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ISSN : 0974-8520

AYUAn International Quarterly Journal of Research in Ayurveda

Website : www.ayujournal.org

Volume 38 / Issue 1-2 / January-June 2017

Spine 5.5 mm

© 2018 AYU (An International Quarterly Journal of Research in Ayurveda) Official publication of Institute for Post Graduate Teaching & Research in Ayurveda, Jamnagar | Published by Wolters Kluwer - Medknow

82

Pharmacological Study

IntroductionAcne vulgaris is one of the commonly encountered skindisorders. It is considered as an adolescent disorderwhichis related to the pilosebaceous follicle of the skin andcharacterizedby formationofopenandclosedcomedones,papules,pustules,nodulesandcysts.Acneaffectsbothmalesandfemalesalthoughmalestendtohavemorewiththeonsetofpuberty.Itaffectsaround9.4%ofthetotalglobalpopulationandistheeighthmostprevalentdiseaseworldwide.[1]Severalfactorssuchasdisturbedhormonal(androgen)balance,excesssebumproductionandhyperkeratinizationareinvolvedinthepathophysiologyofacne.Varietiesofinflammatorymediatorsarealsoinvolvedinthepathogenesisofacnewhichisproducedasaresultofimmunostimulationcausedbycolonizationof

Propionibacteriumacnesintheductofthesebaceousfollicle.Various noninflammatory lesions such as comedones andinflammatorylesionssuchaspapulesandcystsareproducedasaresultofthisprocessleadingtothedevelopmentofacnevulgaris.[2]

Inmodernmedicine,severaltreatmentsareavailableforacnevulgaris,but treatmentmustcomplywithtypeandseverityof the lesions.Treatmentmainly includesprolongeduseof

Evaluation of anti‑inflammatory and antimicrobial activity of AHPL/AYTOP/0213 cream

Sanjay U. Nipanikar1, Dheeraj Nagore1, Soham S. Chitlange2, Devashree Buzruk1

1R and D Center, Ari Healthcare Private Limited, Unit No. 401, International Biotech Park, BTS 2 Building, Chrysalis Enclave, 4th Floor, Plot No – 2A, MIDC Phase II, Hinjewadi, 2Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune, Maharashtra, India

Background:Acnevulgarisisalmostawidespreaddiseaseoccurringinallraces.Propionibacteriumacnes initiateacneandinflammatorymediators aggravate it.Conventional therapies for acne include comedolytic, anti‑inflammatory, and anti‑biotic agents.Due to adverseeffectsofthesetherapies,peoplearesearchingforalternativeoptions.Inthiscontext,apolyherbalformulationAHPL/AYTOP/0213creamwasdevelopedforthetreatmentofAcne.Objective:Theobjectiveofthisstudyistostudyanti‑inflammatoryandantimicrobialactivitiesofAHPL/AYTOP/0213cream.MaterialsandMethods:SkinirritationstudywasconductedonAHPL/AYTOP/0213creamasperOECDguidelines.(1)Anti‑inflammatoryactivity:Anti‑inflammatoryactivityofAHPL/AYTOP/0213creamincomparisonwithdiclofenacsodiumcreamwasassessedincarrageenan‑inducedratpawedemamodel.(2)AntimicrobialactivityforP.acnes:P.acneswereincubatedunderanaerobicconditions.Aliquotsofmoltenbrain–heartinfusionwithglucoseagarwereusedastheagarbase.Formulationandclindamycin(10mg/ml)wereintroducedintotheAgarwellsrandomly.(3)AntimicrobialactivityforStaphylococcusepidermidisandStaphylococcusaureus:bacteriawereincubatedunderaerobicconditionsat37°C.Trypticsoybrothwithglucoseagarwasusedastheagarbase.Avolumeof0.5mlofformulationandclindamycin(10mg/ml)wereintroducedintothewellsrandomly.Theantibacterialactivitywasevaluatedbymeasuringzonesofinhibition(inmm).Results:AHPL/AYTOP/0213creamisnonirritant.Significantreductioninratpawedema(43%)wasobservedwithAHPL/AYTOP/0213whichwas also comparable to diclofenac sodium cream (56.09%).Zone of inhibition for formulationwas20.68mm,28.20mm,and21.40mmforP.acnes,S.epidermidisandS.aureus,respectively,whichwascomparabletoclindamycin.TheminimuminhibitoryconcentrationofformulationAHPL/AYTOP/0213obtainedinanti‑microbialstudywas2.5mg/mL.Conclusion:AHPL/AYTOP/0213creamisnonirritantandpossessessignificantanti‑inflammatoryandantimicrobialactivities,whichfurtherjustifiesitsroleinthemanagementofacnevulgaris.

Keywords:Acnevulgaris,AHPL/AYTOP/0213,antiandrogenic,antibacterial,herbal

Access this article online

Quick Response Code:Website:www.ayujournal.org

DOI:10.4103/ayu.AYU_150_17

Abstract

Address for correspondence: Dr. Sanjay U. Nipanikar, Ari Healthcare Private Limited, R and D Center, Unit No. 401, International

Biotech Park, BTS 2 Building, Chrysalis Enclave, 4th Floor, Plot No – 2A, MIDC Phase II, Hinjewadi, Pune - 411 057, Maharashtra, India.

E-mail: [email protected]

How to cite this article: Nipanikar SU,Chitlange SS,NagoreDH,BuzrukD.Evaluationofanti‑inflammatoryandantimicrobialactivityofAHPL/AYTOP/0213cream.Ayu2017;38:82‑7.

This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms.

For reprints contact: [email protected]

Nipanikar, et al.: Anti-inflammatory and antimicrobial activity of AHPL/AYTOP/0213

83AYU|Volume38|Issue1‑2|January‑June2017

oralandortopicalantibiotics(doxycycline,clindamycinanderythromycin),comedolytic(retinoid)andantiinflammatoryagents.Althoughthesemedicinesarebettertreatmentoptionsforacnemanagement, thesideeffectsof thesemedicationssuchas increasedskindryness, scaling,erythema,burning,stinging,itchingandbacterialresistancearenoticeable.Hence,peoplearelookingforalternativetreatmentoptionsforacnevulgaris.[3,4]

AHPL/AYTOP/0213 cream is a polyherbal formulationdeveloped byAriHealthcare PrivateLimited, in the formof cream for effective management of acne vulgaris,hyperpigmentationandvariousskindisorders.Daruharidraextract(Berberis aristataDC.,Family‑Berberidaceae),[5]Lodhraextract(Symplocos racemosa Roxb.,Family‑Symplocaceae),[6]Yashtimadhuka extract (Glycyrrhiza glabra Linn.Family‑ Leguminosae),[7,8] Jatiphala extract (Myristica fragrans Houtt, Family‑Myristicaceae),[9]Manjishthaextract (Rubia cordifolia Linn., Family‑ Rubiaceae),[10]Vacha extract (Acarus calamusLinn., Family‑Araceae),[11] Dhanyaka ex t rac t (Coriandrum sa t ivum L inn . ,Family‑Umbelliferae)[12] andShalmali extract (Salmalia malabaricaSchott.andEndl.Family‑Bombacaceae)[13]havebeenusedinAHPL/AYTOP/0213cream.

Thus,thepresentstudywasplannedtoevaluateantiinflammatory (in vivo) potentialofAHPL/AYTOP/0213creamincomparisonwithstandarddiclofenacsodiumcreamincarrageenan‑inducedrat paw edema and antimicrobial activity in comparisonwithstandardclindamycinagainstP.acnes,Staphylococcusepidermidis and nonitalic Staphylococcus aureus. SkinirritationstudywasalsoconductedonAHPL/AYTOP/0213creamasperOECDguidelines.

Materials and MethodsAcute dermal toxicity studyThisstudywasconductedtoassesstheprimaryskinirritanteffectofAHPL/AYTOP/0213creaminalbinorats.Thedetailsareasfollows:

Wistaralbinoratspeciesofage/weight180‑250gofeithersex,preferablyfemales,wereusedinthisstudy.

MethodologyEthics committee approvalAtotalofsixanimalswereapprovedbytheInstitutionalAnimalEthicsCommittee(IAEC)inthemeetingheldonDecember31,2013.Theprotocolno.approvedwasDYPIPSR/IAEC/13–14/P‑01.SkinirritationstudyofAHPL/AYTOP/0213creamwasperformedasperOECDguidelineno.402.

ProcedureThebackofthealbinoratswasshavedtoremovethefurcarefully24hbefore applicationof the sample.AHPL/AYTOP/0213creamwasappliedontheskinpatchesofalbinoratsandthesiteoftheapplicationwasexaminedat24,48and72hforchangesinanydermalreactions.Theirritationindexwascalculatedto

assess the irritationpotentialofAHPL/AYTOP/0213Creamthroughdraizescoringcriteriaasfollows:

Erythema and escher formationNoerythema 0Veryslighterythema(barelyperceptible) 1Well‑definederythema 2Moderate‑to‑severeerythema 3Severeerythema(beefredness)toEscherformationPreventinggradingoferythema 4

Edema formationNoedema 0Veryslightedema(barelyperceptible) 1Slightedema(edgesofareawelldefinedbydefiniteraising) 2Moderateedema(raisedapproximately1mm) 3Severeedema(raisedmorethan1mmandextendingbeyondAreaofexposure 4

Anti‑inflammatory activity of AHPL/AYTOP/0213 creamThis studywas conducted to evaluate anti‑inflammatoryactivityofAHPL/AYTOP/0213creaminWistaralbinoratsandthedetailsofthestudyaregivenbelow:

Ethics committee approvalIAEChasapprovedthestudy.Theprotocolno.wasDYPIPSR/IAEC/13–14/P‑01.

Anti-inflammatory activity of AHPL/AYTOP/0213 cream against carrageenan induced paw edema in ratsEighteenWistarratsofeithersex6–8weeksageandweighing150–250 gwere taken and divided into three groupswithsix animals in each group.Animals fromGroup 1wereappliedtopicallywithspecifiedquantityof1gofdiclofenacsodium cream as standard drug.Animals fromGroup 2were applied topicallywith specified quantity of 1 g ofAHPL/AYTOP/0213creamasatestdrug.GroupIIIwastakenasacontrol.Thetestandstandarddrugswereappliedtotheplantarsurfaceofthelefthindpawbygentlyrubbing5timeswith the indexfinger 30min before carrageenan injection(Irishmoss).After30min,1%w/vof0.05mlcarrageenanwasinjectedsubcutaneously.Thepawwasmarkedwithinkattheleveloflateralmalleolusandimmersedinmercuryupto lateralmalleolusmark.The pawvolumewasmeasuredplethysmographicallyimmediatelyafterinjectionat1,2,3,4,and5heventually24hafterdrugapplication.[14,15]

Antimicrobial activity of AHPL/AYTOP/0213 creamAntimicrobial activity of AHPL/AYTOP/0213 cream in comparison with standard clindamycin against Propionibacterium acnesThe antibacterial activity ofAHPL/AYTOP/0213 cream incomparisonwith standard clindamycinwas determined bymodifiedagarwelldiffusionmethod.P.acnes wereincubatedinbrain–heartinfusion(BHI)mediumwith1%glucosefor48hunderanaerobicconditionsandadjustedtoyieldapproximately


84 AYU|Volume38|Issue1‑2|January‑June2017

1.0×108CFU/ml.AliquotofmoltenBHIwithglucoseagarwasusedastheagarbase.Preparedinoculumwasaddedtothemoltenagar,mixedandpouredoverthesurfaceoftheagarbaseandlefttosolidity.Asterile8mmborerwasusedtocutwellsofequidistanceineachofplates;0.5mlofsolutionsofAHPL/AYTOP/0213andstandardclindamycin(10mg/ml)wereintroducedintothewellsrandomly.Theplateswerethenincubatedat37°Cfor48hunderanaerobicconditionsinananaerobicjar(Hi‑media)withgaspackandindicatorstripandthejarwaskeptinanincubatorfor48hat37°C±1°C.Gaspackscontainingcitricacid,sodiumcarbonateandsodiumborohydridewereusedtomaintainandchecktheanaerobiosis.Citricacid releasescarbondioxideandsodiumborohydridereleaseshydrogenwhentheycomeincontactwithoxygen.Anindicatorstripofmethyleneblue,whenintroducedintothejar,changesincolorfromwhitetoblueintheabsenceofanaerobiosis.Thezonesofinhibitionofformulationandstandardwerecalculatedbyformula1.[16,17]

Formula1

Zone of inhibition of formulationAIZone of inhibition obtained for standard

=

P.I.=Activityindex×100s

where,

A.I.‑Activityindex;P.I.‑Percentinhibition

Antimicrobial activity of AHPL/AYTOP/0213 cream in comparison with standard clindamycin against Staphylococcus epidermidis and Staphylococcus aureusThe antimicrobial activity ofAHPL/AYTOP/0213 incomparisonwith standard clindamycinwas determined bymodified agarwell‑diffusionmethod.S. epidermidis andS.aureuswereincubatedseparatelyintrypticsoybroth(TSB)with1%glucosefor24hunderaerobicconditionsat37°Candadjustedtoyieldapproximately1.0×108CFU/ml.TSBwithglucoseagarwasusedastheagarbaseinboththecases.Preparedinoculumswereaddedtothemoltenagar,mixed,andpouredoverthesurfaceoftheagarbaseandlefttosolidity.Asterile8mmborerwasusedtocutwellsofequidistancein each of plates; 0.5ml of solutions of formulation andstandard clindamycin (10mg/ml)were introduced into thewellsrandomly.Theantibacterialactivitywasevaluatedbymeasuringthediameterofzonesofinhibition(inmm).Three

experimentswereperformedseparatelyinboththecases.ThezoneofinhibitionforAHPL/AYTOP/0213andstandardwerecalculatedbyformula1.[16,17]

ResultsAcute dermal toxicity studyRats skinwhereAHPL/AYTOP/0213 creamwas appliedshowedno erythemaor edema.Theprimary skin irritationindexofthecreamwascalculatedas0.00.

Anti‑inflammatory activity of AHPL/AYTOP/0213 creamSignificant reduction in rat paw edemawas observed inAHPL/AYTOP/0213 and diclofenac sodium creamgroupsas compared to control group.The percentage inhibitionof rat paw edema forAHPL/AYTOP/0213was found tobe 43%,while percentage inhibition of rat pawedema forstandarddiclofenacsodiumcreamwas56.09%.ThedetailsarepresentedinTable1.

Antimicrobial activity of AHPL/AYTOP/0213 creamAntimicrobial activity against Propionibacterium acnes (in vitro)The zones of inhibition forAHPL/AYTOP/0213 creamand standard clindamycinwere 20.68mmand26.80mm,respectively.TheA.I.forAHPL/AYTOP/0213creamwas0.77andpercentageinhibitionwasfoundtobe77.19[Figure1].

Table 1: Anti‑inflammatory activity of AHPL/AYTOP/0213 cream

Groups Mean difference in paw volume (ml) (percentage inhibition)

Time (h)

Initial 1st h 2nd h 3rd h 4th h 5th h 24th hControl 2.63±0.55 2.81±0.83 2.91±0.59 3.15±0.92 3.38±0.68 3.55±1.05 2.05±1.00Standard 2.32±0.79 2.19±0.93 2.14±0.75 2.06±1.02 1.94±0.65 1.81±0.82 0.90±0.88Percentageinhibition ‑ 22.06 26.46 34.60 42.60 49.01 56.09TestAHPL/AYTOP/0213 2.36±0.78 2.33±0.70 2.29±1.09 2.22±0.68 2.15±0.89 2.06±0.97 1.15±1.32Percentageinhibition ‑ 17.08 21.30 29.52 39.04 41.97 43.90

Figure 1: Zone of inhibition against Propionibacterium acne (at column width)



Theminimum inhibitory concentration of formulationAHPL/AYTOP/0213 obtained in antimicrobial studywas2.5mg/mL.ThedetailsarepresentedinTable2.

Antimicrobial activity against Staphylococcus aureusThezonesofinhibitionforAHPL/AYTOP/0213andstandardclindamycinwere21.40mm,22.40mmrespectively.TheA.I.forAHPL/AYTOP/0213was0.95andpercentageinhibitionwasfoundtobe95.33[Figure2].ThedetailsarepresentedinTable3.

Antimicrobial activity against Staphylococcus epidermidisThezonesofinhibitionforAHPL/AYTOP/0213andstandardclindamycinwere 28.20mmand 32.40mm, respectively.TheA.I. forAHPL/AYTOP/0213was0.87 andpercentageinhibitionwasfoundtobe87.03[Figure3].ThedetailsarepresentedinTable4.

DiscussionPathogenesis of acne involves increased production ofandrogenhormonesthatstimulateexcessivesebumsecretiononface,neckandbackofthechest.Accumulationofsebum,epithelialcellsandkeratininthesebaceousfollicleleadstoformationof non‑inflammatorymicroscopic lesions.WhenP.acnes grow in these follicles, cytokines are released inresponse to immunostimulant reaction.This leads to thedevelopmentofinflammatoryacnelesionssuchaspapules,pustules,nodulesandcysts.[2]

Currently,variousoralandtopicalantibioticagentssuchasdoxycycline,clindamycinandtopicalcomedolyticagentssuchasretinoidsandevenoralcontraceptivepillsareeffectivelyutilized in the treatment of acne. However, associatedadverse events such as increased skin dryness, scaling andphotosensitivitylimittheirwidespreadandlong‑termuse.[5]Themajorproblemaffectingantibiotic therapyofacnehasalso been the increasing bacterial resistance to standarddrugs.Moreover, tetracycline, doxycycline,may lead to

adverseeffectssuchasgastricdisturbances,tinnitus,vertigoand discoloration of the teeth.While the use of retinoidshastobedonewithcaution,theyaresaidtobeteratogens.Cheilitis, dry skin andmucousmembranes, elevated livertransaminaselevels,hypertriglyceridemiaanddecreasednightvisionarecommonadverseeffectsassociatedwithretinoids.Therearereportedcasesofdepressionthatstartedwiththeuseofisotretinoin.[2]Therefore,increasingtrendfortheuseofalternative treatments foracne isobservedsince the last10years.

Table 4: Antimicrobial activity against Staphylococcus epidermidis

Name of sample Staphylococcus epidermidis

Zone of inhibition (mm)

Activity index

Percentage inhibition

AHPL/AYTOP/0213cream 28.20 0.8703 87.03Clindamycin 32.40 ‑ ‑

Table 3: Antimicrobial activity against Staphylococcus aureus

Name of sample Staphylococcus aureus


Activity index



Table 2: Antimicrobial activity against Propionibacterium acnes (in vitro)

Name of sample Propionibacterium acnes


Activity index



Figure 2: Zone of inhibition against Staphylococcus aureus (at column width)

Figure 3: Zone of inhibition against Staphylococcus epidermis (at column width)


86 AYU|Volume38|Issue1‑2|January‑June2017

Inthiscontext,apolyherbalformulationAHPL/AYTOP/0213creamwasdevelopedbyAriHealthcarePrivateLimited,forthetreatmentofacnevulgaris,hyperpigmentationandvariousskindisorders.ItcontainsDaruharidraextract(Berberis aristata),[18]Lodhra extract (Symplocos racemosa) Yashtimadhukaextract(Glycyrrhiza glabra),[7,8]Jatiphalaextract(Myristica fragrans),[9]Manjishthaextract(Rubia cordifolia),[10]Vachaextract(Acorus calamus),[11]Dhanyakaextract(Coriandrumsativum)[12]andShalmaliextract(Salmalia malabarica).[13]

SkinirritationstudywasconductedonAHPL/AYTOP/0213cream as per OECD guidelines and it was found thatAHPL/AYTOP/0213creamisnonirritantandsafeforuseasalocalapplication.

ThepresentstudyconductedtoassesstheantimicrobialactivityoftheAHPL/AYTOP/0213creamshowedthatthisformulationpossessantimicrobialactivityagainstP.acnes,S.epidermidisandS.aureusorganism.TheantimicrobialactivityofAHPL/AYTOP/0213creamwasclosetothatofstandardantibiotici.e.,clindamycin.

ItisknownthatvariousingredientsofAHPL/AYTOP/0213creamsuchasDaruharidra(Berberis aristata),[18]Lodhra(Symplocos racemosa),[6] Yashtimadhuka (Glycyrrhiza glabra),[7]Jatiphala (Myristica fragrans)[9] andManjishtha (Rubia cordifolia)[10]possessantibacterialactivityagainstP.acnes.Lodhra (Symplocos racemosa)[6] also possess antibacterialactivityagainstS.epidermiswhichisknowntobeoneoftheacne‑causingbacteria.Dhanyaka (Coriandrum sativum) isconsideredtobeusefulasanantisepticforthepreventionandtreatmentofskininfectionswithGram‑positivebacteria.ItisreportedtohaveantibacterialactivityagainstStreptococcuspyogenes, S. aureus andmethicillin‑resistant S. aureuscombinedwithexcellentskintolerance.[12]

In another study, anti‑inflammatory activity (in vivo) ofAHPL/AYTOP/0213 cream in comparisonwith standarddiclofenac sodium cream in carrageenan induced ratpaw edemamodel was assessed, and it was observedthatAHPL/AYTOP/0213 cream possess significant anti‑inflammatory activity. Furthermore, the anti‑inflammatoryactivityofAHPL/AYTOP/0213creamwascomparabletothatofdiclofenacsodiumcream.

MostoftheingredientsofAHPL/AYTOP/0213creampossessantiinflammatoryactivity.[9,11,19,20]Rubia cordifoliaspecificallyshows anti‑inflammatory activity by suppressingP.acnes inducedROSandpro‑inflammatorycytokines,thetwoimportantinflammatorymediators in acne pathogenesis.[10]Acorus calamusisknowntoinhibittheproductionofpro‑inflammatorycytokinesthroughmultiplemechanismsandmaybeanovelandeffectiveanti‑inflammatoryagentforthetreatmentofskindiseases.[11]Berberis aristataalsopossessanti‑inflammatoryand analgesic activity and promoteswound healing.[20] Itis believed that the synergistic actionof these herbs couldhave attributed to the overall antiinflammatory activity offormulation.

Glycyrrhiza glabrapossessesanti‑androgenicactivity;[8]hence,itmaybebeneficialinregulatingexcesssebumproduction,whichprimarily dependson androgen levels and androgensensitivity. Excess sebumproduction leads to the growthof bacteria responsible for the pathogenesis of acne henceYashtimadhuka isbelievedtobeeffectiveinAcne.

ItisevidentfromtheabovediscussionthatthesynergisticeffectoftheseplantsmayhavecontributedtotheoverallantimicrobialeffectagainstP.acnes,S.epidermidisandS.aureusorganismsaswellasanti‑inflammatoryactivityofAHPL/AYTOP/0213cream.ItcanbestatedthatAHPL/AYTOP/0213creamcanbeagoodtreatmentoptionforeffectivemanagementofacnevulgarisandvariousskindisorders.

ConclusionAHPL/AYTOP/0213cream isnonirritant and safe foruseasalocalapplication.AHPL/AYTOP/0213creamasalocalapplicationpossessessignificantanti‑inflammatoryactivityincarrageenaninducedpawedemainratsandantimicrobialactivityagainstP.acnes,S.epidermidisandS.aureus.Thus,AHPL/AYTOP/0213creamcanbeusedasaneffectivetopicaltreatmentoptionforacnevulgarisandvariousskindisorders.

AcknowledgmentAuthorssincerelythankalltrialsitesstaffatPadm.Dr.D.Y.PatilInstituteofPharmaceuticalSciencesandResearch,Pune‑18,for their contribution in conduct of the present study.Authors are extremely grateful toMr. SanjeevanKanjilal(ManagingDirector)andDr.AnishaKanjilal(Director)ofAriHealthcarePvt.Ltd.,forprovidingalltheresearchfacilities,guidance,courageandmoralsupport.

Financial support and sponsorshipNil.

Conflicts of interestTherearenoconflictsofinterest.

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Spine 5.5 mm ISSN : 0974-8520 AYU - Amarantha Ayurveda€¦ · agents. Although these medicines are better treatment options for acne management, the side effects of these medications