WO2008102998A1

WO2008102998A1 - Compositions for improving skin conditions comprising carvacrol as an active ingredient

Info

Publication number: WO2008102998A1
Application number: PCT/KR2008/001022
Authority: WO
Inventors: Deok-Hoon Park; Eun-Sun Jung; Jong-Sung Lee
Original assignee: Biospectrum Inc.
Priority date: 2007-02-21
Filing date: 2008-02-21
Publication date: 2008-08-28
Also published as: KR20080077768A; KR100893162B1

Abstract

The present invention relates to a composition for improving skin conditions comprising carvacrol as an active ingredient. Carvacrol used as an active ingredient, has lower cytotoxicity compared to retinol used as anti-wrinkle agents and exhibits the inhibition effect on collagenase activity and promotion effect on collagen biosynthesis at a molecular level, contributing to excellent efficacy in improvement of skin wrinkles. In addition, carvacrol exhibits the excellent anti-oxidation, skin growth promotion or hair loss prevention effects. In addition, the composition of this invention can be applied to cosmetic, pharmaceutical and food composition having no cytotoxicities and side effects.

Description

COMPOSITIONS FOR IMPROVING SKIN CONDITIONS COMPRISING CARVACROL AS AN ACTIVE INGREDIENT

FIELD OF THE INVENTION The present invention relates to a composition comprising carvacrol as an active ingredient, exhibiting excellent skin wrinkles improvement, skin growth promotion or hair loss prevention and anti-obesity effects with stability and safety.

DESCRIPTION OF THE RELATED ART Wrinkles are caused from skin aging and aged skin is a result of natural changes related to aging process. Skin aging can be broadly divided into physiological aging and photo aging. The former represents changes of skin function, structure or shape associated with aging in entire skin surface, and the latter is induced by ultraviolet radiation. The changes in dermis become remarkable with aging and dermis atrophy is one of representative phenomena after the age of 70. The decrease in both the number of fibroblasts and their biosynthetic potential results in changes of biomolecules having large molecular weights in extracellular matrix, so that the dermis change comes to appear. The changes include segregation of collagen bundle, reduction of mucopolysaccharide synthesis, decrease in the number and diameter of collagen and elastin, decomposition of collagen and elastin, and expansion of blood vessel. Generally, among several intricate causes such as moisture content of skin, collagen content and immune responsiveness to external environments, the main factor of wrinkle formation involves the expression and activity of collagenase, a collagen-degradting enzyme to reduce synthesis and content of collagen. Effect of carvacrol on collagnen and collagenase has not been clarified, it was founded that carvacrol had the enhancement effect on collagen synthesis and the inhibition effect on collagenase activity through this experiment. Hormone imbalance has become increasingly worse due to environmental pollution and auto exhausts. Because of this, the incidence rate of hair loss has become higher, the incidence age is lowered and a variety of skin diseases such as atopy and psoriasis have occurred due to inducing disharmony of skin immune system. In addition, number of young obesity patients has rapidly went on increasing because of the change of dietary life such as meat and instant food centered diets.

Accordingly, studies have been intensively made to develop substances capable of effectively resolving several phenomena {i.e., intrinsic aging and incidence of wrinkles by UV, obesity, and hair loss) which have became serious social problems.

Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVENTION

The present inventors have made intensive researches to develop a novel active substance having activities of improvement in wrinkle, prevention in obesity and hair loss with high stability and safety without side effects on skin. As a result, the present inventors have found that compositions comprising carvacrol as an active ingredient allowed to provide compositions for improving skin wrinkles, anti-obesity, preventing hair loss or promoting hair growth, having excellent effects and safety.

Accordingly, it is an object of this invention to provide a composition for improving skin condition comprising carvacrol as an active ingredient. It is another object of this invention to provide a composition for anti-obesity.

It is still another object of this invention to provide a method for improving skin condition.

It is another object of this invention to provide a method for suppressing obesity. Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.

In one aspect of the present invention, there is provided a composition for improving skin condition comprising carvacrol as an active ingredient, wherein the skin condition improvement is wrinkles improvement, hair growth promotion or hair loss prevention. In another aspect of the present invention, there is provided a method for improving skin conditions, which comprises administering to a subject a composition comprising carvacrol as an active ingredient

In still another aspect of the present invention, there is provided a use of carvacrol for manufacturing a composition for improving skin conditions. In another aspect of the present invention, there is provided a composition for anti-obesity comprising carvacrol as an active ingredient.

In still another aspect of the present invention, there is provided a method for suppressing obesity, which comprises administering to a subject a composition comprising carvacrol as an active ingredient. In another aspect of the present invention, there is provided a use of carvacrol for manufacturing a composition for anti-obesity.

The present inventors have made intensive researches to develop a novel active substance having activities of improvement in wrinkle, prevention in obesity and hair loss with high stability and safety without side effects on skin. As a result, the present inventors have found that compositions comprising carvacrol as an active ingredient allowed to provide compositions for improving skin wrinkles, anti-obesity, preventing hair loss or promoting hair growth, having excellent effects and safety. Carvacrol used as an active ingredient in the present compositon is representative aromatic components in essential oil extracted from oil such as Cinnamomum camphora Sieb, Origanum hirtum, bergamot and thyme. IL⁾PAC name of carvacrol is 2-methyl-5-propan-2-yl-phenol or 2-methyl-5-(l-methylethyl)phenol, and plant-derived compound represented by the following formula I. It has been known that carvacrol has insect-repellent effect, anti-microbial effect on microorganism such as fungi or bacteria and even anti-cancer effect.

Carvacrol used as an active ingredient in compositions of this invention may be extracted from natural source, Cinnamomum camphora Sieb, Origanum hirtum, bergamot and thyme. In detail, the carvacrol may be obtained using conventional various extraction methods. Preferably, the carvacrol may be obtained using various extraction solvents, e.g., (a) water, (b) absolute or hydrous lower alcohol containing 1- 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) mixture of lower alcohol and water, (d) acetone, (e) ethyl acetate, (f) chloroform or (g) 1,3- butyleneglycol.

If necessary, carvacrol used in the present invention may be subjected to additional purification by the well-known methods in the art as well as those obtained by extraction. For instance, it could be appreciated that carvacrol obtained using a variety of additional purification methods such as ultrafiltration with defined molecular weight cut-off value and various chromatography (designed for purification dependent upon size, charge, hydrophobicity and affinity) may be used in the present invention. In addition, Essential oil of origanum hirtum is collected by steam distillation and the carvacrol of this invention may be isolated from the collected essential oil by vacuum procedure.

Furthermore, carvacrol of this invention may be artificially prepared by reacting cymol sulfonic acid to caustic potash (KOH).

The compositions of the present invention have novel use to improve skin wrinkles. The compositions of the present invention have lower cytotoxicity compared to retinol used as anti-wrinkle agents and exhibit the inhibition effect on collagenase activity and promotion effect on collagen biosynthesis at a molecular level, and as a result, excellent efficacy in improvement of skin wrinkles. Such effects and efficacies are demonstrated in Examples described hereunder. It would be appreciated that the improvement of skin wrinkles covers general uses of skin protection {e.g., prevention of skin wrinkles, removal of skin wrinkles and prevention of skin aging). The compositions for improving skin conditions of this invention contribute very effectively to the prevention of hair loss or the promotion of hairs growth. The terms "hair loss prevention" and "hairs growth promotion" used herein have the same meaning.

Moreover, as shown in the following example clearly, the compostion of this invention have an excellent anti-obesity effect.

It is obvious to one skilled in the art that carvacrol represented by Formula I include derivatives obtained by chemical processes with substituents performed conventionally in one skilled in the art. The derivatives show the effects of wrinkles . improvement by promotion of collagen synthesis and/or inhibition of collagenase (MMP-I) activity, or skin growth promotion or hair loss prevention, and anti-obesity effects. According to a preferred embodiment, the active ingredient of carvacrol in the composition is present in the amount of 0.00001-15.0 wt%, more preferably, 0.0001- 10 wt%, most preferably, 0.0001-5 wt% based on the total weight of the composition. If the amount of the active ingredient of carvacrol is lower than 0.00001 wt%, the effect of the composition may be negligible; in the case of exceeding 15.0 wt%, some adverse effects such as skin irritation and instability in formulation are very likely to occur.

According to the preferred embodiment, the composition of the present invention is a cosmetic composition.

The cosmetic compositions of the present invention may contain auxiliaries as well as carrier in addition to the carvacrol as an active ingredient. The non-limiting examples of auxiliaries include antioxidants, stabilizers, solubiϋzers, vitamins, colorants, odor improvers or mixtures of these ingredients. In addition, the cosmetic compositions may additionally comprise promoting materials of skin absorption to enhance the effects.

The cosmetic compositions of this invention may be formulated in a wide variety of form, for non-limited example, including 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. In detail, the cosmetic composition of the present invention can be provided in a form of skin softener (skin lotion), nutrient emulsion (milk lotion), nutrient cream, message cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, facial pack, spray or powder. The cosmetically acceptable carrier contained in the present cosmetic composition, may be varied depending on the type of the formulation. For example, the formulation of pastes, creams or gels may comprise animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide or mixtures of these ingredients.

In the formulation of powder or spray, it may comprise lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or mixtures of these ingredients. Spray may additionally comprise the customary propellants, for example, chlorofluorohydrocarbons, propane/butane or dimethyl ether.

The formulation of solution and emulsion may comprise solvent, solubilizer or emulsifier, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol oils, glycerol fatty esters, polyethylene glycol, fatty acid esters of sorbitan or mixtures of these ingredients.

The formulation of suspension may comprise liquid diluents, for example water, ethanol or propylene glycol, suspending agents, for example ethoxylated isosteary alcohols, polyoxyethylene sorbitol esters and poly oxyethylene sorbitan esters, micocrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures of these ingredients.

The formulation of cleansing compositions with surfactant may comprise aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosucinnate monoester, isothinate, imidazolium derivatives, methyltaurate, sarcocinate, fatty acid amide ether sulfate, alkyl amido betain, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanoline derivatives, ethoxylated glycerol fatty acid ester or mixtures of these ingredients.

The composition of this invention may be prepared as a pharmaceutical composition, and the pharmaceutically acceptable carrier as well as the active ingredient contained in the pharmaceutical composition. The pharmaceutically acceptable carrier, which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of this invention may be administered to mammals such as rat, mouse, domestic animals and human via various routes, for example oral administration, rectal administration or intravenous injection, intramuscular injection, subcutaneous injection, intrauterine injection or intracerebroventricular injection, preferably subcutaneous injection, more preferably topical application.

A suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition, and physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment. In case of oral formulation, a suitable dosage unit may be administered once to several times a day with 0.001-100 mg/kg on the basis of adult. In case of preparation for external use, a suitable dosage unit may be administered by applying once to five times a day in amounts of 1.0 to 3.0 ml on the basis of adult and it has better use for more than 1 month. However, the dosage unit does not limit the scope of this invention.

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition of the present invention may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, oral formulation such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup and an aerosol, preparation for external use such as an ointment and a cream, a suppository and sterile injection solution, and may further comprise a dispersion agent or a stabilizer.

The composition of this invention may be prepared as a food composition. The food composition of this invention may comprise conventional additives for preparing food compositions, e.g., protein, carbohydrates, lipids, nutritive substances and flavors.

Non-limiting examples of carbohydrates described above include, but not limited to, monosaccharide {e.g., glucose and fructose); disaccharide (e.g., maltose, sucrose and oligosaccharide); and polysaccharide {e.g., dextrin and cyclodextrin); and sugar alcohol (e.g., xylitol, sorbitol and erithritol). Non-limiting examples of Flavors include, but not limited to, natural flavors [thaumatin and extract of stevia {e.g., rebaudioside A and glycyrrhizin)] and synthetic flavors {e.g., saccharin and aspartame).

For example, where the food composition of this invention is provided as a drink, it may further comprise citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, extract of eucommia ulmoides oliv, jujube extract or extract of glycyrrhiza uralensis.

Meanwhile, experiment results of a cumulative skin irritation elucidated that carvacrol as natural substance was harmless to human body in specific example of the present invention. Therefore, carvacrol of the present invention may be used with confidence for long period due to not or a little having toxicities and side effects, particularly may be applied to cosmetic, pharmaceutical and food composition with safety as described above.

The summary of features and advantages of this invention is as follows: (i) The composition of the present invention comprises carvacrol as an active ingredient.

(ii) Carvacrol has lower cytotoxicity compared to retinol used as anti-wrinkle agents and exhibits the inhibition effect on collagenase activity and promotion effect on collagen biosynthesis at a molecular level, contributing to excellent efficacy in improvement of skin wrinkles. In addition, carvacrol exhibits the excellent anti- oxidation, skin growth promotion or hair loss prevention effects.

(iii) In addition, the composition of this invention can be applied to cosmetic, pharmaceutical and food composition having no cytotoxicities and side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph showing that carvacrol as an active ingredient of this invention has lower cytotoxicity against human fibroblasts compared to RA. RA represents retinoic acid. RA and carvacrol were treated in amounts of 1 μM, 10 μM and 50 μM, respectively.

Fig. 2 is a graph showing the promotion effect on collagen (Type 1 collagen) biosynthesis by carvacrol. RA was treated in amounts of 1 μM.

Fig. 3 represents the inhibition effect on collagenase (MMP-I) activity as the concentration of carvacrol as an active ingredient of this invention increases. MMP-I and PMA show Type 1 collagenase and phorbol myristate acetate, respectively. PMA was treated in amounts of 100 nM.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES Example 1: Measurement of effect of carvacrol on wrinkles improvement Test of wrinkle improvement effect can be generally measured through collagen biosynthesis ability, collagenase degradation inhibitory ability and clinical test to human. Human fibroblasts (commercially available from pacific) were seeded into a 6- well plate (2 x 10⁵ cells/well) and the well plate was incubated in a 5% CO₂ incubator for 24 hr at 37⁰C. After 24 hr, the medium in each well was removed and samples were treated with various concentrations, followed by incubating again for 24 hr. Following incubation, the cell medium was collected and was used as samples. Hg. 1 represents that samples have no cytotoxicity in case of being treated in amounts of 1 μM, 5 μM and 10 μM.

The extent of collagen synthesis was determined by measuring the amount of procollagen type I C-peptide (PICP) in cell medium using Procollagen Type I C-peptide EIA kit (MKlOl, Takara, Kyoto, Japan). The method was performed in accordance with manufacturer's protocol.

As a method of measuring the activity of collagenase, an enzyme that decomposes collagen, an antibody against collagenase was used. As material inducing collagenase activity, PMA (phorbol myristate acetate, Sigma) was treated. For measuring collagenase activity, a Type 1 collagenase assay kit (Amersham Biosciences, RPN2629) was used, and the absorbance was measured using an ELISA reader (Bio- Tek ELx808™ Series Ultra Microplate Reader, U. K). The measured average values were represented as mean ± standard deviation. A T-test with SPSS/PC+ was conducted to determine significance, and the result is shown in Table 1 and Figs. 2-3. Table 1

Carvacrol Ca rvacrolCarvacrol

Test items (l μM) (10 μM) (50 μM)

Increasing rate of collagen synthesis (%) _20_ j>6 25

Inhibition of collagenase (%) 13 20 29

As shown in Table 1 and Figs. 2-3, it was founded that carvacrol enhanced collagen synthesis and inhibited collagenase activity. Therefore, these results demonstrate that carvacrol has effect of wrinkles improvement. In addition, this enhancement of collagen synthesis and inhibition of collagenase activity exhibited in carvacrol concentration-dependent manner as a whole.

Furthermore, as indicated in Hg. 2, carvacrol showed a similar increasing rate to collagen synthesis to positive control group, RA (retinoic acid).

Example 2: Measurement of effects of cosmetics containing carvacrol on the wrinkles improvement

The effects of cosmetics containing carvacrol on the improvement of wrinkles were measured in a clinical demonstration. The nutrient creams prepared in Preparation Example A (nutrient creams containing carvacrol in amounts of 1%, 2% and 5%, respectively) and Comparative Preparation Example (a nutrient cream containing purified water) were used

The effects of wrinkle improvement were evaluated by measuring the changes in the elasticity of the skin. Measurements were conducted on 30 healthy female test subjects (aged 25 to 35) in a stable environment of temperature ranging from 24⁰C to

26⁰C and humidity ranging from 38% to 40%. After 3 types of nutrient creams of

Preparation Example A and the nutrient cream of Comparative Preparation Example were applied to the facial skin of test subjects twice a day for 3 months, the elasticity was measured using a Cutometer SEM 474 (Courage+Khazaka, Cologne, Germany). Relative grades were set forth for skin elasticity within a range from zero for no elasticity to 5 for the highest elasticity measured, and the results are shown in Table 2.

Table 2

As shown in Table 2, the preparation example A of the present invention showed significantly greater effects on the improvement of wrinkles compared to the comparative preparation example, and skin elasticity enhanced as the concentration of carvacrol increased.

Preparation Example A: Preparation of cream containing carvacrol

A nutrient cream (preparation example A) containing carvacrol were prepared as indicated in Table 3. Aqueous phases including purified water, triethanolamine and propylene glycol, and oil phases including fatty acids, oil components, emulsifiers, and preservatives were heated to 7O⁰C and mixed for emulsification. After completion of the emulsification, the emulsion was cooled to 45⁰C. Carvacrol and perfumes were added and dispersed before cooling to 3O⁰C.

Table 3

Components and contents of nutrient cream containing carvacrol

Example 3: Measurement of effects of carvacrol on inhibition of melanin production

After measuring the inhibition of melanin production by carvacrol using B16 mouse melanoma cells (Korean Cell Line Bank), the measurement was compared with that for the inhibition of melanin production by arbutin, known as a melanin production inhibitor.

B16 mouse melanoma cells FlO (Korean Cell Line Bank) were seeded into each well of a 6-well plate (1 x 10⁵ cells/well) in DMEM (Dulbecco's modified Eagle's media) containing 10% FBS (fetal bovine serum) and the cells were cultured to about more than 80% confluence by incubating in a CO₂ incubator under conditions of 37⁰C and 5.0% CO₂. After cultivation, the medium was removed and samples were replaced in medium diluted at suitable concentration, followed by incubating for 3 days under conditions of 37⁰C and 5.0% CO₂. The concentration of carvacrol was determined with 1 μM, 10 μM and 50 μM, which did not show cytotoxicity. The cells removing medium were washed with PBS (phosphate buffer saline) and treated with trypsin to collect cells. Number of the collected cells was calculated using hematocytometer (Tiefe Depth Profondeur 0.100 mm, Paul Marienfeld GmbH & Co. KG, D.E), centrifuged at 5,000 to 10,000 rpm for 10 min and the supernatant was eliminated, thereby obtaining pellets. This cell pellets were dried at 6O⁰C, 100 ml of IM NaOH containing 10% DMSO was added to thereto, and intracellular melanin was obtained in incubator at 6O⁰C. Then, the cell solution was measured for absorbance at 490 nm using microplate reader (Bio-Tek ELx8081U, U. S) and the amount of melanin per cell constant number was estimated. The experiment results were summarized in Table 4. Table 4

Inhibitory rate of melanin production (%)

23

14

As indicated in Table 4, carvacrol showed significantly greater effects on the inhibition of melanin production compared to albutin. In addition, it was founded that carvacrol inhibited melanin production in concentration-dependent manner.

Example 4: Inhibitory effect on tyrosinase activity by carvacrol

After measuring the inhibition of melanin production by carvacrol using B16 mouse melanoma cells (Korean Cell Line Bank), the measurement was compared with that for the inhibition of intracellular tyrosinase activities by arbutin, known as a melanin production inhibitor.

Murine melanoma (B-16 Fl) cells were seeded into each well of a 6-well plate (1 x 10⁵ cells/well) in DMEM containing 10% FBS (fetal bovine serum) and the cells were cultured to about more than 80% adherence by incubating in a CO₂ incubator under conditions of 37⁰C and 5.0% CO₂. After cultivation, the medium was removed and samples were replaced in medium diluted at suitable concentration, followed by incubating for 3 days under conditions of 37⁰C and 5.0% CO₂. The concentration of carvacrol was determined with 1 μM, 10 μM and 50 μM, which did not show cytotoxicity. The cells removing medium were washed with PBS (phosphate buffer saline) and treated with trypsin to collect cells. Number of the collected cells was calculated using hematocytometer, centrifuged at 5,000 to 10,000 rpm for 10 min and the supernatant was eliminated, thereby obtaining pellets. This cell pellets were lysated using lysis buffer, centrifuged at 12,000 rpm for 10 min and the supernatant was collected. Then, the cell solution was measured for absorbance at 492 nm using microplate reader and the activity of tyrosinase per cell constant number was estimated. The results were summarized in Table 5. Table 5 Sample Treatmene concentration (μM) Inhibitory rate of intracellular tyrosinase activity (% )

Albutin 100 29

1 11

Carvacro 10 43

50 57

As shown in Table 5, the results demonstrate that carvacrol inhibited significantly greater effects on the inhibition of intracellular tyrosinase activities than albutin. In addition, it was founded that carvacrol inhibited intracellular tyrosinase activities in concentration-dependent manner.

Example 5: Evaluation of skin whitening effect in animal level

A whitening effect of carvacrol was measured using brown guinea pigs (Charles River Laboratories, Inc.), known to increase its pigmentation upon exposure to ultraviolet light, like in humans.

To cause pigmentation in the brown guinea pig by ultraviolet (UV), aluminum foil with square windows of 3x3 cm² was adhered to hair-removed abdominal skin of brown guinea pig, and then UV light was irradiated thereon with a SE lamp (wavelength 290-320 nm, Toshiba) (total irradiation energy = 1350 mJ/cm²). After UV irradiation, the aluminum foil was removed and samples (carvacrol or albutin) were applied as the following method. Increased pigmentation was observed at 2 or 3 days after UV irradiation and reached a maximum after about 2 weeks. From the maximum, samples were applied. Applications performed once or twice a day for 50 days. The samples were dissolved or diluted in a certain solvent (Propylene glycol : ethanol : water = 5 : 3 : 2) and applied by a swab. The control with only the solvent was applied to another site. Occurrence of cumulative irritation also was examined.

The degree of pigmentation of skin was determined using a chromameter (CR2002, MINOLTA, JP) to estimate the effects of applied samples. The results are shown in Table 6 below. L^*a^*b^* colorimetric system was used to classify color and L^* value was used as standard in the present invention. The L^* value was corrected using white board standard and was measured more than five times at one site, repeatedly. Pigmentation was evenly distributed. Skin color differences (ΔL^*) between application initial point and application terminal point were obtained and then using these values, their effects of the applied samples were estimated. These experiment results were summarized in the following Table 6. Equation 1

ΔL^* = L^* value at 00 days after application-L^* value at application initial day.

ΔL^* values were obtained both at sample application site and control application site and compared, whereby the effects of the whitening substances can be estimated. Table 6

Samples Treatment concentration (%) Whitening effe

0.2 0.41

Carvacro

1.0 0.55

Albutin 1.0 0.46

Control - 0.35

As described in Table 6, carvacrol showed more excellent whitening effects than albutin, and also had a safety due to not observing the occurrence of cumulative irritation.

Example 6: Safety test of carvacrol on human skin

In order to find out whether carvacrol are safe for use on human skin, a skin safety test was conducted. Suitable for this was a cumulative skin irritation test.

Squalene-based preparations containing carvacrol in amounts of 1%, 5%, and 10% were applied in patches 9 times to the upper arms of 30 healthy adults once every other day for a total time period of 24 hours. This 24-hour cumulative patch test was conducted to determine whether carvacrol irritates the skin or not.

The Finn chamber (Epitest Ltd, Finland) was chosen as the patching method.

The above preparation for external use on the skin was dropped into each chamber in an amount of 15 μl, and a patch was applied. The level of reaction on the skin for each test was scored using the following Equation 1, and the result was shown in Table 7. Equation 2

Average reaction level = [{(Reaction index x reaction Ievel)/No. of test subjects x maximum points (4 points)} x 100] ÷ No. of test (9 times)

Points were marked in accordance with reaction level, for example, ± for 1 point, + for 2 points, and ++ for four points. The composition can be considered safe if the average reaction level is below 3. Table 7

In the Table 7, the number of people is 1, 2, and 2, respectively, for ±, + and ++ all in Tests 1, 2 and 3, the average reaction level was calculated to be 0.09, 0.18 and 0.18, respectively. As the average reaction level is below 3, carvacrol was proven to be a safe substance for human skin, not showing any significant cumulative irritation.

Example 7: Anti-oxidation effect

The scavenging activity of samples may be directly examined by observing the color change of DPPH (1, l-diphenyl-2-picrylhydrazyl, Sigma) as color-developing radicals. 20 μl of sample dissolving in solvents was added to 96-well plate and each 180 μl of DPPH solution was added to thereto, followed by incubating for 30 min at room temperature. The remaining amount of DPPH was determined by measuring the absorbance at 540 nm. The analysis results of anti-oxidation activity are summarized in Table 8. The anti-oxidation effects were determined by observing the scavenging rate of free radicals by sample. Table 8

As shown in Table 8, carvacrol of the present invention exerted the anti- oxidation effects in concentration-dependant manner.

Example 8: Evaluation of anti-inflammatory effects

To determine whether carvacrol had anti-inflammatory effects, the experiment of COX (cyclooxygenase) inhibitory ability was carried out using human monocytic cell, THP-I cell (Korean Cell Line Bank) according to conventional method. The COX activity was measured in accordance with Methods in Enzymology Λ3:9 (1994) published by F. J. Van de Ouderaaa and Muytenhek. THP-I cell line was cultivated and aliquoted into 24-well plate. The incubation volume per well was adjusted to 500 μl, 2 μl of the sample compound, which dissolved in lipopolysaccahride (1 μg/ml, Sigma) and solvents described in the following Table 9 respectively, was added and cultivated for 24-48 hr under the same condition. After 24-48 hr, calcium ionophore (Sigma) and [1- 14C] arachidonic acid 1 μl (in EtOH, 0.1 μCi/ml, Sigma) were added to each well, and cultivated for 10 min under the same condition. Following the cultivation, citric acid was added to each well for adjusting to pH 3.5, shaked, each 500 μl of cultivation solution taken from the plate was aliquoted into micro centrifuge tube, 700 μl of ethylacetate was added to thereto, and the solution was shaking extracted for 10 min. 500 μl of ethylacetate layer was concentrated using speed vacuum dryer for 20 min, 20 μl of the residual was dissolved in ethylacetate, and the resultant was employed with authentic standard in TLC plate. The radioactive band was identified by authentic eicosanoid standards, the radioactivity of the identified band was measured using BAS 2000 bio-imaging analyzer (Fuji, JP). Table 9

As described in Table 9, the results address that carvacrol effectively inhibited COX activities. From these results, it was founded that carvacrol have the inflammatory inhibitory effects.

Example 9: Evaluation of Immunosuppressive effects

For examining whether the samples used in this example suppressed the immune response related to inflammatory, the experiment of interleukin-2 luciferase reporter activity was performed. Interleukin-2 promoter was reported to play an important role in the generation of cytokine related to inflammatory. The activity of interleukin-2 luciferase was measured using the following method: Human T lymphocytes cell line, 1 x 10⁶ of Jurkat cell (Korean Cell Line Bank) were aliquoted into each well of 6-wells, IL-2 luciferase reporter plasmid DNA (Stratagene) was transfected using superfect transfection reagent (In vitrogen). 24 hr after transfection, PHA (Phytohemaglutinin, Sigma) (100 ng/ml) was treated to activate Jurkat cell and each samples was treated with varying concentration. Following 24 hr, the cell was collected and the luciferase activity was measured using luminometer (Berthold Technologies GmbH&Co.KG, Germany). The results were summarized in Table 10. Table 10

Samples IL-2 luciferase activities

PHA (100 ng/ml) 100

PHA(IOO ng/ml)+ Carvacrol (1 uM) 94

PHA(IOO ng/ml)+ Carvacrol (10 uM) 85

PHA(IOO ng/ml)+ Carvacrol (50 uM) 76

As shown in Table 10, the results address that carvacrol is capable of inhibiting the immuno-response by inhibiting the IL-2 expression.

Example 10: Anti-obesity effect

An obesity inhibition test was performed by the following method using well- known animals. Table 11 shows the results.

Method for determining obesity-inhibitory activity was as follows:

Crj : ICR male mice (obtained from Charles River Japan Ltd) aged 7 weeks were preliminarily fed for 1 week, then classified into groups each having 7 animals and subjected to the test. The animals were fed in a thermo-hygrostat at a temperature of 23±1°C, and a humidity of 55±5% under illumination for 12 hours per day. They were fed with a feed Labo MR (manufactured by Nippon Nosan) and allowed to take water ad libitum. The carvacrol was in the form of liposome in 5% lecithin to become 0.1% and 1%. The concentration of each sample solution was regulated so that 0.1 ml of the solution was given per 10 g body weight of mice. The doses employed were 1.5 g/kg and 1 g/kg. To a control group, 5% lecithin emulsion was administered. After fasting the mice, the sample was administered once by force on the next day. During the test period over 2 weeks, the body weight and general conditions were monitored. Table 11

As shown in Table 11, the body weight gain was inhibited in carvacrol. It was observed to the excellence of the effects, as administered concentration was high.

Example 11: Effect of hair loss prevention and hairs growth promotion

For measuring the effect of hair loss prevention and hairs growth promotion, the samples were prepared in the form of hydrogel base containing only viscosity- increasing agent and preservative and test was performed. Each 3 cc of liquids for external use for promoting hairs growth prepared were applied to the hair loss sites of 10 baldness patients twice a day for 3 months. As a result, the liquids containing the samples showed an excellent effect such as the generation of the root of hair from 8 baldness patients. The experiment results were as follows. The control group used the moxidil commercially available from Hanmi pharmaceutical. Co. Ltd. Table 12

As indicated in Table 12, the results demonstrate that carvacrol of this invention exerted a similar hairs growth effect to commercially available moxidil as hairs growth formulations. As described hereinabove, the present invention provides a composition comprising carvacrol as an active ingredient. The carvacrol used as an active ingredient of this invention, has lower cytotoxicity compared to retinol used as anti- wrinkle agents and exhibits the inhibition effect on collagenase activity and promotion effect on collagen biosynthesis at a molecular level, and as a result, excellent efficacy in improvement of skin wrinkles. In addition, the carvacrol exhibits the excellent anti- oxidation, skin growth promotion or hair loss prevention effects. In addition, the composition of this invention can be applied to cosmetic, pharmaceutical and food composition due to having no cytotoxicities and side effects.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A composition for improving skin condition comprising carvacrol as an active ingredient, wherein the skin condition improvement is wrinkles improvement, skin growth promotion or hair loss prevention.

2. A composition for anti-obesity comprising carvacrol as an active ingredient.

3. The composition according to claims 1 or 2, wherein the carvacrol is present in the amount of 0.0001-10 wt% based on the total weight of the composition.

4. The composition according to any one of claims 1 to 3, wherein the composition is a cosmetic composition.

5. The composition according to claim 4, wherein the composition is in the form of one 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.

6. The composition according to any one of claims 1 to 4, wherein the composition is a pharmaceutical composition.

7. The composition according to any one of claims 1 to 4, wherein the composition is a food composition.

8. A method for improving skin condition, which comprises administering to a subject a compositon comprising carvacrol as an active ingredient.

9. The method according to claim 8, wherein the skin condition improvement is wrinkles improvement, hair growth promotion or hair loss prevention.

10. A method for suppressing obesity, which comprises administering to a subject a compositon comprising carvacrol as an active ingredient.

11. The method according to any one of claims 8 to 10, wherein the carvacrol is present in the amount of 0.0001-10 wt% based on the total weight of the composition.

12. The method according to any one of claims 8 to 11, wherein the composition is a cosmetic composition.

13. The method according to claim 12, wherein the composition is in the form of one 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.

14. The method according to any one of claims 8 to 11, wherein the composition is a pharmaceutical composition.

15. The method according to any one of claims 8 to 11, wherein the composition is a food composition.

16. A use of carvacrol for manufacturing a composition for improving skin condition.

17. The use of the carvacrol according to claim 16, wherein the skin condition improvement is wrinkles improvement, hair growth promotion or hair loss prevention.

18. A use of carvacrol for manufacturing a composition for anti-obesity.

19. The use of the carvacrol according to any one of claims 16 to 18, wherein the carvacrol is present in the amount of 0.0001-10 wt% based on the total weight of the composition.

20. The use of the carvacrol according to any one of claims 16 to 19, wherein the composition is a cosmetic composition.

21. The use of the carvacrol according to claim 20, wherein the composition is in the form of one 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.

22. The use of the carvacrol according to any one of claims 16 to 19, wherein the composition is a pharmaceutical composition.

23. The use of the carvacrol according to any one of claims 16 to 19, wherein the composition is a food composition.