Preparation method of perigord truffle extract and application of perigord truffle extract in skin care products

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method of a perigord truffle extract with whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin barrier repairing effects and application of the perigord truffle extract in skin care products. According to the invention, perigord truffle is taken as a raw material, is extracted by an ethanol aqueous solution, is subjected to vacuum concentration, and is separated and recrystallized by using macroporous resin, so that the perigord truffle extract with rapeseed sterol and ergosterol as main active components is obtained. The perigord truffle extract prepared by the preparation method disclosed by the invention has a remarkable effect of inhibiting the formation of melanin, and meanwhile, the perigord truffle extract also has the effects of resisting inflammation, resisting light aging, resisting wrinkles and repairing skin. The perigord truffle extract provided by the invention can be used for relieving 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 of a black truffle extract having whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin barrier repairing effects, and application of the extract in skin care products. Background Art

[0002] Skin, as the largest organ of the human body, constantly interacts with and is affected by the external environment. Under UV radiation, the skin produces more melanin for self-protection, resulting in tanning. Free radicals and reactive oxygen species triggered by UV rays not only induce acute inflammation such as sun spots, but also penetrate into the dermis of the skin, damaging collagen and elastic fibers, leading to sagging skin and the formation of wrinkles. Photoaging caused by long-term UV exposure can cause discomfort such as dryness, itching, stinging and tightness on the skin, accompanied by problems such as aging, wrinkles, pigmentation and darkening of skin color, which seriously threatens skin health.

[0003] At present, there are many kinds of skin care products on the market, such as whitening, moisturizing, anti-aging, anti-wrinkle, etc. The skin care principles mainly include inhibiting melanin production, repairing skin barrier, controlling free radicals, promoting cell renewal, replenishing collagen and elastin, preventing and repairing UV damage, inhibiting non-enzymatic glycosylation, enhancing cell vitality, promoting blood circulation, etc. However, it is difficult for existing skin care products to effectively combine multiple skin care methods, and the effects are relatively single, making it difficult to achieve the best skin care effect.

[0004] Natural active substances are favored for their safety, effectiveness and non-toxic side effects. They can slow down melanin deposition, enhance skin self-protection, reduce sensitivity and comprehensively protect the skin. Black truffle (Tubersinense), as a natural resource, is rich in nutrients such as protein, fat, polysaccharides, vitamins, organic acids, minerals and phenols, as well as a variety of bioactive ingredients such as anthocyanins, oligosaccharides, flavonoids, pheromones and steroids. The unique aroma of black truffle comes from its complex volatile aromatic compounds, including diacetyl, butyrate, dimethyl disulfide, methyl thiomethane and hexanone. These ingredients not only give black truffles a unique flavor, but also make them have potential application value in the field of skin care.

[0005] Patent document CN113754731A discloses a black truffle antioxidant polypeptide and its preparation method and application. The black truffle antioxidant polypeptide is LC10 and NC10 polypeptides alone or in combination, which has the advantages of good antioxidant performance, good solubility, high stability and low production cost, and can be used in antioxidant drugs and cosmetics.

[0006] Patent document CN114848542A discloses a black truffle cosmetic composition, a black truffle face cream and a preparation method thereof. The black truffle cosmetic composition is a compound of black truffle extract, compound amino acids, ergothioneine aqueous solution, magnolia bark extract, α-arbutin, ascorbic acid, tranexamic acid, and mandelic acid, and various substances are used to play a repairing role. It can play a role for different skin problems alone, and can also synergize the same skin problem. It takes multiple approaches to efficiently repair the skin barrier and return the skin to a healthy state of bright, smooth, and moisturized.

[0007] At present, most of the black truffle extracts obtained by existing technology are crude black truffle extracts or antioxidant peptides. The composition of crude black truffle extracts is complex and unclear, and its skin care effect cannot be guaranteed. The skin care effect of black truffle antioxidant peptides is relatively single, and it cannot take into account multiple skin care effects such as whitening, anti-inflammation, 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 black truffle extract and the obtained product, and in particular, proposes the use of the black truffle extract in the preparation of skin care products for whitening, anti-inflammatory, repairing skin barrier, resisting photoaging and improving skin wrinkles. The black truffle extract provided by the present invention is a black truffle extract with rapeseed sterol and ergosterol as the main active ingredients. It has been confirmed by experiments that the black truffle extract can significantly reduce the melanin content in a zebrafish whitening model. At the same time, the black truffle extract also has the effects of anti-inflammatory, anti-photoaging, anti-wrinkle and repairing skin barrier.

[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 black truffle extract, comprising the following steps:

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

[0012] Step S2, eluting and separating the concentrated solution obtained in step S1 using a macroporous adsorption resin, collecting the eluate, freeze-drying to obtain a crude extract, dissolving the crude extract in an ethanol aqueous solution with a mass concentration of 60% to 95%, and subjecting it to 2 to 3 recrystallizations to obtain the product.

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

[0014] Furthermore, the extraction method 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, and 95% ethanol aqueous solution respectively, collecting the 95% ethanol aqueous solution elution part, and freeze-drying.

[0017] Furthermore, the ethanol aqueous solution in step S2 has a mass concentration of 60% to 95%, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc., preferably an ethanol aqueous solution with a mass concentration of 95%.

[0018] The present invention also claims protection for a black truffle extract obtained by the above-mentioned method for preparing the black truffle extract.

[0019] Furthermore, the total content of rapeseed sterol and ergosterol in the black truffle extract is greater than 60%.

[0020] In addition, the present invention also provides the use of the black truffle extract in the preparation of whitening skin care products, specifically for the preparation of skin care products that inhibit the formation of melanin.

[0021] Furthermore, the present invention also provides the use of the black truffle extract in the preparation of anti-inflammatory and skin barrier repairing skin care products, specifically for the preparation of skin care products that inhibit TNF-α gene expression and promote TGM1 gene expression.

[0022] Furthermore, the present invention also provides the use of the black truffle extract in the preparation of anti-photoaging skin care products, specifically for the preparation of skin care products that remove active oxygen and promote the production of SIRT1 protein.

[0023] Furthermore, the present invention also provides the use of the black truffle extract in the preparation of wrinkle-improving cosmetics, specifically for the preparation of skin care products 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.

[0024] Furthermore, the skin care products 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, skin repair and other effects.

[0025] The preparation method of the black truffle extract provided by the present invention is to use black truffle as a raw material, extract with an ethanol aqueous solution, concentrate under reduced pressure, separate with a macroporous resin, and recrystallize to obtain a black truffle extract with rapeseed sterol and ergosterol as main active components. The black truffle extract prepared by the present invention has a significant effect of inhibiting the formation of melanin, and at the same time, the black truffle extract also has the effects of anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair.

[0026] Specifically, it was found through experiments that when the mass concentration of the black truffle extract (SL) prepared by the present invention was 0.02%, the melanin signal intensity of the zebrafish head was compared with that of the normal control group, and the melanin content was reduced by 38%, which provided strong experimental support for the whitening effect of the black truffle extract. At the same time, the relative expression of the TNF-α gene of the black truffle extract (SL) provided by the present invention was significantly reduced compared with that of the model control group, revealing that the black truffle extract has an anti-inflammatory effect, and the relative expression of the transglutaminase TGM1 gene in the black truffle extract (SL) group was significantly increased compared with that of the normal control group, revealing that the black truffle extract has a barrier repair effect. In addition, the ROS fluorescence intensity of the black truffle extract (SL) group provided by the present invention was significantly weakened compared with that of the model control group; and the SIRT1 protein content was significantly increased compared with that of the model control group, revealing that the black truffle extract has an anti-photoaging effect. The type I collagen content, COL3A1 and ELN gene relative expression levels of the black truffle extract (SL) group were significantly increased compared with the model control group; while the relative expression level of the MMP-1 gene was significantly reduced compared with the model control group, revealing that the black truffle extract has anti-wrinkle effects. It is confirmed that the black truffle extract provided by the present invention can alleviate complex skin care problems such as darkening of skin color, skin damage, photoaging, wrinkles, etc. caused by ultraviolet rays from multiple levels, achieving a comprehensive skin-beautifying effect of whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair.

[0027] In short, compared with the prior art, the preparation method of the black truffle extract provided by the present invention has the advantages of simple operation, low cost and high repeatability. At the same time, the black truffle extract with rapeseed sterol and ergosterol as the main active ingredients prepared by the present invention has the effects of whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and repairing skin barrier, 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, overcomes the defects of the black truffle extract in the prior art that the ingredients are complex and unclear, or the skin care effect is relatively single, and provides a skin care product that can take into account the whitening, anti-inflammatory, anti-photoaging, anti-wrinkle and skin repair effects at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Flow chart for preparing black truffle extract in Example 2.

[0029] Figure 2 This is a graph showing the results of high performance liquid chromatography analysis of the black truffle extract obtained in Example 2.

[0030] Figure 3 This is a graph showing the inhibitory effect of the black truffle extract (SL) prepared in Example 2 on zebrafish melanin.

[0031] Figure 4 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression level of the TNF-α gene.

[0032] Figure 5 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression level of the TGM1 gene.

[0033] Figure 6 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the ROS clearance rate.

[0034] Figure 7 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the SIRT1 protein content.

[0035] Figure 8 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression level of the COL3A1 gene.

[0036] Fig. 9 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression level of the ELN gene.

[0037] Fig.10 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression level of the MMP-1 gene.

[0038] Fig.11This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the content of type I collagen. DETAILED DESCRIPTION

[0039] 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.

[0040] Example 1: Preparation of a black truffle extract

[0041] Step S1, slicing wild black truffles, air-drying, crushing, and ultrasonically extracting twice with an ethanol aqueous solution with a mass concentration of 85%, the power of the ultrasonic extraction being 300 W, each time for 3 hours, combining the extracts, and concentrating under reduced pressure to obtain a concentrated solution;

[0042] Step S2, separating the concentrated solution obtained in step S1 by using HPD-100 macroporous adsorption resin, eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 95% ethanol aqueous solution, respectively, collecting the 95% ethanol aqueous solution elution portion, freeze-drying to obtain a crude extract, dissolving the crude extract in 95% ethanol aqueous solution, and recrystallizing twice to obtain.

[0043] Example 2: Preparation of a black truffle extract

[0044] Step S1, slicing wild black truffles, air-drying, crushing, and ultrasonically extracting with a 95% ethanol aqueous solution for 3 times, the ultrasonic extraction power is 200W, each time for 2 hours, combining the extracts, and concentrating under reduced pressure to obtain a concentrated solution;

[0045] Step S2, separating the concentrated solution obtained in step S1 by using D101 macroporous adsorption resin, eluting with water, 30% ethanol aqueous solution, 60% ethanol aqueous solution, and 95% ethanol aqueous solution, respectively, collecting the 95% ethanol aqueous solution elution portion, freeze-drying to obtain a crude extract, dissolving the crude extract in 95% ethanol aqueous solution, and recrystallizing twice to obtain.

[0046] The preparation process flow chart of the black truffle extract of the present invention is as follows Figure 1 shown.

[0047] Experimental Example 1: Detection of Active Ingredients in Black Truffle Extract

[0048] 1. Experimental materials:

[0049] The black truffle extract prepared in Example 2.

[0050] 2. Experimental methods:

[0051] The black truffle extract prepared in Example 2 was subjected to high performance liquid chromatography-nuclear magnetic resonance analysis to analyze the active ingredients of the black truffle extract.

[0052] 3. Experimental results:

[0053] 3.1. The results of HPLC analysis of the black truffle extract prepared in Example 2 are as follows: Figure 2 shown.

[0054] Figure 2 The figure is the result of HPLC analysis of the black truffle extract obtained in Example 2. Figure 2 It can be seen that the main active ingredients of the black truffle extract prepared in Example 2 of the present invention are brassicasterol and ergosterol. Moreover, the total content of brassicasterol and ergosterol in the black truffle extract is greater than 60%.

[0055] 3.2. The NMR carbon spectrum and mass spectrum data of rapeseed sterol and ergosterol in the black truffle extract prepared in Example 2 are as follows:

[0056] (1) Brassicasterol, 13 C-NMR (600MHz, CDCl 3 ): 140.76,135.84,131.72,121.72,71.82,56.85,56.01,50.16,42.81,42.30,42.24,40.17,39.68,37.26,3 6.52,33.10,31.90,31.66,30.94,28.54,24.29,21.07,20.96,19.96,19.65,19.41,17.62,12.08; HREIMS:C 28 H 46 O,m / z:398.3545(calculated for 398.3543).

[0057] (2) Ergosterol, 13 C-NMR (600MHz, CDCl 3): 141.59,139.99,135.78,132.18,119.80,116.49,70.67,55.93,54.77,46.45,43.03,43.03,41.00,40 .65,39.29,38.58,37.24,33.30,32.20,28.51,23.21,21.32,21.32,20.17,19.86,17.82,16.49,12.26.C 28 H 44 O,m / z:396.3388(calculated for 396.3387).

[0058] Experimental Example 2: Whitening Effect Test of Black Truffle Extract

[0059] 1. Experimental materials:

[0060] The black truffle extract prepared in Example 2 (denoted as SL).

[0061] 2. System and sample size:

[0062] Experimental system: Wild-type AB strain zebrafish.

[0063] Zebrafish age: 6 hours post fertilization (6hpf).

[0064] The sample size of each experimental group was 6 (N=3).

[0065] 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).

[0066] 3. Experimental steps:

[0067] (1) Zebrafish were randomly selected and placed in a 12-well plate, with 6 zebrafish in each well.

[0068] (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.

[0069] (3) Incubate at 28°C in the dark for 45 h.

[0070] (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.

[0071]

[0072] 4. Experimental results:

[0073] The experimental results are shown in Table 1 and Figure 3 shown.

[0074] 4.1. The whitening efficacy test results of black truffle extract (SL) are shown in Table 1.

[0075] Table 1 Whitening effect test results of black truffle extract (SL)

[0076]

[0077] 4.2. The results of the inhibitory effect of black truffle extract (SL) on zebrafish melanin are as follows Figure 3 shown.

[0078] From Table 1 and Figure 3 It can be seen that when the mass concentration of the black truffle extract (SL) prepared in Example 2 of the present invention is 0.02%, the melanin signal intensity of the zebrafish head is compared with that of the normal control group, and the melanin content is reduced by 38%, which has a significant whitening effect.

[0079] Experimental Example 3: Anti-inflammatory Effect Test of Black Truffle Extract

[0080] 1. Experimental materials:

[0081] The black truffle extract prepared in Example 2 (denoted as SL).

[0082] 2. Experimental system:

[0083] Cell line: human immortalized keratinocytes (HaCaT).

[0084] Cell generation number: at least 2 generations.

[0085] The number of cells per group: 6×10 5 Pieces / hole.

[0086] 3. Experimental steps:

[0087] (1) Inoculate cells into 6-well plates (6×10 5 / well), at 37°C, 5% CO 2 Incubate for 24h.

[0088] (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 ).

[0089] (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. The culture medium was continued at 37°C and 5% CO. 2 Incubate for 24h.

[0090] (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.

[0091] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0092] Relative RNA expression = 2 ΔΔC(t)

[0093]

[0094] 4. Experimental results:

[0095] The experimental results are as follows Figure 4 shown.

[0096] Figure 4 The figure is the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression of TNF-α gene. Figure 4 It can be seen that the relative expression level of TNF-α gene in the black truffle extract (SL) group prepared in Example 2 was significantly reduced compared with that in the model control group, revealing that the black truffle extract (SL) has an anti-inflammatory effect.

[0097] Experimental Example 4: Test on the Barrier Repair Effect of Black Truffle Extract

[0098] 1. Experimental materials:

[0099] The black truffle extract prepared in Example 2 (denoted as SL).

[0100] 2. System and sample size:

[0101] Cell line: human immortalized keratinocytes (HaCaT).

[0102] Cell generation number: at least 2 generations.

[0103] The number of cells per group: 6×10 5 Pieces / hole.

[0104] 3. Experimental steps:

[0105] (1) Inoculate cells in a 6-well plate (6×10 5 / well), at 37°C, 5% CO 2 Culture for 24 hours.

[0106] (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 the corresponding concentration of sample was added to the sample group. The cells were incubated at 37°C and 5% CO 2 Culture for 24 hours.

[0107] (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.

[0108] (4) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0109] Relative RNA expression = 2 ΔΔC(t)

[0110]

[0111] ΔC(t)=C(t) 目的基因 -C(t) β-actin

[0112] 4. Experimental results:

[0113] The experimental results are as follows Figure 5 shown.

[0114] Figure 5 The figure is the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression of the TGM1 gene. Figure 5 It can be seen that the relative expression level of the TGM1 gene in the black truffle extract (SL) group prepared in Example 2 was significantly increased compared with that in the normal control group, revealing that the black truffle extract (SL) has a barrier repair effect.

[0115] Experimental Example 5: Anti-photoaging effect test of black truffle extract

[0116] 1. Experimental materials:

[0117] The black truffle extract prepared in Example 2 (denoted as SL).

[0118] 2. Experimental system:

[0119] Cell line: human dermal fibroblasts.

[0120] Cell generation number: at least 2 generations.

[0121] ROS clearance rate Experimental cell number per group: 5×10 4 Pieces / hole.

[0122] SIRT1 protein content Experimental cell quantity per group: 6×10 5 Pieces / hole.

[0123] 3. Experimental steps:

[0124] 3.1 ROS clearance rate:

[0125] (1) Inoculate cells in 12-well plates (5×10 4 / well), at 37°C, 5% CO 2 Incubate for 24 h.

[0126] (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. Continue to incubate at 37°C and 5% CO 2 Incubate for 24 h.

[0127] (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.

[0128] (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.

[0129]

[0130] 3.2. SIRT1 protein content:

[0131] (1) Inoculate cells in a 6-well plate (6×10 5 / well), at 37°C, 5% CO 2 Incubate for 24h.

[0132] (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 ).

[0133] (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. The culture medium was continued at 37°C and 5% CO. 2 Incubate for 24h.

[0134] (4) After incubation, the culture medium was removed, the cells were washed three times with D-Hanks buffer, lysed with RIPA buffer, centrifuged at 12,000 g for 5 min, the supernatant was collected, and the SIRT1 protein content was detected by ELISA.

[0135]

[0136] 4. Experimental results:

[0137] The experimental results are as follows Figure 6 and Figure 7 shown.

[0138] Figure 6 The figure is the effect of the black truffle extract (SL) prepared in Example 2 on the ROS clearance rate. Figure 6 It can be seen that the ROS fluorescence intensity of the black truffle extract (SL) group prepared in Example 2 was significantly weakened compared with the model control group. Figure 7 The figure is the result of the effect of the black truffle extract (SL) prepared in Example 2 on the SIRT1 protein content. Figure 7 It can be seen that the SIRT1 protein content in the black truffle extract (SL) group prepared in Example 2 was significantly increased compared with the model control group.

[0139] The above results revealed that the black truffle extract (SL) has anti-photoaging effects.

[0140] Experimental Example 6: Anti-wrinkle efficacy test of black truffle extract

[0141] 1. Experimental materials:

[0142] The black truffle extract prepared in Example 2 (denoted as SL).

[0143] 2. System and sample size:

[0144] Cell lines: human dermal fibroblasts, human immortalized keratinocytes.

[0145] Cell generation number: at least 2 generations.

[0146] Type I collagen content Experimental cell quantity per group: 2×10 4 Pieces / hole

[0147] COL3A1, ELN and MMP-1 gene expression. Cell number for each experiment: 3×10 5 Pieces / hole.

[0148] 3. Experimental steps:

[0149] 3.1. COL3A1, ELN and MMP-1 gene expression:

[0150] (1) Inoculate cells in 6-well plates (3×10 5 / well), at 37°C, 5% CO 2 Incubate for 24h.

[0151] (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 ).

[0152] (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. The culture medium was continued at 37°C and 5% CO. 2 Incubate for 24h.

[0153] (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.

[0154] (5) Using β-actin as an internal reference for gene expression, calculate the relative RNA expression of the target gene.

[0155] Relative RNA expression = 2 ΔΔC(t)

[0156]

[0157] ΔC(t)=C(t) 目的基因 -C(t) β-actin

[0158] 3.2. Type I collagen content:

[0159] (1) Inoculate cells in 96-well plates (2×10 4 / well), at 37°C, 5% CO 2 Culture for 24 hours.

[0160] (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 ).

[0161] (3) After the irradiation, the D-Hanks tube was removed, and 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. The cells were kept at 37°C and 5% CO. 2 Culture for 24 hours.

[0162] (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.

[0163]

[0164] 4. Experimental results:

[0165] The experimental results are as follows Figure 8 to Figure 11 shown. Figure 8 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression of the COL3A1 gene. Fig. 9This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression of the ELN gene. Fig.10 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the relative expression of the MMP-1 gene. Fig.11 This is a graph showing the effect of the black truffle extract (SL) prepared in Example 2 on the content of type I collagen.

[0166] from Figure 8 to Figure 11 It can be seen that the type I collagen content, COL3A1 and ELN gene relative expressions of the black truffle extract (SL) 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 decreased compared with that of the model control group, revealing that the black truffle extract (SL) prepared in Example 2 has anti-wrinkle effects.

[0167] 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 black truffle extract, characterized in that: The following steps are involved: Step S1, slicing wild black truffles, air-drying, crushing, and extracting with ethanol aqueous solution for 2 to 3 times, each time for 2 to 3 hours, combining the extracts, and concentrating under reduced pressure to obtain a concentrated solution; Step S2, eluting and separating the concentrated solution obtained in step S1 using a macroporous adsorption resin, collecting the eluate, freeze-drying to obtain a crude extract, dissolving the crude extract in an ethanol aqueous solution with a mass concentration of 60% to 95%, and subjecting it to 2 to 3 recrystallizations to obtain the product.

2. The method for preparing the black truffle extract 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 the extraction method is hydrothermal reflux extraction or ultrasonic extraction.

3. The method for preparing the black truffle extract 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.

4. The method for preparing the black truffle extract 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 and 95% ethanol aqueous solution respectively, collecting the 95% ethanol aqueous solution elution part and freeze-drying it.

5. A black truffle extract obtained by the method for preparing the black truffle extract according to any one of claims 1 to 4.

6. The black truffle extract according to claim 5, characterized in that The total content of rapeseed sterol and ergosterol in the black truffle extract is greater than 60%.

7. The use of the black truffle extract according to claim 5 or 6 in preparing whitening skin care products, characterized in that: Used to prepare skin care products that inhibit melanin formation.

8. Use of the black truffle extract according to claim 5 or 6 in the preparation of anti-inflammatory and skin barrier repairing skin care products, characterized in that: Used to prepare skin care products that inhibit TNF-α gene expression and promote TGM1 gene expression.

9. The use of the black truffle extract according to claim 5 or 6 in the preparation of anti-photoaging skin care products, characterized in that: Used to prepare skin care products that remove reactive oxygen and promote the production of SIRT1 protein.

10. Use of the black truffle extract according to claim 5 or 6 in preparing a wrinkle-improving skin care product, characterized in that: Used to prepare skin care products 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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