Preparation method and application of tricholoma matsutake extract with whitening and anti-wrinkle effects

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of a tricholoma matsutake extract with whitening, anti-inflammatory, skin barrier repairing, anti-photoaging and anti-wrinkle effects. According to the invention, tricholoma matsutake is used as a raw material, is extracted with an ethanol aqueous solution, is subjected to vacuum concentration, is separated with macroporous resin, and is subjected to 90% ethanol aqueous solution elution and freeze drying to obtain the tricholoma matsutake extract. The tricholoma matsutake extract prepared by the invention has a remarkable effect of inhibiting melanin formation, and meanwhile, the tricholoma matsutake extract is found to be capable of remarkably improving the brightness of the human skin and improving the skin condition through an ultraviolet-induced human skin blackening model test. In addition, the tricholoma matsutake extract also has the effects of resisting inflammation, repairing skin barrier, resisting light aging and improving skin wrinkles. The tricholoma matsutake extract provided by the invention can relieve complex skin care problems such as skin color darkening, skin damage, light aging and wrinkles caused by ultraviolet rays from multiple levels, and a relatively ideal skin beautifying effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and specifically relates to a preparation method and application of a matsutake extract having whitening, anti-inflammatory, skin barrier repair, anti-photoaging and anti-wrinkle effects. Background Art

[0002] Human skin is constantly in contact with the external environment and is constantly stimulated by it. When exposed to ultraviolet rays, the skin will undergo certain changes to avoid damage, such as melanocytes producing more melanin, which causes the skin to become tanned. In addition, the free radicals and reactive oxygen species produced by ultraviolet rays not only induce inflammation, but also damage the skin to the dermis, causing collagen damage and elastic fiber problems, making the skin loose and wrinkles appear. If long-term exposure to the sun causes photoaging, the skin will experience symptoms such as dryness, itching, stinging, and tightness, accompanied by aging, wrinkles, spots, darkening of the skin color, and other problems, which seriously affect skin health.

[0003] Active substances from natural sources have attracted widespread attention because they are safe, effective, non-toxic, harmless and have no side effects. They can effectively slow down the deposition of melanin, enhance the skin's self-protection ability, reduce skin sensitivity, and protect the skin in all directions. Tricholoma matsutake, also known as pine mushroom, pine mushroom, and pine mushroom. Tricholoma matsutake is a species endemic to Asia, mainly distributed in Japan and Northeast, Southwest and Taiwan Province of my country. Tricholoma matsutake contains a variety of active ingredients, including proteins, polysaccharides, amino acids, polypeptides, steroids, terpenes, steroids, volatile oils, phenols and vitamins. According to literature reports, Tricholoma matsutake has multiple functions such as whitening, anti-oxidation, anti-tumor, anti-mutation, anti-radiation, anti-bacterial, lowering cholesterol, lowering blood sugar, and promoting gastrointestinal motility.

[0004] Patent document CN106265254A discloses a pine mushroom extract that can whiten, moisturize and repair the skin, as well as its preparation method and application. The preparation method is to first extract the alcohol- and water-soluble components in the pine mushroom with ethanol and water, recover the ethanol and filter it, then use butanediol and water to dissolve the water- and alcohol-soluble components in the pine mushroom respectively, then mix them evenly, and concentrate and purify them to obtain the product. The product adopts a full-ingredient extraction method, which can retain the active ingredients of natural pine mushroom to the maximum extent, has the effects of whitening, repairing and moisturizing, and can be used in skin care product compositions.

[0005] Patent document CN118045011A discloses the use of a matsutake extract in the preparation of a whitening beauty product. The matsutake extract can be used as a tyrosinase inhibitor and a melanin synthesis inhibitor in the preparation of a whitening skin care product.

[0006] Patent document CN118593390A discloses a pine mushroom extract with whitening effect and a preparation method thereof. The method uses pine mushroom as raw material and obtains it through supercritical fluid extraction. The extract has very excellent biological activity, can reduce melanin secretion, has significant whitening effect, and can be further applied to the development of skin care cosmetics.

[0007] However, the skin care effects of the pine mushroom extract products disclosed in the prior art are relatively single and cannot meet the consumer's demand for comprehensive skin care products. Therefore, it is of great market value to research and develop a pine mushroom extract product that can simultaneously take into account multiple skin care effects such as whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair. Summary of the invention

[0008] In order to solve the defects of the prior art, the present invention proposes a method for preparing a pine mushroom extract with whitening and anti-wrinkle effects and the obtained product, especially the use of pine mushroom extract in the preparation of cosmetics for whitening, anti-inflammatory, skin barrier repair, anti-photoaging and wrinkle improvement. Experiments have confirmed that the pine mushroom extract prepared by the present invention can significantly reduce the melanin content and has a significant whitening effect; in the ultraviolet induced human skin melanization model, the skin brightness of the volunteers was improved and the condition was significantly improved. At the same time, the pine mushroom extract also has the effects of anti-inflammatory, anti-photoaging, anti-wrinkle and skin barrier repair, which can fully meet the needs of consumers for comprehensive skin care products.

[0009] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for preparing a pine mushroom extract having whitening and anti-wrinkle effects, comprising the following steps:

[0011] Step S1, slicing wild matsutake mushrooms, air-drying them, crushing them, and then extracting them with ethanol aqueous solution for 2 to 3 times, each time for 2 to 3 hours, combining the extracts, and concentrating them under reduced pressure to obtain a concentrated solution;

[0012] Step S2, using a macroporous adsorption resin to elute and separate the concentrated solution obtained in step S1, collecting the eluate, and freeze-drying to obtain.

[0013] Furthermore, in step S1, the mass concentration of ethanol in the ethanol aqueous solution is 60% to 95%, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc., and preferably, the mass concentration of ethanol in the ethanol aqueous solution is 95%.

[0014] Furthermore, the extraction method in step S1 is a hydrothermal reflux extraction method or an ultrasonic extraction method, preferably an ultrasonic extraction method. The extraction temperature of the hydrothermal reflux extraction is 40 to 60°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, etc., and the extraction power of the ultrasonic extraction is 100 to 300W, such as 100W, 200W, 300W, etc.

[0015] Furthermore, in step S2, the macroporous adsorption resin is a non-polar macroporous adsorption resin or a medium-polar macroporous adsorption resin, selected from one or more of the following: HPD-100 macroporous adsorption resin, HPD400 macroporous adsorption resin, D101 macroporous adsorption resin, D4020 macroporous adsorption resin, LSA-21 macroporous adsorption resin, DM301 macroporous adsorption resin, HZ816 macroporous adsorption resin or HP-20 macroporous adsorption resin, preferably D-101 macroporous adsorption resin.

[0016] Furthermore, the elution step in step S2 is: eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution respectively, collecting the 90% ethanol aqueous solution elution part, and freeze-drying.

[0017] The present invention also claims protection for a Tricholoma matsutake extract obtained by the preparation method of the Tricholoma matsutake extract having whitening and anti-wrinkle effects.

[0018] In addition, the present invention also provides the use of the Tricholoma matsutake extract in the preparation of whitening cosmetics, specifically for the preparation of cosmetics that inhibit the formation of melanin.

[0019] Furthermore, the present invention also provides the use of the Tricholoma matsutake extract in the preparation of anti-inflammatory and skin barrier repairing cosmetics, specifically for the preparation of cosmetics that inhibit TNF-α gene expression and promote TGM1 gene expression.

[0020] Furthermore, the present invention also provides the use of the Tricholoma matsutake extract in the preparation of anti-photoaging cosmetics, specifically for the preparation of cosmetics that remove active oxygen and promote the production of SIRT1 protein.

[0021] Furthermore, the present invention also provides the use of the Tricholoma matsutake extract in the preparation of wrinkle-improving cosmetics, specifically for the preparation of cosmetics that promote the production of type I collagen, promote the expression of COL3A1 gene and ELN gene, and inhibit the expression of MMP-1 gene.

[0022] Furthermore, the cosmetics are skin care products such as creams, lotions, toners, facial cleansers, gels, facial masks or essences that have whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair effects.

[0023] The preparation method of the pine mushroom extract with whitening and anti-wrinkle effects provided by the present invention is to use pine mushroom as raw material, extract with ethanol aqueous solution, concentrate under reduced pressure, separate with macroporous resin, collect the 90% ethanol aqueous solution elution part, and freeze-dry to obtain. The pine mushroom extract prepared by the present invention has a significant effect of inhibiting the formation of melanin, and at the same time, the pine mushroom extract also has the effects of anti-inflammation, repairing skin barrier, resisting photoaging and improving skin wrinkles.

[0024] Specifically, it was found through experiments that when the mass concentration of the matsutake extract (SR) provided by the present invention was 0.01%, the melanin signal intensity of the zebrafish head was 58% lower than that of the normal control group, revealing that the matsutake extract has a significant whitening effect. At the same time, in the ultraviolet-induced human skin melanization model, after volunteers applied the lotion containing matsutake extract for 4 weeks, the difference in skin ITA° value before and after application was significantly different from that of the negative control (P < 0.05); the difference in skin MI value before and after application was significantly different from that of the negative control (P < 0.05); the difference in visual skin color grade before and after application was significantly different from that of the negative control (P < 0.05); compared with the negative control, the regression coefficients of skin ITA°, MI and visual skin color grade of the matsutake extract test product were significantly different (P < 0.05), proving that the matsutake extract test product has the effect of removing spots and whitening.

[0025] Further, the relative expression of TNF-α gene in the pine mushroom extract (SR) group provided by the present invention was significantly reduced compared with the model control group, revealing that the pine mushroom extract has an anti-inflammatory effect. At the same time, the relative expression of transglutaminase TGM1 gene in the pine mushroom extract (SR) group was significantly increased compared with the normal control group, revealing that the pine mushroom extract has a barrier repair effect. In addition, the ROS fluorescence intensity of the pine mushroom extract (SR) group provided by the present invention was significantly weakened compared with the model control group; while the SIRT1 protein content was significantly increased compared with the model control group, revealing that the pine mushroom extract has an anti-photoaging effect. The type I collagen content, COL3A1 and ELN gene relative expression of the pine mushroom extract (SR) group provided by the present invention were significantly increased compared with the model control group; while the MMP-1 gene relative expression was significantly reduced compared with the model control group, revealing that the pine mushroom extract has an anti-wrinkle effect. It is confirmed that the pine mushroom extract provided by the present invention can alleviate the complex skin care problems such as darkening of skin color, skin damage, photoaging, wrinkles, etc. caused by ultraviolet rays from multiple levels, and achieve a comprehensive skin-beautifying effect of whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair.

[0026] In summary, compared with the prior art, the preparation method of the pine mushroom extract with whitening and anti-wrinkle effects provided by the present invention has the advantages of simple operation, low cost and high repeatability. At the same time, the pine mushroom extract prepared by the present invention has the effects of whitening, anti-inflammatory, repairing skin barrier, anti-photoaging and improving skin wrinkles, and can alleviate the complex skin care problems such as darkening of skin color, skin damage, photoaging, wrinkles, etc. caused by ultraviolet rays from multiple levels, solving the technical defect of the prior art that it is impossible to reasonably combine multiple skin beautification methods to achieve an ideal skin beautification effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of preparing the Matsutake extract in Example 2.

[0028] Figure 2 This is a graph showing the inhibitory effect of the Matsutake extract (SR) prepared in Example 2 on zebrafish melanin.

[0029] Figure 3 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression level of the TNF-α gene.

[0030] Figure 4 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression level of the TGM1 gene.

[0031] Figure 5 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the ROS clearance rate.

[0032] Figure 6 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the SIRT1 protein content.

[0033] Figure 7 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression level of the COL3A1 gene.

[0034] Figure 8 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression level of the ELN gene.

[0035] Fig. 9 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression level of the MMP-1 gene.

[0036] Fig.10 This is a graph showing the effect of the Matsutake extract (SR) prepared in Example 2 on the content of type I collagen. DETAILED DESCRIPTION

[0037] The present invention is further described below by describing specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention. The materials involved in the present invention can be obtained by commercially available or conventional technical means in the art.

[0038] Example 1: Preparation of a Tricholoma matsutake extract with whitening and anti-wrinkle effects

[0039] Step S1, slicing wild matsutake mushrooms, air-drying them, crushing them, and then ultrasonically extracting them twice with an ethanol aqueous solution with a mass concentration of 80%, the power of the ultrasonic extraction being 100 W, each time for 3 hours, combining the extracts, and concentrating them under reduced pressure to obtain a concentrate;

[0040] Step S2, separating the concentrated solution obtained in step S1 by using HPD400 macroporous adsorption resin, and eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution, respectively, collecting the 90% ethanol aqueous solution elution portion, and freeze-drying to obtain.

[0041] Example 2: Preparation of a Tricholoma matsutake extract with whitening and anti-wrinkle effects

[0042] Step S1, slicing wild matsutake mushrooms, air-drying them, crushing them, and then ultrasonically extracting them three times with an ethanol aqueous solution having a mass concentration of 95%, the power of the ultrasonic extraction being 200 W, each time for 2 hours, combining the extracts, and concentrating them under reduced pressure to obtain a concentrate;

[0043] Step S2, separating the concentrated solution obtained in step S1 by using D-101 macroporous adsorption resin, and eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution, respectively, collecting the 90% ethanol aqueous solution elution portion, and freeze-drying to obtain.

[0044] The preparation process flow chart of the Tricholoma matsutake extract of the present invention is as follows Figure 1 shown.

[0045] Experimental Example 1: Whitening effect test of Matsutake extract

[0046] 1. Experimental materials:

[0047] The Tricholoma matsutake extract prepared in Example 2 (denoted as SR).

[0048] 2. System and sample size:

[0049] Experimental system: Wild-type AB strain zebrafish.

[0050] Zebrafish age: 6 hours post fertilization (6hpf).

[0051] The sample size of each experimental group was 6 (N=3).

[0052] Adult fish rearing and breeding methods: follow laboratory standard rearing and breeding methods, in line with the requirements of the international AAALAC certification (certification number: 001458).

[0053] 3. Experimental steps:

[0054] (1) Zebrafish were randomly selected and placed in a 12-well plate, with 6 zebrafish in each well.

[0055] (2) Water-soluble samples were administered and a normal control group was set up at the same time. The volume of each well was 1 mL.

[0056] (3) Incubate at 28°C in the dark for 45 h.

[0057] (4) Three zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The melanin signal intensity (S) of the zebrafish head was analyzed and the formula was used to calculate and determine whether the sample had a whitening effect.

[0058]

[0059] 4. Experimental results:

[0060] The experimental results are shown in Table 1 and Figure 2 shown.

[0061] 4.1. The results of the whitening efficacy test of Tricholoma matsutake extract (SR) are shown in Table 1.

[0062] Table 1 Whitening effect test results of Tricholoma matsutake extract (SR)

[0063]

[0064] 4.2. The results of the inhibitory effect of Matsutake extract (SR) on zebrafish melanin are as follows Figure 2 shown.

[0065] From Table 1 and Figure 2 It can be seen that when the mass concentration of the Tricholoma matsutake extract (SR) prepared in Example 2 of the present invention is 0.01%, the melanin signal intensity of the zebrafish head is compared with that of the normal control group, and the melanin content is reduced by 58%, which has a significant whitening effect.

[0066] Experimental Example 2: Anti-inflammatory Effect Test of Matsutake Extract

[0067] 1. Experimental materials:

[0068] The Tricholoma matsutake extract prepared in Example 2 (denoted as SR).

[0069] 2. Experimental system:

[0070] Cell line: human immortalized keratinocytes (HaCaT).

[0071] Cell generation number: at least 2 generations.

[0072] The number of cells per group: 6×10 5 Pieces / hole.

[0073] 3. Experimental steps:

[0074] (1) Inoculate cells into 6-well plates (6×10 5 / well) and incubated at 37°C, 5% CO2 for 24 h.

[0075] (2) After the incubation, the culture medium was removed, and the D-Hanks wash was performed 1 to 2 times. Then, the sample group and the model control group were exposed to ultraviolet light (80 mJ / cm 2 ).

[0076] (3) After modeling, the normal control group and the model control group were replaced with fresh culture medium, and the sample group was added with fresh culture medium containing samples, and incubated at 37°C, 5% CO2 for 24 h.

[0077] (4) Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0078] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0079]

[0080] 4. Experimental results:

[0081] The experimental results are as follows Figure 3 shown.

[0082] Figure 3 The figure is the result of the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression of TNF-α gene. Figure 3 It can be seen that the relative expression level of TNF-α gene in the Tricholoma matsutake extract (SR) group prepared in Example 2 was significantly reduced compared with that in the model control group, indicating that the Tricholoma matsutake extract (SR) has an anti-inflammatory effect.

[0083] Experimental Example 3: Test on the Barrier Repair Effect of Matsutake Extract

[0084] 1. Experimental materials:

[0085] The Tricholoma matsutake extract prepared in Example 2 (denoted as SR).

[0086] 2. System and sample size:

[0087] Cell line: human immortalized keratinocytes (HaCaT).

[0088] Cell generation number: at least 2 generations.

[0089] The number of cells per group: 6×10 5 Pieces / hole.

[0090] 3. Experimental steps:

[0091] (1) Inoculate cells in a 6-well plate (6×10 5 / well) and cultured at 37°C, 5% CO2 for 24 h.

[0092] (2) After the incubation, the cells were gently rinsed once or twice with D-Hanks, fresh culture medium was added to the normal control group, and fresh culture medium containing samples of corresponding concentrations was added to the sample group, and cultured at 37°C, 5% CO2 for 24 h.

[0093] (3) Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0094] (4) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0095]

[0096] 4. Experimental results:

[0097] The experimental results are as follows Figure 4 shown.

[0098] Figure 4 The figure is the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression of the TGM1 gene. Figure 4 It can be seen that the relative expression level of the TGM1 gene in the Tricholoma matsutake extract (SR) group prepared in Example 2 was significantly increased compared with that in the normal control group, revealing that the Tricholoma matsutake extract (SR) has a barrier repair effect.

[0099] Experimental Example 4: Anti-photoaging effect test of Matsutake extract

[0100] 1. Experimental materials:

[0101] The Tricholoma matsutake extract prepared in Example 2 (denoted as SR).

[0102] 2. Experimental system:

[0103] Cell line: human dermal fibroblasts.

[0104] Cell generation number: at least 2 generations.

[0105] ROS clearance rate Experimental cell number per group: 5×10 4 Pieces / hole.

[0106] SIRT1 protein content Experimental cell quantity per group: 6×10 5 Pieces / hole.

[0107] 3. Experimental steps:

[0108] 3.1 ROS clearance rate:

[0109] (1) Inoculate cells in 12-well plates (5×10 4 / well) and incubated at 37°C, 5% CO2 for 24 h.

[0110] (2) Sample administration: Remove the culture medium and gently rinse the cells once or twice with D-Hanks. Replace the culture medium with fresh medium for the normal control group and the model control group, and replace the culture medium with fresh medium containing the sample for the sample group, and continue to incubate at 37°C, 5% CO2 for 24 hours.

[0111] (3) Modeling: After the incubation, the culture medium was removed, the cells were gently rinsed once or twice with D-Hanks, and then the modeling process was performed. The model control group and the sample group were irradiated with UVA (5 J / cm 2 ), and the normal control group was placed in the dark.

[0112] (4) After modeling, the cells were stained with a reactive oxygen species detection kit and photographed under a fluorescence microscope. Image J was used to analyze the fluorescence intensity (S) of each group of cells, and the ROS clearance rate of the sample group was calculated according to the formula.

[0113]

[0114] 3.2. SIRT1 protein content:

[0115] (1) Inoculate cells in a 6-well plate (6×10 5 / well) and incubated at 37°C, 5% CO2 for 24 h.

[0116] (2) After the incubation, the culture medium was removed, and the cells were washed with D-Hanks for 1 to 2 times. Then, the sample group and the model control group were irradiated with UVA (5 J / cm 2 ).

[0117] (3) After modeling, the normal control group and the model control group were replaced with fresh culture medium, and the sample group was added with fresh culture medium containing samples, and incubated at 37°C, 5% CO2 for 24 h.

[0118] (4) After incubation, the culture medium was removed, the cells were washed three times with D-Hanks, RIPA lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000g for 5 min. The supernatant was collected and the SIRT1 protein content was detected by ELISA.

[0119]

[0120] 4. Experimental results:

[0121] The experimental results are as follows Figure 5 and Figure 6 shown.

[0122] Figure 5 The figure is the effect of the pine mushroom extract (SR) prepared in Example 2 on the ROS clearance rate. Figure 5 It can be seen that the ROS fluorescence intensity of the Tricholoma matsutake extract (SR) group prepared in Example 2 was significantly weakened compared with the model control group. Figure 6 The figure is the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the SIRT1 protein content. Figure 6 It can be seen that the SIRT1 protein content in the Tricholoma matsutake extract (SR) group prepared in Example 2 was significantly increased compared with the model control group.

[0123] The above results revealed that the Matsutake extract (SR) has anti-photoaging effects.

[0124] Experimental Example 5: Anti-wrinkle efficacy test of Matsutake extract

[0125] 1. Experimental materials:

[0126] The Tricholoma matsutake extract prepared in Example 2 (denoted as SR).

[0127] 2. System and sample size:

[0128] Cell lines: human dermal fibroblasts, human immortalized keratinocytes.

[0129] Cell generation number: at least 2 generations.

[0130] Type I collagen content Experimental cell quantity per group: 2×10 4 Pieces / hole

[0131] COL3A1, ELN and MMP-1 gene expression. Cell number for each experiment: 3×10 5 Pieces / hole.

[0132] 3. Experimental steps:

[0133] 3.1. COL3A1, ELN and MMP-1 gene expression:

[0134] (1) Inoculate cells in 6-well plates (3×10 5 / well) and incubated at 37°C, 5% CO2 for 24 h.

[0135] (2) After the incubation, the culture medium was removed, and the cells were washed with D-Hanks for 1 to 2 times. Then, the sample group and the model control group were irradiated with UVA (5 J / cm 2 ).

[0136] (3) After modeling, the normal control group and the model control group were replaced with fresh culture medium, and fresh culture medium containing samples was added to the sample wells, and incubated at 37°C, 5% CO2 for 24 h.

[0137] (4) Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0138] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0139]

[0140] 3.2. Type I collagen content:

[0141] (1) Inoculate cells in 96-well plates (2×10 4 / well) and cultured at 37°C, 5% CO2 for 24 h.

[0142] (2) After the incubation, the culture medium was removed and washed with D-Hanks 1-2 times. 200 μL of D-Hanks was added to the wells to immerse the cells. The normal control group was wrapped with tin foil and placed in a dark place. The model control group and the sample group were irradiated with UVA (5 J / cm 2 ).

[0143] (3) After the radiation, the D-Hanks were removed, fresh culture medium was added to the normal control group and the model control group, and fresh culture medium containing the sample was added to the sample group, and the cells were cultured at 37°C and 5% CO2 for 24 h.

[0144] (4) After the incubation, 200 μL of cell culture supernatant was collected from each well, and the type I collagen content (T) was detected by ELISA.

[0145]

[0146] 4. Experimental results:

[0147] The experimental results are as follows Figure 7 to Figure 10 shown. Figure 7 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression of the COL3A1 gene. Figure 8 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression of the ELN gene. Fig. 9 This is a graph showing the effect of the Tricholoma matsutake extract (SR) prepared in Example 2 on the relative expression of the MMP-1 gene. Fig.10 This is a graph showing the effect of the Matsutake extract (SR) prepared in Example 2 on the content of type I collagen.

[0148] from Figure 7 to Figure 10 It can be seen that the type I collagen content, COL3A1 and ELN gene relative expressions of the Matsutake extract (SR) group prepared in Example 2 were significantly increased compared with those of the model control group; the relative expression of the MMP-1 gene was significantly reduced compared with that of the model control group, revealing that the Matsutake extract (SR) has anti-wrinkle effects.

[0149] Experimental Example 6: Effect of Matsutake Extract on Ultraviolet-Induced Melanization of Human Skin

[0150] 1. Experimental materials:

[0151] Test product: an emulsion containing the Tricholoma matsutake extract (SR) prepared in Example 2.

[0152] The emulsion is composed of the following ingredients and their mass percentages: 0.3% of the matsutake extract (SR) prepared in Example 2, 0.5% of the German Shumei PE9010 preservative, 1.5% of the SSE20 emulsifier, 2% of polydimethylsiloxane, 5% of coconut oil alcohol-caprylate / caprate, 2% of glycerol, 0.3% of SEPINOV EMT10 thickener, 0.3% of polyacrylate cross-linked polymer-6, 65% of glycerol polyether-2, 0.03% of disodium EDTA, and 83.07% of water.

[0153] Negative control: blank control of blackened area.

[0154] Positive control: 7% ascorbic acid (vitamin C) preparation, 7% ascorbic acid prepared according to the formula in Appendix I.

[0155] Subjects: A total of 20 subjects, including 3 males and 17 females, aged 23 to 57 years old, with an average age of 45.70±9.34 years old, met the voluntary inclusion criteria.

[0156] 2. Experimental methods:

[0157] (1) Tests were conducted in accordance with the specific requirements of the "Technical Specifications for Safety of Cosmetics" (2015 edition). The non-exposed areas of the subjects' thighs or backs were selected as the test sites. The minimum erythema dose (MED value) of the subjects' skin to ultraviolet radiation was predicted 24 hours before the test. The subjects were irradiated once a day at the same irradiation point with a xenon arc lamp using a daylight simulator at a dose of 0.75 times the MED for 4 consecutive days. The 4 days after the irradiation were the skin melanization period and no treatment was performed. On the 5th day after the irradiation, the skin color of each test area was visually evaluated and instrumentally tested, and the test areas with poor consistency (areas where the difference between the ITA° value and the mean of all test areas was greater than 5) were eliminated. Starting from the same day, the corresponding test substance was applied to each melanization test area according to the random table. The test substance was applied for 4 consecutive weeks. The skin color was visually evaluated and instrumentally tested 1 week, 2 weeks, 3 weeks and 4 weeks after the application, and the results were recorded.

[0158] (2) Statistical methods: Statistical analysis software was used for statistical analysis of data. Measurement data were expressed as mean ± standard deviation and tested for normal distribution. If the data met the normal distribution requirements, the paired t test was used for comparison before and after the data, otherwise the two related sample rank sum test was used. The two related sample rank sum test was used for comparison before and after the rank data. The independent sample t test or rank sum test was used for comparison between the test product and the control group. At the same time, the regression coefficient of each parameter changing over time was calculated, and the significance level was P < 0.05.

[0159] 3. Experimental results:

[0160] The experimental results are shown in Tables 2 to 4.

[0161] 3.1. The effects of the Tricholoma matsutake extract (SR) prepared in Example 2 on the individual type angle, melanin index and visual skin color grade in the ultraviolet-induced human skin melanization model are shown in Table 2.

[0162] Table 2 Effects of Matsutake extract (SR) on individual type angle, melanin index and visual skin color grade in the UV-induced human skin melanization model

[0163]

[0164] 3.2. The results of the time difference analysis between the test product (SR) and the negative control before and after application are shown in Table 3.

[0165] Table 3 Analysis results of time difference between the test product (SR) and the negative control before and after application

[0166]

[0167] 3.3. The regression coefficient analysis results of the test product (SR) and the negative control are shown in Table 4.

[0168] Table 4 Regression coefficient analysis results of test product (SR) and negative control

[0169]

[0170] It can be seen from Tables 2 to 4 that after the test product (SR) was applied for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in skin ITA° value before and after application was significantly different from that of the negative control (P < 0.05); after the test product (SR) was applied for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in MI before and after application was significantly different from that of the negative control (P < 0.05); after the test product (SR) was applied for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in visual skin color grade before and after application was significantly different from that of the negative control (P < 0.05); compared with the negative control, the regression coefficients of ITA°, MI and visual skin color grade of the test product (SR) were significantly different (P < 0.05), which proved that the test product (SR) has the effect of removing spots and whitening.

[0171] The above are only further embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which belong to the protection scope of the present invention.

Claims

1. A method for preparing a pine mushroom extract having whitening and anti-wrinkle effects, characterized in that: The following steps are involved: Step S1, slicing wild matsutake mushrooms, air-drying them, crushing them, and then extracting them with ethanol aqueous solution for 2 to 3 times, each time for 2 to 3 hours, combining the extracts, and concentrating them under reduced pressure to obtain a concentrated solution; Step S2, using a macroporous adsorption resin to elute and separate the concentrated solution obtained in step S1, collecting the eluate, and freeze-drying to obtain.

2. The method for preparing the Tricholoma matsutake extract with whitening and anti-wrinkle effects according to claim 1, characterized in that: The mass concentration of ethanol in the ethanol aqueous solution in step S1 is 60% to 95%, and preferably the mass concentration of ethanol in the ethanol aqueous solution is 95%.

3. The method for preparing the Tricholoma matsutake extract with whitening and anti-wrinkle effects according to claim 1, characterized in that: The extraction method in step S1 is hydrothermal reflux extraction or ultrasonic extraction, preferably ultrasonic extraction.

4. The method for preparing the Tricholoma matsutake extract with whitening and anti-wrinkle effects according to claim 1, characterized in that: In step S2, the macroporous adsorption resin is a non-polar macroporous adsorption resin or a medium-polar macroporous adsorption resin, selected from one or more of the following: HPD-100 macroporous adsorption resin, HPD400 macroporous adsorption resin, D101 macroporous adsorption resin, D4020 macroporous adsorption resin, LSA-21 macroporous adsorption resin, DM301 macroporous adsorption resin, HZ816 macroporous adsorption resin or HP-20 macroporous adsorption resin, preferably D-101 macroporous adsorption resin.

5. The method for preparing the Tricholoma matsutake extract with whitening and anti-wrinkle effects according to claim 1, characterized in that: The elution step in step S2 is: eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, 90% ethanol aqueous solution, and 95% ethanol aqueous solution respectively, collecting the 90% ethanol aqueous solution elution part, and freeze-drying.

6. A Tricholoma matsutake extract obtained by the method for preparing a Tricholoma matsutake extract with whitening and anti-wrinkle effects as claimed in any one of claims 1 to 5.

7. The use of the Tricholoma matsutake extract in the preparation of whitening cosmetics according to claim 6, characterized in that: Used to prepare cosmetics that inhibit melanin formation.

8. Use of the Tricholoma matsutake extract according to claim 6 in the preparation of anti-inflammatory and skin barrier repairing cosmetics, characterized in that: Used to prepare cosmetics that inhibit TNF-α gene expression and promote TGM1 gene expression.

9. The use of the Tricholoma matsutake extract in the preparation of anti-photoaging cosmetics according to claim 6, characterized in that: Used to prepare cosmetics that remove reactive oxygen species and promote the production of SIRT1 protein.

10. The use of the Tricholoma matsutake extract in the preparation of wrinkle-improving cosmetics according to claim 6, characterized in that: Used to prepare cosmetics that promote the production of type I collagen, promote the expression of COL3A1 gene and ELN gene, and inhibit the expression of MMP-1 gene.

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