Development and Characterization of Cinnamon Leaf Oil Nanocream for Topical ApplicationN. A. ZAINOL, T. S. MING AND Y. DARWIS*Discipline of Pharmaceutical Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia
Zainol, et al.: Cinnamon Leaf Oil Nanocream for Topical Application
Cinnamon leaf oil contains a high percentage of eugenol and has antimicrobial, antioxidant and antiinflammatory properties. However, the undiluted oil can cause irritation to the skin. Therefore, the aims of this study were to develop and evaluate cinnamon leaf oil nanocream using palm oil. Nanocream base was prepared using different ratios of oil, surfactants and water. The surfactant used were mixture of Tween 80:Carbitol or Tween 80:Span 65 at different hydrophile‑lipophile balance values. The pseudoternary phase diagrams were constructed to identify the nanocream base areas and the results showed that the nanocream bases using Span 65 as co‑surfactant produced bigger cream area. Fifteen formulations using mixtures of Tween 80:Span 65 were further evaluated for accelerated stability test, droplet size, zeta potential, rheological properties and apparent viscosity. The nanocream base which had an average droplet size of 219 nm and had plastic flow with thixotropic behavior was selected for incorporation of 2% cinnamon leaf oil. The nanocream containing cinnamon leaf oil had the average size of 286 nm and good rheological characteristics. The in vitro release study demonstrated that eugenol as the main constituent of cinnamon leaf oil was released for about 81% in 10 h. The short‑term stability study conducted for 6 months showed that the cinnamon leaf oil nanocream was stable at a temperature of 25° and thus, cinnamon leaf oil nanocream is a promising natural based preparation to be used for topical application.
Nanocream or semisolid emulsion is one of thepharmaceutical topical formulations that are appliedexternally[1,2].Thenanocreamcanbepreparedbyusinghigh energymethods such ashigh shear stirring, highpressure homogenizers or ultrasound generators[3].Generally, ananocream isveryuseful inpersonal careand cosmetics because the small size of the dropletswhich are in the nano range of 100–600 nm[4] allowthem todeposit uniformlyonto the skin andenhancesthe efficientdeliveryof active ingredients through theskin[5,6].Basically, thecreamcontainsvariousdrugs fordifferent remedialproperties inanappropriatesemisolidbaseeitherhydrophobicorhydrophilic incharacter[7].
Cinnamon (Cinnamomum zeylanicum) leaf oilcontains a high percentage of eugenol and hascharacteristically strong astringent properties,antibacterial[8], antiparasitic, antispasmodic[9] andantidiarrhea[10,3].Thus, theseherbshavebeenused forhealing a number of diseases, such as cardiovascular,respiratory, digestive, immune, urinary, lymphatic,
reproductive, nervous system complaints and severalother disorders. In addition, cinnamon leaf oil alsoshows very effectivemosquito repelling effect[11,12].However, the undiluted oil can cause irritationif directly applied onto the skin[13,14]. Gosh et al.reported the use of cinnamon leaf oilmicroemulsionformulation forwound healing[15],but no study hasbeen done on the preparation of cinnamon leaf oilnanocream.Therefore, the objective of the presentstudywas to prepare cinnamon leaf oil nanocreamusing palm oil as oil phase. Palm oil has beenusedmainly in food industry and its application asa pharmaceutical excipient is not widely studied.Palmoil has advantages because it has high contentof antioxidants such as tocotrienol which prevent
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oxidationof oil and triglycerideswhichmay functionas natural surface active agents.
MATERIALS AND METHODS
Palm oil (SeriMurni) was purchased fromTecsohypermarket (Malaysia), polysorbate 80 (Tween 80),cetostearyl alcohol and cinnamon leaf oil werepurchased from Euro Chemo-Pharma Sdn Bhd(Malaysia), sorbitan tristearate (Span 65) waspurchased from Fluka (USA), propyl paraben,methyl paraben and dethylene glycol monoethylether (Carbitol)were purchased fromSigma-Aldrich(USA),Sodiumcitrate andcitric acidwerepurchasedfrom R&M Chemicals (UK). Cellulose acetatemembrane of 0.2µmwas purchased fromSterlitech(USA), potassium dihydrogen phosphate anddi‑potassium hydrogen phosphatewere supplied byR andMChemicals (UK).
Pseudo ternary phase diagram construction:Phase diagrams of amixture containing palm oil,surfactants of differentHLBvalues andwaterwereconstructed using the water titrationmethod. Thesurfactantsused in this studyweremixturesofTween80:Carbitol atHLB values of 13.92 (90:10), 12.84(70:30) and 10.64 (60:40) orTween 80:Span 65 atHLBvaluesof 13.71 (90:10), 11.17 (70:30) and9.84(60:40).
Oil and surfactantmixturewere prepared at ratiosof 9.0:1.0, 8.0:2.0, 7.0:3.0, 6.0:4.0, 5.0:5.0, 4.0:6.0,3.0:7.0, 2.0:8.0, and 1.0:9.0 in a separated universalbottle. Oneml of distilledwater was added everyfifteenminutes and the changes in themixtureswererecorded.Themixtureswere kept for 24 h at roomtemperature to achieve equilibrium.Then, the finalvisual observation was recorded according to theclassification shown in theTable 1.The conductivityof resultingmixtureswasmeasured using electricalconductometer to classify them as anO/WemulsionorW/O emulsion. The results were plotted in thepseudoternaryphasediagram.
Preparation of primary nanocream base:The primary nanocream base formulation wasprepared by heating the oil andwater phase in thewater bath separately in twodifferent beakers at 55°with continuous stirring at 350 rpm for 30minusingamagnetic stirrer. The oil phase consists of palmoil, propyl paraben (0.05%), and Span 65while thewater phase containingTween80, buffer pH5.5 andmethyl paraben (0.1%).The oil phasewas dispersedin thewater phase then continuouslymixed using amagnetic stirrer at 350 rpmwith the aid of spatulato overcome the formation of a liquid crystallinephase.After a while, the mixture was stirred at1500 rpm for 30min and homogenized usingT25Ultra-Turrax (IKA,USA) at 19,100 rpm for 2min atroom temperature for further characterization.
Preparation of cinnamon leaf oil nanocream:The properties of selected primary nanocream basewere further improvedby adding cetostrearyl alcoholas a rheologicalmodifier.Thenanocreambaseswereprepared according to themethod used to prepareprimary nanocream base and subjected to furthercharacterization.Thebestnanocreambase formulationwas selected for incorporation of 2% cinnamon leafoil. The oil phase of cinnamon leaf oil nanocreamformulationconsistedof cetostearyl alcohol, cinnamonleaf oil, palm oil, Span 65 and propyl paraben(0.05%)while thewater phase consisted ofTween80 and buffer pH5.5. Similarmethod asmentionedabovewas also used for preparing cinnamon leaf oilnanocream formulations.
Accelerated stability study:Twomethodswere used in the accelerated stabilitystudy: centrifugation and heating cooling cycle. Incentrifugationmethod, cream formulationwasplacedin the graduated tubes and centrifuged at 4000 rpmfor 30min (Eppendorf centrifuge 5702 R). In theheating cooling cyclemethod, the nanocream basesample was repeatedly subjected to two differenttemperatures.Firstly, thecreamformulationwasplacedin agraduated tubeand freezedat temperature -8° for24h followedby storing at 45° for 24h to complete1 cycle.The experimentwas repeated for 6 cycles todetermine the stabilityof thenanocreambyobservingseparationandcoagulation in thenanocream.
Droplet size measurement:The droplet sizes of the formulationweremeasuredusing Zeta Sizer 1000HSA, (Malvern Instrument,
TABLE 1: VISUAL OBSERVATION CLASSIFICATIONClassification DescriptionMicroemulsion It is transparent or translucent and can flow easilyLiquid crystal It is transparent or translucent and nonflowable
when inverted at 90°Emulsion It is milky or cloudy and can flow easilyEmollient gel or cream
It is milky or cloudy and nonflowable when inverted at 90°
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UK)which is basedon the basic principle of photoncorrelation spectroscopy.The samplewasdilutedwiththe buffer to get theK count in between 50-200 asrequired bymachine consistency before reading thedroplet size.
Zeta potential measurement:Zetapotential of the formulationwasmeasuredusingZetasizer Nano ZS (Malvern, UK). Zeta potentialof the formulated nanocreamwas determined toensure that they arewithin the limit of ±30 becausewithin thisvalue thedropletsusuallydonot coalesce.The formulationswere dilutedwith the same buffersolution used as the external phase in the formula tofix the ionic strength and reduce the droplet count.Bubbleswere eliminated from the samples beforemeasurement toprevent change in themobilityof thedroplets in the samples.
Rheological and apparent viscosity measurements:The rheologicalmeasurementswere carriedout usingrheometer (rheological instrumentAB, Sweden).The system was equipped with a cone and platemeasuringhead (platediameter40mm).About0.5gof the samplewas placed on the plate and left toequilibratewith the controlled temperature (25°±0.1)for 3min before bringing down the cone. Excesssamplewas swept awaywith tissuepapers.The shearstress was applied in an increasingmanner at therateof10Pascal/sec and the ratemeasurementswererecorded.Rheogramswere drawn by plotting shearstress on the abscissa and shear rate on theordinate.
As the creamsusually exhibitednonNewtonianflow,the rheological behaviorswere studied according tothe following equation:LogG=N log (S-F)–Logη…(Eq. 1).Where,G is the shear rate in sec-1, S is theshear stress in Pascal, F is the yield value,η is theviscosity andN is the slope of Log (S-F) againstlogGplot.WhenN is 1, plasticflowwithBinghammodel is indicated.
Transmission electron microscopy:The size and morphology of the cinnamon leafoil nanocream was studied using FEI CM 12high resolution TEM (Philips, Electron Optics,Eindhoven, Netherlands). The cinnamon leaf oilnanocream samplewas placed on collodion formvarcarbon film-coated 400mesh copper grid heldwithself-locking fine forceps, and then a drop of 2%methylamine tungstate as a negative stain solution
was added to the surface of the grid.The excess ofstained solution on the samplewas gentlywiped offusing filter paper. The gridwas placed on a Petridish linedwith filter paper and left to dry for about10minat room temperaturebeforeexaminationunderthemicroscope.
In vitro release study:The in vitro drug transport through the artificialcellulose acetatemembranewas carried out usinghorizontally static type Franz diffusion cell. TheFranzdiffusioncell consistedof aneffectivediffusionsurface areaof 0.636 cm2 and a receptor cell volumeof 5ml.The static receptor cellwasfilledwith 5mlphosphate buffered saline (pH 5.7) containing 1%Tween80and stirredwitha smallmagneticbar at thespeed of 500 rpm for uniformmixing.The receptorcompartment was maintained at 37±0.5° using acirculatingwater bath.Cinnamon leaf oil nanocream(40 mg) was placed on the cellulose membranesurface facingdonorcompartment and400µl sampleswerewithdrawn from the receptor compartment atpredetermined timepointsof0.5, 1, 1.5, 2, 3, 4, 6, 8,10,12and24h.The samplewithdrawnwas replacedwith 400µl of phosphate buffer saline (pH 5.7)containing 1%Tween 80.The drug content in thecollected sampleswas determined using a validatedHPLC method. The mobile phase consisted ofmethanol andwater (75:25v/v)delivered at1ml/minin C18 Phenomenex column (250 mm×4.6 mm,5µm).TheUV/Visdetectorwasset at thewavelengthof280nmand the injectionvolumewas20µl.
Stability study:The stability studywas conducted at two differenttemperatures,40±2°/75±5%RHand room temperature(25±2°/65±5%RH). The samples at temperature40±2°/75±5%RHwere placed in a stability chamberwhile samples at room temperaturewere left on ashelf.At periodic intervals of 1, 2, 3 and 6months,all the sampleswhichwere stored at 40±2°/75±5%RH and at room temperature were studied forconductivity, pH, droplet size, apparent viscosity,yieldvalue,flowcharacteristics, total eugenol contentand in vitro release.The eugenolwas assayed usingHPLCmethoddescribed above.
Statistical analysis:All parameters except in vitro release studywere evaluated using one-wayANOVA and foridentificationofmeans that are significantlydifferent
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from each other, aposthocTukey’sHSD testwasperformed.Thedifferencewas statistically significantif P<0.05. The in vitro release study statisticalanalysis was performed using a post hoc Dunnetttest. SPSS version 20.0 softwarewere used for thisanalysis and all values are expressed asmean±SD.
RESULTS AND DISCUSSIONS
Construction of pseudoternary phase diagrams is thebestway to study all kinds of formulations that canbe derived from themixingof surfactants,water andoilbecause thediagramscancoverallprobabilitiesofmixing ratios and possible areas of finding cream[16].Figs. 1a-c is the pseudoternary phase diagrams for amixtureofpalmoil,water,Tween80as the surfactantand Carbitol as a cosurfactantwith differentHLBvalues of 10.68, 12.84 and13.92.Fig. 1a showednocreamareapresent,butexhibited largerO/WemulsionandW/Omicroemulsion areas.This couldbebecausethe amount ofTween 80was not enough to form a
surfactant layer at the interface that is responsiblefor producing a cream system[17]. In contrast, thepseudoternary phase diagram represented in fig. 1band c illustrated formation of small cream areas.Increase in the concentration of surfactant (Tween80) and reduce the concentration of cosurfactant(Carbitol) resulted inagradual increase increamarea.However, the combination ofTween80 andCarbitolwas theworst surfactantmixture because it produceda small cream region and the texture of the cream inthis regionwasdifficult to spread, sticky anddidnothave good skin feel.Therefore, it is not a suitablecombinationof surfactant in cream formulation.Thesesurfactants combinationswere excluded from furtherstudy. Pseudoternary phase diagrams formixtures ofpalmoil,water,Tween80andSpan65withdifferentHLB values of 13.71, 11.13 and 9.84 are depictedin figs. 2a-c.All the phase diagrams ofmixtures ofTween 80 and Span 65 showed a larger cream areacompared toTween80 andCarbitol.The creamareawas formedwhenwater content in the systemwas
Fig. 1: Pseudoternary phase diagram with Tween and Carbitol.Pseudoternary phase diagram for a mixture of palm oil, water and Tween 80 as the surfactant and Carbitol as a cosurfactant at HLB 10.68 (a), HLB 12.84 (b), HLB 13.92 (c). O/W ME: Oil in water microemulsion, O/W LC: oil in water liquid crystal, O/W CRM: oil in water cream, O/W EMULSION: oil in water emulsion, W/O ME: water in oil microemulsion.
ba
c
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in the range of 25 to 60%. Itwas found thatwatercontent below 25%was insufficient to hydrate thepolyoxyethylene groupswhichwere critical for theswelling of surfactant chains to demonstrate a creamor gel structure[17]. Increase in cosurfactant (Span65)concentration fromHLB13.71 toHLB9.84,wouldincrease the interfacial tensionof interfacialfilmanda larger creamareawas formed[18,19].The larger creamareawas formedwhen suitable combinationofTween80as surfactant andSpan65ascosurfactantwasusedin a cream formulation.Conductivitymeasurementsrevealed that each point in the cream areawas oftheO/W type because it conducted the electricity.Thus, this study suggested that Span 65 showedbetter cosurfactant action compared to theCarbitolbecause it produced larger cream area as shown inpseudoternaryphasediagrams.Thus, itwas a suitablecombinationwithTween 80 in producing the stablecream formulation. Fifteen cream formulationswererandomly selected from the combination ofTween
80 andSpan65withHLB13.71, 11.13 and9.84 andsubjected to further studyusinganacceleratedstabilitytest to select thebest andmost stable formulation.
Centrifugation is an excellent tool for the productionof phase separation for accelerated stability study ofnanocreams.The result of the centrifugation testwasshown inTable2.Someof the formulationsunderwentphase separation into twophaseswhichwascreamyatthe top and clear solution at thebottom. Itmayhaveoccurreddue toOstwald ripening inwhichmoleculesmove as amonomer, and the coalescence of smalldroplets resulted in the formation of larger dropletsby diffusion processes driven by the gain in surfacefree energy[19].Among the formulations tested, theformulations codedwithA1,B2,B4, andC1 showednophase separation, creaming, cracking, coalescenceor phase inversion during this centrifugation test.These formulationswere considered to have passedthe test andwere then further examinedusinganother
Fig. 2: Pseudoternary phase diagram with Tween and Span 65.Pseudoternary phase diagram for a mixture of palm oil, water and Tween 80 as the surfactant and Span 65 as a cosurfactant at HLB 13.71 (a), HLB 11.13 (b), HLB 9.84 (c). O/W ME: Oil in water microemulsion, O/W LC: oil in water liquid crystal, O/W CRM: oil in water cream, O/W EMULSION: oil in water emulsion, W/O ME: water in oil microemulsion.
b
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a
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accelerated stability test, heating cooling cycle.Afterundergoing heating and cooling for six cycles, somesamples had separated into two layers, whichwascreamy at the top and clear solution at the bottom.However, samplesB2andB4werepartially separated(Table 3).This phase separationmay have occurreddue to the temperature quench during heating andcooling cycles[19].
Lastly, the formulationsB2 andB4weremodifiedusing cetostearyl alcohol. The percentages ofcetostearyl alcohol were calculated from palm oilcontent in the primary formulations and the newformulationsare shown inTable4. In this formulation,cetostearyl alcohol actedas a stabilizer and thickeningagent[20].Normally, it is usedwidely in a variety ofcosmetics and pharmaceutical emulsions.The newformulationswere coded asB2(1),B2(2),B4(1) andB4(2).Theacceleratedstability testusingcentrifugationand heating cooling cyclemethodswere also carriedout on these formulations.All the samples exceptsampleB4(1)were found to be stable as no phaseseparation occurred.All formulations that passed theacceleratedstability testwere furtheranalyzed in termsofdroplet size, zetapotential andapparentviscosity.
Table 5 shows the results of average droplet size(below 250 nm), polydispersity (less than 1 andzeta potential measurement (about 30 mV) ofsamplesB2(1),B2(2) andB4(2) after homogenizing.Increasing duration of homogenizing at constantspeedwould reduce thedroplet sizewhileprolongingthe homogenizing time to 2.5minwould increasethe droplet size. Longer homogenizing timesmaycause instability of particles due to high input ofenergy that leads to aggregation of the droplets intoa larger ones[21].Therewas a significant difference inthe droplet size of samplesB2(2),B2(1) andB4(2).Among the formulations, B2(2) had the smallestdroplet size.All the formulations have higher zetapotential valueswhereby the repulsion force isbiggerthan the attraction force, so the cream is stable[22].Therewere no significant difference in zeta potentialof sampleB2(2),B2(1) andB4(2).
The rheological characteristic of the prepared creamsis important in technical applications includingmanufacturing, pumping, filling and storage.Yieldvalue is known as the minimum shear stressrequired to produceflow[23] and below this point thematerials will behave as solid. The yield value of
pharmaceutical andcosmeticmaterials shouldbehigh,so they do not flow out from the container whenplaced in an upside-down position[24].The apparentviscosity was calculated using Eq. 1. The yieldvalues and apparent viscosity of the formulationsB4(2),B2(2)andB2(1)are shown inTable6.SampleB2(2) had the highest yield value compared to the
TABLE 5: NANOCREAM FORMULATIONSFormulation code
Droplet size (nm)
Polydispersity index
Zeta potential (mV)
B2(1) 240.5±4.57 0.197±0.113 −33.8±0.493B2(2) 219.3±2.93 0.054±0.039 −31.3±0.85B4(2) 243.13±2.9 0.26±0.13 −29.3±3.86The table provides results of nanocream formulations homogenized at speed 19 100 rpm for 2.0 min. Mean±SD, n=3. SD: Standard deviation
TABLE 2: RESULTS OF ACCELERATED STABILITY TEST USING CENTRIFUGATION METHODSSmix ratio Formulation
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other formulations,whichmay due to the optimumsurfactant concentration and high percentage of thecetostearyl alcohol used in that formulationwhichformedmorehydrogenbondswith theaqueousphase.The apparent viscosity of formulationB2(1)was thelowest compared to the other formulations, hence, itis themostunstable formulation.Lowyieldvalueandlow apparent viscositymade the formulation easilyspill out from the container. FormulationsB4(2) hadlower yield value and higher apparent viscosity thanformulationB2(2). Thiswas due to the higher oilcontent in formulationB4(2)whichwould increasethe apparent viscosity of the formulation, and henceitwould be difficult to remove the nanocream fromthe container.Among all formulations studied thebest nanocreamwasB2(2) because it had high yieldvalue and good apparent viscosity.Thus,B2(2)wassuitable tobeused as a nanocreambase in cosmeticsand pharmaceutical applications because itwill notspill outwhen the container isplaced inupsidedownposition and beside that it is also easier to take thenanocreamout from the container.
The rheological properties of samplesB4(2),B2(2)andB2(1) are shown infig. 3 and all the rheograms
have yield value, which mean all formulationshave plastic flow properties.All the rheogramsof formulations studied have the same curvepattern which formed hysteresis-loop type withthe down curves to the left of the up curves. Thiscurve pattern is called thixotropic behaviour. Thethixotrophic behavior is a favourable characteristicof cosmetics, pharmaceutical creams and gelemulsions[25]. Since formulationB2(2) had the bestcharacteristics, it was chosen as the nanocreambase for cinnamon leaf oil. The cinnamon leaf oilnanocreamwas further examined in terms of dropletsize, zeta potential, apparent viscosity and flowcharacteristics.
Thedroplet sizeof thenanocreamafter incorporationof cinnamon leaf oil was increased from219.3±2.93 nm to 286.4±2.15 nm and the apparentviscositywas reduced from 11812±128.22 Pa.s. to10473.14±230.39Pa.s.Thisoccurancemaybedue to2%of palmoil being replaced by cinnamon leaf oilin the formulation.Different types of oilmay affectdroplets size and apparent viscosity of nanocream,however the yield value and zeta potentialwere stillquite high.Thus, it still produced a stable cinnamon
TABLE 6: RHEOLOGICAL PARAMETER OF THE FORMULATED NANOCREAMFormulation code Cetostearyl alcohol (%) Oil (%) Smix (%) Water (%) Apparent viscosity (Pa.S) Yield value (Pa)
Fig. 3: Rheograms of nanocream base formulations at HLB 11.13.Rheograms of (a) nanocream base formulations B4(2), (b) nanocream base formulations B2(2), (c) nanocream base formulations B2(1), at HLB 11.13.
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leaf oil nanocream.The rheogramcurveof cinnamonleaf oil nanocream showed in fig. 4 demonstratedplastic flow properties as it has yield value. It alsohas hysteresis-loopwith the down curves to the leftof theup curveswhere it is called thixotrophy that isimportant for creamapplication.
Fig. 5 shows the image of oil droplets in cinnamonleaf nanocream taken using high resolutiontransmission electronmicroscope (TEM). The oildroplets in nanocream formulation are of a darkcolour and have a spherical shapewith average sizeless than 300 nm.Thus, thisfinding further supportsthe results obtained using Zeta Sizer 1000 HSA,(Malvern Instrument,UK) that the droplet size is inthenano range.
Fig. 6 shows the in vitro release profile of eugenolfrom the optimized cinnamon leaf oil nanocreamformulation through the cellulose acetatemembrane.Almost81%ofeugenol is released from thecinnamonleaf oil nanocream formulation after 24 h. Thepercentageof eugenol released fromcinnamon leafoilnanocream increasedwith timeuntil 10h anddidnotincrease thereafter.Theprolongedeugenol releasecouldbe attributed to embedment of eugenol in the cream.Increased releasedof eugenolmaybe contributed bythe large surface areaof nanosizedparticles andhighsolubility of eugenol in the permeationmedium.Thesmall size of particles is one of the factorswhichcontribute to the increased penetration of skin[7].Besides that, the presence of a surfactant (i.eTween80) in the formulation also contributed to the higherpercentageof eugenol released.The surfactant canactas achemical enhancer in the penetration of eugenolinto the skinwhere50%ofeugenolwas released fromthe formulationwithin5h.
The cinnamon leaf oil nanocream formulationwaspacked into30gglassointment jarswith tight-fittingclosures.This containerwas selected for use sinceitwas easy tomeasure formulation parameters suchas viscosity and pH directly from the ointment jarfollowing storage of the samples for the requiredtime[26].
Based on visual observation, therewas no changeof themilky yellow colour of the cinnamon leaf oilnanocream upon storage at 40±2°/75±5%RH androom temperature (25±2°/65±5%RH) for 6months.After storage of the nanocream in the stability
chamber at either of the two temperatures, thestrong odour of cinnamon leaf oilwas still present.
0
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80
90
0 5 10 15 20 25 30
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Fig. 6: Mean in vitro release profiles of eugenol from nanocream formulation.Mean of in vitro release profiles of eugenol from cinnamon leaf oil nanocream formulation through cellulose acetate membrane. Mean ± SD, n=3.
Fig. 5: Transmission electron micrograph of particle cinnamon leaf oil nanocream. Transmission electron micrograph of image particle cinnamon leaf oil nanocream, under 40 000 magnifications.
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In addition, there was no contamination of fungiand molds in the nanocream at both conditions(25±2°/65±5%RH) and40±2°/75±5%RH). Itmightbe due to the presence of preservatives (methyland propyl paraben) in the nanocream.The resultssuggested that the nanocreamwas stable in bothconditionsover the specified timeof observation.
The results of the conductivity of cinnamon leaf oilnanocreamat twodifferent temperatures,40±2°/75±5%RH and room temperature (25±2°/65±5% RH)after 6months storage are shown inTable 7. Theinitial conductivity of the nanocream stored atroom temperature was 1240.6±3.1µS and after 6months, conductivitywas 1244.7±3.79µS. It wasfound that there was no significant change in theconductivitymeasurement after 6months storageat room temperature.This indicated that the bottomof the container contains the same amount of oilphasewithin the time frameof the stability study[27].Thus, the results suggested that the cinnamon leafoil nanocreamwas stable at this temperature as nocreaming or sedimentation in the nanocreamwasdetected during the period of the study. In contrast,a significant change occurred for the nanocreamstored in the stability chamber at 40°±2°/75±5%RHfor 6monthswhereby the conductivity increased to1260.7±7.5µS.The significant changewas due tothe upwardmovement of the oil phase. Thus, theconductivity increasedbecauseof the lowernumberofoil droplets at thebottomof thenanocreamcontainer.
The pH of a freshly prepared formulation was5.70 and after 6months, the pH of the nanocreamstored at room temperature (25±2°/65±5% RH)was 5.63±0.05 while the pH of the nanocreamstored at 40±2°/75±5%RHwas5.66±0.06 (Table 7).Therewere no significant changes found for bothnanocreams stored either at 40±2°/75±5%RH or
25±2°/65±5%RH.ThepHvaluesofbothnanocreamswhichwere unchanged could be due to the stabilityof thecompounds in thecinnamon leafoilnanocreamformulation. Thus, this indicated that there wasno degradation or ionization of chemicals in theformulation at both temperatures during the periodof study.
Themeandroplet sizesofnanocreamduring6monthsstability study at twodifferent temperatures is shownin Table 7. The freshly prepared nanocream hadan average droplet size of 285.33±1.06 nm andafter six months of storage at room temperature(25±2°/65±5% RH) or at 40±2°/75±5% RH, thedroplets sizes increased to 292.47±4.81 nm and505.73±16.85nm, respectively.Thedroplet sizeof thenanocream stored at room temperature (25±2°/65±5%RH) did not change significantly during the durationof the stability study.Thismay be due tominimalfree energy available in the system, hence noaggregation and coalescence occurred.The size ofdroplets in the nanocream stored at temperature40±2°/75±5%RH increased graduallywith time andtherewasa significant changeafter6months stabilitystudy.These results suggested that this increase ofdroplet size couldbedue to the free energy availablewhich caused freemoveable droplets to collide andcoalescewith each other in the system and henceincrease the droplet size.Thus, it can be concludedfrom this study, that the nanocream stored at roomtemperature (25±2°/65±5%RH) was more stablecompared to the nanocream stored at 40±2°/75±5%RH.Moreover,no significant changes inzetapotentialvalues were observed in all samples throughoutthe study at this temperature (25±2°/65±5%RH).However, thevalueof zeta potential at 40±2°/75±5%RH significantly decreased to 24.43 mV after 6months. Low zeta potential may be due to thecoalescenceof droplets in thenanocream.
21.74Yield value (Pa) 293±11.5 280±10 283±15 290±10 290±10 270±10 286±11.5 286±11.5 290±10Eugenol content (%) 101.04±0.78 100.49±0.45 100.43±0.51 99.82±1.38 99.36±0.16 97.51±1.38 98.99±0.54 97.12±2.34 91.1±1.06T50% (h) 5.65±0.39 5.00±0.16 5.29±0.16 5.39±0.05 6.63±0.06 5.65±0.30 6.91±0.37 5.61±0.17 6.87±0.03T50%: Time of 50% eugenol release, mean±SD, n=3. SD: Standard deviation, Pa: Pascal, Pa.s: Pascal second
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The apparent viscosity of the freshly preparedformulationwas 10499.98 Pa.s and after 6monthsof storage at different temperatures of 25±2°/65±5%RH and 40±2°/75±5%RH, the apparent viscositieswere 10847.63 Pa.s and 9440.63 Pa.s, respectively(Table7).Therewasno significant change inapparentviscosity at room temperature but, at 40±2°/75±5%RH the apparent viscosity decreased significantly.The insignificant change in apparent viscosity at25±2°/65±5%RHmight be due to the intactnessof hydrogen bonds between the polyoxyethylenechains of the surfactants[28]. A significant dropin the apparent viscosity value after storage forsixmonths at a temperature 40±2°/75±5%RHmaybe caused by themovement of a small number ofsurfactantmolecules from the interface to the surface,which affected the structure of the nanocream[29] orowing to the freemovement of droplets resultingin collisionwith each other (Brownianmovement)and coalescence. Thus, this study, suggested thatcinnamon leaf oil nanocreamwasmore stable atlower temperature (25±2°/65±5%RH) compared tothehigher temperature (40±2°/75±5%RH).
The yield valuemeasurement of freshly preparedcinnamon leaf oil nanocream at room temperaturewas 293±11.5 Pa.After 6 months storage at twodifferent temperatures 25±2°/65±5% RH and40±2°/75±5% RH, the values were 290±10 Pafor both temperatures (Table 7). There were nosignificant changes in yield values at bothtemperatures after 6months storage. Even thoughthe apparent viscositywas changed significantly at40±2°/75±5% RH after 6 months stability study,the yield value of the cinnamon leaf oil nanocreamwas not affected. In addition, therewere no changesof the flow characteristic of the nanocream after6months stability study at 25±2°/65±5%RH and40±2°/75±5%RH (fig. 7). The unchanged plastic
flow characteristics and insignificant difference inyield value of cinnamon leaf oil nanocreammightbe due to insignificant physical changes attributed tothenanocreamover the entire stability study forbothtemperatures.Therefore, the slight liquefactionof thenanocream stored at 40±2°/75±5%RHdid not affectits rheological flow.
The eugenol content in the samples (91-101%)waswithin the range of the original eugenol content.There was no significant change in the eugenolcontent in the nanocream after 6months stabilitystudy at room temperature (25±2°/65±5% RH).However, at a temperatureof 40±2°/75±5%RH therewas a significant difference in eugenol content in thenanocream after 1month compared to the freshlyprepared sample (0 month). The eugenol contentin the formulation dropped from 101 to 97% after1month (Table 7).Therefore, it can be concludedthat the cinnamon leaf oil nanocream is stable atroom temperature (25±2°65±5%RH)while at highertemperature it started todegrade after 1monthof thestability study.Reduction of the eugenol content inthe nanocreammay be due to increased degradationof volatile cinnamon leaf oil constituents at thetemperatureof 40±2°/75±5%RH.
During the stability study, the in vitro release ofeugenol from formulations of cinnamon leaf oilnanocreamswereobservedat0, 1, 2, 3 and6monthsat two different temperatures 25±2°/65±5%RH and40±2°/75±5%RH.Thepercentageof eugenol releasewas calculated based on the total drug content at theevaluatedpoint.
Fig. 8 shows the graph of the in vitro releaseprofiles of eugenol from cinnamon leaf oilnanocream through the cellulose acetatemembraneat different temperatures (25±2°/65±5% RH and
Fig. 7: Rheogram of cinnamon leaf oil nanocream after 6 months.Rheogram of cinnamon leaf oil nanocream stored at 25±2°/65±5% RH (a) and at 40±2°/75±5% RH (b) for 6 months, Mean±SD, n =3.
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40±2°/75±5% RH) over the entire test period(0, 1, 2, 3 and 6 months). It was revealed thatthe cumulative release of eugenol from freshlyprepared nanocream at room temperaturewas 81%for 24 h.After 6 months stability study at roomtemperature, the cumulative release of eugenolwas78.6%.Therewas no significant difference in thecumulative release of eugenol from the nanocreamformulation after 6months storage. In contrast, atthe temperature of 40±2°/75±5%RH, the cumulativerelease of eugenol from the nanocream formulationdecreased significantly to 74.1% after 6 monthsstorage.The in vitro release profile of eugenol fromcinnamon leaf oil nanocream remained relativelyconstant at room temperature (25±2°/65±5%RH)but shows a slight decrease at 40±2°/75±5% RHafter 6months storage. Based on this study, itwasconfirmed that the release of eugenol from thecinnamon leafoilnanocreamwasnotaffectedat roomtemperature compared to 40±2°/75±5%RH.
Table7 shows themean timeof50%eugenol releases(T50%) across the cellulose acetatemembraneover thetimeperiod (0,1,2,3and6months)of stabilitystudy.TheT50%of eugenol release from freshly prepared
cinnamon leaf oil nanocreamwas 5.65±0.39h.After6months stability study at room temperature, theT50%of eugenol releasewas 5.61±0.17 h.Therewasno significant change in theT50%of eugenol in theformulationwhen stored at 25±2°/65±5%RH after6months stability study.However,T50%ofeugenolwassignificantly increased to 6.87±0.03h after 6monthsstability studyat40±2°/75±5%RHstorageconditions.Adecrease in cumulative releaseof eugenol from thenanocream formulation after 6months stability studyand increasedT50% of eugenol at 40±2°/75±5%RHmight be attributed to the physical changes of thedroplet size and viscosity of the formulationwhichaffected thepenetrationofeugenolacross thecelluloseacetatemembrane.
In conclusions, thenanocreambase formulationB2(2)consisted of aqueous phase (pH 5.5), palm oil asthe oil phase and themixture ofTween 80:Span 65(70:30)HLB11.13 at the ratio of 47.4:15.8:36.8waschosen as the best nanocreambase formulation.Theselected formulation had rheological characteristicssuitable for topical application. Droplet size afterincorporating cinnamon leaf oil determined by zetasizerwas around 286.4 nm and the zeta potential of-29millivoltswhichcouldhinder the coalescence andaggregation of the oil droplets and produced stablenanocream formulation.Cinnamon leafoil nanocreamwasmost stable at room temperature compared to thehigher temperature (40±2°/75±5%RH).
AcknowledgementsThe authors would like to thank Universiti SainsMalaysia for providing a short term research grant(304/PFarmasi/6312023) to support thiswork.Theauthor (NorAzah Zainol) gratefully acknowledgesMajlis Amanah Rakyat for the granting of ascholarship.
Financial support and sponsorship:The author (Nor Azah Zainol) gratefullyacknowledgesMajlisAmanahRakyat for thegrantingof a scholarship.
Conflicts of interest:There are no conflicts of interest.
Fig. 8: In vitro release profiles of eugenol from nanocream during stability period.In vitro release profiles of eugenol from cinnamon leaf oil nanocream at 25±2°/65±5% RH (a) and at 40±2°/75±5% RH (b) for 0 month( ), 1 month ( ), 2 month ( ), 3 month ( ), 6 month ( ) through cellulose acetate membrane, Mean±SD, n =3.
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