WO2008094000A1

WO2008094000A1 - Herbal composition for preventing and improving baldness disorder

Info

Publication number: WO2008094000A1
Application number: PCT/KR2008/000576
Authority: WO
Inventors: Hee Kyoung Kang; Jung Il Kang; Sang Cheol Kim; Jae Hee Hyun; Elvira Kim; Eun Sook Yoo; Jin Won Hyun; Moon Jae Cho; Deok Bae Park; Ki Ho Kim; Young Heui Kim
Original assignee: Bioland Ltd; Industry Academic Cooperation Foundation of Jeju National University
Current assignee: Industry Academic Cooperation Foundation of Jeju National University; Hyundai Bioland Co Ltd
Priority date: 2007-01-30
Filing date: 2008-01-30
Publication date: 2008-08-07
Anticipated expiration: 2009-07-30
Also published as: KR100841923B1

Abstract

The present invention relates to a composition comprising an extract of Schisandra nigra Max. showing preventing activity of baldness disorder and stimulating activity of hair growth being confirmed by several experiments such as the growth rate test using by vibrissa follicle, PCNA and TGF-β2 expression test in rat hair follicle. Accordingly, the inventive extract can be used as a pharmaceutical composition and health functional food for the treatment of baldness disorder and stimulation of hair growth.

Description

HERBAL COMPOSITION FOR PREVENTING AND IMPROVING BALDNESS DISORDER

Technical Field

[1] The present invention relates to a composition comprising an extract of Schisandra nigra Max. showing preventing activity of baldness disorder and stimulating activity of hair growth.

[2]

Background Art

[3] Hair follicle, a very complex organ, consists of an inner root sheath (IRS), outer root sheath (ORS), hair shaft, hair matrix cell (Paus et al., J. Invest. Dermatol., 113. pp523-532, 1999), and dermal papilla cell, which plays an important role in hair growth among various kinds of the cells forming the hair follicle (Ferraris et al., Exp. Cell Res., K), pp37-4ζ 1997).

[4] Hair follicle develops through the interaction between the mesenchymal cell and epithelial cell during an embryogenesis. Normal hair growth consists of repeated three phases, i.e., anagen, catagen and telogen stages (Paus et al., N. Engl. J. Med., 341. pp491-497, 1999). Especially, human hair has the longer growth period than any other animal hair or the other part hairs of the human body (anagen, 2-5 years; catagen, several days-several weeks; telogen, three months). During the anagen stage, the cell consisting of hair follicle including the keratinocytic cell of matrix surrounding the dermal papilla, proliferates and grows hair. During the catagen stage, within the hair follicle, apoptosis, condensation of the dermal papilla, and the reconstitution of extracellular matrix serially occur (Paus et al., J. Invest. Dermatol., 113. pp523-532, 1999) to stop the cell division and slow the hair growth. The hair follicle at telogen stage falls out except the permanently lasting parts comprising the bulge region of hair follicle at the state of quiescence of growth to restart new anagen (Hardy MH., Trends Genet., &, pp55-61, 1992).

[5] Currently, there have been reported that various kinds of cytokines and growth factors are involved in the development and formation of hair follicle (Stenn et al., Physiol. Rev., 81, pp449-494, 2001); for example, tumor necrosis factor-α (TNF-α), interleukin-lα (IL- lα), interleukin-lβ (IL- lβ), interferon-γ (IFN-γ), fibroblast growth factor-5 (FGF5), hepatocyte growth factor (HGF), vascular epithelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II) and epithelial growth factor receptor (EGFR). Among them, TNF- α increases cell death in the matrix cell and inhibits the elongation of hair shaft (Soma et. al., J. Invest. Dermatol. , Ui, pp948-954, 1998). IL-lα and IL- lβ also inhibits the growth of the hair follicle (Mahe et al., Skin Pharmacol., 9£6), pp366-375, 1996; Xiong et al., J. Interferon Cytokine Res. , 12, ppl51-157, 1997). The over-expression of IFN-γ induces baldness (Carroll et al., J. Invest. Dermatol., 108(4). pp412-422, 1997), FGF5 induces the telogen in hair follicle of rats (Hebert et al., Cell, 78, ppl017-1025, 1994} while HGF (Jindo et al., J. Invest. Dermatol. , JjO, pp338-342, 1998) and VEGF (Yano et al., J. Clin. Invest., 107, pp409-417, 2001) promotes the growth of hair follicle. Also, IGF-I and IGF-II promotes the growth of hair follicle in a dose-dependent manner when incubating the hair follicle lacking insulin (Philpott et al., J. Invest. Dermatol., 102. pp857-861, 1994). It has been reported that necrosis oxurs and hair follicle disappears in EGF-receptor removed transgenic mouse (Murillas et al., EMBO J., 14(2D. pp5216-5223, 1995).

[6] Several drugs promoting hair growth approved by the Food and Drug Administration

(FDA) have been developed, for example, 'Minoxidil' (Buhl et al., J. Invest. Dermatol., 92(3). pp315-320, 1989) and 'Finasteride' (Van Neste et al., Br. J. Dermatol., 143(4). pp804-810, 2000).

[7] TGF- β has been known as a cytokine to regulate various procedures, such as growth and differentiation (Massague et al., Cancer Surv., \2, pp81-103, 1992). TGF-β is classified into 3 types in mammals, i.e., TGF-βl, TGF-β2 and TGF-β3 (Roberts et al., Heidelberg, Springer- Verlag., pp419-472, 1990), and recently the location of TGF-β expression of the 3 types in anagen and catagen hair follicle has been reported. TGF- βl has been found at the hair cuticle and the connective tissue sheath of human anagen hair follicle, TGF- β2, at the outer cellular layer of the outer root sheath of late anagen and TGF-β3, at the keratinization-matrix cells (Soma et al., J. Invest. Dermatol., 118. pp993-997, 2002). Recently, among the isotypes, TGF-β2 has been studied actively. The incubation of hair follicle with TGF-β2 is reported to activate caspase-3 and caspase-9, which are known to induce apoptosis from outer root sheath cell and lower germinative matrix cell (Tsuji et al., JID Symposium. Proceedings, &, pp65-68, 2003). Furthermore, it is reported to inhibit thymidine incorporation, resulting in the inhibition of the growth of hair length in a dose dependent manner to TGF-β2 concentration (Soma et al., J. Invest. Dermatol., I l l, pp948-954, 1998). On the other hand, fetuin, a TGF-β antagonist, elongates the growth of hair follicle. Therefore, TGF- β2 is seemed to have a key role in inducing the catagen of hair follicle and in- hibiting the hair follicle growth (Soma et al., J. Invest Dermatol., 118. pp993-997, 2002).

[8] As an alternative approach, there have been attempts to develop new therapy using herb extracts having hair growth stimulating effect: for example, plant extract belonging to Orchidaceae disclosed in Korea Patent Registration No, 015667; vitex seed and bear fat in Korea Patent Registration No. 0161338; an extract of mixed crude drug consisting of Pinellia ternate , Eugenia caryophyllata, Rubus coreanus, Zanthoxylum piperatum, Vitex rotundifolia , Salvia miltiorrhiza , and Thujae semen in Korea Patent Registration No. 259037. However, there have been still needs to develop new drugs or substances showing more potent activity without adverse action till now.

[9] Therefore, the inventors of the present invention evaluated the treating effect of the extract of Schisandra nigra Max. on baldness disorder by observing the preventing activity of baldness disorder and stimulating activity of hair growth.

[10] Schisandra nigra Max. is belonged to Schisandraceae consisting of 2 families and 22 species, for example, Schisandra nigra Max., Schisandra chinensis, Kadsura japonica, Schisandra nigra var., Schisandra viridicarpa and etc It is scarcely distributed at the height of 600-1,400 m of Mt. Hanra located in Korea and the extract thereof has been known to contain schizandronic acid, schizandrolic acid, schizandronol, oplodiol, (+)-catechin-7-β-D-glucopyranoside, androsin, β-sitosteryl glucoside, schizandriside etc (Takani et al., Chem. Pharm. Bull., 27, pp 1422- 1425.1979).

[11] However, there has been not reported or disclosed about the hair growing activity of the extract of Schisandra nigra Max in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[12] Therefore, the inventors of the present invention carried out various experiments to confirm the effect of the extract of Schisandra nigra Max. on hair growth, for example, the growth rate test using by vibrissa follicle, PCNA expression test and TGF-β2 in rat hair follicle, and finally completed the present invention by confirming the potent activity of hair growth.

[13]

Disclosure of Invention Technical Problem

[14] Accordingly, it is an object of the present invention to provide a pharmaceutical composition and health functional food comprising the extract of Schisandra nigra Max., as an active ingredient in an effective amount to prevent baldness disorder and to stimulate hair growth. [15]

Technical Solution

[16] It is an object of the present invention to provide a pharmaceutical composition comprising the extract of Schisandra nigra Max., as an active ingredient in an effective amount to prevent baldness disorder and to stimulate hair growth.

[17] It is an object of the present invention to provide a health functional food comprising the extract of Schisandra nigra Max., as an active ingredient in an effective amount to prevent baldness disorder and stimulate hair growth.

[18] The term 'extract' disclosed herein includes solvent selected from the group consisting of water, C i - C₄ lower alcohol and the mixture thereof, preferably, the mixture solvent with water and ethanol, more preferably 70 to 90% ethanol.

[19] The term 'extract of Schisandra nigra Max.' disclosed herein comprises the flesh, peel, seed or root of Schisandra nigra Max.

[20] The term 'baldness disorder' disclosed herein comprises androgentic alopecia, alopecia seniles, alopecia areata and the like.

[21] The present invention also provided a use of the extract of Schisandra nigraMax. for the preparation of therapeutic agent for the treatment and prevention of baldness disorderin mammal or human.

[22] The present invention also provided a pharmaceutical composition comprising the extract of Schisandra nigra Max., and a pharmaceutically acceptable carrier thereof as an active ingredient for treating and preventing baldness disorder.

[23] It is an object of the present invention to provide a method of treating or preventing baldness disorder in mammal or human in need thereof comprising administering to said mammal or human with an effective amount of the extract of Schisandra nigra Max., together with a pharmaceutically acceptable carrier thereof.

[24]

[25] Hereinafter, the present invention is described in detail.

[26]

[27] An inventive extract of the present invention can be prepared in detail by following procedures,

[28]

[29] The inventive extract of Schisandra nigra Max. can be prepared by the followings; one-day-dried Schisandra nigra Max. were cut and approximately 1 to 10-fold volume, preferably 1 to 3-fold volume of water, lower alcohols such as methanol, ethanol and the like or the mixtures thereof, preferably ethanol, more preferably 70 to 90% ethanol were added and extracted at the temperature range from 2O⁰C to 100⁰C, preferable 5O⁰C to 100⁰C for approximately 1 to 5 days, preferably 1 to 3 days; the upper layer is extracted by hot water, cold water, reflux extraction or ultra-sonication extraction; afterwards filtered, concentrated and dried to obtain the powdered extract of Schisandra nigra Max. of the present invention.

[30] The present invention also provided a use of the extract of Schisandra nigra Max., prepared from the above-described methodfor the preparation of therapeutic agent for the treatment and prevention of baldness disorderin mammal or human.

[31] The present invention also provided a pharmaceutical composition comprising the extract of Schisandra nigra Max, prepared from the above-described method and a pharmaceutically acceptable carrier thereof as an active ingredient for treating and preventing baldness disorder.

[32] It is an object of the present invention to provide a method of treating or preventing baldness disorder in mammal or human in need thereof comprising administering to said mammal or human with an effective amount of the extract of Schisandra nigra Max., prepared from the above-described method together with a pharmaceutically acceptable carrier thereof.

[33] The inventive composition for preventing baldness disorder and stimulating hair growth may comprise the above-described extract as 0.1-50% by weight based on the total weight of the composition.

[34] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co., Easton PA).

[35] Hereinafter, the following formulation methods and exripients are merely exemplary and in no way limit the invention.

[36] The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

[37] For example, the compositions of the present invention can be dissolved in oils, propylene glyx>l or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glyx>l, ethanol, vegetable oils, isopropyl myristate, etc, but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

[38] Pharmaceutical compositions containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion). In particular, the composition of the present invention may be applied to topical regions such as skin or hair as a form of topical preparation, for example ointment, lotion, liniment, pasta, cataplasma and etc which are not intended to limit thereto.

[39] The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

[40] The desirable dose of the inventive extract of the present composition varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.0001 to 100 mg/kg, preferably 0.001 to 10 mg/kg by weight/ day of the inventive extract or compounds of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract or compound should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

[41] The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection.

[42]

[43] The inventive extract of the present invention also can be used as a main component or additive and aiding agent in the preparation of various functional health functional food and health care food.

[44] The health functional food of the present comprises the above-described extract as

0.01 to 80%, preferable 1 to 50% by weight based on the total weight of the composition.

[45] The health functional food of the present invention can be contained in health functional food or beverage, used in the form of powder, granule, tablet, chewing tablet, capsule or liquid.

[46] Providing that the health beverage composition of present invention contains the above-described extract as the essential component in indicated ratio, there is no particular limitation on other liquid component, wherein the other component can be various deodorants, natural carbohydrates and etc Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose; disaccharide such as maltose, sucrose; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, erythritol. Examples of aforementioned deodorants are natural deodorant such as taumatin; stevia extract such as levaudioside A, gly_yrrhizin; and synthetic deodorant such as saccharin, aspartame. The amount of the above-described natural carbohydrate generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ml of beverage composition.

[47] The other components than aforementioned are various nutrients, vitamin, mineral or electrolyte, synthetic flavoring agent, coloring agent and improving agent in the case of cheese, chocolate and etc such as pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, preservative, gljcerin, alcohol, carbonizing agent used in carbonate beverage. Also components may be used independently of in combination in fruit juice for preparing natural fruit juice or vegetable beverage. The ratio of the components are not so important but generally range from approximately 0 to 20 w/w% per 100 w/w% of present composition.

[48] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural antioxidants such as polyphenol, catechin, α-tocopherol, rosemary extract, vitamin C licorice root extract, chitosan, tannic acid, pytic acid etc

Advantageous Effects

[49] The present invention relates to a pharmaceutical composition comprising the extract of Schisandra nigra Max. showing preventing activity of baldness disorder and stimulating activity of hair growth.

[50] The present invention also relates to a health functional food comprising the extract of Schisandra nigra Max. showing preventing activity of baldness disorder and stimulating activity of hair growth.

[51]

Brief Description of the Drawings

[52] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[53]

[54] Fig. 1 shows the effect of inventive extract on PCNA expression of the hair follicle;

[55] Fig. 2 shows the effect of inventive extract on TGF-β2 expression of the hair follicle.

[56]

Best Mode for Carrying Out the Invention

[57] The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

[58]

[59] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[60]

Mode for the Invention

[61] Example 1. Prepaprtion of the extract of Schisandra nigra Max.

[62] 1 Kg of Schisandra nigra Max. (Jeju-do) was washed with tap water, dried for one day, cut and mixed with 3 L of 85% (v/v) ethanol. The solution was extracted for 3 days with reflux extraction and the residue was filtered. The filtrate was concentrated with rotary evaporator (Evaporator, JP/N-N, EYELA) at 35⁰C for 4 hours, and then dried for 48 hours to obtain powdered extract of Schisandra nigra Max. (67.65 g). The powder was dissolved in DMSO (Dimethyl sulfoxide, Sigma, MO, USA) to use as samples in the following Experimental Examples. The extract was stored in -2O⁰C and diluted to the extent that the final concentration reached to 0.1% DMSO in each experiment.

[63]

[64] Experimental Example 1. Incubation and isolation of rat vibrissa follicles

[65] To confirm the effect of inventive extract on the preventing activity of baldness disorder and stimulating activity of hair growth, the organ culture method was performed according to the method disclosed in the reference (Jindo et al., J. Dermatol ., 20(10). pp756-762, 1993).

[66] 3 weeks-old Wistar male rat (Japan SLC Shizuoka, Japan) anesthetized with ethyl ether was sacrificed to isolate rat vibrissa follicles. The left and right Mystacial pads were incubated with E/P buffer solution (50% Earle's balanced salts solution (EBSS) (Sigma MO, USA) + 50% PBS (Sigma MO, USA) supplemented with 100 unitsM of penicillin and 100 μg/m# of streptomydn (Gibco Inc, NY, USA) and the rat vibrissa follicle was isolated with observing by microscopy. The vibrissa follicle were placed in a Petri dish containing the E/P buffer solution and incubated at 37⁰C, in 5% CO ₂ incubator for 2 hours. 500 jΛ of William E medium (Gibco Inc, NY, USA) containing 2 mM L-glutamine (Gibco Inc, NY, USA), 10 μg/τd of insulin (Sigma MO, USA), 50 nM hydrocortisone (Sigma MO, USA) and 100 units/ mi of penicillin- 100 μg/m# of streptomydn were added to each well of 24- well plate and one vibrissa follicle was placed to each well. The well was further incubated at 37⁰C, in 5% CO ₂ incubator. The media was exchanged in every 3 days and treated with the test sample at the concentration of 5, 10, 20 and 50 μg/m#, and minoxidil sulfate (Sigma, USA) as positive controls at the concentration of 0.1, 1 and 10 μM respectively to compare with each other.

[67] The hair growth of hair follicle was observed by image analyzer (DP controller,

Olympus) at the 0, 7^th, 14^th and 21^st day to compare the growth of hair follicle length with each other and the result was shown in the following Table 1.

[68]

[69] Table 1 [Table 1] [Table ]

[70] [71] As shown in Table 1, the growth of hair follicle in each group at the 21 ^st day was calculated by the difference in length (%) of each hair follicle compared with control group, and the result was shown in the following Table 2.

[72] [73] Table 2 [Table 2] [Table ]

[74] [75] As shown in Table 2, the treatment with minoxidil sulfate 10 μM showed growth activity by about 51% of the hair follicle comparing with control group. The treatment with inventive extract prepared in Example 1 at the concentration of 20 βg/md, and 5 μg/m# showed more potent growth of hair follicle by 122% and 35%, respectively.

[76] At the result, the inventive extract prepared in Example 1 showed the most effective growth activity of rat vibrissa follicles.

[77] [78] Experimental Example 2. Expression of PCNA (Proliferating cell nuclear antigen) in rat vibrissa follicles

[79] To confirm the effect of inventive extract of the expression of PCNA (Proliferating cell nuclear antigen), following experiment was preformed. To examine the cell proliferation (Assy et al., J. Hepatol. , 2ϋ_pp945-952, 1997), immunohistochemistry staining assay was performed according to Santa Cruz Biotechnology manual.

[80] 500 [d of William E medium (Gibco Inc, NY, USA) supplemented with 2 mM L- glutamine (Gibco Inc, NY, USA^ 10 μglmk of Insulin (Sigma MO, USA)^ 50 nM Hydrocortisone (Sigma MO, USA) and 100 units/ m# of penicillin- 100 μg/mi of streptomydn was added to each well of the 24- well plate and one vibrissa follicle prepared in the Experimental Example 1 was added thereto to incubate at 37⁰C, in 5% CO₂ incubator. The media was exchanged in every 3 days and treated with the inventive extract at the concentration of 20 βglrύ, and minoxidil sulfate (Sigma, USA) as a positive control at the concentration of 10 μM. At the 0 and 7 ^ day after the separation of hair follicle, the vibrissa follicle was fixed for 24 hours with 4% paraformaldehyde and washed with 20% sucrose twice. Then, the vibrissa follicle was dehydrated, and solidified to form a parafilm block through transparency or parafilm penetration process using by an automatic tissue processor (Leica, Germany). The parafilm blocks were sliced in the width of 4 μm, attached to a silane coating slide (Muto Chemicals, Japan) and dried. The slides were treated with xylene for 3 times to remove parafilm, hydrated and immunohistochemistry was performed thereto according to ABC Staining Kit (Santa cruz biotechnology, Inc, USA). 1% Hydrogen peroxide (H₂O₂) was treated to inhibit the action of endogenous enzyme, and 1.5% blocking serum was treated for an hour to prevent nonspecific binding with primary antibody. To detect the expression of PCNA protein, rabbit anti-PCNA antibody (Santa Cruz Biotechnology, Inc, USA) diluted to 1: 200 was used as a primary antibody. After the treatment of primary antibody overnight, the well was washed with PBS for 3 times, and treated with anti-rabbit IgG for 30 minutes as the secondary antibody. After washing with PBS for 3 times, avidin-biotinylated horseradish peroxidase enzyme reagent (HRP) was treated thereto for 30 minutes, and to observe the positive reaction, the color was developed with diaminobenzidine (DAB) for 1 minute. The well was treated with Hematoxylin Gill's Formulation #2 (Santa Cruz Biotechnology, Inc, USA) for 10 seconds to perform the comparative staining, mounted with non-aqueous permount (Fisherchemicals, USA) and observed by microscopy.

[81] At the result as shown in Fig. 1, on the day of separation, there showed significant

PCNA expression at the bulb matrix region surrounding dermal papilla in vibrissa follicles. At the 7¹^ day, PCNA expression was not observed in the control group, while the PCNA expression in the test group treated with 20 βg/md, of inventive extract was significant in the bulb matrix region of vibrissa follicles. The group treated with 10 μM minoxidil sulfate also showed significant PCNA expression at the 7 ^ day. Therefore, it has been confirmed that the inventive extract prepared in Example 1 increases the PCNA expression and involves in the hair growth in bulb matrix region of rat vibrissa follicles (See Fig. 1).

[82]

[83] Experimental Example 3. Expression of TGF-β2in rat vibrissa follicles

[84] To confirm the effect of inventive extract on the expression of TGF-β2, following experiment was performed (Assy et al., J. Hepatol, 26, pp945-952, 1997).

[85] 500 fd of William E medium (Gibco Inc, NY, USA) supplemented with 2 mM L- glutamine (Gibco Inc, NY, USA^ 10 μg/τd Insulin (Sigma MO, USA)^ 50 nM Hydrocortisone (Sigma MO, USA) and 100 units/ mi of penicillin- 100 μg/m# of streptomyin was added to each well of the 24- well plate and one vibrissa follicle prepared in Experimental Example 1 was added thereto to incubate at 37⁰C, in 5% CO₂ incubator. The media was exchanged in every 3 days and treated with the inventive extract at the concentration of 20 μg/m-6, and minoxidil sulfate (Sigma, USA) as a positive control at the concentration of 10 μM. At the 0 and 7 * day after the separation of hair follicle, the vibrissa follicle was fixed for 24 hours with 4% paraformaldehyde and washed with 20% sucrose twice. Then, the vibrissa follicle was dehydrated, and solidified to form a parafilm block through transparency or parafilm penetration process using by an automatic tissue processor (Leica, Germany). The parafilm blocks were sliced in the width of 4 μm, attached to a silane coating slide (Muto Chemicals, Japan) and dried. The slides were treated with xylene for 3 times to remove parafilm, hydrated and im- munohistochemistray was performed according to ABC Staining Kit (Santa cruz biotechnology, Inc, USA). 1% Hydrogen peroxide (H₂O₂) was treated to inhibit the action of endogenous enzyme, and 1.5% blocking serum was treated for an hour to prevent nonspecific binding with primary antibody. To detect the expression of TGF-β2, rabbit anti-TGF-β2 antibody (Santa Cruz Biotechnology, Inc, USA) diluted to 1: 200 was used as a primary antibody. After the treatment of primary antibody overnight, the well was washed with PBS for 3 times, and treated with the secondary antibody, anti-rabbit IgG for 30 minutes. After washing with PBS for 3 times, avidin-biotinylated horseradish peroxidase enzyme reagent (HRP) was treated thereto for 30 minutes, and to observe the positive reaction, the color was developed with diaminobenzidine (DAB) for 1 minute. The well was treated with Hematoxylin Gill's Formulation #2 (Santa Cruz Biotechnology, Inc, USA) for 10 seconds to perform the comparative staining, mounted with non-aqueous permount (Fisherchemicals, USA) and observed by microscopy.

[86]

[87] At the results as shown in Fig. 2, TGF-β2 expression was not observed on the day of isolation of the vibrissa follicle, and the group treated with 20 βg/md, of the inventive extract at the 7 * day in the bulb matrix region surrounding the dermal papilla of the hair follicle did not also showed TGF- β2 expression. The group treated with 10 μM minoxidil sulfate at the 7^th day also showed no expression of TGF-β2. Therefore, it has been confirmed that the inventive extract prepared in Example 1 inhibited the TGF- β2 expression relevant to the progression from anagen to catagen and it showed potent stimulating activity of hair growth by acting on anagen phase ( See Fig. 2).

[88]

[89] Hereinafter, the formulating methods and kinds of exάpients comprising the extract of Schisandra nigra Max. will be described, but the present invention is not limited to them. The representative preparation examples are described as follows.

[90]

[91] Preparation of injection

[92] Extract of Example 1 50 mg

[93] Sodium metadisulfite 3.0 mg

[94] Methylparaben 0.8 mg

[95] Propylparaben 0.1 mg

[96] Distilled water for injection optimum amount

[97] Injection preparation was prepared by mixing the above components to a total volume of 2 ml by the conventional method, filling in an ample and sterilizing by conventional injection preparation method.

[98]

[99] Preparation of tablet

[100] Extract of Example 1 50 mg

[101] Oorn Starch 100 mg

[102] Lactose 100 mg

[103] Magnesium Stearate 2 mg

[104] Tablet preparation was prepared by mixing the above components and entabletting.

[105]

[106] Preparation of capsule

[107] Extract of Example 1 100 mg

[108] Oorn Starch 100 mg

[109] Lactose 100 mg

[110] Talc 2 mg

[111] Magnesium Stearate optimum amount

[112] Tablet preparation was prepared by mixing the above components and filling in a gelatin capsule by conventional gelatin preparation method.

[113]

[114] Preparation of liquid [115] Extract of Example 1 100 mg

[116] Sugar 2O g

[117] Fructose 2O g

[118] Lemon flavor optimum amount

[119] Distilled water 100 ml

[120] liquid preparation was prepared by mixing the above components, filling in a 100 ml brown bottle and sterilizing by conventional liquid preparation method. [121]

[122] Preparation of health care food

[123] Extract of Example 1 1000 mg

[124] Vitamin mixture 20 g

[125] Vitamin A acetate 70 μg

[126] Vitamin E 1.0 mg

[127] Vitamin Bl 0.13 mg

[128] Vitamin B2 0.15 mg

[129] Vitamin B6 0.5 mg

[130] Vitamin C 10 mg

[131] Biotin lO μg

[132] Amide nicotinic acid 1.7 mg

[133] Folic acid 50 μg

[134] Calcium pantothenic acid 0.5 mg

[135] Mineral mixure optimum amount

[136] Ferrous sulfate 1.75 mg

[137] Zinc oxide 0.82 mg

[138] Magnesium carbonate 25.3 mg

[139] Monopotassium phosphate 15 mg

[140] Dicalcium phosphate 55 mg

[141] Potassium citrate 90 mg

[142] Calcium carbonates 100 mg

[143] Magnesium chloride 24.8 mg

[144] The above-mentioned vitamin and mineral mixture may be varied in many ways.

Such variations are not to be regarded as a departure from the spirit and scope of the present invention. [145]

[146] Preparation of health beverage [147] Extract of Example 1 1000 mg

[148] αtric add 100 mg

[149] Oligosaccharide 100 g

[150] Apricot concentration 2 g

[151] Taurine 1 g

[152] Distilled water 900 ml

[153] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85⁰C for 1 hour, filtered and then filling all the components in a 2000 ml ample and sterilizing by conventional health beverage preparation method.

[154]

[155] The invention being thus described as will be obvious that it may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to those skilled in art are intended to be included within the scope of the following claims.

[156]

Industrial Applicability

[157] As described in the present invention, the extract of Schisandra nigra Max showed potent preventing activity of baldness disorder and stimulating activity of hair growth being confirmed by several experiments such as the growth rate test using by vibrissa follicle, PCNA and TGF-β2 expression test in rat hair follicle. Accordingly, the inventive extract can be used as a pharmaceutical composition and health functional food for the treatment of baldness disorder and stimulation of hair growth.

Claims

[1] A pharmaceutical composition comprising the extract of Schisandra nigra Max., as an active ingredient in an effective amount to prevent baldness disorder and to stimulate hair growth.

[2] The pharmaceutical composition according to the claim 1, wherein said extract is extracted with the solvent selected from the group consisting of water, C i - C₄ lower alcohol and the mixture thereof.

[3] The pharmaceutical composition according to the claim 1, wherein said extract may comprise as 0.1-50% by weight based on the total weight of the composition.

[4] The pharmaceutical composition according to the claim 1, wherein said composition is in an oral dosage form such as powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule; or topical preparation such as cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like; or injectable preparation such as solution, suspension or emulsion.

[5] A health functional food comprising the extract of Schisandra nigra Max., as an active ingredient in an effective amount to prevent baldness disorder and stimulate hair growth.

[6] The health functional food according to the claim 5, wherein said composition is used in the form of powder, granule, tablet, chewing tablet, capsule or liquid.

[7] A use of the extract of Schisandra nigra Max. for the preparation of therapeutic agent for the treatment and prevention of baldness disorder in mammal or human.

[8] A method of treating or preventing baldness disorder in mammal or human in need thereof comprising administering to said mammal or human with an effective amount of the extract of Schisandra nigra Max., together with a pharmaceutically acceptable carrier thereof.