Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 386734 7 pageshttpdxdoiorg1011552013386734

Research ArticleThe Effect of Camellia sinensis onWound Healing Potential in an Animal Model

Fatemeh Hajiaghaalipour1 M S Kanthimathi1

Mahmood Ameen Abdulla2 and Junedah Sanusi3

1 Department of Molecular Medicine University of Malaya Centre for Proteomics Research (UMCPR) Faculty of MedicineUniversity of Malaya 50603 Kuala Lumpur Malaysia

2 Department of Biomedical Science Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia3 Department of Anatomy Neuroscience Research Group Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to M S Kanthimathi kanthiummcedumy

Received 14 March 2013 Accepted 4 June 2013

Academic Editor Chun Tao Che

Copyright copy 2013 Fatemeh Hajiaghaalipour et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Camellia sinensis (tea) is reported to have health benefits including the building of healthy skin This study evaluated the effectsof topical application of Camellia sinensis extract on the rate of wound closure and the histology of wound area A uniform area of200 cm in diameter was excised from the neck of adult male Sprague Dawley rats The animals were topically treated with 02mLof vehicle (CMC) Intrasite gel (positive control) or 200 and 400mgmL of extract Wounds dressed with the extract and Intrasitegel healed significantly earlier than those with vehicle Histological analysis of the wound area after 10 days showed that woundsdressed with the extract had less scar width when compared to the control The tissue contained less inflammatory cells and morecollagen and angiogenesis compared to wounds dressed with vehicle In this study Camellia sinensis showed high potential inwound healing activity

1 Introduction

Asource of active and efficientmedicinal agents has been pro-vided in nature Based on the usage of natural products intraditional medicine a large number of modern medicineshave been extracted from natural sources [1]

The use of herbal therapies for caring of wounds andinjuries has been popular since ancient civilisations Incontrast to only 1ndash3 of modern drugs being used for thetreatment of wounds and skin disorders almost one-third ofall traditional medicines in use are for this treatment [2]

Wounds are physical injuries that result in breakage ofthe skin Wound healing is a natural response to tissueinjury consisting of a multifactorial and complex cascade ofevents involving various cellular molecular and biochemicalprocesses and resulting in the healing of the wound and therestoration of the intact functional barrier [3 4] Inflamma-tion cell proliferation angiogenesis epithelialisation wound

contraction andmatrix remodeling is the sequence of severalbasic processes This multifactorial sequence of processesstarts from the moment of injury and continues for varyingperiods of time the time period depending on the extent ofthe injured area and the health status of the injured individual[5]

The wound healing process is generally categorised intothree integrated and overlapping phases the inflammatoryphase which consists of the establishment of homeostasisand inflammation the proliferative phase which consistsof granulation contraction and epithelialisation and theremodelling phase or resolution which eventually deter-mines the strength and appearance of the healed tissue [2]Studies have shown that several natural products containingactive compounds like triterpenes alkaloids flavonoids andother biomolecules promote the process of wound healing byinfluencing one or more phases of the healing process [4 6]Another study reported that medicinal plants affect the many

2 Evidence-Based Complementary and Alternative Medicine

phases of the healing process such as coagulation inflamma-tion fibroplasia collagenation epithelialization and woundcontraction [7]

Second only to water tea is one of the most consumedbeverages in the world Tea has been reported to contain anumber of chemical constituents with medicinal and phar-macological properties [8] Tea contains relatively high levelsof flavonoids including catechins and other polyphenolsThis antioxidant and free radical scavenging activity hasbeen suggested to be the cause of the various health benefitsassociated with tea drinkingThe tea plant Camellia sinensisis a member of theTheaceae family and white green oolongand black teas are produced from its leaves

White tea is an unfermented tea made from the budsand young tea leaves which are harvested once a year inthe early spring Unlike black and oolong teas white andgreen tea production does not oxidise the young tea leaves[9 10] White and green teas are produced by steaming freshleaves at high temperatures leaving the polyphenol contentintact but inactivating enzymes that would oxidise the teas[11] Oolong tea undergoes partial fermentation before dryingand has a taste and color somewhere between green andblack teas Black tea however undergoes fermentation whilegreen tea does not [12 13] Numerous in vitro and in vivostudies have reported beneficial health properties of tea andtheir phenolic compounds [14 15] However there has notbeen much attention given to the properties of white tea Inour ongoing studies white tea has demonstrated significantantioxidant and anticarcinogenic properties Therefore thepresent study was undertaken to evaluate the effectiveness oftopical application of the methanolic extract of Silver Needlewhite tea on the rate of wound closure and the histology ofthe wound area

2 Materials and Methods

21 Dressing Intrasite gel (purchased from the Universityof Malaya Medical Centre Pharmacy) was used as a pos-itive control Intrasite gel (trademark Smith and NephewHealthcare Ltd) is a colorless transparent aqueous gel con-taining 23 of a modified carboxymethyl cellulose polymertogether with propylene glycol (20) It functions as ahumectant and preservative When placed in contact with awound the dressing absorbs excess exudates and producesa moist environment at the surface of the wound with-out causing tissue maceration Intrasite gel an amorphoushydrogel gently rehydrates necrotic tissue and facilitatesautolytic debridement while loosening and absorbing sloughand exudates and thus clearing the way for effective woundhealing It is designed for wounds that are granulating andepithelialising It is also used to provide the optimum moistwound management environment during the later stages ofwound closure It is nonadherent and does not harm viabletissue or the skin surrounding the woundThis makes the useof Intrasite gel ideal for every stage in thewoundmanagementprocess [16]

Lignocaine HCL (2 100mg5mL) a local anaestheticwas purchased from the Experimental Animal House Fac-ulty of Medicine University of Malaya (Delta Veterinary

Laboratory PTY LTD NSW) A 1mL of lignocaine was injec-ted subcutaneously

22 Tea Extract Preparation Silver Needle white tea waspurchased from the localmarket Tea infusionswere preparedby placing 2 g of tea leaves in 100mL methanol at roomtemperature for 2 hours and filtered through Whatman filterpaper no 1 The resulting solvent extract was concentratedusing a rotary evaporator The concentrated extracts werestored at minus20∘C until further analysis

23 Experimental Animals Twenty-four healthy adult maleSprague Dawley rats 8 weeks old and weighing 200 to 250 gwere obtained from the Experimental Animal House Facultyof Medicine University of MalayaThis study was carried outaccording to the criteria outlined in the ldquoGuide for the Careand Use of Laboratory Animalsrdquo published by the NationalInstitutes of Health The rats were divided randomly into 4groups of 6 rats each Each rat was housed separately (onerat per cage) and the animals were maintained on standardpellet diet and tapwaterThe studywas approved by the EthicsCommittee for animal experimentation Faculty ofMedicineUniversity of Malaya Ethics no PM22112011FH (R)

24 Experimentally Induced Excision Wounds The ratswere inflicted with excision wounds as described byMughrabi et al [17] Briefly the animals were anaesthetisedwith 009mL of ketamine by im injection (30mgkg100mgmL) and 001mL of Xylazil by im injection(3mgkg 100mgmL) prior to creation of the wounds Theskin was shaved using an electrical clipper disinfected with70 alcohol and injected with 1mL of lignocaine HCl scinjection (2 100mg5mL) An area of uniform wound200 cm in diameter was excised using a circular stamp andsterile scissors from the depilated ethanol-sterilised napeof the dorsal neck of the 24 rats Incision of the muscle layerwas avoided during the procedure so that the tension of theskin would not be affected The wound area was measuredimmediately by placing transparent paper over the woundand tracing out the area This was subsequently placed ona 1mm2 graph sheet The area of the counted squares wascalculated in square millimeters and the area was recordedas described by Zahra et al [18]

25 Topical Wound Application Wounds of Groups I and IIanimals were topically treated with 02mL of 200 (high dose)and 100 (low dose) mgmL of a methanolic extract of whitetea in vehicle respectively Wounds of Groups III animalswere topically treated with 02mL Intrasite gel twice dailyas a reference standard control Wounds of Group IV ratswere topically treated with 02mL of vehicle carboxymethylcellulose (CMC) in normal saline (2) twice daily as controlgroup [19]

26 Estimation of Wound Healing (Wound Closure) Thewound closure area of each animal was assessed by tracing theoutline of the wound on days 5 and 10 after wounding surgeryand the wound closure rate was expressed as the percentage

Evidence-Based Complementary and Alternative Medicine 3

of wound area compared with that on postoperative dayas described previously [18] The wound closure rate wasexpressed as the percentage of wound area compared withthat on post-operative day that is the change in wound sizewas expressed as a percentage of the original wound size

contraction

=

Initial wound size minus Specific day wound sizeInitial wound size

times 100

(1)

27 Histological Evaluation of Healed Wounds Skin tissuesamples of all groups were immediately fixed after ani-mal dissection in 10 buffered formalin dehydrated andprocessed using a paraffin tissue processing machine forparaffin sectioning The wound tissues were cut 5 120583m thickperpendicular to the wound stained with standard haema-toxylin and eosin (HampE) and Massonrsquos trichrome (MT) andexamined using an Olympus BX60 light microscope andcaptured using anOlympus XC10 camera (Tokyo Japan)Theevaluated parameters were epithelialisation inflammatorycell infiltration fibroblast proliferation neovascularisationand collagen deposition which were assessed individuallyby using a numerical scale Stained sections were examinedunder a light microscope and were graded in a blind fashionon 3 slides per animal using the modified 0 to 4 numericalscale as described by other researchers [20 21] The scoreswere 0 for absence 1 for occasional presence 2 for lightscattering 3 for abundance and 4 for confluence of cells orfibers

28 Statistical Analysis Experimental results are presented asmeans plusmn SD of six animals in each group Statistical analysiswas performed by using SPSS (1800 version) statisticalsoftware The effect of extracts on the wound was analysedby one-way ANOVA 119875 values lt 005 were considered signi-ficant

3 Results

Figure 1 shows the macroscopic appearance of the excisedskin wound after surgery The macroscopic examination onday 10 after surgery is presented in Figure 2 As shown in thefigure the 3 groups (high-dose treated low-dose treated andIntrasite gel-treated that is Figures 2(a) 2(b) and 2(c) resp)showed a narrow scar at wound closure compared with thecontrol group that is wound dressed with placebo (CMC)Figure 2(d) which showed incomplete wound healing

The change in the wound size was expressed as a percent-age of thewound area on days 5 and 10 after surgery comparedwith the wound area on day 0 (100) in the differentgroups (Table 1) Wounds dressed with white tea extract orwith reference standard control showed considerable signsof dermal healing and significantly healed faster comparedto the group that received the placebo control treatment(CMC in normal saline) Throughout the experiment thepercentage of wound healing in the control (CMC) groupwassignificantly lower than that in the treated groups or that in

Figure 1 Excision skin wound (200 cm) on surgery day beforetreatment

Table 1 Effect ofmethanolic extract ofCamellia sinensis on percent-age () of wound healing in Sprague Dawley rats

Animalgroups Treatment

Percentage of wound healing(mean plusmn SD) on days after

surgery

Day 5 Day 10

Group 1 High dose200mgmL 619 plusmn 670a 977 plusmn 094a

Group 2 Low dose100mgmL 623 plusmn 399a 945 plusmn 112b

Group 3 Intrasite gel 603 plusmn 670a 954 plusmn 129b

Group 4 CMC in normalsaline 450 plusmn 198b 865 plusmn 240c

Mean values (119899 = 6) followed by different letters (a b and c) in a column aresignificantly different (119875 lt 005)

the group treated with Intrasite gel (standard) on day 5 aftersurgery (Table 1) The percentage of healing in the high dosegroup was significantly higher than those of the low-dose-treated group the reference standard group and the controlgroup on day 10 after surgery although the percentage ofhealing in the placebo (CMC) control group was significantlylower than those of the extract-treated groups and thereference standard control (119875lt005)Therewas no significantdifference between low-dose-treated groups and standardcontrol group on day 10 after surgery (119875 gt 005)The effect oflow dose was similar to that of the standard

The histology of wound area on day 10 after surgeryshowed that wounds dressed with the extract showed com-paratively less scar width at wound closure compared to thecontrol group (Figures 3 and 4) In all the groups with respectto the control group inflammatory cell infiltration was lessintense and the difference between the groupswas statisticallysignificant (119875 lt 005) In Groups I II and III there was nosignificant difference in inflammatory cell infiltration (119875 gt005) In extract-treated groups and the standard treatedgroup comparativelymore collagen andfibroblastswere seen

4 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

Figure 2 Macroscopic appearance of wound on day 10 after surgery (a) A 200mgmL of white tea treated group (b) 100mgmL of white teatreated group (c) Intrasite gel treated group and (d) control group (wound dressed with placebo CMC)

500120583m 500120583m 500120583m 500120583m

25120583m 25120583m 25120583m 25120583m

10120583m 10120583m 10120583m 10120583m

(G1) (G2) (G3) (G4)

The interface between normal skin and the regenerated woundBlood capillaries in the second row

FibroblastsCollagen

Inflammatory cells

Figure 3Histology of wound area stainedwith hematoxylin and eosin on day 10 after surgery Photomicrographs of wound tissues in differentmagnification are shown in the rows

(Figure 3) Proliferation of blood capillaries and angiogenesiswere significantly more pronounced in Groups I II and IIIwhen compared with group IV Figure 4 shows Massonrsquostrichome staining of the wound after 10 days In both groupsII (low-dose treated) and III (Intrasite gel treated) thepresence of collagen and fibroblasts is similar to Group I(high-dose treated) although the photomicrograph of GroupI shows a higher density of collagen andmore fibroblast cellsthough this was not significant (Table 2) The CMC-treatedgroup (Group IV) has significantly (119875 lt 005) less collagenless fibroblasts and less blood capillaries in comparison withthe other groups

4 Discussion

In this study an excision wound model was used for theassessment of the wound healing activity ofCamellia sinensis

specifically white tea Wound healing progression is com-prised of a systematic process of events starting from themoment of injury that is the inflammatory phase (the estab-lishment of homeostasis and inflammation) the proliferationphase (granulation contraction and epithelialisation) andfinally the remodeling phase which determines the strengthand appearance of the healed tissue [3 22]

The outcome of this study demonstrates that the woundcontracting ability of the white tea extract on an excisionwoundmodel was significantly greater than that of the vehicleitself

Histopathological studies of the wound healing processare normally used for evaluating the efficacy of pharma-cological products which promote and accelerate dermalskin substitutes The histological results of this study showthat the granulation tissue in tea extract-treated groups con-tained comparatively less inflammation and more collagen

Evidence-Based Complementary and Alternative Medicine 5

D

E

500 120583mD

E

500 120583m

D

E

500 120583m D

E

500 120583m

25 120583m 25 120583m 25 120583m 25 120583m

(G1) (G2) (G3) (G4)

Figure 4 Histological sections of healed wound on day 10 after-surgery stained with Massonrsquos trichrome Different magnifications of thewound are shown in the rows E epidermis D dermis The arrows in the upper row show the different levels of collagen depositionrepresented by the intensity of the green color

Table 2Themedian histopathologic scores of wound healing were determined in the extract-treated and control groups by using a modified0 to 4 numerical scale The scores were 0 for absence 1 for occasional presence 2 for light scattering 3 for abundance and 4 for confluenceof cells or fibres

Groups Epithelialisation Inflammatorycell infiltration

Fibroblastproliferation Neovascularisation Collagen deposition

G1 (high dose) 383 plusmn 041a 150 plusmn 055b 350 plusmn 055a 267 plusmn 052a 383 plusmn 041a

G2 (low dose) 317 plusmn 041a 183 plusmn 075b 317 plusmn 075a 200 plusmn 063a 333 plusmn 052a

G3 (Intrasite gel) 350 plusmn 055a 167 plusmn 052b 333 plusmn 082a 217 plusmn 075a 367 plusmn 052a

G4 (CMC control) 083 plusmn 041b 367 plusmn 052a 100 plusmn 000b 083 plusmn 075b 117 plusmn 041b

Data are expressed as mean plusmn standard error (119899 = 6) Different letters (a and b) in a column indicate significant difference (119875 lt 005)

and angiogenesis The histopathological examination of thewound sections in the control group exhibited a wide areaof ulceration (presumably containing fibrinous exudates) andinflammatory cells and a mild degree of vascularisation withcongestion in the dermis which indicate that healing wasnot complete It can be concluded that topical application ofthe tea extract at a high concentration (200mgmL) signifi-cantly increased the fibroblast growth collagen synthesis andthus the healing process by accelerating the rate of woundhealing

In the present study haematoxylin and eosin (HampE)staining was used (Figure 3) as it is the most commonmethod in histopathological studies Histological sectionsstained with haematoxylin and eosin (HampE) on day 10 after-surgery showed that wounds dressed with the extract showedcomparatively less scar width at wound closure compared tothe control group (Figure 3) The histology of wound areain Intrasite gel- and low-dose extract-treated groups showedsimilar results In both groups the extent of epitheliali-sation neovascularisation and fibroblast proliferation andthe presence of collagen are similar Histological analysisshowed that the intensities of the collagen (green colour) ofGroups I II and III are different but not significant (119875 gt005) Proliferation of blood capillaries and angiogenesis weresignificantly higher in Groups I II and III when comparedwith Group IV (119875 lt 005)

Although HampE staining is the most popular stainingmethod the stain is not able to differentiate importanthistopathological changes in the wound healing processsuch as collagen deposition An alternative staining methodmodified Massonrsquos trichrome staining (MT) was used todifferentiate the important morphological parameters forwound healing assessment The results of the MT staining inthis study confirm that the presence of collagen in Intrasitegel- and white tea extract-treated groups is significantlyhigher when compared to the control group As shown inFigure 4 for the results of MT staining (presented at twodifferent magnifications 2x and 40x) the intensity of thegreen color represents collagen density Fibroblasts producecollagen in skin which plays an important role in preserv-ing the anatomic integrity of wound healing MT stainingshowed differences but this was not significant for fibroblastproliferation collagen deposition and neovascularisation inIntrasite gel- and white tea extract-treated groups (119875 gt 005)However the difference in collagen deposition between thetreated groups mentioned above and in the CMC-treatedgroup is significant The inflammatory cells in Groups IndashIIIwere significantly less (119875 lt 005) than those in Group IV

The results showed a trend of progressive constructionof tissues surrounding the wound from day 5 onwards(Table 1) especially for the Intrasite gel- and tea extract-treated groups This was significantly different (119875 lt 005)

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

phases of the healing process such as coagulation inflamma-tion fibroplasia collagenation epithelialization and woundcontraction [7]

Second only to water tea is one of the most consumedbeverages in the world Tea has been reported to contain anumber of chemical constituents with medicinal and phar-macological properties [8] Tea contains relatively high levelsof flavonoids including catechins and other polyphenolsThis antioxidant and free radical scavenging activity hasbeen suggested to be the cause of the various health benefitsassociated with tea drinkingThe tea plant Camellia sinensisis a member of theTheaceae family and white green oolongand black teas are produced from its leaves

White tea is an unfermented tea made from the budsand young tea leaves which are harvested once a year inthe early spring Unlike black and oolong teas white andgreen tea production does not oxidise the young tea leaves[9 10] White and green teas are produced by steaming freshleaves at high temperatures leaving the polyphenol contentintact but inactivating enzymes that would oxidise the teas[11] Oolong tea undergoes partial fermentation before dryingand has a taste and color somewhere between green andblack teas Black tea however undergoes fermentation whilegreen tea does not [12 13] Numerous in vitro and in vivostudies have reported beneficial health properties of tea andtheir phenolic compounds [14 15] However there has notbeen much attention given to the properties of white tea Inour ongoing studies white tea has demonstrated significantantioxidant and anticarcinogenic properties Therefore thepresent study was undertaken to evaluate the effectiveness oftopical application of the methanolic extract of Silver Needlewhite tea on the rate of wound closure and the histology ofthe wound area

2 Materials and Methods

21 Dressing Intrasite gel (purchased from the Universityof Malaya Medical Centre Pharmacy) was used as a pos-itive control Intrasite gel (trademark Smith and NephewHealthcare Ltd) is a colorless transparent aqueous gel con-taining 23 of a modified carboxymethyl cellulose polymertogether with propylene glycol (20) It functions as ahumectant and preservative When placed in contact with awound the dressing absorbs excess exudates and producesa moist environment at the surface of the wound with-out causing tissue maceration Intrasite gel an amorphoushydrogel gently rehydrates necrotic tissue and facilitatesautolytic debridement while loosening and absorbing sloughand exudates and thus clearing the way for effective woundhealing It is designed for wounds that are granulating andepithelialising It is also used to provide the optimum moistwound management environment during the later stages ofwound closure It is nonadherent and does not harm viabletissue or the skin surrounding the woundThis makes the useof Intrasite gel ideal for every stage in thewoundmanagementprocess [16]

Lignocaine HCL (2 100mg5mL) a local anaestheticwas purchased from the Experimental Animal House Fac-ulty of Medicine University of Malaya (Delta Veterinary

Laboratory PTY LTD NSW) A 1mL of lignocaine was injec-ted subcutaneously

22 Tea Extract Preparation Silver Needle white tea waspurchased from the localmarket Tea infusionswere preparedby placing 2 g of tea leaves in 100mL methanol at roomtemperature for 2 hours and filtered through Whatman filterpaper no 1 The resulting solvent extract was concentratedusing a rotary evaporator The concentrated extracts werestored at minus20∘C until further analysis

23 Experimental Animals Twenty-four healthy adult maleSprague Dawley rats 8 weeks old and weighing 200 to 250 gwere obtained from the Experimental Animal House Facultyof Medicine University of MalayaThis study was carried outaccording to the criteria outlined in the ldquoGuide for the Careand Use of Laboratory Animalsrdquo published by the NationalInstitutes of Health The rats were divided randomly into 4groups of 6 rats each Each rat was housed separately (onerat per cage) and the animals were maintained on standardpellet diet and tapwaterThe studywas approved by the EthicsCommittee for animal experimentation Faculty ofMedicineUniversity of Malaya Ethics no PM22112011FH (R)

24 Experimentally Induced Excision Wounds The ratswere inflicted with excision wounds as described byMughrabi et al [17] Briefly the animals were anaesthetisedwith 009mL of ketamine by im injection (30mgkg100mgmL) and 001mL of Xylazil by im injection(3mgkg 100mgmL) prior to creation of the wounds Theskin was shaved using an electrical clipper disinfected with70 alcohol and injected with 1mL of lignocaine HCl scinjection (2 100mg5mL) An area of uniform wound200 cm in diameter was excised using a circular stamp andsterile scissors from the depilated ethanol-sterilised napeof the dorsal neck of the 24 rats Incision of the muscle layerwas avoided during the procedure so that the tension of theskin would not be affected The wound area was measuredimmediately by placing transparent paper over the woundand tracing out the area This was subsequently placed ona 1mm2 graph sheet The area of the counted squares wascalculated in square millimeters and the area was recordedas described by Zahra et al [18]

25 Topical Wound Application Wounds of Groups I and IIanimals were topically treated with 02mL of 200 (high dose)and 100 (low dose) mgmL of a methanolic extract of whitetea in vehicle respectively Wounds of Groups III animalswere topically treated with 02mL Intrasite gel twice dailyas a reference standard control Wounds of Group IV ratswere topically treated with 02mL of vehicle carboxymethylcellulose (CMC) in normal saline (2) twice daily as controlgroup [19]

26 Estimation of Wound Healing (Wound Closure) Thewound closure area of each animal was assessed by tracing theoutline of the wound on days 5 and 10 after wounding surgeryand the wound closure rate was expressed as the percentage

Evidence-Based Complementary and Alternative Medicine 3

of wound area compared with that on postoperative dayas described previously [18] The wound closure rate wasexpressed as the percentage of wound area compared withthat on post-operative day that is the change in wound sizewas expressed as a percentage of the original wound size

contraction

=

Initial wound size minus Specific day wound sizeInitial wound size

times 100

(1)

27 Histological Evaluation of Healed Wounds Skin tissuesamples of all groups were immediately fixed after ani-mal dissection in 10 buffered formalin dehydrated andprocessed using a paraffin tissue processing machine forparaffin sectioning The wound tissues were cut 5 120583m thickperpendicular to the wound stained with standard haema-toxylin and eosin (HampE) and Massonrsquos trichrome (MT) andexamined using an Olympus BX60 light microscope andcaptured using anOlympus XC10 camera (Tokyo Japan)Theevaluated parameters were epithelialisation inflammatorycell infiltration fibroblast proliferation neovascularisationand collagen deposition which were assessed individuallyby using a numerical scale Stained sections were examinedunder a light microscope and were graded in a blind fashionon 3 slides per animal using the modified 0 to 4 numericalscale as described by other researchers [20 21] The scoreswere 0 for absence 1 for occasional presence 2 for lightscattering 3 for abundance and 4 for confluence of cells orfibers

28 Statistical Analysis Experimental results are presented asmeans plusmn SD of six animals in each group Statistical analysiswas performed by using SPSS (1800 version) statisticalsoftware The effect of extracts on the wound was analysedby one-way ANOVA 119875 values lt 005 were considered signi-ficant

3 Results

Figure 1 shows the macroscopic appearance of the excisedskin wound after surgery The macroscopic examination onday 10 after surgery is presented in Figure 2 As shown in thefigure the 3 groups (high-dose treated low-dose treated andIntrasite gel-treated that is Figures 2(a) 2(b) and 2(c) resp)showed a narrow scar at wound closure compared with thecontrol group that is wound dressed with placebo (CMC)Figure 2(d) which showed incomplete wound healing

The change in the wound size was expressed as a percent-age of thewound area on days 5 and 10 after surgery comparedwith the wound area on day 0 (100) in the differentgroups (Table 1) Wounds dressed with white tea extract orwith reference standard control showed considerable signsof dermal healing and significantly healed faster comparedto the group that received the placebo control treatment(CMC in normal saline) Throughout the experiment thepercentage of wound healing in the control (CMC) groupwassignificantly lower than that in the treated groups or that in

Figure 1 Excision skin wound (200 cm) on surgery day beforetreatment

Table 1 Effect ofmethanolic extract ofCamellia sinensis on percent-age () of wound healing in Sprague Dawley rats

Animalgroups Treatment

Percentage of wound healing(mean plusmn SD) on days after

surgery

Day 5 Day 10

Group 1 High dose200mgmL 619 plusmn 670a 977 plusmn 094a

Group 2 Low dose100mgmL 623 plusmn 399a 945 plusmn 112b

Group 3 Intrasite gel 603 plusmn 670a 954 plusmn 129b

Group 4 CMC in normalsaline 450 plusmn 198b 865 plusmn 240c

Mean values (119899 = 6) followed by different letters (a b and c) in a column aresignificantly different (119875 lt 005)

the group treated with Intrasite gel (standard) on day 5 aftersurgery (Table 1) The percentage of healing in the high dosegroup was significantly higher than those of the low-dose-treated group the reference standard group and the controlgroup on day 10 after surgery although the percentage ofhealing in the placebo (CMC) control group was significantlylower than those of the extract-treated groups and thereference standard control (119875lt005)Therewas no significantdifference between low-dose-treated groups and standardcontrol group on day 10 after surgery (119875 gt 005)The effect oflow dose was similar to that of the standard

The histology of wound area on day 10 after surgeryshowed that wounds dressed with the extract showed com-paratively less scar width at wound closure compared to thecontrol group (Figures 3 and 4) In all the groups with respectto the control group inflammatory cell infiltration was lessintense and the difference between the groupswas statisticallysignificant (119875 lt 005) In Groups I II and III there was nosignificant difference in inflammatory cell infiltration (119875 gt005) In extract-treated groups and the standard treatedgroup comparativelymore collagen andfibroblastswere seen

4 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

Figure 2 Macroscopic appearance of wound on day 10 after surgery (a) A 200mgmL of white tea treated group (b) 100mgmL of white teatreated group (c) Intrasite gel treated group and (d) control group (wound dressed with placebo CMC)

500120583m 500120583m 500120583m 500120583m

25120583m 25120583m 25120583m 25120583m

10120583m 10120583m 10120583m 10120583m

(G1) (G2) (G3) (G4)

The interface between normal skin and the regenerated woundBlood capillaries in the second row

FibroblastsCollagen

Inflammatory cells

Figure 3Histology of wound area stainedwith hematoxylin and eosin on day 10 after surgery Photomicrographs of wound tissues in differentmagnification are shown in the rows

(Figure 3) Proliferation of blood capillaries and angiogenesiswere significantly more pronounced in Groups I II and IIIwhen compared with group IV Figure 4 shows Massonrsquostrichome staining of the wound after 10 days In both groupsII (low-dose treated) and III (Intrasite gel treated) thepresence of collagen and fibroblasts is similar to Group I(high-dose treated) although the photomicrograph of GroupI shows a higher density of collagen andmore fibroblast cellsthough this was not significant (Table 2) The CMC-treatedgroup (Group IV) has significantly (119875 lt 005) less collagenless fibroblasts and less blood capillaries in comparison withthe other groups

4 Discussion

In this study an excision wound model was used for theassessment of the wound healing activity ofCamellia sinensis

specifically white tea Wound healing progression is com-prised of a systematic process of events starting from themoment of injury that is the inflammatory phase (the estab-lishment of homeostasis and inflammation) the proliferationphase (granulation contraction and epithelialisation) andfinally the remodeling phase which determines the strengthand appearance of the healed tissue [3 22]

The outcome of this study demonstrates that the woundcontracting ability of the white tea extract on an excisionwoundmodel was significantly greater than that of the vehicleitself

Histopathological studies of the wound healing processare normally used for evaluating the efficacy of pharma-cological products which promote and accelerate dermalskin substitutes The histological results of this study showthat the granulation tissue in tea extract-treated groups con-tained comparatively less inflammation and more collagen

Evidence-Based Complementary and Alternative Medicine 5

D

E

500 120583mD

E

500 120583m

D

E

500 120583m D

E

500 120583m

25 120583m 25 120583m 25 120583m 25 120583m

(G1) (G2) (G3) (G4)

Figure 4 Histological sections of healed wound on day 10 after-surgery stained with Massonrsquos trichrome Different magnifications of thewound are shown in the rows E epidermis D dermis The arrows in the upper row show the different levels of collagen depositionrepresented by the intensity of the green color

Table 2Themedian histopathologic scores of wound healing were determined in the extract-treated and control groups by using a modified0 to 4 numerical scale The scores were 0 for absence 1 for occasional presence 2 for light scattering 3 for abundance and 4 for confluenceof cells or fibres

Groups Epithelialisation Inflammatorycell infiltration

Fibroblastproliferation Neovascularisation Collagen deposition

G1 (high dose) 383 plusmn 041a 150 plusmn 055b 350 plusmn 055a 267 plusmn 052a 383 plusmn 041a

G2 (low dose) 317 plusmn 041a 183 plusmn 075b 317 plusmn 075a 200 plusmn 063a 333 plusmn 052a

G3 (Intrasite gel) 350 plusmn 055a 167 plusmn 052b 333 plusmn 082a 217 plusmn 075a 367 plusmn 052a

G4 (CMC control) 083 plusmn 041b 367 plusmn 052a 100 plusmn 000b 083 plusmn 075b 117 plusmn 041b

Data are expressed as mean plusmn standard error (119899 = 6) Different letters (a and b) in a column indicate significant difference (119875 lt 005)

and angiogenesis The histopathological examination of thewound sections in the control group exhibited a wide areaof ulceration (presumably containing fibrinous exudates) andinflammatory cells and a mild degree of vascularisation withcongestion in the dermis which indicate that healing wasnot complete It can be concluded that topical application ofthe tea extract at a high concentration (200mgmL) signifi-cantly increased the fibroblast growth collagen synthesis andthus the healing process by accelerating the rate of woundhealing

In the present study haematoxylin and eosin (HampE)staining was used (Figure 3) as it is the most commonmethod in histopathological studies Histological sectionsstained with haematoxylin and eosin (HampE) on day 10 after-surgery showed that wounds dressed with the extract showedcomparatively less scar width at wound closure compared tothe control group (Figure 3) The histology of wound areain Intrasite gel- and low-dose extract-treated groups showedsimilar results In both groups the extent of epitheliali-sation neovascularisation and fibroblast proliferation andthe presence of collagen are similar Histological analysisshowed that the intensities of the collagen (green colour) ofGroups I II and III are different but not significant (119875 gt005) Proliferation of blood capillaries and angiogenesis weresignificantly higher in Groups I II and III when comparedwith Group IV (119875 lt 005)

Although HampE staining is the most popular stainingmethod the stain is not able to differentiate importanthistopathological changes in the wound healing processsuch as collagen deposition An alternative staining methodmodified Massonrsquos trichrome staining (MT) was used todifferentiate the important morphological parameters forwound healing assessment The results of the MT staining inthis study confirm that the presence of collagen in Intrasitegel- and white tea extract-treated groups is significantlyhigher when compared to the control group As shown inFigure 4 for the results of MT staining (presented at twodifferent magnifications 2x and 40x) the intensity of thegreen color represents collagen density Fibroblasts producecollagen in skin which plays an important role in preserv-ing the anatomic integrity of wound healing MT stainingshowed differences but this was not significant for fibroblastproliferation collagen deposition and neovascularisation inIntrasite gel- and white tea extract-treated groups (119875 gt 005)However the difference in collagen deposition between thetreated groups mentioned above and in the CMC-treatedgroup is significant The inflammatory cells in Groups IndashIIIwere significantly less (119875 lt 005) than those in Group IV

The results showed a trend of progressive constructionof tissues surrounding the wound from day 5 onwards(Table 1) especially for the Intrasite gel- and tea extract-treated groups This was significantly different (119875 lt 005)

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

of wound area compared with that on postoperative dayas described previously [18] The wound closure rate wasexpressed as the percentage of wound area compared withthat on post-operative day that is the change in wound sizewas expressed as a percentage of the original wound size

contraction

=

Initial wound size minus Specific day wound sizeInitial wound size

times 100

(1)

27 Histological Evaluation of Healed Wounds Skin tissuesamples of all groups were immediately fixed after ani-mal dissection in 10 buffered formalin dehydrated andprocessed using a paraffin tissue processing machine forparaffin sectioning The wound tissues were cut 5 120583m thickperpendicular to the wound stained with standard haema-toxylin and eosin (HampE) and Massonrsquos trichrome (MT) andexamined using an Olympus BX60 light microscope andcaptured using anOlympus XC10 camera (Tokyo Japan)Theevaluated parameters were epithelialisation inflammatorycell infiltration fibroblast proliferation neovascularisationand collagen deposition which were assessed individuallyby using a numerical scale Stained sections were examinedunder a light microscope and were graded in a blind fashionon 3 slides per animal using the modified 0 to 4 numericalscale as described by other researchers [20 21] The scoreswere 0 for absence 1 for occasional presence 2 for lightscattering 3 for abundance and 4 for confluence of cells orfibers

28 Statistical Analysis Experimental results are presented asmeans plusmn SD of six animals in each group Statistical analysiswas performed by using SPSS (1800 version) statisticalsoftware The effect of extracts on the wound was analysedby one-way ANOVA 119875 values lt 005 were considered signi-ficant

3 Results

Figure 1 shows the macroscopic appearance of the excisedskin wound after surgery The macroscopic examination onday 10 after surgery is presented in Figure 2 As shown in thefigure the 3 groups (high-dose treated low-dose treated andIntrasite gel-treated that is Figures 2(a) 2(b) and 2(c) resp)showed a narrow scar at wound closure compared with thecontrol group that is wound dressed with placebo (CMC)Figure 2(d) which showed incomplete wound healing

The change in the wound size was expressed as a percent-age of thewound area on days 5 and 10 after surgery comparedwith the wound area on day 0 (100) in the differentgroups (Table 1) Wounds dressed with white tea extract orwith reference standard control showed considerable signsof dermal healing and significantly healed faster comparedto the group that received the placebo control treatment(CMC in normal saline) Throughout the experiment thepercentage of wound healing in the control (CMC) groupwassignificantly lower than that in the treated groups or that in

Figure 1 Excision skin wound (200 cm) on surgery day beforetreatment

Table 1 Effect ofmethanolic extract ofCamellia sinensis on percent-age () of wound healing in Sprague Dawley rats

Animalgroups Treatment

Percentage of wound healing(mean plusmn SD) on days after

surgery

Day 5 Day 10

Group 1 High dose200mgmL 619 plusmn 670a 977 plusmn 094a

Group 2 Low dose100mgmL 623 plusmn 399a 945 plusmn 112b

Group 3 Intrasite gel 603 plusmn 670a 954 plusmn 129b

Group 4 CMC in normalsaline 450 plusmn 198b 865 plusmn 240c

Mean values (119899 = 6) followed by different letters (a b and c) in a column aresignificantly different (119875 lt 005)

the group treated with Intrasite gel (standard) on day 5 aftersurgery (Table 1) The percentage of healing in the high dosegroup was significantly higher than those of the low-dose-treated group the reference standard group and the controlgroup on day 10 after surgery although the percentage ofhealing in the placebo (CMC) control group was significantlylower than those of the extract-treated groups and thereference standard control (119875lt005)Therewas no significantdifference between low-dose-treated groups and standardcontrol group on day 10 after surgery (119875 gt 005)The effect oflow dose was similar to that of the standard

The histology of wound area on day 10 after surgeryshowed that wounds dressed with the extract showed com-paratively less scar width at wound closure compared to thecontrol group (Figures 3 and 4) In all the groups with respectto the control group inflammatory cell infiltration was lessintense and the difference between the groupswas statisticallysignificant (119875 lt 005) In Groups I II and III there was nosignificant difference in inflammatory cell infiltration (119875 gt005) In extract-treated groups and the standard treatedgroup comparativelymore collagen andfibroblastswere seen

4 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

Figure 2 Macroscopic appearance of wound on day 10 after surgery (a) A 200mgmL of white tea treated group (b) 100mgmL of white teatreated group (c) Intrasite gel treated group and (d) control group (wound dressed with placebo CMC)

500120583m 500120583m 500120583m 500120583m

25120583m 25120583m 25120583m 25120583m

10120583m 10120583m 10120583m 10120583m

(G1) (G2) (G3) (G4)

The interface between normal skin and the regenerated woundBlood capillaries in the second row

FibroblastsCollagen

Inflammatory cells

Figure 3Histology of wound area stainedwith hematoxylin and eosin on day 10 after surgery Photomicrographs of wound tissues in differentmagnification are shown in the rows

(Figure 3) Proliferation of blood capillaries and angiogenesiswere significantly more pronounced in Groups I II and IIIwhen compared with group IV Figure 4 shows Massonrsquostrichome staining of the wound after 10 days In both groupsII (low-dose treated) and III (Intrasite gel treated) thepresence of collagen and fibroblasts is similar to Group I(high-dose treated) although the photomicrograph of GroupI shows a higher density of collagen andmore fibroblast cellsthough this was not significant (Table 2) The CMC-treatedgroup (Group IV) has significantly (119875 lt 005) less collagenless fibroblasts and less blood capillaries in comparison withthe other groups

4 Discussion

In this study an excision wound model was used for theassessment of the wound healing activity ofCamellia sinensis

specifically white tea Wound healing progression is com-prised of a systematic process of events starting from themoment of injury that is the inflammatory phase (the estab-lishment of homeostasis and inflammation) the proliferationphase (granulation contraction and epithelialisation) andfinally the remodeling phase which determines the strengthand appearance of the healed tissue [3 22]

The outcome of this study demonstrates that the woundcontracting ability of the white tea extract on an excisionwoundmodel was significantly greater than that of the vehicleitself

Histopathological studies of the wound healing processare normally used for evaluating the efficacy of pharma-cological products which promote and accelerate dermalskin substitutes The histological results of this study showthat the granulation tissue in tea extract-treated groups con-tained comparatively less inflammation and more collagen

Evidence-Based Complementary and Alternative Medicine 5

D

E

500 120583mD

E

500 120583m

D

E

500 120583m D

E

500 120583m

25 120583m 25 120583m 25 120583m 25 120583m

(G1) (G2) (G3) (G4)

Figure 4 Histological sections of healed wound on day 10 after-surgery stained with Massonrsquos trichrome Different magnifications of thewound are shown in the rows E epidermis D dermis The arrows in the upper row show the different levels of collagen depositionrepresented by the intensity of the green color

Table 2Themedian histopathologic scores of wound healing were determined in the extract-treated and control groups by using a modified0 to 4 numerical scale The scores were 0 for absence 1 for occasional presence 2 for light scattering 3 for abundance and 4 for confluenceof cells or fibres

Groups Epithelialisation Inflammatorycell infiltration

Fibroblastproliferation Neovascularisation Collagen deposition

G1 (high dose) 383 plusmn 041a 150 plusmn 055b 350 plusmn 055a 267 plusmn 052a 383 plusmn 041a

G2 (low dose) 317 plusmn 041a 183 plusmn 075b 317 plusmn 075a 200 plusmn 063a 333 plusmn 052a

G3 (Intrasite gel) 350 plusmn 055a 167 plusmn 052b 333 plusmn 082a 217 plusmn 075a 367 plusmn 052a

G4 (CMC control) 083 plusmn 041b 367 plusmn 052a 100 plusmn 000b 083 plusmn 075b 117 plusmn 041b

Data are expressed as mean plusmn standard error (119899 = 6) Different letters (a and b) in a column indicate significant difference (119875 lt 005)

and angiogenesis The histopathological examination of thewound sections in the control group exhibited a wide areaof ulceration (presumably containing fibrinous exudates) andinflammatory cells and a mild degree of vascularisation withcongestion in the dermis which indicate that healing wasnot complete It can be concluded that topical application ofthe tea extract at a high concentration (200mgmL) signifi-cantly increased the fibroblast growth collagen synthesis andthus the healing process by accelerating the rate of woundhealing

In the present study haematoxylin and eosin (HampE)staining was used (Figure 3) as it is the most commonmethod in histopathological studies Histological sectionsstained with haematoxylin and eosin (HampE) on day 10 after-surgery showed that wounds dressed with the extract showedcomparatively less scar width at wound closure compared tothe control group (Figure 3) The histology of wound areain Intrasite gel- and low-dose extract-treated groups showedsimilar results In both groups the extent of epitheliali-sation neovascularisation and fibroblast proliferation andthe presence of collagen are similar Histological analysisshowed that the intensities of the collagen (green colour) ofGroups I II and III are different but not significant (119875 gt005) Proliferation of blood capillaries and angiogenesis weresignificantly higher in Groups I II and III when comparedwith Group IV (119875 lt 005)

Although HampE staining is the most popular stainingmethod the stain is not able to differentiate importanthistopathological changes in the wound healing processsuch as collagen deposition An alternative staining methodmodified Massonrsquos trichrome staining (MT) was used todifferentiate the important morphological parameters forwound healing assessment The results of the MT staining inthis study confirm that the presence of collagen in Intrasitegel- and white tea extract-treated groups is significantlyhigher when compared to the control group As shown inFigure 4 for the results of MT staining (presented at twodifferent magnifications 2x and 40x) the intensity of thegreen color represents collagen density Fibroblasts producecollagen in skin which plays an important role in preserv-ing the anatomic integrity of wound healing MT stainingshowed differences but this was not significant for fibroblastproliferation collagen deposition and neovascularisation inIntrasite gel- and white tea extract-treated groups (119875 gt 005)However the difference in collagen deposition between thetreated groups mentioned above and in the CMC-treatedgroup is significant The inflammatory cells in Groups IndashIIIwere significantly less (119875 lt 005) than those in Group IV

The results showed a trend of progressive constructionof tissues surrounding the wound from day 5 onwards(Table 1) especially for the Intrasite gel- and tea extract-treated groups This was significantly different (119875 lt 005)

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c) (d)

Figure 2 Macroscopic appearance of wound on day 10 after surgery (a) A 200mgmL of white tea treated group (b) 100mgmL of white teatreated group (c) Intrasite gel treated group and (d) control group (wound dressed with placebo CMC)

500120583m 500120583m 500120583m 500120583m

25120583m 25120583m 25120583m 25120583m

10120583m 10120583m 10120583m 10120583m

(G1) (G2) (G3) (G4)

The interface between normal skin and the regenerated woundBlood capillaries in the second row

FibroblastsCollagen

Inflammatory cells

Figure 3Histology of wound area stainedwith hematoxylin and eosin on day 10 after surgery Photomicrographs of wound tissues in differentmagnification are shown in the rows

(Figure 3) Proliferation of blood capillaries and angiogenesiswere significantly more pronounced in Groups I II and IIIwhen compared with group IV Figure 4 shows Massonrsquostrichome staining of the wound after 10 days In both groupsII (low-dose treated) and III (Intrasite gel treated) thepresence of collagen and fibroblasts is similar to Group I(high-dose treated) although the photomicrograph of GroupI shows a higher density of collagen andmore fibroblast cellsthough this was not significant (Table 2) The CMC-treatedgroup (Group IV) has significantly (119875 lt 005) less collagenless fibroblasts and less blood capillaries in comparison withthe other groups

4 Discussion

In this study an excision wound model was used for theassessment of the wound healing activity ofCamellia sinensis

specifically white tea Wound healing progression is com-prised of a systematic process of events starting from themoment of injury that is the inflammatory phase (the estab-lishment of homeostasis and inflammation) the proliferationphase (granulation contraction and epithelialisation) andfinally the remodeling phase which determines the strengthand appearance of the healed tissue [3 22]

The outcome of this study demonstrates that the woundcontracting ability of the white tea extract on an excisionwoundmodel was significantly greater than that of the vehicleitself

Histopathological studies of the wound healing processare normally used for evaluating the efficacy of pharma-cological products which promote and accelerate dermalskin substitutes The histological results of this study showthat the granulation tissue in tea extract-treated groups con-tained comparatively less inflammation and more collagen

Evidence-Based Complementary and Alternative Medicine 5

D

E

500 120583mD

E

500 120583m

D

E

500 120583m D

E

500 120583m

25 120583m 25 120583m 25 120583m 25 120583m

(G1) (G2) (G3) (G4)

Figure 4 Histological sections of healed wound on day 10 after-surgery stained with Massonrsquos trichrome Different magnifications of thewound are shown in the rows E epidermis D dermis The arrows in the upper row show the different levels of collagen depositionrepresented by the intensity of the green color

Table 2Themedian histopathologic scores of wound healing were determined in the extract-treated and control groups by using a modified0 to 4 numerical scale The scores were 0 for absence 1 for occasional presence 2 for light scattering 3 for abundance and 4 for confluenceof cells or fibres

Groups Epithelialisation Inflammatorycell infiltration

Fibroblastproliferation Neovascularisation Collagen deposition

G1 (high dose) 383 plusmn 041a 150 plusmn 055b 350 plusmn 055a 267 plusmn 052a 383 plusmn 041a

G2 (low dose) 317 plusmn 041a 183 plusmn 075b 317 plusmn 075a 200 plusmn 063a 333 plusmn 052a

G3 (Intrasite gel) 350 plusmn 055a 167 plusmn 052b 333 plusmn 082a 217 plusmn 075a 367 plusmn 052a

G4 (CMC control) 083 plusmn 041b 367 plusmn 052a 100 plusmn 000b 083 plusmn 075b 117 plusmn 041b

Data are expressed as mean plusmn standard error (119899 = 6) Different letters (a and b) in a column indicate significant difference (119875 lt 005)

and angiogenesis The histopathological examination of thewound sections in the control group exhibited a wide areaof ulceration (presumably containing fibrinous exudates) andinflammatory cells and a mild degree of vascularisation withcongestion in the dermis which indicate that healing wasnot complete It can be concluded that topical application ofthe tea extract at a high concentration (200mgmL) signifi-cantly increased the fibroblast growth collagen synthesis andthus the healing process by accelerating the rate of woundhealing

In the present study haematoxylin and eosin (HampE)staining was used (Figure 3) as it is the most commonmethod in histopathological studies Histological sectionsstained with haematoxylin and eosin (HampE) on day 10 after-surgery showed that wounds dressed with the extract showedcomparatively less scar width at wound closure compared tothe control group (Figure 3) The histology of wound areain Intrasite gel- and low-dose extract-treated groups showedsimilar results In both groups the extent of epitheliali-sation neovascularisation and fibroblast proliferation andthe presence of collagen are similar Histological analysisshowed that the intensities of the collagen (green colour) ofGroups I II and III are different but not significant (119875 gt005) Proliferation of blood capillaries and angiogenesis weresignificantly higher in Groups I II and III when comparedwith Group IV (119875 lt 005)

Although HampE staining is the most popular stainingmethod the stain is not able to differentiate importanthistopathological changes in the wound healing processsuch as collagen deposition An alternative staining methodmodified Massonrsquos trichrome staining (MT) was used todifferentiate the important morphological parameters forwound healing assessment The results of the MT staining inthis study confirm that the presence of collagen in Intrasitegel- and white tea extract-treated groups is significantlyhigher when compared to the control group As shown inFigure 4 for the results of MT staining (presented at twodifferent magnifications 2x and 40x) the intensity of thegreen color represents collagen density Fibroblasts producecollagen in skin which plays an important role in preserv-ing the anatomic integrity of wound healing MT stainingshowed differences but this was not significant for fibroblastproliferation collagen deposition and neovascularisation inIntrasite gel- and white tea extract-treated groups (119875 gt 005)However the difference in collagen deposition between thetreated groups mentioned above and in the CMC-treatedgroup is significant The inflammatory cells in Groups IndashIIIwere significantly less (119875 lt 005) than those in Group IV

The results showed a trend of progressive constructionof tissues surrounding the wound from day 5 onwards(Table 1) especially for the Intrasite gel- and tea extract-treated groups This was significantly different (119875 lt 005)

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

D

E

500 120583mD

E

500 120583m

D

E

500 120583m D

E

500 120583m

25 120583m 25 120583m 25 120583m 25 120583m

(G1) (G2) (G3) (G4)

Figure 4 Histological sections of healed wound on day 10 after-surgery stained with Massonrsquos trichrome Different magnifications of thewound are shown in the rows E epidermis D dermis The arrows in the upper row show the different levels of collagen depositionrepresented by the intensity of the green color

Table 2Themedian histopathologic scores of wound healing were determined in the extract-treated and control groups by using a modified0 to 4 numerical scale The scores were 0 for absence 1 for occasional presence 2 for light scattering 3 for abundance and 4 for confluenceof cells or fibres

Groups Epithelialisation Inflammatorycell infiltration

Fibroblastproliferation Neovascularisation Collagen deposition

G1 (high dose) 383 plusmn 041a 150 plusmn 055b 350 plusmn 055a 267 plusmn 052a 383 plusmn 041a

G2 (low dose) 317 plusmn 041a 183 plusmn 075b 317 plusmn 075a 200 plusmn 063a 333 plusmn 052a

G3 (Intrasite gel) 350 plusmn 055a 167 plusmn 052b 333 plusmn 082a 217 plusmn 075a 367 plusmn 052a

G4 (CMC control) 083 plusmn 041b 367 plusmn 052a 100 plusmn 000b 083 plusmn 075b 117 plusmn 041b

Data are expressed as mean plusmn standard error (119899 = 6) Different letters (a and b) in a column indicate significant difference (119875 lt 005)

and angiogenesis The histopathological examination of thewound sections in the control group exhibited a wide areaof ulceration (presumably containing fibrinous exudates) andinflammatory cells and a mild degree of vascularisation withcongestion in the dermis which indicate that healing wasnot complete It can be concluded that topical application ofthe tea extract at a high concentration (200mgmL) signifi-cantly increased the fibroblast growth collagen synthesis andthus the healing process by accelerating the rate of woundhealing

In the present study haematoxylin and eosin (HampE)staining was used (Figure 3) as it is the most commonmethod in histopathological studies Histological sectionsstained with haematoxylin and eosin (HampE) on day 10 after-surgery showed that wounds dressed with the extract showedcomparatively less scar width at wound closure compared tothe control group (Figure 3) The histology of wound areain Intrasite gel- and low-dose extract-treated groups showedsimilar results In both groups the extent of epitheliali-sation neovascularisation and fibroblast proliferation andthe presence of collagen are similar Histological analysisshowed that the intensities of the collagen (green colour) ofGroups I II and III are different but not significant (119875 gt005) Proliferation of blood capillaries and angiogenesis weresignificantly higher in Groups I II and III when comparedwith Group IV (119875 lt 005)

Although HampE staining is the most popular stainingmethod the stain is not able to differentiate importanthistopathological changes in the wound healing processsuch as collagen deposition An alternative staining methodmodified Massonrsquos trichrome staining (MT) was used todifferentiate the important morphological parameters forwound healing assessment The results of the MT staining inthis study confirm that the presence of collagen in Intrasitegel- and white tea extract-treated groups is significantlyhigher when compared to the control group As shown inFigure 4 for the results of MT staining (presented at twodifferent magnifications 2x and 40x) the intensity of thegreen color represents collagen density Fibroblasts producecollagen in skin which plays an important role in preserv-ing the anatomic integrity of wound healing MT stainingshowed differences but this was not significant for fibroblastproliferation collagen deposition and neovascularisation inIntrasite gel- and white tea extract-treated groups (119875 gt 005)However the difference in collagen deposition between thetreated groups mentioned above and in the CMC-treatedgroup is significant The inflammatory cells in Groups IndashIIIwere significantly less (119875 lt 005) than those in Group IV

The results showed a trend of progressive constructionof tissues surrounding the wound from day 5 onwards(Table 1) especially for the Intrasite gel- and tea extract-treated groups This was significantly different (119875 lt 005)

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

from the control groupThere was no significant difference inthe semiquantitative histopathological evaluation of woundhealing between the groups treated with Intrasite gel andextract treated groups as shown in Table 2

Inflammation is the first response during the healingperiod as a defence mechanism of the tissue although along duration in the inflammatory phase can cause a delayin the healing process [23] The results of this study suggestthat application of Camellia sinensis extract shortens theinflammation period in order to shorten the healing time

Transforming growth factor-beta1 (TGF-1205731) has beenshown to be expressed during the wound healing processTGF-1205731 acts by the stimulation of fibroblast proliferation anddifferentiation collagen production wound contraction andconnective tissue growth factor gene expression [24] 12057231205731integrin has also been shown to be highly expressed duringreepithelialization [25] Epigallocatechin-3-gallate (EGCG) amajor component of tea has been shown to enhance the roleand expression of TGF-1205731 [26] EGCG has also been reportedto enhance the expression of vascular endothelial cell growthfactor and angiopoietin-1 protein expression [27]

A previous study has shown that healing can be accel-erated and enhanced by the use of specific wound dressingor care product and techniques and that it is not a passiveprocess [28] It has been observed that plant constituentscan significantly accelerate the healing process and improvethe quality of wound healing [29] Numerous studies haveshown that plant compounds could potentially be therapeuticagents to treat wounds [30 31] The findings of this study arealso in line with previous studies reported by various authors[18 19 31]

It is generally accepted that reactive oxygen species(ROS) because of their harmful effects on cells and tissuesare noxious for the wound healing process [32] Antioxi-dantsfree radical scavengers play a significant role in theprocess of wound healing by improving the healing timeand the appearance of the healed tissue while protectingtissues from oxidative damage The wound healing action ofCamellia sinensismay be due to the phytoconstituents presentin the plant (high flavonoid and phenolic content) and alsohigh free radical scavenging activity

The chemical constituents of tea leaves include polyphe-nols (catechins and flavonoids) alkaloids (caffeine theo-bromine theophylline etc) volatile oils polysaccharidesamino acids lipids vitamins (eg vitamin C) and inorganicelements (eg aluminium fluorine and manganese) Thepolyphenols are the group of compounds that are primarilyresponsible for the beneficial healthful properties of tea Theflavonoids have antioxidant anti-inflammatory antiallergicand antimicrobial effects All of these constituents playimportant roles in the maintenance of human health and alsoin wound healing [14]

The oral bioavailability of catechins from tea has beenreported to be about 5 to 50 times less than that in invitro studies [33] although an increase in human plasmaantioxidant activity has been shown after the consumptionof tea [34] Catechins were present in plasma at micromolarlevels after oral consumption of tea Catechins are metabo-lized and circulate as sulfated methylated or glucuronidated

derivatives epigallocatechin gallate and epicatechin gallatewere mostly present in plasma as the free form

As our results show that treated groups healed fastershowed less scarring more blood capillaries less inflamma-tory cells and more fibroblasts and collagen we proposethat white tea might enhance the wound healing process byincreasing the rate of various phases such as cell proliferationangiogenesis and collagen formation

5 Conclusion

This study showed that the wound area of wounds dressedwith white tea extract had relatively less scar width at woundclosure Granulation tissue contained less inflammatory cellsand more collagen with angiogenesis compared to woundsdressed with vehicle Camellia sinensis shows high potentialin wound healing activity especially at higher concentrations

Conflict of Interests

The authors have no conflict of interests and have nocommercial or financial interest in the product described inthis paper

Acknowledgments

The authors would like to thank Parastoo MojtahedzadehDepartment of Anatomy Faculty of Medicine and PouyaHasandarvishDepartment ofMolecularMedicine Faculty ofMedicine University of Malaya for help in animal manage-ment This study was supported by the University of MalayaResearch Grants PVO322011A and RG34111HTM

References

[1] G M Cragg and D J Newman ldquoDrugs from nature pastachievements future prospectsrdquo in Ethnomedicine and DrugDiscovery pp 23ndash37 Elsevier Science AmsterdamTheNether-lands 2002

[2] D Mantle M A Gok and T W J Lennard ldquoAdverse andbeneficial effects of plant extracts on skin and skin disordersrdquoAdverse Drug Reactions and Toxicological Reviews vol 20 no2 pp 89ndash103 2001

[3] T Kondo ldquoTiming of skin woundsrdquo Legal Medicine vol 9 no2 pp 109ndash114 2007

[4] J D Phillipson ldquoPhytochemistry and medicinal plantsrdquo Phyto-chemistry vol 56 no 3 pp 237ndash243 2001

[5] R A Govindarajan P Pushpangadan B Kumara and MVijayakumar ldquoEthno-pharmacological approachestowound-healing-exploring medicinalplantsof Indiardquo Journal of Ethno-pharmacology vol 114 pp 103ndash113 2007

[6] G Soma S Amalesh B Nirup B Sukdeb and C DebprasadldquoEvaluation of the wound healing activity of methanol extractof Pedilanthus tithymaloides (L) Poit leaf and its isolated activeconstituents in topical formulationrdquo Journal of Ethnopharma-cology vol 142 pp 714ndash722 2012

[7] A A Hemmati and F Mohammadian ldquoAn investigation intothe effects of mucilage of quince seeds on wound healing inrabbitrdquo Journal of Herbs Spices and Medicinal Plants vol 7no 4 pp 41ndash46 2000

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

[8] M Toda S Okubo H Ikigai T Suzuki Y Suzuki and TShimamura ldquoThe protective activity of tea against infection byVibrio cholerae O1rdquo Journal of Applied Bacteriology vol 70no 2 pp 109ndash112 1991

[9] Y Hilal and U Engelhardt ldquoCharacterization of white tea-comparison to green and black teardquo Journal of ConsumerProtection and Food Safety vol 2 pp 414ndash421 2007

[10] E Venditti T Bacchetti L Tiano P Carloni L Greci and EDamiani ldquoHot vs cold water steeping of different teas do theyaffect antioxidant activityrdquo Food Chemistry vol 119 no 4 pp1597ndash1604 2010

[11] Y Mizukami Y Sawai and Y Yamaguchi ldquoSimultaneous ana-lysis of catechins gallic acid strictinin and purine alkaloids ingreen tea by using catechol as an internal standardrdquo Journal ofAgricultural and Food Chemistry vol 55 no 13 pp 4957ndash49642007

[12] D Del Rio A J Stewart W Mullen et al ldquoHPLC-MSn analysisof phenolic compounds and purine alkaloids in green and blackteardquo Journal of Agricultural and Food Chemistry vol 52 no 10pp 2807ndash2815 2004

[13] G Rusak D Komes S Likic D Horzic and M KovacldquoPhenolic content and antioxidative capacity of green and whitetea extracts depending on extraction conditions and the solventusedrdquo Food Chemistry vol 110 no 4 pp 852ndash858 2008

[14] S I Trevisanato ldquoTea and healthrdquoNutrition Reviews vol 58 no1 pp 1ndash10 2000

[15] J L Bushman ldquoGreen tea and cancer in humans a review of theliteraturerdquoNutrition and Cancer vol 31 no 3 pp 151ndash159 1998

[16] C Williams ldquoIntrasite Gel a hydrogel dressingrdquo British Journalof Nursing vol 3 no 16 pp 843ndash846 1994

[17] F F Mughrabi H Hashim M Ameen H Khaledi H M Aliand S Ismail ldquoEffect of Bis [benzyl N1015840-(indol-3-ylmethylene)-hydrazinecarbodithioato]-zinc(II) derivatives on wound heal-ing in Sprague Dawley ratsrdquo Indian Journal of ExperimentalBiology vol 49 no 1 pp 50ndash55 2011

[18] A A Zahra F A Kadir A A Mahmood et al ldquoAcute toxicitystudy and wound healing potential of Gynura procumbens leafxtract in ratsrdquo Journal of Medicinal Plant Research vol 5 no 12pp 2551ndash2558 2011

[19] A A Mahmood A A Mariod S Ibrahim Abdelwahab SIsmail and F Al-Bayaty ldquoPotential activity of ethanolic extractof Boesenbergia rotunda (L) rhizomes extract in acceleratingwound healing in ratsrdquo Journal ofMedicinal Plants Research vol4 pp 1570ndash1576 2010

[20] H P Ehrlich H Tarver and T K Hunt ldquoEffects of vitamin Aand glucocorticoids upon inflammation and collagen synthe-sisrdquo Annals of Surgery vol 177 no 2 pp 222ndash227 1973

[21] T Hunt and R Mueller ldquoWound healingrdquo in Current SurgicalDiagnosis and Treatment L W Way Ed pp 80ndash93 Appletonand Lange Paramus NJ USA 10th edition 1994

[22] S Q Wasman A A Mahmood H Salehhuddin A A Zahraand I Salmah ldquoCytoprotective activities of Polygonum minusaqueous leaf extract on ethanol-induced gastric ulcer in ratsrdquoJournal of Medicinal Plant Research vol 4 no 24 pp 2658ndash2665 2010

[23] R Clark ldquoCutaneous wound repairrdquo in Biochemistry and Phys-iology of the Skin L A Goldsmith Ed pp 576ndash601 OxfordUniversity Press New York NY USA 1991

[24] G S Ashcroft J Dodsworth E Van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] A Moustakas S Souchelnytskyi and C-H Heldin ldquoSmad reg-ulation in TGF-120573 signal transductionrdquo Journal of Cell Sciencevol 114 no 24 pp 4359ndash4369 2001

[26] B R Klass O A Branford A O Grobbelaar and K JRolfe ldquoThe effect of epigallocatechin-3-gallate a constituent ofgreen tea on transforming growth factor-1205731-stimulated woundcontractionrdquoWound Repair and Regeneration vol 18 no 1 pp80ndash88 2010

[27] J-G Leu S-A Chen H-M Chen et al ldquoThe effects of goldnanoparticles in wound healing with antioxidant epigallocate-chin gallate and 120572-lipoic acidrdquo Nanomedicine NanotechnologyBiology and Medicine vol 8 pp 767ndash775 2012

[28] D M Cooper ldquoOptimizing wound healing A practice withinnursingrsquos domainrdquoNursing Clinics of North America vol 25 no1 pp 165ndash180 1990

[29] F H Al-Bayaty M A Abdulla M I A Hassan and H MAli ldquoEffect of Andrographis paniculata leaf extract on woundhealing in ratsrdquoNatural Product Research vol 26 no 5 pp 423ndash429 2012

[30] E K Akkol U Koca I Pesin D Yilmazer G Toker andE Yesilada ldquoExploring the wound healing activity of Arnebiadensiflora (Nordm) Ledeb by in vivomodelsrdquo Journal of Ethno-pharmacology vol 124 no 1 pp 137ndash141 2009

[31] D Csupor G Blazso A Balogh and J Hohmann ldquoThetraditional Hungarian medicinal plant Centaurea sadlerianaJanka accelerates wound healing in ratsrdquo Journal of Ethnophar-macology vol 127 no 1 pp 193ndash195 2010

[32] G Bodeker and M A Hughes ldquoWound healing traditionaltreatments and research policyrdquo in Plants For Food and Med-icine pp 245ndash359 Kew Press London UK 1998

[33] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[34] A Rietveld and S Wiseman ldquoAntioxidant effects of tea evi-dence from human clinical trialsrdquo Journal of Nutrition vol 133no 10 pp 3285Sndash3292S 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom