Immunomodulatory activity of Moringa oleifera L. leaf ethanol extract on normal lymphocytes and leukaemic cell lines

Moringa oleifera (M. oleifera), a member of the family Moringaceae, is a smallmedium sized tree, 10-15m high, widely cultivated in East and Southeast Asia, West Indies and Polynesia. Indians have been using leaves, fruits and flowers of M. oleifera as part of their routine diet as this 'wonder' herb also was used in ancient Ayurveda and Siddha medicine for nearly 2000 years. Phytochemical and animal studies have shown that the therapeutic activities of M. oleifera have largely depended on its main polyphenols such as quercetin glucosides, kaempferol glycosides, rutin, and chlorogenic acid. To date, M. oleifera has been studied for their anti-oxidative, antidiabetic, anti-inflammatory and anticancer activity, yet their role in modulating the immune system is still elusive. The present study, therefore, aimed to investigate the in vitro immunomodulatory effect of M. oleifera leaves' ethanol extracts (MOETE) on healthy peripheral blood mononuclear cells (PBMCs) and leukaemic cell lines. Fresh and healthy leaves of M. oleifera were collected from an herbal farm located at Kampar, Selangor. Extracts of M. oleifera leaves were obtained using the mixture of ethanol and water at ratios of 100:0 (100% ethanol), 70:30 (70% ethanol), 50:50 (50% ethanol) and 0:100 (aqueous) as extraction solvents. Healthy donors were used as a source of primary lymphocytes while Jurkat and BV173 cells were utilised as transformed cell lines of T cells and B cells, respectively. The cytotoxicity of aqueous and ethanolic extracts of M. oleifera leaves on the cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay while the marker compounds (quercetin, and kaempferol 3-O-glucoside) in M. oleifera extract were identified and quantified using High Performance Liquid Chromatography (HPLC). The immunomodulatory effect was evaluated through cell proliferation assays, cell cycle analysis and apoptosis assays. The antitumour potential of the extract on Jurkat and BV173 cells was further explored via global secretome and apoptotic proteins proteome arrays. From the cytotoxicity analysis, 70% ethanol M. oleifera leaves extract exerted a dose-dependent stimulatory effect on PBMCs with an EC50 of 28±3 g/mL as well as cytotoxic effects on BV173 (IC50 = 125±6 g/mL) and Jurkat cells (IC50 = 262±3 g/mL). Also, from the HPLC analysis, kaempferol 3-O-glucoside (standard Rt = 32.689 vs sample mean Rt = 32.671), and quercetin (standard Rt = 42.020 vs sample mean Rt = 41.981) were identified. The extract enhanced the viability and proliferation of PBMCs by committing the cells into the cell cycle and reducing apoptosis while exerting anti-proliferative effects, cell cycle arrest and apoptosis in tumour cell lines. Also, the extract induced overexpression of pro-apoptotic cytokines and proteins but suppressed the expression of angiogenic factors and prosurvival proteins in tumour cell lines. Pathway enrichment analysis revealed that MOETE induced apoptosis in BV173 and Jurkat cells mainly through activation of the mitochondrial apoptotic pathway by upregulation of mitochondrial pro-apoptotic B cell lymphoma 2 (BCL2) family of proteins like the BCL-2-associated X protein (BAX) and the BCL-2 homologous antagonist killer (BAK) while downregulation of the anti-apoptotic protein, BCL-2. M. oleifera ethanol extract has immunostimulatory properties on normal lymphocytes and antitumour activity on leukemic cell lines. These abilities can be exploited in developing herbal supplements that strengthen the immune system to support aged and immunocompromised individuals as well as serve as adjuvants in therapies against blood cancers.

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