An active peptide for promoting skin microecological balance

Abstract

The present invention discloses an active peptide for promoting skin microecological balance, and has mainly developed a variety of okra active peptides, which are combined with plant extracts, aiming to obtain a new product that is pure natural, additive-free, can repair the skin, maintain the water-oil balance, has a good moisturizing effect, keeps the skin moist, and has antioxidant and anti-aging effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to an active peptide for promoting skin microecological balance. Background Art

[0002] Cosmetics refer to chemical industrial products or fine chemical products that are applied, sprayed or otherwise distributed on any part of the human body surface, such as skin, hair, fingernails, lips and teeth, etc., for the purpose of cleaning, maintaining, beautifying, modifying and changing appearance, or correcting body odor and maintaining a good state.

[0003] With the continuous improvement of living standards, people's requirements for skin care are also constantly increasing. During the metabolism process of the human body, various free radicals are formed. Among them, 95% of the free radicals are harmful oxygen free radicals. They attack cells, produce oxidation, damage cell functions, reduce human vitality, and cause aging phenomena such as wrinkles and age spots on the face, making the skin dull. Moreover, when the temperature is high in summer, the skin loses a large amount of moisture, and the skin will secrete a large amount of oil to prevent further loss of moisture. When there is a lot of oil on the face, it will cause enlarged pores, shiny and greasy skin, a feeling of greasiness and stickiness, and easily make people feel irritable and depressed. And currently, urban pollution is relatively serious. Long-term excessive oiliness is likely to block pores, and it is also easy to adhere to dust and dirt, causing skin infections and acne, etc.

[0004] To maintain skin health, it is necessary to first maintain the water-oil balance of the skin. The water-oil balance of the skin means that the skin is in the healthiest and most ideal state of being neither oily nor dry, with a pH value between 5 and 5.6, and the secretion amounts of sweat glands and sebaceous glands are both moderate, in a neutral state of being neither greasy nor dry. Such skin is ruddy and delicate and not very sensitive to external stimuli.

[0005] Okra contains various nutrients such as amino acids, proteins, trace elements, polysaccharide compounds, polyphenol components, flavonoid compounds, oils and vitamins, etc., and has the effects of promoting skin microcirculation, promoting the absorption of nutrients by the skin and the excretion of metabolites, and reducing skin relaxation and removing wrinkles. Through research on non-patent literature on okra extraction in China, it is found that current research on okra extraction mostly focuses on food and beverages, and there is less research on its application in cosmetics. However, international brands such as Kiehl's, Origins, and Fresh all have products containing okra extracts, which mainly feature functions such as repair, anti-aging, and moisturizing. As a natural, non-toxic, and multi-functional plant extract, the application of okra extract in cosmetics should be emphasized (Liu Suting, Wang Xiujuan, Wang Meng, Yan Xin. Patent Strategy Analysis of Okra Extraction Technology [J]. Chemical Research, 2023, 34(02)).

[0006] Therefore, the present invention focuses on the analysis of functional polypeptides in okra. Through contemporary biological means such as computer simulation by bioinformatics, the beneficial components therein, especially small molecule peptides, are developed purposefully. A variety of polysaccharide compounds, polyphenolic components, and flavonoid compounds are also obtained in the research. Subsequently, the cosmetic application prospects of the above compounds will be analyzed. Summary of the Invention

[0007] The purpose of the present invention is to provide an active peptide that promotes the balance of skin microecology, focusing on developing a variety of okra active peptides, and cooperating with plant extracts, aiming to provide a new product that is pure natural, additive-free, can repair the skin, maintain the water-oil balance, has a good moisturizing effect, keeps the skin moist, and has antioxidant and anti-aging effects.

[0008] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0009] On the one hand, the present invention provides an active peptide that promotes the balance of skin microecology, including peptides such as MIGDHSFPQSSLIA, NTVEFVRSDDQY, and RFFDIIPTVDS. The active peptide is obtained by enzymatic hydrolysis, extraction, separation, and purification of the fermentation extracts of Lactobacillus plantarum and yeast from okra.

[0010] The enzymes for enzymatic hydrolysis to obtain the active peptide include pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease, and acidic protease.

[0011] The fermentation extracts of Lactobacillus plantarum and yeast from okra are prepared by the following method: Using okra as the raw material, taking out the seed liquids of Lactobacillus plantarum and yeast in a culture medium added with glucose, NaCl, and urea, adding them to the fermentation medium for fermentation culture, setting the fermentation temperature, and adjusting the pH, inoculum size, shaker speed, and fermentation liquid loading amount according to the fermentation situation. The fermentation time is 48h - 72h; obtaining the fermentation broth, centrifuging to take the supernatant and filtering and purifying it to obtain the protein extract, and freeze-drying it under vacuum.

[0012] The preparation method of the active peptide includes: using okra protein freeze-dried powder as the raw material, adding water, stirring evenly, and sequentially adding pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease, and acidic protease. After adding each enzyme, sufficient stirring is required (during the fermentation and enzymatic hydrolysis processes, the type of fermentation medium and the selection of each parameter can be selected according to conventional values, as long as it does not affect the protein extraction and enzymatic hydrolysis). After sufficient enzymatic hydrolysis, the temperature is raised to sterilize and inactivate the enzyme, and a horizontal screw centrifuge is used for centrifugal filtration to remove the enzymatic hydrolysis residue. Then, filtration and purification are carried out to remove insoluble substances. The centrifugate is filtered through a filter membrane to retain small peptides with a molecular weight below 10,000 Daltons, followed by sequencing, bioinformatics analysis, and computer simulation to obtain the active peptide with predicted functions. That is, the product is sent to BGI for sequencing, and protein homology modeling and molecular docking methods are used for analysis (genes related to skin barrier function, such as OCLN, KLK7, COL17A1, SPRR1B), and then bioinformatics analysis is carried out to obtain the top-ranked active peptides MIGDHSFPQSSLIA, NTVEFVRSDDQY, RFFDIIPTVDS.

[0013] The active peptide can also be used in combination with plant extracts.

[0014] The plant extracts include mistletoe extract, purslane extract, chamomile extract, cogongrass rhizome extract, alpine wormwood extract, cedar leaf extract, chlorella extract, peony extract, mint extract, lime tree extract, licorice extract, lotus leaf extract, chicory stem extract, and bletilla striata extract.

[0015] The active peptide can also be used in combination with copper tripeptide, salicylic acid, bifida ferment lysate, lactobacillus / vigna radiata seed extract, hydrolyzed sodium hyaluronate, and resveratrol.

[0016] On the one hand, the present invention provides a complex for promoting skin microecological balance by adding active peptides, which includes an active substance and a cosmetic base substance. The active substance is the addition of active peptides in cosmetics

[0017] Preferably, the content of each component in the complex is as follows: 10 - 20 parts of active peptide, 1 - 10 parts of each plant extract (mistletoe extract, purslane extract, chamomile extract, cogongrass rhizome extract, alpine wormwood extract, cedar leaf extract, chlorella extract, peony extract, mint extract, lime tree extract, licorice extract, lotus leaf extract, chicory stem extract, bletilla striata extract), 0.01 - 0.05 parts of copper tripeptide, 0.01 - 0.05 parts of salicylic acid, 1 - 5 parts of bifida ferment lysate, 1 - 10 parts of lactobacillus / vigna radiata seed extract ferment filtrate, 1 - 5 parts of hydrolyzed sodium hyaluronate, 1 - 5 parts of resveratrol, and the basic matrix is 1 - 10 parts of 1,3 - propanediol, 1 - 10 parts of butanediol, 1 - 5 parts of panthenol, and the rest is water.

[0018] The third aspect of the present invention provides the application of the above-mentioned complex, which can be used in the application of products for promoting skin balance, repairing the skin, and anti-aging, anti-inflammatory and antioxidant. It is characterized in that the complex can be prepared into water, milk, oil, or paste form, and natural preservatives such as bergamot and geranium can be added. The products that can be prepared from the complex include toner, facial cleanser, lotion, and repair cream.

[0019] Technical effects

[0020] The present invention provides an active peptide for promoting skin microecological balance, combined with plant extracts, aiming to provide a pure natural and additive-free cosmetic that can repair the skin, maintain the water-oil balance, has a good moisturizing effect, keeps the skin moist, and has antioxidant, anti-inflammatory and anti-aging effects, making up for the deficiencies in the domestic market. Through modern biological means, it precisely targets the field of skin balance repair, deeply penetrates to the gene repair level, and changes the situation of blind addition in the cosmetic field and foreign occupation of the market.

[0021] The active peptide provided by the present invention can also be used alone, without cell irritation, and has multiple functions such as antioxidant, antibacterial, anti-inflammatory, promoting collagen biosynthesis, promoting cell repair, and relieving irritation.

[0022] Resveratrol has strong antioxidant and free radical scavenging effects, good anti-senility effects, and at the same time has a good effect on maintaining the water-oil balance of the skin; the mistletoe extract in the plant extract is rich in flavonoids, has the functions of scavenging free radicals, enhancing the body's immune function, inhibiting melanin production, and has antioxidant, anti-aging and whitening effects; the purslane extract contains phenolic substances, a large amount of potassium ions, amino acids, nitrogen-containing substances, alkaloids, coumarins, flavonoids, cardiac glycosides, and anthraquinone glycosides. The purslane extract, chamomile extract, licorice extract, cogongrass rhizome extract, alpine wormwood extract, cedar leaf extract, chlorella extract, and peony extract have the functions of cleaning, water-oil balance, and soothing, and can also relieve skin flushing, inhibit skin inflammation, and anti-allergy. The skin curative effects of mint and lime tree extract can condition unclean and clogged skin, and can shrink capillaries, relieve itching, inflammation and burns and other symptoms. The lotus leaf extract has the functions of protection, purification and antioxidant; the bletilla striata extract has the effects of whitening and freckle removal. The chicory stem extract is an odor inhibitor in cosmetics.

[0023] The filtrate of bifida ferment lysate and the filtrate of lactobacillus / vigna radiata seed extract fermentation product contain rich amino acids, polypeptides, minerals and various vitamins, can effectively improve the skin conditions, have a good skin repair effect, and have a certain effect on maintaining the water-oil balance of the skin. The present invention further adds copper tripeptide and salicylic acid to achieve the effects of assisting wound healing and anti-acne.

[0024] In summary, the present invention is composed of a combination of various plant extracts, without adding ingredients harmful to the skin such as pigments, mineral oils, artificial fragrances, and surfactants. It expands the uses of okra in cosmetics, can improve skin metabolism and delay aging, has no cell irritation, and has multiple functions such as antioxidant, antibacterial, anti-inflammatory, promoting collagen biosynthesis, promoting cell repair, and relieving irritation. It provides a natural and additive-free cosmetic that can repair the skin, maintain the water-oil balance, has a good moisturizing effect, keeps the skin moist, and has antioxidant, anti-inflammatory, and anti-aging effects. Detailed implementation mode

[0025] The following further illustrates the implementation mode of the present invention in conjunction with examples.

[0026] The experiment of the present invention is divided into 5 groups. The first group is the blank group with the basic matrix. The second group of basic matrix + active peptide (10 g, the same below) Same as above The third group is the basic matrix + plant extracts (mistletoe extract, purslane extract, chamomile extract, cogongrass rhizome extract, alpine wormwood extract, cedar leaf extract, chlorella extract, peony extract, mint extract, lime tree extract, licorice extract, lotus leaf extract, chicory stem extract, bletilla striata extract, 1 g of each of the above substances), the fourth group is the basic matrix + active peptide + plant extracts (the same as above), and the fifth group is the basic matrix + active peptide + plant extracts (the same as above) + additives (0.01 g of copper tripeptide, 0.01 g of salicylic acid, 1 g of bifida ferment filtrate, 10 g of lactic acid bacteria / mung bean seed extract ferment filtrate, 1 g of hydrolyzed sodium hyaluronate, 1 g of resveratrol). Some experimental results are recorded as integers. The basic matrix is 10 g of 1,3-propanediol, 10 g of butanediol, 5 g of panthenol, and the rest is water (so that each group is 100 g), which are common basic matrices of cosmetics. If not otherwise specified, the concentration used in each group in the experiment to verify the function in the medium is 5%.

[0027] Example 1 Cell irritation

[0028] To confirm the irritation of each experimental group to cells, the inventor referred to the common cell irritation test method and conducted a cytotoxicity experiment using keratinocytes (HaCat) as the object.

[0029] The keratinocytes (HaCat) were cultured in the medium, and each experimental substance was added to the medium. After culturing for 48 hours, 10% EZ-Cytox was mixed in the DMEM medium to measure cell viability and proliferation. After culturing for 2 hours, the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0030] Cell survival rate (%) = (Absorbance of experimental groups 2 - 5 / Absorbance of experimental group 1) × 100 Results: Experimental group 2 showed 2.25% cytotoxicity, experimental group 3 showed 4.25% cytotoxicity, experimental group 4 showed 3.85% cytotoxicity, and experimental group 5 showed 4.15% cytotoxicity. The above groups all had low cytotoxicity below 5.0%, and the above groups could be used as materials for cosmetic safety.

[0031] Example 2 Enhancement of DNA repair system

[0032] Referring to common DNA repair test methods, thymine dimers are generated after UV irradiation, which can be repaired by the DNA repair system in the human body, and when the DNA repair system works effectively, thymine uptake increases. Therefore, the ability to repair DNA can be evaluated by measuring the uptake of bromodeoxyuridine (BrdU), a thymidine analogue.

[0033] Take the mammary gland skin of Wistar female rats, apply the experimental substances of each group, irradiate continuously with HEV blue light for 2 hours, measure the content of bromodeoxyuridine (BrdU), and the results are shown in Table 1.

[0034] Table 1 Results of the determination of the content of bromodeoxyuridine (BrdU).

[0035] Sample Content of bromodeoxyuridine (BrdU) Experimental group 1 0.12±0.02 Experimental group 2 0.20±0.05 Experimental group 3 0.18±0.02 Experimental group 4 0.25±0.04 Experimental group 5 0.25±0.06

[0036] Results: Experimental groups 2 - 5 showed significant DNA repair effects.

[0037] Example 3 Mitigation effect on cell stimulation

[0038] Referring to common cell stimulation mitigation test methods, human fibroblasts were cultured in a medium until the cell confluence reached 80%. The cells were transferred to a 96-well plate at a density of 5 × 10 4 cells / well, the experimental substances of each group were added to the medium, and cultured for 48 hours. After culturing, the cell adhesion and growth status were observed under a microscope. Cultured using lactic acid (0.2%) as a skin irritant. Add 3 mg / ml of MTT solution, and then use an ELISA reader (measure the absorbance at 570 nm) to compare the cell survival rates of each group. Take the cell survival rate of experimental group 1 as 100%.

[0039] Results: The cell survival rate of experimental group 2 was 175%, that of experimental group 3 was 127%, that of experimental group 4 was 190%, and that of experimental group 5 was 225%. Microscopic examination showed that the cell states were at different levels from good to active. The cells in each group survived well and were in a proliferative state, not affected by cell stimulation.

[0040] Example 4 Effect of promoting collagen biosynthesis

[0041] Referring to the common collagen biosynthesis test methods, human fibroblasts were seeded in a 48-well culture plate containing DMEM at a density of 1×10 6 cells / well. Each experimental substance was added to the culture medium, and the cells were cultured for 24 hours. Then, the previous culture medium was replaced with serum-free DMEM medium and cultured for another 24 hours. After culturing, the supernatant of each well was collected, and the level of carboxyl-terminal propeptide of type I procollagen (PICP) in human was measured using a kit (Takara) to calculate the amount of newly synthesized collagen. The PICP content of experimental group 1 was defined as 100%. Ascorbic acid was used as a positive control.

[0042] Results: The PICP content of experimental group 2 increased to 156%, that of experimental group 3 was 115%, that of experimental group 4 was 174%, that of experimental group 5 was 175%, and the PICP content of ascorbic acid increased to 123%.

[0043] Example 5 Antioxidant effect

[0044] Using the cell culture method of Example 4, the scavenging rates of hydroxyl and superoxide anion radicals in cells were detected according to the kit instructions, and the results are shown in Table 2. The scavenging rates of hydroxyl and superoxide anion radicals in the initial state of each group of cells were calculated as 0%.

[0045] Table 2 Determination results of scavenging rates of hydroxyl and superoxide anion radicals.

[0046]

[0047] Results: Experimental groups 2 - 5 all had the ability to scavenge hydroxyl and superoxide anion radicals.

[0048] Example 6 Promotion of the synthesis of beneficial bacteria and inhibition of the formation of harmful bacteria

[0049] Using the cell culture method of Example 4, the size of the inhibition zone against Staphylococcus aureus was observed, and the number of Leuconostoc mesenteroides at 10 CFU / ML was counted. The results are shown in Table 3.

[0050] Table 3 Determination results of the size of the inhibition zone and the number of Leuconostoc mesenteroides

[0051] Sample S.aureus (mm) Leuconostoc mesenteroides (number) Experimental group 1 20.05±0.15 <![CDATA[3.05x10 7 > Experimental group 2 8.05±0.20 <![CDATA[3.65x10 7 > Experimental group 3 12.10±0.55 <![CDATA[3.74x10 7 > Experimental group 4 7.55±0.45 <![CDATA[4.08x10 7 > Experimental group 5 8.05±0.35 <![CDATA[4.51x10 7 >

[0052] Example 7 Performance test:

[0053] Select 200 volunteers aged 20 - 45 (regardless of gender) as the trial subjects. The trial subjects are tested with the company's skin detector. Those identified as having healthy skin and no history of skin allergies are grouped into one group of 100, and those with damaged and dehydrated skin in need of repair are grouped into another group of 100. Then each group is randomly divided into 5 subgroups of 20 people each. The usage method is as follows: After the trial subjects clean their faces, evenly apply the experimental toner on the face and gently pat until fully absorbed. Use it once in the morning and once in the evening every day for 50 consecutive days. Then score the refreshingness, anti-sensitivity, anti-wrinkle property, and moisturizing and smoothness of the experimental product on the skin. The total score for each usage effect is 5 points. The following are the average scores for each item. The results are shown in Table 4.

[0054] Table 4 Scoring results of the refreshingness, sensitivity, anti-wrinkle property, and moisturizing and smoothness of the group with healthy skin and no history of skin allergies.

[0055] Sample Refreshing property Anti-sensitivity Anti-wrinkle property Hydrating and smooth property Experimental group 1 4.7 4.5 4.3 4.3 Experimental group 2 5 4.7 4.6 4.9 Experimental group 3 5 4.5 4.4 4.5 Experimental group 4 5 4.7 4.6 5 Experimental group 5 5 4.8 4.6 4.9

[0056] Table 5 Scoring results of the refreshingness, sensitivity, anti-wrinkle property, and moisturizing and smoothness of the group with damaged and dehydrated skin in need of repair.

[0057] Sample Refreshing property Anti-sensitivity Anti-wrinkle property Hydrating and smooth property Experimental group 1 4.4 3.0 4.0 3.9 Experimental group 2 4.7 4.3 4.1 4.3 Experimental group 3 4.5 3.5 3.9 4.0 Experimental group 4 4.7 4.4 4.5 5 Experimental group 5 4.6 4.7 4.7 4.9

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An active peptide for promoting skin microecological balance, characterized in that, it comprises peptides with sequences of MIGDHSFPQSSLIA, NTVEFVRSDDQY and RFFDIIPTVDS.

2. The active peptide for promoting skin microecological balance according to claim 1, characterized in that, the active peptide is obtained by enzymatic hydrolysis, extraction, separation and purification of the fermentation extract of Lactobacillus plantarum and yeast from okra.

3. The active peptide for promoting skin microecological balance according to claim 2, wherein the enzymes for enzymatic hydrolysis to obtain the active peptide include pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease.

4. The active peptide for promoting skin microecological balance according to claim 2, wherein the fermentation extract of Lactobacillus plantarum and yeast from okra is prepared by the following method: using okra as raw material, taking the seed liquids of Lactobacillus plantarum and yeast in a culture medium added with glucose, NaCl and urea, adding them to the fermentation medium for fermentation culture, setting the fermentation temperature, adjusting the pH, inoculum size, shaker speed and fermentation liquid loading amount according to the fermentation situation, and the fermentation time is 48h - 72h; obtaining the fermentation broth, centrifuging to take the supernatant and filtering and purifying it to obtain the protein extract, and freeze-drying it under vacuum.

5. The preparation method of the active peptide for promoting skin microecological balance according to claim 3, comprises: using okra protein freeze-dried powder as raw material, adding water and stirring evenly, successively adding pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease, and fully stirring after adding each enzyme. After sufficient enzymatic hydrolysis, heating to sterilize and inactivate the enzymes, centrifuging and filtering with a horizontal spiral centrifuge to remove the enzymatic hydrolysis residues, filtering and purifying to remove insoluble substances, filtering the centrifugate through a filter membrane to retain small peptides with a molecular weight below 10,000 Daltons, performing sequencing, bioinformatics analysis and computer simulation to obtain the active peptide with function prediction.

6. The active peptide for promoting skin microecological balance according to any one of claims 1 - 5, wherein the active peptide can also be used in combination with plant extracts.

7. The active peptide for promoting skin microecological balance according to claim 6, wherein the plant extracts include mistletoe extract, purslane extract, chamomile extract, cogongrass rhizome extract, alpine wormwood extract, cedar leaf extract, chlorella extract, peony extract, mint extract, lime tree extract, licorice extract, lotus leaf extract, chicory stem extract, bletilla striata extract.

8. The active peptide for promoting skin microecological balance according to any one of claims 1 - 5, wherein the active peptide can also be used in combination with copper tripeptide.

9. The active peptide for promoting skin microecological balance according to any one of claims 1 - 5, wherein the active peptide can also be used in combination with salicylic acid.

10. The active peptide for promoting skin microecological balance according to any one of claims 1 - 5, wherein the active peptide can also be used in combination with the filtrate of bifida yeast fermentation product.

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