Interaction of Cationic CTAB Surfactant with Curcumin ...

PHYSICAL CHEMISTRY

2013 Carl Hanser Verlag, Munich, Germany

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Rakesh Sharma and Dipti Jani

Interaction of Cationic CTAB Surfactant with Curcumin, an Anticarcinogenic Drug: Spectroscopic Investigation Curcumin, the most active polyphenolic constituent of turmeric cucuminoids obtained from rhizome Curcuma longa, holds a high place in ayurvedic medicine but its role in conventional disease management is also established. Unfortunately, the compound has poor aqueous solubility, which results in poor bioavailability following high doses by oral administration. In order to enhance its effectiveness and improve bioavailability, surfactant assemblies as the colloidal drug carriers with desired properties have been largely utilized. The interaction of curcumin with cetyltrimethylammonium bromide (CTAB) surfactant has been investigated by absorption spectroscopy as a function of surfactant concentration in pre-micellar and micellar range at acidic pH of 6.4. The pre-micellar and micellar region of pure CTAB surfactant at acidic pH of 6.4 is examined through tensiometry and conductometry techniques. Spectral data shows that in presence of curcumin at lower CCTAB, the change in absorbance and peak form initially was assigned to attraction of positive head group of CTAB towards the b-diketone group of drug. In micellar region including CMC, the type of interaction corresponds to the attachment of C16 chains of CTAB to nonpolar aryl groups of drug and simultaneously displacement of polar head group from b-diketone group of the drug. Finally at post micellar CCTAB, the encapsulation of the curcumin into micelles, predominantly in intact monomeric form is observed with the sharp peak at kmax = 423 nm. Key words: Curcumin, CTAB surfactant, CMC, drug-surfactant interaction, UV-Visible spectroscopy Wechselwirkung zwischen mit kationischen Tensid CTAB und dem antikarzinogenen Wirkstoff Curcumin: Spektroskopische Untersuchung. Curcumin, die aktivste polyphenolische Verbindung unter den Gelbwurzel Cucminoiden, wird aus dem Wurzelstock Curcuma longa erhalten und hat in der ayurvedischen Medizin einen hohen Stellenwert; ist aber auch in der konventionellen Krankenbehandlung etabliert. Leider ist die Verbindung schlecht wasserlöslich, was zu einer schlechten Bioverfügbarkeit und daher zu hoher Dosierung bei der oralen Verabreichung führt. Zur Erhöhung seiner Wirksamkeit und zur Verbesserung der Bioverfügbarkeit wurden Tensidaggregate als kolloidale Wirkstoffträger mit gewünschten Eigenschaften umfangreich eingesetzt. Die Wechselwirkung von Curcumin mit Cetyltrimethylammoniumbromid (CTAB) wurde abhängig von der Tensidkonzentration im vormizellaren und mizellaren Bereich bei pH 6,4 mit der Absorptionsspektroskopie untersucht. Der vormizellare und mizellare Bereich des reinen CTAB wurde bei pH 6,4 tensiometrisch und konduktometrisch untersucht. Die spektralen Daten zeigen, dass bei Anwesenheit von Curcumin bei niedrigen CTAB-Konzentrationen die Veränderung von Extinktion und ursprünglicher Peakform von der Anziehung der positiven CTAB-Kopfgruppe zur b-Diketongruppe des Wirkstoffs bestimmt wurde. Im mizellaren Bereich einschließlich der CMC, entspricht der Wechselswirkungstyp der Anlagerung der C16-Ketten des CTAB an die unpolaren Arylgruppen des Wirkstoffs bei

Tenside Surf. Det. 50 (2013) 4

gleichzeitiger Entfernung der polaren Kopfgruppen von den bDiketongruppen des Wirkstoffs. Letzlich wird in der post-mizellaren Region des CTAB aufgrund des scharfen Peaks bei kmax = 423 nm beobachtet, dass Curcumin überwiegend in der intakten monomeren Form in die Mizellen eingeschlossen ist. Stichwörter: Curcumin, CTAB, CMC, Wirkstoff-Tensid-Wechselwirkung, UV-Vis-Spektroskopie

1 Introduction

The Indian solid gold, Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadien-3,5-dione; commonly called as diferuloylmethane), is a bioactive constituent of turmeric, the Indian spice, which is a member of the ginger family obtained from the rhizome \Curcuma longa" and has been known for centuries as a household remedy to many ailments [1 – 4]. It contains two ferulic acid molecules linked via a methylene bridge at the carbon atoms of the carboxyl groups. Curcumin is a lipophilic molecule with phenolic groups and conjugated double bonds which exhibits ketoenol tautomerism (shown in Scheme 1). Recently, curcumin has attracted much interest because several experimental studies have demonstrated that this natural polyphenol has anti-tumor, anti-oxidant, anti-arthritic, anti-amyloid, anti-ischemic, anti-cancer and anti-inflammatory effects and is currently subject to numerous clinical trials in humans [6 – 8]. The photophysical and photochemical properties of curcumin have been studied through the years by several groups for its better applications in health sciences [2, 5 – 12]. Clinical development of

Scheme 1

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Chemical structure of Curcumin drug

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Rakesh Sharma and Dipti Jani: Interaction of cationic CTAB surfactant with curcumin, an anticarcinogenic drug

curcumin is mainly hindered due to its low aqueous solubility (20 lg/mL) which can be improved by increasing the pH of the solution. But this undergoes rapid degradation first hydrolysis, followed by molecular fragmentation with the product being trans-6-(4’-hydroxy-3-methoxy phenyl)-2,4-dioxo-5-hexenal, vanillin, ferulic acid and feruloyl methane, which restricts its use in intravenous administration and is associated with poor absorption in the intestine upon oral administration [13]. Aggregates or micellar systems of surfactants can solubilize poorly soluble drugs and increasing its bioavailability prominently. Surfactant micelles are largely used as colloidal drug carriers by encapsulation of the drugs, in order to ensure the transport to specific sites of action, to minimize drug degradation and loss, to prevent harmful side effects, thus improving the treatment efficacy [14]. Therefore, the study of drug–surfactant interactions has received an increased attention in the last period of time. The alkaline hydrolysis of curcumin was examined by Kee et al. [15] in micellar solutions of cationic surfactants (CTAB and DTAB) and the anionic surfactant (SDS). At pH 13, curcumin was quickly hydrolyzed in micellar SDS solution while it is greatly suppressed in CTAB or DTAB micellar solutions. Results reveal that the drug remains encapsulated in CTAB and DTAB micelles and dissociated from the SDS micelles in the aqueous phase at alkaline medium. Wang et al. [16] have studied the interaction of DTAB with curcumin by spectroscopic techniques and proposed the mechanisms of drug-surfactant interaction at low, intermediate, and high surfactant concentration region, which is relating to interaction forces, surfactant aggregations, as well as structural alterations of curcumin. Thermodynamic parameters for dissolving curcumin in CTAB solutions were examined by Iwunze [17]. A partition coefficient KX, for the distribution of curcumin between the micellar pseudo-phase and aqueous phase was 3.66 · 105 with a binding constant, KS, of 6.59 · 103 M–1. A free energy change for the observed binding, DGb, of –21.79 kJ/mol and additionally, a free energy of transfer, lt, from the aqueous phase to the micellar pseudophase was estimated to be –13.74 kJ/mol. These parameters suggest a favourable thermodynamic stability of curcumin in the micellar system used. Therefore, studies related to curcumin with cationic surfactants, specifically CTAB, has always been important. In the present work, we report the interaction between cetyltrimethylammonium bromide (CTAB) and curcumin in acidic buffer of pH 6.4 using UV-Vis absorption spectroscopy technique. The CTAB-curcumin molecular interaction was determined on the basis of shift in absorption spectra of the drug when going from an aqueous to a more hydrophobic environment at various concentrations of surfactant. The simple mechanism of CTAB-curcumin interaction have been proposed at pre-micelles, intermediate, and post-micellar surfactant concentrations regime, which relating to various attractive and repulsive forces arises, structure and aggregations of surfactant, as well as structural alterations of drug. The present study may used as model for several biochemical and pharmacological systems where old age medicine curcumin is utilized. 2 Experimental 2.1

Materials

The cationic surfactant, cetyltrimethylammonium bromide (CTAB) (Scheme 2), and curcumin from curcuma longa (Turmeric) was AR product of Sigma. Solvents like ethanol,

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Scheme 2

Molecular Structure of cationic CTAB Surfactant

methanol are of AnalaR grade. Phosphate buffer solution (PBS) of pH 6.4 is prepared in the laboratory by dissolving 2.72 g of potassium dihydrogen phosphate in 0.8 L of water adjusting the pH with 1.0 M potassium hydroxide and diluting to 1.0 L with water. Triple distilled deionized water for sample preparation and all-PyrexTM glass apparatus was always used. 2.2 2.2.1

Methods Tensiometry

The surface tension of cationic surfactant solutions was measured by drop weight method using a modified Stalagmometer [18]. The error in ST values was within € 0.05 mNm–1. 2.2.2 Conductometry

Conductance measurements were taken in digital conductivity meter (Kalico, India) using a dip-type cell at 298 K. The cell constant was 1.0 cm–1. All measurements were performed in jacketed vessel, which was maintained at the appropriate temperature € 0.1 8C. The error in conductance measurement was within € 0.5 %. The conductance was measured after proper mixing and temperature equilibrium. 2.2.3 UV-Visible Absorption Spectroscopy

Absorption spectra of curcumin (at fixed 10 lM concentration) in the range of 200 to 600 nm were recorded at various CTAB concentration in PBS of pH 6.4 using a UV-double beam spectrophotometer (2 450, Shimadzu) equipped with thermostatted quartz cells (1 cm path length) using a validated method. Blank curcumin calibration curves were developed by dissolving a given amount of curcumin in ethanol followed by the required dilution by water. The presence of ethanol did not alter either the extinction co-efficient or the specific wavelength at which the maximum in UV absorbance appeared. All the measurements were conducted at 298 K by circulating water through the thermostatted cuvette holder. Each sample was determined in triplicate and the results are reported as the mean of the three. 3 Results and Discussion

Firstly, the micellar behaviour of CTAB at acidic pH of 6.4 using tensiometry and electrical conductivity measurements were investigated at 298 K (Figures 1a and 1b). The surface tension (ST) vs. Log CCTAB plot has the sigmoidal shape with a rapid decrease of ST below the CMC and is constant at higher surfactant concentration, while the specific conductivity (K) has a higher increasing rate below the CMC. Above the CMC rate slightly decreases with CCTAB, respectively. As shown in Figures 1a and 1b, the CMC of CTAB can be taken as the concentrations corresponding to the interceptions of two straight lines in the curves of ST vs. Log CCTAB and K vs. CCTAB. The values of CMC observed are 1.21 and 1.16 mM respectively, in agreement with the reported ones [19, 20]. The surface active parameters were calculated ac-


cording to Xiaoli et al. [21] and presented in Table 1. Results of these experiments cleared the pre-micellar (below CMC) and post-micellar regions (above CMC) of CTAB surfactant in acidic pH of 6.4, which may possibly exhibit different interactions with curcumin drug. The UV-Visible absorbance spectra of blank curcumin, (without CTAB surfactant), in water: ethanol medium at various concentrations are shown in Figure 2a. Curcumin has the absorption peak at 423 nm, which indicates an increase in intensities with drug concentrations [15]. Calibration with

dilute solutions of the drug in water : ethanol gave satisfactory Beer–Lambert plot (Figure 2b) with R2 = 0.99927. Figure 3a depicts the absorption spectra of curcumin at various CCTAB in PBS of pH 6.4 at 298 K. It is noted that the absorption spectrum of drug has striking changes upon addition of CTAB as CCTAB increases the intensity and sharpness of the peak upto the studied 2.5 mM concentration, which indicates the finally solubilization of curcumin into the CTAB solutions. The absorbance versus CCTAB plots (Figure 3b) clearly indicate the almost constant and very fluctuate absorption in pre-micellar region at CCTAB = 0.02 to 0.4 mM, while abrupt increasing at intermediate region CCTAB = 0.4 to 1.4 mM where CMC of surfactant involved and once again almost same or slightly even decreased at higher CCTAB, the post-micellar region of CCTAB = 1.4 to 2.5 mM. It indicates that conjugated structure of curcumin was disturbed at lower surfactant concentrations while it intacts with high absorption in above CMC of CTAB. To understand such spectroscopic changes of the the curcumin and how micellar behaviour of CTAB takes part into the interaction with it, the UV-Vis spectra of curcumin drug are specified at three surfactant concentration ranges and compiled spectra were presented in Figure 4.


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Figure 1 (a) Surface active behavior of CTAB in PBS of pH.6.4 at 298 K. (b) Conductive behavior of CTAB in PBS of pH.6.4 at 298 K

Cationic CTAB Surfactant blank 1.21(ST)

CMC/mM

1.16(COND) smax/mol cm

–2

1.956

Amin/cm2

87.56

DG8ad/kJ mol–1

–42.38

DG8mic/kJ mol–1

–39.75

smax, Gibbs’ surface excess; Amin, area of exclusion per surfactant monomer; DG8ad, standard free energy of adsorption; DG8mic , standard free energy of micellization Table 1

Surface active parameters of CTAB in PBS of pH.6.4 at 298 K


Figure 2 (a) UV-Visible spectra of blank curcumin in ethanol-water mixtures at 298 K. (b) Calibration plot of curcumin at 298 K

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Although the molecular structure of curcumin remains un-ionized or nondegraded under the experimental conditions of pH 6.4, which is lower than the smallest pKa value of curcumin (pKa = 8.38), the b-diketone group was calculated to have maximum electron density in the curcumin molecule [22, 23]. In Figure 4a, at low surfactant concentrations of CCTAB (0.02 to 0.4 mM), the intensity of shoulder peak at kmax = 423 nm (conjugated structure of curcumin) decreased, while the shoulder at around kmax = 350 to 360 nm (feruloyl unit of curcumin) is increased. It is noticed that the positively charged head group of CTAB may electrostatically interact with the b-diketone group of curcumin having a high electron density, forming CTAB/curcumin complexes. The binding of surfactant molecule on the central methylene bridge of curcumin decreases the interactions between two feruloyl moieties. So, as CTAB increases, the formation of CTAB/ curcumin complexes may reduce the absorption of conjugated p-bond characteristics at 423 nm and the increasing in the shoulder at around 350 to 360 nm was due to the feruloyl units of drug. Because of the bound CTAB molecules remain at their monomeric state, there is almost no aggregation among CTAB molecules. At intermediate surfactant concentrations (Fig. 4b), CCTAB = 0.4 to 1.4 mM, the intensity of shoulder peak at kmax = 423 nm once again started to increase and the shoulder at around kmax = 350 to 360 nm disappeared. It is noted that due to the hydrophobic interac-

tion, the long alkyl chains of added CTAB may bind with the aromatic groups of curcumin. The hydrophobic interaction among the alkyl chains of CTAB may overcome the electrostatic interaction of head group of CTAB with the b-diketone group of curcumin, which may gradually lead the head group of CTAB to leave the b-diketone group of curcumin. At the same time, the hydrophobic alkyl chains of different CTAB molecules bounded at the aryl group of curcumin may start to aggregate into small CTAB pre-micelles. The release of b-diketone group of curcumin will help to recover the conjugated structure of curcumin, leading to the enhanced absorption of curcumin at 423 nm. Finally at high surfactant concentrations, CCTAB = 1.4 to 2.5 mM, as shown in Fig. 4c, the CTAB micelles are already present and the absorption showed, that curcumin is located inside CTAB micelles, which is consistent with the reported systems of cur-


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Figure 3 (a) UV-Visible spectra of curcumin(fixed 10 lM) as the function of CTAB concentration in PBS of pH.6.4 at 298 K. (b) Maximum absorbance at kmax = 423 nm of curcumin at various CTAB concentrations

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Figure 4 Compiled UV-Visible Spectra of curcumin at pre-micellar, micellar and post-micellar regions of CTAB


cumin with surfactant micelles [15, 22, 24]. In CTAB micelles, the curcumin molecule totally recovers the conjugated structure, which will sharp increase the absorption at 423 nm due to the high hydrophobic micellar environment. Curcumin is likely to be trapped inside CTAB micelles in the trans-type orientation [16], that is, one phenoxy group resides close to the micellar surface, while the other phenoxy group and the b-diketone group are located near the hydrophobic core of the micelles. In addition to the hydrogen bonding of the phenoxy group with the head group of CTAB, there are hydrophobic interactions between surfactant alkyl chains and the aryl group of curcumin drug. Scheme 3 shows the interaction mechanisms of CTAB with curcumin at pre-micellar, micellar and post micellar regions of CTAB surfactant, which are coincident with the micellization of CTAB without curcumin drug. The results of the absorption suggest that the interaction of CTAB with curcumin is different at surfactant concentrations where CTAB molecules remain in the monomeric form, start to aggregate into CTAB premicelles, and finally form general CTAB micelles [15]. Representation clearly indicated the initially attraction of positive head group of surfactant on drug molecule because of b-diketone group followed the more attachment of nonpolar chain of surfactant towards aromatic moiety of drug and finally micelles of surfactant encapsulated the drug molecule and it solubilized with intact monomeric form. 4 Conclusions

The interaction of curcumin with cationic surfactant, CTAB was investigated using UV-Visible absorption spectroscopy. The results have outlined three distinct processes depending on the surfactant concentration. In the pre-micellar range the variation of the absorbance and peak was assigned to attraction of initially positive head group towards the b-diketone group of curcumin. At CTAB concentration in intermediate/micellar region including CMC, a second type of interaction is observed, corresponding to the attachment of alkyl C16-chains of surfactant to aryl groups of drug and displacement of head group from b-diketone group of the cur-

cumin drug. Finally at CTAB concentration higher than the CMC, in the postmicellar region, a type of interaction is observed, which corresponds to the encapsulation/solubilization of the curcumin drug into micelles, predominantly in monomeric form. Acknowledgements

Financial Assistance from University Grants commission (UGC), New Delhi Project no. F.41-1327/2012(SR) is gratefully acknowledged. References 1. Aggarwal, B. B., Sundaram, C., Malani, N. and Ichikawa, H.: Curcumin: The Indian Solid Gold, Adv. Exp. Med. Biol. 595 (2007) 1 – 75. 2. Tang, C. H., Lee, C. Y. and Huang, M. T.: Phenolic Compounds in Food and Their Effects on Health I., Washington DC, ACS Symposium Series 506, American Chemical Society (1992). 3. Kita, T., Imai, S., Sawada, H., Kumagai, H. and Seto, H.: Biosci. Biotechnol. Biochem. 72 (2008) 1789 – 1798. 4. Anand, P., Sundaram, C., Jhurani, S., Kunnumakkara, A. B. and Aggarwal, B. B.: Curcumin and Cancer: An \old-age" disease with an \age-old" solution, Cancer Lett. 267 (2008) 133 – 164. 5. Teiten, M. H., Eifes, S., Dicato, M. and Diederich, M.: Curcumin-The Paradigm of a Multi-Target Natural Compound with Applications in Cancer Prevention and Treatment, Toxins 2 (2010) 128 – 162. 6. Hatcher, H., Planalp, R., Cho, J., Torti, F. M. and Torti, S. V.: Cell. Mol. Life Sci. 65 (2008) 1631 – 1652. 7. Sharma, R. A., Gescher, A. J. and Steward, W. P.: Eur. J. Cancer 41 (2005) 1955 – 1968. 8. Nardo, L., Andreini, A., Masson, M., Haukvik, T. and Tønnesen, H. H.: J. Fluoresc. 21 (2011) 627 – 635. 9. Nardo, L., Andreini, A. and Tønnesen, H. H.: In Hydrogen Bonding and Transfer in the Excited State; New York, John Wiley & Sons (2010) pp. 353 – 375. 10. Patra, D. and Barakat, C.: Spectrochim. Acta A 79 (2011) 1034 – 1041. 11. Bong, P. H.: Bull. Korean Chem. Soc. 1 (2000) 81 – 86. 12. Chignell, C. F., Bilski, P., Reszka, K. J., Motten, A. G., Sik, R. H. and Dahl, T. A.: Photochem. Photobiol. 59 (1994) 295 – 302. 13. Wang, Y. J., Pan, M. H., Cheng, A. L., Lin, L. I., Ho, Y. S., Hsich, C. Y. and Lin, J. K.: J. Pharm. Biomed. Anal. 15 (1997) 1867. 14. Rangel-Yagui, C., Pessoa, Jr. A. and Tavares, L. C.: J. Pharm. Pharmaceut. Sci. 8 (2005) 147 – 163. 15. Leung, M. H. M., Colangelo, H. and Kee, T. W.: Langmuir 24 (2008) 5672 – 5675. 16. Dan, K., Wang, X, Yang, Q., Niu, Y., Chai, S., Chen, Z., An, X. and Shen, W.: Langmuir 27 (2011) 14112 – 14117. 17. Iwunze, M. O.: J. Mol. Liquids 111 (2004) 161 – 165. 18. Jain, D. V. S. and Singh, S.: Indian J. Chem. 10 (1972) 629. 19. Lah, J., Pohar, C. and Vesnaver, G.: J. Phys. Chem. B 104 (2000) 2522. 20. Tedeschi, A. M., Franco, L., Ruzzi, M., Paduano, L., Corvajaand, C. and D’Errico, G.: Phys. Chem. Chem. Phys. 5 (2003) 4204 – 4209.


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Scheme 3 Schematic presentation of mechanism of interaction between curcumin and CTAB


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21. Xiaoli, T., Zhang, L., Zhao, S., Jiayong, Y. and Jingyi, A.: J. of Surfactant Detergents 7 (2004) 2. 22. Tonnesen, H. H.: Pharmazie 57(12) (2002) 820 – 824. 23. Zsila, F., Bikadi, Z. and Simonyi, M.: Tetrahedron Asymmetry 14 (2003) 2433 – 2444. 24. Wang, Z. F., Leung, M. H. M., Kee, T. W. and English, D. S.: Langmuir 26 (2010) 5520 – 5526. Received: 24. 02. 2013 Revised: 28. 03. 2013

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Correspondence address Dr. Rakesh K. Sharma Assistant Professor Applied Chemistry Department Faculty of Technology & Engineering The Maharaja Sayajirao University of Baroda Post Box no. 51, Kala Bhavan Vadodara-390001 (Gujarat) India Tel.: (O) +9126 52 43 4188 (M) +9192 28 49 92 73 Fax: 0 26 5242 38 98 E-Mail: [email protected]

APPARATUS INFORMATION

The authors of this paper Dr. Rakesh K. Sharma, M. Sc., M. Phil., Ph. D., is the Assistant Professor in the Applied Chemistry Department, Faculty of Technology & Engineering, The M. S. University of Baroda, Vadodara, Gujarat, India. His research interests are in aggregation and phase behavior of surfactants and EO-PO based block copolymers and its application in drug delivery and detergency. He has published more than ten research papers in reputed international journals. Dipti Jani, M. Sc., is a project student in the Applied Chemistry Department, Faculty of Technology & Engineering, The M. S. University of Baroda, Vadodara, Gujarat, India. She is working in the area of applications of surfactants in pharmaceutical sciences.

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