Composition with anti-oxidation, whitening, anti-photoaging and anti-inflammatory effects, preparation method, product and application thereof, and method for analyzing composition based on network pharmacology

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a composition with anti-oxidation, whitening, anti-photoaging and anti-inflammatory effects, a preparation method, a product and application thereof, and a method for analyzing the composition based on network pharmacology. The composition is prepared from the following components in percentage by weight: 40 to 50 percent of peach kernel, 15 to 35 percent of liquorice root, 0.5 to 17 percent of tribulus terrestris, 0.5 to 10.5 percent of radix notoginseng, 0.4 to 1.8 percent of herba leonuri, 1 to 7 percent of silkworm larva, 0.5 to 4.5 percent of rhizoma chuanxiong, 1 to 7 percent of radix angelicae sinensis, 2 to 7 percent of semen coicis, 1 to 8 percent of radix scutellariae, 0.5 to 4 percent of radix ampelopsis and 0.3 to 1.8 percent of semen armeniacae amarae. On the basis of a network pharmacology analysis method, a skin anti-aging effect target protein-protein interaction network is constructed, key action targets of effective components of traditional Chinese medicines are screened and analyzed by utilizing a molecular docking technology, and the composition with the effects of resisting oxidation, whitening, resisting light aging and resisting inflammation is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, a preparation method thereof, a product and application thereof, and a method for analyzing the composition based on network pharmacology. Background Art

[0002] As a unique compatibility principle, "monarch, minister, assistant and messenger" aims to clarify the primary and secondary relationships and interactions of each single medicine in a traditional Chinese medicine compound, so as to achieve accurate treatment of different syndromes and achieve the purpose of syndrome differentiation and treatment. "Monarch medicine" is a medicine that plays a major therapeutic role for the main disease or main symptom. It is the leading component of the formula and plays a decisive role. "Minister medicine" has two meanings: one is a medicine that assists the monarch medicine to strengthen the treatment of the main disease or main symptom; the other is a medicine that plays a major therapeutic role for important concomitant diseases or concomitant symptoms. "Adjuvant medicine" refers to a medicine that cooperates with the monarch medicine and minister medicine to strengthen the therapeutic effect, or directly treats minor concomitant symptoms. "Messenger medicine" is a harmonizing medicine that can guide the medicines in the prescription to reach the lesion, or a medicine that has the effect of harmonizing the medicines in the prescription. The significance of the compatibility of monarch, minister, assistant and messenger is that it stipulates that the medicines in the prescription are orderly, coordinated and restrained. There is both a clear division of labor and close cooperation, which is very rigorous.

[0003] Traditional Chinese medicine focuses on holistic treatment, but the research methods are relatively inefficient. The emergence of network pharmacology provides a new method for TCM research by constructing a "drug-target-disease" network. As an interdisciplinary subject, network pharmacology integrates research methods from multiple fields such as network science, systems biology, computer science, bioinformatics and statistics, thus breaking through the limitations of traditional drug research and comprehensively examining the relationship between disease and drug from a multi-target and multi-component perspective.

[0004] Given the commonality of the mechanisms of action of plant extracts in drugs and cosmetics on the skin, it can be considered that network pharmacology is also applicable to studying the mechanisms of action of plant-based cosmetics on the skin. Currently, the development of cosmetic plant-based raw materials mainly relies on in vitro tests, which have a large number of screenings and low efficiency. Most Chinese medicine compositions have a single skin care effect, and compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects have not yet been reported.

[0005] Therefore, there is an urgent need in this field to study the skin care mechanism of traditional Chinese medicine compound through network pharmacology and cell biology methods, so as to obtain a composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, a preparation method thereof, a product and application thereof, and a method for analyzing the composition based on network pharmacology.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects comprises the following components by weight percentage: 40-50% of peach kernel, 15-35% of liquorice, 0.5-17% of tribulus terrestris, 0.5-10.5% of notoginseng, 0.4-1.8% of motherwort, 1-7% of bombyx batryticatus, 0.5-4.5% of ligusticum chuanxiong, 1-7% of angelica, 2-7% of coix seed, 1-8% of scutellaria baicalensis, 0.5-4% of white ampelopsis and 0.3-1.8% of bitter almond.

[0009] Preferably, the invention comprises the following components by weight percentage: 42-48% peach kernel, 20-35% liquorice, 0.5-5% tribulus terrestris, 0.5-3% panax notoginseng, 0.4-1.5% leonurus, 1.5-7% bombyx batryticatus, 0.5-4% chuanxiong, 1.5-7% angelica, 2-5.5% coix seed, 1-4% scutellaria, 0.5-2% white ampelopsis and 0.3-1% bitter almond.

[0010] More preferably, the composition comprises the following components by weight percentage: 43-45% peach kernel, 30-32% liquorice, 1.5-

[0011] 2.5%, Panax notoginseng 1-2%, Leonurus japonicus 1-1.5%, Bombyx batryticatus 1.5-2%, Ligusticum chuanxiong 0.5-1%, Angelica sinensis 6-7%, Coix lachryma-jobi 2-3%, Scutellaria baicalensis 3-4%, Bletilla striata 1-2% and Apricot kernel 0.5-1%.

[0012] Further preferably, the following components are included by weight percentage: 44.44% peach kernel, 33.33% liquorice, 2.3% tribulus terrestris, 1.53% panax notoginseng, 1.15% motherwort, 1.64% bombyx batryticatus, 0.73% chuanxiong, 6.56% angelica, 2.19% coix seed, 3.83% scutellaria, 1.53% white ampelopsis and 0.77% bitter almond.

[0013] The present invention also provides a method for preparing the above composition, comprising the following steps:

[0014] Peach kernel, liquorice, tribulus terrestris, notoginseng, motherwort, bombyx batryticatus, ligusticum chuanxiong, angelica, coix seed, scutellaria baicalensis, white ampel and bitter almond are crushed and mixed, ethanol is added for reflux extraction, filtered, ethanol is added to the filter residue, reflux extraction is performed, and the extracts are combined to obtain the product.

[0015] Preferably, after the pulverization, the product needs to be sieved through a 60-80 mesh sieve, the volume fraction of the ethanol is 50-70%, and the solid-liquid ratio of the added ethanol is 1g:10-15ml.

[0016] Preferably, the temperature of the reflux extraction is 70-80° C., the time of the reflux extraction is 1-2 h, the reflux extraction requires ultrasound assistance, and the power of the ultrasound is 20-50 KHZ.

[0017] Preferably, after the combined extract is finished, rotary evaporation is further performed, the temperature of the rotary evaporation is 50-60° C., and the pressure of the rotary evaporation is less than 0.01 MPa.

[0018] The present invention also provides a product with antioxidant, whitening, anti-photoaging and anti-inflammatory effects, and the product comprises the above composition or the composition prepared by the above preparation method.

[0019] The present invention also provides the use of the above composition or the composition prepared by the above preparation method in preparing products with anti-oxidation, whitening, anti-photoaging and anti-inflammatory effects.

[0020] The present invention also provides a method for analyzing the anti-oxidation, whitening, anti-photoaging and anti-inflammatory effects of the above composition on the skin based on network pharmacology, comprising the following steps:

[0021] (1) Obtain a dataset of effective ingredients of traditional Chinese medicine with antioxidant, whitening and anti-photoaging effects and their potential targets;

[0022] (2) Obtain a dataset of targets related to skin anti-aging efficacy;

[0023] (3) Construct the interaction network of traditional Chinese medicine-active ingredient-target and the interaction network of traditional Chinese medicine-active ingredient-target-skin anti-aging effect;

[0024] (4) Constructing a protein-protein interaction network of target sites to obtain the key targets of the effective ingredients of traditional Chinese medicine;

[0025] (5) Analyze the mechanism of action of key targets of effective ingredients of traditional Chinese medicine;

[0026] (6) Based on molecular docking technology, the effective ingredients of traditional Chinese medicine are docked with the targets related to skin anti-aging effects.

[0027] Preferably, step (1) comprises the following specific steps: using the Chinese medicine systems pharmacology, TCMSP, SymMap and HERB databases, with drug-like properties ≥ 0.18 as the screening condition, the main active ingredients and potential targets of Glycyrrhiza uralensis, Scutellaria baicalensis, Bletilla striata, Apricot kernel, Leonurus japonicus, Tribulus terrestris, Panax notoginseng, Bombyx batryticatus, Ligusticum chuanxiong, Angelica sinensis, Peach kernel and Coix seed are obtained.

[0028] Preferably, the method for obtaining skin anti-aging efficacy-related targets in step (2) comprises: using antioxidant, anti-ultraviolet and whitening as keywords, searching DisGeNET, OMIM and GeneCards databases, and merging and removing duplicates from the search results.

[0029] Preferably, step (3) comprises the following specific steps: constructing a Chinese medicine-active ingredient-target interaction network by using Cytoscape v3.10.0 software, importing the potential target data set of the Chinese medicine active ingredient of step (1) and the skin anti-aging efficacy-related target data set of step (2) into Venny 2.1.0, obtaining the intersection target related to the skin anti-aging efficacy of the Chinese medicine active ingredient, importing the obtained intersection target and the corresponding Chinese medicine active ingredient into Cytoscape v3.10.0 software, and constructing the Chinese medicine-active ingredient-target-skin anti-aging efficacy interaction network.

[0030] Preferably, the method of constructing a target protein-protein interaction network in step (4) to obtain the key target of the effective ingredient of the traditional Chinese medicine comprises: importing the intersection target of step (3) into the STRING database and Cytoscape v3.10.0 software in sequence.

[0031] Preferably, the method for analyzing the mechanism of action of the key target of the effective ingredient of traditional Chinese medicine in step (5) comprises: performing GO enrichment analysis, KEGG pathway enrichment analysis and GEO difference analysis on the target protein-protein interaction network.

[0032] Preferably, step (6) comprises the following specific steps: using the four active ingredients with the highest degree values ​​in the interaction network of traditional Chinese medicine-active ingredient-target-anti-aging skin efficacy in step (3) as ligands for molecular docking, downloading the ligand structure for molecular docking from the TCMSP database, selecting the four proteins with the highest degree values ​​in the target protein-protein interaction network as receptors for molecular docking, downloading the molecular docking receptor structure from the PDB database, using PyMOl software to perform necessary modifications on the structures of the ligand and receptor before molecular docking, and then performing molecular docking using AutoDock software.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention is based on the network pharmacology analysis method, constructs a target protein-protein interaction network, uses molecular docking technology to screen and analyze the key target points of the effective ingredients of traditional Chinese medicine, and obtains a composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects. The composition has excellent antioxidant, whitening, anti-photoaging and anti-inflammatory effects.

[0035] (2) The present invention networks and systematizes the research methods of the drug's skin antioxidant, whitening, anti-photoaging and anti-inflammatory effects. Compared with traditional research methods, the research scope is narrowed and the research purpose is precise, thereby saving drug research and development costs. At the same time, the efficiency of drug screening and prediction is greatly improved, providing a new reference for the research and development of traditional Chinese medicine compound skin care products, and having a breakthrough significance for the development and industrialization of traditional Chinese medicine resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of the cell tyrosinase activity experiment (compared with the control group, * P<0.05, ** P<0.01, *** P < 0.001,

[0037] **** P<0.0001).

[0038] Figure 2 The results of the experiment on the content of melanin production in cells (compared with the control group, * P<0.05, ** P<0.01, *** P < 0.001,

[0039] **** P<0.0001).

[0040] Figure 3 The results of the cell H2O2 oxidative damage pre-protection experiment (compared with the control group, #### P<0.0001; compared with the model group, **** P<0.0001).

[0041] Figure 4 Results of the cell H2O2 oxidative damage repair experiment (compared with the control group, ### P<0.001; compared with the model group,

[0042] * P<0.05, ** P<0.01, *** P<0.001).

[0043] Figure 5 Results of the UVA light damage repair experiment on cells (compared with the control group, # P<0.05, ## P<0.01; compared with the model group, ** P<0.01, *** P < 0.001, **** P<0.0001). DETAILED DESCRIPTION

[0044] It is worth noting that the raw materials used in the present invention are all common commercial products, among which ethanol, with a content of 95%, was purchased from Shanghai Suishi Chemical Technology Co., Ltd.

[0045] Example 1

[0046] Network pharmacology analysis

[0047] 1. Prediction of Targets of Twelve Traditional Chinese Medicines

[0048] The main active ingredients and potential targets of twelve Chinese medicines (Glycyrrhiza uralensis, Scutellaria baicalensis, Bletilla striata, Apricot kernel, Leonurus japonicus, Tribulus terrestris, Panax notoginseng, Bombyx batryticatus, Ligusticum chuanxiong, Angelica sinensis, Semen persica and Semen coix) were screened by using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), SymMap database and HERB database, with drug-like properties ≥ 0.18 as the screening condition. The gene names of potential targets were obtained by converting potential protein targets into Gene symbols through the Uniport database, and the dataset of active ingredients of Chinese medicines and their corresponding targets was obtained.

[0049] 2 Prediction of skin anti-aging efficacy related targets

[0050] DisGeNET, the Online Mendelian Inheritance in Man (OMIM), and the GeneCards database were used to search for keywords related to antioxidant, anti-ultraviolet, and skin whitening anti-aging effects (antioxidant, antioxidant, antioxidant, oxidative stress, oxidative, oxidation, oxidant, stress response, Melanosis gene, Melanosis, Pigmentation gene, Pigmentation, skinwhitening, skin pigmentation, cosmetology, and sunspots) to obtain the targets of relevant skin anti-aging effects. After removing duplicate values, the relevant target data set was obtained.

[0051] 3. Construction of twelve Chinese medicine-active ingredient-target interaction networks

[0052] The names of the above twelve Chinese medicines, the retrieved active ingredients, and the gene ID numbers of the potential targets corresponding to the active ingredients were input into Cytoscape-v3.10.0 software to draw a Chinese medicine-active ingredient-target interaction map.

[0053] 4. Construction of the interactive network of twelve traditional Chinese medicines-active ingredients-targets-skin anti-aging effects

[0054] The active ingredient prediction target dataset and related action target dataset of the above twelve traditional Chinese medicines were imported into Venny-2.1.0 to obtain the intersection targets of the active ingredients and the targets related to skin anti-aging efficacy. The names, active ingredients, obtained intersection targets, and the efficacy corresponding to the intersection targets of the twelve traditional Chinese medicines were imported into Cytoscape-v3.10.0 software to draw the interaction network of traditional Chinese medicine-active ingredients-action targets-skin anti-aging efficacy. The four active ingredients with the highest degree values ​​were selected for subsequent molecular docking.

[0055] 5. Construction of target protein-protein interaction network (PPI network)

[0056] The intersection targets obtained above were imported into the STRING database, the species were restricted to Homo sapiens, and the connection score was set to >0.9 to obtain the protein interactions corresponding to the intersection targets. The interaction files were obtained and imported into the Cytoscape-v3.10.0 software to draw the PPI network, thereby obtaining the ranking of important protein targets of the active ingredients, and the four targets with the highest degree values ​​were selected for molecular docking.

[0057] 6. GO enrichment analysis and KEGG pathway enrichment analysis of target

[0058] The top 20 targets with the highest degree values ​​in the intersection target PPI network interaction graph were imported into the DAVID database, and the species was set to Homo sapiens and P>0.05. KEGG and GO enrichment analysis was performed to preliminarily obtain differentially expressed genes.

[0059] 7. GEO differential analysis of target sites

[0060] In the GEO database, we found a gene dataset of melasma (GES72140) with a high similarity to the target, and intersected it with the target in the Venny-2.1.0 database. We then used the GEO database's online software GEO2R to perform differential analysis on the intersection of the GES72140 gene dataset and the target and draw a volcano map.

[0061] 8. Molecular Docking

[0062] The four active ingredients with the highest degree values ​​in the interaction network of traditional Chinese medicine-active ingredient-target-anti-aging skin efficacy were selected as ligands for molecular docking, and the ligand structures for molecular docking were downloaded from the TCMSP database. The four target proteins with the highest degree values ​​in the PPI network were selected as receptors for molecular docking, and the receptor structures for molecular docking were downloaded from the PDB database. PyMOl software was used to modify the structures of ligands and receptors before molecular docking, and then molecular docking was performed using AutoDock software.

[0063] According to network pharmacology analysis, a composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects is obtained, as shown in Examples 2-5.

[0064] Example 2

[0065] A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, comprising the following components by weight percentage:

[0066] Peach kernel 44.44%, licorice 33.33%, tribulus terrestris 2.3%, Panax notoginseng 1.53%, Leonurus japonicus 1.15%, Bombyx batryticatus 1.64%, Ligusticum chuanxiong 0.73%, Angelica sinensis 6.56%, Coix seed 2.19%, Scutellaria baicalensis 3.83%, Bletilla striata 1.53% and Apricot kernel 0.77%.

[0067] The preparation method of the above-mentioned composition having the effects of anti-oxidation, whitening, anti-photoaging and anti-inflammatory is as follows:

[0068] (1) First, 12 kinds of Chinese medicinal materials including peach kernel, liquorice, tribulus terrestris, Panax notoginseng, motherwort, bombyx batryticatus, ligusticum chuanxiong, angelica sinensis, coix seed, scutellaria baicalensis, white amphibians and bitter almond were crushed and sieved through a 60-mesh sieve, mixed according to the above weight percentages, added with 60% ethanol by volume at a solid-liquid ratio of 1 g:12 ml, and refluxed at 75° C. for 1.5 h with 40 kHz ultrasound assistance. After the reflux was completed, the mixture was filtered and the filtrate was collected.

[0069] (2) The extracted medicinal residue was added with 60% ethanol by volume at a solid-liquid ratio of 1 g:12 ml, and refluxed at 75° C. with 40 kHz ultrasound for 1.5 h. After reflux, the filtrate was filtered and the filtrate was collected.

[0070] (3) Finally, the extracts from the two ultrasonic refluxes were mixed and evaporated in a rotary evaporator at a vacuum degree of 0.01 MPa and a water bath of 55° C. until no ethanol was present.

[0071] Example 3

[0072] A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, comprising the following components by weight percentage:

[0073] Peach kernel 44.44%, licorice 33.3%, tribulus terrestris 0.93%, panax notoginseng 0.62%, motherwort 0.46%, bombyx batryticatus 6.64%, chuanxiong 4%, angelica 1.78%, coix seed 5.33%, scutellaria 1.54%, white ampel 0.62% and bitter almond 0.31%.

[0074] The preparation method of the above-mentioned composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects is the same as that of Example 2.

[0075] Example 4

[0076] A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, comprising the following components by weight percentage:

[0077] Peach kernel 44.44%, licorice 16.67%, tribulus terrestris 16.67%, Panax notoginseng 2.3%, Leonurus japonicus 0.77%, Bombyx batryticatus 1.15%, Ligusticum chuanxiong 4%, Angelica sinensis 1.78%, Coix seed 5.33%, Scutellaria baicalensis 1.53%, Bletilla striata 3.83% and Apricot kernel 1.53%.

[0078] The preparation method of the above-mentioned composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects is the same as that of Example 2.

[0079] Example 5

[0080] A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, comprising the following components by weight percentage:

[0081] Peach kernel 44.44%, licorice 13.43%, tribulus terrestris 9.72%, Panax notoginseng 10.19%, Leonurus japonicus 1.71%, Bombyx batryticatus 1.15%, Ligusticum chuanxiong 4%, Angelica sinensis 1.78%, Coix seed 5.33%, Scutellaria baicalensis 7.35%, Bletilla striata 1.03% and Apricot kernel 0.68%.

[0082] The preparation method of the above-mentioned composition with antioxidant, whitening, anti-photoaging and anti-inflammatory effects is the same as that of Example 2.

[0083] Table 1 Components and ratios of compositions of Examples 2-5

[0084] Name of Chinese medicine Example 2 Example 3 Example 4 Example 5 Peach Kernel 44.44％ 44.44％ 44.44％ 44.44％ Licorice 33.33％ 33.33％ 16.67％ 13.43％ Tribulus 2.3％ 0.93％ 16.67％ 9.72％ Panax notoginseng 1.53％ 0.62％ 2.3％ 10.19％ Motherwort 1.15％ 0.46％ 0.77％ 1.71％ White silkworm 1.64％ 6.64％ 1.15％ 1.15％ Chuanxiong 0.73％ 4％ 4％ 4％ Angelica 6.56％ 1.78％ 1.78％ 1.78％ Job's tears 2.19％ 5.33％ 5.33％ 5.33％ Scutellaria baicalensis 3.83％ 1.54％ 1.53％ 7.35％ White smilax 1.53％ 0.62％ 3.83％ 1.03％ Bitter almonds 0.77％ 0.31％ 1.53％ 0.68％

[0085] Comparative Example 1

[0086] The same as Example 2, the only difference is that the proportion of each Chinese medicine component is 8.33%.

[0087] A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, comprising the following components by weight percentage:

[0088] Peach kernel 8.33%, licorice 8.33%, tribulus terrestris 8.33%, Panax notoginseng 8.33%, motherwort 8.33%, bombyx batryticatus 8.33%, ligusticum chuanxiong 8.33%, angelica 8.33%, coix seed 8.33%, scutellaria baicalensis 8.33%, white ampel 8.33% and bitter almond 8.33%.

[0089] Test Example 1

[0090] The compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects prepared in Examples 2-5 and Comparative Example 1 were respectively dissolved in pure water, fixed to a certain concentration, filtered through a 1 μm filter membrane for later use, and subjected to antioxidant effect testing.

[0091] 1.DPPH free radical scavenging experiment

[0092] 1.1 Experimental methods:

[0093] First, take 100μl of samples with concentrations of 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mg / ml, and add 100μl of 0.1mol / l DPPH anhydrous ethanol solution respectively under light-proof conditions. Then react for 30 minutes under light-proof conditions at 37℃, and measure the absorbance at 517nm. The absorbance of the reaction between DPPH alcohol solution and sample is recorded as A1; the absorbance is recorded as A2 when the DPPH alcohol solution is replaced by anhydrous ethanol solution; the absorbance is recorded as A0 when the sample is replaced by ultrapure water. Finally, the DPPH free radical scavenging rate is calculated using the formula "DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0]×100%".

[0094] 1.2 Experimental results:

[0095] The experimental results are shown in Table 2. As can be seen from Table 2, Example 2 has the strongest DPPH radical scavenging ability, followed by Example 4 > Example 5 > Example 3 > Comparative Example 1. The DPPH radical scavenging abilities of Examples 3-5 and Comparative Example 1 are enhanced with the increase of the dosage concentration, and there is a good linear relationship between the dosage concentration and the clearance rate in each group.

[0096] Table 2 DPPH free radical scavenging rate at different dosage concentrations

[0097]

[0098] 2. ABTS free radical scavenging experiment

[0099] 2.1 Experimental methods:

[0100] First, 7 mmol / l ABTS solution and 2.45 mmol / l potassium persulfate solution were prepared with PBS (pH = 7.4) solution, respectively. 2 ml of ABTS solution and potassium persulfate solution were taken respectively, mixed, and stored in the dark at 37°C for 16 h. The mixed solution was further diluted with PBS (pH = 7.4) solution until its absorbance at 734 nm was 0.700 ± 0.025 to obtain a mixed solution containing ABTS for later use. Then, 100 μl of samples with concentrations of 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 mg / ml were taken respectively, and 100 μl of the mixed solution containing ABTS was added under the condition of protecting from light. The mixture was reacted at 37°C for 30 min, and the absorbance was measured at 734 nm. The absorbance of the reaction between the mixed solution containing ABTS and the sample is recorded as A1; the sample solution is replaced by PBS (pH = 7.4) solution, and the absorbance is recorded as A0; the ABTS solution is replaced by PBS (pH = 7.4) solution, and the absorbance is recorded as A2. Finally, the ABTS free radical scavenging rate is calculated by the formula "ABTS free radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%".

[0101] 2.2 Experimental results:

[0102] The experimental results are shown in Table 3. As can be seen from Table 3, Example 2 has the strongest ABTS free radical scavenging ability, followed by Example 3 > Example 4 > Example 5 > Comparative Example 1. The ABTS free radical scavenging ability of Examples 2-5 and Comparative Example 1 increases with the increase of the administration concentration, and there is a good linear relationship between each concentration and the clearance rate.

[0103] The scavenging ability was ranked according to IC50 value as follows: Example 2 (0.1979) > Example 3 (0.3501) > Example 4 (0.6487) > Example 5 (0.8127) > Comparative Example 1 (0.8632).

[0104] Table 3 ABTS free radical scavenging rate at different dosage concentrations

[0105]

[0106]

[0107] Test Example 2

[0108] The compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects prepared in Examples 2-5 and Comparative Example 1 were respectively dissolved in pure water, fixed to a certain concentration, filtered through a 1 μm filter membrane for later use, and subjected to a cell whitening effect test.

[0109] 1. Cellular tyrosinase activity assay

[0110] 1.1 Experimental methods:

[0111] B16F10 cells (purchased from ATCC) were cultured for 2-3 generations with DMEM / 1640 complete medium (volume ratio of 1:1) until the B16F10 cells turned gray-black. The gray-black B16F10 cells were suspended in DMEM complete medium and diluted to contain 1×10 per 100 μl of cell suspension. 4 cells, and then 1×10 4 The cells were seeded in a 96-well plate at a density of 100 / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The drug was diluted to the experimental concentration using DMEM culture medium. Taking Example 2 as an example: the drug group concentrations were set to 0.25, 0.5, 1, 2, 4 and 6 mg / ml. 100 μl of drug was given to each well, and the control group was a drug-free DMEM culture medium. Three replicate wells were set for each concentration and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The original culture medium was discarded, and the cells were washed twice with 100 μl / well of PBS. Then 100 μl / well of 1% Triton X-100 solution was added, and placed in a -80°C refrigerator for 1 hour. The above-mentioned well plate was placed at room temperature to melt, and 20 μl of 0.5% levodopa solution was added to each well and oscillated at room temperature for 4-6 hours. The absorbance was measured at a wavelength of 490 nm, and the formula "Tyrosinase activity = A 给药孔 / A 对照孔 × 100%” to calculate cell tyrosinase activity.

[0112] 1.2 Experimental results:

[0113] The experimental results are as follows Figure 1 As shown by Figure 1 It can be seen that the treatment groups of Example 2 and Example 5 can significantly increase the activity of tyrosinase at low concentrations.

[0114] Compared with the control group, when the administration concentrations of Example 2 were 2, 4 and 6 mg / ml, the tyrosinase activity was reduced to 94.7%, 81.7% and 82.0%, respectively; and the tyrosinase activity of Example 5 was reduced to 97.7%, 85.0% and 84.0%.

[0115] While the treatment groups of Example 3 and Comparative Example 1 had no significant effect on tyrosinase activity at low concentrations, high concentrations showed an inhibitory effect on tyrosinase activity. When the administration concentrations of Example 3 were 2, 4, 8 and 12 mg / ml, the tyrosinase activity was reduced to 92.7%, 89.0%, 82.3% and 52.0%, respectively; when the administration concentrations of Comparative Example 1 were 2, 4, 8, 16 and 24 mg / ml, the tyrosinase activity was reduced to 98.0%, 93.7%, 92.7%, 82.0% and 71.7%, respectively. The inhibitory effect of Example 4 on tyrosinase was not significant and unstable. When the administration concentrations were 2, 8, 16 and 24 mg / ml, the tyrosinase activity was only reduced to 91.3%, 95.3%, 97.7% and 93.0%.

[0116] The IC50 values ​​of the composition formulas on tyrosinase activity are arranged as follows: Example 2 (12.43) > Example 3 (13.01) > Example 5 (27.72) > Comparative Example 1 (49.23), among which the IC50 value of Example 4 cannot be calculated. It can be seen that Example 2 has the best inhibitory effect on tyrosinase activity, followed by Example 3. This shows that Examples 2-5 have a certain inhibitory effect on the activity of tyrosinase in B16F10 cells and have the potential to exert whitening effects.

[0117] 2. Intracellular melanin production content experiment

[0118] 2.1 Experimental methods:

[0119] The cell culture, plating method, grouping and drug administration methods in this experimental method are the same as those in the "Cell Tyrosinase Activity Experiment". After treating the cells with drugs for 24 hours, the original culture medium was discarded and washed twice with 100μl / well PBS. Then, 50μl of 1mol / l NaOH solution containing 10% DMSO was added to each well, placed in a 90℃ water bath for 1 hour, and the absorbance value was measured at a wavelength of 405nm. Then, according to the formula "Melanin content (%) = A 给药孔 / A 对照孔 × 100%” to calculate the melanin content.

[0120] 2.2 Experimental results:

[0121] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that when the concentration is higher than 0.375 mg / ml, the treatment groups of Examples 2-5 and Comparative Example 1 can reduce the production of melanin in B16F10 cells. And within the concentration range of 3 mg / ml, the trend of the effect on melanin production is basically that the effect of inhibiting melanin production is stronger with increasing concentration, and the maximum inhibitory effect is achieved at a concentration of 3 mg / ml.

[0122] Compared with the control group, when the administration concentrations of Example 2 were 0.09375, 0.1875, 0.375, 0.75, 1.5 and 3 mg / ml, the melanin production contents were 93.0%, 92.0%, 83.7%, 87.7%, 75.7% and 73.3%, respectively;

[0123] The melanin production contents of Example 3 were 93.7%, 96.7%, 86.3%, 82.3%, 89.7% and 75.3% respectively;

[0124] The melanin production contents of Example 4 were 100.3%, 97.7%, 87.0%, 81.3%, 85.0% and 79.3% respectively;

[0125] The melanin production contents of Example 5 were 105.0%, 98.3%, 97.0%, 90.7%, 101.0% and 78.7%, respectively.

[0126] The melanin production contents of Comparative Example 1 were 82.3%, 87.3%, 88.0%, 88.0%, 86.0% and 76.0%, respectively.

[0127] The average values ​​of the percentage of melanin production content are respectively: Example 2 (73.3%) < Example 3 (75.3%) < Comparative Example 1 (76.0%) < Example 5 (78.7%) < Example 4 (79.3%).

[0128] It can be seen that Example 2 has the best inhibitory effect on cell melanin production, followed by Example 3. This shows that Examples 2-5 and Comparative Example 1 have a certain inhibitory effect on cell melanin production and have whitening potential.

[0129] Test Example 3

[0130] The compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects prepared in Examples 2-5 and Comparative Example 1 were respectively dissolved in pure water, fixed to a certain concentration, filtered through a 1 μm filter membrane for use, and subjected to an anti-inflammatory effect test.

[0131] 1. Elastase Inhibition Experiment

[0132] 1.1 Experimental methods:

[0133] The elastase reaction substrate N-succinyl-l-alanyl-l-alanyl-l-alanine-4-nitroanilide was dissolved in a Tris-HCl buffer solution (pH=8.0) with a concentration of 0.1 mmol / l to a concentration of 1.015 mmol / l, which was called the elastase substrate solution. Then prepare sample solutions with concentrations of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg / ml respectively; add 130 μl of elastase substrate solution and 10 μl of samples of various concentrations to each well of a 96-well plate, then add 15 μl of elastase with a titer of 0.5 U / ml, place on a shaker for shaking culture for 40 minutes, measure the absorbance value at a wavelength of 405 nm and record it as A1; use Tris-HCl buffer solution instead of elastase substrate solution for reaction, and record the absorbance as A2; use ultrapure water instead of sample solution for reaction, and record the absorbance as A3, and calculate the elastase inhibition rate according to the formula "elastase inhibition rate (%) = (A3-A1+A2) / A3×100%".

[0134] 1.2 Experimental results:

[0135] The experimental results are shown in Table 4. As can be seen from Table 4, Examples 2-5 and Comparative Example 1 all achieved the maximum inhibition rate at the highest dose of 5.0 mg / ml. The IC50 values ​​of the composition formulas for inhibiting the activity of elastase are arranged as follows: Example 3 (0.5001) > Example 2 (0.5015) > Example 4 (6.949) > Comparative Example 1 (10.3) > Example 5 (13.55).

[0136] It can be seen that Example 3 has the best inhibitory effect on elastase activity, followed by Example 2, and Examples 2-5 all have obvious anti-inflammatory effects.

[0137] Table 4 Elastase activity inhibition rate at different drug concentrations

[0138]

[0139] Test Example 4

[0140] The compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects prepared in Examples 2-5 and Comparative Example 1 were respectively dissolved in pure water, fixed to a certain dosing concentration, and set aside for intracellular antioxidant effect test.

[0141] 1. Cellular H2O2 Oxidative Damage Pre-protection Experiment

[0142] 1.1 Experimental methods:

[0143] L929 cells (purchased from ATCC) with good growth conditions were cultured in DMEM complete medium until the logarithmic growth phase, digested with trypsin (containing 0.25% EDTA) and centrifuged, suspended in DMEM complete medium and diluted to contain 1×10 cells per 100 μl of cell suspension. 4 cells, and then 1×10 4 Cells were seeded in a 96-well plate at a density of 100 μl / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h. The drug was diluted to the experimental dosing concentration with DMEM medium, and 100 μl of samples of different dosing concentrations were added to each well of the 96-well plate, which was recorded as the dosing group; 100 μl of DMEM medium was added to each well, which was recorded as the control group; and 100 μl of DMEM medium (later oxidized with H2O2) was added to each well, which was recorded as the model group. Three replicate wells were set for each group and each concentration of the drug-treated group. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h, the culture medium was discarded, and the cells were washed three times with 100 μl / well of PBS. 100 μl of H2O2 solution diluted to 1.0 mmol / l with DMEM culture medium was added to each well of the drug-treated group and the model group. The control group was replaced with an equal amount of DMEM culture medium at the same time. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h, and the culture medium was discarded. 100 μl of MTT reagent with a concentration of 0.5 mg / ml was added to all groups. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 h, and the MTT reagent was discarded. 120 μl of DMSO was added to each well, and the cells were shaken on a shaker for 10 min. The absorbance value was measured at 490 nm, and the formula "cell survival rate (%) = A 实验孔 / A 对照孔 × 100%” to calculate the cell survival rate.

[0144] 1.2 Experimental results:

[0145] The experimental results are as follows Figure 3 As shown by Figure 3 It can be seen that compared with the model group (i.e., H2O2-treated group), the cell survival rate in the drug pre-protection group was significantly improved.

[0146] Compared with the control group, after 1.0mM H2O2 injury, the cell survival rate of the model group decreased to 43.8%; after 0.75, 1.5 and 3mg / ml drug protection was given in advance, the cell survival rates of Example 2 were increased to 79.3%, 114.3% and 134.0%, respectively. The cell survival rate was not only improved, but also had the effect of promoting cell growth above the 1.5mg / ml dose; under the same concentration of drug protection in advance, the cell survival rates of Example 3 increased to 88.0%, 96.3% and 99.3%, respectively; the cell survival rates of Example 4 increased to 68.7%, 89.7% and 98.7%, respectively; the cell survival rates of Example 5 increased to 78.7%, 86.3% and 93.3%, respectively; the comparative example 1 also had a significant protective effect, and the cell survival rates increased to 83.7%, 96.3% and 99.3%, respectively.

[0147] 92.3% and 91.7%.

[0148] This shows that the combination formula can effectively protect L929 cells under H2O2 damage conditions and has significant antioxidant capacity.

[0149] 2. Cellular H2O2 Oxidative Damage Repair Experiment

[0150] 2.1 Experimental methods:

[0151] The cell culture, plate seeding method, grouping and drug administration in this experimental method are the same as those in the "Cell H2O2 Oxidative Damage Pre-protection Experiment". 4 The cells were seeded in a 96-well plate at a density of 100 μl / well, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, and then the culture medium was discarded. After washing once with 100 μl / well PBS, 100 μl of H2O2 solution diluted to 0.5 mmol / l with DMEM medium was added to each well of the drug group and the model group. The control group was replaced with an equal amount of DMEM medium at the same time, and incubated in a cell culture incubator at 37°C and 5% CO2 for 4 hours. The culture medium was discarded, and washed three times with 100 μl / well PBS. 100 μl of samples with different drug concentrations were added to each well of the drug group; 100 μl of DMEM medium was added to the model group, and the control group was replaced with an equal amount of DMEM medium again. Three replicate wells were set for each group and each concentration of the drug group, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The culture medium was then discarded, and 100 μl of 0.5 mg / ml MTT reagent was added to all groups. The cells were incubated at 37°C and 5% CO2 for 4 h. The MTT reagent was discarded, and 120 μl of DMSO was added to each well. The cells were shaken for 10 min, and the absorbance was measured at 490 nm. The formula "Cell survival rate (%) = A 实验孔 / A 对照孔 × 100%” to calculate the cell survival rate.

[0152] 2.2 Experimental results:

[0153] The experimental results are as follows Figure 4 As shown by Figure 4 It can be seen that compared with the model group (ie, H2O2 treated group), the cell survival rates of Examples 2-4 and Comparative Example 1 were partially improved when the composition was used to repair damaged cells after oxidative damage, while Example 5 had no obvious repair effect.

[0154] Compared with the control group, after being damaged by 0.5mM H2O2, the cell survival rate of the model group decreased to 91.8%; while after the 0.75, 1.5 and 3mg / ml drug protection was given in advance in Example 2, the cell survival rates were increased to 108.7%, 105.3% and 113.0%, respectively. Similarly, the cell survival rates were not only improved, but also had the effect of promoting cell growth; the cell survival rates of Example 3 increased to 94.7%, 97.3% and 102%, respectively; the cell survival rates of Example 4 increased to 94.7%, 97.3% and 102%, respectively; the cell survival rates of Example 5 decreased to 89.3%, 89.7% and 87.7%, respectively, that is, it did not show the ability to repair cells after oxidative damage; Comparative Example 1 also had a repair effect at high doses, and the cell survival rates were 89.3%, 92.3% and 100.3%, respectively.

[0155] This shows that most of the composition formulas can effectively repair L929 cells damaged by H2O2 and have a certain ability to resist oxidative damage and repair. Among them, Example 2 has the best effect, while Example 5 has no effect.

[0156] Test Example 5

[0157] The compositions with antioxidant, whitening, anti-photoaging and anti-inflammatory effects prepared in Examples 2-5 and Comparative Example 1 were respectively dissolved in pure water, fixed to a certain dosage concentration, and set aside for testing the cell anti-ultraviolet damage efficacy.

[0158] 1. Cell UVA light damage repair experiment

[0159] 1.1 Experimental methods:

[0160] L929 cells with good growth conditions were cultured in DMEM complete medium until the logarithmic growth phase, digested with trypsin (containing 0.25% EDTA) and centrifuged, suspended in DMEM complete medium and diluted to contain 1×10 cells per 100 μl of cell suspension. 4 cells, and then 1×10 4Cells were seeded in a 96-well plate at a density of 100 / well and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The culture medium was discarded and washed once with 100 μl / well of PBS. 50-100 μl of PBS was added to all experimental wells. The control group was shielded with tin cardboard and not irradiated with UVA. The other experimental wells were irradiated with 30 J / cm 3 UVA irradiation. After irradiation, the culture medium was discarded, and the cells were washed twice with 100 μl / well PBS. 100 μl of samples of different drug concentrations diluted with DMEM were added to each well of the drug-treated group; 100 μl of DMEM culture medium was added to the model group; and an equal amount of DMEM culture medium was added to the control group. Three replicate wells were set up for each group and each concentration of the drug-treated group, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The culture medium was then discarded, and 100 μl of MTT reagent with a concentration of 0.5 mg / ml was added to all groups. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 4 hours, the MTT reagent was discarded, and 120 μl of DMSO was added to each well. The cells were shaken on a shaker for 10 minutes, and the absorbance was measured at 490 nm. The formula "Cell survival rate (%) = A 实验孔 / A 对照孔 × 100%” to calculate the cell survival rate.

[0161] 1.2 Experimental results:

[0162] The experimental results are as follows Figure 5 As shown by Figure 5 It can be seen that compared with the model group after UVA irradiation, some concentrations of the drug-treated groups showed an effect of repairing against UV damage.

[0163] If 30J / cm 3 After UVA irradiation, the cell survival rate in the model group decreased to 92.5% compared with the control group;

[0164] After the drug protection of 0.75, 1.5 and 3 mg / ml was given in advance in Example 2, the cell survival rates were increased to 108.7%, 102.0% and 104.7%, respectively. The cell survival rates were not only improved, but also had the effect of promoting cell growth; the cell survival rates of Example 3 were increased to 94.7% and 98.7%, respectively; the cell survival rates of Example 4 were increased to 96.0%, 95.7% and 95.0%, respectively; the cell survival rate of Example 5 was 94.7% under the advance protection of 1.5 mg / ml of the drug; the cell survival rates of Comparative Example 1 were 95.3% and 93.0%, respectively, under the advance protection of 1.5 and 3 mg / ml of the drug.

[0165] This indicates that the composition formula has a certain effect of resisting UVA damage.

[0166] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composition having antioxidant, whitening, anti-photoaging and anti-inflammatory effects, characterized in that: Calculated by weight percentage, the invention comprises the following components: 40-50% of peach kernel, 15-35% of liquorice, 0.5-17% of tribulus terrestris, 0.5-10.5% of panax notoginseng, 0.4-1.8% of leonurus japonicus, 1-7% of bombyx batryticatus, 0.5-4.5% of ligusticum chuanxiong, 1-7% of angelica sinensis, 2-7% of coix seed, 1-8% of scutellaria baicalensis, 0.5-4% of white ampelopsis and 0.3-1.8% of bitter almond.

2. The composition according to claim 1, characterized in that Calculated by weight percentage, the invention comprises the following components: 42-48% of peach kernel, 20-35% of liquorice, 0.5-5% of tribulus terrestris, 0.5-3% of panax notoginseng, 0.4-1.5% of leonurus japonicus, 1.5-7% of bombyx batryticatus, 0.5-4% of chuanxiong, 1.5-7% of angelica sinensis, 2-5.5% of coix seed, 1-4% of scutellaria baicalensis, 0.5-2% of white ampelopsis bud and 0.3-1% of bitter almond.

3. The composition according to claim 2, characterized in that Calculated by weight percentage, the invention comprises the following components: 43-45% of peach kernel, 30-32% of liquorice, 1.5-2.5% of tribulus terrestris, 1-2% of panax notoginseng, 1-1.5% of leonurus japonicus, 1.5-2% of bombyx batryticatus, 0.5-1% of chuanxiong, 6-7% of angelica sinensis, 2-3% of coix seed, 3-4% of scutellaria baicalensis, 1-2% of white ampelopsis bud and 0.5-1% of bitter almond.

4. A method for preparing the composition according to any one of claims 1 to 3, characterized in that: The steps include: Peach kernel, licorice, tribulus terrestris, Panax notoginseng, motherwort, Bombyx batryticatus, Ligusticum chuanxiong, angelica, coix seed, scutellaria baicalensis, white ampel and bitter almond are crushed and mixed, ethanol is added for reflux extraction, filtered, ethanol is added to the filter residue for reflux extraction, and the extracts are combined to obtain the product.

5. The preparation method according to claim 4, characterized in that: After the pulverization, the powder needs to be sieved through a 60-80 mesh sieve. The volume fraction of the ethanol is 50-70%, and the solid-liquid ratio of the added ethanol is 1g:10-15ml.

6. The preparation method according to claim 4, characterized in that: The temperature of the reflux extraction is 70-80° C., the time of the reflux extraction is 1-2 h, and the reflux extraction requires ultrasound assistance, and the power of the ultrasound is 20-50 KHZ.

7. The preparation method according to claim 4, characterized in that: After the combined extract is completed, rotary evaporation is performed, the temperature of the rotary evaporation is 50-60° C., and the pressure of the rotary evaporation is less than 0.01 MPa.

8. A product with antioxidant, whitening, anti-photoaging and anti-inflammatory effects, characterized in that: The product comprises the composition according to any one of claims 1 to 3 or the composition prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the composition according to any one of claims 1 to 3 or the composition prepared by the preparation method according to any one of claims 4 to 7 in preparing products with antioxidant, whitening, anti-photoaging and anti-inflammatory effects.

10. A method for analyzing the antioxidant, whitening, anti-photoaging and anti-inflammatory effects of the composition of any one of claims 1 to 3 on the skin based on network pharmacology, characterized in that: The steps include: (1) Obtain a dataset of effective ingredients of traditional Chinese medicine with antioxidant, whitening and anti-photoaging effects and their potential targets; (2) Obtain a dataset of targets related to skin anti-aging efficacy; (3) Construct the interaction network of traditional Chinese medicine-active ingredient-target and the interaction network of traditional Chinese medicine-active ingredient-target-skin anti-aging effect; (4) Constructing a protein-protein interaction network of target sites to obtain the key targets of the active ingredients of traditional Chinese medicine; (5) Analyze the mechanism of action of key targets of effective ingredients of traditional Chinese medicine; (6) Based on molecular docking technology, the effective ingredients of traditional Chinese medicine are docked with the targets related to skin anti-aging effects.

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