ABSTRACT
This study was carried out to evaluate the hepatoprotective effects of the methanolic extracts of white grubs (anterior and posterior portions) on liver injury induced by carbon tetrachloride (CCl4) in rat (Rattusnorvegicus). The anterior methanolic extract (AME) and posterior methanolic extract (PME) were obtained from the incision of white grub’s abdomen and extracted by using methanol. Male albino rats weighing between 150g- 200g were used. Ten groups of rats (n=6) were administered orally, once in a day for five days with normal saline (negative control), 10mg/kg of silymarin (positive control) or 50mg/kg and 100mg/kg of posterior methanolic extract (PME) and anterior methanolic extract (AME) of white grubs respectively followed by hepatotoxicity induction using CCl4 on day 2 and 3 of the experiment. Blood and liver samples were collected for biochemical and histopathological studies. The extract was also subjected to antioxidant study using 2, 2-diphenyl-1-picrylhadrazyl (DPPH) radical scavenging assay. Rats pretreated with silymarim, PME (50mg/kg) and AME (100mg/kg) and normal control groups showed a significant decrease (p ≤ 0.05) in the levels of aspartate amino transferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP). Histopathologicalstudy showed intense vacuolation, hepatic necrosis and haemorrhage in CCl4 (negative control), PME (100mg/kg), AME (50mg/kg) pretreated groups;while normal hepatic structure was observed in groups pretreated with silymarin, PME (50mg/kg), AME (100mg/kg) and normal control groups. PME exhibited antioxidant activity. The study shows that PME (50mg/kg) and AME (100mg/kg) have hepatopotective effects against CCl4–induced liver damage.
CHAPTER ONE
1.0 INTRODUCTION
1.1 General Introduction
Traditional medicine has a long history. It is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses. The terms complementary/alternative/non-conventional medicine are used interchangeably with traditional medicine in some countries (WHO, 2013).
In some Asian and African countries, 80% of the population depends on traditional medicine for primary health care. In many developed countries, 70% to 80% of the population use some form of alternative or complementary medicine (e.g. acupuncture)(WHO, 2008). Herbal treatments are the most popular form of traditional medicine, and are highly lucrative in the international market. Annual revenues in Western Europe reached US$ 5 billion in 2003-2004 (Richter, 2003). In China, sales of such products totaled US$ 14 billion in 2005. Herbal medicine revenue in Brazil was US$ 160 million in 2007(WHO, 2008).
The healing of human ailments by using therapeutics based on medicines obtained from animals or ultimately derived from them is known as zootherapy (Costa-Neto, 2005). In modern society, zootherapy constitutes an important alternative among many other known therapies practiced worldwide (Alves and Rosa, 2005). Research interest and activities in the areas of ethnobiology and ethnomedicine have increased tremendously in the last
decade. Since the inception of the disciplines, scientific researches in ethnobiology and ethnomedicine have made important contributions to understanding traditional subsistence and medical knowledge and practice (Pieroni, 2005). Since ancient time animals, their parts and their products have constituted part of the inventory of medicinal substances used in various cultures (Mahawarand Jaroli,2007).
Entire organism and their body parts like flesh, bones, teeth, bone marrow, fat, shells musk, secretions, tentacles, as well as products like urine and excreta (dung) are used in traditional medicine. They are used either alone or in combination with other herbs and minerals. Some species of mammals have proved a vital source of traditional medicine (Padmanabhan and Sujana, 2008).Among the domesticated animals, the products of cow, buffalo, camel, goat, sheep and pigs are mostly used, while among the wild animals, the product of tiger, panther, leopard, monkey mongoose, fox, hare, samba deer, porcupine and wild boar are used (Adeola, 1992; Anagelettiet al., 1992). Body parts mostly used are exoskeleton, tissues, blood, fat, bones and gastrointestinal tracts. Similarly, insect larvae (White grubs) is used in the treatment of jaundice, fever, and general body weakness and as food ingredients by various communities in northern Nigeria (Alhassanet al., 2009).
White grub (WG) is the larval stage of many species of beetle. Dung beetles play a crucial role by burying dung in natural savannah and grassland used for cattle grazing in Africa (Brussard and Hijdrin, 1986). In addition to their effect on plants, other species biodegrade, environmental wastes. A functional group found in soil consists of a group of organisms that have the same function and similar impact on soil and are called macro fauna and white grub is one of them (Gitay and Nobel, 1997).
True white grubs are the larvae of May beetles (also called June Beetles) found in the genus Phyllophaga, of which there are over 100 different species. Phyllophaga larvae and other larvae of the family Scarabaeidae are often referred to as "white grubs", including larvae of the Japanese beetle (Popillia japonica Newman), annual white grubs (Cyclocephala spp.), and the green June beetle (Cotinisnitida Linnaeus) (Brian, 2013).
June beetles (Phyllophagaspecies) have a three-year lifecycle and cause most damage in the second year. In June, the adult lays eggs in the soil and within two weeks the white grubs emerge (Watschkeet al., 1995). The grubs feed on grass roots during the warm summer months, and then move deep into the soil for the winter. The grubs continue feeding the second summer, then transformed into the adult beetle during the third year (Hebda, 2010).
Many of the white grub species look similar to each other but vary in size. Mature grubs range in size from 1cm - 5cm. In general, grubs are C-shaped and have three pair of thoracic legs. The head capsule is dark, but the body is usually creamy white in color. The arrangement of hairs and spines on the posterior end of the grub, called the raster, is a distinguishing feature between white grubs’ species (Hodgson, 2007).
The May/June beetles require three years to complete one generation. Adults are usually larger in size compared to the masked chafer and Japanese beetle and range from tan to black in colour (Ralph, 1998). Adults start emerging from turf grass in May and June and are strongly attracted to lights like the masked chafer. During the first summer, mated females deposit eggs in turf grass. Eggs hatch into small white grubs that feed on small turf grass roots until the temperature cools. Small grubs are the overwintering stage until the
next spring when they become active again. During the second summer, grubs continue to grow while feeding on larger turf grass roots until they are almost fully-developed. The large grubs are the overwintering stage. During the third summer, grubs pupate in the soil and adults emerge in May and June (Hodgson, 2007).
The larvae of May/June beetle have a large, fleshy, white to translucent body with a brown head capsule, large jaws with three pairs of brown legs on the thorax. The posterior (hind) portion of the abdomen is slightly enlarge and appears darker. While the anterior portion of the abdomen is whitish in colour. Two parallel rows of spines on the ventral segment of the abdominal end distinguish true white grubs from other larvae (Hill, 2008).
White grubs among Hausa – Fulani is called ―Gwazarma or Doli – doli‖ and is used in the treatment of jaundice, fever and general body weakness, and as food ingredient by various communities in northern Nigeria (Alhassanet al., 2009). According to Murtalaet al.(2011) guinea pigs treated with 0.834g/kg aqueous white grubs extract for 48 and 96 hours had serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) very close to those of normal control but significantly decreased (at P>0.05) as compared to toxicity control . This would probably be due to hepatocurative effect of aqueous white grubs extract on chemically induced hepatocellular damage(Murtalaet al., 2011).Nevertheless, the serum cholesterol and triglyceride levels in guinea pigs after 48 and 96 hours of treatment with aqueous white grubs extract showed a significant increase in serum cholesterol and triglyceride compared to toxicity control (at p<0 .05="" alp="" alt="" and="" are="" ast="" cholesterol="" compared="" control="" decrease="" font="" in="" increase="" indications="" levels="" of="" serum="" tests="" the="" to="" toxicity="" triglyceride="">
possible curative effect of aqueous white grubs extract on CCl4 induced liver damage. Alhassan and Sule (2011) reported the percentage of humic substances (fulvic acid, humic acid and humin) in white grubs (WG) as 16.05 ± 4.28% fulvic acid, 9.50 ± 2.06% humic acid and 74. 65 ± 11.31% humin and that the mineral contents of white grub in terms of relative concentration was found to be in rank of K > Mg > Na >Ca>Mn> Zn > Fe > Cu.White grubs obtain these humic substances and mineral elements from the environmental waste and were found in appreciable amount to meet nutritional requirements and therefore, could be nutritionally and medically relevant (Alhassan and Sule, 2011).
The assumed hepatoprotective property was ascribed to any of the two white grub abdominal fluids. The antioxidant and hepatoprotective effects of the two abdominal components, of white grubs extract have not been investigated and therefore not known.
Liver is one of the largest organs in human body and the site for intense metabolism andexcretion. Thus, to maintain a healthy liver is a crucial factor for overall health and well-being. But it is continuously and variedly exposed to environmental toxins, and abused by poor drug habits, and alcohol and prescribed and over-the-counter drugs, which can eventually lead to various liver ailments like hepatitis, cirrhosis and alcoholic liver disease (Subranonium andPushpangadan, 1999; Sharma et al., 1991).
Carbon tetrachloride (CCl4) is widely used for experimental induction of liver damage. The principle cause of CCl4 induced hepatic damage in lipid peroxidation and decreased activities of anticooxidant enzymes and generation of free radicals (Shahjahanet al., 2004).
In long term repeated dose studies in animals, the liver has been shown to be the most sensitive organ regarding toxicity (Shahjahanet al., 2004).
1.2 Statement of the Research Problem
Whole white grub fluid is known to protect liver from injury induced by CCl4; whether this is conferred by one or all the two components of the white grub fluid has not been clearly determined and thus not known.
1.3 Justification
White grub is traditionally used in the treatment of jaundice, fever and general body weakness, and as food ingredient by various communities in northern Nigeria. The result of this study could add value or otherwise to the use of white grubs in traditional medications. It will also provide baseline information about hepatoprotective potentials of the two components (posterior methanolic extract and anterior methanolic extract) of white grubs extract. Moreover it provide basis by which bioactive compounds could be extracted from the grubs and characterized to produce drugs.
1.4 Aim of the study
To evaluate the prophylactic effect of the two abdominal fluids of white grub on liver injury in albino rats in order to standardized their therapeutic use.
To determine:
The protective effect of the anterior and posterior abdominal components of white grubs’ methanolic extracts (PME and AME) on liver injury.
The antioxidant activities of posterior abdominal component of white grubs extract using 2, 2-diphenyl-1-picrylhydrazil (DPPH) assay.
1.6 Hypotheses
The anterior and posterior methanolic extracts (AMEand PME) have no protective effect on liver injury.
The posterior methanol extracts (PME) have no antioxidant activities in DPPH assay.
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