Discussion

5. DISCUSSION
Metals, a major category of globally distributed pollutants, are natural elements that have been extracted from the earth and harnessed for human, industry and products for millennia (Foulkes et al., 1990). Vanadium, a natural constituent of the earth, extensively in nature is a transitional metal that exists in different oxidative states in fossil fuels, industrial and environmentally degraded area (Pepato et al., 2008). 5.1 COURSE OF VANADIUM INTOXICATION Toxic metal pollution is a serious and insidious problem, as these are intrinsic component of the environment (Kerkvliet, 1995). Heavy metals are included in the main category of environmental pollutants as they can remain in the environment for long periods, their accumulation is potentially hazardous to humans, animals and plants (Benavides et al., 2005; Gbaruko and Friday, 2007). Last few decades have witnessed a dramatic, worrisome increase in contamination of the environment, including soil, air and water (Azevedo and Azevedo, 2006). These metals can enter the plant system, accumulate and later may enter the food chain and cause harm to animals and humans (Vitoria et al., 2001). There has been a significant application of metals in the various industries due to a general global increase in industrial activity over the past few decades. This in turn causes a great escalation of metal in the environment (Gupta et al., 2008). Although some metals are essential to human health, these may act as toxicant when humans or animals are exposed to high concentration (Kowalczyk et al., 2002). Even those metals that are essential, have the potential to turn into harmful at very high level of exposure is a reflection of very basic tenect of toxicology- the dose makes the poison (Howard, 2002). The primary source of anthropogenic sources of heavy metals are point sources such as mines, foundries, smelters, coal burning power plants, as well as diffuse sources such as combustion by products and vehicle emissions (Goyer, 1996; Raikwar et al., 2008). Human also affect the natural geological and biological redistribution of heavy metals by altering the chemical form of heavy metal released to the environment. Such alteration after effect a heavy

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metals toxicity by allowing it to bioaccumulate in plants and animals, bioconcentrate in the food chain or attacks specific organs of the body (Miranda et al., 2005; Mohamed et al., 2010). Humans are often exposed to heavy metals in various ways- mainly through the inhalation of metal in the work place or through the ingestion of food (Mor et al., 2009). The term heavy metal refers to any metallic chemical element that has relatively high density and is toxic at low concentration (Vasak, 2005). As level of these heavy metal rise in air, body, water and top soil it also get increased in human body and contribute in acute and chronic disorders (Canli et al., 1998; Boonchan et al., 2000). Heavy metal toxicity can result in damaged or reduced mental and central nervous function, low energy levels and damage to blood composition, lung, kidneys, liver and other vital organs (Kwiterovich, 1997; Dsouza et al., 2003) (Fig. 19). Recent studies have shown that metal such as iron, copper, cadmium, lead, mercury, nickel and vanadium exhibit ability to produce reactive oxygen species, resulting in lipid per oxidation, DNA damage, depletion of sulfhydryls and altered calcium homeostasis (Stohs and Bagchi, 1995; Flora et al., 2008).
Toxic metals

Damage to antioxidant defense system

Depletion of thiol status

ROS

Lipid

Proteins

DNA

Lipid peroxidation

Protein oxidation

Oxidized nucleic acids

Membrane damage

Protein dysfucntion

Impaired DNA repair

Cell death

Mutagenicity carcinogenesis

Fig. 19: Possible mechanism for metal induced oxidative stress (71)

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Vanadium is the 23 rd element of the periodic table and is widely distributed in the environment (Noda et al., 2003; Ghanny, 2007). Vanadium enters the organism by inhalation, skin and gastrointestinal tract and accumulates mainly in the liver, kidney, spleen, bones and lungs (Chandra et al., 2007; Rashwan and AlFirdous, 2011). The toxicity of vanadium is low, increases with the increase in valancy (Adachi et al., 2000; Jain et al., 2007). Vanadium compounds exert a variety of toxic effects depending on the nature of species, dose, route and duration of administration (Krosniak et al., 2009). Vanadium pentaoxide is the most common commercial form of vanadium and is the primary form found in industrial exposure situations (Awofolu, 2004; Uche et al., 2008). Vanadium pentaoxide is one of the most stable salts of vanadium which is a relatively abundant element with a very wide spectrum (Rivedal et al., 1990; Rojas et al., 1996). It occurs in the minerals as vanadinite, chileite, patronite and carnotite (Budavari et al., 1996). Exposure to vanadium pentaoxide in the work place occur during the cleaning of oil-fired boiler and furnaces, the handling of catalysts in the chemical manufacturing industry, and during the refining, processing, burning of vanadiumrich mineral ores of fossil fuels (Zenz, 1994). Vanadium pentaoxide is used in the production of aniline black dye, to dye ceremics and as a mordant in colouring textiles (Lewis, 1997). The entry of vanadium into the organism depends on the routes of administration. Once it has entered, vanadium (V) is reduced to cationic vanadyl (IV) form by glutathione of erythrocytes or by ascorbic acid in stomach before being absorbed in the duodenum (Sabbioni and Marafante, 1981; Hirano and Suzuki, 1996; Evangelou, 2002; Shukla et al., 2006). Again, vanadium in its anionic vanadate form has been found to absorb at much higher quantities (about five times more than vanadyl form) through anionic transport system (Heinz et al., 1982; Li et al., 1996). Again vanadate after reaching the blood stream is converted into vanadyl ion, although the vanadate form also exists (Yanardag and Tunali, 2006). These vanadate (by transferrin) and vanadyl (by albumin and transferrin) forms are rapidly transported by blood proteins to various tissues (Sabbioni et al., 1993; Fantus et al., 1995; Shrivastava et al., 2011). Unabsorbed vanadium is excreted in feces (Setyawati et al., 1998). Vanadium has been reported to be excreted through bile and through

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urine (Alimonti et al., 2000). Significant amount of vanadium removed from the body through feces (Mukherjee et al., 2004) (Fig. 20).

Fig. 20: ADME of vanadium compounds Vanadium has been reported to possess the ability to cross the placental barrier and thus accumulate in the fetus (Underwood, 1971), is responsible for a number of effects like headache, diarrhea, emphysema, pneumonia, irritation in skin, eyes and nervousness, and also for manic depression (Shrivastava et al., 2007), besides being potent inhibitor of membrane bound ATPase (Nechay, 1984). It is recognised as industrial hazard affecting human and animal reproductive health adversely (Chandra et al., 2007). 5.2 LIPID METABOLISM FOLLOWING VANADIUM PENTAOXIDE INTOXICATION Environmental pollution is a world wide problem we are facing, it is the contamination of the ecosystem that causes instability, disorder, harm or discomfort to the living organisms (Al-Attar, 2011). Toxic heavy metals in water, air and soil are global problems that are growing threat to humanity. Heavy metals constitute some of

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the hazardous substance that are resistant to enviormental condition and they have tendency to accumulate in plants, animals and human tissues (Tikare et al., 2012). Heavy metals are common in industrial applications such as their use in manufacture of pesticides, batteries, alloys, electroplated metal parts, textile, dyes, steel industries etc. As result of human economic activity, the natural circulation of elments (including harmful ones) is subject to various distortions (Kowalczyk et al., 2002). Physiology of an individual is the symbol of health status which mainly consists of enzymological, biochemical and haematological functions (Miranda et al., 2005). Any alteration in physiological parameters can cause severe adverse effects. However, biochemical functions have an edge over others. Biochemical functions include protein formation, cholesterol formation, triglycerides formation and their status maintenance. Adverse changes in individuals biochemistry under heavy metal stress are directly linked with its metabolic activity and health status (Purcell et al., 2001). Liver is the largest metabolic organ of the body and is positioned beneath the diaphragm in the right hypochondrium of the abdominal cavity (Kumar et al., 2000; El-Maraghy et al., 2009). Being the major drug-metabolizing and drug detoxifying organ of the body, it is continuously and widely exposed to xenobiotics and hepatotoxics (Taju et al., 2011). Liver plays a central role in transforming and cleaning chemicals or xenobiotics and is susceptible to the toxicity induced by these agents (Dreher et al., 1997). The unique property of liver to metabolize substances and its close proximity with the gastrointestinal tract, it becomes highly susceptible to get injured by the xenobiotics, heavy metals in particular. Approximately 75% of blood reaching the liver arrives directly from gastrointestinal organs and then spleen through portal veins which bring xenobiotics in concentrated form. Numerous mechanisms may be cited to be responsible for either inducing hepatic injury worsening the damage process. It appears to involve 2 pathways- Direct hepatotoxicity and adverse immune reactions. In most instances, hepatic injury, is initiated by the activation of drugs or heavy metals to chemically reactive metabolites, which have the ability to interact with cellular macromolecules such as proteins, lipids and nucleic acids, leading to protein dysfunction, lipid peroxidation, DNA damage and oxidative stress (Chattopadhyay, 2003). Additionally, these

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reactive metabolites may induce disruption of ionic gradients and intracellular calcium stores, resulting in mitochondrial dysfunction and loss of energy production. Its dysfunction releases excessive amount of oxidants which in turn injures hepatic cells (Higuchi and Gores, 2003). Injury to hepatocytes and bile duct cells lead to accumulation of bile inside liver, this promotes further liver damage. This impairment of cellular function can culminate in cell death and possible liver failure. Hepatic cellular dysfunction and death also have the ability to initiate immunological reactions, including innate and adaptive immune responses. Stress and damage to hepatocytes result in the release of signals that stimulate activation of other cells, particularly those of the innate immune system, including kupffer cells (KC), natural killer (NK) cells, and NKT cells (Kashaw et al., 2011). These cells contribute to the progression of liver injury by producing proinflammatory mediators and secreting chemokines to further recruit inflammotry cells to the liver. It has been demonstrated that various inflammatory cytokines, such as tumor necrosis factors (TNF)-, interferon (IFN)- and interleukin (IL)-1 produced during hepatic injury are involved in promoting tissue damage (Bourdi et al., 2002) (Fig. 21).

Apoptosis Or Necrosis

NK or NKI cells

IFN IL-4

IL-12 IL-18

Kupffer cells

Oxidative stress

Glutathione depletion

Protein adduction

Reactive metabolite CYP450

Heavy metal

Fig. 21: Mechanism of liver injury

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Discussion

Most of the hepatotoxic substance (xenobiotics) damage liver cells mainly by inducing lipid peroxidation and other oxidative stress (Cini et al., 1994; Lynch and Price, 2007). Vanadium, a heavy metal with increased environmental circulation (Ani et al., 2007) due to anthropogenic activities, is of great public concern due to its toxicity and accumulative behaviour at specific target organs, the liver and kidney and brings about oxidative damage, lipid peroxidation and changes in hematological, reproductive and respiratory systems (Mukherjee et al., 2004; Kamal et al., 2012) by way of free radical production resulting in lipid peroxidation, which is a chemical mechanism capable of disrupting the structure and function of the biological membranes that occur as a result of free radical attack on lipids (Rehman et al., 2003; Samipillai et al., 2009). Free radicals are fundamental to many biochemical processes and represent an essential part of aerobic life and metabolism. Free radicals affect all important molecules of the cell such as lipids, proteins, DNA and carbohydrates (Bryan, 2006). Cellular components involving polyunsaturated fatty acid residues of phospholipids are highly sensitive to oxidation. A typical example of such chain reaction is the process of lipid peroxidation that may be initiated by a radical (eghydroxyl radical-OH) in membranes of cytoplasm, mitochondria, nucleus and endoplasmic reticulum (Pala and Tabakcioglu, 2007). The resulting, carbon-centered radical (L) adds rapidly to O2, generate a lipid peroxyl radical (LOO) that propogates the chain reacting with neighboring lipid molecule to generate another L and a lipid hydroperoxide. Peroxidation of lipid causes increase in membrane permeability and at the end cellular damage (Valko et al., 2007). Further, lipid peroxidation has the capability to get catalyze by itself (Droge, 2002). Alteration in the concentration of major lipids like cholesterol, high density lipoprotein (HDL) and triglycerides can give useful information on the lipid metabolism as well as predisposition of animals to atherosclerosis and its associated coronary heart diseases (Abolaji et al., 2007). Reactive oxygen species mediated oxidative damage to macromolecules such as lipids, proteins and DNA lead to alterations in aforesaid parameters. Heavy metal accumulation in various tissues is associated with increase in todays biggest killers: Cardiovascular disease and caner.

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Again, lipid an important biochemical component it forms cell membrane, several hormones and necessary for other cellular functions viz energy storage providing protective cushion and structural support to prevent injury to vital organs such as the heart, liver, kidney and spleen (Gupta et al., 2008) (Fig. 22)..

Lipids ingestion

Lipids stored as adipose tissue

Stored as energy reserve

Digestion and absorption

Synthesized from carbohydrates and proteins

Insulation Support vital organs

Lipids in blood as lipoproteins

Generate heat

Oxidized for energy

Excreted in faeces

Convert to brain and nerve tissue

Fig. 22: Lipid metabolism In the present study serum lipids show increasing trend after both acute and subacute treatment with vanadium pentaoxide. Vanadium pentaoxide on entering the cell gets transformed into highly toxic metabolite which brings morphological and biochemical changes. These active metabolites then bind to transferrin or albumin proteins and get transported to different tissues via blood and deplete glutathione and protein bound sulfhydryl group resulting in enhanced productions of reactive oxygen species which results in increased lipid peroxidation (Stohs et al., 2000; Rao et al., 2006).Vanadium pentaoxide induce ROS formation and raise ALT and AST levels (Shrivastava et al., 2007) due to hepatocellular necrosis or tissue lysosomal disruption and acute cellular injury (Cam et al., 1993; Patrick, 2002; Srinivasan and Ramprasath, 2012). Because of this cellular injury, the cell contents like lipids

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leakout from the tissue into the blood (Upasani and Balaraman, 2001), perhaps the most probable reason for elevated lipids in serum. Cholesterol which is an essential component of the animal lipids as primary component in the makeup of all cells, helps in developing the membrane besides being a source of energy. In the present investigation increase in serum cholesterol has been observed after acute and subacute vanadium pentaoxide intoxication and is in affirmation to Shrivastava et al., 2007. It has been reported that vanadium accumulates in various tissues such as liver kidney and brain. Vanadium pentaoxide induce lipid peroxidation process by which free radical production takes place. These free radicals may change the level of membrane bound enzymes having SH groups. The fluidity of the membrane is determined by fatty acid. Increased enzyme activity has been reported with change in levels of cholesterol and phospholipids. It has been found that liver gets necrotized by heavy metal. Fatty changes with the partial necrosis in the liver get elevated. Necrosis results in the cell lysis due to this synthesized cholesterol within the tissue, get mobilized into the blood stream, which perhaps seem to be the most plausible explanation for enhancement of cholesterol in serum. Another reason assigned for the enhancement of cholesterol is that vanadium pentaoxide exerts its effect by altering the gene expression of some hepatic enzyme like HMG-CoA which participates in cholesterol synthesis. Increase in cholesterol concentration might be due to increase in the concentration of acetyl CoA arising probably from induced -oxidation of fatty acids, since acetyl CoA is a key substrate in the biosynthesis of cholesterol (Rang et al., 1995; Toyin et al., 2008; Almasiova et al., 2012). Phospholipids are the major constituent of cell membranes and necessary for all vital cell processes. They are most important membrane building compounds and are amphiphillic molecules with unique physiochemical properties having both hydrophilic and hydrophobic parts to the molecules (Semalty et al., 2010). Phospholipids also play a significant role in intracellular signaling through membrane. Enhancement in serum phospholipids is revealed after acute and subacute vanadium pentaoxide stress in the present investigation. Vanadium pentaoxide

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depletes levels of -ketoglutarate (-KG) and other kreb cycle intermediates (Subramanian, 2006) and thus elevate levels of acetyl CoA which in turn enhances phospholipids (Velvizhi et al., 2002). The observation made in the present investigation is in affirmation to Essa et al. (2010). Further, increase of phospholipids is also due to interaction of vanadium pentaoxide with reactive biological entities to manifest their physiological, biochemical or pathological effects (Cortizo et al., 2000). The prime target sites for heavy metals are surface membrane possibly due to their exposed location and chemical reactivity (Reul et al., 1999; Aureliano and Gandara, 2005). The interaction of toxic metal with lipids or protein components alter membrane fluidity, vanadium effects indicated initial membrane damage. Further, it is well known that hydroxyl radicals are capable of membrane damage leading to a variety of pathologies including lipid peroxidation (Quiles and Lopez, 2004). ROS produced by vanadium pentaoxide caused alteration in lipid levels which change the membrane fluidity, ATPase activity and cellular functions. Cellular deterioration leads to release of phospholipids in serum (Ani et al., 2007; Morsy et al., 2012). Triglycerides are a type of fat that body uses to store energy and gives energy to muscles. In the present investigation triglycerides show increasing trend after vanadium pentaoxide treatment (Yadav et al., 2004). Liver is the largest metabolic organ of the body and widely exposed to heavy metals (Imranulla and Dev, 2008). Vanadium penatoxide is known to induce formation of reactive oxygen species (ROS) and consequently enhanced lipid peroxidation (Barceloux, 1999; Osuji and Adesiyan, 2005; Obianime et al., 2009). Enhance production of reactive oxygen species (ROS) are the byproducts of many degenerative reaction in many tissue, which affect the regular metabolism by damaging the cellular component (Dreher et al., 1997; Foyer and Noctor, 2002). Dietary fats, including cholesterol, are absorbed from the small intestine and transported into the liver by lipoproteins called chylomicrons which are large droplets of lipids with a thin shell of phospholipids, cholesterol and protein (Imafidon, 2010). Once chylomicrons enter the bloodstream, an enzyme called lipoprotein lipase break down the triglycerides into fatty acids and glycerol (Allouche et al., 2011). Increase in triglycerides level after vanadium pentaoxide intoxication may be attributed to hypoactivity of lipoprotein lipase in

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blood vessels which metabolises triglycerides. Present finding gain support by the observation made by Upasani and Balaraman (2001) Yowundu et al. (2011) after administration of lead and diesel petroleum respectively in albino rats. Another reason assigned for the increament of triglycerides is that vanadium pentaoxide may deplete the level of -KG and other kreb cycle intermediate and this could elevate level of acetyl CoA ( Dakshayani et al., 2002). This acetyl CoA may be used for the synthesis of fatty acids and cholesterol, since fatty acids of different sources are used as substrate for synthesizing triglycerides. Elevated level of acetyl CoA may increase triglycerides level. Present findings are in affirmation to Velvizhi et al. (2002) Essa et al. (2010) after ammonium acetate and ammonium chloride intoxication in rats respectively. Elevated triglycerides along with decreased absorption of fatty acids by adipose tissue is further supplicated with a low level of HDL, insulin resistance and increased risk of atherosclerosis (Terasawa et al., 2000). High density lipoprotein (HDL) is a lipoprotein (a combination of fat and protein) found in the blood and it carries about one-third to one-fourth of the total blood cholesterol. Further, lipoproteins are molecules that carry cholesterol through bloodstream. HDL being a good cholesterol removes excess cholesterol from the blood and takes it to the liver, where it is eliminated from the body. In the present investigation HDL showed decreasing trend after both acute and subacute vanadium pentaoxide treatment. Vanadium pentaoxide induce fall in HDL due to change in gene expression of some hepatic enzymes like HMG-CoA reductase (hydroxylmethyl-glutaryl-CoA), which in turn depresses LDL-receptor gene expression (Kantola et al., 1998; Kojima et al., 2004). Present findings are in accordance to Anad, 2005; Gupta et al., 2008 who also revealed similar results after chromium and heavy metal (Nickel II and chromium VI) respectively in rats. Low density lipoprotein (LDL) carries mostly fat and only a small amount of protein from the liver to other parts of the body. LDL referred as bad cholesterol because it builds up slowly in the walls of arteries feeding the heart and brain (Jackson, 1996). As a result of this, it forms plaque that clots the arteries thereby causing atherosclerosis and increasing the risk of high blood pressure (Durak et al., 2004).

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Very low density low protein (VLDL) contains very little protein. The main purpose of VLDL is to distribute the triglyceride produced by liver. High VLDL level can cause the buildup of cholesterol in arteries and increases risk of heart disease and stroke. Increasing trend has been seen in present study after acute and subacute vanadium pentaoxide intoxication. Increased concentration of serum lipids may also be attributed to increased lipolysis, mediated by increased nor-epinephrine release which act through interference with the intracellular function of Ca2+ in the cytoplasm, is in affirmation to Anad (2005). Another reason assigned for increase in serum lipids is biologically possible and could be due to either increased synthesis or due to impaired feedback inhibition. Present observation gains support by Newiary and Abdou (2009) who observed enhancement in serum lipids in albino rats after lead acetate intoxication. The cholesterol to HDL cholesterol ratio is a number that is a marker in predicting individual's risk of developing atherosclerosis. The number is obtained by dividing the cholesterol value by the value of the HDL cholesterol. High ratios indicate higher risks of heart attacks, low ratios indicate lower risk (Olatunji-Bello et al., 2001; Badal et al., 2011.The ratio of cholesterol to HDL referred to atherogenic index has also been used as indicator of cardiovascular disease (Manzella et al., 2001; Panagiotakos et al., 2003). Present investigation showed increasing trend in cholesterol/HDL ratio after acute and subacute vanadium pentaoxide treatment. The cut-offs for high risk of atherosclerosis was put at atherogenic index of greater than 5 (Ng et al., 1997). Since the values for all the treated groups are more than 5, it could be inferred that the vanadium possess positive risk for atherogenesis and needs amelioration for minimizing the raised levels of the serum biochemical parameters (vide supra). 5.3 BIOCHEMICAL MARKERS ASSESSING VANADIUM PENTAOXIDE INTOXICATION Industrial population is the biggest health hazard in the 21st century which dramatically increasing the overall environment load of heavy metal toxins in the environment (WHO, 2000; Mahour and Saxena, 2008). Heavy metal constitutes a heterogenous group of elements widely varied in chemical properties, biological

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category due to their toxic effects on plants, animals and human beings (ATSDR, 1999; Patrick, 2002). Heavy metal interferes with variety of body processes and is toxic to the body systems including the cardiovascular reproductive, haemopoitic, gastrointestinal, renal and nervous system (Kwiterovich, 1997; Leonard et al., 2004). Toxic heavy metals target sites such as membrane or structural proteins, enzymes or DNA molecules (Florea and Busselberg, 2005). Contamination of heavy metals in the environment is a major global concern because of their toxicity and threat to human life and environment (Ceribasi and Yetis, 2001). Vanadium is widely distributed in the earths crust in a wide range of minerals and in fossil fuels, the chemical gets into the air, water and soil when fuel oil is burnt, or when rocks and soil containing vanadium are broken down (Olopade et al., 2011). Vanadium in mammalian organism usually has two oxidative states: (IV and V). The possibility of changes in the oxidative states by vanadium can cause positive and negative effects on the organism. The direction of this variation was strongly associated with certain parameters of the antioxidant defence and the vanadium complex and dose of this compound (Liboiron et al., 2005). Vanadium administration produced severe alterations in various blood/serum biochemical variables and in cellular components (Shrivastava et al., 2007). The blood, an important liquid connective tissue, flowing in the body performs the role of distribution of oxygen and various tissues with simultaneous expulsion of carbon dioxide. Serum, component of blood had pleothera of enzymatic activities in terms of transaminases (AST and ALT) and phosphatase (ALP) which reflects changes understress of vanadium. Serum enzymes measurement therefore, provides a valuable tool for clinical diagnosis of liver damage as well as toxicity studies (Ashafa et al., 2009). Transaminases (AST and ALT) are important critical enzymes in the biological processes and are an important class of enzymes linking carbohydrate and amino acid metabolism, the relationship between the intermediate of citric acid cycle is well established (Mehana et al., 2010). Their estimation in serum is a useful quantitative marker of extent and type of heaptocellular damage (Mitra et al., 1998). Liver, responsible for maintaining the bodys metabolic homeostasis has been considered as the target organ for the toxicity of xenobiotics and drugs, because most of the orally ingested chemicals and drugs first go to the liver where they are

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metablolized into toxic intermediates (Higuchi and Gores, 2003). Transaminases play important role in metabolism of non-essential amino acids. These enzymes commonly employed as diagnostic tools in the assessment of liver damage in clinical practice (Ha et al., 2001; Emeka and Funme, 2011). Transaminases transfer an amino group from an alpha-amino acid to an alpha-keto acid (transdeaminase). As a result of which different alpha-amino and a different alpha-keto acids are formed. Further, aminotransferase require pyridoxal-5-phosphate as a cofactor which is present in adequate amounts normally but it may be deficient in some pathological states leading to a reduced enzyme activity under stressful condition. In the present investigation the stress is vanadium pentaoxide. In the hepatocytes AST has two isozymes, one being cytoplasmic and other being bound to mitochondrial membrane (Rajesh and Latha, 2004; Baset and Reheem, 2009). ALT is only found in cytoplasm (Sallie et al., 1991; Senthil et al., 2003; Durgut et al., 2008). The membrane composition of hepatocytes is crucial for its function and targeting by toxicants. Vanadium pentaoxide inside the body produces reactive oxygen species (ROS) (Dill et al., 2004). This reactive oxygen species cause peroxidation of unsaturated fatty acids in biological membranes (plasmamembranes and mitochondrial membranes) leading to decrease of membrane fluidity and finally to the disruption of membrane fluidity and function (Halliwell, 1994; Saxena et al., 2009; Morsy et al., 2012). Disruption of thiol protein by vanadium is also a crucial part of cell injury, the enzymes (AST and ALT) leakout from hepatocytes to the tissue fluid and finally reaches the blood (Kaneko et al., 1997; Koriem, 2009). Present finding gains support by the observation made by Cam et al., 1993; Sharma et al., 2002; Nair, 2006; Shrivastava et al., 2007; Saxena et al., 2009; Saxena et al., 2010; Kilikdar et al., 2011; Ibrahim et al., 2012 who observed enhancement in ALT and AST activity after vanadyl sulphate, mercury, cadmium chloride, vanadium, arsenic trioxide, cobalt chloride, lead and lead acetate respectively. Phosphatases is a complex enzyme which performs multiple cellular and metabolicc functions such as growth differentiation, protein synthesis of certain enzymes and transport of phosphorylated intermediates across cell membrane and bone mineralization (Mahour and Saxena, 2008). Alkaline phosphatase (ALP) mainly

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arises from the lining of canaliculi and also from the sinusoidal surface of hepatocytes and excreted via bile. ALP is closely connected with proximal tubules and osteoblast and is involved in the active transport across the capillary walls (Naduka, 1999; Muriel and Escobar, 2003; Shrivastava et al., 2007). In the present investigation elevation in Alkaline phosphatase (ALP) activity shows significant increase after acute and subacute treatment of vanadium pentaoxide. Increased enzyme activity of ALP in serum could be due to damage to the cell membrane of tissues, where this enzyme is firmly attached to the cell membrane joining the biliary canalicules and sinusoidal border of parenchyma cells. Reactive oxygen species alter the functional integrity of cell membrane, thus necrosis or membrane damage which release the enzyme in circulation (Naduka, 1999; Mathur et al., 2002; Muriel and Escobar, 2003; Adedapo et al., 2007b). The increase in ALP level after metallic compound treatment are in accordance to Kumar et al., 2005; Moussa and Bashandy, 2008; Kansal et al., 2011; Gora et al., 2012; Shukla et al., 2012 who observed increament in ALP activity after mercury, lead, lead nitrate, arsenic and vanadium pentaoxide respectively (Fig. 23).

Vanadium compound (oral administration)

Free radical production

Peroxidation of unsaturated fatty acid in biological membrane Leads to Disruption of membrane fluidity, integrity and function

Leakout of transaminases and phosphatases in peripheral blood Fig. 23: Mechanism of the enhancement of serum transaminases and phosphatases in albino rat after V2O5 intoxication (84)

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5.4 ROLE OF LIV.52 IN MODULATION OF VANADIUM PENTAOXIDE INTOXICATION Plants have been important sources of medicine for thousand of years. Even today, the world health organization estimates that upto 80% of people still rely on traditional remedies such as herbs for their medicines. Ayurveda is a traditional and most commonly practiced form of medicine in India (Agnivesa, 2002). The concept of ayurveda is based on combined study of body (Sharira), sense organs (Indriyas), mind (Manas) and soul (Atman) (Sharma and Samhita, 2001). Equilibrium of all these is related to health. When an imbalance exists among anyone of the three doshas, ayurveda suggests a unique combination of food, exercise, meditation and herbs. The use of herbal medicine is widespread, which are used by the people for the treatment of dispartate diseases even at this modern era. There are diverse medicinal plants in the world, which are the impending sources of the drugs. These drugs are invariably single plant extract or fractions or mixtures of extracts/fractions from different plants, which have been carefully standardized for their safety and efficacy (Saeed et al., 2008; Naveen and Sree, 2012). The plant kingdom still holds many species of plants containing substances of medicinal value. Traditional use of any plant for medicinal purpose warrants the safety of such plants, particularly with regard to mutagenicity, nephrotoxicity, carcinogenicity and hepatotoxicity (Ashafa et al., 2009). Ayurvedic herbs stimulate the function of specific organs in the body, possibly by altering hormones, affecting immunity and neurotransmitters and conveying antioxidant properties (Millar, 1998). Cardiovascular problems have been dealt in detail in ayurveda, which describes Heart (Hridaya) as a body organ governing emotionals and circulatory blood to keep a person alive and healthy. It is now becoming a major health problem even in developing countries (Kannel, 1997). Ayurved has given different plants and formulations, which are useful in managing heart diseases (Murthy, 2001). A number of medicinal plants and their formulations are used to cure hepatic disorders in traditional systems of medicine (Saeed et al., 2008). Plant derived natural products have received considerable attention in recent years due to their diverse pharmacological actions including antioxidant and hepatoprotective activity (Wang et al., 2004).

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Heavy metals in the body may stimulate the formation of free radicals and reactive oxygen species, perhaps resulting in oxidative stress (Dietz et al., 1999). Free radicals are atoms or groups of atoms containing atleast one unpaired electron in their orbitals and can be formed when oxygen interacts with certain molecules. Once formed, these highly reactive radicals can start a chain reaction. Thats why it becomes necessary to remove toxic metals from the body or inhibit the production of free radicals. Use of antioxidant becomes necessary. Natural antioxidants are suggested as superior agent to reduce the oxidative damage in comparison to synthetic ones, when the internal enzymetic mechanism fails or are inadequately efficient (Goze et al., 2009) Liv.52, an indigenous preparation is reported to be a powerful hepatic stimulant which increases the functional capacity of the liver, protects the hepatic parenchyma by the way of its antioxidant properties and acts as a powerful detoxification agent (Baijal et al., 2004). Active ingredients of Liv.52 includeCapparis spinosa, Cichorium intybus, Solanum nigrum, Terminalia arjuna, Cassia occidentalis, Achillea millifolium, Tamarix gallica and mandur bhasma (Table-19).
Table-19 Liv.52 components belonging to plant for extract preparation and previous citation Capparis spinosa Cichorium intybus Solanum nigrum Terminalia arjuna Cassia occidentali s Achillea millifolium Tamarix gallica Mandur bhasm Part used Roots Seeds Seeds Bark Seeds Aerial Parts Whole Plant Ash Main constituents Alkaloids, flavonoid, rutin, quercetin Chicorine, choline, lactucin Glycoalkaloids, glycoproteins, gallic acid, naringenin Catechin, Flavones, glycosides Anthraquinones, glycosides, terpenoids, steroids Achilleine, camphor, eugenol, triterpenes Tamarixin, troupin, isoflavonones Calcinating iron rust Activity Antioxidant, antiinflammatory, anticarcinogenic Astringent, detoxicant Emollient, antitumrous, cardiotonic Astringent, diuretic, antimutagenic Antibacterial, antifungalm purgative Antiseptic, antispasmodic, stimulant, vasodilator, analgesic Antiallergic, antithrombotic, cardioprotective Hematinic, tonic Reference Mohammad (2011) Helal et al. (2011) Jain et al. (2011) Doorika and Ananthi (2012) Daniyan et al. (2011) Kumar et al. (2011) Chaturvedi et al. (2012) Devarshi et al. (1986)

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Roots of caper are used in Liv.52 contain considerable amounts of alkaloids, antioxidant, flavonoid compounds rutin and quercetin (Aghel et al., 2007; Behnaz et al., 2012)). Both these compounds are powerful antioxidant (Gadgoli and Mishra, 1999). Quercetin has antibacterial, anti-carcinogenic, analgesic and anti-inflammatory properties. Rutin strengthen capillaries and inhibits platelet clump formation in the blood vessels (Mohammad, 2011). Both these actions of rutin help in smooth circulation (Behnaz et al., 2012). It has also been found to reduce LDL cholesterol level, and exhibits a potent lipid lowering activity for (Eddouks et al., 2005; Shukla, 2007; Aniyathi et al., 2009). treating arteriosclerosis

Rutin

Quercitin

Cichorium intybus seeds are used as ingredient in Liv.52, it contains inulin, chicorine, choline, tannin, lactucin, chichoric acid, aminated acids, starch, protids, minerals and vitamins (B, C, K, P) (Nandagopal and Kumari, 2007; Heibatollah et al., 2008; Naseem, 2011). The tannin works as an astringent, disinfectant, detoxicant tonic (Milala et al., 2009; Wenying and Jingui, 2012). Chicory helps the body to better absorb calcium and other minerals. Chicory is effective in treating hepatitis. Due to the active compounds the glucose from the blood accumulates faster under the aspect of glycogen in the liver. This process leads to decrease in glycemia and cholesterol which reduces diabetes and artery sclerosis (Ahmed et al., 2003; Jamshidzadeh et al., 2004; Rahman et al., 2008; Helal et al., 2011).

Lactucin

Chicorine

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Solanum nigrum (black nightshade) possesses various compounds that are responsible for diverse activities (Lee and Lim, 2003; Atanu et al., 2011). The major active components are glycoalkaloids, glycoproteins, polysaccharides and vitamin A & C. It also contains polyphenolic compounds such as gallic acid, catechin, protocatechuic acid (PCA), caeic acid, epicatechin, rutin, and naringenin (Sikdar and Dutta, 2008; Gogoi and Islam, 2012). Solanum nigrum plant credited with emollient, diuretic, antiseptic, antitumrous, anti-inflammatory, cardiotonic and support healthy liver, kidney and bladder (Dhellot et al., 2006; Jain et al., 2011; Khattak et al., 2012).

Gallic acid

Naringenin

The bark of Terminalia arjuna contains tannin, flavonoids, alkaloids, catechin and saponin glycosides which may be the primary sources for arjunas beneficial effects on the heart (Sehrawat and Sultana, 2006; Doorika and Ananthi, 2012)). It is mainly used for both prevention and treatment of heart disease including angina and hypercholesterolemia (Bajpai et al., 2005; Islam et al., 2011). It is also used as astringent, diuretic, antioxidant, antibacterial and antimutagenic (Paarakh, 2010; Nema et al., 2012).

Catechin

Flavone

Seeds of Cassia occidentalis used in Liv.52, contain anthraquinones, carbohydrates, glycosides, cardiac glycosides, steroids, terpenoids, flavonoids, saponins and phytosterols (Sathya et al., 2012; Saqiq et al., 2012). These phytoactive

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compounds are well known for their wide pharmacological activities ranging from antibacterial and antifungal. The seed is bitter and has purgative properties, diuretic, liver detoxifier as an hepatotonic (Balances and Strengthens the liver). The capacity of phenolic compounds to chelate transition metals also lower the reactivity of metals ion by forming an inert metals-ligand complex (Egharevba et al., 2010; Daniyan et al., 2011).

Anthraquinones

Glycoside

Achillia millifolium (aerial parts) used in Liv.52 and contain active biological compounds achilleine, camphor, eugenol, saponins, proazulene, sterols, sesquiterpene lactones, tannins and triterpenes (Rangari, 2004; Vitalini et al., 2011)). Achillia millifolium considered to be antiseptic, antispasmodic, astringent, stimulant, vasodilator, hepatoprotective, anti-inflammatory, analgesic and anti-hyperlipidemic (Kumar et al., 2011; Armand, 2012).

Achilleine

Eugenol

Whole plant of Tamarix gallica used in Liv.52 syrup, it is rich in antioxidants such as tannin, tamarixin, tamarixetin and troupin (Drabu et al., 2012). Tamarix is found to be rich in polyphenolic compounds such as flavonoids, isoflavonones, phenolic acid, quercetin glycosides and terpenoid antioxidant compounds exhibit a wide spectrum of medicinal properties such as antiallergic, antiinflammatory, anti-thrombotic, cardioprotective and vasodilatory effects (Lefahal et al., 2010; Chaturvedi et al., 2012).

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Tamarixin

Isoflavones

Mandur is especially useful in anemia, amenorrhoea, dysmenorrhea, menorrhagia, chlorosis, hepatic and splenic disorders (Kapoor, 2010). It is a powerful hematinic and tonic and is valuable in the treatment of hemolytic jaundice and microcytic anemia (Devarshi et al., 1986). These plants produce an amazing diversity of secondary metabolites, tannins, terpenoids, alkaloids and flavonoids, phenolic compounds which posses antihyperlipidemic, antifungal (Ali-shtayeh, 1999; Mahasneh, 2002), antibacterial (Perumal et al., 1998), hepatoprotective (Sultana et al., 1995; Bin-Hafeez et al., 2001; Mun et al., 2002;) and antioxidant properties (Munasinghe et al., 2001; Germano et al., 2002). Mandur bhasma, an ayurvedic preparation of iron (prepared using complex ayurvedic processes like shodhana, marana etc.) has long been used in the treatment of liver diseases (Sharma, 1977; Devarshi et al., 1986) In recent years, flavonoids and other phenolic compounds of plant origin have received increasing attention, especially in the field of pharmaceutical sciences and medicine, due to their potential to prevent numerous chronic and degenerative diseases including cancer and cardiovascular diseases (Havsteen, 2002; Ross and Kasum, 2002; Surh, 2003; Boudet, 2007). These compounds have been shown to exert a wide range of antioxidant effect including in vitro, and most of their health-promoting effects have been attributed to their antioxidant effect including their ability to scavenge reactive oxygen species (ROS), chelate metal ions, hepatoprotective and terminate free radical reactions (Fang et al., 2002; Sanmugapriya and Venkataraman, 2006; Akachi et al., 2010; Liu et al., 2010). Phenolics (especially flavonoids) are able to alter peroxidation kinetics by modifying the lipid packing order (Arora et al., 2000). They stabilize membranes by decreasing membrane fluidity (in concentration-dependent manner) and hinder the diffusion of free radicals and restrict peroxidative reactions

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(Blokhina et al., 2003). Flavonols can interact with membrane phospholipids through hydrogen bonding to the polar head groups of phospholipids (Verstraeten et al., 2003). As a consequence, these compounds can be accumulated at the membranes surface both outside and inside the cells. Through this kind of interaction, flavonoids help to maintain membrane integrity by preventing access of deleterious molecules to the hydrophobic region of the bilayers, including those that can affect membrane rheology and those that induce oxidative damage to the membrane components (Michalak, 2006). Flavonoids exhibit different biological activties influencing numerous metabolic pathways. Due to their radical scavenging, antioxidant, antiinflammatory, antiallergic, anticancer, antiatherosclerotic, antiaggregational and detoxification activities they might be useful for prevention and treatment of many human diseases. There is increasing interest in antioxidants, the defense system of the body against free radicals. The main reason for the interest is the protection of cells, their organelles (especially membranes) and metabolic pathways against oxygen free radicals and their reactive derivatives (ROS). ROS participate in formation of chronic inflammation states and other diseases associated with oxidative stress, such as cancer, hypertension, atherosclerosis, cardiovascular and neurodegenerative diseases (Tiwari, 2001). Damage occurs when the capacity of antioxidant processes and detoxification mechanisms are lower than the amount of ROS production. Antioxidants interact with free radicals and stop them before vital molecules are damaged (Menghani et al., 2011). The role of antioxidants consists of scavenging free radicals and quenching singlet oxygen, disconnection of radical reactions, chelate metals which catalyze the oxidation process (Majewska et al., 2011). The liver is central metabolizing organ, as it is more susceptible to metabolism dependent injury (Singh et al., 2011). Vanadium produced ROS which induce oxidative stress. Oxidative stress occurs when there is an imbalance between free radical production and antioxidant defenses resulting deregulation of cellular functions (Kumar et al., 2004). Increased oxidative stress has been suggested in the pathogenesis and progression of many diseases like hyperlipidemia and their associated complications such as atherosclerosis, myocardial infarctions, neuropathy, nephropathy, retinopathy, micro and macrovascular damage and poor wound healing

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(Majumder et al., 2011). Hyperlipidemia is a metabolic disorder specifically characterized by alteration occurring in serum lipid and lipoprotein profile due to increased conditions of total cholesterol (TC), LDL, VLDL, Triglycerides (TGs) with a concominant decrease in the concentration of HDL in the blood circulation (Dhuley et al., 1999). Hyperlipidemia is a major cause of atherosclerosis and atherosclerosisassociated 2001). Medicinal plants have been found to play major role in antihyerlipidemic activity (Kaliora et al., 2006). Thus there is considerable interest on development of lipid lowering drugs from natural products in the recent years (Venukumar and Latha, 2002). Liv.52 contains substantial amount of antioxidants such as carotenoids, flavonoids, ascorbic acid, polyphenols, tannins terpenoids and -tocopherol (vide supra), which are believed to exert their antihyperlipidemic effect through their antioxidant property (Balakrishnan et al., 2012). Pre and post treatment of Liv.52 reduced serum cholesterol level which has been attributed to be due to reduction in the concentration of acetyl CoA resulting from decreased -oxidation of fatty acids since acetyl CoA is a key substrate in the biosynthesis of cholesterol (Rang et al., 1995). Further, hypocholestrolemia following Liv.52 could be due to presence of saponins present in Liv.52 ( Solanum nigrum, Capparis spinosa, Terminalia arjuna, Cassia occidentalis ). These saponins lower blood cholesterol by binding with it in the intestinal lumen thereby preventing its absorption (McDonald et al., 2005). Further, it also binds with bile acids causing a reduction in the enthrohepatic circulation of bile acids and increases fecal excretion of cholesterol (Sidhu and Oakenful, 1986). Increased bile acid excretion is offset by enhanced bile acid synthesis from cholesterol in the liver and the consequent lowering of serum cholesterol (Oakenful and Sidhu, 1995). Saponins may also compete with cholesterol biosynthesis in the liver (Lansky, 1993). Triglycerides are the main storage form of fatty acids. Liv.52 pre and post treatment showed reduction in triglycerides level, can be adduced to inhibition of conditions, such as coronary heart disease (CHD), ischemic cerebrovascular disease and peripheral vascular disease (Hardman and Limbird,

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lipolysis (Owoyele et al., 2005). It can also be attributed to the antioxidant activities of saponins which might have interfered with the oxidation of fatty acids (Rao, 1993). Vanadium pentaoxide is known to induce formation of reactive oxygen species (ROS) and consequently enhanced lipid peroxidation (Quiles and Lopez, 2004). ROS produced by vanadium pentaoxide caused alteration in lipid levels which changes the membrane fluidity, ATPase activity and cellular functions. Cellular deterioration leads to release of phospholipids in serum (Morsy et al., 2012). Hperlipidemia and elevated lipid peroxidation are inter-related (Varga et al., 1997). Hyperlipidemic condition probably stimulate the catabolic pathway via oxidative breakdown (Loeper et al., 1983). Pre and Post treatment of indigenous drug Liv.52 acts as liver tonic and shows protective effect by stabilizing plasmamembrane, thereby preserving the structural integrity of cell as well as the repair of hepatic tissue damage. Present investigation is in affirmation to Pari and Murugan (2004) and Girish et al. (2009). Liv.52 pre and post treatment caused significant increase in the level of HDL, HDL is considered to have anti-atherogenic properties. HDL concentration correlates inversely with coronary heart disease (Philips, 1995). This is because HDL removes cellular cholesterol and transports it to the liver where it gets converted to bile acids and eventually excreted out from the body (Mayes, 1996). Vanadium pentaoxide induces hyperlipidemia and hyperlipidemia enhances lipid peroxidation (Loeper et al., 1983) causing hepatotoxicty by increasing the free radical formation which inturn increase the level of peroxides in hepatic tissue (Mahendran and Devi, 2001) which consequently increases serum lipids (LDL and VLDL). Increased concentration of serum lipids has also been attributed to increased lipolysis, mediated by increased nor-epinephrine release which act through interference with the intracellular function of Ca 2+ in the cytoplasm, is in affirmation to Anad (2005). Another reason assigned for increased serum lipids could be due to either increased synthesis or to impaired feedback inhibition (Newiary and Abdou, 2009). Pre and post treatment of Liv.52 inhibit the lipid peroxidation and bring parameters towards normalcy (Rao and Subbarao, 1971; Mathur et al., 1987).

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Another reason that has been advanced towards normalcy of lipoproteins after pre and posttreatment of Liv.52 is understandable on the basis of reduction in cholesterol that finally results in reduction of LDL, since LDL represents the final stage in cholesterol catabolism (Mayes, 1996). LDL decreased significantly in dose dependent manner and this is due to increase in HDL concentration in the Liv.52 pre and post treated groups, as HDL major function is in the efflux of cholesterol from tissue thereby reducing the amount of cholesterol (Brown and Goldstein, 1984; Yokozawa et al., 2006). The ratio of cholesterol to HDL referred to as atherogenic index, has also been used as indicator of cardiovascular diseses (Pangiotakos et al., 2003). The old ratio was calculated and the cut-offs for high risk in the case of atherogenic index was >5 (Ng et al., 1997). Since the value for all the calculated atherogenic indices was less than 5, it could be inferred that extract Liv.52 does not possess positive risk for atherogenesis. Therefore, the calculated atherogenic index might be an indication that Liv.52 may not predispose to atherosclerosis and associated coronary heart disease. Liv.52 possesses antihyperlipidemic properties and this may help in reducing the incidence of cardiovascular disease like atherosclerosis under V2O5 stress. The Pearsons correlation reveals relationship of various parameters of lipid profile under stress of vanadium pentaoxide, V 2O5 and Liv. 52 and Liv. 52 and V 2O5 which is depicted in Tables numbers 15-17 and which further exhibits the response of lipid profile parameters in three different sets of experiments. Lipid profile gets severely affected even after the remedy of Liv.52, however, fluctuations in their content have been minimized and this minimization has been better seen with pre treatment of Liv.52. Correlation studies further supplicate that vanadium exerts its effect on lipid metabolism. Liv.52 minimizes this effect to considerable extent which can be utilized as a tool considering other heavy metals. Vanadium induces oxidative damage in different tissue by enhancing peroxidation of membrane lipids in tissue and altering the antioxidant systems of the cells (Subramaniam et al., 1994). The peroxidative damage to the cell membrane further causes injury to cellular components due to the interaction of metal ions with the cell organelles (Sarkar et al., 1995; Johnson and Krocning, 1998). Vanadium

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induced hepatic damage leads to substantial increase in the serum activities (AST, ALT and ALP). Free radicals generated by this metal contribute more than one hundred disorders in animals. Oxidation process is one of the important routes for producing free radicals in living system (Pourmorad et al., 2006). Generation of these free radicals and reactive oxygen species is stimulated in the presence of metals (Halliwel and Gutteridge, 1993) and this can seriously disrupt normal metabolism through oxidative damage to cellular components (Zengin and Munzuroglu, 2005; Chowdhury, 2009). To mitigate and repair the damage, plants are blessed with specific status of possessing complex antioxidant system. These antioxidant play an important role in the cellular defense strategy against oxidative stress, inducing resistance to metals by protecting labile macromolecule (Zhang and Kirkham, 1994; Galli et al., 1996). Liv.52 is an indigenous multiherbal hepatotonic where key ingredients are combined to achieve safe and synergistic effect (Kataria and Singh, 1997; Huseini et al., 2005). One active ingredient may be buffered by others, while certain ingredients play the role as adjuvants by strengthening the digestive system or helping to remove wastes or toxic substances (Chauhan and Kulkarni, 1971; Mayuren et al., 2010). Liv.52 a blend of various pure herbs and natural ingredients ( Capparis spinosa, Cichorium intybus, Solanum nigrum, Cassia occidentalis, Terminalia arjuna, Achillea millefolium and Tamarix gallica and mandur bhasma) which possess phenolic compounds, flavonoids, saponin, tannin and antioxidants, have been recognized as excellent scavengers of superoxide, hydroxyl ion and peroxyl radicals and as potent inhibitors of lipid peroxidation (Mathur et al., 1986; Bem et al., 1988; Klaassen and Liu et al., 1997; Mitra, 2000). Solanum nigrum, chichorium intybus and capparis spinosa are the important ingredients of Liv.52, possess various active components like bioflavonoids, glycoalkaloids, glycoproteins, antioxidants and polyphenolic compounds such as gallic acid, rutin and catechin which confers significant protection against vanadium pentaoxide induced liver injury and probable mechanism is by maintenance of structural integrity of hepatocyte cell membrane and due to their ability to suppress the oxidative degradation (Kulkarni, 1976; Rathore and Verma, 1987; Sultana et al., 1995). Mandur bhasma, an ayurvedic preparation of iron (prepared using complex ayurvedic processes like shodhana, marana etc.) has

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long been used in the treatment of liver diseases (Sharma, 1977) Liv.52 restored the increased levels of the hepatic enzymes, indicating repair of the hepatic tissue damage caused by heavy metals (Goel et al., 1991; Sandhir and Gill, 1999). Inhibition of lipid peroxidation and free radical scavenging activity has been suggested as a possible mechanism of action (Desai, 1976; Badhan et al., 1985). Administration of Liv.52 before and after vanadium pentaoxide treatment prevented liver damage. This finding indicates that probably Liv.52 conditioned the hepatic cells and prevented further damage to the liver parenchyma by virtue of which the leakage of the enzymes into the circulation was prevented (Prasad, 1975; Mandal, 1983; Sisodia and Bhatnagar, 2009). It is further added that Liv.52 provided quicker regeneration of hepatic parenchyma and its stimulating action markedly increase the functional efficiency of liver (Subbarao, 1976; Meyer and Kulkarni, 2001). Pretreatment of Liv.52 has an edge over post treatment as pretreatment alerts all metabolic pathways in a reactive state which combine efficiently with vanadium ions and play active role in minimizing the plight of this heavy metal compared to posttreatment.

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