Musella lasiocarpa Extract and Its Application in Preparing Products with Antioxidant, Anti-aging and Whitening Effects

Abstract

The present invention relates to the technical field of plant extracts, and particularly to the application of extracts of Musella lasiocarpa in the preparation of products with antioxidant, anti-aging, and whitening effects. The present invention provides the application of a preparation containing at least one of C1-C6 compounds in the preparation of products with antioxidant, whitening, and / or anti-aging effects. Research shows that the crude extracts of the flowers and flower buds of Musella lasiocarpa, their refined components, and the compounds C1-C6 therein can effectively scavenge DPPH free radicals, down-regulate the expression of genes and proteins related to aging (such as MMP1 gene and MMP1 protein), up-regulate the expression of anti-aging genes (COL1 gene), and inhibit the activity of tyrosinase, having good antioxidant, whitening, and anti-aging effects, and having broad application prospects in the fields of skin care products, health care products, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and particularly to extracts of Musella lasiocarpa and their application in the preparation of products with antioxidant, anti-aging, and whitening effects. Background Art

[0002] Free radicals refer to molecules or atoms with unpaired electrons. Common free radicals include superoxide radicals (O2⁻), hydroxyl radicals (OH·), and lipid peroxidation radicals, etc. In modern life, more and more external factors lead to an increase in our free radicals. For example, environmental toxins such as air pollution, heavy metals, and industrial waste, as well as dietary health imbalances, increased life stress, smoking, second-hand smoke, radiation, and so on. These free radicals are extremely active and will wantonly attack biological macromolecules such as lipids, proteins, and DNA in skin cells, accelerating skin aging and causing problems such as skin dullness, relaxation, and the appearance of fine lines. Antioxidation, whitening, and anti-aging have always been the three key efficacy demands that have attracted much attention.

[0003] Currently, there are a wide variety of whitening and anti-aging products on the market, and their efficacy and quality vary. With the continuous increase in consumers' requirements for skin health and appearance, and the in-depth penetration of technology in the cosmetics industry, the research and development of highly efficient and safe antioxidant, whitening, and anti-aging products have become a hot topic in the industry.

[0004] Therefore, there is still a broad market prospect for providing a product with significant antioxidant, whitening, and anti-aging effects and high safety. Summary of the Invention

[0005] In view of this, the present invention provides extracts of Musella lasiocarpa and their application in the preparation of products with antioxidant, anti-aging, and whitening effects.

[0006] The present invention provides the application of extracts of Musella lasiocarpa containing at least one of C1-C6 compounds in the preparation of antioxidant, whitening, and / or anti-aging products:

[0007] C1 compound: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumaroyl sucrose;

[0008] C2 compound: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose;

[0009] C3 compound: mumeose K;

[0010] C4 compound: daucosterol linoleate;

[0011] C5 compound: glycerol-1-eicosanoate-3-(4E,7E,10E-hexadecatrienoic acid);

[0012] C6 compound: 2-linolenoyl-rac-glycerol;

[0013] The Musella lasiocarpa extract is an ethanol extract of the flowers and flower buds of Musella lasiocarpa.

[0014] In the present invention, in C1-C6 compounds, C is the abbreviation of compound, and C1-C6 compounds are compound1-compound6.

[0015] In the present invention, the Musella lasiocarpa extract includes a Musella lasiocarpa crude extract or a Musella lasiocarpa refined component.

[0016] In some embodiments, the Musella lasiocarpa extract is a Musella lasiocarpa crude extract, which is prepared by the following method: taking the flowers and flower buds of Musella lasiocarpa, extracting them by cold maceration with 75% ethanol, and concentrating them under reduced pressure to dryness to obtain the Musella lasiocarpa crude extract.

[0017] In some specific embodiments, the preparation method of the Musella lasiocarpa crude extract includes: taking the flowers and flower buds of Musella lasiocarpa, extracting them by cold maceration with 3 times the volume of 75% ethanol three times, 24 hours each time, and concentrating the extract to dryness.

[0018] In some embodiments, the concentration of the Musella lasiocarpa crude extract is 0.1-10 mg / ml, specifically 0.1 mg / ml, 0.2 mg / ml, 0.37 mg / ml, and also includes other values between 0.1-10 mg / ml.

[0019] In some other specific embodiments, the Musella lasiocarpa extract is a Musella lasiocarpa refined component, denoted as RC1-RC6 in the present invention, where RC is the abbreviation of Refined component. The RC1-RC6 are prepared by the following method: extracting the flowers and flower buds of Musella lasiocarpa by cold maceration with 3 times the volume of 75% ethanol three times, 24 hours each time, and concentrating the extract to dryness to obtain the Musella lasiocarpa crude extract; loading the Musella lasiocarpa crude extract onto an MCI column and performing gradient elution successively with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution, and 100% methanol aqueous solution to obtain the Musella lasiocarpa refined components RC1-RC6 respectively.

[0020] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation;

[0021] RC2 is the fraction eluted with 20% methanol aqueous solution after MCI chromatography separation;

[0022] RC3 is the fraction eluted with 40% methanol aqueous solution after MCI chromatography separation;

[0023] RC4 is the fraction eluted with 60% methanol aqueous solution after MCI chromatography separation;

[0024] RC5 is the fraction eluted with 80% aqueous methanol solution after MCI chromatography separation;

[0025] RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0026] In some other embodiments, the preparation is one or a combination of two or more of the C1-C6 compounds. In a specific embodiment of the present invention, the C1-C6 compounds are separated from the above-mentioned RC2, RC3, RC4, RC5, and RC6. The specific separation method is as follows:

[0027] The refined component RC2 is loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9:1 - 8:2), petroleum ether-isopropanol (volume ratio 8:2 - 7:3), ethyl acetate:methanol 9.8:0.2 to separate C4.

[0028] The refined component RC4 is loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2 - 8:2), dichloromethane-isopropanol (volume ratio 9.8:0.2 - 9:1), petroleum ether-isopropanol (volume ratio 8:2 - 6:4); then loaded onto a Sephdex column and eluted with methanol to separate C3.

[0029] The refined component RC5 is loaded onto a silica gel column and eluted with dichloromethane-acetone (volume ratio 9:1 - 8:2), dichloromethane-methanol (volume ratio 9:1), petroleum ether-acetone (volume ratio 8:2 - 7:3) to separate C1.

[0030] The refined component RC6 is loaded onto a silica gel column and eluted with dichloromethane-methanol (volume ratio 9.8:0.2 - 9:1), dichloromethane-acetone, petroleum ether-acetone (volume ratio 9:1 - 7:3), petroleum ether-ethyl acetate (volume ratio 9:1 - 8:2); then loaded onto a Sephdex column and eluted with dichloromethane-methanol (volume ratio 1:1) for multiple separations to obtain the refined compounds C2, C5, and C6.

[0031] Specifically, the name of compound C1 is (1,2',3',4',6'-O-pentaacetyl-3-O-trans-p-coumaroylsucrose, CAS No: 1392307-46-8), that is, Mumeose D, Chinese name: 1, 2', 3', 4', 6'-penta-O-acetyl-3-O-(E)-p-coumaroylsucrose, structural formula: C 31 H 38 O 18 , and the specific structure is as follows:

[0032] .

[0033] Compound C2: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose, CAS No: 1603815-30-0), Chinese name: 2', 3', 4', 6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose, structural formula: C 25 H 32 O 15 , and the specific structure is as follows:

[0034] .

[0035] Compound C3: mumeose K, CAS No: 2132384-01-9,

[0036] structural formula: C 25 H 32 O 15 , and the specific structure is as follows:

[0037] .

[0038] Compound C4: daucosterol linoleate, CAS No: 79380-30-6,

[0039] Chinese name: daucosterol linoleate, 3-O-(6'-O-linoleoyl-β-D-glucosyl)-β-sitosterol; structural formula: C 53 H 90 O7, and the specific structure is as follows:

[0040] .

[0041] Compound C5: Glycerol 1-eicosanoate 3-(4E,7E,10E-hexadecatrienoate), CASNo: 1415934-71-2, name: Glycerol-1-eicosanoate 3-(4E,7E,10E-hexadecatrienoate); structural formula: C 39 H 70 O5,

[0042] The structural formula is as follows:

[0043] .

[0044] Compound C6: 1,3-dihydroxylpropyl-(9Z,12Z)-octadeca-9,12-dienate, CAS No:3443-82-1;

[0045] Chinese name: Befunolol, 2-linolenoyl-rac-glycerol;

[0046] Structural formula: C 21 H 38 O4, and the specific structure is as follows:

[0047] .

[0048] The present invention also provides the use of one or a combination of two or more of the C1-C6 compounds in the preparation of products with antioxidant, whitening / or anti-aging effects:

[0049] C1 compound: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumaroyl sucrose;

[0050] C2 compound: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumaroyl sucrose;

[0051] C3 compound: mumeose K;

[0052] C4 compound: daucosterol linoleate;

[0053] C5 compound: glycerol-1-eicosanoate-3-(4E,7E,10E-hexadecatrienoic acid);

[0054] C6 compound: 2-linolenoyl-rac-glycerol.

[0055] In the present invention, the C1-C6 compounds are extracted and separated from the extract of Musella lasiocarpa.

[0056] In some specific embodiments, the present invention uses the following preparations: the crude extract of the ethanol extract of Musella lasiocarpa (the crude extract of Musella lasiocarpa), the refined components (RC1-RC6) and the C1-C6 compounds for in vitro tests, including the DPPH free radical scavenging rate determination experiment, qPCR detection, and Western blot (WB) experiment. The results show that the crude extract of Musella lasiocarpa all exhibits excellent DPPH free radical scavenging rate, down-regulates the expression of genes and proteins related to aging (such as MMP1 gene and MMP1 protein), and up-regulates the expression of anti-aging genes ( COL1 gene), showing significant antioxidant and anti-aging effects.

[0057] The present invention also conducted in vitro mushroom tyrosinase activity experiments and in vivo tyrosinase activity experiments using the above-mentioned preparations. The results showed that the crude extract of Musella lasiocarpa exhibited excellent tyrosinase activity inhibition rate, RC1 and RC6 had certain tyrosinase activity inhibition rates, while C2, C4, C5, and C6 showed good tyrosinase inhibition rates, and thus had good whitening effects.

[0058] In some embodiments, the product includes skin care products with antioxidant, whitening, and / or anti-aging effects, or health care products that contribute to antioxidant effects.

[0059] In some embodiments, the antioxidant includes scavenging DPPH free radicals.

[0060] In some embodiments, the anti-aging includes at least one of the following I) - II):

[0061] Ⅰ. Inhibiting MMP1 the transcription of genes and / or inhibiting the expression of MMP1 protein;

[0062] Ⅱ. Promoting COL1 the transcription of genes.

[0063] In some embodiments, the whitening includes inhibiting the activity of tyrosinase.

[0064] The present invention also provides a product with antioxidant, whitening, and / or anti-aging effects, including excipients and a product containing at least one of the C1 - C6 compounds;

[0065] The product includes the alcohol extract of Musella lasiocarpa flower and flower bud as described above or a combination of one or more of the C1 - C6 compounds.

[0066] The dosage form of the product of the present invention includes skin care products or health care products; the dosage form of the skin care products includes creams, lotions, solutions, sprays, gels, or powders; the dosage form of the health care products includes but is not limited to tablets, emulsions, granules, pills, liquid preparations, ointments, or patches, etc.

[0067] In some embodiments, the excipients are excipients acceptable in skin care products, including but not limited to: preservatives, pigments, fragrances, thickeners, moisturizers, surfactants, waxes, esters, lipids, oils, chelating agents, pH regulators, purified water, antioxidants.

[0068] The present invention also provides a method for antioxidant, anti-aging, and whitening, including: administering the preparation of the present invention. The present invention has no special restrictions on the administration method of the preparation, and an appropriate method can be determined according to the conventional methods in the art in combination with the preparation types as described above.

[0069] The present invention provides the use of a preparation containing at least one of C1-C6 compounds in the preparation of an antioxidant, whitening / or anti-aging product. Research shows that the crude extracts of Musella lasiocarpa and its buds, their refined components, and the compounds C1-C6 therein can effectively scavenge DPPH free radicals, down-regulate the expression of senescence-related genes and proteins (such as MMP1 gene and MMP1 protein), up-regulate the expression of anti-aging genes ( COL1 gene), and inhibit the activity of tyrosinase, having good antioxidant, whitening and anti-aging effects, and having broad application prospects in the fields of skin care products, health care products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shows the DPPH free radical scavenging rate of the crude extract of Musella lasiocarpa, the refined components of Musella lasiocarpa and the refined compounds;

[0071] Figure 2 Shows the MMP1 relative mRNA levels of genes of the crude extract of Musella lasiocarpa, the refined components of Musella lasiocarpa and the refined compounds at different concentrations;

[0072] Figure 3 Shows the COL1 relative mRNA levels of genes of the crude extract of Musella lasiocarpa, the refined components of Musella lasiocarpa and the refined compounds at different concentrations;

[0073] Figure 4 Shows the relative expression levels of MMP1 protein of the crude extract of Musella lasiocarpa at different concentrations;

[0074] Figure 5 Shows the relative expression levels of MMP1 protein of the refined components RC1, RC2, and RC3 of Musella lasiocarpa;

[0075] Figure 6 Shows the relative expression levels of MMP1 protein of the refined components RC4, RC5, and RC6 of Musella lasiocarpa;

[0076] Figure 7 Shows the relative expression levels of MMP1 protein of the refined compounds C1, C2, and C3 of Musella lasiocarpa;

[0077] Figure 8 Shows the relative expression levels of MMP1 protein of the refined compounds C4, C5, and C6 of Musella lasiocarpa;

[0078] Figure 9 Shows the in vitro mushroom tyrosinase inhibition rate of the crude extract of Musella lasiocarpa at different concentrations;

[0079] Figure 10 Shows the in vivo tyrosinase inhibition rate of the crude extract of Musella lasiocarpa, the refined components of Musella lasiocarpa and the refined compounds. Detailed implementation mode

[0080] The present invention provides an extract of Musella lasiocarpa and its application in the preparation of products with antioxidant, anti-aging, and whitening effects. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can clearly make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0081] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market.

[0082] In a specific embodiment of the present invention, the crude extract of Musella lasiocarpa is prepared by the following method:

[0083] Take the flowers and flower buds of Musella lasiocarpa, and extract them by cold soaking with 3 times the volume of 75% ethanol aqueous solution for 3 times, 24 hours each time. Concentrate the extract to dryness to obtain the crude extract of Musella lasiocarpa.

[0084] After preparing the crude extract of Musella lasiocarpa into a sample solution with the required concentration, subsequent experiments are carried out.

[0085] In a specific embodiment of the present invention, the preparation method of the refined components RC1-RC6 of Musella lasiocarpa includes:

[0086] Take the flowers and flower buds of Musella lasiocarpa, and extract them by cold soaking with 3 times the volume of 75% ethanol aqueous solution for 3 times, 24 hours each time. Concentrate the extract to dryness to obtain the crude extract of Musella lasiocarpa;

[0087] Load the crude extract of Musella lasiocarpa onto an MCI column, and elute it successively with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution, and 100% methanol solution to obtain the refined components RC1-RC6 of Musella lasiocarpa respectively.

[0088] In the above elution, each gradient is eluted for 3 column volumes.

[0089] Specifically, RC1 is the part eluted with water after MCI chromatography separation;

[0090] RC2 is the part eluted with 20% methanol aqueous solution after MCI chromatography separation;

[0091] RC3 is the part eluted with 40% methanol aqueous solution after MCI chromatography separation;

[0092] RC4 is the part eluted with 60% methanol aqueous solution after MCI chromatography separation;

[0093] RC5 is the fraction eluted with 80% aqueous methanol solution after MCI chromatography separation;

[0094] RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0095] The prepared refined components RC1 - RC6 of Musella lasiocarpa are made into sample solutions with required concentrations and then subsequent experiments are carried out.

[0096] The present invention will be further described below in conjunction with embodiments:

[0097] Example 1

[0098] 1. Determination of antioxidant effect by DPPH free radical scavenging rate experiment

[0099] 1.1 Experimental process for determination of DPPH free radical scavenging rate

[0100] (1) Preparation of stock solutions: 95% ethanol: Take 95 ml of ethanol in a graduated cylinder and add ultrapure water to 100 ml to prepare. 0.03% DPPH stock solution: Weigh 15 mg of DPPH powder and place it in a 50 ml brown volumetric flask, add about 40 ml of 95% ethanol, ultrasonically dissolve it completely, make up the volume to the scale line, and shake well to obtain the DPPH stock solution:

[0101] (2) Preparation of 0.03‰ DPPH working solution: According to the required amount of DPPH solution, measure 1 / 10 volume of 0.03% DPPH stock solution and dilute it to prepare 0.03‰ DPPH working solution.

[0102] (3) Dilute the positive control Trolex with pure water into a 0.025 mM sample solution to verify the test system.

[0103] (4) Prepare the test substances of the following experimental groups according to the method described above: crude extract of Musella lasiocarpa, refined components RC1 - RC6, compounds C1 - C6. Dilute the test substances with pure water into sample solutions with multiple concentrations. Referring to Table 1, set up sample tubes (T), sample backgrounds (T0), DPPH tubes (C) and solvent backgrounds (C0) in a 96 - well plate. For each test concentration of each sample, 3 parallel wells need to be set up for the sample well (T), and at the same time, 3 parallel wells also need to be set up for the DPPH tube (C).

[0104] (5) Add 50 μl of the same - concentration sample solution and 100 μl of pure water to the sample tube (T) and the sample background (T0) respectively, and add 150 μl of pure water to the DPPH tube (C) and the solvent background (C0) respectively.

[0105] (6) Add 50 μl of DPPH ethanol solution to each of the sample tube (T) and the DPPH tube (C), and replace the sample background (T0) and the solvent background (C0) with 50 μl of 95% ethanol.

[0106] (7) Let it react statically at room temperature for 5 min, and measure OD517.

[0107] (8) Calculate the DPPH free radical scavenging rate:

[0108] (9) Among them, in the formula, T is the absorbance value of the sample well, that is, the absorbance value of the solution after the sample reacts with DPPH; T0 is the absorbance value of the sample background; C is the average value of the absorbance values of the DPPH wells for 3 times, that is, the absorbance value of the DPPH solution without adding the sample; C0 is the absorbance value of the solvent background.

[0109] (10) Statistical analysis and graphing: Statistical analysis and graphing are both performed using GraphPad Prism 9 software. If the data meet the normal distribution and homogeneity of variance, paired t-tests are used to compare before and after the experiment within the group, and one-way ANOVA is used to compare the means between groups. P <0.05 indicates that the difference is statistically significant. The results are shown in Table 2 and Figure 1 .

[0110] Table 1 Experimental sample formulation table for the determination of DPPH free radical scavenging rate

[0111]

[0112] 1.2 Antioxidant effects (DPPH free radical scavenging rate) of the crude extract, refined components and purified compounds of Musella lasiocarpa

[0113] Table 2 Numerical table of DPPH free radical scavenging rates of the crude extract, refined components and purified compounds C1 - C6 of Musella lasiocarpa

[0114]

[0115]

[0116] Summary: Using the positive standard 0.025 mM Trolex showed a good DPPH free radical scavenging rate, indicating that the experimental system was successful. The crude extract of Musella lasiocarpa, 6 refined components of Musella lasiocarpa and 5 purified compounds of Musella lasiocarpa (C1 - C5) all showed excellent DPPH free radical scavenging rates, and showed a certain concentration dependence, with a strong anti-aging effect.

[0117] 2. Detection and analysis of anti-aging genes (qPCR)

[0118] 2.1 qPCR detection steps

[0119] (1) The HFF fibroblasts grown in 12-well plates were modeled by UVA stimulation.

[0120] (2) The cells were collected 24 h after modeling.

[0121] (3) Total cellular RNA was extracted using an RNA extraction kit, and cDNA was synthesized by reverse transcription using a reverse transcription kit.

[0122] (4) Amplification was performed using SYBR Green qPCR Master Mix on a fluorescence quantitative PCR instrument. The specificity of the primers was checked by melting curve.

[0123] (5) The 2^-ΔΔCt method was used to calculate the relative expression levels of the target genes (matrix metalloproteinase type I MMP1 and collagen type I COL1 ), GAPDH using GAPDH as the internal reference gene (

[0124] Primer sequences: F: 5'-ACAACTTTGGTATCGTGGAAGG-3'; R: 5'-GCCATCACGCCACAGTTTC-3')

[0124] 2.2 Anti-aging effects of refined components and purified compounds of Musella lasiocarpa (qPCR)

[0125] Table 3 qPCR experimental design table

[0126]

[0127] Note: The selected concentrations of the crude extract, refined components, and purified compounds of Musella lasiocarpa are based on the maximum safe concentration for fibroblasts.

[0128] 2.2.1 MMP1 PCR

[0129] MMP1 Matrix metalloproteinase-1 (MMP-1) is an important member of the matrix metalloproteinase family, including types I, II, and III. Its main function is to break down interstitial collagen. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, leading to the appearance of wrinkles. Therefore, after treating the test substance on the cell model, by detecting MMP1 changes in gene expression, it can be used to evaluate the anti-wrinkle effect of the test substance. Matrix metallopeptidase 1 (MMP1) The detection primers for

[0130] F: 5'-AAAATTACACGCCAGATTTGCC-3';

[0131] R: 5'-GGTGTGACATTACTCCAGAGTTG-3'.

[0132] The results are shown in Table 4 and Figure 2 .

[0133] Table 4 Numerical table of relative mRNA levels of MMP1 gene of crude extracts of Musella lasiocarpa, refined components of Musella lasiocarpa and purified compounds

[0134]

[0135] Summary: After UVA treatment, compared with BC, MMP1 the relative mRNA level of the gene was significantly up-regulated, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, using the positive control TGF-β, MMP1 the relative mRNA level of the gene was significantly decreased, indicating that the positive control of this experiment was effective. Compared with the group treated with UVA alone, after treatment with the crude extract of Musella lasiocarpa at 0.2 mg / ml, MMP1 the relative mRNA level of the gene was significantly decreased, and this result indicates that the crude extract of Musella lasiocarpa can significantly inhibit MMP1 the expression of the gene and has a significant anti-aging effect. Compared with the group treated with UVA alone, after treatment with the refined components RC2, RC3, RC4, RC5, RC6 of Musella lasiocarpa, MMP1 the relative mRNA level of the gene was significantly decreased, and this result indicates that these 5 refined components of Musella lasiocarpa can significantly inhibit MMP1 the expression of the gene and has a significant anti-aging effect. Compared with the group treated with UVA alone, after treatment with the purified compounds C1, C2, C3, C4 of Musella lasiocarpa, MMP1 the relative mRNA level of the gene was significantly decreased, and this result indicates that these 4 purified compounds of Musella lasiocarpa can significantly inhibit MMP1 the expression of the gene and has a significant anti-aging effect.

[0136] 2.22 COL1 PCR

[0137] Collagen I (COL1) Primers for

[0138] F: 5'- GTGCGATGACGTGATCTGTGA-3'

[0139] R: 5'- CGGTGGTTTCTTGGTCGGT-3'

[0140] COL1 is type I collagen, which accounts for about 80% of the dermis of the skin. Type I collagen helps the dermis (the middle layer of the skin) form fibroblasts, helps new cells grow, provides structure, strength and support for the skin, makes the skin plump and full, promotes the expression of COL1 protein, can play a role in resisting the generation of wrinkles to a certain extent, and thus achieves the effect of anti-aging. The results are shown in Table 5 and Figure 3 。

[0141] Table 5 Numerical table of relative mRNA levels of COL1 gene of crude extracts of Musella lasiocarpa, refined components of Musella lasiocarpa and purified compounds at different concentrations

[0142]

[0143] Summary: After UVA treatment, compared with BC, COL1 the relative mRNA level of the gene decreased significantly, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, using the positive control TGF-β, COL1 the relative mRNA level of the gene increased significantly, indicating that the positive control of this experiment was effective. Compared with the group treated with UVA alone, after treatment with the crude extract of Musella lasiocarpa at 0.2 mg / ml, COL1 the relative mRNA level of the gene increased significantly. This result shows that the crude extract of Musella lasiocarpa can significantly increase COL1 the expression of the gene and has a significant anti-aging effect. Compared with the group treated with UVA alone, after treatment with the refined components RC2, RC4, RC6 of Musella lasiocarpa, COL1 the relative mRNA level of the gene increased significantly. This result shows that these 3 refined components of Musella lasiocarpa can significantly increase COL1 the expression of the gene and has a significant anti-aging effect. Compared with the group treated with UVA alone, after treatment with the purified compounds C1, C2, C3, C4, C5 of Musella lasiocarpa, COL1 the relative mRNA level of the gene increased significantly. This result shows that these 5 purified compounds of Musella lasiocarpa can significantly increase COL1 the expression of the gene and has a significant anti-aging effect.

[0144] 3. Detection and analysis of anti-aging proteins (WB)

[0145] 3.1 WB experimental procedure

[0146] (1) HFF fibroblasts growing in 12-well plates were modeled by UVA stimulation.

[0147] After 24 hours of modeling, the cells were collected. RIPA cell lysis buffer was added to extract the cells.

[0148] (3) Protein quantification: Use a protein quantification method (such as the BCA method) to determine the concentration of the extracted protein.

[0149] (4)SDS-PAGE electrophoresis: Use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein samples according to their molecular weight.

[0150] (5)Transfer membrane: Assemble the gel, membrane, and transfer buffer in a transfer device, and use a wet transfer device to transfer the separated proteins from the gel to a PVDF membrane.

[0151] (6)Blocking: Place the membrane in 5% non-fat milk powder and incubate for 1 h.

[0152] (7)Primary antibody incubation: Add the primary antibody (specific antibody) of the target protein to 5% non-fat milk powder and incubate overnight to allow it to fully bind to the target protein in the membrane.

[0153] (8)Washing: Wash the membrane with buffer TBST to remove unbound primary antibody.

[0154] (9)Secondary antibody incubation: Add the secondary antibody corresponding to the source of the primary antibody (such as goat anti-rabbit IgG) to 5% non-fat milk powder and incubate for 2 h to allow it to bind to the primary antibody. The secondary antibody usually carries a labeled enzyme (such as horseradish peroxidase - HRP) or a fluorescent dye.

[0155] (10)Washing: Wash the membrane with buffer TBST to remove unbound secondary antibody.

[0156] (11)Signal detection: Use the substrate chemiluminescence ECL method to detect the protein labeled with the secondary antibody, and use the ChemiDoc imaging system to image and save the target protein.

[0157] (12)Result analysis: Use ImageJ to perform quantitative analysis of the target protein based on the intensity and location of the signal.

[0158] Table 6 WB experimental design table

[0159]

[0160]

[0161] Note: The three selected concentrations of the crude extract, refined fraction, and purified compound of Musella lasiocarpa are based on the maximum safe concentration for fibroblasts and are successively diluted 3-fold.

[0162] 3.2 Analysis of the anti-aging efficacy (WB) of the refined fraction and purified compound of Musella lasiocarpa

[0163] MMP1 WB results:

[0164] MMP1 is a matrix metalloproteinase, and its main function is to decompose interstitial collagens, including types I, II, and III. Collagen is the main structural protein in the dermis of the skin. When collagen is degraded, the mechanical support of the skin is lost, leading to the appearance of wrinkles. After treating the cell model with the test substance, the anti-wrinkle efficacy of the test substance is evaluated by detecting the change in the expression of MMP1 protein. The results of different test substances are shown in Tables 7 to 11 and Figures 4 - 8 。

[0165] Table 7 Numerical table of relative expression levels of MMP1 protein in crude extracts of Musella lasiocarpa at different concentrations

[0166]

[0167] Summary: After UVA treatment, compared with BC, the expression level of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, when using the positive control TGF-β, the expression level of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the group treated with UVA alone, after treating with crude extracts of Musella lasiocarpa at different concentrations, the expression level of MMP1 protein decreased significantly. This result indicates that the crude extracts of Musella lasiocarpa can significantly inhibit the expression of MMP1 protein and have a significant anti-aging effect.

[0168] Table 8 Numerical table of relative expression levels of MMP1 protein in refined components RC1, RC2, and RC3 of Musella lasiocarpa

[0169]

[0170] Summary: After UVA treatment, compared with BC, the expression level of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, when using the positive control TGF-β, the expression level of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the group treated with UVA alone, when treated with 0.012 mg / ml of RC3, 0.022 mg / ml, and 0.066 mg / ml of RC2 respectively, the expression level of MMP1 protein decreased significantly. This result indicates that the refined components RC2 and RC3 of Musella lasiocarpa can inhibit the expression of MMP1 protein to a certain extent and have a certain anti-aging effect.

[0171] Table 9 Numerical table of relative expression levels of MMP1 protein in refined components RC4, RC5, and RC6 of Musella lasiocarpa

[0172]

[0173] Summary: After UVA treatment, compared with BC, the expression level of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, when using the positive control TGF-β, the expression level of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA-alone treatment group, when treated with RC5 at 0.0003 mg / ml and 0.0008 mg / ml and RC6 at 0.012 mg / ml and 0.037 mg / ml respectively, the expression level of MMP1 protein decreased significantly. This result shows that the refined components RC5 and RC6 of Musella lasiocarpa can inhibit the expression of MMP1 protein to a certain extent and have a certain anti-aging effect.

[0174] Table 10 Numerical table of relative expression levels of MMP1 protein of the refined compounds C1, C2, and C3 of Musella lasiocarpa

[0175]

[0176] Summary: After UVA treatment, compared with BC, the expression level of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under the condition of UVA stimulation, when using the positive control TGF-β, the expression level of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA-alone treatment group, when treated with certain concentrations of C1 (0.0833 mg / ml, 0.25 mg / ml), C2 (0.0278 mg / ml, 0.0833 mg / ml), and C3 (0.0417 mg / ml, 0.125 mg / ml) respectively, the expression level of MMP1 protein decreased significantly. This result shows that the three refined compounds C1, C2, and C3 of Musella lasiocarpa can all inhibit the expression of MMP1 protein and have a certain anti-aging effect.

[0177] Table 11 Numerical table of relative expression levels of MMP1 protein of the refined compounds C4, C5, and C6 of Musella lasiocarpa

[0178]

[0179] Summary: After UVA treatment, compared with BC, the expression level of MMP1 protein increased, indicating the successful establishment of the anti-aging model. Under the condition of UVA stimulation, when using the positive control TGF-β, the expression level of MMP1 protein decreased significantly, indicating the effectiveness of the positive control in this experiment. Compared with the group treated with UVA alone, when treated with different concentrations of C4 (0.009mg / ml, 0.028mg / ml, 0.0083mg / ml), C5 (0.009mg / ml, 0.028mg / ml, 0.0083mg / ml) and C6 (0.009mg / ml, 0.028mg / ml, 0.0083mg / ml) respectively, the expression level of MMP1 protein decreased significantly. This result shows that the three refined compounds C4, C5 and C6 of Musella lasiocarpa can inhibit the expression of MMP1 protein and have significant anti-aging effects.

[0180] 4. Determination of whitening effect by tyrosinase activity experiment

[0181] 4.1 Inhibitory effect of crude extract of Musella lasiocarpa on the activity of mushroom tyrosinase in vitro

[0182] 4.1.1 Experimental procedure for the activity of mushroom tyrosinase in vitro

[0183] (1) Preparation of stock solutions: 1) Mushroom tyrosinase solution: Prepared as 100u / ml with pH6.8 PBS; 2) L-DOPA solution: Prepared as 1mg / ml with pH6.8 PBS; 3) Kojic acid (positive control): Diluted with pH6.8 PBS to: 0.3mg / ml.

[0184] (2) Add 100ul of mushroom tyrosinase solution to each sample tube, cover the lid, vortex and centrifuge briefly the sample tubes and the background tube, and then incubate in a 37°C water bath for 10 minutes.

[0185] (3) Add 400ul of L-DOPA solution to each tube in sequence, cover the lid and vortex and centrifuge briefly, and control the reaction time for each tube to be 5 minutes.

[0186] (4) Transfer the reaction solutions of each tube into a 96-well plate and measure the absorbance at 475nm.

[0187] (5) Calculate the inhibition rate of mushroom tyrosinase:

[0188] (6) Where in the formula, T is the absorbance value of the sample well, that is, the absorbance value of the solution after the reaction of the sample and mushroom tyrosinase; T0 is the absorbance value of the sample background; C is the average of the absorbance values of the enzyme reaction tubes for 3 times, that is, the absorbance of the reaction of mushroom tyrosinase and dopa without adding the sample; C0 is the absorbance value of the solvent background.

[0189] (7) Statistical analysis and graphing: All statistical analyses and graphing were performed using GraphPad Prism 9 software. If the data met the normal distribution and homogeneity of variance, paired t-tests were used to compare the data before and after the experiment within the group, and one-way ANOVA was used to compare the means between groups. P <0.05 indicates that the difference is statistically significant. The results are shown in Table 13 and Figure 9 .

[0190] Table 12 Preparation table of experimental samples for the determination of the inhibition rate of mushroom tyrosinase in vitro

[0191]

[0192] 4.1.2 Results of the whitening effect (mushroom tyrosinase activity in vitro) of the crude extract of Musella lasiocarpa

[0193] Table 13 Numerical table of the inhibition rate of mushroom tyrosinase by crude extracts of Musella lasiocarpa at different concentrations

[0194]

[0195] Summary: Using kojic acid at 0.3 mg / ml as the positive control showed a good inhibition rate of tyrosinase, indicating the success of the experimental system. The crude extracts of Musella lasiocarpa at different concentrations showed a good inhibition rate of tyrosinase and had a concentration-dependent relationship, indicating that the crude extracts of Musella lasiocarpa have a good whitening effect.

[0196] 4.2 Effects of the crude extract of Musella lasiocarpa, the refined components of Musella lasiocarpa, and the refined compounds on tyrosinase activity in vivo

[0197] 4.2.1 Experimental procedure for tyrosinase activity in vivo

[0198] (1) Preparation of stock solutions: 1) Lysis buffer: Prepare a 1% Triton X-100 solution with PBS at pH 6.8. 2) L-DOPA solution: Prepare a 10 mM solution with PBS at pH 6.8; 3) Kojic acid (positive control): Dilute it with PBS at pH 6.8 to 0.1 mg / ml.

[0199] (2) Culture the B16-F10 melanoma cells in the 12-well plate in RPMI1640 medium for 24 h.

[0200] (3) Add samples at different concentrations and the positive control for drug administration.

[0201] (4) After 48 h of treatment, collect the cells. Add lysis buffer to extract the cells.

[0202] (5) Protein quantification: Use the protein quantification method (BCA method) to determine the concentration of the extracted protein.

[0203] (6) According to the protein content detection results, use lysis buffer to unify the total protein amount.

[0204] (7) After taking the unified total protein amount of each sample, add 90 μl of the solution to the corresponding wells of a 96-well plate, and add 10 μl of L-DOPA solution to each well, and incubate at 37 °C for 40 - 60 min.

[0205] (8) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 475 nm.

[0206] (9) Calculate the tyrosinase inhibition rate:

[0207] (10) Among them, in the formula, T is the absorbance value of the sample well, that is, the absorbance value of the solution after the reaction of the sample with tyrosinase; C is the absorbance value of the blank control.

[0208] (11) Statistical analysis and graphing: Statistical analysis and graphing were performed using GraphPad Prism 9 software. If the data meet the normal distribution and homogeneity of variance, paired t-tests were used to compare before and after the experiment within the group, and one-way analysis of variance was used to compare the means between groups. P <0.05 indicates that the difference is statistically significant. The results are shown in Table 14 and Figure 10 .

[0209] 4.2.2 Results of the whitening effect (tyrosinase activity in vivo) of the refined components and refined compounds of Musella lasiocarpa

[0210] Table 14 Numerical table of tyrosinase inhibition rates in vivo of the crude extract, refined components and refined compounds of Musella lasiocarpa

[0211]

[0212] Note: The selected concentrations of the crude extract, refined components and refined compounds of Musella lasiocarpa are based on the maximum safe concentration for B16 cells.

[0213] Summary: Using kojic acid at 0.1 mg / ml showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. The crude extract of Musella lasiocarpa showed an excellent tyrosinase activity inhibition rate, RC1 and RC6 had a certain tyrosinase activity inhibition rate, while C2, C4, C5 and C6 showed a good tyrosinase inhibition rate, indicating that the crude extract of Musella lasiocarpa and two refined components and four refined compounds of Musella lasiocarpa have good whitening effects.

[0214] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of C4 compounds in the preparation of whitening skin care products: C4 compound: carotene linoleate.

2. The use according to claim 1, characterized in that: The whitening includes inhibiting the activity of tyrosinase.

3. The use according to claim 1, characterized in that: The C4 compound is extracted and separated from the ground jellyfish.

4. The use according to any one of claims 1 to 3, characterized in that: The C4 compound is prepared by the following method: The flowers and buds of the Herba Lycoris Radiatae are cold-extracted with 3 times the volume of 75% ethanol for three times, each time for 24 hours, and the extract is concentrated to dryness to obtain a Herba Lycoris Radiatae crude extract; the Herba Lycoris Radiatae crude extract is loaded onto an MCI column, and gradient eluted with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol aqueous solution to obtain Herba Lycoris Radiatae refined components RC1-RC6 respectively; The refined component RC2 was placed on a silica gel column and eluted with dichloromethane-methanol in a volume ratio of 9:1-8:2, petroleum ether-isopropanol in a volume ratio of 8:2-7:3, and ethyl acetate-methanol in a volume ratio of 9.8:0.2 to separate and obtain the C4 compound.

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