Hydroxytyrosol-containing composition with function of inhibiting multiple types of matrix metalloproteinases of skin and application of hydroxytyrosol-containing composition

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a hydroxytyrosol-containing composition with a function of inhibiting multiple types of matrix metalloproteinases of skin and application of the hydroxytyrosol-containing composition. The composition is prepared from the following components: hydroxytyrosol, folium cortex eucommiae extract, polygonum hydropiper extract and creeping rostellularia herb extract. The composition has the effects of inhibiting expression of multiple types of matrix metalloproteinases in human skin immortalized keratinocytes, increasing the content of elastin and inhibiting elastase, and has no obvious side effect. The composition disclosed by the invention can be used for preparing various cosmetics, functional foods and health-care products related to skin aging resistance (especially skin photoaging), 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 composition containing hydroxytyrosol and having the function of inhibiting various types of matrix metalloproteinases in skin, and a use thereof. Background Art

[0002] The skin is the largest organ in the human body and its main function is to protect internal tissues from the external environment. However, with age and the influence of external factors such as ultraviolet (UV) radiation, the skin will gradually show signs of aging, such as wrinkles, spots, and sagging. This skin aging is mainly caused by the degradation of structural proteins such as collagen and elastin in the skin. The reduction of elastin, which plays a supporting role in the skin, will cause the skin to lose elasticity, which in turn causes the skin to sag. The loss or lack of elastin can also affect the overall appearance of the skin.

[0003] Matrix metalloproteinase (MMP) is a class of proteins that contain active Zn 2+ and depends on Ca 2+ The enzyme can degrade structural proteins such as collagen and elastin, leading to aging symptoms of the skin. The mainstream view now is that MMP will increase with age, leading to an increased risk of inducing wrinkles, sagging skin, and dilation of microvessels. Inhibiting the activity of MMP can effectively prevent skin aging. There are 25 subtypes of human MMP that have been discovered so far. According to their structural characteristics, they can be divided into collagenase (MMP-1, MMP-8, MMP-13, MMP-18), gelatinase (MMP-2, MMP-9), matrix hydrolases (MMP-3, MMP-10, MMP-11), matrix lysins (MMP-7, MMP-26) and membrane MMP (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.

[0004] The existing solutions mainly achieve the purpose of anti-skin aging by using chemically synthesized MMP inhibitors. However, these single chemically synthesized inhibitors may have side effects, such as causing inflammatory reactions, allergic reactions, etc., and the production costs are generally high. In addition, the effects of these inhibitors are not satisfactory, because they can often only inhibit the activity of one or several MMPs, but have poor inhibitory effects on multiple types of MMPs. When used in cosmetics, functional foods and health products, chemically synthesized MMP inhibitors may affect the safety and effectiveness of the products.

[0005] 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 can simultaneously inhibit the expression of multiple types of MMPs in skin keratinocytes, increase the elastin content, and inhibit the action of elastase, and the effect of the combination is greater than that of a single ingredient. Summary of the invention

[0006] 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 MMPs in skin keratinocytes, increase the elastin content, and inhibit the action of elastase.

[0007] Specifically, the present invention is realized through the following technical solutions:

[0008] In a first aspect, the present invention provides a composition for inhibiting the functions of various types of matrix metalloproteinases in the skin, the composition comprising the following components: hydroxytyrosol, eucommia leaf extract, polygonum erythrorhizon extract and acanthus monnieri extract.

[0009] As an optional mode, in the above composition, the composition is made of the following components: hydroxytyrosol, eucommia leaf extract, polygonum erythrorhizon 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, promotes the production of elastin, and inhibits the activity of elastase.

[0013] As an optional mode, in the above composition, the usage ratio of hydroxytyrosol: eucommia leaf extract: polygonum erythrorhizon extract: acanthus monnieri extract is (0.5-20) μM: (0.125-5) mg / mL: (2.5-250) μg / mL: (2.5-250) μg / mL.

[0014] As an optional mode, in the above composition, the usage ratio of hydroxytyrosol:eucommia leaf extract:polygonum erythrorhizon extract:acanthus monnieri extract is 5 μM:0.125 mg / mL:2.5 μg / mL:2.5 μg / mL.

[0015] As an optional mode, in the above composition, the usage ratio of hydroxytyrosol:eucommia leaf extract:polygonum erythrorhizon extract:acanthus monnieri extract is 0.5 μM:0.5 mg / mL:10 μg / mL:10 μg / mL.

[0016] As an optional mode, in the above composition, the usage ratio of hydroxytyrosol:eucommia leaf extract:polygonum erythrorhizon extract:acanthus monnieri extract is 1.0 μM:2.5 mg / mL:50 μg / mL:50 μg / mL.

[0017] As an optional mode, in the above composition, the usage ratio of hydroxytyrosol:eucommia leaf extract:polygonum erythrorhizon extract:acanthus monnieri extract is 2.0 μM:5 mg / mL:250 μg / mL:250 μg / mL.

[0018] In a second aspect, the present invention provides use of the composition of the first aspect in preparing an anti-aging product.

[0019] As an optional mode, in the above-mentioned use, the anti-aging includes alleviating or preventing skin aging, cell aging or photoaging.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The composition of the present invention is a combination of hydroxytyrosol with eucommia leaf extract, red polygonum extract and acanthus extract, which has a significant inhibitory effect on UV-induced skin keratinocyte MMP increase and elastin degradation, and inhibits the activity of elastase. The effect is better than using hydroxytyrosol, eucommia leaf extract, red polygonum extract or acanthus extract alone, and is safe and has no adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Inhibitory effects of single substances and combinations on the expression of multiple MMP proteins.

[0026] Figure 2 : The promoting effects of single substances and combinations on elastin expression.

[0027] Figure 3 : Inhibition rate of elastase activity by single substances and combinations. DETAILED DESCRIPTION

[0028] In the above summary of the invention and the specific embodiments, as well as in the claims, specific features of the present invention are mentioned, and it should be understood that all possible combinations including 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 a specific claim, the feature may also be used as much as possible and / or in combination with other specific aspects and embodiments of the present invention.

[0029] 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.

[0030] The following is a brief description of several active ingredients involved in the present invention:

[0031] The present invention provides four substances capable of inhibiting the function of MMP, and provides a compounding method containing the combination of the above four substances, after the above four substances are combined, the inhibitory effect on the function of MMP is significantly enhanced. The above four substances include hydroxytyrosol, eucommia leaf extract, red polygonum extract, and acanthus extract.

[0032] Hydroxytyrosol is a natural polyphenol antioxidant, which is widely found in the leaves and fruits of plants of the genus Olea in the family Oleaceae. Hydroxytyrosol is an amphiphilic molecule with good fat solubility and water solubility. Its molecular formula is C8H 10O3 can be used as a safe and efficient antioxidant in beauty products and health products, effectively enhancing skin elasticity and moisture, improving the body's absorption of minerals, enhancing absorption effects, maintaining bone density, reducing osteoporosis, promoting metabolism, promoting wound healing, eliminating free radicals in the body, restoring the health of human organs, preventing brain failure, delaying aging, etc. It has great development value. The structural formula of hydroxytyrosol is:

[0033] Eucommia leaf extract is the dry leaf extract of Eucommia ulmoides Oliv, a plant of the Eucommia family. Studies have shown that Eucommia leaf extract can reduce the accumulation of triglycerides in fat cells, lower cholesterol levels, reduce blood lipid content and regulate blood lipid metabolism in the body. It also has broad-spectrum antibacterial, antioxidant, free radical scavenging and immunity-enhancing effects. It can accelerate the metabolism of human collagen, prevent or delay skin wrinkling and aging, increase skin luster, improve hair texture, reduce hair loss, delay aging, and prevent Alzheimer's disease.

[0034] Persicaria orientalis (L.) Spach is an annual herbaceous plant of the Polygonaceae family. According to the Xinjiang Chinese Herbal Medicine Handbook, Persicaria orientalis has the effects of dispelling wind and dampness, and treating rheumatoid arthritis. Its medicinal functions include dispelling wind and dampness, clearing away heat and detoxifying, promoting blood circulation, and stopping malaria. It is used to treat rheumatism, arthralgia, dysentery, diarrhea, vomiting, diarrhea, cramps, edema, athlete's foot, carbuncle, furuncle, snake and insect bites, malnutrition and hernia in children, traumatic injuries, and malaria. Its effect in resisting skin photoaging has not been reported.

[0035] Rostellularia procumbens extract is an extract of the annual creeping herb Rostellularia procumbens (L.) Nees. As a traditional Chinese medicine, a large number of studies have found that it contains a variety of chemical components and has good pharmacological effects such as anti-tumor and anti-platelet aggregation. It also has the effects of clearing away heat and detoxification, diuresis and swelling, and anti-malaria. It is used for colds and fever, malaria, sore throat, malnutrition in children, dysentery, enteritis, nephritic edema, urinary tract infection, and chyluria; it is used externally to treat carbuncles, furunculosis, and traumatic injuries. However, no studies have shown that Rostellularia procumbens extract can affect the function of MMP in the skin.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Human skin immortalized keratinocytes (HaCaT) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. Hydroxytyrosol was purchased from MedChemExpress; Eucommia ulmoides leaf extract was purchased from Guangdong Qingyunshan Pharmaceutical Co., Ltd.; Polygonum erythrorhizon extract was purchased from Shaanxi Mufei Biotechnology Co., Ltd.; and Acanthus monnieri extract was purchased from Xi'an Sai'ao Biotechnology Co., Ltd.

[0040] Example:

[0041] 1. Cell culture:

[0042] The experiment used human skin immortalized keratinocytes (HaCaT) and mixed them with DMEM complete medium. 5 / mL was inoculated in a 6cm cell culture dish and cultured in a cell culture incubator at 37℃, 5% CO2 and saturated humidity. The cells adhered to the wall and began to be subcultured when they grew to cover 80% of the bottom area of ​​the culture dish. During subculture, the original culture medium was aspirated, washed twice with PBS, digested with 0.25% trypsin, and left to stand in the cell culture incubator for 7 minutes until the cells shrank and rounded and gradually fell off the culture bottle. Then, 2 times the volume of trypsin-containing complete culture medium was added to stop digestion, and the cell suspension was collected, centrifuged, and resuspended with an appropriate amount of complete culture medium. After counting, a certain concentration of cells was transferred to a new culture dish, 10mL of culture medium was added, and the cells were mixed to make them evenly spread in the dish.

[0043] 2. Cell processing:

[0044] When the cells reached the logarithmic growth phase, they were digested and counted, with 7×10 5 The cells were inoculated in a 6 cm culture dish and placed in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for 24 hours. After the cells adhered to the wall, the model group and the active ingredient treatment group were exposed to ultraviolet radiation for 80 minutes using an ultraviolet aging box UVA 95% + UVB 5% to simulate natural light, and then the culture medium was replaced. The model group was added with an equal amount of complete culture medium, and the active ingredient treatment group was added with complete culture medium of each active ingredient at the corresponding concentration, and continued to be placed in a cell culture incubator for the required time for subsequent experimental testing.

[0045] 3. Detection of the effects of four single substances and combinations on the expression of various types of MMP proteins:

[0046] Human immortalized keratinocytes were treated with hydroxytyrosol (20 μM), Eucommia leaf extract (500 μg / mL), Polygonum erythrorhizon extract (10 μg / mL), Acanthus monnieri extract (10 μg / mL) and four active ingredients in a quarter concentration ratio for 18 hours. The cells were then collected, proteins were extracted, and used for WB (Western Blot) experiments. The present invention detects a relatively comprehensive variety of MMPs including collagenase MMP-1, gelatinase MMP-2, and MMP-9. By comparing with the control group, it can be judged whether the model group is successfully established. If the expression of MMP protein in the model group is significantly increased compared with the control group, the model is successfully established, otherwise it is unsuccessful. At the same time, the expression of MMP protein in the experimental group is compared to see whether it is reduced to judge whether the four single substances and the combination have an inhibitory effect on MMP protein expression.

[0047] The results showed that the four single substances had different inhibitory effects on MMP-1, MMP-2, and MMP-9. Some of the four single substances had stronger inhibitory effects on some MMPs and weaker inhibitory effects on some MMPs. Among them, single substances hydroxytyrosol and Eucommia leaf extract had inhibitory effects on the expression of MMP-1, MMP-2, and MMP-9 proteins, Polygonum multiflorum extract and Acanthus chinensis extract had no significant inhibitory effects on MMP-1, but had inhibitory effects on MMP-2 and MMP-9. The inhibitory effects of the combination of the four substances on MMP-1, MMP-2, and MMP-9 were all greater than those of the single substances.

[0048] 4. Detection of the effects of four single substances and combinations on cell secretion of elastin:

[0049] Human immortalized keratinocytes were treated with hydroxytyrosol (20 μM), Eucommia leaf extract (500 μg / mL), Polygonum erythrorhizon extract (10 μg / mL), Acanthus monnieri extract (10 μg / mL) and four active ingredients at a quarter concentration ratio for 18 h. Cells were then collected, proteins were extracted, and used for WB (Western Blot) experiments. The present invention detects the amount of elastin in cells. By comparing with the control group, it can be judged whether the model group is successfully established. If the expression of elastin in the model group is significantly reduced compared with the control group, the model is successfully established, otherwise it is unsuccessful. At the same time, the expression of elastin in the experimental group is compared to determine whether it rises, to determine whether the four single substances and the combination have a promoting effect on the expression of elastin.

[0050] The results showed that the four single substances had no significant promoting effect on elastin, but the combination treatment group after the four substances were combined in proportion had a significant promoting effect on elastin compared with the model group.

[0051] 5. Detection of the inhibitory effect of four single substances at various concentrations and the combination of four substances on elastase activity:

[0052] Elastase, also known as elastase, is an enzyme from the protease class in molecular biology that can decompose elastin. Elastin plays an important role in maintaining the toughness and firmness of the skin. When elastin is lacking in the skin, the skin will become loose, sagging and fine wrinkles. The present invention detects the effects of four single substances and four combinations of substances on the activity of elastase, and further reflects the content of elastin in the skin.

[0053] The present invention designs four different concentrations of single substances to detect the inhibitory effect on elastase, and combines them in order from low to high concentrations. Hydroxytyrosol concentrations (0.5 μM, 1.0 μM, 2.0 μM), Eucommia leaf extract concentrations (0.5 mg / mL, 2.5 mg / mL, 5.0 mg / mL), Polygonum aviculare extract concentrations (10 μg / mL, 50 μg / mL, 250 μg / mL), Acanthus monnieri extract concentrations (10 μg / mL, 50 μg / mL, 250 μg / mL), Composition 1 (Hydroxytyrosol: Eucommia leaf extract: Polygonum aviculare extract: Acanthus monnieri extract at concentrations of 0.5 μM: 0.5 mg / mL: 10 μg / mL) : 10μg / mL, each single substance accounts for one quarter of the total volume); Combination 2 (Hydroxytyrosol: Eucommia leaf extract: Polygonum erythrorhizon extract: Acanthus monnieri extract at a concentration of 1.0μM: 2.5mg / mL: 50μg / mL: 50μg / mL, each single substance accounts for one quarter of the total volume); Combination 3 (Hydroxytyrosol: Eucommia leaf extract: Polygonum erythrorhizon extract: Acanthus monnieri extract at a concentration of 2.0μM: 5.0mg / mL: 250μg / mL: 250μg / mL, each single substance accounts for one quarter of the total volume).

[0054] The results showed that among the four single substances, hydroxytyrosol and eucommia leaf extract inhibited elastase activity more with the increase of active ingredient concentration, while Polygonum erythrorhizon extract and Acanthus chinensis extract had no significant change. The inhibitory effect of the four substances combined on elastase activity was significantly higher than that of the single substances at the corresponding concentrations, indicating that the combination of the four substances in the present invention has a strong inhibitory effect on elastase and can reduce the degradation of elastin in skin keratinocytes (CAS number of the positive inhibitor: 70967-90-7).

[0055] In summary, the following conclusions can be drawn through the effect experiment:

[0056] (1) The present invention first discovered that the single substances Polygonum multiflorum extract and Acanthus monnieri extract in the composition of the present invention can inhibit the function of skin MMP;

[0057] (2) The combination of substances in the composition of the present invention has a significant inhibitory effect on the increased expression of multiple MMP proteins in skin keratinocytes, and is stronger than a single substance;

[0058] (3) The composition of the present invention can significantly inhibit the decrease in elastin content caused by UV light aging stimulation;

[0059] (4) The combination 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 a single substance;

[0060] (5) The composition of the present invention provides a natural and safe formula that can inhibit the degradation of elastin in skin keratinocytes and inhibit MMP.

[0061] 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 for inhibiting the functions of various types of matrix metalloproteinases in the skin, characterized in that: The composition comprises the following components: hydroxytyrosol, eucommia leaf extract, polygonum erythrorhizon extract and acanthus monnieri extract.

2. The composition according to claim 1, characterized in that: The composition is prepared from the following components: hydroxytyrosol, eucommia leaf extract, polygonum erythrorhizon 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, promotes the production of elastin, 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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