A method for preparing active peptides for promoting skin microecological balance

Abstract

The present invention discloses a method for preparing active peptides for promoting skin microecological balance. The active peptides are obtained by enzymatically extracting, separating and purifying plant lactobacillus and yeast fermentation extracts of okra. A variety of okra active peptides are developed, and plant extracts are combined to obtain a new product that is pure, natural and additive-free, can repair the skin, maintain water-oil balance, has good moisturizing effect, keeps the skin hydrated, and has antioxidant and anti-aging properties.

Description

Technical Field

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

[0002] Cosmetics refer to chemical industrial products or fine chemical products that are applied to 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 human body odor and maintenance of good condition.

[0003] As living standards continue to improve, people's demands for skin care are also increasing. The human body generates various free radicals during metabolism. 95% of these free radicals are harmful oxygen free radicals. They attack cells, causing oxidation and impairing cell function, leading to decreased vitality, wrinkles, dark spots, and other signs of aging, and dulling the skin. Furthermore, during the high temperatures of summer, the skin loses significant moisture, and it produces large amounts of oil to prevent further moisture loss. Excessive oil production leads to enlarged pores, a greasy, shiny complexion, and a sticky, sticky feeling, which can easily cause irritability and depression. Furthermore, with today's severe urban pollution, chronic excessive oil production can easily clog pores and trap dust and dirt, causing skin infections and acne.

[0004] To maintain healthy skin, we 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 most ideal state, neither oily nor dry. The pH value is between 5-5.6, and the secretion of sweat glands and sebaceous glands is moderate, in a neutral state that is neither greasy nor dry. Such skin is rosy and delicate, and is 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 has the effect of reducing skin sagging and removing wrinkles. Through the investigation of 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 and multi-functional plant extract, okra extract should be paid attention to 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 methods such as bioinformatics computer simulation, it specifically develops the beneficial ingredients therein, especially small molecule peptides. In the research, a variety of polysaccharide compounds, polyphenol components, and flavonoid compounds were 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 a method for preparing active peptides that promote skin microecological balance. The active peptides are obtained by enzymatic extraction, separation and purification of Lactobacillus plantarum and yeast fermentation extracts of okra, focusing on the development of a variety of okra active peptides, and combining them with plant extracts, striving to provide a pure, natural, additive-free complex that can repair skin, maintain water-oil balance, and has a good moisturizing effect, keeps skin hydrated, and has antioxidant 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 a method for preparing an active peptide for promoting skin microecological balance. The active peptide is obtained by enzymatically extracting, separating and purifying an extract of plant lactobacillus and yeast fermented from okra.

[0010] The enzymes for obtaining active peptides by enzymatic hydrolysis include pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease.

[0011] The okra Lactobacillus plantarum and yeast fermentation extract is prepared by the following method: taking okra as a raw material, taking out Lactobacillus plantarum and yeast seed liquid from a culture medium supplemented with glucose, NaCl and urea, adding the seed liquid to a fermentation culture medium for fermentation and culturing, setting a fermentation temperature, adjusting the pH, inoculation amount, shaking table speed and fermentation liquid volume according to the fermentation situation, and fermenting for 48 hours to 72 hours; obtaining a fermentation liquid, centrifuging the supernatant, filtering and purifying the supernatant to obtain a protein extract, and vacuum freeze-drying and freeze-drying.

[0012] The preparation method of the active peptide comprises: using okra protein freeze-dried powder as a raw material, adding water, stirring evenly, sequentially adding pepsin, trypsin, carboxypeptidase, aminopeptidase, neutral protease, alkaline protease and acidic protease, and fully stirring each time an enzyme is added (during the fermentation and enzymatic hydrolysis process, the type of fermentation medium and the selection of various parameters can be selected according to conventional values ​​so as not to affect the extraction and enzymatic hydrolysis of the protein), after sufficient enzymatic hydrolysis, heating and sterilizing to inactivate the enzyme, centrifuging with a horizontal centrifuge to remove the enzymatic hydrolysis residue, filtering and purifying, and then filtering with a filter membrane to intercept small molecular peptides below 10,000 Daltons, and performing sequencing, bioinformatics analysis and computer simulation 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 bioinformatics analysis was performed to obtain the top-ranked active peptides MIGDHSFPQSSLIA, NTVEFVRSDDQY, and RFFDIIPTVDS.

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

[0014] Plant extracts include mistletoe extract, purslane extract, chamomile extract, imperata root extract, artemisia alpinia 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 obtained active peptide can also be used in combination with copper tripeptide, salicylic acid, bifid yeast fermentation product filtrate, lactic acid bacteria / mung bean seed extract, hydrolyzed sodium hyaluronate, and resveratrol.

[0016] 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 material, wherein the active substance is a cosmetic compound to which active peptides are added.

[0017] The preferred contents of the components in the complex are 10-20 parts of active peptides, 1-10 parts of each plant extract (mistletoe extract, purslane extract, chamomile extract, imperata root extract, artemisia alpinia 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.

[0018] A third aspect of the present invention provides applications of the aforementioned complex, which can be used to promote skin balance, repair skin, and develop anti-aging, anti-inflammatory, and antioxidant products. The complex can be formulated into lotions, emulsions, oils, and pastes, and can contain natural preservatives such as bergamot and geranium. Products that can be formulated with the complex include toners, cleansers, lotions, and repair creams.

[0019] Technical Effects

[0020] 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 and additive-free cosmetic that can repair the skin, maintain water and oil balance, and has good moisturizing effect, keeps the skin hydrated, and has anti-oxidation, anti-inflammatory and anti-aging effects. It makes up for the shortcomings of the domestic market, accurately targets the field of skin balance and repair through modern biological means, deeply penetrates to the level of gene repair, and changes the situation in the cosmetics field where blind additives are used and the market is dominated by foreign countries.

[0021] 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, antibacterial, anti-inflammatory, promoting collagen biosynthesis, promoting cell repair, and alleviating irritation.

[0022] Resveratrol has strong antioxidant and anti-free radical properties, offering excellent sun-resistant and anti-aging benefits. It also helps maintain the skin's water-oil balance. Among plant extracts, mistletoe extract is rich in flavonoids, which scavenge free radicals, enhance immunity, and inhibit melanin production, offering antioxidant, anti-aging, and whitening properties. Portulaca oleracea extract contains phenolic compounds, a large amount of potassium ions, amino acids, nitrogen-containing substances, alkaloids, coumarins, flavonoids, cardiac glycosides, and anthraquinone glycosides. Portulaca oleracea extract, chamomile extract, licorice extract, imperata root extract, artemisia alpinia extract, cedar leaf extract, chlorella extract, and peony extract all have cleansing, water-oil balancing, and soothing properties. They also reduce redness, inhibit skin inflammation, and have anti-allergic properties. Peppermint and lime tree extracts have skin benefits, conditioning impure and congested skin, constricting capillaries, and relieving itching, inflammation, and burns. Lotus leaf extract has protective, purifying, and antioxidant properties. Bletilla striata extract whitens and removes freckles. Chicory stem extract is an odor suppressant in cosmetics.

[0023] The filtrate of the bifid yeast fermentation product and the lactic acid bacteria / mung bean seed extract fermentation product are rich in amino acids, peptides, minerals, and various vitamins. They can effectively improve skin condition and repair the skin, and play a role in maintaining the skin's water and oil balance. This product also contains copper tripeptide and salicylic acid to promote wound healing and anti-acne effects.

[0024] In summary, the present invention combines multiple plant extracts without the addition of pigments, mineral oils, artificial fragrances, surfactants, or other ingredients that are harmful to the skin. This expands the use of okra in cosmetics, improves skin metabolism, delays aging, and is non-irritating to cells. It exhibits antioxidant, antibacterial, anti-inflammatory properties, promotes collagen biosynthesis, promotes cell repair, and alleviates irritation. This provides a pure, natural, additive-free cosmetic that repairs the skin, maintains water-oil balance, and provides excellent moisturizing properties, keeping the skin hydrated and providing antioxidant, anti-inflammatory, and anti-aging benefits. DETAILED DESCRIPTION

[0025] The embodiments of the present invention will be further described below with reference to the examples.

[0026] 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 consisted of a base matrix plus plant extracts (1g each of mistletoe extract, purslane extract, chamomile extract, imperata root extract, artemisia alpinia 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). The fourth group consisted of a base matrix plus active peptides plus plant extracts (same as above). The fifth group consisted of a base matrix plus active peptides plus plant extracts (same as above) plus additives (0.01g copper tripeptide, 0.01g salicylic acid, 1g bifid yeast fermentation filtrate, 10g lactic acid bacteria / mung bean seed extract fermentation filtrate, 1g hydrolyzed sodium hyaluronate, and 1g resveratrol). Some experimental results are rounded up. The base matrix consisted of 10g 1,3-propylene glycol, 10g butylene glycol, 5g panthenol, and the remainder water (making each group 100g). These are commonly used cosmetic base matrices. Unless otherwise specified, the concentration used in the culture medium for each group in the experiments to verify the function of the examples was 5%.

[0027] Example 1 Cell Stimulation

[0028] In order to confirm the irritation of each experimental group to cells, the inventors conducted a cytotoxicity experiment using keratinocytes (HaCat) as the subjects, referring to common cell irritation test methods.

[0029] Keratinocytes (HaCat) were cultured in culture medium, and each experimental substance was added to the culture medium. After 48 hours of culture, 10% EZ-Cytox was mixed with DMEM medium to measure cell viability and proliferation. After 2 hours of culture, absorbance was measured at 450 nm using a microplate reader.

[0030] Cell viability (%) = (absorbance of experimental groups 2-5 / absorbance of experimental group 1) × 100. Results: Experimental group 2 exhibited 2.25% cytotoxicity, experimental group 3 exhibited 4.25% cytotoxicity, experimental group 4 exhibited 3.85% cytotoxicity, and experimental group 5 exhibited 4.15% cytotoxicity. All of these groups exhibited low cytotoxicity of 5.0% or less, indicating that these groups can be used as safe materials for cosmetics.

[0031] Example 2 DNA repair system enhancement

[0032] Referring to common DNA repair testing methods, UV irradiation generates thymine dimers, which can be repaired by the body's DNA repair system. When the DNA repair system is working effectively, thymine uptake increases. Therefore, the ability to repair DNA can be assessed by measuring the uptake of bromodeoxyuridine (BrdU), a thymidine analog.

[0033] The breast skin of female Wistar rats was smeared with the experimental substances in each group and irradiated with HEV blue light for 2 hours. The bromodeoxyuridine (BrdU) content was measured. The results are shown in Table 1.

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

[0035] sample Bromodeoxyuridine (BrdU) content 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 to 5 showed significant DNA repair effects.

[0037] Example 3 Alleviation of Cell Stimulation

[0038] Referring to the common cell stimulation relief test method, human fibroblasts were cultured in culture medium until the cell confluence reached 80%. 4 Cells were transferred to a 96-well plate at a density of 100 cells / well. Each experimental group was added to the culture medium and cultured for 48 hours. Following incubation, cell adhesion and growth were observed under a microscope. Lactic acid (0.2%) was used as a skin irritant. MTT solution (3 mg / ml) was added, and cell viability was compared between groups using an ELISA reader (measuring absorbance at 570 nm). The cell viability of experimental group 1 was assumed to be 100%.

[0039] Results: The cell viability was 175% in experimental group 2, 127% in experimental group 3, 190% in experimental group 4, and 225% in experimental group 5. Microscopic examination revealed that the cells were in varying states of good to active, with cells in each group surviving well and proliferating, unaffected by cell stimulation.

[0040] Example 4: Promoting collagen biosynthesis

[0041] Referring to the common collagen biosynthesis test method, human fibroblasts were seeded in a 48-well culture plate containing DMEM, and 1×10 6 Each group of experimental substances was added to the culture medium and cultured for 24 hours. The culture medium was then replaced with serum-free DMEM medium and cultured for 24 hours. After culture, the supernatant from 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.

[0042] 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%.

[0043] Example 5 Antioxidant Effect

[0044] The cell culture method of Example 4 was used to detect the hydroxyl and superoxide anion free radical scavenging rates in the cells according to the kit instructions. The results are shown in Table 2. The calculation was performed based on the hydroxyl and superoxide anion free radical scavenging rates of each group of cells in the initial state being 0%.

[0045] Table 2 Results of hydroxyl and superoxide anion free radical scavenging rates.

[0046]

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

[0048] Example 6: Promoting the synthesis of beneficial bacteria and inhibiting the formation of harmful bacteria

[0049] 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 of Leuconostoc mesenteroides. The results are shown in Table 3.

[0050] Table 3 Inhibition zone size and Leuconostoc mesenteroides count determination results

[0051] 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 ]]>

[0052] Example 7 Performance Test:

[0053] 200 volunteers aged 20-45 (both male and female) were selected as trial subjects. The trial subjects were tested using the company's skin tester. One group of 100 people were identified as having healthy skin and no history of skin allergies, and another group of 100 people were identified as having damaged, dehydrated skin that needed repair. Each group was then randomly divided into 5 small groups of 20 people each. The method of use was as follows: After cleansing the face, the trial subjects evenly applied the test toner to the face, gently patting until fully absorbed. Use once a day, morning and evening, for 50 consecutive days. The test product was then rated for its effects on skin freshness, anti-sensitivity, anti-wrinkle, and hydration and smoothness. The total score for each effect was 5 points. The following is the average score for each item. The results are shown in Table 4.

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

[0055] sample Freshness Anti-sensitivity Anti-wrinkle Hydrating and smooth 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 freshness, sensitivity, anti-wrinkle and moisturizing smoothness of the damaged and dehydrated skin requiring repair group.

[0057] sample Freshness Anti-sensitivity Anti-wrinkle Hydrating and smooth Experimental Group 1 4.4 3.0 4.0 3.9 Experimental Group 2 4.7 4.S 4.1 4.3 Experimental Group 3 4.5 3.5 S.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, 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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various embodiments of the present invention.

Claims

1. A method for preparing a complex for promoting skin microecological balance by adding active peptides, comprising an active substance and a basic cosmetic matrix, wherein the active substance is a cosmetic added with an active peptide, wherein the active peptide is a peptide with the sequence MIGDHSFPQSSLIA, NTVEFVRSDDQY and RFFDIIPTVDS, and the content of each component in the complex is 10-20 parts of the active peptide, 1-10 parts of each plant extract, 0.01-0.05 parts of copper tripeptide, 0.01-0.05 parts of salicylic acid, and 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, 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. The plant extracts are mistletoe extract, purslane extract, chamomile extract, imperata root extract, artemisia alpinia 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.

Images