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Encapsulation of Curcumin in Cationic Micelles Suppresses Alkaline Hydrolysis Mandy H. M. Leung, Hannah Colangelo, and Tak W. Kee* School of Chemistry and Physics, UniVersity of Adelaide, Adelaide, South Australia 5005, Australia ReceiVed March 12, 2008. ReVised Manuscript ReceiVed April 10, 2008 The alkaline hydrolysis of curcumin was studied in three types of micelles composed of the cationic surfactants cetyl trimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB) and the anionic surfactant sodium dodecyl sulfate (SDS). At pH 13, curcumin undergoes rapid degradation by alkaline hydrolysis in the SDS micellar solution. In contrast, alkaline hydrolysis of curcumin is greatly suppressed in the presence of either CTAB or DTAB micelles, with a yield of suppression close to 90%. The results from fluorescence spectroscopic studies reveal that while curcumin remains encapsulated in CTAB and DTAB micelles at pH 13, curcumin is dissociated from the SDS micelles to the aqueous phase at this pH. The absence of encapsulation and stabilization in the SDS micellar solution results in rapid hydrolysis of curcumin.
Introduction Curcumin is a naturally occurring yellow-orange pigment found in the Indian spice turmeric, and its medicinal properties have been documented in ancient literature.1 A recent study shows that curcumin exists predominantly in the keto-enol form, of which the chemical structure is shown in Figure 1.2 The upsurge in research activities over the past decade on curcumin is largely due to the discovery that curcumin possesses effective antioxidant,3 anti-inflammatory,4 and anticancer5–7 properties. Recent work has also demonstrated that curcumin possesses the ability to prevent protein aggregation in debilitating diseases such as Alzheimer’s8 and Parkinson’s.9 A major challenge in using curcumin for treatment of diseases is the poor aqueous solubility (∼20 µg/mL), which significantly limits its availability in biological systems. For the fraction of curcumin that is aqueous soluble, another main challenge to widespread clinical application is the lack of stability. Curcumin undergoes rapid degradation first by hydrolysis, which is then followed by molecular fragmentation.10 The aqueous solubility of curcumin can be improved by increasing the pH of the solution. However, this approach leads to an undesirable outcome: a high rate of degradation by alkaline hydrolysis.10–12 An attractive alternative approach to addressing the poor aqueous solubility issue is to encapsulate curcumin in surfactant micelles; several studies have * To whom correspondence should be addressed. E-mail: tak.kee@ adelaide.edu.au. (1) Goel, A.; Kunnumakkara, A. B.; Aggarwal, B. B. Biochem. Pharmacol. 2008, 75, 787. (2) Payton, F.; Sandusky, P.; Alworth, W. L. J. Nat. Prod. 2007, 70, 143. (3) Ruby, A. J.; Kuttan, G.; Babu, K. D.; Rajasekharan, K. N.; Kuttan, R. Cancer Lett. 1995, 94, 79. (4) Lantz, R. C.; Chen, G. J.; Solyom, A. M.; Jolad, S. D.; Timmermann, B. N. Phytomedicine 2005, 12, 445. (5) Aggarwal, B. B.; Kumar, A.; Bharti, A. C. Anticancer Res. 2003, 23, 363. (6) Shi, M.; Cai, Q.; Yao, L.; Mao, Y.; Ming, Y.; Ouyang, G. Cell Biol. Int. 2006, 30, 221. (7) Surh, Y. J. Food Chem. Toxicol. 2002, 40, 1091. (8) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem. 2005, 280, 5892. (9) Masuda, M.; Suzuki, N.; Taniguchi, S.; Oikawa, T.; Nonaka, T.; Iwatsubo, T.; Hisanaga, S.i.; Goedert, M.; Hasegawa, M. Biochemistry 2006, 45, 6085. (10) Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.; Hsieh, C. Y.; Lin, J. K. J. Pharm. Biomed. Anal. 1997, 15, 1867. (11) Bernabe-Pineda, M.; Ramirez-Silva, M. T.; Romero-Romo, M.; GonzalezVergara, E.; Rojas-Hernandez, A. Spectrochim. Acta, Part A 2004, 60A, 1091. (12) Tønnesen, H. H.; Karlsen, J. Z. Lebensm.-Unters. Forsch. 1985, 180, 402.
Figure 1. Chemical structure of the keto-enol form of curcumin. The pKa values at 8.31, 10.0, and 10.2 in aqueous solution correspond to deprotonation of the three hydroxyl groups of curcumin,21 with the (a) enolic and (b and c) phenolic protons highlighted.
shown that curcumin has a significantly higher solubility (∼740 µg/mL) in micellar solutions.13,14 When curcumin is encapsulated in a micelle, it is segregated from the aqueous phase, and that leads to a central question: what is the effect of micelle encapsulation on the stability of curcumin against alkaline hydrolysis? Despite the vast literature on curcumin’s potent medicinal properties,3–9 there is surprisingly a low number of studies on the physicochemical properties of curcumin in micellar systems.13–16 Within this limited number of studies, Tønnesen has investigated the chemical stability of curcumin at pH 5 and 8 in a number of surfactant solutions, including sodium dodecyl sulfate (SDS), Triton X-100 (TX-100), tetradecyl trimethylammonium bromide (TTAB).13 The results indicate that SDS and TX-100 micelles are highly effective in stabilizing curcumin, which increases the chemical stability by nearly 1800 times relative to a solution where these micelles are absent. Additionally, the study shows that TTAB micelles are not nearly as effective as SDS and TX-100 micelles in stabilizing curcumin at pH 8. While this work provides important insights into the degradation of curcumin in a slightly alkaline condition, the application of micelles on stabilizing curcumin at a higher pH has not been reported. There is significance in stabilizing curcumin in micelles (13) Tønnesen, H. H. Pharmazie 2002, 57, 820. (14) Chignell, C. F.; Bilski, P.; Reszka, K. J.; Motten, A. G.; Sik, R. H.; Dahl, T. A. Photochem. Photobiol. 1994, 59, 295. (15) Bruzell, E. M.; Morisbak, E.; Tonnesen, H. H. Photochem. Photobiol. Sci. 2005, 4, 523. (16) Iwunze, M. O. J. Mol. Liq. 2004, 111, 161.
10.1021/la800780w CCC: $40.75 2008 American Chemical Society Published on Web 05/07/2008
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at a high pH (>11) because of the high rate of degradation.11,12 Moreover, in the studies of the anti-inflammatory17 and inhibitory18 effects of curcumin, a 0.5 M NaOH (pH 13.7) solution was used initially to dissolve curcumin and some of the experiments were conducted at high pH (>11). Due to the high rate of degradation at these pH values, it is unclear whether curcumin or the degradation products were responsible for the observed effects. Therefore, the ability to stabilize curcumin at high pH can address this important issue. In this paper, we present results from our recent studies on using micelle encapsulation as an effective method to suppress alkaline hydrolysis of curcumin at pH 13. In particular, we demonstrate that micelles composed of cationic surfactants suppress alkaline hydrolysis with a yield close to 90%. It has been shown that curcumin has three pKa values at 8.38, 9.88, and 10.51 in aqueous solution, corresponding to deprotonation of the three hydroxyl groups (Figure 1);11 therefore, curcumin is fully deprotonated at pH 13 to form the highly negatively charged species, Cur3-. Micellar systems of the cationic surfactants cetyl trimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB) and the anionic surfactant sodium dodecyl sulfate (SDS) were selected for our studies because these systems are well characterized, with known critical micelle concentrations (CMC) and surfactant aggregation numbers.19,20
Experimental Section Materials. Curcumin (from Curcuma longa, or Turmeric), purity ∼70% by HPLC assay, and DTAB (∼99%) were obtained from Sigma. CTAB (g99% by AT assay) and SDS (g99% by GC assay) were purchased from Fluka. NaOH pellets (AR grade, g 97%) from Chem-Supply and methanol (AR grade, g 99.5%) from Merck were used as received. Solutions were prepared with neat water from a Millipore Milli-Q NANOpure water system. UV-Visible Absorption Studies. Micellar solutions of 30.6 mM DTAB, 16.2 mM SDS, and 1.84 mM CTAB in neat water were prepared. The surfactant concentrations are twice the CMC and thus ensure the formation of micelles. A solution of 5 mg/mL curcumin in methanol was used as stock. A small quantity of the curcumin stock solution (11.05 or 15.8 µL) was added to 3 mL of micellar solution to achieve a final concentration of 50 or 60 µM for curcumin. Absorbance readings were taken from 300 to 700 nm using a Cary 5000 UV-vis-NIR spectrophotometer (Varian). A volume of 111.3 µL of 2.7 M NaOH was added to the cuvette, and the pH was measured to be 13. Fluorescence Studies. Micellar solutions were made with the same surfactant concentrations. A solution of 1 mg/mL curcumin in methanol was used as stock. A 4 µL volume of this stock solution was added to 3 mL of micellar solution to achieve a concentration of 4.7 µM for curcumin. The pH 7 and 13 curcumin-micellar solutions were prepared using the methods highlighted above. Fluorescence spectra were taken from 480 to 750 nm using a Cary Eclipse Fluorescence spectrophotometer (Varian) with the excitation and emission slit widths set at 5 nm. The excitation wavelength for each solution was 420 nm for water, 428 nm for SDS, and 421 nm for both CTAB and DTAB, whereas the excitation wavelength for each of the 0.1 M NaOH solutions was 468 nm for water and SDS and 470 nm for both CTAB and DTAB.
Results and Discussions UV-Vis Absorption Spectra of Curcumin and Cur3-. Curcumin exhibits an intense optical absorption in the UV-visible spectral region in neat water and micellar solutions with an (17) Chan, M. M.-Y.; Huang, H. I.; Fenton, M. R.; Fong, D. Biochem. Pharmacol. 1998, 55, 1955. (18) Kurien, B. T.; Scofield, R. H. J. Ethnopharmacol. 2007, 110, 368. (19) Hansson, P.; Joensson, B.; Stroem, C.; Soederman, O. J. Phys. Chem. B 2000, 104, 3496. (20) Jalsenjak, N.; Tezak, D. Chem.sEur. J. 2004, 10, 5000.
Figure 2. UV-vis absorption spectra of curcumin (50 µM) and Cur3in (a) water, (b) CTAB, (c) DTAB, and (d) SDS micelles.
absorption peak around 420 nm and molar extinction coefficients ranging from 25 000 to 60 000 M-1 cm-1, shown as solid lines in Figure 2. The UV-vis absorption spectrum exhibits a structure with the presence of three peaks (approximately 400, 420, and 450 nm) for curcumin in the cationic micelles, but this structure is absent in SDS micelles and water. At pH 13 (0.1 M NaOH), Cur3- produces a red-shifted spectrum (λmax ≈ 480 nm) with a similar or higher molar extinction coefficient compared to the keto-enol form of curcumin, as shown in Figure 2 (dotted curves). This characteristic red-shift occurs in aqueous solution as well as in the micellar solutions. Interestingly, Cur3- produces identical spectra in aqueous solution and in the presence of SDS micelles, as will be discussed further. The large molar extinction coefficients of curcumin and Cur3- in these solutions indicate that it is an ideal system to be investigated using UV-vis absorption spectroscopy. Alkaline Hydrolysis of Curcumin. The degradation of curcumin in aqueous solution has been linked to hydrolysis, and studies have shown that the hydrolytic degradation process occurs rapidly at a pH above neutral.10,12,21 In alkaline hydrolysis, curcumin is initially deprotonated and subsequently fragmented into smaller molecules; Wang et al. have used high performance liquid chromatography and mass spectrometry to show that curcumin degrades to form trans-6-(4′-hydroxy-3′-methoxyphenyl)-2,4-dioxo-5-hexanal as the main product, which further decomposes to vanillin, ferulic acid, and feruloyl methane.10 While Cur3- has an intense visible absorption as mentioned above, there have been concerns that condensation of molecular fragments might introduce errors in spectrophotometric measurements.21 In our studies, however, we determined that the contributions from the condensation products to the total absorption signal are relatively minor (see the Supporting Information). Thus, the decrease of the visible absorbance over time, signifying the disappearance of Cur3-, can be used as a measurement of degradation.
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Letters Table 1. Rate of Degradation of Cur3- in Aqueous and Micellar Solutions and Yield of Suppression of Degradation by the Cationic Micelles micelle
rate (nM h-1)
yield of suppression
(neat water) SDS CTAB DTAB
844 ( 104 1150 ( 30.3a 102 ( 71.8 93.6 ( 85.6
-a 87.4 ( 16.4%b 88.1 ( 16.2%b
a The slightly faster rate of degradation in pH 13 SDS micellar solution suggests that the degradation pathway may be different from that of the pH 13 aqueous solution, and hence, no yield of suppression of degradation is given for the SDS micellar solution. Errors of the rate were determined by standard deviations in three independent measurements. b Yield ) [(rateneat water - ratemicelle)/rateneat water] × 100%. The standard errors were estimated using error propagation.
Figure 3. UV-Vis absorption spectra of Cur3- in (a) water, (b) CTAB, (c) DTAB, and (d) SDS micelles over a course of 20 h. Insets show the decays of the Cur3- absorption maxima due to degradation.
The kinetics of degradation of Cur3- were investigated in the following solutions: neat water, DTAB, CTAB, and SDS micelles. It has been shown that the presence of ions lowers the CMC,22,23 and hence, this effect further promotes the formation of micelles in our studies due to a concentration of 0.1 M NaOH24 in the solutions. Degradation of Cur3- was recorded over approximately 20 h at 1 h intervals by monitoring the decrease in the absorption maximum. The results of the kinetic studies are shown in Figure 3. In aqueous solution, the absorption maximum decays to approximately 67% of the original value in 20 h (Figure 3a). In the CTAB and DTAB micellar solutions, however, the decays are minimal (Figure 3b and c). In the SDS micellar solution, the absorption signal decreases to approximately the 60% point (Figure 3d), similar to the decay in aqueous solution. The timedependent change in absorbance (∆A) of each solution is given in the insets. The decay kinetics of Cur3- in aqueous solution (inset of Figure 3a) fit well to a pseudozero order model, with a rate of degradation of 844 ( 104 nM h-1 which is in agreement with a previous study at a comparable pH.11 Similarly, the decay curves of Cur3- in CTAB, DTAB, and SDS micellar solutions are also consistent with pseudozero order kinetics, giving rise to rates of degradation which are summarized in Table 1. Suppression of Degradation of Cur3- by Cationic Micelles. The rate of degradation of Cur3- is significantly lower in cationic micelles than in aqueous solution. In CTAB and DTAB micelles, the rates of degradation have values of 101 ( 71.8 and 93.6 ( 85.6 nM h-1, respectively; these values are nearly within the experimental errors. The ratio between the rates of degradation in aqueous solution and CTAB micelles indicates that the degradation is approximately 8 times slower in CTAB micelles. In other words, the presence of CTAB micelles suppresses the degradation of Cur3- with a yield of 87.4 ( 16.4%; see Table (21) Tønnesen, H. H.; Karlsen, J. Z. Lebensm.-Unters. Forsch. 1985, 180, 132. (22) Lebedeva, N. V.; Shahine, A.; Bales, B. L. J. Phys. Chem. B 2005, 109, 19806. (23) Thevenot, C.; Grassl, B.; Bastiat, G.; Binana, W. Colloids Surf., A 2005, 252, 105. (24) Abe, M.; Kato, K.; Ogino, K. J. Colloid Interface Sci. 1989, 127, 328.
1. Using the same analysis, a similar suppression yield of 88.1 ( 16.2% was measured in DTAB micelles. To the authors’ knowledge, these results are the first work to demonstrate the ability of cationic micelles to stabilize Cur3- and suppress degradation. While CTAB and DTAB micelles suppress degradation of Cur3-, anionic micelles composed of SDS are ineffectiVe in lowering the rate of degradation. In fact, our results indicate that degradation of Cur3- in SDS micelles occurs at a slightly higher rate than in aqueous solution, with a value of 1150 ( 30.3 nM h-1. The presence of a statistically significant difference (Table 1) between the rates of degradation in SDS micelles and aqueous solution implies that the two solutions may have slightly different degradation pathways. In short, the cationic micelles composed of CTAB and DTAB are highly effective in suppressing degradation of Cur3-. The SDS micelles, however, do not inhibit the degradation process. Interestingly, results from a previous study by Tønnesen13 show an opposite trend in a weakly alkaline solution. At pH 8, curcumin undergoes rapid degradation in CTAB micelles. Additionally, SDS micelles are remarkably effective in preventing degradation of curcumin. The sharp contrast between these results at pH 8 and our results reported herein (pH 13) signifies the following. First, the strong dependence of the degradation rate of curcumin in CTAB micelles on pH indicates that the degradation pathway is highly pH sensitive. There is a general agreement in the literature that the pKa value of 8.31 corresponds to the loss of the enolic proton (Figure 1) of curcumin,11,25 and hence, approximately 33% of curcumin at pH 8 is deprotonated to this level according to the Henderson-Hasselbalch Equation. At pH 13, however, curcumin is fully deprotonated. The combination of results from ref 13 and our results strongly indicates that degradation of curcumin in CTAB micelles is highly dependent on the level of deprotonation of curcumin. This effect is expected because a similar pH-dependent degradation has been observed in aqueous solution.12 Second, the ability of SDS micelles to prevent degradation of curcumin at pH 8 has been attributed to the electrostatic repulsion between the OH- ions and the negatively charged surfactant headgroup.13 It follows that curcumin remains segregated from the OH- ions and hence avoids undergoing degradation at this pH. At pH 13, however, Cur3- is formed, and its rapid degradation in SDS micelles will be discussed below. Micelle Encapsulation of Cur3-. The suppression of Cur3degradation in cationic micelles (Table 1) and the identical UV-vis spectra of Cur3- in aqueous solution and SDS micelles (dotted curves in Figure 2a and d) lead to the following hypotheses. First, effective suppression of Cur3- degradation is due to efficient (25) Shen, L.; Ji, H. F. Spectrochim. Acta, Part A 2007, 67, 619.
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on Cur3-, it has the potential to mediate attractive electrostatic interactions with the positively charged headgroups of CTAB and DTAB micelles. These important attractive interactions allow Cur3- to remain encapsulated and contribute greatly to its stabilization in the cationic micelles. In contrast, Cur3- interacts repulsively with the negatively charged headgroups of SDS micelles. The presence of electrostatic repulsion destabilizes Cur3in the SDS micelle, leading to dissociation from the micelle (see the Supporting Information). As discussed earlier, once curcumin is liberated to the aqueous phase, alkaline hydrolysis will proceed and lead to degradation. Figure 4. Fluorescence spectra of Cur3- in the micellar and aqueous solutions. The spectra of Cur3- in aqueous and SDS micelle solutions are virtually identical, and they are 1 order of magnitude less intense than those of Cur3- in the cationic micellar solutions.
encapsulation in cationic micelles. Second, rapid degradation of Cur3- in the presence of SDS micelles indicates that Cur3- is dissociated from the micelle to the aqueous phase. In order to test these hypotheses, we performed fluorimetric measurements of Cur3- in aqueous and micellar solutions, and the results are shown in Figure 4. The fluorescence intensities of Cur3- in DTAB and CTAB micelles are 1 order of magnitude higher than that in aqueous solution, and these results are consistent with the fluorescence properties of curcumin at neutral pH.14,16,26,27 The results strongly support that Cur3- remains encapsulated in DTAB and CTAB micelles, which effectively suppresses degradation. Additionally, the fluorescence spectrum of Cur3- in the SDS micellar solution is virtually identical to that in aqueous solution, which unequivocally supports that Cur3- is dissociated from the SDS micelles and liberated to the aqueous phase. Since Cur3is dissociated from SDS micelles, it is subject to alkaline hydrolysis due to its presence in the aqueous phase. Electrostatic Interactions of Cur3- with the Micelle Headgroup. Here, we address the issue as to why Cur3- is dissociated from SDS micelles. Because of the negative charges (26) Iwunze, M. O. Tenside, Surfactants, Deterg. 2003, 40, 96. (27) Kunwar, A.; Barik, A.; Pandey, R.; Priyadarsini, K. I. Biochim. Biophys. Acta 2006, 1760, 1513.
Conclusion We have presented strong experimental evidence to show that the encapsulation of curcumin in cationic micelles composed of DTAB and CTAB surfactants is highly effective in suppressing alkaline hydrolysis at pH 13. Our studies concluded that the ability of cationic micelles to stabilize the deprotonated curcumin (Cur3-) is due to its attractive electrostatic interactions with the CTAB and DTAB headgroups. These important interactions permit Cur3- to be encapsulated in the micelles, leading to suppression of degradation. The SDS micelles, however, are unable to prevent alkaline hydrolysis because electrostatic repulsions between Cur3- and the negatively charged headgroups result in the dissociation of Cur3- from the micelle. Once liberated, Cur3- undergoes alkaline hydrolysis in the aqueous phase of the solution. Acknowledgment. This work was supported in part by a startup grant provided by the School of Chemistry and Physics and a research grant from the Australian Research Council and National Health and Medical Research Council Network “Fluorescence Applications in Biotechnology and Life Sciences”. Supporting Information Available: Minor contributions from the condensation products in the degradation of Cur3-, and fluorescence spectra of curcumin in SDS micelles titrated with 0.1 M NaOH. This material is available free of charge via the Internet at http://pubs.acs.org. LA800780W
