Journal of Ethnopharmacology 131 (2010) 443450

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Healing fuwound ice

MariamFaculty of Scien 5113

a r t i c l

Article history:Received 3 FebReceived in reAccepted 6 JulAvailable onlin

Keywords:Rosmarinus ofDiabetes mellitusWound healingExcision woundHistological analysis

s (Rosresene aerness rs of twn of25

wounds were visually observed; blood glucose level, body weight, regenerated granulation tissue weightand the percentage of wound contraction were measured. On days 6 and 15 after wounding, the animalswere sacriced and the histology of wound area was examined.Results: Signicant positive differences (p

444 M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450

ing skin diseases such as hair loss, infections and healing ofwounds(Abu-rmaileh and A, 2000; Pieroni et al., 2004; Heinrich et al.,2006).

Healing of wounds is considered necessary for the removal ofdamaged tias to restorceral defectwound heainteraction(ECM) molecoagulationMasters et a

Diabeteshealth proband economneous wounand is causof cytokinebial infectioHirsch et al.plications ldeformitiesdevelopmebe complic(Lerman etthreateningcancer andsynthetic r2004).

Healingproblem fofore a new etreatment sis the use oexplore newmanageme

The poteRosmarinusnew medicno studies for extensivout to assesofcinalis ohealing pro

2. Materia

2.1. Plant c

Rosmarinal-Bayt Unithe herbwalogical Sciespecimen (Nof BiologicaMafraq, Jord

2.2. Plant e

Essentiaaerial partsIn which, wmaterial toand condenwas collect

For aqueous extract preparation, fresh and cleaned aerial parts ofthe herb (100g) were grounded into solution with 300ml of dis-tilled water using an electric blender. After centrifugation of themixture for 10min at 4100 rpm, the supernatant was collected and

thrial oiat 4

ima

le BAhe anuk Ueniche ind carndaratize

iabet

e webeforitonAldin 0utivein the, wia w

of thebloodbeticmend

ound

r 24d w, S.Aerfo7). Bic mi0% etter)wing sny).cm oessinonitooun

ice g

rworoup

s nonessemic

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r 3 cssentssues and/or invaded pathogens from the body as welle the continuity and architecture of a cutaneous or vis-(Matsuda et al., 1998; Lerman et al., 2003). Cutaneousling is a complex process that involves coordinatedbetween a number of cell types, extracellular matrixcules, and growth factors in three overlapping phases:and inammation, proliferation and remodeling (Bohll., 2002; Eming et al., 2007).mellitus constitutes one of the most important publiclems due to its high prevalence and enormous socialic consequences (Silva et al., 2007). Delayed cuta-d healing is a chronic complication in diabetic patients

ed primarily by hyperglycemia, diminished expressions, oxidative stress, vascular insufciency and micro-ns (Lerman et al., 2003; Sivan-Loukianova et al., 2003;, 2008;Abu-Al-Basal, 2009). Several other diabetic com-ike neuropathy, nephropathy, atherosclerosis and footcontribute to the severity of the disease and in the

nt of chronic wounds in diabetic patients, that mightated leading to ulceration, necrosis and amputational., 2003; Hirsch et al., 2008). Diabetes mellitus is life-and becomes the third largest killer of humans aftercardiovascular diseases, even with the use of severalecent drugs for effective treatment options (Li et al.,

impairment of diabetic patients is still a serious clinicalr physicians worldwide due to unclear etiology; there-ra inwound healing research is required involving newtrategies to deal with this emerging issue. One of thesef medicinal plants as a source for natural products totherapeutic agents/tools intended for diabeticwounds

nt and treatment.nt antioxidant and ethnopharmacological properties ofofcinalis make it an excellent source for searching a

inal target as wound healing, since currently there areor the inuence of such herb on healing chronicwoundse burns. Therefore, the present study has been carrieds the efcacy of the ethnobotanical use of Rosmarinusn various parameters and stages of cutaneous woundcess in diabetic BALB/c mice.

ls and methods

ollection

us ofcinalis herb was collected in April, 2008 from Al-versity campus, Mafraq, Jordan. Taxonomic identity ofs conrmed at theHerbariumof theDepartment of Bio-nces, University of Jordan, Amman, Jordan. A vouchero. RMJD-4-008) has been deposited in the Departmentl Sciences, Faculty of Science, Al-al-Bayt University,an for future reference.

xtract preparation

l oil was extracted from 500g of fresh and cleanedof Rosmarinus ofcinalis by steam distillation method.ater was heated to produce steam that pass on the herbcarry the most volatile chemicals. Following by coolingsation of the vapor mixture and the resulting distillateed and separated from the layer of water by extraction.

lteredessentvessels

2.3. An

Mafrom tYarmopathogwith tuse anon staacclim

2.4. D

Micminedintrape(Sigmasolvedconsecsalineon micglycemlevelswith aful diarecom

2.5. W

Aftethetizechemiewere pal., 200diabetwith 7diameline usGermaleast 1any drwas mafter w

2.6. M

Aftethree gbesideand/orBALB/cof expout tretreatedwater.intrapeday fopure eough Whatman lter paper no. 2 under vacuum. Bothl and aqueous extract were separately kept in closedC in a refrigerator for further use.

ls

LB/c mice 6 weeks of age (1820g) were obtainedimal house of the Department of Biological Sciences,niversity, Irbid, Jordan. Mice were kept under specic-free conditions, housed, fed and treated in accordanceternational guidelines principles of laboratory animale (Hedrich and Bullock, 2006). They were maintainedd pellet diet and water ad libitum for 2 weeks to bed prior to the investigation.

es induction

re weighed and their fasting blood glucose was deter-re inducing diabetes. After fasting for 18h, mice wereeally injected with 0.2ml of alloxan monohydraterich Canada, Oakville, Ontario, Canada), freshly dis-.90% saline at a dose of 100mg/kg body weight for 3days. Normal control mice only received physiological

e same way. Symptoms of diabetic mellitus appearedithin 1 week after the third injection of alloxan. Hyper-as conrmed by measuring fasting blood serum glucosemice through three consecutive determinations. Miceglucose level above 16.7mMwere considered success-model of hyperglycemia and used for experiments ased previously (Zhang et al., 2009).

ing

h of hyperglycemia conrmation, mice were anaes-ith 350g/kg body weight of chloral hydrate (Scharlu., Barcelona, Spain). Full-thickness excision skinwoundsrmed as described previously (Gutirrez-Fernndez etriey, hairs on the dorsum of diabetic and normal non-ce were shaved, the exposed skin area were cleanedhanol, and two full-thickness round skinwounds (4mmere created on the samemouse along the dorsalmiddle

terile biopsy punch equipment (Revolving punch pliers,The two wounds were separated from each other by atf unwounded skin. The wounds were left open withoutg material for the duration of the study. Wound healingred by taking photographs on 0, 3, 6, 10, and 15 days

ding.

rouping and treatment

und creation, diabeticmicewere randomly divided intos of twenty each, according to similar body weights,-diabetic normal group. Various doses of plant extractntial oil were preliminary tested for their tolerance ine to select the optimum dose intended for treatmentental animals. Group I: non-diabetic mice left with-ent as normal control. Group II: diabetic control miceh vehicle by intraperitoneal injection of 0.2ml distilledp III: diabetic mice treated with aqueous extract byeal injection of 0.2ml at a dose of 10% (v/v) once a

onsecutive days. Group IV: diabetic mice treated withial oil by topical application of 25l per each excision

M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450 445

Table 1Effect of Rosmarinus ofcinalis aqueous extract and essential oil on blood glucose level in alloxan-diabetic mice for 15 days after cutaneous full-thickness excision woundcreation.

Group Day after wounding

I 0.2II 0.2III 0.2IV 0.3

Data are expre contrat a dose of 10 , twice

* Statisticall** Statisticall

wound of thhoused,mawhich showthe study.

2.7. Body w

Body wediately befosamples (10mental micaccording tokept on iceusing microserum wasdase/peroxiBarcelona, S

2.8. Wound

Micewetreatment w6, 10 and 1sured fromby planimeof woundtion= (A0 is the area oet al., 2007)

2.9. Granul

The granthe excisionwet weight

2.10. Histol

Two micwounding.a depth susue, adjacena margin oflin, processthen embedwith hemattional sectiwith Massomaturation

tatis

ults. Comts t-onsid

ults

ood g

ica-indurintracte levuntrentialon d

dy w

y weicen ofor e

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anul

re weightat v

eightto the. Thediab0 3 6

Blood glucose level (mM)05.82 0.34 05.93 0.27 06.2522.36 1.00* 22.33 0.87* 23.1322.13 1.28* 22.57 1.04* 20.8823.48 1.03* 20.53 0.61* 15.98

ssed as means SEM for six mice in each group. I: non-diabetic control; II: diabetic% for 3 days); IV: essential oil-treated diabetic group (T.A. of 25l/excision woundy signicant when compared to non-diabetic control group (I) at p

446 M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450

Table 2Effect of Rosmarinus ofcinalis aqueous extract and essential oil on body weight of alloxan-diabetic mice for 15 days after cutaneous full-thickness excision wound creation.

Group Day after wounding

0 3 6 10 15

Body weight (g)I 20.87 0.26 20.77 0.20 21.03 0.1II 19.70 0.30* 18.22 0.24* 17.00 0.2III 19.38 0.28* 17.95 0.26* 17.93 0.1IV 19.47 0.28* 18.38 0.18* 18.73 0.1

Data are expressed as means SEM for six mice in each group. I: non-diabetic control; II: diabetic contrat a dose of 10% for 3 days); IV: essential oil-treated diabetic group (T.A. of 25l/excision wound, twice

* Statistically signicant when compared to non-diabetic control group (I) at p

M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450 447

Fig. 1. Hematowounding. (Aextract; (G anwound. Promiwhite arrow h

insulin secrdrate metaan improvetreated mic(Tsuneki etous studiesin crude exsuch as carverbenoneet al., 2004tionship ofeffect has binalis leaveserum insuxylin and eosin staining histological sections of cutaneous wound site obtained from theand B) Untreated diabetic wound; (C and D) untreated normal wound (non-diabetic); (d H) diabetic wound treated with Rosmarinus ofcinalis essential oil. Thicken and immanent and dilated blood vessels (b); immature granulation tissue area (g) showing invasioead). Scale bars: 50m (A, C, E, and G); 200m (B, D, F, and H).

etion. This later may recover the disorders in carbohy-bolism and/or intracellular glucose utilization, causedment in body weight gained nearly to normal in thee compared with the untreated diabetic mice (Table 2)al., 2004; Mallick et al., 2006; Zhang et al., 2009). Previ-conrmed the presence of powerful antioxidant agentstracts and essential oil of the aerial part of the herbnosol, rosmanol, epirosmanol, carnosic acid, borneol,and rosmarinic acid (Calabrese et al., 2000; Angioni; Ramrez et al., 2004; Moreno et al., 2006). The rela-antioxidant potential of the herb and its hypoglycemiceen reported. The ethanol extracts of Rosmarinus ofc-s signicantly lower blood glucose level and increaselin concentration in alloxan-induced diabetic rabbits,

suggestingand activat

Woundskin cells torepair damaoccur in thrproliferatioinammatiized by plathe damagthe woundincluding resis of debrigrowth factcontrols and Rosmarinus ofcinalis treated BALB/c mice on day 6 afterE and F) diabetic wound treated with Rosmarinus ofcinalis aqueousture epidermis (e) with debridement crust overlying the area of then of inammatory cells (neutrophill: black arrow head; macrophage:

a capability of the extract to inhibit lipid peroxidatione antioxidant enzymes (Bakirel et al., 2008).healing is a complex natural regeneration process ofminimize or eliminate scarring as well to help heal andge. The normal subsequent events of cutaneous healingee overlapping phases: coagulation and inammation,n and remodeling (Eming et al., 2007). Coagulation andon begin immediately after injury and are character-telet aggregation to control excessive blood loss fromed vessels and the inux of inammatory cells intosite. These cells play multiple roles in wound healing,lease of protease for wound debridement, phagocyto-s and bacteria, and secretion of various cytokines andors.Which, in turn, causemigration and division of cells

448 M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450

Fig. 2. Hematoxylin and eosin staining histological sections of cutaneous wound site obtained from the controls and Rosmarinus ofcinalis treated BALB/c mice, revealingepidermal and dermal architecture of the diabetic wounds on day 15 after wounding. (A) Untreated diabetic wound; (B) untreated normal wound (non-diabetic); (C) diabeticwound treated with Rosmarinus ofcinalis aqueous extract; (D) diabetic wound treated with Rosmarinus ofcinalis essential oil. Epidermis (e); dermis (d); granulation tissue(g); blood vessels (black arrow head). Scale bar: 50m (AD).

involved inthis phase,newly formmacrophagextracellulatissue integever, the remand reorgan

lls n

marihe wnt aing,s 3 anwas

Fig. 3. Massonarchitecture owith Rosmarinbroblasts (blathe proliferative phase (Stipcevic et al., 2006). Duringangiogenesis, collagen deposition, epithelialization anded granulation tissue, consisting of endothelial cells,es, broblasts and the components of a new provisionalrmatrixbegin tocoverandll thewoundarea to restorerity (Midwood et al., 2004; Eming et al., 2007). How-odeling phase of repair involves collagen cross linking

ization, evolution of granulation tissue into scar tissue

and ce2005).

Roserate tdifferewound(Tabletial oiltrichrome staining histological sections of cutaneous wound site obtained from the conf late granulation tissue on day 15 after wounding. (A) Untreated diabetic wound; (B) uus ofcinalis aqueous extract; (D) diabetic wound treated with Rosmarinus ofcinalis esck arrow head); inammatory cells (white arrow head). Scale bar: 200m (AD).o longer needed are removed by apoptosis (Rai et al.,

nus ofcinalis aqueous extract and essential oil accel-ound healing in diabetic BALB/c mice by inuencing

spects of the healing process at various days afterunder the conditions of the present investigationd 4 and Figs. 13). However, the healing effect of essen-more effective than that observed with the extract.trols and Rosmarinus ofcinalis treated BALB/c mice, revealing dermalntreated normal wound (non-diabetic); (C) diabetic wound treated

sential oil. Deposition and arrangement of collagen bers/bands (c);

M.A. Abu-Al-Basal / Journal of Ethnopharmacology 131 (2010) 443450 449

Potential healing of Rosmarinus ofcinalis was high-lighted byreduced inammation and wound debridement (Fig. 1), closureof the wound area due to rapid wound contraction (Table 4),full-thickness epidermal regeneration and organization (Fig. 2),increase inmaturationlular matrixand Figs. 2normal cutthat appearcapability othe impaireuntreated dinammatioThis could binammatoabnormalitimacrophagure in the rapoptosis (In additionof growth fin diabetesplete epitheleading to iand scarcelLoukianovaEming et al

Healingon the basiOluwatnyi eantioxidantthe herb threported thdisrupted aing the proacute inamfactors thatlonged, as tmatrix synt2007; Gutiicals at or arhealing thromatrix elemthe herb mation and tisdepositiongranulation

The mainorganic exttial effect onresponse anpolyphenol(Calabrese eTakaki et aloil depend odistilled (Aet al., 2009produce vaoils, these pare - andand limoneever there awith the napinene, camal., 2001; L

species chemotypes has a different essential oil composition andhence, adifferent rangeof therapeutic benets (Celiktas et al., 2005;Sotomayor et al., 2009). In this study, a representative sample ofRosmarinus ofcinalis was harvested from different locations in Al-

t Unitilizetypeto contialns, a

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Basal,. SeedJournaia, A.,nal ofaileh, B374yeh, Mn areahnophl, S.A.,onal Jon, S., 2, Jordi, M.Rinus orimenA., Baas, P.,antifuurnalT., Baktidiaban-disters,ts of nnd hee, V.,hemicthe wet weight of granulation tissue indicating rapiddue to high vascularization and deposition of extracel-elements involving collagen and broblasts (Table 3and 3). Furthermore, histological ndings revealed

aneous architecture of the treated wounds more thaned in the untreated non-diabetic wounds. The healingf Rosmarinus ofcinalis was evident when compared tod healing process of the wounds administered to theiabetic mice that showed a delay in coagulation andn phase of healing on day 6 after wounding (Fig. 1).e associated with an increase in the number of acutery cells, impaired leukocytes function, the metabolices of diabetes, inadequatemigration of neutrophils andes to the wound along with reduced chemotaxis, fail-esolution of inammation and a defect in neutrophilGutirrez-Fernndez et al., 2007; Hirsch et al., 2008)., reduced microcirculation and diminished expressionactors contribute to the disruption of wound healing, such as impairment in wound closure and/or com-lialization (Table 4 and Fig. 2), broblasts proliferationnadequate deposition of brous collagen tissue (Fig. 3)y formed granulation tissue (Table 3 and Fig. 3) (Sivan-et al., 2003; Lerman et al., 2003; Cianfarani et al., 2006;., 2007; Silva et al., 2007).potential of Rosmarinus ofcinalis could be explaineds of the powerful anti-microbial (Angioni et al., 2004;t al., 2004), anti-inammatory (Takaki et al., 2008) and(Calabrese et al., 2000; Moreno et al., 2006) effects ofat are well documented in the literature. It has beenat if a wound becomes infected, the normal healing iss the inammatory phase becomes chronic suppress-liferation phase of healing (Yates et al., 2007). Thematory during the early stages of wounding generatesare essential for tissue growth and repair. When pro-

he case in diabetes, preventing wound remodeling andhesis, leading to delay in wound closure (Eming et al.,rrez-Fernndez et al., 2007). The production of free rad-ound thewoundbedmaycontribute todelays inwoundugh the destruction of lipids, proteins and extracellularents (Calabrese et al., 2000). Due to these properties,y facilitate wound healing by reducing local inamma-sue destruction, increasing angiogenesis and collagenleading to improvements in both local circulation andtissue formation.bioactive compounds isolated from both aqueous and

racts of the aerial parts of Rosemary having poten-inhibiting pathogenic growth, reducing inammatoryd preserving viable tissue are mostly terpenoids and

s such as carnosol, carnosic acid and rosmarinic acidt al., 2000; Oluwatnyi et al., 2004; Moreno et al., 2006;., 2008). The quality and chemical composition of then how and where the plant was grown, harvested, and

ngioni et al., 2004; De Mastro et al., 2004; Sotomayor). When conditions cause the plants to permanentlyriations in the chemical composition of their essentiallants are known as chemotypes. The main constituents-pinene, 1, 8-cineole, camphor, verbenone, borneolne. Often all these chemicals can exist in the oil, how-re principal chemotypes (CT) of Rosmarinus ofcinalis,mes given by one of the main constituents, such as -phor/borneol, cineole, and verbenone CT (Pintore et

ahlou and Berrada, 2003). Each one of the Rosemary

al-Baysoil, ferchemofutureof esselocatio

5. Con

Essited suwhening varstudyuse ofever, futhe ramary cinterva

Conic

The

Ackno

Thetechniessentmenthelp inDepartversityout the

Refere

Abu-Al-skinies. P

Abu-Al-Linncan

Abu-RabJour

Abu-rm27, 5

Ali-Shtatiniaof Et

Al-Khalinati

Al-Quraback

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Angioni,CabrandL. Jo

Bakirel,of anallox

Bohl MaEffecwou

CalabresBiocversity campus due to differences in the quality of thersand irrigation thatmayrepresentat leastoneormore

s. Therefore, further studies should be followed in thenrm and assess the range of wound healing potentialoils of Rosemary chemotypes collected from differentt several time intervals, in Jordan.

ions

l oil from the aerial parts of Rosmarinus ofcinalis exhib-or signicant healing effect over the aqueous extract,ally applied on the wound of diabetic mice, by affect-stages of the healing process. The result of the presents scientic pharmacological evidence on the folkloricarinus ofcinalis aerial parts for healing wounds. How-r scientic verication is required to conrm and assessf wound healing potential of essential oils of Rose-

otypes collected fromdifferent locations, at several timeJordan.

interest

or has declared that no conict of interest exists.

gments

or acknowledge Was Al-bekearat for his support andssistance, Suhail El-Suhaile, who kindly prepared thel of Rosmarinus ofcinalis, Al-al-Bayt University, Depart-ological Sciences, Mafraq, Jordan, Ismail Zaid for thetographing histological sections, Yarmouk University,t of Biological Sciences, Irbid, Jordan andAl-al-Bayt Uni-fraq, Jordan, for providing necessary facilities to carryy.

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Healing potential of Rosmarinus officinalis L. on full-thickness excision cutaneous wounds in alloxan-induced-diabetic BAL...IntroductionMaterials and methodsPlant collectionPlant extract preparationAnimalsDiabetes inductionWoundingMice grouping and treatmentBody weight and serum glucose measurementWound contractionGranulation tissueHistologyStatistical analysis

ResultsBlood glucose levelBody weightGranulation tissueWound contractionHistological evaluation

DiscussionConclusionsConflict of interestAcknowledgmentsReferences