in vitro antioxidant, antiproliferative, and …...4 biomedresearchinternational 1.2 0.8 0.4 0 1...

Research ArticleIn Vitro Antioxidant Antiproliferative and PhytochemicalStudy in Different Extracts of Nyctanthes arbortristis Flowers

Manjulatha Khanapur Ravi K Avadhanula and Oruganti H Setty

Department of Biochemistry School of Life Sciences University of Hyderabad Professor C R Rao Road GachibowliHyderabad Andhra Pradesh 500 046 India

Correspondence should be addressed to Oruganti H Setty ohssluohydernetin

Received 28 February 2014 Revised 16 April 2014 Accepted 16 April 2014 Published 20 May 2014

Academic Editor Aramati B Reddy

Copyright copy 2014 Manjulatha Khanapur 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

Nyctanthes arbortristis L (Oleaceae) is widely used in the Indian system of traditional medicine and is reported to have variousbiological activities The present study was intended to evaluate the antioxidant and antiproliferative activities of flower extracts ofNyctanthes arbortristis The shade dried flowers were extracted with 95 ethanol under sonication and the antioxidant activitieswere investigated using in vitro assays along with the determination of phytochemical constituents (total polyphenol and totalflavonoid) Arborside C and 120573-monogentiobioside ester of 120572-Crocetin were identified in crude active extracts through LCMSMSanalysis The antiproliferative activity was carried out by MTT assay by employing different human cancer cell lines The lowestIC50

value of 2456plusmn 663 120583gmL was observed against Colo 205 cell line The extract exhibited significant antioxidant andantiproliferative properties and the observed biological activities in this study provide scientific validation of ethnomedicinal useof this plant

1 Introduction

Cancer is responsible for 12 of the worldrsquos mortality andthe second-leading cause of death in the world [1] In spiteof much progress in the recent past in the cancer treatmentsa key problem in tumor therapy with established cytostaticcompounds is the development of drug resistance and acuteside effects Most available drugs suffer from insufficientspecificity toward tumor cells [2] Hence the identificationof better antitumor drugs is the need of the hour Overthe last two decades number of studies has investigated thediverse health benefits and protective effects of natural sub-stances present in the plants particularly having antioxidantand antiproliferative properties The scientific evaluation ofmedicinal plants used in the preparation of folk remedieshas providedmodernmedicinewith effective pharmaceuticaldrugs for the treatment of many infectious and chronicdiseases including cancer [3] Between 1983 and 1994 morethan 60 of the approved anticancer drugs in the UnitedStates of America were from natural origin Most of theanticancer agents have been shown to possess antioxidant

potential that can play an important role in the protection ofsome forms of cancer [4 5]

Plants as master chemists accumulate a wealth of intri-cate secondary metabolites which further deliver effectivetreatments for a plethora of human diseases Alternative orcomplementary traditional therapies are used to cure manydiseases potentially that are plant derived or other naturalsources In traditional system of medication the importanceto be considered is its broad range of outcomes whichoften treat the whole person rather than a specific symptomor disease These therapies also make accessibility to thepopulation living in diverse environmental conditions suchas geographical and climatic Thus make the accessibilityto the person(s) in need with less efforts and dependenceon other resources which in turn could be economicallybeneficial as well [6] The phytochemicals found in plant-based foods also possess biological properties along withtheir antioxidant property Consuming of antioxidant-richfoods has several health benefits that help to prevent manydiseases [7 8] The secondary metabolites such as alkaloidsflavonoids coumarins and steroids have been shown to

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 291271 10 pageshttpdxdoiorg1011552014291271

2 BioMed Research International

possess antioxidant and anticancer activities in both in vivoand in vitro models [9ndash11] Phytomedicine could be in theform of crude preparations (extracts tinctures and essentialoils) containing a wide variety of compounds or could bepuremolecules with a strong and specific activityThe naturalproducts symbolize safety in contrast to the synthetic drugsbut still there is need to check their efficacy with systematicstudies Therefore the need for new therapeutic optionshas prompted many researchers to evaluate the efficacy ofcompounds found in natural products [12]

Nyctanthes arbortristis (Oleaceae) is a mythological plantand possesses high medicinal values in Ayurveda The pop-ular medicinal uses of N arbortristis include antihelminthicand antipyretic besides it is used in disorders like rheuma-tism and skin ailments and as a sedative Phytochemicalinvestigations of N arbortristis indicated the presence of alarge number of phenolic compounds iridoids and carote-noids such as arbortristoside (A B C) with many biologicalactivities like anticancer antileishmania anti-inflammatoryantiallergic immunomodulatory and antiviral [13]The flow-ers of this sacred plant have not been explored for antipro-liferative activity till date Therefore the present study wasinitiated with the aim of investigating the antioxidant andantiproliferative activities of the flower extracts

2 Materials and Methods

21 Reagents Doxorubicin DPPH quercetin and gallicacid were purchased from Sigma Aldrich USA Organicsolvents and HCl hexamethylenetetramine sodium nitritealuminum chloride NaOH NaCO

3 and Folin-Ciocalteu

reagent were purchased fromMerck India

22 Plant Material The flowers fromwell grown and healthyplants of N arbortristis were collected in and around theUniversity of Hyderabad Hyderabad A voucher specimen(UoHMDPNA-00005) has been preserved in our labora-tory for future reference

23 Preparation of Extract and Phytochemical ScreeningThe flowers were shade dried and coarsely powdered usingelectric blender The powdered material was then extractedwith 95 ethanol under sonication The ethanol solvent wasremoved under reduced pressure using a rotary vacuumevaporator (Buchii USA) and dark reddish gummy ethanolicextract of N arbortristis flowers (NafE) was obtained Thisethanolic extract was taken in double distilledwater (ddH

2O)

andpartitionedwith hexane ethyl acetate andn-butanol suc-cessively and subsequently the organic solvents were recov-ered under reduced pressure and concentrated Insoluble partobtained on partitioning between aqueous and organic layerswas also collected and concentrated under reduced pressureFinally the remaining aqueous part was also concentrated topolar extract by lyophilisationThusN arbortristis ethanolicextract (NafE) hexane extract (NafEHx) ethyl acetate extract(NafEEa) n-butanol extract (NafEBu) insoluble part extract(NafEIn) and aqueous extract (NafEW) total six extractswere obtained from extraction process and preserved atminus20∘C for further analysis Phytochemical screening of

secondary metabolites in all six extracts was carried out asdescribed by Harbone [14]

24 Determination of Phytoconstituents

241 Determination of Total Phenolic Contents The amountof total soluble phenolic content in all six extracts (NafENafEHx NafEEa NafEBu NafEIn and NafEW) was deter-mined according to Folin-Ciocalteu method with minormodifications [15] Briefly 10 120583L of extracts from the stocksolution was mixed with 100 120583L of Folin-Ciocalteu reagentAfter 10min of incubation at room temperature 300 120583Lof 20 Na

2CO3solution was added and the volume was

adjusted to 1mL using dH2O The mixture was incu-

bated in the dark for 2 h and the absorbance was mea-sured at 765 nm using a UV-Vis spectrophotometer againstblank sample The total phenolic content was measuredas gallic acid equivalents (mg GAE)gm of dry weight(dw) and the values were presented as means of triplicateanalysis

242 Determination of Flavonoid Contents Total flavonoidcontent in extracts was estimated by a colorimetricmethod asdescribed by Veronica et al [16] with minor modifications bytaking 20 120583L of each extractmixedwith 500 120583L distilled waterand 30 120583L of 5 NaNO

2solution After 5min of incubation

at room temperature 60120583L of 10 AlCl3solution was added

Subsequently 350 120583L of 1M NaOH and 40 120583L of distilledwater were added to make the final volume of 1mL Sampleswere further incubated for 15min at room temperature andthe absorbance of the samples was measured at 510 nm Thetotal flavonoids were determined as quercetin equivalents(mg QE)g of dw and the values were expressed as means oftriplicate analysis

25 Antioxidant Assays

251 Total Antioxidant Capacity The total antioxidant activ-ity of test extracts was evaluated by green phosphomolybde-num complex according to themethod of Prieto et al [17] Analiquot of 10 120583L of extracts was mixed with 1mL of reagentsolution (06M sulphuric acid 28mM sodium phosphateand 4mMammoniummolybdate) in Eppendorf tubes Tubeswere incubated in a dry thermal bath at 95∘C for 90minAfter cooling the absorbance of themixture was measured at695 nm against a blank Ascorbic acid was used for referenceand the reducing capacities of the analyzed extracts wereexpressed as mg of ascorbic acid equivalents (mg AAE)g ofdw

252 DPPH∙ Radical Scavenging Activity The hydrogen-donating abilities of extracts were examined according to themethod of Cuendet et al [18] with some modifications using2 2-diphenyl-2-picrylhydrazyl hydrate (DPPH) as reagentthat offers a convenient and accurate method for titratingthe oxidizable groups of natural or synthetic antioxidantsBriefly 0004wv of DPPH radical solution was preparedin methanol and then 900 120583L of this solution was mixed with

BioMed Research International 3

100 120583L of extract solution containing 20ndash360 120583gmL of driedextractsThe absorbancewasmeasured at 517 nmafter 30minof incubation Methanol (95) DPPH solution and ascorbicacid were used as blank control and reference respectivelyThe IC

50value represents the concentration of extracts that

inhibits 50 of the radical Scavenging concentration for 50of DPPH free radical (IC

50) was calculated from logarithmic

regression equation obtained from the values of at least fivedilutions of the primary concentration

26 Evaluation of Antiproliferative Activity The six differentcell lines that were used in study are colorectal adenocar-cinoma (Colo 205) retinoblastoma (Y79) chronic myel-ogenous leukemia (K562) breast adenocarcinoma (MCF7)breast adenocarcinoma (MDAMB231) The cells lines wereobtained from the National Centre for Cell Sciences PuneIndia and were cultured at a seeding density of 02 times106 in DMEMRPMI medium supplemented with 10 FBS100UmL penicillin and 100 120583gmL streptomycin respec-tively and maintained in a humidified atmosphere with 5CO2at 37∘C The samples were dissolved in dimethylsul-

foxide (DMSO not exceeding the final concentration of001) and further diluted in cell culture medium Theantiproliferative response of extract was assessed by MTTassay [19] Cells (sim10000) were plated in 200120583L growthmedium in the presence or absence of the extract (25 50100 and 200120583gmL) in 96-well culture plates for 24 h Thenthe culture plates were centrifuged at 2000 rpm for 10minat room temperature 100 120583L of supernatant was discardedand 20120583L of MTT (5mgmL in PBS) was added to each welland incubated for 4 h at 37∘C The viability of the cells wasdetermined using a spectrophotometer at 570 nm The IC

50

that is the concentration of the extract required to inhibit cellgrowth by 50 was determined

27 Chromatography Profile High Performance Liquid Chro-matography and Liquid Mass Spectrometry (HPLCMSMS)Agilent 1200 series coupled with DAD-UV detector thatwas equipped with Agilent Technologies 6520 with AccurateMass Q-TOF mode was used to perform mass spectrometryand Zorbax SB-C18 column rapid resolution (35 120583m 46times 150mm) The flow rate was 045mLmin and the injec-tion volume was 3 120583L The analyses were performed usingbinary gradients of Milli-Q water (with 01 formic acid +10mM ammonium formate) (solvent A) and HPLC gradeacetonitrile (with 01 formic acid) (solvent B) with thefollowing elution profile from 0min 35 (B) in (A) 10min55 (B) in (A) 25min 95 (B) in (A) 35min 35 (B) in(A)

28 Statistical Analysis Data were presented as means stan-dard deviation (SD) Statistical analysis was performed usingStudentrsquos 119905-test analysis and one-way analysis of variance(ANOVA) The results were considered statistically signifi-cant when 119875 lt 005 The Dictionary of Natural Productson DVD software (CRC Press Taylor and Francis Grouphttpsnetbeansorg) was used to analyze the chromatogra-phy profiling data

Table 1 Phytochemical screening of flower extracts of N arbortris-tis

Type of extract Phytochemical constituentsS A F P Sp G T

NafE + minus + + + + +NafEHx + minus minus minus minus minus +NafEEa minus minus + + minus minus +NafEBu minus minus + + + + +NafEIn minus minus + + minus minus +NafEW minus minus + + + + +Note +ve represents presence and minusve represents absence of phytochemicalS steroids A alkaloids F flavonoids P phenolics Sp saponins Gglycosides T terpenoids

1200

1000

800

600

400

200

0

(mg

g dw

)

Phytoconstituents

Total polyphenolic contentTotal flavonoid content

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW

Different extracts of N arbortritis

Figure 1 Phytoconstituents content (total flavonoid and totalphenolic) in different extracts of N arbortristis flower

3 Results

31 Extraction Preliminary Phytochemical Screening Phyto-constituents Assay and LCMSMS Analysis In the presentstudy the extraction was carried out under ultrasonicationusing 95 ethanol as the solvent followed by fractionation ofsame extract with various solvents with increasing polarityand the final extracts were designated as NafE NafEHxNafEEa NafEBu NafEIn and NafEW These six differentextracts were subjected to phytochemical screening to checkthe presence of different phytoconstituents and results aretabulated in Table 1 The UV profile of NafE NafEa andNafBu chromatograms analysed at all wave lengths demon-strated two 120582max in the region of 240ndash280 nm and 300ndash380 nm thus suggesting the presence of flavonoids [20]Theresults of phytoconstituents in different flower extracts ofN arbortristis are presented in Table 1 and Figure 1 Theflavonoid content in different extracts (NafE NafEa andNafBu) was found to be in the order of 640 plusmn 209mgQE100 g 590plusmn109mgQE100 g and 235plusmn181mgQE100 g


12

08

04

0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times102

DAD1-GSig = 2808 NAFM POS MSMSd

Response units versus acquisition time (min)

(a)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times106

1

1

1

23

5

7

9

Counts versus acquisition time (min)

+ESI TIC scan frag = 1440V NAFM POS MSMSd

(b)

Figure 2 (a) LC-UV chromatogram at 280 nm of ethanolic extract of flowers of N arbortristis (b) LC-MS total ion chromatogram of theethanolic extract of flowers of N arbortristis Compounds 1 (RT = 51) Arborside C and 2 (RT = 19603) Crocin-3

1

09

08

07

06

05

04

03

02

01

0

08

06

04

02

0

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

485 490 495 500 505 510 515 520 525 530 535

times102

times102

HO

HO

O

OOO

OO

O

OHOH

OH

5111807(M + H)+

Counts () versus mass-to-charge (mz)


+ESI product ion (5101min) frag = 1750V CID400 (5111808[z = 1]rarrlowastlowast) NAFM POS MSMSd

Figure 3 Mass spectrum (TOF MS ES+) of peak number 1 in N arbortristis flowers ethanol extract (identified as Arborside C)

respectively and by Folin-Ciocalteu method for total pheno-lic content of NafE NafEa and NafBu extracts was shownas 991 plusmn 05mg GAE100 g 781 plusmn 102mg GAE100 g and591 plusmn 007mg GAE100 g respectively

The NafE was subjected to LC-DAD-ESI-MS to iden-tify the phytochemical(s) by absorption peaks in UV(Figure 2(a)) and with molecular ion in Q-TOF and bytheir fragmentation (MSMS) using the positive ionisationmode and observed many peaks (Figures 2(b) 3 and 4)however only two peaks were identified Peak 1 (Figure 2(b))(RT = 51min 120582 = 280 nm and MW = 510494)(Figure 3) had [M + H]+ at mz 511 and was identifiedas Arborside C [21] namely 6 120573-hydroxyguanine with O-benzoyl substitution with loss of benzoyl that ion appearedatmz 105 benzoatemz 121 and glucosidemz 165 and 6 120573-hydroxyguanine atmz 244 228 and 212 Peak 2 (Figure 2(b))(RT = 20509min 120582 = 440 nm and MW = 65227)(Figure 4) had [M+H]+ atmz 6531994 andwas identified ascarotenoid glycosides namely 120573-monogentiobioside ester of120572-Crocetin (or Crocin-3) with loss of 15-anhydro-D-glucitolthat ion appeared at mz 1650651 and mz 491 carotenoidester with other fragments at mz 459 315 and 147 [22]The reddish-orange coloured tubular calyx of flower of Narbortristis is due to carotenoid pigments (Crocetin and itsderivatives) which are reported from the flowers of this plant[22 23]

32 Antioxidant and Free Radical Scavenging Ability AssaysPhosphomolybdenum assay is a quantitative method to eval-uate the antioxidant capacity indicated by electron donatingcapacity [17]The results showed that all the extracts exhibiteddifferent degrees of activity as presented in Figure 5(a) Thehighest antioxidant capacity was observed in NafEEa with3011 plusmn 177 of AAE100 g dw of plant material followedby NafE and NafEBu with 2876 plusmn 151 AAE100 g dw and2466 plusmn 209 AAE100 g dw respectively NafEHx NafEInand NafEW were found to be lt500 plusmn 212 AAE100 g dwwhich were considered to be ineffective The phosphomolyb-denum method is quantitative since the total antioxidantactivity is expressed as the number of equivalents of ascorbicacid N arbortristis flower ethyl acetate butanol and ethanolextracts range between 3011 and 2466mg AAEg dw andother extracts were very low in concentration

DPPH assay is a very sensitive qualitative assay forradical scavenging property and the experiment was carriedout on the present study and its results can indicate thepresence of antioxidant compounds in plant extracts [24]Figure 5(b) illustrates a significant (119875 lt 005) decrease inthe concentration of DPPH due to the scavenging activitiesof the extract samples The samples showed concentrationdependent DPPH radical scavenging activities The IC

50val-

ues of NafE NafEHx NafEEa NafEBu NafEIn and NafEWwere found to be at 3271 plusmn 132 120583gmL 32837 plusmn 225 120583gmL


1

2

1

0

09

08

07

06

05

04

03

02

01

0

times102

times103

HO

HO

HO

HO

HO

HO

OO

OO

OO

OHOH


100 120 140 160 180 200

200

220 240 260 280 300 320 340 360 380 400

400

420 440 460 480 500 520 540 560 580 600

600

620 640 660



C26 H23 N2 O84911435

6531880

800 1000 1200 1400 1600

Figure 4 Mass spectrum (TOF MS ES+) of peak number 2 in N arbortristis flowers ethanol extract (identified as Crocin-3)

40

30

20

10

0

(mg

AA

Eg

dw)

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW

Total antioxidant capacity


(a)

120

100

80

60

40

20

00 40 80 120 160 200 240 280 320 360

Inhi

bitio

n (

)DPPH scavenging activity

Concentration (120583gmL)

NafENafEHxNafEEa

NafEBuNafEInNafEW

(b)

Figure 5 (a) Total antioxidant content in different extracts All values are expressed as the means plusmn SEM (b) DPPH radical scavengingactivity of N arbortristis flower in different extracts All values are expressed as the means plusmn SEM

2398plusmn105 120583gmL 3029plusmn178 120583gmL 10411plusmn151 120583gmLand 40115 plusmn 129 120583gmL respectively Percentage inhibitionat 40 120583gmL of NafE NafEEa and NafEBu was found to be5253 plusmn 286 6925 plusmn 396 and 6798 plusmn 354 respectivelyAssessment of free radicals scavenging by DPPH method forantioxidant potential is known to be accurate convenientand rapid N arbortristis flowers ethyl acetate butanol andethanol extracts could scavenge DPPH radical effectively 50ndash70 respectively at the highest concentration of 360120583gmLThere are reports on antioxidant property of this plant withrespect to its leaves flowers and stem Extensive work onleaves has been carried out but has not been much studiedon flowers Earlier report suggests that antioxidant activitiesfrom leaves stem and flower extracts were significantlyhigher in the extracts from lower polarity over the extractsfrom higher polarity solvent [13] This supports our present

data where the lower polarity extracts were more active thanthe higher polarity solvents

33 Evaluation of Antiproliferative Activity In order to eval-uate N arbortristis as a potential candidate for cancertherapy the above extracts were assayed against a panel offive different human tumor cells colorectal adenocarcinoma(Colo 205) retinoblastoma (Y79) chronic myelogenousleukemia (K562) breast adenocarcinoma (MCF-7) breastadenocarcinoma (MDAMB-231) and the chemotherapeuticdrug Doxorubicin as a positive control Out of six extractstested only two ethanolic extract (NafE) and ethyl acetateextract (NafEEa) were found to be active whereas NafEHxNafEBu NafEIn and NafEW extracts did not inhibit theproliferation of tumor cells thus indicating their noncyto-toxicities against the above cancer cell lines The MTT assay


100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)

Figure 6 Antiproliferative activity of N arbortristis flower extracts against (a) colorectal adenocarcinomamdashColo 205 cell line (b)retinoblastomamdashY79 cell line (c) chronic myelogenous leukemiamdashK562 cell line (d) breast adenocarcinomamdashMCF7 cell line (e) breastadenocarcinomamdashMDAMB231 cell line and (f) human embryonic kidney cellsmdashHEK cell line Significant values ( lowastlowastlowast119875 lt 0001 lowastlowast119875 lt 001and lowast119875 lt 005) were obtained by Studentrsquos 119905-test analysis Composite treatments were compared using one-way analysis of variances(ANOVA) and probability values were found to be equal to or less than 005 for all the six cell lines


Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)

7201plusmn9402456plusmn663lowastlowastlowast8182plusmn2115587plusmn719lowastlowast6182plusmn9605363plusmn684lowastlowast5424plusmn53918436plusmn539lowast6780plusmn0965097plusmn310lowastlowast

NafEE

a(200120583gmL)

7067plusmn5302579plusmn269lowastlowastlowast6806plusmn2025950plusmn341lowastlowast5848plusmn5209802plusmn747lowast4657plusmn06421473plusmn064lowast5265plusmn20210081plusmn050lowast

Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003

8942plusmn202032plusmn0108942plusmn202036plusmn0029233plusmn045036plusmn0099065plusmn241045plusmn011

DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour

replicatesplusmnstandard

deviation(SD)Sign

ificant119875values

(lowastlowastlowast119875lt0001lowastlowast119875lt001andlowast119875lt005)w

ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com

positetreatmentswerec

ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines


that measures the formazan product at 570 nm clearly provesthe cytotoxicity of the tested extracts Figures 6(a)ndash6(e) showthe cytotoxicity values of two active extracts in tested celllines in comparison with normal human embryonic kidneycells (Figure 6(f)) the IC

50values are presented in Table 2

The NafE and NafEEa were found to be cytotoxic to testedcell lines These extracts significantly inhibited the growth ofcancer cells in a concentration dependent manner as theycaused significant cell death in both sensitive and resistanthuman cancer cell lines NafE extract has shown the mostpotent cytotoxicity on all five cancer cell linesThe percentageinhibition shown by NafE was found to be in the range of5424 plusmn 339 to 8181 plusmn 211 (119875 lt 005) against all fivecell lines at the highest concentration of 200120583gmL Thelowest IC

50value was observed against Colo 205 cell line

(2456 plusmn 663 120583gmL) On the other hand NafEEa extract atthe same concentration exhibited slightly lesser percentageinhibition across the cell lines tested (4657 plusmn 064 to 7066 plusmn530 119875 lt 005) with lowest IC

50values found against

Colo 205 cell line (2579 plusmn 269 120583gmL) The difference inthe antiproliferative effects between different extracts mayhave resulted from the different phytoconstituents and theirconcentrations contained in the extracts due to the sensitivityto the solvent used and mode of extraction The cytotoxiceffect of NafE and NafEEa was also studied in normalembryonic kidney cell line using the MTT method Theresults clearly indicated that these two extracts were nontoxicand had no inhibitory effect on cell proliferation in HEK-293 and there was minimal reduction in cell survivability(Figure 6(f)) The percentage viability was above 95 at thehighest concentration of 200120583gmLThis advocates thatNafEand NafEEa extracts did not show any kind of toxic effect onthe normal cells Hence the cytotoxicity of the active extractswas found to be highly selective against the cancer cell linesused

Crocetin carotenoid is an active component of mostancient expensive spice saffron (Crocus sativus [25]) thatis also reported to possess anticancer properties [26] Indimethylbenzanthracene (DMBA) induced skin tumorige-nesis the hydroalcoholic extract of leaves of this plant at250mgkg was found to be as chemopreventive [27] and4-hydroxyhexahydrobenzofuran-7-one isolated from leavesat 20mgkg which inhibited the cell growth of Ehrlichascites carcinoma cells by 4327 and did not have anycytotoxic effect [28] Arbortristoside A and B and iridoidglycosides are reported from seeds at 25mgkg in micewhich possess anticancer activity againstmethylcholanthreneinduced fibrosarcoma [29] Iridoids and carotenoids aremostfrequent compounds identified in N arbortristis and theyhave been reported for various biological activities [30 31]

It was an understandable interest to know how highlevels of phenolics exhibited high antioxidant activity andalso influence the anticancer activity in different extractsWe expected that the extracts with the high content of thetotal phenolics possessing antioxidant potential possibilityhave high anticancer activity Cytotoxicity screening mod-els provide important preliminary data to help selectingplant extracts with potential antineoplastic properties forfuture work [32 33] As discussed earlier several plant

species that are rich in flavonoids are reported to preventand possess therapeutic properties [32ndash35] The flowers ofthis plant were reported with rich phytochemicals diter-penoids nycanthin flavonoids anthocyanins essential oil6 120573-hydroxyguanine carotenoids 120573-monogentiobioside 120573-digentiobioside and various biological activities [13] Withthe aid of hyphenated techniques LCMSMS Arborside Cand Crocin and 120573-monogentiobioside ester of 120572-Crocetin(881015840-Diapocarotenedioic acid) were identified Possibly theantioxidant and anticancer activities of ethanolic and ethylacetate extracts of N arbortristis are influenced by thepresence of phytoconstituents which is in accordance withthe findings of phytochemical evaluation which indicated thepresence of flavonoids phenolics Crocin-3 and Arborside Cin extracts with promising activity

4 Conclusion

N arbortristis is known for its varied medicinal propertiesin traditional ayurvedic medicine and reported for vari-ous bioactive phytoconstituents In this study an attemptwas made to investigate antiproliferative effects of differentextracts of N arbortristis in different human cancer celllines apart from its antioxidant potential The present studyindicated that the ethyl acetate and ethanol extracts of Narbortristis possessed the significant phenolic content andexhibited potent antioxidant and antiproliferative activitieswhich were comparable to the commercial antioxidant gallicacid and the anticancer drug Doxorubicin This seems thatthe N arbortristis flower extracts can be considered as goodsources for drug discovery Further investigation is beingcarried out to identify and characterize the inherent bioactivecompounds responsible for the antioxidant and anticanceractivities from the ethyl acetate and ethanol extracts of Narbortristis

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

This research was gratefully supported by UGC-UPE IIManjulatha Khanapur acknowledges UoH DBT-CREEB forfinancial assistance as postdoctoral fellow Ravi K Avadhan-ula is thankful to UGC-UPE II for financial assistance inthe form of fellowship The authors are thankful to Mr MirZahoor Gul Research Scholar from the Department of PlantSciences School of Life Sciences UoH and Mr PrashanthfromMetabolomics facility UoH for their technical support

References

[1] T Efferth S Kahl K Paulus et al ldquoPhytochemistry and phar-macogenomics of natural products derived from traditionalChinese medicine and Chinese materia medica with activityagainst tumor cellsrdquo Molecular Cancer Therapeutics vol 7 no1 pp 152ndash161 2008


[2] S Umthong P Phuwapraisirisan S Puthong andC ChanchaoldquoIn vitro antiproliferative activity of partially purified Trigonalaeviceps propolis from Thailand on human cancer cell linesrdquoBMC Complementary and Alternative Medicine vol 11 article37 2011

[3] C Stevigny C Bailly and J Quetin-Leclercq ldquoCytotoxic andantitumor potentialities of aporphinoid alkaloidsrdquo CurrentMedicinal ChemistrymdashAnti-Cancer Agents vol 5 no 2 pp 173ndash182 2005

[4] G Block ldquoMicronutrients and cancer time for actionrdquo Journalof theNational Cancer Institute vol 85 no 11 pp 846ndash848 1993

[5] Y Rajeshwar M Gupta and U K Mazumder ldquoAntitumoractivity and in vivo antioxidant status of Mucuna pruriens(Fabaceae) seeds against Ehrlich ascites carcinoma in Swissalbinomicerdquo Iranian Journal of Pharmacology andTherapeuticsvol 4 no 1 pp 46ndash53 2005

[6] M J Balunas and A D Kinghorn ldquoDrug discovery frommedi-cinal plantsrdquo Life Sciences vol 78 no 5 pp 431ndash441 2005

[7] S M Nabavi M A Ebrahimzadeh S F Nabavi A Hamidiniaand A R Bekhradnia ldquoDetermination of antioxidant activityphenol and flavonoid content of Parrotia persicaMeyrdquo Pharma-cologyonline vol 2 pp 560ndash567 2008

[8] K P Mishra L Ganju M Sairam P K Banerjee and R CSawhney ldquoA review of high throughput technology for thescreening of natural productsrdquo Biomedicine and Pharmacother-apy vol 62 no 2 pp 94ndash98 2008

[9] SMishima KMatsumoto Y Futamura et al ldquoAntitumor effectof stilbenoids fromVateria indica against allografted sarcoma S-180 in animal modelrdquo Journal of Experimental Therapeutics andOncology vol 3 no 5 pp 283ndash288 2003

[10] M Gupta U K Mazumber R S Kumar and T S KumarldquoAntitumor activity and antioxident role of Bauhinia racemosaagainst Ehrlich ascites carcinoma in Swiss albino micerdquo ActaPharmacologica Sinica vol 25 no 8 pp 1070ndash1076 2004

[11] M Z Gul F Ahmad A K Kondapi I A Qureshi and I AGhazi ldquoAntioxidant and antiproliferative activities ofAbrus pre-catorius leaf extractsmdashan in vitro studyrdquo BMC Complementaryand Alternative Medicine vol 13 article 53 2013

[12] M Z Gul V Attuluri I A Qureshi and I A Ghazi ldquoAntioxi-dant and 120572-glucosidase inhibitory activities ofMurraya koenigiileaf extractsrdquo Pharmacognosy Journal vol 4 no 32 pp 65ndash722012

[13] J Agrawal and A Pal ldquoNyctanthes arbortristis Linnmdasha criticalethnopharmacological reviewrdquo Journal of Ethnopharmacologyvol 146 no 3 pp 645ndash658 2013

[14] J B Harborne Phytochemical Methods A Guide to ModernTechniques of Plant Analysis Chapman amp Hall New York NYUSA 1998

[15] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998

[16] D Veronica W Xianzhong K K Adom and R H LiuldquoThermal processing enhances the nutritional value of tomatoesby increasing total antioxidant activityrdquo Journal of Agriculturaland Food Chemistry vol 50 no 10 pp 3010ndash3014 2002

[17] P Prieto M Pineda and M Aguilar ldquoSpectrophotometricquantitation of antioxidant capacity through the formation ofa phosphomolybdenum complex specific application to thedetermination of vitamin Erdquo Analytical Biochemistry vol 269no 2 pp 337ndash341 1999

[18] M Cuendet K Hostettmann and O Potterat ldquoIridoid glu-cosides with free radical scavenging properties from Fragreablumeirdquo Helvetica Chimica Acta vol 80 no 4 pp 1144ndash11511997

[19] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[20] R Singh B Wu L Tang Z Liu and M Hu ldquoIdentification ofthe position of mono-O-glucuronide of flavones and flavonolsby analyzing shift in onlineUV spectrum (120582

119898119886119909) generated from

an online diode array detectorrdquo Journal of Agricultural and FoodChemistry vol 58 no 17 pp 9384ndash9395 2010

[21] V Srivastava A Rathore S M Ali and J S Tandon ldquoNew ben-zoic esters of loganin and 6120573-hydroxyloganin from Nyctanthesarbortristisrdquo Journal of Natural Products vol 53 no 2 pp 303ndash308 1990

[22] V K Dhingra T R Seshadri and S K Mukerjee ldquoCarotenoidglycosides of Nyctanthes arbortristis Linnrdquo Indian Journal ofChemistry B Organic Chemistry IncludingMedicinal Chemistryvol 14 pp 231ndash232 1976

[23] C Gadgoli and S Shelke ldquoCrocetin from the tubular calyx ofNyctanthes arbortristisrdquo Natural Product Research vol 24 no17 pp 1610ndash1615 2010

[24] F Sharififar G Dehghn-Nudeh and M Mirtajaldini ldquoMajorflavonoids with antioxidant activity from Teucrium polium LrdquoFood Chemistry vol 112 no 4 pp 885ndash888 2009

[25] M Giaccio ldquoCrocetin from saffron an active component of anancient spicerdquo Critical Reviews in Food Science and Nutritionvol 44 no 3 pp 155ndash172 2004

[26] A Dhar S Mehta G Dhar et al ldquoCrocetin inhibits pancreaticcancer cell proliferation and tumor progression in a xenograftmouse modelrdquoMolecular Cancer Therapeutics vol 8 no 2 pp315ndash323 2009

[27] M Dinamani S Savaiah and A R Rao ldquoChemoprevention ofDMBA induced skin tumorigenesis in swiss albinomice a studywithNyctanthes arbortristis leaf extractrdquoThe Bioscan vol 4 no2 pp 295ndash299 2009

[28] N A Khatune E Islam A A Rahman A Mosaddik and EHaque ldquoIn-vivo cytotoxic evaluation of a new benzofuran deri-vative isolated from Nyctanthes arbortristis L on Ehrlich ascitecarcinoma cell (EAC) in micerdquo Journal of Medicinal Sciencesvol 3 no 2 pp 169ndash173 2003

[29] T Susan A Muzaffer and K K Purushothaman ldquoInhibitoryactivity of arbortristoside A on fibrosarcoma in albino ratsrdquoArogya vol 12 pp 122ndash130 1986

[30] E L Ghisalberti ldquoBiological and pharmacological activity ofnaturally occurring iridoids and secoiridoidsrdquo Phytomedicinevol 5 no 2 pp 147ndash163 1998

[31] C M Lee A C Boileau T W M Boileau et al ldquoReview ofanimal models in carotenoid researchrdquo Journal of Nutrition vol129 no 12 pp 2271ndash2277 1999

[32] P Srinivasan M V Vadhanam J M Arif and R C Gupta ldquoArapid screening assay for antioxidant potential of natural andsynthetic agents in vitrordquo International Journal of Oncology vol20 no 5 pp 983ndash986 2002

[33] J H Cardellina II R W Fuller W R Gamble et al ldquoEvolvingstrategies for the selection dereplication and prioritization ofantitumor and HIV-inhibitory natural products extractsrdquo inBioassaay Methods in Natural Product Research and Develop-ment L Bohlin and J G Bruhn Eds pp 25ndash36 Kluwer Acade-mic Publishers Dordrecht The Netherlands 1999


[34] TMHardy and TO Tollefsbol ldquoEpigenetic diet impact on theepigenome and cancerrdquo Epigenomics vol 3 no 4 pp 503ndash5182011

[35] P J Ferguson E Kurowska D J Freeman A F Chambersand D J Koropatnick ldquoA flavonoid fraction from cranberryextract inhibits proliferation of human tumor cell linesrdquo Journalof Nutrition vol 134 no 6 pp 1529ndash1535 2004


possess antioxidant and anticancer activities in both in vivoand in vitro models [9ndash11] Phytomedicine could be in theform of crude preparations (extracts tinctures and essentialoils) containing a wide variety of compounds or could bepuremolecules with a strong and specific activityThe naturalproducts symbolize safety in contrast to the synthetic drugsbut still there is need to check their efficacy with systematicstudies Therefore the need for new therapeutic optionshas prompted many researchers to evaluate the efficacy ofcompounds found in natural products [12]

Nyctanthes arbortristis (Oleaceae) is a mythological plantand possesses high medicinal values in Ayurveda The pop-ular medicinal uses of N arbortristis include antihelminthicand antipyretic besides it is used in disorders like rheuma-tism and skin ailments and as a sedative Phytochemicalinvestigations of N arbortristis indicated the presence of alarge number of phenolic compounds iridoids and carote-noids such as arbortristoside (A B C) with many biologicalactivities like anticancer antileishmania anti-inflammatoryantiallergic immunomodulatory and antiviral [13]The flow-ers of this sacred plant have not been explored for antipro-liferative activity till date Therefore the present study wasinitiated with the aim of investigating the antioxidant andantiproliferative activities of the flower extracts

2 Materials and Methods

21 Reagents Doxorubicin DPPH quercetin and gallicacid were purchased from Sigma Aldrich USA Organicsolvents and HCl hexamethylenetetramine sodium nitritealuminum chloride NaOH NaCO

3 and Folin-Ciocalteu

reagent were purchased fromMerck India

22 Plant Material The flowers fromwell grown and healthyplants of N arbortristis were collected in and around theUniversity of Hyderabad Hyderabad A voucher specimen(UoHMDPNA-00005) has been preserved in our labora-tory for future reference

23 Preparation of Extract and Phytochemical ScreeningThe flowers were shade dried and coarsely powdered usingelectric blender The powdered material was then extractedwith 95 ethanol under sonication The ethanol solvent wasremoved under reduced pressure using a rotary vacuumevaporator (Buchii USA) and dark reddish gummy ethanolicextract of N arbortristis flowers (NafE) was obtained Thisethanolic extract was taken in double distilledwater (ddH

2O)

andpartitionedwith hexane ethyl acetate andn-butanol suc-cessively and subsequently the organic solvents were recov-ered under reduced pressure and concentrated Insoluble partobtained on partitioning between aqueous and organic layerswas also collected and concentrated under reduced pressureFinally the remaining aqueous part was also concentrated topolar extract by lyophilisationThusN arbortristis ethanolicextract (NafE) hexane extract (NafEHx) ethyl acetate extract(NafEEa) n-butanol extract (NafEBu) insoluble part extract(NafEIn) and aqueous extract (NafEW) total six extractswere obtained from extraction process and preserved atminus20∘C for further analysis Phytochemical screening of

secondary metabolites in all six extracts was carried out asdescribed by Harbone [14]

24 Determination of Phytoconstituents

241 Determination of Total Phenolic Contents The amountof total soluble phenolic content in all six extracts (NafENafEHx NafEEa NafEBu NafEIn and NafEW) was deter-mined according to Folin-Ciocalteu method with minormodifications [15] Briefly 10 120583L of extracts from the stocksolution was mixed with 100 120583L of Folin-Ciocalteu reagentAfter 10min of incubation at room temperature 300 120583Lof 20 Na

2CO3solution was added and the volume was

adjusted to 1mL using dH2O The mixture was incu-

bated in the dark for 2 h and the absorbance was mea-sured at 765 nm using a UV-Vis spectrophotometer againstblank sample The total phenolic content was measuredas gallic acid equivalents (mg GAE)gm of dry weight(dw) and the values were presented as means of triplicateanalysis

242 Determination of Flavonoid Contents Total flavonoidcontent in extracts was estimated by a colorimetricmethod asdescribed by Veronica et al [16] with minor modifications bytaking 20 120583L of each extractmixedwith 500 120583L distilled waterand 30 120583L of 5 NaNO

2solution After 5min of incubation

at room temperature 60120583L of 10 AlCl3solution was added

Subsequently 350 120583L of 1M NaOH and 40 120583L of distilledwater were added to make the final volume of 1mL Sampleswere further incubated for 15min at room temperature andthe absorbance of the samples was measured at 510 nm Thetotal flavonoids were determined as quercetin equivalents(mg QE)g of dw and the values were expressed as means oftriplicate analysis

25 Antioxidant Assays

251 Total Antioxidant Capacity The total antioxidant activ-ity of test extracts was evaluated by green phosphomolybde-num complex according to themethod of Prieto et al [17] Analiquot of 10 120583L of extracts was mixed with 1mL of reagentsolution (06M sulphuric acid 28mM sodium phosphateand 4mMammoniummolybdate) in Eppendorf tubes Tubeswere incubated in a dry thermal bath at 95∘C for 90minAfter cooling the absorbance of themixture was measured at695 nm against a blank Ascorbic acid was used for referenceand the reducing capacities of the analyzed extracts wereexpressed as mg of ascorbic acid equivalents (mg AAE)g ofdw

252 DPPH∙ Radical Scavenging Activity The hydrogen-donating abilities of extracts were examined according to themethod of Cuendet et al [18] with some modifications using2 2-diphenyl-2-picrylhydrazyl hydrate (DPPH) as reagentthat offers a convenient and accurate method for titratingthe oxidizable groups of natural or synthetic antioxidantsBriefly 0004wv of DPPH radical solution was preparedin methanol and then 900 120583L of this solution was mixed with









50







1200

1000

800

600

400

200

0

(mg

g dw

)

Phytoconstituents


Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



3 Results



12

08

04

0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times102



(a)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times106

1

1

1

23

5

7

9



(b)


1

09

08

07

06

05

04

03

02

01

0

08

06

04

02

0

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

485 490 495 500 505 510 515 520 525 530 535

times102

times102

HO

HO

O

OOO

OO

O

OHOH

OH

5111807(M + H)+









50val-



1

2

1

0

09

08

07

06

05

04

03

02

01

0

times102

times103

HO

HO

HO

HO

HO

HO

OO

OO

OO

OHOH


100 120 140 160 180 200

200

220 240 260 280 300 320 340 360 380 400

400

420 440 460 480 500 520 540 560 580 600

600

620 640 660



C26 H23 N2 O84911435

6531880

800 1000 1200 1400 1600


40

30

20

10

0

(mg

AA

Eg

dw)

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



(a)

120

100

80

60

40

20

00 40 80 120 160 200 240 280 320 360

Inhi

bitio

n (



NafENafEHxNafEEa

NafEBuNafEInNafEW

(b)






100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)



Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from


























50







1200

1000

800

600

400

200

0

(mg

g dw

)

Phytoconstituents


Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



3 Results



12

08

04

0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times102



(a)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times106

1

1

1

23

5

7

9



(b)


1

09

08

07

06

05

04

03

02

01

0

08

06

04

02

0

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

485 490 495 500 505 510 515 520 525 530 535

times102

times102

HO

HO

O

OOO

OO

O

OHOH

OH

5111807(M + H)+









50val-



1

2

1

0

09

08

07

06

05

04

03

02

01

0

times102

times103

HO

HO

HO

HO

HO

HO

OO

OO

OO

OHOH


100 120 140 160 180 200

200

220 240 260 280 300 320 340 360 380 400

400

420 440 460 480 500 520 540 560 580 600

600

620 640 660



C26 H23 N2 O84911435

6531880

800 1000 1200 1400 1600


40

30

20

10

0

(mg

AA

Eg

dw)

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



(a)

120

100

80

60

40

20

00 40 80 120 160 200 240 280 320 360

Inhi

bitio

n (



NafENafEHxNafEEa

NafEBuNafEInNafEW

(b)






100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)



Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from



















12

08

04

0

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times102



(a)

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39

times106

1

1

1

23

5

7

9



(b)


1

09

08

07

06

05

04

03

02

01

0

08

06

04

02

0

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

485 490 495 500 505 510 515 520 525 530 535

times102

times102

HO

HO

O

OOO

OO

O

OHOH

OH

5111807(M + H)+









50val-



1

2

1

0

09

08

07

06

05

04

03

02

01

0

times102

times103

HO

HO

HO

HO

HO

HO

OO

OO

OO

OHOH


100 120 140 160 180 200

200

220 240 260 280 300 320 340 360 380 400

400

420 440 460 480 500 520 540 560 580 600

600

620 640 660



C26 H23 N2 O84911435

6531880

800 1000 1200 1400 1600


40

30

20

10

0

(mg

AA

Eg

dw)

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



(a)

120

100

80

60

40

20

00 40 80 120 160 200 240 280 320 360

Inhi

bitio

n (



NafENafEHxNafEEa

NafEBuNafEInNafEW

(b)






100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)



Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from



















1

2

1

0

09

08

07

06

05

04

03

02

01

0

times102

times103

HO

HO

HO

HO

HO

HO

OO

OO

OO

OHOH


100 120 140 160 180 200

200

220 240 260 280 300 320 340 360 380 400

400

420 440 460 480 500 520 540 560 580 600

600

620 640 660



C26 H23 N2 O84911435

6531880

800 1000 1200 1400 1600


40

30

20

10

0

(mg

AA

Eg

dw)

Naf

E

Naf

EHx

Naf

EEa

Naf

EBu

Naf

EIn

Naf

EW



(a)

120

100

80

60

40

20

00 40 80 120 160 200 240 280 320 360

Inhi

bitio

n (



NafENafEHxNafEEa

NafEBuNafEInNafEW

(b)






100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)



Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from



















100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Colo 205

NafENafEEa

DoxorubicinDMSO

(a)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


Y79

NafENafEEa

DoxorubicinDMSO

(b)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


K562

NafENafEEa

DoxorubicinDMSO

(c)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MCF-7

NafENafEEa

DoxorubicinDMSO

(d)

100

80

60

40

20

0

Inhi

bitio

n (

)

25 50

100

200

05 1 5 10

DM

SO


MDA-MB 231

NafENafEEa

DoxorubicinDMSO

(e)

120

100

80

60

40

20

0

Cel

l pop

ulat

ion

()

25 50 100 200


HEK-293

NafENafEEa

(f)



Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from



















Table2Percentage

inhibitio

nof

cancer

cellproliferatio

nandIC

50values

Sample

Celltype

Colo205

Y79

K562

MCF

MDA-

MB

inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50inhibitio

nIC

50NafE

(200120583gmL)


NafEE

a(200120583gmL)


Doxorub

icin

(10120583

gmL)

9233plusmn045

039plusmn003


DMSO

2(Solvent

Cntl)

371plusmn056

mdash373plusmn153

mdash376plusmn100

mdash444plusmn170

mdash402plusmn117

mdash

Values

werethe

means

offour


deviation(SD)Sign

ificant119875values


ereo

btainedby

Stud

entrsquos119905-te

stanalysis

Com


ompared

usingon

e-way

analysisof

varia

nces

(ANOVA

)and

prob

abilityvalues

werefou

ndto

beequaltoor

lessthan

005

fora

llthefou

rcell

lines












4 Conclusion




Acknowledgments


References






















119898119886119909) generated from





























4 Conclusion




Acknowledgments


References






















119898119886119909) generated from






































119898119886119909) generated from


















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