WO2006118364A1

WO2006118364A1 - COSMETIC COMPOSITION CONTAINING (20S)-GINSENOSIDE Rg3 FOR PREVENTING AND IMPROVING SKIN WRINKLES

Info

Publication number: WO2006118364A1
Application number: PCT/KR2005/002298
Authority: WO
Inventors: Soon Keun Lee; Wie-Jong Kwak; Yong-Baik Cho; Chang-Kyun Han; Jeong Joo Shin; Joo Hyun Kim; Eun Ju Lee; Chil Mann Jung; Se Jun Yun
Original assignee: Sk Chemicals Co., Ltd.
Priority date: 2005-05-02
Filing date: 2005-07-18
Publication date: 2006-11-09
Also published as: KR20060114774A

Abstract

The present invention relates to a cosmetic composition for preventing and improving skin wrinkles comprising 20(S)-ginsenoside Rg3, and more particularly to a cosmetic composition comprising 20(S)-ginsenoside Rg3, which is superior in proliferating fibroblast, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds, thus enabling to effectively prevent and improve skin wrinkles.

Description

COSMETIC COMPOSITION CONTAINING (20S)-GINSENOSIDE Rg3 FOR PREVENTING AND IMPROVING SKIN WRINKLES

TECHNICAL FIELD The present invention relates to a cosmetic composition for preventing and improving skin wrinkles comprising 20(S)-ginsenoside Rg3, and more particularly to a cosmetic composition comprising 20(S)-ginsenoside Rg3, which is superior in proliferating fibroblasts, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds, thus enabling to effectively prevent and improve skin wrinkles.

RELATED PRIOR ART

Vitamin A (retinol) and retinyl palmitate are commonly used as cosmetic compounds for preventing and improving skin wrinkles. However, cosmetics comprising these compounds may easily deteriorate with time, and may also cause serious skin irritation when they are penetrated or absorbed into the skin exceeding their effective amounts. Thus, there is a need for developing a new compound having superior effects in improving skin wrinkles, drug stability and safety on skin.

Panax Ginseng (briefly 'Ginseng' hereinafter) is a medicinal herb belonging to Araliaceae family, which has been widely used in oriental medicine and also folk remedies. The ginseng is largely divided into three different kinds according to the status as green, white and red ginseng. White ginseng is prepared by removing rootlets of green ginseng (i.e. undried ginseng) followed by drying under the sun. Red ginseng is prepared by steaming the white ginseng. Saponin in ginseng is known to have an anti-oxidizing effect by promoting secretion of catecholamine in adrenal gland and inhibiting lipid peroxidation, along with other effects of protecting against radioactive rays, preventing abnormalities or micronuclei caused by alkylating agents, and lowering mutations in culturing cells. According to a WTO's report, main active ingredients of ginseng are saponin ginsenosides. More specifically, green ginseng, white ginseng and red ginseng commonly contains 18 compounds of Ro, Ra₁, Ra2, Ra3, Rb₁, Rb2, Rb3, Rc, Rd, Re, Rf, Rg₁, Rg2, Rg3, Rhi, 2OgIc-Rf, Q-R₁ and N-R₁ as active ingredients. However, 4 compounds of Malonyl-Rbi, Malonyl-Rb2, Malonyl-Rcand and Malonyl-Rd are contained only in green ginseng and white ginseng, while 8 compounds of Rs₁, Rs2, Rg3(S), Rh2, N-R₄, Rg2(R), Rh₁(R) and Rh₄ are contained only in red ginseng.

According to the structure of saponin aglycon, ginseng saponin may be divided into panaxadiol-type ginsenosides (Ra₁, Ra2, Ra3, Rb₁, Rb2, Rb3, Rc, Rd and Rg3)_/ ginsenosides (Re, Rf, Rgi and Rg₂) and oleanolic acid-type ginsenosides. Ginseng roots comprise 48-60% of panaxadiol-type saponin, 30-35% of panaxatriol-type saponin and small quantity of oleanolic acid-type saponin.

One of minor ginsenosides, Rg3, is produced during the process of steaming ginseng. A few patents have already disclosed that Rg3 has activities of inhibiting cancer cell metastasis, inhibiting platelet aggregation, inducing vasorelaxation, decreasing tolerance to anticancer drugs, inhibiting experimental liver damage and antithrombotic activity. However, the pharmaceutical activities in the aforementioned patents were restricted to orally administered Rg3, and there has been no report about the activities of topically applied Rg3 for improving skin wrinkles.

In general, ginsenoside Rg3 is extracted and separated from ginseng by extracting crude saponin with an organic solvent and condensing the crude saponin, followed by separation with silica gel or repeated purification with preparative liquid chromatography. However, the extracts also comprise saponin compounds in ginsenoside Rg family, having very similar molecular weight and physiochemical properties, which remains in small quantity even after the final purification. Further, the yield of ginsenoside Rg3 is not so high and the repeated purification process for high purity requires a considerable amount of time and cost. The biotransformation process using acidic and basic hydrolysis and glycosidase still bring us other problems such as complexity in purification process and remnant solvents.

The glycosidic bonds on the structurally unstable ginsenoside aglicone C-20 position are easily cleaved by hydrolysis by performing heating and steaming ginseng thus expediting brown change, and C-20(S) and C-20(R) isomers may be produced as hydroxyl group causes inversion equilibrium. Thus, characteristic components (Rh₁, Rh₂, Rs₁, Rs₂, Rg3, and Rgs), called as minor ginsenosides, may be extracted by heating or steaming ginseng. Further, ginsenoside Rg3, a prosapogenin, is contained more than 50 times of that in green ginseng or white ginseng.

The present inventors found that ginsenoside Rg3 has an activity of improving skin wrinkles, and filed a patent application claiming cosmetic composition comprising ginsenoside Rg3 (KR patent application no. 2003-65882). In addition, the skin improvement may be further accomplished by controlling the amount of ginsenoside Rg3 and the ratio of SR isomers to a specific range.

Specifically, the present inventors found that the effects of proliferating fibroblast, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds vary depending on the amount of ginsenoside Rg3 and the ratio of 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3. More specifically, the present inventors found the aforementioned effects may be maximized by controlling the amount of 20(S)-ginsenoside Rg3 to 40-80 wt% and as the purity of S isomer increase up to 100%, thus completing the present invention.

SUMMARY OF INVENTION

Therefore, in an embodiment of the present invention there is provided a cosmetic composition for preventing or improving skin wrinkles, which comprises 20(S)-ginsenoside Rg3.

DETAILED DESCRIPTION

The present invention relates to a cosmetic composition for preventing or improving skin wrinkles, which comprises 20(S)-ginsenoside Rg3 as active ingredient.

Hereunder is provided a detailed description of the present invention. The present invention relates to a cosmetic composition comprising 20(S)-ginsenoside Rg₃, which is superior in proliferating fibroblast, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds, thus enabling to effectively prevent or improve skin wrinkles.

The skin improvement effect varies depending on the amount of ginsenoside Rg₃ and the ratio of 20(S)-ginsenoside Rg₃ and 20(R)-ginsenoside Rg₃, which are prepared from ginseng by acidic hydrolysis extraction method.

The purity of ginsenoside Rg₃ and the ratio of the RS isomers may be controlled by repeating silica gel column chromatography (chloroform : methanol : distilled water = 9:3:1). Specifically, the amount of ginsenoside Rg₃ may be controlled in the range of 20-95 wt%. Further, the present inventors verified that the effects of proliferating fibroblast, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds, thus enabling to effectively prevent and improve skin wrinkles, may be maximized by controlling the amount of 20(S)-ginsenoside Rg₃ to 40-80 wt% and using nearly 100% pure S isomer.

Thus, the amount of 20(S)-ginsenoside Rg₃ is preferred to be in the range of 40-80 wt %. The effects are weakened when the amount is below 40 wt%, whereas the amount of above 80 wt% would incur high cost during the extraction and purification. Meanwhile, the side effect of the ginseng-derived ginsenoside Rg3 is lower than any other synthetic material, and the skin toxicity test also showed that the extract herein has no harmful effect on the skin.

Therefore, the ginseng-derived ginsenoside Rg3, when applied to cosmetics, may be formulated into skin softeners, astringents, lotions, essence, creams, gels, packs, soaps, essence sheets, hydrogel patches or masks. The ginseng-derived ginsenoside Rg3 is preferred to be comprised in the amount of 0.000001-20.0 wt% with respect to the total cosmetic composition. The aforementioned effects may not be obtained when the amount is below 0.000001 wt%, however, when the amount is above 20 wt% the effect of improvement may not be satisfactory considering the increase in the amount and the formulation may be unstable.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a HPLC result of 20(S)-ginsenoside Rg3 prepared in Example 1. Figure 2 is a photograph of skin of hairless mouse after inducing artificial wrinkles.

Figure 3 is a photograph showing the effect of improving wrinkles after applying 2500 IU/ g of retinol for two weeks.

Figure 4 is a photograph showing the effect of improving wrinkles after applying 0.01 % of specimen 9 prepared in Example 2 for two weeks.

Figure 5 is a photograph showing the effect of improving wrinkles after applying 0.01% of the extracts of Example 1 for two weeks.

Figure 6 is a photograph showing the effect of improving elastic fiber after applying negative control group for two weeks. The black stripe is elastic fiber. Figure 7 is a photograph showing the effect of improving elastic fiber after applying 2500 IU/ g of retinol for two weeks.

Figure 8 is a photograph showing the effect of improving elastic fiber after applying specimen 9 prepared in Example 2 for two weeks. Figure 9 is a photograph showing the effect of improving elastic fiber after applying the compound prepared in Example 1 for two weeks.

Figure 10 is a photograph showing the effect of improving collagen cells after applying a negative control group for two weeks. The blue part is collagen, the dark brown is cell nucleus, and the red is cytoplasm/ muscle/ keratin.

Figure 11 is a photograph showing the effect of improving collagen cells after applying 2500 IU/ g of retinol for two weeks.

Figure 12 is a photograph showing the effect of improving collagen cells after applying specimen 9 prepared in Example 2 for two weeks. Figure 13 is a photograph showing the effect of improving collagen cells after applying the extracts of Example 1 for two weeks.

EXAMPLES

The present invention is described in greater detail with reference to the accompanying Examples. The examples herein are meant only to illustrate the present invention, and they should not be construed as limiting the scope off the claimed invention.

Example 1: Preparation and separation of ginsenoside Rg3 RS by acidic hydrolysis 2 kg of ginseng was minced into pieces and extracted under reflux by using

70% methanol, which is 5-10 times the weight of the ginseng, at 60-80 ⁰C for 5 hours. The extracts were concentrated under reduced pressure at 50 ⁰C and 368 g of ginseng extracts were obtained. This extracts were dissolved in 5 L of distilled water and washed with 2 L of n-hexane three times. The aqueous layer was extracted three times with 2 L of n-butanol, thereby obtaining 214 g of butanol fraction. The butanol fraction was dissolved in 1 L of methanol while heating, and macerated to remove the precipitates. Then, 1.2 g of a mixture of 20(R)-ginsenoside Rg3 and 20(S)-ginsenoside Rg3 was obtained by performing silica gel column chromatography (chloroform : methanol : distilled water = 9:3:1). After suspending this mixture in 24 mL of methanol while heating and removing precipitates by cooling the suspension, the remnant liquid was concentrated under reduced pressure. 576 mg of concentrates was dissolved in 20 mL of pyridine and ice-cooled with addition of 20 mL of acetic anhydride, followed by stirring at room temperature for 5 hours. The products were extracted two times with 100 mL of ethyl acetate and washed with 100 mL of 5% chloric acid and distilled water three times, respectively. The ethyl acetate fraction was concentrated under reduced pressure and 341 mg of concentrates were applied to silica gel column chromatography (dichloromethane : ethyl acetate = 8:2). 238 mg of the separated 20(S)-ginsenoside Rg3 peracetate was dissolved in 40 mL of mixture of 5% chloric acid and methanol and stirred at room temperature for 3 hours. The solution added with 100 mL of distilled water was concentrated under reduced pressure at 35 ⁰C to remove methanol, was further extracted with 50 mL of butanol three times. The butanol fraction was concentrated under reduced pressure and applied to silica gel chromatography (chloroform : methanol : distilled water = from 9:3:1 to 4:3:1 to increase polarity), thereby obtaining 158 mg of ginsenoside Rg3 (50±1.0 %).

Further, 20(S)-ginsenoside and 20(R)-ginsenoside Rg3 were separated by varying the solubility toward water (polarity). It was verified that so prepared ginsenoside Rg3 comprising stereoisomers are superior in proliferating fibroblasts, expediting collagen biosynthesis and elastin synthesis, enhancing skin immunity, functions as oxidants and healing skin wounds, thus being capable of preventing and improving skin wrinkles.

The purity of ginsenoside Rg3 may be increased by repeating purification and separation with silica gel column chromatography (chloroform : methanol : distilled water = 9:3:1), and the ratio of RS isomers may also be controlled. Example 2: Change in the amount of ginsenoside Rg3 and the ratio of RS isomers

The final products of Example 1 was applied to repeated separation and purification with silica gel column chromatography (chloroform : methanol : distilled water = from 9:3:1 to 4:3:1), where the polarity was gradually increased, thereby obtaining a mixture of 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 with a different ratio.

The structure of 20(S)-ginsenoside Rg3 is shown in Formula 1. The amounts of 20(S)-ginsenoside Rg3 and the ratios are shown in Table 1.

Formula 1

Table 1. Amounts of Ginsenoside Rg3 and Ratios of RS isomer

Conditions for liquid chromatography analysis

(i) Column: J'sphere ODS-80H(250 x 4.6 mm LD, S-4 μm,YMC)

(ii) UV absorbance: at 205 nm

(iii) Mobile phase (Gradient system)

(iv) Flow rate: 1 mL/min

Reference Example 1: Toxicity Test

(1) Single dose toxicity study by intravenous and oral administration Single dose toxicity study was performed by applying intravenous and oral administration to a 5-week-old Wistar male rat. 50 mg/kg of final product (Example 1) and 100 mg/kg of specimen 1-15 (Example 2) were intravenously administered and 200 mg/kg of the ginsenoside Rg3 was orally administered. Neither death nor abnormality was observed within 14 days after the administration, thus verifying the safety of the extracted powder prepared in Examples.

(2) Repeated dose toxicity study via oral administration for 3 months Repeated dose toxicity study was performed by applying oral administration to a 5-week-old Sprague Dawley male rat for 3 months. 100 mg/kg of the final product of Example 1 and specimen 1-15 of Example 2 were orally administered every day for 3 months. General conditions were observed every day, and weight was measured at the intervals of 7 days. Quantitative and qualitative investigations of urine and hematological examination at the end of administration, postmortem observation and long-term weight measurement showed no change due to the administration of the extract powder prepared in Examples, thus verifying their safety.

Reference Example 2; Skin irritation test

(1) Primary skin irritation test

Primary skin irritation test was performed by using white rabbit (New Zealand). Hairs (3 cm x 10 cm) in left and right regions of spine were removed, and each region was divided into two, thus having a total of four test regions. Right top and left bottom regions were stuck by 18-gauge syringe to provide #-shaped wound, the other two regions were left unhurt. The final product was dissolved in butylene glycol to provide 20% solution, and 0.5 niL of the solution was absorbed in two sheets of 2.5 cm x 2.5 cm lint. Two additional sheets of lint were absorbed only with butylene glycol. The prior lints and the latter lints were attached to the right and left regions, respectively, and it was observed if there was any abnormality at 24, 48 and 72 hours after attachment.

As a result, no skin abnormality such as rubefaction and edema was observed in both the wound and unhurt regions. (2) Continuous skin irritation test

Continuous skin irritation test was performed by using about 350 g of Hartley Guinea pig. The extract powder of Example 1 was dissolved in butylene glycol to provide 5% solution, and 0.1 mL of the solution was open-coated on the left abdomen of the pig one time a day for 4 weeks (6 times a week). Abnormalities were observed every day, and skin irritation of the final product (Example 1) was evaluated based on the result by comparing with the positive control group, which was coated with 2% sodium lauryl sulfate solution.

As a result, no skin irritation was observed by the repeatedly coated final product of Example 1.

(3) Human patch test

According to the standard issued by Japanese Patch Test Study group, human patch test was performed by coating the extract of Example 1 on the back of 30 healthy male adults, followed by attaching the coated region for 48 hours. Skin irritation was observed twice, i.e., 2 hours and 72 hours after removing the patch.

Further, photo patch test was performed by attaching the patch for 24 hours and, at

2 hours after removing the patch, the region was irradiated with DERMARAY UV-A ray (BLB 10 etc.) for 7 minutes at a distance of 20 cm. Specimen was attached on the back for 2 hours and the rash response was determined 10 minutes after removing the specimen.

As a result, no skin irritation was observed by repeated coating the extract of

Example 1. Thus, the extract of Example 1 comprising ginseng-derived ginsenoside

Rg3 was verified to be very stable when applied to the skin.

Experimental Example 1: Effect of proliferating fibroblast

Human normal fibroblast cells were dispensed in 96-well micro plate so that each well contains 5000 cells, and cultured in DMDM (Dulbeccos Modified Eagles

Media) comprising fetal bovin serum at 37 ⁰C for 24 hours. The medium was diluted ten times with the extract of Example 1 and the specimen of Example 2, and cultured at 37 ⁰C for 3 days. Each well was added with 30 μL of 0.2% MTT (3-(4_/5-dimethylthiazole-2-yl)-2_/5-diphenyltetrazolum bromide) solution, and cultured for 4 hours. After removing the medium, each well was added 100 μL of DMSO (dimethyl sulfoxide) solution, and shaken for 10 minutes. Absorbance of the upper fraction was measured at 570 ran by using a microplate reader. After the results of the treated and the control groups were compared, the values of cell proliferation were calculated by applying Equation 1 as provided in Table 2.

Equation 1

Absorbance {treated) - Absorbance {control)

Cell proliferation (%) ^: x 100 Absorbance {control)

Table 2

Example 2

Compared to untreated negative control group, the groups treated with the final product of Example 1 and the specimen of Example 2 showed maximum 98% of fibroblast proliferation, i.e., much improved in fibroblast proliferation without toxicity. This verifies that cell proliferation effect improves as the amount ginseng-derived ginsenoside Rg3 and the ratio of the S-form isomer comes close to 100%. Specifically, for the enhancement of the cell proliferation effect, the amount of (20S)-ginsenoside Rg₃ is preferred to be within 40-80 wt% and the ratio is 100%, and more preferably the amount of ginsenoside Rg₃ is 60 wt% and the ratio of the S-form isomer is about 100%.

Experimental Example 2: Effect of biosynthesis of collagen

Human normal fibroblast was dispensed in 96-well microplate for cell culture, and cultured under the condition of 37 °C, 5% CO2 for 24 hours in the medium comprising 5% FBS (fetal bovine serum), followed by washing with PBS

(phosphate buffer saline). The medium comprising cultured fibroblast and 1% FBS was treated with specimen 9, which showed good cell proliferation effects in

Examples 1 and 2; specimens 7 and 8 in the same amount as that of specimen 9; and specimens 3, 6, 12 and 1, which have the same composition but with different amount so that they can be 1, 5, 10 μM, respectively. The medium was added with 30 μg/mL of L[2,3,4,5-H]fluorine 5 μCi, ascorbic acid, and cultured under the condition of 37 ⁰C, 5% CO2 for 24 hours. The medium was divided into two fractions, and only one of them was treated with type 1 collagenase. After protein was precipitated by adding 5% TCA (trichloroacetic acid), the amount of collagen produced by the radioactive difference of the fractions was measured.

By comparing with the untreated negative control group, the collagen biosynthesis effect of each specimen was evaluated and converted into the values of the relative collagen biosynthesis (RCB) of the experimental groups along with the positive control group treated with vitamin A (retinol). The values of increase in the relative collagen biosynthesis were provided in Table 3.

Table 3

* Example 2

Compared to the vitamin A-treated group, the collagen biosynthesis was promoted when the amount of ginsenoside Rg3 was about 40-80 wt% and the ratio of S-form isomer approached 100% (Examples 1 & 2; specimen 6, 9, 12 & 15).

Experimental Example 3: Effect of synthesis of elastin

Human normal fibroblast was added in 96-well microplate for cell culture, and cultured for 24 hours. The medium was treated with 1-10 μM of specimen and cultured for 2 days. The amount of elastin secreted by fibroblast was measured by using fastin elastin assay kit, and the values amended with cell number or protein amount are provided in Table 4. Table 4. Effect of proliferating elastin

* Example 2

As shown in Table 4, Example 1 and Example 2 show similar effects of collagen biosynthesis promotion depending on the amount of ginsenoside Rg3 and the ratio of S-form isomer. Experimental Example 4: Effect of enhancing skin immunity

Hairs on the abdomen of an 8-week-old white mouse were removed, and the region was irradiated with 2.5 KJ/ m² of UBV by using a sun lamp, thus causing contact hypersensitivity and inhibition of skin immunity. This region was coated with solution prepared by the extract of Example 1 and the specimen 6, 9 and 12 of Example 2 in 70% ethanol. After 3 days, the irradiated region was sensitized with 3% trinitrochlorobenzene, and the abdomen was coated with 1% trinitrobenzene after 5 days. Ear edema was evaluated by measuring the ear thickness of the experimental and the control groups. Skin immunity effect was calculated by using Equation 2, and the results are provided in Table 5.

Equation 2

Ear thickness {coated, irradiated) - Ear thickness (irradiated)

Skin immunity effect (%) = x 100 Ear thickness {untreated) - Ear thickness {irradiated)

Table 5

* Example 2

The effect of skin immunity enhancement varies widely depending on the amount of ginsenoside Rg3, and specimen in Example 2 showed the highest enhancement effect.

Experimental Example 5: Antioxidative Effect Test

Lipid peroxidation caused by lipoxygenase was inhibited by the extract of Example 1 and the specimen of Example 2. The degree of inhibition was measured, and vitamin C was used as a positive control group.

12 mL of sodium phosphate buffer solution (pH 6.8, 50 mM) was placed in a test tube. 0.1 mL of 0.1% (v/v) specimen solution dissolved in sodium phosphate buffer solution (50 mM) and 0.1 mL of arachidonic acid (6.6 mM, Sigma) were also added in the test tube. 0.1 mL of lipoxigenase dissolved in 50 mM sodium-phosphate buffer solution was added in each test tube, and reaction was performed at 25 °C for 10 minutes (the final concentration of specimen in each reaction solution was 50 μg/mL). For a negative control group, 0.1 mL of sodium-phosphate buffer solution (50 mM) was used instead of specimen solution. After reaction completed, 1 mL of 20% (v/v) trichloroactic acid and 2 mL of 0.67% (v/v) thiobarbituric acid was added. After boiling water bath for 10 minutes and cooling down to room temperature, the upper-layer fraction was separated by centrifugation (1800 rpm, 3 minutes), and absorbance was observed at 535 nm. Lipid peroxidation inhibition was calculated by using Equation 3, and the results were provided in Table 6. Equation 3

Absorbance {treated)

Lipid peroxidation inbition (%) = ^■ x lOO Absorbance {negative control)

Table 6

* Example 2

Example 1 and specimen 6, 9 & 12 were verified to be superior to the positive control group (Vitamin C) in inhibiting skin ageing.

Prescription Examples 1-4; Preparation of Cosmetics (beauty wash solution)

Beauty wash solutions comprising the extracts herein are prepared in Prescription Examples 1-4 with their contents shown in Table 7.

Table 7

Preparation method

(1) Pre-weighed ingredients 2, 3 & 4 were dissolved in distilled water 1 at room temperature, thus providing solution 1.

(2) Pre-weighed ingredients 5, 6, 7, 8 & 9 were admixed and added in solution 1, thus providing solution 2.

(3) Ingredient 10 and the final product and specimen prepared in Examples 1 and 2, respectively, were dissolved in solution 2 followed by stirring and filtration, thereby obtaining beauty wash solutions.

Prescription Example 5-7: Preparation of Lotion

Lotions comprising the extracts herein are prepared in Prescription Examples 5-7 with their contents shown in Table 8.

Table 8

Preparation method

(1) Pre-weighed ingredients 1-12 were placed in a heatable oily-phase tank, and dissolved with stirring at 75 ⁰C.

(2) Pre-weighed ingredients 13-18 were placed in an aqueous-phase tank, and dissolved with stirring at 75 ⁰C.

(3) Solution(2) was moved to an emulsifying tank under vacuum, and solution(l) was also transferred to the emulsifying tank, followed by emulsification at 3500 rpm for 5 minutes.

(4) 1% solution of the ingredients 19-20 was injected into the emulsifying tank, uniformly admixed at 2500 rpm. The solution was vacuumed while removing bubbles, and cooled to 55 °C under agitation at 25 rpm.

(5) Pre-weighed extracts in Examples were dissolved in ethanol at room temperature and uniformly admixed with pre-weighed perfume in the emulsifying tank. The solution was cooled to 30 °C and lotion was prepared.

Prescription Example 8-10 and Comparative Prescription Example 1: Preparation of Cream

Creams comprising the extracts herein are prepared in Prescription

Examples 8-10, and those without including the extract were prepared in Comparative Prescription Example 1, as shown in Table 9.

Table 9

Preparation method

(1) Pre-weighed ingredients 1-12 were placed in a heatable oily-phase tank, and dissolved with stirring at 75 ⁰C. (2) Pre-weighed ingredients 13-18 were placed in an aqueous-phase tank, and dissolved with stirring at 75 ⁰C.

(3) The solution(2) was moved to an emulsifying tank under vacuum, and the solution(l) was also transferred to the emulsifying tank, followed by emulsification at 3500 rpm for 5 minutes.

(4) 1% solution of the ingredients 19-20 was injected into the emulsifying tank, uniformly admixed at 2500 rpm. The solution was vacuumed while removing bubbles, and cooled to 55 ⁰C under agitation at 25 rpm.

(5) Pre-weighed extracts in Examples were dissolved in ethanol at room temperature and uniformly admixed with pre-weighed perfume in the emulsifying tank. The solution was cooled to 30 ⁰C and cream was prepared.

Prescription Example 11-13; Preparation of hydrogel patch

Hydrogel patch comprising the extracts herein was prepared in Prescription Examples 11-13, as shown in Table 10.

Table 10

Preparation method

(1) Pre-weighed ingredients 1-6 were placed in a dissolving tank and uniformly stirred at room temperature, to provide viscous liquid. Ingredients 7-9 were further dissolved at room temperature and added into the dissolving tank, followed uniform stirring.

(2) Ingredients 10-14 were dissolved at 60 ⁰C and admixed with ingredients 15-16. A small amount of the mixture was injected into the dissolving tank each time, thus producing gel .

(3) The obtained hydrogel was uniformly coated on siliconated polyester film (1 m x 1 m, 700 g/m²) by using a coating machine equipped with an applicator gap, and laminated with nonporous polyethylene non-woven fabric. The multi-layered laminates were double-cut into an appropriate size for application on face or under eyes, and packed in laminated films of paper, polyethylene and aluminum.

Experimental Example 6: Induction of artificial wrinkles on hairless mouse by using squalane-OOH method 1 L of squalane was irradiated with UVA at a maximum strength of 30 J/m²/sec for 4 weeks. The irradiated squalane was admixed with 1 L of methanol in a shaker at 2,000 rpm for 2 hours. Upper-layer fraction was separated by centrifugation at 2,000 rpm for 3 minutes, thereby obtaining squalane-OOH. The oxidized squalane was diluted with ethanol to the ratio of 1:3, and this wrinkle-inducing solution was coated on the skin of 8-week-old hairless mouse twice a day for 6 weeks (10 μL each time).

The extracts and specimen herein were coated for 2 weeks and the wrinkle improvement is shown in Figures 3-5.

Experimental Example 7: Promotion of elastin synthesis observed by Verhoeff stain method

After applying the specimen herein to wrinkle-induced hairless mouse, the effect of elastin synthesis promotion was observed by elastin stain method. The wrinkle-induced eighty mice were divided into eight groups, and six groups were treated with extract (Ex. 1) and specimen 3, 6, 9, 12 and 15 (Ex. 15) ethanol solution (0.01%). One group was treated with retinol (2500 IU/g)/ethanol solution (50%) as a positive control group. The other group was treated with 50% ethanol aqueous solution as a negative control group. After 2-week treatments, the separated skin of the hairless mouse was observed by using Verhoeff stain method.

The improvements were shown in Figures 7-9 as compared with the negative control group in Figure 6. The extract (Ex. 1) and the specimen 9 in Ex. 2) were verified to be superior in promoting elastin synthesis to retinol in the positive control group.

Experimental Example 8; Promotion of collagen synthesis observed by Masson trichrome stain method

After applying the specimen herein to a wrinkle-induced hairless mouse, the effect of collagen synthesis promotion was observed by collagen stain method. The wrinkle-induced eighty mice were divided into eight groups, and six groups were treated with extract (Ex. 1) and specimen 3, 6, 9, 12 and 15 (Ex. 15) ethanol solution (0.01%). One group was treated with retinol (2500 IU/g)/ethanol solution (50%) as a positive control group. The other group was treated with 50% ethanol aqueous solution as a negative control group. After a 2- week treatment, the separated skin of the hairless mouse was observed by using Masson trichrome stain method.

The improvements were shown in Figures 11-13 as compared with the negative control group in Figure 10. The extract (Ex. 1) and the specimen 9 in Ex. 2) were verified to be superior in promoting collagen synthesis to retinol in the positive control group.

Experimental Example 9: Wounds healing effect in rats

Hairs on the back of 8-week-old Wistar male rats were removed, and the skin on the back was cut with a scalpel under an anesthesia and satured. Creams (0.01 % of the specimen in Prescription Examples 8-10 with various ginsenoside Rg3 quantity), creams (0.01% of Centella asiatica), creams according to Comparative

Prescription Example 1 were coated on the wounds once a day (0.5 g each time) for 6 days. After the rats were euthanized, skins around the wounds were separated to provide three samples per each test animal (width: 2 cm).

Tensile strength was measured, and the relative results compared with Comparative Prescription Example 1 were provided in Table 11.

Table 11

Creams comprising the extracts herein were verified to be superior in healing wounds compared with the positive control group.

Experimental Example 10; Effect of human skin wrinkle improvement Effect of human skin wrinkle improvement was observed in fifteen healthy women, who are 30-45 years old, by using the creams of Prescription Examples 8, 9 & 10 and the creams of Comparative Prescription Example 1.

The aforementioned four creams were applied to the skin below eyes twice a day (in the morning and before sleep) for 8 weeks. The improvement was evaluated by using questionnaires (Table 15) and image analysis. The test subjects could select one of four degrees of improvement (i.e. no, low, medium and much). Image analysis was performed by preparing replica of the skin around the eyes after sufficient skin stabilization under a constant condition of 22 ⁰C with relative humidity of 50%. Surface was measured by using an image analyzer system, based on which a relative change of wrinkles was obtained.

Table 12

Wrinkle improvement was remarkable with Prescription Examples herein. Especially, cream of Prescription Examples showed noticeable skin improvement as compared with Comparative Prescription Example 1 according to more than 70% of the test subjects.

Table 13

Wrinkles were observed to decrease down to 30% or less in their density when the creams of Prescription Examples herein were used.

INDUSTRIAL APPLICABILITY

As set forth above, the effect of skin wrinkle improvements varies depending on the amount ginseng-derived ginsenoside Rg3 herein and the ratio of stereoisomers. Especially, the effects of proliferating fibroblast, promoting collagen biosynthesis and elastin synthesis, enhancing skin immunity, and functioning as antioxidants and healing skin wounds may be maximized by controlling the amount of 20(S)-ginsenoside Rg3 to 40-80 wt% and as the purity of S isomer increase up to 100%, thus completed the present invention.

Claims

CLAIMSWhat is claimed is:

1. A cosmetic composition for preventing and improving skin wrinkles, wherein said composition comprises 20(S)-ginsenoside Rg3.

2. The cosmetic composition of claim 1, wherein the amount of said 20(S)-ginsenoside Rg₃ is contained in the amount of from 40 to 80 wt% with respect to that of ginseng extract powder.

3. The cosmetic composition of claim 1, wherein said composition further comprises 20(R)-ginsenoside Rg₃, wherein the ratio of the 20(S)-ginsenoside Rg₃ and the 20(R)-ginsenoside Rg₃ is 10:0-5:5.

4. The cosmetic composition of claim 1, wherein the 20(S)-ginsenoside Rg₃ is separated from ginseng by acidic hydrolysis.

5. The cosmetic composition of claim 1, wherein the amount of said 20(S)-ginsenoside Rg₃ is contained in the amount of from 0.000001 to 20 wt% with respect to the amount of the total cosmetic composition.