Anti-aging active peptide with complexion improving effect

Abstract

The invention relates to the field of skin repair and improvement, in particular to an active peptide with complexion improving activity and related application thereof.

Description

Technical Field

[0001] The present application relates to the field of skin repair and improvement, and specifically to an active peptide having the activity of improving complexion and its related uses. Background Art

[0002] For thousands of years, Orientals, especially Chinese, have never stopped pursuing the "beauty of skin color". The ancients praised healthy skin color. For example, "Hands like soft catkins, skin like solidified fat, neck like cicada, teeth like gourd rhinoceros" is used to describe a woman's skin as smooth and delicate as solidified grease; another example is "face like peach blossoms, eyes like autumn water, lips red without makeup, eyebrows green without painting" is used to describe a woman's skin as delicate as peach blossoms. According to the "Yellow Emperor's Internal Classic", "The qi of the five internal organs is read on the face", which shows that the skin color can reflect the health status of human organs. Therefore, "Oriental complexion" emphasizes the health status that matches the skin characteristics of Oriental people. That is, it has a good state in terms of skin physiology, skin optics, and skin color. Modern science has a complete set of evaluation systems for "Oriental complexion". The "Oriental complexion" that meets the characteristics of "healthy skin color" mainly includes the following characteristics:

[0003] 1. Skin physiological indicators: crow's feet (few), nasolabial folds (few), fine lines under the eyes (few), high skin elasticity and firmness, etc.

[0004] 2. Skin optical indicators: glossiness (high), roughness (small), pigmentation area and red area (small), etc.

[0005] 3. Skin color indicators: large L value, small a value, small b value, large ITA value, small MI.

[0006] As the pace of life of modern people is getting faster and faster, their skin problems are getting more and more serious. The younger generation's perception of aging is gradually advanced. 94% of the "post-00s" have felt skin aging before the age of 25. The age of "post-95s" and "post-90s" feeling skin aging is concentrated between 26 and 30 years old, and most of the "post-85s" feel skin aging around the age of 30. According to the latest research by scientists at the Chando R&D Center: In the past nearly three years, the turning point of skin aging in Chinese women has been advanced by about two years. The facial aging phenotypes of Chinese women's skin, such as crow's feet, nasolabial folds, and tear grooves, have shown an obvious acceleration turning point between the ages of 24 and 33. Based on the above situation, there are a large number of unmet consumer demands in society to improve skin quality, delay skin aging, and restore a healthy "oriental complexion".

[0007] To delay skin aging, NAD regulation is receiving more and more attention. The full name of NAD in Chinese is "nicotinamide adenine dinucleotide". NAD+ is the oxidized state of NAD, an important coenzyme found in cells. It plays an important role in regulating cell function, neuroprotection, DNA repair and anti-aging. Scientists at the National Center for Aging Research in the United States have found that supplementing NAD+ in the human body can ultimately prolong life through mitochondrial and DNA repair, and improve health levels.

[0008] As we age, the level of NAD+ in the human body decreases rapidly. Data show that the NAD+ content in the human body decreases by 50% every 20 years. At around 40 years old, the NAD+ content in the human body is only 25% of that in children. At present, the ways to increase NAD+ intake can be roughly divided into oral health products and intravenous injections. Users believe that this method can increase the level of NAD+ in cells, thereby improving the energy metabolism efficiency of cells and increasing the energy supply of the human body; therefore, in the past few years, NAD+ supplements have become a "cellular anti-aging" product favored by more elites.

[0009] The inventors of this application studied the AT1R receptor on venous endothelial cells (AT1R is the main regulatory receptor for vasoconstriction) as a target receptor. We first obtained the AT1R crystal structure from the PDB database, and continued to screen potential polypeptide sequences from the genomes of traditional Chinese plants that regulate complexion (including Salvia miltiorrhiza, peach blossom, wolfberry, etc.), and potential polypeptides that can bind to it, and then performed computer-based sequence structure simulation optimization to obtain candidate polypeptides. Finally, the anti-dark circle ability of the target polypeptide was verified through experiments, and a patent was applied for and authorized for the relevant polypeptide (CN114920799B). The research field of this application is limited to anti-dark circles. On the basis of the existing patents, we have conducted a deeper study on the relevant polypeptides and confirmed that it has the efficacy of significantly improving the sub-health state of the skin, improving skin gloss, reducing roughness, reducing the area of ​​spots and sensitive red areas, reducing crow's feet, nasolabial folds and fine lines under the eyes, improving skin elasticity and firmness, and anti-aging. This application is specially proposed. Summary of the invention

[0010] Based on the above-mentioned defects of the prior art, the present invention provides an active peptide that can improve the sub-healthy state of the skin, increase skin gloss, reduce roughness, reduce the area of ​​spots and red areas, reduce crow's feet, nasolabial folds and fine lines under the eyes, and improve skin elasticity and firmness. The specific structure is as follows:

[0011] A biologically active polypeptide, whose amino acid sequence is shown in SEQ ID NO: 1, and whose specific amino acid sequence is: GRTYKHPA. The above polypeptide can be synthesized by conventional methods.

[0012] The second aspect of the present invention provides a method for preparing the biologically active polypeptide, which can be artificially synthesized by genetic engineering methods, directly obtained from cells by separation and purification methods, or more directly prepared by chemical synthesis.

[0013] The third aspect of the present invention provides the use of the bioactive polypeptide in the preparation of active peptide drugs or cosmetics that can improve the sub-health state of the skin, increase skin gloss, reduce roughness, reduce the area of ​​spots and red areas, reduce crow's feet, nasolabial folds and fine lines under the eyes, increase skin elasticity and firmness, anti-aging, accelerate skin renewal, strengthen the skin barrier, effectively stimulate keratinocyte formation and mitochondrial metabolism, enhance skin defense and the like.

[0014] In some embodiments, the bioactive polypeptide also has the effect of inhibiting the NF-κB signaling pathway and promoting the content of type I collagen.

[0015] In some embodiments, the bioactive polypeptide also has the efficacy of inhibiting tyrosinase activity and reducing melanin content.

[0016] In some embodiments, the bioactive polypeptide also has the effect of inhibiting the expression of related inflammatory factors.

[0017] In some embodiments, the bioactive polypeptide also has the effect of promoting vasodilation.

[0018] In a fourth aspect, the present invention provides a product comprising the bioactive polypeptide or its derivatives; the bioactive polypeptide derivatives refer to polypeptide derivatives obtained by modifying the amino acid side chain groups, amino terminus or carboxyl terminus of the bioactive polypeptide by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation, while not affecting its biological activity.

[0019] In a fifth aspect, the present invention provides a pharmaceutical composition comprising a cosmetically or pharmaceutically effective amount of at least one bioactive peptide according to the foregoing and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant.

[0020] In some embodiments, the dosage form of the pharmaceutical composition includes cream, lotion, water, gel, oil, powder, mud, wax-based, patch, film or lyophilized.

[0021] In some embodiments, the excipients include: hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, polyacrylamide, polyacrylic acid, polyvinyl alcohol, dextran, ammonia, lactic acid, citric acid, pyrrolidone carboxylic acid, glycerol, urea, sorbitol, amino acids, lanolin, petrolatum.

[0022] In some embodiments, the adjuvant includes: solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, flavoring agents, surfactants, water, ionic or non-ionic emulsifiers, fillers, chelating agents, chelating agents, preservatives, vitamins, retardants, wetting agents, essential oils, dyes, pigments.

[0023] In the sixth aspect of the present invention, the bioactive peptides of the present application can be used in combination with other ingredients, including but not limited to: vasodilators, antioxidants, reactive carbonyl species scavengers, anti-glycation agents, antihistamines, antivirals, antiparasitic agents, emulsifiers, moisturizers, anti-wrinkle agents and / or anti-aging agents, etc.

[0024] In the first to sixth aspects of the present invention, the content of the bioactive peptide (e.g., acGA-8-2) is selected from: 1-100ppm, preferably 2ppm, 5ppm, 10ppm, 15ppm, 20ppm, 25ppm, 30ppm, 35ppm, 40ppm, 45ppm, 50ppm, 55ppm, 60ppm, 65ppm, 70ppm, 75ppm, 80ppm, 85ppm, 90ppm, 95ppm, 100ppm; most preferably 15ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :Schematic diagram of the basic scheme of acGA-8-2 active peptide skin efficacy test.

[0026] Figure 2 : Experimental results of acGA-8-2 active peptide brightening skin color (from the perspective of skin color science: ITA value, L value, MI value).

[0027] Figure 3 : Experimental results of acGA-8-2 active peptide reducing wrinkles (from the perspective of skin physiology: crow's feet).

[0028] Figure 4 : Experimental results of acGA-8-2 active peptide reducing wrinkles (from the perspective of skin physiology: crow's feet).

[0029] Figure 5 : The experimental results of acGA-8-2 active peptide reducing wrinkles (from the perspective of skin physiology: nasolabial folds and fine lines under the eyes).

[0030] Figure 6 : Experimental results of acGA-8-2 active peptide reducing wrinkles (from the perspective of skin physiology: R5, R7, etc.).

[0031] Figure 7 :AcGA-8-2 active peptide improves pigmentation (skin optical perspective: skin luster, spots, etc.) experimental results.

[0032] Figure 8 :The experimental results of changes in vasodilation of chick chorioallantoic membrane.

[0033] Fig. 9 : Experiment on the effect of acGA-8-2 on promoting the content of type Ⅰ collagen.

[0034] Fig.10 :AcGA-8-2 can inhibit NF-κB experimental results.

[0035] Fig.11 : Whitening efficacy test results.

[0036] Fig.12 : In vitro soothing test results.

[0037] Fig.13 : Results of in vitro antioxidant test.

[0038] Fig.14 : Test results on cell energy and skin efficacy. Example:

[0039] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values ​​shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "1 to 10" recorded should be considered to include any and all sub-ranges between the minimum value 1 and the maximum value 10 (including the endpoints); that is, all sub-ranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum value of 10 or less, such as 5.5 to 10.

[0040] The polypeptides involved in this application are as follows:

[0041] Bioactive Peptides sequence acGA-8-2 GRTYKHPA

[0042] Related noun meanings:

[0043] ITA value: A parameter that characterizes human skin color. The larger the ITA value, the lighter the skin color, and vice versa.

[0044] L value: represents the brightness of the skin. The larger the L value, the lighter the skin color.

[0045] MI value: represents the melanin index of the skin. The larger the MI value, the darker the skin color, and vice versa.

[0046] R5 value: net elasticity. The larger the R5, the better the skin elasticity.

[0047] R7 value: the amount of elasticity of the rebound part / the total amount of elasticity of the stretching part. The larger the R7, the better the skin elasticity.

[0048] R0 value: the maximum amplitude of the first cycle. The smaller the R0, the tighter the skin.

[0049] F4 value: represents the firmness of the skin. The smaller the F4 value, the tighter the skin, and vice versa.

[0050] Control: Normally cultured cells without any treatment.

[0051] Example 1: acGA-8-2 human testing method

[0052] Experimental plan: Valid subjects: 30 people, age range: (43.4±8.2) years old;

[0053] Skin condition of the subjects: Dull skin, pigmentation, poor skin firmness and sensitivity.

[0054] Test reagents: emulsion (containing 15ppm acGA-8-2) vs. emulsion (not containing 15ppm acGA-8-2)

[0055] Main testing indicators: crow's feet, nasolabial folds, fine lines under the eyes, elasticity, firmness, anisotropy, glossiness, roughness, pigmentation area, red area, Lab value, ITA value, MI value, etc.

[0056] Experimental design: Each subject was randomly assigned to the left and right face for comparison (code 1 or 2). The specific experimental plan was as follows: the subject washed the face with a cleaning product under specified environmental conditions, dried it with dandruff-free absorbent paper, and sat in the test environment for 30 minutes before undergoing a skin basal value test. The test side and the control side were selected according to the random table (Table 1). The test side used lotion (containing 15ppm acGA-8-2) and the control side used lotion (not containing 15ppm acGA-8-2). The tests were performed on day 0 (D0), 30min (T30min), day 3 (D3), day 14 (D14), and day 28 (D28) (see Figure 1 ).

[0057] Table 1: Randomization table of products used by subjects

[0058]

[0059]

[0060] The experimental results show that (see Figure 2-Figure 7): The acGA-8-2 active peptide of the present application can effectively brighten the skin tone, reduce wrinkles, especially crow's feet and nasolabial folds, improve skin gloss, reduce the area of ​​spots, reduce the area of ​​red areas, and improve skin firmness. It is very effective in improving / enhancing the oriental complexion and has achieved unexpected technical effects.

[0061] In addition, we have also conducted in vitro experiments on anti-wrinkle, whitening, soothing, and promoting vasodilation. Preliminary experimental results show that acGA8-2 has the following effects:

[0062] 1. It can inhibit the NF-κB signaling pathway and promote the content of type Ⅰ collagen.

[0063] 2. It can inhibit tyrosinase activity and reduce melanin content.

[0064] 3. It can inhibit the expression of related inflammatory factors.

[0065] 4. It can promote vasodilation.

[0066] The following is described in more detail through specific embodiments:

[0067] Example 2: Experiment on the effect of acGA-8-2 on vasodilation

[0068] Chicken chorioallantoic membrane test

[0069] Chicken embryos that met the quality standards (Xinxing Dahua Agricultural and Poultry Egg Co., Ltd.) were selected, and 300 μL of 10 μM angiotensin II (ANGⅡ) was used to induce vasoconstriction. After 5 min, 300 μL of samples of different concentrations were added, including 2 ppm acGA-8-2, 5 ppm acGA-8-2, 10 ppm acGA-8-2, and 10 μM candesartan.

[0070] The experimental results show that (see Figure 8 ): Adding angiotensin II (ANGⅡ) for 5 minutes caused vasoconstriction; adding samples of different concentrations caused vasodilation after 5 minutes. This indicates that acGA-8-2 has a good effect of vasodilation.

[0071] Example 3: Experiment on the promotion of type I collagen content by acGA-8-2

[0072] Using the fibroblast HFF-1 cell model, 10μM angiotensin II (ANGⅡ) was used to induce the cells, and different concentrations of samples were added to treat the cells, namely 10μM candesartan, 2ppm acGA-8-2, 5ppm acGA-8-2, and 10ppm acGA-8-2. The experimental results showed (see Fig. 9):ANGⅡ group significantly reduced the fluorescence intensity of type I collagen, 2ppm acGA-8-2, 5ppm acGA-8-2, and 10ppm acGA-8-2 all significantly increased the fluorescence intensity of type I collagen induced by ANGⅡ, further confirming that acGA-8-2 has a good anti-wrinkle effect.

[0073] Example 4: acGA-8-2 can inhibit the NF-κB signaling pathway

[0074] Based on the steps similar to Example 3, the expression of NF-κB was detected, and the results showed that ( Fig.10 ):ANGⅡ group significantly increased the fluorescence intensity of NF-κB, and 2ppm acGA-8-2 and 5ppm acGA-8-2 significantly reduced the fluorescence intensity of NF-κB induced by ANGⅡ, further confirming that acGA-8-2 has a good anti-wrinkle effect.

[0075] Example 5: Experimental results of the whitening effect of acGA-8-2

[0076] Using the melanoma B16 cell model, the cells were induced with 10 μM angiotensin II (ANGⅡ), and different concentrations of samples were added to treat the cells, namely 2 ppm acGA-8-2, 5 ppm acGA-8-2, 10 ppm acGA-8-2, and 4 μM 377 (phenylethyl resorcinol, Shanghai Yuanshang Science and Technology Co., Ltd.).

[0077] Fig.11 The experimental results of (left) melanin content and (right) tyrosinase activity are shown. It can be seen that: (left) ANGⅡ group significantly increased melanin content, 5ppm acGA-8-2 and 10ppm acGA-8-2 significantly reduced the melanin content after ANGⅡ induction, indicating the whitening effect of acGA-8-2. (right) ANGⅡ group significantly increased tyrosinase activity, 5ppm acGA-8-2 and 10ppm acGA-8-2 significantly reduced the tyrosinase activity after ANGⅡ induction, indicating the whitening effect of acGA-8-2.

[0078] Example 6: In vitro relief test

[0079] Using the RAW264.7 cell model, the cells were induced with 10 μM angiotensin II (ANGⅡ), and different concentrations of samples were added to treat the cells, namely 2 ppm acGA-8-2, 5 ppm acGA-8-2, and 10 ppm acGA-8-2.

[0080] Fig.12 The relevant experimental results are shown: (left) IL-6 content; (right) TNF-α content;

[0081] (Left) The ANGⅡ group significantly increased the inflammatory factor IL-6 content, and 2ppm acGA-8-2, 5ppm acGA-8-2, and 10ppm acGA-8-2 all significantly reduced the inflammatory factor IL-6 content induced by ANGⅡ, indicating the soothing effect of acGA-8-2. (Right) The ANGⅡ group significantly increased the TNF-α content, and 2ppm acGA-8-2, 5ppm acGA-8-2, and 10ppm acGA-8-2 all significantly reduced the TNF-α content induced by ANGⅡ, also indicating the soothing effect of acGA-8-2.

[0082] Example 7: In vitro antioxidant test

[0083] Using the RAW264.7 cell model, 10μM angiotensin II (ANGⅡ) was used to induce the cells, and different concentrations of samples were added to treat the cells, namely 2ppm acGA-8-2 and 5ppm acGA-8-2. The experimental results showed (see Fig.13 ):ANGⅡ group significantly increased the ROS fluorescence intensity, and 2ppm acGA-8-2 and 5ppm acGA-8-2 both reduced the ROS fluorescence intensity after ANGⅡ induction, indicating the antioxidant effect of acGA-8-2.

[0084] Example 8: Cell energy promotion test

[0085] Using the HFF-1 cell model, the cells were induced with 10 μM angiotensin II (ANGⅡ), and different concentrations of samples were added to treat the cells, namely 10 μM candesartan, 2 ppm acGA-8-2, and 5 ppm acGA-8-2.

[0086] The experimental results show that (see Fig.14 ): (Left) ANGⅡ group significantly inhibited AND+ level, 2ppm acGA-8-2, 5ppm acGA-8-2 significantly promoted NAD+ level after ANGⅡ induction, indicating that acGA-8-2 promotes cell energy. (Right) ANGⅡ group significantly inhibited AMPK, 2ppm acGA-8-2, 5ppm acGA-8-2 significantly promoted AMPK level after ANGⅡ induction, indicating that acGA-8-2 promotes cell energy, and has the effect of accelerating skin renewal, strengthening skin barrier, and effectively stimulating keratinocyte formation and mitochondrial metabolism, enhancing skin defense.

[0087] The above description is only a preferred embodiment, which is only used as an example and does not limit the combination of features necessary to implement the present invention. The titles provided are not intended to limit the various embodiments of the present invention. Terms such as "comprising", "including" and "including" are not intended to be limiting. In addition, unless otherwise specified, plural forms are included when there is no numeral modification, and "or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

Claims

1. A bioactive polypeptide for use in the preparation of a medicine or cosmetic having the effects of improving sub-healthy skin conditions, increasing skin gloss, reducing roughness, reducing the area of ​​pigmentation and red areas, reducing crow's feet, nasolabial folds and fine lines under the eyes, increasing skin elasticity and firmness, anti-wrinkle and firming, accelerating skin renewal, brightening skin tone, improving elasticity, soothing sensitivity, strengthening skin barrier and enhancing skin defense, wherein the amino acid sequence of the bioactive polypeptide is as shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the derivative of the biologically active polypeptide is a polypeptide derivative obtained by modifying the amino acid side chain group, amino terminus or carboxyl terminus of the biologically active polypeptide according to claim 1 by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation, while not affecting its biological activity.

3. A pharmaceutical composition comprising the bioactive peptide according to any one of claims 1 to 2 and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant.

4. The pharmaceutical composition of claim 3, wherein the excipient comprises: Hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyethylene glycol, polyacrylamide, polyacrylic acid, polyvinyl alcohol, dextran, ammonia, lactic acid, citric acid, pyrrolidone carboxylic acid, glycerin, urea, sorbitol, amino acids, lanolin, petrolatum.

5. The pharmaceutical composition of claim 4, wherein the adjuvant comprises: Solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, flavoring agents, surfactants, water, ionic or non-ionic emulsifiers, fillers, chelating agents, sequestrants, preservatives, vitamins, retardants, wetting agents, essential oils, dyes, pigments.

6. A method for preparing the bioactive polypeptide according to any one of claims 1 to 5, comprising synthesizing it by genetic engineering, and obtaining it from cells by separation and purification or by chemical synthesis.

7. The use according to any one of claims 1-2, wherein the content of the bioactive polypeptide is selected from: 1-100 ppm, preferably 2ppm, 5ppm, 10ppm, 15ppm, 20ppm, 25ppm, 30ppm, 35ppm, 40ppm, 45ppm, 50ppm, 55ppm, 60ppm, 65ppm, 70ppm, 75ppm, 80ppm, 85ppm, 90ppm, 95ppm, 100ppm; most preferably 15ppm.

8. The pharmaceutical composition of any one of claims 3-5, wherein the content of the bioactive polypeptide is selected from: 1-100 ppm, preferably 2 ppm, 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm, 40 ppm, 45 ppm, 50 ppm, 55 ppm, 60 ppm, 65 ppm, 70 ppm, 75 ppm, 80 ppm, 85 ppm, 90 ppm, 95 ppm, 100 ppm; most preferably 15 ppm.

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