Hemostatic and Wound Healing Properties of Chromolaena ...downloads.hindawi.com/journals/isrn.dermatology/2013/168269.pdf6 ISRNDermatology (A.U.) 0 0.5 1 1.5 2 2.5 3 Thromboxane synthase

Hindawi Publishing CorporationISRN DermatologyVolume 2013 Article ID 168269 8 pageshttpdxdoiorg1011552013168269

Research ArticleHemostatic and Wound Healing Properties ofChromolaena odorata Leaf Extract

Hataichanok Pandith12 Xiaobo Zhang1 Jason Liggett1 Kyung-Won Min1

Wandee Gritsanapan2 and Seung Joon Baek13

1 Department of Biomedical and Diagnostic Sciences The University of Tennessee Knoxville TN 37996 USA2Department of Pharmacognosy Faculty of Pharmacy Mahidol University 447 Sri Ayutthaya Road RatchathewiBangkok 10400 Thailand

3Department of Biomedical and Diagnostic Sciences College of Veterinary Medicine University of Tennessee2407 River Drive Knoxville TN 37996-4542 USA

Correspondence should be addressed to Wandee Gritsanapan wandeegrimahidolacth and Seung Joon Baek sbaek2utkedu

Received 30 May 2013 Accepted 9 July 2013

Academic Editors S-C Chao C Feliciani P D Shenefelt and Y Tuzun

Copyright copy 2013 Hataichanok Pandith et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Chromolaena odorata (L) King and Robinson (Siam weed) extract has been used to stop bleeding and in wound healing inmany tropical countries However its detailed mechanisms have not been elucidated In this study we examined the molecularmechanisms by which Siam weed extract (SWE) affected hemostatic and wound healing activities SWE promoted Balbc 3T3fibroblast cell migration and proliferation Subsequently we found that heme oxygenase-1 (HO-1) the accelerating wound healingenzyme was increased at the transcriptional and translational levels by SWE treatments The HO-1 promoter analyzed withluciferase assay was also increased by treatment of SWE in a dose-dependent manner This induction may be mediated by severalkinase pathways including MEK p38MAPK AKT and JNK Quantitative real-time PCR using undifferentiated promonocytic celllines revealed that thromboxane synthase (TXS) a potent vasoconstrictor and platelet aggregator was increased and MMP-9 ananti platelet aggregator was decreased in the presence of SWE Our studies presented that SWE accelerated hemostatic and woundhealing activities by altering the expression of genes including HO-1 TXS and MMP-9

1 Introduction

Wound healing is an intricate process in which usually theskin repairs itself after injury The process is divided intofour overlapping phases hemostasis (cessation of bleeding)inflammation proliferation and remodeling [1] Hemostasisis mainly controlled by thromboxane synthase (TXS) whichconverts prostaglandin H

2into thromboxane A

2 a potent

vasoconstrictor and platelet aggregator [2] Plasminogen acti-vator inhibitor type 1 (PAI-1) also plays a role in hemostasisby inhibition of fibrinolysis which prevents failure of thehemostatic process [3] Subsequently neutrophils release freeradicals to kill bacteria in the inflammation phase [4 5] andheme and heme proteins also accumulate at the local site ofthe woundThese heme and heme proteins have prooxidativeand proinflammatory properties by inducing the expression

of adhesion molecules causing vascular permeability andleukocyte infiltration These actions initiate wound healingprocess Heme oxygenase-1 (HO-1) has antiinflammatoryand antioxidant activities and is responsible for a widerange of wound healing functions It converts heme intobiliverdinbilirubin iron and carbon monoxide which arepotent antioxidant products The overexpression of HO-1helps to accelerate wound healing such as amelioration ofinflammation proliferation and protection against endothe-lial cell apoptosis [6]Matrixmetalloproteinases (MMPs) alsoplay a major role in wound healing by extracellular matrix(ECM) remodeling [7] and MMP-9 is key effector amongthose [8]

Siam weed (Chromolaena odorata (L) King and Robin-son) is a perennial scandent or semiwoody shrub in theAster-aceae family It has been used for a variety of ailments inmany

2 ISRN Dermatology

tropical countries for a long time especially to stop bleedingNumerous studies have demonstrated that Siam weed extract(SWE) accelerates hemostasis [9ndash11] and wound healing [12ndash14] However themolecular targets of SWE in wound healingactivity have not been identified In this study we investigatedthe effect of 70 ethanolic SWE (C1) and its bioactive com-pounds scutellarin tetramethyl ether (scu) and stigmasterolon enhancedwound healing activity Scu and stigmasterol arethemajor components of SWE as assessed by thin-layer chro-matography In rat models the 70 ethanolic extract pos-sesses the highest effectiveness to stop bleeding comparedto other solvent extracts [15] Scu and stigmasterol exhibithemostatic [16] and antiinflammatory activities [17 18]

In this study we examined the molecular targets ofSWE (C1) in the wound healing process and found that theexpression of HO-1 and other proteins played a pivotal rolein SWE-induced wound healing activity Our data suggestthat SWE affects the wound healing process by using novelmolecular targets

2 Materials and Methods

21 Reagents All antibodies were purchased fromSantaCruzBiotechnology (Santa Cruz CA USA) CellTiter 96 AqueousOne Solution Cell Proliferation Assay was purchased fromPromega (Madison WI USA) Scutellarin tetramethyl ether(scu) and stigmasterol were purchased from ChromaDexInc (Irvine CA USA) and Sigma Aldrich (St Louis MOUSA) respectively All chemicals were purchased fromFisherScientific (Pittsburgh PA USA) unless otherwise specified

22 Preparation of Standard Solutions 12-O-tetradecanoyl-phorbol-13-acetate (TPA) scu and stigmasterol were dis-solved in DMSO and were kept at minus80∘C until used

23 Plant Materials and Plant Extract Preparation Siamweed mature leaves were collected from Samut Sakhonprovince Thailand in December 2009The plant sample wasidentified by DrWandee Gritsanapan and the voucher spec-imen (no CO-003) was deposited at the Department of Phar-macognosy Faculty of Pharmacy Mahidol University Theleaves were dried in a hot air oven at 60∘C for 24 h The driedsample was ground into moderate powder Then 100 g ofthe powder was exhaustively extracted with 70 vv ethanol(EtOH) (1 10 wv) while shaking at 25∘C 120 rpm for 12 h ona shaker The mixture was filtered through Whatman filterpaper no 1 and the filtrate was concentrated using a rotaryevaporator at 40∘C The dark green viscous ethanolic extract(C1) was kept in a tightly closed brown vial at 4∘C until used

A stock solution of the extract was prepared by completedissolving of 342mg of the ethanolic extract in 1 mL EtOHusing sonication for 30min The stock solution was kept atminus80∘C until used The working solutions were prepared bydiluting the stock solution with EtOH to the concentrationsof 342 342 and 100mgmL

24 Cell Cultures Balbc 3T3 and U937 cells (ATCC Man-assas VA USA) were maintained in Dulbeccorsquos modified

Eaglersquos medium (DMEM) and RPMI 1640 medium respec-tively Both culture media were supplemented with 10 fetalbovine serum (FBS) penicillin G (100 UmL) streptomycin(100 120583gmL) and amphotericin B (025 120583gmL) at 37∘C and5 CO

2in a humidified incubator

25 Quantitative Real-Time-PCR and Reverse-TranscriptasePCR U937 cells were grown to 80ndash90 confluence in 10 cmplates and treated with EtOH as vehicle TPA 100 nM aspositive control SWE 342 and 100 120583gmL and scu 10and 20120583M in the presence of 10 serum for 12 h TotalRNA was prepared using the Total RNA kit (Omega Bio-Tek Norcross GA USA) and reverse transcripted witha verso cDNA kit (Thermo Scientific Surrey KT USA)according to the manufacturersrsquo instruction Three repli-cates of real-time PCR were carried out using MyiQ ther-mal cycler (Bio-RAD Hercules CA USA) for 25 cyclesfor human thromboxane synthase (Tx) sense strand 51015840-GCCAAATGGAGCTCAGAAAG-31015840 antisense strand 51015840-TGCAGTAGCACCTCTGGATG-31015840 human PAI-1 sensestrand 51015840-CACGAGTCTTTCAGACCAAGAG-31015840 antisensestrand 51015840-CACACAAAAGCTCCTGTAAGC-31015840 MMP-9sense strand 51015840-GCTCTTCCCTGGAGACCTG-31015840 antisensestrand 51015840-ACACGCGAGTGAAGGTGAG-31015840 and humanGAPDH sense strand 51015840-GACCACAGTCCATGCCAT-CACT-31015840 antisense strand 51015840-TCCACCACCCTGTTGCTG-TAG-31015840 at 94∘C for 30 s 55∘C for 30 s and 72∘C for 1minusing the ABsolute QPCR SYBR-Green mix (ABgene HouseEpsom UK) with specific primers for human Tx PAI-1MMP-9 and GAPDH The quantification of each gene wascalculated For the reverse-transcriptase PCR the reactionwas carried out for 25 cycles for mouse HO-1 sense strand51015840-CCCACGCATATACCCGCTAC-31015840 antisense strand 51015840-CTAGCAGGCCTCTGACGAAG-31015840 and mouse GAPDHsense strand 51015840-CAGGAGCGAGACCCCACTAACAT-31015840antisense strand 51015840-GTCAGATCCACGACGGACACATT-31015840 at 94∘C for 30 s 55∘C for 30 s and 72∘C for 1min usingReadyMix Tag polymerase (Sigma Aldrich) with specificprimers Each PCR product was electrophoresed on 1agarose gel and visualized by ethidium bromide staining

26 Cell Migration Assay Balbc 3T3 cells were grown inDMEM supplemented with 10 FBS until confluence in 6 cmplates The cells were starved with DMEM in an absence ofserum for 24 h Cell monolayer was scratched with a sterilepipette tip Any cellular debris was removed by washing with1 X phosphate buffer saline (PBS) and media were replacedwith 1 DMEM containing EtOH as a control and 342342 and 100120583gmL of SWE The cells were viewed using anOlympus IMT2 microscope (Lake Success NY USA) at 40xtotal magnification and captured using a Sony model XC-ST50 camera (Park Ridge NJ USA) at 0 6 12 and 24 h Thedegree of cell migration was observed by the gap betweencontrol and treatment

27 Cell Proliferation Assay The effect of SWE on cell prolif-eration inBalbc 3T3 cells was investigated using theCellTiter96AqueousOne SolutionCell ProliferationAssay (Promega)

ISRN Dermatology 3

Cells were seeded at a density of 20 times 104 cellswell in 96-well tissue culture plates in four replicates and grown for 24 hCells were then treated with EtOH 342 342 and 100120583gmLof SWE in the presence of 10 serum At 12 h after treatment20120583L CellTiter 96 Aqueous One Solution was added to eachwell and the plate was incubated for 1 h at 37∘C Absorbanceat 490 nmwas recorded in an enzyme-linked immunosorbentassay (ELISA) plate reader (Bio-Tek Instruments WinooskiVT USA)

28 Western Blot Analysis Balbc 3T3 cells were grown to80ndash90 confluence in 6 cm plates The cells were starved for24 h before treatment with 342 342 and 100120583gmL SWEand Siam weedrsquos bioactive compounds scu and stigmasterol10 and 20120583M using EtOHorDMSO as vehicle controls in theabsence of serum for 12 h Total cell lysates were isolated usingRIPA buffer (1 x PBS 1 NP-40 05 sodium deoxycholateand 01 SDS) supplemented with protease inhibitors (1mMphenylmethylsulfonyl fluoride 5 120583gmL aprotinin and leu-peptin) and phosphatase inhibitors (1mMNa

3VO4and 1mM

NaF) Protein concentration was determined by the BCAprotein assay (Pierce Rockford IL USA) Protein (30 120583g)was separated on sodium dodecyl sulfate-polyacrylamidegel and transferred into a nitrocellulose membrane (PallLife Sciences Pensacola FL USA) The blots were blockedwith 5 skim milkTris buffer salineTween 005 (TBS-T) for 1 h and probed with a specific primary antibody in5 skim milk (HO-1 antibody was hybridized with 1 skimmilk)005 TBS-T at 4∘C overnight After three washes withTBS-T the blots were incubatedwith horseradish peroxidase-conjugated secondary antibody for 1 h and washed with TBS-T several times Proteins were detected by the enhancedchemiluminescence system (Thermo Scientific Rockford ILUSA)

29 Transient Transfection and Luciferase Promoter AssayTransient transfection was performed using TransIT-LT1(Mirus Bio LLC Madison WI USA) according to themanufacturerrsquos protocol Briefly Balbc 3T3 cells were platedin 12-well plates at a density of 20 times 105 cellswell and grownto 50 confluence The plasmid pHO-1GL394 luciferaseconstruct was kindly provided byDr AnupamAgarwal (Uni-versity of Alabama at Birmingham) The plasmid mixturescontaining 1120583gmL HO-194-Luc plasmid and 01 120583gmLpRL-null vector were cotransfected for 24 h Transfected cellswere pretreated with 342 342 and 100120583gmL of SWE 10and 20120583M of scu compound and 10120583M of stigmasterol for24 h in the presence of 10 serum EtOH and DMSO wereused as vehicle controls for extract and bioactive compoundsrespectively Cells were harvested in 1 x luciferase lysis buffer(Promega Madison WI USA) and luciferase activity wasmeasured and normalized to the pRL-null luciferase activityusing a dual luciferase assay kit (Promega Madison WIUSA)

210 Statistic Analysis Statistical significance was calculatedusing theNewman-KeulsMultiple Comparison Test compar-ing all pairs of means (119875 lt 005)

3 Results and Discussions

31 SWE Promotes Fibroblasts Cell Migration and Prolifer-ation In wound healing cell migration is an importantfactor that is directed and modulated by a wide range ofcellular signals Among them chemical stimuli have beenshown to play significant roles in guiding the direction oflocomotion and in the accumulation of cells To investigatethe wound healing activity of SWE we employed fibroblastcell lines (Balbc 3T3 cells) to perform cell migration andproliferation assays As shown in Figure 1(a) SWE facilitatedcell migration of Balbc 3T3 fibroblasts compared to EtOH-treated cells The gap between cells was nearly closed after24 h with 342 120583gmL of SWE compared to gap in vehicle-treated cells which was still significant after 24 h Howeverthe cells began to die off when 100 120583gmL SWEwas added for24 h Proliferation of Balbc 3T3 fibroblasts was analyzed toevaluate cellular responses to SWE SWE modestly but sig-nificantly stimulated cell proliferation with a strong responseat the doses of 342 and 100 120583gmL (Figure 1(b)) Interestingly100 120583gmL SWE with 1 serum caused cell death in a scratchassay whereas 100120583gmL SWE with 10 serum facilitatedcell growth Cell migration and proliferation are importantmechanisms of tissue regeneration by which SWE facilitatesthe wound healing process

32 SWE Increased HO-1 Protein and mRNA To determinemolecular targets of SWE in the wound healing processtranscription factors and angiogenesis factors were examinedbecause they are involved in the wound healing processVEGF and bFGF stimulates wound healing through angio-genesis The VEGF also promotes collagen deposition andepithelialization [19] Treatment with SWE and its majorbioactive components was done to examine the expressionof these proteins As shown in Figure 2(a) SWE at 342and 100 120583gmL increased HO-1 expression but VEGF andbFGF expressions were not changed We observed that HO-1expression increased in a dose-dependent manner with thehighest expression at the 100 120583gmL dose for 12 h treatmentWe alsomeasured two bioactive components in SWE scu andstigmasterol and found that only HO-1 protein expressionincreased with 20120583M of stigmasterol (Figure 2(b)) FurtherHO-1 mRNA expression was also investigated As a result wefound that SWE increased HO-1 mRNA in a dose-dependentmanner whereas scu and stigmasterol did not affect mRNAexpression (Figure 2(c)) It has been shown that HO-1 isnecessary for efficient wound closure and neovascularization[20]Thus our results suggest that SWE facilitated the woundhealing process through the induction of HO-1 expressionThe antioxidative activity of HO-1 is one of pathway whichaccelerates wound healing The antioxidative effect of SWEagainst oxidizing agents on human dermal fibroblasts andepidermal keratinocytes [21] might come from the increasingof HO-1 level Interestingly SWE increased HO-1 at thetranscriptional level whereas stigmasterol may affect HO-1 expression at the translational level It has been reportedthat SWE and its flavonoids exhibited inhibitory activity ofplatelet-activating factor (PAF) receptor binding [22] ThePAF is important mediator in the inflammatory response

4 ISRN Dermatology

EtO

HC1

342120583

gm

LC1

100120583

gm

L

6 h 12 h0 h 24 h

C1342120583

gm

L

(a)

Cel

l via

bilit

y ra

tio (f

old

with

EtO

H)

0

02

04

06

08

1

12

14

cb

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

C1342120583

gm

L

(b)

Figure 1 Cell migration and proliferation by SWE (a) Balbc 3T3 fibroblasts were scratched using a pipette tip to make gaps between cellsbefore treatment At 6 12 and 24 h of treatment the plates were photographed under a light microscope (40x) This picture is representativeof five independent fields It is notable that 100 120583gmL SWE for 24 h is toxic to the cells (b) Balbc 3T3 cell proliferation was measured usingthe CellTiter96 Aqueous One Solution Cell Proliferation Assay Values are expressed as mean plusmn SD of four replicates Different letters (a band c) represent statistically significant differences of means between groups (119875 lt 005)

C1 (6 h) C1 (12 h)EtOH 342 342 100 EtOH 342 342 100

HO-1

VEGF

bFGF

Actin

(120583gmL)

(a)

10 20 10 20DMSO

Scu Stigmasterol

HO-1

VEGF

bFGF

Actin

(120583M)

(b)

10 20 10 20DMSO

Scu Stigmasterol

GAPDH GAPDH

342 100EtOH

C1

mHO-1 mHO-1(120583M)(120583gmL)

(c)

Figure 2 Expression of genes involved in wound healing activity (a) Balbc 3T3 fibroblasts were grown in the presence of indicated SWEfor 6 h and 12 h The cell lysates were electrophoresed in SDS-PAGE and several genes and proteins were examined Actin was used as acontrol (b) Scu and stigmasterol were added to Balbc 3T3 cells for 12 h Western blot was carried out as described in the Materials andMethods section (c) Balbc 3T3 cells were treated with SWE for 12 h and HO-1 mRNA expression was measured Reverse-transcriptase PCRwas performed and the data represent three independent experiments GAPDH was used as control

ISRN Dermatology 5

LUCminus94 Kb

(a)

c

b

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

C1342120583

gm

L

(b)

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

aa

aa

DM

SO

Scu10120583

M

Scu20120583

M

Stig

mas

tero

l10120583

M

(c)

C1342 120583gmL

EtO

H

PD98

05940120583

M (M

EKE

RK)

SB20

3580

10120583

M(p

38 M

APK

)

SP60

0125

10120583

M (J

NK)

LY29

4002

50120583

M (A

KT)

Rapa

myc

in 1

00 nM

(mTO

R)

U01

262120583

M (M

EK)

+++++++minus

(d)

Figure 3 SWE-induced HO-1 expression (a) Schematic diagram of human HO-1 promoter HO-1 promoter construct was transfected intofibroblast cells and luciferase was measured after treatment of indicated SWE (b) or single compound (c) Values are expressed as mean plusmn SDof four replicates Different letters (a b and c) represent statistically significant difference of means compared to the values of controls andcompared between groups (119875 lt 005) (d) Western blot analysis of HO-1 expression in the presence of kinase inhibitors Actin was used forcontrol The cells were pretreated with indicated kinase inhibitors for 1 h followed by SWE treatment for 12 h Cell lysates were subjected toWestern blot

Therefore SWE and its flavonoids could play a role inhemostatic and wound healing activity via PAF pathway butfurther analysis remains to be investigated

33 SWE Increased HO-1 Promoter Activity To further pro-vide evidence that SWE affects HO-1 expression at thetranscriptional level we examined the promoter activity of

HO-1 As shown in Figure 3(a) 94 kb HO-1 promoter wasused to test HO-1 activity in fibroblasts All concentrationsof SWE increased HO-1 promoter activity however singlecompounds did not affect promoter activity (Figures 3(b) and3(c)) These results are consistent with the mRNA expressionshown in Figure 2(c) To further elucidate signaling pathwaysaffected by SWE-induced HO-1 expression we treated cellswith different kinase inhibitors in the presence of SWE The

6 ISRN Dermatology

(AU

)

0

05

1

15

2

25

3Thromboxane synthase

aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)

Figure 4 Expression of (a) TXS (b) MMP-9 and (c) PAI-1 in U937 cells U937 cells were treated with SWE TPA or scu for 12 h and geneexpression was analyzed by real-time PCR performed using a specific primer described in Section 2 Values are expressed as mean plusmn SDof four replicates Different letters (a b and c) represent statistically significant difference of means compared to the values of controls andcompared between groups (119875 lt 005)

result revealed that many kinase pathways could affect SWE-induced HO-1 expression to varying degrees except for themTOR pathway (Figure 3(d)) Indeed our result is consistentwith a recent report indicating thatHO-1 is regulated byAKTp38MAPK and JNK [23]

34 SWE Affected the TXS PAI-1 and MMP-9 mRNA Pro-duction Human promonocytic cell line U937 is well knownto be differentiated by 12-O-tetradecanoyl-phorbol-13-acetate(TPA or PMA) accompanied with increased expression ofTXS [24] PAI-1 [25] andMMP-9 [26] Since these genes playan important role in wound healing we measured alterationsof them in the presence of SWE U937 cells were treated witheither TPA as a control or compounds and TXS PAI-1 andMMP-9 genes were examined by real-time PCR As shownin Figure 4(a) SWE at the dose of 100 120583gmL stimulated

TXS mRNA expression without TPA but PAI-1 expressionwas not altered by compounds (Figure 4(b)) InterestinglyMMP-9 mRNA expression was decreased in the presence of100 120583gmL SWE (Figure 4(c)) It has been shown thatMMP-9is not only awound healing stimulator but also an antiplateletaggregator by inhibition of thrombin and collagen-inducedplatelet aggregation [27 28] Moreover increased MMP-9expression predicts poor wound healing [29] Thus MMP-9 may be involved in wound healing activity by SWE as anantiplatelet aggregator Neither scu nor stigmasterol alteredthe mRNA expression of these three genes

4 Conclusions

In summary our results indicate that SWE facilitated woundhealing activity as assessed by cellmigration and proliferationassaysThis wound healing activitymay bemediated byHO-1

ISRN Dermatology 7

(Siam weed) extract

MEK p38MAPK AKT JNKTXS

Plateletaggregation Antiplatelet

aggregation

Promoter

Hemostasis Inflammation Cell proliferation

Wound healing

- Neovascularization- Cell migration

Chromolaena odorata L

MMP-9

HO-1 gene

Figure 5 Schematic diagram showing that C odorata extract stimulated the hemostatic activity by stimulation of TXS and repression ofMMP-9 expressions It also activates MEK p38MAPK AKT and JNK kinase pathways which initiated the expression of HO-1The inductionof HO-1 will inhibit an inflammation and stimulate cell proliferation thereby enhancing neovascularization and cell migration that helps toaccomplish wound healing

induction in fibroblasts whereas TXS induction and MMP-9 suppression in U937 cells may also contribute to SWE-induced wound healing activity (Figure 5)

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors thank Misty Bailey for her critical reading ofthis paper This work was supported by The University ofTennessee Center of Excellence in Livestock Diseases andHuman Health grant and PhD Sandwich Program in theStrategic Scholarships Fellowships Frontier (CHE-PhD-SW)no CHE510780 Office of The Higher Education Commis-sion Thailand

References

[1] D T Nguyen D P Orgrill and G F Murphy ldquoThe pathophysi-ologic basis for wound healing and cutaneous regenerationrdquo inBiomaterials for Threating Skin Loss D Orgill and G BlancoEds pp 25ndash57 Woodhead Publishing Limited CambridgeUK 2009

[2] R Vezza A M Mezzasoma G Venditti and P GreseleldquoProstaglandin endoperoxides and thromboxane A

2activate

the same receptor isoforms in human plateletsrdquoThrombosis andHaemostasis vol 87 no 1 pp 114ndash121 2002

[3] Y Aso ldquoPlasminogen activator inhibitor (PAI)-1 in vascularinflammation and thrombosisrdquo Frontiers in Bioscience vol 12no 8 pp 2957ndash2966 2007

[4] PMartin and S J Leibovich ldquoInflammatory cells duringwoundrepair the good the bad and the uglyrdquo Trends in Cell Biologyvol 15 no 11 pp 599ndash607 2005

[5] L Fialkow Y Wang and G P Downey ldquoReactive oxygen andnitrogen species as signaling molecules regulating neutrophilfunctionrdquo Free Radical Biology and Medicine vol 42 no 2 pp153ndash164 2007

[6] F A D T GWagener H E van Beurden JW von denHoff GJ Adema andCG Figdor ldquoTheheme-heme oxygenase systema molecular switch in wound healingrdquo Blood vol 102 no 2 pp521ndash528 2003

[7] I Stamenkovic ldquoExtracellular matrix remodelling the role ofmatrix metalloproteinasesrdquo The Journal of Pathology vol 200no 4 pp 448ndash464 2003

[8] Y Hirose K Chiba T Karasugi et al ldquoA functional polymor-phism in THBS2 that affects alternative splicing and MMPbinding is associated with lumbar-disc herniationrdquo AmericanJournal of Human Genetics vol 82 no 5 pp 1122ndash1129 2008

[9] P A Akah ldquoMechanism of hemostatic activity of Eupatoriumodoratumrdquo International Journal of Crude Drug Research vol28 no 4 pp 253ndash256 1990

[10] Y Wongkrajang S Muagklum P Peungvicha P Jaiarj andN Opartkiattikul ldquoEupatorium odoratum linn an enhancerof hemostasisrdquo Mahidol University Journal of PharmaceuticalSciences vol 17 pp 9ndash13 1990

[11] Y Wongkrajang S Thongpraditchote S Nakornchai WChuakul K Muangklum and P Jaiarj ldquoHemostatic activities ofEupatorium odoratum Linn calcium removal extractrdquoMahidolUniversity Journal of Pharmaceutical Sciences vol 21 pp 143ndash148 1994

[12] T T Phan M A Hughes and G W Cherry ldquoEnhancedproliferation of fibroblasts and endothelial cells treated withan extract of the leaves of Chromolaena odorata (Eupolin) anherbal remedy for treating woundsrdquo Plastic and ReconstructiveSurgery vol 101 no 3 pp 756ndash765 1998

8 ISRN Dermatology

[13] T T Phan J Allen M A Hughes G Cherry and F Woj-narowska ldquoUpregulation of adhesion complex proteins andfibronectin by human keratinocytes treated with an aqueousextract from the leaves of Chromolaena odorata (Eupolin)rdquoEuropean Journal of Dermatology vol 10 no 7 pp 522ndash5272000

[14] T T Phan M A Hughes and G W Cherry ldquoEffects ofan aqueous extract from the leaves of Chromolaena odorata(Eupolin) on the proliferation of human keratinocytes and ontheir migration in an in vitro model of reepithelializationrdquoWound Repair and Regeneration vol 9 no 4 pp 305ndash313 2001

[15] H Pandith S Thongpraditchote Y Wongkrajang and WGritsanapan ldquoIn vivo and in vitro hemostatic activity of Chro-molaena odorata leaf extractrdquo Pharmaceutical Biology vol 50no 9 pp 1073ndash1077 2012

[16] T Triratana R Suwannuraks andW Naengchomnong ldquoEffectof Eupatorium odoratum on blood coagulationrdquo Journal of theMedical Association ofThailand vol 74 no 5 pp 283ndash287 1991

[17] O Gabay C Sanchez C Salvat et al ldquoStigmasterol a phytos-terol with potential anti-osteoarthritic propertiesrdquo Osteoarthri-tis and Cartilage vol 18 no 1 pp 106ndash116 2010

[18] H Pandith X Zhang S Thongpraditchote Y WongkrajangW Gritsanapan and S J Baek ldquoEffect of Siam weed extractand its bioactive component scutellarein tetramethyl ether onanti-inflammatory activity through NF-KB pathwayrdquo Journal ofEthnopharmacology vol 147 no 2 pp 434ndash441 2013

[19] P Bao A Kodra M Tomic-Canic M S Golinko H P Ehrlichand H Brem ldquoThe role of vascular endothelial growth factor inwound healingrdquo Journal of Surgical Research vol 153 no 2 pp347ndash358 2009

[20] A Grochot-Przeczek R Lach J Mis et al ldquoHeme oxygenase-1accelerates cutaneous wound healing inmicerdquo PLoS One vol 4no 6 Article ID e5803 2009

[21] P T Thang S Patrick L S Teik and C S Yung ldquoAnti-oxidanteffects of the extracts from the leaves of Chromolaena odorataon human dermal fibroblasts and epidermal keratinocytesagainst hydrogen peroxide and hypoxanthine-xanthine oxidaseinduced damagerdquo Burns vol 27 no 4 pp 319ndash327 2001

[22] S K Ling M M Pisar and S Man ldquoPlatelet-activating factor(PAF) receptor binding antagonist activity of the methanolextracts and isolated flavonoids from Chromolaena odorata (L)King and Robinsonrdquo Biological and Pharmaceutical Bulletinvol 30 no 6 pp 1150ndash1152 2007

[23] A Zuniga-Toala O Medina-Campo S Espada A Cuadradoand J Pedraza-Chaverri ldquoNordihydroguaiaretic acid inducesheme oxygenase-1 (HO-1) and cytoprotection in a phosphatidylinositol 3 kinase (PI3K)-dependent way in renal epithelial(LLC-PK1) cellsrdquo Journal ofMedicinal Plants Research vol 7 no5 pp 186ndash190 2013

[24] T Nanayama S Hara H Inoue C Yokohama and T TanabeldquoRegulation of two isozymes of prostaglandin endoperoxidesynthase and thromboxane synthase in human monoblastoidcell line U937rdquo Prostaglandins vol 49 no 6 pp 371ndash382 1995

[25] V Ellis T-C Wun N Behrendt E Ronne and K Dano ldquoInhi-bition of receptor-bound urokinase by plasminogen-activatorinhibitorsrdquo Journal of Biological Chemistry vol 265 no 17 pp9904ndash9908 1990

[26] H Watanabe I Nakanishi K Yamashita T Hayakawa andY Okada ldquoMatrix metalloproteinase-9 (92 kDa gelatinasetypeIV collagenase) fromU937monoblastoid cells correlation withcellular invasionrdquo Journal of Cell Science vol 104 no 4 pp 991ndash999 1993

[27] P Jurasz A W Y Chung A Radomski and M W RadomskildquoNonremodeling properties of matrix metalloproteinases theplatelet connectionrdquo Circulation Research vol 90 no 10 pp1041ndash1043 2002

[28] C Fernandez-Patron M A Martinez-Cuesta E Salas etal ldquoDifferential regulation of platelet aggregation by matrixmetalloproteinases-9 and -2rdquoThrombosis and Haemostasis vol82 no 6 pp 1730ndash1735 1999

[29] Y Liu D Min T Bolton et al ldquoIncreased matrix metallopro-teinase-9 predicts poor wound healing in diabetic foot ulcersrdquoDiabetes Care vol 32 no 1 pp 117ndash119 2009

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

2 ISRN Dermatology

tropical countries for a long time especially to stop bleedingNumerous studies have demonstrated that Siam weed extract(SWE) accelerates hemostasis [9ndash11] and wound healing [12ndash14] However themolecular targets of SWE in wound healingactivity have not been identified In this study we investigatedthe effect of 70 ethanolic SWE (C1) and its bioactive com-pounds scutellarin tetramethyl ether (scu) and stigmasterolon enhancedwound healing activity Scu and stigmasterol arethemajor components of SWE as assessed by thin-layer chro-matography In rat models the 70 ethanolic extract pos-sesses the highest effectiveness to stop bleeding comparedto other solvent extracts [15] Scu and stigmasterol exhibithemostatic [16] and antiinflammatory activities [17 18]

In this study we examined the molecular targets ofSWE (C1) in the wound healing process and found that theexpression of HO-1 and other proteins played a pivotal rolein SWE-induced wound healing activity Our data suggestthat SWE affects the wound healing process by using novelmolecular targets

2 Materials and Methods

21 Reagents All antibodies were purchased fromSantaCruzBiotechnology (Santa Cruz CA USA) CellTiter 96 AqueousOne Solution Cell Proliferation Assay was purchased fromPromega (Madison WI USA) Scutellarin tetramethyl ether(scu) and stigmasterol were purchased from ChromaDexInc (Irvine CA USA) and Sigma Aldrich (St Louis MOUSA) respectively All chemicals were purchased fromFisherScientific (Pittsburgh PA USA) unless otherwise specified

22 Preparation of Standard Solutions 12-O-tetradecanoyl-phorbol-13-acetate (TPA) scu and stigmasterol were dis-solved in DMSO and were kept at minus80∘C until used

23 Plant Materials and Plant Extract Preparation Siamweed mature leaves were collected from Samut Sakhonprovince Thailand in December 2009The plant sample wasidentified by DrWandee Gritsanapan and the voucher spec-imen (no CO-003) was deposited at the Department of Phar-macognosy Faculty of Pharmacy Mahidol University Theleaves were dried in a hot air oven at 60∘C for 24 h The driedsample was ground into moderate powder Then 100 g ofthe powder was exhaustively extracted with 70 vv ethanol(EtOH) (1 10 wv) while shaking at 25∘C 120 rpm for 12 h ona shaker The mixture was filtered through Whatman filterpaper no 1 and the filtrate was concentrated using a rotaryevaporator at 40∘C The dark green viscous ethanolic extract(C1) was kept in a tightly closed brown vial at 4∘C until used

A stock solution of the extract was prepared by completedissolving of 342mg of the ethanolic extract in 1 mL EtOHusing sonication for 30min The stock solution was kept atminus80∘C until used The working solutions were prepared bydiluting the stock solution with EtOH to the concentrationsof 342 342 and 100mgmL

24 Cell Cultures Balbc 3T3 and U937 cells (ATCC Man-assas VA USA) were maintained in Dulbeccorsquos modified

Eaglersquos medium (DMEM) and RPMI 1640 medium respec-tively Both culture media were supplemented with 10 fetalbovine serum (FBS) penicillin G (100 UmL) streptomycin(100 120583gmL) and amphotericin B (025 120583gmL) at 37∘C and5 CO

2in a humidified incubator

25 Quantitative Real-Time-PCR and Reverse-TranscriptasePCR U937 cells were grown to 80ndash90 confluence in 10 cmplates and treated with EtOH as vehicle TPA 100 nM aspositive control SWE 342 and 100 120583gmL and scu 10and 20120583M in the presence of 10 serum for 12 h TotalRNA was prepared using the Total RNA kit (Omega Bio-Tek Norcross GA USA) and reverse transcripted witha verso cDNA kit (Thermo Scientific Surrey KT USA)according to the manufacturersrsquo instruction Three repli-cates of real-time PCR were carried out using MyiQ ther-mal cycler (Bio-RAD Hercules CA USA) for 25 cyclesfor human thromboxane synthase (Tx) sense strand 51015840-GCCAAATGGAGCTCAGAAAG-31015840 antisense strand 51015840-TGCAGTAGCACCTCTGGATG-31015840 human PAI-1 sensestrand 51015840-CACGAGTCTTTCAGACCAAGAG-31015840 antisensestrand 51015840-CACACAAAAGCTCCTGTAAGC-31015840 MMP-9sense strand 51015840-GCTCTTCCCTGGAGACCTG-31015840 antisensestrand 51015840-ACACGCGAGTGAAGGTGAG-31015840 and humanGAPDH sense strand 51015840-GACCACAGTCCATGCCAT-CACT-31015840 antisense strand 51015840-TCCACCACCCTGTTGCTG-TAG-31015840 at 94∘C for 30 s 55∘C for 30 s and 72∘C for 1minusing the ABsolute QPCR SYBR-Green mix (ABgene HouseEpsom UK) with specific primers for human Tx PAI-1MMP-9 and GAPDH The quantification of each gene wascalculated For the reverse-transcriptase PCR the reactionwas carried out for 25 cycles for mouse HO-1 sense strand51015840-CCCACGCATATACCCGCTAC-31015840 antisense strand 51015840-CTAGCAGGCCTCTGACGAAG-31015840 and mouse GAPDHsense strand 51015840-CAGGAGCGAGACCCCACTAACAT-31015840antisense strand 51015840-GTCAGATCCACGACGGACACATT-31015840 at 94∘C for 30 s 55∘C for 30 s and 72∘C for 1min usingReadyMix Tag polymerase (Sigma Aldrich) with specificprimers Each PCR product was electrophoresed on 1agarose gel and visualized by ethidium bromide staining

26 Cell Migration Assay Balbc 3T3 cells were grown inDMEM supplemented with 10 FBS until confluence in 6 cmplates The cells were starved with DMEM in an absence ofserum for 24 h Cell monolayer was scratched with a sterilepipette tip Any cellular debris was removed by washing with1 X phosphate buffer saline (PBS) and media were replacedwith 1 DMEM containing EtOH as a control and 342342 and 100120583gmL of SWE The cells were viewed using anOlympus IMT2 microscope (Lake Success NY USA) at 40xtotal magnification and captured using a Sony model XC-ST50 camera (Park Ridge NJ USA) at 0 6 12 and 24 h Thedegree of cell migration was observed by the gap betweencontrol and treatment

27 Cell Proliferation Assay The effect of SWE on cell prolif-eration inBalbc 3T3 cells was investigated using theCellTiter96AqueousOne SolutionCell ProliferationAssay (Promega)

ISRN Dermatology 3



3VO4and 1mM







4 ISRN Dermatology

EtO

HC1

342120583

gm

LC1

100120583

gm

L

6 h 12 h0 h 24 h

C1342120583

gm

L

(a)

Cel

l via

bilit

y ra

tio (f

old

with

EtO

H)

0

02

04

06

08

1

12

14

cb

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

C1342120583

gm

L

(b)


C1 (6 h) C1 (12 h)EtOH 342 342 100 EtOH 342 342 100

HO-1

VEGF

bFGF

Actin

(120583gmL)

(a)

10 20 10 20DMSO

Scu Stigmasterol

HO-1

VEGF

bFGF

Actin

(120583M)

(b)

10 20 10 20DMSO

Scu Stigmasterol

GAPDH GAPDH

342 100EtOH

C1

mHO-1 mHO-1(120583M)(120583gmL)

(c)


ISRN Dermatology 5

LUCminus94 Kb

(a)

c

b

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

C1342120583

gm

L

(b)

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

aa

aa

DM

SO

Scu10120583

M

Scu20120583

M

Stig

mas

tero

l10120583

M

(c)

C1342 120583gmL

EtO

H

PD98

05940120583

M (M

EKE

RK)

SB20

3580

10120583

M(p

38 M

APK

)

SP60

0125

10120583

M (J

NK)

LY29

4002

50120583

M (A

KT)

Rapa

myc

in 1

00 nM

(mTO

R)

U01

262120583

M (M

EK)

+++++++minus

(d)





6 ISRN Dermatology

(AU

)

0

05

1

15

2

25


aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)





4 Conclusions


ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ISRN Dermatology 3



3VO4and 1mM







4 ISRN Dermatology

EtO

HC1

342120583

gm

LC1

100120583

gm

L

6 h 12 h0 h 24 h

C1342120583

gm

L

(a)

Cel

l via

bilit

y ra

tio (f

old

with

EtO

H)

0

02

04

06

08

1

12

14

cb

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

C1342120583

gm

L

(b)


C1 (6 h) C1 (12 h)EtOH 342 342 100 EtOH 342 342 100

HO-1

VEGF

bFGF

Actin

(120583gmL)

(a)

10 20 10 20DMSO

Scu Stigmasterol

HO-1

VEGF

bFGF

Actin

(120583M)

(b)

10 20 10 20DMSO

Scu Stigmasterol

GAPDH GAPDH

342 100EtOH

C1

mHO-1 mHO-1(120583M)(120583gmL)

(c)


ISRN Dermatology 5

LUCminus94 Kb

(a)

c

b

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

C1342120583

gm

L

(b)

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

aa

aa

DM

SO

Scu10120583

M

Scu20120583

M

Stig

mas

tero

l10120583

M

(c)

C1342 120583gmL

EtO

H

PD98

05940120583

M (M

EKE

RK)

SB20

3580

10120583

M(p

38 M

APK

)

SP60

0125

10120583

M (J

NK)

LY29

4002

50120583

M (A

KT)

Rapa

myc

in 1

00 nM

(mTO

R)

U01

262120583

M (M

EK)

+++++++minus

(d)





6 ISRN Dermatology

(AU

)

0

05

1

15

2

25


aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)





4 Conclusions


ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 ISRN Dermatology

EtO

HC1

342120583

gm

LC1

100120583

gm

L

6 h 12 h0 h 24 h

C1342120583

gm

L

(a)

Cel

l via

bilit

y ra

tio (f

old

with

EtO

H)

0

02

04

06

08

1

12

14

cb

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

C1342120583

gm

L

(b)


C1 (6 h) C1 (12 h)EtOH 342 342 100 EtOH 342 342 100

HO-1

VEGF

bFGF

Actin

(120583gmL)

(a)

10 20 10 20DMSO

Scu Stigmasterol

HO-1

VEGF

bFGF

Actin

(120583M)

(b)

10 20 10 20DMSO

Scu Stigmasterol

GAPDH GAPDH

342 100EtOH

C1

mHO-1 mHO-1(120583M)(120583gmL)

(c)


ISRN Dermatology 5

LUCminus94 Kb

(a)

c

b

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

C1342120583

gm

L

(b)

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

aa

aa

DM

SO

Scu10120583

M

Scu20120583

M

Stig

mas

tero

l10120583

M

(c)

C1342 120583gmL

EtO

H

PD98

05940120583

M (M

EKE

RK)

SB20

3580

10120583

M(p

38 M

APK

)

SP60

0125

10120583

M (J

NK)

LY29

4002

50120583

M (A

KT)

Rapa

myc

in 1

00 nM

(mTO

R)

U01

262120583

M (M

EK)

+++++++minus

(d)





6 ISRN Dermatology

(AU

)

0

05

1

15

2

25


aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)





4 Conclusions


ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ISRN Dermatology 5

LUCminus94 Kb

(a)

c

b

b

a

EtO

H

C1342120583

gm

L

C1100120583

gm

L

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

C1342120583

gm

L

(b)

0000

0500

1000

1500

2000

2500

3000

3500

Luci

fera

se ac

tivity

(fol

d)

aa

aa

DM

SO

Scu10120583

M

Scu20120583

M

Stig

mas

tero

l10120583

M

(c)

C1342 120583gmL

EtO

H

PD98

05940120583

M (M

EKE

RK)

SB20

3580

10120583

M(p

38 M

APK

)

SP60

0125

10120583

M (J

NK)

LY29

4002

50120583

M (A

KT)

Rapa

myc

in 1

00 nM

(mTO

R)

U01

262120583

M (M

EK)

+++++++minus

(d)





6 ISRN Dermatology

(AU

)

0

05

1

15

2

25


aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)





4 Conclusions


ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 ISRN Dermatology

(AU

)

0

05

1

15

2

25


aa

a

a

c

b

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M(a)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M

000

200

400

600

800

1000

1200

1400

b

a a ac a

(AU

)

MMP-9

(b)

EtO

H

TPA100

nM

C1342120583

gm

L

C1100120583

gm

L

Scu10120583

M

Scu20120583

M0000

10000

20000

30000

40000

50000

60000

aaa

b

aa

(AU

)

PAI-1

(c)





4 Conclusions


ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ISRN Dermatology 7

(Siam weed) extract



aggregation

Promoter


Wound healing



MMP-9

HO-1 gene





Acknowledgments


References



2activate












8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















8 ISRN Dermatology























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Hemostatic and Wound Healing Properties of Chromolaena ...downloads.hindawi.com/journals/isrn.dermatology/2013/168269.pdf6 ISRNDermatology (A.U.) 0 0.5 1 1.5 2 2.5 3 Thromboxane synthase