phytochemical study and evaluation of the cytotoxic...

Research ArticlePhytochemical Study and Evaluation of the Cytotoxic Propertiesof Methanolic Extract from Baccharis obtusifolia

Juan Carlos Romero-Benavides 1 Gina C Ortega-Torres 1 Javier Villacis2

Sara L Vivanco-Jaramillo1 Karla I Galarza-Urgileacutes 1 and Natalia Bailon-Moscoso2

1Departamento de Quımica y Ciencias Exactas Universidad Tecnica Particular de Loja (UTPL) San Cayetano Alto SN1101608 Loja Ecuador2Departamento de Ciencias de la Salud Universidad Tecnica Particular de Loja (UTPL) San Cayetano Alto SN1101608 Loja Ecuador

Correspondence should be addressed to Juan Carlos Romero-Benavides jcromerobutpleduec

Received 2 March 2018 Accepted 19 June 2018 Published 1 August 2018

Academic Editor Iftikhar Ali

Copyright copy 2018 Juan Carlos Romero-Benavides et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Some species of the Baccharis genus have been shown to possess important biomedical properties including cytotoxic activityIn this study we examined the cytotoxic effect of methanol extract from Baccharis obtusifolia (Asteraceae) in cancer cell lines ofprostate (PC-3) colon (RKO) astrocytoma (D-384) and breast (MCF-7) The methanolic extract displayed the largest substantialcytotoxic effect in lines of colon cancer (RKO) and cerebral astrocytoma (D-384) Chromatographic purification of the Bobtusifolia methanolic extract led to the isolation and identification of 541015840-dihydroxy-7-methoxyflavone (1) and 5-hydroxy-741015840-dimethoxyflavone (2) compounds of the flavonoid type

1 Introduction

Baccharis is the largest genus in the family Asteraceaewith over 500 species distributed throughout North andSouth America [1] The largest American genus Baccharis(Asteraceae) includes about 400 species Of these species20 are locally used for medical purposes or to a lesserextent as food or as rawmaterial for different local industries[2] The most prominent compounds in Baccharis are diter-penoids phenolic compounds like flavonoids and coumarinsand triterpenoids among others Flavonoids are importantcompounds isolated from a wide range of plants [3] Dietswith a high flavonoid content are associated with positivehealth effects and the prevention of several diseases [4] Addi-tionally pharmacological studies have demonstrated the anti-inflammatory effects [3] and antioxidant capacity [5] of sev-eral flavonoids and some flavonoids have been demonstratedto possess cytotoxic antifungal antiviral and antibacterialproperties [5ndash7] The most prominent biomedical applica-tions of the Baccharis obtusifolia HBK commonly knownas ldquoChilca redondardquo include the treatment of rheumatism

liver disease wounds and ulcers [1] In the present studywe performed a chemical composition analysis of the activephytometabolites obtained from themethanolic extract of theleaves ofBaccharis obtusifoliaWe also evaluated the cytotoxicactivity of this extract on human cancer cell lines

2 Materials and Methods

21 Preparation of the Extracts The leaves from the Baccharisobtusifolia species were collected on Villonaco (04∘0110158402510158401015840Lat S 79∘1410158404510158401015840 Long O 2849masl) mountain of theLoja Province of Ecuador A sample specimen (PPN-as-014) was deposited and identified in the Herbarium ofDepartamento deQuımica of Universidad Tecnica Particularof Loja Ecuador

The collected leaves were subjected to a dehydrationprocess in a drying tray with airflow at a temperature of 32∘Cfor seven days (final humidity 68)

To obtain the extract we used 145 g of dried leaves andcold methanol (4-5∘C) The method employed was dynamicmaceration for 5 hours in a light-free environment This

HindawiInternational Journal of Medicinal ChemistryVolume 2018 Article ID 8908435 5 pageshttpsdoiorg10115520188908435

2 International Journal of Medicinal Chemistry

O

OH O

OH

O

OH O

（3 （3

（3

54-dihydroxy-7-methoxyflavone () 5-hydroxy-74-dimethoxyflavone ()

Figure 1 Structure of compounds isolated from Baccharis obtusifolia

procedure was repeated three times The extract was thenconcentrated at 50mbar and 37∘C on a rotary evaporator(Buchi R210 Switzerland Flawil) and stored at 4∘C

22 Isolation of Secondary Metabolites The methanolicextract (874 g) was filtrated to remove chlorophylls with areverse phase silica gel RP-18 65 cm (CCMerck DarmstadtGermany) using specific mixtures of solvents fraction 1MeOHH

2O 8515 (1000ml) fraction 2 MeOHH

2O 9010

(250ml) fraction 3 100 MeOH (500ml) and fraction 4100 dichloromethane (500ml)

To separate the components from the extract withoutchlorophylls (fraction 1) a silica gel-60 F254 chromatographycolumn (CC Merck) was used Mixtures of hexane and ethylacetate solvents were used in polarity starting with hexane(100) to separate the compounds Fractions of 200ml eachwere collected using a vacuum pump and the solvent wasthen removed on a rotary evaporator and the residue wasrecovered with dichloromethaneThin layer chromatography(TLC Merck) was performed on each fraction to detect thecompounds The compounds were visualized by sprayingthe solutions with a mixture of vanillin ethanol and acidsulfuric followed by heating on a hot plate Fractions witha similar profile were pooled and purified by conventionalprocedures Fraction 40-43 was crystallized using hexane andethyl acetate and fraction 70-79 was crystallized using hexaneand petroleum ether

23 Characterization and Identification of Secondary Metabo-lites We followed the methods of Bailon-Moscoso et al(2016) [8] Melting points were determined using a Fisher-Johns apparatusThe 1Hand 13CNMR spectra were recordedat 400MHz and 100MHz respectively on Varian 400MHz-Premium Shielded equipment (Varian Massachusetts USA)using tetramethylsilane as an internal reference CDCl

3

and DMSO-d6 were used as solvents chemical shifts wereexpressed in parts per million (ppm) and coupling constants(J) were reported in Hz Mass spectra (MS) were determinedby a gas chromatograph (Agilent Technologies 6890 NWilmington DE) coupled to a mass spectrometer (AgilentTechnologies 5973 inert)

24 Cell Culture Procedures Four human cancer cell lineswere used PC-3 RKO D-384 and MCF-7 The cells werecultured in RPMI-1640 medium supplemented with 10fetal bovine serum (FBS Invitrogen Karlsruhe Germany)1 antibiotic-antimitotic solution (100 unitsml penicillin

G 100 120583gml streptomycin and 025 120583gml amphotericinB Gibco Grand Island NY) and 1 L-glutamine (2mMGibco) The cells were incubated at 37∘C in a 5 CO

2

atmosphere The viable cells were counted using the trypanblue exclusion method in a hemocytometer [8]

25 Cell Viability Analysis via MTS Assay The MTS (5-[3-(carboxymethoxy)phenyl]-3-(45-dimethyl-2-thiazolyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt) cell viabilityassay was used to assess the inhibitory effects of the extractson the survival of human cancer cell lines A total of 3ndash5times 103 cellswell were seeded into 96-well plates and wereallowed to adhere for 24 h The cells were then treatedwith 50 120583gml of whole extract to yield a final volume of2ml Each concentrationassay was performed three timesDimethyl sulfoxide (DMSO) was used as a negative controlat a final concentration of 01 vv and 1 120583M doxorubicinwas used as a positive control The cells were incubated withthe treatments for 48 h after which 20120583L MTS (5mgmlAqueous One Solution Reagent Gibco) was added andfurther incubated for 4 h at 37∘C The absorbance wasmeasured at 570 nm The data obtained with cells treatedwith DMSO were considered to represent 100 viability[8] In cell lines with an inhibition percentage over 30doses of 15 45 60 75 and 100 120583gml of methanolic extractwere applied and the MTS assay was used to measurethe inhibitory concentration 50 (IC

50) using nonlinear

regression

26 Statistical Analysis All of the data were reported asmeans plusmn standard error of the mean (SEM) of three indepen-dent experiments The nonlinear regression was determinedby GraphPad Prism 5 (GraphPad Software San Diego CA)

3 Results

31 Isolation of Secondary Metabolites from B Obtusi-folia From the methanolic extract without chlorophyllstwo representative fractions were obtained F 70-79 elutedin HexEtOAc 8020 from which we isolated 80mg ofa white crystalline solid identified as sakuranetin (541015840-dihydroxy-7-methoxyflavanone) (1) and the fraction F 43-45 eluted in HexEtOAc 9010 from which we isolated112mg of a yellow crystalline solid identified as 5-hydroxy-741015840-dimethoxyflavone (2) (Figure 1) All of the compoundswere identified based on physical and spectroscopic data viacomparisons with the literature [9ndash11]

International Journal of Medicinal Chemistry 3

Table 1 Effect of Baccharis obtusifolia extract on the growth of human cancer cell lines

Treatment of inhibition plusmn SEMa

Human cancer cell linesMCF-7 (breast cancer) PC3 (prostate cancer) RKO(colon cancer) D-384 (astrocytoma)

Methanolic extract 202 plusmn 07 183 plusmn 12 892 plusmn 13 606 plusmn 05Doxorubicin (1120583M) 409 plusmn 74 548 plusmn 19 892 plusmn 40 790 plusmn 46aMean and standard error (SEM) of at least three independent experiments Control cells were considered to exhibit 100 viability

Table 2 Half maximal inhibitory concentration (IC50) of the compounds isolated from Baccharis obtusifolia on cancer cell lines

CompoundIC50plusmn SEMa (120583M)

MCF-7(breast cancer)

PC-3(prostate cancer)

RKO(colon cancer)

D-384(astrocytoma)

54-dihydroxy-7-methoxyflavone (1) gt100 gt100 6811 plusmn 108 gt1005-hydroxy-741015840-dimethoxyflavone (2) gt100 gt100 3430 plusmn 134 gt100aEach datum is given as the mean and its standard error (SEM) of at least three independent experiments

32 Characterization and Identification of Secondary Metabo-lites Physical and spectroscopic constants from 541015840-dihy-droxy-7-methoxyflavone (1) mp 240ndash243∘C EIMS mz ()286 (M+ 100) 269 (36) 241 (16) 167 (13) 166 (14) 138 (14)128 (16) 121 (7) 118 (10) 1HNMR (DMSO-d

6) 120575 ppm 28 (1H

dd) 31 (1H dd) 389 (3H s) 640 (1H d J=23Hz) 679 (1Hd J=23Hz) 685 (1H s) 696 (2H d J=93Hz) 798 (2H dJ=93Hz) 1295 (1H s) 13C NMR (DMSO-d

6) 120575 ppm 79 (C-

2) 431 (C-3) 1960 (C-4) 1641 (C-5) 951 (C-6) 1680 (C-7)942 (C-8) 1628 (C-9) 1035 (C-10) 1305 (C-1rsquo) 1280 (C-2rsquo)1142 (C-3rsquo) 1561 (C-4rsquo) 1156 (C-5rsquo) 1280 (C-6rsquo) 556 (OCH

3-

7)Physical and spectroscopic constants from 5-hydroxy-

741015840-dimethoxyflavone (2) mp 160ndash163∘C EIMS mz ()298 (M+ 100) 269 (28) 255 (14) 166 (11) 138 (16) 135 (10)132 (16) 1H NMR (CDCI

3) 120575 ppm 389 390 (3H each s)

637 (1H d J= 24Hz) 649 (1H d J=24Hz) 658 (1H s)702 (2H d J=93Hz) 785 (2H d J=93Hz) 1280 (1H s)13C NMR (CDCl

3) 120575 ppm 1640 (C-2) 1043 (C-3) 1824 (C-

4) 1654 (C-5) 980 (C-6) 1654 (C-7) 926 (C-8) 1621 (C-9)1043 (C-10) 1235 (C-1rsquo) 1280 (C- 2rsquo) 1144 (C-3rsquo) 1576 (C-4rsquo)1145 (C-5rsquo) 1283 (C-6rsquo) 556 (OCH

37rsquo) 557 (OCH

3-4rsquo)

33 Cytotoxic Effects of Methanolic Extract and IsolatedCompounds on Human Cancer Cell Lines The effect of themethanolic extract on cell viability was determined via anMTS assay using human cancer cell lines treated for 48 hwith thewhole extract ofB obtusifolia (50 120583gml)The extractexhibited a strong inhibitory effect on RKO 892 as in D-384 cells with 606 However the MCF-7 and PC3 cell linesexhibited moderate inhibitory activity with 202 and 183respectively (Table 1)

Compounds 1 and 2 presented higher IC50

to 100 120583Mfor MCF-7 D-384 and PC-3 cell lines for the RKO cellline we obtained IC

50of 6811 120583M (1948120583gml) and 3430

120583M (1022 120583gml) for compounds 1 and 2 respectivelyCompounds 1 and 2 were specific for the RKO line andcompound 2 was more effective than compound 1 (Table 2)

4 Discussion

The species belonging to the Asteraceae family are char-acterized by their abundant biological activity [12] whichresults from their chemical composition including secondarymetabolites such as terpenes diterpenes flavonoids andcoumarins [13] In the present study two flavonoid com-pounds were isolated 541015840-dihydroxy-7-methoxyflavanone(1) and 5-hydroxy-741015840-dimethoxyflavone (2) These com-pounds have been identified for the first time in Baccharisobtusifolia but they have been reported in other species com-pound 1 in Chromolaena subscandens [14] Trixis vauthieri[15] and Trigona spinipes [16] and compound 2 in Baccharispolycephala [4] Thymus vulgaris [9] Combretum erythro-phyllum [17] Kaempferia parviflora [18] and Boesenbergiapandurata [19]

Our evaluation of the biological activity of methanolicextract revealed a high percentage of inhibition in RKO celllines (892) followed by D-384 cells (606) This activitycan be attributed to the presence of flavonoids in the extractSome flavonoids act on these stages in very different celltypes which may lead to the use of these compounds ascytostatic agents in the later stages of carcinogenesis ratherthan as early-stage preventive elements [20 21]

IC50of flavonoids 1 and 2 shows similar values to the other

compounds of the same type [22ndash24] Numerous studies havesuggested that flavonoids may play a protective role in theprevention of cancer coronary heart diseases bone loss andmany other age-related diseases [21] The activity found inthe methanol extract may be due to the presence of isolatedflavonoids

Chemically flavones are substances of phenolic natureand are characterized by having two benzene aromatic ringsunited by a bridge with three carbon atoms They have thegeneral structure C

6-C3-C6 which may form a third ring

Natural flavonoids are often present in at least three phenolichydroxyls [25] The NMR 1H data from compounds 1 and2 show a substitution pattern ldquoparardquo in the ring B [26]


substitutions in these flavones are found in the C-41015840 positionof the B ring in the case of compound 1 the substituentcorresponds to a hydroxyl group (OH) and for compound2 the substituent corresponds to a methoxy group (OCH

3)

In the NMR data of 13C from compound 1 in the aliphaticzone 120575 (556 ppm) there appears a signal that belongs to amethoxy group The signal in 120575 (431 ppm) is assigned toC-3 [26] and the signal in 1035 ppm is assigned to C-10 ofhydroxylated flavones which are included in the structureof our compound and confirmed in the 1H NMR spectrumThe spectral data of compound 2 show two signals in 120575(556ndash557 ppm) belonging to twomethoxy groups present inthis structure and these two groups are also observed in the1H NMR spectrum

Flavonoids have characteristics that make them attrac-tive for anticancer research They act in vitro via variousmechanisms in the oncogenic process which renders thempossible useful agents in the early stages of cancer or inthe inhibition of later stages of progression or invasionThis activity may be explained by studying the Structure-Activity Relationship (SAR) between several flavonoids andcancer [27] An example is the study of Zhang (2005) whichfocused on activity of flavonoids and breast cancer cells Thisauthor found that the double bond between C-2 and C-3 the ring B connected to C-2 the hydroxyl group in C-5 the nonhydroxylation in C-3 and the presence of apolarsubstituents in C-6 C-7 C-8 or C-41015840 were structural char-acteristics important for the interaction between flavonoidsand breast cancer resistance protein (BCRP) [21]The structure5-hydroxy-741015840-dimethoxyflavone (2) possesses importantstructural characteristics like the double bond between C-2and C-3 and the presence of an OH group at the 5-positionand the carbonyl group at the 4-position Previous studieshave suggested that the position number and substitution ofhydroxyl groups in rings A and B as well as the saturationin the C2-C3 bond may be important factors that increasethe cytotoxic or antiproliferative activities of flavonoids [28]which would explain the cytotoxic action of 5-hydroxy-741015840-dimethoxyflavone in RKO cells On the contrary the flavonesakuranetin (1) does not exhibit the double bond at C-2 andC-3 which inmono- or dihydroxylated flavones results in theloss of cellular growth inhibitory activity [28] It would beinteresting to continue studies of this species and the isolatedcompounds Additional studies of themolecularmechanismsunderlying the effect of these secondary metabolites oncancer cell survival are accordingly necessary

Data Availability

The dataset supporting this article is included in themanuscript

Disclosure

The authors alone are responsible for the content and writingof the paper

Conflicts of Interest

The authors report no conflicts of interest

Acknowledgments

This work was partially supported by Universidad TecnicaParticular de Loja Ecuador (Grant PROY FIN QUI 0008)

References

[1] M J Abad and P Bermejo ldquoBaccharis (Compositae) a reviewupdaterdquo Arkivoc vol 7 pp 76ndash96 2007

[2] M Abad A Bessa B Ballarin O Aragon E Gonzales and PBermejo ldquoAnti-inflammatory activity of four Bolivian Baccha-ris species (Compositae)rdquo Journal of Ethnopharmacology vol103 no 3 pp 338ndash344 2006

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

[4] M Davila O Sterner and N Hinojosa ldquoFlavonoids fromBaccharis Polycephala Weddellrdquo Revista Boliviana de Quimicavol 3 p 137 2013

[5] A Seyoum K Asres and F K El-Fiky ldquoStructurendashradicalscavenging activity relationships of flavonoidsrdquo Phytochemistryvol 67 no 18 pp 2058ndash2070 2006

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

[7] E Valarezo J Rosales V Morocho et al ldquoChemical compo-sition and biological activity of the essential oil of Baccharisobtusifolia Kunth from Loja Ecuadorrdquo Journal of Essential OilResearch vol 27 no 3 pp 212ndash216 2015

[8] N Bailon-Moscoso J C Romero-Benavides M Sordo et alldquoPhytochemical study and evaluation of cytotoxic and geno-toxic properties of extracts from Clusia latipes leavesrdquo RevistaBrasileira de Farmacognosia vol 26 no 1 pp 44ndash49 2016

[9] KMiura andNNakatani ldquoAntioxidativeActivity of Flavonoidsfrom Thyme (Thymus Vulgaris l)rdquo Agricultural and BiologicalChemistry vol 53 no 11 pp 3043ndash3045 1989

[10] B Achari C Chaudhuri C R Saha P K Dutta and S CPakrashi ldquoA clerodane diterpene and other constituents ofClerodendron inermerdquo Phytochemistry vol 29 no 11 pp 3671ndash3673 1990

[11] A G Gonzalez Z E Aguiar J G Luis A G Ravelo JT Vazquez and X A Domınguez ldquoFlavonoids from Salviatexanardquo Phytochemistry vol 28 no 10 pp 2871-2872 1989

[12] M C A da Silva and S R Paiva ldquoAntioxidant activity andflavonoid content of Clusia fluminensis PlanchampTrianardquoAnaisda Academia Brasileira de Ciencias vol 84 no 3 pp 609ndash6162012

[13] J Prada L L Ord E Coy-Barrera and L L Orduz-Dıaz ldquoBac-charis latifoliaUnaAsteraceae poco valorada conpotencialidadQuımica y Biologica en el Neotropicordquo Revista Facultad deCiencias Basicas vol 1 pp 92ndash105 2016

[14] A Guzma and O E Rodriguez ldquoFlavonoides de Chromolaenasubscandens (Hieronrdquo RM King vol 2 p 25 2008

[15] A Ribeiro D Pilo-Veloso A J Romanha and C L ZanildquoTrypanocidal flavonoids from Trixis vauthierirdquo Journal ofNatural Products vol 60 no 8 pp 836ndash838 1997

[16] M O Freitas F A F Ponte M A S Lima and E R SilveiraldquoFlavonoids and triterpenes from the nest of the stingless beeTrigona spinipesrdquo Journal of the Brazilian Chemical Society vol19 no 3 pp 532ndash535 2008


[17] N D Martini D R P Katerere and J N Eloff ldquoBiologicalactivity of five antibacterial flavonoids from Combretum ery-throphyllum (Combretaceae)rdquo Journal of Ethnopharmacologyvol 93 no 2-3 pp 207ndash212 2004

[18] C Yenjai K Prasanphen S Daodee V Wongpanich and PKittakoop ldquoBioactive flavonoids from Kaempferia parviflorardquoFitoterapia vol 75 no 1 pp 89ndash92 2004

[19] T Jaipetch V Reutrakul P Tuntiwachwuttikul and T SantisukldquoFlavonoids in the black rhizomes of Boesenbergia pandutardquoPhytochemistry vol 22 no 2 pp 625-626 1983

[20] M E Morris and S Zhang ldquoFlavonoid-drug interactionsEffects of flavonoids on ABC transportersrdquo Life Sciences vol 78no 18 pp 2116ndash2130 2006

[21] S Zhang X Yang R A Coburn and M E Morris ldquoStructureactivity relationships and quantitative structure activity rela-tionships for the flavonoid-mediated inhibition of breast cancerresistance proteinrdquoBiochemical Pharmacology vol 70 no 4 pp627ndash639 2005

[22] L Wang L Kuang J A Hitron et al ldquoApigenin suppressesmigration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expressionrdquo Toxicol-ogy and Applied Pharmacology vol 272 no 1 pp 108ndash116 2013

[23] K Sak ldquoCytotoxicity of dietary flavonoids on different humancancer typesrdquoPharmacognosy Reviews vol 8 no 16 p 122 2014

[24] S Srivastava R R Somasagara M Hegde et al ldquoQuercetina natural flavonoid interacts with DNA arrests cell cycle andcauses tumor regression by activating mitochondrial pathwayof apoptosisrdquo Scientific Reports vol 6 Article ID 24049 2016

[25] O Cartaya and I Reynaldo ldquoFlavonoides Caracterısticasquımicas y aplicacionesrdquo Cultivos Tropicales vol 2 no 1 pp 5ndash14 2001

[26] J C Herrera A J Rosas Romero O E Crescente M Acostaand S Pekerar ldquoAnalysis of 5-hydroxy-7-methoxyflavones bynormal-phase high-performance liquid chromatographyrdquo Jour-nal of Chromatography A vol 740 no 2 pp 201ndash206 1996

[27] E C Alvarez and F C Orallo ldquoActividad biologica de losflavonoides (I)rdquoAccion frente al cancer Offarm Farm Y Soc vol22 pp 130ndash140 2003

[28] C Martınez J Yanez J Rodrıguez M Canteras M Alcarazand V Vicente ldquoInhibicion del crecimiento de la lıneade melanoma murino B16F1O por diferentes compuestospolifenolicosrdquo Revista Espanola de Patologıa vol 34 pp 317ndash324 2001

TribologyAdvances in

Hindawiwwwhindawicom Volume 2018


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Journal of

Chemistry


Advances inPhysical Chemistry

Hindawiwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2018

Bioinorganic Chemistry and ApplicationsHindawiwwwhindawicom Volume 2018

SpectroscopyInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Medicinal ChemistryInternational Journal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of

Applied ChemistryJournal of



Biochemistry Research International


Enzyme Research


Journal of

SpectroscopyAnalytical ChemistryInternational Journal of


MaterialsJournal of



BioMed Research International Electrochemistry

International Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom


O

OH O

OH

O

OH O

（3 （3

（3

54-dihydroxy-7-methoxyflavone () 5-hydroxy-74-dimethoxyflavone ()

Figure 1 Structure of compounds isolated from Baccharis obtusifolia

procedure was repeated three times The extract was thenconcentrated at 50mbar and 37∘C on a rotary evaporator(Buchi R210 Switzerland Flawil) and stored at 4∘C

22 Isolation of Secondary Metabolites The methanolicextract (874 g) was filtrated to remove chlorophylls with areverse phase silica gel RP-18 65 cm (CCMerck DarmstadtGermany) using specific mixtures of solvents fraction 1MeOHH

2O 8515 (1000ml) fraction 2 MeOHH

2O 9010

(250ml) fraction 3 100 MeOH (500ml) and fraction 4100 dichloromethane (500ml)

To separate the components from the extract withoutchlorophylls (fraction 1) a silica gel-60 F254 chromatographycolumn (CC Merck) was used Mixtures of hexane and ethylacetate solvents were used in polarity starting with hexane(100) to separate the compounds Fractions of 200ml eachwere collected using a vacuum pump and the solvent wasthen removed on a rotary evaporator and the residue wasrecovered with dichloromethaneThin layer chromatography(TLC Merck) was performed on each fraction to detect thecompounds The compounds were visualized by sprayingthe solutions with a mixture of vanillin ethanol and acidsulfuric followed by heating on a hot plate Fractions witha similar profile were pooled and purified by conventionalprocedures Fraction 40-43 was crystallized using hexane andethyl acetate and fraction 70-79 was crystallized using hexaneand petroleum ether

23 Characterization and Identification of Secondary Metabo-lites We followed the methods of Bailon-Moscoso et al(2016) [8] Melting points were determined using a Fisher-Johns apparatusThe 1Hand 13CNMR spectra were recordedat 400MHz and 100MHz respectively on Varian 400MHz-Premium Shielded equipment (Varian Massachusetts USA)using tetramethylsilane as an internal reference CDCl

3

and DMSO-d6 were used as solvents chemical shifts wereexpressed in parts per million (ppm) and coupling constants(J) were reported in Hz Mass spectra (MS) were determinedby a gas chromatograph (Agilent Technologies 6890 NWilmington DE) coupled to a mass spectrometer (AgilentTechnologies 5973 inert)

24 Cell Culture Procedures Four human cancer cell lineswere used PC-3 RKO D-384 and MCF-7 The cells werecultured in RPMI-1640 medium supplemented with 10fetal bovine serum (FBS Invitrogen Karlsruhe Germany)1 antibiotic-antimitotic solution (100 unitsml penicillin

G 100 120583gml streptomycin and 025 120583gml amphotericinB Gibco Grand Island NY) and 1 L-glutamine (2mMGibco) The cells were incubated at 37∘C in a 5 CO

2

atmosphere The viable cells were counted using the trypanblue exclusion method in a hemocytometer [8]

25 Cell Viability Analysis via MTS Assay The MTS (5-[3-(carboxymethoxy)phenyl]-3-(45-dimethyl-2-thiazolyl)-2-(4-sulfo-phenyl)-2H-tetrazolium inner salt) cell viabilityassay was used to assess the inhibitory effects of the extractson the survival of human cancer cell lines A total of 3ndash5times 103 cellswell were seeded into 96-well plates and wereallowed to adhere for 24 h The cells were then treatedwith 50 120583gml of whole extract to yield a final volume of2ml Each concentrationassay was performed three timesDimethyl sulfoxide (DMSO) was used as a negative controlat a final concentration of 01 vv and 1 120583M doxorubicinwas used as a positive control The cells were incubated withthe treatments for 48 h after which 20120583L MTS (5mgmlAqueous One Solution Reagent Gibco) was added andfurther incubated for 4 h at 37∘C The absorbance wasmeasured at 570 nm The data obtained with cells treatedwith DMSO were considered to represent 100 viability[8] In cell lines with an inhibition percentage over 30doses of 15 45 60 75 and 100 120583gml of methanolic extractwere applied and the MTS assay was used to measurethe inhibitory concentration 50 (IC

50) using nonlinear

regression

26 Statistical Analysis All of the data were reported asmeans plusmn standard error of the mean (SEM) of three indepen-dent experiments The nonlinear regression was determinedby GraphPad Prism 5 (GraphPad Software San Diego CA)

3 Results

31 Isolation of Secondary Metabolites from B Obtusi-folia From the methanolic extract without chlorophyllstwo representative fractions were obtained F 70-79 elutedin HexEtOAc 8020 from which we isolated 80mg ofa white crystalline solid identified as sakuranetin (541015840-dihydroxy-7-methoxyflavanone) (1) and the fraction F 43-45 eluted in HexEtOAc 9010 from which we isolated112mg of a yellow crystalline solid identified as 5-hydroxy-741015840-dimethoxyflavone (2) (Figure 1) All of the compoundswere identified based on physical and spectroscopic data viacomparisons with the literature [9ndash11]










RKO(colon cancer)

D-384(astrocytoma)



6) 120575 ppm 28 (1H


6) 120575 ppm 79 (C-


3-



3) 120575 ppm 389 390 (3H each s)


3) 120575 ppm 1640 (C-2) 1043 (C-3) 1824 (C-


37rsquo) 557 (OCH

3-4rsquo)




50of 6811 120583M (1948120583gml) and 3430


4 Discussion










3)



Data Availability


Disclosure




Acknowledgments


References



































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls













RKO(colon cancer)

D-384(astrocytoma)



6) 120575 ppm 28 (1H


6) 120575 ppm 79 (C-


3-



3) 120575 ppm 389 390 (3H each s)


3) 120575 ppm 1640 (C-2) 1043 (C-3) 1824 (C-


37rsquo) 557 (OCH

3-4rsquo)




50of 6811 120583M (1948120583gml) and 3430


4 Discussion










3)



Data Availability


Disclosure




Acknowledgments


References



































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






3)



Data Availability


Disclosure




Acknowledgments


References



































Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






Na

nom

ate

ria

ls






















Journal of

Chemistry





Journal of

Volume 2018






Volume 2018




Journal of






Enzyme Research


Journal of



MaterialsJournal of






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ate

ria

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phytochemical study and evaluation of the cytotoxic...

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