Self-assembled cardanol azo derivatives as antifungal agent with chitin-binding ability

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ARTICLE IN PRESSG ModelIOMAC 4341 1–7

International Journal of Biological Macromolecules xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

elf-assembled cardanol azo derivatives as antifungal agent withhitin-binding ability

enial Mahata1, Santi M. Mandal1, Rashmi Bharti, Vinay Krishna Gupta,ahitosh Mandal, Ahindra Nag, Golok B. Nando ∗

ubber Technology Centre, Central Research Facility, Department of Chemistry, School of Medical Science and Technology, Indian Institute of Technologyharagpur, Kharagpur 721302, WB, India

r t i c l e i n f o

rticle history:eceived 17 March 2014eceived in revised form 14 April 2014ccepted 3 May 2014vailable online xxx

a b s t r a c t

Cardanol is a non-isoprenoic phenolic lipid-mixture of distilled cashew nut shell liquid obtained fromAnacardium occidentale. Herein, cardanol is purified from cashew nut shell liquid (CNSL) and synthesizedto new compounds with different azo amphiphiles. These synthesized compounds are allowed to self-assembled in hydrophobic environment and checked antifungal activity against Candida albicans. Self-assembled structure of CABA showed higher antifungal activity (16 �g/mL) and chitin-binding ability in

eywords:ntifungal hydrogelardanolhitin-binding assayelf-assembly

comparison to CAP and CANB. Furthermore, the self-assembled azo amphiphiles are immobilized withsilver ions to prepare hydrogel which showed eight folds enhanced antifungal activity. Toxicity is reducedby several folds of self-assembled or hydrogel structure in comparison to pure compounds. Thus, the self-assembled structure of amphiphiles and their hydrogels have been found to be new macromolecules ofinterest with potential use as antifungal drugs.

© 2014 Published by Elsevier B.V.

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. Introduction

Design and development of non-covalent self-assemblyupramolecules from renewable resources have received remark-ble attention due to their potential application as bioactive agentsn tissue engineering to drug development [1,2]. Several studiesave shown the potentiality of self-assembled value added mate-ials as ranging from coating on medical device to antibacterialctivity [3,2]. The increased incidences of multi-drug resistantungal pathogens are of great threat in infection control. Numerousttempts have been made to design or identify novel antifungalompounds with unique characteristics. Fungal infections areften serious with an associated fatality rates ranging from 50%o 100%. Most fungal infections are caused by Candida albicans, ofral-gastro-intestinal track of man and other warm-blooded ani-als [4,5]. Chitin is a homopolymer of �1,4-N-acetylglucosamine

Please cite this article in press as: D. Mahata, et al., Int. J. Biol. Macrom

GlcNAc) and takes part in cell wall synthesis of almost all fungi6–8].

∗ Corresponding author. Tel.: +91 3222 282291/3222 283194.E-mail addresses: [email protected] (D. Mahata),

[email protected] (G.B. Nando).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.ijbiomac.2014.05.017141-8130/© 2014 Published by Elsevier B.V.

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Cardanol is one of the promising renewable natural resources,a waste from the cashew industry, obtained CNSL [9]. It is yel-low to brown colored phenolic lipid carrying a C15 side chainat meta-position with various degrees of unsaturation, knownas m-pentadecenylphenol (Fig. 1a). Distillate CNSL is a mixtureof non-isoprenoid phenolic lipid, obtained from roasting shellswhich contains cardanol (60–65%) with cardol (15–20%) andother polymeric material (10%) [10]. The major structural advan-tages of cardanol having reactive phenolic group and unsaturatedhydrophobic alkyl side chain at the meta-position of phenolicgroup. Due to this unique nature, cardanol and its derivative couldbe recognized as amphiphilic building block with supramoleculararchitecture [11].

Cardanol display several biological activities including antimi-crobial [12,13] antioxidant [14,15] and antitumor [16] howeverstrong cytotoxicity of this kind of compound limits its appli-cation as lack of biocompatibility [17,18]. In order to decreasethe toxicity of cardanol derivatives and try to improve antifun-gal activity, our aim was extended to develop self-assembledsupramolecular structure. In the present study, we have designedand synthesized cardanol-based three different functional polar

ol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.05.017

azo amphiphiles, 4-[(4′-cardanyl)azo] benzoic acid (CABA), 4-[(4′-cardanyl)azo] phenol (CAP), 4-[(4′-cardanyl)azo] nitro benzene(CANB). The antifungal activities of all synthesized self-assembledcompounds along with their silver immobilized hydrogel have

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dx.doi.org/10.1016/j.ijbiomac.2014.05.017


http://www.sciencedirect.com/science/journal/01418130

http://www.elsevier.com/locate/ijbiomac

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mailto:[email protected]


ARTICLE IN PRESSG ModelBIOMAC 4341 1–7

2 D. Mahata et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx

Fig. 1. Images for cardanol purification and characterization. Image of cashew fruit and chemical structure of cardanol (a), HPLC chromatogram of technical CNSL (blue) andQ8p (1); trF ). (Fort

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ure cardanol (pink) (b). The peaks are mentioned in the chromatogram as cardol

T-IR (c), 1H NMR (d), 13C NMR (e), MALDI-MS spectrum: [cardanol + potassium]+ (fo the web version of the article.)

een investigated against C. albicans, with chitin (N-acetyl-d-lucosamine) binding abilities.

. Materials and methods

.1. Chemicals and reagents

Technical standard Cashew nut shell liquid was purchased fromatya Cashew Industry (India). Para amino Benzoic acid, para aminohenol, para amino nitro benzene, sodium nitrite and sodiumydroxide were purchased from Sigma Aldrich (USA). Chitin, Triton-100, potassium phosphate buffer, glutaraldehyde, Epon-Aralditeesin and hydrogen peroxide were from Merk (India). All solventsere of analytical grade from Merck (India).

.2. Instrumentation

.2.1. High performance liquid chromatography (HPLC)


The HPLC analysis was done on an Agilent HPLC system (1100eries), comprising of two reciprocating pumps, a 481 variable-avelength detector, an injector with 20 �L loop, and ZORBAX

B C-18 (4.6 mm × 150 mm, particle size 5 �m) Agilent column

iene (2); diene (3); and monoene (4). Analytical characterization of cardanol with interpretation of the references to color in this figure legend, the reader is referred

was used. The mobile phase was acetonitrile/water/acetic acid(80:20:1) at a flow rate of 1 mL/min. Absorbance was monitoredat 250 nm. Each analysis was carried out of 5 mg/mL in acetonitrileand filtering through a C18 Sep-Pak cartridge (Water Associates,Milford, MA).

2.2.2. Ultraviolet–visible spectroscopy (UV–vis)The UV–vis absorbance spectra were scanned (300–600 nm) and

recorded on a Shimadzu UV-1601 against only solvent as blankreference. Experiments are performed by keeping concentration0.02 mM samples. In all experiments, solutions were taken in quartzcuvette of 1-cm path length.

2.2.3. Fourier transform infrared (FTIR)For IR experiment, samples were dissolved in chloroform and


placed onto a KBr pellet and dried. The dried specimen wasrecorded on Shimadzu 8400 FT-IR spectrophotometer. Absorbancespectra were obtained from 4000 to 400 cm−1 with a 4 cm−1 reso-lution, Background spectra were also collected and subtracted.

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ARTICLEIOMAC 4341 1–7

D. Mahata et al. / International Journal of

.2.4. Nuclear magnetic Resonance (NMR)The 1H and 13C spectra were taken on a Bruker DPX200

500 MHz for 13C and 200 MHz for 1H) in CDCl3 using tetram-thylsilane (TMS) as an internal standard. Both cases 2 mg/mLoncentrated solutions were prepared. All signals were referencedo TMS to within ±0.1 ppm.

.2.5. MALDI-TOF-MSTo obtain MALDI mass spectra, a Voyager time-of-flight mass

pectrometer (Applied Biosystem, USA), equipped with 337 nm N2aser was used and operated in accelerating voltage 20 kV. The spec-ra were recorded in positive ion linear mode. Reproducibility of thepectrum was checked four times from separately spotted samples.

.2.6. Circular dichroism (CD)The circular dichroism (CD) spectrum was recorded at room

emperature using a Jasco J-815 spectropolarimeter equipped with Jasco PTC-423 S Peltier temperature controller. The scanningate was 50 nm min−1 with a response time of 2 s. Spectrum wasecorded at standard sensitivity (100 mdeg) with a data-pitch of.5 nmin continuous mode. The scanning range was 260–190 nmnd spectrum was the average of two consequent accumulations.he baseline was corrected by subtracting the corresponding bufferlank.

.2.7. Scanning electron microscopy (SEM)To record the SEM images of self-assembled and organogels,

amples were dissolved in dichloromethane, 5–10 �L solutionlaced on the glass cover slip following drop-caste method. Sam-les were then fixed onto a graphite stub and kept in an auto sputteroater (E5200, Bio-Rad) under low vacuum for gold coating up to20 s. Surface morphology was studied by using a scanning electronicroscope (JEOL JSM 5800) with an accelerated voltage between

and 20 kV.

.2.8. Transmission electron microscopy (TEM)The TEM was performed in JEOL JEM 2100 instrument. A small

ortion of self-assembled samples were drop cast on a Cu-grid.fter drying the grid at ambient temperature, samples were directly

maged under TEM.

.2.9. Isothermal titration calorimetry (ITC)The titration was performed using a ITC200 Systems (GE

ealthcare, USA) coupled with non-reactive Hastelloy® cells forhemical resistance. The titrations were carried out 0.25 × 10−3 molelf-assembled solution with 0.1 × 10−3 mol chitin in dimethylulphoxide (DMSO). All solutions were degassed right before thexperimental runs in same condition at 25 ◦C and at 180 s intervalstilizing a stir speed of 310 rpm. Blank ITC experiments were doneo correct heat of dilution effects. Origin 7.0 (OriginLab Corp., MA)as used to analyze the ITC data to determine the binding con-

tant (K) and enthalpy of binding (�H) directly from the bindinghermograms.

.2.10. Fluorescence activated cell sorter (FACS)A FACS flow cytometer (FACS Caliber flow cytometer, Becton-

ickinson, USA) was used to obtain the data with a wavelengthf laser excitation at 520 nm. Data were analyzed by Cell QuestPro

oftware attached with FACS Caliber.

.3. Purification of cardanol


Cardanol was extracted and purified from technical CNSLccording to procedure Kumar et al. [10]. Technical CNSL (100 gm)as dissolved in methanol (320 mL), and ammonium hydroxide

25%, 200 mL) was added, stirred for 15 min at room temperature.

PRESSical Macromolecules xxx (2014) xxx–xxx 3

Now the solution was extracted with hexane (4× 200 mL). Theorganic layer was washed with 5% HCl (100 mL) followed by dis-tilled water (100 mL). Activated charcoal (10 g) was added to theorganic layer, stirred for 10 min, and filtered through Cellite (15 g).The filtrate was dried over anhydrous sodium sulfate, and con-centrated to get pure cardanol (60 g). The purity of unsaturatedcardanol was confirmed by HPLC and characterized using FTIR, 1HNMR, 13C NMR and MALDI-MS analysis.

2.4. General synthesis of cardanol diazonium amphiphiles

Syntheses of three different cardanol-diazonium compoundswere carried out following to electrophilic substitution reaction[19]. Commercially available three different aniline derivativessuch as p-aminobenzoic acid, p-aminophenol, p-nitrophenol wasused. Aniline derivatives (25 mmol) were dissolved in 50 mL 1(N)HCl solution and diazotized with sodium nitrite (1.7 g, 25 mmolin 10 mL of water) solution at 0–4 ◦C with constant stirring sep-arately. The solution was diluted with chilled methanol. Cardanol(7.4 g, 25 mmol) was dissolved in a chilled 5% methanolic potassiumhydroxide solution and added dropwise to the diazonium salt solu-tion. The red dye formed was stirred for a further period of 6 h andpoured into dilute HCl solution with stirring. The red dye was sepa-rated, washed thoroughly with water, and dried. The dye was thenpurified by column chromatography on silica gel (60–120 mesh)using petroleum ether and ethyl acetate (10:1) as eluent. The puritywas confirmed by using UV–vis, FTIR, NMR and MALDI-MS analysisand described details characterization in supplementary section.

2.5. Preparation of self-assembly

Ten mg/mL solution of compound CABA, CAP, and CANB wereprepared separately in hydrophobic (chloroform) solvent. All solu-tions were rotated by vortex shaker for 24 h with 1000 rpm at roomtemperature for the preparation of self-assembled structure.

2.6. Preparation of hydrogel

The self-assembled samples (40 mg) were dispersed in 1 mL ofethanol. Freshly prepared silver nitrate solution was added to theethanolic solution of each self-assembled compounds separatelyat room temperature. Concentration of AgNO3 was maintained at1 mM in final solution. The mixture was kept without any disturb-ance for 25–30 min at room temperature to become a viscous gel.

2.7. Chitin-binding assay

Ten mg of chitin (N-acetyl-d-glucosamine) was suspended in the2 mL of 3% hydrogen peroxide for 3 h and dissolved it by the additionof 3 mL 55% H2SO4 [20]. After complete dissolution of chitin, 5 mLof 25% sodium hydroxide was added to achieve pH 5–6 and cen-trifuged the solution to remove the salt of sodium sulphate. Finally,220 �L chitin solution is diluted with DMSO to make 0.1 mM solu-tions and titrated with 0.25 mM all self-assembled amphiphiles forITC experiments.

2.8. Flow cytometry analysis

The fluorescence intensity of Calcofluor White (CFW) congu-jated fluorescein isothiocyanate (FITC) was measured by flowcytometry analysis. In brief, C. albicans cells were grown to mid log


phase, centrifuged, washed, and resuspended in buffer to obtain2 × 104 CFU/mL. The fungal suspension was diluted to 104 CFU/mLin the PBS buffer. All compounds of 8 �g/mL (as the MIC value ofhydrogel of CABA) concentrations was incubated for 1 h and after

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hat, cells were washed gently twice with PBS buffer, further incu-ated with CFW-FITC stained in dark for 10 min. Cells without anyreatment used as control.

.9. SEM observations of Candida cells

One milliliter of the C. albicans cell suspension at the concen-ration of 1 × 107 cells/mL was inoculated on a Sabouraud dextrosegar plate and incubated at 37 ◦C for 12 h. The cells were collectedfter centrifugation and washed with PBS (1×) buffer. A 10% DMSOreated culture was used as a control and samples with 8 �g/mL wasreated the cells for 1 h. After that, cells of Candida was withdrawnrom the treated sample after centrifugation at 5000 rpm for 3 minnd fixed for SEM study before coated with gold and viewed underEM. For cross section of Candida cells, samples were fixed in 3%lutaraldehyde, rinsed in buffer, post fixed in 1% osmium tetrox-de in 0.1 M potassium phosphate for 2 h at 4 ◦C, dehydrated inthanol and embedded in Epon-Araldite resin. Ultra thin sectionsere observed in FESEM.

.10. Cytotoxicity assay

MTT [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-henyl)-2-(4-sulfophenyl)-2H-tetrazolium)], assay was per-ormed to determine cell cytotoxicity following the methodescribed earlier by Mandal et al. [21]. Human Embryonic Kidney93 (HEK cells (2.0 × 103) were seeded in 100 �L complete DMEMedium per well in 96 well plates. Plates were incubated at

7 ◦C in 5% CO2 for 24 h for cell attachment. Cells were treatedith individual compounds with variable concentration from 5

o 100 �g/mL and incubated at 37 ◦C in 5% CO2 for 48 h. Threeells were used in the 96 well plates for each derivative and

epeated three times. For the MTT assay, thiazolyl blue tetrazoliumromide solution (100 �L; 1 mg/mL) in incomplete medium wasdded and this mixture incubated for 4 h. After that, 100 �L ofimethylsulphoxide (DMSO) was added and the plates wereotate for 5 min. Optical density was recorded at 550 nm withMSO as the blank. Percentage of cell viability was plotted againstoncentrations of derivatives. Cells treated without any compoundsed as control.

.11. Antifungal assay

The minimum inhibitory concentration (MIC) of all synthe-ized compounds against planktonic cells of Candida strain wasetermined according to the guidelines of the Clinical and Lab-ratory Standards Institute [CLSI (M27-A2 document, NCCLS]. A00 �L of total volume in each well was made by amendinghe growth medium (RPMI 1640) with different concentrationsf individual compounds (512–1.0 �g/mL) and fungal inoculum3.5 × 106 CFU/mL). Two wells of the plate used as growth (withoutntifungal) and sterility (without inoculum) controls. The platesere incubated at 30 ◦C for 18 h. Experiments were carried out in

6-well plate in triplicate on three different sets [22].

.12. Statistical analysis


Data were analyzed as the mean and standard deviationmean ± SD) of three test replicates for each sample. The statisti-al analysis was performed by analysis of variance (ANOVA) usingraph pad Prism 5 Software at the 0.05 significance level.

PRESSical Macromolecules xxx (2014) xxx–xxx

3. Result and discussion

3.1. Characterization of pure cardanol

Reversed phase HPLC was used to purify cardanol, the obtainedchromatogram showed the separation and purification of cardanolfrom crude CSNL (Fig. 1b). Pure cardanol showed three peaks inHPLC chromatogram corresponding to three unsaturated com-ponents: 74.2% 3-[8(Z),11(Z),14-pentadecatrienyl] phenol, l 1.9%3-[8(Z),11(Z),14-pentadecadienyl] phenol, 14.8% 3-[8(Z),11(Z),14-pentadecadecenyl] phenol. The purified cardanol was furthercharacterized by FTIR, 1H NMR, 13C NMR, and mass spectroscopy. InIR spectrum (Fig. 1c), peaks observed at 1264 and 1152 cm−1 weredue to symmetric and asymmetric stretching of vinyl unsaturationin the side chain, respectively. The absorption bands at 3349 cm−1

for O H and 1588 cm−1 for Ph O stretching indicate the presenceof phenolic group in cardanol. 1H NMR analysis (Fig. 1d) shows aro-matic proton peaks at ı 6.5–7.2 ppm range. The peaks in the rangeı 4.9–5.4 ppm represents to vinyl unsaturation and upfield region,i.e., between ı values of 3.0 and 0.8 ppm, suggesting the presenceof alkyl chain of unsaturation. In 13C NMR (Fig. 1e), the peaks due toolefinic unsaturation appear at ı values in the range 114–132 ppm.There are also aromatic carbon peaks in the range 112–128 ppm.The peak at 156 ppm is due to the aromatic carbon attached to oxy-gen atom. The aliphatic carbons of the side chain appear in the rangeı 14–36 ppm. Further, the mass spectra (Fig. 1f) give base peak at338 with potassium ion confirms the purity of cardanol.

3.2. Self-assembled and hydrogel structure characterization

The molecular interactions involved in the self-assembled com-pounds have been investigated by spectroscopic analysis. FT-IRanalysis was performed for pure, self-assembled and hydro-gel structures of CABA, CAP, and CANB in chloroform solution(Fig. 2). The results suggest that the unsaturated alkyl side chainexhibits carbon–carbon symmetric stretching frequency (around1233–1219 cm−1) for CABA, CAP, and CANB shifted by 13, 10and 3 cm−1, respectively toward lower frequency due to the �–�stacking in the unsaturated side chain. In addition, the aromatic car-bon–carbon double bond stretching also shifted from 1477 cm−1

to 1452 cm−1 and 1465 cm−1 to 1457 cm−1, suggests the forma-tion of �–� stacking for CABA and CAP, respectively. Interestingly,CANB showed opposite result by shifting the 6 cm−1 carbon–carbondouble bond toward higher frequency due to the strong repul-sion between terminal nitro group for assembly compared to pureCANB. The opposite results are observed at silver based hydrogel,where silver incorporated into the double bonds and reduces the�–� stacking interaction. It is interesting to note that, two types ofhydrogen bond stretching are observed at 3308 and 3412 cm−1 inthe assembly and 3297, 3429 cm−1 in the gel but pure compoundshows only a single peak at 3303 cm−1 for compound CABA suggestsextensive hydrogen bonding between acid group at 3308 cm−1 andnew peak at 3412 cm−1 between hydroxy group. The terminal acidgroup plays a vital role in the formation of rolling cylindrical-likestructure. The broadening of hydrogen bonding peak at 3391 cm−1

in the assembly and 3426 cm−1 in hydrogel indicate the formationof beta sheet-like structure in CAP, whereas incorporation of termi-nal nitro group in CANB shows there is no such shifting of hydroxylstretching. The peak at 3445 cm−1 indicates absence of extensiveterminal hydrogen bonding units in CANB assembly.

CD is a valuable spectroscopic technique for studying the con-formation of secondary structures as alpha-helix and beta-sheet


[23]. The broad negative bands are observed at 215 and 212 nmcorresponding to n–�* transition of self-assembled CABA and CAP,respectively. Moreover, shift of 7 nm in same region of CABA and4 nm in CAP indicates the aggregation of hydrophobic moiety

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D. Mahata et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx 5

F -[(4′-cardanyl)azo] benzoic acid (CABA, i); 4-[(4′-cardanyl)azo] phenol (CAP, ii); 4-[(4′-c B (d), pure (black, i); self-assembled (blue, ii) and hydrogel gel (red, iii). (For interpretationo sion of the article.)

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Table 1Antifungal activity of all synthesized compounds against C. albicans.

Compounds name MIC (�g/mL)

Cardanol 64CABA 32CAP 64CANB 64Self-assembled CABA 16Self-assembled CAP 64Self-assembled CANB 64

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ig. 2. Scheme for derivatives synthesis from cardanol. Scheme for synthesis of 4ardanyl)azo] nitro benzene (CANB, iii) (a) and FTIR spectra of CABA (b), CAP (c), CANf the references to color in this figure legend, the reader is referred to the web ver

y �–� stacking interaction. A broad positive band at 209 nmnd negative band near 193 nm are responsible for random coilonformation in CANB (Fig. S1). The terminal nitro group haso hydrogen bonding unit and created strong negative dipolarepulsion between themselves therefore only entanglement ofydrophobic chains act as driving force for formation of randomoil structure.

The morphology of self-assembled and hydrogel structuresharacterized by field emission scanning electron microscopyFE-SEM) exhibits fibrillar to thallus like features (Fig. 3). The trans-

ission electron microscopy (TEM) of self-assembled structureseveals nanofiber structure with variable shape and sizes (Fig. S2).ABA shows a highly ordered and symmetrical arrangement ofolling cylindrical structure with planar sheet formation in theirurface. The aggregation of fibers show highly ordered and sym-etrical arrangement may be due to the strong hydrogen bonding

etween terminal acid group and three types of �–� stackingnteraction at different positions indicating the layer sheet type

orphology of CABA. CAP exhibits sheet like layered structureith some disorder aggregation because of their weak terminalydrogen bonding interaction, whereas CANB shows highly dis-rder random aggregation on their hydrophobic side chain by �–�tacking interaction. TEM images revealed also show that layerheet fibers are rolled into regular pattern, indicating the forma-


ion of rolling sheet with 500 nm diameter in CABA. Interestingly,he lamellar type layer by layer fiber sheet is formed in CAP with aiameter of 83 nm but the CANB shows a solid nanorod of diameter2 nm.

CABA hydrogel 8CAP hydrogel 32CANB hydrogel 32

3.3. Antifungal activity

All the synthesized compounds their self-assembled structuresand hydrogels were tested against a multi-drug resistant humanfungal pathogen, C. albicans SJ11. The strain, SJ11 is a hospital isolateand characterized earlier with culture conditions and activity assay[24]. It was observed that the hydrogel of CABA was more activeamong all of them. Silver based hydrogel was two folds more activethan self-assembled compounds which showed further two foldsmore potent than pure compounds (Table 1).


3.4. Chitin-binding ability with self-assembled compounds

The probable mechanism of action is evaluated with their chitin-binding properties. Only the self-assembled compounds were used

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ig. 3. FE-SEM images of prepared self-assembled and hydrogels. Images were capydrogel (f), from CANB self-assembled (g) and hydrogel (i). Macroscopic view of

btained from CABA (b), CAP (e) and CANB (h).

o evaluate their chitin binding abilities rather than their hydrogelo normalize the action of silver ions. ITC experiment was per-ormed to compare their binding abilities with chitin that makes

rigid layer surrounding the membrane, the cell wall composi-ion of C. albicans. The binding isotherm for the chitin fragment–GlcNAc–) is shown in Fig. 4. The interactions are based on bothydrophobic as well as hydrophilic nature and entropically derivedecause the values of �S and �H are positive (Table S1). The

arge surface area of highly symmetrical sheet-type aggregationnd formation of efficient hydrogen bond of carboxyl group inelf-assembled CABA have higher magnitude of chitin binding abil-


ty than self-assembled CAP due to the weak hydrogen bondingbility of hydroxyl group. However, weak aggregation behaviorf self-assembled CANB shows lower binding ability than CABAut slightly higher than CAP may be the higher hydrogen bonding

ig. 4. ITC based binding isotherm plot of self-assembled compounds with chitin. Isotherc).

from CABA self-assembled (a) and hydrogel (c), from CAP self-assembled (d) andssembled and hydrogel solution in glass vial as (i) and (ii), respectively. Hydrogel

ability of nitro group than hydroxyl group. Additionally, fluores-cence analysis demonstrates that self-assembled compounds havechitin-binding ability by the quenching of CFW-FITC dye fluores-cence dye in C. albicans SJ11 (Fig. S3). Cells without any treatmentshowed the fluorescence intensity of 97.63%, whereas intensity wasdecreased when the cells were treated with self-assembled com-pounds before incubation with CFW-FITC dye. Maximum reduction(37.23%) was observed for CABA in comparison to CAP (21.76%)and CANB (22.34%). The higher fluorescence quenching suggeststhat CABA have higher chitin-binding affinity than the CAP orCANB. Moreover, CAP and CANB, both have more or less similar


fluorescence quenching ability as well as similar chitin-bindingability. Both experiments, ITC and FACS results indicate that all self-assembled compounds are efficient for binding with chitin and alsovery good agreement for antifungal activities.

m fittings are obtained from interaction of chitin with CABA (a), CAP (b) and CANB

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.5. Cross-sectional characterization of Candida cells

The anti-candidal activity of hydrogels was also confirmed fromE-SEM study. The SEM photomicrographs of the untreated cells of. albicans showed the usual structure of nucleus and vacuoles. Theffect of hydrogels derived from CABA over Candida revealed thatells were dense with vesicles and membranous bodies (Fig. S4).ost remarkable alterations was observed in cell membrane and

ell wall with drastic damage. The cells were less damaged for CAPnd CANB compared to CABA. After damage in cell wall, cells wereound to be collapsed and lysed followed by an outflow of the cyto-lasmic material caused fungal growth inhibition [25]. Moreover,he unsaturated lipid chain of cardanol increased the permeabilityf liposomal bilayer membrane resulting leakage of potassium ionsrom cell [13], Therefore, enhancement of membrane permeabil-ty was another major target of compounds for antifungal activity.he activity was increased for self-assembled structure due to theirtrong outer hydrophobic environment which easily binds to mem-rane lipids, activity was also more increased for hydrogel mighte due to the presence of additional silver ions which have knowntrong antimicrobial properties [26].

.6. Cytotoxicity

MTT assay was performed to examine the compatibility andfficacy of the compounds in biological systems. The results showhat pure compounds have highest toxicity in comparison withelf-assembled or hydrogel derivatives. The self-assembled struc-ure showed less toxicity than hydrogel (Fig. S5 and Table S2). Thelightly more toxic nature of hydrogel might be due to the presencef silver ions. These results are in good agreement for less-toxicature of self-assembled and their silver based hydrogel for safe uset their required concentrations. During self-assembly, all the func-ional moieties interact with each other, therefore the hydrophobicnteraction results to reduce the toxicity.

. Conclusion

In conclusion, the present study demonstrates the self-ssembled structure of cardanol based on azo-amphiphilesontaining carboxy, hydroxy and nitro functional moieties. Theelf-assembled structure was formed due to strong hydrogen bond-ng and �–� stacking with hydrophobic interaction among them.elf-assembled compounds represent the amphiphiles nature and


oves to macromolecular organization from liquids to semi-solidels. Self-assembled and hydrogel of 4-[(4′-cardanyl)azo] benzoiccid showed higher antifungal activity against multi-drug resistant. albicans with chitin-binding properties in comparison to other

[[[

PRESSical Macromolecules xxx (2014) xxx–xxx 7

tested synthesized complexes. Thus, toxic cardanol turned to less-toxic self-assembled or hydrogel macromolecular organization asa potential antifungal agent.

Acknowledgment

D. Mahata acknowledges University Grant Commission (UGC),

New Delhi, India for providing financial assistance as JuniorResearch fellowship.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2014.05.017.

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Self-assembled cardanol azo derivatives as antifungal agent with chitin-binding ability