The College of Pharmacy discussed the MSc thesis entitled “Phytochemical Investigation & Evaluation of the Cytotoxic Effect of Clitoria ternatea (Fabaceae) Cultivated in Iraq” by the student Duha Nabhan Saleh and the supervisor, Assist. Prof. Dr. Amjed Haseeb Khamees at the Pharmacognosy and Medicinal Plant Department.
The study aimed to investigate the phytochemicals of Clitorea ternatea L. cultivated in Iraq and to evaluate the cytotoxic effect of the methanolic extract on glioblastoma (AMGM5) cells relative to normal human derived fibroblasts (NHF), since there was no previous study concerning this cultivated Iraqi species.
The study included defatting the plant material by maceration using hexane. Subsequently, the plant material was extracted using a Soxhlet apparatus with 85% methanol. The obtained extract was then fractionated using solvents of different polarities, including petroleum ether, chloroform, and ethyl acetate. Various bioactive constituents present in these fractions were identified through preliminary phytochemical tests, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). In addition, gas chromatography–mass spectrometry (GC–MS) was employed to analyze the hexane fraction. Four pure compounds were isolated using preparative high-performance liquid chromatography (preparative HPLC), while a new compound was isolated using preparative thin-layer chromatography (preparative TLC) and a CombiFlash chromatography system. The isolated compounds were further characterized using several analytical techniques, including thin-layer chromatography, purity analysis by high-performance liquid chromatography, infrared spectroscopy (IR), liquid chromatography-mass spectrometry (LC-MS), and proton nuclear magnetic resonance spectroscopy (^1H-NMR). Furthermore, the cytotoxic activity of the crude extract was evaluated using a colorimetric cell proliferation assay on glioblastoma cells and normal human fibroblast cells. The cells were treated with different concentrations of the extract (1000, 500, 250, 125, 62.5, and 31.75 µg/mL) for 72 hours, after which cell viability was assessed.
The results showed the presence of the following compounds: Stigmasterol, ursolic acid, lupeol, para-coumaric acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, catechin, apigenin, theophylline, scoparone, and 7α-voacangine hydroxyindolenine. The compounds ursolic acid, lupeol, para-coumaric acid, and theophylline compounds were isolated by several runs of HPLC analysis, while an unknown coumarin was separated by preparative layer chromatography (PLC), and the second unknown, which is an indole-type alkaloid, was isolated using CombiFlash chromatography. The crude methanolic extract showed concentration-dependent cytotoxic impact on AMGM5 cells, with an IC₅₀ value of about 621.8 µg/mL. NHF cells maintained > 80% viability at all tested dosages, with no notable cytotoxicity below 500 µg/mL.
The study recommended that Iraqi Clitorea ternarea be a rich natural source of phytochemicals with potential use in pharmacology and pharmaceutical research.
