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DOI 10.1002/mnfr.201500681
RESEARCH ARTICLE
Redox modulation of curcumin stability: Redox active antioxidants increase chemical stability of curcumin Yoshiki Nimiya1,2∗ , Weicang Wang1∗ , Zheyuan Du1 , Elvira Sukamtoh1 , Julia Zhu1 , Eric Decker1 and Guodong Zhang1 1 2
Department of Food Science, University of Massachusetts, Amherst, MA, USA Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan
Scope: Substantial studies have shown that curcumin, a dietary compound from turmeric, has beneficial effects on many diseases. However, curcumin rapidly degrades at physiological pH, making it difficult to interpret whether the observed actions of curcumin are from curcumin itself or its degradation products. Therefore, it is important to better understand the mechanisms involved in curcumin degradation and the roles of degradation in its biological actions. Methods and results: Here, we show that a series of redox active antioxidants with diverse chemical structures, including gallic acid, ascorbate (vitamin C), tert-butylhydroquinone (TBHQ), caffeic acid, rosmarinic acid, and Trolox (a water-soluble analog of vitamin E), dramatically increased curcumin stability in phosphate buffer at physiological pH. When treated in basal cell culture medium in MC38 colon cancer cells, curcumin rapidly degraded with a half-life of several minutes and showed a weak antiproliferative effect; co-addition of antioxidants enhanced stability and antiproliferative effect of curcumin. Finally, co-administration of antioxidant significantly increased plasma level of curcumin in animal models. Conclusion: Together, these studies strongly suggest that a redox-dependent mechanism plays a critical role in mediating curcumin degradation. In addition, curcumin itself, instead of its degradation products, is largely responsible for the observed biological actions of curcumin.
Received: August 31, 2015 Revised: November 10, 2015 Accepted: November 12, 2015
Keywords: Antioxidant / Curcumin / Stability
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Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction
Substantial human and preclinical studies have shown that curcumin, a dietary compound from turmeric, has potent anticancer, anti-inflammatory and antioxidative effects [1]. For example, in terms of cancer, a Phase II␣ human clinical trial of colorectal cancer showed that daily intake of 4 g of curcumin for a month caused 40% reduction of aberrant crypt foci [2]. In another Phase II human clinical trial, daily intake of 8 g of curcumin demonstrates anticancer efficacy in some patients with advanced pancreatic cancer [3]. Currently, the therapeutic effects of curcumin are being evaluated in over 100 human clinical trials, targeting human Correspondence: Guodong Zhang E-mail: [email protected] Abbreviations: none
i.p., intraperitoneal; TBHQ, tert-butylhydroqui-
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
diseases including but not limited to cancers, cardiovascular diseases, and inflammatory diseases such as arthritis and colitis [4]. A problem to develop curcumin-based therapeutics is its poor chemical stability: its half-life in aqueous buffer at physiological pH is only several minutes, leading to rapid formation of various degradation products, such as ferulic acid and feruloyl methane [5], and recently discovered bicyclopentadione derivatives of curcumin [6, 7]. Due to the short half-life of curcumin in aqueous medium, it is difficult to determine whether the observed biological actions of curcumin in vitro are due to curcumin itself or its degradation products, hampering mechanistic understanding of curcumin biology [4]. Previous research has suggested two mechanisms to explain curcumin ∗ These authors contributed equally to this work. Colour online: See the article online to view Figs. 1, 2, 4, 5 in colour.
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degradation in aqueous buffer (see Supporting Information Fig. S1): (1) hydroxyl ion mechanism, in which hydroxyl ion (OH− ) attacks the carbonyl group of curcumin, generating break-down products such as ferulic acid and feruloyl methane [5]; and (2) phenolic radical mechanism, in which curcumin is first converted to a phenolic radical, which then migrates to the conjugated heptadienedione chain and initiates a chain reaction of curcumin degradation to generate cyclized compounds such as bicyclopentadione derivatives of curcumin [4, 6, 7]. Recent research showed that the bicyclopentadione derivatives of curcumin, instead of ferulic acid and feruloyl methane, are the major degradation products, suggesting a critical role of the phenolic radical mechanism in curcumin degradation [4, 6, 7]. This leads to our hypothesis that redox active antioxidants could increase the chemical stability and biological activity of curcumin, through suppressing the formation of curcumin phenolic radicals or by regenerating oxidized curcumin. To test this hypothesis, here we systematically studied the effects of a wide range of redox active antioxidants on the chemical stability of curcumin.
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Materials and methods
2.1 Chemicals and materials Antioxidants, including gallic acid, ascorbate (vitamin C), tert-butylhydroquinone (TBHQ), caffeic acid, rosmarinic acid, Trolox (a water-soluble analog of vitamin E), and disodium EDTA, were purchased from Thermal Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). HPLC solvents were purchased from Thermal Fisher Scientific.
2.2 Chemical synthesis of curcumin and its analogs Curcumin (>98% purity) was purchased from Thermal Fisher Scientific. Since many commercial samples of curcumin contain other curcuminoids, pure curcumin was prepared by chemical synthesis as described [8]. Briefly, vanillin (3.04 g) and tributyl borate (10.8 mL) were added into a solution of acetylacetone (1.03 mL) and boric anhydride (0.35 g) in anhydrous ethyl acetate at 50˚C, then n-butylamine (0.4 mL) dissolved in ethyl acetate was drop-wise added and the reaction was stirred overnight. Hydrochloric acid (1 N, 30 mL) was added and stirred for another 30 min. The reaction mixture was extracted with ethyl acetate, and pure curcumin was obtained after methanol re-crystallization. Dimethoxy curcumin [1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5dione] was synthesized using the same strategy of curcumin synthesis, except vanillin was replaced with 3,4dimethoxybenzaldehyde. The structure and purity of synthesized curcumin and its analog were confirmed by NMR, TLC and HPLC, as reported previously [9]. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.3 Colorimetric assay of curcumin stability in phosphate buffer To assess curcumin stability using UV spectroscopy, a 25 M curcumin solution in 0.1 M phosphate buffer (2.5 g/L KH2 PO4 and 11.5 g/L Na2 HPO4 , pH = 7.4) was freshly prepared in UV quartz cuvettes, then UV spectrum (210–550 nm) was continuously recorded at different time points. To study the effects of antioxidants on curcumin stability in the buffer, a phosphate solution of 25 M curcumin, with or without antioxidant, was freshly prepared and its absorbance at 420 nm was continuously recorded using a plate reader (Molecular Devices, Sunnyvale, CA, USA).
2.4 HPLC analysis of curcumin stability in phosphate buffer A 25 M curcumin solution, with or without antioxidant, was freshly prepared in 0.1 M phosphate buffer (pH = 7.4), and curcumin concentration in the buffer was analyzed by HPLC on an Agilent 1100 HPLC system, using an Agilent TC-C18(2) column (4.6 × 250 mm, 5 m) eluted with a mobile phase of 80% methanol with 0.1% acetic acid and 20% water with 0.1% acetic acid, flow rate = 1 mL/min, detection wavelength at 420 nm.
2.5 Cancer cell proliferation assay MC38 colon cancer cells (a gift from Professor Ajit Varki at the University of California, San Diego) were plated into 96-well plates (6000 cells per well) in 100 L DMEM (purchased from Lonza, Allendale, NJ, USA) fortified with 10% fetal bovine serum (purchased from Corning Inc., Corning, NY, USA) and allowed to attach overnight. The cells were then treated with curcumin, with or without redox active antioxidants, in DMEM basal medium for 24 h. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay.
2.6 Analysis of plasma levels of curcumin in mice The animal experiment was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of University of Massachusetts Amherst. Briefly, 20 mg/kg curcumin, with or without 40 mg/kg TBHQ, dissolved in 50 L of DMSO was intraperitoneally (i.p.) injected into 6-wk-old male Swiss Webster mice. After 1 h, the mice were sacrificed to harvest the blood. The harvested blood was centrifuged at 1200 × g for 10 min at 4˚C to prepare the plasma fraction; then 200 L plasma was immediately extracted with 400 L ethyl acetate, the ethyl acetate extract was dried and reconstituted in methanol for HPLC analysis. The HPLC was performed on an Agilent 1100 www.mnf-journal.com
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HPLC system, using an Agilent TC-C18(2) column (4.6 × 250 mm, 5 m), flow rate = 1 mL/min, detection wavelength at 420 nm. The mobile phase consisted methanol with 0.1% acetic acid (mobile phase B) and water with 0.1% acetic acid (mobile phase A). The extracted sample was eluted on a gradient starting with 65% B for 5 min, increasing to 75% B in 5 min and holding at 75% B for 5 min, decreasing to 65% B in 1 min and holding at 65% B for 4 min. 2.7 Statistics Group comparisons were carried out using one-way analysis of variance or Student’s t-test. p-Values less than 0.05 were considered statistically significant.
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Results
3.1 Curcumin has a poor stability in aqueous buffer at physiological pH Consistent with previous studies [5, 7], we found that curcumin has a poor stability in aqueous buffer at physiological pH. UV spectroscopy analysis showed that at a concentration of 25 M, curcumin rapidly degraded in phosphate buffer (pH = 7.4), with a time-dependent decrease of absorbance at max of curcumin (420 nm) (Fig. 1A). This is consistent with previous studies, which showed that the degradation products of curcumin do not exhibit absorbance at 420 nm [5–7]. To further validate the results obtained from spectroscopic analysis, we used RP HPLC to measure curcumin concentration in the phosphate buffer. HPLC analysis showed that after 12-min incubation, there is 80–90% degradation of curcumin in the buffer, followed with a slower degradation of the remaining curcumin (Fig. 1B). At a lower concentration (e.g. 1 M), curcumin also rapidly degraded in phosphate buffer (see Supporting Information Fig. S2). Together, these results confirm the poor chemical stability of curcumin in aqueous buffer at physiological pH. 3.2 Redox active antioxidants increase chemical stability of curcumin After we established the poor stability of curcumin in aqueous buffer, we studied the effects of antioxidants on curcumin stability, using a colorimetric assay and HPLC analysis. As shown in Fig. 1A, curcumin degradation in the buffer is correlated with a decrease of absorbance at its max (420 nm); therefore, we used a 96-well-plate-based colorimetric assay to rapidly screen antioxidants on their effects to modulate curcumin stability. When incubated alone in phosphate buffer, curcumin rapidly degraded, while co-addition of a wide range of redox active antioxidants dramatically increased curcumin stability (Fig. 2A). These antioxidants possessed diverse chemical structures, including gallic acid, ascorbate (vitamin C), TBHQ, caffeic acid, rosmarinic acid, and Trolox C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(a water-soluble analog of vitamin E). Co-addition of these antioxidants, such as ascorbate or Trolox, did not change pH of the phosphate buffer (data not shown), indicating the protective effects were not due to altered pH. Next, we performed HPLC analysis to further validate the results obtained from the colorimetric assays. Consistent with the colorimetric assays, co-addition of above redox active antioxidants dramatically decreased curcumin degradation in the phosphate buffer within a 1-h incubation period (Fig. 2B), confirming the protective effects of antioxidants on curcumin stability. We also tested EDTA, which is a widely used metal-chelating antioxidant that does not have radical scavenging activity. The HPLC analysis showed that EDTA, even at a dose as high as 200 M, had little protective effect on curcumin stability (Fig. 2A). We further characterized the dose–response effects of these antioxidants on curcumin stability, and the actions of antioxidants on long-term stability of curcumin. The dose– response experiment showed that these redox active antioxidants significantly increased curcumin stability at doses as low as 1 M (Supporting Information Fig. S3), demonstrating potent protective effects of these antioxidants on curcumin stability. Regarding the long-term stability, co-addition of ascorbate significantly increased long-term stability of curcumin: after 24-h and 48-h incubation, there was, respectively, 74.7 and 53.7% of curcumin remaining in the phosphate buffer. In contrast, when curcumin was incubated alone, there was only 5.9 and 4.2% curcumin remaining after 24-h and 48-h incubation, respectively (Fig. 3). Considering the half-life of curcumin in phosphate buffer was less than 10 min (see Fig. 1), co-addition of ascorbate caused a >200-fold increase of the half-life of curcumin. Besides ascorbate, other antioxidants, such as gallic acid, TBHQ and Trolox, also significantly increased long-term stability of curcumin (Fig. 3), suggesting that co-administration of redox active antioxidants could be a practical strategy to enhance curcumin stability. Our results above strongly suggest that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a critical role in mediating curcumin degradation. To further test which mechanism plays a major role, we compared the stability of curcumin with its structural analog dimethoxy curcumin (see chemical structures in Supporting Information Fig. S4). Compared with curcumin, dimethoxy curcumin does not have the radical-initiating phenolic groups. HPLC analysis showed that in phosphate buffer, curcumin rapidly degraded, while dimethoxy curcumin was very stable. This result further supports that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a major role in mediating curcumin degradation.
3.3 Roles of degradation in the antiproliferative activity of curcumin in vitro Because of the poor stability of curcumin in aqueous medium, it remains unclear whether the observed biological activities www.mnf-journal.com
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of curcumin in vitro are derived from curcumin itself or its degradation products, hampering mechanistic understanding of curcumin [4]. After we demonstrated that redox active antioxidants stabilize curcumin in aqueous medium, we used these antioxidants to probe the roles of degradation in the biological activity of curcumin. We first conducted our cellular assays in serum-free basal DMEM medium. Curcumin rapidly degraded in the basal medium with a half-life of several minutes (Supporting Information Fig. S5A); after 24-h treatment in the basal medium in MC38 colon cancer cells, curcumin showed a weak inhibitory effect on cancer cell proliferation: at a dose as high as 20 M, curcumin suppressed 20% inhibition of cell proliferation. Co-addition of ascorbate or Trolox increased curcumin stability in basal DMEM medium (data not shown); and co-addition of these two antioxidants dramatically enhanced the actions of curcumin, with a 40–80% inhibition of MC38 cell proliferation. As a control, treatment with ascorbate or Trolox alone had little effect on MC38 proliferation (Fig. 4A–C). Besides ascorbate and Trolox, we also tested other antioxidants, and found all redox active antioxidants used in our study significantly increased antiproliferative effect of curcumin in MC38 colon cancer cells (Fig. 4D). Next, we performed similar assays in complete medium (containing 10% serum). Previous studies have shown that with the presence of 10% serum, the half-life of curcumin increased to 8 h [5]. Co-addition of antioxidant did not further enhance the biological activity of curcumin C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Curcumin rapidly degrades in phosphate buffer at physiological pH. (A) (Left) Representative UV spectrum of a 25 M curcumin solution in 0.1 M phosphate buffer (pH = 7.4). (Right) Quantification of absorbance at 420 nm. (B) (Left) Representative HPLC chromatography of a 25 M curcumin solution in 0.1 M phosphate buffer, detection wavelength at 420 nm. (Right) Quantification of curcumin concentration in the phosphate buffer by using HPLC.
(Supporting Information Fig. S5B), most likely because curcumin was already stabilized in the complete medium.
3.4 Antioxidant TBHQ increases circulating levels of curcumin in animal models Finally, we tested whether co-administration of antioxidant increased the circulating level of curcumin in animal models. We used DMSO as the solvent to dissolve curcumin for i.p. injection into the mice, because curcumin is highly stable in DMSO (data not shown), minimizing curcumin degradation prior the i.p. injection. Consistent with previous studies [10,11], HPLC analysis showed that 1 h after i.p. injection of 20 mg/kg curcumin, the plasma concentration of curcumin was 14.8 ± 16 nM. Co-administration of curcumin with TBHQ (dose = 40 mg/kg), a phenolic antioxidant widely used in food systems, increased the plasma concentration of curcumin to 87.6 ± 32.3 nM (approximately sixfold increase; Fig. 5).
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Here, our central finding is that a wide range of redox active antioxidants with diverse chemical structures significantly increased chemical stability of curcumin in aqueous medium, supporting a redox-dependent mechanism to play a major www.mnf-journal.com
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Figure 2. Antioxidants increase curcumin stability in phosphate buffer. (A) A colorimetric assay shows that co-addition of antioxidants (dose of antioxidants = 15 and 150 M) increased curcumin stability in phosphate buffer. (B) HPLC analysis confirms that these antioxidants (dose of antioxidant = 10 M), except EDTA, increased curcumin stability in phosphate buffer. (Left) Quantification of curcumin concentration in the phosphate buffer by using HPLC, the results are expressed as percentage of curcumin concentration at a specific time point to that at time 0. (Right) Representative HPLC chromatography, detection wavelength at 420 nm. The results are mean ± SD.
role in mediating curcumin degradation. In addition, we found that when curcumin was stabilized by co-addition of antioxidants, its biological activity was significantly enhanced, suggesting that curcumin itself, instead of its degradation products, is largely responsible for the observed biological actions of curcumin. Previous research has suggested two mechanisms to explain curcumin degradation in aqueous buffer: hydroxyl ion mechanism and phenolic radical mechanism (see scheme in Supporting Information Fig. S1) [4–7]. Here, our experiments using redox active antioxidants and curcumin analog strongly support that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a major role in mediating curcumin degradation. Together, our study and previous investigations [4, 6, 7] suggest that curcumin degradation in aqueous buffer could happen through a mechanism comparable to that of lipid peroxidation. The first step of curcumin degradation is hydrogen dissociation from the phenolic group to form a phenolic radical, which then migrates to the conjugated heptadienedione chain and leads to formation of cyclized compounds such as the recently identified bicyclopentadione derivatives of curcumin [4, 6, 7]. The radical could C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
be then transferred to another curcumin molecule, resulting in a chain reaction to cause a rapid and massive curcumin degradation [4]. Both the phenolic group and the conjugated heptadienedione chain seem to play critical roles in curcumin degradation. Indeed, converting the radical-initiating phenolic (–OH) groups to methoxy groups (–OCH3 ), or reduction of the carbon–carbon double bonds in the conjugated heptadienedione chain, significantly increased curcumin stability (see Supporting Information Fig. S4 and ref. 10). The redox active antioxidants increase chemical stability of curcumin, at least in part, through directly suppressing formation of the curcumin phenolic radical. The redox active antioxidants used in our study have been shown to have lower hydrogen dissociation energies compared with that of the phenolic group of curcumin [12]. Therefore, these antioxidants can efficiently reduce the curcumin phenolic radicals, and terminate chain reaction of curcumin degradation. Together, these results suggest a redox-dependent mechanism as a major mechanism to mediate curcumin degradation. Because of the poor stability of curcumin in aqueous medium, it remains unclear whether the observed biological activities of curcumin in vitro are derived from curcumin www.mnf-journal.com
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Figure 3. Antioxidants increase long-term stability of curcumin in phosphate buffer. (Left) Quantification of curcumin concentration after 24-h and 48-h incubations. To study the effects of antioxidants on long-term stability of curcumin, a 25 M curcumin solution in 0.1 M phosphate buffer, with or without antioxidants (dose of antioxidant = 150 M), was freshly prepared, and the concentration of curcumin at different time point was analyzed by HPLC. The results are expressed as percentage of curcumin concentration at a specific time point to that at time 0. (Right) Representative HPLC chromatography. The results are mean ± SD.
itself or its degradation products [4, 5]. Here, our results support that curcumin, rather than its degradation products, is largely responsible for the observed biological activities of curcumin. Indeed, in serum-free medium, curcumin rapidly de-
graded with a half-life of several minutes; therefore, treatment with curcumin alone in basal medium would actually reflect the biological effects of the curcumin degradation products. Our result showed that even at a high dose (20 M), curcumin
Figure 4. Antioxidants increase antiproliferative effect of curcumin in MC38 colon cancer cells. (A) Representative microscope images of the treated cells. Treatment with curcumin alone, or Trolox or ascorbate alone, had little effect on cell density or morphology; in contrast, their combination dramatically reduced cell density and changed cell morphology. (B) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different doses of ascorbate (3.9– 62.5 M), or a combination of curcumin and ascorbate, for 24 h. (C) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different doses of Trolox (3.9–62.5 M), or a combination of curcumin and Trolox, for 24 h. (D) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different antioxidant (dose of each antioxidant = 3.9 M), or a combination of curcumin and antioxidant, for 24 h. The results are mean ± SD. The samples designated with different letters are statistically different (p < 0.05). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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had a poor effect to suppress MC38 cell proliferation (20% inhibition, see Fig. 4). Co-addition of cellular antioxidants stabilized curcumin and dramatically enhanced the antiproliferative effects of curcumin (Fig. 4). These results showed that curcumin itself, rather than its degradation products, has potent biological activities. The process of curcumin degradation leads to formation of less-active or inactive compounds, therefore is unlikely to contribute to the biological actions of curcumin. In complete medium (containing 10% serum), the half-life of curcumin increased to 8 h [5]; since curcumin was highly stable with the presence of serum, co-addition of antioxidants did not further enhance the actions of curcumin (Supporting Information Fig. S5). We need to point out the antiproliferative effect of curcumin was much more potent in complete medium compared that in basal medium (Fig. 4 and Supporting Information Fig. S5), further supporting that curcumin itself, rather than its degradation products, had potent biological activities. Together, these results support that curcumin, instead of its degradation products, contributed to the observed biological actions of curcumin. Further studies are needed to further clarify this point, as a previous study has shown that the oxidized products of curcumin, but not curcumin itself, had potent inhibitory effect on human type II topoisomerases [13]. To date, whether curcumin degradation occurs in vivo remains unknown [4]. Here, we showed that co-administration of antioxidant TBHQ significantly increased circulating level of curcumin in animal models (Fig. 5). This result indicates that chemical degradation of curcumin could happen in vivo, and redox active antioxidants could increase curcumin stability in vivo through suppressing the chemical degradation of curcumin. In our study, we used TBHQ because it can be co-solubilized with curcumin in DMSO (curcumin is highly stable in DMSO, thus minimizing curcumin degradation prior the i.p. injection into mice); and TBHQ is a widely C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
used phenolic antioxidant in the food system. However, the limitation of our experiment is that we only tested one antioxidant; more studies are therefore needed to test whether other types of antioxidants could enhance the pharmacokinetics profile of orally administered curcumin in vivo. Consistent with our findings, previous studies have shown that coadministration of redox active antioxidants, such as ascorbate (vitamin C), enhanced biological activities of curcumin in several different disease models in vivo [14, 15]. For example, in a cadmium-induced hepatotoxicity model, administration of 200–400 mg/kg curcumin alone or 400 mg/kg ascorbate alone had no effect, while their combination dramatically inhibited cadmium-induced hepatotoxicity in rats [15]. It remains to decide whether ascorbate interacted with curcumin through enhancing the chemical stability and circulating level of curcumin in vivo. A major barrier to develop curcumin-based therapeutics is its poor pharmacokinetics profile in vivo. Indeed, a previous human study has shown that after a single oral dose of 10 or 12 g of curcumin, free form of curcumin was barely detected in human plasma [16]. High doses of curcumin were required to exert its health-promoting biological activities in animal and human studies. In a Phase II␣ trial of colorectal cancer, curcumin had no effect at a dose of 2 g/day; and only inhibited aberrant crypt foci at a dose as high as 4 g/day [2]. Further studies are needed to test whether our finding could serve as a practical strategy to enhance pharmacokinetics profiles and biological activities of curcumin in vivo. In summary, here we demonstrate, for the first time, that a wide range of redox active antioxidants dramatically increased chemical stability of curcumin in aqueous medium. This further supports a previously proposed phenolic radical mechanism as a major mechanism that mediates curcumin degradation [4, 6, 7]. Further studies are needed to explore the applications of this finding in developing more effective curcumin-based functional foods or therapeutics. www.mnf-journal.com
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This work was supported by a faculty start-up fund and Armstrong Fund of Science Award from University of Massachusetts Amherst (G.Z.). Potential conflict of interest statement: The authors have submitted a provisional US patent to stabilize curcumin using antioxidants, which has been assigned to UMass-Amherst.
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[1] Aggarwal, B. B., Kumar, A., Bharti, A. C., Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res. 2003, 23, 363–398. [2] Carroll, R. E., Benya, R. V., Turgeon, D. K., Vareed, S. et al., Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia. Cancer Prev. Res. 2011, 4, 354–364. [3] Dhillon, N., Aggarwal, B. B., Newman, R. A., Wolff, R. A. et al., Phase II trial of curcumin in patients with advanced pancreatic cancer. Clin. Cancer Res. 2008, 14, 4491–4499. [4] Schneider, C., Gordon, O. N., Edwards, R. L., Luis, P. B., Degradation of curcumin: From mechanism to biological implications. J. Agric. Food Chem. 2015, 63, 7606–7614. [5] Wang, Y. J., Pan, M. H., Cheng, A. L., Lin, L. I. et al., Stability of curcumin in buffer solutions and characterization of its degradation products. J. Pharm. Biomed. Anal. 1997, 15, 1867–1876.
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[8] Wichitnithad, W., Nimmannit, U., Wacharasindhu, S., Rojsitthisak, P., Synthesis, characterization and biological evaluation of succinate prodrugs of curcuminoids for colon cancer treatment. Molecules 2011, 16, 1888–1900. [9] Chen, W. F., Deng, S. L., Zhou, B., Yang, L. et al ., Curcumin and its analogues as potent inhibitors of low density lipoprotein oxidation: H-atom abstraction from the phenolic groups and possible involvement of the 4-hydroxy3-methoxyphenyl groups. Free Radic Biol. Med. 2006, 40, 526–535. [10] Pan, M. H., Huang, T. M., Lin, J. K., Biotransformation of curcumin through reduction and glucuronidation in mice. Drug Metab. Dispos. 1999, 27, 486–494. [11] Tamvakopoulos, C., Dimas, K., Sofianos, Z. D., Hatziantoniou, S. et al., Metabolism and anticancer activity of the curcumin analogue, dimethoxycurcumin. Clin. Cancer Res. 2007, 13, 1269–1277. [12] Jha, N. S., Mishra, S., Jha, S. K., Surolia, A., Antioxidant activity and electrochemical elucidation of the enigmatic redox behavior of curcumin and its structurally modified analogues. Electrochim. Acta 2015, 151, 574–583. [13] Ketron, A. C., Gordon, O. N., Schneider, C., Osheroff, N., Oxidative metabolites of curcumin poison human type II topoisomerases. Biochemistry 2013, 52, 221–227. [14] Patumraj, S., Wongeakin, N., Sridulyakul, P., Jariyapongskul, A. et al., Combined effects of curcumin and vitamin C to protect endothelial dysfunction in the iris tissue of STZ-induced diabetic rats. Clin. Hemorheol. Microcirc. 2006, 35, 481–489.
[6] Gordon, O. N., Luis, P. B., Sintim, H. O., Schneider, C., Unraveling curcumin degradation: autoxidation proceeds through spiroepoxide and vinylether intermediates en route to the main bicyclopentadione. J. Biol. Chem. 2015, 290, 4817– 4828.
[15] Tarasub, N., Junseecha, T., Tarasub, C., Na Ayutthaya, W. D., Protective effects of curcumin, vitamin C, or their combination on cadmium-induced hepatotoxicity. J. Basic Clin. Pharm. 2012, 3, 273–281.
[7] Griesser, M., Pistis, V., Suzuki, T., Tejera, N. et al., Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin. J. Biol. Chem. 2011, 286, 1114–1124.
[16] Vareed, S. K., Kakarala, M., Ruffin, M. T., Crowell, J. A. et al., Pharmacokinetics of curcumin conjugate metabolites in healthy human subjects. Cancer Epidemiol. Biomarkers Prev. 2008, 17, 1411–1417.
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DOI 10.1002/mnfr.201500681
RESEARCH ARTICLE
Redox modulation of curcumin stability: Redox active antioxidants increase chemical stability of curcumin Yoshiki Nimiya1,2∗ , Weicang Wang1∗ , Zheyuan Du1 , Elvira Sukamtoh1 , Julia Zhu1 , Eric Decker1 and Guodong Zhang1 1 2
Department of Food Science, University of Massachusetts, Amherst, MA, USA Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo, Japan
Scope: Substantial studies have shown that curcumin, a dietary compound from turmeric, has beneficial effects on many diseases. However, curcumin rapidly degrades at physiological pH, making it difficult to interpret whether the observed actions of curcumin are from curcumin itself or its degradation products. Therefore, it is important to better understand the mechanisms involved in curcumin degradation and the roles of degradation in its biological actions. Methods and results: Here, we show that a series of redox active antioxidants with diverse chemical structures, including gallic acid, ascorbate (vitamin C), tert-butylhydroquinone (TBHQ), caffeic acid, rosmarinic acid, and Trolox (a water-soluble analog of vitamin E), dramatically increased curcumin stability in phosphate buffer at physiological pH. When treated in basal cell culture medium in MC38 colon cancer cells, curcumin rapidly degraded with a half-life of several minutes and showed a weak antiproliferative effect; co-addition of antioxidants enhanced stability and antiproliferative effect of curcumin. Finally, co-administration of antioxidant significantly increased plasma level of curcumin in animal models. Conclusion: Together, these studies strongly suggest that a redox-dependent mechanism plays a critical role in mediating curcumin degradation. In addition, curcumin itself, instead of its degradation products, is largely responsible for the observed biological actions of curcumin.
Received: August 31, 2015 Revised: November 10, 2015 Accepted: November 12, 2015
Keywords: Antioxidant / Curcumin / Stability
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Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction
Substantial human and preclinical studies have shown that curcumin, a dietary compound from turmeric, has potent anticancer, anti-inflammatory and antioxidative effects [1]. For example, in terms of cancer, a Phase II␣ human clinical trial of colorectal cancer showed that daily intake of 4 g of curcumin for a month caused 40% reduction of aberrant crypt foci [2]. In another Phase II human clinical trial, daily intake of 8 g of curcumin demonstrates anticancer efficacy in some patients with advanced pancreatic cancer [3]. Currently, the therapeutic effects of curcumin are being evaluated in over 100 human clinical trials, targeting human Correspondence: Guodong Zhang E-mail: [email protected] Abbreviations: none
i.p., intraperitoneal; TBHQ, tert-butylhydroqui-
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diseases including but not limited to cancers, cardiovascular diseases, and inflammatory diseases such as arthritis and colitis [4]. A problem to develop curcumin-based therapeutics is its poor chemical stability: its half-life in aqueous buffer at physiological pH is only several minutes, leading to rapid formation of various degradation products, such as ferulic acid and feruloyl methane [5], and recently discovered bicyclopentadione derivatives of curcumin [6, 7]. Due to the short half-life of curcumin in aqueous medium, it is difficult to determine whether the observed biological actions of curcumin in vitro are due to curcumin itself or its degradation products, hampering mechanistic understanding of curcumin biology [4]. Previous research has suggested two mechanisms to explain curcumin ∗ These authors contributed equally to this work. Colour online: See the article online to view Figs. 1, 2, 4, 5 in colour.
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degradation in aqueous buffer (see Supporting Information Fig. S1): (1) hydroxyl ion mechanism, in which hydroxyl ion (OH− ) attacks the carbonyl group of curcumin, generating break-down products such as ferulic acid and feruloyl methane [5]; and (2) phenolic radical mechanism, in which curcumin is first converted to a phenolic radical, which then migrates to the conjugated heptadienedione chain and initiates a chain reaction of curcumin degradation to generate cyclized compounds such as bicyclopentadione derivatives of curcumin [4, 6, 7]. Recent research showed that the bicyclopentadione derivatives of curcumin, instead of ferulic acid and feruloyl methane, are the major degradation products, suggesting a critical role of the phenolic radical mechanism in curcumin degradation [4, 6, 7]. This leads to our hypothesis that redox active antioxidants could increase the chemical stability and biological activity of curcumin, through suppressing the formation of curcumin phenolic radicals or by regenerating oxidized curcumin. To test this hypothesis, here we systematically studied the effects of a wide range of redox active antioxidants on the chemical stability of curcumin.
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Materials and methods
2.1 Chemicals and materials Antioxidants, including gallic acid, ascorbate (vitamin C), tert-butylhydroquinone (TBHQ), caffeic acid, rosmarinic acid, Trolox (a water-soluble analog of vitamin E), and disodium EDTA, were purchased from Thermal Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). HPLC solvents were purchased from Thermal Fisher Scientific.
2.2 Chemical synthesis of curcumin and its analogs Curcumin (>98% purity) was purchased from Thermal Fisher Scientific. Since many commercial samples of curcumin contain other curcuminoids, pure curcumin was prepared by chemical synthesis as described [8]. Briefly, vanillin (3.04 g) and tributyl borate (10.8 mL) were added into a solution of acetylacetone (1.03 mL) and boric anhydride (0.35 g) in anhydrous ethyl acetate at 50˚C, then n-butylamine (0.4 mL) dissolved in ethyl acetate was drop-wise added and the reaction was stirred overnight. Hydrochloric acid (1 N, 30 mL) was added and stirred for another 30 min. The reaction mixture was extracted with ethyl acetate, and pure curcumin was obtained after methanol re-crystallization. Dimethoxy curcumin [1,7-bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5dione] was synthesized using the same strategy of curcumin synthesis, except vanillin was replaced with 3,4dimethoxybenzaldehyde. The structure and purity of synthesized curcumin and its analog were confirmed by NMR, TLC and HPLC, as reported previously [9]. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.3 Colorimetric assay of curcumin stability in phosphate buffer To assess curcumin stability using UV spectroscopy, a 25 M curcumin solution in 0.1 M phosphate buffer (2.5 g/L KH2 PO4 and 11.5 g/L Na2 HPO4 , pH = 7.4) was freshly prepared in UV quartz cuvettes, then UV spectrum (210–550 nm) was continuously recorded at different time points. To study the effects of antioxidants on curcumin stability in the buffer, a phosphate solution of 25 M curcumin, with or without antioxidant, was freshly prepared and its absorbance at 420 nm was continuously recorded using a plate reader (Molecular Devices, Sunnyvale, CA, USA).
2.4 HPLC analysis of curcumin stability in phosphate buffer A 25 M curcumin solution, with or without antioxidant, was freshly prepared in 0.1 M phosphate buffer (pH = 7.4), and curcumin concentration in the buffer was analyzed by HPLC on an Agilent 1100 HPLC system, using an Agilent TC-C18(2) column (4.6 × 250 mm, 5 m) eluted with a mobile phase of 80% methanol with 0.1% acetic acid and 20% water with 0.1% acetic acid, flow rate = 1 mL/min, detection wavelength at 420 nm.
2.5 Cancer cell proliferation assay MC38 colon cancer cells (a gift from Professor Ajit Varki at the University of California, San Diego) were plated into 96-well plates (6000 cells per well) in 100 L DMEM (purchased from Lonza, Allendale, NJ, USA) fortified with 10% fetal bovine serum (purchased from Corning Inc., Corning, NY, USA) and allowed to attach overnight. The cells were then treated with curcumin, with or without redox active antioxidants, in DMEM basal medium for 24 h. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay.
2.6 Analysis of plasma levels of curcumin in mice The animal experiment was conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of University of Massachusetts Amherst. Briefly, 20 mg/kg curcumin, with or without 40 mg/kg TBHQ, dissolved in 50 L of DMSO was intraperitoneally (i.p.) injected into 6-wk-old male Swiss Webster mice. After 1 h, the mice were sacrificed to harvest the blood. The harvested blood was centrifuged at 1200 × g for 10 min at 4˚C to prepare the plasma fraction; then 200 L plasma was immediately extracted with 400 L ethyl acetate, the ethyl acetate extract was dried and reconstituted in methanol for HPLC analysis. The HPLC was performed on an Agilent 1100 www.mnf-journal.com
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HPLC system, using an Agilent TC-C18(2) column (4.6 × 250 mm, 5 m), flow rate = 1 mL/min, detection wavelength at 420 nm. The mobile phase consisted methanol with 0.1% acetic acid (mobile phase B) and water with 0.1% acetic acid (mobile phase A). The extracted sample was eluted on a gradient starting with 65% B for 5 min, increasing to 75% B in 5 min and holding at 75% B for 5 min, decreasing to 65% B in 1 min and holding at 65% B for 4 min. 2.7 Statistics Group comparisons were carried out using one-way analysis of variance or Student’s t-test. p-Values less than 0.05 were considered statistically significant.
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Results
3.1 Curcumin has a poor stability in aqueous buffer at physiological pH Consistent with previous studies [5, 7], we found that curcumin has a poor stability in aqueous buffer at physiological pH. UV spectroscopy analysis showed that at a concentration of 25 M, curcumin rapidly degraded in phosphate buffer (pH = 7.4), with a time-dependent decrease of absorbance at max of curcumin (420 nm) (Fig. 1A). This is consistent with previous studies, which showed that the degradation products of curcumin do not exhibit absorbance at 420 nm [5–7]. To further validate the results obtained from spectroscopic analysis, we used RP HPLC to measure curcumin concentration in the phosphate buffer. HPLC analysis showed that after 12-min incubation, there is 80–90% degradation of curcumin in the buffer, followed with a slower degradation of the remaining curcumin (Fig. 1B). At a lower concentration (e.g. 1 M), curcumin also rapidly degraded in phosphate buffer (see Supporting Information Fig. S2). Together, these results confirm the poor chemical stability of curcumin in aqueous buffer at physiological pH. 3.2 Redox active antioxidants increase chemical stability of curcumin After we established the poor stability of curcumin in aqueous buffer, we studied the effects of antioxidants on curcumin stability, using a colorimetric assay and HPLC analysis. As shown in Fig. 1A, curcumin degradation in the buffer is correlated with a decrease of absorbance at its max (420 nm); therefore, we used a 96-well-plate-based colorimetric assay to rapidly screen antioxidants on their effects to modulate curcumin stability. When incubated alone in phosphate buffer, curcumin rapidly degraded, while co-addition of a wide range of redox active antioxidants dramatically increased curcumin stability (Fig. 2A). These antioxidants possessed diverse chemical structures, including gallic acid, ascorbate (vitamin C), TBHQ, caffeic acid, rosmarinic acid, and Trolox C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(a water-soluble analog of vitamin E). Co-addition of these antioxidants, such as ascorbate or Trolox, did not change pH of the phosphate buffer (data not shown), indicating the protective effects were not due to altered pH. Next, we performed HPLC analysis to further validate the results obtained from the colorimetric assays. Consistent with the colorimetric assays, co-addition of above redox active antioxidants dramatically decreased curcumin degradation in the phosphate buffer within a 1-h incubation period (Fig. 2B), confirming the protective effects of antioxidants on curcumin stability. We also tested EDTA, which is a widely used metal-chelating antioxidant that does not have radical scavenging activity. The HPLC analysis showed that EDTA, even at a dose as high as 200 M, had little protective effect on curcumin stability (Fig. 2A). We further characterized the dose–response effects of these antioxidants on curcumin stability, and the actions of antioxidants on long-term stability of curcumin. The dose– response experiment showed that these redox active antioxidants significantly increased curcumin stability at doses as low as 1 M (Supporting Information Fig. S3), demonstrating potent protective effects of these antioxidants on curcumin stability. Regarding the long-term stability, co-addition of ascorbate significantly increased long-term stability of curcumin: after 24-h and 48-h incubation, there was, respectively, 74.7 and 53.7% of curcumin remaining in the phosphate buffer. In contrast, when curcumin was incubated alone, there was only 5.9 and 4.2% curcumin remaining after 24-h and 48-h incubation, respectively (Fig. 3). Considering the half-life of curcumin in phosphate buffer was less than 10 min (see Fig. 1), co-addition of ascorbate caused a >200-fold increase of the half-life of curcumin. Besides ascorbate, other antioxidants, such as gallic acid, TBHQ and Trolox, also significantly increased long-term stability of curcumin (Fig. 3), suggesting that co-administration of redox active antioxidants could be a practical strategy to enhance curcumin stability. Our results above strongly suggest that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a critical role in mediating curcumin degradation. To further test which mechanism plays a major role, we compared the stability of curcumin with its structural analog dimethoxy curcumin (see chemical structures in Supporting Information Fig. S4). Compared with curcumin, dimethoxy curcumin does not have the radical-initiating phenolic groups. HPLC analysis showed that in phosphate buffer, curcumin rapidly degraded, while dimethoxy curcumin was very stable. This result further supports that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a major role in mediating curcumin degradation.
3.3 Roles of degradation in the antiproliferative activity of curcumin in vitro Because of the poor stability of curcumin in aqueous medium, it remains unclear whether the observed biological activities www.mnf-journal.com
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of curcumin in vitro are derived from curcumin itself or its degradation products, hampering mechanistic understanding of curcumin [4]. After we demonstrated that redox active antioxidants stabilize curcumin in aqueous medium, we used these antioxidants to probe the roles of degradation in the biological activity of curcumin. We first conducted our cellular assays in serum-free basal DMEM medium. Curcumin rapidly degraded in the basal medium with a half-life of several minutes (Supporting Information Fig. S5A); after 24-h treatment in the basal medium in MC38 colon cancer cells, curcumin showed a weak inhibitory effect on cancer cell proliferation: at a dose as high as 20 M, curcumin suppressed 20% inhibition of cell proliferation. Co-addition of ascorbate or Trolox increased curcumin stability in basal DMEM medium (data not shown); and co-addition of these two antioxidants dramatically enhanced the actions of curcumin, with a 40–80% inhibition of MC38 cell proliferation. As a control, treatment with ascorbate or Trolox alone had little effect on MC38 proliferation (Fig. 4A–C). Besides ascorbate and Trolox, we also tested other antioxidants, and found all redox active antioxidants used in our study significantly increased antiproliferative effect of curcumin in MC38 colon cancer cells (Fig. 4D). Next, we performed similar assays in complete medium (containing 10% serum). Previous studies have shown that with the presence of 10% serum, the half-life of curcumin increased to 8 h [5]. Co-addition of antioxidant did not further enhance the biological activity of curcumin C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Curcumin rapidly degrades in phosphate buffer at physiological pH. (A) (Left) Representative UV spectrum of a 25 M curcumin solution in 0.1 M phosphate buffer (pH = 7.4). (Right) Quantification of absorbance at 420 nm. (B) (Left) Representative HPLC chromatography of a 25 M curcumin solution in 0.1 M phosphate buffer, detection wavelength at 420 nm. (Right) Quantification of curcumin concentration in the phosphate buffer by using HPLC.
(Supporting Information Fig. S5B), most likely because curcumin was already stabilized in the complete medium.
3.4 Antioxidant TBHQ increases circulating levels of curcumin in animal models Finally, we tested whether co-administration of antioxidant increased the circulating level of curcumin in animal models. We used DMSO as the solvent to dissolve curcumin for i.p. injection into the mice, because curcumin is highly stable in DMSO (data not shown), minimizing curcumin degradation prior the i.p. injection. Consistent with previous studies [10,11], HPLC analysis showed that 1 h after i.p. injection of 20 mg/kg curcumin, the plasma concentration of curcumin was 14.8 ± 16 nM. Co-administration of curcumin with TBHQ (dose = 40 mg/kg), a phenolic antioxidant widely used in food systems, increased the plasma concentration of curcumin to 87.6 ± 32.3 nM (approximately sixfold increase; Fig. 5).
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Discussion
Here, our central finding is that a wide range of redox active antioxidants with diverse chemical structures significantly increased chemical stability of curcumin in aqueous medium, supporting a redox-dependent mechanism to play a major www.mnf-journal.com
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role in mediating curcumin degradation. In addition, we found that when curcumin was stabilized by co-addition of antioxidants, its biological activity was significantly enhanced, suggesting that curcumin itself, instead of its degradation products, is largely responsible for the observed biological actions of curcumin. Previous research has suggested two mechanisms to explain curcumin degradation in aqueous buffer: hydroxyl ion mechanism and phenolic radical mechanism (see scheme in Supporting Information Fig. S1) [4–7]. Here, our experiments using redox active antioxidants and curcumin analog strongly support that the phenolic radical mechanism, instead of the hydroxyl ion mechanism, plays a major role in mediating curcumin degradation. Together, our study and previous investigations [4, 6, 7] suggest that curcumin degradation in aqueous buffer could happen through a mechanism comparable to that of lipid peroxidation. The first step of curcumin degradation is hydrogen dissociation from the phenolic group to form a phenolic radical, which then migrates to the conjugated heptadienedione chain and leads to formation of cyclized compounds such as the recently identified bicyclopentadione derivatives of curcumin [4, 6, 7]. The radical could C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
be then transferred to another curcumin molecule, resulting in a chain reaction to cause a rapid and massive curcumin degradation [4]. Both the phenolic group and the conjugated heptadienedione chain seem to play critical roles in curcumin degradation. Indeed, converting the radical-initiating phenolic (–OH) groups to methoxy groups (–OCH3 ), or reduction of the carbon–carbon double bonds in the conjugated heptadienedione chain, significantly increased curcumin stability (see Supporting Information Fig. S4 and ref. 10). The redox active antioxidants increase chemical stability of curcumin, at least in part, through directly suppressing formation of the curcumin phenolic radical. The redox active antioxidants used in our study have been shown to have lower hydrogen dissociation energies compared with that of the phenolic group of curcumin [12]. Therefore, these antioxidants can efficiently reduce the curcumin phenolic radicals, and terminate chain reaction of curcumin degradation. Together, these results suggest a redox-dependent mechanism as a major mechanism to mediate curcumin degradation. Because of the poor stability of curcumin in aqueous medium, it remains unclear whether the observed biological activities of curcumin in vitro are derived from curcumin www.mnf-journal.com
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itself or its degradation products [4, 5]. Here, our results support that curcumin, rather than its degradation products, is largely responsible for the observed biological activities of curcumin. Indeed, in serum-free medium, curcumin rapidly de-
graded with a half-life of several minutes; therefore, treatment with curcumin alone in basal medium would actually reflect the biological effects of the curcumin degradation products. Our result showed that even at a high dose (20 M), curcumin
Figure 4. Antioxidants increase antiproliferative effect of curcumin in MC38 colon cancer cells. (A) Representative microscope images of the treated cells. Treatment with curcumin alone, or Trolox or ascorbate alone, had little effect on cell density or morphology; in contrast, their combination dramatically reduced cell density and changed cell morphology. (B) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different doses of ascorbate (3.9– 62.5 M), or a combination of curcumin and ascorbate, for 24 h. (C) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different doses of Trolox (3.9–62.5 M), or a combination of curcumin and Trolox, for 24 h. (D) Quantification of cell proliferation of MC38 cells treated with curcumin (20 M), or several different antioxidant (dose of each antioxidant = 3.9 M), or a combination of curcumin and antioxidant, for 24 h. The results are mean ± SD. The samples designated with different letters are statistically different (p < 0.05). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 5. Co-administration of antioxidant increases plasma concentration of curcumin. (Left) Representative HPLC chromatography of the plasma from mice treated with control (20 mg/kg curcumin alone), or a combination of 20 mg/kg curcumin and 40 mg/kg TBHQ. (Right) Quantification of plasma concentration of curcumin. n = 4 mice per group; the results are mean ± SD.
had a poor effect to suppress MC38 cell proliferation (20% inhibition, see Fig. 4). Co-addition of cellular antioxidants stabilized curcumin and dramatically enhanced the antiproliferative effects of curcumin (Fig. 4). These results showed that curcumin itself, rather than its degradation products, has potent biological activities. The process of curcumin degradation leads to formation of less-active or inactive compounds, therefore is unlikely to contribute to the biological actions of curcumin. In complete medium (containing 10% serum), the half-life of curcumin increased to 8 h [5]; since curcumin was highly stable with the presence of serum, co-addition of antioxidants did not further enhance the actions of curcumin (Supporting Information Fig. S5). We need to point out the antiproliferative effect of curcumin was much more potent in complete medium compared that in basal medium (Fig. 4 and Supporting Information Fig. S5), further supporting that curcumin itself, rather than its degradation products, had potent biological activities. Together, these results support that curcumin, instead of its degradation products, contributed to the observed biological actions of curcumin. Further studies are needed to further clarify this point, as a previous study has shown that the oxidized products of curcumin, but not curcumin itself, had potent inhibitory effect on human type II topoisomerases [13]. To date, whether curcumin degradation occurs in vivo remains unknown [4]. Here, we showed that co-administration of antioxidant TBHQ significantly increased circulating level of curcumin in animal models (Fig. 5). This result indicates that chemical degradation of curcumin could happen in vivo, and redox active antioxidants could increase curcumin stability in vivo through suppressing the chemical degradation of curcumin. In our study, we used TBHQ because it can be co-solubilized with curcumin in DMSO (curcumin is highly stable in DMSO, thus minimizing curcumin degradation prior the i.p. injection into mice); and TBHQ is a widely C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
used phenolic antioxidant in the food system. However, the limitation of our experiment is that we only tested one antioxidant; more studies are therefore needed to test whether other types of antioxidants could enhance the pharmacokinetics profile of orally administered curcumin in vivo. Consistent with our findings, previous studies have shown that coadministration of redox active antioxidants, such as ascorbate (vitamin C), enhanced biological activities of curcumin in several different disease models in vivo [14, 15]. For example, in a cadmium-induced hepatotoxicity model, administration of 200–400 mg/kg curcumin alone or 400 mg/kg ascorbate alone had no effect, while their combination dramatically inhibited cadmium-induced hepatotoxicity in rats [15]. It remains to decide whether ascorbate interacted with curcumin through enhancing the chemical stability and circulating level of curcumin in vivo. A major barrier to develop curcumin-based therapeutics is its poor pharmacokinetics profile in vivo. Indeed, a previous human study has shown that after a single oral dose of 10 or 12 g of curcumin, free form of curcumin was barely detected in human plasma [16]. High doses of curcumin were required to exert its health-promoting biological activities in animal and human studies. In a Phase II␣ trial of colorectal cancer, curcumin had no effect at a dose of 2 g/day; and only inhibited aberrant crypt foci at a dose as high as 4 g/day [2]. Further studies are needed to test whether our finding could serve as a practical strategy to enhance pharmacokinetics profiles and biological activities of curcumin in vivo. In summary, here we demonstrate, for the first time, that a wide range of redox active antioxidants dramatically increased chemical stability of curcumin in aqueous medium. This further supports a previously proposed phenolic radical mechanism as a major mechanism that mediates curcumin degradation [4, 6, 7]. Further studies are needed to explore the applications of this finding in developing more effective curcumin-based functional foods or therapeutics. www.mnf-journal.com
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This work was supported by a faculty start-up fund and Armstrong Fund of Science Award from University of Massachusetts Amherst (G.Z.). Potential conflict of interest statement: The authors have submitted a provisional US patent to stabilize curcumin using antioxidants, which has been assigned to UMass-Amherst.
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