Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 709865 10 pageshttpdxdoiorg1011552013709865

Research ArticleEvaluation of the Wound Healing Potential of Resina Draconis(Dracaena cochinchinensis) in Animal Models

Huihui Liu1 Shaohui Lin2 Dan Xiao1 Xiao Zheng1 Yan Gu1 and Shanyu Guo1

1 Department of General Surgery Ninth Peoplersquos Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 China2Department of Geriatrics Ninth Peoplersquos Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200011 China

Correspondence should be addressed to Shanyu Guo guoshyusinacom

Received 5 February 2013 Revised 7 April 2013 Accepted 8 April 2013

Academic Editor Yong Qing Yang

Copyright copy 2013 Huihui Liu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ResinaDraconis (RD) is a type of dragonrsquos blood resin obtained fromDracaena cochinchinensis (Lour) SC Chen (Yunnan China) Ithas been used as amedicine since ancient times bymany culturesThe ethanolic extract ofResinaDraconis (RDEE)was evaluated forits wound-healing activity using excision and incision woundmodels in rats Group I the control group was treated with ointmentbase Group II which served as a reference standard was treated withmoist exposed burn ointment (MEBO) Group III was treatedwith RDEEThe parameters observed were percentage of wound contraction epithelialization period tensile strength histopatho-logical studies microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF) and transforminggrowth factor-1205731 (TGF-1205731) The group treated with RDEE showed significantly better wound contraction and better skin-breakingstrength as compared with the control group The results of histopathological examination MVD and the expression levels ofgrowth factors supported the outcome of the wound models as well The present study provided a scientific rationale for the tradi-tional use of RD in the management of wounds

1 Introduction

Skin healing is a complex process that involves inflammationreepithelization angiogenesis granulation tissue formationand deposition of interstitial matrix beside other events car-ried out by different types of cells such as keratinocytes fibro-blasts inflammatory cells and endothelial cells There arethree stages of the process of wound healing inflammationproliferation and remodelingThe proliferative phase is char-acterized by angiogenesis collagen deposition granulationtissue formation epithelization and wound contractionAngiogenesis involves new blood vessel growth from endo-thelial cells In fibroplasia and granulation tissue formationfibroblasts excrete collagen and fibronectin to form a newextracellularmatrix Subsequently epithelial cells crawl acrossthe wound bed to cover it and the wound is contracted bymyofibroblasts which grip the wound edges and undergocontraction using a mechanism similar to that in smoothmuscle cells The final stage of wound healing is remodelingor maturation of the granulation tissue into mature connec-tive tissue andor scar Alterations in any of these steps can

lead to healing delay or even the inability to heal completely[1] Current methods used to treat wounds include debride-ment irrigation antibiotics tissue grafts and proteolyticenzymes which possess major drawbacks and unwanted sideeffects The use of traditional medicinal remedies and plantsin the treatment of burns and wounds is an important aspectof healthmanagement and at the same time is an effective wayto provide cheaper healthcare options [2]

Since ancient times people have used plants and prepa-rations thereof to accelerate the wound-healing process [3]Recently the interest of using alternative therapies and nat-ural remedies in wound management has rapidly increasedThere are hundreds of medicinal plants that have longhistories of curative properties against various diseases andailments However their use is merely based on traditionwithout any scientific evidence of their efficacy or knowledgeabout putative active compounds or their mode of actionsResina Draconis is a red resin from tree stem of Dracaenacochinchinensis (Lour) SC Chen growing in Yunnan andGuangxi provinces in China belonging to theLiliaceae fam-ily genus Dracaena It was discovered by Cai and Xu [4] in

2 Evidence-Based Complementary and Alternative Medicine

1979 that it could serve as a substitute for Sanguis Draconisa precious crude medicine recorded in the official Chinesepharmacopoeia named as ldquodragonrsquos bloodrdquo [5] In Chinesemedicine Resina Draconis is a major component of the well-known hemostatic preparation ldquoYun Nan Bai Yaordquo so it isconsidered important for its potential application in Chinesemedical practice As a ldquopanacea of blood activatingrdquo resin RDhas great medicinal value and the main biological activitycomes from phenolic compounds [6] Pharmacological stud-ies have showed that RD has positive effects on treatmentof blood stasis syndrome trauma tumors inflammationgynecopathy allergic dermatitis and so on It can promoteblood circulation and serve as an antithrombotic antioxi-dant antiseptic and anti-inflammation compound [7] Theethanolic extracts of Resina Draconis possessed potentialantithrombotic properties affecting platelet aggregation andthus having anticoagulation activities [8] All these studiesindicate that Resina Draconis has enormous potential forfurther study

It is well known that one kind of traditional Chinesemedicine (TCM) usually contains a great number of compo-nents which all together contribute to therapeutic effectsDespite that Chen et al [9] have studied the wound-healingactivity of dragonrsquos blood (Croton lechleri sap) no scientificinvestigation is conducted on Resina Draconisrsquos wound-heal-ing potential Hence in this study we aimed to further inves-tigate the wound-healing effects of RD using the excision andincision wound models and to explore the possible mecha-nisms

2 Materials and Methods

21 Preparation of Plant Extracts Crude Resina Draconiswasprovided by the pharmacy department of No 9 PeoplersquosHospital Affiliated to Shanghai Jiao TongUniversity School ofMedicine RD (30 g)was dissolved in absolute alcohol at roomtemperature for 48 h under shade and then was filtered Thesolvent was concentrated under reduced pressure to ensurethat no residual methanol was left behind furnishing amethanol extract Normally 457 g of dried powder can beobtained from 30 g of RD The sample was stored at minus80∘Cuntil usedThe extract cream was formulated using ointmentas the vehicle The ointment consisted of propylene gly-col liquid paraffin (6 1) and applied topically onto the testanimals Extracts were prepared as 5 in the ointment

22 Phytochemical Analysis Preliminary phytochemicalanalysis was carried out using standard procedures to identifythe constituents as described by Evans and Trease [10] andHarborne [11]

23 Experimental Animals All study protocols were ap-proved by the Shanghai Jiao Tong University Medical CenterInstitutional Animal Care and Use Committee HealthySprague-Dawley male rats weighing between 180 and 200 gwere used The rats were housed in polypropylene cage andmaintained in standard laboratory conditions of temperature

(22 plusmn 2∘C) and light-dark cycle of 12 h 12 hThey were main-tained on standard pellet diet and provided with waterad-libitum throughout the experiment At the end of theexperiment the animals were sacrificed under anesthesia

231 Excision Wound Model The anesthetized rats wereinflicted with excision wounds as described by Morton andMalone [12] The dorsal fur of the animals was shaved withan electric clipper and the area of the wound to be createdwas outlined on the back of the animals with methylene blueusing a circular stainless steel stencil A full thickness of theexcision wound of circular area of 254mm2 and 2mm depthwas created along the markings with a surgical blade Theanimals were randomly divided into 3 groups of 24 for eachGroup I (control group) where animals were applied withsimple ointment base [13] Animals of Group II (standardgroup) were applied with a thin layer of moist exposed burnointment (MEBO) Group IIIrsquos (experimental group) animalswere applied with a thin layer of the extract mixed with oint-ment All vehicles were applied once daily till the day ofepithelization The wound tissue was removed from controlMEBO and RRDEE treated rats by sacrificing the animalson the 3rd 7th 11th and 15th day after wound creationAdditionally the rats of the three groupsweremaintained andtreated as above for calculating of the rate of contraction andperiod of epithelialization

232 Incision Wound Model The animals were randomlydivided into three groups of six Two 6 cm long paravertebralincisions were made using surgical blade (No 15) throughthe entire thickness of skin at a distance of about 2 cm fromthe midline on each side of the depilated back of the ratAfter the incision surgical sutures were applied to the partedskin at intervals of one centimeter The wounds were leftundressed then ointment base MEBO and RDEE ointmentwere applied daily up to 10 days When wounds were curedthoroughly the sutures were removed on day 10 and thetensile strength of cured wound skin was measured

24Measurement ofWoundContraction Woundmarginwastraced after wound creation by using transparent paper andthe area was measured by graph paper Wound contractionwas measured every two days interval throughout the moni-toring period

Wound contraction (100)

=Initial wound size minus Specific day wound size

Initial wound sizetimes 100

(1)

25 Epithelialization Period The epithelialization time wasmeasured from the initial day to the day when the scab felloff from the wound surface exclusive of leaving a raw woundbehind [14]

26 Measurement of Tensile Strength The force required toopen the healing action is known as the tensile strength

Evidence-Based Complementary and Alternative Medicine 3

It indicates how much the repaired tissue resists breakingunder tension and may indicate in part the quality of therepaired tissue The maximum load (tensile strength) tol-erated by wounds was measured blindly on coded samplesusing a biomechanical analyzing instrument (Instron Can-ton MA USA) Skin strips were stretched at a constantrate (1mmmin) until disruption occurred Wound breakingstrength was expressed as the meanmaximum level of tensilestrength in newton (N) before separation of wounds

27 Histopathological Studies Granulation tissues from con-trol and treatment groupswere taken on the 3rd 7th 11th and15th day after wound creation The specimens were formalinfixed and paraffin embedded according to the routine labora-torial techniques Subsequently serial 5120583mthick sectionswasobtained and stained with Masson-trichrome (for detectionof collagen fibers) and hematoxylin and eosin (HampE) (forgeneral morphological observations) Slides were examinedqualitatively under a light microscope for collagen forma-tion fibroblast proliferation angiogenesis and granulationtissue formation [1 15]

28 CD31 Immunohistochemistry Analysis and MicrovesselDensity (MVD) Angiogenesis was assessed by CD31 (Epit-omics CA USA) immunohistochemical in all the casesImmunohistochemical staining was performed on paraffinsections using the SP method After the sections were rinsedin distilled water the endogenous peroxidase was inactivatedwith 30 hydrogen peroxide in distilled water for 10minutesat room temperature After rinsing the sections in phosphate-buffered saline (PBS pH 74) the nonspecific binding site wasblocked with 10 normal goat serum for 20 minutes at roomtemperatureThe blocking serumwas discarded and then theprimary antibodies were added directly Rabbit anti-rat CD31monoclonal antibody (Epitomics CA USA) was diluted to1 500 in BSAThe sectionswere incubatedwith primary anti-bodies in a humid chamber at 4∘C overnight Sections werewashed three times in phosphate buffer solution (PBS) andgoat anti-rabbit polymer-peroxidase complex was added andthe sections were incubated for 30 minutes at room temper-ature After rinsing with PBS Streptavidin-horseradish per-oxidase conjugate was added and the peroxidase activity wasmade visible with diaminobenzidine and counterstainedwithhematoxylin for 30 sec then dehydrated and mountedQuantifications were performed by Image-Pro Plus 60 anal-ysis system to calculate the integral optical density (IOD) ofeach field

Themicrovessel density (MVD) was measured accordingto the method described by Zhen et al [16] Briefly in areasof the most intense CD31 positive neovascularization indi-vidual MVDs were made on a times200 magnification field Anyendothelial cell or endothelial cell cluster was considereda single countable microvessel MVD was expressed as theabsolute number of microvessels per times200 field for each case

29 RNA Extraction and Quantitative Real-Time PCR Theexpression patterns of TGF-1205731 and VEGF of rat wound tissueon the 3rd 7th 11th and 15th day after wound creation

were analyzed by quantitative real-time PCR Total RNA wasextracted from the Granulation tissue sample with TRIzol(Invitrogen Carlsbad CA USA) and then was further puri-fied step using the RNeasy Mini Kit (Qiagen) in each casefollowing the manufacturerrsquos instructionsThe concentrationof the total RNA was detected Total RNA (1mg) in a 20mLreaction volumewas reverse transcribed into cDNAusing thePrimeScript RT reagent kit (Takara Dalian China) Real-time PCR in 96-well optical plates was performed and anal-yzed with a Stratagene Mx3000P (Stratagene La Jolla CAUSA)The reactionswere performed in a 20mL volume usinga SYBR Green reaction mix (Takara Dalian China) with2mL cDNA The primers were TGF-1205731 forward 51015840-CGCAAC AAC GCA ATC TAT G-31015840 and reverse 51015840-ACC AAGGTAACGCCAGGA-31015840 VEGF forward 51015840-TCA CCAAAGCCAGCACATAGGAGA-31015840 and reverse 51015840-TTACACGTCTGC GGA TCT TGG ACA-31015840 GAPDH forward 51015840-GAACGG GAA GCT CAC TGG C-31015840 and reverse 51015840-GCA TGTCAGATCCACAACGG-31015840The thermal cycling consisted ofdenaturation for 30 sec at 95∘C followed by 40 cycles of 30 secat 95∘C and 30 sec at 60∘C The threshold cycle (CT) valuesof target genes were normalized with GAPDH of the samesample and expressed as they were relative to controls

210Western Blot Analysis Granulation tissues were homog-enized in RIPA lysis buffer (150mM NaCl 1 Nonidet P-4001 SDS 50mMTris-HCl pH 74 1mMEDTA 1mMPMSFand 1 times Roche complete Mini Protease Inhibitor Cock-tail) Protein concentration was determined using a BCAProtein Assay Kit Equal amounts of protein were separatedby 10 SDS gel electrophoresis (SDS-PAGE) under denatur-ing and nonreducing conditions and then transferred to anitrocellulose membrane The membrane was blocked with5 nonfat milk in TBST at room temperature for 1 h and thenincubated with anti-VEGF antibody (Abcam UK 1 200) at4∘C overnight After washing in TBST the blots were incu-bated with a horseradish-coupled secondary antibody Thesignals were visualized using the enhancement system (ECL)GAPDH was used as the internal control and treated withthe same protocol The amount of proteins in gel slabs wasquantified using a densitometer (Image Pro Plus 60 MediaCybernetics)

211 Statistical Analysis The data were expressed as mean plusmnSD and performed using SPSS (Version190 Chicago ILUSA) Significance was assessed by using the one-wayANOVA followed by 119905-test Values were considered statisti-cally significant when 119875 value is less than 005

3 Results

31 Phytochemical Analysis The phytochemical analysis ofthe extract by qualitative method showed the presence offlavonoids triterpenoids steroids cardiac glycosides anthra-quinones carbohydrates saponins and phenols (Table 1)

32 Wound Contraction Wound contraction is an essentialprocess in healing that leads to wound closure The rate

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Results of the phytochemical analysis of RD extract

Constituents ResultCardiac glycosides +Flavonoids +Catechin tannins minus

Triterpenes +Carotenoids minus

Anthraquinones +Carbohydrates +Saponins +Phenols +Steroids ++ presence minus absence

Day 3 Day 7 Day 11 Day 150

20

40

60

80

100

120

Control MEBO RDEE

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Wou

nd co

ntra

ctio

n (

)

Figure 1 The rate of contraction in control MEBO and RDEEtreated wound is shown here Values are expressed as mean plusmn SD(119899 = 6 animals) lowastlowast119875 lt 001 versus control

of contraction of the control group MEBO group and theRDEE treated group wounds is shown in (Figure 1) Theresults revealed that treatment with RDEE and MEBOresulted in much faster contraction of wound (119875 lt 005)

33 Epithelialization Time The epithelialization time wasmeasured from the first day The epithelialization time wasfound to be significantly (119875 lt 005) reduced in MEBO groupand RDEE group as depicted in (Figure 2) Mean time toreepithelialization was 1867 days (range 18ndash20 days) controlgroup 1416 days (range 13ndash15 days) MEBO group and 1512days (range 14ndash16 days) RDEE group There was no signifi-cant difference in the duration of wound healing between thegroups treated with MEBO and RDEE both healed by about15 days The control group however needed around 19 daysto heal about four days longer than the wound-healing timeneeded under MEBO and RDEE treatments

34 Tensile Strength of Incision Wound Model The results ofthe measurement of skin breaking strength on the 10th day

Control MEBO RDEE0

5

10

15

20

Animal groups

Epith

eliz

atio

n pe

riod

(day

s) lowastlowastlowastlowast

Figure 2 Period of epithelialization in control MEBO and RDEEtreated wounds is shown Values are expressed asmean plusmn SD (119899 = 6animals) lowastlowast119875 lt 001 versus control

Control MEBO RDEE0

3

6

9

12

15Te

nsile

stre

ngth

(N)

Animal groups

lowastlowastlowastlowast

Figure 3 Tensile strength measurements of 10th day wound tissueof control MEBO and RDEE treated rats Values are expressed asmean plusmn SD (119899 = 6 animals) lowastlowast119875 lt 001 versus control

after operation in incision wound-healing model weredepicted in (Figure 3) A significant increase in the woundbreaking strength (1153 plusmn 079N) was observed when com-pared with the controls (885 plusmn 048N)

35 Histopathological Study Histopathological examinationsof the healed wounds are shown in Figures 4 and 5 Two typesof stains were used Hematoxylin and Eosin (HampE) stainsand Masson-Trichrome stains for general morphology HampEstains collagen fibers pale pink cytoplasmpurple nuclei blueand red blood cells cherry red Masson-Trichrome stains col-lagen blue while cytoplasm red blood cells and muscle arestained red and is typically used to assess the advancementof collagen deposition during the formation of granulationtissue and matrix remodeling [17] The blue colour stainingintensity corresponds to the relative quantity of collagen fiber

Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c)

Figure 4 Photomicrograph of cutaneous wounds in rats at 7 days after wounding HampE stains (a) Control group (b) MEBO group and (c)RDEE group GT granulation tissue BV blood vessel F fibroblasts cells CF collagen fibers

deposit which reflects the process of synthesis and degrada-tion and remodeling [18] Histological sections of granulationtissue from RDEE treated rats showed more proliferat-ing blood capillaries collagen fibres and fibroblasts cells(Figure 4) which was similar to the effect of MEBO treatedgroup 7 days after wound creation when compared withthe control group On the 21st day after wound creation allgroups of experimental rats showed complete epithelializa-tion of the wound area Masson staining revealed that thecollagen bundles were thicker denser disorganized andmoreabundant in the control groups By contrast collage fiberswere decreased and more regularly ranged in the groupstreated with MEBO and RDEE Otherwise we observedsebaceous gland in the center (Figure 5)

36 Immunohistochemistry Analysis and MVD Vascularendothelial cells were detected using a mouse anti-rat CD31monoclonal antibody CD31 expressionwasmainly present inthe cytoplasm and membrane of endothelial cell or clusterBeing different from the control group condensed shortand twisted blood vessels were observed in the RDEE treatedgroup (Figure 6(a)) In quantitative analysis the intensityof CD31 was increased as compared with the control groupon the 3rd 7th and 11th day (Figure 6(b)) The microvessel

density (MVD) of the RDEE treated wounds was significantlyhigher than that of the control group (Table 2)

37 Gene Expression Analysis To investigate the molecularmechanism of RDEE-induced wound-healing activity theexpression levels of related genes were examined TGF-1205731and VEGF are the major genes that are generally involvedin wound healing Following several days of treatment asignificant increase in expression of TGF-1205731 and VEGF in thewound tissue of extract treated rats was noted as comparedwith the control animals (119875 lt 005)The results showed a day-dependent effect on TGF-1205731 and VEGF mRNA expressionThe mRNA expression levels of TGF-1205731 of the three groupsincreased from day 3 after injury reaching pick levels at day11 and then it declined constantly the mRNA expression ofVEGF was also increased from day 3 and correspondinglypeaked at day 7 (Figures 7(a) and 7(b)) TGF-1205731 and VEGFmRNA expression significantly correlated with the woundcontraction

38 Protein Levels by Western Blotting We further examinedthe protein levels of VEGF in the granulation tissues of ratsfrom the three groups by Western blotting Western blotanalysis showed an upregulated expression of VEGF in RDEE

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

1979 that it could serve as a substitute for Sanguis Draconisa precious crude medicine recorded in the official Chinesepharmacopoeia named as ldquodragonrsquos bloodrdquo [5] In Chinesemedicine Resina Draconis is a major component of the well-known hemostatic preparation ldquoYun Nan Bai Yaordquo so it isconsidered important for its potential application in Chinesemedical practice As a ldquopanacea of blood activatingrdquo resin RDhas great medicinal value and the main biological activitycomes from phenolic compounds [6] Pharmacological stud-ies have showed that RD has positive effects on treatmentof blood stasis syndrome trauma tumors inflammationgynecopathy allergic dermatitis and so on It can promoteblood circulation and serve as an antithrombotic antioxi-dant antiseptic and anti-inflammation compound [7] Theethanolic extracts of Resina Draconis possessed potentialantithrombotic properties affecting platelet aggregation andthus having anticoagulation activities [8] All these studiesindicate that Resina Draconis has enormous potential forfurther study

It is well known that one kind of traditional Chinesemedicine (TCM) usually contains a great number of compo-nents which all together contribute to therapeutic effectsDespite that Chen et al [9] have studied the wound-healingactivity of dragonrsquos blood (Croton lechleri sap) no scientificinvestigation is conducted on Resina Draconisrsquos wound-heal-ing potential Hence in this study we aimed to further inves-tigate the wound-healing effects of RD using the excision andincision wound models and to explore the possible mecha-nisms

2 Materials and Methods

21 Preparation of Plant Extracts Crude Resina Draconiswasprovided by the pharmacy department of No 9 PeoplersquosHospital Affiliated to Shanghai Jiao TongUniversity School ofMedicine RD (30 g)was dissolved in absolute alcohol at roomtemperature for 48 h under shade and then was filtered Thesolvent was concentrated under reduced pressure to ensurethat no residual methanol was left behind furnishing amethanol extract Normally 457 g of dried powder can beobtained from 30 g of RD The sample was stored at minus80∘Cuntil usedThe extract cream was formulated using ointmentas the vehicle The ointment consisted of propylene gly-col liquid paraffin (6 1) and applied topically onto the testanimals Extracts were prepared as 5 in the ointment

22 Phytochemical Analysis Preliminary phytochemicalanalysis was carried out using standard procedures to identifythe constituents as described by Evans and Trease [10] andHarborne [11]

23 Experimental Animals All study protocols were ap-proved by the Shanghai Jiao Tong University Medical CenterInstitutional Animal Care and Use Committee HealthySprague-Dawley male rats weighing between 180 and 200 gwere used The rats were housed in polypropylene cage andmaintained in standard laboratory conditions of temperature

(22 plusmn 2∘C) and light-dark cycle of 12 h 12 hThey were main-tained on standard pellet diet and provided with waterad-libitum throughout the experiment At the end of theexperiment the animals were sacrificed under anesthesia

231 Excision Wound Model The anesthetized rats wereinflicted with excision wounds as described by Morton andMalone [12] The dorsal fur of the animals was shaved withan electric clipper and the area of the wound to be createdwas outlined on the back of the animals with methylene blueusing a circular stainless steel stencil A full thickness of theexcision wound of circular area of 254mm2 and 2mm depthwas created along the markings with a surgical blade Theanimals were randomly divided into 3 groups of 24 for eachGroup I (control group) where animals were applied withsimple ointment base [13] Animals of Group II (standardgroup) were applied with a thin layer of moist exposed burnointment (MEBO) Group IIIrsquos (experimental group) animalswere applied with a thin layer of the extract mixed with oint-ment All vehicles were applied once daily till the day ofepithelization The wound tissue was removed from controlMEBO and RRDEE treated rats by sacrificing the animalson the 3rd 7th 11th and 15th day after wound creationAdditionally the rats of the three groupsweremaintained andtreated as above for calculating of the rate of contraction andperiod of epithelialization

232 Incision Wound Model The animals were randomlydivided into three groups of six Two 6 cm long paravertebralincisions were made using surgical blade (No 15) throughthe entire thickness of skin at a distance of about 2 cm fromthe midline on each side of the depilated back of the ratAfter the incision surgical sutures were applied to the partedskin at intervals of one centimeter The wounds were leftundressed then ointment base MEBO and RDEE ointmentwere applied daily up to 10 days When wounds were curedthoroughly the sutures were removed on day 10 and thetensile strength of cured wound skin was measured

24Measurement ofWoundContraction Woundmarginwastraced after wound creation by using transparent paper andthe area was measured by graph paper Wound contractionwas measured every two days interval throughout the moni-toring period

Wound contraction (100)

=Initial wound size minus Specific day wound size

Initial wound sizetimes 100

(1)

25 Epithelialization Period The epithelialization time wasmeasured from the initial day to the day when the scab felloff from the wound surface exclusive of leaving a raw woundbehind [14]

26 Measurement of Tensile Strength The force required toopen the healing action is known as the tensile strength

Evidence-Based Complementary and Alternative Medicine 3

It indicates how much the repaired tissue resists breakingunder tension and may indicate in part the quality of therepaired tissue The maximum load (tensile strength) tol-erated by wounds was measured blindly on coded samplesusing a biomechanical analyzing instrument (Instron Can-ton MA USA) Skin strips were stretched at a constantrate (1mmmin) until disruption occurred Wound breakingstrength was expressed as the meanmaximum level of tensilestrength in newton (N) before separation of wounds

27 Histopathological Studies Granulation tissues from con-trol and treatment groupswere taken on the 3rd 7th 11th and15th day after wound creation The specimens were formalinfixed and paraffin embedded according to the routine labora-torial techniques Subsequently serial 5120583mthick sectionswasobtained and stained with Masson-trichrome (for detectionof collagen fibers) and hematoxylin and eosin (HampE) (forgeneral morphological observations) Slides were examinedqualitatively under a light microscope for collagen forma-tion fibroblast proliferation angiogenesis and granulationtissue formation [1 15]

28 CD31 Immunohistochemistry Analysis and MicrovesselDensity (MVD) Angiogenesis was assessed by CD31 (Epit-omics CA USA) immunohistochemical in all the casesImmunohistochemical staining was performed on paraffinsections using the SP method After the sections were rinsedin distilled water the endogenous peroxidase was inactivatedwith 30 hydrogen peroxide in distilled water for 10minutesat room temperature After rinsing the sections in phosphate-buffered saline (PBS pH 74) the nonspecific binding site wasblocked with 10 normal goat serum for 20 minutes at roomtemperatureThe blocking serumwas discarded and then theprimary antibodies were added directly Rabbit anti-rat CD31monoclonal antibody (Epitomics CA USA) was diluted to1 500 in BSAThe sectionswere incubatedwith primary anti-bodies in a humid chamber at 4∘C overnight Sections werewashed three times in phosphate buffer solution (PBS) andgoat anti-rabbit polymer-peroxidase complex was added andthe sections were incubated for 30 minutes at room temper-ature After rinsing with PBS Streptavidin-horseradish per-oxidase conjugate was added and the peroxidase activity wasmade visible with diaminobenzidine and counterstainedwithhematoxylin for 30 sec then dehydrated and mountedQuantifications were performed by Image-Pro Plus 60 anal-ysis system to calculate the integral optical density (IOD) ofeach field

Themicrovessel density (MVD) was measured accordingto the method described by Zhen et al [16] Briefly in areasof the most intense CD31 positive neovascularization indi-vidual MVDs were made on a times200 magnification field Anyendothelial cell or endothelial cell cluster was considereda single countable microvessel MVD was expressed as theabsolute number of microvessels per times200 field for each case

29 RNA Extraction and Quantitative Real-Time PCR Theexpression patterns of TGF-1205731 and VEGF of rat wound tissueon the 3rd 7th 11th and 15th day after wound creation

were analyzed by quantitative real-time PCR Total RNA wasextracted from the Granulation tissue sample with TRIzol(Invitrogen Carlsbad CA USA) and then was further puri-fied step using the RNeasy Mini Kit (Qiagen) in each casefollowing the manufacturerrsquos instructionsThe concentrationof the total RNA was detected Total RNA (1mg) in a 20mLreaction volumewas reverse transcribed into cDNAusing thePrimeScript RT reagent kit (Takara Dalian China) Real-time PCR in 96-well optical plates was performed and anal-yzed with a Stratagene Mx3000P (Stratagene La Jolla CAUSA)The reactionswere performed in a 20mL volume usinga SYBR Green reaction mix (Takara Dalian China) with2mL cDNA The primers were TGF-1205731 forward 51015840-CGCAAC AAC GCA ATC TAT G-31015840 and reverse 51015840-ACC AAGGTAACGCCAGGA-31015840 VEGF forward 51015840-TCA CCAAAGCCAGCACATAGGAGA-31015840 and reverse 51015840-TTACACGTCTGC GGA TCT TGG ACA-31015840 GAPDH forward 51015840-GAACGG GAA GCT CAC TGG C-31015840 and reverse 51015840-GCA TGTCAGATCCACAACGG-31015840The thermal cycling consisted ofdenaturation for 30 sec at 95∘C followed by 40 cycles of 30 secat 95∘C and 30 sec at 60∘C The threshold cycle (CT) valuesof target genes were normalized with GAPDH of the samesample and expressed as they were relative to controls

210Western Blot Analysis Granulation tissues were homog-enized in RIPA lysis buffer (150mM NaCl 1 Nonidet P-4001 SDS 50mMTris-HCl pH 74 1mMEDTA 1mMPMSFand 1 times Roche complete Mini Protease Inhibitor Cock-tail) Protein concentration was determined using a BCAProtein Assay Kit Equal amounts of protein were separatedby 10 SDS gel electrophoresis (SDS-PAGE) under denatur-ing and nonreducing conditions and then transferred to anitrocellulose membrane The membrane was blocked with5 nonfat milk in TBST at room temperature for 1 h and thenincubated with anti-VEGF antibody (Abcam UK 1 200) at4∘C overnight After washing in TBST the blots were incu-bated with a horseradish-coupled secondary antibody Thesignals were visualized using the enhancement system (ECL)GAPDH was used as the internal control and treated withthe same protocol The amount of proteins in gel slabs wasquantified using a densitometer (Image Pro Plus 60 MediaCybernetics)

211 Statistical Analysis The data were expressed as mean plusmnSD and performed using SPSS (Version190 Chicago ILUSA) Significance was assessed by using the one-wayANOVA followed by 119905-test Values were considered statisti-cally significant when 119875 value is less than 005

3 Results

31 Phytochemical Analysis The phytochemical analysis ofthe extract by qualitative method showed the presence offlavonoids triterpenoids steroids cardiac glycosides anthra-quinones carbohydrates saponins and phenols (Table 1)

32 Wound Contraction Wound contraction is an essentialprocess in healing that leads to wound closure The rate

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Results of the phytochemical analysis of RD extract

Constituents ResultCardiac glycosides +Flavonoids +Catechin tannins minus

Triterpenes +Carotenoids minus

Anthraquinones +Carbohydrates +Saponins +Phenols +Steroids ++ presence minus absence

Day 3 Day 7 Day 11 Day 150

20

40

60

80

100

120

Control MEBO RDEE

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Wou

nd co

ntra

ctio

n (

)

Figure 1 The rate of contraction in control MEBO and RDEEtreated wound is shown here Values are expressed as mean plusmn SD(119899 = 6 animals) lowastlowast119875 lt 001 versus control

of contraction of the control group MEBO group and theRDEE treated group wounds is shown in (Figure 1) Theresults revealed that treatment with RDEE and MEBOresulted in much faster contraction of wound (119875 lt 005)

33 Epithelialization Time The epithelialization time wasmeasured from the first day The epithelialization time wasfound to be significantly (119875 lt 005) reduced in MEBO groupand RDEE group as depicted in (Figure 2) Mean time toreepithelialization was 1867 days (range 18ndash20 days) controlgroup 1416 days (range 13ndash15 days) MEBO group and 1512days (range 14ndash16 days) RDEE group There was no signifi-cant difference in the duration of wound healing between thegroups treated with MEBO and RDEE both healed by about15 days The control group however needed around 19 daysto heal about four days longer than the wound-healing timeneeded under MEBO and RDEE treatments

34 Tensile Strength of Incision Wound Model The results ofthe measurement of skin breaking strength on the 10th day

Control MEBO RDEE0

5

10

15

20

Animal groups

Epith

eliz

atio

n pe

riod

(day

s) lowastlowastlowastlowast

Figure 2 Period of epithelialization in control MEBO and RDEEtreated wounds is shown Values are expressed asmean plusmn SD (119899 = 6animals) lowastlowast119875 lt 001 versus control

Control MEBO RDEE0

3

6

9

12

15Te

nsile

stre

ngth

(N)

Animal groups

lowastlowastlowastlowast

Figure 3 Tensile strength measurements of 10th day wound tissueof control MEBO and RDEE treated rats Values are expressed asmean plusmn SD (119899 = 6 animals) lowastlowast119875 lt 001 versus control

after operation in incision wound-healing model weredepicted in (Figure 3) A significant increase in the woundbreaking strength (1153 plusmn 079N) was observed when com-pared with the controls (885 plusmn 048N)

35 Histopathological Study Histopathological examinationsof the healed wounds are shown in Figures 4 and 5 Two typesof stains were used Hematoxylin and Eosin (HampE) stainsand Masson-Trichrome stains for general morphology HampEstains collagen fibers pale pink cytoplasmpurple nuclei blueand red blood cells cherry red Masson-Trichrome stains col-lagen blue while cytoplasm red blood cells and muscle arestained red and is typically used to assess the advancementof collagen deposition during the formation of granulationtissue and matrix remodeling [17] The blue colour stainingintensity corresponds to the relative quantity of collagen fiber

Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c)

Figure 4 Photomicrograph of cutaneous wounds in rats at 7 days after wounding HampE stains (a) Control group (b) MEBO group and (c)RDEE group GT granulation tissue BV blood vessel F fibroblasts cells CF collagen fibers

deposit which reflects the process of synthesis and degrada-tion and remodeling [18] Histological sections of granulationtissue from RDEE treated rats showed more proliferat-ing blood capillaries collagen fibres and fibroblasts cells(Figure 4) which was similar to the effect of MEBO treatedgroup 7 days after wound creation when compared withthe control group On the 21st day after wound creation allgroups of experimental rats showed complete epithelializa-tion of the wound area Masson staining revealed that thecollagen bundles were thicker denser disorganized andmoreabundant in the control groups By contrast collage fiberswere decreased and more regularly ranged in the groupstreated with MEBO and RDEE Otherwise we observedsebaceous gland in the center (Figure 5)

36 Immunohistochemistry Analysis and MVD Vascularendothelial cells were detected using a mouse anti-rat CD31monoclonal antibody CD31 expressionwasmainly present inthe cytoplasm and membrane of endothelial cell or clusterBeing different from the control group condensed shortand twisted blood vessels were observed in the RDEE treatedgroup (Figure 6(a)) In quantitative analysis the intensityof CD31 was increased as compared with the control groupon the 3rd 7th and 11th day (Figure 6(b)) The microvessel

density (MVD) of the RDEE treated wounds was significantlyhigher than that of the control group (Table 2)

37 Gene Expression Analysis To investigate the molecularmechanism of RDEE-induced wound-healing activity theexpression levels of related genes were examined TGF-1205731and VEGF are the major genes that are generally involvedin wound healing Following several days of treatment asignificant increase in expression of TGF-1205731 and VEGF in thewound tissue of extract treated rats was noted as comparedwith the control animals (119875 lt 005)The results showed a day-dependent effect on TGF-1205731 and VEGF mRNA expressionThe mRNA expression levels of TGF-1205731 of the three groupsincreased from day 3 after injury reaching pick levels at day11 and then it declined constantly the mRNA expression ofVEGF was also increased from day 3 and correspondinglypeaked at day 7 (Figures 7(a) and 7(b)) TGF-1205731 and VEGFmRNA expression significantly correlated with the woundcontraction

38 Protein Levels by Western Blotting We further examinedthe protein levels of VEGF in the granulation tissues of ratsfrom the three groups by Western blotting Western blotanalysis showed an upregulated expression of VEGF in RDEE

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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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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 3

It indicates how much the repaired tissue resists breakingunder tension and may indicate in part the quality of therepaired tissue The maximum load (tensile strength) tol-erated by wounds was measured blindly on coded samplesusing a biomechanical analyzing instrument (Instron Can-ton MA USA) Skin strips were stretched at a constantrate (1mmmin) until disruption occurred Wound breakingstrength was expressed as the meanmaximum level of tensilestrength in newton (N) before separation of wounds

27 Histopathological Studies Granulation tissues from con-trol and treatment groupswere taken on the 3rd 7th 11th and15th day after wound creation The specimens were formalinfixed and paraffin embedded according to the routine labora-torial techniques Subsequently serial 5120583mthick sectionswasobtained and stained with Masson-trichrome (for detectionof collagen fibers) and hematoxylin and eosin (HampE) (forgeneral morphological observations) Slides were examinedqualitatively under a light microscope for collagen forma-tion fibroblast proliferation angiogenesis and granulationtissue formation [1 15]

28 CD31 Immunohistochemistry Analysis and MicrovesselDensity (MVD) Angiogenesis was assessed by CD31 (Epit-omics CA USA) immunohistochemical in all the casesImmunohistochemical staining was performed on paraffinsections using the SP method After the sections were rinsedin distilled water the endogenous peroxidase was inactivatedwith 30 hydrogen peroxide in distilled water for 10minutesat room temperature After rinsing the sections in phosphate-buffered saline (PBS pH 74) the nonspecific binding site wasblocked with 10 normal goat serum for 20 minutes at roomtemperatureThe blocking serumwas discarded and then theprimary antibodies were added directly Rabbit anti-rat CD31monoclonal antibody (Epitomics CA USA) was diluted to1 500 in BSAThe sectionswere incubatedwith primary anti-bodies in a humid chamber at 4∘C overnight Sections werewashed three times in phosphate buffer solution (PBS) andgoat anti-rabbit polymer-peroxidase complex was added andthe sections were incubated for 30 minutes at room temper-ature After rinsing with PBS Streptavidin-horseradish per-oxidase conjugate was added and the peroxidase activity wasmade visible with diaminobenzidine and counterstainedwithhematoxylin for 30 sec then dehydrated and mountedQuantifications were performed by Image-Pro Plus 60 anal-ysis system to calculate the integral optical density (IOD) ofeach field

Themicrovessel density (MVD) was measured accordingto the method described by Zhen et al [16] Briefly in areasof the most intense CD31 positive neovascularization indi-vidual MVDs were made on a times200 magnification field Anyendothelial cell or endothelial cell cluster was considereda single countable microvessel MVD was expressed as theabsolute number of microvessels per times200 field for each case

29 RNA Extraction and Quantitative Real-Time PCR Theexpression patterns of TGF-1205731 and VEGF of rat wound tissueon the 3rd 7th 11th and 15th day after wound creation

were analyzed by quantitative real-time PCR Total RNA wasextracted from the Granulation tissue sample with TRIzol(Invitrogen Carlsbad CA USA) and then was further puri-fied step using the RNeasy Mini Kit (Qiagen) in each casefollowing the manufacturerrsquos instructionsThe concentrationof the total RNA was detected Total RNA (1mg) in a 20mLreaction volumewas reverse transcribed into cDNAusing thePrimeScript RT reagent kit (Takara Dalian China) Real-time PCR in 96-well optical plates was performed and anal-yzed with a Stratagene Mx3000P (Stratagene La Jolla CAUSA)The reactionswere performed in a 20mL volume usinga SYBR Green reaction mix (Takara Dalian China) with2mL cDNA The primers were TGF-1205731 forward 51015840-CGCAAC AAC GCA ATC TAT G-31015840 and reverse 51015840-ACC AAGGTAACGCCAGGA-31015840 VEGF forward 51015840-TCA CCAAAGCCAGCACATAGGAGA-31015840 and reverse 51015840-TTACACGTCTGC GGA TCT TGG ACA-31015840 GAPDH forward 51015840-GAACGG GAA GCT CAC TGG C-31015840 and reverse 51015840-GCA TGTCAGATCCACAACGG-31015840The thermal cycling consisted ofdenaturation for 30 sec at 95∘C followed by 40 cycles of 30 secat 95∘C and 30 sec at 60∘C The threshold cycle (CT) valuesof target genes were normalized with GAPDH of the samesample and expressed as they were relative to controls

210Western Blot Analysis Granulation tissues were homog-enized in RIPA lysis buffer (150mM NaCl 1 Nonidet P-4001 SDS 50mMTris-HCl pH 74 1mMEDTA 1mMPMSFand 1 times Roche complete Mini Protease Inhibitor Cock-tail) Protein concentration was determined using a BCAProtein Assay Kit Equal amounts of protein were separatedby 10 SDS gel electrophoresis (SDS-PAGE) under denatur-ing and nonreducing conditions and then transferred to anitrocellulose membrane The membrane was blocked with5 nonfat milk in TBST at room temperature for 1 h and thenincubated with anti-VEGF antibody (Abcam UK 1 200) at4∘C overnight After washing in TBST the blots were incu-bated with a horseradish-coupled secondary antibody Thesignals were visualized using the enhancement system (ECL)GAPDH was used as the internal control and treated withthe same protocol The amount of proteins in gel slabs wasquantified using a densitometer (Image Pro Plus 60 MediaCybernetics)

211 Statistical Analysis The data were expressed as mean plusmnSD and performed using SPSS (Version190 Chicago ILUSA) Significance was assessed by using the one-wayANOVA followed by 119905-test Values were considered statisti-cally significant when 119875 value is less than 005

3 Results

31 Phytochemical Analysis The phytochemical analysis ofthe extract by qualitative method showed the presence offlavonoids triterpenoids steroids cardiac glycosides anthra-quinones carbohydrates saponins and phenols (Table 1)

32 Wound Contraction Wound contraction is an essentialprocess in healing that leads to wound closure The rate

4 Evidence-Based Complementary and Alternative Medicine

Table 1 Results of the phytochemical analysis of RD extract

Constituents ResultCardiac glycosides +Flavonoids +Catechin tannins minus

Triterpenes +Carotenoids minus

Anthraquinones +Carbohydrates +Saponins +Phenols +Steroids ++ presence minus absence

Day 3 Day 7 Day 11 Day 150

20

40

60

80

100

120

Control MEBO RDEE

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Wou

nd co

ntra

ctio

n (

)

Figure 1 The rate of contraction in control MEBO and RDEEtreated wound is shown here Values are expressed as mean plusmn SD(119899 = 6 animals) lowastlowast119875 lt 001 versus control

of contraction of the control group MEBO group and theRDEE treated group wounds is shown in (Figure 1) Theresults revealed that treatment with RDEE and MEBOresulted in much faster contraction of wound (119875 lt 005)

33 Epithelialization Time The epithelialization time wasmeasured from the first day The epithelialization time wasfound to be significantly (119875 lt 005) reduced in MEBO groupand RDEE group as depicted in (Figure 2) Mean time toreepithelialization was 1867 days (range 18ndash20 days) controlgroup 1416 days (range 13ndash15 days) MEBO group and 1512days (range 14ndash16 days) RDEE group There was no signifi-cant difference in the duration of wound healing between thegroups treated with MEBO and RDEE both healed by about15 days The control group however needed around 19 daysto heal about four days longer than the wound-healing timeneeded under MEBO and RDEE treatments

34 Tensile Strength of Incision Wound Model The results ofthe measurement of skin breaking strength on the 10th day

Control MEBO RDEE0

5

10

15

20

Animal groups

Epith

eliz

atio

n pe

riod

(day

s) lowastlowastlowastlowast

Figure 2 Period of epithelialization in control MEBO and RDEEtreated wounds is shown Values are expressed asmean plusmn SD (119899 = 6animals) lowastlowast119875 lt 001 versus control

Control MEBO RDEE0

3

6

9

12

15Te

nsile

stre

ngth

(N)

Animal groups

lowastlowastlowastlowast

Figure 3 Tensile strength measurements of 10th day wound tissueof control MEBO and RDEE treated rats Values are expressed asmean plusmn SD (119899 = 6 animals) lowastlowast119875 lt 001 versus control

after operation in incision wound-healing model weredepicted in (Figure 3) A significant increase in the woundbreaking strength (1153 plusmn 079N) was observed when com-pared with the controls (885 plusmn 048N)

35 Histopathological Study Histopathological examinationsof the healed wounds are shown in Figures 4 and 5 Two typesof stains were used Hematoxylin and Eosin (HampE) stainsand Masson-Trichrome stains for general morphology HampEstains collagen fibers pale pink cytoplasmpurple nuclei blueand red blood cells cherry red Masson-Trichrome stains col-lagen blue while cytoplasm red blood cells and muscle arestained red and is typically used to assess the advancementof collagen deposition during the formation of granulationtissue and matrix remodeling [17] The blue colour stainingintensity corresponds to the relative quantity of collagen fiber

Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c)

Figure 4 Photomicrograph of cutaneous wounds in rats at 7 days after wounding HampE stains (a) Control group (b) MEBO group and (c)RDEE group GT granulation tissue BV blood vessel F fibroblasts cells CF collagen fibers

deposit which reflects the process of synthesis and degrada-tion and remodeling [18] Histological sections of granulationtissue from RDEE treated rats showed more proliferat-ing blood capillaries collagen fibres and fibroblasts cells(Figure 4) which was similar to the effect of MEBO treatedgroup 7 days after wound creation when compared withthe control group On the 21st day after wound creation allgroups of experimental rats showed complete epithelializa-tion of the wound area Masson staining revealed that thecollagen bundles were thicker denser disorganized andmoreabundant in the control groups By contrast collage fiberswere decreased and more regularly ranged in the groupstreated with MEBO and RDEE Otherwise we observedsebaceous gland in the center (Figure 5)

36 Immunohistochemistry Analysis and MVD Vascularendothelial cells were detected using a mouse anti-rat CD31monoclonal antibody CD31 expressionwasmainly present inthe cytoplasm and membrane of endothelial cell or clusterBeing different from the control group condensed shortand twisted blood vessels were observed in the RDEE treatedgroup (Figure 6(a)) In quantitative analysis the intensityof CD31 was increased as compared with the control groupon the 3rd 7th and 11th day (Figure 6(b)) The microvessel

density (MVD) of the RDEE treated wounds was significantlyhigher than that of the control group (Table 2)

37 Gene Expression Analysis To investigate the molecularmechanism of RDEE-induced wound-healing activity theexpression levels of related genes were examined TGF-1205731and VEGF are the major genes that are generally involvedin wound healing Following several days of treatment asignificant increase in expression of TGF-1205731 and VEGF in thewound tissue of extract treated rats was noted as comparedwith the control animals (119875 lt 005)The results showed a day-dependent effect on TGF-1205731 and VEGF mRNA expressionThe mRNA expression levels of TGF-1205731 of the three groupsincreased from day 3 after injury reaching pick levels at day11 and then it declined constantly the mRNA expression ofVEGF was also increased from day 3 and correspondinglypeaked at day 7 (Figures 7(a) and 7(b)) TGF-1205731 and VEGFmRNA expression significantly correlated with the woundcontraction

38 Protein Levels by Western Blotting We further examinedthe protein levels of VEGF in the granulation tissues of ratsfrom the three groups by Western blotting Western blotanalysis showed an upregulated expression of VEGF in RDEE

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

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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

Table 1 Results of the phytochemical analysis of RD extract

Constituents ResultCardiac glycosides +Flavonoids +Catechin tannins minus

Triterpenes +Carotenoids minus

Anthraquinones +Carbohydrates +Saponins +Phenols +Steroids ++ presence minus absence

Day 3 Day 7 Day 11 Day 150

20

40

60

80

100

120

Control MEBO RDEE

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Wou

nd co

ntra

ctio

n (

)

Figure 1 The rate of contraction in control MEBO and RDEEtreated wound is shown here Values are expressed as mean plusmn SD(119899 = 6 animals) lowastlowast119875 lt 001 versus control

of contraction of the control group MEBO group and theRDEE treated group wounds is shown in (Figure 1) Theresults revealed that treatment with RDEE and MEBOresulted in much faster contraction of wound (119875 lt 005)

33 Epithelialization Time The epithelialization time wasmeasured from the first day The epithelialization time wasfound to be significantly (119875 lt 005) reduced in MEBO groupand RDEE group as depicted in (Figure 2) Mean time toreepithelialization was 1867 days (range 18ndash20 days) controlgroup 1416 days (range 13ndash15 days) MEBO group and 1512days (range 14ndash16 days) RDEE group There was no signifi-cant difference in the duration of wound healing between thegroups treated with MEBO and RDEE both healed by about15 days The control group however needed around 19 daysto heal about four days longer than the wound-healing timeneeded under MEBO and RDEE treatments

34 Tensile Strength of Incision Wound Model The results ofthe measurement of skin breaking strength on the 10th day

Control MEBO RDEE0

5

10

15

20

Animal groups

Epith

eliz

atio

n pe

riod

(day

s) lowastlowastlowastlowast

Figure 2 Period of epithelialization in control MEBO and RDEEtreated wounds is shown Values are expressed asmean plusmn SD (119899 = 6animals) lowastlowast119875 lt 001 versus control

Control MEBO RDEE0

3

6

9

12

15Te

nsile

stre

ngth

(N)

Animal groups

lowastlowastlowastlowast

Figure 3 Tensile strength measurements of 10th day wound tissueof control MEBO and RDEE treated rats Values are expressed asmean plusmn SD (119899 = 6 animals) lowastlowast119875 lt 001 versus control

after operation in incision wound-healing model weredepicted in (Figure 3) A significant increase in the woundbreaking strength (1153 plusmn 079N) was observed when com-pared with the controls (885 plusmn 048N)

35 Histopathological Study Histopathological examinationsof the healed wounds are shown in Figures 4 and 5 Two typesof stains were used Hematoxylin and Eosin (HampE) stainsand Masson-Trichrome stains for general morphology HampEstains collagen fibers pale pink cytoplasmpurple nuclei blueand red blood cells cherry red Masson-Trichrome stains col-lagen blue while cytoplasm red blood cells and muscle arestained red and is typically used to assess the advancementof collagen deposition during the formation of granulationtissue and matrix remodeling [17] The blue colour stainingintensity corresponds to the relative quantity of collagen fiber

Evidence-Based Complementary and Alternative Medicine 5

(a) (b) (c)

Figure 4 Photomicrograph of cutaneous wounds in rats at 7 days after wounding HampE stains (a) Control group (b) MEBO group and (c)RDEE group GT granulation tissue BV blood vessel F fibroblasts cells CF collagen fibers

deposit which reflects the process of synthesis and degrada-tion and remodeling [18] Histological sections of granulationtissue from RDEE treated rats showed more proliferat-ing blood capillaries collagen fibres and fibroblasts cells(Figure 4) which was similar to the effect of MEBO treatedgroup 7 days after wound creation when compared withthe control group On the 21st day after wound creation allgroups of experimental rats showed complete epithelializa-tion of the wound area Masson staining revealed that thecollagen bundles were thicker denser disorganized andmoreabundant in the control groups By contrast collage fiberswere decreased and more regularly ranged in the groupstreated with MEBO and RDEE Otherwise we observedsebaceous gland in the center (Figure 5)

36 Immunohistochemistry Analysis and MVD Vascularendothelial cells were detected using a mouse anti-rat CD31monoclonal antibody CD31 expressionwasmainly present inthe cytoplasm and membrane of endothelial cell or clusterBeing different from the control group condensed shortand twisted blood vessels were observed in the RDEE treatedgroup (Figure 6(a)) In quantitative analysis the intensityof CD31 was increased as compared with the control groupon the 3rd 7th and 11th day (Figure 6(b)) The microvessel

density (MVD) of the RDEE treated wounds was significantlyhigher than that of the control group (Table 2)

37 Gene Expression Analysis To investigate the molecularmechanism of RDEE-induced wound-healing activity theexpression levels of related genes were examined TGF-1205731and VEGF are the major genes that are generally involvedin wound healing Following several days of treatment asignificant increase in expression of TGF-1205731 and VEGF in thewound tissue of extract treated rats was noted as comparedwith the control animals (119875 lt 005)The results showed a day-dependent effect on TGF-1205731 and VEGF mRNA expressionThe mRNA expression levels of TGF-1205731 of the three groupsincreased from day 3 after injury reaching pick levels at day11 and then it declined constantly the mRNA expression ofVEGF was also increased from day 3 and correspondinglypeaked at day 7 (Figures 7(a) and 7(b)) TGF-1205731 and VEGFmRNA expression significantly correlated with the woundcontraction

38 Protein Levels by Western Blotting We further examinedthe protein levels of VEGF in the granulation tissues of ratsfrom the three groups by Western blotting Western blotanalysis showed an upregulated expression of VEGF in RDEE

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

(a) (b) (c)

Figure 4 Photomicrograph of cutaneous wounds in rats at 7 days after wounding HampE stains (a) Control group (b) MEBO group and (c)RDEE group GT granulation tissue BV blood vessel F fibroblasts cells CF collagen fibers

deposit which reflects the process of synthesis and degrada-tion and remodeling [18] Histological sections of granulationtissue from RDEE treated rats showed more proliferat-ing blood capillaries collagen fibres and fibroblasts cells(Figure 4) which was similar to the effect of MEBO treatedgroup 7 days after wound creation when compared withthe control group On the 21st day after wound creation allgroups of experimental rats showed complete epithelializa-tion of the wound area Masson staining revealed that thecollagen bundles were thicker denser disorganized andmoreabundant in the control groups By contrast collage fiberswere decreased and more regularly ranged in the groupstreated with MEBO and RDEE Otherwise we observedsebaceous gland in the center (Figure 5)

36 Immunohistochemistry Analysis and MVD Vascularendothelial cells were detected using a mouse anti-rat CD31monoclonal antibody CD31 expressionwasmainly present inthe cytoplasm and membrane of endothelial cell or clusterBeing different from the control group condensed shortand twisted blood vessels were observed in the RDEE treatedgroup (Figure 6(a)) In quantitative analysis the intensityof CD31 was increased as compared with the control groupon the 3rd 7th and 11th day (Figure 6(b)) The microvessel

density (MVD) of the RDEE treated wounds was significantlyhigher than that of the control group (Table 2)

37 Gene Expression Analysis To investigate the molecularmechanism of RDEE-induced wound-healing activity theexpression levels of related genes were examined TGF-1205731and VEGF are the major genes that are generally involvedin wound healing Following several days of treatment asignificant increase in expression of TGF-1205731 and VEGF in thewound tissue of extract treated rats was noted as comparedwith the control animals (119875 lt 005)The results showed a day-dependent effect on TGF-1205731 and VEGF mRNA expressionThe mRNA expression levels of TGF-1205731 of the three groupsincreased from day 3 after injury reaching pick levels at day11 and then it declined constantly the mRNA expression ofVEGF was also increased from day 3 and correspondinglypeaked at day 7 (Figures 7(a) and 7(b)) TGF-1205731 and VEGFmRNA expression significantly correlated with the woundcontraction

38 Protein Levels by Western Blotting We further examinedthe protein levels of VEGF in the granulation tissues of ratsfrom the three groups by Western blotting Western blotanalysis showed an upregulated expression of VEGF in RDEE

6 Evidence-Based Complementary and Alternative Medicine

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

(a) (b) (c)

Figure 5 Photomicrograph showing histopathological changes of healed skin wounds on day 21 of postwounding (stained with hematoxylin-eosin and masson-trichrome) Collagen fibers were arranged more regularly and sparse than those of the scar tissue in the control group (a)Control group (b) MEBO group and (c) RDEE group SG sebaceous gland

Table 2 The comparison of MVD (mean plusmn SD) between each group

Group MVD (mean plusmn SD)3 d 7 d 11 d 15 d

Control 2000 plusmn 228 2467 plusmn 450 4600 plusmn 587 5500 plusmn 405MEBO 3200 plusmn 335lowastlowast 6083 plusmn 354lowastlowast 6900 plusmn 358lowastlowast 4433 plusmn 413lowastlowast

RDEE 3533 plusmn 388lowastlowast 6250 plusmn 356lowastlowast 7233 plusmn 432lowastlowast 5150 plusmn 836All values are expressed as the mean plusmn SD Means labeled with superscripts were significantly different lowastlowast119875 lt 001 versus control

treated group as compared with the control group On the3rd 7th and 11th day after wound creation the protein levelsof VEGF were significantly increased in RDEE treated group(119875 lt 005) (Figure 8(a)) Quantified protein level and the foldchanges were shown in Figure 8(b)

4 Discussion

Wound healing is a complicated process The aim of woundhealing is to promote rapid wound closure and recoverfunctional properties Hence in this study excision and inci-sion woundmodels were used to evaluate the effects of RDEE

on wound healing The significant reduction in wound sizeand mean epithelization time as well as the higher expressionof growth factors in the RDEE treated group as comparedwith those from vehicle group corroborate with the histo-pathological findings of increased epithelization activityangiogenesis and higher collagen fibers formation Thesefindings imply that RDEE promoted wound-healing activityvia angiogenesis collagen deposition epithelization andwound contraction

Excisional and incisionalwounds are the twomainwoundmodels in wound research which allowed the determinationof the wound-healing phases The excisional wound is foundto be more suitable for histological evaluation due to the

Evidence-Based Complementary and Alternative Medicine 7

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

(A) (B) (C)

(a)

Day 3 Day 7 Day 11 Day 150

5

10

CD31

pos

itive

stai

ning

(IO

D f

old

chan

ge)

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowastlowastlowast

Control MEBO RDEE

(b)

Figure 6 The histologic anti-rat CD31 staining of wound tissue samples (a) Endothelial cells stained with the antibody were represented bybrown colour (A) Control group (B) MEBO group and (C) RDEE group BV blood vessel (b) Quantitative analysis of the CD31 stain wascalculated All data were expressed as mean plusmn SD lowastlowast119875 lt 001 versus control

broadermorphological changes occurring during the processof wound healing Wound contraction is an essential processin healingwhich leads towound closureThus visible appear-ances and measurements of wound contraction become reli-able parameters inmacroscopic evaluation for wound healing[19] This study showed that RDEE significantly stimulatedthe contraction of wounds as seen from the percentage ofwound contraction (Figure 1) Reepithelialization is impor-tant as it restores the integrity of the skin making it lessvulnerable to infection The extract-treated animals showeda decreased time to epithelialization (Figure 2) comparedwith the control group In incision wound the increase intensile strength of RDEE treated wounds may be due to theincrease in collagen concentration and stabilization of thefibres [20] Since incision wound treated with RDEE showedgreater tensile strength it might be speculated that it not onlyincreased collagen synthesis per cell but also aided in cross-linking of the protein Histological analysis further revealedthat topical application of RDEE significantly increased thefibroblast growth collagen synthesis and the healing process

Histological evaluation showed that healing process of thewounded tissue in RDEE treated group was comparably close

to the reference MEBO treated group whereas significantdifference was observed in negative control group Granula-tion tissue primarily contains fibroblasts collagen fibres veryless edema and newly generated blood vessels which wereobserved in RDEE treated group of animals (Figure 4) Thishistopathological observation provided additional evidencefor the experimental wound-healing studies based on thecontraction value of wound areas and the measurementof tensile strength Enhanced healing activity has beenattributed to increased collagen deposition and angiogenesis[21] Collagen plays a central role in the healing of woundsand it is a principal component of connective tissue andprovides a structural framework for the regenerating tissueHistopathological study showed better proliferation of col-lagen fibers in the RDEE group compared with the controlgroup (Figure 4) In addition the slim defined and well-organized collagen fibers in the RDEE treated group reinforcethe improved quality of the final remodeling of the wound(Figure 5) Angiogenesis during wound repair serves the dualfunction of providing the nutrients required by supplyingessential nutrients and oxygen to the wound site and promot-ing granulation tissue formation [22] Histological evaluation

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

8 Evidence-Based Complementary and Alternative Medicine

Day 3 Day 7 Day 11 Day 150

1

2

3

4

5TG

F-120573

1 m

RNA

relat

ive l

evel

s (fo

ld ch

ange

)

lowast lowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

Control MEBO RDEE

(a)

Day 3 Day 7 Day 11 Day 150

05

1

15

2

VEG

F m

RNA

relat

ive l

evels

(fold

chan

ge) lowast lowast lowast lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

Control MEBO RDEE

(b)

Figure 7 TGF-1205731 and VEGF mRNA expression in cutaneouswounds (a) TGF-1205731 (b) VEGF Normalization relative to GAPDHwas performed Results presented in bar graph are the mean plusmn SDlowast119875 lt 005 lowastlowast119875 lt 001 versus control

showed an increase in the number of blood vessels in thegranulation tissue of the rats treated with RDEE Enhancedexpression of CD31 as revealed through immunohistochem-istry in RDEE treated group might be responsible for thisactivity In the present study the treatment group was foundto increase angiogenesis as evidenced by MVD (Table 2)

Many types of cytokines and growth factors are respon-sible for inflammation reepithelialization the formation ofgranulation tissue and neovascularization during the woundhealing process [23] Transforming growth factor beta isan important growth factor that regulates different cellularfunctions in all phases of wound healing TGF-1205731 producedby fibroblasts as a multifunctional cytokine acts on thesecells [24] and enhances granulation tissue formation andcollagen formation in wound-healing process [25] TGF-1205731has also been reported to encourage wound contraction

VEGFGAPDH

(A) (B) (C)

3d 7d 11d 15d 3d 7d 11d15d 3d 7d 11d 15d

(a)

Day 3 Day 7 Day 11 Day 150

6

12

18

VEG

FG

APD

H ra

tion

Control MEBO RDEE

lowast

lowast

lowastlowastlowast

(b)

Figure 8 (a) Western blot results for vascular endothelial growthfactor (VEGF) protein in the three groups (b) Quantification ofdensitometry analysis of protein levels All data were expressed asmean plusmn SD lowast119875 lt 005 lowastlowast119875 lt 001 versus control

through its direct induction of alpha smooth muscle actinexpression in fibroblasts [26] Otherwise VEGF appears tobe a key factor in pathological situations such as tissuerepair which involves neovascularization and increased vas-cular permeability VEGF improves angiogenesis during theprocess of wound healing by stimulating the migration ofendothelial cells through the extracellular matrix [27] VEGFalso has been demonstrated to mediate angiogenic activityduring the proliferative phase of wound healing [28] Inaddition VEGF mediates vascular hyperpermeability andpromotes the secretion of active growth factors and cytokinesnecessary for wound repair [29] The animals treated withRDEE significantly enhanced the expression of VEGF whichis the most potent angiogenic factor during wound healingthereby stimulating the formation of new blood vessels [30]In the present study our results showed that the expressionof VEGF was significantly higher in the RDEE treated groupand peaked on the 7th day (Figures 7(b) and 8)

The wound-healing property of RD may be attributed tothe phytoconstituents present in the resina and the quickerprocess of wound healing could be a function of either theindividual or the additive effects of the phytoconstituents Inethnopharmacological studies the effects of dragonrsquos bloodon various biological activities such as attenuate visceralnociception antiviral antibacterial and antifungal have beenreported [31 32] Our preliminary phytochemical screening

Evidence-Based Complementary and Alternative Medicine 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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 9

of ethanolic extract of RD showed the presence of flavonoidstriterpenoids steroids cardiac glycosides anthraquinonescarbohydrates saponins and saponins (Table 1) Recent stud-ies with other plant extracts have shown that phytochemicalconstituents such as flavonoids [33] and triterpenoids [34] areknown to promote the wound-healing process mainly due totheir astringent and antimicrobial properties which appearto be responsible for wound contraction and increased rate ofepithelization Previous studies have revealed that the resin isrich in flavonoids sterols and terpenoids [35 36] Possiblythe wound-healing action of RD may probably be due tothe presence of phytoconstituents in the plant or could be afunction of either the individual or the additive effects of thephytoconstituents however further phytochemical studiesare needed to isolate the active compound(s) responsible forthese pharmacological activities

5 Conclusion

We have shown that the RDEE facilitates wound healing inthe experimental animal model There is a need for furtherstudies in order to isolate the active ingredients in the plantthat are responsible for its biological activities and to elucidatethe mechanisms of actions of these active ingredients

Authorsrsquo Contribution

Huihui Liu and Shaohui Lin contributed equally to the paperas joint first authors

Acknowledgments

This study was supported by the Science and TechnologyCommission of Shanghai Municipality (no 09DZ1970400)The authors sincerely thank Zhicheng Song and Xueyi Fengfor their excellent technical assistance during this work

References

[1] 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

[2] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivowoundhealing potential ofHypericumperforatumLrdquoJournal of Ethnopharmacology vol 127 no 2 pp 468ndash477 2010

[3] M Fronza B HeinzmannMHamburger S Laufer and IMer-fort ldquoDetermination of the wound healing effect of Calendulaextracts using the scratch assay with 3T3 fibroblastsrdquo Journalof Ethnopharmacology vol 126 no 3 pp 463ndash467 2009

[4] X Cai and Z Xu ldquoStudies on the plant origin of ChineseDragonrsquos bloodrdquo Acta Botanica Yunnanica vol 1 pp 1ndash9 1979

[5] C P Committee Pharmacopoeia of the Peoplersquos Republic ofChina vol 88 China Chemical Industry Press Beijing China2005

[6] Z Zhihong W Jinliang and Y Chongren ldquoChemical con-stituents of Sanguis Draxonis made in Chinardquo Zhong Cao Yaovol 32 no 6 pp 484ndash486 2001

[7] C S Choy C M Hu W T Chiu et al ldquoSuppression of lipo-polysaccharide-induced of inducible nitric oxide synthase andcyclooxygenase-2 by Sanguis Draconis a dragonrsquos blood resinin RAW 2647 cellsrdquo Journal of Ethnopharmacology vol 115 no3 pp 455ndash462 2007

[8] N Xin Y J Li Y Li et al ldquoDragonrsquos Blood extract has anti-thrombotic properties affecting platelet aggregation functionsand anticoagulation activitiesrdquo Journal of Ethnopharmacologyvol 135 no 2 pp 510ndash514 2011

[9] Z P Chen Y Cai and J D Phillipson ldquoStudies on theanti-tumour anti-bacterial and wound-healing properties ofDragonrsquos bloodrdquo PlantaMedica vol 60 no 6 pp 541ndash545 1994

[10] W Evans and G Trease Pharmacognosy Bailliere TindalLondon UK 1989

[11] J Harborne ldquoMethods of plant analysisrdquo in PhytochemicalMethods vol 132 1973

[12] J J Morton and M H Malone ldquoEvaluation of vulneray activityby an open wound procedure in ratsrdquo Archives Internationalesde Pharmacodynamie et de Therapie vol 196 no 1 pp 117ndash1261972

[13] A Asif G Kakub S Mehmood R Khunum and M GulfrazldquoWound healing activity of root extracts of Berberis lyceumRoyle in ratsrdquo Phytotherapy Research vol 21 no 6 pp 589ndash5912007

[14] A N Rashed F U Afifi and A M Disi ldquoSimple evaluationof the wound healing activity of a crude extract of Portulacaoleracea L (growing in Jordan) inMus musculus JVI-1rdquo Journalof Ethnopharmacology vol 88 no 2-3 pp 131ndash136 2003

[15] G J Kaur and D S Arora ldquoAntibacterial and phytochemicalscreening of Anethum graveolens Foeniculum vulgare andTrachyspermum ammirdquo BMC Complementary and AlternativeMedicine vol 9 article 30 2009

[16] H N Zhen X Zhang P Z Hu et al ldquoSurvivin expression andits relation with proliferation apoptosis and angiogenesis inbrain gliomasrdquo Cancer vol 104 no 12 pp 2775ndash2783 2005

[17] L Braiman-Wiksman I Solomonik R Spira and T Tennen-baum ldquoNovel insights into wound healing sequence of eventsrdquoToxicologic Pathology vol 35 no 6 pp 767ndash779 2007

[18] P Aramwit and A Sangcakul ldquoThe effects of sericin cream onwound healing in ratsrdquo Bioscience Biotechnology and Biochem-istry vol 71 no 10 pp 2473ndash2477 2007

[19] P Gal R Kilik M Mokry et al ldquoSimple method of open skinwound healing model in corticosteroid-treated and diabeticrats standardization of semi-quantitative and quantitative his-tological assessmentsrdquo Veterinarni Medicina vol 53 no 12 pp652ndash659 2008

[20] A L Udupa D R Kulkarni and S L Udupa ldquoEffect of Tridaxprocumbens extracts on wound healingrdquo International Journalof Pharmacognosy vol 33 no 1 pp 37ndash40 1995

[21] A Shukla A M Rasik and B N Dhawan ldquoAsiaticoside-induced elevation of antioxidant levels in healing woundsrdquoPhytotherapy Research vol 13 no 1 pp 50ndash54 1999

[22] H Roy S Bhardwaj and S Yla-Herttuala ldquoBiology of vascularendothelial growth factorsrdquo FEBS Letters vol 580 no 12 pp2879ndash2887 2006

[23] J S Lim and G Yoo ldquoEffects of adipose-derived stromal cellsand of their extract on wound healing in a mouse modelrdquo Jour-nal of Korean Medical Science vol 25 no 5 pp 746ndash751 2010

[24] P Y Lee S Chesnoy and L Huang ldquoElectroporatic deliveryof TGF-1205731 gene works synergistically with electric therapy toenhance diabetic wound healing in dbdb micerdquo Journal ofInvestigative Dermatology vol 123 no 4 pp 791ndash798 2004

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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

10 Evidence-Based Complementary and Alternative Medicine

[25] S Werner T Krieg and H Smola ldquoKeratinocyte-fibroblastinteractions in wound healingrdquo Journal of Investigative Derma-tology vol 127 no 5 pp 998ndash1008 2007

[26] A Desmouliere A Geinoz F Gabbiani and G GabbianildquoTransforming growth factor-1205731 induces 120572-smooth muscleactin expression in granulation tissuemyofibroblasts and in qui-escent and growing cultured fibroblastsrdquo Journal of Cell Biologyvol 122 no 1 pp 103ndash111 1993

[27] S Constantino Rosa Santos C Miguel I Domingues et alldquoVEGF and VEGFR-2 (KDR) internalization is required forendothelial recovery during wound healingrdquo Experimental CellResearch vol 313 no 8 pp 1561ndash1574 2007

[28] N N Nissen P J Polverini A E Koch M V Volin R LGamelli and L ADiPietro ldquoVascular endothelial growth factormediates angiogenic activity during the proliferative phase ofwound healingrdquo American Journal of Pathology vol 152 no 6pp 1445ndash1452 1998

[29] C J Corral A Siddiqui LWu C L Farrell D Lyons and T AMustoe ldquoVascular endothelial growth factor is more importantthan basic fibroblastic growth factor during ischemic woundhealingrdquo Archives of Surgery vol 134 no 2 pp 200ndash205 1999

[30] B Romana-Souza A P Nascimento and AMonte-Alto-CostaldquoPropranolol improves cutaneous wound healing in streptozo-tocin-induced diabetic ratsrdquoEuropean Journal of Pharmacologyvol 611 no 1ndash3 pp 77ndash84 2009

[31] M T L P Peres F Delle Monache A B Cruz M G Pizzolattiand R A Yunes ldquoChemical composition and antimicrobialactivity of Croton urucurana Baillon (Euphorbiaceae)rdquo Journalof Ethnopharmacology vol 56 no 3 pp 223ndash226 1997

[32] L A Gurgel J J C Sidrim D T Martins V C Filho and V SRao ldquoin vitro antifungal activity of dragonrsquos blood from Crotonurucurana against dermatophytesrdquo Journal of Ethnopharmacol-ogy vol 97 no 2 pp 409ndash412 2005

[33] H Tsuchiya M Sato T Miyazaki et al ldquoComparative study onthe antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureusrdquo Journal of Ethno-pharmacology vol 50 no 1 pp 27ndash34 1996

[34] M Scortichini and M P Rossi ldquoPreliminary in vitro evaluationof the antimicrobial activity of terpenes and terpenoids towardsErwinia amylovora (Burrill) Winslow et alrdquo Journal of AppliedBacteriology vol 71 no 2 pp 109ndash112 1991

[35] A Vachalkova L Novotny M Nejedlikova and V SuchyldquoPotential carcinogenicity of homoisoflavonoids and flavonoidsfrom Resina sanguinis draconis (Dracaena cinnabari Balf)rdquoNeoplasma vol 42 no 6 pp 313ndash316 1995

[36] M Masaoud J Schmidt and G Adam ldquoSterols and triter-penoids from Dracaena cinnabarirdquo Phytochemistry vol 38 no3 pp 795ndash796 1995

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