Mart. & Zucc. (Malvaceae) in Candida Spp.downloads.hindawi.com/journals/bmri/2015/807670.pdf · Malvaceae are a family consisting of herbs, subshrubs, shrubs, lianas, and small and

Research ArticlePhytochemical Analysis and Modulation of AntibioticActivity by Luehea paniculata Mart amp Zucc (Malvaceae) inMultiresistant Clinical Isolates of Candida Spp

Joatildeo T Calixto Juacutenior123 Selene M Morais12 Cleacutecio G Martins1 Larissa G Vieira1

Maria Flaviana B Morais-Braga4 Joara N P Carneiro4 Antonio J P Machado4

Irwin R A Menezes5 Saulo R Tintino4 and Henrique D M Coutinho4

1Laboratorio de Produtos Naturais Universidade Estadual do Ceara (UECE) 60740-000 Fortaleza CE Brazil2Rede Nordeste de Biotecnologia (RENORBIO) 60740-000 Fortaleza CE Brazil3Faculdade de Juazeiro do Nort (FJN) 63010-210 Juazeiro do Norte CE Brazil4Laboratorio de Microbiologia e Biologia Molecular Universidade Regional do Cariri (URCA) 63105-000 Crato CE Brazil5Laboratorio de Farmacologia e Quımica Molecular Universidade Regional do Cariri (URCA) 63105-000 Crato CE Brazil

Correspondence should be addressed to Henrique D M Coutinho hdmcoutinhogmailcom

Received 31 August 2014 Revised 16 January 2015 Accepted 16 January 2015

Academic Editor Thean-Hock Tang

Copyright copy 2015 Joao T Calixto Junior 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

The high incidence of fungal infections has led to the continuous search for new drugs Extracts of Luehea paniculata a tree ofmultiple medicinal uses were evaluated for anti-Candida activity as well as its modulator potential of the Fluconazole antibioticChemical prospecting of ethanol extracts of leaf and bark was carried out the quantification of total phenols and flavonoidscharacterized by the HPLC-DAD technique The rosmarinic acid and the vitexin flavonoid were observed as major constituents inELELP and ESWELP respectively Antioxidant activity was also evaluated by the method of scavenging the free radical DPPH andquercetinwas used as standard obtaining IC

50values 0341 (mgmL) for ELELP and 0235 (mgmL) for ESWELPThemicrodilution

assay was performed for antifungal activity against strains of Candida albicans C krusei and C tropicalis and showed minimuminhibitory concentrations values ge1024 120583gmL In the modulator action of extracts on Fluconazole against multiresistant clinicalisolates of Candida (subinhibitory concentration minimum of 128120583gmL) a significant synergism was observed indicating thatthe extracts potentiated the antifungal effect against C tropicalis where antioxidant flavonoids could be responsibleThis is the firstreport about modifying activity of the antibiotic action of a species of the genus Luehea

1 Introduction

Northeastern Brazil presents a huge variety of plants manyof which display biological activities [1] Essential oils fromCaatinga biome plants as Lippia sidoides and Croton sppspecies have shown antifungal activity against Candida sppandMicrosporum canis [2 3]

Opportunistic fungal infections have increased in recentyears mostly occurring in hospital settings and in immuno-compromised individuals Yeasts of the genus Candidacaused themost common superficial and invasive infections[4]These infections comprise a big problem nowadays since

they are increasingly common in immunosuppressive dis-eases in which these fungi turn into opportunistic pathogenspotentially responsible for highmortality [5 6]Themortalityrate even after drug treatment is high ranging from40 to 60[7]The incidence of fungal infections has led to a continuoussearch for new drugs which besides having a biocide effectwould entail no side effects This is configured as a majorchallenge since fungi are like humans eukaryotes and as aresult of the similarity between the cells many commercialdrugs have caused cytotoxicity in the liver and kidneys of thehost [8]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 807670 10 pageshttpdxdoiorg1011552015807670

2 BioMed Research International

According to Sorg [9] diseases such as cancer emphy-sema cirrhosis arthritis and inflammation in addition tothe aging process itself are related to free radicals ElejaldeGuerra [10] explained that antioxidant therapy and dietenriched with antioxidants appear to prevent or at leastmitigate the organic deterioration by oxidative stress It canbe observed that in recent decades there has been growthof scientific research in this area involving the effect ofcrude extracts purified fractions or isolated componentshighlighting phenolic compounds that in many studies haveshown this activity [1 11] Therefore antioxidant activitywhich protects the live organism against deterioration couldplay an important role for an antifungal agent

Malvaceae are a family consisting of herbs subshrubsshrubs lianas and small and large trees with about 250genera and 4200 species and in Brazil there are about 80genera and 400 species [12] According to the List of Speciesof Flora of Brazil 69 genera and 754 species are pointed out[13] distributed in 30 genera and 393 taxa of the Malvaceaesubfamily [14] The genus Luehea is one of 252 genera of theMalvaceae familyThe leaves of Luehea aremarketed as herbalagainst dysentery leucorrhoea rheumatism gonorrhea andtumors the infusion of the flowers is used against bronchitisand the root is depurative [15] Braga [16] pointed out theastringency of the barks of L speciosa Willd (botanicalsynonym of L divaricata) and also recorded the occurrenceof the species L candicansMart et Zucc (Luehea uniflora StHil) and L paniculata Mart presenting the same use as thefirst named

In this context the aim of this work was to investigatethe antifungal effect of ethanol extract from different partsof Luehea paniculata Mart amp Zucc a medicinal tree ofmultiple effects individually and in combination with a com-mercial drug against clinical isolates of multidrug-resistantstrains of Candida An evaluation of free radical-scavengingactivity and phytochemical analysis of the extracts werealso performed in order to relate the existence of phenoliccompounds with activities determined in this study

2 Material and Methods

21 Place of Collection of the Plant Material and ObtainingExtracts Leaves and sapwood of three specimens of Lpaniculatawere collected on November 19 2012 (dry season)around 1730 hours from the Cerrado located in the center ofCaatinga area Lavras daMangabeira city centralMesoregionin South of Ceara Northeastern Brazil

The area is characterized as a vegetation relic [17]presenting itself in deep soil on a tabular relief on longdissected surfaces with penetration of the savannah floraThepatch of savannah in question is located in hills of 280 to600m in height at the following coordinates 06∘721015840243210158401015840Sand 38∘971015840739610158401015840WThe botanical material was collected anddeposited in the Prisco Bezerra Herbarium of the FederalUniversity of Ceara under number 54641

The plant material was dried at 60∘C weighed (Table 1)and subjected to maceration with ethanol PA for seven daysAfter this period filtration and concentration of the extract

Table 1 Dry weight and yield of the crude ethanol extracts of Lpaniculata

Naturalproduct Solvent Dry matter Crude extract Extract yields

ELELP Ethanol 595 g 61649 g 1036ESWELP Ethanol 195 g 36621 g 1878ELELP ethanol extract of leaf of L paniculata ESWELP ethanolextract ofsapwood of L paniculata

were made distilling all the solvent on a rotary evaporatorunder reduced pressure (40 rpm at 60∘C) and then in a waterbath at 60∘C obtaining the crude extracts

22 Evaluation of Antioxidant Activity Using the methoddeveloped by Yepez et al [18] 01mL of methanol solutionof sample (10 000ndash1 ppm) was added to a test tube contain-ing 39mL of 65 10minus5M of 11-diphenyl-2-picryl-hydrazyl(DPPH) methanol solutionThe test was performed in tripli-cate and the results were considered positive if the absorbancedecreased with time After 10 h the UV absorbance of themixture was measured at 515 nm To calculate the samplepotential for DPPH inhibition in terms of percentage (IP)the following equation was used

IP =119860DPPH minus 119860Sample

119860DPPHtimes 100 (1)

The inhibitory potential () was applied in the Origin70 statistic program to calculate the 50 inhibitory con-centration (IC

50) where 119860DPPH is the absorbance of the

DPPH solution and 119860Sample is the absorbance of the solutioncontaining the extract at a particular concentration

23 Quantification of Flavonoids and Total Phenols in theExtracts The extracts were subjected to determination offlavonoids according to themethodology proposed by Funariand Ferro [19] A stock solution was prepared by addingto a round bottom flask 12mg of extract and completingthe volume to 10mL with methanol (70) 2mL of thissolution was transferred to another 10mL flask 1mL ofaluminum chloride (AlCl

3) chloride 15 was added and the

volume was completed to 10mL with methanol (70) Thesolution was allowed to stand for 30 minutes and then thespectrophotometric reading was performed at 425 nm Thistest was performed in triplicate

Total phenolics content was determined by the spec-trophotometric method using the Folin-Ciocalteu reagentand gallic acid as reference standard [20] Ethanol extracts(75mg) were dissolved in methanol and transferred to a25mL volumetric flask and final volume was completed withmethanol An aliquot of 100 120583L of the latter solution wasshaken with 500120583L of Folin-Ciocalteu reagent and 6mL ofdistilled water for 1min after this time 2mL of 15 Na

2CO3

was added to the mixture and stirred for 30 s Finally thesolution was settled to 10mL volume with distilled waterAfter 2 hours of incubation the absorbance of the sampleswas measured at 750 nm

BioMed Research International 3

24 Chemical Apparatus and General Procedures All chem-icals were of analytical grade Methanol formic acid gallicacid chlorogenic acid and rosmarinic acid were purchasedfrom Merck (Darmstadt Germany) Vitexin luteolin andrutinwere acquired fromSigmaChemical Co (St LouisMOUSA) High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence AutoSampler (SIL-20A) HPLC system (Shimadzu Kyoto Japan)equippedwith Shimadzu LC-20AT reciprocating pumps con-nected to a DGU 20A5 degasser with a CBM 20A integratorSPD-M20A diode array detector and LC solution 122 SP1software

25 Quantification of Compounds by HPLC-DAD Reversephase chromatographic analyses were carried out undergradient conditions using C

18column (46mm times 150mm)

packed with 5 120583m diameter particles the mobile phase waswater containing 1 formic acid (A) and methanol (B) andthe composition gradient was 12 of B until 10min andwas changed to obtain 20 30 50 60 70 20and 10 of B at 20 30 40 50 60 70 and 80minutesrespectively following themethod described by Boligon et al[21] with slightmodifications L paniculata ethanolic extractsand mobile phase were filtered through 045 120583m membranefilter (Millipore) and then degassed by ultrasonic bath priorto use and the extracts were analyzed at a concentrationof 10mgmL The flow rate was 08mLmin and injectionvolume was 40 120583L and the wavelength was 271 nm for gallicacid 327 nm for chlorogenic acid and rosmarinic acid337 nm for vitexin and 365 nm for luteolin and rutin Allthe samples and mobile phase were filtered through 045 120583mmembrane filter (Millipore) and then degassed by ultrasonicbath prior to use Stock solutions of standards referenceswere prepared in the HPLC mobile phase at a concentrationrange of 0025ndash0250mgmL for luteolin vitexin and rutinand 0030ndash0350mgmL for gallic acid chlorogenic acidand rosmarinic acid Chromatography peaks were confirmedby comparing their retention time with those of referencestandards and by DAD spectra (200 to 500 nm) Calibrationcurve for gallic acid is 119884 = 12509119909 + 13548 (119903 = 09996)chlorogenic acid 119884 = 11948119909 + 12565 (119903 = 09999) vitexin119884 = 12647119909 + 13469 (119903 = 09997) rosmarinic acid 119884 =13476119909 + 11976 (119903 = 09999) rutin 119884 = 12706119909 + 13845(119903 = 09999) and luteolin 119884 = 11865119909+12596 (119903 = 09998)All chromatography operations were carried out at ambienttemperature and in triplicate

The limit of detection (LOD) and limit of quantification(LOQ)were calculated based on the standard deviation of theresponses and the slope using three independent analyticalcurves LOD and LOQ were calculated as 33 and 10120590119878respectively where 120590 is the standard deviation of the responseand 119878 is the slope of the calibration curve [22]

26 Antifungal Assay Preparation of the Initial Solution Thepreparation of the starting solution of the samples was madeby weighing 10mg of extracts and diluting them in 1mL ofdimethyl sulfoxide (DMSO Merck Darmstadt Germany) toachieve an initial concentration From this concentration

dilution was made in sterile distilled water to achieve aconcentration of 2048mgmL (solution test)

27 Strains Drugs and Culture Media Used The microor-ganisms used in sensitivity to natural products testing wereobtained from the Mycology Laboratory (LM) of the Fed-eral University of Paraıba (UFPB) and the Laboratory ofMicrobiology and Molecular Biology (LMBM) the RegionalUniversity of Cariri (URCA) Three standard yeast Candidaalbicans (CA 77) Candida krusei (CK 01) and Candidatropicalis (CT 23) strains were used besides these collectionsof multiresistant clinical isolates of the three species of fungicited were used CA 40006 CK 40095 and CT 40042 TheFluconazole antibiotic was used as the reference drug in themodulation of the commercial drug test

The yeasts were first grown on solid medium Sabourauddextrose agar (SDA) and then transferred to test tubescontaining 5mL of sterile saline The concentration of thestrains was diluted in 09 saline solution until a concen-tration of 105UFCmL was obtained and compared withthe nephelometric McFarland scale [23] This procedureprovided a standard yeast suspension containing 1 times 105 cellspermL [24] Inmicrodilution tests Sabourauddextrose broth(CSD) was the medium used

28 Test Minimum Inhibitory Concentration (MIC) Eppen-dorf tubes were prepared for distribution in microdilutionplate each containing 15mL of solution containing 1350 to150 120583LCSD 120583L of fungal suspensionThe plate was completedby adding 100 120583L of this solution to each well (96-well plates)and then proceeding to serial microdilution with a solutionof 100 120583L of the natural product (extract) varying concentra-tions 1024ndash8mgmLwith the lastwell for growth controlTheplates were put inside the incubator for 24 hours at 35∘C [25]and thereafter readings were taken in a spectrophotometricELISA (Thermoplate) with a wavelength of 630 nm deviceTheMICwas defined as the lowest concentration at which nogrowth was observed All tests were performed in triplicate

29 Test Modulating Action of Antifungal To investigate theeffect of the modulator antifungal action of the front linesthe method proposed by Coutinho et al [26] was usedwhere the solution of the extract was tested at subinhibitoryconcentration (MIC8) Eppendorf tubes were prepared eachcontaining 15mL of the culture medium containing CSD150 120583L of the fungal suspension and the natural productconcentration MIC8 For the control Eppendorf tubeswith 15mL of solution containing 1350120583L and 150 120583L CSDsuspension of microorganisms were prepared The ELISAplate was completed by adding 100 120583L of this solution toeach well Then 100 120583L of drug (antifungal agent) was mixedwell to the first proceeding in the broth microdilution seriesuntil the penultimate cavityThe concentrations of antifungalgradually decreased from 1024 to 1mL

210 Statistical Analysis The determinations were made intriplicate and the results were normalized by calculating thegeometric mean The results were compared using analysis


Table 2 Antiradical activity (IC50) and total phenols and flavonoids in the extracts of L paniculata

Extracts Phenolic compounds content(mg EAGgminus1 of extract)

Flavonoids(mg EAGgminus1 of extract) IC50 (mgmLminus1) 119877

2

ELELP 171036 plusmn 00143 63123 plusmn 00311 0341 0989ESWELP 89021 plusmn 00098 3001 plusmn 00059 0235 0986Quercetin mdash mdash 0203 0952mdash substance not subjected to the test EAG gallic acid equivalent IC50 concentration of sample able to sequester 50 of DPPH radicals 1198772 coefficient ofdetermination ELELP ethanolic leaf extract of L paniculata ESWELP ethanolic extract of sapwood of L paniculata

Table 3 Phenolics and flavonoids composition of L paniculata ethanolic extracts

Compounds Leaves Sapwood LOD LOQmgg mgg 120583gmL 120583gmL

Gallic acid 114 plusmn 003a 011 111 plusmn 001a 011 0017 0056Chlorogenic acid 039 plusmn 001b 003 009 plusmn 002b 0 0026 0090Vitexin 237 plusmn 002c 023 498 plusmn 001c 049 0032 0105Rosmarinic acid 1006 plusmn 001d 1 416 plusmn 001d 041 0028 0096Rutin 427 plusmn 001e 042 293 plusmn 002e 029 0009 0034Luteolin 795 plusmn 003f 079 285 plusmn 001e 028 0015 0049Results are expressed as mean plusmn standard deviations (SD) of three determinations Averages followed by different letters differ by Tukeyrsquos test at P lt 005 Limitof detection (LOD) and limit of quantification (LOQ)

of variance (ANOVA) and the comparison between thegeometric means was performed according to Tukeyrsquos testwhich was considered significant when 119875 lt 005 [27]

3 Results

31 Antioxidant Activity and Relation with Phenolic Com-pounds The results of the antioxidant activity content oftotal phenols and flavonoids and IC

50values (concentration

of the sample can sequester 50 of the DPPH radicals)determined for extracts of L paniculata and the positivecontrol (quercetin) are shown in Table 2 and Figure 1 Thecontent of total phenols and of flavonoids was higher in theleaf extract than in the extract of sapwood

32 HPLC Analysis HPLC fingerprinting of L paniculataethanolic extracts revealed the presence of the gallic acid(119905119877= 037min peak 1) chlorogenic acid (119905

119877= 2019min

peak 2) vitexin (119905119877= 4178min peak 3) rosmarinic acid (119905

119877

= 4815 peak 4) rutin (119905119877= 4987min peak 5) and luteolin

(119905119877= 5712min peak 6) (Figure 2 and Table 3)

33 Anti-Candida Activity and Modulation of the AntibioticIn this study the L paniculata ethanolic leaf extract (ELELP)tested against six strains of Candida demonstrated clinicallyirrelevant activity for the yeast C krusei CK 40095 and CK01 (MIC ge 1024) In the other strains (CA 77 CA 40006CT 23 and CT 40042) there was an insignificant effect andMIC 1024 120583gmL was also observed The ESWELP presentedMIC 1024 120583gmL for all strains tested being considered ofinhibitory effect without relevance to the yeasts (Figure 3)

34 Statistical Analysis Statistically significant differences byTukeyrsquos test (119875 gt 005) for both extracts were observed

5 10 1005000

02

04

06

08

Concentration (mgmL)

ESWELPELELP

Abso

rban

ce (5

15

nm)

Quercetin

Figure 1 Antioxidant activity in different concentrations of Lueheapaniculata extracts ELELP L paniculata ethanolic leaf extractESWELP L paniculata ethanolic extract of sapwood

for the amounts related to growth inhibition in C albi-cans and C tropicalis compared with the curve of deathexpressed by Fluconazole indicating irrelevant effects whenused alone against the yeast strains Relative to C kruseisignificant differences (119875 gt 005) were not observed havinga similar rate of growth inhibition to antifungal althoughneither Fluconazole nor natural products exhibit clinicallyrelevant activity whereasMIC value is ge1024120583gmL for both


(mAU

)

(min)50025000

0

750

500

250

6

5

4

32

1

(a)

(mAU

)

(min)

0

750

500

250

50025000

6543

21

(b)

Figure 2 Representative high performance liquid chromatography profile of L paniculata ethanolic extracts ((a) EEFLP (b) EEECLP) Gallicacid (peak 1) chlorogenic acid (peak 2) vitexin (peak 3) rosmarinic acid (peak 4) rutin (peak 5) and luteolin (peak 6)

100

75

50

25

015 25 3020

Candida krusei

Gro

wth

()

Log concentration (120583gmL)

(a)

100

75

50

25

015 25 353020

C albicansG

row

th (

)


(b)

100

75

50

25

015 25 3020

C tropicalis

Gro

wth

()

FluconazoleEEECLPEEFLP

Positive control (100 growth)Negative control


(c)

Figure 3 Values of MIC of natural products of L paniculata and antibiotic Fluconazole next to the strains of Candida graphics ELELP Lpaniculata ethanolic leaf extract ESWELP L paniculata ethanolic extract of sapwood


100

75

50

25

0150500 10 25 3020

Gro

wth

()


Control positive (100 growth)Control negative (0 growth)


C krusei

(a)

100

75

50

25

0

Gro

wth

()

150500 10 25 3020Log concentration (120583gmL)

C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()


Fluconazole tropFluconazole AlbiFluconazole kruz

Fluconazole

(d)

Figure 4 Modulatory effect of natural products of L paniculata in association with antifungal Fluconazole next to multiresistant strains ofCandida graphics ELELP L paniculata ethanolic leaf extract ESWELP L paniculata ethanolic extract of sapwood

The proximity expressed in the graph between the growthrates in lower concentrations cannot be substantiated byrelevant clinical activity of both natural products of Lpaniculata as Fluconazole for C krusei

This work is the first recorded piece of research onthe potentialization of Fluconazole using natural productsfrom L paniculata In the tests carried out to measure thismodulating effect it was observed that natural products doindeed potentialize the effect of the antifungal agent againstthree strains of Candida in particular C tropicalis and Calbicans and that there was therefore synergism when theextracts were combined with Fluconazole (Figure 4)

Using the Tukey test (119875 gt 005) statistically significantdifferences were observed between the absorbance valuesof 119909 growth of C tropicalis indicating that the action ofFluconazole alone was much lower than that in associationwith L paniculata extracts Significant 119875 lt 00001 (ANOVA)

statistical values were also observed for the modulatoryaction of C tropicalis For C albicans there was a significantdifference (119875 gt 005) between the results of Fluconazoleand L paniculata ethanolic extract of sapwood (ESWELP)However the same difference was not observed between theresults of Fluconazole and ELELP indicating that this extractdid not alter the effect of the antibiotic C krusei and nostatistical difference was observed (119875 gt 005)

4 Discussions

Phenolic compounds and some of their derivatives are con-sidered effective agents in preventing oxidation and inhibit-ing or eliminating free radicals that affect organ systemsMany of them are very good at radical scavenging and mostof these are found as esters or glycosides which are solublein water and polar organic solvents [28] Phytochemical


studies with other species of the genus Luehea collected inother regions of Brazil have shown the presence of phenoliccompounds especially flavonoids [15 29ndash32]

The fact that the content of total phenols and flavonoidsis higher in the leaf extract than in the extract of the sapwoodis related to various environmental factors that influencethe production of flavonoids in plants such as temperaturenutrition injury radiation quality and the metabolism ofsugar and nitrogen [33 34] since leaves are more exposedto environmental stress For Oliveira [35] solar radiation isa factor that is usually related to quantitative variation Somestudies have shown a quantitative increase of flavonoids inorgans exposed to light compared to those subjected to lower

The ethanol extract of sapwood (ESWELP) displayed anantioxidant activity value (IC

50= 0235mgmL) similar to

the standard quercetin (IC50

= 0203mgmL) mainly dueto the presence of the flavonoids vitexin rosmarinic acidrutin and luteolin as characterized by HPLC comparisonwith correspondent standards The ELELP also resulted inoptimal antioxidant activity close to the standard (IC

50=

0341mgmL) relating this activity to flavonoids especiallyrosmarinic acid luteolin and rutin Good antioxidant activ-ity of the ethanol extract of leaves of other species of thegenus L divaricata has been cited by Muller [36] Using theDPPHmethod the author observed the crude extract and thefractions ethyl acetate and butanol with antioxidant activitysimilar to quercetin

According to Afanasrsquoev et al [37] who investigated theantioxidant activity of rutin this flavonoid has a therapeuticaction in diseases involving free radicals and is not consideredtoxic Several other activities of rutin have been elucidatedsuch as antihyperlipidemic activity [38] suppression ofcellular immunity [39] and anti-inflammatory [40] andanticonvulsant effect in rats [41] Their effectiveness in thetreatment of diseases caused by Candida albicans was inves-tigated and confirmed by Han [42] Phenolic compoundsincluding flavonoids have demonstrated their therapeuticpotential as anti-inflammatory antifungal antimicrobialantioxidant and wound healing agents [43] Rosmarinic acidalso has significant antioxidant activity [44] reducing thebody numerous deleterious events such as the formationof reactive oxygen species lipid peroxidation and DNAfragmentation [45 46]

Akio Tanaka et al [15] observed that Candida albicansC krusei C parapsilosis and C tropicalis were resistant toextracts of other species of the genus Luehea divaricataCoelho de Souza et al [47] in an ethnopharmacologicalstudy evaluated the antimicrobial potential of some plants incommon use in the Rio Grande do Sul State Brazil amongthem L divaricata Using the agar diffusion method theauthors found that the methanol extract of leaves showedno activity against C albicans The antifungal activity of Ldivaricata was evaluated by Zacchino et al [48] As a resultthe authors reported a moderate action of dichloromethaneextract in inhibiting the growth of fungal hyphae of dermato-phytes

Silva [49] studying L candicans in the region of PuertoRico Parana State Brazil evaluated antifungal activity ofcrude extracts fractions and isolated compoundsThe crude

extract of the leaves showed antifungal activity against Ckrusei strain at a concentration of 125mgmL (CMF) andthe fungistatic concentration of 625mgmL (MIC) Marqueset al [50] studied 23 species of Cerrado another vegetationtype region in Brazil (Midwest) and found in relation toL paniculata a good inhibitory of microorganisms amongthem strains of Candida

Literature data show that of the constituents found inhigher percentages only quercetin does not present antifun-gal activity when tested against standard strains ofC albicansATCC 40227 and C krusei ATCC 6538 [6 51] Rutin whentested against standard strains ofC albicans C krusei andCtropicalis (ATCC 13803) demonstrated similar action to thediluent DMSO [6 52]

The potentiation of Fluconazole activity reported in thisstudy mainly observed for C tropicalis (both extracts) andC albicans (ethanol extract of sapwood) can be attributedto the synergistic activity of flavonoid constituents presentin extracts This can be inferred in view of the provenantimicrobial activity [53] and antibiotics modulator [54ndash56]by flavonoids There are other studies which demonstrateFluconazole modulator activity of natural products Endoet al [57] reported potent antifungal activity of extractsand pure compound isolated from Punica granatum andsynergism with fluconazole against Candida albicans Thecombination of these may alter the membrane permeabilityof the fungal strains favoring the intracellular passage ofthe antifungal which inhibits the synthesis of ergosterolcausing an increase in death of the microorganisms at alower concentration According to Wynn et al [58] themechanism of action of azoles such as Fluconazole is theinhibition of ergosterol biosynthesis by interaction with theenzyme lanosterol demethylase responsible for the conver-sion of lanosterol to ergosterol an essential component ofthe fungal membrane Inhibition of ergosterol synthesis cancause changes in permeability of the plasma membrane andinhibition of proliferation at all stages of the life cycle of thefungus [59]

The combination of Fluconazole with natural productscan be an alternative way of minimizing side effects of theantibiotic since it leads to a significant synergistic effectreducing the MIC of the antifungal agent thus reducing thedose which is necessary for its therapeutic use [56]

The mechanisms by which the extracts can interfere withthe growth of microorganisms are varied and may in part berelated to the chemical nature of some of their components[27] As a result they may show greater interaction with thelipid bilayer of the cellular membrane which affects the res-piratory chain and energy production [60] or even provideincreased permeability of the cell to antibiotics leading tothe disruption of cellular activity [61 62] The use of extractsas antimicrobial agents has a low risk of increased microbialresistance to their action because they are complex mixturesproviding greater difficulties for microbial adaptability [63]

Despite the search for new substances in plant extractsthrough isolation and identification some results seem tobe related to the combination of the compounds containedin these complex mixtures which characterize these extracts[27] Veras et al [51] suggest that quercetin and isoquercitrin


when tested alone had no significant effect demonstratingan antagonistic effect on the results of modulation antibioticCoutinho et al [26 54] stated that these mechanisms ofaction may be related to the combination of antibiotics withextracts of a subinhibitory concentration addeddirectly to theculture medium

This strategy is called ldquoherbal shotgunrdquo or ldquosynergisticmultieffect targetingrdquo and refers to plants and the use ofdrugs in a combined approach using single or multipleextracts which can affect not only a single target but alsoseveral different therapeutic compounds which act togetherin a synergistic or antagonistic manner Not only does thisprocedure result from the combination of extracts but also itis due to combinations of natural and synthetic products orantibiotics [55 64 65]

A final approach in relation to antioxidant action ofphenolic compounds is due to the mechanism of modulatingaction of extracts on bacterial antibiotic susceptibility whichprobably involves antioxidant preferentially iron-chelatingor prooxidant properties of polyphenols [66]

5 Conclusions

Chemical analysis performed on the natural products ofL paniculata revealed the presence of flavonoids such asrutin and vitexin and phenolic acids such as rosmarinicacid justifying the significant values in the evaluation ofantioxidant potentialThe results of this study also imply thatstem bark and leaf extracts of L paniculata have no clinicallyrelevant antifungal activity however in combination withan antibiotic for evaluating the interference of the fungalresistance profile a significant synergistic effect for C trop-icalis is indicated representing interesting alternative effortsin combating candidiasis

Conflict of Interests

The authors declare they have no conflict of interests

Acknowledgments

The authors acknowledge the support and cooperationreceived from RENORBIOUECE (Northeast BiotechnologyNetworkState University of Ceara) FJN (Faculty of Juazeirodo Norte Ceara State) URCALMBM (Regional Universityof Cariri CEMicrobiology Laboratory and Molecular Biol-ogy) UECELPN (Laboratory ofNatural Products StateUni-versity of Ceara) and PADETEC (Technology DevelopmentPark)

References

[1] S M Morais KS B Lima S M C Siqueira et al ldquoCorrelacaoentre as atividades antiradical antiacetilcolinesterase e teor defenois totais de extratos de plantas medicinais de farmaciasvivasrdquo Revista Brasileira de Plantas Medicinais vol 15 no 4 pp575ndash582 2013

[2] L Bertini S Proietti C Caporale F Mondello A Cassone andC Caruso ldquoTherapeutic potential of plant bioactive moleculesrdquoJournal of Biotechnology vol 150 supplement 463 pages 2010

[3] Y A R Menezes Extracao caracterizacao e atividadesbiologicas e proteınas da especie Cnidoscolus urens (L) Arthur[Dissertacao Mestrado em Ciencias Farmaceuticas] Universi-dade Federal do Rio Grande do Norte Natal Brazil 2013

[4] J J C Sidrin and M F G Rocha Micologia medica a luz deautores contemporaneos Guanabara Koogan Rio de JaneiroBrazil 2010

[5] A R T Pupulin P G Carvalho L Nishi et al ldquoEntero-patogenos relacionados a diarreia em pacientes HIV que fazemuso de terapia anti-retroviralrdquoRevista da Sociedade Brasileira deMedicina Tropical vol 42 pp 551ndash555 2009

[6] M F B Morais-Braga D L Sales J N P Carneiro et al ldquoEfeitoantifungico e atividade moduladora de Lygodium venustumSWrdquo Revista Ouricuri vol 3 no 2 pp 146ndash159 2013

[7] R A Barnes ldquoEarly diagnosis of fungal infection in immuno-compromised patientsrdquo Journal of Antimicrobial Chemotherapyvol 61 supplement 1 pp 3ndash6 2008

[8] T Rossi M A B Lozovoy R V Silva et al ldquoInteracoes entreCandida albicans e hospedeirordquo Semina vol 32 no 1 pp 15ndash28 2011

[9] O Sorg ldquoOxidative stress a theoretical model or a biologicalrealityrdquo Comptes RendusmdashBiologies vol 327 no 7 pp 649ndash662 2004

[10] J I Elejalde Guerra ldquoOxidative stress diseases and antioxidanttreatmentrdquo Anales de Medicina Interna vol 18 no 6 pp 326ndash335 2001

[11] M Skerget P Kotnik M Hadolin A R Hras M Simonic andZ Knez ldquoPhenols proanthocyanidins flavones and flavonolsin some plant materials and their antioxidant activitiesrdquo FoodChemistry vol 89 no 2 pp 191ndash198 2005

[12] V C Souza and H Lorenzi Botanica sistematica guia ilustradopara identificacao das famılias de Angiospermas da florabrasileira baseado em APG II Instituto Plantarum NovaOdessa Brazil 2005

[13] M G Bovini G Esteves M C Duarte C Takeuchi and JKuntz ldquoMalvaceaerdquo in Lista de Especies da Flora do BrasilJardim Botanico do Rio de Janeiro Rio de Janeiro Brazil 2013httpfloradobrasiljbrjgovbrjabotfloradobrasilFB156

[14] M G Bovini ldquoMalvaceae s str na Reserva Rio das PedrasMangaratiba Rio de Janeiro Brasilrdquo Rodriguesia vol 61 no 2pp 289ndash301 2010

[15] J C Akio Tanaka C C Da Silva B P Dias Filho C VNakamura J E De Carvalho and M A Foglio ldquoChemicalconstituents of Luehea divaricata Mart (Tiliaceae)rdquo QuimicaNova vol 28 no 5 pp 834ndash837 2005

[16] R Braga ldquoPlantas do Nordeste Especialmente de Cearardquo inCongresso Brasileiro de Florestas Tropicais p 9 Mossoro Brazil1976

[17] M A Figueiredo and A Fernandes ldquoEncraves de cerrado nointerior do Cearardquo Ciencia Agronomica vol 18 pp 103ndash1061987

[18] B Yepez M Espinosa S Lopez and G Bolanos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002


[19] C S Funari and V O Ferro ldquoAnalise de propolisrdquo Ciencia eTecnologia de Alimentos vol 26 no 1 pp 171ndash178 2006

[20] C M M Sousa H R Silva G M Vieira Junior et al ldquoFenoistotais e atividade antioxidante de cinco plantas medicinaisrdquoQuımica Nova vol 30 no 2 pp 351ndash355 2007

[21] A A Boligon T F Kubica D N Mario et al ldquoAntimicrobialand antiviral activity-guided fractionation from Scutia buxifoliaReissek extractsrdquoActa Physiologiae Plantarum vol 35 no 7 pp2229ndash2239 2013

[22] R P Pereira A A Boligon A S Appel et al ldquoChemicalcomposition antioxidant and anticholinesterase activity ofMelissa officinalisrdquo Industrial Crops and Products vol 53 pp34ndash45 2014

[23] NCCLSmdashNational Committee for Clinical Laboratory Stan-dardsMethods for Determining Bactericidal Activity of Antimi-crobial Agents Approved Guide Line M26-A NCCLSmdashNationalCommittee for Clinical Laboratory StandardsWayne Pa USA1999

[24] CLSImdashClinical and Laboratory Standards Institute ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing ofFilamentous Fungi Aproved Standard M27-A2 CLSImdashClinicaland Laboratory Standards Institute Wayne Pa USA 2002

[25] NCCLSmdashNational Committee for Clinical Laboratory Stan-dards ldquoMethods for dilution antimicrobial susceptibility testsfor bacteria that grow aerobically approved standardrdquo TechRep NCCLS document M7-A6 National Committee for Clini-cal Laboratory Standards Philadelphia Pa USA 2003

[26] H D M Coutinho J G M Costa V S Falcao-Silva J PSiqueira-Junior andEO Lima ldquoEffect ofMomordica charantiaL in the resistance to aminoglycosides in methicilin-resistantStaphylococcus aureusrdquo Comparative Immunology Microbiol-ogy and Infectious Diseases vol 33 no 6 pp 467ndash471 2010

[27] E F FMatias E F Alves B S Santos et al ldquoBiological activitiesand chemical characterization of Cordia verbenacea DC astool to validate the ethnobiological usagerdquo Evidence-BasedComplementary and Alternative Medicine vol 2013 Article ID164215 7 pages 2013

[28] C M O Simoes E P Schenkel G Gosmann et al Farmacog-nosia da planta ao medicamento Editora da UFRGSEditora daUFSC Porto Alegre Brazil 2010

[29] C B Alice G A D A B Silva N C S Siqueira andL A Mentz ldquoLevantamento fitoquımico de alguns vegetaisutilizados na medicina popular do Rio Grande do Sul parte IrdquoCadernos de Farmacia vol 1 no 2 pp 83ndash94 1985

[30] E Lopes ldquoAvaliacao das atividades biologicas de Luehea divari-catardquo in SemanaAcademica de Estudos Farmaceuticos Resumosvol 1 p 10 Concurso Academico de Pesquisa Cientıfica daFaculdade de Farmacia (UFRGS) Porto Alegre Brazil 1990

[31] V M Vargas R R Guidobono and J A P HenriquesldquoGenotoxicity of plant extractsrdquoMemorias do Instituto OswaldoCruz vol 86 supplement 2 pp 67ndash70 1991

[32] D A Da Silva V G Alves D M M Franco et al ldquoAntiprolif-erative activity of Luehea candicans Mart et Zucc (Tiliaceae)rdquoNatural Product Research Formerly Natural Product Letters vol26 no 4 pp 364ndash369 2012

[33] L Gobbo-Neto and N P Lopes ldquoPlantas medicinais fatores deinfluencia no conteudo de metabolitos secundariosrdquo QuımicaNova vol 30 no 2 pp 374ndash381 2007

[34] H S Rosa Caracterizacao e determinacao da atividadeantifungica in vitro de extratos obtidos de Sida tuberculata REFRIES (MALVACEAE) [Mestrado em Bioquımica] Universi-dade Federal do Pampa Uruguaiana Brazil 2013

[35] A P Oliveira Avaliacao da atividade gastroprotetora do extratometanolico e desenvolvimento tecnologico de preparacoes extra-tivas das partes aereas da Marrubium vulgare L (Lamiaceae)[Dissertacao (Mestrado em Ciencias Farmaceuticas)] Universi-dade do Vale do Itajaı 2011

[36] J B Muller Avaliacao das atividades antimicrobiana antioxi-dante e antinociceptiva das folhas da Luehea divaricata Martius[Mestrado em Ciencias Farmaceuticas] UFSM Santa MariaBrazil 2006

[37] I B Afanasrsquoev A I Dorozhko A V Brodskii V A Kostyukand A I Potapovitch ldquoChelating and free radical scavengingmechanisms of inhibitory action of rutin and quercetin in lipidperoxidationrdquo Biochemical Pharmacology vol 38 no 11 pp1763ndash1769 1989

[38] K F R Santos T T Oliveira T J Nagem A S Pinto And andM G A Oliveira ldquoHypolipidaemic effects of naringenin rutinnicotinic acid and their associationsrdquoPharmacological Researchvol 40 no 6 pp 493ndash496 1999

[39] E Middleton Jr C Kandaswami and T C Theoharides ldquoTheeffects of plant flavonoids on mammalian cells Implicationsfor inflammation heart disease and cancerrdquo PharmacologicalReviews vol 52 no 4 pp 673ndash751 2000

[40] T Guardia A E Rotelli A O Juarez and L E Pelzer ldquoAnti-inflammatory properties of plant flavonoids Effects of rutinquercetin and hesperidin on adjuvant arthritis in ratrdquo Farmacovol 56 no 9 pp 683ndash687 2001

[41] M Nassiri-Asl S Shariati-Rad and F Zamansoltani ldquoAnti-convulsive effects of intracerebroventricular administrationof rutin in ratsrdquo Progress in Neuro-Psychopharmacology andBiological Psychiatry vol 32 no 4 pp 989ndash993 2008

[42] Y Han ldquoRutin has therapeutic effect on septic arthritis causedby Candida albicansrdquo International Immunopharmacology vol9 no 2 pp 207ndash211 2009

[43] S C Santos and J C P Mello ldquoTaninosrdquo in FarmacognosiaDa Planta ao medicamento C M O Simoes E P Schenkel GGosmann et al Eds pp 527ndash554 Editora da UFRGSEditoraUFSC Porto Alegre Brazil 2004

[44] L Gu T Wu and Z Wang ldquoTLC bioautography-guidedisolation of antioxidants from fruit of Perilla frutescens varacutardquo LWTmdashFood Science and Technology vol 42 no 1 pp131ndash136 2009

[45] A A Izzo and F Capasso ldquoHerbal medicines to treat Alz-heimerrsquos diseaserdquo Trends in Pharmacological Sciences vol 28no 2 pp 47ndash48 2007

[46] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008

[47] G Coelho de Souza A P S Haas G L von Poser E E SSchapoval and E Elisabetsky ldquoEthnopharmacological studiesof antimicrobial remedies in the south of Brazilrdquo Journal ofEthnopharmacology vol 90 no 1 pp 135ndash143 2004

[48] S Zacchino C Santecchia S Lopez et al ldquoIn vitro antifungalevaluation and studies on mode of action of eight selected


species from the Argentina florardquo Phytomedicine vol 5 pp389ndash395 1998

[49] DA SilvaEstudoQuımico eAvaliacao deAtividadeAntifungicae Antiproliferativa da Especie Luehea candicansMART et ZUCC(Tiliaceae) [Dissertacao (Mestrado em Quımica)] UniversidadeEstadual de Maringa Maringa Brazil 2004

[50] M C S Marques L Hamerski F R Garcez et al ldquoIn vitrobiological screening and evaluation of free radical scavengingactivities of medicinal plants from the Brazilian CerradordquoJournal of Medicinal Plants Research vol 7 no 15 pp 957ndash9622013

[51] H N H Veras I J M dos Santos A C B dos Santos et alldquoComparative evaluation of antibiotic and antibiotic modifyingactivity of quercetin and isoquercetin in vitrordquoCurrent Topics inNutraceutical Research vol 9 no 1-2 pp 25ndash30 2011

[52] M K A Araruna S A Brito M F B Morais-Braga et alldquoEvaluation of antibiotic amp antibiotic modifying activity ofpilocarpine amp rutinrdquo Indian Journal of Medical Research vol135 no 2 pp 252ndash254 2012

[53] M L Falcone Ferreyra S P Rius and P Casati ldquoFlavonoidsbiosynthesis biological functions and biotechnological appli-cationsrdquo Frontiers in Plant Science vol 3 article 222 2012

[54] H D M Coutinho A Vasconcellos M A Lima G GAlmeida-Filho and R R N Alves ldquoTermite usage associ-ated with antibiotic therapy enhancement of aminoglycosideantibiotic activity by natural products of Nasutitermes corniger(Motschulsky 1855)rdquo BMC Complementary and AlternativeMedicine vol 9 article 35 2009

[55] H N H Veras F F G Rodrigues A V Colares et alldquoSynergistic antibiotic activity of volatile compounds from theessential oil of Lippia sidoides and thymolrdquo Fitoterapia vol 83no 3 pp 508ndash512 2012

[56] F G Figueredo E O Ferreira B F F Lucena et al ldquoModulationof the antibiotic activity by extracts from Amburana cearensisA C Smith and Anadenanthera macrocarpa (Benth) BrenanrdquoBioMed Research International vol 2013 Article ID 640682 5pages 2013

[57] E H Endo D A Garcia Cortez T Ueda-Nakamura C VNakamura and B P Dias Filho ldquoPotent antifungal activityof extracts and pure compound isolated from pomegranatepeels and synergismwith fluconazole againstCandida albicansrdquoResearch in Microbiology vol 161 no 7 pp 534ndash540 2010

[58] R L Wynn M A Jabra-Rizk and T F Meiller ldquoFungal drugresistance biofilms and new antifungalsrdquo General Dentistryvol 51 no 2 pp 94ndash98 2003

[59] J A Urbina ldquoErgosterol biosynthesis and drug development forChagas diseaserdquoMemorias do Instituto Oswaldo Cruz vol 104supplement 1 pp 311ndash318 2009

[60] K Nicolson G Evans and P W OrsquoToole ldquoPotentiation ofmethicillin activity against methicillin-resistant Staphylococcusaureus by diterpenesrdquo FEMS Microbiology Letters vol 179 no2 pp 233ndash239 1999

[61] B J Juven J Kanner F Schved and H Weisslowicz ldquoFactorsthat interact with the antibacterial action of thyme essential oiland its active constituentsrdquoThe Journal of Applied Bacteriologyvol 76 no 6 pp 626ndash631 1994

[62] S Burt ldquoEssential oils their antibacterial properties and poten-tial applications in foodsmdasha reviewrdquo International Journal ofFood Microbiology vol 94 no 3 pp 223ndash253 2004

[63] D J Daferera B N Ziogas and M G Polissiou ldquoTheeffectiveness of plant essential oils on the growth of Botrytiscinerea Fusarium sp and Clavibacter michiganensis subspMichiganensisrdquo Crop Protection vol 22 no 1 pp 39ndash44 2003

[64] S H Hemaiswarya A K Kruthiventi and M Doble ldquoSyner-gism between natural products and antibiotics against infec-tious diseasesrdquo Phytomedicine vol 15 no 8 pp 639ndash652 2008

[65] H Wagner and G Ulrich-Merzenich ldquoSynergy researchapproaching a new generation of phytopharmaceuticalsrdquo Phy-tomedicine vol 16 no 2-3 pp 97ndash110 2009

[66] Z Samoilova G Smirnova N Muzyka and O OktyabrskyldquoMedicinal plant extracts variously modulate susceptibilityof Escherichia coli to different antibioticsrdquo MicrobiologicalResearch vol 169 no 4 pp 307ndash313 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


According to Sorg [9] diseases such as cancer emphy-sema cirrhosis arthritis and inflammation in addition tothe aging process itself are related to free radicals ElejaldeGuerra [10] explained that antioxidant therapy and dietenriched with antioxidants appear to prevent or at leastmitigate the organic deterioration by oxidative stress It canbe observed that in recent decades there has been growthof scientific research in this area involving the effect ofcrude extracts purified fractions or isolated componentshighlighting phenolic compounds that in many studies haveshown this activity [1 11] Therefore antioxidant activitywhich protects the live organism against deterioration couldplay an important role for an antifungal agent

Malvaceae are a family consisting of herbs subshrubsshrubs lianas and small and large trees with about 250genera and 4200 species and in Brazil there are about 80genera and 400 species [12] According to the List of Speciesof Flora of Brazil 69 genera and 754 species are pointed out[13] distributed in 30 genera and 393 taxa of the Malvaceaesubfamily [14] The genus Luehea is one of 252 genera of theMalvaceae familyThe leaves of Luehea aremarketed as herbalagainst dysentery leucorrhoea rheumatism gonorrhea andtumors the infusion of the flowers is used against bronchitisand the root is depurative [15] Braga [16] pointed out theastringency of the barks of L speciosa Willd (botanicalsynonym of L divaricata) and also recorded the occurrenceof the species L candicansMart et Zucc (Luehea uniflora StHil) and L paniculata Mart presenting the same use as thefirst named

In this context the aim of this work was to investigatethe antifungal effect of ethanol extract from different partsof Luehea paniculata Mart amp Zucc a medicinal tree ofmultiple effects individually and in combination with a com-mercial drug against clinical isolates of multidrug-resistantstrains of Candida An evaluation of free radical-scavengingactivity and phytochemical analysis of the extracts werealso performed in order to relate the existence of phenoliccompounds with activities determined in this study

2 Material and Methods

21 Place of Collection of the Plant Material and ObtainingExtracts Leaves and sapwood of three specimens of Lpaniculatawere collected on November 19 2012 (dry season)around 1730 hours from the Cerrado located in the center ofCaatinga area Lavras daMangabeira city centralMesoregionin South of Ceara Northeastern Brazil

The area is characterized as a vegetation relic [17]presenting itself in deep soil on a tabular relief on longdissected surfaces with penetration of the savannah floraThepatch of savannah in question is located in hills of 280 to600m in height at the following coordinates 06∘721015840243210158401015840Sand 38∘971015840739610158401015840WThe botanical material was collected anddeposited in the Prisco Bezerra Herbarium of the FederalUniversity of Ceara under number 54641

The plant material was dried at 60∘C weighed (Table 1)and subjected to maceration with ethanol PA for seven daysAfter this period filtration and concentration of the extract

Table 1 Dry weight and yield of the crude ethanol extracts of Lpaniculata

Naturalproduct Solvent Dry matter Crude extract Extract yields

ELELP Ethanol 595 g 61649 g 1036ESWELP Ethanol 195 g 36621 g 1878ELELP ethanol extract of leaf of L paniculata ESWELP ethanolextract ofsapwood of L paniculata

were made distilling all the solvent on a rotary evaporatorunder reduced pressure (40 rpm at 60∘C) and then in a waterbath at 60∘C obtaining the crude extracts

22 Evaluation of Antioxidant Activity Using the methoddeveloped by Yepez et al [18] 01mL of methanol solutionof sample (10 000ndash1 ppm) was added to a test tube contain-ing 39mL of 65 10minus5M of 11-diphenyl-2-picryl-hydrazyl(DPPH) methanol solutionThe test was performed in tripli-cate and the results were considered positive if the absorbancedecreased with time After 10 h the UV absorbance of themixture was measured at 515 nm To calculate the samplepotential for DPPH inhibition in terms of percentage (IP)the following equation was used

IP =119860DPPH minus 119860Sample

119860DPPHtimes 100 (1)

The inhibitory potential () was applied in the Origin70 statistic program to calculate the 50 inhibitory con-centration (IC

50) where 119860DPPH is the absorbance of the

DPPH solution and 119860Sample is the absorbance of the solutioncontaining the extract at a particular concentration

23 Quantification of Flavonoids and Total Phenols in theExtracts The extracts were subjected to determination offlavonoids according to themethodology proposed by Funariand Ferro [19] A stock solution was prepared by addingto a round bottom flask 12mg of extract and completingthe volume to 10mL with methanol (70) 2mL of thissolution was transferred to another 10mL flask 1mL ofaluminum chloride (AlCl

3) chloride 15 was added and the

volume was completed to 10mL with methanol (70) Thesolution was allowed to stand for 30 minutes and then thespectrophotometric reading was performed at 425 nm Thistest was performed in triplicate

Total phenolics content was determined by the spec-trophotometric method using the Folin-Ciocalteu reagentand gallic acid as reference standard [20] Ethanol extracts(75mg) were dissolved in methanol and transferred to a25mL volumetric flask and final volume was completed withmethanol An aliquot of 100 120583L of the latter solution wasshaken with 500120583L of Folin-Ciocalteu reagent and 6mL ofdistilled water for 1min after this time 2mL of 15 Na

2CO3

was added to the mixture and stirred for 30 s Finally thesolution was settled to 10mL volume with distilled waterAfter 2 hours of incubation the absorbance of the sampleswas measured at 750 nm


















2






3 Results





119877= 2019min


119877





5 10 1005000

02

04

06

08


ESWELPELELP

Abso

rban

ce (5

15

nm)

Quercetin




(mAU

)

(min)50025000

0

750

500

250

6

5

4

32

1

(a)

(mAU

)

(min)

0

750

500

250

50025000

6543

21

(b)


100

75

50

25

015 25 3020

Candida krusei

Gro

wth

()


(a)

100

75

50

25

015 25 353020

C albicansG

row

th (

)


(b)

100

75

50

25

015 25 3020

C tropicalis

Gro

wth

()




(c)



100

75

50

25

0150500 10 25 3020

Gro

wth

()




C krusei

(a)

100

75

50

25

0

Gro

wth

()


C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()



Fluconazole

(d)






4 Discussions









50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































2






3 Results





119877= 2019min


119877





5 10 1005000

02

04

06

08


ESWELPELELP

Abso

rban

ce (5

15

nm)

Quercetin




(mAU

)

(min)50025000

0

750

500

250

6

5

4

32

1

(a)

(mAU

)

(min)

0

750

500

250

50025000

6543

21

(b)


100

75

50

25

015 25 3020

Candida krusei

Gro

wth

()


(a)

100

75

50

25

015 25 353020

C albicansG

row

th (

)


(b)

100

75

50

25

015 25 3020

C tropicalis

Gro

wth

()




(c)



100

75

50

25

0150500 10 25 3020

Gro

wth

()




C krusei

(a)

100

75

50

25

0

Gro

wth

()


C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()



Fluconazole

(d)






4 Discussions









50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2






3 Results





119877= 2019min


119877





5 10 1005000

02

04

06

08


ESWELPELELP

Abso

rban

ce (5

15

nm)

Quercetin




(mAU

)

(min)50025000

0

750

500

250

6

5

4

32

1

(a)

(mAU

)

(min)

0

750

500

250

50025000

6543

21

(b)


100

75

50

25

015 25 3020

Candida krusei

Gro

wth

()


(a)

100

75

50

25

015 25 353020

C albicansG

row

th (

)


(b)

100

75

50

25

015 25 3020

C tropicalis

Gro

wth

()




(c)



100

75

50

25

0150500 10 25 3020

Gro

wth

()




C krusei

(a)

100

75

50

25

0

Gro

wth

()


C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()



Fluconazole

(d)






4 Discussions









50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(mAU

)

(min)50025000

0

750

500

250

6

5

4

32

1

(a)

(mAU

)

(min)

0

750

500

250

50025000

6543

21

(b)


100

75

50

25

015 25 3020

Candida krusei

Gro

wth

()


(a)

100

75

50

25

015 25 353020

C albicansG

row

th (

)


(b)

100

75

50

25

015 25 3020

C tropicalis

Gro

wth

()




(c)



100

75

50

25

0150500 10 25 3020

Gro

wth

()




C krusei

(a)

100

75

50

25

0

Gro

wth

()


C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()



Fluconazole

(d)






4 Discussions









50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

75

50

25

0150500 10 25 3020

Gro

wth

()




C krusei

(a)

100

75

50

25

0

Gro

wth

()


C albicans



(b)

100

75

50

25

0

Gro

wth

()




C tropicalis

(c)

100

75

50

25

0

Gro

wth

()



Fluconazole

(d)






4 Discussions









50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























50=















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusions




Acknowledgments


References











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Mart. & Zucc. (Malvaceae) in Candida Spp.downloads.hindawi.com/journals/bmri/2015/807670.pdf · Malvaceae are a family consisting of herbs, subshrubs, shrubs, lianas, and small and