A skin external composition and its application as a blue light absorber and antioxidant

Abstract

The present invention discloses a composition for external use on skin and its application as a blue light absorber and antioxidant. The composition for external use on skin comprises a terminalia chebula fruit extract, EGCG and a solvent, wherein the mass ratio of the terminalia chebula fruit extract to the EGCG is (0.01-300):1; in the composition, the mass fraction of the terminalia chebula fruit extract is 0.005%-1%, and the mass fraction refers to the mass ratio of the terminalia chebula fruit extract to the solvent. The composition of the present invention has better blue light absorption and antioxidant effects than a single component terminalia chebula fruit extract of the same mass, and has a good antioxidant effect compared to a single component EGCG of the same mass.

Description

Technical Field

[0001] The present application relates to a composition for external use on skin and its application as a blue light absorber and an antioxidant. Background Art

[0002] Humans are exposed to sunlight for a long time. Usually, sunlight can be divided into three parts according to the spectrum: ultraviolet rays (wavelength 200~400nm), visible light (wavelength 400~800nm) and infrared rays (800nm~1mm). Among them, ultraviolet rays and visible light are the most harmful to the human body. Ultraviolet rays can be divided into UVA (320~400nm), UVB (290~320nm) and UVC (200~290nm) according to their wavelength. The shorter the wavelength, the greater the biological hazard. UVC is absorbed by the ozone layer, so it will not reach the surface of the earth and cause damage to the skin. However, UVA and UVB can reach the dermis and epidermis of the skin respectively, inducing lipid peroxidation, protein oxidation modification, etc., causing different degrees of damage to the skin and leading to premature aging of the skin. It is reported that UVB is the most destructive component of solar radiation reaching the earth's surface. If the skin is widely exposed to UVB, it will bring various adverse effects to the skin, such as edema, sunburn, hyperplasia, inflammation, erythema and immunosuppression, and may even develop into skin cancer. At present, the molecular mechanisms related to skin damage caused by UVB have been studied, including oxidative stress, inflammatory response and cell apoptosis. Among them, oxidative stress is crucial to skin damage, accounting for 50% of skin damage caused by ultraviolet radiation. Among several oxidative stressors, reactive oxygen species (ROS) are considered to be closely related to the skin damage process caused by ultraviolet radiation.

[0003] Reactive oxygen species (ROS) are products of cellular aerobic reactions and play an important role in the phagocytic and killing functions of phagocytes. Generally speaking, the level of ROS in the human body is in a dynamic balance. However, when the body is subjected to external stimuli, such as in the UVB-induced skin damage model, some major antioxidant enzymes, including catalase, glutathione peroxidase and superoxide dismutase, as well as the limiting enzyme glutathione are depleted, leading to aerobic metabolic disorders in the body's internal environment and abnormal ROS homeostasis in the body, which will cause oxidative stress. Oxidative stress damages cellular macromolecules, which are mainly responsible for regulating signal transduction and differentiation processes related to cell damage and death. [7] , for example: damage to cellular components (proteins, lipids and DNA), genomic instability (increased gene mutations), cell death and a series of life processes such as transcription and translation of genetic information.

[0004] In addition to the ultraviolet rays mentioned above, the main physical medium for generating ROS is visible light, which can also cause certain damage to the skin. The most penetrating visible light is the high-energy band with a wavelength of 400~500nm - blue light. Especially in recent years, the popularity of electronic devices has greatly facilitated people's daily lives, but at the same time, people are exposed to more and more opportunities for blue light, which in turn causes certain harm to the eyes and skin of the human body. Blue light can penetrate the epidermis to reach the dermis, induce mitochondria in cells to produce reactive oxygen free radicals, cause DNA damage, cell damage, and reduce extracellular matrix, thereby accelerating skin aging. Ju Ah et al. found that blue light irradiation induced the activation of TRPV1 in keratinocytes, thereby inducing the production of ROS, and as the energy of blue light increased, the level of ROS generated in cells increased, showing an energy-dependent trend. Earlier, the Swiss Rahn company also used blue light to induce human keratinocytes to produce ROS to verify the ability of its three cosmetic ingredients to resist blue light. The results also proved that these three active substances can effectively reduce the ROS produced by HACAT cells induced by blue light. At the same time, since long-vessel rosacea is believed to be caused by elevated levels of ROS in the skin, this condition leads to the formation and dilation of small blood vessels, increased microcirculation, redness of the skin and even an inflammatory state, especially in the malar area. This condition may be exacerbated by skin exposure to blue light. Therefore, adding antioxidants to cosmetics can effectively reduce the level of ROS in the skin and alleviate the phenomenon of skin redness and rosacea.

[0005] In summary, if human skin is to be protected from damage caused by elevated ROS levels, cosmetic ingredients should preferably contain active substances that can reduce the additional ROS generated by ultraviolet and blue light exposure. Currently, the search for safe and effective chemical preventive compounds or natural products to protect people from the harm of solar radiation has attracted widespread attention.

[0006] Terminalia chebula fruit extract is a natural source and is the active ingredient in the fruit of Terminalia chebula, a plant of the family Clivia. It is particularly common in Mongolian and Tibetan medicines and has good antibacterial, antiallergic and antiviral effects. It also contains 30% to 40% tannins and has good antioxidant effects. Currently, blue light absorbers on the market are mostly used in the industrial field, and are less common in the cosmetic field. Plant-based blue light absorbers are even rarer, and there is no report on the blue light absorption of Terminalia chebula. Therefore, the use of Terminalia chebula in the cosmetics market has great promotion potential.

[0007] Epigallocatechin gallate (EGCG) is the main component of green tea polyphenols. It is a catechin monomer extracted from tea leaves and has anti-inflammatory, antibacterial, and anti-cancer effects. The antioxidant activity of EGCG is also often reported. However, due to its instability during storage and the possibility of skin allergy at high concentrations, the aim is to achieve better antioxidant effects while reducing the concentration of EGCG by compounding a natural ingredient. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the defects of the prior art that epigallocatechin gallate is sensitizing at high concentrations and limits its use, and to provide a skin topical composition and its use as a blue light absorber and antioxidant. The skin topical composition of the present invention has better blue light absorption and antioxidant effect than the single component Terminalia chebula fruit extract of the same mass, and has a good antioxidant effect compared to the single component EGCG of the same mass.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] A composition for external use on skin, comprising a terminalia chebula fruit extract, EGCG and a solvent, wherein the mass ratio of the terminalia chebula fruit extract to the EGCG is (0.01-300):1;

[0011] In the skin external composition, the mass fraction of the Terminalia chebula fruit extract is 0.005% to 1%, and the mass fraction refers to the mass ratio of the Terminalia chebula fruit extract to the solvent.

[0012] In the present invention, the EGCG refers to epigallocatechin gallate.

[0013] In the present invention, the EGCG can be conventional in the art, such as EGCG with a purity of more than 99%, or a plant extract containing EGCG, such as tea extract, as long as an excellent skin topical composition with blue light absorption and antioxidant effects can be obtained. Wherein, the mass ratio of the terminalia chebula fruit extract to the EGCG is (0.01-300): 1 The mass of "the EGCG" is based on the mass of pure EGCG.

[0014] In the present invention, the mass ratio of the terminalia chebula fruit extract to the EGCG is preferably (0.33-100):1, for example, 2:1, 3.33:1, 1.67:1, 10:1, 15:1, 20:1, 23.3:1 or 50:1.

[0015] In the present invention, the mass fraction of the Terminalia chebula fruit extract is preferably 0.005% to 0.3%, such as 0.01%, 0.03%, 0.07%, 0.1% or 0.2%.

[0016] In the present invention, the Terminalia chebula fruit extract preferably contains 30% to 40% tannins, where % is the percentage of the mass of tannins to the total mass of the Terminalia chebula fruit extract.

[0017] In the present invention, the terminalia chebula fruit extract can be conventional in the art, for example, commercially available or homemade.

[0018] Among them, the commercially available one is, for example, purchased from Sytheon Corporation in the United States.

[0019] Among them, the preparation method of the homemade terminalia chebula fruit extract preferably includes the following steps: extracting terminalia chebula fruit powder with water to obtain a residue and a filtrate, extracting the residue with water, and combining the filtrates obtained from the two water extractions; the solid-liquid ratio during the water extraction is 1: (9-11), for example 1:10.

[0020] The terminalia chebula fruit powder is obtained by, for example, crushing dry terminalia chebula fruits from the Pan-Himalayan region and then passing through a 60-mesh sieve.

[0021] The water extraction temperature is preferably 90-100°C, for example 90°C.

[0022] The water extraction time is preferably 1.5 to 2.5 hours, for example 2 hours.

[0023] In the present invention, the mass fraction of the EGCG is preferably 0.001% to 0.5%, for example 0.005%, 0.003%, 0.002%, 0.001% or 0.3%, and the mass fraction refers to the mass ratio of the EGCG to the solvent.

[0024] In the present invention, the EGCG can be commercially available EGCG commonly used in the art, for example, purchased from Shanghai Huilang Company.

[0025] In the present invention, the solvent may be conventional in the art, generally a solvent commonly used in cosmetics, such as one or more of ethanol, isopropanol and water. The water is generally deionized water.

[0026] In the present invention, the skin external preparation generally further comprises a dispersant.

[0027] In the present invention, preferably, the mass fraction of the terminalia chebula fruit extract is 0.1% to 0.2%, and the mass fraction of the EGCG is 0.001% to 0.002%. Compared with a single component, this specific combination can achieve a better blue light absorption effect.

[0028] In the present invention, preferably, the mass fraction of the terminalia chebula fruit extract is 0.005% to 0.01%, and the mass fraction of the EGCG is 0.003% to 0.005%. This specific combination has stronger antioxidant properties than a single component.

[0029] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.005%, the mass fraction of the EGCG is 0.005%, and the solvent is, for example, deionized water.

[0030] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.01%, the mass fraction of the EGCG is 0.003%, and the solvent is, for example, deionized water.

[0031] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.03%, the mass fraction of the EGCG is 0.002%, and the solvent is, for example, deionized water.

[0032] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.07%, the mass fraction of the EGCG is 0.003%, and the solvent is, for example, deionized water.

[0033] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.1%, the mass fraction of the EGCG is 0.001%, and the solvent is, for example, deionized water.

[0034] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.2%, the mass fraction of the EGCG is 0.002%, and the solvent is, for example, deionized water.

[0035] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.01%, the mass fraction of the EGCG is 0.005%, and the solvent is, for example, deionized water.

[0036] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.01%, the mass fraction of the EGCG is 0.001%, and the solvent is, for example, deionized water.

[0037] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.005%, the mass fraction of the EGCG is 0.003%. The solvent is, for example, deionized water.

[0038] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.1%, the mass fraction of the EGCG is 0.03%, and the solvent is, for example, deionized water.

[0039] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.1%, the mass fraction of the EGCG is 0.3%, and the solvent is, for example, deionized water.

[0040] In a preferred embodiment of the present invention, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 1%, the mass fraction of the EGCG is 0.3%, and the solvent is, for example, deionized water.

[0041] In the present invention, the preparation method of the skin external composition can be conventional in the art, and the method comprises mixing the terminalia chebula fruit extract, the EGCG and the solvent.

[0042] The mixing order may be, for example, first mixing the terminalia chebula fruit extract with the EGCG and then mixing with the solvent; or first mixing the terminalia chebula fruit extract and the EGCG with the solvent separately and then combining and mixing them.

[0043] Those skilled in the art can dilute the solution of the terminalia chebula fruit extract and the solution of EGCG to obtain the composition containing the terminalia chebula fruit extract that meets the experimental requirements.

[0044] The present invention also provides an application of the skin external composition as a blue light absorber or an antioxidant.

[0045] The present invention also provides a use of the skin external composition as a blue light absorbing component or an antioxidant component in the preparation of skin external preparations.

[0046] The present invention also provides a skin external preparation, which comprises the skin external preparation.

[0047] In the present invention, the skin preparation is preferably a cosmetic that does not have the purpose of treating diseases, and the cosmetic can be a conventional cosmetic in the art, preferably one or more of lotion, essence, emulsion and cream. In the skin preparation, except for the skin composition of the present invention, the remaining components are conventional components in the art, such as deionized water and one or more of functional active ingredients, moisturizers, oils, emulsifiers, thickeners, preservatives, stabilizers, fragrances, pigments and pH regulators.

[0048] In the present invention, the content of the skin external composition in the skin external preparation is preferably 0.001-2%, for example 0.13%, where the percentage is the percentage of the dry weight of the skin external composition to the total weight of the skin external preparation.

[0049] When the skin topical preparation is a lotion, the lotion, for example, includes the following components: 5% glycerin, 3-6% polyol A, 1% betaine, 0.5% panthenol, 0.05-0.1% dipotassium glycyrrhizinate, 0.5% polyethylene glycol-32, 0.15-0.2% surfactant A, 0.4-0.45% preservative A, 0-0.15% xanthan gum, 0.03% fragrance and 0.0001%-10% fermentation supernatant, 0.001-2% of the skin topical composition; the rest is supplemented to 100% with deionized water. Wherein, the polyol A is one or more of butylene glycol, 1,2-pentanediol and dipropylene glycol. The surfactant A is one or more of polysorbate-20 and PEG-40 hydrogenated castor oil. The preservative A is methylparaben and phenoxyethanol.

[0050] When the skin topical preparation is an essence, the essence, for example, includes the following components: 5% glycerol, 3-6% polyol B, 1% panthenol, 0.1% dipotassium glycyrrhizinate, 0.1% allantoin, 0.05-3.6% surfactant B, 0.25-0.45% preservative B, 0.2-0.4% xanthan gum, 0.3% acrylic resin Pemulen TR-2, 0.05% fragrance, 0.0001%-10% fermentation supernatant, 0.001~2% of the skin topical composition, and the rest is supplemented to 100% with deionized water. Wherein, the polyol B is butylene glycol and / or 1,3-propylene glycol. The surfactant B is polyglycerol-2 oleate and / or polyglycerol-10 oleate. The preservative B is any two or three of propylparaben, methylparaben and phenoxyethanol. The acrylic resin Pemulen TR-2 is an acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer.

[0051] When the skin topical preparation is an emulsion, the emulsion, for example, includes the following components: 5% glycerol, 5% 1,3-propylene glycol, 1% panthenol, 0.5% tocopherol acetate, 2% polydimethylsiloxane, 2% emulsifier A165, 2% oil C, 3% isopropyl isostearate, 0.5% cetearyl alcohol, 0.3% preservative C, 0.5% xanthocollagen, 0.1% disodium EDTA, 0.0001%-10% fermentation supernatant, 0.001~2% of the skin topical composition; the rest is supplemented to 100% with deionized water. The skin topical preparation also contains triethanolamine, and the content of the triethanolamine is based on adjusting the pH value of the skin topical preparation to 5.5-7; wherein the oil C is mineral oil and / or isohexadecane. The preservative C is methylparaben and / or propylparaben. The emulsifier A165 is a commercially available product, which contains glyceryl stearate and PEG-100 stearate. The xanthan gum is a sugar, an extracellular microbial polysaccharide produced by fermentation of Xanthomonas.

[0052] When the skin topical preparation is a cream, the cream, for example, includes the following components: 5% glycerol, 5% 1,3-propylene glycol, 1% panthenol, 0.5% tocopheryl acetate, 2% polydimethylsiloxane, 2% emulsifier A165, 2% oil D, 3% isopropyl isostearate, 2% cetearyl alcohol, 0.3% preservative D, 2% hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, 0.1% disodium EDTA, 0.0001%-10% fermentation supernatant, 0.001~2% of the skin topical composition; the rest is supplemented to 100% with deionized water. The skin topical preparation also contains triethanolamine, and the content of the triethanolamine is based on adjusting the pH value of the skin topical preparation to 5.5-7; wherein the oil D is mineral oil and / or isohexadecane; the preservative D is methylparaben and / or propylparaben.

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0054] The reagents and raw materials used in the present invention are commercially available.

[0055] The positive and progressive effect of the present invention is that the application of the terminalia chebula fruit extract and the compound of terminalia chebula fruit extract and EGCG in blue light absorption has not been reported, and experiments have shown that the compound has an enhanced effect in anti-oxidation, and has the dual effects of blue light absorption and anti-oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : is the blue light absorption spectrum of the composition for external use on skin in Effect Example 1.

[0057] Figure 2 The results of the DPPH free radical scavenging experiment of the skin external composition in Effect Example 2 are shown.

[0058] Figure 3 The effect of the skin external application composition in Effect Example 3 on the production of ROS by HACAT cells induced by H2O2.

[0059] Figure 4 In Effect Example 3, the change in ROS concentration in HACAT cells under the action of the skin external application composition of the present invention was analyzed by flow cytometry.

[0060] Figure 5 The effect of the skin external application composition in Effect Example 4 on the production of ROS by UVB-induced HACAT cells.

[0061] Figure 6 The DPPH free radical scavenging test result of the skin external composition with a mass fraction of 0.0103% in Effect Example 2. DETAILED DESCRIPTION

[0062] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0063] Reagents and equipment:

[0064] DPPH, Sigma, USA;

[0065] Epigallocatechin gallate (EGCG), Shanghai Huilang Company;

[0066] L-ascorbic acid (VC), Sinopharm Chemical Reagent Co., Ltd.;

[0067] DMEM medium and Sijiqing serum were from Thermofisher, USA;

[0068] Reactive oxygen detection kit, Shanghai Bio-Tech Biotechnology Co., Ltd.;

[0069] HACAT cells, ATCC, USA;

[0070] UV-visible spectrophotometer, Beijing Puxi General Instrument Co., Ltd.;

[0071] Fluorescence microplate reader, Perkin Elmer, USA;

[0072] Flow cytometer, Thermofisher, USA.

[0073] Bio Sun UV irradiation system, VILBER, France;

[0074] Terminalia chebula fruit extract (hereinafter referred to as: Terminalia chebula), Sytheon Company, USA;

[0075] The terminalia chebula fruit extract can also be prepared by itself. The specific preparation process is as follows: 100 g of dry terminalia chebula fruit produced in the Pan-Himalayan region is taken, cleaned, dried, crushed by a high-speed grinder, and passed through a 60-mesh sieve, 50 g of powder is weighed in a round-bottom flask, and 500 mL of deionized water is added according to a solid-liquid ratio of 1:10, and stirred for extraction, the extraction temperature is 90°C, and the extraction time is 2 hours. The filtrate is collected by centrifugation, and 500 mL of deionized water is added to the filter residue, and stirred for extraction for 2 hours at a temperature of 90°C, and the filtrate is collected by centrifugation. The two filtrates are combined, and the filtrate is freeze-dried to obtain the terminalia chebula fruit extract.

[0076] Statistical analysis: All experiments were performed three times, with at least three parallel samples in each experimental group. Data are presented as mean ± standard deviation (Mean ± SD). Calculated by two-tailed t test P value, P <0.05 was considered to be significantly different. P <0.01; "***" represents P <0.001; “****” represents P <0.0001. Example

[0077] 40 mg of Terminalia chebula fruit extract and 1 mg of EGCG were weighed and added to 10 g of deionized water respectively to prepare a test solution with a mass fraction of 0.4% of Terminalia chebula fruit extract and 0.01% of EGCG. The two test solutions were then diluted according to specific experimental requirements and mixed to obtain a composition as a blue light absorber and antioxidant.

[0078] Effect Example 1 Blue light spectrum absorption

[0079] The test solution with a mass fraction of 0.4% Terminalia chebula fruit extract and the test solution with a mass fraction of 0.01% EGCG in Example 1 were diluted and finally formulated into the compositions shown in Table 1 below. The baseline was adjusted with water, and the blue light spectra of each sample of the same volume were scanned in the range of 400-500 nm.

[0080] Table 1

[0081]

[0082] Note: “0.005%+0.005%” means that the mass fraction of the Terminalia chebula fruit extract and the mass fraction of EGCG in the composition is 0.005%, and the same meaning shall apply to other compositions.

[0083] Depend on Figure 1 It can be seen that the composition of the present invention has a strong absorption capacity for blue light with a wavelength of 400-450nm and a high absorbance, and the 400-450nm band is a band in blue light that is harmful to human skin. As the concentration of the terminalia chebula fruit extract in the composition increases, the absorbance increases, and the absorbance is positively correlated with the concentration. When the concentration of the terminalia chebula fruit extract is 0.2% and the concentration of EGCG is 0.002%, the absorbance can reach 1.41, indicating that the composition of the terminalia chebula fruit extract and EGCG has a good ability to absorb blue light, and achieves a blue light protection effect by reducing the transmittance of blue light.

[0084] The skin senses blue light through opsin photoreceptors on cells. Excessive exposure to blue light can cause opsin aggregation in cells, causing cell damage and delaying barrier repair.

[0085] The composition plays a role in blue light protection in two important aspects: first, the chemical structure of the terminalia chebula fruit extract contains conjugated double bonds, and the unique chemical structures of the various compounds contained in the terminalia chebula fruit extract and their cooperation can be more conducive to the absorption of blue light bands, have a filtering effect on blue light, and can weaken the intensity of blue light. As a filter for high-energy blue light, it directly absorbs blue light and plays a protective role. Second, as an antioxidant, it plays a role in anti-oxidative damage by removing UVA, UVB, and blue light-induced active oxygen species.

[0086] Effect Example 2 Scavenging DPPH Free Radicals

[0087] At present, the most commonly used experiment in in vitro antioxidant research is the DPPH free radical scavenging experiment. The free radical has the maximum absorbance at 517nm. When antioxidant substances are added, the antioxidant capacity of the test substance is compared by observing the degree of light change in the solution color.

[0088] The specific experimental process is as follows:

[0089] (1) Preparation of DPPH test solution: Weigh 4 mg of DPPH reagent and dilute to 100 mL with anhydrous ethanol to obtain a 0.1 mmol / L DPPH solution. Store in a brown bottle for later use.

[0090] (2) Sample group preparation: The samples were dissolved in pure water to prepare 0.01% and 0.005% Terminalia chebula fruit extract by mass; 0.005%, 0.003% and 0.001% EGCG by mass; finally, the sample solution of the composition shown in Table 2 was prepared, and then 0.2 mL of the sample solution was taken and 2.8 mL of DPPH reaction solution was added to each sample group; 0.2 mL of the sample solution was taken in the blank group and 2.8 mL of anhydrous ethanol was added; 0.2 mL of pure water was taken in the control group and 2.8 mL of DPPH reaction solution was added; after mixing, the mixture was placed in a dark place at room temperature for 30 min, and the absorbance at 517 nm was measured.

[0091] (3) Preparation of the positive group: 2 mg / mL vitamin C solution (VC solution) was prepared into different concentrations of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, and 2.8 mL of DPPH reaction solution was added to compare the clearance rate with the sample.

[0092] (4) Data processing: DPPH clearance rate (%) = (1-(A1-A 空 ) / (A2-A 空 ))×100

[0093] Where A1 is the absorbance of the sample solution at 517 nm, A 空 is the absorbance value of the blank group at 517nm, and A2 is the absorbance value of the reference substance at 517nm. The test results are shown in Table 2 and Figure 2 shown.

[0094] Table 2

[0095]

[0096] According to the above table and Figure 2 It can be seen that the combination of a specific content of Terminalia chebula fruit extract and EGCG has a significant improvement in the DPPH clearance rate compared to the individual components under the same conditions, which can be as high as more than 90%.

[0097] In order to compare the DPPH scavenging effect of the composition with that of the monomer at the same concentration, a DPPH free radical scavenging experiment was further conducted with 0.0103% of Terminalia chebula and 0.0103% of EGCG. The test results are shown in Tables 3 and Figure 6 shown.

[0098] Table 3

[0099]

[0100] Effect Example 3 Active Oxygen Scavenging

[0101] Reactive oxygen species (ROS) were detected by fluorescent probe DCFH-DA to compare the antioxidant effects of single components of Terminalia chebula fruit extract and EGCG and their mixtures.

[0102] The specific experimental process is as follows:

[0103] (1) Sample solution preparation: The sample was dissolved in pure water and prepared into 0.01% and 0.005% of Terminalia chebula fruit extract and 0.005% and 0.003% of EGCG, respectively; and finally, the sample solution of the composition shown in Table 4 was prepared.

[0104] (2) Cell culture: Normal human keratinocytes HACAT were grown in T175 flasks using DMEM medium + 10% sera from Sijiqing + 1% penicillin / streptomycin / amphotericin. For the ELISA quantitative experiment and flow cytometry experiment, 2×10 4 The density of 10 cells / well was used in 96-well plates and 2×10 6 The cells were seeded at a density of 100 μg / well in a 6-well plate. After 24 hours, the cells were pretreated with serum-free medium to prepare different concentrations of sample solution for 2 hours, then the original culture medium was removed, and the fluorescent probe DCFH-DA with a final concentration of 10 μM / L was added to the cells for 30 minutes, and then the subsequent reactive oxygen induction and detection experiments were carried out. The cells were kept incubated in an incubator at 37°C with 5% CO2.

[0105] (3) H2O2-induced ROS experiment: After the probe incubation, a certain concentration of H2O2 was added to treat the cells for 30 min. The absorbance was measured at 485 / 535 nm using a fluorescence microplate reader. For the cells that were subsequently processed by flow cytometry, they were digested with trypsin, washed with PBS, and then tested on the flow cytometer.

[0106] In order to reflect the inhibitory effect of single chebula fruit extract and EGCG and the compound of chebula fruit extract and EGCG on ROS, the present invention sets single chebula fruit extract and EGCG and their compound as experimental groups. In addition, a blank experimental group (abbreviated as NT) and a control group (i.e. VC, concentration of 0.02%) are also set up to represent the ROS level before H2O2 induction and the inhibitory effect of VC on ROS. The test results are shown in Table 4 below.

[0107] Table 4

[0108]

[0109] like Figure 3 As shown, in the H2O2-induced oxidative damage model, all experimental groups effectively reduced the ROS produced by H2O2 stimulation, and the inhibitory effects of Terminalia chebula extract, EGCG, and the combination of the above substances were better than those of the positive control VC.

[0110] Specifically, the inhibition rates of the two groups of compound products, 0.01% Terminalia chebula fruit extract + 0.003% EGCG and 0.005% Terminalia chebula fruit extract + 0.005% EGCG, were 76% and 67%, respectively. Under the same conditions, the inhibition rates of their respective single components were 52% and 46%, respectively. It can be found that the inhibitory effect of the combination on ROS levels is far better than that of a single component.

[0111] In order to further verify the antioxidant effect of the composition, we performed flow cytometry analysis.

[0112] like Figure 4 As shown, when not stimulated with H2O2 (NT group), the fluorescence intensity of the blank group was 10 3 When stimulated by H2O2, the fluorescence intensity of the cells shifted to the right, indicating that a higher level of ROS was produced in the cells. The experimental groups showed that the cells in the experimental groups tended to shift to the initial state, especially the 0.01% Terminalia chebula fruit extract + 0.003% EGCG compound group ( Figure 4 The 0.01+0.003 Terminalia chebula+EG group) was closest to the state of cells without any treatment, proving that the composition containing Terminalia chebula fruit extract had a significant effect in inhibiting reactive oxygen species.

[0113] Effect Example 4 UV-induced ROS experiment

[0114] The UVB-induced cell photooxidation model was used to expand the antioxidant application of Terminalia chebula fruit extract and EGCG.

[0115] By the same method as in Example 3, after the probe incubation, 15 mJ UVB was used to induce the generation of reactive oxygen species, followed by PBS washing, and the absorbance was measured at 485 / 535 nm by a fluorescent microplate reader. The test results are shown in FIG. Figure 5 The specific fluorescence intensity data of each experimental group are shown in Table 5 below.

[0116] Table 5

[0117]

[0118] The results showed that, consistent with the results of the H2O2-induced oxidative damage model, in the photo-oxidation model, all experimental groups significantly eliminated UVB-induced ROS. When the different concentrations of the compound were compared with their respective monomers, the compound of 0.01% chebula fruit extract + 0.003% EGCG and 0.005% chebula fruit extract + 0.005% EGCG showed significant protective effects on cells in the photo-oxidation model.

[0119] The present invention uses an ultraviolet-visible spectrophotometer to confirm that the composition of the present invention has the function of absorbing blue light through the absorption capacity of the test object in the 400-500nm band, and the composition of the present invention is prepared into solutions of different concentrations to detect the scavenging effect of DPPH free radicals and the inhibitory effect of reactive oxygen species (ROS), and the results show that the composition of the present invention has enhanced antioxidant effect.

Claims

1. A composition for external use on skin, characterized in that: It includes terminalia chebula fruit extract, EGCG and a solvent; The mass fraction of the terminalia chebula fruit extract is 0.01%, and the mass fraction of the EGCG is 0.003%; Alternatively, the mass fraction of the Terminalia chebula fruit extract is 0.005%, and the mass fraction of the EGCG is 0.005%.

2. The composition for external use on skin according to claim 1, wherein The terminalia chebula fruit extract contains 30% to 40% tannins, where % is the percentage of the mass of tannins to the total mass of the terminalia chebula fruit extract; The preparation method of the terminalia chebula fruit extract comprises the following steps: extracting terminalia chebula fruit powder with water to obtain a filter residue and a filtrate, extracting the filter residue with water, and combining the filtrates obtained by the two water extractions; the solid-liquid ratio during the water extraction is 1:(9-11).

3. The composition for external use on skin according to claim 2, wherein The solid-liquid ratio during the water extraction is 1:

10.

4. The composition for external use on skin according to claim 2, characterized in that The water extraction temperature is 90-100°C.

5. The composition for external use on skin according to claim 4, characterized in that The temperature of the water extraction was 90°C.

6. The composition for external use on skin according to claim 2, wherein The water extraction time is 1.5 to 2.5 hours.

7. The composition for external use on skin according to claim 6, wherein The water extraction time is 2h.

8. The composition for external use on skin according to claim 1, wherein The skin external composition comprises the following components: a terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.005%, the mass fraction of the EGCG is 0.005%; the solvent is deionized water; Alternatively, the skin topical composition comprises the following components: terminalia chebula fruit extract, EGCG and a solvent; the mass fraction of the terminalia chebula fruit extract is 0.01%, the mass fraction of the EGCG is 0.003%; and the solvent is deionized water.

9. The composition for external use on skin according to any one of claims 1 to 8, wherein The method for preparing the skin external composition comprises the following steps: mixing the terminalia chebula fruit extract, the EGCG and the solvent.

10. The composition for external use on skin according to claim 9, wherein The mixing is firstly mixing the Terminalia chebula fruit extract with the EGCG and then mixing with the solvent; Alternatively, the mixing is performed by mixing the Terminalia chebula fruit extract and the EGCG with the solvent respectively, and then combining and mixing them together.

11. Use of the composition for external use on skin according to any one of claims 1 to 10 as a blue light absorbing component and / or an antioxidant component in the preparation of an external preparation for skin.

12. A skin external preparation, characterized in that: The composition comprises the composition for external use on skin according to any one of claims 1 to 10.

13. The external skin preparation according to claim 12, wherein The skin external preparation is one or more of lotion, essence, emulsion and cream; In the skin external preparation, the content of the skin external composition is 0.001% to 2%, where % is the percentage of the dry weight of the skin external composition to the total weight of the skin external preparation.

14. The external skin preparation according to claim 13, wherein In the skin external preparation, the content of the skin external composition is 0.13%.

Images