title: Cytokine and eicosanoid modulation properties of southern African medicinal plants used traditionally to treat pain and inflammation
Creators: name: Khumalo, Gugulethu P
Publisher: name: Griffith University
Year: 2023
Subjects: subject: medicinal plants
subject: southern Africa
subject: cytokines
subject: inflammation
subject: pain

description: Selected southern African medicinal plants used traditionally to treat pain and inflammation were investigated for immunomodulatory and anti-inflammatory properties by evaluating their regulatory effects against cytokines, chemokines and lipid autacoids under two conditions. Firstly, cytokine levels in unstimulated RAW 264.7 cells were used to determine the basal cytokine release upon exposure to the plant extracts. The effects of the extracts and controls on cellular immunity were also examined by activating an inflammatory response in RAW 264.7 cells with lipopolysaccharide (LPS) under the same conditions. The effects on the release of anti-inflammatory cytokines (interleukin (IL)-2 and IL-10), the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, tumour necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and the chemokines monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein (MIP)-2 were determined using the cytokine multiplex-bead assays. Generally, the cells treated with ethanol extracts were more potent cytokine inhibitors compared to the aqueous extracts. The LPS-stimulated cells treated with the ethanol extracts of Erythrina lysistemon Hutch., Pterocelastrus rostratus Walp. Syzygium cordatum Hochst. ex Krauss and Warburgia salutaris (G. Bertol.) Chiov., demonstrated significant inhibition (p&lt;0.005) of &gt;85% of IL-1β, IL-6 and TNF-α secretion. The aqueous bark extracts of P. rostratus and S. cordatum also significantly decreased the release of all of the tested cytokines and chemokines. Of the leaf extracts tested, the ethanol extracts of Melianthus comosus Vahl, Tetradenia riparia (Hochst.) Codd and W. salutaris, demonstrated the greatest inhibitory activity, each inducing 50-fold inhibition of IL-1β, IL-6 and TNF-α levels in LPS-stimulated RAW 264.7 macrophages. The aqueous extract of M. comosus also significantly inhibited the secretion of all the tested pro- inflammatory cytokines and chemokines. The RAW 264.7 cells treated with plant extracts in the absence of LPS demonstrated significant upregulation of anti-inflammatory cytokines. The aqueous bark extract of E. lysistemon, as well as the leaves of W. salutaris and Zantedeschia albomaculata (Hook.) Baill., were the strongest stimulators of the secretion of the anti- inflammatory cytokines IL-10 and IL-2 in RAW 264.7 cells in the absence of LPS stimulation. The bark and leaf extracts of the medicinal plants were also evaluated for modulatory effects against some key lipid mediators of the arachidonic acid metabolism in LPS-induced RAW 264.7 cells. The inhibitory properties of aqueous and ethanol extracts on the release of prostaglandin E2 (PGE2), leukotriene-B4 (LTB4), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB) in LPS-activated macrophages were determined using ELISA assays. All of the ethanol extracts significantly down-regulated cytosolic COX-2 enzyme levels, with the highest activity noted from the leaves of Mentha longifolia (L.) Huds., T. riparia, and W. salutaris, each demonstrating over 20-fold inhibition of cytosolic COX-2 levels. Furthermore, the leaf extracts of M. longifolia, T. riparia, Terminalia sericea Burch. ex DC., and Z. albomaculata, substantially decreased cytosolic NF-κB levels. The bark extracts also substantially down-regulated cytosolic COX-2 and NF-κB levels. The ethanol bark extract of Ocotea bullata (Burch.) Baill., demonstrated the greatest NF-κB inhibitory activity, with up to a 20-fold decrease seen in LPS-induced RAW 264.7 cells. The ethanol bark extract of Warburgia salutaris (Bertol.f.) Chiov., exhibited the greatest inhibition of COX-2 levels. Chemical characterisation of the ethanol bark extract of S. cordatum led to the isolation and identification of four bioactive ellagic acid derivatives: ellagic acid 4-O-α-rhamnopyranoside (1), ellagic acid 4-O-α-4''-acetylrhamnopyranoside (2), 3-O-methylellagic acid 4'-O-α-3''-O- acetylrhamnopyranoside (3) and 3-O-methylellagic acid 4'-O-α-4''-O-acetylrhamnopyranoside (4). Their structures were confirmed by NMR spectroscopy, mass spectrometry, and comparison with published data. In conclusion, southern African medicinal plants demonstrate significant anti-inflammatory and immunomodulatory properties by significantly decreasing the levels of pro-inflammatory cytokine, chemokines, and lipid mediators, whilst substantially up regulating the levels of anti-inflammatory cytokines in RAW 264.7 cells. The MTS assay was used to determine the cell viability and 50% cytotoxic concentration (IC50) in RAW 264.7 and human dermal fibroblast (HDF) cell lines. The aqueous extracts showed minimal cytotoxicity at the highest concentration screened (5mg/mL). The studies presented herein highlight the potential of selected southern African plants to alleviate inflammation and pain. Further studies are required to comprehensively understand the immunomodulatory and anti- inflammatory mechanisms of these plants. Notably, most of the medicinal plant extracts tested in this thesis were evaluated for the first time in anti-inflammatory assays, despite being widely used in southern African traditional medicine.