Silkworm chrysalis cordyceps isolated polysaccharide with high oxidation resistance and moisturizing performance as well as preparation method and application of silkworm chrysalis cordyceps isolated polysaccharide

Abstract

The invention belongs to the field of natural product extraction and separation, and particularly relates to preparation and product application of cordyceps militaris separation polysaccharide. The total sugar content of the silkworm chrysalis cordyceps polysaccharide extracted and purified by the technical scheme of the invention is high (84.07 + / -3.69%); the silkworm chrysalis cordyceps militaris separated polysaccharide-2 obtained through further separation is high in antioxidant activity, and the scavenging rate of DPPH free radicals is 98.66 + / -2.22% when the concentration is 0.5 mg / mL, and even is slightly higher than that of L-ascorbic acid (97.04 + / -1.43%) with the same concentration; meanwhile, the hygroscopicity of the cordyceps militaris isolated polysaccharide-2 is obviously higher than that of the same type of humectant sodium alginate, and the moisture retention of the cordyceps militaris isolated polysaccharide-2 is obviously higher than that of the sodium alginate and the classic humectant glycerol. The silkworm chrysalis cordyceps isolated polysaccharide-2 prepared by the technical scheme of the invention can be applied to preparation of anti-aging and moisturizing skin care products such as emulsions, cream, gels, film agents and the like.

Description

Technical Field

[0001] The invention belongs to the field of natural product preparation, and specifically relates to a silkworm chrysalis cordyceps isolated polysaccharide with high antioxidant and moisturizing properties, a preparation method thereof, and skin care uses thereof. Background Art

[0002] Polysaccharides are a type of macromolecular natural product composed of more than 10 monosaccharides. They are widely distributed in animals and plants, have diverse physiological and pharmacological functions, and are used in food, medicine, and related fields. Natural polysaccharides have extremely high medicinal value. For example, Lentinan injection, an immunomodulator, has been clinically used as an adjuvant treatment for malignant tumors; heparin, an anticoagulant, is used to prevent and treat thrombosis or embolic diseases; hyaluronic acid, a moisturizer, is used for corneal epithelial damage and skin moisturizing. It is expected that the market share of bioactive polysaccharides will exceed US$28.5 billion in 2025.

[0003] Natural polysaccharides are also widely used in the skin care industry, especially in the field of functional cosmetics. The rich hydroxyl groups and loose porous morphology of polysaccharides are conducive to the adsorption and retention of water, and play a role in moisture absorption and moisturizing (Arabian Journal of Chemistry, 2022, 15: 104-163); they can quench free radicals, play an antioxidant role, and delay aging (Carbohydrate Polymers, 2023, 314: 120-965). As effective ingredients, many plant polysaccharides are added to cosmetic products, such as Bletilla striata polysaccharide (Daily Chemicals Science, 2022, 45: 30-34, 38), Ganoderma lucidum polysaccharide (Chinese Journal of Aesthetic Medicine, 2021, 30: 178-182), ginseng polysaccharide (Contemporary Chemical Research, 2020, 9: 97-98), etc.

[0004] Natural polysaccharides have complex structures and large molecular weights, which poses challenges to efficient extraction (Foods, 2019, 8: 304). In addition to the extraction method, further purification and separation will also affect the yield and biological activity of polysaccharides (Trends in Food Science & Technology, 2018, 81: 108-115). Ethanol precipitation is a common method for enriching polysaccharides from extracts. As a preliminary and necessary purification method, ethanol precipitation has the advantages of low cost, mild conditions, and simple operation (Arabian Journal of Chemistry, 2022, 15: 103770). More importantly, by changing the ethanol concentration, polysaccharides with different physical and chemical properties and biological activities can be precipitated. Combined with further analysis and comparison, the purification technology scheme for obtaining highly active polysaccharides can be determined (International Journal of Biological Macromolecules, 2024, 267: 131278).

[0005] Cordyceps militaris, also known as Cordyceps militaris and Cordyceps sinensis, is a fungus of the genus Cordyceps in the family Ergotaceae. It was approved as a new resource food in 2009 and is an important edible and medicinal fungus in my country. Cordycepin and cordycepic acid are the characteristic components of Cordyceps militaris. In addition, Cordyceps militaris is also rich in polysaccharides, with the yield reported in the literature ranging from 4.37% to 21.6% (Guangzhou Chemical Industry, 2024, 52: 17-20; Food and Oils, 2024, 37: 92-96, 113). Cordyceps militaris polysaccharides have beneficial pharmacological effects such as lowering blood sugar, enhancing immunity, and anti-tumor (Food Bioscience, 2022, 45: 101503), but its beauty and skin care research is very limited. Although patent CN 109864910B discloses an anti-aging skin care product with added Cordyceps militaris water extract, patent CN105902442A discloses a skin care product with added Cordyceps militaris crude polysaccharide, and CN 118027236 A discloses a method and use for the flash-type efficient extraction of Cordyceps militaris polysaccharide with high hypoglycemic activity, the above-mentioned technical inventions are all water extracts or crude polysaccharides obtained at a fixed ethanol concentration, and do not explore the effects of other concentrations of ethanol on the functions of polysaccharides, especially the antioxidant and hygroscopic and moisturizing properties; lacking effective separation and even basic structural characterization, it is impossible to determine whether the polysaccharide is the main active contributing component.

[0006] Excessive oxidative stress is the culprit leading to aging and many diseases, and anti-oxidation is the basis and key to delay skin aging. In the screening of antioxidant active substances, 2,2-biphenyl-1-picrylhydrazyl (DPPH) radical is a commonly used screening model, because DPPH radical is a relatively stable free radical in nature, if the test substance can effectively quench DPPH radical, it means that the antioxidant capacity of the substance is strong (Food Research International, 2015, 76: 359-365). Therefore, the present invention uses DPPH radical as a screening model, utilizes antioxidant activity-guided strategy to purify and separate silkworm chrysalis cordyceps polysaccharide, provides a kind of silkworm chrysalis cordyceps separated polysaccharide with high antioxidant activity, has moisture absorption and moisturizing effect, effectively solves the purification and separation problem of silkworm chrysalis cordyceps skin care polysaccharide, and provides technical solutions for the development of precise targeted high-end skin care products. Summary of the invention

[0007] The object of the present invention is to provide a method for extracting and separating isolated polysaccharides of silkworm chrysalis and Cordyceps militaris. The isolated polysaccharides of silkworm chrysalis and Cordyceps militaris prepared by the method have significant antioxidant and hygroscopic moisturizing properties, especially the moisturizing property is significantly higher than that of sodium alginate and the classic moisturizer glycerin, and can be used in the preparation of anti-aging and moisturizing skin care products.

[0008] According to the first aspect of the present invention, the present invention provides a method for preparing isolated polysaccharides from silkworm pupa and Cordyceps militaris: using defatted powder of silkworm pupa and Cordyceps militaris fruiting bodies as raw material, a hot water extraction method is adopted to prepare crude polysaccharides; and the isolated polysaccharides from silkworm pupa and Cordyceps militaris are obtained through filtration, concentration, ethanol precipitation, deproteinization, ion exchange separation, dialysis and freeze drying.

[0009] The method for preparing isolated polysaccharides from silkworm chrysalis and Cordyceps militaris specifically comprises the following steps:

[0010] The dried product of silkworm pupa Cordyceps militaris fruiting body was crushed and passed through a 40-mesh sieve for later use; petroleum ether was added to degrease in the dark for 12 hours, and the degreased powder of silkworm pupa Cordyceps militaris was obtained by vacuum filtration. 40g of silkworm pupa Cordyceps militaris degreased powder was put into a beaker, 1200mL of distilled water was added at a solid-liquid ratio of 1:30g / mL, extracted at 80℃ for 2.5h, and filtered with gauze; 800mL of distilled water was added to the filter residue and extracted again at 80℃ for 1.5h. The two filtrates were combined and filtered under reduced pressure to obtain the water extract of silkworm pupa Cordyceps militaris. The extract was concentrated to 20% of the original volume, and then anhydrous ethanol was added to adjust the final ethanol concentration in the filtrate to a certain concentration (v / v), precipitated at 4℃ for 12h, and the precipitate was collected by centrifugation. Use an appropriate amount of distilled water to redissolve the precipitate to obtain a crude polysaccharide solution of Cordyceps militaris, add 1 / 4 volume of the crude polysaccharide solution of Cordyceps militaris Sevag reagent (chloroform: n-butanol = 4:1, v / v), mix and shake for 15 minutes, centrifuge and discard the bottom organic phase and the middle layer of milky denatured protein, repeat the above operation until no obvious protein is produced. Take out the top layer of polysaccharide solution and freeze-dry to obtain Cordyceps militaris polysaccharide;

[0011] Preferably, adjusting the final ethanol concentration in the filtrate to a certain concentration means that the final ethanol concentration in the filtrate is 60 to 90% (v / v), more preferably 80% (v / v);

[0012] Preferably, the final concentration of ethanol in the filtrate is adjusted to 80% (v / v). Under this condition, the total sugar content of the silkworm Cordyceps polysaccharide is 84.07±3.69%, which is significantly higher than the silkworm Cordyceps polysaccharide obtained by precipitation with other concentrations of ethanol (P<0.05 or P<0.01).

[0013] The method for preparing isolated polysaccharides of silkworm pupa and Cordyceps militaris described in the present invention also includes separating the prepared polysaccharides of silkworm pupa and Cordyceps militaris. The specific method is: prepare the prepared polysaccharides of silkworm pupa and Cordyceps militaris into a 5 mg / mL deionized water solution, and load it onto a diethylaminoethyl (DEAE)-52 cellulose chromatography column (30×400mm); after the column is balanced, first elute with deionized water, and then elute with NaCl solutions of different concentrations in sequence, control the flow rate to about 1 mL / min, collect the eluates respectively, and concentrate to 20% of the original volume; dialyze 3 times (molecular weight cutoff 7000Da, change deionized water every 12 hours), and freeze-dry the dialysate to obtain isolated polysaccharides of silkworm pupa and Cordyceps militaris;

[0014] Preferably, the concentration of the NaCl eluent is 0.1 to 0.5 mol / L, more preferably 0.1 mol / L;

[0015] Preferably, the elution is first performed with deionized water and then with a 0.1 mol / L NaCl solution. Under this condition, the antioxidant activity of the isolated polysaccharide of Cordyceps militaris is the highest, and the scavenging rate of DPPH free radicals reaches 98.66±2.22% at 0.5 mg / mL, which is significantly higher than other components (P<0.05 or P<0.01), and even slightly higher than the positive control L-ascorbic acid at the same concentration (97.04±1.43%, P>0.05).

[0016] According to the second aspect of the present invention, the present invention provides a silkworm pupa cordyceps isolated polysaccharide, wherein the total sugar content of the silkworm pupa cordyceps isolated polysaccharide is 92.15±1.68%, the protein content is 0.26±0.11%, the uronic acid content is 0.97±0.21%, and the sulfate content is 1.86±0.29%; the silkworm pupa cordyceps isolated polysaccharide is composed of arabinose (2.51%), galactose (5.97%), glucose (81.48%), xylose (2.53%) and mannose (7.51%); the silkworm pupa cordyceps isolated polysaccharide is a heterogeneous polysaccharide with an average molecular weight of 121.82kDa; the average particle size of the silkworm pupa cordyceps isolated polysaccharide is 576.1nm; the average potential of the silkworm pupa cordyceps isolated polysaccharide is -11.6mV; The surface of the silkworm pupa cordyceps isolated polysaccharide presents loose and irregular morphological characteristics, and is also dispersed with diamond or spherical particles of different sizes and shapes; the silkworm pupa cordyceps isolated polysaccharide not only has high antioxidant activity, but also has excellent moisture absorption and moisture retention properties; the silkworm pupa cordyceps isolated polysaccharide has a moisture absorption rate of 36.48±1.90% at 48h in a 43% humidity environment, which is significantly higher than the same type of commercially available moisturizer sodium alginate (22.95±2.14%, P<0.01); the silkworm pupa cordyceps isolated polysaccharide has a moisture retention rate of 85.67±2.66% at 48h in a dry environment, which is significantly higher than sodium alginate (76.9±4.43%, P<0.05) and the classic moisturizer glycerin (49.29±1.61%, P<0.01). The infrared spectrum of the silkworm pupa cordyceps isolated polysaccharide is basically as follows Figure 8 shown.

[0017] According to the third aspect of the present invention, the present invention provides the use of the isolated polysaccharide of silkworm pupa and Cordyceps militaris in the preparation of anti-aging and moisturizing skin care products; it can be used to prepare emulsions, creams, gels, films and other products.

[0018] The present invention has the following beneficial effects:

[0019] ① The total sugar content of the isolated polysaccharide of silkworm chrysalis and Cordyceps militaris provided by the present invention exceeds 90%, and the structural characteristics are clear, which is different from the crude polysaccharide of Cordyceps militaris disclosed in patent CN 105902442 A, and can be used for the development of precision targeted skin care products.

[0020] ② The isolated polysaccharide of silkworm chrysalis and Cordyceps militaris provided by the present invention has significant antioxidant activity. The scavenging rate of DPPH free radicals at a low concentration of 0.5 mg / mL reaches 98.66±2.22%. The scavenging effect of DPPH free radicals is higher than that of the Cordyceps militaris water extract disclosed in patent CN109864910 A, and slightly higher than L-ascorbic acid, which is recognized to have the highest antioxidant activity.

[0021] ③ The silkworm chrysalis cordyceps isolated polysaccharide provided by the present invention has excellent moisture absorption and moisture retention properties. The moisture absorption is significantly higher than the same type of moisturizing agent sodium alginate; the moisture retention is significantly higher than sodium alginate and the classic moisturizing agent glycerin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .DEAE-52 elution curve of Cordyceps militaris polysaccharide-80;

[0023] Figure 2 . The scavenging rate of different polysaccharide components isolated from Cordyceps militaris polysaccharide-80 on DPPH free radicals at 0.5 mg / mL (different letters above the error bars represent statistical differences);

[0024] Figure 3 .The moisture absorption performance of the isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris within 48 hours;

[0025] Figure 4 . Moisturizing properties of Cordyceps militaris polysaccharide-2 within 48 hours;

[0026] Figure 5 . Ion chromatogram of monosaccharide composition of polysaccharide-2 isolated from silkworm chrysalis (peak 1-arabinose; peak 2-galactose; peak 3-glucose; peak 4-xylose; peak 5-mannose);

[0027] Figure 6 . Molecular weight distribution of isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris;

[0028] Figure 7 . Particle size (A) and potential (B) distribution diagram of polysaccharide-2 isolated from silkworm chrysalis and Cordyceps militaris;

[0029] Figure 8 .Infrared spectrum of polysaccharide-2 isolated from silkworm chrysalis and Cordyceps militaris;

[0030] Fig. 9 .Scanning electron micrograph of polysaccharide-2 isolated from silkworm chrysalis. DETAILED DESCRIPTION

[0031] The dried fruiting bodies of Cordyceps militaris were purchased from Anguo Medicinal Materials Wholesale Market; the molecular weight was determined by Shimadzu's high performance liquid chromatograph, model LC-16; the monosaccharide composition was determined by Dionex's ion chromatograph, model ICS-5000; the infrared spectrum was determined by Bruker's infrared spectrometer, model TensorⅡ; the scanning electron microscope (SEM) was determined by HITACHI ultra-high resolution field emission scanning electron microscope, model Regulus 8100; the data of three parallel experiments were expressed as mean ± SD, and statistical tests were performed using one-way analysis of variance (ANOVA), and P < 0.05 indicated statistical differences.

[0032] Example 1 Preparation of Silkworm Cordyceps Polysaccharide

[0033] The dried fruiting bodies of silkworm pupa and Cordyceps militaris were crushed and passed through a 40-mesh sieve for later use; petroleum ether was added to degrease for 12 hours in the dark, and degreased silkworm pupa and Cordyceps militaris powder was obtained by vacuum filtration. 40g of degreased silkworm pupa and Cordyceps militaris powder was put into a beaker, and 1200mL of distilled water was added at a solid-liquid ratio of 1:30g / mL, and extracted at 80℃ for 2.5h, and filtered with gauze; 800mL of distilled water was added to the filter residue and extracted again at 80℃ for 1.5h. The two filtrates were combined and filtered under reduced pressure to obtain the water extract of silkworm pupa and Cordyceps militaris.

[0034] The extract was concentrated to 20% of the original volume, and then anhydrous ethanol was added to adjust the final ethanol concentration in the filtrate to 80% (v / v). The solution was precipitated at 4°C for 12 hours and the precipitate was collected by centrifugation. The precipitate was re-dissolved with an appropriate amount of distilled water to obtain a crude polysaccharide solution of Cordyceps militaris. Sevag reagent (chloroform: n-butanol = 4:1, v / v) with a volume of 1 / 4 of the crude polysaccharide solution of Cordyceps militaris was added, mixed and shaken for 15 minutes, and the bottom organic phase and the milky denatured protein in the middle layer were discarded after centrifugation. The above operation was repeated until no obvious protein was produced. The top layer of polysaccharide solution was taken out and freeze-dried to obtain Cordyceps militaris polysaccharide-80. The phenol-sulfuric acid method was used, and the glucose standard curve (Y = 12.611X + 0.0681, R 2 =0.9992) to calculate the total sugar content to be 84.07±3.69%.

[0035] Comparative Example

[0036] Refer to Example 1, except that the final concentration of ethanol in the filtrate is adjusted to 60% (v / v), and Cordyceps militaris polysaccharide-60 is precipitated, with a total sugar content of 71.25±2.04%. Other experimental steps are the same as in Example 1.

[0037] Refer to Example 1, except that the final concentration of ethanol in the filtrate is adjusted to 70% (v / v), and Cordyceps militaris polysaccharide-70 is precipitated, with a total sugar content of 65.73±2.18%. Other experimental steps are the same as Example 1.

[0038] Refer to Example 1, except that the final concentration of ethanol in the filtrate is adjusted to 90% (v / v), and Cordyceps militaris polysaccharide-90 is precipitated, with a total sugar content of 52.41±0.84%. Other experimental steps are the same as in Example 1.

[0039] By comparison, it can be seen that the total sugar content of the Cordyceps militaris polysaccharide (Cordyceps militaris polysaccharide-80) obtained by ethanol precipitation with a final concentration of 80% (v / v) is the highest, which is significantly higher than the Cordyceps militaris polysaccharides obtained by precipitation with other concentrations of ethanol (P<0.05 or P<0.01). The reason may be that lower concentrations of ethanol mainly precipitate polysaccharides with larger molecular weights; while higher concentrations of ethanol mainly precipitate polysaccharides and oligosaccharides with smaller molecular weights; when the ethanol concentration exceeds a certain threshold, more small molecular impurities will be precipitated, thereby reducing the relative content of total sugars in the polysaccharide. Since the total sugar content of Cordyceps militaris polysaccharide-80 is the highest (84.07±3.69%) and the impurities are the least, it is further separated.

[0040] Example 2 Separation of Silkworm Cordyceps Polysaccharide-80

[0041] The above-mentioned Cordyceps militaris polysaccharide-80 was prepared into a 5 mg / mL deionized water solution and loaded onto a diethylaminoethyl (DEAE)-52 cellulose chromatography column (30×400 mm). After the column was balanced, it was first eluted with deionized water, and then eluted with 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl solutions in sequence, with a flow rate of about 1 mL / min. The eluents were collected and the elution curve was drawn ( Figure 1 ).

[0042] like Figure 1 As shown, silkworm Cordyceps polysaccharide-80 is mainly composed of 5 components (components 1-5 correspond to deionized water and 0.1, 0.2, 0.3, 0.4, 0.5 mol / L NaCl solution elution), among which components 1 and 2 eluted with deionized water and 0.1 mol / L NaCl account for a large proportion. Components 1 to 5 were collected separately, concentrated to 20% of the original volume, and dialyzed 3 times (molecular weight cutoff 7000Da, deionized water was replaced every 12 hours). The dialyzate was freeze-dried to obtain silkworm Cordyceps isolated polysaccharide-1, silkworm Cordyceps isolated polysaccharide-2, silkworm Cordyceps isolated polysaccharide-3, silkworm Cordyceps isolated polysaccharide-4 and silkworm Cordyceps isolated polysaccharide-5.

[0043] Example 3 Comparison of Antioxidant Activity of Isolated Polysaccharides from Silkworm Cordyceps

[0044] The antioxidant activity of different isolated polysaccharides from Cordyceps militaris was evaluated using DPPH free radical as a model. Specifically, different isolated polysaccharides from Cordyceps militaris were prepared into 0.5 mg / mL distilled water solutions; 2 mL of the test solution was added to 2 mL of DPPH-ethanol solution (0.1 mmol / L), mixed evenly, and allowed to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. L-ascorbic acid (LAA) was used as a positive control; the clearance rate was calculated according to formula (1).

[0045] DPPH clearance rate (%) = [1-(A1-A2) / A3] × 100 Formula (1)

[0046] Among them, A1 refers to the absorbance of the isolated polysaccharide sample of Cordyceps militaris; A2 refers to the absorbance of the control group in which anhydrous ethanol is used instead of DPPH; A3 refers to the absorbance of the blank group in which distilled water is used instead of the sample.

[0047] like Figure 2 As shown in the figure, different isolated polysaccharides from silkworm chrysalis and Cordyceps militaris exhibit different scavenging activities against DPPH free radicals. Among them, the isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris eluted with 0.1mol / L NaCl had the highest scavenging rate against DPPH free radicals, reaching 98.66±2.22% at 0.5mg / mL, which was significantly higher than other isolated polysaccharides (P<0.05 or P<0.01), and slightly higher than LAA at the same concentration (97.04±1.43%, P>0.05). The above results show that isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris is the main antioxidant activity contributing component of polysaccharide-80 from silkworm chrysalis and Cordyceps militaris.

[0048] Example 4 Evaluation of the Moisture Absorption and Moisture Retention Properties of Silkworm Cordyceps and Cordyceps Sinensis Isolated Polysaccharide-2

[0049] The antioxidant activity of polysaccharide-2 isolated from Cordyceps militaris was the highest, so its hygroscopic and moisturizing potential was further evaluated.

[0050] The hygroscopicity evaluation is as follows: the Cordyceps militaris isolated polysaccharide-2 is placed in a vacuum drying oven, dried at 60°C to constant weight, and then 100 mg is taken out and placed in a sealed container pre-filled with a saturated sodium carbonate solution (RH is 43%); at room temperature, it is weighed at 4, 8, 12, 24, and 48 hours. Glycerol and sodium alginate are used as controls; the hygroscopicity is calculated according to formula (2).

[0051] Moisture absorption rate (%) = [(Wt-W0) / W0] × 100 Formula (2)

[0052] Wherein, Wt is the weight (mg) of the polysaccharide-2 isolated from silkworm chrysalis and Cordyceps militaris at a specified time; W0 is the weight (mg) of the polysaccharide-2 isolated from silkworm chrysalis and Cordyceps militaris before placement.

[0053] like Figure 3As shown in the data, under a humidity environment of 43%, the moisture absorption rate of the polysaccharide-2 isolated from silkworm pupa and Cordyceps sinensis gradually increased with time, reaching 36.48±1.90% at 48h. Although it was lower than glycerol (59.66±5.25%, P<0.01), it was significantly higher than the same type of commercially available moisturizer sodium alginate (22.95±2.14%, P<0.01).

[0054] The moisturizing evaluation is as follows: place the Cordyceps militaris polysaccharide-2 in a vacuum drying oven and dry it at 60°C to constant weight, then take out 100 mg and put it into a weighing bottle, mix it evenly with 1 mL of distilled water, and place it in a closed desiccator filled with color-changing silica gel; weigh it at room temperature at 4, 8, 12, 24, and 48 hours. Glycerol and sodium alginate are used as controls; calculate the moisturizing rate according to formula (3).

[0055] Moisture retention rate (%) = Hn / H0×100 Formula (3)

[0056] Wherein, H0 is the weight of water before being placed in the drying oven (mg); Hn is the weight of water at a specified time (mg).

[0057] like Figure 4 As shown in the data, under dry conditions, the moisture retention rate of the polysaccharide-2 isolated from silkworm pupa and Cordyceps militaris gradually decreased with time, falling to 85.67±2.66% at 48h, but was significantly higher than that of sodium alginate (76.9±4.43%, P<0.05) and glycerol (49.29±1.61%, P<0.01).

[0058] In summary, the Cordyceps militaris isolated polysaccharide-2 not only has good hygroscopic properties, but also has excellent moisturizing properties, both of which are superior to the same type of commercially available moisturizer sodium alginate.

[0059] Example 5 Structural characteristics of isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris

[0060] The total sugar content of polysaccharide-2 isolated from silkworm pupa Cordyceps militaris was determined by phenol-sulfuric acid method and was 92.15±1.68%; the protein content was determined by Coomassie Brilliant Blue method and was 0.26±0.11%; the uronic acid content was determined by m-hydroxybiphenyl method and was 0.97±0.21%; and the sulfate content was determined by barium chloride gelatin turbidimetry and was 1.86±0.29%.

[0061] Determination of monosaccharide composition: Weigh 5 mg of Cordyceps militaris polysaccharide-2, add 1 mL of 2M trifluoroacetic acid and hydrolyze at 120°C for 2 h. After cooling, dilute to 50 mL with distilled water. Instrument and chromatographic conditions: injection volume is 20 μL; Dion ICS-5000 ion chromatograph; chromatographic column is CarboPac PA20 (6.5 μm, 3 mm × 150 mm); detector is pulsed amperometric detector. Flow rate is 0.6 mL / min; 0-21 min mobile phase is: 97% water + 3% sodium hydroxide solution (250 mM); 21.1-30 min mobile phase is: 94% water + 2% sodium hydroxide solution (250 mM) + 4% sodium acetate solution (1 M); 30.1-50 min mobile phase is: 20% water + 80% sodium hydroxide solution (250 mM).

[0062] like Figure 5 As shown, by comparing with standard sugars, it was determined that the silkworm chrysalis Cordyceps militaris isolated polysaccharide-2 was mainly composed of arabinose (2.51%), galactose (5.97%), glucose (81.48%), xylose (2.53%) and mannose (7.51%).

[0063] Molecular weight determination: The polysaccharide-2 isolated from Cordyceps militaris was prepared into a 2 mg / mL deionized water solution. Instrument and chromatographic conditions: injection volume 20 μL; Shimadzu LC-16 high performance liquid chromatograph; chromatographic column Ultrahydrogel TM Linear (2μm, 7.8mm×300mm); the detector is a differential refractometer; the mobile phase is a 0.1M NaNO3 deionized water solution; the flow rate is 1.0mL / min. Dextran with different molecular weights is used as the standard sugar, and a third-order calibration curve is established based on the molecular weight and retention time.

[0064] like Figure 6 As shown, the molecular weight of the isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris is mainly concentrated at 4.23 kDa (peak 2), accounting for 95.02%, and another 4.91% is concentrated at 2399.17 kDa (peak 1), which is a non-uniform polysaccharide. Through calculation, it can be seen that the average molecular weight of the isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris is: 4.23 kDa × 95.02% + 2399.17 kDa × 4.91% = 121.82 kDa.

[0065] Particle size and potential analysis: The particle size and potential of Cordyceps militaris isolated polysaccharide-2 were determined using a MALVERN ZETASIZERNANOZS nanoparticle size and zeta potential analyzer.

[0066] like Figure 7 As shown in A, the average particle size of the isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris is 576.1 nm; Figure 7As shown in B, the average potential of the isolated polysaccharide-2 from Cordyceps militaris is -11.6 mV.

[0067] Infrared spectral analysis: 2 mg of dried Cordyceps militaris polysaccharide-2 was weighed and mixed with 100 mg of dried spectral grade potassium bromide, pressed into tablets, and then placed in a Bruker TensorⅡ infrared spectrometer for measurement in the scanning range of 4000-400 cm -1 .

[0068] like Figure 8 As shown, 3444cm -1 The strong absorption peak near 2926cm is caused by the stretching vibration of OH; -1 It is the stretching or bending vibration of CH; 1639cm -1 and 1400cm -1 They are the asymmetric stretching vibration and symmetric stretching vibration of C=O; 1078cm -1 The absorption peak is caused by the skeleton vibration of COC; 778cm -1 The absorption peak at is the characteristic peak of α-type glycosidic bond. Infrared spectrum further confirmed that the isolated polysaccharide-2 from Cordyceps militaris conforms to the structural characteristics of polysaccharide.

[0069] Scanning electron microscopy analysis: Hitachi Regulus 8100 field emission scanning electron microscope was used to observe the morphological characteristics of the isolated polysaccharide-2 from Cordyceps militaris. Before observation, the sample was treated with foil spraying using an ion sputtering instrument.

[0070] like Fig. 9 As shown, at a magnification of ×500, the surface of the silkworm pupa Cordyceps militaris isolated polysaccharide-2 presents a loose and irregular morphology, and there are also scattered diamond or spherical particles of different sizes and shapes.

[0071] Example 6 Preparation of Silkworm Cordyceps Isolated Polysaccharide-2 Emulsion

[0072] 0.5 g of isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris was dissolved in 10 mL of pure water (phase A), and then slowly added into 5 mL of perilla oil (phase B); after homogenization at 20000 r / min for 5 min, an emulsion of isolated polysaccharide-2 from silkworm chrysalis and Cordyceps militaris was obtained.

[0073] Example 7 Preparation of Silkworm Cordyceps Isolated Polysaccharide-2 Cream

[0074] Dissolve 0.5g of Cordyceps militaris polysaccharide-2, 0.1g of xanthan gum, and 1mL of butylene glycol in 10mL of pure water for later use (phase A); mix 5g of myristic acid glyceryl, 3g of PEG-100 stearic acid, and 1g of cetyl stearic acid for later use (phase B). Add phase A to phase B, heat and homogenize at 85°C for 10 minutes, then transfer to a mortar and grind to room temperature until the oil and water are not separated to obtain the Cordyceps militaris polysaccharide-2 cream.

[0075] Example 8 Preparation of Silkworm Cordyceps Isolated Polysaccharide-2 Gel

[0076] 0.5 g of Carbomer-940 was added to 50 mL of purified water for swelling and set aside; 0.5 g of Cordyceps militaris polysaccharide-2 was dissolved in 10 mL of purified water and slowly added to the Carbomer-940 matrix; then 2 mL of ethylparaben ethanol solution (3%, w / v) and 1 mL of triethanolamine were added, and the mixture was fully mixed to obtain a Cordyceps militaris polysaccharide-2 gel.

[0077] Example 9 Preparation of Silkworm Cordyceps Isolated Polysaccharide-2 Film

[0078] Dissolve 0.5g of Cordyceps militaris polysaccharide-2 in 10mL of pure water, add 0.5g of glucomannan, and stir well (phase A); dissolve 0.2g of sorbitol, 0.5g of glycerol, and 0.1g of vitamin C in 100mL of purified water, and stir well (phase B). While stirring, slowly add phase A to phase B, mix well to obtain a film of Cordyceps militaris polysaccharide-2.

[0079] 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 made without departing from the spirit and principles of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A polysaccharide isolated from silkworm chrysalis and Cordyceps militaris, wherein the polysaccharide isolated from silkworm chrysalis and Cordyceps militaris has a total sugar content of 92.15±1.68%, a protein content of 0.26±0.11%, a uronic acid content of 0.97±0.21%, and a sulfate content of 1.86±0.29%; The silkworm chrysalis cordyceps isolated polysaccharide is composed of arabinose (2.51%), galactose (5.97%), glucose (81.48%), xylose (2.53%) and mannose (7.51%); The silkworm chrysalis cordyceps isolated polysaccharide is a heterogeneous polysaccharide with an average molecular weight of 121.82 kDa; The average particle size of the isolated polysaccharide of silkworm chrysalis and Cordyceps militaris is 576.1 nm; The average potential of the isolated polysaccharide of silkworm chrysalis and Cordyceps militaris is -11.6 mV; The surface of the isolated polysaccharide from silkworm chrysalis and Cordyceps militaris presents a loose and irregular morphological feature, and is also dispersed with rhombus or spherical particles of different sizes and shapes; And / or, the infrared spectrum of the isolated polysaccharide from silkworm chrysalis and Cordyceps militaris is substantially as shown in FIG8 .

2. The isolated polysaccharide of silkworm chrysalis and Cordyceps militaris according to claim 1, wherein the scavenging rate of the isolated polysaccharide of silkworm chrysalis and Cordyceps militaris on DPPH free radicals is 98.66±2.22% at 0.5 mg / mL; And / or, the moisture retention rate of the silkworm chrysalis Cordyceps militaris isolated polysaccharide in a dry environment is 85.67±2.66% at 48 hours; And / or, under a 43% humidity environment, the moisture absorption rate at 48 hours is 36.48±1.90%.

3. An isolated polysaccharide of silkworm chrysalis and Cordyceps militaris according to claim 1 or 2, which is prepared by hot water extraction using defatted powder of silkworm chrysalis and Cordyceps militaris fruiting bodies as raw material; and the isolated polysaccharide of silkworm chrysalis and Cordyceps militaris is obtained by filtration, concentration, ethanol precipitation, deproteinization, ion exchange separation, dialysis, and freeze drying.

4. The method according to claim 3, wherein the method for preparing isolated polysaccharides from silkworm chrysalis and Cordyceps militaris comprises the following steps: The dried fruiting bodies of silkworm pupa and Cordyceps militaris were crushed and passed through a 40-mesh sieve for later use; petroleum ether was added to degrease in the dark for 12 hours, and degreased silkworm pupa and Cordyceps militaris powder was obtained by vacuum filtration; 40g of degreased silkworm pupa and Cordyceps militaris powder was put into a beaker, and 1200mL of distilled water was added at a solid-liquid ratio of 1:30g / mL, and the mixture was extracted at 80°C for 2.5 hours, and filtered with gauze; 800mL of distilled water was added to the filter residue and extracted again at 80°C for 1.5 hours; the two filtrates were combined and filtered under reduced pressure to obtain a water extract of silkworm pupa and Cordyceps militaris; the extract was concentrated to 20% of the original volume, and then anhydrous ethanol was added to adjust the final ethanol concentration in the filtrate to a certain concentration (v / v ), precipitate at 4℃ for 12h, collect the precipitate by centrifugation; re-dissolve the precipitate with appropriate amount of distilled water to obtain crude polysaccharide solution of Cordyceps militaris, add Sevag reagent (chloroform: n-butanol = 4:1, v / v) with 1 / 4 volume of crude polysaccharide solution of Cordyceps militaris, shake for 15min after mixing, discard the bottom organic phase and the middle layer of milky denatured protein after centrifugation, repeat the above operation until no obvious protein is produced; take out the top layer of polysaccharide solution, and freeze-dry to obtain Cordyceps militaris polysaccharide.

5. The method according to claim 4, wherein adjusting the final ethanol concentration in the filtrate to a certain concentration means that the final ethanol concentration in the filtrate is 60 to 90% (v / v), more preferably 80% (v / v).

6. The method according to claim 4 further comprises separating the prepared silkworm chrysalis and Cordyceps militaris polysaccharide; the specific method is: preparing the prepared silkworm chrysalis and Cordyceps militaris polysaccharide into a 5 mg / mL deionized water solution, loading it onto a DEAE-52 cellulose chromatography column (30×400 mm); after the column is balanced, eluting it with deionized water first, and then eluting it with NaCl solutions of different concentrations in sequence, controlling the flow rate to about 1 mL / min, collecting the eluates respectively, and concentrating them to 20% of the original volume; dialyzing 3 times, and freeze-drying the dialyzate to obtain the separated silkworm chrysalis and Cordyceps militaris polysaccharide.

7. The method according to claim 6, wherein the concentration of the NaCl eluent is 0.1-0.5 mol / L, more preferably 0.1 mol / L.

8. Use of the isolated polysaccharide from silkworm chrysalis and Cordyceps militaris according to claim 1 or 2 in the preparation of anti-aging and moisturizing skin care products.

9. The use according to claim 7, wherein the anti-aging and moisturizing skin care products are emulsions, creams, gels, and film products.

10. The use according to claim 9, wherein the preparation method of the emulsion, cream, gel or film product is: Dissolve 0.5 g of isolated polysaccharide from silkworm chrysalis and Cordyceps militaris in 10 mL of pure water, then slowly add into 5 mL of perilla oil; homogenize at 20000 r / min for 5 min to obtain an emulsion of isolated polysaccharide from silkworm chrysalis and Cordyceps militaris; Dissolve 0.5g of isolated polysaccharide from silkworm chrysalis and Cordyceps militaris, 0.1g of xanthan gum and 1mL of butylene glycol in 10mL of pure water to obtain phase A; mix 5g of myristic acid glyceryl, 3g of PEG-100 stearic acid and 1g of cetyl stearic acid to obtain phase B; add phase A to phase B, heat and homogenize at 85°C for 10min, and then grind in a mortar until the mixture is at room temperature and the oil and water are not separated, thereby obtaining the isolated polysaccharide cream from silkworm chrysalis and Cordyceps militaris; 0.5 g of Carbomer-940 was added to 50 mL of purified water for swelling and set aside; 0.5 g of Cordyceps militaris polysaccharide was dissolved in 10 mL of purified water and slowly added to the Carbomer-940 matrix; then 2 mL of ethylparaben ethanol solution (3%, w / v) and 1 mL of triethanolamine were added; and the mixture was thoroughly mixed to obtain a Cordyceps militaris polysaccharide gel; Dissolve 0.5 g of isolated polysaccharide from silkworm chrysalis and Cordyceps militaris in 10 mL of pure water, add 0.5 g of glucomannan, and stir well to obtain phase A; dissolve 0.2 g of sorbitol, 0.5 g of glycerol, and 0.1 g of vitamin C in 100 mL of purified water, and stir well to obtain phase B; while stirring, slowly add phase A into phase B, mix well to obtain isolated polysaccharide film from silkworm chrysalis and Cordyceps militaris.

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