research article chemical assessment and antimicrobial and

Research ArticleChemical Assessment and Antimicrobial andAntioxidant Activities of Endophytic Fungi Extracts Isolatedfrom Costus spiralis (Jacq) Roscoe (Costaceae)

Poliana Guerino Marson Ascecircncio1 Seacutergio Donizeti Ascecircncio1 Aline Aires Aguiar1

Adriana Fiorini2 and Raphael Sanzio Pimenta3

1Research Laboratory of Natural Products Federal University of Tocantins Avenida NS 15 109 Norte 77020-210 Palmas TO Brazil2Department of Clinical Analysis and Biomedicine State University of Maringa Avenida Colombo 579087020-900 Maringa PR Brazil3Laboratory of General and Applied Microbiology Federal University of Tocantins Avenida NS 15 109 Norte77020-210 Palmas TO Brazil

Correspondence should be addressed to Poliana Guerino Marson Ascencio polianamarsonuftedubr

Received 10 November 2014 Accepted 28 November 2014 Published 17 December 2014

Academic Editor Vincenzo De Feo

Copyright copy 2014 Poliana Guerino Marson Ascencio et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Costus spiralis (Costaceae) is a species native to the Amazon region and is used in traditional medicine The endophytic fungi usedin this studywere obtained from leaves of this plant 13 strains were selected to obtain hydroethanolic extracts andwere submitted tohydroalcoholic extraction and evaluated for antioxidant activity by DPPH (22-difenil-1-picrilhidrazil) and FRAP (ferric reducingantioxidant power) and all of the fungi had positive resultsThe antimicrobial action of crude extracts had a good range of activitiesAll extracts had inhibitory activities against the yeasts of Candida albicans and C parapsilosis with 125 to 500 120583gmL MIC Eightextracts had antimicrobial activities against Bacillus subtilis (MIC from 624 to 125 120583gmL) 5 against Pseudomonas aeruginosa (MICfrom 125 to 500 120583gmL) 2 against Salmonella enterica (MIC from 125 to 625 120583gmL) and 2 against Enterococcus faecalis (MIC from500 to 125 120583gmL) The presence of secondary metabolites including coumarins was observed during chemical evaluation by thinlayer chromatography Total phenol content was estimated and a strong positive correlation to antioxidant activity was observedaccording to its Pearson coefficientThis is the first report of the bioactive potential of endophytic fungi isolated from the Costaceaefamily in Brazilian ecosystems

1 Introduction

Costus spiralis is a Brazilian Amazon plant that is recognizedfor its medical and ornamental values This plant is com-monly used in popular medicine to treat urinary infectionsand kidney stones and for diabetes management The plantalso acts as an antioxidant antibacterial agent and diureticand to promote wound healing [1ndash3] Normally plants fromtropical regions have a higher diversity of endophytic micro-organisms compared with those found in temperate regions[4] Endophytic fungi are microorganisms that colonize thevegetal tissues either inter- or intracellularly without causingany damage to the vegetal host There are few studies thatfocus on the diversity ecology and other factors involved in

endophyte plant interaction [5ndash7]Thesemicroorganisms area good resource for identifying new bioactive products withmore than 20000 substances described to date [8] Amongthese substances 51 had novel structures and 80 exhibitedsome biological activity making endophytes a promisingresource for the identification of new bioactive molecules [910] A variety of pharmacological activities have already beendescribed from endophytic fungi including antibacterial [11ndash13] antifungal [14ndash16] antiparasitic [17] trypanocidal [18 19]leishmanicidal [19] antimalarial [20] anti-inflammatory[21] neuroprotective [22] antioxidant [7] immunosuppres-sion [23] antiviral [23 24] anticolinesterasic [12] antineo-plastic [25ndash29] and cytotoxic [12 22 30] properties

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 190543 10 pageshttpdxdoiorg1011552014190543

2 Evidence-Based Complementary and Alternative Medicine

Despite scientific advances infectious diseases remain amajor contributor tomortality andmorbidity in public healthThemain explanation for this is the ability ofmicroorganismsto acquire resistance against antimicrobial substances Thischaracteristic results in a constant need to discover anddevelop new drugs [31]

Oxygen radicals and superoxide anions play importantroles in biochemicalphysiological reactions in the humanbody However when these species are produced in excessdue to pathophysiological processes or environmental inter-ference they can promote tissue damage and result in disease[32] Currently few antioxidant substances can be used inclinical situations thus underscoring the necessity to identifynew safe and efficient molecules for this purpose [33]Bioactive molecules from new sources such as endophyticfungi deserve attention because they may lead to drugs withdifferent pathologies food additives or cosmetics

2 Materials and Methods

21 Endophytic Fungi Endophytic fungi were isolated fromhealthy leaves of C spiralis From each leaf three 05 cm2pieces were cut The leaf sample fragments were surface ster-ilized by successive dipping in 2 Extran detergent (Sigma-Aldrich) for 2min 70 ethanol for 1min and 2 sodiumhypochlorite for 3min and then by washing with steriledistilled water for 2min they were then placed on potatodextrose agar (PDA Difco) containing 100mgmL chloram-phenicol The sterile water wash was plated on PDA to con-firm external disinfection Plates were incubated for up to 60days at 25∘CThemycelia from themargins of fungal coloniesgrowing from the leaf fragments were transferred to freshPDA and further purified by transferring hyphal plugs to newPDA plates Thirteen endophytic fungi were selected fromleaves of C spiralis These microorganisms were previouslytested with phytopathogen controls (data not shown) and arepreserved in Culture Collection from the Laboratory ofGeneral and Applied Microbiology at the Federal Universityof Tocantins Brazil

22 Molecular Identification

221 Fungal DNA Extraction Endophytic fungi were cul-tured on Sabouraud dextrose agar slants for 7 days at 25∘CTheDNA extraction was performed according to themethoddescribed by de Hoog et al [34] Briefly approximately 01 gof fungal mycelia was transferred to 15mL tubes containing400 120583L of lysis buffer (50mM Tris-HCl pH 80 5mM EDTApH 80 100mMNaCl 1 SDS) and kept at minus20∘C for 10minThe fungi cells weremixedwith 1mmglass beads and agitatedusing a Vortex for 30 sec followed by a 30 sec interval onice then repeated Cells were incubated for 30min at 60∘Cwith 5 120583L 20mgmL Proteinase K The samples were thenincubated for 10min at 65∘C with 162 120583L of CTAB solution(200mM Tris-HCl pH 75 200mM Na-EDTA 82 NaCl2CTAB) Following incubation 570 120583L chloroform isoam-ylic alcohol (24 1 vv) was added and the tubes were kept for10min on ice The homogenate was centrifuged at 14500timesg

for 10min at 4∘C Then the supernatant was transferred toa new tube and 10 of the residual volume of 3M sodiumacetate was added The mixture was then incubated on icefor 30min Precipitates were removed by centrifugation andthe DNAwas recovered by isopropanol precipitation washedwith 70 (vv) ethanol allowed to air dry and resuspendedin 50120583L of TE buffer (10mM Tris-HCl pH 80 and 01mMEDTA) The concentration was measured by monitoring theUV absorbance at 260 nm using Nanodrop 2000 (ThermoUSA) and the DNA was diluted to 10 ng120583L with ultrapurewater

222 DNA Amplification and Sequencing DNA sequencingwas performed using a BigDye Direct Cycle Sequencing Kit(Life Technologies) For PCR amplification 1120583L of dilutedDNA (10 ng120583L) wasmixedwith 50120583L of BigDyeDirect PCRMaster Mix 075 120583L of each forward ITS1-M13 (51015840-TGTAAA-ACGACGGCCAGTTCCGTAGGTGAACCTGCGG-31015840) andreverse ITS4-M13 (51015840-CAGGAAACAGCTATGACCTCC-TCCGCTTATTGATATGC-31015840) primers at a 10 120583M concen-tration which include the M13 universal primer sequencesand 24 120583L of ultrapure water PCR cycling conditions were asfollows initial denaturation of 10min at 94∘C followed by 35cycles (30 s) of denaturation at 96∘C annealing for 15 sec at62∘C and extension for 30 sec at 72∘C Following PCR 3 120583Lof BigDye Direct SequencingMaster Mix and 1 120583L of forwardBigDye Direct M13 forward primer (provided) were addeddirectly to the PCR sample put back into the PCR thermalcycler and incubated as follows 15min at 37∘C 2min at 80∘Cand 1min at 96∘C followed by 25 cycles of 10 sec at 96∘C5 sec at 50∘C and 75 sec at 60∘C

At the completion of the sequencing reaction the sequen-cing products were purified following the ethanolEDTApro-tocol for BigDyeTerminator version 31 Cycle SequencingKitElectrophoresis was performed on the Applied Biosystems3500xLGenetic Analyzer Electropherogramswere proofreadwith the software BioEdit Sequence Alignment Editor (1997ndash2013) The sequencing results for each sample were analyzedusing BLASTn (Basic Local Alignment Search Tool version2215 of BLAST 20) to verify the results and to identify thefungus for each sample

23 Crude Extract Acquisition The 13 endophytes weremultiplicated in PDA at 25∘C plusmn 2 for 5 days Subsequently6mm discs were placed in the center of 4 Petri dishes withPDA agar and incubated at 25∘C plusmn 2 for 14 days These cul-tures were extracted (1 20 pv) by hydroethanolicmaceration(1 3 vv) for 48 h at 25∘C under agitation The samples werecentrifuged at 2400timesg for 20min at 8∘C and then filteredThe solvent from each supernatant was removed on a rotaryevaporator at 35∘C lyophilized and stored in a desiccatorwithout lightThe same extraction process was performed for4 sterile BDA Petri dishes that were used as negative control

24 Antimicrobial Activity

241Microorganisms Allmicroorganismswere obtained fromthe American Type Culture Collection (ATCC Rockville

Evidence-Based Complementary and Alternative Medicine 3

MD USA) Extracts were tested against the Gram-positive(G+ve) bacteria Bacillus subtilis 6623 Enterococcus fae-calis 29212 and Staphylococcus aureus 29213 Gram-negative(Gminusve) bacteria Escherichia coli 25922 Pseudomonas aerug-inosa 27853 Klebsiella pneumoniae 700603 and Salmonellaenterica subsp enterica serovar Typhi 19430 and the yeastsCandida albicans 10231 and C parapsilosis 22019

242 Antibacterial Susceptibility Test Theminimal inhibitoryconcentrations (MICs) of all extracts and the referenceantibiotics tetracycline (Sigma T3258) and penicillin (SigmaP3032) were determined using microdilution techniques inMueller-Hinton broth (Merck) following the protocol estab-lished for bacteria [35] Inoculates were prepared in the samemedium at a density adjusted to a 05 McFarland turbiditystandard (108 colony-forming units (CFU)mL) and diluted1 10 for the broth microdilution procedure Microtiter plateswere incubated at 37∘C and the MICs were recorded after24 h of incubation The lyophilized crude extracts of endo-phytes were diluted in dimethylsulfoxide (Sigma D8418)filtered in sterilemembranes and tested at 1000 500 250 125625 31 25 78 39 and 19 120583gmL concentrations The MICwas defined as the lowest concentration of crude extracts thatinhibit the target microorganismsrsquo growth

The minimal bactericidal concentrations (MBCs) weredetermined from the results obtained in MIC For this analiquot of 10 120583L from the wells that had inhibition was spreadin Petri dishes with Mueller-Hinton agar (Fluka 70191) andincubated at 37∘C for 24 h The MBCs were defined as thelowest concentration of crude extract that resulted in nogrowthwhen the treated culture was spread on antibiotic-freemedium plates after the incubation All tests were repeatedand confirmed

243 Antifungal Susceptibility Test The MICs of crudeextracts were determined against yeasts using broth microdi-lution techniques according to the method described byCLSI [36] MICs were determined in RPMI 1640 medium(Sigma R6504) pH 70 The starting inoculum was 10 times106 CFUmL Microtiter plates were incubated at 37∘C in adark humid chamber andMICswere recorded after 48 hThelyophilized hydroethanolic extracts from endophytes werediluted in dimethylsulfoxide (SigmaD8418) filtered in sterilemembranes and tested at 1000 500 250 125 625 3125 7839 and 19 120583gmL concentrations The MIC was defined asthe lowest concentration of compounds withoutmicroorgan-ism growth Nystatin (Sigma N6261) was used as the drugcontrolTheminimal fungicidal concentrations (MFCs) wereobtained from the MIC results For this an aliquot of 10 120583Lfrom the wells that showed inhibition was spread in Petridishes with Sabouraud agar (Merck 105438) and incubated at37∘C for 24 h The MFCs were defined as the lowest concen-tration of crude extract that resulted in no growth when thetreated culture was spread on plates with antibiotic-freemedium after the incubation All tests were repeated andconfirmed

25 Antioxidant Assay

251 Thin Layer Chromatography (TLC) The antioxidantactivity from the hydroethanolic extracts of the 13 fungistrains was evaluated by thin layer chromatography (TLC)The extracts were dissolved in a hydroethanolic solution(3 1 vv) to achieve a final concentration of 20mgmL Thesolutionwas loaded onto aTLCplate (20times 20 cmMerck) andeluted with ethyl acetate formic acid acetic acid and water(100 11 11 27 vv) A methanolic solution of 22-difenil-1-picrilhidrazil (DPPH) (Aldrich D9132) at 02 was used tovisualize the products Quercetin (Sigma Q0125) and ascor-bic acid (Sigma A0278) were used as standards Positivesamples showed yellow bands on a purple background inchromatograms

252 Determination of Antioxidant Activities by DPPHMethod Antioxidant activity was determined in accordancewith the published methods [37 38] Ascorbic acid was usedas a standard (2ndash10 120583gmL Sigma A0278) Fungi extracts andnegative controls were diluted to 25 50 100 150 and200120583gmL A methanolic solution of DPPH (01mM 2mL)was added to 1mL of each dilution For each experimentsolutions with 1mL sample and 2mL ethanol were used toestablish a baseline Controls were performed with 1mL ofethanol and 2mL of methanolic solution of DPPH (01mM)These solutions were homogenized and kept in the dark for30min at 25∘C and the absorbance was obtained at 517 nmwith a spectrophotometer (BiochromModel Biowave II) Alltests were conducted in triplicate

Antioxidant activity of each extract concentration wasdetermined using the following equation

AA = 100 minus [Abssample minus Absblank] times100

Abscontr (1)

where AA is the total antioxidant activity Abssample is theabsorbance of samples or standard Absblank is the baselineabsorbance and Abscontr is the absorbance of the controlsolution

Linear regression curves were obtained from the AAresults and their respective results and predictive equationswere expressed as CE

50

253 Determining the Antioxidant Activity by Ferric ReducingAntioxidant Power (FRAP) FRAP analyses were performedin accordance with the published protocols [39] An acetatebuffer solution (03M pH 36) a 10mM solution of 246-tris(2piridil)-s-triazine (TPTZ) in 40mMHCl and an aqueoussolution of ferric chloride (20mM)were preparedThe FRAPreagentwas prepared fewminutes beforemixing the solutionsfor analysis (10 1 1 vv)

The total antioxidant activity (TAA) for each of the crudeextracts was determined from three methanolic dilutions(05 025 and 0125mgmL) and 6 aqueous dilutions usingferrous sulfate (Sigma-Aldrich 61230) as a standard in 500and 2000120583Mconcentrations For each sample 01mL of eachdilution was added to 29mL of FRAP and kept in the dark at37∘C for 30min The absorbance of each solution was


Table 1 Identification of endophytic filamentous fungi from Costus spiralis plant

UFT code Reference species GenBank access number of identity Number of bases Species identification4427 Phomopsis sp GU0666931 99 564 Phomopsis sp4416 Diaporthe phaseolorum JQ5141501 99 603 Diaporthe phaseolorum4432 Diaporthe sp FJ7999411 98 602 Diaporthe sp4409 Diaporthe sp EF423554 99 534 Diaporthe sp4419 Phomopsis sp AY7459861 99 561 Phomopsis sp4426 Not identified mdash mdash mdash Not identified4417 Diaporthe sp FJ7999411 99 602 Diaporthe sp4420 Sordariomycetes sp JX1741461 99 549 Sordariomycetes sp4400 Not identified mdash mdash mdash Not identified4435 Phomopsis sp EU9772191 97 559 Phomopsis sp4405 Diaporthe phaseolorum AY5778151 99 602 Diaporthe phaseolorum4410 Cochliobolus sp JQ7539611 96 572 Cochliobolus ssp4403 Sordariomycetes sp JX1741461 99 549 Sordariomycetes spUFT code Culture Collection of the Federal University of Tocantins Brazil

determined at 593 nmusing a spectrophotometer (BiochromModel Biowave II) and FRAP as an internal reference Fromthese results calibration curves were calculated for eachsample The result of TAA from each sample was expressedin millimole equivalents of iron per mg of lyophilized fungiextract (mM Fe+2mg Ext)

26 Chemical Evaluation of the Extracts by TLC The sec-ondary metabolites from the 13 hydroethanolic extractsand from the negative control were evaluated by TLCusing different mobile phases and visualizing agents in alu-minum sheets (20 times 20 cm) preactivated covered by silicagelGF254

(Merck)The samples were dissolved in hydroethanolicsolution (1 3 vv) The sheets were placed in a chromatog-raphy chamber eluted along a 16 cm path in one direc-tion and observed by luminescence (UV365 nm) Using thepreviously described techniques and parameters for identifi-cation [40] and identical standards we were able to evaluatethe following classes of secondary metabolites alkaloidswith quinine standard (Aldrich 145904) and caffeine (Cal-biochem 205548) monoterpenoids sesquiterpenoids andditerpenoids using a thymol standard (Sigma T0501)triterpenoids and steroids with a stigmasterol standard(Sigma S2424) phenolic compounds with a tannic acidstandard (Sigma-Aldrich 403040) rutin (Sigma R5143)and scopoletin (Sigma S104) coumarin using scopoletinstandards (Sigma S104) and coumarin (Sigma C4261)anthraquinones naphthoquinones and anthocyanins with4-hydroxyanthraquinone and lapachol standards (Aldrich142905) saponins and polyketides through a saponin stan-dard (Sigma 47036) thymol (Sigma T0501) and stigmasterol(Sigma S2424) condensed proanthocyanidins and leucoan-thocyanidins with a catechin standard (Fluka 43412)

27 Quantification of Total Phenolic Compounds The quan-tification of phenols in hydroethanolic extracts was realizedusing the Folin-Ciocalteu method [41] using tannic acid(Sigma-Aldrich 403040) as a standard An aliquot (150 120583L)from each sample (10mgmL) was added to 250 120583L of

Folin-Ciocalteu reagent 500120583L of sodiumcarbonate solution(75 wv) was then added to the mixture The sample wasdiluted to 50mL with water and left to stand in the dark for30min at 25∘C Each sample had its absorbance verified at760 nm in spectrophotometer (Biochrom Model Biowave II)using water to establish a baseline

The calibration curve was calculated using tannic acid at01ndash05 120583gmL as a standard The total phenol content wasexpressed in milligram equivalents of tannic acid per gramof extract (mg EATg) using linear equations The assayswere conducted in triplicate and the experimental designwascompletely analyzed using the Tukey test (ANOVA) with theASSISTAT 76 beta software

Pearson correlation analysis was performed using theBioEstat 53 software and graphics were prepared withOrigimPro 86 We used the following ranges of the Pearsoncoefficient (r) to represent an absent or very weak correlation(000 to 019) a weak correlation (020 to 039) a moderatecorrelation (040 to 059) a strong correlation (060 to 079)and a very strong correlation (080 to 1) [42]

3 Results and Discussions

31 Molecular Identification Molecular identification wasachieved by sequencing the Internal Transcribed Spacer (ITS)region Sequences with 99 or more similarity were con-sidered to be from the same species Those with similaritiesbetween 93 and 98 were considered to be from the samegenus Sequences below 93 similarity were considered tobe a previously unidentified strain [43] Among the thirteenendophyte fungi analyzed 11 were identified as being from thePhomopsisDiaportheCochliobolus or Sordariomycetes genus(Table 1)

The unidentified fungi (4426 and 4400) may representnew species and further studies are needed to determinetheir phylogenetic classification

32 Antibacterial and Antifungal Activities The endophyticfungi extracts were tested against G+ve and Gminusve bacteria


Table 2 Minimum inhibitory concentration (MIC in 120583gmL) of crude extracts obtained from endophytic fungi isolated from Costus spiralisagainst yeasts and bacteria

Extract code Endophytic species Yeasts BacteriaCa Cp Ec Pa Kp Se Bs Sa Ef

CEBP1 Phomopsis sp 125 125 a 125 a a 125 a aCEDp2 Diaporthe phaseolorum 500 500 a 500 a a a a aCED3 Diaporthe sp 500 500 a a a a a a aCED4 Diaporthe sp 500 500 a a a a a a aCEP4 Phomopsis sp 125 125 a a a a 625 a aCE6 Not identified 500 500 a a a a a a aCED7 Diaporthe sp 250 250 a a a a 125 a aCES8 Sordariomycetes sp 250 250 a a a a 125 a aCE9 Not identified 500 500 a 125 a a 125 a aCEP10 Phomopsis sp 500 500 a a a a a a aCEDp11 Diaporthe phaseolorum 125 125 a 250 a 125 625 a 500CEC12 Cochliobolus ssp 125 125 a 125 a 625 125 a 125CES12 Sordariomycetes sp 250 250 a a a a 125 a aCECn a a a a a a a a aNystatin 19 19 mdash mdash mdash mdash mdash mdash mdashPenicillin mdash mdash mdash mdash mdash 19 39 39 39Tetracycline mdash mdash 19 39 156 mdash mdash mdash mdashNote Ca Candida albicans Cp C parapsilosis Ec Escherichia coli Pa Pseudomonas aeruginosa Kp Klebsiella pneumoniae Se Salmonella enterica subspenterica serovar Typhi Bs Bacillus subtilis Sa Staphylococcus aureus and Ef Enterococcus faecalisa MICMFCMBC above 1000120583gmL mdash not available CECn crude extract control negative

and yeasts These microorganisms were selected accordingto their medical importance The minimum inhibitory con-centration (MIC) of extracts was determined using themicrodilution method (Table 2) [44]

In the literature there are no representative criteria forthe MIC of endophytic fungi extracts Therefore we used thecriteria cited in Table 3 for comparison [45]

TheMIC values obtained for C albicans and C parapsilo-sis show moderate activities for all of the extracts tested TheCEBP1CEP4CEDp11 andCEC12 had aminimum fungicidalconcentration (MFC) equal to 125120583gmL for both yeastsshowing a fungicidal effect in the Phomopsis sp D phaseolo-rum and Cochliobolus sp extracts All other extracts showedno fungicidal activity

For the antibacterial evaluation the tested extracts wereactive against G+ve and Gminusve bacteria Eight extracts wereable to inhibit the growth ofB subtilis withMICbetween 625and 125 120583gmLB subtiliswas themicroorganismmost inhib-ited by the tested extracts followed by P aeruginosa whichwas inhibited by five extracts and S enterica and E faecaliswhich were both inhibited by two extracts The lower MICs(625120583gmL)which are considered to be good activities wereobtained from CEC12 against S enterica and from CEP5 andCEDp11 against B subtilis the last of which showed an MBCof 625 120583gmL The other antibacterial assays did not reportMBC Strains CEDp11 and CEC12 inhibited more pathogens(C albicans C parapsilosis P aeruginosa S enterica Bsubtilis and E faecalis)

Antimicrobial activities from endophytic fungi of Cspiralis have not previously been reported in the literature

Table 3 Criteria of selection of positive antimicrobial activities ofcrude extracts

MIC of crude extract ResultBelow 100 120583gmL Good antimicrobial activityBetween 100 and 500 120583gmL Moderate antimicrobial activityBetween 500 and 1000 120583gmL Weak antimicrobial activityAbove 1000 120583gmL InactiveMIC = minimum inhibitory concentration

Our results thus demonstrate the biotechnological potentialof these strains

33 Chemical Evaluation of Extracts Different classes ofsecondary metabolites were found by evaluating the 13fungi extracts We observed variations between the detectedcompounds and the fungi species (Table 4) Particularlynoteworthy are the presence of triterpenes in five extracts andthe presence of steroids and coumarins and the absence ofalkaloids and condensable tannins in all analyzed extracts

We conducted TLC using different mobile phases for the13 analyzed extracts The best resolution among the com-pounds was observedwith toluene ethyl-acetate (75 25 vv)observed by UV at 365 nm The fingerprints of all fungiextracts showed compounds with common characteristics ofcoumarins by emission of blue or green fluorescence andthese results were confirmed by NEU visualization atUV365 nm (Figure 1)


Table 4 TLC results of secondary metabolites in the fungal extracts

Crudeextract code Endophytic species UV

365 nm1 NEU2 Chloridricvanillin3

KOH104

KOH105 Dragendorf6 Sulfuric

vanillin7Sulfuricvanillin8

Liebermann-Burchard9


CEP1 Phomopsis sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (+) (+)CEDp2 Diaporthe phaseolorum (+) (+) (minus) (+) (minus) (minus) (minus) (minus) (+) (+)CED3 Diaporthe sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (+) (+)CED4 Diaporthe sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CEP4 Phomopsis sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CE6 Not identified (+) (+) (minus) (+) (minus) (minus) (+) (+) (+) (+)CED7 Diaporthe sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CES8 Sordariomycetes sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CE9 Not identified (+) (+) (minus) (+) (minus) (minus) (+) (minus) (+) (+)CEP10 Phomopsis sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CEDp11 Diaporthe phaseolorum (+) (+) (minus) (+) (+) (minus) (+) (minus) (minus) (+)CEC12 Cochliobolus ssp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CES13 Sordariomycetes sp (+) (+) (minus) (+) (minus) (minus) (+) (minus) (minus) (+)CECn (minus) (minus) (minus) (minus) (minus) (minus) (minus) (minus) (minus) (minus)(+) positive (minus) negative CECn crude extract control negative1Coumarins 2phenolic compounds 3protoanthocyanins leucoanthocyanins 4coumarins (mobile phase to coumarins) 5anthraquinones naphthoquinoneand anthocyanins (mobile phase to anthraquinones) 6alkaloids 7phenols sterols terpenes higher alcohols polyketides and saponins (mobile phase tosaponins) 8phenols sterols terpenes higher alcohols polyketides and saponins (mobile phase to mono- sesqui- and diterpenes) 9triterpenes (pink-reddishband) 10sterols (gray band)

Figure 1 Fingerprints of 13 fungi extracts obtained by TLC observed by UV at 365 nm Mobile phase toluene ethyl acetate (75 25 vv) 1CEP1 2CEDp2 3CED3 4CED4 5CEP5 6CE6 7CED7 8CES8 9CE9 10CEP10 11CEDp11 12CEC12 13CES13 C- negative control

Thefingerprints show the presence of one complexmatrixof compounds in the extracts With respect to the molecularcomposition we observed differences and a single profile forCE6 CEDp11 CEC12 and CES13 and we observed similar-ities between CEP1 and CEDp2 the samples CED3 CED4and CEP5 and the samples CED7 CES8 and CE9 It wasalso possible to observe variations in this matrix of com-pounds between different strains from D phaseolorumspecies (CEDp2 and CEDp11)

Experiments that showed differences in the production ofsecondary metabolites were observed with Streptomyces sp[46] These results confirmed the importance of using fin-gerprint acquisition and chemical screening as a tool for thesimple selection of chemical characteristics in bioprospectionof fungi strains

The coumarins are secondary metabolites found in dif-ferent organisms such as vegetables bacteria fungi lichens

and others This class of compounds was related to severalbiological activities including protease inhibition acetyl-cholinesterase K vitamin antagonism antimicrobial growthregulation antiallergenic antimalaric antiviral immuno-suppression hypolipidemic hypotensor antispasmodic andantioxidant activities [30 47ndash50]

To obtain more information about the chemical com-pounds observed in the fingerprints we conducted thefractionation of the crude extracts by liquid-liquid partitionusing dichloromethane as the nonpolar organic phase andwater as the polar phase Fractions were analyzed by TLC andcompared with the crude extracts The results from CEDp11and CEC12 show the nonpolar nature of most of the com-pounds in the extracts similar to the coumarins identified inprevious assays

Further the organic fractions show higher resolutionamong the compounds being visualized in higher number


Figure 2 TLC of CEDp11 and CEC12 samples and their respectivefractions visualized at UV365 nm Mobile phase toluene ethylacetate (75 25 vv) 11B crude extract 11Or organic fraction 11Aqaqueous fraction 12B crude extract 12Or organic fraction 12Aqaqueous fraction P quercetin standard

when compared with the crude extracts These data showthe complex matrix of compounds present in the extractsobtained from these fungi species This complex matrixcan increase the interest in the possible biotechnologicalapplications of these extracts and encourage the elucidationof the chemical structures of these molecules (Figure 2)

The CEC12 fingerprint (ethyl acetate toluene 25 75 vv)demonstrated 10 different fluorescent bands in the organicfraction Bands 2 3 and 6 (119877

119891034 042 and 062) represent

compounds that are exclusive to this extract (Table 5)When the mobile phase was used to separate anthracenic

compounds different from the other endophytic speciesthe sample CEDp11 from D phaseolorum showed peculiarcompounds The CEDp11 fingerprint from the organic frac-tion had 5 unique compounds 3 of which were colored byvisible light in TLC (Table 5) The presence of orange andyellow bands (119877

119891045 055 and 091) is characteristic of

anthraquinones Typical fluorescence bands for coumarinswere visualized in this sample (Table 5)

These results show the complexity of the analyzed sam-ples particularly the organic fractions of CEDp11 and CEC12

34 Quantification of Phenolic Compounds and Verificationof Antioxidant Activity The quantification of phenolic com-pounds from the 13 crude extracts and negative controlswas conducted Significant differences were found betweenthe samples (119875 lt 001) and the negative control (Table 6)Phenolic compounds have been very well known for theirantioxidant properties owing to their unique ability to act asfree radical scavengers which in turn is an outstandingattribute of their unique biochemical structure [51]

TLC screening revealed the presence of antioxidant sub-stances in all samples by the presence of yellow bands inpurple background resulting from the reduction of theDPPHradical As a result of these analyses the samples were sub-mitted for characterization of their antioxidant activity usingthe DPPH and FRAP methods The results were significant

Table 5 119877119891values of bands from organic subfractions of CEDp11

(D phaseolorum) and CEC12 (Cochliobolus ssp) in TLC

Band 119877119891

CharacteristicCEDp11lowast

1 019 Blue fluorescence (UV 365 nm)2 045 Orange (visible) red fluorescence (UV 365 nm)3 052 Green fluorescence (UV 365 nm)4 055 Orange (visible)5 091 Yellow (visible) fluorescence (UV 365 nm)

CEC12lowastlowast

2 034 Yellow fluorescence (UV 365 nm)mdashstrong3 042 Blue fluorescence (UV 365 nm)mdashstrong4 049 Blue fluorescence (UV 365 nm)mdashstrong5 055 Blue fluorescence (UV 365 nm)mdashstrong6 062 Blue fluorescence (UV 365 nm)mdashstrong7 068 Blue fluorescence (UV 365 nm)mdashweak8 076 Blue fluorescence (UV 365 nm)mdashstrong9 092 Blue fluorescence (UV 365 nm)mdashweak10 093 Blue fluorescence (UV 365 nm)mdashweaklowastMobile phase ethyl acetate methanol water (100 13 5 10 (vv))lowastlowastMobile phase ethyl acetate toluene (25 75 (vv))

particularly for CEC12 CE6 CES13 CEDp11 and CEP1(Table 6)

The average of values of antioxidant activities obtainedfrom the FRAP and DPPH methods had a very strongPearson correlation (119903 = 0901 119875 lt 005) showing that thesemethods have a positive correlation to the evaluated samplesThis correlation suggests that these compounds act ashydrogen sources for DPPH radical and block the electrondonation resulting in iron complex reduction in the FRAPmethodThere was also a strong positive correlation betweenthe phenolic compound contents in samples according to theFRAP and DPPH results (119903 = 072 119875 lt 005 119903 = 070119875 lt 005 resp) possibly attributed to the antioxidant activityof phenolic substances including coumarins

Extracts that had higher antioxidant activities were sub-mitted to liquid-liquid fractionation with dichloromethaneIn the resulting organic fractions the antioxidant activitiesincreased (Table 6) showing the nonpolar characteristicof the antioxidant compounds present as expected forcoumarins This is the first report of antioxidant activitiesfrom endophytic fungi from the Costaceae plants familyCoumarins that contain dihydroxyl groups in the ortho posi-tion such as fraxetin (78-di-hydroxy-6-methoxycoumarin)esculetin (67-di-hydroxy-coumarin) and 4-methyl esculetin(67-di-hydroxy-4-methylcoumarin) are considered power-ful lipid peroxidation inhibitors and can eliminate the super-oxide anion radical to promote iron chelation These propri-eties make these substances very interesting as antioxidantswith possible applications in radial-free disease prevention[50]

The extracts from endophytic fungi from C spiralishad antioxidant activities and the important and complexchemicals can be explored for biotechnological purposes


Table 6 Total phenol content and antioxidant activities by DPPH (CE50) and FRAP from hydroethanolic crude extracts and organicsubfractions from endophytic fungi

Code Endophytic speciesAverage of total

phenolic compoundsconcentrations(120583gTAEmg)lowast

FRAP assay (120583MFe2SO4mg)lowastlowastcrude extract

FRAP assay (120583MFe2SO4mg)lowastlowast

organic subfraction

DPPH assayIC50 (120583gmL)lowastlowastcrude extract

DPPH assayIC50 (120583gmL)lowastlowast

organic subfraction

CEP1 Phomopsis sp 420 d 36486 plusmn 238 4816 plusmn 123 66034 plusmn 271 45676 plusmn 113CEDp2 Diaporthe phaseolorum 371 d 29043 plusmn 121 NC 113691 plusmn 57 NCCED3 Diaporthe sp 349 d 23371 plusmn 023 NC 137596 plusmn 201 NCCED4 Diaporthe sp 365 d 17346 plusmn 034 NC 130900 plusmn 123 NCCEP4 Phomopsis sp 358 d 12005 plusmn 010 NC 142516 plusmn 210 NCCE6 Not identified 489 c 44529 plusmn 098 54714 plusmn 109 45201 plusmn 014 21356 plusmn 123CED7 Diaporthe sp 368 d 12025 plusmn 190 NC 176530 plusmn 076 NCCES8 Sordariomycetes sp 365 d 14389 plusmn 043 NC 123534 plusmn 210 NCCE9 Not identified 355 d 20014 plusmn 087 NC 101475 plusmn 234 NCCEP10 Phomopsis sp 371 d 13374 plusmn 029 NC 180226 plusmn 123 NCCEDp11 Diaporthe phaseolorum 567 b 29496 plusmn 109 4372 plusmn 098 65942 plusmn 076 42386 plusmn 076CEC12 Cochliobolus ssp 528 bc 47115 plusmn 287 5435 plusmn 058 37801 plusmn 087 18978 plusmn 123CES13 Sordariomycetes sp 710 a 41301 plusmn 176 5236 plusmn 123 62849 plusmn 156 39998 plusmn 014CECn 006 e NA NC NA NCAA mdash mdash mdash 641 641CE50 = concentration able to reduce in 50 the DPPH radical lowastaverages followed by the same letter are not significantly different (Tukeyrsquos test 119875 gt 001)lowastlowastvalues average plusmn standard deviation 119899 = 3 NA = not active NC not conducted CECn = crude extract control negative AA = ascorbic acid TAE = tannicacid equivalent

4 Conclusions

The biotechnological potential of pharmacological sub-stances from endophytic fungi extracts of C spiralis wasdemonstrated All extracts had inhibitory activities againstthe yeastsCandida albicans andC parapsilosis Some extractshad antimicrobial activities against the following bacteriaBacillus subtilis Pseudomonas aeruginosa Salmonella enter-ica and Enterococcus faecalis The antioxidant activities weremeasured by DPPH and FRAP and all fungi had positiveresults

The TLC results show very diverse chemicals in boththe number of compounds and the metabolic class whereascoumarins were present in all of the analyzed extracts Thefingerprint of CEDp11 (D phaseolorum) was different from allothers tested including the sample CEDp2 which was froma different strain of the same fungi species showing that eachstrain exhibits a unique chemical composition These resultshave not been previously reported in endophytic fungi fromthe Costaceae plants present in Brazilian ecosystems

Conflict of Interests

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

Acknowledgments

The authors thank Fundacao Oswaldo Cruz (FIOCRUZ)for reference microorganism donation Ilsamar Mendes for

collaboration with data tabulation and Fundacao de Amparoa Pesquisa do Estado do Tocantins (FAPT) and Con-selho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq) for financial support

References

[1] M Habsah M Amran M M Mackeen et al ldquoScreening ofZingiberaceae extracts for antimicrobial and antioxidant activ-itiesrdquo Journal of Ethnopharmacology vol 72 no 3 pp 403ndash4102000

[2] F G BragaM LM Bouzada R L Fabri et al ldquoAntileishmanialand antifungal activity of plants used in traditional medicine inBrazilrdquo Journal of Ethnopharmacology vol 111 no 2 pp 396ndash402 2007

[3] R R Hafidh A S Abdulamir F Abu Bakar F Abas F Jahan-shiri and Z Sekawi ldquoAntioxidant research in Asia in the periodfrom 2000ndash2008rdquo American Journal of Pharmacology and Toxi-cology vol 4 no 3 pp 48ndash66 2009

[4] B Schulz and C Boyle ldquoThe endophytic continuumrdquoMycolog-ical Research vol 109 no 6 pp 661ndash686 2005

[5] O Petrini ldquoFungal endophytes of tree leavesrdquo in MicrobialEcology of Leaves J Andrews and S S Hirano Eds pp 179ndash197 Springer New York NY USA 1991

[6] D Wilson ldquoEndophytemdashthe evolution of a term and clarifica-tion of its use and definitionrdquo Oikos vol 73 no 2 pp 274ndash2761995

[7] R B Jalgaonwala B V Mohite and R T Mahajan ldquoNaturalproducts from plant associated endophytic fungi a reviewrdquoJournal of Microbiology and Biotechnology Research vol 1 pp21ndash32 2011


[8] B H Ownley K D Gwinn and F E Vega ldquoEndophytic fungalentomopathogens with activity against plant pathogens ecol-ogy and evolutionrdquo BioControl vol 55 no 1 pp 113ndash128 2010

[9] P A Paranagama E M K Wijeratne and A A L GunatilakaldquoUncovering biosynthetic potential of plant-associated fungieffect of culture conditions on metabolite production by Para-phaeosphaeria quadriseptata andChaetomium chiversiirdquo Journalof Natural Products vol 70 no 12 pp 1939ndash1945 2007

[10] X-L Yang J-Z Zhang and D-Q Luo ldquoThe taxonomy biologyand chemistry of the fungal Pestalotiopsis genusrdquo NaturalProduct Reports vol 29 no 6 pp 622ndash641 2012

[11] K Nithya and J Muthumary ldquoSecondary metabolite fromPhomopsis sp isolated from Plumeria acutifolia Poiretrdquo RecentResearch in Science and Technology vol 2 no 4 pp 99ndash1032010

[12] A H Aly A Debbab and P Proksch ldquoFungal endophytesunique plant inhabitants with great promisesrdquo Applied Micro-biology and Biotechnology vol 90 no 6 pp 1829ndash1845 2011

[13] L W Wang B G Xu J Y Wang et al ldquoBioactive metabolitesfrom Phoma species an endophytic fungus from the Chinesemedicinal plant Arisaema erubescensrdquo Applied Microbiologyand Biotechnology vol 93 no 3 pp 1231ndash1239 2012

[14] GH SilvaH L Teles LM Zanardi et al ldquoCadinane sesquiter-penoids of Phomopsis cassiae an endophytic fungus associatedwith Cassia spectabilis (Leguminosae)rdquo Phytochemistry vol 67no 17 pp 1964ndash1969 2006

[15] Z Huang X Cai C Shao et al ldquoChemistry and weak antimi-crobial activities of phomopsins produced by mangrove endo-phytic fungus Phomopsis sp ZSU-H76rdquo Phytochemistry vol 69no 7 pp 1604ndash1608 2008

[16] R S Pimenta J F M Da Silva J S Buyer andW J JanisiewiczldquoEndophytic fungi from plums (Prunus domestica) and theirantifungal activity againstMonilinia fructicolardquo Journal of FoodProtection vol 75 pp 1883ndash1889 2012

[17] J G Ondeyka G L Helms O D Hensens et al ldquoNodulisporicacid A a novel and potent insecticide from a NodulisporiumSp isolation structure determination and chemical transfor-mationsrdquo Journal of the American Chemical Society vol 119 no38 pp 8809ndash8816 1997

[18] M Verza N S Arakawa N P Lopes et al ldquoBiotransformationof a tetra hydrofuran lignan by the endophytic fungus Phomop-sis sprdquo Journal of the Brazilian Chemical Society vol 20 no 1pp 195ndash200 2009

[19] L H Rosa V N Goncalves R B Caligiorne et al ldquoLeishmani-cidal trypanocidal and cytotoxic activities of endophytic fungiassociated with bioactive plants in Brazilrdquo Brazilian Journal ofMicrobiology vol 41 no 2 pp 420ndash430 2010

[20] C Hemtasin S Kanokmedhakul K Kanokmedhakul et alldquoCytotoxic pentacyclic and tetracyclic aromatic sesquiterpenesfromPhomopsis archerirdquo Journal of Natural Products vol 74 no4 pp 609ndash613 2011

[21] D Weber O Sterner T Anke S Gorzalczancy V Martino andC Acevedo ldquoPhomol a new antiinflammatory metabolite froman endophyte of the medicinal plant Erythrina crista-gallirdquoTheJournal of Antibiotics vol 57 no 9 pp 559ndash563 2004

[22] A H Aly A Debbab J Kjer and P Proksch ldquoFungal endo-phytes from higher plants a prolific source of phytochemicalsand other bioactive natural productsrdquo Fungal Diversity vol 41pp 1ndash16 2010

[23] G Strobel B Daisy U Castillo and JHarper ldquoNatural productsfrom endophytic microorganismsrdquo Journal of Natural Productsvol 67 no 2 pp 257ndash268 2004

[24] B Guo J R Dai S Ng et al ldquoCytonic acids A and B noveltridepside inhibitors of hCMV protease from the endophyticfungus Cytonaema speciesrdquo Journal of Natural Products vol 63no 5 pp 602ndash604 2000

[25] S Shweta S Zuehlke B T Ramesha et al ldquoEndophytic fungalstrains of Fusarium solani from Apodytes dimidiata E Mey exArn (Icacinaceae) produce camptothecin 10-hydroxycampt-othecin and 9-methoxycamptothecinrdquo Phytochemistry vol 71no 1 pp 117ndash122 2010

[26] HW Zhang Y C Song and R X Tan ldquoBiology and chemistryof endophytesrdquo Natural Product Reports vol 23 no 5 pp 753ndash771 2006

[27] Y Huang J Zhao L Zhou et al ldquoAntimicrobial compoundsfrom the endophytic fungus Fusarium sp Ppf4 isolated fromthe medicinal plant Paris polyphylla var yunnanensisrdquo NaturalProduct Communications vol 4 no 11 pp 1455ndash1458 2009

[28] T Bunyapaiboonsri S Yoiprommarat P Srikitikulchai KSrichomthong and S Lumyong ldquoOblongolides from the endo-phytic fungus Phomopsissp BCC 9789rdquo Journal of NaturalProducts vol 73 no 1 pp 55ndash59 2010

[29] P Mohana Kumara S Zuehlke V Priti et al ldquoFusarium prolif-eratum an endophytic fungus from Dysoxylum binectariferumHookf produces rohitukine a chromane alkaloid possessinganti-cancer activityrdquo Antonie van Leeuwenhoek vol 101 no 2pp 323ndash329 2012

[30] V M Chapla C R Biasetto and A R Araujo ldquoFungosendofıticos uma fonte inexplorada e sustentavel de novos ebioativos produtos naturaisrdquo Revista Virtual de Quımica vol 5no 3 2012

[31] M C C Ayres M S Brandao G M Vieira-Junior et alldquoAntibacterial activity of useful plants and chemical con-stituents of the roots of Copernicia pruniferardquo Brazilian Journalof Pharmacognosy vol 18 no 1 pp 90ndash97 2008

[32] D Huang O U Boxin and R L Prior ldquoThe chemistry behindantioxidant capacity assaysrdquo Journal of Agricultural and FoodChemistry vol 53 no 6 pp 1841ndash1856 2005

[33] S Kaul S Gupta M Ahmed and M K Dhar ldquoEndophyticfungi from medicinal plants a treasure hunt for bioactivemetabolitesrdquo Phytochemistry Reviews vol 11 no 4 pp 487ndash5052012

[34] G S de Hoog E Gottlich G Platas O Genilloud G Leottaand J van Brummelen ldquoEvolution taxonomy and ecology ofthe genusThelebolus in Antarcticardquo Studies in Mycology vol 51pp 29ndash66 2005

[35] Clinical and Laboratory Standards Institute (CLSI) Methodsfor Dilution Antimicrobial Susceptibility Tests for Bacteria ThatGrow Aerobically Approved Standard M07-A8 CLSI WaynePa USA 2009

[36] Clinical and Laboratory Standards Institute (CLSI) ReferenceMethod for Broth Dilution Antifungal Susceptibility Testing ofYeasts Approved Standard M27-A3 NCCLS Wayne Pa USA2008

[37] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWTmdashFoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[38] H H Moresco G S Queiroz M G Pizzolatti and I M CBrighente ldquoChemical constituents and evaluation of the toxicand antioxidant activities ofAverrhoa carambola leavesrdquo Brazil-ian Journal of Pharmacognosy vol 22 no 2 pp 319ndash324 2012

[39] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquoThe FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996


[40] H Wagner and S Bladt Plant Drug Analysis A Thin LayerChromatography Atlas Springer Berlin Germany 2nd edition2001

[41] E L CAmorim J ENascimento JMMonteiro T J S PeixotoSobrinho T A S Araujo and U P Albuquerque ldquoA simple andaccurate procedure for the determination of tannin andflavonoid levels and some applications in ethnobotany andethnopharmacologyrdquo Functional Ecosystems and Communitiesvol 2 no 1 pp 88ndash94 2008

[42] M J Campbell Statistics at SquareOne BMJ PublishingGroup9th edition 1997 httpwwwbmjcomabout-bmjresources-readerspublicationsstatistics-square-one

[43] J I Pounder K E Simmon C A Barton S L Hohmann ME Brandt and C A Petti ldquoDiscovering potential pathogensamong fungi identified as nonsporulating moldsrdquo Journal ofClinical Microbiology vol 45 no 2 pp 568ndash571 2007

[44] E G Alves A H C Vinholis L A Casemiro et al ldquoEstudocomparativo de tecnicas de screening para avaliacao da ativi-dade anti-bacteriana de extratos brutos de especies vegetais e desubstancias purasrdquo Quımica Nova vol 31 no 5 pp 1224ndash12292008

[45] F B Holetz G L Pessini N R Sanches D A G Cortez C VNakamura and B P Dias Filho ldquoScreening of some plants usedin the Brazilian folk medicine for the treatment of infectiousdiseasesrdquoMemorias do Instituto Oswaldo Cruz vol 97 no 7 pp1027ndash1031 2002

[46] A Taddei M Valderrama J Giarrizzo M Rey and C CastellildquoChemical screening a simple approach to visualizing Strep-tomyces diversity for drug discovery and further researchrdquoResearch in Microbiology vol 157 no 3 pp 291ndash297 2006

[47] M Kuramata S Fujioka A Shimada T Kawano and YKimura ldquoCitrinolactones A B and C and sclerotinin C plantgrowth regulators from Penicillium citrinumrdquo Bioscience Bio-technology and Biochemistry vol 71 no 2 pp 499ndash503 2007

[48] K G Guimaraes J D de Souza Filho T R dosMares-Guia andF C Braga ldquoDihydroisocoumarin from Xyris pterygoblepharaactive against dermatophyte fungirdquo Phytochemistry vol 69 no2 pp 439ndash444 2008

[49] C M Oliveira L O Regasini G H Silva et al ldquoDihydroi-socoumarins produced by Xylaria sp and Penicillium spendophytic fungi associated with Piper aduncum and Alibertiamacrophyllardquo Phytochemistry Letters vol 4 no 2 pp 93ndash952011

[50] C M O Simoes E P Schenkel G Gosmann J C P de MelloL A Mentz and P R Petrovick Farmacognosia Da Planta aoMedicamento UFSC UFRGS Florianopolis Brazil 6th edi-tion 2010

[51] M A Soobrattee V S Neergheen A Luximon-Ramma O IAruoma and T Bahorun ldquoPhenolics as potential antioxi-dant therapeutic agents mechanism and actionsrdquo MutationResearch Fundamental and Molecular Mechanisms of Mutage-nesis vol 579 no 1-2 pp 200ndash213 2005

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


Despite scientific advances infectious diseases remain amajor contributor tomortality andmorbidity in public healthThemain explanation for this is the ability ofmicroorganismsto acquire resistance against antimicrobial substances Thischaracteristic results in a constant need to discover anddevelop new drugs [31]

Oxygen radicals and superoxide anions play importantroles in biochemicalphysiological reactions in the humanbody However when these species are produced in excessdue to pathophysiological processes or environmental inter-ference they can promote tissue damage and result in disease[32] Currently few antioxidant substances can be used inclinical situations thus underscoring the necessity to identifynew safe and efficient molecules for this purpose [33]Bioactive molecules from new sources such as endophyticfungi deserve attention because they may lead to drugs withdifferent pathologies food additives or cosmetics

2 Materials and Methods

21 Endophytic Fungi Endophytic fungi were isolated fromhealthy leaves of C spiralis From each leaf three 05 cm2pieces were cut The leaf sample fragments were surface ster-ilized by successive dipping in 2 Extran detergent (Sigma-Aldrich) for 2min 70 ethanol for 1min and 2 sodiumhypochlorite for 3min and then by washing with steriledistilled water for 2min they were then placed on potatodextrose agar (PDA Difco) containing 100mgmL chloram-phenicol The sterile water wash was plated on PDA to con-firm external disinfection Plates were incubated for up to 60days at 25∘CThemycelia from themargins of fungal coloniesgrowing from the leaf fragments were transferred to freshPDA and further purified by transferring hyphal plugs to newPDA plates Thirteen endophytic fungi were selected fromleaves of C spiralis These microorganisms were previouslytested with phytopathogen controls (data not shown) and arepreserved in Culture Collection from the Laboratory ofGeneral and Applied Microbiology at the Federal Universityof Tocantins Brazil

22 Molecular Identification

221 Fungal DNA Extraction Endophytic fungi were cul-tured on Sabouraud dextrose agar slants for 7 days at 25∘CTheDNA extraction was performed according to themethoddescribed by de Hoog et al [34] Briefly approximately 01 gof fungal mycelia was transferred to 15mL tubes containing400 120583L of lysis buffer (50mM Tris-HCl pH 80 5mM EDTApH 80 100mMNaCl 1 SDS) and kept at minus20∘C for 10minThe fungi cells weremixedwith 1mmglass beads and agitatedusing a Vortex for 30 sec followed by a 30 sec interval onice then repeated Cells were incubated for 30min at 60∘Cwith 5 120583L 20mgmL Proteinase K The samples were thenincubated for 10min at 65∘C with 162 120583L of CTAB solution(200mM Tris-HCl pH 75 200mM Na-EDTA 82 NaCl2CTAB) Following incubation 570 120583L chloroform isoam-ylic alcohol (24 1 vv) was added and the tubes were kept for10min on ice The homogenate was centrifuged at 14500timesg

for 10min at 4∘C Then the supernatant was transferred toa new tube and 10 of the residual volume of 3M sodiumacetate was added The mixture was then incubated on icefor 30min Precipitates were removed by centrifugation andthe DNAwas recovered by isopropanol precipitation washedwith 70 (vv) ethanol allowed to air dry and resuspendedin 50120583L of TE buffer (10mM Tris-HCl pH 80 and 01mMEDTA) The concentration was measured by monitoring theUV absorbance at 260 nm using Nanodrop 2000 (ThermoUSA) and the DNA was diluted to 10 ng120583L with ultrapurewater

222 DNA Amplification and Sequencing DNA sequencingwas performed using a BigDye Direct Cycle Sequencing Kit(Life Technologies) For PCR amplification 1120583L of dilutedDNA (10 ng120583L) wasmixedwith 50120583L of BigDyeDirect PCRMaster Mix 075 120583L of each forward ITS1-M13 (51015840-TGTAAA-ACGACGGCCAGTTCCGTAGGTGAACCTGCGG-31015840) andreverse ITS4-M13 (51015840-CAGGAAACAGCTATGACCTCC-TCCGCTTATTGATATGC-31015840) primers at a 10 120583M concen-tration which include the M13 universal primer sequencesand 24 120583L of ultrapure water PCR cycling conditions were asfollows initial denaturation of 10min at 94∘C followed by 35cycles (30 s) of denaturation at 96∘C annealing for 15 sec at62∘C and extension for 30 sec at 72∘C Following PCR 3 120583Lof BigDye Direct SequencingMaster Mix and 1 120583L of forwardBigDye Direct M13 forward primer (provided) were addeddirectly to the PCR sample put back into the PCR thermalcycler and incubated as follows 15min at 37∘C 2min at 80∘Cand 1min at 96∘C followed by 25 cycles of 10 sec at 96∘C5 sec at 50∘C and 75 sec at 60∘C

At the completion of the sequencing reaction the sequen-cing products were purified following the ethanolEDTApro-tocol for BigDyeTerminator version 31 Cycle SequencingKitElectrophoresis was performed on the Applied Biosystems3500xLGenetic Analyzer Electropherogramswere proofreadwith the software BioEdit Sequence Alignment Editor (1997ndash2013) The sequencing results for each sample were analyzedusing BLASTn (Basic Local Alignment Search Tool version2215 of BLAST 20) to verify the results and to identify thefungus for each sample

23 Crude Extract Acquisition The 13 endophytes weremultiplicated in PDA at 25∘C plusmn 2 for 5 days Subsequently6mm discs were placed in the center of 4 Petri dishes withPDA agar and incubated at 25∘C plusmn 2 for 14 days These cul-tures were extracted (1 20 pv) by hydroethanolicmaceration(1 3 vv) for 48 h at 25∘C under agitation The samples werecentrifuged at 2400timesg for 20min at 8∘C and then filteredThe solvent from each supernatant was removed on a rotaryevaporator at 35∘C lyophilized and stored in a desiccatorwithout lightThe same extraction process was performed for4 sterile BDA Petri dishes that were used as negative control

24 Antimicrobial Activity

241Microorganisms Allmicroorganismswere obtained fromthe American Type Culture Collection (ATCC Rockville











Abscontr (1)



50



































KOH104

















119891034 042 and 062) represent










Band 119877119891



CEC12lowastlowast












organic subfraction



organic subfraction


4 Conclusions





Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Abscontr (1)



50



































KOH104

















119891034 042 and 062) represent










Band 119877119891



CEC12lowastlowast












organic subfraction



organic subfraction


4 Conclusions





Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































KOH104

















119891034 042 and 062) represent










Band 119877119891



CEC12lowastlowast












organic subfraction



organic subfraction


4 Conclusions





Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















119891034 042 and 062) represent










Band 119877119891



CEC12lowastlowast












organic subfraction



organic subfraction


4 Conclusions





Acknowledgments



References



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















organic subfraction



organic subfraction


4 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
















research article chemical assessment and antimicrobial and

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