A kind of Hydrangea latifolia extract and its preparation and application

Abstract

The present invention belongs to the technical field of daily cosmetics, and specifically relates to a preparation method and application of a broadleaf hydrangea extract. The broadleaf hydrangea extract adopts water extraction and specific enzymes such as β-galactosidase enzymolysis, which significantly improves the transfer rate of active ingredients such as polysaccharides. After subsequent purification using a specific type of macroporous resin, the Gardner color value of active ingredients such as polysaccharides is significantly reduced. In addition, the prepared broadleaf hydrangea extract showed the effect of inhibiting the secretion of inflammatory factors, keratinocyte migration, and collagen loss in cell experiments, which is conducive to wound healing and repair, and provides new ideas for the preparation of skin care and beauty products with medical and cosmetic post-operative repair and barrier repair effects.

Description

Technical Field

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

[0002] Sparassiscrispa Fr. belongs to the class Basidiomycetes, family Sparassidaceae. There are numerous varieties of Sparassis, also known as hydrangea mushrooms and cauliflower. As of January 8, 2024, Index Fungorum (http: / / www.indexfungorum.org) lists 29 species of the genus. The fruiting body of Sparassis consists of a short, thick stalk with branching petals. The petals resemble ginkgo leaves, with thin, uneven edges. They are fleshy in early growth, become tough in later stages, and become hard and brittle with age. The fruiting body is annual and is mostly white, bleached white, or pale yellow. Because of its resemblance to a hydrangea, it is also known as white hydrangea. The appearance, color, and nutritional content of Sparassis also vary depending on its origin and climatic conditions.

[0003] Sparassis is a fungus used both as food and medicine. It primarily grows in pine forests or at the base of pine trees, making its wild resources extremely rare. While wild wood-rotting fungi typically thrive in shaded areas or environments with limited sunlight, Sparassis requires at least 10 hours of sunlight daily, making it the only "sunshine mushroom" to date. There are differing opinions on the origin of Sparassis in my country. The widely accepted theory was that it originated from Sparassis crispa (Wulf.) Fr. However, further research revealed significant morphological differences between Sparassis collected in East Asia and those from Europe, leading to the designation of the Sparassis from Northeast my country as Sparassis latifolia.

[0004] As of February 2025, only Sparassis Crispa extracts were included in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)", and Sparassis latifolia was not included. However, with the hot application of new cosmetic raw materials, new applications for Sparassis latifolia in cosmetics will be provided.

[0005] Among the many light medical aesthetics projects, botulinum toxin and hyaluronic acid are the two most mature categories in the anti-aging field. However, with the approval of several new materials in recent years, a wider range of materials have entered the light medical field, providing consumers with more choices, such as polycaprolactone (PCL), poly-L-lactic acid (PLL), and collagen. Everything has two sides, and light medical aesthetics is no exception. On the one hand, it opens the skin barrier, allowing active ingredients to easily enter the skin and take effect. However, on the other hand, a damaged skin barrier increases the risk of invasion by bacteria, viruses, and other pathogens. Post-operative repair products further repair damaged skin, avoiding potential risks after surgery.

[0006] Therefore, it is urgent to provide a new cosmetic and skin care product derived from natural materials that can be used for post-operative repair of medical aesthetics. Summary of the Invention

[0007] The present invention provides a Hydrangea latifolia extract applicable to post-operative restoration of medical aesthetics and application thereof.

[0008] In a first aspect of the present invention, a Sparassis latifolia extract is provided. 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 components of the extract is 80-98%.

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

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

[0011] In another preferred embodiment, the Hydrangea latifolia extract further has one or more characteristics selected from the following group:

[0012] (1) The molar mass percentage of fucose in the total polysaccharide component is 1-5%, preferably 2-3%;

[0013] (2) The molar mass percentage of galactose in the total polysaccharide component is 3-8%, preferably 5-6%;

[0014] (3) The molar mass percentage of galactose in the total polysaccharide component is 2-6%, preferably 2-3%;

[0015] (4) The average molecular weight of the extract is 1 kDa to 5 kDa, preferably 2 kDa to 4 kDa, more preferably 2.5 to 3.5 kDa; and / or

[0016] (4) The Gardner color value of the extract is ≤5, preferably ≤4.

[0017] In a second aspect of the present invention, a method for preparing the Hydrangea latifolia extract according to the first aspect of the present invention is provided, the method comprising the steps of:

[0018] (1) Extraction: Using water as solvent, enzymatically hydrolyze the raw material of Hydrangea latifolia, then inactivate the enzyme, filter, and concentrate to obtain a crude extract;

[0019] (2) Purification: The crude extract is purified and decolorized using a macroporous resin, and the eluate is collected, concentrated, and dried to obtain the Hydrangea latifolia extract;

[0020] wherein in step (1), the enzyme used for enzymatic hydrolysis is selected from the group consisting of β-glucanase, β-galactosidase, xylanase, amylase and cellulase;

[0021] 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.

[0022] In another preferred embodiment, the raw material of Hydrangea latifolia in the method is the fruiting body of Hydrangea latifolia.

[0023] In another preferred embodiment, the amount of the macroporous resin used in the method is 1 to 4 times the mass of the crude extract, preferably 1 to 2 times.

[0024] In another preferred embodiment, in the step (2), the elution is performed using deionized water.

[0025] In another preferred embodiment, the enzyme in the method is β-galactosidase.

[0026] In another preferred embodiment, the amount of enzyme used in the method is 2-8% of the mass of the raw material Hydrangea latifolia, preferably 4-6%.

[0027] In another preferred embodiment, the polysaccharide transfer rate in the method is ≥115%, preferably ≥140%, and more preferably ≥160%.

[0028] In another preferred embodiment, the macroporous resin in the method is LS-109D.

[0029] In another preferred embodiment, the polysaccharide retention rate in the method is ≥30%, preferably ≥35%, more preferably ≥30%.

[0030] In another preferred embodiment, the polysaccharide retention rate in the method is calculated by the following formula:

[0031] Polysaccharide retention rate % = (polysaccharide content in the effluent + water wash sample effluent) / crude polysaccharide content * 100% (Formula I).

[0032] In another preferred embodiment, the conditions extracted in the method have one or more characteristics selected from the following group:

[0033] (1) The enzymatic hydrolysis temperature is 40~60℃, preferably 50~60℃;

[0034] (2) The liquid-to-material ratio of water to raw material is 5-40:1 mL / g, preferably 15-25:1 mL / g;

[0035] (3) Enzymatic hydrolysis time is 1~5h, preferably 1~2h;

[0036] (4) The inactivation temperature is 70-120°C, preferably 85-120°C; and / or

[0037] (5) The number of enzymatic hydrolysis is 1 to 5 times, preferably 2 times.

[0038] In another aspect of the present invention, use of a Sparassis crispa (Wulf.) Fr. extract in cosmetics is provided.

[0039] In another preferred embodiment, the hydrangea extract is an extract of Sparassis latifolia.

[0040] In another preferred embodiment, the Hydrangea latifolia extract is prepared by the method described above.

[0041] In another preferred embodiment, the cosmetics are medical and cosmetic repair cosmetics.

[0042] In a third aspect of the present invention, there is provided a use of the Hydrangea latifolia extract as described in the first aspect of the present invention for preparing a daily product or daily chemical product with medical and aesthetic post-operative repair and / or barrier repair effects.

[0043] In another preferred embodiment, the product further has one or more characteristics selected from the following group:

[0044] (1) Inhibit the secretion of inflammatory factors;

[0045] (2) Repair skin scratch damage;

[0046] (3) Enhance keratinocyte migration; and / or

[0047] (4) Reduce collagen loss.

[0048] In a fourth aspect of the present invention, a composition is provided, comprising: (1) the Hydrangea latifolia extract according to the first aspect of the present invention; and (2) additional ingredients that can be used in daily chemical products.

[0049] In another preferred embodiment, the amount of the Hydrangea latifolia extract in the composition is 0.05 to 99 wt %.

[0050] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The molecular weight test graph of Hydrangea latifolia polysaccharide is shown.

[0052] Figure 2 Ion chromatograms of monosaccharide standards are shown.

[0053] Figure 3 Shown are the monosaccharide test ion chromatograms of Hydrangea latifolia polysaccharide.

[0054] Figure 4 A flow chart for determining the maximum safe concentration of a sample in Efficacy Example 1 is shown.

[0055] Figure 5 The effect of the Hydrangea latifolia extract in Efficacy Example 1 on the viability of macrophage Raw264.7 cells is shown.

[0056] Figure 6 This is a flow chart showing the effect of samples on LPS-induced TNF-α and IL-6 secretion in macrophage Raw264.7 cells in Efficacy Example 1.

[0057] Figure 7 The effect of the Hydrangea latifolia extract of Example 1 on TNF-α was shown.

[0058] Figure 8 The effect of the Hydrangea latifolia extract of Example 1 on IL-6 is shown.

[0059] Figure 9 A flow chart for determining the maximum safe concentration of a sample in Efficacy Example 2 is shown.

[0060] Figure 10 The effect of the Hydrangea latifolia extract in Efficacy Example 2 on the cell viability of keratinocytes is shown.

[0061] Figure 11 The flowchart of the experiment for determining the effect of the Hydrangea latifolia extract on cell scratching in Efficacy Example 2 is shown.

[0062] Figure 12 The migration rate of keratinocytes by the Hydrangea latifolia extract in Efficacy Example 2 is shown.

[0063] Figure 13 The graph shows the scratch marks of keratinocytes by the Hydrangea latifolia extract in Efficacy Example 2.

[0064] Figure 14 A flow chart for determining the maximum safe concentration of a sample in Efficacy Example 3 is shown.

[0065] Figure 15 The effect of the Hydrangea latifolia extract on fibroblast viability in Efficacy Example 3 is shown.

[0066] Figure 16 A flow chart showing the method for measuring the effect of a sample on collagen in Efficacy Example 3 is shown.

[0067] Figure 17 The effect of the Hydrangea latifolia extract on type I collagen in Efficacy Example 3 is shown.

[0068] Figure 18 The effect of the Hydrangea latifolia extract on type III collagen in Efficacy Example 3 is shown. DETAILED DESCRIPTION

[0069] After extensive and in-depth research and a large number of experimental screenings, the inventors unexpectedly discovered for the first time that hydrangea extracts can be applied to the field of cosmetics, with the preferred example being hydrangea latifolia extract. The preparation method of the hydrangea latifolia extract includes: water extraction, β-galactosidase hydrolysis and macroporous resin purification. Compared with existing technologies such as ordinary water extraction, the method significantly improves the transfer rate of active ingredients such as polysaccharides and significantly reduces the Gardner chroma of polysaccharides. In the present invention, it was also found that the hydrangea latifolia extract can inhibit the secretion of inflammatory factors, keratinocyte migration, and collagen loss, providing a new idea for the preparation of skin care or beauty products with medical and aesthetic post-repair and barrier repair effects. On this basis, the inventors completed the present invention.

[0070] the term

[0071] As described herein, "Edgeococcus latifolius extract", "Edgeococcus latifolius polysaccharide" and "Edgeococcus latifolius polysaccharide extract" can be used interchangeably, all referring to an extract composed of four monosaccharides: fucose Fuc, galactose Gal, glucose Glc and mannose Man. In a preferred embodiment, it is the extract obtained in Example 15.

[0072] Preparation method of Hydrangea latifolia extract

[0073] 1. Extraction: Weigh the raw material, Hydrangea latifolia, add 10 times (raw material mass kg / solvent volume L) of water, and 5% of the raw material mass of β-galactosidase. Hydrolyze at 40°C-55°C for 2 hours. Heat to boiling (85°C-100°C) and extract twice. Filter and concentrate under reduced pressure (55°C-80°C, vacuum pressure 0.060-0.080 MPa) to recover the water and obtain a 1x crude extract.

[0074] 2. Purification: The crude extract was purified with 2 times the feed volume of LS-109D macroporous resin. The crude extract was mixed with 1 volume of water, loaded onto the sample, and washed with 4 BV of water. The load effluent and the washing solution were collected.

[0075] 3. Concentration: The collected sample solution is concentrated to obtain a concentrated sample effluent, which is then combined with the water wash to obtain an extract. The extract is then vacuum-dried or freeze-dried to obtain a powdered Hydrangea macrophylla extract. Alternatively, the extract can be dispersed with 1,2-propylene glycol, 1,3-propylene glycol, butylene glycol, and glycerol to obtain a Hydrangea macrophylla extract solution.

[0076] The powder or liquid extract of Hydrangea latifolia can be further prepared into daily chemical or daily use products.

[0077] Hydrangea Extract and Wound Healing and Repair

[0078] Wound healing and repair is a complex and dynamic process, which is generally divided into four stages: bleeding and hemostasis, inflammation, proliferation and tissue remodeling, and each stage is highly coordinated.

[0079] During the bleeding and hemostasis phase, platelets bind to fibrin to form a fibrin clot, which causes blood to coagulate and inhibits local bleeding. Furthermore, activated platelets release a variety of cytokines, such as PDGF, CXCL4, TGF-β, and IGF, through degranulation, promoting subsequent injury repair.

[0080] Inflammatory phase: Neutrophils and macrophages are the primary effector cells during the inflammatory phase. These immune cells release a variety of cytokines and chemokines, recruiting cells from the blood and the edge of the wound into the wound. Furthermore, the secreted cytokines can further promote cell proliferation at the site of injury. However, excessive levels of inflammatory factors can lead to persistent redness and swelling, and slow healing.

[0081] Proliferation: Angiogenesis is key to wound repair. Macrophages and damaged endothelial cells release FGF-2 and VEGF, promoting angiogenesis. Growth factors such as EGF and TGF-β stimulate keratinocyte proliferation and migration at the wound edge, leading to re-epithelialization of the damaged dermis. After re-epithelialization is complete, keratinocytes differentiate, restoring the epidermal barrier function. This cell proliferation can be measured by the wound healing rate.

[0082] Tissue remodeling: The granulation tissue formed during the early repair process is gradually replaced by newly formed collagen-rich dermal matrix. Normal skin tissue is primarily composed of type III collagen, forming a basket-weave structure, while damaged areas are remodeled to coarser type I collagen, forming a dense parallel structure.

[0083] The hydrangea extract can be effective in all three stages of wound healing and repair. During the inflammatory stage, it can inhibit the secretion of inflammatory factors to prevent wound redness, swelling, and inflammation; during the proliferation stage, it can accelerate keratinocyte migration and promote the recovery of epidermal barrier function; and during the tissue reconstruction stage, it can reduce collagen loss and help tissue remodel rapidly. Therefore, the hydrangea extract described in this application has opened up new ideas for the preparation of medical and cosmetic post-operative repair products derived from natural products.

[0084] Application of Hydrangea latifolia Extract

[0085] The extract of Hydrangea latifolia has the function of repairing the skin barrier, is non-phototoxic and non-sensitizing, and can be used to prepare medical and cosmetic post-operative repair daily chemical products. The amount of the extract in the product is 0.05 to 99 wt%.

[0086] The main advantages of the present invention are:

[0087] 1. The extraction method of the extract of the present invention uses water as the extraction solvent, and the solvent is safe and non-toxic.

[0088] 2. The preparation method of the extract of the present invention also includes enzymatic extraction, which is simple, requires low equipment, is low in cost, and is suitable for industrial production.

[0089] 3. The enzymatic hydrolysis method of the present invention uses an appropriate amount of β-galactosidase, such as 5% of the feed amount, to hydrolyze the active ingredients such as polysaccharides. Compared with general techniques such as water extraction, the transfer rate of active ingredients such as polysaccharides is significantly improved, reaching as high as 161.93%.

[0090] 4. After the extract purification method of the present invention uses macroporous resin purification, the Gardner color value of active ingredients such as polysaccharides is significantly reduced, decreasing by 56.15% compared with the extract not purified by macroporous resin.

[0091] 5. The Hydrangea latifolia extract of the present invention is non-phototoxic, non-sensitizing, and non-irritating to the skin.

[0092] 6. The Hydrangea latifolia extract of the present invention can be further prepared into products with medical and cosmetic post-operative repair and barrier repair effects in daily chemical products.

[0093] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

[0094] Example 1 Raw material extraction

[0095] The raw material, Hydrangea latifolia, was crushed and passed through a 24-mesh sieve. Accurately weigh 1.0828 g of the powder (m) after passing through the 24-mesh sieve, adding 100 mL of deionized water and weighing it as mo. Extract in a water bath at 90°C for 2.0 hours, cool, and make up to mo with deionized water. Shake well. After centrifugation, transfer 1.0 mL of the supernatant to a 10 mL colorimetric tube, add deionized water to the mark, and shake well. The polysaccharide content (by dry weight) was determined using UV spectrophotometry.

[0096] Table 1 Polysaccharide content of aqueous extract of Hydrangea latifolia

[0097]

[0098] Enzyme type selection

[0099] 10g of Hydrangea latifolia powder was extracted twice at 55°C for 1.0h at a liquid-to-solid ratio of 20:1 mL / g water. The extracts were then heated to boiling (85-100°C) and the combined extracts were combined to investigate the effects of 5% β-glucanase, 5% β-galactosidase, 5% xylanase, 5% amylase, 5% cellulase, and water on the extraction yield of polysaccharides from Hydrangea latifolia. After centrifugation, 0.2mL of the enzymatically hydrolyzed crude polysaccharide was transferred to a 10mL colorimetric tube, brought to volume with deionized water, and shaken to obtain the product. The polysaccharide content was determined by UV spectrophotometry.

[0100] Table 2 Enzyme type screening results

[0101]

[0102] Compared to traditional water extraction, enzymatic extraction increased the transfer rate of active polysaccharides, with β-galactosidase achieving the highest rate of 161.93%. Therefore, β-galactosidase was selected as the enzyme for subsequent extraction.

[0103] Enzyme dosage selection

[0104] The preparation method of enzyme selection comprises the following steps:

[0105] Ten grams of Hydrangea latifolia powder was extracted twice at 55°C for 1.0 h with a liquid-to-solid ratio of 20:1 mL / g. The extracts were then heated to boiling (85°C–100°C) and the combined extracts were combined. The effects of β-galactosidase addition at 5%, 4%, 2%, 1%, and 0.5% on the extraction yield of polysaccharides from Hydrangea latifolia were investigated. Polysaccharide content was determined by UV spectrophotometry.

[0106] Table 3 Enzyme dosage screening results

[0107]

[0108] The results showed that the polysaccharide transfer rate was the highest at 5% β-galactosidase, reaching 187.82%. 5% β-galactosidase was subsequently selected as the enzyme dosage for extraction.

[0109] Purification resin screening

[0110] Take the crude extract obtained in Example 3, weigh 1 part each of different types of pretreated resins AB-8, D101, HP20, LS-109D, LS-305, D301T, D941, D900 and LS-308, 40g each, and place them in columns respectively. Add 20g of crude extract respectively. After mixing the crude extract with 1 volume of water, load the sample, first wash with 4BV water, collect the sample effluent and the washing liquid. Combine the sample effluent and the washing liquid. Then use the ultraviolet spectrophotometer polysaccharide method to test the filtrate polysaccharide content, calculate the polysaccharide retention rate, and then test the Gardner color value.

[0111] Polysaccharide retention rate% = (polysaccharide content in effluent + water wash sample effluent) / crude polysaccharide content * 100%

[0112] Table 4 Resin model screening results

[0113]

[0114] The experimental results show that when LS-109D resin is used 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 is subsequently used to purify the crude polysaccharide extract.

[0115] Characterization of Hydrangea latifolia Extract

[0116] The polysaccharide extract prepared in Example 15 was subjected to chromatographic detection.

[0117] The gel chromatography-differential refractive index system was used, the liquid phase system was U3000 (Thermo, USA), and the differential refractive index detector was Optilab T-rEX (Wyatt technology, CA, USA). The average molecular mass of the polysaccharide of Hydrangea latifolia was determined to be 3.055 kDa according to the standard curve. Figure 1 shown.

[0118] Monosaccharide components were analyzed and detected using a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) with an electrochemical detector. Quantification was performed using the external standard method. Standards of varying concentrations were prepared and fitted using Chromeleon software to obtain a standard curve. The ion chromatogram of the monosaccharide standard is shown in Figure 2. Figure 2 shown.

[0119] The analysis showed that the polysaccharide of Hydrangea latifolia is composed of four monosaccharides: fucose Fuc, galactose Gal, glucose Glc and mannose Man, with molar mass ratios of 2.19%, 5.65%, 89.23% and 2.94% respectively. Figure 3 shown.

[0120] The results showed that the polysaccharide of Hydrangea latifolia was a polysaccharide with different chemical compositions, in which the molar mass ratio of Fuc:Gal:Glc:Man was approximately 1:2.58:40.74:1.34.

[0121] Safety Implementation Case 1: Phototoxicity Test of Hydrangea latifolia Extract

[0122] Test method: Determine phototoxicity according to OECD TG439 method.

[0123] 1. Preparation of chlorpromazine hydrochloride (CPZ) positive control: Accurately weigh chlorpromazine hydrochloride powder as the initial concentration for the light-treated group (+Irr) and the no-light-treated group (-Irr), respectively. Perform a 2-fold serial dilution with PBS to prepare 8 concentrations in total for later use.

[0124] 2. Hydrangea latifolia extract test sample: After dissolving in aqueous solution, the sample is prepared into a 1 mg / mL stock solution. After sterilization by filtration through a 0.22 μm membrane, it is diluted 2-fold with PBS to prepare a total of 8 concentrations for later use. The specific concentrations are shown in the table below.

[0125] 3. According to the experimental design, prepare the test substances at different concentrations using PBS buffer:

[0126] Table 5 Concentration grouping table

[0127]

[0128] 4. Add medicine

[0129] 4.1 Normal control group (NC): Set up 6 replicate wells, remove the culture medium in the wells, and add 100 μL PBS buffer to each well.

[0130] 4.2 Experimental group: 8 concentrations of each sample were set up, and 6 replicate wells were set up for each concentration. The culture medium in the wells was removed, and 100 μL of PBS buffer was added to each well to prepare different concentrations of samples.

[0131] 4.3 Zero wells: Select wells without cells at the edge, set up 6 replicate wells, and add 100 μL PBS buffer to each well.

[0132] 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, 5% CO2 for 18-22 hours.

[0133] 6. Testing

[0134] The 96-well plate was placed in a microplate reader, the shake time was set to 10 seconds, and the absorbance was measured at a wavelength of 540 nm.

[0135] 7. Calculate the cell survival rate under each sample treatment.

[0136] 8. The evaluation criteria are shown in Table 6.

[0137] According to: Cell survival rate (%) =

[0138] Table 6 Light stimulation evaluation criteria

[0139]

[0140] The experimental results are shown in the table below. The test results are consistent with the experimental evaluation, that is, the IC of the test substance (Hydrangea latifolia extract) cannot be calculated at the maximum allowed concentration (1000 μg / mL). 50 (+lrr) and IC 50 (-lrr) value, which indicates that the test substance has no potential phototoxicity, expressed as "PIF=*1.0000". In summary, the Hydrangea latifolia extract has no phototoxicity.

[0141] Table 7 Light stimulation evaluation criteria

[0142]

[0143] Effective implementation case 1

[0144] To evaluate the effect of Hydrangea latifolia extract on LPS-induced TNF-α and IL-6 secretion in Raw264.7 macrophage cells.

[0145] 1. Determination of the maximum safe concentration of samples by MTT method

[0146] 1.1 Experimental Methods

[0147] Experimental methods such as Figure 4 shown.

[0148] 1.1.2 Experimental Grouping: A blank control group and an experimental group were set up, and three cell-free wells were set up as zero wells. Six concentration gradients were set up for each sample in the experimental group, and three replicate wells were set up under each concentration gradient;

[0149] 1.1.3 Solution preparation: According to the experimental design, different concentrations of the test substances were prepared in basal culture medium. The concentrations are shown in the following table.

[0150] Table 8 Concentration groups

[0151]

[0152] 1.1.4 Dosing: Add 200 μL of basal medium to each well of the zero-conditioning well and the blank control group, and 200 μL of sample prepared with basal medium to each well of the experimental group. Then return the cells to the incubator (37°C, 5% CO2).

[0153] 1.1.5 Detection: Shake for 10 minutes and measure the absorbance (OD value) at 490 nm.

[0154] 1.1.6 Calculation of relative cell viability: Cell viability % = (OD value of experimental group - OD value of zero-adjusted well) / (OD value of blank control group - OD value of zero-adjusted well) * 100%

[0155] The experimental results are as follows Figure 5 As shown in the results, when the concentration of Hydrangea latifolia extract was ≤0.3125 mg / mL, the cell viability was >90%. Appropriate concentrations could be selected for determination within the concentration range of ≤0.3125 mg / mL.

[0156] 2. Evaluate the effect of Hydrangea latifolia extract on LPS-induced TNF-α and IL-6 secretion in Raw264.7 macrophage cells

[0157] 2.1 Experimental Methods

[0158] Experimental methods such as Figure 6 shown.

[0159] 2.1.1 Liquid preparation

[0160] According to the experimental design, different concentrations of test substances were prepared, and the concentrations are shown in the following table.

[0161] Table 9 Concentration groups

[0162]

[0163] 2.1.2 Inoculation: After inoculation, place the cells back into the incubator for incubation (37°C, 5% CO2).

[0164] 2.1.3 Experimental groups: Group according to the table above.

[0165] 2.1.4 Solution preparation: Based on the obtained cell viability test results, select an appropriate sample concentration as the test concentration and prepare the sample solution to be tested.

[0166] 2.1.5 Drug administration: Perform 3 replicate wells per group and per concentration, then return to the incubator for incubation.

[0167] 2.1.6 Collect the cell supernatant, centrifuge at 1000 rpm for 10 min, collect the supernatant and place it in a 1.5 mL centrifuge tube, and store at -20°C until use.

[0168] 2.1.7 Measure the cell supernatant using ELISA kit.

[0169] 2.1.8 Results Analysis

[0170] Graphad Prism was used for graphing, and t-test was used for statistical analysis for comparison among the groups. P < 0.05 indicated a significant difference, P < 0.01 indicated a significant difference, and P < 0.001 indicated a very significant difference.

[0171] The experimental results are as follows Figure 7 and Figure 8 As shown in the results, the inhibition rates of 0.3 mg / mL and 0.075 mg / mL of Hydrangea latifolia extract on TNF-α were 91.40% and 91.57%, respectively. The inhibition rates of 0.3 mg / mL and 0.075 mg / mL of Hydrangea latifolia extract on IL-6 were 30.14% and 22.44%, respectively.

[0172] Effective implementation case 2

[0173] To evaluate the effects of Hydrangea latifolia extract on scratch injury in keratinocytes.

[0174] Determination of maximum safe concentration of samples by MTT method

[0175] 1.1 Experimental Methods

[0176] Experimental methods such as Figure 9 shown.

[0177] 1.2 Inoculation: Inoculate cells into 96-well plates at a volume of 200 μL per well and place the plates back into the incubator for incubation (37°C, 5% CO2).

[0178] 1.3 Experimental grouping: The experiment set up a negative control group and an experimental group.

[0179] 1.4 Solution preparation: Prepare different concentrations of the test substance using basal culture medium according to the experimental design.

[0180] Table 10 Concentration groups

[0181]

[0182] 1.5 Dosing: Plate the cells in triplicate for each concentration and group. Return the cells to the incubator (37°C, 5% CO2).

[0183] 1.6 Detection: Shake for 10 minutes and measure the absorbance (OD value) at a wavelength of 490 nm.

[0184] 1.7 Calculation of relative cell viability: Cell viability % = (OD value of experimental group - OD value of zero-adjusted well) / (OD value of blank control group - OD value of zero-adjusted well) * 100%

[0185] The experimental results are as follows Figure 10 As shown, the cell viability of hydrangea extract was greater than 90% in the concentration range of 0.015625%-0.5%, and the appropriate concentration could be selected in the concentration range of ≤0.5% for the next experiment.

[0186] 2. Effects of Hydrangea latifolia extract on cell scratch damage

[0187] 2.1 Experimental process, experimental design according to the table below.

[0188] Experimental methods such as Figure 11 shown.

[0189] Table 11 Experimental groups

[0190]

[0191] (1) Cell plating

[0192] When the cell density was ≥70%, the cells were digested with trypsin (0.25%) and resuspended, centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded. 1 mL of complete culture medium was added again, pipetting and mixing was performed, and the cells were diluted to an appropriate multiple and counted. The cell density was adjusted to 3.5 x 10 based on the counting results. 5 cell / mL, set aside.

[0193] (2) Cell scratching and drug treatment

[0194] Cell scratching: 24 hours after changing the medium, remove the 6-well plate, discard the old medium, re-add 1 mL of PBS to each well, and use a 200 μL pipette tip to scratch the cells perpendicular to the horizontal line on the back, with 3 scratches per well; rinse the cells 3 times with PBS to rinse off the scratched cell debris, and then take pictures under a microscope to record the scratches.

[0195] Drug treatment and LPS stimulation: 1.99 mL of MEM basal medium and 10 μL of 200 μg / mL LPS were added to each well of the blank control group and the negative control group; 1.99 mL of TGF-β1 (100 ng / mL) prepared with MEM basal medium and 10 μL of 200 μg / mL LPS were added to each well of the positive control group; 1.99 mL of the test solution with a concentration of 2% or 0.5% and 10 μL of 200 μg / mL LPS were added to each well of the experimental group, with 2 replicates for each concentration in each group.

[0196] (3) Take photos of scratches

[0197] Pictures of the cell scratches in each group at 0 h and 24 h after drug addition were taken under a microscope to record the changes in the scratches.

[0198] (4) Data processing

[0199]

[0200] Image Pro Plus was used to analyze the cell scratches, and the cell scratch areas at 0 h and 24 h were recorded. The scratch healing rate (%) was calculated according to the formula, and the differences in scratch healing rates among the groups were compared.

[0201] Figure 13 The cell scratch pictures are shown in the figure. Figure 12 As shown, compared with the blank control group (BC), the cell migration rate of the negative control group (NC, LPS stimulation) was significantly reduced (***P < 0.001); compared with the negative control group (NC), the positive control group (TGF-β1) can significantly increase the cell migration rate (###P < 0.001), and the difference is statistically significant, indicating that the experimental system is effective; compared with the negative control group (NC), the Hydrangea latifolia extract can significantly increase the cell migration rate at a concentration of 0.5%, and the migration rate is 28.90% (#P < 0.05).

[0202] Effective implementation case 3

[0203] To evaluate the effect of Hydrangea latifolia extract on the expression of type I and type III collagen in 3T3 mouse fibroblasts.

[0204] Determination of maximum safe concentration of samples by MTT method

[0205] 1.1 Experimental Methods

[0206] Experimental methods such as Figure 14 shown.

[0207] (1) Inoculation: When the cell density is ≥70%, trypsinize the cells and resuspend them, centrifuge at 1000 rpm for 4 minutes, and discard the supernatant; add 1 mL of complete culture medium, pipette to mix, dilute to an appropriate multiple and count. According to the counting results, adjust the cell density to 8x10 4 Cells were seeded into 96-well plates at a volume of 200 μL per well and placed back into the incubator for incubation (37°C, 5% CO2).

[0208] (2) Experimental grouping: The experiment set up a blank control group and an experimental group, and set up 3 cell-free wells as zero wells. The samples in the experimental group were set up with 6 concentration gradients, and 3 replicate wells were set up under each concentration gradient.

[0209] (3) Solution preparation: According to the experimental design in Table 12, different concentrations of the test substances were prepared using basal culture medium.

[0210] Table 12 Test concentrations of Hydrangea latifolia extract

[0211]

[0212] (4) Administration: After 24 hours, remove the 96-well plate, discard the old culture medium, add 200 μL of basal culture medium to each well of the zero-well and blank control group, and add 200 μL of sample prepared with basal culture medium to each well of the experimental group. Plate the plate with three replicates per group and concentration. Then return the plate to the incubator (37°C, 5% CO2).

[0213] (5) Detection: 24 h after drug treatment, remove the 96-well plate, add 20 μL of MTT working solution (stock solution 5 mg / mL) to each well, return the plate to the incubator and continue culturing for 4 h, and then return the plate to the incubator (37°C, 5% CO2).

[0214] 1.2 Detection: 24 h after drug treatment, remove the 96-well plate and add 20 μL of MTT working solution (stock solution 5 mg / mL) to each well. Return the plate to the incubator and continue incubation for 4 h. Then, discard the liquid in the wells and add 150 μL of DMSO to each well. After shaking for 10 min, measure the absorbance (OD value) at a wavelength of 490 nm.

[0215] (6) Calculation of relative cell viability: Cell viability % = (OD value of experimental group - OD value of zero-adjusted well) / (OD value of blank control group - OD value of zero-adjusted well) * 100%

[0216] The experimental results are as follows Figure 15As shown, the cell viability of the Hydrangea latifolia extract was greater than 90% in the concentration range of 0.015625-0.5%, and the appropriate concentration could be selected in the concentration range of ≤0.5% for the next experiment.

[0217] 2. Effect of Hydrangea latifolia extract on UVA-induced collagen type I expression in 3T3 mouse fibroblasts

[0218] 2.1 Methods

[0219] Experimental methods such as Figure 16 shown.

[0220] (1) Inoculation: When the cell density is ≥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 culture medium again, pipette to mix, dilute to an appropriate multiple and count. According to the counting results, adjust the cell density to 2.5x10 5 Cells were seeded into 6-well plates at a volume of 2 mL per well and placed back into the incubator for incubation (37°C, 5% CO2).

[0221] (2) Experimental grouping: Group the cells according to the table below, and arrange the plates with 4 replicate wells per concentration per group.

[0222] Table 13 Experimental groups

[0223]

[0224] (3) Solution preparation: Based on 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 a positive control for the experiment.

[0225] (4) Administration: After 24 hours, the 6-well plate was removed and the old culture medium was discarded. Samples were added to 4 replicate wells of each concentration in each group. 2 mL of basal culture medium was added to each well of the blank control group (BC). 2 mL of vitamin E solution (0.02%) was added to each well of the positive control group. 4 replicate wells of each concentration in the experimental group were added to each well, and 2 mL of the corresponding concentration sample was added to each well. The plates were then returned to the incubator for incubation (37°C, 5% CO2).

[0226] (5) Fluorescent staining of type I and type III collagen

[0227] Fixation: After 24 h, remove the 6-well plate, wash the cells three times with PBS, add 500 μL of 4% paraformaldehyde to each well, and fix at room temperature for 15 min.

[0228] Permeabilization: Remove the supernatant. Wash the cells three times with PBS, add 500 μL of 0.1% Triton X-ray (in PBS) to each well, and incubate at room temperature for 10 minutes.

[0229] Blocking: Remove the supernatant, wash the cells three times with PBS, add 500 μL of 10% goat blocking serum (diluted with PBS) to each well, and block at room temperature for 1 hour.

[0230] Incubation with primary antibody: discard the blocking goat serum, do not wash, add 500 μL of appropriately diluted type I collagen primary antibody (diluted with 10% blocking goat serum, dilution ratio 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, dilution ratio 1:1000) to each of the other 2 wells. Incubate at 4°C in the dark overnight.

[0231] Incubation with secondary antibody: discard the supernatant, wash the cells three 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 hour.

[0232] Nuclear staining: discard the supernatant, wash the cells three times with PBS, add 500 μL Hochest33342 to each well, and incubate at room temperature for 5 min.

[0233] Fluorescence microscopy: Discard the supernatant, wash the cells three times with PBS, add 500 μL of PBS to each well, and photograph under a fluorescence microscope. Select five different locations in each well to photograph. When photographing, try to select locations with similar cell numbers.

[0234] (6) Data processing: Image J was used to analyze the fluorescence expression intensity of type I collagen in each group, and GraphPad Prism software was used to draw graphs. T-test was used for statistical analysis between groups. #P < 0.05 indicated a significant difference, ##P < 0.01 indicated a significant difference, and ###P < 0.001 indicated an extremely significant difference.

[0235] The experimental results are as follows Figure 17 As shown in the data, compared with the blank control group (BC), the type I collagen content 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 content in the 0.5% and 0.2% concentrations of Hydrangea latifolia extract treatment groups 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 difference was statistically significant (**P < 0.01).

[0236] The experimental results are as follows Figure 18As shown in the data, compared with the blank control group (BC), the type III collagen content 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 content in the 0.5% and 0.2% concentrations of Hydrangea latifolia extract treatment groups 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 difference was statistically significant (**P < 0.01).

[0237] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An extract of Sparassis latifolia, characterized in that The Hydrangea latifolia extract comprises: fucose Fuc, galactose Gal, glucose Glc and mannose Man, wherein the molar mass percentage of glucose in the total polysaccharide components is 80-98%; the molar mass percentage of fucose is 1-5%; and the molar mass percentage of galactose is 3-8%. The extraction method of the Hydrangea latifolia extract comprises the following steps: (1) Extraction: Using water as solvent, enzymatically hydrolyze the raw material of Hydrangea latifolia, then inactivate the enzyme, filter, and concentrate to obtain a crude extract; (2) Purification: The crude extract is purified and decolorized using a macroporous resin, and the eluate is collected, concentrated, and dried to obtain the Hydrangea latifolia extract; Wherein, in step (1), the enzyme used for enzymatic hydrolysis is β-galactosidase; The macroporous resin in the method is LS-109D; In step (1), the enzymatic hydrolysis temperature is 40-60°C; the inactivation temperature is 70-120°C; The amount of enzyme used in step (1) is 2-8% of the mass of the Hydrangea latifolia raw material; The amount of macroporous resin used in step (2) is 1 to 4 times the mass of the crude extract; The Hydrangea latifolia extract has the characteristics of an average molecular mass of 1-5 kda and a Gardner color value of ≤5.

2. The Hydrangea latifolia extract according to claim 1, wherein The Hydrangea latifolia extract also has one or more characteristics selected from the following group: (1) The molar mass percentage of fucose is 2~3%; (2) The molar mass percentage of galactose is 5-6%; and / or (3) The molar mass percentage of glucose is 85~95%.

3. The Hydrangea latifolia extract according to claim 1, wherein In the method, the liquid-to-material ratio of water to raw material is 5-40:1 mL / g.

4. The Hydrangea latifolia extract according to claim 1, wherein In the method, the raw material of Hydrangea latifolia is the fruiting body of Hydrangea latifolia.

5. The Hydrangea latifolia extract according to claim 1, wherein The amount of enzyme used in the method is 4-6% of the mass of the raw material Hydrangea latifolia.

6. The Hydrangea latifolia extract according to claim 1, wherein In the method, the polysaccharide retention rate is ≥30%.

7. The Hydrangea latifolia extract according to claim 1, wherein The conditions extracted in the method have one or more characteristics selected from the following group: (1) The enzymatic hydrolysis temperature is 50~60℃; (2) The liquid-to-material ratio of water to raw material is 15-25:1 mL / g; (3) Enzymatic hydrolysis time is 1~5h; (4) The inactivation temperature is 85-120°C; and / or (5) The number of enzymatic hydrolysis is 1 to 5 times.

8. The use of the Hydrangea latifolia extract according to claim 1, wherein Used for preparing daily products with medical and cosmetic post-operative repair and / or barrier repair effects.

9. The use according to claim 8, characterized in that The product also has one or more characteristics selected from the following group: (1) Inhibit the secretion of inflammatory factors; (2) Repair skin scratch damage; (3) Enhance keratinocyte migration; (4) Reduce collagen loss.

10. The use of the Hydrangea latifolia extract according to claim 1, wherein Used for preparing daily chemical products with medical cosmetic post-operative repair and / or barrier repair effects.

11. A composition, characterized in that The composition comprises: (1) the Hydrangea latifolia extract according to claim 1; and (2) additional ingredients that can be used in daily chemical products.

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