HEPATO CURATIVE AND ANTIOXIDANT EFFECT OF ETHYL-ACETATE AND N-BUTANOL FRACTIONS OF DETARIUM MICROCARPUM STEM BARK INDUCED LIVER DAMAGE IN WISTAR RATS

HEPATO CURATIVE AND ANTIOXIDANT EFFECT OF ETHYL-ACETATE AND N-BUTANOL FRACTIONS OF Detarium microcarpum STEM BARK INDUCED LIVER DAMAGE IN WISTAR RATS

ABSTRACT

Spondias mombin is used in folk medicine in Nigeria for the treatment of hepatitis. This study comparatively evaluates the in vivo hepato‑curative and antioxidant effects of Spondias mombin leaf (SML) and Spondias mombin stem (SMS) methanolic extracts in a rat model of hepatotoxicity. Methods: 42 rats were equally divided into seven groups of six animals each.  Group A received water, Group B water, Groups C and D received SML at 500 and 1000 mg/ kg bw, respectively, Groups E and F received SMS 500 and 1000 mg/kg bw, respectively, and Group G received silymarin at 100 mg/kg.  All extracts and drugs were administered daily by oral gavage for a total of seven days, and then for Groups B to G acute hepatotoxicity was induced by administering CCl₄. After 48 hours rats were sacrificed and assayed for histological and biochemical indices of hepatotoxicity. Results: CCl₄ treatment induced liver injury, with significantly increased levels of markers of hepatocellular injury alanine aminotransferase (ALT), aspartate transaminase (AST) total bilirubin (TBIL) and conjugated bilirubin (CBIL), as well as a significant reduction of total circulatory protein.  SML or SMS plant extracts at 500 and 1000 mg/kg prior to CCl₄ treatment significantly ameliorated liver injury, and decreased the levels of ALT, AST, TBIL, and CBIL. SML or SMS extracts significantly increased cellular levels of glutathione, the activities of catalase and superoxide dismutase, and significantly decreased thiobarbituric acid reactive substances. Conclusion: This study provides preliminary evidence supporting the potential benefit of Spondias mombin for treatment of xenobiotic‑induced hepatotoxicity.

CHAPTER ONE

INTRODUCTION

Background to study

The liver plays an important role in regulating various physiological processes. It is essential in the body for maintenance, performance and regulating homeostatic functions. It is involved with almost all the biochemical pathways for growth, fight against diseases, nutrient supply, energy provision and reproduction. In addition, it aids metabolism of carbohydrate, protein and fat, detoxification, secretion of bile and storage of vitamins (Ahsan et al., 2009). Because of its central role in drug metabolism, it is the most vulnerable tissue for drug toxicity (Sunil et al., 2012). The role played by the liver in the removal of substances from the portal circulation makes it susceptible to persistent attack by offending foreign compounds, culminating in liver dysfunction (Bodkhe and Ram, 2007). The liver secretes bile, prothrombin, fibrinogen, blood-clotting factors and heparin, a mucopolysaccharide sulfuric acid ester that prevents blood from clotting within the circulatory system (Bhawna and Kumar, 2010). Toxic chemicals, xenobiotics, alcohol consumption, malnutrition, anaemia, medications, autoimmune disorders (Marina, 2006), viral infections (hepatitis A, B, C, D, etc.) and microbial infections (Sharma and Ahuja, 1997) are harmful and cause damage to the hepatocytes.

Liver diseases contribute markedly to the global burden of diseases and are major causes of illness and death worldwide.^[1-4] Liver diseases remain a public health challenge, for which the development of new pharmaceutical treatments are required.  The evaluation of the hepatoprotective benefits of medicinal plants using laboratory animals is a useful initial step in determining drug safety of new biomolecules. ^[5-9] Natural products from ethnomedicine provide safe and effective alternative treatments for hepatotoxicity. Many previous report have associated these hepato-protective effects with endogenous phytoextracts or phyto-compounds that are rich in natural antioxidants. an increasing number of bioactive compounds and evaluated for hepato-protective and antioxidant.

Antioxidants are broadly divided into enzymic antioxidants and non enzymic antioxidants. Enzymic antioxidants include the superoxide dismutases, glutathione peroxidase and catalase (Klaunig and Kamendulis, 2004). Non-enzymic antioxidants, which include vitamin E, vitamin C, β-carotene, reduced glutathione, and coenzyme Q function to quench reactive oxygen species (Clarkson and Thompson, 2000). Antioxidants have various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction and radical scavenging (Rao et al., 2004). Many chemicals damage mitochondria, an intracellular organelle that produces energy, its dysfunction release excessive amount of oxidants which in turn damage hepatic cells. Activation of some enzymes in the cytochrome P450 system, such as CYP2E1, also leads to oxidative stress (Jaeschke et al., 2002).

Carbon tetrachloride (CCl4) is a well known hepatotoxin used in diverse experimental models (Singh et al., 2008). In addition to hepatic problems, it causes dysfunction of the kidneys, lungs, testis, brain, and blood by generating free radicals (Ozturk et al., 2003; Khan et al., 2009). Carbon tetrachloride (CCl4) is rapidly transformed to trichloromethyl radical (CCl3*) and its derivative trichloromethyl peroxy radical (CCl3OO*), generated by cytochrome P450 of liver microsomes (Brent and Rumack, 1993). These free radicals react with membrane lipids leading to their peroxidation (Singh et al., 2008). Membrane disintegration of hepatocytes with subsequent release of membrane associated enzymes and necrosis are some of the consequences of CCl4 induced liver damage.

1.2 Statement of Research Problem

It has been suggested that natural antioxidants in food, such as phenolic compounds or flavonoids, might play an essential role in the prevention of oxidative stress-related disorders and diseases, and in the reduction of premature mortality. ^[21-22] Flavonoids are certainly ubiquitous in the epidermal cells of plant parts such as the flowers, leaves, stems, roots, seeds, and fruits, and exist in glycosidic and non-glucosidic forms.^[23]

Spondias mombin L. (Anacardiaceae) is commonly known as (English), akika (Yoruba), ijikara (Igbo), tsadarmaser (Hausa), chabbuh (Fulani), nsukakara (Efik) and “atoa” (Ashanti).^[24] It is a deciduous erect tree, which grows up to 15-20 meters in height with a trunk 60-75 cm wide. ^[25-26] Spondias mombin  is commonly found in the tropical Americas, including the West Indies, and has been naturalized in parts of Africa, including Ghana, and some parts of Asia.^[26]

In ethnomedicine, Spondias mombin  plant parts including the  stem-bark, leaves, and roots have used for the treatment of various conditions. Spondias mombin possesses anti-microbial^[27,28], and anti-viral activities^[29], with leaves used, for example, for their anti-inflammatory^[30], anthelmintic activity^[31], haematinic^[32], and sedative^[33]activities, and stem-bark has an anti-mycobacterial^[34] activity Phytochemical screening indicates that the Spondias mombin leaf (SML) contains tannins, saponins, alkaloids, flavonoids and phenols.^[35] The leaves are also rich in ascorbic acid, niacin, and contain riboflavin and thiamine.^[35]Although the hepatoprotective effects of SML and Ocimum gratissimum have been evaluated in rats after intoxication with dimethyl nitrosamine,^[36] the effects of SML or SMS on CCl₄-induced hepatotoxicity has yet to be assessed.

1.3 Justification

Liver diseases remain one of the major threat to public health and a worldwide problem (Asha and Pushpangadan, 1998). The use of medicinal plants with high level of antioxidant constituents has been proposed as an effective therapeutic approach for hepatic damages (Govind, 2011).

Many antioxidants have been used to protect organs from the free radical challenges. Several researches are attempting to explore the possibility of using herbs containing antioxidants as organ curative agents. In view of severe undesirable side effects of synthetic agents, there is a growing focus to follow systematic research methodology and to evaluate scientific basis for the traditional herbal medicines that are claimed to possess hepatocurative activity.

Though the stem bark of Detarium microcarpum is used traditionally in Nigeria as a valuable remedy to treat liver diseases, no detailed pharmacological investigation have been done to that respect. This is why the present study is being undertaken to investigate it‟s hepato curative and antioxidant activity.

 1.4 AIM AND OBJECTIVES

1.4.1 Aim

The aim of this study was to establish if SML and SMS methanolic extracts are hepato-protective to CCl₄-induced hepatotoxicity in rats.

1.4.2 Specific objectives

1.    To carry out a phytochemical screening of the SML and SMS extract.

2.    To determine the Effect of Spondias mombin (leaf) and Spondias mombin (stem) on biochemical parameters

3.    To determine the Effect   of       SML and     SMS on           haematological parameters

4.    To determine Effect of SML and SMS on hepatic biomarkers

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