Stability and anti-inflammatory activity of the reduction-resistant curcumin analog, 2,6-dimethyl-curcumin

Abstract

The efficacy of the curry spice compound curcumin as a natural anti-inflammatory agent is limited by its rapid reductive metabolism in vivo. A recent report described a novel synthetic derivative, 2,6-dimethyl-curcumin, with increased stability against reduction in vitro and in vivo. It is also known that curcumin is unstable at physiological pH in vitro and undergoes rapid autoxidative transformation. Since the oxidation products may contribute to the biological effects of curcumin, we tested oxidative stability of 2,6-dimethyl-curcumin in buffer (pH 7.5). The rate of degradation was similar to curcumin. The degradation products were identified as a one-carbon chain-shortened alcohol, vanillin, and two isomeric epoxides that underwent cleavage to vanillin and a corresponding hydroxylated cleavage product. 2,6-Dimethyl-curcumin was more potent than curcumin in inhibiting NF-κB activity but less potent in inhibiting expression of cyclooxygenase-2 in LPS-activated RAW264.7 cells. 2,6-Dimethyl-curcumin and some of its degradation products covalently bound to a peptide that contains the redox-sensitive cysteine of IKKβ kinase, the activating kinase upstream of NF-κB, providing a mechanism for the anti-inflammatory activity. In RAW264.7 cells vanillin, the chain-shortened alcohol, and reduced 2,6-dimethyl-curcumin were detected as major metabolites. These studies provide new insight into the oxidative transformation mechanism of curcumin and related compounds. The products resulting from oxidative transformation contribute to the anti-inflammatory activity of 2,6-dimethyl-curcumin in addition to its enhanced resistance against enzymatic reduction.

Figure 1

Chemical structures of curcumin, 2,6-dimethyl-curcumin, and the curcumin metabolites tetrahydrocurcumin and bicyclopentadione.

Fig. 2

Degradation of 2,6-dimethyl-curcumin. (a) Stability of 2,6-dimethyl-curcumin at pH 7.5. 2,6-Dimethyl-curcumin (25 μM) was diluted in 20 mM phosphate buffer pH 7.5, and the sample was analyzed by repetitive scanning every 1 min or (b) in the time drive mode at 410 nm in a UV/Vis spectrophotometer. (c) Incubation reactions of 2,6-dimethyl-curcumin in 20 mM phosphate buffer pH 8 were extracted after 1 h or (d) 5 h and analyzed using RP-HPLC with diode array detection. The chromatograms were recorded at UV 205 nm. (e) Isolated and identified products of degradation of 2,6-dimethyl-curcumin.

Fig. 3

Time course of the degradation of 2,6-dimethyl-curcumin. (a) The disappearance of 2,6-dimethyl-curcumin is plotted as % remaining of the starting amount on the right axis. Formation of products in % of total products is plotted on the left axis. 2,6-Dimethyl-curcumin (25 μM) was dissolved in 20 mM phosphate buffer pH 8 in an HPLC autosampler vial. Aliquots (10 μl) were injected every 1 h for 10 h. The mean ± SD of three independent replicates is shown. (b) RP-HPLC analysis of the incubation of isolated epoxide 2 in buffer pH 7.5 for 1 h. (c) RP-HPLC analysis of the incubation of isolated epoxide 3. The chromatograms were recorded at UV 205 nm.

Fig. 4

Anti-inflammatory activity of 2,6-dimethyl-curcumin. (a) RAW264.7 cells stably expressing luciferase downstream of an NF-κB response element were treated with vehicle or with 2,6-dimethyl-curcumin (1-50 μM) 1 h prior to LPS (100 ng/mL). Cells were harvested 4 h after LPS addition, and luciferase activity was measured in the cell supernatant. (b) Western blot detection of COX-2 in RAW264.7 cells treated with 2,6-dimethyl-curcumin or curcumin 1 h prior to stimulation with LPS (100 ng/mL). The cells were harvested after 5 h, and protein was resolved by SDS-PAGE. The image shown is representative of three replicates with identical results.

Fig. 5

Peptide adduction by 2,6-dimethyl-curcumin and its degradation products. A 27-amino acid peptide containing redox-regulated Cys179 of Inhibitor of nuclear factor κ-B kinase subunit β (IKKβ) (10 μM) was reacted with 2,6-dimethyl-curcumin (50 μM) at 37°C for 1 h. An aliquot of the reaction was analyzed using LC-MS. (a) The first panel shows the total ion chromatogram (TIC) and the following panels show extracted ion chromatograms for peptide adducts with 2,6-dimethyl-curcumin (+396; m/z 1703), epoxides 2 and 3 (+412; m/z 1711), methoxyphenol (+122; m/z 1567), 1-hydroxy-ethyl-methoxyphenol (+166; m/z 1589), and for the unreacted peptide and its dimer, respectively, (m/z 1506). (b) Total ion chromatogram for analysis of the IKKβ peptide before incubation with 2,6-dimethyl-curcumin and of a Cys179Ala mutant IKKβ peptide (10 μM) reacted with 2,6-dimethyl-curcumin (50 μM) for 1 h.

Fig. 6

Metabolism of 2,6-dimethyl-curcumin in RAW264.7 cells. RAW264.7 cells were incubated with 2,6-dimethyl-curcumin (10 μM) for 30 min and extracted. LC-SRM-MS analysis was performed in the positive ion mode. The ion chromatograms for detection of vanillin, product 5, tetrahydro-2,6-dimethyl-curcumin, and 2,6-dimethyl-curcumin are shown.

Fig. 7

Proposed structures of 2,6-dimethyl-curcumin and its degradation products adducting to the IKKβ peptide. The red arrow indicates predicted sites of reaction with Cys179 of the 27-amino acid peptide of IKKβ.

Scheme 1

Proposed mechanisms of oxidative transformation of 2,6-dimethyl-curcumin. (a) Direct pathway of formation of vanillin 1 via β-cleavage of a dioxetane radical intermediate. (b) Formation of epoxides 2 and 3 via peroxide mediated dimerization of 2,6-dimethyl-curcumin. The first step is formation of a peroxyl radical (ROO•). In the second step the peroxyl radical adds to the double bond of a second molecule of 2,6-dimethyl-curcumin (blue). Addition can occur at either C-6 or C-7 and results in the formation of the same epoxides, 2 and 3 via homolytic intramolecular substitution S_Hi or addition of the alkoxyl radical to the double bond, respectively. (c) Formation of chain-shortened alcohol 5 by phenyl 1,2-migration rearrangement of a peroxide-linked dimer of 2,6-dimethyl-curcumin.