Polyphenols from Silybum marianum inhibit in vitro the oxidant response of equine neutrophils and myeloperoxidase activity

Abstract

A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100 nm and 100 μm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release from 10 μm. Classical MPO assay showed quercetin as the most potent inhibitor, followed by taxifolin, dehydrosilybin and silybin. SIEFED MPO assay highlighting the binding of tested compounds to MPO showed that only quercetin and taxifolin maintained an efficient inhibition above 90% at 10 μm. Altogether, our results showed a strong inhibition of PMN activation by planar compounds such as quercetin and dehydrosilybin and a strong inhibition of MPO activity by the smallest molecules, quercetin and taxifolin. In conclusion, the compounds from silymarin may be useful for modulating the oxidative response of PMNs, involved in the pathogenesis of laminitis, but further in vivo studies are needed.

Flavonoid composition, cellular antioxidant activity and (myelo)peroxidase inhibition of a Bryonia alba L. (Cucurbitaceae) leaves extract

“…The next step in testing the peroxidase inhibitory potential of the leaves of B. alba and their isolated compounds consisted in the assessment of their inhibitory activity on the MPO. The effect of samples on the activity of MPO was assessed by direct quantification . Tested concentrations ranged between 2 and 100 μg/ml (Table ).…”

“…The effect of samples on the activity of MPO was assessed by direct quantification. [22][23][24][25] Tested concentrations ranged between 2 and 100 lg/ml ( Table 1). The method showed important inhibition of the MPO both for the crude extract and its isolated flavonoids.…”

“…Blank consisted in the dilution buffer. Fluorescence was detected for excitation and emission wavelenghts (λexc = 573 nm and λem = 587 nm), on a Fluoroskan Ascent reader (30 min, 37°C) …”

“…Equine neutrophils were isolated from the blood of healthy horses, as described by Zholobenko et al . Toxicity of the samples on isolated neutrophils was assessed using the Trypan blue test . For ROS assay, one million neutrophils were added in each well of a white 96‐well microplate and incubated with samples (the crude extract and its isolated flavonoids at concentrations ranging between 2 and 100 μg/ml – Table ) for 10 min at 37°C.…”

“…Fluorescence was detected for excitation and emission wavelenghts (kexc = 573 nm and kem = 587 nm), on a Fluoroskan Ascent reader (30 min, 37°C). [22][23][24][25] Effects on the total ROS produced by PMAactivated neutrophils Equine neutrophils were isolated from the blood of healthy horses, as described by Zholobenko et al Toxicity of the samples on isolated neutrophils was assessed using the Trypan blue test. [25] For ROS assay, one million neutrophils were added in each well of a white 96-well microplate and incubated with samples (the crude extract and its isolated flavonoids at concentrations ranging between 2 and 100 lg/ml - Table 1) for 10 min at 37°C.…”