Phytochemical Studies And Evaluation Of The Antitrypanosomal Activity Of Vitex Simplicifolia Oliv. (verbenaceae) Leaf

PHYTOCHEMICAL STUDIES AND EVALUATION OF THE ANTITRYPANOSOMAL ACTIVITY OF VITEX SIMPLICIFOLIA OLIV. (Verbenaceae) LEAF

BSTRACT
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.
x
xi
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.

CHAPTER ONE
1.0 INTRODUCTION
Nigeria’s biodiversity is rich in medicinal plants. The World Health Organization
(WHO) reported that 70─90 % of the world’s population relies chiefly on
traditional medicine and a major part of the traditional therapies involve the use
of plant extracts or their active constituents. Many plants have therefore become
sources of important drugs and as such the pharmaceutical industries have
exploited traditional medicine as a source of bioactive agents that can be used in
the preparation of synthetic medicines. Natural products play important roles in
drug discovery and development process, particularly in the field of infectious
diseases, where 75 % of these drugs are of natural origin.
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 rhodensiense 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. In Africa where
trypanosomiasis is endemic, plants have been used for generations. Natural
products derived from them offer novel possibilities to obtain new drugs that are
active against trypanosomes. The disease is caused by flagellate parasites –
protozoa belonging to the genus trypanosome and family trypanosidae.
2
1.1Human African Trypanosomiasis
African trypanosomiasis or sleeping sickness is a parasitic disease of humans and
other animals. [1] It is caused by protozoa of the species Trypanosoma brucei [2].
There are two types that infect humans; Trypanosoma brucei gambiense (T.b.g.)
and Typanosma brucei rhodesiense (T.b.r). T.b.g is usually transmitted by the bite
of an infected tse tse fly and is most common in rural areas. Initially, in the first
stage of the disease, there are fevers, headaches, itchiness, and joint pains [1].
This begins one to three weeks after the bite [3]. Weeks to months later the
second stage begins with confusion, poor co-ordination, numbness and trouble
sleeping [1, 3].
Diagnosis is via finding the parasite in a blood smear or in the fluid of a lymph
node [3]. A lumber puncture is often needed to tell the differences between first
and second stage disease.
History of Discovery
Although the symptoms of African sleeping sickness were documented by Atkins
in 1742, the association of the clinical syndrome with its etiological agent, the
trypanosome, was not documented until 1902 by Forde [4]. In the School of
tropical medicine, Forde chronicled his treatment of a 42 year –old European
male colonialist who presented to his practice in the Gambia colony in May 1901.
The patient complained of fever and malaise, bading Forde to make a preliminary
diagnosis of malaria. He initiated anti-malaria treatment, but days later the
patient’s condition had yet to improve. Slides of the patient blood were prepared.
3
This examination ruled out malaria due to lack of malaria parasites found in the
blood. Only later, Dutton a second physician from the Liverpool School of
Tropical Medicine, made the identification of Trypanosoma brucei in the patients
blood . Due to the probable location of the patient’s inoculation, this case can be
attributed to the species T.b gambiense.
The identification of T.b rhodesiense as another species of trypanosome to cause
African sleeping sickness was not documented until 1910. Stephens and Fantham
describe a strain of trypanosome observed in a blood smear of a patient who
presented with symptoms of African trypansomiasis. The patient had no history
of travel within a region known to be endemic with T.b brucei, yet his blood
smear clearly indicated trypanosome infection. The novel morphology was
believed to be a new species of T. brucei. Because the patient was believed to
have been infected in Rhodesia (present day Zimbabwe), the new parasite was
thus named- T. b rhodesiense [5].
Experiments published in 1912 by Kinghorn and Yorke proved that T.b
rhodesiense could be transmitted from human to animals by tsetse fly. They also
concluded through their research that many game animals in East Africa,
including water buck, hartebeest, impala, and warhog, served as reservoirs for T.b
rhodesiese in this region of the continent [6].