Application of flavonoid piperazine ester or salt compound thereof in cosmetics

Abstract

The invention provides a healthy, safe and effective new raw material for cosmetics, namely flavone piperil ester or a salt compound thereof. The flavonoid piperazine ester can well inhibit the activity of hyaluronidase and increase the content of collagen in cells, so that the flavonoid piperazine ester has the effects of relieving, moisturizing and resisting wrinkles, meanwhile, the flavonoid piperazine ester shows remarkable DPPH free radical scavenging capacity, and the flavonoid piperazine ester is a good antioxidant. The composition can be used for preparing skin external preparations and is a good cosmetic raw material.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a compound flavoxate for moisturizing, soothing, anti-wrinkle, firming and anti-inflammatory. The present invention also relates to a cosmetic composition comprising flavoxate or a salt thereof. Technical Background

[0002] Flavoxate, chemical name: 3-methylflavone-8-carboxylic acid-2-piperidinyl ethyl ester, is a class of synthetic flavonoids. Hydrochloride is the common application form of flavoxate. Flavoxate hydrochloride is the first antispasmodic drug approved by the U.S. Food and Drug Administration for the treatment of overactive bladder. Its efficacy and safety have been tested in several clinical trials. 1 .

[0003] So far, there is no report on the application of flavopiprate in cosmetics in the prior art, nor is there any report on its moisturizing, soothing, anti-wrinkle, firming and anti-inflammatory activities.

[0004] 【1】Arcaniolo D, Conquy S, Tarcan T. Flavoxate: present and future. Eur RevMed Pharmacol Sci. 2015; 19(5): 719-31. PMID: 25807422. Summary of the invention

[0005] The object of the present invention is to provide a flavoxate or its salt compound having multiple functions such as moisturizing, soothing, anti-wrinkle, firming, anti-inflammatory and the like, and its application in cosmetics.

[0006] Flavonoxate or its salt compounds are currently only used in the pharmaceutical industry. However, the inventors have found that the use of flavoxate or its salts in cosmetics can inhibit the activity of hyaluronidase. It can increase the collagen content in cells. It can significantly remove DPPH free radicals. Therefore, flavoxate or its salt compounds have multiple skin beautifying functions, such as moisturizing, soothing, anti-wrinkle, firming, and anti-inflammatory.

[0007] Therefore, one purpose of the present invention is to directly add flavoxate or its salt compounds into cosmetics.

[0008] According to a preferred embodiment of the present invention, the cosmetics are in the form of moisturizing cosmetics, anti-inflammatory cosmetics, anti-acne cosmetics, anti-aging cosmetics or other functional cosmetics.

[0009] According to a preferred embodiment of the present invention, the cosmetics include soap, facial cleanser, shampoo, shower gel, skin softener, skin care gel, skin care lotion, skin care cream, essence, eye cream, facial mask and other cosmetic forms. These forms can be prepared by methods well known to those skilled in the art.

[0010] According to a preferred embodiment of the present invention, flavoxate or its salt compounds can be used as auxiliary materials in the field of cosmetics. The auxiliary materials acceptable in the field of cosmetics are: solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, moisturizers, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / propellants, flavors and pigments.

[0011] According to a preferred embodiment of the present invention, the concentration of the flavoxate or its salt compound used as a cosmetic or cosmetic excipient is any concentration from 0.1 mg / ml to 200 mg / ml.

[0012] According to the present invention, it is found that flavoxate or its salt compound can be used directly for skin beautification, and can also be added to cosmetics as an effective additive. Therefore, another object of the present invention is to provide a cosmetic composition, which contains flavoxate or its salt compound and an excipient acceptable in the cosmetic field.

[0013] The cosmetic auxiliary materials are: solvent, solubilizer, preservative, antioxidant, pH regulator, penetration enhancer, liposome, moisturizer, thickener, chelating agent, skin feel regulator, surfactant, emulsifier, propellant / propellant, flavor and pigment.

[0014] According to an embodiment of the present invention, the flavoxate or its salt compound accounts for 0.01% to 20% of the total weight of the above-mentioned composition. In a preferred embodiment, the flavoxate or its salt compound accounts for 0.1% to 10% of the total weight of the composition, more preferably 0.5% to 5%, and most preferably 0.5% to 2%. In summary, flavoxate or its salt can be used to prepare skin external preparations and is a good cosmetic raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a data table showing the cell viability of cells treated with flavoxate in Example 1;

[0016] Figure 2 is a data table of the inhibition rate of hyaluronidase by the test samples in Example 2;

[0017] Figure 3 is a data table of the expression of collagen I content of the test samples in Example 3;

[0018] Figure 4This is a data table showing the scavenging of DPPH free radicals by flavoxate in Example 3. DETAILED DESCRIPTION

[0019] In order to make the objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental reagents and materials used in the present invention can be obtained commercially.

[0021] Example 1 Cytotoxicity Experiment

[0022] The CCK-8 assay is a method for detecting cell survival and growth. The measured OD value is proportional to the cell activity. The higher the cell activity, the greater the value. Conversely, when the cell activity is significantly less than 80%, it is considered to have obvious cytotoxicity.

[0023] Reagents and materials:

[0024] Cell counting kit-8 (CCK8), dimethyl sulfoxide (DMSO), DMEM culture medium, fetal bovine serum, phosphate buffered saline (PBS).

[0025] instrument:

[0026] ELISA reader, CO2 incubator, clean bench.

[0027] Cell lines:

[0028] Human keratinocytes (HaCaT).

[0029] Samples to be tested:

[0030] Drug group: flavoxate

[0031] Blank control group: PBS

[0032] Experimental methods:

[0033] 1. Collect cells in logarithmic growth phase, adjust the concentration of cell suspension, add 100ul to each well of 96-well plate, and make the cell density to be tested 3*10^3 cells / well.

[0034] 2. Culture the cells at 5% CO2 and 37°C for 24 and 48 hours.

[0035] 3. Add 10ul of different concentrations of flavonoids and incubate the culture plate in the incubator for 24 hours

[0036] 4. Add 10ul CCK-8 solution to each well.

[0037] 5. Continue incubating in the cell culture incubator for 1.5 hours.

[0038] 6. Measure the absorbance of each well at 450 nm using an enzyme reader.

[0039]

[0040] like Figure 1 As shown, the cell viability rates of 0.005%, 0.01%, 0.02% and 0.04% flavoxate in 24 hours were 98.78%, 95.12%, 95.54% and 93.53%, respectively, all of which were within normal levels. It can be seen that flavoxate has no toxic effect on cells, and its safety as a raw material for skin cosmetics is guaranteed.

[0041] Example 2 Hyaluronidase inhibition experiment

[0042] Experimental instruments and reagents:

[0043] Spectrophotometer, phosphate buffered saline (PBS), hyaluronic acid, hyaluronidase, CaCl 2 , acetylacetone solution, anhydrous ethanol, Ehrlich reagent.

[0044] Experimental methods:

[0045] The experiment was divided into four groups: the total reaction volume of each group was the same

[0046] Blank control group A1 PBS+hyaluronidase A2, blank control group without enzyme PBS + solvent without hyaluronidase Sample Group B1 Test sample + hyaluronidase Sample group B2 without enzyme Test sample + solvent without hyaluronidase

[0047] According to the group, 100 μL of CaCl 2 (2.5mmol / L) was mixed with 500μL hyaluronidase (500U / mL) or a solvent without hyaluronidase, and 20μL was placed in a 37℃ constant temperature air bath for 20min; 20μL PBS was added to the blank control group and placed in a 37℃ constant temperature air bath for 20min; 20μL flavopiprate solution of relevant concentration was added to the sample group and placed in a 37℃ constant temperature air bath for 20min; then 40μL hyaluronic acid was added to each group and placed in a 37℃ constant temperature air bath for 40min; then 10μL acetylacetone solution was added and placed in a 50℃ constant temperature air bath for 10min, and then dried in a 75℃ oven for 40min and a 50℃ oven for 10min. After the reaction was completed and cooled, the plastic wrap was quickly torn off to avoid the generation of water vapor, and then 100μL 96% anhydrous ethanol and 10μL Ehrlich reagent were added, and the absorbance was measured at 492nm.

[0048]

[0049] Experimental results:

[0050] Hyaluronidase can decompose hyaluronic acid in the body, turning it into a low molecular weight acidic irritant, thereby causing histamine release and inducing sensitive symptoms in the body. In addition, the reduction of hyaluronic acid will destroy the barrier function of the skin and cause water loss inside the skin. Therefore, inhibiting the activity of hyaluronidase can achieve the effects of soothing, moisturizing, and skin repair. The results of the test samples' effects on the inhibition rate of hyaluronidase are shown in Figure 2 The results showed that the inhibition rate at 0.01% was 15.88%, and the inhibition rate at 0.02% was 31.48%. It can be seen that flavoxate has the effect of inhibiting the activity of hyaluronidase, which proves that flavoxate has the effects of soothing, moisturizing and skin repairing.

[0051] Example 3 Collagen content test

[0052] Reagents and materials:

[0053] Fetal bovine serum, DMEM medium, phosphate buffer, trypsin, RIPA lysis buffer, collagen I ELISA kit, BCA protein kit.

[0054] instrument:

[0055] ELISA reader, CO 2 Incubator, clean bench.

[0056] Cell lines:

[0057] Human keratinocytes (HaCaT).

[0058] Experimental methods:

[0059] Take HaCaT cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to make the adherent cells fall off, and count 1-4×10 6 / mL to prepare a cell suspension.

[0060] Follow 10 5 The cells were seeded in 6-well plates until they grew to about 80% confluence to establish a UV light aging model.

[0061] The blank control group was added with 200 μL PBS, supplemented with culture medium to 800 μL, and no UV irradiation was performed;

[0062] UV group and sample group, add appropriate amount of PBS to wash repeatedly until colorless, add 200μL PBS, place under 80mJ / cm2 UV lamp for irradiation, the distance between the lamp source and the culture bottle is 15cm. After irradiation, discard PBS, add PBS solution and culture medium to 800μL in UV group, add culture medium and relevant concentration sample to 800μL in sample group. Blank control group, UV group, sample group continue to be 37℃, 5%CO 2 Incubate in the incubator for 48 h.

[0063] After the culture, scrape the cells with a cell scraper, mix well by pipetting, extract cell protein with RIPA, quantify with BCA to 1 mg / mL, and operate according to the collagen I ELISA operating instructions. Measure the OD value of each well at 450 nm with a microplate reader within 15 minutes.

[0064] Experimental results:

[0065] Collagen is a structural protein and the most abundant protein in the human body. It is present in tissues such as skin, bones, and muscles, and has the functions of supporting tissue structure and protecting skin. In an ultraviolet overexposure environment, the activities of collagenase and elastase increase significantly, elastin is hydrolyzed, and the synthesis of collagen is also inhibited. In this experiment, the test sample was used to treat cells irradiated with ultraviolet rays, and the collagen I content in the corresponding cells was detected to determine whether the flavopiridol of the present invention can promote the generation of collagen.

[0066] The results of the test samples on the collagen I content are shown in Figure 3 The results showed that compared with the blank control group, the collagen content in the UV group was reduced; compared with the UV group, flavopiprate significantly increased the expression of collagen I (p<0.01), promoting collagen production.

[0067] It can be seen from this that flavopiprate can increase the collagen content in cells after ultraviolet radiation, promote collagen production, thereby increasing skin elasticity and skin firmness, and delaying skin aging.

[0068] Example 4 DPPH free radical scavenging experiment

[0069] Free radicals are a class of active chemical substances with strong oxidative capacity. A large number of free radicals produced in the body can damage the skin and accelerate aging.

[0070] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH is weakened due to pairing with its single electron. The degree of fading of DPPH ethanol solution is quantitatively related to the number of electrons it accepts, which can be used to evaluate the ability of a substance to scavenge free radicals.

[0071] Flavonoids were mixed with DPPH (final concentration of 100 μM) for reaction, 3 replicate wells were set, and a blank control without drug was set at the same time, at 30°C for 1 hour, the OD value was measured by an enzyme marker, and the detection wavelength was 517 nm. The antioxidant rate was calculated.

[0072]

[0073] Experimental results:

[0074] like Figure 4 As shown in the experiment, flavonoids have a strong ability to scavenge free radicals, and their application in cosmetics can effectively improve skin aging problems caused by free radicals.

[0075] Example 5: Preparation of Essence Water

[0076]

[0077] Process: Mix phase A, stir evenly, and filter to obtain.

[0078] Example 6: Preparation of emulsion

[0079]

[0080]

[0081] Process: Heat phase A to 80°C, slowly add phase B which has been pre-mixed and heated to isothermal under homogenization, homogenize at a speed of 10,000 r / min for 3 minutes, add phase C to adjust pH 6-7, stir and cool to 45°C, add phase D which has been pre-heated to isothermal, stir and cool to 30°C.

[0082] Example 7: Preparation of facial mask:

[0083]

[0084] Process: Heat phase A to 80°C and keep warm for 10 minutes. Weigh phase B, stir and heat to 80°C. When phase B becomes a uniform solution, mix phase C, which has been pre-mixed and heated to 80°C, with phase B. After phase B and phase C are evenly mixed, add phase A and mix well. Then add phase D to adjust the pH to 6-7, stir and cool to 45°C, add phase E, which has been pre-heated to isotherm, stir and cool to 30°C.

[0085] Example 8: Preparation of spray

[0086]

[0087] Process: Heat deionized water to 60°C and add methylparaben. After dissolving, cool to 40°C, add other substances and stir until clear.

[0088] Example 9: Preparation of facial cream

[0089]

[0090] Process: Heat phase A to 80°C, slowly add phase B which has been pre-mixed and heated to isothermal under homogenization, homogenize at a speed of 10,000 r / min for 3 minutes, add phase C to adjust pH 6-7, stir and cool to 45°C, add phase D which has been pre-heated to isothermal, stir and cool to 30°C.

[0091] Example 10: Preparation of Shampoo

[0092]

[0093] Process: Heat phase A to 80°C, keep warm for 10 minutes, add phase B and stir evenly. Cool the uniform mixture of phase A and phase B to 45°C, add phase C to adjust pH to 6-7, add phase D preheated to 45°C, stir, and cool to 30°C.

[0094] Comparative Example 1

[0095] The essence water does not contain flavoxate hydrochloride, and the rest is the same as in Example 5, specifically:

[0096]

[0097] Process: Mix phase A, stir evenly, and filter to obtain.

[0098] The flavoxate hydrochloride compositions described in Examples 5-10 above have all been subjected to ① stability testing, in which the materials were placed under -20°C, 4°C, room temperature, 48°C, cycle (continuously changing four temperature conditions), and light conditions for 1 week, and the color and morphology of each material remained stable; ② skin safety testing, which confirmed that it was non-irritating to the skin and would not cause adverse reactions such as erythema, desquamation, stinging and burning, and could be used with confidence;

[0099] The repair effect on damaged skin was evaluated:

[0100] Specifically, volunteers were used for testing: 6 testers were recruited, half male and half female, aged 20-35 years old, and the test method was as follows:

[0101] 1) Tape irritation test: The tape was repeatedly applied to the inner skin of the forearm 15 times to damage the skin;

[0102] 2) Each tester applied Control Example 1 on his left hand and Example 5 on his right hand;

[0103] 3) After 15 minutes, clean and observe the evaluation records;

[0104] Scoring: A 5-point system is used, with 1 point - redness is more severe, 2 points - no improvement, 3 points - slight improvement, but obvious redness is still visible, 4 points - redness has basically disappeared, and 5 points - redness has completely disappeared.

[0105] No. 1 No. 2 No.3 No. 4 No. 5 No. 6 Example 5 4 4 5 4 5 4 Comparative Example 1 2 2 3 2 2 2

[0106] It can be seen from the above table that, compared with the control example 1, the embodiment 5 of the present invention can effectively repair the damaged skin, indicating that flavoxate hydrochloride has the effects of soothing, anti-inflammatory and repairing, and can be used as a skin protectant.

[0107] The above experimental results show that flavoxate has moisturizing, soothing, anti-wrinkle, firming and anti-inflammatory effects.

[0108] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Use of flavoxate or its salts in cosmetics, which can inhibit the activity of hyaluronidase.

2. The use of flavopiprate or its salts in cosmetics can increase the collagen content in cells.

3. The use of flavonoids or their salts in cosmetics can significantly scavenge DPPH free radicals.

4. The use of ketoxicate or its salt in cosmetics according to any one of claims 1 to 3, characterized in that: The flavoxate salts include flavoxate hydrochloride.

5. The use of flavoxate or its salt in cosmetics according to any one of claims 1 to 3, characterized in that: The cosmetic is one of moisturizing cosmetics, anti-inflammatory cosmetics, anti-acne cosmetics or anti-aging cosmetics.

6. The use of flavoxate or its salt in cosmetics according to any one of claims 1 to 3, characterized in that: The cosmetic is in the form of one of soap, facial cleanser, shampoo, shower gel, skin softener, skin care gel, skin care lotion, skin care cream, essence, eye cream, and facial mask.

7. The use of flavoxate or its salts in cosmetics according to claim 5, characterized in that: The cosmetics also contain auxiliary materials acceptable in the cosmetics field.

8. The use of flavoxate or its salts in cosmetics according to claim 7, characterized in that: The excipients acceptable in the cosmetic field are: solvents, solubilizers, preservatives, antioxidants, pH adjusters, penetration enhancers, liposomes, moisturizers, thickeners, chelating agents, skin feel regulators, skin protectants, surfactants, emulsifiers, propellants / propellants, flavors and pigments.

9. The use of flavoxate or its salts in cosmetics as claimed in claim 5, characterized in that: The final usage concentration of the flavoxate or its salt in any cosmetic is any concentration of 0.1 mg / ml to 200 mg / ml.

10. The use of flavoxate or its salts in cosmetics according to claim 7, characterized in that: The final usage concentration of the flavoxate or its salt compound in any cosmetic auxiliary material is any concentration of 0.1 mg / ml to 200 mg / ml.

11. A cosmetic composition, characterized in that The invention contains flavopiprate or its salt compounds, and excipients acceptable in the cosmetic field.

12. The cosmetic composition according to claim 11, characterized in that The proportion of flavoxate or its salt compound in the composition is 0.01% to 20%, and the proportion is by weight.

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