Wound healing effect of Euphorbia hirta linn.

14 Pages·2017·2.05 MB·English

by  Riazul Haque Tuhin

Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423
DOI10.1186/s12906-017-1930-x
RESEARCH ARTICLE Open Access
Euphorbia hirta
Wound healing effect of
linn.
(Euphorbiaceae) in alloxan induced
diabetic rats
Riazul Haque Tuhin1, Mst. Marium Begum2, Md. Sohanur Rahman3, Rubaba Karim1, Taslima Begum1,
Siraj Uddin Ahmed1, Ronia Mostofa1, Amir Hossain1, Mohamed Abdel-Daim4 and Rayhana Begum1*
Abstract
Background: Euphorbia hirta linn., isaspeciesofEuphorbiaceaefamily.Theyareknownasasthmaplant,barokhervi.
TheplantE.hirtaisfamousforitsmedicinalimportanceamongthetribalpopulation.Itisacommonpracticetouse
thewholetohealwounds.Severalpharmacologicalpropertiesincludingantiseptic,anti-inflammatory,antidibetic,
antispasmodic,antibacterial,antiviral,antifungal,anticonvulsant,nootropic,antifertilityandaphrodisiacpropertieshave
alreadybeenreportedforthisplant.Theaimofpresentworkwastoevaluatethewoundhealingpropertyindiabetic
animalsbyoralandtopicaladministrationofethanolicextractofE.hirtawholeplant.
Methods:TheethanolicextractofE.hirtawassubjectedtodeterminethetotalphenoliccontentandtotalflavonoid
contentusinggalicacidandquercetin,respectivelyasstandard.Asingleinjectionofalloxanmonohydrate(120mg/kg,
i.p.)preparedinnormalsalinewasadministeredtoproducediabetesinrats,afterovernightfasting.Foranalyzingthe
rateofcontractionofwound,excisionwoundssized4.90cm2andof2mmdepthwereused.Oral(100,200and400mg/
kg/day;p.o.)andtopicaltreatmentwiththeextract(5%and10%ointment50mg/kg/day)andstandard(5%povidone
iodineointment50mg/kg/day)wasstartedonthedayofinductionofwoundandcontinuedupto16days.Themeans
ofwoundareameasurementbetweengroupsatdifferenttimeintervalswerecomparedusingANOVAandDunnet’stest.
Thediabeticwoundhealingmechanismwasstudiedbymeasuringtheplasmalevelofglucose,malondialdehyde(MDA)
andnitricoxide(NO)inbothcontrolandtreatedgroups.Fortheconfirmationofactivity,histopathologyofthewounds
tissuesfromexcisionwoundmodelwasperformed.
Results:Phytochemicalinvestigationsshowedthepresenceofvariousphytoconstituents(carbohydrates,saponins,
alkaloids,glycosides,steroids,flavonoids,tannins).IntheethanolicextractofE.hirtathetotalphenolcontentwas285
±3.22mg/gwhereasthetotalflavonoidcontentwas118.46±1.85mg/g.Inthepresentstudy,E.hirtacausedsignificant
woundcloserbothorally(35.92%,44.69%and61.42%atthedosesof100,200and400,respectively)andtopically
(32.86%and36.32%atthedosesof5%and10%)treatedgroupsascomparedtodiabeticcontrol.However,theorally
treatedgroupsshowedmoresignificanteffectthanthetopicallytreatedgroups.Moreover,oraladministrationofE.hirta
ethanolicextractsignificantlyreducedthebloodglucoselevelsindiabeticwoundrats(p<0.01)onday8andday16as
comparedtothediabeticwoundcontrol(p<0.01).Ontheotherhand,topicalapplicationofE.hirtadidnotinfluence
thebloodglucoselevelsindiabeticrats(p>0.05).Italsodemonstratedasignificantdecreaseintheplasmalevelsoflipid
malondialdehydeandnitricoxide.Theresultsofbiochemicalparameterswerefurthersupportedbythehistopathological
changesofdifferentorgans(liver,pancrease,kidney,heartandskinfromwoundarea)whichwereevidencedthrougha
decreaseininflammatorycellinfiltration.
(Continuedonnextpage)
*Correspondence:[email protected]
1DepartmentofPharmacy,PrimeasiaUniversity,Dhaka1213,Bangladesh
Fulllistofauthorinformationisavailableattheendofthearticle
©TheAuthor(s).2017OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
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Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page2of14
(Continuedfrompreviouspage)
Conclusion:The present study demonstrates that E. hirta whole plant extract promotes healing of wounds
more significantly as compared to diabetic control rats, where healing is otherwise delayed.
Keywords: Euphorbia hirta, Wound healing, Alloxan, Nitric oxide, Malondialdehyde,
Background and anti-inflammatory activities [11]. Preliminary phyto-
Wound may be defined as a loss or breaking of cellular chemicalscreeningofethanolicextractofEuphorbiahirta
and anatomic or functional continuity of living tissue [1] shows the presence of plant tannins, flavonoids, alkaloids,
which is generally classified according to the depth of tis- cardiac glycosides along with other anti-oxidants and ab-
sue loss. Wounds are recognized as one of the major senceofsaponin,cyanogenicglycosides[14].
problemsindevelopingcountriesresultingseverecompli- In traditional medicine, herbal-based drugs have been
cations in many cases that leads to high cost for therapy reported to exhibit curative value for various disorders
[2]. The healing process generally occurs in three stages [15].Formanyyears,plantshavebeenusedastheremedy
including inflammation, proliferation, and remodeling de- for various skin and dermatological disorders especially
pending upon the extent of damage and the host’s ability cut, wounds, burns etc. [16]. Although distinct treatment
to repair tissue [3]. Improper wound healing control may options are available (analgesics, antibiotics, anti-
give rise to diabetic foot ulcer or amputation in extreme inflammatory drugs etc) for wound healing management,
cases[4].AccordingtotheWHOreport,diabetesissetto most of these therapies are responsible for undesirable
elevate from 171 million to 366 million worldwide by the side effects [17]. Herbal medicines play promising role in
year 2030 [5]. It is believed that the main reason for the wound management by facilitating disinfection, debride-
increased rate in morbidity and mortality of diabetes is ment and provision of suitable environment for the
due to the development of macro- and micro-vascular natural healing process [18]. These medications are less
complications which include failure of the wound healing toxic and provide fewer side effects compared to that of
process [6]. Poorly repaired wound may give rise to se- conventionalmedications.Therefore,itisimportanttoes-
quence of events involving inflammation, proliferation tablish a scientific validation for the medicinal effect of
and migration of different cell types [7]. However, recov- plantsastraditionalmedicineinwoundhealing[2].
eryoftheearlyphaseofwound(inflammatoryphase)will The evidence of antidibetic properties of E. hirta have
greatly influence the overall integrity of the healing already been reported in the previous study [19]. Moreover,
process[8].Formerstudiesshowedthathighglucosecon- due to the presence of flavonoid compounds in the whole
centration leads to formation of sugar-derived substances plant extract it was found to induce anti-inflammatory and
called advanced glycation end products (AGEs) that in- wound healing effects [20, 21] which lead us to assess the
hibitthewoundhealingprocessbydelayinginflammatory diabetic wound healing activity of E. hirta. Hence, the
andproliferationphasesoftheprocess[6].Thiseventually presentworkinvolves evaluation ofwoundhealing property
leads to generation of free radicals resulting imbalance in in alloxan induced diabetic animals by oral and topical ad-
the level of free radicals and antioxidants which induces ministrationofethanolicextractofE.hirtawholeplant.
oxidative stress and tissue damage followed by delayed With regards to the above mentioned facts, the
wound healing [9]. Therefore, elimination of ROS (react- present study was therefore, undertaken to evaluate the
ive oxygen species) could be a significant strategy in wound healing property in alloxan induced diabetic ani-
healingwounds[10]. mals by oral and topical administration of ethanolic
Euphorbia hirta linn. (Family: Euphorbiaceae, Species: extract ofE.hirtawholeplant.
Euphorbia hirta) is a small, erect or ascending annual
herb reaching up to 50 cm, with hairy stems. It is known Methods
as baridudhi in Hindi and barokhervi in Bengali [11]. Plantmaterial
Euphorbia hirta linn. is an official drug included in Whole plants of Euphorbia hirta were collected on May,
African pharmacopeia of 1985 [12]. E. hirta is distributed 2015 from the road side (Road 83, Gulshan 2, Dhaka)
throughout the hotter parts of India and Australia where situated in front of Saudi Arabian Embassy in Dhaka,
it is often found in waste places along the roadsides [13]. Bangladesh. Authentication of Euphorbia hirta was done
In traditional Indian medicinal systems, leaves of Euphor- from Bangladesh National Herbarium and the voucher
bia hirta used in the treatment of coryza, cough, asthma, specimen(authenticationnumber: 43,100) was deposited
bronchial infections, bowel complaints, helminthic infes- in the herbarium for future references. Collected plants
tations, wounds, kidney stones and abscesses [11]. The were washed properly and then the leaves were separated
aqueous extract exhibits anxiolytic, analgesic, antipyretic, from the plant and other contamination, separated leaves
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page3of14
ware then Shade dried. The dried plant parts were then light-dark cycle, and were allowed to acclimatize for one
grinded into coarse powder materials by milling from week before the experiments. They were given free ac-
Pharmacognosy & Phytochemistry Lab of Primeasia Uni- cess to standard laboratory animal feed and water ad
versityinordertobeusedforexperiment. libitum. The rats were fasted over night before the ex-
periment. All surgery was performed under isoflurane
Drugsandchemicals (5% in 100% oxygen) anesthesia, and all efforts were
Povidone iodine ointment was obtained from the pharma- made to minimize suffering. Animal care and research
ceutical industry Eskayef Bangladesh Limited. Alloxan protocols were based on principles and guidelines ap-
monohydratewasobtainedfromSigmaAldrichChemicals, proved by the Guide for the Care and Use of Laboratory
Germany. All other chemicals were obtained from Merck Animals (NIH publication No: 85–23, revised in 1985)
(Darmstadt,Germany)andwereofanalyticalgrade. [27]. The prior approval for conducting the experiments
on rats was obtained from the Departmental Ethics
Extractionprocedure Committee of Biomedical Research Center Dhaka
Extraction was done by Macration, About 200 g of pre- University(Refno. DPT/BMRC/2015–2016/313).
pared coarse powder of leaves of Euphorbia hirta was
soaked by 95% Ethanol (600 ml) in water in a conical Acuteskinirritationstudyofointmentformulation
flask and plugged with cotton and then covered with The ethanolic extract of E. hirta was evaluated for anti-
aluminium foil for seven days with constant stirring. inflammatory activity that was performed on animals by
After seven days the preparation was filtered and filtrate usingastandardprotocol[21].Ondoingthis,onedayprior
was collected for the purpose of preparing extract. The to the experiment the animals were isolated in order to
filtrate was evaporated by rotary evaporator at 78.37 °C avoid contact with other rats and their dorsal hairs at the
and then the remaining part was kept in normal air for back were removed. One square cm area of whole and
few days to evaporate any remaining solvent. The resi- wounded skin of animals was subjected to different con-
due of Euphorbia hirta (39 g, 19.5%) was then collected, centrationsof50mgOintmentandtheywereobservedfor
weighedandstored inaclose container. seven days to identify any signs of oedema and erythema.
0.8%formalinsolutionwasusedasstandardirritant.
Phytochemicalanalysis
Plants contain many chemical constituents which are Inductionofseverediabetes
therapeutically active or inactive like carbohydrates, tri- The animals were injected by intraperitoneal route with
terpenoids, alkaloids, glycosides, tannins, flavonoids, es- a single dose of Alloxan monohydrate (120 mg/kg, body
sential oils and other similar secondary metabolites. weight) in normal saline [28]. After 3 days Fasting blood
Different qualitative chemical tests were performed for glucose level was measured by Accu chek glucometer to
the present work to establish the profile of ethanolic ex- confirm the diabetic status of the animals. The animals
tract for its chemical composition. Qualitative phyto- showing diabetes (Blood glucose level > 200 mg/dL) was
chemical analyses were done using the standard selected forwoundhealingstudy usingstandard.
procedures [22, 23]. The total phenolic content (TPC) of
Ethanolic Extract of Euphorbia hirta was determined Excisionwoundmodel
using the Folin-Ciocalteu reagent [24] and total flavon- All surgical affairs were conducted under sterile condi-
oid content (TFC) was determined by the method of tion. The rats were anaesthetized prior to development
Raoetal. (2010) [25]. of the wounds and hairs from the dorsal thoracic central
region of anaesthetized rats were removed followed by
Preparationofointment marking the area of the wound at the back of the ani-
Hydrophobic ointments of povidone iodine and Euphorbi mals [29]. The wound was created along these markings
hirtawas preparedbystandardmethod [26]. Detail ofin- by using toothed forceps, a surgical blade and pointed
gredients for the formulation of ointment from E. hirta scissors. The thickness of the excision wound was
leavesispresentedinTable1. 2.5 cm in width (circular area 4.90 cm2) and 0.2 cm
depth which left open [30, 31]. The measurements of
Pharmacologicalevaluation the wound areas of the excision wound model were
Experimentalanimal taken on 1st, 4th, 8th, 12th and 16th day following the
Female Swiss albino rats weighing between 180 to 200 g initial wound using transparent paper and a permanent
were used. The rats were collected from Pharmacy De- marker. The recorded wound areas were measured by
partment of Jahangirnagar University. They were housed tracing the wound margin on graph paper. The wound
in polypropylene cages in groups of six rats per cage and contraction was calculated as percent reduction in
were keptin aroom maintained at 25 ± 2°C with a12 h woundarea givenintheformulabelow [32].
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page4of14
Table1PreparationofmedicatedformulationswithethanolicextractofE.hirtaleaves
Formula Ingridients Amount
5%w/wointmentPreparation Povidoneiodine/EthanolicExtractofEuphorbiahirtawholeplants. 5g
Petroleumjelly 70g
Cetostearylalcohl 10g
PEG6000 5g
Liquidparaffin 10g
Methylparaben 1Drop
Total=100g
10%w/wointmentPreparation Povidoneiodine/EthanolicExtractofEuphorbiahirtaleaves 10g
Petroleumjelly 65g
Cetostearylalcohl 10g
PEG6000 5g
Liquidparaffin 10g
Methylparaben 1Drop
Total=100g
healed area Collectionofbloodsample
%Wound contraction¼ (cid:2)100
Total wound area After 16 days of the induction of diabetes, all animals of
ðHealed area¼originalwound area−presentwoundareaÞ different groups were fasted overnight before collection of
blood by retro orbital puncture into heparinized tubes
Epithelialisation time (day of fall of eschar and scar under light and under anesthetic condition. The plasma
area) was noted as a number of days after wounding wasobtainedbycentrifugationat5000rpmfor5min.The
[7]. Significance in wound healing of the test groups plasma was later on used for the determination of
has been derived by comparing healed wound area biochemical parameters like plasma nitric oxide and
with that of the control group within the respective malondialdehydelevel.
days. The experimental animals were divided into the
following groups and received the subsequent treat-
Evaluationofnitricoxideinplasma
ments accordingly:
As nitrite and nitrate are formed as end products of
reactive nitrogen intermediates, the measurement of
Groups Treatment
nitrite (free radicals) in rat plasma by using Griess
GroupI 5ml/kg/daydistilledwater,p.o.;(Normalcontrol,NC)
method since the reagent acts as a marker to determine
GroupII Alloxanmonohydrate(120mg/kg,bodyweight)induced the formation of NO (an oxygen derived radical) [33].
diabetes+Excisionwoundmodel(Diabeticwoundcontrol) 750 μl of each experimental sample was taken in a test
GroupIII Alloxanmonohydrate(120mg/kg,bodyweight)induced tube in duplicate and 750 μl of standard sodium nitrite
diabetes+Excisionwoundmodel+100mg/kg/dayEthanolic
extractofEhirta,p.o. (50, 25, 12.5, 6.25, 3.125 and 1.56 μM/ml) was placed in
different test tube, in duplicate. After this 750 μl of the
Group Alloxanmonohydrate(120mg/kg,bodyweight)induced
IV diabetes+Excisionwoundmodel+200mg/kg/dayEthanolic sulphanilamide solution (1% Sulphanilamide in 5%
extractofE.hirta,p.o. phosphoric acid) was dispensed to all the test tube
GroupV Alloxanmonohydrate(120mg/kg,bodyweight)induced containing experimental samples and nitrite standards
diabetes+Excisionwoundmodel+400mg/kg/dayEthanolic which were incubated for 5–10 min at room
extractofE.hirta,p.o. temperature. Following this 750 μl of the NED, 0.1% N-
Group Alloxanmonohydrate(120mg/kg,bodyweight)induced 1-Napthylethylenediamine dihydrochloride in distilled
VI diabetes+Excisionwoundmodel+5%PovidoneIodine
water solution, was dispensed in all the test tube. The
ointment
standard and samples were incubated again for 5–10 min
Group Alloxanmonohydrate(120mg/kg,bodyweight)induced
VII diabetes+Excisionwoundmodel+5%Ointmentof at room temperature. A purple/magenta color appeared
ethanolicextractofE.hirta immediately. Absorbance was measured within 30 min in
Group Alloxanmonohydrate(120mg/kg,bodyweight)induced an ELISA plate reader with a filter of 520 nm and was
VIII diabetes+Excisionwoundmodel+10%Ointmentof expressed as μmol/ml. NO concentration was calculated
ethanolicextractofE.hirta.
byusingstandardcurveequation.
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page5of14
Evaluationofmalondialdehyde(MDA)inplasma Table2PreliminaryphytochemicalanalysisofE.hirtaleaves
Malondialdehyde, a naturally occuring end product of extract
membrane lipid peroxidation, is one of the most Phytoconstituents EthanolicextractofE.hirta
frequently used biomarker for free radical mediated Carbohydrates +
damage [34, 35]. In the present study, plasma MDA level
Saponins +
of rats was measured by TBARS (Thiobarbituric Acid
Alkaloids +
Reacting Substances) method [36]. 1.5 ml of 0.67%
Glycosides -
thiobarbituric acid, 1.5 ml of 20% trichloroacetic acid and
700μl of phosphatebuffer, (pH 7.4)wereadded to300μl Terpenoids +
of serum and mixed followed by incubation in a water Steroids +
bath at 90 °C for 30 min. The reaction was stopped by Flavonoids +
dipping the test tubes into ice for 10 min. Samples were
PhenolicsandTannins +
centrifuged at 3000 rpm. The supernatant was removed
Anthraquinone -
and absorbance was measured spectrophotometrically at
Coumarins +
535 nm in a 1 cm cuvette. The concentration of MDA
wascalculatedbasedontheabsorbance. +=Present,-=Absent
Histopathology R2 = 0.9823. The content of total flavonoids in dry
Forhistopathologicalexamination,theratsweresacrificed extracts was estimated and expressed as quercetin
after 16 days before which blood was collected to obtain equivalents (QE) per gram of dry extracts. Total
serumandexcisedimmediately.Organs(heart,lung,liver, flavonoids content of EEH was 153.88 ± 2.15 mg QE/g
kidney,pancrease)andaspecimensampleoftissue(5μm of dry extract. The standard curve equation of
thick) from the healed wound is isolated from each quercetin was y = 0.0043× + 0.3437 with a correlation
experimental group of rats and fixed in 10% buffer coefficient of R2 = 0.97.
formalin for histopathological examination. After that
histopathological evaluation of these organs and skin Acuteskinirritationstudy
samplesweredoneinEximBankHospital,Departmentof All ointment formulations did not produce any skin
histopathology,Dhaka,Bangladesh. irritation,i.e.,erythemaandedemaforaboutaweekwhen
Statisticalanalysis
The values are represented as mean ± S.E.M, and
statistical significance between treated and control
groups was analyzed using one-way analysis of variance
(ANOVA), followed by Dunnett’s test where P < 0.05
wasconsideredstatistically significant.
Results
Preliminaryphytochemicalanalysis
The traditional use of the species was scientifically
validatedthrough the identificationof the phytochemicals
responsible for their use in indigenous systems of health
care. The result of qualitative chemical analysis of the
ethanolicextractofE.hirtaistabulatedinTable2.
Quantitativeevaluationfortotalpolyphenolicsand
flavonoidcontents
The plant is rich in phenolics, alkaloids, steroids,
terpenoids and flavonoids as shown by preliminary
Fig.1PhotographicrepresentationofeffectofE.hirtaointmentat
phytochemical screening. The content of total phenols
thedifferentconcentrationsoverskinafter7dayswherea=Group
in dry extracts was estimated and expressed as gallic 1:Aqueoussolutionof0.8%formalin,erythemaandedemaskinrash
acid equivalents (GAE) per gram of dry extract. Total andinflammationwasobserved.b=Group2:50mgof5%w/w
phenolic content of was 585 ± 2.18 mg GAE/g of dry ethanolicextractofE.hirtawholeplantointment,nolesionoccur.
c=Group3:50mgof10%w/wethanolicextractofE.hirtawhole
extract. The standard curve equation of gallic acid was
plantointment,nolesionoccur
y = 0.003× + 0.1559 with a correlation coefficient of
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page6of14
Fig.2PhotographicrepresentationofeffectofE.hitaonthewoundcontractionattheendofthestudyshowingwoundhealingafteroraland
topicaltreatmentattheendofthestudy.a:GroupII=Diabeticwoundcontrol,b:GroupIII=Diabeticwoundcontrol+100mgethanolicextract
ofE.hirta,c:GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,d:GroupV=Diabeticwoundcontrol+400mgethanolic
extractofE.hirta,e:GroupVI=Diabeticwoundcontrol+5%Povidoneiodine,f:GroupVII=Diabeticwoundcontrol+5%ethanolicextractofE.
hirta,g:GroupVIII=Diabeticwoundcontrol+10%ethanolicextractofE.hirta
Table3EffectofethanolicextractofE.hirtawholeplantonexcisionwoundinalloxaninduceddiabeticrats
Group/ Woundarea(cm2) %ofwoundcontractionattheend
Postwounding ofthestudy
days
1stday 4thday 8thday 12thday 16thday
GroupI N/A N/A N/A N/A N/A N/A
GroupII 4.90±1.13 4.90±1.20 4.85±0.88 4.72±0.89 4.60±0.93 6.12
GroupIII 4.90±1.11 4.83±1.05† 4.58±1.17† 3.76±0.59† 3.14±0.36† 35.92
GroupIV 4.90±1.03 4.70±0.91† 4.50±0.86† 3.23±0.65† 2.71±0.40* 44.69
GroupV 4.90±1.10 4.58±1.07† 4.16±0.88† 2.48±0.33* 1.89±0.35** 61.42
GroupVI 4.90±1.12 4.51±1.01† 4.35±0.76† 3.26±0.33† 2.95±0.54* 39.79
GroupVII 4.90±1.16 4.83±1.21† 4.55±0.73† 3.92±0.58† 3.29±0.37† 32.86
GroupVIII 4.90±0.88 4.75±1.03† 4.47±0.68† 3.41±0.56† 3.12±0.49† 36.32
EachvalueisMean±S.E.M(n=3).(*)indicatesstatisticallysignificantdifferencefromthediabeticwoundcontrolusingANOVA,followedbyDunnett’smultiple
comparisontest(**p<0.01).(†)indicatesstatisticallynosignificantdifferencefromthediabeticwoundcontrolusingANOVA,followedbyDunnett’smultiple
comparisontest(p>0.05).GroupI=normalcontrol,GroupII=Diabeticwoundcontrol,GroupIII=Diabeticwoundcontrol+100mgethanolicextractofE.hirta,
GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,GroupV=Diabeticwoundcontrol+400mgethanolicextractofE.hirta,GroupVI=Diabetic
woundcontrol+5%Povidoneiodine,GroupVII=Diabeticwoundcontrol+5%ethanolicextractofE.hirta,GroupVIII=Diabeticwoundcontrol+10%ethanolicextract
ofE.hirta
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page7of14
Fig.3Histopathologicalchangesintheskin;a:GroupI=Normalcontrol,b:GroupII=Diabeticwoundcontrol,c:GroupIII=Diabeticwound
control+100mgethanolicextractofE.hirta,d:GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,e:GroupV=Diabetic
woundcontrol+400mgethanolicextractofE.hirta,f:GroupVI=Diabeticwoundcontrol+5%Povidoneiodine,g:GroupVII=Diabeticwound
control+5%ethanolicextractofE.hirta,h:GroupVIII=Diabeticwoundcontrol+10%ethanolicextractofE.hirta.Eachgroupwasassessedat
400Xmagnification,scalebar:40μm
applied over the skin. However, 0.8% formalin group contraction of E. hirta whole plant extract in alloxan
showedsevererednessandedemaasshowninFig.1. induceddiabeticratsareshowninTable3.
The statistical analysis showed that reference drugs and
WoundhealingeffectofE.hirtaondiabeticexcision extract (200 and 400 mg/kg, p.o) caused a significant
woundmodel contraction of the wound as compared to diabetic wound
Oral administration (100, 200 and 400 mg/kg, p.o.) and control group ((p < 0.5 p < 0.01)) at the end of the
topical application (5% and 10% ointment) of E. hirta study period. There was significant increase in wound
wholeplantextractpromotesthecontractionofwoundin contraction observed with the oral administration of E.
diabetic rats, when compared to diabetic control (Fig. 2). hirtawholeplantextractatthedosesof200and400mg/
The percentage of wound contraction on 16 day of kg, p.o. in contrast with diabetic control group (p < 0.05
observation showed that in diabetic control it was 6.12%. and p < 0.01, respectively). However, oral administration
Intheorallytreatedgroupsitwasfoundtobe35.92,44.69 of E. hirta whole plant extract at the higher dose
and 61.42% at different doses (100, 200 and 400 mg/kg). promotedwoundcontractionbetterthanpovidoneiodine
Moreover, topical application of 5 and 10% of E. hirta treatedgroupbutnosignificantdifferencewasobserved.
whole plant extract the percentage of wound contraction
was 32.86 and 36.32%, respectively whereas in standard Histopathologicalevaluation
drug, povidone iodine ointment, it was 39.79%. EffectofEHEEonhistopathologyofskin,pancreas,kidney,
Observation showed that on 8th day onwards, the oral liverandheart
administration and topical applications of ethanolic
extractofE.hirtapromotedthewoundcontractionfaster Skin Histology of excision biopsy of skin wound at day
than diabetic control. Wound contraction progressed 16 showed healed skin structures with normal
much faster in orally treated groups than the topically epithelization, restoration of adnexa and fibrosis within
treated group and diabetic control group. The results of the dermis in the treated groups while the diabetic
wound healing activity and % (percentage) of wound control group lags behind treated group in formation of
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page8of14
Fig.4Histopathologicalchangesinthepancrease;a:GroupI=Normalcontrol,b:GroupII=Diabeticwoundcontrol,c:GroupIII=Diabetic
woundcontrol+100mgethanolicextractofE.hirta,d:GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,e:Group
V=Diabeticwoundcontrol+400mgethanolicextractofE.hirta,f:GroupVI=Diabeticwoundcontrol+5%Povidoneiodine,g:Group
VII=Diabeticwoundcontrol+5%ethanolicextractofE.hirta,h:GroupVIII=Diabeticwoundcontrol+10%ethanolicextractofE.hirta.Each
groupwasassessedat400Xmagnification,scalebar:40μm
the amount of ground substance in the granulation treated with EHEE at the dose of 400 mg/kg, p.o. did not
tissue, asobservedintissue sections. show any significant change of pancreas, when compared
E. hirta at the dose of 100 mg/kg, p.o. showed minimal with normal pancreas. However, ointment treated groups
amount of granulation tissue, large number of did not significantly reduced cellular damage and showed
mononuclearinflammatorycells,anddelayedepithelization mild to moderate multifocal mononuclear cell infiltration
whereas E. hirta at the dose of 200 mg/kg showing inthepancreas(Fig.4a-f).
moderate amount of granulation tissue, few number of
mononuclear inflammatory cells, and early epithelization. Liver Liver tissue of diabetic rat showed distortion in
E. hirta at the dose of 400 mg/kg showing healed skin the arrangement of cells around the central vein,
structureswithwell-formed,neartonormalepidermis,res- enlargement and thickening of the walls of veins,
toration of extensive fibrosis and collagen tissue within the capillaries, and development of fibrosis in the
dermis. However, orally treated groups showed minimal degenerated cells. EHEE (400 mg/kg, p.o.) treatment
amount of granulation tissue, mild to moderate number of almost restored the cellular arrangement of hepatocytes
mononuclearinflammatorycellsinfiltrationascomparedto around the central vein and reduced fibrosis. It also
thetopicallytreatedgroups(Fig.3a-f). helped to bring the blood vessels to normal condition.
On the other hand, ointment treated groups did not
PancreasNormalcontrolratexhibitednormalhistological effectively restored the cellular arrangement of
architecture. Group received alloxane without treatment, hepatocytes(Fig. 5a-f).
demonstrated cellular damage to the pancreatic acini and
islets,whichshowedpancreaticβ-celldamageanddegener- Kidney Morphological features of kidney remains
ation. Diabetic rats treated with the EHEE at the doses of normal in the control group like prominent glomeruli,
100 and 200 mg/kg exhibited degenerative and atrophic collecting ducts, tubules and ascending and descending
changes characterized by β-cell vacuolation and marked to loops. Aloxane-induced DM group showed presence of
moderate reduction in their size, respectively. The groups crystal deposition along with destructed glomeruli and
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page9of14
Fig.5Histopathologicalchangesintheliver;a:GroupI=Normalcontrol,b:GroupII=Diabeticwoundcontrol,c:GroupIII=Diabeticwound
control+100mgethanolicextractofE.hirta,d:GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,e:GroupV=Diabetic
woundcontrol+400mgethanolicextractofE.hirta,f:GroupVI=Diabeticwoundcontrol+5%Povidoneiodine,g:GroupVII=Diabeticwound
control+5%ethanolicextractofE.hirta,h:GroupVIII=Diabeticwoundcontrol+10%ethanolicextractofE.hirta.Eachgroupwasassessedat
400Xmagnification,scalebar:40μm
infiltration of red blood cells. Groups received the EHEE normal control group. However, oral administration of
orally demonstrated the reversal of these pathological E. hirta ethanolic extract significantly reduced the blood
destructions as apparent by the cell regeneration and glucose levels in diabetic wound rats (p < 0.01) on day 8
moderate to minimal infiltration of inflammatory cells. and day 16 as compared to the diabetic wound control
However, ointment treated groups did not significantly (p < 0.01). On the other hand, topical application of E.
reduced the pathological destructions showing inflam- hirta did not influence the blood glucose levels in
matory cellsinfiltration (Fig. 6a-f). diabetic rats(p>0.05) (Table4).
Heart Normal control group showed a regular
arrangement of cardiac myocytes. Alloxane-induced Estimation of free radicals (MDA and NO levels) In
DMratsdemonstrated alargeinfarctareawithpromin- contrast with the normal control group, diabetic
ent lymphocyte infiltration and fibrosis. Oral adminis- wound significantly increased free radicals (LPO and
tration of EHEE reversed these morphological changes NO level) levels in diabetic wound control group.
in a dose dependent manner and better result was ob- However, treatment with ethanolic extract of E. hirta
served in the orally treated group as compared to the showed significant decrease in the level of free
ointment treated groups (Fig. 7a-f). radicals, LPO and NO as compared with diabetic
wound control. Moreover, in oral administration of
EffectofethanolicextractofE.hirtaonbiochemical ethanolic extract of E. hirta showed significant
parameters decrease in all the groups (p < 0.01). On the other
hand, compared to the ointment formulation orally
FastingbloodglucoselevelThe administration of single administered ethanolic extract of E. hirta resulted in
dose of alloxan produced significant increase in the better inhibition on the elevated levels of MDA and
blood glucose levels in allthe groups as compared tothe NO has been shown in Fig. 8.
Tuhinetal.BMCComplementaryandAlternativeMedicine (2017) 17:423 Page10of14
Fig.6Histopathologicalchangesintherightkidney;a:GroupI=Normalcontrol,b:GroupII=Diabeticwoundcontrol,c:GroupIII=Diabetic
woundcontrol+100mgethanolicextractofE.hirta,d:GroupIV=Diabeticwoundcontrol+200mgethanolicextractofE.hirta,e:GroupV=Diabetic
woundcontrol+400mgethanolicextractofE.hirta,f:GroupVI=Diabeticwoundcontrol+5%Povidoneiodine,g:GroupVII=Diabeticwound
control+5%ethanolicextractofE.hirta,h:GroupVIII=Diabeticwoundcontrol+10%ethanolicextractofE.hirta.Eachgroupwasassessedat400X
magnification,scalebar:40μm
Discussion agent that will improve new tissueformation without pro-
On phytochemical screening, EHEE showed the presence ducing any deleterious side effect. Plant products are uni-
ofalkaloids, phenols, steroids, triterpinoids and flavonoids. versally accepted as potential wound healers for years due
A number of scientific studies have reported that certain to theirprofoundavailabilitywithminimaltoxicity, lackof
terpenoids, steroids and phenolic compounds (tannins, undesirablesideeffectsandtheirpotentialityascrudprep-
coumarins and flavonoids) have protective effects due to aration[39].Inourpresentstudyinvolvingexcisionwound
their antioxidant properties [37] These active constituents models in diabetic condition which included observation
support the process of wound healing by increasing the of different physical, histological and biochemical parame-
viability of collagen fibrils, by enhancing the strength of ters, indicated the wound healing activity in the ethanolic
collagen fibres either by augmenting the circulation or by extract of E. hirta. The study revealed significant increase
preventing the cell damage or by promoting DNA inbloodglucoselevelwasobservedinalloxaninduceddia-
synthesis [38]. The major constituents like flavonoids, betic rats. Alloxan cause immense reduction in insulin re-
triterpenoids and alkaloids present in EEH might play a lease by the destruction of beta cells of the islets of
major role in the process of wound healing, however langerhans and caused hyperglycemia. The adipose tissue
further phytochemical studies are needed to find out the and skeletal muscle are unable to uptake glucose from
active compound(s) responsible for wound healing serum in the absence of insulin so glucose conversion to
promotingactivity. fat and glycogen is blocked in the adipose tissue and skel-
Wound healing is associated with a series of etal muscles, resulting in increase of blood glucose level
physiological processes that include inflammation, cell [41]. In the current study, the healing of skin wounds was
proliferationandmigration,angiogenesis,matrixsynthesis, delayed significantly in diabetic rats, which may be corre-
re-epithelialization and formation of granulation tissues lated with the elevated glucose levels measured in the
[39].Thenewtissuesarecomposedoffibroblasts,collagen, blood plasma of these animals. As expected, the diabetic
edema and new small blood vessels [40]. A safe treatment conditions of the rats favored in delaying the wound heal-
for wound healing should ideally consider a therapeutic ingprocessbyabnormalphysiologicalresponse.Significant