research article isolation, characterization, crystal...

Research ArticleIsolation Characterization Crystal StructureElucidation and Anticancer Study of Dimethyl CardamoninIsolated from Syzygium campanulatum Korth

Abdul Hakeem Memon1 Zhari Ismail1 Abdalrahim F A Aisha1

Fouad Saleih Resq Al-Suede2 Mohammad Shahrul Ridzuan Hamil1

Suzana Hashim2 Mohammed Ali Ahmed Saeed1

Madeeha Laghari3 and Amin Malik Shah Abdul Majid2

1 Department of Pharmaceutical Chemistry School of Pharmaceutical Sciences Universiti Sains Malaysia11800 Minden Penang Malaysia

2 EMAN Research and Testing Laboratory School of Pharmaceutical Sciences Universiti Sains Malaysia11800 Minden Penang Malaysia

3 Department of Pharmaceutical Technology School of Pharmaceutical Sciences Universiti Sains Malaysia11800 Minden Penang Malaysia

Correspondence should be addressed to Zhari Ismail ismailzharigmailcom

Received 22 June 2014 Revised 2 August 2014 Accepted 14 August 2014 Published 28 October 2014

Academic Editor Muhammad Nabeel Ghayur

Copyright copy 2014 Abdul Hakeem Memon et alThis 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

Syzygium campanulatumKorth is an equatorial evergreen aboriginal shrub ofMalaysia Conventionally it has been used as a stom-achic However in the currently conducted study dimethyl cardamonin or 2101584041015840-dihydroxy-61015840-methoxy-3101584051015840-dimethylchalcone(DMC) was isolated from S campanulatum Korth leaf extract The structural characterization of DMC was carried out bymaking use of various techniques including UV IR NMR spectral followed by LC-MS and X-ray crystallographic techniquesFor determining the purity of compound highly effective techniques including TLC HPLC and melting point were used Thecytotoxicity of DMC and three different extracts of S campanulatum was evaluated against human colon cancer cell line (HT-29)by three different assays DMC and ethanolic extract revealed potent and dose-dependent cytotoxic activity on the cancer cell linewith IC

50126 and 901 120583gmL respectively Quite astonishingly to our knowledge this is the very first report on S campanulatum

as being a rich source (35) of DMC X-ray crystallography and anticancer activity on human colon cancer cells

1 Introduction

Chalcone is merely one of the many types of flavonoidphytochemical group Chalcones are assumed to be precur-sors of flavones in the biosynthesis of flavonoids Struc-turally chalcones possess 13-diaryl-2-propen-1-one moietyin which the two aryl groups are linked together by threecarbons (carbonyl and an 120572 120573-unsaturated system) [1]Chalcones are yellow-coloured compounds having numer-ous biological activities including cytotoxic [2] anticancer[3] anti-inflammatory antioxidant analgesic antibacterialantifungal and antiprotozoal [4] Chalcones reserves are

inexhaustible in all parts of edible plants Syzygium campanu-latumKorth (Myrtaceae) is a shrubby evergreen angiospermwhich is dicotyledonous tropical or subtropical and mon-soonal with a height ranging from 2 to 20m indigenousto Malaysia and Thailand It is a commonly found orna-mental tree planted along roads parks and public places Oncrushing its leaves produce a fragrance that is like that ofcinnamon It is known by sundry vernacular names suchas pokok kelat paya ubah laut (East Malaysia) Chinesered-wood (Chinese name) red lip wild cinnamon Aus-tralian brush cherry and kelat oil [5] Albeit traditionallybeing used as stomachic to date no pharmacognostical and

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 470179 11 pageshttpdxdoiorg1011552014470179

2 Evidence-Based Complementary and Alternative Medicine

phytochemical profiling is reported on S campanulatumexcept isolation of betulinic acid [6] The compound DMChas been isolated from C operculatus [7] M serrata [8]and S samarangense [9] DMC possessed hepatoprotective[10] cytoprotective [11] anti-inflammatory [12] antifungalantibacterial [8] anti-TB [13] antiviral [14] antispasmodic[15] antidiarrhoeal [16] antihyperglycaemic [17] anti-MDR[18 19] and antiapoptotic effect [20] DMC has been evalu-ated for its cytotoxic activity on various human cancer cellsincluding liver cancer (SMMC-7721s) HeLa (SPC-A-1 95-D and GBC-SD) [7] colon (SW-480) [9] leukemia (K562)[21] colorectal carcinoma (HCT-116 and LOVO) [22] breast(MDA-MB-453) and ovarian [23] cell lines

In the currently ongoing study for the first time DMCwas isolated from S campanulatum leaf extract at highpercentage yield The compound was characterized usingNMR and LC-MS and its cytotoxic activity was performedon human breast cancer cells Chemical structure of DMCwas further authenticated by X-ray crystallography Threedifferent extracts of S campanulatum were also subjectedto phytochemical profiling versus alkaloids tannins thetotal flavonoids polyphenols saponins polysaccharides andproteins content In addition to that the extracts were alsoscreened for antioxidant and cytotoxic activity in vitro Inpresent work DMC was evaluated for its potential antipro-liferative effect against human colon cancer cell lines Fur-thermore an attempt was made to understand the potency ofDMC cytotoxicity three different assays that is cell viabilityassay migration and clonogenicity assays were performedon selected human colon cancer cells

2 Materials and Methods

21 Plant Material The green leaves of S campanulatumKorth were collected in March 2013 from the main campusof University Sains Malaysia Penang Malaysia The plantwas authenticated by the Herbarium of School of BiologicalSciences USM where a voucher specimen was deposited(Ref number 11047)

22 Chemicals and Reagents RPMI 1640 medium trypsinand heat inactivated fetal bovine serum (HIFBS) were ob-tained fromGibco UK and 5-fluorouracil 99HPLC gradephosphate-buffered saline (PBS) penicillinstreptomycin(PS) solution 3-(45-dimethylthiazol-2-yl)-2-5-diphenyltet-razolium bromide (MTT) reagent and crystal violet werepurchased from Sigma-Aldrich USA

23 Cell Lines and Culture Conditions Human colon adeno-carcinoma cell line (HT-29 ATTC HTB-38) was purchasedfromATCC (Rockville MD USA)The cells were cultured inRPMI supplemented with 10 HIFBS and 1 PS Cells werecultured in a 5 CO

2in a humidified atmosphere at 37∘C

231 Extraction The green leaves were washed under tapwater and dried at 45∘C in oven for 3 days and ground tofine powder using electric grinder (Retsch Germany) The

powdered leaves (500 g) were separately extracted by vari-ous methods including soxhlet with 1 1 n-hexane-methanol(extract 1) and ethanol (extract 2) maceration with 1 1ethanol-water (extract 3) and refluxwithwater (extract 4) for24 h The extracts were filtered using Whatman filter papernumber 1 and the filtrates were evaporated to dryness at40∘C by using rotary evaporator (Buchi USA) The crudeextracts were kept for 12 h at 45∘C in oven to ensure completedryness Extract 1 was selected for isolation of DMC whilethe remaining three extracts were selected for evaluation ofcytotoxicity and phytochemical profile of S campanulatumplant Stock solutions at 10mgmL of extracts 2 3 and 4 wereprepared in 100 dimethyl sulfoxide (DMSO) and furtherdiluted with cell culture medium

232 Isolation of DMC The crude n-hexane-methanol (1 1)extract (20 g) of S campanulatum was ground into fine pow-der usingmortar and pestle Fine powdered crude extract wassubjected to flash column chromatography under vacuumusing column (10 times 7 cm) packed with silica gel with particlesize of 0063ndash0200mm 230ndash400mesh The mixture of n-hexane-ethyl acetate (250mL) by decreasing having hexaneratios 100 925 60 30 15 and 0 was used for obtaining 17fractions of the extract Each fractionwas collected separatelyin (500mL) conical flask and kept in fume hood at roomtemperature (25∘C) for evaporation for 3-4 days A yelloworange solid (38 g) appeared in fractions 7 and 8 and wascollected for further investigation

233 Crystallization Solid obtained from fractions 7 and 8was dissolved separately in n-hexane-ethyl acetate (15 1) andallowed to evaporate at room temperature (25∘C) Crystalsappeared with some impurities which were washed slowlyin pure n-hexane To obtain more pure crystals solidswere dissolved in n-hexane by adding 2 drops of methanolSolution was kept in fume hood to evaporate and fine yelloworange crystalswere obtainedThese crystalswere redissolvedin methanol (2mL) and allowed to stand 24 h at roomtemperature (25∘C) to obtain fine needle type orange crystalsPurity of crystals was analyzed by performing melting pointHPTLC HPLC UV IR NMR and X-ray crystallography

234 Characterization of DMC The purified orange yel-low crystals of DMC were characterized by FTIR spec-trophotometer (Perkin Elmer Spectrum one FTIR spectrom-eter USA) using potassium bromide (KBr) disc methodThe IR spectrum was scanned at infrared region of 400ndash4000 cmminus1 The sample was prepared in HPLC grademethanol (10mgmL) and was filtered through 045-micronfilter The spectrophotometer was controlled by computerwith UV WinLab 25 software Furthermore the purifiedDMC was analyzed by 2D-NMR spectra (FT-NMR spec-trometer Bruker 500MHz) in deuteratedmethanol (MeOD)The NMR peaks were labelled as singlet (s) doublet (d)triplet (t) and multiplet (m) chemical shifts were referencedwith respect to solvent signals The single crystals obtainedwere analysed by Bruker SMART APEX2 CCD area detector

Evidence-Based Complementary and Alternative Medicine 3

C18A

C4A

C3AC2A

C1A

C6AC7A

C8AC5A C9A

C10AC11A

C12A

C13AC14AC15A

C16AC17A

O1AO2A

O3A O4A

(a)

H-bonding

(b)

Figure 1 (a) Stereochemical structures of dimethyl cardamonin (b) Crystal packing of dimethyl cardamonin The molecules packed inmonoclinic crystal system through intermolecular hydrogen bonds shown as dashed lines

diffractometer (Figure 1) The molecular graphics were con-structed by Mercury 31 Development (Build RC5) software

Mass spectra (119899 = 5) of DMC were determined using amicrOTOF-Q ESI mass spectrometer (Waters) coupled withAgilent HPLC 1200 series USA The sample was prepared inMSgrademethanol and 2120583L samplewas injected directly intothe ES source at a flow rate of 5 120583Lmin The MS conditionswere column ACQUITY UPLC BEH C18 17120583 (WatersIreland) mobile phase solvents A and B doubled distilleddeionised water and acetonitrile respectively both with01 formic acid (100 120583L100mL) at a flow rate 03mLminpositive ion mode gas (N

2) temperature 350∘C flow rate

10 Lmin nebulizer pressure 15 psi sample infusion flowrate 20120583Lmin HV voltage 40 kV octopole RF amplitude150Vpp skim 1 voltage minus388 V skim 2 voltage minus60Vcap (KV) 3V sampling cone 40 extraction cone 4 andscan range mz 100ndash1000 units Acquired mass spectrarepresented the average of 119899 = 5 spectra

235 Quantification of DMC in Different Extracts of Scampanulatum HPLC analysis was performed using AgilentHPLC 1260 system on ZORBAX Eclipse Plus Phenyl-Hexylcolumn (46 times 250mm 5 microns) The mobile phaseconsisted of A (acetonitrile) and B (01 H

3PO4in water)

The elution programwas isocratic at 60 (A) and 40 (B) for20min at 1mLmin flow rate Sample injection volume was

10 120583L (500 ppm) methanolic DMC solution at 120582max 330 nmThe purity of DMC was assured from chromatogram andplant extracts were analyzed for the presence of DMC and theresults were calculated as ww

236 Phytochemical Analysis Preliminary tests for alka-loids tannins flavonoids glycosides phenols steroids andterpenoids on all three extracts of S campanulatum wereperformed as described in [24ndash26]The above phytochemicalgroups were found in all three extracts except water extract inwhich flavonoids steroids and terpenoids were not present

237 Estimation of Total Phenolics Flavonoids SaponinsPolysaccharides Proteins and Antioxidant Effect of Three Dif-ferent Extracts of S campanulatum Total phenolic contents(TPC) were estimated using a colorimetric assay [27] Twentymicroliters of extracts and gallic acid (GA) in methanol(5mgmL) were added to 158mL distilled water followed by100 120583L of Folin-Ciocalteau reagent and incubated for 8min inthe dark at RT Subsequently 300 120583L of sodium bicarbonate(20) was added and incubated in the dark at 30∘C for 2 hand absorbance was measured at 765 nm GA was used asa standard (003ndash5mgmL) and the results are expressedin Table 1 as ww GA equivalents using GA calibrationequation (119910 = 0000119909 + 0074 1198772 = 09997)


Table 1 Total phenols glycosaponins polysaccharides proteins and antioxidant values of different extracts of S campanulatum

S number Extract Phenols( plusmn SD)

Glycosaponins( plusmn SD)

Polysaccharides( plusmn SD)

Proteins( plusmn SD)

Flavonoids( plusmn SD)

IC50 DPPH(120583gmL plusmn SD)

1 Water 312 plusmn 09 87 plusmn 008 165 plusmn 02 253 plusmn 002 449 plusmn 081 240 plusmn 102 Ethanolic 314 plusmn 036 16 plusmn 038 068 plusmn 003 305 plusmn 004 688 plusmn 011 128 plusmn 0713 Ethanolic 50 392 plusmn 037 527 plusmn 018 104 plusmn 013 252 plusmn 001 366 plusmn 022 951 plusmn 097

Total flavonoids content (TFC) was determined usingquercetin as a standard [28] The standard and extracts(500120583L of 5mgmL) were added to 01mL 10 (wv) ofaluminium chloride 01mL of 1M potassium acetate 15mLof methanol and 28mL of water The reaction mixture wasincubated for 30min at RT and absorbance was taken at415 nm All samples were prepared in triplicate In blankpreparation distilled deionised water was used as constituentof 10 aluminium chloride Total concentration of TFC wascalculated as mg of quercetin (06ndash156mgmL) equivalent byusing quercetin calibration curve (119910 = 0008119909 + 0004 1198772 =0999)

DPPH scavenging activity was performed as described in[24] DPPH (075mL) at a final concentration of 0005 wvwas added to (025mL) three different plant extracts of 1times103to 1 times 10minus6 120583gmL The mixtures were incubated in the darkat 30∘C for 30min Subsequently absorbance was measuredat 517 nm and DPPH scavenging effect was calculated asfollows

DPPH scavenging effect = (1 minus (absorbance of samples-blank)(absorbance of negative control-blank))times 100 [6]Theresults are presented asmean percentage inhibitionplusmn SD (119899 =3)

Total glycosaponins (TGS) total polysaccharides (TPS)and total proteins were estimated from water ethanol andethanol 50 extracts of S campanulatum using methods asdescribed in [29] Results are summarized in Table 1

238 TLC Analysis of DMC in S campanulatum ExtractsTLC technique was performed to check the purity of isolatedDMC and presence of DMC in S campanulatum extractsTLC plates coated with 025mm silica gel 60 F254 (MerckGermany) were used The methanolic solution 10 120583L of1mgmL DMC and three plant extracts were applied onplate using microsyringe Plate was developed in saturatedchamber using n-hexane-ethyl acetate-formic acid (7 3 01)The plate was dried at 110∘C for 30min and examined at 256and 350 nm wavelengths

239 Colour Reactions of DMC DMC possesses carbonylgroup (C=O) of the chromogen (ArndashCOCH=CHndashAr) asan intermediate which can produce deeper colour duringthe reaction by a phenomenon known as ldquoHalochromyrdquo[30] The chemicals used for colour tests of DMC are FeCl

3

(alcoholic) H2SO4concentrated H

2SO4-HNO

3(mixture)

H2SO4-(CH3COO)

2O (mixture) NaBH

4-HCl SbCl

5 and

H3BO3-C6H8O7or Wilsonrsquos boric test

FeCl3 (Alcoholic) Colour Complex with DMC DMC (15mg)was dissolved in ethanol (1mL) followed by two drops of

freshly prepared alcoholic FeCl3solution Instantaneously

definite shades of colours (ie blue wine red blue blackviolet or green colours) were produced

H2SO4 (Concentrated) Colour ComplexwithDMCDMCpro-duced transient intense red colour carbonium ion complexwhen dissolved in H

2SO4(concentrated) DMC exhibits a

decent halochromic effect when wetted with concentratedH2SO4 The resonance must be considered as extended to the

benzene ring

H2SO4-HNO3 (Mixture) Colour Complex with DMC On theaddition of concentratedHNO

3to solution ofDMC inH

2SO4

(concentrated) no any little characteristic change in colourwas observed This is due to nitration of chalcone on carbon31015840 rather than oxidation and the resulting nitrochalcone Inthis test DMC has (ndashCH

3) group on 31015840-C so nitronium ion

complex with DMC is not possible

H2SO4-(CH3COO)2O (Mixture) Colour Complex with DMCSubstituted (ndashOCH

3and ndashOH) chalcones in CH

3COOH

solution yield deep colour (orange to purple) when treatedwith a drop or two of H

2SO4(concentrated) Two drops of

H2SO4(concentrated) added to (02 wv) acetic anhydride

solution of DMC (5mg) at 10ndash15∘C DMC changes fromorange to purple colour Due to the addition of acetic anhy-dride to the DMC in H

2SO4(concentrated) bathochromic

shift has been reorganized in terms of stability conferredon the carbonium ion (IIrarr III) by acetylation with aceticanhydride (Figure 2)

NaBH4-HCl (Mixture) Color Complex with DMC Na (orK) BH

4is a selective reagent for the reduction of carbonyl

(ndashCHO and C=O) group to the corresponding alcohol DMCwith NaBH

4-HCl (mixture) produced instantly red colour In

a 25mL Erlenmeyer flask NaBH4(33mg) was added in 1mL

of methanolic DMC (133mg) in small portions with swirlingthat the temperature did not exceed 45∘C The reaction isexothermic so NaBH

4(33mg) should be added slowly After

addition of NaBH4(33mg) mixture was boiled for 2min

The mixture was allowed to cool and red colour DMC ioncomplex was produced on addition of ethanolic-HClmixture(Figure 2)

SbCl5 Colour Complex with DMC DMC treated with SbCl5

in CCl4produced intense red or violet precipitates which

are characteristically different from the yellow or orangeprecipitates produced by flavonols flavones and flavanonesDMC (5mg) dissolved in 5mL of anhydrous CCl

4and 1mL

(2 anhydrous CCl4) solution of SbCl

5was added


O

O OH

OH O

O O

O

33

DMC

O

O

OH

OH

OH

OH

DMC

O O(ii)

(II)

Colour DMC-carbonium ion complex

O

O

O

O

DMC

OO

O

ODMC

H

O

O

(I)

(i)

(III)Purple colour DMC-acetic acid complex ion

O

ODMC 4

O4

O

Red colour DMC complex ion

4

HCl

O

ODMC

O

O

Red colour DMC-ion complex with antimony pentachloride

O

ODMC

O

O

Red colour DMC-ion complex with borocitric acid reagent

O

OHOH OH OH

OHOH

OH

OH

OH OHOHOHOH

OH OH OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

FeCl3 +3H + Fe

minus

minus

minus

minus

Browngreen color DMC-FeCl3-complex ion

+ 3HCl

3 minus

(1)

(2)

(3)

(4)

(5)

(6)

(7)

H2SO4 (conc)

NO2

CH3

(CH3COO)2O

NaBH4+NaCl +B(OH)3 + H2O

SbCl5SbCl5

ndashBNa

DMC (no color change because of ndashCH3 on carbon 3998400 positon

H3BO3ndashC6H8O7 H3BO3ndashC6H8O7

Figure 2 Schematic diagram of colour reactions of dimethyl cardamonin with different chemical reagents

H3BO3-C6H8O7 (Wilsonrsquos Boric Test) Colour Complex withDMC Chalcones with an ortho-(ndashOCH

3or ndashOH) group give

positive colour reaction with borocitric acid reagent [31]Two separate solutions were prepared in absolute acetone (a)boric acid (saturated solution) and (b) dry citric acid (05 gm)in absolute acetone (5mL) Citric acid was allowed in air (30ndash40∘C) for complete efflorescence and then heated in a thinlayer for 2 h at 100∘C DMC (5mg) was dissolved in 1mL

of dry acetone and then divided into two equal portionsIn the first portion 2mL of boric acid-citric acid-acetonereagent (solutions C andD) and in the second portion 05mLof solution D were added After few minutes the coloursof both portions were compared and stronger red colourin the first portion containing boric acid-citric acid reagentwas regarded as a positive reaction for 5-(ndashOH or ndashOCH

3)

chalcone (DMC)


2310 Cell Viability Assay Viability of the HT-29 cells wasdetermined by the MTT test as described by [32 33] Cells(70ndash80 confluency) were treated with various concentra-tions of extract andDMC Similarly 5-fluorouracil with serialconcentration is used as standard reference drug After 48 hincubation 20120583L of MTT solution (5mgmL in PBS) wasadded and incubated for an additional 3-4 h Subsequentlythe medium was aspirated carefully and 150120583L of dimethylsulfoxide (DMSO) was added After incubation for 15minthe optical density was measured at 570 nm using a high-endTecanM200Promultimodemicroplate readerDatawererecorded and analyzed for the assessment of the effects ofthe test substance on cell viability and growth inhibitionThe IC

50values were calculated using regression equation as

explained before [34]The results are presented as the averagepercentage viability to the negative control (1 DMSO) Thepercentage of cell viability was calculated using the followingformula cell viability = (absorbance of treatedabsorbanceof untreated) times 100

The percentage of inhibition was plotted against theconcentration in Microsoft excel and the IC

50was calculated

using the regression equation

2311 Migration Assay of HT-29 Cells The effect of DMC onthe migration of HT-29 cells was examined by the woundhealing assay HT-29 cells were maintained in 6-well plate inRPMI until 100 confluent monolayer growth was obtainedAs previously described [35] the monolayer was scratchedwith a sterile 200 120583L micropipette tip and then washed withPBS to remove the unattached cells and to smoothen theedges of the scratch After that 2mL of RPMI was added toeach well followed by addition of DMC at 10 and 20 120583gmLfinal concentrations 5-Fluorouracil (20120583gmL) and vehicle(1 DMSO) were added as positive and negative controlsrespectively Subsequently 6ndash8 microscopic fields per wellwere photographed at 0 12 and 24 h using an AMG EVOSfI digital microscope The width of the cell-free area wasmeasured using Leica Qwin software

The percentage of wound closure was then calculatedrelative to zero time using the following formula

wound closure = (1 minus (the width at the indicatedtimes (h)the width at zero time)) times 100The resultsare displayed as average plusmn SD (119899 = 6)

2312 Clonogenicity Assay The effect of DMC on HT-29colony formation was evaluated as described by [36] HT-29 cells in log phase growth were prepared in a single-cell suspension before being placed in a 6-well plate (500cellswell) The cells were incubated for 24 h to facilitatetheir attachment and treated with various concentrations ofethanol extract DMC 5-fluorouracil (3 120583gmL) as a positivecontrol and 1 DMSO as a negative control Followingadditional 48 h incubation treatments were removed andcells were incubated in a drug-free medium for an additional10 days until large colonies were produced Colonies werethen fixed with 4 paraformaldehyde for 30min and stainedwith 02 (wv) crystal violet for 20min The colonieswith gt50 cells were counted under a stereomicroscope and

the plating efficiency (PE) and survival fraction (SF) werecalculated Results are expressed as the mean plusmn SD (119899 = 3)

3 Results and Discussion

31 Extraction and Isolation of the Active Compound Driedgreen leaves of S campanulatum were extracted using soxh-let with n-hexane-methanol (1 1) and crude extract wasobtained The crude extract was subjected to flash columnchromatography using increasing concentration of ethylacetate in n-hexane starting with 100 n-hexane Seventeenfractions were obtained yellow solid mass of DMC appearedin fractions 7 and 8 (n-hexane-ethyl acetate 85 15) and wasrecrystallized using a mixture of methanol-n-hexane (1 9)

32 Spectroscopy DMC was obtained as orange yellowishneedle type crystals having MP 126∘C The UV-Vis spectraof DMC showed absorption at 120582max 332 nm indicating itschalcone characteristics [37] FTIR spectrum showed a strongand sharp vibrational band at 3401 cmminus1 that indicated thepresence of (ndashOH) group 2932 cmminus1 (CH

2) [38] 1623 cmminus1

(C=O) and 1545 cmminus1 (CH=CH) [39] The presence of alkylgroups was assigned by 2850 and 2939 cmminus1 two vibrationalbands [40]These prominent characteristic functional groupsindicated the presence of chalcone a class of compoundsbased on trans-13-diaryl-2-propen-1-ones backbone [41 42]DMC was also characterized by 1H and 13C NMR The13C DEPT-135 and 145 NMR spectra recorded in MeOD at12575MHz at 25∘C are shown in Figures S1 S2-A and S2-Brespectively (see the SupplementaryMaterial available onlineat httpdxdoiorg1011552014470179) The 2D-HSQC andHMBCNMRspectra ofDMCare shown in Figures S3 and S4respectively Supplementary Figures S5 and S6 illustrate thecharacteristic diagonal component and cross peaks of DMCfound in 2D-TOCSY and 2D-COSY NMR spectra respec-tively The NMR data of these spectra were also matchedwith previous reported NMR spectra [43 44] The molecularweight of DMC was determined by liquid chromatography-mass spectroscopy (LC-MS) one fragmented molecular ionpeak was observed at 195 because of cleavage of moleculeat 120572-unsaturated carbon as the main fragmentation and onepeak at 299 as the main compound shown in supplementaryFigures S7-A and B

33 Crystallography DMCsingle crystal was found appropri-ate for X-ray crystallographic study The crystals appeared asorange yellow needle type structure A perspective view of thecrystal structure is illustrated in Figure 1 DMC intramolecu-lar geometry was analyzed using monoclinic space group P21(number 4) a knowledge base molecular geometry obtainedfrom Cambridge Structural Database Each unit of DMCconsists of two benzene rings one carbonyl carbon and 120572-120573-unsaturated carbons In addition two hydroxyl methyland one methoxy groups are attached on one benzene ringCrystal data and structure refinement details of DMC are asfollows formula C18 H18 O4 formula weight 29832 crystalsystem monoclinic space group P21 (number 4) a 108917


020406080

100120140160180200

DMC Waterextract

Ethanolic waterextract

Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)

Figure 3 (a) Cell viability () plusmn SD (119899 = 3) tested on DMC and three different extracts of S campanulatum at two selected concentrations(b) Dose-dependent antiproliferative effect of S campanulatum ethanolic extract and DMC on HT-29 cell lines was assessed by MTT assay

(4) b 246366 (10) and c 114428 (5) (A) respectively andalpha 90∘ beta 99387∘ (2) gamma 90∘ V (A3) 30294 (2)Z 8 D (calc) 1308 gcm3 abs coeff Mu (CuKa) 0752mmF (000) 1264 crystal size 009times014times052mm temperature100K radiation Cu Ka 154178 A 120579 min max 39 693∘dataset minus13 13 minus29 29 minus13 13 tot uniq data 28817 R(int) 0045 observed data [119868 gt 20 sigma (119868)] 9904 8774Nref Npar 9904 806 and R wR2 S 00851 02586 103Theselected bond lengths and angles ofDMCcrystallographicallyare shown in Figure 1 and are listed in supplementary data 1The crystal packing (Figure 1) shows that the DMCmoleculesare connected in such a way they form a crisscross chain byCndashHsdot sdot sdot 120587(arene)sdot sdot sdotH bonds along the 119888 axis There are alsointermolecular hydrogen bonding interactions in which C-11(donor for H-bond) via H-11ndashO in neighboring molecules at119909 1 minus 119910 1 minus 119911 to produce a three-dimensional network [45]In chalcones dihedral angle between the two benzene ringsis 130(1)∘ and the dihedral angle from the plane of C7ndashC9 tothe benzene rings (C1ndashC6) is 138(1)∘ and (C10ndashC15) is 26(1)∘This indicates that the central C7ndashC9 portion lies nearly inthe benzene ring plane of C10ndashC15 but is quite displaced outof the other benzene ring of C1ndashC6 [42]

34 Chromatography The purity of DMC found to be 98using HPLC by injecting 10 120583L (500 ppm) methanolic DMCsolution was checked by performing HPLC analysis usingisocratic water-acetonitrile (4 6) solvent system DMC wasdetected at a 120582max of 330 nm with a retention time of 99minand 98 purity (Supplementary Figure S8)

35 Chemical Reactions Characterization of different func-tional groups that is ndashOH ndashCH

3 ndashOCH

3 gtC=O and

CH=CH present in DMC molecule was also confirmed byperforming various colour reactions for chalcones Figure 2

indicates that the DMC forms colour complex ions (CCI)and is characterised by strong halochromism due to the factthat the (gtC=O) group activated by the (ndashCH=CHndash) declaresthe presence of the (ndashCOCH=CHndash) group linked with twophenyl rings

Different colour complex ions (CCI) of DMC indicatedthat alc FeCl

3produce CCI only with (ndashOH) chalcones

while NaBH4-HCl and Ac

2O-H2SO4reagents indicate that

the chromogen involved in formation CCI of chalcone is(ArndashCOndashCH=CHndashAr) This reaction involves the attack by(ndashC+=O) at the carbonyl carbon of the chalcones chromogenproducing (ndashC+ndash(OAc)ndashCH=CHndash) CCI [31] The H

2SO4-

concentrated HNO3reagent did not produce CCI because

in DMC molecule 31015840-C is not free and occupied by (ndashCH3)

group

36 Inhibition of HT-29 Proliferation by Ethanolic Extract ofS campanulatum and Isolated DMC TheMTT cell prolifer-ation assay was executed with great care in order to study theeffect of three different extracts andDMC isolated compoundfrom the leaves of S campanulatum on HT-29 cell line Thescreening on ethanolic water and 50 ethanol extracts of Scampanulatum and DMC compound isolated from S cam-panulatum leaves at two selected concentrations that were50 120583gmL and 100 120583gmL confirmed that ethanolic extractand DMC have strong cytotoxic effect against HT-29 cells ascompared to water and 50 ethanolic extract (Figure 3(a))In correspondence to Figure 3(b) both ethanolic extract ofS campanulatum and DMC inhibited the proliferation onHT-29 in dose-dependent manner The median inhibitoryconcentrations (IC

50s) for ethanolic extract of S campan-ulatum and DMC were then calculated authentically andwith the accurate precision from the dose response curvesand were found to be 90 and 126 120583gmL respectively For


Figure 4 Migration assay of HT-29 cells treated with different concentrations of DMC and ethanolic extract of S campanulatum at 0 12 and48 h

further advancement and accuracy 5-fluorouracil a standardtherapeutic drug for cancer was used as a positive controland it showed significant toxicity towards HT-29 cells withan IC

50of 276120583gmL In comparison to previous study in

which hexane and methanolic extracts of S campanulatumwere reported active against HCT-116 HT-29 MCF-7 T47Dand rat hepatoma cells using cell-enzyme based in vitroassay [46] unlikely in present study ethanolic extract of Scampanulatum and pure DMC are studied for the first timeagainst HT-29 cell line using three different assays

37 DMC Inhibited Migration of HT-29 Cells Wound healingassay was used to examine the effects of DMC and ethanolicextract of S campanulatum on the migration of HT-29 cellsFor improved results this was executed on the experimentallines the percentage of inhibition of wound closure in thepresence and absence of DMC was calculated after every12 and 48 h relative to the zero time The results of thepercentage of inhibition of wound healing assay are asdepicted in Figure 4 The results sharply outlined the factthat the percentage of wound closure after 48 h was almost


00

200

400

600

800

1000

12h 24hTime (hours)

5-Fu 3120583gmLDMC 3120583gmLDMC 6120583gmL

SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60

Concentration (120583gmL)

(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)

Figure 5 (a) The inhibitory effects of DMC and ethanolic extract of S campanulatum on the migration property of HT-29 cells whichis a characteristic of metastasis of the cancer cells (b) Clonogenic cell survival () of HT-29 treated with 1 DMSO as a negative control3 120583gmL of 5-fluorouracil as positive control and indicated concentrations of S campanulatum ethanolic extract andDMC (c)The percentageof surviving fraction obtained after the treatment with DMC and ethanolic extract of S campanulatum The percentage of survivingfraction of the HT-29 colonies was decreased with increasing concentration of DMC and ethanolic extract S campanulatum mean plusmn SD(119899 = either 6 or 10)

100 in the untreated cells whereas in treated group thewound remained open even after 48 h period time Thepercentage of inhibition of migration of HT-29 cancer cells isshown in Figure 5(a) DMC (3 120583gmL) at every 12 and 48 hinhibited themigration ofHT-29 cells up to 60 and 75while6 120583gmL concentration of DMC inhibited cell migration at 80and 90 at the same duration of 12 and 48 h respectivelyThe ethanolic extract of S campanulatum after 12 and 48 hat 30 and 60120583gmL inhibited the HT-29 migration 30ndash60and 50ndash90 respectively The positive control 5-fluorouracil(3 120583gmL) showed 40 and 80 inhibition inmigration ofHT-29 cells after 12 and 48 h respectively

38 DMC Obstructed HT-29 Colony Formation Treatmentof HT-29 cells with DMC in the concentration range of3ndash6 120583gmL caused dose-dependent inhibition of the HT-29 clonogenicity The cell survival () graph for DMCis depicted in Figure 5(b) At concentrations of 3 120583gmLDMC inhibited the colony formation of HT-29 At 6120583gmLconcentrations DMC significantly inhibited the formation ofcolonies the survival fraction (SF) at 6 and 3 120583gmL of DMCwas 15 plusmn 48 and 21 plusmn 26 and 25 plusmn 44 70 plusmn 40 afterethanol extract treatment at the concentration of 60 120583gmLand 30 120583gmL respectively At high concentration ethanolicextract showed cytotoxic effect while at low concentration it


was cytostatic as evident by decreased survival fraction (SF)These results can be compared with the standard reference5-fluorouracil at 3 120583gmL that showed 20 survival fraction(Figure 5(c))

In the last 10 years about ninety chalcones with antitumoractivities were reported [1] through induction of apoptosiscell signal transduction redifferentiation and induction ofapoptosis under hypoxia [1 21 22 47] However in thepresent study the cell migration study was conducted torepresent important step in metastasis of cancer cells Theresult is shown as percentage of inhibition of migrating cellsrelative to negative control Significant reduction in HT-29cell mobility was achieved 100 after 12 h at 6120583gmL forDMC and for ethanolic extract of S campanulatum after 48 hat 60120583gmL of treatment 984 relative to negative control

4 Conclusions

In the present study the isolation of DMC from driedleaves of S campanulatum with its purification and detailedchemical structural characterization and anticancer activityis reported It has been observed that dimethyl cardamonin(DMC) and ethanolic extract of S campanulatum possessstrong cytotoxic activity against human colon cancer cellsFurther in vivo experiments will then be conducted toevaluate the potential of DMC and ethanolic extract of Scampanulatum for the treatment of colon cancer in animalsin order to include DMC and ethanolic extract in the clinicaltrials

Disclosure

X-ray crystallographic data of the structures have beendeposited with the Cambridge Crystallographic Data CenterCCDC 971050 for dimethyl cardamonin This data can beobtained free of charge fromCCDC via httpwwwccdccamacukdata requestcif

Conflict of Interests

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

Acknowledgments

The author would like to thank Universiti Sains Malaysia(USM) andMinistry ofAgriculture andAgro-Based IndustryMalaysia (Grant no 304PFarmasi650582K123) for finan-cial support of this research work

References

[1] E H Zhang R F Wang S Z Guo and B Liu ldquoAn update onantitumor activity of naturally occurring chalconesrdquo Evidence-Based Complementary and Alternative Medicine vol 2013Article ID 815621 22 pages 2013

[2] C C Yit and N P Das ldquoCytotoxic effect of butein on humancolon adenocarcinoma cell proliferationrdquo Cancer Letters vol82 no 1 pp 65ndash72 1994

[3] L W Wattenberg J B Coccia and A R Galbraith ldquoInhibitionof carcinogen-induced pulmonary and mammary carcino-genesis by chalcone administered subsequent to carcinogenexposurerdquo Cancer Letters vol 83 no 1-2 pp 165ndash169 1994

[4] O A Oyedapo C O Adewunmi E O Iwalewa and VO Makanju ldquoAnalgesic antioxidant and anti-inflammatoryrelated activities of 21-hydroxy-241-dimethoxychalcone and 4-hydroxychalcone in micerdquo Journal of Biological Sciences vol 8no 1 pp 131ndash136 2008

[5] A N M Roseli T F Ying and M F Ramlan ldquoGrowthinhibition of syzygium campanulatum korth for containerplanting by the application of uniconazolerdquo Pertanika Journalof Tropical Agricultural Science vol 33 no 1 pp 1ndash6 2010

[6] A F A Aisha Z Ismail K M Abu-Salah J M Siddiqui GGhafar and A M S Abdul Majid ldquoSyzygium campanulatumkorth methanolic extract inhibits angiogenesis and tumorgrowth in nude micerdquo BMC Complementary and AlternativeMedicine vol 13 article 168 2013

[7] C-L Ye J-W Liu D-Z Wei Y-H Lu and F Qian ldquoInvitro anti-tumor activity of 2101584041015840-dihydroxy-61015840- methoxy-3101584051015840-dimethylchalcone against six established human cancer celllinesrdquo Pharmacological Research vol 50 no 5 pp 505ndash5102004

[8] S Gafner J-L Wolfender S Mavi and K HostettmannldquoAntifungal and antibacterial chalcones from Myrica serratardquoPlanta Medica vol 62 no 1 pp 67ndash69 1996

[9] M J Simirgiotis S Adachi S To et al ldquoCytotoxic chalcones andantioxidants from the fruits of Syzygium samarangense (WaxJambu)rdquo Food Chemistry vol 107 no 2 pp 813ndash819 2008

[10] W-G Yu J Qian and Y-H Lu ldquoHepatoprotective effects of2101584041015840-dihydroxy-61015840-methoxy- 3101584051015840-dimethylchalcone on CCl

4-

induced acute liver injury in micerdquo Journal of Agricultural andFood Chemistry vol 59 no 24 pp 12821ndash12829 2011

[11] M-Y Su H-Y Huang L Li and Y-H Lu ldquoProtective effects of2101584041015840-dihydroxy-61015840-methoxy- 3101584051015840-dimethylchalcone to PC12cells against cytotoxicity induced by hydrogen peroxiderdquo Jour-nal of Agricultural and Food Chemistry vol 59 no 2 pp 521ndash527 2011

[12] Y-J Kim H Ko J-S Park et al ldquoDimethyl cardamonininhibits lipopolysaccharide-induced inflammatory factorsthrough blocking NF-120581B p65 activationrdquo InternationalImmunopharmacology vol 10 no 9 pp 1127ndash1134 2010

[13] R L Pavan C Q F Leite R G Coelho et al ldquoEvaluationof anti-mycobacterium tuberculosis activity of campomanesiaadamantium (myrtaceae)rdquo Quımica Nova vol 32 no 5 pp1222ndash1226 2009

[14] B H Havsteen ldquoThe biochemistry and medical significance ofthe flavonoidsrdquo Pharmacology amp Therapeutics vol 96 no 2-3pp 67ndash202 2002

[15] E C Amor I M Villasenor M N Ghayur A H Gilaniand M I Choudhary ldquoSpasmolytic flavonoids from Syzygiumsamarangense (Blume) Merr amp LM Perryrdquo Zeitschrift furNaturforschung C vol 60 no 1-2 pp 67ndash71 2005

[16] M N Ghayur A H Gilani A Khan E C Amor I MVillasenor and M I Choudhary ldquoPresence of calcium antag-onist activity explains the use of Syzygium samarangense indiarrhoeardquo Phytotherapy Research vol 20 no 1 pp 49ndash522006

[17] S Shahreen J Banik A Hafiz et al ldquoAntihyperglycemic activi-ties of leaves of three edible fruit plants (Averrhoa carambolaFicus hispida and Syzygium samarangense) of Bangladeshrdquo


African Journal of Traditional Complementary and AlternativeMedicines vol 9 no 2 pp 287ndash291 2011

[18] H-Y Huang J-L Niu L-M Zhao and Y-H Lu ldquoReversaleffect of 2101584041015840-dihydroxy-61015840-methoxy-31015840 51015840-dimethylchalconeon multi-drug resistance in resistant human hepatocellularcarcinoma cell line BEL-74025-FUrdquo Phytomedicine vol 18 no12 pp 1086ndash1092 2011

[19] H-Y Huang J-L Niu and Y-H Lu ldquoMultidrug resistancereversal effect of DMC derived from buds of Cleistocalyxoperculatus in human hepatocellular tumor xenograft modelrdquoJournal of the Science of Food and Agriculture vol 92 no 1 pp135ndash140 2012

[20] Y Luo and Y Lu ldquo2101584041015840-Dihydroxy-61015840-methoxy-3101584051015840-dimethylchalcone inhibits apoptosis of MIN6 cells viaimproving mitochondrial functionrdquo Pharmazie vol 67 no 9pp 798ndash803 2012

[21] C-L Ye F Qian D-Z Wei Y-H Lu and J-W Liu ldquoInductionof apoptosis in K562 human leukemia cells by 2101584041015840- dihydroxy-61015840-methoxy-3101584051015840-dimethylchalconerdquo Leukemia Research vol29 no 8 pp 887ndash892 2005

[22] H Ko Y-J Kim E C Amor et al ldquoInduction of autophagy bydimethyl cardamonin is associated with proliferative arrest inhuman colorectal carcinoma HCT116 and LOVO cellsrdquo Journalof Cellular Biochemistry vol 112 no 9 pp 2471ndash2479 2011

[23] D-D Li X-Q Wu J Tang X-Y Wei and X-F Zhu ldquoON-III inhibits erbB-2 tyrosine kinase receptor signal pathway andtriggers apoptosis through induction of Bim in breast cancercellsrdquo Cancer Biology and Therapy vol 8 no 8 pp 739ndash7432009

[24] O P Sharma andT K Bhat ldquoDPPHantioxidant assay revisitedrdquoFood Chemistry vol 113 no 4 pp 1202ndash1205 2009

[25] K Mehta B N Patel and B K Jain ldquoPhytochemical analysis ofleaf extract of Phyllanthus fraternusrdquo Research Journal of RecentSciences vol 2 pp 12ndash15 2013

[26] A Imtiyaz T Jagrati S Manik U Lone and J Rabia ldquoPre-liminary phytochemical studies of the miracle herb of thecentury Nigella sativa L (Black Seed)rdquo Indo American Journalof Pharmaceutical Research vol 3 no 4 pp 3000ndash3007 2013

[27] J C Argoti S Salido P J Linares-Palomino B Ramırez BInsuasty and J Altarejos ldquoAntioxidant activity and free radical-scavenging capacity of a selection of wild-growing Colombianplantsrdquo Journal of the Science of Food andAgriculture vol 91 no13 pp 2399ndash2406 2011

[28] I Kosalec M Bakmaz S Pepeljnjak and S Vladimir-KnezevicldquoQuantitative analysis of the flavonoids in raw propolis fromnorthern CroatiardquoActa Pharmaceutica vol 54 no 1 pp 65ndash722004

[29] K Hussain Z Ismail A Sadikun and P Ibrahim ldquoAnal-ysis of proteins polysaccharides glycosaponins contents ofPiper sarmentosum Roxb and anti-TB evaluation for bio-enhancinginteraction effects of leaf extracts with Isoniazid(INH)rdquo Natural Product Radiance vol 7 no 5 pp 402ndash4082008

[30] P H Hermans Theoritical Organic Chemistry Elsevier NewYork NY USA 1st edition 1954

[31] B Sharma S C Agrawal and K C Gupta ldquoColour reactions ofchalcones and their mechanism (a review)rdquo Oriental Journal ofChemistry vol 24 no 1 pp 289ndash294 2008

[32] 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

[33] M B K Ahamed A F A Aisha Z D Nassar et al ldquoCatrsquoswhiskers tea (orthosiphon stamineus) extract inhibits growthof colon tumor in nude mice and angiogenesis in endothelialcells via suppressing VEGFR phosphorylationrdquo Nutrition andCancer vol 64 no 1 pp 89ndash99 2012

[34] O S A Al-Salahi C Kit-LamAM S AbdulMajid et al ldquoAnti-angiogenic quassinoid-rich fraction from Eurycoma longifoliamodulates endothelial cell functionrdquo Microvascular Researchvol 90 pp 30ndash39 2013

[35] H M Baharetha Z D Nassar A F Aisha et al ldquoProapoptoticand antimetastatic properties of supercritical CO2 extract ofNigella sativa Linn against breast cancer cellsrdquo Journal ofMedicinal Food vol 16 pp 1121ndash1130 2013

[36] Z D Nassar A F A Aisha F S R Al Suede A S A Majid andA M S A Majid ldquoIn vitro antimetastatic activity of koetjapicacid against breast cancer cellsrdquo Biological and PharmaceuticalBulletin vol 35 no 4 pp 503ndash508 2012

[37] S V Tsukerman V D Orlov and V F Lavrushin ldquoUltra-violet absorption spectra of chalcone analogs containing aselenophene nucleusrdquo Chemistry of Heterocyclic Compoundsvol 5 no 1 pp 55ndash57 1971

[38] D E Okwu and N Ukanwa ldquoIsolation and characterizationof flavonoids chalcones and anthocynidines from Brideliaferruginea benthrdquo Der Chemica Sinica vol 1 no 2 pp 21ndash282010

[39] A N Vanita S B Zangade R S Shinde and S G PatilldquoSynthesis of some new chalcones flavones and screening fortheir antimicrobial activityrdquo Der Pharmacia Lettre vol 2 no 5pp 245ndash250 2010

[40] M A Iqbal R A Haque M B K Ahamed A M S A Majidand S S Al-Rawi ldquoSynthesis and anticancer activity of para-xylyl linked bis-benzimidazolium salts and respective Ag(I) N-heterocyclic carbene complexesrdquoMedicinal Chemistry Researchvol 22 no 5 pp 2455ndash2466 2013

[41] L F Motta A C Gaudio and Y Takahata ldquoQuantitativestructure-activity relationships of a series of chalcone deriva-tives (1 3-diphenyl-2-propen-1-one) as anti-Plasmodium falci-parum agents (antimalaria agents)rdquo Internet Electronic Journalof Molecular Design vol 5 pp 555ndash569 2006

[42] M A Rahman ldquoChalcone a valuable insight into the recentadvances and potential pharmacological activitiesrdquo ChemicalSciences pp 1ndash16 2011

[43] F R Pavan C Q F Leite R G Coelho et al ldquoEvaluationof anti-mycobacterium tuberculosis activity of Campomanesiaadamantium (Myrtaceae)rdquo Quımica Nova vol 32 no 5 pp1222ndash1226 2009

[44] J K Adesanwo F O Shode O Aiyelaagbe R T Oyede andH Baijnath ldquoIsolation and characterization of a new chalconefrom the leaves of Heteropyxis natalensisrdquo International Journalof Medicine and Medical Sciences vol 1 no 2 pp 28ndash32 2009

[45] M-H Wu X-H Yang W-D Zou W-J Liu and C LildquoRefinement of the crystal structure of (E)-13-diphenyl-2-propen-1-one C15H12Ordquo Zeitschrift fur Kristallographie vol221 no 3 pp 323ndash324 2006

[46] A F A Aisha K M Abu-Salah Y Darwis and A M SAbdul Majid ldquoScreening of antiangiogenic activity of sometropical plants by rat aorta ring assayrdquo International Journal ofPharmacology vol 5 no 6 pp 370ndash376 2009

[47] Y Meng J Zhong and H Sun ldquoEffects of Ashitaba chalcone oncaspase-3 and Bax protein expressions of mouse hepatocarci-noma cellsrdquo CarcinogenesisTeratogenesis and Mut agenesis vol23 no 1 pp 50ndash53 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


phytochemical profiling is reported on S campanulatumexcept isolation of betulinic acid [6] The compound DMChas been isolated from C operculatus [7] M serrata [8]and S samarangense [9] DMC possessed hepatoprotective[10] cytoprotective [11] anti-inflammatory [12] antifungalantibacterial [8] anti-TB [13] antiviral [14] antispasmodic[15] antidiarrhoeal [16] antihyperglycaemic [17] anti-MDR[18 19] and antiapoptotic effect [20] DMC has been evalu-ated for its cytotoxic activity on various human cancer cellsincluding liver cancer (SMMC-7721s) HeLa (SPC-A-1 95-D and GBC-SD) [7] colon (SW-480) [9] leukemia (K562)[21] colorectal carcinoma (HCT-116 and LOVO) [22] breast(MDA-MB-453) and ovarian [23] cell lines

In the currently ongoing study for the first time DMCwas isolated from S campanulatum leaf extract at highpercentage yield The compound was characterized usingNMR and LC-MS and its cytotoxic activity was performedon human breast cancer cells Chemical structure of DMCwas further authenticated by X-ray crystallography Threedifferent extracts of S campanulatum were also subjectedto phytochemical profiling versus alkaloids tannins thetotal flavonoids polyphenols saponins polysaccharides andproteins content In addition to that the extracts were alsoscreened for antioxidant and cytotoxic activity in vitro Inpresent work DMC was evaluated for its potential antipro-liferative effect against human colon cancer cell lines Fur-thermore an attempt was made to understand the potency ofDMC cytotoxicity three different assays that is cell viabilityassay migration and clonogenicity assays were performedon selected human colon cancer cells

2 Materials and Methods

21 Plant Material The green leaves of S campanulatumKorth were collected in March 2013 from the main campusof University Sains Malaysia Penang Malaysia The plantwas authenticated by the Herbarium of School of BiologicalSciences USM where a voucher specimen was deposited(Ref number 11047)

22 Chemicals and Reagents RPMI 1640 medium trypsinand heat inactivated fetal bovine serum (HIFBS) were ob-tained fromGibco UK and 5-fluorouracil 99HPLC gradephosphate-buffered saline (PBS) penicillinstreptomycin(PS) solution 3-(45-dimethylthiazol-2-yl)-2-5-diphenyltet-razolium bromide (MTT) reagent and crystal violet werepurchased from Sigma-Aldrich USA

23 Cell Lines and Culture Conditions Human colon adeno-carcinoma cell line (HT-29 ATTC HTB-38) was purchasedfromATCC (Rockville MD USA)The cells were cultured inRPMI supplemented with 10 HIFBS and 1 PS Cells werecultured in a 5 CO

2in a humidified atmosphere at 37∘C

231 Extraction The green leaves were washed under tapwater and dried at 45∘C in oven for 3 days and ground tofine powder using electric grinder (Retsch Germany) The

powdered leaves (500 g) were separately extracted by vari-ous methods including soxhlet with 1 1 n-hexane-methanol(extract 1) and ethanol (extract 2) maceration with 1 1ethanol-water (extract 3) and refluxwithwater (extract 4) for24 h The extracts were filtered using Whatman filter papernumber 1 and the filtrates were evaporated to dryness at40∘C by using rotary evaporator (Buchi USA) The crudeextracts were kept for 12 h at 45∘C in oven to ensure completedryness Extract 1 was selected for isolation of DMC whilethe remaining three extracts were selected for evaluation ofcytotoxicity and phytochemical profile of S campanulatumplant Stock solutions at 10mgmL of extracts 2 3 and 4 wereprepared in 100 dimethyl sulfoxide (DMSO) and furtherdiluted with cell culture medium

232 Isolation of DMC The crude n-hexane-methanol (1 1)extract (20 g) of S campanulatum was ground into fine pow-der usingmortar and pestle Fine powdered crude extract wassubjected to flash column chromatography under vacuumusing column (10 times 7 cm) packed with silica gel with particlesize of 0063ndash0200mm 230ndash400mesh The mixture of n-hexane-ethyl acetate (250mL) by decreasing having hexaneratios 100 925 60 30 15 and 0 was used for obtaining 17fractions of the extract Each fractionwas collected separatelyin (500mL) conical flask and kept in fume hood at roomtemperature (25∘C) for evaporation for 3-4 days A yelloworange solid (38 g) appeared in fractions 7 and 8 and wascollected for further investigation

233 Crystallization Solid obtained from fractions 7 and 8was dissolved separately in n-hexane-ethyl acetate (15 1) andallowed to evaporate at room temperature (25∘C) Crystalsappeared with some impurities which were washed slowlyin pure n-hexane To obtain more pure crystals solidswere dissolved in n-hexane by adding 2 drops of methanolSolution was kept in fume hood to evaporate and fine yelloworange crystalswere obtainedThese crystalswere redissolvedin methanol (2mL) and allowed to stand 24 h at roomtemperature (25∘C) to obtain fine needle type orange crystalsPurity of crystals was analyzed by performing melting pointHPTLC HPLC UV IR NMR and X-ray crystallography

234 Characterization of DMC The purified orange yel-low crystals of DMC were characterized by FTIR spec-trophotometer (Perkin Elmer Spectrum one FTIR spectrom-eter USA) using potassium bromide (KBr) disc methodThe IR spectrum was scanned at infrared region of 400ndash4000 cmminus1 The sample was prepared in HPLC grademethanol (10mgmL) and was filtered through 045-micronfilter The spectrophotometer was controlled by computerwith UV WinLab 25 software Furthermore the purifiedDMC was analyzed by 2D-NMR spectra (FT-NMR spec-trometer Bruker 500MHz) in deuteratedmethanol (MeOD)The NMR peaks were labelled as singlet (s) doublet (d)triplet (t) and multiplet (m) chemical shifts were referencedwith respect to solvent signals The single crystals obtainedwere analysed by Bruker SMART APEX2 CCD area detector


C18A

C4A

C3AC2A

C1A

C6AC7A

C8AC5A C9A

C10AC11A

C12A

C13AC14AC15A

C16AC17A

O1AO2A

O3A O4A

(a)

H-bonding

(b)







3PO4in water)




















3


2SO4-HNO

3(mixture)

H2SO4-(CH3COO)

2O (mixture) NaBH

4-HCl SbCl

5 and








benzene ring



2SO4






3COOH



















4and 1mL


5was added


O

O OH

OH O

O O

O

33

DMC

O

O

OH

OH

OH

OH

DMC

O O(ii)

(II)


O

O

O

O

DMC

OO

O

ODMC

H

O

O

(I)

(i)


O

ODMC 4

O4

O


4

HCl

O

ODMC

O

O


O

ODMC

O

O


O

OHOH OH OH

OHOH

OH

OH

OH OHOHOHOH

OH OH OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

FeCl3 +3H + Fe

minus

minus

minus

minus


+ 3HCl

3 minus

(1)

(2)

(3)

(4)

(5)

(6)

(7)

H2SO4 (conc)

NO2

CH3

(CH3COO)2O


SbCl5SbCl5

ndashBNa








3)

chalcone (DMC)






50was calculated










2) [38] 1623 cmminus1




020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C18A

C4A

C3AC2A

C1A

C6AC7A

C8AC5A C9A

C10AC11A

C12A

C13AC14AC15A

C16AC17A

O1AO2A

O3A O4A

(a)

H-bonding

(b)







3PO4in water)




















3


2SO4-HNO

3(mixture)

H2SO4-(CH3COO)

2O (mixture) NaBH

4-HCl SbCl

5 and








benzene ring



2SO4






3COOH



















4and 1mL


5was added


O

O OH

OH O

O O

O

33

DMC

O

O

OH

OH

OH

OH

DMC

O O(ii)

(II)


O

O

O

O

DMC

OO

O

ODMC

H

O

O

(I)

(i)


O

ODMC 4

O4

O


4

HCl

O

ODMC

O

O


O

ODMC

O

O


O

OHOH OH OH

OHOH

OH

OH

OH OHOHOHOH

OH OH OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

FeCl3 +3H + Fe

minus

minus

minus

minus


+ 3HCl

3 minus

(1)

(2)

(3)

(4)

(5)

(6)

(7)

H2SO4 (conc)

NO2

CH3

(CH3COO)2O


SbCl5SbCl5

ndashBNa








3)

chalcone (DMC)






50was calculated










2) [38] 1623 cmminus1




020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3


2SO4-HNO

3(mixture)

H2SO4-(CH3COO)

2O (mixture) NaBH

4-HCl SbCl

5 and








benzene ring



2SO4






3COOH



















4and 1mL


5was added


O

O OH

OH O

O O

O

33

DMC

O

O

OH

OH

OH

OH

DMC

O O(ii)

(II)


O

O

O

O

DMC

OO

O

ODMC

H

O

O

(I)

(i)


O

ODMC 4

O4

O


4

HCl

O

ODMC

O

O


O

ODMC

O

O


O

OHOH OH OH

OHOH

OH

OH

OH OHOHOHOH

OH OH OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

FeCl3 +3H + Fe

minus

minus

minus

minus


+ 3HCl

3 minus

(1)

(2)

(3)

(4)

(5)

(6)

(7)

H2SO4 (conc)

NO2

CH3

(CH3COO)2O


SbCl5SbCl5

ndashBNa








3)

chalcone (DMC)






50was calculated










2) [38] 1623 cmminus1




020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O OH

OH O

O O

O

33

DMC

O

O

OH

OH

OH

OH

DMC

O O(ii)

(II)


O

O

O

O

DMC

OO

O

ODMC

H

O

O

(I)

(i)


O

ODMC 4

O4

O


4

HCl

O

ODMC

O

O


O

ODMC

O

O


O

OHOH OH OH

OHOH

OH

OH

OH OHOHOHOH

OH OH OH

OH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

FeCl3 +3H + Fe

minus

minus

minus

minus


+ 3HCl

3 minus

(1)

(2)

(3)

(4)

(5)

(6)

(7)

H2SO4 (conc)

NO2

CH3

(CH3COO)2O


SbCl5SbCl5

ndashBNa








3)

chalcone (DMC)






50was calculated










2) [38] 1623 cmminus1




020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















50was calculated










2) [38] 1623 cmminus1




020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















020406080

100120140160180200

DMC Waterextract


Cel

l via

bilit

y (

)

50120583gmL100120583gmL

Ethanolicextract

(a) (b)





3 ndashOCH

3 gtC=O and








2SO4-



group










00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























00

200

400

600

800

1000

12h 24hTime (hours)


SE 30120583gmLSE 60120583gmL

mig

ratio

n (

)In

hibi

tion

of ce

ll

(a)

0

20

40

60

80

100

120

Negative

Cel

l sur

viva

l (

)

5-Fu3

DMC3

DMC6

SE30

SE60


(b)

0

20

40

60

80

100

120

Surv

ivin

g fr

actio

n (

)

Negative 5-Fu3

DMC3

DMC6

SE30

SE60


(c)







4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Conclusions


Disclosure




Acknowledgments


References











4-














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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