CHAPTER 1

INTRODUCTION

1.0 General Introduction

Human African Trypanosomiasis (HAT) is one of the Neglected Tropical Diseases (NTDs) listed by

WHO. HAT, also known as sleeping sickness in human and nagana in cattle is affected by kinetoplastid

protozoa parasite from the genus of Trypanosoma sp. (Brun et al., 2010; Bucher et al., 2017). This endemic

disease remains to cause a major health and economic problems in rural sub-Saharan Africa. Even though

Current chemotherapy of HAT rely on five registered drugs, pentamidine, suramin, melarsoprol,

nifurtimox, and eflornithine. However the usage of these drugs has reported with their numerous side

effects, difficulty in administration and a certain loss of efficacy (Burri, 2010). Hence, there is an urgent

need for new drugs to treat the diseases that are safe, effective, affordable and easy-to-apply, mainly

those with demonstrating new in the mode of actions.

T. brucei subspecies are transmitted to mammalian hosts by the bite of tsetse flies from the family of

Glossina spp. The tsetse flies become infected when taking their blood meal on an infected mammalian

host and transferred to another. After the development cycle in salivary gland, the trypanosome

transforms into metacyclic form. The metacyclic trypanosomes are injected into the skin of mammalian

host during the feeding process and transformed into bloodstream trypomastigotes and multiply for a

few days before, invading the lymphatic system which defined as the first stage of HAT. Early symptoms

are rather nonspecific, including general malaise, high fever headache, and diarrhea. The second stage

start when the parasite crosses the brain and provoke neurological function breakdown, including

psychological changes and also disruption to the sleeping patterns, which refer to common name of
‘sleeping sickness’ used for the disease and finally if leaf untreated, the patient will be coma and death

(Kayser et al., 2003).

Despite human trypanosome, a variety of other trypanosome species also attribute to animal

trypanosomiasis in a wide geographic distribution. Trypanosoma evansi is the causative agent responsible

for bovine trypanosomiasis and surra. The disease became a great concern to the tropical country mainly

Asian region. Malaysia is reported as endemic for this disease (Zainal Abidin 1992). A case of surra was

found in various livestock in early 2012 in Malaysia. Four animal species such as deer, cattle, buffalo, and

pig in Perak state affected for trypanosomiasis (Nurulaini et al., 2013). Tabanid fly is the main vector for

this disease that transmits the protozoa parasite to the animal hosts (Nurulaini et al., 2007). Even though

human infection by animal species of trypanosome is impossible but, there are some reported cases

describe the patient diagnosed with T. evansi and T.lewisi (a common parasite in rat) (Shrivastava et al.,

1974; Joshi, 2005, 2013; Truc et al., 2013).

Trypanosome and some related parasitic protozoa are affected by a form of programmed cell death

(PCD), which showed the hallmark of apoptosis. According to Nomenclature Committee on Cell Death

(NCCD), the term apoptosis describes a specific morphological aspect of cell death. PCD in trypanosome

parasites allows an efficient regulation of the size of parasites population (cell density) and also act as a

mechanism to maintain genetic stability and differentiation (Welburn et al., 2006). Even though, there are

lacking studies of PCD in protozoa, there are still reports found that substances as staurosporine,

Concanavalin A and prostaglandin A were observed be trigger to induce PCD in Trypanosoma brucei sp.

(Welburn et al., 1996; Figarella et al., 2006 & Barth et al., 2014).

Medicinal chemists have synthesized a number of drugs to treat parasitic diseases. However, a major

problem occurred when many of these drugs were developed many years ago and some parasite strains

have become resistant to them. The development of new antiparasitic drugs has not been a priority choice
for the pharmaceutical industries because many of parasitic diseases were happen in poor countries,

which they can’t afford to pay the expensive drugs. Thus, there will be a risk to invest in drug development

against the parasitic disease.

Alkaloids are one of the most important classes of the natural product providing drugs for humans as

most of them are well known because of their toxicity (cocaine, morphine and etc). Other than that, many

alkaloids also reported having an effect on the treatment of parasitic infection. The example is quinine,

isolated compound from Cinchona succirubra was used to treat malaria for more than three centuries. In

addition, few studies have been reported, the presence of indole moiety from alkaloid derivative could

display important antiprotozoal activities. With regard to this, alkaloid could be remarkable potential to

provide pharmaceutical and biological agents contributing to the development of future antiparasitic

drugs.

D. costulata Hook, known as jelutong in Malaysia, is a tall forest tree spread in Southern Thailand,

Malaysia, and Sumatra. Some of the natural chemical compounds including alkaloid, triterpene, and

flavanoid are reported to be present in D. costulata (Mirand et al., 1983; Reanmongkol et al., 2002).

Ochrolifuanines is one of the compounds belong to alkaloid group was first discovered from leaf extracts

of D. costulata (Mirand et al. 1983). This compound has also been reported to be present in root bark of

Strychnos potatorum (Massiot et al., 1992). However, bioactivity of these compound was not fully been

studied.

The rich of biodiversity of Malaysia has remained untouched as far as the discovery of new chemical

entities is concerned. The previous study also discovered that has shown that Malaysia plant species has

potential antitrypanosomal activity in vitro (Norhayati et al., 2013). Therefore, in search of the potential

antrypanosomal compound from D. costulata was performed using bioassay-guided isolation approach to
identify the active compound. Various chromatography techniques were conducted until the active

compound obtained and followed by the structure elucidation to determine the active compound.

Whereas, mechanism of action studies on apoptosis were performed by using the terminal

deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and cell cycle arrest study being

performed using Flow Cytometry.

The objectives of this study are:

i. To isolate bisindole alkaloid compound, ochrolifuanine from Dyera costulata.

ii. To determine the antitrypanosomal activity of the compound against Trypanosoma brucei brucei

strain BS221 and determination of toxicity effect of the compound on normal cell and its selectivity toward

trypanosome protozoa.

iii. To investigate the cell death mechanism via apoptosis induced by the compound in trypanosome

protozoa