PHYTOCHEMICAL AND ANTI SNAKE VENOM STUDIES ON THE METHANOL AERIAL PART EXTRACT OF TACAZZEA APICULATA OLIV (PERIPLOCACEAE)

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Department of Pharmaceutical Sciences

ABSTRACT
Tacazzea apiculata (Olive) is a woody climber indigenous to Tropical Africa often used in traditional medicine for the treatment of pain, inflammation, skin infections, diabetes, epilepsy and snakebite. This study aimed to isolate and characterize some of the phytochemical constituent(s) present in aerial part extract of T. apiculata and to scientifically validate the ethno medicinal claim of the use of the plant in the management of snakebite envenomation. The powdered aerial part of T. apiculata was extracted with methanol using maceration method which afforded methanol aerial part extract (MAE). Portion of this was treated with n-hexane, chloroform and ethyl acetate to obtain n-hexane (HEF), chloroform (CHF) and ethyl acetate fractions (EAF). Preliminary phytochemical screening of the methanol extract revealed the presence of secondary metabolites including alkaloids, flavonoids, saponins, tannins, and steroids/triterpenes. Silica gel chromatographic separation of chloroform fraction (CHF) afforded total of eleven major fractions C1 – C11, fractions C2 and C5 were subjected to further silica gel chromatographic separation which resulted to C2A and C5C sub fractions respectively. Repeated purification of C2A using silica gel packed column (micro pipette) eluted with n-hexane 100 % isocratic led to the isolation of a brown oily compound Tam 4. Similarly, repeated purification of C5C using silica gel packed column (micro pipette) eluted with n-hexane: ethyl acetate (8:2) isocratic resulted in the isolation of a white powdered compound Tam 2. The structures of the compounds were elucidated using physico-chemical tests and spectroscopic techniques (IR, 1D and 2D NMR) and by comparison with reference spectral data. The oral and intraperitoneal route LD50 of the methanol aerial part extract (MAE) in mice were estimated to be 5000 and 894 mg/kg, respectively. The LD99 of the Naja nigricollis venom was estimated to be 4.6 mg/kg. Antivenin studies suggest that methanol extract possess significant antivenin activity against Naja nigricollis venom both ex-vivo and in-vivo with maximum protections level of 100 % survival at 80 mg/kg dose and 16.67 % survival at 2000 mg/kg dose respectively. Spectral analysis of both Tam 4 and Tam 2 by way of NMR showed Tam 4 to be Squalene and Tam 2 β-sitosterol. The structures were confirmed with the literature data and methanol extract demonstrated significant antivenin activity and lends credence to traditional use of the plant in the management of snakebite.