Research Article Antioxidant and Antiadipogenic Activities

Research ArticleAntioxidant and Antiadipogenic Activities of Galkeun-Tanga Traditional Korean Herbal Formula

Soo-Jin Jeong Sae-Rom Yoo Ohn-Soon Kim Chang-Seob Seo and Hyeun-Kyoo Shin

Herbal Medicine Formulation Research Group Herbal Medicine Research Division Korea Institute of Oriental MedicineDaejeon 305-811 Republic of Korea

Correspondence should be addressed to Hyeun-Kyoo Shin hkshinkiomrekr

Received 1 August 2014 Revised 18 November 2014 Accepted 23 November 2014 Published 10 December 2014

Academic Editor Hyunsu Bae

Copyright copy 2014 Soo-Jin Jeong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Galkeun-tang (GKTGalgen-tang in Chinese and Kakkon-to in Japanese) a traditional herbal formula has been used for treatmentof the common cold Here we report in vitro antioxidant and antiadipogenic effects of GKT GKT increased the activities ofscavenging 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 22-diphenyl-1-picrylhydrazyl (DPPH) radicalsGKT also significantly reduced the malondialdehyde (MDA) generation during low-density lipoprotein (LDL) oxidation and theelectrophoretic mobility of oxidized LDL indicating inhibitory effects of GKT on Cu2+-mediated oxidation of LDL Regardingantiadipogenic activity GKT treatment significantly suppressed lipid accumulation triglyceride production and glycerol-3-phosphate dehydrogenase (GPDH) activity in differentiated 3T3-L1 adipocytes Consistent with this GKT significantly reducedthe secretion of leptin a major adipokine in differentiated 3T3-L1 adipocytes Overall our findings suggest that GKT has thepotential for antioxidative and antiadipogenic properties

1 Introduction

Oxidative stress is an imbalance between the production ofreactive oxygen species (ROS) and antioxidative defenses[1] Disruption in normal redox signaling can mediate toxiceffects and thus is closely associated with various humandiseases [2] Obesity is a metabolic disorder caused by excessfat accumulation in adipose tissue [3] Recent papers haveconfirmed a critical role for oxidative stress in the pathogen-esis of obesity or its associated diseases [4ndash6] A change inprotection against antioxidativemechanisms was observed inan obese rodentmodel and in obese humanmales [7 8]Thusthe development of antioxidants could be a valuable approachfor treating and preventing obesity or its related diseasesAntioxidative activities of synthetic or natural productshave been reported in obesity models Natural products areconsidered particularly attractive antiobesity drug candidatesbecause of their higher efficacies and fewer side effects

Many traditional herbalmedicines have attracted increas-ing attention for their complementary therapeutic effectswith few or no side effects compared withWesternmedicines[9 10] Galkeun-tang (GKT Galgen-tang in Chinese and

Kakkon-to in Japanese) a traditional herbal formula hasbeen used widely for treatment of the common cold fluand fever In recent research papers several pharmacologicalactivities of GKT have been reported including antiviralanti-inflammatory and immune-regulating activities [11ndash15]However the effect of GKT on obesity or obesity-relateddiseases has not been reported The pathogenesis of obesityand its associated diseases is closely related to an inflam-matory reaction caused by nutrient excess and imbalancethat has been termed ldquometainflammationrdquo [16] Thus wehypothesized that the previously reported anti-inflammatoryactivity of GKT may influence obesity or its related diseases

We therefore investigated the antioxidant and antiadi-pogenic effects of GKT We analyzed its scavenging activi-ties on 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) and 22-diphenyl-1-picrylhydrazyl (DPPH) radicalsin in vitro systems The effect on low-density lipoprotein(LDL) oxidation was assessed by measuring productionof malondialdehyde (MDA) In addition its inhibitoryeffects on adipogenesis the process by which preadipocytesbecome differentiated adipocytes [17] were determined byOil Red O staining and assays for triglyceride content

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 763494 9 pageshttpdxdoiorg1011552014763494

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Composition of GKT

Herbal medicine Scientific name Supplier Source Amount (g)Puerariae Radix Pueraria lobata Omniherb Jecheon Korea 90Cinnamomi Ramulus Cinnamomi cassia HMAX China 60Ephedrae Herba Ephedra sinica HMAX China 60Paeoniae Radix Paeonia lactiflora Omniherb Hwasun Korea 60Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis HMAX China 60Zingiberis Rhizoma Crudus Zingiber officinale Omniherb Yeongcheon Korea 60Zizyphi Fructus Ziziphus jujuba Omniherb Yeongcheon Korea 50

Total amount 440

glycerol-3-phosphate dehydrogenase (GPDH) activity andleptin production in differentiated 3T3-L1 adipocytes

2 Materials and Methods

21 Plant Materials The seven herbal medicines formingGKT were purchased from Omniherb (Yeongcheon Korea)and HMAX (Jecheon Korea) The origin of these herbalmedicines was taxonomically confirmed by Professor JeHyun Lee Dongguk University Gyeongju Korea A voucherspecimen (2008-KE02-1simKE02-7) has been deposited at theHerbal Medicine Formulation Research Group Korea Insti-tute of Oriental Medicine

22 Preparation of GKT Water Extract A GKT decoctionconsisting of seven herbal medicines including PuerariaeRadix Cinnamomi Ramulus Ephedrae Herba PaeoniaeRadix Glycyrrhizae Radix et Rhizoma Zingiberis RhizomaCrudus and Zizyphi Fructus was mixed (Table 1 35 kg440 g times 80) and extracted in a 10-fold mass of water at 100∘Cfor 2 h under pressure (1 kgfcm2) using an electric extractor(COSMOS-660 Kyungseo Machine Co Incheon Korea)The water extract was then filtered through a standard sieve(number 270 53120583mChungGye SangGong Sa Seoul Korea)and the solution was evaporated to dryness and freeze-driedto give a powder The yield of GKT water extract was 126(4416 g)

23 ABTS Radical Scavenging Activity The free radical scav-enging activity of GKT extract on ABTS was assessed by themethod described by Re et al [18] with slight modificationsABTS radical cation was produced by reacting 7mM ABTSsolution with 245mM potassium persulfate in the dark atroom temperature for 16 h Absorbance of the reactant waslater adjusted to 07 at a wavelength of 734 nm A 100 120583Laliquot of GKT solution at different concentrations (125ndash200120583gmL) was mixed with 100120583L ABTS∙+ solution Thereactionmixture was incubated for 5min in the dark at roomtemperature The absorbance of the resulting solution wasmeasured at 734 nm with a spectrophotometer (BenchmarkPlus Bio-RadHercules CA)The radical scavenging capacityof the tested samples was calculated using the followingequation

Scavenging activity ()

= (1 minus

Absorbance of sampleAbsorbance of control

) times 100

(1)

24 DPPH Radical Scavenging Activity The free radicalscavenging activity of GKT extract on DPPH was assessedusing the method described by Moreno et al [19] In briefa 100 120583L aliquot of GKT sample solution at different con-centrations (50ndash400120583gmL) was mixed with 100120583L DPPHsolution (015mM in methanol) The reaction mixture wasincubated for 30min in the dark at room temperatureThe absorbance of the resulting solution was measured at517 nmwith a spectrophotometer (Benchmark Plus Bio-RadHercules CA) The radical scavenging capacity of the testedsamples was calculated using the above formula

25 Oxidation of LDL by CuSO4 LDL samples (500 120583g

proteinmL Biomedical Technologies Stoughton MA) wereprepared at 37∘C in a medium containing 10mM phosphatebuffer (pH 74) and various concentrations (157ndash250120583gmL)of GKT extract After 5min oxidation was initiated bythe addition of CuSO

4(25 120583M) After 6 h of oxidation

lipid peroxidation (Section 26) and electrophoretic mobility(Section 27) of the LDL were measured as described below

26 Determination of MDA Using TBARS Assay Lipid per-oxidation of LDL was estimated by the determination of thelevel ofMDAusing a TBARS assay kit (Bioassay Systems LLCHayward CA) according to the manufacturerrsquos protocolsAfter oxidation 50 120583g of LDLs was mixed with 200120583L ofthiobarbituric acid (TBA) and incubated at 100∘C for 30minAfter completing the reaction the absorbance at 535 nm wasmeasured using a spectrophotometer

27 Relative Electrophoretic Mobility (REM) Assay The elec-trophoretic mobility of LDLs was measured using agarose gel(08 agarose in TAE buffer) electrophoresis and CoomassieBrilliant Blue R-250 staining Electrophoresis was performedat 100V for 30min REM was defined as the ratio of thedistances migrated from the origin by oxidized LDL (oxLDL)versus native LDL

28 Cell Culture andDifferentiation Mouse preadipocyte cellline 3T3-L1 was obtained from the American Type CultureCollection (ATCC CL-173 Rockville MD) The cells werecultured in DMEM (Gibco BRL Carlsbad CA) supple-mented with 10 newborn calf serum (NCS Gibco BRLCarlsbad CA) at 37∘C For adipocyte differentiation the cellswere stimulated with 3T3-L1 differentiation medium con-taining isobutylmethylxanthine dexamethasone and insulin

Evidence-Based Complementary and Alternative Medicine 3

(MDI) (Zenbio Inc Research Triangle Park NC) for 48 hafter reaching confluence The medium was switched toDMEM containing 10 fetal bovine serum (FBS) and1 120583gmL insulin after 2 days and then changed to DMEMcontaining 10 FBS for an additional 4 days Cells weretreated with GKT extract (0 25 50 100 200 or 400 120583gmL)during 8 days of the differentiation period GW9662 (SigmaSt Louis MO) a peroxisome proliferator-activated receptor-gamma (PPAR-120574) antagonist was used as a positive control

29 Cytotoxicity Assay Undifferentiated 3T3-L1 cells weretreated with various concentrations of GKT for 24 h Toobtain differentiated adipocytes 3T3-L1 preadipocytes weredifferentiated for 8 days in the presence of GKT The cellswere incubated with GKT extract at 37∘C under 5 CO

2

Cell counting kit- (CCK-) 8 solution (Dojindo KumamotoJapan) was added and incubated for 4 h After incubationthe absorbance was read at 450 nm using a microplate reader(Benchmark Plus Bio-Rad Hercules CA)

210 Oil Red O Staining 3T3-L1 preadipocytes were differ-entiated into adipocytes by adding isobutylmethylxanthinedexamethasone and insulin (MDI) for 8 days The cellswere treated with or without GKT (200120583gmL) or GW9662(20120583M) during the differentiation period The differentiated3T3-L1 cells were fixed with 10 formalin for 15min at roomtemperature and washed with 70 ethanol and phosphate-buffered saline (PBS) The cells were stained with Oil RedO (Sigma St Louis MO) for 5min and then washed withPBS Cell images were collected using an Olympus CKX41inverted microscope (Olympus Tokyo Japan) Stained oildroplets were dissolved in isopropyl alcohol andmeasured byreading the absorbance at 520 nm

211 Triglyceride Quantification Assay 3T3-L1 preadipocyteswere differentiated into adipocytes by addingMDI for 8 daysThe cells were treated with or without GKT (25 50 100 200or 400120583gmL) orGW9662 (20120583M)during the differentiationperiod The triglyceride content of cells was enzymaticallymeasured using a commercial kit (BioVision Inc MilpitasCA) Briefly the 3T3-L1 adipocytes treated with GKT werehomogenized in 5 NP-40 assay buffer and the sample wasslowly heated to solubilize all triglycerides The samples weremixed with lipase and triglyceride reaction mixture After1 h of incubation the sample absorbance was measured at570 nm

212 GPDH Activity Assay After the induction of adipocytedifferentiation in the presence of GKT 3T3-L1 cells werewashed twice with PBS GPDH activity was determined byusing a commercial kit (TAKARA Tokyo Japan) and moni-toring the dihydroxyacetone phosphate-dependent oxidationof nicotinamide adenine dinucleotide (NADH) at 340 nmand the results were expressed as unitmg of protein

213 Leptin Immunoassay Leptin levels were assayed using amouse leptin immunoassay kit (RampD Systems MinneapolisMN) according to the manufacturerrsquos instructions In briefthe culture supernatant was collected from the differentiated

3T3-L1 with or without GKT treatment An equal ratio ofthe supernatants (50120583L) and assay diluent RD1W (50 120583L)was added to wells of a 96-well plate and incubated for 2 hat room temperature After washing 5 times with 400120583L ofwash buffer 100 120583L of mouse leptin conjugate was added toeach well and incubated for 2 h at room temperature Afterwashing 5 times 100120583L of substrate solution was added toeach well and incubated for 30min at room temperature inthe dark Finally 100 120583L of stop solution was added to eachwell and the absorbance was measured at 450 nm

214 Chemicals and Reagents for HPLC Analysis Cin-namaldehyde cinnamic acid glycyrrhizin liquiritin paeoni-florin and puerarin (all purity ge 980) were purchasedfromWako Fine Chemicals (Osaka Japan)TheHPLC-gradereagents methanol acetonitrile and water were obtainedfrom J T Baker (Phillipsburg NJ) Glacial acetic acid wasobtained from Junsei Chemical Co (Tokyo Japan)

215 Preparation of Standard and Sample Solutions for HPLCAnalysis Methanol standard stock solutions were madecontaining each of cinnamic acid glycyrrhizin liquiritinpaeoniflorin and puerarin at 10mgmL Cinnamaldehydewas dissolved in methanol at a concentration of 105mgmLThe stock solutions were stored below 4∘C

For HPLC analysis lyophilized GKT extract was weighed(200mg) into a 20mL flask and distilled water was addedto the volumetric mark and then the mixture was passedthrough a 02120583msyringe filter before injection into theHPLCsystem

216 HPLC Analysis of GKT HPLC was performed on a Shi-madzu LC-20A HPLC system (Shimadzu Co Kyoto Japan)consisting of a solvent delivery unit an on-line degasser acolumn oven an autosampler and a photodiode array (PDA)detector The data processor employed LCsolution software(Version 124) The analytical column used was a Gemini C

18

(250 times 46mm particle size 5120583m Phenomenex TorranceCA) maintained at 40∘C The mobile consisted of solventA (10 vv acetic acid in H

2O) and solvent B (10 vv

acetic acid in acetonitrile) The gradient flow was as follows5ndash70 B for 0ndash40min 70ndash100 B for 40ndash45min 100B for 45ndash50min and 100ndash5 B for 55min The flow ratewas 10mLmin and the injection volume was 10 120583L Thequantitative analysis of the six compounds was carried out at230 nm (paeoniflorin) 254 nm (glycyrrhizin and puerarin)and 280 nm (cinnamaldehyde cinnamic acid and liquiritin)

217 Statistical Analysis All data were presented as mean plusmnstandard error of the mean (SEM) Group differences wereassessed by one-way ANOVA and post hoc Tukeyrsquos multiplecomparison test using Graphpad InStat ver310 (GraphpadSoftware Inc San Diego CA) Significance of differencesfrom the normal control was taken as 119875 lt 005

3 Results

31 HPLC Analysis of GKT All calibration curves wereobtained by assessment of peak areas from standard


Table 2 Regression data linear range correlation coefficient LOD and LOQ for marker compounds (119899 = 3)

Compound Linear range (120583gmL) Regression equationa Correlation coefficient (1198772) LODb (120583gmL) LOQc (120583gmL)Puerarin 234ndash30000 119910 = 3981406x + 3597626 10000 004 015Paeoniflorin 078ndash10000 119910 = 3873830x minus 1527319 09999 005 015Liquiritin 156ndash20000 119910 = 1849504x + 1206272 10000 003 011Cinnamic acid 234ndash30000 119910 = 2733838x + 3146188 10000 002 006Cinnamaldehyde 164ndash10500 119910 = 12200031x + 6258719 10000 001 003Glycyrrhizin 234ndash30000 119910 = 826643x + 584351 10000 036 121ay peak area (mAU) of compounds 119909 concentration (120583gmL) of compoundsbLOD = 3 times signal-to-noise ratiocLOQ = 10 times signal-to-noise ratio

Table 3 Contents of six compounds in the GKT by HPLC (119899 = 3)

Compound Mean (mgg) SD RSD () SourcePuerarin 1029 002 021 Puerariae RadixPaeoniflorin 355 002 069 Paeoniae RadixLiquiritin 664 005 075 Glycyrrhizae Radix et RhizomaCinnamic acid 201 005 244 Cinnamomi RamulusCinnamaldehyde 730 004 050 Cinnamomi RamulusGlycyrrhizin 1217 009 076 Glycyrrhizae Radix et Rhizoma

solutions in the concentration ranges puerarin cinnamicacid and glycyrrhizin 234ndash30000 120583gmL paeoniflorinliquiritin and cinnamaldehyde 078ndash10000120583gmL 156ndash20000120583gmL and 164ndash10500 120583gmL respectively Theretention times were 1381 1572 1765 2577 2808 and3355min for puerarin paeoniflorin liquiritin cinnamicacid cinnamaldehyde and glycyrrhizin respectively Thecalibration curves correlation coefficients (1198772) limit ofdetection (LOD) and limit of quantification (LOQ) of thesix marker compounds are summarized in Table 2 Usingoptimized chromatography conditions a three-dimensionalchromatogram was obtained using the HPLC-PDA detector(Figure 1) The concentrations of the six marker compoundswere 201ndash1217mgg and are summarized in Table 3

32 Antioxidant Activity of GKT To evaluate the antioxidantactivity of GKT we tested its scavenging activities on ABTSand DPPH radicals The ABTS radical scavenging activity ofGKT is presented in Table 4 The extracts of GKT showeda dose-dependent radical scavenging activity The concen-tration of GKT required for 50 inhibition (IC

50) of ABTS

radicals was 5116 120583gmL while the IC50

value of ascorbicacid as a positive control was 322120583gmL The antioxidantactivities obtained for GKT by the DPPH method are shownin Table 5 Similar to the ABTS assay GKT reduced DPPHradical formation in a concentration-dependent mannerTheIC50of GKT against DPPH radicals was 24296120583gmL while

the IC50value of ascorbic acid was 1043 120583gmL

33 Effect of GKT on Cu2+-Mediated Oxidation of LDL Thegeneration of MDA equivalents during LDL oxidation wasestimated by the TBARS assay As shown in Figure 2(a)when LDL was incubated with CuSO

4for 6 h a significant

Table 4 Scavenging effects of GKT on ABTS∙+

Sample Concentration(120583gmL)

Scavenging effect()

IC501

(120583gmL)

GKT2

125 1924 plusmn 119

5116 plusmn 26725 3285 plusmn 28050 5808 plusmn 351100 8235 plusmn 026200 9746 plusmn 030

AA3125 2115 plusmn 019

322 plusmn 00625 4061 plusmn 1445 7586 plusmn 106

1Concentration required for 50 reduction of ABTS∙+ at 5min reaction2Galkeun-tang and 3ascorbic acidEach value is the mean plusmn SEM of triplicate determinations

Table 5 Scavenging effects of GKT on DPPH


Scavenging effect()

IC501

(120583gmL)

GKT2

50 633 plusmn 014

24296 plusmn 266100 2264 plusmn 049200 4766 plusmn 107400 7870 plusmn 060

AA3125 2115 plusmn 019


1Concentration required for 50 reduction of DPPH at 30min reaction2Galgeun-tang and 3ascorbic acidEach value is the mean plusmn SEM of triplicate determinations


1000

minus10

200

250

300

350

Wavelength (nm

)10000

20000

30000

40000

0

200

400

600

800

1000

Time (min)

Inte

nsity

(mAU

)

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

O

O

O OO

O

O

OO

O

OO

O

O

O O

O

O

O

O

HOHO

HO HO

HOHO

HOHO

HO

HOHO

CH

PaeoniflorinLiquiritin

Cinnamic acidCinnamaldehyde

Glycyrrhizin

HOOC

HOOC

HOOC

Puerarin

H

Figure 1 Three-dimensional chromatogram of GKT by HPLC-PDA

minus20

0

20

40

60

80

100

120

LDL OxLDL 156 313 625 125 250

GKT(120583gmL)

TBA

RS (

OxL

DL)

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

(a)

08

09

1

11

12

13

14

15

LDL OxLDL 156 3125 625 125 250

GKT

REM

(AU

)



(120583gmL)

LDL 0 157 3125 625 125 250

OxLDL + GKT (120583gmL)

(b)

Figure 2 Inhibitory effects of GKT on Cu2+-induced LDL oxidation The indicated concentrations of GKT and LDLs were incubated withCuSO

4for 6 h at 37∘CThe TBARS level (a) and electrophoretic mobility (b) of LDLs were measured using a TBARS assay kit and agarose gel

electrophoresis respectively The data are presented as the mean values of three experimentsrsquo plusmn SEM lowastlowast119875 lt 001 compared with the oxLDLgroup


0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)

Figure 3 Cytotoxic effects of GKT extract in 3T3-L1 cells (a) Undifferentiated 3T3-L1 cells were treated with various concentrations of GKT(0 78125 15625 3125 625 125 250 or 500120583gmL) for 24 h (b) Adipocyte differentiation was induced by adding isobutylmethylxanthinedexamethasone and insulin (MDI) to 3T3-L1 preadipocytes for 8 days The cells were exposed to various concentrations of GKT (0 625125 250 500 or 1000120583gmL) during the differentiation period Cell viability was determined using a CCK-8 assay kit by measuring theabsorbance at 450 nm Data are presented as the mean plusmn SEM

increase in TBARS was detected In contrast GKT signif-icantly reduced the amount of TBARS formed in a dose-dependent manner (IC

50 5323 120583gmL) The alteration of

mobility in agarose gel electrophoresis reflects the increasein the negative charge of LDL particles which occurs duringoxidation [20] When the oxidation was carried out in thepresence of GKT the increase in electrophoretic mobility ofoxLDL was significantly reduced (Figure 2(b)) These datasuggest that GKT has an inhibitory effect on LDL oxidation

34 Cytotoxic Effects of GKT in 3T3-L1 Cells To evaluate thepossible cytotoxicity of GKT against 3T3-L1 preadipocytesthe cells were treated with various concentrations of GKTfor 24 h As shown in Figure 3(a) GKT had no cytotoxicityagainst 3T3-L1 preadipocytes Additionally the cytotoxicityof GKT was assessed in the differentiated adipocytes Duringdifferentiation for 8 days the cells were exposed to variousconcentrations of GKT No significant cytotoxic effect wasobserved in GKT-treated adipocytes (Figure 3(b))

35 The Inhibitory Effects of GKT on Adipogenesis of 3T3-L1Adipocytes Triglyceride production is one of the importantevents which occurs during adipogenesis [21] Oil Red Ostaining was carried out to examine lipid accumulation inthe differentiated 3T3-L1 adipocytes The number of lipiddroplets detectable with Oil Red O staining was obvi-ously increased in adipocytes compared with preadipocytes(Figure 4(a)) However GKT treatment significantly reducedlipid accumulation compared with the untreated differenti-ated cells In addition the intracellular triglyceride contentwas measured in 3T3-L1 adipocytes treated with GKT Con-sistent with the results of Oil Red O staining triglycerideproduction was significantly increased in the differentiatedadipocytes compared with preadipocytes but GKT sig-nificantly inhibited triglyceride production compared withuntreated differentiated cells (Figure 4(b))

GPDH is an enzyme that generates glycerol-3-phosphatefrom dihydroxyacetone phosphate for lipid biosynthesis inadipocytes [22] As shown in Figure 5(a) treatment withGKT at the concentration of 25sim400120583gmL significantlyinhibited the activity of GPDH compared with untreateddifferentiated adipocytes Consistent with this GKT alsohad a suppressive effect on secretion of leptin a keyadipokine [23] compared with the untreated differentiatedcells (Figure 5(b)) Similarly treatment with GW9662 thepositive control dramatically inhibited adipogenesis in 3T3-L1 cells

4 Discussion

In the present study we demonstrate that a traditionalherbal medicine GKT has antioxidant and antiadipogenesisproperties For quality control of the GKT extract weperformed a quantitative determination of the six maincomponents in GKT using HPLC coupled with a PDA Theinvestigated components were as follows puerarin formPuerariae Radix cinnamaldehyde and cinnamic acid fromCinnamomi Ramulus paeoniflorin from Paeoniae Radixand liquiritin and glycyrrhizin from Glycyrrhizae Radix etRhizoma The optimized HPLC-PDA method was appliedfor simultaneous quantitation of the six components inGKT (Figure 1) Among these components puerarin andglycyrrhizin which are marker components of PuerariaeRadix and Glycyrrhizae Radix et Rhizoma were detected at1029mgg and 1217mgg respectively as the major compo-nents ofGKT (Table 3)The establishment of thisHPLC-PDAmethod will be helpful in improving quality control of GKT

In in vitro assay systems GKT showed significant scav-enging effects againstABTS andDPPHradicals (Table 4) andit inhibited LDL oxidation mediated by CuSO

4in a dose-

dependent manner (Figure 2) In addition GKT revealedantiadipogenic activity by blocking lipid accumulation


Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT

lowastlowastlowastlowastlowastlowast

(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)

Figure 4 Inhibitory effect of GKT extract on triglyceride accumulation in 3T3-L1 adipocytes 3T3-L1 preadipocytes were differentiated intoadipocytes by adding isobutylmethylxanthine dexamethasone and insulin (MDI) for 8 days The cells were treated with or without GKT orGW9662 (20120583M) during the differentiation period (a and b) Lipid accumulation in the cells was analyzed by Oil Red O staining (a) Thecells stained with Oil Red O were visualized using an Olympus CKX41 inverted microscope at times200 magnification (left panel) Stained oildroplets were dissolved in isopropyl alcohol and measured by reading absorbance at 520 nm (right panel) (b)The content of triglyceride wasenzymatically measured at 570 nm using a commercial kit (BioVision Inc Milpitas CA) Data are presented as the mean plusmn SEM lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001 compared with the differentiated control

0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)

Figure 5 Inhibitory effects of GKT on adipogenesis-related factors in 3T3-L1 adipocytes 3T3-L1 preadipocytes were differentiated intoadipocytes by adding isobutylmethylxanthine dexamethasone and insulin (MDI) for 8 daysThe cells were exposed to various concentrationsof GKT during the differentiation period (a) GPDH activity of the cells was assessed by measuring the decrease in NADH at 340 nm usingthe TAKARA GPDH activity assay kit (b) Culture supernatant was collected from the GKT-treated cells Leptin production was determinedby ELISA at 540 nm subtracted from 450 nm using a mouse leptin immunoassay kit (RampD Systems) Data are presented as the mean plusmn SEMlowastlowastlowast

119875 lt 0001 lowastlowast119875 lt 001 and lowast119875 lt 005 compared with the differentiated control


triglyceride production GPDH activity and leptin produc-tion in adipocytes without cytotoxic effects (Figures 3ndash5)

Oxidative stress plays an important role in the pathogen-esis of various diseases including obesityThus the ldquooxidativestress paradigmrdquo is an appealing concept for developingnovel therapeutics [24] Excess oxidative stress in obesepatients coincides with fat accumulation in adipocytes [4]We induced the cellular differentiation of 3T3-L1 mousepreadipocytes into adipocytes This cell line is useful forstudying adipogenesis [25] After adipocyte differentiationa significant increase of intracellular lipid accumulationcompared with untreated preadipocytes was observed byOil Red O staining In contrast GKT significantly inhibitedthe amount of lipid accumulation compared with untreateddifferentiated adipocytes (Figure 4(a)) Microscopy furtherconfirmed the inhibitory effect of GKT on adipogenesisGKT treatmentmarkedly reduced the increase in the numberand size of adipocytes a hallmark of adipocyte endocrinefunction [26] Lipid droplets consist of a core of lipid estersand a surface lined with a phospholipid monolayer and inadipocytes the lipid ester core contains triglycerides [27ndash29] Consistent with the results of Oil Red O stainingGKT significantly decreased the content of triglyceride inadipocytes (Figure 4(b)) Furthermore we evaluated theGPDH activity in differentiated 3T3-L1 adipocytes GPDHis activated strongly in mature adipocytes and plays a rolein the triglyceride biosynthesis pathway [30 31] GPDHenzyme activity was significantly decreased in GKT-treatedadipocytes (Figure 5(a)) The level of another key factor inadipogenesis leptin was measured in adipocytes differen-tiated in the absence or presence of GKT Leptin is anadipokine exclusively produced by adipocytes in proportionto triglyceride accumulation [32] As expected GKT signif-icantly inhibited the level of secretion of leptin in differen-tiated 3T3-L1 adipocytes (Figure 5(b)) Similar to our studyseveral groups have reported dual effects of natural productsincluding purple sweet potato [33] safflower seed [34] andbuckwheat sprout [35] on oxidative stress and adipogenesisTogether these results suggest the potential of natural prod-ucts including herbal medicines as adipogenesis-preventingsubstances with antioxidant properties

Interestingly several herbal components of GKT havebeen reported to have antioxidant properties or antiobe-sity effects Pueraria lobata extract ameliorated impairedglucose and lipid metabolism in obese mice [36] Puer-aria lobata extract also inhibited tert-butyl hydroperoxide-induced ROS generation [37] An extract of Cinnamomumcassia twigs inhibited adipocyte differentiation via activationof the insulin signaling pathway in 3T3-L1 preadipocytes[38] Ephedra sinica extract exerted antioxidant effects byaccelerating free radical scavenging activity and oxidantreducing power [39] These data could support antiadi-pogenicantioxidant activities of GKT Additional studies willbe required to confirm the antioxidantantiobesity effects ofGKT using a high fat diet-fed obese animal model

Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgment

Thisworkwas supported by aGrant from theKorean Instituteof Oriental Medicine (no K14030)

References

[1] D J Betteridge ldquoWhat is oxidative stressrdquoMetabolism Clinicaland Experimental vol 49 no 2 supplement 1 pp 3ndash8 2000

[2] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[3] N Rasouli B Molavi S C Elbein and P A Kern ldquoEctopic fataccumulation and metabolic syndromerdquo Diabetes Obesity andMetabolism vol 9 no 1 pp 1ndash10 2007

[4] S Furukawa T FujitaM Shimabukuro et al ldquoIncreased oxida-tive stress in obesity and its impact onmetabolic syndromerdquoTheJournal of Clinical Investigation vol 114 no 12 pp 1752ndash17612004

[5] C K Roberts R J Barnard R K Sindhu M Jurczak AEhdaie and N D Vaziri ldquoOxidative stress and dysregulationof NAD(P)H oxidase and antioxidant enzymes in diet-inducedmetabolic syndromerdquo Metabolism vol 55 no 7 pp 928ndash9342006

[6] E Tumova W Sun P H Jones M Vrablik C M Ballantyneand R C Hoogeveen ldquoThe impact of rapid weight loss onoxidative stress markers and the expression of the metabolicsyndrome in obese individualsrdquo Journal of Obesity vol 2013Article ID 729515 10 pages 2013

[7] I D Capel and H M Dorrell ldquoAbnormal antioxidant defencein some tissues of congenitally obesemicerdquoBiochemical Journalvol 219 no 1 pp 41ndash49 1984

[8] M Ozata M Mergen C Oktenli et al ldquoIncreased oxidativestress and hypozincemia in male obesityrdquo Clinical Biochemistryvol 35 no 8 pp 627ndash631 2002

[9] T Xue and R Roy ldquoStudying traditional Chinese medicinerdquoScience vol 300 no 5620 pp 740ndash741 2003

[10] D Normile ldquoThe new face of traditional Chinese medicinerdquoScience vol 299 no 5604 pp 188ndash190 2003

[11] M Kurokawa M Tsurita J Brown Y Fukuda and K ShirakildquoEffect of interleukin-12 level augmented byKakkon-to a herbalmedicine on the early stage of influenza infection in micerdquoAntiviral Research vol 56 no 2 pp 183ndash188 2002

[12] M SWuH R Yen CW Chang et al ldquoMechanism of action ofthe suppression of influenza virus replication by Ko-Ken Tangthrough inhibition of the phosphatidylinositol 3-kinaseAktsignaling pathway and viral RNP nuclear exportrdquo Journal ofEthnopharmacology vol 134 no 3 pp 614ndash623 2011

[13] K Nagasaka M Kurokawa M Imakita K Terasawa and KShiraki ldquoEfficacy of Kakkon-to a traditional herb medicine inherpes simplex virus type 1 infection inmicerdquo Journal ofMedicalVirology vol 46 no 1 pp 28ndash34 1995

[14] K Muraoka S Yoshida K Hasegawa et al ldquoA pharmacologicstudy on the mechanism of action of Kakkon-to body tem-perature elevation and phagocytic activation of macrophages indogsrdquo Journal of Alternative and Complementary Medicine vol10 no 5 pp 841ndash849 2004

[15] H Yano S Hiraki and S Hayasaka ldquoEffects of Kakkon-toand Sairei-to on experimental elevation of aqueous flare inpigmented rabbitsrdquo Japanese Journal of Ophthalmology vol 43no 4 pp 279ndash284 1999


[16] G S Hotamisligil ldquoInflammation and metabolic disordersrdquoNature vol 444 no 7121 pp 860ndash867 2006

[17] M I Lefterova and M A Lazar ldquoNew developments inadipogenesisrdquo Trends in Endocrinology amp Metabolism vol 20no 3 pp 107ndash114 2009

[18] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[19] M I N Moreno M I Isla A R Sampietro and M AVattuone ldquoComparison of the free radical-scavenging activityof propolis from several regions of Argentinardquo Journal ofEthnopharmacology vol 71 no 1-2 pp 109ndash114 2000

[20] D L Sparks and M C Phillips ldquoQuantitative measurementof lipoprotein surface charge by agarose gel electrophoresisrdquoJournal of Lipid Research vol 33 no 1 pp 123ndash130 1992

[21] DW Haslam andW P T James ldquoObesityrdquoTheLancet vol 366no 9492 pp 1197ndash1209 2005

[22] L P Kozak and J T Jensen ldquoGenetic and developmental controlof multiple forms of L-glycerol 3-phosphate dehydrogenaserdquoThe Journal of Biological Chemistry vol 249 no 24 pp 7775ndash7781 1974

[23] F Zhang M B Basinski J M Beals et al ldquoCrystal structureof the obese protein leptin-E100rdquo Nature vol 387 no 6629 pp206ndash209 1997

[24] H Sies ldquoOxidative stress oxidants and antioxidantsrdquo Experi-mental Physiology vol 82 no 2 pp 291ndash295 1997

[25] H Green and M Meuth ldquoAn established pre-adipose cell lineand its differentiation in culturerdquo Cell vol 3 no 2 pp 127ndash1331974

[26] T Tsuda F Horio K Uchida H Aoki and T Osawa ldquoDietarycyanidin 3-O-120573-D-glucoside-rich purple corn color preventsobesity and ameliorates hyperglycemia in micerdquo Journal ofNutrition vol 133 no 7 pp 2125ndash2130 2003

[27] D J Murphy and J Vance ldquoMechanisms of lipid-body forma-tionrdquo Trends in Biochemical Sciences vol 24 no 3 pp 109ndash1151999

[28] K Tauchi-Sato S Ozeki T Houjou R Taguchi and T Fuji-moto ldquoThe surface of lipid droplets is a phospholipidmonolayerwith a unique fatty acid compositionrdquoThe Journal of BiologicalChemistry vol 277 no 46 pp 44507ndash44512 2002

[29] T Fujimoto Y Ohsaki J Cheng M Suzuki and Y ShinoharaldquoLipid droplets a classic organelle with new outfitsrdquoHistochem-istry and Cell Biology vol 130 no 2 pp 263ndash279 2008

[30] J Pairault and H Green ldquoA study of the adipose conversion ofsuspended 3T3 cells by using glycerophosphate dehydrogenaseas differentiation markerrdquo Proceedings of the National Academyof Sciences of the United States of America vol 76 no 10 pp5138ndash5142 1979

[31] L S Wise and H Green ldquoParticipation of one isozyme ofcytosolic glycerophosphate dehydrogenase in the adipose con-version of 3T3 cellsrdquo The Journal of Biological Chemistry vol254 no 2 pp 273ndash275 1979

[32] H C Chen and R V Farese Jr ldquoDetermination of adipocytesize by computer image analysisrdquo Journal of Lipid Research vol43 no 6 pp 986ndash989 2002

[33] J-H Ju H-S Yoon H-J Park et al ldquoAnti-obesity andantioxidative effects of purple sweet potato extract in 3T3-L1adipocytes in vitrordquo Journal of Medicinal Food vol 14 no 10pp 1097ndash1106 2011

[34] S-Y Yu Y-J Lee J-D Kim et al ldquoPhenolic compositionantioxidant activity and anti-adipogenic effect of hot waterextract from safflower (Carthamus tinctorius L) seedrdquo Nutri-ents vol 5 no 12 pp 4894ndash4907 2013

[35] Y-J Lee K-J Kim K-J Park B-R Yoon J-H Lim and O-H Lee ldquoBuckwheat (Fagopyrum esculentumM) sprout treatedwithmethyl jasmonate (MeJA) improved anti-adipogenic activ-ity associated with the oxidative stress system in 3T3-L1adipocytesrdquo International Journal of Molecular Sciences vol 14no 1 pp 1428ndash1442 2013

[36] J K Prasain N Peng R Rajbhandari and J M Wyss ldquoTheChinese Pueraria root extract (Pueraria lobata) amelioratesimpaired glucose and lipid metabolism in obese micerdquo Phy-tomedicine vol 20 no 1 pp 17ndash23 2012

[37] S E Jin Y K Son B-S Min H A Jung and J S Choi ldquoAnti-inflammatory and antioxidant activities of constituents isolatedfromPueraria lobata rootsrdquoArchives of Pharmacal Research vol35 no 5 pp 823ndash837 2012

[38] Y Han H W Jung H S Bae J-S Kang and Y-K ParkldquoThe extract of Cinnamomum cassia twigs inhibits adipocytedifferentiation via activation of the insulin signaling pathwayin 3T3-L1 preadipocytesrdquo Pharmaceutical Biology vol 51 no 8pp 961ndash967 2013

[39] F L Song R Y Gan Y Zhang Q Xiao L Kuang and H B LildquoTotal phenolic contents and antioxidant capacities of selectedchinese medicinal plantsrdquo International Journal of MolecularSciences vol 11 no 6 pp 2362ndash2372 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Composition of GKT

Herbal medicine Scientific name Supplier Source Amount (g)Puerariae Radix Pueraria lobata Omniherb Jecheon Korea 90Cinnamomi Ramulus Cinnamomi cassia HMAX China 60Ephedrae Herba Ephedra sinica HMAX China 60Paeoniae Radix Paeonia lactiflora Omniherb Hwasun Korea 60Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis HMAX China 60Zingiberis Rhizoma Crudus Zingiber officinale Omniherb Yeongcheon Korea 60Zizyphi Fructus Ziziphus jujuba Omniherb Yeongcheon Korea 50

Total amount 440

glycerol-3-phosphate dehydrogenase (GPDH) activity andleptin production in differentiated 3T3-L1 adipocytes

2 Materials and Methods

21 Plant Materials The seven herbal medicines formingGKT were purchased from Omniherb (Yeongcheon Korea)and HMAX (Jecheon Korea) The origin of these herbalmedicines was taxonomically confirmed by Professor JeHyun Lee Dongguk University Gyeongju Korea A voucherspecimen (2008-KE02-1simKE02-7) has been deposited at theHerbal Medicine Formulation Research Group Korea Insti-tute of Oriental Medicine

22 Preparation of GKT Water Extract A GKT decoctionconsisting of seven herbal medicines including PuerariaeRadix Cinnamomi Ramulus Ephedrae Herba PaeoniaeRadix Glycyrrhizae Radix et Rhizoma Zingiberis RhizomaCrudus and Zizyphi Fructus was mixed (Table 1 35 kg440 g times 80) and extracted in a 10-fold mass of water at 100∘Cfor 2 h under pressure (1 kgfcm2) using an electric extractor(COSMOS-660 Kyungseo Machine Co Incheon Korea)The water extract was then filtered through a standard sieve(number 270 53120583mChungGye SangGong Sa Seoul Korea)and the solution was evaporated to dryness and freeze-driedto give a powder The yield of GKT water extract was 126(4416 g)

23 ABTS Radical Scavenging Activity The free radical scav-enging activity of GKT extract on ABTS was assessed by themethod described by Re et al [18] with slight modificationsABTS radical cation was produced by reacting 7mM ABTSsolution with 245mM potassium persulfate in the dark atroom temperature for 16 h Absorbance of the reactant waslater adjusted to 07 at a wavelength of 734 nm A 100 120583Laliquot of GKT solution at different concentrations (125ndash200120583gmL) was mixed with 100120583L ABTS∙+ solution Thereactionmixture was incubated for 5min in the dark at roomtemperature The absorbance of the resulting solution wasmeasured at 734 nm with a spectrophotometer (BenchmarkPlus Bio-RadHercules CA)The radical scavenging capacityof the tested samples was calculated using the followingequation

Scavenging activity ()

= (1 minus

Absorbance of sampleAbsorbance of control

) times 100

(1)

24 DPPH Radical Scavenging Activity The free radicalscavenging activity of GKT extract on DPPH was assessedusing the method described by Moreno et al [19] In briefa 100 120583L aliquot of GKT sample solution at different con-centrations (50ndash400120583gmL) was mixed with 100120583L DPPHsolution (015mM in methanol) The reaction mixture wasincubated for 30min in the dark at room temperatureThe absorbance of the resulting solution was measured at517 nmwith a spectrophotometer (Benchmark Plus Bio-RadHercules CA) The radical scavenging capacity of the testedsamples was calculated using the above formula

25 Oxidation of LDL by CuSO4 LDL samples (500 120583g

proteinmL Biomedical Technologies Stoughton MA) wereprepared at 37∘C in a medium containing 10mM phosphatebuffer (pH 74) and various concentrations (157ndash250120583gmL)of GKT extract After 5min oxidation was initiated bythe addition of CuSO

4(25 120583M) After 6 h of oxidation

lipid peroxidation (Section 26) and electrophoretic mobility(Section 27) of the LDL were measured as described below

26 Determination of MDA Using TBARS Assay Lipid per-oxidation of LDL was estimated by the determination of thelevel ofMDAusing a TBARS assay kit (Bioassay Systems LLCHayward CA) according to the manufacturerrsquos protocolsAfter oxidation 50 120583g of LDLs was mixed with 200120583L ofthiobarbituric acid (TBA) and incubated at 100∘C for 30minAfter completing the reaction the absorbance at 535 nm wasmeasured using a spectrophotometer

27 Relative Electrophoretic Mobility (REM) Assay The elec-trophoretic mobility of LDLs was measured using agarose gel(08 agarose in TAE buffer) electrophoresis and CoomassieBrilliant Blue R-250 staining Electrophoresis was performedat 100V for 30min REM was defined as the ratio of thedistances migrated from the origin by oxidized LDL (oxLDL)versus native LDL

28 Cell Culture andDifferentiation Mouse preadipocyte cellline 3T3-L1 was obtained from the American Type CultureCollection (ATCC CL-173 Rockville MD) The cells werecultured in DMEM (Gibco BRL Carlsbad CA) supple-mented with 10 newborn calf serum (NCS Gibco BRLCarlsbad CA) at 37∘C For adipocyte differentiation the cellswere stimulated with 3T3-L1 differentiation medium con-taining isobutylmethylxanthine dexamethasone and insulin




2











18





3 Results









50) of ABTS








Scavenging effect()

IC501

(120583gmL)

GKT2

125 1924 plusmn 119


AA3125 2115 plusmn 019





Scavenging effect()

IC501

(120583gmL)

GKT2

50 633 plusmn 014


AA3125 2115 plusmn 019




1000

minus10

200

250

300

350

Wavelength (nm

)10000

20000

30000

40000

0

200

400

600

800

1000

Time (min)

Inte

nsity

(mAU

)

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

O

O

O OO

O

O

OO

O

OO

O

O

O O

O

O

O

O

HOHO

HO HO

HOHO

HOHO

HO

HOHO

CH



Glycyrrhizin

HOOC

HOOC

HOOC

Puerarin

H


minus20

0

20

40

60

80

100

120

LDL OxLDL 156 313 625 125 250

GKT(120583gmL)

TBA

RS (

OxL

DL)

lowastlowast

lowastlowast

lowastlowast


(a)

08

09

1

11

12

13

14

15

LDL OxLDL 156 3125 625 125 250

GKT

REM

(AU

)



(120583gmL)

LDL 0 157 3125 625 125 250


(b)





0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)








4 Discussion



4in a dose-



Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2











18





3 Results









50) of ABTS








Scavenging effect()

IC501

(120583gmL)

GKT2

125 1924 plusmn 119


AA3125 2115 plusmn 019





Scavenging effect()

IC501

(120583gmL)

GKT2

50 633 plusmn 014


AA3125 2115 plusmn 019




1000

minus10

200

250

300

350

Wavelength (nm

)10000

20000

30000

40000

0

200

400

600

800

1000

Time (min)

Inte

nsity

(mAU

)

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

O

O

O OO

O

O

OO

O

OO

O

O

O O

O

O

O

O

HOHO

HO HO

HOHO

HOHO

HO

HOHO

CH



Glycyrrhizin

HOOC

HOOC

HOOC

Puerarin

H


minus20

0

20

40

60

80

100

120

LDL OxLDL 156 313 625 125 250

GKT(120583gmL)

TBA

RS (

OxL

DL)

lowastlowast

lowastlowast

lowastlowast


(a)

08

09

1

11

12

13

14

15

LDL OxLDL 156 3125 625 125 250

GKT

REM

(AU

)



(120583gmL)

LDL 0 157 3125 625 125 250


(b)





0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)








4 Discussion



4in a dose-



Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























50) of ABTS








Scavenging effect()

IC501

(120583gmL)

GKT2

125 1924 plusmn 119


AA3125 2115 plusmn 019





Scavenging effect()

IC501

(120583gmL)

GKT2

50 633 plusmn 014


AA3125 2115 plusmn 019




1000

minus10

200

250

300

350

Wavelength (nm

)10000

20000

30000

40000

0

200

400

600

800

1000

Time (min)

Inte

nsity

(mAU

)

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

O

O

O OO

O

O

OO

O

OO

O

O

O O

O

O

O

O

HOHO

HO HO

HOHO

HOHO

HO

HOHO

CH



Glycyrrhizin

HOOC

HOOC

HOOC

Puerarin

H


minus20

0

20

40

60

80

100

120

LDL OxLDL 156 313 625 125 250

GKT(120583gmL)

TBA

RS (

OxL

DL)

lowastlowast

lowastlowast

lowastlowast


(a)

08

09

1

11

12

13

14

15

LDL OxLDL 156 3125 625 125 250

GKT

REM

(AU

)



(120583gmL)

LDL 0 157 3125 625 125 250


(b)





0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)








4 Discussion



4in a dose-



Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1000

minus10

200

250

300

350

Wavelength (nm

)10000

20000

30000

40000

0

200

400

600

800

1000

Time (min)

Inte

nsity

(mAU

)

OH

OH

OH

OH

OHOH

OH

OH

OH

OH

OH

O

O

O OO

O

O

OO

O

OO

O

O

O O

O

O

O

O

HOHO

HO HO

HOHO

HOHO

HO

HOHO

CH



Glycyrrhizin

HOOC

HOOC

HOOC

Puerarin

H


minus20

0

20

40

60

80

100

120

LDL OxLDL 156 313 625 125 250

GKT(120583gmL)

TBA

RS (

OxL

DL)

lowastlowast

lowastlowast

lowastlowast


(a)

08

09

1

11

12

13

14

15

LDL OxLDL 156 3125 625 125 250

GKT

REM

(AU

)



(120583gmL)

LDL 0 157 3125 625 125 250


(b)





0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)








4 Discussion



4in a dose-



Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

0 78125 15625 3125 625 125 250 500

Cel

l via

bilit

y (

of c

ontro

l)

GKT (120583gmL)

(a)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

0 625 125 250 500 1000

GKT (120583gmL)

(b)








4 Discussion



4in a dose-



Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Preadipocytes

Prea

dipo

cyte

s

Adipocytes

ORO staining

Cell morphology

0

50

100

150

Lipi

d ac

cum

ulat

ion

( o

f con

trol)

Adipocytes

Con

trol

GW9662

GKT

Control GW9662 GKT


(a)

00

50

50 100 200 40025

100150200250300350

Trig

lyce

ride (

nmol

L)

lowastlowastlowast

lowastlowast

GW9662

GKT (120583gmL)

(b)


0

1

2

3

4

5

6

GPD

H ac

tivity

(uni

tmg)

0 50 100 200 40025 GW9662GKT (120583gmL)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

(a)

0

100

200

300

400

500

Lept

in (p

gm

L)

0 50 100 200 40025 GW9662GKT

lowastlowastlowast

lowastlowastlowast

lowastlowast

(120583gmL)

(b)









Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgment


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Antioxidant and Antiadipogenic Activities