Research Article Cytotoxic Constituents from the Rhizomes...

Research ArticleCytotoxic Constituents from the Rhizomes of Curcuma zedoaria

Omer Abdalla Ahmed Hamdi12 Syarifah Nur Syed Abdul Rahman3

Khalijah Awang12 Norhanom Abdul Wahab14 Chung Yeng Looi15

Noel Francis Thomas2 and Sri Nurestri Abd Malek13

1 Center for Natural Products and Drugs Discovery (CENAR) University of Malaya 50603 Kuala Lumpur Malaysia2 Department of Chemistry Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia3 Institute of Biological Sciences Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia4 Biology Division Center for Foundation Studies in Science University of Malaya 50603 Kuala Lumpur Malaysia5 Department of Pharmacology Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia

Correspondence should be addressed to Sri Nurestri Abd Malek srimalekumedumy

Received 14 February 2014 Accepted 8 June 2014 Published 13 July 2014

Academic Editor Xin Ming

Copyright copy 2014 Omer Abdalla Ahmed Hamdi 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

Curcuma zedoaria also known as Temu putih is traditionally used in food preparations and treatment of various ailments includingcancer The cytotoxic activity of hexane dichloromethane ethyl acetate methanol and the methanol-soxhlet extracts of Curcumazedoaria rhizomes was tested on two human cancer cell lines (Ca Ski and MCF-7) and a noncancer cell line (HUVEC) usingMTT assay Investigation on the chemical components in the hexane and dichloromethane fractions gave 19 compounds namelylabda-8(17)12 diene-1516 dial (1) dehydrocurdione (2) curcumenone (3) comosone II (4) curcumenol (5) procurcumenol (6)germacrone (7) zerumbone epoxide (8) zederone (9) 9-isopropylidene-26-dimethyl-11-oxatricyclo[621015]undec-6-en-8-ol(10) furanodiene (11) germacrone-45-epoxide (12) calcaratarin A (13) isoprocurcumenol (14) germacrone-110-epoxide (15)zerumin A (16) curcumanolide A (17) curcuzedoalide (18) and gweicurculactone (19) Compounds (1ndash19) were evaluated fortheir antiproliferative effect using MTT assay against four cancer cell lines (Ca Ski MCF-7 PC-3 and HT-29) Curcumenone (3)and curcumenol (5) displayed strong antiproliferative activity (IC

50= 83 plusmn 10 and 93 plusmn 03 120583gmL resp) and were found to

induce apoptotic cell death on MCF-7 cells using phase contrast and Hoechst 33342PI double-staining assay Thus the presentstudy provides basis for the ethnomedical application of Curcuma zedoaria in the treatment of breast cancer

1 Introduction

It is widely reported that more than 35000 plant speciesare used for medicinal purposes worldwide Of these 1200and 2000 plant species from Peninsular Malaysia and EastMalaysia respectively are used in folklore medicine [1] Onesuch plant is Curcuma zedoaria (Berg) Rosc belonging tothe Zingiberaceae family and known by the locals as Temuputih or Kunyit putihThe leaf blades are 80 cm long usuallywith a purple-brown flush running along the midrib on bothsurfaces of the leaf In the young plants the rhizomes ofCurcuma zedoaria are easily confused with those of Curcumaaeruginosa and Curcuma mangga because both have almostsimilar yellow colorHowever a cross-section of the rhizomes

of the mature plants of Curcuma aeruginosa is slightly darkpurplish whilst Curcuma mangga have brighter yellow color[2] Temu putih is used by the Malays in the preparationof traditional medicinemdashconsumed either on their own orin mixtures with other plant species They are also widelyconsumed as spices as flavors in native dishes and as foodpreparations in postpartum confinement [2ndash4] Curcumazedoaria also called Er-chu in Chinese is clinically usedfor the treatment of cervical cancer [5] In Japan it hasalso been used as an aromatic stomachic [6] Whilst in theAyurvedic medicine it is used for the treatment of fevers(cooling) antiseptic mild expectorant and deodorizer [7]In Indonesia Curcuma zedoaria is widely consumed in theform of ldquojamurdquo for the treatment of breast and cervical

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 321943 11 pageshttpdxdoiorg1011552014321943

2 The Scientific World Journal

cancers [8] Medicinal plants are used widely especially inAsia as an alternative medicine for cancer-related diseasesbecause it is believed for having active natural occurringcompounds in killing cancer [9 10] However there are onlylimited studies on the efficacy on the use of medicinal plants[11] It is therefore important to identify the componentswhich are responsible for the chemotherapeutic effects andthe molecular pathway by which these compounds affectcancer cell death Up to date there are numerous reportedarticles on the cytotoxic components of Curcuma zedoariaand the mechanism of cell death exerted by some of thesecompounds [5ndash8 12ndash20] In our continuous effort to studythe bioactive and theirmode of actions frommedicinal plantsof Curcuma species this communication reports the isolationof 19 compounds and among these two bioactive compounds(curcumenone and curcumenol) were identified as cytotoxiccomponents and were able to induce apoptosis

2 Materials and Methods

21 Plant Samples Curcuma zedoaria rhizomes were col-lected from Tawamangu Indonesia and a voucher specimen(KL 5764) was deposited at the herbarium of the Departmentof Chemistry Faculty of Science University of Malaya KualaLumpur Malaysia

22 Extraction of Plant Sample Briefly the washed and driedrhizomes of Curcuma zedoaria were finely ground The finepowders of Curcuma zedoaria (10 kg) were soaked in n-hexane for 3 days Then the solvent containing extract wasdecanted and filtered (were repeated twice each time withfive liters of n-hexane) All the filtrates were combined andevaporated using a rotary evaporator (Buchi Switzerland) togive the n-hexane extract The n-hexane-insoluble residuewas further extracted with CH

2Cl2to give the CH

2Cl2-

soluble extract and CH2Cl2-insoluble residue The CH

2Cl2-

insoluble residue was further extracted with EtOAc to givethe EtOAc-soluble and EtOAc-insoluble extract The EtOAc-insoluble extract was then extracted with MeOH to give theMeOH extract The insoluble residue obtained after MeOHextraction was further subjected to soxhlet extraction usingmethanol to give the MeOH SE extract after evaporation ofexcess solvent All the extracts were weighed after solventevaporation

23 Cell Culture The human cell lines MCF-7 (breast can-cer) Ca Ski (cervical cancer) and HT-29 (colon cancer)were cultured as monolayer in RPMI 1640 growth mediaHUVEC (human umbilical vein endothelial cells) and PC-3 (prostate cancer) cells were cultured in DMEM All cellswere purchased from theAmericanTissueCultureCollection(ATCC USA) except for human umbilical vein endothelialcells (HUVEC)whichwere obtained fromScienCell ResearchLaboratories (Carlsbad CA) All the media were supple-mented with 10 vv foetal bovine serum (FBS) 100 120583gmLpenicillinstreptomycin and 50 120583gmL amphotericin B Thecells were cultured in a 5 CO

2incubator at 37∘C

24 MTT Cytotoxicity Assay Cell viability was investigatedusing 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazoliumbromide (MTT) assay Cells were detached from the 25 cm3tissue culture flask when it achieved 80 confluency Thedetached cells were pellet by centrifugation (1000 rpm 5minutes) Cells (30times104 cellsmL)were seeded onto a 96-wellmicrotiter plate (Nunc)The cells were incubated at 37∘CCO

2

incubator for 24 h to give adherent cells The test compounds(1ndash100 120583gmL) were added onto the 96-well microtiter platecontaining adherent cells The untreated cells were incubatedin 10 media containing 05 DMSO (without addition ofany test compoundsextracts) This mixture was regardedas the negative control whereas doxorubicin as the positivecontrol The plates were incubated for 72 h at 37∘C in a5 CO

2incubator After 72 h the media were removed

and 100 120583L of fresh medium and 20120583L of MTT (Sigmafilter sterile 5mgmL) were added to each well and furtherincubated for 4 hours (37∘C) after which the media weresubstituted with 150 120583L DMSOThe 96-well microtiter plateswere then agitated at room temperature onto an incubatorshaker to dissolve the formazan crystals The absorbance (119860)of the content of the plates was measured at 540 nm using amicroplate reader The percentage of inhibition of each testsample was calculated according to the following formulaPercentage of inhibition () = (119860control minus 119860 sample)119860control times100The average of three replicates was then obtained TheIC50

for each extract was extrapolated from the graphs ofthe percentage inhibition versus concentration of test agentsCytotoxicity of each test agent is expressed as IC

50value The

IC50value is the concentration of test agents that cause 50

inhibition or cell death averaged from the three experiments[21ndash23] The selectivity index (SI) was also calculated asdescribed by [24 25] using the ratio between IC

50of the

extract or compounds on normal cell lines (HUVEC) andIC50

of the tested extract or compounds on cancerous celllines Selectivity index (SI) values equal or greater than threewere considered to have a high selectivity towards cancerouscells An SI value denotes the selectivity of the sample to thetested cell lines [24 25]

25 General Methods on Characterization of the Active Prin-ciples TLC techniques were used to monitor the purity ofisolated compounds Analytical TLC was performed on theprecoated plates with silica gel 60 F

254(Merck of 2025mm)

(normal phase) HPTLC and PTLC were carried out on theprecoated plates with silica gel 60 GF

254(Merck 2025mm)

Spots were detected by UV (254 360 nm) and by sprayingof vanillin-H

2SO4or anisaldehyde-H

2SO4followed by gentle

heating CCwas carried out onKieselgel 60 (0043ndash0063mmand 0063ndash0200mm) (Merck) and Sephadex LH 20 (25ndash100m) (Merck) HPLC was used to isolate and purify thecompounds HPLC was performed using Waters Systemequipped with Binary Gradient Module (Waters 2545) Sys-tem Fluidics Organizer and PhotodiodeArrayDetector (190ndash400 nm Waters 2998) and Sample Manager (Waters 2767)The column used was Waters XBridge Prep C18 5 120583M (10 times250mm) column with Waters XBridge Prep C18 5120583M (10 times10mm) column guard cartridge The data were collected

The Scientific World Journal 3

and analyzed by MassLynx software 1D NMR (1H 13CDept 135) and 2D NMR (HSQC HMBC COSY NOESY)spectra were recorded from a JEOL 400MHz FT NMRspectrometer at 400MHz for 1H-NMR and at 100MHz for13C-NMR Chemical shifts in ppm were referenced to theinternal standard TMS (120575 = 0 ppm) for use in 1H-NMRandCDCl

3(120575 770 ppm) 13C-NMR spectra respectivelyThe

GC-MS analyses were performed using Shimadzu QP2010Series gas chromatography and operated in the split lessmodeat 275∘C The column used was DM 5MS (5 diphenyl95dimethyl polysiloxane) capillary column (300m times 025mmtimes 025 120583m) with helium as carrier gas at a flow rate of1mLminminus1 The column temperature was programmed asfollows initially at 60∘C then increased to 250∘C at 5∘Cper minute and then held for 1 minute The total ionchromatogram was obtained by autointegration using ChemStation and the components were identified by comparingtheir mass spectral data with the accompanying SpectralDatabase (NIST 05 Mass Spectral Library USA) wheneverpossible IR spectra were obtained on a Perkin Elmer 1600Series FT-IR infrared spectrophotometer with chloroform assolvent The wavelength is indicated in cmminus1 Mass spectraof LC-MS were recorded using Agilent Technologies 6530Accurate-Mass Q-TOF LC-MS

26 Extraction and Isolation of Pure Compounds The pow-dered rhizomes (10 kg) were initially extracted with hexaneto give the hexane extract (242 g 24) The hexane extract(200 g) was then subjected to silica gel column chromatog-raphy (CC) eluting initially with hexane followed by hex-ane enriched with increasing percentages of ethyl acetate(EtOAc) Fractions were then combined according to similar-ity of thin layer chromatography (TLC) spots to give 21 frac-tions (fractions 1ndash21) Germacrone-4 5 epoxide (12 124mg)and germacrone-1 10 epoxide (15 80mg) were isolated fromfraction 5 throughmicro CC and preparative thin layer chro-matography (PTLC) Fraction 6 afforded germacrone (7 216mg) and furanodiene (11 88mg) upon purification with CCand PTLC Fraction 7 was further chromatographed usingvarious isolation techniques such as Sephadex-LH20 PTLCand high performance thin layer chromatography (HPTLC)to afford dehydrocurdione (2 345mg) curcumanolideA (1749mg) and two labdanes namely labda-8 (17) 12 diene-15 16 dial (1 162 mg) and labda-8(17) 12 diene-15 15-dimethoxy-16-al or calcaratarin A (13 226mg) Fraction8 was further purified using PTLC to give curcumenol (5155mg) and zerumin A (16 98mg) Isoprocurcumenol (14102mg) was isolated from fraction 9 using two successivePTLC Fraction 10was chromatographed and further purifiedby HPTLC to afford a second monoclinic modification ofcurcumenol as a crystallized dimer elucidated by single crys-tal X-ray diffraction analysisThis dimer is 9-isopropylidene-2 6-dimethyl-11-oxatricyclo [621015] undec-6-en-8-ol (1054mg) as previously described [20] whilst curcuzedoalide(18 134mg) was isolated from fraction 10 using HPLCCurcumenone (3 164mg) was purified from fractions 12 and13 Procurcumenol (6 89 mg) and zerumbone epoxide (8119mg) were isolated from fractions 15 and 16 respectively

using micro CC and PTLC The CH2Cl2extract (10 g) was

then subjected to silica gel CC with initial elution of 5EtOAc-hexane and gradually increasing the polarity to 100EtOAc andfinallywithMeOH Fractionswere then combinedaccording to similarity of TLC spots to give 23 fractions(fractions 1ndash23) Fraction 2 was subjected to micro CC toafford comosone II (4 66mg) zederone (9 244mg) andgweicurculactone (19 36mg) upon purification with HPLCAll the isolated compounds were identified using NMRspectroscopy and other supportive data (MS IR and UV)and results obtained were consistent with reported data [14ndash18 20 23 26ndash32] The structures of isolated compounds areshown in Figure 1 The method of isolation is summarized inFigure 2

27 Phase Contrast Microscopy Briefly MCF-7 cells (5 times10

5) were grown in a tissue culture dishes (60mm) for

overnightThen the cells were treated with curcumenone (3)and curcumenol (5) at a concentration of 125 and 25 120583gmLrespectively After 48 hours cells were gently rinsedwith PBSTheobservation ofmorphological changes of apoptoticMCF-7 cells after treatment with the two bioactive compounds wasviewed using an inverted phase contrast microscope (LeicaDMI 3000B Germany) at 400x magnification according tothe method [33]

28 Fluorescence Microscopy The morphological features ofMCF-7 cells upon treatment by the test compounds alsoobserved by double staining of Hoechst 33342PI assay[33ndash37] using the inverted fluorescence microscope (LeicaDM16000B) Briefly 5 times 105 cells were grown overnightand treated with 125 and 25 120583gmL of curcumenone (3)and curcumenol (5) After treatment of cells with the testcompounds for 48 hours both floating and adherent cellswere collected by centrifugation and washed once with coldPBS Then Hoechst 33342 solutions (10120583gmL) were addedand incubated at 37∘C for 7 minutes The cells were thenstained with PI (25120583gmL) and further incubated in thedark for 15 minutes Cell suspension (100 120583L) was mountedonto glassmicroscope slides andobservedunder fluorescencemicroscope using UV488 dual excitation (460 nm emissionof Hoechst 33342 575 nm emission of PI) Approximately atotal of 200 target cells were calculated and themorphologicalcharacteristics of the nuclei were analyzed for quantificationof apoptosis and necrosis [37] The percentage of apoptoticnecrotic and dead cells was determined according to theformula described by [37]

29 Statistical Analysis All data were presented as mean plusmnstandard deviation All experiments were conducted in tripli-catesThe data were subjected to one-way analysis of variance(ANOVA) with the significant differences between groupsdetermined by Duncanrsquos multiple range tests (DMRT) at 95significant difference (119875 lt 005) using STATGRAPHICS Plussoftware (version 30 Statistical Graphics Corp PrincetonNJ USA)


(1)

CHO

CHO

O

O

(2)

O

H

O

(3) (4)

O

HOH

(5)

H

O

HOH

(6)

O

OO

(9)

OH

H

(10)

(16)(15)(14)(13)

(18) (19)

O

(7)

O

O

(8)

O

(11)

O

O

(12)

H

CHO

O

H

OH H

O

COOH

CHO

OO

(17)

O

H

O

O

O

O

O

O

H3CO

O

CH2

OCH3

Figure 1 The structures of isolated compounds


Fr 5Fr 13Fr 12Fr 6 Fr 7 Fr 8 Fr 9 Fr 15 Fr16

12 15 7 11 1 2 13 17 5 16 14 3 6 8

Fr 10

10 18

23 fractions

21 fractions

Fraction 2

4 9 19

Hexane extracts (20 g)

CC silica gel

CC silica gel

n-hexaneEtOAc (100 0-0 100) rarr MeOH

Hex EtOAc (95 5) rarr 100 EtOAc rarr MeOH

Dichloromethane extract (10 g)

Figure 2 Schematic represents the isolation method of the bioactive compounds from Curcuma zedoaria

Table 1 Antiproliferative activity [IC50 values (120583gmL)] and selectivity index of crude and fractionated extracts of Curcuma zedoaria againsthuman cancer and noncancer (HUVEC) cell lines

Extracts IC50 (120583gmL)a

MCF-7 SIb Ca Ski SIb HUVECHexane 184 plusmn 16 54 190 plusmn 15 53 gt1000Dichloromethane 406 plusmn 23 25 835 plusmn 27 12 gt1000Ethyl acetate gt1000 10 gt1000 10 gt1000Methanol gt1000 10 gt1000 10 gt1000Methanol (soxhlet extraction) gt1000 10 gt1000 10 gt1000aData are presented as mean plusmn standard deviation (SD) of three replicatesbSI is the selectivity index SI values ge30 denote high selectivity towards cancerous cells

3 Results and Discussion

31 Detection of Cell Viability by MTT Assay The antipro-liferative activity of crude and extracts of Curcuma zedoariawas analysed using MTT assay The IC

50values (120583gmL)

were evaluated for these crude extracts averaged from threeexperiments against two human cancer cell lines (Ca Ski andMCF-7) and a noncancer cell (HUVEC) and the result issummarized inTable 1 A plant extractwith IC

50le 20120583gmL

is considered active [21ndash23] The hexane extract showedhigh inhibitory activity against Ca Ski and MCF-7 cellswhilst the dichloromethane (CH

2Cl2) extract possessedmild

cytotoxicity against MCF-7 and exhibited weak cytotoxicityagainst Ca Ski The extracts of Curcuma zedoaria altogethershowed to be essentially ineffective on the normal cellsSelectivity indexes (SI) of the antiproliferative activity of

Curcuma zedoaria extracts were evaluated by the ratio of thecytotoxic activity (IC

50) of each extracts against the cancer

cells with the normal cells (HUVEC)The SI with greater or equal value of three was considered

to be highly selective towards cancer cells [24 25 38 39]As shown in Table 1 the hexane extract showed selectiveactivity towards Ca Ski and MCF-7 cells with SI valuesof 53 and 54 respectively Thus the data have revealedthat the hexane extracts exhibited antiproliferative effect andpossessed selective activity towards Ca Ski and MCF-7 cellsin reference to normal cells (HUVEC)

32 Isolation of Active Principles The isolation of the activeprinciples (compounds 1ndash19) has been described extensivelyin section methodology These chemical components were


Table 2 Antiproliferative activity [IC50 values (120583gmL)]a and selectivity index of isolated compounds against selected human cancer cell linesand human umbilical vein endothelial cells (HUVEC)

CompoundsIC50 (120583gmL)

SIb

MCF-7 Ca Ski PC-3 HT-29 HUVEClabda-8(17) 12 diene-15 16 dial (1) 163 plusmn 02 (28) 145 plusmn 01 (31) 263 plusmn 24 (17) 215 plusmn 31 (21) 453 plusmn 19dehydrocurdione (2) 330 plusmn 11 (07) 217 plusmn 11 (11) 191 plusmn 28 (13) 227 plusmn 24 (11) 240 plusmn 21curcumenone (3) 83 plusmn 10 (60) gt1000 (05) 398 plusmn 42 (13) 433 plusmn 62 (12) 500 plusmn 86comosone II (4) gt1000 760 plusmn 12 na na nacurcumenol (5) 93 plusmn 03 (28) 185 plusmn 10 (14) 173 plusmn 12 (15) 248 plusmn 27 (10) 259 plusmn 14procurcumenol (6) 161 plusmn 22 (10) 624 plusmn 03 (03) 133 plusmn 17 (12) 155 plusmn 23 (11) 163 plusmn 10germacrone (7) 591 plusmn 29 (12) 393 plusmn 12 (19) 552 plusmn 49 (13) 429 plusmn 41 (17) 737 plusmn 03zerumbone epoxide (8) 241 plusmn 01 (06) 345 plusmn 06 (04) 108 plusmn 19 (13) 137 plusmn 27 (10) 142 plusmn 11zederone (9) gt1000 (04) gt1000 (04) 270 plusmn 19 (16) 191 plusmn 25 (22) 421 plusmn 27second monoclinic curcumenol (10) gt1000 (07) gt1000 (07) na na 717 plusmn 61furanodiene (11) 365 plusmn 26 (11) na 395 plusmn 45 (10) 472 plusmn 44 (09) 409 plusmn 26germacrone-4 5-epoxide (12) 372 plusmn 40 (13) na 439 plusmn 72 (11) 396 plusmn 46 (12) 484 plusmn 47calcaratarin A (13) 625 plusmn 48 (08) na 417 plusmn 34 (11) 483 plusmn 51 (10) 473 plusmn 42isoprocurcumenol (14) 588 plusmn 42 (08) na 374 plusmn 45 (12) 516 plusmn 39 (09) 451 plusmn 30germacrone-1 10-epoxide (15) 612 plusmn 58 (09) na 532 plusmn 49 (10) 728 plusmn 83 (08) 555 plusmn 16zerumin A (16) 223 plusmn 11 (12) na 219 plusmn 16 (12) 174 plusmn 20 (15) 258 plusmn 19curcumanolide A (17) 298 plusmn 31 (07) na 188 plusmn 24 (12) 213 plusmn 32 (10) 217 plusmn 70curcuzedoalide (18) 498 plusmn 36 (09) na 621 plusmn 81 (07) 582 plusmn 35 (08) 453 plusmn 78gweicurculactone (19) 312 plusmn 32 (23) na 383 plusmn 22 (19) 357 plusmn 58 717 plusmn 61 (20)doxorubicinlowast 01 plusmn 00 (40) 02 plusmn 10 (20) na na 14 plusmn 00aData are presented as mean plusmn standard deviation (SD) of three replicatesbSI is the selectivity index SI ge30 denotes high selectivity towards cancerous cellslowastna-not available

identified using spectroscopic (NMR IR and UV) andspectrometric studies (GS-MS LC-MS and MS) and werefound to be in agreement with reported data [14ndash18 20 2326ndash32]

33 Antiproliferative Activity of Compounds (1ndash19) The com-pounds (1ndash19) isolated from the hexane and dichloromethaneextracts were further evaluated on four selected cancer celllines (MCF-7 Ca Ski HT-29 and PC-3) and a normal humanumbilical vein endothelial cell (HUVEC) Many researchershave utilizedHUVEC cell lines in determining cytotoxicity oftest samples against normal cells [40ndash44] Similar approachhas also widely employed in high-throughput screening indrug discovery [45] Test samples showingmild or no toxicitytowards normal cell lines (HUVEC) would be a potentiallygood candidate for drug development The antiproliferativeactivity of the compounds (1ndash19) is presented in Table 2The isolated terpenoids from Curcuma zedoaria were foundto possess moderate antiproliferative effect against the fourselected human carcinoma In the present study only cur-cumenone (3) and curcumenol (5) demonstrated strongantiproliferative activity against MCF-7 Curcumenone (3)was selectively toxic to MCF-7 cells whilst curcumenol (5)displayed appreciable selectivity towards MCF-7 in referenceto HUVECs with SI values of 60 and 28 respectively

It appeared therefore that compounds (3) and (5) haveselective activity towards MCF-7 cell line To the best ofour knowledge there is no report on the cytotoxicity ofcompounds (3) (5) and (10) against human MCF-7 CaSki and PC-3 cell lines In a study by [14] curcumenoneisolated from the rhizomes of Curcuma zedoaria has beenreported to have protective effect on alcohol-treated miceand acceleration of liver alcohol dehydrogenase activity Itis also been found as an effective protective effect on D-galactosaminelipopolysaccharide-induced acute liver injury[5] In a previous reported publication [46] claimed thatcurcumenol is widely used to treat cancer and inflammationand also known as an antibiotic or anticancer drug In theirstudy [46] found that curcumenol is not a mechanism-basedinhibitor through time- and NADPH-dependent inhibitionsand also suggested that curcumenol may be safely used with-out inducing metabolic drug-drug interaction through P450inhibition Labda-8(17) 12 diene-15 16 dial (1) displayed highselective activity towards Ca Ski and appreciable selectivityto MCF-7 but only exhibited moderate cytotoxicity againstthe cancer cells Procurcumenol (6) and zerumbone epoxide(8) exhibited good cytotoxic effect against PC-3 and HT-29 cell lines but were not selective on these tumor cellsPreviously [26] described that zerumbone epoxide isolatedfrom the rhizomes of Curcuma zedoaria possessed cytotoxiceffects Thus this is in a good agreement with our current


Control

Curcumenone Curcumenol

125 120583gmL

25120583gmL

Figure 3 Morphological analysis of MCF-7 cells treated with curcumenone and curcumenol as observed under inverted phase contrastmicroscope (400x)

study Compounds (1ndash10) displayed appreciable to weakcytotoxic activity against Ca Ski (IC

50values ranging from

145 plusmn 01 to 1000 120583gmL respectively) Only curcumenone(3) did not show any antiproliferative activity against CaSki Other compounds (1 2 5 9 16 and 17) also exhibitedmoderate inhibitory activity against the tested carcinomaPC-3 and HT-29 cell lines All tested compounds showedmild cytotoxicity towards the normal cell lines (HUVEC)Curcumenone (3) and zerumbone epoxide (8) were foundto have slight toxicity towards the normal cell Althoughthe pure compounds are not as effective as doxorubicin ininhibiting the proliferation of the cancer cells they inflictless damage to the noncancerous cells To the best of ourknowledge it is important to note that compounds (1 8 12-13 and 19) are reported here for the first time from Curcumazedoaria In this study zerumin A (16) was isolated fromthe hexane fraction and displayed moderate cytotoxic effecton MCF-7 Ca Ski and PC-3 cell lines This is in agreementwith that reported by [23] whereby zerumin A isolated fromCurcuma mangga exhibited antiproliferative effect on Ca SkiandMCF-7 displaying IC

50of 87plusmn029 and 142plusmn006 120583gmL

respectively In our previous study [19] curzerenone andalismol were also reported present in Curcuma zedoariawhich were not found in the present study The reason forthis difference is possibly due to the source of the plantsamples The plant sample in the present study was obtainedfrom Tawamangu Java Indonesia whilst those in the earlierreport were collected from Jogjakarta Indonesia In additionit is also important to note that diterpenoids (compounds1 13 and 16) were not detected in the sample obtainedfrom Jogjakarta and the isolation of curzerenone and alismolwere based on bioassay-guided procedure The cytotoxicityassay used in the present study could only provide importantpreliminary data to help select isolated compounds withpotential anticancer properties Further studies on the effectof curcumenone (3) and curcumenol (5) on the mode ofMCF-7 cell death were thus pursued

34 Induction of Apoptosis The result from the cytotoxicityassay provides important preliminary data that may helpselect compounds with promising anticancer effects for


Control


125 120583gmL

25120583gmL

Figure 4 Apoptosis-inducing effect of curcumenone and curcumenol on MCF-7 cells by double staining using Hoechst 33342PI andvisualized under fluorescence microscope (630x)

further work A detailed investigation on the underlyingmechanism involved in cell death would provide a moreconvincing evidence of anticancer effect Thus apoptosisinduction of the active compounds in the cancer cells wasinvestigated Apoptosis is described as programmed celldeath It is an essential process that enables the removalof cells from tissues thus maintaining the proper functionof multicellular organisms In the average human adultabout 50ndash70 billion cells die by apoptosis each day Howeverdiseases such as cancer resulted when cells fail to die Aseries of events is involved in the process of apoptosis Theevents start with cell dehydration which leads to cytoplasmcondensation and alteration in cell shape and size The nextevent is chromatin condensation which starts at the nuclearperiphery and results in the concave shape of the nucleusfollowed by nuclear membrane integration and nuclear frag-mentation Nuclear fragmentation and other organelles ofthe apoptotic cells are enveloped by fragments of cytoplasmand form apoptotic bodies which are phagocytosized byneighboring cells thus preventing inflammatory reactionThese events can be observed in an inverted phase contrastand fluorescence microscope Thus apoptosis was first andis still best described morphologically [37] Induction ofapoptosis based on biochemical changes or flow cytometric

analyses should always be backed up with morphologicalstudies [37]

35 Morphology of MCF-7 Cells Treated with Curcumenoneand Curcumenol as Observed under Inverted Phase ContrastMicroscope In this study the apoptosis inducing capacityof the two bioactive compounds namely curcumenone (3)and curcumenol (5) on MCF-7 cells was thus investigatedusing inverted phase contrast microscope MCF-7 cells wereincubated for 48 h with 125 and 25120583gmL of curcumenone(3) and curcumenol (5) respectively Exposure of MCF-7cells to the compounds led to cell shrinkage loss of contactwith adjacent cells and decrease in cell numbers (Figure 3)In comparison the untreated (control) cells were observed asintact and were cuboids or polygonal in shape Floating cellsdetached from the surface of the tissue culture dishes (notshown) were also observed

36 Hoechst 33342PI Staining of MCF-7 Cells upon Treat-ment with Curcumenone and Curcumenol Dual staining byHoechst 33342propidium iodide (PI) of MCF-7 and Ca Skicells revealed that induction of apoptotic death occurred after48 h incubation with curcumenone (3) and curcumenol (5)


Control

Apoptotic cells ()

125 120583gmL0

102030405060

CurcumenoneCurcumenol

lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)

Figure 5 Apoptotic index and percentage of necrotic cells Values expressed aremeans plusmn standard deviation (sd) of triplicatemeasurementsThe asterisks (lowast) denote significant differences between groups (119875 lt 005)

The untreated cells displayed intact regular form and werehomogenously stained with a dimmer blue color Afterthe cells were treated (48 h) with 125 and 25 120583gmL ofcurcumenone (3) and curcumenol (5) respectively apoptoticnuclei emitted much brighter blue fluorescence due to thehighly condensed chromatin As in Figure 4 crescents wereobserved around the periphery of the nucleus due to chro-matin condensation Cells that were in late apoptosis emittedpink fluorescenceThe organized structure of pink chromatindenoted dead cells with normal nuclei Dead cells withapoptotic nuclei showed highly condensed and fragmentedbright pink chromatin Necrotic cells were swollen withirregularmembranes and fluorescence bright pink chromatin(due to PI)There was a significant increase in the percentageof apoptotic cells due to increasing dose of tested compoundsCurcumenol (5) revealed better inducing apoptosis capacityin comparison to curcumenone (3) as observed in Figure 5

4 Conclusions

In this study Curcuma zedoariawas shown to possess severalcompounds that have antiproliferative effect on four cancercell lines (MCF-7 Ca Ski PC-3 and HT-29) Amongst thesetwo compounds namely curcumenone (3) and curcumenol(5) present in the hexane extract were able to induceapoptosis in MCF-7 cells by inhibiting the proliferation ofthe cancer cells However further investigations are necessary

to determine their mode of action It is noteworthy tomention that the hexane extract and the two compoundscurcumenone and curcumenol showed low toxicity towardsthe normal cell line (HUVEC) If this also occurs in vivothen this plant has the potential to be developed as anticanceragent

Conflict of Interests

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

Acknowledgments

Theauthorswish to acknowledge theMinistry ofHigher Edu-cation of Malaysia and the University of Malaya for finan-cial assistance received through the following grants UMHigh Impact Research Grant UM-MOHEUMC6251 HIRMOHESC02 RG015-09BIO PS3442009B and PV0362011B

References

[1] I Jantan ldquoConservation of medicinal plants and their tradi-tional knowledgerdquo in Proceedings of the seminar UPMonMedic-inal Plants Cure for the 21st Century (Biodiversity Conservationand Utilization of Medicinal Plants) M N B Nair and GNathan Eds pp 20ndash24 Serdang Malaysia October 1998


[2] S N Malek F Abdullah N M Ali H Ibrahim and M NJalil ldquoAnalysis of essential oil of Curcuma zedoariardquo Journal ofTropical Medicinal Plants vol 5 no 1 pp 29ndash32 2004

[3] H Burkill A Dictionary of the Economic Products of theMalay Peninsula Ministry of Agriculture and Cooperative 2ndedition 1966

[4] K Larsen H Ibrahim S H Khaw and L G Saw Gingers ofPeninsular Malaysia and Singapore Natural History Publica-tions Borneo Sdn Bhd Borneo Malaysia 1999

[5] H Matsuda K Ninomiya T Morikawa and M YoshikawaldquoInhibitory effect and actionmechanismof sesquiterpenes fromzedoariae rhizoma on D-galactosaminelipopolysaccharide-induced liver injuryrdquo Bioorganic and Medicinal Chemistry Let-ters vol 8 no 4 pp 339ndash344 1998

[6] S Park J Jung H Lee et al ldquoZedoariae rhizoma and curcumininhibits platelet-derived growth factor-induced proliferation ofhuman hepatic myofibroblastsrdquo International Immunopharma-cology vol 5 no 3 pp 555ndash569 2005

[7] S Lakshmi G Padmaja and P Remani ldquoAntitumour effects ofisocurcumenol isolated from Curcuma zedoaria rhizomes onhuman and murine cancer cellsrdquo vol 2011 Article ID 25396213 pages 2011

[8] W Tuti and L Andriani ldquoThe use of Curcuma zedoaria Roscmeal to reduce abdominal fat and meat cholesterol in broilerrdquoin Proceedings of the International Symposium Modern AnimalHusbandry-Food Safety and Socio-Economic Development vol53 pp 126ndash129 Universitatea de Stiinte Agricole si MedicinaVeterinara Ion Ionescu de la Brad April 2010

[9] N Hasima L I L Aun M N Azmi et al ldquo11015840S-11015840-Acetoxyeugenol acetate a new chemotherapeutic natural com-pound against MCF-7 human breast cancer cellsrdquo Phy-tomedicine vol 17 no 12 pp 935ndash939 2010

[10] K Awang M Nurul Azmi L I Lian Aun A Nazif Aziz HIbrahim and N Hasima Nagoor ldquoThe apoptotic effect of 1rsquoS-1rsquo-Acetoxychavicol acetate from Alpinia conchigera on humancancer cellsrdquoMolecules vol 15 no 11 pp 8048ndash8059 2010

[11] R Othman H Ibrahim M A Mohd M R Mustafa andK Awang ldquoBioassay-guided isolation of a vasorelaxant activecompound from Kaempferia galanga Lrdquo Phytomedicine vol 13no 1-2 pp 61ndash66 2006

[12] R Lobo K S Prabhu and A Shirwaikar ldquoCurcuma zedoariaRosc (white turmeric) a review of its chemical pharmacolog-ical and ethnomedicinal propertiesrdquo Journal of Pharmacy andPharmacology vol 61 no 1 pp 13ndash21 2009

[13] O-J Oh H Y Min and S K Lee ldquoInhibition of inducibleprostaglandin E

2production and cyclooxy-genase-2 expression

by curdione from Curcuma zedoariardquo Archives of PharmacalResearch vol 30 no 10 pp 1226ndash1239 2007

[14] Y KimuraM Sumiyoshi and T Tamaki ldquoEffects of the extractsand an active compound curcumenone isolated from Curcumazedoaria rhizomes on alcohol-induced drunkenness in micerdquoFitoterapia vol 84 no 1 pp 163ndash169 2013

[15] J Lu Y Dang M Huang W Xu X Chen and Y WangldquoAnti-cancer properties of terpenoids isolated from RhizomaCurcumaemdasha reviewrdquo Journal of Ethnopharmacology vol 143no 2 pp 406ndash411 2012

[16] M K Jang H J Lee J S Kim and J Ryu ldquoA curcuminoidand two sesquiterpenoids from Curcuma zedoaria as inhibitorsof nitric oxide synthesis in activated macrophagesrdquo Archives ofPharmacal Research vol 27 no 12 pp 1220ndash1225 2004

[17] H W D Matthes B Luu and G Ourisson ldquoCytotoxiccomponents of Zingiber zerumbet Curcuma zedoaria and Cdomesticardquo Phytochemistry vol 19 no 12 pp 2643ndash2650 1980

[18] Y Shiobara Y Asakawa M Kodama K Yasuda and T Take-moto ldquoCurcumenone curcumanolide A and curcumanolide Bthree sesquiterpenoids from Curcuma zedoariardquo Phytochem-istry vol 24 no 11 pp 2629ndash2633 1985

[19] S N Syed Abdul Rahman N Abdul Wahab and S NAbd Malek ldquoIn vitro morphological assessment of apoptosisinduced by antiproliferative constituents from the rhizomesof Curcuma zedoariardquo Evidence-Based Complementary andAlternativeMedicine vol 2013 Article ID 257108 14 pages 2013

[20] O A Ahmed Hamdi K Awang A H A Hadi D R Syamsirand S W Ng ldquoCurcumenol from Curcuma zedoaria a secondmonoclinic modificationrdquo Acta Crystallographica E vol 66 no11 p o2844 2010

[21] R I Geran N H Greenberg M M Mcdonald A M Schu-macher and B J Abbott ldquoProtocols for screening chemicalagents and natural products against animal tumor and otherbiological systemsrdquoCancer ChemotherapyReports vol 3 pp 17ndash19 1972

[22] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorptionrdquoToxicology Letters vol 24 no 2-3 pp 119ndash124 1985

[23] S N A Malek G S Lee S L Hong et al ldquoPhytochemicaland cytotoxic investigations of Curcuma mangga rhizomesrdquoMolecules vol 16 no 6 pp 4539ndash4548 2011

[24] P M Herst T Petersen P Jerram J Baty and M V BerridgeldquoThe antiproliferative effects of phenoxodiol are associatedwith inhibition of plasma membrane electron transport intumour cell lines and primary immune cellsrdquo BiochemicalPharmacology vol 74 no 11 pp 1587ndash1595 2007

[25] S Ramasamy N A Wahab N Z Abidin S Manickam andZ Zakaria ldquoGrowth inhibition of human gynecologic andcolon cancer cells by Phyllanthus watsonii through apoptosisinductionrdquo PLoS ONE vol 7 no 4 Article ID e34793 pp 1ndash152012

[26] F Xu S Nakamura Y Qu et al ldquoStructures of new sesquiter-penes from Curcuma comosardquo Chemical and PharmaceuticalBulletin vol 56 no 12 pp 1710ndash1716 2008

[27] P M Giang and P T Son ldquoIsolation of sesquiterpenoids fromthe rhizomes of Vietnamese Curcuma aromatica salisbrdquo Journalof Chemistry vol 38 pp 96ndash99 2000

[28] K Firman T Kinoshita A Itai and U Sankawa ldquoTerpenoidsfrom Curcuma heyneanardquo Phytochemistry vol 27 no 12 pp3887ndash3891 1988

[29] M Kuroyanagi A Ueno K Ujiie and S Sato ldquoStructures ofsesquiterpenes from Curcuma aromatica SALISBrdquo Chemicaland Pharmaceutical Bulletin vol 35 no 1 pp 53ndash59 1987

[30] D Chavez L A Acevedo and R Mata ldquoJimenezin a novelannonaceous acetogenin from the seeds of Rollinia mucosacontaining adjacent tetrahydrofuran-tetrahydropyran ring sys-temsrdquo Journal of Natural Products vol 61 no 4 pp 419ndash4211998

[31] A Dekebo E Dagne and O Sterner ldquoFuranosesquiterpenesfrom Commiphora sphaerocarpa and related adulterants of truemyrrhrdquo Fitoterapia vol 73 no 1 pp 48ndash55 2002

[32] M Kuroyanagi A Ueno K Koyama and S Natori ldquoStructuresof sesquiterpenes ofCurcumaaromatica SALISBmdashII Studies onminor sesquiterpenesrdquo Chemical and Pharmaceutical Bulletinvol 38 no 1 pp 55ndash58 1990


[33] P Moongkarndi N Kosem S Kaslungka O Luanratana NPongpan and N Neungton ldquoAntiproliferation antioxidationand induction of apoptosis by Garcinia mangostana (mangos-teen) on SKBR3 human breast cancer cell linerdquo Journal ofEthnopharmacology vol 90 no 1 pp 161ndash166 2004

[34] S I AbdelWahab A B Abdul A S Alzubairi M M Elhassanand SMohan ldquoIn vitro ultramorphological assessment of apop-tosis induced by Zerumbone on (HeLa)rdquo Journal of Biomedicineand Biotechnology vol 2009 Article ID 769568 10 pages 2009

[35] J M Brown and L D Attardi ldquoThe role of apoptosis in cancerdevelopment and treatment responserdquo Nature Reviews Cancervol 5 no 3 pp 231ndash237 2005

[36] G Thuret C Chiquet S Herrag et al ldquoMechanisms of stau-rosporine induced apoptosis in a human corneal endothelial celllinerdquo British Journal of Ophthalmology vol 87 no 3 pp 346ndash352 2003

[37] H J M BradyApoptosis Methods and Protocols Humana PressTotowa NJ USA 2004

[38] J R Dimmock P Kumar A J Nazarali et al ldquoCytotoxic 26-bis(arylidene)cyclohexanones and related compoundsrdquo Euro-pean Journal of Medicinal Chemistry vol 35 no 11 pp 967ndash9772000

[39] W Mahavorasirikul V Viyanant W Chaijaroenkul A Itharatand K Na-Bangchang ldquoCytotoxic activity of Thai medicinalplants against human cholangiocarcinoma laryngeal and hep-atocarcinoma cells in vitrordquo BMC Complementary and Alterna-tive Medicine vol 10 no 55 pp 1ndash8 2010

[40] H Xuan Z Li H Yan et al ldquoAntitumor activity of Chi-nese propolis in human breast cancer MCF-7 and MDA-MB-231 cellsrdquo Evidence-Based Complementary and AlternativeMedicine vol 2014 Article ID 280120 11 pages 2014

[41] D Su Y Cheng M Liu et al ldquoComparison of piceid andresveratrol in antioxidation and antiproliferation activities invitrordquo PLoS ONE vol 8 no 1 Article ID e54505 2013

[42] J Parada-Turska R PaduchMMajdanM Kandefer-SzerszenandWRzeski ldquoAntiproliferative activity of parthenolide againstthree human cancer cell lines and human umbilical veinendothelial cellsrdquo Pharmacological Reports vol 59 no 2 pp233ndash237 2007

[43] Y K Yong J J Tan S S Teh et al ldquoClinacanthus nutansextracts are antioxidant with antiproliferative effect on culturedhuman cancer cell linesrdquo Evidence-Based Complementary andAlternative Medicine vol 2013 Article ID 462751 8 pages 2013

[44] T R L SiekmannKM BurgazliMA BobrichGNoll andAErdogan ldquoThe antiproliferative effect of pinostrobin on humanumbilical vein endothelial cells (HUVEC)rdquoEuropeanReview forMedical and Pharmacological Sciences vol 17 no 5 pp 668ndash672 2013

[45] The Native Antigen Company 2088 httpwwwthenativeanti-gencompanycomnative-antigen-services-drug-testingasp

[46] D Sun Z Fang Y Zhang Y Cao L Yang and J YinldquoInhibitory effects of curcumenol on human liver cytochromeP450 enzymesrdquo Phytotherapy Research vol 24 no 8 pp 1213ndash1216 2010

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


cancers [8] Medicinal plants are used widely especially inAsia as an alternative medicine for cancer-related diseasesbecause it is believed for having active natural occurringcompounds in killing cancer [9 10] However there are onlylimited studies on the efficacy on the use of medicinal plants[11] It is therefore important to identify the componentswhich are responsible for the chemotherapeutic effects andthe molecular pathway by which these compounds affectcancer cell death Up to date there are numerous reportedarticles on the cytotoxic components of Curcuma zedoariaand the mechanism of cell death exerted by some of thesecompounds [5ndash8 12ndash20] In our continuous effort to studythe bioactive and theirmode of actions frommedicinal plantsof Curcuma species this communication reports the isolationof 19 compounds and among these two bioactive compounds(curcumenone and curcumenol) were identified as cytotoxiccomponents and were able to induce apoptosis

2 Materials and Methods

21 Plant Samples Curcuma zedoaria rhizomes were col-lected from Tawamangu Indonesia and a voucher specimen(KL 5764) was deposited at the herbarium of the Departmentof Chemistry Faculty of Science University of Malaya KualaLumpur Malaysia

22 Extraction of Plant Sample Briefly the washed and driedrhizomes of Curcuma zedoaria were finely ground The finepowders of Curcuma zedoaria (10 kg) were soaked in n-hexane for 3 days Then the solvent containing extract wasdecanted and filtered (were repeated twice each time withfive liters of n-hexane) All the filtrates were combined andevaporated using a rotary evaporator (Buchi Switzerland) togive the n-hexane extract The n-hexane-insoluble residuewas further extracted with CH

2Cl2to give the CH

2Cl2-

soluble extract and CH2Cl2-insoluble residue The CH

2Cl2-

insoluble residue was further extracted with EtOAc to givethe EtOAc-soluble and EtOAc-insoluble extract The EtOAc-insoluble extract was then extracted with MeOH to give theMeOH extract The insoluble residue obtained after MeOHextraction was further subjected to soxhlet extraction usingmethanol to give the MeOH SE extract after evaporation ofexcess solvent All the extracts were weighed after solventevaporation

23 Cell Culture The human cell lines MCF-7 (breast can-cer) Ca Ski (cervical cancer) and HT-29 (colon cancer)were cultured as monolayer in RPMI 1640 growth mediaHUVEC (human umbilical vein endothelial cells) and PC-3 (prostate cancer) cells were cultured in DMEM All cellswere purchased from theAmericanTissueCultureCollection(ATCC USA) except for human umbilical vein endothelialcells (HUVEC)whichwere obtained fromScienCell ResearchLaboratories (Carlsbad CA) All the media were supple-mented with 10 vv foetal bovine serum (FBS) 100 120583gmLpenicillinstreptomycin and 50 120583gmL amphotericin B Thecells were cultured in a 5 CO

2incubator at 37∘C

24 MTT Cytotoxicity Assay Cell viability was investigatedusing 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazoliumbromide (MTT) assay Cells were detached from the 25 cm3tissue culture flask when it achieved 80 confluency Thedetached cells were pellet by centrifugation (1000 rpm 5minutes) Cells (30times104 cellsmL)were seeded onto a 96-wellmicrotiter plate (Nunc)The cells were incubated at 37∘CCO

2

incubator for 24 h to give adherent cells The test compounds(1ndash100 120583gmL) were added onto the 96-well microtiter platecontaining adherent cells The untreated cells were incubatedin 10 media containing 05 DMSO (without addition ofany test compoundsextracts) This mixture was regardedas the negative control whereas doxorubicin as the positivecontrol The plates were incubated for 72 h at 37∘C in a5 CO

2incubator After 72 h the media were removed

and 100 120583L of fresh medium and 20120583L of MTT (Sigmafilter sterile 5mgmL) were added to each well and furtherincubated for 4 hours (37∘C) after which the media weresubstituted with 150 120583L DMSOThe 96-well microtiter plateswere then agitated at room temperature onto an incubatorshaker to dissolve the formazan crystals The absorbance (119860)of the content of the plates was measured at 540 nm using amicroplate reader The percentage of inhibition of each testsample was calculated according to the following formulaPercentage of inhibition () = (119860control minus 119860 sample)119860control times100The average of three replicates was then obtained TheIC50

for each extract was extrapolated from the graphs ofthe percentage inhibition versus concentration of test agentsCytotoxicity of each test agent is expressed as IC

50value The

IC50value is the concentration of test agents that cause 50

inhibition or cell death averaged from the three experiments[21ndash23] The selectivity index (SI) was also calculated asdescribed by [24 25] using the ratio between IC

50of the

extract or compounds on normal cell lines (HUVEC) andIC50

of the tested extract or compounds on cancerous celllines Selectivity index (SI) values equal or greater than threewere considered to have a high selectivity towards cancerouscells An SI value denotes the selectivity of the sample to thetested cell lines [24 25]

25 General Methods on Characterization of the Active Prin-ciples TLC techniques were used to monitor the purity ofisolated compounds Analytical TLC was performed on theprecoated plates with silica gel 60 F

254(Merck of 2025mm)

(normal phase) HPTLC and PTLC were carried out on theprecoated plates with silica gel 60 GF

254(Merck 2025mm)

Spots were detected by UV (254 360 nm) and by sprayingof vanillin-H

2SO4or anisaldehyde-H

2SO4followed by gentle

heating CCwas carried out onKieselgel 60 (0043ndash0063mmand 0063ndash0200mm) (Merck) and Sephadex LH 20 (25ndash100m) (Merck) HPLC was used to isolate and purify thecompounds HPLC was performed using Waters Systemequipped with Binary Gradient Module (Waters 2545) Sys-tem Fluidics Organizer and PhotodiodeArrayDetector (190ndash400 nm Waters 2998) and Sample Manager (Waters 2767)The column used was Waters XBridge Prep C18 5 120583M (10 times250mm) column with Waters XBridge Prep C18 5120583M (10 times10mm) column guard cartridge The data were collected














(1)

CHO

CHO

O

O

(2)

O

H

O

(3) (4)

O

HOH

(5)

H

O

HOH

(6)

O

OO

(9)

OH

H

(10)

(16)(15)(14)(13)

(18) (19)

O

(7)

O

O

(8)

O

(11)

O

O

(12)

H

CHO

O

H

OH H

O

COOH

CHO

OO

(17)

O

H

O

O

O

O

O

O

H3CO

O

CH2

OCH3




12 15 7 11 1 2 13 17 5 16 14 3 6 8

Fr 10

10 18

23 fractions

21 fractions

Fraction 2

4 9 19


CC silica gel

CC silica gel












50le 20120583gmL












SIb






Control


125 120583gmL

25120583gmL









Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of














(1)

CHO

CHO

O

O

(2)

O

H

O

(3) (4)

O

HOH

(5)

H

O

HOH

(6)

O

OO

(9)

OH

H

(10)

(16)(15)(14)(13)

(18) (19)

O

(7)

O

O

(8)

O

(11)

O

O

(12)

H

CHO

O

H

OH H

O

COOH

CHO

OO

(17)

O

H

O

O

O

O

O

O

H3CO

O

CH2

OCH3




12 15 7 11 1 2 13 17 5 16 14 3 6 8

Fr 10

10 18

23 fractions

21 fractions

Fraction 2

4 9 19


CC silica gel

CC silica gel












50le 20120583gmL












SIb






Control


125 120583gmL

25120583gmL









Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



12 15 7 11 1 2 13 17 5 16 14 3 6 8

Fr 10

10 18

23 fractions

21 fractions

Fraction 2

4 9 19


CC silica gel

CC silica gel












50le 20120583gmL












SIb






Control


125 120583gmL

25120583gmL









Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




SIb






Control


125 120583gmL

25120583gmL









Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control


125 120583gmL

25120583gmL









Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control


125 120583gmL

25120583gmL







Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control

Apoptotic cells ()

125 120583gmL0

102030405060


lowastlowast

lowast

lowast

250 120583gmL

(a)

Control

Necrotic cells ()

125 120583gmL01234567


lowast lowast

lowast

lowast

250 120583gmL

(b)

Control

Dead cells ()

125 120583gmL 25120583gmL0123456

87


lowast lowast

lowast

lowast

(c)



4 Conclusions





Acknowledgments


References























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Cytotoxic Constituents from the Rhizomes...

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Transcript of Research Article Cytotoxic Constituents from the Rhizomes...