PHARMACEUTICAL COMPOSITION FOR PREVENTING HAIR LOSS OR PROMOTING HAIR GROWTH, COMPRISING GINSENOSIDE RG4 OR MIXTURE (RGX 365) OF GINSENOSIDES RG2, RG4, RG6, AND RH1 AS ACTIVE INGREDIENT

Abstract

The present invention relates to a composition for preventing hair loss or promoting hair growth, comprising ginsenoside Rg4 or a mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1 as an active ingredient. Treatment with ginsenoside Rg4 or the mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1 in human hair follicle dermal papilla cells exhibits a desirable effect of forming three-dimensional spheres capable of inducing hair follicle formation and growth and increasing the size thereof, exhibits a desirable effect of activating Wnt/β-catenin signaling, which is important for hair follicle cell activation and regeneration, and exhibits a desirable effect of increasing the expression levels of ALPL, VCAN, BMP2, and FGF7 genes, which are important for hair follicle formation and growth. Thus, ginsenoside Rg4 or the mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1 can be usefully utilized as a composition for preventing hair loss and promoting hair growth.

Claims

1. A pharmaceutical composition comprising ginsenoside Rg4 as an active ingredient for preventing hair loss or promoting hair growth.

2. The pharmaceutical composition of claim 1, wherein the active ingredient further comprises ginsenosides Rg2, Rg6, and Rh1 as a mixture with the ginsenoside Rg4.

3. The pharmaceutical composition of claim 2, wherein the mixture comprises 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

4. A cosmetic composition comprising ginsenoside Rg4 as an active ingredient for preventing hair loss or promoting hair growth.

5. The cosmetic composition of claim 4, wherein the active ingredient further comprises ginsenosides Rg2, Rg6, and Rh1 as a mixture with the ginsenoside Rg4.

6. The cosmetic composition of claim 5, wherein the mixture comprises 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

7. The cosmetic composition of claim, wherein the cosmetic composition is prepared into a formulation selected from the group consisting of a solution, an emulsion, a suspension, a paste, a gel, a powder, a skin lotion, a lotion, a cream, an essence, a spray, a shampoo, a conditioner, and a soap.

8. A health functional food comprising ginsenoside Rg4 as an active ingredient for preventing hair loss or promoting hair growth.

9. The health functional food of claim 8, wherein the active ingredient further comprises ginsenosides Rg2, Rg6, and Rh1 as a mixture with the ginsenoside Rg4.

10. The health functional food of claim 9, wherein the mixture comprises 35-45% by weight of ginsenoside Rg2, 30-40% by weight of ginsenoside Rg4, 10-20% by weight of ginsenoside Rg6, and 1-5% by weight of ginsenoside Rh1.

11. The health functional food of claim 8, wherein the health functional food is prepared into a formulation selected from the group consisting of a tablet, a capsule, a pill, and a solution.

12. A method for preventing hair loss or promoting hair growth in a subject, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 1.

13. A method for preventing hair loss or promoting hair growth in a subject, comprising administering to the subject in need thereof an effective amount of the cosmetic composition of claim 4.

14. A method for preventing hair loss or promoting hair growth in a subject, comprising administering to the subject in need thereof an effective amount of the health functional food of claim 8.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0037] FIGS. 1A and 1B show assay results for cell viability of human hair follicle dermal papilla cells cultured in a 2D (monolayer) and a 3D (spheroid) manner according to treatment concentrations of Example 1 of the present disclosure.

[0038] FIGS. 2A and 2B show assay results for cell viability of human hair follicle dermal papilla cells cultured in a 2D (monolayer) and a 3D (spheroid) manner according to treatment concentrations of Example 2 (Rgx 365) of the present disclosure.

[0039] FIGS. 3A, 3B, and 3C shows the formation of 3D spheroids of human hair follicle dermal papilla cells cultured in a 3D pattern according to the treatment of Example 1 (ginsenoside Rg4) of the present disclosure or Comparative Example 1 (ginsenoside Rg2) in contrast-phase microscope images and graphs depicted on the basis of digitized data.

[0040] FIG. 4 shows assay results for the sizes of 3D spheroid of the human hair follicle dermal papilla cells cultured in a 3D pattern in the presence or absence of Example 2 (Rgx 365) of the present disclosure.

[0041] FIG. 5A shows assay results for TCF/LEF activity in HEK293 cells according to the treatment of Example 1 (ginsenoside Rg4) of the present disclosure and FIGS. 5B, 5C, and 5D show mRNA expression levels of β-catenin, WNT5A, and LEF1 in human hair follicle dermal papilla cells according to the treatment of Example 1 (ginsenoside Rg4) of the present disclosure.

[0042] FIGS. 6A, 6B, 6C, and 6D show assay results for mRNA expression levels of ALPL, VCAN, FGF7, and BMP2 in human hair follicle dermal papilla cells cultured in a 3D pattern according to the treatment of Example 1 (ginsenoside Rg4) of the present disclosure.

[0043] FIGS. 7A, 7B, and 7C show assay results for mRNA expression levels of ALPL, VCAN, and FGF7 in 3D cultured human hair follicle dermal papilla cells according to the treatment of Example 2 (Rgx 365) of the present disclosure.

BEST MODE FOR CARRYING OUT THE INVENTION

[0044] Hereinafter, the present disclosure will be described in detail with reference to exemplary embodiments. However, these exemplary embodiments are for specifically illustrating the present disclosure and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1: Preparation of Ginsenoside Rg4

[0045] With reference to Korean Patent No. 10-1783295, ginsenoside Rg4 was prepared from ginsenoside Re.

Example 2: Preparation of Mixture (Rgx 365) of Ginsenosides Rg2, Rg4, Rg6, and Rh1

[0046] With reference to Korean Patent Application No. 10-2019-0041458, a mixture (Rgx 365) of ginsenosides Rg2, Rg4, Rg6, and Rh1 was prepared.

Experimental Example 1: Cell Culture

[0047] With reference to the documents [Topouzi, H. et al., Exp Dermatol., 26(6), 491-496, 2017; Higgins, C. A. et al., Proc Natl Acad Sci USA., 110(49), 19679-19688, 2013; Higgins, C. A. et al., Exp Dermatol., 19(6), 546-548, 2010], human hair follicle dermal papilla (HDP) cells were incubated in a general 2D monolayer culture manner and in a 3D spheroid culture manner as in the actual human follicle structure.

[0048] First, for 2D monolayer culture, human hair follicle dermal papilla cells were cultured in a mesenchymal stem cell medium (MSCM) (Innoprot, Bizakaia, Spain) supplemented with 5% fetal bovine serum (FBS, Thermo Fisher Scientific, Inc., Waltham, Mass., USA) in an incubator maintaining the condition of 37° C. and 5% CO.sub.2.

[0049] Next, for 3D spheroid culture, human hair follicle dermal papilla cells were cultured in a plate (round-bottom ultra-low attachment microplate, Corning, N.Y., USA) containing the stem cell medium supplemented with 5% fetal bovine serum at 37° C. under a 5% CO.sub.2 atmosphere.

[0050] HEK293 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 U/ml) under the condition of 37° C. and 5% CO.sub.2.

Experimental Example 2: Assay for Cell Viability

[0051] Cell viability of the human hair follicle dermal papilla cells treated according to the Examples of the present disclosure was examined by WST-1 assay (WST-1 assay kit, water soluble tetrazolium salt, Daeil Lab Service Co. Ltd, Korea).

[0052] First, the human hair follicle dermal papilla cells cultured in a 2D or 3D manner in Experimental Example 1 were seeded at a density of 1×10.sup.4 cells/well into 96-well plates (96-well round-bottom ultra-low attachment microplates) and incubated at 37° C. for 24 hours in a 5% CO.sub.2 atmosphere. After removal of the medium, the cells were cultured for 48 hours in media containing various concentrations of Example 1 or 2. Then, 10 μl of the WST-1 solution was added to the culture and incubated for 30 min before reading absorbance at 450 nm on an iMark microplate reader (Bio-Rad Laboratories, Hercules, Calif., USA). For reliability, experiments were conducted in triplicate. Experiment results are depicted in FIGS. 1 and 2.

[0053] With reference to FIGS. 1 and 2, Examples 1 and 2 exhibited the same cell viability of the 2D or 3D cultured human hair follicle dermal papilla cells as in the non-treated group irrespective of the concentrations thereof, which demonstrates that Examples 1 and 2 are materials free of cytotoxicity.

Experimental Example 3: Effect of Hair Loss Prevention and Hair Growth Promotion

Experimental Example 3-1. Formation of 3D Spheroid

[0054] Referring to the document [Higgins, C. A. et al., Exp Dermatol., 19(6), 546-548, 2010; Higgins, C. A. et al., Proc Natl Acad Sci USA., 110(49), 19679-19688, 2013; Kang, B. M. et al., J Invest Dermatol., 132(1), 237-239, 2012], a 3D spheroid culture of human hair follicle dermal papilla cells is reported to induce better formation and growth of hair follicles, compared to a 2D culture of human hair follicle dermal papilla cells. In addition, the document [Whiting, D. A., J Am Acad Dermatol., 45(3 Suppl), S81-S86, 2001; de Lacharriere, O. et al., Arch Dermatol., 137(5), 641-646, 2001; Jahoda, C. A., Exp Dermatol., 7(5), 235-248, 1998] reports that a decrease in the size of dermal papilla may cause follicle miniaturization in patients with hair loss. The Examples of the present disclosure were examined for effects of preventing hair loss and promoting hair growth by monitoring the formation and size of 3D spheroids after application to the 3D spheroid culture of Experimental Example 1.

[0055] First, the formation and size change of 3D spheroids of human hair follicle dermal papilla cells were observed. In this regard, the 3D spheroid culture of cells in Experimental Example 1 was seeded at a density of 5×10.sup.4 cells/well into 96-well plates (96-well round-bottom ultra-low attachment microplates) and incubated for 48 hours with predetermined concentrations of Examples 1 and 2 and Comparative Example 1 (ginsenoside Rg2). Thereafter, the human dermal papilla cells were observed under a phase-contrast microscope to see whether the cells formed 3D spheroids and to measure sizes of the spheroids. The results are given in FIGS. 3 and 4.

[0056] With reference to FIGS. 3 and 4, the 3D culture of human hair follicle dermal papilla cells treated with Example 1 or 2 was observed to increase the number and size of 3D spheroids in a concentration-dependent manner, as opposed to the non-treated group.

[0057] Particularly turning to FIG. 3C, when applied to the 3D culture of human hair follicle dermal papilla cells, ginsenoside Rg4 of Example 1 increased the number and size of the 3D spheroids two to three folds, compared to ginsenoside Rg2 of Comparative Example 1, which implies that ginsenoside Rg4 of Example 1 is more selective for the effect of preventing hair loss and promoting hair growth.

[0058] It was understood from the result that ginsenoside Rg4 or a mixture (Rgx 365) of ginsenosides Rg2, Rg4, Rg6, and Rh1 is superb in terms of inductive property for hair growth promotion and hair loss prevention.

Experimental Example 3-2. Activation of Wnt/β-Catenin Signaling Pathway

[0059] According to the document [Andl, T. et al., Dev Cell., 2(5), 643-653, 2002; Kishimoto, J. et al., Genes 8 Dev., 14(10), 1181-1185, 2000], it is reported that the Wnt/β-catenin signaling pathway plays an important role in promoting the formation of hair follicles and maintaining and activating genes expressed during the anagen stage of the hair cycle. Thus, Wnt/β-catenin is in a very important position in activating and regenerating hair follicle cells. In this regard, when treated with the Example of the present disclosure, the cells cultured in Experimental Example 1 were examined for Wnt/β-catenin signaling activation, which leads to the effect of preventing hair loss and promoting hair growth.

[0060] Whether the Wnt/β-catenin signaling is activated or not was determined using a TCF/LEF luciferase reporter plasmid (TOPFlash, Merck-Millipore, Darmstadt, Germany) designed to specifically react with the transcription factor TCF/LEF activated by β-catenin, and a reporter plasmid having a mutation caused in the promoter thereof (FOPFlash, Merck-Millipore, Darmstadt, Germany).

[0061] First, the TCF/LEF luciferase reporter plasmid (TOPFlash (TCF/LEF-wt) or FOPFlash (TCF/LEF-mut)) and a β-galactosidase plasmid were transfected into the HEK293 cells cultured in Experimental Example 1 with the aid of Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Inc.). After being incubated with 20 μg/ml of Example 1 for 24 hours, the transfected cells were lysed with a passive lysis buffer (Promega, Madison, Wis., USA). The cell lysate thus formed was added with D-luciferin and measured for luciferase activity by Glomax 96 Microplate Luminometer (Turner Biosystem, Sunnyvale, Calif., USA). β-Galactosidase activity was measured with the aid of Luminescent β-galactosidase Detection Kit II (Clontech Laboratories Inc., USA). On the basis of the data, luciferase activity values were derived, and are depicted in FIG. 5A. For reliability, experiments were conducted in triplicate.

[0062] Next, in order to confirm the luciferase activity results, mRNA analysis was performed on β-catenin, WNT5A, and LEF1, which are involved in the Wnt/β-catenin signaling. First, a 3D spheroid culture of human hair follicle dermal papilla cells was treated with Example 1 (20 μg/ml) for 48 hours, followed by RNA extraction with a TRIzol solution (Invitrogen, Thermo Fisher Scientific). From 2 μg of the RNA extract, cDNA was synthesized in the presence of M-MLV reverse transcriptase. The synthesized cDNA was mixed with HOT FIREPol EvaGreen qPCR Mix Plus solution (Solis BioDyne, Tartu, Estonia) and each of primer sets for the Wnt/β-catenin target genes β-catenin, WNT5A, and LEF1 and subjected to Step OnePlus Real-Time PCR (Applied Biosystems; Thermo Fisher Scientific). Relative to the expression of β-actin housekeeping gene, an expression level of each gene was derived. Analysis results of mRNA expressions are depicted in FIGS. 5B to 5D.

[0063] With reference to FIG. 5, Example 1 increased the activation of TCF/LEF and the mRNA expression levels of β-catenin, WNT5A, and LEF1 two folds or more, compared to the non-treated group (control). Exhibiting the excellent effect of activating the Wnt/β-catanin signaling, therefore, Example 1 (ginsenoside Rg4) of the present disclosure can be used in a composition for promoting hair growth.

Experimental Example 3-3. Dermal Papilla Signature Gene Expression and Marker Associated with Hair Follicle Neogenesis

[0064] Signature genes expressed in dermal papilla cells include genes coding for ALP (alkaline phosphatase) which significantly increase in expression level upon 3D culture and plays an important role in the neogenesis and growth of hair follicles, VCAN (versican) which plays an essential role in follicle neogenesis, BMP2 (bone morphogenetic protein 2) which is indispensable for dermal papilla cell functions, and FGF7 (fibroblast growth factor 7) which is involved in hair growth. After being applied to the 3D spheroid culture of human hair follicle dermal papilla cells in Experimental Example 1, the Examples of the present disclosure were examined for the effect of preventing hair loss and promoting hair growth by monitoring mRNA expression levels of ALPL, VCAN, BMP2, and FGF7.

[0065] The mRNA expression analysis of ALPL, VCAN, BMP2, and FGF7 was carried out in the same manner as for β-catanin, WNT5A, and LEF1 in Experimental Example 3-2, with the exception of using primer sets for ALPL, VCAN, FGF7, and BMP2 instead of those for β-catenin, WNT5A, and LEF1. The mRNA expression analysis data are depicted in FIGS. 6 and 7.

[0066] Referring to FIGS. 6 and 7, the 3D spheroid culture of human hair follicle dermal papilla cells treated with Example 1 or 2 of the present disclosure increased in expression levels of all of ALPL, VCAN, BMP2, and FGF7, compared to the non-treated control, demonstrating that the Examples have an excellent effect of preventing hair loss and promoting hair growth.

[0067] From the result, it is understood that ginsenoside Rg4 or a mixture of ginsenosides Rg2, Rg4, Rg6, and Rh1 according to the present disclosure can be advantageously used in a composition for preventing hair loss or promoting hair growth.

Preparation Example 1: Hydrophilic Ointment Formulation

[0068] A hydrophilic ointment formulation was prepared by a typical method using the mixture (Rgx 365) of ginsenosides Rg2, Rg4, Rg6, and Rh1 of Example 2 of the present disclosure according to the composition listed in Table 1, below.

TABLE-US-00001 TABLE 1 Material name Content (% by weight) Example 2 8.0 White Vaseline 35.0 Stearyl alcohol 30.0 Ethyl (or methyl) Trace p-oxybenzoate Propylene glycol 20.0 Sodium lauryl sulfate 3.4 Propyl p-oxybenzoate Trace

Preparation Example 2: Preparation of Skin Lotion Formulation

[0069] A skin lotion formulation was prepared by a typical method using the mixture (Rgx 365) of ginsenosides Rg2, Rg4, Rg6, and Rh1 of Example 2 of the present disclosure according to the composition listed in Table 2, below.

TABLE-US-00002 TABLE 2 Material name Content (% by weight) Example 2 4.0 Butylene glycol 3.5 Glycerin 2.5 Polyoxyethylene 0.1 hydrogenated castor oil Ethanol 2.5 Betaine 1.0 Citric acid 0.01 Sodium citrate 0.03 Preservative Suitable amount Fragrance Suitable amount Purified water to 100

Preparation Example 3: Preparation of Cream Formulation

[0070] A cream formulation was prepared by a typical method using the mixture (Rgx 365) of ginsenosides Rg2, Rg4, Rg6, and Rh1 of Example 2 of the present disclosure according to the composition listed in Table 3, below.

TABLE-US-00003 TABLE 3 Material name % by weight (w/w) Example 2 7.0 Cetosteary alcohol 3.0 Self-emulsifying 1.5 monostearic acid Lipophilic monostearic acid 1.5 Bee wax 0.5 Liquid paraffin 8.0 Squalane 7.0 Isocetyl octanoate 4.0 Purified jojoba oil 4.0 Dimethyl siloxane 0.3 Sorbitan monostearate 1.0 Ethylene glycol monostearate 1.2 Glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 Preservative 0.25 Fragrance Suitable amount Pigment Suitable amount Purified water to 100

Preparation Example 4: Preparation of Health Function Food

[0071] Granules were prepared by mixing 20 g of Example 2 (ginsenoside Rg2, Rg4, Rg6, and Rh1 mixture, Rgx 365), a suitable amount of a vitamin complex, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, 10 mg of vitamin C, 10 μg of biotin, 1.7 mg of nicotinamide, 50 μg of folic acid, 0.5 mg of calcium pantothenate, a suitable amount of an inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of calcium phosphate dibasic, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride, and can be modified into various formulations. In addition, any modification may be imparted to the composition ratio of vitamin and mineral mixtures and the ingredients may be mixed according to typical preparation methods for health function foods.

Preparation Example 5: Preparation of Health Functional Beverage

[0072] A beverage was prepared by mixing 1 g of Example 2 of the present disclosure (ginsenoside Rg2, Rg4, Rg6, and Rh1 mixture, Rgx 365), 0.1 g of citric acid, 100 g of fructooligosaccharide, and 900 g of purified water, and stirring, heating, filtering, sterilizing, and refrigerating the mixture according to a typical beverage preparation method.