Research Article Evaluation of Wound Healing Properties of Grape Seed ... - Semantic Scholar

12 Pages·2016·1.97 MB·English

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2016, Article ID 7965689, 12 pages
http://dx.doi.org/10.1155/2016/7965689
Research Article
Evaluation of Wound Healing Properties of Grape Seed,
Sesame, and Fenugreek Oils
DorsafMoallaRekik,1SamehBenKhedir,2KamiliaKsoudaMoalla,1
NazihaGratiKammoun,3TarekRebai,2andZouheirSahnoun1
1LaboratoryofPharmacology,FacultyofMedicineofSfax,UniversityofSfax,Sfax,Tunisia
2LaboratoriesofHistologyandEmbryology,FacultyofMedicineofSfax,UniversityofSfax,Sfax,Tunisia
3OlivierInstitute,BP1087,3000Sfax,Tunisia
CorrespondenceshouldbeaddressedtoDorsafMoallaRekik;[email protected]
Received18August2016;Accepted4October2016
AcademicEditor:LutfunNahar
Copyright©2016DorsafMoallaRekiketal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution
License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly
cited.
Background.Medicinalplantshaveprovedatalltimestobeapowerfulremedyforhealthcare.Accordingly,grapeseed,sesame,and
fenugreekextractedoilswithpharmacologicalpropertiesareinvestigatedaswoundtreatments.Thisstudyassessesthepotential
ofouroilsforhealingwoundsinducedonrats.Methods.Phytochemicalanalysesofoilshaveinvolved:qualityvalue,polyphenol,
chlorophylls,carotene,andfattyacids.Antibacterialactivitywascarriedout.Antioxidantactivitywasevaluated:thescavenging
effectonDPPHradicals,thereducingpower,and𝛽-carotenediscoloration.Uniformwoundexcisionwasinducedonratsdorsum
randomlydividedintofivegroups:groupstreatedwith“CICAFLORA(cid:2)”andtestedoilsanduntreatedone.Theposthealingbiopsies
werehistologicallyassessed.Results.Woundbiopsiestreatedwithoilsshowedthebesttissueregenerationcomparedtocontrol
groups.Groupstreatedwithouroilsand“CICAFLORA”hadhigherwoundcontractionpercentage.Polyunsaturatedfattyacids
inoilsactasinflammatorymediatorsincreasingneovascularization,extracellularremodeling,migration,andcelldifferentiation.
Woundhealingeffectwasattributedtoantibacterialandantioxidantsynergy.Conclusion.Accordingtofindings,oilsshowedbetter
activityinwoundhealingcomparedto“CICAFLORA”duetoaphytoconstituentssynergy.However,clinicaltrialsonhumansare
necessarytoconfirmefficacyonhumanpathology.
1.Background this study in order to explore their phytochemical compo-
sitions, to evaluate their wound healing effect and to better
Wounds regardless of their types and causes are common explainthemechanismonwoundhealing.
diseases that constitute a major problem of public health at Trigonella foenum graecum L. (Fenugreek) is an annual
the global level and mainly in countries in the process of herbthatbelongstotheFabaceaefamily,commonlyusedin
development. However, despite the impressive progress in orientalcountriesasaspiceinfoodpreparations.Theseeds
modernmedicine,drugsdispensedtotreattheskinrepresent arereportedtohavenutritiveandrestorativepropertiesand
3%oftheintendedonesandarenotyetreallyeffective. canstimulatedigestiveprocesses[1];moreover,theyareused
Various medicinal plants, mainly their oils, have always asatraditionalremedyforthetreatmentofdiabetes[2].
been used to treat different kinds of wounds. The literature SesamumindicumL.(Sesame)isapioneercultivatedself-
presents various herbal formulations and natural extracts pollinatingannualplant,originatingfromAfrica,belonging
with several phytochemical compounds (vitamins, phenols, tothePedaliaceaefamily.Sesametakesanimportantrolein
sterols, etc.), in healing plants for the application of wound human nutrition and the seeds are essentially used for the
care.Awholelistofthosemedicinalplantsaretraditionally productionofoil.
used in folk medicine, including our three selected plants, Thefruitisamicronizedandoblongcapsulecontaining
grapeseed,fenugreek,andsesame,whichareinvestigatedin numerous seeds [3]. This oilseed has numerous nutritional
2 Evidence-BasedComplementaryandAlternativeMedicine
[4], ethnobotanical, pharmaceutical, and medical applica- ISO3960/2001method.Theindexofperoxideofafattyacid
tions.Infact,itislaxative,emollient,anddemulcent[5].This is the number of milliequivalents of active oxygen contents
urgesustofurtherexplorethisoilseedandfocusourconcern in1kgoftheproductandoxidizingtheiodineofpotassium
ondermalrepair,stressingourresearchonwoundhealingin withtheliberationofiodineandtitrationofthisonebythe
particular. thiosulfateofsodium.
Vitis vinifera L. (grapes) belongs to the Vitaceae family.
Grapes have been a traditional treatment in Europe for (ii) Acidity Value. The acidity value indicates the content
thousandsofyears.Thegrapeseedoilisrichinunsaturated of free fatty acids present in the tested oils, expressed in
fatty acids, especially linoleic acid [6] which constitutes a oleic acid. It represents an important quality parameter for
considerableproportionoftheseed. the commercial classification of the product according to
Woundhealing,thesignificantconcerninpathologylike ISO 660/2003 method. 5g of oil was dissolved in 30mL of
inpostsurgeries,burns,andscars,isadynamicandcomplex equalvolumesofethanol/ether(1/1)neutralized.Thefreecar-
process that involves biochemical and physiological phe- boxylicfunctionsweremeasuredbyasolutionofpotassium
nomena from inflammation to proliferation to remodeling, hydroxideinthepresenceof1%phenolphthalein.Theendof
behaving in a harmonious way to ensure tissue repair [7]. theexperienceismarkedbytheappearanceofapinkcolor.
Duringtheinflammatoryphase,thisprocessishamperedby
theproductionofahighleveloffreeradicals.Ifnotcontrolled (iii)TheSaponificationValue.Thesaponificationvalueisan
bytheantioxidativecapacityofthehost,theinhibitionofboth indirect measure that allows classifying the oil according
thecellmigrationandtheproliferationtakesplace,whichcan to the length of fatty acid chains; the criterion was bound
damage the surrounding, wound tissue [8]. So, the present to the molecular weight of fatty acids. This measure can
studyevaluatesthewoundhealingeffectofthreemedicinal turn out useful because this value gives an idea about the
plants previously referred to (the fenugreek seed oil, the qualityofoilsaccordingtoISO3657/2002method.Thevalue
sesameseedoil,andthegrapeseedoil)viatheexplorationof of saponification represents the quantity in milligrams of
phytochemicalcompositionandthedifferentparametersof necessary potassium hydroxide to transform the free fatty
antibacterialandantioxidantactivitiestoexplainthecrucial acids and the glycerides contained in 1g of fat into soap
mechanismofwoundhealing. andisdeterminedbymixingavolumeofoilwithpotassium
hydroxideandtitratedwithhydrochloricacid.
2.Methods
(iv)SpecificExtinctionCoefficientat232nmand270nm.The
determinationoftheUV-specific extinctionvaluesgivesan
2.1.PlantMaterialandReagents
approximation of the oxidation process in unsaturated oils
2.1.1. Reagents. The (DPPH) 1,1-diphenyl-2-picrylhydrazyl [9] according to COI/T.20/Doc (No 19/Rev.2 of November
was purchased from Sigma Chemical Co. (St. Louis, MO, 2008) method. 0.25g of the oil was dissolved in 25mL of
USA). Butylated-hydroxytoluen (BHT) and all the other cyclohexane.Theabsorbanceofthesolutionoffattyoilwas
chemicalswereofanalyticalgrade. measuredbyUV/visiblespectrophotometeratspecificwave
Fortheevaluationofthewoundhealing,“CICAFLORA” lengthof270nm.Theextinctionat270nmofarawfatcan
was used as a reference product. It consists in an emulsion giveanideaaboutthelevelofsecondaryoxidation.
oil-waterthatcontainsMimosaasamainactivecomponent
(v) Free Fatty Acid Percentage. The free fatty acid method,
andismarketedinacreamform.
COI/T.20/Doc(No34,November2015),determinesthefree
fatty acids in oils by chromatography in gas phase. The
2.1.2.Materials. Theanalysisofthemethylestersoffattyacids
content of free fatty acids is expressed in acidity, calculated
wasmadebychromatographyingasphase(C.P.G)bymeans
as the percentage of oleic acid. It consists in diluting 0.2g
ofaUNICAM610chromatograph,equippedwithadetector
of extracted oil in 3mL hexane and 0.3mL of methanolic
(FID)allowingthedetectionofcompounds,acolumn(15m
potassium hydroxide. The reactive mixture is shaken in a
inlengthand0.22mmindiameter)linedwithafilm(0.25𝜇m
hangingwhirlpoolfor2minandthenspin-dried.Thus,the
thick) of a polar phase (50% cyanopropylmethyl and 50%
upper phase contains the esters of fatty acids dissolved in
phenylmethyl-polysiloxane), and an injector divisor. The
∘ ∘ thehexaneandthelowerphaseistrainedbythefractionof
detectorwasatatemperatureof250 C,thecolumnat180 C,
∘ glycerinandtheminorconstituentsoftheblankoil.
and the injector at 220 C. The quantity of injected oil was
0.2𝜇L. (2)QuantificationofPolyphenols.Thecontentoftotalphenols
ofthevarioussamplesisdeterminedaccordingtothemethod
2.2.AnalyticalMethods describedbyTsaietal.[10].InthismethodFolin-Ciocalteu
reagentwasadded tothetest oil.Afteragitation,asolution
2.2.1.PhytochemicalAnalysis of washing soda is added. After 2h of incubation, the
absorbanceismeasuredat760nm.
(1)QualityValueandFattyAcids
(3) Chlorophylls and Carotene. The contents of chlorophylls
(i) Peroxide Value. The peroxide value of any oil is an pigments and carotenoids in oils were calculated after the
importantindicatorofprimaryoxidationlevelaccordingto readingoftheopticaldensitiesat670nmand470nmusing
Evidence-BasedComplementaryandAlternativeMedicine 3
coefficientsofextinctionforcaroteneandtotalchlorophylls been inoculated with the tested microorganisms. The Petri
∘
[11]. dishes were kept at 4 C for 2h. The plates were inoculated
∘
withthebacteriaandincubatedat37 Cfor24h.Thediame-
2.2.2.AntioxidantActivities tersofalltheinhibitionzonesweremeasuredinmillimeters.
Allthetestswereperformedinduplicate.
(1)DPPHFreeRadical-ScavengingAssay.Thisassayhasbeen
used to investigate the scavenging activity of antioxidant (3)DeterminationsoftheMinimumInhibitoryConcentration
compounds.Infact,DPPHisastablefreeradicalthatcanbe (MIC)andMinimumBactericidalConcentration(MBC).The
reducedbyaproton-donatingsubstratelikeanantioxidant, microdilutionmethodwasusedtoinvestigatetheminimum
causing the discoloration of DPPH and reduction of the inhibitory concentration (MIC) and the minimum bacteri-
absorbanceat517nm. cidalconcentration(MBC)accordingtotheNationalCom-
TheDPPHfreeradical-scavengingpotentialofthethree mitteeforClinicalLaboratoryStandards(NCCLS,2001).All
studied oils was determined according to the reports of tests were performed in Mueller Hinton Broth (MHB). The
Bersuderetal.[12].Radical-scavengingactivitywasexpressed oils were dissolved in 20% dimethylsulfoxide (DMSO) and
as the inhibition percentage and was calculated using the then diluted from the highest concentration to the lowest
equationofDPPHradicalscavengingactivity. one. A serial doubling dilution of the oils was prepared
in a 96-well plate. Overnight broth cultures of each strain
DPPH radical-scavenging activity(%) ∘
were prepared. Petri dishes were kept at 4 C for 2h. Then,
∘
𝐴 −𝐴 (1) bacteria were incubated at 37 C for 24h. The microbial
= control sample ×100. growth was determined by absorbance at 600nm using the
𝐴
control universal microplate reader [16]. To evaluate MBC, broth
𝐴controlistheabsorbanceofthecontrolreactionand𝐴sampleis fromeachwellwa∘stakenandinoculatedinMuellerHinton
Agar(MHA)at37 Cfor48hforthebacteria.
theabsorbanceofoils/standardBHTsamples.TheIC50value
(mg sample/mL) is the effective concentration at which the
(4)MBC/MICRatio.TheMBC/MICratio[17]cangiveaclear
DPPH radicals are scavenged by 50%. The test was carried
ideaabouttheeffectofoilsunderstudyonbacteria.Indeed,
outinduplicate.
iftheratioishigherthan4,theoilissaidtobebacteriostatic
andiftheoilisendowedwithabactericideeffect,theratiois
(2) Ferric Reducing Antioxidant Power FRAP. The ability of
lowerthan4.
the oils (0.06mg/mL to 1mg/mL) to reduce iron (III) was
determined according to the method of Yildirim et al. [13].
TheIC50value(mgsample/mL)istheeffectiveconcentration 2.2.4.WoundHealingActivityTest
atwhichtheabsorbanceis0.5forthereducingpower.BHT
is used for comparison and all data values are the mean of (1) Experimental Animals. ThirtyWistarmaleratsweighing
duplicateanalysis. 175 ± 3.98g were used for the experiment. They were
randomly housed in clean polyethylene cages individually
(3) 𝛽-Carotene Bleaching Assay. This spectrophotometric under controlled conditions of 22–25∘C, 60–70% relative
technique in the ultraviolet ray was developed by Marco humidity, and 12 hours of dark-light cycle with free access
[14] and then slightly modified by Miller [15]. It consists in to water and food. Procedures and animal comfort were
a measurement at 470nm. The discoloration of 𝛽-carotene controlledbytheInternationalGuidelinesforAnimalCare.
resultsfromanoxidationbythelinoleicacid.
(2) Circular Excision Wound Model. After anesthesia with
ketamine (100mg/kg body weight) by intramuscular injec-
2.2.3.AntibacterialActivity
2
tion,afullthicknessofellipticareaofapproximately200mm
(1)MicrobialStrains.Theantimicrobialactivityofthestudied woundwasinducedontheshavedrats’dorsalinterscapular
oilswasevaluatedusingarangeoflaboratorycontrolstains: region [18]. The day on which wound was created was
two Gram-positive bacteria: Bacillus subtilis (JN 934392) consideredasday0andallthewoundswerecoveredwitha
andStaphylococcusaureus(ATCC6538);twoGram-negative gauzedressingandweretreateduntiltheycompletelyhealed.
bacteria: Escherichia coli (ATCC 25922) and Salmonella
enteritidis(ATCC43972). (3) Excision Wounds Treatment. The rats were divided into
five groups consisting of six rats each. Group number (I)
(2) Determination of Antibacterial Activity by the Disc Dif- wasuntreatedandservedasacontrol(thewoundswerejust
fusionMethod.Theoilsweretestedforantibacterialactivity cleanedwithaphysiologicsaline).Group(II)wastreatedwith
by the method of disc diffusion according to the National fenugreek oil, group (III) with sesame oil, and group (IV)
CommitteeforClinicalLaboratoryStandards(NCCLS,2001) withgrapeseedoilandservedasthetestgroups,whilegroup
using suspension of the tested microorganisms. Mueller- (V) was treated with “CICAFLORA” cream and served as a
Hinton agar (MHA), sterilized in a flask and cooled, was standardreference(positivecontrol).
distributed to sterilized Petri dishes. The filter paper discs After rinsing the wounds with the physiologic saline,
(6mm in diameter) were individually impregnated with oil the test samples (fenugreek oil, grape seed oil, and sesame
and then placed onto the agar plates which had previously oil) and the “CICAFLORA” cream were applied, in a fine
4 Evidence-BasedComplementaryandAlternativeMedicine
Table1:Valuesofphytochemicaltests.
Phytochemicaltest
Oils Acidityvalue UVconstants Peroxidevalue Saponificationvalue
% K232 K270 MeqO2/Kg 170<𝑥<255
Grapeseed 1.83 3.0113 2.1710 12 175.32
Sesame 0.4 3.0103 1.1547 0.9 173.91
Fenugreek 0.2 3.0079 0.4373 11 172.32
layer covering all over the surface of the wound, every two showthemorphologyoftissues:organization,epithelialpro-
days until the wound completely healed. So, the treatment liferation and granuloma tissue formation, collagenisation,
wasstoppedwhenthewoundsofanyfirstgroupcompletely newlycapillariesformed,andscarformationindermis.
healed; then the rats were sacrificed and the granulation The studied criteria in histopathological sections con-
tissues were excised from animals. A part of wet tissue was sisted in the reepithelialization, cornification of the epithe-
fixed in formalin at 10% (v/v), embedded in paraffin, and lium, fibroblast, and collagen contents. Furthermore, histo-
presentedforhistologicalobservation. logical biopsies examined for advanced tissue regeneration
werecharacterizedbythepresenceofwell-organizedstratum
(4) Wound Healing Evaluation Parameters. To evaluate the ofbothepidermisandderma.
process of wound healing for the 5 study groups, we relied
ontwoclinicalmacroscopiccriteriaincludingthequalitative
2.2.5.StatisticalAnalysis. Statisticalanalyseswereperformed
(colorofwound)andthequantitativecriteria(woundclosure
usingSPSSversion17(SPSSInc.,Chicago,Il,USA).
rate)andonemicroscopiccriterion(histologicalevaluation).
Student test was applied to compare weights averages
before and after treatment between oil-treated groups and
(5)ChromaticStudy.Superficialwoundstendtolightenfrom
bothnegativeandpositivecontrolgroupstodetectascertain
redtopalepinkandbecomemorehomogeneousandmore
significant differences. Nonparametric tests, Kruskal Wallis
consistent in texture when they heal. The chromatic evalu-
test,andMannWhitneytestwereusedtocomparedifferent
ationofthehealingprocesswasdonethroughphotography
groups. Raw data were shown with median IQR for each
ofwounds.Thisstudyconsistsofcodingthewoundofeach
group.Differenceswereconsideredtobestatisticallysignif-
rat:brightredforbloodthatcoversthewound,darkredfor icantat𝑝<0.05.
coagulatedbloodinepidermis,redforgranulationtissue,and
finallypinkforepithelializationphase[19].
3.Results
(6) Rate of Wound Closure and Epithelialization Time. The
rateofclosureofeachindividualwoundfrombothcontrols 3.1.PhytochemicalAnalysis
and the treated groups was used as an indicator of wound
3.1.1. Quality Value and Fatty Acids. Table 1 presents the
healing.Awoundmarginwastracedafterthewoundincision
valuesofacidity,specificextinctioncoefficient,peroxide,and
using transparent paper and then the area was measured
thesaponification.
through the Mayrovitz rule [20]. Wound contraction was
Table 2 shows a different percentage of polyunsatured
measured on the 3rd, 5th, 7th, 9th, and 10th days until
acidsespeciallyoleic,linoleic,andlinolenicacidsthatreach
complete wound healing and expressed in percentage of
together 89.37%, 82.5%, and 84.28%, respectively, for grape
healedarea.Woundcontraction,thepercentageofreduction
seedoil,sesameoil,andfenugreekoil.
oftheoriginalwoundsize,wascalculatedusingthefollowing
expression:
3.1.2. Quantitative Polyphenols, Chlorophylls, and Carotene.
(𝐴 −𝐴 ) Thequantitativedosageoftotalphenoliccompoundsofoils
Wound closure(%)= 𝑇𝐴 𝐷 ×100. (2) showshighvaluesforoils,butgrapeseedoilandfenugreekoil
𝑇
haveahigherlevelofchlorophylls(8.078ppm)andcarotene
Theinitialwoundareaonday0andthewoundareaonallthe (56.78ppm)(Table3).
followingdaysarerepresentedas𝐴𝑇and𝐴𝐷,respectively.
Theepithelizationperiodwasconsideredasthenumber 3.2.AntioxidantActivities
of required days to fall of scab without any residual raw
wound[21]. 3.2.1. DPPH Free Radical-Scavenging Assay. The antioxidiz-
ing efficiency increases with the concentration of the oil
(7) Histological Examination. All of the skin samples were (Figure1).Thethreeoilsseemtohaveapotentialantioxidant
fixedin10%neutralbufferedformalin.Followingthefixation, activity compared to the BHT activity. The percentages of
3𝜇m sections of paraffin were perpendicularly made to the inhibitionare68.12%,84.59%,and84.6%forgrapeseedoil,
surfaceofskinincludingthewholethicknessofskin.Serial sesameoil,andfenugreekoil,respectively,and54.6%forBHT
sections were stained with hematoxylin-eosin (HE) [22] to ataconcentrationof1mg/g.
Evidence-BasedComplementaryandAlternativeMedicine 5
Table2:Fattyacidscomponents.
Fattyacid
Oil C16:0 C18:0 C18:1 C18:2 C18:3
Palmiticacid% Stearicacid% Oleicacid%(Ω9) Linoleicacid%(Ω6) 𝛼-Linolenicacid%(Ω3)
Grapeseed 6.69 3.57 19.75 69.33 0.29
Sesame 10.44 3.18 40.19 41.94 0.37
Fenugreek 8.36 3.60 19.19 37.71 27.38
Table3:Polyphenols,chlorophylls,andcarotenevalues. 0.8
0.7
Polyphenols Chlorophylls Carotene
Oils
0.6
(ppm) (ppm) (ppm)
m 0.5
Grapeseed 191.1 8.078 15.24 n
Sesame 191.3 0.005 3.98 700 0.4
Fenugreek 210.3 0.229 56.78 DO 0.3
0.2
0.1
90
0
80
0 g g g g g
H / / / / /
P 70 mg mg mg mg mg
P
D 60 6 5 5 5 1
n of 50 0,0 0,12 0,2 0,
hibitio 3400 GFernaupge rseeeekd SVeitsaammien C
% in 20
10 Figure2:Ferricreducingpowerassay.
0
0 g g g g g
/ / / / /
mg mg mg mg mg 90
0,06 0,125 0,25 0,5 1 n 7800
o 60
Fenugreek Sesame biti 50
Grape seed BHT hi
n 40
Figure1:ActivityofDPPHfreeradicalscavenging. % of i 2300
10
0
0 L L L L L
3.2.2. Ferric Reducing Antioxidant Power FRAP. Figure 2 m m m m m
/ / / / /
illustratesthereducingcapacityofoilsonironIII. 𝜇g 𝜇g mg mg mg
0,1 1 0,01 0,1 1
3.2.3.𝛽-CaroteneBleachingAssay. Figure3demonstratesthe
𝛽-carotenebleachingassaysresults. Fenugreek Sesame
Grape seed BHT
Figure3:Activityofinhibitionofbleachingof𝛽-carotene.
3.3.AntibacterialActivity
3.3.1.DiameterofInhibitionZone. Theantimicrobialactivity
ofoilstestedondifferentbacteriagram(+)andgram(−)was 3.4.WoundHealingActivityTest. Thewoundhealingprocess
presentedinTable4. was carried out by a chromatic study on the basis of the
progressive changes in wound color during the different
3.3.2.DeterminationofMinimumInhibitoryandBactericidal phases of cicatrization for each group: fenugreek oil, grape
Concentrations(MICandMBC𝜇g/mLMH). Table5points seedoil,sesameoil,“CICAFLORA,”andphysiologicserum.
totheconcentrationsofMICandMBC.
3.4.1. Wound Healing Evaluation Parameters. Weights of
3.3.3. Ratio = MBC/MIC. This ratio was used in order WistarratswereillustratedinTable7.Thestatisticalcompar-
to verify the antimicrobial potential of oils, and the ratio ison of their average weight for the same group before and
MBC/MICisrequired(Table6). aftertreatmentwasnotsignificant(𝑝<0.05).
6 Evidence-BasedComplementaryandAlternativeMedicine
Table4:Diameterofinhibitionzone(mm).
Stains
Gram(+) Gram(−)
Oils
Bacillussubtilis Staphylococcusaureus Escherichiacoli Salmonellaenteritidis
JN934392 ATCC6538 ATCC25922 ATCC43972
Grapeseed 17±1.0 — 11±1.0 11±1.0
Sesame 12±0.5 — — —
Fenugreek 10±1.0 — — —
Table5:Concentrationofbacteria(𝜇g/mLMH).
Stains
Gram(+) Gram(−)
Oils Bacillussubtilis Staphylococcusaureus Escherichiacoli Salmonellaenteritidis
JN934392 ATCC6538 ATCC25922 ATCC43972
MIC MBC MIC MBC MIC MBC MIC MBC
Grapeseed 6.25 12.5 — 6.25 12.5 6.25 12.5
Sesame 6.25 25 — — —
Fenugreek 3.125 25 — — —
MIC:minimalinhibitoryconcentration,MBC:minimalbactericideconcentration.
Table6:Bactericideeffectoftestedoils.
Stains
Gram(+) Gram(−)
Oils
Bacillussubtilis Staphylococcusaureus Escherichiacoli Salmonellaenteritidis
JN934392 ATCC6538 ATCC25922 ATCC43972
Grapeseed Bactericide — Bactericide Bactericide
Sesame Bacteriostatic — — —
Fenugreek Bacteriostatic — — —
Table7:Averageweightsbeforeandaftertreatmentwithcontrolsandoils.
Averageweight, Averageweight, Averageweight,grape Averageweight, Averageweight,
Day
untreated(g) CICAFLORA(g) seedoil(g) fenugreek(g) sesame(g)
Beforetreatment 182,5±2.271 184.5±2,271 176.5±2.121 193,5±2,121 172,33±2,926
Aftertreatment 183±2.265 184.33±2.559 176±2.212 193±2.265 171±2.849
𝑝value 0.25 0.085 0.25 0.25 0.66
Student’s𝑡-testwasappliedtodetectandascertainsignificantdifferences.
3.4.2. Chromatic Study. Wound photography of the same Towardsthe7thday,thescabsallowedtheapparitionof
groupratswasillustratedinFigure4.Thechosendays(0/3/ aredcolorationthatcorrespondedtothetissuegranulation
5/7/9 and 10) were corresponding to the wound induction with spread sides of the wound for the rats of the control
day, inflammatory phase, granulation tissue formation, and group(untreatedgroup).
reepithelialization,respectively. Fromthe10thday,thesescabsinratstreatedwithoilsand
The chromatic study of the wounds showed a similar referenceproductbegantofalltoletapinkishcolorappear
colorationduringthefirstthreedays.Infact,thebrightred that characterized an epithelialization ending after 11 days.
colorationobservedonthewoundingdayreflectedthecolor Thewoundcontractingabilityoftheoilsand“CICAFLORA”
ofthebloodcoveringtheunderlyingmusclesafterexcisionof was more significant than those of the control groups. The
theskin.Thiscolorationbecamedarkredonthesecondday rateofwoundclosurebyfenugreekoil,sesameoil,grapeseed
whichgaveevidenceoftheformationofabloodclot.Thisclot oil,andCICAFLORAandinthecontrolgroupisillustrated
enabledthebloodcoagulation.Fromthe3rdday,theblood inTable8andFigure5.Theirratesofwoundclosureat11th
clot was converted to a scab which retracted in the treated daywere,respectively,99.84%,99.83%,99.84%,94.82%,and
rats. 86.05%.
Evidence-BasedComplementaryandAlternativeMedicine 7
Day 0 Day 3 Day 5 Day 7 Day 9 Day 10
(a)
Day 0 Day 3 Day 5 Day 7 Day 9 Day 10
(b)
Day 0 Day 3 Day 5 Day 7 Day 9 Day 10
(c)
Day 0 Day 3 Day 5 Day 7 Day 9 Day 10
(d)
Day 0 Day 3 Day 5 Day 7 Day 9 Day 10
(e)
Figure4:Visualobservationofthewoundhealingexperimentondays0,3,5,7,9,and10.(a)Untreatedgroup,(b)grouptreatedwith
“CICAFLORA,”(c)grouptreatedwithgrapeseedoil,(d)grouptreatedwithsesameoil,and(e)grouptreatedwithfenugreekoil.
3.4.3.HistologicalExamination. Epidermalregenerationcov- 4.Discussion
eringoverthewoundsurfacetreatedbythefenugreek,grape
seed,andsesameoilswascoloredbythehematoxylin-eosinto Variousplants,essentiallytheiroils,havebeenusedtotreat
investigatetheepitheliumandtissueorganization(Figure6). wounds. The literature presents several phytochemical con-
We noticed fibroconnective tissue regeneration in the stituents, various herbal formulations, and natural extracts
referencebiopsiesandthoseofthethreetestedoils,without from medicinal plants to the application for wound care.
hairy adnexal or glandular structures. However, this tissue Someofthosemedicinalplantsaretraditionallyusedinfolk
regeneration was lower in the biopsies of control rats. The medicine,includingourplants,grape,fenugreek,andsesame,
microscopic examination of the scar zones handled by var- whichareinvestigatedinthisstudyinordertoexploretheir
ious oils highlighted an epidermic complete regeneration phytochemicalcompositions,toevaluatetheirwoundhealing
andwellorganized,whichwasconsiderednormal.Also,the effect,andtobetterunderstandtheirmechanismonwound
thicknessoftheepitheliumwasmoreimportantforthegrape healing. Consequently, topical application of the tested oils
seed oil, sesame oil, and fenugreek oil than “CICAFLORA” and the cicatrizing reference drug seems to accelerate the
cream. healingofwoundsandthecontractionoftheskinborderstoa
8 Evidence-BasedComplementaryandAlternativeMedicine
Table8:Statisticalstudy:medianIQRandnonparametrictests.
1 3 5 7 9 11
0.90(0.84–1.12) 0.56(0.56–0.60) 0.18(0.15–0.18) 0.04(0.02–0.06) 0.002(0.002–0.003)
Grapeseed 1.29(1.12–1.38)
(b∗) (b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗)
0.94(0.84–0.97) 0.42(0.42–0.43) 0.18(0.18–0.21) 0.08(0.04–0.09) 0.003(0.001–0.003)
Fenugreek 1.38(1.17–1.38)
(a∗,b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗)
0.94(0.94–1.12) 0.56(0.56–0.84) 0.18(0.16–0.21) 0.05(0.023–0.094) 0.002(0.001–0.003)
Sesame 1.29(1.03–1.31)
(b∗) (b∗) (a∗∗,b∗∗) (a∗∗,b∗∗) (a∗∗,b∗∗)
0.77(0.56–0.77) 0.38(0.37–0.41) 0.18(0.17–0.19) 0.06(0.06–0.07)
CICAFLORA 1.17(1.00–1.50) 1.03(0.96–1.12)
(b∗) (b∗) (b∗∗) (b∗∗)
Untreated 1.17(1.15–1.41) 1.22(0.99–1.38) 0.91(0.81–0.96) 0.50(0.38–0.50) 0.32(0.27–0.32) 0.21(0.11–0.21)
Nonparametrictests:KruskalWallisandMannWhitneytests.RawdatashownwithmedianIQR(𝑛=6)foreachgroup.
∗𝑝<0.05,∗∗𝑝<0.001.a:comparedtoCICAFLORA;b:comparedtountreated.
Rate of wound healing they kept a good quality over this work. The specific UV
120 absorbance values at 232nm, a primary oxidation indicator
ofoils,were3.01andtheK270 valueswerelower.According
100 to common standards, the UV absorbance at 232nm for
extravirginoliveoilmustbeunder2.5[25].Thesefindings
were concordant with the previous peroxide values. Both
80
n parameters,reflectingthedegreeoftheoilautooxidation,can
o
cti increasewiththeageofoilsandtheirexposuretosunlightor
a
ntr 60 hightemperatures.
o
of c Tested oils contain a significant amount of unsaturated
% 40 fatty acids that reach 89.37%, 82.5%, and 84.28% for grape
seedoil,sesameoil,andfenugreekoil,respectively.Thehigh
level of polyunsaturated fatty acids can make it extremely
20
susceptible to oxidation [26]. However, the oils were very
stable due to the presence of a number of antioxidants like
0 polyphenols, carotene, and chlorophylls. The antioxidants
Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 described in the present study can explain the lower values
of the autooxidation parameters. In fact, several researches
Grape seed CICAFLORA
reportedthatmanyvegetableoilswereanimportantnatural
Fenugreek Untreated
Sesame sourceofcarotenoidssuchasPistacialentiscusthatpresented
a value ranging between 5.8 and 10.57mg/kg oil [27], but
Figure5:Rateofwoundclosure. ourstudiedoilshavesubstantiallyhigheramountsespecially
in fenugreek and sesame oils 56.78mg/kg and 15.24mg/kg,
respectively. Carotenoids are the most important source of
fastrecoverycomparedtothecontrol.Thebesthealingactiv-
vitaminA.
itywouldbeattributedtotheirphysicochemicalproperties, Theautooxidationisthemajorcauseofthedeterioration
antioxidant,andantibacterialactivities. of oil during the storage. It depends on several factors
So,oilacidityistheresultofthedegreeoftriacylglycerol as the initial composition of the oil and the presence in
distribution due to a lipolysis reaction, in which free fatty minor compounds with pro- or antioxidizing activities like
acidsareformed.Theaciditymeasuredintheoilsampleswas chlorophylls[28].Inourstudy,grapeseedoilpresentedthe
low in the order of 0.2% for the fenugreek oil, 0.4% for the highestvalue(8.078mg/kg).
sesameoil,and1.83%forthegrapeseedoil.Allthestudied The autooxidation distorts edible oils by degradation of
oilswereofacidicpHthatpromotedtheinhibitioncapacity theessentialfattyacidsandconsequentlythereductioninthe
ofbacteriagrowthandacceleratedthewoundhealingprocess nutritional value and the formation of products of decom-
[23]. position[29,30].Thephenoliccompoundsinthethreeoils
Peroxidationisabeginningtofatautoxidation,whichis (197.56±11.2mg/kg),duetotheirantioxidizingactivity,can
an inevitably slow phenomenon. The manipulation of oils contributetothepreservationofthequalityofthisone[31].
andthemannerofstoragecanreduceautooxidationeffects. DPPHisastablefreeradicalwhichisreducedinthepresence
According to common regulations, the peroxide values of of an antioxidant, mostly by the phenolic compounds [32].
extravirginoliveoilmustbeunder20MeqO2/kg[24],which Indeed,thechemicalstructureofpolyphenolsenablesthem
concordswiththeresultsofthethreeoils.Thoselowperoxide to trap this free radical by hydrogen transfer. Our results
valuesindicatethatthetestedoilswerenewlyharvestedand demonstrated that our tested oils reacted strongly against
extractedandthenstoredingoodconditions,suggestingthat the DPPH radicals. This finding would be related to high
Evidence-BasedComplementaryandAlternativeMedicine 9
Oil G×200 G×400 Oil G×200 G×400
d (IV) ep ep k (II) der
Grape see der der Fenugree der ep
ep
(a) (b)
Oil G×200 G×400
Oil G×200 G×400
e (III) ep ep RA (V) ep ep
m O
a L der der
Ses der AF
C
der CI
(c) (d)
Oil G×200 G×400
e (I)
n
ali
c s
gi
o
ol der
ysi der
h
P
(e)
Figure6:Histologicalinvestigationoftreatedanduntreatedbiopsies:epidermal(ep)anddermal(der)architectureofwoundsafterthe11th
day.(a)Grouptreatedwithgrapeseedoil,(b)grouptreatedwithfenugreekoil,(c)grouptreatedwithsesameoil,(d)grouptreatedwith
“CICAFLORA,”and(e)untreatedgroup(Gr200andGr400).
concentrations of polyphenols in oils and explain their injury, they initiate a prolonged inflammatory phase that
relativestabilityandlowerautooxidation. resultsindelayeddepositionofmatrixcomponents,wound
Otherantioxidantsinourstudiedoilsdescribedinliter- remodeling,andclosure[37].
aturecanalsocontributetotheirstabilityandtothewound Fromthe3rdday,aproliferativephasehasbeentriggered
healingphenomenonsuchasvitaminEandsterols[33]. and characterized by the formation of granulation tissue,
Ourstudyshowedatotalclosureofthewoundstreated including angiogenesis, the migration of fibroblasts, and
bytheoilsand“CICAFLORA”after11dayswithanadvanced collagen synthesis [38]. In our study, from the 3rd day, the
tissue regeneration characterized by the presence of well- blood clot was converted to a scab which retracted in the
organized stratum of derma and epidermis in comparison treated rats. But an inflammatory reaction, manifested by
withthoseofthecontrolbiopsieswhosetissueneoformation an edema and oozing on wounds, seems more important
wasincomplete.Accordingtoliterature,thenaturalcontrac- in the rats of the control groups than those of all the three
tionofwoundstakesplacebythe21stday[34].Thisfinding treated ones. Towards the 7th day, the scabs allowed the
underlines the capacity of studied oils to accelerate the apparitionofaredcolorationthatcorrespondedtothetissue
proliferationcontributingtoafastrecovery.Thesamehealing granulationwithspreadsidesofthewoundfortheratsofthe
period was also observed with two medicinal plant oils: control groups. In the treated groups, an important wound
Cucurbitapepo.L(Cucurbitaceae)andLinumusitatissimum contractionwasobservedwithanadvancedreepithelization.
(Linaceae)[35]. Theepithelialcellsofthewoundbordersproliferatedtowards
The chromatic study of the wounds showed a similar thecenterandledacompletewoundclosuretowardsthe11th
coloration during the first three days corresponding to the day(Figure5).
formationofabloodclotwithdebrisofcellfillingthebreach The phytoconstituents of the studied oils could explain
in the course of the initial inflammatory phase. So, fibrin, the mechanisms of the skin wound healing process. So,
once stabilized in blood clot, is a key element in the initial their considerable amount of polyunsaturated fatty acids
processofskinhealing.Itallowstherecruitmentoffibroblasts includedoleicacid,linoleicacid,andlinolenicacid.Linoleic
by chemotactic effect and stimulates the production of acid, a precursor of arachidonic acid, is important in the
collagen [36]. When inflammatory cells arrive at the site of inflammatory cascade (prostaglandins, thromboxanes, and
10 Evidence-BasedComplementaryandAlternativeMedicine
leukotrienes) [39]. These substances act as inflammatory its later amendments and approved by the Committee of
mediators and accelerate the inflammatory process. Thus, AnimalEthics(Protocolno.94-1939).
they increase local neovascularization, the remodeling of
theextracellularmatrix,migration,andfibroblasticcelldif-
CompetingInterests
ferentiation [38], which accelerates the healing of wounds.
Fattyacidshavebeen reportedto havetheabilityto reduce Theauthorsdeclarethattheyhavenocompetinginterests.
transepidermal water loss and increase skin hydration and
supportiveenvironmentforacceleratedskinwoundhealing
Authors’Contributions
[40].
In addition, the wound healing effect would also be
DorsafMoallaRekikandSamehBenKhedirconceivedand
attributedtoasynergybetweenanantibacterialandantiox-
designedtheexperiments.DorsafMoallaRekik,SamehBen
idant action observed in our study and described in the
Khedir,NazihaGratiKammoun,andKamiliaKsoudaMoalla
literature.
performedtheexperiments.DorsafMoallaRekik,SamehBen
Generally,bacterialspecieshaveoptimummoisturecon-
Khedir,NazihaGratiKammoun,andKamiliaKsoudaMoalla
tentclosedtoneutrality(6.5 < pH < 7.5).Habitually,bac-
analyzedthedata.DorsafMoallaRekik,SamehBenKhedir,
terianeedapHrangingfrom5.5to8.0togrow;otherwise,
Naziha Grati Kammoun, Kamilia Ksouda Moalla, and
there is a slowdown in their development activity, reaching
Zouheir Sahnoun contributed reagents/materials/analysis
acompletegrowthcessationatapHunder4.5orabove9.0.
tools. Dorsaf Moalla Rekik, Kamilia Ksouda Moalla, and
AlltheoilsshowedacidicpHthatpromotedtheinhibitionof
Sameh Ben Khedir wrote the paper. All authors read and
bacterialgrowthandacceleratedthewoundhealingprocess
approvedthefinalmanuscript.
especiallyintheinflammationphase.AcidicpHcontributed
totheidealenvironmentforfibroblasticactivity,cellmigra-
Acknowledgments
tion,cellproliferation,andreorganizationofcollagen,which
resultedinthestimulationofwoundhealing[41].
ThisworkwassupportedbytheMinistryofHigherEducation
PolyphenolsandcarotenoidstogetherwithvitaminEand
and Scientific Research, Tunisia. The authors are grateful
sterols demonstrated a beneficial effect on wound healing
to Madiha JALLOULI, English Professor at the Faculty of
andcollagensynthesisbypreventingdamagingeffectsoffree
MedicineofSfax,forhavingproofreadthemanuscript.This
radicalsandensuringthestabilityandintegrityofbiological
research was supported by the Tunisian Ministry of Higher
membranes [42]. Palmieri et al. [43] also described how
EducationandScientificResearchviaSfaxUniversity.
vitaminEhasahumectanteffectonskinwoundscarring.
In addition, sterols are powerful compounds that can
help to reduce systemic inflammation [44]. They can speed References
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