Chemical Physics Letters 501 (2010) 118–122

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Chemical Physics Letters

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A traditional painkiller as a probe for microheterogeneity in 1-propanol–watermixtures

Sreeja Chakraborty a, Esha Sehanobish b, Munna Sarkar a,⇑a Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF, Bidhannagar, Kolkata 700064, West Bengal, Indiab Department of Biochemistry, Ballygunge Science College, University of Calcutta, Kolkata 700019, West Bengal, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 June 2010In final form 22 October 2010Available online 26 October 2010

0009-2614/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.cplett.2010.10.050

⇑ Corresponding author. Fax: +91 33 23374637.E-mail address: munna.sarkar@saha.ac.in (M. Sark

Photo physical and spectral property of indomethacin has been harnessed to sense the cluster structuresthat exist at the microscopic level in binary mixtures of 1-propanol–water. The ability of indomethacin toswitch from a fluorescent complex with alcohol self-association cluster at low water content, to a non-fluorescent complex with hydrogen-bonded water cluster at high water content, imparts the drug withits unique microheterogeneity sensing property. Similar results are obtained for methanol–water and1-butanol–water binary mixtures.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

Painkillers belonging to the Non-Steroidal Anti-InflammatoryDrugs (NSAID) have been drawing renewed interest amongresearchers, mainly because of their alternate functions which in-clude anticancer effects, protective effects against neurodegenera-tion, inducer of membrane fusion, etc. [1,2]. Some of these NSAIDsalso have interesting spectroscopic properties that are sensitive totheir microenvironment and can be used effectively as reporter oftheir interaction with biomolecules [3–5]. Indomethacin (N-(para-chlorobenzoyl)-5-methoxy-2-methylindoole-3-acetic acid) (Figure 1)is a traditional painkiller whose spectroscopic and photochemicalproperties have been well studied [6]. Recently, we have shownthat for this traditional painkiller, the absorption spectrum in theUV–visible range and the fluorescent spectrum is extremely sensi-tive to the microheterogeneity that exist in the binary mixtures ofethanol–water [7]. Whether this heterogeneity sensing property ofindomethacin is a general property of the drug, or is it restrictedonly to ethanol–water mixtures is a question that needs to be an-swered to establish this property as a new function of a traditionalpainkiller.

In this study, we have used the spectral properties of indometh-acin to probe primarily the heterogeneity that exists in binarymixtures of 1-propanol–water. We have presented some keydata for methanol–water mixtures and have included data for1-butanol–water mixtures as Supplementary material, to showthat indomethacin can sense the microheterogeneity that existin other binary mixtures of water and alcohol. Binary mixturesof 1-butanol–water is not completely miscible in all propor-tions and shows a two-phase region at 1-butanol concentrations

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ar).

6.4 6 [BuOH] 6 80.2% at 20 �C. Therefore, the result of indometha-cin in 1-butanol–water binary mixtures, in the region where themixture shows single phase, is provided as Supplementary mate-rial. It is known that binary of alcohol–water do not form idealmixtures. Primary alcohols like ethanol, methanol and 1-propanolare miscible with water at all proportions. Macroscopically thesemixtures appear homogeneous, but at microscopic level differenttypes of clusters are formed. The two predominant type of clustersthat exist are alcohol self-association clusters and hydrogen-bonded water clusters. These involve both water and alcohol mol-ecules at various stoichiometry depending on the amount of waterin alcohol [8–11]. The microheterogeneity that exists in these bin-ary mixtures could be sensed only with advanced techniques likeneutron scattering [12], mass spectroscopy [10,11], Raman spec-troscopy [13], etc. Our results will demonstrate how simple tech-niques like UV–visible absorption and fluorescence spectroscopycan also be used to sense the same heterogeneity when we usethe photo physical property of the traditional painkiller indometh-acin as a reporter.

2. Materials and methods

Highest available quality (purity > 99%) indomethacin was pur-chased from Sigma Aldrich Fluka and was used without furtherpurification. UV spectroscopy grade 1-propanol, methanol and 1-butanol were purchased from Spectrochem, India. They were driedand stored over 3A molecular sieves (Merck, India). Quartz distilledwater was thrice distilled before use. Thermo Spectronic spectro-photometer Unicam UV500 was used to record UV–visible absorp-tion spectra. Steady state fluorescence measurements were donewith Hitachi F-7000 spectrofluorimeter.

Fluorescence lifetimes were determined by using a Time Corre-lated Single-Photon Counting (TCSPC) technique from Horiba Jobin

Figure 1. Structure of indomethacin.

S. Chakraborty et al. / Chemical Physics Letters 501 (2010) 118–122 119

Yvon Inc. NJ, USA using a 800 ps pulsed diode excitation source(Nano LED) and a MCP-PMT detection system. The single-photoncounting controller (Fluoro Hub) was used to record the data usingthe software Data Station V2.5. The excitation wavelength used inthis study is 280 nm and the emission wavelength was fixed at310 nm. Fluorescence intensity decay curves could be fitted to atri-exponential series using DAS6 decay analysis software that isprovided with the system.

IðtÞ ¼ B0 þ B1 expð�t=s1Þ þ B2 expð�t=s2Þ þ B3 expð�t=s3Þ

where B1, B2, B3 are pre-exponential factors and B0 is a constant.The goodness of fit is evaluated by reduced v2 values. The

instrument response function (IRF) has a full width at half maxi-mum (FWHM) at 800 ps. Hence, lifetime components <0.8 ns couldnot be considered for our analysis.

All experiments were done with 35 lM of indomethacin andthe temperature was kept constant at 298 K using a circulatingwater bath. Freshly prepared solutions were used in each experi-ment to avoid photoreaction [6]. The error in data points is ex-pressed as the standard deviation of each data point for fourseparate experiments. Since it was difficult to prevent water frombeing absorbed continuously in dehydrated methanol during sam-ple transfer and handling, measurement of actual water content inmethanol gave uncertain results. To overcome this problem, allsample handling and transfer for methanol–water data sets weredone in a specially dehumidified room.

3. Results and discussion

Figure 2 shows the absorption spectra of indomethacin in 1-propanol water mixtures with increasing wt.% of water. Theabsorption spectrum in pure 1-propanol (0 wt.% water) is similarto other N-benzoyl indole derivatives showing a peak at 230 nm

Figure 2. Absorption spectra of indomethacin in 1-propanol–water mixtures withincreasing weight percentages of water.

and a shoulder around 260–280 nm [14]. A weaker peak is ob-served around 320 nm. Increasing water concentration results ina decrease in the peak at 230 nm with a concomitant increase ofthe shoulder around 270 nm giving a clear isosbestic point at254 nm as indicated in the figure. The peak at 320 nm remains al-most unaffected by the increase in the concentration of the waterin the mixtures. The presence of the isosbestic point indicates theexistence of two ground states complexes of indomethacin. As wehave shown in case of ethanol–water mixtures [7], the most likelycandidates of such complexes involve indomethacin with differentcluster structures present in the mixtures. An interesting point tonote is that the presence of isosbestic point also shows that theabsorption spectrum can sense indomethacin complexed with onlytwo different types of clusters present in the mixtures at low andhigh water concentration. Both in dehydrated pure 1-propanoland in mixtures containing 93 wt.% water, there is significantabsorption at 270 nm. This shows that both types of ground statecomplexes have significant contribution at 270 nm.

In order to identify the nature of the two types of clusters withwhich the drug forms ground state complexes, indomethacin wasexcited at 270 nm and the fluorescence spectra were monitoredwith increasing water concentration (Figure 3). Care was taken toavoid any photochemical changes and aliquot of fresh solutionwas taken for each scan. Figure 3 shows a strong dependence ofthe fluorescence intensity on the water content in the binary mix-tures. A plot of the fluorescence intensity at 330 nm with increas-ing wt.% of water is shown in Figure 3 (inset). Even though there isa large scatter in the data points, it is clear that the band intensityshows a sharp decrease after 60 wt.% water. Similar type of de-crease in band intensity has already been observed by us for etha-nol–water [7] after 50 wt.% water instead of 60 wt.% (Figure 3inset).

The excitation spectra of indomethacin in the binary mixtureswere monitored at 330 nm. Figure 4a shows a plot of excitationintensity at 270 nm that decreases with increasing concentrationof water in the mixtures. Contrary to Figure 4a, the absorptionband at 270 nm (Figure 2) increases with water content. This270 nm band arises from both types of ground state complex ofindomethacin irrespective of whether they are fluorescent ornon-fluorescent and the increase is due to the preferential solva-tion of the non-fluorescent complex with increasing water content.

Figure 3. Fluorescence spectra of indomethacin monitored at kex = 270 nm in 1-propanol–water mixtures with varying water content. Inset: Fluorescence intensityof indomethacin monitored at 330 nm in 1-propanol–water mixtures withincreasing weight percentages of water.

Figure 4. (a) Plot of excitation intensity at 270 nm band with varying waterconcentration in 1-propanol–water mixtures. The excitation spectra were moni-tored at 330 nm. (b) Plot of absorbance at 232 nm with increasing water content in1-propanol–water mixtures.

Figure 5. (a) Absorption spectra of indomethacin in methanol–water mixtures withincreasing weight percentages of water; (b) fluorescence spectra of indomethacinmonitored at kex = 270 nm in methanol–water mixtures with varying watercontent. Inset: Fluorescence intensity of indomethacin monitored at 336 nm inmethanol–water mixtures with increasing weight percentages of water.

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The excitation band at 270 nm, on the other hand, arise from thosefluorescent ground state species that contribute to the intensity ofthe 330 nm band of the emission spectra (Figure 3). Hence thepreferential solvation of the non-fluorescent species will not be re-flected in the excitation band at 270 nm (Figure 4a). The decreasein intensity of the excitation band at 270 nm reflects the changein concentration of the fluorescent ground state complex due toits conversion to the non-fluorescent complex with increasingwater in the binary mixtures. Figure 4b shows a plot of the absorp-tion peak at 232 nm with increasing water. Interestingly, it followsthe same trend as seen for the emission band at 330 nm (Figure 3)indicating that the fluorescence contribution comes from the bandcentered around 230 nm. The changes in the absorption spectra,the emission intensity at 330 nm, the excitation peak at 270 nmand the absorption peak at 232 nm with increasing water, all pointto the fact that indomethacin forms a fluorescent complex and anon-fluorescent complex. The fluorescent complex dominates till60 wt.% water whereas non-fluorescent complex dominates athigher water concentration. Similar results were seen by us in caseof indomethacin in ethanol–water mixtures [7].

This is because the fundamental properties of the cluster struc-tures in the 1-propanol–water mixtures are similar to those ob-served in the ethanol–water mixtures. At lower waterconcentration, the molecular association is controlled by the 1-

propanol self-association structures. As the water concentrationis increased, the alcohol clustering structures changes to hydrogenbonding network intrinsic to pure water. The change from alcoholself-association clusters to hydrogen-bonded water clusters withincreasing water, results in marked change in the microenviron-ment of indomethacin. The decrease in fluorescence intensity indi-cates that up to 60 wt.% water, indomethacin forms a fluorescentcomplex with the predominant 1-propanol self-association clus-ters. With further increase in water concentration, as the natureof the clusters changes to hydrogen-bonded water clusters, thiscomplex is converted to a weakly fluorescent/non-fluorescentcomplex as seen in Figure 3. The water clusters disintegrates atlower 1-propanol concentrations compared with the water–etha-nol mixtures [10,11]. This could account for the decrease in bandintensity after 60 wt.% water in this case compared to the50 wt.% water as was previously observed in ethanol–water mix-tures [7].

Figure 5a shows the absorption spectra of indomethacin withincreasing wt.% of water in methanol–water mixtures and Figure 5bshows the change in the fluorescence spectra with increasingwater content. The inset shows the plot of the 336 nm fluorescence

Table 1Fluorescence lifetime data of indomethacin in 1-propanol containing differentpercentages of water (kex = 280 nm, kem = 310 nm).

wt.% of water inn-propanol

B1 (s1 ns) B2 (s2 ns) B3 (s3 ns) v2

0 15.11 (0.06) 67.68 (4.86) 17.20 (9.27) 0.7212.15 14.7 (0.06) 74.65 (5.4) 10.63 (13) 0.6929.30 16.1 (0.047) 75.21 (5.96) 8.69 (18.0) 0.8740.13 13.39 (0.04) 78.1 (6) 8.4 (20) 1.250.45 18.54 (0.04) 70.6 (5.9) 10.85 (18) 1.0660.34 21.36 (0.05) 70.6 (6) 8.04 (25) 0.9669.80 20.34 (0.08) 72.5 (6) 7.13 (37) 1.2178.87 32.8 (0.05) 62.9 (6) 4.29 (35) 0.75

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intensity with varying concentration of water. As in case of 1-pro-panol–water mixtures, the absorption spectra show a decrease inthe 230 nm band along with an increase in the 270 nm band, givinga clear isosbestic point that indicates the presence of two groundstate complexes of indomethacin. The changes in the fluorescencespectra seen in Figure 5b are similar to that seen in Figure 3 for 1-propanol–water mixtures. The only difference is that the fluores-cence intensity shows a sharp decrease after 40 wt.% water in caseof methanol–water mixtures, instead of 60 wt.% as seen in Figure 3.

For indomethacin in 1-butanol–water mixtures, absorptionspectra recorded in the alcohol rich and water rich single phases,do not show any isosbestic point (Figure S1a). The emission spectraat low water content and that in high water content resemble thatof other alcohol–water binary mixtures and a plot of fluorescenceintensity at 330 nm with increasing wt.% water clearly show simi-lar trend with just a gap in the region where the mixture is bipha-sic. The data is provided as Supplementary material (Figures S1band c).

Interesting point to note is that the sharp decrease in the emis-sion intensity occurs from 40 wt.%, 50 wt.% [7], and 60 wt.% waterfor methanol, ethanol and 1-propanol mixtures with water respec-tively. This correlates with the fact that with an increase in thehydrophobicity of the alcohol, alcohol self-association clustersform in water at lower concentration [10,11]. It should also bepointed out, that in all the three binary mixtures, viz. methanol–water, ethanol–water [7] and 1-propanol–water, the absorptionspectra shows a clear isosbestic point indicating the existence oftwo types of complexes of indomethacin in two types of microen-vironment. This is consistent with the fact that in all the three bin-ary mixtures, the hydrogen-bonding water clusters and alcoholself-association clusters do not co-exist. The hydrogen-bondedwater structures get disrupted when the alcohol self-associationclusters are formed, which makes it possible to maintain a singlephase even when alcohol self-association clusters are formed[11]. This makes methanol, ethanol and 1-propanol completelymiscible with water. Whereas, it is known that in the 1-butanol–water mixtures, 1-butanol self-association clusters are formedwithout disintegrating the hydrogen-bonded network of watereven at low alcohol concentration. It is the coexistence of twotypes of clusters that leads to phase separation [11]. Coexistenceof both types of clusters even in the alcohol rich and water rich sin-gle phases, results in the absence of isosbestic point due to thecoexistence of fluorescent and non-fluorescent complexes of indo-methacin with varying numbers. However, the predominance ofalcohol self-association cluster in alcohol rich single phase and thatof water cluster in water rich single phase, is clearly seen in the dif-ference in emission profile of indomethacin in 1-butanol–watermixtures.

It should be mentioned, that the physicochemical processes ofdifferent solutes are also found to be affected by the clusterformation in alcohol–water mixtures. For example, the fluores-cence lifetimes of acridine, the rate constant for hydrolysis oftert-butylchloride are controlled by the preferential solvation ofthese solutes in alcohol, which in turn is modulated by the micro-scopic structures in alcohol–water mixtures [10]. Non-Markovianeffects were seen in ethanol–water mixtures on the non-radiativedecay rates of the first singlet state of Rhodamine 3B. This is themanifestation of clustering of ethanol molecules in low ethanolcontent in the binary mixtures [15]. Proton transfer from the ex-cited states of 1- and 2-napthol, to the water solvent was studiedin ethanol–water mixtures. The clustering effects in the binarymixtures have been found to play a dominant role in the protonhydration dynamics. For both the napthols, a water cluster of4 ± 1 molecules act as a proton acceptor in each case [16].

Time resolved fluorescence was used to see how the changingalcohol–water cluster structures with increasing water content in

the binary mixtures affected the lifetime components of indometh-acin. A different time resolved setup with a resolution limit of0.8 ns was used in this study. For our study with ethanol–watermixtures, we were limited by a setup having a resolution limit of1.2 ns [7]. However, we had found a long lifetime componentwhose lifetime changes with increasing water, correlated well withthe expected changes in microviscosity of the ethanol–water mix-tures. For indomethacin in 1-propanol–water mixtures, the life-time data could be fitted to a tri-exponential decay. The lifetimesand pre-exponentials at different wt.% water are shown in Table 1.One of the components [B1 (s1 ns)] has a very short lifetime <0.8 ns,the resolution limit of the instrument and will therefore not be dis-cussed. The lifetime of the second component remains more or lessunaffected by the change in water concentration with the averagevalue at 6 ns. The pre-exponential factors changes, increasing till40 wt.% water and then decreases. As seen by mass spectroscopy,alcohol self-association clusters form and grow only in presenceof water, for both ethanol–water and 1-propanol–water mixtures,reaching a maximum at 40–50 wt.% alcohol and then decreases.The changes in the pre-exponential factor (B2 in Table 1) followthis trend in growth and decay of these clusters. We are thereforetempted to assign the 6 ns component to indomethacin in alcoholself-association clusters. The change in the pre-exponential for thethird lifetime component does not show any systematic variationin its lifetime values and pre-exponential term with increasingwater content indicating that this lifetime component is unaffectedby clusters. The time resolved data does not give any clear inter-pretation in this case.

To sum up our results, indomethacin complexed with alcoholself-association clusters is the fluorescent complex and indometh-acin complexed with hydrogen-bonded water clusters is the non-fluorescent complex. The ability of indomethacin to switchoverfrom fluorescent to non-fluorescent complex with the variationof its microenvironment in alcohol self-association cluster to waterclusters, imparts the drug with the unique microheterogeneitysensing property and clearly establish indomethacin as a sensorfor cluster structures in alcohol–water mixtures.

Acknowledgements

We are thankful to Mr. Ajoy Das of Chemical Sciences Divisionfor his instrumental help. One of the authors, Sreeja Chakraborty,thanks Council of Scientific and Industrial Research for providingher the fellowship for her Ph.D. program.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.cplett.2010.10.050.

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