PHARMAWAVE 10/17 Formulation And Characterization Of An .
PHARMAWAVE 10/17Formulation and characterization of an analgesic ointment indicated for pediatric useUttam Kumar Mandal*; Anis Arabi Hashmi; Bappaditya ChatterjeeIKOP Sdn. Bhd., Kulliyyah of Pharmacy, International Islamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah,Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia.*Corresponding author:Dr. Uttam Kumar Mandal, HOD (R&D Department), IKOP Sdn. Bhd., Kulliyyah of Pharmacy, InternationalIslamic University Malaysia (IIUM), Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200, Kuantan,Pahang Darul Makmur, Malaysia.E-mail: mandalju2007@gmail.com, Mobile: 60 109062750, Fax: 60 9571 6775AbstractThis research work deals with formulation development and characterization of an analgesic pediatricointment. The ointment was developed with menthol and camphor as active ingredients at lowerconcentrations than used for an adult formulation. The formulation was novel as it replaced theoleaginous hydrocarbon petroleum jelly base of conventional ointment by olive oil, a more acceptableexcipient to the pediatric patients. The developed formulation was compared against iKOOL, acommercial analgesic ointment with similar composition produced by IKOP Sdn. Bhd., IIUM, Malaysiawith respect to various quality control tests. The developed formulation showed encouraging results,butneeds further investigation to convert it into a commercial product.Keywords: Analgesic ointment, Menthol, Camphor, Formulation development.Running title: Analgesic ointment formulation.INTRODUCTIONAs compared to oral administration, topicaladministration of analgesic medicine provides somedistinct and important advantages. It overcomesgastric irritation and undesirable effects oncognition and level of consciousness associatedwith its oral administration [1]. Topicaladministration ensures a more even and continuousapplication of the dosage for a long period directlyto the site of the pain. Topical ointment containingmenthol and camphor has been used safely formany decades for analgesic relief of minor achesand pains of muscles and joints [2-4]. Additionally,they are indicated for general relief and suppressionof common cold symptoms as cough, sore throat,nasal congestion together with Their natural originhas helped them to be used commercially as safeand efficacious as compared to their syntheticcounterparts. Menthol (C10H20O; molecular weight,156; a terpene alcohol) is obtained either fromMandal et al., Pharmawave, 10:2017naturally occurring peppermint oil of synthesizedby hydrogenation of thymol. In lowerconcentrations (1% or less), menthol depressescutaneous sensory receptors, while at higherconcentrations (1.25% to 16%), it stimulatessensory receptors and thus it gives its counterirritant action [5]. Camphor (a terpenoid class ofcompound) is obtained from the wood ofCinnamomum Camphora L. [2]. The analgesic andcounter irritant effect of camphor is sm is not yet fully understood [6]. In onestudy, Moqrich et al. [7] found that camphoractivates TRPV3, a member of the transientreceptor channel that causes excitation anddesensitization of sensory nerves. When mentholand camphor are used together as analgesic, theyprovide synergistic action [8]. In few reports,menthol was found to cause allergic contactdermatitis [9,10] and systemic allergic reactions4
PHARMAWAVE 10/17[11]. Camphor occurs in in two enantiomer form;natural one is dextrorotatory (D-camphor), whilethe synthetic one form laevorotatory (L-camphor).As compared to the natural one, synthetic camphoris more toxic orally as claimed by toxicity study inmice [12].There are many branded products, both as over-thecounter (OTC) and prescription, in the form ofointment, liniment, and cream available in themarkets containing the active ingredients likementhol and camphor. However, these commercialproducts contain high amount of active drugs (5 to10% menthol and 5% camphor) as well asexcipients. As a result, these products are not safewhen applied to the pediatric population. Topicaltherapies, especially, analgesic and counter irritantmedications that are widely used for pediatricpatients are often misused. Their unique type ofskin which is quite immature with respect to barrierproperty is more susceptible to permeation of drugsas well as excipients as compared to the adult skin;this may often lead to undesirable toxicity [13].Skin surface area to body weight ratio in infants andchildren is significantly higher than the adults,which pose a great risk of accumulation of drugs intheir body. US Food and Drug Administration(FDA) does not entertain the use of commonlyprescribed topical medicines to pediatric patients asaround 70% of them are devoid of any pediatriclabelling [13].At this juncture, the objective of the present workwas to explore the feasibility of formulation andcharacterization of an ointment containing areduced amount of menthol (2% w/w) and camphor(2% w/w) for pediatric patients. An attempt wasmade to reduce the amount mineral oil as ointmentbase and replaced it with live oil. This wasfundamentally proposed for safety and patientcompliance reasons, as olive oil is being historicallyused to soothe baby skin rashes in addition to itsMandal et al., Pharmawave, 10:2017well-known skin moisturizer effect. A analgesicointment formulation, iKOOL produced by IKOPSdn Bhd., IIUM, Malaysia was used as referenceformulation to compare with the developedformulation.MATERIALS & METHODSMaterialsOlive oil, menthol crystal, camphor, eucalyptus oil,and peppermint oil were obtained as generous giftfrom IKOP Sdn., IIUM, Malayisa. For GC analysis,camphor ( 95.50 %), and menthol ( 98.20 %)(Sigma-Aldrich, USA), and petroleum benzine(boiling point 80 - 100 ⁰C) (Fischer scientific Co.,UK) were generously donated by IKOP Sdn Bhd.,Malaysia. Analgesic ointment iKOOL used asreference sample was also obtained from IKOPSdn. Bhd., IIUM, Malaysia. Instrumental facility toprepare the ointment and characterize like waterbath, rotational viscometer, centrifuge, stabilitychamber, gas chromatography etc were alsoprovided by IKOP Sdn. Bhd.Manufacturing MethodComposition of the ointment formulation is shownin Table 1. Water bath (WNB 8 Memmert GmbH Co. KG) was adjusted to 750 C. Beeswax andolive oil were transferred in a glass beaker placedon that water bath. Beeswax was allowed to meltand mixed with olive oil completely. Then camphorwas added to the mixture with consistent stirring.The mixture was cooled down by lowering the bathtemperature until 600C. Immediately, menthol,eucalyptus oil, and peppermint oil were added to themixture and allowed to cool down to 280C until itforms an ointment with the required consistency.5
PHARMAWAVE 10/17Table 1: Compositionointment.Material nameOlive oilBees waxMenthol crystalCamphorEucalyptus oilPeppermint oilTotalof the analgesic babyQuantity (g/100g)60 to 73%17 to 30%2233100gmOptimization of the Ointment BaseIn order to optimize the viscosity of theformulation, few experimental trials were carriedout with different ratios of beeswax and olive oil asshown in Table 2. iKOOL ointment formulationprepared by IKOP Sdn. Bhd was taken as thereference product to optimize the developedformulation.Table 2: Various experimental ratios of beeswaxand olive oil for analgesic baby ointmentformulation.Number Ointment baseof trials BeeswaxOlive oil% (w/w)% (w/w)1306022565320704157551773Characterization of the Developed OintmentViscosity MeasurementThe viscosity of the developed formulation wasdetermined by Brookfield Rotational Viscometer(DV-II PRO Digital viscometer). A 200 ml testsample was taken in a clean and dry 500 ml beakerand the viscosity of the test sample was determinedby using spindle no 6 at speeds of 20,50,100 and150 r.p.m. During the measurement, the spindle wasMandal et al., Pharmawave, 10:2017lowered up to a point where the spindle does not hitthe bottom of the beaker. The temperature was keptuniform for all the test samples.Centrifugation Stressed TestThe stability of the optimized formulated and thereference ointment formulation was evaluated usingthe forced centrifugation test. This test was carriedout as per the method described by Baie & Sheikh[14]. Both the formulations were subjected to speedranged from (2000 to 14000) rpm for 15 minutes ina centrifuge apparatus (Heraeus Megafuge 8Centrifuge, Fisher Thermo Scientific, Model No.FB15067) and phase separation of the formulation,if any, was evaluated by visual observation.Microbial Contamination TestDeveloped ointment formulation was tested for bothTAMC (total aerobic microbial count) and TYMC(total combined yeast/mould count). An amount of10 g of the sample was suspended in sodiumchloride peptone solution (PH 7). One (1) ml of theprepared previous solution was added to a sterilepetri dish. Casein Soya bean digestive agar mediumand sabouraud dextrose agar were prepared forcultivation of bacteria and fungi respectively, bypouring them into the prepared petri dishes. Thenthe petri dishes were incubated at 300-350 forbacteria and 200-250 for fungi for five days. One (1)ml of the buffered sodium chloride was used as anegative control.After the incubation period, colonies were countedusing the manual colony counter. Not more than 104for bacteria and not more than 102 for fungi per gm/ml was considered as the limit of the microbialcontamination test.Assay for the Developed Formulation for Mentholand Mamphor by Gas Chromatography Both thetest and reference formulations were tested for thecontent of menthol and camphor by gas6
PHARMAWAVE 10/17chromatography method. According to USPpharmacopeia, the formulation should contain notless than 90.0 percent and not more than 110.0percent of the labeled amounts.A GasChromatography(GC)system(AgilentTechnologies, 7890B GC System) The test wasperformed according to the following procedure:Preparation of Solutions:Diluent: Petroleum benzeneBlank: 1ml of the diluent was transferred into GCvial after filtration through 0.45 µm filter paper.Standard stock solution of camphor and menthol: 1g of camphor was weighted and transferred into a100ml volumetric flask, containing 60ml of diluent,and after sonication takes place, the volume toppedup with the diluent. The same method was adoptedfor menthol stock preparation using 1g of thementhol material.Working Standard solution: 1 ml of both camphorand menthol standard stock solution was transferredinto 10ml volumetric flask containing 5 ml diluent,mixed well and the volume was topped up, andfinally transferred into the GC vials after passingthem through a 0.45 µm membrane filter paperSample stock solution: 1 g of the ointment samplewas transferred into 100ml volumetric flaskcontaining 60ml diluent, then sonicated to ensuresample dissolution, and make up the volume withdiluent.Working sample solution: 1 ml of the sample stocksolution is transferred into 10ml volumetric flaskcontaining 5 ml diluent, mixed well and the volumewas topped up. Finally, they were transferred intoGC vial bypassing a µm 45.0 filter paper.Chromatographic parameters:GC detector: Flame ionization detector (FID)Gas carrier: HeliumColumn: VF-WAXmsMandal et al., Pharmawave, 10:2017Length: 30.0 mInternal diameter: 250.00 µmFilm thickness: 0.25 µmMode: Split (Ratio 20:1)Injection volume: 1µLRun time: 14 MinuteThe content of active ingredient was calculatedbased on the following equation: .equation 1Where, AT: Area of peak response due to analytecontent in sample solutionAS: Area of peak response due to analytecontent in standard solutionWT: Weight of sample takenP: % of purity of analyte standardStability StudyAn accelerated stability study was carried outaccording to the ICH guidelines at 400C/75% RH.Due to time constraint, only one sample of threemonths was analysed. The formulation was filled inthe aluminum collapsible tubes; physicalappearance, viscosity, and drug assay were analysedas evaluation parameters.RESULTS AND DISCUSSIONSeveral experimental trials were carried out toinvestigate the best ratio of olive oil and beeswaxfor the required viscosity of the formulation. Theresults are shown in Table 3 and Fig. 1 to Fig. 4based on the highest obtained torque values. It wasfound that the ratio of 17:73 w/w for beeswax andolive oil was just enough to provide the requiredviscosity like the reference product. Theformulation with this composition of beeswax andolive oil (17:73 w/w) was selected as the optimized7
PHARMAWAVE 10/17formulation. This selection was based on in itsconsistency and viscosity resemblance to thecommercial iKOOL formulation prepared by IKOPSdn. Bhd., IIUM, Malaysia. The higher theconcentration of beeswax, the higher was theviscosity. The optimized formulation arison with the commercial product. Table 4shows the results of centrifugation stressed testsubjected to the optimized test and referenceformulation. As such, both the formulations resultedsimilar type of physical stability profile; theywithstand physical separation by centrifugationforce exerted at 6000 RPM for 15 minutes, initiateseparation at 8000 RPM, but completely separate at10000 RPM. No bacteria or fungi growth wasrevealed after microbial contamination testperformed on the optimized formulation. 3 monthsof accelerated stability study at 400 C and 75% RHfor three months showed good stability profile forthe optimized formulation. The formulation wasphysical stable without any kind of phaseseparation. As evident from the GC chromatograms(Fig. 5 and Fig. 6), menthol and camphor were wellseparated without any kind of interference. Assayresults for both active pharmaceutical ingredientsafter 3 months of accelerated stability study isshown in Table 5. It indicates that both camphor(2.12 %) and menthol (1.96 %) were within theaccepted range ( 10% of the label claim) of theircontent percentage (2% w/w).Mandal et al., Pharmawave, 10:2017Table 3: Viscosity measurement results based onthe highest obtained torque value.Number 61.7/2003085.00r.p.m5iKOOL65.6/5013120.00r.p.mFig. 1: Viscosity of test formulation (beeswax andolive oil ratio-17:73) and reference formulation(iKOOL).89
PHARMAWAVE 10/17Fig. 2: Viscosity of test formulation (beeswax andolive oil ratio-15:75) and reference formulation(iKOOL).Fig. 3: Viscosity of test formulation (beeswax andolive oil ratio-20:70) and reference formulation(iKOOL).Mandal et al., Pharmawave, 10:2017Fig. 4: Viscosity of test ointment (beeswax andolive oil ratio-25:65) and reference product(Original iKOOL)Table 4: Results of centrifugation stressed testsubjected to optimized test and referenceformulationCentrifugation ation9
PHARMAWAVE 10/17Table 5: Assay results for menthol and camphoranalytical results in mix standard and optimized testSampleidentityMentholin mixedstandardMentholin testsampleCamphor inmixedstandardCamphor in ion after 3 months of accelerated stability study.subjected to 3 months accelerated stability study.Fig. 5: Representative GC chromatograms showingthe separation of menthol (2.758 minutes) andcamphor (3.213 minutes) in mix standard.Mandal et al., Pharmawave, 10:2017Fig. 6: Representative GC chromatograms showingthe separation of menthol (2.757 minutes) andcamphor (3.214 minutes) in test sampleCONCLUSIONAn optimized analgesic ointment formulation with acomposition of 2% menthol and 2% camphortogether with beeswax and olive oil mixture (17: 73w/w) as a base was developed for pediatric patients.The various evaluation parameters carried out toassess the quality of the developed pediatricointment formulation and they showed satisfactoryresults when compared with the reference iKOOLointment prepared by IKOP Sdn. Bhd, IIUM,Malaysia. The viscosity of the optimizedformulation was also satisfactory when comparedwith the reference product. Moreover, the assayresults for both the active ingredients after a periodof 3 months accelerated stability study was withinthe accepted range for the active pharmaceuticalingredients. The developed analgesic pediatricointment containing menthol and camphor may befurther investigated to commercialize in future as a10
replacement of the conventionalointments available in the market.analgesicREFERENCES[1]H.K. Archer, M.S. Pettit, Analgesic andantiphlogistic compositions and therapeutic wrap fortopical delivery, U.S. Patent No. 5, 976, 547. Washington,DC: U.S. Patent and Trademark Office, 1999.[2]P. Zuccarini, Camphor: risks and benefits of awidely used natural product, J. Appl. Sci. Environ. Manag. 3(2009) 69-74.[3]C. Gaudioso, J. Hao, M.F. Martin-Eauclaire, M.Gabriac, P. Delmas, Menthol pain relief through cumulativeinactivation of voltage-gated sodium channels, Pain 153(2012) 473-484.[4]P. Johar, V. Grover, R. Topp, D.G. Behm, Acomparison of topical menthol to ice on pain evokedtetanic and voluntary force during delayed onset musclesoreness, Int. J. Sports Phys Ther. 7 (2012) 314-322.[5]T. Patel, Y. Ishiuji, G. Yosipovitch, Menthol: arefreshing look at this ancient compound, J. Am. Acad.Dermatol. 57 (2007) 873-878.[6]H. Xu, N.T. Blair, D.E. Clapham, Camphor activatesand strongly desensitizes the transient receptor potentialvanilloid subtype 1 channel in a vanilloid-independentmechanism, J. Neurosci. 25 (2005) 8924-8937.[7]A. Moqrich, S.W. Hwang, T.J. Earley, M.J. Petrus,A.N. Murray, K.S. Spencer, M. Andahazy, G.M. Story, A.Patapoutian, Impaired thermosensation in mice lackingTRPV3, a heat and camphor sensor in the skin, Science 307(2005) 1468-1472.[8]T.M. Wang, L.Q. Ding, H.J. Jin, R. Shi, J.S. Wu, L. Zhu,Y.Q. Jia, Y.M. Ma, Simultaneous quantification of multiplevolatile active components in rat plasma using aheadspace-solid phase dynamic extraction scopy: application in a pharmacokinetic study ofLonghu Rendan pills, RSC Adv. 5 (2015) 29631-29638.[9]A. Aguirre, J.M. Oleaga, R. Zabala, R. Izu, J.L.Díaz‐Pérez, Allergic contact dermatitis from Reflex spray, Contact Derm. 30 (1994) 52-53.[10]S.M. Wilkinson, M.H. Beck, Allergic contactdermatitis from menthol in peppermint, Contact Derm. 30(1994) 42-43.[11]E.M.McGowan, Menthol urticarial,Arch. Dermatol. 94 (1966) 62–63.Mandal et al., Pharmawave, 10:2017PHARMAWAVE 10/17[12]N. Chatterjie, G.J. Alexander, Anticonvulsantproperties of spirohydantoins derived from opticalisomers of camphor, Neurochem. Res. 11 (1986)16691676.[13]D.W. Metry, A.A. Hebert, Topical therapies andmedications in the pediatric patient, Pediatr. Clin. NorthAm. 47 (2000) 867-876.[14]S. H. Baie, K.A. Sheikh, The wound healingproperties of Channa striatus-cetrimide cream-woundcontraction and glycosaminoglycan measurement, J.Ethnopharmacol. 73 (2000) 15-30.11
a centrifuge apparatus (Heraeus Megafuge 8 Centrifuge, Fisher Thermo Scientific, Model No. FB15067) and phase separation of the formulation, if any, was evaluated by visual observation. Microbial Contamination Test Developed ointment formulation was test
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uate the quality of grain damaged by rodents with respect to nutritional value, infection by moulds and aflatoxin contamination. 2 Materials and methods 2.1 Study area The study was conducted in Mwarakaya ward (03 49.17́'S; 039 41.498′E) located in Kilifi-south sub-county, in the low landtropical(LLT)zoneofKenya.Thisstudy site wasselect-
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uniformity of films, surface pH, disintegration time and in-vitro dissolution studies. The formulation F5 has disintegration time of 56 seconds and is more promising and showed drug release of 99.89%; hence formulation F5 was selected as best formulation. Keywords: Oral Films, Metoprolol Succinate, Solvent Casting Method. INTRODUCTION
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Ravindran et al. / Formulation and Evaluation IJPCR, Volume 8, Issue 9: September 2016 Page 1306 Tween 80 Figure 1: Fruits of Dimocarpus longan. Formulation 1 Formulation 2 Figure 2: Formulated antioxidant creams The added to a free radical Chemicals 2,2 -Diphenyl-1-picryl hydrazyl (DPPH) was obtained from Sigma Aldrich Co, St Louis, USA .
mixed. Since formulation containing antimicrobial agents as active moiety, it is likely to protect from microbial growth 8, 9. To determine the activity of formulation is subject to study the prepared formulation with standard method called standard cup plate method and the inhibition zone diameters were measured with the help of zone reader.
Thus it might seem that Scrum, the Agile process often used for software development, would not be appropriate for hardware development. However, most of the obvious differences between hardware and software development have to do with the nature and sequencing of deliverables, rather than unique attributes of the work that constrain the process. The research conducted for this paper indicates .
