Eudragit EPO Based Nanoparticle Suspension of ... · C. Bothiraja 1, Atmaram P. Pawar 1,...

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^{Eudragit (R) EPO Based Nanoparticle Suspension of}

^{Andrographolide In Vitro and In Vivo}

^{Article in Nanoscience and Nanotechnology Letters middot December 2009}

^{DOI 101166nnl20091029}

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^{4 authors}

^{Bothiraja Chellam}

^{Bharati Vidyapeeth Deemed University}

^{46 PUBLICATIONS 291 CITATIONS}

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^{Prakash Pawar}


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^{Karimunnisa Shaikh}

^{Savitribai Phule Pune University}


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^{Praveen Sher}

^{University of Minho}


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^{Copyright copy 2009 American Scientific PublishersAll rights reservedPrinted in the United States of America}

^{Nanoscience andNanotechnology LettersVol 1 156ndash164 2009}

^{Eudragitreg EPO Based Nanoparticle Suspension ofAndrographolide In Vitro and In Vivo}

^{C Bothiraja1 Atmaram P Pawar1lowast Karimunnisa S Shaikh1 and Praveen Sher21Department of Pharmaceutics Bharati Vidyapeeth University Poona College of Pharmacy}

^{Erandwane Pune 411038 Maharashtra India2Polymer Sciences and Engineering Division National Chemical Laboratory Pune 411045 Maharashtra India}

^{Characteristic formulation of an isolated active phytoconstituent is of great therapeutic significanceThe purpose of this work was to develop Eudragitreg EPO based nanoparticle suspension of isolatedandrographolide in order to increase its solubility and efficacy Andrographolide was isolated fromAndrographis paniculata and Its Nanoparticle suspension was prepared by precipitation techniqueThe 32 factorial design was used to study the effect of Eudragitreg EPO and Pluronicreg F-68 on char-acteristics of nanoparticle suspension The optimized formulation was subjected to lyophilizationThe nanoparticle suspension lyophilized suspension and the redispersed lyophilized nanoparticlesuspension were characterized by drug content encapsulation efficiency particle size electroki-netic properties DSC PXRD FTIR TEM and in vitro drug release Optimized batch showed particlesize 255plusmn9 nm with encapsulation efficiency 938plusmn067 and zeta potential 293plusmn34 mV Therewas marked increase in drug dissolution with complete drug release within 10 minutes in nanopar-ticle suspension and redispersed naoparticle suspension as compared to pure drug Howeverlyophilization retarded the drug release The nanoparticle suspension and redispersed nanoparti-cle suspension also showed improved hepatoprotectivity as compared to andrographolide againstCCl4-induced hepatotoxicity in rats The results were confirmed by histopathological studies onhepatic lesions Thus particle engineering can be used to improve pharmaceutical properties ofphytoconstituents}

^{Keywords Andrographolide Eudragitreg EPO Pluronicreg F-68 Nanoparticle SuspensionHepatoprotective Activity}

^{Isolation and identification of phytoconstituent from plantextracts are of great help in pharmaceuticals The isolatedconstituent having pharmacological significance possessa definite physicochemical characteristic as compared toits extract and so can be easily exploited in developinga dosage form Andrographolide an important diterpenelactone present in Andrographis paniculata Nees (Acan-thaceae family) It is also known as ldquoking of bittersrdquoAndrographolide has several pharmacological actionsincluding hepatoprotectant anti-inflammatory anti-viralanti-thrombotic anti-cancer and hypoglycaemic activity1ndash6}

^{Andrographolide has been formulated in various dosageforms like tablet capsules and mixture using its extractalone or in combination7 According to our previousstudy it has an experimental log P value of 2632plusmn0135 and aqueous solubility of 329plusmn 073 gml Due}

^{lowastAuthor to whom correspondence should be addressed}

^{to its low aqueous solubility andrographolide has a lowbioavailability (44) after oral administration leading topoor therapeutic application89 In pursuit for this com-pound many researchers have attempted making cyclodex-trin complexes and liposomes to increase solubility andefficacy1011 Nevertheless the use of cyclodextrin is asso-ciated with a risk of nephrotoxicity and employing lipo-some might incur stability problems during storage1213}

^{Considering these limitations the efficient delivery methodis warranted at this stage to enhance its therapeuticefficacyIn order to improve its dissolution and therapeutic effect}

^{use the development of polymeric nanoparticle suspen-sion composed of Eudragitreg EPO as polymeric matrix andPluronicreg F-68 as surfactant was considered The selectedpolycationic Eudragitreg EPO which was supposed toimprove the interaction with the negatively-charged mucusof the gastro-intestinal tract to achieve therapeutic efficacy}

^{156 Nanosci Nanotechnol Lett 2009 Vol 1 No 3 1941-490020091156009 doi101166nnl20091029}

^{Bothiraja et al Eudragitreg EPO Based Nanoparticle Suspension of Andrographolide In Vitro and In Vivo}

^{of andrographolide Andrographolide isolated from theextract was used to obtain a novel oral nanoparticulatesystem by nanoprecipitation technology14 The amount ofpolymer Eudragitreg EPO (X1 and stabilizer Pluronicreg}

^{F-68 (X2 were optimized by employing 32 factorialdesign The optimized batch was further processed toobtain lyophilized powder and aqueous suspension of thelyophilized powder which were evaluated for differen-tial scanning calorimetry (DSC) powder X-ray diffraction(PXRD) fourier transform infrared spectroscopy (FTIR)transmission electron microscopy (TEM) in vitro releasein vivo hepatoprotective activity and storage stability studyThe fresh leaf material of Andrographis paniculata was}

^{obtained from Shri Shail Medi Farms Nagpur (Maha-rashtra India) Standard andrographolide was purchasedfrom Research Organic Chennai India Eudragitreg EPOand HPMC K-15 were gifts from Cipla PharmaceuticalsMumbai India Pluronicreg F-68 gifted from Alembic phar-maceuticals Mumbai India Methanol GR grade carbontetrachloride and olive oil were purchased from the MerckChemicals Mumbai India All other chemical reagentswere of analytical gradeIsolation of andrographolide was carried out by cold}

^{maceration method15 The powdered leaves (100 g) ofAndrographis paniculata were extracted with a 11 mixtureof dichloromethane (75 ml) and methanol (75 ml) Thestructure and purity were checked by various spectralstudies and compared with standard andrographolideSolubility profile of andrographolide was obtained by dis-persing 300 mg of drug in 100 ml of 01 N HCl purifiedwater and various organic solvents under constant stirring(100 rpm) using magnetic stirrer at 37plusmn05 C for 24 hrat the end of which samples were withdrawn and filteredthrough 045-m membrane filters The filtrates were suit-able diluted and analyzed using a UV spectrophotometer(Jasco V-500) at 227 nmNanoparticle suspension of isolated andrographolide}

^{was prepared by precipitation method12 Andrographolide(120 mg) and specific amount of Eudragitreg EPO were}

^{Table I Coded levels and actual values of the variables along with the measured responses of 32 factorial design}

^{Concentration of Concentration of EncapsulationCoded levels Eudragitreg EPO Pluronicreg F-68 Drug efficiencya Zeta potential Mean particle Polydispersitya}

^{Batches (X1 X2) ( wv) ( wv) contenta () () (mV)a size (nm)a}

^{AG mdash mdash mdash mdash mdash mdash 49461plusmn7 1590plusmn0280S1 minus1 minus1 03 04 613plusmn217 481plusmn088 113plusmn52 166plusmn5 0361plusmn0049S2 minus1 0 03 05 676plusmn197 543plusmn085 117plusmn46 173plusmn6 0341 plusmn 0039S3 minus1 1 03 06 724plusmn214 604plusmn060 121plusmn51 181plusmn7 0328plusmn0037S4 0 minus1 045 04 827plusmn223 826plusmn074 197plusmn58 227plusmn8 0295plusmn0037S5 0 0 045 05 863plusmn109 885plusmn063 268plusmn37 235plusmn8 0291plusmn0032S6 0 1 045 06 884plusmn107 938plusmn067 293plusmn34 255plusmn9 0274plusmn0013S7 1 minus1 06 04 852plusmn212 904plusmn082 213plusmn42 309plusmn5 0242plusmn0022S8 1 0 06 05 876plusmn132 922plusmn075 186plusmn38 328plusmn7 0228plusmn0027S9 1 1 06 06 893plusmn191 947plusmn090 177plusmn43 334plusmn6 0223plusmn0024S6 (Blank) mdash mdash mdash mdash mdash 336plusmn38 241plusmn6 0281plusmn0011}

^{aAll the determinations were performed in triplicate and values were expressed as meanplusmnSD n= 3 X1 Polymer Eudragitreg EPO X2 Stabilizer Pluronicreg F-68}

^{dissolved in 15 ml methanol The organic solution wasquickly injected in 30 ml of aqueous solution containingPluronicreg F-68 under stirring at 2000 rpm Stirring wascontinued for 2 hr at 40 C for complete evaporation ofmethanol The volume was adjusted up to 40 ml with aque-ous solution of 200 mg of HPMC K-15 to obtain nanopar-ticle suspension The 32 factorial design was used tostudy the effect of Eudragitreg EPO and Pluronicreg F-68 oncharacteristics of nanoparticle suspension The optimizednanoparticle suspension was lyophilized (Khera Instru-ments Mumbai India) at minus42 C for 72 hr and whichwas also redispersed in water to get aqueous nanoparticlesuspensionA prior knowledge and understanding of the process}

^{and the process variables under investigation led to pre-liminary experiments Based on this preliminary data the32 factorial design was adopted to optimize the amountof Eudragitreg EPO (X1 and Pluronicreg F-68 (X2 identi-fied as the independent variables affecting the drug contentand the percent drug encapsulation efficiency (dependentvariables) The response surfaces of the obtained resultswere also plotted The coded and the actual values ofthe experimental design are given in Table I The dataanalysis of values obtained from various batches for drugcontent and encapsulation efficiency were subjected tomultiple regression analysis using PCP Disso softwareThe equation fitted was}

^{Y = 0+1X1+2X2+11X21 +22X}

^{22 +12X1X2}

^{where Y is the measured response X is the levels of fac-tors is the coefficient computed from the responses ofthe formulationsPhysical mixtures of the drug Eudragitreg EPO}

^{Pluronicreg F-68 and HPMC K-15 were prepared by dryblending using same ratios as that used for preparation ofoptimized batch of nanoparticle suspension The drug con-tent of nanoparticle suspension was determined by dissolv-ing nanoparticle suspension in methanol and measuring the}

^{Nanosci Nanotechnol Lett 1 156ndash164 2009 157}

^{Eudragitreg EPO Based Nanoparticle Suspension of Andrographolide In Vitro and In Vivo Bothiraja et al}

^{absorbance by UV spectrophotometer The drug contentwas calculated as}

^{Drug content = CAtimesVA}

^WA

^times100

^{where CA is the total concentration of andrographolide innanoparticle suspension WA is the theoretical amount ofandrographolide added VA is the volume of nanoparticlesuspension Non-encapsulated andrographolide was sepa-rated from the nanoparticle suspension by centrifugation(Eppendorf 5810R Germany) at 14000 rpm for 2 hr16}

^{Encapsulation efficiency of andrographolide was calcu-lated by the equation}

^{Encapsulation efficiency = CAVAminus FAVA}

^CAVA

^times100

^{where FA was the concentration of non-encapsulatedandrographolideZeta potential of nanoparticle suspension (n = 3)}

^{was determined by electrophoretic light scattering usingMalvern Zetasizer 3000 (UK) The average particle sizeand polydispersity index of andrographolide nanoparti-cle suspension and redispersed nanoparticle suspensionwere determined after dispersing in water using Malvern(Malvern Instruments UK)The differential scanning calorimetry thermograms of}

^{andrographolide Eudragitreg EPO Pluronicreg F-68 physi-cal mixture and lyophilized nanoparticle suspension wereobtained using a Mettler Toledo DSC 821e (Switzerland)instrument equipped with an intra cooler Instrument wascalibrated for DSC temperature and enthalpy using Indiumstandard The samples were hermetically sealed in per-forated aluminium pans and heated at constant rate of10 Cmin over the temperature range of 0ndash250 CThe system was purged with nitrogen gas at the rate of100 mlmin to maintain inert atmospherePowder X-ray diffraction patterns of andrographolide}

^{Eudragitreg EPO Pluronicreg F-68 physical mixtureand lyophilized nanoparticle suspension were recordedby using a Philips PW 1729 X-ray diffractometer(Netherlands) using Cu K radiation (1542 Aring) with avoltage of 30 kV and a current of 30 mA Samples werescanned from 5 to 50 2 FT-IR measurement of andro-grapholide Eudragitreg EPO Pluronicreg F-68 physical mix-ture and lyophilized nanoparticle suspension were obtainedon FTIR-8400 (Shimadzu Corporation Japan) using theKBr disk technique (about 10 mg sample for 100 mgdry KBr) External morphology of nanoparticle suspen-sion was determined by Transmission Electron Microscopy(Philipps EM-CM 12 80 kV) The sample for TEM wasmade by placing one drop of the nanoparticle suspensionon a copper grid and dried under vacuum pressureDissolution studies of andrographolide physical mix-}

^{ture nanoparticle suspension lyophilized and redispersednanoparticle suspension were performed in 01 N HCl}

^{Formulation equivalent to 20 mg of andrographolide wereplaced in 500 ml of dissolution medium which was stirredwith rotating paddle at 100 rpm with temperature adjustedto 37plusmn 02 C At selected time intervals sample wasremoved and replaced with fresh dissolution medium16}

^{The sample was filtered through 02 m membrane fil-ter and analyzed by UV spectrophotometer Optimizednanoparticle suspension was subjected to elevated temper-ature and humidity conditions of 40plusmn 2 C75plusmn 5 RHover the period of 3 months and the effect of temperatureand humidity on suspension was studied by measuring drugcontent encapsulation efficiency zeta potential and parti-cle size which were compared with initial measurementsThe results of the above evaluations were analyzed statis-tically using the Ktuskal-Wallis test followed by Dunnettrsquospost test p lt 005 denoted significance in all casesThe hepatoprotective activity of nanoparticle suspen-}

^{sion was determined in comparison with andrographolidein Wistar albino rats 150ndash200 g obtained from Yashfarm Pune India) Thirty six male Wistar albino rats werehoused in polypropylene cages with free access to standardlaboratory diet and water They were kept at 23plusmn2 C and55plusmn 10 relative humidity (RH) with a 12 h lightndashdarkcycle Animal handing routines were performed accord-ing to Good Laboratory Practice The research protocol ofthe animal experimentation was approved by InstitutionalAnimal Ethics Committee (IAEC-3108) Acute toxicitystudy was performed for andrographolide nanoparticle sus-pension according to OECD guidelines17}

^{Prior to testing rats were evaluated by clinical obser-vations and body weight determination to assure freedomfrom potential confounding variables Thirty six healthymale rats were randomly assigned to six experimentalgroups of six rats each Male rats have less variations inphysiology than female rats in present study only malerats are usedGroup I received distilled water and served as the}

^{controlGroup II received a 7-days repeated oral dose of distilled}

^{water and served as toxic groupGroup III received a 7-days repeated oral dose 20 mgkg}

^{isolated andrographolide suspended in aqueous solution of05 HPMC K15Group IV and V were received a 7-days repeated oral}

^{dose of 20 mgkg and 10 mgkg of nanoparticle suspen-sion respectively The effective doses of andrographolide(20 mgkg) were based on earlier reports and clinicaldosages18}

^{Group VI received a 7-days repeated oral dose of10 mgkg of redispersed nanoparticle suspension At theend of 7 days all the groups except Group I were receiveda single oral dose of 125 mlkg mixture of 11 (vv) CCl4and olive oil19}

^{Blood samples were drawn 24 h after administrationof CCl4 from retroorbital plexus under ether anesthe-sia The samples were centrifuged at 7000 rpm at 4 C}

^{158 Nanosci Nanotechnol Lett 1 156ndash164 2009}


^{for 20 min within 1 h after collection The sera werestored in the refrigerator till further analysis Concentrationof SGPT (Serum glutamic-pyruvate transaminase) SGOT(Serum glutamic-oxaloacetic transaminase) ALP (Serumalkaline phosphatase) and triglycerides in blood serumwere evaluated by an autoanalyzer (Shimadzu CL-7200Shimadzu Japan) The results are expressed as meanplusmnSD and statistical analysis was carried out using Kruskal-Wallis following By Dunnettrsquos test and P lt 005 was takenas significant Histological examination was performedusing two animals of each group Liver samples were takenfrom the distal portion of the left lateral lobe The tissuewas fixed in 10 formalin for 24 h The samples werethen embedded in paraffin cut into 5 m section andstained with haematoxylin and eosin for examination bylight micrographySpectral data of the isolated andrographolide and stan-}

^{dard andrographolide confirmed the identity of the iso-lated compound as andrographolide15 Solubility studies ofandrographolide in different solvents were in the increas-ing order of Water lt 01 N HCl lt Ethyl acetate lt Butyllactatelt Benzyl alcohollt EthanolltAcetoneltMethanol(Table II) Nanoprecipitation technology was selected forthe production of submicron particle complying with thelow aqueous solubility of andrographolide On the basisof drug solubility (11514plusmn 12 mgml) and miscibilityin aqueous phase methanol was selected as a choice ofsolvent The rapid diffusion of methanol from disperseddroplets into aqueous phase with subsequent evaporationleads to fast precipitation of dissolved drug and polymerin the form of nanoparticles20}

^{Drug content and encapsulation efficiency of nanopar-ticle suspensions were in the range of 60 to 90 and48 to 94 respectively (Table I) which were mainlyinfluenced by polymer concentration The curvilinearrelation observed between the drug content encapsula-tion efficiency with Eudragitreg EPO concentration It canbe explained on the basis of lipophilicndashlipophilic inter-action between andrographolide (log P 2632plusmn 0135)and Eudragitreg EPO (log P 207plusmn 034) Consequently}

^{Table II Solubility profile of andrographolide in various solvents at25 C and miscibility of solvents in water}

^{AG in organic Organic solvents inSolvents solvent (mgml)a water ( ww)b}

^{Acetone 9002plusmn15 sim1000}

^{Benzyl alcohol 4953plusmn9 35Butyl lactate 3259plusmn7 77Ethanol 7716plusmn17 sim1000Ethyl acetate 1947plusmn8 80Methanol 11514plusmn12 sim1000Water 0003plusmn0007 mdash01 N HCl 0007plusmn0002 mdash}

^{aExperimental data bData from Ref [36] AGmdashAndrographolide}

^{with increase in the Eudragitreg EPO amount andro-grapholide gets preferentially dispersed in the internalorganic phase21 Pluronicreg F-68 also displayed similartrend and increase in encapsulation efficiency which canbe due to the formation of interpenetrated network chainbetween the hydrophobic portion of Pluronicreg F-68 withEudragitreg EPO during precipitation22 It is also predictedby the regression values of X1 and X2 as shown inTable III The particle size also shown similar effects Neg-ative influence of polymerndashpolymer interaction as compareto polymerndashpluronic interaction signifies the stabilizingeffect of the latter by minimizing dispersion and distribu-tion of drug outside the matrix Response surface graphsfor drug content and encapsulation efficiency are shown inFigure 1As shown in Table I particle size of the nanoparti-}

^{cle suspension was in range of 166 to 308 nm whichwas almost 200 times smaller than the andrographolideThe increase in particle size of nanoparticle suspensionwith decrease in polydispersity index was observed withincrease in polymer content The smaller particle sizeobtained at low polymer content may be due to high distri-bution efficiency of the internal polymer-solvent phase intothe external phase23ndash25 Increase in the viscosity of inter-nal phase with increased amount of polymer also providesresistance for mass transfer in turn diffusion of polymer-solvent phase into the external phase leading to particleenlargementThe zeta potential values of the nanoparticle suspen-}

^{sion are presented in Table I All formulations exhibitedstrongly positive zeta potential values due to polycationicEudragitreg EPO comprising of various ammonium groupsThe increased zeta potential values in initial batches maybe attributed to Eudragitreg EPO available at the surfaceof the particles due to high viscosity of external aqueousphase The subsequent decline in values of zeta potentialis an inverse function of particle size26}

^{As solid state pharmaceutics have many advantages overliquid formulation mainly improved physicochemical sta-bility and less susceptibility to microbial contaminationattempts were made to obtain dry powder nanoparticle}

^{Table III Estimation of regression coefficients for different responsevariables}

^Responses

^{Coefficient Yield Encapsulation efficiency}

^{0 8580 87661 1037 19422 348 46611 minus832 minus142322 mdash mdash12 minus145 minus157R2 09947 09988}

^{P lt 005}



^{Fig 1 Response surface plot showing effect of factorial variables ondrug content and encapsulation efficiency}

^{suspension by lyophilization technique Based on theresults of the factorial design batch S6 having drug con-tent of 884plusmn 107 encapsulation efficiency of 938plusmn067 zeta potential of 293plusmn 34 mV was further pro-cessed to obtain dry powder When it was compared withblank batch no significant variations in particle size andzeta potential were observed (Table I) Aqueous dispersionof the lyophilized powder have the average particle size of6342plusmn104 nm Almost twice increase in size of particlescould be due to changes in the internal structure of the par-ticles originated during the freeze drying process causedby the formation of ice crystal in the water phase or morelikely to particle aggregation during freeze-drying result-ing in poor redispersion27}

^{Figure 2 shows the DSC thermograms of andro-grapholide Pluronicreg F-68 Eudragitreg EPO lyophilizednanoparticle suspension and its physical mixture Andro-grapholide and Pluronicreg F-68 exhibit a sharp melt-ing endotherm at 23535 C (H 26265 Jg) and5599 C (H 16615 Jg) respectively whereas Eudragitreg}

^{Fig 2 DSC curves for (A) Andrographolide (B) Pluronicreg F-68(C) Eudragitreg EPO (D) Physical mixture and (E) Lyophilized nanopar-ticle suspension}

^{EPO showed a broad melting endotherm at 5626 C(H 6359 Jg) The physical mixture exhibited a sharpendotherm at 5424 C (H 4940 Jg) corresponding tothe melting of Pluronicreg F-68 and Eudragitreg EPO anda low intensity endotherm at 22085 C which may beattributed to the melting of undissolved crystalline andro-grapholide The thermograme of lyophilized nanoparti-cle suspension displayed a sharp endotherm at 5272 C(H 3409 Jg) corresponding to Pluronicreg F-68 with asmall shoulder endotherm of Eudragitreg EPO but no drugpeak It explains monotectic behavior of the system wheredrug gets completely dissolved below its melting temper-ature in molten mass of the excipients The similar behav-ior was also reported for the nifedipine with Pluronicreg}

^{F-68 Gelucire and paracetamol with PEG2829 The PXRDdiffraction patterns as shown in Figure 3 reveal char-acteristic peaks at 2 of 98 119 148 158 184}

^{and 267 which can be inferred to traits of a high crys-talline structure The complete disappearance of peaksin lyophilized powder may be due to formation of anamorphous complex while undergoing the nanoprecipita-tion with intermolecular interaction occurring within thematrix Peaks of reduced intensity were observed in phys-ical mixtureThe intermolecular interaction in nanoparticle suspen-}

^{sion was established by FT-IR shown in Figure 4 Andro-grapholide exhibits the characteristics intensities of C Oabsorption band at 1674 cmminus1 and the OH stretch at3398 cmminus1 The spectra of physical mixtures seemedto be a summation of spectra of drug and excipientsindicating no intermolecular interaction However FTIRspectra of the lyophilized powder showed shifting of}



^{Fig 3 PXRD patterns of (A) Andrographolide (B) Pluronicreg F-68(C) Eudragitreg EPO (D) Physical mixture and (E) Lyophilized nanopar-ticle suspension}

^{C O absorption band of andrographolide towards lowerwave number at 1647 cmminus1 and completely disappearanceof the OH stretching These result suggested occurrenceof intermolecular hydrogen bonding in the nanoparticlesuspension which might be stronger than the physical mix-tures Furthermore the formation of hydrogen bondingin nanoparticle suspension has been correlated with theformation of amorphous form30 The surface topographyof the nanoparticle suspension was studied using TEMwhich displayed uniform sized spherical shaped nanopar-ticles with size range correlating with particle size studies(Fig 5)The dissolution profile of andrographolide physical}

^{mixture nanoparticle suspension lyophilized nanoparticlesuspension and redispersed lyophilized nanoparticle sus-pension in 01 N HCl is shown in Figure 6 As comparedwith pure drug and physical mixture the nanoparticlesupensionredispersed nanoparticle suspension showed sig-nificant increase in dissolution rate with complete drugrelease within 10 minutes This could be due to thehigh mass transfer caused by increase in surface areaof the drug The increased saturation solubility which}

^{Fig 4 FT-IR spectra of (A) Andrographolide (B) Pluronicreg F-68(C) Eudragitreg EPO (D) Physical mixture and (E) Lyophilized nanopar-ticle suspension}

^{Fig 5 TEM photograph of the andrographolide nanoparticle suspen-sion system magnified 30000times}



⁰

²⁰

⁴⁰

⁶⁰

⁸⁰

¹⁰⁰

^{0 20 40 60 80 100 120}

^{Time (Mins)}

^Dru

^{g re}

^leas

^{ed (}

⁾

^{Fig 6 Dissolution profile of andrographolide () physical mixture ()andrographolide nanoparticle suspension () lyophilized nanoparti-cle suspension (mdash) and redispersed nanoparticle suspension () in01 N HCl Values were expressed as meanplusmnSD n= 3}

^{is inverse function of particle size especially for parti-cles below 2 m might also have contributed for rapiddrug release31 However lyophilization retarded the drugrelease where the t90 for nanoparticle suspension andlyophilized nanoparticle suspension was 5 min and 20 minrespectively The decreased dissolution of lyophilized for-mulation is probability due to the aggregation of the par-ticles in lyophilization but still particles exhibited sizebelow 1 m The optimized nanoparticle suspension sub-jected to stability study at 45 plusmn2 C75plusmn5 RH Duringstability study no significant difference in drug con-tent (8573plusmn 167) encapsulation efficiency (9137plusmn082) zeta potential (2729plusmn193 mV) and particle size(2583plusmn 438 nm) was observed over the period of three}

^{Table IV Hepatoprotective evaluation of andrographolide and andrographolide nanoparticle suspension against CCl4 induced hepatotoxicity in rats}

^{SGPT SGOT ALP Triglycerides Hepatocyte Inflammatory Fatty SinusoidalGroups (IUL) (IUL) (IUL) (mgdl) necrosis cell change dilatation}

^{Control 457plusmn68 1348plusmn207 1625plusmn180 896plusmn237 mdash mdash mdash mdashCCL4 6797plusmn365b 17131plusmn3341b 5290plusmn236b 2574plusmn114b +++ +++ ++ +++AG 20 mgkg 1939plusmn238b c 2578plusmn786c 2186plusmn202b c 1484plusmn133a c + ++ + +AGNS 20 mgkg 1026plusmn115b c 1682plusmn621c 1775plusmn92c 1064plusmn133c mdash mdash mdash mdashAGNS 10 mgkg 2383plusmn158b c 3093plusmn332a c 2705plusmn115b c 1797plusmn332b c + ++ + +RNS 10 mgkg 2527plusmn255b c 3223plusmn493a c 2887plusmn146b c 1869plusmn212b c ++ ++ + +CCl4mdashCarbon tetrachloride SGPTmdashSerum glutamic-pyruvate transaminase SGOTmdashSerum glutamic-oxaloacetic transaminase ALPmdashSerum alkaline phosphataseAGmdashAndrographolide AGNSmdashAndrographolide nanoparticle suspension RNSmdashRedispersed nanoparticle suspension Each value represents the meanplusmnSD observation insix rats abSignificant difference at P lt 005 and P lt 0001 levels as compared with the control group respectively cSignificant difference at P lt 0001 levels comparedwith the CCL4 group Grades are as follows mdash (normal) + (mild) ++ (moderate) +++ (severe)}

^{months as compared to freshly prepared nanoparticle sus-pension (p gt 005)The results of acute toxicity study revealed that the}

^{nanoparticle suspension was safe up to 2000 mgkg whichis as per OECD guidelines Pure drug its nanoparti-cle suspension and redispersed nanoparticle suspensionwere tested for comparative hepatoprotective effect furthernanoparticle suspension was used in two different concen-trations Blood sampling for biochemical testing was done24 h after CCl4 intoxication and the test observations arein accordance with those of the previous reports3233 Themarked release of SGOT SGPT ALP and Triglyceridesinto circulation indicated severe damage to hepatic tissuemembrane during CCl4 intoxication34 A single oral doseof CCl4 at 125 mlkg caused a dramatic elevation in serumenzymes and triglyceride indicating an acute hepatotoxi-city (Table IV) The toxic effects were also confirmed byhistopathological study revealing extensive hepatocellu-lar degeneration and necrosis fatty changes inflammatorycell infiltration and sinusoidal dilatation (Table IV Fig 7)The hepatoprotective effect of the andrographolide for-}

^{mulations is due to the intoxication of hepatotoxinReduction in the levels of SGOT and SGPT towards thenormal value is an indication of stabilization of plasmamembrane as well as repair of hepatic tissue damageswhereas reduction of ALP levels suggests the stabil-ity of the biliary function When given in the samedose (20 mgkg) nanoparticle osuspension showed bet-ter hepatoprotection as compared to andrographolide Theenhanced hepatoprotective activity of submicron drug par-ticles and polymerndashmucous interactions between polyca-tionic polymers and negatively-charged gastro-intestinalmucous may be responsible for better efficacy of nanopar-ticle suspension35 Furthermore the nanoparticle suspen-sion showed comparable hepatoprotective effect in halfdose (10 mgkg) as that of pure andrographolide Theaqueous dispersion of lyophilized nanoparticle suspensionalso showed same efficacy as that of nanoparticle suspen-sion These observations were also confirmed by decreasein the incidence and severity of histopathological hepaticlesions}



^{Fig 7 Representative photographs of liver section stained with hema-toxylin and eosin (A) Control (B) CCl4 (C) Andrographolide(D) Andrographolide nanoparticle suspension (20 mgkg) (E) Andro-grapholide nanoparticle suspension (10 mgkg) (F) Redispersed nanopar-ticle suspension (10 mgkg) treated groups [larrHepatocyte necrosis darrInflammatory cell Fatty changes+Sinusoidal dilatation]}

^{The study utilizes the particle engineering to improveprimary properties of the phytoconstituent Andro-grapholide The bioavailability of andrographolide whichis dissolution rate limited can be improved by produc-ing nanoparticles using nanoprecipitation technique Thepolycationic polymer Eudragitreg EPO and Pluronicreg F-68as stabilizer can be used to obtain physicochemicallystable nanoparticle suspension In addition ionic inter-actions between cationic polymer with GI mucosa mayimprove bioavailability As the nanoparticle suspensionand lyophilized nanoparticle suspension produced almostsame improvement in hepatoprotective activity of andro-grapholide lyophilization of nanoparticle suspension canbe attempted to improve physicochemical biological aswell as pharmaceutical properties of phytoconstituents}

^{Acknowledgments The authors are thankful to AllIndia Council for Technical Education New DelhiIndia for providing financial support in form of Quality}

^{Improvement Programme Fellowship to Bothiraja Authorsare also thankful to Professor S L Bodhankar and SachinBadole for their help during in vivo study}

^{References and Notes}

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^{2 Y C Shenv C F Chen and W F Chiou Br J Pharmacol 135399 (2002)}

^{3 C Calabrese S H Berman J G Babish X Ma L Shinto M DorrK Wells C A Wenner and L J Standish Phytotherapy Res 14333 (2000)}

^{4 H Y Zhao and W Y Fang Chin Med J 104 770 (1991)5 T Matsuda M Kuroyanagi S Sugiyama K Umehara A Ueno}

^{and K Nish Chem Pharm Bull (Tokyo) 42 1216 (1994)6 X F Zhang and B K Tan Clin Exp Pharmacol Physiol 27 358}

^{(2000)7 P Thisoda N Rangkadilok N Pholphana L Worasuttayangkurn}

^{S Ruchirawat and J Satayavivad Eur J Pharmacol 553 39(2006)}

^{8 Z Y Zang Y D Wang and HeGx Clin Trad Herb Drugs 13 41(1982)}

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^{Received 3 September 2009 Accepted 23 September 2009}


^{View publication statsView publication stats}

^{Copyright copy 2009 American Scientific PublishersAll rights reservedPrinted in the United States of America}

^{Nanoscience andNanotechnology LettersVol 1 156ndash164 2009}

^{Eudragitreg EPO Based Nanoparticle Suspension ofAndrographolide In Vitro and In Vivo}

^{C Bothiraja1 Atmaram P Pawar1lowast Karimunnisa S Shaikh1 and Praveen Sher21Department of Pharmaceutics Bharati Vidyapeeth University Poona College of Pharmacy}

^{Erandwane Pune 411038 Maharashtra India2Polymer Sciences and Engineering Division National Chemical Laboratory Pune 411045 Maharashtra India}

^{Characteristic formulation of an isolated active phytoconstituent is of great therapeutic significanceThe purpose of this work was to develop Eudragitreg EPO based nanoparticle suspension of isolatedandrographolide in order to increase its solubility and efficacy Andrographolide was isolated fromAndrographis paniculata and Its Nanoparticle suspension was prepared by precipitation techniqueThe 32 factorial design was used to study the effect of Eudragitreg EPO and Pluronicreg F-68 on char-acteristics of nanoparticle suspension The optimized formulation was subjected to lyophilizationThe nanoparticle suspension lyophilized suspension and the redispersed lyophilized nanoparticlesuspension were characterized by drug content encapsulation efficiency particle size electroki-netic properties DSC PXRD FTIR TEM and in vitro drug release Optimized batch showed particlesize 255plusmn9 nm with encapsulation efficiency 938plusmn067 and zeta potential 293plusmn34 mV Therewas marked increase in drug dissolution with complete drug release within 10 minutes in nanopar-ticle suspension and redispersed naoparticle suspension as compared to pure drug Howeverlyophilization retarded the drug release The nanoparticle suspension and redispersed nanoparti-cle suspension also showed improved hepatoprotectivity as compared to andrographolide againstCCl4-induced hepatotoxicity in rats The results were confirmed by histopathological studies onhepatic lesions Thus particle engineering can be used to improve pharmaceutical properties ofphytoconstituents}

^{Keywords Andrographolide Eudragitreg EPO Pluronicreg F-68 Nanoparticle SuspensionHepatoprotective Activity}

^{Isolation and identification of phytoconstituent from plantextracts are of great help in pharmaceuticals The isolatedconstituent having pharmacological significance possessa definite physicochemical characteristic as compared toits extract and so can be easily exploited in developinga dosage form Andrographolide an important diterpenelactone present in Andrographis paniculata Nees (Acan-thaceae family) It is also known as ldquoking of bittersrdquoAndrographolide has several pharmacological actionsincluding hepatoprotectant anti-inflammatory anti-viralanti-thrombotic anti-cancer and hypoglycaemic activity1ndash6}

^{Andrographolide has been formulated in various dosageforms like tablet capsules and mixture using its extractalone or in combination7 According to our previousstudy it has an experimental log P value of 2632plusmn0135 and aqueous solubility of 329plusmn 073 gml Due}

^{lowastAuthor to whom correspondence should be addressed}

^{to its low aqueous solubility andrographolide has a lowbioavailability (44) after oral administration leading topoor therapeutic application89 In pursuit for this com-pound many researchers have attempted making cyclodex-trin complexes and liposomes to increase solubility andefficacy1011 Nevertheless the use of cyclodextrin is asso-ciated with a risk of nephrotoxicity and employing lipo-some might incur stability problems during storage1213}

^{Considering these limitations the efficient delivery methodis warranted at this stage to enhance its therapeuticefficacyIn order to improve its dissolution and therapeutic effect}

^{use the development of polymeric nanoparticle suspen-sion composed of Eudragitreg EPO as polymeric matrix andPluronicreg F-68 as surfactant was considered The selectedpolycationic Eudragitreg EPO which was supposed toimprove the interaction with the negatively-charged mucusof the gastro-intestinal tract to achieve therapeutic efficacy}

^{156 Nanosci Nanotechnol Lett 2009 Vol 1 No 3 1941-490020091156009 doi101166nnl20091029}














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Eudragit EPO Based Nanoparticle Suspension of ... · C. Bothiraja 1, Atmaram P. Pawar 1,...

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