Pharmacognostic Standardization and Anti- Sickling Activities of Bioactive Compounds from Seeds of Picralima Nitida Stapf (Apocynaceae)

Abstract

This present study was to establish the pharmacognostic standards of the Picralima nitida, to investigate the anti-sickling effects of crude methanol seed extract and fractions of Picralima nitida, to isolate and elucidate the structure of the bioactive constituents. Phytochemical analysis of the extract, fractions and isolates were carried out using standard procedures. Pharmacognostical profile and proximate/numerical standards were also evaluated. The anti-sickling effect was studied using sodium methabisulphite-induced sickling of the HbSS red blood cells obtained from confirmed non-crisis state (steady state) sickle cell patients. The effects of P. nitida on biochemical parameters of HbSS blood were studied. The effect of the extract on the rheology and viscosity of sickle cell blood were studied using various concentrations (2.5, 5 and 10 mg/ml) at 30 minutes intervals through 180 minutes of incubation. Comparison of the anti-sickling effect of methanol seed extract of P. nitida, fractions and isolates on sickled red blood cells was done using a known standard drug, p-hydroxybenzoic acid. The microscopy of the powdered seeds revealed the presence of sclereids, parenchyma and epidermal cells, calcium oxalate crystal and fat globules. Qualitative and quantitative phytochemical analyses of the extract showed the presence of alkaloids (5.84 g), tannins (0.065), flavonoids (7.03 g), glycosides (4.58 g), terpenoids (4.08 g), protein (11.63 g), steroids, resins, reducing sugars, carbohydrates, fats and oils. The analytical standards gave 3.0, 11.5, 3.5, 4.0, 12.5, 20.4 and 8.0 % for moisture content, total ash, acid insoluble ash, water soluble ash, sulphated ash, alcohol soluble and water soluble extractives respectively. The acute toxicity test showed that the extract was safe at dose 3500 mg/kg. The study showed significant (p < 0.05) antisickling activities of the fractions on HbSS blood and were given to be in order CHCl3 ≤ CH2CL2 < EtOAc < Aqueous at the tested concentrations. The Aqueous Fraction showed highest degree of inhibition (85%) and was comparable to P-hydroxybenzoic acid (100%). The negative control (normal saline) showed no change, Compound 1 at 2.5 mg/ml showed the best anti-sickling activity (100% reversal of sickling at 120 min) compared to that of the standard drug p-hydroxybenzoic acid (100 % reversal of sickling at 180 min). The anti-sickling activity of the compounds are in the order CP1 (100 %) > CP3 (95 %) > CP2 (92 %) unsickling at 120 min. However compound 3 and compound 2 at 5 mg/ml showed 100 % reversal of sickling at 180 min.The isolated compounds are Ajmalicine → (19 α)-16, 17-didehydro – 19 – methyl – 2 –oxayohimbin – 16- carboxylic acid methyl ester, was obtained as white powder. Its molecular formula was derived as C21H24O3 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 353.1867. (1) And Ajmalicine Oxindole B → (19 α) -19-methyl -2-oxoformosanan -16- carboxylic acid methyl ester, was obtained as light yellow powder. Its molecular formula was derived as C21H24O4 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 369.1815. (2). The results of the study suggest that the methanol extract and fractions of P. nitida possess antisickling effect and justify’s the ethnomedicinal claims