www.iajpr.com Page483 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876 FORMULATION AND EVALUATION OF NOVEL HERBAL HAND SANITIZER Mithun A. Thombare*, Babaso V. Udugade, Tushar P. Hol, Manoj B. Mulik, Dnyaneshwar A. PawadeSatara College of Pharmacy, Satara - 415004. (M. S.) India. Corresponding authorMithun A. Thombare Satara College of Pharmacy, New Additional MIDC, A/P- Degaon, Satara- 415004, (M. S.) India. mithunthombare@gmail.com +91-9423616476. Copyright2015ThisisanOpenAccessarticledistributedunderthetermsoftheIndoAmericanjournalofPharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE INFOABSTRACT Article history Received 20/01/2015 Available online 30/01/2015 Keywords Hand Hygiene, Extraction,Antimicrobial Activity,Herbal Sanitizer. HerbalhandsanitizerwaspreparedusingleavesextractsofOcimumsanctum,Eugenia caryophyllus and Cymbopogon flexuous. The antibiotic sensitivity test of the prepared herbal handsanitizeragainstskinpathogenswascheckedusingdiscdiffusionmethodandresults werecomparedwithcommerciallyavailablesynthetichandsanitizer.Resultsdepictedthat theherbalhandsanitizergiveslargerinhibitionzonethanthecommerciallyavailable synthetichandsanitizeragainstStaphylococcusaureusandPseudomonasaeruginosa.The efficacyofherbalhandsanitizerwasevaluatedusingmicroorganismsuspensions;which revealedthattheherbalhandsanitizerisefficientinreducinghighernumberof microorganismfromthehandsascomparedtocommercialsynthetichandsanitizer.Thus, owingtohigherantimicrobialactivityandefficacytheseherbalextractscanbeusedinthe preparation of herbal hand sanitizers on commercial scale. Please cite this article in press as Mithun A. Thombareet al. Formulation and Evaluation of Novel Herbal Hand Sanitizer. Indo American Journal of Pharm Research.2015:5(01). www.iajpr.com Page484 Vol 5, Issue 01, 2015.Mithun A. Thombare etal. ISSN NO: 2231-6876 INTRODUCTION Hygieneisdefinedasmaintenanceofcleanlinesspracticewhichcarriesutmostimportanceinmaintenanceofwell-being. Keepingbodilyhygieneandusageofcleanserarerequisiteofhealthyliving.Theseconceptshighlighttheneedofmaintaining hygiene in prevention of diseases.[1] Although good and simple hygiene technique is single most important, easy and less expensive meansofpreventinghealthcareassociatedinfectionsandthespreadofantimicrobialmultidrugresistance;but,unfortunatelypoor handhygienepracticearestillobservedduetolackofscientificawareness,lackofknowledgeofriskandunavailabilityofhand hygiene facilities. Nosocomial infections are those which acquired or originated in a hospital or health care setting and are prevalence ofcompromisedhosts,efficientmechanismsoftransmissionfrompatienttopatient.Thus,occurrenceofnosocomialinfectionsis alarminglyincreasingand hasemergedasaseriousconcernin hospitalcareoutcome;resulting inprolongedhospitalization,ample diseaseandmortality,andexcessivecosts.Escherichiacoli,manyPseudomonasspp.andStaphylococcusaureusarecommonly involved opportunistic microorganisms that primarily cause nosocomial infections. Generally infectious sites are urinary tract, surgical wounds,respiratorytract,skin,blood,gastrointestinaltractandcentralnervoussystem.Thesepathogensalsotendtobecome incorporatedintonormalfloraofhealthcareworkers.Pseudomonasaeruginosaisthemostcommonlydetectedmicroorganismin hospitalized patients and immunosuppressed people. Opportunistic fungal infections havebecomevery important, especially in HIV patients.Thehighestfrequenciesofopportunisticfungalinfectionsdocumentedarecandidacies,aspergillosisandcryptococcosis. Usually,microbesresidingon hands aredividedinto residentand transientflora.Residentflora(e.g.Corynebactariumdiphtheriae, Staphylococcusepidermidis,Streptococcusviridians)colonizingdeeperskinlayersaremoreresistanttomechanicalremoval,has lowerpathogenicpotential.Whiletransientflora(e.g.Staphylococcusaureus,Gram-negativebacilli,Candidaspp.)colonizesthe superficialskinlayerforshortertimeperiods,isusuallyacquiredbycontactwithapatientorcontaminatedenvironmentandare responsible for most health care associated infection and the spread of antimicrobial resistant, but these microorganisms can be easily removed by mechanical means such as hand washing. In the scenario mechanized life style; a consumer will always prefer readymade formulation of alcohol hand rub rather than hand washing (application of a non-antimicrobial or antimicrobial soap; and mechanical friction isgeneratedbyrubbingthe handstogetherfor1minute,followedbyrinsingwithwaterandthendryingthoroughlywitha disposabletowel).Handsanitizerorhandantisepticisasupplementoralternativetohandwashingwithsoapandwater.Vari ous preparations are available including gel, foam and liquid solution. The active ingredient in hand sanitizer is mostly alcohol, inactive ingredientsincludesathickeningagentsuchapolyacrylicacidforalcoholgel,humectantssuchasglycerinforliquidrubs, polypropyleneglycolandessentialoilsofplants.Alcoholbasedhandsanitizeraremoreeffectivethatkillsmostmicroorganism compared to soap and do not dry out hands as much. The level of alcohol varies between 60-85% (most commonly used level is 62%). Alcoholrubsanitizerskillmostbacteria,fungiandstopsomevirusestoo.Alcoholrubsanitizerscontainingatleast70%alcohol (mainly ethyl alcohol) kill 99.9% of the bacteria on hand 30 seconds after application and 99.999% in 1 minute. [2] Traditional healers have long used plant to prevent or cure infectious conditions. Plants are rich in wide variety of secondary metabolitessuchastannins,terpenoids,alkaloids,andflavonoidsetc.whichhavebeenfoundtopossessin-vitroantimicrobial properties.Consideringthisultimatum;anattempthasbeenmadetoscreenclassicalliteraturefortheherbswithantimicrobial propertiesandithasbeenfoundthatOcimumsanctum(tulsileaves),Eugeniacaryophyllus(clove),Cymbopogonflexuous(lemon grass), and Aloe barbadensis (aloe) holds that antimicrobial potency.[3] Thus, we aimed to formulate and evaluate herbal hand sanitizer comprising of alcoholic extracts combination of these astonishing herbs using other suitable excipients; which can be used as ready to use herbal hand sanitizer. MATERIALS Leavesoftulsi(Ocimumsanctum),lemongrass(Cymbopogonflexuous),aloe(Aloebarbadensis)andfloweringbudsof clove(Eugeniacaryophyllus)werecollectedfromthelocalareaofSatararegion,Maharashtra,Indiaandauthenticatedbyexpert botanistfromDepartmentofBotanyofYashvantraoChavanInstituteofScience,Satara.Nutrientagar(forbacterialcultivation), marketed preparation (Dettol Hand Sanitizer) and other excipients were purchased from market. Escherichia coli (Gramve), Pseudomonas aeruginosa (Gram ve), Staphylococcus aureus (Gram +ve) and Basilus subtilis (Gram +ve) pathogens were selected for evaluation of antimicrobial activity of the herbal hand sanitizer. METHODS Crude Drug Extraction [4] 10 gm of well dried crude drug (tulsi, clove and lemon grass) was taken. Grinded into fine powder. The powder of each plant material was added separately to 100 ml of methanol. www.iajpr.com Page485 Vol 5, Issue 01, 2015.Mithun A. Thombare etal. ISSN NO: 2231-6876

The mixture was heated on water bath at 60C for 1 h, then filtered and plant extract filtrate was collected. Formulation of Herbal Hand Sanitizer Thecompositionsofalcoholbasedandnon-alcoholherbalhandsanitizerspreparedwereasgiveninTable1and2 respectively.Carbopol940wasaddedtodeionizedwaterwithconstantstirring.Afteruniformmixing,triethanolamine(TEA)was addedwithslowstirringtoavoidformationofpossibleairbubblesintheproductandtheblendwaskeptasidefor24h.Allthe extracts were added to denatured alcohol along with glycerin, polysorbate 20 and were then mixed with aqueous phase. Finally 0.25% eachofmethylandpropylparabenwasadded aspreservativeand0.5%ofperfumewasmixedwithslowstirringtoobtainunifor m product. Prepared product was stored in air tight containers. [5] Table 1: Composition of alcohol based herbal hand sanitizer formulation. Sr. No.Ingredients andExcipients Quantity Given (%) Quantity Taken (gm/ml) Uses 1Deionized water30.009.0Vehicle 2Alcohol (denatured)62.0018.6Antibacterial 3Tulsi leaves extract1.000.3Antibacterial 4Lemon grass oil1.000.3Antibacterial 5Clove extract1.000.3Antibacterial 6Carbopol 9400.500.15Thickening agent 7Triethanol amine0.700.21Solubilizing agent 8Glycerin2.300.69Emollient 9Polysorbate 200.500.15Emulsifier 10Preservative0.500.15Preservative 11Perfume0.500.15Fragrance Table 2: Composition of non-alcohol herbal hand sanitizer formulation. Sr. No.Ingredients and ExcipientsQuantity (ml) 1Aloe Vera gelq. s. 2Tulsi leaves extract1 3Lemon grass oil1 4Clove extract1 Antibacterial Activity The screening of antibacterial activity of the extracts against pathogens was performed using disc diffusion method. Nutrient agar mediawasprepared(Table3),sterilizedandasepticallyspreadedonfoursetsofPetriplates(eachsetcontaining threeplates) whichwerepreviouslymarkedasAHS,ANHSandCHS(formulationcoding).MicroorganismsusedwereStaphylococcusaureus, Bacillussubtilis,EscherichiacoliandPseudomonasaeruginosa.Theplateswereinoculatedwithmicroorganismsuspensionand incubated at 37C for 24 h. Next day filter paper discs loaded with alcohol based herbal hand sanitizer, non-alcohol hand sanitizer and synthetic hand sanitizer were placed in the respectively marked plates. It was taken care that the sterile discs completely absorbs the formulation.After24htestresultswereobservedtodeterminetheefficacyofformulationsintermsofzoneofinhibitionof microorganism. Higher the zone of inhibition, the more effective is the test formulation. [6] Table 3: Composition of nutrient agar culture media. IngredientsQuantity (gm/lit) Peptic digest of animal tissue5.0 Sodium chloride5.0 Beef extract1.5 Yeast extract1.5 Agar15.0 Final pH at 250C7.40.2 www.iajpr.com Page486 Vol 5, Issue 01, 2015.Mithun A. Thombare etal. ISSN NO: 2231-6876 RESULTS AND DISCUSSION Totalfourbacterialspecieswereselectedinthepresentstudytoevaluatetheantimicrobialactivityoftheprepared formulations. The antimicrobial potencyof prepared formulations wasevaluated using disc diffusion method (as mentioned before). AfterincubationtheresultsobservedintermsofzoneofinhibitionagainstmicroorganismsarequotedinTable4.Alcoholbased herbalhandsanitizer(atconcentration400g/ml)showedgreaterinhibitionzonesagainstbacterialspeciesEscherichiacoli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus in comparison to commercially available hand sanitizer. Table 4: Antimicrobial study results. Name of the Microorganism Zone of Inhibition (diameter in mm) AHSANHSCHSStd.(SLS) Escherichia coli36273232 Pseudomonas aeruginosa30192537 Bacillus subtilis32253031 Staphylococcus aureus32252930 Fig No. 1: Zone of inhibition in diameter on P. aeruginosa. Fig. No. 2: Zone of inhibition in diameter on E. coli. www.iajpr.com Page487 Vol 5, Issue 01, 2015.Mithun A. Thombare etal. ISSN NO: 2231-6876 Fig. No. 3: Zone of inhibition in diameter on B. subtilis. Fig. No. 4: Zone of inhibition in diameter on S. aureus. Fig. No. 5: Result of antimicrobial study of Hand-sanitizer against various pathogens. DISCUSSION The prepared formulation of herbal hand sanitizer showed significant results against three bacterial species. The significance wasfoundtobemoreincomparisontothecommerciallyavailablehandsanitizerorhanddisinfectant.Thecomposition(Ocimum sanctumEugenia caryophyllus and cymbopogonflexuous) hasbeenattributingwithpropertieslikeanthelmintic, antimicrobial, anti-inflammatoryandanalgesicetc.Thealcoholshaveexcellent,rapid(withinfewseconds)germicidalactivityagainstvegetative bacteria, fungi, and many viruses and antimicrobial activity is based on protein denaturation of microorganisms. Alcohol based hand. sanitizersarehighlyeffectiveagainstmycobacteria(thebacteriamostresistanttothedisinfectionprocess)andmultidrug-resistant pathogens. Alcohol based herbal hand sanitizer are approximately 100 times more effective against microorganism than commercially available hand sanitizer. Herbal hand sanitizer offer numerous advantages over commercially available hand sanitizer onto both hands, www.iajpr.com Page488 Vol 5, Issue 01, 2015.Mithun A. Thombare etal. ISSN NO: 2231-6876 usuallyrequiresonly15to30seconds.Whereasvigorousfriction,rinsingwithwater,anddryingwithatowelarenotneededlike hand disinfectants or soaps. CONCLUSION Hands are the most common mode of transmission of pathogens to patients and proper hand hygiene can prevent health care-associatedinfectionsandthespreadofantimicrobialresistance.Scientificevidenceandeaseofusesupportofalcohol-basedhand sanitizers during patient care. It maybe concluded that Herbal Hand Sanitizer has a significant anti-microbial effect on the specified microorganismsexceptP.aeruginosa. Thus,thereisimmensepotentialinestablishing theuseofantimicrobial herbal productsasa measuretocontrolthemultidrug resistant microbesaswellastochecktheirspreadthrough handsfromonegeographical region to another. REFERENCES 1.Vyaset P., Antimicrobial Activity of Ayurvedic Hand Sanitizer, International Journal of Pharmaceutical and Biological Archives 2011; 2:2: 766. 2.http://en.wikipedia.org/wiki/Hand_sanitizer. 3.KokateCK,PurohitAP,GokhaleSB.TextbookofPharmacognosy.50thed.Pune:NiraliPrakashan;2014.p.14.67-14.68,14.85,14.52. 4.JoshiMG.,KamatDV.,KamatSD.,EvaluationofHerbalHandWashFormulation,NaturalProductRadiance2008;7:5:413-415. 5.WaniNS.,BhaleroaAK.,RanawareVP.,ZanjeR.,FormulationandEvaluationofHandSanitizer,InternationalJournalof PharmTech Research 2013; 5:1: 40-43. 6.Kokare CR. Pharmaceutical microbiology experiments and techniques. 2nd ed. Nashik: Career Publication; 2008.p. 201-202.

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