Research Article Antibacterial and Cytotoxic Activity of...

Research ArticleAntibacterial and Cytotoxic Activity of CompoundsIsolated from Flourensia oolepis

Mariana Beleacuten Joray1 Lucas Daniel Trucco2 Mariacutea Laura Gonzaacutelez1

Georgina Natalia Diacuteaz Napal1 Sara Mariacutea Palacios1 Joseacute Luis Bocco2

and Mariacutea Cecilia Carpinella1

1Fine Chemicals and Natural Products Laboratory School of Chemistry Catholic University of CordobaAvda Armada Argentina 3555 X5016DHK Cordoba Argentina2CIBICI CONICET and Department of Clinical Biochemistry Faculty of Chemical Science National University of CordobaHaya de la Torre and Medina Allende Cordoba Argentina

Correspondence should be addressed to Marıa Cecilia Carpinella ceciliacarpinellaucceduar

Received 16 September 2015 Revised 29 November 2015 Accepted 30 November 2015

Academic Editor Jairo Kennup Bastos

Copyright copy 2015 Mariana Belen Joray 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 antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluatedBioguided fractionation led to five flavonoids identified as 2101584041015840-dihydroxychalcone (1) isoliquiritigenin (2) pinocembrin (3)7-hydroxyflavanone (4) and 741015840-dihydroxy-31015840-methoxyflavanone (5) Compound 1 showed the highest antibacterial effect withminimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250120583gmL against Gram-positive and Gram-negative bacteria respectively On further assays the cytotoxic effect of compounds 1ndash5 was determined by MTT assay onacute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR)phenotypes Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC

50= 66ndash99 120583M) and a lower effect against

CML cells (IC50= 275ndash300 120583M) Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and

by Annexin VPI staining respectively Upon treatment 1 induced cell cycle arrest in the G2M phase accompanied by a strong

induction of apoptosis These results describe for the first time the antibacterial metabolites of F oolepis extract with 1 being themost effective This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells highlightingits potential as a lead compound

1 Introduction

Since the discovery of chemotherapy scientists have devel-oped a vast arsenal of bioactive agents enabling the successfultreatment of a huge variety of diseases including bacterialinfections and cancer However over time the treatment ofthese ailments is frequently associated with side effects andthe development of resistance rendering the drugs useless[1 2]This has increased the importance of the search for newentities with antibacterial and anticancer properties

The role of natural products in drug discovery remainscritical Sixty-five percent of the 118 new chemical enti-ties (NCE) approved between 1981 and 2010 for indicationas antibacterial drugs and 34 of the 128 approved as

anticancer drugs corresponded to natural products andsemisynthetic derivatives obtained from these natural pre-cursors [3] The fact that natural products have 40 morechemical scaffolds than synthetic chemistry means that theyhave a considerable advantage in continuing to provide newcommercial drug leads [4] The key is to efficiently andeffectively access this diversity [4] Among sources of naturalactive compounds plants are a promising starting point[5] Approximately one-third of the top-selling drugs havebeen derived from plant metabolites [6] and there are 190examples of clinical trials involving pure or combined plantcompounds for treating different ailments currently reported(httpswwwclinicaltrialsgov US Institute of Health queryldquoplant drugsrdquo only herbal extracts or plant-derived pure

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 912484 11 pageshttpdxdoiorg1011552015912484

2 Evidence-Based Complementary and Alternative Medicine

compounds administered as therapeutic agents were consid-ered) The plant world is still far from being totally exploredhowever particularly the native flora from Argentina [7]Flourensia oolepis S F Blake (Asteraceae) commonly knownas ldquochilcardquo is a woody bush widely distributed in the highareas of the provinces of Cordoba and San Luis in centralArgentina [8]The antibacterial activity of the ethanol extractobtained from this plant was previously reported by ourgroup [7] The current study aims to gain insight into themetabolites responsible for this effect

The cytotoxic activity of the compounds isolated againstleukemic cell lines and their multidrug resistant (MDR)counterparts was also investigated and this was extended todetermine the mechanism involved in leukemic cell toxicity

To our knowledge this is the first detailed study aboutthe constituents of F oolepis that show these pharmacologicalproperties

2 Materials and Methods

21 Plant Material and Extract Preparation Aerial parts ofthe native plant F oolepis S F Blake were collected from thehills of Cordoba Province Argentina in December 2005 Avoucher specimen was deposited in the ldquoMarcelino SayagordquoHerbarium of the School of Agricultural Science CatholicUniversity of Cordoba (UCCOR 23) The authorization forthe use of the plant is available from the authors Crushedair-dried material (200 g) was extracted by 48 h macerationwith 700mL of ethanol and the yield of the extract obtainedafter solvent removal and expressed as percentage weight ofair-dried crushed plant material was 23 g

22 Chemicals Equipment and Reagents 3-(45-dimethyl-thiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) waspurchased from Sigma-Aldrich CO (St Louis MO USA)Doxorubicin hydrochloride 998 (DOX Synbias PharmaLtd) was purchased from Nanox Release Technology(Buenos Aires Argentina)

Gentamicin sulfate (potency 550ndash590 120583gmg) and ery-thromycin (potency 863 120583gmg) were provided by Labora-torio Fabra SA Buenos Aires Argentina and UnifarmaBuenos Aires Argentina respectively1H- and 13C-NMR and two-dimensional spectra were

recorded with a Bruker AVANCE II 400 spectrometer(Bruker Corporation Ettlingen Germany) with tetramethyl-silane (TMS) as the internal reference HPLC was performedon a Shimadzu LC-10 AS (Shimadzu Corp Tokyo Japan)equipped with a Phenomenex Prodigy 5 120583 ODS (46mmidtimes 250mm) reversed-phase columnThemobile phase waswatermethanoltrifluoroacetic acid 65 35 1 with detectionat 365 nm

Flow cytometry analysis was performed in a Becton-Dickinson (BD) FACS Canto II flow cytometer (BD Bio-sciences USA)

23 Bioguided Isolation of theActive Principles fromFlourensiaoolepis The antibacterial ethanol extract of F oolepis (12 g)was initially subjected to vacuum liquid chromatography on

silica gel (622 g 63ndash200120583m 110 times 240 cm Macherey ampNagel) eluted with a step gradient of hexanediethyl ether(Et2O)methanol (MeOH) to yield 26 fractions which were

combined in 6 groups according to their thin layer chro-matography (TLC) profile (F1 to F6) Of these fraction F3eluted with hexaneEt

2O 50 50 and F5 eluted with 100

Et2O demonstrated bactericidal activity at 2mgmL and

were therefore submitted to additional separation methodsfor further purification F3 was processed by radial prepar-ative chromatography using an isocratic mobile phase ofhexaneEt

2O 70 30 The fractions obtained were combined

in 5 groups in accordance with the TLC analysis (F31 to

F35) From fraction F

34 compound 3 was obtained by

spontaneous crystallization (978 purity by HPLC) Thepure compound the remaining F

34 containing traces of 3 and

another substance as well as the rest of the fractions weretested for antibacterial activity Only 3 and the remaining F

34

exerted bactericidal effects at 2mgmLThe latter was furtherfractionated by preparative TLC (Analtech DE USA mobilephase tolueneethyl acetateglacial acetic acid 8 25 04) tofinally obtain compound 1 (999 purity by HPLC)

Fraction F5 was subjected to column chromatography(130 g 35ndash70 120583m 30 times 60 cm Fluka) using 100 Et

2O as

the mobile phase The resulting fractions were placed in 14groups according to TLC monitoring (F

51 to F514) Of these

F52 showed bactericidal activity and was further fractionated

by column chromatography on silica gel (60 g 35ndash70 120583m30 times 60 cm Fluka) performed with hexaneEt

2Oglacial

acetic acid 8 2 04 The fractions obtained were combinedin 7 groups according to their TLC profile (F

521ndashF527)

Their antibacterial activity was evaluated at 2mgmL andF522 and F

525 were seen to be active Both groups were

separately submitted to solid phase extraction (Sep-Pak plusC18Waters Ireland) using waterMEOHtrifluoroacetic acid65 35 1 as the mobile phase to afford compound 2 (991purity by HPLC) from F

522 and compounds 4 (964 purity

by HPLC) and 5 (972 purity by HPLC) from F525

Compounds 1ndash5 were identified by 1H NMR and 13CNMR comparing their spectra (copies of the original spectraare obtainable from the corresponding author) with referencedata from literature as 2101584041015840-dihydroxychalcone C

15H12O3

(1 mz 2402) [9 10] isoliquiritigenin C15H12O4(2 mz

2562) [11] pinocembrin C15H12O4(3 mz 2562) [8] 7-

hydroxyflavanone C15H12O3(4 mz 2402) [12] and 741015840-

dihydroxy-31015840-methoxyflavanone C16H14O5(5 mz 2863)

[13] The compounds were quantified by HPLC and theiryields in g per 100 g of dried and crushed plant material were124 006 114 03 and 006 for 1ndash5 respectively

24 Microorganisms and Preparation of Inocula The antibac-terial activity assays were carried out on strains of Entero-coccus faecalis (Andrews and Horder) Scheifer and Klipper-Balz (ATCC 29212) Escherichia coli (Migula) Castellaniand Chalmers (ATCC 25922) Pseudomonas aeruginosa(Schroeter)Migula (ATCC 27853) and Staphylococcus aureussubsp aureus Rosenbach (ATCC 6538) and on a clinical iso-late of tetracycline erythromycin and polymyxin-resistantProteus mirabilis (Pr2921) [14] This isolate was identified

Evidence-Based Complementary and Alternative Medicine 3

by conventional biochemical assays and by the commercialsystem API20E (bioMerieux SA Mercy LrsquoEtoile France)Antimicrobial resistances were determined by disk diffusionsusceptibility testing (Kirby-Bauer Method) according toCLSI [15] Bacterial suspensions were prepared on sterilesaline from each organism grown overnight Turbidity wasspectrophotometrically adjusted to the McFarland 05 stan-dard Dilutions were carried out with sterile saline to give anadjusted concentration of 15 times 107 colony-forming unitsmL

25 Antibacterial Activity Test and Determination of MICsand MBCs MICs were determined by the agar dilution testaccording to CLSI [15] with some modifications [7] Brieflythe plate count agar medium (PCA Oxoid Ltd UK) wasadded in duplicate to the suitable amount of fraction orpure compound previously dissolved in ethanol The finalconcentration of solvent was 2 (no adverse effects wereobserved at this concentration) After solidification of theagar in 6-well plates (Greiner Bio-One Germany) 2120583L ofeach bacterial suspension was placed on the agar surfaceThree independent experiments were carried out Plates wereincubated in ambient air at 37∘C for 24 h For readingvisual observations were carried out Nitro blue tetrazolium(NBT Sigma-Aldrich CO USA) solution in saline phosphatebuffer (PBS) was applied on one spot for confirmation Platescontaining only the culture medium with or without theaddition of the dissolution solvent were used as controlsPositive controls with commercial gentamicin sulfate or ery-thromycin dissolved in sterile water or ethanol respectivelywere simultaneously carried out The minimum inhibitoryconcentration (MIC) was defined as the lowest concentrationthat completely inhibits growth of the microorganism

The bactericidal effect was further studied from eachconcentration of product showing growth inhibition For thisstudy 05mm portions of agar coincident with the placeat which inocula had been placed and showing negativegrowth at 24 hr (without NBT) were cut and transferred tobrain-heart infusion broth (BHI Oxoid Ltd UK) Tubes wereincubated in ambient air at 37∘C for five days At the end ofthis period the minimum bactericidal concentration (MBC)values defined as the lowest concentration with absence ofturbidity were recorded

26 Cell Lines and Culture Conditions CCRF-CEM acutelymphoblastic leukemia (ALL) [16] and K562 chronicmyeloid leukemia (CML) [17] cells and their MDR P-glycoprotein overexpressing variants [18 19] CEMADR5000and Lucena 1 respectively were a generous gift from Dr TEfferth (Institute of Pharmacy and Biochemistry JohannesGutenberg University Mainz Germany) and Dr V Rum-janek (Instituto de Bioquımica Medica Leopoldo de MeisUniversidade Federal do Rio de Janeiro Rio de JaneiroBrazil) respectively The HEK293T non-tumor derived cellline [20] andhuman isolatedmononuclear cells (PBMC)werealso tested Leukemic and PBMC cells were grown in RoswellPark Memorial Institute (RPMI) 1640 medium (InvitrogenLife Technologies CA USA) while the HEK293T cell linewas grown in Dulbeccorsquos Modified Eaglersquos medium (DMEM

Invitrogen Life Technologies CA USA) both of these mediasupplemented with 10 heat-inactivated fetal bovine serum(FBS PAA Laboratories Pasching Austria) 2mM glutamine(Invitrogen Life Technologies CA USA) and penicillin(100 unitsmL) streptomycin (100 120583gmL) (Invitrogen LifeTechnologies CA USA) Cells were cultured at 37∘C in a 5CO2humidified environment

CEMADR5000 cells were exposed once a week togradually increasing doses of DOX from 17 to 86120583M Thelatter concentration was then used for maintenance of thecells Lucena 1 were continuously cultured in the presenceof 60 nM DOX Both cell lines were grown in drug-freemedium 3-4 days before the experiments Overexpressionof P-glycoprotein (P-gp) in both MDR variants was verifiedby flow cytometry using FITC-labeled mouse anti-human P-gp (BD Pharmingen USA) Cells were subcultured twice aweek from frozen stocks and used before the 20th passageAll experiments were performed with cells in the logarithmicgrowth phase with cell viabilities over 90 determined bytrypan blue staining

27 Isolation of Human Mononuclear Cells from PeripheralBlood Peripheral blood mononuclear cells (PBMC) werecollected from fresh heparinized blood and separated by den-sity gradient centrifugation (Ficoll) as described by Rennoet al [21] As the current study required samples from healthyhuman volunteer donors ethical approval was provided bythe Catholic University of Cordoba Research Ethics BoardWritten signed consents were obtained from donors

28 Cell Proliferation Assay To investigate the cytotoxicpotential of the isolated compounds the MTT colorimetricassay was performed [22] Briefly 5 times 104 cells suspendedin 100 120583L of growth medium were seeded in 96-well plates(Greiner Bio-One Germany) containing 100 120583L of mediumin the presence of serial twofold dilutions of each tested com-pound previously dissolved in ethanol (1 vv no adverseeffects on cell growth were observed at this concentration)The compounds were evaluated at a final maximum concen-tration of 100 120583M After 72 hours 20120583L of 5mgmL solutionof MTT in sterile PBS was added to each well and furtherincubated for 4 h Then the supernatants were removed andreplaced with 100120583LDMSO to solubilize the resulting purpleformazan crystals produced from metabolically viable cellsAbsorbance was measured with an iMark micro-plate reader(Bio-Rad USA) at 595 nm Two wells were used for eachconcentration of the products assayed and three independentexperiments were performed Untreated and ethanol (1)treated cells were used as controls while DOX (maximumtested concentration 69120583M) was used as reference Thepercentage of cytotoxic activity of the assayed compoundswas determined by the following formula cytotoxicity () =[1 minus (optical density of treated cells minus optical densityDMSO)(optical density of ethanol control cells minus opticaldensity DMSO)] times 100

Medium inhibitory concentrations (IC50) represent the

concentrations of the tested samples required to inhibit 50cell proliferation and were calculated from the mean valuesof data from wells


To confirm the effect of compound 1 on the proliferationof CCRF-CEM and K562 cell suspensions (5 times 104 cellswell)were treated in culture mediumwith different concentrationsof 1 (final volume of 200120583L) and incubated in triplicate for 2448 and 72 h Two independent experiments were performedThe number of viable cells at the different time points wascounted by the trypan blue dye exclusion method using ahemocytometer (Neubaur USA)

29 Cell Cycle Distribution by Flow Cytometry CCRF-CEMand K562 cells placed in supplemented RPMI 1640 mediumin 12-well plates (Greiner Bio-One Germany) at a density of5 times 105mL were treated with 1 at a concentration equivalentto two times its IC

50for 72 h Control cells were devoid of 1

and negative controls contained 1 ethanol After treatmentcells were harvested and washed twice with ice cold PBSThen cells were fixed with 70 cold ethanol and kept at4∘C for 24 h Before analysis the cells were washed twicewith PBS and stained with a solution containing propidiumiodide (PI 50120583gmL Sigma-Aldrich CO USA) and RNaseA (100120583gmL Sigma-Aldrich CO USA) in PBS (pH 74)for 30min in the dark DNA content was evaluated by flowcytometryThe relative distribution of at least 20000 cells wasanalyzed using FlowJo software version 762 (Tree Star IncOR USA)

210 Assessment of Apoptosis by Annexin VPI Double Stain-ing Assay CCRF-CEM and K562 cells (5 times 105 cellsmL)placed in supplemented RPMI 1640 medium in 12-well plates(Greiner Bio-One Germany) were treated for 72 h withcompound 1 at two times its IC

50value After incubation

cells were harvested washed and suspended in cold PBSThe proportion of cells undergoing apoptosis was examinedby double staining using allophycocyanin (APC) conjugatedAnnexin-V (BD Pharmingen USA) and propidium iodide(Sigma-Aldrich CO USA) Over the following hour thefluorescence of ten thousand cells including that of controlgroups was assayed and populations were analyzed withFlowJo software version 762 (Tree Star Inc OR USA)Percentages of Annexin-V positive cells (PIminus or PI+) indicateearly or late apoptosis respectively

211 Statistical Analysis Data were analyzed by one-wayanalysis of variance (ANOVA) and means were comparedusing Bonferronirsquos comparison test with 119901 le 005 consideredas statistically significantThe inhibitory concentration (IC

50)

was calculated by log-Probit analysis responding to at leastfive concentrations at the 95 confidence level with upperand lower confidence limits

3 Results and Discussion

In the current study five compounds 2101584041015840-dihydroxychal-cone (1) isoliquiritigenin (2) pinocembrin (3) 7-hydroxy-flavanone (4) and 741015840-dihydroxy-31015840-methoxyflavanone (5)(Figure 1) were obtained by bioguided fractionation from theethanol extract of the aerial parts of Flourensia oolepis Thisextract was selected among 51 extracts assayed by our group

after demonstrating an effective growth inhibition of a panelof pathogenic bacteria [7] As far as we know there was noinformation up to now about the metabolites responsible forthe antibacterial activity of this extract These results supportthe popular use of F oolepis as an herb used for the treatmentof respiratory tract infections such as bronchitis [23]

The presence of compounds 1 3 and 4 in F oolepis hasbeen previously reported [8 24] but the presence of com-pounds 2 and 5 is reported here for the first time It shouldbe noted that while compounds 1ndash4 have been frequentlydescribed in nature this is not the case for compound 5In fact although there is considerable information availableabout the presence of its corresponding flavone (geraldone)[25 26] there is very little describing the presence of 5 inplants and there is a total absence of information concerningspecies native to Argentina

As shown in Table 1 compound 1 was the most effectivefollowed by 3 Both metabolites showed inhibitory effectsagainst Gram-positive referents with remarkableMIC valuesranging from 15 to 62120583gmL In relation to their bactericidalactivity it should be noted that the MBCs of 1 and 3 againstS aureus were similar to those obtained with the commercialantibiotics (31 62 10 and 60 120583gmL for 1 3 gentamicin anderythromycin resp)

As previously established in the literature metaboliteswith MICs le 100 120583gmL are considered noteworthy [27 28]Based on this criterion compound 1was clearly active againstE faecalis S aureus and P mirabilis while 3 was effectiveagainst both positive strains (Table 1) Since S aureus isassociated with respiratory diseases like bronchitis [29] theactivity of compounds 1 and 3 against this pathogen supportsthe popular use of F oolepis for the treatment of theseaffections [30]

On the other hand the bacteriostatic activity of 1 againstthe resistant strain of P mirabilis which was comparable toor even better than that of the commercial antibiotics usedas referents is highly encouraging It should be pointed outthat P mirabilis is an important pathogen that has acquiredresistance to clinically used antibiotics being the secondmostcommon cause of urinary tract infections [14]

The antibacterial activity of compounds 1ndash4 has beenpreviously described [31ndash34] however as far as we knowthere are no reports of this inhibitory property for 5

Certain structural features can be linked to the majorantibacterial efficacy displayed by the chalcone 1 It has beenreported that the carbonyl region is part of the active site offlavonoids [31] with the hydroxylations at positions 21015840 and41015840 being important structural features for the antibacterialeffect of chalcones [31 35] Interestingly the other chalconeisolated 21015840410158404-trihydroxychalcone commonly known asisoliquiritigenin (2) showed lower activity than its dihydrox-ylated counterpart (Table 1) demonstrating that the presenceof a hydroxyl group at position 4 reduces effectiveness aphenomenon that was also described by M A Alvarez et al[35] In addition 1 was more active for inhibiting bacterialgrowth than its respective flavanone 4

With respect to flavanones when comparing the activityof 3 and 4 against the Gram-positive strains it was observedthat the presence of a hydroxyl group at position 5 in


1

HO OH

OH

O2

3

HO O

O4

HO

OH

HO

A

B

O

120573

120572120573998400

2

3

4

5

62998400

3998400

4998400

5998400

6998400

OH

O

O

2

345

6

78

2998400

3998400

4998400

5998400

6998400

A

B

C

HO

OH

O O

O

5

CH3

Figure 1 Chemical structures of 2101584041015840-dihydroxychalcone (1) isoliquiritigenin (2) pinocembrin (3) 7-hydroxyflavanone (4) and 741015840-dihydroxy-31015840-methoxyflavanone (5)

Table 1 Antibacterial activity of compounds 1ndash5 isolated from Flourensia oolepis extract

Compound MIC (MBC) (120583gmL)E coli P aeruginosa Pmirabilis E faecalis S aureus

2101584041015840-Dihydroxychalcone (1) 250(500)

250(gt500)

62(125)

62(125)

31(31)

Isoliquiritigenin (2) gt500(gt500)

gt500(gt500)

gt500(gt500)

500(gt500)

250(500)

Pinocembrin (3) 500(500)

gt500(gt500)

gt500(gt500)

62(125)

15(62)

7-Hydroxyflavanone (4) 500(500)

500(gt500)

gt500(gt500)

250(500)

250(250)

741015840-Dihydroxy-31015840-methoxyflavanone (5) gt500(gt500)

gt500(gt500)

gt500(gt500)

500(gt500)

250(gt500)

Gentamicin 4(8)

4(4)

10(10)

8(10)

8(10)

Erythromycin 125(gt500)

62(500)

500(gt500)

1(30)

1(60)

compound 3 may be responsible for its improved activitysince this substitution pattern constitutes the only structuraldifference with respect to 4 This agrees with a study of anumber of structurally different flavonoids which showedthat hydroxylation at position 7 of flavanones is important fortheir antibacterial activity and the presence of an additional

hydroxyl group at position 5 enhances the activity [31] Onthe other hand it was also reported that the presence ofmethoxy groups drastically reduces the antibacterial activityof flavonoids [31 36]This may be the reason why compound5 showed the weakest bacteriostatic activity among theflavanones


Table 2 Anticancer effects of compounds 1ndash5 isolated from Flourensia oolepis extract toward sensitive and resistant leukemic cells

Compounds IC50(120583M) values and 95 confidence limits (lower upper)

CCRF-CEM CEMADR5000 K562 Lucena 12101584041015840-Dihydroxychalcone (1) 66 (33ndash141) 99 (58ndash183) 275 (146ndash520) 300 (146ndash649)Isoliquiritigenin (2) 581 (226ndash1487) 519 (269ndash991) gt100 gt100Pinocembrin (3) 589 (164ndash2103) 535 (195ndash1459) gt100 gt1007-Hydroxyflavanone (4) 728 (300ndash1752) 662 (275ndash1607) gt100 gt100741015840-Dihydroxy-31015840-methoxyflavanone (5) 248 (108ndash573) 304 (154ndash601) gt100 gt100Doxorubicin 0133 (00ndash03) gt69 41 (16ndash114) gt69

lowast

lowast

lowast

lowast

lowast

lowast

Control

00

05

10

15

20

Cel

l num

ber (times10

6)

24 48 720Incubation time (h)

Compound 1 26 120583MCompound 1 52 120583MCompound 1 104 120583M

(a)

lowast

lowastlowastlowast

Control

00

05

10

15

Cel

l num

ber (times10

6)



(b)

Figure 2 Time- and dose-dependent effects of compound 1 on CCRF-CEM (a) and K562 (b) cells Cell viability was quantified by using thetrypan blue dye exclusion method The results are expressed as mean plusmn SEM Significant differences were labeled with asterisks (119901 lt 005)compared to 1 ethanol control group at each time (ANOVA followed by Bonferronirsquos test)

The anticancer activity of compounds 1ndash5 against a panelof two leukemia cell lines and their respective drug-resistantphenotypes was further evaluated by conducting MTT cellviability assays As observed in Table 2 compound 1 showeda strong cytotoxic effect against CCRF-CEM cells and theirMDR counterpart CEMADR5000 as is evident from theIC50

values obtained which were below 10 120583M the valueestablished by the US National Cancer Institute plant screen-ing program for considering a pure compound as cytotoxic[37] It is interesting to note that 1 also showed a moderateinhibitory effect against K562 and Lucena 1 (Table 2) withIC50

values lower than 30 120583M As observed a lack of cross-resistance on P-gp-overexpressing cell lines was evidenced bythe toxic effect exerted by 1 over these (resistant index (RI) =IC50

resistant cellsIC50

sensitive cells = 15 and 11 againstCEMADR5000 and Lucena 1 cells resp) in contrast to thatfound when cells were exposed to DOX (RI ge 5188 and 168against CEMADR5000 and Lucena 1 resp) These resultshighlight the potential of 1 as a therapeutic agent for thetreatment of resistant phenotypes Although its toxic effectstoward different human andmurine tumor cell types such asMCF-7 (breast) [38] MGC-803 (gastric) [39] 26-L5 (colon)B16-BL6 (melanoma) A549 and LLC (lung) HeLa (cervix)HT-1080 (fibrosarcoma) HL 60 (promyelocytic leukemia)

and PANC-1 (pancreatic) [40ndash42] have been described thisis the first report about the cytotoxicity of 1 against ALL andCML cells and particularly against their MDRP-gp sublinesas a way to circumvent the reduced response of these resistantphenotypes to chemotherapy

The toxic effect of 1 was also determined against thenoncancerous rapidly dividing cell line HEK293T [43] andagainst PBMC From the IC

50values obtained we saw that

1 was highly selective against ALL cells as evidenced by theselectivity indexes calculated (SI ratio between IC

50value

in nontarget cells to the IC50

value in tumor cells) withvalues greater than 6 taking into consideration previousclassifications [44 45] When the medium growth inhibitoryconcentration of 1 toward HEK293T (IC

50= 670120583M) and

PBMC (IC50

= 828 120583M) was compared to that of CCRF-CEM (IC

50= 66 120583M) SIs of 101 and 125 were respectively

observed while in the case of CEMADR5000 (IC5099 120583M)

the IC50

ratios were 68 and 84 respectively On the otherhand compounds with SIs between 3 and 6 were rated asmoderately selective [44 45] According to the ratios betweenthe IC

50obtained for PBMC and those obtained for K562

cells (IC50= 275 120583M) and for Lucena 1 (IC

50= 300 120583M) cells

compound 1 meets this criterion (SI = 30 and 28 resp) Itis worth noting that 1 also showed a low toxic effect when


0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)

Figure 3 CCRF-CEM and K562 cells treated with 1 ethanol (controls (a) and (c) resp) or with 1 at 2 times IC50((b) and (d) resp) for 72 h

were stained with PI for cell cycle phase distribution analysis by flow cytometry The percentages of cells in each phase including sub-G1(hypodiploid) are indicated

assayed against normal hepatocytes (IC50

34 120583gmL by thetrypan blue exclusion method) [46]

The selective activity of 5 is also interesting showingmoderate toxicity against CCRF-CEM and CEMADR5000in contrast to its complete lack of effectiveness (IC

50gt

100 120583M) on the rest of the assayed cellsComparing the cytotoxic activity of chalcones 1 and 2

the presence of an additional hydroxyl group at position 4of compound 2 would not prove favorable for its toxicityas reflected by its higher IC

50values (519ndashgt100 120583M) with

respect to 1 (66ndash300120583M)This is in complete agreementwith

Li et al [40] who tested these compounds against a panelof different tumor cell lines and reported that the toxicityexerted by 1 broadly exceeded that of compound 2 From ourresults we can infer that the presence of 4-OH negativelyinfluences both cytotoxic and antibacterial activity

The strong cytotoxic activity exhibited by 1 in comparisonto the low potency exerted by 4 matches that reported byPouget et al [38] who found that 1 was more effective atinhibiting MCF-7 cell proliferation than its correspondingflavanone 4 The presence of a hydroxyl group at position C-5 made no difference to the activity of compounds 3 and 4


100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)

Figure 4 Effects of compound 1 on cell death process CCRF-CEM and K562 cells were incubated for 72 h with 1 ethanol (controls (a) and(c) resp) or with 2 times IC

50of 1 ((b) and (d) resp) Apoptotic effect of 1 was evaluated by flow cytometry analysis after double staining with

Annexin V-APC and PI Annexin V-APC intensity is represented on the 119909-axis and PI fluorescence intensity is represented on the 119910-axis

in contrast to that observed with respect to the antibacterialeffect exerted by these compounds The substitution patternof the B-ring in flavanone 5 with respect to 4 increasedthe compoundrsquos toxicity against CCRF-CEM and its MDRcounterpart (see Table 2)

When the number of viable cells was determined at differ-ent time points by a trypan blue exclusion test it was observedthat 1 exerted a negative effect on normal cell proliferationof both CCRF-CEM (Figure 2(a)) and K562 (Figure 2(b))cell lines in a time- and dose-dependent manner As shown

in Figure 2(a) compound 1 at 26 120583M showed a significantdifference (119901 lt 005) in the number of CCRF-CEM viablecells with respect to the ethanol control at 72 h from thebeginningwhile this effectwas already observed at 48 hwhen52 120583M of 1 was added When K562 cells were treated with1 at 208120583M for 48 h a significant decrease (119901 lt 005)was observed in the number of viable cells compared tothe ethanol control and when the concentration of 1 wasincreased to 416 120583M this difference (119901 lt 005) was alreadyobserved at 24 h (Figure 2(b))


When exposed to cytotoxic agents cells may suffer deathor a stop in their cell cycle which in turn can triggerdeath by apoptosis if cells cannot overcome the damage[47] Apoptosis is a common mechanism involved in thetoxicity of many anticancer drugs including those fromnatural origin [48] Hence due to the strong compound 1-induced cytotoxicity and since the mechanism underlyingthis effect in the leukemic cells CCRF-CEM and K562 cellswas uncertain we performed flow cytometric experiments todissect the possible mode of action beyond the toxic effect of1 As depicted in Figure 3(b) analysis of the DNA contentsof CCRF-CEM cells indicated that treatment with 1 at 2 timesIC50

for 72 h produced a cell cycle arrest of 34 of the cellsin the G

2M phase compared to 7 of cells in this phase

in the ethanol control (Figure 3(a)) K562 cells also showedan increase in the proportion of cells in the G

2M phase

(19) (Figure 3(d)) compared to the ethanol control (6)(Figure 3(c)) Given the key role that the G

2M checkpoint

plays in the maintenance of chromosomal integrity allowingcells to repair DNA damage before entering mitosis [49]blockage of the cell cycle at this stage by compound 1emerges as a potential pharmacological strategy for thetreatment of leukemia The arrest observed was also accom-panied by an increase in the hypodiploid sub-G1 populationfrom 1 in the ethanol controls (Figures 3(a) and 3(c)) to10 and 25 for CCRF-CEM and K562 cells respectively(Figures 3(b) and 3(d)) indicating the presence of celldeath

To further determine the induction of apoptosis theflipping of phosphatidylserine to the outer membrane asa hallmark of apoptosis was analyzed using Annexin Vstaining As shown in Figures 4(b) and 4(d) compound 1 at2 times IC

50induced apoptosis in CCRF-CEM and in K562 at

72 h of treatment reaching 267 and 687 respectively ofearly and late apoptotic cells

In agreement with our results Lou et al [50 51] reportedthat the exposure of the human gastric cancer cell line MGC-803 to 1 resulted in a dose-dependent G

2M phase cell cycle

arrest accompanied by apoptosis but these effects have notbeen described yet for ALL and CML cells

4 Conclusions

This work extends the currently available information aboutthe chemical composition and activity of F oolepis and offersa set of bioactive molecules that can be used as a modelfor the design of improved antibacterial and cytotoxic com-pounds The antibacterial activity observed for compound 1in addition to its strong and selective cytotoxicity highlightsits potential as a lead compound to fight against pathogenicbacteria and ALL and CML cells including their MDRphenotypes

Conflict of Interests

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

Acknowledgments

This work was supported by the Catholic University ofCordoba FONCYT (BID 1728 33593 PICTO CRUP 6-31396 and PICT 0141) MINCyT Cordoba (GRF 2008) andCONICET (PIP 11220100100236) The authors thank JossHeywood for revising the English language They also thankDr V Rumjanek and Dr T Efferth for providing K562 and itsderivate Lucena 1 and CCRF-CEM and CEMADR5000 cellsrespectively

References

[1] V Kuete S Alibert-Franco K O Eyong et al ldquoAntibacterialactivity of some natural products against bacteria express-ing a multidrug-resistant phenotyperdquo International Journal ofAntimicrobial Agents vol 37 no 2 pp 156ndash161 2011

[2] L Hu Z-R Li J-N Li J Qu J-D Jiang and D W Boykinldquo3-(21015840-Bromopropionylamino)-benzamides as novel S-phasearrest agentsrdquo Bioorganic ampMedicinal Chemistry Letters vol 17no 24 pp 6847ndash6852 2007

[3] D J Newman and G M Cragg ldquoNatural products as sourcesof new drugs over the 30 years from 1981 to 2010rdquo Journal ofNatural Products vol 75 no 3 pp 311ndash335 2012

[4] A Harvey ldquoStrategies for discovering drugs from previouslyunexplored natural productsrdquoDrug Discovery Today vol 5 no7 pp 294ndash300 2000

[5] M C Carpinella and M Rai Novel Therapeutic Agents fromPlants Science Publishers Enfield NH USA 2009

[6] X H Ma C J Zheng L Y Han et al ldquoSynergistic ther-apeutic actions of herbal ingredients and their mechanismsfrom molecular interaction and network perspectivesrdquo DrugDiscovery Today vol 14 no 11-12 pp 579ndash588 2009

[7] M B Joray M R del Rollan G M Ruiz S M Palaciosand M C Carpinella ldquoAntibacterial activity of extracts fromplants of central Argentinamdashisolation of an active principlefrom Achyrocline satureioidesrdquo Planta Medica vol 77 no 1 pp95ndash100 2011

[8] G N D Napal M C Carpinella and S M Palacios ldquoAnti-feedant activity of ethanolic extract from Flourensia oolepis andisolation of pinocembrin as its active principle compoundrdquoBioresource Technology vol 100 no 14 pp 3669ndash3673 2009

[9] A F Barrero M M Herrador P Arteaga I Rodriguez-Garciaand M Garcia-Moreno ldquoResorcinol derivatives and flavonoidsof Ononis natrix subspecies ramosissimardquo Journal of NaturalProducts vol 60 no 2 pp 65ndash68 1997

[10] E Wollenweber and D S Seigler ldquoFlavonoids from the exudateof Acacia neovernicosardquo Phytochemistry vol 21 no 5 pp 1063ndash1066 1982

[11] Z-P Zheng K-WCheng J Chao JWu andMWang ldquoTyrosi-nase inhibitors from papermulberry (Broussonetia papyrifera)rdquoFood Chemistry vol 106 no 2 pp 529ndash535 2008

[12] E Kostrzewa-Susłow and T Janeczko ldquoMicrobial transforma-tions of 7-hydroxyflavanonerdquo The Scientific World Journal vol2012 Article ID 254929 8 pages 2012

[13] C A Calanasan and J KMacLeod ldquoA diterpenoid sulphate andflavonoids fromWedelia asperrimardquo Phytochemistry vol 47 no6 pp 1093ndash1099 1998

[14] M C Carpinella L De Bellis M B Joray V Sosa PM Zuninoand S M Palacios ldquoInhibition of development swarmingdifferentiation and virulence factors in Proteus mirabilis by an


extract of Lithrea molleoides and its active principle (ZZ)-5-(trideca-4101584071015840-dienyl)-resorcinolrdquo Phytomedicine vol 18 no 11pp 994ndash997 2011

[15] CLSIMethods for Dilution Antimicrobial Susceptibility Tests forBacteria That Grow Aerobically Approved Standard Clinicaland Laboratory Standards Institute Wayne Pa USA 2012

[16] T Efferth M Davey A Olbrich G Rucker E Gebhartand R Davey ldquoActivity of drugs from traditional chinesemedicine toward sensitive andMDR1- or MRP1-overexpressingmultidrug-resistant human CCRF-CEM leukemia cellsrdquo BloodCells Molecules and Diseases vol 28 no 2 pp 160ndash168 2002

[17] V M Rumjanek R S Vidal and R C Maia ldquoMultidrugresistance in chronic myeloid leukaemia how much can welearn from MDR-CML cell linesrdquo Bioscience Reports vol 33no 6 Article ID e00081 2013

[18] A Kimmig V Gekeler M Neumann et al ldquoSusceptibilityof multidrug-resistant human leukemia cell lines to humaninterleukin 2-activated killer-cellsrdquoCancer Research vol 50 no21 pp 6793ndash6799 1990

[19] M A M Moreira C Bagni M B de Pinho et al ldquoChangesin gene expression profile in two multidrug resistant cell linesderived froma samedrug sensitive cell linerdquoLeukemiaResearchvol 38 no 8 pp 983ndash987 2014

[20] L D Trucco V Andreoli N G Nunez M Maccioni andJ L Bocco ldquoKruppel-like factor 6 interferes with cellulartransformation induced by the H-ras oncogenerdquo The FASEBJournal vol 28 no 12 pp 5262ndash7276 2014

[21] M N Renno G M Barbosa P Zancan et al ldquoCrudeethanol extract from babassu (Orbignya speciosa) cytotoxicityon tumoral and non-tumoral cell linesrdquo Anais da AcademiaBrasileira de Ciencias vol 80 no 3 pp 467ndash476 2008

[22] M B Joray M L Gonzalez S M Palacios and M CCarpinella ldquoAntibacterial activity of the plant-derived com-pounds 23-methyl-6-O-desmethylauricepyrone and (ZZ)-5-(trideca-47-dienyl)resorcinol and their synergy with antibi-otics against methicillin-susceptible and -resistant Staphylococ-cus aureusrdquo Journal of Agricultural and Food Chemistry vol 59no 21 pp 11534ndash11542 2011

[23] A Sersic A Cocucci S Vieiras et al Flores del Centro deArgentinaUnaGuıa Ilustrada ParaConocer 141 Especies TıpicasAcademia Nacional de Ciencias Cordoba Argentina 2006

[24] E Guerreiro J Kavka O S Giordano and E G GrosldquoSesquiterpenoids and flavonoids from Flourensia oolepisrdquo Phy-tochemistry vol 18 no 7 pp 1235ndash1237 1979

[25] E Wong and G Latch ldquoEffect of fungal diseases on phenoliccontents of white cloverrdquo New Zealand Journal of AgriculturalResearch vol 14 no 3 pp 633ndash638 1971

[26] E Wong and C M Francis ldquoFlavonoids in genotypes ofTrifolium subterraneummdashIThe normal flavonoid pattern of theGeraldton varietyrdquo Phytochemistry vol 7 no 12 pp 2123ndash21291968

[27] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[28] T P T Cushnie and A J Lamb ldquoRecent advances in under-standing the antibacterial properties of flavonoidsrdquo Interna-tional Journal of Antimicrobial Agents vol 38 no 2 pp 99ndash1072011

[29] J-F T K Akoachere R N Ndip E B Chenwi L M NdipT E Njock and D N Anong ldquoAntibacterial effect of Zingiberofficinale andGarcinia kola on respiratory tract pathogensrdquo EastAfrican Medical Journal vol 79 no 11 pp 588ndash592 2002

[30] A Sersic A Cocucci S Benıtez-Vieyra et al Flores del Centrode Argentina Una Guıa Ilustrada Para Conocer 141 EspeciesTıpicas Academia Nacional de Ciencias Cordoba Argentina2006

[31] L E Alcaraz S E Blanco O N Puig F Tomas and F HFerretti ldquoAntibacterial activity of flavonoids againstmethicillin-resistant Staphylococcus aureus strainsrdquo Journal of TheoreticalBiology vol 205 no 2 pp 231ndash240 2000

[32] I C Zampini M A Vattuone and M I Isla ldquoAntibacterialactivity of Zuccagnia punctata Cav ethanolic extractsrdquo Journalof Ethnopharmacology vol 102 no 3 pp 450ndash456 2005

[33] H Koo P L Rosalen J A Cury Y K Park and W HBowen ldquoEffects of compounds found in propolis on Strep-tococcus mutans growth and on glucosyltransferase activityrdquoAntimicrobial Agents andChemotherapy vol 46 no 5 pp 1302ndash1309 2002

[34] H P Avila E D F A Smania F D Monache and A SmaniaJr ldquoStructure-activity relationship of antibacterial chalconesrdquoBioorganic and Medicinal Chemistry vol 16 no 22 pp 9790ndash9794 2008

[35] M A Alvarez V E P Zarelli N B Pappano and N BDebattista ldquoBacteriostatic action of synthetic polyhydroxylatedchalcones against Escherichia colirdquo Biocell vol 28 no 1 pp 31ndash34 2004

[36] T P T Cushnie and A J Lamb ldquoAntimicrobial activity offlavonoidsrdquo International Journal of Antimicrobial Agents vol26 no 5 pp 343ndash356 2005

[37] V Kuete P Y Ango S O Yeboah et al ldquoCytotoxicity of fourAframomum species (A arundinaceum A alboviolaceum Akayserianum and A polyanthum) towards multi-factorial drugresistant cancer cell linesrdquoBMCComplementary andAlternativeMedicine vol 14 no 1 article 340 2014

[38] C Pouget F Lauthier A Simon et al ldquoFlavonoids structuralrequirements for antiproliferative activity on breast cancercellsrdquo Bioorganic and Medicinal Chemistry Letters vol 11 no24 pp 3095ndash3097 2001

[39] C Lou M Wang G Yang et al ldquoPreliminary studies on anti-tumor activity of 2101584041015840-dihydroxychalcone isolated from HerbaOxytropis in human gastric cancer MGC-803 cellsrdquo Toxicologyin Vitro vol 23 no 5 pp 906ndash910 2009

[40] F Li S Awale Y Tezuka and S Kadota ldquoCytotoxic constituentsfromBrazilian red propolis and their structure-activity relation-shiprdquo Bioorganic and Medicinal Chemistry vol 16 no 10 pp5434ndash5440 2008

[41] S Awale T Miyamoto T Z Linn et al ldquoCytotoxic constituentsof Soymida febrifuga from Myanmarrdquo Journal of Natural Prod-ucts vol 72 no 9 pp 1631ndash1636 2009

[42] A S Sufian K Ramasamy N Ahmat Z A Zakaria and MI M Yusof ldquoIsolation and identification of antibacterial andcytotoxic compounds from the leaves ofMuntingia calabura LrdquoJournal of Ethnopharmacology vol 146 no 1 pp 198ndash204 2013

[43] J McNulty J J Nair C Codina et al ldquoSelective apoptosis-inducing activity of crinum-type Amaryllidaceae alkaloidsrdquoPhytochemistry vol 68 no 7 pp 1068ndash1074 2007

[44] CM Viau D J Moura V A Facundo and J Saffi ldquoThe naturaltriterpene 3120573612057316120573-trihydroxy-lup-20(29)-ene obtained fromthe flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cellsrdquo BMC Complementary and AlternativeMedicine vol 14 no 1 article 280 2014

[45] E M Acton V L Narayanan P A Risbood R H ShoemakerD T Vistica and M R Boyd ldquoAnticancer specificity of some


ellipticinium salts against human brain tumors in vitrordquo Journalof Medicinal Chemistry vol 37 no 14 pp 2185ndash2189 1994

[46] O Sabzevari G Galati M Y Moridani A Siraki and PJ OrsquoBrien ldquoMolecular cytotoxic mechanisms of anticancerhydroxychalconesrdquo Chemico-Biological Interactions vol 148no 1-2 pp 57ndash67 2004

[47] LG LeonO J Donadel C E Tonn and JM Padron ldquoTessaricacid derivatives induce G2M cell cycle arrest in human solidtumor cell linesrdquo Bioorganic and Medicinal Chemistry vol 17no 17 pp 6251ndash6256 2009

[48] C Pieme S KumarMDongmo et al ldquoAntiproliferative activityand induction of apoptosis by Annona muricata (Annonaceae)extract on human cancer cellsrdquo BMC Complementary andAlternative Medicine vol 14 no 1 article 516 2014

[49] J-G Lee J-H Kim J-H Ahn K-T Lee N-I Baek and J-H Choi ldquoJaceosidin Isolated from dietary mugwort (Artemisiaprinceps) Induces G2M cell cycle arrest by inactivatingcdc25C-cdc2 via ATM-Chk12 activationrdquo Food and ChemicalToxicology vol 55 pp 214ndash221 2013


[51] C Lou G Yang H Cai et al ldquo2101584041015840-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803 cells viadown-regulation of survivin mRNArdquo Toxicology in Vitro vol24 no 5 pp 1333ndash1337 2010

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


compounds administered as therapeutic agents were consid-ered) The plant world is still far from being totally exploredhowever particularly the native flora from Argentina [7]Flourensia oolepis S F Blake (Asteraceae) commonly knownas ldquochilcardquo is a woody bush widely distributed in the highareas of the provinces of Cordoba and San Luis in centralArgentina [8]The antibacterial activity of the ethanol extractobtained from this plant was previously reported by ourgroup [7] The current study aims to gain insight into themetabolites responsible for this effect

The cytotoxic activity of the compounds isolated againstleukemic cell lines and their multidrug resistant (MDR)counterparts was also investigated and this was extended todetermine the mechanism involved in leukemic cell toxicity

To our knowledge this is the first detailed study aboutthe constituents of F oolepis that show these pharmacologicalproperties

2 Materials and Methods

21 Plant Material and Extract Preparation Aerial parts ofthe native plant F oolepis S F Blake were collected from thehills of Cordoba Province Argentina in December 2005 Avoucher specimen was deposited in the ldquoMarcelino SayagordquoHerbarium of the School of Agricultural Science CatholicUniversity of Cordoba (UCCOR 23) The authorization forthe use of the plant is available from the authors Crushedair-dried material (200 g) was extracted by 48 h macerationwith 700mL of ethanol and the yield of the extract obtainedafter solvent removal and expressed as percentage weight ofair-dried crushed plant material was 23 g

22 Chemicals Equipment and Reagents 3-(45-dimethyl-thiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) waspurchased from Sigma-Aldrich CO (St Louis MO USA)Doxorubicin hydrochloride 998 (DOX Synbias PharmaLtd) was purchased from Nanox Release Technology(Buenos Aires Argentina)

Gentamicin sulfate (potency 550ndash590 120583gmg) and ery-thromycin (potency 863 120583gmg) were provided by Labora-torio Fabra SA Buenos Aires Argentina and UnifarmaBuenos Aires Argentina respectively1H- and 13C-NMR and two-dimensional spectra were

recorded with a Bruker AVANCE II 400 spectrometer(Bruker Corporation Ettlingen Germany) with tetramethyl-silane (TMS) as the internal reference HPLC was performedon a Shimadzu LC-10 AS (Shimadzu Corp Tokyo Japan)equipped with a Phenomenex Prodigy 5 120583 ODS (46mmidtimes 250mm) reversed-phase columnThemobile phase waswatermethanoltrifluoroacetic acid 65 35 1 with detectionat 365 nm

Flow cytometry analysis was performed in a Becton-Dickinson (BD) FACS Canto II flow cytometer (BD Bio-sciences USA)

23 Bioguided Isolation of theActive Principles fromFlourensiaoolepis The antibacterial ethanol extract of F oolepis (12 g)was initially subjected to vacuum liquid chromatography on

silica gel (622 g 63ndash200120583m 110 times 240 cm Macherey ampNagel) eluted with a step gradient of hexanediethyl ether(Et2O)methanol (MeOH) to yield 26 fractions which were

combined in 6 groups according to their thin layer chro-matography (TLC) profile (F1 to F6) Of these fraction F3eluted with hexaneEt

2O 50 50 and F5 eluted with 100

Et2O demonstrated bactericidal activity at 2mgmL and

were therefore submitted to additional separation methodsfor further purification F3 was processed by radial prepar-ative chromatography using an isocratic mobile phase ofhexaneEt

2O 70 30 The fractions obtained were combined

in 5 groups in accordance with the TLC analysis (F31 to

F35) From fraction F

34 compound 3 was obtained by

spontaneous crystallization (978 purity by HPLC) Thepure compound the remaining F

34 containing traces of 3 and

another substance as well as the rest of the fractions weretested for antibacterial activity Only 3 and the remaining F

34

exerted bactericidal effects at 2mgmLThe latter was furtherfractionated by preparative TLC (Analtech DE USA mobilephase tolueneethyl acetateglacial acetic acid 8 25 04) tofinally obtain compound 1 (999 purity by HPLC)

Fraction F5 was subjected to column chromatography(130 g 35ndash70 120583m 30 times 60 cm Fluka) using 100 Et

2O as

the mobile phase The resulting fractions were placed in 14groups according to TLC monitoring (F

51 to F514) Of these

F52 showed bactericidal activity and was further fractionated

by column chromatography on silica gel (60 g 35ndash70 120583m30 times 60 cm Fluka) performed with hexaneEt

2Oglacial

acetic acid 8 2 04 The fractions obtained were combinedin 7 groups according to their TLC profile (F

521ndashF527)

Their antibacterial activity was evaluated at 2mgmL andF522 and F

525 were seen to be active Both groups were

separately submitted to solid phase extraction (Sep-Pak plusC18Waters Ireland) using waterMEOHtrifluoroacetic acid65 35 1 as the mobile phase to afford compound 2 (991purity by HPLC) from F

522 and compounds 4 (964 purity

by HPLC) and 5 (972 purity by HPLC) from F525

Compounds 1ndash5 were identified by 1H NMR and 13CNMR comparing their spectra (copies of the original spectraare obtainable from the corresponding author) with referencedata from literature as 2101584041015840-dihydroxychalcone C

15H12O3

(1 mz 2402) [9 10] isoliquiritigenin C15H12O4(2 mz

2562) [11] pinocembrin C15H12O4(3 mz 2562) [8] 7-

hydroxyflavanone C15H12O3(4 mz 2402) [12] and 741015840-

dihydroxy-31015840-methoxyflavanone C16H14O5(5 mz 2863)

[13] The compounds were quantified by HPLC and theiryields in g per 100 g of dried and crushed plant material were124 006 114 03 and 006 for 1ndash5 respectively

24 Microorganisms and Preparation of Inocula The antibac-terial activity assays were carried out on strains of Entero-coccus faecalis (Andrews and Horder) Scheifer and Klipper-Balz (ATCC 29212) Escherichia coli (Migula) Castellaniand Chalmers (ATCC 25922) Pseudomonas aeruginosa(Schroeter)Migula (ATCC 27853) and Staphylococcus aureussubsp aureus Rosenbach (ATCC 6538) and on a clinical iso-late of tetracycline erythromycin and polymyxin-resistantProteus mirabilis (Pr2921) [14] This isolate was identified





















50)













1

HO OH

OH

O2

3

HO O

O4

HO

OH

HO

A

B

O

120573

120572120573998400

2

3

4

5

62998400

3998400

4998400

5998400

6998400

OH

O

O

2

345

6

78

2998400

3998400

4998400

5998400

6998400

A

B

C

HO

OH

O O

O

5

CH3





250(gt500)

62(125)

62(125)

31(31)


gt500(gt500)

gt500(gt500)

500(gt500)

250(500)


gt500(gt500)

gt500(gt500)

62(125)

15(62)


500(gt500)

gt500(gt500)

250(500)

250(250)


gt500(gt500)

gt500(gt500)

500(gt500)

250(gt500)

Gentamicin 4(8)

4(4)

10(10)

8(10)

8(10)


62(500)

500(gt500)

1(30)

1(60)







lowast

lowast

lowast

lowast

lowast

lowast

Control

00

05

10

15

20

Cel

l num

ber (times10

6)



(a)

lowast

lowastlowastlowast

Control

00

05

10

15

Cel

l num

ber (times10

6)



(b)





resistant cellsIC50






50value



50= 670120583M) and

PBMC (IC50




the IC50




50= 300 120583M) cells



0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)






50gt








100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































50)













1

HO OH

OH

O2

3

HO O

O4

HO

OH

HO

A

B

O

120573

120572120573998400

2

3

4

5

62998400

3998400

4998400

5998400

6998400

OH

O

O

2

345

6

78

2998400

3998400

4998400

5998400

6998400

A

B

C

HO

OH

O O

O

5

CH3





250(gt500)

62(125)

62(125)

31(31)


gt500(gt500)

gt500(gt500)

500(gt500)

250(500)


gt500(gt500)

gt500(gt500)

62(125)

15(62)


500(gt500)

gt500(gt500)

250(500)

250(250)


gt500(gt500)

gt500(gt500)

500(gt500)

250(gt500)

Gentamicin 4(8)

4(4)

10(10)

8(10)

8(10)


62(500)

500(gt500)

1(30)

1(60)







lowast

lowast

lowast

lowast

lowast

lowast

Control

00

05

10

15

20

Cel

l num

ber (times10

6)



(a)

lowast

lowastlowastlowast

Control

00

05

10

15

Cel

l num

ber (times10

6)



(b)





resistant cellsIC50






50value



50= 670120583M) and

PBMC (IC50




the IC50




50= 300 120583M) cells



0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)






50gt








100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

HO OH

OH

O2

3

HO O

O4

HO

OH

HO

A

B

O

120573

120572120573998400

2

3

4

5

62998400

3998400

4998400

5998400

6998400

OH

O

O

2

345

6

78

2998400

3998400

4998400

5998400

6998400

A

B

C

HO

OH

O O

O

5

CH3





250(gt500)

62(125)

62(125)

31(31)


gt500(gt500)

gt500(gt500)

500(gt500)

250(500)


gt500(gt500)

gt500(gt500)

62(125)

15(62)


500(gt500)

gt500(gt500)

250(500)

250(250)


gt500(gt500)

gt500(gt500)

500(gt500)

250(gt500)

Gentamicin 4(8)

4(4)

10(10)

8(10)

8(10)


62(500)

500(gt500)

1(30)

1(60)







lowast

lowast

lowast

lowast

lowast

lowast

Control

00

05

10

15

20

Cel

l num

ber (times10

6)



(a)

lowast

lowastlowastlowast

Control

00

05

10

15

Cel

l num

ber (times10

6)



(b)





resistant cellsIC50






50value



50= 670120583M) and

PBMC (IC50




the IC50




50= 300 120583M) cells



0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)






50gt








100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















lowast

lowast

lowast

lowast

lowast

lowast

Control

00

05

10

15

20

Cel

l num

ber (times10

6)



(a)

lowast

lowastlowastlowast

Control

00

05

10

15

Cel

l num

ber (times10

6)



(b)





resistant cellsIC50






50value



50= 670120583M) and

PBMC (IC50




the IC50




50= 300 120583M) cells



0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)






50gt








100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

G0G1 = 51S = 42G2M = 7

Sub-G1

1

(a)

0

500

1000

1500

2000

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

10

G0G1 = 35S = 31G2M = 34

(b)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

1

G0G1 = 53S = 41G2M = 6

(c)

0

300

600

900

1200

Cel

l cou

nt

200 400 600 800 1k0

PI fluorescence

Sub-G1

25

G0G1 = 40S = 41G2M = 19

(d)






50gt








100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

104PI

fluo

resc

ence

101 102 103100 104

Annexin V-APC

03 04

08

(a)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

09 44

223

(b)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

02 23

28

(c)

100

101

102

103

104

PI fl

uore

scen

ce

101 102 103100 104

Annexin V-APC

32 444

243

(d)












2M phase


2M checkpoint






2M phase cell cycle


4 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2M phase


2M checkpoint






2M phase cell cycle


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 Antibacterial and Cytotoxic Activity of...

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