research article crotonis fructus and its...

Research ArticleCrotonis Fructus and Its Constituent Croton OilStimulate Lipolysis in OP9 Adipocytes

Mi-Seong Kim1 Ha-Rim Kim12 Hong-Seob So12 Young-Rae Lee13 Hyoung-Chul Moon4

Do-Gon Ryu5 Sei-Hoon Yang6 Guem-San Lee7 Je-Ho Song8 and Kang-Beom Kwon125

1 Center for Metabolic Function Regulation Wonkwang University School of Medicine No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

2 BK21 Plus Program and Department of Smart Life-Care Convergence Graduate School Wonkwang UniversityNo 460 Iksan-Daero Iksan Jeonbuk 570-749 Republic of Korea

3 Department of Oral Biochemistry Wonkwang University School of Dentistry No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

4 Institute of Customized Physical Therapy No 217 Wallgye-ro Gwangju Metropolitan City 506-303 Republic of Korea5 Department of Korean Physiology Wonkwang University School of Korean Medicine No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

6Department of Internal Medicine Wonkwang University School of Medicine No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

7Department of Herbology Wonkwang University School of Korean Medicine No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

8Department of Sports Industry ampWelfare Wonkwang University School of Natural Science No 460 Iksan-Daero IksanJeonbuk 570-749 Republic of Korea

Correspondence should be addressed to Je-Ho Song sjhaowonkwangackr and Kang-Beom Kwon dessonwkuackr

Received 4 August 2014 Accepted 23 October 2014 Published 11 November 2014

Academic Editor Waris Qidwai

Copyright copy 2014 Mi-Seong Kim et alThis 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

Introduction Crotonis fructus (CF) is the mature fruit of Croton tiglium L and has been used for the treatment of gastrointestinaldisturbance in Asia It is well known that the main component of CF is croton oil (CO) The present study is to investigate theeffects of CF extracts (CFE) and CO on lipolysis in OP9 adipocytesMethods Glycerol release to the culture supernatants was usedas a marker of adipocyte lipolysis Results Treatment with various concentrations of CFE and CO stimulates glycerol release in adose-dependent mannerThe increase in glycerol release by CFE is more potent than isoproterenol which is a 120573-adrenergic agonistas a positive control in our system The increased lipolysis by CFE and CO was accompanied by an increase of phosphorylatedhormone sensitive lipase (pHSL) but not nonphosphorylated HSL protein and mRNA Pretreatment with H89 which is a proteinkinase A inhibitor significantly abolished the CFE- and CO-induced glycerol release in OP9 adipocytes These results suggest thatCFE and CO may be a candidate for the development of a lipolysis-stimulating agent in adipocytes

1 Introduction

Croton tigliumL (family Euphorbiaceae) is a plant distributedin the tropical and subtropical zones of Asia The fruit ofCroton tiglium L is Crotonis Fructus (CF) which is oneof several medicines used for attenuating gastrointestinaldiseases such as constipation visceral pain and intestinal

inflammation [1 2] Additionally the crude extracts of CFhave been reported to activate M3 muscarinic receptor andCa2+ influx through the L-typeCa2+ channel in isolated rabbitjejunum [3] It is also well known that the main componentof CF is the croton oil (CO)There is an abundance of linoleicacid oleic acid and eicosenoic acid in a methyl-esterifiedsample of CO [4] It has been reported that CO may regulate

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 780385 5 pageshttpdxdoiorg1011552014780385

2 Evidence-Based Complementary and Alternative Medicine

gastrointestinal motility and induce intestinal inflammationrelated to immunological milieu and motor activity in mice[5]

Obesity is a medical condition in which excess bodyfat has accumulated to the extent that is associated witha variety of diseases including metabolic syndrome andcardiovascular disease White adipose tissue is the major fatreservoir in mammals storing the excess energy from thediet as triacylglycerol (TAG) a neural lipid composed ofthree fatty acids which is bound to the carbon backbone ofa glycerol molecule [6] One of the therapeutic methods fortreating obesity is to reduce TAG in white adipocytes whichis termed lipolysis Lipolysis is a complex process that ishighly regulated and involves the coordinated participation ofseveral lipases [7 8] Lipolysis occurs through the sequentialhydrolysis of TAG by the action of three lipases adiposetriglyceride lipase (ATGL) hormone-sensitive lipase (HSL)and monoglyceride lipase (MGL) [9] Among them HSL isa key enzyme in the mobilization of fatty acids in adipocytesand its activity is regulated posttranscriptionally by reversiblephosphorylation by protein kinase A (PKA) [10]

In this study we used OP9 mouse stromal cells firstreported by Bickel and colleagues as a useful new modelof adipocyte metabolism in 2006 [11] In their study OP9cells initiated the same events including lipid metabolisminsulin signaling and glucose transport much like 3T3-L1cells [11] We already reported about the inhibitory effects ofPericarpium Zanthoxyli extract on adipocyte differentiationby using OP9 cells [12]

In the present study the effects of CF extract (CFE) andCO on lipolysis in OP9 cells were investigated by measuringglycerol release and evaluating HSL expression level

2 Materials and Methods

21 Reagents OP9 cells were purchased from the AmericanType Culture Collection (Manassas VA USA) Minimumessential medium alpha (MEM120572) and fetal bovine serum(FBS) were purchased from Invitrogen (Carlsbad CA USA)Insulin 3-isobutyl-1-methylxanthine (IBMX) dexametha-sone (DEXA) isoguanosine and croton oil (CO) were pur-chased from Sigma chemical (St Louis MO USA) HA89a protein kinase inhibitor was purchased from Calbiochem(San Diego CA) Antibodies against HSL and 120573-actin werepurchased from Santa Cruz Biotechnology (Santa Cruz CAUSA) Antibody against pHSL was obtained from Cell Sig-naling Technology (Beverly MA USA) All of the chemicalswere of analytical grade

22 Preparation of Crotonis Fructus Extracts (CFE) Maturefruits of Croton tinglium L were purchased fromKwangmyu-ngdangMedicinal Herbs Co Ltd (Ulsan Republic of Korea)and authenticated by Professor GS Lee one of the authorsA voucher specimen (WKU030305-CT201305E) has beendeposited at the Department of Herbology College of KoreanMedicine Wonkwang University Iksan Korea

The powdered Crotonis Fructus (100 g) was extractedusing refluxmethodwith 1000mLof 70aqueous ethanol for2 hours The extract was evaporated under 40mmHg using

a rotary evaporator and then freeze-dried The yield of thefinal extract was 1148 (ww)

23 Cell Culture and Adipocyte Differentiation InductionOP9 cells were cultured inMEM120572 containing 20FBS 2mMl-glutamine 100UmL penicillin and 100120583gmL strepto-mycin at 37∘C in a 5 CO

2incubator To induce differ-

entiation 1-day postconfluent preadipocytes were incubatedin a differentiation medium containing 10 FBS 05mMIBMX 025 120583M DEXA 175 nM insulin 2mM l-glutamine100UmL penicillin and 100 120583gmL streptomycin for 2 daysThe medium was then changed to MEM120572 containing 10FBS 2mM l-glutamine and 175 nM insulin and the cellswere cultured for 3 days

24 Determination of Cell Viability The effect of CFE onOP9cell viability was determined using an establishedMTT assayThe attached cells were kept untreated or treated with variousconcentrations of CFE (list the concentrations in parenthe-ses) for 24 h at 37∘C The cells were washed with phosphate-buffered saline (PBS) prior to adding MTT (05mgmL PBS)and incubated at 37∘C for 30min Formazan crystals weredissolved with dimethyl sulfoxide (100120583Lwell) and detectedat OD

570with a model Emax (Molecular Devices Sunnyvale

CA USA)

25 Glycerol Release After treatment of differentiated OP9cells with various concentrations of CFE and CO freeglycerol content in cell supernatants was quantified using aglycerol quantification kit according to the manufacturerrsquosinstructions (Sigma Chemicals) Glycerol was quantified atOD540 with a model Emax (Molecular Devices) Isopro-terenol (Merck Darmstadt Germany) a known stimulatorof lipolysis was used as a positive control

26 Western Blot Analysis The differentiated OP9 cells werepretreated with 20 120583M H89 for 2 h and then treated with20120583gmL CFE and 10 120583gmL CO for 12 h at 37∘C Cells werelysed with ice-cold M-PER mammalian protein extractionreagent (Pierce Biotechnology Rockford IL USA) and theprotein concentration in the lysate was determined usingthe Bradford method [13] Samples (20120583g) were separatedby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis with 10 acrylamide and transferred to Hybond-Ppolyvinylidene fluoride (PVDF) membranes (GE HealthcareLife Sciences Buckinghamshire UK) using a western blotapparatus Protein expression levels were determined bysignal analysis using an image analyzer (Fuji-Film TokyoJapan)

27 Quantitative Real-TimePolymerase ChainReaction (PCR)Total RNA was extracted from cells using a FastPure RNAkit (TaKaRa Shiga Japan) The RNA concentration andpurity were determined by absorbance at 260280 nm cDNAwas synthesized from 1120583g of total RNA using a Prime-Script RT reagent kit (TaKaRa) Adipocyte differentiation-related gene mRNA expressions were determined by real-time PCR using the ABI PRISM 7900 Sequence Detection

Evidence-Based Complementary and Alternative Medicine 3

CON ISO 20 30 40 500

50

100

150

200

250

300

350

Glycerol release ()Viability ()

CFE (120583gmL)

lowast

lowastlowastlowastlowast lowastlowast

lowastlowast

(a)


CON ISO 2 5 10 200

20

40

60

80

100

120

140

160

180

CO (120583gmL)

lowast

lowast lowastlowast

lowast

(b)Figure 1 Effects of Crotonis Fructus extracts (CEF) and croton oil (CO) on glycerol release and viability Differentiated OP9 adipocytes weretreated with various concentrations of CFE (20ndash50120583gmL) CO (2ndash20120583gmL) and 200 nM isoproterenol (ISO) for 12 h Glycerol release(black bar) was determined with a glycerol quantification kit and cell viability (gray bar) was quantified with MTT assay as described inMaterials and Methods Data are expressed as the mean plusmn SD of four independent experiments and a percentage of vehicle (DMSO-)treatedcontrol cells (CON) lowast119875 lt 005 lowastlowast119875 lt 001 versus CON

System and SYBR Green I (Applied Biosystems FosterCity CA USA) The forward and the reverse primers forHSL were 51015840-GGAGCACTACAAACGCAACGA-31015840 and 51015840-TCGGCCACCGGTAAAGAG-31015840 respectively the primersfor GAPDH were 51015840-CGTCCCGTAGACAAAATGGT-31015840and 51015840-TTGATGGCAACAATCTCCAC-31015840 respectively Allresults were normalized to the GAPDH housekeeping geneto control for variation in mRNA concentrations Relativequantification was performed using the comparative ΔΔ119862

119905

method according to the manufacturerrsquos instructions

28 Statistical Analysis Statistical analysis was performedusing OriginPro 80 software One way analysis of variance(ANOVA) followed by Duncanrsquos test Data were expressed inmeans plusmn SD Differences with 119875 values less than 005 wereconsidered statistically significant

3 Results and Discussion

Crotonis Fructus (CF) is the mature fruit of Croton tigliumL which has been used in traditional medicine for the treat-ment of gastrointestinal (GI) diseases including constipationabdominal pain peptic ulcer and intestinal inflammationin Asia [2 3 5] The main components of CF comprise thegreat parts of the extracted essential oil from seed and bark(fruit) of Croton tiglium which is named by Croton tigliumoil (CO) and modulate intestinal transit in mice [5] Therewere many reports about the effects of CF extracts and COon GI problems [2 3] Kim et al [14] also reported thatisoguanosine from Croton tiglium L has antitumor activityagainst implanted S-180 ascitic tumor in mice Howeverthere are no studies on their lipolytic effects on adipocytesparticularly on the underlying mechanism

A significant increase in the amount of glycerol releasedinto the media was observed in those OP9 adipocytes treatedwith CF extracts (CFE) (20ndash50120583gmL 119875 lt 001) for 12 h(Figure 1(a)) in a dose-dependent manner The increase inglycerol release by 20 120583gmL CFE was observed after 6 hof treatment (16-fold 119875 lt 005) and peaked after 12 h oftreatment (22-fold 119875 lt 001) (data not shown) Lipolysisof TAG into glycerol and fatty acids is activated by the 120573-adrenergic receptor agonist isoproterenol (ISO) and is used asa positive control in our system ISO also increased glycerolrelease after 12 h of treatment (14-fold 119875 lt 005) Howeverthe effect of CFE on lipolysis is more potent than ISO even ata concentration of 20120583gmL We also checked the effects ofCFE on ISO-induced lipolysis and the data revealed that CFEdid not have any additional effect on the lipolytic effect of ISO(data not shown) And then we want to know what the mainconstituent of CFE increasing glycerol release is As shown inFigure 1(b) CO significantly increased glycerol release intothe media at all concentrations tested but it was less effectivethan CFE We also tested the effects of isoguanosine onlipolysis and there was none (data not shown) We could notexclude the lipolytic effects of other compounds inCFENoneof CFE CO and isoguanosine at the concentration rangestested was cytotoxic according to the MTT (Figure 1)

In murine adipocytes PKA phosphorylates HSL at sev-eral serine residues (563 659 and 660) resulting in increasedtranslocation ofHSL to the lipid droplet surface and increasedlipolytic activity [15] To better elucidate the mechanismsunderlying the lipolytic actions of CFE and CO we inves-tigated the effects of CFE and CO on HSL phosphorylationin Ser563 As shown in Figure 2 CFE (20120583gmL) and CO(10 120583gmL) treatment for 12 h did not modify total proteinand mRNA contents of HSL but significantly increased the


Glycerol release ()HSL mRNA ()

0

25

50

75

100

125

150

175

200

225

250

HSL

CON

ISO

CFE

20

CO 1

0

lowastlowast lowast

lowastlowast

120573-Actin

CFE20

+H89

CO10

+H89

$

p-HSLS563

Figure 2 Signaling pathway involved in the lipolytic effects of CFEand CO Differentiated OP9 adipocytes were pretreated with 20120583MH89 and then treated with 20120583gmL of CFE 10 120583gmL of CO and200 nM ISO for 12 h Glycerol release (black bar) quantificationis the same as Figure 1 legends and real-time PCR for hormonesensitive lipase (HSL) mRNA (gray bar) was carried out using aspecific primer for HSL as described in Materials and MethodsProtein expression levels for p-HSLS563 andHSL (lower panel) weresubjected to western blot analysis by using specific antibodies Dataare expressed as the mean plusmn SD of four independent experimentsand a percentage of vehicle (DMSO-)treated control cells (CON)lowast119875 lt 005 lowastlowast119875 lt 001 versus CON 119875 lt 001 versus CFE-treatedgroup and $119875 lt 005 versus CO-treated group

phosphorylation of HSL at Ser563 However the CFE- andCO-induced glycerol release and phosphorylation of HSLat Ser563 were significantly inhibited in the presence of thePKA inhibitor H89 These data suggest that CFE and COstimulate lipolysis through HSL phosphorylation at Ser563by PKA activation In addition to the PKA-mediated phos-phorylation HSL may be phosphorylated by other kinasessuch as extracellular signal-regulated kinase (ERK12) whichactivates HSL by phosphorylation [16] Furthermore AMP-activated protein kinase (AMPK) phosphorylates HSL atSer565 which prevents phosphorylation induced PKA [17]Other kinases such as ERK12 and AMPKmay be involved inthe underlyingmechanism of CFE- and CO-induced glycerolrelease from lipid droplets

Collectively the present data demonstrate the ability ofCFE to stimulate lipolysis in OP9 adipocytes with CO asits major constituent The lipolytic actions of CFE and CO

are mainly mediated by the phosphorylation of HSL throughPKA activation These findings explain a mechanism bywhichCFE andCO induce lipolysis in adipocytesThis wouldfurther aid in the development of therapeutic strategy inpreventing obesity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Mi-Seong Kim and Ha-Rim Kim contributed equally to thisstudy

Acknowledgment

This research was supported by a National Research Foun-dation of Korea (NRF) Grant funded by the Korean Gov-ernment (MEST) (no 2011-0030130) Republic of Korea andby the Basic Science Research Program through the NationalResearch Foundation of Korea (NRF) funded by theMinistryof Education Science and Technology (no 2011-0013528)

References

[1] J-C Tsai S Tsai and W-C Chang ldquoEffect of ethanol extractsof three Chinesemedicinal plants with anti-diarrheal propertieson ion transport of the rat intestinal epitheliardquo Journal ofPharmacological Sciences vol 94 no 1 pp 60ndash66 2004

[2] X Wang M Lan H-P Wu et al ldquoDirect effect of croton oilon intestinal epithelial cells and colonic smooth muscle cellsrdquoWorld Journal of Gastroenterology vol 8 no 1 pp 103ndash107 2002

[3] J Hu W-Y Gao L Ma S-L Man L-Q Huang and C-X LiuldquoActivation ofM3muscarinic receptor and Ca2+ influx by crudefraction from Crotonis Fructus in isolated rabbit jejunumrdquoJournal of Ethnopharmacology vol 139 no 1 pp 136ndash141 2012

[4] M Lan PWan Z-YWang and X-L Huang ldquoGC-MS analysisof chemical components in seeds oil from Croton tigliumrdquoJournal of Chinese Medicinal Materials vol 35 no 7 pp 1105ndash1108 2012

[5] X Wang F Zhang Z Liu et al ldquoEffects of essential oil fromCroton tiglium L on intestinal transit in micerdquo Journal ofEthnopharmacology vol 117 no 1 pp 102ndash107 2008

[6] K Jaworski E Sarkadi-Nagy R E Duncan M Ahmadian andH S Sul ldquoRegulation of triglyceride metabolism IV Hormonalregulation of lipolysis in adipose tissuerdquo American Journal ofPhysiology Gastrointestinal and Liver Physiology vol 293 no1 pp G1ndashG4 2007

[7] R E Duncan M Ahmadian K Jaworski E Sarkadi-Nagy andH S Sul ldquoRegulation of lipolysis in adipocytesrdquoAnnual Reviewof Nutrition vol 27 pp 79ndash101 2007

[8] M J Watt and L L Spriet ldquoTriacylglycerol lipases and metabo-lic control implications for health and diseaserdquo AmericanJournal of PhysiologymdashEndocrinology and Metabolism vol 299no 2 pp E162ndashE168 2010

[9] A Lass R Zimmermann M Oberer and R Zechner ldquoLipo-lysismdasha highly regulated multi-enzyme complex mediates the


catabolism of cellular fat storesrdquo Progress in Lipid Research vol50 no 1 pp 14ndash27 2011

[10] C Holm ldquoMolecular mechanisms regulating hormone-sensi-tive lipase and lipolysisrdquo Biochemical Society Transactions vol31 no 6 pp 1120ndash1124 2003

[11] N E Wolins B K Quaynor J R Skinner et al ldquoOP9 mousestromal cells rapidly differentiate into adipocytes characteriza-tion of a useful new model of adipogenesisrdquo Journal of LipidResearch vol 47 no 2 pp 450ndash460 2006

[12] H-R Kim J-M Kim M-S Kim et al ldquoInhibitory effects ofPericarpium zanthoxyli extract on adipocyte differentiationrdquoInternational Journal of Molecular Medicine vol 33 no 5 pp1140ndash1146 2014

[13] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[14] J H Kim S J Lee Y B Han J J Moon and J B Kim ldquoIsolationof isoguanosine fromCroton tiglium and its antitumor activityrdquoArchives of Pharmacal Research vol 17 no 2 pp 115ndash118 1994

[15] M J Watt A G Holmes S K Pinnamaneni et al ldquoRegulationof HSL serine phosphorylation in skeletal muscle and adiposetissuerdquo American Journal of Physiology Endocrinology andMetabolism vol 290 no 3 pp E500ndashE509 2006

[16] A S Greenberg W-J Shen K Muliro et al ldquoStimulationof lipolysis and hormone-sensitive lipase via the extracellularsignal-regulated kinase pathwayrdquo The Journal of BiologicalChemistry vol 276 no 48 pp 45456ndash45461 2001

[17] N M Anthony M P Gaidhu and R B Ceddia ldquoRegulation ofvisceral and subcutaneous adipocyte lipolysis by acute AICAR-induced AMPK activationrdquo Obesity vol 17 no 7 pp 1312ndash13172009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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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

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


gastrointestinal motility and induce intestinal inflammationrelated to immunological milieu and motor activity in mice[5]

Obesity is a medical condition in which excess bodyfat has accumulated to the extent that is associated witha variety of diseases including metabolic syndrome andcardiovascular disease White adipose tissue is the major fatreservoir in mammals storing the excess energy from thediet as triacylglycerol (TAG) a neural lipid composed ofthree fatty acids which is bound to the carbon backbone ofa glycerol molecule [6] One of the therapeutic methods fortreating obesity is to reduce TAG in white adipocytes whichis termed lipolysis Lipolysis is a complex process that ishighly regulated and involves the coordinated participation ofseveral lipases [7 8] Lipolysis occurs through the sequentialhydrolysis of TAG by the action of three lipases adiposetriglyceride lipase (ATGL) hormone-sensitive lipase (HSL)and monoglyceride lipase (MGL) [9] Among them HSL isa key enzyme in the mobilization of fatty acids in adipocytesand its activity is regulated posttranscriptionally by reversiblephosphorylation by protein kinase A (PKA) [10]

In this study we used OP9 mouse stromal cells firstreported by Bickel and colleagues as a useful new modelof adipocyte metabolism in 2006 [11] In their study OP9cells initiated the same events including lipid metabolisminsulin signaling and glucose transport much like 3T3-L1cells [11] We already reported about the inhibitory effects ofPericarpium Zanthoxyli extract on adipocyte differentiationby using OP9 cells [12]

In the present study the effects of CF extract (CFE) andCO on lipolysis in OP9 cells were investigated by measuringglycerol release and evaluating HSL expression level

2 Materials and Methods

21 Reagents OP9 cells were purchased from the AmericanType Culture Collection (Manassas VA USA) Minimumessential medium alpha (MEM120572) and fetal bovine serum(FBS) were purchased from Invitrogen (Carlsbad CA USA)Insulin 3-isobutyl-1-methylxanthine (IBMX) dexametha-sone (DEXA) isoguanosine and croton oil (CO) were pur-chased from Sigma chemical (St Louis MO USA) HA89a protein kinase inhibitor was purchased from Calbiochem(San Diego CA) Antibodies against HSL and 120573-actin werepurchased from Santa Cruz Biotechnology (Santa Cruz CAUSA) Antibody against pHSL was obtained from Cell Sig-naling Technology (Beverly MA USA) All of the chemicalswere of analytical grade

22 Preparation of Crotonis Fructus Extracts (CFE) Maturefruits of Croton tinglium L were purchased fromKwangmyu-ngdangMedicinal Herbs Co Ltd (Ulsan Republic of Korea)and authenticated by Professor GS Lee one of the authorsA voucher specimen (WKU030305-CT201305E) has beendeposited at the Department of Herbology College of KoreanMedicine Wonkwang University Iksan Korea

The powdered Crotonis Fructus (100 g) was extractedusing refluxmethodwith 1000mLof 70aqueous ethanol for2 hours The extract was evaporated under 40mmHg using

a rotary evaporator and then freeze-dried The yield of thefinal extract was 1148 (ww)

23 Cell Culture and Adipocyte Differentiation InductionOP9 cells were cultured inMEM120572 containing 20FBS 2mMl-glutamine 100UmL penicillin and 100120583gmL strepto-mycin at 37∘C in a 5 CO

2incubator To induce differ-

entiation 1-day postconfluent preadipocytes were incubatedin a differentiation medium containing 10 FBS 05mMIBMX 025 120583M DEXA 175 nM insulin 2mM l-glutamine100UmL penicillin and 100 120583gmL streptomycin for 2 daysThe medium was then changed to MEM120572 containing 10FBS 2mM l-glutamine and 175 nM insulin and the cellswere cultured for 3 days

24 Determination of Cell Viability The effect of CFE onOP9cell viability was determined using an establishedMTT assayThe attached cells were kept untreated or treated with variousconcentrations of CFE (list the concentrations in parenthe-ses) for 24 h at 37∘C The cells were washed with phosphate-buffered saline (PBS) prior to adding MTT (05mgmL PBS)and incubated at 37∘C for 30min Formazan crystals weredissolved with dimethyl sulfoxide (100120583Lwell) and detectedat OD

570with a model Emax (Molecular Devices Sunnyvale

CA USA)

25 Glycerol Release After treatment of differentiated OP9cells with various concentrations of CFE and CO freeglycerol content in cell supernatants was quantified using aglycerol quantification kit according to the manufacturerrsquosinstructions (Sigma Chemicals) Glycerol was quantified atOD540 with a model Emax (Molecular Devices) Isopro-terenol (Merck Darmstadt Germany) a known stimulatorof lipolysis was used as a positive control

26 Western Blot Analysis The differentiated OP9 cells werepretreated with 20 120583M H89 for 2 h and then treated with20120583gmL CFE and 10 120583gmL CO for 12 h at 37∘C Cells werelysed with ice-cold M-PER mammalian protein extractionreagent (Pierce Biotechnology Rockford IL USA) and theprotein concentration in the lysate was determined usingthe Bradford method [13] Samples (20120583g) were separatedby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis with 10 acrylamide and transferred to Hybond-Ppolyvinylidene fluoride (PVDF) membranes (GE HealthcareLife Sciences Buckinghamshire UK) using a western blotapparatus Protein expression levels were determined bysignal analysis using an image analyzer (Fuji-Film TokyoJapan)

27 Quantitative Real-TimePolymerase ChainReaction (PCR)Total RNA was extracted from cells using a FastPure RNAkit (TaKaRa Shiga Japan) The RNA concentration andpurity were determined by absorbance at 260280 nm cDNAwas synthesized from 1120583g of total RNA using a Prime-Script RT reagent kit (TaKaRa) Adipocyte differentiation-related gene mRNA expressions were determined by real-time PCR using the ABI PRISM 7900 Sequence Detection


CON ISO 20 30 40 500

50

100

150

200

250

300

350


CFE (120583gmL)

lowast


lowastlowast

(a)


CON ISO 2 5 10 200

20

40

60

80

100

120

140

160

180

CO (120583gmL)

lowast

lowast lowastlowast

lowast



119905









0

25

50

75

100

125

150

175

200

225

250

HSL

CON

ISO

CFE

20

CO 1

0

lowastlowast lowast

lowastlowast

120573-Actin

CFE20

+H89

CO10

+H89

$

p-HSLS563









Acknowledgment


References

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CON ISO 20 30 40 500

50

100

150

200

250

300

350


CFE (120583gmL)

lowast


lowastlowast

(a)


CON ISO 2 5 10 200

20

40

60

80

100

120

140

160

180

CO (120583gmL)

lowast

lowast lowastlowast

lowast



119905









0

25

50

75

100

125

150

175

200

225

250

HSL

CON

ISO

CFE

20

CO 1

0

lowastlowast lowast

lowastlowast

120573-Actin

CFE20

+H89

CO10

+H89

$

p-HSLS563









Acknowledgment


References

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















0

25

50

75

100

125

150

175

200

225

250

HSL

CON

ISO

CFE

20

CO 1

0

lowastlowast lowast

lowastlowast

120573-Actin

CFE20

+H89

CO10

+H89

$

p-HSLS563









Acknowledgment


References

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article crotonis fructus and its...

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