A composition containing hydroxytyrosol and having the function of inhibiting various types of matrix metalloproteinases in skin and its use

Abstract

The present invention belongs to the field of cosmetics technology and specifically relates to a composition containing hydroxytyrosol that inhibits various types of matrix metalloproteinases in the skin, and its uses. The composition comprises the following components: hydroxytyrosol, an extract of Eucommia ulmoides leaves, an extract of Polygonum multiflorum, and an extract of Acanthus monnieri. The composition simultaneously inhibits the expression of various types of matrix metalloproteinases in immortalized keratinocytes of human skin, increases elastin content, and inhibits elastase, without significant side effects. The composition can be used to prepare various cosmetics, functional foods, and health products related to anti-skin aging (particularly photoaging), and has broad application prospects.

Description

Technical Field

[0001] The present 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 the skin, and uses thereof. Background Art

[0002] The skin is the largest organ in the human body, and its primary function is to protect internal tissues from external environmental influences. However, with aging and the effects of external factors such as ultraviolet (UV) radiation, the skin gradually develops signs of aging, such as wrinkles, dark spots, and sagging. This aging is primarily caused by the degradation of structural proteins such as collagen and elastin in the skin. A decrease in elastin, the skin's supporting protein, leads to a loss of elasticity, resulting in sagging. The loss or deficiency of elastin also affects the skin's overall appearance.

[0003] Matrix metalloproteinase (MMP) is a class of proteins containing active Zn 2+ and depends on Ca 2+ The enzymes that degrade structural proteins such as collagen and elastin lead to the appearance of skin aging symptoms. The current mainstream view is that MMPs increase in an age-dependent manner, leading to an increased risk of wrinkles, sagging skin, and dilation of microvessels. Inhibiting MMP activity 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.

[0004] Existing solutions primarily rely on the use of chemically synthesized MMP inhibitors to combat skin aging. However, these single chemically synthesized inhibitors can have side effects, such as inflammatory and allergic reactions, and are generally expensive to produce. Furthermore, the effectiveness of these inhibitors is unsatisfactory, as they often only inhibit the activity of one or a few MMPs and are ineffective against multiple types of MMPs. Chemically synthesized MMP inhibitors, when used in cosmetics, functional foods, and health supplements, may impact product safety and efficacy.

[0005] In view of this, the inventors screened and compounded a large number of medicinal and edible substances and functional food raw materials, and obtained a composition that can simultaneously inhibit the expression of multiple types of MMPs in skin keratinocytes, increase elastin content, and inhibit the action of elastase. 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 that can simultaneously inhibit the expression of multiple types of MMPs in skin keratinocytes, increase elastin content, and inhibit the action of elastase.

[0007] Specifically, the present invention is achieved 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 barbata leaf extract, polygonum multiflorum extract, and acanthus monnieri extract.

[0009] As an optional mode, in the above composition, the composition is made of the following components: hydroxytyrosol, Eucommia ulmoides leaf extract, Polygonum multiflorum 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 manner, in the above composition, the usage ratio of hydroxytyrosol: Eucommia ulmoides leaf extract: Polygonum multiflorum 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 ulmoides 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 ulmoides 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 ulmoides 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 ulmoides leaf extract: Polygonum erythrorhizon extract: Acanthus mongholicus 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 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 use 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, nutritional product, 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, polygonum multiflorum extract and acanthus monnieri extract. It has a significant inhibitory effect on UV-induced increase in MMP in skin keratinocytes and degradation of elastin, and inhibits the activity of elastase. The effect is better than using hydroxytyrosol, eucommia leaf extract, polygonum multiflorum extract or acanthus monnieri 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 substance and combination. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

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

[0031] The present invention provides four substances capable of inhibiting MMP function, and provides a compound method containing a combination of the four substances. The combination of the four substances significantly enhances the inhibitory effect on MMP function. The four substances include hydroxytyrosol, eucommia leaf extract, Polygonum multiflorum extract, and Acanthus monnieri extract.

[0032] Hydroxytyrosol is a natural polyphenol antioxidant that is widely found in the leaves and fruits of the genus 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 effective antioxidant in beauty products and health care products. It can effectively enhance skin elasticity and moisturizing, improve the body's absorption of minerals, enhance absorption effect, maintain bone density, reduce osteoporosis, promote metabolism, promote wound healing, eliminate free radicals in the body, restore the health of human organs, prevent brain degeneration, and delay aging. It has great development value. The structural formula of hydroxytyrosol is:

[0033] Eucommia leaf extract is an extract from the dried leaves 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 lipids, and regulate lipid metabolism. It also has broad-spectrum antibacterial and antioxidant properties, scavenges free radicals, and enhances immunity. It can accelerate collagen metabolism, prevent or delay skin wrinkling and aging, increase skin radiance, improve hair texture, reduce hair loss, slow aging, and prevent Alzheimer's disease.

[0034] Persicaria orientalis (L.) Spach is an annual herbaceous plant of the Polygonum genus, Polygonaceae. According to the Xinjiang Handbook of Traditional Chinese Medicine, Persicaria orientalis has the efficacy of dispelling wind and dampness, and treating rheumatoid arthritis. Its medicinal properties include dispelling wind and dampness, clearing heat and detoxifying, promoting blood circulation, and preventing malaria. It is also effective in treating rheumatic pain, dysentery, diarrhea, vomiting, diarrhea, cramps, edema, athlete's foot, carbuncles, furuncles, snake and insect bites, malnutrition and hernia in children, traumatic injuries, and malaria. However, its efficacy in combating photoaging has not been reported.

[0035] Acanthus extract is an extract of the annual creeping herb Rostellularia procumbens (L.) Nees. As a traditional Chinese medicine, numerous studies have shown that it contains a variety of chemical components with promising anti-tumor and anti-platelet aggregation effects. It also has heat-clearing and detoxifying properties, acts as a diuretic and detumescent, and has antimalarial properties. It is used for colds and fevers, malaria, sore throats, malnutrition in children, dysentery, enteritis, nephritic edema, urinary tract infections, and chyluria. It is also used topically to treat carbuncles, boils, and injuries from falls. However, no studies have shown that Acanthus extract can affect the function of MMPs in the skin.

[0036] 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.

[0037] 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.

[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 multiflorum 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 into a 6cm cell culture dish and cultured in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Cells were allowed to adhere to the culture dish and subcultured when they grew to cover 80% of the bottom area of ​​the culture dish. During subculture, the original culture medium was aspirated, the cells were washed twice with PBS, and then digested with 0.25% trypsin. The cells were allowed to stand in the cell culture incubator for 7 minutes until they shrank, rounded, and gradually detached from the culture flask. At this time, 2 volumes of trypsin-containing complete culture medium were added to terminate the digestion. The cell suspension was collected, centrifuged, and resuspended in an appropriate amount of complete culture medium. After counting, the cells were transferred to a new culture dish at a desired concentration. 10mL of culture medium was added and the cells were mixed to evenly distribute the cells in the dish.

[0043] 2. Cell treatment:

[0044] When the cells reached the logarithmic growth phase, they were digested and counted, with a count of 7×10 5 Cells were seeded into 6-cm culture dishes and cultured in a CO2 incubator at 37°C, 5% CO2, and saturated humidity for 24 hours. After the cells adhered, the model and active ingredient-treated groups were exposed to ultraviolet radiation (UVA 95% + UVB 5%) for 80 minutes, simulating natural light. The culture medium was then replaced. An equal amount of complete culture medium was added to the model group, while the corresponding concentrations of complete culture medium for each active ingredient were added to the active ingredient-treated groups. The cells were then cultured in the cell incubator for the desired time before 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 ulmoides 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 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 determined whether the model group was successfully established. If the MMP protein expression in the model group was significantly increased compared with the control group, the model was successfully established, otherwise it was unsuccessful. At the same time, the expression of MMP protein in the experimental group was compared to determine whether it was reduced to determine whether the four single substances and the combination had 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 certain MMPs and weaker inhibitory effects on others. Hydroxytyrosol and Eucommia ulmoides leaf extract inhibited the expression of MMP-1, MMP-2, and MMP-9 proteins. Polygonum multiflorum extract and Acanthus monnieri extract had no significant inhibitory effect on MMP-1, but had inhibitory effects on MMP-2 and MMP-9. The inhibitory effects of the four substances combined 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 post-treated with hydroxytyrosol (20 μM), Eucommia ulmoides 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 hours. The cells were then collected, protein extracted, and used for Western Blot (WB) experiments. The present invention detects the amount of elastin in the cells. By comparing with the control group, it can be determined whether the model group was successfully established. If the elastin expression in the model group was significantly reduced compared with the control group, the model was successfully established; otherwise, it was unsuccessful. At the same time, the expression of elastin in the experimental group was compared to determine whether it increased, to determine whether the four single substances and the combination had a promoting effect on elastin expression.

[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 effects of four single substances at various concentrations and four combinations of substances on elastase activity:

[0052] Elastase, also known as elastase, is an enzyme in molecular biology that belongs to the protease family and breaks down elastin. Elastin plays an important role in maintaining the skin's resilience and firmness. A lack of elastin in the skin can cause it to sag, sag, and develop fine wrinkles. This study examined the effects of four single substances and combinations of these substances on elastase activity, further revealing the elastin content in the skin.

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

[0054] The results showed that, among the four individual substances, hydroxytyrosol and Eucommia ulmoides leaf extract showed enhanced inhibition of elastase activity with increasing concentration of the active ingredients, while Polygonum erythrorhizon extract and Acanthus monnieri extract showed no significant change. The combined effect of the four substances on elastase activity was significantly greater than that of the individual substances at corresponding concentrations, demonstrating that the four-substance combination of the present invention has a strong inhibitory effect on elastase and can reduce elastin degradation in skin keratinocytes (CAS number for 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 skin elastase activity, 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 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 multiple types of matrix metalloproteinases in the skin, characterized in that: The composition is prepared from the following components: hydroxytyrosol, eucommia leaf extract, polygonum multiflorum extract and acanthus monnieri extract. In the composition, the dosage ratio of hydroxytyrosol: eucommia leaf extract: polygonum multiflorum extract: acanthus monnieri extract is (0.5-20) μM: (0.125-5) mg / mL: (2.5-250) μg / mL: (2.5-250) μg / mL. The composition inhibits the expression of matrix metalloproteinases, promotes elastin production, and inhibits elastase activity. The types of the matrix metalloproteinases are MMP1, MMP2 and MMP9.

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

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

4. The use according to claim 2, characterized in that: The product is a cosmetic product.

5. The use according to claim 2, characterized in that: The product is a facial mask, a facial cream, an essence, a lotion, a toner or a makeup.

6. The use according to claim 4, characterized in that: The product also contains auxiliary materials commonly used in cosmetics. The amount of the composition in the product is 0.5%-20% by weight.

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