Rooperol as an antioxidant and its role in the innate immune system: an in vitro study

Biologically active rooperol is formed when the glucose subunits of the nontoxic glycoside, hypoxoside, are cleaved by β-glucosidase. Hypoxoside is isolated from Hypoxis, a medicinal plant genus frequently used by the indigenous people of South Africa as an immune system booster. The aim of this study was to investigate rooperol's antioxidant and anti-inflammatory properties using the ferric reducing ability of plasma (FRAP) assay, NO and ROS production, and phagocytosis.

Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages was induced using 10-100 nM 1,25(OH)(2)D(3) and PMA over 72 h. Differentiation was confirmed by light microscopy and flow cytometry. Undifferentiated and/or differentiated cells were treated with DMSO (0.25 v/v%, vehicle control), hypoxoside (50 μg/mL), rooperol (20 μg/mL) or PMA (10/20 nM, positive control). ROS production was measured in undifferentiated and differentiated monocyte-macrophages using DCFH-DA and flow cytometry. Phagocytosis of pHrodo™ Escherichia coli BioParticles(®) was measured using pre-treated monocyte-macrophage differentiated U937 cells. NO production was measured in monocyte-macrophage differentiated U937 cells using DAF-2 DA and flow cytometry.

Rooperol was shown to have similar or greater antioxidant potential than ascorbic acid. Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages were confirmed morphologically (cell attachment, clump- and pseudopodia-formation) and biochemically (CD11b and CD14 cell surface marker expression). Rooperol significantly increased ROS and NO production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukemia cells. Hypoxoside had no or very little effect on ROS and NO production, and phagocytosis.

This study confirms previous reports that hypoxoside has to be converted to rooperol to be biologically active. The FRAP assay confirms the antioxidant capacity of rooperol seen in previous studies, whereas rooperol's induction of ROS and NO production, and phagocytosis constitute novel findings. Possible mode(s) of action for the in vitro anti-inflammatory activities of rooperol may be explained by ROS and NO production, and phagocytosis.