Natural-source composition with function of inhibiting multiple types of matrix metalloproteinases of skin and application of natural-source composition

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a natural-source composition with a function of inhibiting multiple types of matrix metalloproteinases of skin and application of the natural-source composition. The composition is prepared from the following components: herba cistanche powder, American ginseng powder, a red-knees herb extract and a creeping rostellularia herb extract. The composition has the effects of inhibiting expression of multiple types of matrix metalloproteinases in skin immortalized keratinocytes, improving elastin synthesis and inhibiting elastase decomposition, and has no obvious side effects. The composition disclosed by the invention 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

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and in particular relates to a natural composition having the function of inhibiting various types of matrix metalloproteinases of the skin and its use. Background Art

[0002] It is well known that wrinkles in the aging skin are closely related to the loss of collagen. In fact, the elasticity and tension of human muscle tissue are not only related to collagen, but also to the role of elastin. Elastin plays a key supporting and elastic role in the skin, which helps maintain the structural integrity and elasticity of the skin. However, with age and the influence of external factors, elastin will gradually degrade, resulting in a decrease in skin elasticity and the appearance of wrinkles. This is mainly the result of the combined effects of natural aging and photoaging.

[0003] Photoaging mainly refers to aging caused by ultraviolet radiation. A large amount of ultraviolet radiation destroys the dynamic balance between elastin and elastase in the body. In addition, a large amount of ultraviolet radiation activates the expression and activity of matrix metalloproteinases (MMPs). Matrix metalloproteinases can degrade almost all molecules in the extracellular matrix, accelerate the degradation of elastin, etc., cause skin tissue aging, and ultimately manifest as decreased skin elasticity and wrinkle formation.

[0004] Matrix metalloproteinase (MMP) is a class of proteins containing active Zn 2+ and depends on Ca 2+ Enzymes that can degrade structural proteins such as collagen and elastin, leading to aging symptoms on the skin. The mainstream view now is that MMPs will increase with age-dependent dependence, leading to an increased risk of inducing wrinkles, sagging skin, and dilation of microvessels. Inhibiting the activity of MMPs can effectively prevent skin aging. There are 25 subtypes of human MMPs that have been discovered so far. According to their structural characteristics, they can be divided into collagenases (MMP-1, MMP-8, MMP-13, MMP-18), gelatinases (MMP-2, MMP-9), matrix hydrolases (MMP-3, MMP-10, MMP-11), matrix lysins (MMP-7, MMP-26) and membrane-type MMPs (MMP-14, MMP-15 to MMP-17, MMP-24, MMP-25). In addition, there are other MMPs (MMP-12, MMP-19 to MMP-23, MMP-27, MMP-28), and their substrates are not the same.

[0005] At present, the research on inhibiting the function of matrix metalloproteinase is basically based on a single substance, and most of them are chemical substances obtained by high-throughput screening experiments. However, these single chemical synthesis inhibitors may have side effects, such as causing inflammatory reactions, allergic reactions, etc., and the production cost is generally high, so there is a problem that they are not suitable for use as cosmetics or functional food raw materials. In addition, the existing technology is insufficient in regulating elastin synthesis and metalloproteinase and elastase activity, and cannot achieve a comprehensive skin health care effect.

[0006] In view of this, the inventors screened and combined a large number of medicinal and edible substances and functional food raw materials, and thus obtained a composition that can simultaneously inhibit the activity of multiple types of matrix metalloproteinases in skin keratinocytes, increase elastin content, and inhibit the action of elastase. Summary of the invention

[0007] In order to overcome the defects of the prior art and enrich the prior art, the present invention provides a composition and its use which can simultaneously inhibit the expression of multiple types of MMP in skin keratinocytes, improve elastin synthesis, and inhibit the decomposition of elastase. The composition of the present invention comprehensively improves the skin elasticity and anti-aging ability, and the development and application of new raw materials and multi-component compositions can improve the utilization rate of raw materials, more effectively regulate the biological activity inside the skin, and bring innovative solutions to the field of skin health care.

[0008] Specifically, the present invention is realized through the following technical solutions: In a first aspect, the present invention provides a natural source composition having the function of inhibiting various types of matrix metalloproteinases in the skin, the composition comprising the following components: Cistanche deserticola powder, American ginseng powder, Polygonum aviculare extract and Acanthus monnieri extract.

[0009] As an optional mode, in the above composition, the composition is made of the following components: Cistanche deserticola powder, American ginseng powder, Polygonum aviculare extract and Acanthus monnieri extract.

[0010] As an optional manner, in the above-mentioned 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.

[0011] As an optional mode, in the above composition, the type of the matrix metalloproteinase is one or more of the following: MMP1, MMP2 or MMP9.

[0012] As an optional mode, in the above composition, the composition inhibits the expression of matrix metalloproteinases, improves elastin synthesis, and inhibits elastase decomposition.

[0013] As an optional mode, in the above composition, the usage ratio of Cistanche deserticola powder: American ginseng powder: Polygonum aviculare extract: Acanthus monnieri extract is 200-800 μg / mL: 200-800 μg / mL: 5-15 μg / mL: 5-15 μg / mL.

[0014] As an optional mode, in the above composition, the usage ratio of Cistanche deserticola powder: American ginseng powder: Polygonum aviculare extract: Acanthus monnieri extract is 500 μg / mL: 500 μg / mL: 10 μg / mL: 10 μg / mL.

[0015] In a second aspect, the present invention provides use of the composition of the first aspect in preparing an anti-aging product.

[0016] As an optional mode, in the above-mentioned use, the anti-aging includes alleviating or preventing skin aging, cell aging or photoaging.

[0017] As an optional mode, in the above use, 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.

[0018] 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, nutrient, snack, energy bar, chewing gum, candy, facial mask, facial cream, essence, lotion, toner or makeup.

[0019] 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.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention screens and compound a large number of medicinal and edible substances and functional food raw materials to obtain for the first time a composition that can inhibit the activity of multiple types of matrix metalloproteinases in skin keratinocytes, increase elastin content, inhibit the action of elastase, and has no obvious side effects.

[0021] (2) The composition of the present invention comprehensively improves skin elasticity and anti-aging ability. The development and application of new raw materials and multi-component compositions can improve the utilization rate of raw materials and more effectively regulate the biological activity inside the skin, bringing innovative solutions to the field of skin health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Inhibitory effects of three compositions of different concentrations on elastase.

[0023] Figure 2 : Effects of preferred compositions on extracellular matrix metalloproteins. DETAILED DESCRIPTION

[0024] Skin aging is a complex process. Macroscopically, skin aging is manifested by fine lines, loss of elasticity, orange peel or dull skin, and reduced thickness of the epidermis and dermis. Microscopically, typical features of skin aging include epidermal atrophy, decreased basal keratinocyte mitotic rate, decreased proliferation capacity and cell senescence, atrophy of the dermal extracellular matrix, and changes in the physiological properties of connective tissue.

[0025] The following is a brief description of several active ingredients involved in the present invention: The present invention provides four substances capable of inhibiting the function of MMP, and provides a combination of various concentrations of the four substances, wherein the combination of the four substances significantly enhances the inhibitory effect on the function of MMP. The four substances include Cistanche deserticola powder, American ginseng powder, Polygonum aviculare extract, and Acanthus monnieri extract.

[0026] As used herein, Cistanche powder is purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd., and is in the form of brown powder, rich in alkaloids, amino acids, minerals and trace elements. It can increase the proliferation reaction of lymphocytes, thereby enhancing the body's immune function. In addition, the phenylpropanol sugar contained in Cistanche powder is the most effective ingredient for delaying aging, and has a significant delaying effect on the aging of the pituitary gland, gonads, thymus and other parts of the human body.

[0027] As used herein, American ginseng powder was purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd. American ginseng is one of the ginseng species, and ginsenosides are the main active substances. Ginsenosides can prolong telomere length and promote telomerase activity during cell aging (see Lei Xiujuan, Feng Kai, Sun Liwei, et al. Research progress on the anti-aging mechanism of ginsenosides [J]. Amino Acids and Biological Resources, 2010, 32(01): 44-47). The ginsenoside content used in this article exceeds 15%, which has a strong anti-aging effect. In addition, American ginseng can lower blood sugar, regulate insulin secretion, promote sugar metabolism and fat metabolism, and has a certain auxiliary effect on the treatment of diabetes.

[0028] As used herein, the extract of Acanthus chinensis was purchased from Xi'an Sai'ao Biotechnology Co., Ltd. Acanthus chinensis is a herbaceous plant of the Acanthaceae family ( Rostellularia procumbens (L.) Nees ) is rich in alkaloids, acanthocyanins C, D, E and other active ingredients, and has antibacterial, antiviral, and anti-tumor effects. It is reported that acanthocyanins may achieve the effect of anti-chronic nephritis by inhibiting the expression of inflammatory proteins in the TLR4 / NF-κB pathway (see, Li Yue. Research on the effective substances of acanthocyanins against chronic nephritis and their mechanism of action [D]. Hubei University of Chinese Medicine, 2019.). However, no research has shown that acanthocyanins extract can affect the function of MMP in the skin.

[0029] As used herein, Polygonum multiflorum extract was purchased from Shaanxi Mufei Biotechnology Co., Ltd. Polygonum multiflorum is a plant of the Polygonaceae family ( Persicaria orientalis (L.) Spach ), the fruit of Polygonum multiflorum is generally used as a Chinese medicine, called water safflower seeds. It is reported that the components of Polygonum multiflorum extract, such as protocatechuic acid, quercetin, kaempferol-glucoside, quercetin, and kaempferol-rhamnoside, are absorbed throughout the intestine. In addition, Polygonum multiflorum extract can improve myocardial cell hypoxia-reoxygenation injury, and its mechanism of action may be related to inhibiting myocardial cell apoptosis, increasing ATPase activity, and protecting mitochondrial function (see, Li Jingliang, Li Ping, Chang Kun, et al. Progress in the Utilization of Medicinal Plant Polygonum multiflorum [J]. Agriculture and Technology, 2023, 43(09): 170-172). The effect of Polygonum multiflorum on resisting skin photoaging has not been reported.

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] 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.

[0038] Example: Example 1: Effects of single drugs and combinations on elastase activity Drug dilution: Dissolve Cistanche deserticola powder, American ginseng powder, Polygonum aviculare extract, and Acanthus monnieri extract in ultrapure water and prepare according to the concentrations shown in Table 1.

[0039] Table 1: Monomer and combination drug concentrations

[0040] Elastase assay: The EnzChek™ Elastase Assay Kit (purchased from Invitrogen, catalog number: E12056) was used in the test. According to the instructions, control wells and sample wells (including combinations 1, 2, 3 and corresponding concentrations of monomer drugs) were set up. 25 μL of elastin and 25 μL of enzyme were added to each test well, and 3 combinations and corresponding concentrations of single drugs were added to the sample wells. The remaining volume was filled with diluent, and the total system was 200 μL. After adding all reagents to the 96-well plate, they were quickly transferred to the microplate reader (protected from light). According to the kinetic detection, Ex / Em was 505 / 515 nm, the total time was set to 1 h, and a reading was taken every 3 min.

[0041] The test results are as follows Figure 1As shown, compared with the blank control group, the three different concentrations of the combination showed good elastase inhibition effects. Compared with the single administration, the inhibition of elastase by combination 1 was better than that of the four single administrations at the corresponding concentrations ( Figure 1 A), while the inhibitory effects of combination 2 and combination 3 on elastase were stronger than those of some single administrations, but their effects were not as good as those of American ginseng powder ( Figure 1 B and Figure 1 C). Therefore, through this experiment, the optimal combination for inhibiting elastase activity was screened out as combination 1 (Cistanche deserticola powder 500 μg / mL, American ginseng powder 500 μg / mL, Polygonum erythrorhizon extract 10 μg / mL, Acanthus monnieri extract 10 μg / mL) Example 2: Effect of the composition on the expression of extracellular matrix proteins in the UV-stimulated mouse skin keratinocyte (HaCaT) photoaging model 1. Cell inoculation: HaCaT cells were grown to the appropriate generation, digested and counted, and then plated at 2×10 6 / mL was inoculated into a 10 cm cell culture dish and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0042] 2. Cell administration: Based on the results of elastase activity, the experiment selected combination 1 in Table 1 and the corresponding concentration of monomer drugs for cell experiments. Specifically, it consists of the following 4 drugs, including 500 μg / mL of Cistanche deserticola powder; 500 μg / mL of American ginseng powder, 10 μg / mL of Polygonum aviculare extract, and 10 μg / mL of Acanthus extract. The experiment set up a blank group (CON), a UV modeling group, and a drug-adding group after modeling (combination 1 and a single drug). When the cells in the 10 cm dish grew to 80% confluence, 10 mL of culture medium containing the corresponding concentration of drugs was added to each dish of the modeling and sample groups, and 10 mL of DMEM complete medium was added to each dish of the blank group. Three replicate dishes were set up for each group; after the drug administration was completed, the 10 cm dish was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24 h.

[0043] 3. Protein extraction and denaturation: Remove the culture medium, wash twice with pre-cooled PBS solution, gently scrape the cells with a cell scraper, transfer to a 15 mL centrifuge tube, centrifuge (3000 rpm, 5 min) and discard the supernatant. Use 300 μL of PMSF cell lysis buffer (PMSF: RIPA = 1: 99) to blow the cells apart and transfer to a 1.5 mL centrifuge tube, lyse on ice for 20 min, and shake up and down vigorously every 10 min to complete the lysis. After the lysis is complete, centrifuge (14,000 r / min, 10 min) and then draw the supernatant into a new 1.5 mL centrifuge tube. The total protein content is determined by BCA kit (purchased from Beyotime). After mixing the qualified protein sample with 5x loading buffer, place it in a 100℃ metal bath for 5 min to denature the protein and store it at -20℃ for later use.

[0044] 4. Western blot test: (1) Electrophoresis: separate proteins of different molecular weights by using different concentrations (8%-15%) of precast gel (purchased from Sangon Biotechnology). After adding the protein sample, perform SDS-PAGE electrophoresis at a constant voltage of 80 V for 90 min; (2) Transfer: after the PVDF membrane is activated by methanol solution for about 2 min, transfer is performed at a constant current of 200 mA for 70 min-90 min (slightly adjusted according to the molecular weight of the protein); (3) Blocking: use protein-free rapid blocking solution (purchased from Yazyme, product number PS108P) to block the PVDF membrane at room temperature for 15 min. After blocking, wash 3 times with 1 x TBST buffer (purchased from Solebol, 10 x TBS is dissolved in 1L ddH2O, and 500 μL Tween-20 is added to prepare 1 x TBST buffer), each time for 5 min; (4) Incubate with primary and secondary antibodies: incubate overnight at 4°C, and then wash with TBST solution for 10 min the next day, for a total of 3 times. After washing, the membrane was placed in the secondary antibody and incubated at room temperature for 70 min. Afterwards, it was washed 3 times with TBST, 10 min each time. (5) Gel imaging: The positive bands that glowed in the membrane were detected using the Odyssey fluorescence imaging instrument. Image J software was used to analyze the grayscale value of the positive bands and calculate the relative expression of the protein.

[0045] Primary antibodies: anti-rabbit Elastin (purchased from Proteintech, dilution ratio 1:1000, size 70 kDa); anti-rabbit MMP1 (purchased from Proteintech, dilution ratio 1:1000, size 54 / 62 kDa); anti-rabbit MMP2 (purchased from Proteintech, dilution ratio 1:500, size 72 kDa); anti-rabbit MMP9 (purchased from Proteintech, dilution ratio 1:500, size 92 kDa); the expression level of anti-mouse GAPDH was used as an internal reference, dilution ratio 1:10000, size 36 kDa.

[0046] Fluorescent secondary antibody: anti-rabbit and anti-mouse HRP-conjugated IgG fluorescent secondary antibodies (exposure ratio 1:50,000) were purchased from Genentech.

[0047] The results are as follows Figure 2 As shown in the figure, compared with the blank control group, the expression levels of MMP1, MMP2, and MMP9 proteins in cells were significantly increased after UV stimulation, and the expression level of Elastin protein was significantly decreased (single-factor ANOVA, p<0.05, p<0.01, p<0.001, p<0.0001), indicating that the UV-stimulated HaCaT cell photoaging model was successfully established. The preferred combination was repaired after injury, and the results showed that compared with the UV modeling group, the expression of MMP1, MMP2, and MMP9 proteins in combination 1 was significantly reduced, and the expression level of Elastin protein was extremely significantly increased (single-factor ANOVA, p<0.05, p<0.01, p<0.001, p<0.0001, and significantly better than the single drug group ( Figure 2 A-2D, T-test, p<0.05, p<0.01).

[0048] In summary, the preferred composition 1 can reduce the production of MMP1, MMP2, and MMP9 in human skin keratinocytes, increase elastin synthesis, inhibit the decomposition of elastase, thereby reducing UV light aging damage and ultimately improving skin aging.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A composition of natural origin having the function of inhibiting various types of matrix metalloproteinases in the skin, characterized in that: The composition comprises the following components: Cistanche deserticola powder, American ginseng powder, Polygonum aviculare extract and Acanthus monnieri extract.

2. The composition according to claim 1, characterized in that: The composition is prepared from the following components: Cistanche deserticola powder, American ginseng powder, Polygonum multiflorum extract and Acanthus monnieri extract.

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 type of the matrix metalloproteinase is one or more of the following: MMP1, MMP2 or MMP9.

5. The composition according to claim 1, characterized in that: The composition inhibits the expression of matrix metalloproteinases, improves elastin synthesis, and inhibits elastase decomposition.

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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