Rev. bras. farmacogn. vol.27 número2

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Engineering

spray-dried

rosemary

extracts

with

improved

physicomechanical

properties:

a

design

of

experiments

issue

Luiza

T.

Chaul

_,

_Edemilson

_C.

_Conceic¸

_ão

_,

_Maria

_Tereza

_F.

_Bara

_,

_José

_R.

_Paula

_,

_Renê

_O.

_Couto

a_{FaculdadedeFarmácia,UniversidadeFederaldeGoiás,Goiânia,GO,Brazil}

b_{DepartamentodeCiênciasFarmacêuticas,CentrodeCiênciasdaSaúde,UniversidadeEstadualdeLondrina,Londrina,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11August2016 Accepted5October2016 Availableonline1December2016

Keywords:

Designofexperiments Phytomedicine Soliddosageform Powdertechnology

Rosmarinusofficinalis

Spraydrying

a

b

s

t

r

a

c

t

A33_{Box–BehnkendesignandResponseSurfaceMethodologywereperformedtoevaluatetheinfluence}

ofextractfeedrate,dryingairinlettemperatureandspraynozzleairflowrateontheprocessyield, sta-bilityparameters(moisturecontentandwateractivity)andonseveralphysicomechanicalproperties ofspray-driedrosemaryextracts.Powderyieldrangedfrom17.1to74.96%.Thespray-driedrosemary extractsshowedmoisturecontentandwateractivitybelow5%and0.5%,respectively,whichindicate theirchemicalandmicrobiologicalstabilities.Evenwithoutusingdryingaids,somesetsof experimen-talconditionsrendereddriedproductswithsuitableflowabilityandcompressibilitycharacteristicsfor directpreparationofsoliddosageforms.AnalysisofvarianceandResponseSurfaceMethodologyproved thatstudiedfactorssignificantlyaffectedmostofthespray-driedrosemaryextractqualityindicatorsat differentlevels.Themainprocessingparameteraffectingthespray-driedrosemaryextract characteris-ticswasinlettemperature.Thebestcombinationofparametersusedtoobtainareasonableyieldofstable dryrosemaryextractswithadequatetechnologicalpropertiesforpharmaceuticalpurposeinvolvesan extractfeedrateof2ml/min,80◦_{Cinlettemperatureand40l/minSA.Thedesignofexperimentsapproach}

isaninterestingstrategyforengineeringspray-driedrosemaryextractswithimprovedcharacteristics forpharmaceuticalindustrialpurpose.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Rosemary(Rosmarinusofficinalis L.,Lamiaceae), nativetothe Mediterranean,isahouseholdspecieusedworldwideasafood preservativeandfood-flavoringagent.Overthelastdecades,the valuablemedicinalpropertiesofthisherbhavebeeninthe spot-lightofthescientificcommunity.Anevidence-based systematic reviewofrosemary’stherapeuticusehasbeenpublished(Ulbricht etal.,2010).Recently,preclinicalsurveyshavecontributedto evi-dence that rosemary hasremarkable anti-inflammatory (Rocha etal.,2015),anti-proliferative(Cattaneoetal.,2015),and

antibi-otic(Barretoetal.,2014)activities,aswellasantidepressant-like,

anxiolytic,andantinociceptiveeffects(Abdelhalimetal.,2015). Moreover,a clinical trialin humans reported the beneficial effectsofrosemaryfortreatmentofopiumwithdrawalsyndrome duringaddictiontreatmentprograms(Solhietal.,2013).Thisbroad rangeofrelevanttherapeuticpropertiesislikelyattributedtothe antioxidantactivityof this herb, whichis a powerful sourceof

∗ _{Correspondingauthor.}

E-mail:rocouto@uel.br(R.O.Couto).

antioxidantcompoundse.g.,rosmarinicandcarnosicacids, cam-phor,and1,8-cineol(Barretoet al.,2014;Cattaneoetal.,2015;

Lemosetal.,2015;Rochaetal.,2015;Lietal.,2016).

To become suitable for further therapeutic applications, herbalrawmaterial typicallyundergoesseveralpharmaceutical processingtechnologiese.g.,theextractionofbioactivecompounds anddryingofplantextracts.Inthelatter,thedryertypeandsetof operatingconditionsusedinthedryingprocessofaliquid feed extractplayimportantrolesindeterminingthepropertiesof co-processedproducts(Oliveiraetal.,2006;Souzaetal.,2008).As spraydryingpresentsseveraladvantagesoverotherdrying tech-niquessuchconventionalairdrying,freeze-dryingandspoutedbed drying,itisthemostcommonlyemployedinthepharmaceutical, foodandflavorindustry(Pateletal.,2014).

Inbrief,spraydryingisathree-stepunitoperation(i.e., atomiza-tion,dehydration,andpowdercollection)inwhichdryparticulate productsarerecoveredfromaliquidsolutionordispersion (sus-pensionoremulsion)bysprayingtheliquidintoastreamofdrying gasunderdeterminedsetof conditions(Oliveiraand Petrovick, 2010).Due to its remarkableoperational flexibility,spray dry-ingoffersaveryaccuratecontroloverpowderparticleproperties such as physicochemical stability, solubility, morphology and

http://dx.doi.org/10.1016/j.bjp.2016.10.006

flowability(AmeriandMaa,2006;Vehring,2008;Cortés-Rojasand

Oliveira,2012).

Theequipment’soperatingvariableshavebeenwidelystudied duringthespraydryingofphytopharmaceuticals.Suchvariables includesolutionordispersionfeedrate,dryingairinlet tempera-ture,dryingairoutlettemperature,spraynozzleairflowrate,spray nozzlediameter,spraynozzletype, dryingairflow rate, aspira-tion rate,drying air inlet humidity,drying air outlethumidity, compressedair flow,compressedair pressure,etc.(Oliveiraand

Petrovick,2010).

Currently,ourresearchgrouphasstruggledgreatlytodevelop rosemary’s phytopharmaceuticals intermediate products with greateraggregatevaluebyspraydrying(Coutoetal.,2012a).The operatingvariablessetupdemonstratedaneffectonthe chemi-calprofileandinvitroantioxidantactivityofspray-driedrosemary extracts (SDRE). From a phytopharmaceutical technology point ofview,besidesthephysicochemicalqualitycontroland biolog-icalactivityassessments,determiningtheeffectsofspraydrying processvariablesinthedryingperformanceand physicomechan-icalpropertiesoftheproducts,isastartingpointforthefulland accuratevalidationandscale-upofrosemary’spowdertechnology process.

Improvingbothchemicalandmicrobiologicalstabilities,aswell as theirflowabilityand compressibility during the preformula-tionstudies,mayturntheSDREintomoreinvitingintermediate productsfordeveloping soliddosageforms (SDF),which repre-sentthemajorityofmedicinesmarketedworldwide.Multifactorial chemometrictoolssuchasthedesignofexperimentsapproachand ResponseSurfaceMethodology(RSM)maybeusefulstrategiesin thispursuit(Materoetal.,2013).

Inthispaper,wereporttheeffectofextractfeedrate(EF), dry-ingairinlettemperature(IT)andspraynozzleairflowrate(SA)in severalphysicomechanicalpropertiesofSDRE.A33_{Box–Behnken} design andRSMwere performed.Thecorrelations betweenthe processadequacyindicatorswerealsoassessed.Byusinga deter-minedsetofexperimentalconditions,weengineeredproductswith improvedlevelsofstability,flowabilityandcompressibility,which mayenablethedirectmanufactureoftabletsand/orcapsulesfilling.

Materialsandmethods

Herbaldrug

Samplesof rosemaryleaves(Rosmarinus officinalisL., Lami-aceae)werecollectedfrom specimenslocated inthe medicinal plantsgardenoftheHospitaldeMedicinaAlternativadaSecretaria EstadualdaSaúdedoEstadodeGoiás(863m,16◦₄₃′_50.3′′_South, 49◦₁₄′_32.9′′_{West/Goiânia,GO,Brazil).Onceidentified,avoucher} specimenwaspreparedanddepositedintheUniversidadeFederal deGoiás(UFG)HerbariumundertheregistrationidentificationUFG –43206.Theleavesweredriedatroomtemperature(25±2◦_C)and groundinaTE-625knifemill(TecnalLtda,Piracicaba,SP,Brazil). Ameanpowdersizeof437.94±7.00␮mwasachieved.Powdered material(herbaldrug)wasstoredshelteredfromlightandmoisture forsubsequentuseintheextractionprocedure.

Feedextract

Thehydroalcoholic feed extract wasobtainedat room tem-perature bypercolation oftheherbaldrug,using assolventan ethanol:watersolution(80:20,v/v)aspreviouslyreported(Couto

etal.,2012a).Theextractwasstoredinclosedflasksprotectedfrom

lightatatemperaturebetween−2and8◦_{Cpriortocharacterization} andfurtheruseinthedryingexperiments.Extractdensityand alco-holiccontentweredeterminedaccordingtothemethodsdescribed

Table1

Codedfactorsandtheirlevelsinthe33_{Box–Behnkenfactorialdesignmatrices.}

Run EF(ml/min) IT(◦_{C) SA(l/min)}

1 −1(2) −1(80) 0(40)

2 +1(6) −1 (80) 0(40)

3 −1(2) +1(140) 0(40)

4 +1(6) +1(140) 0(40)

5 −1(2) 0(110) −1(30)

6 +1(6) 0(110) −1(30)

7 −1(2) 0(110) +1(50)

8 +1(6) 0(110) +1(50)

9 0(4) −1 (80) −1 (30)

10 0(4) +1(140) −1(30)

11 0(4) −1(80) +1(50)

12 0(4) +1(140) +1(50)

13 0(4) 0(110) 0(40)

14 0(4) 0(110) 0(40)

15 0(4) 0(110) 0(40)

−1,0and+1:low,averageandhighcodedvalues,respectively,inthefactorialdesign

matrices.

EF,extractfeedrate;IT,dryingairinlettemperature;SA,spraynozzleairflowrate.

intheBrazilianPharmacopoeia5thedition(FarmacopeiaBrasileira,

2010).Solidcontent(CS,w.b.)ofa1gsamplewasmeasuredwith agravimetricmethodinanMB35halogenlampanalyzer(Ohaus Inc.,PineBrook,NJ,USA).Theextractviscositywasmeasuredat roomtemperatureusing a DV–III+viscometer(Brookfield Engi-neeringLaboratories,Inc.,Middleboro,MA,USA).Allexperiments wereperformedintriplicate.

Designofexperiments

The spray drying experiments followed a 33 _{Box–Behnken} designwiththreefactorsinthree levelsandthreecentralpoint replicatesaspresentedinTable1,whichshowsthedesign matri-ceswiththecodedandnon-codedvaluesofeachfactorstudied.The criticalprocessparametersinvestigated(factors)were:(i)extract feedrate(EF,ml/min);(ii)dryingairinlettemperature(IT,◦_C)and (iii)sprayingairflowrate(SA,l/min).Theirselectionwasbasedon ourpreviousexperiences.

Thedryingprocesseswereperformedatroomtemperaturein alaboratory-scalespraydryermodelMSD1.0(LabmaqdoBrasil Ltda.,RibeirãoPreto,SP,Brazil)withconcurrentflowregime.The liquidextractfeedsystemwascomposedofaperistalticpumpand apneumatic(twofluid)spraynozzlewithaninletorificediameter of1.2mm.Thecylindricaldryingchamberwasmadeof borosili-categlass,160mmindiameterand645mminheight.Thedrying airwassuppliedbyablower(nominalflowrateof1m3_/min)and electricallyheated.Thetemperaturewasmaintainedbyadigital PIDcontroller.

Thefollowingsetofconditionsremainedfixedforall experi-ments:nozzleairpressureof0.4MPa;massofextractportionfeed (WE)of300gandaspirationairflowof100%.Beforetheextractwas fedintothechamber,thedrierwasstartedwithdistilledwater inordertoreachthermalequilibrium.Then,iftheinletand out-lettemperatureswereconstant,thefeedextractwassprayedinto thechamber.Dryingairoutlettemperatures(OT,◦_C)ineachrun wererecordedinordertobetterunderstandtherelationsbetween factorsstudiedandpowderproperties.

TheSDREwereseparatedfromairbyastainlesssteelcyclone andcollectedinaglassflask.TheSDREwereweighed,storedin closedflasksprotectedfromlightandkeptinadesiccatoratroom temperatureforfurthercharacterization.

CharacterizationoftheSDRE

ofpowder(drybasis)collectedintheflask(W)totheWEandits solidcontent(CS,%w.b.)byEq.(1).

PY= W

WE×CS

×100%, (1)

Themoisturecontentof powder(MC,%w.b.) wasmeasured froma0.5gofsampleemployinganMB35halogenlampanalyzer (OhausInc.,PineBrook,NJ,USA).Wateractivity(WA)was mea-suredusingaTesto650thermohygrometer(TestoAG,Lenzkirch, Germany)andahermeticchamber.

Bulk(b,g/cm3)andtapped (t, g/cm3)densities, Hausner’s ratio(HR)andCarr’sorCompressibilityIndex(CI,%)were deter-mined according to the methods described in the American Pharmacopoeia30thedition(USP,2007).Theproductbwas deter-minedbypouring5gofdrypowderintoa25mlgraduatedcylinder andmeasuringitsvolume.Thetwasdeterminedbycontrolled tappingofthecylinderusingasieveshaker(BertelLtda,Caieiras, SP,Brazil)untilaconstantvolumewasachieved.Hence,HRandCI werecalculatedaccordingtotheirdefinitions:

HR(−)= t b

(2)

CI(%)= t−b t

×100 (3)

Anglesofrepose(,◦_{)weredeterminedaccordingtothemethod} describedelsewhere(Araújoetal.,2010).Themeanpowder par-ticlediameter(D50,␮m)wasmeasuredfromthecumulativesize distribution,bysieving20gofpowderwithTylerseriessieves(710, 355,300,250,180,106and53␮m)andanelectro-magnetic sieve-shaker(BertelLtda,Caieiras,SP,Brazil)for20min(Brazil,2010).

RSM

InTable1,thefactorslevelswerecodedtoallowtheAnalysisof

Variance(ANOVA)byRSMfollowingthecodingrulegivenbyEq.

(4):

Coded.value=(uncode.value−0.5×(high.value+low.value)) 0.5×(high.value−low.value)

(4)

ANOVA/RSMontheexperimentaldatawasperformedusingthe Statistica7software(StatsoftInc.,Tulsa,OK,USA).Onlythefactors withp≤0.05wereconsideredsignificant.Theresponsefunction appliedwasaquadraticpolynomialequation,givenbyEq.(5):

Y=ˇ0+ˇ1X1+ˇ2X2+ˇ3X3+ˇ11X₁2+ˇ22X₂2+ˇ33X₃2

+ˇ12X1X2+ˇ13X1X3+ˇ23X2X3 (5)

InEq.(5),Yisthepredictedresponse(dependentvariable);ˇ0 isthemodelconstant;X1,X2andX3areindependentvariables;ˇ1, ˇ2andˇ3arelinearcoefficients;ˇ12,ˇ13andˇ23arecrossproduct coefficients;andˇ11,ˇ22andˇ33arethequadraticcoefficients.

Scanningelectronicmicroscopy(SEM)

Themorphologyofthespray-driedpowderthatpresentedthe mostimprovedsetofphysicomechanical properties was evalu-ated using a scanning electron microscope (JEOL,JSM – 6610) equippedwithenergydispersiveX-rayspectroscopy(Thermo Sci-entificNSSSpectral Imagging).BeforeSEManalysis,thesample wasmountedonametalstubwithdouble-sidedadhesivetapeand recoveredwithagoldlayerof200 ˚Athicknessundervacuumusing ametallisator(DentonVacuum,DeskV).Photomicrographswith

magnificationsof5500×,15,000×and25,000×wererecordedat 5kV.

Resultsanddiscussion

Characterizationofthefeedextract

Theconcentratedhydroalcoholicextractpresentedasolid con-tentof9.7±0.1(%w.b.),densityof0.964±0.002g/cm3_,viscosityof 5.2±0.1mPasandalcoholiccontentof38.2±0.5(%v/v).Generally, inaspraydryingprocessthecharacteristicsofthedriedparticles dependonthecharacteristicsoftheatomizeddroplets.Theyare closelyrelatedtothePY,astothepackagingandflowing charac-teristicsofthedriedpowders.Ithasbeenreportedthatthesolid content,density,viscosityandsurfacetensionofthefeed mate-rialinfluencethedropletsizeandshapeandconsequentlythesize, shapeandthedensityofthedryparticles(Goulaetal.,2004;Goula

andAdamopoulos,2004;Huntington,2004).Accordingly,suchfeed

extractcharacteristicsmusttobeconsideredduringthespray dry-ingexperimentaldesignsince theymaydrivethechoiceofthe dryer’soperationalparameterlevelsinordertoobtainproducts withimprovedphysicomechanical,physicochemicaland pharma-cologicalproperties.

Criticalprocessparametersaffectthephysicomechanical propertiesoftheSDRE

Thespraydryingprocessadequacywasestimatedintermsof severalphysicomechanicalcharacteristicsof thedried products. Productrecovery(yield)mayindicatethesuccessand affordabil-ityoftheprocess.Powdermoisturecontentprovidesinformation asregardstothesolventremovalefficiencyandmayinfluencethe cohesivenessandsizedistributionofthedriedparticles.WA corre-lateswiththeproduct’schemical andmicrobiologicalstabilities andconsequentlyitsshelf life.TheHR, CI,angleof repose,and meanpowderparticlesdiameterwereusedtoindirectlyestimate thecompressibilityandflowability(cohesiveness)characteristics ofthedriedpowders.

Evaluating allthese particulate materialproperties is a pre-requisitefor the rationale of developing intermediate products for furtherSDF manufacturingby applying specificunit opera-tionse.g.,mixing,granulation(powderagglomeration),tableting, coatingandcapsulefilling(Materoetal.,2013).Additional infor-mationconcerningthedryingbehaviorasaresultofthebinomial feedmaterial/drier’ssetupwassuppliedbytheOT.Theresultsof completeproductcharacterizationaredisplayedinTable2,which showsthattheSDREhaddiversecharacteristicswhendifferentsets ofconditionswereappliedinthedryingprocess.

ANOVAand correlationanalyseswereperformedby RSMin ordertoaccurately determinetheeffects of thecriticalprocess parametersstudiedinthedriedproductproperties.Thetableswith completeANOVAsforeachpowderpropertyareoverlooked,buta summaryofthemaineffects(boldvalues)andtheirsignificance arelistedinTable3.Table3alsodisplayscommentsonthetypes oftheeffectsasrepresentedbypositive(directlyproportional)and negative(inverselyproportional)signs.

Table2

Resultsofspray-driedproductscharacteristics.

Run OT(◦_{C) PY(%w/w) MC(%w/w) WA(}

−) HR(−) CI(%) (◦_{) D}

50(␮m)

1 50 52.81 2.85 0.22 1.08 8.16 25 387.07

2 45 63.58 2.74 0.23 1.32 24.53 42.33 330.62

3 90 17.1 1.26 0.24 1.38 27.5 45 157.63

4 79 33.92 1.17 0.26 1.35 26.09 43.87 262.1

5 77 64.98 2.19 0.23 1.27 21.05 39.33 300.65

6 62 74.96 2.61 0.21 1.42 29.54 47 342.43

7 70 26.28 1.38 0.26 1.14 12.5 28.67 163.81

8 60 59.86 2.21 0.22 1.33 25 42.67 348.03

9 49 69.33 3.46 0.23 1.37 26.83 44.03 354.62

10 87 37.32 1.91 0.23 1.17 14.63 33.67 335.23

11 50 51.92 2.51 0.24 1.18 15.09 35 262.7

12 80 21.67 1.49 0.24 1.23 18.6 38.32 133.82

13 64.3 62.63 2.18 0.22 1.28 22.22 39.93 277.79

14 65 63.54 2.11 0.24 1.29 22.92 40.72 240.8

15 66.7 57.53 2.11 0.22 1.29 22.45 40.17 235.61

OT,dryingairoutlettemperature;PY,processyield;MC,moisturecontent;WA,wateractivity;HR,HausnerRatio;CI,Carr’sIndex;,angleofrepose;D50,meanpowder

particlessize.

Table3

SummaryofRSManalysis.

Factor OT(◦

C) PY(%w/w) MC(%w/w) CI(%) (◦

) D50(␮m)

Intercept 65.33c _61.23c _2.13c _22.5c _40.27c _251.4c

EF −5.13c _+8.89b _{0.13 +4.49}a _+4.73a _34.25

EF2 _{0.71 −3.96 −0.19 1.14 0.22 25.05}

IT +17.75c _−15.95c _−0.72c _{1.53 1.81 −55.78}a

IT2 _{−0.04 −15.42}b _{0.06 −2.1 −1.44 7.91}

SA −1.88a _−10.86c _−0.32a _{−2.61 −2.42 −53.07}a

SA2 _{1.21 −0.75 0.15 −1.65 −1.08 12.28}

EF×IT −1.5 1.51 0.01 −4.45 −4.62 40.23

EF×SA 1.25 5.9 0.1 1.00 1.58 35.61

IT×SA 2.0 0.44 0.13 3.93 3.42 −27.37

R2 _{0.99 0.98 0.95 0.78 0.77 0.87}

Boldvaluesrepresenttheeffectsofthefittedpolynomialequationsthatprovedtobesignificantatp<0.05. a_p<0.01.

b_p<0.001.

c _{Positive(+)andnegativesigns(−)indicatedirectlyandinverselyproportionaleffect,respectively;EF,extractfeedrate(ml/min);IT,dryingairinlettemperature(}◦_C);SA, spraynozzleairflowrate(l/min);OT,dryingairoutlettemperature;PY,processyield;MC,moisturecontent;CI,Carr’sIndex;,angleofrepose;D50,meanpowderparticles size;R2_{,determinationcoefficientforthefittedpredictionmodels.}

cannotbesetwithatemperatureregulator.Hence,itresultsfroma combinationofthein-processparametersi.e.,feedextract proper-ties,pumpsetting,IT,atomizerpressure,spraynozzleinletorifice diameter,compressedairflowrateintheatomizer,aswellasthe flowrateofdryingair.ANOVA/RSMprovedthatEF,ITandSAall affectedOTwithpvaluesof0.0007,0.000002and0.041, respec-tively(Table3).IncreasedITresultedinincreasedOTbecauseof increasedsupplyofheatenergy(Table3,Fig.1aand c).Onthe otherhand,increasedEFandSAresultedindecreasedOTdueto increasedsolventevaporationrate(Table3,Fig.1a–c).Thefitted predictionmodel,withR2_{=0.99,isgivenby:}

OT(◦_C)

=65.33−5.13

2

+17.75

30

−1.88

10

(6)

ThePYvaluesrangedfrom17.1to74.96%.Evenwithoutusing anydryingaidonlytwo(15.4%)ofthethirteendifferent experi-mentalconditionsstudiedleadtopowderrecoverieslowerthan 20%.PowderrecoverydependedontheEFparameteratp=0.003

(Table3).ItcanbeobservedintheresponsesurfaceshowninFig.2a

andbthatincreasingEFhadapositivelinearinfluenceonPY,which meansthatthehighertheEF,thehigherthePY.BothITandits squaredterm(IT2_{)exertednegativenonlineareffectsonpowder} recovery(Table3,Fig.2aandc),withpvaluesof0.0002and0.0014, respectively.Conversely,SAexertedanegativelineareffectonPY

withp=0.0012(Table3,Fig.2bandc).Thefittedpredictionmodel, withR2_{=0.98,isgivenby:}

PY(%)=61.23+8.89

−15.95

30

−15.42

IT2−110 30

−10.86

10

(7)

Increasingthefeedrateofthesprayingmaterialanddecreasing bothITandSApresumablyleadtoanincreaseinthedropletsize andviscosityand,subsequently,particlesizeanddensity.Inturn, thelossoffineandlightparticlesintheexhaustedairthatpasses throughthe cyclonedecreases,which raisesthepowder recov-eryalongthedryingprocess.Thesepromisingproductrecoveries achievedmayalsobeattributedtolowadherenceofthepowders onthedryingchamberandcyclonewalls.Avalidexplanationto this isthat theresultantdryingtemperaturestayed lowerthan theextract’sglasstransitiontemperatureinmostsetsofoperating conditions.

Duetotheinherentcomplexityofamultifactorialunit oper-ation such as thespray drying process, there is a broad range ofin-processvariables thatareassumedtoaffectthePYduring thedevelopmentofpharmaceuticalproducts.Besidestheeffects ofcriticalprocessingparameteradjustments(JangamandThorat,

2010; León-Martínezet al.,2010; Coutoetal., 2013), thereare

100

90

80

70

60

50

100

90

80

70

60

50

30 40 50 140

110

80

2 6

80

110

140

4

2

4

6

EF (ml/min) EF (ml/min)

IT (ºC) SA (

I/min)

IT(ºC) _{SA (I/min)}

O T (ºC)

O T (ºC)

100

90

80

70

60

50

90

40

50 O

T (ºC)

a

b

C

Fig.1.Surfaceplotofdryingairoutlettemperatureasafunctionof:(A)extractfeedrateanddryingairinlettemperature;(B)extractfeedrateandspraynozzleairflowrate; (C)dryingairinlettemperatureandspraynozzleairflowrate.

chamberandcycloneoflab-scalespraydryers (Ameriand Maa,

2006).

Thetype,amountandincorporationtimeofdryingaids(Couto

etal.,2012b,2011),andeventheenvironmentalfactors(Ciesielski

andZbicinski,2010)havealsobeenprovedtoexertakeyrolein

thePY.Despitetheeffortsinevaluatingtheimpactofthese param-etersinspraydryingperformance,theliteraturedoesnotpresent aconsensusregardingtheacceptablerangeofpowderrecoveryfor bothbench-topandindustrialspraydryingprocesssofar.

Inourspraydryingruns,theMCrangedfrom1.17%to3.46% (w/w),andforallSDREtheWAvalueswerebelow0.3(Table2). ResidualmoistureandWAvaluesbelow5%(w/w)and0.5, respec-tively,meetthepharmaceuticalrequirementsfordrymaterialsat particulatelevel(USP,2007).Therefore,itispossibletoassertthat alltheproductsshowninTable2presentedsatisfactorylevelsof MCandWA.

AccordingtoANOVA/RSM,noneofthestudiedfactors signifi-cantlyaffectedWA.However,bothITandSAhadlinearnegative influenceonMCwithpvalues of0.0005and 0.02, respectively

(Table3).Inotherwords,anincreaseinbothITandSAresulted

inreducedvaluesofmoisturecontent.Thesurfaceresponseofthe MCasafunctionofITandSAisshowninFig.3.Thefittedprediction model,withR2_{=0.95,isgivenby:}

MC(%)=2.13−0.72

−0.32

10

(8)

Inaspraydryingsystem,removalofmoisturefromthesprayed particles depends upon the temperature of the drying air. The greaterthedrying airtemperature,thegreaterthetemperature

differencebetweentheparticleandthedryingair,themore effi-cientistheheattransferphenomenaand,therefore,thegreaterthe evaporationrate.Synergistically,anincreaseindryingairflowrate helpsinremovalofmoistureatahigherrate.Thishappenssince foragivenamountoffeedextract,atanygiventime,increasingthe SAgeneratessmalleratomizeddropletsintothechamber.Hence, thecontactsurfacebetweentheparticleandtheairwouldrise,a largeramountofhotairwouldbeavailableandlessenergywould berequiredformoistureremoval.

Althoughseemingtobeverycloseinname,residualmoisture andWAaredistinctphysicalparametersthat,butnot necessar-ily,oftencorrelatewitheachother.Whereasthemoisturecontent representsthewholewatercompositionin adriedsystem,WA isameasurementoftheavailabilityoffreewaterthatis respon-siblefor anychemical (hydrationinteractions)and biochemical (enzymatic)reaction,andalsomicrobiologicalgrowth(Queketal., 2007).Hence,thelowertheWA,thelowerthechemicalpotentialof waterandthedrivingforceininteractionsinvolvingwater,which notnecessarilydirectlydependsonthemoisturecontentinthe product.

80

70 60

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10 0 80

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70 60

50

40

30

20

10 0 50

40

30 11

0

14

0 2

4

2

4

6

EF (ml/min) EF (ml/min) 6

IT (ºC) SA (I/min)

SA (I/mi

n)

PY (%

W/W)

80

70

60

50

40

30

20

10 0 90

40

50 140

110

80

IT (ºC)

PY (%

W/W)

PY (%

W/W)

b

c

a

Fig.2. Surfaceplotofprocessyieldasafunctionof:(A)extractfeedrateanddryingairinlettemperature;(B)extractfeedrateandspraynozzleairflowrate;(C)dryingair inlettemperatureandspraynozzleairflowrate.

3.6

3.0

2.4

1.8

1.2

30

40

50 140

110

80

IT(ºC) SA (I/

min) MC (%

W/W)

Fig.3.Surfaceplotofmoisturecontentasafunctionofdryingairinlettemperature andspraynozzleairflowrate.

450

375

300

225

150

90

40

50 ₈₀

110

140

IT (ºC)

SA (I/min) D

50 ( µ_m)

Fig.5. Micrographsofspray-driedrosemaryextractrecordedat5kVwithmagnificationof:(A)5500×,(B)15,000×and(C)25,000×.

bemaintainedinthedriedpowderandconsideredduringthemass lossdetermination.

TheHRvariedbetween1.08and1.42.SinceHRlowerthan1.25 indicatesnon-cohesiveand low internal friction powders(USP, 2007),satisfactoryrheologicalpropertieswhereachievedinfive (38.5%)ofthethirteendifferentexperimentalruns.Noneofthe studiedfactorssignificantlyaffectedtheHRatp<0.05.TheCI var-iedfrom8.16%to29.54%.Forthisqualityindicator,theliterature proposesthefollowingclassificationofflowingproperties: excel-lent(CI<10%),good(11%<CI<15%),fair(16%<CI<20%),passable (21%<CI<25%),poor(26%<CI<31%),verypoor(32%<CI<37%)and very,verypoor(CI>38%)(USP,2007).

Therefore,asobservedfor theHRparameter,fiveconditions studied produced powders with suitable flowability (CI<20%). ANOVAshowedthatonlytheEFinfluencedtheCI,withapvalue of0.047.IncreasingtheEFlinearlyincreasedtheCI.Thismaybe attributedtoanincreaseinMCinthepowderswhenusingupper levelsofEF.Raisingtheresidualmoisturemayincreasethe cohe-siveforcesbetweentheparticlesduetohydrogenand/orVander Waal’sinteractionsandhenceimpairsthepowderflowability.The fittedpredictionmodel,withR2_{=0.78,isgivenby:}

CI(%)=22.5+4.49

(9)

Theanglesofreposerangedfrom25.0◦_upto47.0◦_.Anglesof reposeintherangeof25◦ _to40◦ _{indicatethatthecohesiveness} ofthepowderproductiswithinthesatisfactorylimitaccordingto thepharmaceuticalneeds(USP,2007).Thelessstablethepacked system,thebettertheflowandsmallerthevalue.Moreover, valuesvaryingwithin41◦_and45◦_{arepassableforaparticulatebed,} butaddingalubricantisrequiredtoimproveitsflowability(USP 30thEd.,2007).Thereforeit,ispossibletoassertthatseven(53.9%) ofourdifferentexperimentalrunsprovidedintermediateproducts withvaluableflowability(25◦_<<40◦_{),in whichtwoareinthe} rangeattributedtoexcellent(25◦_<<30◦_{),two othersaregood} (31◦_<<35◦_{)andthreeindicatefairlygood(36}◦_<<40◦_)flowing properties.AsfortheCI,theangleofreposedependsonlyonthe

EFwithapvalueof0.041,inawaythatincreasingEFincreasedthe

(Table3).AdirectcorrelationbetweenCIandcanbeseenand

resultscanbeexplainedalongthesamelineasthatoftheCI.The fittedpredictionmodel,withR2_{=0.77,isgivenby:}

(◦₎

=40.27+4.73

2

(10)

Non-cohesivefree-flowingpowdersarerequiredforSDF man-ufacturing. Together with exerting a key role in the efficiency ofmanuallyorindustriallyfilling ofcapsules,theyare determi-nantforasuccessful“diefilling”(fillingthematrixcompression) andcompressionstagesalongtheindustrialproductionoftablets. Accuratelyaccomplishingtheseunitoperationsarecompulsoryto assurethecontentuniformityanddosageofthedrugcontained ineachformulation.Furthermore,itcaninfluencethefinal hard-nessoftablets,whichmayaffectthephysicalstabilityofitsdosage formduringfurthercoating,packing,transport,storageand hand-lingbytheuser,aswellasintheavailabilityofthedrugtobefurther absorbedandreachitsactionsite.

Itisnoteworthythatevenwithoutusinganydryingaidmost ofourSDREpresentedbetterflowabilityandcompressibilitythan thoseobtainedusingcolloidalsilicondioxide(Tixosil333®)and maltodextrinDE14asdryingcarrier(40:20proportioninrelation tothetotalsolidscontent)inthestudyofsprayandspoutedbed dryingofrosemaryextract(Souzaetal.,2008).

The mean particle diameter (D50) ranged from 133.82 to 387.07␮m.Inadditiontoitsmoisturecontentanddensity,thesize distributionandshapedisplayaremarkableeffectonthe compres-sionandflowofapharmaceuticalpowder,becauseitinfluences theparticlepackinggeometryandtheparticle-to-particle interac-tionlevels(cohesion).Typically,thesmallerandmoresphericalare theparticles,thegreaterwillbethestabilityofthepackedsystem, whichdecreasetheparticlesbedcompressibilityandflowability.

Table4

Correlationmatrixbetweenthequalityindicatorsofthespray-driedproducts.a

Qualityindicator OT(◦_{C) PY(%w/w) MC(%w/w) WA(}

−) HR(−) CI(%) (◦_{) D}

50(␮m)

OT(◦_{C) 1}

−0.45 −0.68 0.13 0.04 0.01 0.01 −0.31

PY(%) −0.45 1 +0.65 −0.45 0.09 0.09 0.08 +0.52

MC(%) −0.68 +0.65 1 −0.39 0.002 0.001 0.001 +0.58

WA(−) 0.13 −0.45 −0.39 1 −0.04 −0.03 −0.03 −0.38

HR(−) 0.04 0.09 0.002 −0.04 1 +0.99 +0.96 0.01

CI(%) 0.01 0.09 0.001 −0.03 +0.99 1 +0.97 0.001

(◦_{) 0.01 0.08 0.001}

−0.03 +0.96 +0.97 1 −0.001

D50(␮m) −0.31 +0.52 +0.58 −0.38 0.01 0.001 −0.001 1

a_{Valuesarethedeterminationcoefficients(R}2_{)fromthecorrelationanalysis;positive(+)andnegativesigns(−)indicatedirectlyandinverselyproportionaleffect,}

respectively;boldvaluesrepresentthemorerelevantcorrelation;OT,dryingairoutlettemperature;PY,processyield;MC,moisturecontent;WA,wateractivity;HR,

HausnerRatio;CI,Carr’sIndex;,angleofrepose;D50,meanpowderparticlessize.

dissolutionrateand,indirectlywiththebioavailability(becauseit

alsodependsondrugpermeability)andclinicalefficacyofadrug

fromapharmaceuticaldosageform.Accordingly,duringthe

ratio-naledevelopmentofSDF,controllingtheparticlesizedistributionis

requiredforreachingequilibriumbetweentheir

physicomechan-icalandbiopharmaceuticalpropertiesinordertoguaranteethe

qualityandefficacyofthemedicine.

ThesurfaceresponseplotofD50asafunctionofITandSAis

pre-sentedinFig.4.Thesurfaceshowsthatthemeanpowderdiameter

increasedwithdecreasingbothITandSA,whichisaresultofthe increaseinthesizeofdropletssprayedintothedryingchamber.The effectofITandSAonthemeanparticlediameterwasconfirmedby ANOVA(Table3),whichdemonstratedpvaluesof0.02and0.024, respectivelyforthesein-processparameters.Thefittedprediction model,withR2_{=0.87,isgivenby:}

D50(␮m)=251.4−55.78

30

−53.07

10

(11)

The results arising from this investigation demonstrate the remarkableimpactoftheprocessparametersonthedrying perfor-manceduringtherosemaryhydroalcoholicextractspraydrying. Thehighdeterminationcoefficientsobservedinthefittedmodels confirmtheirabilitytodescribetheexperimentalresultsand pre-dictresponses(Table3).FromtheresultsdisplayedinTable3,it canbeseenthatITisthemainfactoraffectingthequality indica-torsoftheSDRE,becausetheeffectvalues(coefficients)forthis independentvariablearegreaterthantheothersthathaveshown tobesignificant.

Ourfindingsemphasizethatthecomprehensionandaccurate selectionof thedryingparameterlevels arethekeyto guaran-teesatisfactorylevelsofprocessingperformancewhiledeveloping SDRE. Under theranges studied,the experimentalrun number

1(Tables 1and2),withalowEF(2ml/min),lowIT(80◦_C)and

intermediate SA(40l/min), provided spray-driedpowders with improvedpropertiesfordirectpreparationoftabletsandcapsules filling.

Theseresults,togetherwiththefactthattheprocesscanbe adjustedtoattainoptimizedperformance,suggestthatthespray drying techniquemaybeattractive andsuitable for developing novel intermediate phytopharmaceutical products of rosemary aimingatthemanufactureofSDF.Tothecompletedevelopment andvalidationofapilotspraydryingprocessitwouldbe neces-sarytoextendthevalidityoftheseresultsbyfurtherperforming complementarypreformulationstudies.Ourfurtherinvestigations willbetargetedtoevaluateotherprocessingparameterse.g.,spray nozzleinletorificediameter,nozzleairpressureanddryingairflow rate,andtheirinteractionswiththepropertiesofdryextractsofR. officinalis.

Howdotheprocessadequacyindicatorscorrelatewitheach other?

Tosimplifyinterpretationoftherelationshipsbetweenthe pro-cessqualityindicators,acorrelationsmatrixwaspreparedandis presentedin Table4.Thedeterminationcoefficients(R2_)values thatweobtainedforthemorerelevantinteractionsarehighlighted (bold).

In summary,theOT hadanappreciablenegative correlation withthe residualmoisturecontent (R2_{=0.68). Thismeansthat} increasingtheOTproportionallydecreasestheMC inthe pow-ders.Inturn,theMCpositivelycorrelatedwithboththeprocess yield(R2_{=0.65)andmeanparticlessize(R}2_{=0.58)i.e.,anincrease} inMCleadstoanincreasein PYandD50.Areasonable interac-tionwasevidencedwithintheseparameters,whichalsopositively correlatedwitheachother(R2_=0.52).

Themajorinterdependencies(0.96≤R2_{≤0.99)wereobserved} betweentheHR,CI andtheangle ofrepose. Thesecorrelations wereexpected,becausetheHR,CIandwereallrelatedtopowder mechanics.Noneoftheothermatrixtermssignificantlycorrelated witheachother.Overall,thesecorrelationslendfurthersupportfor thevalidityofthecommentsaddressedforthetrendspresentedin theprevioussection,andreinforcethehypothesisthatthevariables ofthespraydryingprocess mustbeanalyzed altogetherduring thepreformulationstudiesforengineeringproductswithimproved properties.

Morphologicalaspectsofthemostpromisingproduct

TheSEM imagesoftheSDREproduced byexperimentalrun number1,areshown inFig.5a–c,withamagnificationlevelof 5500×,15,000×and25,000×,respectively.BySEMtheSDRE parti-clesweremostlyobservedasspherical(Fig.5aandb)andshoweda smoothsurface,butsomedepressionsandcrackswerealsopresent (Fig.5c).Thesecharacteristicsmeetthoseobservedforspray-dried particlesofotherindustrialcropssuchassoybean(Georgettietal.,

2008),ac¸aí(Tononetal.,2008),chicory(Tonelietal.,2010),Bindens

pilosa(Cortés-RojasandOliveira,2012),clove(Cortés-Rojasetal., 2014)andAchilleamillefolium(Vladicetal.,2016).Asthemajority ofthefeedmaterialshavesingulardryingbehavior,determining theeffectofprocessvariablesuponparticlemorphologyingeneral termshasbeenreportedtobeadifficulttask.Thisissuehasbeen extensivelyrevisedelsewhere(Walton,2000).

Conclusion

theprocessqualityindicatorsistheIT.Thebestsetofconditions toachieveagreaterrecoveryofdrypowderofR.officinalisextract withlowlevelsofmoisturecontentandWA,aswellassuitable flowabilityandcompressibility,requiresalowRF(2ml/min),low IT(80◦_{C)andintermediateSA(40l/min).}

Authors’contributions

LTC(PhDstudent)contributedinobtainingtheherbaldrug, run-ningthelaboratorywork,analysisofthedataanddraftedthepaper. JRPcontributedinplantidentification,herbariumconfectionand tocriticalreadingofthemanuscript.MTFBcontributedtocritical readingofthemanuscript.ECCandROCdesignedthestudy, super-visedthelaboratorywork,analyzedthedataandcontributedto criticalreadingofthemanuscript.Alltheauthorshavereadthe finalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors gratefully acknowledge financial support from CNPqandgrants fromCAPES;HospitaldeMedicinaAlternativa (SecretariaEstadualdaSaúde,Goiânia,GO,Brazil)for supplying herbalmaterial;andtheMulti-userLaboratoryofHighResolution MicroscopyfromInstituteofPhysics–FederalUniversityofthe GoiásStateforthefacilitiesusedintheSEManalysis.Wethank JohnCarpenter(RibeirãoPreto,SP,Brazil)fortheEnglishlanguage revision.

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