Application of Active Peptides for Promoting Skin Microecological Balance

Abstract

The present invention discloses an application of an active peptide in promoting skin microecological balance, which is obtained by enzymolysis extraction, separation and purification of the Lactobacillus plantarum and yeast fermentation extracts of okra. A variety of okra active peptides are developed with emphasis, and plant extracts are incorporated, aiming to obtain a new product that is 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 properties.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to the application of active peptides for promoting skin microecological balance. Background Art

[0002] Cosmetics refer to chemical industrial products or fine chemical products that are spread on any part of the human body surface, such as skin, hair, nails, lips and teeth, by smearing, spraying or other similar methods, for the purpose of cleaning, maintenance, beautification, modification and change of appearance, or correction of body odor and maintenance of good condition.

[0003] With the continuous improvement of living standards, people's requirements for skin care are also increasing. In the process of metabolism, the human body forms various free radicals. Among them, 95% of free radicals are oxygen free radicals that are harmful to us. They attack cells, produce oxidation, damage cell function, reduce human vitality, and cause wrinkles, spots and other aging phenomena on the face, and the skin becomes dull. In addition, when the temperature is high in summer, the skin loses a lot of moisture, and the skin will secrete a lot of oil to prevent further loss of moisture. When there is a lot of oil on the face, it will cause enlarged pores, greasy and shiny skin, and a greasy and sticky feeling, which can easily make people feel irritable and depressed. In addition, urban pollution is relatively serious nowadays. Excessive oil production for a long time can easily clog pores, and it is also easy to adhere to dust and dirt, causing skin infections and acne.

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

[0005] Okra contains a variety of nutrients such as amino acids, proteins, trace elements, polysaccharides, polyphenols, flavonoids, oils and vitamins. It can promote skin microcirculation, promote the skin's absorption of nutrients and the excretion of metabolites, and reduce skin sagging and remove wrinkles. Through research on domestic non-patent literature on okra extraction, it was found that the current research on okra extraction is mostly focused 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 extract, which mainly have repair, anti-aging, moisturizing and other effects. As a natural, non-toxic, multi-functional plant extract, okra extract should be taken seriously in its application in cosmetics (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, and through contemporary biological means such as computer simulation using bioinformatics, the beneficial ingredients therein, especially small molecule peptides, are developed in a targeted and purposeful manner. In the research, a variety of polysaccharide compounds, polyphenol components, and flavonoid compounds are also obtained. The cosmetic application prospects of the above compounds will be analyzed later. Summary of the invention

[0007] The purpose of the present invention is to provide an application of active peptides for promoting skin microecological balance. Okra is used as a raw material, and the plant lactobacillus and yeast fermentation extracts of okra are obtained by enzymatic extraction, separation and purification. A variety of okra active peptides are developed, and plant extracts are combined to strive to provide a pure, natural, additive-free complex that can repair the skin, maintain water-oil balance, and has a good moisturizing effect, keeps the skin hydrated, and has anti-oxidation and anti-aging effects.

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

[0009] On one hand, the present invention provides use of an active peptide in preparing a cosmetic for promoting skin microecological balance, wherein the active peptide is a peptide with a sequence of MIGDHSFPQSSLIA, NTVEFVRSDDQY, and RFFDIIPTVDS.

[0010] Active peptides can also be used alone without cell irritation. They have antioxidant, antibacterial, anti-inflammatory, collagen biosynthesis promotion, cell repair promotion, irritation relief, anti-aging and other effects.

[0011] Another aspect of the present invention provides the use of active peptides in preparing cosmetics for promoting skin microecological balance, wherein the cosmetics are prepared in the form of water, milk, oil, or paste, and natural preservatives such as bergamot and geranium may be added. The products that can be prepared include toner, cleanser, lotion, and repair cream.

[0012] The active peptide is obtained by extracting, separating and purifying the fermented extract of Lactobacillus plantarum and yeast of okra through enzymatic hydrolysis.

[0013] The enzymes for obtaining active peptides by enzymolysis include pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease.

[0014] The plant lactobacillus and yeast fermentation extract of okra is prepared by the following method: taking okra as a raw material, taking out the seed liquid of plant lactobacillus and yeast from a culture medium added with glucose, NaCl and urea, adding the seed liquid of plant lactobacillus and yeast into a fermentation culture medium for fermentation and cultivation, setting the fermentation temperature, adjusting the pH, inoculation amount, shaking table speed and fermentation liquid amount according to the fermentation situation, and the fermentation time is 48h-72h; obtaining the fermentation liquid, centrifuging and taking the supernatant for filtration and purification to obtain the protein extract, and vacuum freeze-drying and freeze-drying.

[0015] The preparation method of the active peptide comprises: taking okra protein freeze-dried powder as raw material, adding water, stirring evenly, adding pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease in sequence, stirring fully each time an enzyme is added (during the fermentation and enzymolysis process, the type of fermentation medium and the selection of various parameters can be selected according to conventional numerical values, so as not to affect the extraction and enzymolysis of the protein), after sufficient enzymolysis, heating and sterilizing to inactivate the enzyme, centrifuging with a horizontal centrifuge to remove the enzymolysis residue, filtering and purifying, and then filtering with a filter membrane to intercept small molecule peptides below 10,000 Daltons, sequencing, bioinformatics analysis and computer simulation are performed to obtain active peptides with predicted functions. The product was sent to BGI for sequencing, and protein homology modeling and molecular docking methods were used for analysis (OCLN, KLK7, COL17A1, SPRR1B, genes related to skin barrier function, and then bioinformatics analysis was performed to obtain the top-ranked active peptides MIGDHSFPQSSLIA, NTVEFVRSDDQY, and RFFDIIPTVDS.

[0016] The obtained active peptides can also be used in combination with plant extracts.

[0017] Plant extracts include mistletoe extract, purslane extract, chamomile extract, imperata root extract, alpine artemisia 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.

[0018] The obtained active peptide can also be used in combination with copper tripeptide, salicylic acid, bifida yeast fermentation product filtrate, lactic acid bacteria / mung bean seed extract, hydrolyzed sodium hyaluronate, and resveratrol.

[0019] On the one hand, the present invention provides a compound for adding active peptides to promote skin microecological balance, comprising an active substance and a cosmetic base substance, wherein the active substance is a cosmetic with active peptides added thereto.

[0020] The preferred contents of the components in the complex are: 10-20 parts of active peptides, 1-10 parts of plant extracts (mistletoe extract, purslane extract, chamomile extract, imperata 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 bifid yeast fermentation product filtrate, 1-10 parts of lactic acid bacteria / mung bean seed extract fermentation product filtrate, 1-5 parts of hydrolyzed sodium hyaluronate, 1-5 parts of resveratrol, and the basic matrix is ​​1-10 parts of 1,3-propylene glycol, 1-10 parts of butylene glycol, 1-5 parts of panthenol, and the rest is water.

[0021] Technical Effects

[0022] The present invention provides an active peptide that promotes the balance of skin microecology, and is combined with plant extracts, striving to provide a pure natural, additive-free cosmetic that can repair the skin and maintain water-oil balance, while having a good moisturizing effect, keeping the skin hydrated, and having anti-oxidation, anti-inflammatory and anti-aging effects, making up for the shortcomings of the domestic market, and accurately targeting the field of skin balance and skin repair through modern biological means, deeply penetrating to the level of gene repair, changing the situation in the field of cosmetics where blind additives are added and foreign countries occupy the market.

[0023] The active peptide provided by the present invention can also be used alone, has no cell irritation, and has multiple effects such as anti-oxidation, anti-bacteria, anti-inflammatory, promoting collagen biosynthesis, promoting cell repair, and alleviating irritation.

[0024] Resveratrol has strong antioxidant and anti-free radical effects, good anti-sun aging effects, and good effects on maintaining the water and oil balance of the skin; mistletoe extract is rich in flavonoids in plant extracts, which can remove free radicals, enhance the body's immune function, inhibit melanin production, and have antioxidant, anti-aging and whitening effects; purslane extract contains phenolic substances, a large number of potassium ions, amino acids, nitrogen-containing substances, alkaloids, coumarins, flavonoids, cardiac glycosides, and anthraquinone glycosides. Purslane extract, chamomile extract, licorice extract, Imperata extract, Artemisia alpina extract, cedar leaf extract, Chlorella extract, and peony extract have the effects of cleaning, water and oil balance, and soothing. They can also relieve skin redness, inhibit skin inflammation, and resist allergies. The skin effects of mint and lime tree extracts can regulate unclean and blocked skin, shrink capillaries, and relieve symptoms such as itching, inflammation, and burns. Lotus leaf extract has the effects of protection, purification, and antioxidant; Bletilla striata extract has the effects of whitening and removing spots. Chicory stem extract is an odor suppressant in cosmetics.

[0025] The filtrate of the fermentation product of bifid yeast and the filtrate of the fermentation product of lactic acid bacteria / mung bean seed extract are rich in amino acids, peptides, minerals and various vitamins, which can effectively improve skin conditions, have a good effect on repairing skin, and have a certain effect on maintaining the water and oil balance of the skin. The present invention further adds copper tripeptide and salicylic acid to achieve the effect of assisting wound healing and anti-acne.

[0026] In summary, the present invention is composed of a combination of multiple plant extracts, without adding pigments, mineral oils, artificial flavors, surfactants and other ingredients that are harmful to the skin, expanding the use of okra in cosmetics, improving skin metabolism and delaying aging, non-cell irritation, and having multiple effects such as anti-oxidation, anti-bacterial, anti-inflammatory, promoting collagen biosynthesis, promoting cell repair, relieving irritation, etc. Provided is a pure natural, additive-free cosmetic that can repair the skin, maintain water-oil balance, and has good moisturizing effect, keeps the skin moisturized, and has anti-oxidation, anti-inflammatory and anti-aging effects. DETAILED DESCRIPTION

[0027] The embodiments of the present invention are further described below in conjunction with the examples.

[0028] The present invention divides the experiment into 5 groups, the first group is a blank group with basic matrix, The second group of basic matrix + active peptide (10g, below same), The third group of basic matrix + plant extracts (mistletoe extract, purslane extract, chamomile extract, Imperata root 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, each of which is 1g), the fourth group of basic matrix + active peptide + plant extract (same as above), the fifth group of basic matrix + active peptide + plant extract (same as above) + additives (0.01g copper tripeptide, 0.01g salicylic acid, 1g bifida yeast fermentation product filtrate, 10g lactic acid bacteria / mung bean seed extract fermentation product filtrate, 1g hydrolyzed sodium hyaluronate, 1g resveratrol), some experimental results are recorded as integers. The basic matrix is ​​10g 1,3-propylene glycol, 10g butylene glycol, 5g panthenol, and the rest is water (so that each group is 100g) and other commonly used cosmetic basic matrices. Unless otherwise specified, the concentration used in the culture medium for each group in the experiments to verify the function in the examples was 5%.

[0029] Example 1 Cell Stimulation

[0030] In order to confirm the irritation of each experimental group to cells, the inventors referred to the common cell irritation test method and conducted a cytotoxicity experiment using keratinocytes (HaCat) as the object.

[0031] Keratinocytes (HaCat) were cultured in a culture medium, and each group of experimental substances was added to the culture medium. After 48 hours of culture, 10% EZ-Cytox was mixed in the DMEM culture medium to measure cell viability and proliferation. After 2 hours of culture, the absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0032] 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. All of the above groups had low cytotoxicity of less than 5.0%, and the above groups can be used as safe materials for cosmetics.

[0033] Example 2 DNA repair system enhancement

[0034] Referring to the common DNA repair test method, UV irradiation generates thymine dimers, 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 analog.

[0035] The breast skin of female Wistar rats was smeared with the experimental substances in each group, and then irradiated with HEV blue light for 2 hours. The content of bromodeoxyuridine (BrdU) was measured. The results are shown in Table 1.

[0036] Table 1. Results of determination of bromodeoxyuridine (BrdU) content.

[0037] sample Bromodeoxyuridine (BrdU) content Experimental Group 1 0.12±0.02 Experimental Group 2 0.20±0.05 Experimental Group 3 0.1S±0.02 Experimental Group 4 0.25±0.04 Experimental Group 5 0.25±0.06

[0038] Results: Experimental groups 2 to 5 showed significant DNA repair effects.

[0039] Example 3 Relief of Cell Stimulation

[0040] Referring to the common cell stimulation relief test method, human fibroblasts were cultured in culture medium until the cell confluence reached 80%. 4 The density of cells / well was transferred to a 96-well plate, and each group of experimental materials was added to the culture medium and cultured for 48 hours. After culture, the cell adhesion and growth status were observed under a microscope. Lactic acid (0.2%) was used as a skin irritant for culture. MTT solution 3mg / ml was added, and then the ELISA reader (measured the absorbance at 570nm) was used to compare the cell survival rate of each group. The cell survival rate of experimental group 1 was 100%.

[0041] Results: The cell survival rate was 175% in experimental group 2, 127% in experimental group 3, 190% in experimental group 4, and 225% in experimental group 5. Microscopic examination showed that the cell status varied from good to active, and the cells in each group survived well and were in a proliferative state, and were not affected by cell stimulation.

[0042] Example 4 Promoting collagen biosynthesis

[0043] Referring to the common collagen biosynthesis test method, human fibroblasts were seeded in a 48-well culture plate containing DMEM, and 1×10 6 Cells / well were added to the culture medium for 24 hours. The culture medium was then replaced with serum-free DMEM culture medium and cultured for 24 hours. After culture, the supernatant of each well was collected and the level of human type I procollagen carboxyl terminal peptide PICP 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.

[0044] Results: The PICP content of experimental group 2 increased to 156%, the PICP content of experimental group 3 was 115%, the PICP content of experimental group 4 was 174%, and the PICP content of experimental group 5 was 175%. The PICP content of ascorbic acid increased to 123%.

[0045] Example 5 Antioxidant Effect

[0046] The cell culture method of Example 4 was used to detect the scavenging rates of hydroxyl and superoxide anion free radicals in the cells according to the instructions of the kit, and the results are shown in Table 2. The scavenging rates of hydroxyl and superoxide anion free radicals in the initial state of each group of cells were calculated as 0%.

[0047] Table 2 Hydroxyl and superoxide anion free radical scavenging rate determination results.

[0048]

[0049] Results: Experimental groups 2-5 all had the ability to scavenge hydroxyl and superoxide anion free radicals.

[0050] Example 6 Promoting the synthesis of beneficial bacteria and inhibiting the formation of harmful bacteria

[0051] The cell culture method of Example 4 was used to observe the size of the inhibition zone of Staphylococcus aureus and to count 10 CFU / ML Leuconostoc mesenteroides. The results are shown in Table 3.

[0052] Table 3 The size of the inhibition zone and the number of Leuconostoc mesenteroides

[0053] sample S. aureus(mm) Leuconostoc mesenteorides (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 ]]>

[0054] Example 7 Performance Test:

[0055] 200 volunteers aged 20-45 (both male and female) were selected as trial subjects. The trial subjects were tested with the company's skin detector, and 100 people were identified as having healthy skin and no history of skin allergies, and 100 people were identified as having damaged and dehydrated skin that needed to be repaired; each group was then randomly divided into 5 small groups, with 20 people in each group. The method of use is: after the trial subject cleans the face, apply the experimental toner evenly on the face, gently pat until it is fully absorbed, use it once a day in the morning and evening, and use it continuously for 50 days. Then the experimental product is scored for its refreshing, anti-sensitivity, anti-wrinkle, and moisturizing and smoothness on the skin. The total score for each use effect is 5 points. The following is the average score for each item. The results are shown in Table 4.

[0056] Table 4 The results of the refreshing, sensitive, anti-wrinkle and moisturizing and smoothness scores for the group with healthy skin and no history of skin allergies.

[0057] sample Freshness Anti-sensitivity Wrinkle resistance Hydration and smoothness 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

[0058] Table 5 The results of the refreshing, sensitive, anti-wrinkle and moisturizing and smoothness scores for the group with damaged and dehydrated skin in need of repair.

[0059] sample Freshness Anti-sensitivity Wrinkle resistance Hydration and smoothness 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

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of an active peptide in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the active peptide is a peptide with the sequences MIGDHSFPQSSLIA, NTVEFVRSDDQY, RFFDIIPTVDS, and the active peptide is obtained by enzymatic hydrolysis, extraction, separation and purification of the fermented extract of Lactobacillus plantarum and yeast from okra. The fermented extract of Lactobacillus plantarum and yeast from okra is prepared by the following method: using okra as the 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, and adjusting the pH, inoculum size, shaker speed, and fermentation liquid filling volume according to the fermentation situation, with a fermentation time of 48h - 72h; obtaining the fermentation broth, centrifuging to take the supernatant and filtering and purifying it to obtain a protein extract, and freeze-drying it under vacuum. The preparation method of the active peptide includes: using okra protein freeze-dried powder as the raw material, adding water and stirring evenly, adding pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease, and acidic protease in stages, and fully stirring after adding each enzyme. After sufficient enzymatic hydrolysis, heating to sterilize and inactivate the enzyme, centrifuging and filtering with a horizontal spiral centrifuge to remove the enzymatic hydrolysis residue, filtering and purifying to remove insoluble substances, and filtering the centrifugate through a filter membrane to retain small peptides with a molecular weight below 10,000 Daltons, and performing sequencing bioinformatics analysis and computer simulation to obtain the active peptide with predicted functions. The promotion of skin microecological balance includes antioxidant, antibacterial, and promoting collagen biosynthesis.

2. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the promotion of skin microecological balance includes anti-inflammatory.

3. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the promotion of skin microecological balance includes promoting cell repair.

4. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the promotion of skin microecological balance includes relieving irritation.

5. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the promotion of skin microecological balance includes anti-aging.

6. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the cosmetic is prepared into a water or milk form.

7. Use of the active peptide according to claim 1 in the preparation of a cosmetic for promoting skin microecological balance, characterized in that, the cosmetic is prepared into an oil or paste form.

Images