PHYTOCHEMICAL STUDIES AND EVALUATION OF THE ANTITRYPANOSOMAL ACTIVITY OF VITEX SIMPLICIFOLIA OLIV. (VERBENACEAE) LEAF
ABSTRACT
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 ے . 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.
