Potent Antioxidant and Genoprotective Effects of Boeravinone G, a Rotenoid Isolated from Boerhaavia diffusa

Background and Aims: Free radicals are implicated in the aetiology of some gastrointestinal disorders such as gastric ulcer, colorectal cancer and inflammatory bowel disease. In the present study we investigated the antioxidant and genoprotective activity of some rotenoids (i.e. boeravinones) isolated from the roots of Boerhaavia diffusa, a plant used in the Ayurvedic medicine for the treatment of diseases affecting the gastrointestinal tract. Methods/Principal Findings: Antioxidant activity has been evaluated using both chemical (Electron Spin Resonance spectroscopy, ESR) and Caco-2 cells-based (TBARS and ROS) assays. DNA damage was evaluated by Comet assay, while pERK 1/2 and phospho-NF-kB p65 levels were estimated by western blot. Boeravinones G, D and H significantly reduced the signal intensity of ESR induced by hydroxyl radicals, suggesting a scavenging activity. Among rotenoids tested, boeravinone G exerted the most potent effect. Boeravinone G inhibited both TBARS and ROS formation induced by Fenton's reagent, increased SOD activity and reduced H 2O 2-induced DNA damage. Finally, boeravinone G reduced the levels of pERK 1 and phospho-NF-kB p65 (but not of pERK 2) increased by Fenton's reagent. Conclusions: It is concluded that boeravinone G exhibits an extraordinary potent antioxidant activity (significant effect in the nanomolar range). The MAP kinase and NF-kB pathways seem to be involved in the antioxidant effect of boeravinone G. Boeravinone G might be considered as lead compound for the development of drugs potentially useful against those pathologies whose aetiology is related to ROS-mediated injuries

Chemical structure of the most potent antioxidant rotenoids.

<p>Rotenoids were obtained from Kupchan partitioning of the methanol extract of <i>B. diffusa</i> root following by sequential silica gel column chromatography and HPLC.</p

Effect of boeravinone G (0.1–1 ng/ml) on Fenton's reagent (H₂O₂/Fe²⁺ 2 mM)-induced reactive species (ROS) production.

<p>Effect observed in differentiated Caco-2 cells after 24-hour boeravinone G exposure. Data represent mean ± SEM of 6 experiments. ^#p<0.001 <i>vs</i> control (vehicle); *p<0.05, **p<0.01 and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of boeravinone G (0.1–1 ng/ml) on superoxide dismutase (SOD) activity.

<p>SOD activity was evaluated in Caco-2 cells exposed to Fenton's reagent (H₂O₂/Fe²⁺ 1 mM) without or with boeravinone G (0.1–1 ng/ml). Data represent mean ± SEM of 4 experiments. ^#p<0.001 <i>vs</i> control (vehicle); *p<0.05 and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone; °p<0.05 <i>vs</i> control.</p

Effect of boeravinone G (0.1–1 ng/ml) on pERK₁ (A) and pERK₂ (B) expression.

<p>Quantitative analysis and representative western blot analysis of pERK₁ and pERK₂ in Caco-2 cells exposed to Fenton's reagent (H₂O₂/Fe²⁺ 1 mM) without or with boeravinone G (0.1–1 ng/ml). The results were normalized with anti-ERK₂ (pERK_1/2/ERK₂). ^#p<0.01 <i>vs</i> control (vehicle); ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of boeravinone G (0.1–1 ng/ml) on Fenton's reagent (H₂O₂/Fe²⁺ 1 mM)-induced malondialdehyde-equivalents (MDA-equivalents) production.

<p>Effect observed in differentiated Caco-2 cells after 24-hour boeravinone G exposure. Data represent mean ± SEM of 6 experiments. ^#p<0.001 <i>vs</i> control (vehicle) and ***p<0.001 <i>vs</i> H₂O₂/Fe²⁺ alone.</p

Effect of 5 mg/ml of <i>Boerhaavia diffusa</i> methanol extract on <i>electron spin resonance spectroscopy</i>.

<p>Representative ESR spectra of DMPO-OH spin adduct signal (A) and DMPO-OH spin adduct signal in the presence of 5 mg/ml of <i>Boerhaavia diffusa</i> methanol extract (B).</p

Effect of boeravinone G (BG, 0.1–1 ng/ml) on DNA damage.

<p>DNA damage (tail intensity) was detected by the Comet assay in Caco-2 cells exposed to 75 µM H₂O₂ for 5 min in absence or presence of boeravinone G. a = control; b = H₂O₂ 75 µM; c = H₂O₂ 75 µM+BG 0.1 ng/ml; d = H₂O₂ 75 µM+BG 0.3 ng/ml; e = H₂O₂ 75 µM+BG 1 ng/ml. Data represent mean ± SEM of 4 experiments. ^#p<0.001 <i>vs</i> control (vehicle) and ***p<0.001 <i>vs</i> H₂O₂ alone.</p

Antioxidant activity (AA), detected using ESR assay, of the fractions obtained from the carbon tetrachloride extract of <i>B. diffusa</i> root.

<p>Antioxidant activity (AA), detected using ESR assay, of the fractions obtained from the carbon tetrachloride extract of <i>B. diffusa</i> root.</p