A kind of matsutake extract with whitening effect and preparation method thereof

Abstract

The present invention provides a pine mushroom extract with whitening effect and a preparation method thereof. Specifically, the pine mushroom extract is obtained by supercritical fluid extraction using pine mushroom as a raw material. The extract has very excellent biological activity, reduces melanin secretion, has significant whitening effect, and can be further applied to the development of skin care cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic application development, and specifically relates to a matsutake extract with whitening effect and a preparation method thereof. Background Art

[0002] As a precious edible wild mushroom, Matsutake has a long history of consumption in China. In recent years, due to its precious morphological characteristics, it has been used by more and more cosmetic companies; currently, the extraction method of Matsutake extract in the field of daily chemicals is mainly the traditional extraction method, and Matsutake extract is mainly used for anti-aging.

[0003] The patent "Whitening effect of a Matsutake extract composition" (CN111110614A) describes a Matsutake extract composition obtained by mixing Matsutake, oats, Tremella fuciformis, and Pueraria lobata with water in proportion, heating and stirring, and filtering to remove impurities; the composition has whitening effect to a certain extent, but its extraction method is traditional water heating extraction, and it belongs to a mixed extraction of the composition.

[0004] The patent "A whitening and freckle-removing cream and its preparation method" (CN115554224A) describes a whitening and freckle-removing cream including ergothioneine pine mushroom extract. Although the invention has whitening and freckle-removing effects, it belongs to the field of cosmetic preparations.

[0005] The patent "A whitening composition with added pine mushroom fermentation extract, preparation method and application" (CN113413336A) describes a method of fermenting pine mushroom raw materials with Lactobacillus rhamnosus to obtain pine mushroom fermentation extract, and mixing it with Yunnan Camellia Sinensis, Yunnan Polygonatum, pomegranate seeds, and Sichuan pepper extracts in proportion to obtain a whitening composition with added pine mushroom fermentation extract; although the invented composition has whitening effect, the fermentation process time is long and the equipment occupancy rate is high. And it belongs to the mixed extraction of the composition.

[0006] Therefore, there is an urgent need in the art to develop a pine mushroom extract that is extracted from a single substance, has a simple extraction process, and has whitening effects. Summary of the invention

[0007] The invention provides a matsutake extract with whitening effect and a preparation method thereof.

[0008] In a first aspect of the present invention, a pine mushroom extract is provided. The pine mushroom extract is prepared from pine mushrooms by supercritical extraction, wherein the content of β-sitosterol acetate in the pine mushroom extract is 25-30%, based on the mass of the pine mushroom extract.

[0009] In another preferred embodiment, the content of quassin hemiacetal C in the Tricholoma matsutake extract is 4-5wt%.

[0010] In another preferred embodiment, the content of 7,7"-dihydroxy-6,6'-dimethoxy-3,3'-dicoumarol in the Tricholoma matsutake extract is 3-4wt%.

[0011] In another preferred embodiment, the content of lactucin in the Tricholoma matsutake extract is 3-4wt%.

[0012] In another preferred embodiment, in the Tricholoma matsutake extract, the content of 1,8-dihydroxy-9,10-anthrone-3-methyl-(2-hydroxy) propionate is 3-4wt%.

[0013] In another preferred embodiment, the content of 6-aldehyde isoflavone A in the Tricholoma matsutake extract is 3-4wt%.

[0014] In another preferred embodiment, the content of vitamin B1 in the Tricholoma matsutake extract is 2.5-3.5wt%.

[0015] In another preferred embodiment, the content of conoside in the Tricholoma matsutake extract is 2.5-3.5wt%.

[0016] In another preferred embodiment, the content of Ophiopogon japonicus flavonoids A in the Tricholoma matsutake extract is 2-3wt%.

[0017] In another preferred embodiment, the content of 1-Acetyl-3-(methoxy-carbonyl)-β-carboline in the Tricholoma matsutake extract is 2-3 wt %.

[0018] In another preferred embodiment, the Tricholoma matsutake extract is used to inhibit tyrosine kinase activity.

[0019] In another preferred embodiment, the inhibition rate of the Tricholoma matsutake extract on tyrosinase is 20-40%.

[0020] In another preferred embodiment, the inhibition rate of the Tricholoma matsutake extract on tyrosinase is in a dose-effect relationship with the concentration.

[0021] In another preferred embodiment, the higher the concentration of the Tricholoma matsutake extract, the higher the inhibition rate of tyrosinase.

[0022] In another preferred embodiment, when the Tricholoma matsutake extract is at a low concentration (<10 μg / mL), the relative cell viability is >95%, preferably >98%, more preferably >99%, and most preferably 100%.

[0023] In another preferred embodiment, when the Tricholoma matsutake extract is at a high concentration (80-120 μg / mL), the relative cell viability is >65%, preferably >68%, more preferably >69%, and most preferably 70%.

[0024] In another preferred embodiment, the Tricholoma matsutake extract has the effect of inhibiting melanin secretion.

[0025] In another preferred embodiment, the Tricholoma matsutake extract has the effect of inhibiting the secretion of melanin by B16-F10 melanoma cells.

[0026] In another preferred embodiment, the melanin reduction amount of the Tricholoma matsutake extract is 40-60%.

[0027] In another preferred embodiment, the melanin reduction amount of the Tricholoma matsutake extract at a concentration of 6.25 μg / mL is 52-60%.

[0028] In another preferred embodiment, the Tricholoma matsutake extract has a whitening effect.

[0029] In another preferred embodiment, the Matsutake extract exhibits significant whitening effect in a zebrafish whitening test model.

[0030] In another preferred embodiment, the whitening effect of the Tricholoma matsutake extract is 20-40%.

[0031] In another preferred embodiment, the whitening effect of the Tricholoma matsutake extract at a low concentration is 20-30%.

[0032] In another preferred embodiment, the low concentration of the Tricholoma matsutake extract is 50 μg / mL.

[0033] In another preferred embodiment, the whitening effect of the Tricholoma matsutake extract at a high concentration is 35-40%.

[0034] In another preferred embodiment, the high concentration of the Tricholoma matsutake extract is 200 μg / mL.

[0035] In another preferred embodiment, the Tricholoma matsutake extract is prepared by the following method:

[0036] The pulverized matsutake powder is subjected to supercritical carbon dioxide extraction, wherein the extraction conditions are as follows: the extraction kettle pressure is 20-30 MPa, the temperature is 45-55°C; the first separation kettle pressure is 7-9 MPa; the second separation kettle pressure is 5-6 MPa; and the CO2 flow rate is 55-65 kg / h.

[0037] In another preferred embodiment, the extraction conditions further include: the extraction cycle time is 1-3h.

[0038] In another preferred embodiment, the Tricholoma matsutake powder is a coarse powder of 20-30 mesh.

[0039] In another preferred embodiment, the method further comprises: before the extraction, forming a dispersion with the Tricholoma matsutake powder and a solvent, and then extracting the dispersion.

[0040] In another preferred embodiment, the solvent is selected from the following group: anhydrous ethanol.

[0041] In another preferred embodiment, the dispersion comprises 5-15 wt % of Tricholoma matsutake powder, preferably 8-12 wt %.

[0042] In a second aspect of the present invention, a method for preparing the Tricholoma matsutake extract according to the first aspect of the present invention is provided, the method comprising the steps of:

[0043] (S1) providing matsutake raw materials;

[0044] (S2) subjecting the Tricholoma matsutake raw material to supercritical extraction to obtain Tricholoma matsutake extract.

[0045] In another preferred embodiment, the Tricholoma matsutake extract is an oil-soluble component of Tricholoma matsutake.

[0046] In another preferred embodiment, the Tricholoma matsutake raw material is dried Tricholoma matsutake slices.

[0047] In another preferred embodiment, the (S1) further comprises the optional step of crushing the Tricholoma matsutake raw material.

[0048] In another preferred embodiment, the method for extracting the Tricholoma matsutake extract further comprises the optional step (S1a) of uniformly mixing the crushed Tricholoma matsutake raw material with 1 to 20% wt of anhydrous ethanol.

[0049] In another preferred embodiment, the method for extracting the Tricholoma matsutake extract further comprises the optional step (S3) of extracting the Tricholoma matsutake raw material again after supercritical extraction to obtain components with greater polarity contained in the Tricholoma matsutake.

[0050] In another preferred embodiment, the re-extraction includes but is not limited to traditional alcohol / water extraction means.

[0051] In another preferred embodiment, in the supercritical extraction described in step (S2), the supercritical extraction agent is CO2, and the CO2 flow rate is 50-70 kg / h.

[0052] In another preferred embodiment, the extraction temperature in the supercritical extraction in step (S2) is 40-60°C, preferably 45-55°C.

[0053] In another preferred embodiment, in the supercritical extraction described in step (S2), the flow rate of supercritical CO2 is 50-70 kg / h, preferably 60 kg / h.

[0054] In another preferred embodiment, the extraction time of the supercritical extraction in step (S2) is 2-3 hours, preferably 2 hours.

[0055] In another preferred embodiment, the extraction pressure of the supercritical extraction in step (S2) is 10-40 MPa, preferably 15-30 MPa, and more preferably 25 MPa.

[0056] In another preferred embodiment, step (S2) includes: passing the used materials through a first separation kettle and a second separation kettle in sequence.

[0057] In another preferred embodiment, in the supercritical extraction, the separation pressure of the first separation kettle is 5-10 MPa, preferably 7-9 MPa;

[0058] The separation pressure of the second separation kettle is 2-10 MPa, preferably 5-6 MPa.

[0059] In the third aspect of the present invention, there is provided a use of the Tricholoma matsutake extract as described in the first aspect of the present invention, which is used for preparing a product containing the Tricholoma matsutake extract.

[0060] In another preferred embodiment, the product is a daily chemical product.

[0061] In another preferred embodiment, the daily chemical product further contains additional ingredients that can be used in daily chemical products.

[0062] In another preferred embodiment, the product includes but is not limited to: lotion, essence, essence milk, facial cream, and toner.

[0063] In another preferred embodiment, the product has a whitening effect.

[0064] In another preferred embodiment, the product has the effect of inhibiting tyrosinase activity.

[0065] In another preferred embodiment, the product has the effect of inhibiting melanin secretion.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The GC / MS positive ion spectrum of supercritical extract A of Tricholoma matsutake is shown.

[0068] Figure 2 The GC / MS negative ion spectrum of supercritical extract A of Tricholoma matsutake is shown.

[0069] Figure 3 Shown is the tyrosinase inhibition assay of different Matsutake extracts.

[0070] Figure 4The effect of Tricholoma matsutake extract on the viability of B16-F10 melanoma cells is shown.

[0071] Figure 5 The effect of Matsutake extract on melanin secretion by B16-F10 melanoma cells is shown.

[0072] Figure 6 Shows the results of the zebrafish skin whitening efficacy test of Matsutake supercritical extract A. DETAILED DESCRIPTION

[0073] After extensive and in-depth research, the inventor unexpectedly developed a matsutake extract with whitening effect for the first time. The extract is prepared by supercritical extraction method, the preparation method is simple and there is no solvent residue, the cytotoxicity is low, and the extract has very excellent biological activity, which can inhibit tyrosinase and reduce the formation of melanin in B16-F10 melanoma cells at extremely low concentrations, and has significant whitening effect, which can be further applied to the development of skin care cosmetics.

[0074] Terminology

[0075] Tyrosinase

[0076] Tyrosinase, also known as polyphenol oxidase, is a copper-containing oxidoreductase of about 75 ku. It is widely present in animals, plants, microorganisms and the human body. It is the rate-limiting enzyme for melanin synthesis and directly affects the synthesis of melanin.

[0077] Tyrosine generates dopa under the action of tyrosinase, and then finally generates melanin through a series of steps. Melanin makes animals appear darker, especially black and brown, and sometimes yellow. The expression and activity of tyrosinase determine the speed and output of melanin production. The higher the activity of tyrosinase, the more melanin is formed in the skin. Under the catalytic action of tyrosinase, tyrosine is oxidized to dopaquinone; dopaquinone is automatically oxidized to generate dopa and dopachrome. Dopa is also a substrate of tyrosinase, which is catalyzed to generate dopaquinone again; the reaction products of dopachrome, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindolecarboxylic acid (DHICA), are oxidized to generate eumelanin, which is the main component of skin pigment; in the presence of cysteine ​​or glutathione, dopaquinone is converted into cysteinyl dopa, and finally generates pheomelanin.

[0078] Tyrosinase has multiple biological functions. In recent years, its application in medicine, beauty, food, environmental protection and other fields has attracted widespread attention at home and abroad.

[0079] B16-F10 melanoma cell line

[0080] The B16-F10 melanoma subcutaneous tumor model is simple to operate, low in cost, highly reproducible, and has a relatively short experimental cycle. It is a commonly used animal model for studying melanoma. It can be used to study the role of target genes, drugs, etc. in the occurrence and development of melanoma.

[0081] Matsutake

[0082] Matsutake is the fruiting body of the fungus Tricholoma matsutake Sing of the family Trichostomaceae. Its chemical composition is also known as pine mushroom and pine fungus. It is named because it grows in pine forests and its buds are shaped like deer antlers. It is recorded in my country's "Jingshi Zhenglei Beiji Bencao", "Jiu Pu", "Bencao Gangmu", and "Botanical Names and Realities Illustrated Long Edition"; and in Japan's Heian period ancient books "The Pillow Book", "Murasaki Shikibu Diary", and "Tosa Diary". In my country, it is mainly produced in the coniferous broad-leaved forest area at an altitude of 3,000m in the northeast region and the border area of ​​Guizhou, Yunnan, and Sichuan. It is a national second-level endangered species and a precious natural wild medicinal mushroom in the world.

[0083] Matsutake is a precious fungus with high nutritional and medicinal value. It has a complex composition, containing polysaccharides, amino acids, proteins, peptides, steroids, terpenes, oils and a variety of enzymes, and has a variety of biological activities. According to literature records, Matsutake has the effects of strengthening the body, tonifying the kidney and strengthening yang, benefiting the stomach and intestines, regulating qi, resolving phlegm and expelling parasites. Modern medicine shows that Matsutake also has anti-tumor, antibacterial, antiviral, diabetes treatment, whitening, and immune regulation.

[0084] Matsutake Extract

[0085] The pine mushroom extract of the present invention is extracted from pine mushrooms using supercritical fluid extraction to obtain a pine mushroom extract extract. The prepared pine mushroom extract extract is used to perform a whitening efficacy test.

[0086] In the present invention, the "Matsutake extract" and "Matsutake extract concentrate" can be used interchangeably.

[0087] Preparation method of pine mushroom extract

[0088] The Tricholoma matsutake extract of the present invention is prepared by the following method:

[0089] (S1) providing matsutake raw materials;

[0090] (S2) subjecting the Tricholoma matsutake raw material to supercritical extraction to obtain Tricholoma matsutake extract.

[0091] In the present invention, the Tricholoma matsutake extract is an oil-soluble component of Tricholoma matsutake.

[0092] In the present invention, the Tricholoma matsutake raw material is dried Tricholoma matsutake slices.

[0093] In the present invention, the (S1) further comprises crushing the Tricholoma matsutake raw material.

[0094] In the present invention, the method for extracting the Tricholoma matsutake extract further comprises the optional step (S1a) of uniformly mixing the crushed Tricholoma matsutake raw material with 1 to 20% wt of anhydrous ethanol.

[0095] In another preferred embodiment, the extraction method of the pine mushroom extract further comprises the optional step (S3) of re-extracting the pine mushroom raw material after supercritical extraction to obtain the components with higher polarity contained in the pine mushroom. The extraction means include but are not limited to the traditional alcohol / water extraction means.

[0096] Uses of Matsutake Extract

[0097] The Tricholoma matsutake extract of the present invention can inhibit tyrosinase activity and reduce melanin secretion of B16-F10 melanoma cells at a relatively low concentration, thereby achieving a whitening effect.

[0098] The Tricholoma matsutake extract of the present invention can be used to prepare products containing the Tricholoma matsutake extract.

[0099] In the present invention, the product is a daily chemical product.

[0100] In the present invention, the daily chemical product further contains additional ingredients that can be used in daily chemical products.

[0101] In the present invention, the products include but are not limited to: lotion, essence, essence milk, face cream, and toner.

[0102] In the present invention, the product has whitening effect.

[0103] In another preferred embodiment, the product has the effect of inhibiting tyrosinase activity.

[0104] In another preferred embodiment, the product has the effect of inhibiting melanin secretion.

[0105] Compared with the prior art, the present invention has the following beneficial effects:

[0106] (1) The extract has excellent biological activity and can inhibit tyrosinase and reduce the formation of melanin in B16-F10 melanoma cells at extremely low concentrations.

[0107] (2) The preparation method of the extract is simple, low-cost, and suitable for industrial expansion production.

[0108] (3) The preparation method of the extract is in a low-temperature state throughout the process, the active ingredients are not destroyed, and the active ingredient extraction efficiency is high.

[0109] (4) The preparation method of the extract is novel, has no traditional solvent residue, is simple to post-process, and has clean ingredients.

[0110] (5) The extract preparation method can also use the traditional alcohol / water extraction method to extract the components with higher polarity from the waste after extraction, thereby achieving "zero" waste of raw materials to the greatest extent.

[0111] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0112] Example 1 Preparation of Tricholoma matsutake extract

[0113] 1.1 Preparation of supercritical extract A of Tricholoma matsutake

[0114] Take 2948g of dry Matsutake slices, crush them into 20-30 mesh coarse powder, add 294.8mL (10%wt) anhydrous ethanol and mix evenly; the pressure of the extraction kettle is 25Mpa, and the temperature is 50°C; the pressure of separation kettle I: 7-9Mpa; the pressure of separation kettle II: 5-6Mpa; CO2 flow rate: 60kg / h; extraction cycle time: 2h; collect the extract, and after rotary evaporation to remove ethanol, obtain Matsutake supercritical extract A, weighing 77.5g, with a yield of 2.63%.

[0115] The mass spectrum of supercritical extract A of Matsutake is shown in Figure 1 and Figure 2 shown.

[0116] 1.2 Preparation of supercritical extract B of Tricholoma matsutake

[0117] Take 3344g of dried Matsutake slices and crush them into 20-30 mesh coarse powder; the pressure of the extraction kettle is 25Mpa and the temperature is 50°C; the pressure of separation kettle I is 7-9Mpa; the pressure of separation kettle II is 5-6Mpa; the CO2 flow rate is 60kg / h; the extraction cycle time is 2h; collect the extract and obtain

[0118] The supercritical extract B of Tricholoma matsutake weighs 5 g and has a yield of 0.45%.

[0119] Comparative Example 1: Matsutake extract obtained by other methods

[0120] 1.3 Matsutake water extract

[0121] Take 50g of dried Matsutake slices and crush them into 20-30 mesh coarse powder; add 500mL of purified water, heat and reflux to extract for 1h, filter and add 500mL of purified water to the residue again, heat and reflux to extract for 1h, filter, combine the two filtrates, concentrate and dry to obtain Matsutake water extract, weighing 14.86g, with a yield of 29.72%.

[0122] 1.4 Matsutake 60% ethanol extract

[0123] Take 50g of dried Matsutake slices and crush them into 20-30 mesh coarse powder; add 500mL of 60% ethanol, heat and reflux to extract for 1h, filter and add 500mL of 60% ethanol to the residue again, heat and reflux to extract for 1h, filter, combine the two filtrates, concentrate and dry to obtain 10.54g of Matsutake water extract, with a yield of 21.08%.

[0124] 1.5 Matsutake 85% ethanol extract

[0125] Take 50g of dried Matsutake slices and crush them into 20-30 mesh coarse powder; add 500mL of 85% ethanol, heat and reflux to extract for 1h, filter and add 500mL of 85% ethanol to the residue again, heat and reflux to extract for 1h, filter, combine the two filtrates, concentrate and dry, and obtain 10.98g of Matsutake water extract with a yield of 21.96%.

[0126] Some extraction conditions of different Matsutake extracts can be seen in Table 1 below.

[0127] Table 1 Extraction conditions of different pine mushroom extracts

[0128]

[0129] Example 2 Component Analysis of Matsutake Extract

[0130] GC / MS was used to analyze component A of supercritical extract of Tricholoma matsutake. The analysis results are shown in Table 2 below.

[0131] Table 2 GC / MS analysis results of supercritical extract A of Tricholoma matsutake

[0132]

[0133]

[0134]

[0135] The analysis results show that supercritical extract A of Tricholoma matsutake is an extract with complex components, containing up to 75 compounds under LC / MS detection conditions.

[0136] Biological Activity Example 1 Tyrosine Inhibition Experiment of Matsutake Extract

[0137] 1. Test reagents and preparation methods

[0138] (1) PBS buffer (0.1 M, pH = 6.8)

[0139] Reagent A: weigh 7.16 g Na2HPO4·12H2O and dilute to 100 mL with distilled water;

[0140] Reagent B: weigh 3.12 g NaH2PO4·2H2O and dilute to 100 mL with distilled water; or weigh 2.72 g KH2PO4 and dilute to 100 mL;

[0141] Take 49mL of reagent A and 51mL of reagent B, mix them evenly, and dilute them by half (add 100mL of distilled water).

[0142] (2) L-Tyrosine solution

[0143] Accurately weigh 4.5 mg of L-tyrosine and dilute to 50 mL with the PBS buffer obtained by the above method.

[0144] (3) Tyrosinase solution

[0145] Accurately weigh 5 mg of tyrosinase and add 10 mL of the PBS buffer obtained by the above method using a pipette.

[0146] (4) 1% β-arbutinol solution (positive control)

[0147] Weigh 0.1 g of β-arbutin and dilute to 10 mL with 10% ethanol solution.

[0148] 1.2 Test methods

[0149] (1) Preparation of test samples

[0150] Use PBS buffer to prepare a solution of appropriate concentration, the specific concentration is shown in Table 3, and then the test can be carried out.

[0151] (2) Detection process control group: Take 250 μL PBS buffer, add 750 μL L-tyrosine solution, and keep warm at 37°C for 30 min; add 50 μL tyrosinase solution, react at 37°C for 30 min, and measure its absorbance at 450 nm.

[0152] Experimental group: Take 25μL, 50μL, 125μL and 250μL of the test solution respectively, make up to 250μL with PBS buffer, add 750μL of L-tyrosine solution, and keep warm at 37℃ for 30min; add 50μL of tyrosinase solution, react at 37℃ for 30min, and measure the absorbance at 450nm.

[0153] Blank group: take 25μL, 50μL, 125μL and 250μL of the test solution respectively, make up to 250μL with PBS buffer, add 750μL of L-tyrosine solution, and keep warm at 37℃ for 30min; add 50μL of PBS buffer, react at 37℃ for 30min, and measure the absorbance at 450nm.

[0154] Positive control group: take 25μL, 50μL, 125μL and 250μL of arbutinol solution respectively, make up to 250μL with PBS buffer, add 750μL of L-tyrosine solution, and keep warm at 37℃ for 30min; add 50μL of tyrosinase solution, react at 37℃ for 30min, and measure the absorbance at 450nm.

[0155] 1.3 Experimental Results

[0156] The experimental results are shown in Table 3 and Figure 3 It can be seen that in the tyrosinase inhibition test, the inhibition rate of Matsutake supercritical extract A and Matsutake 85% ethanol extract was higher at the final test concentration, and the inhibition rate showed an increasing trend with the increase of the test concentration, which had a certain dose-effect relationship.

[0157] Table 3 Inhibitory effects of different pine mushroom extracts on tyrosinase

[0158]

[0159] Biological Activity Example 2 Evaluation of the Effect of Matsutake Extract on Cell Viability

[0160] The MTT method was used to determine the maximum safe concentration of the extract.

[0161] (1) Inoculation: When the cell density is ≥70%, discard the old culture medium, add 5 mL of PBS to wash the cells, digest with trypsin for 1 min, pipette the cells to resuspend, add RPMI-1640 complete culture medium to terminate digestion, mix well and transfer to a 2 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, discard the supernatant; add 1 mL of complete culture medium again, pipette and mix well, dilute appropriately and count, and adjust the cell density to 8x10 based on the counting results. 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% CO 2 ).

[0162] (2) Experimental grouping: A negative control group and an experimental group were set up, and two wells without cells were set up as zero wells. Six concentration gradients were set for the samples in the experimental group, and two replicate wells were set up under each concentration gradient;

[0163] (3) Solution preparation: According to the experimental design, different concentrations of the test substances were prepared in the basal culture medium. The specific concentrations are shown in Table 4 below:

[0164] Table 4 Concentration settings of different pine mushroom extracts

[0165]

[0166] (4) Administration: After 24 hours, take out the 96-well plate, discard the old culture medium, add 200 μL of basal culture medium to each well of the zero well and the blank control group, and add 200 μL of the sample prepared with basal culture medium to each well of the experimental group. Plate the plate according to 2 replicate wells per group and per concentration. Then return to the incubator for culture (37°C, 5% CO2);

[0167] (5) Detection: 24 h after drug treatment, remove the 96-well plate, add 20 μL of MTT working 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, add 150 μL of DMSO to each well, shake for 10 min, and measure the absorbance (OD value) at a wavelength of 490 nm.

[0168] (6) Calculation of relative cell viability: Cell viability % = (OD value of the experimental group - OD value of the zero-adjustment well) / (OD value of the blank control group - OD value of the zero-adjustment group) * 100%.

[0169] The experimental results are as follows Figure 4 As shown, supercritical extract A of Tricholoma matsutake showed low toxicity, and the relative cell viability was close to 100% at low concentration (6.25 μg / mL), and it could also maintain a high relative cell viability of more than 70% at high concentration (100 μg / mL). Compared with supercritical extract A of Tricholoma matsutake, 85% alcohol extract of Tricholoma matsutake showed higher cytotoxicity, which may be due to the influence of residual organic solvents.

[0170] Biological Activity Example 3 Evaluation of the Effect of Matsutake Extract on Melanin Secretion

[0171] The B16-F10 melanoma mouse cells were used as a model to evaluate the effect of Tricholoma matsutake extract on melanin secretion.

[0172] 3.1 Experimental steps (1) Experimental groups: The specific experimental groups in the experiment are shown in Table 5.

[0173] Table 5 Experimental grouping

[0174]

[0175] (2) Inoculation: When the cell density is ≥70%, discard the old culture medium, add 5 mL of PBS to wash the cells, digest with trypsin for 1 min, pipette the cells to resuspend, add RPMI-1640 complete culture medium to terminate digestion, mix and transfer to a 2 mL centrifuge tube, centrifuge at 1000 rpm for 4 minutes, discard the supernatant; add 1 mL of complete culture medium again, pipette and mix, dilute appropriately and count, adjust the cell density to 3.5 x 105 cells / mL based on the counting results, inoculate the cells into a 6-well plate at a volume of 2 mL per well, and return to the incubator for incubation (37°C, 5% CO2)

[0176] (3) Plating: Grouping was performed as shown in Table 5, and plating was performed with 2 replicate wells per group and per concentration;

[0177] (4) Solution preparation: According to the cell viability test results obtained in Biological Activity Example 2, select an appropriate concentration as the test concentration of the experimental group samples; use β-arbutin (200 μg / mL) and kojic acid (100 μg / mL) as positive controls for the experiment.

[0178] (5) Administration: After 24 hours, the 6-well plate was removed, the old culture medium was discarded, the cells were washed with PBS, and then the samples were added to 2 replicate wells per group at each concentration. The negative control group (NC) was divided into 2 replicate wells, and 2 mL of basal culture medium was added to each well. The positive control group (PC) was divided into 2 replicate wells, and 2 mL of β-arbutin (200 μg / mL) and kojic acid (100 μg / mL) were added to each well. The experimental group was divided into 2 replicate wells per concentration, and 2 mL of the corresponding concentration sample was added to each well. Then the cells were returned to the incubator for co-culture (37°C, 5% CO2).

[0179] (6) Melanoma cell collection: 24 hours after administration, the 6-well plate was removed and the supernatant was discarded. After washing twice with PBS, 500 μL of trypsin was added to each well. After 1 min, the cells were blown to resuspend. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 10 min. The supernatant was discarded and 200 μL of 1 mol / L sodium hydroxide solution was added to each tube. The tube was placed in an 80°C water bath for 1 h and vortexed to mix. 150 μL of the plate was taken from each well and placed in a 96-well plate. The 1 mol / L sodium hydroxide solution was used as a blank and the absorbance (OD) was measured at a wavelength of 405 nm.

[0180] (7) Result settlement: Melanin content = (OD value of experimental group - OD value of blank well) / (OD value of negative control group - OD value of blank well) * 100%.

[0181] 3.2 Experimental Results

[0182] The results are as follows Figure 5As shown in Table 6 below, supercritical extract A of Tricholoma matsutake can significantly inhibit the secretion of melanin by melanoma cells at a low test concentration. The reduction in melanin by supercritical extract A of Tricholoma matsutake at a test concentration of 6.25 μg / mL can reach the efficacy of 85% ethanol extract of Tricholoma matsutake at a test concentration of 50 μg / mL.

[0183] Table 6 Melanin inhibition test results

[0184]

[0185] Biological Activity Example 4 Evaluation of the Whitening Effect of Matsutake Extract

[0186] The skin-whitening efficacy of Tricholoma matsutake extract was evaluated using a zebrafish model.

[0187] 4.1 System and samples

[0188] (1) Experimental system: wild-type AB strain zebrafish.

[0189] (2) Zebrafish age: 6 hours post fertilization (6 hpf).

[0190] (3) Sample size for each experiment: 15 (N=10).

[0191] (4) Adult fish rearing and breeding methods: The methods are in accordance with the company's laboratory standard rearing and breeding methods, which meet the requirements of the international AAALAC certification (certification number: 001458).

[0192] 4.2 Principle and method

[0193] The zebrafish is transparent in the early stages of development, and melanin begins to grow from the retinal epithelium at 24 hours of embryonic development. Pigment cells originate from a group of cells differentiated from the dorsal ectoderm - neural crest cells, which then proliferate, migrate, and differentiate into pigment mother cells. Intervention in the process of melanin formation can inhibit the formation of melanin. The whiteness of zebrafish skin is used to evaluate whether the sample has whitening effect.

[0194] 4.3 Experimental procedures (1) Randomly select zebrafish and place them in a 6-well plate, with 15 zebrafish in each well.

[0195] (2) The samples were dispersed and administered, and a normal control group was set up at the same time. The volume of each well was 3 mL.

[0196] (3) Incubate at 28°C in the dark for 45 h.

[0197] (4) Ten 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 whitening efficacy. (Judgment basis: statistical analysis p < 0.05, efficacy value ≥ 20%, determined to be effective.)

[0198]

[0199] 4.4 Experimental Results

[0200] The experimental results are shown in Table 7 and Figure 6 As shown, supercritical extract A of Matsutake mushroom showed significant whitening effect in the zebrafish whitening test model. It had a certain whitening effect at a low concentration of 50μg / mL, and as the test concentration increased, the effect increased, showing a dose-effect relationship.

[0201] Table 7 Whitening effect of Matsutake extract

[0202]

[0203] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A Tricholoma matsutake extract, characterized in that The Tricholoma matsutake extract is prepared by supercritical extraction using Tricholoma matsutake as a raw material, and the Tricholoma matsutake extract is prepared by the following method: Take 2948g of dried Matsutake slices, crush them into 20-30 mesh coarse powder, add 294.8mL of anhydrous ethanol and mix evenly; the pressure of the extraction kettle is 25Mpa, and the temperature is 50℃; the pressure of separation kettle I: 7-9Mpa; the pressure of separation kettle II: 5-6Mpa; CO2 flow rate: 60kg / h; extraction cycle time: 2h; collect the extract, and after rotary evaporation to remove ethanol, obtain Matsutake extract.

2. The Tricholoma matsutake extract according to claim 1, characterized in that The matsutake extract has the activity of inhibiting tyrosine kinase; The pine mushroom extract has the effect of inhibiting the secretion of melanin, and the pine mushroom extract has the effect of inhibiting the secretion of melanin by B16-F10 melanoma cells.

3. A method for preparing a Tricholoma matsutake extract as claimed in claim 1, characterized in that: The method comprises the steps of: Take 2948g of dried Matsutake slices, crush them into 20-30 mesh coarse powder, add 294.8mL of anhydrous ethanol and mix evenly; the pressure of the extraction kettle is 25Mpa, and the temperature is 50℃; the pressure of separation kettle I: 7-9Mpa; the pressure of separation kettle II: 5-6Mpa; CO2 flow rate: 60kg / h; extraction cycle time: 2h; collect the extract, and after rotary evaporation to remove ethanol, obtain Matsutake extract.

4. The method according to claim 3, characterized in that The extraction method of the pine mushroom extract also includes: extracting the pine mushroom raw material after supercritical extraction again to obtain the water-soluble components contained in the pine mushroom.

5. A use of the Tricholoma matsutake extract as claimed in any one of claims 1 to 2, characterized in that: Used for preparing products containing pine mushroom extract; the products are daily chemical products selected from the following group: lotion, facial cream, lotion.

6. The use according to claim 5, characterized in that The daily chemical product is an essence.

7. The use according to claim 5, characterized in that The daily chemical product is an essence lotion.

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