International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 3, Issue 1, 2011

ResearchArticle

FORMULATIONDEVELOPMENTFORTREATMENTANDMANAGEMENTOFHIVAIDS
*AMOLB.CHOUDHARI1,VINODD.RANGARI1,VAIBHAVM.DARVEKAR2
1

J.L.ChaturvediCollegeofPharmacy,Electroniczone,MIDC,Hingnaroad,Nagpur,2PatalDhamalWadhavaniCollegeofPharmacy, Dhamangoanroad,YavatmalEmail:amolchoudhari5@rediffmail.com Received:30Sep2010,RevisedandAccepted:29Oct2010

ABSTRACT The formulations contains a plants which have a potent immunomodulator activity namely Andrographis paniculata, Momardica charantia, Phyllanthusniruri,Terminalliachebula,GlycyrrhizaglabraandPunicagranatum.IthasgoodimpactinthetreatmentandmanagementofHIVAIDS because these formulations not only treat disease but also enhance the body vitality and immunity. The humoral and cellmediated immune responsewasobservedthroughDelayedTypeHypersensitivity(DTH)andMacrophagesPhagocytosisbyCarbonclearancetest.Theseformulation have studied on Swiss albino mice in normal and suppressed immune system by Cyclophosphamide (CP) in three different doses, it shown maximum decrease in foot paw oedema from 2.15mm to 0.35 mm, similarly maximum increase in the WBC and Platelet count up to 10.40.07 thousand/mm3 and96142142inrespectively.SimilarlytheseformulationshaveshownmaximumincreaseinmeanPhagocyticindexincarbon clearancetestinnormalandsuppressedconditionupto0.0190in(F1)and0.0151anditshownsignificantstatisticalanalysisP<0.001. Keywords:HIVAIDS,immunity,immuneresponse,Humoralandcellmediatedimmunity. INTRODUCTION HIV virus attacks and impairs the bodys natural defense system againstdiseaseandinfection.Theunderlyingproblemidentifiedasa severe depression of immune system that is cause by the nearly completelackofoneclassofTlymphocyte,whicharecalledhelper cell that are needed for initiating and maintaining many immune responses.1,2. Theworldhealthorganizationestimatedthat,intheyear2007,the cumulative total infected were 33.2 millions. Numbers of people newly infected in each year are 2.5 million and 2.1 million people dying by AIDS in each year. Every day over 6800 persons become infectedwithHIVandover5700personsdyingfromAIDS. 3 Sothere

is a need to control the death of people and this formulation have negligible side effect and have potential to strengthen immune systemandsuppressedthevirussoitcannotdetectinthebloodand itprolongthelifeofhumanbeings. MATERIALANDMETHODS Selection of the crude drugs have been done after the extensive reviewoftheliteraturetakingintoconsiderationthespecificactivity of active constituent present in the medicinal plant. Some of the crude drugs have also been added for their immunostimulant or toniceffect,orsimplyasabioavailabilityenhancer4,5,6. Themedicinalplantsselectedaregivenintable1.

Table1:Themedicinalplantsusedinformulations Sr No. 1 2 3 4 5 6 Preparationofherbalformulation The quantity of extracts required for formulating herbal drug formulation(Table 2) arecalculated on the basisofhumandose of

Nameofplant Andrographis paniculata Momardica charantia Phyllanthus niruri Terminallia chebula Glycyrrhiza glabra Punicagranatum

Plantpart used Aerialpart Seeds Wholeplant Fruit Roots Bark

Activeconstituents Andrographolide,bicyclicditerpenoid lactonesandkalmeghin MAP30(MomardicaAntiHIVprotein),Alpha momarcharin,MRK29(RIP), momardicosides Phyllanthin,Hypophyllanthin, RapendusinicacidAmonosodiumsalt(RA) Gallicacid,Ellagicacid,Chebulicacid, Galloylglucoses Glycyrrhizin PunicalinandPunicoretin

Activity ImmunostimulantInhabitedsyncytiumformation, AntiHIVactivity. RTactivity,inhibitsHIV1infection,syncytium formation,herpessimplexvirus,HIV1integrase, Betaglucosidaseinhibitory. HIV1inhibitor, InhibitedHIV1RT, HIV1Integrase, InhibitorofHIV1Protease AntiHIVactivity, InhibiteHIVinduceplaqueformation. HIV1RTactivity.

powder form and percentage practical yield of respective crude drugs. Three formulations are prepared using 2% w/v gum tragacanth as suspending agent and considered as Lower dose, AveragedoseandHigherdoseformulation7.

Table2:QuantityofplantextractsusedforpreparingherbalformulationsF1,F2,F3. Sr.No. 1 2 3 4 5 6 ExtractName Andrographispaniculata Momardicacharantia Phyllanthusniruri Terminalliachebula Glycyrrhizaglabra Punicagranatum QuantityofExtract mg/kg(F1) 155 130 550 425 524 513 QuantityofExtract mg/kg(F2) 312 392 1100 851 1049 1026 QuantityofExtract mg/kg(F3) 467 654 1650 1277 1573 1539

Choudharietal. IntJPharmPharmSci,Vol3,Issue1,105108 Animal The experimental protocol was submitted and approved by Institutional Ethical Committee (IAEC No. 648/02/C/CPCSEA), J. L. C.Collegepharmacy,Nagpur. Albino mice (Swiss) of either sex weighing between 2025 g were employed in this investigation. They were housed under standard conditions of temperature 22 0 C ( 30 C) humidity 35 % to 60 %, and light (12:12 hr light/dark cycle) in polypropylenemicecage. Material Cyclophosphamide (Highmedia.) immunosuppressant. diluted eight times in a dose of 10 was used as standard Preparationofsuspensionofdoseofherbalformulation ThreeformulationswerepreparedconsideringLower(F1),Average (F2), and Higher (F3) in distilled water using 2% w/v gum tragacanthasasuspendingagent. PreparationofsolutionofCyclophosphamide 30 mg / kg solution of cyclophosphamide was prepared in sterile normalsaline8. Pharmacologicalstudy ToxicityStudy Toxicity studies of Herbal drug formulations were carried out in Swiss albino mice according to OECD guideline 423. Dose ranging between 500, 1000, 2000, 4000 and 5000 mg/kg of body wt. of formulation were administered stepwise to the mice according to their weights. The mice were observed individually after at least onceduringthefirst30minutes,periodicallyduringthefirst24hrs, therewasnomortalityfoundtilldoseof5000mg/kgbodyweightin formulationF1,F2andF38. Delayedtypehypersensitivitymodel Procedure OndayzeroCyclophosphamide(CP)30mg/kgwasadministratedIP totheanimalofgroupv,vi,viiandviii2hrsbeforesensitizingwith 1x106SRBCs,asantigen,throughIProute. Allthe groupwere treated as per thetable3 for nextfive days, i.e. day 1 to day 5. All the animals were maintained on same diet and environment throughout the duration of the experiment. Administration of extract was done by oral route using animal feedingneedleand1mlsyringe. On day 5 the left hind paw thickness was measured for all the animals. The animals were then challenged with the same antigen SRBCs, 1x106 in 0.1 ml, in the left hind paw by SC route. The Paw thicknessmeasurementwasagaindoneatintervalof24,48,72and 96hrsfromthechallenge. On the day 10 all mice were anaesthetized by putting in an anesthetic ether chamber. Blood was collected from the retro obituaryveinforWBCandTotalPlateletCount8.

Carbon ink suspension Pelikan 4001, Germany black ink was

l /gm body weight of mice

(Bafana, A., 2004). Antigenic material The sheep RBCs (SRBCs) were used as antigenic material. The sheep blood was withdrawn from external jugular vein of sheep (Government Vetneiry college, Nagpur). It was mixed in 1:1 proportion in Alsevers solution & storedat20to80Cinrefrigerator. Procedure Swiss albino mice (HA strain) weighing between 2030gm were brought. All mice were marked with picric acid and randomly divided into eight groups, each group comprising of six animals. Weight of individual mice was taken on electrical signal pan balance and numberingwasdonetoeachmice. PreparationofSRBCsuspension Sheep blood was collected from Veterinary College in Alsevars solution(1:1)andcentrifugedat25003000rpmfor10minutes. Supernatant was removed with Pasture pipette, and packed SRBCs were washed thrice with sterile Alsevars solution. The resulting SRBCs were suspended in sterile Alsevars solution to obtainacelldensityof106 SRBCs/mm 3 ,usingimprovedNeubaur chamber8.

Table3:ExperimentaldesignofDelayedtypehypersensitivityinmice Day0 Day1 To Day5 Day5 Day6to Day9 Day10

GroupVVIII GroupIVIII GroupIandV GroupIIandVI GroupIIIandVII GroupIVandVIII GroupIVIII GroupIVIII GroupIVIII

30mg/kgofCPbyIProute,2hrsbeforeSensitization Sensitizationwith1x106 SRBCsbyIProute 2%w/vgumtragacanth F1:Lowerdose F2:Averagedose F2:Higherdose MeasurementofPawthickness Challengewith1x106SRBCsbySCroute MeasurementofPawthickness CollectionofBloodforWBCandTotal PlateletCount

Table4:EffectofPreparedHerbalFormulationsonMeanFootPadOedemainDTHmodel Gp.No. I. II. III. IV. V. VI. VII. VIII. Gp.Description Control F1 F2 F3 CP CP+F1 CP+F2 CP+F3 MeanFootPawOedema 24hrs 48hrs 1.6+0.07* 1.01+0.08* 1.31+0.08* 0.76+0.12* 0.85+0.05 1.46+0.09 0.89+0.08 1.51+0.14 1.75+0.02* 2.15+0.13* 1.20+0.02* 1.68+0.07* 1.24+0.05 1.90+0.05 1.45+0.05 2.0+0.10 72hrs 0.99+0.08* 0.65+0.07* 0.75+0.03 0.80+0.06 1.20+0.13* 0.95+0.07* 1.0+0.07 1.05+0.05 96hrs 0.55+0.55* 0.35+0.07* 0.40+0.05 0.45+0.05 0.75+0.04* 0.60+0.08* 0.65+0.04 0.68+0.04

106

Choudharietal. IntJPharmPharmSci,Vol3,Issue1,105108

[[

Table5:EffectofpreparedformulationonleukocyteandplateletcountinDTHmodel Sr.No. I. II. III. IV. V. VI. VII. VIII.

Groups Control F1 F2 F3 CP CP+F1 CP+F2 CP+F3

HaematologicalParameters LeukocyteCount (Thousand/mm3) 8.40.1* 10.40.4* 9.60.2 9.10.1 5.520.27* 7.520.27* 6.87+0.32 6.32+ 0.37

PlateletCount (Thousand/mm3) 76680220* 96142142* 90292170 79831190 55669310* 75167250* 65038+275 60391+290

ExperimentalDesign:CarbonClearanceTestinmice Table6:EffectofPreparedHerbalFormulationsonMicrophage PhagocytosisbyCarbonClearanceMethod Sr.No. I. II. III. IV. V. VI. VII. VIII. GroupDescription Control F1 F2 F3 CP CP+F1 CP+F2 CP+F3

MeanPhagocyticIndex 0.01210.0005* 0.01900.0009* 0.01810.0007 0.01710.0010 0.00900.0015* 0.01510.0012* 0.0141+0.0022 0.0133+0.0025

RESULTSANDDISCUSSION The formulations have shown immunostimulant activity in DTH model, when used alone and also a significant immunostimulant activity in the animals whose immunity was suppressed using cyclophosphamide. The phagocytic activity was also found to increaseinCarbonClearancetestwhentheformulationusedalone aswellasinthesuppressedimmunesystem. The crude drugs viz. namely Andrographis paniculata, Momardica charantia,Phyllanthusniruri,Terminalliachebula,Glycyrrhizaglabra andPunicagranatumwhichhavebeencollectedandauthenticated.

The crude drugs were shredded and powder to a coarse powder consistency, which was subjected to extraction by using suitable solvents. The solvent and method of extraction was selected such that the resultant extract contains the active constituents. The extract obtained were concentrated and dried in desiccator or by sundrayingprocess. All the individual dried extracts were checked for their active ingredients by proximate chemical analysis. It showed that all extractcontainsrequiredactiveconstituent. All the extracts were subjected to chromatographic evaluation to check possible number of component in respective extracts using 107

Choudharietal. IntJPharmPharmSci,Vol3,Issue1,105108 thin layerchromatographytechnique. Theextracts of Andrographis paniculatawascomparedwithstandardsamplesofA.paniculataby their Rf values. For the design and preparation of an effective immunostimulant herbal formulation, all the individual dried extracts were mixed in a requisite amount and 2% w/v Gum tragacanthwasaddedtotheformulationasasuspendingagent.The quantitiesofextractswerecalculatedonthebasisofhumandoseof powderformofdrugsandtheirrespectivepercentageyield. Thepreparedformulationweresubjectedtotoxicitystudyandwere found tobe safeuptodailydoseof5000mg/kgofbodywt./mice withnotoxicreactionbeingobserved. The immunostimulant activity of the prepared herbal formulations was studied using delayed type hypersensitivity (DTH) model and carbon clearance test in mice. The results obtained from the DTH model have indicated significant decrease in the mean foot paw oedema,afterchallengingwith106SRBC,whenplottedagainsttime. TheCPreceivinggrouphasshownmaximumoedemaof2.150.13 mmafter24hrs,ofchallenge,deceasingto0.750.04mmafter96 hrs.ThegroupreceivingF1haveshownsignificantlylowervaluesof mean foot paw oedema as compared with the controlled group, indicating a strong immunostimulant effect of formulation F1 than thatF2andF3.ThegroupreceivingCP+F1hasalsoshownmarked decreased in the mean foot paw oedema as compared with the CP receiving group confirming the immunostimulant effect of the formulationsinthesuppressedimmunesystem.ResultsofWBCand Platelet counts for the animals receiving F1, F2 and F3 have also shown significant increasing in count as compared to the control group animals. The results of CP + F1, CP + F2 and CP+F3 groups haveshownamarkedincreaseinthePlateletcountsrespectively,as comparedwiththeCPtreatedgroup.Thisclearlyindicatesthatthe formulation has a strong immunostimulant activity on the suppressedimmunesystem.Theresultobtainedduringthepresent investigationshowedthatthereissignificantantibodyproductionin responsetoSRBCsinformulationF1. The Phagocytic Index of the above formulations was studied by using Carbon Clearance test. The results obtained from the Carbon Clearance test have indicated significant increase in Phagocytic Indexwhenplottedagainsttime.ThegroupreceivingCPhasshown minimum Phagocytic Index (O.0090 0.0015) after 15 min. the groupreceivingF1havesignificantincreaseinthePhagocyticIndex whencomparedwiththecontrolgroupindicatingimmunostimulant activity.ThegroupsreceivingCP+F1,CP+F2andCP+F3havealso shown marked increase in the Phagocytic Index when compared with group receiving CP. This indicates the immunostimulant activityoftheformulationsinsuppressedimmunesystem. Theincreaseincarbonclearanceindexreflectstheenhancementof phagocytic function of mononuclear macrophages and nonspecific immunity. Phagocytosis by macrophages is important against pathogenic microorganism and its effectiveness is markedly enhanced by opsonisation of parasite with antibodies and complement C3 b leading to more rapid clearance of parasite from blood. CONCLUSION The herbal formulation designed and developed as potential immunostimulanthasbeenfoundtobesafeuptoaveryhighdoseof 5000 mg/kg/day/mice during toxicity studies. The formulations have shown immunostimulant activity in DTH model, when used aloneandalsoasignificantimmunostimulantactivityintheanimals whose immunity was suppressed using cyclophosphamide. The phagocytic activity was also found to increase in Carbon Clearance test when the formulation used alone as well as in the suppressed immunesystem.Thustheformulationsunderstudyhavetheability 18. 19. 20. 21. torecoverthesuppressedimmunesystem.Thereitcanbeusedfor thetreatmentandmanagementofAIDSandAIDSrelatedcomplexin which the immune system is suppressed or deranged by the HIV infection. ACKNOWLEDGEMENT The author feels to express sincere thanks to Shilpa Deshpande, Nishant Jain and Nilesh Dabarde for their cooperation during projectwork. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Rangari VD, Dumbre R, and Dumbre MR. HIVAIDS and Bioactive Natural Product, Published by Studium press, LLC, PrintedbySalasarImagingSystemDelhi110035,2008. Sahil K, Bhattacharya, Prasun K, Das. Parantap Sen, Churchill BI,TextbookofPharmacology,LivingStonePvtLtd.NewDelhi, 2003. http//:www.google.com, AIDS epidemic update December 2007,(Accessed,07Aug2008). Kokate CK, Purohit AP, and Gokhale SB, Textbook of Pharmacognosy;EightEdition,NiraliPrakashan,Pune,1997. Rangari VD, A text Book of Pharmacognosy and phytochemistry; Vol. I and II, Second Edition, career publication,2002. Kirtikar KR, and Basu BD, Indian Medicinal Plants. Second Edition, International Book Distributors, Dehradun. Volume I IV,1987. HandaSS,IndianHerbalPharmacopoeia;RevisedNewEdition, PublishedbyIndiandrug ManufacturersAssociation,Mumbai, 2002. Bafna AR, Mishra SH,Immunomodulatoryactivity of menthol extract of flower heads of Sphaeranthus indicus Linn. Ars Pharmaceutica,2004.45(3),281291. Wilson K, and Waugh A, Ross and Wilson Anatomy and Physiology in Health and Illness. Eight Edition , Churchill Livingstone,NewYork.,1996. LachmanLeon,LiebermanHA,KanigJL,ATextBookofTheory and Practice of Industrial Pharmacy; THIRD EDITION, VARGHESEPUBLICATIONHOUSE,Bombay1987. OECD: Guideline, 423, acute oral toxicity: Environmental HealthandSafetyMonographseriesonTestingandAssesment No.24,2000. HARSHMOHAN,ATextbookofPathology;Fifthedition,JAYPEE BROTHERSMedicalPublisher,NewDelhi.2005,7476. Tortora GJ, and Grabowski SR, Principal of Anatomy and Physiology; Eight Edition, Harper Collins College Publishers, NewYork.,1996. NadkarniKM,IndianMaterialMedica;PopularPrakashanPvt. Ltd.NewDelhi1999. WealthofIndia,RawMaterials.VolumeI:AZ,PID,CSIR,New Delhi.1985. Khandelwal K, Practical Pharmacognosy. Nirali publication, Pune,2000,7988. Satoskar RS, Bhandarkar SD, In Pharmacology and Pharmcotherapeutic; Part I, 8 th edn, Popular Prakashan BombayIndia,1983. StahlE,ThinLayerChromatography;ALaboratory Handbook, SpringerVerlag,Berlin,1969,175179. PharmacopiaofIndia;MinistryofHealth,GovernmentofIndia, SecondEdition,1966. Merk and Co, The Merk Index, New Jersey, Twelfth Edition, 1996. Kulkarni SK, Handbook of Experimental Pharmacology; First Edition,VallabhPrakashan,Delhi,1987,8889.

108