Project Abstract

Phytochemical Studies and Evaluation of the Antitrypanosomal Activity of Vitex simplicifolia Oliv. (Verbenaceae) Leaf Abstract
Trypanosomiasis, caused by protozoan parasites of the genus Trypanosoma, remains a significant health concern in many parts of sub-Saharan Africa. The limited efficacy and toxicity of existing drugs have necessitated the search for alternative treatments from natural sources. Vitex simplicifolia is a plant traditionally used in African herbal medicine for various ailments. This study aimed to investigate the phytochemical composition of Vitex simplicifolia leaf extract and evaluate its antitrypanosomal activity. The phytochemical analysis of the leaf extract revealed the presence of alkaloids, flavonoids, tannins, saponins, and terpenoids. These phytoconstituents are known for their diverse biological activities, including antimicrobial and antiparasitic properties. The antitrypanosomal activity of the leaf extract was evaluated against Trypanosoma brucei brucei, the causative agent of animal African trypanosomiasis. The in vitro assays showed significant dose-dependent inhibition of trypanosomal growth by the leaf extract. Further investigations into the mechanism of action revealed that the Vitex simplicifolia leaf extract exhibited trypanocidal effects by inducing structural damage to the parasites' cell membrane and interfering with their metabolic processes. This dual mode of action suggests the potential of the extract as a promising antitrypanosomal agent. The cytotoxicity of the leaf extract was also assessed using mammalian cells, and the results indicated a selective toxicity profile against the trypanosomes with minimal impact on the host cells. This selective toxicity is crucial for the development of safe and effective antitrypanosomal agents. Overall, this study demonstrates the potential of Vitex simplicifolia leaf extract as a source of antitrypanosomal compounds. The presence of bioactive phytochemicals and the observed antitrypanosomal activity warrant further investigation to isolate and characterize the active compounds responsible for the therapeutic effects. Future studies should focus on optimizing extraction methods, elucidating the mechanism of action at the molecular level, and conducting in vivo studies to evaluate the efficacy of the leaf extract as a potential treatment for trypanosomiasis.

Project Overview

Background

Trypanosomiasis, a disease of major importance in human and animals has continued to threaten human health and economic development. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense as the etiological agents of trypanosomiasis affect millions of people in sub-saharan Africa and are responsible for the death of about half a million patients per year. Another name for the human form of the disease is sleeping sickness while that of cattle is nagana. The World Health Organization reported that 70-90% of the world’s population relies on the use of plant extracts or their active constituents. Many plants have therefore become sources of important drugs. There has been several claims by the traditional medical practitioners that Vitex simplicifolia Oliv. cures trypanosomiasis. This informed the reason for investigating the plant.

Method

The dried leaves (500 g) of Vitex simplicifolia were macerated with 3.0 L of 100 % methanol and extracted at room temperature for 24 h. with agitation. The resulting methanol was removed by rotary evaporation at 40 ºC under reduced pressure. The crude methanol extract (13.34 g, 2.668 %) was dissolved in 300 ml of 10 % methanol in water and the resulting mixture (i.e., the aqueous layer) partitioned with 3.0 L n-hexane (6 x 500 ml), 3.0 L of Dichloromethane( DCM )(6 x 500 ml), ethyl acetate (6 x 500 ml) and 1.0 L n-butanol (2 x 500 ml) using separating funnel to obtain n-hexane (HF, 1.06g, 7.95 %), DCM (2.98 g, 22.34 %), ethyl acetate (EF, 1.08 g, 8.10 %), n-butanol (BF, 5.75 g, 43.10%) and water (WF, 1.69 g, 12.67 %) fractions respectively. The DCM fraction (2.98 g) was subjected to vacuum liquid chromatography (VLC) using the following mixtures DCM: MeOH (9:1), DCM: MeOH (7:3), DCM: MeOH (1:1), DCM: MeOH (3:7), DCM: MeOH (1:9), MeOH 100%. The DCM : MeOH (7:3) yielded 49.5 mg and it was further purified using semi-preparative high pressure liquid chromatography (HPLC) to obtain 2.2 mg of the isolate which was code named DCM1. Phytochemical analysis was done using standard methods. Both in vivo and in vitro assay were carried out. Statistical analysis was also done and the results were expressed as mean ±SD using student’s t-test. The difference between the treated group and the control group is significant at P 0 Û’ . 05. Acute toxicity (LD50) of the methanol extract was estimated (p.o) in swiss albino mice weighing between 20-30 g using a standard method. The difference within means was analyzed using the one –way ANOVA.

Results

The phytochemical analysis revealed the presence of mainly alkaloids, flavonoids, steroids and protein. The acute toxicity result showed that the (LD50) was above 5000 mg/kg. The results of the parasitology testing revealed that the bioactive compound showed activity during the in vivo and in vitro assay. Ultra violet (UV) and nuclear magnetic resonance (NMR) analysis were done and the spectra data obtained show similarity with literature data.