Sparassis crispa extract as well as preparation and application thereof

Abstract

The invention belongs to the technical field of daily cosmetics, and particularly relates to a preparation method and application of a sparassis crispa extract. The sparassis crispa extract is subjected to water extraction and enzymolysis with a specific enzyme such as beta-galactosidase, so that the transfer rate of active ingredients such as polysaccharide is remarkably increased. After a specific type of macroporous resin is subsequently adopted for purification, the Gardner chromatic value of active ingredients such as polysaccharide is obviously reduced, in addition, the prepared sparassis crispa extract shows the effects of inhibiting inflammatory factor secretion, keratinocyte migration and collagen loss in a cell experiment, wound healing and repairing are facilitated, and the application prospect is wide. A new thought is provided for preparing products such as skin care and beauty makeup products with post-medical repair and barrier repair effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of daily-use cosmetics, and particularly relates to a preparation method and application of Sparassis latifolia extract Background Art

[0002] Sparassis belongs to Basidiomycetes, Sparassidaceae, and Sparassis (Sparassis crispa Fr.). There are many varieties of Sparassis, also known as Hydnum repandum and cauliflower mushroom. As of January 8, 2024, Index fungorum (website link: http: / / www.indexfungorum.org) records 29 names of Sparassis. The fruiting body of Sparassis is formed by a short and thick stalk and petal branches on the stalk. The petal-like pieces are similar to ginkgo leaves, with thin and uneven edges. It is fleshy in the early growth stage, tough in the later stage, and hard and brittle when old. The fruiting body is annual, mostly white, dirty white or light yellow. Because its overall shape resembles a hydrangea flower, it is also known as white hydrangea. The appearance, color and nutritional components of Sparassis also vary according to factors such as the origin and climate conditions of Sparassis

[0003] Sparassis is an edible and medicinal fungus. Sparassis mostly grows in pine forests or at the base of pine trees. Its wild resources are very scarce. Generally, wild wood-rotting fungi mostly grow in shady places or environments with little sunlight, while Sparassis requires more than 10 hours of sunlight per day and is the only "sunshine mushroom" so far. Regarding the origin of Sparassis in China, the early widely accepted view was that Sparassis in China was Sparassis crispa (Wulf.) Fr. However, with further research, it was found that Sparassis collected in East Asia was very different from Sparassis in Europe in morphological characteristics, so Sparassis originating from Northeast China was named Sparassis latifolia

[0004] As of February 2025, only the extract of Sparassis crispa is included in the List of Names of Raw Materials Used in Cosmetics (2021 Edition), and the extract of Sparassis latifolia is not included. However, with the booming application for new cosmetic raw materials, it will provide new applications for Sparassis latifolia in cosmetics

[0005] Among numerous light medical aesthetic projects, botulinum toxin and hyaluronic acid are the two most mature categories in the anti-aging field. However, in recent years, with the approval of some new materials, more different materials have entered the track in the field of light medicine, bringing more choices to consumers, such as polycaprolactone (PCL), poly-L-lactic acid (PLLA), and collagen. Everything has two sides, and light medical aesthetics is no exception. On the one hand, it opens the skin barrier, enabling active ingredients to easily enter the skin and exert their effects. However, on the other hand, the damage to the skin barrier increases the risk of invasion by bacteria, viruses, etc. Post-operative repair products further repair the damaged skin, avoiding potential post-operative risks.

[0006] Therefore, there is an urgent need to provide a new cosmetic and skincare product derived from natural materials and applicable to post-operative repair in medical aesthetics. Summary of the Invention

[0007] The present invention provides an extract of Sparassis latifolia applicable to post-operative repair in medical aesthetics and its application.

[0008] In the first aspect of the present invention, there is provided an extract of Sparassis latifolia, which includes fucose (Fuc), galactose (Gal), glucose (Glc), and mannose (Man), wherein the molar mass percentage of glucose in the total polysaccharide component of the extract is 80 - 98%.

[0009] In another preferred embodiment, the extract of Sparassis latifolia is a polysaccharide extract.

[0010] In another preferred embodiment, the molar mass ratio of glucose in the total polysaccharide component of the extract is 85 - 95%.

[0011] In another preferred embodiment, the extract of Sparassis latifolia further has one or more of the following characteristics: (1) The molar mass percentage of fucose in the total polysaccharide component is 1 - 5%, preferably 2 - 3%; (2) The molar mass percentage of galactose in the total polysaccharide component is 3 - 8%, preferably 5 - 6%; (3) The molar mass percentage of mannose in the total polysaccharide component is 2 - 6%, preferably 2 - 3%; (4) The average molecular weight of the extract is 1 kDa - 5 kDa, preferably 2 kDa - 4 kDa, more preferably 2.5 - 3.5 kDa; and / or (4) The Gardner color value of the extract ≤ 5, preferably ≤ 4.

[0012] In a second aspect of the present invention, there is provided a method for preparing the Sparassis latifolia extract as described in the first aspect of the present invention, the method comprising the steps of: (1) Extraction: Using water as a solvent, enzymatically hydrolyze the Sparassis latifolia raw material, then inactivate the enzyme, filter, and concentrate to obtain a crude extract; (2) Purification: Purify and decolorize the crude extract with macroporous resin, collect the eluate, concentrate and dry to obtain the Sparassis latifolia extract; Wherein in the step (1), the enzyme used for enzymatic hydrolysis is selected from the group consisting of: β-glucanase, β-galactosidase, xylanase, amylase, and cellulase; Wherein the macroporous resin in the method is selected from the group consisting of: AB-8, D101, HP20, LS-109D, LS-305, D301T, D941, D900, and LS-308.

[0013] In another preferred example, the Sparassis latifolia raw material in the method is the Sparassis latifolia fruit body.

[0014] In another preferred example, the amount of macroporous resin used in the method is 1 to 4 times the mass of the crude extract, preferably 1 to 2 times.

[0015] In another preferred example, in the step (2), the elution is carried out with deionized water.

[0016] In another preferred example, the enzyme in the method is β-galactosidase.

[0017] In another preferred example, the amount of enzyme used in the method is 2 to 8% of the mass of the raw Sparassis latifolia, preferably 4 to 6%.

[0018] In another preferred example, the polysaccharide transfer rate in the method is ≥115%, preferably ≥140%, more preferably ≥160%.

[0019] In another preferred example, the macroporous resin in the method is LS-109D.

[0020] In another preferred example, the polysaccharide retention rate in the method is ≥30%, preferably ≥35%, more preferably ≥30%.

[0021] In another preferred example, the polysaccharide retention rate in the method is calculated by the following formula: Polysaccharide retention rate % = (polysaccharide content in the effluent + polysaccharide content in the effluent from the water-washed sample loaded) / crude polysaccharide content * 100% (Formula I).

[0022] In another preferred example, the extraction conditions in the method have one or more characteristics selected from the group consisting of: (1) The enzymatic hydrolysis temperature is 40 to 60 °C, preferably 50 to 60 °C; (2) The liquid ratio of water to raw materials is 5 - 40:1 mL / g, preferably 15 - 25:1 mL / g; (3) The enzymolysis time is 1 - 5 h, preferably 1 - 2 h; (4) The inactivation temperature is 70 - 120 °C, preferably 85 - 120 °C; and / or (5) The number of enzymolysis times is 1 - 5 times, preferably 2 times.

[0023] In another aspect of the present invention, there is provided the use of Sparassis crispa (Wulf.) Fr. extract in cosmetics.

[0024] In another preferred example, the Sparassis crispa extract is Sparassis latifolia extract.

[0025] In another preferred example, the Sparassis latifolia extract is prepared by the method described above.

[0026] In another preferred example, the cosmetic is a medical aesthetic repair cosmetic.

[0027] In the third aspect of the present invention, there is provided that the use of the Sparassis latifolia extract as described in the first aspect of the present invention is for preparing a daily product or a daily chemical product with the efficacy of medical aesthetic post - repair and / or barrier repair.

[0028] In another preferred example, the product further has one or more characteristics selected from the following group: (1) Inhibiting the secretion of inflammatory factors; (2) Repairing skin scratch injury; (3) Promoting keratinocyte migration; and / or (4) Reducing collagen loss.

[0029] In the fourth aspect of the present invention, there is provided a composition, which comprises: (1) the Sparassis latifolia extract as described in the first aspect of the present invention; and (2) additional components that can be used in daily chemical products.

[0030] In another preferred example, the dosage of the Sparassis latifolia extract in the composition is 0.05 - 99 wt%.

[0031] It should be understood that within the scope of the present invention, the above - mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1Shows the molecular weight test spectrum of Sparassis latifolia polysaccharide.

[0033] Figure 2 Shows the ion chromatogram of monosaccharide standard.

[0034] Figure 3 Shows the ion chromatogram of monosaccharide test of Sparassis latifolia polysaccharide.

[0035] Figure 4 Shows the flow chart for determining the maximum safe concentration of the sample in Functional Example 1.

[0036] Figure 5 Shows the effect of Sparassis latifolia extract on the viability of macrophage Raw264.7 cells in Functional Example 1.

[0037] Figure 6 Shows the flow chart for determining the effect of the sample on the secretion of TNF-α and IL-6 by LPS-induced macrophage Raw264.7 cells in Functional Example 1.

[0038] Figure 7 Shows the effect of Sparassis latifolia extract on TNF-α in Functional Example 1.

[0039] Figure 8 Shows the effect of Sparassis latifolia extract on IL-6 in Functional Example 1.

[0040] Figure 9 Shows the flow chart for determining the maximum safe concentration of the sample in Functional Example 2.

[0041] Figure 10 Shows the effect of Sparassis latifolia extract on the viability of keratinocytes in Functional Example 2.

[0042] Figure 11 Shows the experimental flow chart for determining the effect of Sparassis latifolia extract on cell scratch in Functional Example 2.

[0043] Figure 12 Shows the migration rate of Sparassis latifolia extract on keratinocytes in Functional Example 2.

[0044] Figure 13 Shows the scratch map of Sparassis latifolia extract on keratinocytes in Functional Example 2.

[0045] Figure 14 Shows the flow chart for determining the maximum safe concentration of the sample in Functional Example 3.

[0046] Figure 15 Shows the effect of Sparassis latifolia extract on the viability of fibroblasts in Functional Example 3.

[0047] Figure 16 Shows the flow chart for measuring the effect of the sample on collagen in the efficacy implementation case 3.

[0048] Figure 17 Shows the effect of Sparassis latifolia extract on type I collagen in the efficacy implementation case 3.

[0049] Figure 18 Shows the effect of Sparassis latifolia extract on type III collagen in the efficacy implementation case 3. Detailed implementation manner

[0050] Through extensive and in-depth research and a large number of experimental screenings, the inventor of the present invention unexpectedly discovers for the first time that Sparassis crispa extract can be applied to the cosmetic field, and the preferred example is Sparassis latifolia extract. The preparation method of the Sparassis latifolia extract includes: water extraction, β-galactosidase enzymolysis and macroporous resin purification. Compared with the existing technologies such as ordinary water extraction, this method significantly improves the transfer rate of active ingredients such as polysaccharides and significantly reduces the Gardner color number of polysaccharides. In the present invention, it is also found that Sparassis latifolia extract can inhibit the secretion of inflammatory factors, the migration of keratinocytes and the loss of collagen, providing new ideas for the preparation of skin care or beauty products with the functions of post-medical aesthetic repair and barrier repair. On this basis, the inventor of the present invention has completed the present invention.

[0051] Terms As described herein, "Sparassis latifolia extract", "Sparassis latifolia polysaccharide" and "Sparassis latifolia polysaccharide extract" can be used interchangeably, and all refer to the extract composed of 4 monosaccharides, namely fucose (Fuc), galactose (Gal), glucose (Glc) and mannose (Man). In the preferred embodiment, it is the extract obtained in Example 15.

[0052] Preparation method of Sparassis latifolia extract 1. Extraction: Weigh the raw material Sparassis latifolia, 10 times (feed amount mass KG / solvent volume L) of water, 5% of the feed amount mass of β-galactosidase, enzymolyze at 40°C - 55°C for 2 h; then heat to boiling (85°C - 100°C) for extraction 2 times; filter; concentrate under reduced pressure to recover water (55°C - 80°C, vacuum pressure 0.060 - 0.080 Mpa) to obtain 1-fold thick extract; 2. Purification: Purify the thick extract with 2-fold feed amount LS-109D macroporous resin. After mixing the thick extract with 1-fold volume of water, load the sample, first wash with 4 BV of water, and collect the loaded sample effluent and washing solution.

[0053] 3. Concentration: The collected sample solution is concentrated to obtain the collected and concentrated sample effluent, and the washing solutions are combined to obtain an extract; the Sparassis latifolia extract powder is obtained by vacuum drying or freeze drying. The extract can also be dispersed in solvents such as 1,2-propanediol, 1,3-propanediol, butanediol and glycerol to form a Sparassis latifolia extract solution.

[0054] The Sparassis latifolia extract powder or extract solution can be further prepared into daily chemical or daily use products.

[0055] Sparassis latifolia extract and wound healing and repair Wound healing and repair is a complex and dynamic process, generally divided into four stages: bleeding and hemostasis, inflammation, proliferation phase and tissue remodeling phase. The stages are highly coordinated.

[0056] Bleeding and hemostasis stage: Platelets bind to fibrin to form a fibrin clot, causing blood coagulation and inhibiting local bleeding. In addition, activated platelets can release various cytokines, such as PDGF, CXCL4, TGF-β, IGF, etc., to promote the subsequent injury repair process.

[0057] Inflammation stage: Neutrophils and macrophages are the main effector cells in the inflammation stage. These immune cells release various cytokines and chemokines to recruit cells in the blood and at the injury margin into the wound local area; at the same time, the secreted cytokines can also further promote the proliferation of cells at the injury site, but if the inflammatory factors are too high, phenomena such as swelling and slow healing will always occur.

[0058] Proliferation: Angiogenesis is the key to wound repair. Macrophages and damaged endothelial cells release FGF-2 and VEGF to promote angiogenesis. Growth factors such as EGF and TGF-β stimulate the proliferation and migration of keratinocytes at the injury margin, enabling re-epithelialization of the damaged dermis. After the re-epithelialization process is completed, the keratinocytes differentiate and the epidermal barrier function is restored again. The cell proliferation can be observed through the scratch healing rate.

[0059] Tissue remodeling phase: The granulation tissue formed during the early repair process is gradually replaced by the newly formed collagen-rich dermal matrix. Normal skin tissue is mainly composed of type III collagen forming a basket-weaving structure, while the damaged area is remodeled into thicker type I collagen and forms a dense parallel structure.

[0060] The Sparassis latifolia extract can exert its efficacy in all three stages of the late wound healing and repair process. In the inflammation stage, it can inhibit the secretion of inflammatory factors and prevent wound swelling and inflammation. In the proliferation stage, it can accelerate the migration of keratinocytes and promote the restoration of the epidermal barrier function. In the tissue remodeling stage, it can reduce the loss of collagen and help the tissue to rapidly remodel. Therefore, the Sparassis latifolia extract described in this application has opened up a new idea for the preparation of post-medical aesthetic repair products derived from natural products.

[0061] Application of Sparassis latifolia Extract The Sparassis latifolia extract has the effect of repairing the skin barrier, and has no phototoxicity and no sensitization, and can be used for the preparation of post-medical aesthetic repair daily chemical products, and its dosage in the products is 0.05-99 wt%.

[0062] Main advantages of the present invention: 1. The extraction method of the extract described in the present invention uses water as the extraction solvent, and the solvent is safe and non-toxic.

[0063] 2. The preparation method of the extract described in the present invention also includes an enzymatic extraction method, which is simple, requires low equipment, and has low cost, and is suitable for industrial production.

[0064] 3. In the enzymatic hydrolysis method described in the present invention, an appropriate amount of β-galactosidase such as 5% of the feeding amount is used for enzymatic hydrolysis. Compared with general techniques such as water extraction, the transfer rate of active ingredients such as polysaccharides is significantly improved, up to 161.93%.

[0065] 4. After the purification method of the extract described in the present invention uses macroporous resin purification, the Gardner color value of active ingredients such as polysaccharides is significantly reduced, and it has decreased by 56.15% compared with the extract without macroporous resin purification.

[0066] 5. The Sparassis latifolia extract described in the present invention has no phototoxicity, no sensitization, and no irritation to the skin.

[0067] 6. The Sparassis latifolia extract described in the present invention can be further prepared into products with post-medical aesthetic repair and barrier repair effects in daily chemicals.

[0068] The following will further illustrate the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0069] Example 1 Raw Material Extraction The raw material Sparassis latifolia was pulverized and passed through a 24-mesh sieve. 1.0828 g of the powder passing through the 24-mesh sieve was precisely weighed as m, and then 100 mL of deionized water was added, and the weight was mo. It was extracted in a water bath at 90 °C for 2.0 hours, cooled, and made up to mo with deionized water, and shaken well. After centrifugation, 1.0 mL of the supernatant was taken into a 10-mL colorimetric tube, and deionized water was added to the scale line and shaken well to obtain the sample. The polysaccharide content (calculated by dry weight) was measured by ultraviolet spectrophotometry.

[0070] Table 1 Polysaccharide Content of Water Extract of Sparassis latifolia

[0071] Enzyme Type Selection 10 g of Sparassis latifolia powder was taken, under the conditions of temperature 55 °C, time 1.0 h, and liquid-solid ratio 20:1 mL / g water, and then extracted 2 times by heating to boiling (85 °C - 100 °C), and the material liquid was combined. The effects of 5% β-glucanase, 5% β-galactosidase, 5% xylanase, 5% amylase, 5% cellulase, and water on the polysaccharide extraction rate of Sparassis latifolia were studied. For the crude polysaccharide of Sparassis latifolia after enzymatic hydrolysis, 0.2 mL of the supernatant was taken into a 10-mL colorimetric tube after centrifugation, and deionized water was added to the scale line and shaken well to obtain the sample. The polysaccharide content was measured by ultraviolet spectrophotometry.

[0072] Table 2 Enzyme Type Screening Results

[0073] Compared with the traditional water extraction method, the transfer and promotion rate of the active ingredient polysaccharide after enzyme extraction has increased. Among them, β-galactosidase is the highest, reaching 161.93%. Therefore, β-galactosidase was selected as the enzyme type for enzyme extraction in the follow-up.

[0074] Enzyme Dosage Selection The preparation method for enzyme selection includes the following steps: 10 g of Sparassis latifolia powder was taken, under the conditions of temperature 55 °C, time 1.0 h, and liquid-solid ratio 20:1 mL / g of water, and then extracted 2 times by heating to boiling (85 °C - 100 °C), and the material liquid was combined. The effects of the addition amounts of β-galactosidase being 5%, 4%, 2%, 1%, and 0.5% on the polysaccharide extraction rate of Sparassis latifolia were studied. The polysaccharide content was measured by ultraviolet spectrophotometry.

[0075] Table 3 Enzyme Dosage Screening Results

[0076] The results showed that when the dosage of β-galactosidase was 5%, the polysaccharide transfer rate was the highest, reaching 187.82%. Subsequently, 5% β-galactosidase was selected as the addition amount of the extraction enzyme.

[0077] Purification resin screening Take the crude extract obtained in Example 3. Weigh 1 portion of each of the pretreated resins of different models, AB-8, D101, HP20, LS-109D, LS-305, D301T, D941, D900, and LS-308, with each portion being 40 g, and place them in columns respectively. Add 20 g of the crude extract to each column. After mixing the crude extract with 1 volume of water, load the sample. First, wash with 4 BV of water, and collect the sample effluent and the washing solution. Then combine the sample effluent and the washing solution. Next, use the ultraviolet spectrophotometer method for polysaccharides to measure the polysaccharide content in the filtrate, calculate the polysaccharide retention rate, and then measure the Gardner color value.

[0078] Polysaccharide retention rate % = (polysaccharide content in the effluent + polysaccharide content in the washing solution of the sample effluent) / crude polysaccharide content * 100% Table 4 Results of resin model screening

[0079] It can be seen from the experimental results that when using LS-109D resin for purification, the polysaccharide retention rate of the crude polysaccharide extract is very high, and at the same time, the Gardner color value is greatly reduced. Therefore, LS-109D resin was used to purify the crude polysaccharide extract subsequently.

[0080] Characterization of Sparassis latifolia extract Perform chromatographic detection on the polysaccharide extract prepared in Example 15.

[0081] Using a gel chromatography - differential refractive index system, the liquid phase system is U3000 (Thermo, USA), and the differential refractive index detector is Optilab T-rEX (Wyatt technology, CA, USA). According to the standard curve, the average molecular weight of the Sparassis latifolia polysaccharide was determined to be 3.055 kDa, as Figure 1 shown.

[0082] Using a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), an electrochemical detector was used to analyze and detect the monosaccharide components. The external standard method was used for quantification. By preparing standard products with different concentrations and using Chromeleon software to fit the standard curve. The ion chromatogram of the monosaccharide standard product is as Figure 2 shown.

[0083] Analysis shows that Sparassis latifolia polysaccharide is composed of four monosaccharides, namely fucose (Fuc), galactose (Gal), glucose (Glc), and mannose (Man), with molar mass ratios of 2.19%, 5.65%, 89.23%, and 2.94%, respectively. The ion chromatogram of the monosaccharide test of Sparassis latifolia polysaccharide is as shown in Figure 3 the following figure.

[0084] The results show that Sparassis latifolia polysaccharide is a polysaccharide with different chemical compositions, and the molar mass ratio of Fuc:Gal:Glc:Man is approximately 1:2.58:40.74:1.34.

[0085] Safety implementation case 1 Phototoxicity test of Sparassis latifolia extract Test method: The phototoxicity was determined with reference to the OECD TG439 method. 1. Preparation of positive control of chlorpromazine hydrochloride (CPZ): Accurately weigh the chlorpromazine hydrochloride powder, which serves as the initial concentration for both the light treatment group (+Irr) and the non-light treatment group (-Irr), and perform 2-fold serial dilutions with PBS to prepare 8 concentrations for standby.

[0086] 2. Test sample of Sparassis latifolia extract: After dissolving in an aqueous solution, the sample was prepared into a 1 mg / mL stock solution, filtered through a 0.22 μm membrane to sterilize, and then serially diluted 2-fold with PBS to prepare 8 concentrations for standby. The specific concentrations are shown in the following table.

[0087] 3. According to the experimental design, prepare test substances with different concentrations using PBS buffer: Table 5 Concentration grouping table

[0088] 4. Drug addition 4.1 Normal control group (NC): Set 6 replicate wells, remove the culture medium in the wells, and add 100 μL of PBS buffer to each well again.

[0089] 4.2 Experimental group: For each sample, there are 8 concentrations, and 6 replicate wells are set for each concentration. Remove the culture medium in the wells, and add 100 μL of samples with different concentrations prepared with PBS buffer to each well.

[0090] 4.3 Zero adjustment wells: Select wells without cells at the edge, set 6 replicate wells, and add 100 μL of PBS buffer to each well.

[0091] 5. Light exposure: After the light exposure, remove the test solution, and add 100 μL of DMEM modified complete medium to each well and continue culturing at 37 °C and 5% CO2 for 18 - 22 h.

[0092] 6. Detection Determined by an enzyme - linked immunosorbent assay (ELISA) reader. Place the 96 - well plate in the ELISA reader, set the plate shaking time to 10 s, and measure the absorbance at a wavelength of 540 nm.

[0093] 7. Calculate the cell viability under each sample treatment.

[0094] 8. The evaluation criteria are shown in Table 6.

[0095] According to: Cell viability (%) =

[0096] Table 6 Light - stimulation evaluation criteria

[0097] The experimental results are shown in the following table. The results of this test meet the test evaluation, that is, for the test substance (extract of Sparassis latifolia), the IC 50 (+lrr) and IC 50 (-lrr) values cannot be calculated at the maximum allowable concentration (1000 μg / mL). This indicates that the test substance has no potential phototoxicity, expressed as "PIF = *1.0000". In summary, the extract of Sparassis latifolia has no phototoxicity.

[0098] Table 7 Light - stimulation evaluation criteria

[0099] Functional implementation case 1 Evaluate the effect of the extract of Sparassis latifolia on the secretion of TNF - α and IL - 6 by LPS - induced macrophage Raw264.7 cells.

[0100] 1. Determine the maximum safe concentration of the sample by the MTT method 1.1 Experimental method The experimental method is as Figure 4 shown.

[0101] 1.1.2 Experimental grouping: Set up a blank control group and an experimental group in the experiment, and set another 3 cell - free wells as zero - adjustment wells. In the experimental group, set 6 concentration gradients for each sample, and set 3 replicate wells for each concentration gradient; 1.1.3 Solution preparation: According to the experimental design, prepare test substances with different concentrations using the basal medium, and the concentrations are shown in the following table.

[0102] Table 8 Concentration grouping

[0103] 1.1.4 Administration: Add 200 μL of basal medium to each well of the zero - adjustment wells and the blank control group, and add 200 μL of the sample prepared with basal medium to each well of the experimental group. Then place it back in the incubator for culture (37 °C, 5% CO2).

[0104] 1.1.5 Detection: After shaking for 10 min, measure the absorbance (OD value) at a wavelength of 490 nm.

[0105] 1.1.6 Calculation of relative cell viability: Cell viability % = (OD value of experimental group - OD value of zero adjustment well) / (OD value of blank control group - OD value of zero adjustment group) * 100% The experimental results are as Figure 5 shown. When the concentration of Sparassis latifolia extract ≤ 0.3125 mg / mL, the cell viability is > 90%. An appropriate concentration can be selected within the concentration range of ≤ 0.3125 mg / mL for determination.

[0106] 2. Evaluation of the effect of Sparassis latifolia extract on the secretion of TNF-α and IL-6 by LPS-induced macrophage Raw264.7 cells 2.1 Experimental method The experimental method is as Figure 6 shown.

[0107] 2.1.1 Solution preparation According to the experimental design, prepare test substances with different concentrations as shown in the following table.

[0108] Table 9 Concentration grouping

[0109] 2.1.2 Inoculation: After inoculation, place it back in the incubator for incubation (37 °C, 5% CO2).

[0110] 2.1.3 Experimental grouping: Group according to the above table.

[0111] 2.1.4 Solution preparation: According to the obtained cell viability test results, select an appropriate sample concentration as the test concentration and prepare the sample solution to be tested.

[0112] 2.1.5 Administration: Perform according to 3 replicate wells for each concentration in each group, and then place it back in the incubator for incubation.

[0113] 2.1.6 Collect cell supernatant, centrifuge at 1000 rpm for 10 min, collect the supernatant and place it in a 1.5 mL centrifuge tube, and store it at -20 °C for later use.

[0114] 2.1.7 Use ELISA kit to measure the cell supernatant.

[0115] 2.1.8 Result analysis Use Graphad Prism to plot graphs. The t-test statistical analysis is used for comparison between groups. P < 0.05 indicates significant difference, P < 0.01 indicates highly significant difference, and P < 0.001 indicates extremely significant difference.

[0116] The experimental results are as follows Figure 7 and Figure 8 shown. The inhibition rates of Sparassis latifolia extract at 0.3 mg / mL and 0.075 mg / mL on TNF-α were 91.40% and 91.57% respectively. The inhibition rates of Sparassis latifolia extract at 0.3 mg / mL and 0.075 mg / mL on IL-6 were 30.14% and 22.44% respectively.

[0117] Functional implementation case 2 Evaluate the effect of Sparassis latifolia extract on the scratch injury of keratinocytes.

[0118] Determination of the maximum safe concentration of the sample by MTT method 1.1 Experimental method The experimental method is as follows Figure 9 shown.

[0119] 1.2 Inoculation: Inoculate cells into a 96-well plate at a volume of 200 μL per well, and place it back in the incubator for incubation (37 °C, 5% CO2).

[0120] 1.3 Experimental grouping: Set a negative control group and an experimental group in the experiment.

[0121] 1.4 Solution preparation: Prepare test substances at different concentrations with basal medium according to the experimental design.

[0122] Table 10 Concentration grouping

[0123] 1.5 Administration: Plate according to 3 replicate wells for each concentration in each group. Then place it back in the incubator for culture (37 °C, 5% CO2).

[0124] 1.6 Detection: Measure the absorbance (OD value) at a wavelength of 490 nm after shaking for 10 min.

[0125] 1.7 Calculation of relative cell viability: Cell viability % = (OD value of experimental group - OD value of zero adjustment well) / (OD value of blank control group - OD value of zero adjustment group) * 100% The experimental results are as follows Figure 10 shown. In the concentration range of 0.015625% - 0.5% of Sparassis crispa extract, the cell viability was > 90%, and an appropriate concentration could be selected within the concentration range of ≤ 0.5% for the next experiment.

[0126] 2. Effect of Sparassis latifolia extract on cell scratch injury 2.1 Experimental procedure, according to the following experimental design.

[0127] The experimental method is as follows Figure 11 shown.

[0128] Table 11 Experimental grouping

[0129] (1) Cell seeding When the cell density ≥ 70%, digest the cells with trypsin (0.25%), resuspend them, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant; add 1 mL of complete medium again, pipette to mix well, dilute by an appropriate multiple and count. Adjust the cell density to 3.5x10 5 cell / mL for use.

[0130] (2) Cell scratch and drug treatment Cell scratch: 24 h after changing the medium, take out the 6-well plate, discard the old medium, add 1 mL of PBS to each well, use a 200 μL pipette tip to make scratches perpendicular to the horizontal line on the back, with 3 scratches in each well; rinse the cells 3 times with PBS to wash away the scratched cell debris, and then take a microscopic photo to record the scratches.

[0131] Drug treatment and LPS stimulation: Add 1.99 mL of MEM basal medium and 10 μL of LPS with a concentration of 200 μg / mL to the blank control group and the negative control group, add 1.99 mL of TGF-β1 (100 ng / mL) prepared with MEM basal medium and 10 μL of LPS with a concentration of 200 μg / mL to the positive control group; add 1.99 mL of the test solution with concentrations of 2% and 0.5% and 10 μL of LPS with a concentration of 200 μg / mL to the experimental group, with 2 replicates for each concentration in each group.

[0132] (3) Scratch photography Take pictures of the corresponding cell scratches in each group at 0 h and 24 h after adding the drug through a microscope, and record the changes in the scratches.

[0133] (4) Data processing

[0134] Analyze the cell scratches with Image Pro Plus, calculate the cell scratch areas at 0 h and 24 h, calculate the scratch healing rate % according to the formula, and compare the differences in the scratch healing rates among groups.

[0135] Figure 13 is a picture of cell scratches, and the experimental results are as Figure 12As shown, compared with the blank control group (BC), the cell migration rate of the negative control group (NC, stimulated with LPS) was significantly decreased (***P < 0.001); compared with the negative control group (NC), the positive control group (TGF-β1) could significantly increase the cell migration rate (P < 0.001), and the difference was statistically significant, indicating that the experimental system was effective; compared with the negative control group (NC), the extract of Sparassis latifolia could significantly increase the cell migration rate at a concentration of 0.5%, and the migration rate was 28.90% (#P < 0.05).

[0136] Functional implementation case 3 Evaluate the effects of Sparassis latifolia extract on the expression of type I and type III collagen in 3T3 mouse fibroblasts.

[0137] Determine the maximum safe concentration of the sample by MTT method 1.1 Experimental methods The experimental methods are as Figure 14 shown.

[0138] (1) Inoculation: When the cell density ≥ 70%, digest the cells with trypsin and resuspend them. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant; add 1 mL of complete medium again, pipette and mix well, dilute by an appropriate multiple and count. According to the counting results, adjust the cell density to 8x10 4 cell / mL, and inoculate the cells into a 96-well plate at a volume of 200 μL per well, and place it back in the incubator for incubation (37 °C, 5% CO2).

[0139] (2) Experimental grouping: The experiment set up a blank control group and an experimental group, and another 3 cell-free wells were used as zero-adjustment wells. Six concentration gradients of the sample were set in the experimental group, and 3 replicates were set at each concentration gradient.

[0140] (3) Solution preparation: According to the experimental design in Table 12, prepare the test substances at different concentrations with basal medium.

[0141] Table 12 Test concentrations of Sparassis latifolia extract

[0142] (4) Administration: Take out the 96-well plate after 24 h, discard the old medium, add 200 μL of basal medium to each zero-adjustment well and blank control group well, and add 200 μL of the sample prepared with basal medium to each experimental group well. Plate according to 3 replicates for each concentration in each group. Then place it back in the incubator for culture (37 °C, 5% CO2); (5) Detection: After 24 h of drug addition treatment, take out the 96-well plate, add 20 μL of MTT working solution (stock solution 5 mg / mL) to each well, place it back in the incubator for continued culture for 4 h, and then place it back in the incubator for culture (37 °C, 5% CO2).

[0143] 1.2 Detection: 24 h after the addition of the drug, take out the 96-well plate, add 20 μL of MTT working solution (stock solution 5 mg / mL) to each well, and put it back into the incubator for continuous culture for 4 h; then discard the liquid in the wells, add 150 μL of DMSO to each well again, shake for 10 min, and measure the absorbance (OD value) at a wavelength of 490 nm.

[0144] (6) Calculation of relative cell viability: Cell viability % = (OD value of the experimental group - OD value of the zero-adjustment well) / (OD value of the blank control group - OD value of the zero-adjustment group) * 100% The experimental results are as Figure 15 shown. In the concentration range of 0.015625 - 0.5% of the Sparassis latifolia extract, the cell viability is > 90%. An appropriate concentration can be selected within the concentration range of ≤ 0.5% for the next experiment.

[0145] 2. Effect of Sparassis latifolia extract on the expression of type I collagen in UVA-induced 3T3 mouse fibroblasts 2.1 Method The experimental method is as Figure 16 shown.

[0146] (1) Seeding: When the cell density ≥ 70%, discard the culture medium, digest the cells with trypsin and resuspend them, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant; add 1 mL of complete medium again, pipette and mix well, dilute by an appropriate multiple and count. According to the counting results, adjust the cell density to 2.5x10 5 cell / mL, and seed the cells into a 6-well plate at a volume of 2 mL per well, and put it back into the incubator for incubation (37 °C, 5% CO2).

[0147] (2) Experimental grouping: Group according to the following table, and plate according to 4 replicates per concentration per group.

[0148] Table 13 Experimental grouping

[0149] (3) Solution preparation: According to the obtained cell viability test results, select an appropriate sample concentration as the test concentration and prepare the sample solution to be tested; use vitamin E (0.02%) as the positive control for the experiment.

[0150] (4) Drug administration: Take out the 6-well plate after 24 h, discard the old culture medium, add samples according to 4 replicates per concentration per group. For the blank control group (BC), add 2 mL of basal medium to each well, for the positive control group, add 2 mL of vitamin E solution (0.02%) to each well, for the experimental group, add 2 mL of the corresponding concentration sample to 4 replicates per concentration, and then put it back into the incubator for culture (37 °C, 5% CO2) incubation.

[0151] (5)Fluorescent staining of type I and type III collagen Fixation: Remove the 6-well plate after 24 h, wash the cells 3 times with PBS, add 500 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min.

[0152] Permeabilization: Discard the supernatant. Wash the cells 3 times with PBS, add 500 μL of 0.1% Triton (prepared with PBS) to each well, and let it stand at room temperature for 10 min.

[0153] Blocking: Remove the supernatant, wash the cells 3 times with PBS, add 500 μL of 10% goat blocking serum (diluted with PBS) to each well, and block at room temperature for 1 h.

[0154] Incubation with primary antibody: Discard the blocking goat serum without washing. Add 500 μL of appropriately diluted type I collagen primary antibody (diluted with 10% blocking goat serum at a dilution ratio of 1:1000) to each of 2 wells, and add 500 μL of appropriately diluted type III collagen primary antibody (diluted with 10% blocking goat serum at a dilution ratio of 1:1000) to each of the other 2 wells. Incubate overnight at 4 °C in the dark.

[0155] Incubation with secondary antibody: Discard the supernatant, wash the cells 3 times with PBS, add 500 μL of appropriately diluted fluorescent secondary antibody (diluted with PBS) to each well, and incubate at room temperature for 1 h.

[0156] Nuclear staining: Discard the supernatant, wash the cells 3 times with PBS, add 500 μL of Hochest33342 to each well, and incubate at room temperature for 5 min.

[0157] Photographing with a fluorescence microscope: Discard the supernatant, wash the cells 3 times with PBS, add 500 μL of PBS to each well, take pictures with a fluorescence microscope, and take 5 different positions in each well. Note that when taking pictures, try to select positions with similar cell numbers.

[0158] (6)Data processing: Analyze the fluorescence expression intensity of type I collagen in each group using Image J, plot the graph using GraphPad Prism software, and perform statistical analysis using T-test between groups. #P < 0.05 indicates significant difference, ##P < 0.01 indicates highly significant difference, and P < 0.001 indicates extremely significant difference.

[0159] The experimental results are as Figure 17As shown, compared with the blank control group (BC), the type I collagen in the vitamin E (PC) (0.02%) treatment group was significantly higher than that in the blank control group, and the difference was statistically significant (**P < 0.01); the type I collagen in the Sparassis latifolia extract treatment groups with concentrations of 0.5% and 0.2% was significantly higher than that in the blank control group, and the promotion rates of type I collagen synthesis were 23.79% and 44.47% respectively, and the differences were statistically significant (**P < 0.01).

[0160] The experimental results are as Figure 18 shown. Compared with the blank control group (BC), the type III collagen in the vitamin E (PC) (0.02%) treatment group was significantly higher than that in the blank control group, and the difference was statistically significant (**P < 0.01); the type III collagen in the Sparassis latifolia extract treatment groups with concentrations of 0.5% and 0.2% was significantly higher than that in the blank control group, and the promotion rates of type III collagen synthesis were 26.11% and 30.05% respectively, and the differences were statistically significant (**P < 0.01).

[0161] All the documents mentioned in the present invention are cited herein by reference as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. An extract of Sparassis latifolia, characterized in that, The Sparassis latifolia extract comprises: fucose (Fuc), galactose (Gal), glucose (Glc) and mannose (Man), wherein the molar mass percentage of glucose in the total polysaccharide component is 80-98%.

2. The Sparassis latifolia extract according to claim 1, wherein The Sparassis latifolia extract further has one or more characteristics selected from the following group: (1) The molar mass percentage of fucose is 1-5%; (2) The molar mass percentage of galactose is 3-8%; (3) The molar mass percentage of mannose is 2-6%; (4) The average molecular mass is 1-5 kDa; and / or (5) The Gardner color value ≤ 5.

3. The preparation method of Sparassis latifolia extract according to claim 1, characterized in that, The method comprises the steps of: (1) Extraction: Using water as a solvent, enzymatically hydrolyzing the Sparassis latifolia raw material, then inactivating the enzyme, filtering, and concentrating to obtain a crude extract; (2) Purification: Purifying and decolorizing the crude extract with macroporous resin, collecting the eluate, concentrating and drying to obtain the Sparassis latifolia extract as described above; Wherein in the step (1), the enzyme used for enzymatic hydrolysis is selected from the following group: β-glucanase, β-galactosidase, xylanase, amylase and cellulase; Wherein the macroporous resin in the method is selected from the following group: AB-8, D101, HP20, LS-109D, LS-305, D301T, D941, D900 and LS-308.

4. The preparation method of Sparassis latifolia extract according to claim 3, characterized in that, The enzyme in the method is β-galactosidase.

5. The preparation method of Sparassis latifolia extract according to claim 3, wherein, The dosage of the enzyme in the method is 2-8% of the mass of the Sparassis latifolia raw material.

6. The preparation method of Sparassis latifolia extract according to claim 3, characterized in that, The macroporous resin in the method is LS-109D.

7. The preparation method of Sparassis latifolia extract according to claim 3, characterized in that, The extraction conditions in the method have one or more characteristics selected from the following group: (1) The enzymatic hydrolysis temperature is 40-60 °C; (2) The liquid-to-solid ratio of water to the raw material is 5-40:1 mL / g; (3) The enzymatic hydrolysis time is 1-5 h; (4) The inactivation temperature is 70-120 °C; and / or (5) The number of enzymatic hydrolysis times is 1-5 times.

8. The use of Sparassis latifolia extract according to claim 1, characterized in that, For preparing daily-use products or daily chemical products with the efficacy of post-medical aesthetic repair and / or barrier repair.

9. The use according to claim 8, wherein, The product as described above further has one or several characteristics selected from the following group: (1) Inhibiting the secretion of inflammatory factors; (2) Repairing skin scratch injuries; (3) Promoting keratinocyte migration; (4) Reducing collagen loss.

10. A composition, characterized in that, The composition comprises: (1) The Sparassis latifolia extract as claimed in claim 1; and (2) Additional ingredients that can be used in daily chemical products.

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