WO2014168429A1

WO2014168429A1 - Pharmaceutical composition and health food for preventing or improving lipid related metabolic disease or the loss of weight and body fat, which comprise the black tea aqueous ethanol enfleurage extract

Info

Publication number: WO2014168429A1
Application number: PCT/KR2014/003114
Authority: WO
Inventors: Mi Won Son; Tae Ho Lee; Nam Joon Baek; Sang Jin Lee; Hyang Hwa Lee; Hyun Sung Park
Original assignee: Dong-A ST Co Ltd
Current assignee: Dong-A ST Co Ltd
Priority date: 2013-04-12
Filing date: 2014-04-10
Publication date: 2014-10-16
Anticipated expiration: 2015-10-12
Also published as: KR20140123626A; KR101501233B1

Abstract

The present invention relates to a composition comprising black tea extracts. More particularly, the herb extract extracted by enfleurage with aqueous ethanol solution of the present invention contains higher concentration of gallic acid which is an active ingredient and an index ingredient of black tea than the herb extract prepared by the conventional hot-water extraction and ethanol extraction, and has excellent effect of reducing body weight and body fat, improving fatty liver, regulating blood sugar, and reducing blood cholesterol in the high fat diet-induced obese mouse model, so that the extract of the present invention can be effectively used as an active ingredient of a pharmaceutical composition and health food for preventing and treating obesity, metabolic disease, diabetes, and hyperlipidemia.

Description

Pharmaceutical composition and health food for preventing or improving lipid related metabolic disease or the loss of weight and body fat/ which comprise the black tea aqueous ethanol enfleurage extract

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a composition comprising the black tea aqueous ethanol enfleurage extract, more precisely a composition for anti-obesity comprising the black tea aqueous ethanol enfleurage extract or a composition for preventing, improving, or treating lipid related metabolic disease comprising the same .

2. Description of the Related Art

Obesity is a kind of biological phenomena, which is resulted from complicated interaction among genetic factors, metabolic factors, environmental factors, and behavioral factors. In general, obesity is recognized as over-weight, but in the field of medicine, when BMI (body mass index) is at least 30 ( at least 30% heavier than standard weight) or BMI is at , least 27 and particularly circulatory diseases such as diabetes, hypertension, and hyperlipidemia are suspected in relation to the over-weight, it is considered as obesity. Obesity is not only a critical factor causing various adult diseases including insulin resistance, diabetes, hypertension, cancer, gallbladder disease, hyperlipidemia, and atherosclerosis but also makes immune system weak in people or animals, which becomes an important social issue drawing our attention. Up to date, obesity has been believed to be mostly attributed to genetic factors (at least 70%) and then other environmental factors such as high fat diet or lack of exercise. However recently, it seems that imbalance in energy precisely between energy intake and energy consumption rises as a major cause. Over the past tens of years, there have been no big changes in genetic factors. Nevertheless, the occurrence rate of obesity has been rapidly increased, suggesting that genetic factors are not the only major reasons. So, complicated factors including genetic factors and various environmental factors breaking energy balance altogether are considered all as major causes. It is still important and required to disclose accurate reason of obesity. Obesity treatment agents known so far are largely classified as appetite suppressants, energy consumption accelerators, and fat absorption inhibitors, which are exemplified by Xenical (Xenical, Roche, Switzerland), Reductil (Reductil, Abbott, USA), and Exolise (Exolise, Arkopharma, France) , etc. Most obesity treatment drugs are appetite suppressants to inhibit appetite by regulating neurotransmitters related to hypothalamus. However, the treatment agents of today have problems of causing side effects including heart disease, circulatory disease, and neurological disease, and inconsistent drug effect. The products under development of today still have a problem of side effects and the treatment effect thereof is still not satisfactory. Therefore, it is necessary to develop a more improved obesity drug.

Lipid related metabolic disease indicates particularly the disease related to blood lipid among many diseases caused by in vivo metabolic disorder. More specifically, fatty liver, type II diabetes, hyperlipidemia , cardiovascular disease, atherosclerosis, and lipid related metabolic syndrome are included in the criteria. The metabolic syndrome herein indicates when various metabolic diseases such as diabetes and obesity are observed simultaneously. It is as importantly required to develop a drug for the said lipid related metabolic disease as to develop a drug for obesity. Black tea is a post - fermented tea that is fermented by a microorganism in a tea plant (Camelia sinensis L.) belonging to Theaceae. Black tea contains gallic acid, caffeine, and catechins as components. Black tea cultivated in China is diverse according to the area of production. For example, puer tea, Chilj abyeongcha , and Tacha are produced in Yunnan Province, while Kangjuncha and Geumchumcha are produced in Sichuan Province. In the meantime,

Bokjeoncha, heukjeoncha, and Whajuncha are all a kind of black tea produced in Hunan Province, and Nocheongbyeongcha is produced in Hubei Province. The black tea produced in Guangxi Province is Ukbocha. The most representative black tea, puer tea, was first favored to drink by minority races residing border areas of China long before, which was produced from Mocha prepared by drying the leaves of Yunnan large leaf tea plant under the sun. At first, minority races enjoyed this tea and later the tea was introduced to the main land of China and designated as gong-cha in 1726. According to the form, the tea is called Sancha which is leaf tea, or brick tea which is steamed chunk. Brick tea on the market is exemplified by Byeongcha, Juncha, Gincha, Bangcha, and Tacha. Most teas produced by natural drying method, so called Gunchang, disappeared in the time of the Cultural Revolution. So, teas of today on the market are mostly Sookbyeongs that are produced by Ginap with the dried Mocha prepared by fast fermentation using a microorganism in the course of a preparation process called Actoe. Chyeongbyeong puer tea produced by the traditional method is limited in production. These days, regular tea users have been in increase since tea was discovered to have the effect of decomposing fat and cholesterol (Food Science and Technology Dictionary, Doosan Encyclopedia Doopedia) .

The conventional procedure of producing puer tea is composed of picking up the tea leaf, drying the tea leaf in the shade to reduce moisture content therein, reducing green color of the tea leaf which is coming back to dark green when the tea leaf is roasted, rubbing the tea leaf with hands to brew the tea well or to accelerate the fermentation of the tea, drying the tea leaf finished with rubbing with hands with spreading it on a bamboo mat in the sun/ ferment ing the tea leaf in high temperature high humidity condition by using a microorganism, which is necessary for the production of ripe tea and at this time the color of the fermented tea after 4 6 weeks becomes red but not necessary for the tea, and compressing the tea leaf in a mold to produce the tea in the shape of Byeongcha or Tacha.

Korean Patent No. 10-0443116 describes that the puer tea extract is obtained by fermenting the picked tea leaves of the large leaf tea plant growing in the region of Yunnan Province, China, for at least 4 years and then performing extraction with the fermented tea using 50% aqueous ethanol solution (w/w) at the volume of 20 times the volume of tea for 3 hours, followed by spray-drying, and that the obtained puer tea extract and the composition comprising the same have a pharmaceutical activity of lowering blood cholesterol and of ant i -obesity , suggesting that they are effective in preventing and treating obesity and hyperlipidemia.

Yamagami T. et . al . also reported that the Chinese black tea hot water extract had the effect of anti -hypercholesterol (Ann Nutr Metab, 53(2008) 33~42), which means the effect of reducing total cholesterol and low density cholesterol and the Chinese black tea water-soluble extract had the effect of reducing blood cholesterol in a rat (Nutrition Research, 28(2008) 450-456) . Hidenori Urata et al . reported that the Chinese black tea water-soluble extract had the effect of reducing abdominal fat and improving obesity index (Nutrition Research, 31(2011) 421~428) . All the above effects described in those reports are of hot-water extract or heat ethanol extract. In the meantime, the concentrations of gallic acid or catechines known as active ingredients of black tea are decreased in a high temperature solution. Particularly, gallic acid having the effect of the loss of weight and fatty liver (British journal of Nutrition 98(2007), 98, 727-735, Chemico-Biological Interactions, 174(2008), 109-117) becomes decomposed fast in a high temperature solution to turn into pyrogallol (Geochimica et Cosmochimica Acta, Vol. 52. pp. 341-344) . The concentration of catechins, known as an active ingredient of black tea, is not changed at the temperature range of 24 "C ~ 37 °C , but is reduced 20 ~ 25% when the catechins stay in a solution at 100°C for 3 hours (Stability of tea theaflavins and catechins, Food Chemistry, Vol. 83 pp.189-195) . The present inventors studied and confirmed that the black tea aqueous ethanol enfleurage extract contained higher concentration of gallic acid, the active ingredient and the marker compound as well, than the conventional black tea extract prepared by hot-water extraction and heat ethanol extraction and had excellent effect of reducing body weight and body fat, improving fatty liver, regulating blood sugar, and reducing blood cholesterol in the high fat diet- induced obese mouse model, leading to the completion of this invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient.

It is another object of the present invention to provide a health food for preventing or improving obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient. To achieve the above objects, the present invention provides a pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient.

The present invention also provides a health food for preventing or improving obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active -ingredient.

The present invention further provides a method for preventing obesity or lipid related metabolic disease which includes the step of administering the black tea aqueous ethanol enfleurage extract to a subj ect .

The present invention also provides a use of the black tea aqueous ethanol enfleurage extract for the preparation of a pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease.

In addition, the present invention provides a use of the black tea aqueous ethanol enfleurage extract for the preparation of a health food for preventing or improving obesity of lipid related metabolic disease. ADVANTAGEOUS EFFECT

As explained hereinbefore, the black tea aqueous ethanol enfleurage extract of the present invention contains higher content of gallic acid, the active ingredient and the marker compound, than the conventional black tea extract prepared by the conventional hot-water extraction and heat ethanol extraction, and has excellent effect of reducing body weight and body fat, improving fatty liver, regulating blood sugar, and reducing blood cholesterol in the high fat diet-induced obese mouse model, so that the black tea aqueous ethanol enfleurage extract of the present invention can be effectively used as an active ingredient of a pharmaceutical composition and of a health food for preventing or treating obesity, metabolic disease, diabetes, and hyperlipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein: Figure 1 is a diagram illustrating the effect of reducing body weight in the high fat diet- induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea hot-water extract. (* p<0.05, ** p<0.01 vs control)

Figure 2 is a diagram illustrating the effect of reducing body fat in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea hot-water extract.

Figure 3 is a Micro- CT photograph and a graph illustrating the fat area and the effect of reducing body fat in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea hot-water extract.

Figure 4 is a diagram illustrating the effect of decreasing blood sugar in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea hot-water extract .

Figure 5 is a diagram illustrating the effect of improving fatty liver in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea hot-water extract . Figure 6 is a diagram illustrating the effect of reducing body weight in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea heat aqueous ethanol extract.

Figure 7 is a diagram illustrating the effect of reducing body fat in the high fat diet- induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea heat aqueous ethanol extract.

Figure 8 is a diagram illustrating the effect of decreasing blood sugar and blood cholesterol in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea heat aqueous ethanol extract.

Figure 9 is a diagram illustrating the effect of improving fatty liver in the high fat diet-induced obese mouse model treated with the black tea aqueous ethanol enfleurage extract or the black tea heat aqueous ethanol extract.

Figure 10 is a diagram illustrating the inhibitory effect of the black tea aqueous ethanol enfleurage extract on adipocyte differentiation. Figure 11 is a diagram illustrating the inhibitory effect of the black tea aqueous ethanol enfleurage extract on lipid accumulation.

Figure 12 is a diagram illustrating the inhibitory effect of the black tea aqueous ethanol enfleurage extract on proliferation and differentiation of preadipocytes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail .

The present invention provides a pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient.

The present invention also provides a method for treating obesity or lipid related metabolic disease which includes the step of administering a pharmaceutically effective dose of the black tea aqueous ethanol enfleurage extract to a subject having obesity or lipid related metabolic disease. The present invention also provides a method for preventing obesity or lipid related metabolic disease which includes the step of administering a pharmaceutically effective dose of the black tea aqueous ethanol enfleurage extract to a subject.

The present invention also provides a use of the black tea aqueous ethanol enfleurage extract for the preparation of a pharmaceutical composition for preventing and treating obesity or lipid related metabolic disease.

The black tea herein can be either cultivated or purchased.

The black tea herein is preferably selected from the group consisting of Byeongcha, Jeoncha, Tacha, Bangcha, Kumkwakongcha , Kangjun, Kumchun, Bokjun, Huekjun, Whajun, Nochunbyeong , Yookbocha, puer tea, and red tea, but not always limited thereto.

The aqueous ethanol herein is preferably 30% 70% aqueous ethanol solution, and more preferably 50% aqueous ethanol solution, but not always limited thereto.

The metabolic disease herein is preferably one or more diseases selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia , cardiovascular disease, atherosclerosis, and lipid related metabolic disease, but not always limited thereto.

The above black tea aqueous ethanol enfleurage extract preferably contains gallic acid at the concentration of 2 ~ 8% (w/w) , but not always limited thereto .

The above black tea aqueous ethanol enfleurage extract is preferably supposed to reduce body weight and more preferably to decrease body fat, particularly abdominal fat or subcutaneous fat, but not always limited thereto. Moreover, the decrease of body fat herein is preferably attributed to the inhibition of adipocyte differentiation, and the inhibition of adipocyte differentiation is not supposed to inhibit the adipogenic, transcription factors C/EBPa and PPARy2 and not supposed to affect the expressions of their target genes FABP4 , FAS, and ACC, either.

The decrease of body fat herein is preferably achieved by the inhibition of proliferation of preadipocytes or adipocytes.

It is preferred for the black tea aqueous ethanol enfleurage extract to improve fatty liver by lowering blood cholesterol or blood sugar, but not always limited thereto. The said subject herein is a vertebrate, preferably a mammal, more preferably a test animal such as rat, rabbit, guinea pig, hamster, dog, and cat, and most preferably an anthropoid such as chimpanzee and gorilla.

In addition, the said black tea aqueous ethanol enfleurage extract preferably does not cause side effects particularly metabolic disease including insulin resistant diabetes resulted from the malfunction of PPARy2 of adipocytes.

The extract can be administered once a day or several times a day. The dose of the extract cannot limit the spirit and scope of the present invention in any aspects.

The black tea aqueous ethanol enfleurage extract is preferably prepared by the method comprising the following steps, but not always limited thereto:

1) mixing black tea and 30% ~ 70% aqueous ethanol solution at the ratio of 1:4 ~ 1:8;

2) extracting the mixture of step 1) for 60 ~ 80 hours ; and

3) filtering the extract of step 2) and then concentrating thereof under reduced pressure. In the above method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the black tea of step 1) is preferably mixed with 50% aqueous ethanol solution, but not always limited thereto.

In the above method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the extraction method is preferably shaking extraction, Soxhelt extraction, or reflux extraction, but not always limited thereto. It is preferred to add the extraction solvent to the dried black tea at the volume of 4 - 8 times the dried black tea weight, and more preferably 6 times the weight. The preferable extraction temperature is 5 35^°C, and more preferably 10 ~ 30°C, but not always limited thereto. The extraction time is preferably 24 ~ 100 hours, and more preferably 60 ~ 80 hours, and most preferably 72 hours, but not always limited thereto. The extraction is preferably performed 1 - 5 times, and more preferably 3 - 4 times repeatedly, and most preferably 3 times, but not always limited thereto.

In the method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the concentration under reduced pressure of step 3) is preferably performed by using vacuum evaporator or rotary evaporator, but not always limited thereto. The drying process is performed preferably by low pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but not always limited thereto.

In a preferred embodiment of the present invention, the present inventors tried to confirm the pharmaceutical effect of the black tea aqueous ethanol enfleurage extract as a composition for preventing and treating obesity or lipid related metabolic disease. To do so, the inventors first obtained the diverse black tea extracts over the ethanol concentrations and extraction hours by reflux extraction. The obtained black tea aqueous ethanol extracts proceeded to HPLC to measure the concentration of gallic acid. As a result, the black tea aqueous ethanol extract obtained by enfleurage with 50% ethanol added at the volume of 6 times dried weight of the tea for 72 hours demonstrated the highest gallic acid content (see Table 1 ~ Table 4 ) .

To investigate the preventive and therapeutic effect on obesity or lipid related metabolic disease of both the black tea aqueous ethanol enfleurage extract and the black tea hot-water extract, the present inventors orally administered the both black tea aqueous ethanol enfleurage extract and the black tea hot-water extract to the high fat diet-induced obese mouse model, followed by investigation of weight, body fat, blood sugar, and histopathologic examination. As a result, the group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage showed the highest weight loss up to 23.5 %, compared with the high fat diet (control) group. The group treated with 300 mg/kg of the black tea 100% hot-water solution showed 18.1% body weight loss, while the group treated with 100 mg/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage showed 13.2% body weight loss. The group treated with 100 mg/kg of the black tea 100% hot-water extract showed only 3.9% reduction in the body weight. From the comparison between before and after the administration, it was confirmed that the body weight of the high fat diet (control) group was approximately 10.24 g increased, while the weight of the mouse group treated with 300 mg/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was approximately 0.22 g decreased, indicating that significant weight decrease was induced by the extract (23.5%), compared with the control group. The group treated with 300 nig/kg of the black tea 100% hot-water extract showed the increase of body weight as much as 1.94 g, indicating the weight reducing effect was 18.1% by the control (see Figure 1 and Table 6) .

It was also confirmed that the said body weight loss was mainly attributed to the decrease of body fat not to the loss of real body weight or lean body mass. The body fat of the high fat diet group was approximately 7.73% increased 8 weeks after the diet, while the body fat of the group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was approximately 7.25% reduced, suggesting that the body fat reduction effect in this group surpassed that of the control. The body fat of the group treated with 300 ing/kg of the black tea 100% hot-water extract was approximately 3.18% decreased, compared with that of the control (see Figure 2 and Table 7) .

Abdominal fat and subcutaneous fat of the control group having been taking high fat diet for 8 weeks were much increased, compared with those of the normal group, confirmed by Micro-CT. However, abdominal fat and subcutaneous fat of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage were not increased and maintained as similar level as to those of the normal group even after taking high fat diet. Abdominal fat and subcutaneous fat of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage were respectively 512.4+77.3 mm³ and 125.9+86.8 mm³, which indicates far better body fat reduction effect than that of the control group showing 3383.4+249.8 mm³ of abdominal fat and 973.0+191.6 mm³ of subcutaneous fat but similar level to those of the normal group showing 442.2+81.9 mm³ of abdominal fat and 77.9+62.3 mm³ of subcutaneous fat (see Figure 3 and Table 8) .

The level of blood sugar of the group treated with 300 iug/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage for 8 weeks was 77.97±9.65 mg/dL, indicating that the level of blood sugar raised by high fat diet was reduced almost half the level lowered in the control group (blood sugar of the high fat diet group: 158.96±20.06 mg/dL) (see Figure 4 and Table 9) .

From the result of the liver histopathologic examination, it was confirmed that fatty liver of the group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage for 8 weeks was significantly improved, compared with that of the high fat diet group, indicating the extract had the effect of improving fatty liver (see Figure 5) .

In a preferred embodiment of the present invention, the present inventors orally administered the black tea aqueous ethanol enfleurage extract and the black tea heat ethanol extract to the high fat diet-induced obese mouse model, followed by investigation of weight, body fat, blood sugar, blood cholesterol and histopathologic examination to investigate the preventive and therapeutic effect of both the black tea aqueous ethanol enfleurage extract and the black tea heat ethanol extract on obesity or lipid related metabolic disease. 4 weeks after the administration, the group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage demonstrated 22.1% weight loss by that of the high fat diet group, and the group treated with 300 nig/kg of the black tea 50% ethanol extract showed 16.5% weight loss. 8 weeks later, the group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage demonstrated 23.9% weight loss by that of the high fat diet group, while the group treated with 300 nig/kg of the black tea 50% ethanol extract showed 19.5% weight loss. From the comparison between before and after the administration, it was confirmed that the body weight of the high fat diet grou was approximately 1.68g increased, while the weight of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was approximately 11.47 g decreased, indicating that significant weight decrease was induced by the extract, compared with the control. The weight of the mouse group treated with

300 nig/kg of the black tea 50% ethanol heat extract was 9.01 g reduced, suggesting that the weight was still reduced compared with that of the control (see

Figure 6 and Table 10) .

In the meantime, body fat of the high fat diet group was increased approximately 0.73% 8 weeks later, compared with before the administration. However, body fat of the mouse group treated with 300 mg/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was approximately 11.63% decreased, indicating excellent body fat reduction effect of the extract. Body fat of the mouse group treated with 300 mg/kg of the black tea 50% ethanol heat extract was approximately 6.48% reduced, compared with that of the control (see Figure 7 and Table 11) .

Blood sugar of the mouse group treated with 300 mg/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was significantly reduced to 32.94+11.23 mg/dL 8 weeks after the administration, compared with that of the control (blood sugar of the high fat diet group: 86.03±14.02 mg/dL) . Blood sugar level of the mouse group treated with 300 nig/kg of the black tea 50% ethanol heat extract was 61.94±8.98 mg/dL, which was lower than that of the control, 8 weeks after the administration, but the blood sugar reduction effect of the black tea 50% aqueous ethanol extract prepared by enfleurage was greater than that.

The level of cholesterol of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enfleurage was 170.29+16.67 mg/dL, which was significantly lower than that of the control (blood sugar of the high fat diet group: 229.16+11.44 mg/dL) . In the meantime, the level of cholesterol of the mouse group treated with 300 nig/kg of the black tea 50% ethanol heat extract was 177.95±10.39 mg/dL, which was almost equal to that reduced by the black tea 50% aqueous ethanol extract prepared by enfleurage (see Figure 8 and Table 12) .

His opathologic examination with the mouse liver was performed 8 weeks after the administration. As a result, fat globules of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol extract prepared by enf leurage were significantly reduced, compared with those of the high fat diet group, suggesting that the extract had fatty liver improving effect (see Figure 9) .

The inhibitory effect of the black tea 50% aqueous ethanol extract prepared by enfleurage on adipocyte differentiation was investigated. As a result, fat formation was inhibited by the black tea 50% aqueous ethanol extract prepared by enfleurage dose-dependently , which was consistent with the result of Oil-Red-0 test. Test animals were treated with the black tea 50% aqueous ethanol extract prepared by enfleurage at different concentrations of 0, 100, 200, 250, 300, and 400 ug/ i. As a result, it was confirmed that the concentration of 200 ~ 300 p.g/ml of the black tea 50% aqueous ethanol extract prepared by enfleurage would be enough to inhibit adipocyte differentiation. The adipocytes treated with the black tea 50% aqueous ethanol extract prepared by enfleurage proceeded to RT-PCR to investigate the changes of adipogenic transcription factors. As a result, even though the adipocyte differentiation was inhibited by the black tea 50% aqueous ethanol extract prepared by enfleurage, C/EBP and PPARy2 mRNA remained unchanged. The expressions of their target genes FABP4, FAS, and ACC were not changed, either.

In the group treated with 50 μg/mi of the black tea 50% aqueous ethanol extract prepared by enfleurage, fat was reduced 50 ~ 70% with maintaining the normal expressions of PPARy2 and C/EBP genes, the master transcription factors necessary for the differentiation of preadipocytes into adipocytes, confirmed by Oil Red-0 test (see Figure 10) .

The inhibitory effect of the black tea 50% aqueous ethanol extract prepared by enfleurage on mature adipocytes was also investigated. As shown in Figure 11, 300

of the black tea 50% aqueous ethanol extract prepared by enfleurage reduced fat in cells. When mature adipocytes were treated with 100 of the black tea 50% aqueous ethanol extract prepared by enfleurage, even previously accumulated fat volume was lowered by 70%. The above result was consistent with that of the quantification using stained Oil Red-O. Changes of adipogenic transcription factors were also observed. As a result, even though adipocyte differentiation was inhibited by the black tea 50% aqueous ethanol extract prepared by enfleurage, the levels of C/EBPa and PPARy mRNA were unchanged and the expressions of their target genes FABP4 , FAS, and ACC were not affected, either (see Figure 11) .

Therefore, it was confirmed that the black tea 50% aqueous ethanol extract prepared by enfleurage had the effect of reducing fat, but did not reduce the expressions of C/EBPa and PPARy, the adipogenic transcription factors. The malfunction of PPARY2 causes various metabolic disorders including insulin resistance. Therefore, the above results indicate that the black tea 50% aqueous ethanol extract prepared by enfleurage does not accompany any side effect caused by the malfunction of PPARy2.

The inhibitory effect of the black tea 50% aqueous ethanol extract prepared by enfleurage on the proliferation of preadipocytes and the differentiation of adipocytes was investigated. As a result, the proliferation in the early stage of adipocyte differentiation was not accomplished normally. When

300 βg/ i of the black tea 50% aqueous ethanol extract prepared by enfleurage was treated to non- differentiated 3T3-L1 preadipocytes, the proliferation of the preadipocytes was also inhibited. In conclusion, the black tea aqueous ethanol extract inhibited adipocyte differentiation and at the same time limited the number of preadipocytes, suggesting that it had the effect of inhibiting adipogenesis (see Figure 12 ) .

The black tea 50% aqueous ethanol extract prepared by enfleurage of the present invention contained higher concentration of gallic acid, the active ingredient and the marker compound of the same, and had the effects of reducing body weight and body fat, improving fatty liver, regulating blood sugar, and reducing blood cholesterol in the high fat diet- induced obese mouse model, so that the extract can be effectively used as an active ingredient of a pharmaceutical composition and of a health food for preventing or treating obesity, metabolic disease, diabetes, and hyperlipidemia.

The composition of the present invention can be administered orally or parenterally (for_* example, external application, intravenous injection, hypodermic injection or peritoneal injection) but oral administration is preferred. For formulations for parenteral administration, powders, granules, tablets, capsules, sterilized suspensions, liquids, water- insoluble excipients, suspensions, emulsions, syrups, suppositories, external use such as aerosols and sterilized injections can be prepared by the conventional method, and preferably skin external pharmaceutical compositions such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or cataplasmas can be prepared, but not always limited thereto. The composition for local administration can be prepared as anhydrous or water- based. Water insoluble excipients and suspensions can contain propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. The composition of the present invention can additionally include, in addition to the black tea aqueous ethanol extract, one or more effective ingredients having the same or similar effect to them. The composition can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration. The pharmaceutically acceptable carrier can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer' s solution, buffered saline, dextrose solution, mal todextrose solution, glycerol and ethanol. Other general additives such as anti -oxidative agent, buffer solution, bacteriostatic agent, etc., can be added. In order to prepare injectable solutions such as aqueous solution, suspension and emulsion, diluents, dispersing agents, surfactants, binders and lubricants can be additionally added.

It is preferred for the pharmaceutically acceptable additive to be included at the concentration of 0.1 ~ 90 weight part in the composition of the present invention.

The effective dosage of the pharmaceutical composition of the present invention can be determined according to absorpt iveness of the active ingredient, age and gender of patient, degree of obesity, etc, but can be regulated properly by those in the art. In the case of oral administration, the pharmaceutical composition can be administered by 0.0001 ~ 100 rag/kg per day for an adult, and more preferably by 0.001 ~ 100 mg/kg per day. The administration frequency is once a day or a few times a day. The dosage cannot limit the scope of the present invention in any aspects.

The composition for treating metabolic disease of the present invention can include, in addition to the black tea aqueous ethanol enfleurage extract, one or more effective ingredients having the same or similar function to the extract.

The present invention also provides a health food for preventing or improving obesity or lipid related metabolic disease comprising the black tea aqueous ethanol enfleurage extract as an active ingredient.

The present invention also provides a use of the black tea aqueous ethanol enfleurage extract for the preparation of a health food for preventing and improving obesity or lipid related metabolic disease. The black tea herein can be either cultivated or purchased.

The black tea herein is preferably selected from the group consisting of Byeongcha, Jeoncha, Tacha, Bangcha, Kumkwakongcha , Kangjun, Kumchun, Bokjun, Huekjun, hajun, Nochunbyeong , Yookbocha, puer tea, and red tea, but not always limited thereto.

The metabolic disease herein is preferably one or more diseases selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, atherosclerosis, and lipid related metabolic disease, but not always limited thereto.

The above black tea aqueous ethanol enfleurage extract preferably contains gallic acid at the concentration of 2 ~ 8% (w/w), but not always limited thereto.

The above black tea aqueous ethanol enfleurage extract is preferably supposed to reduce body weight and more preferably to decrease body fat, particularly abdominal fat or subcutaneous fat, but not always limited thereto. Moreover, the decrease of body fat herein is preferably attributed to the inhibition of adipocyte differentiation, and the inhibition of adipocyte differentiation is not supposed to inhibit the adipogenic transcription factors C/EBPa and PPARy2 and not supposed to affect the expressions of their target genes FABP4, .FAS, and ACC, either.

The decrease of body fat herein is preferably achieved by the inhibition of proliferation of preadipocytes or adipocytes .

It is preferred for the black tea aqueous ethanol enfleurage extract to improve fatty liver by lowering blood cholesterol or blood sugar, but not always limited thereto.

The black tea aqueous ethanol enfleurage extract is preferably prepared by the method . comprising the following steps, but not always limited thereto:

1) mixing black tea and 30% ~ 70% aqueous ethanol solution at the ratio of 1:4 ~ 1:8; 2) extracting the mixture of step 1) for 60 ~ 80 hours ; and

3) filtering the extract of step 2) and then concentrating thereof under reduced pressure.

In the above method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the black tea of step 1) is preferably- mixed with 50% aqueous ethanol solution, but not always limited thereto.

In the above method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the extraction method is preferably shaking extraction, Soxhelt extraction, or reflux extraction, but not always limited thereto. It is preferred to add the extraction solvent to the dried black tea at the volume of 4 - 8 times the dried black tea weight, and more preferably 6 times the weight. The preferable extraction temperature is 5 35°C, and more preferably 10 ~ 30 °C , but not always limited thereto. The extraction time is preferably 24 ~ 100 hours, and more preferably 60 ~ 80 hours, and most preferably 72 hours, but not always limited thereto. The extraction is preferably performed 1 - 5 times, and more preferably 3 - 4 times repeatedly, and most preferably 3 times, but not always limited thereto. In the method for preparing the black tea aqueous ethanol enfleurage extract of the present invention, the concentration under reduced pressure of step 3) is preferably performed by using vacuum evaporator or rotary evaporator, but not always limited thereto. The drying process is performed preferably by low pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but not always limited thereto.

The black tea aqueous ethanol enfleurage extract of the present invention contained higher concentration of gallic acid, the active ingredient and the marker compound of the same, and had the effects of reducing body weight and body fat, improving fatty liver, regulating blood sugar, and reducing blood cholesterol in the high fat diet- induced obese mouse model, so that, the extract can be effectively used as an active ingredient of a pharmaceutical composition and of a health food for preventing or treating obesity, metabolic disease, diabetes, and hyperlipidemxa.

The black tea aqueous ethanol enfleurage extract of the present invention can be provided as a food composition after mixing with a food nutritionally acceptable carrier. It is preferred for the black tea aqueous ethanol extract or lysate to be added to the total composition at the concentration of 0.01 ~ 99.9 weight%, but not always limited thereto.

The black tea aqueous ethanol enf ieurage extract of the present invention can be used as a food additive. In that case, the black tea aqueous ethanol enfleurage extract of the present invention can be added as it is or as mixed with other food components according to the conventional method. The mixing ratio of the black tea aqueous ethanol enfleurage extract of the present invention can be regulated according to the purpose of use (prevention, health enhancement or treatment) . If long term administration is required for health and hygiene or regulating health condition, long term administration of the black tea aqueous ethanol enfleurage extract of the present invention is possible since the extract barely has toxicity or side effects.

The food herein is not limited. For example, the black tea aqueous ethanol enfleurage extract of the present invention can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns , flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included. The health beverages containing the black tea aqueous ethanol extract of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol. The content of the natural carbohydrate is preferably 1 ~ 20 g and more preferably 5 ~ 12 g in

100 ml of the composition. Besides, natural sweetening agents (thaumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic sweetening agents (saccharin, aspartame, etc.) can be included as a sweetening agent.

In addition to the ingredients mentioned above, the food composition of the present invention can include in a variety of nutrients, vitamins, minerals (electrolytes), flavors including natural flavors and synthetic flavors, coloring agents and extenders (cheese, chocolate, etc.) , pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosif iers , pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The food composition of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 0.001 ~ 50 weight part for the total weight of the food composition of the present invention.

In another preferred embodiment of the present invention, the black tea aqueous ethanol enfleurage extract of the present invention can be provided in the form of powder, granule, tablet, capsule, syrup, or beverages.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples, Experimental Examples and Manufacturing Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. Example 1 : Preparation of black tea ethanol extracts according to different solvent concentrations

Black tea (post- fermented tea obtained from Camellia sinensis L.) produced and processed from the 1^st grade tea leaves in Yunnan Province, China, in 2006, was purchased. After eliminating impurities, the well-dried herb was used for the experiment. 120 ml of 15%, 30%, 50%, 70%, 85%, or 96% aqueous ethanol solution (v/v) was added to 20 g of black tea, followed by extraction at 10 ~ 30 ^°C (room temperature) for 72 hours. The extract was filtered and concentrated under reduced pressure at 45 ~ 60°C, followed by freeze-drying . As a result, 0.54 ~ 3.46 g of the black tea aqueous ethanol enfleurage extract was obtained. The content of gallic acid in the obtained black tea aqueous ethanol enfleurage extract was measured by HPLC.

As a result, as shown in [Table 1] , the higher the ethanol concentration, the higher the gallic acid content. However, considering yield in mass production and industrial efficiency and gallic acid content together, the optimum extraction solvent solution was determined as 50% aqueous ethanol solution (v/v) (Table 1) . Example 2 : Preparation of black tea ethanol extracts according to different solvent volumes

Black tea (post - fermented tea obtained from Camellia sinensis L.) produced and processed from the 1^st grade tea leaves in Yunnan Province, China, in 2006, was purchased. After eliminating impurities, the well-dried herb was used for the experiment. 80 mi, 120 mi, 160 mi, or 200 ml of 50% aqueous ethanol solution (v/v) was added to 20 g of black tea, followed by extraction at 10 ~ 30 °C (room temperature) for 72 hours. The extract was filtered and concentrated under reduced pressure at 45 ~ 60°C, followed by freeze-drying . As a result, 1.54 ~ 3.46 g of the black tea aqueous ethanol enfleurage extract was obtained. The content of gallic acid in the obtained black tea aqueous ethanol enfleurage extract was measured by HPLC.

As a result, as shown in [Table 2] , the optimum extraction method was determined as the one using aqueous ethanol solution (v/v) 6 times the volume of black tea with considering yield and gallic acid content (Table 2) . Example 3 : Preparation of black tea ethanol extracts according to different extraction hours

Black tea (post- fermented tea obtained from Camel l ia, sinensis L.) produced and processed from the 1^st grade tea leaves in Yunnan Province, China, in 2006, was purchased. After eliminating impurities, the well-dried herb was used for the experiment. 120 vai of 50% aqueous ethanol solution (v/v) was added to 20 g of black tea, followed by extraction at 10 ~ 30°C (room temperature) for 24, 48, 72 or 96 hours. The extract was filtered and concentrated under reduced pressure at 45 ~ 60°C, followed by freeze-drying . As a result, 2. SO ~ 3.46 g of the black tea aqueous ethanol enfleurage extract was obtained. The content of gallic acid in the obtained black tea aqueous ethanol enfleurage extract was measured by HPLC.

As a result, as shown in [Table 3] , the optimum extraction time was determined as 72 hours with considering yield and gallic acid content together (Table 3) .

Example 4 : Preparation of black tea heat reflux extract Black tea heat extracts were prepared by using 50% aqueous ethanol solution and 100% hot-water. Then, the content of gallic acid therein was compared.

Black tea (post - fermented tea obtained from Camellia sinensis L.) produced and processed from the 1^st grade tea leaves in Yunnan Province, China, in 2006, was purchased. After eliminating impurities, the well-dried herb was used for the experiment. 1000 mi of 0% or 50% aqueous ethanol solution (v/v) was added to 50 g of black tea, followed by heat reflux extraction for 3 hours. The extract was filtered and concentrated under reduced pressure at 45 ~ 60 °C , followed by freeze-drying . As a result, 8.00 g and 8.50 g of the black tea heat reflux extracts were obtained. The content of gallic acid in the obtained black tea heat reflux extract was measured by HPLC.

As a result, as shown in [Table 4] , the gallic acid content in the black tea heat ethanol extract was less than that in the black tea aqueous ethanol enfleurage extract of the present invention (Table 4) .

Example 5: Investigation of gallic acid content in black tea extract High-performance liquid chromatography (HPLC) was performed to analyze the contents of gallic acid in the black tea aqueous ethanol enfleurage extracts and the black tea heat reflu extracts obtained in Example 1 - Example 4.

HPLC was performed by using CAPCELL PAK MG (4.6 x 250mm, 5μπι, Shiseido) column (moving phase (A) : 0.6% formic acid, moving phase (B) : acetonitrile ) according to the conditions presented in [Table 5] . UV280 nm detector was used and the temperature of the column was set as 30°C . The samples were loaded at the flow rate of 0.8 i /min, 10 βΐ at a time.

[Table 5]

Conditions of HPLC moving phase

<5-l> Pre- treatment of samples

Stock standard solution was prepared for HPLC. Particularly, 10.0 nig of standard gallic acid measured accurately was loaded in a 10 mi measuring flask, to which 50% aqueous ethanol solution (v/v) was added, followed by stirring to prepare the stock standard solution. The prepared stock standard solution was diluted stepwise in the range of 0.01 ~ 0.2 ing/ra to prepare working standard solution for quantification.

<5-2> Preparation of test solution

Exactly 30 mg of the above black tea aqueous ethanol was loaded in a 10 mi measuring flask, to which 50% aqueous ethanol solution (v/v) was added, followed by ultrasonic vibration for 20 minutes. After cooling the mixed solution, gauge mark was made with 50% aqueous ethanol solution (v/v) . The mixture was well mixed by shaking. 1 mi of the mixture was transferred into a 1.5 ml microcentrifuge tube, followed by centrifugation at 13,000 rpm for 5 minutes. The supernatant was obtained and used as test solution.

<5-3> Quantification

The gallic acid contents in the stock standard solution and the test solution prepared in Example <5- 1> and Example <5-2> were measured by HPLC. <5-4> Calculation of content

The contents of gallic acid in the test solution and the standard solution were calculated by

Mathematical Formula 1.

[Mathematical Formula 1]

Gallic acid content (%) = Ct/Wt x 1000 x P

Ct : gallic acid concentration in the sample

(mg/mL)

Wt : sample amount (mg)

P : purity of standard gallic acid (%/l00)

[Table l]

Yield and gallic acid content in black tea aqueous ethanol enfleurage extract according to solvent concentration

EtOH Temper days

ature

20g 50% 120 mi Room 3 3.46 17.3 3.24

EtOH Temper days

ature

20g 30% 120 m Room 3 3.16 15.8 3.11

EtOH Temper days

ature

20g 15% 120 ml Room 3 3.06 15.3 3.25

EtOH Temper days

ature

[Table 2]

Yield and gallic acid content in black aqueous ethanol enfleurage extract according solvent times

[Table 3]

Yield and gallic acid content in black aqueous ethanol enfleurage extract according extraction hours

[Table 4]

Yield and gallic acid content in black tea heat reflux extract Black Solven Solven Tempe Time Produc Yield Galli tea t t ratur t (g) (%) c Amount Amount e Acid

(%)

50g 50% 1,000 95-10 3 8.00 16.0 2.04

EtOH mi 0°C hours

50g 100% 1,000 95-10 3 8.50 17.0 2.98 water ml 0°C hours

As a result, it was confirmed that the content of gallic acid in the black tea aqueous ethanol enfleurage extract extracted using 50% aqueous ethanol solution (v/v) 6 times the volume of black tea at 10 -

30^°C (room temperature) for 72 hours was higher than the content of gallic acid in the black tea 100% hot- water extract, suggesting that the extraction method of the present invention was excellent.

Experimental Example 1: Effects of black tea 50% aqueous ethanol solution enfleurage extract and 100% hot-water extract in high fat diet-induced obese mouse model

Anti-obesity effect of the black tea 50% aqueous ethanol enfleurage extract prepared in Example 1 was compared with that of the black tea 100% hot-water extract obtained in Example 4 in the high fat diet- induced obese mouse model. The high fat diet-induced obese mouse model used in this experiment was prepared as follows. Male C57/BL6 mice at 3 weeks (Dae Han Biolink, Korea) were raised for 1 week at 22 ~ 24°C with 60 ~ 80% humidity for adaptation, during which standard diet and water were provided. Then, the mice were raised with high fat diet (Research diet, USA) for 7 weeks. Body weight of each mouse was measured and top 70% were selected as test animals and the down 30% were eliminated. The top 70% selected above were grouped according to weight and body fat and each group had 8 mice. The animals were orally administered with each test composition every morning between 9 am ~ 11 am for 8 weeks.

<1-1> Changes of body weight

Weight of each test animal was measured once a week. The result was presented as the weight (g) after the last administration (8 weeks later) by the weight before the administration.

As a result, as shown in Figure 1 and Table 6, the group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract demonstrated 23.5% weight loss, compared with the high fat diet (control) group, which was the highest weight reducing effect. In the meantime, the group treated with 300 nig/kg of the black tea 100% hot-water extract showed 18.1% body- weight decrease, while the group treated with 100 mg/kg of the black tea 50% aqueous ethanol enfleurage extract showed 13.2% weight decrease. The group treated with 100 mg/kg of the black tea 100% hot-water extract showed 3.9% weight decrease.

As shown in Table 6, body weight of the high fat diet group after 8 weeks of the treatment was increased approximately as much as 10.24 g, compared with before the administration. In the meantime, body weight of the group treated with 300 nig/ kg of the black tea 50% aqueous ethanol enfleurage extract was reduced 0.22 g. The result indicates that the experimental group showed significantly more weight loss than that of the control (23.5% decrease) . The group treated with 300 mg/kg of the black tea 100% hot-water extract showed increased body weight (1.94 g) , which indicated 18.1% weight decrease, compared with that of the control group (Figure 1 and Table 6) .

[Table 6]

Reducing effect of black tea hot-water extract and black tea aqueous ethanol enfleurage extract on weight of high fat diet-induced obese mouse model Experimental group Initial Weight Rate of Rate of weight after 8 change change

(g) weeks (%) to

⁽g⁾ control

(%)

Normal diet group 27.68 ± 29.99 ± +8.3%

0.66 0.86

High fat diet group 35.26 ± 45. ± +29.1%

(Control ) 1.02 1.43

Black tea 100% hot- 35.07 ± 43.70 ± +24.4% -3.9% water extract 100 0.91 1.55

mg/kg

Black tea 100% hot- 35.31 ± 37.25 ± +4.1% -18.1% water extract 300 1.21 2.12

Black tea 50% 35.23 ± 39.47 ± +12.3% -13.2% aqueous ethanol 1.18 0.83

enfleurage extract

100 mg/kg

Black tea 50% 35.04 ± 34.81 ± -1.0% -23.5% aqueous ethanol 1.03 1.73

enfleurage extract

300 mg/kg

<l-2> Changes of body fat

Minispec LF90 live animal analyzer (Bruker, Germany) is a NMR machine using nuclear magnetic resonance, which realizes fast and accurate analysis and quantification of internal components such as fats, non-fats, and body fluid without killing or dissecting an animal. After weight the mouse using a scale, the motile mouse was placed in Minispec LF90 and body fat and lean body mass were measured without administering any anesthetic and sedative to the mouse .

As a result, as shown in Figure 2 and [Table 7] , changes of body weight were mostly attributed to changes of body fat not to changes of lean body mass.

As shown in [Table 7] , body fat of the high fat diet group (Control) was 7.73% increased 8 weeks later, but body fat of the group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract was 7.25% decreased, which was significant decrease compared with that of the control. Body fat of the group treated with 300 mg/kg of the black tea 100% hot-water extract was 3.18% reduced, indicating that the decrease of body fat was more than that of the control (Figure 2 and Table 7) . [Table 7]

Reducing effect of black tea hot-water extract and black tea aqueous ethanol enfleurage extract on body fat of high fat diet-induced obese mouse model

(%) weeks (%) (%P)

Normal Diet Group 6.38±0.76 5.91±0.76 -0.47%P

High fat diet group 24.24 ± 31.97 ± +7.73%P (Control) 1.39 0.80

Black tea 100% hot- 24.12 ± 28.50 ± +4.38%P water extract 100 nig/kg 1.23 1.56

Black tea 100% hot- 24.27 ± 21.08 ± -3.18%P water extract 300 nig/kg 1.26 3.25

Black tea 50% aqueous 24.31 ± 26.85 ± +2.54%P ethanol enfleurage 1.13 1.13

extract 100 mg/kg

Black tea 50% aqueous 24.34 ± 17.10 ± -7.25%P ethanol enfleurage 1.11 2.09

extract 300 mg/kg

<l-3> Microtomoraphy or computed tomography (Micro-CT) etamine (Huons, 1.5 mi/kg) and xylazine (Bayer, 0.5 ml/kg) were injected intraperitoneally to anesthetize the mouse. Imaging of body was performed by using in-vivo Micro-CT (Skyscan 1076, Bruker AXS , Germany) for the evaluation of mouse abdominal adipose tissues. Abdominal subcutaneous fat and visceral fat in the area from the 2^nd lumbar vertebra to the 5^th lumbar vertebra were measured by using SkyScan CT analyzer version 1.11 (Bruker ASX, Germany) .

As a result, as shown in figure 3, abdominal fat and subcutaneous fat of the high fat diet-induced obesity model (Control) taking high fat diet for 8 weeks were all increased significantly, compared with those of the normal group. In the meantime, abdominal fat and subcutaneous fat of the mouse administered with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract were almost similar to those of the normal group even after taking high fat diet.

As shown in Table 8, abdominal fat and subcutaneous fat of the mouse treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract were 512.4±77.3 mm³ and 125.9+86.8 mm³, which indicated significant body fat reducing effect, compared with those of the high fat diet group (Control) (abdominal fat: 3383.4+249.8 mm³, subcutaneous fat: 973.0+191.6 mm³), which were almost as similar to those of the normal group (abdominal fat: 442.2+81.9 mm³, subcutaneous fat: 77.9±62.3 mm³) (Figure 3 and Table 8) .

[Table 8]

Reducing effect of black tea hot-water extract and black tea aqueous ethanol enfleurage extract on abdominal fat and subcutaneous fat of high fat diet- induced obese mouse model

Experimental Group Abdominal fat Subcutaneous (mm³) fat (mm³)

Normal Diet Group 442.2±81.9 77.9±62.3

High fat diet group 3383. ±249.8 973.0±191.6 (Control)

Black tea 100% hot-water 1945.3+6.7 780.9±403.6 extract 300 nig/ kg

Black tea 50% aqueous 512. ±77.3 125.9±86.8 ethanol enfleurage

extract 300 mg/kg

<l-4> Changes of blood sugar

1 mi of blood was taken from abdominal vein of the mouse for hematological examination. EDTA-2K, the anticoagulant, was added to prevent blood clotting. Plasma was separated from the blood by centrifugation and the biochemical profiles of the blood plasma were investigated by using a blood biochemical automatic analyzer (20i, KoneLab) .

As a result, as shown in Figure 4 and [Table 9] , blood sugar of the mouse treated with 300 mg/kg of the black tea 50% aqueous ethanol enfleurage extract after 8 weeks of administration was 77.97+9.65 mg/dL, suggesting that blood sugar raised by high fat diet was lowered almost half the level of the control (blood sugar of high fat diet group: 158.96±20.06 mg/dL) (Figure 4 and Table 9) . [Table 9]

Reducing effect of black tea hot-water extract and black tea aqueous ethanol enfleurage extract on blood sugar of high fat diet- induced obese mouse model

<l-5> His topathological test of liver

Mouse liver was extracted, followed by fixing in 10% neutral buffered formalin (Sigma, HT50-1-640) for at least 24 hours. The fixed tissues were treated by vacuum tissue processor ASP300 (Leica) . Then, paraffin blocks were prepared by using embedding center EG1150H (Leica) . The paraffin block was cut into 4 pm . thick sections by using microtome RM2145 (Leica) , which proceeded to H&E staining by using autostainner XL (Leica) . The stained tissue samples were observed under optical microscope (Axioskop 2, Zeiss ) .

As a result, as shown in Figure 5, the result of the histopathological test with the mouse liver extracted 8 weeks after the administration confirmed that fat globules of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract were significantly decreased, compared with those of the high fat diet group, indicating that the extract of the present invention had the effect of improving fatty liver (Figure 5) .

Experimental Example 2: Comparison of the effect of black tea 50% aqueous ethanol enfleurage extract with the effect of black tea 50% aqueous ethanol heat extract in high fat diet-induced obese mouse model

Anti-obesity effect of the black tea 50% aqueous ethanol enfleurage extract prepared in Example 1 was compared with that of the black tea 50% aqueous ethanol heat extract prepared in Example 4 in the high fat diet-induced obese mouse model. The high fat diet-induced obese mouse model used in this experiment was prepared as follows. Male C57/BL6 mice at 3 weeks (Dae Han Biolink, Korea) were raised for 1 week at 22 ~ 24°C with 60 ~ 80% humidity for adaptation, during which standard diet and water were provided. Then, the mice were raised with high fat diet (Research diet, USA) for 24 weeks. Body weight of each mouse was measured and top 70% were selected as test animals and down 30% were eliminated. The top 70% selected above were grouped according to weight and body fat and each group had 8 mice. The animals were orally administered with each test composition every morning between 9 am ~ 11 am for_.8 weeks.

<2-l> Changes of body weight

Body weight and body fat of each test animal were measured once a week. The result was presented as the body weight (g) after the last administration (8 weeks later) by the weight before the administration.

As a result, as shown in Figure 6 and [Table 10] , the body weight of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract was 22.1% decreased 4 weeks after the administration, compared with that of the high fat diet-induced obese positive control group. Body weight of the group treated with 300 nig/kg of 50% aqueous ethanol heat extract was 16.5% decreased. 8 weeks later, body weight of the group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract was 23.9% decreased, while body weight of the group treated with 300 mg/kg of 50% aqueous ethanol heat extract was 19.5% reduced. That is, body weight reducing effect of the black tea 50% aqueous ethanol enfleurage extract was greater than that of the black tea 50% aqueous ethanol heat extract.

As shown in [Table 10] , body weights before and after the administration were compared. 8 weeks after the administration, the weight of the high fat diet group (Control) was approximately 1.68 g increased, but the weight of the mouse group treated with 300 mg/kg of the black tea 50% aqueous ethanol enfleurage extract was 11.47 g reduced, indicating that the administration group showed far greater weight reducing effect than the control. In the meantime, the weight of the group treated with 300 mg/kg of 50% aqueous ethanol heat extract was 9.01 g reduced, indicating that the extract had weight reducing effect as well, compared with the control (Figure 6 and Table 10) .

[Table 10]

Reducing effect of black tea aqueous ethanol enfleurage extract and black tea aqueous ethanol heat extract on body weight of high fat diet- induced obese mouse model

<2-2> Changes of body fat

Body fat of each mouse group was measured as shown in Example <l-2>.

As a result, as shown in Figure 7 and [Table 11] , it was confirmed that the weight loss shown in Example <2-l> was mostly attributed to the decrease of body fat. As shown in [Table 11] , body fat of the fat diet group (Control) was approximately 0.73% increased 8 weeks after the administration, compared with before the administration. However, body fat of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract was approximately 11.63% decreased, suggesting that body fat reducing effect of the extract was far greater than that of the control. In the meantime, body fat of the group treated with 300 mg/kg of the black tea 50% aqueous ethanol heat extract was approximately 6.48% reduced, indicating the extract had body fat reducing effect as well, compared with the control (Figure 7 and Table 11) .

[Table ll]

Reducing effect of black tea aqueous ethanol enfleurage extract and black tea aqueous ethanol heat extract on body fat of high fat diet-induced obese mouse model

Black tea 50% 29.53±0.83 17.91±2.49 -11.63%P aqueous ethanol

enfleurage extract

300 mg/kg

Black tea 50% 29.57±0.79 23.09±1.83 -6.48%P aqueous ethanol

heat extract

300 mg/kg

<2-3> Changes of blood sugar and cholesterol

Blood sugar and blood cholesterol were measured by the same manner as described in Example <l-4>.

As a result, as shown in Figure 8 and [Table 12] , blood sugar of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol enfleurage extract was significantly reduced (32.94+11.23 mg/dL) 8 weeks after the administration, compared with that of the control (blood sugar of high fat diet group (Control) : 86.03+14.02 mg/dL) . Blood sugar of the mouse group treated with 300 mg/kg of the black tea 50% aqueous ethanol heat extract was 61.94±8.98 mg/dL, which was also lower than that of the control, but the blood sugar reducing effect of the black tea 50% aqueous ethanol enfleurage extract was much greater.

Blood cholesterol of the group treated with 300 mg/kg of the black tea 50% aqueous ethanol enfleurage extract was 170.29±16.67 rag/dL, which was significantly lower level than that of the control (blood cholesterol of high fat diet group (Control) : 229.16±11.44 mg/dL) . Blood cholesterol of the group treated with 300 rag/kg of the black tea 50% aqueous ethanol heat extract was 177.95+10.39 mg/dL, indicating that this extract also reduced blood cholesterol as low as the black tea 50% aqueous ethanol enfleurage extract could do (Figure 8 and Table 12) .

[Table 12]

Reducing effect of black tea aqueous ethanol enfleurage extract and black tea aqueous ethanol heat extract on blood sugar and blood cholesterol of high fat diet- induced obese mouse model

300 mg/kg

<2-4> Histopathological test of liver

Histopathological test with the mouse liver was performed by the same manner as described in Example <l-5>.

As a result, as shown in Figure 9, the result of the histopathological test with the mouse liver extracted 8 weeks after the administration confirmed that fat globules of the mouse group treated with 300 nig/kg of the black tea 50% aqueous ethanol enf leurage extract were significantly decreased, compared with those of the high fat diet group, indicating that the extract of the present invention had the effect of improving fatty liver (Figure 9) .

Experimental Example 3: Inhibitory effect of black tea ethanol enfleurage extract on adipocyte differentiation

<3-l> Measurement of neutral fat in adipocytes

The inhibitory effect of the black tea 50% ethanol enfleurage extract obtained in Example 1 on adipocyte differentiation was measured.

Particularly, 3T3-L1 preadipocyt es were distributed at the density of 5 x 10³ cells/cm², followed by culture for 4 days. When the cell population grew enough to stop the proliferation because of contact inhibition, the cells were treated with differentiation hormone as follows, followed by further culture for 6 days .

Day 0: 10% FBS, 5 μg/ml Insulin, , 2 μΜ Dexamethasone (Dex) , 0.5 mM isobutylmethylxanthine (IBMX) ,

Day 2: 10% FBS, 5 /χΆΐ Insulin,

Day 4 : 10% FBS ,

Day 6: observation of differentiation, and

Day 6 ~ Day 10: Mature adipocytes finished with differentiation were cultured in the medium supplemented with 10% FBS for 4 days. The black tea 50% ethanol enfleurage extract was dissolved in the medium at the concentration of 20 mg/mt, which was filtered with 0.2 μΜ filter. Serial dilution was performed to prepare the extract diluents at proper concentrations. The non-differentiated preadipocytes (3T3-L1) were treated with adipocyte differentiation inducing hormones (0.5 mM 1 - isobutyl - 3 - methylxanthine ( IBMX) , 2 M dexamethasone (Dex) , 5 g/ml insulin ; MDI) to induce the differentiation into adipocytes (Kirkland et al . , 1990), during which the black tea 50% ethanol enfleurage extract was treated thereto at different concentrations of 0, 10, 25, 50, 100, and 200 ^g/m^. Then, cell differentiation was observed. On the 6^th day of the adipocyte differentiation, the medium was removed and the cells were washed with PBS twice and then fixed in 10% formaldehyde at room temperature for 1 hour. To stain the formed neutral fat, Oil Red 0 (Sigma #00625) was dissolved in isopropanol (0.5%) , which was diluted in distilled water at the ratio of 3:2. Oil Red O solution was added to the fixed 3T3-L1 cells in order for the cells to be submerged, leading to staining at

37°C for 30 minutes. Upon completion of staining, the stained Oil Red O was extracted by using isopropanol. Then, OD₅io was measured, followed by quantification.

As a result, as shown in Figure 10, adipogenesis was inhibited by the black tea 50% ethanol enfleurage extract dose-dependently (Figure 10A) . This result was consistent with the result of quantification with extracted Oil-Red O (Figure 10B) . The black tea 50% ethanol enfleurage extract was treated to the cells at different concentrations of 0, 100, 200, 250, 300, and 400 yg/mH. As a result, it was confirmed that 200 ~ 300 yg/m of the black tea 50% ethanol enfleurage extract would be enough amount to inhibit adipocyte differentiation (Figure 10 C) . <3-2> Measurement of adipogenic transcription factor

Upon completion of differentiation, total RNA was extracted by using RNeasy spin column (Qiagen. Chatsworth, CA, Cat. No. 74106) . The extracted RNA

(2ug) was reverse - transcribed at 37^°C for 1 hour with

M-MLV reverse transcriptase (Promega, Madison WI, Cat.

No. M170B) , dNTP and hexamer random primer. The synthesized cDNA (20 ng) was quantified with ABI 7000 Real-Time PCR System using Power SYBR Green PCR Master

Mix (Applied Biosystems, Cat. No. 4367659) .

Quantitative PCR was performed as follows: step 1)

50°C, 2 minutes; step 2) 95^°C, 10 minutes; step 3)

95°C, 15 seconds; step 4) 60°C, 1 minute; and step 5) repeating step 3) ~ step 4) 40 times. The primer sequences used herein were as follows ((F) : Forward primer and (R) : Reverse primer) .

C/EBPa (F) 5 ' -AGCCAAGAAGTCGGTGGACA- 3 ' (SEQ. ID.

NO : 1 ) ,

C/EBPa (R) 5 ' - CAGCCTAGAGATCCAGCGACC- 3 (SEQ. ID.

NO : 2 ) ;

PPARy2 (F) 5 CTGATGCACTGCCTATGAGCA- 3 ' (SEQ. ID. NO : 3 ) ,

PPARy2 (R) ' -ATGCGAGTGGTCTTCCATCAC - 3¹ (SEQ. ID. NO : 4 ) ; ACC (F) 5 ' -TAACAGAATCGACACTGGCTGGCT- 3 ' (SEQ. ID. NO : 5 ) ,

ACC (R) 5 ' -ATGCTGTTCCTCAGGCTCACATCT- 3 ' (SEQ. ID. NO: 6) ;

FAS (F) 5¹ -TGCTCCCAGCTGCAGGC- 3¹ (SEQ. ID. NO: 7),

FAS (R) 5 ' - GCCCCGTAGCTCTGGGTGTA- 3 ' (SEQ. ID. NO:

8) ;

FABP4 (F) 5 ' - GATGAAATCACCGCAGACGACA- 3 ' (SEQ. ID. NO: 9) ,

FABP4 (R) 5 ' -ATTGTGGTCGACTTTCCATCCC- 3 ' (SEQ. ID.

NO : 10 ) ;

18s rRNA (F) 5 ' -ACCGCAGCTAGGAATAATGGAATA- 3 ' (SEQ. ID. NO: 11) ,

18s rRNA (R) 5¹ - CTTTCGCTCTGGTCCGTCTT- 3 ' (SEQ . ID. NO: 12) .

Changes of adipogenic transcription factors were confirmed by using RT-PCR with the cells of Experimental Example <3-l>. The expressions of C/EBPa and P PAR Y2, known as the master transcription factors, are very important in the process of adipocyte differentiation.

As a result, as shown in Figure 10, even though adipocyte differentiation was inhibited by the black tea 50% ethanol enfleurage extract, the levels of C/ΕΒΡα and PPARy2 mRNA were maintained regularly and the expressions of their target genes FABP4 , FAS and ACC were not affected either (Figure 10 D) . When 50 ^g/mi of the black tea 50% ethanol enf leurage extract was treated, the amount of fat was 50 ~ 70% reduced, confirmed by Oil Red-0 quantification, without changing the expressions of PPARy2 and C/EBPa, the master transcription factors necessary for the differentiation of preadxpocytes into adipocytes.

Experimental Example 4: Inhibitory effect of black tea ethanol enf leurage extract on mature adipocytes

<4-l> Measurement of neutral fat in adipocytes

To investigate the inhibitory effect of the black tea 50% ethanol enfleurage extract obtained in Example 1 on mature adipocytes, the mature 3T3-L1 adipocytes were treated with the black tea ethanol extract at different concentrations of 100, 200, 300, 500, 750, and 1000 βg/Wίi. 4 days later, neutral fat in the cells was stained with Oil Red-O, followed by investigation.

As a result, as shown in Figure 11, it was confirmed that the intracellular fat was reduced by the treatment of 300 /.g/mi of the black tea 50% ethanol enfleurage extract (Figure 11A) . When the mature adipocytes were treated 100 //g/m£ of the black tea 50% ethanol enfleurage extract, the existing fat was approximately 70% reduced. These results were consistent with the result of quantification via Oil Red-0 staining (Figure 11B) .

<4-2> Measurement of adipogenic transcription factor

Changes of adipogenic transcription factors were confirmed by using RT-PCR as described in Example <3- 2> with the cells of Experimental Example <4-l>.

As a result, as shown in Figure 11, even though adipocyte differentiation was inhibited by the black tea 50% ethanol enfleurage extract, the levels of C/EBPoi and PPARy2 mRNA were maintained regularly and the expressions of their target genes FABP4 , FAS, and ACC were not changed, either (Figure 11C) .

Therefore, it was confirmed that the black tea 50% ethanol enfleurage extract had the effect of reducing fat but had no effect on the expressions of PPARy2 and C/EBP . The malfunction of PPARy2 could cause various metabolic diseases including insulin resistance. Therefore, the above results indicate that the black tea 50% ethanol enfleurage extract does not carry any side effect caused by PPARy2 malfunctioning. Experimental Example 5: Inhibitory effect of black tea 50% ethanol enfleurage extract on proliferation during adipocyte differentiation

To investigate whether or not the black tea 50% ethanol enfleurage extract obtained in Example 1 could inhibit the proliferation of preadipocytes, 3T3-L1 cells were treated with 300 yg/v of the black tea 50% 50% ethanol enfleurage extract in the period of adipocyte differentiation. Then, cell morphology was observed under optical microscope and the live cells were counted.

As a result, as shown in Figure 12, the proliferation was inhibited in the early differentiation stage, unlike in the control (Figure 12 A and Figure 12 B) . When non-differentiated 3T3-L1 preadipocytes were treated with 300 iig / v of the black tea 50% ethanol enfleurage extract, the proliferation of preadipocytes was also inhibited (Figure 12 C) .

Therefore, it was confirmed that the extract of the present invention inhibited adipocyte differentiation and at the same time inhibited the proliferation of preadipocytes in order to inhibit adipogenesis . Manufacturing Example 1: Preparation of injectable solutions

The black tea aqueous ethanol enfleurage extract of the present invention 100 nig Sodium metabisulphi te 3.0 nig

Methylparaben 0.8 nig

Propylparaben 0.1 nig

NaCl 14 nig

Sterilized distilled water proper amount Injectable solutions were prepared by mixing all the above components, putting the mixture into 2 mi ampoules and sterilizing thereof by the conventional method for preparing injectable solutions. Manufacturing Example 2: Preparation of tablets

The black tea aqueous ethanol enfleurage extract of the present invention 200 nig

Lactose 100 nig

Macrocrystalline cellulose 50 nig Crospovidone 20 nig

Magnesium stearate proper amount

Tablets were prepared by mixing all the above components by the conventional method for preparing tablets. Manufacturing Example 3: Preparation of capsules

The black tea aqueous ethanol enfleurage extract of the present invention 100 mg

Lactose 50 mg Low substituted hydroxypropylcellulose 20 mg

Silicon dioxide 5 mg

Talc 2 mg

Magnesium stearate proper amount

Capsules were prepared by mixing all the above components, which filled gelatin capsules according to the conventional method for preparing capsules.

Manufacturing Example 4: Preparation of liquid formulations

The black tea aqueous ethanol enfleurage extract of the present invention 1000 mg

Sugar 20 g

Anhydrite citric acid 3 g

Fructose 20 g Lemon flavor proper amount

All the above components were dissolved in purified water. After adding lemon flavor, total volume was adjusted to be 100 ml by adding purified water. Liquid formulations were prepared by putting the mixture into 100 ml brown bottles and sterilizing thereof by the conventional method for preparing liquid formulations.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient.

2. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea is preferably selected from the group consisting of Byeongcha, Jeoncha, Tacha, Bangcha, Kumkwakongcha , Kangjun, Kumchun, Bokjun, Huekjun, Whajun,

Nochunbyeong , Yookbocha, puer tea, and red tea.

3. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the aqueous ethanol is preferably 30% ~ 70% aqueous ethanol solution.

4. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract is prepared by the method comprising the following steps: 1) mixing black tea and 30% - 70% aqueous ethanol solution at the ratio of 1:4 ~ 1:8;

2) extracting the mixture of step 1) for 60 ~ 80 hours ; and

5. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract is prepared by the method comprising the following steps:

1) mixing black tea and 50% aqueous ethanol solution at the ratio of 1:6;

2) extracting the mixture of step 1) at 10 ~ 30 ^°C for 72 hours; and

6. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the metabolic disease is one or more diseases selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia , cardiovascular disease, atherosclerosis, and lipid related metabolic disease.

7. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract contains gallic acid at the concentration of 2 ~ 8% (w/w) .

8. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract is characterized by reducing body weight.

9. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract is characterized by reducing body fat.

10. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 9, wherein the decrease of body fat is characteristically the decrease of abdominal fat or subcutaneous fat.

11. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 9, wherein the decrease of body fat is attributed to the inhibition of adipocyte differentiation .

12. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 11, wherein the inhibition of adipocyte differentiation affects neither the expressions of C/EBP and P PARY2, the adipogenic transcription factors, nor the expressions of their target genes FABP4, FAS, and ACC as well.

13. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 9, wherein the decrease of body fat is induced by the suppression of proliferation of preadipocytes or adipocytes .

14. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract characteristically improves fatty liver.

15. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract characteristically reduces blood cholesterol.

16. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract characteristically reduces blood sugar.

17. The pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease according to claim 1, wherein the black tea aqueous ethanol enfleurage extract is characterized by having no such side effects as metabolic disease mediated by PPARy2 malfunctioning including insulin resistant diabetes.

18. A health food for preventing or improving obesity or lipid related metabolic disease which comprises the black tea aqueous ethanol enfleurage extract as an active ingredient.

19. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea is preferably selected from the group consisting of Byeongcha, Jeoncha, Tacha, Bangcha, Kumkwakongcha , Kangjun, Kumchun, Bokjun, Huekjun, Whajun, Nochunbyeong, Yookbocha, puer tea, and red tea.

20. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the aqueous ethanol is preferably 30% ~ 70% aqueous ethanol solution.

21. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract is prepared by the method comprising the following steps:

1) mixing black tea and 30% ~ 70% aqueous ethanol solution at the ratio of 1:4 ~ 1:8; 2) extracting the mixture of step 1) for 60 ~ 80 hours; and

22. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract is prepared by the method comprising the following steps:

1) mixing black tea and 50% aqueous ethanol solution at the ratio of 1:6;

2) extracting the mixture of step 1) at 10 ~ 30 °C for 72 hours; and

23. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the metabolic disease is one or more diseases selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia , cardiovascular disease, atherosclerosis, and lipid related metabolic disease.

24. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract contains gallic acid at the concentration of 2 ~ 8% (w/w) .

25. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract is characterized by reducing body weight.

26. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract is characterized by reducing body fat .

27. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the decrease of body fat is characteristically the decrease of abdominal fat or subcutaneous fat .

28. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 26, wherein the decrease of body fat is attributed to the inhibition of adipocyte differentiation.

29. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 28, wherein the inhibition of adipocyte differentiation affects neither the expressions of C/EBPa and PPARy2, the adipogenic transcription factors, nor the expressions of their target genes FABP4, FAS, and ACC as well.

30. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 26, wherein the decrease of body fat is induced by the suppression of proliferation of preadipocytes or adipocytes.

31. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract characteristically improves fatty liver.

32. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract characteristically reduces blood cholesterol .

33. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract characteristically reduces blood sugar .

34. The health food for preventing or improving obesity or lipid related metabolic disease according to claim 18, wherein the black tea aqueous ethanol enfleurage extract is characterized by having no such side effects as metabolic disease mediated by PPARy2 malfunctioning including insulin resistant diabetes.

35. A method for preventing obesity or lipid related metabolic disease which includes the step of administering a pharmaceutically effective dose of the black tea aqueous ethanol enfleurage extract to a subject.

36. A method for treating obesity or lipid related metabolic disease which includes the step of administering a pharmaceutically effective dose of the black tea aqueous ethanol enfleurage extract to a subject having obesity or lipid related metabolic disease .

37. A use of the black tea aqueous ethanol enfleurage extract for the preparation of a pharmaceutical composition for preventing or treating obesity or lipid related metabolic disease.

38. A use of the black tea aqueous ethanol enfleurage extract for the preparation of a health food for preventing or improving obesity or lipid related metabolic disease.