Abstract
Clinacanthus nutans has had a long history of use in folk medicine in Malaysia and Southeast Asia; mostly in the relief of inflammatory conditions. In this study, we investigated the effects of different extracts of C. nutans upon lipopolysaccharide (LPS) induced inflammation in order to identify its mechanism of action. Extracts of leaves and stem bark of C. nutans were prepared using polar and non-polar solvents to produce four extracts, namely polar leaf extract (LP), non-polar leaf extract (LN), polar stem extract (SP), and non-polar stem extracts (SN). The extracts were standardized by determining its total phenolic and total flavonoid contents. Its anti-inflammatory effects were assessed on LPS induced nitrite release in RAW264.7 macrophages and Toll-like receptor (TLR-4) activation in TLR-4 transfected human embryonic kidney cells (HEK-BlueTM-hTLR4 cells). The levels of inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-12p40, and IL-17) in treated RAW264.7 macrophages were quantified to verify its anti-inflammatory effects. Western blotting was used to investigate the effect of the most potent extract (LP) on TLR-4 related inflammatory proteins (p65, p38, ERK, JNK, IRF3) in RAW264.7 macrophages. All four extracts produced a significant, concentration-dependent reduction in LPS-stimulated nitric oxide, LPS-induced TLR-4 activation in HEK-BlueTM-hTLR4 cells and LPS-stimulated cytokines production in RAW264.7 macrophages. The most potent extract, LP, also inhibited all LPS-induced TLR-4 inflammatory proteins. These results provide a basis for understanding the mechanisms underlying the previously demonstrated anti-inflammatory activity of C. nutans extracts.
Highlights
Clinacanthus nutans (‘Belalai Gajah’ in Malay, or ‘You Dun Cao’ in Mandarin) has long been used in Thailand to improve bladder function and has been used traditionally in Malaysia as a folk medicine for kidney and bladder disease (Low et al, 2011)
In order to examine the anti-inflammatory effects of C. nutans, we investigated its effects on human embryonic kidney (HEK) cells stably transfected with human TLR (HEK-BlueTM-4) and in murine macrophages (RAW264.7) challenged with LPS, the Gram-negative bacterial cell wall component, a well-established activator of Toll-like receptor-4 (TLR-4) (Mai et al, 2013)
The viability of HEK cells (HEK-BlueTM-hTLR4) and murine macrophages (RAW 264.7) were not significantly reduced by any of the extracts compared to cells treated with 0.1% Dimethyl sulfoxide (DMSO) (Figures 1A,B)
Summary
Clinacanthus nutans (‘Belalai Gajah’ in Malay, or ‘You Dun Cao’ in Mandarin) has long been used in Thailand to improve bladder function and has been used traditionally in Malaysia as a folk medicine for kidney and bladder disease (Low et al, 2011). C. nutans have been consumed as crude extract of the leaves or stem bark. The leaf extracts of C. nutans has been reported to possess antioxidant (Khoo et al, 2015), analgesic and anti-inflammatory activities against varicella zoster virus (Thawaranantha et al, 1992) as well as inhibitory activity against scorpion venom-induced fibroblast lysis (Uawonggul et al, 2006). Kittisiripornkul (1984) reported the anti-inflammatory activity of a n-butanol-soluble fraction from the leaves (Kittisiripornkul, 1984) and a methanol extract of the whole plant reduced carrageenan-induced paw oedema and ethyl phenylpropiolateinduced ear oedema in rats (Wanikiat et al, 2008). The exact cellular mechanisms underlying the anti-inflammatory actions of C. nutans extracts remain unknown
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