Oxidative Transformation of Demethoxy- and Bisdemethoxycurcumin: Products, Mechanism of Formation, and Poisoning of Human Topoisomerase IIα

Abstract

Extracts from the rhizome of the turmeric plant are widely consumed as anti-inflammatory dietary supplements. Turmeric extract contains the three curcuminoids, curcumin (≈80% relative abundance), demethoxycurcumin (DMC; ≈15%), and bisdemethoxycurcumin (BDMC; ≈5%). A distinct feature of pure curcumin is its instability at physiological pH, resulting in rapid autoxidation to a bicyclopentadione within 10-15 min. Here, we describe oxidative transformation of turmeric extract, DMC, and BDMC and the identification of their oxidation products using LC-MS and NMR analyses. DMC autoxidized over the course of 24 h to the expected bicyclopentadione diastereomers. BDMC was resistant to autoxidation, and oxidative transformation required catalysis by horseradish peroxidase and H2O2 or potassium ferricyanide. The product of BDMC oxidation was a stable spiroepoxide that was equivalent to a reaction intermediate in the autoxidation of curcumin. The ability of DMC and BDMC to poison recombinant human topoisomerase IIα was significantly increased in the presence of potassium ferricyanide, indicating that oxidative transformation was required to achieve full DNA cleavage activity. DMC and BDMC are less prone to autoxidation than curcumin and contribute to the enhanced stability of turmeric extract at physiological pH. Their oxidative metabolites may contribute to the biological effects of turmeric extract.

Figure 1

Stability of turmeric extract and curcumin at pH 7.5. A 50 μM solution of (A) turmeric extract or (B) curcumin in 10 mM NH₄OAc buffer pH 7.5 was scanned from 700 to 200 nm every 2 min for 20 min.

Figure 2

Horseradish peroxidase (HRP)-catalyzed transformation of DMC and BDMC. (A) DMC or (B) BDMC (50 μM) were diluted in 500 μl 10 mM NH₄OAc buffer pH 7.5 in a spectrophotometer cuvette and the UV/Vis spectra (700 to 200 nm) were recorded every min. HRP and H₂O₂ (40 μM) were added after the first (A) or second (B) scan.

Figure 3

RP-HPLC analysis of DMC autoxidation products. (A) 100 μl of a reaction of 50 μM DMC in 1.5 ml 10 mM NH₄OAc buffer pH 7.5 were injected at 48 h reaction time without prior extraction. The chromatogram was recorded at UV 205 nm using a diode array detector. The inset shows the UV spectrum of peak 1a recorded online during HPLC analysis. (B) Selected carbon chemical shifts and HMBC correlations (arrows) of demethoxy-bicyclopentadione diastereomer 1a.

Figure 4

RP-HPLC analysis of HRP-H₂O₂-catalyzed transformation of BDMC. (A) 100 μl of a reaction of 50 μM DMC in 1.5 ml 10 mM NH₄OAc buffer pH 7.5 containing horseradish peroxidase (0.01 U/ml) and H₂O₂ (40 μM) were injected at 10 min reaction time without prior extraction. The chromatogram was recorded at UV 205 nm using a diode array detector. The inset shows the UV spectrum of peak 2a recorded online during HPLC analysis. (B) Selected carbon chemical shifts and HMBC correlations (arrows) of demethoxy-spiroepoxide diastereomer 2a.

Figure 5

Negative ion LC-ESI-MS analysis of ¹⁸O incorporation in (A) demethoxy bicyclopentadione 1a (B) bisdemethoxy spiroepoxide 2a. DMC and BDMC, respectively, were oxidized using HRP/H₂O₂ in 10 mM NH₄OAc buffer pH 7.5 containing 50% H₂¹⁸O. Expanded MS1 spectra (negative ion mode) of (A) 1a from m/z 360 to 380, and (B) 2a from m/z 330 to 350 are shown.

Figure 6

Degradation of turmeric extract analyzed by LC-ESI-MS. Turmeric extract (25 μM) was incubated in 10 mM NH₄OAc buffer pH 7.5 for the indicated time points, extracted, and analyzed using LC-ESI-MRM-MS in the positive ion mode. The time course of the degradation and formation of (A) curcumin and bicyclopentadione, (B) DMC and DMC bicyclopentadione, and (C) BDMC were quantified using d₆-curcumin and d₆-bicyclopentadione, respectively, as internal standards. The average of three independent reactions is shown with the error bars representing standard deviations.

Figure 7

Effects of DMC and BDMC on DNA cleavage mediated by human topoisomerase IIα. Reactions were carried out in the absence of oxidant (open symbols) or in the presence of 50 μM K₃Fe(CN)₆ (closed symbols). The left panel shows the effects of DMC (circles) and BDMC (squares) on DNA cleavage. The right panel shows control reactions carried out in the absence of compounds (No Drug) or in the presence of 100 μM etoposide (Etop) or curcumin (Curc). Error bars represent the standard deviations for at least three independent experiments. The top shows a representative ethidium bromide-stained agarose gel of DNA cleavage reactions that contain 0–100 μM DMC and 50 μM K₃Fe(CN)₆. The first lane contains only negatively supercoiled DNA. The positions of negatively supercoiled (FI), nicked (FII), and linear (FIII) DNA are indicated. Baseline levels of enzyme-mediated DNA cleavage in the absence of oxidant were ~2%.

Scheme 1

Proposed mechanism of oxidative transformation of DMC and BDMC.

Chart 1

Structures of curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), and curcumin bicyclopentadione.