PHYTOCHEMICAL STUDIES AND EVALUATION OF THE ANTITRYPANOSOMAL ACTIVITY OF VITEX SIMPLICIFOLIA OLIV. (VERBENACEAE) LEAF
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CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF STUDY
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.
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