antimicrobial activity, growth inhibition of human tumour ... 180.pdf ·...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 659183 9 pageshttpdxdoiorg1011552013659183

Research ArticleAntimicrobial Activity Growth Inhibition of Human TumourCell Lines and Phytochemical Characterization ofthe Hydromethanolic Extract Obtained from Sapindussaponaria L Aerial Parts

Khaled N Rashed1 Ana SiriT2 Jasmina GlamoIlija2 Ricardo C Calhelha34

Isabel C F R Ferreira3 and Marina SokoviT2

1 Pharmacognosy Department National Research Centre Dokki Giza 12311 Egypt2 Institute for Biological Research ldquoSinisa Stankovicrdquo University of Belgrade Boulevard Despota Stefana 142 11000 Belgrade Serbia3Montain Research Center (CIMO) ESA Polytechnic Institute of Braganca Campus de Santa Apolonia Apartado 11725301-855 Braganca Portugal

4 Centre of Chemistry University of Minho Campus de Gualtar 4710-057 Braga Portugal

Correspondence should be addressed to Marina Sokovic mrisibissbgacrs

Received 26 April 2013 Revised 7 October 2013 Accepted 9 October 2013

Academic Editor Ziad Daoud

Copyright copy 2013 Khaled N Rashed 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 hydromethanolic extract of Sapindus saponaria L aerial parts was investigated for antimicrobial activity (against several Gram-positive and Gram-negative bacteria and fungi) and capacity to inhibit the growth of different human tumor cell lines as alsonontumor liver cells The evaluated extract was further characterized in terms of phytochemicals using UV 1H-NMR 13C-NMRand MS spectroscopic tools The extract has shown a significant antimicrobial activity on all tested bacterial and fungal speciesThe best activity was achieved against Bacillus cereus and Staphylococcus aureus among bacteria and against all three Penicilliumspecies tested It also revealed cytotoxicity against human colon (HCT-15) cervical (HeLa) breast (MCF-7) and lung (NCI-H460)carcinoma cell lines with HeLa being the most susceptible tumor cell line The extract was not toxic for nontumor liver cellsChromatographic separation of the extract resulted in the isolation and identification of stigmasterol oleanolic acid luteolinluteolin 8-119862-120573-glucoside (orientin) luteolin 6-119862-120573-glucoside (isoorientin) luteolin 7-119874-120573-glucuronide and rutin The results ofthe present findings may be useful for the discovery of novel antitumor and antimicrobial agents from plant origin

1 Introduction

Plants are considered as one of the main sources of biologi-cally active compounds In spite of the recent domination ofthe synthetic chemistry as a method to discover and producedrugs the potential of bioactive plants or their extracts toprovide new and novel products for disease treatment andprevention is still enormous [1]

The discovery of antibiotics has decreased the spread andseverity of a wide variety of diseases However and as a resultof their uncontrolled use the efficiency of many antibiotics isbeing threatened by the emergence of microbial resistance toexisting chemotherapeutic agents [2]While bioactive natural

compounds have been isolatedmainly from cultivablemicro-bial strains untapped biologically active metabolites of dif-ferent resources including plants remain to be investigated toalleviate or help responding to current health care situations[3] Plant-derived natural products represent an attractivesource of antimicrobial agents since they are natural havemanageable side effects and are available at affordable prices[4] Furthermore those natural products may have differentmechanisms from conventional drugs and could be of clinicalimportance in health care improvement [5]

Cancer is characterized by uncontrolled growth andspread of abnormal cells [6] For many years scientistswere searching for chemically synthesized compounds with

2 BioMed Research International

antitumor properties In the last few decades research hasfocused on the use of natural products crude plant extractsor a combination of different phytochemicals for cancertherapy this trend is based upon first the synergistic effectof the different plant metabolites in the crude extract andsecond the multiple points of intervention of such extracts[7]

Sapindus saponaria L is a tree from Sapindaceae familyand it is popularly known as soapberry being distributed inCentral and South America The bark root and fruits areused in popular medicine as tranquilizer astringent diureticexpectorant tonic blood cleanser healing and to countercough [8] being also a neutralizer of hemorrhage [9] Itsfruits have activity as antifungal [10] and larvicidal [11]Abdel-Wahab and Selim [12] detected the presence of car-bohydrates steroids flavonoids and saponins in leaves andstems of S saponaria

The objective of the present study was to investigateantimicrobial activity of the hydromethanolic extract ofS saponaria aerial parts and also its capacity to inhibitthe growth of different human tumor cell lines The mainphytochemicals present in the extract were completely char-acterized by UV 1H-NMR 13C-NMR and MS

2 Materials and Methods

21 Plant Material and Preparation of the HydromethanolicExtract Sapindus saponaria L aerial parts were collectedfromAl-Zohiriya garden Giza Egypt inApril 2011The plantwas identified by Dr Mohammed El-Gebaly Departmentof Botany National Research Centre (NRC) and by MrsTereez Labib Consultant of plant taxonomy at the Ministryof Agriculture andDirector ofOrman botanical garden GizaEgypt A voucher specimen number 20532ss was deposited inthe herbarium of Al-Zohiriya garden Giza Egypt

For the extract preparation 12 kg of air dried powder ofS saponaria aerial parts was extracted with methanol water80 20 (vv) at room temperature several times until exhaus-tionThe extract was concentrated under reduced pressure togive 68 g of crude extract

22 Evaluation of Antimicrobial Activity ofthe Hydromethanolic Extract

Antibacterial Activity The following Gram (minus) (Enterobac-ter cloacae human isolate Escherichia coli ATCC 35210Pseudomonas aeruginosa ATCC 27853 and Salmonellatyphimurium ATCC 13311) and Gram (+) bacteria (Bacilluscereus clinical isolate Listeria monocytogenes NCTC 7973Micrococcus flavus ATCC 10240 and Staphylococcus aureusATCC 6538) were used The organisms were obtained fromMycological Laboratory Department of Plant PhysiologyInstitute for Biological Research ldquoSinisa Stankovicrdquo Univer-sity of Belgrade Serbia The antibacterial assay was carriedout by microdilution method [13] The bacterial suspensionswere adjusted with sterile saline to a concentration of 10 times105 CFUmL The inocula were prepared daily and storedat 4∘C until use Dilutions of the inocula were cultured

on solid medium to verify the absence of contaminationand to check the validity of the inoculum The minimuminhibitory and bactericidal concentrations (MICs andMBCs)were determined using 96-well microtitre plates The extractwas diluted in 5 of DMSO (10mgmL) and added to trypticsoy broth (TSB) medium (100 120583L) with bacterial inoculum(10times 104 CFUperwell) to achieve thewanted concentrationsThe microplates were incubated at rotary shaker (160 rpm)for 24 h at 37∘C The following day 30 120583L of 02mgmLsolution of INT (119901-iodonitrotetrazolium violet) was addedand the plates were returned to the incubator for at leastone-half hour to ensure adequate color reaction Inhibitionof growth was indicated by a clear solution or a definitedecrease in color reaction [14] The lowest concentrationswithout visible growth (at the binocular microscope) weredefined as concentrations that completely inhibited bacterialgrowth (MICs) The MBCs were determined by serial sub-cultivation of 2120583L into microtitre plates containing 100120583L ofbroth per well and further incubation for 24 h The lowestconcentration with no visible growth was defined as theMBC indicating 995 killing of the original inoculum Theoptical density of each well was measured at a wavelength of655 nm by microplate manager 40 (Bio-Rad Laboratories)and compared with a blank and the positive control Theantibiotics streptomycin and ampicillin were used as positivecontrols (1mgmL in sterile physiological saline) Threeindependent experiments were performed in triplicate

Antifungal Activity The used fungi are Aspergillus fumigatus(ATCC 1022)Aspergillus versicolor (ATCC 11730)Aspergillusochraceus (ATCC 12066) Aspergillus niger (ATCC 6275)Trichoderma viride (IAM 5061) Penicillium funiculosum(ATCC 36839) Penicillium ochrochloron (ATCC 9112) andPenicillium verrucosum var cyclopium were obtained fromMycological Laboratory Department of Plant PhysiologyInstitute for Biological Research ldquoSinisa Stankovicrdquo Univer-sity of Belgrade Serbia The micromycetes were maintainedon malt agar and the cultures were stored at 4∘C and sub-cultured once a month The antifungal assay was carriedout by modified microdilution technique [15 16] The fungalspores were washed from the surface of agar plates withsterile 085 saline containing 01Tween 80 (vv)The sporesuspension was adjusted with sterile saline to a concentrationof approximately 10 times 105 in a final volume of 100120583L perwellThe inocula were stored at 4∘C for further use Dilutionsof the inoculum were cultured on solid malt agar to verifythe absence of contamination and to check the validity ofthe inoculum MIC determinations were performed by aserial dilution technique using 96-well microtiter plates Theextract was diluted in 5 of DMSO (10mgmL) and added inbroth Malt Medium (MA) with inoculum The microplateswere incubated at rotary shaker (160 rpm) for 72 h at 28∘CThe lowest concentrations without visible growth (at thebinocular microscope) were defined as MICs The fungicidalconcentrations (MFCs) were determined by serial subculti-vation of 2120583L of tested fractions dissolved in medium andinoculated for 72 h into microtiter plates containing 100120583L ofbroth per well and further incubation for 72 h at 28∘C Thelowest concentration with no visible growth was defined as

BioMed Research International 3

MFC indicating 995 killing of the original inoculum Thefungicides bifonazole and ketoconazole were used as positivecontrols (1ndash3500 120583gmL) Three independent experimentswere performed in duplicate

23 Evaluation of Cells Growth Inhibition Capacity of theHydromethanolic Extract Five human tumor cell lines wereused MCF-7 (breast adenocarcinoma) NCI-H460 (non-small cell lung cancer) HCT-15 (colon carcinoma) HeLa(cervical carcinoma) and HepG2 (hepatocellular carci-noma) Cells were routinely maintained as adherent cell cul-tures in RPMI-1640medium containing 10 heat-inactivatedfetal bovine serum (FBS) and 2mM glutamine (MCF-7 NCI-H460 and HCT-15) or in DMEM supplementedwith 10 FBS 2mM glutamine 100UmL penicillin and100mgmL streptomycin (HeLa and HepG2 cells) at 37∘Cin a humidified air incubator containing 5 CO

2 Each cell

line was plated at an appropriate density (75 times 103 cellswellfor MCF-7 NCI-H460 and HCT-15 or 10 times 104 cellswellfor HeLa and HepG2) in 96-well plates and allowed to beattached for 24 h Sulforhodamine B assay was performedaccording to a procedure previously described by the authors[17] Briefly cells were then treated for 48 h with variousextract concentrations Following this incubation period theadherent cells were fixed by adding 10 cold trichloroaceticacid (TCA 100 120583L) and incubated for 60min at 4∘C Plateswere then washed with deionized water and dried sulforho-damine B solution (01 in 1 acetic acid 100120583L) was thenadded to each plate well and incubated for 30min at roomtemperature Unbound SRB was removed by washing with1 acetic acid Plates were air dried the bound SRB wassolubilised with 10mM Tris (200120583L) and the absorbancewas measured at 540 nm in the microplate reader mentionedabove

For hepatotoxicity evaluation a cell culture was preparedfrom a freshly harvested porcine liver obtained from a localslaughter house according to a procedure established by theauthors [17] it was designed as PLP2 Cultivation of the cellswas continuedwith directmonitoring every two to three daysusing a phase contrast microscope Before confluence wasreached cells were subcultured and plated in 96-well platesat a density of 10 times 104 cellswell and cultivated in DMEMmedium with 10 FBS 100UmL penicillin and 100 120583gmLstreptomycin Ellipticine was used as positive control (024ndash652120583gmL)

Three independent experiments were performed in tripli-cate and the results were expressed asmean valuesplusmn standarddeviation (SD)

24 Phytochemical Characterization ofthe Hydromethanolic Extract

Experimental UVVIS ShimadzuUV-visible recording spec-trophotometer model-UV 240 (NRC Egypt) Spectroscopicdata NMRmdashVarian 400MHz MS (Finnigan MAT SSQ7000 70 ev) Silica gel (60ndash200mesh Merck) Sephadex LH-20 (Sigma) thin layer chromatography (TLC) precoatedsheets of silica gel 60 F

254(Merck)

Isolation of the Bioactive Compounds The crude hydrometh-anolic extract (68 g) was defatted with petroleum ether (60ndash80∘C) the defatted extract (52 g) was subjected to silica gelcolumn chromatography elutingwith dichloromethane ethylacetate and methanol gradually The fractions that showedsimilar thin layer chromatography (TLC) were collected andaccording to that four fractions were obtained Fraction 1(17 g) elutedwith dichloromethane ethyl acetate (80 20 vv)gave compound (1) (stigmasterol 19mg) and further elu-tion with dichloromethane ethyl acetate (60 40 vv) gavecompound (2) (oleanolic acid 22mg) Fraction 2 (14 g)eluted with ethyl acetate gave compound (3) (luteolin 12mg)Fraction 3 (900mg) eluted with ethyl acetate methanol(95 5 vv) gave compound (4) (luteolin 8-119862-120573-glucoside(orientin) 24mg) and compound (5) (luteolin 6-119862-120573-glucoside (isoorientin) 27mg) and further elutionwith ethylacetate and methanol (90 10 and 80 20 vv) respectivelygave compound (6) (luteolin 7-119874-120573-glucuronide 29mg) andcompound (7) (quercetin 3-119874-120572-rutinoside 22mg) obtainedfrom fraction 4 (175mg) All compounds were purified onsephadex LH-20 column

Acid Hydrolysis of Flavonoids Solutions of 5mg of com-pounds (4) (5) (6) and (7) in 5mL 10 HCl were heatedfor 5 hThe reactionmixture was extracted with ethyl acetateThe ethyl acetate fraction (aglycone) and the aqueous fraction(sugars) were concentrated for identification The sugarswere identified by TLC (acetonitrile water 85 15 vv) bycomparison with authentic standards

3 Results and Discussion

31 Antimicrobial Activity Theresults of antibacterial activityof hydromethanolic extract of S saponaria are presentedin Table 1 and Figure 1 Inhibitory activity was achieved atconcentration of 03ndash125mgmL while bactericidal effectwas obtained at 125ndash25mgmL The most sensitive bacte-rial species were Bacillus cereus and Staphylococcus aureuswhile Listeria monocytogenes and Salmonella typhimuriumwere the most resistant species to tested extract In gen-eral both antibiotics showed better activity than the testedextract Streptomycin possessed MICs of 005ndash025mgmLand MBCs of 010ndash050mgmL while ampicillin revealedMICs of 010ndash030mgmL and MBCs of 015ndash050mgmL

All microfungi were sensitive to hydromethanolic extractof S saponaria (Table 2 and Figure 2) The extract inhibitedall microfungi at 0075ndash06mgmL (MIC) and completelystopped the growth (MFC) at 03ndash125mgmL The extractexhibited inhibitory concentrations at 0075ndash03mgmL andfungicidal effects at 03ndash50mgmL The most sensitivemicrofungi were Penicillium species on the other handAspergillus fumigatus was the most resistant to the testedextract Commercial antifungal agents bifonazole (MIC010ndash020mgmL MFC 020ndash025mgmL) and ketoconazole(MIC 015ndash250mgmL MFC 020ndash350mgmL) were ingeneral more active than the investigated extract with anexception in case of Penicillium species The extract showedhigher inhibitory activity effect towards all three Penicillium


Table 1 Antibacterial activity of the hydromethanolic extract of Sapindus saponaria aerial parts and standards

BacteriaExtract

MIC (mgmL)MBC (mgmL)

StreptomycinMIC (mgmL)MBC (mgmL)

AmpicillinMIC (mgmL)MBC (mgmL)

Bacillus cereus 03 plusmn 000 005 plusmn 0005 01 plusmn 005

125 plusmn 010 01 plusmn 003 015 plusmn 005

Micrococcus flavus 03 plusmn 001 0125 plusmn 0015 01 plusmn 000


Staphylococcus aureus 03 plusmn 001 025 plusmn 0003 01 plusmn 005


Listeria monocytogenes 125 plusmn 010 015 plusmn 005 015 plusmn 003


Escherichia coli 06 plusmn 000 010 plusmn 003 030 plusmn 005


Pseudomonas aeruginosa 06 plusmn 006 005 plusmn 0006 010 plusmn 000


Enterobacter cloacae 06 plusmn 000 005 plusmn 0005 015 plusmn 000


Salmonella typhimurium 125 plusmn 025 005 plusmn 0006 015 plusmn 003


MIC minimum inhibitory concentration MBC minimum bactericidal concentration

species tested than both mycotics Also the tested extractexhibited higher fungicidal activity against P ochrochloronand P verrucosum than ketoconazole

According to the existing literature the investigation ofSapindus saponaria extracts is limited but there is moreinformation about isolated compounds Extracts from thedried pericarp of S saponaria fruits were investigated fortheir antifungal activity against clinical isolates of yeastsCandida albicans and 119862 non-albicans from vaginal secre-tions of women with vulvovaginal candidiasis From alltested extracts the n-BuOH and one of its fractions showedstrong activity against all Candida isolates tested [10] Theacetylated saponin 3-b-O-[a-L-rhamnopyranosyl-(1-3)-b-D-glucopyranosyl] hederagenin was detected by Lemos et al[18] who observed antimicrobial activity against Pseu-domonas aeruginosa Bacillus subtilis and Cryptococcus neo-formans Triterpenoid saponins with hederagenin or oleano-lic acid as aglycone have been found to possess antifungalactivity against C glabrata C albicans Trichosporon beigeliiPenicillium avelaneum UC-4376 Pyricularia oryzae Crypto-coccus neoformans Coccidioides immitis and Saccharomycescerevisiae as well as against the dermatophytes Microsporumcanis and Trichophyton mentagrophytes [19 20] Triterpeneacids (ursolic and oleanolic) isolated from Miconia speciesshowed a significant antibacterial activity against some bac-terial strains [21] Luteolin showed selectively antibacterialactivity against MRSA and methicillin-sensitive S aureusstrains with MIC 39 to 156 and 625 to 125120583gmL respec-tively [22] Also luteolin 7-119874-glucosides possess antibacterialactivity [23] and rutin has been shown to exhibit antimicro-bial activity [24]

Fromprevious results it could be noticed that all flavonoidaglycones (myricetin quercetin and luteolin) showed better

antibacterial and antifungal activities than other flavonoidglycosides [25 26] Luteolin exhibited relatively higher activi-ties than the other compounds andwasmore effective againstfungi than against bacteria [27 28] These authors presentedthat certain features relating to flavonoid structure andantimicrobial activity can be identifiedThe active flavonoidswere polyhydroxylated (myricetin datiscetin quercetin lute-olin and kaempferol) except for flavone which does notcontain any hydroxyl group These active flavonoids havein common the obligatory C-4 keto group and hydroxylgroup substitutions at C-3 C-5 and C-7 and have at leastone hydroxyl group on ring B These observations indicatethat the more hydrophilic flavonols or flavones are betterinhibitors than less hydrophilic ones These observationssuggest that the hydroxyl group at C-3 is required for activityThis requirement however is not necessary for the flavoneskeleton as in the case of luteolin Antimicrobial activity oftotal extract (although low compared to others) may be dueto the presence of some aglycones unstable flavonoid gly-cosides or some other bioactive secondary metabolites Thisis in agreementwith the literature that plant extracts generallycontain flavonoids in glycosidic formThis may be the reasonwhy the plant extract did not produce asmarked inhibition assome fractionated extracts or asmany of the pure compounds[29]

32 Growth Inhibition of Human Tumor Cell Lines Thehydromethanolic extract of S saponaria revealed cytotoxicityagainst human colon cervical breast and lung carcinomacell lines (Table 3) Among the tested human tumor celllines themost susceptible onewasHeLa (cervical carcinomaGI50

= 258120583gmL) showing similar results to the other


HO

HO

HOH

H

H

H

H

H

CH3

CH3

CH3

CH3

CH3

CH3

HO

OH

(2) Oleanolic acid

O

O

O

O

O

OH

OH

HO

HO

HO

OHOH

OH

OH

OH

(3) Luteolin

O O O

O

OH

OH

HO

HO

HO OH

OH

(5) Isoorientin (6) Luteolin 7-O-120573-glucuronide

O

O

O

OH

OH

OH

OHHO

O

OH

OH

O

O

HO

OH

OH

OH OH

OHOH

OOH

HO

HO

HO

O

O

(7) Rutin

H3C

H3C

H3C

H3C H3C

H3C

(1) Stigmasterol

CH2OH

(4) Orientin

H3C

Figure 1 Chemical structure of the compounds isolated and identified in the hydromethanolic extract of Sapindus saponaria aerial parts

tested tumor cell lines The extract up to 800 120583gmL didnot show capacity to inhibit the growth of HepG2 cell line(hepatocellular carcinoma) Moreover the hydromethanolicextract of S saponaria up to 800120583gmL was not toxic fornontumor liver cells Ellipticine was used as positive control

but the comparisonwith the extract results should be avoidedbecause it is an individualpurified compound and not amix-ture like the extract (in the crude extract the concentrationof each individual bioactive compound is certainly muchlower and in the range of the pure compound) Furthermore


Table 2 Antifungal activity of the hydromethanolic extract of Sapindus saponaria aerial parts and standards

FungiExtract

MIC (mgmL)MFC (mgmL)

BifonazoleMIC (mgmL)MFC (mgmL)

KetoconazoleMIC (mgmL)MFC (mgmL)

Aspergillus fumigatus 06 plusmn 006 015 plusmn 003 020 plusmn 003


Aspergillus versicolor 03 plusmn 006 010 plusmn 005 020 plusmn 003


Aspergillus ochraceus 02 plusmn 003 015 plusmn 003 015 plusmn 003


Aspergillus niger 02 plusmn 003 015 plusmn 003 020 plusmn 006


Penicillium verucosumvar cyclopium



Penicillium ochrochloron 0075 plusmn 000 020 plusmn 006 020 plusmn 003


Penicillium funiculosum 0075 plusmn 000 020 plusmn 003 25 plusmn 03


Trichoderma viride 03 plusmn 006 010 plusmn 005 020 plusmn 003


MIC minimum inhibitory concentration MFC minimum fungicidal concentration

Figure 2 Antibacterial activity of the hydromethanolic extract ofSapindus saponaria aerial parts by microdilution method The rowsrepresent different bacterial species and columns different extractconcentrations (03ndash25mgmL) Comparison between control-bacterial growth (K-red color) blank control (B-yellow) and treatedsamples (yellow color MIC and MBC) and treated samples with noactivity (red color) in INT assay

besides ellipticine showing very lowGI50values for tumor cell

lines it also presents high toxicity for normal cells (nontumorcells)

There is no valuable data about previous investigation ofthis plant extracts Some of the compounds isolated fromthis extract were already tested for some biological activityFlavonoids are generally regarded to have a wide rangeof pharmacological activity (antioxidant anti-inflammatoryantimicrobial antiviral and antitumor) among these com-pounds apigenin and luteolin have been confirmed to haveantitumor activity [22] Stigmasterol a constituent isolatedfrom Bacopa monnieri Linn aerial parts showed therapeuticefficacy against Ehrlich ascites carcinoma in mice [30]Oleanolic acid is a pentacyclic triterpenoidwidely distributed

in nature and possessing various important bioactivities suchas antitumor and hepatoprotection [31]

The findings revealed that the antimicrobial and antitu-mor properties of the hydromethanolic extract of S saponariaprovide preliminary scientific validation for the traditionalmedicinal use of this plant as a potential phytotherapeuticagent in certain diseases and for the control of bacteriaand fungi in the environment However the extracts andactive compound isolated from S saponaria should be furtherstudied in animal models in order to evaluate their in vitroefficacy and toxicity

33 Phytochemical Characterization Chromatographic sep-aration and purification of the hydromethanolic extract ofS saponaria aerial parts allowed the identification of sevenbioactive compounds stigmasterol oleanolic acid luteolinluteolin 8-119862-120573-glucoside (orientin) luteolin 6-119862-120573-glucoside(isoorientin) luteolin 7-119874-120573-glucuronide and rutin (Fig-ure 3) Their structures were elucidated on the basis of UV1H-NMR 13C-NMR and MS analyses

Compound (1) was identified as stigmasterol white nee-dle crystals 1H-NMR (400MHz CDCl

3) 120575 532 (IH m H-

6) 511 (1H dd 119869 = 142 82Hz H-22) 504 (1H dd 119869 = 14282Hz H-23) 354 (IH m H-3) 104 (3H s CH

3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)

1218 (C-6) 719 (C-3) 567 (C-17) 569 (C-14) 509 (C-9)507 (C-24) 426 (C-13 4) 396 (C-12) 374 (C-1) 402 (C-20) 367 (C-10) 314 (C-8 7) 317 (C-2) 309 (C-25) 288 (C-16) 248 (C-15) 247 (C-28) 215 (C-11) 208 (C-26) 204 (C-19) 197 (C-27) 191 (C-21) It gave dark spot under short UV


Table 3 Cytotoxicity of hydromethanolic extract of Sapindus saponaria aerial parts and ellipticine (standard) in human tumor cell lines andin nontumor liver primary culture

Extract (GI50 120583gmL) Ellipticine (GI50 120583gmL)HCT-15 (colon carcinoma) 36276 plusmn 1464 191 plusmn 006

HepG2 (hepatocellular carcinoma) gt800 322 plusmn 067

HeLa (cervical carcinoma) 25858 plusmn 340 114 plusmn 021

MCF-7 (breast carcinoma) 37634 plusmn 1966 091 plusmn 004

NCI-H460 (lung carcinoma) 36829 plusmn 1894 142 plusmn 000

PLP2 (nontumor liver cells) gt800 206 plusmn 003

GI50 values correspond to the sample concentration achieving 50 of growth inhibition in human tumor cell lines or in liver primary culture PLP2

Figure 3 Antifungal activity of the hydromethanolic extract ofSapindus saponaria aerial parts by microdilution method The rowsrepresent different fungal species and columns different extractconcentrations (0075ndash50mgmL) Comparison between control(K-fungal growth) blank control (B) and treated samples (MIC andMFC wells with no fungal growth) and treated samples with noactivity

light that changed to violet colour on spraying with vanillinsulphuric and heating in an oven at 110∘C for 5min NMRspectral data has shown signals very close to that reported byYinusa et al [32] for stigmasterol

Compound (2) was identified as oleanolic acid whiteamorphous powder 1H-NMR (CDCl

3 400MHz) 120575 523 (IH

t 119869 = 34 H-12) 317 (1H dd 119869 = 10 42Hz H-3) 274 (1Hdd 119869 = 125 4 Hz H-18) 095 (3H sMe-23) 076 (3H sMe-24) 085 (3H s Me-25) 077 (3H s Me-26) 123 (3H s Me-27) 089 (3H sMe-29) 095 (3H sMe-30) (+) ESI-MS119898119911456 [MndashH]+This compoundwasmonitored by TLC andwasdetected by heating the plate at 110∘C after spraying with 119901-anisaldehyde-sulfuric acid furthermore spectral results werein agreement with the published data [33]

Compound (3) was identified as luteolin pale yellowcrystals UV 120582max (nm) (MeOH) 254 350 (NaOMe) 273411 (AlCl

3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-

NMR 120575 129 (s 1H 5-OH) 736 (d 119869 = 79Hz 1H H-61015840) 734(d 119869 = 2Hz 1H H-21015840) 682 (d 119869 = 8Hz 1H H-51015840) 664(s 1H H-3) 642 (d 119869 = 2Hz 1H H-8) 617 (d 119869 = 2Hz1H H-6) EI-MS 119898119911 286 This compound was isolated asa deep purple spot and with spraying with AlCl

3 it gave a

yellow fluorescence color under UV light UV spectral dataconfirm that it is a flavone with free OH groups at positions31015840 41015840 (ring B) and 5 7 (ring A) 1H-NMR and MS are very

similar to the characteristics of luteolin reported by Saeidniaet al [34]

Compound (4) was identified as luteolin 8-119862-120573-glucoside(orientin) yellow crystals UV 120582max (nm) (MeOH) 252266 348 (NaOMe) 268 403 (AlCl

3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355

(NaOAcH3BO3) 262 371 1H NMR (DMSO-d6 400MHz)

120575 ppm 752 (1H dd 119869 = 2 82Hz H-61015840) 745 (1H d 119869 = 2HzH-21015840) 684 (1H d 119869 = 82Hz H-51015840) 665 (1H s H-3) 622(1H s H-6) 464 (1H d 119869 = 95Hz H-110158401015840) 32ndash41 (the restof sugar protons) 13CNMRspectrum (DMSO-d6 100MHz)120575 ppm 18165 (C-4) 16695 (C-2) 16385 (C-7) 16034 (C-5)15625 (C-9) 1512 (C-41015840) 14645 (C-31015840) 12198 (C-11015840) 11925(C-61015840) 11578 (C-51015840) 11347 (C-21015840) 10458 (C-8) 10318 (C-10)10185 (C-3) 9854 (C-6) 8186 (C-510158401015840) 7875 (C-210158401015840) 7362 (C-110158401015840) 7084 (C-310158401015840) 7065 (C-410158401015840) 6164 (C-610158401015840)

Compound (5) was identified as luteolin 6-119862-120573-glucoside(isoorientin) yellow crystals UV 120582max (nm) (MeOH) 245267 345 (NaOMe) 225 264 406 (AlCl

3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294

340 354 (NaOAcH3BO3) 271 307 352 1H NMR (DMSO-

d6 270MHz) 120575 ppm 745 (1H dd 119869 = 25 82Hz H-61015840)74 (1H d 119869 = 25Hz H-21015840) 69 (1H d 119869 = 82Hz H-51015840)664 (1H s H-3) 647 (1H s H-8) 462 (1H d 119869 = 95HzH-110158401015840) 31ndash406 ppm (the rest of sugar protons) 13C NMR(DMSO-d6 100MHz) 120575 ppm 18245 (C-4) 16474 (C-7)16435 (C-2) 16162 (C-9) 15698 (C-5) 15055 (C-41015840) 14664(C-31015840) 12218 (C-11015840) 11965 (C-61015840) 11682 (C-51015840) 11394 (C-21015840)10956 (C-6) 10425 (C-10) 10348 (C-3) 9435 (C-8) 8215(C-510158401015840) 7985 (C-210158401015840) 7375 (C-110158401015840) 7148 (C-310158401015840) 7094 (C-410158401015840)6235 (C-610158401015840) Compounds (4) and (5) showed deep purplespots under UV light which changed to yellowwith ammoniavapor indicating that they are flavones with free 5-OH and41015840-OH [35] Complete acid hydrolysis of the two compoundsrevealed that no sugars were detected meaning that theyremained without change in addition to the appearance ofan additional spot on the chromatogram (Wessely-Moserrearrangement between C-6 and C-8) which may be dueto acid isomerization [36] indicating that the compoundsaremono-C-glycosidesThus the compounds were subjectedto ferric chloride degradation and cochromatographed withauthentic sugars samples where glucose was detected UVspectral data and NMR signals for the two compounds arevery similar to the ones reported for orientin and isoorientinrespectively [37 38]


Compound (6) was identified as luteolin 7-119874-120573-glucuronide 1H-NMR (400MHz DMSO-119889

6) 120575 ppm 1292

(1H s 5-OH) 745 (1H d 119869 = 20Hz H-21015840) 738 (1H dd119869 = 84Hz 119869 = 20Hz H-61015840) 682 (1H d 119869 = 84Hz H-51015840)678 (1H d 119869 = 19Hz H-8) 672 (1H s H-3) 637 (1H d119869 = 19Hz H-6) 512 (1H d 119869 = 75Hz H-110158401015840) 32ndash41 ppm(rest of sugar protons) 13CNMR (100MHz DMSO-119889

6) d

1827 (C-4) 17254 (C-610158401015840) 16517 (C-2) 16368 (C-7) 16175(C-5) 15762 (C-9) 15086 (C-41015840) 14663 (C-31015840) 12177 (C-11015840)11978 (C-61015840) 11671 (C-51015840) 11419 (C-21015840) 10595 (C-10) 10362(C-3) 10025 (C-6 C-110158401015840) 9524 (C-8) 7718 (C-310158401015840) 7455(C-510158401015840) 7363 (C-210158401015840) 7265 (C-410158401015840) (minus) ESIMS 119898119911 461[MndashH]minus

Compound (7) was identified as quercetin 3-119874-rutinoside (rutin) 1H-NMR (DMSO 270MHz) 120575 1275 (s1H 5-OH) 758 (2H m 119869 = 8Hz H-21015840 61015840) 684 (d 1H119869 = 85Hz H-51015840) 637 (1H d 119869 = 22Hz H-8) 619 (1H d119869 = 22Hz H-6) 525 (1H d 119869 = 75Hz H-110158401015840) 448 (1H d119869 = 22Hz H-1101584010158401015840) 115 (d 119869 = 6Hz CH

3-rhamnosyl) 13C

NMR (CD3OD 100MHz) quercetin 120575 17928 (C-4) 1660

(C-7) 16248 (C-5) 15926 (C-9) 15848 (C-2) 14984 (C-41015840)14576 (C-31015840) 13558 (C-3) 12342 (C-61015840) 12322 (C-11015840)11758 (C-21015840) 11626 (C-51015840) 10557 (C-10) 9874 (C-6) 9492(C-8) glucose sugar 120575 10485 (C-1101584010158401015840) 7815 (C-510158401015840) 7735(C-310158401015840) 7564 (C-21015840101584010158401015840) 7173 (C-410158401015840) 6864 (C-610158401015840) rhamnosesugar 120575 10253 (C-1101584010158401015840) 7385 (C-4101584010158401015840) 7265 (C-2101584010158401015840) 7235(C-3101584010158401015840) 694 (C-5101584010158401015840) 1765 (C-6101584010158401015840) (minus) ESIMS 119898119911 609[MndashH]minus Compounds (6) and (7) showed deep purple spotsunder UV light and after spraying with AlCl

3gave a yellow

flurorescent color EI-MS data and 1H- and 13C-NMR datawere in agreement with the results reported in the literaturefor luteolin 7-119874-120573-glucuronide [39] and rutin [40]

4 Conclusion

The extract has shown a significant antimicrobial activitytowards all bacterial and fungal species tested It can beconcluded that tested extract showed strong antibacterialand antifungal effect against human and animal pathogenicspecies food spoilage and contaminators and mycotoxinproducers It also revealed some cytotoxicity against humancolon (HCT-15) cervical (HeLa) breast (MCF-7) and lung(NCI-H460) carcinoma cell lines with HeLa being the mostsusceptible tumor cell line The extract was not toxic fornontumor liver cells Chromatographic separation of theextract resulted in the isolation of stigmasterol oleanolicacid luteolin luteolin 8-119862-120573-glucoside (orientin) luteolin6-119862-120573-glucoside (isoorientin) luteolin 7-119874-120573-glucuronideand rutin The compounds were identified by UV 1H-NMR13C-NMR and MS spectroscopic tools The results reportedhereinmay be useful for the discovery of novel anticancer andantimicrobial agents from plant origin

Conflict of Interests

There is no conflict of interests associated with the authors ofthis paper

Acknowledgments

The authors are grateful to the strategic project PEst-OEAGRUI06902011 for financial support to CIMO Ricardo CCalhelha thanks FCT POPH-QREN and FSE for his Grant(SFRHBD783072011) and Serbian Ministry of Educationand Science Grant no 173032 for financial support

References

[1] I Raskin D M Ribnicky S Komarnytsky et al ldquoPlants andhuman health in the twenty-first centuryrdquo Trends in Biotechnol-ogy vol 20 no 12 pp 522ndash531 2002

[2] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[3] E N Quiroga A R Sampietro andM A Vattuone ldquoScreeningantifungal activities of selected medicinal plantsrdquo Journal ofEthnopharmacology vol 74 no 1 pp 89ndash96 2001

[4] A Ghosh B K Das A Roy B Mandal and G ChandraldquoAntibacterial activity of somemedicinal plant extractsrdquo Journalof Natural Medicines vol 62 no 2 pp 259ndash262 2008

[5] J N Eloff ldquoWhich extractant should be used for the screeningand isolation of antimicrobial components from plantsrdquo Jour-nal of Ethnopharmacology vol 60 no 1 pp 1ndash8 1998

[6] G Karp Cell and Molecular BiologymdashConcepts and Experi-ments John Wiley amp Sons Hoboken NJ USA 2nd edition1999

[7] V S Neergheen T Bahorun E W Taylor L S Jen andO I Aruoma ldquoTargeting specific cell signaling transductionpathways by dietary and medicinal phytochemicals in cancerchemopreventionrdquoToxicology vol 278 no 2 pp 229ndash241 2010

[8] M L A Albiero M E Bacchi and M S K Mourao ldquoCarac-terizacao anatomica das folhas frutos e sementes de Sapindussaponaria L (Sapindaceae)rdquo Acta Science vol 23 no 5 pp549ndash560 2001

[9] O Castro JMGutierrezM Barrios I CastroM Romero andE Umana ldquoNeutralizacion del efecto hemorragico inducidopor veneno deBothrops asper (Serpentes Viperidae) por extrac-tos de plantas tropicalesrdquoRevista de Biologıa Tropical vol 47 no3 pp 605ndash615 1999

[10] J K Tsuzuki T I E Svidzinski C S Shinobu et al ldquoAntifungalactivity of the extracts and saponins from Sapindus saponariaLrdquo Anais da Academia Brasileira de Ciencias vol 79 no 4 pp577ndash583 2007

[11] F C Barreto M G Cavasin G H H Silva and G I SilvaldquoEstudo das alteracoes morfo-histologicas em larvas de Aedesaegypti (diptera culicidae) submetidas ao extrato bruto etano-lico de Sapindus saponaria Lin (SAPINDACEAE)rdquo Revista dePatalogıa Tropical vol 35 pp 37ndash57 2006

[12] M S Abdel-Wahab and A M Selim ldquoLipids and flavonoids ofSapindus saponariardquo Fitoterapia vol 56 no 3 pp 167ndash168 1985

[13] Clinical and Laboratory Standards Institute ldquoMethods fordilution antimicrobial susceptibility tests for bacteria that growaerobicallyrdquo Approved standard 8th ed CLSI publication M07-A8 Wayne PA USA 2009

[14] T Tsukatani H Suenaga M Shiga et al ldquoComparison of theWST-8 colorimetric method and the CLSI broth microdilutionmethod for susceptibility testing against drug-resistant bacte-riardquo Journal of Microbiological Methods vol 90 no 3 pp 160ndash166 2012


[15] H Hanel and W Raether ldquoA more sophisticated method ofdetermining the fungicidal effect of water-insoluble prepara-tions with a cell harvester using miconazole as an examplerdquoMycoses vol 31 no 3 pp 148ndash154 1988

[16] A Espinel-Ingroff ldquoComparison of the E-test with the NCCLSM38-P method for antifungal susceptibility testing of commonand emerging pathogenic filamentous fungirdquo Journal of ClinicalMicrobiology vol 39 no 4 pp 1360ndash1367 2001

[17] R Guimaraes L Barros M Duenas et al ldquoNutrients phyto-chemicals and bioactivity of wildRoman chamomile a compari-son between the herb and its preparationsrdquo Food Chemistry vol136 no 2 pp 718ndash725 2013

[18] T L G Lemos A L Mendes M P Sousa and R Braz-FilholdquoNew saponin from Sapindus saponariardquo Fitoterapia vol 63 no6 pp 515ndash517 1992

[19] Z Du N Zhu N Ze-Ren-Wang-Mu and Y Shen ldquoTwo newantifungal saponins from the Tibetan herbal medicine Clematistanguticardquo Planta Medica vol 69 no 6 pp 547ndash551 2003

[20] M W Lee S U Kim and D R Hahn ldquoAntifungal activity ofmodified hederagenin glycosides from the leaves of Kalopanaxpictum var chinenserdquo Biological and Pharmaceutical Bulletinvol 24 no 6 pp 718ndash719 2001 (Chinese)

[21] L C Scalon Cunha M L Andrade E Silva N A J CardosoFurtado et al ldquoAntibacterial activity of triterpene acids andsemi-synthetic derivatives against oral pathogensrdquo Zeitschriftfur Naturforschung C vol 62 no 9-10 pp 668ndash672 2007

[22] S S Guo Y J Shi Y J Gao D Su and X L Cui ldquoThe cytologymechanismof anti-parainfluenza virus infection of total flavoneof Scutellaria barbatardquo Yao-xue Xue-bao (Acta PharmaceuticaSinica) vol 44 no 12 pp 1348ndash1352 2009

[23] C Nishino N Enoki and S Tawata ldquoAntibacterial activityof flavonoids against Staphylococcus epidermidis a skin bac-teriumrdquo Agricultural and Biological Chemistry vol 51 no 1 pp139ndash143 1987

[24] H P Baise T S Walker F R Stermitz R S Hufbauer andJ M Vivanco ldquoEnantiometric dependent phytotoxic and anti-microbial activity of (plusmn) catechin a rhizosecreted racemicmixture fromCentaurea maculosa(spotted knapweed)rdquo PlantPhysiology vol 128 pp 1173ndash1177 2002

[25] I Aljancic V Vajs N Menkovic et al ldquoFlavones and sesquiter-pene lactones from Achillea atrata subsp multifida antimicro-bial activityrdquo Journal of Natural Products vol 62 no 6 pp 909ndash911 1999

[26] T E Tshikalange J J MMeyer and A A Hussein ldquoAntimicro-bial activity toxicity and the isolation of a bioactive compoundfrom plants used to treat sexually transmitted diseasesrdquo Journalof Ethnopharmacology vol 96 no 3 pp 515ndash519 2005

[27] J Kukic V Popovic S Petrovic et al ldquoAntioxidant and antimi-crobial activity ofCynara cardunculus extractsrdquoFoodChemistryvol 107 no 2 pp 861ndash868 2008

[28] X Zhu H Zhang and R Lo ldquoPhenolic compounds from theleaf extract of artichoke (Cynara scolymus L) and their antimi-crobial activitiesrdquo Journal of Agricultural and Food Chemistryvol 52 no 24 pp 7272ndash7278 2004

[29] M Sokovic P D Marin and D Brkic ldquoAntifungal activityof ethanolic extract from Phlomis fruticosa Lrdquo Archives ofBiological Sciences vol 52 pp 203ndash208 2000

[30] G Tirtha M K Tapan and J Singh ldquoEvaluation of antitumoractivity of stigmasterol a constituent isolated from Bacopamonnieri Linn aerial parts against ehrlich ascites carcinoma inmicerdquo Orient Pharmaceutical and Experimental Medicine vol11 no 1 pp 41ndash49 2011

[31] Z Ovesna A Vachalkova K Horvathova and D TothovaldquoPentacyclic triterpenic acids new chemoprotective com-poundsrdquo Neoplasma vol 51 pp 327ndash333 2004

[32] I Yinusa N G Ilogbulem and A O Joseph ldquoIsolation ofstigmasterol from serial plant part of Spillanthes acmellaMurrrdquoWorld Journal of Life Sciences and Medical Research vol 2 no2 pp 77ndash80 2012

[33] M Y Baek J G Cho D Y Lee E M Ahn T S Jeong and N IBaek ldquoIsolation of triterpenoids from the stem bark of Albiziajulibrissin and their inhibition activity onACAT-1 andACAT-2rdquoJournal of Applied Biological Chemistry vol 53 no 3 pp 310ndash315 2010

[34] S Saeidnia N Yassa R Rezaeipoor et al ldquoImmunosuppressiveprinciples from achillea talagonica an endemic species of IranrdquoDaru vol 17 no 1 pp 37ndash41 2009

[35] J T Mabry R K Markham and B M ThomasThe SystematicIdentification of Flavonoids Springer Berlin Germany 1970

[36] B J Harborne J T Mabry and H Mabry The FlavonoidsChapman and Hall London UK 1975

[37] K P Agrawal 13C-NMR of Flavonoids vol 39 of Studies inOrganic Chemistry Elsevier Amesterdam Netherlands 1989

[38] A Kuruuzum-Uz Z Guvenalp K Stroch L O Demirezer andA Zeeck ldquoAntioxidant potency of flavonoids from Vitex agnus-castus L growing in Turkeyrdquo Fabad Journal of PharmaceuticalSciences vol 33 no 1 pp 11ndash16 2008

[39] Y Lu and L Yeap Foo ldquoFlavonoid and phenolic glycosides fromSalvia officinalisrdquo Phytochemistry vol 55 no 3 pp 263ndash2672000

[40] Z Guvenalp N Kilic C Kazaz Y Kaya and L O DemirezerldquoChemical constituents of Galium tortumenserdquo Turkish Journalof Chemistry vol 30 no 4 pp 515ndash523 2006

Impact Factor 173028 Days Fast Track Peer ReviewAll Subject Areas of ScienceSubmit at httpwwwtswjcom

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawi Publishing Corporation httpwwwhindawicom Volume 2013

The Scientific World Journal


antitumor properties In the last few decades research hasfocused on the use of natural products crude plant extractsor a combination of different phytochemicals for cancertherapy this trend is based upon first the synergistic effectof the different plant metabolites in the crude extract andsecond the multiple points of intervention of such extracts[7]

Sapindus saponaria L is a tree from Sapindaceae familyand it is popularly known as soapberry being distributed inCentral and South America The bark root and fruits areused in popular medicine as tranquilizer astringent diureticexpectorant tonic blood cleanser healing and to countercough [8] being also a neutralizer of hemorrhage [9] Itsfruits have activity as antifungal [10] and larvicidal [11]Abdel-Wahab and Selim [12] detected the presence of car-bohydrates steroids flavonoids and saponins in leaves andstems of S saponaria

The objective of the present study was to investigateantimicrobial activity of the hydromethanolic extract ofS saponaria aerial parts and also its capacity to inhibitthe growth of different human tumor cell lines The mainphytochemicals present in the extract were completely char-acterized by UV 1H-NMR 13C-NMR and MS

2 Materials and Methods

21 Plant Material and Preparation of the HydromethanolicExtract Sapindus saponaria L aerial parts were collectedfromAl-Zohiriya garden Giza Egypt inApril 2011The plantwas identified by Dr Mohammed El-Gebaly Departmentof Botany National Research Centre (NRC) and by MrsTereez Labib Consultant of plant taxonomy at the Ministryof Agriculture andDirector ofOrman botanical garden GizaEgypt A voucher specimen number 20532ss was deposited inthe herbarium of Al-Zohiriya garden Giza Egypt

For the extract preparation 12 kg of air dried powder ofS saponaria aerial parts was extracted with methanol water80 20 (vv) at room temperature several times until exhaus-tionThe extract was concentrated under reduced pressure togive 68 g of crude extract

22 Evaluation of Antimicrobial Activity ofthe Hydromethanolic Extract

Antibacterial Activity The following Gram (minus) (Enterobac-ter cloacae human isolate Escherichia coli ATCC 35210Pseudomonas aeruginosa ATCC 27853 and Salmonellatyphimurium ATCC 13311) and Gram (+) bacteria (Bacilluscereus clinical isolate Listeria monocytogenes NCTC 7973Micrococcus flavus ATCC 10240 and Staphylococcus aureusATCC 6538) were used The organisms were obtained fromMycological Laboratory Department of Plant PhysiologyInstitute for Biological Research ldquoSinisa Stankovicrdquo Univer-sity of Belgrade Serbia The antibacterial assay was carriedout by microdilution method [13] The bacterial suspensionswere adjusted with sterile saline to a concentration of 10 times105 CFUmL The inocula were prepared daily and storedat 4∘C until use Dilutions of the inocula were cultured

on solid medium to verify the absence of contaminationand to check the validity of the inoculum The minimuminhibitory and bactericidal concentrations (MICs andMBCs)were determined using 96-well microtitre plates The extractwas diluted in 5 of DMSO (10mgmL) and added to trypticsoy broth (TSB) medium (100 120583L) with bacterial inoculum(10times 104 CFUperwell) to achieve thewanted concentrationsThe microplates were incubated at rotary shaker (160 rpm)for 24 h at 37∘C The following day 30 120583L of 02mgmLsolution of INT (119901-iodonitrotetrazolium violet) was addedand the plates were returned to the incubator for at leastone-half hour to ensure adequate color reaction Inhibitionof growth was indicated by a clear solution or a definitedecrease in color reaction [14] The lowest concentrationswithout visible growth (at the binocular microscope) weredefined as concentrations that completely inhibited bacterialgrowth (MICs) The MBCs were determined by serial sub-cultivation of 2120583L into microtitre plates containing 100120583L ofbroth per well and further incubation for 24 h The lowestconcentration with no visible growth was defined as theMBC indicating 995 killing of the original inoculum Theoptical density of each well was measured at a wavelength of655 nm by microplate manager 40 (Bio-Rad Laboratories)and compared with a blank and the positive control Theantibiotics streptomycin and ampicillin were used as positivecontrols (1mgmL in sterile physiological saline) Threeindependent experiments were performed in triplicate

Antifungal Activity The used fungi are Aspergillus fumigatus(ATCC 1022)Aspergillus versicolor (ATCC 11730)Aspergillusochraceus (ATCC 12066) Aspergillus niger (ATCC 6275)Trichoderma viride (IAM 5061) Penicillium funiculosum(ATCC 36839) Penicillium ochrochloron (ATCC 9112) andPenicillium verrucosum var cyclopium were obtained fromMycological Laboratory Department of Plant PhysiologyInstitute for Biological Research ldquoSinisa Stankovicrdquo Univer-sity of Belgrade Serbia The micromycetes were maintainedon malt agar and the cultures were stored at 4∘C and sub-cultured once a month The antifungal assay was carriedout by modified microdilution technique [15 16] The fungalspores were washed from the surface of agar plates withsterile 085 saline containing 01Tween 80 (vv)The sporesuspension was adjusted with sterile saline to a concentrationof approximately 10 times 105 in a final volume of 100120583L perwellThe inocula were stored at 4∘C for further use Dilutionsof the inoculum were cultured on solid malt agar to verifythe absence of contamination and to check the validity ofthe inoculum MIC determinations were performed by aserial dilution technique using 96-well microtiter plates Theextract was diluted in 5 of DMSO (10mgmL) and added inbroth Malt Medium (MA) with inoculum The microplateswere incubated at rotary shaker (160 rpm) for 72 h at 28∘CThe lowest concentrations without visible growth (at thebinocular microscope) were defined as MICs The fungicidalconcentrations (MFCs) were determined by serial subculti-vation of 2120583L of tested fractions dissolved in medium andinoculated for 72 h into microtiter plates containing 100120583L ofbroth per well and further incubation for 72 h at 28∘C Thelowest concentration with no visible growth was defined as




2 Each cell






254(Merck)








BacteriaExtract




























HO

HO

HOH

H

H

H

H

H

CH3

CH3

CH3

CH3

CH3

CH3

HO

OH

(2) Oleanolic acid

O

O

O

O

O

OH

OH

HO

HO

HO

OHOH

OH

OH

OH

(3) Luteolin

O O O

O

OH

OH

HO

HO

HO OH

OH


O

O

O

OH

OH

OH

OHHO

O

OH

OH

O

O

HO

OH

OH

OH OH

OHOH

OOH

HO

HO

HO

O

O

(7) Rutin

H3C

H3C

H3C

H3C H3C

H3C

(1) Stigmasterol

CH2OH

(4) Orientin

H3C






FungiExtract






























3) 120575 532 (IH m H-


3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)














3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References
















































2 Each cell






254(Merck)








BacteriaExtract




























HO

HO

HOH

H

H

H

H

H

CH3

CH3

CH3

CH3

CH3

CH3

HO

OH

(2) Oleanolic acid

O

O

O

O

O

OH

OH

HO

HO

HO

OHOH

OH

OH

OH

(3) Luteolin

O O O

O

OH

OH

HO

HO

HO OH

OH


O

O

O

OH

OH

OH

OHHO

O

OH

OH

O

O

HO

OH

OH

OH OH

OHOH

OOH

HO

HO

HO

O

O

(7) Rutin

H3C

H3C

H3C

H3C H3C

H3C

(1) Stigmasterol

CH2OH

(4) Orientin

H3C






FungiExtract






























3) 120575 532 (IH m H-


3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)














3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References















































BacteriaExtract




























HO

HO

HOH

H

H

H

H

H

CH3

CH3

CH3

CH3

CH3

CH3

HO

OH

(2) Oleanolic acid

O

O

O

O

O

OH

OH

HO

HO

HO

OHOH

OH

OH

OH

(3) Luteolin

O O O

O

OH

OH

HO

HO

HO OH

OH


O

O

O

OH

OH

OH

OHHO

O

OH

OH

O

O

HO

OH

OH

OH OH

OHOH

OOH

HO

HO

HO

O

O

(7) Rutin

H3C

H3C

H3C

H3C H3C

H3C

(1) Stigmasterol

CH2OH

(4) Orientin

H3C






FungiExtract






























3) 120575 532 (IH m H-


3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)














3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References














































HO

HO

HOH

H

H

H

H

H

CH3

CH3

CH3

CH3

CH3

CH3

HO

OH

(2) Oleanolic acid

O

O

O

O

O

OH

OH

HO

HO

HO

OHOH

OH

OH

OH

(3) Luteolin

O O O

O

OH

OH

HO

HO

HO OH

OH


O

O

O

OH

OH

OH

OHHO

O

OH

OH

O

O

HO

OH

OH

OH OH

OHOH

OOH

HO

HO

HO

O

O

(7) Rutin

H3C

H3C

H3C

H3C H3C

H3C

(1) Stigmasterol

CH2OH

(4) Orientin

H3C






FungiExtract






























3) 120575 532 (IH m H-


3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)














3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References















































FungiExtract






























3) 120575 532 (IH m H-


3-10) 09

(3H d 119869 = 65 CH3-20) 084 (3H d 119869 = 74 CH

3-27) 082

(3H d 119869 = 74 CH3-26) 068 (3H s CH

3-13) 13C-NMR

(100MHz CDCl3) 120575 1406 (C-5) 1384 (C-22) 1291 (C-23)














3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References

























































3 400MHz) 120575 523 (IH



3) 272 330 343 421 (AlCl

3+ HCl) 258 277 386

(NaOAc) 266 296 367 (NaOAc+H3BO3) 260 306 370 1H-


3 it gave a




3) 272 302 332

422 (AlCl3HCl) 268 302 357 384 (NaOAc) 267 355




3) 225 262 365

(AlCl3HCl) 263 282 296 358 365 (NaOAc) 258 268 294





6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References















































6) 120575 ppm 1292


6) d



3-rhamnosyl) 13C



3gave a yellow


4 Conclusion




Acknowledgments


References













































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