Isolation and Characterization of Antimalarial Compounds from Root Bark of Callichilia Stenopetala Stapf. (Family Apocynaceae)
A Thesis Submitted to the School of Postgraduate Studies, University of Lagos
Plants and their metabolic products have served as therapeutic weapons for treatment of various human and animal diseases. Callichilia stenopetala Stapf (family Apocynaceae) has been used by traditional practitioners in the South-eastern part of Nigeria as a remedy for the treatment of various ailments including different types of resistant malaria with their “re-current” fever. Malaria with emergence of its drug resistant parasites is a major health issue in the tropics and subtropical areas of the world. This study was aimed at evaluating in vivo antiplasmodial activities of methanolic extracts of some Nigerian medicinal plants. Antioxidant activity was included as a supporting factor, considering the relationship between oxidative stress and chronic diseases. Seven plants were initially evaluated for their antimalarial and antioxidant activities and the plant with the most promising activity was studied further. Phytochemical analysis and oral acute toxicity of methanolic extract of C. stenopetala was carried out using standard procedures. The methanolic extract of C. stenopetala was fractionated with water and organic solvents viz:- hexane, chloroform and ethyl acetate successively to obtain various fractions. Antiplasmodial activities of the crude extract and the obtained fractions of C. stenopetala were studied in detail against chloroquine sensitive Plasmodium berghei berghei NK 65 – infected mice using 4-day suppressive test procedure. The result was compared with some standard antimalarial agents, chloroquine phosphate and artesunate. An attempt was also made towards isolating and elucidating the structure(s) of constituent(s) thought to be responsible for the antimalarial activity of the plant. Bio-activity guided chromatographic isolation of two most active fractions – hexane and chloroform was done. The isolates obtained were subjected to spectral analysis for characterization using various spectrometric and chromatographic techniques: one-Dimensional (1D) and two-Dimensional (2D) NMR techniques, Attached Proton Test (APT), Correlation Spectroscopy (COSY), Nuclear Overhauser Effect Spectroscopy (NOESY), Heteronuclear Multiple Bond Correlation (HMBC), Heteronuclear Single Quantum Coherence (HSQC), Infra-red (IR), Gas Chromatography-Mass Spectroscopy (GC-MS) and X-ray crystallography. In vitro anti-oxidant activity of the crude extract, the obtained fractions and the pure isolates were also assessed using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content (TPC). The root bark of C. stenopetala was selected for the study based on its promising bioactivity. Results of its phytochemical analysis test showed presence of alkaloid, saponins, tannins, triterpenes and phenolic compounds. No mortality or evidence of adverse effects was observed in acute toxicity test. The crude extract, the hexane fraction and chloroform fraction demonstrated intrinsic antimalarial properties that were dose-dependent. A comparative analysis indicated that at dose 250 mg kg-1, the crude extract, hexane and the chloroform fractions produced 69.3, 82.24 and 39.9% suppression of parasitaemia respectively against 82.1% for chloroquine (5 mg kg-1) and 73.80% for artesunate (10 mg kg-1). Three active compounds, structurally elucidated as vobtusine, α-amyrin acetate and lupeol acetate were isolated and they exhibited significant (P< 0.05) chemosuppression at 7 – 10 mg kg-1 (62.2, 67.61 and 68.65% respectively) compared with the standard drugs chloroquine and artesunate. The antioxidant activities of the crude extract and the fractions were dose-dependent, and the effect resides mainly in the aqueous and ethyl acetate fractions. Only vobtusine exhibited a significant free radical scavenging effect (68.7%) at 0.2 mg mL-1. These results suggest that C. stenopetala root bark has antimalarial and antioxidant activities which may be due to the isolated compounds. This is the first report on antimalarial constituents of C. stenopetala, to the best of my knowledge.