IN VIVO EFFICACY OF METHANOLIC EXTRACTS OF ROOT AND LEAF OF MORINDA LUCIDA IN PLASMODIUM BERGHEI – INFECTED MICE

ABSTRACT

Therapeutic effects of the methanolic root extract and a combination of leaf and root extracts of Morinda lucida were evaluated for antiplasmodial activities. The phytochemical  screening  of  the  methanolic  root  extract  showed  the  presence  of saponins, flavonoids, alkaloids, phenolic nucleus and phlobotannins. The LD50 of the crude methanolic root extract was calculated to be 3807.89mg/kg and percentage suppression of parasitaemia was 56.30, 59.84, 67.72 and 81.80% for doses of 100, 200, 400mg/kg body weight (b.w) and 5mg/kg chloroquine respectively. Effective dose dependent inhibitions of parasitaemia were also observed in the curative test and the mean survival period in days were 15.00 ± 0.70, 18.75 ± 0.5, 19.75 ± 1.39, 23.25 ± 1.38 and  8.75  ±  1.25,  for  100,  200,  400mg/kg  body  weight  of  the  extract,  5mg/kg chloroquine, and untreated control respectively. Percentage prophylaxis was calculated to be 24.26, 39.41, 43.67 and 84.91% for doses of 100, 200, 400mg/kg body weight of the methanolic root extract, and 5mg/kg chloroquine respectively. The crude methanolic extract of the root was partially purified by Column chromatography to give fractions 1- 4. Fractions 3 and 4, exhibited a significant curative effect in established infection. There was no significant difference at p>0.05 in antimalarial activity of fraction 4 and the  crude  extract.  Remarkable  parasitaemia  inhibition  by the  extracts  resulted  into longer mouse survival relative to the control, as demonstrated in the mean survival time of the mice (29.25±1.43, 11.25±0.75, 11.75±1.60, 24.25±1.11, 28.50±1.32, 28.00±0.00 and 5.75±2.14, for crude methanolic root extract, fraction 1, fraction 2, fraction 3, fraction 4 of the crude methanolic root extract, chloroquine and control groups respectively).  For the chloroquine group,  two  of the mice out  of four used  in  the experiment, survived beyond 30 days. In the combination study, it was observed that, the antimalarial activity for leaf and root was slightly more, compared to that of each of the extract used singly, as seen in parasite inhibition, after 5days of treatment (26.00, 20.00, 25.28, 21.35, 27.00, 19.50, 8.5 and 85.00), for 100, 200mg/kg leaf extract alone, 100 and 200mg/kg root extract alone, 50, 100mg/kg leaf and root extracts, 5mg/kg chloroquine and control groups respectively. The effect of methanolic root extract on some serum and liver enzymes in mice infected with Plasmodium berghei were also studied. Specific activities of Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate transaminase (GPT), Alkaline Phosphatase (ALP) and Gamma glutamyl transferase (GGT) were significantly (p<0.05) high in the infected not treated group, compared to the positive control (not infected, not treated). It is concluded that the methanolic root extract of Morinda lucida is potentially useful for the development of antimalarial drug.

CHAPTER ONE

1.0   INTRODUCTION

Malaria is a mosquito-borne infectious disease caused by a eukaryotic protist of the genus Plasmodium. It is widespread in the tropical and subtropical regions, including parts  of  the  Americas,  Asia,  and  Africa.  According  to  the  World  Malaria  Report, malaria is prevalent in 108 countries of the tropical and semitropical world, with 35 countries in central Africa bearing the highest burden of cases and deaths. Of the 35 countries that account globally for ~98% of malaria deaths, 30 are located in sub- Saharan Africa, accounting for 98.5% of the deaths in Africa, with four countries alone accounting for ~50% of deaths  on  the continent  (Nigeria,  Democratic  Republic  of Congo, Uganda and Ethiopia) (Rowe, 2006).

In sub-Saharan Africa,  approximately 365 million cases occurred in 2002 and 963 thousand deaths in 2000, equating to 71% of worldwide cases and 85.7% of worldwide deaths. Almost 1 out of 5 deaths of children under 5 in Africa is due to malaria. With increased efforts in controlling malaria in Africa in the recent years, it is reported that a total of 11 countries and one area in the African Region showed a reduction of more than 50% in either confirmed malaria cases or malaria admissions and deaths (Algeria, Botswana,  Cape  Verde,  Eritrea,  Madagascar,  Namibia,  Rwanda,  Sao  Tome  and Principe,  South  Africa,  Swaziland,  Zambia,  and  Zanzibar,  United  Republic  of Tanzania), whereas there was evidence of an increase in malaria cases in 3 countries in 2009 (Rwanda, Sao Tome and Principe, and Zambia) (World Malaria Report, 2010 and Rowe, 2006). However, these claims of improved malaria situation in Africa, as presented by the World Health Organisation (WHO), have been challenged (Roberts and Tren (2011) and Rowe, 2009).

Malaria is naturally transmitted by the bite of a female Anopheles mosquito. When a mosquito bites an infected person, a small amount of blood is taken, which contains malaria parasites. These develop within the mosquito, and about one week later, when the mosquito takes its next blood meal, the parasites are injected with the mosquito’s saliva into the person being bitten. After a period of between two weeks and several months (occasionally years) spent in the liver, the malaria parasites start to multiply within red blood cells, causing symptoms that include fever and headache. In severe cases, the disease worsens, leading to coma and death (Snow et al., 2005).

The vast majority of malaria deaths occur in Africa, south of the Sahara, where malaria also presents major obstacles to social and economic development. Each year, there are approximately 350–500 million cases of malaria (CDC), killing between one and three million people, the majority of whom are young children in sub-Saharan Africa (Snow et al., 2005).

Malaria is Africa’s leading cause of under-five mortality (20%) and constitutes 10% of the continent’s overall disease burden. It accounts for 40% of public health expenditure, 30-50% of inpatient admissions, and up to 50% of outpatient visits in areas with high malaria transmission. Malaria is commonly associated with poverty, but is also a cause of poverty and a major hindrance to economic development. Malaria has been estimated to cost Africa more than US$ 12 billion every year in lost Gross Domestic Product (GDP), even though it could be controlled for a fraction of that sum (World Malaria Report, 2010).

There are several reasons why Africa bears an overwhelming proportion of the malaria burden.  Most  malaria  infections  in  Africa  south  of  the  Sahara  are  caused  by Plasmodium falciparum, the most severe and life-threatening form of the disease. This region  is  also  home  to  the  most  efficient,  and  therefore  deadly,  species  of  the mosquitoes which transmit the disease. Moreover, many countries in Africa lacked the infrastructures and resources necessary to mount sustainable campaigns against malaria and as a result few benefited from historical efforts to eradicate malaria.

One of the greatest challenges facing Africa in the fight against malaria is drug resistance. Resistance to chloroquine, (Wellems, 2002) the cheapest and most widely used antimalarial, is common throughout Africa (particularly in southern and eastern parts of the continent). Resistance to sulfadoxine-pyrimethamine (SP), often seen as the alternative to chloroquine, is also increasing in east and southern Africa. As a result of these trends, many countries have no choice, but to change their treatment policies and use drugs which are more expensive, including combinations of drugs, which is hopefully believed will slow the development of resistance (Wellems, 2002).

Malaria remains a major public health problem in Nigeria, accounting for nearly 110 million clinically diagnosed cases per year, 60 per cent of outpatient health-care visits and 30 per cent of hospitalizations. At least 200,000 children die of malaria each year, and up to 11 per cent of maternal mortality is caused by the disease (Geoffrey and Paula (2009)).

Nation-wide in Nigeria, malaria accounts for one in every five deaths of children and 1 in 10 deaths of pregnant women. In addition to its direct health impact, the disease imposes a heavy social and economic burden: an estimated $900 million is lost to malaria annually in prevention and treatment costs and productivity loss (UNICEF, 2009).

Growing political commitment by African leaders for action on malaria was given a boost by the founding of the Roll Back Malaria (RBM) global partnership in 1998. Less than two years later African Heads of State and their representatives met in Abuja, Nigeria to translate RBM’s goal of halving the malaria burden by 2010 into tangible political action. The Abuja Declaration, signed in April 2000 endorsed a concerted strategy to tackle the problem of malaria across Africa. The Abuja Declaration endorsed RBM’s goal and established a series of interim targets for the number of people having access to treatment, protective measures or, in the case of pregnant women, receiving intermittent preventive treatment to ensure that progress would be made towards the goal and malaria-endemic countries and other RBM partners held responsible.

In Nigeria, Morinda lucida is one of the 4 most used traditional medicines against fever. It grows in grassland, exposed hillsides, thickets and forests. It is widely known and used, especially the root and leaf parts, in the southern and north-central parts of Nigeria as  a  remedy against  various  ailments,  including  malaria  fever  (Makinde  and  Obih (1985)).

Research Aim and Objectives

Aim: To investigate the in vivo efficacy of methanolic root and leaf extracts of Morinda lucida in Plasmodium berghei infected mice.

Objectives:

i.      Screening of crude extracts of Morinda lucida against Plasmodium berghei

ii.      Fractionation of crude extract by Column chromatography

iii.     Screening of fractions for antiplasmodial activity

iv.      Evaluation of polytherapy for antiplasmodial activity

Justification for this Research

Given the future scenario challenging the health sector such as inadequate health care, cost of producing drugs, additional research is needed in order to realize the full benefits of natural plants and respond to the health needs of people, especially in developing countries like Nigeria. History reveals that plants have always been considered as an important source of medicine against malaria: both quinine and artemisinin have been derived from traditional medicine and plant extracts. Artemisinin derivatives are now recommended by World Health Organization (WHO) worldwide, in combination with other drugs, such as lumefantrine, amodiaquine, mefloquine, sulphadoxine- pyrimethamine, as the first line of treatment of malaria. This fact has encouraged the continuing search for new natural product-derived anti-malarial drugs. One of the strategies in the search for new antimalarial compounds is a study of active constituents of medicinal plants. It is important, therefore, to investigate the antimalarial activities of Morinda lucida in order to determine the potential as source of new antimalarial agent. The plant parts (root and leaf) of Morinda lucida, “Ugigo” (local name in Ebira tribe), have been used as traditional remedy for the treatment of symptomatic malaria by the tribal population of Ebira land, Kogi State North – Central Nigeria, as such, the choice for their antimalarial screening in this work.

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