Anti-Obesity Potential of Gallic Acid from Labisia pumila, through Augmentation of Adipokines in High Fat Diet Induced Obesity in C57BL/6 Mice

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1 Advnces in Reserch 2(10): , 2014, Article no. AIR SCIENCEDOMAIN interntionl Anti-Oesity Potentil of Gllic Acid from Lisi pumil, through Augmenttion of Adipokines in High Ft Diet Induced Oesity in C57BL/6 Mice Anjli Pndey1, Srng Bni1* nd Pyre Ll Sngwn2 1 Deprtment of Phrmcology, Indin Institute of Integrtive Medicine, Jmmu Twi, Indi. Nturl Product Chemistry Division, Indin Institute of Integrtive Medicine, Jmmu Twi, Indi. 2 Authors contriutions This work ws crried out in collortion etween ll uthors. All uthors red nd pproved the finl mnuscript. th Originl Reserch Article Received 18 Mrch 2014 th Accepted 15 My 2014 th Pulished 16 June 2014 ABSTRACT Aims: The incidence of oesity hs incresed t n lrming rte in recent yers, ecoming worldwide helth prolem. Anti-oesity drugs ville hve hzrdous sideeffects, thus, wide vriety of nturl mterils re eing explored for their nti-oesity potentil. This study ws undertken to investigte the ctivity of Gllic cid (GA), compound isolted from queous lef extrct from Lisi pumil (LPPM/A003) on its potentil to prevent oesity. Plce nd Durtion of Study: The study ws crried out t Deprtment of Phrmcology, Indin Institute of Integrtive Medicine, Council of Scientific nd industril reserch, Jmmu (J nd K) for durtion of 12 months. Methodology: All the test mterils were initilly screened for In-vitro dipocyte differentition. The ctive smple ws selected for In-vivo nti-oesity effect in high ft diet induced oesity in C57BL/6 mice nd the iochemicl nd moleculr prmeters were mesured. Results: In In-vitro screening, oth LPPM/A003 nd GA hd inhiitory effect on ft droplet formtion nd triglyceride ccumultion. The concentrtion t which GA showed 50% inhiition ws 19.86μg/ml. The In-vivo studies in oese mice decresed the weight in GA *Corresponding uthor: E-mil: srngni@rediffmil.com;

2 treted nimls. Excessive secretion of leptin in cse of oesity leds to disrupted functions of hypothlmus nd GA showed promise y inhiiting Leptin. Oesity is considered s stte of low-grde inflmmtion nd inflmmtion is regrded s cuse or consequence of oesity. It lso inhiited the incresed expression of TNF- α nd IL-6 in serum of treted nimls. GA inhiited the increse in serum levels of Triglyceride, LDL nd VLDL. HDL levels were elevted t ll the dose levels with significnt increse t 8mg/kg dose. Conclusion: Gllic cid is n effective compound cple of modulting diet induced weight gin in oese mice. Reserch in this field pves the wy to discover new tretments for oesity. Keywords: Gllic cid; high ft diet induced oesity; dipokines; leptin; Lisi pumil. ABBREVIATIONS GA: Gllic Acid; LPPM/A003: Aqueous Extrct of leves of Lisi pumil; PG: Pyrogllol C57BL/6: C57 lck 6 inred strin of lortory mice; TNF-α: Tumour necrosis fctorlph; IL-6: Interleukin-6; ORO: Oil-Red-O; IC50: Concentrtion t which 50% inhiition; HDL: High Density Lipoprotein; LDL: Low density lipoprotein; VLDL: Very Low Density Lipoprotein. 1. INTRODUCTION Good nutrition, physicl ctivity, nd helthy ody weight re essentil prts of person s overll helth nd well-eing. Our current oesogenic environment promotes incresed food intke, non-helthful foods nd physicl inctivity, so weight gin nd disese re relly nturl nd expected consequences of this oesogenic environment. Nturl product (nutrceuticl) interventions re currently eing investigted on lrge-scle sis s potentil tretments for oesity nd weight mngement. Ethno otnicl clims indicte tht progress hs een mde concerning our knowledge of ioctive components in trditionl plnts nd their links to oesity. With glol increse in the prevlence of oesity, nutrceuticls plys key role in its prevention nd tretment. Oesity is n excess of ody ft tht frequently results in significnt impirment of helth. It is the most prevlent nutritionl disorder nd is descried s glol epidemic. Oesity results when the size or numer of ft cells in person's ody increses resulting s possile gtewy for vrious chronic physicl nd psychologicl prolems. It increses the likelihood of vrious diseses tht include hert disese, type 2 dietes, ostructive sleep pne, certin types of cncer, nd osteorthritis [1]. It is most commonly cused y comintion of excessive food energy intke, lck of physicl ctivity nd genetic susceptiility, lthough, few cses re cused primrily y endocrine disorders. Oesity is chrcterized y incresed dipose tissue mss tht results from oth incresed ft - cell numer (hyperplsi) nd incresed ft-cell size (hypertrophy) [2]. The numer of dipocytes present in n orgnism is determined to lrge degree y the dipocyte differentition process, which genertes mture dipocytes from firolst-like predipocytes. A ft cell develops s internlly produced lipid droplets colesce into single lrge mss. Eventully, cellulite results due to enhnced dipogenesis nd ccumultion of dipocytes under the skin. 557

3 Adipose tissue, once thought to function primrily s pssive depot for the storge of excess lipid, is now understood to ply much more ctive role in metolic regultion, secreting vriety of metolic hormones [3]. Adipose tissue is currently known to secrete lrge numer of proteins termed dipokines tht ct in n utocrine, prcrine, or endocrine fshion to control vrious metolic functions. Leptin is n importnt dipocyte hormone tht, influences food intke through direct effect on the hypothlmus. In oese individuls, leptin concentrtions re lredy high ecuse of the incresed mount of leptin-secreting dipose tissue [4]. Eexcessive secretion of leptin s in cse of oesity or experimentl models of induced oesity leds to disrupted functions of hypothlmic centres tht n oese suject tends to go on over feeding mode. Hence, it ecomes impertive to ring out effective reduction of the over expressed levels of leptin in oesity. Severl pro-inflmmtory fctors re produced in dipose tissue with incresing oesity. Compred with tht of len individuls, dipose tissue in oese persons shows higher expression of pro-inflmmtory proteins, including TNF-α, interleukin 6 (IL-6), monocyte chemotctic protein 1, inducile nitric oxide synthse etc. [5]. Mcrophge numers in dipose tissue lso increse with oesity [6], where they pprently function to scvenge dipocytes, which increse drmticlly with oesity [7]. Mcrophges re responsile for most of the cytokine production in oese dipose tissue. Although triglycerides re essentil for norml physiology, excess triglyceride ccumultion occurs in oesity nd is lso ssocited with insulin resistnce. Lisi pumil or more commonly known s Kcip Ftimh hs een used widely in South Est Asin communities for vriety of illnesses nd in food supplements. This plnt hs een widely used y Mlysin women for genertions for its postprtum rejuventing properties nd toning of dominl muscles [8]. In literture the weight regulting ctivity of Lisi queous extrct is reported in ovriectomized rts [9]. We wnted to look for the weight mngement effect in high ft diet induced oese mice. During the course of study we found, queous extrct of Lisi pumil (LPPM/A003) to hve nti-oesity potentil nd further study ws undertken to identify the compound present in LPPM/A003 responsile for nti-oesity potentil. LPPM/A003 nd two compound i.e., Gllic cid (GA) nd Pyrogllol (PG) isolted from this extrct were screened through in-vitro experimenttion for nti-oesity potentil where GA ws found to hve most significnt effect nd ws then tken up for detiled In vivo studies in high ft diet induced oese mice to look into the vrious different spects nd trgets relted to oesity. 2. MATERIALS 2.1 Plnt Mteril Plnt mteril ws collected from Kwsn Tsik Bnding, Grik, Perk Mlysi on Novemer 2007nd uthenticted y Forest Reserch Institute of Mlysi. A voucher smple (Accession no ) is retined nd deposited t the Herrium of this institute Preprtion of LPPM/A003 extrct Dry Powdered leves of L. pumil (500g) were soked in distilled wter (4.0 litre) nd the contents were stirred t 70ºC for 3hrs. The contents were then filtered nd the mrc tken in 558

4 fresh 4.0 litres of wter nd the process ws repeted twice (totl extrctions =three) keeping the temperture nd time prmeters constnt. The totl filtrte ~ 10 litres ws concentrted t 50ºC nd reduced to two litres volume nd then freeze dried to get 47.5g light rownish free flowing powder leled s LPPM/A003. The drug-extrct rtio (DER) ws 100:9.5 (w/w) HPLC of LPPM/A003 The HPLC nlysis ws done on Bruker Dltonics Esquire 3000 (LC) Agilent 1100 series º instrument using RP-18 (Merck, 5µm, 4x250mm) column, temp 30 C t wvelength 270nm with flow rte of 0.5ml/min. Moile phse ws used in grdient s solvent A (wter contining 0.1% formic cid) nd solvent B (cetonitrile), Time (mins): Solvent B (%): The queous extrct ws stndrdized on the sis of mrker compounds Gllic cid (GA) nd Pyrogllol (PG) isolted nd well chrcterized from the lef extrct of plnt. On quntifiction, the concentrtion of Gllic cid nd Pyrogllol ws found to e 1.98% nd % respectively. However, the extrct ws found to e rich in polyphenolic compounds. HPLC of LPPM/A003 is shown in Fig. 1. The HPLC grph of stndrd gllic cid (Rt=4.60min) nd pyrogllol (Rt=6.07 min) t wvelength 270nm nd 254nm is lso shown in the Fig. 1 [10]. Fig. 1. HPLC grph of LPPM/A003 with seprtion of gllic cid (Rt=4.64 min) nd pyrogllol (Rt=6.31) t two different wvelengths 2.2 Animls C57BL/6 mle mice ged 3 weeks old were tken nd were fed with High ft diet (60% Kcl % Ft) upto 19 weeks; these were then tken for the study to ssess nti-oesity potentil of the test mteril. Animls were housed under stndrd lortory conditions with dequte 559

5 fresh ir supply (Air chnges per hour), room temperture ºC, reltive humidity 55-61%, with 12 hours light /drk cycle. All experimentl procedures used in present study were in ccordnce with institutionl guidelines for niml reserch (CPCSEA, 2003). The study protocols were pproved y the Institutionl Animl Use nd Cre Committee of Indin Institute of Integrtive Medicine, Jmmu. 3. EXPERIMENTAL PROCEDURES 3.1 Adipogenesis Inhiitory Assy in 3T3-L1 Mouse Adipocytes (In vitro) A terminl differentition of dipocytes is ccompnied y the ccumultion of gret mounts of lipids in lrge cytoplsmic vesicles. A common ssy to mesure dipocyte differentition in cell culture is with the dye Oil Red-O (ORO). ORO is lipid-solule right red dye which is relile indictor of dipocyte differentition [11]. Oil Red O (Solvent Red 27, Sudn Red 5B, C.I , nd C26H24N4O) is lysochrome (ft-solule dye) dizo 4 dye used for stining of neutrl triglycerides nd lipids. 3T3-L1 cells pproximtely 60X10 cells were seeded for 48-72hrs to get 70-80% confluence. After 48 hrs 200µl of AIM (Adipogenesis induction medium) freshly prepred ws dded. 72hrs lter 200μl APM (Adipogenesis progression medium) with the test smples in different concentrtions ws dded to the wells. The cells were incuted for 48hrs in humidified tmosphere (37ºC) of 5% CO2 nd 95% ir. [12]. Cells were fixed y dding 100μl of 10% formlin nd ORO stining ws done. OD ws red t 492 nm in microplte reder nd the percentge inhiition ws clculted. The results were expressed s IC50 vlues using Grphpd prism softwre nd the percentge inhiition of dipogenesis in test drug treted smples were clculted [13]. 3.2 Induction of Oesity Ten nimls were llotted to Group 1 nd served s Norml Diet Control Group nd fed with Diet (10% Kcl % Ft ) for 19 weeks. Forty nimls were llotted to Group 2 nd fed with High Ft Diet (60% Kcl % Ft) for 19 weeks to induce oesity [14]. Oesity induction ws confirmed sed on the comprison of High Ft Diet nimls ody weights with Norml Diet nimls. The nimls which were found non oese were eliminted from the study. To ssess the therpeutic effect, GA ws dministered to oese mice dily for period of 28 dys t 2, 4 nd 8mg/kg dose levels. Weekly twice ody weights nd feed intke ws recorded. 3.3 Study Design Eight nimls from Norml Diet group selected for the min study were ssigned s Control Group. Thirty two oese nimls from High Ft Diet groups were selected nd llotted to four groups (8 nimls per group) s High Ft Diet Control Group, Test drug groups included dministrtion of GA t dose levels of 2, 4 nd 8mg/kg respectively (Tle 1). 560

6 Tle 1. Study design Experimentl Groups Norml Control High Ft Control High Ft Diet + GA-2 High Ft Diet + GA-4 High Ft Diet + GA-8 Dose (mg/kg) No. of Animls Men Body Weight Individul ody weight ws recorded once on the dy of commencement of tretment nd on th dys 4, 8, 11, 15, 18, 22, 25 nd 28 of the experimentl period. 3.5 Feed Consumption The food consumption ws mesured on the dy of ody weight mesurement during tretment period. The cge wise verge food intke (g/mouse/dy) ws clculted. 3.6 Blood Collection nd Biomrker Estimtion On Dy 29 of experiment, lood ws collected from ll nimls in the groups through retro oritl plexus. Serum ws seprted from whole lood y centrifugtion nd processed for lipid profile nlysis nd moleculr trget estimtion. 3.7 Estimtion of Leptin Estimtion of Leptin ws done y ELISA kits from R nd D systems ccording to mnufcturer s informtion in the serum smples of the experimentl nimls. 3.8 Serum Biochemistry The serum lipid profile ws nlyzed using the EM-360 Fully utomted clinicl chemistry nlyzer (Trnssi Bio-Medicls Ltd., Indi). The totl cholesterol, Triglycerides, HDL (High density lipoprotein), LDL (Low density lipoprotein) nd VLDL (Very low density lipoprotein) were mesured in mg/dl. 3.9 Estimtion of Pro-Inflmmtory Mrkers Serum ws collected from the experimentl nimls nd estimtion of pro-inflmmtory mrkers viz. TNF-lph nd IL-6 ws estimted y ELISA kits from R nd D systems ccording to mnufcturer s informtion Acute Sfety Study The cute toxicity study ws performed in complince with Schedule Y [15] nd Good Lortory Prctices requirements of Govt. of Indi. In the cute studies, mle nd femle Swiss lino mice were used. In experiment designed to determine orl LD50 of GA in swiss lino mice (6/sex/group; weeks of ge; ody weight rnge: mles 28-35g, femles 561

7 25 33g) were dministered single orl (gvge) dose of GA t 50, 500, 1000 nd 2000 mg/kg ody weight nd nimls were oserved for 14 dys for ny signs of moridity or mortlity. Similr set of experimenttion ws performed in wistr rts (6/sex/group; weeks of ge; ody weight rnge: mles g, femles g) Sttisticl Evlution The men nd stndrd error (S.E.) of the men for ech group ws clculted nd the results were expressed s percent inhiition compred with the control group. The significnce ws determined sttisticlly y pplying Student s t-test. 4. RESULTS 4.1 Adipogenesis Inhiitory Assy in 3T3-L1 Mouse dipocytes (In vitro) A rod concentrtion rnge of 3.12, 6.25, 12.5, 25, 50 nd 100 μg/ml ws tested. The concentrtion t which LPPM/A003 showed 50% inhiition (IC50) ws 94.51μg/ml nd tht of GA ws 19.86μg/ml, however, IC50 vlue ws not otined for PG upto the highest workle concentrtion (Tle 2). Lower the IC50 (μg/ml) vlue, etter is the efficcy. Fig. 2 shows ORO treted control cells nd inhiition of dipogenesis in smple treted cells. During dipocytes differentition GA hd significnt inhiitory effect on ft droplet formtion nd triglyceride ccumultion Tle 2. Inhiition of dipocyte differentition y different concentrtions of test smples in 3T3 L1 dipocyte cells Smple LPPM/A003 (Aqueous extrct) PYROGALLOL (PG) GALLIC ACID (GA) Percent inhiition t grded concentrtion (μg/ml) NIL6.24% 11.7% 28.15% 41.17% IC50 μg/ml % NIL- 8.91% 9. 58% 24.21% 26.66% 31.44% % 21.67% 37.76% 53.55% 70.12% 76.45% The results re expressed s IC50 vlues using Grphpd prism softwre. This is the concentrtion t which 50% inhiition of dipogenesis tkes plce. Lower the IC 50 (μg/ml) vlue, etter is the efficcy 4.2 Men Body Weight The dt otined from Adipogenesis inhiitory ssy in 3T3-L1 mouse dipocytes showed GA to hve the most significnt ctivity when compred to PG nd LPPM/A003. Detiled study ws tken up to look into the nti-oesity effect of GA (In vivo) nd scertin the nti oesity ctivity to this mrker compound. 562

8 () Control () LPPM/A μg/ml (c) GA-100 μg/ml Fig. 2. ORO stined mouse 3T3L1 cells () Control cells () Cells treted with LPPM/A μg/ml (c) Cells treted with GA-100μg/ml There ws significnt increse in ody weight of high ft diet fed group nimls when compred to norml control group (Tle 3). Mice on high ft diet when treted with GA (2, 4 nd 8mg/kg, p.o.) showed significnt decrese in ody weight when compred to ody weights of mice in high ft diet control group nimls (Fig. 3). Brod rnge of doses ws tken for the study (dt not shown) ut the effect ws prominent t the dose level of 2, 4 nd 8mg/kg p. o. 4.3 Effect on Feed Consumption The food consumption ws mesured on the dy of ody weight mesurement during tretment period. There were no significnt chnges in verge food intke (g/mouse/dy) oserved t tested dose levels of test item for 28 dys in therpeutic dose when compred to High ft diet control (Tle 4). 563

9 Fig. 3. The figure represents Men ody weight in grms of norml control group, high ft diet control group nd GA treted test groups = HFD Vs HFD + Test item treted (p < 0.05); GA: Gllic cid t 2, 4 nd 8mg/kg p. o. n = 8 per group 4.4 Inhiition of Leptin Expression in GA Administered Oese Rts A decrese in levels of leptin concentrtion in groups treted with grded doses of GA ws oserved when compred to High ft diet fed group (Fig. 4). GA ws found effective in reducing of the over expressed levels of leptin in oese experimentl nimls. Fig. 4. Figure represents concentrtion of Leptin produced in norml control group, high ft diet control group nd GA treted test groups *= p<0.05; ** = p<0.01; GA: Gllic cid t 2, 4 nd 8 mg/kg p. o. n=8 per group 564

10 4.5 Effect of Orl Administrtion of GA on Lipid Profile in Test Groups Oesity or high-ft diet is ssocited with insulin resistnce which in turn is ssocited with n increse in plsm concentrtions of cholesterol, triglycerides, or oth nd is cused y defects in the metolism of the lipoprotein clsses, very-low-density lipoprotein (VLDL), low density lipoprotein (LDL), nd high-density lipoprotein (HDL). Chronic oesity resulted in significnt increse in serum cholesterol nd triglyceride concentrtions in high ft diet fed nimls. In GA (2, 4 nd 8mg/kg) treted groups, serum cholesterol levels were reduced t 8 mg/kg in comprison with high ft diet (HFD) control. Compred with norml control-group nimls, oese-group nimls hd higher concentrtions of cholesterol in totl serum ( mg/dl) nd in VLDL (30.35mg/dl) nd LDL (86.45 mg/dl) nd decrese levels of HDL (28.5 mg/dl). Significnt reduction ws oserved in serum Triglyceride nd VLDL levels t tested dose levels. HDL levels were slightly elevted t 4 nd 8mg/kg nd significnt increse ws oserved t 8mg/kg dose nd lso the elevted levels of LDL ws decresed y dministrtion of GA t grded doses (Tle 5). 4.6 Effect of Grded Doses of GA on Pro-Inflmmtory Cytokines Associted With Oesity The serum from the tretment groups ws collected nd sujected to quntikine estimtion of TNF-α nd IL-6 y R nd D Systems ELISA kit. GA significntly decresed the TNF-α level in dose dependent mnner showing significnt inhiition t higher dose levels of 8 mg / kg per orl. Predictly we noted higher percentge expression of ±16.79GA/ml of TNF-α in high ft diet control group when compred to 25.52±1.83% expression in nïve control. Mximum suppression ws oserved t 8mg/kg p.o. dose (Fig. 5). Up-regultion of IL-6 is considered to e involved in the pthologicl process of oesity. IL-6 increses lipolysis nd ft oxidtion in humns [16]. This study clerly show tht high ft diet induced oese mice given GA hd considerly reduced mounts of IL-6 protein expressed y the dipocytes in the peripherl lood. The expression of IL-6 in GA 4 nd 8mg/kg dose ws 15.32±2.14 nd 14.77±2.76 respectively (Fig. 6). This finding ws in shrp contrst to high ft diet control group mice where higher mounts of IL-6 (25.5±1.89) were detected in ll of the oese nimls nlyzed. Thus orl dministrtion of GA t dose levels of 2, 4 nd 8 mg/kg ppers to inhiit the production of IL-6 in the serum smples of experimentl mice. 4.7 Acute Sfety Study Aqueous extrct of Lisi pumil is sfe [17]. The 14-dy oservtion period during the cute orl toxicity study nd ody weight mesurements did not revel ny toxic effects in either species. In the cute toxicity studies, orl LD50 of GA in Swiss lino mice ws greter thn 1000mg/kg ody weight, LD50 dose otined from series of experiments ws 1580mg/kg in swiss lino mice. GA did not show ny chnge in gross generl ehviour of these test nimls t the lower doses. 565

11 Tle 3. Effect on men ody weight y orl dministrtion of GA t grded doses Tretment groups Norml Control High Ft Control High Ft Diet + GA 2 mg/kg High Ft Diet + GA 4 mg/kg High Ft Diet + GA 8 mg/kg Body weights (g) Dy 15 Dy ± ± ± ± ± ±0.39 Dy ± ± ±0.86 Dy ± ± ±0.48 Dy ± ± ±0.27 Dy ± ± ±0.24 Dy ± ± ±0.41 Dy ± ± ±0.49 Dy ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.83 Vlues re expressed s Men ± SEM; n= 8; = Norml Vs HFD (p<0.05); = HFD Vs HFD + Test item treted (p<0.05) Tle 4. Tle represents group men feed consumption Tretment groups Norml Control High Ft Control High Ft Diet + GA 2mg/kg High Ft Diet + GA 4mg/kg High Ft Diet + GA 8mg/kg FEED consumption (g) Dy Dy ± ± ± ± ± ±0.24 Dy ± ± ±0.8 Dy ± ± ±1.08 Dy ± ± ± ± ± ± ± ± ± ± ±0.45 Dy ± ± ±0.39 Dy ± ± ±0.19 Dy ± ± ± ± ± ± ± ± ± ± ±0.17 Vlues re expressed s Men ± SEM; GA: Gllic cid t 2,4 nd 8 mg/kg p.o.; n = 8 per group Tle 5. Tle represents serum lipid profile of the experimentl groups Tretment groups Norml Control High Ft Control High Ft Diet + GA 2 mg/kg High Ft Diet + GA 4 mg/kg High Ft Diet + GA 8 mg/kg Cholesterol (mg/dl) Triglycerides (mg/dl) HDL (mg/dl) LDL (mg/dl) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±10.24 VLDL (mg/dl) 12.73± ± ± ± ±2.17 VLDL= Very-low-density lipoprotein cholesterol. HDL = High-density lipoprotein. LDL = Low-density lipoprotein; Vlues re expressed s Men ± SEM; n= 8 = Norml Vs HFD (p<0.05) = HFD Vs HFD + Test item treted (p<0.05) 566

12 Fig. 5. Expression of TNF-α in serum from high ft diet induced oese C57BL/6 mice treted with different concentrtion of GA *= p<0.05; ** = p<0.01; GA: Gllic cid t 2, 4 nd 8mg/kg p.o; n = 8 per group Fig. 6. Expression of IL-6 in serum from high ft diet induced oese C57BL/6 mice treted with different concentrtion of GA *= p<0.05; ** = p<0.01; GA: Gllic cid t 2, 4 nd 8 mg/kg p.o.; n = 8 per group 5. DISCUSSION The present study demonstrtes tht Gllic cid (GA) isolted from queous extrct of Lisi pumil significntly reduced weight gin in diet induced oese C57BL/6 mice. Before proceeding to the In vivo efficcy study on C57Bl/6 mice the in vitro nti-oesity potentil of GA ws evluted in mouse 3T3L1 cells. A common ssy to mesure dipocyte differentition in cell culture is with the dye Oil Red-O (ORO). During dipocytes differentition, oth LPPM/A003 nd GA hd inhiitory effect on ft droplet formtion nd triglyceride ccumultion. The concentrtion t which LPPM/A003 showed 50% inhiition ws 94.51μg/ml nd tht of GA ws 19.86μg/ml, however, IC50 vlue ws not otined for PG upto the highest workle concentrtion (Tle 2) (Fig. 2). 567

13 GA ws then tken up for further investigtion for estlishing its In vivo nti-oesity study. Mice on high ft diet when treted with GA (2, 4 nd 8 mg/kg, p. o.) showed significnt decrese in ody weight when compred to ody weights of mice in high ft diet control group nimls (Tle 3 nd Fig. 3). It ws shown tht dministrtion of GA t grded doses did not lter food intke in C57BL/6 mice indicting tht the prevention of weight gin induced y the test smple is not due to reduction in energy intke (Tle 4). Leptin is hormone with oth centrl nd peripherl effects on metolism nd energy lnce. However, in most oese individuls, leptin concentrtions re lredy high ecuse of the incresed mount of leptin-secreting dipose tissue [4]. It ppers tht with incresing leptin concentrtions, the hormone induces trget cells to ecome resistnt to its ctions. Excessive secretion of leptin s in cse of oesity or experimentl models of induced oesity leds to disrupted functions of hypothlmic centers tht n oese suject tends to go on over feeding mode. Hence, it ecomes impertive to ring out effective reduction of the over expressed levels of leptin in oesity nd vrious doses of GA shows promise in this re s indicted in Fig. 4. Significnt reduction ws oserved in serum Triglyceride nd VLDL levels t tested dose levels. HDL levels were slightly elevted t 2 nd 4mg/kg nd significnt increse ws oserved t 8 mg/kg dose nd lso the elevted levels of LDL ws decresed y dministrtion of GA t grded doses (Tle 5). Chronic inflmmtion ppers to e cliniclly importnt chnge tht occurs in dipose tissue when it ecomes oese [18] nd orl dministrtion of GA t dose levels of 2, 4 nd 8mg/kg inhiited the expression of TNF-α nd IL-6 in the serum of experimentl mice (Figs. 5 nd 6). Inflmmtion is thought to contriute to the development of the sequel of oesity. Adipose tissue is now considered to e n ctive endocrine orgn tht secretes vrious humorl fctors (dipokines), nd its shift to production of pro-inflmmtory cytokines like TNF lph nd IL-6 in oesity likely contriutes to the low-level systemic inflmmtion tht is seen in metolic syndrome-ssocited chronic pthologies such s oesity. Recent studies hve shown tht oesity induces chronic locl inflmmtion in dipose tissue, nd tht cells of the innte immune system, prticulrly mcrophges, re crucilly involved in dipose inflmmtion nd systemic metolic normlities. Adipose tissue TNF-α concentrtion is correlted with oesity [19,20]. TNF-α my increse systemic insulin resistnce y promoting the relese of ftty cids from dipose tissue into the loodstrem to ct on tissues such s muscle nd liver [21]. IL-6 expression is lso incresed in oese dipose tissue; IL-6 expression in dipose tissue from oese individuls is 10-fold tht in dipose tissue from len individuls if normlized for the numer of dipocytes present [22]. Plsm concentrtions of IL-6 increse with oesity, elevted IL-6 concentrtion is predictor for development of type 2 dietes nd for myocrdil infrction [23,24]. These dt indicte tht GA from queous extrct of Lisi pumil ppers to e n effective compound cple of modulting weight gin in high ft diet induced oese mice without inducing ny dverse effects. These effects pper to e medited through the reduction of incresed leptin levels, rised triglycerides nd cholesterol levels nd suppression of low level systemic inflmmtion. 5. CONCLUSION The findings demonstrte GA to hve potent nti-oesity ctivity nd this is suggestive of its possile therpeutic usefulness. Its pprent sfety over long term dministrtion is encourging enough to wrrnt further studies to explore its possile role in modern clinicl prctice. Optimizing helth with the inclusion of nutrceuticls, will llow for more individuls 568

14 to e le to control their oesity, nd decrese the urden tht it my plce on them physiclly, socilly, nd psychologiclly. ACKNOWLEDGEMENT The uthors re thnkful to Dr. Rjen M. nd Ms. Li Fong from Holist Biotech, Mlysi for providing the plnt mteril. COMPETING INTERESTS Authors hve declred tht no competing interests exist. REFERENCES Hslm DW, Jmes WP. Oesity. Lncet. 2005;366 (9492): Couillrd C, Muriege P, Imeult P, Prud homme D, Ndeu A, Tremly A, Bouchrd C, Despres JP. Hyperleptinemi is more closely ssocited with dipose cell hypertrophy thn with dipose tissue hyperplsi. Int J Oes Relt Met Disord. 2000;24: Heidi SC, Ren D, Leff T. Adipogenesis nd ft-cell function in oesity nd dietes. Trends Mol Med. 2002;8(9): Considine RV, Sinh MK, Heimn ML, Kriuciuns A, Stephens TW, Nyce MR, Ohnnesin JP, Mrco CC, McKee LJ, Buer TL. Serum immunorective-leptin concentrtions in norml-weight nd oese humns. N Engl J Med. 1996;1;334(5): Hotmisligil GS, Shrgill NS, Spiegelmn BM. Adipose expression of tumor necrosis fctor-lph: direct role in oesity-linked insulin resistnce. Science. 1993;259: Weiserg SP, McCnn D, Desi M, Rosenum M, Leiel RL, Ferrnte AW Jr. Oesity is ssocited with mcrophge ccumultion in dipose tissue. J Clin Invest. 2003;112: Cinti S, Mitchell G, Brtelli G, Murno I, Ceresi E, Floi E, Wng S, Fortier M, Greenerg AS, Oin MS. Adipocyte deth defines mcrophge locliztion nd function in dipose tissue of oese mice nd humns. J Lipid Res. 2005;46: Burkill IH. A Dictionry of the Economic Products of the Mly Peninsul, Crown Agent, London, UK; Fzlin M, Nzimoon WM, Gu HF, Ostenson CG. Lisi pumil extrct regultes ody weight nd dipokines in ovriectomized rts. Mturits. 2009;20: Kour K, Shrm N, Chndn BK, Koul S, Sngwn PL, Bni S. Protective effect of Lisi pumil on stress-induced ehviorl, iochemicl, nd immunologicl ltertions. Plnt Med. 2010;6(14): Wu Z, Xie Y, Morrison RF, Bucher NLR, Frmer SR. PPAR γ induces the Insulindependent Glucose Trnsporter GLUT4 in the sence of C/EBP during the conversion of 3T3 firolsts into dipocytes. J Clin Invest. 1998;101: Olivo LAS, F Cstro-Munozledo, Kuri-Hrcuch W. A predipose 3T3 cell vrint highly sensitive to dipogenic fctors nd to humn growth hormone. J. Cell Sci. 1995;108(5): Fu M, Tingwn Sun, Angie Bookout L, Michel Downes, Ruth Yu T, Ronld M. Evns nd Dvid J. Mngelsdorf. A Nucler Receptor Atls: 3T3-L1 Adipogenesis Mol Endocrinol. 2005;19(10):

15 14. Lin S, Thoms TC, Storlien LH, Hung XF. Development of high ft diet-induced oesity nd leptin resistnce in C57l/6J mice. Int J Oes. 2000;24: Drugs nd Cosmetics Act of 1988 (Schedule Y ). Drugs Controller Generl of Indi (DCGI), New Delhi, Director-Generl of Indin Council of Medicl Reserch, New Delhi; Hll vn G, Steenserg A, Scchetti M, Fischer C, Keller C, Schjerling P, Hiscock N, Møller K, Sltin B, Mrk A. Ferio, Bente K. Pedersen. Interleukin-6 stimultes lipolysis nd ft oxidtion in humns. J Clin Endocrinol Met. 2003;88: Singh GD, Gnjoo M, Youssouf MS, Koul A, Shrm R, Singh S, Sngwn PL, Koul S, Ahmd DB, Johri RK. Su-cute toxicity evlution of n queous extrct of Lisi pumil, Mlysin her. Food Chem. Toxicol. 2009;47: Hotmisligil GS. Inflmmtion nd metolic disorders. Nture. 2006;444(7121): Hotmisligil GS, Spiegelmn BM. Tumor necrosis fctor lph: key component of the oesity-dietes link. Dietes. 1994;43: Kern PA, Rngnthn S, Li C, Wood L, Rngnthn G. Adipose tissue tumor necrosis fctor nd interleukin-6 expression in humn oesity nd insulin resistnce. Am J Physiol Endocrinol Met. 2001;280 E Tilg H, Moschen AR. Inflmmtory mechnisms in the regultion of insulin resistnce. Mol Med. 2008;14(3-4): Fried SK, Bunkin DA, Greenerg AS. Omentl nd sucutneous dipose tissues of oese sujects relese interleukin-6: Depot difference nd regultion y glucocorticoid. J Clin Endocrinol Met. 1998;83: Prdhn AD, Mnson JE, Rifi N, Buring JE, Ridker PM. C-rective protein, interleukin 6, nd risk of developing type 2 dietes mellitus. J Am Med Assoc. 2001;286: Ridker PM, Rifi N, Stmpfer MJ, Hennekens CH. Plsm concentrtion of interleukin6 nd the risk of future myocrdil infrction mong pprently helthy men. Circultion 2000;101: Pndey et l.; This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Peer-review history: The peer review history for this pper cn e ccessed here: 570

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