EVALUATION OF THE MECHANISMS OF ANTINEUROINFLAMMATORY EFFECTS OF ETHANOL EXTRACT OF Moringa oleifera LAM (MORINGACEAE) LEAVES

Abstract

Neuroinflammation is the hallmark of neurodegenerative diseases which causes dementia and ataxia reducing the quality of life in the aged population. Conventional treatments are not very effective in targeting the underlying pathology of the diseases. Moringa oleifera (MO) has been used for centuries to treat a variety of diseases whose pathogenesis have lately been established to be inflammatory. The study was designed to evaluate the antineuroinflammatory mechanisms of ethanol extract of MO leaves (EMOL). Moringa oleifera obtained from a domestic garden at Ojoo lbadan was authenticated at Forest Herbarium lbadan with voucher number FHI 109601. Pulverized leaves (500g) of MO were extracted by maceration in 50% aqueous ethanol at room temperature. Twenty-five Swiss male mice (18.22 g) were allotted into 5 treatment groups (n=5): 5% Tween80 (10 mL/kg), EMOL (250, 500, 1000 and 2000 mg/kg) were used for central nervous system studies. In Lipopolysaccharide (LPS) cognitive deficit (LCD), thirty male mice were distributed into groups 1-5 (n=6) and treated orally for 7 days: 5% Tween80 (10 mL/kg), LPS and EMOL (100, 200, 400 mg/kg) before intraperitoneal administration of 250 ug/kg LPS to groups 2-5. The LCD was assessed by Y-maze test. The EMOL was partitioned into 20%, 50%, 80% and 100% methanol fractions (F20, F50, F80, and F100), respectively. Bioactivities of the fractions were evaluated using MTT and nitrite assays. The F50 was further purified to isolate compounds using HPLC, ¹H NMR and ¹³C NMR. Isolated compounds were screened by MTT assay in the presence of compounds on murine microglia (BV-2) and macrophages (RAW 264.7). Lipopolysaccharides was used to induce inflammation either in the presence and absence of various EMOL (100, 150 and 200 ug/mL), fractions (12.5, 25 and 50 ug/mL) and three compounds (12.5, 12.5 and 25uM) BV-2 cells. Nitric oxide (NO), cytokine (TNF-a) and PGE² production were evaluated in the supernatants using spectrophotometry and ELISA. Expression of cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS) and p38 proteins were determined using western blots. The effect of the isolated compounds on NF-xB transactivation was evaluated using luciferase reporter gene assay. Data were analysed using descriptive statistics and ANOVA at alpha 0.05. The EMOL (100-400 mg/kg) significantly increased % alternation in LCD (61.55±1.162, 59.68±1.948, 64.25±1.938) compared with LPS (49.13±1.225). The MTT assay revealed that EMOL, fractions (F20 and F50) and the compounds (kaempferol, quercetin and rutin), had no effect on viability of BV-2 and RAW 264.7 cells. The EMOL (130 and 200 ug/mL) and karmpferol (12.5 uM) significantly reduced NO (43.82±4.23, 38.68±12.71), (16.39±1.48); PGE₂ (45.05±1.30, 59.30±3.20), (51.73±1.48); and TNF-a (57.67±2.38. 60.43±8.07), (42.31±5.1) compared with LPS. Kaempferol, quercetin and rutin inhibited COX-2 and iNOs protein expressions in LPS stimulated BV-2 cells. Kaempferol, quercetin and rutin significantly reduced NF-xB transcriptional activation (40.49±10.01, 20.74±7.54, 41.68±8.32) in HEK 293 cells compared with TNF-a, and also significantly inhibited p-38 expression (58.06±18.17, 52.78±11.81, 26.86±3.96) in RAW 264.7 cells, respectively. The antineuroinflammatory effect of Moringa oleifera leaves was mediated via inhibition of p-38 protein expression, nuclear factor kappa-B transactivation and tumor necrosis factor-a release.