1336 | New J. Chem., 2014, 38, 1336--1345 This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

Cite this: NewJ.Chem., 2014,

38, 1336

p-Sulfonatocalix[4]arene as a carrier forcurcumin†

Paulpandian Muthu Mareeswaran,ab Eththilu Babu,a Veerasamy Sathish,a

Byoungkook Kim,c Seong Ihl Woob and Seenivasan Rajagopal*a

The encapsulation of curcumin using p-sulfonatocalix[4]arene (p-SC4) is an attempt to increase the

bioavailability of curcumin by increasing water solubility. The degradation of curcumin due to the basicity of

p-SC4 is circumvented by maintaining the pH at 3 using 2% hydrochloric acid. The interaction is studied using

UV-visible absorption, emission, transient absorption and excited state lifetime methods. The encapsulation of

curcumin with p-SC4 increases the excited lifetime of curcumin, as well as the lifetime of the transients (triplet

state of curcumin and phenoxyl radical of curcumin) produced upon irradiation. The mode of interaction is

studied using 1H NMR and ROESY spectral techniques. The stability of curcumin in the presence of p-SC4

and the 2 : 1 ratio of p-SC4 binding with curcumin is established using HR-MS and MALDI-TOF analysis.

The amount of enhancement in solubility is studied using the HTLC technique.

Introduction

Calixarenes are well established supramolecules having exten-sive host properties.1–4 The upper and lower rims of calixarenescan be synthetically modified as per our needs.4 Sulfonationof the calixarene at the upper rim facilitates water solubility.5

Even though many water soluble calixarenes carrying a car-boxylic acid group and an amino group at the para-position areavailable,5 para-sulfonatocalix[4]arene (p-SC4) is syntheticallyvery facile and it has intriguing properties with regard toincoming guest molecules.6–8 The cone like structure of p-SC4is stabilized by the planar H-bonding of the O–H groups atthe narrow ends of the molecule (Chart 1).9 p-SC4, with ap-electron-rich hydrophobic cavity and the strong hydrophilicnature of the upper and lower rims has become increasinglyimportant in the fields of supramolecular chemistry and crystalengineering.10,11 p-SC4 shows interesting inclusion propertiesand forms a wide range of metal coordination complexes, bothin solution and in the solid state.12–16 There are many reportson the binding of p-SC4 with biologically important molecules,like amino acids, peptides and proteins.17–19

Curcumin (diferuloylmethane), a polyphenol and a crucialIndian medicinal and food ingredient, exhibits a wide range of

applications from the food industry to medicine and its activitiesrange from interesting photophysics to anticancer agent.20–24

It has been used in Indian medicinal systems such as Siddhaand Ayurveda for wound healing, rheumatism and anorexia.25

The pharmacological activities of curcumin, like antioxidant,anti-inflammation, anti-angiogenesis and apoptosis, emphasize itsmultifaceted role as a medicine.26 Curcumin is a scavenger of manyreactive oxygen species, like superoxide anion radicals, hydroxylradicals, nitrogen dioxide radicals.27 Curcumin has the abilityto prevent protein aggregation, which is the main cause forAlzheimer’s and Parkinson’s diseases.28–30 The problem withcurcumin as drug is its poor solubility in water.31 Many approaches

Chart 1 The structures of p-SC4 and curcumin.

a School of Chemistry, Madurai Kamaraj University, Madurai, Tamil Nadu, India.

E-mail: rajagopalseenivasan@yahoo.comb Department of Chemical and Biomolecular Engineering,

Korea Advanced Institute of Science and Technology, Daejeon, South Koreac Research Analysis Centre, Korea Advanced Institute of Science and Technology,

Daejeon, South Korea

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nj00935a

Received (in Montpellier, France)14th August 2013,Accepted 10th December 2013

DOI: 10.1039/c3nj00935a

www.rsc.org/njc

NJC

PAPER

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article OnlineView Journal | View Issue

This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 1336--1345 | 1337

to improve the solubility have been reported: nanoformulationof curcumin,32–34 synthetic modifications by introducing watersoluble polar groups35 and encapsulation of curcumin with ahost molecule which can act as a cargo vehicle.36–39

The synthetic modification and nanoformulation have theirown disadvantages, like the change in the activity of modifiedcurcumin and dispersion of nanoparticles.40 The best way toincrease the solubility is encapsulation of curcumin with awater soluble host molecule.41 Harada et al., examined theencapsulation of curcumin with cyclodextrin.42 However, aprevious report points out that when cyclodextrin is presentinside the cell, it leads to deformation of the cell structure.43

Interestingly, recent studies show that upon cell transfectioncalixarenes do not affect the structure of the cell.43–45 Inaddition to this advantage p-SC4 has medicinal properties, likeantitumor, antiviral and antiinflammatory activities.45 Thuswe envisage that the water soluble host candidate for theencapsulation of curcumin is p-SC4. Further, the spectralproperties of curcumin are favourable for use as an imagingagent to monitor the physiological functions.46 Curcumin isprone to undergo hydrolysis depending on the pH and environ-ment.47,48 Therefore we have to ensure that the encapsulationof curcumin in p-SC4 does not affect the activities and structureof curcumin.49,50 To realize the effect of encapsulation ofcurcumin by p-SC4, we have studied the spectral properties ofcurcumin in the presence of p-SC4 at pH 3. The excited statebehaviour of curcumin in the presence of a host moleculereveals the interaction of the host molecule with the transientsproduced upon excitation of curcumin.42 In this paper wepresent our results on the binding of curcumin with p-SC4 byusing absorption, emission, excited state lifetime, transientabsorption, time correlated single photon counting (TCSP),NMR, mass spectrometry and hybrid tandem liquid chromato-graphy (HTLC) techniques.

Experimental section

The host p-SC4 is synthesized using the literature procedure.51,52

Curcumin was procured from Merck. Deuterium oxide, acetonitrile-d3 and acetic acid-d4 were procured from Sigma-Aldrich. Deionized-double distilled water and HPLC grade acetonitrile were used assolvents. On dissolving p-SC4, the pH is raised to 9.2. Sincecurcumin has a tendency to degrade even at neutral pH, in thiswork, the pH was adjusted to 3.0 using 2% HCl, and the studiesare carried out at both pH 9.2 and 3.0.

Determination of binding constant of curcumin

UV-Vis spectral titration. By keeping the concentration ofthe guest, curcumin, fixed at 1 � 10�6 M and varying the con-centration of the host, p-SC4, from 1 � 10�5 M to 9 � 10�5 M atboth pH 3.0 and 9.2, the UV-visible absorption spectrum ofcurcumin was recorded using an Analytik Jena Specord S100spectrophotometer using a 1 cm path length cuvette. The valueof the binding constant (Ka) of curcumin with p-SC4 was

evaluated with the aid of the Benesi–Hildebrand equation,53,54

(eqn (1)), from the plot of 1/DA vs. 1/[G].

1/DA = 1/KaDe [H] + 1/De [G] (1)

Here, DA is the change in the absorbance of the curcumin onthe addition of p-SC4. De is the difference in the molar extinc-tion coefficient between the free curcumin and p-SC4–curcu-min adduct, [H] is the total concentration of p-SC4 and [G] isthe total concentration of curcumin. The plot of 1/DA vs. 1/[G]gives a good straight line. From the slope of the line thebinding constant Ka is calculated.

Fluorescence spectral titrations

For this titration, by fixing the concentration of the guest,curcumin, at 1 � 10�6 M, the concentration of the host, p-SC4s,is varied from 1 � 10�6 to 9 � 10�6 M at both pH 3.0 and 9.2 andthe fluorescence spectra of curcumin are recorded in the absenceand in the presence of various concentrations of p-SC4 on a JASCOFP 6300 spectrofluorimeter. The binding constant for the bindingof curcumin with p-SC4 is estimated based on the enhancementof fluorescence intensity with the change of concentration ofp-SC4. We have calculated the binding constant using themodified Benesi–Hildebrand equation,55 (eqn (2))

I0/(I � I0) = b/(a � b) � [1/Ka[H] + 1] (2)

where, I0 is the luminescence intensity of the guest in theabsence of host, I is the luminescence intensity of the complexin the presence of host, [H] is the concentration of the host, andKa is the binding constant for the binding of the host withguest. In the eqn (2), a and b are constants. The values of I0 andI are available from luminescence measurements. The value ofKa can be determined by plotting I0/I � I0 against the inverse ofthe concentration of the host, (M�1).

Excited state lifetime measurement

Fluorescence decays were recorded using the correlated singlephoton counting (TCSPC) method with the following set up.A diode pumped millena CW laser (Spectra Physics) 532 nm wasused to pump a Ti:sapphire rod in a Tsunami picosecond modelocked laser system (Spectra Physics). The 750 nm (8 MHz) line wastaken from the Ti:sapphire laser and passed through a pulse picker(Spectra Physics, 3980 2s) to generate 80 kHz pulses. The secondharmonic output (375 nm) was generated by a flexible harmonicgenerator (Spectra Physics, GWU 23 ps). The vertically polarised375 nm laser was used to excite the sample. The fluorescenceemission at the magic angle (54.71) was dispersed in a mono-chromator ( f/3 aperture), counted by a MCP PMT (Hamamatsu R3809) and processed through constant fraction discriminator(CFD), time-to-amplitude converter (TAC) and multi-channelanalyzer (MCA). The instrument response function for thissystem is E52 ps and the fluorescence decay was analyzed byusing the software provided by IBH (DAS-6) and PTI globalanalysis software. The average lifetime of the components hasbeen calculated by the following equation (eqn (3)).56

tav =P

tiAi/P

Ai (3)

Paper NJC

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

1338 | New J. Chem., 2014, 38, 1336--1345 This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

tav is the average lifetime, ti is the lifetime of a particularcomponent, and Ai is the relative amplitude of the respectivecomponent.

Transient absorption spectra

Transient absorption measurements were made with a laser flashphotolysis technique using an Applied Photophysics SP-Quanta RayGCR-2(10) Nd:YAG laser as the excitation source.57,58 The timedependence of the luminescence decay is observed using a Czerny–Turner monochromator with a stepper motor control and aHamamatsu R-928 photomultiplier tube. The production of theexcited state on exposure to light of wavelength 355 nmwas measured by monitoring (pulsed xenon lamp of 250 W)the absorbance change. The change in the absorbance of thesample on laser irradiation is used to calculate the rate con-stant as well as to record the time-resolved absorption transientspectrum. The change in the absorbance on flash photolysis iscalculated using the equations, (eqn (4) and (5))

DA = log I0/(I0 � DI) (4)

DI = (I � It) (5)

where DA is the change in the absorbance at time t, I0, I and It

are the voltage after flash, the pretrigger voltage and the voltageat a particular time, respectively. A plot of ln(DAt � DAN) vs.time gives a straight line. The slope of the straight line gives therate constant for the decay and the reciprocal of the rateconstant gives the lifetime of the triplet. The time-resolvedtransient absorption spectrum is recorded by plotting thechange in absorbance at a particular time vs. wavelength.

NMR spectral analysis

The NMR spectral analyses are carried out using Agilent 400 MHz4 mm NMR DD2. The solvent mixture of deuterium oxide/acetonitrile-d3 (70/30%) is used for 1H NMR titration androtating frame nuclear Overhauser effect (ROESY) spectraltechniques. The solution is acidified to approximately pH 3by adding a few drops of CD3COOD.

Mass spectrometry measurements

Mass spectrometry analysis is carried out by two methods. Thecompounds having a lower molecular weight (less than 1000m/z values) are analyzed using a Bruker Daltonik (microTOF-QII)HR-MS spectrometer. The compounds having a higher molecularweight (greater than 1000 m/z values) are analyzed using a Brukerautoflex III MALDI-TOF-MS spectrometer.

Hybrid tandem LC analysis

Hybrid tandem LC (HTLC) analysis is carried using microTOF-QII,Bruker Daltonik. The analytical column C18 is used. The columndiameter is 2.1 mm, particle size is 1.7 mm and signals detectedbetween 190–500 nm. The mobile phase is mixture of acetonitrile–water (70 : 30, v/v). The injection volume is 10 ml, retention time isfive minutes and flow rate is 0.2 ml min�1. The linearity ofanalysis is evaluated using standard curcumin solutions ofconcentration from 0.1 M to 0.7 M in acetonitrile.

Results and discussionEnhancement of solubility of curcumin with p-SC4

The solubility of 5 mg of curcumin is tested with water,acetonitrile, p-SC4 solutions (10�3 M, pH 3.0 and 9.2) and,p-SC4 solutions (10�3 M, pH 3 and 9.2) with 1% acetonitrile.Since curcumin is having tendency to degrade even in slightlybasic pH like physiological pH, the pH 3 is selected to establishthe interaction. The pH 9.2 is the pH of p-SC4 dissolved inwater. The interaction and solubility of curcumin is depicted inFig. 1. Curcumin is completely soluble in acetonitrile and lesssoluble in water. The interaction of curcumin with p-SC4 in waterin the presence of 1% acetonitrile as well as without acetonitrile atpH 9.2 gives a red colour, which indicates the degradation ofcurcumin. The interaction of curcumin with p-SC4 in water in thepresence of 1% acetonitrile, as well as without acetonitrile at pH 3,produced a minimal colour change. The absorption spectra ofthe above solutions are shown Fig. 2. The absorption spectraconfirm that the pure water solution has a very low absorption,on the other hand both the p-SC4 solutions at pH 3 and 9.2

Fig. 1 (A) 5 mg of curcumin in ACN (10 ml), (B) 5 mg of curcumin in water(10 ml), (C) 5 mg of curcumin in HCl (0.1 N, 10 ml) (D) 5 mg of curcuminin aqueous NaOH solution (0.1 N, 10 ml) (E) 5 mg of curcumin in aqueousp-SC4 solution (10�3 M, 10 ml) at pH 3, (F) 5 mg of curcumin in aqueousp-SC4 solution (10�3 M, 10 ml) at pH 3 with 1% ACN (G) 5 mg of curcuminin aqueous p-SC4 solution (10�3 M, 10 ml) at pH 9.3, (H) 5 mg of curcuminin aqueous p-SC4 solution (10�3 M, 10 ml) at pH 9.2 with 1% ACN.

Fig. 2 UV-visible absorption spectra of (a) 5 mg of curcumin in ACN (10 ml),(b) 5 mg of curcumin in water (10 ml), (c) 5 mg of curcumin in p-SC4 in water(10�3 M, 10 ml) at pH 9.2 with 1% ACN, (d) 5 mg of curcumin in p-SC4(10�3 M, 10 ml) at pH 9.2, (e) 5 mg of curcumin in p-SC4 (10�3 M, 10 ml) atpH 3 with 1% ACN (f) 5 mg of curcumin in p-SC4 (10�3 M, 10 ml) at pH 3.

NJC Paper

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 1336--1345 | 1339

have considerable absorptions. Hence, the water solubility ofcurcumin is enhanced in the presence of p-SC4. The depositsobserved in the solution with pure water without acetonitrile isdue to the lack of solubility.

Steady state methods

Although the aqueous solution of p-SC4 has the tendency tosolubilize curcumin, the turbid nature of the solution leads toinaccurate results when it is used to study the binding ofcurcumin with p-SC4. Therefore, we have used p-SC4 solutionswith 99%/1% water–acetonitrile (v/v) to study the interactionwith curcumin.

Absorption spectral study

The absorption spectrum of p-SC4 at 1 � 10�4 M is given inFig. S1 (ESI†). The absorption maximum of p-SC4 is around200 nm. The absorption spectrum of curcumin at 1 � 10�6 M isgiven in Fig. S2 (ESI†). It has two absorption maxima at 261 and426 nm and these two peaks correspond to the phenolic moietyand b-diketone moiety of substituted benzenes.59 In order tounderstand the nature of the ground state interaction betweencurcumin and p-SC4, a titration is carried out by fixingthe concentration of curcumin at 1 � 10�6 M and varying theconcentration of p-SC4 from 1� 10�5 M to 9� 10�5 M at pH 9.2and 3.0. The results of the titration of the curcumin–p-SC4system are shown Fig. 3. The intensity of the peak at 426 nmdecreases but the peak at 261 nm increases upon addition ofincreasing concentrations of p-SC4 at pH 9.2. The decrease ofabsorbance at 426 nm is due to the degradation of curcumin byp-SC4 and the increase of peak at 261 is due to the associationof degraded aromatic products with p-SC4.

On the other hand, the increase in the concentration ofp-SC4 at pH 3 increases the absorbance at both 261 and 426 nm.There is little change in the shape of the spectrum. This showsthat there is little degradation upon increasing the concentrationof p-SC4 at pH 3. The binding constant from this titration hasbeen calculated using the Benesi–Hildebrand method.53 TheBenesi–Hildebrand plot is given in Fig. S3 (ESI†). The bindingconstant of curcumin with p-SC4 determined using the absorp-tion spectral titration is 4.5 � 0.53 � 104 M�1. The Job’s plot(Fig. 4) shows that the increase in the concentration of p-SC4starts with a 1 : 1 binding ratio and further increase in concen-tration of p-SC4 leads to a 2 : 1 binding ratio. The binding

constant value of curcumin with p-SC4 is comparable to thosewith cyclodextrin and curcurbituril reported previously.38,39,60

Since cyclodextrin and curcurbituril have their own disadvantages,like cytotoxicity and a rigid nature,43 binding with the flexible andbenign p-SC4 could be of biological importance.

Emission spectral study

The advantage with curcumin is that it is highly luminescent.The lmax of the emission is around 546 nm upon excitationat 426 nm. The emission spectrum of curcumin is shown inFig. S4 (ESI†). Even though the p-SC4 is an aromatic host, it isnot fluorescent in nature. Thus we can use emission spectro-scopy conveniently for the determination of the binding con-stant of curcumin with p-SC4. The concentration of curcumin isfixed at 1 � 10�6 M for the emission spectral titration. Theconcentration of p-SC4 is varied from 1� 10�6 M to 9� 10�6 M atboth pH 9.2 and 3. The emission spectra of curcumin at differentconcentrations of p-SC4 are shown in Fig. 5. Interestingly, aconsiderable enhancement of emission intensity of curcumin inthe presence of p-SC4 is observed at pH 9.2, but the enhancementis not consistent with the increase in the concentration of p-SC4.On the other hand, the emission intensity of curcumin increasesconsistently with the increase in the concentration of p-SC4 atpH 3. This increase in the emission intensity with the increase inthe concentration of p-SC4, is used to calculate the bindingconstant. The modified Benesi–Hildebrand equation is used tocalculate the binding constant of curcumin with p-SC4 fromemission spectral technique.55 The I0/(I � I0) is plotted with theinverse concentration to get the Benesi–Hildebrand plot. TheBenesi–Hildebrand plot is given in Fig. S5 (ESI†). The binding

Fig. 3 Absorption spectra of curcumin (1 � 10�6 M) in the presence of (a)p-SC4 varying from 1 � 10�5 M to 9 � 10�5 M at pH 9.2 and (b) p-SC4varying from 1 � 10�5 M to 9 � 10�5 M at pH 3.

Fig. 4 The Job’s plot for curcumin–p-SC4 complex formation.

Fig. 5 The emission spectra of curcumin (1 � 10�6 M) in the presence of(a) p-SC4 varying from 1 � 10�6 M to 6 � 10�6 M at pH 9.2 and (b) p-SC4varying from 1 � 10�6 M to 9 � 10�6 M at pH 3.

Paper NJC

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

1340 | New J. Chem., 2014, 38, 1336--1345 This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

constant value calculated from the slope of the Benesi–Hildebrandplot is 5.2 � 0.73 � 104 M�1. Thus the binding constant valuecalculated by absorption as well as by emission spectral techniquesis very close, confirming the reliability of the value. There aremany reports on the binding of p-SC4 with biologically impor-tant compounds using emission spectroscopy.61 The bindingconstant values of various biologically important compoundswith p-SC4 are comparable with the binding constant value ofcurcumin with p-SC4.19

Time resolved methods

From the results observed from the absorption and emissionspectral studies, it is evident that p-SC4 stabilizes the curcumin inaqueous solution. From the binding constant values the efficiency ofbinding is established in the ground state. The biological activities ofthe curcumin are related to the presence of phenolic –OH groupsand keto–enol tautomerism.62 The free radical chemistry ofcurcumin is focused on phenolic rings.63,64 However, as thisstudy is carried out entirely in water, the intermolecularH-bonding character of water with curcumin will force thetautomeric equilibrium towards the diketone form. There areconsiderable reports also available focusing on the b-diketonemoiety.63 It is necessary to study the excited state dynamics ofthe curcumin in presence of p-SC4 using time resolved methodsto investigate these aspects in the presence of p-SC4.

Excited state lifetime study using TCSPC

The excited state lifetime of curcumin has been measuredusing the TCSPC technique. The concentration of curcumin isfixed at 2 � 10�6 M. The concentration of p-SC4 is 2 � 10�6 Mand 5 � 10�6 M at pH 3. The excitation wavelength is fixed at400 nm in a Ti:sapphire laser. 10 000 counts have been fixed forcounting. The decay of the excited state is shown in Fig. 6 andthe lifetime data are collected in Table 1. The data are fittedwith double exponential decays showing two lifetimes in thepicosecond time range. These lifetimes are closely related to thereported lifetimes elsewhere.56 This two life times are due to thepresence of dynamic cis–trans forms of curcumin in solutionstate.56 The average lifetime (tav) of the curcumin is calculated

using eqn (3).56 The increase in tav of curcumin from 412 ps to425 ps in the presence of p-SC4 is observed. Further increase in theconcentration of p-SC4 increases the tav to 445 ps. These resultsshow that the excited state lifetime of curcumin is stabilized uponbinding with p-SC4 without any degradation at pH 3.

Transient absorption spectral studies

The transient absorption spectrum is recorded using ms laserpulses of a Nd:YAG laser. The concentration of curcumin isfixed at 5 � 10�6 M and the concentration of p-SC4 is alsofixed at 5 � 10�6 M at pH 3. Transient absorption spectra ofcurcumin in the presence of p-SC4 are shown in Fig. 7. Thetransient absorption spectrum of curcumin alone possess twopeaks at 500 nm and 680 nm, respectively. Since the medium ofstudy is water, the curcumin will be present in the b-diketoneform. Therefore the peak at 500 nm corresponds to the phenoxylradical of curcumin.63 The p-SC4 has four phenolic units and thetransient of p-SC4 has no any change in absorption. This is dueto the strong intramolecular H-bonding of the lower rim –OHgroups of p-SC4.19 The peak at 680 nm corresponds to the tripletexcited state of curcumin.63

The intensity of the absorption of the peak at 500 nmincreases upon addition of p-SC4. This is due to the bindingof p-SC4 with the phenolic part of curcumin. The phenolic unitsinteract with the sulfonato groups on the upper rim via H-bonds,therefore the –OH bonds of the phenolic unit become weak,which facilitates the phenoxyl radical formation.19 The traces ofthe transient at 500 nm are shown in Fig. S6 (ESI†). The lifetimeof the transient at 500 nm is collected in Table 2. The presence ofp-SC4 increases the lifetime of phenoxyl radical. Therefore thephenoxyl radical formation is stabilized by p-SC4. The peak at680 nm also increases in the presence of p-SC4. The traces of

Fig. 6 The excited state lifetimes of 2 � 10�6 M of curcumin (’)using TCSPC in the presence of 2 � 10�6 M of p-SC4 ( ) and 5 � 10�6 Mof p-SC4 ( ) at pH 3.

Fig. 7 Transient absorption spectra of p-SC4 (5 � 10�6 M) (’), curcumin(5 � 10�6 M) ( ), and mixture of p-SC4 (1 � 10�5 M) and curcumin(1 � 10�5 M) ( ) at pH 3.

Table 1 Excited state lifetimes of curcumin–p-SC4 complexes at pH 3using the TCSPC technique

p-SC4 t1 (ps) t2 (ps) tav (ps)

— 440 279 4122 � 10�6 M 440 338 4255 � 10�6 M 458 372 445

NJC Paper

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 1336--1345 | 1341

transient at 680 nm are shown in Fig. S7 (ESI†). The lifetime ofthe transient 680 nm is also collected in Table 2. The lifetimevalues show that the presence of p-SC4 also stabilized the tripletstate of the curcumin. The transient absorption spectrum ofcurcumin alone and in the presence of p-SC4 at various timescalesare shown in Fig. S8 (ESI†). The structure of transient spectra isnot changed at various timescales; therefore, the addition of p-SC4will not reduce the biological activity of curcumin.

NMR analysis

The mode of binding of curcumin with p-SC4 is studied using1H NMR titration and ROESY spectral techniques. For the sakeof convenience the protons are named using capital letters andthe naming is shown in Fig. S9 (ESI†). As curcumin and p-SC4are soluble in acetonitrile and water, respectively, the solventD2O/CD3CN (70/30%) is used with few drops of CD3COOD tomaintain an acidic pH. The individual 1H NMR spectra of p-SC4and curcumin are shown Fig. S10 and S11 (ESI†). The 1H NMRspectrum of curcumin–p-SC4 mixture is shown in Fig. S12 (ESI†).The expansion of the 1H NMR spectrum of the aromatic region isshown in Fig. 8. The chemical shift values are given Table 3.

Fig. S12 and S8 (ESI†) show that the aromatic part of curcuminis shifted to the upfield region. There is no new peak appearance orsplitting observed in this spectrum. These chemical shifts are dueto the binding of the aromatic part of curcumin with the electronrich aromatic cavity of p-SC4 and also, the binding is similar andequal. On the other hand, the peaks corresponding to p-SC4 areshifted to the downfield region. Since the binding of curcumin withp-SC4 inside the cavity is via p–p interactions, the aromatic protonsof p-SC4 are shifted to the downfield region. This observationnullifies the outside binding of curcumin with p-SC4 and confirmsthe binding in the cavity of p-SC4.

To rationalize the above observations from the 1H NMRtitration we fixed the concentration of curcumin at 4 � 10�3 Mand titrated with p-SC4 by varying concentrations from 0–8 �10�3 M. The spectral titration is given in Fig. 9. In the spectraltitration the presence of 2 � 10�3 M of p-SC4 (ratio 1 : 0.5) has

Table 2 Rates of decay and lifetimes of transient species at 500 nm and680 nm in the presence of p-SC4 at pH 3

Trace at 500 nm Trace at 680 nm

K t (s) K t (s)

Curcumin 3.9 � 104 2.5 � 10�5 3.9 � 106 2.5 � 10�7

Curcumin–p-SC4 9.9 � 103 1.1 � 10�4 6.9 � 106 1.4 � 10�7

Fig. 8 1H NMR spectra of curcumin and the p-SC4–curcumin mixture inD2O/CD3CN (70/30%) with two drops of CD3COOD. (Expansion of thearomatic region.)

Table 3 Chemical shift values (ppm) of the 1H NMR titration of curcuminand the p-SC4–curcumin mixture in D2O/CD3CN (70/30%) with a fewdrops of CD3COOD

Proton Curcumin p-SC4 Curcumin–p-SC4 Change in ppm

CACA0 4.19 4.07 �0.12CBCB0 — 5.52 —CDCD0 7.56 7.44 �0.12CECE0 7.46 7.35 �0.11CFCF0 7.01 6.89 �0.12CGCG0 7.89 7.77 �0.12CHCH0 7.19 7.08 �0.11SA 7.37 7.57 0.2SB 3.78 4.24 0.46

Fig. 9 1H NMR spectral titration of (a) curcumin (4 � 10�3 M) alone, (b)curcumin (4 � 10�3 M) with p-SC4 (2 � 10�3 M), (c) curcumin (4 � 10�3 M)with p-SC4 (4 � 10�3 M) (d) curcumin (4 � 10�3 M) with p-SC4 (6 �10�3 M) and (e) curcumin (4 � 10�3 M) with p-SC4 (8 � 10�3 M) in D2O/CD3CN (70/30%) with two drops of CD3COOD.

Paper NJC

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

1342 | New J. Chem., 2014, 38, 1336--1345 This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

shifted the peaks slightly towards the downfield region (Fig. 9b).The second and third increase of concentrations also (ratio 1 : 1and 1 : 1.5) have shifted the peaks toward the downfield region(Fig. 9c and d). When the concentration of p-SC4 is increased to8� 10�3 M, all the peaks are again shifted to the upfield region andthe chemical shifts are similar to values of Fig. 8. This experimentconfirms the observation using Job’s plot (Fig. 4) using absorptionspectroscopy that the binding starts with 1 : 1 binding ratio andfurther increasing the concentration leads to 1 : 2 binding ratio.

The interaction of protons of curcumin with protons of p-SC4 isstudied using 2D spectral techniques. Since this study involves host–guest interactions, the interaction is monitored using rotating framenuclear Overhauser effect spectra (ROESY spectra). The ROESYspectrum is shown in Fig. 10. The coupling of aromatic protons ofp-SC4 (SA) with curcumin protons (CACA0, CFCF0 and CGCG0) areillustrated by cross peaks a, d and h. Since the CACA0 proton iscoupling with SA, it is clear that the binding mode is cavity binding.The SA proton also seems have cross peaks with CECE0 and CDCD0,but the proximity of peak is too close to resolve. The proton SB iscoupling with CGCG0 with cross peak g. There are a number of crosspeaks (b, c, e and f) observed corresponding to the intramolecularlong range coupling of protons in the curcumin itself.

Mass spectrometry studies

The degradation products of curcumin in the presence of p-SC4at pH 9.2 were identified and the stability of curcumin in thepresence of p-SC4 at pH 3 was established using electron sprayionization mass spectrometry technique (ESI-MS). To establishthe association product of curcumin with p-SC4 the MALDI-TOF technique is used, since the association products havemass values more than 1000 m/z.

Mass spectrometry studies using ESI-MS

The ESI-MS spectra of curcumin in the presence of p-SC4 atpH 9.2 and 3 are shown in Fig. S13 (ESI†). The m/z values are

collected in Table 3. The ESI-MS spectrum of curcumin in thepresence of p-SC4 at pH 9.2 possess the peaks corresponding tovanillin, vanillic acid, ferulic acid and feruloyl methane alongwith the peak corresponding to p-SC4. These are the majordegradation products of curcumin. The peak at 369.1252 corres-ponding to curcumin is also present in the spectrum, but itsintensity is very low compared to the intensity of the peaks ofother products, whereas the ESI-MS spectrum of curcumin in thepresence of p-SC4 at pH 3 possesses the peak corresponding tocurcumin with high intensity. There are no other predominantpeaks except vanillin + Na and ferulic aldehyde peaks withlow intensity compared to curcumin, along with the peakcorresponding to p-SC4. Thus the degradation of curcumin isnegligible in the presence of p-SC4 at pH 3.

Mass spectrometry studies using MALDI-TOF

The MALDI-TOF spectra of curcumin in the presence of p-SC4at pH 9.2 and 3 are shown in Fig. 11. The m/z values arecollected in Table 4. The MALDI-TOF spectra of curcumin in thepresence of p-SC4 at pH 9.2 possess only one peak at 1027.070.This corresponds to the association of p-SC4 + 4Na-ferulic alde-hyde. The ferulic aldehyde is a degradation product of curcumin,which is associated with p-SC4 at pH 9.2. There is no otherpredominant peak in this spectrum. The MALDI-TOF spectrumof curcumin in the presence of p-SC4 possesses three peaks at1112.057, 1201.1017 and 1849.6753 corresponding to p-SC4 +H-curcumin, p-SC4 + 4Na-curcumin and 2p-SC4–curcumin,respectively. This spectrum confirms the presence of bindingof p-SC4 with curcumin. The ratio of binding is 1 : 1 as well as2 : 1, confirmed by this mass spectrometry study.

HTLC analysis

The degradation of curcumin in the presence of p-SC4 at pH 9.2and the stability and enhancement in water solubility ofcurcumin with p-SC4 are studied using the HTLC technique.

Fig. 10 ROESY spectra of curcumin and the p-SC4–curcumin mixture in D2O/CD3CN (70/30%) with a few drops of CD3COOD.

NJC Paper

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 1336--1345 | 1343

5 mg of curcumin is dissolved in 10 ml of water, with 10�3 M ofp-SC4 at pH 9.2 and at pH 3. The solutions are shaken well for10 minutes and the undissolved particles are allowed to settle.The 10 ml of supernatant liquid is injected in to the column.From the standard solutions of curcumin it is observed that theretention time of curcumin is 2.4 min. The concentrations ofthe curcumin present in water and p-SC4 solutions both at

pH 9.2 and 3 are calculated using peak area of the chromatogramat retention time 2.4 min. Hence, the amount of curcuminpresent in the solution is also calculated. The HTLC chromato-grams are shown in the Fig. S14 (ESI†). The amount of curcuminpresent in the water is 9.8 � 10�4 g. On the other hand theamount of curcumin present in the p-SC4 solution at pH 3 is3.1 � 10�3 g. Therefore, there is a 32% increase in the solubilityof curcumin observed in the presence of p-SC4 solution at pH 3.The chromatogram of curcumin solution in the presence ofp-SC4 at pH 9.2 various peaks corresponds to the degradedproducts, including curcumin. Since the degraded products areknown from the mass spectrometry studies, the individualstandard degraded products are run through the HTLC columnunder the same conditions and the retention is recorded. Fromthe retention time the individual peaks of the correspondingproducts in p-SC4 solution at pH 9.2 are identified in the HTLCchromatogram (Fig. S14, ESI†).

Conclusions

The binding constant value determined using absorption andemission techniques is 5.0 � 104 M�1, and it shows that thebinding of curcumin with p-SC4 is efficient. The average life-time of curcumin in the aqueous medium is increased uponaddition of p-SC4. The transient absorption spectral studiesshow that the phenoxyl radical is stabilized upon addition ofp-SC4. The NMR spectral titration and ROESY spectral studiesalso confirm that the binding is via the cavity of p-SC4 with thearomatic part of curcumin. The association peaks in MALDI-TOFanalysis confirm the curcumin carrying ability of p-SC4. HTLCanalysis quantifies that the water solubility of curcumin isenhanced in the presence of p-SC4 by 32%. The physiologicalconcentration of curcumin is 1.5 g per day per person,65 sincethe bioavailability is very less due to the poor water solubility.Since p-SC4 is increasing the water solubility by 32%, it can beused to increase the water solubility and bioavailability ofcurcumin. As p-SC4 itself has biological activities and due to

Table 4 Mass values of 1 : 1 mixtures of p-SC4 (10�3 M) and curcumin (10�3 M) using ESI-MS and MALDI-TOF techniques

Compounds

Mass spectrum (m/z)

pH 3 pH 9.2

ESI-MSCurcumin 368.1219 (0.9 � 104) 369.1252 (0.17 � 104)Curcumin + Na 391.1088 (1.2 � 104) —Vanillin — 152.1179 (0.18 � 104)Vanillin + Na 177.042 (0.11 � 104) 174.9427 (0.98 � 104)Vanillic acid — 312.0102 (0.19 � 104)Ferulic aldehyde 248.2128 (0.13 � 104) —Feruloyl methane — 208.2113 (0.29 � 104)Feruloyl methane + Na — 285.1022 (0.17 � 104)p-SC4 + 4Na 832.5767 (0.12 � 104) 831.2753 (0.18 � 104)

MALDI-TOFp-SC4 + 4H-curcumin 1112.057 (1213.957) —p-SC4 + 4Na-curcumin 1201.1017 (281.098) —2(p-SC4)-curcumin 1849.6753 (659.355) —p-SC4 + 4Na-ferulic aldehyde — 1027.070 (590.2)

p-SC4 + 4H – protonated p-SC4, p-SC4 + 4Na – p-SC4 associated with Na.

Fig. 11 MALDI-TOF analysis of a 1 : 1 mixture of p-SC4 (10�3 M)–curcumin(10�3 M) at (a) pH 9.2 and (b) pH 3.

Paper NJC

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

1344 | New J. Chem., 2014, 38, 1336--1345 This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014

its low cytotoxicity, p-SC4 can be envisaged as drug deliveryvehicle for curcumin.

Acknowledgements

We gratefully thank Prof. P. Ramamurthy, National Centre forUltrafast Processes (NCUFP), University of Madras, Taramanicampus, Chennai for his help in time resolved studies. Wethank UGC-UPE for financial support.

References

1 C. D. Gutsche, Calixarenes: An Introduction, RSC Publishing,2008.

2 L. Mandolini and R. Ungaro, Calixarenes in Action, ImperialCollege Press, 2000.

3 W. Sliwa and C. Kozlowski, Calixarenes and Resorcinarenes,Wiley, 2009.

4 C. D. Gutsche, Calixarenes Revisited, Royal Society of Chem-istry, 1998.

5 S. Shinkai, K. Araki, T. Matsuda, N. Nishiyama, H. Ikeda,I. Takasu and M. Iwamoto, J. Am. Chem. Soc., 1990, 112,9053–9058.

6 C. D. Gutsche and P. F. Pagoria, J. Org. Chem., 1985, 50,5795–5802.

7 C. D. Gutsche and I. Alam, Tetrahedron, 1988, 44, 4689–4694.8 D.-S. Guo, S. Chen, H. Qian, H.-Q. Zhang and Y. Liu, Chem.

Commun., 2010, 46, 2620–2622.9 J. L. Atwood, L. J. Barbour, A. Jerga and B. L. Schottel,

Science, 2002, 298, 1000–1002.10 M. Makha, Y. Alias, C. L. Raston and A. N. Sobolev, New

J. Chem., 2007, 31, 662–668.11 T. Schrader, Nat. Chem., 2012, 4, 519–520.12 G.-S. Wang, H.-Y. Zhang, D. Li, P.-Y. Wang and Y. Liu,

Supramol. Chem., 2011, 23, 441–446.13 G.-S. Wang, H.-Y. Zhang, F. Ding and Y. Liu, J. Inclusion

Phenom. Macrocyclic Chem., 2011, 69, 85–89.14 S. J. Dalgarno, M. J. Hardie, M. Makha and C. L. Raston,

Chem.–Eur. J., 2003, 9, 2834–2839.15 V. Ball, M. Winterhalter, F. Perret, G. Esposito and

A. W. Coleman, Chem. Commun., 2001, 2276–2277.16 W. Yang and M. M. de Villiers, J. Pharm. Pharmacol., 2004,

56, 703–708.17 R. V. Pinjari and S. P. Gejji, J. Phys. Chem. A, 2010, 114,

2338–2343.18 N. Hontama, Y. Inokuchi, T. Ebata, C. Dedonder-Lardeux,

C. Jouvet and S. S. Xantheas, J. Phys. Chem. A, 2009, 114,2967–2972.

19 P. Muthu Mareeswaran, M. Prakash, V. Subramanian andS. Rajagopal, J. Phys. Org. Chem., 2012, 25, 1217–1227.

20 T. Esatbeyoglu, P. Huebbe, I. M. Ernst, D. Chin,A. E. Wagner and G. Rimbach, Angew. Chem., 2012, 51,5308–5332.

21 B. T. Kurien and R. H. Scofield, Mol. Nutr. Food Res., 2009,53, 939–940.

22 J. Dujic, S. Kippenberger, A. Ramirez-Bosca, J. Diaz-Alperi,J. Bereiter-Hahn, R. Kaufmann, A. Bernd and M. Hofmann,Int. J. Cancer, 2009, 124, 1422–1428.

23 L. Alappat and A. B. Awad, Nutr. Rev., 2010, 68, 729–738.24 D. K. Agrawal and P. K. Mishra, Med. Res. Rev., 2010, 30,

818–860.25 P. Anand, C. Sundaram, S. Jhurani, A. B. Kunnumakkara

and B. B. Aggarwal, Cancer Lett., 2008, 267, 133–164.26 A. Goel, S. Jhurani and B. B. Aggarwal, Mol. Nutr. Food Res.,

2008, 52, 1010–1030.27 A. Perez-Garrido, A. M. Helguera, J. M. Ruiz and

P. Z. Rentero, Eur. J. Med. Chem., 2012, 49, 86–94.28 J. W. Yan, Y. P. Li, W. J. Ye, S. B. Chen, J. Q. Hou, J. H. Tan,

T. M. Ou, D. Li, L. Q. Gu and Z. S. Huang, Bioorg. Med.Chem., 2012, 20, 2527–2534.

29 P. Anand, S. G. Thomas, A. B. Kunnumakkara, C. Sundaram,K. B. Harikumar, B. Sung, S. T. Tharakan, K. Misra,I. K. Priyadarsini, K. N. Rajasekharan and B. B. Aggarwal,Biochem. Pharmacol., 2008, 76, 1590–1611.

30 M. Masuda, N. Suzuki, S. Taniguchi, T. Oikawa, T. Nonaka,T. Iwatsubo, S.-i. Hisanaga, M. Goedert and M. Hasegawa,Biochemistry, 2006, 45, 6085–6094.

31 P. Anand, A. B. Kunnumakkara, R. A. Newman andB. B. Aggarwal, Mol. Pharmaceutics, 2007, 4, 807–818.

32 H. Yu and Q. Huang, J. Agric. Food Chem., 2012, 60, 5373–5379.33 Bhawana, R. K. Basniwal, H. S. Buttar, V. K. Jain and N. Jain,

J. Agric. Food Chem., 2011, 59, 2056–2061.34 S. Ganta and M. Amiji, Mol. Pharmaceutics, 2009, 6, 928–939.35 A. Safavy, K. P. Raisch, S. Mantena, L. L. Sanford,

S. W. Sham, N. R. Krishna and J. A. Bonner, J. Med. Chem.,2007, 50, 6284–6288.

36 A. L. Koner, I. Ghosh, N. i. Saleh and W. M. Nau, Can.J. Chem., 2011, 89, 139–147.

37 M. Takahashi, S. Uechi, K. Takara, Y. Asikin and K. Wada,J. Agric. Food Chem., 2009, 57, 9141–9146.

38 B. Tang, L. Ma, H.-y. Wang and G.-y. Zhang, J. Agric. FoodChem., 2002, 50, 1355–1361.

39 M. A. Rankin and B. D. Wagner, Supramol. Chem., 2004, 16,513–519.

40 A. Mukerjee and J. K. Vishvanatha, Anticancer Res., 2009, 29,3867–3875.

41 Z. Wang, M. H. Leung, T. W. Kee and D. S. English, Lang-muir, 2010, 26, 5520–5526.

42 T. Harada, D. T. Pham, M. H. Leung, H. T. Ngo,S. F. Lincoln, C. J. Easton and T. W. Kee, J. Phys. Chem. B,2011, 115, 1268–1274.

43 R. Lalor, H. Baillie-Johnson, C. Redshaw, S. E. Matthews andA. Mueller, J. Am. Chem. Soc., 2008, 130, 2892–2893.

44 R. Lalor, J. L. DiGesso, A. Mueller and S. E. Matthews, Chem.Commun., 2007, 4907–4909.

45 F. Perret, A. N. Lazar and A. W. Coleman, Chem. Commun.,2006, 2425–2438.

46 S. Dutta, S. Padhye, K. I. Priyadarsini and C. Newton, Bioorg.Med. Chem. Lett., 2005, 15, 2738–2744.

47 V. A. Marcolino, G. M. Zanin, L. R. Durrant, T. Benassi Mdeand G. Matioli, J. Agric. Food Chem., 2011, 59, 3348–3357.

NJC Paper

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online

This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 1336--1345 | 1345

48 R. Ghosh, J. A. Mondal and D. K. Palit, J. Phys. Chem. B,2010, 114, 12129–12143.

49 J. Barry, M. Fritz, J. R. Brender, P. E. S. Smith, D.-K. Lee andA. Ramamoorthy, J. Am. Chem. Soc., 2009, 131, 4490–4498.

50 M. H. M. Leung, H. Colangelo and T. W. Kee, Langmuir,2008, 24, 5672–5675.

51 D. Xiong, M. Chen and H. Li, Chem. Commun., 2008,880–882.

52 S. Shinkai, S. Mori, H. Koreishi, T. Tsubaki and O. Manabe,J. Am. Chem. Soc., 1986, 108, 2409–2416.

53 J. R. Lakowicz, Principles of Fluorescence Spectroscopy,Springer, 2006.

54 P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305–1323.55 S. P. Upadhyay, R. R. S. Pissurlenkar, E. C. Coutinho and

A. V. Karnik, J. Org. Chem., 2007, 72, 5709–5714.56 L. Nardo, A. Andreoni, M. Masson, T. Haukvik and

H. H. Tonnesen, J. Fluoresc., 2011, 21, 627–635.

57 E. Rajkumar and S. Rajagopal, Photochem. Photobiol. Sci.,2008, 7, 1407–1414.

58 P. Muthu Mareeswaran, E. Babu and S. Rajagopal,J. Fluoresc., 2013, 23, 997–1006.

59 S. Gunasekaran, R. K. Natarajan, S. Natarajan andR. Rathikha, Asian J. Chem., 2008, 20, 2903–2913.

60 S. Swaroop, B. Mishra and K. I. Priyadarsini, BARC Newsl.,2007, 208–212.

61 M. H. Leung and T. W. Kee, Langmuir, 2009, 25, 5773–5777.62 Y. Sugiyama, S. Kawakishi and T. Osawa, Biochem. Pharma-

col., 1996, 52, 519–525.63 F. Ortica and M. A. J. Rodgers, Photochem. Photobiol., 2001,

74, 745–751.64 R. Adhikary, P. J. Carlson, T. W. Kee and J. W. Petrich,

J. Phys. Chem. B, 2010, 114, 2997–3004.65 R. A. Sharma, A. J. Gescher and W. P. Steward, Eur. J. Cancer,

2005, 41, 1955–1968.

Paper NJC

Publ

ishe

d on

10

Dec

embe

r 20

13. D

ownl

oade

d by

Uni

vers

ity o

f W

inds

or o

n 29

/10/

2014

03:

10:3

6.

View Article Online