pubs.acs.org/Langmuir © 2009 American Chemical Society
Effective Stabilization of Curcumin by Association to Plasma Proteins: Human Serum Albumin and Fibrinogen Mandy H. M. Leung and Tak W. Kee* School of Chemistry and Physics, University of Adelaide, Adelaide, South Australia 5005, Australia Received December 22, 2008. Revised Manuscript Received January 23, 2009 The use of curcumin as an effective wound healing agent is of significant interest currently. It is well established that curcumin undergoes rapid degradation in physiological buffer by hydrolysis. The means by which curcumin is stabilized at the wound site to enable healing is poorly understood because blood plasma is composed of approximately 92% water. Plasma proteins, which constitute the remaining 6-8%, has been shown to stabilize curcumin. It is, however, still unclear which proteins are responsible for this phenomenon. In this study, the effects of major plasma proteins, which include human serum albumin (HSA), fibrinogen, immunoglobulin G (IgG), and transferrin, on stabilizing curcumin are investigated. In particular, we investigate their effects on the hydrolysis of curcumin at pH 7.4. In the presence of both transferrin and IgG, curcumin continues to undergo rapid hydrolysis but this reaction is suppressed by the presence of either HSA or fibrinogen with an impressive yield of approximately 95%. Furthermore, the binding constants of curcumin to HSA and fibrinogen are on the order of 104 and 105 M-1, respectively. The binding constants of transferrin and IgG, however, are at least 1 order of magnitude less than those of HSA and fibrinogen. The results support that strong binding occurs at the hydrophobic moieties of HSA and fibrinogen, excluding water access. Therefore, strong interactions with HSA and fibrinogen inhibit hydrolysis of curcumin and in turn lead to effective suppression of degradation.
Introduction Curcumin, the yellow pigment isolated from the rhizomes of the Indian spice plant turmeric, exhibits a large number of medicinal properties. The low incidence of digestive tract cancers in the Indian subcontinent1,2 has motivated researchers to investigate the anticancer properties of curcumin. Studies have shown that curcumin not only possesses anticancer properties, 3,4 but it also has anti-inflammatory,5 antioxidant,6 anti-Alzheimer’s disease,7 anti-cystic fibrosis,8 and wound healing effects. 9-11 Additionally, the potential of curcumin as a photodynamic therapy agent in skin cancer treatment was demonstrated in a number of studies.12,13 A recent study shows that excited-state intramolecular hydrogen atom transfer is a major photophysical process in curcumin upon photoexcitation.14 This result *To whom correspondence should be addressed. E-mail: tak.kee@ adelaide.edu.au. (1) Mohandas, K. M.; Desai, D. C. Indian J. Gastroenterol. 1999, 18, 118. (2) Moragoda, L.; Jaszewski, R.; Majumdar, A. P. N. Anticancer Res. 2001, 21, 873. (3) Aggarwal, B. B.; Kumar, A.; Bharti, A. C. Anticancer Res. 2003, 23, 363. (4) Shi, M.; Cai, Q.; Yao, L.; Mao, Y.; Ming, Y.; Ouyang, G. Cell Biol. Int. 2006, 30, 221. (5) Lantz, R. C.; Chen, G. J.; Solyom, A. M.; Jolad, S. D.; Timmermann, B. N. Phytomedicine 2005, 12, 445. (6) Ruby, A. J.; Kuttan, G.; Babu, K. D.; Rajasekharan, K. N.; Kuttan, R. Cancer Lett. 1995, 94, 79. (7) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem. 2005, 280, 5892. (8) Egan, M. E.; Pearson, M.; Weiner, S. A.; Rajendran, V.; Rubin, D.; Glockner-Pagel, J.; Canny, S.; Du, K.; Lukacs, G. L.; Caplan, M. J. Science 2004, 304, 600. (9) Jagetia, G. C.; Rajanikant, G. K. Plast. Reconstr. Surg. 2005, 115, 515. (10) Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Life Sci. 2006, 78, 2081. (11) Gopinath, D.; Ahmed, M. R.; Gomathi, K.; Chitra, K.; Sehgal, P. K.; Jayakumar, R. Biomaterials 2004, 25, 1911. (12) Koon, H.; Leung, A. W. N.; Yue, K. K. M.; Mak, N. K. J. Environ. Pathol., Toxicol. Oncol. 2006, 25, 205. (13) Park, K.; Lee, J.-H. Oncol. Rep. 2007, 17, 537. (14) Adhikary, R.; Mukherjee, P.; Kee, T. W.; Petrich, J. W. J. Phys. Chem. B 2009, in press.
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is expected because the keto-enol form of curcumin, shown in Figure 1, is the predominant one of the two tautomeric forms in solution, in which intramolecular hydrogen bonding is present; Payton et al. showed that the diketo tautomer is present at less than 1% concentration.15 Although curcumin has potential for extensive use in disease treatment, there are two major challenges that limit this target. First, curcumin has low solubility in aqueous solution (approximately 20 μg/mL), which poses a severe limitation on the achievable concentration in biological systems. Second, the soluble portion undergoes rapid degradation at physiological pH.16 Several methods have been proposed to address the issues of low solubility and stability. Encapsulation in polymer nanoparticles,17 which are aggregates of cross-linked and random copolymers, has shown promise as a potentially effective method of delivering curcumin. In addition, aggregation of surfactants, including sodium dodecyl sulfate (SDS), cetyl trimethylammonium bromide (CTAB), and Triton X-100, into micelles also provides desirable effects in solubilizing and stabilizing curcumin.18-20 In particular, we have previously shown that cationic micelles are capable of stabilizing curcumin at elevated pH, which is the condition often used in the studies of curcumin’s medicinal effects.19 In addition, preferential binding to lipid bilayers and vesicles has been suggested as a delivery system for administration of curcumin as a drug.21-23 Recently, the use of serum albumin as (15) Payton, F.; Sandusky, P.; Alworth, W. L. J. Nat. Prod. 2007, 70, 143. (16) 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. (17) Bisht, S.; Feldmann, G.; Soni, S.; Ravi, R.; Karikar, C.; Maitra, A.; Maitra, A. J. Nanobiotechnol. 2007, 5, 3. (18) Iwunze, M. O. J. Mol. Liq. 2004, 111, 161. (19) Leung, M. H. M.; Colangelo, H.; Kee, T. W. Langmuir 2008, 24, 5672. (20) Toennesen, H. H. Pharmazie 2002, 57, 820. (21) Thangapazham, R. L.; Puri, A.; Tele, S.; Blumenthal, R.; Maheshwari, R. K. Int. J. Oncol. 2008, 32, 1119. (22) Kunwar, A.; Barik, A.; Pandey, R.; Priyadarsini, K. I. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 1513. (23) Sun, Y.; Lee, C.-C.; Hung, W.-C.; Chen, F.-Y.; Lee, M.-T.; Huang, H. W. Biophys. J. 2008, 95, 2318.
Published on Web 3/25/2009
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Experimental Section
Figure 1. Chemical structure of the keto-enol form of curcumin. The dashed line indicates the intramolecular hydrogen bonding.
a carrier for curcumin has been reported,22,24-28 implying that this protein has the ability to stabilize curcumin. This implication is consistent with the results by Wang et al.,16 indicating that degradation of curcumin in buffer solutions is suppressed in the presence of serum, of which the major components are water (∼92%) and proteins (6-8%). This result leads to an important question: which proteins are responsible for stabilizing curcumin? Plasma proteins are a family of proteins in blood plasma, which include serum proteins and the proteins responsible for forming blood clots. The major proteins in blood plasma are human serum albumin (HSA), immunoglobulin G (IgG), transferrin, and fibrinogen.29 Approximately 60% of the proteins in the human blood plasma is HSA, which plays the role of maintaining the osmotic pressure of blood vessels. Additionally, HSA also acts as a carrier protein, transporting molecules including lipids, steroid hormones, and drugs. Immunoglobulins (Ig), which are also known as antibodies, constitute approximately 35% of plasma proteins, and they play key roles in identifying and targeting harmful pathogens. There are five classes of Ig, namely, IgA, D, E, G, and M, and the most abundant class is IgG, composing about 75% of the total Ig. The glycoprotein fibrinogen, which is present in blood plasma at 4% concentration, contributes significantly to the formation of blood clots. When bleeding occurs due to tissue damage, fibrinogen is converted to fibrin by the clotting enzyme thrombin to eventually form a clot around the wound. Transferrin,30 the glycoprotein that functions as an iron (Fe3+) carrier, and other regulatory proteins including enzymes and hormones make up the final protein component in blood plasma. In this study, we focus on investigating the role of plasma proteins in stabilizing curcumin in phosphate buffer solution. The central aim of this study is to provide important insight into the role of plasma protein stabilization which in turn results in the effectiveness of curcumin as a wound healing agent. In the absence of plasma proteins, curcumin undergoes rapid degradation in phosphate buffer, as has been observed in a previous study.16 Here, we report that while low to moderate level of curcumin stabilization takes place in the presence of IgG and transferrin, curcumin is greatly stabilized in the presence of HSA and fibrinogen. The results imply that the association of curcumin to HSA and fibrinogen contributes significantly to its stability in an environment that mimics the wound site. (24) Reddy, A. C. P.; Sudharshan, E.; Rao, A. G. A.; Lokesh, B. R. Lipids 1999, 34, 1025. (25) Sahoo, B. K.; Ghosh, K. S.; Dasgupta, S. Biophys. Chem. 2008, 132, 81. (26) Barik, A.; Mishra, B.; Kunwar, A.; Priyadarsini, K. I. Chem. Phys. Lett. 2007, 436, 239. (27) Zsila, F.; Bikadi, Z.; Simonyi, M. Biochem. Biophys. Res. Commun. 2003, 301, 776. (28) Barik, A.; Priyadarsini, K. I.; Mohan, H. Photochem. Photobiol. 2003, 77, 597. (29) Putnam, F. W. The Plasma Proteins: Structure, Function, and Genetic Control, 2nd ed.; Academic Press: New York, 1975; Vol. 1, p 481. (30) Welch, S. Transferrin: The Iron Carrier; CRC Press: Boca Raton, FL, 1992; p 304.
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Materials. Curcumin (from Curcuma longa, or Turmeric), purity ∼70% by high performance liquid chromatography (HPLC) assay, human serum albumin (97-99%, agarose gel electrophoresis), human fibrinogen type I (∼67% protein, 91% clottable protein), human transferrin (g80%), and immunoglobulin G (reagent grade, g95%, SDS-PAGE or HPLC) were obtained from Sigma Aldrich. Ethanol (AR grade, 99.5%) from Ajax Finechem Pty Ltd. was used as received. Phosphate buffer solutions (50 mM) were prepared with neat water from a Millipore Milli-Q NANOpure water system, and the pH was adjusted to 7.4 with HCl. UV-Visible Absorption and Fluorescence Spectra of Curcumin. HSA, fibrinogen, human transferrin, and IgG in pH 7.4 phosphate buffer were prepared with [HSA] = 12.8 μM, [fibrinogen] = 11.2 μM, [transferrin] = 8.7 μM, and [IgG] = 10.0 μM. A solution of 5 mg/mL curcumin in ethanol was used as stock. A small quantity of the curcumin stock solution was added to 2 mL of protein solution to achieve a final concentration of curcumin to protein concentration ratio of 1:2. In the buffer solution with no protein, [curcumin] = 20 μM. Absorbance readings were taken from 300 to 600 nm using a Cary 5000 UV-vis/NIR spectrophotometer (Varian). In the experiments where degradation of curcumin was recorded, the UV-vis absorption spectra were collected over 20 min at 2 min intervals. Fluorescence spectra were taken from 450 to 700 nm using a Cary Eclipse Fluorescence spectrophotometer (Varian) with the excitation and emission slit widths set at 5 nm. The excitation wavelength for each 1 μM curcumin solution was 420 nm. The concentrations of proteins are as follows: [HSA] = 5.4 μM, [fibrinogen] = 0.4 μM, [transferrin] = 1.6 μM, and [IgG] = 0.8 μM. All experiments were performed at 25 °C except those on the denatured proteins, which were done at 50 °C. Determination of Curcumin-Protein Binding Constants. In order to prevent quenching of fluorescence due to high protein concentration, the absorbance of these solutions was kept at a value of approximately 0.1 at 290 nm. Therefore, protein solutions with [HSA] = 5.4 μM, [fibrinogen] = 0.4 μM, [transferrin] = 1.6 μM, and [IgG] = 0.8 μM were prepared in pH 7.4 phosphate buffer. A solution of curcumin in ethanol (0.68 mM) was used as stock. Curcumin was titrated into the protein solution at 0.2 μM intervals from 0 to 1 μM and then at 1 μM intervals afterward. Fluorescence spectra were recorded from 300 to 550 nm with an excitation wavelength of 280 nm. The binding process is described by the following equilibrium K
protein þ nðcurcuminÞ h ½protein-curcuminn
ð1Þ
where the protein binds with n equivalents of curcumin to form a protein-curcumin complex, [protein-curcuminn]. The extent to which the binding occurs is indicated by the binding constant K. In order to develop detailed understanding of the effects of binding (eq 1) on the suppression of degradation, quantification of K for the association between curcumin and plasma proteins is essential. Using a well-established method,28,31 which involves quenching the intrinsic fluorescence of the protein by known concentrations of curcumin, K can be quantified using eq 2. log10
F0 -F ¼ n log10 ½curcumin þ log10 K F
ð2Þ
In this equation, F0 is the initial fluorescence intensity prior to addition of curcumin, whereas F is the fluorescence intensity at a specific curcumin concentration, [curcumin]. By titration of curcumin and plotting the left-hand side of eq 2 against (31) Feng, X.-Z.; Lin, Z.; Yang, L.-J.; Wang, C.; Bai, C.-l. Talanta 1998, 47, 1223.
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Figure 2. UV-vis absorption (solid) and fluorescence (dash) spectra of curcumin in (a) pH 7.4 phosphate buffer, (b) HSA, (c) fibrinogen, (d) human transferrin, and (e) IgG. For UV-vis absorption spectra, the concentration ratio of protein to curcumin is 2:1, where [HSA], [fibrinogen], [transferrin], and [IgG] are 12.8, 11.2, 8.7, and 10.0 μM, respectively. For fluorescence spectra, while [curcumin] is kept at 1 μM, [HSA] = 5.4 μM, [fibrinogen] = 0.4 μM, [transferrin] = 1.6 μM, and [IgG] = 0.8 μM.
log10[curcumin], a linear curve is obtained with a slope of n. The y-intercept of log10 K provides quantification of K.
Results and Discussions UV-Visible Absorption and Fluorescence Spectra of Curcumin. Curcumin shows intense absorption in the UVvis region in phosphate buffer and protein solutions with absorption maxima close to 420 nm, as shown in Figure 2 (solid curves). Furthermore, the UV-vis absorption spectra of curcumin bound to HSA and fibrinogen have a shoulder around 450 nm (as indicated by solid arrows in Figure 2), which is also present in the spectrum of curcumin encapsulated in the cationic micelle, as shown in our previous study.19 The identical spectral shoulder indicates that the local environment of HSA and fibrinogen bound curcumin is similar to the palisade layer of the micelle, where curcumin is located upon encapsulation, as suggested by a previous study.32 This 450 nm shoulder structure, however, is absent in phosphate buffer and IgG, suggesting that curcumin is in a vastly different environment. In addition, these spectra are almost identical, with a spectral shoulder at 350 nm (as indicated by dashed arrows in Figure 2). These results imply that curcumin is present in the aqueous phase, and this is further supported by other experimental results, as will be shown below. The fluorescence intensity of curcumin in phosphate buffer and IgG is less than half of that in HSA, fibrinogen, and transferrin, as shown in Figure 2 (dashed curves). These results are consistent with the notion that curcumin in the IgG solution is in an environment different from that of HSA, fibrinogen, and transferrin. Previous studies have shown that the fluorescence (32) Khopde, S. M.; Priyadarsini, K. I.; Palit, D. K.; Mukherjee, T. Photochem. Photobiol. 2000, 72, 625.
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yield of curcumin is substantially lower in the polar aqueous environment than in nonpolar surroundings.19,22,28 The fluorescence intensity of curcumin in IgG (Figure 2e) is similar to that of curcumin in phosphate buffer (Figure 2a), further supporting that curcumin is present largely in the aqueous phase and implying that it is only weakly associated with IgG. On the other hand, the high fluorescence yields in HSA, fibrinogen, and transferrin imply that curcumin is segregated from the aqueous solvent. The absorption and fluorescence spectra of curcumin show a Stokes shift of about 80 nm in HSA, fibrinogen, and transferrin, but this shift increases to approximately 120 nm in phosphate buffer solution and IgG. The Stokes shifts of curcumin in HSA, fibrinogen, and transferrin are close to that in micelles (see the Supporting Information),18,19 which further suggests the similarity between the local environments. Additionally, the nearly identical Stokes shift between curcumin in phosphate buffer and IgG agrees with the implication that curcumin is present in the aqueous phase and the interaction between curcumin and IgG is weak. Hydrolytic Degradation of Curcumin. A study by Wang et al. has shown that alkaline hydrolysis is the main process in the degradation of curcumin in buffer solution.16 The results indicate that curcumin is partially deprotonated initially, which is followed by fragmentation into trans-6-(40 -hydroxy-30 methoxyphenyl)-2,4-dioxo-5-hexanal, as determined by HPLC and mass spectrometry.16 This product is then further decomposed into smaller molecules such as vanillin, feruloyl methane, and ferulic acid; these molecules contribute negligibly to the absorption of 420 nm light.16,19,33 In addition, we have also established that plasma proteins exhibit negligible absorption at this wavelength (see the Supporting Information). Therefore, the decrease in absorbance signifies the decrease of curcumin concentration solely. In this study, the kinetics of curcumin degradation were investigated in the following solutions: phosphate buffer, HSA, fibrinogen, transferrin, and IgG. The results on degradation of curcumin are shown in Figure 3. In phosphate buffer solution, the degradation is accompanied by a substantial decrease in the UV-vis absorption intensity, as shown in Figure 3a. The inset shows the decrease at the absorption maximum, which decays to approximately 60% of the initial value in 10 min. A considerable degradation of curcumin is also present in both transferrin and IgG solutions (Figure 3d and e), where the absorption at maximum decreases to roughly 65% of the original value. In contrast, the decays in HSA and fibrinogen are negligible, as shown in Figure 3b and c, respectively. These results clearly highlight the intrinsic ability of HSA and fibrinogen to stabilize curcumin. The insets in Figure 3 illustrate the time dependent decays of curcumin in the corresponding solutions, which show linear behavior and hence fit well to a pseudo-zero-order model, in agreement with previous reports.19,33 The rate of degradation is determined from the initial slope of the best-fit linear curve, and Table 1 summarizes the rates of degradation of curcumin in buffer and protein solutions. At pH 7.4, curcumin degrades initially at a rapid rate of 3.45 ( 0.34% min-1 (for the first 10 min) in phosphate buffer solution and then the degradation decreases drastically afterward, showing a biphasic behavior. In the presence of HSA and fibrinogen, however, the rates of degradation are suppressed to the low values of 0.20 ( 0.10 and 0.22 ( 0.13% min-1, respectively. The time dependent decay kinetics (33) Bernabe-Pineda, M.; Ramirez-Silva, M. T.; Romero-Romo, M.; GonzalezVergara, E.; Rojas-Hernandez, A. Spectrochim. Acta, Part A 2004, 60A, 1091.
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Figure 3. UV-vis absorption spectra of curcumin in (a) pH 7.4 phosphate buffer, (b) HSA, (c) fibrinogen, (d) transferrin, and (e) IgG over the course of 20 min. The concentration ratio of protein to curcumin is 2:1, where [HSA], [fibrinogen], [transferrin], and [IgG] are 12.8, 11.2, 8.7, and 10.0 μM, respectively. The insets show the decay of curcumin at the absorption maxima. The rate of degradation is determined from the initial slope of the best-fit linear curve.
for transferrin and IgG are similar to those for curcumin in buffer solution, exhibiting a biphasic characteristic with a fast decay followed by a slow degradation. The fast decay rate is 1.80 ( 0.31% min-1 for transferrin and 2.92 ( 0.91% min-1 for IgG. Suppression of Degradation of Curcumin. The rates of degradation of curcumin in protein solutions are generally lower than that in phosphate buffer. It is evident that degradation of curcumin is suppressed by plasma proteins. The ratio between the rates of degradation in phosphate buffer and protein solutions indicates that the degradation occurs approximately 16 times slower in the presence of HSA and fibrinogen, roughly 2 times slower with transferrin but with no apparent suppression with IgG. In other words, the degradation of curcumin is suppressed with an impressive yield of 94.2 ( 13.9% by HSA and 93.6 ( 14.0% by fibrinogen. Additionally, the yield of suppression of degradation by transferrin is 47.8 ( 14.1% (Table 1). To the authors’ knowledge, these results are the first work to demonstrate and quantify the ability of specific proteins in blood plasma to stabilize curcumin. The effective inhibition of the rapid degradation of curcumin at physiological pH leads to a central question: what is the underlying reason for the effectiveness of HSA and fibrinogen in suppressing the degradation of curcumin? Binding between Curcumin and Plasma Proteins. The UV-vis absorption spectral features and fluorescence intensity of curcumin in the plasma protein solutions imply that curcumin has preferential interactions with HSA, fibrinogen, and transferrin. The results in Figure 3d, however, indicate that transferrin is ineffective in suppressing the degradation of curcumin, which prompted the investigations on the binding between curcumin and plasma proteins. Using the method described in the Experimental Section, the binding constants of curcumin to plasma proteins were measured and are summarized in Table 1. 5776
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The fluorescence quenching results are available in the Supporting Information. Briefly, fibrinogen and HSA exhibit high binding constants on the order of 104-105 M-1, whereas transferrin and IgG have binding constants in the 103 M-1 range. The magnitude of the binding constants for the association of curcumin to HSA and IgG agree with the values obtained in previous studies.22,26,34 Here, we report the binding constants for the association of curcumin to fibrinogen and transferrin for the first time, which have values of (5.99 ( 1.75) 104 and (5.15 ( 1.05) 103 M-1, respectively. The crystal structures of HSA and fibrinogen show that these proteins have hydrophobic moieties and they are often involved in binding of small molecules.35,36 The binding constants for the association of curcumin to HSA and fibrinogen, with values on the order of 104-105 M-1, are consistent with the binding constants for similar molecules to these proteins.37,38 Previous studies have attributed binding constants of this magnitude to hydrophobic interactions between the protein and the small molecule, where binding takes place in the hydrophobic “pocket” of the protein. Strong association of curcumin to the hydrophobic domains of HSA and fibrinogen inhibits interactions with the surrounding water molecules, leading to suppression of degradation due to hydrolysis of curcumin. The curcumin-transferrin and curcumin-IgG binding constants are approximately 103 M-1, which is 1 order of magnitude lower than those of fibrinogen and HSA. The lower binding constants indicate that there are weaker hydrophobic interactions between curcumin and these proteins. These binding constants agree well with those of the associations of transferrin and IgG to 8-anilino-1-naphthalene sulfonate (ANS),39,40 which is a standard fluorescence probe molecule for investigations of hydrophobic interactions. Transferrin and IgG exhibit weak binding to small hydrophobic molecules probably because these proteins are specialized in binding with metal ions (Fe3+ and Al3+)30 and macromolecules such as pathogens, respectively. The weak binding to these plasma proteins signifies that curcumin has an increased level of interactions with the surrounding water molecules, which leads to lower yields of suppression of degradation than those of HSA and fibrinogen. In addition, the higher yield of suppression of transferrin (47.8 ( 14.1%) than that of IgG (15.4 ( 28.2%) also corresponds well to a higher value of binding constant of transferrin of (5.15 ( 1.05) 103 M-1 than that of IgG of (1.27 ( 0.29) 103 M-1. These results further support the importance of hydrophobic interactions in suppressing degradation of curcumin. Weak Suppression of Degradation with Denatured HSA. To illustrate the significance of protein conformation for suppressing degradation of curcumin, similar experiments were carried out using phosphate buffer solution and denatured HSA at T = 50 °C, for which the results are summarized in Table 1. The rates of degradation of curcumin in buffer solution and in the presence of HSA increase to 23.2 ( 1.4 and 11.6 ( 5.0% min-1 at 50 °C, respectively. At this temperature, the ability of HSA to suppress degradation of curcumin is greatly (34) Liu, Y.; Yang, Z.; Du, J.; Yao, X.; Lei, R.; Zheng, X.; Liu, J.; Hu, H.; Li, H. Immunobiology 2008, 213, 651. (35) Cohen, C.; Tooney, N. M. Nature (London) 1974, 251, 659. (36) He, X. M.; Carter, D. C. Nature (London) 1992, 358, 209. (37) Goncalves, S.; Santos, N. C.; Martins-Silva, J.; Saldanha, C. J. Photochem. Photobiol., B 2007, 86, 170. (38) Qi, Z.; Zhang, Y.; Liao, F. L.; Ou-Yang, Y. W.; Liu, Y.; Yang, X. J. Pharm. Biomed. Anal. 2008, 46, 699. (39) Egea, M. A.; Garcıa, M. L.; Alsina, M. A.; Mestres, C.; Reig, F. Colloid Polym. Sci. 1994, 272, 570. (40) Rispens, T.; Lakemond, C. M. M.; Derksen, N. I. L.; Aalberse, R. C. Anal. Biochem. 2008, 380, 303.
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Table 1. Rate of Degradation of Curcumin in Buffer and Protein Solutions, Yield of Suppression of Degradation by Proteins, and CurcuminProtein Association Constant rate of degradation (% min-1)
protein
yield of suppressiona
binding constant (M-1)
3.45 ( 0.34, (6.65 ( 0.64) 10-3 phosphate buffer (pH 7.4)b HSA 0.20 ( 0.10 94.2 ( 13.9% (1.22 ( 0.35) 105 fibrinogen 0.22 ( 0.13 93.6 ( 14.0% (5.99 ( 1.75) 104 transferrinb 1.80 ( 0.31, 0.18 ( 0.10 47.8 ( 14.1% (5.15 ( 1.05) 103 IgGb 2.92 ( 0.91, 0.58 ( 0.62 15.4 ( 28.2% (1.27 ( 0.29) 103 phosphate buffer (pH 7.4)b,c 23.2 ( 1.4, 0.35 ( 0.48 11.6 ( 5.0, (1.35 ( 0.05) 10-2 50.2 ( 30.5% HSAb,c a Yield = [(ratebuffer - rateprotein)/ratebuffer] 100%. The standard errors were estimated using error propagation. b The results indicate that there are two distinct rates of degradation. The fast rate is used for calculating the yield of suppression. c Measurements were taken at 50 °C.
diminished; the yield of suppression of degradation decreases to merely 50.2 ( 30.5%, relative to 94.2 ( 13.9% at 25 °C. As a consequence of denaturation, HSA is likely to expose its hydrophobic moiety, which leads to a weaker association with curcumin and in turn a higher rate of degradation by hydrolysis. The same experiment was also performed on fibrinogen at 50 °C, but no results were obtained due to interference of measurements, which arises from the formation of insoluble aggregates by the denatured proteins. In short, the results strongly support hydrophobic interactions as the important curcumin-protein association that gives rise to effective suppression of degradation.
Conclusion We have established that the plasma proteins HSA and fibrinogen play a critical role in stabilizing curcumin. These proteins exhibit the impressive ability to suppress degradation of curcumin by hydrolysis with a yield of approximately 95%. The results suggest that the stabilization effects of these proteins enable curcumin to maintain its medicinal properties at the wound site to promote healing. The stabilization effects of HSA and fibrinogen have been attributed to the strong binding of curcumin to these proteins, with binding constants on the order
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of 104-105 M-1. The results indicate that binding of curcumin to the hydrophobic moiety of these proteins suppresses the degradation process effectively. Acknowledgment. This work was supported by a research grant from the Australian Research Council and National Health and Medical Research Council Network “Fluorescence Applications in Biotechnology and Life Sciences” (FABLS). M. H.M.L. was supported by a research assistantship funded by the Faculty of Sciences at the University of Adelaide through the Research Development Scheme. The authors thank Dr. Heath Ecroyd for his assistance in measurements of binding constants and the studies on degradation of curcumin in the presence of denatured proteins. Supporting Information Available: Results on quenching of intrinsic fluorescence of plasma protein by curcumin, which were used for determining binding constants, fluorescence spectrum of curcumin in SDS micelles, and UV-vis absorption and fluorescence spectra of plasma proteins. This material is available free of charge via the Internet at http:// pubs.acs.org.
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