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. 2011 Jan 14;286(2):1114-24.
doi: 10.1074/jbc.M110.178806. Epub 2010 Nov 11.

Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

Markus Griesser  1 , Valentina Pistis, Takashi Suzuki, Noemi Tejera, Derek A Pratt, Claus Schneider
Affiliations

Autoxidative and cyclooxygenase-2 catalyzed transformation of the dietary chemopreventive agent curcumin

Markus Griesser et al. J Biol Chem. .

Abstract

The efficacy of the diphenol curcumin as a cancer chemopreventive agent is limited by its chemical and metabolic instability. Non-enzymatic degradation has been described to yield vanillin, ferulic acid, and feruloylmethane through cleavage of the heptadienone chain connecting the phenolic rings. Here we provide evidence for an alternative mechanism, resulting in autoxidative cyclization of the heptadienone moiety as a major pathway of degradation. Autoxidative transformation of curcumin was pH-dependent with the highest rate at pH 8 (2.2 μM/min) and associated with stoichiometric uptake of O(2). Oxidation was also catalyzed by recombinant cyclooxygenase-2 (COX-2) (50 nm; 7.5 μM/min), and the rate was increased ≈10-fold by the addition of 300 μM H(2)O(2). The COX-2 catalyzed transformation was inhibited by acetaminophen but not indomethacin, suggesting catalysis occurred by the peroxidase activity. We propose a mechanism of enzymatic or autoxidative hydrogen abstraction from a phenolic hydroxyl to give a quinone methide and a delocalized radical in the heptadienone chain that undergoes 5-exo cyclization and oxygenation. Hydration of the quinone methide (measured by the incorporation of O-18 from H(2)(18)O) and rearrangement under loss of water gives the final dioxygenated bicyclopentadione product. When curcumin was added to RAW264.7 cells, the bicyclopentadione was increased 1.8-fold in cells activated by LPS; vanillin and other putative cleavage products were negligible. Oxidation to a reactive quinone methide is the mechanistic basis of many phenolic anti-cancer drugs. It is possible, therefore, that oxidative transformation of curcumin, a prominent but previously unrecognized reaction, contributes to its cancer chemopreventive activity.

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Figures

FIGURE 1.
FIGURE 1.
Effect of pH on the transformation of curcumin. A, extended conjugation of the seven-carbon heptadienone chain connecting the two phenolic rings results in the yellow-orange color of curcumin. In 4′-methoxy- and 4′,4′-dimethoxycurcumin the phenolic hydroxyl group is replaced by a methoxy group. B, curcumin was added to buffer of the pH indicated and the decrease in absorbance at 430 nm at room temperature was monitored for 20 min using a UV/Vis spectrophotometer. For all reactions 70 μm curcumin was added except for pH 9.0 where 50 μm curcumin was added. The buffers pH 5 to 8 were generated using mixtures of 200 mm Na2HPO4 and 100 mm citric acid, pH 8.5 and 9.0 were 100 mm K2HPO4 adjusted with NaOH. C, repetitive scans in 2-min intervals of a solution of 70 μm curcumin in 100 mm Tris-HCl buffer, pH 8.
FIGURE 2.
FIGURE 2.
Oxygen consumption during the transformation of curcumin. A, 1 ml of Na-phosphate/citrate buffer pH 8.0 was placed in a Clark-type oxygen electrode and allowed to adjust to 30 °C. Curcumin (60 μm) or vehicle (ethanol) was added after 2 min (arrow), and the oxygen concentration was monitored for 10 min. B, curcumin (60 μm) was added to 1 ml of Na-phosphate/citrate buffer pH 8.0 in a spectrophotometer cuvette, and the absorbance at 430 nm was recorded for 10 min.
FIGURE 3.
FIGURE 3.
RP-HPLC analysis of the transformation products of curcumin. The transformation reaction of curcumin as shown in Fig. 1B was extracted and analyzed by RP-HPLC with detection by (A) UV 205 nm and (B and C) LC-ESI-MS. Three products 1, 2, and 3 were detected in the UV chromatogram and gave a signal in the ion trace m/z 399 (panel B), equivalent to a molecular weight of 400 for all three products. C, unreacted curcumin was detected as the [M-H]− molecular ion in the ion trace m/z 367.
FIGURE 4.
FIGURE 4.
RP-HPLC comparison of the products of curcumin autoxidation with the putative degradation products, vanillin, ferulic acid, and feruloylmethane. A, 500 ng each of vanillin and ferulic acid and 1 μg of feruloylmethane were injected on a Waters Symmetry 5-μm column (4.6 × 250 mm) eluted with a linear gradient of acetonitrile/water/acetic acid (20:80:0.01, by vol) to (80:20:0.01, by vol) in 20 min at a flow rate of 1 ml/min and diode array detection. B, analysis of autoxidized curcumin showing the elution of products 1, 2, and 3. C, co-chromatographic analysis of a mixture of the sample in B and 500 ng each of vanillin, ferulic acid, and feruloylmethane. The chromatograms shown were at recorded at UV 205 nm.
FIGURE 5.
FIGURE 5.
1H and H,H-COSY NMR spectra of product 1 (bicyclopentadione) isolated from autoxidation of curcumin. The 1H spectrum at the top shows five signals corresponding to the hydrogens at carbons 1, 2, 4, 6, and 7 of the original heptadienone chain of curcumin and the signals of the aromatic protons. The H,H-COSY spectrum (below) shows a cross peak between H6 and H7 as well as between H6 and H2. Note the lack of a cross-peak between the neighboring H1 and H2 due to unfavorable arrangement of the two spins at the ring structures. In addition, H1 shows weak coupling (J = 3.5 Hz) with the hydroxyl group at C3 (δ 5.76). H2′ of the aromatic ring showed a cross-peak with C1′ at 148.4 ppm in the HMBC experiments (not shown) that allowed assignment of all six aromatic protons to the corresponding phenolic ring.
FIGURE 6.
FIGURE 6.
Spectrophotometric analysis of the COX-2-catalyzed transformation of curcumin. A, transformation of curcumin was accelerated by the presence of 50 nm COX-2 compared with autoxidation. Addition of 300 μm H2O2 further accelerated the rate of COX-2-catalyzed transformation whereas it had only a small effect on autoxidation. B, addition of 10 μm indomethacin did not change the rate of COX-2 (50 nm) catalyzed transformation of curcumin. C, addition of acetaminophen (ApAP) dose-dependently inhibited the COX-2 (50 nm) catalyzed transformation of curcumin. All reactions were conducted using 100 μm curcumin and 1 μm hematin in 100 mm potassium phosphate buffer pH 9 in the absence or presence of 50 nm COX-2 as indicated. The absorbance at 430 nm was recorded in the time-drive mode using a UV/Vis spectrophotometer.
FIGURE 7.
FIGURE 7.
LC-ESI-MS/MS analysis of the bicyclopentadione 1 obtained by autoxidation of curcumin in regular and H218O-buffer. MS1 spectrum of 1 obtained form the incubation of curcumin in (A) regular buffer and (B) buffer containing 92% H218O. The MS2 spectra obtained by CID of the molecular ions m/z 399 and 401 are shown in supplemental Fig. S2. MS3 spectra obtained by CID of (C) m/z 247 and (D) m/z 249. The solid arrows indicate fragments derived from m/z 247 and m/z 249, respectively. The dashed arrows indicate the fragments obtained by CID of m/z 232 (in panel C) and m/z 234 (in panel D), respectively. E, proposed fragmentation of 1 in the CID experiments. The asterisk denotes the oxygen atom derived from H218O and the fragments that contain 18O. The MSn analyses were performed using an ion trap instrument as described under “Experimental Procedures.”
FIGURE 8.
FIGURE 8.
RP-HPLC analysis of the incubation of curcumin with LPS-activated RAW264.7 cells. RAW264.7 cells were activated with 100 ng/ml LPS for 5 h in DMEM adjusted to pH 7.6 using 20 mm sodium phosphate buffer in the absence of FBS. Curcumin (20 μm) was added to the RAW264.7 cells and incubated for additional 2 h. The medium was removed, acidified to pH 4, and extracted twice with 2.5 ml of ethyl acetate/isopropanol (90:10, by vol). An aliquot was injected on RP-HPLC with diode array detection using the same conditions as in Fig. 4. The peak identified as hexahydrocurcumin (structure sown) contained an unidentified product that eluted as a front shoulder. The chromatogram was recorded at 205 nm.
FIGURE 9.
FIGURE 9.
Proposed mechanism of autoxidative and COX-2 catalyzed transformation of curcumin. The reaction steps are explained in the “Discussion.” Products 2 and 3 are isomers of 1 differing in the configuration of carbons 1 and 7, respectively. R = -OCH3.
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