KR101505895B1 - A composition for inhibiting adipogenesis comprising 3,4`-Dihydroxyflavone - Google Patents

Abstract

The present invention relates to a composition for inhibiting adipocyte formation comprising 3,4'-dihydroxyflavone as an active ingredient.

Description

A composition for inhibiting adipocyte formation comprising 3,4'-dihydroxyflavone as an active ingredient {Compositions for inhibiting adipogenesis comprising 3,4-Dihydroxyflavone}

The present invention relates to a composition for inhibiting adipocyte formation comprising 3,4'-dihydroxyflavone as an active ingredient.

Globally, overweight and obese populations are increasing rapidly, leading to several complications such as type II diabetes, hypertension and cardiovascular disease, thus causing macroeconomic burdens in many countries [Hausman DB, DiGirolamo M , Bartness TJ, Hausman GJ, Martin RJ. 2001. Obes Rev 2: 239-254.]. In overweight and obesity, adipocytes and adipose tissue play an important role. Fat cells accumulate excess energy in the form of triglycerides and secrete free fatty acids in response to energy needs. Adipose tissue is mainly composed of adipocytes, they are metabolic organs that integrate several physiological pathways, endocrine organs that secrete hormones and cytokines, and are essential for systemic insulin sensitivity and energy production [Rosen ED, Spiegelman BM. 2006. Nature 444: 847-853). Adipogenesis is a key regulator of adipose tissue production and adipocyte production [Camp HS, Ren D, Leff T. 2002. Trends Mol Med 8: 442-447.]. It is precisely regulated by positive and negative stimuli, involving molecules and multiple transcription factors involved in the insulin signaling pathway, which require fusion of multiple stimuli including nutrients and hormones [Koutnikova H, Auwerx J. 2001. Ann Med. 33: 556-561].

Previous studies have identified peroxisome proliferator-activated receptor gamma (PPAR) gamma and cytidine-cytidine- adenosine- adenosine- thymidine / enhancer-binding proteins (C / EBPs) Farmer SR. 2006. Cell Metab 4: 263-273]. Insulin-like factor I (IGF-I) and epidermal growth factor (EGF) are also known as adipocyte regulatory proteins [Boney CM, Smith RM, Gruppuso PA. 1998. Endocrinology 139: 1638-1644].

Flavonoids, plant-derived polyphenolic compounds, are widely distributed in fruits and vegetables and are therefore routinely consumed in human diets [Di Carlo G, Mascolo N, Izzo AA, Capasso F. 1999. Life Sci 65: 337-353]. They are characterized by a diphenylpropane (C ₆ C ₃ C ₆ ) skeleton. There is a physiological advantage in flavonoids, including protection from cardiovascular disease and cancer and oxidative stress [Di et al., 1999].

In general, the MEK / ERK (mitogen-activated protein kinase / extracellular signal-regulated kinase) signaling pathway is associated with adipocyte formation (Prusty D, Park BH, Davis KE, Farmer SR. 2002. J Biol Chem 277: 46226-46232). The ERK signaling pathway plays a diverse role in adipocyte formation essential to the major adipocyte formation regulator [Bost F, Aouadi M, Caron L, Binetruy B. 2005. Biochimie 87: 51-56].

With respect to overweight and obesity issues, the present inventors have focused on flavonoids that regulate the ERK signaling pathway as a safer and more effective natural substance that inhibits or retards adipocyte formation.

[Related Prior Art Patent Literature]

Korean Patent Application No. 10-2011-0109130

It is an object of the present invention to provide a flavonoid that inhibits or inhibits adipocyte formation.

In order to achieve the above object, the present invention provides a composition for inhibiting adipocyte formation comprising 3,4'-dihydroxyflavone as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing and treating obesity comprising 3,4'-dihydroxyflavone as an active ingredient.

The present invention also provides a food composition for improving obesity comprising 3,4-dihydroxyflavone as an active ingredient.

The present invention also provides a composition for inhibiting the expression of PPAR gamma (peroxisome proliferator-activated receptor gamma) comprising 3,4'-dihydroxyflavone as an active ingredient.

The present invention also provides a method for inhibiting the differentiation of precursor adipocytes by treating 3,4'-dihydroxyflavone with precursor adipocytes.

The present invention also provides a method for inhibiting lipid accumulation in a subject by treating 3,4'-dihydroxyflavone with total fat adipocytes.

The present invention also provides a method for inhibiting the expression of PPAR gamma (peroxisome proliferator-activated receptor gamma) of a cell by treating 3,4'-dihydroxyflavone with pro-adipocytes.

In the present invention, "pharmaceutically acceptable" can be administered to a patient without causing undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition containing it. The carrier may be selected to minimize any degradation of the active ingredient, as is well known to those skilled in the art, and to minimize any deleterious side effects in the patient.

The compositions of the present invention may be administered topically, orally, or parenterally. For example, the composition can be administered extracorporeally, into the skull, into the vagina, into the rectum, into the skin, into the heart, into the heart, into the stomach, intravenously, intramuscularly, intraperitoneally, percutaneously, intranasally, ≪ / RTI > As used herein, "intracranial administration" refers to administration of a substance directly to the brain via a catheter or needle, such as through an intrathecal, intracisternal, intracisternal, or transsphenoidal ).

The parenteral administration of the composition, when used, is generally characterized by injection. The injectable can be prepared in conventional form, as a liquid solution or suspension, in the form of a solid suitable for suspension in liquid before injection, or as an emulsion. More recently, the approach for improved parenteral administration involves living to maintain a constant dose by using a slow release or sustained release system (see U.S. Patent No. 3,610,795).

The exact amount of composition required will depend on the patient, age, weight and general condition of the patient, the severity of the allergic disorder being treated, the manner of administration, etc., depending on the patient. It is therefore impossible to determine the exact amount for all compositions. However, the appropriate amount can also be determined by those skilled in the art using only basic experimentation according to the teachings provided in the art.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other therapeutically active compounds as well as in a suitable set.

And may be formulated in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions. Examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose , Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The present invention provides a health supplement comprising a compound of the present invention and a pharmaceutically acceptable food supplementary additive exhibiting an obesity suppressing and improving effect. Examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in powder, granule, tablet, have.

It can also be added to food or beverage for the purpose of promoting the improvement of obesity. At this time, the amount of the compound in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, have.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the compound of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described.

In the present invention, the present inventors investigated whether flavonoids have the ability to regulate adipocyte formation. The present inventors screened with hydroxyl (OH) substituents having various numbers and positions of flavonoids. The present inventors analyzed the effect of flavonoids on cell growth of various cell lines. Next, flavonoids were pretreated with 3T3-L1 preadipocytes to assess the effect of flavonoids on adipocyte formation. The present inventors have found that the treatment of 3,4'-dihydroxyflavone (3,4'-DHF) reduces expression of adipocyte formation markers and lipid accumulation. Finally, we observed that the 3, 4'-DHF-pre-treated cells inhibited MEK / ERK phosphorylation. These results suggest that 3,4'-DHF inhibits adipocyte formation through the MEK / ERK signaling pathway.

Hereinafter, the present invention will be described in detail.

Effect of Flavoid on Cell Survival Rate of Preadipocytes

We evaluated the effect of plavoids on cell viability of various cell lines including 3T3-L1 precursor adipocytes, NIH-3T3 fibroblasts. Substantially, 3T3-L1 precursor adipocytes and NIH-3T3 fibroblasts were cloned from heterogenenous Swiss 3T3 cells [Green H, Meuth M. 1974. Cell 3: 127-133]. 3T3-L1 cells differentiate into adipocytes under conditioned cell culture conditions [Green et al., 1974]. The differentiated 3T3-L1 adipocytes have morphological and biochemical characteristics of adipocytes. Thus, 3T3-L1 cells are able to investigate the regulation mechanism of adipocyte differentiation [Green H, Kehinde O. 1976. Cell 7: 105-113].

Treatment of 3,4'-DHF did not show a significant decrease in the proliferation of 3T3-L1, NIH-3T3 (Fig. 2B). On the other hand, 3-HF caused a significant decrease in cell proliferation including 3T3-L1, NIH-3T3 cells. Treatment of 3,2'-DHF and 3,3'-DHF also resulted in a significant reduction in cell proliferation of 3T3-L1 and NIH-3T3. These results confirmed that the OH moiety plays an important role in the biological function of flavonoids in the B ring in the C ₆ C ₃ C ₆ skeleton.

The present inventors have investigated the effect of Plavoid on the adipocyte formation of 3T3-L1 precursor adipocytes. First, we performed induction of adipocyte formation of 3T3-L1 cells (Fig. 1A-a) and observed time-dependent expression of a typical adipocyte-forming marker PPARgamma that regulates adipocyte-specific gene expression and terminal differentiation I could. Regarding the induction of adipocyte formation, pre-adipocyte factor (Pref 1) was expressed in differentiated 3T3-L1 in addition to the typical adipocyte formation markers PPAR gamma, C / EBP beta and C / EBP alpha (Fig. 1A-b). For oil red O staining, we were able to detect accumulation of protoplasmic droplets and obtain a significant increase (approximately 7-fold) of Oil red O staining (Fig. 1B), which successfully induced 3T3- Suggesting that L1 cells were differentiated.

Effect of 3,4'-DHF on progenitor adipocyte differentiation

We evaluated the effect of flavonoids on adipocyte differentiation of 3T3-L1 cells. Because 3,4'-DHF did not show deleterious effects on the proliferation of 3T3-L1 cells, we performed induction of adipocyte formation on 3,4'-DHF pretreated 3T3-L1 cells (Figure 3A) . The role of 3,4'-DHF during adipocyte formation was examined by intracellular lipids (Fig. 3B). In oil red O staining, the present inventors were able to detect a time-dependent accumulation of intracellular lipids and found that lipid accumulation was reduced by 3,4'-DHF pretreatment (FIG. 3B). When quantifying the amount of staining, the data also showed a clear inhibition of oil red O staining (more than two-fold) by 3,4'-DHF pretreatment. Flavonoid-pretreated 3T3-L1 cells also showed a significant decrease in the production of intracellular triglycerides 6 days and 8 days after induction of adipocyte formation. These results indicate that 3,4'-DHF has an effect on lipid accumulation and adipocyte formation.

3,4'-DHF inhibits adipocyte differentiation by inhibiting MEK / ERK activation in 3T3-L1 cells

The role of 3,4'-DHF in adipocyte formation and its correlation with MEK / ERK signaling were investigated. We have found that the expression of PPAR gamma is reduced by 3,4'-DHF pretreatment, which correlates with reduced accumulation of triglycerides and decreased expression of Oil red O in differentiated 3T3-L1 cells. Next, ERK and MEK activation during adipocyte formation was investigated in the presence and absence of 3,4'-DHF. The present inventors observed that although the phosphor-MEK and phosphor-ERK levels were increased as adipogenic differentiation proceeded, activation of MEK and ERK was markedly inhibited by 3,4'-DHF pretreatment.

Finally, PPAR gamma phosphor-MEK / ERK and total MEK / ERK expression during adipocyte formation were investigated at days 0, 2, 4, 6 and 8. Without the 3, 4 ' -DHF treatment, when adipocyte-forming hormone was added on day 2, MEK phosphorylation began to increase (left panel of FIG. 4B). Phosphor-ERK levels were observed from 4 days and increased until 8 days. In the early adipocyte differentiation, MEK / ERK phosphorylation and PPAR gamma expression began to increase (FIG. 4B). On the other hand, when the precursor adipocytes were treated with 3, 4'-DHF, significant low PPAR gamma expression and MEK / ERK phosphorylation were detected (right panel of FIG. 4B). These results suggest that 3,4'-DHF pre-treatment not only inhibits MEK and ERK phosphorylation but also inhibits the expression of adipocyte transcription factors such as PPAR gamma.

 As can be seen from the present invention, 3,4'-DHF reduces MEK / ERK activation and inhibits adipocyte formation. Because 3,4'-DHF has an anti-adipogenic effect and adipocyte regulation is important in fat tissue expansion, this compound can provide a new alternative to overweight or obesity treatments.

Fig. 1 is a diagram derived from 3T3-L1 precursor adipocytes for adipocyte formation. Fig. A: RT-PCR shows only PPAR gamma which is expressed in time-dependent manner. b, PPAR gamma, C / EBP beta, C / EBP alpha and pref 1 (preadipocyte marker) were expressed after induction of differentiation. B: Percentage of oil red O staining showed approximately 300% intracellular lipid accumulation (* p < 0.05) on differentiated 3T3-L1 cells compared to that for preadipocytes
Figure 2 shows the flavonoid effect on cell viability of 3T3-L1 (precursor adipocytes) and NIH-3T3 (fibroblast) cells. A: 3-hydroxyflavone (3-HF), 3,2'-dihydroxyflavone (3,2'-DHF), 3,3'-dihydroxyflavone (3,3'- 4'-DHF). B: Effect of four flavonoids on 3T3-L1, NIH-3T3 cell proliferation. Cell proliferation was compared to the control and marked at different levels * (p <0.05). All experiments were performed three times.
Figure 3 shows the effect of 3,4'-DHF on 3T3-L1 progenitor adipocyte differentiation. A: Scheme showing timeline of cell differentiation. B: In the presence of 3,4'-DHF, intracellular lipids were less accumulated and the percentage of Oil red O staining was significantly reduced compared to the case without 3,4'-DHF pretreatment (p <0.05, * Display). C: The cells were disrupted for the triglyceride assay and the ratio of triglyceride amount was significantly reduced by 3,4'-DHF treatment. All experiments were performed three times.
Figure 4 shows that 3,4'-DHF inhibits PPAR gamma and MEK / ERK phosphorylation. A: The MEK / ERK pathway is activated during 3T3-L1 differentiation. The expression of PPAR gamma expresses the process of adipocyte formation by inducing adipocyte formation. 3,4`-DHF treatment reduces PPAR gamma level as well as MEK / ERK phosphorylation level. B: In the control group (without 3,4`-DHF treatment), phosphorylated MEK and ERK were observed at days 2 and 4, respectively. PPARgamma induces adipocyte induction hormone addition 2. From day 4, the downstream of the MEK / ERK signaling pathway is activated. MEK and ERK phosphorylation was delayed to 8 days and decreased PPAR gamma activity was observed.
Figure 5 is an illustration of 3,4'-DHF inhibition of adipocyte formation in 3T3-L1. 3,4'-DHF inhibits PPARgamma and MEK / ERK signaling pathways in adipocyte formation.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

The flavonoids of the present invention (3-hydroxyflavone (3-HF), 3,2'-dihydroxyflavone (3,2'-DHF), 3,3'-dihydroxyflavone dihydroxyflavone (3,4'-DHF)) was purchased from Indofine Chemical Inc. (Hillsborough, NJ) and dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich).

Example 1: Cell culture and adipocyte differentiation

3T3-L1 precursor adipocytes and NIH-3T3 fibroblasts were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen) and 100 U / ml penicillin-streptomycin (Sigma-Aldrich) . The adipocyte formation is schematically illustrated in FIG. 3A and described previously [Harrington M, Pond-Tor S, Boney CM. 2007. Obesity 15: 563-571]. Briefly, precursor adipocytes were cultured with flavonoid (30 μM) for 24 hours. The medium was replaced with adipocyte induction medium consisting of 1 μM dexamethasone (DEX, Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich) and 5 μg / ml insulin And cultured for 2 days. DEX and IBMX were recovered after 2 days of exposure and insulin was recovered after 4 days. The medium was then changed to the normal culture medium and kept for 2 days.

Example 2: RNA isolation and RT-PCR

Total RNA was isolated from 3T3-L1 cells by Trizol (Sigma-Aldrich) at each time point after induction of adipocyte formation. cDNA was synthesized with M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. The reverse transcription reaction was performed with total RNA and the PCR products were separated by electrophoresis on 1% agarose gel. The primers used for PCR were: preadipocyte factor 1 (Pref-1), forward (F) 5'-CTAACCCATGCGAGAACGAT-3`, Reverse (R) 5`-GCTTGCACAGACTCGAAG-3`; PPAR gamma F 5`-CAAGCCCTTTACCACAGTTG-3`, R 5`-CCCTTGCATCCTTCACAAG-3`; C / EBP alpha F 5`-CAAGCCGAGCAAGAAGCCGG-3`, R 5`-GCTGCGGTTCCGGCGCGGGCGGCGGAC-3`; C / EBP beta F 5 '-GAACCTGGAAGCTTGTCGCC-3`, R 5`-ACCAGCTTGTCACCATCTCG-3`; GAPDH, F5`-TGCTTCTGGTGATGGCTACTGCCTTC-3` and R5`-GGTACTCCTGGAAG ATGTCCACC-3`. Each oligonucleotide set was designed to span two different exons to avoid genomic DNA contamination.

Example 3: Cell viability assay

Cell proliferation was assessed using a conventional MTT assay [Hsu CL, Yen GC. 2007. J Agric Food Chem 55: 1730-1736. Specifically, 3T3-L1 cells were plated in 96-well plates at a density of 1 x ¹⁰ cells. After 24 hours, the culture medium was replaced with a medium containing flavonoids (3-HF, 3,2'-DHF, 3,3'-DHF, or 3,4'-DHF) and cultured for 24 hours. Cells were treated with MTT solution (Sigma-Aldrich; final concentration, 0.25 mg / ml) for 4 hours at 37 ° C. Formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using an enzyme-linked immune-sorbent essay reader (ELISA; Bio-Rad). Cell viability was expressed as percentage (%) of cell growth compared to control.

Example  4: Intracellular  Lipid accumulation analysis

Adipocyte formation is induced in adipocyte induction medium, and is induced by the previously described [Fu M, Sun T, Bookout AL, Downes M, Yu RT, Evans RM, Mangelsdorf DJ. 2005. Mol Endocrinol 19: 2437-2450], as described above, using Oil Red O staining. In summary, the cells were washed twice with PBS and fixed with 10% formaldehyde for 20 minutes. After washing twice with PBS, the cells were incubated with Oil Red O reagent (0.1 mg / ml, Sigma-Aldrich) for 1 hour and washed twice with distilled water. The dyes from the cells were extracted with isopropyl alcohol and the amount of dye quantified at 510 nm with an ELISA reader.

Example 5: Analysis of the amount of triglyceride

Cells were harvested and disrupted in disruption buffer (5% Triton X-100) and the amount of intracellular total triglyceride was determined using a conventional triglyceride assay kit (DiaSys Diagnostic Systems GmbH, Germany).

Example 6: Western blot analysis

Cells were treated with 1% Triton X-100, 100 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM p- nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride and protease inhibitors -Aldrich). &Lt; / RTI &gt; The protein concentration was measured with a Bradford protein assay reagent (Bio-Rad). Equal amounts of proteins were loaded into each well and separated by 10% SDS PAGE. Protein was transferred to nitrocellulose membrane (Millipore) and blocked in 5% skim milk powder. Samples were probed with the following primary antibodies: phosphor-ERK (Santa Cruz), ERK (Santa Cruz), phosphor-MEK (Cell Signaling technology), MEK (Santa Cruz), PPAR gamma (Santa Cruz), GAPDH ), And beta-actin (Santa Cruz). Secondary antibodies were either goat anti-mouse IgG- or anti-rabbit IgG-peroxidase attachment (Amersham Biosciences) and their protein bands detected by enhanced chemiluminescence (Amersham Biosciences).

The data of the present invention are expressed as the mean ± standard deviation of the mean (SEM). Statistical analysis was performed on an Excel program (Microsoft, Redmond, WA) using one-way ANOVA (Analysis of variance) or two-tailed Student's t-test. Significant differences were considered at P <0.05.

Claims (7)

delete  A pharmaceutical composition for preventing and treating obesity comprising 3,4'-dihydroxyflavone as an active ingredient. A dietary composition for improving obesity comprising 3,4-dihydroxyflavone as an active ingredient. delete A method of inhibiting the differentiation of precursor adipocytes in vitro by treating 3,4'-dihydroxyflavone with total fat cells in vitro. 3,4'-Dihydroxyflavone is treated in vitro to precursor adipocytes to inhibit lipid accumulation of the cells in vitro. A method of inhibiting the expression of PPAR gamma (peroxisome proliferator-activated receptor gamma) in the in vitro by treating 3,4'-dihydroxyflavone with total fat cells in vitro.

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