WO2014003224A1 - Compositions for whitening skin comprising madecassoside - Google Patents

Compositions for whitening skin comprising madecassoside Download PDF

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Publication number
WO2014003224A1
WO2014003224A1 PCT/KR2012/005211 KR2012005211W WO2014003224A1 WO 2014003224 A1 WO2014003224 A1 WO 2014003224A1 KR 2012005211 W KR2012005211 W KR 2012005211W WO 2014003224 A1 WO2014003224 A1 WO 2014003224A1
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WIPO (PCT)
Prior art keywords
madecassoside
skin
composition
whitening
present
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PCT/KR2012/005211
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French (fr)
Inventor
Deok Hoon Park
Eun Sun Jung
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Bio Spectrum, Inc.
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Application filed by Bio Spectrum, Inc. filed Critical Bio Spectrum, Inc.
Priority to JP2014522735A priority Critical patent/JP2014520166A/en
Priority to PCT/KR2012/005211 priority patent/WO2014003224A1/en
Priority to CN201280021188.7A priority patent/CN103796631A/en
Publication of WO2014003224A1 publication Critical patent/WO2014003224A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the present invention relates to a skin whitening composition comprising madecassoside, and more particularly to a skin whitening composition comprising madecassoside that has excellent stability without side effects on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin generation.
  • Human skin color is determined according to the concentration and distribution of melanin in the skin, and affected by environmental or physiological factors such as ultraviolet rays of the sun, fatigue, or stress as well as hereditary factors.
  • the melanin pigment that is produced in the melanocytes of the human skin is a phenolic polymer consisting of a complex of a black pigment and a protein and plays an important role in preventing skin damage caused by UV light.
  • the activity of tyrosinase present in melanocytes was reported to be the most important factor in melanin biosynthesis. Tyrosinase plays an important role in the skin darkening process by converting tyrosine into DOPA and DOPA-quinone, which are intermediate products of melanin polymer production.
  • cytokines are involved in the interaction between keratinocytes and melanocytes. Particularly among them, PGE2 which is secreted from keratinocytes has been reported to play a major role in stimulating melanin pigmentation by increasing the production of cAMP in melanocytes. Substances capable of inhibiting such PGE2 can also act as whitening substances effective in preventing skin pigmentation caused by UV rays.
  • Overproduction of the melanin may induce discoloration, melasma, freckles or the like. Accordingly, the inhibition of melanin overproduction improves skin hyperpigmentation such as melasma and freckles due to UV, hormone, and hereditary factors, as well as skin brightening and whitening effects.
  • hydroquinone has problems in that only an extremely restricted amount should be used due to the severe skin irritation, even though it exhibits skin whitening effects.
  • Ascorbic acid is easily oxidized, so that cosmetics blended with it have problems of color and odor changes, and kojic acid is unstable in a solution, which requires a complicated preparation prccess.
  • thiol based compounds such as glutathione and cysteine have unique unpleasant odors as well as problems in transdermal absorption, and glycosides and derivatives thereof have problems in that they cannot be appropriately used as mixed ingredients of cosmetics due to their high polarities.
  • Vitamin C is disadvantageous in that it is easily oxidized in an aqueous solution, so as not to continuously exhibit its effect.
  • skin-whitening compositions containing extracts of natural medicinal herbs have been developed. However, since most of them are colored, there are limitations in blending. Further, since their effective ingredients are not identified, consistent effects cannot be expected in products.
  • It is an object of the present invention to provide a skin whitening composition comprising madecassoside that exhibits whitening effects not only by inhibiting the production of melanin in skin melanocytes, but also by inhibiting the skin darkening cytokine PGE2 that is secreted from skin keratinocytes.
  • composition for whitening skin comprising madecassoside as an active ingredient.
  • a cosmetic composition comprising said composition for whitening skin.
  • a method of whitening the skin of a subject by administering said composition for whitening skin to the subject in a dermatologically acceptable way.
  • madecassoside exhibits skin whitening effects not only by inhibiting skin darkening cytokine, which is secreted from keratinocytes, but also by inhibiting the production of melanin in melanocytes.
  • madecassoside caused neither toxicity nor irritation, suggesting that it is safe for human use.
  • madecassoside of the present invention can be advantageously used in skin whitening cosmetic products, drugs or foods.
  • FIG. 1 shows a system for coculturing skin keratinocytes with melanocytes.
  • FIG. 2 shows a comparison of melanin density between a UV-irradiated artificial skin, treated with 0.5% madecassoside, and a UV-irradiated control skin.
  • Madecassoside which is used as an active ingredient in the composition of the present invention is a member of the triterpenoid family, which is found mainly in Centella asiatica , and is known to improve wound healing by reducing the production of keloid. It has a structure represented by the following formula 1.
  • This madecassoside can be extracted from natural sources, including plants of Centellaasiatica(L.) and the like.
  • madecassoside can be obtained from the above natural sources us ing various extract ion methods known in the art.
  • it can be extracted using an extraction solvent selected from among (a) water, (b) an anhydrous or hydrated lower alcohol containing 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol with water, (d) acetone, (e) ethylacetate, (f) chloroform, (g) 1,3-butylene glycol, or a mixture of two or more thereof. More preferably, it can be extracted using an aqueous solution of methanol, ethanol and butanol, and most preferably, it can be extracted using an aqueous solution of butanol.
  • the extracts of the present invention include, in addition to the extracts obtained using the above-described extraction solvents, extracts obtained through conventional purification processes. For example, fractions obtained through various additional purification methods, such as separation with an ultra filtration membrane having a given molecular weight cut-off or separation by various chromatography systems (manufactured for separation according to size, charge, hydrophobicity or affinity), are also included in the scope of the extract of the present invention.
  • madecassoside that is used in the present invention may be prepared by chemical synthesis or may be a commercially available product (for example, madecassoside available from Chromadex or Sigma).
  • madecassoside of the present invention acts on all skin melanocytes and keratinocytes to exhibit the effects of inhibiting intracellular melanogenesis and skin pigmentation.
  • madecassoside examples include, in addition to the madecassoside compound of formula 1, madecassoside derivatives, which show the effects of suppresing melanin synthesis and/or cytokines inducing skin darkening to exhibit of whitening skin, among derivatives obtained by adding substituents to madecassoside or substituting madecassoside according to a conventional method known in the art. More specifically, examples of madecassoside that may be used in the present invention include, in addition to the compound of formula 1, derivatives obtained by linking various substituents to the structure of formula 1 as a nucleus according to a method known in the art.
  • the content of madecassoside may be preferably 0.0001 to 15 wt%, based on the total weight of the composition. If the content is less than 0.0001 wt%, the whitening effect is not sufficient, and if the content is more than 15 wt%, the whitening effect is slightly increased, whereas there is a problem in the stability upon formulation. More preferably, madecassoside is contained in an amount of 0.0001 to 10 wt%, based on the total weight of the composition.
  • the skin-whitening composition of the present invention may be used as a cosmetic composition for whitening skin.
  • the cosmetic composition of the present invention may contain, in addition to madecassoside as an effective ingredient, components that are conventionally used in cosmetic compositions, for examples, conventional auxiliaries, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. Further, the cosmetic composition may further contain a skin absorption enhancer to improve skin-whitening effect.
  • the cosmetic composition of the present invention may be prepared as any type of formulations as well known in the related art, including, if not specifically limited to, 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 powdered foundation, an emulsion foundation, a wax foundation, a spray, or the like. More specifically, the cosmetic composition may be formulated into a softening toner, a nutrient toner, nutrient cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, and a powder.
  • the formulation of the present invention is paste, cream, or gel, it may contain, as carrier components, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, and so forth.
  • the formulation of the present invention is a powder or spray formulation, it may contain, as carrier components, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder.
  • a propellant such as chlorofluorohydrocarbon, propane/butane, or dimethyl ether.
  • the formulation of the present invention is a solution or an emulsion, it may contain, as carrier components, a solvent, a solubilizing agent or an emulsifier.
  • a solvent e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol-aliphatic ester, polyethylene glycol, or sorbitan-aliphatic ester.
  • the formulation of the present invention is a suspension
  • it may contain, as carrier components, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, or the like.
  • a liquid diluent such as water, ethanol, or propylene glycol
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester
  • microcrystalline cellulose aluminum metahydroxide
  • bentonite agar
  • tragacanth gum or the like.
  • the formulation of the present invention is a surfactant-containing cleanser
  • it may contain, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ester sulfate, sulfosuccinic acid monoester, isethionate, imidazolium derivatives, methyl taurate, sarcosinate, aliphatic acid amide ether sulfate, alkyl amidobetaine, aliphatic alcohol, aliphatic glyceride, aliphatic acid diethanol amide, vegetable oil, lanolin derivatives, or ethoxylated glycerol aliphatic acid ester.
  • the content of madecassoside may be 0.0001 to 15 wt%, and preferably 0.001 to 10 wt%, based on the total weight of the cosmetic composition.
  • the skin-whitening composition of the present invention may be used as a pharmaceutical composition for whitening skin.
  • the content of madecassoside may be 0.0001 to 15 wt%, and preferably 0.001 to 10 wt%, based on the total weight of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluent commonly used in the preparation of pharmaceutical compositions.
  • the pharmaceutical composition of the present invention may be formulated into any suitable formulation including oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup and aerosol; external preparation such as ointment and cream; suppository, and strerilized solution for injection.
  • oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup and aerosol
  • external preparation such as ointment and cream
  • suppository suppository, and strerilized solution for injection.
  • the pharmaceutical composition of the present invention may be administered to mammals such as rat, mouse, livestock, and human via various routes such as oral and parenteral route (e.g., oral, rectal or intravenous, intramuscular, subcutaneous or intraepidural injection), preferably transcutaneous route among parenteral routes, and more preferably topical application.
  • oral and parenteral route e.g., oral, rectal or intravenous, intramuscular, subcutaneous or intraepidural injection
  • transcutaneous route among parenteral routes preferably topical application.
  • the effective dosage of the pharmaceutical composition of the present invention may be determined depending on the patient s age, gender, body weight, symptom, and the route of administration.
  • madecassoside may be preferably administered at a daily dosage of 1.0 to 3.0 ml for adult one to five times a day for at least one month.
  • madecassoside may be preferably administered at a daily dosage of 0.1 to 100 mg/kg for adults one time or several times, depending on the patient's age, gender, and body weight.
  • the dosage may be adjusted depending on the route of administration, severity of the disease, the patient's gender, body weight, and age.
  • the scope of the present invention is not limited to the dosage.
  • the skin-whitening composition of the present invention may be used as a food composition for whitening skin.
  • the food composition of the present invention may contain, in addition to madecassoside as an effective ingredient, conventional additives for preparing food compositions, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors.
  • carbohydrates include conventional sugars, such as monosaccharides (e.g., glucose and fructose), disaccharides (e.g., maltose, sucrose and oligosaccharide), and polysaccharides (e.g., dextrin and cyclodextrin); and sugar alcohols (e.g., xylitol, sorbitol and erithritol).
  • flavors include natural flavors [thaumatin and extract of stevia (e.g., rebaudioside A and glycyrrhizin)] and synthetic flavors (e.g., saccharin and aspartame).
  • the food composition of the present invention may include not only the madecassoside of the present invention but also other additives, such as, for example, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and so forth.
  • other additives such as, for example, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and so forth.
  • madecassoside proved to be a natural substance harmless to the human body in the cumulative skin irritation test for madecassoside.
  • the madecassoside of the present invention free from toxicity and adverse side effects, can be safely used for long-term use, particularly in the aforementioned applications, such as cosmetic, pharmaceutical or food compositions.
  • the present invention also provides a method for whitening the skin of a subject by administering said composition to the subject in a dermatologically acceptable way.
  • the term "subject” refers to a mammal including human, such as, for example, dogs, pigs, horses, cows, etc. that need to improve or prevent skin darkening or hyperpigmentation caused by ultraviolet rays of the sun, hormone, hereditary factors, or the like.
  • the term "administration or administering” refer to introducing a defined substance into an object by an appropriate method.
  • the composition of the present invention may be used by any known route of administration, oral or non-oral, for deliver to a target site of tissue. Further, the composition of the present invention may be administered by way of any device for delivering an active substance to a target site, such as cells.
  • Example 1 Evaluation of inhibitory effect of madecassoside on melanin synthesis in murine B16 melanoma cells
  • Murine B16 melanoma cells were used to measure the inhibitory effect of madecassoside(Sigma) on melanin synthesis, which was compared with the inhibitory effect of vitamin C on melanin synthesis.
  • murine melanoma(B-16 F10) cells were inoculated on 6-well plates containing DMEM media supplemented with 10% FBS(fetal bovine serum) (1 X 10 6 cells per well),and cultured in a 5% CO 2 incubator at 37°C until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO 2 incubator at 37°C for 3 days. The treatment concentration of madecassoside was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity.
  • the cell pellets were dried at 60 °C, and then 100 ⁇ l of 1 M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in a 60 °C incubator, thereby obtaining intracellular melanin.
  • the resulting solution was measured for absorbance at 490 nm using a microplate reader, thus determining the amount of melanin per cell count. The results are shown in the following Table 1.
  • madecassoside exhibited higher inhibition rate of melanin synthesis than vitamin C at the same treatment concentration.
  • Example 2 Evaluation of inhibitory effect of madecassoside on melanin synthesis in human melanocyte
  • Human melanocytes were used to measure the inhibitory effect of madecassoside(Sigma) on melanin synthesis, which was compared with the inhibitory effect of vitamin C on melanin synthesis.
  • human epidermal melanocytes (derived from neonates) purchased from Cascade Biologics were cultured in Medium 254 containing a human melanocytes growth supplement purchased from Cascade Biologics.
  • human epidermal melanocytes were inoculated on 6-well plates at a density of 1 X 10 5 cells/well and cultured in a 5% CO 2 incubator at 37°C for 5 days until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO 2 incubator at 37°C for 5 days while replacing medium at two-day intervals.
  • the treatment concentration of madecassoside was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity.
  • the medium was removed, and the cells were washed with PBS (phosphated buffer saline) and treated with trypsin, followed by collecting the cells.
  • the collected cells were counted using a hematocytometer, and then the cells of the treatment groups were adjusted to the same number, and the cells of each group were placed in a 2 mL tube. Then, the cells were centrifuged at 5,000-10,000 rpm for 10 minutes, and the supernatant was removed to obtain pellets.
  • the cell pellets were dried at 60 °C, and then 100 ⁇ l of 1M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in a 60 °C incubator, thereby obtaining intracellular melanin.
  • the resulting solution was measured for absorbance at 490 nm using a microplate reader, thus determining the amount of melanin per cell count. The results are shown in the following Table 2.
  • madecassoside exhibited higher inhibition rate of melanin synthesis than vitamin C at the same treatment concentration.
  • Example 3 Measurement of effect on inhibition of UV-induced production of PGE2 in keratinocytes
  • human epidermal keratinocytes (derived from newborn infants) purchased from Cascade Biologics Inc. were cultured in EpiLife medium (Cascade Biologics Inc.) containing a human keratinocyte growth supplement.
  • keratinocytes were inoculated into a 6-well plate at a density of 1 X 10 5 cells/well, and then cultured under the conditions of 5 % CO 2 and 37 °C until about 80% or more of the cells adhered to the well bottom. After the culture, the medium was removed, and the cells were treated with medium containing a sample diluted to suitable concentration.
  • the cells were incubated under the conditions of 5% CO 2 and 37 °C for 24 hours, after which the medium was replaced with PBS (phosphate buffered saline), and then the cells were irradiated with artificial UV light (20 mJ of UVB). After completion of UV irradiation, the PBS was removed and replaced with each of keratinocyte culture media treated with madecassoside at various concentrations, followed by culture for 24 hours. Then, the cell culture medium was collected, and the amount of secreted PGE2 in the culture medium was measured using a commercially available ELISA kit (R&D). The results of the measurement are shown in Table 3 below.
  • Example 4 Measurement of effect on inhibition of UV-induced melanin production in keratinocyte-melanocyte coculture system
  • the effect of madecassoside on the inhibition of melanin production was measured. Specifically, melanocytes were inoculated into a 12-well plate, while keratinocytes were inoculated into Millicell. The adhesion of the keratinocytes inoculated into the Millicell was confirmed, and then the keratinocytes were treated with madecassoside at various concentrations. The next day, the culture medium was removed and replaced with PBS, and then the cells were irradiated with UVB.
  • the PBS was replaced with each of media treated with madecassoside at various concentrations, and the Millicell was placed on the 12-well plate inoculated with the melanocytes.
  • the culture media were transferred to each other through the filter membrane of the Millicell and were changed by substances contained therein.
  • the media were removed, and the cells were washed with PBS (phosphate buffered saline) and collected by treatment with trypsin.
  • the collected cells were counted with a hematocytometer, and then the number of the cells was adjusted to be the same between the treated groups.
  • the cells were dispensed into 2 mL tubes and centrifuged at 5,000-10,000 rpm for 10 minutes, and the supernatant was removed, thus obtaining cell pellets.
  • the cell pellets were dried at 60 °C, and 100 ⁇ l of a 1M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in an incubator at 60 °C, thereby obtaining intracellular melanin.
  • the resulting solution was measured for absorbance at 490 nm using a microplate reader, thereby determining the amount of melanin per cell number. The results of the measurement are shown in Table 4 below.
  • Example 5 Measurement of whitening effects in artificial skin
  • MEL-300-B (MaTek, USA) as an artificial skin was purchased and treated for 2 weeks with the artificial skin culture medium EPI-100-LLMM (MaTek, USA) containing each of the components shown in Table 5 below, while the medium as the sample was replaced at 3-day intervals.
  • the tissue was irradiated with 50 mJ of UVB.
  • the treated tissue was fixed in 10% formaldehyde, after which it was embedded in paraffin and sectioned.
  • the tissue sections were subjected to Fontana-Masson stain of melanin. Then, the amount of melanin per unit area was determined using Image-pro plus program (MediaCybernetics, U.S.A.). The results of the measurement are shown in Table 5 and FIG. 2.
  • irradiation with UV light can promote a skin darkening process
  • whether a test substance has a whitening effect can be determined by irradiating animals with UV light, administering the test substance orally or transdermally into the animals and measuring the effect of the test substance.
  • the whitening effect of madecassoside was measured using Tortoiseshell guinea pigs (brown guinea pigs) known to undergo pigmentation by UV light similarly to humans.
  • Tortoiseshell guinea pigs brown guinea pigs
  • UV/madecassoside group each consisting of 8 animals, and were bred during the test period.
  • the normal group and the UV control group were administered orally with 0.5 ml of physiological saline
  • the UV/madecassoside group was administered orally with a solution of 100 mg (per kg of bodyweight on a solid basis) in 0.5 ml of saline using a liquid syringe.
  • the administration was carried out at the same time five times a week for a total of 4 weeks.
  • the UV control group and the UV/madecassoside group were irradiated with UV light using a SE lamp (wavelength of 290-320 nm; Toshiba) (total dose of irradiation: 1350 mJ/cm 2 ).
  • a SE lamp wavelength of 290-320 nm; Toshiba
  • total dose of irradiation 1350 mJ/cm 2
  • the degree of skin pigmentation was determined using a chromameter (CR2002 chromameter, Minolta, Japan) to evaluate the effects. The results are shown in Table 6 below.
  • the L*A*B* colorimetric system was used to classify color, and an L* value was used as a standard in this Example.
  • the L* value was corrected using white board standard, and measured more than five times at one region, repeatedly. Pigmentation was evenly distributed.
  • Skin external preparations containing madecassoside and vitamin C were prepared using the components shown in Table 7 below.
  • a skin external preparation was prepared without a tyrosinase inhibitor.
  • the experimental groups 1 and 2 are skin external preparations containing madecassoside and vitamin C, respectively.
  • Example 7-1 Each skin external preparation prepared in Example 7-1 was applied every other day to the forearms of 30 healthy adults, and left for 24 hours, and this was repeated so that each subject was treated with 9 fresh patches in totla, so as to confirm whether skin irritation is caused by madecassoside.
  • the patch test was performed using a Finn chamber(Epitest Ltd, Finland). The external preparations were loaded dropwise in an amount of 15 ⁇ l per patch on the chamber. At every round of the patch application, the response of the skin was scored using the following Mathematical Equation. The results are shown in the following Table 8.
  • Carboxyvinyl polymer 0.1
  • Triethanolamine 0.1
  • Sorbitan sesquioleate 0.5
  • Liquid paraffin 5.0
  • Carboxyvinyl polymer 0.1
  • Sorbitan sesquioleate 0.5
  • Sorbitan sesquioleate 0.8
  • the above ingredients were mixed, and then tabletted according to a conventional preparation method to prepare a tablet formulation.
  • the above ingredients were mixed, and then sealed in a gelatin capsule according to a conventional preparation method to prepare a capsule formulation.

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Abstract

The present invention relates to a skin whitening composition comprising madecassoside as an active ingredient. The composition comprising madecassoside can be safely on the skin without causing side effects and has excellent effects of inhibiting the UV-induced production of skin darkening cytokine in keratinocytes and inhibiting the production of melanin in melanocytes to inhibit pigmentation. Thus, the composition comprising madecassoside as an active ingredient is very effective in inhibiting UV-induced pigmentation, inhibiting dark skin discoloration and whitening the skin.

Description

COMPOSITIONS FOR WHITENING SKIN COMPRISING MADECASSOSIDE
The present invention relates to a skin whitening composition comprising madecassoside, and more particularly to a skin whitening composition comprising madecassoside that has excellent stability without side effects on skin, and has a superior effect to inhibit pigmentation on skin by restraining melanin generation.
Human skin color is determined according to the concentration and distribution of melanin in the skin, and affected by environmental or physiological factors such as ultraviolet rays of the sun, fatigue, or stress as well as hereditary factors. The melanin pigment that is produced in the melanocytes of the human skin is a phenolic polymer consisting of a complex of a black pigment and a protein and plays an important role in preventing skin damage caused by UV light. The activity of tyrosinase present in melanocytes was reported to be the most important factor in melanin biosynthesis. Tyrosinase plays an important role in the skin darkening process by converting tyrosine into DOPA and DOPA-quinone, which are intermediate products of melanin polymer production.
However, the mechanism inducing melanin synthesis before tyrosinase works is not clarified yet in detail while the process through which melanin is made is disclosed(KoreanJ.MedicinalCropSci.18(2):79.85.2010).
Meanwhile, a variety of skin whitening substances developed to date exhibit whitening effects by inhibiting tyrosinase, which plays an important role in melanin synthesis, and inhibiting a series of melanin synthesis pathways. However, in recent years, studies on the development of skin whitening agents have been conducted for the purpose of exhibiting whitening effects not only by inhibiting melanin production in melanocytes, but also inhibiting the activity of keratinocytes, which are adjacent to melanocytes and secrete melanin-stimulating factors by UV rays (Exp Dermatol. 2008 Nov; 17 (11) : 946-52).
A variety of cytokines are involved in the interaction between keratinocytes and melanocytes. Particularly among them, PGE2 which is secreted from keratinocytes has been reported to play a major role in stimulating melanin pigmentation by increasing the production of cAMP in melanocytes. Substances capable of inhibiting such PGE2 can also act as whitening substances effective in preventing skin pigmentation caused by UV rays.
Overproduction of the melanin may induce discoloration, melasma, freckles or the like. Accordingly, the inhibition of melanin overproduction improves skin hyperpigmentation such as melasma and freckles due to UV, hormone, and hereditary factors, as well as skin brightening and whitening effects.
On the other hand, conventional materials having tyrosinase inhibiting activities such as hydroquinone, ascorbic acid, kojic acid, and glutathione have been used for improving skin whitening effect or hyperpigmentation. However, hydroquinone has problems in that only an extremely restricted amount should be used due to the severe skin irritation, even though it exhibits skin whitening effects. Ascorbic acid is easily oxidized, so that cosmetics blended with it have problems of color and odor changes, and kojic acid is unstable in a solution, which requires a complicated preparation prccess. In addition, thiol based compounds such as glutathione and cysteine have unique unpleasant odors as well as problems in transdermal absorption, and glycosides and derivatives thereof have problems in that they cannot be appropriately used as mixed ingredients of cosmetics due to their high polarities. Vitamin C is disadvantageous in that it is easily oxidized in an aqueous solution, so as not to continuously exhibit its effect. In recent years, skin-whitening compositions containing extracts of natural medicinal herbs have been developed. However, since most of them are colored, there are limitations in blending. Further, since their effective ingredients are not identified, consistent effects cannot be expected in products.
Under such circumstances, the present inventors have made extensive efforts to develop novel natural substances, which has excellent whitening effect and stability without side effect on skin, thereby completing the present invention.
It is an object of the present invention to provide a skin whitening composition comprising madecassoside that exhibits whitening effects not only by inhibiting the production of melanin in skin melanocytes, but also by inhibiting the skin darkening cytokine PGE2 that is secreted from skin keratinocytes.
To achieve the above objects, in accordance with one aspect of the present invention, there is provided a composition for whitening skin, comprising madecassoside as an active ingredient.
In accordance with another aspect of the present invention, there is provided a cosmetic composition, a pharmaceutical composition, or a food composition, comprising said composition for whitening skin.
In accordance with still another aspect of the present invention, there is provided a method of whitening the skin of a subject by administering said composition for whitening skin to the subject in a dermatologically acceptable way.
According to the present invention, madecassoside exhibits skin whitening effects not only by inhibiting skin darkening cytokine, which is secreted from keratinocytes, but also by inhibiting the production of melanin in melanocytes. In addition, it was confirmed that madecassoside caused neither toxicity nor irritation, suggesting that it is safe for human use. Thus, madecassoside of the present invention can be advantageously used in skin whitening cosmetic products, drugs or foods.
FIG. 1 shows a system for coculturing skin keratinocytes with melanocytes.
FIG. 2 shows a comparison of melanin density between a UV-irradiated artificial skin, treated with 0.5% madecassoside, and a UV-irradiated control skin.
Madecassoside which is used as an active ingredient in the composition of the present invention is a member of the triterpenoid family, which is found mainly in Centella asiatica, and is known to improve wound healing by reducing the production of keloid. It has a structure represented by the following formula 1.
[Chemical formula 1]
[Rectified under Rule 91 30.08.2012]
Figure WO-DOC-FIGURE-18
This madecassoside can be extracted from natural sources, including plants of Centellaasiatica(L.) and the like. Specifically, madecassoside can be obtained from the above natural sources us ing various extract ion methods known in the art. Preferably, it can be extracted using an extraction solvent selected from among (a) water, (b) an anhydrous or hydrated lower alcohol containing 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol with water, (d) acetone, (e) ethylacetate, (f) chloroform, (g) 1,3-butylene glycol, or a mixture of two or more thereof. More preferably, it can be extracted using an aqueous solution of methanol, ethanol and butanol, and most preferably, it can be extracted using an aqueous solution of butanol.
It will be obvious to a person skilled in the art that, even when extraction solvents other than the above-mentioned extraction solvents are used, extracts that exhibit substantially the same effects as the above extraction solvents can be obtained. In addition to the extraction solvent, specific extraction conditions (temperature, time, etc.) can be suitably selected by a person skilled in the art depending on the properties of the extraction solvent.
The extracts of the present invention include, in addition to the extracts obtained using the above-described extraction solvents, extracts obtained through conventional purification processes. For example, fractions obtained through various additional purification methods, such as separation with an ultra filtration membrane having a given molecular weight cut-off or separation by various chromatography systems (manufactured for separation according to size, charge, hydrophobicity or affinity), are also included in the scope of the extract of the present invention. In addition, madecassoside that is used in the present invention may be prepared by chemical synthesis or may be a commercially available product (for example, madecassoside available from Chromadex or Sigma).
The effects of madecassoside of the present invention on the inhibition of intracellular tyrosinase activity and melanin production, which are generally measured to screen whitening substances, are at least 50% higher than those of vitamin C which has been used in whitening cosmetic products. In addition, madecassoside of the present invention has the effect of inhibiting the production of PEG2, a skin darkening-related cytokine which is secreted from skin keratinocytes. Thus, madecassoside of the present invention acts on all skin melanocytes and keratinocytes to exhibit the effects of inhibiting intracellular melanogenesis and skin pigmentation.
It will be obvious to a person skilled in the art that examples of madecassoside that may be used in the present invention include, in addition to the madecassoside compound of formula 1, madecassoside derivatives, which show the effects of suppresing melanin synthesis and/or cytokines inducing skin darkening to exhibit of whitening skin, among derivatives obtained by adding substituents to madecassoside or substituting madecassoside according to a conventional method known in the art. More specifically, examples of madecassoside that may be used in the present invention include, in addition to the compound of formula 1, derivatives obtained by linking various substituents to the structure of formula 1 as a nucleus according to a method known in the art. For example, it is believed that derivatives obtained by linking a acetyl group, a hydroxyl group, a halo group, a nitro group or a C1-3 alkyl group to the compound of formula 1 are likely to exhibit effects identical or similar to the compound of formula 1. Thus, these derivatives also fall with in the scope of the present invention.
In the skin-whitening composition of the present invention, the content of madecassoside may be preferably 0.0001 to 15 wt%, based on the total weight of the composition. If the content is less than 0.0001 wt%, the whitening effect is not sufficient, and if the content is more than 15 wt%, the whitening effect is slightly increased, whereas there is a problem in the stability upon formulation. More preferably, madecassoside is contained in an amount of 0.0001 to 10 wt%, based on the total weight of the composition.
In a preferred embodiment of the present invention, the skin-whitening composition of the present invention may be used as a cosmetic composition for whitening skin.
The cosmetic composition of the present invention may contain, in addition to madecassoside as an effective ingredient, components that are conventionally used in cosmetic compositions, for examples, conventional auxiliaries, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. Further, the cosmetic composition may further contain a skin absorption enhancer to improve skin-whitening effect.
The cosmetic composition of the present invention may be prepared as any type of formulations as well known in the related art, including, if not specifically limited to, 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 powdered foundation, an emulsion foundation, a wax foundation, a spray, or the like. More specifically, the cosmetic composition may be formulated into a softening toner, a nutrient toner, nutrient cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, and a powder.
If the formulation of the present invention is paste, cream, or gel, it may contain, as carrier components, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, and so forth.
If the formulation of the present invention is a powder or spray formulation, it may contain, as carrier components, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder. In particular, if it is spray, it may additionally contain a propellant, such as chlorofluorohydrocarbon, propane/butane, or dimethyl ether.
If the formulation of the present invention is a solution or an emulsion, it may contain, as carrier components, a solvent, a solubilizing agent or an emulsifier. The examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol-aliphatic ester, polyethylene glycol, or sorbitan-aliphatic ester.
If the formulation of the present invention is a suspension, it may contain, as carrier components, a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, or the like.
If the formulation of the present invention is a surfactant-containing cleanser, it may contain, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ester sulfate, sulfosuccinic acid monoester, isethionate, imidazolium derivatives, methyl taurate, sarcosinate, aliphatic acid amide ether sulfate, alkyl amidobetaine, aliphatic alcohol, aliphatic glyceride, aliphatic acid diethanol amide, vegetable oil, lanolin derivatives, or ethoxylated glycerol aliphatic acid ester.
In the cosmetic composition of the present invention, the content of madecassoside may be 0.0001 to 15 wt%, and preferably 0.001 to 10 wt%, based on the total weight of the cosmetic composition.
In another preferred embodiment of the present invention, the skin-whitening composition of the present invention may be used as a pharmaceutical composition for whitening skin.
In the pharmaceutical composition of the present invention, the content of madecassoside may be 0.0001 to 15 wt%, and preferably 0.001 to 10 wt%, based on the total weight of the pharmaceutical composition.
The pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluent commonly used in the preparation of pharmaceutical compositions.
According to a method known to those skilled in the art, the pharmaceutical composition of the present invention may be formulated into any suitable formulation including oral formulation such as powder, granule, tablet, capsule, suspension, emulsion, syrup and aerosol; external preparation such as ointment and cream; suppository, and strerilized solution for injection.
The pharmaceutical composition of the present invention may be administered to mammals such as rat, mouse, livestock, and human via various routes such as oral and parenteral route (e.g., oral, rectal or intravenous, intramuscular, subcutaneous or intraepidural injection), preferably transcutaneous route among parenteral routes, and more preferably topical application.
The effective dosage of the pharmaceutical composition of the present invention may be determined depending on the patient s age, gender, body weight, symptom, and the route of administration. For topical administration, madecassoside may be preferably administered at a daily dosage of 1.0 to 3.0 ml for adult one to five times a day for at least one month.
Further, for oral administration, madecassoside may be preferably administered at a daily dosage of 0.1 to 100 mg/kg for adults one time or several times, depending on the patient's age, gender, and body weight. The dosage may be adjusted depending on the route of administration, severity of the disease, the patient's gender, body weight, and age. However, the scope of the present invention is not limited to the dosage.
In another preferred embodiment of the present invention, the skin-whitening composition of the present invention may be used as a food composition for whitening skin.
The food composition of the present invention may contain, in addition to madecassoside as an effective ingredient, conventional additives for preparing food compositions, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the above-described carbohydrates include conventional sugars, such as monosaccharides (e.g., glucose and fructose), disaccharides (e.g., maltose, sucrose and oligosaccharide), and polysaccharides (e.g., dextrin and cyclodextrin); and sugar alcohols (e.g., xylitol, sorbitol and erithritol). Examples of flavors include 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 the present invention is provided as a drink, it may include not only the madecassoside of the present invention but also other additives, such as, for example, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and so forth.
In the specified examples of the present invention, madecassoside proved to be a natural substance harmless to the human body in the cumulative skin irritation test for madecassoside. Thus, the madecassoside of the present invention, free from toxicity and adverse side effects, can be safely used for long-term use, particularly in the aforementioned applications, such as cosmetic, pharmaceutical or food compositions.
Meanwhile, the present invention also provides a method for whitening the skin of a subject by administering said composition to the subject in a dermatologically acceptable way.
As used herein, the term "subject" refers to a mammal including human, such as, for example, dogs, pigs, horses, cows, etc. that need to improve or prevent skin darkening or hyperpigmentation caused by ultraviolet rays of the sun, hormone, hereditary factors, or the like.
As used in the present invention, the term "administration or administering refer to introducing a defined substance into an object by an appropriate method. The composition of the present invention may be used by any known route of administration, oral or non-oral, for deliver to a target site of tissue. Further, the composition of the present invention may be administered by way of any device for delivering an active substance to a target site, such as cells.
Those skilled in the art will understand from the above description and the following examples that skin darkening or hyperpigmentation can be improved, ameliorated, prevented or treated by administering the composition of the present invention to a subject using the above-described methods
Hereinafter, the present invention will be described in further detail with the following examples. It is apparent to those skilled in the art that the examples are only for illustrative purposes and are not intended to limit the scope of the present invention.
Example 1: Evaluation of inhibitory effect of madecassoside on melanin synthesis in murine B16 melanoma cells
Murine B16 melanoma cells were used to measure the inhibitory effect of madecassoside(Sigma) on melanin synthesis, which was compared with the inhibitory effect of vitamin C on melanin synthesis.
Specifically, murine melanoma(B-16 F10) cells were inoculated on 6-well plates containing DMEM media supplemented with 10% FBS(fetal bovine serum) (1 X 106cells per well),and cultured in a 5% CO2 incubator at 37℃ until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO2 incubator at 37℃ for 3 days. The treatment concentration of madecassoside was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity. Media was removed from the cells, and the cells were washed with PBS(phosphated buffer saline) and treated with trypsin, followed by collecting the cells. After completion of the treatment, the medium was removed, and the cells were washed with PBS (phosphated buffer saline) and treated with trypsin, followed by collecting the cells. The collected cells were counted using a hematocytometer, and then the cells of the treatment groups were adjusted to the same number, and the cells of each group were placed in a 2 mL tube. Then, the cells were centrifuged at 5,000-10,000 rpm for 10 minutes, and the supernatant was removed to obtain pellets. The cell pellets were dried at 60 ℃, and then 100 ㎕ of 1 M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in a 60 ℃ incubator, thereby obtaining intracellular melanin. The resulting solution was measured for absorbance at 490 nm using a microplate reader, thus determining the amount of melanin per cell count. The results are shown in the following Table 1.
Table 1
Sample Treatment concentration (uM) Inhibition rate of melanin synthesis (%)
Vitamin C 1 0
Vitamin C 5 0
Vitamin C 10 2
Madecassoside 1 5
Madecassoside 5 55
Madecassoside 10 65
As shown Table 1, madecassoside exhibited higher inhibition rate of melanin synthesis than vitamin C at the same treatment concentration.
Example 2: Evaluation of inhibitory effect of madecassoside on melanin synthesis in human melanocyte
Human melanocytes were used to measure the inhibitory effect of madecassoside(Sigma) on melanin synthesis, which was compared with the inhibitory effect of vitamin C on melanin synthesis.
In the experiments, human epidermal melanocytes (derived from neonates) purchased from Cascade Biologics were cultured in Medium 254 containing a human melanocytes growth supplement purchased from Cascade Biologics. Specifically, human epidermal melanocytes were inoculated on 6-well plates at a density of 1 X 105cells/well and cultured in a 5% CO2 incubator at 37℃ for 5 days until reaching about 80% confluency. Then, media was removed from the cells, replaced with diluted fresh media, and cultured in a 5% CO2 incubator at 37℃ for 5 days while replacing medium at two-day intervals. The treatment concentration of madecassoside was determined over a range of 1 uM, 10 uM and 50 uM, which show no cytotoxicity. After completion of the treatment, the medium was removed, and the cells were washed with PBS (phosphated buffer saline) and treated with trypsin, followed by collecting the cells. The collected cells were counted using a hematocytometer, and then the cells of the treatment groups were adjusted to the same number, and the cells of each group were placed in a 2 mL tube. Then, the cells were centrifuged at 5,000-10,000 rpm for 10 minutes, and the supernatant was removed to obtain pellets. The cell pellets were dried at 60 ℃, and then 100 ㎕ of 1M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in a 60 ℃ incubator, thereby obtaining intracellular melanin. The resulting solution was measured for absorbance at 490 nm using a microplate reader, thus determining the amount of melanin per cell count. The results are shown in the following Table 2.
Table 2
Sample Treatment concentration (uM) Inhibition rate of melanin synthesis (%)
Vitamin C 1 0
Vitamin C 5 2
Vitamin C 10 6
Madecassoside 1 19
Madecassoside 5 28
Madecassoside 10 51
As shown Table 2, madecassoside exhibited higher inhibition rate of melanin synthesis than vitamin C at the same treatment concentration.
Example 3: Measurement of effect on inhibition of UV-induced production of PGE2 in keratinocytes
Using human keratinocytes, the effect of madecassoside on the inhibition of PGE2 that is a melanin-induced cytokine was measured.
Specifically, human epidermal keratinocytes (derived from newborn infants) purchased from Cascade Biologics Inc. were cultured in EpiLife medium (Cascade Biologics Inc.) containing a human keratinocyte growth supplement. Specifically, keratinocytes were inoculated into a 6-well plate at a density of 1 X 105 cells/well, and then cultured under the conditions of 5 % CO2 and 37 ℃ until about 80% or more of the cells adhered to the well bottom. After the culture, the medium was removed, and the cells were treated with medium containing a sample diluted to suitable concentration. Then, the cells were incubated under the conditions of 5% CO2 and 37 ℃ for 24 hours, after which the medium was replaced with PBS (phosphate buffered saline), and then the cells were irradiated with artificial UV light (20 mJ of UVB). After completion of UV irradiation, the PBS was removed and replaced with each of keratinocyte culture media treated with madecassoside at various concentrations, followed by culture for 24 hours. Then, the cell culture medium was collected, and the amount of secreted PGE2 in the culture medium was measured using a commercially available ELISA kit (R&D). The results of the measurement are shown in Table 3 below.
Table 3
Sample Treatment concentration (uM) PGE2 (ng/mL)
Non-UV irradiated group Control - 19
UV-irradiated groups Control - 54
Madecassoside 10 55
Madecassoside 50 45
Madecassoside 100 39
As can be seen in Table 3 above, madecassoside concentration-dependently reduced PGE2 production induced by UV irradiation.
Example 4: Measurement of effect on inhibition of UV-induced melanin production in keratinocyte-melanocyte coculture system
Using the keratinocyte-melanocyte coculture system shown in FIG. 1, the effect of madecassoside on the inhibition of melanin production was measured. Specifically, melanocytes were inoculated into a 12-well plate, while keratinocytes were inoculated into Millicell. The adhesion of the keratinocytes inoculated into the Millicell was confirmed, and then the keratinocytes were treated with madecassoside at various concentrations. The next day, the culture medium was removed and replaced with PBS, and then the cells were irradiated with UVB. Then, the PBS was replaced with each of media treated with madecassoside at various concentrations, and the Millicell was placed on the 12-well plate inoculated with the melanocytes. In this coculture system, it could be observed that the culture media were transferred to each other through the filter membrane of the Millicell and were changed by substances contained therein.
After the treatment, the media were removed, and the cells were washed with PBS (phosphate buffered saline) and collected by treatment with trypsin. The collected cells were counted with a hematocytometer, and then the number of the cells was adjusted to be the same between the treated groups. Then, the cells were dispensed into 2 mL tubes and centrifuged at 5,000-10,000 rpm for 10 minutes, and the supernatant was removed, thus obtaining cell pellets. The cell pellets were dried at 60 ℃, and 100 ㎕ of a 1M sodium hydroxide solution containing 10% DMSO was added to the cell pellets in an incubator at 60 ℃, thereby obtaining intracellular melanin. The resulting solution was measured for absorbance at 490 nm using a microplate reader, thereby determining the amount of melanin per cell number. The results of the measurement are shown in Table 4 below.
Table 4
Sample Treatment concentration (uM) Inhibition rate of melanin synthesis (%)
Madecassoside 10 19
Madecassoside 50 28
Madecassoside 100 51
As can be seen in Table 4 above, madecassoside inhibited melanin production in a concentration-dependent manner.
Example 5: Measurement of whitening effects in artificial skin
MEL-300-B (MaTek, USA) as an artificial skin was purchased and treated for 2 weeks with the artificial skin culture medium EPI-100-LLMM (MaTek, USA) containing each of the components shown in Table 5 below, while the medium as the sample was replaced at 3-day intervals. Before the sample was replaced, the tissue was irradiated with 50 mJ of UVB. The treated tissue was fixed in 10% formaldehyde, after which it was embedded in paraffin and sectioned. The tissue sections were subjected to Fontana-Masson stain of melanin. Then, the amount of melanin per unit area was determined using Image-pro plus program (MediaCybernetics, U.S.A.). The results of the measurement are shown in Table 5 and FIG. 2.
Table 5
Test item control Kojic acid (0.5%) madecassoside (0.1%) madecassoside (0.5%)
melanin density 4.9 3.8 4.3 3.7
As can be seen in Table 5 above, the groups treated with madecassoside showed a decrease in melanin density in a manner dependent on the concentration of madecassoside.
Example 6: Whitening effect of oral administration
Because irradiation with UV light can promote a skin darkening process, whether a test substance has a whitening effect can be determined by irradiating animals with UV light, administering the test substance orally or transdermally into the animals and measuring the effect of the test substance.
Thus, in this Example, the whitening effect of madecassoside was measured using Tortoiseshell guinea pigs (brown guinea pigs) known to undergo pigmentation by UV light similarly to humans. Specifically, 6-7 week-old guinea pigs were divided into a normal group, a UV control group and an UV/madecassoside group, each consisting of 8 animals, and were bred during the test period. The normal group and the UV control group were administered orally with 0.5 ml of physiological saline, and the UV/madecassoside group was administered orally with a solution of 100 mg (per kg of bodyweight on a solid basis) in 0.5 ml of saline using a liquid syringe.
The administration was carried out at the same time five times a week for a total of 4 weeks. 2 weeks after the oral administration, the UV control group and the UV/madecassoside group were irradiated with UV light using a SE lamp (wavelength of 290-320 nm; Toshiba) (total dose of irradiation: 1350 mJ/cm2). 2 or 3 days after UV irradiation, pigmentation appeared, and after about 2 weeks, a change in the skin color was measured.
The degree of skin pigmentation was determined using a chromameter (CR2002 chromameter, Minolta, Japan) to evaluate the effects. The results are shown in Table 6 below. The L*A*B* colorimetric system was used to classify color, and an L* value was used as a standard in this Example. The L* value was corrected using white board standard, and measured more than five times at one region, repeatedly. Pigmentation was evenly distributed.
Table 6
Sample Whitening effect (L*)
UV Control group 0.52±0.09
UV/Madecassoside 0.85±0.08
As can be seen in Table 6 above, the L* value was greater in the UV/madecassoside group than in the UV control group, suggesting that the skin color was brighter in the UV/madecassoside group.
Example 7: Safety test of madecassoside on human skin
7-1. Skin external preparations containing madecassoside
In order to confirm the safety of madecassoside on human skin, skin external preparations containing madecassoside and vitamin C were prepared, and safety test of the external preparations was performed.
Skin external preparations containing madecassoside and vitamin C were prepared using the components shown in Table 7 below. As a control group, a skin external preparation was prepared without a tyrosinase inhibitor. The experimental groups 1 and 2 are skin external preparations containing madecassoside and vitamin C, respectively.
To prepare the skin external preparations, purified water, glycerin, and butylene glycol were mixed and solved at 70℃ (aqueous phase). The remaining components except for the above three components and trimethanolamine were dissolved at 70℃ (oil phase). The oil phase was added to the aqueous phase, and stirred with a Homomixer (Tokushu Kika company, Japan) to primarily emulsify. Finally, trimethanolamine was added thereto. Foam produced in the mixed solution was removed, and then cooled to room temperature to prepare skin external preparations.
Table 7
Component Content (wt%)
Control group Experimental group 1 Experimental group 2
Purified water 72 72 72
Glycerin 8.0 8.0 8.0
Butylene glycol 4.0 4.0 4.0
Madecassoside 0 1.0 0
Vitamin C 0 0 1.0
Caprylic / capric triglyceride 8.0 8.0 8.0
Squalane 5.0 5.0 5.0
Cetearyl glucoside 1.5 1.5 1.5
Sorbitan stearate 0.4 0.4 0.4
Cetearyl alcohol 1.0 1.0 1.0
Trimethanolamine 0.1 0.1 0.1
Total weight 100 100 100
7-2. Cumulative irritation test
Each skin external preparation prepared in Example 7-1 was applied every other day to the forearms of 30 healthy adults, and left for 24 hours, and this was repeated so that each subject was treated with 9 fresh patches in totla, so as to confirm whether skin irritation is caused by madecassoside.
The patch test was performed using a Finn chamber(Epitest Ltd, Finland). The external preparations were loaded dropwise in an amount of 15 ㎕ per patch on the chamber. At every round of the patch application, the response of the skin was scored using the following Mathematical Equation. The results are shown in the following Table 8.
[Mathematical Equation]
Average response degree =
[[Response index X Response degree / Total number of subjects X Highest score (4 points)] X 100] ÷ Times of examination (9 rounds)
In regard to the response degree, 1 point was provided for ±, 2 points for +, 4 points for ++. When the average response degree was less than 3, the composition was determined to be safe for the skin.
[Rectified under Rule 91 30.08.2012]
Table 8
Figure WO-DOC-TABLE-8
As shown in Table 8 above, in Experimental group 1, the subjects corresponding to ±, +, ++ numbered 1, 0 and 0, respectively, while the others showed no response. Further, the average response degree was calculated to be 0.13 ([(1x1)/(20x4)]x 100/9=0.13), which is less than 3, demonstrating that the composition of the present invention is safe for human skin. Accordingly, the external preparation containing madecassoside (Experimental group 1) causes no noticeable cumulative irritation, like the control group and the external preparation containing vitamin C. In such results, madecassoside according to the present invention was found to be safe for human skin.
Hereinafter, formulation examples for the composition of the present invention will be described.
Formulation Example 1: Cosmetic formulations
1. Softening toner : (Content : wt%)
Madecassoside: 0.01
Glycerin: 3.0
Butylene glycol: 2.0
Propylene glycol: 2.0
Carboxyvinyl polymer: 0.1
Ethanol: 10.0
Triethanolamine: 0.1
Preservative, trace pigment, trace fragrance and trace purified water: balance
Total sum: 100.0
2. Nutrient toner (content : wt%)
Madecassoside: 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, trace pigment, trace fragrance and trace purified water: balance
Total sum: 100.0
3. Nutrient cream (content: wt%)
Madecassoside: 0.005
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, trace pigment, trace fragrance and trace purified water: balance
Total sum: 100
4. Massage cream (content: wt%)
Madecassoside: 0.005
Beeswax: 10.0
Polysorbate 60: 1.5
Sorbitan sesquioleate: 0.8
Liquid paraffin: 40.0
Squalane: 5.0
Caprylic/capric triglyceride: 5.0
Glycerin: 3.0
Butylene glycol 3.0
Propylene glycol: 3.0
Triethanolamine: 0.2
Preservative, trace pigment, trace fragrance and trace purified water: balance
Total sum: 100.0
5. Pack (content: wt%)
Madecassoside: 0.005
Polyvinylalcohol: 13.0
Sodiumcarboxymethylcellulose: 0.2
Allantoin: 0.1
Ethanol: 5.0
Nonylphenyl ether: 0.3
Preservative, trace pigment, trace fragrance and trace purified water: balance
Total sum: 100.0
Formulation Example 2: Pharmaceutical preparation
1. Preparation of powder formulation
Madecassoside: 2g
Lactose : 1g
The above ingredients were mixed, and charged in an air-tight package to prepare a powder formulation.
2. Preparation of tablet formulation
Madecassoside: 100㎎
Corn starch: 100㎎
Lactose: 100㎎
Magnesium stearate: 2㎎
The above ingredients were mixed, and then tabletted according to a conventional preparation method to prepare a tablet formulation.
3. Preparation of capsule formulation
Madecassoside: 100㎎
Corn starch: 100㎎
Lactose: 100㎎
Magnesium stearate: 2㎎
The above ingredients were mixed, and then sealed in a gelatin capsule according to a conventional preparation method to prepare a capsule formulation.

Claims (5)

  1. A composition for whitening skin, comprising madecassoside as an active ingredient.
  2. The composition for whitening skin according to claim 1, wherein the madecassoside that exhibits whitening effects not only by inhibiting the production of melanin in skin melanocytes, but also by inhibiting the skin darkening cytokine PGE2 that is secreted from skin keratinocytes.
  3. The composition for whitening skin according to claims 1 or 2, wherein the content of madecassoside is 0.0001 to 15.0 wt%, based on the total weight of the composition.
  4. The composition for whitening skin according to claim 1, wherein the composition is used to cosmetic composition, pharmaceutical composition, or food composition for the purpose of skin-whitening.
  5. The composition for whitening skin according to claim 4, wherein the cosmetic 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.
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