IDOKO EMMANUEL EMEKA PG/M.Sc/11/59494

PHYTOCHEMICAL STUDIES AND EVALUATION OF THE ANTITRYPANOSOMAL ACTIVITY OF

VITEX SIMPLICIFOLIA OLIV. (Verbenaceae) LEAF

FACULTY OF PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY

Ebere Omeje Digitally Signed by: Content manager’s Name DN : CN = Webmaster’s name O= University of Nigeria, Nsukka OU = Innovation Centre

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PHYTOCHEMICAL STUDIES AND EVALUATION OF THE ANTITRYPANOSOMAL ACTIVITY OF VITEX SIMPLICIFOLIA OLIV.

(Verbenaceae) LEAF

BY

IDOKO EMMANUEL EMEKA PG/M.Sc/11/59494

DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY

FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA

SEPTEMBER, 2015

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TITLE PAGE PHYTOCHEMICAL STUDIES AND EVALUATION OF THE

ANTI TRYPANOSOMAL ACTIVITY OF VITEX SIMPLICIFOLIA OLIV. (Verbenaceae) LEAF

BY

IDOKO EMMANUEL EMEKA PG/M.Sc/11/59494

A THESIS PRESENTED TO THE DEPARTMENT OF PHARMACEUTICAL AND MEDICINAL CHEMISTRY, FACULTY OF PHARMACEUTICAL SCIENCES OF THE UNIVERSITY OF NIGERI A,

NSUKKA IN PARTIAL FULFILLMENT FOR THE AWARD OF MASTERS DEGREE IN PHARMACEUTICAL AND MEDICINAL

CHEMISTRY.

SUPERVISOR(S) DR. (MRS) N. J. NWODO

DR.W.O. OBONGA

SEPTEMBER, 2015.

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CERTIFICATION

This is to certify that IDOKO, EMMANUEL EMEKA, a post graduate student in

the Department of Pharmaceutical and Medicinal Chemistry, with registration

number: PG/M.Sc/11/59494 has satisfactorily completed the requirements for the

award of Masters degree in Pharmaceutical and Medicinal Chemistry. The work

embodied in this project is original and has not been submitted in part or full for

any other diploma or degree of this or any other university.

DR. (MRS). N.J. NWODO PROF.C.J. MBAH (HEAD OF DEPARTMENT)

DR. W.O OBONGA

(SUPERVISOR(S)

EXTERNAL EXAMINER

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DEDICATION

Dedicated to the memory of my late father.

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ACKNOWLEDGEMENT

I wish to express my sincere gratitude to my supervisor, Dr. (Mrs) N. J. Nwodo

for her dedication and encouragement through out this work.

My gratitude also goes to my co-supervisor, Dr. W. O. Obonga for his assistance

during the research.

I am also grateful to Prof. C. J. Mbah for his contribution. My unreserved

appreciation goes to both Prof. Peter Proksch and Prof. Reto Brun of the Swiss

Tropical and Public Health Institute, Switzerland for the laboratory analysis they

carried out during the research.

My special thanks go to Dr. M. O. Agbo, Pharm. Philip Uzor and Pharm. Charles

Nnadi for the enormous contributions they made during the research.

My special thanks also go to my classmates and all those (not mentioned) who

contributed either spiritually or physically to the success of my work.

Idoko, E. E.

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TABLE OF CONTENT

TITLEPAGE - - - - - - - - - i

CERTIFICATION - - - - - - - - - ii

DEDICATION -- - - - - - - - - iii

ACKNOWLEDGEMENT - - - - - - - iv

TABLE OF CONTENT - - - - - - - - v

LIST OF FIGURES - - - - - - - - viii

LIST OF TABLES - - - - - - - - - ix

ABSTRACT - - - - - - - - - x

1.0 INTRODUCTION - - - - - - - - 1

1.1. Human African Trypanosomiasis - - - - - 2

1.1.1 Epidemiology of Human Trypanosomiasis - - - - 4

1.1.2 Pathogenicity of Trypanosomiasis - - - - - 4

1.1.3 Diagnosis of Human Trypanosomiasis - - - - - 6

1.1.4 Geographical Distribution of Trypanosomiasis - - - 7

1.1.5 Morphology and Characteristics - - - - - 8

1.1.6 Taxonomy of Trypanosomes - - - - - - 9

1.1.7 Life Cycle of Trypanosomes in the Host - - - - 10

1.1.8 Vectors of Trypanosomes - - - - - - 11

1.1.9 Treatments of Human Trypanosomiasis - - - - 14

1.2 The Use of plants in Phytomedicine - - - - - 20

1.2.1 Taxonomy of the genus Vitex - - - - - - 23

1.2.2 The plant: (Vitex simplicifolia Oliv.) - - - - - 25

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1.2.3 Ethnomedicinal uses of Vitex simplicifolia Oliv - - - 26

1.2.4 Geographical Distribution of Vitex simplicifolia Oliv - - 29

1.2.5 Table 1: Previous antitrypanosomal studies on medicinal plants - 30

1.2.6 Aims and Objectives of the Study - - - - - 34

2.1 Materials - - - - - - - - - 35

2.1.1Chemicals and Reagents - -- - - - - - 35

2.1.2 Equipment - - - - - - - - - 35

2.1.3 Plant Material - - - - - - - - 35

2.1.4 Experimental Animals - - - - - - - 35

2.2 Parasites - - - - - - - - - 36

2.2.1 Methods - - - - - - - - - 36

2.2.2 Extraction, Fractionation and Isolation Procedure - - - 36 2.2.3 Phytochemical Analysis - - - -- - - - 38

2.2.4 Acute Toxicity Test of the Crude Extract - - - - 38

2.2.5 In-vivo Anti-trypanosomal Activity - - - - - 38

2.2.6 In-vitro Anti-trypanosomal Activity Test - - - - 41

2.2.7 Statistical Analysis - - - - - - - - 42 3.1 Extraction/Fractionation Yield - - - - - - 43

3.1.1 Results of parasitology testing - - - - - - 45

3.1.2 Effects of Vitex simplicifolia Oliv. on Parasitaemia Level - - 45

3.1.3 Effects of Vitex simplicifolia Oliv. on Weight of Rats - - 47

3.2 Effects of Vitex simplicifolia Oliv. on Packed Cell Volume (PCV) - 50

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4.1 DISCUSSION - - - - - - - - - 57

4.2 CONCLUSION - - - - - - - - 58

REFERENCES - - - - - - - - 60

APPENDICES - - - - - - - - - 71

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LIST OF FIGURES

Figure 1: Life Cycle of African Trypanosomes - - - - 11

Figure 2: Chemical Structure of Suramin - - - - - 16

Figure 3: chemical Structure of Pentamidine - - - - - 16

Figure 4: Chemical Structure of Melarsoprol - - - - - 17

Figure 5: Chemical structure of Enantiomer R of Eflornithine and

S-Eflornithine - - - - - - - - 18

Figure 6: Chemical structures of caratuberside - - - - 19

Figure 7: Chemical structures of some compounds isolated from Vitex simplicifolia - - - - - - - - 20

Figure 8: The plant (Vitex simplicifolia Oliv.) - - - - 25

Figure 9: Flow Chart of the general separating procedure - - - 37

Figure 10:Shows chemical structure of bioactive isolate (DCM1) - 52

Figure 11: Effect of the extract/fraction of V.simplicifolia on the

parasitaemia level of trypanosomal-infected rats - - 54

Figure 12: Effects of the extract/fraction of V.simplicifolia on the

weight of trypanosomal-infected rats - - - - 55

Figure 13: Effect of the extract/fraction of V.simplicifolia on the PCV

of trypanosomal-infected rats - - - - - 56

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LIST OF TABLES

Table 1: Previous antitrypanosomal studies on medicinal plants- - - - 30

Table 2: Previous antitrypanosomal studies on Vitex simplicifolia Oliv.- -33

Table 3: Percentage Yield of Extracts/Fractions-- - - - - -43

Table 4: Phytoconstituents of methanolic extract (ME) - - - -44

Table 5: Shows physico chemical properties of the bioactive isolate

DCM 1 from the V.simplicifolia Oliv. DCM fraction- - - -44

Table 6: Comparative Response of Methanolic Extracts/Fractions to

Parasitaemia- - - - - - - - - - 47

Table 7: Effect of ME and Solvent Fractions of Vitex simplicifolia Oliv. On

Mean Weight of Treated Rats- - - - - - - 49

Table 8: Percentage Response of ME and Fractions to Packed

Cell Volume (PCV)- - - - - - - - -51

Table 9: Shows the results of in vitro antitrypanosomal assay of

the DCM 1 fraction- - - - - - - - -52

Table 10 showing chemical shift - - - - - - 53

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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 P05 .0 ے. 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.

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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.

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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.

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1.1 Human 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.

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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].

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1.1.1 Epidemiology of Human Trypanosomiasis

As of 2010 it caused around 9,000 deaths down from 34,000 in 1990 [7]. As of

2000, the disability- adjusted life year ( 9 to 10 years) cost due to sleeping

sickness are 2.0 million [8]. Over 60 million people living in some 250 locations

are at risk of contracting the disease and under 10,000 new cases were reported in

2009 [9].

The disease has been recorded as occurring in 37 countries, all in sub-saharan

Africa. It occurs regularly in South East Uganda and Western Kenya and killed

more than 48000 Africans in 2008 [10]; the population at risk being about 69

million with one third of this number being at a very high to moderate risk and

remaining two third at a “low” to “very low” risk [11].

1.1.2 Pathogenicity of Trypanosomiasis

In the usual scheme of classification of trypanosomes, Trypanosoma lewisi

occupies the position of the type of species of a number of non-pathogenic

trypanosomes [12]. While this usage is justifiable in the present state of our

knowledge of these organisms, one must not lose sight of the fact that there is

abundant evidence to show that Trypanosoma lewisi is not strictly non-

pathogenic, but occasionally manifests a decided virulence for rats, especially

young ones. Apart from such frequent disturbances as fever, anemia and loss of

weight, a considerable mortality may occur among infected rats. Perhaps the best

instance that can be cited is that reported by Jurgens [13] who noted a mortality

of 29.3 percent (16 out of 47) among young rats. Other authors have noted a

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slight mortality, or no mortality resulting from infections of Trypanosoma lewisi

[14].

These differences in pathogenicity indicate that there are strains of trypanosome

that differ fundamentally as regards their virulence. Delanoe [14] has added

support to this conception of pathogenic and non-pathogenic strains of

Tryanosoma lewisi by showing that while certain strains or organism from certain

sources, are incapable of infecting mice, and other strains may infect even a

considerable percentage (40 %) of the mice inoculated. Further, Roudsky[15] has

shown by his “reinforced virus” that the virulence of a given strain is not

absolutely fixed, but that it can be markedly increased for both rats and mice.

Finally, Wendelstadt and Fellmer[16] have succeeded in raising the virulence of

Trypanosoma lewisi by passage through cold-blooded animals.

These facts surfice to show the existence of strains of Trypanosoma lewisi and

indicate that possibly all strains possess potential pathogenic properties.

Unfortunately, the known facts regarding its pathogencity are too meager to

warrant any generalizations. Since the natural host of these organisms make it

peculiarly suited to laboratory study, further work upon this subject seems highly

desirable, as a clearer comprehension of the conditions governing its

pathogenicity would probably aid materially in advancing our knowledge of the

more important group of organisms, the pathogenic trypanosomes.

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1.1.3 Diagnosis of Human Trypanosomiasis

The diagnosis of African Trypanosomiasis is made through laboratory methods,

because the clinical features of infection are not sufficiently specific. The

diagnosis rests on finding the parasite in body fluid or tissue by microscopy. The

parasite load in T.b. rhodesiense infection is substantially higher than the level in

T.b gambiense infection. T.b rhodesiense parasite can easily be found in blood.

They can also be found in lymph node fluid or in fluid or biopsy of a chancre.

Serologic testing is not widely available and is not used in the diagnosis, since the

microscopic detection of the parasite is straight forward.

The classic method for diagnosing T.b gambiense infection is by microsopic

examination of lymph node aspirate, usually from a posterior node [17]. It is

often difficult to detect T.b gambiense in the blood. Concentration techniques and

serial examinations are frequently needed. Serologic testing is available outside

the U.S for T.b gambiense, however, it is normally used for screening purposes

only and the definitive diagnosis rests on microscopic observation of the parasite.

All patients diagnosed with African Trypanosomiasis must have their

cerebrospinal fluid examined to determine whether there is involvement of the

central nervous system, since the choice of treatment drug(s) will depend on the

disease stage. The World Health Organization criteria for central nervous system

involvement include increased protein in cerebrospinal fluid and a white cell

count of more than 5 [17, 18]. Trypanosomes can often be observed in

cerebrospinal fluid in persons with second stage infection.

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1.1.4 Geographical Distribution of Trypanosomiasis

The distribution of African trypanosomiasis is completely linked to the range of

the vector, the tsetse fly. Due to the tsetse fly’s climatic restrictions the disease is

restricted between the 14th latitude north and the 29th latitude south on the African

continent [17]. According to the WHO, countries where the disease is currently

epidermic include Angola, Democratic Republic of Congo, Cote d’Ivorie, Central

African Republic, Guinea, Mozambique, Tanzania and Chad. African sleeping

sickness can also be found in low endemic levels in Benin, Burkina Faso, Gabon,

Ghana, Equatorial Guniea, Kenya, Mali, Nigeria, Togo and Zambia. Because of

poor disease surveillance and reporting, epidemiological information in Burundi,

Botswana, Ethiopia, Liberia, Mamibia, Rwanda, Senegal and Sierra Leone is

poorly understood.

The disease is a threat to more than 60 million people throughout Africa.

However only 3 to 4 million of these people are under surveillance, leading to the

reporting of only 45,000 cases in 1999. Epidemiologists estimate that between

300,000 and 500,000 cases actually occurred during that same time period [17].

Surveillance is not only essential to track disease trends to determine possible

interventions but also to identify infected individual so that treatment may be

initiated before the disease progresses to less treatment state.

There have been three major epidemics in Africa in the last century, one between

1896 and 1906 in Uganda and the Congo basin. Another one is 1920 that

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incorporated several African countries; and finally, one that started in 1970 and is

still in progress across much of Africa [17].

1.1.5 Morphology and Characteristics

A sound knowledge of the basic features of the various trypanosomes enables the

identification of each species and so the exact cause of the disease. Once the basic

features possessed by all trypanosomes are appreciated, the diagnostic differences

can be recognized and the species identified.

Basic Morphology of Trypanosomes

The trypanosome (trypamastigote) consists of a single cell varying in size from 8

to over 50µm. All the activities associated with a living organism take place

within this unicellular organism- nutrition, respiration, excretion, reproduction.

The substance of which all living cells consist, the proptoplasm, coprises three

parts; an outer protective and retaning layer, the pellicle = cell envelope=cell

membrane; within which the cypoplasm forms the bulk of the contents.

Suspended in the cytoplasm are various structures, the most prominent being the

nucleus, which may be regarded as the command centre of the cell and which also

plays a major part in reproduction. It contains DNA (deoxyribonucleic acid),

which is arranged in form of genes and chromosomes; it represents the genetic

information and is responsible for the manufacture of enzymes and other proteins

of the cell. Small granules, (formerly called “volutin granules”) can sometimes be

seen in the cypoplasm; they may have various origin, they may be food or nuclear

9

reserves, or result from reaction between the trypanosomes and the host’s

immune system.

Specific Morphology

The sub-genus Nannomonas (T. congolense) is the smallest of the pathogenic

trypanosomes, with a length of 9-22µm. The stained blood smear of the above

sub genus show monomorphic forms in that they lack free flagellum. Generally,

two variants are to be seen, a shorter form (9-18µm) the typical congolense type

and a longer form (up to 25µm) with individuals intermediate in length between

the two. The proportion of long and short forms varies in different cases. There

is evidence which indicate that strains with the longer forms, the so called

“dimorphic” strains, cause a more servere form of trypanosomiasis.

1.1.6 Taxonomy of Trypanosomes

Taxonomical classification [19]

Kingdom – Protozoa

Phylum – Sarcomastigoohora

Sub phylum – Mastigoohora

Class – Zoomastigoohora

Order - Kineplastida

Family - Trypanosomatidae

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Section – Salivaria

Genus – Trypanosoma

Species – Brucei

Subspecies - Gambiense, Rhodesiense

Trypanosoma brucei subspecies: African sleeping sickness

Trypanosoma Cruzi: Chagas Disease

Plasmodium Species: Malaria

SchistosomaSpecies: Blood Flukes

1.1.7 Life Cycle of Trypanosomes in the Host

The entire life cycle of African typanosome is represented by extracellular stages.

A tsetse fly becomes infected with blood stream trypomastigotes when taking a

blood meal on an infected mammalian host. In the fly’s mid gut, the parasites,

transform into procyclic trypomastigotes multiply by binary fission, leave the mid

gut and transform into epimastigotes. These reach the fly’s salivary glands and

continue mutiplication by binary fission.

The entire life cycle takes about three weeks. In addition to the bite of the fly, the

disease can be transmitted by:

- Mother to child infection [20]

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- Laboratory accidental infection, through the handling of blood of infected

person.

- Blood transfusion

- Sexual contact. [21]

Fig.1: Life Cycle of African Trypanosomes

1.1.8 Vectors of Trypanosomes

The vector for both types of African trypanosomiasis is tse-tsefly (genus

Glossina). It is a large brown biting fly that serves as both a host and vector for

trypanosome parasites. Biologist has identified 23 different species of Glossina,

12

of which all but three will transmit the trypanosomal infection to mammals. The

flies generally measure 7 to 14mm in length. Currently, this species of flies are

restricted to sub-saharan Africa north of the Kalahari Desert, which currently

restricts the transmission of the disease to within this region. However, with rapid

and frequent intercontinental travel, the introduction of this specie to naïve

regions poses a threat. Tsetse flies are haematophagous-dependant on blood

sucking to derive nutrients. Different species of Glossina have different

preferences for the sources of their blood meal with some specifically perferring

human blood and are therefore important vectors of the disease in human

populations. Both male and female flies feed on blood and are both vectors of the

parasites.

Horse flies (Tabanidae) and stable flies (Muscidae) possibly play a role in

transmission of nagana (the animal form of sleeping sickness) and the human

disease form [22].

Clinical Signs and Symptoms:

African trypanosomiasis symptoms occur in two stages. The first stage, known as

the haemolymphatic phase, is characterized by fever, headaches, joint pains, and

itching. Fever is intermittent, with attacks lasting from a day to a week, separated

by intervals of a few days to a month or longer. Invasion of the circulatory and

lymphatic systems by the parasites is associated with severe swelling of lymph

nodes, often to tremendous sizes. Winterbottom's sign, the tell-tale swollen lymph

13

nodes along the back of the neck, may appear. Occasionally, a red sore called a

chancre will develop at the location of the tsetse fly bite. If left untreated, the

disease overcomes the host's defenses and can cause more extensive damage,

broadening symptoms to include anemia, endocrine, cardiac, and kidney

dysfunctions. The second, neurological phase, begins when the parasite invades

the central nervous system by passing through the blood–brain barrier. Disruption

of the sleep cycle is a leading symptom of this stage and is the one that gave the

disease the name 'sleeping sickness.' Infected individuals experience a

disorganized and fragmented 24-hour rhythm of the sleep-wake cycle, resulting in

daytime sleep episodes and nighttime periods of wakefulness.

Other neurological symptoms include confusion, tremor, general muscle

weakness, hemiparesis and paralysis of a limb. Parkinson-like movements might

arise due to non-specific movement disorders and speech disorders. Individuals

may also exhibit psychiatric symptoms such as irritability, psychotic reactions,

aggressive behaviour, or apathy which can sometimes dominate the clinical

diagnosis. Without treatment, the disease is invariably fatal, with progressive

mental deterioration leading to coma, systemic organ failure, and death. An

untreated infection with T. b. rhodesiense will cause death within months whereas

an untreated infection with T. b. gambiense will cause death after several years.

Damage caused in the neurological phase is irreversible. The Gold standard for

diagnosis is identification of trypanosomes in a patient sample by microscopic

examination. Patient samples that can be used for diagnosis include chancre fluid,

14

lymph node aspirates, blood, bone marrow, and, during the neurological stage,

cerebrospinal fluid. Detection of trypanosome-specific antibodies can be used for

diagnosis, but the sensitivity and specificity of these methods are too variable to

be used alone for clinical diagnosis. Further, seroconversion occurs after the onset

of clinical symptoms during a T. b. rhodesiense infection, so is of limited

diagnostic use.

1.1.9 Treatments of Human Trypanosomiasis

Currently there are few medically related prevention options for African

Trypanosomiasis (i.e. no vaccine exists for immunity). Although the risk of

infection from a tsetse fly bite is minor (estimated at less than 0.1 %), the use of

insect repellants, wearing long-sleeved clothing, avoiding tsetse-dense areas,

implementing bush clearance methods and wild game culling are the best options

to avoid infection available for local residents of affected areas.

Treatment options for trypanosomiasis include:

First stage: The current treatment for first-stage disease is intravenous or

intramuscular pentamidine for T. b. gambiense or intravenous suramin for T. b.

rhodesiense.

Second stage: For T. b. gambiense intravenous Eflornithine or the combination of

Nifurtimox and eflornithine appear to be more effective and easier to give. These

treatments may replace melarsoprol when available with the combination being

first line. Intravenous melarsoprol was previously the standard treatment for

second-stage (neurological phase) disease and is effective for both types. It is the

15

only treatment for second stage T. b. rhodesiense however, it causes death in 5 %

of people who take it. Resistance to melarsoprol can occur.All persons diagnosed

with African Trypanosomiasis should receive treatment. The specific drug and

treatment course will depend on the type of infection (T.b gambiense or T.b

rhodesiense) and the disease stage (ie whether the central nervous system has

been invaded by the parasite. There are no tests of cure for African

trypansosomiasis. After treatment, patients need to have several examinations of

their cerebrospinal fluid for 2 years, so that relapse can be detected if it occurs. In

trypanosomiasis, chemotherapy is used to prevent the onset of the second stage

and its irreversible neurological disorders, elimination of parasite and subsequent

prevention of transmission.

Suramin

Suramin (fig.2) was discovered coincidentally in an attempt to develop a colorless

drug from colored dye, trypan red and trypan blue which were found to possess

trypanostatic action. It is the drug of choice in the early stage of sleeping

sickness. Its action is attributed to its ability to precipitate parasitic protein

constituents of the trypanosomes. Suramin is a potent retroviral reverse

transcriptase enzyme inhibitor but is ineffective in HIV infection and proved fatal

when used.

The most frequent adverse reactions are nausea and vomiting. About 90 % of

patients will get an urticarial rash that disappears in a few days without needing to

stop treatment. There is a greater than 50 % chance of adrenal cortical damage,

16

but only a smaller proportion will require lifelong corticosteroid replacement. It is

common for patients to get a tingling or crawling sensation of the skin with

suramin. Kidney damage and exfoliative dermatitis occur less commonly.

Fig.2 Chemical Structure of Suramin

Pentamidine:

The drug (fig.2) is the mainstay of treatment for stage I infection with

Trypanosoma brucei gambiense (West African Trypanosomiasis). It is a

diamidine structure and is more useful because of its relative stability and lower

toxicity profile. Serious side effects include kidney damage, hepatic impairment,

anemia and hypoglycemia.

Fig.3 chemical Structure of Pentamidine

Melarsoprol:

17

The organoarsenical melarsoprol (Arsobal) (fig.3) developed in the 1940s is

effective for patients with second-stage sleeping sickness. However, 3–10 % of

those injected have reactive encephalopathy (convulsions, progressive coma, or

psychotic reactions), and 10–70 % of such cases result in death; it can cause brain

damage in those who survive the encephalopathy. However, due to its

effectiveness, Melarsoprol is still used today. Resistance to melarsoprol is

increasing, and combination therapy with Nifurtimox is currently under research.

Fig.4 Chemical Structure of Melarsoprol

Eflornithine:

Eflornithine (difluoromethylornithine or DFMO) (fig.4), the most modern

treatment, was developed in the 1970s by Albert Sjoerdsma and underwent

clinical trials in the 1980s. The drug was approved by the United States Food and

Drug Administration in 1990, but Aventis, the company responsible for its

manufacture, halted production in 1999. In 2001, however, Aventis, in

association with Médecins Sans Frontières and the World Health Organization,

signed a long-term agreement to manufacture and donate the drug.

18

Fig.5 chemical structure of Enantiomer R of eflornithine and S-eflornithine

19

Fig.6 chemical structures of caratuberside

Penicilloside Caratuberside (fig.5) and penicilloside are pregnane glycosides previously isolated from the genus caralluma [19] These compounds are pregnane glycosides previously isolated from the genus caralluma [23] Structure of isolated antitrypanosomal compounds from Vitex simplicifolia

Flavon-5-ol

3 6 7 8 3’ 4’ 5’

2 OCH3 H OH H H OH OCH3

3 OCH3 OCH3 OCH3 H H OH H

R1O

20

4 OCH3 H OCH3 H H OH H

5 OH H OH H H OH H

6 OCH3 H OH H H OCH3 OCH3

Fig.7 Chemical structure of some compounds isolated from Vitex simplicifolia [24]. 1.2 The Use of plants in Phytomedicine

The past decade has witnessed a tremendous resurgence in the interest and use of

medicinal plant products especially in North America. Surveys of plant medical

usage by the American public have shown an increase from just about 3% or the

population in 1991 to over 37 % in 1998. [24, 25]. The North American market

for sales of plant medicinals has declined to about $3 billion/year [26]. Once the

domain of health food and specialty stores, phytomedicines have clearly re-

merged into the mainstream as evidenced by their availability for sale of a wide

range of retail outlets, the extent of their advertisement in the popular media, and

the recent entrance of several major pharmaceutical companies into the business

of producing phytomedicinal products [25, 27]. No doubt a major contributing

factor to this great increase in phytomedicinal use in the U.S has been the passing

of Federal Legislation in 1994(Dietary Supplement Health and Education Act or

“DSHEA”) that facilitated the production and marketing of phytomedicinal

products [25].

The past decade has also witnessed intense interest in “nutraceuticals” (or

“functional foods”) in which phytochemical constituents can have long term

health promoting or medicinal qualities. Although the distinction between

21

medicinal plants and nutraceuticals can sometimes be vague, a primary

charateristics of the later is that nutraceuticals having a nutritional role in the diet

and benefits to health may arise from long term use as foods. (ie chemoprevent)

[26]. In contrast, many medicinal plants exert specific medicinal actions without

serving a nutritional role in the human diet and may be used in response to

specific health problems over a short or long term intervals.

For many of the medicinal plants of current interest, a primary focus of research

to date has been in the area of phytochemistry, pharmacognosy and horticulture.

In the area of phytochemistry, medicinal plants have been characterized for their

possible bioactive compounds, which have been separated and subjected to

detailed structural analysis. Research in the pharmacognosy of medicinal plants

has also involved assays of bio-activity, identification of potential modes of

action and target sites for active phytomedicinal compounds. Horticultural

research in medicinal plants has focused on developing the capacity for optional

growth in cultivation. This has been especially pertinent as many medicinal plants

are still harvested in the wild, and conditions for growth in cultivation have not

been optimized. Wild harvesting of medicinal plants can be problematic in terms

of bio-diversity loss, potential variation in medicinal plants quality, and

occasionally, in proper plant identification with potential tragic consequences.

From the perspective of plant physiology, extensive opportunities exist for basic

research on medicinal plants and the study of their phytomedicinal chemical

production. This reviews a discussion on some fundamental aspects of

22

phytomedicinal chemical production by plant cells with an overview of several

medicinal plants that have received considerable attention over the past decades.

The Benefits of Phytomedicine:

In contrast to synthetic pharmaceuticals based upon single chemicals, many

phytomedicines exert their beneficial effects through the additive or synergistic

action of several chemical compound acting at single or multiple target sites

associated with a physiological process. As pointed out [26, 28], this synergistic

or additive pharmacological effect can be beneficial by eliminating the

problematic Side effects associated with the predominance of a single xenobiotic

compound in the body. In this respect, [29], extensively documented low

synergistic interaction underlies the effectiveness of a number of phytomedicine.

This theme of multiple chemicals acting in an addition or synergistic manner

likely has its origin in the functional role of secondary products in promoting

plant survival.

Of the vast number of medicinal plants used in western and non- Western medical

approaches, a small number has received considerable interest and use in North

America over the past few years. What follows is an overview of a medicinal

plant of current interest focusing on its biochemical characteristics and

pharmacological actions of their plant secondary product chemicals.

Ginseng:

The name”ginseng” often leads to some confusion due to its use for different

plants; with different phytochemical constituents. True ginsengs are plants in the

23

genus (Panax quinquefolium) have received the most interest for photomedicinal

use [30-31]. However, there is evidence that extracts of ginseng and

eleutherococcus sp. can have immunostimulatory effect in humans, and this may

contribute to the adaptogen or tonic effects of these plants [31- 34, 35]. The major

secondary products present in ginseng roots are an array of triterpene saponins

collectively called gensenosides [33, 36]. The gensenosides are glycosylated

derivatives of two major aglycones panaxadiol and panaxatriol [30, 36]. At

present, 30 ginsenoside have been identified of which the ginsenosides, Rb1,

Rb2, Rd, Re, Rt, Rg1 and Rg 2 are considered to be the most for pharmacological

activity [30, 34, 35]. Different ginseng species have different proportions of

ginsenoside in root tissue and this may relate to reported differences in the

pharmacological properties of these plant materials [37].

1.2.1 Taxonomy of the genus Vitex

Taxonomic name: Vitex rotundifolia L.F

Synonyms: Vitex agnus- castus var ovata (Thunb) Makino, Vitex Ovata (Thund)

Makino, Vitex repens Blanco, Vitex trifolia sub sp. Litoralis Stenis, vitex trifolia

var obovata Bentham, Vitex trifolia var ovata (Thunb) Merrill, Vitex Ovata var.

repens Ridley, Vitex trifolia var. simplicifolia (cham.) Vitex trifolia var unifoliata

schauer.

Common names: Beach vitex (English)

Chasteberry, cloister pepper, dan ye mam

24

Jing (Chinese –China) hamago (Japan),

Hinahina kolo (Hawaiian-Hawaii) Kolokolo Kahakai (Hawaiian-Hwaii), man

hyung jar (Korea), mawana (Hawaiian-Hawaii), monk’s pepper, pohinahima

(Hawaiian-Hwaii) Polinalina (Hawaiian –Hawaii) round leaf chaste tree, single

leaf chaste tree.

25

1.2.2 The plant: (Vitex simplicifolia Oliv.)

Fig.8 The plant (Vitex simplicifolia Oliv.)

Name: Vitex simplicifolia

Family: Labiatae, Verbenaceae

Synonyms: Vitex madiensis Oliv.

Subsp. Madiensis

26

Description: A small tree or shrub with dense, pale indumentum and mauve

flowers; in Savanna. It grows to a height of approx.8m

Ecology: In Savanna

Young twigs are used as tooth picks in Nigeria

1.2.3 Ethnomedicinal uses of Vitex simplicifolia Oliv.

The initialization of plant based product in food supplements and health

industries were increased tremendously for the past four to five years. This was

believed to be due to carcinogenic related problem with the usage of artificial or

chemical based products. Therefore, a lot of studies have been done by the

research all over the world to determine the active bio-component in plant, which

could replace artificial produce. For example, uses of Punica granatum [37], uses

of coconut shell and [38] uses of Carica papaya.[39] Many other researches have

also embarked on the study to identify the possibility of using plant components

to solve human health problems [40,]. Besides a lot of studies were also under

taken by researchers- to use plant materials to handle environmental pollution;

for example using maize cob to remove heavy metals in industrial waste water.

Similarly Moringa oleifera could be used as a natual absorbent and antimicrobial

agent in water treatment.

Vitex trifolia var. simplicifolia is basically a sea side shrub from the family

lamiaceae or verbenaceae. The vitex genus family consists of about 250 specie of

27

shrubs and trees; its widely cultivated in warm temperate and subtropical regions

[41].

One of them is Vitex trifolia species with variety of simplicifolia. The plant was

used to prepare traditional dessert among Siamese communities in kelanthan

called “khanom Bai kunthi”. The ingredients were rice flour salt and extract of

Vitex trifolia var simplicifolia leaf. Extracts from leaves of Vitex trifolia var

simplicifolia will give colour, flavour and fragrance to the dessert. The factor that

determines the colour is the plant pigments such as chlorophyll, xantophyll,

carotene, flavone, flavonol and anthocyanin. Chlorophyll can be destroyed after

certain temperature. However, as the chlorophyll is destroyed, the other pigments

such as carotenoid and anthocyanin are expressed. Anthocyanins are oxidant

flavonoids which improve human health condition. Besides, antioxidant

supplementation can block NF-KB (unclear factor kappa-light-drain enhancer of

activated B cells) N-F-KB inhibits cancer

Wound Healing and Antibacterial Properties:

Vitex simplicifolia Oliv is used as internal and external remedies to treat disease

such as dermatitis, migrains fever, aches, amoebiasis, sore teeth, and infant

tetanus. Ethno botanical investigations have revealed that the plant is also used in

the treatment of skin infections and wound healing. In Burkina Faso, infectious

disease are the leading cause of infant mortality (2.37 % and maternal (14.6 %)

therefore they constitute public health problems. The treatment of skin disease

28

with plant materials dates back to acient times. About 30% of the traditional

remedies are used to treat wounds and skin lesions, compared to only 1-3% of

modern drugs [42-45]. The healing process is an immune response that begins

after injury and takes place in three stages: vascular and inflammatory stage, the

phase of tissue repair and phase of maturation. A drug having simultaneously the

pontential antioxidant and antimicrobial activities may be a good therapeutic

agent to accelerate cicatrisation and wound healing [46-49]. Aroma therapy is

now considered to be another alternative way in healing people, and therapeutic

values of aromatic plants lie in their volatile constituents such as

monoterpenoids, sesquiterpenoids and phenolic compounds that produce a

definite physiological action on the human body.[50] The genus, Vitex

simplicifolia Oliv also has antitrypanasomial and antinflammatory activities [45].

Medicinal Uses of Vitex negundo linn:

A perfect example of medicinal plant credited with innumerable medicinal

qualities validated by modern science and used since ancient times is Vitex Linn

(family-verbenaceae). The genus consists of 250 species of which about 14

species are found in India and some have commercial and medicinal importance.

Vitex negundo Linn, commonly known as five-leave chaste tree or Monk’s pepper

is used as medicine fairly through out the greater part of India and found mostly

at warmer zones and ascending to an alttitude of 1500 m in outer Western

Himalayas [51, 52].

29

The plant is a large aromatic shrub or sometimes a smaller slender tree with

quadrangular densely whitish tomentose branchlets up to 4.5-5.5m in height. Bark

thin, yellowish grey, leaves 3-5 forliolate; leaflets lanceolate; terminal leaflets 5-

10x1.6-2.3cm, lateral one smaller, all nearly glabrous. Upper surface of the leaves

are green and the lower surface are silvery in colour. Flower, bluish purple, roots

are cylindrical [53].

The plant is bitter, arid, astringent, cephalic, stomachic, antiseptic, alterant,

thermogenic, depurative, rejuvenating, ophthalmic anti-gonorrhoeic, anti-

inflammatory, antipyretic and useful in bronchitis, asthma and enlargement of

spleen. Roots are tonic and are useful as demulcent in dysentery. Bark is useful in

ophthalmopathy. Leaves have antinfammatory, antipyretic and tranquillizer.

Flowers are useful in haemorrhagic and cardiac disorders.

Other medicinal uses of Vitex simplicifolia Oliv.:

Apart from antitrypanosomal, antibacterial, and anti-oxidant properties, the bark

is used to treat swellings, oedema and oral treatment [54].

Vitex rotundifolia was historically used to surpress sexual desire in women and

for similar reasons become a culinary spice in monastries hence the common

name Monk’s pepper. Some of the active chemical compounds have been linked

to female hormone balance, female reproductive organs, menopause, actions on

the pituitary glands, and treatment for acne. In Korea, it has been used for the

rehabilitation and land scaping in sea board areas.

30

1.2.4 Geographical Distribution of Vitex simplicifolia Oliv.

Vitex is the largest genus in the family verbenaceae which comprises 250 species

distributed all over the world [55]. The vitex species are deciduous shrubs. The

species used in medicine are V. simplicifolia Oliv. V. agnus –castus Linn and, V.

negundo linn. V. agnus-castus (chaste tree) is widespread on river banks and on

shores in the meditarranean region, southern Europe and in central Asia [56]. V.

negundo chiefly occurs in Pakistan, India, and Sri-lanka [57], V. rotundifolia linn

is distributed in the Mediterranean region, central Asia and along the sea coast

from South to North of China [58-59] V. trifolia occurs in Asian countries and in

Vietnam. [60].

In Africa, it is distributed from Mali to Ivory Coast to Nigeria, Cameroon, and

Central Africa extending to Egypt, Sudan and Uganda

1.2.5 Table 1: Previous antitrypanosomal studies on medicinal plants.

S/N Botanical name of plant

Family Part of plant investigated

Study design in vivo/in vitro

Activity Reference

1. Zapoteca portoricensis

Fabaceae Powdered roots

In vivo/in vitro

Active 61

2. Annona senegalensis

Annonaceae Leaves, root and stem

in vivo Active 62

31

3. Lychnophora salicifolia

Asteraceae Leaves In vivo Active 63

4. Lychnophora granmongolense

Asteraceae Leaves Active 64

5. Morinda lucida Rubiaceae Leaves In vivo Active 65

6. Morinda lucida Leaves In vivo `active 66

7. Senna occidentalis

Fabaceae Leaves In vivo/in vitro

Active 67

8. Aspilia platyphylla

Asteraceae-heliantheae

Roots In vivo Active 68

9. Nauclea diderrichii

Rubiaceae Leaves, stem and roots

In vivo Active 69

10. Vitex simplicifolia

Verbenaceae Leaves In vitro

Active 20

11. Baccharis retina Asteraceae Leaves In vitro

Active 70

12. Saussurea costus Asteraceae Roots In vivo/in vitro

Active 71

13. Morinda morindiodes

Rubiaceae Roots In vivo Active 72

14 Kaya senegalensis

Maliaceae Leaves In vivo Active 73

15 Securidaca longepedunculata

Polygalaceae Roots In vivo Active 74

32

16 Combretum racemosun

Combretaceae Leaves In vitro/in vivro

Active 75

17. Terminalia avicennioides

Combretaceae Leaves In vitro/in vivo

Active 76

18. Ceiba pentandra Bombacaceae Stem In vitro/in vivo

Active 77

33

Table 2: Previous antitrypanosomal studies on Vitex simplicifolia Oliv.

More work needs to be done on this plant to isolate more bioactive compounds

hence this study.

S/N BOTANICAL

NAME OF

PLANT

FAMILY PART

OF

PLANT

STUDY

DESIGN

IN

VIVO/IN

VITRO

Activity Reference

1. Vitex

simplicifolia

Oliv.

Verbenaceae Leaves In vitro Active 24

34

1.2.6 Aim and Objectives of the Study

(i) Aim

� To evaluate the antitrypanosomal activity of the leaf extract of Vitex

simplicifolia Oliv.

� Objectives

� To determine the toxicity of V. simplicifolia Oliv.

� To carry out both in vivo and in vitro antitrypanosomial assays

� To determine the phytochemicals present in the plant

� To determine the nature of the bioactive constituents

35

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Chemicals and Reagents

Distilled water, Water for injection, Analytical grade methanol, Diminazene

aceturate, Dimethylsulfoxide (BDH, England) and many more. All the chemicals

used for the experiment were sourced from reputable chemical company and used

without further purification

2.1.2 Equipment

Weighing balance, Syringe of different capacities (1ml, 5ml and 10ml) and

needles, L 3000 microscope (China), Slides and cover slips, M 24 Haematocrit

reader (China), M 901 Multipurpose Centrifuge (England), Heparinized capillary

tubes, Beakers, Scissors, Stirrer, Refrigerator, Funnel and filter cloth.

2.1.3 Plant Material

Fresh leaves of Vitex simplicifolia were collected from Orba in Enugu State in

January 2012. The plant material was identified and authenticated by Mr. A. O.

Ozioko of the Bioresources Conservation and Development Programme (BCDP)

Nsukka. A voucher specimen was deposited at the herbarium unit of the Institute.

2.1.4 Experimental Animals

A total of fifty five (55) rats and thirteen (13) mice of both sexes obtained from

the animal house of the Faculty of Veterinary Medicine, University of Nigeria

Nsukka were used for the trypanocidal screening of both the crude methanolic

extracts and various solvent fractions and acute toxicity tests respectively. The

36

animals were kept in plastic baskets and had unrestricted access to food and water

for the duration of the experiment.

2.2 Parasites

The parasite, Trypanosoma brucei obtained from an infected rat in the

Department of Parasitology, Faculty of Veterinary Medicine, University of

Nigeria, Nsukka were used to infect the experimental animals. This was done by

bleeding the rats through the tip of the tail and collecting the infected blood using

heparinized capillary tube and diluting it with normal saline which serves as

inoculum. Healthy rats were infected with 0.1 ml of the inoculums containing

about 103 trypanosomes/ml and maintained in the laboratory by continuous

passage of infected blood into the rats.

2.2.1 Methods

2.2.2 Extraction, Fractionation and Isolation Procedure

The dried leaves (500 g) of Vitex simplicifolia were macerated with 3.0 L of 100

% methanol and extracted at room temperature for 24 hours 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 (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 %), dichloromethane (DCMF, 2.98 g,

22.34 %), ethyl acetate (EF, 1.08 g, 8.10 %), n-butanol (BF, 5.75 g, 43.10%) and

37

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. The work flow chat is shown fig.9

Fig.9 Flow Chart of the general separating procedure.

38

2.2.3 Phytochemical Analysis

The phytochemical analysis was done using standard method[78,79]

2.2.4 Acute Toxicity Test of the Crude Extract

The acute toxicity (LD50) of the methanol extract was estimated (p.o) in Swiss

albino mice weighing between 20-30 g following Lorke’s method. [80]. The

methanol extract was dissolved in 10 % v/v Tween 80. Dose levels used ranged

from 10 – 5000 mg/kg of the methanol extract. The test comprises two phases.

First phase: Nine (9) mice randomly divided into three (3) groups of three mice

each. Each group of three mice received 10 mg/kg, 100 mg/kg and 1000 mg/kg of

the crude extract. After administration of methanol extract, signs of toxicity such

as death, change in physical appearance and behavioural changes were observed

for 24 h.

Second phase: Four mice each received individually different doses of 1600,

2900 and 5000 mg/kg body weight per oral of the methanol extract respectively.

The mice were monitored for 24h. for lethality. The LD50 was calculated as the

geometric mean of the maximum dose of the extract that caused zero percent

lethality (0% death) and the maximum dose that resulted in 100 % lethality.

2.2.5 In-Vivo Anti-trypanosomal Activity

The fifty- five rats were grouped into eleven groups of 5 rats each. They were

labeled using picric acid and gentian violet (GV) Each group was housed in a

separate basket. The grouping was as follows:

39

Group A: Infected and treated with 400 mg/kg of Vitex simplicifolia crude extract

per kg body weight.

Group B: Infected and treated with 200 mg/kg of Vitex simplicifolia crude extract

per kg of body weight.

Group C: Infected and treated with 100 mg/kg of Vitex simplicifolia crude extract

per kg of body weight.

Group D: Infected and treated with 100 mg/kg of BF Vitex simplicifolia fraction

per kg body weight.

Group E: infected and treated with 100 mg/kg of HF Vitex simplicifolia fraction

per kg body weight

Group F: Infected and treated with 100 mg/kg of EF Vitex simplicifolia fraction

per kg body weight

Group G: Infected and treated with 100mg/kg of WF Vitex simplicifolia fraction

per kg body weight

Group H: Infected and treated with 100 mg/kg of DCMF Vitex simplicifolia

fraction per kg body weight

Group I: Infected and treated with 3.5 mg of diminazene aceturate per kg body

weight

Group J: Infected and untreated.

Group K: uninfected (control).

Animals in groups A to J were infected with 0.2ml of infected blood diluted with

normal saline by intraperitoneal injection.

40

Study Parameters: Parasite pathogenicity and animal response to treatment were

monitored using three parameters viz:

• Level of parasitaemia

• Weight of animals

• Packed cell volume (PCV)

The animals PCV and body weights were measured prior to infection to obtain

base line values. On the 3rd day post infection, the levels of parasitaemia of the

animals were measured. This process was repeated on the 4th and 5th day post

infection. Treatment with the extracts began on the 5th day post infection. The

body weights and PCV were also measured before treatment post infection. Body

weight and PCV were taken every 5-10 days within the treatment period and 10

days after. The level of parasitaemia was estimated using the rapid matching

method while viewing the blood smear under microscope [81]. In this method,

blood from the tip of the tail of the rat was used to make a smear on the slide. The

smear was covered with a cover slip and then viewed under a microscope. The

image observed was compared to a reference and the matching level was taken as

the correct level of parasitaemia. A centrifuge and haematocrit reader was used to

determine the PCV. In this method, the animals were bled through the tip of their

tail using a sharp scissors and the blood collected with heparinized capillary tube.

The unfilled end of the tube was sealed with plasticine. The capillary tubes were

then placed in the micro haematocrit and centrifuged at a speed of 10,000

41

revolutions per minute for 5 minutes. The haematocrit reader was then used to

obtain the percentage PCV.

Body weights of the animals were obtained using a lever balance in a room. The

body weight was taken before infection and every 5-10 days post infection and

throughout the duration of treatment.

Solution of the extract was made by weighing out the appropriate weight of the

extract and dissolving in 10 % DMSO. Adequate dilutions were made to enable

withdrawal of appropriate doses for treatment using the available syringes. The

intraperitoneal route was employed for treatment of animals. Diminazene

aceturate was also diluted using water to obtain a dilution that enabled the

withdrawal of 3.5 mg/kg dose using a 1ml syringe. In each case, care was taken

not to pierce the abdomen of the animals during drug administration. This was to

ensure eliminating death due to injury to the animals.

2.2.6 In-Vitro Anti-trypanosomal Activity Test

In vitro antitrypanosomal activity was performed according to method described

by Atawodi et al [82], with slight modification. In vitro trypanocidal activity was

performed in duplicates in 96 well micro titer plates (Flow laboratories Inc.,

McLean, Virginia 22101, USA). Blood (10 μL) containing about 126-130

parasites per field, after dilution with Phosphate-Buffered Saline with Glucose

(PBSG) in a ratio of 4:1, was mixed with 10 μL of extract solution of 80.0, 40.0

and 20.0 mg/ml to produce effective test concentrations of 40 , 20 and 10 mg/ml,

respectively. To ensure that the effect monitored was that of the extract alone, the

42

untreated blood in PBSG was monitored as well. Reference tests were also

performed with two concentrations (40 and 20 mg/ml) of Samoricide® plus (1.05

g diminazene diaceturate+1.31 g antipyrine+1 mg vitamin B12)-a commercial

trypanocidal drug. Under this in vitro system adopted, parasites survived for

about 4 h when no extract was present. Cessation or drop in motility of the

parasites in extract-treated blood compared to that of parasite-loaded control

blood without extract was taken as a measure of trypanocidal activity.

2.2.7 Statistical Analysis 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<0.05.The

difference within means was analyzed using the one-way ANOVA

43

CHAPTER THREE

RESULTS

3.1 Extraction/Fractionation Yield

The percentage yield of the methanolic extract used for the study is shown in

Table 3. The result shows that Vitex simplicifolia methanol extract had the

percentage yield of 2.668 % w/w .

Table 3: Percentage Yield of Extracts/Fractions

Extracts/Fractions Yield (g) %

Yield

Methanol (ME) 13.340 2.668

Aqueous (WF) 1.690 2.67

n-Hexane (HF) 1.060 7.98

Ethylacetate (EF) 1.080 8.10

n-Butanol (BF) 5.750 43.10

Dichloromethane (DCMF) 2.980 22.34

44

Table 4: Shows the Phytoconstituents of Methanolic Extracts (ME) and DCMF

TEST RESULT DCMF

Alkaloid + +

Steroid + +

Tannins + +

Flavonoid + +

Protein + -

Saponin + +

Carbohydrate + -

Phenol + -

Glycoside + +

Table 5: Shows physicochemical properties of the bioactive isolate (DCM 1)

from the V.simplicifolia Oliv. (DCM) fraction.

Molecular weight 480 g/mole

Appearance Light yellow

UV spectrum 247.1 nm and 321.1nm

Retention time 19,360

Impression: Steroid, flavonoid

45

3.1.1 Results of parasitology testing

After the experiments were conducted, results were obtained for the

parasitological findings using parasitaemia level, body weight and packed cell

volume (PCV) as monitoring parameters. It took exactly 5 days for the level of

parasitaemia to come up to levels that can be properly measured. On this day the

animals showed visible signs of trypanosomiasis such as decreased movement

and dullness. Treatment began on this day and lasted for five days. The observed

changes as recorded for parasitaemia level, weight variations and changes in PCV

are shown in the Table 6- 8 and graphical representation can be seen in Figure 1

1-13.

3.1.2 Effects of Vitex simplicifolia Oliv. on Parasitaemia Level

The comparative trypanocidal activity of the test extract (ME), different fractions

and the standard drugs as seen in the parasitaemia level is shown specifically in

Table 6 and Fig.11.

In group A treated with 400 mg of ME per kg body weight, there was a marked

reduction in parasitemia in most of the animals and no deaths were recorded

within the duration of the experiment and three animals attained complete

clearance. In group B, treated with 200 mg of ME per kg body weight there was a

steady decrease in parasitaemia level although not as prominent as that recorded

in group A. Two animals in this group attained complete clearance at the end of

the experiment. One animal died in this group. This may be attributed to drug

toxicity or high susceptibility of the animal to the infection. In group C, treated

46

with 100 mg of ME per kg body weight, reduction in parasitaemia levels was less

pronounced and although no deaths were recorded after the experiment, the

reduction obtained was smaller than that obtained with lower doses. Of all

Groups D-H treated with different fractions, only groups D and H showed various

degrees of reduction in parasitaemia levels.

The parasitaemia of infected, untreated and infected but treated groups are as

shown in Fig. 11. In all the groups, parasites were first sighted 4 days post

infection. In Group J (infected, untreated), there were progressive increases

infection (0 % survival). Results showed that parasites in the blood stream of rats

treated with Diminazene aceturate (positive control, Group I) were completely

eliminated on day 7. The rats remained aparasitaemic and survived beyond the 14

– day observation period (100 % survival). The extract did not affect the onset of

parasitaemia, but was able to reduce its level and prolong the lifespan of the

treated rats.

47

Table 6: Comparative Response of Methanolic Extracts/Fractions to

Parasitaemia

Animal

Groups

*Mean Parasite Count

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8

A 0.0 0.0 0.0 7.8±0.2 7.5±0.4 7.4±0.3 6.8±0.4 6.7±0.3 6.8±0.9

B 0.0 0.0 0.0 7.8±0.1 7.5±0.3 7.0±0.2 6.9±0.2 6.8±0.2 6.7±0.4

C 0.0 0.0 0.0 8.1±0.5 8.0±0.4 7.5±0.1 7.8±0.2 7.2±0.5 7.6±0.2

D 0.0 0.0 0.0 7.5±0.1 7.2±0.1 7.0±0.4 7.0±0.4 6.9±0.7 2.6±0.4

E 0.0 0.0 0.0 8.1±0.1 8.0±0.4 8.2±0.2 8.0±0.2 7.8±0.1 7.8±0.1

F 0.0 0.0 0.0 8.1±0.2 8.0±0.5 7.9±0.4 7.8±0.7 7.9±0.1 7.8±0.4

G 0.0 0.0 0.0 7.8±0.2 7.8±0.2 7.7±0.1 7.8±0.4 7.6±0.1 7.7±0.1

H 0.0 0.0 0.0 8.0±0.4 7.8±0.1 7.3±0.1 7.0±0.1 6.9±0.4 3.5±0.3

I 0.0 0.0 0.0 7.8±0.1 7.5±0.3 4.0±0.1 2.5±0.1 0.0 0.0

J 0.0 0.0 0.0 7.9±0.3 8.0±0.3 8.2±0.1 7.9±0.5 8.3±0.1 8.4±0.1

K 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

*values expressed as mean±SD, n=5

3.1.3 Effects of Vitex simplicifolia Oliv. on Weight of Rats

Change in body weight of the treated animals was monitored during the period of

the study as shown in Table 7. It was observed that animals treated with 400 , 200

mg/kg, BF and DCMF of the plant extract on average maintained their body

weight post treatment while the animals treated with 100 mg/kg, WF, EF and HF

showed reduced body weights. There was significant difference between 400 and

48

100 mg/kg. For the negative controls, the animals lost a lot of body weights and

survived for only 30 days. In contrast, the animals treated with diminazene

aceturate slightly increased their body weights and maintained their weight after

30 days.

49

Table 7: Effect of ME and Solvent Fractions of Vitex simplicifolia on Weight

of Treated Rats

Animal

Groups

*Mean Body weight (g)

Day 0 Day 5 Day 10 Day 15 Day 20 Day 30 Day 40 Day 50

A 120.0±0.0 121.0±1.1 120.5±0.4 120.0±0.2 122.0±0.1 120.5±0.2 122.0±0.8 121.5±1.2

B 118.5±0.3 119.0±0.0 118.5±0.2 118.0±0.6 119.0±0.4 120.0±0.5 119.5±0.1 119.0±0.3

C 125.0±0.2 125.0±0.9 124.5±0.5 128.0±0.2 122.5±0.2 121.0±0.3 119.0±0.0 115.0±0.4

D 138.0±0.5 138.0±0.2 139.5±0.5 140.0±0.0 142.0±0.4 144.0±0.9 144.0±0.6 147.0±0.2

E 119.0±0.9 119.5±0.1 118.5±0.3 118.0±0.0 118.5±0.1 117.0±0.1 115.0±0.4 105.0±0.8

F 129.0±0.2 129.0±0.2 128.5±0.2 125.5±0.4 123.0±0.8 120.5±0.0 119.0±0.3 111.0±0.7

G 137.0±0.1 130.5±0.3 130.0±0.5 128.5±0.2 125.5±1.0 120.0±0.9 120.5±0.2 120.0±0.3

H 115.5±0.4 116.0±0.1 117.0±0.1 120.0±0.1 126.0±0.0 126.0±0.2 129.0±0.5 139.0±0.5

I 126.0±0.2 128.5±0.8 127.0±0.4 126.0±0.3 127.5±0.4 129.0±0.4 130.5±0.7 130.0±0.6

J 132.0±0.0 131.0±1.0 128.0±0.1 125.5±0.5 120.0±0.3 117.5±0.1 112.5±0.3 100.0±0.3

K 140.0±0.5 140.0±0.3 140.0±.0.3 139.0±0.3 141.0±0.2 140.5±0.4 142.0±0.8 142.5±0.7

*values expressed as mean±SEM of 5 different determinations

50

3.2 Effects of Vitex simplicifolia Oliv. on Packed Cell Volume (PCV)

The study on packed cell volume (PCV) analysis gave results that were consistent

with the observations made on parasitaemia. As shown in Figure 14, the PCV of

rats treated with 100-400 mg/kg ME was on average above 43 % which was

fairly within the reference values of 42 - 52 for males. The PCV of animal treated

with 100 mg/kg DCMF was 42%. The rats treated with 100 mg/kg EF and WF

relative to negative controls, their PCVs were below the reference values (42-52

%). The animals treated with standard drug have their PCVs within the accepted

limits (42 – 44 %) (Fig.13). The PCV of rats treated with ME (100-400 mg/kg),

different solvent fractions (100 mg/kg), negative control (vehicle), uninfected

untreated group and positive controls (diminazene aceturate, 3.5 mg/kg) were

monitored post treatment for 60 days.

51

Table 8: Percentage Response of ME and Fractions to Packed Cell Volume

(PCV).

Animal

Groups

*Mean PCV (%)

Day 0 Day 5 Day 10 Day 15 Day 20 Day 30 Day 40 Day 50 Day 60

A 42.5±0.4 42.2±0.1 42.6±0.3 43.8±0.4 42.0±0.8 42.9±0.3 43.8±0.9 42.7±0.3 42.8±0.0

B 43.1±0.2 43.2±1.0 42.1±0.8 42.9±0.2 42.4±0.9 42.7±0.2 43.8±1.1 42.6±0.5 42.1±0.3

C 42.8±0.1 42.3±0.4 43.8±0.1 42.1±0.6 42.8±0.1 42.7±0.5 42.6±0.4 42.0±0.8 42.4±0.1

D 42.5±1.1 44.5±0.4 43.9±0.5 43.8±0.5 42.9±0.4 44.3±0.7 43.8±0.3 43.0±0.1 42.8±0.5

E 45.9±1.0 45.2±1.1 42.3±0.2 40.4±0.5 39.9±1.1 40.3±0.1 38.3±0.9 38.0±0.2 38.1±0.5

F 43.8±0.9 42.7±1.7 40.9±0.4 38.8±1.0 39.2±0.4 37.0±0.4 36.4±0.3 35.2±0.4 35.3±0.1

G 44.1±0.6 42.9±0.5 41.8±0.1 39.0±1.3 37.2±0.3 36.1±0.7 34.9±0.5 33.2±0.6 35.2±0.1

H 43.2±0.4 43.2±0.5 42.9±0.4 42.7±0.1 42.0±0.9 42.8±0.3 43.9±0.5 44.6±1.2 42.8±0.8

I 42.7±0.5 42.8±0.1 43.8±0.2 42.9±0.3 42.9±0.1 43.2±0.1 43.8±0.2 43.0±0.4 44.1±0.8

J 44.8±0.6 42.9±1.4 40.8±0.9 39.8±0.5 37.8±0.6 36.4±0.1 35.9±0.5 35.4±0.2 33.8±0.6

K 42.8±0.2 42.8±0.2 42.9±0.1 42.2±1.1 42.7±1.2 42.5±0.3 42.4±0.9 42.6±1.0 42.6±0.5

*value expressed as mean±SD, n=5

52

3.2.1: Table 9: Shows the results of in vitro antitrypanosomal assay on the

DCM 1 fraction

Sample T.b. rhod.

IC50 (µg/ml)

T.cruzi.

IC50(µg/ml)

Cytotox L6 IC50

Melarsoprol

Podophyllotoxin

0.003 0.407 0.008

DCM1 10.12 46.05 >100

Fig. 10. Possible chemical structure of the bioactive isolate (DCM1). 23,3R,5R,10R,14S)-2,3.14-trihydroxy-10,13-dimethyl-17-[(2S)-2,5,6-trihydro-6-methylheptan-2-yl)-2,3,4,5,11,12,13,14,15,16,17-dodecahydro-]H-cyclophenanthren-6(10H)-one

53

Table 10: Showing Chemical Shift

N/S Chem. shift Of Cpd 113C ppm

Chem. shift of cpd 2 13C ppm……

∆ value H1NMR Cpd1

H1NMR Cpd2

∆ value

1. 39.41 38.04 1.37 2. 68.56 68.08 0.48 3.96 4.24 0.28 3. 68.85 68.17 0.68 3.85 4.22 0.37 4. 31.84 32.48 0.64 5. 51.87 51.42 0.45 2.40 2.97 0.57 6. 191.18 203.42 12.24 7. 121.68 121.66 0.08 5.81 6.23 0.42 8. 165.51 166.07 0.44 9. 35.21 34.48 0.73 10. 37.45 38.66 1.21 1.44 11. 21.06 21.13 0.07 12. 31.84 31.75 0.09 2.13 1.83-1.95 0.28 13. 50.56 48.10 2.46 3.35 2.56 0.79 14. 85.38 84.15 1.23 15. 32.66 31.99 0.67 2.13 2.07-2.19 0.06 16 21.79 17 50.00 50.06 0.06 2.41 2.97 0.56 18 18.19 17.90 0.29 0.88 1.20 0.32 19 24.55 24.49 0.06 0.95 1.08 0.13 20 72.03 76.78 4.75 21 21.53 21.53 0.00 1.71 1.58 0.13 22 77.96 78.26 0.30 23 32.98 32.99 0.01 24 78.57 80.31 1.74 3.33 4.19 0.86 25 71.44 72.25 0.19 26 27.37 26.85 0.52 1.19 1.45 0.26 27 24.32 25.34 1.02 0.99 1.50 0.51

54

Figure 11: Effect of the extract/fraction of V.simplicifolia on the parasitaemia

level of trypanosomal-infected rats.

AC=ME;400,200,100mg/kg DH=BF,HF,EF,WF,DCMF;100mg/kg

55

Figure 12: Effects of the extract/fraction of V.simplicifolia on the weight of

trypanosomal-infected rats.

AC=ME;400,200,100mg/kg DH=BF,HF,EF,WF,DCMF;100mg/kg

56

Figure 13: Effect of the extract/fraction of V.simplicifolia on the PCV of

trypanosomal-infected rats.

A-C=ME;400,200,100mg/kg D-H=BF,HF,EF,WF,DF;100mg/kg

57

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSSION

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,

58

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 100

mg/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 in

crease activity.

The phytochemical constituents of the plant’s crude extract and DCM fraction

revealed the presence of alkaloids, steroid and flavonoid together with other

phytochemicals. The isolate appeared as a base peak in the DCM fraction. It also

59

appeared as a base peak in the butanol fraction. DCM1 having been found to be

either a steroid or a flavonoid is in agreement with previous work done on the

plant [24]. This goes a long way to ascribe the antitrypanosomal activity of this

plant to either the steroid or the flavonoid.

Table 10 which is the table for chemical shift is closely related to the one in

literature. The Table shows the comparison of the structure of DCM1 with the

NMR spectra data of similar structure in the literature [84]. It shows that the

structures are closely related.

The NMR (1H and 13C) data of DCM1 are similar to the ecdysteroids in the

literature. The UV spectrum in table 5 and the NMR spectra in appendix C

suggest that fig. 10 is likely the structure of DCM1.

4.2 CONCLUSION

The present study has shown that Vitex simplicifolia possesses antitrypanosmal

activity. Apart from being a source of antitrypanosomal drug, it provides a natural

remedy for such ailment for the rural dwellers who ordinarily can not afford the

commercial drugs in the market.

This study justifies the ethnobotanical use of Vitex simiplicifolia in the treatment

of trypanosomiasis.

60

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APPENDIX A

0,0 10,0 20,0 30,0 40,0 50,0 60,0-200

250

600 NJ121003 #3 VSL-DCMGB UV_VIS_1mAU

min1

- 1

9,3

60

WVL:235 nm

Figure A1.1….HPLC chromatogram of DCM1 at 235 nm

Peak #1 100% at 19.36 min

-10,0

70,0

200 250 300 350 400 450 500 550 595

%

nm

247.1

321.1

No spectra library hits found!

Figure A1.2….UV spectrum of DCM1

69

Figure A1.3…..Positive and negative modes of DCM1

APPENDIX B

7070

71

71