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Research ArticleAssessment of Antioxidant Activity ofSpray Dried Extracts of Psidium guajava Leaves by DPPH andChemiluminescence Inhibition in Human Neutrophils

M R V Fernandes A E C S Azzolini M L L Martinez C R F SouzaY M Lucisano-Valim and W P Oliveira

Faculty of Pharmaceutical Sciences of Ribeirao Preto Laboratory of RampD on Pharmaceutical Processes-LAPROFARUniversity of Sao Paulo Avenue do Cafe SN BL Q 14040-903 Ribeirao Preto SP Brazil

Correspondence should be addressed to W P Oliveira wpolivfcfrpuspbr

Received 6 February 2014 Accepted 26 March 2014 Published 13 April 2014

Academic Editor Qaisar Mahmood

Copyright copy 2014 M R V Fernandes et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava Lleaves Different drying carriers namely maltodextrin colloidal silicon dioxide Arabic gum and 120573-cyclodextrin at concentrationsof 40 and 80 relative to solids content were added to drying composition SDE were characterized through determination ofthe total phenolic tannins and flavonoid content Antioxidant potential of the SDE was assessed by two assays cellular testthat measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristateacetate (PMA) and the DPPH radical scavenging (DPPHlowast method) In both assays the antioxidant activity of the SDE occurredin a concentration-dependent manner and showed no toxicity to the cells Using the CLlum method the IC

50ranged from 542

to 650 120583gmL The IC50

of the SDE ranged from 796 to 811 120583gmL using the DPPH∙ method Psidium guajava SDE presentedsignificant antioxidant activity thus they show high potential as an active phytopharmaceutical ingredient Our findings in humanneutrophils are pharmacologically relevant since they indicate that P guajava SDE is a potential antioxidant and anti-inflammatoryagent in human cells

1 Introduction

Currently there is a growing interest in herbal products withantioxidant properties which have the potential to protect thebody against free radical damage and degenerative diseasesSeveral studies have been conducted to find new sources ofcompounds capable to inactivate free radicals generated bymetabolic pathways in human tissues and cells producedmainly by human neutrophils [1] These cells are the mostabundant circulating leukocytes and are the first line ofdefense againstmicroorganisms cells infected by viruses andtumor cells [2]

Standardized plant preparations are generally adminis-tered in the form of liquid extracts viscous products andpowders resulting from the drying and comminuting of plantmaterial or by drying an extract [3] Among these optionsthe tendency in the pharmaceutical industry is to use dry

extracts Products in solid form show significant advantagesover conventional liquid forms including chemical physical-chemical and microbiological stability ease of standard-ization higher concentration of active compounds easeof transportation less space for product storage less riskof microbial contamination and the capacity to transformpowders into different solid pharmaceutical forms Trans-forming a plant extract into dry extract is also widely usedin developing herbal medicinal products [4]

Drying processes generally used in the preparation of dryextracts spray drying spouted bed drying and freeze-dryinginclude Due to the complex composition of herbal extractsthe drying methodmust be selected based on its physical andchemical properties thermal stability and the intended useof same [5]

Several authors have used the spray drying technique forstandardization of active phytopharmaceutical ingredients

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 382891 10 pageshttpdxdoiorg1011552014382891

2 BioMed Research International

and thereby guaranteeing the efficacy quality and safety ofthe product [6] In general the powders obtained by thisdrying method have good reconstitution characteristics andlow water activity and are suitable for transport and storage[7] therefore they are frequently used as intermediate andfinal products particularly in solid dosage forms Variousdrying carriers are used for drying including maltodextrinmodified starch cyclodextrins Arabic gum microcrystallinecellulose and colloidal silicon dioxide in order to improveprocess performance and product quality Drying carrierscan be used individually or as blends and the proportionsestablished are specific to each case considering both physicaland chemical properties of extract to be dried and the dryingtechnology to be used [5]

The aim of this work was to produce and characterizespray dried extracts from Psidium guajava leaves using differ-ent drying carriers and evaluate their antioxidant potentialthrough their inhibitory effect on the generation of reactiveoxygen species (ROS) by stimulated neutrophils and by ROSscavenging properties in a cell-free system using the DPPHradical methodology

Psidium guajava L (Myrtaceae) popularly known asguava is originally from Central and South America andis cultivated in all tropical and subtropical countries [8]Extracts and metabolites of this plant particularly from theleaves and fruits possess useful pharmacological activitiessuch as antispasmodic and antimicrobial properties and havebeen used in the treatment of diarrhea Other reported usesinclude its function as a hypoglycemic antitussive anti-inflammatory and antioxidant agent thereby reinforcing itstraditional use [9] This plant has been used in traditionaland folk medicine in many parts of the world In Brazilit is inserted in the list of plant species selected for studyby the Research Program on Medicinal Plants (Programa dePesquisas de Plantas Medicinais PPPM) and included in theNational Report of Medicinal Plants of Interest to the PublicHealth System [10]

2 Material and Methods

21 Chemicals The chemicals used in this study are asfollows methanol (A108501BJ) aluminum chloride(C103601AG) ethanol (A108401BJ) and hydrochloricacid (A102801BJ Labsynth Diadema SP Brazil) sodiumtungstate-2-hydrate (230) triton X-100 and phosphomo-lybdic acid (443 Vetec Quımica Fina Ltda Duque deCaxias RJ Brazil) gallic acid monohydrate (27645-250G-R)11-diphenyl-2-picrylhydrazyl (DPPH∙ D9132) luminol (5-amino-23-dihydro-14-phthalazinedione A8511-5G) phor-bol-12-myristate-13-acetate (PMA P8139) and Trypan blue(T6146-25G Sigma-Aldrich St Louis MO USA) gelatin(microbiological grade 214320 Difco Laboratories DetroitMN USA) dimethyl sulfoxide (DMSO 3317275) (Merck-Schuchardt Hohenbrunn DE) ldquoKitrdquo to determine lactatedehydrogenase enzyme (LDH Liquiform 86-230mdashLabtestDiagnostica Lagoa Santa MG Brazil) Drying carriers usedare colloidal silicon dioxide (Aerosil 200 Evonik DegussaHanau Germany) maltodextrin (MOR-REX 1910 CornProducts do Brasil) 120573-cyclodextrin (Kleptose Roquette

Table 1 Drying carriers added to feed compositions

Samples code Drying Aids Ratio ()MA 80 Maltodextrin DE10 aerosil 7 1MDEA 80 Maltodextrin DE10 gum Arabic aerosil 5 2 1120573CD 80 120573-Cyclodextrin 8

Lestrem FR) and gumArabic (Encapsia NEXIRA do BrasilSao Paulo SP Brazil)

22 Plant Material Psidium guajava leaves (Pedro Satocultivar) were collected in February 2011 at the farm andindustry Casa da Goiaba in Lavras MG (21∘13101584032910158401015840 S44∘591015840060510158401015840W and altitude of 883m) The plant materialwas identified by ProfessorDrMarcelo Polo and the exsiccatewas deposited in the Herbarium of the Federal University ofAlfenas (UALF-01505)The fresh leaves were dried in an ovenwith circulating air at a temperature of 45∘C until a constantweight was achieved and ground in aMarconi knifemill (MAmod 680) using 20 mesh (833 120583m)

23 Extraction of Bioactive Compounds The dried andground leaves were extracted by dynamic maceration underconstant stirring for 60min using ethanol 70 (vv) at 50∘CThe plantsolvent ratio was set at 1 10 (wv) The extractionsolution obtained was filtered and concentrated in a rotaryevaporator (Fisatom mod 802) at 50∘C under vacuum of650mmHg until solid contents reached 107The extractivesolution and concentrated extract were analyzed for totalphenolics total tannins and total flavonoid contents

24 Spray Drying Drying carriers were added to the con-centrated extract (8 wet base) before spray drying Thedrying carriers used and the codes of the dried extracts (DE)obtained are presented in Table 1

A benchtop spray dryer (model SD 05 Lab-Plant UK)with a drying chamber measuring 215mm in diameter and500mm in height with a concurrent flow regime was usedin the drying runs The equipment has as peripherals asuspension supply system consisting of a peristaltic pump adual fluid atomizer (1mm diameter inlet orifice) and an aircompressor a supply system for the drying gas consisting ofa compressor and an air filter a temperature control systemfor the drying gas and a powder product collection system(cyclone)

The operating parameters were set according to previousstudies carried out by our research group [11ndash13] inlet airdrying temperature 119879gi = 150

∘C drying air flow 119882119892=

60m3h extract feed flow rate 119882susp = 4 gmin atomizingair pressure 119875atm = 15 bar and atomizing air flow rate119882atm = 15 lpm During the drying experiments the roomtemperature and relative humidity () were monitored byusing a digital hygrometer Minipa MTH 1361

Dryer performance was evaluated by determining theproduct recovery (119877) and thermal efficiency of the system (120578)Table 2 presents the equations used in these determinations[3]

BioMed Research International 3

Table 2 Equations used to determine the spray dryer performance

Parameter Equation Unit Equation

Productrecovery (119877) 119877 =

Mc sdot (1 minus 119883119901)

119882119904sdot 119862119904sdot 120579times 100 () gg (1)

Thermalefficiency (120578) 120578 =

(119879gi minus 119879go)

(119879gi minus 119879amb)times 100 (mdash) (2)

Mc mass collected (g) 119883119901 product moisture content (gg) 119882

119904 flow rate

of feed composition (gmin) 119862119904 concentration of total solids (gg) 120579

processing time (min) 119879gi inlet gas temperature (∘C) 119879go outlet gastemperature (∘C) 119879amb environment temperature (∘C)

25 Characterization of the Spray Dried Product The spraydried product was characterized by quantification of theproduct moisture content water activity particle size sol-ubility in water total polyphenols tannins and flavonoidcontents and antioxidant activityThemethods used in thesecharacterizations are presented in the following

251 Product Moisture Content and Water Activity Productmoisture content was determined by Karl Fischerrsquos volu-metric method (Karl Fischer-model 870 KF Titrino plusMetrohm Switzerland)

The water activity of the dried extract was determinedusing the equipment Aqua Lab 4TEV (Decagon DevicesUSA) using the dew-point sensor at 25∘C immediately afterthe drying processThe results are expressed as the average ofthree determinations

252 Solubility in Water The water solubility of the driedproducts was determined according to Cano-Chauca et al[14] with some modifications Samples of 100mg of powderwere exactly weighted and added to 10mL of distilled waterbeing vigorously stirred for 10min in a magnetic stirrerThe samples were centrifuged for 5min at 3000 rpm in alaboratory centrifuge (Fanem Model 206 Sao Paulo Brazil)An aliquot of approximately 2mL of the supernatant wastransferred to a previously weighted Petri dish The solidconcentration was determined by the oven drying methodpreviously described Results were expressed as grams ofsoluble powder per 100 g of water (119899 = 3)

253 Particle Size and Distribution Particle size distribu-tion was determined using the image analysis by opticalmicroscopy Samples of the dried extracts were dispersed onthe surface of microscopy laminas and images of the pow-ders were obtained at 50x magnification using an Olympusmicroscope (model BX60MIV) coupled with a digital camera(model 320 SPOT Insight Diagnostic Instruments) andanalyzed using Image-Pro Plus 70 software The measure-ments obtained were used to determine the mean particlediameter and the cumulative frequency distribution [15]The process was repeated using at least three laminas anda minimum of 1000 particles were measured to ensure thereliability and repeatability of the method [16]

254 Determination of Total Polyphenols Tannins and Fla-vonoids Contents Total polyphenols (TP) and tannins (TT)contents were quantified using the Folin-Denis meth-od which involves the reduction of phosphomolybdic-phosphotungstic acid by phenolics in alkalinemedium result-ing in an intense blue color being the absorvance measuredbyUV-vis spectrophotometry at a wavelength of 750 nm aftera reaction time of 2min [3 17] The results are expressed asgallic acid equivalents per gram of extract or gram of plantmaterial using an analytical curveThe samples were analyzedin triplicate

The total flavonoids content (TF) was quantified usinga spectrophotometric method based on the displacement ofthe wavelength at 425 nm following the addition of 05AlCl3(wv) with a reaction time of 30min Absorbance was

measured at 425 nm using a UVVIS HP 8453 spectropho-tometer (Agilent Technologies Waldbronn Germany) Totalflavonoids content was expressed in milligrams of quercetinper gram of dried product as determined by an analyticalcurve The samples were analyzed in triplicate

255 Evaluation of Antioxidant Activity Evaluation of theantioxidant activity (119860

119860) of original extractive solution

concentrated extract and the spray dried products wasdetermined by two distinct in vitro assays that quanti-fied free radical scavenging activity using the 11-diphenyl-2-picrylhydrazyl radical [18] and the chemiluminescenceamplified by luminol (CLlum) in a cell medium [19]

(1) Antioxidant Activity Using the DPPH∙ Method DPPHradical scavenging activity was measured using the Bloismethod [20] described by Georgetti et al [18] Ten 120583L ofsolutions of the concentrated extract and of the spray driedproducts were assayed at different concentrations (10 2040 50 60 and 80 120583gmL) These were added to a reactionmixture containing 10mL of 01M acetate buffer (pH 55)10mL ethanol and 05mL of DPPH∙ 0250mM ethanolicsolution The change in absorbance was measured after20min at 517 nm at 25∘C The results were expressed as IC

50

(120583gmL) which reflects the substrate concentration requiredto produce a 50 reduction in DPPH∙ and are presentedas the mean and standard deviation obtained in triplicatemeasurements

(2) Assays in Cell Models

Isolation of Human Neutrophils Individuals healthy volun-teers (119899 = 10) of both sexes aged between 18 and 40years old who were not using medications were recruitedin accordance with the protocol approved by the EthicsCommittee on Human Research (CEP) of the Faculty ofPharmaceutical Sciences of Ribeirao Preto (FCFRP) of theUniversity of Sao Paulo (CEPFCFRP number 1082011)The blood was collected from volunteers by venipunctureusing Alsever solution pH 61 (vv) as an anticoagulantNeutrophils were isolated according to the Henson method[21] modified by Lucisano andMantovani [22] Cell viabilitywas determined by the Trypan blue exclusion assay whichyielded cell preparations with viability greater than 87


Table 3 Physicochemical characterization of the extractive solution (ES) and of concentrated extract (CE) of P guajava

Samples 119862119904() 120588 (gcm3) pH (mdash) Alcoholic grade (∘GL) TF (mgmdb) TP ( mmdb) TT ( mmdb) DPPH∙1 (120583gmL)

SE 327 (003) 089 (000) 594 (002) 70 2355 (001) 2629 (003) 1131 (002) 394 (010)CE 107 (012) 093 (000) 490 (007) 0 2252 (001) 2317 (007) 1261 (010) 334 (011)119862119904 concentration of solids 120588 density TF total flavonoids TP total polyphenols TT total tannins db dry base 1antioxidant activity by the DPPH∙ method

expressed as IC50 Values are presented as the mean (standard deviation)

(3) Evaluation of ROS Production by ChemiluminescenceAntioxidant activity in the cellular system evaluatedby chemiluminescence (CL) was performed according toLucisano-Valim et al [19] CLmeasurements were performedin a final volume of 05mL The chemiluminescent probeluminol (028mM) and 5 120583L of different concentrations ofthe extracts evaluated or 20 DMSO (control) was addedto 250 120583L of neutrophil solution (1 times 106 cellsmL) and themixture was incubated for 3min at 37∘C The cells were thenstimulated with 50 120583L of 10minus7M phorbol-12-myristate-13-acetate (PMA)The chemiluminescence produced by the cellswas monitored for 20min in a luminometer (Auto Lumat LB953 EG amp G Berthold Bad Wildbad Baden-WurttembergGermany) in which the light output was recorded as cpm(counted photons per min) The integrated area values(area under the curve) were calculated from curves of CLversus time The percentage inhibition of CL promoted byeach sample was calculated using the following formula100 minus [(ASAC) times 100] where AS is the integrated area ofeach sample evaluated at each concentration and AC is theintegrated area of the control sample IC

50values (substrate

concentration that inhibits 50 of CL) were calculated bynonlinear regression and used to compare the inhibitoryeffects Three independent experiments were performed induplicate

(4) Cytotoxicity Study Evaluation of the toxicity of the con-centrate extract and dried products was performed to checkwhether the modulatory activities obtained on neutrophilswere due to the toxic effects on the cells Analyses wereperformed as described by Kabeya et al [23] Aliquots of theneutrophil suspension (1 times 106 cellsmL) were incubated for20min at 37∘C in Hanks solution (negative control) 02Triton X-100 (positive control) dimethylsulfoxide (DMSO)(control solvent 025 or 1) or the samples under evaluationat concentrations of 5 and 50120583gmL dissolved in DMSOthe final reaction volume was 1mL Following centrifugation(755timesg 10min 4∘C) the cell sediments were resuspended inHanks solution containing 01 gelatin and cell viability wasassessed by the Trypan blue exclusion assay based on countsof 200 cells

The activity of lactate dehydrogenase (LDH) released inthe supernatant was calculated after measuring the decreasein absorbance at 340 nm for 3min at 37∘C using a LiquiformLDHkit Total lysis of the cells (positive control) was achievedwith 02 (vv) Triton X-100 Toxicity was evaluated in threeindependent experiments measured in duplicate

26 Statistical Analysis The statistical analysis of the exper-imental data was carried out with the aid of SAS software

version 90 The Kruskal-Wallis test was also proposedfollowed by Dunnrsquos posttest to reveal statistical differencesbetween groups [24 25] using a significance level of 5Theresults were obtained with the aid of the software R 2140using the PGIRMESS library of functions [26]

Analysis of variance (ANOVA) followed by the Tukeytest were carried out for the experimental data of the assaysinvolving cells with the aid of the GraphPad Prism software(version 30 for Windows GraphPad Software Inc SanDiego CA USA)

3 Results and Discussion

31 Characterization of the Extraction Solution and Con-centrated Extract The extractive solution and concentratedextracts were analyzed for solids (119862

119904) density (120588) pH alcohol

content total flavonoids (TF) total polyphenols (TP) andtotal tannins (TT) contents and antioxidant activity (119860

119860)

using the DPPH∙ method (Table 3)Concentration of the extractive solution was performed

to increase the concentration of solids to approximately107 more suitable for drying [16] Table 3 indicates thatno significant loss of active substances or antioxidant activityoccurred during the concentration step It can be seenthat both extractive solution and concentrated extract showremarkable free scavenging potential presenting an IC

50

lower than 40 120583gmL

32 Spray Drying The properties of the feed compositionweremodified by the addition of different drying carriers andtheir effects on drying performance physical and chemicalproduct properties and antioxidant activity are presented inthe following

33 Drying Performance Table 4 shows the experimentalresults obtained for spray drying performance in terms ofoutlet drying gas temperature (119879go) product recovery (119877)and process thermal efficiency (120578) The experimental resultsof product recovery are consistent with the ones reported byTonon et al [27] which shows 119877 between 34 and 55 whenmaltodextrin was used as drying carrier These values wereinfluenced by changes in the process operating conditionssuch as inlet gas temperature (138ndash202∘C) maltodextrin con-centration (10ndash30) and feed flow rate of feed composition(5ndash25 gmin) According to Goula and Adamopoulos [28]one source of material loss occurs in the cyclone Since thecyclone used had a cut diameter near 39 120583m lower particleswould be carried out with the exhaust gas Oliveira et al [16]evaluated the powder losses in both spray and spouted bed


Table 4 Spray dryer performance

Samples 119879go (∘C) 119879amb (

∘C) RH () 119877 () 120578 ()MA 80 1090 289 421 5138 339MDEA 80 1000 212 466 5963 388120573CD 80 1020 202 581 6148 370119879go outlet gas temperature 119879rm room temperature RH relative humidity 119877 product recovery 120578 thermal efficiency

Table 5 Physical characterization of Psidium guajava spray dried extracts

Samples 119883119901() 119886

119908(mdash) Water solub () 119889

119901

lowast (120583m)MA 80 434 (018) 016 (003) 944 (19) 133 (120)MDEA 80 582 (036) 019 (003) 877 (57) 121 (127)120573CD 80 393 (005) 018 (003) 834 (16) 158 (164)119883119901 product moisture (Karl-Fischer titration) 119886

119908 water activity lowast119889

119901 mean diameter of the particles (deviation of particle size distribution not the mean

particle size) Values are presented as the mean (standard deviation)

drying systems presenting results almost similar to the onesobtained in this work

Thermal efficiency is a parameter commonly used tooptimize the drying process since it is linked with the energyconsumption In this study this parameter was estimated bythe energy balance of the drying gas at the inlet and outlet ofthe dryer It can be seen in the data presented in Table 4 thatthe drying carriers used did not present significant effect onspray drying thermal efficiency Souza et al [3] showed thatthe thermal efficiency tended to be increased conversely withthe flow rate of feed composition with the other variablesmaintained constant

34 Physical Properties of P guajava Spray Dried ExtractsTable 5 presents the physical properties of the spray driedpowders of Psidium guajava engineered with different dryingcarriers

Product moisture content (119883KF) from 393 to 582 wasobtained which can be considered as adequate assumingthe maximum moisture content recommended by the USPharmacopoeia [29] for dry extracts of medicinal plants(le5)The values obtained in thiswork are in agreementwiththose reported by Gallo et al [30] which ranged from 241(plusmn008) to 472 (plusmn028)

The water activity (119886119908) of dried product is an important

parameter which is linkedwithmicrobiological and chemicalproduct stability Since it is a determining factor for valuesof 119886119908lower than 05 and does not permit the growth of

microorganisms [30 31] As can be seen in Table 5 in thiswork the spray dried powders showed 119886

119908values lower

than 05 which favors the physicochemical stability of theproducts

Regarding the water solubility the spray dried productscontaining maltodextrin and aerosil (MA 80) maltodextrinaerosil and gum Arabic (MDEA 80) and 120573-cyclodextrin(120573-CD) were quite soluble and did not show statisticallysignificant differences (119875 gt 005) between themThese resultsare in agreement with the ones reported by Cano-Chaucaet al [14] who used maltodextrin as a drying carrier for

mango juice and obtained solubility values greater than 90This trend was also observed for spray dried preparations ofBidens pilosa [12] Analysis of the results revealed that themost water soluble dried products were obtained from com-positions containing 120573-cyclodextrin and the maltodextrin-aerosil mixture as drying carriers

It can be seen in Table 5 that the spray dried powdersshowed mean particle ranging from 121 to 158120583m thereforewithin the range expected for spray dried powders from5 to 150 120583m [32] The results did not evidence significanteffects of the feed composition on the mean particle sizeSimilar results were reported by Souza et al [3] duringspray drying of Rosmarinus officinalis in presence of colloidalsilicon dioxide and maltodextrin using the same setup andsimilar drying conditions which obtained powder particleswith mean diameters ranging from 9 to 13 120583m In generalthe product diameter is affected by several processing andfeed composition factors such as drying operating condi-tions type and concentration of drying carriers and dryerconfiguration including the powder collecting system

341 Total Polyphenols Tannins and Flavonoids ContentsOne way to standardize herbal products involves controllingthe concentration of market compounds that may or may notbe related to their biological activityThe compounds selectedas markers in this study have been reported as responsiblefor the antioxidant antimicrobial and antidiarrheal activitiesof the plant Psidium guajava [9] Comparisons betweenthe concentrations of chemical markers (total flavonoidstotal polyphenols and total tannins) of the concentratedextract with the spray dried ones yielded similar valueswhile revealing some statistical difference between the groups(Figures 1 2 and 3)

Among the drying compositions the maltodextrin andaerosil (MA 80) mixture and the formulation preparedonly with 120573-cyclodextrin (120573CD 80) were the ones with thelowest reduction of the contents of chemical markers Theseresults are consistent with the recent work of Cortes-Rojasand Oliveira [12] who compared several drying carriers


0EC MA 80 MDEA 80 BCD 80

5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb

Figure 1 Comparisons between the total polyphenols from theconcentrated extract and spray dried extracts using the Kruskal-Wallis test (119875 lt 005) lowastabStatistical difference determined by Dunnrsquosposttest

EC MA 80 MDEA 80 BCD 800

3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b

Figure 2 Comparisons between the total tannins from the concen-trated extract and spray dried extracts using the Kruskal-Wallis test(119875 lt 005) abStatistical difference determined by Dunnrsquos posttest

and observed that the drying composition based on 120573-cyclodextrin showed the highest flavonoid concentration Inthe pharmaceutical field cyclodextrins are useful due to theirinteractionwith drugmolecules to form inclusion complexesDuring the formation of a complex the physicochemical andbiological properties of the pharmaceutical can be alteredincluding improving its solubility and its physical and chem-ical stability [33] Maltodextrin is often used in the spraydrying process because of its low cost high water solubilityand low viscosity in solution [34] Tonon et al [27] studiedthe effect of maltodextrin DE10 and reported that this dryingcarrier retained around 80 of the anthocyanidins presentin acai juice following spray drying which is in agreementwith results observed in this work in which the maltodextrinand aerosil formulation (MA 80) retainedmost of themarkercompounds

Table 6 Antioxidant activity of the dry extracts of P guajava usingthe DPPH∙ method

Samples IC50 (120583gmL) InhibitionMA 80 811 (005) 885 (05)MDEA 80 976 (021)lowast 859 (09)CD 80 796 (018) 861 (08)Conc Ext 334 (011)lowast 873 (06)Quercetin 096 (001) 862 (09)119875 lt 005 lowastStatistical difference determined by Dunnrsquos posttestValues are presented as the mean (standard deviation)


5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab

Figure 3 Comparisons between the total flavonoids from theconcentrated extract and spray dried extracts using the Kruskal-Wallis test (119875 lt 005) abStatistical difference determined by Dunnrsquosposttest

342 Antioxidant Activity of P guajava Extract and SprayDried Products The antioxidant activity of extractive solu-tion concentrated extract and spray dried powders wasdetermined by two different methodologies DPPH∙ methodand evaluation of ROSproduction in a cellmediummeasuredby chemiluminescence assay

(1) Antioxidant Activity by DPPH∙ Method Table 6 showsthe experimental results of antioxidant activity measuredby the DPPH∙ method of the spray dried powders and ofconcentrated extract (control) The quercetin is a naturalantioxidant flavonoid used as a reference standard IC

50

values (concentration required to inhibit 50 of the oxidativereaction) were used to present the experimental data

The percentage of maximum inhibition of the samplesevaluated showed similar values ranging from 859 to 885A slight decrease in antioxidant activity occurred character-ized by higher IC

50values for the spray dried product com-

pared with the concentrated extract Statistically significantdifferences (119875 le 005) were only observed for the samplesof concentrated extract and MDEA 80 Georgetti et al [13]reported maximum inhibition for DPPH∙ of 528 (plusmn097)for spray dried soybean extracts with maltodextrin as dryingcarrier while the concentrated extract showed approximately75 of maximum DPPH∙ inhibition The authors also used


100

80

60

40

20

00

times109

Concentration (120583gmL)C 50 25 5 25 125 HBSS

Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)

Figure 4 Modulatory effect of different concentrations of the extracts studied on ROS production in human neutrophils stimulated withPMA determined by luminol-dependent chemiluminescence assay (a) Conc Ext (concentrated extract) (b) MA 80 (c) MDEA 80 and (d)120573CD 80 Data expressed as the integrated area of CL 0ndash20min The figure is representative of three independent experiments with similarprofiles performed in duplicate

colloidal silicon dioxide and starch as spray drying carriersand verified a correlation between antioxidant activity andthe total polyphenols content of the extract The resultssuggest that chemical composition and antioxidant activityare affected by the drying temperature Thus compoundscapable of providing a synergistic effect for antioxidantactivity can be damaged or eliminated by the drying processcontributing to a reduction in the antioxidant effectiveness ofthe dried product [35]

(2) Evaluation of ROS Production by ChemiluminescenceAssays involving CL have been widely used for monitor-ing ROS generated by enzymes cells and tissues due toits sensitivity and reproducibility [36] In this study totalROS production by neutrophils stimulated with phorbol-12-myristate-13-acetate (PMA) was measured by chemilu-minescence assay with a luminol probe (CLlum) PMA is asoluble stimulus synthetic and known for its tumor promot-ing action Neutrophils activated by this stimulus produceluminol-dependent chemiluminescence profiles immediately

following the addition of the stimulus that persistent for over20min [36] as confirmed in this study In all the assaysthe concentrated extract and spray dried products weresolubilized in 25 DMSO obtaining a final solvent concen-tration of 025 Laboratory studies have shown this DMSOconcentration does not interfere with CLlum production(data not shown) The concentrated extract and spray driedproduct showed a concentration-dependent inhibitory effectof CLlum on PMA stimulated neutrophils (Figure 4)

A comparative study of the inhibitory effect of CL onthe concentrated extract and spray dried products was con-ducted based on the calculation of the IC

50values (Table 7)

The results obtained showed that the concentrated extractand spray dried product evaluated were able to modulateROS production by neutrophils activated with PMA in aconcentration-dependent manner

Several studies using different plant extracts have report-ed a modulatory effect on stimulated neutrophil samplesIn the work of Paula et al [36] a modulatory effect of anextract of the fruit pulp of Tamarindus indica was observed


Table 7 Inhibitory effect of the concentrated extract and spray dried extracts ofPsidium guajava onCLlum produced by neutrophils stimulatedwith PMA

Conc (120583gmL)a Percentage inhibition ()b

Conc Ext MA 80 MDEA 80 120573CD 80125 2240 (1077) 2646 (156) 2636 (473) 2798 (492)25 3523 (669) 320 (679) 3754 (789) 346 (860)50 4825 (522) 5152 (868) 5072 (963) 4762 (911)250 7437 (402) 774 (855) 7727 (840) 7729 (100)500 8639 (302) 8756 (599) 8876 (556) 886 (795)IC50 603 (177) 554 (224) 542 (285) 650 (360)n 3 3 3 3Quercetin IC50 167 (157)aFinal concentration of the concentrated extract and spray dried product evaluated bpercentage inhibition of CLlum produced by neutrophils stimulated withPMAValues are presented as the mean (standard deviation)IC50 sample concentration that promotes 50 inhibition of chemiluminescence119899 number of independent experiments performed in duplicateIC50 119875 = 008 (no statistically significant difference was determined by Dunnrsquos posttest)

Table 8 Analysis of the cytotoxicity of concentrated extract and spray dried extracts of Psidium guajava on stimulated neutrophils

Samples Viable cells ()c Trypan blue LDHd activity (UmL) Liberated LDH ( positive control)e

Positive controla mdash 01516 (000) 100Negative controlb 965 (071) 00126 (001) 840 (585)DMSO control (02) 970 (071) 00166 (0002) 1110 (155)Conc Ext 9400 (283) 00137 (002) 758 (876)MA 80 942 (318) 00158 (001) 1053 (368)MDEA 80 967 (106) 00153 (0001) 1020 (047)120573CD 80 932 (106) 00166 (000) 1107 (019)Values are presented as the mean (standard deviation) a1 times 106 neutrophilsmL lysed with 02 Triton X-100 b1 times 106 neutrophilsmL incubated with HanksDMSO solvent control c of viable cells determined by Trypan blue exclusion assay based on counts of 200 cells dLDH enzyme activity evaluated by theLiquiform (Labtest Diagnostica) LDH kit epercentage of LDH liberated in the supernatant compared with neutrophils completely lysed with Triton X-100

on neutrophils stimulated with PMA and this effect wasalso concentration dependent The authors reported thatthe extract was more effective as an inhibitor of neutrophiloxidative metabolism induced by PMA compared with otherstimuli Studying the antioxidant activity of an extract ofCalendula officinalis Braga et al [1] also evaluated theinhibitory effects in human neutrophils CLlum activated byPMA They verified that the extract exerted its anti-ROSactivity in a concentration-dependent manner and that itpresented a significant effect even at low concentrations(160 120583gmL)

The results obtained here showed that both the concen-trated extract and the spray dried products promoted aninhibitory effect on luminol-dependent chemiluminescenceThis observed effect suggests a ROS scavenger effect whichwas confirmed by in vitro evaluation of free radical scaveng-ing activity (DPPH∙) Since this study was conducted usingcrude extract with no investigation of isolated compounds itwas not possible to elucidate which of themetabolites presentin the extract are responsible for the effects observed Despitethis limitation studies with crude extracts are importantbecause the biological activities of a natural product are notusually due to a single compound but to the synergismbetween various substances [37] No statistically significantdifferences were verified between the concentrated extract

and spray dried products which indicates that neither thedrying process nor the drying carrier significantly interferedwith the inhibitory effect presented by the samples studied

(3) Evaluation of the Cytotoxicity of Psidium guajava Thecytotoxicity of the concentrated extract and the spray driedproducts was verified by two assays the Trypan blue exclu-sion assay anddetermination of lactate dehydrogenase (LDH)enzyme activity The aim was to evaluate whether the modu-lator effect presented by the samples on the neutrophils wasassociated with diminished neutrophil function determinedby decreasing CLlum or whether it was related to a toxiceffect on the cells of these samples Initially the concentratedextract was assessed for toxicity against neutrophils at con-centrations of 5120583gmL (data not shown) and 50120583gmL Laterthe same test was performedwith the spray dried product andconcentrated extract at a concentration of 50 120583gmL In theTrypan blue exclusion assay in the presence of the extractsthe cells obtained viabilities greater than 93 and usingthe kit to determine LDH enzyme activity the percentagerelease of the enzyme relative to the positive control (TritonX-100) was low compared with the negative control valuesThese results suggest that all the extracts evaluated showedno cytotoxic activity on neutrophils using these assays(Table 8)


4 Conclusion

Standardized dried extracts from P guajava leaves containingvarious drying carriers were successfully engineered by spraydrying Product recoveries of around 57 were verified dur-ing the drying runs however the operating parameters werefixed in order to permit quantifiable comparisons betweenthe different feed compositions No significant changes inthe concentrations of marker compounds (total polyphenolstannins and flavonoids) were observed

Product properties in terms of moisture content andwater activity were inside the recommended ranges in orderto minimize product spoilage due to chemical degradingreactions or microbial growth The product also presentedhigh water solubility (from 83 to 94) and an increase in sol-ubility was observed when maltodextrin was used as dryingcarrier The concentrated extract and the spray dried extractspresented pronounced free radical scavenging activity in boththe DPPH method and in assay involving PMA-stimulatedhuman neutrophilsThese findings provide information con-cerning the potential in vivomodulatory effect of the producton the effect or functions of human neutrophils

In conclusion analysis of the results obtained confirmedthe effective antioxidant activity of spray dried Psidium gua-java extracts indicating his potential to be used as an activephytopharmaceutical ingredient for food and pharmaceuticaluses

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful to FAFESP for the financial aid(Process nos 200803588-0 and 201110333-1) and to CNPqfor providing a PhD scholarship for M R V Fernandes(Process no 1409512010-5)

References

[1] P C Braga M Dal Sasso M Culici et al ldquoAntioxidant activityof Calendula officinalis extract inhibitory effects on chemilu-minescence of human neutrophil bursts and electron paramag-netic resonance spectroscopyrdquo Pharmacology vol 83 no 6 pp348ndash355 2009

[2] R Selvatici S Falzarano A Mollica and S Spisani ldquoSignaltransduction pathways triggered by selective formylpeptideanalogues in human neutrophilsrdquo European Journal of Pharma-cology vol 534 no 1ndash3 pp 1ndash11 2006

[3] C R F Souza I A Schiavetto F C F Thomazini and W POliveira ldquoProcessing of Rosmarinus officinalis Linne extract onspray and spouted bed dryersrdquo Brazilian Journal of ChemicalEngineering vol 25 no 1 pp 59ndash69 2008

[4] W P Oliveira C R F Souza L E Kurozawa and K J ParkldquoSpray drying of food and herbal productsrdquo in Spray DryingTechnology M W Woo A S Mujundar and W R W DaudEds Chapter 5 pp 113ndash156 2010

[5] W P Oliveira L A P Freitas and J T Freire ldquoDrying of phar-maceutical productsrdquo in Transport Phenomena in ParticulateSystems J T Freire A M Silveira and M C Ferreira Eds pp48ndash171 Bentham Science Oak Park Ill USA 2012

[6] R F Bott T P Labuza and W P Oliveira ldquoStability testingof spray- and spouted bed-dried extracts of Passiflora alatardquoDrying Technology vol 28 no 11 pp 1255ndash1265 2010

[7] T C Kha M H Nguyen and P D Roach ldquoEffects of spraydrying conditions on the physicochemical and antioxidantproperties of the Gac (Momordica cochinchinensis) fruit arilpowderrdquo Journal of Food Engineering vol 98 no 3 pp 385ndash3922010

[8] PM Alves P H A S Leite J V Pereira et al ldquoAntifungal activ-ity of the extract of Psidium guajava Linn (ldquogoiabeirardquo) uponleavens of Candida of the oral cavity an in vitro evaluationrdquoBrazilian Journal of Pharmacognosy vol 16 pp 192ndash196 2006

[9] R M P Gutierrez S Mitchell and R V Solis ldquoPsidium guajavaa review of its traditional uses phytochemistry and pharmacol-ogyrdquo Journal of Ethnopharmacology vol 117 no 1 pp 1ndash27 2008

[10] BRASIL ldquoRelacao Nacional de Plantas Medicinais de Interesseao SUS (RENISUS)rdquo 2009 httpportalsaudegovbrportalarquivospdfRENISUSpdf

[11] C R F Souza andW POliveira ldquoPowder properties and systembehavior during spray drying of Bauhinia forficata link extractrdquoDrying Technology vol 24 no 6 pp 735ndash749 2006

[12] D F Cortes-Rojas andW P Oliveira ldquoPhysicochemical proper-ties of phytopharmaceutical preparations as affected by dryingmethods and carriersrdquo Drying Technology vol 30 pp 921ndash9342012

[13] S R Georgetti R Casagrande C R F SouzaW P Oliveira andM J V Fonseca ldquoSpray drying of the soybean extract effectson chemical properties and antioxidant activityrdquo LWTmdashFoodScience and Technology vol 41 no 8 pp 1521ndash1527 2008

[14] M Cano-Chauca P C Stringheta A M Ramos and J Cal-Vidal ldquoEffect of the carriers on the microstructure of mangopowder obtained by spray drying and its functional characteri-zationrdquo Innovative Food Science and Emerging Technologies vol6 no 4 pp 420ndash428 2005

[15] C R F Souza and W P Oliveira ldquoSpouted bed drying ofBauhinia forficata link extract the effects of feed atomizer posi-tion and operating conditions on equipment performance andproduct propertiesrdquo Brazilian Journal of Chemical Engineeringvol 22 no 2 pp 239ndash247 2005

[16] W P Oliveira R F Bott and C R F Souza ldquoManufacture ofstandardized dried extracts from medicinal Brazilian plantsrdquoDrying Technology vol 24 no 4 pp 523ndash533 2006

[17] O Folin andWDenis ldquoOnphosphotungstic-phosphomolybdiccompounds as color regentsrdquo Journal of Biological Chemistryvol 12 pp 239ndash243 1912

[18] S R Georgetti R Casagrande F T Moura-de-Carvalho Vicen-tini W A Verri Jr and M J V Fonseca ldquoEvaluation of theantioxidant activity of soybean extract by different in vitromethods and investigation of this activity after its incorporationin topical formulationsrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 64 no 1 pp 99ndash106 2006

[19] Y M Lucisano-Valim L M Kabeya A Kanashiro et al ldquoAsimple method to study the activity of natural compoundson the chemiluminescence of neutrophils upon stimulation byimmune complexesrdquo Journal of Pharmacological and Toxicolog-ical Methods vol 47 no 1 pp 53ndash58 2002

[20] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958


[21] P M Henson ldquoThe immunologic release of constituents fromneutrophil leukocytes I The role of antibody and complementon nonphagocytosable surfaces or phagocytosable particlesrdquoJournal of Immunology vol 107 no 6 pp 1535ndash1546 1971

[22] Y M Lucisano and B Mantovani ldquoLysosomal enzyme releasefrom polymorphonuclear leukocytes induced by immune com-plexes of IgM and of IgGrdquo Journal of Immunology vol 132 no4 pp 2015ndash2020 1984

[23] L M Kabeya C H T P da Silva A Kanashiro et al ldquoInhi-bition of immune complex-mediated neutrophil oxidativemetabolism a pharmacophore model for 3-phenylcoumarinderivatives using GRIND-based 3D-QSAR and 2D-QSAR pro-ceduresrdquo European Journal of Medicinal Chemistry vol 43 no5 pp 996ndash1007 2008

[24] M Pagano and K Gauvreau Princıpios de BioestatısticaThom-son Sao Paulo Brazil 2004

[25] M Hollander and D A Wolfe Nonparametrics Statistics Meth-ods Wiley series in probability and statistics Applied probabil-ity and statistic section JohnWileyamp SonsNewYorkNYUSA2nd edition 1999

[26] R Development Core Team R Foundation For Statistical Com-puting Vienna Austria 2011

[27] R V Tonon C Brabet and M D Hubinger ldquoInfluence ofprocess conditions on the physicochemical properties of acai(Euterpe oleraceae Mart) powder produced by spray dryingrdquoJournal of Food Engineering vol 88 no 3 pp 411ndash418 2008

[28] A M Goula and K G Adamopoulos ldquoSpray drying of tomatopulp effect of feed concentrationrdquo Drying Technology vol 22no 10 pp 2309ndash2330 2004

[29] USP XXX United States Pharmacopeia United States Pharma-copeial Convention Rockville Md USA 30th edition 2007

[30] L Gallo J M Llabot D Allemandi V Bucala and J PinaldquoInfluence of spray-drying operating conditions on Rhamnuspurshiana (Cascara sagrada) extract powder physical proper-tiesrdquo Powder Technology vol 208 no 1 pp 205ndash214 2011

[31] R R Araujo C C C Teixeira and L A P Freitas ldquoThe prepa-ration of ternary solid dispersions of an herbal drug via spraydrying of liquid feedrdquoDrying Technology vol 28 no 3 pp 412ndash421 2010

[32] C S Favaro-Trindade S C Pinho and G A Rocha ldquoReviewmicroencapsulation of food ingredientsrdquo Brazilian Journal ofFood Technology vol 11 pp 103ndash112 2008

[33] V J Stella andQHe ldquoCyclodextrinsrdquoToxicologic Pathology vol36 no 1 pp 30ndash42 2008

[34] C Turchiuli M Fuchs M Bohin et al ldquoOil encapsulation byspray drying and fluidised bed agglomerationrdquo Innovative FoodScience and Emerging Technologies vol 6 no 1 pp 29ndash35 2005

[35] M Cousins J Adelberg F Chen and J Rieck ldquoAntioxidantcapacity of fresh and dried rhizomes from four clones ofturmeric (Curcuma longa L) grown in vitrordquo Industrial Cropsand Products vol 25 no 2 pp 129ndash135 2007

[36] F S Paula L M Kabeya A Kanashiro et al ldquoModulation ofhuman neutrophil oxidative metabolism and degranulation byextract of Tamarindus indica L fruit pulprdquo Food and ChemicalToxicology vol 47 no 1 pp 163ndash170 2009

[37] R Verpoorte Y H Choi and H K Kim ldquoEthnopharmacologyand systems biology a perfect holistic matchrdquo Journal ofEthnopharmacology vol 100 no 1-2 pp 53ndash56 2005

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


and thereby guaranteeing the efficacy quality and safety ofthe product [6] In general the powders obtained by thisdrying method have good reconstitution characteristics andlow water activity and are suitable for transport and storage[7] therefore they are frequently used as intermediate andfinal products particularly in solid dosage forms Variousdrying carriers are used for drying including maltodextrinmodified starch cyclodextrins Arabic gum microcrystallinecellulose and colloidal silicon dioxide in order to improveprocess performance and product quality Drying carrierscan be used individually or as blends and the proportionsestablished are specific to each case considering both physicaland chemical properties of extract to be dried and the dryingtechnology to be used [5]

The aim of this work was to produce and characterizespray dried extracts from Psidium guajava leaves using differ-ent drying carriers and evaluate their antioxidant potentialthrough their inhibitory effect on the generation of reactiveoxygen species (ROS) by stimulated neutrophils and by ROSscavenging properties in a cell-free system using the DPPHradical methodology

Psidium guajava L (Myrtaceae) popularly known asguava is originally from Central and South America andis cultivated in all tropical and subtropical countries [8]Extracts and metabolites of this plant particularly from theleaves and fruits possess useful pharmacological activitiessuch as antispasmodic and antimicrobial properties and havebeen used in the treatment of diarrhea Other reported usesinclude its function as a hypoglycemic antitussive anti-inflammatory and antioxidant agent thereby reinforcing itstraditional use [9] This plant has been used in traditionaland folk medicine in many parts of the world In Brazilit is inserted in the list of plant species selected for studyby the Research Program on Medicinal Plants (Programa dePesquisas de Plantas Medicinais PPPM) and included in theNational Report of Medicinal Plants of Interest to the PublicHealth System [10]

2 Material and Methods

21 Chemicals The chemicals used in this study are asfollows methanol (A108501BJ) aluminum chloride(C103601AG) ethanol (A108401BJ) and hydrochloricacid (A102801BJ Labsynth Diadema SP Brazil) sodiumtungstate-2-hydrate (230) triton X-100 and phosphomo-lybdic acid (443 Vetec Quımica Fina Ltda Duque deCaxias RJ Brazil) gallic acid monohydrate (27645-250G-R)11-diphenyl-2-picrylhydrazyl (DPPH∙ D9132) luminol (5-amino-23-dihydro-14-phthalazinedione A8511-5G) phor-bol-12-myristate-13-acetate (PMA P8139) and Trypan blue(T6146-25G Sigma-Aldrich St Louis MO USA) gelatin(microbiological grade 214320 Difco Laboratories DetroitMN USA) dimethyl sulfoxide (DMSO 3317275) (Merck-Schuchardt Hohenbrunn DE) ldquoKitrdquo to determine lactatedehydrogenase enzyme (LDH Liquiform 86-230mdashLabtestDiagnostica Lagoa Santa MG Brazil) Drying carriers usedare colloidal silicon dioxide (Aerosil 200 Evonik DegussaHanau Germany) maltodextrin (MOR-REX 1910 CornProducts do Brasil) 120573-cyclodextrin (Kleptose Roquette

Table 1 Drying carriers added to feed compositions

Samples code Drying Aids Ratio ()MA 80 Maltodextrin DE10 aerosil 7 1MDEA 80 Maltodextrin DE10 gum Arabic aerosil 5 2 1120573CD 80 120573-Cyclodextrin 8

Lestrem FR) and gumArabic (Encapsia NEXIRA do BrasilSao Paulo SP Brazil)

22 Plant Material Psidium guajava leaves (Pedro Satocultivar) were collected in February 2011 at the farm andindustry Casa da Goiaba in Lavras MG (21∘13101584032910158401015840 S44∘591015840060510158401015840W and altitude of 883m) The plant materialwas identified by ProfessorDrMarcelo Polo and the exsiccatewas deposited in the Herbarium of the Federal University ofAlfenas (UALF-01505)The fresh leaves were dried in an ovenwith circulating air at a temperature of 45∘C until a constantweight was achieved and ground in aMarconi knifemill (MAmod 680) using 20 mesh (833 120583m)

23 Extraction of Bioactive Compounds The dried andground leaves were extracted by dynamic maceration underconstant stirring for 60min using ethanol 70 (vv) at 50∘CThe plantsolvent ratio was set at 1 10 (wv) The extractionsolution obtained was filtered and concentrated in a rotaryevaporator (Fisatom mod 802) at 50∘C under vacuum of650mmHg until solid contents reached 107The extractivesolution and concentrated extract were analyzed for totalphenolics total tannins and total flavonoid contents

24 Spray Drying Drying carriers were added to the con-centrated extract (8 wet base) before spray drying Thedrying carriers used and the codes of the dried extracts (DE)obtained are presented in Table 1

A benchtop spray dryer (model SD 05 Lab-Plant UK)with a drying chamber measuring 215mm in diameter and500mm in height with a concurrent flow regime was usedin the drying runs The equipment has as peripherals asuspension supply system consisting of a peristaltic pump adual fluid atomizer (1mm diameter inlet orifice) and an aircompressor a supply system for the drying gas consisting ofa compressor and an air filter a temperature control systemfor the drying gas and a powder product collection system(cyclone)

The operating parameters were set according to previousstudies carried out by our research group [11ndash13] inlet airdrying temperature 119879gi = 150

∘C drying air flow 119882119892=

60m3h extract feed flow rate 119882susp = 4 gmin atomizingair pressure 119875atm = 15 bar and atomizing air flow rate119882atm = 15 lpm During the drying experiments the roomtemperature and relative humidity () were monitored byusing a digital hygrometer Minipa MTH 1361

Dryer performance was evaluated by determining theproduct recovery (119877) and thermal efficiency of the system (120578)Table 2 presents the equations used in these determinations[3]





Mc sdot (1 minus 119883119901)



(119879gi minus 119879go)



119904 flow rate















50










50values (substrate











119860)



50






Samples 119879go (∘C) 119879amb (



Samples 119883119901() 119886


119901












119908values lower









5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb



3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b






5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab




50






100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Mc sdot (1 minus 119883119901)



(119879gi minus 119879go)



119904 flow rate















50










50values (substrate











119860)



50






Samples 119879go (∘C) 119879amb (



Samples 119883119901() 119886


119901












119908values lower









5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb



3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b






5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab




50






100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







50values (substrate











119860)



50






Samples 119879go (∘C) 119879amb (



Samples 119883119901() 119886


119901












119908values lower









5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb



3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b






5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab




50






100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Samples 119879go (∘C) 119879amb (



Samples 119883119901() 119886


119901












119908values lower









5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb



3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b






5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab




50






100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



5

10

15

20

25

Tota

l pol

yphe

nols

( m

m)

a lowast

lowastb



3

6

9

12

15

18

Tota

l tan

nins

( m

m) a

b






5

10

15

20

25

Tota

l flav

onoi

ds (q

uerc

etin

mg

g)

ab




50






100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(a)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(b)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(c)

100

80

60

40

20

00

times109


Inte

grat

ed ar

ea o

f QL

(0ndash20

min

)

(d)






50values (Table 7)















4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


4 Conclusion






Acknowledgments


References











































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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