Effect Of Aqueous Extract Of Lawsonia Inermis Leaves On The Liver Following Acute Ethanol-Induced Hepatic Damage In Wistar Rats
ABSTRACT
Lawsonia inermis commonly known as henna has been used traditionally, especially in ayurvedic medicine, for various conditions including liver ailments, and reported to have hepatoprotective properties. This study aims to study the effect of aqueous extract of L. inermis leaves in acute ethanol induced hepatic damage in adult Wistar rats. Thirty (30) female rats were equally divided into five (5) groups (I-V). Group I which served as negative control received distilled water (2 ml) for 8 days. Group II which served as positive control received 40 % ethanol (20 ml/kg) on the 8th day after seven days of distilled water. Group III which served as prophylactic group received 400 mg/kg of aqueous extract of Lawsonia inermis for seven days, and 40 % ethanol (20 ml/kg) on the 8th. Group IV served as the therapeutic group, and received 400 mg/kg of the extract for seven days after receiving 20 ml/kg of 40 % ethanol on the first. Group V received silymarin (70 mg/kg) for seven days before 20 ml/kg of 40 % ethanol on the 8th day, to serve as the reference drug. All animals were sacrificed on the ninth (9) day. Body weight changes and liver body weight index were determined. Liver tissues were collected for assessment of oxidative stress markers (superoxide dismutase, catalase, reduced glutathione and malondialdehyde), protein concentration, DNA fragmentation, and also for haematoxylin and eosin staining, and acridine orange-ethidium bromide staining. Body weight increased in all groups from initial mean weight of 121.5 g, though significantly (p<0.05) only in Group I and Group IV. Liver body weight index was highest in Group II, and was significantly (p<0.05) different from Group I and Group IV. Ethanol administration reduced levels of Superoxide dismutase, catalase, reduced glutathione and caused an increase in lipid peroxidation (Malondialdehyde), though insignificantly. Increase in levels of Superoxide dismutase, catalase, reduced glutathione and decrease in lipid peroxidation (Malondialdehyde) was observed in the groups receiving extract, more so prophylactically, though statistically insignificant. Silymarin significantly increased the level of Superoxide dismutase in comparison to Group II, levels of catalase, reduced glutathione, and Malondialdehyde were insignificantly less than Groups III and IV. Protein concentration was significantly higher in Group II, Group III and Group V, with Group II having the highest. Group III and Group V also differed significantly (p<0.05) from Group II. DNA fragmentation was observed to be most in Group II, while Group III and Group V had Fragmentation pattern comparable to that of Group I. H&E staining revealed attenuation of the effects of ethanol administration by the extract and silymarin, though the extract proved more effective prophylactically at 400 mg/kg dose and duration of seven days. Acridine orange-ethidium bromide (AO-EB) staining revealed reduced necrotic and apoptotic cells in Groups III, IV and V. The extract of Lawsonia inermis at 400 mg/kg for seven days proved to have more effective hepatoprotection prophylactically than therapeutically, and provided comparable hepatoprotection with that of silymarin.
CHAPTER ONE
Background
Alcohol consumption is customary in most cultures and alcohol abuse is common worldwide. For example, more than 50 % of Americans consume alcohol and 23 % of Americans participated in heavy and/or binge drinking at least once in a month (Substance Abuse and Mental Health Services Administration, 2011). The most commonly recognized symptoms of alcohol consumption are associated with chronic alcoholism, and it is a casual/risk factor in about 60 major types of diseases (Massey and Arteel, 2012). These, and other effects of alcohol consumption, has made alcohol the third leading risk factor globally for disease and disability (WHO, 2011).
Acute Alcohol exposure or binge drinking is defined in National Survey on Drug Use and Health (NSDUH) 2010 as five or more drinks on a single occasion within a time period of three hours (Massey and Arteel, 2012). However, it can also include a period of heavy drinking that may span several days or periods of intermittent, repeated episodes of heavy drinking (Massey and Arteel, 2012). Reports by NSDUH show that more than 58 million Americans participated in binge drinking on at least one occasion within thirty days in 2010 (Substance Abuse and Mental Health Services Administration, 2011) and acute alcohol consumption is a major underlying cause of morbidity and mortality during hospital admittance (Jones et al., 1991). Due to this, and increasing prevalence of binge drinking in people aged 18-25 (Massey and Arteel, 2012), greater interest has turned towards the effect of acute alcohol exposure. The liver is an organ of immense complexity that has fascinated mankind since antiquity. It is essential for survival as no other organ can compensate for its multiplicity of functions (Sirica et al., 2008). Ethanol is toxic, and the body begins to dispose of it immediately upon its consumption (Shakashiri, 2009). The liver, and to a lesser extent, the gastrointestinal tract are the main sites of alcohol metabolism (Lieber, 1970), with 90 % of it occurring in the liver (Shakhashiri, 2009). The susceptibility of the liver to alcohol-induced toxicity is due to both the high concentration of alcohol found in the portal blood (versus systemic), as well as the metabolic consequence of ethanol metabolism (Massey and Arteel, 2012). Potential mechanisms by which acute alcohol consumption causes damage include steatosis, dysregulated immunity and inflammation, and altered gut permeability (Massey and Arteel, 2012).
Medicinal plants are a part of human society to combat diseases, from the dawn of civilization (Bandyopadhyay et al., 2002). Among the estimated 2,500,000 higher plant species on earth, only 35,000 to 70,000 species (less than 1%) have been used for medicinal purpose (Ponnu et al., 2003). Medicinal plants have contributed immensely to health care in Nigeria (Adebolu and Oladimeji, 2005). This is due in part to the recognition of the value of traditional medical systems, and the identification of medicinal plants from indigenous pharmacopoeias, which have significant healing power (Adebolu and Oladimeji, 2005). Plants provide an alternative strategy in the search for new drugs. There is a rich abundance of plants reputed in traditional medicine to possess protective and therapeutic properties. It is likely that plants will continue to be a valuable source of new molecules which may, after possible chemical manipulation, provide new and improved drugs (Shah et al., 2006). There are plenty of chances to find out a new compound of plant origin (Farnsworth, 1988), including those used as ornamentals, amongst which is Lawsonia inermis (Latha et al., 2004). L. inermis commonly called henna is used all over the world, and is abundantly available in tropical and subtropical areas (Simon et al., 1984; Rao et al., 2005). Henna is a flowering plant, two to six meters in height. It‟s the sole specie in the genus Lawsonia in the family Lythraceae (Endrini et al., 2012). Some common names of L. inermis in different languages are: Henna (English), lalle (Hausa), lali (Yoruba), mehndi/heena (Urdu), mehndi (Hindi). Ancient history of India describes diverse uses and appreciable role of henna in Ayurvedic or natural health medicines (Lavhate et al., 2007). It has been used both cosmetically and medicinally for over 9,000 years. Traditionally in India, it is applied to hands and feet as a symbol of fertility (Chopra et al., 1956; Chetty, 2008).
1.2 Statement of Problem
Alcohol consumption is on the rise and health care is becoming less affordable, especially to majority of people in the third world countries. Thus a cheap, readily available way for management of liver damage caused by alcohol consumption is needed.
1.3 Justification
Worldwide, about 16 % of drinkers engage in heavy episodic drinking, often referred to as binge-drinking, which is the most harmful to health (WHO, 2014). Alcohol consumption increases the risk of developing more than 200 diseases (WHO, 2011). Lower-income groups are most affected by social and health consequences of alcohol, they often lack quality health care and are less protected by functional family or community networks (WHO, 2014). An alternative way of management of liver damage that is cheap and affordable, as well as easily accessible can be developed especially for the places where poverty is prevalent and tertiary health care is less affordable, using a plant that is cheap and found almost all over the world.
1.4 Significance of Study
Medicinal plants may serve as vital sources of potentially useful new compounds for the
development of effective therapies to combat a variety of health problems (Adebolu and Oladimeji, 2005). Different parts of L. inermis have been used traditionally in ayurvedic medicine in management and treatment of ailments including liver ailments (Lavhate et al., 2007). This study will help to determine if L. inermis can be used to provide a cheap affordable and easily available way of management of liver damage caused by alcohol consumption.
1.5 Aim and Objectives of the Study
1.5.1 Aim of the study
This study aims to investigate the effects of Lawsonia inermis leaves in acute Ethanol-induced liver injury in rats.
1.5.2 Objectives of the study
The study will investigate the following:
i. changes in histology of the liver, in Wistar rats administered aqeous extract of Lawsonia inermis (AELI) leaves and rats not treated with AELI, in acute ethanol-induced liver injury, using haemotoxylin and eosin stains.
ii. the presence of apoptotic, necrotic and normal hepatocytes histologically, in Wistar rats administered AELI and rats not treated with AELI, in acute ethanol-induced liver injury, using acridine orange and ethidium bromide stains.
iii. changes in superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and reduced glutathione (GSH) in the liver lysate of Wistar rats administered AELI and rats not treated with AELI, in acute ethanol-induced liver injury, as markers of oxidative stress using antioxidant assays.
iv. the level of DNA damage in the hepatocytes, in Wistar rats administered AELI and rats not treated with AELI in acute ethanol-induced liver injury, using gel electrophoresis.
v. the changes in protein concentration of the hepatocytes in Wistar rats administered AELI and rats not treated with AELI in acute ethanol-induced liver injury, using Bradford reagent.
1.6 Hypothesis
Lawsonia inermis provides hepatoprotection in acute ethanol-induced liver injury.
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