreased intensity of CO and COH may indicate a coordination reaction between Zn and the H atom on the -OH group, revealing the importance of the hydroxyl group. Therefore, the four peaks in the O1s plot of CMP-Zn, combined with C1s and Zn 2p, suggest that the product obtained by CMP-Zn complexation is a complex rather than a physical mixture. Simulations of possible Zn complexation sites show that Zn primarily coordinates with the H atom of the hydroxyl group on the C-chain, forming Zn-O, which then links with a free hydroxyl group to form Zn-OH. The H atom of the hydroxyl group on each C-chain can be substituted indefinitely. This also demonstrates that Zn modification only alters the hydroxyl linkage on the sugar ring and does not change the α-D-Glc... p The basic configuration.

[0101] 2.3.2 In vitro antioxidant activity of CMP and CMP-Zn Characterization of CMP-Zn confirmed that the Zn complex pairs with α-D-Glc p The main structure of the structural CMP was not affected. Therefore, further research is needed on its biological functions.

[0102] First, antioxidant activity was tested, such as... Figure 14 As shown, for α-D-Glc p The DPPH radical scavenging activities of CMP and CMP-Zn structures were respectively assessed. Figure 14 -A), ABTS + Free radical scavenging activity ( Figure 14 -B) and total reducing power ( Figure 14 -C) determination. CMP showed average ability in all three antioxidant indicators.

[0103] In this study, the scavenging activity of CMP was approximately 16%, while the scavenging activity of CMP-Zn was the highest, exceeding 30%, which was significantly higher than that of CMP (P<0.05). Meanwhile, ABTS... + The scavenging activity and total reducing power were significantly higher than those of CMP (P<0.05). However, there was still a significant gap compared with Vc, so CMP-Zn improved the antioxidant capacity of CMP to some extent.

[0104] 2.3.3 In vitro hypoglycemic activity of CMP and CMP-Zn Inhibition of α-amylase and α-glucosidase delays carbohydrate digestion. Therefore, less glucose is absorbed, resulting in lower blood glucose levels. Thus, as... Figure 15 and 16 As shown, the enzyme inhibitory activities of CMP and CMP-Zn were evaluated, with acarbose used as a sample control. Figure 15It was found that CMP and CMP-Zn had the ability to inhibit α-amylase, and this ability increased with increasing concentration. At 0.8 mg / mL, the inhibitory activity of CMP-Zn reached over 75%, significantly higher than that of CMP and the positive control group (P<0.05). Meanwhile, from... Figure 16 It was found that at low concentrations, neither CMP nor CMP-Zn showed significant inhibition of α-glucosidase activity. However, at high concentrations, CMP-Zn exhibited an inhibitory activity of 63%, significantly higher than that of CMP and the positive control group (P<0.05). This indicates that chemical modification is beneficial for enhancing the original biological functions of polysaccharides. The significant increase in the inhibition rates of both CMP-Zn and CMP-Zn suggests that chemical modification can improve the hypoglycemic function of CMP.

[0105] 2.3.4 In vivo hypoglycemic activity of CMP and CMP-Zn To more accurately demonstrate the hypoglycemic function of CMP and modified CMP, in vivo experiments were conducted for further confirmation. The maximum tolerated concentration (MTC) of zebrafish was investigated to determine the maximum tolerated concentration of CMP and CMP-Zn in zebrafish, and the results are shown in Table 6.

[0106] As shown in Table 6, when the concentrations of CMP and CMP-Zn were in the range of 5-20 μg / mL, no significant abnormalities were observed in the toxic phenotype as the concentrations increased exponentially, similar to the state of the HG group. The mortality rate of 45 zebrafish in each group was 0%. However, when the concentrations of CMP and CMP-Zn increased to above 62.5 μg / mL, all 45 zebrafish died. Therefore, the MTC of zebrafish was determined to be 20 μg / mL.

[0107] Figure 17 The glucose level in the HG group was 0.98±0.04 mmol / L, significantly higher than the CK group's glucose level of 0.15±0.03 mmol / L, indicating the successful establishment of the hyperglycemic model. The glucose level in zebrafish in the HG+MTF group was 0.49±0.01 mmol / L, lower than the model control group's 2.10±0.03 mmol / L, with a hypoglycemic efficacy of 50%. This indicates that at this concentration, dimethoprim has a certain hypoglycemic effect on hyperglycemic zebrafish. When the concentrations of CMP and CMP-Zn were below 20 μg / mL, the glucose level in zebrafish was significantly lower than in the HG group (P<0.05). Simultaneously, at MTC=20 μg / mL, the glucose level in zebrafish treated with CMP-Zn was 0.26±0.023 mmol / L, also lower than the HG group, with a hypoglycemic efficacy of 52%. Therefore, in vivo experiments show that CMP-Zn enhances the blood sugar-lowering effect, which is consistent with the results of in vitro experiments.

[0108] Table 6. Maximum Tolerable Concentration (MTC) Results for CMP and CMP-Zn

[0109] 2.3.5 Detection of key gene expression regulating blood glucose lowering in zebrafish In order to improve the blood sugar lowering mechanism of CMP and make its biological functions clearer and more comprehensive, this invention further analyzes the blood sugar lowering mechanism of CMP through gene experiments.

[0110] like Figure 18 As shown, after CMP-Zn processing sirtuin 1 ( sirt1 ) and insulin receptor ( insr The expression of ) increased ( Figure 18 Compared with the control, the HG group and the CMP-Zn treatment group showed a 5-fold and 6.6-fold decrease, respectively, while the CMP-Zn treatment group showed a significantly higher decrease than the control group, with increases of 4.6-fold and 1.5-fold, indicating that this is the main contributing factor to the reduction of hyperglycemia (P<0.05).

[0111] CMP-Zn processing increases sirt1 This indicates that CMP-Zn enhances insulin sensitivity in hyperglycemic zebrafish by reducing lactate formation. Compared to HG, lactate dehydrogenase A (… Ldha ), inhibiting hypoxia-inducible factor 1 subunit A ( Hif1a ) and pyruvate dehydrogenase kinase subtype 2 ( Pdk2 The expression level decreased after treatment with CMP and CMP-Zn. Figure 18 -C, D, and E). Among them hif1a It increased 24.3 times after hyperglycemia, which indicates hif1a It can survive by being expressed in the hypoxic environment that develops in the later stages of hyperglycemia. hif1a The expression of [something] increases in the later stages, which may be due to lower oxygen supply or higher oxygen consumption in the later stages of hyperglycemia.

[0112] HG Group pdk2 Genes increased 11-fold under high-glucose conditions. hif1a Expression induction pdk2 Expression of this gene leads to inhibition of mitochondrial glucose oxidation. Additionally, the gene responsible for converting pyruvate to lactate... ldha It also increased 8.5-fold in the HG group, and its expression pattern and hif1a as well as pdk2 similar.

[0113] After CMP-Zn processing hif1a Compared to HG, it has decreased by 8 times. pdk2 Compared to HG, it has decreased by 11 times. ldhaCompared to HG, it decreased by 8 times, significantly lower than HG (P<0.05). The relative reduction of the three genes demonstrates their role in lowering hyperglycemia. pdk2 Upregulation of expression is predicted to reduce pyruvate dehydrogenase 1 (PDH1) pdha1 The activity of ) pdha1 It catalyzes the conversion of pyruvate to acetyl-CoA.

[0114] Analysis of the expression levels of three genes revealed that, under CMP-Zn treatment in hyperglycemic zebrafish, reducing... pdk2 Expression to reverse pdha1 Activity, by reducing ldha The activity is enhanced, and glucose oxidation is further increased through the tricarboxylic acid cycle (TCA), thereby reducing lactate levels. Therefore, CMP-Zn treatment... pdk2 The decreased expression level indicates an improvement in the hyperglycemic status of zebrafish.

[0115] In summary, the hypoglycemic mechanism of CMP involves regulating various genes to inhibit glycolysis, promote glucose oxidation, and reduce lactic acid. Meanwhile, the hypoglycemic mechanism of CMP-Zn remains unchanged, and its gene expression level is even higher than that of CMP. This mechanism has not been extensively studied in the hypoglycemic effects of polysaccharides, and the hypoglycemic mechanism of CMP in this invention plays an important role in improving the biological functions of polysaccharides.

[0116] 4.2.6 Identification of 16S rRNA-producing microbial community and gene function After performing paired-end sequencing on the libraries using the Illumina NovaSeq sequencing platform, an average of 88,192.7 raw PE reads (Tags) were obtained per sample group. After splicing and filtering, an average of 77,171.7 effective tags were obtained per group, with an average validity rate of 87.50% and an average goods coverage of 97.84% per group. This indicates that the amount of sequencing data was reasonable, and the effectiveness and coverage were appropriate.

[0117] Analysis of inter-group differences using the alpha diversity index, such as Figure 19 The Chao1 index and the number of observed observations were used for each group. The Chao1 index and the number of observed observations in the CK were significantly higher than those in the experimental groups, indicating that the species richness of the gut microbiota in each experimental group was reduced. Figure 20The Simpson and Shannon indices for each group were shown. The Shannon and Simpson indices in the CK group were higher than those in the experimental groups. This indicates that hyperglycemia treatment reduced gut microbiota diversity in all experimental groups. However, the MTF-treated group restored some richness and diversity in the gut compared to the HG group. Similarly, CMP-Zn treatment alleviated the decline in richness and diversity compared to the CMP group. Therefore, CMP-Zn treatment is more effective than CMP in restoring gut microbiota diversity and richness in hyperglycemic zebrafish. In previous studies, β-configuration pumpkin polysaccharides alleviated gut microbiota dysbiosis and increased diversity and richness in mice, consistent with the results of this experiment (Wu et al., 2021). Therefore, the effects of CMP on the gut are similar to those of β-configuration polysaccharides, and CMP-Zn may even be more effective.

[0118] illustrate: Figure 19 In / 20 / 21 / 22 / 23, C, H, M, CM, and Z refer to the control group (CK), high glucose treatment group (HG), positive control group (HG+MTF), polysaccharide treatment group (CMP), and polysaccharide zinc treatment group (CMP-Zn), respectively.

[0119] Depend on Figure 21 The Venn diagram shows that the total number of ASVs in the CK, HG, MTF, CMP, and Zn groups were 1103, 287, 669, 304, and 381, respectively. Among these, 23 were common ASVs, accounting for 2.08%, 8.01%, 3.44%, 7.57%, and 6.04% of each group, respectively. The number of unique ASVs in each group were 920, 154, 523, 185, and 248, accounting for 83.41%, 53.66%, 78.18%, 60.86%, and 65.09% of each group, respectively. These results indicate that after CMP and CMP-Zn treatment, compared to HG, the proportion of unique genes increased, more closely resembling the proportion in CK, suggesting that treatment alleviated the loss of unique genes after high glucose exposure. Simultaneously, it reduced microbial diversity and abundance.

[0120] like Figure 22 As shown in -A, a door is displayed ( phylum The top ten most abundant phyla of gut microbiota at the horizontal level. The dominant phylum in the gut of the CK group was Proteobacteria (…). Proteobacteria Bacteroidetes ( Bacteroidota Firmicutes ( Firmicutes ), Actinobacteria ( Actinobacteriota ), Cyanobacteria ( Cyanobacteria ), Green Curvature ( Chloroflexi ), Iron bacteria ( Fusobacteriota ), Acidobacteria ( Acidobacteriota Campylobacteria ( Campylobacterota ) and Archaea ( Euryarchaeota ).

[0121] In the HG group, the relative abundance of Proteobacteria increased compared to the control group (CK), with an average increase of 69.59%, while the abundance of other phyla decreased, with Bacteroidetes and Firmicutes decreasing by an average of 32.05% and 98.91%, respectively. In the MTF group, the relative abundance of Proteobacteria decreased by 22.43% compared to HG, while the relative abundance of Actinobacteria increased by 566.29%. The relative abundance of Firmicutes and Bacteroidetes both increased to varying degrees. In the CMP-Zn group, compared to the HG group, the relative abundance of Proteobacteria decreased by an average of 11.72%, while Actinobacteria increased by an average of 59.94%. Meanwhile, Bacteroidetes increased by an average of 99.35%, and Firmicutes increased by approximately 35.54%. These findings suggest that CMP-Zn intervention can alleviate the microbial imbalance caused by diabetes, although its effect is lower than that of the MTF group.

[0122] Figure 22 -B shows the genus ( genus The top ten genera in terms of relative abundance of gut microbiota at the CK level. The dominant genera in the gut of the CK group were Shewanella (…). Shewanella ), genus *Streptococcus* Herbaspirillum Aeromonas spp. Aeromonas ), Neosphingolipids ( Novosphingobium ), Pseudomonas spp. Pseudomonas ), Achromobacterium spp. Achromobacter ), genus *Pseudomonas* Plesiomonas ), Curvularia ( Flectobacillus ), Vibrio genus ( Vibrio ), Enterobacter.

[0123] In the HG group, Shewanella decreased by 100%, while the relative abundance of *Syntrophus*, *Neosphophytum*, *Pseudomonas*, and *Campobacter* all increased compared to the control group, with average increases of 52%, 860.94%, 1041.02%, and 2141.44%, respectively. All other bacterial genera decreased, with *Enterobacter* decreasing by 100%, *Aeromonas* by 24.4%, and *Pseudomonas* by 11.32%. High glucose significantly affected the composition of the gut microbiota compared to normal zebrafish. In the MTF group, compared to the HG group, the abundance of Shewanella, *Aeromonas*, and *Enterobacter* all increased. In the CMP-Zn group, compared to the HG group, the abundance of Shewanella increased to 29.52%, and the relative abundance of Spirulina, Pseudomonas, Achromobacterium, and Enterobacter all increased, with average increases of 2693.75%, 1044.38%, 100%, and 8499.36%, respectively. All other bacterial genera decreased. Notably, the abundance of Enterobacter was higher than in the HG group, indicating that CMP-Zn improved bacterial genus abundance to some extent. In summary, considering the bacterial abundance, it can be concluded that CMP-Zn can improve intestinal flora imbalance caused by high glucose intake, and its effect is superior to that of the original CMP.

[0124] like Figure 23 As shown, gene functions were annotated using the KEEG database. Among the 35 richest entries in the KEEG homology analysis (KO), a large number of ABC transporter family genes were found, including ABC.PA.S (K02028), ABC.PA.P (K02029), ABC.PA.S (K02030), ABC.SN.A (K02049), ABC.SN.P (K02050), ABC.SN.S (K02051), and ABC.SP.S (K02055), as well as ABC transporter system-related genes livG (K01995), livF (K01996), livH (K01997), livM (K01998), and livK (K01999). These genes are mainly involved in the ABC transporter system (map2010) and are related to the input and output of exogenous substances, and the metabolism of drugs and exotoxins.

[0125] Among all entries, genes involved in glycolipid synthesis pathways, namely fabG (K00059), ABC.MS.P (K02025), ABC.MS.S (K02026), and ABC.PA.S (K02027), were also found. These genes are related to fatty acid biosynthesis (map00061), lipopolysaccharide biosynthesis (map00540), and polysaccharide transport. Additionally, K03832 was discovered, and its expression requires pyruvate accumulation. Furthermore, an entry related to oxidative stress, K00799, was found, defined as glutathione-S-transferase, which is involved in pathways related to reactive oxygen species. Of the functions mentioned above, except for K02025, K02026, K02027, K03832, and K00799, the expression of the remaining functional genes increased after the addition of CMP-Zn. This indicates that the expression of ABC transporter protein increased and fatty acid synthesis increased, which may be related to Zn metal stimulation.

[0126] It is noteworthy that K03832 and K00799 showed significantly downregulated functions after CMP-Zn addition compared to the HG group. Firstly, regarding K03832, previous studies have shown that the upstream gene regulating K03832 expression is regulated by pyruvate, and its expression level is positively correlated with pyruvate levels (Noinaj et al., 2010). This study mentions that... pdha1 CMP-Zn catalyzes the conversion of pyruvate to acetyl-CoA, thereby enhancing the tricarboxylic acid cycle and promoting glucose oxidation. Therefore, the addition of CMP-Zn reduces pyruvate levels in zebrafish, preventing the expression of genes regulating K03832 and thus limiting its function. Secondly, K00799, as an oxidative stress-related gene, has been clearly identified in previous studies as a key factor in insulin resistance (Cui et al., 2018). Furthermore, in a high-glucose environment, cellular inflammation may be enhanced, and inflammatory cells can produce large amounts of oxidants, making oxidative stress possible (Rammos et al., 2013). Therefore, the reduced K00799 function in CMP-Zn compared to the HG group indicates that CMP-Zn addition inhibits K00799 expression, thereby reducing insulin resistance. This also supports the previous studies on the hypoglycemic mechanism. The above data suggest that CMP also has a hypoglycemic effect in vivo, and the mechanism involves glycolysis and glucose oxidation; however, the hypoglycemic function of CMP is not as strong as that of modified CMP.

[0127] In summary, this embodiment developed a simple chemical method to modify CMP. The results showed that Zn complexation had little impact on the structure of CMP; the main Zn complexation site was the hydroxyl group on the sugar ring, which bound to the free hydroxyl group. Secondly, the traditional biological activities of CMP were tested, and the results indicated that the antioxidant capacity of CMP was significantly improved after modification. In vivo and in vitro experiments both demonstrated a significant improvement in the hypoglycemic effect of complexed CMP. Finally, to refine the functional mechanism of CMP, gene experiments were used to study the hypoglycemic mechanism. The results showed that CMP mainly accelerates glucose oxidation and reduces lactate accumulation by activating insulin receptor expression, inhibiting glycolysis, accelerating the conversion of pyruvate to acetyl-CoA, and thus through the tricarboxylic acid cycle. Furthermore, Zn modification did not affect the mechanism and even enhanced it. Therefore, this study developed a chemical modification method to enhance the original biological functions of CMP and refined the relevant mechanisms of CMP's biological function. It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A zinc-modified Cordyceps militaris polysaccharide, characterized in that, The zinc-modified Cordyceps militaris polysaccharide is a Cordyceps militaris polysaccharide-zinc complex (CMP-Zn), wherein the Cordyceps militaris polysaccharide (CMP) is in the form of α-D-pyranoglobin (α-D-Glc). p The polysaccharide with the main chain structure of Glc has a weight-average molecular weight of 9.6-9.7 kDa and a Glc content of ≥92% in the monosaccharide composition; the CMP-Zn complex has a weight-average molecular weight of 1.5-1.6 kDa and a zinc content of 3.8-4.2% (w / w).

2. The zinc-modified Cordyceps militaris polysaccharide according to claim 1, characterized in that, The glycosidic bond types of the CMP include: →4)-Glc p -(1→、→3,4)-Glc p -(1→、→4,6)-Glc p -(1→、→3)-Glc p -(1→and Glc p -(1→, where →4)-Glc p -(1→ is the main chain skeleton.) 3. A method for preparing any of the zinc-modified Cordyceps militaris polysaccharides as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Dissolve Cordyceps militaris polysaccharide (CMP) and trisodium citrate in water at a mass ratio of 0.5:0.6, and pre-stir at 45-55℃ to form a transparent solution; S2: Slowly add ZnCl2 solution dropwise to the solution obtained in step S1, with the mass ratio of CMP to ZnCl2 being 0.5:1.0, and adjust the pH to 5.0-5.5 with NaOH; S3: The mixture obtained in step S2 was subjected to hydrothermal reaction at 50°C for 2.5 hours under ultrasonic power of 300 W and frequency of 40 kHz. S4: The reaction solution obtained in step S3 is subjected to ethanol precipitation, centrifugation, rotary evaporation to remove alcohol and freeze drying to obtain the CMP-Zn complex.

4. The preparation method according to claim 3, characterized in that, In step S1, the pre-stirring temperature is 50℃ and the time is 10 min; in step S2, the ZnCl2 solution concentration is 100 mg / mL and the dropping rate is slow; in step S3, sonication and stirring are performed simultaneously.

5. The application of zinc-modified Cordyceps militaris polysaccharide as described in claim 1 or 2, or zinc-modified Cordyceps militaris polysaccharide prepared by the method described in any one of claims 3-4, in the preparation of products with antioxidant, hypoglycemic, or high-sugar-induced intestinal flora imbalance functions.

6. The application according to claim 5, characterized in that, The antioxidant function is manifested in CMP-Zn increasing the scavenging rate of DPPH free radicals and ABTS. + Free radical scavenging rate and total reducing power.

7. The application according to claim 5, characterized in that, The blood sugar lowering function is achieved through one or more of the following pathways: (1) In vitro inhibition of α-amylase and α-glucosidase activities, with CMP-Zn inhibiting α-amylase by ≥75% at a concentration of 0.8 mg / mL; and inhibiting α-glucosidase by 63% at high concentrations. (2) It lowers blood glucose levels in zebrafish with high glucose levels in vivo, with a hypoglycemic effect of 52% at a concentration of 20 μg / mL; (3) Upregulation of insulin receptor gene Insr and the gene of silence information regulator 1 Sirt1 Expression of lactate dehydrogenase A gene is downregulated. Ldha Hypoxia-inducible factor 1 subunit A gene Hif1a and pyruvate dehydrogenase kinase subtype 2 gene Pdk2 The expression of [something] promotes glucose oxidation and reduces lactic acid accumulation.

8. The application according to claim 5, characterized in that, The restoration of gut microbiota imbalance induced by high glucose is manifested in: restoring the Shannon index of gut microbiota to more than 85% of the normal level, and increasing the proportion of Proteobacteria (…). Proteobacteria The abundance of Bacteroidetes decreased by at least 11.72%, making the abundance of Bacteroidetes (Bacteroidetes) Bacteroidetes The abundance of Firmicutes increased by at least 99.35%, making Firmicutes ( Firmicutes ) and Actinobacteria ( Actinobacteriota They rebounded by 35.54% and 59.94% respectively.

9. The application of zinc-modified Cordyceps militaris polysaccharide as described in claim 1 or 2, or zinc-modified Cordyceps militaris polysaccharide prepared by the method described in any one of claims 3-4, in lowering blood sugar.

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