Hepatoprotective Antioxidant Activity Coccinia Grandis Root Extract Rats Biology Essay

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India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world. Plants have been used in traditional medicine for several thousand years. A vast majority of population particularly those living in villages depend largely on herbal medicines6, 18.

Medicinal plants form the backbone of traditional system of medicine in India. Pharmacological studies have acknowledged the value of medicinal plants as potential source of bioactive compounds. Phytochemicals from medicinal plants serve as lead compounds in drug discovery and design. Medicinal plants are rich source of novel drugs that forms the ingredients in traditional systems of medicine, nutraceuticals, modern medicines, food supplements, pharmaceutical intermediates, folk medicines, bioactive principles and lead compounds in synthetic drugs. WHO pointed out that more than 80% of world's population depends on plants to meet their primary healthcare needs47.

Drug-induced liver injury is a major health problem that challenges not only health care professionals but also the pharmaceutical industry and drug regulatory agencies. According to the United States Acute Liver Failure Study Group, drug-induced liver injury accounts for more than 50% of acute liver failure, including hepatotoxicity caused by overdose of acetaminophen (39%) and idiosyncratic liver injury triggered by other drugs (13%). Drugs are an important cause on liver injury. Approximately 75% of the idiosyncratic drug reaction results in liver transplantation or death51.

Liver disease is still a worldwide health problem about 20000 deaths found every year due to liver disorders. Unfortunately, conventional or synthetic drugs used in the treatment of liver disease are inadequate and sometime can have serious side effects10,58.

In India, about 40 poly herbal commercial formulations reputed to have hepatoprotective action are being used. It has been reported that 160 phyto-constituents from 101 brands have hepatoprotective activity. Liver protective herbal drugs containing a variety of chemical constituents like phenol, flavonoids, coumarins, alkaloids, lignans, glycosides, essential oils, carotenoids, mono terpenes, organic acids, lipids, and xanthenes. Plant extracts of many crude drugs are also used for the treatment of liver disorders. Extract of different plants of about 25 plants have been reported to cure liver disorders58.

Many formulations containing herbal extracts are sold in the Indian market for liver disorders. But management of liver disorders by a simple and precise herbal drug is still an intriguing problem. Several Indian medicinal plants have been extensively used in the Indian traditional system of medicine for the management of liver disorder. Some of these plants have already been reported to possess strong antioxidant activity58.

1.1 LIVER3

The liver is a large, solid gland situated in the right upper quadrant of the abdominal cavity. The liver is most completely covered by visceral peritoneum and by dense irregular connective tissue layer that lies deep to the peritoneum. In the living subject the liver is reddish brown in colour, soft in consistency and very friable. It is the largest gland in the body. It secretes bile and performs various other metabolic functions. It weighs about 1500 g in male and about 1300 g in females.

1.1.1 LIVER LOBES

The liver is the divided in to two principle lobes-a large right lobes and a smaller left lobe-by the faciform ligament, a fold of the peritoneum.

1.1.2 BLOOD SUPPLY

The liver receives 20% of its blood supply through the hepatic artery and 80% through portal vein. Before entering the liver, both the hepatic artery and the portal vein divided into left and right branches.

1.1.3 NERVE SUPPLY

The liver receives its nerve supply from the hepatic plexus, which contain both sympathetic and parasympathetic (vagal) fibers. Nerves also reach the liver through its various peritoneal ligaments.

1.1.4 HISTOLOGY OF LIVER35

The lobes of the liver are made up of many functional units called lobules. A lobule is typically a six-sided structure (hexagon) that consists of specialized epithelial cells, called hepatocytes (hepat- = liver; -cytes = cells), arranged in irregular, branching, interconnected plates around a central vein. Instead of capillaries, the liver has larger, endothelium-lined spaces called sinusoids, through which blood passes. Also present in the sinusoids are fixed phagocytes called stellate reticulo endothelial (Kupffer) cells, which destroy worn-out red blood cells and white blood cells, bacteria, and other foreign matter in the venous blood draining from the gastrointestinal tract.

Bile, which is secreted by hepatocytes, enters bile canaliculi (kan'-a-LIK-u'li = small canals), which are narrow intercellular canals that empty into small bile ductules. The ductules pass bile into bile ducts at the periphery of the lobules. The bile ducts merge and eventually form the larger right and left hepatic ducts, which unite and exit the liver as the common hepatic duct. Farther on, the common hepatic duct joins the cystic duct (cystic = bladder) from the gallbladder to form the common bile duct. Bile enters the cystic duct and is temporarily stored in the gallbladder.

1.1.5 EXTRA HEPATIC BILIARY APPARATUS

The biliary apparatus collect bile from the liver, stores it in the gall bladder and transmits it to the second part of the duodenum.

The apparatus consist of

The right and left hepatic ducts

The common hepatic ducts

The gall bladder

The cystic ducts

The bile ducts

1.1.6 HEPATIC DUCTS

The right and left hepatic ducts emerge at the porta hepatics from the right and left lobes of the liver.

1.1.7 COMMOM HEPATIC DUCTS

It is formed by the union of right and left hepatic ducts near the right end of the porta hepatis. It runs down wards and is joined by the cystic duct to the form the bile ducts.

1.1.8 GALL BLADDER

This is a pear-shaped reservoir of the situated in a fossa on the inferior surface of the right lobe of the liver. The gall bladder is 7 to 10 cm long 3 cm broad at its widest part, and about to 30 to 50 ml in capacity.

1.1.9 CYSTIC DUCT

This ducts about 3 to 4 cm long. It begins at the neck of the gall bladder, runs down wards, backwards and to the left, and ends by joining the common hepatic duct at an acute angle to form the bile duct.

1.1.10 BILE DUCT

It is formed by the union of cystic and common hepatic ducts near the port a hepatis. It is 8 cm long and has a diameter of about 6mm.Near the middle of the second part of the duodenum, it comes in contact with pancreatic duct and accompanies it through the wall of the duodenum.

1.1.11 FUNCTIONS OF LIVER

Metabolic functions: Liver helps in the metabolism of carbohydrates, proteins, lipids, vitamins, and many hormones.

Storage functions: Liver storage site for glycogen, certain vitamins (A, B12, D, E and K) and minerals (iron and copper).

Secretion of bile: Liver secretes bile, which contain bile salt, bile pigment, cholesterol, fatty acids and lecithin.

Synthetic function: Liver produces glucose by gluconeogenesis.

Excretory function: Liver excretes cholesterol, bile pigments, heavy metals (Pb, As, Bi, etc….), toxins, bacteria like typhoid and viruses (Hey fever).

Inactivation of hormones and drugs: liver catabolizes hormones such as growth hormones, parathromone, cortisol, insulin, glycogen and estragon. It also inactivates the drugs particularly the fat soluble drugs.

1.1.12 DISEASES OF THE LIVER

Acute hepatitis

Areas of necrosis develop as groups of hepatocytes die and the eventual outcome depends on the size and the number of the areas.

Viral hepatitis

Viral infections are the commonest cause of acute liver injury and include Type A, Type B and Type C hepatitis. The viruses enter the liver cells, causing degenerative changes leading to inflammatory reactions, accompanied by production of exudates containing plasma cells, lymphocytes and granulocytes.

Chronic hepatitis

This is defined as any form of hepatitis which persists for more than 6 months. It may be caused by viruses or drugs, but in some cases the cause is unknown.

Chronic persistent hepatitis

This is mild, persistent inflammation following acute viral hepatitis. There is usually little or no fibrosis.

Chronic active hepatitis

This is a continuing progressive inflammation with cell necrosis and the formation of fibrous tissue that may be lead to cirrhosis of liver.

1.1.13 HEPATOTOXICITY7

The liver is an important organ for metabolizing and detoxifying drugs. However, drugs and their metabolites can damage the liver. Some hepatotoxins are found in nature as fungal or bacterial metabolism, product of plants or as minerals. Many are product of pharmaceutical or chemical industry, still others are industrial by product or waste materials there by polluting environment and May again access to humans. Drug induced hepatotoxicity often resembles naturally occurring liver disease.

The mechanism of drug induced Hepatotoxicity3

Bioactivation Detoxification with

Cytochrome P450 Glutathione epoxide

Enzymes hydrolase

Drug Reactive metabolite Stable non-reactive

metabolism

Free Radical Electrophilic Excreted

Metabolites

Lipid peroxidation covalent binding to

Macromolecules

Membrane damage Neo antigen

Direct cell death Sensitized lymphocyte

1.2 PARACETAMOL

Paracetamol is a widely used anti-pyretic /analgesic and produces acute liver damage if overdoses are consumed and is mainly metabolized in liver to excretable glucuronide and sulphate conjugates . However, the hepatotoxicity of paracetamol has been attributed to the formation of toxic metabolites when a part of paracetamol is activated by hepatic cytochrome P-450 to a highly reactive metabolite N-acetyl-Pbenzoquinoneimine (NAPQI). NAPQI is initially detoxified by conjugation with reduced glutathione (GSH) to form mercapturic acid. However, when the rate of NAPQI formation exceeds the rate of detoxification by GSH, it oxidizes tissue macromolecules such as lipid or -SH group of protein and alters the homeostasis of calcium after depleting GSH. Paracetamol, an analgesic and antipyretic, is assumed to be safe in recommended doses; overdoses, however, produce hepatic necrosis. Small doses are eliminated by conjugation followed by excretion, but when the conjugation enzymes are saturated, the drug is diverted to an alternative metabolic pathway, resulting in the formation of a hydroxylamine derivative by cytochrome P-450 enzyme. The hydroxylamine derivative, a reactive electrophilic agent, reacts non- enzymatically with glutathione and detoxifies. When the hepatic reserves of glutathione depletes, the hydroxylamine reacts with macromolecules and disrupts their structure and function. Extensive liver damage by paracetamol itself decreases its rate of metabolism and other substrates for hepatic microsomal enzymes. Induction of cytochrome P-450 or depletion of hepatic glutathione is a prerequisite for paracetamol-inducedtoxicity24.

Paracetamol is a well-known anti pyretic, which produce hepatic necrosis at higher doses. This hepatotoxicant is primarily metabolized by sulfation and glucuronidation to reactive metabolites and then activated by the enzyme cytochrome P-450 system to produce liver injury. Its mode of action is by covalent binding of its toxic metabolite, n-acetyl p-benzo quinine-amine to tissue macromolecules, resulting in cell necrosis.

Paracetamol converted in to reactive toxic metabolites by hepatic microsomal cytochrome P-450. These free radicals bind covalently to un saturated lipid membrane, provoking a sharp increase of lipid peroxides following by pathological changes such as elevated level of serum marker enzyme like SGOT, SGPT and SALP, decreased protein synthesis, depletion of GSH, triglyceride accumulation, increased lipid peroxidation, destruction Ca2+ homeostasis and finally hepatocyte damage59.

1.3 HEPATOPROTECTION

The liver disorder are one of the serious health problem, throughout the world more than 350 million peoples were affected with chronic hepatitis infection and India about 20,000 deaths found every year due to liver disorder. Despite its frequent occurrence, high morbidity and high mortality, its medical management is currently inadequate, so far not get any therapy has successfully prevented the progression of hepatic disease, even though newly developed drugs have been used to treat chronic liver disorders, these drugs have often side effects5.

Herbal medicine derived from plant extracts increasingly utilized to treat a wide variety of clinical diseases. There is a growing interest in the pharmacological evaluation of various plants used in Indian traditional system of medicine. Many researches have been directed towards the provision of empirical proof to back up the use of many plants in traditional medical practices. Scientific evaluation of medicinal plants is important for the discovery of novel drugs and also helps to assess the risks of toxicity associated with the use of either herbal preparations or conventional drugs of plants origin.7

Free radicals and lipid peroxidative metabolites also cause damage to hepatocytes, leading severe necrosis, sepsis or endotoxemia. Paracetamol is a widely used hepatotoxin in rodents and its toxicity induced in rat liver closely resembles to human cirrhosis and hence it is an acceptable animal model for analysing hepatoprotective agents.

1.4 Free radicals25

free radicals are the chemical species (molecules or molecular fragments) that possess one or more unpaired electrons and have an independent existence. Free radical is conventionally represented by a superscript dot (R.). They are genetically unstable and very reactive. A free radical is easily formed when a covalent bond between entities is broken and one electron remains with each newly formed atom.

These free radicals are produced when the body uses oxygen for energy. This are also created when the body is exposed to pollution, machinery exhaust, cigarette smoke, other harmful environmental toxins, and aerobic metabolism.

Free radicals are highly reactive due to the presence of unpaired electrons. Any free radicals involving oxygen can be referred to as reactive oxygen species (ROS). Reactive Oxygen Species can damage cell by oxidising membrane phospholipids, proteins and nucleic acids. These damaging effects of ROS are normally kept under control by endogenous anti-oxidant system including Superoxide dismutase, Catalase, Peroxidase, Glutathione peroxidise.

NADPH oxidase on the plasma membrane and cytoplasmic enzymes such as xanthene oxidase and nitric oxide synthase can all generate super oxide anions (O2.). In addition, mitochondrial area a major source of ROS. Super oxide anion is produced by the electron transport chain on the inner mitochondrial membrane, and the rate of production is dependent on mitochondria protection. In the presence of mitochondria SOD, O2. can be converted to hydrogen peroxide (H2O2), which can then diffuse out of mitochondria in to cytoplasm. In the presence of high ion concentration, H2O2 can form the highly reactive hydroxyl radical (OH.) via the fention reaction. O2.radical can also react with nitric oxide to form the highly reactive peroxynitrate (ONOO.).

1.4.1 CAUSES OF FREE RADICALS

Alcohol

Smoking

Radiation

Cholesterol

Fat

Meat

Mental distress

Environmental pollution.

Food preservatives and pesticides.

1.4.2 CHARECTERISTIC OF FREE RADICALS

Extreme reactivity.

Damage to various tissues.

Short life span.

Generation of new ROS by chain reaction.

1.4.3 GENERAL FEATURES OF A FREE RADICAL REACTION35

Free radical reactions take three distinct identifiable steps.

Initiation step: Formation of radicals.

Propagation step: It is the heart of free radical reaction. In this step, the required free radical is regenerated repeatedly, which would take the reaction to complete.

Termination step: It involves the destruction of free radicals.

Initiation step:

RH R. + H

Organic Free

Compound radical

Propagation step

R. + O2 RO2.

RO2.+ RH RO. + OH. + R.

Hydroxy radical

Termination step

R. + H RH + I.

Stable free radical

RO2. + X Inactive products

(Termination by Inhibitor)

1.4.4 GENERATION OF FREE RADICALS

The free radicals are produced in the body in the following manner:

They are constantly produced during the normal oxidation of food stuffs, due to leaks in the electron transport chain in mitochondria. About 1-4% of oxygen taken up in the body is converted as free radicals.

Some enzymes such as aldehyde oxidase and xanthene oxidase form super oxide anion radical or H2O2.

NADPH oxidase enzyme in the inflammatory cells (Neutrophils, eosinophils, monocyte and macrophages) produce super oxide anion by a process respiratory worst during phagocytosis. The super oxide is converted to H2O2 then to Hypochlorous acid (HOLO) with the help of myeloperoxide (MPO) and super oxide dismutase (SOD). The super oxide and hypochlorous ions are the final effects of bactericidal action. This is a deliberate production of free radicals by the body. About 10% of oxygen uptake by macrophage is used for free radical generation along with the activation of macrophages, the consumption of oxygen by the cell is increases drastically this is called respiratory worst.

Macrophages also produce nitric oxide from Arginine by the enzyme nitric oxide synthase. This is also important anti-bacterial mechanism.

Peroxidation is also catalysed by Lipo-oxygenase in platelets and leucocytes.

Ionising radiation damages tissues by producing hydroxyl radicals, H2O2 anion reaction.

Light of appropriate wavelength can cause photolysis of oxygen to produce singlet oxygen. The capacity to produce tissue damage by H2O2 in minimal.

When compared to other free radicals (by definition H2O2 is not a free radicals).

But in presence free ion, H2O2 can generate hydroxyl radical, which is highly reactive. However in the body ion is always bound to proteins (transferrin and ferritin), minimising its catalytic role in hydroxyl radical production.

Cigarette smoke containing high concentration of various free radicals. Other toxic compounds such as carbon tetrachloride. Drugs and inhalation of air pollutants will increase the production of free radicals.

1.4.5 TYPES OF FREE RADICALS44

The most important free radicals in the body are the derivatives of oxygen and they are:

Superoxide anion (O2.)

Singlet oxygen (O.)

Hydroxyl radical (OH.)

Lipid peroxyl radicals (LO2.)

Hypochlorous acid (HOCl.)

Hydrogen peroxide (H2O2)

Nitric oxide (NO.)

Peroxy nitrate (ONOO.)

Other common free radical

Alkoxyl (RO.)

Peroxyl (RO2.)

Hydroperoxyl (HO2.)

Carbon centred radicals (CCl3.)

Hydrogen centred radicals (H.)

Sulphur centred radicals (RS.)

Superoxide anions (O2·)

Superoxide anion is a reduced form of molecular oxygen created by receiving one electron. Superoxide anion is an initial free radical formed from mitochondrial electron transport systems. Mitochondria generate energy using 4 electrons, reducing oxygen to water. Some of the electrons escaping from the chain reaction of mitochondria directly react with oxygen and form the superoxide anions. The superoxide anion plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide (H2O2), hydroxyl radical (OH.) or singlet oxygen (O.) in living systems. The superoxide anion can react with nitric oxide (NO.) and form peroxynitrite (ONOO.), which can generate toxic compounds such as hydroxyl radical and nitric dioxide.

(ONOO- +H+ .OH + .NO2)

Hydrogen peroxide (H2O2)

Hydrogen peroxide is a most stable reactive oxygen species. H2O2 is the primary product of the reduction of oxygen by various oxidase such as uricase, xanthine oxidase, α-hydroxyl acid oxidase and D-amino acid oxidase localized in peroxisome. Research shows that the H2O2 is the most effective species for cellular injury. It plays an important role in the production of more ROS molecules including HOCL (hypochlorousacid ) by the action of myeloperoxidase an enzyme present in the phagosomes of neutrophil and most importantly, formation of OH.via oxidation of transition metals.

H+ + Cl- + H2O2 HOCl + H2O

The well-known Fenton reaction is initiated when Fe2+ comes in contact with H2O2 ions of Co, Cu, and Ni can also participate in a similar reaction.

Fe2+ + H2O2 Fe3+ +.OH + OH-

H2O2 also react with O2. to initiate with Haber-Weiss reaction producing OH. in presents of Fe2+.

O2. + H2O2 O2 +.OH + OH-

The most important function of H2O2 is its role as an intercellular signalling molecule.

Hydroxyl radicals (OH.)

It is highly reactive and it may react with any molecule present in the cells and hence they are short lived. The life span of hydroxyl radicals at 370 C is 10-9 sec. The hydroxyl radical is formed from hydrogen peroxide in a reaction catalysed by metal ions (Fe+ or Cu+), often bound in complex with different proteins or other molecules. This is known as the Fenton reaction.

H2O2+ Cu+ / Fe2+ OH. + OH. + Cu+ / Fe3+

Superoxide also plays an important role in connection with reaction 1, by recycling the metal ions.

Cu2+ / Fe3+ + O2. Cu+ / Fe2+ + O2

The sum of reaction1 and 2 is the Haber-Weiss reaction; transition metals thus play an important role in the formation of hydroxyl radicals. Lipid is very sensitive to OH. attack and initiates LPO (Lipid peroxidation). The hydroxyl radicals are responsible for DNA damage and LPO.

Nitric oxide (NO.)

Nitric oxide is an inorganic free radical containing odd number of electrons and can form a covalent link with other molecules by sharing a pair of electrons. It is synthesized by an enzyme nitric oxide synthase located in various tissues and plays an active role in free radical tumour biology. NO is synthesized enzymatically from L-arginine by NO synthase.

L-arginine + O2 + NADPH L-citrulline + NO + NADP+

Nitric oxide is another free radical that has an important biological role. NO. produced in the body relaxes muscles in blood vessels, and lowers blood pressure. But, excess NO. produced in cases of severe infection can be harmful. Unlike HO. or O2., NO. is a much slower reacting radical and it combines with other free radical and inhibits further reaction.

1.4.6 DISEASES CAUSED BY THE FREE RADICALS25,44,36

The free radicals are generated during the normal metabolic reaction in the body. Free radicals are very unstable and react quickly with other compounds, trying to capture the needed electron to gain stability.

Some free radicals arise normally during metabolism sometimes the body's immune system cells purpose fully create free radicals to neutralise bacteria and virus. However environmental factors such as pollution, cigarette smoke and herbicide can also produce free radical. Normally the body can handle free radicals but if antioxidants are unavailable or if the free radical production is excess then damage can occur.

The formation of free radicals and the occurrence of oxidative stress is a common component of Parkinson's disease. Patients with Parkinson's disease have reduced glutathione levels and free radical damage is found in the form of increased lipid peroxidation and oxidation of DNA bases.

Cancer

Free radical act as promoting agents of cancer. This is attributed to gross chromosomal damage, altered gene expression, inhibition of DNA repair process etc.

Cardiovascular disease (CHD)

Oxidised low density lipoprotein (LDL), formed by the action of free radicals, have been implicated in the onset of CHD. ROS-induced oxidative stress plays a role in various cardio vascular diseases such as hypertension, ischemic heart disease, atherosclerosis, cardio myopathies, cardio hypertrophy and congestive heart failure.

Rheumatoid arthritis

Rheumatoid arthritis is an autoimmune disease that causes chronic inflammation of the joints and tissue around the joints with infiltration of macrophages and activated T cells. The pathogenesis of this disease is linked predominantly with the formation of free radicals at the site of inflammation. T cells isolated from the synovial fluid of patients with rheumatoid arthritis show signs of decreased intracellular GSH level.

Diabetic mellitus

It is a syndrome characterised by a loss of glucose haemostasis. Destruction of islets of pancreas due to the accumulation of free radicals in one of the causes in the pathogenesis of insulin dependent diabetes mellitus.

Neurological disorders

The brain is particularly exposed to oxidative damage because of its high oxygen utilisation, its high content of oxidizable polyunsaturated fatty acids and the presence of redox-active metals (Cu, Fe). Oxidative stress increase with age and therefore it can be considered as an important causative factor in several neurodegenerative diseases like Alzheimer's and Parkinson's disease especially for older individual.

Lung disease

ROS through oxidative changes exert a number of toxic effects in different biological systems. Such ROS induced events in lung disease is arachidonic acid (AA) release and metabolism of active products. In the lugs, the release of arachidonic acid from membrane bound phospholipids is by phospholipase A2. This reaction occurs due to specific stimuli by hormones such as epinephrine or bradykinin.

Infertility

Living spermatozoa produce ROS. Its excess production and long persistence could be a significant cause of male infertility. They produce less motile sperm, which are ineffective for fermentation. The abnormal spermatozoa are a primary source of free radicals.

Aging

Aging in humans is associated with changes in physical characteristics and the decline of many physiological functions. Increased accumulation of free radicals increases the risk of variety of oxidative stress in older individuals. These radicals are capable of causing apoptosis, necrosis and cell death.

1.5 ANTIOXIDANTS

Antioxidants are any substance, present at a lower concentration compared to that of oxidizable substance that delay or inhibits oxidative damage to a target molecule. Antioxidants neutralize free radicals by donating one of their own electrons, editing the carbon-stealing reaction. They act as scavengers, helping to prevent cell and tissue damage that could lead to cellular damage and disease. They are substances that protect other chemicals of the body from damaging oxidation reactions by reacting with free radicals and other reactive oxygen species within the body. One antioxidant molecule can only react with single free radical. There for there is a constant need to replenish antioxidant resources, whether endogenously or through supplementation.

ANTIOXIDANT

Non-enzymatic antioxidants

Enzymatic antioxidant

Flavonoids

Anthocyanidins cyaniding,

Pelagonidin

Isoflavanoids

Genistein

Hydroxyl, Cinnamic acid, Ferulic p-coumaric

Hydroxyl, Benzoic acids, Gallic acid, Ellagic acid

Flavonols, Chrysin

Flavonols, Hespertin

Flavonols, catechin, EGCG

Flavonols, Quercetin

Kaemferol

Phenolic aids

Antioxidant co-factor

Co-enzyme Q10

Low molecular weight antioxidants, glutathione, uric acid

Oragano sulphur compounds allium, alkyl sulphide, indoles

Carotenoids, β-carotene, lycopene, lutein, zeaxanthine

Vitamins A,C,E,K

Minerals Zn, Se

Poly Phenols

Secondary enzymes, Glutathione reductase,

G-6PDH

Primary enzymes, SOD, Catalase, Glutathione

1.5.1 CLASSIFICATION OF ANTIOXIDANTS

Antioxidants are broadly classified into two groups

Enzymatic antioxidants

Non-Enzymatic antioxidants

Enzymatic Antioxidants40

Superoxide dismutase (SOD)

Superoxide dismutase is an endogenously produced intracellular enzyme present essentially in every cell in the body. Cellular SOD is actually represented by a group of metalloenzymes with various prosthetic groups. The prevalent enzyme is cuprozine (CuZn) SOD which is a stable dimeric protein (32, 0000). SOD appears in 3 forms according to the catalytic metal present in the active site.

Cu-Zn SOD in the cytoplasm and contains copper and zinc as metal cofactors.

Mn-SOD in the mitochondria and contain Mn.

Extracellular SOD recently has been described contain copper (Cu-SOD)

2O2.2H++ SOD H2O2 + O2

SOD scavenges both intracellular and extracellular superoxide radical and prevents the lipid peroxidation of plasma membrane. However it should be conjugated with catalase or GPx to prevent the action of H2O2, which promotes the formation of hydroxyl radicals. SOD also prevents hyper activation and capitation induced by superoxide radicals.

Glutathione peroxidase (GPx) reductase/enzyme (GSH)

It is a tetrameric protein with MW of 84,000 and has very high activity towards H2O2 and hydro peroxides. It is found in both cytosol (70%) and mitochondria (30%) of various tissues. Glutathione peroxidase reduces H2O2 to H2O by oxidizing glutathione (GSH) reproduction of the oxidized form of glutathione (GSSH) and then catalysed by glutathione reductase. These enzymes also requires trace metal cofactors for maximal efficiency, including selenium for glutathione peroxidase; copper, zinc or manganese for SOD; and iron for catalase.

H2O2 + 2GSH GSSG + 2H2O (Glutathione peroxide)

GSSG + NADPH + H + 2GSH + NADP+ (Glutathione reductase)

GSSG : Oxidised glutathione

GSH : Reduced glutathione

Catalase

Catalase is a protein enzyme present in most aerobic cells in animal tissues and also present in all body organs being especially concentrated in liver and erythrocytes, while heart, brain, skeletal muscle contains only low amounts. Catalase and glutathione peroxidase decomposes hydrogen peroxide to water and molecular oxygen.

catalase

2H2O2 H2O + ½ O2

Non Enzymatic Antioxidants

Vitamin C (Ascorbic acid)

Ascorbic acid is the most abundant water soluble antioxidant in the body and acts primarily in cellular fluid. It is present in potatoes, citrus fruits and green leafy vegetables. Humans are unable to synthesise L-ascorbic acid from d-glucose, due to absence of the enzyme L-gulanolacetone oxidase. Thus vitamin C is obtained through the diet. Vitamin C is the most powerful antioxidant vitamin for the liver and reduces toxic damage to the liver cells from chemical overload. It neutralise free radicals generated during the phase 1 detoxification pathway in the liver.

Vitamin E (α-Tocopherol)

Vitamin E is the most abundant fat-soluble antioxidant in the body and the most efficient chain-braking antioxidant available. Natural vitamin E is biologically more active than synthetic vitamin E. Vitamin E is a powerful antioxidant that protects fats from damage. Since cell membranes are composed of fats, vitamin E is the best protector of cell membranes.

Major dietary sources of vitamin E are nuts, vegetable oils, seeds, wheat germ and whole grains. Approximately 40% of the ingested tocopherol is absorbed. The main function of tocopherol is to prevent the peroxidation of membrane phospholipids and avoid cell membrane damage through its antioxidant action.

α-tocophenol + LOO. α-tocophenol. + LOOH

α-tocophenol + LOO LOO- α- tocophenol

Because of antioxidant properties vitamin E neutralise ROMs reduce oxidative DNA damage and genetic mutations.

Natural carotenoids

Carotenoids such as beta-carotene are most commonly found in fruits and vegetables and are most significant for human health. It is important to take only natural sources of beta-carotene and other carotenoids. Beta-carotene gets converted in the body to vitamin A and yet has none of the toxic side effects of high doses of vitamin A. Large population studies have shown that low intakes of beta-carotene are associated with a higher incidence of cancer. Beta-carotene is a powerful protective antioxidant.

Thiamine (vitamin B1)

Thiamine is having antioxidant properties and is helpful in reducing the toxic effects of smoking, alcohol and lead. Thiamine deficiency is common in those who consume excessive alcohol.

Riboflavin (vitamin B2)

Riboflavin is required during phase one detoxification in the liver, and is crucial in the production of body energy.

Nicotinamide (vitamin B3)

This is also known as niacinamide, and is required by the liver on detoxification system; it is needed for the metabolism of fats and helps to keep cholesterol levels under control.

Pyridoxine (vitamin B6)

Pyridoxine is required for effective phase one liver detoxification. Pyridoxine inhibits the formation of a toxic chemical called homocysteine, which accelerates cardiovascular disease.

Zinc

The mineral zinc has antioxidant properties and is part of the powerful antioxidant enzyme called superoxide dismutase (SOD).

Cruciferous vegetables

Cruciferous vegetables such as cabbage, cauliflower, broccoli, kale, mustard greens and radish, contain important substance such as indoles, thiols and sulphur compounds which enhance the liver's phase one and two detoxification pathways.

Green tea

Green tea exerts strong antioxidant actions and is also able to inhibit cancer cell growth. The Chinese, who are large drinkers of green tea, have a 60% less chance of oesophageal cancer.

1.5.2 MECHASISM OF ACTION OF ANTIOXIDANTS

These are:

Physical barriers preventing ROS generation or ROS access to important biological sites. E.g. UV filters, cell membranes

Chemical traps / sinks 'absorb' energy and electrons quenching ROS. E.g. carotenoids, anthocyanidins.

Catalytic systems neutralize or divert ROS. E.g. SOD, catalase and glutathione peroxidase

Binding / inactivation of metal ion prevents generation of ROS by Haber-Weiss reaction. E.g. Ferritin, catechins.

Sacrificial and chain breaking antioxidants scavenge and destroy ROS. E.g. Ascorbic acid, tocopherol, flavonoids, glutathione, uric acid.

LIST OF ABBREVIATIONS

ALP - Alkaline Phosphatase

ANOVA - Analysis of Variance

BSI - Botanical Survey of India

CCl3. - Trichloro Methyl Free Radical

CAT - Catalase

CPCSEA - Committee for the Purpose of Control and Supervision of Experiments on Animals

CHD - Cardio Vascular Disease

oC - Degree Celsius

GPx - Glutathione Peroxidase

GSH - Reduced Glutathione

GSSH - Oxidized Glutathione

hr - hour

HOCl - Hypochlorous acid

H2O2 - Hydrogen peroxide

HO2. - Hydroperoxy radical

Kg - kilogram

LDH - Lactate Dehydrogenase

LDL - Low Density Lipoprotein

LPO - Lipid peroxidation

LO2. - Lipid peroxyl radicals

µL - Micro litre

µmol - Micromole

µg - Micro gram

mM - Milli molar

ml - milliliter

Na2 HPO4 - Disodium hydrogen phosphate

NADP - Nicotinamide Adenine Dinucleotide Phosphate

NADPH - Nicotinamiide Adenine Dinucleotide Phosphate (reduced form)

NAPQI - N-acetyl-P benzoquinoneimine

_ - Negative

IAEC - Institutional Animal Ethics Committee

NO - Nitric Oxide

O2. - Superoxide radical

OH. - Hydroxyl radical

% - Percentage

PCM - Paracetamol

+ - Positive

ROI - Reactive Oxygen Intermediate

ROS - Reactive Oxygen Species

rpm - revolution per minute

SOD - Superoxide Dismutase

S.E.M - Standard Error Mean

SGOT - Serum Glutamate Oxaloacetate Transaminase

SGPT - Serum Glutamate Pyruvate Transaminase

TCA - Trichloro Acetic Acid

TP - Total Protein

UV - Ultra violet

WHO - World Health Organisation

LIST OF TABLES

SL. NO.

TITLE

PAGE NO.

1

Chemicals and Reagents

46

2

Instrument Used

47

3

Result of Phytochemical Screening

52

4

Biochemical Estimation of Coccinia grandis Root Extract

54

5

Antioxidant Activity and Total Protein of Coccinia grandis Root Extract

58

LIST OF FIGURES

SL. NO.

TITLE

PAGE NO.

1

Liver

3

2

Mechanism of Drug Induced Hepatotoxicity

8

3

Steps for Free Radical Reaction

13

4

Types of Antioxidants

21

5

Coccinia grandis Root

36

6

Coccinia grandis Leaves

36

7

Glutamate Oxaloacetate Transaminase (SGOT) Activity of Coccinia grandis Root Extract

55

8

Serum Glutamate Pyruvate Transaminase (SGPT) Activity of Coccinia grandis Root Extract

55

9

Alkaline Phosphatase (ALP) Activity of Coccinia grandis Root Extract

56

10

Total Protein Activity of Coccinia grandis Root Extract

56

11

Total Bilirubin Activity of Coccinia grandis Root extract

57

12

Direct Bilirubin Activity of Coccinia grandis Root Extract

57

13

Super Oxide Dismutase (SOD) Activity of Coccinia grandis Root Extract

59

14

Catalase (CAT) Activity of Coccinia grandis Root Extract

59

15

Peroxidase (Px) Activity of Coccinia grandis Root Extract

60

16

Glutathione Peroxidase (GPx) Activity of Coccinia grandis Root Extract

60

17

Reduced Glutathione (GSH) Activity of Coccinia grandis Root Extract

61

18

Histopathology of Control Group

62

19

Histopathology of Toxic Control Group

62

20

Histopathology of Standard (Silymarin) Group

62

21

Histopathology of 200 mg/kg (Coccinia grandis root) Extract Group

63

22

Histopathology of 400 mg/kg (Coccinia grandis root) Extract Group

63

CONTENTS

SL. No.

TOPIC

PAGE NO.

1

Introduction

1-26

2

Review of Literature

27-35

3

Plant Profile

36-39

4

Objective and Plan of Work

40-41

5

Materials and Methods

Phytochemical Analysis

Biochemical Estimation

Antioxidant Activity Study

Histopathological Study

42-51

6

Results

52-63

7

Discussion and Conclusion

64-66

8

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