Anti-allergic soothing cosmetic and preparation method thereof

Abstract

The invention relates to an anti-allergic soothing cosmetic and a preparation method thereof. The invention discloses an N-Ala-Phe-Cys-C aqueous solution or an external cosmetic containing N-Ala-Phe-Cys-C for the first time, wherein the N-Ala-Phe-Cys-C aqueous solution or the external cosmetic has anti-allergic soothing effects. The eye cream has excellent single-time and multi-time skin external application safety, and is free of irritation to eyes. Compared with a blank control group, the cosmetics prepared from the 0.01-10% N-Ala-Phe-Cys-C aqueous solution and the formulas 1-4 of the embodiments 5-7 have a remarkable itching relieving effect, and have an inhibiting effect on allergic itching caused by histamine phosphate. Compared with a blank control group, the 0.05%-10% N-Ala-Phe-Cys-C aqueous solution and various formula cosmetics in embodiments 5-7 have a remarkable inhibition effect on auricle swelling caused by xylene.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a tripeptide compound with anti-allergic and soothing effects, cosmetics containing the compound and a preparation process thereof. Background Art

[0002] The impact of modern industry on the environment and the change of social living habits have led to a gradual increase in the incidence of human allergic diseases. Allergic diseases seriously affect the quality of life, work and study of patients, and even cause huge economic losses. Allergic diseases have become a common disease worldwide. Although allergic diseases do not pose the most direct threat to human health like cardiovascular diseases and cancer, they still cause long-term distress to humans, cause a variety of complications, and may even endanger human life.

[0003] Allergic reaction, also known as hypersensitivity reaction or allergic reaction, refers to the abnormal or pathological immune response produced by the body after the body comes into contact with the same antigen again. According to the occurrence mechanism and clinical characteristics of allergic reaction, it can be divided into two types: immediate type and delayed type. Immediate type patients mainly show allergic symptoms within 1 hour after contact with allergens, while delayed type patients will take about 1 to 2 hours to develop related allergic symptoms after ingesting allergens, and some even last up to 24 to 48 hours.

[0004] Climate change, air pollution, and the complexity of cosmetic types and ingredients have increased the possibility of sensitive skin and irritation. As people's awareness of skin health increases, the perception rate and attention to sensitive skin are gradually increasing. Researchers in the cosmetics industry are aware of this phenomenon and have placed products for sensitive skin in an increasingly important position. They have developed active substances for various causes and manifestations of sensitive skin, as well as many anti-allergic activity evaluation experiments with different focuses. However, since researchers often cannot accurately grasp the concept of sensitive skin and confuse it with skin allergies and irritation, the development of sensitive skin-specific cosmetics and the efficacy evaluation system lack systematicity and logical rules, which has brought inconvenience to product development.

[0005] Skin allergy is generally considered not to be a skin disease in medicine, but a skin condition that is susceptible to multiple factors and induces subjective sensory symptoms, that is, the skin is highly reactive, poorly tolerant, and even allergic. Sensitive skin mainly occurs on the face, but may also appear in other parts of the body such as the hands, feet, and scalp. In daily life, there is no objective evidence of skin lesions or skin diseases on sensitive skin. Its main characteristics are drier, tighter, and redder. Slight stimulation can cause itching, desquamation, and even burning and stinging sensations. In severe cases, negative emotions or mental symptoms may occur. Due to the complexity of the causes and mechanisms of sensitive skin, there is currently no specific treatment method, so the use of targeted cosmetics and conscious daily skin care has become very necessary.

[0006] The commonly mentioned "skin allergy" is ACD, also known as allergic contact dermatitis, which is an antigen-specific skin allergic reaction mediated by thymus-dependent T lymphocytes. The occurrence of ACD includes two stages: sensitization and stimulation. In the sensitization stage, when the skin first contacts the hapten, it is carried by the carrier protein of the epidermal cell membrane or phagocytosed by Langerhans' cells (LC), and moves from the epidermis into the lymph, sensitizing T lymphocytes to form effector T cells and memory T cells, which then spread through the blood and throughout the body, which takes about 10 days; in the stimulation stage, when the skin contacts the same hapten again, the memory T cells rapidly differentiate into a large number of effector T lymphocytes, and an inflammatory reaction mediated by helper T cells (CD4+Th1) and a cytotoxic effect mediated by cytotoxic T cells (CD8+CTL) occur in 1 to 2 days.

[0007] The commonly referred to "skin irritation" is ICD, also known as secondary allergy, which refers to tissue cell damage caused by normal skin contact stimulation. The pathogenic pathway of ICD is as follows: the irritant enters the stratum corneum of the skin, causing keratinocyte activation or slight damage, producing cytokines IL-1, IL-2 and IFN-γ, etc., directly inducing dose-related leukocyte chemotaxis and even nonspecific T lymphocyte activation. ICD has a wide range of morphological changes. In the acute stage, it is mainly manifested as skin erythema, edema, desquamation and keratinocyte vesicle-like changes; in the chronic stage, it is mainly manifested as skin fissures, lichenification and excessive keratinization.

[0008] Table 1 Differences and connections between sensitive skin, skin irritation and skin allergy

[0009]

[0010] (Reference: Fan Lina, Jia Yan, et al., Analysis of the causes of sensitive skin and progress in the evaluation of anti-allergic activity of cosmetics, Surfactant Detergent Industry, 2015, 45(7), 409-414).

[0011] What is commonly referred to as "anti-allergic" refers to a series of repair effects on sensitive skin, including daily care and improvement, inhibiting irritation reactions, and relieving inflammation and allergies.

[0012] Many plants contain flavonoids, glycosides, and volatile oils with anti-allergic activity, which provides sufficient resources for the development of anti-allergic drugs and cosmetics. There have been many studies on the anti-allergic activity of plant extracts. Middleton et al. first reported the anti-allergic effect of citrus; Hirai Koichi conducted research on Guangxi sweet tea, hawthorn, mint, papaya, etc. in 1997, and the results showed that sweet tea, mint, and hawthorn had strong anti-allergic activity. Hirai Koichi further studied that the anti-allergic active ingredient of Guangxi sweet tea was GOD-type ellagitannin polymers; Huang Li et al. used in vitro hyaluronidase anti-allergic test experiments to study the anti-allergic activity of perilla leaves, indicating that the flavonoids it contains are the main anti-allergic ingredients; Qiu Jiangyun et al. extracted flavonoids from Houttuynia cordata, and used hyaluronidase test method and histamine-induced increase in mouse capillary permeability (verified its anti-allergic activity; Li Fei et al.'s research showed that honeysuckle water extract has a certain therapeutic effect on type I allergy in ovalbumin-sensitized mice. In addition to the anti-allergic effects of the water-soluble components of the above plants, the volatile oils of the plants also have anti-allergic effects. Li Xiaoli et al. used the guinea pig isolated ileum contraction experiment caused by histamine phosphate (HA) and acetylcholine chloride (Ach), the guinea pig isolated ileum sensitized by ovalbumin (OA) and the rat mast cell degranulation experiment to verify that the volatile oil of magnolia has strong anti-allergic activity; Jiang Han et al. used the delayed hypersensitivity reaction induced by 2,4-dinitrochlorobenzene to verify the anti-allergic activity of Qi Ai volatile oil.

[0013] Currently, the cosmetics industry has conducted extensive research on natural anti-allergic substances. Table 2 lists some active substances with anti-allergic and anti-irritation properties that are generally recognized in the industry and their efficacy characteristics.

[0014] Table 2 Anti-allergic irritation plant extracts and their efficacy characteristics

[0015]

[0016] Common skin allergic reactions are medically known as hypersensitivity reactions, also known as allergic reactions. They refer to abnormal specific immune responses that are mainly characterized by physiological dysfunction or tissue cell damage when the body is stimulated by certain antigens. Its occurrence depends on two factors: the stimulation of the antigen and the reactivity of the body. People with allergic physiques are generally referred to as "allergic physiques" because they are prone to allergic reactions and allergic diseases but cannot find the cause of the disease. Antigens that induce allergic reactions are called allergens. There are hundreds of antigenic substances that cause allergic reactions, and they sensitize the body through inhalation, ingestion, injection or contact.

[0017] According to the relevant explanations of skin allergies in traditional Chinese medicine, Western medicine and biology, and referring to the principle of "comprehensive prevention and treatment" in traditional Chinese medicine, the common ways for cosmetics to prevent and treat skin sensitivity and irritation are: ① Stay away from allergens and reduce the irritation of allergens themselves; ② Inhibit the release of inflammatory mediators such as histamine; ③ Enhance the body's barrier function; Blocking any of the above prevention and treatment approaches will achieve a certain preventive and therapeutic effect, but to achieve a good anti-allergic effect, we should learn from and apply the concept of "holistic prevention and treatment" of traditional Chinese medicine, and form a comprehensive and network-like prevention and treatment to achieve excellent anti-allergic effects.

[0018] M.Rajeswari Prabha and B.Ramachandramurty reported, Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A.Juss, International Journal of Peptides Volume 2013, Article ID 629549, 6 pages http: / / dx.doi.org / 10.1155 / 2013 / 629549. In the literature, the authors extracted and isolated a new branched tripeptide compound from the young leaves of neem, which is N-Ala-Phe-Cys-C, cas number: 1574218-99-07, relative molecular mass: 339.41, and the chemical structure is as follows:

[0019]

[0020] The separation method is given in the literature as follows:

[0021] Preparation of crude extract: Young leaves of A. indica were homogenized with warm 80% ethanol (1 g / 10 mL). The extract was filtered through Whatmann No. 1 filter paper. The extract was partitioned three times with an equal volume of petroleum ether to remove the pigment. The alcoholic portion without pigment was evaporated to dryness on a boiling water bath. The residue obtained was treated with 2% polyvinylpyrrolidone (1 mL per gram of leaves as starting material) and centrifuged at 3000 g for 10 minutes at 4°C to remove phenolic compounds. The clear supernatant obtained was used as a crude product of small peptides.

[0022] Preparation of Cu(II)-ninhydrin reagent: Prepare Cu(II)-ninhydrin reagent by dissolving copper nitrate (25 mmol / L) and ninhydrin (1% w / v) in a mixture of minimal amount of water and glacial acetic acid (3:1 v / v) and dilute with required amount of acetone.

[0023] Circular paper chromatography: Spot the crude extract on the center of Whatman No. 1 filter paper and make a pencil mark around the spot. The diameter of the spot sample is limited to 0.5 cm. After drying with hot air, repeat the spot. Sample spotting can be repeated 15 to 20 times to ensure sufficient concentration of the components to be detected.

[0024] Chromatography was performed in an isopropanol: water (4:1, v / v) solvent system by connecting the filter paper core to the solvent through the hole in the center of the round paper. After approximately 20-40 minutes, the chromatogram was dried at ambient temperature for 30 minutes. The chromatogram was evenly sprayed with Cu(II)-ninhydrin reagent and then dried at 60°C for 10 minutes to form an air-dried chromatogram.

[0025] Purification of peptides: In order to perform various spectroscopic studies on the compounds, a simple and inexpensive purification procedure was followed. Two circular chromatograms of these compounds were analyzed simultaneously using Whatman filter paper disk No. 1 (12 cm). One was developed with Cu(II)-ninhydrin reagent. The corresponding area of ​​the paper on the other chromatogram containing Cu(II)-ninhydrin positive compounds was cut into pieces and eluted in 80% ethanol. The compounds so obtained were used for spectroscopic studies. The plant extracts were run on circular paper chromatograms using isopropanol:water (4:1) solvent system and developed with Cu(II)-ninhydrin reagent. The production of yellow chromophore indicated the presence of Cu(II)-ninhydrin positive compounds. The obtained compounds were purified and detected using inexpensive paper chromatography as described above. 2 μg concentration of peptides was obtained from 1 g of leaf extract.

[0026] However, the above document does not provide any use of the tripeptide. After searching, no document has reported any use of the above tripeptide compound. Summary of the invention

[0027] In view of the current high incidence of skin allergies, there is an urgent need to improve the irritation, itching, red blood streaks and other symptoms caused by skin allergies. The applicant has conducted in-depth research on compounds with potential anti-skin allergy effects and unexpectedly found that the above-mentioned tripeptide compound N-Ala-Phe-Cys-C has significant skin allergy inhibitory activity and can be administered transdermally to play an anti-allergic and soothing role.

[0028] N-Ala-Phe-Cys-C is mild, non-irritating, has good water solubility, and is safe for external skin use. It can be further prepared into anti-allergic external cosmetics to soothe the skin and improve skin allergy symptoms on the face, back and other parts of the body.

[0029] The present invention further provides cosmetics comprising N-Ala-Phe-Cys-C, which can be facial masks, facial creams, skin creams, essences, or any other cosmetics that can be used on the skin to play an anti-allergic and soothing role.

[0030] N-Ala-Phe-Cys-C is usually prepared together with additives into external preparations, external medicines, quasi-drugs or skin cosmetics for application.

[0031] The additives include matrix, such as purified water, deionized water, ethanol, polyols, surfactants, viscosity regulators, film formers, adsorbents, pH regulators, oily solvents, vitamins, antibacterial and bactericides, cooling agents, moisturizers, metal ion chelating agents, fragrances, etc.

[0032] As a film-forming agent, it is mainly used to form a film after the cosmetics are applied to the skin, completely covering the skin, such as polyvinyl alcohol, hydroxypropyl methylcellulose, etc.

[0033] Adsorbents are used in cosmetics to absorb oil, etc., so that the skin feels refreshed after using the mask, such as kaolin, honey, etc.

[0034] Examples of the polyhydric alcohol include glycerin, 1,3-butylene glycol, and propylene glycol.

[0035] The surfactant is not particularly limited, and nonionic, anionic, cationic, or amphoteric surfactants can be appropriately used, for example, polysorbate 80, collagen solution, and glyceryl monostearate.

[0036] As viscosity regulators, hyaluronic acid, sodium alginate, glyceryl monostearate, guar gum, and hydroxypropyl cellulose can be used.

[0037] As the pH adjuster, citric acid or its salt, tartaric acid or its salt can be selected.

[0038] As the oily solvent, olive oil, wheat germ oil, isopropyl palmitate, castor oil, and stearic acid triglyceride can be selected.

[0039] Examples of the vitamins include vitamin E, vitamin C, benzyl nicotinate, niacinamide, D-panthenol, panthenol ethyl ether, biotin, pyridoxine hydrochloride, and riboflavin.

[0040] Examples of the antibacterial and bactericidal agent include climbazole, ethyl paraben, diazolidinyl urea, borax, methylisothiazolinone, trichlorohydroxydiphenyl ether, hibiscus alcohol, triclosan, chlorhexidine gluconate, phenoxyethanol, resorcinol, isopropyl methylphenol, salicylic acid, zinc pyrithione, benzalkonium chloride, photosensitizer No. 301, and sodium mononitroguaiacol.

[0041] Examples of the cooling agent include 1-menthol, camphor, and mannitol.

[0042] Examples of the moisturizer include L-pyrrolidonecarboxylic acid, sodium hyaluronate, sorbitol, chondroitin sulfate, cordyceps sinensis extract, and saffron extract.

[0043] Examples of the antioxidant include butylated hydroxyanisole, isopropyl gallate, propyl gallate, and isoascorbic acid.

[0044] Examples of the metal ion chelating agent include ethylenediaminetetraacetic acid or a salt thereof.

[0045] Examples of the fragrance include natural fragrances such as orange oil, lemon oil, bergamot oil, lime oil, lemongrass oil, and lavender oil; and synthetic fragrances such as menthol, rose oxide, linalool, citral, and linalyl acetate.

[0046] The matrix of the semisolid or solid dosage form includes vaseline, solid paraffin, vegetable oil, mineral oil, cetyl alcohol, lanolin, waxes, polyethylene glycol, cholesterol, white oil, etc., and the aforementioned additives, emulsifiers such as lecithin, lower alcohols such as ethanol and isopropanol, etc. can be added as needed.

[0047] The N-Ala-Phe-Cys-C and the cosmetics containing N-Ala-Phe-Cys-C described in the present application have good anti-allergic and soothing effects through skin application, excellent safety for single and multiple skin external use, and no irritation to the eyes. Compared with the blank control group, the 0.01-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics obtained by formula 1-4 of Examples 5-7 have significant antipruritic effects and have an inhibitory effect on allergic itching caused by histamine phosphate. And the cosmetics containing 0.5-10% N-Ala-Phe-Cys-C aqueous solution and formula 2-4 of Examples 5-7 have a more significant inhibitory effect on allergic itching caused by histamine phosphate compared with the positive control group Jieryin.

[0048] Compared with the blank control group, the 0.05%-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics of each formula of Examples 5-7 have a significant inhibitory effect on the auricle swelling caused by xylene, and compared with the positive control group (fluocinolone acetonide ointment), the 0.5%-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics of Formulas 2-4 of Examples 5-7 (i.e., containing 0.5%-10% N-Ala-Phe-Cys-C) have a more significant inhibitory effect on the auricle swelling caused by xylene. DETAILED DESCRIPTION

[0049] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Example 1 Single and multiple skin irritation of N-Ala-Phe-Cys-C aqueous solution (in accordance with the 2015 edition of the Cosmetic Safety Technical Specifications)

[0051] 1. Materials and Methods

[0052] 1. The test substance is 10% N-Ala-Phe-Cys-C aqueous solution

[0053] 2. Test animals: 8 ordinary white New Zealand rabbits, which were adaptively raised for 3 days before the test.

[0054] 3. Single skin irritation test steps:

[0055] Take 4 test animals, and 24 hours before the test, cut the hair on both sides of the spine of the experimental animals, without damaging the epidermis, and the hair removal range is about 3cm×3cm on the left and right. During the test, take about 0.5mL of the test substance and apply it directly on the skin, with an application area of ​​2.5cm×2.5cm, then use two layers of gauze (3cm×3cm) and a layer of cellophane, and then fix it with non-irritating tape and bandage. The skin on the other side is used as a control. Use a closed test, and the application time is 4h.

[0056] After the test, use warm water or non-irritating solvent to remove the residual test substance.

[0057] Observe the skin reaction at the application site 1, 24, 48 and 72 hours after removing the test substance, and score the skin reaction according to Table 3. Make a comprehensive evaluation based on the average score of the test animals. Determine the skin irritation intensity according to Table 4 based on the average of the highest scores at each observation time point of 24, 48 and 72 hours.

[0058] 4. Multiple skin irritation test steps

[0059] Four experimental animals were selected. Before the test, the hair on both sides of the spine of the experimental animals' backs were trimmed, with the hair removal area being 3 cm × 3 cm each, and the application area being 2.5 cm × 2.5 cm.

[0060] Take about 0.5mL of the test substance and apply it on one side of the skin, and apply purified water (solvent) on the other side as a control. Apply once a day for 14 consecutive days. Starting from the second day, the hair should be trimmed before each application, and the residual test substance should be removed with water. Observe the results after one hour and score according to Table 4. The control area and the test area are treated in the same way. Result evaluation: Calculate the average score of each animal per day according to the following formula, and determine the skin irritation intensity according to Table 4.

[0061]

[0062] Table 3 Skin irritation reaction scores

[0063]

[0064] Table 4 Skin irritation intensity classification

[0065] Integral mean strength 0-＜0.5 Non-irritating 0.5-＜2.0 Mild irritation 2.0-＜6.0 Moderate irritation 6.0-8.0 Strong irritation

[0066] .

[0067] 5. Test results

[0068] Table 5 Single skin irritation test results

[0069]

[0070] Table 6 Results of multiple skin irritation tests

[0071]

[0072]

[0073] As can be seen from the data in Tables 5 and 6 above, the 10% N-Ala-Phe-Cys-C aqueous solution has no irritation to the skin of the test animals, whether in a single test or multiple tests.

[0074] Example 2 Acute eye irritation test (refer to the 2015 edition of the Cosmetic Safety Technical Specifications)

[0075] 1. Test substance: 10% N-Ala-Phe-Cys-C aqueous solution.

[0076] 2. Experimental animals and feeding environment: 3 healthy adult white rabbits. The animals should be fed in the experimental animal room for 3 days before the experiment.

[0077] Both eyes of the test animals were examined within 24 hours before the start of the test (including the use of sodium fluorescein for examination). Animals with eye irritation, corneal defects and conjunctival damage cannot be used for the test, and new test animals should be selected. Standard compound feed was used, and drinking water was not restricted.

[0078] 3 Test steps

[0079] Gently pull open the lower eyelid of one eye of the rabbit, drop 0.1 mL of the test substance into the conjunctival sac, and passively close the upper and lower eyelids for 1 second to prevent the loss of the test substance. The other eye is left untreated as a self-control. Do not rinse the eye within 24 hours after dropping the test substance.

[0080] The animal eyes are examined at 1, 24, 48, 72 hours, and on the 4th and 7th days after the instillation of the test substance. If no irritation reaction occurs within 72 hours, the test can be terminated. If the cornea is found to be involved or there are other eye irritation effects, and it does not recover within 7 days, the observation time needs to be extended to determine the reversibility or irreversibility of the damage, generally not more than 21 days, and observation reports for 7 days, 14 days and 21 days should be provided. In addition to observing the cornea, iris and conjunctiva, other damaging effects should be recorded and reported. In each examination, the score of the eye irritation reaction should be recorded according to the scoring criteria for eye damage in Table 7.

[0081] The irritation intensity of the test substance to the eye is determined by evaluating the mean value of the irritation response integral of the cornea, iris or conjunctiva of the animals at 24, 48 and 72 hours after administration of the test substance and the recovery time, and by the eye irritation response classification in Table 8.

[0082] After the 24-hour observation and recording, the eyes of all animals were further examined using sodium fluorescein.

[0083] Table 7 Scoring criteria for eye damage

[0084]

[0085]

[0086] Table 8 Grading of eye irritation reactions

[0087]

[0088] The results of the acute eye irritation test are shown in the following table:

[0089]

[0090] As shown in the table above, no eye irritation was found in the test animals within 72 days after the sample was administered. Therefore, it is considered that the test sample has no eye irritation in the acute eye irritation test in rabbits.

[0091] Example 3 Anti-allergic test of N-Ala-Phe-Cys-C aqueous solution (hyaluronidase in vitro inhibition test) (implemented with reference to Chinese patent CN 104784297B)

[0092] 1. The relationship between hyaluronidase and allergy

[0093] Kakegawa et al. reported that various drugs that affect the release of histamine from mast cells can regulate the activity of hyaluronidase, and there is a good correlation between the inhibition of hyaluronidase activity and the inhibition of histamine release from mast cells (Kakegawa H, Inhibitory effects of some natural products on the activation of hyaluridse and their antiallergic actions [J]. Hen Pharm Bull, 1991, 40 (6): 1439-1422). Yoo et al. reported that hyaluronidase participates in the process of tissue inflammation and is a participant in type I allergic reactions. In the third stage of allergic reactions, it participates in the reaction and causes mast cell degranulation (Yoo, Effects of allergy-related drugs on hyaluridase action and histamine by rat peritoneal mast cell. Tachan Sacngri Hakboechi. 1988, 22: 59-72). Therefore, the hyaluronidase in vitro inhibition test can be used as a simple biochemical method to evaluate the antiallergic properties of samples. Since the hyaluronidase in vitro model is simple and easy, this biological experimental method is widely used as an indicator for studying anti-allergic effects.

[0094] 2. Anti-allergic activity detection: Elson-Morgan modified method: Hyaluronidase is a participant in type I allergic reactions. Studies have shown that hyaluronidase is strongly correlated with inflammation and allergies. Kakegawa has reported that various drugs that release histamine from mast cells can regulate the activity of hyaluronidase.

[0095] 3. Reagent preparation

[0096] (1) Hyaluronidase: Use acetate buffer solution as solvent to prepare hyaluronidase at a concentration of 500u / mL. It should be prepared and used immediately and should not be kept overnight.

[0097] (2) Sodium hyaluronate: Use acetate buffer solution as solvent to prepare a 0.5 mg / mL sodium hyaluronate solution.

[0098] (3) Buffer: 4.8 mL of solution A (0.2 mol / L acetic acid aqueous solution) and 45.2 mL of solution B (0.2 mol / L sodium acetate aqueous solution) were mixed and diluted to 100 mL to prepare an acetate buffer solution with a pH of 5.6.

[0099] (4) Acetylacetone solution: Mix 50 mL of 1.0 mol / L sodium carbonate solution and 3.5 mL of acetylacetone solution evenly. (Prepare immediately before use)

[0100] (5) Preparation of P-DAB color developer: 0.8 g of p-dimethylaminobenzaldehyde, 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol were mixed evenly for use.

[0101] (6) CaCl2 solution: 2.5 mol / L.

[0102] (7) NaOH solution: 5 mol / L.

[0103] (8) Prepare N-Ala-Phe-Cys-C into aqueous solutions of different concentrations for later use.

[0104] Experimental steps:

[0105]

[0106] Calculation of antiallergic activity:

[0107] Hyaluronidase inhibition rate (%) = [(CD) - (AB)] / (CD) × 100%

[0108] Where: A—OD value of (hyaluronidase + sample + hyaluronidase sodium) sample solution

[0109] B—OD value of (acetic acid buffer + sample + acetate buffer) sample blank

[0110] C—OD value of (hyaluronidase + deionized water + hyaluronidase sodium) control solution

[0111] D—(acetic acid buffer + deionized water + acetate buffer) OD value of the control blank solution

[0112] The results are shown in Table 9:

[0113] Table 9 Effects of different concentrations of N-Ala-Phe-Cys-C aqueous solution on hyaluronidase

[0114] Sample concentration Hyaluronidase inhibition rate (%) Sample concentration Hyaluronidase inhibition rate (%) 0.01％ 10.3 1.0％ 70.6 0.05％ 17.3 2.0％ 80.1 0.1％ 30.7 5.0％ 88.4 0.5％ 50.6 10.0％ 92.5 .

[0115] Hyaluronidase is a participant in type I allergic reactions. Studies have shown that hyaluronidase is strongly correlated with inflammation and allergies. The experimental results show that N-Ala-Phe-Cys-C aqueous solution has a significant inhibitory effect on hyaluronidase. When the concentration of N-Ala-Phe-Cys-C is 0.01%, there is a significant inhibitory activity, and the inhibition rate increases with the increase in concentration. When the concentration is 10%, the inhibition rate of hyaluronidase can reach 92.5%. Therefore, it is believed that N-Ala-Phe-Cys-C aqueous solution can inhibit allergic reactions by inhibiting hyaluronidase.

[0116] Example 5 Preparation of anti-allergic soothing essence

[0117] Components Recipe 1 Recipe 2 Recipe 3 Recipe 4 N-Ala-Phe-Cys-C 0.01 0.5 5 10 Hyaluronic acid 6.0 6.0 6.0 6.0 Trehalose 5.0 5.0 5.0 5.0 Mannitol 2.5 2.5 2.5 2.5 5% Benzalkonium Bromide Aqueous Solution 2ml 2ml 2ml 2ml Deionized water Up to 100ml Up to 100ml Up to 100ml Up to 100ml .

[0118] Note: The data in the above table are in g for solids and ml for liquids.

[0119] Preparation method:

[0120] Take 70% of the prescribed amount of deionized water, add 5% benzalkonium bromide aqueous solution, mannitol, trehalose, N-Ala-Phe-Cys-C and hyaluronic acid in sequence, stir and dissolve to obtain a clear and transparent solution, which is the anti-allergic essence.

[0121] Example 6 Preparation of N-Ala-Phe-Cys-C Anti-allergic Soothing Facial Mask

[0122] The whitening facial mask of this embodiment is composed of the following components by mass percentage:

[0123]

[0124] Note: The data in the above table are in g for solids and ml for liquids.

[0125] The preparation steps are as follows:

[0126] Step 1) Weigh polyvinyl alcohol and sodium alginate, add 70% of the prescribed amount of deionized water, and heat to completely swell to form a film-forming material;

[0127] Step 2) Weighing zinc oxide and kaolin, grinding and mixing with glycerin and olive oil, and adding to the film-forming material prepared in step 1) as a matrix;

[0128] Step 3) Add honey, N-Ala-Phe-Cys-C, 7% sorbitol solution, and ethyl p-hydroxyformate ethanol solution to the matrix obtained in step 2), dilute to 100 ml with deionized water, and stir to mix evenly.

[0129] Example 7 Preparation of N-Ala-Phe-Cys-C Anti-allergic Soothing Skin Cream

[0130]

[0131]

[0132] Note: The data in the above table are in g for solids and ml for liquids.

[0133] Preparation method:

[0134] Step 1) Mix wheat germ oil, vitamin E, olive oil, glyceryl monostearate, and ethyl paraben in ethanol, heat, and stir to dissolve to obtain an oil phase;

[0135] Step 2) taking a mixture of N-Ala-Phe-Cys-C, collagen aqueous solution, glycerol, and polysorbate 80, stirring and dissolving to obtain an aqueous phase;

[0136] Step 3) Add the aqueous phase obtained in step 2 to the oil phase obtained in step 1), stir, emulsify, cool to room temperature, make up to volume with deionized water, stir evenly, and obtain the finished product.

[0137] Example 8 Effects of N-Ala-Phe-Cys-C aqueous solution and cosmetics of various formulas in Examples 5-7 on local skin itching in guinea pigs (implemented with reference to Chinese patent CN104784297B)

[0138] Test animals: guinea pigs, half male and half female, weighing 160-190 g;

[0139] Experimental groups: The groups were described as follows:

[0140] Group 1 was the blank control group;

[0141] Group 2 was the positive control group using Jieryin;

[0142] Group 3 was the experimental group with 0.01% N-Ala-Phe-Cys-C aqueous solution;

[0143] Group 4 was the experimental group with 0.5% N-Ala-Phe-Cys-C aqueous solution;

[0144] Group 5 was the experimental group with 5% N-Ala-Phe-Cys-C aqueous solution;

[0145] Group 6 was the experimental group with 10% N-Ala-Phe-Cys-C aqueous solution;

[0146] Group 7 was the test group Example 5 Formulation 1;

[0147] Group 8 was the test group Example 5 Formulation 2;

[0148] Group 9 was the test group Example 5 Formulation 3;

[0149] Group 10 was the test group Example 5 Formula 4;

[0150] Group 11 was the test group, Example 6, Formulation 1;

[0151] Group 12 was the test group, Example 6, Formulation 2;

[0152] Group 13 was the test group, Example 6, Formulation 3;

[0153] Group 14 was the test group, Example 6, Formula 4;

[0154] Group 15 was the test group Example 7 Formulation 1;

[0155] Group 16 was the test group, Example 7, Formulation 2;

[0156] Group 17 was the test group, Example 7, Formulation 3;

[0157] Group 18 was the test group, Example 7, formulation 4.

[0158] Three days before the experiment, the dorsum of the right hind paw of each group of guinea pigs was shaved and externally applied with distilled water, Jie'eryin, 0.01% N-Ala-Phe-Cys-C aqueous solution, 0.5% N-Ala-Phe-Cys-C aqueous solution, 5% N-Ala-Phe-Cys-C aqueous solution, 10% N-Ala-Phe-Cys-C aqueous solution, and the cosmetics obtained from formulas 1-4 of Examples 5-7, respectively. The dosage was 0.5 ml / pig. On the day of the experiment, the shaved area of ​​the right hind foot was scratched with coarse sandpaper, with an area of ​​1 square centimeter. The topical application was repeated once. Ten minutes after the last application, 0.01% histamine phosphate solution was dripped on the wound surface at 0.05 ml / guinea pig. After that, the concentration was increased in sequence of 0.02%, 0.03%, and 0.04% every 3 minutes, with 0.05 ml / guinea pig each time, until the guinea pig turned back to lick the right hind foot. The total amount of histamine phosphate administered when the guinea pig finally turned back to lick the right hind foot was taken as the itch threshold. The itch threshold of each group was recorded and compared. The results are shown in Table 10.

[0159] Table 10 Effects of N-Ala-Phe-Cys-C aqueous solution and various formula cosmetics of Examples 5-7 on the itch domain reaction caused by histamine phosphate (n=10, mean)

[0160] Group Itch threshold (ug) Group Itch threshold (ug) Blank control group 59.36 Example 5 Formulation 4 288.60 Jieeryin 171.31 Example 6 Formulation 1 122.46 0.01% N-Ala-Phe-Cys-C aqueous solution 125.96 Example 6 Formulation 2 178.06 0.5% N-Ala-Phe-Cys-C aqueous solution 184.72 Example 6 Formulation 3 277.33 5% N-Ala-Phe-Cys-C aqueous solution 255.56 Example 6 Formulation 4 285.38 10% N-Ala-Phe-Cys-C aqueous solution 280.95 Example 7 Formulation 1 128.17 Example 5 Formulation 1 136.15 Example 7 Formulation 2 176.53 Example 5 Formulation 2 182.84 Example 7 Formulation 3 268.27 Example 5 Formulation 3 262.96 Example 7 Formulation 4 285.58 .

[0161] Allergic reactions are generally manifested as skin itching. The experimental results show that, compared with the blank control group, the 0.01-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics obtained from the formula 1-4 of Examples 5-7 have significant antipruritic effects and have an inhibitory effect on allergic itching caused by histamine phosphate. In addition, the cosmetics containing 0.5-10% N-Ala-Phe-Cys-C aqueous solution and the formula 2-4 of Examples 5-7 have a more significant inhibitory effect on allergic itching caused by histamine phosphate compared with the positive control group Jieryin.

[0162] Example 9 Effects of N-Ala-Phe-Cys-C aqueous solution and cosmetics of various formulas in Examples 5-7 on xylene-induced ear swelling in mice (implemented with reference to Chinese patent CN104784297)

[0163] 180 healthy male mice weighing 20-22g were randomly divided into 18 groups, 10 mice in each group. The experimental grouping was the same as in Example 8, and the blank control group was treated with 0.03ml of distilled water per mouse, once a day. The remaining groups were respectively treated with fluocinolone acetonide ointment and the samples of the experimental groups, 0.03g per mouse each time, once a day.

[0164] Dosage method: Apply fluocinolone acetate ointment or other test substances or distilled water to the right auricle of mice for 3 consecutive days. One hour after the last application of the drug, inject 0.5% Evans blue saline 0.1ml / 10g through the tail vein of the mouse, and then drip 0.03ml of xylene into the right auricle so that it covers the inside and outside of the auricle. After 40 minutes, the mice were killed by dislocating the cervical vertebra. Cut off the left and right auricles and use a 2mm diameter puncher to punch round ear pieces at the same position of both ears. Weigh with a balance, the difference in weight between the left and right ear pieces is the swelling degree, and the swelling inhibition rate (%) is calculated according to the following formula.

[0165] Swelling inhibition rate (%) = (average swelling degree of the control group - average swelling degree of the drug group) / average swelling degree of the control group × 100%

[0166] The results are shown in Table 11.

[0167] Table 11 Effects of N-Ala-Phe-Cys-C aqueous solution and various formula cosmetics of Examples 5-7 on ear swelling caused by xylene (n=10, mean)

[0168]

[0169]

[0170] The experimental results show that there are significant differences between the positive control group, the test group (administered with different concentrations of N-Ala-Phe-Cys-C aqueous solution or the cosmetics of each formula of Examples 5-7) and the blank control group. Compared with the blank control group, the 0.05%-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics of each formula of Examples 5-7 have a significant inhibitory effect on the auricle swelling caused by xylene, and compared with the positive control group (fluocinolone acetonide ointment), the 0.5%-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics of Formula 2-4 of Examples 5-7 (i.e., containing 0.5%-10% N-Ala-Phe-Cys-C) have a more significant inhibitory effect on the auricle swelling caused by xylene, indicating that the 0.05%-10% N-Ala-Phe-Cys-C aqueous solution and the cosmetics of each formula of Examples 5-7 achieve anti-allergic effects by inhibiting inflammation.

Claims

1. Use of the tripeptide compound N-alanine-phenylalanine-cysteine-C for preparing cosmetics with anti-allergic and soothing effects.

2. The use according to claim 1, characterized in that The chemical structure of the tripeptide compound N-alanine-phenylalanine-cysteine-C is as follows:

3. The use according to claim 1, characterized in that The cosmetic is administered transdermally.

4. The use according to claim 1, characterized in that The cosmetic is administered transdermally through the skin of the face, back, etc.

5. The use according to claim 1, characterized in that The cosmetics are facial masks, facial creams, skin care creams, essences, or any other cosmetics that can be used on the skin to play an anti-allergic and soothing role.

6. The use according to claim 1, characterized in that The cosmetic is prepared by N-alanine-phenylalanine-cysteine-C and an additive.

7. The use according to claim 1, characterized in that The mass percentage concentration of N-alanine-phenylalanine-cysteine-C in the cosmetic is 0.01%-10%.

8. The use according to claim 1, characterized in that The mass percentage concentration of N-alanine-phenylalanine-cysteine-C in the cosmetic is 0.5%-5%.

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