Sulfated mulberry and silkworm chrysalis cordyceps polysaccharide as well as preparation method and application thereof

Abstract

The invention provides sulfated mulberry silkworm chrysalis cordyceps polysaccharide. In the sulfated mulberry silkworm chrysalis cordyceps polysaccharide, the total sugar content is 50.52 + / -2.62%, the uronic acid content is 1.77 + / -0.10%, the sulfate group content is 13.45 + / -0.36%, and the substitution degree is 1.193. The sulfated mulberry and silkworm chrysalis cordyceps polysaccharide provided by the invention has a remarkable bacteriostatic effect, and the minimum inhibitory concentration (MIC) of the sulfated mulberry and silkworm chrysalis cordyceps polysaccharide to escherichia coli (ATCC 25922) and bacillus subtilis (ATCC 6633) is 2.0 mg / mL; and when the concentration is 2.0 mg / mL, the inhibition rate on alpha-glucosidase reaches 92.5 + / -2.08%, which is higher than that of acarbose with the same concentration. The compound can be used for preparing related products such as spray, external gel and oral liquid.

Description

Technical Field

[0001] This invention belongs to the field of natural product preparation and application, specifically relating to a sulfated silkworm pupa cordyceps polysaccharide, its preparation method, and its antibacterial and hypoglycemic uses. Background Technology

[0002] Cordyceps militaris is a precious tonic medicinal herb in my country, possessing immunomodulatory and anti-tumor activities. Its artificial cultivation is mainly achieved through two pathways: one is based on mycelial fermentation to produce metabolites, and the other is biomimetic cultivation using heterologous insects as hosts. Among these, the silkworm, as an insect with significant medicinal value, has become an excellent host for artificially cultivated Cordyceps due to its wide application and development potential in the medical field. Studies have shown that Cordyceps cultivated using silkworm pupae as a substrate has a significantly higher total amino acid content (especially the eight essential amino acids for humans) than wild-type and similar artificially cultivated Cordyceps. Component analysis reveals that the artificially cultivated Cordyceps militaris mycelium is rich in various biomolecules such as polysaccharides, proteins, nucleosides, lectins, fibrinolytic enzymes, and superoxide dismutase. Among these, Cordyceps militaris polysaccharides, as the core active ingredient, possess multiple biological activities including antioxidant, immunomodulatory, and anti-tumor effects, demonstrating broad prospects for medicinal development.

[0003] Existing studies on the sulfation modification of Cordyceps militaris mainly focus on Cordyceps militaris and Cordyceps sinensis (Journal of Agricultural and Food Chemistry, 2015, 63:3464-3471; Molecules, 2013, 18:167-177). However, the larvae and pupae of the silkworm (B. mori), a traditional medicinal insect, are rich in active ingredients such as proteins and polysaccharides. They have a history of use in traditional Chinese medicine for tonifying and strengthening the body. Furthermore, silkworm farming technology is mature, and large-scale production is cost-effective. In recent years, Cordyceps militaris has been shown to possess similar active ingredients to Cordyceps militaris (such as cordycepin and polysaccharides), demonstrating significant potential for medicinal development. However, the sulfation modification of its polysaccharides has not yet been reported.

[0004] In recent years, the comorbidity of diabetes with bacterial infectious diseases (such as skin and soft tissue infections and intestinal flora imbalance-related infections) has received increasing attention, and the development of functional products with both hypoglycemic and antibacterial activities has important clinical value. Studies have shown that α-glucosidase inhibitors can delay carbohydrate breakdown and significantly reduce postprandial blood glucose by competitively inhibiting the activity of small intestinal brush border enzymes; while bacterial infections (such as Escherichia coli and Staphylococcus aureus) are closely related to complications such as diabetic foot ulcers and urinary tract infections. (Journal of Wenzhou University (Natural Science Edition), 2025, 46(3): 50-62); Zhu Xiaoyan et al. further confirmed that Escherichia coli is a common pathogen in diabetic patients, and infection can easily lead to complications and aggravate disease progression (Medical Animal Control, 2025, 41(5): 501-505). Therefore, polysaccharide components with both hypoglycemic and antibacterial activities have become a hot topic in the research and development of functional products. Summary of the Invention

[0005] This invention is the first to employ the aminosulfonic acid method for modification, which, compared to the traditional chlorosulfonic acid-pyridine method, offers milder conditions and higher safety. This invention provides a method for preparing sulfated silkworm pupa cordyceps polysaccharide, its structural characteristics, and its antibacterial and hypoglycemic applications, providing a clear active ingredient basis for the development of novel antibacterial and hypoglycemic functional products.

[0006] The purpose of this invention is to provide a sulfated silkworm pupa cordyceps polysaccharide, which has highly efficient antibacterial activity. The minimum inhibitory concentration (MIC) against Escherichia coli (ATCC 25922) and Bacillus subtilis (ATCC6633) is 2.0 mg / mL, which is significantly better than that of unmodified polysaccharide. At a concentration of 2.0 mg / mL, the inhibition rate against α-glucosidase reaches 92.5 ± 2.08%, which is higher than that of acarbose at the same concentration.

[0007] According to a first aspect of the present invention, the present invention provides a sulfated Cordyceps militaris polysaccharide, wherein the sulfated Cordyceps militaris polysaccharide is obtained by sulfated Cordyceps militaris polysaccharide with aminosulfonic acid; wherein the degree of sulfate substitution of the sulfated Cordyceps militaris polysaccharide is 0.356 to 1.193.

[0008] According to a second aspect of the present invention, the present invention provides a method for preparing sulfated silkworm pupa cordyceps polysaccharide, comprising the following steps:

[0009] Silkworm pupa cordyceps polysaccharide and aminosulfonic acid were placed in an organic solvent and heated for a certain time. After cooling to room temperature, the reaction was quenched with an alkaline solution. The pH of the reaction solution was adjusted to neutral, and ethanol was used to precipitate the product. The precipitate was redissolved in distilled water, dialyzed, and freeze-dried to obtain sulfated silkworm pupa cordyceps polysaccharide with different degrees of substitution.

[0010] In certain specific embodiments of the present invention, the heating reaction for a certain time refers to heating the reaction in stages for a certain time; preferably, the reaction is carried out at 60°C for 1 hour, and then the temperature is raised to 80°C for 2 hours.

[0011] In certain specific embodiments of the present invention, the specific steps may be as follows: Weigh 1.0 g of silkworm pupa cordyceps polysaccharide (Bm-CMP) and place it in a round-bottom flask. Add a certain amount of aminosulfonic acid according to different mass ratios. Then add an appropriate amount of N,N-dimethylformamide (DMF) solvent and urea catalyst. Stir magnetically until fully mixed. React at 60°C for 1 h, then raise the temperature to 80°C and react for 2 h. After the reaction is completed, cool to room temperature and add 10 wt% NaOH aqueous solution to quench the reaction. Adjust the pH value of the reaction to 7.0-8.0. Then add 1, 2, and 3 times the volume of anhydrous ethanol in three portions each time and precipitate at 4°C for 12 h. The obtained precipitate is re-dissolved in distilled water, dialyzed three times with distilled water, and freeze-dried to obtain Bm-CMP with different degrees of substitution.

[0012] Preferably, the mass ratio of the silkworm pupa cordyceps polysaccharide to aminosulfonic acid is 1:2 to 1:5, and more preferably 1:3;

[0013] Preferably, the sulfated silkworm pupa cordyceps polysaccharide has a degree of sulfate substitution of 0.356 to 1.193;

[0014] Preferably, the mass ratio of the silkworm pupa cordyceps polysaccharide to aminosulfonic acid is 1:3, and the degree of sulfate substitution of the obtained sulfated silkworm pupa cordyceps polysaccharide is 1.193. The sulfated Cordyceps militaris polysaccharide prepared at this time has a total sugar content of 50.52±2.62%, a uronic acid content of 1.77±0.10%, a sulfate group content of 13.45±0.36%, and a sulfate group substitution degree of 1.193. The monosaccharide composition and content of the obtained sulfated Cordyceps militaris polysaccharide are as follows: arabinose 1.52%, glucosamine 0.50%, galactose 15.02%, glucose 55.60%, xylose 3.01%, mannose 23.65%, and galacturonic acid 0.70%. The molecular weight of the sulfated Cordyceps militaris polysaccharide is 2.65 kDa. The particle size of the sulfated Cordyceps militaris polysaccharide is 48.52 nm, and the potential is -24.9 mV.

[0015] Preferably, in the preparation process of the sulfated silkworm pupa cordyceps polysaccharide, Bm-CMP is a purified polysaccharide, comprising the following steps:

[0016] 1) Extraction of crude polysaccharides from silkworm pupae and cordyceps

[0017] The silkworm pupa cordyceps was dried at 75℃ to constant weight, pulverized, and passed through a 60-mesh sieve to obtain silkworm pupa cordyceps powder for later use. Petroleum ether was added for defatting in the dark for 12 hours, and the powder was then filtered under reduced pressure to obtain defatted silkworm pupa cordyceps powder. 20g of the defatted silkworm pupa cordyceps powder was weighed and soaked in water at a liquid-to-solid ratio of 30:1 (mL / g) for 4 hours, then extracted in a heating mantle at 70℃ for 3 hours. After cooling, the mixture was filtered, and the filtrate was concentrated under reduced pressure to 1 / 4 of its original volume. Three times the volume of anhydrous ethanol was added, and precipitation was carried out at 4℃ for 12 hours. The precipitate was freeze-dried to obtain crude silkworm pupa cordyceps polysaccharide, with an extraction rate of 28.95±3.32%. The crude polysaccharide extraction rate (%) = m(g) / M(g)×100, where m is the mass of crude silkworm pupa cordyceps polysaccharide and M is the mass of defatted cordyceps powder.

[0018] 2) Purification of crude polysaccharide from silkworm pupae and Cordyceps sinensis

[0019] The polysaccharide was deproteinized three times using the Sevag method (Sevag reagent consisted of n-butanol and chloroform in a 4:1 volume ratio); after removing residual Sevag reagent, it was dialyzed three times (dialyzed with distilled water for 72 h, with water changed every 12 h); the dialysate was freeze-dried to obtain deproteinized polysaccharide. 100 mg of deproteinized polysaccharide was weighed and dissolved in 20 mL of deionized water, and loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm); 0.5 mol / L NaCl solution was used as the eluent, the flow rate was 1 mL / min, and 5 mL was used per tube; the eluent was detected using the phenol-sulfuric acid method. After the polysaccharide was completely eluted, the eluents were combined, concentrated, dialyzed, and freeze-dried to obtain purified polysaccharide from *Bombyx mori-hosted* Cordyceps militaris polysaccharides, named Bm-CMP (Bombyx mori-hosted Cordyceps militaris polysaccharides). After deproteinization, dialysis, and DEAE column chromatography purification, the polysaccharide content of Bm-CMP was 50.52±2.62%.

[0020] According to another aspect of the present invention, the present invention provides the use of the sulfated silkworm pupa cordyceps polysaccharide in antibacterial and hypoglycemic functional products; it can be used to prepare functional products such as sprays, topical gels, and oral liquids.

[0021] Preferably, the spray contains sulfated silkworm pupa cordyceps polysaccharide, propylene glycol, benzalkonium chloride, Tween-80 and water.

[0022] Preferably, the topical gel contains sulfated silkworm pupa cordyceps polysaccharide, carbomer, glycerin, triethanolamine and water.

[0023] Preferably, the oral liquid contains sulfated silkworm pupa cordyceps polysaccharide, propylene glycol, benzalkonium chloride, Tween-80, erythritol and water.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] ① Sulfated polysaccharides of silkworm pupa and Cordyceps sinensis with a degree of substitution of 0.356 to 1.193 were prepared by a staged heating reaction using aminosulfonic acid method. In particular, the antibacterial activity was the highest when the degree of substitution of sulfate groups was 1.193.

[0026] ② The sulfated silkworm pupa cordyceps polysaccharide provided by this invention has a minimum inhibitory concentration (MIC) of 2.0 mg / mL against Escherichia coli (ATCC 25922) and Bacillus subtilis (ATCC6633), which is significantly better than that of the unmodified polysaccharide; at a concentration of 2.0 mg / mL, the inhibition rate against α-glucosidase reaches 92.5±2.08%, which is higher than that of acarbose at the same concentration. Attached Figure Description

[0027] Figure 1 Chromatogram of monosaccharide composition of Bm-sCMP-3 (peaks in the standard: 1-fucose; 2-arabinose; 3-glucosamine; 4-galactose; 5-glucose; 6-xylose; 7-mannose; 8-ribose; 9-galacturonic acid; 10-glucuronic acid);

[0028] Figure 2 Molecular weight distribution diagram of Bm-sCMP-3;

[0029] Figure 3 Infrared spectrum of Bm-sCMP-3;

[0030] Figure 4 Scanning electron microscope image of Bm-sCMP-3;

[0031] Figure 5 Particle size distribution of Bm-sCMP-3;

[0032] Figure 6 Potentiogram of Bm-sCMP-3;

[0033] Figure 7 ΔOD of 2 mg / mL sample 600 value;

[0034] Figure 8 Inhibitory effect of 2 mg / mL sample on α-glucosidase (different superscript lowercase letters indicate statistical differences between groups). Detailed Implementation

[0035] Cordyceps militaris was provided by Suzhou Jiahe Sericulture Biotechnology Co., Ltd.; monosaccharide standards and T-series dextran standards were purchased from Sinopharm Chemical Reagent Co., Ltd.; molecular weight was determined using a Waters HPLC system (model 1525); monosaccharide composition was determined using a Dionex ICS-5000 ion chromatograph; infrared spectroscopy was performed using a PerkinElmer Fourier transform infrared microscope (model Spotlight200i); scanning electron microscopy (SEM) was performed using a HITACHI super-resolution field emission scanning electron microscope (model Regulus8100). All experiments were performed in triplicate, and data are expressed as mean ± SD. Statistical analysis was performed using t-tests or ANOVA, with P < 0.05 considered statistically significant.

[0036] Example 1: Extraction of crude polysaccharides from silkworm pupae and Cordyceps sinensis

[0037] Silkworm pupae and Cordyceps sinensis were dried at 75℃ to constant weight, pulverized, and passed through a 60-mesh sieve to obtain Cordyceps sinensis powder for later use. Petroleum ether was added for defatting in the dark for 12 hours, followed by vacuum filtration to obtain defatted Cordyceps sinensis powder. 20g of the defatted Cordyceps sinensis powder was weighed and soaked in water at a liquid-to-solid ratio of 30:1 (mL / g) for 4 hours, then extracted in a heating mantle at 70℃ for 3 hours. After cooling, the mixture was filtered, and the filtrate was concentrated under reduced pressure to 1 / 4 of its original volume. Three times the volume of anhydrous ethanol was added, and precipitation was carried out at 4℃ for 12 hours. The precipitate was freeze-dried to obtain crude Cordyceps sinensis polysaccharide, with an extraction rate of 28.95±3.32%. The crude polysaccharide extraction rate (%) = m(g) / M(g)×100, where m is the mass of crude Cordyceps sinensis polysaccharide and M is the mass of defatted Cordyceps sinensis powder.

[0038] Example 2: Purification of crude polysaccharide from silkworm pupae and Cordyceps sinensis

[0039] The polysaccharide was deproteinized three times using the Sevag method (Sevag reagent consisted of n-butanol and chloroform in a 4:1 volume ratio). After removing residual Sevag reagent, the polysaccharide was dialyzed three times (dialyzed with distilled water for 72 hours, with water changed every 12 hours). The dialysate was freeze-dried to obtain deproteinized polysaccharide. 100 mg of deproteinized polysaccharide was weighed and dissolved in 20 mL of deionized water, and loaded onto a diethylaminoethyl (DEAE) cellulose-52 column (2.6 cm × 60 cm). 0.5 mol / L NaCl solution was used as the eluent, with a flow rate of 1 mL / min and 5 mL per tube. The eluent was analyzed using the phenol-sulfuric acid method. After the polysaccharide was completely eluted, the eluents were combined, concentrated, dialyzed, and freeze-dried to obtain purified silkworm pupa cordyceps polysaccharide Bm-CMP. The polysaccharide content after deproteinization, dialysis, and DEAE column chromatography was 50.52 ± 2.62%.

[0040] Example 3: Preparation of Sulfated Cordyceps Polysaccharide from Silkworm Pupae

[0041] Weigh 100 mg of Bm-CMP into a round-bottom flask, and add 200 mg, 300 mg, 400 mg, and 500 mg of aminosulfonic acid (SA) respectively. Then add appropriate amounts of N,N-dimethylformamide (DMF) solvent and urea catalyst. Stir magnetically until fully mixed. React at 60°C for 1 hour, then raise the temperature to 80°C and react for another 2 hours. After the reaction is complete, cool to room temperature, add 10 wt% NaOH solution to quench the reaction, and adjust the pH to 7.0-8.0. Then, perform the reaction in three separate batches, each batch containing... Add 1, 2, and 3 times the volume of anhydrous ethanol and precipitate at 4°C for 12 h; the precipitate is redissolved with distilled water, dialyzed three times with distilled water, and freeze-dried to obtain sulfated Bm-CMP, which are named Bm-sCMP-2 (Bm-CMP:SA = 1:2), Bm-sCMP-3 (Bm-CMP:SA = 1:3), Bm-sCMP-4 (Bm-CMP:SA = 1:4), and Bm-sCMP-5 (Bm-CMP:SA = 1:5) respectively.

[0042] Comparative Example

[0043] Based on the optimal process obtained in Example 3 (i.e., when the feed ratio of Bm-CMP to aminosulfonic acid (SA) is 1:3, the sulfation product Bm-sCMP-3 has the highest degree of substitution, which is 1.193), in order to prove that the staged heating method provided by this invention is superior to isothermal heating, the following comparative experiment was conducted:

[0044] Option A: Aminosulfonic acid method: react at a constant temperature of 60℃ for 3 hours.

[0045] Weigh 100 mg of Bm-CMP and place it in a round-bottom flask. Add 300 mg of aminosulfonic acid (SA), followed by an appropriate amount of N,N-dimethylformamide (DMF) solvent and urea catalyst. Stir magnetically until fully mixed and react at 60 °C for 3 h. After the reaction is complete, cool to room temperature and add 10 wt% NaOH solution to quench the reaction. Adjust the pH of the reaction to 7.0-8.0. Then add 1, 2, and 3 times the volume of anhydrous ethanol respectively and precipitate at 4 °C for 12 h. The precipitate is redissolved with distilled water, dialyzed three times with distilled water, and freeze-dried to obtain sulfated Bm-CMP, named Bm-sCMP-6. Its degree of sulfate substitution was measured to be 0.386.

[0046] Option B: Aminosulfonic acid method: Reaction at a constant temperature of 80℃ for 3 hours

[0047] Weigh 100 mg of Bm-CMP and place it in a round-bottom flask. Add 300 mg of aminosulfonic acid (SA), followed by an appropriate amount of N,N-dimethylformamide (DMF) solvent and urea catalyst. Stir magnetically until fully mixed and react at 80 °C for 3 h. After the reaction is complete, cool to room temperature and add 10 wt% NaOH solution to quench the reaction. Adjust the pH of the reaction to 7.0-8.0. Then add 1, 2, and 3 times the volume of anhydrous ethanol respectively and precipitate at 4 °C for 12 h. The precipitate is redissolved with distilled water, dialyzed three times with distilled water, and freeze-dried to obtain sulfated Bm-CMP, named Bm-sCMP-7. Its degree of sulfate substitution was measured to be 0.985.

[0048] Option C: Chlorosulfonic acid-pyridine method: reaction at a constant temperature of 70℃ for 2 hours

[0049] 100 mg Bm-CMP was dissolved in 20 mL of anhydrous formamide and stirred at room temperature until the polysaccharide was completely dissolved. 20 mL of sulfonating reagent (chlorosulfonic acid: anhydrous pyridine = 1:1, v / v) was added while heating, and the reaction was carried out at 70 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the product was neutralized with 2 mol / L NaOH solution. The pH was adjusted to 7.0-8.0, and then 1, 2, and 3 times the volume of anhydrous ethanol were added each time, and precipitation was carried out at 4 °C for 12 h. The precipitate was reconstituted with distilled water, dialyzed three times with distilled water, and freeze-dried to obtain sulfated Bm-CMP, named Bm-sCMP-8, with a sulfate degree of substitution of 0.213.

[0050] Option D: Chlorosulfonic acid-pyridine method: reaction at a constant temperature of 60℃ for 3 hours

[0051] 100 mg Bm-CMP was dissolved in 20 mL of anhydrous formamide and stirred at room temperature until the polysaccharide was completely dissolved. 20 mL of sulfonating reagent (chlorosulfonic acid: anhydrous pyridine = 1:1, v / v) was added while heating, and the reaction was carried out at 60 °C for 3 h. After the reaction was complete, the mixture was cooled to room temperature, and the product was neutralized with 2 mol / L NaOH solution. The pH was adjusted to 7.0-8.0, and then 1, 2, and 3 times the volume of anhydrous ethanol were added, respectively, and the mixture was precipitated at 4 °C for 12 h. The precipitate was reconstituted with distilled water, dialyzed three times with distilled water, and freeze-dried to obtain sulfated Bm-CMP, named Bm-sCMP-9, with a sulfate degree of substitution of 0.307.

[0052] The results showed that the sulfated polysaccharides prepared by the aminosulfonic acid method had a higher degree of sulfate substitution than those prepared by the chlorosulfonic acid-pyridine method. In the aminosulfonic acid sulfation reaction, staged heating was more beneficial than constant temperature heating in increasing the degree of sulfate substitution.

[0053] Example 4: Determination of the antibacterial activity of sulfated Bm-CMP and Bm-CMP

[0054] The Bm-sCMP-X (2-5) prepared in Example 3 was prepared into a sterile solution of 16 mg / mL. 100 μL of sterile beef extract peptone medium was added to wells 1-10 of a sterile 96-well plate. 100 μL of the prepared polysaccharide solution was added to well 1 and mixed. Then, 100 μL of the mixture was added from well 1 to well 2 and mixed. 100 μL of the mixture was then added from well 2 to well 3 and mixed, and so on, for a 2-fold dilution until well 10. After mixing, 100 μL was aspirated from well 10 and discarded. At this point, the final concentrations of the polysaccharide solution in wells 1-10 of the 96-well plate were 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg / mL, respectively (three parallel wells were set up for each concentration). Then, 100 μL of Escherichia coli and Bacillus subtilis bacterial suspension in the logarithmic growth phase was added to wells 1-10. Immediately afterwards, the 96-well plate was placed in a microplate reader to measure the OD of each well. 600 The values ​​were recorded; the 96-well plate was then placed in a 37°C incubator for 24 hours, and the OD values ​​of each well were measured again. 600 Record the data and calculate the absorbance difference (ΔOD) before and after culture in each well. 600 ), can enable OD 600 The lowest concentration of polysaccharide solution with a concentration ≤0.05 (i.e., significantly inhibiting bacterial growth) can be determined as the minimum inhibitory concentration (MIC). Using Gram-negative Escherichia coli (ATCC25922) and Gram-positive Bacillus subtilis (ATCC6633) as test strains, the ΔOD of Bm-sCMP-X (2–5) at different concentrations was measured. 600 See Table 1 below.

[0055] Table 1. ΔOD of Bm-sCMP-X and Bm-CMP on two strains. 600 Value (24h incubation)

[0056]

[0057]

[0058] Note: Data are mean ± SD (n = 3).

[0059] As shown in Table 1, the antibacterial activity of sulfated Bm-CMP was superior to that of the unsulfated samples. Bm-sCMP-3 exhibited the highest antibacterial activity, with a minimum inhibitory concentration (MIC) of 2000 μg / mL (2.0 mg / mL) against both *Escherichia coli* (ATCC 25922) and *Bacillus subtilis* (ATCC 6633). Bm-sCMP-4 and Bm-sCMP-5 both had MICs of 4000 μg / mL (4.0 mg / mL) against both strains. The ΔOD of the 2 mg / mL sample was... 600 Values ​​such as Figure 7 As shown.

[0060] Example 4: Determination of the hypoglycemic activity of sulfated Bm-CMP and Bm-CMP

[0061] Bm-sCMP-X (2-5) and Bm-CMP were dissolved in distilled water to prepare solutions with a concentration of 2.0 mg / mL. 100 μL of the polysaccharide solution was mixed thoroughly with 300 μL of 0.24 U / mL α-glucosidase solution, followed by 600 μL of pH 6.8 phosphate-buffered saline (PBS). The mixture was vortexed and incubated at 37°C for 15 min to allow for full interaction between the polysaccharide and enzyme. Then, 2.74 mg / mL of 4-nitrophenyl-β-D-galactopyranoside (PNPG) solution was added and thoroughly mixed. The mixture was incubated at 37°C for another 20 min to allow the enzyme to catalyze the substrate reaction. Finally, 4 mL of Na₂CO₃ solution was added to terminate the reaction, and the absorbance of the system was measured at 400 nm.

[0062]

[0063] Table 2 below shows the reaction system for the α-glucosidase inhibitory activity experiment.

[0064] Table 2. Experimental reaction system for α-glucosidase inhibitory activity.

[0065]

[0066] Experimental results are as follows Figure 8 As shown, compared with acarbose, the α-glucosidase inhibitory activity of Bm-sCMP-X increases with the increase of aminosulfonic acid (SA). Similar to the antibacterial activity, the α-glucosidase inhibitory activity is strongest when it is Bm-sCMP-3, which is higher than that of acarbose at the same concentration, reaching 92.5±2.08%.

[0067] Example 5: Compositional Analysis of Sulfated Cordyceps militaris Polysaccharide from Silkworm Pupae

[0068] Total sugar content was determined by the phenol-sulfuric acid colorimetric method; uronic acid content was quantitatively analyzed by the m-hydroxybiphenyl colorimetric method; sulfate content was determined by the barium chloride-gelatin turbidity method; the degree of sulfate substitution (DS) was calculated as DS = 1.62 × S / (32 - 1.02 × S), where S represents the mass percentage of sulfate groups; the potential parameters and particle size distribution of the samples were characterized using a Malvern Zetasizer Nano ZS nanoparticle size and Zeta potential analyzer.

[0069] Table 3. Compositional analysis results of Bm-CMP and its sulfates

[0070]

[0071] Different superscript letters indicate statistical differences between groups, P < 0.05 or P < 0.01.

[0072] Example 6: Further Characterization of Bm-sCMP-3

[0073] Because Bm-sCMP-3 has high antibacterial and hypoglycemic activities, its structure was further characterized.

[0074] Monosaccharide composition analysis: Accurately weigh 5.0 mg of Bm-sCMP-3 sample into a 5 mL stoppered graduated test tube, add 1 mL of 2 mol / L trifluoroacetic acid solution, seal, and hydrolyze in a 121℃ oven for 2 h. After cooling to room temperature, dilute to 50 mL with ultrapure water, filter through a 0.45 μm microporous membrane, and use the filtrate as the test solution. The injection volume is 20 μL. Separation and analysis were performed using a Dionex ICS-5000 ion chromatograph (equipped with a pulsed amperometric detector). The chromatographic column was CarboPac PA20 (particle size 6.5 μm, 3 mm × 150 mm). The gradient elution program was as follows: 0–21 min: mobile phase 98% ultrapure water + 2% 250 mM NaOH solution; 21.1–30 min: mobile phase adjusted to 93% ultrapure water + 2% 250 mM NaOH solution. NaOH solution + 5% 1M NaAc solution, the mobile phase is switched to 20% ultrapure water + 80% 250mM NaOH solution for 30.1–50 min, and the flow rate is controlled at 0.5 mL / min.

[0075] Molecular weight analysis: A 2.0 mg / mL solution of Bm-sCMP-3 was prepared, filtered through a 0.45 μm microporous membrane, and 20 μL was injected. The chromatogram was performed using a Waters 1525 high-performance liquid chromatograph (equipped with a 2414 differential refractive index detector and an Empower3 workstation) with an Ultrahydrogel column. TMSeparation was performed using linear (2 μm, 7.8 mm × 300 mm) chromatography. A series of dextran were used as standards, and a third-order calibration curve for the determination of polysaccharide molecular weight was established based on retention time and molecular weight values. Chromatographic conditions: mobile phase: 0.1 M NaNO3 solution; flow rate: 0.9 mL / min; column temperature: 45 °C.

[0076] Infrared spectroscopy analysis: 1.0 mg of dried Bm-sCMP-3 and 100 mg of dried potassium bromide powder were thoroughly ground and mixed in an agate mortar. After being compressed into tablets, the mixture was placed in an FTIR-650 infrared spectrometer and analyzed at 4000 cm⁻¹. -1 ~400cm -1 Spectral scanning was performed within the wavenumber range.

[0077] Scanning electron microscopy analysis: The dried Bm-sCMP-3 sample was placed on the sample stage, fixed with conductive adhesive, and then sputtered with gold using an ion sputtering instrument (accelerating voltage 10-15kV, sputtering time 30-60s). Finally, the sample surface morphology was observed and images were acquired under a scanning electron microscope.

[0078] Depend on Figure 1 It can be seen that, by comparing with the standard, Bm-sCMP-3 is composed of seven monosaccharides: arabinose (1.52%), glucosamine (0.50%), galactose (15.02%), glucose (55.60%), xylose (3.01%), mannose (23.65%), and galacturonic acid (0.70%).

[0079] Depend on Figure 2 It can be seen that Bm-sCMP-3 is a homogeneous polysaccharide with a molecular weight of 2.65 kDa.

[0080] like Figure 3 As shown in the infrared spectrum of Bm-sCMP-3, at 3440 cm⁻¹ -1 The strong and broad absorption peak is caused by the stretching vibration of -OH, which is a typical characteristic of polysaccharide compounds; 2963 cm⁻¹ -1 It is the CH stretching vibration of the methyl or methylene group in a carbohydrate molecule; 1051 cm⁻¹ -1 and 1002cm -1 The absorption peak at 1261 cm⁻¹ corresponds to the asymmetric and symmetric stretching vibrations of the COC bonds in the pyranose ring, confirming the existence of the pyranose ring structure and clarifying the polysaccharide backbone; -1 and 811cm -1 The strong absorption peaks at 1153 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of the S=O bond in the sulfate group, which are typical characteristics of the sulfate group. -1The absorption peak at that location may be due to the stretching vibration of the COS bond formed between the sulfate group and the sugar ring. The above infrared spectroscopy analysis indicates that Bm-sCMP-3 fully conforms to the structural characteristics of sulfated polysaccharides.

[0081] Depend on Figure 4 It can be seen that at a magnification of ×2.0k, the microstructure of Bm-sCMP-3 is diverse and complex. Among them, granular structures are widely distributed, with approximately elliptical, circular or irregular shapes, smooth surfaces, and both independent distribution and aggregation phenomena exist among them, which is speculated to be structural units formed by the aggregation of polysaccharide molecular chains through intermolecular forces.

[0082] The particle size of Bm-sCMP-3 is 48.52 nm. Figure 5 The potential is -24.90mV. Figure 6 ).

[0083] Example 7: Preparation of Bm-sCMP-3 spray

[0084] Take 1.0g Bm-sCMP-3, 70.0mL propylene glycol, 0.2g benzalkonium chloride and 0.5mL Tween-80, add an appropriate amount of purified water and stir to dissolve. Finally, add water to make up to 100mL, mix well and filter through a 0.22μm microporous membrane for sterilization. Then, bottle it in a spray bottle to obtain Bm-sCMP-3 spray.

[0085] Example 8: Preparation of Bm-sCMP-3 Topical Gel

[0086] Take 1.0g Bm-sCMP-3, 1.0g carbomer 940, 10.0mL glycerol and 0.3g ethylparaben, add an appropriate amount of purified water and stir to disperse; separately, place carbomer 940 in an appropriate amount of purified water and let it swell overnight. Mix the two phases and add 0.2mL benzalkonium chloride, heat to 60℃ and stir to dissolve, adjust the pH to 6.5 with triethanolamine, and finally add water to make up to 100mL to obtain Bm-sCMP-3 topical gel.

[0087] Example 9: Preparation of Bm-sCMP-3 Oral Solution

[0088] Take 1.0g Bm-sCMP-3, 5.0mL propylene glycol, 0.02g benzalkonium chloride, 0.5mL Tween-80 and 2.0g erythritol, add them to 50mL of purified water at 40-50℃ and stir to dissolve. Adjust the pH to 6.5±0.2 with 0.1M NaOH or HCl solution, make up to 100mL, sterilize through a 0.22μm filter membrane and aseptically fill to obtain Bm-sCMP-3 oral solution.

[0089] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A sulfated silkworm pupa cordyceps polysaccharide, characterized in that: It was prepared by reacting silkworm pupa cordyceps polysaccharide and aminosulfonic acid in N,N-dimethylformamide through a staged heating reaction. The reaction conditions were: first react at 60℃ for 1 h, then raise the temperature to 80℃ and react for 2 h.

2. The sulfated silkworm pupa cordyceps polysaccharide according to claim 1, characterized in that: The sulfated silkworm pupa cordyceps polysaccharide has a degree of sulfate substitution of 0.356 to 1.

193.

3. The sulfated silkworm pupa cordyceps polysaccharide according to claim 2, characterized in that: The sulfated silkworm pupa cordyceps polysaccharide has a sulfate substitution degree of 1.

193.

4. A method for preparing sulfated Cordyceps militaris polysaccharide, comprising the following steps: placing Cordyceps militaris polysaccharide and aminosulfonic acid in an organic solvent, heating and reacting in stages for a certain time; cooling to room temperature, quenching the reaction with an alkaline solution, adjusting the pH of the reaction solution to neutral, precipitating with ethanol to obtain a precipitate, redissolving the precipitate with distilled water, dialyzing, and freeze-drying to obtain sulfated Cordyceps militaris polysaccharide with different degrees of substitution.

5. The method according to claim 4, characterized in that: The mass ratio of the silkworm pupa cordyceps polysaccharide to aminosulfonic acid is 1:2 to 1:

5.

6. The method according to claim 5, characterized in that: The sulfated silkworm pupa cordyceps polysaccharide has a degree of sulfate substitution of 0.356 to 1.

193.

7. The method according to claim 6, characterized in that: The mass ratio of the silkworm pupa cordyceps polysaccharide to aminosulfonic acid is 1:3, and the degree of sulfate substitution of the obtained sulfated silkworm pupa cordyceps polysaccharide is 1.

193. And / or, the segmented heating refers to reacting at 60°C for 1 hour, and then raising the temperature to 80°C for 2 hours.

8. The method according to claim 7, characterized in that: The monosaccharide composition and content of the obtained sulfated Cordyceps militaris polysaccharide are as follows: arabinose 1.52%, glucosamine 0.50%, galactose 15.02%, glucose 55.60%, xylose 3.01%, mannose 23.65%, and galacturonic acid 0.70%; the total sugar content of the sulfated Cordyceps militaris polysaccharide is 50.52±2.62%, the uronic acid content is 1.77±0.10%, and the sulfate group content is 13.45±0.36%; the molecular weight of the sulfated Cordyceps militaris polysaccharide is 2.65 kDa; the particle size of the sulfated Cordyceps militaris polysaccharide is 48.52 nm, and the potential is -24.90 mV.

9. A use of sulfated silkworm pupa cordyceps polysaccharide, wherein the sulfated silkworm pupa cordyceps polysaccharide is prepared by the method of claim 7, and is used in the preparation of antibacterial and hypoglycemic products.

10. The use according to claim 9, wherein the product is a spray, a topical gel, or an oral liquid.

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