A composition of natural origin having the function of inhibiting various types of matrix metalloproteinases in the skin and its uses

Abstract

The present invention belongs to the technical field of cosmetics, and particularly relates to a natural source composition having the function of inhibiting various types of matrix metalloproteinases in the skin and its uses. The composition comprises the following components: cistanche powder, american ginseng powder, polygonum orientale extract and justicia procumbens extract. The composition of the present invention can simultaneously inhibit the expression of various types of matrix metalloproteinases in immortalized keratinocytes of the skin, improve the synthesis of elastin, and inhibit the decomposition of elastase, and has no obvious side effects. The composition of the present invention can be used for preparing various cosmetics, functional foods and health products related to anti-skin aging, and has broad application prospects.

Description

Technical Field

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

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

[0003] Photoaging mainly refers to the aging caused by ultraviolet radiation. A large amount of ultraviolet radiation disrupts 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 a decrease in skin elasticity and the formation of wrinkles.

[0004] Matrix metalloproteinases (MMPs) are a class of enzymes containing active Zn 2+ and dependent on Ca 2+ that can degrade structural proteins such as collagen and elastin, thereby causing aging symptoms in the skin. The current mainstream view is that MMPs increase with age dependence, leading to an increased risk of inducing wrinkles, skin laxity, and microvascular dilation. Inhibiting the activity of MMPs can effectively prevent skin aging. A total of 25 subtypes of human MMPs 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), stromelysins (MMP-3, MMP-10, MMP-11), matrilysins (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] Currently, the research on inhibiting the function of matrix metalloproteinases is basically based on single substances, and most of them are chemical substances obtained through 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 costs are generally high. Therefore, there are problems that they are not suitable for use as raw materials for cosmetics or functional foods. In addition, the existing technologies have deficiencies in regulating elastin synthesis and the activities of matrix metalloproteinases and elastases, and cannot achieve comprehensive skin health care effects.

[0006] In view of this, the present inventors screened and combined a large number of substances homologous to medicine and food and raw materials for functional foods, and then obtained a composition that simultaneously inhibits the activities of various types of matrix metalloproteinases in skin keratinocytes, increases the content of elastin, and inhibits the action of elastase. Summary of the Invention

[0007] In order to overcome the defects of the existing technology and enrich the existing technology, the present invention provides a composition having the functions of inhibiting the expression of various types of MMPs in skin keratinocytes, increasing elastin synthesis, and inhibiting elastase decomposition, and its uses. The composition of the present invention comprehensively improves 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 activities inside the skin, and bring innovative solutions to the field of skin health care.

[0008] Specifically, the present invention is achieved through the following technical solutions:

[0009] In the first aspect, the present invention provides a composition of natural origin having the function of inhibiting various types of matrix metalloproteinases in the skin, and the composition comprises the following components: cistanche powder, american ginseng powder, polygonum orientale extract, and justicia procumbens extract.

[0010] As an optional manner, in the above composition, the composition is made of the following components: cistanche powder, american ginseng powder, polygonum orientale extract, and justicia procumbens extract.

[0011] As an optional manner, in the above composition, the types of the matrix metalloproteinases are 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.

[0012] As an alternative, in the above composition, the type of the matrix metalloproteinase is one or more of the following: MMP1, MMP2 or MMP9.

[0013] As an alternative, in the above composition, the composition inhibits the expression of matrix metalloproteinase, enhances elastin synthesis and inhibits elastase degradation.

[0014] As an alternative, in the above composition, the dosage ratio of cistanche powder: american ginseng powder: polygonum orientale extract: justicia procumbens extract is 200 - 800 μg / mL: 200 - 800 μg / mL: 5 - 15 μg / mL: 5 - 15 μg / mL.

[0015] As an alternative, in the above composition, the dosage ratio of cistanche powder: american ginseng powder: polygonum orientale extract: justicia procumbens extract is 500 μg / mL: 500 μg / mL: 10 μg / mL: 10 μg / mL.

[0016] In a second aspect, the present invention provides the use of the composition described in the first aspect above in the preparation of an anti - aging product.

[0017] As an alternative, in the above use, the anti - aging includes alleviating or preventing skin aging, cell aging or photo - aging.

[0018] As an alternative, 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.

[0019] As an alternative, in the above use, the product is an oral solution, a liquid suspension, an injection, a tablet, a pill, a granule, a powder, a film, a capsule, a spray, an aerosol, a syrup, a beverage, a nutritional product, a snack, an energy bar, a chewing gum, a candy, a facial mask, a cream, an essence, a lotion, a toner or a makeup.

[0020] As an alternative, in the above use, the product further contains excipients or vehicles commonly used in cosmetics, functional foods, beverages or nutritional supplements. By weight percentage, the dosage of the composition in the product is 0.5% - 20%.

[0021] The present invention has the following beneficial effects compared with the prior art:

[0022] (1) By screening and compounding a large number of substances homologous to medicine and food and functional food raw materials, the present invention first obtains a composition that simultaneously inhibits the activities of multiple types of matrix metalloproteinases in skin keratinocytes, increases the content of elastin, inhibits the action of elastase and has no obvious side effects.

[0023] (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

[0024] Figure 1 : Inhibitory effects of three compositions of different concentrations on elastase.

[0025] Figure 2 : Effects of preferred compositions on extracellular matrix metalloproteins. DETAILED DESCRIPTION

[0026] 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.

[0027] The following is a brief description of several active ingredients involved in the present invention:

[0028] 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.

[0029] 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.

[0030] 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.

[0031] As used herein, the Rostellularia procumbens extract was purchased from Xi'an Sai'ao Biotechnology Co., Ltd. Rostellularia procumbens is the whole herb of the herbaceous plant Rostellularia procumbens ( Rostellularia procumbens (L.) Nees ), which is rich in active ingredients such as alkaloids, rostellarin C, D, E, etc., and has antibacterial, antiviral, antitumor and other effects. It has been reported that Rostellularia procumbens may achieve the effect of anti-chronic nephritis by inhibiting the expression of inflammatory proteins in the TLR4 / NF-κB pathway (see, Li Yue. Study on the effective substances and mechanism of Rostellularia procumbens against chronic nephritis [D]. Hubei University of Chinese Medicine, 2019.). However, there is currently no study showing that the Rostellularia procumbens extract can affect the function of MMP in the skin.

[0032] As used herein, the Polygonum orientale extract was purchased from Shaanxi Mufei Biotechnology Co., Ltd. Polygonum orientale is a plant of the Polygonaceae family ( Persicaria orientalis (L.) Spach ), and generally the fruit of Polygonum orientale is used as traditional Chinese medicine, called Fructus Polygoni Orientalis. It has been reported that components such as protocatechuic acid, taxifolin, kaempferol-glucoside, quercitrin, kaempferol-rhamnoside, etc. in the Polygonum orientale extract are absorbed throughout the intestinal tract. In addition, the Polygonum orientale extract can improve the hypoxia-reoxygenation injury of cardiomyocytes, and the mechanism of action may be related to inhibiting cardiomyocyte apoptosis, increasing ATPase activity, protecting mitochondrial function, etc. (see, Li Liangliang, Li Ping, Chang Kun, etc. Research progress on the utilization of the medicinal plant Polygonum orientale [J]. Agriculture and Technology, 2023, 43(09): 170-172). The effect of Polygonum orientale in resisting skin photoaging has not been reported yet.

[0033] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0037] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are intended to include but not be limited to.

[0038] 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 explain the present invention and are not used to limit the scope of the present invention.

[0039] For those without specific technologies or conditions indicated in the examples, they are carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular channels.

[0040] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0041] Example:

[0042] Example 1: Effects of single drugs and compositions on elastase activity

[0043] Drug dilution: Dissolve cistanche powder, american ginseng powder, polygonum orientale extract, and justicia procumbens extract in ultrapure water and configure them according to the concentrations shown in Table 1.

[0044] Table 1: Concentrations of monomeric and combined drugs

[0045]

[0046] Elastase determination: The EnzChek™ Elastase Assay Kit (purchased from Invitrogen, catalog number: E12056) was used in the experiment. According to the instructions, set up control wells and sample wells (including Combinations 1, 2, 3 and the monomeric drugs at corresponding concentrations). Add 25 μL of elastin and 25 μL of enzyme to each detection well. Add 3 kinds of compositions and the monomeric drugs at corresponding concentrations to the sample wells, and make up the remaining volume with diluent. The total system is 200 μL. After adding all the reagents to the 96-well plate, quickly transfer it to the microplate reader (in the dark). According to kinetic detection, Ex / Em is 505 / 515 nm, set the total duration to 1 h, and take readings every 3 min.

[0047] The test results are asFigure 1 As shown, compared with the blank control group, the three different concentrations of the composition all showed good elastase inhibitory effects. Compared with single administration, the inhibitory effect of Combination 1 on elastase 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 powder 500 μg / mL, American ginseng powder 500 μg / mL, polygonum orientale extract 10 μg / mL, justicia procumbens extract 10 μg / mL)

[0048] Example 2: Effect of the composition on the expression of extracellular matrix proteins in a photoaging model of UV-stimulated mouse skin keratinocytes (HaCaT)

[0049] 1. Cell seeding: When the HaCaT cells grew to the appropriate passage number, after digestion and counting, they were seeded in a 10 cm cell culture dish at a density of 2×10 6 / mL and placed in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0050] 2. Cell administration: Based on the results of elastase activity, Combination 1 and the corresponding concentration of monomer drugs in Table 1 were selected for cell experiments. Specifically, it consisted of the following four drugs, including cistanche powder 500 μg / mL; American ginseng powder 500 μg / mL, polygonum orientale extract 10 μg / mL, justicia procumbens extract 10 μg / mL. The experiment set up a blank group (CON), a UV modeling group, and a group with drug addition after modeling (Combination 1 and single drugs). When the cells in the 10 cm dish grew to 80% confluence, 10 mL of the culture medium containing the corresponding concentration of the drug was added to each dish in the modeling and sample groups, and 10 mL of DMEM complete medium was added to each dish in the blank group. Each group was set with 3 replicate dishes; after drug administration, the 10 cm dishes were placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity and cultured for 24 h.

[0051] 3. Protein extraction and denaturation: Remove the culture medium, wash the cells twice with pre-cooled PBS solution, gently scrape the cells with a cell scraper, transfer them into a 15 mL centrifuge tube, discard the supernatant after centrifugation (3000 rpm, 5 min). Resuspend the cells with 300 μL of cell lysis buffer containing PMSF (PMSF: RIPA = 1:99), transfer them to a 1.5 mL centrifuge tube, and lyse the cells on ice for 20 min, with vigorous shaking up and down every 10 min to ensure complete lysis. After lysis, centrifuge (14,000 r / min, 10 min) and aspirate the supernatant into a new 1.5 mL centrifuge tube. The total protein content is determined using a BCA kit (purchased from Beyotime). After mixing the qualified protein sample with 5x loading buffer, place it in a 100 °C metal bath for 5 min to denature the protein, and store it at -20 °C for later use.

[0052] 4. Western blot assay: (1) Electrophoresis: Separate proteins with different molecular weights using precast gels with different concentrations (8%-15%) (purchased from Sangon Biotech). After adding the protein sample, perform SDS-PAGE electrophoresis at a constant voltage of 80 V for 90 min; (2) Blotting: Activate the PVDF membrane with methanol solution for about 2 min, and then perform blotting at a constant current of 200 mA for 70-90 min (slightly adjusted according to the protein molecular weight); (3) Blocking: Use a protein-free rapid blocking solution (purchased from Yaenzyme, product number PS108P) to block the PVDF membrane at room temperature for 15 min. After blocking, wash the membrane 3 times with 1x TBST buffer (purchased from Solarbio, dissolve 10x TBS in 1L ddH2O and add 500 μL of Tween-20 to prepare 1x TBST buffer), 5 min each time; (4) Incubation with primary and secondary antibodies: Incubate overnight at 4 °C, and then wash with TBST solution for 10 min the next day, 3 times in total. After washing, place the membrane in the secondary antibody and incubate at room temperature for 70 min. After completion, wash with TBST 3 times, 10 min each time. (5) Gel imaging: Use an Odyssey fluorescence imaging instrument to detect the positive bands emitting light in the membrane. Analyze the gray value of the positive bands using Image J software and calculate the relative protein expression level.

[0053] 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.

[0054] Fluorescent secondary antibodies: Anti-rabbit and anti-mouse HRP-conjugated IgG fluorescent secondary antibodies (dilution ratio 1:50000) were purchased from Gene Company.

[0055] The results are as Figure 2 shown. Compared with the blank control group, the protein expression levels of MMP1, MMP2, and MMP9 in cells were significantly increased after UV stimulation, while the protein expression level of Elastin was significantly decreased (one-way ANOVA, p < 0.05, p < 0.01, p < 0.001, p < 0.0001), indicating that the photoaging model of HaCaT cells induced by UV was successfully established. After repairing the damage with the optimized composition, the results showed that compared with the UV-induced model group, the protein expressions of MMP1, MMP2, and MMP9 in Composition 1 were significantly decreased, and the protein expression level of Elastin was extremely significantly increased (one-way ANOVA, p < 0.05, p < 0.01, p < 0.001, p < 0.0001), and it was significantly better than each group administered alone ( Figure 2 A-2D, T test, p < 0.05, p < 0.01).

[0056] In summary, the optimized 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 the damage of UV photoaging, and ultimately improving skin aging.

[0057] Obviously, those skilled in the art can make various modifications and variations 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 equivalent technologies, 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 is made from the following components: cistanche powder, american ginseng powder, polygonum orientale extract, and justicia procumbens extract; in the composition, the dosage ratio of cistanche powder: american ginseng powder: polygonum orientale extract: justicia procumbens extract is 500 μg / mL: 500 μg / mL: 10 μg / mL: 10 μg / mL; the matrix metalloproteinases are MMP2 and MMP9.

2. Use of the composition according to claim 1 in the preparation of anti-aging cosmetics.

3. The use according to claim 2, characterized in that: The anti-aging includes alleviating or preventing skin aging, cell aging, or photoaging.

4. The use according to claim 2, characterized in that: The cosmetics are facial masks, creams, essences, lotions, toners, or makeup.

5. The use according to claim 2, wherein: The cosmetics further contain common excipients or vehicle agents; calculated by weight percentage, the dosage of the composition in the cosmetics is 0.5% - 20%.

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