TOXICITY AND IMMUNOMODULATORY PROPERTIES OF METHANOL EXTRACT OF THE STEM BARK OF LANNEA ACIDA A. RICH. (ANACARDIACEAE) IN WISTAR RATS

ABSTRACT

Lannea acidaA. Rich.belongs to the family of Anacardiaceae; and the bark, leaves and roots have been reported to possess many medicinal properties, including immunomodulatory activity.Despite this widespread belief, data on the immunomodulatory properties and safety of this plant is still lacking. The aim of this work was to determine the toxicity and immunomodulatory properties of the stem bark of Lannea acida in Wistar rats. Methanol extract of Lannea acida A. Rich.was screened for phytochemical properties using standard methods. The acute and the subacute (28-day daily repeated dose) toxicity studies were performed using the OECD Fixed Dose Procedure guideline 420 and OECD guideline 407 respectively. To evaluate the immunomodulatory properties, 12 male and 12 female Wistar rats were pre-sensitised with 0.1ml sheep red blood cell intraperitoneally, and subsequently divided into 4 groups of 6 animals each (3 males, 3 females). The first group served as the control while the remaining three groups received daily oral doses of 250, 500 and 1,000 mg/kg body weight of methanol extract of L. acida, respectively for 14 days.The humoral response (IgM and IgG) to sheep red blood cells was determined by ELISA method and read by microplate reader. The cellular immune response was determined by estimation of the CD4+ and CD8+T-cell counts using flow cytometer, and measuring the delayed type hypersensitivity reaction from the paw thickness of rats given subcutaneous injection of SRBC. The methanol extract of the stem bark of L. acida was found to contain carbohydrate, reducing sugars, alkaloids, glycosides, anthraquinones, triterpenes, cardiac glycosides and tannins; and had an acute lethal dose that was> 3,000 mg/kg. The 28-day oral toxicity study showed a significant decrease (p < 0.05) in the relative organ weight percent of the spleen and kidneys of rats that received a dose of 1,000 mg/kg body weight compared with the control; and a significant decrease(p < 0.05) in the serum potassium levels across all the doses compared with the control. There was also a statistically significant increase(p < 0.05) in serum alanine transaminase level at the dose of 500 mg/kg.Histological examination of the organs following 28-day daily oral administration showed hepatic sinusoidal vascular congestion and peri-portal inflammation; splenic red pulp hemorrhage; kidney glomerular necrosis and tubulo-interstitial inflammation; and thymocyte depletion in the experimental groups compared to the control.There was significant decrease(p < 0.05)in the levels of serum immunoglobulin at the doses of 1,000 mg/kg for IgM and the doses of 500 and 1,000mg/kg for IgG. In conclusion, methanol extract of the stem bark of L. acida has relatively low acute toxicity but when given as a daily dosing for 28 days, it has hepatotoxic, nephrotoxic and immunotoxic effects and hence, potential for multi-organ toxicity when employed for sub-acute and chronic uses.

CHAPTER ONE

1.0 INTRODUCTION

1.1 Preamble

The use of medicinal plants for the treatment of various ailments has been part of mankind since antiquity. Despite the advancement of orthodox and pharmaceutical medicine, herbal medicines are still popular in Europe and North America because they are believed to be natural and safe. In the African folklore, many ailments have been treated and continue to be treated with herbal medicines. Many of today‟s pharmaceutical products have their precursors in medicinal plants (Cowan, 1999;Newman et al., 2000; Butler, 2004).

Lannea acida belongs to the family of Anacardiaceae, a major group of angiosperms (flowering plants). In the African traditional folklore, the bark, leaves and roots of L. acida areused for the managementof various ailments such as: conjunctivitis, sores, diarrhoea, stomach aches, gonorrhoea, rheumatism, fever, malaria, skin diseases, cough and dysentery (Koné et al., 2004; Ouattara et al., 2011a). The plant has also been suggested to have antibacterial properties against drug-resistant gram-positive organisms (Koné et al.,2004),as well as antidiarrhoeal (Etuk et al., 2009), immunomodulatory and anti-Mycobacterium H37Rv activities (Ouattara et al., 2011b). The stem bark also enhances the count, morphology and motility of sperm as well as the testosterone levels in male Wistar rats(Ahmed et al., 2010).

1.2 Statement of Research Problems

Many traditional herbal preparations find their way for use in humans without the empirical knowledge of their pharmacological properties and safety. This inadvertently worsens the health of the individuals who receive them. In most cases, the preparations do not possess the pharmacological properties and the efficacies that are attributed to them (Firenzouli and Gori, 2007). Preparations containing Lannea acida are used in humans for their immunomodulatory properties despite the paucity of data about their safety and efficacy. Therefore, there is the need to properly elucidate the safety and immunomodulatory properties of the plant Lannea acida.

1.3 Justification

Most African countries including Nigeria are saddled with high prevalence of diseases and the accessibility of drugs to combat these is still problematic. Pharmaceutical products are generally unaffordable and unavailable to the common people who suffer the greatest brunt of these diseases. The World Health Organization (WHO) estimates that about 80% of the population in developing countries still depends on traditional medicine for their primary health care needs (Bodeker et al., 2005),because they are accessible, available and affordable by most consumers.

The immune system has been noted to play a key role in the pathogenesis of many human diseases such as arthritis, ulcerative colitis, allergy, asthma, infections including HIV; and parasitic infestations, cancers and other diseases such as prion disease (Hansson et al., 2002; Opitz et al., 2010; Fong, 2014). The use of conventional drug products that stimulate or suppress immune systems (immunomodulators) have played a crucial role in the treatment of most of these diseases, but they are limited by their accessibility, costs and side-effects which make the sufferers of these diseases to resort to the traditional herbal medicine for their healing. However, the herbal medicine systems are not devoid of problems. There are safety and efficacy issues associated with their use. These concerns made the World Health Assembly (WHA) in her resolution 42.43 of 1989 to state inter alia that “member states should make a comprehensive evaluation of their traditional systems of medicine, to make a systematic inventory and assessment (pre-clinical and clinical) of the medicinal plants used by the traditional practice individuals and by the population; to introduce measures for the regulation and control of medicinal plant products and for the establishment and maintenance of suitable standards; and to identify those medicinal plants, or remedies derived from them, which have a satisfactory efficacy/side-effect ratio and which should be included in national formularies or pharmacopoeias.” (WHA, 1989).

Despite the report that L. acida may possess some immunostimulatory properties (Ouattara et al., 2011b), and that preparations containing the herb extracts may enhance immunity in HIV patients (USP, 2010), its specific role in immune system has not been well elucidated. This study therefore attempts to determine the safety, and immunomodulatory properties of L. acida.

1.4 Aim and Objectives

1.4.1 Aim:

The aim of this study is to determine the toxicity and immunomodulatory properties of methanol extract of the stem bark of L. acida in Wistar rats.

1.4.2 Specific Objectives:

The specific objectives of this study are:

1. To determine the phytochemical constituents of methanol extract of the stem bark of L. acida

2. To determine the acute oral toxicity of the methanol extract of the stem bark of L.acida in Wistar rats.

3. To determine the subacute oral toxicity of methanol extract of L.acida in Wistar rats.

4. To evaluate the immunological parameters (IgM, IgG, CD4+ and CD8+T-cell Counts)of Wistar rats administered with methanol extract of L. acida.

1.5 Research Hypothesis

Null Hypothesis:

Methanol stem bark extract of L. acida does not possess immunomodulatory properties and is not safe at oral dose of ≤ 3, 000mg/Kg in Wistar rats. Alternative Hypothesis:

Methanol stem bark extract of L. acida possesses immunomodulatory property and is safe at oral dose of ≤3, 000mg/Kg in Wistar rats.

1.6 Scope of the Work

This research involved the determination of the acute oral toxicity of L.acida using the fixed-dose method(OECD, 2002),and the sub-acute (28-day daily dose) oral toxicity (OECD, 2008).

The immunomodulatory properties of L.acida were determined by measuring the humoral (IgM and IgG) and cellular(CD4+ and CD8+T-lymphocyte) immune responses as well as the delayed type hypersensitivity response.