research article ixora coccinea enhances cutaneous wound...

Research ArticleIxora coccinea Enhances Cutaneous WoundHealing by Upregulating the Expression of Collagenand Basic Fibroblast Growth Factor

Aadesh Upadhyay12 Pronobesh Chattopadhyay1 Danswrang Goyary1

Papiya Mitra Mazumder2 and Vijay Veer1

1 Division of Pharmaceutical Technology Defence Research Laboratory DRDO Tezpur Assam 784001 India2Department of Pharmaceutical Sciences Birla Institute of Technology Mesra Ranchi Jharkhand 835215 India

Correspondence should be addressed to Pronobesh Chattopadhyay chattopadhyaydrlgmailcom

Received 4 September 2013 Accepted 5 December 2013 Published 29 January 2014

Academic Editors R Couture S Cuzzocrea A Fernandez-Guasti and B-N Wu

Copyright copy 2014 Aadesh Upadhyay 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

Background Ixora coccinea L (Rubiaceae) has been documented for traditional use in hypertension menstrual irregularitiessprain chronic ulcer and skin diseases In the present study I coccinea was subjected to in vitro and in vivo wound healinginvestigation Methods Petroleum ether chloroform methanol and water sequential I coccinea leaves extracts were evaluatedfor in vitro antioxidant antimicrobial and fibroblast proliferation activities The promising I coccinea methanol extract (IxME)was screened for in vivo wound healing activity in Wistar rat using circular excision model Wound contraction measurementhydroxyproline quantification and western blot for collagen type III (COL3A1) basic fibroblast growth factor (bFGF) and Smad-2-3 -4 and -7 was performedwith 7-day postoperative wound granulation tissue Gentamicin sulfate (001ww) hydrogel was usedas reference standard Results IxME showed the potent antimicrobial antioxidant activities with significant fibroblast proliferationinducing activity as compared to all other extracts In vivo study confirmed the wound healing accelerating potential of IxME asevidenced by fasterwound contraction higher hydroxyproline content and improved histopathology of granulation tissueWesternblot analysis revealed that the topical application of I coccineamethanol extract stimulates the fibroblast growth factor and Smadmediated collagen production in wound tissue

1 Background

The World Health Organization estimated that 80 of theworldrsquos population still relies on plant-based medicines fortheir primary health care and skin related disorders speciallywounds which is the most common reason for medicalvisits in the developing countries Wounds and wound man-agement therapy have a long history and in the differentAyurvedic classics of India like Charaka Samhita SushrutaSamhita and Ayurveda Siksha approximately 70 of thewound healing medicines are of plant origin In the lastfew decades traditional wound healing plants have receivedenough attention for scientific investigations [1ndash3] wherepathophysiological process of wound healing and variousrelated activities such as fibroblast growth stimulation andantioxidant and antimicrobial activities has been extensively

studied and correlated to the rationale of the traditional plantmedicines [1 4 5] In thrust of finding for an effective woundhealing herb Ixora coccinea L (Rubiaceae) was selected forthe in vitro and in vivo wound healing investigations I coc-cinea is a small-medium evergreen shrub widely cultivatedornamental plant throughout South Asian regions and usedin the treatment of various ailments like infection hyper-tension menstrual irregularities sprain chronic ulcer andskin diseases including cutaneous wounds [6ndash8] The plantshave been reported for cytotoxic antitumor antimicrobialand anti-inflammatory activity [7 9ndash13] The earlier reportsof I coccinea indicated the preliminary wound healing andantimicrobial properties of flower and root extracts [6 8] butthe effect of I coccinea leaf was not properly investigated onvarious aspects of wound healing Therefore the presentstudy was aimed to investigate the effect of I coccinea leaves

Hindawi Publishing CorporationISRN PharmacologyVolume 2014 Article ID 751824 9 pageshttpdxdoiorg1011552014751824

2 ISRN Pharmacology

extract on fibroblast proliferation and related growth factorsinvolved in collagen production pathway in wound granula-tion tissue

2 Materials and Methods

21 Cell Line Bacterial Culture and Chemicals Humandermal fibroblast (HDF10605) cell line was procured fromSigma Aldrich The bacterial strains Bacillus subtilis (MTCC441) Staphylococcus aureus (MTCC 3160) Streptococcusmutans (MTCC 890) Escherichia coli (MTCC 443) Kleb-siella pneumoniae (MTCC 109) and Pseudomonas aerugi-nosa (MTCC 741) were obtained from Institute of Micro-bial Technology (IMTECH) Chandigarh Mueller-Hintonbroth butylated hydroxytoluene (BHT) gallic acid 22-diphenyl-1-picrylhydrazyl scavenging (DPPH) and nitrob-lue tetrazolium (NBT) were purchased from Sigma TheFolin-Ciocalteu reagent phenazine methosulfate (PMS)120573-nicotinamide adenine dinucleotide (NADH) potassiumferrocyanide trichloroacetic acid (TCA) ferric chloridehydrogen peroxide (H

2O2) phenazine methosulfate fluoride

(PMSF) dimethyl sulphoxide (DMSO) and 3-(45-dimethyl-thiazol-2-yl)-25-diphenyltetrazolium bromide salt (MTT)purchased were from Himedia (Mumbai India) All primaryand secondary antibodies and BCIP (5-bromo-4-chloro-31015840-indolyphosphate p-toluidine salt)-NBT reagent were pur-chased from Santa Cruz Biotech Inc (Texas USA) The sol-vents and chemicals which are notmentioned in the text wereof analytical grade

22 Preparation of Extracts I coccinea leaves were collectedduring September-October (2011) from campus garden ofDefence Research Laboratory Tezpur Assam (India) andauthenticated at Botanical Survey of India Shillong (India)and the specimen sample deposited (Acc no 081168) About100 g of shade dried leaves powder was successively extractedwith petroleum ether chloroform methanol and water at1500 lb at room temperature in Accelerated Solvent Extractor(ASE 15 Dionex USA)The extraction was considered com-plete when the initial color of the percolate gradually changedto colorless Each solvent extract was concentrated in rotaryevaporator (Rotavac Heidolph2 Germany) under reducedpressure and the water extract was freeze-dried Preliminaryphytochemical screening was performed as described earlier[14]

23 Antimicrobial and Antioxidant Wound Healing RelevantAssays Agar broth dilution technique was used in the deter-mination of minimum inhibitory concentration (MIC) inantimicrobial screening according to Hayouni et al [15]

Antioxidant evaluations including DPPH free radical andsuperoxide anion radical scavenging activity (SRSA) ferricion reducing antioxidant power (FRAP) and total phenoliccontent were performed as described earlier [16ndash19]

24 Fibroblast Proliferation Assay Human dermal fibrob-last cells were cultured in DMEM containing 10 FetalBovine Serum (FBS) and antibiotics (100UmLpenicillin and

100UmL streptomycin) in a humidified CO2incubator with

5 CO2at 37∘C Media were replaced every alternate day

Cells were harvested on confluency using 005 Trypsin-EDTA and subcultured in fresh media for producing singlecell suspension

The fibroblast proliferation assay was performed asdescribed by Adetutu et al [4] The cells were counted usinga Scepter 20 Automated Cell counter (Millipore USA) andseeded at a density of 1 times 103 cells per well in 96-well plateexcluding the first row and maintained at 37∘C in humidified5CO

2atmospherewith 10FBSThemediumwas replaced

after 24 h incubation with DMEM containing 05 FBS andextract samples All samples were dissolved in DMSO Thefinal concentrations of extracts ranged from 156 to 100120583gmLand forDMSO04 vv DMEM05FBS (withDMSO) andDMEM10 FBS serve as normal control and growth stim-ulation controls After 48 h of incubation cell viability wasassayed byMTT assaymethodTheMTT solution (5mgmL)was added to each well before 4 h of culture terminationDMSO was added to each well and optical density was mea-sured at 570 nm using a microplate reader (SpectraMax Plus384 Molecular Devices USA) Each sample was assayed intriplicate and three independent tests were performed

25 Hydrogen Peroxide Induced Oxidative Stress Hydrogenperoxide (10 times 10minus4M) was used to induce oxidative stressas described by Annan and Houghton [5] Dermal fibroblastcells were seeded in 96-well plates (5 times 103 cellswell) con-tainingDMEM10 FBS incubated at 37∘C in humidified 5CO2atmosphere After 24 h the growthmediumwas replaced

with fresh DMEM containing different concentrations ofextracts (156ndash100 120583gmL) and simultaneously exposed to10 times 10minus4Mhydrogen peroxide and incubated for 3 h at 37∘CCatalase (250UmL) was used as positive control After theincubation cell viability was assessed by MTT assay methodEach sample was assayed in triplicate and three independenttests were performed

26 Wound Healing Activity

261 Animals Healthy adult Swiss albino male mice (20ndash25 g) and male Wistar rats (250ndash300 g) housed in DefenceResearch Laboratory (DRL) Tezpur Assam (India) wereacclimatized for 3 days They were given free access tofood and water ad libitum The experiments were performedaccording to the Institutional Animal Ethical Committeeguidelines (IAECDRL05July2011) of DRL Tezpur

262 Acute Skin Irritation and Toxicity Study The acute skinirritation and toxicity study was performed for 1 and 25(ww) IxME hydrogel according to the OECD guidelines402 (OECD guidelines 1987) [20] Hydrogel was applied onthe shaved back of the mice and monitored for 14 days forabnormal skin response including irritation redness itchinginflammation and other related symptoms

263 Animal Grouping and Excision Wound Creation AllWistar rats were anesthetized with sodium phenobarbitone

ISRN Pharmacology 3

(40mgkg) intraperitoneally (ip) Circular 20mm diameterwounds were caused on dorsal skin of each animal up to thedepth of loose subcutaneous tissue using surgical scissor andforceps Animals were randomly divided into four groupsnontreated (group I) vehicle control (Carbopol 934 con-taining 5 propylene glycol group II) I coccinea methanolextract (IxME 25 ww group III) and gentamicin sulfate(001 ww group IV) Each group contains 20 animals andhydrogel formulations were applied topically once daily untilcomplete epithelializationOn the 7th postoperative day one-third of animals were euthanized and wound granulation tis-sues (excluding any underlyingmuscle and extraneous tissue)were harvested A portion of harvested tissue was immedi-ately stored in liquid nitrogen for further analysis and anotherportion was fixed in 4 formaldehyde for histopathologicalevaluations Half of the remaining animals were euthanizedon day 15 after injury the entire granulation tissue was usedfor histopathological assessment and remaining animals wereobserved until complete epithelialization [21]

264 Wound Contraction Rate and Hydroxyproline ContentEstimation The progressive changes of wounded area werephotographed (Nikon Coolpix-S3000 camera) and evaluatedby using special size analysis softwaremdashImageJ (NationalInstitutes of Health Maryland USA)mdashas described earlier[21]

Hydroxyproline content was analyzed on day 7 afterinjury granulation tissue as described by Upadhyay et al [21]Tissue hydrolysate samples were mixed with 1mL of 10mMCuSO

4followed by the addition of 1mL of 25N NaOH and

then 1mL of 6H2O2The solutionwasmixed and incubated

at 80∘C for 5min with frequent vigorous shaking Uponcooling 4mL of 3NH

2SO4was addedwith agitation Finally

2mL of 5 p-dimethyl amino benzaldehyde was added andincubated at 70∘C for 15min Absorbance was measured at500 nmusing aUV-VIS spectrophotometer (CE7200 CECILUSA)

265 Histopathological Evaluation Granulation tissues weresectioned (6120583m thick) and stained with hematoxylin-eosin(HE) and Massonrsquos trichrome (MT) stains Tissues sectionswere examined for epithelialization inflammatory cell infil-tration fibroblast proliferation neovascularization and col-lagen deposition

266 Western Blot Analysis Western blot analysis was per-formed as described by Upadhyay et al [21] Protein con-centration was estimated in tissue homogenate using Brad-ford reagent (Sigma Germany) Primary antibody COL3A1-mouse monoclonal IgG

1(sc-271249) bFGF-mouse mono-

clonal IgG2a (sc-74413) Smad 2-goat polyclonal IgG (sc-

6200) Smad 4-rabbit polyclonal IgG (sc-7154) Smad 7-mouse monoclonal IgG

1(sc-365846) 120573-Actin-mouse mon-

oclonal IgG1(sc-47778) and respective secondary antibody

goat anti-mouse IgG-AP (sc-2008) rabbit anti-goat IgG-HRP (sc-2768) and goat anti-rabbit IgG-AP (sc-2007) werepurchased from Santa Cruz Biotech (USA) Smad 3-rabbitpolyclonal IgG (Cat-10832) was purchased from Cayman

Table 1 Antibacterial activity of different I coccinea leaves extractsexpressed as minimal inhibitory concentration (MIC) in mgmL

Bacteria MIC (mgmL)IxPE IxCE IxME IxWE

Bacillus subtilis (MTCC111) 2 gt2 0125 1Staphylococcus aureus (MTCC3160) gt2 gt2 1 gt2Streptococcus mutant (MTCC890) gt2 1 2 2Escherichia coli (MTCC443) 2 1 2 gt2Klebsiella pneumoniae (MTCC109) gt2 gt2 025 gt2Pseudomonas aeruginosa (MTCC741) 2 2 05 2IxPE I coccinea petroleum ether extract IxCE I coccinea chloroformextract IxME I coccinea methanol extract IxWE I coccinea water extractChloramphenicol was used as positive control (MICs lt 90120583gmL)

Chemicals (USA) Equal amount of protein was electropho-resed on 12 SDS-PAGE with 4 stacking gel (Mini Trans-Blot BioRad Laboratories Inc USA) at 80V for 45minProteins were transblotted onto the PVDF membrane (Milli-pore Corp USA) and processed with COL3A1 bFGF Smad-2 -3 -4 -7 and 120573-Actin primary antibodies (1 1000) andcorresponding secondary antibodies (1 2000) The desiredproteins were detected by BCIP-NBT solution and westernMax-HRP-Chromogenic detection kit (Amresco USA) 120573-actin was estimated as internal control

27 Statistical Analysis The results are expressed as means plusmnstandard deviation (SD) Data were statistically analyzedusing Dunnett test A 119875 value lt005 was considered sta-tistically significant as compared to nontreated and vehicletreated group

3 Results

31 Extract and Phytochemicals The yield of the I coc-cinea petroleum ether (IxPE) chloroform (IxCE) methanol(IxME) and water (IxWE) was found 331 222 1633 and1092 (ww) respectively The preliminary phytochemicalscreening of I coccinea extracts showed the presence ofalkaloid flavanoids terpenes phenolic carbohydrate andsaponins in different extracts

32 Antimicrobial and Antioxidant Properties In the antimi-crobial assay methanol extract was found very active (MIC0125ndash2mgmL) as compared to other solvent extracts wherethe gram positive B subtilis and gram negative K pneu-moniae were found the most sensitive (MIC 0125 and025mgmL resp) For chloroform extract MIC values wereranging between 1 and 2mgmL Petroleum ether and waterextracts showed poor antimicrobial activity against selectedpathogens (Table 1)

On the other hand in DPPH superoxide radical scaveng-ing activity (SRSA) IxPE and IxCE were found inactive andshowing 05 and 683 gallic acid equivalents respectively inFCR assay (Table 2) IxME and IxWEwere potent antioxidantextracts in which IxME showed higher DPPH scavengingproperty in comparison to IxWE with IC

50(120583gmL) values

4 ISRN Pharmacology

Table 2 Scavenging activity of different I coccinea extracts against DPPH and superoxide anion free radicals and their Folin-Ciocalteureagent (FCR) reducing capacity

Ixora coccinea Scavenging activity againstDPPH radical (IC50 plusmn SD)

Scavenging activity againstsuperoxide radical (IC50 plusmn SD)

FCR reducing capacity(mgGAcgextract)

IxPE NA NA 05 plusmn 004

IxCE NA NA 683 plusmn 05

IxME 963 plusmn 122 83803 plusmn 2104 2775 plusmn 055

IxWE 2601 plusmn 266 65506 plusmn 1783 1232 plusmn 043

Gallic acid 110 plusmn 039 15686 plusmn 3639 mdashIC50 values are expressed in 120583gmL NA not active SD standard deviation IC50 amount of antioxidant necessary to scavenge the initial DPPHsuperoxideradical by 50 mgGAcgextract mg of gallic acid equivalentgram of extract GAc gallic acid IxPE I coccinea petroleum ether extract IxCE I coccineachloroform extract IxME I coccineamethanol extract IxWE I coccinea water extract

0

05

1

15

2

25

3

35

4

45

00 05 10 15 20 25Concentration (mgmL)

IxPE IxCEIxME IxWEGallic acid

Abso

rban

ce at

700

nm

Figure 1 Ferric ion reducing antioxidant power (FRAP) of differentI coccinea extracts

963 and 2601 respectively On the other hand IxWE (IC50=

65506120583gmL) possess the higher superoxide radical scav-enging strength than IxME (IC

50= 83803 120583gmL) FRAP and

FCR assay data further revealed the antioxidant potency ofthe IxME and IxWE (Figure 1 and Table 2)

33 Fibroblast Proliferation and Viability Among all theextracts IxPE decreased the cell viability from 8402to 4499 in a concentration dependant manner (156ndash100 120583gmL) (Figure 2(a))The IxCE IxME and IxWEextractsshowed the biphasic proliferation response where the cellviability increased at low concentrations and decreased athigher concentrations The IxCE exposure for 48 h increasedthe cell viability from 8995 to 9855 (156 to 625120583gmL)however decreased at higher concentrations (Figure 2(a))The IxME showed the significant fibroblast proliferationactivity which was almost close to the level of positive control(11316 at 125 120583gmL) On the other hand IxWE decreasedthe cell viability from 10152 to 8433 (312 to 100 120583gmL)

The exposure of H2O2(10 times 10minus4M) for 3 h decreased the

cell viability (4812) IxPE synergies the effect of H2O2and

decreased the cell viability from 4703 to 3532 with increas-ing concentration (Figure 2(b)) Potent antioxidant IxME andIxWE showed dose dependent protection (4733ndash7471 and4785ndash6947 resp) However IxCE showed 4836ndash6009cell viability at 156ndash100 120583gmL

34 Wound Healing

341 Acute Skin Irritation and Toxicity In skin irritation andtoxicity assay any sign of inflammation and irritation werenot observed in both 1 and 25 (ww) IxME hydrogelTherefore high concentration 25 (ww) IxMEhydrogel wasselected for in vivo wound healing study

342 Wound Contraction The whole wound area wasreduced in parallel to postwound days (Figure 3(a)) Non-treated and vehicle treated animal groups showed 513and 5425 wound contraction on 21st postoperative daysrespectively On the other hand IxME treated animal groupshowed 9678 and gentamicin sulfate treated group 8959wound contraction The contraction rate was significantlyhigher in IxME treated group as compared to nontreated andvehicle treated control group

343 Hydroxyproline Content IxME and gentamicin sulfatetreated group showed significantly increased level of hydrox-yproline as compared to nontreated and vehicle treatedgroups (Figure 3(b)) Although the hydroxyproline content ofIxME treatment groupwas higher as compared to gentamicinsulfate treated group but the data were statistically not sig-nificant

344 Histopathological Observations IxME and gentamicinsulfate treated animal groups showed well organized woundhealing processes (inflammation proliferation and remod-eling) in postoperative days On the other hand vehicle andnontreated animal groups depict slow rate of epithelializationand dermal layer with lesser collagen bundles (Table 3) His-topathological section of day 7 showed mild to moderateedema and ulcer in nontreated and vehicle treated groupswith abundant polymorphonuclear cell (PMC) The infiltra-tion rate of mononuclear and fibroblast cells were observedlow (Figure 4) However 7-day IxME treated wound tissue

ISRN Pharmacology 5

0

20

40

60

80

100

120

1400

5

FBS

10

FBS 156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

Concentration (120583mmL)

(a)

0

25

50

75

100

156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

lowastlowast

lowast

lowast

lowast

H2O2

10

mM

Cata

lase

250

IUm

L


(b)

Figure 2 (a) Effect of I coccinea extracts on human dermal fibroblast proliferation (b) Protection of human dermal fibroblast cells againstH2O2-induced damage with simultaneous application of different I coccinea extracts FBS Fetal Bovine Serum Cat-Catalase Values are

expressed as mean plusmn SD Asterisk (lowast) indicates significantly different (119875 lt 005) as compared to H2O2treated negative treated groups

Table 3 Histopathological evaluation of wound healing process in different treatment groups

Groups Wound healing processS U Ed PMC MNC FP RE CD

Day 7Nontreated ++ ++ ++ +++ + + minus minus

Vehicle control ++ ++ ++ +++ + + +minus minus+IxME (25) + minus minus ++ ++ +++ ++ ++Gentamicin sulfate (001) + minus minus ++ ++ ++ + +

Day 15Nontreated ++ + minus+ ++ +++ ++ + ++Vehicle control + + minus ++ +++ ++ ++ ++IxME (25) minus minus minus minus + +++ +++ +++Gentamicin sulfate (001) minus minus minus minus+ + ++ ++ ++

HE and MT staining were scored as mild (+) moderate (++) and severe (+++) for epidermal andor dermal re-modeling S scab U ulcer Ed edema PMCpolymorphonuclear cells MNC mononuclear cells FP fibroblast proliferation CD collagen deposition RE reepithelialization IxME I coccinea methanolextract

showed increased density of mononuclear cells with distinctonset of reepithelialization Edema and ulcerous area wereabsent in both IxME and gentamicin sulfate treatments 15-day IxME treatment significantly accelerated the cutaneouswound healing as depicted by thick well organized re-epithelialized layer dermis with compact collagen layeringand faster keratinization with intraepithelial cornificationwhereas slow reepithelialization with minor ulcer area wasnoticed in nontreated animal group Massonrsquos trichrome(400times) staining of 7- and 15-day postoperative wound granu-lation tissue depicted the clearer picture of wound healingwhere IxME treatment showed increased macrophage andfibroblast density with higher collagen deposition On theother hand gentamicin treatment although showed completereepithelialization with irregular packing of collagen fibersand minor to moderate macrophages infiltration (Figure 4)

In vehicle and nontreated animal groups collagen bundleswere loosely packed and granulation tissues were moderatelycellular with mononuclear and fibroblast cells

345 Effect of IxME on Granulation Tissue Protein West-ern blotting showed significantly increased expression ofCOL3A1 and bFGF protein in IxME and gentamicin sulfatetreated wound granulation tissue (Figure 5) The expressionlevels of signal transducer protein Smad-2 -3 and -4 werealso significantly increased whereas the inhibitory proteinSmad-7 was found unaltered Moreover the expression levelsof the COL3A1 bFGF and Smad proteins (2 3 and 4) weresignificantly higher in IxME treated animals groups in com-parison of gentamicin sulphate treated groupThe120573-actinwasused as an internal control

6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days

Nontreated Vehicle control

IxME (25 ww) Gentamicin sulfate (001 ww)

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Nontreated Vehiclecontrol

IxME Gentamicinsulfate

Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)

Figure 3 Effect of I coccinea methanol extract (IxME) on wound healing (a) Pictorial representation of wound closure in wistar rat(b) Wound contraction rate (c) Hydroxyproline content Values are expressed as mean plusmn SD Asterisk (lowast) indicates significantly different(119875 lt 005) as compared to the nontreated and vehicle treated groups

4 Discussion

Cutaneous wound healing is a complex cascade of tissueregenerative and restorative events including chemotaxiscell division neovascularization synthesis and maturationof new extracellular matrix and remodelling of scar Theseevents can be broadly categorized into inflammation pro-liferation and remodeling phases of wound healing whichare regulated by several mediators including cytokines andvarious secreted growth factors

Although the wound healing cascade takes place by itselfand does not require much help but various risk factors suchas infection have serious impact B subtilis S aureus E coliK pneumoniae and P aeruginosa are the most wound infect-ing pathogens as the open wound provides the favorable

conditions formicrobial growth [22] Our study indicated theantimicrobial potency of I coccineamethanol extract (IxME)against most common wound infecting pathogens for exam-ple B subtilis (MIC = 0125mgmL) K pneumoniae (MIC =025mgmL) and P aeruginosa (MIC = 050mgmL) as com-pared to other solvent extracts (Table 1) corroborating withthe previous reports of antimicrobial potential [8 11 13]

Invading microbes prolonged the inflammatory phase ofwound healing by producing toxins and wound exudates andsubsequently delayed the granulation tissue formation [22]Activated polymorphonuclear cells including neutrophilesleukocytes and mononuclear cell (MNC) like lymphocytesmonocytes and macrophages phagocytize and kill all theinfecting pathogen Reactive oxygen species (ROS) are thekey product for bactericidal activity of these activated cells

ISRN Pharmacology 7

Nontreated Vehicle treated IxME Gentamicin sulfate

7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)

Figure 4 Microscopic view of healing wound granulation tissue and remodeling epidermisdermis in (1) nontreated (2) vehicle control (3)IxME and (4) gentamicin sulfate treated animal groups Section shows the hematoxylin and eosin stained epidermis and dermis in (A) and(C) (100x) and Massonrsquos trichrome stained dermis in (B) and (D) (400x) of 7- and 15-day postoperative treated animal groups respectivelyThe arrows point to the events of wound healingmdashS scab U ulcer Re reepithelization F fibroblast PMC polymorphonuclear cells MNCmononuclear cells C collagen and NV neovascularization

ROS like HOminus2 HOminus and O2minus although in low concentra-tions sterile the wound fibrin clot matrix and are the impor-tant determinant of wound angiogenesis [23] Whereas theprofuse ROS not only damage extracellular structure pro-teins lipids and DNA but also stimulate signal transductionpathways to prolong the inflammatory phase of wound heal-ing Many scientific publications have proven the beneficialeffect of different plant-based antioxidants on wound repairprocess [24 25] and our findings of free radical scavengingactivity (DPPH SRSA FRAP and FCR) in methanol andwater I coccinea extracts revealed that this plant may beuseful in wound healing (Figure 1 and Table 2)

The proliferation phase of wound healing involves for-mation of granulation tissue synthesis and deposition ofcollagen fibers and reepithelialization In the late phase ofinflammation the activatedmacrophages initiate the prolifer-ation phase that actively progressed by the infiltrating fibrob-lasts Fibroblast cells synthesize the collagen fibers and othercytoskeleton matrix components [26 27] In vitro fibroblast

proliferation assay for I coccinea revealed the toxic effect ofIxPE and IxCE as the cell viability decreased (lt80 to05 FBS) with increasing concentrations (Figure 2(a)) IxPEdecreased cell population could be due to the presence ofproliferation inhibitory components in I coccinea petroleumether extract which also synergized the H

2O2-induced

fibroblast cell death Whereas IxCE at 100 120583gmL decreasedthe cell population below 80was considered as cytotoxic Inthe biphasic proliferation response of IxCE IxME and IxWEthe inhibitory effect at higher concentrations might be due toaccumulation of growth inhibitory components of respectivecrude extract (Figure 2(a)) IxME and IxWE showed the con-centration dependent protection and effectively antagonizedthe H2O2-induced cell death (Figure 2(b)) and this protective

effect might be due to antioxidant properties of the extracts(Table 2) The above findings of in vitro fibroblast prolifera-tion and protection against H

2O2coincide with the previous

reports of different medicinal plants [1 3ndash5] However theIxCE did not show any in vitro antioxidant activity (DPPH

8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1

NontreatedVehicle control

IxMEGentamicin sulfate

bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4

Smad-2 Smad-3 Smad-4 Smad-7

lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)

Figure 5 Effect of I coccineamethanol extract (IxME) on COL3A1 and bFGF Smad-2 -3 -4 and -7 protein expressions on day 7 (seven) inwound tissues detected by western blot Lane (1) nontreated (2) vehicle control (3) IxME and (4) gentamicin sulfate treated animal grouprespectively Values are expressed as mean plusmn SD Asterisk (lowast) indicates significantly different (119875 lt 005) as compared to the nontreated andvehicle treated groups Hash () indicates significantly different (119875 lt 005) as compared to gentamicin sulfate treated group

SRSA andFRAP) thereby the protective effect against H2O2

induced oxidative stress could be due to the intracellularantioxidant enzymes (superoxide dismutase glutathione per-oxidase catalase etc) inducing activity

From the above in vitro antimicrobial antioxidant andfibroblast cell proliferation studies it was observed that I coc-cineamethanol extract possesses the highest activity as com-pared to other extracts Keeping in view of the above findingsIxME (25 ww) was further evaluated for in vivo woundhealing activity in Wistar rat in circular excision model

In wound repair process the centripetal movements ofsurrounding epithelial tissues (rallied by the maturing extra-cellular matrix) close the wound opening [27] Collagen is amajor component of extracellular matrix and wound repairprocess depends on the regulated production and deposi-tionmaturation of new collagen Hydroxyproline is a basicconstituent of collagen structure and its content is an indexof collagen synthesis The periodic assessment of wound areashowed that the topical application of IxME significantlyaccelerated the wound contraction rate and hydroxyprolinecontent as compared to nontreated and hydrogel vehicletreated groups The wound contraction rate for IxME waseven faster than gentamicin treated group although the datawas not significant (Figure 3) Fibroblasts in granulation tis-sue regulate the production deposition and their subsequentmaturation of collagen fibers that impart physical strength tothe tissueHistopathological examination of IxME treated tis-sue on 7 and 15 postoperative days showed the well organizedwound healing with prominent macrophage and fibroblastinfiltration Reepithelialization was also higher with perfusecollagen deposition in mature dermis as compared to non-treated and vehicle control both (Figure 4)

During proliferation phase various secreted chemotacticmolecules and growth factors such as transforming growth

factor-120573 (TGF-120573) basic fibroblast growth factor (bFGF) andplatelet derive growth factor (PDGF) control the motion ofmacrophagefibroblasts [27 28] bFGF promotes the neovas-cularization in granulation tissue to increase oxygen sup-ply that facilitates the collagen production and maturationReleased TGF-120573 bounds to the extracellular fibroblast TGF-120573 receptors and initiates the TGF-120573-Smad mediated collagenproduction A complex interplay of Smad family proteins(Smad-2 -3 -4 and -7 TGF-120573 type I receptor kinases sub-strate) transduced the receptor signals to specific target geneand regulate the synthesis of collagen in granulation tissue[28 29] The mechanism of collagen production throughSmad mediated signaling pathway in the granulation fibrob-last has been revealed earlier [22 28 29] and the presentfindings of western blot analysis showed that IxME hydrogeltopical application significantly upregulated the expressionof bFGF COL3A1 and Smad-2 and -4 proteins as com-pared to gentamicin sulfate and control groups whereas theexpression of Smad-7 the inhibitory protein was unaltered(Figure 5) Histopathological examination and expression oftissue protein (bFGF and COL3A1) supported the in vitrofindings of fibroblast proliferation potential of IxME

5 Conclusion

The present study revealed that I coccinea methanol extractmediated wound healing activity may be a combine effect ofantimicrobial antioxidant and fibroblast proliferating prop-erties which is also supported by the in vitro and in vivoexperimental studies

Conflict of Interests

The authors declare that they have no conflict of interests

ISRN Pharmacology 9

Acknowledgments

Financial support to A Upadhyay in the form of ResearchFellowship by DRDO Ministry of Defence Government ofIndia is gratefully acknowledged Authors are also thankfulto Defence Research Laboratory Tezpur Assam India andBirla Institute of Technology Mesra Ranchi India forproviding necessary support for carrying out the researchwork

References

[1] P J Houghton P J Hylands A YMensah A Hensel andAMDeters ldquoIn vitro tests and ethnopharmacological investigationswound healing as an examplerdquo Journal of Ethnopharmacologyvol 100 no 1-2 pp 100ndash107 2005

[2] B Kumar M Vijayakumar R Govindarajan and P Pushpan-gadan ldquoEthnopharmacological approaches to wound healing-exploring medicinal plants of Indiardquo Journal of Ethnopharma-cology vol 114 no 2 pp 103ndash113 2007

[3] R Thakur N Jain R Pathak and S S Sandhu ldquoPractices inwound healing studies of plantsrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2011 Article ID 438056 17pages 2011

[4] A Adetutu W A Morgan and O Corcoran ldquoAntibacterialantioxidant and fibroblast growth stimulation activity of crudeextracts of Bridelia ferruginea leaf a wound-healing plant ofNigeriardquo Journal of Ethnopharmacology vol 133 no 1 pp 116ndash119 2011

[5] K Annan and P J Houghton ldquoAntibacterial antioxidant andfibroblast growth stimulation of aqueous extracts of Ficus asper-ifoliaMiq andGossypiumarboreumL wound-healing plants ofGhanardquo Journal of Ethnopharmacology vol 119 no 1 pp 141ndash144 2008

[6] B S Nayak A L Udupa and S L Udupa ldquoEffect of Ixora coc-cinea flowers on dead space wound healing in ratsrdquo Fitoterapiavol 70 no 3 pp 233ndash236 1999

[7] W D Ratnasooriya S A Deraniyagala G Galhena S S PLiyanage S D N K Bathige and J R A C Jayakody ldquoAnti-inflammatory activity of the aqueous leaf extract of Ixora coc-cineardquo Pharmaceutical Biology vol 43 no 2 pp 147ndash152 2005

[8] N Selvaraj B Lakshmanan PMMazumderM KaruppasamyS S Jena and A K Pattnaik ldquoEvaluation of wound healing andantimicrobial potentials of Ixora coccinea root extractrdquo AsianPacific Journal of Tropical Medicine vol 4 no 12 pp 959ndash9632011

[9] P G Latha andK R Panikkar ldquoCytotoxic and antitumour prin-ciples from Ixora caccinea flowersrdquo Cancer Letters vol 130 no1-2 pp 197ndash202 1998

[10] J Annapurna P V S Amarnath D A Kumar S V Ramakr-ishna and K V Raghavan ldquoAntimicrobial activity of Ixora cac-cinea leavesrdquo Fitoterapia vol 74 no 3 pp 291ndash293 2003

[11] T O Idowu A O Ogundaini A O Salau E M Obuotor MBezabih and B M Abegaz ldquoDoubly linked A-type proantho-cyanidin trimer and other constituents of Ixora caccinea leavesand their antioxidant and antibacterial propertiesrdquo Phytochem-istry vol 71 no 17-18 pp 2092ndash2098 2010

[12] A Torey S Sasidharan L Y Latha S Sudhakaran and SRamanathan ldquoAntioxidant activity and total phenolic contentof methanol extracts of Ixora caccineardquo Pharmaceutical Biologyvol 48 no 10 pp 1119ndash1123 2010

[13] L Y Latha I Darah K Jain and S Sasidharan ldquoPharmaco-logical screening of methanolic extract of Ixora speciesrdquo Asian

Pacific Journal of Tropical Biomedicine vol 2 no 2 pp 149ndash1512012

[14] K R Khandelwal Practical Pharmacognosy Techniques ampExperiments Nirali Prakashan 11th edition 2004

[15] E A Hayouni K Miled S Boubaker et al ldquoHydroalcoholicextract based-ointment from Punica granatum L peels withenhanced in vivo healing potential on dermal woundsrdquo Phy-tomedicine vol 18 no 11 pp 976ndash984 2011

[16] I M C Brighente M Dias L G Verdi and M G PizzolattildquoAntioxidant activity and total phenolic content of some Brazil-ian speciesrdquo Pharmaceutical Biology vol 45 no 2 pp 156ndash1612007

[17] I Suntar E K Akkol F S Enol H Keles and I E OrhanldquoInvestigating wound healing tyrosinase inhibitory and antiox-idant activities of the ethanol extracts of Salvia cryptanthaand Salvia cyanescens using in vivo and in vitro experimentalmodelsrdquo Journal of Ethnopharmacology vol 135 no 1 pp 71ndash77 2011

[18] X H Wang and J T Dai ldquoA comparative study on antioxidantactivity of water and ethanol extracts of ten Chinese herbsrdquoJournal ofMedicinal Plants Research vol 6 no 11 pp 2210ndash22152012

[19] S Maurya and D Singh ldquoQuantitative analysis of total phenoliccontent in Adhatoda vasica nees extractsrdquo International Journalof PharmTech Research vol 2 no 4 pp 2403ndash2406 2010

[20] OECD guideline for testing of chemicals-402 Acute DermalToxicity 1987

[21] A Upadhyay P Chattopadhyay D Goyary P M Mazumderand V Veer ldquoEleutherine indica L accelerates in vivo cutaneouswoundhealing by stimulating Smad-mediated collagen produc-tionrdquo Journal of Ethnopharmacology vol 146 no 2 pp 490ndash494 2013

[22] P G Bowler B I Duerden and D G Armstrong ldquoWoundmicrobiology and associated approaches to wound manage-mentrdquoClinical Microbiology Reviews vol 14 no 2 pp 244ndash2692001

[23] M Schafer and S Werner ldquoOxidative stress in normal andimpaired wound repairrdquo Pharmacological Research vol 58 no2 pp 165ndash171 2008

[24] A Shukla AMRasik andGK Patnaik ldquoDepletion of reducedglutathione ascorbic acid vitamin E and antioxidant defenceenzymes in a healing cutaneous woundrdquo Free Radical Researchvol 26 no 2 pp 93ndash101 1997

[25] B P Mudge C Harris R R Gilmont B S Adamson andR S Rees ldquoRole of glutathione redox dysfunction in diabeticwoundsrdquoWound Repair and Regeneration vol 10 no 1 pp 52ndash58 2002

[26] Y Mimura H Ihn M Jinnin Y Asano K Yamane and KTamaki ldquoEpidermal growth factor induces fibronectin expres-sion in human dermal fibroblasts via protein kinase C 120575 signal-ing pathwayrdquoThe Journal of Investigative Dermatology vol 122no 6 pp 1390ndash1398 2004

[27] T Kondo and Y Ishida ldquoMolecular pathology of wound heal-ingrdquo Forensic Science International vol 203 no 1ndash3 pp 93ndash982010

[28] M Schiller D Javelaud and A Mauviel ldquoTGF-120573-inducedSMAD signaling and gene regulation consequences for extra-cellular matrix remodeling and wound healingrdquo Journal ofDermatological Science vol 35 no 2 pp 83ndash92 2004

[29] J Massague and R R Gomis ldquoThe logic of TGF-120573 signalingrdquoFEBS Letters vol 580 no 12 pp 2811ndash2820 2006

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of

2 ISRN Pharmacology

extract on fibroblast proliferation and related growth factorsinvolved in collagen production pathway in wound granula-tion tissue

2 Materials and Methods

21 Cell Line Bacterial Culture and Chemicals Humandermal fibroblast (HDF10605) cell line was procured fromSigma Aldrich The bacterial strains Bacillus subtilis (MTCC441) Staphylococcus aureus (MTCC 3160) Streptococcusmutans (MTCC 890) Escherichia coli (MTCC 443) Kleb-siella pneumoniae (MTCC 109) and Pseudomonas aerugi-nosa (MTCC 741) were obtained from Institute of Micro-bial Technology (IMTECH) Chandigarh Mueller-Hintonbroth butylated hydroxytoluene (BHT) gallic acid 22-diphenyl-1-picrylhydrazyl scavenging (DPPH) and nitrob-lue tetrazolium (NBT) were purchased from Sigma TheFolin-Ciocalteu reagent phenazine methosulfate (PMS)120573-nicotinamide adenine dinucleotide (NADH) potassiumferrocyanide trichloroacetic acid (TCA) ferric chloridehydrogen peroxide (H

2O2) phenazine methosulfate fluoride

(PMSF) dimethyl sulphoxide (DMSO) and 3-(45-dimethyl-thiazol-2-yl)-25-diphenyltetrazolium bromide salt (MTT)purchased were from Himedia (Mumbai India) All primaryand secondary antibodies and BCIP (5-bromo-4-chloro-31015840-indolyphosphate p-toluidine salt)-NBT reagent were pur-chased from Santa Cruz Biotech Inc (Texas USA) The sol-vents and chemicals which are notmentioned in the text wereof analytical grade

22 Preparation of Extracts I coccinea leaves were collectedduring September-October (2011) from campus garden ofDefence Research Laboratory Tezpur Assam (India) andauthenticated at Botanical Survey of India Shillong (India)and the specimen sample deposited (Acc no 081168) About100 g of shade dried leaves powder was successively extractedwith petroleum ether chloroform methanol and water at1500 lb at room temperature in Accelerated Solvent Extractor(ASE 15 Dionex USA)The extraction was considered com-plete when the initial color of the percolate gradually changedto colorless Each solvent extract was concentrated in rotaryevaporator (Rotavac Heidolph2 Germany) under reducedpressure and the water extract was freeze-dried Preliminaryphytochemical screening was performed as described earlier[14]

23 Antimicrobial and Antioxidant Wound Healing RelevantAssays Agar broth dilution technique was used in the deter-mination of minimum inhibitory concentration (MIC) inantimicrobial screening according to Hayouni et al [15]

Antioxidant evaluations including DPPH free radical andsuperoxide anion radical scavenging activity (SRSA) ferricion reducing antioxidant power (FRAP) and total phenoliccontent were performed as described earlier [16ndash19]

24 Fibroblast Proliferation Assay Human dermal fibrob-last cells were cultured in DMEM containing 10 FetalBovine Serum (FBS) and antibiotics (100UmLpenicillin and

100UmL streptomycin) in a humidified CO2incubator with

5 CO2at 37∘C Media were replaced every alternate day

Cells were harvested on confluency using 005 Trypsin-EDTA and subcultured in fresh media for producing singlecell suspension

The fibroblast proliferation assay was performed asdescribed by Adetutu et al [4] The cells were counted usinga Scepter 20 Automated Cell counter (Millipore USA) andseeded at a density of 1 times 103 cells per well in 96-well plateexcluding the first row and maintained at 37∘C in humidified5CO

2atmospherewith 10FBSThemediumwas replaced

after 24 h incubation with DMEM containing 05 FBS andextract samples All samples were dissolved in DMSO Thefinal concentrations of extracts ranged from 156 to 100120583gmLand forDMSO04 vv DMEM05FBS (withDMSO) andDMEM10 FBS serve as normal control and growth stim-ulation controls After 48 h of incubation cell viability wasassayed byMTT assaymethodTheMTT solution (5mgmL)was added to each well before 4 h of culture terminationDMSO was added to each well and optical density was mea-sured at 570 nm using a microplate reader (SpectraMax Plus384 Molecular Devices USA) Each sample was assayed intriplicate and three independent tests were performed

25 Hydrogen Peroxide Induced Oxidative Stress Hydrogenperoxide (10 times 10minus4M) was used to induce oxidative stressas described by Annan and Houghton [5] Dermal fibroblastcells were seeded in 96-well plates (5 times 103 cellswell) con-tainingDMEM10 FBS incubated at 37∘C in humidified 5CO2atmosphere After 24 h the growthmediumwas replaced

with fresh DMEM containing different concentrations ofextracts (156ndash100 120583gmL) and simultaneously exposed to10 times 10minus4Mhydrogen peroxide and incubated for 3 h at 37∘CCatalase (250UmL) was used as positive control After theincubation cell viability was assessed by MTT assay methodEach sample was assayed in triplicate and three independenttests were performed

26 Wound Healing Activity

261 Animals Healthy adult Swiss albino male mice (20ndash25 g) and male Wistar rats (250ndash300 g) housed in DefenceResearch Laboratory (DRL) Tezpur Assam (India) wereacclimatized for 3 days They were given free access tofood and water ad libitum The experiments were performedaccording to the Institutional Animal Ethical Committeeguidelines (IAECDRL05July2011) of DRL Tezpur

262 Acute Skin Irritation and Toxicity Study The acute skinirritation and toxicity study was performed for 1 and 25(ww) IxME hydrogel according to the OECD guidelines402 (OECD guidelines 1987) [20] Hydrogel was applied onthe shaved back of the mice and monitored for 14 days forabnormal skin response including irritation redness itchinginflammation and other related symptoms

263 Animal Grouping and Excision Wound Creation AllWistar rats were anesthetized with sodium phenobarbitone

ISRN Pharmacology 3






















3 Results





4 ISRN Pharmacology










0

05

1

15

2

25

3

35

4

45



Abso

rban

ce at

700

nm




50= 83803 120583gmL) FRAP and






34 Wound Healing





ISRN Pharmacology 5

0

20

40

60

80

100

120

1400

5

FBS

10

FBS 156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE


(a)

0

25

50

75

100

156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

lowastlowast

lowast

lowast

lowast

H2O2

10

mM

Cata

lase

250

IUm

L


(b)












6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days



lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)


4 Discussion





ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 3






















3 Results





4 ISRN Pharmacology










0

05

1

15

2

25

3

35

4

45



Abso

rban

ce at

700

nm




50= 83803 120583gmL) FRAP and






34 Wound Healing





ISRN Pharmacology 5

0

20

40

60

80

100

120

1400

5

FBS

10

FBS 156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE


(a)

0

25

50

75

100

156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

lowastlowast

lowast

lowast

lowast

H2O2

10

mM

Cata

lase

250

IUm

L


(b)












6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days



lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)


4 Discussion





ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

4 ISRN Pharmacology










0

05

1

15

2

25

3

35

4

45



Abso

rban

ce at

700

nm




50= 83803 120583gmL) FRAP and






34 Wound Healing





ISRN Pharmacology 5

0

20

40

60

80

100

120

1400

5

FBS

10

FBS 156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE


(a)

0

25

50

75

100

156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

lowastlowast

lowast

lowast

lowast

H2O2

10

mM

Cata

lase

250

IUm

L


(b)












6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days



lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)


4 Discussion





ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 5

0

20

40

60

80

100

120

1400

5

FBS

10

FBS 156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE


(a)

0

25

50

75

100

156

312

625

125 25 50 100

Cel

l via

bilit

y (

)

IxPE IxCEIxME IxWE

lowastlowast

lowast

lowast

lowast

H2O2

10

mM

Cata

lase

250

IUm

L


(b)












6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days



lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)


4 Discussion





ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

6 ISRN Pharmacology

Postoperative days

Non

treat

ed

cont

rol

Vehi

cle

cont

rol

IxM

EG

enta

mic

in

sulfa

te

0 6 12 18 24

(a)

0

20

40

60

80

100

3 6 9 12 15 18 21 24 28

Wou

nd co

ntra

ctio

n (

)

Postoperative days



lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowast

lowast lowastlowast

lowastlowast lowast

(b)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000



Hyd

roxy

prol

ine (

120583g100

mg

tissu

e)

lowast

lowast

(c)


4 Discussion





ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 7


7 da

ys15

day

s

1A 2A 3A 4A

1B 2B 3B 4B

1C 2C 3C 4C

1D 2D 3D 4D

MT

(400times

)H

E (100times

)M

T (400times

)H

E (100times

)





2O2-induced





8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

8 ISRN Pharmacology

0

05

1

1

15

2

2 3 4 1 2 3 4 1 2 3 4

25

COL3A1



bFGF 120573-Actin

lowast

lowast

lowast

lowast

Rela

tive d

ensit

y

(a)

005

115

225

335

4


lowast

lowast

lowast

lowast

lowast

lowast

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4


Rela

tive d

ensit

y


(b)








5 Conclusion




ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

ISRN Pharmacology 9

Acknowledgments


References



































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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