Composition for inhibiting functions of various types of matrix metalloproteinases in skin and use thereof

Abstract

A composition for inhibiting functions of various types of matrix metalloproteinases in skin and a use thereof. The composition comprises one or more of the following components: epigallocatechin gallate (EGCG), dihydroquercetin, sugarcane polyphenol, coix seed powder, ginseng, or galangal. The composition has the effects of inhibiting expression of various types of matrix metalloproteinases in skin fibroblasts, inhibiting activity of matrix metalloproteinases, improving collagen content, and inhibiting elastase, and does not have obvious side effects. The composition can be used for preparing various cosmetics, functional foods and health care products related to skin aging resistance, and has a wide application prospect.

Description

Composition for inhibiting functions of various types of matrix metalloproteinases in skin and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese application No. 202311308518.7, filed on October 11, 2023. Said application No. 202311308518.7 is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention belongs to the technical field of cosmetics, and in particular relates to a composition for inhibiting the functions of various types of matrix metalloproteinases in the skin and its use. Background Art

[0004] The aging of the skin's deeper layers is closely linked to the loss of collagen. Collagen makes up 80% of the dermis and forms a reticular support structure with a strong ability to lock in moisture. Macroscopically, there are many factors that contribute to collagen degradation, including age, ultraviolet rays, unhealthy lifestyle habits and stress, environmental pollution, and electronic radiation. These external and internal factors can all contribute to collagen loss. A decrease in collagen levels in the skin can cause dryness, roughness, and sagging, leading to wrinkles. It can also enlarge pores, accompanied by a loss of radiance, dark spots, and other signs of aging. In addition to collagen, elastin also plays a supporting role in the skin. A decrease in elastin in the skin can lead to a loss of elasticity. Elastin helps slow and reduce sagging, and the loss or lack of elastin can affect the skin's appearance.

[0005] Matrix metalloproteinase (MMP) is a class of proteins containing Zn 2+ and is dependent on Ca 2+ A class of enzymes that degrade the extracellular matrix, particularly collagen, leading to skin aging. Therefore, the current mainstream view is that MMPs induce wrinkles, sagging skin, and dilation of microvessels, and that the production of matrix metalloproteinases increases with age. Twenty-five subtypes of human MMPs have been discovered, which can be divided into collagenases (MMP1, MMP8, MMP13, MMP18), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP10, MMP11), matrix dissolving factors (MMP7, MMP26), and membrane-type matrix metalloproteinases (MMP14, MMP15 to MMP17, MMP24, MMP25), based on their structure and substrate specificity. In addition, there are other MMPs (MMP12, MMP19 to MMP23, MMP27, MMP28).

[0006] Current research on MMP inhibition is primarily based on single substances, most of which are derived through high-throughput screening. These substances may have high production costs or strong toxic side effects, making them unsuitable for use as cosmetics or functional food ingredients.

[0007] In view of this, the inventors screened and compounded a large number of medicinal and edible substances and functional food raw materials, and thus obtained a composition that simultaneously inhibits the expression of multiple types of matrix metalloproteinases in skin fibroblasts, inhibits the activity of matrix metalloproteinases, increases collagen content, and inhibits elastase.

[0008] Summary of the Invention

[0009] In order to overcome the defects of the prior art and enrich the prior art, the present invention provides a composition and its use that can simultaneously inhibit the expression of multiple types of matrix metalloproteinases in skin fibroblasts, inhibit the activity of matrix metalloproteinases, increase collagen content, and inhibit elastase.

[0010] Specifically, the present invention is achieved through the following technical solutions:

[0011] In a first aspect, the present invention provides a composition for inhibiting the functions of multiple types of matrix metalloproteinases in the skin, the composition comprising one or more of the following components: epigallocatechin gallate (EGCG), dihydroquercetin, sugarcane polyphenols, coix seed powder, ginseng or galangal.

[0012] As an optional mode, in the above composition, the composition consists of the following components: EGCG, dihydroquercetin, sugarcane polyphenols, coix seed powder, ginseng and galangal.

[0013] As an optional manner, in the above composition, the type of matrix metalloproteinase is selected from one or more of the following: MMP1, MMP8, MMP13, MMP18, MMP2, MMP9, MMP3, MMP10, MMP11, MMP7, MMP26, MMP14, MMP15, MMP16, MMP17, MMP24, MMP25, MMP12, MMP19, MMP20, MMP21, MMP22, MMP23, MMP27 or MMP28.

[0014] As an optional mode, in the above composition, the type of the matrix metalloproteinase is one or more of the following: MMP1, MMP2, MMP3 or MMP8.

[0015] As an optional mode, in the above composition, the composition inhibits the expression, secretion and / or activation of matrix metalloproteinases, and the composition promotes collagen secretion and inhibits elastase activity.

[0016] As an optional method, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is (0.2-1.2) μM: (2-10) μM: (0.2-28) μg / mL: (0.01-20) μg / mL: (0.05-2) μg / mL: (0.01-1) μg / mL.

[0017] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 0.50 μM: 4.00 μM: 12.00 μg / mL: 8.00 μg / mL: 0.80 μg / mL: 0.01 μg / mL.

[0018] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 0.31 μM: 8.34 μM: 25.01 μg / mL: 16.67 μg / mL: 1.67 μg / mL: 0.02 μg / mL.

[0019] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 1.04 μM: 2.50 μM: 25.01 μg / mL: 16.67 μg / mL: 1.67 μg / mL: 0.02 μg / mL.

[0020] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 1.04 μM: 8.34 μM: 25.01 μg / mL: 5.00 μg / mL: 1.67 μg / mL: 0.02 μg / mL.

[0021] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 1.04 μM: 8.335 μM: 0.834 μg / mL: 0.03 μg / mL: 0.167 μg / mL: 0.834 μg / mL.

[0022] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 0.625 μM: 5 μM: 0.5 μg / mL: 0.02 μg / mL: 0.1 μg / mL: 0.5 μg / mL.

[0023] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 0.5 μM: 4 μM: 0.4 μg / mL: 0.016 μg / mL: 0.08 μg / mL: 0.4 μg / mL.

[0024] As an optional manner, in the above composition, the dosage ratio of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal is 16.67 μM: 20 μM: 200 μg / mL: 520.83 μg / mL: 1.67 μg / mL: 0.19 μg / mL.

[0025] In a second aspect, the present invention provides use of the composition of the first aspect in preparing an anti-aging product.

[0026] As an optional mode, in the above use, the anti-aging includes alleviating or preventing skin aging, cell aging or photoaging.

[0027] As an alternative, in the above use, the product is an oral product or an external use product, and the product is a cosmetic, a functional food, a beverage or a nutritional supplement.

[0028] As an optional mode, in the above-mentioned use, the product is an oral solution, liquid suspension, injection, tablet, pill, granule, powder, film, capsule, spray, aerosol, syrup, beverage, nutritional product, snack, energy bar, chewing gum, candy, facial mask, facial cream, essence, lotion, toner or makeup.

[0029] As an optional mode, in the above-mentioned use, the product further comprises auxiliary materials or excipients commonly used in cosmetics, functional foods, beverages or nutritional supplements, and the amount of the composition in the product is 0.5%-20% by weight.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The inventors screened and compounded a large number of medicinal and edible substances and functional food raw materials, and for the first time obtained a composition that can inhibit the expression of multiple types of matrix metalloproteinases in skin fibroblasts, inhibit the activity of matrix metalloproteinases, increase collagen content, inhibit elastase, and has no obvious side effects.

[0032] (2) Compared with the prior art, the present invention is a combination of multiple substances. Compared with the existing single substances, the composition of the present invention has multiple targeting effects on multiple types of MMP (collagenase, gelatinase, stroma lytic enzyme), which is manifested as an inhibitory effect on the expression of MMP1, MMP2, MMP3 and MMP8 proteins in fibroblasts, and a significant inhibitory effect on the activity of MMP2 enzyme, a protective effect on the reduction of collagen simulated by external stimulation, and at the same time, it can significantly inhibit the activity of skin elastase. The innovation of the present invention is mainly reflected in the function of the combination of EGCG, dihydroquercetin, coix seed powder, ginseng, sugarcane polyphenols and galangal in inhibiting the activity of skin elastase. It comprehensively emphasizes the role of the various combinations of the six substances in inhibiting MMP proteins, protecting collagen loss, inhibiting MMP2 enzyme activity, and inhibiting elastase, and further emphasizes the role of the combination of the above six substances in inhibiting skin aging.

[0033] (3) The composition of the present invention can be used to prepare various cosmetics, functional foods and health products related to anti-skin aging, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: Toxicity test of single substances on mouse skin fibroblasts.

[0035] Figure 2: Toxicity test of different combinations of several substances on mouse skin fibroblasts.

[0036] Figure 3: Inhibition rates of single substances and combined ratios on the expression of multiple MMP proteins.

[0037] Figure 4: Effects of single substances and combinations on the hydroxyproline content secreted after cell stimulation.

[0038] Figure 5: Inhibition rate of MMP2 enzyme activity by single substance and combination ratio.

[0039] Figure 6: Inhibition rate of elastase activity by single substances and combination ratios. DETAILED DESCRIPTION

[0040] In the above summary of the invention and detailed description, as well as in the claims, specific features of the present invention are mentioned. It should be understood that all possible combinations of these specific features are disclosed in the specification of the present invention. For example, when a specific feature is disclosed in a specific aspect or embodiment of the present invention or in a specific claim, this feature can also be used as much as possible and / or in combination with other specific aspects and embodiments of the present invention.

[0041] Skin aging is a complex process. Macroscopically, it manifests as fine lines, loss of elasticity, orange peel or dull skin, and decreased epidermal and dermal thickness. Microscopically, typical features of skin aging include epidermal atrophy, decreased basal keratinocyte mitotic rate, decreased proliferation and cellular senescence, atrophy of the dermal extracellular matrix, and altered connective tissue physiology.

[0042] The following is a brief description of the several active ingredients involved in the present invention:

[0043] The present invention provides six substances capable of inhibiting MMP function, and provides a compound formulation containing one or more combinations of these six substances. The combination of these six substances significantly enhances the inhibitory effect on MMP function. These six substances include the medicinal and edible substance coix seed powder; the Chinese medicinal herbs ginseng and galangal; the polyphenols EGCG (epigallocatechin gallate) and sugarcane polyphenols; and the flavonoid dihydroquercetin (taxifolin).

[0044] As used herein, “EGCG (Epigallocatechin gallate)” is the main component of tea polyphenols, isolated from green tea, and has a molecular formula of C 22 H 18 O 11 , with multiple effects such as antibacterial, antiviral, and antioxidant. Due to its unique chemical structure, EGCG has a very strong antioxidant effect and a strong ability to scavenge oxygen free radicals. EGCG can inhibit hyaluronidase and thus inhibit the degradation of hyaluronic acid. EGCG can also inhibit the increase in the mRNA expression level of matrix metalloproteinases in human skin fibroblasts induced by UVA. The structural formula of EGCG is:

[0045] As used herein, “dihydroquercetin”, also known as quercetin, CAS registration number: 480-41-1, its chemical name is 3,3',4',5,7-pentahydroxyflavone, chemical formula: C 15 H 10O7. Quercetin is a flavonol compound that is widely distributed in the plant kingdom and has multiple biological activities. Quercetin is also found in many foods, such as onions, apples, mangoes, etc. In addition, about 100 kinds of medicinal plants (such as Sophora japonica, Platycladus orientalis leaves, Alpinia officinalis, Coltsfoot flower, Mulberry parasite, Panax notoginseng, Ginkgo biloba, Elderberry, etc.) contain this ingredient. Quercetin is a polyhydroxyflavonoid compound with antioxidant and free radical scavenging effects. Studies have shown that quercetin can promote the proliferation of human skin fibroblasts and stimulate their secretion of collagen and hyaluronic acid, which is of great significance for maintaining skin elasticity, reducing skin wrinkle formation, and delaying aging. Quercetin is non-toxic, therefore, it is of great significance for the treatment and prevention of cancer, aging, and cardiovascular diseases, and has great development value. The structural formula of dihydroquercetin is:

[0046] As used in this article, "Job's tears powder" is an ancient substance with medicinal and edible properties. Job's tears powder mainly contains rich nutrients, vitamins and esters, including proteins, fatty acids, carbohydrates, sugars, a small amount of vitamin B1, leucine, lysine, arginine, tyrosine, as well as compounds such as coixol, coix ester, coix lactone, α-β-sitosterol, triterpenes, etc. Job's tears powder is also rich in calcium, phosphorus, potassium, selenium, phosphorus, sodium, magnesium and other trace elements essential to the human body. Job's tears powder can prevent hyperlipidemia, hypertension, stroke, cardiovascular disease and heart disease. Job's tears powder is rich in water-soluble fiber, which can absorb bile salts, which are responsible for digesting fat. Therefore, after taking job's tears powder, the intestinal absorption rate of fat becomes worse, which can lower blood lipids and blood sugar. Job's tears powder can promote the metabolism of blood and water in the body, has diuretic and anti-edema effects, and can help defecation and help lose weight. Job's tears powder, rich in protein, can lighten skin spots and brighten the skin. It can be drunk or used as a facial mask. However, no research has shown that Job's tears powder can affect the function of MMPs in the skin.

[0047] As used herein, "ginseng" refers to the root of the Araliaceae plant Panax ginseng, which contains over 100 active ingredients and is suitable for treating vital energy deficiency. Consuming ginseng can nourish the spleen and lungs, promote the production of body fluids, and quench thirst. Ginseng also has a calming effect. Ginsenosides are the primary active substances in ginseng, possessing anti-aging and anti-infective properties. Combined with ginseng's polysaccharides, minerals, and vitamins, these nutrients effectively combat cholesterol, aid metabolism, promote blood circulation in subcutaneous microvessels, and increase the skin's nutrient supply. Therefore, ginseng can delay skin aging and make the skin appear rosy and lustrous. Currently, there are skin care products containing ginseng extract, which can achieve anti-photoaging effects by regulating the MAPK and NF-κB pathways in human fibroblasts. Ginseng extract also has anti-inflammatory properties and can activate cells, significantly increasing skin moisture content and elasticity, and reducing wrinkles.

[0048] As used in this article, "sugarcane polyphenols" are made from sugarcane, which is squeezed, filtered, extracted, and the excess sugar and salt are removed to obtain sugarcane molasses, and then water and ethanol are used as extraction solvents. It is filtered, vacuum concentrated, ion exchanged and spray dried to form a powder; or it is a liquid substance made by filtering, vacuum distillation and other processes. The main characteristic component is total polyphenols. The National Health Commission's Announcement No. 2 of 2022 officially defined sugarcane polyphenols as a "new food raw material." Sugarcane polyphenols can be antioxidant, have strong antioxidant and free radical scavenging effects, and are also antimicrobial and have anti-inflammatory effects. Sugarcane polyphenols have inhibitory effects on enzymes related to skin aging and can promote the synthesis of collagen.

[0049] As used herein, "galangal" refers to the rhizome of the ginger plant, Alpinia officinalis, which regulates gastrointestinal function, nourishes the spleen and warms the stomach, and is used to treat loss of appetite and indigestion. It also has anti-inflammatory and analgesic properties, as well as antibacterial and antifungal properties. It significantly promotes hematopoiesis. Some skincare products contain galangal extract, which has been shown to improve skin elasticity and improve skin sagging.

[0050] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0052] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0053] Example:

[0054] Since collagen in the skin is mainly produced and secreted by fibroblasts, making the skin more elastic and resilient, the present invention uses mouse skin fibroblasts for experiments. The present invention detects whether treating skin fibroblasts with several substances can inhibit the expression of MMP proteins in skin fibroblasts. Based on the results, the present invention combines and proportions the substances and then detects the inhibitory effect of MMP protein expression in skin fibroblasts.

[0055] The experimental materials were C57BL / 6J P0 mice. The processing method was to remove the head, tail and limbs, leaving the trunk, and cut the mouse from the neck along the hip with surgical scissors to obtain skin tissue. The skin tissue was digested with skin tissue digestion fluid and mouse skin fibroblasts were isolated.

[0056] 1. Toxicity test of 6 substances on mouse skin fibroblasts:

[0057] EGCG (purchased from Zhejiang Tiancao Biotechnology Co., Ltd., Lot 202210121), dihydroquercetin (purchased from Jilin Jianwei Natural Biotechnology Co., Ltd., Lot 2308002), coix seed powder (purchased from Huangshan Hualvyuan Biotechnology Co., Ltd., Lot 220318), ginseng (purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd., Lot 20220601), sugarcane polyphenols (purchased from Shaanxi Baichuan Kangze Biotechnology Co., Ltd., Lot BCKZ221109-2) and galangal (purchased from Yulin Shengding Juyuan Chinese Herbal Medicine Co., Ltd., Lot 20221216) were prepared according to the concentrations in Table 1 below.

[0058] Table 1: Substance concentration table

[0059] In addition, the 6 substances were combined in different ways to evaluate their toxicity to mouse skin fibroblasts. The specific concentrations and ratios are shown in Table 2 below.

[0060] Table 2: Combination of 6 substances

[0061] 10,000 mouse skin fibroblasts were seeded in a 96-well cell culture plate. The next day, the cells were treated for 18 hours according to the protocol described in Tables 1 and 2. Since ATP (adenosine triphosphate) is a constant substance within cells and the amount of endogenous ATP reflects cell activity, intracellular ATP levels were measured and used as an indicator to evaluate the cytotoxicity of the above substances, both individually and in combination. The results are shown in Figures 1 and 2. Concentrations that showed no toxicity or less than 80% toxicity to skin fibroblasts were selected for subsequent experiments.

[0062] 2. Six substances have inhibitory effects on the expression of multiple types of MMP proteins:

[0063] Mouse skin fibroblasts were inoculated into culture dishes and cultured for 24 hours. A control group and a model group were selected to establish a model of increased expression of MMP protein in skin fibroblasts using doxorubicin. Doxorubicin is an antibiotic drug with a chemical formula of C 27 H 29 NO 11It binds to nucleic acids by inserting into the DNA double helix and stabilizing the topoisomerase II cleavage activation complex, resulting in DNA chain breaks at specific doxorubicin-induced sites. Doxorubicin can inhibit the synthesis of RNA and DNA and is often used to establish cell damage or cell aging models.

[0064] The experimental groups treated skin fibroblasts with various concentrations of the six substances shown in Table 3 below for 18 hours. The cells were then collected, and protein was extracted for Western blotting. The present invention comprehensively detects multiple types of MMPs, including collagenases MMP1 and MMP8, gelatinase MMP2, and stroma lytic enzyme MMP3. The success of the model group can be determined by comparing it with the control group. If MMP protein expression in the model group is significantly increased compared to the control group, the model is successfully established; otherwise, it is unsuccessful. Simultaneously, the experimental groups were compared to determine whether MMP protein expression was reduced to determine whether the six substances had an inhibitory effect on MMP protein expression.

[0065] Three concentrations were set for each of the six substances from low to high, and the expression levels of MMP1, MMP2, MMP3 and MMP8 proteins were detected respectively.

[0066] The results showed that the six substances had different inhibitory effects on MMP1, MMP2, MMP3, and MMP8. Some of the six substances had stronger inhibitory effects on certain MMPs, weaker inhibitory effects on others, and some concentrations of the six substances had no inhibitory effects on any MMPs (see Table 3). The strongest inhibitory effect on MMP1 was achieved with 150 μg / mL sugarcane polyphenol, while the strongest inhibitory effect on MMP2 was achieved with 10 μg / mL ginseng, the strongest inhibitory effect on MMP3 was achieved with 10 μg / mL ginseng, and the strongest inhibitory effect on MMP8 was achieved with 150 μg / mL sugarcane polyphenol.

[0067] In order to evaluate the comprehensive effects of the six substances on multiple types of MMPs in skin fibroblasts, the inhibitory efficiency of each substance at each concentration on multiple MMPs was averaged to obtain the optimal inhibitory concentration of each substance.

[0068] The results showed that in the designed concentration gradient, 6.25 μM EGCG, 50 μM dihydroquercetin, 100 μg / mL coix seed powder, 10 μg / mL ginseng, 150 μg / mL sugarcane polyphenols, and 0.125 μg / mL galangal had a higher average inhibition rate on MMP protein, so this concentration was selected as the concentration for subsequent experiments.

[0069] Table 3: Inhibition rates of 6 substances at different concentrations on the expression of 4 MMP proteins in skin fibroblasts

[0070] 3. The combination of the six substances has a significant inhibitory effect on MMP protein expression:

[0071] Mouse skin fibroblasts were seeded in culture dishes, and a control group (control) and a model group (doxorubicin) were set up. Six substances (EGCG, dihydroquercetin, coix seed powder, ginseng, sugarcane polyphenols, and galangal) were added at optimal concentrations according to the concentrations listed in Table 4 below. The cells were cultured for 18 hours, after which the cells were harvested, proteins extracted, and Western blotting was performed. Comparison with the model group allowed evaluation of whether the combination of these six substances had an inhibitory effect on MMP protein expression, and whether the combined combination exhibited significant inhibitory efficacy compared to a single substance alone.

[0072] Table 4: Combination of 6 substances

[0073] The single substance group and the combination compound data were subjected to normal distribution test and variance homogeneity test, and then T test was performed. The P value of the two groups of data was less than 0.05, indicating that there was a significant difference, which is indicated by * in the figure. The P value was less than 0.01, indicating that there was an extremely significant difference, which is indicated by ** in the figure.

[0074] The results are shown in Figure 3. The results show that the combination of the six substances has a significant inhibitory effect on the expression of MMP1, MMP2, MMP3, and MMP8 proteins in mouse skin fibroblasts. The inhibition rate of the six-substance combination group on the expression of MMP1, MMP2, and MMP3 proteins is significantly higher than that of the single substance group. The combination group has a significant difference in the expression of MMP8 protein compared with the single substance group. Among the several combinations of the six substances, combination 3 has a stronger inhibitory effect on multiple MMPs than other combination methods. In addition, compared with the broad-spectrum MMP inhibitor GM6001 (Med Chem Express, HY-15768), the substance combination of the composition of the present invention shows a more significant inhibitory effect.

[0075] 4. Detection of the effects of 6 single substances and 6 combinations of substances on cell collagen secretion:

[0076] Mouse skin fibroblasts were inoculated into culture dishes, and a control group (Control) and a model group (doxorubicin) were set up. 6.25 μM EGCG, 50 μM dihydroquercetin, 100 μg / mL coix seed powder, 10 μg / mL ginseng, 150 μg / mL sugarcane polyphenols, and 0.125 μg / mL galangal were added, respectively, and compounded according to Table 4. The cells were cultured for 22 hours, and the culture medium was taken to detect the content of collagen secreted by the cells.

[0077] Since hydroxyproline is a hallmark amino acid in collagen, the hydroxyproline content in the culture medium was selected to calibrate the amount of total collagen. The results are shown in Figure 4. Through detection, it was found that the amount of collagen secreted into the culture medium by the model group (doxorubicin, 3μM) was significantly reduced compared with the control group. After adding EGCG, coix seed powder, ginseng and galangal, the amount of collagen secreted by the cells was higher than that of the model group. After adding the six substances in different ways of compounding, the amount of collagen secreted by the cells was also higher than that of the model group. Among them, the collagen content of combination 4, combination 5 and combination 6 was higher than that of a single substance. The data of the six-substance compound group and the single substance group were subjected to normal distribution and T test. The results showed that the compound group was significantly higher than the single drug group. This shows that the six-substance compound combination 4, combination 5 and combination 6 can significantly restore the decrease in the content of collagen secreted by cells due to stimulation, that is, the compound combination 4, combination 5 and combination 6 can enhance the content of collagen.

[0078] 5. Detection of the inhibitory effects of six single substances at various concentrations and six combinations of substances on MMP2 enzyme activity:

[0079] The above test results prove that EGCG, dihydroquercetin, coix seed powder, ginseng, sugarcane polyphenols, and galangal all have inhibitory effects on the expression of multiple matrix metalloproteinases (MMPs). Based on the above results, it is speculated that the above six substances may have inhibitory effects on the enzyme activity of MMPs, so the MMP2 protease activity was tested.

[0080] The individual substances were tested at varying concentrations, from low to high, to verify that they inhibited MMP2 enzyme activity to varying degrees. Appropriate concentrations of the six substances were selected for combination (Table 5), and the selected concentrations of the individual substances were also tested. The results are shown in Figure 5. The results show that three combinations of the six substances had a significant inhibitory effect on MMP2 enzyme activity, with inhibition rates as high as 57%-65%. Statistical analysis of the data from the combined and single substance groups was performed, and a T-test was performed after normal distribution tests and homogeneity of variance tests. The statistical P value was less than 0.01, indicated by ** in the figure, indicating that the combined group had a more significant inhibitory effect on MMP2 enzyme activity than the single substance group.

[0081] Table 5: Combination ratio of 6 substances used to detect MMP2 enzyme activity

[0082] 6. Detection of the inhibitory effects of six single substances at various concentrations and six combinations of substances on elastase activity:

[0083] Elastase, also known as elastase, is an enzyme from the protease class in molecular biology that can break down elastin. Elastin is an elastic fiber that, together with collagen, determines the properties of connective tissue and can maintain the toughness and firmness of the skin. High collagen content in the skin makes the skin plumper and firmer, but a lack of collagen can make the skin less elastic. The present invention detected the effects of six single substances and six combinations of substances on elastase activity, which can then reflect the content of elastin in the skin.

[0084] The present invention designed different concentrations of six single substances to test their inhibitory effects on elastase. The concentration with the strongest inhibitory effect on elastase was selected to form a combination of the six substances, which was designated as combination 13 (the dosage ratios of EGCG: dihydroquercetin: sugarcane polyphenols: coix seed powder: ginseng: galangal were 16.67 μM: 20 μM: 200 μg / mL: 520.83 μg / mL: 1.67 μg / mL: 0.19 μg / mL). The results are shown in FIG6 . The experimental results show that among the six substances, the concentrations with the highest inhibitory effects on elastase activity were 100 μM EGCG, 120 μM dihydroquercetin, 3125 μg / mL coix seed powder, 10 μg / mL ginseng, 1200 μg / mL sugarcane polyphenols, and 1.125 μg / mL galangal. The combination of the six substances has a significantly higher inhibitory effect on elastase activity than a single substance, indicating that the combination of the six substances in the present invention has a strong inhibitory effect on elastase and can reduce the degradation of elastin in skin fibroblasts (CAS number of the positive inhibitor is 70967-90-7).

[0085] In summary, the following conclusions can be drawn from the effect experiments: (1) The composition of the present invention is non-toxic to cells within the selected concentration range; (2) The present invention first discovered that the single substances sugarcane polyphenols and dihydroquercetin in the composition of the present invention can inhibit the function of skin MMP; (3) The combination ratio of substances in the composition of the present invention has a significant inhibitory effect on the increased expression of multiple MMP proteins in skin fibroblasts, and is stronger than that of a single substance; (4) The combination ratio of substances in the composition of the present invention has a significant inhibitory effect on the activity of MMP2 enzyme; (5) The composition of the present invention can significantly inhibit the decrease in collagen content caused by stimulation; (6) There is currently no literature mentioning that EGCG, dihydroquercetin, coix seed powder, ginseng, sugarcane polyphenols and galangal in the composition of the present invention can inhibit the activity of skin elastase; (7) The combination ratio of substances in the composition of the present invention has a significant inhibitory effect on the activity of skin elastase, and is significantly better than that of a single substance; (8) The composition of the present invention provides a natural and safe formula that can inhibit the degradation of collagen and elastin in skin fibroblasts.

[0086] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A composition for inhibiting the functions of various types of matrix metalloproteinases in the skin, characterized in that: The composition comprises epigallocatechin gallate, dihydroquercetin, sugarcane polyphenols, coix seed powder, ginseng and galangal.

2. The composition according to claim 1, characterized in that: The composition consists of the following components: epigallocatechin gallate, dihydroquercetin, sugarcane polyphenols, coix seed powder, ginseng and galangal.

3. The composition according to claim 1, characterized in that: The type of matrix metalloproteinase is selected from one or more of the following: MMP1, MMP8, MMP13, MMP18, MMP2, MMP9, MMP3, MMP10, MMP11, MMP7, MMP26, MMP14, MMP15, MMP16, MMP17, MMP24, MMP25, MMP12, MMP19, MMP20, MMP21, MMP22, MMP23, MMP27 or MMP28.

4. The composition according to claim 3, characterized in that: The types of matrix metalloproteinases are MMP1, MMP2, MMP3 and MMP8.

5. The composition according to claim 1, characterized in that: The composition inhibits the expression, secretion and / or activation of matrix metalloproteinases, and the composition promotes collagen secretion and inhibits the activity of elastase.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of anti-aging products.

7. The use according to claim 6, characterized in that: The anti-aging includes alleviating or preventing skin aging, cell aging or photoaging.

8. The use according to claim 6, characterized in that: The product is an oral product or an external product, and the product is a cosmetic, a functional food, a beverage or a nutritional supplement.

9. The use according to claim 6, characterized in that: The product is an oral solution, liquid suspension, injection, tablet, pill, granule, powder, film, capsule, spray, aerosol, syrup, beverage, nutritional supplement, snack, energy bar, chewing gum, candy, facial mask, facial cream, essence, lotion, toner or makeup.

10. The use according to claim 6, characterized in that: The product also contains auxiliary materials or excipients commonly used in cosmetics, functional foods, beverages or nutritional supplements. The amount of the composition in the product is 0.5%-20% by weight.

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