Method of using composition containing <i>Hovenia dulcis </i>Thunb. extract as active ingredient for prevention and treatment of bone diseases

Abstract

The invention relates to a composition containing extracts of the fruit of Hovenia dulcis Thunb., gallocatechin, and methyl vanillate as active ingredients for preventing and treating bone disease. The extracts of the fruit of Hovenia dulcis Thunb., the gallocatechin, and the methyl vanillate according to the present invention are natural materials and thus cause no side effects, and may activate the Wnt/β-catenin pathway to promote bone formation rather than inhibiting bone decomposition, and therefore may be effective in treating osteoporosis and related diseases. In addition, the extracts of the fruit of Hovenia dulcis Thunb., the gallocatechin, and the methyl vanillate according to the present invention may have the effects regrowing hair and preventing and treating wounds and metabolic diseases related to the activation of Wnt/β-catenin pathway.

Claims

1. A method of treating a subject having an osteoporosis, the method comprising administering a composition comprising an effective amount of methyl vanillate as an active ingredient, wherein the methyl vanillate promotes a bone formation by activating a Wnt/β-catenin signaling pathway and inducing osteoblast differentiation.

2. The method of claim 1, wherein the composition is administered via oral, parenteral, intraarterial, intradermal, transdermal, intramuscular, intraperitoneal, intravenous or subcutaneous routes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result of the screening of the natural compound which activates Wnt/β-catenin signaling pathway.

(2) FIG. 2 shows the result of the measurement of the transcription of Hovenia dulcis Thunb. extract by β-catenin in HEK-TOP.

(3) FIG. 3 is the result showing an increase of protein in β-catenin through Hovenia dulcis Thunb. extract.

(4) FIG. 4 is a photograph confirming an increase of β-catenin via immunohistochemical staining.

(5) FIG. 5 is a photograph showing an increase in thickness of the calvaria of a Hovenia dulcis Thunb. extract-treated group.

(6) FIG. 6 is a photograph showing an increase in thickness of the calvaria of a Hovenia dulcis Thunb. extract-treated group.

(7) FIG. 7 is a photograph showing the growth of bone by gallocatechin and methyl vanillate.

(8) FIG. 8 is a photograph showing the induction of differentiation of POB by gallocatechin (a gallocatechin-treated group: (+), a non-treated group: (−)).

(9) FIG. 9 and FIG. 10 are a photograph and a graph showing the effects of ALP activation of gallocatechin and methyl vanillate.

DETAILED DESCRIPTION

(10) Hereinafter, the present invention will be described in detail with reference to the following examples. However, these examples are only intended to illustrate the invention and the content of the present invention should not be construed to be limited to the examples.

Example 1

(11) Production and Screening of Hovenia dulcis Thunb. Extract

(12) HEK-TOP is a stable cell line which has a Topflash capable of measuring the reporter of β-catenin in HEK293. The cells were treated with more than 300 natural compounds at a concentration of 1 ug/ml. After 24 hours, the transcription by β-catenin was measured via a reporter (FIG. 1). Hovenia dulcis Thunb. was selected as a compound which increases the transcription by β-catenin through screening. For the selected Hovenia dulcis Thunb., the transcription by β-catenin in HEK-TOP was measured again and the results showed that the reporter value was increased (FIG. 2). Hovenia dulcis Thunb. was purchased from the Korean plant extract bank and extracted using a distilled water.

Example 2

(13) Confirmation of the Efficacy of Hovenia dulcis Thunb. Extract

(14) In the calvaria of 4-day-old postnatal mice supplied by Orient Bio, POB (primary osteoblast) was separated and then treated with 5 μg/ml of Hovenia dulcis Thunb. extract for 24 hours. The results showed that β-catenin protein was increased FIG. 3).

(15) While inducing the differentiation in the calvaria of 4-day-old postnatal rat, 5 μg/ml of Hovenia dulcis Thunb. extract was treated for a week. As a result, it was confirmed through the immunohistochemical staining that β-catenin was increased in the Hovenia dulcis Thunb. extract-treated group (FIG. 4). In addition, the results of the measurement of the thickness showed that the thickness of the calvaria was increased in the Hovenia dulcis Thunb. extract-treated group as compared to the control group (FIG. 5 and FIG. 6).

Example 3

(16) Confirmation of the Efficacy of Gallocatechin and Methyl Vanillate

(17) Gallocatechin and methyl vanillate are known as one component of Hovenia dulcis Thunb. extract capable of activating Wnt/β-catenin. By changing the concentrations of gallocatechin and methyl vanillate, the effects on the bone growth were investigated (Chem Pharm Bull (Tokyo), 1996 September; 44 (9): 1736-43; Yakugaku Zasshi. 1997 February; 117 (2): 108-18; Arch Pharm Res. 2005 July; 28 (7): 804-9).

(18) While inducing the differentiation in the calvaria of 4-day-old postnatal mice supplied by Orient Bio. 2 nM of gallocatechin and 0.2 nM of methyl vanillate were treated for one week. The result of the measurement of the thickness showed that the thickness of the calvaria was increased in the gallocatechin and methyl vanillate-treated groups (FIG. 7). Also, it was confirmed that the thickness of the calvaria was increased in a small amount as compared to the positive control group (Hovenia dulcis Thunb. extract).

(19) In addition, in order to confirm the activity of ALP (alkaline phosphatase) showing the activation of bones, 20 μM of gallocatechin was treated with primary osteoblast (POB) cells, and allowed to differentiate and stain for 9 days. The results showed that the expression of ALP was increased in the gallocatechin-treated group (FIG. 8). In addition, in order to confirm the activity of ALP (alkaline phosphatase) in the gallocatechin and methyl vanillate-treated groups, the analysis was conducted and the results showed that the activity of ALP was increased within the concentration gradient (FIG. 9 and FIG. 10).

Example 4

(20) In-Vivo Administration of the Extract for the Treatment of Bone Diseases

(21) 6-week old specific pathogen-free (SPF) SD-based rat supplied from the DAEHAN Experiment Supply Center was used and the acute toxicity experiments were conducted as follows.

(22) The Hovenia dulcis Thunb. extract, gallocatechin and methyl vanillate obtained in Example 1 were orally administered once at a dose of 1 g/kg to two animals in each group. The animal mortality, clinical symptoms, and change in the body weight were observed and then hematologic testing and blood biochemical tests were conducted. The autopsy was conducted and abnormalities in the thoracic and cavity organs were observed with the unaided eye. As a result of the experiments, the specific clinical symptoms and died animals were not found in all animals treated with experimental substances, Even in change in the body weight, blood tests, blood biochemical tests and autopsy findings, the toxic changes were not observed. As a result, it was confirmed that the extract of the invention showed no toxic changes up to 1 g/kg in rats, and that the minimum lethal dose (LD.sub.50) upon oral administration was 1 g/kg or more, suggesting a safe substance.

(23) When viewed on the basis of the above results, it has been determined that the above described Hovenia dulcis Thunb. extract, gallocatechin and methyl vanillate were effective in the prevention and/or treatment of a alopecia (see, WNT signals are required for the initiation of hair follicle development Andl T. et al. (2002) Dev Cell. 2: 643-653), a wound (see, Effect of Wnt signaling pathway on wound healing. Zhang D L, et al (2009) Biochem Biophys Res Commun. 378: 149-151) and a metabolic disease (Wnt Signaling Inhibits Adipogenesis through β-Catenin-dependent and -independent Mechanisms Jennifer A K, et al. (2005)) which are associated with the activity of Wnt/β-catenin signaling pathway.

(24) The present invention was described in detail with reference to exemplary embodiments so far, but it will be understood to those skilled in the art to which the present invention pertains that various modification and changes can be made and the elements thereof can be replaced with their equivalents, without departing from the scope of the invention. Moreover, it will be possible to conduct a number of variations without departing from the essential categories of the present invention, thus applying a particular situation or material to the teachings of the present invention. Accordingly, it should be construed that the invention is not limited to the particular embodiment disclosed as the best mode that is planned to implement the present invention and that the invention includes all embodiments falling within the scope of the appended claims.