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.
Conclusion
Vitex simplicifolia has anti trypanosomal activity. The bioactive compound (DCM1) is either a steroid or a flavonoid.
DISCUSSION AND SUMMARY
The acute toxicity test carried out with the crude extract of the
plant showed that no lethality was observed in the mice upon oral
administration, even doses as high as 5000 mg/kg, signifying that the
extract was relatively safe [80]. The observed parasitological relief of
the animals during the in vivo test explains the antitrypanosomal
potentials of the plant. This is because the control groups that were
infected and not treated died few days after infection. The reduction in
parasitaemia was dose dependent since there were more reduction in
parasitaemia at higher doses. The three parameters monitored in the in
vivo test showed significant improvement on administration of crude
extract/fractions thereby substantiating the antitrypanosomal potentials
of the plant. The death of one animal in group B when 200 mg/kg body
weight of ME was administered could be attributed to either toxicity or
high susceptibility of the animal to the infection.
However, two animals in the same group attained complete clearance
with the same dose level. The drop in parastaemia level on
administration of DCM and B fractions when compared with almost zero
effect of the other three fractions confers activity on the two as shown
in fig.11. The effects of extract/fraction on body weight of the
treated animals showed that animals treated with 400, 200 mg/kg ME and
100 mg/kg, BF and DCMF of the plant extract on the average maintained
their body weights post treatment while those treated with 100 mg/kg,
WF, EF and HF showed reduced body weights. This ascribes
antitrypanosomal activity on both BF and DCMF as shown in the graph
(fig. 12). The animals in the negative control lost a lot of body weight
and survived only for 30 days. The packed cell volume (PCV) analysis
result was consistent with observations made on parasitaemia. Animals
treated with 100-400 mg/kg ME was on the average above 43% which was
within the reference values 42-52 for males. Those treated with 100
mg/kg DCMF was 42% while those treated with 100mg/kg, EF and WF fell
below reference values. This again confers activity on DCM fraction. But
generally, extract/fraction had no pronounced effect on PCV.
The IC50 value of DCM 1 10.12 μg/ml when compared with the commercial
drug, melarsoprol against trypanosoma brucei rhodesiense and 46.05
μg/ml against typanosoma cruzi is appreciable [83]. Both the methanolic
extract and fractions were found to be effective against the resistant
strain of Trypanosoma brucei brucei in vivo and Trypanosoma brucei
rhodesiense in vitro. Cytotoxicity for L6 mammalian cell is greater than
100 (Table 9). This implies that is a bit toxic. A future comprehensive
work on the structure – activity relationship on DCM 1 may take care of
the toxicity and also increase activity.