KR20190028618A - Pharmarceutical composition for prevention and treatment of canities comprising extracts or fractions of Longanae Arillus as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating canities containing a Longanae Arillus extract or fraction thereof as an active component. The Longanae Arillus extract, extracted by at least one solvent selected from a group consisting of C_2-3 alcohol and a mixed solution of C_2-3 alcohol and water, or a fraction thereof increases the survival rate of melanoblast cells, the cell differentiation/melanin content of melanoblast, the cell migration rate, and tyrosinase activity, and also significantly increases the content of melanin in hair, thereby being usefully used for preventing or treating canities.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for treating or preventing white rot,

The present invention relates to a process for the production of longan- Arillus extract or a fraction thereof as an active ingredient.

Whitehead disease is whitening and whitening of hair caused by melanin reduction caused by melanocytes in hair moths such as hair, eyebrows and eyelashes. Is known to be caused by the decrease in the number of melanocytes forming melanin pigment in hair follicles and the loss of pigment due to failure to transfer melanin to surrounding keratinocytes.

The upper part of the hair follicle extends from the epidermis to the deep layer of the dermis in a tubular form embedded in the epidermis. The underside or hair bulb of the hair follicle includes an indentation located in the papilla. The area around the dermal papilla on the lower part of the follicle is the area where matrix cells with a high degree of proliferation are distributed. These cells are precursors of horny cells that make up the hair. The cells that result from the proliferation of these precursors migrate vertically in the parent hair, gradually keratinize the upper part of the parent hair, and the keratinized cells gather to form hair stalk.

In addition, the color of head hair and bodily hair is based, in part, on the amount and ratio of two melanin groups; Uelanins (brown and black pigments) and pheomelanins (red and yellow pigments). Pigmentation of hair and hair must have a melanocyte in the hair follicle. The melanin produced at melanocytes is transferred to keratinocytes to form a hairy or hairy strand of melanin. This structure is known as "follicular pigmentation unit ".

Furthermore, melanogenesis in mammals involves tyrosinase, DOPAchrome tautomerase (TRP-2) and DHICA oxidase (TRP-1), which are at least three enzymes, The activity of the three enzymes is known to be essential for the maximum activation of melanin biosynthesis.

First, the tyrosinase is referred to as an enzyme that initiates the biosynthesis of melanin or restricts the formation of melanin. Tyrosinase also catalyzes oxidation of the waveguide in tyrosine and then oxidation to dopaquinone. The dopaquinone compound is naturally transformed into a dopachrome or cysteinyldopa derivative in the presence of cysteine.

The TRP-2 catalyzes the tautomerization of dipachrome with 5,6-dihydroxyindole-2-carboxylic acid (DHICA). In the presence of TRP-2, dipachroma naturally decarboxylates to form 5,6-dihydroxyindole (DHI).

In addition, TRP-1 oxidizes the DHICA compound to form a quinone derivative.

It is influenced by both melanocyte and melanocyte precursor cells of hair follicles and is associated with a specific and gradual depletion of hair melanocytes (Commo et al., Br. J. Dermatol., 2004, 150, 435-443 ). Other types of cells present in hair follicles are unaffected. In addition, the depletion of these melanocytes is not observed in the epidermis. The cause of this gradual and specific depletion of melanocytes and melanocyte precursors in hair follicles has not yet been determined.

In addition, the hair and hairs have a cycle. These cycles consist of the anagenic phase, the catagenic phase, and the telogenic phase, and then develop into a new growth phase. As a result of this hair cycle, the hair follicle pigmentation unit must also be periodically resumed. During the transition from man to the growth phase, enzymes necessary for melanin synthesis, such as tyrosinase and TRP-1, except for TRP-2, located around the maternal papilla in the generative stage, part of the inactive melanocyte proliferates (Pigment Cell Res. 2004 Oct; 17: 488-497). In parallel, the remaining quiescent melanocytes remain in the inactive state in the upper layer of the hair follicles. Tyrosinase and TRP-1 enzymes are expressed in the parental melanocytes during the growth phase but are no longer expressed in the melanocytes during the retrogressive and resting periods. Thus, the normal cycle of melanocytes in human hair follicles requires the presence of a melanocyte precursor, which is present on top of the hair follicle, which can be activated periodically to regenerate hair folliculate pigmentation units.

Patent Document 1 discloses a composition containing, as an active ingredient, ellagic acid or derivatives thereof for treating white rot.

The inventors of the present invention have conducted studies to develop an effective therapeutic agent for the treatment of white mucus, and found that the extract or fraction thereof of yongtan meat has a high survival rate of melanoblast, cell differentiation / melanin content of melanoblast, melanoblast cell migration rate, tyrosinase activity But also the melanin content in the hair is remarkably increased, so that it can be usefully used for prevention or treatment of white mastication. Thus, the present invention has been completed.

European Published Patent EP 1870081

It is an object of the present invention to provide a longan The present invention also provides a pharmaceutical composition for preventing or treating canities containing an extract of Arillus or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for preventing or ameliorating white rot, comprising a canola extract or a fraction thereof as an active ingredient.

It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating wheat germ which contains a canola extract or a fraction thereof as an active ingredient.

In order to achieve the above object,

The present invention relates to a process for the production of C _2-3 alcohols; And a mixed solution of C _2-3 alcohol and water; and a pharmaceutical composition for preventing or treating white moxibustion containing the extract of Longanae Arillus or its fraction as an active ingredient. to provide.

The present invention also relates to a process for the production of a _{compound of} formula And a mixture solution of C _2-3 alcohol and water. The present invention provides a cosmetic composition for preventing or ameliorating white moxibustion which contains, as an active ingredient, an extract of Yugang canola extract or a fraction thereof extracted with at least one solvent selected from the group consisting of

Further, the present invention relates to a process for _{producing a} polyhydroxyalkanoate, And a mixed solution of C _2-3 alcohol and water. The present invention also provides a health functional food composition for preventing or ameliorating white mastication comprising the extract of Yongtan Extract or its fraction as an active ingredient.

The C _2-3 alcohols of the present invention; And a mixed solution of C _2-3 alcohol and water. The extract of the Yongtan extract or its fractions extracted from at least one solvent selected from the group consisting of the survival rate of melanoblast cells, the cell differentiation / melanin content of melanoblasts, the cell migration rate, Not only increases the activity of anazase but also significantly increases the content of melanin in hair, and thus can be usefully used for preventing or treating white rot.

FIG. 1 is a graph showing the cell survival rate according to the kind of solvent (negative control: DMSO-treated negative control and α-MSH: positive control (α-Melanocyte-stimulating hormone) .
2 is a graph showing the content of melanin in the extract of yin yang, according to the kind of solvent.
FIG. 3 is a graph showing the cell migration rate of the eye lysolecule extract according to the type of solvent.
Fig. 4 is a graph showing tyrosinase activity of a lucerolytic extract according to a solvent type.
FIG. 5 is a graph showing the cell survival rate of the extract of Yongtan extract according to the concentration of ethanol.
6 is a graph showing the content of melanin in the extract of Yongtan extract according to the concentration of ethanol.
FIG. 7 is a graph showing the cell migration rate of the yin yang extract according to the concentration of ethanol.
FIG. 8 is a photomicrograph of the melanoblast cell migration according to the concentration of ethanol in the extract of the yin yang.
9 is a graph showing the tyrosinase activity of the extract of Yongtan extract according to the concentration of ethanol.
FIG. 10 is a graph showing tyrosinase activity and melanin content in mouse hair caused by a kind of eyedrop extract according to the type or concentration of a solvent.
FIG. 11 is a representative photograph of a phytotricogram of human white rice with ethanol extract of canola oil.
FIG. 12 is a graph showing the effect of ethanol extract of canine leukocyte on human white rot reduction for 24 weeks.
13 is a graph showing the effect on the cell migration rate of the luciferin fraction.
FIG. 14 is a graph showing the effect of melanin content and tyrosinase content in C3H / Hej mouse hair on the yellowness fraction.
15 is a schematic diagram showing the mechanism of white mold.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the production of C _2-3 alcohols; And a mixed solution of C _2-3 alcohol and water; and a pharmaceutical composition for preventing or treating white moxibustion containing the extract of Longanae Arillus or its fraction as an active ingredient do.

Hereinafter, the pharmaceutical composition for the prevention or treatment of white moth disease according to the present invention will be described in detail.

Lonaganae Arillus is an evergreen tree of the Sapindaceae and refers to the flesh of the fruit in the fruit. In Yongan pharmacopoeia, Yongan is an irregular flaky rupture, which is ruptured longitudinally with the husks of Yongan. It is usually sticky with several sticks, 2 ~ 4cm in length, 1 ~ 2cm in width, 2 ~ 4mm in thickness, ~ Translucent in dark brown color, one side is wrinkled and uneven, the other side is shiny, there is vertical wrinkles, the vagina is soft and tacky, there is a slight peculiar smell, and the taste is moon.

The yongcheng, which is the target of extraction of the yongtan extract, which is the active ingredient of the present invention, may be cultivated or marketed as defined in Korean Pharmacopoeia without limitation.

The extract of yun yuan, which is an active ingredient of the present invention, can be obtained through a conventional extraction method, but a solvent is important for extracting an effective ingredient for achieving the purpose of treatment or prevention of white rot of the present invention. ₂ or C ₃ alcohol; And a mixed solution of C ₂ or C ₃ alcohol and water. Also, the solvent may be ethanol; Isopropanol; A mixed solution of ethanol and water; And a mixed solution of isopropanol and water, more preferably at least one selected from the group consisting of ethanol, ethanol and water, and isopropanol. At this time, the mixed solution concentration of the C _2-3 alcohol and water is preferably 20 vol% or more, preferably 30 vol% or more and 40 vol% or more, more preferably 50 vol% or more, based on C _2-3 alcohol having a purity of 96% More preferable. In this case, the concentration of the mixed solution is 100 vol.%, Which means that C _2-3 alcohol alone is used instead of the mixed solution with water. Also, the inclusion of other substances that affect the purity of the C _2-3 alcohol produced or marketed is within the scope of not affecting the desired extract components of the present invention.

Specifically, the extract of Yongtan, which is an active ingredient of the present invention, has a melanin content of about 1.5 times higher than that of the positive control group of α-MSH when the solvent is ethanol or isopropanol, and the number of melanoblasts And the tyrosinase activity was significantly increased compared with the positive control. In addition, in the case of the same type of daily dose, the cell migration rate and tyrosyanase activity But the melanin content was remarkably increased (see Experimental Example below).

For example, the solvent may be added in an amount of 5 to 15 times or more of the volume of the dried eyeball, and the solvent may be added to the eyeball, followed by shaking extraction, Soxhlet extraction, ultrasonic extraction, and reflux cooling extraction. The temperature at the time of extraction is preferably 5 to 95 ° C, more preferably 15 to 80 ° C, but is not limited thereto. Further, the extraction time is preferably 10 hours to 15 days, more preferably 20 hours to 5 days, and the extraction time is preferably 1 to 5 times, more preferably 3 times or more. In addition, after filtration of the yinmei extract, the filtrate is concentrated under reduced pressure and dried to produce yinmei extract. At this time, the drying may be performed by vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying.

The number of melanocytes in the hairy spindle (melanocyte) is significantly decreased, and the activity of making melanocytes is decreased, so that the melanin of the mosquitoes and follicles is lost, It turns white and becomes white hair (gray hair). The location of the onset is mainly hair, eyebrows, eyelashes, beard, etc. The cause of the disease may be a natural aging process as the body ages, but genetic factors, high fever, mental shock, drugs, diabetes, Thyroid disease may be the cause. Since the development of white moth by aging progresses gradually, it may appear as various intermediate colors between black and white, and white mood in the present invention includes such phenomenon, discoloration of hair, shedding and the like.

A C _2-3 alcohol according to the present invention; And a mixed solution of C _2-3 alcohol and water; the extract of Yongtan extract extracted with at least one solvent increases the survival rate of the melanoblast cell line melb-a cell and the cell differentiation / melanin content of melanoblast , The tyrosinase activity was improved, the melanoblast cell migration rate was excellent, and the incidence of white mold was significantly slower than that of the placebo group (see Experimental Examples below).

Therefore, the canola extract of the present invention can be used for the treatment or prevention of white mastitis.

In addition, the composition of the present invention may include a preparation of a drag-eye extract in the form of a pharmaceutically acceptable salt. Such pharmaceutically acceptable salts are useful as addition salts formed by free acids. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid. Examples of the organic acids include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, There may be mentioned fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid or aspartic acid Etc. may be used. Furthermore, it may include both hydrates and solvates as well as pharmaceutically acceptable salts.

In addition, the composition of the present invention may optionally contain one or more of the fractions of the eye lotion extract, and may further contain one or more active ingredients showing the same or similar functions.

Specifically, the fractions of the canine extract include C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water; and the organic solvent is an organic solvent selected from the group consisting of hexane, chloroform or ethyl acetate . In the present invention, the ethanol extract of canola oil is suspended in water, and then the mixture is divided into hexane, chloroform or ethyl acetate, and concentrated under reduced pressure to prepare a fraction. The fraction obtained by fractionating hexane, chloroform and ethylacetate Or more fractions.

The fractions of the canola extract according to the present invention showed an increase in the melanoblast cell migration effect and the increase in the melanin content and tyrosinase content in the C3H / Hej mouse hair in the order of the chloroform fraction, the hexane fraction and the ethyl acetate fraction in the order of the negative control group (See Experimental Example 5 below).

Therefore, the fractions of the canola extract according to the present invention can be useful for the treatment or prevention of white rot.

The composition of the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration in actual clinical administration, and when formulated, it may further include pharmaceutically acceptable additives. The pharmaceutically acceptable additives may be diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are usually used. Examples of the additives include starch, gelatinized starch, microcrystalline cellulose, lactose, Corn starch, corn starch, powdered cellulose, hydroxypropylcellulose, opaques, starch glycolate sodium, carnauba wax, synthetic aluminum silicate, calcium phosphate, calcium phosphate, calcium hydrogen phosphate, lactose, mannitol, , Stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

The pharmaceutical compositions of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powders, ointments, lotions, lozenges, or drops) to humans and other animals, Oral, oral or nasal spray, and the like. In certain embodiments, a compound of the invention is administered in an amount of from about 0.01 mg / kg to about 50 mg / kg, such as from about 1 mg / kg to about 25 mg / kg body weight of a subject, / Day < / RTI > at a dosage level of < RTI ID = 0.0 >

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents of the following examples commonly used in the art: water or other solvents, solubilizing agents, and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate , Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (for example cottonseed, peanut, corn, germ, olive, castor, and pine oil), glycerol, Fatty acid esters of furfuryl alcohol, polyethylene glycol and sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous or lipid productive suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions or emulsions in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. And isotonic sodium chloride solution. In addition, sterilized, fixed oils are conventionally used as a solvent or dispersion medium. For this purpose, any blend stationary oil may be utilized, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by incorporation of the active agent in the form of sterile solid compositions that are filtered or dissolved prior to use in a sterile water or other sterile injectable medium prior to use, have.

The topical or transdermal dosage forms of the pharmaceutical compositions of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed with the pharmaceutically acceptable carrier and any necessary preservatives or buffers under sterile conditions, as required. Ophthalmic formulations, ear drops, and eye drops are also contemplated to be within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches with the added benefit of providing controlled cleavage of the compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound across the skin. The speed can be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of white fever.

The present invention also relates to a process for the production of a _{compound of} formula And a mixed solution of C _2-3 alcohol and water; and a cosmetic composition for preventing or ameliorating hypercholesterolemia comprising the extract of Yongtan Extract or its fraction as an active ingredient.

Hereinafter, the cosmetic composition for prevention or improvement of white moth disease according to the present invention will be described in detail.

The cosmetic composition can be produced in the form of a cosmetic product of a general emulsified formulation and a solubilized formulation. Cosmetics of the emulsified form include nutrient lotion, cream, essence and the like, and the cosmetics of the solubilized formulation include the soft lotion. In addition to cosmetics containing the extract of the present invention, it can also be prepared in the form of an adjuvant which can be applied topically or systemically, which is conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.

Suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing the oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) And may be provided in the form of a fecal dispersant, a soap, a cream, a skin, a lotion, a powder, an ointment, a spray or a conceal stick.

It can also be prepared in the form of a foam or an aerosol (spray) composition further containing a compressed propellant. The composition of the present invention can further comprise at least one of a fatty substance, an organic solvent, a solubilizing agent, a thickening agent and a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a perfume, a surfactant, Any other ingredients commonly used in cosmetics, such as nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as < RTI ID = 0.0 > cosmetics < / RTI > or dermatology. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

In the cosmetic composition containing the eye drop of the present invention, the extract of the present invention may be added to the cosmetic composition usually contained in an amount of 1 to 15% by weight, preferably 2 to 10% by weight.

Further, the present invention relates to a process for _{producing a} polyhydroxyalkanoate, And a mixture solution of C _2-3 alcohol and water. The present invention provides a health functional food composition for preventing or ameliorating white moth disease, which comprises, as an active ingredient, a yellowness extract or a fraction thereof extracted with at least one solvent selected from the group consisting of

Hereinafter, the health functional food composition for preventing or ameliorating white moth disease according to the present invention will be described in detail.

The health functional food composition according to the present invention comprises C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water; or a fraction thereof may be directly added to the food or may be used together with other food or food ingredients. At this time, the content of the active ingredient to be added may be determined depending on the purpose. Generally, the content in the health functional food may be 0.01 to 90 parts by weight of the total food weight.

There is no particular limitation on the form and the kind of the health functional food composition. The form of the health functional food to which the substance is added may be a tablet, a capsule, a powder, a granule, a liquid, a ring and the like. The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient such as a normal health functional food. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, and the like. These components may be used independently or in combination. The proportion of such additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to the following examples.

However, the following examples illustrate the present invention in detail, and the present invention is not limited by the following examples.

< Example  1> Yonggak ( Longanae Arillus ) Preparation of extract

Example  1-1: Isopropanol extract

Flesh of Rice ( Lonaganae Arillus ) was dried at 60 ° C for 6 hours, and then extracted with 500 g of canola oil and 1000 ml of isopropanol (purity: 99%, manufacturer: MERCK) as an extractant in an extraction vessel and then filtered with filter paper to obtain an extract. The solvent was then concentrated under reduced pressure and dried to obtain 100 g of an extract.

Example  1-2: Ethanol extract

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that ethanol (purity: 99%, manufacturer: MERCK) was used instead of isopropanol as a solvent.

Example  One- 3: 95 volume%  Ethanol extract of concentration

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that ethanol (a mixed solution of ethanol and distilled water used in Example 2) of 95% by volume concentration was used instead of isopropanol as a solvent Respectively.

Example  One- 4: 70 volume%  Ethanol extract of concentration

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that ethanol (a mixed solution of ethanol and distilled water used in Example 2) at a concentration of 70% by volume was used instead of isopropanol as a solvent Respectively.

Example  One- 5: 30 volume%  Ethanol extract of concentration

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that ethanol (a mixed solution of ethanol and distilled water used in Example 2) was used at a concentration of 30 vol% instead of isopropanol as a solvent Respectively.

< Comparative Example  1> Yonggak ( Longanae Arillus ) Preparation of extract

Comparative Example 1-1: Methanol extract

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that methanol (purity: 99%, manufacturer: MERCK) was used instead of isopropanol as a solvent.

Comparative Example 1-2: Water extract

In Example 1, 100 g of an extract was obtained in the same manner as in Example 1, except that distilled water was used instead of isopropanol as a solvent.

< Example  2> Yonggak  For ethanol extracts Fraction  Produce

300 ml of water was added to 100 g of the ethanol extract of canola oil prepared in Example 1-2 and suspended in the same amount of hexane to separate the water layer and the hexane layer. In the same manner, chloroform and ethyl acetate were sequentially fractionated and concentrated under reduced pressure to obtain hexane soluble extract, chloroform soluble extract and ethyl acetate soluble extract, respectively.

< Experimental Example  1> Depending on the type of solvent Yonggak  Evaluation of efficacy of extract

In order to evaluate the efficacy of Yonggak Extract according to the type of solvent, the effect on the viability, melanin content and cell migration of melanoblast cells was examined as follows.

Melano blast ( melanoblast ) Cell culture

Melb-a (melanoblast) was purchased from Functional Genomics Cell Bank (London, UK) and Welcome Trust. Melb-a was cultured in a 10% CO ₂ incubator at 37 ° C and cultured in RPMI 1640 medium supplemented with 1% penicillin (Invitrogen Corp. (CA, USA)) / streptomycin (Invitrogen Corp. (CA, USA) (Sigma Chemical Co., St. Louis, USA), 1 ng / ml bFGF (Fibroblast growth factor-basic; PeproTech), 5% fetal bovine serum (Fetal bovine serum; Invitrogen Corp., CA, USA), and 2 nM L-glutamine.

The cells are treated with trypsin / ethylenediaminetetraacetic acid (trypsin-EDTA; Invitrogen Corp., CA, USA), precipitated and suspended in complete RPMI 1640 medium (Invitrogen Corp. (CA, USA)). The number of living cells was measured using a trypan blue exclusion method and a hemocytometer. In a 10 cm cell culture dish, complete RPMI 1640 medium was added and inoculated at a concentration of 2 × 10 ⁵ cells / dish.

One. Melano blast  Confirm cell viability

The following experiments were carried out to confirm the survival rate of melb-a cells by the extracts of canine yogurts prepared in Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2.

Specifically, 200 μl of Melb-a cells (4 × 10 ³ cells / well) was added to each well of a 96-well plate, followed by culture at 37 ° C. in a 10% CO ₂ incubator for 24 hours. -1, 1-2, and Comparative Examples 1-1 and 1-2 were added to the culture medium containing 50 ppm of the extract of the Yongtan extract prepared in Comparative Examples 1-1 and 1-2, and then the culture was changed every two days for 4 days (total sample was treated twice) . DMSO, a negative control, and α-MSH (α-Melanocyte-stimulating hormone), a positive control, were treated with 0.8 μM. After 96 hours, 5 mg / ml MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide) was added to the cultured cells; Sigma Chemical Co. (St Louis, USA)) was dissolved in DMSO, and the resultant was measured with an ELISA microplate reader at 540 nm in an ELISA microplate reader. The results are shown in FIG. 1 and Table 1 below.

FIG. 1 is a graph showing the cell survival rate of the extract of Yongtan extract according to the kind of solvent.

Cell survival rate Negative control group 100.00 α-MSH 120.00 Comparative Example 1-2 115.00 Comparative Example 1-1 110.00 Examples 1-2 120.00 Example 1-1 115.00

As shown in Table 1 and FIG. 1, it can be confirmed that the extract of Yongtan extract according to the type of solvent has similar or increased cell survival rate as compared with the negative control, so that it is not toxic to cells.

2. Melano blast  Cell differentiation / melanin assay (melanin assay)

Melanoblast is a non-pigmented cell in which tyrosinase function is stopped. Melanoblast differentiation can be measured by using melanin content. The following experiments were carried out to confirm the content of melanin by the extracts of canine yogurt prepared in Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2.

Specifically, after 96 hours of incubation of the cells cultured in the same manner as that of the melanoblast cell line, the culture medium was removed, and the cells were washed twice with PBS (phosphate buffered saline; Invitrogen Corp., CA, USA) -EDTA was added to collect the cells. The collected cells were resuspended in 1.5 ml Eppendorf tubes and centrifuged to obtain cell pellets. 100 μl of a 1 N NaOH solution containing 10% DMSO was added to each of the cells thus collected, suspended, and reacted at 80 ° C for 1 hour. The melanin in the reacted and dissolved cells was measured using a plate reader at a wavelength of 405 nm in comparison with the control group, and the results are shown in FIG. 2 and Table 2.

FIG. 2 is a graph showing intracellular melanin content of a canola extract according to the type of a solvent.

Melanin content Negative control group 100.00 α-MSH 160.00 Comparative Example 1-2 180.00 Comparative Example 1-1 150.00 Examples 1-2 220.00 Example 1-1 190.00

As shown in FIG. 2 and Table 2, the content of melanin by the extract of Comparative Example 1-1, in which the solvent is methanol, was lower than that of the positive control group of? -MSH, and the content of melanin by the extract of Comparative Example 1-2, -MSH, while the content of melanin by the extract of Example 1-2, in which the solvent was ethanol, increased about 1.5-fold over that of -MSH, and the content of melanin by the extract of Example 1-1 in which the solvent was isopropanol was lower than that of the solvent Which is higher than that of Comparative Example 1-2, which is water.

Therefore, it was confirmed that the solvent had an excellent melanin content in the order of methanol, water, isopropanol and ethanol.

3. Melano blast  Confirmation of cell migration effect

Transwell migration assay was performed to examine the effects of melanoblast migration by the extracts of canine yogurts prepared in Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2.

Specifically, cell migration was measured using a Transwell cell culture chamber. The Transwell cell culture chamber (Costar 3422) had a 0.8 μm polyvinylpyrrolidone-free polycarbonate filter and was coated with 1% gelatin in the filter. After coating the coated cell insert, 600 μl of serum-free RPMI medium was added to the bottom of the chamber, 100 μl of melb-a cells (2 × 10 ⁶ cells / ml) was inoculated on the chamber and incubated for 24 hours. The concentration of the test sample of the present invention was 50 ppm, and the positive control group, α-MSH, was treated with 0.8 μM. After 24 hours, the filter is cut out and fixed with methanol. After staining with hematoxylin and eosin, wipe with a cotton swab to remove unmoved cells on the filter. To view the transferred cells, the lower part of the filter was observed under a microscope. To measure the number of cells, the filter was divided into four equal parts and magnified 40 times by a microscope. The experiment was repeated three times under the respective conditions, and the results are shown in FIG. 3 and Table 3.

FIG. 3 is a graph showing the cell migration rate of the eye lysolecule extract according to the type of solvent.

Cell migration rate Negative control group 100.00 α-MSH 130.00 Comparative Example 1-2 150.00 Comparative Example 1-1 130.00 Examples 1-2 220.00 Example 1-1 210.00

As shown in Fig. 3 and Table 3, the number of melanoblasts moved on the lower surface of the filter treated with the Yukunobuchi extract prepared in each of Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2, The cell migration rate of the extract of Comparative Example 1-1 in which the solvent was methanol and the extract of Comparative Example 1-2 in which the solvent was water was increased to less than about 1.5 times as compared with that of the negative control. However, in Example 1 where the solvent was isopropanol -1 extract and the extract of Example 1-2 in which the solvent was ethanol were increased about twice as compared to the negative control.

Therefore, it was confirmed that the solvent had an excellent melanoblast cell migration rate in the order of methanol, water, isopropanol and ethanol.

4. Identification of tyrosinase activity effect

In order to examine the effect of the extracts of canine lucerne prepared in Examples 1-1 and 1-2 and Comparative Examples 1-1 and 1-2 on induction of tyrosinase, intracellular tyrosinase assay was performed as follows .

The intracellular tyrosinase activity was analyzed by measuring the DOPA oxidizing activity. Melb-a cells were inoculated in 60 dishes at a concentration of 2 x 10 ⁴ cells / dish. The cells were incubated 24 hours before the addition of the same amount of culture medium containing the test sample to the adherent cells and then exchanged every two days for 4 days (treatment of the total sample twice). After 96 hours, the cells were washed with cold PBS, the cells were separated from the dish using a scraper, transferred into a 1.5 ml Eppendorf tube, and the cells were precipitated by centrifugation, and the supernatant was removed. 100 μl of a lysis buffer (0.1 M phosphate buffer, pH 6.8, 1% (w / v) Triton X-100) was added to the precipitated cells and reacted for 30 minutes at 4 ° C on a sonicator. The cells were centrifuged at 13000 rpm for 10 minutes to remove cell debris and the amount of protein in the sample was measured using a Bio-Rad protein assay kit. The extracted protein was added to a 96-well plate, and 2.5 mM L-DOPA (3,4-dihydroxy-Lphenyl-alanine; Sigma Chemical Co., St. Louis, USA) dissolved in PBS was added thereto. After the reaction, the resultant was measured with an ELISA reader at 475 nm and compared with the control group. The results are shown in FIG. 4 and Table 4.

Fig. 4 is a graph showing tyrosinase activity by the extract of yin yang depending on the type of solvent.

Tyrosinase Negative control group 100.00 ± 18.00 α-MSH 140.00 ± 17.00 Comparative Example 1-2 160.00 ± 21.00 Comparative Example 1-1 130.00 ± 13.00 Examples 1-2 180.00 ± 29.00 Example 1-1 170.00 ± 22.00

As shown in Fig. 4 and Table 4, the tiresanase activity of the extract of Comparative Example 1-1, in which the solvent was methanol, was lower than that of the positive control group? -MSH, and the extract of Comparative Example 1-2 in which the solvent was water and the isopropanol The tyrosinase activity of the extract of Example 1-2, in which the extract and the solvent were ethanol, was increased compared to the positive control, and the increase in the tyrosinase activity of the ethanol extract was found to be the greatest.

Therefore, it was confirmed that the solvent had excellent tyrosinase activity by the extract in the order of methanol, water, isopropanol and ethanol.

< Experimental Example  2> Depending on the concentration of ethanol Yonggak  Evaluation of efficacy of extract

In order to evaluate the efficacy of the extract of Yongtan extract according to the ethanol concentration, the effect on the viability, melanin content, cell migration rate and tyrosinase activity of melanoblast cells was examined as follows.

One. Melano blast  Confirm cell viability

In order to confirm the survival rate of melb-a cells by the extracts of canine yogurts prepared in Examples 1-2, 1-3, 1-4, and 1-5, a method similar to that of Experimental Example 1 , And the results are shown in FIG. 5 and Table 5.

FIG. 5 is a graph showing the cell survival rate according to the ethanol concentration.

Cell survival rate Negative control group  100.00 5.00 α-MSH 120.00 ± 3.00 Examples 1-5 110.00 6.20 Examples 1-4 115.00 ± 6.50 Example 1-3 120.00 ± 7.00 Examples 1-2 120.00 6.80

As shown in FIG. 5 and Table 5, it can be confirmed that the extract of Yongtan extract according to the concentration of ethanol has a higher cell survival rate than that of the negative control and is not toxic to the cells.

2. Melano blast  Cell differentiation / melanin assay (melanin assay)

Experiments were carried out in the same manner as in Experimental Example 1 to confirm the content of melanin by the extracts of canine yogurts prepared in Examples 1-2, 1-3, 1-4 and 1-5. The results are shown in Fig.

6 is a graph showing the content of melanin by the extract of the yin yang depending on the concentration of ethanol.

Melanin content Negative control group  100.00 ± 13.00 α-MSH 160.00 ± 12.00 Examples 1-5 170.00 ± 16.00 Examples 1-4 195.00 ± 20.0 Example 1-3 220.00 ± 22.00 Examples 1-2 230.00 ± 23.00

As shown in FIG. 6 and Table 6, the melanin content of the 30 vol.% Ethanol lanceolate extract prepared in Example 1-5 was similar to the melanin content by the positive control group, .alpha.-MSH, It was found that the melanin content by the 70 vol.% Ethanol lanolin extract was further increased. The 95 vol.% Ethanol lanolin extract prepared in Example 1-3 and the 100 vol.% Ethanol lanolin extract prepared in Example 1-2 The melanin content was significantly increased more than two times in comparison with the negative control group.

Therefore, it can be confirmed that the content of melanin is significantly increased as the concentration of ethanol as a solvent increases.

3. Melano blast  Confirmation of cell migration effect

In order to examine the effect of the extract of the yangyang extract prepared in Examples 1-2, 1-3, 1-4, and 1-5 on the movement of melanoblast, a method similar to that of Experimental Example 1, 3 . The results are shown in Figs. 7-8 and Table 7.

FIG. 7 is a graph showing the cell migration rate of the yin yang extract according to the concentration of ethanol.

FIG. 8 is a photomicrograph of the melanoblast cell migration according to the concentration of ethanol in the extract of the yin yang.

Cell migration rate Negative control group  100.00 α-MSH 140.00 Examples 1-5 220.00 Examples 1-4 230.00 Example 1-3 240.00 Examples 1-2 245.00

As shown in Figs. 7-8 and Table 7, the extracts of the yogurt extracts prepared in Examples 1-2, 1-3, 1-4, and 1-5, The cell migration rate according to the number of melanoblasts is about twice that of the negative control, and the cell migration rate is similar even when the concentration of ethanol is increased.

4. Identification of tyrosinase activity effect

In order to examine the effect of the extracts of canine leukocyte prepared in Examples 1-2, 1-3, 1-4 and 1-5 on tyrosinase induction, Experiments were carried out, and the results are shown in FIG. 9 and Table 8.

9 is a graph showing the tyrosinase activity of the extract of Yongtan extract according to the concentration of ethanol.

Tyrosinase Negative control group  100.00 ± 18.00 α-MSH 140.00 ± 17.00 Examples 1-5 150.00 ± 21.00 Examples 1-4 160.00 ± 23.00 Example 1-3 180.00 ± 29.00 Examples 1-2 190.00 ± 28.00

As shown in FIGS. 9 and 8, it can be seen that the activity of tyrosinase increases as the concentration of ethanol increases.

< Experimental Example 3>  The extract C3H / Hej  Melanin in mouse hair Generation  And confirming the enhancement of tyrosinase activity

The test agent (6% of canine lanceolate extract, 30% of ethanol, 2% of polyoxyxylated hydrogenated castor oil, and 2% of diol) were used as the extracts of canine lye prepared in Examples 1-1 to 1-5 and Comparative Examples 1-1 and 1-2 0.03% of sodium dithiophosphate, 0.03% of grapefruit seed extract, 0.5% of citric acid, and purified water) was prepared and used as a sample to test the effect of melanin production and tyrosinase activity in mouse hair.

Specifically, hair of the back part of C3H / Hej mice at 7 weeks of age was removed, and the skin of the back part was cleanly selected. Five mice were selected for each substance group, and 100 μl of the sample material was applied daily for 4 weeks Α-MSH (α-Melanocyte-stimulating hormone) was used as a positive control at a concentration of 1 mg / kg. After the end of the test, the animals were sacrificed and the newly-emerged hairs were collected. The amount of melanin in the hair and the absorbance of the hair were measured, and some of them were stored in a 1 mM L-Tyrosin solution and the tyrosinase activity was measured. Respectively.

FIG. 10 is a graph showing tyrosinase activity and melanin content in mouse hair caused by a kind of eyedrop extract according to the type or concentration of a solvent.

As shown in FIG. 10, the activity of tyrosinase in the hair by the extract of Yongguk Yoon was rather lower than that of the negative control. The extracts of methanol, 30 vol.% Ethanol, 70 vol.% Ethanol, 100 vol. Ethanol and isopropanol extracts were increased compared to the negative control, but the increase was similar.

The melanin content in the hair by the extract of yangyang extract was 115% in the extract of Comparative Example 1-2 prepared by using water as a solvent and 111% in the extract of Comparative Example 1-1 prepared by using methanol as a solvent compared with the negative control While that of the extract of Example 1-2 prepared with ethanol as a solvent was remarkably increased to 160% and that of Example 1-1 prepared with isopropanol as a solvent was 153%.

Therefore, the effect of the extract of Yongtan extract on the hair is similar to that of tyrosinase activity in hair, but the effect on the melanin content of hair varies depending on the kind of solvent, and water, methanol, ethanol and isopropanol, And when the solvent was ethanol, the melanin content in the hair was significantly increased as the concentration was increased.

< Experimental Example  4> Yonggak  Effect of Ethanol Extract on the Reduction of Human White Rice

The ethanol extract of yuyuan yukang prepared in Example 1-2 was prepared as a test preparation and the effect of reducing white hair in human hair was tested using the extract.

Specifically, 10 healthy subjects with white moths were treated daily with 1 ml of sample every other day for 6 months. A negative control group was used to compare the results with placebo preparations containing no yongguk extract Respectively. After completion of the test, the degree of occurrence of white moth was evaluated by phototricogram as compared to before execution, and the results are shown in Table 9 and FIG.

Placebo agent Yongcheng extract group On start 100% 100% 4 weeks 112% 109% 12 weeks 121% 112% 24 weeks 136% 118%

FIG. 11 is a representative photograph of a phytotricogram of human white rice with ethanol extract of canola oil.

FIG. 12 is a graph showing the effect of ethanol extract of canine leukocyte on human white rot reduction for 24 weeks.

As shown in Table 9 and FIG. 12, when the degree of white mold generation in human hair was compared with that before execution, the results of white mold generation in the hair coated with the test preparation containing the canola oil ethanol extract prepared in Example 1-2 And the frequency was decreased.

<Experimental Example 5> Confirmation of effect on cell migration rate, fraction of melanin and tyrosinase content in C3H / Hej mouse hair,

In order to examine the effect of melanoblast cell migration of water, hexane, chloroform and ethyl acetate fractions on the ethanol extract of canola oil prepared in Example 1-2, The results are shown in Figs. 13-14 and Table 10-11.

13 is a graph showing the effect on the cell migration rate of the luciferase fraction.

FIG. 14 is a graph showing the effect of melanin content and tyrosinase content in C3H / Hej mouse hair on the yellowness fraction.

Cell migration rate Negative control group 100 ± 14 α-MSH 124 ± 14 Ethanol extract 150 ± 12 Hexane fraction 165 ± 22 Chloroform fraction 171 ± 21 Ethyl acetate fraction 157 ± 18 Water fraction 108 ± 16

Melanin content Tyrosinase Negative control group 100 ± 15 100 ± 14 α-MSH 134 ± 12 117 ± 14 Ethanol extract 136 ± 17 121 ± 12 Hexane fraction 181 ± 28 154 ± 22 Chloroform fraction 202 ± 15 186 ± 21 Ethyl acetate fraction 166 ± 11 142 ± 18 Water fraction 105 ± 15 106 ± 16

As a result, as shown in FIGS. 13-14 and 10-11, the hexane fraction of the ethanol extract of canola oil increased melanoblast migration by 165%, 171% of the chloroform fraction, and 157% of the ethyl acetate fraction, Respectively. The melanoblast treated with α-MSH, a positive control, was increased by 124%. Melanin content and tyrosinase content in C3H / Hej mouse hair were also increased by 181% and 154%, respectively. Melanin content and tyrosinase content of chloroform fraction increased by 202% and 186%, respectively. , Ethyl acetate fraction showed a melanin content of 166% and tyrosinase content of 142%. (Figs. 13 and 14).

Claims (9)

C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water; and at least one of the following ingredients : Longanae ( Longanae) extracted with at least one solvent selected from the group consisting of Arillus ) extract as an active ingredient.
C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water; and a cosmetic composition for preventing or ameliorating white mastication containing the extract of Yongtan Extract extracted with at least one solvent selected from the group consisting of:
C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water. The present invention relates to a health functional food composition for preventing or ameliorating wheat wilt comprising an extract of Yongtan extract extracted from at least one solvent selected from the group consisting of:
4. The method according to any one of claims 1 to 3,
Wherein the C ₃ alcohol is isopropanol.
4. The method according to any one of claims 1 to 3,
_Wherein the mixed solution comprises at least 20% by volume of C _2-3 alcohol.
C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water. The pharmaceutical composition according to claim 1, wherein the organic solvent fraction is at least one selected from the group consisting of:
C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water. The cosmetic composition for preventing or ameliorating white moth disease according to any one of claims 1 to 4, wherein the organic solvent fraction of the extract is at least one selected from the group consisting of:
C _2-3 alcohol; And a mixed solution of C _2-3 alcohol and water. The composition according to claim 1, wherein the organic solvent fraction of the extract is at least one selected from the group consisting of:
9. The method according to any one of claims 6 to 8,
Wherein the organic solvent fraction is at least one selected from the group consisting of hexane fraction, chloroform fraction and ethyl acetate obtained by sequentially fractionating with hexane, chloroform or ethyl acetate.

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