Synthesis, characterization, in vitro antimicrobial and

Synthesis, characterization, in vitro antimicrobial and anticancer evaluation of random copolyesters bearing biscoumarin units in the main chains Narendran Kandaswamy & Nanthini Raveendiran

Research on Chemical Intermediates ISSN 0922-6168 Volume 41 Number 10 Res Chem Intermed (2015) 41:7189-7206 DOI 10.1007/s11164-014-1806-3

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Author's personal copy Res Chem Intermed (2015) 41:7189–7206 DOI 10.1007/s11164-014-1806-3

Synthesis, characterization, in vitro antimicrobial and anticancer evaluation of random copolyesters bearing biscoumarin units in the main chains Narendran Kandaswamy • Nanthini Raveendiran

Received: 9 June 2014 / Accepted: 2 September 2014 / Published online: 24 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract A series of aliphatic–aromatic random copolyesters bearing a biscoumarin group were synthesized by phase-transfer-catalyzed interfacial polycondensation of 3,30 -methylene-bis(4-hydroxycoumarin)1 and aromatic diols such as hydroquinone, resorcinol, ethyl resorcinol, bisphenol-A, and curcumin with sebacoyl chloride. These copolyesters were obtained with yields in the range between 76 and 85 % and the inherent viscosity between 0.22 and 0.33 dl/g. All the copolyesters were found to be soluble in chlorinated and polar aprotic solvents. The chemical structures of the copolyesters were analyzed by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance (1H-NMR) spectroscopy. The physical properties of copolyesters obtained by altering the aromatic diols 3, were characterized by thermogravimetric analysis, differential scanning calorimetry, gel permeation chromatography, and X-ray diffraction (XRD) technique. Copolyesters exhibited good thermal stability having a decomposition temperature above 223 °C. Copolyesters with the bisphenol group as backbone exhibited higher thermal stability than others. Results revealed that the copolyesters exhibit a glass transition temperature in the range of -11 to 54 °C. It was found that copolyester with ethyl resorcinol and curcumin shown low and high Tg values, respectively. XRD measurement revealed the amorphous nature of copolyester with a low degree of crystallinity. Agar disc diffusion method was employed to study the antimicrobial activity of these random copolyesters. The synthesized copolyester was subjected to in vitro anticancer activity against lung cancer (Hep-2) cell line. Keywords

Biscoumarin copolyesters Antimicrobial Anticancer Amorphous

N. Kandaswamy (&) N. Raveendiran Postgraduate and Research Department of Chemistry, Pachaiyappa’s College, Chennai 600 030, India e-mail: [email protected]

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Author's personal copy 7190

N. Kandaswamy, N. Raveendiran

Introduction Polyesters are of great importance because of their low glass transition temperature, high thermal stability, and excellent mechanical property, which can withstand extreme conditions. Of particular interest, aromatic aliphatic polymers find versatile applications in various fields. A probe through the literature indicates that there has been rising interest in synthesizing aromatic aliphatic copolyester [1–6] as biomaterials [7, 8] in a variety of medical disciplines. In spite of the unique properties such as thermal stability, good chemical resistance, and excellent mechanical strength, polymers have been significantly limited by their fabrication process due to the high glass transition temperature and poor solubility in common organic solvents. Extensive studies have been made to sort out the problems of solubility and processability of copolyesters. The main focus of research is to develop polymers by using suitable monomers, stoichiometry, and experimental conditions to improve the thermal properties, mechanical properties, and processability. The incorporation of bulky monomers [3, 9] or the use of bent monomers [3, 10] containing biscoumarin units such as 3,30 -methylene-bis(4-hydroxycoumarin) 1, containing polar carbonyl groups or the use of meta oriented aromatic units along with introduction of flexible spacer [3, 10–15] in the polymer chain, tends to reduce the interaction between the polymer chains and eventually leads to an increase in free volume and solubility with enhance processability by retaining its thermal stability. When considering a biomaterial for implantation, the first and foremost requirement is nontoxic and chemically inert/active and sustainable. Biocompatible means that the biomaterials should not form thrombi in the blood system, which ultimately leads to tumor in surround tissues, instantly declined by the body. Implantation of surgical devices such as screws, plates, sutures, prosthesis anchor, staples, and valves has the potential to infect patients with various microbes. To encounter this problem it is required to develop a drug-delivery device capable of delivering an antimicrobial drug in the vicinity of surgical implants or the implantation of antimicrobial polymer-coated medical devices has to be introduced. Often, surgical implantation leads to adverse side effects such as pain and swelling. Sometimes long-term inflammation leads to development of dysplasia. Coumarin and its derivatives are one of the active classes of organic compounds possessing a wide spectrum of biological activity including anticoagulant, estrogenic, antimicrobial, analgesic, antibacterial, antifungal, anti-inflammatory, antitumor, and antiHIV. Of particular interest 3,30 -methylene-bis(4-hydroxycoumarin) 1 exhibits antiproliferative activity with less toxicity. By considering the above facts, it is required to develop a biomaterial possessing both antimicrobial and anticancer activity. The aim of the present investigation is to synthesize and characterize a series of random copolyesters containing a bioactive biscoumarin group and different aromatic diols through aliphatic diacid chloride by interfacial polycondensation technique [16]. The synthesis of proposed random copolyesters is to investigate the influence of the nonlinear and bulky biscoumarin group along with para or meta-oriented aromatic units on the properties such as solubility and thermal behavior of the copolyesters. The derived copolyesters were characterized by inherent viscosity measurements, solubility tests, FTIR, 1H-NMR

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Author's personal copy Random copolyesters bearing biscoumarin units

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spectroscopy, X-ray diffraction analysis (XRD), gel permeation chromatography (GPC), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The synthesized copolyester was subjected to in vitro anticancer activity against lung cancer (Hep-2) cell line and antimicrobial studies.

Experimental Materials and methods 4-Hydroxycoamarin (Aldrich) was used as such. Formaldehyde, curcumin, resorcinol, ethylresorcinol, hydroquinone, bisphenol-A, sebacoyl chloride (Aldrich) were used as received. Tetra-n-butylammonium bromide (TBAB) and sodium hydroxide (Merck, India) were used as received. Solvents were purified according to the standard procedure in the literature [17]. Solvents like chloroform (CHCl3), dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAc) and N-methylpyrrolidone (NMP) were used for determining the solubility behavior of copolyesters. The melting point of 3,30 -methylene-bis(4-hydroxycoumarin) was determined by open capillary method. Inherent viscosity was measured at a polymer concentration of 0.5 g/dl in NMP at 30 °C using an Ubbelohde suspended-level viscometer. 5 % (w/v) solutions were taken as a criterion for solubility of copolyesters in various solvents at room temperature or upon heating. FTIR spectra were recorded on PerkinElmer 883 spectrophotometer. 1H-NMR spectra for monomer and polymers were recorded on a Bruker 400-MHz spectrometer using CDCl3 or DMSO-d6 as a solvent at room temperature. Molecular weight of copolyesters was measured on a Waters 501 GPC equipped with a polystyrene-divinylbenzene column and a differential refractive index detector. Polystyrene was employed as calibration standard and chloroform as solvent. TGA was performed in nitrogen at 10 °C min-1 with a TGA instrument SDT Q600. DSC was performed with a DSC 200 F3 Maia instrument at a heating rate of 10 °C min-1 under a nitrogen atmosphere. XRD measurements were recorded on a Bruker XRD D8 FOCUS using Cu-Ka radiation source. Antibacterial activity of random copolyesters was determined by disc diffusion method on Mueller-Hinton agar (MHA) medium and Sabouraud dextrose agar (SDA) medium, respectively. Both MHA and SDA media were poured into the Petri plate. After solidification, the inoculums were spread on the solid plates with a sterile swab moistened with the bacterial suspension. The discs were placed in MHA and SDA plates and 20 ll of sample concentration was added. Each sample was placed in the disc. The plates were incubated for 24 h at 37 °C. The antimicrobial activity was then determined by measuring the diameter of zone of inhibition. Standard antibiotic ampicillin was used as a reference. Fresh bacterial cultures of Gram-negative bacteria namely Escherichia coli and Salmonella typhi and Grampositive bacteria Bacillus subtilis and Staphylococcus aureus were used for the antibacterial test. Antifungal activity of extracts was determined by disc diffusion method on SDA medium. Disc diffusion assay (DDA) can also be performed for

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screening by standard method. Stock cultures were maintained at 4 °C on Nutrient agar Slant. Active cultures for experiments were prepared by transferring a loop full of culture from the stock cultures into the test-tubes containing Sabouraud dextrose broth, which were incubated at 24 h at 37 °C. Aspergillus niger and Aspergillus flavus were the fungi that were used for the study. Amphotericin-B was used as standard. Hep-2 cell lines were obtained from National Centre for Cell Sciences, Pune (NCCS). The cells were maintained in Minimal Essential Medium (MEM) supplemented with 10 % FBS, penicillin (100 lg/ml), and streptomycin (100 lg/ml) in a humidified atmosphere of 50 lg/ml CO2 at 37 °C. MEM was purchased from Hi Media Laboratories. Fetal bovine serum (FBS) was purchased from Cistron Laboratories. Trypsin, methylthiazolyl diphenyl-tetrazolium bromide (MTT), and DMSO were purchased from Sisco research laboratory chemicals Mumbai. Synthesis of 3,30 -methylene-bis(4-hydroxycoumarin)1 A mixture of 40 % formaldehyde solution (0.8 g, 1.0 mmol) and 4-hydroxycoumarin (3 g, 2.0 mmol) was taken in 100 ml of distilled water in a round-bottomed flask. With vigorous stirring, the reaction temperature was maintained at 70–75 °C. After 1 h, the aliquot was tested by TLC and found that there were no monomers present, which indicates the completion of the reaction. The solid obtained was filtered, dried, and recrystallized using DCM/methanol (25:75) to give a white powder (2.6 g, 83.8 %). Rf: 0.25 (n-hexane: EtOAc-1:1). mp: 272–275 °C. 1H-NMR (400 MHz, CDCl3): d (ppm): 11.32 (s, 2H); 7.988–8.00 (d, 2H); 7.57–7.61 (t, 2H); 7.34–7.39 (t, 4H); 3.84 (s, 2H). FTIR: c (cm-1) 3,647, 3,064, 1,650, 1,596, 1,565, 1,453, 1,344, 765. Copolymerization procedure In a three-neck round-bottomed flask (500 cm3 in volume) equipped with a mechanical stirrer, dry nitrogen inlet, outlet and dropper, a mixture of 3,30 methylene-bis(4-hydroxycoumarin) 1 and aromatic diol 3 each (0.002 mol) was taken. Sodium hydroxide (0.008 mol) dissolved in 80 ml of distilled water was added and vigorously stirred for 5 min until the appearance of clear solution and then 2 ml of 2 % TBAB was added. An amount of 0.004 mol of appropriate acid dichloride, dissolved in 30 ml of dry methylene chloride was slowly added for 20 min at room temperature with vigorous stirring and the stirring was continued for a further period of 1.5 h at 2,000 rpm. The reaction mixture was then poured into methanol; the precipitated polymer was filtered and washed several times with water. The polymer was dissolved in chloroform and reprecipitated in methanol to remove methylated ester impurity and dried under reduced pressure at 80 °C for 24 h. In vitro assay for anticancer activity (MTT assay) The MTT assay was performed as first described by Mosmann with the modifications suggested by Denizot and Lang. Cells (1 9 105/well) were plated

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in 24-well plates and incubated in 37 °C with 5 % CO2 condition. After the cell reaches the confluence, the various concentrations of copolyester were added and incubated for 24 h. After incubation, the sample was removed from the well and washed with phosphate-buffered saline (pH 7.4) or MEM without serum. An amount of 100 ll/well (5 mg/ml) of 0.5 % 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl–tetrazolium bromide (MTT) was added and incubated for 4 h. After incubation, 1 ml of DMSO was added in all the wells. The absorbance at 570 nm was measured with a UV spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50 % inhibition (IC50) was determined graphically. The % cell viability was calculated using the following formula: % cell viability ¼ ðA570 of treated cells=A570 of control cellsÞ 100 Graphs were plotted using the % of cell viability in the Y-axis and concentration of the sample in the X-axis. Cell control and sample control were included in each assay to compare the full cell viability in cytotoxicity and anticancer activity assessments.

Results and discussion Synthesis of copolyesters A new series of copolyesters containing biscoumarin units was synthesized by phase-transfer catalyzed interfacial polycondensation technique as shown in Scheme 1. This involves a reaction of one equivalent of 3,30 -methylene-bis(4hydroxycoumarin) 1 and one equivalent of aromatic diol 3 with two equivalents of aliphatic acid dichloride in dichloromethane-aqueous sodium hydroxide system using TBAB as a phase-transfer catalyst. During the progress of reaction, the reaction mixture became hazy and the system was temporarily homogeneous and the polymer became precipitated during the polycondensation process. The yield of the copolyesters was found to be quantitative (76–85 %) as shown in Table 1. From GPC that the number average molecular weight (Mn) of the copolyester 4a and 4d are 10,400 and 11,912 and weight average molecular weight (Mw) are 12,012 and 12,824 with 1.15 and 1.07 as polydispersity index values, respectively. This interfacial technique was chosen rather than a homogeneous system because it has marked advantages such as easy operation and simplicity in product isolation.

Polymer characterization FTIR analysis Structural assignments of the peaks coincide with the spectral data of the FTIR analysis. All the copolyesters show characteristic absorption bands due to carbonyl

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OH

OH +

O

OO

O Cl C

O C Cl

CH2

+

HO Ar' OH

8

O

1

3a - 3e

2

NaOH Dichloromethane

TBAB

O O C

O

O

OO

CH2

O C O Ar' O 8

n

O

4a - 4e

CH3 Ar' :

, a

,

,

CH3 c

b O

d O

e O

O

Scheme 1 Synthesis of aliphatic aromatic random copolyesters 4a–e

stretching of the ester group at around 1,722–1,725 cm-1. However, it is interesting to find that all the spectra show the absorption band at 1,648 cm-1, corresponding to the stretching of the carbonyl group of 3,30 -methylene-bis(4-hydroxycoumarin) 1 molecule. Therefore it can be inferred that the biscoumarin molecule is an integral part of the polymer backbone. Stretching vibration of ester C–O appears at 1,080–1,083 cm-1. All the copolyesters show absorption around 2,850–2,950 cm-1 due to the presence of the alkyl group in the polymer chain. No significant absorption band in the OH stretching region was detected, confirming that the dihydroxy monomers effectively got involved in polymerization.

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Table 1 Physical properties of aliphatic aromatic copolyesters derived from 3,30 -methylene-bis(4-hydroxycoumarin) 1 with various aromatic diols 3a–e Yield (%)

ginh (dl/ g)a

IDT (°C)b

T10 (°C)c

Tmax (°C)d

Tg (°C)e

Tm (°C)e

Copolyester

Aromatic diols

4a

3a

85

0.26

231

275

321

17.8

63

4b

3b

78

0.24

229

262

371

-0.6

61

4c

3c

72

0.22

223

267

355

4d

3d

82

0.33

263

292

407

18.1

64

4e

3e

76

0.30

221

259

310

54.3

110

-11

43

a

ginh of the copolyesters were measured in NMP at 30 °C at the concentration of 0.5 g/dl

b

Initial decomposition temperature determined by TGA in nitrogen at heating rate 10 °C min-1

c

Temperature of 10 % weight loss

d

Maximum polymer decomposition temperature

e

Glass transition and melting temperature measured by DSC in nitrogen atmosphere at heating rate 10 °C min-1

Fig. 1 1

1

H-NMR spectrum of copolyester 4d in CDCl3

H-NMR analysis

The 1H-NMR spectrum of the copolyester 4d and 4e is highlighted in Figs. 1 and 2. The 4e spectrum shows characteristic signals in the range of 8.02–7.06 ppm due to the aromatic protons of biscoumarin and curcumin. The a, b unsaturated protons of the curcumin group expected to appear at about 7.53 ppm, buried along with the aromatic region corresponds to the b proton of the unsaturated carbon (signal b) and a signal at 6.85–6.90 ppm corresponds to a proton attached to the carbonyl group

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Fig. 2


1

H-NMR spectrum of copolyester 4e in DMSO

(signal c). The signal exceptionally appeared at 6.17 ppm (signal e) correspond to methyne proton of the enol structure. The methoxy protons (signal f) of the curcumin group and the bridged carbon bearing two protons (signal d) of the biscoumarin group appeared at 3.88 and 3.85 ppm, respectively, thus confirming that both curcumin and biscoumarin effectively get involved in polymerization. The signal characterizing the methylene groups of suberoyl unit (CH2, signal a) appears at 2.39–1.27 ppm. The characteristic signals regarding the chemical structure of copolyester 4a–d are as follows. 4a:1H-NMR (400 MHz, CDCl3, ppm): d 7.23–7.52 (m, Ar–H), 7.08 (s, Ar–H), 3.82 (s, biscoumarin bridged –CH2–), 1.25–2.76 (m, sebacoyl unit). 4b:1H-NMR (400 MHz, CDCl3, ppm): d 6.90–7.52 (m, Ar–H), 3.82 (s, biscoumarin bridged –CH2–), 1.25–2.76 (m, sebacoyl unit). 4c:1H-NMR (400 MHz, CDCl3, ppm): d 6.90–7.52 (m, Ar–H), 3.82 (s, biscoumarin bridged –CH2–), 2.32–2.36 (q, ethyl –CH2–), 1.17 (t, ethyl –CH3), 1.34–2.78 (m, sebacoyl unit). 4d: 1H-NMR (400 MHz, CDCl3, ppm): d 6.75–6.95 (d, Ar–H), 7.22–7.26 (d, Ar–H), 7.30–7.53 (m, biscoumarin Ar–H), 3.83 (s, biscoumarin bridged –CH2–), 1.65 (s, gem dimethyl unit), 1.25–2.76 (m, sebacoyl unit). Polymer solubility The solubility parameter is an important criterion that has to be considered for polymer processing. The solubility data (Table 2) shows that the copolyester 4a–

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Table 2 Solubility behavior of random copolyesters Polymer

DMSO

DMF

DCM

CHCl3

THF

Dioxane

DMAc

Acetone

NMP

4a

??

??

??

??

?

?

??

-

?

4b

??

??

??

??

?

?

??

-

??

4c

??

??

??

??

?

?

??

-

??

4d

??

??

??

??

?-

?-

??

-

??

4e

??

??

??

??

?-

?-

??

-

??

Solubility: ??, soluble at room temperature; ?, soluble on heating; ? -, partial soluble or swollen on heating; -, insoluble even on heating

e exhibited good solubility in chlorinated and polar aprotic solvents, which is attributed to the presence of non-linear biscoumarin moiety along with different flexible spacer leads to reduced rigidity of polymer chains [6]. All the copolyesters are readily soluble in most of the polar solvents, which might be due to the presence of the carbonyl group of biscoumarin unit in the polymer chain. Inherent viscosity The inherent viscosity of the copolyesters was determined in NMP at 30 °C at the concentration of 0.5 g/dl using a Ubbelohde suspended-level viscometer. Polymers with high molecular weights show higher viscosity values. In polymers with comparable molecular weight, the rigid polymers exhibit relatively higher viscosity values than flexible ones. From the values, it is evident that the polymer with the bisphenol backbone shows comparatively high ginh than other polymers. From Table 1, it is evident that the copolyesters with para-oriented aromatic diol 3a show higher viscosity values than the meta-oriented one 3b, which is attributed to the fact that meta orientation of the benzene ring reduces the rigidity, and hence effective volume, leading to lower viscosity values. The effect of introduction of flexible alkyl groups into the polymer backbone is very little, but measurable. Thermal properties The thermal properties of copolyesters were investigated by TGA at a heating rate of 10 °C min-1 in nitrogen atmosphere and the thermograms of the copolyesters 4a–e are shown in Fig. 3. TGA data of all the copolyesters are summarized in Table 1. The initial decomposition temperature (IDT) of all the copolyesters ranged from 221 to 263 °C and the T10 values were in the range of 259–292 °C. Generally, copolyesters with para-oriented monomeric diol are more thermally stable than their respective meta-oriented types, which may be attributed to the compact packing of polymer chains [18]. In addition, the length of the flexible spacer also has a significant effect in deciding the thermal stability of copolyesters and it is explicit from Table 1. Copolyester 4a is thermally more stable than 4b, which is attributed to the difference in the orientation of the aromatic unit. It is observed that the random copolyester 4d bearing bisphenol group shows higher thermal stability than the others.

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4a 4b 4c 4d 4e

100

Weight (%)

80

60

40

20

0 100

200

300

400

500

600

Temperature (°C) Fig. 3 TGA curves of biscoumarin copolyesters 4a–e

Glass transition temperature The glass transition temperature (Tg) of random copolyesters is determined by DSC at a heating rate of 10 °C min-1 under nitrogen atmosphere and presented in Table 1. The thermograms are shown in Fig. 4. The glass transition temperature is in the range of -11 to 54 °C. In general, incorporation of a bulky unit in the polymer chain restricts free rotation and enhances the Tg value, but the presence of a flexible spacer reduces the rigidity of the polymer chain and ultimately leads to lower Tg values, which is explicit from Table 1. The bulky biscoumarin unit 1 and the various aromatic diols 3a–e forming copolyesters along with longer flexible spacers result in lower Tg values [1, 19]. Copolyester 4a containing hydroquinone unit shows highest Tg value than its corresponding copolyester 4b bearing resorcinol unit and this could be attributed to the difference in stereoregularity along the polymer segments. Thus, the copolyester 4a maintains its segmental symmetry (para orientation) there by enhancing its rigidity and forms a closed-pack arrangement [20, 21] and ultimately leads to a higher Tg value than its unsymmetrical segmented (meta orientation) [22, 23] copolyester 4b. It is interesting to observe that among the series of random copolyesters, the polymer having a pendant ethyl unit in the resorcinol group exhibited lower Tg values, whereas polymers with curcumin groups at the backbone show higher Tg values. X-ray diffraction X-ray diffraction patterns of copolyesters corroborated the semicrystalline and amorphous character of random copolyesters. All the copolyesters showed wide

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4e

Tg=54.3 4d

Heat flow endo

Tg=18.1

4c

Tg=-11 4a

Tg=17.8

-20

0

20

40

60

80

100

120

Temperature (°C)

Intensity (a. u)

Fig. 4 DSC thermograms of random copolyesters

4a 4b

4d 4e

10

20

30

40

50

2θ Fig. 5 X-ray diffractograms of copolyesters

123

123

9

14

15

–

4c

4d

4e

Stda

33

15

14

10

13

Ampicillin (100 lg/ml) as standard

11

4b

12

10

–

17

15

10

13

12

10

9

–

14

13

13

12

12

38

15

14

13

13

14

9

100 lg

–

17

15

15

14

14

11

150 lg

–

18

15

11

12

12

8

50 lg

B. subtilis

40

20

17

12

13

13

10

100 lg

–

22

20

14

13

13

10

150 lg

–

17

16

12

11

12

8

50 lg

S. aureus

37

19

16

13

11

12

8

100 lg

–

19

18

14

13

13

10

150 lg

7200

a

9

10

4a

50 lg

150 lg

50 lg

100 lg

s. typhi

E. coli

1

Compound

Table 3 Zones of inhibition (in mm) of the compounds against various bacteria

Author's personal copy N. Kandaswamy, N. Raveendiran


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halos over the range of 2h = 10–40 (Fig. 5), suggesting essentially the amorphous nature of the derived polyesters. This could be attributed to the presence of bulky biscoumarin group into the polymer backbone, which hinders the dense chain packing of polymer chains, resulting in the amorphous nature of these polyesters, which is also reflected in their improved solubility. However, the copolyesters 4a, 4b, and 4d exhibit some degree of crystallinity, showing peaks at 2h & 17.5° and 20.1°. Antibacterial activity The results of antimicrobial activity are presented in Table 3 and the comparative activities of monomer 1 and copolyesters against various microbes are shown in Figs. 6, 7, 8, and 9. The monomer and the copolyesters are active against microbes such as Gram-negative species, viz, E. coli and S. typhi, Gram-positive species viz, B. subtilis and S. aureus with inhibitory zone ranges of 10.0–17.0 and 10.0–22.0 mm, respectively. All the copolyester exhibiting enhanced antibacterial activity compared with the monomer 1 and this is due to the lipophilicity of the copolyesters. In general, more lipophilic compounds can penetrate greater the lipophilic cell membranes of Gram-positive bacteria, while less lipophilic compound are more liable to penetrate the cell wall of Gram-negative bacteria. It is explicit from Table 3 that of all the copolyesters, 4e exhibits extreme antibacterial activity and this is attributed to the ability of a,b-unsaturated ketone from curcumin, undergo a conjugated addition to a nucleophilic group like thiol group in an essential protein of the microorganism. The presence of more lipophilic methoxy moiety at the position 4,40 of curcumin and small percentage of end phenolic and hydroxyl group of coumarin has contributed wide spectrum of antibacterial activity. From the above discussion, it is clear that the structure of the copolyester has a distinct effect on the antibacterial activities, thus proving that these could act as efficient antibacterial polymers.

Fig. 6 Antibacterial activity against E. coli

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Fig. 7 Antibacterial activity against S. typi

Fig. 8 Antibacterial activity against B. subtilis

Antifungal activity The antifungal activity of copolyesters was studied against two fungal species, viz., A. niger and A. flavus, and is graphically depicted in Figs. 10 and 11. Antifungal activity of both monomer and copolyesters at various concentrations is shown in Table 4. Both the copolyesters 4d and 4e exhibited very good activity against A. niger, and A. flavus. Increased lipophilicity enhances the penetration of the copolyester into lipid membrane and these copolyesters also disturb the respiration process of the cell and thus block protein synthesis, which restricts further growth of the organism. It is inferred that the lipophilicity character of copolyester enhances the antifungal activity of the copolyesters.

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Fig. 9 Antibacterial activity against S. aureus

Fig. 10 Antifungal activity against A. niger

Anticancer evaluation of copolyesters against lung cancer (Hep-2) cell line Viable cells were determined by absorbance. The copolyester concentration required for a 50 % inhibition of viability (IC50) was determined graphically from Fig. 12. The effect of the copolyesters on the proliferation of Hep-2 cell line was expressed as the % cell viability. The affected Hep-2 cell line at different concentrations is shown in Table 5. The anticancer activity results indicated that most of the copolyesters exhibited inhibition activity against with varying intensities on cancer cell line. Copolyesters 4c and 4e exhibited significant antitumor activity (IC50 = 64, 34.4 lg/ml) and possess ethyl resorcinol and curcumin moiety, which is known to exhibit antitumor activity along with the biscoumarin unit. The covalent conjugation of curcumin and biscoumarin through ester linkage increases its

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Fig. 11 Antifungal activity against A. flavus

Table 4 Inhibition zone (mm) of the compounds against fungus Compound

A. niger 50 lg

A. flavus 100 lg

150 lg

50 lg

100 lg

150 lg

1

–

9

9

–

–

10

4a

9

9

11

10

10

12

4b

10

10

11

9

10

10

4c

11

11

12

10

10

12

4d

13

13

15

12

13

13

4e

15

15

17

14

15

18

Stda a

18

Amphotericin (20 lg/ml) as standard

Fig. 12 Inhibitory effect of copolyesters concentration on Hep-2 cell line

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16


7205

Table 5 In vitro anticancer activity of synthesized compounds Compound

IC50 (lg/ml)a Hep-2b

Viability % Sample concentration (lg/ml) 1,000

500

250

125

62.5

31.2

15.6

7.8

Cell control

1

11.53

23.07

36.53

46.15

61.53

69.23

82.69

92.30

100

108.2

4a

13.46

26.92

38.46

50.00

63.46

82.69

90.38

94.23

100

128.9

4b

13.46

25.00

36.53

50.00

61.53

73.07

82.69

90.38

100

118.2

4c

7.69

13.46

25.00

38.46

50.00

63.46

75.00

86.53

100

67.4

4d

11.53

21.15

36.53

48.07

61.53

73.07

82.69

92.30

100

120.3

4e

5.76

15.38

26.92

40.38

46.15

51.92

61.53

73.07

100

34.4

a

‘‘IC50, compound concentration required to inhibit tumor cell proliferation by 50 %’’

b

Lung cancer cell line (Hep-2)

molecular size and steric hindrance may improve its cellular permeability and restrict its cancer progression [24, 25]. It is explicit from Table 5 that a low concentration of copolyester 4e and 4c induced greater anticarcinogenic activity effects on the Hep-2 cell line than the individual agents.

Conclusions In summary, aliphatic aromatic biscoumarin random copolyesters have been synthesized via phase transfer-catalyzed interfacial polycondensation of 3,30 methylene-bis(4-hydroxycoumarin) and various aromatic diols with sebacoyl chloride. All the copolyesters exhibited very good solubility in various organic solvents and this is attributed to the incorporation of the bulky biscoumarin unit. Inherent viscosities were in the range of 0.22–0.33 dl/g. The structures of the random copolyesters were confirmed by FTIR and 1H-NMR spectroscopy. Low glass transition temperatures observed from DSC and XRD patterns of the copolyesters reveal that all these copolyesters are found to be amorphous in nature. The copolyesters derived from 3,30 -methylene-bis(4-hydroxycoumarin) show no weight loss below 223 °C, indicating its good thermal stability, and have a low glass transition temperature value in the range -11 to 54 °C. Of particular interest, copolyester 4d with stereoregularity and low glass transition temperature there by maintaining good thermal stability are preferable as biomaterials in various biomedical applications. On the basis of above-mentioned physical properties and antimicrobial and anticancer results, these copolyesters exhibited promising results in the field of biomaterials for further development. Acknowledgments The authors would like to thank CLRI technical staff for their analytical support.

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