In vitro nitric oxide inhibition of selected south African medicinal plants : a bio-guided purification of anti-inflammatory compounds from Conyza scabrida

Abstract

This study aimed to investigate the anti-inflammatory activity of plant species by in vitro screening of extracts, fractions, and pure compounds on LPS-activated RAW 246.7 mouse macrophages. Plants were sourced from the plant repository in the Biodiscovery Centre, at the University of Pretoria. The plant materials were ground into fine powdered and extracted with dichloromethane: methanol (DCM:MeOH) (1:1) followed by 100 % MeOH, filtered, concentrated, and dried to generate extracts. Twelve extracts were fractionated using positive pressure solid phase extraction (ppSPE) Gilson liquid hander workstation, resulting in seven fractions per plant extract. The observed anti-inflammatory activity was mainly attributed to specific ppSPE fractions of the plant extracts rather than the entire crude extract.The most active plants species identified with 90 % inhibition at 25 μg/mL, were Dodonaea. viscosa, Buxus natalensis, Flacourtia. indica, and Conyza scabrida. The crude extract and ppSPE fractions (6 and 7) of C. scabrida demonstrated strong anti-inflammatory activity at 50 μg/mL with 97.8 % and 97.0 % inhibition, respectively. The Ultra-Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Mass Spectrometry Mass Spectrometry (UPLC-QTOF-MS) analysis of the active ppSPE fractions was used to tentatively identify two major compounds 5,3′,4′-trihydroxy-3,6,7,8-tetramethoxyflavone (C1) and hautriwaic acid (C2) and were subsequently purified using preparative high-performance liquid chromatography (prep-HPLC-MS) and confirmed by Nuclear Magnetic Resonance (NMR). At a concentration of 100 μM, they inhibited NO production by 96.6 % and 59.2 %, respectively. We have provided scientific preliminary evidence supporting ethnopharmacological claims of twelve South African medicinal plant species traditionally used to treat inflammatory diseases.