Ensella lasiocarpa extract and application thereof in preparation of antioxidant, anti-aging and whitening products

Abstract

The invention relates to the technical field of plant extracts, in particular to application of an ensexual euphorbia herb extract in preparation of antioxidant, anti-aging and whitening products. The invention provides an application of a preparation containing at least one of C1-C6 compounds in preparation of antioxidant, whitening and / or anti-aging products. Researches show that the crude extract of the flowers and buds of the euphorbia lasiocarpa, the refined components of the crude extract and the refined components of the euphorbia lasiocarpa and compounds C1-C6 in the crude extract can effectively remove DPPH free radicals, down-regulate expression of senescence-related genes and proteins (such as MMP1 genes and MMP1 proteins), up-regulate expression of anti-aging genes (COL1 genes) and inhibit the activity of tyrosinase, and the euphorbia lasiocarpa has good anti-oxidation, whitening and anti-aging effects, and can be used for preparing the traditional Chinese medicine composition. Wide application prospects are realized in the fields of skin care products, health care products and the like.

Description

Technical Field

[0001] The invention relates to the technical field of plant extracts, in particular to a nephrolepis troglodytes extract and application thereof in preparing antioxidant, anti-aging and whitening products. Background Art

[0002] Free radicals are molecules or atoms with unpaired electrons. Common free radicals include superoxide radicals (O2 - ), hydroxyl radicals (OH·) and lipid peroxidation free radicals. In modern life, more and more external factors lead to the increase of free radicals, such as environmental toxins, such as air pollution, heavy metals, industrial waste, unbalanced diet, increased life pressure, smoking, secondhand smoke, radiation, etc. These free radicals are very active and will attack lipids, proteins, DNA and other biological macromolecules in skin cells, accelerate skin aging, and cause skin dullness, sagging, fine lines and other problems. Anti-oxidation, whitening and anti-aging have always been the three key efficacy claims that have attracted much attention.

[0003] At present, there are a wide variety of whitening and anti-aging products on the market, with varying efficacy and quality. With consumers' increasing demands for skin health and appearance, and the deep penetration of technology in the cosmetics industry, the development of efficient and safe antioxidant whitening and anti-aging products has become a hot topic in the industry.

[0004] Therefore, providing a product that has significant and safe antioxidant, whitening and anti-aging effects still has broad market prospects. Summary of the invention

[0005] In view of this, the present invention provides a Herba Lysimachiae extract and application thereof in preparing antioxidant, anti-aging and whitening products.

[0006] The present invention provides the use of a Herba Lysimachiae extract containing at least one of C1 to C6 compounds in the preparation of an antioxidant, whitening, or anti-aging product: Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose; Compound C2: 2',3',4',6'-tetra- O -acetyl-3- O -( E )- p -coumarylsucrose; C3 compound: mumeose K; C4 compound: carotene linoleate; C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid); C6 compound: 2-linolenoyl-rac-glycerol; The extract of the ground jellyfish is an ethanol extract of the flower and bud of the ground jellyfish.

[0007] In the present invention, among the C1-C6 compounds, C is the abbreviation of compound, and the C1-C6 compounds are compound1-compound6.

[0008] In the present invention, the extract of the Herba Lycoris Radiatae includes a crude Herba Lycoris Radiatae extract or a refined Herba Lycoris Radiatae component.

[0009] In some embodiments, the extract of the Herba Lycoris Radiatae is a crude Herba Lycoris Radiatae extract, which is prepared by the following method: taking the flowers and buds of the Herba Lycoris Radiatae, cold-immersing and extracting them with 75% ethanol, and concentrating them to dryness under reduced pressure to obtain the crude Herba Lycoris Radiatae extract.

[0010] In some specific embodiments, the method for preparing the crude extract of the ground jellyfish comprises: taking the flowers and buds of the ground jellyfish, cold-immersing and extracting them three times with 3 volumes of 75% ethanol, each time for 24 hours, and concentrating the extract to dryness.

[0011] In some embodiments, the concentration of the crude extract of the Herba Lycoris Radiatae 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.

[0012] In some other specific embodiments, the extract of the Herba Lycoris Radiatae is a refined component of the Herba Lycoris Radiatae, which is recorded as RC1 to RC6 in the present invention, wherein RC is the abbreviation of Refined Component. The RC1 to RC6 are prepared by the following method: cold extraction with 3 volumes of 75% ethanol for three times, each time for 24 hours, and the extract is concentrated to dryness to obtain a crude extract of the Herba Lycoris Radiatae; the crude extract of the Herba Lycoris Radiatae 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 in sequence to obtain refined components of the Herba Lycoris Radiatae RC1-RC6, respectively.

[0013] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation; RC2 is the fraction eluted with 20% methanol in water after MCI chromatography separation; RC3 is the fraction eluted with 40% methanol in water after MCI chromatography separation; RC4 is the fraction eluted with 60% methanol in water after MCI chromatography separation; RC5 is the fraction eluted with 80% methanol in water after MCI chromatography separation; RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0014] In 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 RC2, RC3, RC4, RC5, and RC6. The specific separation method is as follows: The refined component RC2 was 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) and ethyl acetate:methanol 9.8:0.2 to separate C4.

[0015] The refined component RC4 was 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), and petroleum ether-isopropanol (volume ratio 8:2-6:4); the purified component RC4 was loaded onto a Sephdex column and eluted with methanol to separate C3.

[0016] The refined component RC5 was loaded onto a silica gel column and eluted with dichloromethane-acetone (volume ratio 9:1-8:2), dichloromethane-methanol (volume ratio 9:1), and petroleum ether-acetone (volume ratio 8:2-7:3) to separate C1.

[0017] The refined component RC6 was 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), and petroleum ether-ethyl acetate (volume ratio 9:1-8:2). It was then loaded onto a Sephdex column and eluted with dichloromethane-methanol (volume ratio 1:1). After multiple separations, refined compounds C2, C5, and C6 were obtained.

[0018] Specifically, the name of compound C1 is (1,2',3',4',6'-O-pentaacetyl-3-O-trans-p-coumaroylsucrose, CAS No: 1392307-46-8), namely 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 , the specific structure is as follows: .

[0019] 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: C25 H 32 O 15 , the specific structure is as follows: .

[0020] Compound C3: mumeose K, CAS No: 2132384-01-9, Structural formula: C 25 H 32 O 15 , the specific structure is as follows: .

[0021] Compound C4: daucosterol linoleate, CAS No: 79380-30-6, Chinese name: Carotene linoleate, 3-O-(6'-O-linoleyl-β-D-glucose)-β-sitosterol; structural formula: C 53 H 90 O7, the specific structure is as follows: .

[0022] Compound C5: Glycerol 1-eicosanoate 3-(4E,7E,10E-hexadecatrienoate), CAS No: 1415934-71-2, Name: Glycerol-1-eicosanoate-3-(4E,7E,10E-hexadecatrienoate); Structural formula: C 39 H 70 O5, The structural formula is as follows: .

[0023] Compound C6: 1,3-dihydroxylpropyl-(9Z,12Z)-octadeca-9,12-dienate, CAS No: 3443-82-1; Chinese name: benfuranolol, 2-linolenoyl-rac-glycerol; Structural formula: C 21 H 38 O4, the specific structure is as follows: .

[0024] 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 antioxidant, whitening and / or anti-aging products: Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose; Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose; C3 compound: mumeose K; C4 compound: carotene linoleate; C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid); C6 compound: 2-linolenoyl-rac-glycerol.

[0025] In the present invention, the C1-C6 compounds are extracted and separated from the extract of Herba Lycoris Radiatae.

[0026] In some specific embodiments, the present invention uses the preparation: crude extract of ethanol extract of Hedysarum schrenkiana (hedysarum crude extract), refined fractions (RC1~RC6) and C1~C6 compounds to conduct in vitro tests, including DPPH free radical scavenging rate determination experiment, qPCR detection, and protein immunoblotting (WB) experiment. The results show that the crude extract of Hedysarum schrenkiana exhibits excellent DPPH free radical scavenging rate, downregulates the expression of aging-related genes and proteins (such as MMP1 gene and MMP1 protein), up-regulating anti-aging genes ( COL1 gene), showing significant antioxidant and anti-aging effects.

[0027] The present invention also uses the above preparation to conduct in vitro mushroom tyrosinase activity experiments and in vivo tyrosinase activity experiments. The results show that the crude extract of the ground jellyfish shows an excellent tyrosinase activity inhibition rate, RC1 and RC6 have a certain tyrosinase activity inhibition rate, and C2, C4, C5 and C6 show a good tyrosinase inhibition rate, so it has a good whitening effect.

[0028] In some embodiments, the product comprises an anti-oxidation, whitening and / or anti-aging skin care product, or a health product that helps with anti-oxidation.

[0029] In some embodiments, the anti-oxidation comprises: scavenging DPPH free radicals.

[0030] In some embodiments, the anti-aging comprises at least one of the following I) to II): Ⅰ. Inhibition MMP1 gene transcription and / or inhibition of MMP1 protein expression; II. Promotion COL1 Transcription of genes.

[0031] In some embodiments, the whitening comprises inhibiting the activity of tyrosinase.

[0032] The present invention also provides a product with antioxidant, whitening and / or anti-aging effects, comprising an auxiliary material and a product containing at least one of the C1 to C6 compounds; The product includes the alcohol extract of the nasturtium and flower buds as described above or one or a combination of two or more of the C1-C6 compounds.

[0033] The dosage forms of the products of the present invention include skin care products or health care products; the dosage forms of the skin care products include creams, emulsions, solutions, sprays, gels or powders; the dosage forms of the health care products include but are not limited to tablets, emulsions, granules, pills, liquid preparations, ointments or patches, etc.

[0034] In some embodiments, the auxiliary materials are acceptable auxiliary materials 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, and antioxidants.

[0035] The present invention also provides an anti-oxidation, anti-aging and whitening method, comprising: administering the preparation of the present invention. The present invention has no special restrictions on the administration method of the preparation, and a suitable method can be determined according to the conventional method in the art combined with the preparation type as described above.

[0036] The present invention provides the use of a preparation containing at least one of the C1-C6 compounds in the preparation of anti-oxidation, whitening and / or anti-aging products. Studies have shown that the crude extracts and refined components of the nasturtium and its flower buds and the compounds C1-C6 therein can effectively scavenge DPPH free radicals and down-regulate the expression of aging-related genes and proteins (such as MMP1 gene and MMP1 protein), up-regulating anti-aging genes ( COL1 It can inhibit the expression of tyrosinase and has good antioxidant, whitening and anti-aging effects. It has broad application prospects in the fields of skin care products and health care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It indicates the DPPH free radical scavenging rate of the crude extract of Hedyssa suberecta, the refined components of Hedyssa suberecta and the refined compounds; Figure 2 The crude extracts of different concentrations of Hedyssa suberecta, the refined components of Hedyssa suberecta and the refined compounds MMP1 Relative mRNA levels of genes; Figure 3 The crude extracts of different concentrations of Hedyssa suberecta, the refined components of Hedyssa suberecta and the refined compounds COL1 Relative mRNA levels of genes; Figure 4 It represents the relative expression of MMP1 protein in crude extracts of different concentrations of Hedyssa centifolia; Figure 5 It represents the relative expression of MMP1 protein in the refined fractions RC1, RC2 and RC3 of the Herba Lycoris Radiatae; Figure 6 It represents the relative expression of MMP1 protein in the refined fractions RC4, RC5 and RC6 of the Herba Lycoris Radiatae; Figure 7 It represents the relative expression of MMP1 protein in compounds C1, C2 and C3 extracted from the extract of Hedyssa officinalis; Figure 8 It represents the relative expression of MMP1 protein in compounds C4, C5 and C6 extracted from the extract of Hedyssa officinalis; Fig. 9 It represents the in vitro mushroom tyrosinase inhibition rate of crude extracts of different concentrations of Hedyssa centifolia; Fig.10 It represents the in vivo tyrosinase inhibition rate of the crude extract of Nymphaeaceae, the refined components of Nymphaeaceae and the refined compounds. DETAILED DESCRIPTION

[0038] The present invention provides a Herba Lysimachiae extract and its application in the preparation of antioxidant, anti-aging and whitening products. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0039] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0040] In a specific embodiment of the present invention, the crude extract of Herba Lycoris Radiatae is prepared by the following method: The flowers and buds of the ground jellyfish were taken, and cold-immersed and extracted three times with 3 times the volume of 75% ethanol aqueous solution, each time for 24 hours, and the extract was concentrated to dryness to obtain a crude extract of the ground jellyfish.

[0041] The prepared crude extract of Nasturtium schreberi was made into a sample solution of required concentration for subsequent experiments.

[0042] In a specific embodiment of the present invention, the preparation method of the refined components RC1 to RC6 of the nephrite comprises: Take the flowers and buds of the ground jellyfish, use 3 times the volume of 75% ethanol aqueous solution to cold-immerse and extract 3 times, each time for 24 hours, and concentrate the extract to dryness to obtain the ground jellyfish crude extract; The crude extract of the Herba Lycoris Radiatae was loaded onto an MCI column and eluted with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol solution in sequence to obtain Herba Lycoris Radiatae refined components RC1 to RC6, respectively.

[0043] In the elution, each gradient elution was performed for 3 column volumes.

[0044] Specifically, RC1 is the fraction eluted with water after MCI chromatography separation; RC2 is the fraction eluted with 20% methanol in water after MCI chromatography separation; RC3 is the fraction eluted with 40% methanol in water after MCI chromatography separation; RC4 is the fraction eluted with 60% methanol in water after MCI chromatography separation; RC5 is the fraction eluted with 80% methanol in water after MCI chromatography separation; RC6 is the fraction eluted with 100% methanol after MCI chromatography separation.

[0045] The obtained refined components RC1~RC6 of the ground jellyfish were made into sample solutions of required concentrations for subsequent experiments.

[0046] The present invention will be further described below in conjunction with embodiments: Example 1 1. DPPH free radical scavenging rate test to determine the antioxidant effect 1.1 Experimental process of DPPH free radical scavenging rate determination (1) Preparation of stock solution: 95% ethanol: Take 95 ml of ethanol in a measuring cylinder and add ultrapure water to 100 ml. 0.03% DPPH stock solution: Weigh 15 mg of DPPH powder into a 50 ml brown volumetric flask, add about 40 ml of 95% ethanol, sonicate to completely dissolve it, dilute to the mark, shake well, and use as DPPH stock solution: (2) Preparation of 0.03‰ DPPH working solution: Based on 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.

[0047] (3) The positive control substance Trolex was diluted with pure water to a 0.025 mM sample solution to verify the test system.

[0048] (4) Prepare the following experimental group test substances according to the method described above: crude extract of Herba Lysimachiae, refined fractions RC1 to RC6, and compounds C1 to C6. Dilute the test substances with pure water to form sample solutions of multiple concentrations. Referring to Table 1, set up sample tubes (T), sample background (T0), DPPH tubes (C), and solvent background (C0) in a 96-well plate. Three parallel wells should be set up for each sample well (T) of each test concentration of each sample, and three parallel wells should also be set up for the DPPH tube (C).

[0049] (5) Add 50 μl of sample solution of the same concentration and 100 μl of pure water to the sample tube (T) and sample background (T0), and add 150 μl of pure water to the DPPH tube (C) and solvent background (C0).

[0050] (6) Add 50 μl of DPPH ethanol solution to each of the sample tube (T) and DPPH tube (C). Replace the sample background (T0) and solvent background (C0) with 50 μl of 95% ethanol.

[0051] (7) Allow the reaction to stand at room temperature for 5 min and measure OD517.

[0052] (8) Calculate DPPH free radical scavenging rate:

[0053] (9) In the formula, T is the absorbance of the sample well, that is, the absorbance of the solution after the sample reacts with DPPH; T0 is the background absorbance of the sample; C is the average of the three absorbances of the DPPH well, that is, the absorbance of the DPPH solution when no sample is added; and C0 is the background absorbance of the solvent.

[0054] (10) Statistical analysis and graphing: GraphPad Prism 9 software was used for statistical analysis and graphing. If the data met the normal distribution and homogeneity of variance, paired t-test was used for pre- and post-experimental comparison within the group, and one-way analysis of variance was used for comparison of means between groups. P <0.05 indicates that the difference is statistically significant. The results are shown in Table 2 and Figure 1 .

[0055] Table 1 DPPH free radical scavenging rate test sample solution table 1.2 Antioxidant efficacy of crude extracts, refined fractions and refined compounds of N. terrestris (DPPH free radical scavenging rate)

[0056] Table 2 DPPH free radical scavenging rate values ​​of crude extract, refined fractions and refined compounds C1-C6 of Nymphaeaceae

[0057]

[0058] Summary: The use of 0.025mM Trolex showed good DPPH free radical scavenging rate, indicating that the experimental system was successful. The crude extract of the scutellaria baicalensis, 6 refined components of the scutellaria baicalensis and 5 refined compounds of the scutellaria baicalensis (C1-C5) all showed excellent DPPH free radical scavenging rate, and showed a certain concentration dependence, with strong anti-aging effect.

[0059] 2. Detection and analysis of anti-aging genes (qPCR) 2.1 qPCR detection steps (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0060] (2) Collect cells 24 hours after modeling.

[0061] (3) Use an RNA total extraction kit to extract cellular RNA, and use a reverse transcription kit to reverse transcribe and synthesize cDNA.

[0062] (4) Amplify using SYBR Green qPCR Master Mix fluorescence quantitative PCR instrument and check the specificity of primers through melting curve.

[0063] (5) Calculate the target gene (type I matrix metalloproteinase) using the 2^-ΔΔCt method MMP1 and type I collagen COL1 ) relative expression levels, GAPDH As an internal reference gene ( GAPDH Primer sequences: F: 5'-ACAACTTTGGTATCGTGGAAGG-3'; R: 5'-GCCATCACGCCACAGTTTC-3') 2.2 Anti-aging effects of refined components and extracted compounds of Rhizoma Dioscoreae (qPCR) Table 3 qPCR experimental design table Note: The selected concentrations of crude extracts of Nymphaeaceae, refined fractions of Nymphaeaceae and refined compounds of Nymphaeaceae are the maximum safe concentrations based on fibroblasts.

[0064] 2.2.1 MMP1 PCR MMP1Matrix Metalloproteinase - 1 is an important member of the matrix metalloproteinase family, including types I, II and III. Its main function is to decompose 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, which will lead to the appearance of wrinkles. Therefore, after the test substance is applied to the cell model, the test MMP1 Changes in gene expression can be used to evaluate the anti-wrinkle efficacy of the test substance. Matrix metallopeptidase 1 (MMP1) The detection primers are: F:5'-AAAATTACACGCCAGATTTGCC-3'; R:5'-GGTGTGACATTACTCCAGAGTTG-3'.

[0065] The results are shown in Table 4 and Figure 2 .

[0066] Table 4 Relative mRNA levels of MMP1 gene in crude extracts of Hedyssa suberectus, refined fractions and refined compounds of Hedyssa suberectus at different concentrations

[0067]

[0068] Conclusion: After UVA treatment, compared with BC, MMP1 The relative mRNA level of the gene was significantly upregulated, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used. MMP1 The relative mRNA level of the gene decreased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group, after treatment with 0.2 mg / ml of the crude extract of the Herba Lycoris Radiatae, MMP1 The relative mRNA level of the gene decreased significantly, indicating that the crude extract of Hedysarum sutchuenensis can significantly inhibit MMP1 Compared with the UVA treatment group, the RC2, RC3, RC4, RC5, and RC6 components of the ground chrysanthemum were treated with MMP1 The relative mRNA level of the gene decreased significantly, indicating that the five refined components of the genus Hedyspermum officinale can significantly inhibit MMP1 Compared with the UVA treatment group, the treatment with compounds C1, C2, C3, and C4 extracted from the ground lotus extract showed significant anti-aging effects. MMP1 The relative mRNA level of the gene decreased significantly, indicating that the four refined compounds of the ground lotus can significantly inhibit MMP1 The expression of genes has significant anti-aging effects.

[0069] 2.22 COL1 PCR

[0070] Collagen I (COL1) Primers: F:5'- GTGCGATGACTGATCTGTGA-3' R:5'- CGGTGGTTTCTTGGTCGGT-3' COL1 is type I collagen, which accounts for about 80% of the dermis. Type I collagen helps the dermis (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 achieve a certain effect of resisting the formation of wrinkles, and thus achieves anti-aging effect. The results are shown in Table 5 and Figure 3 .

[0071] Table 5 Relative mRNA levels of COL1 gene in crude extracts of N. schrenkiana, refined fractions and refined compounds of N. schrenkiana at different concentrations

[0072]

[0073] Conclusion: 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 UVA stimulation, the positive control TGF-β was used. COL1 The relative mRNA level of the gene increased significantly, indicating that the positive control of the experiment was effective. Compared with the UVA treatment group, after treatment with 0.2 mg / ml of the crude extract of the Herba Lycoris Radiatae, COL1 The relative mRNA level of the gene increased significantly, indicating that the crude extract of Hedysarum truncatum can significantly improve COL1 Compared with the UVA treatment group, the RC2, RC4, and RC6 components of the ground chrysanthemum were treated with COL1 The relative mRNA level of the gene increased significantly, indicating that the three refined components of the ground lotus can significantly improve COL1 Compared with the UVA treatment group, the treatment with compounds C1, C2, C3, C4, and C5 extracted from the ground lotus extract showed significant anti-aging effects. COL1 The relative mRNA level of the gene increased significantly, indicating that the five refined compounds of the ground lotus can significantly improve COL1 The expression of genes has significant anti-aging effects.

[0074] 3. Detection and analysis of anti-aging proteins (WB) 3.1 WB experimental process

[0075] (1) HFF fibroblasts grown in 12-well plates were stimulated with UVA to establish the model.

[0076] (2) 24 hours after modeling, collect cells and add RIPA cell lysis buffer to extract cells.

[0077] (3) Protein quantification: Use a protein quantification method (such as the BCA method) to determine the extracted protein concentration.

[0078] (4) SDS-PAGE electrophoresis: Use polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein samples according to molecular weight.

[0079] (5) Transfer: Assemble the gel, membrane and transfer buffer in a transfer apparatus, and use a wet transfer apparatus to transfer the separated proteins from the gel to the PVDF membrane.

[0080] (6) Blocking: Place the membrane in 5% skimmed milk powder and incubate for 1 hour.

[0081] (7) Primary antibody incubation: Add the primary antibody (specific antibody) of the target protein to 5% skimmed milk powder and incubate overnight to allow it to fully bind to the target protein in the membrane.

[0082] (8) Washing: Wash the membrane with TBST buffer to remove unbound primary antibody.

[0083] (9) Secondary antibody incubation: Add the secondary antibody corresponding to the source of the primary antibody (e.g. goat anti-rabbit IgG) to a solution containing 5% skim milk powder and incubate for 2 hours to allow it to bind to the primary antibody. The secondary antibody is usually labeled with an enzyme (e.g. horseradish peroxidase-HRP) or a fluorescent dye.

[0084] (10) Washing: Wash the membrane with TBST buffer to remove unbound secondary antibody.

[0085] (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.

[0086] (12) Result analysis: ImageJ was used to perform quantitative analysis of the target protein based on the signal intensity and location.

[0087] Table 6 WB experiment design table

[0088]

[0089]

[0090] Note: The three concentrations selected for the crude extract of Nymphaeaceae, the refined fraction of Nymphaeaceae and the refined compound of Nymphaeaceae are the maximum safe concentrations based on fibroblasts, and are obtained by diluting them 3 times in sequence.

[0091] 3.2 Analysis of the anti-aging effects of refined components and extracted compounds of Rhizoma Dioscoreae (WB) MMP1 WB results: MMP1 is a matrix metalloproteinase, which mainly decomposes interstitial collagen, 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, which leads to the appearance of wrinkles. After the test object acts on the cell model, the anti-wrinkle effect of the test object is evaluated by detecting the changes in the expression of MMP1 protein. The results of different test objects are shown in Tables 7 to 11 and Figure 4~Figure 8 .

[0092] Table 7 Relative expression values ​​of MMP1 protein in crude extracts of different concentrations of Hedysarum sphaerocephalum Summary: After UVA treatment, compared with BC, the expression of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, the expression of MMP1 protein decreased significantly after treatment with different concentrations of the crude extract of the Herba Lycoris Radiatae. This result shows that the crude extract of the Herba Lycoris Radiatae can significantly inhibit the expression of MMP1 protein and has a significant anti-aging effect.

[0093] Table 8 Relative expression values ​​of MMP1 protein in the refined fractions RC1, RC2 and RC3 of Nymphaeaceae Summary: After UVA treatment, compared with BC, the expression of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA treatment group alone, the expression of MMP1 protein decreased significantly after treatment with 0.012 mg / ml RC3, 0.022 mg / ml and 0.066 mg / ml RC2, respectively. This result shows that the refined components RC2 and RC3 of the Herba Lycoris can inhibit the expression of MMP1 protein to a certain extent and have a certain anti-aging effect.

[0094] Table 9 Relative expression values ​​of MMP1 protein in the refined fractions RC4, RC5 and RC6 of the Herba Lycopodii

[0095] Summary: After UVA treatment, compared with BC, the expression of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA treatment group alone, the expression of MMP1 protein decreased significantly after treatment with 0.0003mg / ml, 0.0008mg / ml of RC5 and 0.012mg / ml, 0.037mg / ml of RC6. This result shows that the refined components RC5 and RC6 of the Herba Lycoris can inhibit the expression of MMP1 protein to a certain extent and have a certain anti-aging effect.

[0096] Table 10 Relative expression values ​​of MMP1 protein in compounds C1, C2, and C3 extracted from the extract of Nymphaeaceae

[0097] Summary: After UVA treatment, compared with BC, the expression of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression 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 a certain concentration of C1 (0.0833mg / ml, 0.25mg / ml), C2 (0.0278mg / ml, 0.0833mg / ml) and C3 (0.0417mg / ml, 0.125mg / ml), the expression of MMP1 protein decreased significantly. This result shows that the three refined compounds C1, C2 and C3 of the ground cypress can inhibit the expression of MMP1 protein and have certain anti-aging effects.

[0098] Table 11 Relative expression values ​​of MMP1 protein in compounds C4, C5, and C6 extracted from the extract of Nymphaeaceae Summary: After UVA treatment, compared with BC, the expression of MMP1 protein increased, indicating that the anti-aging model was successfully established. Under UVA stimulation, the positive control TGF-β was used, and the expression of MMP1 protein decreased significantly, indicating that the positive control of this experiment was effective. Compared with the UVA alone treatment group, when 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) were used for treatment, the expression of MMP1 protein decreased significantly. This result shows that the three refined compounds C4, C5 and C6 of the ground cypress can inhibit the expression of MMP1 protein and have significant anti-aging effects.

[0099] 4. Tyrosinase activity test to determine the whitening effect 4.1 Inhibitory effect of crude extract of T. sutchuenensis on mushroom tyrosinase activity in vitro 4.1.1 In vitro mushroom tyrosinase activity test process (1) Preparation of stock solutions: 1) Mushroom tyrosinase solution: Prepared with pH 6.8 PBS to 100u / ml; 2) Levodopa solution: Prepared with pH 6.8 PBS to 1mg / ml; 3) Kojic acid (positive control): Diluted with pH 6.8 PBS to 0.3mg / ml.

[0100] (2) Add 100 μl of mushroom tyrosinase solution to each sample tube, cover the lid, vortex and separate the sample tube and background tube, and then incubate in a 37°C water bath for 10 minutes.

[0101] (3) Add 400 μl of L-DOPA solution to each tube in turn, cover the tube, vortex and spin down immediately, and control the reaction time of each tube to 5 minutes.

[0102] (4) Transfer the reaction solution in each tube into a 96-well plate and measure the absorbance at 475 nm.

[0103] (5) Calculation of mushroom tyrosinase inhibition rate:

[0104] (6) In the formula, T is the absorbance of the sample well, i.e., the absorbance of the solution after the sample reacts with mushroom tyrosinase; T0 is the background absorbance of the sample; C is the average of three absorbances of the enzyme reaction tube, i.e., the absorbance of the reaction between mushroom tyrosinase and DOPA when no sample is added; and C0 is the background absorbance of the solvent.

[0105] (7) Statistical analysis and graphing: GraphPad Prism 9 software was used for statistical analysis and graphing. If the data met the normal distribution and homogeneity of variance, paired t-test was used for pre- and post-experimental comparison within the group, and one-way analysis of variance was used for comparison of means between groups. P <0.05 indicates that the difference is statistically significant. The results are shown in Table 13 and Fig. 9 .

[0106] Table 12 In vitro mushroom tyrosinase inhibition rate determination experimental sample preparation table 4.1.2 Results of the whitening effect of the crude extract of the herbaceous jellyfish (in vitro mushroom tyrosinase activity) Table 13 Numerical table of mushroom tyrosinase inhibition rates of different concentrations of crude extracts of Hedysarum truncatum Summary: The use of 0.3mg / ml kojic acid showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. Different concentrations of the crude extract of the ground jellyfish showed a good tyrosinase inhibition rate, and it was concentration-dependent, indicating that the crude extract of the ground jellyfish has a good whitening effect.

[0107] 4.2 Effects of crude extracts of N. terrestris, refined fractions and refined compounds of N. terrestris on tyrosinase activity in vivo 4.2.1 In vivo tyrosinase activity experimental process

[0108] (1) Preparation of stock solution: 1) Lysis buffer: Prepare 1% Triton X-100 solution in pH 6.8 PBS. 2) Levodopa solution: Prepare 10 mM in pH 6.8 PBS. 3) Kojic acid (positive control): Dilute to 0.1 mg / ml in pH 6.8 PBS.

[0109] (2) B16-F10 melanocytes in a 12-well plate were cultured in RPMI1640 medium for 24 h.

[0110] (3) Add samples of different concentrations and positive controls for drug treatment.

[0111] (4) After 48 hours of treatment, collect the cells and add lysis buffer to extract the cells.

[0112] (5) Protein quantification: Use protein quantification method (BCA method) to determine the extracted protein concentration.

[0113] (6) Based on the protein content test results, use lysis buffer to unify the total protein amount.

[0114] (7) Take the same total protein amount from each sample and add 90 μl of the solution to the corresponding wells of a 96-well plate. Add 10 μl of L-DOPA solution to each well and incubate at 37°C for 40-60 min.

[0115] (8) Use an ELISA reader to measure the absorbance at 475 nm.

[0116] (9) Calculation of tyrosinase inhibition rate:

[0117] (10) In the formula, T is the absorbance of the sample well, that is, the absorbance of the solution after the sample reacts with tyrosinase; C is the absorbance of the blank control.

[0118] (11) Statistical analysis and graphing: GraphPad Prism 9 software was used for statistical analysis and graphing. If the data met the normal distribution and homogeneity of variance, paired t-test was used for pre- and post-experimental comparison within the group, and one-way analysis of variance was used for comparison of means between groups.P <0.05 indicates that the difference is statistically significant. The results are shown in Table 14 and Fig.10 .

[0119] 4.2.2 Results of the whitening effect of the refined components and extracted compounds of the Herba Lycoris Radiatae (tyrosinase activity in vivo) Table 14 In vivo tyrosinase inhibition rate values ​​of crude extracts of Hedyssa suberectus, refined components of Hedyssa suberectus and refined compounds Note: The selected concentrations of crude extracts of Nymphaeaceae, refined fractions of Nymphaeaceae and refined compounds of Nymphaeaceae are the maximum safe concentrations based on B16 cells.

[0120] Summary: The positive standard 0.1mg / ml kojic acid showed a good tyrosinase inhibition rate, indicating that the experimental system was successful. The crude extract of the scutellaria baicalensis showed an excellent tyrosinase activity inhibition rate, RC1 and RC6 had a certain tyrosinase activity inhibition rate, and C2, C4, C5 and C6 showed a good tyrosinase inhibition rate, indicating that the crude extract of the scutellaria baicalensis and the two refined components and four refined compounds of the scutellaria baicalensis have good whitening effects.

[0121] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a Herba Lysimachiae extract containing at least one of C1 to C6 compounds in the preparation of antioxidant, whitening and / or anti-aging products: Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose; Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose; C3 compound: mumeose K; C4 compound: carotene linoleate; C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid); C6 compound: 2-linolenoyl-rac-glycerol; The extract of the ground jellyfish is an ethanol extract of the flower and bud of the ground jellyfish.

2. The use according to claim 1, characterized in that: The extract of the nephrite is prepared by the following method: taking the flowers and buds of the nephrite, cold-immersing and extracting them with 75% ethanol, and concentrating them to dryness under reduced pressure to obtain a crude extract of the nephrite.

3. The use according to claim 1, characterized in that: The extract of the ground jellyfish is prepared by the following method: taking the flowers and buds of the ground jellyfish, cold-immersing and extracting them with 75% ethanol, and concentrating them to dryness under reduced pressure to obtain a crude extract of the ground jellyfish; applying the crude extract of the ground jellyfish to an MCI column, and gradient eluting them with water, 20% methanol solution, 40% methanol solution, 60% methanol solution, 80% methanol solution and 100% methanol-water solution in sequence to obtain refined components RC1-RC6 of the ground jellyfish respectively.

4. Application of one or a combination of two or more of the C1-C6 compounds in the preparation of antioxidant, whitening and / or anti-aging products: Compound C1: 1,2',3',4',6'-penta-O-acetyl-3-O-(E)-p-coumarylsucrose; Compound C2: 2',3',4',6'-tetra-O-acetyl-3-O-(E)-p-coumarylsucrose; C3 compound: mumeose K; C4 compound: carotene linoleate; C5 compound: glycerol-1-eicosanoic acid-3-(4E, 7E, 10E-hexadecanoic acid); C6 compound: 2-linolenoyl-rac-glycerol.

5. The use according to any one of claims 1 to 4, characterized in that: The products include anti-oxidation, whitening and / or anti-aging skin care products, or health products or foods that help anti-oxidation.

6. The use according to any one of claims 1 to 4, characterized in that: The anti-oxidation comprises: removing DPPH free radicals.

7. The use according to any one of claims 1 to 4, characterized in that: The anti-aging includes at least one of the following I to II: Ⅰ. Inhibition MMP1 Gene transcription and / or inhibition of MMP1 protein expression; II. Promotion COL1 Transcription of genes.

8. The use according to any one of claims 1 to 4, characterized in that: The whitening includes inhibiting the activity of tyrosinase.

9. A product having antioxidant, whitening and / or anti-aging effects, characterized in that: Including auxiliary materials and any of the following (1) to (2): (1) Extract of Herba Lycoris Radiatae; (2) One or a combination of two or more of the C1 to C6 compounds; The preparation method of the Herba Lycopodii extract comprises: Take the flowers and buds of the ground jellyfish, extract them with 75% ethanol, and then concentrate them to dryness under reduced pressure to obtain a crude extract of the ground jellyfish; or The method comprises: taking the flowers and buds of the Herba Lycoris Radiatae, cold-immersing and extracting them with 75% ethanol, separating the extracts by MCI chromatography, collecting the water elution part and the elution part of methanol aqueous solution with volume fractions of 20%, 40%, 60%, 80% and 100% in sequence, respectively, to obtain Herba Lycoris Radiatae refined extract components; The C1-C6 compounds are extracted and separated from the extract of the Herba Lycoris Radiatae.

10. The product according to claim 9, characterized in that The dosage forms of the products include skin care products or health care products; the dosage forms of the skin care products include creams, lotions, solutions, sprays, facial masks, essence water, gels or powders; the dosage forms of the health care products include tablets, emulsions, granules, pills, liquid preparations, ointments or patches.

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