EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITIES OF EXTRACTS AND FLAVONOID-RICH FRACTION FROM Ocimum gratissimum LINN.(LAMIACEAE) LEAF

Abstract

Inflammatory responses are among causes of ill health, impaired quality of life and untimely death. Naturally occurring phenolics are sale phytochemicals that provides combinatorial multitargeted therapy in chronic inflammatory diseases. Ocimum gratissimum (Og) leaves due to its richness in phenolic compounds, have been used in managing inflammation-related diseases. This study was designed to evaluate the anti-inflammatory activity of extracts from Og and the mechanisms of its flavonoid-rich fraction.

Ocimum gratissimum leaves obtained from a domestic garden at lkoloba Ibadan was authenticated at Forest Herbarium Ibadan (FHI 110191). Powdered Og leaf was sequentially extracted in n-hexane (HE0g), chloroform (CE0g) and methanol (ME0g) by maceration. Anti-inflammatory activity of the extracts were screened in 25 female Wistar rats, divided into five groups (n=5) of control (1% tween 80), Indometttacin (10 mg/kg), HEOg, CEOg and MEOg at 400 mg/kg using carrageenan-induced paw oedema. The flavonoid-rich fraction (EAFOg) was obtained from MEOg by partitioning with ethylacctate, and flavonoids identified by High Performance Liquid Chromatography using authentic standards. Air pouch was induced for 6 days in 60 female Wistar rats randomly allotted into 12 treatment groups. Groups 1-6 were divided into saline, carrageenan, EAFOg (50, 100 mg/kg), SC58125 (1 mg/kg: COX-2 inhibitor) and indomethacin (5 mg/kg) while groups 7-12 were divided into saline, lipopolysaccharide, EAFOg (25, 50, 100 mg/kg), indomethacin (5 mg/kg). Groups 2-6 were challenged with carrageenan for 24 h and 8-12 with LPS for 6 h. Pouch exudates were measured and leucocytes counts determined by microscopy. Nitric Oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH) levels and activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) were determined spectrophotometrically. Tumour Necrosis Factor-alpha (TNF-α) and Prostaglandin E₂ (PGE₂) were determined by ELISA. In vitro inhibitory effect on NO, Interleukin (IL)-1 1β-10, TNF-α release were assayed in LPS stimulated RAW 264.7 cells. Inhibitory activity on cyclooxygenase-1/2 and 15-lipoxygenase enzymes were determined spectrophotometrically. Data were analysed using descriptive statistics and ANOVA at α0.05. The MEOg reduced oedema volume (0.54 ±0.09 mL) relative to control (1.37 ±0:15 mL). The HPLC revealed presence of rutin (1.131 ug/mg), ellagic acid (11.655 ug/mg), myricetin (6.535 ug/mg) and morin (0.073 ug/mg) in EAFOg. The EAFOg (50, 100 mg/kg) reduced exudates volume (1.67± 0.07, 0.77 ± 0.12 mL) and neutrophil counts (23.26 ± 3.32, 18.58 ± 0.74) compared to carrageenan-only (2.60 ± 0.06mL 62.09 ± 7.47), respectively. The EAFOg (25, 50, 100 mg/kg) significantly reduced neutrophil count (3.53 ± 0.28, 1.99 ± 0.34, 2.06 ± 0.29) and PGE₂ (223.20 ± 5.01, 187.90± 8.69, 165.20 ± 14.93 pg/mL) in LPS air pouch relative to LPS-only (4.45 ± 0.28; 242.20 ± 8.33 pg/mL), respectively. The ENFOg reduced TNT- α, NO, MPO, MDA but increased GSH and SOD compared to control. In vitro, EAFOg reduced NO, IL-Iβ, IL-10 and TNF- α release in LPS-stimulated RAW 264.7 cells and preferentially inhibited COX-2 (IC₅₀ =48.86) relative to COX- 1 and I5-LO (IC₅₀ >100). The flavonoid-rich fraction of Ocimum gratissimum leaves demonstrated potent anti inflammatory activities through mechanisms related to inhibition of neutrophil migration, pro-inflammatory cytokines, cyclooxygenase-2 and oxidative stress.