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journal of functional foods 12 (2015) 208–218

Available online at

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff

Aqueous extracts of hulled barley containing
coumaric acid and ferulic acid inhibit
adipogenesis in vitro and obesity in vivo
Cho-Rong Seo a, BoRa Yi a, Sumi Oh a, So-Mi Kwon a, Suji Kim a,
No-Joon Song a, Jae Youl Cho b, Ki-Moon Park a, Jee-Yin Ahn c,
Joung-Woo Hong d, Mi-Ja Kim a, JaeHwan Lee a, Kye Won Park a,*

Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Korea
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea
Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute,
Sungkyunkwan University School of Medicine, Suwon 440-746, Korea
Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Republic of Korea




Article history:

World epidemic obesity is a major contributing factor to metabolic diseases including insulin

Received 4 June 2014

resistance and cardiovascular diseases. In this study, aqueous and ethanolic extracts of hulled

Received in revised form 23

barley with roasting temperatures of up to 250 °C were investigated for their anti-

November 2014

adipogenic effects in vitro and in vivo. An aqueous extract of hulled barley roasted at 210 °C

Accepted 26 November 2014

(AHB210) effectively inhibited adipocyte differentiation. Intraperitoneal injections of 15 or

Available online 10 December 2014

50 mg/kg AHB210 dose dependently prevented body weight gain, fat mass increase, and
dysregulated lipid profiles in high fat diet-induced obese male mice. In addition, oral ad-


ministration of AHB210 to ovariectomized rats also prevented body weight gain. A high

Hulled barley

performance liquid chromatographic analysis identified coumaric acid and ferulic acid as

Adipocyte differentiation

primary anti-obesity mediators. The presence of beta glucan in AHB210 was less likely to


be responsible for the lipid accumulating actions. Taken together, AHB210 may be useful

Coumaric acid

to prevent obesity and its related metabolic diseases.

Ferulic acid

© 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon 440-746, Korea. Tel.: (+82) 31
290 7804; fax: (+82) 31 290 7882.
E-mail address: (K.W. Park).
Abbreviations: AHB, aqueous extracts of hulled barley; EHB, ethanol extracts of hulled barley; AHB−, aqueous extracts of hulled barley
without roasting; EHB−, ethanol extracts of hulled barley without roasting; AHB170, aqueous extracts of hulled barley roasted at 170 °C;
EHB170, ethanol extracts of hulled barley roasted at 170 °C; AHB210, aqueous extracts of hulled barley roasted at 210 °C; EHB210, ethanol
extracts of hulled barley roasted at 210 °C; AHB250, aqueous extracts of hulled barley roasted at 250 °C; EHB250, ethanol extracts of hulled
barley roasted at 250 °C; PPARγ, peroxisome proliferator-activated receptor γ; C/EBPα, CCAAT-enhancer-binding proteins α; aP2, adipocyte
protein 2; CD36, cluster of differentiation 36; MSC, mesenchymal stem cells; DMI, dexamethasone; IBMX, insulin; NCD, normal chow diet;
HFD, high fat diet; CA, coumaric acid; FA, ferulic acid; PCA, protocatechuic acid; ChA, chlorogenic acid; HA, 4-hydroxybenzoic acid; VA,
vanillic acid
1756-4646/© 2014 Elsevier Ltd. All rights reserved.

journal of functional foods 12 (2015) 208–218 1. 2006). Cyperus rotundus. and cardiovascular diseases (Behall et al. Logan. Finally. and hulled barley (or covered barley). The PPARγ ligand GW7845 (20 nM. Saitama. chlorogenic acid. However. Barley is generally classified as hulless barley (or naked barley) separated from the husk. Tontonoz. Gupta. further suggesting that these compounds are primary anti-obesity mediators. Drotleff. It has various preventive and therapeutic functions against hypertension. Treatment of diabetic mice with peroxisome proliferator-activated receptor γ (PPARγ) agonists increases the number of smaller adipocytes. liver.. Other bioactive compounds in barley include phenolic acids such as ferulic acid. Ltd.. Japan). Korea). Gyinggido. 2006). & Hallfrisch. kindly provided by Dr. USA) supplemented with 10% fetal bovine serum (FBS) and antibiotics. hydroxybenzoic acid. ferutinin. Scholfield. Moon. Remesar. atherosclerosis. Mixtures of roasted hulled barley with 90 °C deionized water were further heated in a drying oven (HYSC Co. & Scherer. 210. 2011). the mixtures were filtered through Whatman #2 filter paper and lyophilized using a freeze-drier (Ilshinbiobase Co. 3T3-L1 preadipocytes were cultured in DMEM supplemented with 10% fetal calf serum (Hyclone) and antibiotics. atherosclerosis. Barley provides various beneficial health effects for obesity. Yu. and genistein also mediate the effects of anti-obesity of herbal extracts (Meydani & Hasan. and stored at −20 °C before use. and 250 °C using a coffee roaster (Model CBR-101. type 2 diabetes.. Scholfield. our data also show that phenolic compounds including coumaric acid and ferulic acid are enriched in the anti-adipogenic water extracts of hulled barley. Cell culture Mesenchymal C3H10T1/2 and preadipocyte 3T3-L1 cells were purchased from the American Type Culture Collection (Manassas. Los Angeles. To induce adipocyte differentiation cells were seeded at 5 × 10 4/ml in 6-well plates. Korea). 1997). Behall. Louis. 2012. 2004. Burley. and 5 µg/ ml insulin (Sigma-Aldrich) for 2 days followed by media containing FBS and insulin. heart. Roasted hulled barley was mixed with 70% ethanol or 90 °C deionized water at the ratio of 1:2 (w/w). Cho. Korea).. 2005. Kim. 2011). Cichorium intybus.. Wang. & Cha. either dietary fiber or polyphenolic compounds mediate the anti-obesity effects of herbal extracts. 2. Briefly. Lee. Yadav. Cade. Genesis Co.2. as it decreases food intake and satiety (Behall. 2004. 2012.. aqueous and ethanol extracts of hulled barley roasted up to 250 °C were prepared and investigated for their anti-lipogenic effects on preadipocytes. Delaney et al. St. C3H10T1/2 cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone. UCLA. 170. & Roh. & Ternes. Preparation of aqueous and ethanol extracts of hulled barley Hulled barley was provided by a local barley supplier (Icheon. 2000). Seoul. & Greenwood. whereas covered barley is used as animal fodder and as a source of fermentable material for beer and certain distilled beverages (Tosh. 2003). Lipid accumulation in muscle. 1997. Additionally. Korea) at 100 °C for 30 min. resveratrol. 2013). These compounds exert their 209 effects in many cell types including adipocytes. Adipocyte hypertrophy is believed to be a major contributing factor in adult onset obesity. suggesting that further understanding of both the number and size of adipocytes are critical issues for obesity and metabolic diseases (Prins & O’Rahilly. protocatechuic acid. 2014). & Dilshad. Song. Tontonoz & Spiegelman. USA) and maintained and differentiated into adipocytes as described previously (Song et al. & Fernandez-Lopez. Ltd. 2. . and obesity (Ahmad. 2013). Plant extracts including Hippophae rhamnosides. 2003). osteoblasts. Anjum. The extracts were resolved in dimethyl sulphoxide (DMSO) at a concentration of 25 mg/ml. & Temelli.) is a member of the grass family and is one of the first cultivated grains (Jacobsen et al. Ltd. Therefore.. UT. Hulled barley was roasted at 0. The medium was also supplemented with beta glucan. MO. indicating the importance of small adipocytes in metabolic diseases (Rangwala & Lazar. Gyeonggi. USA). Panax japonicas. Vasanthan. studies also suggest the importance of hyperplasia in high-fat diet (HFD) fed mice and humans. Kusminski. Mixtures of roasted hulled barley with 70% ethanol were mixed for 2 h using a vertically moving mixer (Taitec Co. Hahn. USA) was further included for adipocyte differentiation of C3H10T1/2 cells. and confluent cells were induced in DMEM supplemented with 10% FBS. Nawaz. Introduction The world’s obesity epidemic can be attributed to excess lipid accumulation in metabolic tissues (Bray. 2004. Barley (Hordeum vulgare L. Park. An aqueous extract of hulled barely roasted at 210 °C was further studied for the preventive effect in an obese animal model. In addition. numerous efforts to prevent obesity are being undertaken but effective strategies and molecular targets remain to be identified (Alemany. and the cardiovascular system can cause insulin resistance and various cardiovascular diseases (Browning & Horton. and Paeonia suffruticosa prevent obesity or its related symptoms (Pichiah. Schneider. 2008).. Zahoor. coumaric acid. Materials and methods 2. & Hallfrisch. Beta glucan is a major bioactive compound in barley accounting for up to 4–5% (w/w). and immune cells (Chuang & McIntosh. 2004. Nelumbo nucifera. 2001. dietary intake and body weight are inversely correlated in epidemiological studies. Ltd. the effects of barley are often not considered to be related to these compounds because of the high beta glucan content in barley. The bioactivities of these plants are attributed to dietary fiber. After the treatment. These bioactivities are often suggested to be related to the beta glucan in barley extracts (Behall. 2011. Naked barley is mainly used as rice and used as a component of various foods. & Scherer. whereas adipocyte hyperplasia is often seen in juvenile onset obesity (Sun. 2010). 2001). 2013). 2012). Tao. Chagnon et al. Gyeonggi. VA. Matsuda et al. Scholfield. and benzoic acid (Jood & Kalra. Bohnsack. & Hallfrisch. type 2 diabetes.1. Bioactive compounds such as isoflavone. CA. 2003). not separated from husk.5 mM isobutyl-1-methylxanthine (Sigma-Aldrich). Moon.. & Sharma. Accordingly. 1 µM dexamethasone (Sigma-Aldrich. 2007. Vasudeva. In this study. 0.

4. The OVX rats were further divided into two groups. the mice were fasted for 16 h. CA. and reverse transcribed using 4 µl 5 × RTase M-MLV buffer. hulled barley extracts were preextracted with ethanol to remove free sugar. and the other group was injected with vehicle (1% DMSO/PBS) as a control. anesthetized with ether. Takara). Rubilar. and abdominal fat were collected. 3. The contents of phenolic compounds from the roasted hulled barley aqueous extract were analyzed by high performance liquid chromatography (Hitachi L-2400. The rats were anesthetized with Rompun and Zoletil (1:3.6. & Martinez. fats. Sineiro. USA). Measurement of beta glucan Beta glucan contents were measured from at least two independent extracts. & Nunez. To quantify the intracellular triacylglycerol content. kidneys. Korea). and ferulic acid were purchased from Sigma-Aldrich. Korea) and were randomly divided into four groups (n = 4–5/group). A P < 0. After a 1 week acclimation. the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 4 h.5.. 3.1.9 mm × 150 mm). Analysis of variance followed by the Student– Newman–Keuls test was used to analyze the differences in gene expression and lipid accumulation. and stained with 0. Total RNA and random primers were mixed and heated at 65 °C for 5 min. Animal studies All animal studies were carried out in accordance with the Animal Research Committee of the College of Biotechnology and Bioengineering at Sungkyunkwan University (protocol number: BT-2012-02). Blood was collected by cardiac puncture. hydroxybenzoic acid. 2. Data are presented as means and standard errors. five rats were sham-operated and nine rats were bilaterally ovariectomized (OVX). Anti-lipogenic effects of the hulled barley aqueous extract on preadipocytes Various extracting conditions such as storage and heating temperature can influence bioactive ingredients. The harvested tissues were fixed in 10% formaldehyde for 2 days and embedded in paraffin for histological studies.05 was considered significant. w/w) and their back was shaved bilaterally. Korea) and housed in controlled temperature (24 ± 2 °C) and humidity (50 ± 10%) conditions under a 12-h light and dark cycle. Takara. chlorogenic acid. v/v/v) under isocratic condition. Korea). Blood was separated by centrifugation at 850 × g for 10 min to collect serum. .. p-coumaric acid. Eight-week-old female Sprague–Dawley rats were purchased (Orient. 2013). and acetic acid (88:10:2. Co. Japan) with a UV detector (Hitachi). The stationary phase was a Waters Nova-Pak C18 column (4 µm. (Seoul. Differences in the animal study results and gene expression for coumaric acid-treated samples were analyzed with the unpaired Student’s t-test. Ireland). Young-in. Ohtsu. and a flow rate of 0. Co. Ltd. Real-time quantitative polymerase chain reaction (PCR) analysis Total RNA was isolated using TRIzol reagent (Invitrogen. the samples were incubated with lichenase enzyme at 40 °C for 1 h followed by further hydrolysis with beta-glucosidase at 40 °C for 15 min.3. Results and discussion 3. After a 1 week acclimation. (Seoul. 2. Gene specific primers were described previously (Song et al. vanillic acid. cDNA was heat inactivated at 70 °C for 10 min and amplified with a thermal cycler dice (Takara) using the Power SYBR Premix Ex Taq (RP041A. Finally. Food intake was measured every 3 days. At the end of 8 weeks. the mice were housed in individual cages in a temperature-controlled room with a 12-h light/dark cycle. The mobile phase was a mixture of ultrapure water. Meydani & Hasan. Then. RTase M-MLV (2640A. Paraffin blocks were sectioned at a thickness of 5 µm and were subjected to hematoxylin and eosin staining. and food intake was determined three times per week. 2005). IL. and oil. Two groups of mice fed the HFD were injected daily with a dose of 15 or 50 mg/kg AHB210. the absorbance was measured with an enzyme-linked immunosorbent assay reader at 510 nm after incubation with GOPOD reagent. These phenolic compounds were detected in the eluent at 280 nm. USA). New Brunswick. 2. 9-week-old female rats were randomized into two groups. 2010. resulting in different antioxidative and anti-obesity effects (Gomez-Plaza.75 ml/min. Mice in one group were fed a normal chow diet (ND) as a negative control for obesity. Gil-Munoz. 2000.. Chicago. The control OVX group was administered an oral gavage once daily with vehicle (water) and the treatment group received 15 mg/kg AHB210 for 8 weeks. Briefly. 2. Lopez-Roca.5% Oil Red O (Sigma-Aldrich) in a mixture of isopropanol and distilled water at a 3:2 ratio for 45 min. Orient. The rats were individually housed and allowed free access to water and food (Solid feed 5L79. while the other three groups were given a HFD containing 60% fat (Research Diets Inc. Pinelo. stained cells from at least two independent experiments were resolved with isopropanol and measured with a spectrophotometer at 520 nm. Other chemicals were purchased from Daejung Chemical Co.210 journal of functional foods 12 (2015) 208–218 vanillic acid. After differentiation. Body weight was measured weekly. 10 mM dNTP mixture. The contents of beta glucan in hulled barley were determined using a mixed-linkage β-glucan kit (Megazyme. Wicklow. Tokyo. Analysis of p-coumaric acid and ferulic acid in the hulled barley aqueous extract Protocatechuic acid. or ferulic acid extract every 2 days. Both ovaries were excised followed by ligation of the uterine tubes. coumaric acid. Japan) at 37 °C for 50 min. LTD. and the liver. and body weight was monitored every 3 days for 8 weeks. Male C57BL/6 (6-week-old) mice were purchased from Central Lab Animal Inc. acetonitrile. Jerez. The tissues were weighed after removing fat and draining the fluid. USA).7. Statistical analysis Statistical analyses were conducted using PASW Statistics 17 (SPSS Inc. Carlsbad. 2. Yong-In. and the concentrations were calculated using a calibration curves of each standard compound. NJ..

2 a 1 abc bcd 0. Water extracts of hulled barley roasted at 0 (AHB−). 50 µM conjugated linoleic acids (CLA). However. 1C). AHB210 showed higher . As expected. AHB170. and AHB210 but not EHB inhibited lipid accumulation during adipocyte differentiation of 3T3-L1 cells (Fig. 1A).5.2 250 °C EHB a a a a a 1 0.4 b b c 0.05).6 0. these data suggest that the hulled barley aqueous extract may be useful to extract anti-lipogenic ingredients. ORO stained cells from at least two independent experiments were quantified by extracting dye with isopropanol and measured absorbance at 520 nm.8 b 0.5 25 Ctrl CLA 250 °C 50 (μg/ml) Lipid accumulation Ctrl - 170 210 250 AHB - 170 210 250 EHB 1.2. Lipid accumulation was assessed by Oil Red O staining.4 0. the aqueous extract of hulled barley roasted at 250 °C (AHB250) at the same concentration did not inhibit lipid accumulation.2 0 CLA 50μM - 170 210 D AHB210 C Ctrl CLA 12. Interestingly. (C) 3T3-L1 cells were treated with 12. Among these. AHB250 did not significantly affect lipogenesis in 3T3-L1 cells. 210 (AHB210). (D) 3T3-L1 cells were treated with 12. (A) C3H10T1/2 cells were treated with 12.6 0. and 250 °C (AHB250). 1). did not affect adipogenesis (Fig. Therefore. Furthermore. 210 (AHB210).8 cd abc abc ab ab bcd d 0. 170 (AHB170). and ethanol extracts (70%) of hulled barley (EHB) with similar roasting conditions were prepared. AHB210 dose dependently suppressed lipid accumulation during adipocyte differentiation (Fig. and AHB210 barley extracts were determined. AHB− . Data are means ± SEM and were analyzed using one-way analysis of variance and the Student–Newman–Keuls test.5 µg/ml of an aqueous extract of hulled barley (AHB) roasted at 0 (AHB−). 1B). various roasting conditions were applied to evaluate the biological actions of hulled barley on adipocyte differentiation. as a positive control. 3. (B) Oil Red O-stained C3H10T1/2 cells were quantified. the aqueous extract (AHB) prepared without roasting (AHB−) or roasting at 170 °C (AHB170) and 210 °C (AHB210) effectively inhibited lipid accumulation (Fig. suggesting that a higher temperature may affect stability or generation of bioactive components from hulled barley (Fig. In agreement with the effects in C3H10T1/2 cells. total phenolic contents of the anti-adipogenic AHB− . 1D). The similar anti-adipogenic effects of the water extract and the lack of an effect by the ethanol extract were verified in preadipocyte 3T3-L1 cells. Mean with different letters at each samples are significantly different (P < 0.5 µg/ml of the AHB or EHB to differentiate them into adipocytes over 6 days followed by quantification. Quantification of lipid accumulation in differentiated cells by measuring the extracted dye at 520 nm.2 0 Ctrl CLA - 170 210 250 AHB - 170 210 250 EHB Fig. and 250 °C (AHB250) or an ethanol extract of hulled barley (EHB) following various roasting conditions to differentiate adipocytes for 7 days. 1 – Aqueous extract of hulled barley inhibits lipid accumulation in mesenchymal C3H10T/12 and preadipocyte 3T3-L1 cells. Each extract was treated with C3H10T1/2 during adipocyte differentiation to investigate the effects on lipid accumulation. Taken together.211 journal of functional foods 12 (2015) 208–218 A B AHB CLA 50μM - 170 a 210 Lipid accumulation Ctrl 1. regardless of the roasting condition. The ethanol extract. prevented strong lipid accumulation induced by adipogenesis. 2010). 170 (AHB170). AHB170. and 50 µg/ml of AHB roasted at 210 °C (AHB210) during differentiation for 6 days. Suppression of adipogenic marker expression by AHB210 during adipocyte differentiation Since total polyphenolic contents are often associated with antiobesity effects (Meydani & Hasan. 25.

Similarly. To exclude this possibility. Vatanparast et al. OVX rats were orally delivered with 15 mg/kg/day (Fig.3.04 mg QE/g total phenolic contents. Up to 100 µg/ml of AHB210 treated for 24 hours did not affect cell viability (Supplementary Fig. D). & Nam. Together. Although total phenolic contents alone are not solely responsible for the anti-adipogenic effects of AHB. total phenolic contents (0. 3A. The AHB210 group administered 15 mg/kg/ day (HFD + AHB15) gained 12.5% less (35 g) and the 50 mg/kg/ day treatment (HFD + AHB50) gained 17. all EHB contained 0. The mRNA expression levels of peroxisome proliferator-activated receptor γ (PPARγ). 0. Data are means ± SEM and were analyzed using one-way analysis of variance and the Student–Newman–Keuls test (*p < 0.212 journal of functional foods 12 (2015) 208–218 3 3 a PPARγ 2 Normalized expression (genes/36b4) b aP2 2 b b bc c 1 a c d 1 0 3 bc 0 3 C/EBPα a 2 CD36 a 2 b bc 1 c b 1 d CLA c d 0 Ctrl bc 12. Preventing body weight increase by AHB210 in HFDinduced obese and estrogen deficient animal models The in vitro anti-adipogenic effects of natural products are often associated with in vivo anti-obesity effects (Meydani & Hasan. 0.5% less (33 g) compared to the body weight gain in the control HFD group (40 g) (Fig. CCAAT-enhancer-binding protein-α (C/EBPα). 2014). and cluster of differentiation 36 (CD36) measured by quantitative real-time polymerase chain reaction. and 50 µg/ml showed a dose dependent decrease in expression of peroxisome proliferatoractivated receptor γ (PPARγ). the anti-adipogenic action of AHB210 further prompted us to investigate the potential anti-obesity effects in vivo. OVX. Taken together. the HFD fed group gained about 42% more body weight compared to that in the control ND fed group (40 vs. S1B). further suggesting that the anti-lipogenic effects of AHB were not attributed to cellular toxicity. expression of adipocyte markers was determined by real-time PCR (Fig. Regardless of roasting condition. Thus.12 mg quercetin equivalents (QE)/g sample) with higher amounts of phenolics observed as roasting temperature increased (AHB−. adipocyte protein 2 (aP2). 28 g).5. CCAAT-enhancer-binding protein α (C/EBPα). Rosen & Spiegelman. Thus. Similar to the effects in HFD-induced obese C57BL6 mice. Similar to CLA. particularly during menopause (Kim. The OVX rats gained significantly more body weight compared to the sham control group (401 vs. OVX rats with oral delivery of AHB210 (OVX + AHB15) prevented weight gain induced by estrogen deficiency (359 g).001. and EHB did not result in toxicity (data not shown). or OVX with daily oral administration of 15 mg/kg of AHB210 (OVX + AHB15) for 8 weeks. The experiments were repeated in triplicate. B). and cluster of differentiation 36 (CD36). 60% fat) for 8 weeks to induce obesity. treatment of cells with AHB170. To expand the effects of AHB210. **p < 0. 315 g). CLA (50 µM) was used as a positive control to show the anti-adipogenic effects. To confirm the anti-adipogenic effects of AHB210. these data suggest the potential use of AHB210 for preventing body . S1C).01 mg QE/g. At the end of 8 weeks. 2005. The rats were sham operated. An increase in body weight can also be induced by estrogen deficiency. ***p < 0. 2009). The anti-adipogenic effects of AHB210 could be attributed to the cytotoxic effects of AHB. 2010.05. AHB210 inhibited the expression of adipocyte differentiation markers and lipid accumulation during adipocyte differentiation.5 25 50 (μg/ml) Fig. AHB210 was chosen for further studies due to its higher total phenolic content and strong anti-lipogenic action. AHB250. No cytotoxic effects were also observed in the C3H10T1/2 cells (Supplementary Fig.5 25 0 50 (μg/ml) Ctrl CLA 12. Seven week old C57BL/6J mice were fed on a high fat diet (HFD.0001). 2). adipocyte protein 2 (aP2).09 mg QE/g). relatively high contents of total phenolics correlated with the antiadipogenic actions. 2 – Aqueous extracts of hulled barley roasted at 210 °C (AHB210) suppress the expression of adipocyte markers in C3H10T1/2 cells. 3C. 3. Ahn. S1A). we performed viability assays in 3T3-L1 cells. 25. Longer treatments with AHB210 (48 hours) also did not affect cell numbers (Supplementary Fig. supporting the idea that phenolic compounds may provide the anti-adipogenic actions to AHB.. AHB170. to further analyze the anti-adipogenic effects. C3H10T1/2 cells treated with AHB210 for 6 days at 12. C3H10T1/2 cells were treated with the indicated concentrations of AHB210 during differentiation for 7 days and expression of adipocyte markers was measured.

05). C57BL/6J mice aged 7 weeks were fed with a HFD (60% fat) for 8 weeks to induce obesity. Consistent with the decreases in body weight. (C) Oral administration of 15 mg/kg AHB210 for 8 weeks to ovariectomized (OVX) rats also decreased body weight increases. total food intake was not different among the groups suggesting that less energy intake may not have been a primary cause of weight reduction in the AHB210 treated group. The anti-obesity effects of AHB210 were further analyzed in HFD fed obese mice. and OVX rats orally administered 15 mg/kg AHB210 for 8 weeks (AHB OVX group. (D) Significant differences in the area under curve (AUC) of the body weight changes in the HFD and AHB210 treated groups. However. Body weights of sham-operated group. n = 5).0001). Data are means ± SEM. In addition. other tissues such as spleen and kidney were not different. Mice were weighed twice per week. serum cholesterol. *Difference between HFD and AHB210 treated groups are indicated (*p < 0. **p < 0. As obesity is positively associated with higher serum glucose levels and increased lipid contents. weight increases associated with hormone deficiency in female rats as well as HFD in male mice.0001). Data were analyzed by analysis of variance (ANOVA) with different letters indicating a significant difference (P < 0. showing the relatively selective actions of AHB210 on metabolic tissues. 4A). Data are means ± SEM. OVX rats orally administered vehicle for 8 weeks (control OVX group. ***p < 0.05. fat mass (epididymal fat) and liver weight gain were significantly lower in the AHB210 treated groups than those in the other groups (Fig. (A) Weight gain by high fat diet (HFD) decreased following daily intraperitoneal injection of 15 mg/kg (HFD + AHB15) or 50 mg/kg (HFD + AHB50). Data are means ± SEM and were analyzed using Student’s t-test. Data are means ± SEM and were analyzed using Student’s t-test. *Difference between the HFD and AHB210 treated groups are indicated (*p < 0. and different letters indicate a significant difference (P < 0. **p < 0.05).001. fatty . n = 4). Significant weight differences following injection of AHB210 compared to vehicle in HFD fed mice were observed (n = 4–5). (B) Significant differences in the area under the curve (AUC) of body weight changes in the HFD and AHB210 treated groups.213 journal of functional foods 12 (2015) 208–218 A (g) 45 40 Body weight B ND (N=5) HFD (N=5) HFD +AHB 15 (N=4) HFD+ AHB 50 (N=4) (AUC) 800 * * 35 * * * * * * * * 600 * * * * * * * * * * ** ** ** ** ** ** ** ** ** ** ** ** * * ** ** ** ** ** ** ** ** * ** ** ** 30 25 a 700 b b b 500 400 300 200 100 0 ND HFD HFD+ AHB15 HFD+ AHB50 20 0 C 2 4 6 8 (weeks) (g) 450 Body weight 400 D Sham (N=5) OVX (N=5) OVX + AHB 15 (N=4) (AUC) 4500 a * 350 * * * * 3000 ab b 1500 300 ** ** ** ** ** ** ** 0 Sham 250 200 0 2 4 6 OVX OVX+AHB15 8 (weeks) Fig. triacylglycerols. Rats were weighed weekly. Data were analyzed by ANOVA. 3 – Aqueous extracts of hulled barley roasted at 210 °C (AHB210) prevent weight gain in diet induced obese C57BL6 male mice and estrogen-deficient female rats. ***p < 0.001.05.

our data clearly show that other bioactive ingredients in AHB besides beta glucan influence the health beneficial effects.. & Chen. (B) Representative hematoxylin and eosin (H&E) sections of epididymal fat from HFD fed mice. respectively. higher effective doses of beta glucan in mice and human studies also suggest that beta glucan alone is not likely to mediate the anti-obesity effects of AHB210. 10 ng/ml (0. 1997. and satiety (Ahmad et al. HDL. Tang. 2006).02%) compared to that in hulled barley and lower than that in AHB250 (0. 3. This is in sharp contrast to our in vivo studies in which daily administration of 15 or 50 mg/kg (corresponds to <1 µg beta glucan/kg mice) AHB210 exhibited antiobesity effects. as determined by glucose tolerance tests (Supplementary Fig. beta glucan contents in hulled barley.07%).5 b a 2 1. Although not significantly different. 1×). (A) Injections of AHB210 into high fat diet (HFD) fed mice prevented fat mass and liver weight increases but not weight of spleen or kidney. Although it is impossible to exclude the possibility that beta glucan is an anti-adipogenic effector. & Song. Smaller adipocytes were observed in the HFD + AHB50 treated group compared to the large adipocytes in the HFD group. 50 µg/ml of AHB210 significantly inhibited lipid accumulation. C).214 journal of functional foods 12 (2015) 208–218 A (g) 3. Shi. Lin. Beta glucan contents in barley can be up to 4–5% (w/w) and have been suggested to decrease the risk of cardiovascular diseases and immune stimulation (Ahmad et al. Hong. Accumulation of lipid in the liver decreased following AHB210 treatment (scale bar = 50 µm). 4B. Additionally. As expected.5 b b 1 a a 0. Jung. (C) Representative H&E sections of livers from HFD fed mice. 2013). Kim. Li. 2006). acids. triacylglycerols and free fatty acids showed a ten- dency toward decreasing in the AHB210 treated groups (Table 1). and AHB250 were determined and their corresponding contents were directly tested for anti-lipogenic effects. daily administration of a few grams of beta glucan (per kg body weight) in mice prevents weight gain and obesity (Choi. glucose tolerance was improved in HFD fed animals receiving AHB. w/w) times higher doses than the expected beta glucan contents in AHB210. Plasma glucose levels decreased from 230 to 227 and 175 mg/dl in the 15 and 50 mg/kg/day treated groups.. A daily dose of 3 g barley beta glucan is recommended for a human health benefit on weight loss. The beta glucan content in AHB210 was low (0. However. Cholesterol. Kim et al. C). 1000× contents in 50 µg/ml of AHB210) failed to inhibit adipogenesis (Fig 5B.5 3 a HFD (N=5) HFD +AHB 15 (N=4) HFD+ AHB 50 (N=4) ab 2. Fat cells from epididymal fat tissues in AHB210 injected HFD mice were smaller in size than those of control HFD fed mice as shown by H&E staining (scale bar = 50 µm). Accordingly. as shown by hematoxylin and eosin staining (Fig. 3. it is possible that the higher beta glucan contents in AHB210 compared to AHB250 mediate the anti-adipogenic effects.02%. . these data suggest that AHB210 treatments prevent weight gain and its associated comorbidities in obese animal models... S4). To test this possibility. hepatic steatosis induced by obesity was greatly alleviated in the HFD + AHB50 treated group. Consistent with our observations. Behall et al.5 0 a a FAT Liver a a Spleen Kidney B HFD HFD+ AHB50 C HFD HFD+ AHB50 Fig. glucose. Furthermore. Similarly. AHB210. Different letters indicate a significant difference (P < 0. glucose control. and high density lipoprotein (HDL) and low density lipoprotein (LDL) levels were also measured. suggesting that beta glucan may not be the primary mediator of the anti-adipogenesis effect (Fig. 5A). Moreover. 4 – Aqueous extracts of hulled barley roasted at 210 °C (AHB210) prevent obesity in diet induced obese C57BL6 male mice. C3H10T1/2 and 3T3-L1 cells were treated with up to 1000 (20%. and LDL levels were also decreased significantly in the 50 mg/kg/day treated group with intermediate reduction in the 15 mg/kg/day treated group.4. 2010. Beta glucans may not be the major components for the anti-adipogenic effects of AHB Strong anti-adipogenic effects of AHB210 but weak antiadipogenic effects of AHB250 suggested that the active ingredients are relatively high in AHB210 but less in AHB250. Identification of coumaric and ferulic acids as potential mediators of the anti-adipogenic effects of AHB Dietary polyphenolic compounds can mediate the various biological effects including anti-adipogenesis (Meydani & Hasan. 2012. Mice were fasted for 12 h and euthanized to harvest organs at the end of experiments (n = 4–5). and 10 µg/ml (20%. 2012. Data are means ± SEM and were analyzed using analysis of variance and the Student– Newman–Keuls test for organ weight. Taken together..05). Kim et al. 1 µg/ ml (2%.5. 100×). To further exclude the possibility that beta glucan in AHB exerted anti-adipogenic effects. about 3% beta glucan (w/w) was found in hulled barley.

compounds by comparison with authentic standard compounds was performed.23 ± 8.79ab 166. Different letters indicate a significant difference (P < 0. Six compounds of coumaric acid (CA).98 ± 5. A correlation between relatively high phenolic contents and the anti-adipogenic effects of AHB210 suggests that polyphenolic compounds found in barley are responsible for the anti-adipogenic effects (Yu et al. free fatty acids (FFA). Differentiated cells were stained with Oil Red O. chlorogenic acid (ChA).77 ± 5. hydroxybenzoic acid (HA). AHB250.06a 3320. and high density lipoprotein (HDL) levels in C57BL/6 mice fed a high fat diet (HFD) and administered a daily dose of 15 mg/kg (HFD + AHB15) or 50 mg/kg (HFD + AHB50) of aqueous extracts of hulled barley roasted at 210 °C. triacylglycerols.60a 32.35 ± 180.54 ± 16. ferulic acid (FA).07% AHB250 Ctrl AHB 50 0. (A) Determination of beta glucan contents in AHB210. Data were analyzed by ANOVA. 5 – Determining of beta glucan.54a 176.215 journal of functional foods 12 (2015) 208–218 Table 1 – Plasma glucose. Values are means ± SEM.27 ± 15.02b 103.99 ± 1. total cholesterol.92 ± 5.70b 125. C) Beta glucan at 1 (0. (D) Quantification of differentiated C3H10T1/2 cells.05). and ferulic acid contents in hulled barley roasted at 210 °C (AHB210).01 1 10 (μg/ml) β-glucan D Lipid accumulation 1.06a 21.08 ± 9. To test this possibility.48 ± 10.98 ± 6. and different letters indicate a significant difference (P < 0. 2001).5 0 Ctrl AHB 50 0.59a 213. ORO stained cells from at least two independent experiments were quantified by extracting dye with isopropanol and measured absorbance at 520 nm.56b 162.02a 41. Other compound previously found in barley 2010).62 ± 117. Data were analyzed by analysis of variance with Student–Newman–Keuls test.01 1 β-glucan 10 (μg/ml) Fig. and hulled barley. AHB210 were used as an anti-adipogenesis control.08a 168. HPLC analysis followed by individual identification of A B β-glucan contents (%) 4 3% 3 Ctrl 2 AHB 50 0. HFD (n = 5). low density lipoprotein (LDL).54a 217. p-coumaric acid.81 ± 5.05). 100 (1%).18a 3847.99b Data were analyzed by one-way ANOVA using SPSS (Duncan’s multiple range test).5 a a a a 1 b 0.65 ± 6.. Groups HFD HFD + AHB 15 (AHB 15 mg/kg) HFD + AHB 50 (AHB 50 mg/kg) Glucose (mg/dl) Cholesterol (mg/dl) Triacylglycerols (mg/dl) FFA (uEq/l) LDL (mg/dl) HDL (mg/dl) 230.85 ± 15.01 1 10 (μg/ml) β-glucan C 1 0. 6A).31 ± 8.01%).38 ± 21.02% 0 Barley flour AHB210 0. protocatechuic acid (PCA). HFD + AHB50 (50 mg/kg): a group of mice fed with HFD and treated with 50 mg/ kg of AHB210 (n = 4).52a 3625. .93a 98. and 1000 (10% w/w) times higher amounts than the expected contents at 50 µg/ml in AHB210 were added to C3H10T1/2 cells (B) or 3T3-L1 cells (C) for differentiation into adipocytes. and vanillic acid (VA) were identified (Fig. Data are means ± SEM. (B. HFD + AHB15 (15 mg/kg): a group of mice fed with HFD and treated with 15 mg/kg of AEB210 (n = 4).35 ± 203.34a 227.88a 111.

ChA. In line with our observations. PCA did not show obvious anti-adipogenic effects (data not shown). protocatechuic acid.5 50 1 AHB210 (μg/ml) 5 10 f 50 100 200 300 500 CA(μM) ab 1 ab b b 0. HA.. at least in part. 6B–D). Although the effective doses of coumaric acid and ferulic acid for antiadipogenesis are about 10 times higher than the expected contents in AHB210. which correlated well with the stronger biological actions of AHB210.4 0. chlorogenic acid.2 0 Ctrl 12. He. and FA contents compared to those of AHB250. recent studies also suggest that a combination of a few dietary . 6 – Identification of coumaric acid (CA) and ferulic acid (FA) as potential mediators of anti-adipogenic effects of the aqueous hulled barley (AHB) extract. PCA. Phenolic compositions and bioavailability in the grains thus can be varied by different extraction and hydrolysis procedures. S3). our data show that these compounds. It is also possible that a combination of these phenolic compounds might additively (or synergistically) contribute to the anti-adipogenic effects of AHB210. 2014). and VA were higher in AHB250 suggesting that these are not primary components responsible for the selective anti-adipogenic actions of AHB210. It will be also worthwhile to investigate whether similar extraction methods or roasting conditions can be applicable for the enhancement of anti-adipogenic effects of other grains and beans (Wang et al.8 0. (C.216 journal of functional foods 12 (2015) 208–218 A (μmol/g) B Ctrl CA(μM) 100 500 Ctrl 100 Contents of compounds 7 AHB210 AHB250 6 5 4 3 FA(μM) 500 2 1 0 CA FA PCA ChA HA VA D 1.05). FA. D) C3H10T1/2 cells were treated with the indicated doses of CA (C) and FA (D) and were induced to differentiate into adipocytes for 7 days. p-coumaric acid. 4-hydroxybenzoic acid. PCA. PCA. Lipid accumulation was assessed by Oil Red O staining.2 a a 1 ab Lipid accumulation Lipid accumulation C 1. the combination of coumaric acid and ferulic acid for anti-adipogenesis was increased.6 c cd cd c cd d 0.4 e ef f 0. & Chen. mediate the actions of AHB210. AHB210 exhibited higher CA. VA.5 50 1 5 AHB210 (μg/ml) 10 50 100 200 300 500 FA(μM) Fig. ORO stained cells from at least two independent experiments were quantified by extracting dye with isopropanol and measured absorbance at 520 nm. CA. ferulic acid. The contents of HA. Our data show that the contents of anti-adipogenic phenolic com- pounds in the water extracts of hulled barley roasted at 210 °C are higher than those from other roasting and extraction conditions. anti-adipogenic effects of CA. such as caffeic acid was not detectable in AHB210 and AHB250.6 c d d d 0. and FA were also investigated. vanillic acid.2 bc 0. ChA. Accordingly. showing the possibility of additive effects in antilipogenesis by co-treatment with coumaric acid and ferulic acid (Supplementary Fig. Most of phenolic compounds in cereal grains exist in soluble and insoluble forms (Wang. Therefore. Different letters indicate a significant difference (P < 0. these studies bring new perspectives for the exploitation in functional food area of hulled barley-based products that will prevent obesity and metabolic diseases. to ensure health-beneficial effects against obesity and metabolic diseases. (A) Contents of polyphenolic compounds in hulled barley roasted at 210 °C (AHB210) and AHB250 based on high performance liquid chromatography analysis.8 0. 2014). In contrast.2 0 Ctrl 12. hulled barley can be standardized based on the contents of ferulic acid and coumaric acid. (B) Coumaric acid (CA) and ferulic acid (FA) inhibited adipocyte differentiation. Consistently. whereas CA and FA both dose dependently suppressed lipid accumulation as assessed by Oil Red O staining (Fig. Data are means ± SEM and were analyzed using analysis of variance with Student–Newman–Keuls test. Further.

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