WO2009128583A1

WO2009128583A1 - Novel compounds and fractions for enhancing ucp expression

Info

Publication number: WO2009128583A1
Application number: PCT/KR2008/003104
Authority: WO
Inventors: Tae-Sun Park; Ha-Won Kim
Original assignee: Industry Academic Cooperation Foundation, Yonsei University
Priority date: 2008-04-16
Filing date: 2008-06-03
Publication date: 2009-10-22

Abstract

The present invention relates to a novel compound, Juniperanol and compositions for preventing or treating hyperlipidemia, fatty liver, diabetes and obesity. In addition, the present invention relates to a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises a fraction of a Juniper tree as an active ingredient, wherein the fraction is prepared by treating a porous adsorbent with an adsorbed extract of the Juniper tree with a mixed solvent of (i) a nonpolar solvent and (ii) water, ethanol, methanol or ethylacetate. The Juniperanol compound or fractions of this invention leads to decreases in body fat weight, visceral fat weight and total cholesterol levels, and increases in expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3) as well as prevention of insulin resistance, thereby exhibiting prominent prevention or treatment efficacies against hyperlipidemia, fatty liver, diabetes and obesity.

Description

NOVEL COMPOUNDS AND FRACTIONS FOR ENHANCING UCP

EXPRESSION

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a novel cedrane-typed compound,

Juniperanol and compositions for preventing or treating hyperlipidemia, fatty liver, diabetes and obesity. In addition, the present invention relates to a novel fraction originated from natural sources for preventing or treating hyperlipidemia, fatty liver, diabetes and obesity.

DESCRIPTION OF THE RELATED ART

Changes in life styles and living environments result in a pathogenic increase of visceral fat obesity in modern people. Frequent occurrence of visceral obesity in turn leads to a rapid increase in development of metabolic syndromes which are accompanied by diabetes, hypertension, lipid metabolism disorders, insulin resistance and the like. These attendant diseases mutually interact to increase the pathogenic risk of individual conditions and are common diseases which are associated with a variety of metabolic changes such as senescence, stress conditions, compromised immune function and the like.

According to the 2005 National Health and Nutrition Survey, 32% of Korean adults above the age of 20 were found to suffer from obesity (35.2% of male adults and 28.3% of female adults). Recently, the incidence of childhood obesity is also soaring in Korea. According to the 2005 survey data, 11.3% of primary schoolchildren, 10.7% of junior high school students, and 16% of senior high school students were diagnosed with obesity (body mass index (BMI) > 25 kg/m²). Further, 17% of overweight (BMI > 23 kg/m²) or obese juveniles showed metabolic syndrome. Then, such an increase in the overweight and obese population contributes to a rise in the prevalence rate of chronic diseases. For example, according to the 2005 survey data on Korean people over the age of 30, the prevalence rates of hypertension (30.2% of male and 25.6% of female), diabetes (9.0% of male and 7.2% of female), and hypercholesterolemia (7.5% of male and 8.8 % of female) were significantly higher than in other countries.

Based on the survey data, obesity may result in an estimated socioeconomic loss of approximately 1 billion U.S. dollars in 2001. To this end, the Korean Health Plan 2010 was released by the Ministry of Health and Welfare as its public health policy. According to this white paper, main goals were established to accomplish an adult obesity rate of less than 20% and a juvenile obesity rate of less than 15%. As an implementation strategy to achieve these goals, an attempt was tried to find a precise definition and method of obesity measurement.

The best therapeutic effects on obesity can be achieved only with a combination of diet therapy, exercise therapy and behavior modification therapy. However, these therapeutic methods require plenty of time and effort in conjunction with difficulty in practice. For these reasons, anti-obesity drugs or diet products are widely used. However, orlistat, which is currently used as an anti-obesity drug, suffers from adverse side effects such as steatorrhea (fatty stools), enteric gas production, and flatus. Another anti-obesity drug, sibutramine, is also known to have adverse side effects such as headache, thirst, anorexia, insomnia, constipation and the like. Further, orlistat inhibits absorption of vitamin D and E, whereas administration of phentermine and sibutramine results in adverse side effects such as an increased heart rate/ palpitations, vertigo, and the like. Recently, values and demands for herbal or natural medicines are increasing in view of adverse side effects of synthetic drugs and limitations of Western medicine in treating chronic diseases. To cope with this trend, the present inventors have screened an anti-obesity substance from a variety of wild or volunteer plants and then gave attention to Juniperus chinensis.

Juniperus chinensis is an evergreen tall tree that belongs to the family Cupressaceae of the Order Coniferales of the Subclass Coniferophytae, which is widely distributed throughout Korea, Japan, China and Mongolia. In folk remedy fields, Juniperus chinensis has been used for the treatment of common cold, urinary tract infection (UTI), urticaria, dysentery, diarrhea, hemorrhage, and rheumatoid arthritis. The stem of Juniperus chinensis Js therapeutically effective for parasitic skin problems and rheumatism, whereas fruits of the plant are known to have therapeutic effects on convulsions, violent coughing and hepatitis. Further, Juniperus chinensis \s used in treatment of wounds and various dermatological diseases due to its antidotal and sterilizing effects and is also effective for alleviation of vomiting, diarrhea, and abdominal pain (Hong Won-Sik, Ingredients and Utilization of Medicinal Herbs, 1999).

Recently, there have been several reports that leave extracts of Juniperus chinensis have anticancer effects (AIi et al, J. Ethnopharmacol. 53(3): 165-169 (1996)), and that Juniperus chinensis extracts exhibit antifungal effects (Ohashi H., Asai T., and Kawai S., Holzforschung, 48:193-198 (1994)).

U.S. Pat. No. 7071195 proposes to treat obesity with amine or amide derivatives acting as ligands for neuropeptide Y Y5. U.S. Pat. No. 702722 discloses thiazolidinedione derivatives for treating diabetes, hyperlipidemia or obesity. U.S. Pat. No. 6987131 describes anti-hyperlipidemia compositions comprising phenylacetylglutamine, phenylacetylisoglutamine or phenylacetic acid. U.S. Pat. No. 6942967 suggests the apobec-1 protein as target molecules for treating arteriosclerosis, hyperlipidemia, obesity and diabetes.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

DETAILED DESCRIPTION OF THIS INVETNION

The present inventors have made intensive researches to develop novel compounds and fractions, which are derived from natural sources and purified with a high yield in large scale for commercialization, exhibit anti-obesity, -hyperlipidemia and/or -diabetes efficacies through enhanced UCP expressions. As a result, we have discovered novel compounds and fractions having efficacies described above by extracting and fractionating Juniper trees. Accordingly, it is an object of this invention to provide a Juniperanol compound.

It is another object of this invention to provide a compositioh for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.

It is still another object of this invention to provide a method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity by use of the Juniperanol compound.

It is further object of this invention to provide a use of the Juniperanol compound for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity. It is still further object of this invention to provide a method for preparing the

Juniperanol compound.

It is another object of this invention to provide a fraction of Juniper trees for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.

It is still another object of this invention to provide a method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity by use of a fraction of Juniper trees.

It is further object of this invention to provide a use of a fraction of Juniper trees for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

In one aspect of this invention, there is provided a compound represented by the following formula I:

The present inventors have made intensive researches to develop natural compounds having anti-obesity, -hyperlipidemia and/or -diabetes. As a result, we have discovered a novel compound having such effects represented by the formula I from Juniper trees. The compound of the present invention has a distinctly different stereostructure from cedrol. In this regard, the present compound is named as Juniperanol.

According to a preferred embodiment, the compound of the present invention has a crystal structure having unit cell dimensions of a = 6.0454±7 A, b = 19.291±2 A and c = 25.910±3 A (α=β=γ= 90°).

Preferably, the crystal of the compound has a PIiI₁I₁ space group and orthorhombic crystal system.

The Juniperanol compound may be purified from various natural sources, preferably Juniper trees.

The term used herein "Juniper tree" is used to intend to encompass Juniperus chinensis and Pterocarpus santalinus, preferably Juniperus chinensis.

The Juniperanol compound of this invention may be chemically synthesized.

In another aspect of this invention, there is provided a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises the Juniperanol compound represented by the following formula I as an active ingredient:

In still another aspect of this invention, there is provided a method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises administering to a subject a pharmaceutically effective amount of a composition comprising the Juniperanol compound as an active ingredient.

In further aspect of this invention, there is provided a use of the Juniperanol compound for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.

Since the present composition comprises the Juniperanol compound described above, the common descriptions between them are omitted in order to avoid undue redundancy leading to the complexity of this specification. The compound represented by the formula I exhibits prevention or treatment efficacies on hyperlipidemia, fatty liver and obesity. As demonstrated in Examples, Juniperanol contributes to decreases in body fat weight, visceral fat weight and total cholesterol levels, and increases in expression of UCP (uncoupling proteins) responsible for thermogenesis and expression and phosphorylation of AMPK (AMP- activated protein kinase) and ACC (acetyl-CoA carboxylase) for fatty acid oxidation, finally exhibiting prevention or treatment efficacies on hyperlipidemia, fatty liver and obesity.

According to a preferred embodiment, the composition increases expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3). As an active ingredient, Juniperanol elevates expression of mitochondrial proteins, such as UCP2 and UCP3 which promote thermogenesis, thereby exerting anti-obesity efficacies.

"UCP2 (uncoupling protein 2)" and "UCP3 (uncoupling protein 3)" are mitochondrial proteins and found mainly in adult adipocytes and skeletal muscles, respectively. These proteins lead to heat generation in mitochondria to increase cellular energy consumption, which act as excellent targets for anti-obesity drugs.

According to a preferred embodiment, Juniperanol as active ingredients is contained in extracts or fractions of Juniperus chinensis.

The extract of Juniperus chinensis may be obtained by extracting Juniperus chinensis (preferably the xylem of Juniperus chinensis) with various extraction solvents: (a) absolute or water-bearing lower alcohol containing 1-4 carbon atoms

(methanol, ethanol, propanol, butanol, /?-propanol, iso-propanol and n -butanonl etc.), (b) mixture of lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethylether, (i) butylacetate, and and (j) water.

The fraction of Juniperus chinensis containing Juniperanol refers to a further isolated or purified form obtained by additionally isolating or purifying the extract of Juniperus chinensis. For instance, it could be appreciated that active fractions obtained using a variety of additional purification methods such as an ultrafiltration with defined molecular weight cut-off value and various chromatography (designed for purification dependent upon size, charge, hydrophobicity and affinity) are included in the present fractions.

The extracts or fractions of this invention can be obtained in the form of powder by use of vacuum distillation, lyophilization or spray drying.

According to a preferred embodiment, the present composition may be provided in a pharmaceutical composition or a food composition.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier. In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methythydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

The pharmaceutical composition of this invention may be administered orally or parenterally, preferably orally.

A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment.

Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight).

According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer. The food composition may contain additional ingredients used in conventional food compositions as well as Juniperanol. The food composition may comprise conventional additives for preparing food compositions, e.g., proteins, carbohydrates, lipids, nutritive substances and flavors. Non-limiting examples of natural carbohydrates include, but not limited to, monosaccharide {e.g., glucose and fructose), disaccharide {e.g., maltose and sucrose), oligosaccharide, polysaccharide {e.g., dextrin and cyclodextrin) and sugar alcohol {e.g., xylitol, sorbitol and erythritol). Non-limiting examples of flavors include, but not limited to, natural flavors {e.g., thaumatin and extract of Stevla) and synthetic flavors {e.g., saccharin and aspartame). For example, where the food composition of this invention is provided as a drink, it may further comprise citric acid, liquefied fructose, sucrose, glucose, acetic acid, malic acid, fruit juices, Eucommia ulmoides extracts, jujube extracts and/or licorice extracts as well as Juniperanol as active ingredients.

The present compositions comprising Juniperanol as active ingredients lead to decreases in body fat weight, visceral fat weight and total cholesterol levels, and increases in expression of UCP (uncoupling proteins) responsible for thermogenesis and expression and phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase) for fatty acid oxidation, finally exhibiting prevention or treatment efficacies on hyperlipidemia, fatty liver and obesity.

The term used herein "hyperlipidemia" refers to diseases induced by elevated blood fat levels associated with abnormalities in fat metabolisms for triglycerides and cholesterol. More specifically, hyperlipidemia refers to conditions with elevated lipid levels such as triglycerides, LDL cholesterol, phospholipids and fatty acids and is often called as hypercholesterolemia. The term used herein "fatty liver" means conditions with excessive accumulation of fats in liver resulting from lesions of fat metabolisms. This condition is a pathological cause for various diseases such as angina pectoris, myocardial infarction, stroke, arteriosclerosis and pancreatitis. The term "diabetes" used herein refers to chronic diseases caused by relative or absolute insulin insufficiency leading to glucose-intolerance. The term "diabetes" is used to intend to encompass all types of diabetes, e.g., Type 1 diabetes, Type 2 diabetes and hereditary diabetes. Type 1 diabetes is insulin-dependent diabetes caused mainly by β-cell disruption. Type 2 diabetes is insulin-independent diabetes caused by insufficient insulin secretion after diet or insulin resistance.

According to a preferred embodiment, the present composition is used in prevention or treatment of Type 2 diabetes, more preferably Type 2 diabetes associated with insulin resistance.

In still further aspect of this invention, there is provided a method for preparing the Juniperanol compound, which comprises the steps of: (a) preparing an extract by contacting an extraction solvent to a Juniper tree; and (b) fractionating the extract to prepare the Juniperanol compound.

The present method will be described in more detail as follows: (a) Preparation of Extracts from Juniper Tree

The Juniper tree useful in the present invention may include any Juniper tree, preferably, Juniperus chinensis, Juniperus chinensis var. sargentii Henry, Juniperus chinensis .var. globosa Hornibr, Juniperus chinensis var. horizontalis Nakai, Juniperus chinensis var. procumbend, Aquilaria agallocha or Pterocarpus santalinus, more preferably Juniperus chinensis, and most preferably the xylem of Juniperus chinensis.

The extraction solvent may include any solvent known to one of skill in the art.

Preferably, the extraction solvent is nonpolar solvents, more preferably, nonpolar solvents selected from the group consisting of dichloromethane, hexane, chloroform and ethylether, most preferably dtchloromethane.

According to a preferred embodiment, the filtration and concentration of extracts is carried out after extraction.

(b) Isolation of Juniperanol by Fractionation of Extracts

The fractionation of Juniper tree extracts may be performed in accordance with conventional fractionation or purification procedures. Most preferably, the fractionation is performed by applying the Juniper tree extract to a silica gel column. According to a preferred embodiment, the fractionation is performed by applying the extract of the step (a) to a first silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity.

More preferably, the step (b) comprises the substeps of (b-1) applying the extract of the step (a) to a first silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity, and (b-2) applying the fraction having anti-obesity activity of the substep (b-1) to a second silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity. Preferably, the elution in the substep (b-1) is performed using dichloromethane and methanol.

Preferably, the elution in the substep (b-2) is performed using normal hexane and ethylacetate.

In another aspect of this invention, there is provided a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises a fraction of a Juniper tree as an active ingredient, wherein the fraction is prepared by treating a porous adsorbent with an adsorbed extract of the Juniper tree with a mixed solvent of (i) a nonpolar solvent and (ii) water, ethanol, methanol or ethylacetate.

In still another aspect of this invention, there is provided a method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises administering to a subject a pharmaceutically effective amount of the composition comprising the fraction of the present invention described above.

In further aspect of this invention, there is provided a use of the fraction of the present invention described above for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity. The Juniper tree useful in the present invention may include any juniper tree, preferably, Juniperus chinensis, Juniperus chinensis var. sargentii Henry, Juniperυs chinensis .var. globosa Hornibr, Juniperus chinensis var. horizontalis Nakai, Juniperus chinensis var. procumbend, Aquilaria agallocha or Pterocarpus santalinus, more preferably Juniperus chinensis, and most preferably the xylem of Juniperus chinensis. The extract of Juniper trees may be obtained using various extraction solvents: (a) absolute or water-bearing lower alcohol containing 1-4 carbon atoms (methanol, ethanol, propanol, butanol, /?-propanol, iso-propanol and n -butanonl etc.), (b) mixture of lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethylether, (i) butylacetate, and and G) water. Preferably, the extraction solvent is nonpolar solvents, more preferably, nonpolar solvents selected from the group consisting of dichloromethane, hexane, normal hexane, cyclohexane, chloroform and ethylether, most preferably dichloromethane.

The fractionation of Juniper tree extracts may be performed in accordance with conventional fractionation or purification procedures. According to the present invention, the fractionation is carried out by applying Juniper tree extracts to a porous adsorbent for adsorption and then eluting adsorbed components from adsorbents.

According to a preferred embodiment, the porous adsorbent useful in the present invention is silica, zeolite, activated alumina, activated carbon, agarose, dextran or cellulose, more preferably silica, agarose, dextran or cellulose, most preferably silica.

The solvent for extraction is a mixed solvent of (i) a nonpolar solvent and (ii) water, ethanol, methanol or ethylacetate. Preferably, the mixed solvent is a mixed solvent of (i) a nonpolar solvent and (ii) methanol. The nonpolar solvent used in the mixed solvent preferably includes dichloromethane, hexane, normal hexane, cyclohexane, chloroform and ethylether, more preferably dichloromethane. The most preferable combination of the mixed solvent is dichloromethane and methanol.

In the mixed solvent, the weight ratio of (i) the nonpolar solvent to (ii) water, ethanol, methanol or ethylacetate preferably ranges from 99:1 to 60:40, more preferably 99:1 - 70:30, still more preferably 99:1 - 80:20, most preferably 99:1 -

90:10.

According to a preferred embodiment, the fraction is carried out by applying

Juniper tree extracts to the adsorbent {e.g., silica gel) and then treating the adsorbent with the mixed solvent with gradient of increasing concentrations of polar solvents such as water, ethanol, methanol and ethylacetate. For instance, the fractionation may be performed by gradient of increasing concentrations of methanol

(starting from CH₂CI₂ with an increasing concentration of methanol, dichloromethane:methanol, 100:0 - 90:10). For this case, components with lower polarity are eluted preferentially. According to a preferred embodiment, the fraction of the present invention comprises the compound (Juniperanol) represented by the following formula I:

According to a preferred embodiment, the Juniperanol compound has a crystal structure having unit cell dimensions of a = 6.0454±7 A, b = 19.291±2 A and c = 25.910±3 A (α=β=γ= 90°). Preferably, the crystal of the Juniperanol compound has a P2₁2₁2₁ space group and orthorhombic crystal system.

According to a preferred embodiment, the fraction of the present invention comprises epicedrol.

More preferably, the fraction of the present invention further comprises at least one component selected from the group consisting of nootkatone, cedr-8-en- 15-ol, globulol, l-methyl-4-((S)-6-methylhept-5-en-2-yl)-benzene, trimethyldodecapentaene, l,l,3α,7-tetramethyl-lα,2,3,3α,4,5,6,7β-octahydro-lH- cyclopropa[a]naphthalene, 7,8-dirnothoxy-2,2-dimethyl-chromene and di(2- ethylhexyl) phthalate. The fraction of the present invention is very effective in prevention or treatment of hyperlipidemia, fatty liver and obesity. As demonstrated in Examples, the fraction of the present invention contributes to decreases in body fat weight, visceral fat weight and total cholesterol levels, and increases in expression of UCP (uncoupling proteins) responsible for thermogenesis, finally exhibiting prevention or treatment efficacies on hyperlipidemia, fatty liver and obesity.

According to a preferred embodiment, the fraction of the present invention increases expressions of the UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3) proteins. The present fraction elevates expressions of mitochondrial proteins, UCP2 and UCP3 to promote thermogenesis, thereby exerting anti-obesity efficacies.

A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment. Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight).

The present composition comprising the fraction from Juniper trees as active ingredients decreases body fat weight, visceral fat weight and total cholesterol levels, and increases expression of UCP (uncoupling proteins) responsible for thermogenesis, finally exhibiting prevention or treatment efficacies on hyperlipidemia, fatty liver, diabetes and obesity.

The features and advantages of the present invention will be summarized as follows: (i) the present invention provides a novel compound Juniperanol represented by the formula I.

(ii) the present composition comprising Juniperanol or fractions as active ingredients contributes to decreases in body fat weight, visceral fat weight and total cholesterol levels, and increases in expression of UCP (uncoupling proteins) responsible for thermogenesis and expression and phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase) for fatty acid oxidation, finally exhibiting prevention or treatment efficacies on hyperlipidemia, fatty liver, diabetes and obesity.

(iii) Interestingly, the present composition has prominent therapeutic efficacies by increasing expressions of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3) responsible for thermogenesis, which is distinctly different action mechanism from existing anti-obesity drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically represents a purification process for CH₂CI₂ extracts of Juniperus chinensis. Fig. 2a represents the stereostructure of Juniperanol revealed by the single crystal X-ray analysis. Fifteen carbon atoms are numbered in Cl - C15, and one oxygen atom is indicated with 01. The carbon atom in CIl orients upward in the plane.

Fig. 2b represents the stereostructure of (+)-Cedrol for comparison revealed by the single crystal X-ray analysis. The carbon atom in CIl orients downward in the plane.

Fig. 3a represents effects of Juniperus chinensis fraction FRlB on body fat levels of C. elegans. The adipocytes of C. elegans emitting red fluorescence in the dark room were imaged under fluorescence microscopes. Numbers denote fat levels. Fig. 3b represents effects of Juniperanol on body fat levels of C. elegans. The adipocytes of C. elegans emitting red fluorescence in the dark room were imaged under fluorescence microscopes. Numbers denote fat levels.

Fig. 4a illustrates changes in body weight of mice fed with either high-fat diet (HFD) or Juniperus chinensis fraction FRlB plus HFD (FRlB). **P < 0.01.

Fig. 4b illustrates changes in body weight of mice fed with either high-fat diet (HFD) or Juniperanol plus HFD (Juniperanol). *P < 0.01.

Fig. 5a illustrates levels of visceral fat in various visceral adipose tissues of mice fed with either high-fat diet (HFD) or Juniperus chinensis fraction FRlB plus HFD (FRlB). *P < 0.05, **P < 0.01.

Fig. 5b illustrates levels of visceral fat in various visceral adipose tissues of mice fed with either high-fat diet (HFD) or Juniperanol plus HFD (Juniperanol). *P < 0.05. Fig. 6a represents the Western blotting analysis results of the expression patterns of the UCP proteins in epididymal adipose tissues of mice fed with either high-fat diet (HFD) or Juniperus chinensis fraction FRlB plus HFD (FRlB). α-Tubulin was used as a housekeeping protein.

Fig. 6b represents the RT-PCR analysis results of the expression patterns of the UCP genes in epididymal adipose tissues of mice fed with either high-fat diet (HFD) or Juniperanol plus HFD (Juniperanol). GAPDH was used as a housekeeping gene.

Fig. 7 illustrates changes in expression levels and phosphorylation rates of AMPK and ACC proteins in epididymal adipose tissues of mice fed with either high-fat diet (HFD) or Juniperanol plus HFD (Juniperanol). ND corresponds to mice fed with normal diet.

The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

EXAMPLES EXAMPLE 1: Fractionation of Juniperus chinensis Extract and Isolation of

Juniperanol

Preparation of Juniperus chinensis Extract

Where water remains in Juniperus chinensis samples to be extracted, the residual water may result in decreased extraction efficiency of active ingredients as well as incorporation of various interfering substances during a purification process. For these reasons, 10 kg of the Juniperus chinensis xylem was ground into powder and dried to eliminate water in a drying oven at 6O⁰C for 24 hr, and then it was allowed to be followed by organic solvent extraction. Twenty five L of dichloromethane (CH₂CI₂) was added to 10 kg of the dried Juniperus chinensis, followed by extraction at 25⁰C for 7 days. The extraction process was repeated two times. The primary extract and the secondary extract were combined and filtered, and the resulting filtrate was concentrated under vacuum to afford 160 g of an extract concentrate. Finally, the concentrate was freeze-dried at -8O⁰C. As 160 g of a desired product was obtained from 10 kg of Juniperus chinensis, the CH₂CI₂ extraction yield was calculated to be 1.6%.

Fractionation and Isolation of Juniperanol

The CH₂CI₂ extracts of Juniperus chinensis obtained above were purified using a 2,200 mL column having a diameter of 6.5 cm and a height of 70 cm and packed with silica gel (230-400 mesh, Merck, Germany) for column chromatography. Fig. 1 illustrates a purification process for CH₂CI₂ extracts oft Juniperus chinensis.

The Juniperus chinensis extract was fractionated using a developing solvent (starting from CH₂CI₂ with an increasing concentration of methanol, dichloromethane:methanol, 99:1 - 95:5). Ingredients with lower polarity were eluted preferentially. According to the elution order, the extract was fractionated into three fractions FRO, FRlA and FRlB. The weight of fractions was 22 g for FRO, 94 g for FRlA, and 30 g for FRlB, respectively. Three fractions of FRO, FRlA and FRlB at varying concentrations were added to Caenorhabditis elegans (C. e/egans) and the body fat weight in C. elegans was examined under a fluorescence microscope in a dark room. As a result, the fraction FRlB exhibited the most significant decrease in the body fat level. Then, 19 g of FRlB was further fractionated through a silica gel column and divided into five fractions, FRlB-I (6.0 g), FR1B-2 (3 g), FR1B-3 (2 g), FR1B-4 (6.1 g), and FR1B-5 (trace). For the second silica gel chromatography, a developing solvent (starting from normal hexane with an increasing concentration of ethylacetate to 7:3 of normal hexane:ethylacetate). Each of five fractions was added at different specified concentrations to C. elegans, and the degree of decrease in the body fat was measured. As a result, the fraction FRlB-I exhibited the most significant reduction in the body fat weight. Of five fractions, only the FRlB-I was composed of a pure single compound and exhibited a significant decrease in the amount of body fat. Therefore, the chemical structure of FRlB-I was determined by instrumental analysis and then designated as "Juniperanol".

EXAMPLE 2: Chemical Characterizations and Structural Analysis of

Juniperanol

Crystallization of Juniperanol

A crystallization of Juniperanol was performed using methanol. Methanol was added to the concentrated Juniperanol. The mixture was heated and allowed to stand at room temperature for 1 to 2 days to gradually induce crystallization of the compound. The title compound was obtained as colorless needle-like crystals, when observation was made under a light microscope. Juniperanol was slowly heated to determine a melting point which was in a range of 63 to 67⁰C.

Solubility of Juniperanol

Various solvents were added to small amounts of Juniperanol crystals and solubility of the compound was observed under a light microscope. Juniperanol was slightly dissolved in distilled water or methanol, which are very highly polar. However, Juniperanol was completely dissolved in ethanol, ethyl acetate, chloroform, n-hexane, or benzene, which has relatively low polarity. Table 1 shows the solubility of Juniperanol in various organic solvents. TABLE 1

Elemental analysis of Juniperanol

2.0 mg of purified Juniperanol was subjected to elemental analysis at 1,100⁰C. 2,5-bis(5-tert-butyl-benzoxazolyl)thiophene (BBOT, C₂₆H_2SN₂O₂S) was used as a standard. According to the elemental analysis results, Juniperanol was revealed to consist of three elements, carbon (C), hydrogen (H) and oxygen (O), as shown in Table 2. An elemental ratio of C: H: O = 78.27%: 10.73%:7.48%. TABLE 2

Mass analysis of Juniperanol

The mass analysis of purified Juniperanol showed that it had a molecular weight of 222. Juniperanol was identified to have the chemical formula of Ci₅H_2SO. Physical characteristics of Juniperanol are summarized in Table 3. TABLE 3

Structural analysis of Juniperanol

The ¹H-NMR analysis of purified Juniperanol was carried out: ¹H-NMR(CDCI₃) δ 0.85(d, J=7, 3H), 1.00(s, 3H), 1.26(s, 3H), 1.27-1.29(d, J=IO, IH), 1.32(s, 3H), 1.35-1.43 (m, 4H), 1.51-1.58(m, 3H), 1.61-1.71(m, 3H), 1.78-1.89(m, 3H).

In addition, the ¹³C-NMR spectra for Juniperanol were obtained: ¹³C-NMR (150 MHz, in CDCI₃): δ(ppm) 25.37(Cl), 37.02(C2), 41.48(03), 54.11(C4), 56.54(05), 43.41(06), 61.06(C7), 75.11(08), 35.37(C9), 31.61(ClO), 41.99(CIl), 30.20(C12), 27.65(C13), 28.93(C14), 15.59(015).

For elucidating an accurate three dimensional structure of Juniperanol, a single crystal X-ray diffractometer was carried out. The analytic results are summarized in Table 4.

TABLE 4. Conditions for single crystal X-ray analysis of Juniperanol and results

The three dimensional stereostructure of Juntperanot was revealed by NMR data and single crystal X-ray data described above. Interestingly, we found that Juniperanol isolated is a novel compound unpublished up to now and is represented by the formula I. The stereostructure of Juniperanol revealed by single crystal X-ray analysis is illustrated in Fig. 2a.

For comparison, (+)-cedrol was X-ray analyzed to elucidate its stereostructure.

(+)-cedrol used was commercially purchased from Fluka (Cat. No. 22135) and has the following characteristics: Molecular formula, Ci₅H₂₅O (molecular weight, 222.37); purity, 99.7%; optical activity, [α]_20/D = +10.5±l; b.p., 273⁰C; m.p., 82-86⁰C (theoretical, 55-59⁰C).

As represented in Fig. 2b, it was concluded that (+)-cedrol has a mirror image of Juniperanol.

EXAMPLE 3: GC-MS Analysis and X-ray Crystal Analysis GC-MS analysis of Juniperanol

A GC-MS (Gas liquid chromatography-mass spectrometer) analysis was performed using Juniperanol isolated in Examples described above. The instrumental conditions for GC-MS analysis were: GC analysis instrument,

Agilent HP6890 GC; mass detector, Agilent 5973N; GC column, Capillary column DB- 5MS 1127 (Agilent Tech.), 30 m x 0.32 mm x 0.25 urn; mobile gas, helium gas; oven initial temperature and maximum temperature, 100⁰C and 32O⁰C; oven temperature increasing rate, 4°C/min; Front inlet initial temperature and pressure: 28O⁰C, 6.55 psi; Front inlet velocity, 9.9 ml/min.

Juniperanol contained in the FRB-I fraction isolated from Juniperus chinensis was detected as a single peak at a retention time of 18.75 min and its purity was analyzed to be 100.0%.

GC-MS analysis of extracts front Juniperus chinensis

The GC-MS analysis was performed for the CH₂CI₂ extract from Juniperus chinensis. All forty four peaks were observed. Each peak was mass-analyzed for its identification. The main component in the extract was detected at a retention time of 18.89 min, which showed the mass fragmentation pattern identical to that of Juniperanol. The content of Juniperanol in the CH₂CI₂ extract was measured to be 49.8%. The components contained in the CH₂CI₂ extract were revealed: Juniperanol (49.8%), (-)-α-cedrene (5.9%), nootkatone (2.9%), α-guaiene (1.5%), cedr-8-en- 15-ol (1.0%) and α-cedrene oxide (0.6%). Table 5 represents chemicals and their contents corresponding to twenty-four peaks in GC-MS analysis for the CH₂CI₂ extract.

TABLE 5a. GC-MS analysis results of CH₂CI₂ extract from Juniperus chinensis

TABLE 5b.

GC-MS analysis of FRIB fraction from Juniperus chinensis

The GC-MS analysis was performed for the FRlB fraction from Juniperus chinensis. All fourteen peaks were observed. The main component was detected at a retention time of 18.84 min, which showed the mass fragmentation pattern identical to that of Juniperanol. The content of Juniperanol in the FRlB fraction was measured to be 56.8%. The components contained in the FRlB fraction were revealed: Juniperanol (56.8%), epicedrol (24.1%), nootkatone (1.8%), cedr-8-en- 15-ol (1.1%) and globulol (0.8%). Table 6 represents compounds and their contents corresponding to eleven peaks in GC-MS analysis for the FRlB fraction. TABLE 6. GC-MS analysis results of FRlB fraction from Juniperus chinensis

The main component contained at about 57% content in the FRlB fraction from Juniperus chinensis was isolated and then subjected to GC-MS analysis, and as a result it was elucidated to be Juniperanol. In addition to this, the main component was further analyzed by X-ray diffraction to elucidate its stereostructure, and consequently we observed the same stereostructure as represented in Fig. 2a, verifying that the main component in the FRlB fraction is Juniperanol.

EXAMPLE 4: Evaluation on Anti-Obesity Effects of Juniperus chinensis Fractions in C. elegans

Adult (L4 stage) C. elegans immediately prior to egg-laying were used in this Example. Therefore, the eggs were hatched after sample treatment and most of adult C. elegans were killed on the 3rd post-treatment day. A fat content was examined in C. elegans which had newly hatched and grown.

Anti-obesity effects of FRO, FRlA and FRlB fractions

Using a dissecting microscope (x 50), C. elegans at the 4th stage (L4, the imaginal stage) was transferred to 3 ml_ of a cholesterol-supplemented S-medium (containing NaCI, K₂HPO₄, KH₂PO₄, cholesterol, citrate, trace metals, CaCI₂ and MgSO₄), and 100 μl of E coll OP50 (OD₆₀₀ = 0.2) liquid-cultured overnight before 1 day of experiment was added thereto. The Juniperus chinensis fraction samples FRO, FRlA and FRlB at various concentrations (0, 10, 50, 100, and 250 μg/mL) were added to the liquid media containing C. elegans (n=3 for each group), followed by rotary culture in a dark room at 16⁰C and 100 rpm. The water-insoluble sample was dissolved in DMSO and added to the culture media. The final concentration of DMSO was adjusted below 1%. 1% DMSO exhibited no effects on adipogenesis and growth of C. elegans. 24 hours after the sample was added to C. elegans, 30 μl of a Nile red dye at a concentration of 10 μg/mL was added (final concentration of Nile red was adjusted to 100 ng/mL) to the media, followed by continuous rotary culture for 2 days.

In order to arrest the locomotory movement of C. elegan, 20 μl of 0.3% NaN₃ was dropped on a slide glass to which 100 μl of C. elegans cultured in the presence of Nile red for 2 days was added. Under a fluorescence microscope, adipocytes of C. elegans emitting red fluorescence in a dark room were micrographed and compared with the control group to thereby determine the amount of fat. A low fat content was indicated by +1, whereas a high fat content was assigned with +4.

As represented in experimental results, Table 7 and Fig. 3a, the fraction FRlB exhibited the most significant fat-reducing effects. The naive control group acquired a score of +4 in the fat content, whereas the FRIB-treated group exhibited significant reduction of the fat content at concentrations of 50-250 μg/mL. TABLE 7

Anti-obesity effects of Juniperanol

The fraction FRlB was separated by a column and divided into FRlB-I (containing a single compound, Juniperanol), FR1B-2, FR1B-3 and FR1B-4. C. elegans was treated with these subfractions at different concentrations of 0, 0.1, 1, 10, and 100 μg/mL. Adipocytes of C. elegans were stained with Nile red and then examined under a fluorescence microscope in a dark room. As indicated in Table 8 and Fig. 3b, the fraction FRlB-I consisting of the single compound Juniperanol exhibited the most pronounced body fat lowering effects at a concentration of 1 μ/mL or higher. TABLE 8

EXAMPLE 5: Anti-Obesity Effects of FRlB Fraction in Mice

Preparation of experimental diet and maintenance of experimental animals

The obesity-inducing diet used in this example was high-fat diet (HFD: 40% fat calorie, 17 g lard + 3% corn oil/100 g diet) developed by the present inventors, whereas the FRlB diet has the same composition as HFD, except that the FRlB fraction is included at a content of 0.5%. The composition of experimental diets is set forth in Table 9. TABLE 9

6-week-old male C57BL/6J mice were allowed to acclimate to a new environment of the breeding room for one week prior to experiments. Animals were fed a solid feed as a laboratory animal chow. According to a randomized block design, the thus-acclimated mice were randomly divided into a control group with administration of high-fat diet (HFD) and a FRlB group, followed by maintaining for a total of 6 weeks. Animals were fed daily solid feed pellets and water ad libitum, between 10 to 11 a.m. The dietary intake was daily measured, whereas the body weight was checked once every three days. In order to avoid a sudden change in the body weight of animals due to ingestion of the feed, a feed trough was removed and the body weight of mice was measured 2 hours later. A food efficiency ratio (FER) was calculated as a ratio of total weight gain/total food intake, the total weight gain being cumulative during the entire experimental period from the start day of feeding the experimental diet to the sacrifice day of animals. After the experimental animals were fasted for 12 hours or more, the blood, liver and various visceral adipose tissues (from epididymal, perirenal, mesenteric and retroperitoneal fat depots) were collected under diethylether anesthesia, and the harvested liver and tissues were washed with 0.1 M PBS (pH 7.4), followed by weighing. The blood collected from the abdominal aorta of animals was centrifuged at 1000 x g for 15 min to separate the plasma.

Evaluation of changes in body weight and visceral fat level When changes in the body weight of animals were investigated after 6-week feeding of the experimental diet, the FRlB fed group, as shown in Fig. 4a, exhibited a 11% loss in the final body weight (P < 0.05), as compared to the high-fat diet control group.

When the amount of visceral fat was examined after 6-week feeding of the experimental diet, the FRlB fed group, as shown in Fig. 5a, exhibited a 37% decrease in the total visceral fat weight (P < 0.05), as compared to the high-fat diet control group, specifically with a 37% decrease in epididymal fat (P < 0.05), a 55% decrease in retroperitoneal fat (P < 0.05), a 33% decrease in perirenal fat (P < 0.05), and a 10% decrease in mesenteric fat, thus verifying that administration of FRlB fraction resulted in decreases of the body weight and visceral fat.

Analysis of expression of the UCP protein

The protein samples were prepared from epididymal fat of mice and the expression patterns of UCP (uncoupling protein) 2 and UCP3 proteins, which play a role in the control of thermogenesis in body, were examined by the Western blotting method.

As shown in the Western blotting results of Fig. 6a, the expression levels of both UCP2 and UCP3 proteins in epididymal fat are significantly elevated in the FRlB fed group compared with the HFD group. These results demonstrate that the FRlB fraction increases expression of mitochondrial heat-generating proteins, UCP2 and UCP3 to promote thermogenesis, thereby decreasing the body weight.

EXAMPLE 6: Prevention and Treatment Efficacies of Fractions of Juniperus chinensis Extract against Hyperlipidemia and Fatty Liver

Plasma total cholesterol, triglyceride, HDL total cholesterol, FFA (free fatty acid) and glucose levels were measured twice using commercial kits (Bio Clinical System), and LDL+VLDL cholesterol level in blood was calculated by subtracting HDL cholesterol level from total cholesterol level. Lipid components were extracted from liver tissues by the Folch et al. method. 0.25 g of liver tissues in 1 mL of distilled water were homogenized using a polytron homogenizer (IKA-WERKE GmbH & Co., Ultra-Turrax, Staufen, Germany). 5 mL of chloroform: methanol solution (2:1, v/v) was added to the homogenate, well agitated and centrifuged for 10 min at 1000 x g to separate a lower layer solution. The upper layer solution was again mixed with 2 mL of chloroform: methanol solution (2:1, v/v) and underwent the same procedures to completely separate lipid components in liver. The lower layer solution was mixed with 3 mL of chloroform: methanol: 0.05% CaCI₂ (3:48:47, v/v/v) for 1 min and centrifuged at 1000 x g for 10 min. The lower layer solution so obtained was dried using nitrogen gas and dissolved in 1 mL methanol for lipid analysis. For measurement of triglyceride and cholesterol levels in lipid extracts of liver tissues, the commercial kits (Bio Clinical system) as used for plasma analysis were used. TABLE 10. Biochemical indices in plasma and liver of mice fed with experimental diets

aIRI (insulin resistance index) = 10^" pmol insulin x mmol glucose x L^"

'Significantly different from the value for ND diet group by Student's t-test at P < 0.05.

Lipid levels in plasma and liver tissues were examined in mice fed with experimental diets for 6 weeks, as shown in Table 10. It was clearly observed that all of total cholesterol, triglyceride and LDL+VLDL cholesterol levels in plasma were significantly reduced in FRIB-fed mice compared with HFD control mice. Similarly, hepatic cholesterol and triglyceride levels were also shown to be significantly reduced in FRIB-fed mice compared with HFD control mice. These results lead us to reason that the FRlB fraction from Juniperus chinensls could effectively alleviate hyperlipidemia and fatty liver symptoms associated with obesity induced by HFD feeding.

EXAMPLE 7: Prevention and Treatment Efficacies of Fractions of Juniperus chinensis Extract against Type 2 Diabetes or Obesity-induced Insulin Resistance

Insulin level in blood samples was measured by ELISA using Mouse Insulin kit (Shibayaki, Japan). It was well known to one of skill in the art that diet-induced obesity animal models or obese humans exhibit Type 2 diabetes showing simultaneously elevated blood insulin and glucose levels in fasted state. A term "metaflammation" was recently coined to indicate inflammations induced by excess supply of nutrients or metabolites and obesity was indicated as chronic and low-level inflammation, highlighting the correlation between obesity and immune system. In particular, various obesity complications {e.g., Type 2 diabetes, insulin resistance, arteriosclerosis, cancers and asthma) have been newly suggested to occur in interactions with the immune system during obesity development. For example, the TLR4 (toll-like receptor 4) molecule responsible for innate immune responses plays a pivotal role in inflammation and insulin resistance pathway in response to dietary fats (particularly, saturated fatty acids) as ligands, as well as in appetite control in the central nervous system. In the Example, mice were fed with HFD or FRlB diet for 6 weeks. It was elucidated that both blood glucose and blood insulin levels in a fast state were considerably decreased, exhibiting significantly decreased IRI (insulin resistance index), in mice fed FRlB diet than in the control mice fed HFD as described in Table 10. In these contexts, it could be concluded that the FRlB faction of the present invention is effective in treating Type 2 diabetes and insulin resistance and also is anticipated to be effective in treating metabolic inflammation closely associated with obesity.

EXAMPLE 8: Anti-Obesity Effects of Juniperanol in Mice

Preparation of experimental diet and maintenance of experimental animals The obesity-inducing diet used in this example was high-fat diet (HFD: 40% fat calorie, 17 g lard + 3% corn oil/100 g diet) developed by the present inventors, whereas the Juniperanol diet has the same composition as HFD, except that Juniperanol is included at a content of 0.2%. The composition of experimental diets is set forth in Table 9. 6-week-old male C57BL/6J mice were allowed to acclimate to a new environment of the breeding room for one week prior to experiments. Animals were fed a solid feed as a laboratory animal chow. According to a randomized block design, the thus-acclimated mice were randomly divided into a control group with administration of high-fat diet (HFD) and a Juniperanol group, followed by maintaining for a total of 6 weeks. Animals were fed daily solid feed pellets and water ad libitum, between 10 to 11 a.m. The dietary intake was daily measured, whereas the body weight was checked once every three days. In order to avoid a sudden change in the body weight of animals due to ingestion of the feed, a feed trough was removed and the body weight of mice was measured 2 hours later. A food efficiency ratio was calculated as a ratio of total weight gain/total food intake, the total weight gain being cumulative during the entire experimental period from the start day of feeding the experimental diet to the sacrifice day of animals. After the experimental animals were fasted for 12 hours or more, the blood, liver and various visceral adipose tissues (from epididymal, perirenal, mesenteric and retroperitoneal fat depots) were collected under diethylether anesthesia, and the harvested liver and tissues were washed with 0.1 M PBS (pH 7.4), followed by weighing. The blood collected from the abdominal aorta of animals was centrifuged at 1000 x g for 15 min to separate the plasma.

Evaluation of changes in body weight and visceral fat level

When changes in the body weight of animals were investigated after 6-week feeding of the experimental diet, the Juniperanol fed group, as shown in Rg. 4b, exhibited a 10% loss in the final body weight (P < 0.05), as compared to the high- fat diet control group.

When the amount of visceral fat was examined after 6-week feeding of the experimental diet, the Juniperanol fed group, as shown in Fig. 5b, exhibited a 37% decrease in the total visceral fat weight (P < 0.05), as compared to the high-fat diet control group, specifically with a 37% decrease in epididymal fat (P < 0.05), a 55% decrease in retroperitoneal fat (P < 0.05), a 50% decrease in perirenal fat (P < 0.05), and a 10% decrease in mesenteric fat, thus verifying that administration of FRlB fraction resulted in decreases of the body weight and visceral fat. Analysis of expression of the UCP gene mRNA was extracted from epididymal adipose tissues of experimental diet-fed mice, and the expression level of UCP (uncoupling protein) responsible for regulation of thermogenesis was measured by RT-PCR analysis. The primer sequences and PCR conditions used are described in Table 11. TABLE 11

According to the results of RT-PCR analysis as shown in Fig. 6b, the expression of UCP2 and UCP3 genes implicated in thermogenesis significantly increased in visceral adipose tissues of the Juniperanol group, as compared to the HFD-fed group. Therefore, it could be understood that Juniperanol increases the expression of mitochondrial thermogenic proteins (UCP2 and UCP3) to thereby facilitate thermogenesis, thus resulting in body weight loss.

Analysis of influences on expression and phosphorylation of AMPK and ACC proteins implicated in fatty acid oxidation

Proteins were extracted from epididymal adipose tissues of laboratory diet-fed mice, and the amounts and phosphorylation of the AMPK (AMP-activated protein kinase) and ACC (Acetyl-CoA Carboxylase) proteins responsible for fatty acid β- oxidation were measured by the Western blot analysis.

As shown in Fig. 7, the p-AMPK/AMPK ratio and p-ACC/ACC ratio were significantly increased in epididymal adipose tissues of the Juniperanol group, as compared to the high-fat diet control group. Furthermore, the quantities of AMPK and ACC also were analyzed to be increased in the Juniperanol group. Therefore, it could be appreciated that Juniperanol not only promotes the expression of AMPK and ACC involved in fatty acid oxidation of visceral adipose tissues, but also activates AMPK and ACC proteins to thereby facilitate the fatty acid oxidation.

EXAMPLE 9: Prevention and Treatment Efficacies of Juniperanol against Hyperlipidemia and Fatty Liver

Plasma total cholesterol, triglyceride, HDL total cholesterol, FFA (free fatty acid) and glucose levels were measured twice using commercial kits (Bio Clinical

System), and LDL+VLDL cholesterol level in blood was calculated by subtracting HDL cholesterol level from total cholesterol level. Lipid components were extracted from liver tissues by the Folch et al. method. 0.25 g of liver tissues in 1 mL of distilled water were homogenized using a polytron homogenizer (IKA-WERKE GmbH & Co., Ultra-Turrax, Staufen, Germany). 5 mL of chloroform: methanol solution (2:1, v/v) was added to the homogenate, well agitated and centrifuged for 10 min at 1000 x g to separate a lower layer solution. The upper layer solution was again mixed with 2 mL of chloroform:methanol solution (2:1, v/v) and underwent the same procedures to completely separate lipid components in liver. The lower layer solution was mixed with 3 mL of chloroform:methanol:0.05% CaCI₂ (3:48:47, v/v/v) for 1 min and centrifuged at 1000 x g for 10 min. The lower layer solution so obtained was dried using nitrogen gas and dissolved in 1 mL methanol for lipid analysis. For measurement of triglyceride and cholesterol levels in lipid extracts of liver tissues, the commercial kits (Bio Clinical system) as used for plasma analysis were used. TABLE 12. Biochemical indices in plasma and liver of mice fed with experimental diets

aIRI (insulin resistance index) = 10^" pmol insulin x mmol glucose x L^"

'Significantly different from the value for ND diet group by Student's t-test at P< 0.05.

Lipid levels in plasma and liver tissues were examined in mice fed with experimental diets for 6 weeks, as shown in Table 12. It was clearly observed that ail of total cholesterol, triglyceride and LDL+VLDL cholesterol levels in plasma were significantly reduced in Juniperanol-fed mice compared with HFD control mice. Similarly, hepatic cholesterol and triglyceride levels were also shown to be significantly reduced in Juniperanol-fed mice compared with HFD control mice. These results lead us to reason that Juniperanol isolated from Juniperus chinensis could effectively alleviate hyperlipidemia and fatty liver symptoms associated with obesity induced by HFD feeding.

EXAMPLE 10: Prevention and Treatment Efficacies of Juniperanol against Type 2 Diabetes or Obesity-induced Insulin Resistance Insulin level in blood samples was measured by ELISA using Mouse Insulin kit

(Shibayaki, Japan). In the Example, mice were fed with HFD or Juniperanol diet for 6 weeks. It was elucidated that both blood glucose and blood insulin levels in a fast state were considerably decreased, exhibiting significantly decreased IRI (insulin resistance index) in mice fed Juniperanol diet than in mice fed HFD as described in

Table 12. In these contexts, it could be concluded that Juniperanol of the present invention is effective in treating Type 2 diabetes and insulin resistance and also is anticipated to be effective in treating metabolic inflammation closely associated with obesity.

Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

Claims

What is claimed is:

1. A compound represented by the following formula I:

2. The compound according to claim 1, wherein the compound has a crystal structure having unit cell dimensions of a = 6.0454 ± 7 A, b = 19.291 ± 2 A and c = 25.910 ± 3 A (α=β=γ= 90°).

3. The compound according to claim 2, wherein the crystal of the compound has a P2_i2₁2₁ space group and orthorhombic crystal system.

4. A composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises the compound of any one of claims 1-3 as an active ingredient.

5. The composition according to claim 4, wherein the composition is a pharmaceutical composition.

6. The composition according to claim 4, wherein the composition is a food composition.

7. The composition according to claim 4, wherein the composition increases expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3).

8. The composition according to claim 4, wherein the active ingredient is contained in extracts or fractions of Juniperus chinensis.

9. A method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises administering to a subject a pharmaceutically effective amount of a composition comprising the compound of any one of claims 1-3 as an active ingredient.

10. The method according to claim 9, wherein the composition increases expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3).

11. The method according to claim 9, wherein the active ingredient is contained in extracts or fractions of Juniperus chinensis.

12. Use of the compound of any one of claims 1-3 for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.

13. The use according to claim 12, wherein the composition increases expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3).

14. The use according to claim 12, wherein the compound is contained in extracts or fractions of Juniperus chinensis.

15. A method for preparing the compound of any one of claims 1-3, which comprises the steps of:

(a) preparing an extract by contacting an extraction solvent to a Juniper tree; and

(b) fractionating the extract to prepare the compound of any one of claims 1-3.

16. The method according to claim 15, wherein the Juniper tree is Juniperus chinensis, Juniperus chinensis var. sargentii Henry, Juniperus chinensis .var. globosa Hornibr, Juniperus chinensis var. horizontalis Nakai, Juniperus chinensis var. procumbend, Aquilaria agallocha or Pterocarpus santalinus.

17. The method according to claim 16, wherein the juniper tree is Juniperus chinensis.

18. The method according to claim 15, wherein the extraction solvent is a nonpolar solvent selected from the group consisting of dichloromethane, hexane, chloroform and ethylether.

19. The method according to claim 18, wherein the extraction solvent is dichloromethane.

20. The method according to claim 18, wherein the step (b) is performed using a silica gel column.

21. The method according to claim 20, wherein the step (b) comprises applying the extract of the step (a) to a first silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity.

22. The method according to claim 20, wherein the step (b) comprises the substeps of (b-1) applying the extract of the step (a) to a first silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity, and (b-2) applying the fraction having anti-obesity activity of the substep (b-1) to a second silica gel column and performing elution under conditions to elute preferentially components with low polarity to obtain a fraction having anti-obesity activity.

23. The method according to claim 22, wherein the elution in the substep (b-1) is performed using dichloromethane and methanol.

24. The method according to claim 22, wherein the elution in the substep (b-2) is performed using normal hexane and ethylacetate.

25. A composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises a fraction of a juniper tree as an active ingredient, wherein the fraction is prepared by treating a porous adsorbent with an adsorbed extract of the Juniper tree with a mixed solvent of (i) a nonpolar solvent and (ii) water, ethanol, methanol or ethylacetate.

26. The composition according to claim 25, wherein the Juniper tree is Juniperus chinensis, Juniperus chinensis var. sargenti/^' Henry, Juniperus chinensis .var. globosa Hornibr, Juniperus chinensis var. horizontalis Nakai, Juniperus chinensis var. procumbend, Aquilaria agallocha or Pterocarpus santalinus.

27. The composition according to claim 26, wherein the Juniper tree is Juniperus chinensis.

28. The composition according to claim 25, wherein the extract of the Juniper tree is obtained from the xylem of the Juniper tree.

29. The composition according to claim 25, wherein the extract of the Juniper tree is obtained using as an extraction solvent a nonpolar solvent selected from the group consisting of dichloromethane, hexane, normal hexane, cyclohexane, chloroform and ethylether.

30. The composition according to claim 29, wherein the extraction solvent is dichloromethane.

31. The composition according to claim 25, wherein the porous adsorbent is silica, zeolite, activated alumina, activated carbon, agarose, dextran or cellulose.

32. The composition according to claim 31, wherein the porous adsorbent is silica.

33. The composition according to claim 25, wherein the mixed solvent is a mixed solvent of (i) a nonpolar solvent and (ii) methanol.

34. The composition according to claim 25, wherein the nonpolar solvent is dichloromethane, hexane, normal hexane, cyclohexane, chloroform and ethylether.

35. The composition according to claim 34, wherein the nonpolar solvent is dichloromethane.

36. The composition according to claim 25, wherein the mixed solvent has 99:1- 60:40 of a weight ratio of (i) the nonpolar solvent to (ii) water, ethanol, methanol or ethylacetate.

37. The composition according to claim 36, wherein the mixed solvent has 99:1- 90:10 of a weight ratio of (i) the nonpolar solvent to (ii) water, ethanol, methanol or ethylacetate.

38. The composition according to claim 25, wherein the fraction comprises the compound represented by the following formula I:

39. The composition according to claim 25 or 38, wherein the fraction comprises epicedrol.

40. The composition according to claim 25 or 38, wherein the fraction further comprises at least one component selected from the group consisting of nootkatone, cedr-8-en-15-ol, globulol, l~methyl-4-((S)-6-methylhept-5-en-2-yl)-benzene, trimethyldodecapentaene, l,l,3α,7-tetramethyl-lα,2,3,3α,4,5,6,7β-octahydro-lH- cyclopropa[a]naphthalene, 7,8-dimothoxy-2,2-dimethyl-chromene and di(2- ethylhexyl) phthalate.

41. The composition according to claim 25, wherein the composition is a pharmaceutical composition.

42. The composition according to claim 25, wherein the composition is a food composition.

43. The composition according to claim 25, wherein the composition increases expression of UCP2 (uncoupling protein 2) or UCP3 (uncoupling protein 3).

44. A method for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity, which comprises administering to a subject a pharmaceutically effective amount of the composition of any one of claims 25-43.

45. Use of the fraction of claim 25 for manufacturing a composition for preventing or treating hyperlipidemia, fatty liver, diabetes or obesity.