The Wound Healing Action of Kakawati Gel from Gliricidia ...iomcworld.com/ijptp/files/Rogie Royce et al... dec 2015.pdfThe Wound Healing Action of Kakawati Gel from Gliricidia sepium

InternationalJournalofPharmacyTeaching&Practices2015,Vol.6,Issue4,2642-2649.

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TheWoundHealingActionofKakawatiGelfromGliricidiasepium(Jacques)Steudel(FamilyFabaceae)

RogieRoyceCarandang*,KristalCieloBuemio,AdelaineLopez

DepartmentofIndustrialPharmacy,CollegeofPharmacy,UniversityofthePhilippinesManila,TaftAvenuecornerPedroGilSt.,Ermita,Manila1000,Philippines

ResearchArticlePlease cite this paper as RogieRoyceCarandang*,KristalCieloBuemio,AdelaineLopez.TheWoundHealingActionofKakawatiGelfromGliricidiasepium(Jacques)Steudel(FamilyFabaceae). IJPTP, 2015, 6(4), 2642-2649.

AbstractObjective: Gliricidia sepium, locally known as kakawati isrecognizedforitswoundhealingproperty.Thestudyaimedtodeterminethewoundhealingactionofasafe,stable,lowcostformulationfromthelyophilizedsapofGsepium.Methods:Theorganoleptic,physical,andchemicalpropertieswere determined. Short term stability testing, as well ascompatibility study with common excipients was performed.Wound healing property was determined by threemethods:(1)measurementoftherateofwoundcontraction,(2)tensilestrength of the healed tissue using a tensiometer and (3)histological examination of collagen deposition. The 7.5% gelformulation was also tested for its efficacy with woundhealing-impairedrats.Results: The pilot-scale manufacture of a gel-basedformulationusingwaterextractofGsepiumwasfeasible.Theextract was compatible with glycerin, sodium CMC, sodiumbenzoate and sodium citrate. Stability testing with commonexcipients showed that the gel formulation was stable at atemperatureof30ºC.ItwasstableatpHvaluesof5to10andinnaturalandartificiallight.Theformulatedproductisagreengel of moderate consistency possessing a natural fragrancewith faint fetid odor. The gel formulation was found to beeffectiveat7.5%concentrationasawoundhealingagent.Conclusion: The gel formulation has been proven to havewoundhealingpropertiesandwasfoundtobesafe,effective,stableandlowcost.Keywords:woundhealing,kakawati,gelformulation,Gliricidiasepium,diabetes

IntroductionExpensive treatment of wound injuriescreatesaheavyburdentopoorFilipinos.Inline with this, the latter resorted toalternative herbal remedies that are notonly tested to heal wounds but are alsoreadily available. One of these herbalremedies is the plant Gliciridia sepiumJacquesSteudel(FamFabaceae)thatcanbefound almost everywhere. The sap of thebark, leaves and roots has been cited byfolkloric literatures as a wound healingpromoter[1].ManyFilipinossuffer fromdiabetesand itscomplications. The long term effects ofdiabetes impair wound healing bydiminishing sensation and arterial inflow.One of the manageable conditions isinfectionfromunhealedwoundscausedbyimpairment of the body’s natural healingmechanism[2].The effects of diabetes on healing arediverse, multifactorial, complex and inter-related.Itisoneofthewell-knownintrinsicfactorswhichaffectwoundhealing.Infact,diabetesaffectsalmostallstagesofwoundhealingtosomeextent[3].WiththeknowledgethatGsepiumgrowsinabundance almost everywhere, thechallenge was to develop the sap of thebark of theplant into a suitable, safe, andlow cost dosage formas an alternative forthe topicalmanagement ofmild to severewounds of normal as well as the woundhealing-impaired individuals, which can be

CorrespondingAuthor:RogieRoyceCarandang,RPh,MPHDepartmentofIndustrialPharmacy,CollegeofPharmacy,UniversityofthePhilippinesManila,TaftAvenuecornerPedroGilSt.,Ermita,Manila1000,PhilippinesEmail:[email protected]


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broughtaboutbydiabetescomplications,burnsandotherskininjuries[4].MaterialandMethodCollectionofPlantMaterial Fresh, matured barks of G sepium werecollected from Lipa, Batangas. Collection may bedone anytime of the year except when it rains inorder to avoid fungal growth on the bark due toabsorptionofwater.ProcessingofPlantMaterial Immediately after collection, the stem andtrunks were washed thoroughly in running waterand allowed to drip and air-dry. The bark wasremoved using a sharp knife. Undesirable plantparts and other extraneousmatterwere removed.Thebarkswere subjected tomechanicalextractionthrough expression using cheesecloth andpurificationofthesapextractwasdonebyfiltration.The 100% sap collected was then lyophilized toremove water and to ensure that the activeprinciplesweretheonlyonepresentinthesample. PhysicochemicalandOtherTestsThe following testswereperformed in the sample:(1) phytochemical screening, (2) organolepticevaluation, (3) foreignorganicmatter, (4)moisturecontent determination and (5) extractives and ashdetermination.PreformulationstudiesThe following tests were conducted: (1)hygroscopicitystudiesand(2)stabilityteststoheat,moisture,lightandpH.CompatibilityTestingMixtures (1:1) of the lyophilized extract with anexcipient placed in sealed and unsealed stopperedvials were stored at refrigerated temperature,ambientroomtemperatureandinoven(Memmert)set at 50°C protected from light for four weeks.Threetrialswereperformedforeachconditionandaqueous extract –excipient mixture. Their physicalappearanceswereobservedweeklyandathinlayerchromatographicprofilewasdoneandcomparedtothecontrolgroup.

TrialformulationThreetrialsof7.5%w/wkakawatigelwereconducted following a basic gel formulaconsisting of the following: viscosity-enhancing agent, humectant, neutralizer,preservatives and appropriate solvent. Thedrug-excipientmixturewas based on theirrespectivecompatibility.FinalformulationThe formulation with the best physicalattributeswasselectedtobetheprototypeformula. It was validated using a largerbatch size. In-process quality control testsfor the aqueous extract were performed.The amount of viscosity-enhancing agentandwaterphasethathadtobestirredandhomogenized necessitated the used ofHeidolphmixer. The finished productwasevaluated based on the followingparameters: color, appearance,consistency,pH,microbialcontent,andTLC.ValidationofWoundHealingActivity.A. MeasurementofWoundContractionAfter surgery, the excision wound marginwas traced at 3-day intervals and the areameasured by the aid of a caliper.Measurementsweretobecontinuedtothe15th day. Wound contraction percentagewas determined from the measurementusingtheformula:%WoundContraction=(HealedArea/TotalArea)x100 B. MeasurementofTensileStrengthTensile is the resistance to breaking undertension.Itindicateshowmuchtherepairedtissue resists breaking under tension andmay indicate in part the quality of therepaired tissue. The instrument used formeasurement is called a tensiometer. Forthe quantitation, one of the edges of thewound was fixed while applying ameasurable force to the other one. Theloadorweightsingramsrequiredtodisrupt


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thewoundisdeterminedaftercompletehealingofthewound.C. Histologicalexamination Theskinincludingthewoundswasremovedfrom each rat for histological examination. Thewounded area was fixed with 10% formaldehyde.Dehydrated samples were embedded into paraffinand cut into thin sections. These sections werestained with eosin and examined under lightmicroscope. The thickness of collagen layerdepositedonthetissuewasmeasured.D. Dataanalysis One-way ANOVA was used to determine iftherewas a significant difference between treatedgroups at different time intervals. Least SquareDifference (LSD) was used to know which doseshavesignificantdifference.Thedatawereanalyzedby considering the following: percent of woundcontraction, tensile strength, and histologicalexaminationofcollagenformation.StabilityTestingandExpirationDatingofKakawatiGelTo determine the products stability to heat, theproductwasstoredinitsfinalpackagingatambientroom temperature for reference, and in ovens(Memmert) set at accelerated temperatures of 40,50 and 60°C. Physical changes were observedvisuallyandassayedbyTLC.ResultsandDiscussionTable-1 presents the summary of thephysicochemical and other tests performed in thesample.Phytochemical screening was conducted todetermine what groups of plant constituents arepresent and could be extracted from the plantmaterial [5]. It was found that tannins, glycosides,reducing substances, plant acids and flavonoidswerepresent in thesamples.Saponins,particularlythesteroidaltypewerealsodetected.ThepresenceofalkaloidalsubstanceswasnotobservedThesapextractobtainedfromthebarkofGsepiumby mechanical expression was green in color butupon standing, separation of green sediments andyellowish-brown solution was noted. This can be

homogenized through agitation. It had anherbal characteristic odor. However, adistinctfetidodoroftheplant,thoughveryfaint, could still be detected. The sapextract was acidic, with a pH of 5.45,specific gravity of 1.0271 and relativeviscosityof1.4334cps.ThepHofthesapissaidtobepreferablebecausethepHofthenormal human skin is also slightly acidic.This is significant since higher acidity oralkalinitymaycauseirritationorcouldevenworsen the dermatologic condition. Thecolorandodorimpartedbythesapextractweresomehowexpectedtoaffectthecolorand odor of the gel since it will comprise7.5%oftheformulation.Table-1:SummaryofthePhysicochemicalandothertestsA.PhytochemicalScreening

Results

1.LitmusPaper2.Tannins3.Glycosides4.ReducingSubstances5.Alkaloids6.PlantAcids7.Saponins8.Flavanoids

SlightlyacidicPresentPresentPresentAbsentPresentPresentPresent

B.Testsperformed Mean*1.pH 5.452.specificgravity 1.02713.viscosity 1.43344.foreignorganicmatter 0.05%5.moisturecontent 5.86%6.water-solubleextractives(hot)

1.85%

7.water-solubleextractives(cold)

1.47%

8.alcohol-solubleextractives(hot)

0.86%

9.alcohol-solubleextractives(cold)

1.13%

10.totalash 13.73%11.acid-insolubleash 0.06%12.water-solubleash 0.14%*Threetrialswereperformed

The determination of foreign organicmatter was performed. The determinationmaybeusedasameasureofpurityandcan


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020406080

100

Percent Wound

Contraction

3 6 9 12 15

Days

Percent Wound Contraction of Normal Rats vs Days

StandardSap extractGelControl

serveasastandardfortheplantmaterial[6].Foreignorganic matter found in the samples includes soil,dead insects and decayed matter. These wereremoved from the samples. The mean percentageforeign organic matter was 0.05%, which wasconsideredlow,indicatingthatthesampleswereofhighpuritypriortouse.

Figure 1: Percent Wound Contraction vs. Days ofNormalRats

020406080

100

Percent Wound Contraction

3 6 9 12 15

Days

Percent Wound Contraction vs Days of Wound Healing Impaired Rats

StandardSap ExtractGelcontrol

Figure2:PercentWoundContractionvs.DaysofWoundhealingImpairedRatsVaryingquantitiesofmoistureareusuallypresentindrugs.Withnaturalproducts,absorbedmoisture isof great concern. The presence ofmoisture in thedrugcouldaffectitsstabilityinstorageasmoisturepromotes mold growth. Moisture is also animportant consideration in formulation since theamountoftheactiveconstituentinaformulationisusuallybasedonthemoisture-freedrug.Themeanpercent moisture content of the samples wasdeterminedtobe5.86%.Itwasexpectedthatalowmoisturecontent shouldbeobtainedsince thesapwasbeen lyophilized.However, the lyophilized saptends to absorb moisture upon exposure to air,thus,properstorageshouldbeconsidered.

Inthedeterminationofextractives,thehotand cold method of water and alcoholsoluble extractives were done. Thesemeasures can be used as a basis for theidentityandpurityoftheplantmaterialandassuch,maybesetasstandards.Theyareapproximatemeasures of the amount of acertain constituents or a group of relatedconstituents the drug contains. In thesamples, the mean percent water-solubleextractive for hot and cold method wasdetermined to be 1.85 and 1.47%respectively, while the mean percentalcohol soluble extractive for hot and coldmethod was determined to be 0.86 and1.13%. Figure 3: Examination of the excised wound under the light microscopetreated with G sepium gel formulation

Figure 4: Examination of the excised wound under the light microscope treated with Solcoseryl® Jelly Ash determinations were also conducted.Thetotalash,acid-insolubleashandwater

Collagen Layer

Collagen Layer


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soluble ash contents were determined. The meanpercenttotalashcontentofthesampleswasfoundto be 13.73%, mean percent acid-insoluble ashcontent was 0.06%, and themean percent watersoluble ash, 0.14%. These represent the inorganicsaltsnaturallyoccurringinthedrugandadheringtoit. However, in some cases, these may includeinorganic matter added for the purpose ofadulteration. Therefore, it may be used as a basisfor cleanliness and purity of the plant material,asidefromservingasabasisforjudgingits identity[7].

Figure 5: Examination of the excised wound under the light microscope using G. sepium extract

Figure 6: Examination of the excised wound under the light microscope (Control) Based on the compatibility testing and trialformulationsdone, final formulationof thegelwasaccomplishedusingthefollowingexcipients:sodiumCMC,glycerin,sodiumcitrate,sodiumbenzoateanddeionizedwater. The adjustment in the amount ofexcipientswasbasedontheobjectivethattherightpH,consistencyandgelstabilitybemet.Aspartofthestabilitystudies,theformulationwassubjectedtovaryingdegreesoftemperaturetoseehow heat affected the integrity of the active

ingredient,aswellastheexcipients.Thegelformulationwasstoredinitsfinalpackagingat an ambient temperature (30°C) andaccelerated conditions of 40, 50 and60°C.No changes were observed for thosesamples stored at a room temperature(30°C). Liquefaction, darkening andincreasedinpHwerenotedat40°Cafter1monthandat50and60°Cafter15daysand10daysrespectively.TLCresultsshowednochanges intheRfvaluesofspotsproducedwhen compared with the standardchromatogram of G sepium and neithertherewerenewspotsproduced.However,at temperatures 40°C, 50°C, 60°C, it wasobserved that during the later part of thestability period, liquefaction occurred.Degradation was fastest at 60°C. TLCshowed disappearance of 3 spots at 40°Cafter1month.At50°C,disappearanceof3spotswas seen on the 15th day. The samewas true at 60°C, wherein there wasdisappearanceof3 spotson the5thday. Itcanbe seen that therewasdecompositionoftheactiveingredientsinceothermarkerscan no longer be detected. TLC profiles ofsamplesstoredattemperatures40°C,50°Cand 60°C were noted. As expected, anincrease in temperature would cause anincrease in the rate of liquefaction, owingto a fall in apparent viscosity andconsistency. The results showed a trendthat more adverse storage conditionscausedearlier signsof instability in thegelformulation.Moisture has an adverse effect on drugstability,aswell.Whenadrugisexposedtoanenvironmenthavingaconsiderablyhighrelativehumidity(RH),thedrugcantakeupmoisture that could result in degradationparticularly if the drug is prone tohydrolysis.Moreover,itcanhastenpossibleinteraction with the other excipients,particularly those that are easily oxidized.Stability tomoisture of the lyophilized sapextract showed no significant changes insamples stored in stoppered vials, exceptforslightdarkeninginasampleexposedat

Collagen Layer

Collagen Layer


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90%RH(KNO3).Ontheotherhand,samplesstoredin unstoppered vials showed slight darkening evenat75%RH (NaCl).TLCof the lyophilizedsapextractrevealedthatthosewithstopperedvialswerestableup to 75% RH after 2 weeks while those withunstoppered vialswere found to be unstable evenat 60% RH. Thus, it follows that an increase in RHfavors decomposition of the active constituentsparticularlywithunstopperedvials.Hygroscopicity study of the gel formulation storedat 45% RH absorbed a mean amount of moisture0.04%of its initialweight;0.04%at60%RH;0.13%at 75% RH; and 0.27% at 90% RH. These datasuggestthatthecontainer-closuresystemofthegelformulation was tightly fitted and impervious tomoisture, thus, the affinity to moisture was lesseralthoughthereisincreasingrelativehumidity.Instability due to light or photodegradation isdependent on two factors: (1) intensity and (2)wavelength. Sunlight encompasses the wavelengthrangeof290-780nm,however,only thehigherUVrange of 290-390 nm can cause photodegradation.Conversely, light from fluorescent light bulbsincludes visible and potentially degrading UVradiation, which includes the wavelength range of320-380 nm. On the other hand, light fromconventional tungsten filament light bulbs,discharge radiation of >390 nm wavelength. Thiscauseslittleornoharmtodrugproducts [8]. Inthestudy,thesamplesofthelyophilizedsapextractandformulated gel were stored under sunlight andfluorescentlightbulbs.Nosignificantchangeswereobserved in all of the samples after twoweeks ofexposure at different conditions. Nonetheless, it isimportantthatthelyophilizedsapextractbestoredin amber-colored bottles which have the highestcapacity to adsorb radiation. The final packagingcontainerwhichisanAluminumcollapsibletubeforthegelformulationpreventsanddelaysphotolysis.Asstatedabove,pHisacriticalfactorthatshouldbeconsidered in the formulation of topicalpreparations.ImbalanceinthepHoftheskinresultsin irritation or subsequent aggravation of anydermatologicconditionpresent.Thus,thepHofthepreparationshouldmatchoratleastdeviateslightlyfromthatoftheskin.However,changesinthepHofthedrugasaresultofadditionofcertainexcipients,can significantly affect its stability. The lyophilized

sap extract were dissolved in differentmediums whose pH were adjusted that ispH1.0,3.0,5.0,7.0and10.0.AtpH1.0,thesolution initially turned brownish coloredfor both stoppered and unstoppered vials.AtpH3.0and5.0, thosewithunstopperedvials showed mold growth after 2 weekswhile with stoppered vials showedturbidity. Result from the study showedthatG. sepium lyophilized sap extractwashighly unstable at pH less than 5.0 assignified by darkening, turbidity and moldgrowth formation. The lyophilized sapextractwas found tobemost stableatpH5.0 topH10.0.However, TLC showed thatatpH10.0therewasdecompositionofthelyophilized sap extract since only one spotwas detected. Only at pH 5.0 and pH 7.0gave the same number of consistentmarkers.ThisisdeemedfavorablesincethepHofnormalhumanskinisslightlyacidic.Part of the objective of this study was toproduce a suitable gel formulation thatwouldbebeneficialnotonlyforindividualswith normal wound healing response butalso to wound healing impaired patientssuch as diabetics. In line with this, twotreatment groups were devised. The firsttreatment group consists of 10 malediabetes- induced rats while the othergroup consists of 10 normal male albinorats. Four wounds were also excised butthistimeweretreatedwithSolcoseryl Jellyasthestandarddrug,puresapextractofGsepium, 7.5% gel formulation and control.Threetestswerealsoconductedamongthetreatmentgroups.Intermsofwoundcontraction(Figures1-2),wound healing impaired rats showedslowerwound contraction as compared tonormal rats. The 7.5% gel formulationexhibited faster rate ofwound contractionon wound-healing impaired rats ascompared to the standard drug, thusshowing a significant difference. On theother hand, Solcoseryl® Jelly exhibited thesame response with that of the gelformulationonnormalrats.Therewasalso


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asignificantdifferencebetween the rateofwoundcontraction of Solcoseryl jelly and the pure sapextract inboth treatmentgroups.Thesame is truewith the 7.5% gel formulation having significantdifference with that of the sap extract. The threeaforementioned treatments showed significantdifferencewiththatofthecontrol.Tensile strength on wound healing-impaired ratsand normal rats revealed that there was nosignificant difference between the standard(Solcoseryl jelly), pure sap extract and gelformulation. However, a significant difference wasobtained between the three treatments and thecontrol.In terms on the thickness of collagen deposition,wound healing-impaired rats showed significantdifference between the standard and pure sapextract.Collagenlayers(Figures3-6)formedbytheusedofthestandarddrugwerethickerascomparedwith that collagen formedby thepure sapextract.No significant difference on thickness of collagenlayerswas revealedbetween the standardand theformulated gel and the latter with the pure sapextract. Likewise, normal rats exhibited nosignificant difference between the standard,formulatedgelandthepuresapextract.Thethreeaforementioned treatment groups showedsignificantdifferencewiththatofthecontrol.Resultsshowedthatthe7.5%formulatedgelhadcomparableefficacyascomparedwiththatofthestandard.Thepuresapextractalsohadthenaturetopromotewoundhealing.However,aformulatedgelwasfoundtobemoreeffectiveintermsoftherateofwoundcontractioninbothtreatmentgroups.ConclusionThe pilot-scale manufacture of a gel-basedformulation using water extract of G sepium isfeasible. The extract is compatible with glycerin,sodiumCMC,sodiumbenzoateandsodiumcitrate.It was proven to have wound healing properties.Stability testing with common excipients showedthatthegelformulationwasstableattemperaturesof30ºC.ItwasstableatpHvaluesof5to10andinnaturalandartificiallight.Theformulatedproductisa green gel of moderate consistency possessing anatural fragrance with faint fetid odor. The gel

formulation was found to be effective at7.5% concentration. The gel formulationcostsaboutPhp20.98perunitbasedontheproduct costing. The gelwas proven to beatparwiththestandardproduct,Solcoserylgel,andmoreeffectivethanthecontrol, inwoundhealingactivityforbothnormalandwoundhealing-impairedrats.AcknowledgementTheresearcherswouldliketoacknowledgeProf. Jocelyn Bautista-Palacpac for herinvaluable support in pursuing thisresearch.References1. Quisumbing E. Medicinal Plants of thePhilippines.Quezon City: Katha Publishing Co.,Inc.,1978.2. O'Meara S,Cullum N,Majid M,Sheldon T.Systematic reviews of wound caremanagement. Health Technology Assessment2000;4(21):1-237.3. Singer J, Clark R. Mechanisms of Disease:Cutaneous Wound Healing. The New EnglandJournalofMedicine1999;341(10):738-746.4. Daemen College Wound Therapy ResearchProgram.BiotechInitiativeAimedatDevelopingFaster, More Effective Wound Treatments[Internet]. 2007. Available fromhttp://www.daemen.edu/news/08-9-05.html5. Gutierrez H. An Illustrated Manual ofPhilippine Materia Medica. Taguig, MetroManila: National Research Council of thePhilippines,1980.6. Santos, A, Aguilar-Santos G, ObligacionMB,Olay L, Fojas F. Philippine Plants and theirContained Natural Products: Biological andPharmacological Research Survey. NationalResearchCouncilofthePhilippines1981;1(4):4673-674.7.Cortes-MarambaN.GuidebookontheProperUseofMedicinalPlants.Taguig,MetroManila:NSTA,1982.8. Gutierrez L, Palacpac J, Buenaventura A,ObusanB.FinalReportoftheResearchonTheAntipruriticActionofGliricidiasepium(Jacques)Steudel (Family Fabaceae): Formulation ofKakawati Lotion (Phase 1 and 2). Philippines:NIHUPManila,2001and2004.


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AUTHORS’CONTRIBUTIONS

Authorscontributedequallytoallaspectsofthestudy.

PEERREVIEW

Notcommissioned;externallypeerreviewed.

CONFLICTSOFINTEREST

Theauthorsdeclarethattheyhavenocompetinginterests.

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The Wound Healing Action of Kakawati Gel from Gliricidia ...iomcworld.com/ijptp/files/Rogie Royce et al... dec 2015.pdfThe Wound Healing Action of Kakawati Gel from Gliricidia sepium