Method for improving skin conditions with veratric acid or acceptable salt thereof as an active ingredient

Abstract

A method for improving skin conditions containing applying a composition containing veratric acid or acceptable salt thereof as an active ingredient. The composition has an excellent effect of reducing skin wrinkles through molecular mechanisms including the promotion of collagen synthesis and inhibition of collagenase activity. Also, the composition has an excellent effect of reducing wrinkles by absorbing UVB to prevent UV rays from penetrating the skin, promotion of collagen synthesis and inhibition of collagenase activity. In addition, the composition has effect of promotion of hair growth and the prevention of hair loss through growth promotion of hair dermal papilla cells, stimulation of IGF-1 secretion, and inhibition of TGF-beta1 secretion. Also, because the composition is derived from plants such as natural materials, it is harmless to the human body and can be safely applied to cosmetic, pharmaceutical, and food compositions.

Claims

1. A method for reducing skin wrinkles, comprising: applying topically a composition comprising veratric acid or a salt thereof as an active ingredient to a person in need of reducing skin wrinkles, wherein the composition contains the veratric acid or salt thereof in an amount of 0.01-1.0 wt % based on the total weight of the composition.

2. The method of claim 1, wherein the composition has a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

3. A method for promoting hair growth comprising: applying topically a composition comprising veratric acid or a salt thereof as an active ingredient to a person in need of promoting hair growth, wherein the composition contains the veratric acid or salt thereof in an amount of 0.01-1.0 wt % based on the total weight of the composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the proliferation rate of hair follicle dermal papilla cells under various concentration of veratirc acid.

(2) FIG. 2 shows the secretion rate of IGF-1 under various concentration of veratric acid.

(3) FIG. 3 shows the inhibition rate of TGF-beta1 as a function of the concentration of veratric acid.

(4) FIG. 4 shows the synthesis of collagen under various concentration of veratric acid when human normal fibroblasts were irradiated with UVB.

(5) FIG. 5 shows the viability of cell under various concentration veratric acid when fibroblasts were irradiated with UVB.

(6) FIG. 6 shows the viability of cell under various concentration veratric acid when keratinocytes were irradiated with UVB.

(7) FIG. 7 is shows collagenase activity under various concentration of veratric acid when human normal fibroblasts were irradiated with UVB.

BEST MODE

(8) Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes and are not intended to limit the scope of the present invention.

Example 1: Preparation of Hair Growth Agent

(9) A hair growth agent containing varatric acid was prepared as a hydrogel base having the component and contents shown in Table 1 below. Concretely, an aqueous phase comprising purified water was dissolved by heating to 70° C., and oil phase comprising a preservative and a thickener was dissolved by heating to 70° C., thus preparing an emulsion using homixer (Tokushu Kika, Japan). Then, the emulsion was cooled to 45° C., and veratric acid was added to an amount of 0.1 and 1.0 wt % on the total weight of the composition and dispersed in the cooled emulsion, followed by cooling to 30° C. In Table 1, test groups 1 and 2 are hair growth agent containing veratric acid.

(10) TABLE-US-00001 TABLE 1 Content (%) Component Test group 1 Test group 2 Veratric acid 0.1 1.0 Preservative (Gramben) 0.5 0.5 Thickener (xanthan-gum) 0.3 0.3 Purified water 99.1 98.2 Total weight 100 100

Example 2: Measurement of Effects on Hair Growth and Hair Loss Prevention of Veratric Acid

(11) 2-1. Measurement of Effects on Hair Growth and Hair Loss Prevention Under Invitro Conditions

(12) Human hair follicle dermal papillar cells (Application, Inc., USA) were seeded into a 6-well micro plate containing DMEM medium (Gibco, USA) with FBS at a density of 3×10.sup.5 cells per well, and then were cultured in a 5% CO.sub.2 incubator at 37° C. for 24 hours. The next day, the cells were washed once with serum-free medium, and the medium was replaced with serum-free medium. Then, the cells were treated with 0, 10 and 100 μM of veratric acid, followed by incubating for 72 hours, after which the cells were washed once with serum-free medium, the medium was replaced with serum-free medium containing 10% MTT, and after 3 hours, the optical density (O.D.) value was measured and calculated as a percentage of control. The results are shown in FIG. 1.

(13) As a result, veratric acid increased the proliferation of human hair follicle dermal papilla cells in a concentration-dependent manner.

(14) 2-2. Measurement of Effects on Hair Growth and Hair Loss Prevention Under Invivo Conditions

(15) 40 hair loss patients (age: 40s to late 60s) were divided into the following four groups each consisting of 10 persons: alopecia patients having follicular atrophy and baldness; typical male-pattern alopecia patients; and acute alopecia areata patients. Each of the hair loss compositions prepared in Example 1 was applied to the hair loss portion of each alopecia patient twice a day in an amount of 3 cc each time for 6 months. The conditions of hair loss and hair growth were observed at 1-month intervals. In a comparative group, Moxidil (Hanmi Pharm Co., Ltd., Korea) was used, and in a control group, 50% ethanol alone was used. The results are shown in Table 2 below.

(16) The criteria of evaluation were as follows:

(17) 4: having high effect=new hairs appear

(18) 3: having moderate effect=new hairs (downy hairs) appear

(19) 2: having slight effect=degree of hair loss decreases

(20) 1: having no effect=no change appears

(21) TABLE-US-00002 TABLE 2 Control Comparative — group group Test group 1 Test group 2 Efficacy/effect 1.6 4.3 2.7 3.4

(22) As shown in Table 2 above, in the alopecia patients treated with the hair loss compositions containing veratric acid of the present invention, bristle hairs including downy hairs started to appear from 1 or 2 months after treatment with the compositions. 6 months after treatment with the compositions, the hair growth effect was shown 80% of the patients. In addition, it was observed that new hairs grew continuously and no hair loss phenomenon was found.

Example 3: Measurement of Effects of Veratric Acid on Inducing Growth Factor IGF-1

(23) Human hair follicle dermal papillar cells (Application, Inc., USA) were seeded into a 24-well micro plate at density of 7.5×10.sup.4 cells per well, and then were cultured in a 5% CO.sub.2 incubator at 37° C. for 24 hours. Then, the medium was replaced with serum-free medium, followed by being cultured for 16 hours. Then, each well was treated with 0, 10 and 100 μM of veratric acid, after which the cells were incubated for 48 hours. Then, the cell culture media were collected.

(24) The secretion rate of growth factor insulin-like growth factor-1 (IGF-1) was determined by measuring the amount of secretion rate of IGF-1 using Insulin-like growth factor-1 quantikine ELISA kit (DG100, R&D system, USA). The results are shown in FIG. 2.

(25) As a result, veratric acid increased the IGF-1 secretion of human hair follicle dermal pailla cells in a concentration-dependent manner.

Example 4: Measurement of Effects of Veratric Acid on the Inhibition of TGF-Beta1 Production

(26) Human hair follicle dermal papillar cells (Application, Inc., USA) were seeded into a 24-well micro plate at a density of 7.5×10.sup.4 cells per well, and then were cultured in a 5% CO.sub.2 incubator at 37° C. for 24 hours. Then, the medium was replaced with serum-free medium, followed by being cultured for 16 hours. Then, each well was treated with 0, 10 and 100 μM of veratric acid, after which the cells were incubated for 48 hours. Then, the cell culture media were collected.

(27) The secretion rate of transforming growth factor-beta1 (TGF-beta1) was determined by measuring the amount of secretion rate of TGF-beta1 using Human LAP TFG-beta1 ELISA kit (ab100647, abcam, USA). The results are shown in FIG. 3.

(28) As a result, veratric acid excellently inhibited TGF-beta1 production, as veratric acid suppressed the expression of TGF-beta1. It is suggested that veratric acid has hair growth promoting effects.

Example 5: Measurement of Wrinkle-Reducing Effect of Veratric Acid

(29) Human normal fibroblasts (Department of Dermatology, Ajou University) were seeded into a 24-well micro plate containing DMEM medium at a density of about 2×10.sup.5 cells per well, and were cultured in a 5% CO.sub.2 incubator at 37° C. for 24 hours. Then, the medium was removed from each well of the plate, and each well was treated with 0, 1, 10 and 100 μM concentrations of veratric acid, after which the cells were cultured for 24 hours. Then, the cell medium was collected, thereby preparing samples.

(30) The amounts of collagen synthesis in the above-prepared samples was determined by measuring the amount of procollagen type I C-peptide (PICP) using a Procollagen Type I C-peptide EIA kit (MK101; Takara, Kyoto, Japan). The results are shown in FIG. 4.

(31) As a result, veratric acid increased collagen synthesis, and collagen synthesis in human normal fibroblasts was increased as the concentration of veratric acid was increased. It is suggested that the collagen synthesis was dependent on the veratric acid concentration.

Example 6: Measurement of UV-Preventing Effect of Veratric Acid

(32) Keratinocyte (HaCaT) and fibroblast were cultured in DMEM medium containing penicillin-streptomycin and serum. Then, the cells were seeded into a 12-well micro plate at density of about 1×10.sup.5 cells per well, and were cultured in a 5% CO.sub.2 incubator at 37° C. until the cells attached about 80% over on well-area. Then, each well was pretreated with 0, 1, 10 and 100 μM of veratric acid, and after 24 hours, the medium was replaced with PBS, and then the cells were irradiated with UV (UVB 20 mJ/cm.sup.2). Then, the UV-irradiated cells were replaced DMEM medium containing penicillin-streptomycin and serum, the cells were incubated for 48 hours. The cell viability was determined by measuring the viability of keratinocyte (HaCaT) and fibroblast using a MTT assay (30-1010K, ATCCTM). The results are shown in FIGS. 5 and 6.

(33) As a result, veratric acid prevented cell death of UV-induced kerationcyte and fibroblasts. Veratric acid increased the preventing rate of cell death in a concentration-dependent manner.

Example 7: Measurement of Effects of Veratric Acid on the Inhibition of Collgenase Production

(34) Human normal fibroblasts (Department of Dermatology, Ajou University) were seeded into a 24-well micro plate containing DMEM medium at a density of about 2×10.sup.5 cells per well, and were then cultured in a 5% CO.sub.2 incubator at 37° C. for 24 hours. Then, the medium was removed from each well, and the cells were treated with 0, 1, 10 and 100 μM concentrations of veratric acid. Then, the cells were cultured for 24 hours, after which the cell culture media were collected, thereby preparing samples.

(35) The inhibition effects of collagenase production was determined by measuring the amount of collagenase using MMP-1 ELISA kit (QIA55, Merck, Germany). The results are shown in FIG. 7.

(36) As a result, veratric acid decreased collgenase production in human normal fibroblasts. Veratric acid decreased the amounts of collagenase production in human normal fibroblast in a concentration-dependent manner.

Example 8: Measurement of Wrinkle-Reducing Effect of Cosmetic Composition Containing Veratric Acid

(37) 8-1. Preparation of Nourishing Creams Containing Veratric Acid

(38) The nourishing cream having the composition and contents shown in Table 3 below was prepared. Specifically, an aqueous phase including purified water, triethanolamine, and propylene glycol was dissolved by heating to 70° C., and an oily phase including fatty acid, oily components, an emulsifier, and a preservative was dissolved by heating to 70° C. and added to the aqueous phase. Then, the resulting solution was cooled to 45° C., and veratric acid was added with in amounts of 0, 0.01, 0.05 and 1 wt % based on the total weight of the composition and was dispersed, followed by cooling to 30° C.

(39) TABLE-US-00003 TABLE 3 Component Content (wt %) veratric acid 0.01, 0.05 or 1.00 Jojoba oil 5.0 Liquid paraffin 7.0 Cetylaryl alcohol 2.0 Polyglyceryl-3-methylglucose 2.0 disterate Glyceryl stearate 0.5 Squalane 3.0 Propylene glycol 4.0 Glycerine 5.0 Triethanolamine 0.3 Carboxyvinylpolymer 0.3 Tocopheryl acetate 0.2 Preservative and fragrance Trace Purifired water Balance Sum 100   

(40) 8-2. Measurement of Wrinkle-Reducing Effect of Cosmetic Composition Containing Veratric Acid

(41) In the measurement of skin elasticity, nourishing creams described in Preparation Example 8-1 was applied to the face of 30 healthy women of 21 to 42 years old twice a day for 3 months. The control group was used nourishing cream containing purified water.

(42) The effect on wrinkle reduction was evaluated by measuring a change in skin elasticity. The measurement of skin elasticity was performed with Coutometer SEM 474 (Courage+Khazaka, Cologne, Germany) under the conditions of constant temperature of 24˜26° C. and constant humidity of 38-40%. The evaluation criteria were as follows 0 indicating no improvement in skin elasticity, and 5 indicating improvement in skin elasticity. The results are shown in Table 4 below.

(43) TABLE-US-00004 TABLE 4 Veratric acid (wt %) 0.01 0.05 1 0 Skin elasticity 2.81 3.12 3.36 2.59

(44) As a result, the use of the nourishing creams containing veratric acid showed significantly excellent skin elasticity compared to the nourishing cream containing no veratric acid. Also, the skin elasticity was increased in a concentration-dependent manner as the concentration veratric acid was increased.

Example 9: Effect of Preventing Wrinkles by Oral Administration

(45) In order to measure the wrinkle-preventing effect of veratric acid, 6-7-week-old hairless mice (Skh: HR-1) were divided into a control group, a UV group and a UV/veratric acid group, each consisting of 8 animals, and were bred during the test period. The control group and the UV group were administered orally with 0.5 ml of saline, and the UV/veratric acid group was administered orally with a solution of 100 mg per kg veratric acid in 0.5 ml of saline.

(46) The above-prepared samples were administered orally at the same point of time for 5 days a week for a total of 5 weeks. From 2 weeks to 5 weeks after oral administration, the UV group and the UV/veratric acid group were irradiated with UV light three times a week such that the total dose of UV radiation reached 600 mJ/cm.sup.2.

(47) To measure the wrinkle-preventing effect of veratric acid, skin replicas were taken from the back of the hairless mice using a silicon polymer before autopsy. In the same manner, after 5 weeks, skin replicas were taken from the groups treated with each of the samples. Using the taken skin replicas, the antiaging effect, i.e., wrinkle-reducing effect of each sample was measured. The results are shown in Table 5 below.

(48) TABLE-US-00005 TABLE 5 UV/veratric acid R-parameter Control group UV group group R1 value 0.435 ± 0.0120 0.481 ± 0.009 0.462 ± 0.013 R2 value 0.357 ± 0.0105 0.424 ± 0.010 0.405 ± 0.011 R3 value 0.212 ± 0.0097 0.237 ± 0.008 0.219 ± 0.012 R4 value 0.098 ± 0.0086 0.138 ± 0.014 0.116 ± 0.010 R5 value 0.231 ± 0.013  0.318 ± 0.010 0.290 ± 0.013 R1 value: Skin roughness, R2 value: Maximum roughness, R3 value: Average roughness, R4 value: Smoothness depth, R5 value: Arithmetic average roughness (International Journal of Cosmetic Science, 2005 Jun; 27(3): 155-60).

(49) As a result, when the hairless mice were irradiated with UV light, all the R1, R2, R3, R4 and R5 values indicative of wrinkles was increased. In addition, it was observed that R-parameter value was reduced in veratric acid-administered hairless mice compared to the UV/control groups.

Example 10: Safety Test of Veratric Acid on Human Skin

(50) 10-1. Preparation of Skin External Formulation Containing Veratric Acid

(51) In order to confirm whether veratric acid is safe for human skin, a veratric acid-containing skin external formulation having the components and contents shown in Table 6 was prepared, and then a skin safety verification test was carried out. Specifically, purified water, glycerin, and butylene glycol were mixed and dissolved at a temperature of 70° C. (aqueous phase). The remaining components except for the above three components and trimethanolamine were dissolved at a temperature of 70° C. (oil phase). The oil phase was added to the aqueous phase, and stirred with a homomixer (Tokushu Kika, Japan) to prepare an emulsion, and trimethanolamine was added thereto. Then, bubbles produced in the mixture were removed, after which the mixture cooled to room temperature, thereby preparing skin external formulations.

(52) TABLE-US-00006 TABLE 6 Content (wt %) Control Component Test group 1 Test group 2 Test group 3 group Purified water 71 71 71 71 Glycerin 8.0 8.0 8.0 8.0 Butylene glycol 4.0 4.0 4.0 4.0 Veratric acid 0.1 0.5 1.0 0 Caprylic/capric 8.0 8.0 8.0 8.0 triglyceride Squalane 5.0 5.0 5.0 5.0 Cetearyl 1.5 1.5 1.5 1.5 glucoside Sorbitan stearate 0.4 0.4 0.4 0.4 Cetearyl alcohol 1.0 1.0 1.0 1.0 Trimethanolamine 0.1 0.1 0.1 0.1 Total 100 100 100 100

(53) 10-2. Cumulative Irritation Test

(54) Each of the skin external formulations prepared in Example 10-1 was applied every other day to the forearms of 30 healthy adults and allowed to stand for 24 hours. This was repeated so that each subject was treated with 9 fresh patches in total, so as to confirm whether veratric acid irritates the skin. Control group was used to a skin external formulation containing no veratric acid on squalane base.

(55) The patch test was performed using a Finn chamber (Epitest Ltd, Finland). The external formulations were added dropwise to the chamber in an amount of 15 μl per patch. At every round of the patch application, the response of the skin was scored using the following empirical formula 1. The results are shown in Table 7 below.
Average response degree=[[Response index×Response degree/Total number of subjects×Highest score (4 points)]×100]/Number of examinations  [empirical formula 1]

(56) In regard to the response degree, 1 point was provided for ±, 2 points for +, and 4 points for ++. When the average response degree was less than 3, the composition was determined to be safe for use on the skin.

(57) TABLE-US-00007 TABLE 7 Number of subjects showing response Week 1 Week 2 Week 3 1.sup.st 2.sup.st 3.sup.st 1.sup.st 2.sup.st 3.sup.st 1.sup.st 2.sup.st Average Test ±/+/ ±/+/ ±/+/ ±/+/ ±/+/ ±/+/ ±/+/ ±/+/ 3.sup.st response material ++ ++ ++ ++ ++ ++ ++ ++ ±/+/+/+ degree Control 1/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− 0.09 group Test −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− 0.00 group 1 Test −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− 0.00 group 2 Test 1/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− −/−/− 0.09 group 3 Number 30 30 30 30 30 30 30 30 30 of subjects

(58) As a result, in the control group, the subjects corresponding to ±, +, and ++ numbered 1, 0 and 0, respectively, in the first patch, and no skin response appeared after the second patch. In the test group 1 and test group 2 to which veratric acid was 0.1 wt % and 0.5 wt % applied, no skin response appeared in all patches. In the test group 3 to which veratric acid was 1.0 wt % applied, the subjects corresponding to ±, +, and ++ numbered 1, 0 and 0, respectively, in the first patch, and no skin response appeared after the second patch. The average reactive levels in control group and test group 3 were 0.09, respectively, and the average reactive levels in test groups 1 and 2 were 0.00, respectively. Control group, test group 1, test group 2 and test group 3 are all less than 3, suggesting that the composition of the present invention is safe for use on human skin.

Formulation Example 1: Cosmetic Formulations

(59) 1-1. Preparation of Skin Softener

(60) Shown in Table 8 below, a skin softener contacting veratric acoid as an active ingredient was prepared according to a conventional method.

(61) TABLE-US-00008 TABLE 8 Component Content (wt %) Veratric acid 0.01 Glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 Ethanol 10.0 Triethanolamine 0.1 Preservative, pigment, fragrance, Balance and purified water Total 100.0

(62) 1-2. Preparation of Nourishing Softener

(63) Shown in Table 9 below, a nourishing softener containing veratric acid as an active ingredient was prepared according to a conventional method.

(64) TABLE-US-00009 TABLE 9 Component Content (wt %) Veratric acid 0.01 Beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 5.0 Squalane 5.0 caprylic/capric triglyceride 5.0 Glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservative, pigment, fragrance, Balance and purified water Total 100.0

(65) 1-3. Preparation of Nourishing Cream

(66) Shown in Table 10 below, a nourishing cream containing veratric acid as an active ingredient was prepared according to a conventional method.

(67) TABLE-US-00010 TABLE 10 Component Content (wt %) Veratric acid 0.01 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Liquid paraffin 10.0 Squalane 5.0 caprylic/capric triglyceride 5.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, pigment, fragrance, Balance and purified water Total 100.0

(68) 1-4. Preparation of Massage Cream

(69) Shown in Table 11 below, a massage cream containing veratric acid as an active ingredient was prepared according to a conventional method.

(70) TABLE-US-00011 TABLE 11 Component Content (wt %) Veratric acid 0.01 Beeswax 10.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 caprylic/capric triglyceride 4.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, pigment, fragrance, Balance and purified water Total 100.0

(71) 1-5: Preparation of Pack

(72) Shown in Table 11 below, a pack containing veratric acid as an active ingredient was prepared according to a conventional method.

(73) TABLE-US-00012 TABLE 12 Component Content (wt %) Veratric acid 0.01 Polyvinyl alcohol 13.0 Sodium carboxymethyl cellulose 0.2 Allantoin 0.1 Ethanol 5.0 Nonyl phenyl ether 0.3 Preservative, pigment, fragrance, Balance and purified water Total 100.0

Preparation Example 2: Preparation of Pharmaceutical Formulations

(74) 2-1: Preparation of Powder Formulation

(75) The components shown in Table 13 below were mixed with each other and then filled in a sealed bag, thereby preparing a powder formulation containing veratric acid as an active ingredient.

(76) TABLE-US-00013 TABLE 13 Component Content (g) Veratric acid 2 Lactose 1

(77) 2-2: Preparation of Tablet Formulation

(78) The components shown in Table 14 below were mixed with each other and then compressed to a tablet according to a conventional method, thereby preparing a tablet formulation containing veratric acid as an active ingredient.

(79) TABLE-US-00014 TABLE 14 Component Content (mg) Veratric acid 100 Corn starch 100 Lactose 100 Magnesium stearate 2

(80) 2-3: Preparation of Capsule Formulation

(81) The components shown in Table 15 below were mixed with each other and then filled into a gelatin capsule according to a conventional method, thereby preparing a capsule formulation containing veratric acid as an active ingredient.

(82) TABLE-US-00015 TABLE 15 Component Content (mg) Veratric acid 100 Corn starch 100 Lactose 100 Magnesium stearate 2