CN118027236A - Method for flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity and application thereof - Google Patents

Abstract

The present invention belongs to the field of natural product preparation and application, and is particularly related to a method and use for efficiently preparing high hypoglycemic activity silkworm pupa cordyceps polysaccharides using flash extraction. The flash extraction technology provided by the present invention can efficiently prepare silkworm pupa cordyceps polysaccharides, with high polysaccharide yield (18.3 ± 1.82%), high inhibition rate of α-glucosidase (83.2 ± 7.64%), low energy consumption and extremely short extraction time (only 1min), significantly better than hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and other methods. The purified polysaccharides of Cordyceps militaris extracted and purified using this technology also have strong hypoglycemic activity. At a concentration of 5mg/mL, the inhibition rate of α-glucosidase reached 99.4 ± 1.25%, which is significantly higher than acarbose at the same concentration. The purified polysaccharides of silkworm pupa cordyceps extracted and purified by the technical solution of the present invention can be used for the preparation of hypoglycemic functional products such as oral liquids, capsules, and tablets.

Description

A method and application for the high-efficiency extraction of silkworm chrysalis and Cordyceps sinensis polysaccharides with high hypoglycemic activity

Technical Field

The invention belongs to the field of natural product preparation, and specifically relates to a method and use for efficiently preparing silkworm chrysalis cordyceps polysaccharide with high hypoglycemic activity by flash extraction.

Background technique

Cordyceps militaris, also known as Cordyceps militaris, is a fungus belonging to the genus Cordyceps of the family Clavicipitaceae. It is native to my country and was approved as a new resource food by the Ministry of Health in 2009. It is a high-value medicinal and edible fungus. Cordyceps militaris is rich in polysaccharides, cordycepin, cordycepic acid and other bioactive substances. Among them, polysaccharides are important active ingredients in Cordyceps militaris, which have a wide range of pharmacological effects such as enhancing immunity, anti-tumor, and lowering blood sugar (Food Bioscience, 2022, 45: 101503), and have high application value in the fields of biomedicine and food.

The low content, low extraction rate and great difficulty of silkworm pupa cordyceps polysaccharide have become bottleneck problems restricting industrial development. At present, the main extraction methods of silkworm pupa cordyceps polysaccharide include hot water extraction method (Food Science, 2017, 38 (14): 91-96; CN106749742A; CN116496424A), ultrasonic assisted method (Food Research and Development, 2023, 44 (11): 159-165; CN107501428A), microwave assisted method (Journal of Jiangsu Agricultural Sciences, 2009, 25 (5): 1143-1150), etc. However, these methods have the disadvantages of long time, high energy consumption and low extraction rate.

Flash extraction technology uses a high-speed shear head to form a high-intensity shear force, has a very high wall-breaking efficiency, and uses strong stirring to allow the solvent to penetrate quickly and the effective ingredients to diffuse rapidly. It has the advantages of short extraction time, low temperature, and no radiation risk (Trends in Food Science & Technology, 2021, 112: 581-591). At present, there are no reports on the preparation of silkworm Cordyceps polysaccharides using flash extraction technology.

Metabolic diseases, led by diabetes, remain one of the major factors endangering public health. The Guidelines for the Prevention and Treatment of Type 2 Diabetes in China reported that the incidence of type 2 diabetes in my country is 10.4%. In addition, the prevalence of people over 60 years old is higher than 20%, and undiagnosed patients account for 63% of the total. Although chemical hypoglycemic drugs such as sulfonylureas and biguanides have played an important role in regulating blood sugar and reducing complications, the adverse reactions and resistance of long-term use have never been solved. Compared with chemical hypoglycemic drugs, natural hypoglycemic products are gradually being recognized by people for their high safety and suitability for long-term use. Inhibition of α-glucosidase and thus reduction of glucose intake is the most important mechanism of polysaccharide hypoglycemic. The oligosaccharide hypoglycemic drugs acarbose and its analogs that have been marketed are classic α-glucosidase inhibitors. Zhu Zhenyuan et al. (Modern Food Science and Technology, 2014, 30(12):55-60) found that the Cordyceps sinensis polysaccharide prepared by hot water extraction can inhibit the activity of α-glucosidase, but did not investigate whether different extraction parameters and methods have an effect on the activity.

Processing voltage is an important technical parameter for flash extraction. People often focus on the effect of voltage on polysaccharide yield (Food Industry, 2020, 41(11):42-45; Food Research and Development, 2023, 44(9):164-170), but ignore the effect of voltage on the biological activity of polysaccharides. The present invention provides a method and use for efficiently preparing silkworm pupa cordyceps polysaccharides with high hypoglycemic activity by flash extraction by exploring the effects of different extraction voltages on the yield and hypoglycemic activity of silkworm pupa cordyceps polysaccharides.

Summary of the invention

The purpose of the present invention is to provide a method for efficiently preparing silkworm pupa cordyceps polysaccharides with high hypoglycemic activity using flash extraction. Compared with traditional hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and ultrasound-assisted extraction, the silkworm pupa cordyceps polysaccharides prepared using the flash extraction technology provided by the present invention have high yield, low energy consumption and short extraction time; and the silkworm pupa cordyceps polysaccharides prepared and purified using this technology have extremely high α-glucosidase inhibitory activity and show excellent hypoglycemic function.

According to the first aspect of the present invention, the present invention provides a method for efficiently preparing silkworm pupa cordyceps polysaccharides by flash extraction, wherein silkworm pupa cordyceps defatted powder is used as raw material, soaked in water, then placed in a flash extractor, and treated under a certain voltage; the silkworm pupa cordyceps polysaccharides are obtained by centrifugation, concentrating the supernatant, ethanol precipitation, and freeze drying.

Preferably, the defatted powder of silkworm pupa and Cordyceps is defatted by petroleum ether, and the specific steps are: drying fresh silkworm pupa and Cordyceps at 60° C. to constant weight, crushing and passing through a 40-mesh sieve to prepare silkworm pupa and Cordyceps powder for later use; adding petroleum ether to defatting in the dark for 12 hours, and filtering under reduced pressure to obtain the defatted powder of silkworm pupa and Cordyceps.

Preferably, the certain processing voltage is 70-150V, more preferably 110V; more preferably, a flash extractor model JHBE-20A produced by Xi'an Taikang Biotechnology Co., Ltd. is used, and the processing voltage is set to 110V.

Preferably, the method for efficiently preparing silkworm pupa cordyceps polysaccharide by flash extraction specifically comprises the following steps: soaking silkworm pupa cordyceps defatted powder in water at a solid-liquid ratio of 1:40 g/mL for 2 hours, then placing it in a flash extractor (model JHBE-20A of Xi'an Taikang Biotechnology Co., Ltd.), and treating it at a voltage of 110 V for 1 minute; centrifuging and taking the supernatant after the end, concentrating the supernatant to 1/4 of the original volume to obtain a concentrated solution, adding 3 times the volume of anhydrous ethanol to precipitate at 4°C for 12 hours, and freeze-drying the precipitate to obtain silkworm pupa cordyceps polysaccharide; under this condition, the yield of silkworm pupa cordyceps polysaccharide is 18.3±1.82%, and the inhibition rate of α-glucosidase at 5 mg/mL is 83.2±7. 64%, which is significantly better than traditional hot water extraction (polysaccharide yield: 10.7±0.26%; inhibition rate of α-glucosidase at 5 mg/mL: 31.5±4.81%), enzyme-assisted extraction (polysaccharide yield: 10.7±0.93%; inhibition rate of α-glucosidase at 5 mg/mL: 35.4±2.95%), microwave-assisted extraction (polysaccharide yield: 14.4±2.03%; inhibition rate of α-glucosidase at 5 mg/mL: 48.6±5.11%) and ultrasound-assisted extraction (polysaccharide yield: 13.6±1.53%; inhibition rate of α-glucosidase at 5 mg/mL: 50.7±4.69%).

The preparation method of silkworm pupa cordyceps polysaccharide of the present invention also comprises the steps of purifying the prepared silkworm pupa cordyceps polysaccharide, deproteinizing by using Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent, freeze-drying the dialyzate to obtain deproteinized polysaccharide, and purifying by using a diethylaminoethyl (DEAE) cellulose chromatographic column to obtain purified silkworm pupa cordyceps polysaccharide; the specific method is: deproteinizing by using Sevag reagent (chloroform: n-butanol=4:1, volume ratio) for 3 times; removing denatured protein and residual Sevag reagent by centrifugation, dialyzing in distilled water for 3 times (dialysis bag molecular weight cutoff is 7000Da, distilled water is replaced once every 12 hours); freeze-drying the dialyzate to obtain the deproteinized polysaccharide. Weigh 100 mg of deproteinized polysaccharide and dissolve it in 20 mL of deionized water, then load it onto a diethylaminoethyl (DEAE) cellulose chromatographic column (2.6 cm × 60 cm); slowly elute with 0.5 mol/L NaCl solution at a flow rate of 0.5 mL/min, 2 mL per tube; determine the absorbance of the eluate in each tube by the phenol-sulfuric acid method, and when all the polysaccharides have been eluted, combine the eluates, concentrate them, and dialyze them three times (the same method as above), and freeze-dry them to obtain the purified polysaccharide of silkworm chrysalis.

According to the second aspect of the present invention, the present invention provides a purified polysaccharide of silkworm pupa and Cordyceps militaris, wherein the total sugar content of the purified polysaccharide of silkworm pupa and Cordyceps militaris is 83.5±1.46%, the protein content is 7.26±2.05%, the uronic acid content is 4.16±0.68%, and the sulfate content is 3.71±0.69%; the purified polysaccharide of silkworm pupa and Cordyceps militaris is composed of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56; the number average molecular weight (Mn) of the purified polysaccharide of silkworm pupa and Cordyceps militaris is 1.85kDa, and the weight average molecular weight (Mw) is 5.09kDa.

According to the third aspect of the present invention, the present invention provides the use of the purified polysaccharide of silkworm chrysalis and Cordyceps militaris in the preparation of a product with hypoglycemic function; it can be used to prepare oral liquids, capsules, tablets and other preparations.

The present invention has the following beneficial effects:

① The flash extraction technology provided by the present invention has significant advantages in shortening the extraction time, reducing energy consumption, and protecting the polysaccharide structure. It can greatly improve the yield and hypoglycemic activity of Cordyceps militaris polysaccharides, and is significantly superior to existing technologies such as hot water extraction.

② The purified polysaccharide of silkworm chrysalis and Cordyceps militaris provided by the present invention has an extremely high inhibition rate on α-glucosidase, which is significantly higher than acarbose at the same concentration at 5 mg/mL, showing excellent hypoglycemic function, and can be used to prepare products for hypoglycemic-related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effects of different treatment voltages on the yield of Cordyceps militaris polysaccharides (different superscript lowercase letters represent statistical differences);

Figure 2. Effects of different treatment voltages on the inhibition of α-glucosidase activity by Cordyceps militaris polysaccharides (different superscript lowercase letters represent statistical differences);

Figure 3. Chromatogram of monosaccharide composition of purified polysaccharide of Cordyceps militaris (peaks in standard: 1-fucose; 2-rhamnose; 3-arabinose; 4-galactose; 5-glucose; 6-xylose; 7-mannose; 8-fructose; 9-galacturonic acid; 10-glucuronic acid);

Figure 4. Molecular weight distribution of purified polysaccharides from silkworm chrysalis and Cordyceps militaris;

Figure 5. Infrared spectrum of purified polysaccharide from silkworm chrysalis and Cordyceps militaris;

Figure 6. Scanning electron micrograph of purified polysaccharide from silkworm chrysalis;

Figure 7. Inhibitory effect of purified polysaccharides from Cordyceps militaris on α-glucosidase (different superscript lowercase letters represent statistical differences within the group; different superscript uppercase letters represent statistical differences between groups at the same concentration).

Detailed ways

Silkworm pupa and Cordyceps were provided by Suzhou Wujiang Jiahe Sericulture Professional Cooperative; the flash extractor was provided by Xi'an Taikang Biotechnology Co., Ltd., model JHBE-20A; the molecular weight was determined by Waters high performance liquid chromatograph, model 1525; the monosaccharide composition was determined by Dionex ion chromatograph, model ICS-5000; the infrared spectrum was determined by PerkinElmer Fourier transform infrared microscope system, model Spotlight200i; the scanning electron microscopy (SEM) was determined by HITACHI ultra-high resolution field emission scanning electron microscope, model Regulus8100; all experiments were performed in triplicate, and the data were expressed as mean ± SD. The statistical analysis of the data was performed by t-test or ANOVA analysis, and P < 0.05 was considered statistically significant.

Example 1 Flash Extraction of Silkworm Cordyceps Polysaccharide and Screening of Treatment Voltage

Fresh silkworm pupa cordyceps is dried at 60℃ to constant weight, crushed and sieved through a 40-mesh sieve to make silkworm pupa cordyceps powder for later use; petroleum ether is added to degrease in the dark for 12 hours, and the silkworm pupa cordyceps defatted powder is obtained by vacuum filtration. The silkworm pupa cordyceps defatted powder is soaked in water at a solid-liquid ratio of 1:40g/mL for 2 hours, then placed in a flash extractor and treated at a certain voltage (70-150V) for 1 minute. After the end, the supernatant is centrifuged and concentrated to 1/4 of the original volume, and 3 times the volume of anhydrous ethanol is added to precipitate at 4℃ for 12 hours. The precipitate is freeze-dried to obtain silkworm pupa cordyceps polysaccharide.

Polysaccharide yield (%) = m/M×100, where: m is the mass of freeze-dried Cordyceps militaris polysaccharide (g); M is the mass of defatted powder of Cordyceps militaris (g). The polysaccharide content was determined using the phenol-sulfuric acid method, and the glucose standard equation was: Y = 9.541X + 0.0075, R ² = 0.9997.

The Cordyceps militaris polysaccharides extracted at different voltages were dissolved in distilled water to prepare 5 mg/mL solutions for the determination of the inhibitory activity against α-glucosidase. The specific determination method is as follows:

Accurately pipette 100 μL of silkworm chrysalis Cordyceps polysaccharide solution and mix with 300 μL of 0.24U/mL α-glucosidase solution, then add 600 μL of phosphate buffered saline (PBS) buffer (pH=6.8); after oscillation, incubate at 37°C for 15 min; add 2.74 mg/mL 4-nitrophenyl-β-D-pyranogalactoside (PNPG) solution and mix thoroughly, continue to incubate at 37°C for 20 min; finally, add 4 mL of sodium carbonate to terminate the reaction, and measure the absorbance at 400 nm. The α-glucosidase inhibition rate is calculated as follows: α-glucosidase inhibition rate (%) = [1-(A _sample -A _background )/(A _negative -A _blank )] × 100; Table 1 is the α-glucosidase inhibition activity experimental reaction system.

Table 1. α-glucosidase inhibitory activity test reaction system

1.1 Effect of different treatment voltages on the yield of polysaccharides from silkworm chrysalis

The solid-liquid ratio was fixed at 1:40 g/mL and the flash extraction time was 1 min. The effect of different treatment voltages (70-150 V) on the yield of Cordyceps militaris polysaccharides was investigated. The results are shown in Figure 1.

As shown in Figure 1, in the range of 70-110V, the polysaccharide yield increases with the increase of voltage value. When the voltage value reaches 110V, the polysaccharide yield is 18.3±1.82%, which is significantly higher than the polysaccharide yields at 70V and 90V (P<0.01); when the voltage value reaches 130V, the polysaccharide yield is the highest, which is 20.4±2.35%. Although it is higher than the polysaccharide yield at 110V, there is no statistical difference (P>0.05); when the voltage value increases to 150V, the polysaccharide yield decreases significantly (P<0.05).

1.2 Effects of different treatment voltages on the inhibitory activity of α-glucosidase by Cordyceps militaris polysaccharides

The solid-liquid ratio was fixed at 1:40 g/mL, the flash extraction time was 1 min, and the concentration of Cordyceps militaris polysaccharide was 5 mg/mL. The effect of different treatment voltages (70-150 V) on the inhibition of α-glucosidase activity by Cordyceps militaris polysaccharide was investigated. The results are shown in Figure 2.

As shown in Figure 2, in the range of 70 to 110 V, the inhibition rate of α-glucosidase increased significantly with the increase of voltage value (P<0.01). When the voltage value reached 110 V, the inhibition rate reached 83.2±7.64%, which was significantly higher than the inhibition rates at 70 V and 90 V (P<0.01). When the voltage value increased to 130 V, the inhibition rate began to decrease significantly (P<0.01). When the voltage value continued to increase to 150 V, the inhibition rate dropped to 53.5±6.69%.

Taking extraction effect, α-glucosidase inhibitory activity, energy consumption and other aspects into consideration, a processing voltage of about 110 V was appropriate, at which the yield of Cordyceps militaris polysaccharide was 18.3±1.82% and the inhibition rate of α-glucosidase was 83.2±7.64%.

Comparative Example

On the basis of Example 1, in order to further prove that the flash extraction technology provided by the present invention can efficiently prepare Cordyceps militaris polysaccharides with high hypoglycemic activity, the present invention implemented the following comparative test:

Option A: Hot water extraction

The defatted powder of silkworm pupa Cordyceps militaris was soaked in water at a solid-liquid ratio of 1:40 g/mL for 2 hours, and then extracted at 80° C. for 2 hours; after cooling, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 hours, and the precipitate was freeze-dried to obtain silkworm pupa Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

The results showed that the yield of Cordyceps militaris polysaccharide obtained by hot water extraction was 7.52±1.63% (2.4 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 31.5±4.81% (2.6 times lower than the technical solution of the present invention).

Option B: Enzyme-assisted extraction

The silkworm pupa cordyceps defatted powder is soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 hours, and then the pH value is adjusted to about 5.0, and 2.0% of the mass of the cordyceps defatted powder is added with cellulase (enzyme activity is 10 U/mg), and the mixture is placed in a shaker at about 50° C. for incubation for 2 hours. After the incubation, the enzymolysis solution is placed in a boiling water bath for 15 minutes to inactivate the enzyme; after cooling, the supernatant is centrifuged to obtain the supernatant, and the supernatant is concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol is added to precipitate at 4° C. for 12 hours, and the precipitate is freeze-dried to obtain silkworm pupa cordyceps polysaccharide; the polysaccharide yield is calculated and the inhibitory activity to α-glucosidase is determined, and the method is the same as that in Example 1.

The results showed that the yield of Cordyceps militaris polysaccharide obtained by enzyme-assisted extraction was 10.7±0.93% (1.7 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 35.4±2.95% (2.4 times lower than the technical solution of the present invention).

Scheme C: Microwave-assisted extraction

The defatted powder of silkworm chrysalis and Cordyceps militaris was soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 h, and then transferred to a microwave-ultrasonic combined synthetic extractor (Beijing Xianghu Technology Development Co., Ltd., model XH-300B); the microwave mode was turned on, and the extraction was carried out at 500 W power and 70° C. for 8 min; after the extraction, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 h, and the precipitate was freeze-dried to obtain silkworm chrysalis and Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

The results showed that the yield of Cordyceps militaris polysaccharides obtained by microwave-assisted extraction was 14.4±2.03% (1.3 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 48.6±5.11% (1.7 times lower than the technical solution of the present invention).

Scheme D: Ultrasound-assisted extraction

The defatted powder of silkworm chrysalis and Cordyceps militaris was soaked in water at a liquid-to-solid ratio of 1:40 g/mL for 2 h, and then transferred to a microwave-ultrasonic combined synthetic extractor (Beijing Xianghu Technology Development Co., Ltd., model XH-300B); the ultrasonic mode was turned on, and the extraction was carried out at 300 W power and 60° C. for 30 min; after the extraction, the supernatant was centrifuged and concentrated to 1/4 of the original volume, 3 times the volume of anhydrous ethanol was added and precipitated at 4° C. for 12 h, and the precipitate was freeze-dried to obtain silkworm chrysalis and Cordyceps militaris polysaccharide; the polysaccharide yield was calculated and the inhibitory activity against α-glucosidase was determined, using the same method as in Example 1.

The results showed that the yield of Cordyceps militaris polysaccharides obtained by ultrasound-assisted extraction was 13.6±1.53% (1.3 times lower than the technical solution of the present invention); the inhibition rate of α-glucosidase at 5 mg/mL was 50.7±4.69% (1.6 times lower than the technical solution of the present invention).

In summary, the yield of silkworm Cordyceps militaris polysaccharides obtained by the flash extraction technology provided by the present invention is higher than that of traditional hot water extraction, enzyme-assisted extraction, microwave-assisted extraction and ultrasound-assisted extraction, and has the advantages of low energy consumption and extremely high efficiency (only 1 minute of extraction is required); the inhibition rate of α-glucosidase is also higher than that of the above methods, indicating that the 110V flash treatment can not only increase the yield of silkworm Cordyceps militaris polysaccharides, but also optimize the polysaccharide structure and improve the hypoglycemic activity.

Example 2 Purification of Silkworm Cordyceps Polysaccharide

Deproteinization was performed 3 times using Sevag reagent (chloroform: n-butanol = 4:1, volume ratio); denatured proteins and residual Sevag reagent were removed by centrifugation and dialyzed 3 times in distilled water (dialysis bag molecular weight cutoff was 7000Da, distilled water was replaced every 12 hours); the dialyzate 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 chromatographic column (2.6 cm × 60 cm); 0.5 mol/L NaCl solution was used for slow elution, with a flow rate of 0.5 mL/min, and 2 mL per tube; the absorbance of each tube of eluate was determined by the phenol-sulfuric acid method, and the eluate was combined when all the polysaccharides were eluted, concentrated, and dialyzed 3 times (the method is the same as above), and freeze-dried to obtain the purified polysaccharide of silkworm chrysalis.

Example 3 Composition Analysis and Characterization of Purified Polysaccharides from Silkworm Cordyceps

The total sugar content in the purified polysaccharide from silkworm pupa Cordyceps militaris was determined by the phenol-sulfuric acid method and was 83.5±1.46%; the protein content was determined by the Coomassie Brilliant Blue method and was 7.26±2.05%; the uronic acid content was determined by the m-hydroxybiphenyl method and was 4.16±0.68%; and the sulfate content was determined by the barium chloride gelatin turbidimetry method and was 3.71±0.69%.

Monosaccharide composition analysis: Weigh 5 mg of purified polysaccharide from Cordyceps militaris into a 5 mL stoppered test tube, add 1 mL of 2 mol/L trifluoroacetic acid, and hydrolyze in an oven at 121°C for 2 h. Then distilled water is diluted to 50 mL, filtered with a 0.45 μm microporous filter membrane for injection analysis, and the injection volume is 20 μL. Instrument and chromatographic conditions: American Dionex ICS-5000 ion chromatograph, equipped with a pulsed amperometric detector, and the chromatographic column is CarboPac PA20 (6.5 μm, 3 mm×150 mm). Gradient elution is used, and the mobile phase is: 98% water + 2% 250 mM NaOH (0-21 min); 93% water + 2% 250 mM NaOH + 5% 1 M NaAc (21.1-30 min); 20% water + 80% 250 mM NaOH (30.1-50 min). The flow rate is 0.5 mL/min.

Molecular weight analysis: The purified polysaccharide of Cordyceps militaris was prepared into a 2 mg/mL solution. The sample was filtered with a 0.45 μm microporous filter membrane before injection. The injection volume was 20 μL. The analytical instrument was a Waters 1525 high performance liquid chromatograph, a 2414 differential refractometer and an Empower 3 workstation. The chromatographic column was an Ultrahydrogel ^TM Linear (2 μm, 7.8 mm × 300 mm). Dextran with different molecular weights was used as a standard sugar to prepare a series of standard sugar solutions with different molecular weights. The third-order calibration curve for the determination of polysaccharide molecular weight was established based on the retention time and molecular weight value. The chromatographic conditions were as follows: mobile phase: 0.1 M NaNO ₃ ; flow rate: 0.9 mL/min; column temperature: 45 °C.

Infrared spectrum analysis: 2 mg of dried purified polysaccharide of Cordyceps militaris was mixed with 100 mg of dried potassium bromide, pressed into tablets, and then placed in an infrared spectrometer for measurement in a scanning range of 4000 cm ^-1 to 400 cm ^-1 .

Scanning electron microscope analysis: Take the dried silkworm pupa Cordyceps purified polysaccharide, stick it on the double-sided conductive adhesive stage with a cotton swab, and use an ear-cleaning bulb to blow away the unsticky polysaccharide powder. Place it in an ion sputtering instrument for foil spraying, and then put the treated samples in order under the scanning electron microscope, and observe their morphology after the instrument settings are stable.

As shown in FIG3 , by comparing with standard sugars, it was determined that the purified polysaccharide of silkworm chrysalis consisted of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56.

As shown in FIG4 , the number average molecular weight (Mn) of the purified polysaccharide of Cordyceps militaris was 1.85 kDa, and the weight average molecular weight (Mw) was 5.09 kDa.

As shown in Figure 5, from the infrared spectrum of the purified polysaccharide from silkworm chrysalis, it can be observed that there is a strong absorption peak near 3422cm ^-1 , which is caused by the stretching vibration of -OH; 2931cm ^-1 is the asymmetric stretching vibration of methyl CH; 1645cm ^-1 is the stretching vibration of carbonyl CO; 1404cm ^-1 is the stretching vibration of CO; the absorption peak near 1063cm ^-1 is the skeleton vibration of COC glycosidic bond; 863cm ^-1 is the characteristic peak of α-glucosidic bond; 598cm ^-1 is the absorption peak of pyranose ring. Infrared spectrum analysis further confirmed that the purified polysaccharide from silkworm chrysalis conforms to the structural characteristics of polysaccharides.

As shown in Figure 6, at a magnification of ×1k, the surface of the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is rough, showing irregular holes, with some particles of different sizes scattered in it.

Example 4 Evaluation of the Inhibition of α-glucosidase Activity by Purified Polysaccharides from Silkworm Cordyceps

In order to investigate the effect of purification on the hypoglycemic activity of Cordyceps militaris polysaccharides, the present invention further evaluated the inhibitory effect of purified Cordyceps militaris polysaccharides on α-glucosidase, and used acarbose (an α-glucosidase inhibitor already on the market) at the same concentration as the positive control, using the same method as Example 1.

As shown in FIG. 7 , within the concentration range of 1 to 5 mg/mL, the inhibitory effect of the purified polysaccharide of silkworm chrysalis on α-glucosidase increased significantly with increasing concentration (P<0.01). When the concentration reached 5 mg/mL, the inhibition rate of α-glucosidase reached 99.4±1.25%, which was significantly higher than that of acarbose at the same concentration (92.5±2.11%, P<0.05); it was also significantly higher than the inhibition rate of α-glucosidase under the treatment voltage of 110 V (83.2±7.64%, P<0.01, see actual results). Example 1) shows that purification can further improve the hypoglycemic activity of silkworm Cordyceps polysaccharides; the half inhibition concentration of silkworm Cordyceps polysaccharides extracted and purified by the technical solution of the present invention on α-glucosidase is 1.48±0.21 mg/mL, which is 2.85 times lower than the 4.22 mg/mL reported in the literature (Modern Food Science and Technology, 2014, 30(12):50-60), indicating that the silkworm Cordyceps polysaccharides extracted and purified by the technical solution of the present invention have extremely high α-glucosidase inhibition and exhibit excellent hypoglycemic function.

Example 5 Preparation of Purified Silkworm Cordyceps Polysaccharide Oral Liquid

Take 2.0g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris, add it into 100mL of purified water, stir it at room temperature until dissolved, add 0.5g of erythritol and 0.2g of potassium sorbate, stir evenly, sterilize it in cans instantly, bottle it, and seal it to obtain purified polysaccharide oral liquid from silkworm chrysalis and Cordyceps militaris.

Example 6 Preparation of Purified Polysaccharide Capsules from Silkworm Chrysalis and Cordyceps

Take 5.0g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris passed through a 40-mesh sieve, spray it with 90% ethanol at a ratio of 1:1, mix evenly, add 10% starch to make a soft material, pass through a 20-mesh sieve to granulate, place it in a 60°C oven to dry for 1h, pass through a 20-mesh sieve to granulate, and fill it into No. 3 empty capsules in an environment with a relative humidity of less than 65% to obtain purified polysaccharide capsules from silkworm chrysalis and Cordyceps militaris.

Example 7 Preparation of Purified Polysaccharide Tablets from Silkworm Chrysalis and Cordyceps

5.0 g of purified polysaccharide from silkworm chrysalis and Cordyceps militaris was mixed with 300 mg of polyvinyl pyrrolidone and 4.0 mg of calcium hydrogen phosphate, ground into powder, and passed through a 100-mesh sieve; 5% of a 95% ethanol solution of povidone K30 was added during stirring to prepare a soft material, and wet granulation was performed (passed through a 16-mesh sieve); the mixture was dried at 60° C., sieved, and granulated; 1% of magnesium stearate and 2% of micro-powdered silica gel were added, the mixture was evenly mixed, and tableting was performed to obtain purified polysaccharide tablets from silkworm chrysalis and Cordyceps militaris.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (9)

1. A method for efficiently preparing silkworm pupa cordyceps polysaccharide with high hypoglycemic activity by flash extraction, which comprises taking silkworm pupa cordyceps defatted powder as raw material, soaking it in water, and then placing it in a flash extractor and treating it under a certain voltage; centrifuging, concentrating the supernatant, ethanol precipitation, and freeze-drying to obtain silkworm pupa cordyceps polysaccharide. 2. The method according to claim 1, wherein the certain processing voltage is 70 to 150V. 3. The method according to claim 1, wherein the silkworm chrysalis and Cordyceps defatted powder is obtained by defatting with petroleum ether. 4. The method according to claim 1 or 2, wherein the certain processing voltage is 110V, and a flash extractor model JHBE-20A of Xi'an Taikang Biotechnology Co., Ltd. is used. 5. The method according to claim 1 or 2, wherein the specific steps are: Dry fresh silkworm pupa cordyceps to constant weight, crush and sieve to make silkworm pupa cordyceps powder for later use; add petroleum ether to defat in dark, and filter under reduced pressure to obtain silkworm pupa cordyceps defatted powder; The defatted powder of silkworm pupa and Cordyceps militaris is soaked in water, and then placed in a flash extractor and processed at a voltage of 110V; after the extraction, the supernatant is centrifuged and concentrated to obtain a concentrated solution, anhydrous ethanol is added for precipitation, and the precipitate is freeze-dried to obtain silkworm pupa and Cordyceps militaris polysaccharide; The yield of the silkworm chrysalis cordyceps polysaccharide is 18.3±1.82%, and the inhibition rate of α-glucosidase at 5 mg/mL is 83.2±7.64%. 6. The method according to claim 1, wherein the preparation method of the silkworm chrysalis Cordyceps polysaccharide further comprises purifying the prepared silkworm chrysalis Cordyceps polysaccharide, deproteinizing with Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent, freeze-drying the dialysate to obtain deproteinized polysaccharide, and purifying with a diethylaminoethyl (DEAE) cellulose chromatographic column to obtain purified silkworm chrysalis Cordyceps polysaccharide. 7. The method according to claim 6, wherein the specific method of the purification is: deproteinizing with Sevag reagent, dialyzing in distilled water after removing residual Sevag reagent; freeze-drying the dialyzate to obtain deproteinized polysaccharide; dissolving the deproteinized polysaccharide in deionized water, and loading it onto a diethylaminoethyl (DEAE) cellulose chromatographic column; slowly eluting with NaCl solution, measuring the absorbance of each tube of eluate with phenol-sulfuric acid method, combining the eluates after all the polysaccharides have been eluted, concentrating, dialyzing, and freeze-drying to obtain purified polysaccharide of Bombyx mori. The purified polysaccharide from silkworm chrysalis and Cordyceps militaris satisfies one or more of the following conditions: 1) the total sugar content is 83.5±1.46%; 2) the protein content is 7.26±2.05%; 3) the uronic acid content is 4.16±0.68%; 4) the sulfate content is 3.71±0.69%; 5) the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is composed of arabinose, galactose, glucose, xylose, galacturonic acid, and glucuronic acid in a molar ratio of 31.6:21.6:274:23.6:1.00:3.56; 6) the number average molecular weight (Mn) of the purified polysaccharide from silkworm chrysalis and Cordyceps militaris is 1.85 kDa; 7) the weight average molecular weight (Mw) is 5.09 kDa. 8. Use of the purified polysaccharide of silkworm chrysalis and Cordyceps militaris obtained by the method of claim 7 for preparing a product with hypoglycemic function. 9. The use according to claim 8, wherein the purified polysaccharide from silkworm chrysalis and Cordyceps militaris can be used to prepare oral liquid, capsule or tablet.