the effects of punica granatum flower extract on skin

Research ArticleThe Effects of Punica granatum Flower Extract onSkin Injuries Induced by Burn in Rats

Ebrahim Nasiri12 Seyed Jalal Hosseinimehr13 Jafar Akbari13

Mohammad Azadbakht13 and Soheil Azizi2

1Traditional and Complementary Medicine Research Center Mazandaran University of Medical Sciences Sari Iran2Faculty of Allied Medical Sciences Mazandaran University of Medical Sciences Sari Iran3Faculty of Pharmacy Mazandaran University of Medical Sciences Sari Iran

Correspondence should be addressed to Seyed Jalal Hosseinimehr sjhosseinimyahoocom

Received 30 August 2016 Revised 5 November 2016 Accepted 26 December 2016 Published 19 January 2017

Academic Editor Orish Ebere Orisakwe

Copyright copy 2017 Ebrahim Nasiri et alThis 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

Background We compared the efficacy of P granatum (P) flower extract with that of silver sulfadiazine (SSD) for treating thermalburn injuries in ratsMethods Ten Wistar rats in each group were topically given base cream normal saline cream containing 1SSD or creams containing 5 or 10 Punica granatum flower extract The treatments were administered once daily until completewound healing was observed The wound area and healing time were assessed In addition percentage wound contraction andhistopathological characteristics such as neovascularization and collagen formation were determined The tannin content in Pgranatum extract was determined Results The decrease in the average size of wounds on day 15 of the treatment was higher inrats treated with creams containing P granatum extract than in rats treated with cream containing SSD (28 plusmn 09 cm2 versus 84 plusmn32 cm2) The wounds completely healed on day 25 of the treatment in rats treated with creams containing P granatum flowerextract compared with those in rats treated with the other agentsConclusionThese results indicated that P granatum flower extractpromoted wound healing in rats and could be used for managing burn injuries

1 Introduction

Burn injury is a public health problem worldwide espe-cially in undeveloped countries that lack adequate medicalfacilities in terms of morbidity long-term disability andmortality [1 2] Infection is the key complication of burnsand is responsible for various health problems [1] Burnsare associated with high healthcare costs multiple operativeprocedures prolonged rehabilitation periods long-term dis-ability prolonged hospitalization loss of body extremitiesinfection and even death [3ndash7] Exposure to hot water is oneof the most frequent causes of burns [6] Healing of burnwounds is a complex process and appropriate wound healingis crucial for the restoration of disrupted anatomical conti-nuity and disturbed functional status of the skin [7] Woundhealing involves three phases namely inflammation tissueformation and tissue remodeling [8] Creams containing1 silver sulfadiazine (SSD) are primarily used for treating

burn injuries because of the antibacterial activity of SSD [1 49 10] However SSD is associated with several disadvantagessuch as delayed wound healing development of resistancerenal toxicity leukopenia and adverse reactions [4 5 10]Therefore it is important to find a more effective drug withfewer side effects for treating burn injuries

The use of natural products for treating burns is animportant part of health management and is a resourcefulmethod for providing more effective and cheaper health-care options [11ndash13] Punica granatum Linn (Punicaceae)commonly known as pomegranate is a small tree nativeto the Mediterranean region [14 15] This tree is calledGolnar in Iranian traditional medicine Several studies haveexamined the efficacy of herbalmedicines for healingwounds[11 13 15] The fruits and flowers of Golnar are used inhome remedies for treating gastric pain gastrointestinalinfection bleeding or gastralgia dysentery and hemorrha-gia and wound healing [15 16] This herbal medicine has

HindawiAdvances in Pharmacological SciencesVolume 2017 Article ID 3059745 8 pageshttpsdoiorg10115520173059745

2 Advances in Pharmacological Sciences

antitumor antidiarrheal antiulcer antifungal antioxidantand hepatoprotective properties [14 17] P granatum extractshave been used for treating diabetes mellitus and microbialinfections [18 19] In addition few studies have shown thatthe extracts of pomegranate flowers have beneficial effectson healing wounds In the present study we compared theefficacy of creams containing P granatum flower extract withthat of a cream containing SSD on healing burn wounds inrats

2 Material and Methods

21 Preparation of P granatum Flower Extract

Plant Material P granatum flowers were purchased from alocal herbal drug market (Sari Iran) in the summer of 2013The identity of these flowers was confirmed by senior botanistDr Mohammad Azadbakht at the Mazandaran Universityof Medical Sciences Iran (Herbarium number 1003) Theflowers were dried at room temperature and were powderedusing a grinder Aqueous ethanol (70) was gradually addedto the powdered flowers and the mixture was kept atroom temperature without exposure to direct light for 72 hAfter filtration the solution was concentrated in a rotaryevaporator under reduced pressure The temperature of therotary evaporator was from 40 to 50∘C The hydroalcoholicextract of the flowers was then powdered in a freeze dryerThe yield of the dried extract was 268

22 HPLC Analysis of P granatum Flower Extract

HPLC The concentration of tannic acid in the P granatumflower extract was analyzed by performing HPLC HPLC wasperformed using HPLC Knauer Smartline system (KnauerAssociation Germany) containing 1000mL pump solventdelivery system sampler injector and photodiode arraydetector (model DAD2800 all purchased fromKnauer Asso-ciation) set at 280 nm and attached to ChromGate software(version 317) Analysis was performed using an ODS-C18column (250 times 4 times 6mm 5120583m particle size NUCLEODURDuren Germany) and a corresponding guard column All thesolvents were filtered and degassed before injecting them intothe column Mobile phase was methanol-water-phosphoricacid (50 50 001) and the flow rate of the mobile phasewas 10mLmin All the measurements were performed atambient temperature

23 Determination of Tannin in P granatum Using Spec-trophotometry Method Tannin contained in P granatumflower extract was determined for spectrophotometry byusing Folin-Denis reagent [20] In this method tan-nic acid reducts Folin-Denis reagent (phosphomolybdic-phosphotungstic reagent) in alkaline solution Absorbance ofthe product was determined at 760 nm by using UV 6505spectrophotometer (Jenway UK) The calibration (standard)curve was prepared using several concentrations of tannicacid (01ndash1mgmL) Three solutions containing 1mgmL Pgranatum flower extract were prepared and their absorbancewas read in the same way as that of the standard solution of

tannic acid The concentration of tannins in the P granatumflower extract was calculated using the calibration curve Allthe reagents used for spectrophotometry were obtained fromMerck (Germany)

24 Preparation and Formulation of Creams Containing 5and 10 P granatum Flower Extract P granatum flowerextract was mixed with liquid paraffin stearyl alcohol cetylalcohol and Span 80 at 70∘C The aqueous phase was pre-pared by adding Tween 80 propyl and methyl paraben andglycerin in distilled water and by heating to 70∘C Next theaqueous and oil phases were mixed and were homogenizedat 500 rpm for 15minThe creamwas allowed to cool at roomtemperature during homogenization All formulations werestored at 4∘C 25∘C and 40∘C for twoweeks and their stabilitywas evaluated

25 Study Animals The experimental protocol for animalstudies was approved by the Ethical and Research Committeeof the Mazandaran University of Medical Sciences (number118-92) Male Wistar rats (119873 = 50 weight 160ndash200 g age8ndash10 weeks) were obtained from the Mazandaran Universityof Medical Sciences They were housed under standardconditions at room temperature with a 12 h lightdark cycleandwere providedwith laboratory food andwater ad libitumThe rats were anesthetized by injecting 50mgkg sodiumthiopental intraperitoneally and their backs were shaved andcleaned with 70 alcohol An insulating box with a base of 2times 5 cm was prepared and was used to induce baseline burnsin rats under controlled conditions The rats were placed inthe box in a supine position with their backs exposed to hotwater at 90∘C for 6 s [4] This method allowed the burningof an exact diameter of the skin and resulted in a regularrectangular second-degree burn injury (approximately 10of total body surface) on the back of the rats This devicehelps to create a controlled burn in the animal model All therats were immediately resuscitated by injecting 5mL normalsaline intraperitoneally [4 6]

The rats with burn injuries were randomly divided intofive groups containing 10 rats each Group 1 was the controlgroup in which the rats were only treated using normal salinewithout any topical treatment Rats in group 2 were treatedwith base cream lacking any effective agent Rats in group3 were treated with a cream containing 1 SSD (BehvarzanPharmaceutical Company Iran) Rats in groups 4 and 5 weretreated with creams containing 5 and 10 P granatumflower extract respectively All the treatments were initiated24 h after the burn injury Wounds were dressed once dailyBefore dressing the wounds in all the groups were washedwith normal saline and the wound was covered by a similarthin layer of the cream in each group After 72 hours all of thegroup wounds were kept open after dressing To quantify therate of wound healing the area of the lesions was determinedon days 1 3 7 10 14 20 25 30 and 33 after the burn injuryWound area was measured in square millimeter on eachexperimental day by a ruler and a photograph Percentagewound contraction at each time point was determined usingthe following formula percentage wound contraction =(initial wound area minus current area)initial wound area times 100

Advances in Pharmacological Sciences 3

Measurements were made daily by the same examiner byusing a ruler and by taking photographs [7 21]

26 Histological Analysis Skin tissue samples for histologicalanalysis were obtained under anesthesia by making a smallexcision in the center region of the wound on days 8 and21 after the burn injury The tissue samples were fixed in10 formalin and were embedded in paraffin The paraffin-embedded tissue blocks were cut into 4120583m thick sections andwere stained with hematoxylin and eosin Light microscopywas performed to assess pathological changes such as angio-genesis granulation tissue (GT) formation and reepithelial-ization in the wounds [4 6 7] The results are expressedas an average of 10 microscopic fields Histopathologicalanalysis was performed by evaluating reepithelializationReepithelialization was determined by measuring the follow-ing parameters thickness of the epidermal layer thicknessof granular cell layer organization of squamous cells andextent of keratin layer and orthokeratin Complete healingwas evaluated by determining the degree of scar formationcollagen organization and innervation and formation of hairfollicles at 3 weeks after the burn injury A score of 0ndash3 wasassigned to all the parameters evaluated 0 = absent 1 =mildlypresent 2 = moderately present and 3 = strongly present[7] Collagen organization was also scored on levels 0ndash3 asfollows

0 = minus lagging down disorganized and poorly ori-ented collagen fibers

1 = + horizontally oriented 10ndash20 collagen fibers

2 = ++ horizontally oriented 30ndash40 collagen fibers

3 = +++ well-formed and horizontally oriented col-lagen fibers

Bacteriological Assessment Swabs were taken from the burnwound area before dressing change on the 4th and 8th daysThe swabs were collected and transferred to the laboratoryfor testing In the quantitative count study 05mL of normalsaline was added to each of the samples Each sample dilutionwas spread onto Blood Agar and MacConkey Agar and theplates were incubated at 37∘C for 24 h Diagnostic test forthe colonies was applied with Novobiocin test by a blindedhistopathologist

27 Statistical Analysis Wound sites were assessed dailyMacroscopic evaluation was performed daily by the directobservation of wounds during dressing Inflamed tissue wascharacterized by the presence of edema secretion rednessdark secretion or pus pain and bleeding during wounddressing Statistical analysis was performed using SPSS soft-ware andMSExcel One-way analysis of variancewas used forcomparing quantitative variables among the groups Scheffepost hoc multiple comparisons test was used to compare themeans among the groupsThe differences between the groupswere considered significant at 119875 lt 005

3 Results

In this study an isocratic elution of methanol in anacidic aqueous medium was used to analyze tannic acidconcentration in the P granatum flower extract Theseparation of tannic acid is shown in Figure 1 Tannic acidstandard has a typical retention time of 37min whichwas 38min in the extract and tannic acid in extract wasconfirmed with peak of pure tannic acid The purity of thetannic acid peak in the HPLC chromatogram was confirmedusing the photodiode array detector The UV spectrum ofpure tannic acid was the same as that of the tannic acid peakin the HPLC chromatogram of P granatum flower extract(Figure 1) Tannin content in P granatum flower extract wasdetermined using a spectrophotometric method Calibrationcurve from standard solution of tannic acid was prepared (119910= 04593119909 + 00684 1198772 = 09693) and with the help of thiscurve the tannin content of herbal extract was estimatedThe mean weight of tannin present in P granatum flowerextract was 0487 plusmn 0035mgmg (119873 = 3) of the extract(4871 plusmn 355mg100mg of the extract ie 487)

The effect of burn injury on weight loss in rats wasrecordedNo significant differenceswere observed among thegroups with respect to weight loss induced by the burn injurySkin lesions were measured on days 1 3 7 10 15 20 25 and30 after the burn injury The average wound sizes on the firstday were 125 plusmn 26 125 plusmn 44 118 plusmn 24 126 plusmn 49 and122 plusmn 57 cm2 in rats treated with cream containing 1 SSDbase cream normal saline cream containing 5 P granatumflower extract and cream containing 10 P granatum flowerextract respectively Wound area in rats treated with creamscontaining P granatum flower extract was lower by 1 cm2 onday 20 after the treatment (Figure 2)

Wound areas were not significantly different among thegroups on days 3 7 and 10 after the burn injury but weresignificantly different on day 15 after the burn injury (119875 lt0001) Post hoc multiple comparisons Scheffe test showedthat treatment of rats with creams containing 5 Punicagranatum with 1 SSD was significant (119875 lt 0001) and 10P granatum flower extract showed smaller wound area onday 15 after the burn injury than rats treated with creamcontaining 1 SSD (119875 lt 0004) The mean wound area ofrats in the different groups is shown in Figure 2 No significantdifference was observed in wound size among rats treatedwith normal saline base cream and cream containing 1SSD However a significant difference was observed in thewound size between rats treated with the base cream andcreams containing 5 and 10 P granatum flower extract(119875 lt 0024) The wound area was not different betweenrats treated with creams containing 5 and 10 P granatumflower extract on day 15 after the burn injury

The percentage wound contraction was significantlyincreased in rats treated with creams containing 5 and 10P granatum flower extract compared with that in rats treatedwith the other agents Rats treated with cream containing1 SSD showed longer healing time than those treated withcreams containing P granatum flower extract These findingsare summarized in Table 1


0

0

(mAU

)

100

200

300

400

500

2 4 6 8 10(Minutes)

12 14 16 18 20 22 24

(a)

0

(mAU

)

100

200

300

400

500

(Minutes)0 2 4 6 8 10 12 14 16 18 20 22 24

(b)

0

200 250 300(nm)

350 400

1000

500

(mAU

)

Punica granatum

0

500

1000

(c)

Tannic acid

200 250 300(nm)

350 400

0

1000

500

0

1000

500

(mAU

)

(d)

Figure 1 HPLC profile of Punica granatum total extract and tannic acid analyzed (a) HPLC chromatogram of Punica granatum extract (b)HPLC chromatogram of tannic acid (retention time 38) (c) UV spectrum of peak Punica granatum extract with a retention time of 32 and(d) UV spectrum of tannic acid

1 3 7 10 15 20 25 30Time (day after burn injury)

SSD 1BCNS

P 5P 10

02468

101214161820

Wou

nd ar

ea (c

m2)

Figure 2 Mean wound area (cm2) of the animal groups treatedwith various topical creams Silver sulfadiazine 1 (SSD 1) normalsaline (NS) base cream (BC) Punica granatum flowers cream 5(P 5) and Punica granatum flowers cream 10 (P 10) P 5 withSSD 1 (119875 lt 0003) and P 10 with SSD 1 (119875 lt 0004)

Complete wound healing was observed on day 25 in ratstreated with creams containing 5 and 10 P granatumflower extract and on day 33 in rats treated with the otheragents (Table 1)

These results indicated that wound healing in rats treatedwith P granatum flower extract occurred 10 days before thatin rats treatedwith the other agentsThe condition of the burnwound on days 12 and 23 after the injury is shown in Figure 3

Visual analysis of burn wounds treated with the basecream and cream containing 1 SSD showed redness andedema in the wound area however this was not observedin wounds treated with creams containing 5 and 10 Pgranatum flower extract and with normal saline Laboratoryassessments showed no evidence of pathological bacteriaOn days 5 and 8 a slight colorless secretion appearedin wounds treated with the base cream 10 P granatumflower extract normal saline and cream containing 1 SSDCulturing of skin tissue samples treated with the aboveagents in Blood Agar yielded large convex round and whitecolonies Novobiocin test is used to differentiate coagulase-negative staphylococci These bacteria were inferred to beStaphylococcus epidermidis which are a part of the normalskin flora Significant neovascularization and fibroblasticproliferation were observed on day 8 in rats treated withcreams containing 5 and 10P granatum flower extract and1 SSD compared with those treated with normal saline andbase cream (Table 2)

GT formation was observed on day 8 in wounds treatedwith creams containing 5 and 10 P granatum flowerextract and 1 SSD compared with wounds treated withnormal saline and base creamGT formation scorewas higherin the 10 P granatum flower extract group than in woundstreated with the other agents (Table 2)


Table 1 Comparison of the percentage of wound contraction between Punica granatum and treated groupslowast

Day P 5 P 10 SSD 1 BC NS1 0 0 0 0 03 minus57 plusmn 58 minus127 plusmn 62 minus98 plusmn 71 minus63 plusmn 53 minus64 plusmn 58

7 155 plusmn 96 38 plusmn 56 minus41 plusmn 69 93 plusmn 62 162 plusmn 73

10 449 plusmn 75 331 plusmn 85 129 plusmn 52 125 plusmn 54 288 plusmn 82



25 997 plusmn 08 100 822 plusmn 21 888 plusmn 42 937 plusmn 35

30 100 mdash 935 plusmn 13 97 plusmn 28 993 plusmn 21

33 mdash mdash 975 plusmn 04 984 plusmn 19 995 plusmn 11lowastSilver sulfadiazine 1 (SSD 1) normal saline (NS) base cream (BC) Punica granatum flowers cream 5 (P 5) and Punica granatum flowers cream 10(P 10)

Table 2 Morphology and histopathology of granulation tissue examined in different groups on the 8th day after burn injurylowast

Componentsgroups SSD 1 NS P 5 P 10 BCMonocytic (macrophage histiocyte infiltration) 2 1 2 2 2Neovascularization 2 1 2 2 1Fibroblastic proliferation 2 1 2 2 2Matrix mucopolysaccharide deposition 2 1 2 2 2Degree of inflammation 3 1 2 3 3Extent of bacterial colonization minus2 minus3 minus1 minus1 minus1

Degree of granulation tissue formation 2 1 2 2 1Sum of scores 11 3 11 12 10lowastSilver sulfadiazine 1 (SSD 1) normal saline (NS) base cream (BC) Punica granatum flowers cream 5 (P 5) and Punica granatum flowers cream 10(P 10) These samples were taken randomly from each group

SSD 1 BCNS P 5 P 10

A=

12

th d

ayB

=23

rd d

ay

Figure 3 Comparison on dorsal wound condition between groups on the 12th day and 23rd day after burning Silver sulfadiazine 1 (SSD1) normal saline (NS) base cream (BC) Punica granatum flowers cream 5 (P 5) and Punica granatum flowers cream 10 (P 10 in A= on the 12th day and B = on the 23rd day after burn injury)

On day 8 macrophage histiocytic infiltration and fibrob-lastic proliferation were similar in wounds treated withcreams containing 5 and 10 P granatum flower extractbase cream and cream containing 1 SSD Histopathologicalanalysis of the dermis on day 21 after the burn injury showedthat the degree of innervation and formation of lymphaticducts in wounds treated with cream containing 10 P grana-tum flower extract were better than those in wounds treated

with the other agentsThe results of histopathological analysisfor reepithelialization showed that on day 21 granular celllayer thickness and epidermal thickness in wounds treatedwith creams containing 10 P granatum flower extract and1 SSD were higher than those in wounds treated with otheragents (Table 3) Furthermore the formation of horizontallyoriented collagen fibers of appropriate tension and strengthin the scar tissue was better in wounds treated with creams


Table 3 Histopathological examination of dermis on 21 days after burn injury in treated groupslowast

Groups Degree of scarformation

Matrix ampcollagenizationorganization

Extent of hairfollicles

Extent oflymphatic ducts

Degree ofinnervation Sum

P 5 3 3 2 1 0 9P 10 3 3 3 1 1 11SSD 1 2 2 0 0 0 4Base C 2 2 0 0 0 4NS 2 2 1 1 0 6lowastSilver sulfadiazine 1 (SSD 1) normal saline (NS) base cream (BC) Punica granatum flowers cream 5 (P 5) and Punica granatum flowers cream 10(P 10) These samples were taken randomly from each group

SSD 1 BC NSP 5 P 10

8th

day

21

st da

y

Figure 4 Comparison of the histopathology of biopsy samples from second-degree burns on 8 and 21 days after treatment with basic cream(BC) no treatment or normal saline (NS) standard silver sulfadiazine treatment (SSD 1) and Punica granatum 5 (P 5) and Punicagranatum 10 (P 10) creams treatment Neovascularization activity and fibroblastic proliferation were better in P 5 P 10 and SSD 1cream compared with NS and BC groups on the 8th day Also degree of granulation tissue was better in these groups The thickness of thegranular cell layer and the epidermal thickness degree in the wound of Punica 10 and SSD 1 wound treatment were better than in othergroups on the 21st day

containing 10 and 5 P granatum flower extract than inthose treated with the other agents (Figure 4)

4 Discussion

In this study the efficacy of creams containing P granatumflower extract was evaluated for treating burn wounds in ratsThe results of this study showed that creams containing 5and 10 P granatum flower extract facilitated the healingof the burned tissue Topical creams containing P granatumflower extract were more effective in inducing wound healingthan creams containing 1 SSD and other agents On day15 after the burn injury the wound size was significantlysmaller in rats treated with creams containing 5 and10 P granatum flower extract than in rats treated withthe other agents P granatum has various pharmacologicalproperties including anti-inflammatory [22] antioxidant[23 24] antibacterial [3] wound healing [25 26] antifungal[14 27] antispasmodic [28] and antiulcer properties [2930] Our study showed that creams containing extracts of Pgranatum flowers weremore effective than creams containing1 SSD for treating burn wounds Treatment with creamscontaining the P granatum flower extract accelerated thedegree of GT formation compared to treatment with basecream and normal saline The extent of scar tissue and hair

follicle formation was higher in wounds treated with creamscontaining the P granatum flower extract than in woundstreated with the other agents The time for wound healingwas shorter for wounds treated with creams containing Pgranatum flower extract than for wounds treated with theother agents Collagen organization was higher in woundstreated with creams containing P granatum flower extractthan in wounds treated with the other agents Prelimi-nary chemical analysis of the P granatum flower extractshowed that it contained high concentrations of tannin(0487mgmg 487) Previous studies have reported that Pgranatum extracts contain polyphenolic compounds such asellagic acid 33101584041015840-tri-O-methyl ellagic acid ethyl brevifolincarboxylate maslinic acid daucosterol and tannins [31ndash33]Singh et al (2002) reported that Punica peel and seed extractshad antioxidant properties and suppressed peroxidation [24]Tannins exert antibacterial effects against many bacteriaTannins are polyphenolic compounds containing hydroxyliccarboxylic and other hydrophilic groups and are consideredto be macromolecules [31 34 35] Although tannins areknown to exert antibacterial effects and promote woundhealing the mechanisms underlying these effects are unclear[36 37] Our study has shown that P granatum flowerextract contains a high level of tanninsThis effectivematerialprobably facilitates wound healing in this research study


Wound healing is a multiphase process characterizedby wound contraction granulation epithelialization andcollagenation Wound healing involves 3 phases that isinflammation proliferation and remodeling [38 39] Pro-liferation is followed by epithelialization angiogenesis andcollagen formation GT is formed at the end of the pro-liferation phase Fibroblasts collagen edema and newblood vessels are formed and undergo maturation in theremodeling phase resulting in the formation of scar tissueCollagen is the main protein that contributes to woundstrength [8 39] The barrier function of the skin is disruptedafter thermal injuries which may result in the develop-ment of infections in the wounded area Infection com-plicates burn wounds and delays their healing Thereforewound dressing should be performed appropriately to pre-vent the entry of environmental microorganisms [31 34ndash40]

We observed that the healing of wounds treated withcreams containing 5 and 10 P granatum flower extractwas faster than that of wounds treated with the otheragents This may be because of the beneficial effects ofP granatum extract on wound healing parameters suchas revascularization fibroplasias wound contraction andcollagen synthesis In addition this beneficial effect of Pgranatum flower extract may be associated with its antibac-terial anti-inflammatory and antioxidant properties Theseproperties of P granatum flower extract probably augmenttogether and promote wound healing compared with theantibacterial effects of standard 1 SSD Creams contain-ing SSD are commonly used for treating burn injuriesbecause of the antibacterial property of SSD Althoughcreams containing SSD are recommended as the standardtreatment for treating burn wounds the use of SSD mayincrease the duration of hospitalization [4 6 40] Becausecreams containing P granatum flower extract promotedfaster wound healing than creams containing 1 SSD theuse of these creams may result in a shorter hospital stayof patients with burn injuries The treatment of woundswith creams containing the P granatum flower extractresulted in better wound contraction than the treatmentof wounds with the other agents On day 15 of the studythe percentage wound contraction in wounds treated withcreams containing the P granatum flower extract was 77-78 compared with 32 40 and 42 for wounds treated withcream containing 1 SSD base cream and normal salinerespectively indicating that the P granatum flower extracthad the best effect onwound healing comparedwith the otheragents

5 Conclusion

The results of this study showed that creams containing the Pgranatum flower extract remarkably improved the healing ofburn wounds compared with creams containing standard 1SSD base cream andnormal salineThiswas probably relatedto tannins of the P granatum flower extract Thus the resultsof this study support the use of Golnar (P granatum) flowersfor treating burn injuries as mentioned in Iranian traditionalmedicine

Competing Interests

The authors declare no potential competing interests withrespect to the authorship andor publication of this study

Acknowledgments

This study was supported by a grant from MazandaranUniversity of Medical Sciences Sari Iran This research wasthe subject of a PhD thesis of Ebrahim Nasiri as a student ofMazandaran University of Medical Sciences

References

[1] K S Priya A Gnanamani N Radhakrishnan and M BabuldquoHealing potential of Datura alba on burn wounds in albinoratsrdquo Journal of Ethnopharmacology vol 83 no 3 pp 193ndash1992002

[2] N K Upadhyay R Kumar S K Mandotra et al ldquoSafety andhealing efficacy of Sea buckthorn (Hippophae rhamnoides L)seed oil on burn wounds in ratsrdquo Food and Chemical Toxicologyvol 47 no 6 pp 1146ndash1153 2009

[3] G Hassanzadeh F Hajmanouchehri A B Roi et al ldquoCompar-ing effects of Silver sulfadiazine sucralfate and brassica oleraceaextract on burn wound healingrdquo Life Science Journal vol 10 no6 pp 852ndash861 2013

[4] S J Hosseinimehr G Khorasani M Azadbakht P ZamaniM Ghasemi and A Ahmadi ldquoEffect of aloe cream versussilver sulfadiazine for healing burn wounds in ratsrdquo ActaDermatovenerologica Croatica vol 18 no 1 pp 2ndash7 2010

[5] G Khorasani S J Hosseinimehr M Azadbakht A Zamaniand M R Mahdavi ldquoAloe versus silver sulfadiazine creamsfor second-degree burns A Randomized Controlled StudyrdquoSurgery Today vol 39 no 7 pp 587ndash591 2009

[6] G Khorasani S J Hosseinimehr P Zamani M Ghasemi andA Ahmadi ldquoThe effect of saffron (Crocus sativus) extract forhealing of second-degree burn wounds in ratsrdquo Keio Journal ofMedicine vol 57 no 4 pp 190ndash195 2008

[7] D D S Tavares Pereira M H M Lima-Ribeiro N T DePontes-Filho A M D A Carneiro-Leao and M T D SCorreia ldquoDevelopment of animal model for studying deepsecond-degree thermal burnsrdquo Journal of Biomedicine andBiotechnology vol 2012 Article ID 460841 7 pages 2012

[8] A J Singer and R A F Clark ldquoCutaneous wound healingrdquoNewEngland Journal of Medicine vol 341 no 10 pp 738ndash746 1999

[9] A F P M Vloemans A M Soesman M Suijker R W Kreisand E Middelkoop ldquoA randomised clinical trial comparing ahydrocolloid-derived dressing and glycerol preserved allograftskin in the management of partial thickness burnsrdquo Burns vol29 no 7 pp 702ndash710 2003

[10] M H E Hermans ldquoResults of a survey on the use of differenttreatment options for partial and full thickness burnsrdquo Burnsvol 24 no 6 pp 539ndash551 1998

[11] I P Suntar E K Akkol D Yilmazer et al ldquoInvestigations onthe in vivo wound healing potential of Hypericum perforatumLrdquo Journal of Ethnopharmacology vol 127 no 2 pp 468ndash4772010

[12] S Shailajan S Menon S Pednekar and A Singh ldquoWoundhealing efficacy of Jatyadi Taila in vivo evaluation in rat usingexcision woundmodelrdquo Journal of Ethnopharmacology vol 138no 1 pp 99ndash104 2011


[13] S Gurung and N Skalko-Basnet ldquoWound healing properties ofCarica papaya latex in vivo evaluation in mice burn modelrdquoJournal of Ethnopharmacology vol 121 no 2 pp 338ndash341 2009

[14] G Kaur Z Jabbar M Athar and M S Alam ldquoPunica grana-tum (pomegranate) flower extract possesses potent antioxidantactivity and abrogates Fe-NTA induced hepatotoxicity in micerdquoFood and Chemical Toxicology vol 44 no 7 pp 984ndash993 2006

[15] S M Momen-Tonkaboni Tohfatol-Momenin (Tohfehe-HakimMomen) Traditional Medicine of Research center of Shahidbeheshti University of Medical Sciences Nashre Shar Consti-tiute Tehran Iran 2008

[16] M A Al Yahya ldquoPreliminary phytochemical and pharmacho-logical and on the rind of Pomegranate (Punica granatume L)rdquoPakistan Journal of Biological Sciences vol 8 no 3 pp 479ndash4812005

[17] N Arun and D Singh ldquoPunica granatum a review on phar-macological and therapeutic propertiesrdquo International Journalof Pharmaceutical Sciences and Research vol 2 no 3 pp 1240ndash1245 2012

[18] M K Reddy S K Gupta M R Jacob S I Khan and DFerreira ldquoAntioxidant antimalarial and antimicrobial activitiesof tannin-rich fractions ellagitannins and phenolic acids fromPunica granatum Lrdquo Planta Medica vol 73 no 5 pp 461ndash4672007

[19] F Fernando Luiz Affonso K R D Silva A P Lopes et alldquoPreclinical evaluation of the crude extract from the fruits ofPunica grantaum L (Punicaceae) for antimicrobial activity inin vitro and ex vivo experimental models a comparative studyrdquoAfrican Journal of Pharmacy and Pharmacology vol 8 no 18pp 479ndash484 2014

[20] K Jaimand M B Rezaee S R Tabaei Aghdaei M NaderyHajibagher Kandy and S Meshkizadeh ldquoDtermination oftannins in rose water wastewater and petal residue of Rosadamascena Millrdquo Iranian Journal of Medicinal and AromaticPlants vol 27 no 2 pp 348ndash357 2011 (Persian)

[21] A P De Oliveira E D S Franco R Rodrigues Barretoet al ldquoEffect of semisolid formulation of persea americanamill (Avocado) oil on wound healing in ratsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID472382 8 pages 2013

[22] M Sarker S C Das S K Saha Z Al Mahmud and S CBachar ldquoAnalgesic and anti-inflammatory activities of flowerextracts of Punica granatum Linn (Punicaceae)rdquo Journal ofApplied Pharmaceutical Science vol 2 no 4 pp 133ndash136 2012

[23] D Prashanth M K Asha and A Amit ldquoAntibacterial activityof Punica granatumrdquo Fitoterapia vol 72 no 2 pp 171ndash173 2001

[24] R P Singh K N Chidambara Murthy and G K JayaprakashaldquoStudies on the antioxidant activity of pomegranate (Punicagranatum) peel and seed extracts using in vitromodelsrdquo Journalof Agricultural and Food Chemistry vol 50 no 1 pp 81ndash862002

[25] S Adiga P Tomar and R R Rajput ldquoEffect of Punica granatumpeel aqueous extract on normal and dexamethasone suppressedwound healing in wistar ratsrdquo International Journal of Pharma-ceutical Sciences Review and Research vol 5 no 2 pp 34ndash372010

[26] A G Pirbalouti A Koohpayeh and I Karimi ldquoThe woundhealing activity of flower extracts ofPunica granatum andAchil-lea kellalensis in Wistar ratsrdquo Acta Poloniae PharmaceuticamdashDrug Research vol 67 no 1 pp 107ndash110 2010

[27] B K Dutta I Rahman and T K Das ldquoAntifungal activity ofIndian plant extractsrdquo Mycoses vol 41 no 11-12 pp 535ndash5361998

[28] A Ahangarpour R Heidari M Abdolahzadeh and A AOroojan ldquoAntispasmodic effects of aqueous and hydroalcoholicpunica granatum flower extracts on the uterus of non-pregnantratsrdquo Journal of Reproduction and Infertility vol 13 no 3 pp140ndash142 2012

[29] R Gautam and S C Sharma ldquoAnti-ulcer activity of Punicagranatum linnin diabetic ratsrdquo International Journal of Phar-macy and Pharmaceutical Sciences vol 4 no 3 pp 459ndash4612012

[30] F Borrelli and A A Izzo ldquoThe plant kingdom as a source ofanti-ulcer remediesrdquo Phytotherapy Research vol 14 no 8 pp581ndash591 2000

[31] P K Ghosh and A Gaba ldquoPhyto-extracts in wound healingrdquoJournal of Pharmacy and Pharmaceutical Sciences vol 16 no 5pp 760ndash820 2013

[32] E P Lansky and R A Newman ldquoPunica granatum(pomegranate) and its potential for prevention and treatmentof inflammation and cancerrdquo Journal of Ethnopharmacologyvol 109 no 2 pp 177ndash206 2007

[33] A G Pirbalouti A Shahrzad K Abed and BHamedi ldquoWoundhealing activity of Malva sylvestris and Punica granatum inalloxan-induced diabetic ratsrdquo Acta Poloniae Pharmaceuticavol 67 no 5 pp 511ndash516 2010

[34] A Banso and S O Adeyemo ldquoEvaluation of antibacterialproperties of tannins isolated from Dichrostachys cinereardquoAfrican Journal of Biotechnology vol 6 no 15 pp 1785ndash17872007

[35] T Okuda ldquoSystematics and health effects of chemically distincttannins in medicinal plantsrdquo Phytochemistry vol 66 no 17 pp2012ndash2031 2005

[36] C K Sen S Khanna G Gordillo D Bagchi M Bagchi andS Roy ldquoOxygen oxidants and antioxidants in wound healingan emerging paradigmrdquo Annals of the New York Academy ofSciences vol 957 pp 239ndash249 2002

[37] K Li Y Diao H Zhang et al ldquoTannin extracts from immaturefruits of Terminalia chebula Fructus Retz promote cutaneouswound healing in ratsrdquo BMC Complementary and AlternativeMedicine vol 11 no 1 article 86 2011

[38] M Ayyanar and S Ignacimuthu ldquoHerbal medicines for woundhealing among tribal people in Southern India ethnobotani-cal and scientific evidencesrdquo International Journal of AppliedResearch in Natural Products vol 2 no 3 pp 29ndash42 2009

[39] K Das ldquoWound healing potential of aqueous crude extract ofStevia rebaudiana inmicerdquo Brazilian Journal of Pharmacognosyvol 23 no 2 pp 351ndash357 2013

[40] G Mohajeri H Masoudpour M Heidarpour et al ldquoTheeffect of dressing with fresh kiwifruit on burn wound healingrdquoSurgery vol 148 no 5 pp 963ndash968 2010

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


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AddictionJournal of



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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


antitumor antidiarrheal antiulcer antifungal antioxidantand hepatoprotective properties [14 17] P granatum extractshave been used for treating diabetes mellitus and microbialinfections [18 19] In addition few studies have shown thatthe extracts of pomegranate flowers have beneficial effectson healing wounds In the present study we compared theefficacy of creams containing P granatum flower extract withthat of a cream containing SSD on healing burn wounds inrats

2 Material and Methods

21 Preparation of P granatum Flower Extract

Plant Material P granatum flowers were purchased from alocal herbal drug market (Sari Iran) in the summer of 2013The identity of these flowers was confirmed by senior botanistDr Mohammad Azadbakht at the Mazandaran Universityof Medical Sciences Iran (Herbarium number 1003) Theflowers were dried at room temperature and were powderedusing a grinder Aqueous ethanol (70) was gradually addedto the powdered flowers and the mixture was kept atroom temperature without exposure to direct light for 72 hAfter filtration the solution was concentrated in a rotaryevaporator under reduced pressure The temperature of therotary evaporator was from 40 to 50∘C The hydroalcoholicextract of the flowers was then powdered in a freeze dryerThe yield of the dried extract was 268

22 HPLC Analysis of P granatum Flower Extract

HPLC The concentration of tannic acid in the P granatumflower extract was analyzed by performing HPLC HPLC wasperformed using HPLC Knauer Smartline system (KnauerAssociation Germany) containing 1000mL pump solventdelivery system sampler injector and photodiode arraydetector (model DAD2800 all purchased fromKnauer Asso-ciation) set at 280 nm and attached to ChromGate software(version 317) Analysis was performed using an ODS-C18column (250 times 4 times 6mm 5120583m particle size NUCLEODURDuren Germany) and a corresponding guard column All thesolvents were filtered and degassed before injecting them intothe column Mobile phase was methanol-water-phosphoricacid (50 50 001) and the flow rate of the mobile phasewas 10mLmin All the measurements were performed atambient temperature

23 Determination of Tannin in P granatum Using Spec-trophotometry Method Tannin contained in P granatumflower extract was determined for spectrophotometry byusing Folin-Denis reagent [20] In this method tan-nic acid reducts Folin-Denis reagent (phosphomolybdic-phosphotungstic reagent) in alkaline solution Absorbance ofthe product was determined at 760 nm by using UV 6505spectrophotometer (Jenway UK) The calibration (standard)curve was prepared using several concentrations of tannicacid (01ndash1mgmL) Three solutions containing 1mgmL Pgranatum flower extract were prepared and their absorbancewas read in the same way as that of the standard solution of

tannic acid The concentration of tannins in the P granatumflower extract was calculated using the calibration curve Allthe reagents used for spectrophotometry were obtained fromMerck (Germany)

24 Preparation and Formulation of Creams Containing 5and 10 P granatum Flower Extract P granatum flowerextract was mixed with liquid paraffin stearyl alcohol cetylalcohol and Span 80 at 70∘C The aqueous phase was pre-pared by adding Tween 80 propyl and methyl paraben andglycerin in distilled water and by heating to 70∘C Next theaqueous and oil phases were mixed and were homogenizedat 500 rpm for 15minThe creamwas allowed to cool at roomtemperature during homogenization All formulations werestored at 4∘C 25∘C and 40∘C for twoweeks and their stabilitywas evaluated

25 Study Animals The experimental protocol for animalstudies was approved by the Ethical and Research Committeeof the Mazandaran University of Medical Sciences (number118-92) Male Wistar rats (119873 = 50 weight 160ndash200 g age8ndash10 weeks) were obtained from the Mazandaran Universityof Medical Sciences They were housed under standardconditions at room temperature with a 12 h lightdark cycleandwere providedwith laboratory food andwater ad libitumThe rats were anesthetized by injecting 50mgkg sodiumthiopental intraperitoneally and their backs were shaved andcleaned with 70 alcohol An insulating box with a base of 2times 5 cm was prepared and was used to induce baseline burnsin rats under controlled conditions The rats were placed inthe box in a supine position with their backs exposed to hotwater at 90∘C for 6 s [4] This method allowed the burningof an exact diameter of the skin and resulted in a regularrectangular second-degree burn injury (approximately 10of total body surface) on the back of the rats This devicehelps to create a controlled burn in the animal model All therats were immediately resuscitated by injecting 5mL normalsaline intraperitoneally [4 6]

The rats with burn injuries were randomly divided intofive groups containing 10 rats each Group 1 was the controlgroup in which the rats were only treated using normal salinewithout any topical treatment Rats in group 2 were treatedwith base cream lacking any effective agent Rats in group3 were treated with a cream containing 1 SSD (BehvarzanPharmaceutical Company Iran) Rats in groups 4 and 5 weretreated with creams containing 5 and 10 P granatumflower extract respectively All the treatments were initiated24 h after the burn injury Wounds were dressed once dailyBefore dressing the wounds in all the groups were washedwith normal saline and the wound was covered by a similarthin layer of the cream in each group After 72 hours all of thegroup wounds were kept open after dressing To quantify therate of wound healing the area of the lesions was determinedon days 1 3 7 10 14 20 25 30 and 33 after the burn injuryWound area was measured in square millimeter on eachexperimental day by a ruler and a photograph Percentagewound contraction at each time point was determined usingthe following formula percentage wound contraction =(initial wound area minus current area)initial wound area times 100










3 Results






0

0

(mAU

)

100

200

300

400

500

2 4 6 8 10(Minutes)

12 14 16 18 20 22 24

(a)

0

(mAU

)

100

200

300

400

500

(Minutes)0 2 4 6 8 10 12 14 16 18 20 22 24

(b)

0

200 250 300(nm)

350 400

1000

500

(mAU

)

Punica granatum

0

500

1000

(c)

Tannic acid

200 250 300(nm)

350 400

0

1000

500

0

1000

500

(mAU

)

(d)



SSD 1BCNS

P 5P 10

02468

101214161820

Wou

nd ar

ea (c

m2)



















SSD 1 BCNS P 5 P 10

A=

12

th d

ayB

=23

rd d

ay












SSD 1 BC NSP 5 P 10

8th

day

21

st da

y



4 Discussion






5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










3 Results






0

0

(mAU

)

100

200

300

400

500

2 4 6 8 10(Minutes)

12 14 16 18 20 22 24

(a)

0

(mAU

)

100

200

300

400

500

(Minutes)0 2 4 6 8 10 12 14 16 18 20 22 24

(b)

0

200 250 300(nm)

350 400

1000

500

(mAU

)

Punica granatum

0

500

1000

(c)

Tannic acid

200 250 300(nm)

350 400

0

1000

500

0

1000

500

(mAU

)

(d)



SSD 1BCNS

P 5P 10

02468

101214161820

Wou

nd ar

ea (c

m2)



















SSD 1 BCNS P 5 P 10

A=

12

th d

ayB

=23

rd d

ay












SSD 1 BC NSP 5 P 10

8th

day

21

st da

y



4 Discussion






5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

0

(mAU

)

100

200

300

400

500

2 4 6 8 10(Minutes)

12 14 16 18 20 22 24

(a)

0

(mAU

)

100

200

300

400

500

(Minutes)0 2 4 6 8 10 12 14 16 18 20 22 24

(b)

0

200 250 300(nm)

350 400

1000

500

(mAU

)

Punica granatum

0

500

1000

(c)

Tannic acid

200 250 300(nm)

350 400

0

1000

500

0

1000

500

(mAU

)

(d)



SSD 1BCNS

P 5P 10

02468

101214161820

Wou

nd ar

ea (c

m2)



















SSD 1 BCNS P 5 P 10

A=

12

th d

ayB

=23

rd d

ay












SSD 1 BC NSP 5 P 10

8th

day

21

st da

y



4 Discussion






5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of














SSD 1 BCNS P 5 P 10

A=

12

th d

ayB

=23

rd d

ay












SSD 1 BC NSP 5 P 10

8th

day

21

st da

y



4 Discussion






5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









SSD 1 BC NSP 5 P 10

8th

day

21

st da

y



4 Discussion






5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




5 Conclusion


Competing Interests


Acknowledgments


References














































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

the effects of punica granatum flower extract on skin

Documents