synthesis, characterization, and in vitro antimicrobial and

Research ArticleSynthesis Characterization and In Vitro Antimicrobialand Anticancer Evaluation of Copolyester Bearing 4-ArylideneCurcumin in the Main Chain

Narendran Kandaswamy and Nanthini Raveendiran

Postgraduate and Research Department of Chemistry Pachaiyapparsquos College Chennai 600 030 India

Correspondence should be addressed to Narendran Kandaswamy knarenchemgmailcom

Received 11 June 2014 Revised 2 August 2014 Accepted 20 August 2014 Published 29 October 2014

Academic Editor Hongwei Duan

Copyright copy 2014 N Kandaswamy and N Raveendiran 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

Synthesis of random copolyester bearing 4-arylidene curcumin M1in the polymer backbone was prepared by solution

polycondensationmethodThe influence of copolyester bearing 4-arylidene curcuminM1unit on the properties of copolyester such

as inherent viscosity solubility and thermal stability was investigated and studied in detailThe inherent viscosity and polydispersityindex of the copolyester were found to be 019 dLg and 138 respectivelyThe chemical structure of the copolyester was investigatedby Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) spectroscopyThe physicalproperties of copolyester were characterized by thermogravimetric analysis (TGA) differential scanning calorimetry (DSC) gelpermeation chromatography (GPC) andX-ray diffraction (XRD) technique Agar disc diffusionmethodwas employed to study theantimicrobial activity of the random copolyester In vitro anticancer activity against lung cancer (Hep-2) cell line was investigated

1 Introduction

Biocompatibility is an important aspect to be considered foran implantable biomaterial The implanted biomaterial intoa body initiates a host response that reflects the first stepsof tissue repair Generally after implantation of biomaterialnumerous types of host responses lead to the develop-ment of adverse effects such as blood-material interactionsacute inflammation chronic inflammation granulation tis-sue development foreign body reaction [1] and biofilmformation due to microbes Sometimes prolonged inflam-mation may lead to tumor in the surrounding tissue Hencethere is a requirement for an improved drug delivery devicethat is capable of delivering a drug having antimicrobialanti-inflammatory and antitumor effects in the vicinity ofa surgical implant over a prolonged period or the usageof coated medical devices Where in the coating includesthe subject polymer matrix and a low solubility prodrugSuch coatings can be applied to surgical implements such asscrews plates prosthesis anchors washers sutures staplesvalves and membranes By considering all these facts it is

required to develop a polymeric biomaterial having very goodantimicrobial and anticancer activity along with it has tomaintain its physical property such as good thermal stabilitymechanical strength solubility and easy fabrication processA probe through the literature indicates that there has been arising interest in synthesizing aliphatic aromatic copolyester[2ndash5] as biomaterials in various medical disciplines Theincorporation of bulks [6 7] such as 4-arylidene curcumincontaining polar carbonyl groups along with introductionof flexible spacer [6 8 9] in the polymer chain tends toreduce the interaction between polymer chains and even-tually leads to an increase in free volume and solubilitywith improvement in processability [10ndash13] by maintainingits thermal stability Curcumin and its derivatives are oneof the important classes of organic compounds possessingintense biological activity including antimicrobial antibacte-rial antifungal anti-inflammatory antitumor and anti-HIVOf particular interest 4-arylidene curcumin possesses betterbioavailability without increasing toxicity

The aim of the present investigation is to synthesiseand characterize random copolyester containing biologically

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 495927 6 pageshttpdxdoiorg1011552014495927

2 International Scholarly Research Notices

active 4-arylidene curcumin by inherent viscosity measure-ments solubility tests FTIR 1H-NMR spectroscopy X-ray diffraction analysis (XRD) thermogravimetric analysis(TGA) differential scanning calorimetry (DSC) and gelpermeation chromatography The synthesized copolyesterwas subjected to in vitro anticancer activity against lungcancer (Hep-2) cell line and antimicrobial studies

2 Experimental

21 Materials and Methods Curcumin (Aldrich) was usedas such p-Tolualdehyde sebacoyl chloride 16-hexanediolDMAP triethylamine DMF and piperidine (Aldrich) wereused as received Solvents were purified according to thestandard procedure in the literature [14] Hep-2 cell lineswere obtained from the National Centre for Cell SciencesPune (NCCS)The cells weremaintained inminimal essentialmedium (MEM) supplemented with 10 FBS streptomycin(100 120583gmL) and penicillin (100 120583gmL) in a humidifiedatmosphere of 50 120583gmL CO

2at 37∘C MEM was purchased

from Hi Media Laboratories Fetal bovine serum (FBS) waspurchased from Cistron Laboratories Trypsin methylthi-azolyl diphenyl-tetrazolium bromide (MTT) and dimethylsulfoxide were purchased from Sisco Research LaboratoryChemicals Mumbai Inherent viscosity was determined at apolymer concentration of 05 gdL in DMF at 30∘C using anUbbelohde suspended level viscometer 3 (wv) solutionswere taken as a standard for solubility of copolyester in var-ious solvents FTIR spectra were recorded on Perkin Elmer883 spectrophotometer 1H-NMR spectra for monomer andpolymer were recorded on a Bruker 400MHz spectrometerusing CDCl

3and DMSO as a solvent Thermogravimetric

analysis (TGA) was performed in nitrogen at 10∘C minminus1with a TGA instrument SDT Q600 Differential scanningcalorimetry (DSC) was performed with DSC 200 F3 Maiainstrument at a heating rate of 10∘C minminus1 under nitrogenatmosphere Molecular weight of copolyester was measuredonWaters 501 gel permeation chromatography equippedwithpolystyrene-divinylbenzene column and a differential refrac-tive index detector Polystyrene was employed as calibrationstandard and chloroform as solvent X-ray diffraction mea-surements were recorded on a Bruker XRDD8 FOCUS usingCu K120572 radiation source Antibacterial activity of randomcopolyester was determined by disc diffusion method onMuller Hinton agar (MHA)medium and Sabouraud dextroseagar (SDA) medium respectively Both Muller Hinton agarand Sabouraud dextrose agar media were poured into thePetri plate After the medium was solidified the inoculumswere spread on the solid plates with sterile swab moistenedwith the bacterial suspension The discs were placed inMHA and SDA plates and 20120583L of sample concentrationwas added Each sample was placed in the disc The plateswere incubated for 24 h at 35∘C Then the antimicrobialactivity was determined by measuring the diameter of zoneof inhibition Standard antibiotic ampicillin (20 120583gdisc) wasused as reference Fresh bacterial cultures of Gram positivebacteria Bacillus subtilis and Staphylococcus aureus and Gramnegative bacteria namely P vulgaris and P aeruginosa were

used for the antibacterial test Antifungal activity of extractswas determined by disc diffusion method on Sabourauddextrose agar medium Disc diffusion assay (DDA) canalso be performed for screening by standard method Stockcultures weremaintained at 4∘C on nutrient agar slant Activecultures for experiments were prepared by transferring a loopfull of culture from the stock cultures into the test tubescontaining Sabouraud dextrose broth which were incubatedfor 24 h at 35∘C C albicans were the fungi which were usedfor the study

TheMTT assay was performed as first described by Mos-mannwith themodifications suggested by Denizot and LangCells (1times 105well) were plated in 24-well plates and incubatedat 37∘C with 5 CO

2condition After the cell reaches the

confluence the various concentrations of copolyester P1

were added and incubated for 24 hours After incubationthe sample was removed from the well and washed withphosphate-buffered saline (pH 74) or MEM without serum100 120583Lwell (5mgmL) of 05 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-tetrazolium bromide (MTT) was added andincubated for 4 hours After incubation 1mL of DMSOwas added in all the wells The absorbance at 570 nm wasmeasured with UV-spectrophotometer using DMSO as theblank Measurements were performed and the concentrationrequired for a 50 inhibition (IC

50) was determined graph-

ically The cell viability was calculated using the followingformula

cell viability = (119860570

of treated cells119860570

of control cells) times 100 (1)

Graphs were plotted using the of cell viability in 119910-axis andconcentration of the sample in119909-axis Cell control and samplecontrol were included in each assay to compare the full cellviability in cytotoxicity and anticancer activity assessments

22 Synthesis of 17-Bis-(4-hydroxy-3-methoxy-phenyl)-4-(4-methyl-benzylidene)-hepta-16-diene-35-dione M

1 In a three-

neck round bottom flask containing curcumin (1mmol 3 g)in a mixture of chloroformmethanol (25mL) A methanolicsolution of p-methylbenzaldehyde (1mmol 0978 g) wasadded with stirring A small amount of catalyst (piperidine5 vv) was added to the mixture which was then stirredat room temperature for further 48 h The progress of thereaction was monitored by TLC [DCM-MeOH (99 1 vv)]After completion of the reaction the productwas precipitatedby adding the reaction mass into a solution of 40mL ofhexane-chloroform (50 50 vv) mixture which resultedin M1 17-bis-(4-hydroxy-3-methoxy-phenyl)-4-(4-methyl-

benzylidene)-hepta-16-diene-35-dione The product wasseparated and washed with hexane to get a pure product(yield 33 g 868) IR (KBr) ] cmminus1 was 3456 3078 28982363 1657 1486 1356 and 11461H NMR (400MHz DMSO-d6) 120575 was 235 (s 3H) 385

(s 6H) 688 (d 2H) 690 (m 4H) 72 (d 2H) 755 (m 2H)788 (m 4H) and 801 (s 1H)

23 Synthesis of Copolyester (P1) A mixture of 4-arylidene

curcumin M1(3 g 1mmol) and hexane diol M

3(075 g

International Scholarly Research Notices 3

4000 3500 3000 2500 2000 1500 1000 50040

50

60

70

80

90

100

2931

1720

CndashH stretch

C = O stretch(ester ) (ester)

( )

1626

1110

C = Ostretch

CndashO stretch

Wave number (cmminus1)

Tran

smitt

ance

()

M1

P1

(alkyl)

Figure 1 FTIR spectra of copolyester P1

11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

Figure 2 1H-NMR spectrum of copolyester P1in CDCl

3

1 mmol) in 30mL of DMF was taken in a three-neck roundbottom flask Temperature of the reaction mass was reducedto 15∘C and charged with triethylamine (128 g 2mmol)and (05 ww) DMAP To this sebacoyl chloride M

2(3 g

2mmol) dissolved in dry DMF (15mL) was added for 15minwith constant stirring for 24 h The resulting reaction masswas filtered and the filtrate was poured into water (250mL)to precipitate the random copolyester It was thenwashed anddried under vacuum to get the product The yield was almostquantitative Spectral data are presented in Figures 1 and 2

3 Results and Discussion

31 FTIR and 1H-NMR Analysis Structural assignmentsof the peaks coincide with the spectral data of the FTIRanalysis shown in Figure 1 The copolyester shows charac-teristic absorption due to carbonyl stretching of the estergroup at around 1720 cmminus1 However it is interesting to findthe peak at 1626 cmminus1 corresponding to the stretching ofcarbonyl group of 120572120573-unsaturated compoundM

1 So it can

be inferred that the 4-arylidene curcumin molecule is anintegral part of the polymer backbone Stretching vibrationof ester CndashO appears at 1080ndash1110 cmminus1 The copolyesterP1exhibits absorption band around 2850ndash2931 cmminus1 this is

attributed to the presence of ndashCndashH stretching vibration ofalkyl group in the polymer chain (see Scheme 1) No signif-icant absorption in the OH stretching region was detectedconfirming that the dihydroxy monomer M

1effectively got

involved in polymerisationIn Figure 2 1H-NMR spectrum of the copolyester P

1

was discussed The characteristic peaks in the range of704ndash780 ppm are due to the aromatic protons of monomerM1 The characteristic peak of curcumin at 970 ppm cor-

responding to the hydroxyl protons completely disappearsafter the reaction The methoxy protons (ndashOCH

3) of the

monomer M1group appeared at 391 ppm respectively The

peak characterising themethylene groups of sebacoyl unitM2

andhexamethylene unitM3at 122ndash258 ppmand the pendant

methyl unit frommonomerM1got merged with the peaks of

aliphatic methylene groups at the region mentioned aboveIt is explicit that the monomerM

1successfully incorporated

into the polymer chains

32 Polymer Solubility and Inherent Viscosity Solubilityparameter is an important criterion for polymer processingThe copolyester showed good solubility in chlorinated sol-vents and polar aprotic solvents this may be due to thepresence of pendant arylidene group in the monomer M

1

which leads to reduced rigidity of polymer chains and hindersclose chain packing thereby reducing chain interactionsThus it is inferred that the incorporation of arylidene pendantunit influence more in solubility The inherent viscosity ofthe copolyester was determined in DMF at 30∘C at theconcentration of 05 gdL using an Ubbelohde suspendedlevel viscometer The inherent viscosity of the copolyesterwas found to be 019 dLg In general inherent viscositiesincrease with increase in molecular weight It is evidentthat copolyester exhibits lower viscosity value because thepresence of unsymmetrical bulky monomer M

1with aryli-

dene pendant unit may tend to reduce the polymer chainlength and hence inherent viscosity value decreases It is alsoreflected in the GPC data The number average molecularweight (M

119899) andweight averagemolecularweight (M

119908) of the

copolyester P1are 4429 and 6127 with 138 as polydispersity

index value respectively It is inferred that the copolyesterformed in a random manner and lower molecular weight ofthe polymer is suitable for the biological application

33 DSC and TGA Studies The influence of polymer struc-ture on the thermal properties of copolyester P

1was investi-

gated by thermogravimetric analysis (TGA) at a heating rateof 10∘C minminus1 in nitrogen atmosphere and the TGA curveis shown in Figure 4 The initial decomposition temperature(IDT) temperature for 10 weight loss (119879

10) and the max-

imum decomposition temperature (119879max) of copolyester are225∘C 240∘C and 320∘C which indicates its good thermalstability The 119879

119892of the copolyester was determined by DSC

at a heating rate of 10∘C minminus1 under nitrogen atmosphereand the DSC thermogram of copolyester P

1shows low glass

transition temperature (119879119892) at 172∘Candmelting temperature

(119879119898) at 675∘C as shown in Figure 3 As expected the lowest119879

119892

value was observed for copolyester which can be attributed


TEADMAP DMF

CH2

CH2

O O

O OO O

O O

OO

8

O O

OH OHO O

+ CCO O

ClCl8

+ HOOH

n

M1 M2

P1

M3

Scheme 1 Synthesis of copolyester bearing 4-arylidene curcumin

50 100 150 200

Hea

t flow

endo

Temperature (∘C)

Tg = 172 ∘C

0

Figure 3 DSC thermogram of random copolyester P1

to the fact that the pendant arylidene unit from curcuminmonomerM

1in the polymeric structure was unable to array

regular and hinders the close chain packing thus reducingthe rigidity of the polymeric structure

34 XRD X-ray diffraction pattern of copolyesterP1exhibits

the essential amorphous and semicrystalline nature Apartfrom this copolyester exhibited some degrees of crystallinityat 2120579 range of about 5∘ 17∘ and 19∘ This observation couldbe attributed to the introduction of methylene unit [6] fromboth acid dichloride M

2and diol M

3as shown in X-ray

diffraction pattern In addition the presence of pendantarylidene unit from monomer M

1reduces coplanarity and

hindered the dense packing of polymer chains and destroyedthe ordered arrangement resulting in amorphous nature ofthese polyesters which is also reflected in their improvedsolubility Copolyester P

1exhibits conspicuous amorphous

halo at 2120579 range of about 15∘ to 30∘ and all these facts areevident from X-ray diffractogram of P

1as shown in Figure 5

200 400 600 800minus20

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)

Figure 4 TGA curve of copolyester P1

35 Antimicrobial Activity The results of antimicrobial activ-ity are presented in Table 1 and the comparative activitiesof monomer M

1and copolyester P

1against microbes are

shown in Figure 6 The monomer and the copolyester areactive against all the microbes such as B subtilis S aureus Pvulgaris P aeruginosa and C albicans with inhibitory zoneranges of 90ndash130mm and 90-140mm respectively All thecopolyesters exhibited enhanced antimicrobial activity com-pared with the monomer and this is due to the lipophilicityof the copolyester In general more lipophilic compoundscan penetrate greater the lipophilic cell membranes of Grampositive bacteria while less lipophilic compounds are moreliable to penetrate the cell wall of Gram negative bacteriaIt is explicit from Figure 6 Copolyester P

1exhibits extreme

antimicrobial activity and this is attributed to the abilityof 120572 120573-unsaturated ketone from curcumin to undergo aconjugated addition to a nucleophilic group like thiol groupin an essential protein of the microorganism The presenceof more lipophilic methoxy moiety at the position 441015840 ofcurcumin and small percentage of end phenolic and hydroxyl


Table 1 Zones of inhibition (in mm) of the compounds against various microbes

Compound B subtilis S aureus P vulgaris P aeruginosa C albicans20 120583L 40 120583L 60 120583L 20 120583L 40120583L 60 120583L 20 120583L 40 120583L 60120583L 20 120583L 40 120583L 60 120583L 20 120583L 40120583L 60 120583L

M1 9 10 11 9 10 12 9 9 10 10 10 11 9 9 12P1 10 11 12 9 11 13 11 13 13 12 14 14 11 13 14

10 20 30 40 50 60 70 80

0

200

400

600

800

1000

Inte

nsity

(au

)

2120579

Figure 5 X-ray diffractogram of copolyester P1

M1

P1

0

2

4

6

8

10

12

14

B su

btili

s

S a

ureu

s

P vu

lgaris

P ae

rugi

nosa

C a

lbica

ns

MonomerPolymer

Figure 6 Comparative activities of monomer M1and copolyester

P1against various microbes

group of coumarin has contributed to the wide spectrumof antibacterial activities thus proving their efficiency asantimicrobial polymers

36 Anticancer Evaluation of Copolyester P1 Viable cells were

determined by the absorbance Measurements were per-formed and the concentration required for a 50 inhibition

Table 2 Anticancer activities of copolyester P1 on Hep-2 cell line

S number Concentration(120583gmL) Dilution Absorbance

(OD)Cell

viability ()1 1000 Neat 007 13462 500 1 1 014 26923 250 1 2 020 38464 125 1 4 027 51925 625 1 8 034 65386 312 1 16 041 78847 156 1 32 045 86538 78 1 64 050 96159 Cell control mdash 052 100

0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)

Concentration (120583gmL)

Figure 7 Graphical representation of activities of copolyester P1in

the MTT assay

of viability (IC50) was determined graphically The effect of

the copolyester on Hep-2 cell line was expressed as the cell viability IC

50of the polymer was determined and was

shown in Table 2 The assessment of cell viability by MTTassay through graphical representation is shown in Figure 7The covalent conjugation of 4-arylidene curcumin throughester linkage increases its molecular size and steric hindrancemay improve its cellular permeability and restrict its cancerprogression [15 16] It is explicit from Table 2 that low con-centration of copolyesterP

1induced greater anticarcinogenic

activity effects on Hep-2 cell line than the individual agentsLiterature survey reveals that the substitution of electrondonating groups at the para position of benzene ring increasesthe antitumor and antioxidant activity of curcumin [17]

4 Conclusion

In summary aliphatic aromatic random copolyester bearing4-arylidene curcumin has been synthesised via solution


polycondensation method The copolyester showed goodsolubility in various organic solvents and this is attributed tothe incorporation of pendant arylidene unit The structureof the random copolyester was confirmed by FTIR and 1H-NMR spectroscopy XRD pattern of the copolyester revealedthat the copolyester was found to be amorphous with somedegrees of crystallinity in natureThe enhanced antimicrobialand anticancer activity of the copolyester revealed that it canfind versatile applications in various biomedical disciplinesOf particular interest it can act as prodrug for the delivery ofarylidene curcumin or prodrug carriers for other therapeuticagents

Conflict of Interests

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

Acknowledgment

The authors would like to thank CLRI technical persons fortheir analytical support

References

[1] J M Anderson A Rodriguez and D T Chang ldquoForeign bodyreaction to biomaterialsrdquo Seminars in Immunology vol 20 no2 pp 86ndash100 2008

[2] Y Chen Y Yang J Su L Tan and Y Wang ldquoPreparation andcharacterization of aliphaticaromatic copolyesters based onbisphenol-A terephthalate hexylene terephthalate and lactidemiotiesrdquo Reactive and Functional Polymers vol 67 no 5 pp396ndash407 2007

[3] G Rabani H Luftmann andAKraft ldquoSynthesis and propertiesof segmented copolymers containing short aramid hard seg-ments and aliphatic polyester or polycarbonate soft segmentsrdquoPolymer vol 46 no 1 pp 27ndash35 2005

[4] M Bagheri K Didehban Z Rezvani and A A EntezamildquoThermotropic polyesters Synthesis characterization and ther-mal transition of poly[440-bis(x-alkoxy)biphenyl isophtha-late]rdquo European Polymer Journal vol 40 no 4 pp 865ndash8712004

[5] H Montes de Oca J E Wilson A Penrose et al ldquoLiquid-crys-talline aromatic-aliphatic copolyester bioresorbable polymersrdquoBiomaterials vol 31 no 30 pp 7599ndash7605 2010

[6] T Vlad-Bubulac and C Hamciuc ldquoAliphatic-aromatic copol-yesters containing phosphorous cyclic bulky groupsrdquo Polymervol 50 no 10 pp 2220ndash2227 2009

[7] D Acierno R Fresa P Iannelli and P Vacca ldquoSegmentedliquid-crystalline polyesters with allyl groups as lateral sub-stituents Synthesis and characterizationrdquo Polymer vol 41 no11 pp 4179ndash4187 2000

[8] T Ranganathan C Ramesh and A Kumar ldquoThermotropicliquid-crystalline polyesters containing biphenyl mesogens inthe main chain the effect of connectivityrdquo Journal of PolymerScience A Polymer Chemistry vol 42 no 11 pp 2734ndash27462004

[9] R M Tejedor L Oriol M Pinol et al ldquoPhotoreactive main-chain liquid-crystalline polyesters synthesis characterization

and photochemistryrdquo Journal of Polymer Science A PolymerChemistry vol 43 no 20 pp 4907ndash4921 2005

[10] G Chen and R W Lenz ldquoLiquid crystal polymers XVIIIComparison of the properties of aromatic polyesters with dif-ferent mesogenic units and a common flexible spacerrdquo Journalof Polymer Science Polymer Chemistry vol 22 no 11 pp 3189ndash3201 1984

[11] M Bagheri K Didehban and A A Entezami ldquoThermotropicpolyesters (part 3) synthesis characterization and thermaltransition of random copolyesters containing terephthalate andisophthalate unitsrdquo Iranian Polymer Journal vol 13 no 4 pp327ndash334 2004

[12] L-L Lin and J-L Hong ldquoSemi-rigid thermotrophic polyestercontaining a rigid bent spirobicromanemoieties-primary char-acterizations and the thermal behaviorrdquo Polymer vol 41 no 12pp 4501ndash4512 2000

[13] L-L Lin and J-L Hong ldquoEffect of isotropization on the ther-mal behavior of two thermotropic copolyesters containingspirobicromanemoietiesrdquo Polymer vol 42 no 3 pp 1009ndash10162001

[14] D D Perrin and W L F Armarego Purification of LaboratoryChemicals Pergamon Press New York NY USA 1989

[15] A Slusarz N S Shenouda M S Sakla et al ldquoCommon botan-ical compounds inhibit the hedgehog signaling pathway inprostate cancerrdquo Cancer Research vol 70 no 8 pp 3382ndash33902010

[16] A Barve T O Khor X Hao et al ldquoMurine prostate cancerinhibition by dietary phytochemicalsmdashcurcumin and pheny-ethylisothiocyanaterdquo Pharmaceutical Research vol 25 no 9 pp2181ndash2189 2008

[17] H Tang C J Murphy B Zhang et al ldquoCurcumin polymers asanticancer conjugatesrdquo Biomaterials vol 31 no 27 pp 7139ndash7149 2010

Submit your manuscripts athttpwwwhindawicom

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Journal ofNanomaterials


active 4-arylidene curcumin by inherent viscosity measure-ments solubility tests FTIR 1H-NMR spectroscopy X-ray diffraction analysis (XRD) thermogravimetric analysis(TGA) differential scanning calorimetry (DSC) and gelpermeation chromatography The synthesized copolyesterwas subjected to in vitro anticancer activity against lungcancer (Hep-2) cell line and antimicrobial studies

2 Experimental

21 Materials and Methods Curcumin (Aldrich) was usedas such p-Tolualdehyde sebacoyl chloride 16-hexanediolDMAP triethylamine DMF and piperidine (Aldrich) wereused as received Solvents were purified according to thestandard procedure in the literature [14] Hep-2 cell lineswere obtained from the National Centre for Cell SciencesPune (NCCS)The cells weremaintained inminimal essentialmedium (MEM) supplemented with 10 FBS streptomycin(100 120583gmL) and penicillin (100 120583gmL) in a humidifiedatmosphere of 50 120583gmL CO

2at 37∘C MEM was purchased

from Hi Media Laboratories Fetal bovine serum (FBS) waspurchased from Cistron Laboratories Trypsin methylthi-azolyl diphenyl-tetrazolium bromide (MTT) and dimethylsulfoxide were purchased from Sisco Research LaboratoryChemicals Mumbai Inherent viscosity was determined at apolymer concentration of 05 gdL in DMF at 30∘C using anUbbelohde suspended level viscometer 3 (wv) solutionswere taken as a standard for solubility of copolyester in var-ious solvents FTIR spectra were recorded on Perkin Elmer883 spectrophotometer 1H-NMR spectra for monomer andpolymer were recorded on a Bruker 400MHz spectrometerusing CDCl

3and DMSO as a solvent Thermogravimetric

analysis (TGA) was performed in nitrogen at 10∘C minminus1with a TGA instrument SDT Q600 Differential scanningcalorimetry (DSC) was performed with DSC 200 F3 Maiainstrument at a heating rate of 10∘C minminus1 under nitrogenatmosphere Molecular weight of copolyester was measuredonWaters 501 gel permeation chromatography equippedwithpolystyrene-divinylbenzene column and a differential refrac-tive index detector Polystyrene was employed as calibrationstandard and chloroform as solvent X-ray diffraction mea-surements were recorded on a Bruker XRDD8 FOCUS usingCu K120572 radiation source Antibacterial activity of randomcopolyester was determined by disc diffusion method onMuller Hinton agar (MHA)medium and Sabouraud dextroseagar (SDA) medium respectively Both Muller Hinton agarand Sabouraud dextrose agar media were poured into thePetri plate After the medium was solidified the inoculumswere spread on the solid plates with sterile swab moistenedwith the bacterial suspension The discs were placed inMHA and SDA plates and 20120583L of sample concentrationwas added Each sample was placed in the disc The plateswere incubated for 24 h at 35∘C Then the antimicrobialactivity was determined by measuring the diameter of zoneof inhibition Standard antibiotic ampicillin (20 120583gdisc) wasused as reference Fresh bacterial cultures of Gram positivebacteria Bacillus subtilis and Staphylococcus aureus and Gramnegative bacteria namely P vulgaris and P aeruginosa were

used for the antibacterial test Antifungal activity of extractswas determined by disc diffusion method on Sabourauddextrose agar medium Disc diffusion assay (DDA) canalso be performed for screening by standard method Stockcultures weremaintained at 4∘C on nutrient agar slant Activecultures for experiments were prepared by transferring a loopfull of culture from the stock cultures into the test tubescontaining Sabouraud dextrose broth which were incubatedfor 24 h at 35∘C C albicans were the fungi which were usedfor the study

TheMTT assay was performed as first described by Mos-mannwith themodifications suggested by Denizot and LangCells (1times 105well) were plated in 24-well plates and incubatedat 37∘C with 5 CO

2condition After the cell reaches the

confluence the various concentrations of copolyester P1

were added and incubated for 24 hours After incubationthe sample was removed from the well and washed withphosphate-buffered saline (pH 74) or MEM without serum100 120583Lwell (5mgmL) of 05 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-tetrazolium bromide (MTT) was added andincubated for 4 hours After incubation 1mL of DMSOwas added in all the wells The absorbance at 570 nm wasmeasured with UV-spectrophotometer using DMSO as theblank Measurements were performed and the concentrationrequired for a 50 inhibition (IC

50) was determined graph-

ically The cell viability was calculated using the followingformula

cell viability = (119860570

of treated cells119860570

of control cells) times 100 (1)

Graphs were plotted using the of cell viability in 119910-axis andconcentration of the sample in119909-axis Cell control and samplecontrol were included in each assay to compare the full cellviability in cytotoxicity and anticancer activity assessments

22 Synthesis of 17-Bis-(4-hydroxy-3-methoxy-phenyl)-4-(4-methyl-benzylidene)-hepta-16-diene-35-dione M

1 In a three-

neck round bottom flask containing curcumin (1mmol 3 g)in a mixture of chloroformmethanol (25mL) A methanolicsolution of p-methylbenzaldehyde (1mmol 0978 g) wasadded with stirring A small amount of catalyst (piperidine5 vv) was added to the mixture which was then stirredat room temperature for further 48 h The progress of thereaction was monitored by TLC [DCM-MeOH (99 1 vv)]After completion of the reaction the productwas precipitatedby adding the reaction mass into a solution of 40mL ofhexane-chloroform (50 50 vv) mixture which resultedin M1 17-bis-(4-hydroxy-3-methoxy-phenyl)-4-(4-methyl-

benzylidene)-hepta-16-diene-35-dione The product wasseparated and washed with hexane to get a pure product(yield 33 g 868) IR (KBr) ] cmminus1 was 3456 3078 28982363 1657 1486 1356 and 11461H NMR (400MHz DMSO-d6) 120575 was 235 (s 3H) 385

(s 6H) 688 (d 2H) 690 (m 4H) 72 (d 2H) 755 (m 2H)788 (m 4H) and 801 (s 1H)

23 Synthesis of Copolyester (P1) A mixture of 4-arylidene

curcumin M1(3 g 1mmol) and hexane diol M

3(075 g


4000 3500 3000 2500 2000 1500 1000 50040

50

60

70

80

90

100

2931

1720

CndashH stretch


( )

1626

1110

C = Ostretch

CndashO stretch


Tran

smitt

ance

()

M1

P1

(alkyl)


11 10 9 8 7 6 5 4 3 2 1 0

(ppm)


3


2(3 g




1 So it can



1effectively got


1



3) of the









1


1with aryli-



119908) of the




1was investi-


10) and the max-




1shows low glass



119892



TEADMAP DMF

CH2

CH2

O O

O OO O

O O

OO

8

O O

OH OHO O

+ CCO O

ClCl8

+ HOOH

n

M1 M2

P1

M3


50 100 150 200

Hea

t flow

endo

Temperature (∘C)

Tg = 172 ∘C

0







2and diol M

3as shown in X-ray







200 400 600 800minus20

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)



1and copolyester P



1exhibits extreme





M1 9 10 11 9 10 12 9 9 10 10 10 11 9 9 12P1 10 11 12 9 11 13 11 13 13 12 14 14 11 13 14

10 20 30 40 50 60 70 80

0

200

400

600

800

1000

Inte

nsity

(au

)

2120579


M1

P1

0

2

4

6

8

10

12

14

B su

btili

s

S a

ureu

s

P vu

lgaris

P ae

rugi

nosa

C a

lbica

ns

MonomerPolymer








(OD)Cell


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)



the MTT assay







4 Conclusion






Acknowledgment


References


























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




4000 3500 3000 2500 2000 1500 1000 50040

50

60

70

80

90

100

2931

1720

CndashH stretch


( )

1626

1110

C = Ostretch

CndashO stretch


Tran

smitt

ance

()

M1

P1

(alkyl)


11 10 9 8 7 6 5 4 3 2 1 0

(ppm)


3


2(3 g




1 So it can



1effectively got


1



3) of the









1


1with aryli-



119908) of the




1was investi-


10) and the max-




1shows low glass



119892



TEADMAP DMF

CH2

CH2

O O

O OO O

O O

OO

8

O O

OH OHO O

+ CCO O

ClCl8

+ HOOH

n

M1 M2

P1

M3


50 100 150 200

Hea

t flow

endo

Temperature (∘C)

Tg = 172 ∘C

0







2and diol M

3as shown in X-ray







200 400 600 800minus20

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)



1and copolyester P



1exhibits extreme





M1 9 10 11 9 10 12 9 9 10 10 10 11 9 9 12P1 10 11 12 9 11 13 11 13 13 12 14 14 11 13 14

10 20 30 40 50 60 70 80

0

200

400

600

800

1000

Inte

nsity

(au

)

2120579


M1

P1

0

2

4

6

8

10

12

14

B su

btili

s

S a

ureu

s

P vu

lgaris

P ae

rugi

nosa

C a

lbica

ns

MonomerPolymer








(OD)Cell


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)



the MTT assay







4 Conclusion






Acknowledgment


References


























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




TEADMAP DMF

CH2

CH2

O O

O OO O

O O

OO

8

O O

OH OHO O

+ CCO O

ClCl8

+ HOOH

n

M1 M2

P1

M3


50 100 150 200

Hea

t flow

endo

Temperature (∘C)

Tg = 172 ∘C

0







2and diol M

3as shown in X-ray







200 400 600 800minus20

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)



1and copolyester P



1exhibits extreme





M1 9 10 11 9 10 12 9 9 10 10 10 11 9 9 12P1 10 11 12 9 11 13 11 13 13 12 14 14 11 13 14

10 20 30 40 50 60 70 80

0

200

400

600

800

1000

Inte

nsity

(au

)

2120579


M1

P1

0

2

4

6

8

10

12

14

B su

btili

s

S a

ureu

s

P vu

lgaris

P ae

rugi

nosa

C a

lbica

ns

MonomerPolymer








(OD)Cell


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)



the MTT assay







4 Conclusion






Acknowledgment


References


























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






M1 9 10 11 9 10 12 9 9 10 10 10 11 9 9 12P1 10 11 12 9 11 13 11 13 13 12 14 14 11 13 14

10 20 30 40 50 60 70 80

0

200

400

600

800

1000

Inte

nsity

(au

)

2120579


M1

P1

0

2

4

6

8

10

12

14

B su

btili

s

S a

ureu

s

P vu

lgaris

P ae

rugi

nosa

C a

lbica

ns

MonomerPolymer








(OD)Cell


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)



the MTT assay







4 Conclusion






Acknowledgment


References


























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







Acknowledgment


References


























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



synthesis, characterization, and in vitro antimicrobial and

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