Method for purely physically extracting sparassis crispa polysaccharide and application of sparassis crispa polysaccharide in skin care

Abstract

The invention discloses a method for purely physically extracting sparassis crispa polysaccharide and application of the sparassis crispa polysaccharide in skin care, and relates to a method for preparing high-purity sparassis crispa polysaccharide by using an amino silane modified silica gel material for physically adsorbing pigment of a crude polysaccharide solution and combining a membrane filtration method. The amino silane reagent modified silica gel material prepared by the invention is used for adsorbing and decolorizing crude polysaccharide, and protein and small molecular compounds in the crude polysaccharide are removed by combining a membrane filtration method, so that the sparassis crispa polysaccharide is refined. The method for purely physically extracting the sparassis crispa polysaccharide has the characteristics of simple process, suitability for factory continuous production, no organic solvent residue, high product purity, stronger antioxidant and skin repair activity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a method for physically extracting polysaccharides from Sparassis and application thereof in skin care. BACKGROUND

[0002] Sparassis is a precious fungus for both medicine and food, which contains abundant active polysaccharides, mainly β-glucan, accounting for 40-50% of the dry weight. A large number of literatures show that Sparassis β-glucan has various biological activities in anti-tumor, immune regulation, improvement of body hematopoietic function, promotion of wound healing, antioxidant and antibacterial activities, but has not been applied in skin care, especially in repair and anti-aging.

[0003] Polysaccharides, as an important class of biological macromolecules, have wide application value in the fields of food, medicine and cosmetics. High-purity polysaccharides are the key to the study of polysaccharide activity and application. However, the naturally extracted polysaccharides are often accompanied by the presence of pigment impurities, which not only affects the study of the structure-activity relationship of polysaccharides, but also has a negative impact on the biological activity of polysaccharides. Therefore, the decolorization treatment of polysaccharides becomes a necessary step in the polysaccharide purification process.

[0004] The existing polysaccharide decolorization mainly includes H2O2 oxidation chemical decolorization method and physical adsorption decolorization method. The H2O2 oxidation decolorization method mainly utilizes the strong oxidizing property of H2O2 to decompose pigments, but H2O2 also causes damage to the structure of polysaccharides while decomposing pigments, and the residual H2O2 also leaves a safety hazard for the application of Sparassis polysaccharides in food and cosmetics. The adsorption and desorption method is a physical decolorization method, which mainly utilizes the large specific surface area and rich void structure of the adsorbent to adsorb pigments for decolorization. The adsorbent includes activated carbon and macroporous resin. The activated carbon has a fast adsorption speed for pigments, but has poor selectivity, which easily leads to high loss rate of polysaccharides, and the activated carbon is difficult to separate from polysaccharides, resulting in the decrease of polysaccharide quality. The macroporous resin has good decolorization effect and low polysaccharide loss, but the adsorption time of the macroporous resin is long, and the adsorption equilibrium time is 2-4 h, which causes low decolorization efficiency and cannot meet the actual production requirements. Based on this, the present application constructs a kind of silica gel material modified by amino silane, which is used for the decolorization of crude polysaccharides from Sparassis, so as to achieve low polysaccharide adsorption and fast decolorization, realize the physical extraction of high-purity Sparassis polysaccharides, and reveal the application prospect of Sparassis polysaccharides extracted by physical adsorption method in skin care through in vitro antioxidant performance, zebrafish tail fin repair and zebrafish anti-inflammatory experiments. SUMMARY

[0005] In order to solve the above technical problems, the present application provides the following technical solutions:

[0006] The present application relates to a method for physically extracting polysaccharides from Sparassis and application thereof in skin care, which comprises the following steps:

[0007] Step one, preparation of adsorption material: put silica gel into a three-necked round flask, add dehydrated toluene, add amino / amine silane reagent, pass high-purity nitrogen as protective gas, mechanically stir under condensation reflux at 50℃ for 24h, after the reaction is completed, cool and filter, sequentially wash with toluene, 3 times of methanol, pre-dry in a 80℃ air oven, dry in a 80℃ vacuum oven;

[0008] Step two, extraction of Floralia polysaccharide: take dry Floralia, wash and soak until complete, prepare Floralia solution according to the mass ratio Floralia: water = 1: (10-40), crush into slurry with a homogenizer, adjust pH = 5 with NaOH, add cellulase, incubate at 50℃ for 2-5h, then increase the solution temperature to 95℃ for 10min to inactivate the cellulase;

[0009] Step three, impurity removal of Floralia polysaccharide: add 0.5%-3% diatomite and 0.5-3‰ modified silica gel to the Floralia slurry, stir uniformly for 1-10min, filter to remove insoluble impurities, collect the filtrate, repeat the filtration for 2-3 times without changing the diatomite and modified silica gel, to obtain clear colorless Floralia crude polysaccharide solution;

[0010] Step four, purification of Floralia polysaccharide: the clear colorless Floralia crude polysaccharide solution is subjected to microfiltration to remove small molecule compounds, to prepare Floralia polysaccharide solution with a purity of more than 90%, the solution is subjected to low-temperature vacuum drying and crushing, to obtain Floralia polysaccharide powder.

[0011] As a preferred technical scheme of the present application, the enzyme specific activity of the cellulase is ≥10000u / g, and the enzyme dosage is 1%-5% of the dry weight of Floralia

[0012] As a preferred technical scheme of the present application, the filter membrane for removing small molecule impurities is selected from 10KDa-0.2um PVDF hollow fiber membrane or 10nm-0.2um ceramic membrane.

[0013] As a preferred technical scheme of the present application, the silica gel material has a particle size of 40-60μm.

[0014] As a preferred technical scheme of the present application, the amino / amine-based silane reagent includes 3-(2-aminoethylamino) propyl triethoxysilane, 3-aminopropyl triethoxysilane, anilinomethyl triethoxysilane, diethylaminomethyl triethoxysilane, N-(6-aminohexyl) aminomethyl triethoxysilane, [3-(1,3-dimethylbutenyl) aminopropyl] triethoxysilane, bis[3-(triethoxysilyl) propyl] amine, triethoxy-3-(2-imidazolin-1-yl) propylsilane, gamma-aminopropyl methyl diethoxysilane, N-(beta-aminoethyl-gamma-aminopropyl) methyl dimethoxysilane, cyclohexylaminopropyl trimethoxysilane, N-methylaminopropyl dimethoxysilane, bis(3-(methylamino) propyl) trimethoxysilane.

[0015] As a preferred technical scheme of the present application, the amino / amine-based silane reagent is used in an amount of m 硅胶 :V 硅烷试剂 = 10:0.5-10:5.

[0016] As a preferred technical scheme of the present application, the enzyme specific activity of the cellulase is greater than or equal to 10000 u / g, and the enzyme is used in an amount of 1%-5% of the dry weight of the flower ball fungus.

[0017] As a preferred technical scheme of the present application, the filter membrane for removing small molecular impurities is selected from a hollow fiber membrane with a pore size of 10KDa-0.2um or a ceramic membrane with a pore size of 10nm-0.2um.

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

[0019] 1. The extraction method of the flower ball fungus polysaccharide and the application in skin care, by constructing a modified silica gel high-efficiency decolorizing agent, realizing rapid physical decolorization of the flower ball fungus crude polysaccharide, and not introducing chemical reagents in the extraction process of the flower ball fungus polysaccharide, the structure and activity of the polysaccharide are not damaged, and high-efficiency continuous production can be realized in industry.

[0020] 2. The physical extraction method of the flower ball fungus polysaccharide and the application in skin care, the flower ball fungus polysaccharide extracted by pure physical extraction has good antioxidant, repair and anti-inflammatory effects, and has good application prospect in antioxidant, anti-aging and repair cosmetic. DETAILED DESCRIPTION

[0021] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, and are used together with embodiments of the present application to explain the present application, and do not constitute a limitation on the present application. In the drawings:

[0022] Figure 1 is a scanning electron microscope image of 3-(2-aminoethylamino) propyl triethoxysilane modified silica gel;

[0023] Figure 2 is a Fourier infrared spectrum of 3-(2-aminoethylamino) propyl triethoxysilane modified silica gel;

[0024] Figure 3 is a graph of the decolorization rate and polysaccharide adsorption rate of a solution of crude polysaccharide of Sphaerocystis globosa on 3-(2-aminoethylamino) propyl triethoxysilane modified silica gel.

[0025] Figure 4 is a schematic diagram of the DPPH free radical scavenging capacity of different concentrations of Sphaerocystis globosa polysaccharides in a pure physical Sphaerocystis globosa polysaccharide extraction method and application in skin repair;

[0026] Figure 5 is a graph of the effect of different concentrations of Sphaerocystis globosa polysaccharides on tail fin growth (A) and area statistics (B) in a pure physical Sphaerocystis globosa polysaccharide extraction method and application in skin repair;

[0027] Figure 6 is a graph of the effect of different concentrations of Sphaerocystis globosa polysaccharides on the number of neutrophils on the surface of zebrafish skin. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present application will be described below with reference to the accompanying drawings, and it should be understood that the preferred embodiments described herein are intended to illustrate and explain the present application, and are not intended to limit the present application.

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] Embodiment 1

[0031] 20 g of silica gel was placed in a 150 mL three-necked round-bottom flask, 50 mL of dehydrated toluene was added, 1.7 mL of 3-(2-aminoethylamino) propyl triethoxysilane reagent was added, high-purity nitrogen gas was used as a protective gas, and mechanical stirring was used under reflux condensation at 50°C for 24 h. After the reaction was completed, cooling and filtration were performed, and 25 mL of toluene and 3 times 25 mL of methanol were used for washing, respectively. Pre-drying was performed in a 80°C air oven, and drying was performed in a 80°C vacuum oven.

[0032] After the dry C. japonica was cleaned and soaked completely, C. japonica solution was prepared according to the mass ratio of C. japonica : water = 1:40, and then broken into slurry by homogenizer. The pH value was adjusted to 5 by NaOH, 5% of 10000u / g cellulase of the dry weight of C. japonica was added, and the solution was incubated and stirred at 50℃ for 2h. Then the temperature of the solution was increased to 95℃ for 10min to inactivate the cellulase.

[0033] 1% diatomite and 1‰ modified silica gel were added into the C. japonica slurry solution and stirred uniformly for 10min. The insoluble impurities were removed by filtration, and the filtrate was collected. The filtration was repeated for 3 times without changing the diatomite and modified silica gel to obtain the clear and colorless C. japonica crude polysaccharide solution. The small molecule compounds were removed by 0.2um PVDF hollow fiber membrane filtration. The solution was dried at low temperature under vacuum and then crushed to obtain C. japonica polysaccharide powder. The yield of C. japonica polysaccharide was 13.5%, the polysaccharide content was 93.3%, the transmittance of 0.5% polysaccharide solution was 95.8%, and the viscosity was 261mPa·S.

[0034] Example 2

[0035] 20g of silica gel was placed in a 150mL three-necked round-bottom flask, 50mL of dehydrated toluene was added, 1.7mL of 3-(2-aminoethylamino)propyltriethoxysilane reagent was added, high-purity nitrogen gas was used as protective gas, and mechanical stirring was used under reflux condensation at 50℃ for 24h. After the reaction was completed, it was cooled and filtered, and then washed with 25mL of toluene and 3 times of 25ml of methanol. It was pre-dried in a 80℃ air oven and dried in a 80℃ vacuum oven.

[0036] After the dry C. japonica was cleaned and soaked completely, C. japonica solution was prepared according to the mass ratio of C. japonica : water = 1:40, and then broken into slurry by homogenizer. The pH value was adjusted to 5 by NaOH, 5% of 10000u / g cellulase of the dry weight of C. japonica was added, and the solution was incubated and stirred at 50℃ for 2h. Then the temperature of the solution was increased to 95℃ for 10min to inactivate the cellulase.

[0037] 3% diatomite and 2‰ modified silica gel were added into the C. japonica slurry solution and stirred uniformly for 5min. The insoluble impurities were removed by filtration, and the filtrate was collected. The filtration was repeated for 2 times without changing the diatomite and modified silica gel to obtain the clear and colorless C. japonica crude polysaccharide solution. The small molecule compounds were removed by 10KDa PVDF hollow fiber membrane filtration. The solution was dried at low temperature under vacuum and then crushed to obtain C. japonica polysaccharide powder. The yield of C. japonica polysaccharide was 19.6%, the polysaccharide content was 95.7%, the transmittance of 0.5% polysaccharide solution was 92.6%, and the viscosity was 181mPa·S.

[0038] Example 3

[0039] Put 20 g of silica gel in a 150 mL three-necked round-bottom flask, add 50 mL of dehydrated toluene, add 1.7 mL of 3-(2-aminoethylamino) propyl triethoxysilane reagent, pass high-purity nitrogen as a protective gas, mechanically stir at 50°C under reflux condensation for 24 h, cool and filter after the reaction is complete, wash with 25 mL of toluene, 3 times with 25 mL of methanol, pre-dry in a 80°C air oven, and dry in a 80°C vacuum oven;

[0040] After the dry C. globosum is completely washed and soaked, it is prepared into a C. globosum solution according to a mass ratio of C. globosum: water = 1:40, broken into a slurry with a homogenizer, adjusted to pH = 5 with NaOH, added with 5% of 50000 u / g of cellulase based on the dry weight of C. globosum, and incubated at 50°C for 2 h, then the temperature of the solution is increased to 95°C for 10 min to inactivate the cellulase;

[0041] 1% of diatomite and 2.5‰ of modified silica gel are added to the C. globosum slurry and stirred uniformly for 10 min, the insoluble impurities are removed by filtration, the filtrate is collected, and the filtration is repeated 3 times without replacing the diatomite and modified silica gel to obtain a clear and colorless C. globosum crude polysaccharide solution, the solution is filtered through a 10 nm ceramic membrane to remove small molecule compounds, and the solution is dried at low temperature under vacuum, pulverized to obtain C. globosum polysaccharide powder. The yield of C. globosum polysaccharide is 18.2%, the polysaccharide content is 90.2%, the transmittance of a 0.5% polysaccharide solution is 92.6%, and the viscosity is 184 mPa·S.

[0042] Example 4

[0043] Based on the above Examples 1-3, further descriptions are made.

[0044] The surface morphology of the adsorbent material (such as Figure 1 ) is analyzed by using an S-4800 field emission scanning electron microscope produced by Hitachi Company, Japan, with an acceleration voltage of 10 kV and a working current of 7 μA.

[0045] After modification, the surface of the PSA becomes rough and the particles are relatively loose. Such a wrinkled surface is conducive to increasing the adsorption sites on the surface of the silica gel and promoting the adsorption of pigment molecules.

[0046] Example 5

[0047] Based on the above Examples 1-3, further descriptions are made.

[0048] The infrared spectrum of the adsorbent material is tested by using a Thermo Nicolet 6700 infrared spectrometer, the sample is mixed and ground with KBr, and then pressed into a tablet for testing, as shown in Figure 2 By comparing the infrared spectra of the original silica gel and the modified silica gel, it can be found that the positions of the main characteristic peaks of the two are basically the same, wherein, the 3674.99 cm-1 The broad absorption peak at 2987.34 cm⁻¹ is attributed to the stretching vibrations of Si–OH and adsorbed water; -1 and 2901.23cm -1 The absorption peak at 1228.90 cm⁻¹ corresponds to the asymmetric and symmetric stretching vibrations of the C–H bond. -1 The peak at 1066.31 cm⁻¹ can be attributed to C–O stretching vibrations. -1 and 797.20cm -1 The strong absorption peaks at 892.8 cm⁻¹ correspond to the asymmetric and symmetric stretching vibrations of Si–O–Si, respectively. -1 The absorption peak at 892.8 cm⁻¹ is attributed to the Si–OH bending vibration, indicating that the modification process did not disrupt the basic framework structure of silica. Notably, the absorption peak at 892.8 cm⁻¹ is... -1 The Si–OH bending vibration peak at the modified silica gel surface weakens after modification. This is mainly because the Si–OH groups on the silica gel surface undergo a condensation reaction with 3-(2-aminoethylamino)propyltriethoxysilane to form Si–O–Si bonds, resulting in a decrease in the number of free Si–OH groups. This indicates that the aminosilane reagent has been successfully grafted onto the silica gel surface.

[0049] Example 6

[0050] Further description is provided based on the above embodiments 1-3.

[0051] Characterization of 3-(2-aminoethylamino)propyltriethoxysilane-modified silica gel materials and their adsorption effects on pigments and polysaccharides.

[0052] Decolorization rate: A certain amount of modified PSA was added to the *Hydrangea macrophylla* polysaccharide solution, and the mixture was then gently stirred. After stirring for 5 minutes, the mixture was centrifuged (8000 r·min). -1 (10 min) Remove modified PSA, collect the supernatant for analysis, and compare the absorbance of different amounts of PSA before and after decolorization of *Hydrangea hyacinthia* polysaccharide using a UV-Vis spectrophotometer at 420 nm. Calculate the decolorization rate of *Hydrangea hyacinthia* polysaccharide according to the formula.

[0053] Decolorization rate = (A O -A e ) / A e ×100% (2-2)

[0054] Where A0 and A e The values ​​are the absorbance values ​​of the crude polysaccharide solution of *Hydrangea macrophylla* before and after decolorization at 420 nm.

[0055] Polysaccharide adsorption rate: Accurately weigh different amounts of modified PSA and add them to the crude polysaccharide solution of *Hydrangea macrophylla*, stir magnetically at 420 rpm for 5 min, and centrifuge at 8000 rpm. -1, 10 min) to remove the modified PSA, and then the content of the polysaccharide was analyzed.

[0056] The content of the polysaccharide in the solution of the Gloeostereum incarnatum polysaccharide before and after decolorization was determined at 490 nm by the phenol-sulfuric acid method using glucose as a standard, and the determination was performed in triplicate. Finally, the adsorption rate of the polysaccharide was calculated according to the following formula based on the concentrations of the Gloeostereum incarnatum polysaccharide solution before and after decolorization.

[0057] Polysaccharide adsorption rate = (C o -C t ) / C o × 100% (2-1)

[0058] Wherein: C0and C t are the contents of the Gloeostereum incarnatum polysaccharide before and after decolorization, respectively.

[0059] Figure 3 It can be seen that the decolorization rate of the Gloeostereum incarnatum polysaccharide solution increases with the increase of the amount of the adsorbent, and the adsorption rate of the polysaccharide also increases. The decolorization rate of the modified silica gel is higher than that of the silica gel raw material, and the adsorption rate of the polysaccharide of the modified silica gel is lower than that of the silica gel raw material at a high usage amount, which indicates that the amino groups of the modified silica gel can increase the adsorption of the pigment, but reduce the adsorption of the polysaccharide. Considering the decolorization efficiency (> 75%) and the polysaccharide retention rate (< 15%), the usage amount of 0.15% of the modified silica gel is selected as the optimal decolorization condition. This selection not only ensures good decolorization effect, but also maximizes the retention of the active ingredients of the polysaccharide.

[0060] Example 7

[0061] Based on the above Examples 1-3, the skin care effect of the Gloeostereum incarnatum polysaccharide is further described. The in vitro antioxidant performance of the Gloeostereum incarnatum polysaccharide, the specific steps and results are as follows:

[0062] In the process of removing DPPH free radicals, first, Gloeostereum incarnatum polysaccharide solutions of different concentrations obtained by different extraction methods, the concentration range includes 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL; Then select 0.1 mM DPPH methanol solution, add 50 μL of polysaccharide solution to 150 μL of DPPH solution, and place it in a dark environment at 25°C for 30 minutes. The absorbance of the reaction system was measured at 517 nm by using a microplate reader, and the absorbance was used to reflect the removal of DPPH free radicals; After measuring the absorbance, ascorbic acid (Vc) was used as a positive control, and the removal rate of DPPH free radicals was calculated according to the following formula:

[0063]

[0064] Wherein, Q(DPPH) is the DPPH free radical scavenging rate of the *Hydrangea macrophylla* polysaccharide solution, A1 is the absorbance of the *Hydrangea macrophylla* polysaccharide solution in DPPH free radical scavenging, A2 is the absorbance of methanol instead of DPPH, and A3 is the absorbance of the control solution (methanol instead of *Hydrangea macrophylla* polysaccharide).

[0065] To better demonstrate the advantages of this method in maintaining the activity of *Hydrangea macrophylla* polysaccharides, *Hydrangea macrophylla* polysaccharides decolorized with H2O2 were selected for comparison to assess their antioxidant activity in scavenging DPPH free radicals.

[0066] The test results of DPPH free radical scavenging ability are as follows: Figure 4

[0067] The DPPH free radical scavenging assay is widely recognized as a method for evaluating the in vitro antioxidant activity of natural substances. Figure 4 As can be seen, the free radical scavenging rate of DPPH increases with the increase of polysaccharide concentration, and the *Hydrangea hydrangea* polysaccharide modified by PSA physical adsorption using this patent has a better ability to scavenge DPPH free radicals than the polysaccharide decolorized by H2O2 chemical method.

[0068] Example 8

[0069] Based on Examples 1-3 above, the skin care effects of *Hydrangea macrophylla* polysaccharide are further described. The specific steps and results of the *Hydrangea macrophylla* polysaccharide's repair effect on zebrafish tail fins are as follows:

[0070] Healthy, malformed, and normally developed AB strain zebrafish at 48 hpf were selected. After anesthetizing the zebrafish in 15 mL of pure water with 10 μL of anesthetic solution, the embryos were dissected using the posterior segment of the pigmentation gap on the ventral side of the caudal fin as a reference. The caudal fin was transversely cut under a stereomicroscope using a sterile scalpel. The zebrafish were then immediately transferred to clean culture water to recover their vitality and then grouped into 6-well plates with 30 zebrafish per well. A model control group, a sample group, and a positive control group were set up. The model control group received 4 mL of culture solution; the sample group received 4 mL of *Hydrangea macrophylla* polysaccharide solution decolorized with modified silica gel adsorbent, with concentration gradients of 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL; and the positive control group received 4 mL of 1.0 mg / mL *Rehmannia glutinosa* culture solution. The plates were covered and incubated at (28.5±1)℃ for 48 hpf. Then, the zebrafish tail fin was photographed under a stereomicroscope, and the zebrafish images were analyzed using the image analysis software ImageJ.

[0071] Zebrafish has a high similarity of up to 87% with the human genome, its skin structure and function are highly similar to the human body, and it has the advantages of small individual differences, short development cycle, transparent embryos easy to observe, and lower test cost, and has become a model animal for evaluating the efficacy of cosmetic raw materials. Based on the repair efficacy of zebrafish caudal fin growth area of spore polysaccharide, as shown in Figure 5 Figure 5 It can be seen from Figure 5 (A) that spore polysaccharide has good repair efficacy on zebrafish caudal fin, and the repair effect is continuously enhanced with the increase of spore polysaccharide concentration. According to the area statistics of zebrafish caudal fin growth, as shown in (B), the spore polysaccharide extracted by the adsorbent synthesized by the present patent has good repair efficacy, and the repair effect of 1.0 mg / mL spore solution is comparable to that of the rehmannia solution control group, and the repair effect is proportional to the polysaccharide concentration. It shows that the spore polysaccharide extracted by the method of the present patent has good application prospect in the field of anti-oxidation, anti-aging and repair efficacy cosmetics.

[0072] Example 9

[0073] Based on the above examples 1-3, the skin care effect of spore polysaccharide is further described. The specific steps and results of the anti-inflammatory performance of spore polysaccharide on zebrafish caudal fin are as follows:

[0074] Healthy, non-malformed, normally developed CZ13 neutrophil green fluorescent labeled zebrafish were selected for anti-inflammatory experiments, and sodium dodecyl sulfate (SLS) was used to induce zebrafish epidermal inflammation. Blank control group, model group, sample group and positive control group were set up. The blank control group was directly cultured with 4 mL E3 culture solution; the model group was added with 4 mL 60 ug / mL sodium dodecyl sulfate (SLS) culture solution; the sample group was added with 4 mL 60 ug / mL SLS and 1 mL spore polysaccharide solution treated by the new adsorbent decolorization, and the concentration gradient of the spore polysaccharide solution was set as: 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL; the positive control group was added with 4 mL 60 ug / mL SLS and 1 mL 0.0625 mg / mL potassium glycyrrhizinate mixed culture solution. The zebrafish were transferred to a 6-well plate, 30 zebrafish were placed in each well, the culture plate cover was covered and wrapped with aluminum foil paper, and incubated at 28.5±1℃ in the dark for 24 h. The whole zebrafish was placed under a body fluorescence microscope for photography, and the abdominal skin surface area of the zebrafish was selected for statistics of the number of neutrophils on the skin surface.

[0075] In the zebrafish model, a significant increase in the number of neutrophils is an important marker of inflammatory response, and the effect of anti-inflammatory drugs can be effectively evaluated by inhibiting the aggregation and migration of neutrophils. Figure 6 The fluorescence neutrophil number statistics chart of zebrafish skin surface of different concentrations of spore polysaccharide is shown inFigure 6 It can be seen that with the increase of the concentration of pure polysaccharide of Sparassis, the number of neutrophils on the surface of zebrafish skin shows a significant downward trend (P<0.05), indicating that the pure polysaccharide of Sparassis has a significant inhibitory effect on inflammation of zebrafish. Compared with the blank group, the number of neutrophils on the surface of zebrafish skin at 0.5 mg / mL, 1.0 mg / mL and 1.5 mg / mL of the pure polysaccharide of Sparassis is reduced by 2.23%, 8.19% and 34.52% respectively, indicating that the anti-inflammatory ability of the polysaccharide of Sparassis on zebrafish is enhanced with the increase of its concentration, and the inhibitory effect on inflammation of zebrafish is the most significant when the concentration of the pure polysaccharide of Sparassis is 1.5 mg / mL (P<0.05).

[0076] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for purely physical extraction of polysaccharides from *Hydrangea macrophylla*, characterized in that, It includes the following steps: Step 1: Place 20g of silica gel in a 150mL three-necked round-bottom flask, add 50mL of dehydrated toluene, add 1.7mL of amino / aminosilane reagent, purge with high-purity nitrogen as a protective gas, and reflux at 50℃ for 24h under mechanical stirring. After the reaction is complete, cool and filter, wash successively with 25mL of toluene and 25mL of methanol three times, pre-dry in an 80℃ forced-air oven, and dry in an 80℃ vacuum oven. Step 2: After thoroughly cleaning and soaking the dried *Hydrangea macrophylla* fungus, prepare a *Hydrangea macrophylla* solution by mixing it with water at a mass ratio of 1:(10-40). After homogenizing the solution into a slurry, adjust the pH to 5 with NaOH. Add 1% to 5% (by weight of *Hydrangea macrophylla*) of cellulase (enzyme activity 10-50 u / mg), keep the solution at 50℃ and stir for 2-5 hours. Then, raise the solution temperature to 95℃ and maintain it for 10 minutes to inactivate the cellulase. Step 3: Add 0.5% to 3% diatomaceous earth and 0.5-3‰ of the modified silica gel synthesized in this patent to the *Hydrangea spores* slurry, stir evenly for 1 to 10 minutes, filter to remove insoluble impurities, collect the filtrate, and repeat the filtration 2 to 3 times without replacing the diatomaceous earth and modified silica gel to obtain a clear and colorless *Hydrangea spores* crude polysaccharide solution. Step 4: The clear and colorless crude polysaccharide solution of *Hydrangea macrophylla* is microfiltered to remove small molecule compounds, thus preparing a polysaccharide solution with a purity of over 90%. The solution is then dried and pulverized under low temperature vacuum to obtain *Hydrangea macrophylla* polysaccharide powder.

2. The method for purely physical extraction of *Hydrangea macrophylla* polysaccharides according to claim 1, characterized in that, The decolorizing material is an amino-modified silica gel.

3. The method for extracting polysaccharides from *Hydrangea macrophylla* using purely physical extraction according to claim 1, characterized in that... The amino / aminosilane reagent includes 3-(2-aminoethylamino)propyltriethoxysilane, 3-aminopropyltriethoxysilane, anilinemethyltriethoxysilane, diethylaminomethyltriethoxysilane, N-(6-aminohexyl)aminomethyltriethoxysilane, [3-(1,3-dimethylbutenyl)aminopropyl]triethoxysilane, bis[3-(triethoxysilyl)propyl]amine, triethoxy-3-(2-imidazolin-1-yl)propylsilane, γ-aminopropylmethyldiethoxysilane, N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane, cyclohexylaminopropyltrimethoxysilane, N-methylaminopropyldimethoxysilane, and bis(3-(methylamino)propyl)trimethoxysilane. The amount of amino / aminosilane reagent used is m. 硅胶 V 硅烷试剂 =10:0.5-10:

5.

4. The method for purely physical extraction of *Hydrangea macrophylla* polysaccharides according to claim 1, characterized in that, The filter membrane for removing small molecule impurities is a hollow fiber membrane of 10 kDa-0.2 μm or a ceramic membrane of 10 nm-0.2 μm. The specific activity of cellulase is ≥10000 u / g, and the amount of enzyme used is 1%-5% of the dry weight of *Hydrangea macrophylla*.

5. The application of the *Hydrangea macrophylla* polysaccharide prepared according to claims 1-4 in skin care for anti-aging, anti-oxidation and repair.

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