Herbs are facing a comeback and people are going back to the naturals with a trust of safety and wellbeing. Many secondary metabolites of medicinal plants are commercially significant and find use in number of pharmaceutical compounds. Flora particularly used in traditional systems of medicine can provide biologically active molecules and helps for the development of modified derivatives with reduced toxicity and enhanced activity(Joy et al., 1998). The important medicinal properties of diverse plants are due to presence of several constituents i.e. saponins, tannins, alkaloids, phenols, glycoalkaloids, flavonoids, terpenoids, glycosides and esters. Among them some act as synergistic and enhance the bioactivity of other compounds (Tiwari, 2008).
On the years there have been numerous studies documenting the antioxidant, antibacterial anti-inflammatory and anticancer properties of plant ingredients (Naili et al., 2009). The factors that influence the useful life of erythrocytes have been much studied. Microbial infections and free radical mediated processes have been implicated in the pathogenesis of hematological disorders (Hill and Thornally, 1981). The antioxidant may be useful in retarding oxidative stress. The natural antimicrobial agents protect living organisms from damages resulting in the prevention of various diseases. Numerous studies have shown that aromatic and medicinal plants are sources of diverse nutrient and non-nutrient molecules, many of which display antioxidant and antimicrobial properties, which can protect the human body against both cellular oxidation reactions and pathogens (Sengul et al., 2009).
1.1. Hematopoietic system
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The hematopoietic system consists of three primary cell components: leukocytes, platelets, and erythrocytes. Blood cells produced from the similar bone marrow stem cells. These eternal, pluripotent, undifferentiated, stem cells generate erythrocytes, leukocytes and platelets. Illustration below demonstrates the different types of blood cells. Leukocytes are a group of allied cell types that concerned in immune function. Leukocytes comprise neutrophils, eosinophils, basophils, lymphocytes and monocytes (Doohan, 2000).
Fig.1 Different types of blood cells
Hemolysis is the breakage of the red blood cells (RBCs) membrane. Hemolysis causes the discharge of hemoglobin from the red blood cells. The primary function of the erythrocyte is to carry oxygen from the lungs to the peripheral tissues. Its optimal design enables efficient oxygen transport via the hemoglobin molecule. The common metabolic state and local factors of patient control oxygen release. Some drugs selectively accumulate in erythrocytes, resulting in substantial differences when comparing blood to plasma drug concentrations. In a few instances, enzymes found in erythrocytes (e.g., aldehyde dehydrogenase) may impact on the systemic metabolism of drugs (Petros and Ericson, 1793). Hemolysis is clearly detected by viewing a pink to red trace in serum due to the prescence of hemoglobin. Hemolysis can be categorized in to in vivo or in vitro (Lena, 2003).
Fig.2. Hemolysed red blood cell and normal red blood cell.
1.2.1. In vivo (Inside the body)
In vivo hemolysis may be resulted by a large number of medical circumstances, including many Gram-positive bacteria (e.g., enterococcus, streptococcus and staphylococcus), parasites (e.g., malaria), autoimmune disorders and some genetic disorders (e.g., G6PD deficiency or sickle-cell disease) (Ponder, 1994).
1.2.2. In vitro (Outside the body)
In vitro hemolysis may occur due to improper techniques and lack of skill during the collection of blood specimens. In vitro hemolysis may also occur by the effects of mechanical processing and bacterial action in cultured blood specimens (Lippi et al., 2009).
1.3. Etiology of hemolysis
The normal life spans of RBCs are about 120 days. After that RBCs are isolated from the circulation. Hemolysis means early destruction, therefore a short RBC life span (< 120 days). Hemolysis may occur because of disorders extrinsic to the RBC or from inherent RBC abnormality. Hemolysis may be produced by three mechanisms such as congenital, acquired and acquired (Bernadette et al., 2007).
Congenital hemolysis is a state active at birth and often prior to birth, or that develop during the earliest month of life despite of causation (Erlandson et al., 1960).
184.108.40.206. Defects of hemoglobin synthesis and structure
Defects of hemoglobin production and formation are one of the most important inborn causes of hemolysis. It includes the diseases like Thalassaemia, Sickle cell disease, and unstable haemoglobin (Dhaliwal et al., 2004).
220.127.116.11. Membrane defects
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Hemolysis may be due to congenital red cell membrane defects, the commonest of which are heritable spherocytosis and heritable elliptocytosis. Other than this red cell enzyme defects, Pyruvate kinase insufficiency and Glucose-6-phosphate dehydrogenase insufficiency can also provoke hemolysis (Searcy et al., 1970).
1.3.2. Acquired (non-immune)
Hemolysis acquired during lifetime due to infections, toxicity etc.
Hypersplenism is characterized by enlarged spleen, destruction of red blood cells and a peripheral blood cytopenia. Hypersplenism can be cured by splenectomy. The mechanism of hypersplenism is multifactorial and includes both pooling of cells within the spleen and speedy destruction of cells within the spleen. The plasma volume is usually augmented in splenomegaly, and a dilutional factor contributes to any anaemia (Dameshek, 1955).
18.104.22.168. Toxic states
Toxic states like Infections, e.g. malaria, Uraemia, Drugs Chemicals, e.g. lead poisoning, Venoms, e.g. cobra bites can induce haemolysis (Greer et al., 2004).
22.214.171.124. Microangiopathic conditions
Pathological conditions like Disseminated intravascular coagulation (DIG), Hemolytic uraemic syndrome (HUS), Thrombotic thrombocytopenic purpura (TTP) and Malignant hypertension (Evans et al., 2010).
126.96.36.199. Trauma to red cells
Trauma to red cells may occur in a number of situations, including cardiac prostheses, hemoglobinuria and burns (Sutera, 1977).
188.8.131.52. Acquired red cell membrane defects
Red blood cell defects may acquire during the life span due to some diseases such as Paroxysmal nocturnal hemoglobinura (PNH), Liver disease, Vitamin E deficiency etc (Rosse et al., 2004).
1.3.3. Acquired (immune)
Hemolysis can also be induced by some immunity related disorders.
184.108.40.206. Lymphoproliferative disorders
Anti-red-cell antibodies may present secondary to malignancies, such as chronic lymphocytic leukaemia. The warm antibodies produced in such circumstances are polyclonal, do not originate from the malignant clone, and are indicative of the overall immune deregulation that occurs in these conditions. However, when cold antibodies are produced in lymphoma they are frequently monoclonal (Kaden et al., 1999).
220.127.116.11. Autoimmune disorders
Hemolysis can occur in association with all the autoimmune diseases but is most common in systemic lupus erythematosus.
Viral infections frequently predate warm antibody in children and young adults. Infectious mononucleosis may lead to production of cold antibodies with blood group specificity and mycoplasma pneumoniae with individual specificity (Clarridge and Zighelbotm-Daum, 1985).
Certain drugs, such as methyldopa, L-dopa and mefanamic acid, induce the production of autoantibody directed against red cell antigens through an unknown mechanism. If haemolysis does occur it usually ceases within 2 weeks of stopping the drug. Some drugs cause haemolysis by acting as a hapten. This occurs most frequently with high-dose parenteral penicillin. It is adsorbed on to the red cell and in that form stimulates antibody production. IgM antibodies are frequently produced without clinical effect, but if IgG antibodies are produced haemolysis may be severe (Garratty, 2009).
18.104.22.168. Complication of blood transfusion
If blood has been incorrectly cross-matched (or not cross matched), or if (more commonly) there has been a clerical error and the patient has antibodies to donor red cell antigens, rapid complement-dependent lysis of the transfused cells may occur. Rarely, immediate haemolysis may also be due to high-liter anti-A or anti-B IgG antibodies in the donor blood, when group O blood is given to A or B recipients (Ting, 2005).
22.214.171.124. Immune hemolytic disease of the newborn
In immune haemolytic disease of the newborn, IgG antibodies produced by the mother against fetal red cell antigens cross the placenta and produce hemolysis in the fetus (Gottstein and Cooke, 2003).
Hemolysis may possibly acute, chronic, or periodic. Chronic hemolysis may be complicated by aplastic emergency (short-term failure of erythropoiesis), frequently caused by an infectivity, often parvovirus. Hemolysis might be extravascular, intravascular, or both.
1.4.1. Extravascular hemolysis
Extravascular hemolysis can be occurring in pathological hemolysis. Normally, injured or unusual red blood cells are separated by occurs when injured or atypical RBCs are removed from the systemic circulation by cells of the bone marrow, liver, and spleen. The spleen cells destroying mildly abnormal RBCs and cells enclosed by warm antibodies. In some pathological conditions spleen may get enlarged and leads to hemolysis of normal RBC. Liver cells will attack only severely abnormal RBCs or RBCs layered with cold antibodies or complement (C3). They are also damaged within the circulation, which can remove injured cells efficiently (Dhaliwal et al., 2004).
1.4.2. Intravascular hemolysis
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Premature RBC destruction was induced intravascularly. Intravascular hemolysis occurs when the cell membrane has been severely injured by autoimmune diseases or trauma or shear stress. Intravascular hemolysis may induce hemoglobinemia when the amount of hemoglobin released into plasma fluid increases the hemoglobin -binding capacity of the plasma-binding protein (Hatherilll et al., 1986).
1.5. Consequences of hemolysis
Unconjugated hyperbilirubinemia and jaundice arise when the translation of hemoglobin to bilirubin cross the liver's capability to conjugate and eliminate bilirubin. Bilirubin catabolism leads to abnormal increase of stercobilin in the stool and urobilinogen in the urine and cholelithiasis. The bone marrow responds to the increased loss of RBCs by accelerating the production and liberation of RBCs, resulting in a reticulocytosis (Daugirdas, 2007).
Fig. 3. Consequences of hemolysis
1.6. Symptoms and signs
Systemic manifestation similar to those of other anemias and comprise pallor, fatigue, dizziness, and hypotension. Hemolytic emergency is unusual; it may be accompanied by symptoms like chills, fever, body pain, prostration, and shock. Rigorous hemolysis may produce jaundice and splenomegaly. Red or reddish-yellow urine is a symptom of Hemoglobinuria (Sibai, 2004).
Microscopical assessment of peripheral smear, quantifying reticulocyte count, serum bilirubin, LDH, and ALT can be used to analyse the hemolytic condition. Amount of hemosiderin in urine and haptoglobin in serum may be used to know the hemolytic crisis. Measurement of RBC survival by means of a radioactive label is used to test the hemolytic condition. Quantitative hemoglobin electrophoresis, , flow cytometry, cold agglutinins, red blood cell enzyme assays and osmotic fragility test are the other tests that may help to detect the causes of hemolysis (Munro, 2009).
Management of hemolysis is based on the specific mechanism of hemolysis. Iron-replacement therapy may require in Hemoglobinuria and hemosiderinuria. Initial treatment of warm antibody autoimmune hemolysis can be start by giving corticosteroids. Long-standing transfusion therapy may leads to iron accumulation, which demand chelation therapy. Splenectomy is advantageous in several situations, especially when splenic enlargement is the foremost cause of RBC destruction. The patient is kept warm in case of cold agglutinin disease. Folate replacement is necessary for patients with ongoing long-term hemolysis (Lichtin, 2009).
1.9. Free radicals
The ability to utilize oxygen has provided humans with the benefit of metabolizing fats, proteins, and carbohydrates for energy. A highly reactive atom Oxygen is capable of becoming part of potentially harmful molecules normally called free radicals .Free radicals take electrons from other substances in order to combine with the unpaired electron and neutralize themselves. Once free radicals generated in the human body, it will attack the molecules and the initial attack makes the first free radical to become stable but the molecule attacked by the free radical will converted in to new free radical, causing a chain reaction. Failure of deactivation of subsequent free radicals leads to progression of number of free radical reactions(Mark, 1998).
1.9.1. Types of free radicals
Free radicals are reactive molecules that can produce injure to the cells. Free radicals can be produced from exogenous and endogenous sources. The exogenous sources consist of electromagnetic radiation, cosmic radiation, ultraviolet radiation, ozone, tobacco smoke, automobile exhaust fumes, air pollutants, industrial effluents, fungal toxins, organic solvents, pesticides, herbicides, etc. (Irshad and Chaudhuri, 2002). Endogenous sources of free radicals include mitochondrial electron transport chain, pro-oxidative enzyme systems, lipid peroxidation, inflammation, peroxisomes, glycoxidation, auto-oxidation of aminoacids, catecholamines, haemoglobin, ischemia-reperfusion injury and stimulation of polymorphonuclear leucocytes and macrophages due to respiratory burst (Sen et al., 2010).
A healthy person is able to face the presence of free radicals by defending the body with an anti-free radical system. This internal anti-free radical system consists of enzymatic and non-enzymatic mechanisms including superoxide dismutase, catalase, carotenoids, polyphenols, and anthocyanines among others (Amira, 2010). Excess of free radical generated in the human body cannot be handled effectively by the normal anti free radical system and the end result is oxidative stress causing cellular damage. Free radicals like reactive nitrogen species (RNS) and reactive oxygen species are products of normal cellular metabolism, which are formed and degraded by all aerobic organisms (Halliwell, 1991).
126.96.36.199. Reactive Oxygen Species (ROS)
Oxygen is the fundamental molecule for the aerobic life processes. Molecular oxygen, O2, is a biradical with two unpaired electrons. Mitochondria consume more than 90% of inhaled oxygen and thus the electron transport chain occur in mitochondria is the main supply of ATP in the mammalian cells. Free radicals obtained from oxygen characterize the most part of the important class of radical species generated in living systems (Cadenas and Davies, 2000).
They can be classified into oxygen-centered radicals and oxygen-centered non-radicals. The primary oxygen-centered free radicals are superoxide anion (O2â€¢-), hydroxyl (OHâ€¢), hydroperoxyl (OOHâ€¢), peroxyl (ROOâ€¢) and alkoxyl (ROâ€¢) radicals. Hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid (HOCl), and singlet oxygen (1O2) are the oxygen-centered non free radicals. These are together termed as reactive oxygen species (ROS). The adding of one electron to an oxygen molecule result in the development of superoxide anion radical (O2â€¢-).super oxide anion is considered as the "primary reactive oxygen species and it can react with other molecules to generate secondary reactive oxygen species (Turrens, 2003).
Superoxide anion radical (O2â€¢-)
Superoxide anion created by the addition of an electron to a molecular oxygen species. It is the initial free radical produced within the inner mitochondrial membrane of a cell during electron transport system. In spite of being a free radical, O2â€¢- is not highly reactive due to its charged state and lacks the ability to penetrate a biological membrane, with the exception of erythrocyte membrane which has an 'anion channel' that helps in its penetration. Superoxide anion radical regulates metabolites capable of signaling and communicating important informations to the cellular genetic machinery. Over production of O2â€¢- takes place in various chronic inflammatory cases, induced by drug, toxin, stress, tissue injury and heavy exercises (Valko et al., 2006).
Superoxide anions have significant role in the formation of other reactive oxygen species such as hydrogen peroxide and singlet oxygen in living systems. Peroxynitrite (ONOO-) can also be produced from superoxide by reacting with nitric oxide. This peroxy nitrate can generate toxic compounds such as hydroxyl radical and nitric dioxide (Fridovich, 1997).
Hydrogen peroxide (H2O2)
Hydrogen peroxide is a non-radical oxygen species, which is the least reactive molecule among ROS. Hydrogen peroxide also acts as an intracellular signalling molecule. H2O2 is mainly generated from superoxide anion through a dismutation reaction by the enzyme superoxide dismutase. It is relatively stable and hence long-lived under physiological pH and temperature in the absence of metal ions. It is poorly reactive because of its weak oxidizing and reducing property (Mc-Cord, 2000).
In a transition-metal-free system, H2O2 shows limited toxicity. H2O2 is highly diffusible and easily crosses the cell membrane and plays a radical forming role as an intermediate in the production of free radicals via oxidation of transition metal ions. Hydrogen peroxide can produce hydroxyl radicals in the presence of superoxide anion and metal ions (Leonard et al., 2004). In the presence of H2O2, hypochlorous acid is formed by the action of myeloperoxidase, an enzyme present in the phagosomes of neutrophils. H2O2 can degrade certain heme proteins, such as hemoglobin, to release iron ions. In living cells, three antioxidant enzymes, namely glutathione peroxidase, catalase and peroxiredoxins act as enzymatic defense system against hydrogen peroxide (Rhee, 1999).
Hydroxyl radical (OH.)
The hydroxyl radical is the nonreactive state of the hydroxide ion. It is the most reactive and very dangerous oxy radical because of its highest 1-electron reduction potential. Due to its strong reactivity, it can react with almost all the cells in the living species and causes more damage to biological membranes than any other ROS. Hydroxyl radical is formed in vivo from hydrogen peroxide and superoxide anion in the presence of trace amounts of transition metal ions like iron or copper. The released Fe2+ ions can generate highly reactive hydroxyl radical through the metal-catalyzed Fenton reaction (Liochev et al., 2002).
Normally, redox-active free iron or copper does not exist in vivo, as these transition metal ions remain bound to proteins, biological membranes, nucleic acids or low-molecular weight chelating agents like citrate, histidine or ATP. However, conditions like stress, ischemia and cellular acidosis release transition metal ions from some metalloproteins by the action of superoxide anion radical, resulting in the generation of hydroxyl radicals as depicted in the above reactions. Aromatic compounds undergo addition reactions with hydroxyl radicals, resulting in the formation of hydroxylated free radicals and peroxyl type radicals. OH radicals abstract a hydrogen atom from saturated compounds to yield a free radical, which can react with oxygen to generate further free radicals. The highly toxic hydroxyl radical can cleave covalent bonds in polypeptides, proteins, lipid and DNA (Valko et al., 2005).
Peroxyl and alkoxyl radicals
Peroxyl (ROOâ€¢) and alkoxyl (ROâ€¢) radicals are the additional reactive radicals derived from oxygen in the living systems (Degrey, 2002). They are good oxidizing agents and are formed by decomposition of alkyl peroxides (ROOH), irradiation of UV light, and homolysis of peroxides in the presence of transition metal ions and direct reaction of oxygen with alkyl radicals. The protonated form of superoxide anion is hydroperoxyl radical (HOOâ€¢), which is the simplest peroxyl radical. Some peroxyl radicals can react with each other to generate singlet oxygen or break down to liberate superoxide anion. It has been demonstrated that the peroxyl and alkoxyl radicals can abstract hydrogen from other molecules and initiate fatty acid lipid peroxidation, resulting in chain reaction (Aikens and Dix, 1991).
Singlet oxygen (1O2)
Singlet oxygen is a non-radical and rather mild oxidant compared with other ROS. It is highly reactive towards any molecule with lone pairs of low ionization energy. 1O2 can be formed from hydrogen peroxide, which reacts with either superoxide anion or HOCl in living tissues. It is involved in the oxidation and degradation of cholesterol and acts against various microorganisms and cancer cells (Stief, 2003).
188.8.131.52. Reactive Nitrogen Species (RNS)
Nitrosative stress is the overproduction of reactive nitrogen species (RNS) like nitric oxide (NOâ€¢), nitric dioxide (NO2â€¢) and peroxynitrite (ONOOâ€¢). This condition can initiate a series of nitrosylation reactions that can alter the structure of proteins leading to normal cellular dysfunction (Bergendi et al., 1999).
Nitric oxide (NOâ€¢)
Nitric oxide is a small lipophilic molecule with a single unpaired electron. NOâ€¢ acts as an important biological signaling molecule as it rapidly undergoes addition, substitution, redox and chain terminating reactions. Its main physiological role includes smooth muscle relaxation, blood pressure regulation, and intracellular messenger by stimulating guanyl cyclase and protein kinases, defense mechanisms and immune regulation. Increased nitric oxide production occurs in septic shock, eclampsia, bronchial asthma, arthritis and ulcerative colitis. Excess NOâ€¢ is believed to be involved in various pathophysiological conditions like ischemia, stroke, gastrointestinal dysfunctions, achalasia, congenital hypertrophic pyloric stenosis, etc. (Bredt, 1999).
Nitric dioxide (NO2â€¢)
Nitric oxide is mainly formed from the reaction of peroxyl radical and NOâ€¢, and in addition from tobacco smoke and polluted air. It initiates lipid peroxidation by abstraction of labile hydrogen atoms from the double bonds and thus generates free radicals (Ridnour et al., 2004).
Peroxynitrite is a potent oxidizing agent similar to hydroxyl radical. It is highly cytotoxic due to its high diffusibility across cell membranes. It acts as an important tissue-damaging species generated during inflammation, neuro degeneration and renal disorders. During oxidative burst, nitric oxide and superoxide anion react together to produce significant amounts of peroxynitrite anion. Peroxynitrite can cause oxidation of proteins, LDL and DNA bases resulting in oxidative stress. This may cause cell death and tissue damage causing neurological disorders and stroke, arthritis, inflammatory bowel.
1.9.2. Reactions Involving Free Radicals
Free radicals are extremely unsteady molecules and by reacting with atoms or molecules present in the cells free radicals attains a added stable state. The free radicals experience four crucial types of chemical reactions that are:
In case of hydrogen abstraction a radical react with another molecule which has a free hydrogen atom. Accordingly, the radical become stable by binding with a hydrogen atom, while the hydrogen contributor is changed to a free radical.
In case of addition reaction the radical react with a stable molecule and the resulting molecule is converted into a free radical.
In termination reaction instead of a stable molecule radical react with another radical to form a stable compound..
In disproportionation reaction identical radicals bind with each other and one of the radicals offer an electron to the next radical because of that two dissimilar molecules are produced, each of which is stable (Defeng et al., 2003).
A series of defense mechanisms has been developed by living organisms against exposure to free radicals. They exert their action either by protecting the antioxidant defense mechanisms or scavenging the reactive oxygen species. Catalytic removal of free radicals by antioxidant enzymes is the primary defense against ROS/RNS and free radical scavengers acts as second line defense and hence can be classified into enzymatic and non-enzymatic antioxidants respectively (Cadenas and Davies, 1997).
The enzymatic antioxidants like superoxide dismutase, catalase, and glutathione peroxidise are produce endogenously. The non-enzymatic antioxidants include reduced glutathione, antioxidant vitamins, minerals, co-factors and phytochemicals, which are obtained from natural plant sources. The antioxidants derived from both natural and synthetic source has anticipated for employed in the management of various human diseases (Lee et al., 2004).
Synthetic antioxidant compounds like butylated hydroxytoluene, butylated hydroxyanisole and tertiary butylhydroquinone are normally used in processed foods. But reports revealed that these compounds have some toxic effects like hepato toxicity and mutagenesis (Nagavani and Rao, 2010) Flavonoids and phenolics extracted from plants was reported as scavengers of free radicals (Oteiza et al., 2005). Because of the low toxicity and much efficacy natural antioxidant source is acquiring much significance.
1.10.1. Enzymatic antioxidants
The word antioxidant means any matter that delays or inhibits oxidative damage. The first line antioxidant defense enzymes (primary enzymes) against free radicals are superoxide dismutase (SOD), glutathione peroxides (GPx), and catalase (CAT). The secondary antioxidant enzyme is glutathione reductase (GSSH) (Maksimenko et al., 2010).
Superoxide dismutase (SOD)
Under normal circumstances, formation of superoxide anion is kept under tight control by SOD enzymes. SOD is a metalloprotein found in both prokaryotic and eukaryotic cells. It converts superoxide to hydrogen peroxide (H2O2) and represents the first line of defense against oxygen toxicity. Three forms of SOD have been described, namely Cu-Zn-SOD, Mn-SOD, and Fe-SOD. Superoxide dismutase is known to be a stress protein and it is produced in response to oxidative stress. SOD has been detected in a large number of tissues and is thought to protect the cell from damage caused by reactive oxygen species. Superoxide anion is the only known substrate for SOD (Desai and Sivakami, 2007).
Glutathione peroxidase (GPx)
Glutathione peroxidase enzyme is a well-known first line defense against oxidative stress, which requires glutathione as a co-factor. It is one of the key enzymes liable for the deprivation of organic peroxides and hydrogen peroxide in the brain cells. GPx catalyses the oxidation of reduced glutathione to oxidized glutathione with the expense of H2O2 (Zhu et al., 2006). By its selenium dependency, GPx can be divided in two isoforms, Se-dependent GPx and Se-independent GPx. The former is a tetramer of MW 84000 with very high activity towards both H2O2 and organic hydroperoxides. It is found in both cytosol (70%) and mitochondria (30%) of various tissues. Since selenium is an integral component of GPx, the measurement of this enzyme has been used as a functional index of selenium level (Rotruck et al., 1973).
Catalase is a heme containing tetrameric enzyme. It is localized mainly in the mitochondria and in sub-cellular respiratory organelles of most mammalian cells. In dismutation process hydrogen peroxide was converted in to molecular oxygen and water and was catalyzed by hydrogen peroxide. One of the main antioxidative functions of catalase is to reduce the formation of hydroxyl radicals from hydrogen peroxide, via the Fenton reaction (Ames et al., 1993). Catalase makes a bond with NADPH and protects the enzyme from inactivation and thus increases its efficacy. GPx and CAT were found to be important in the inactivation of many environmental mutagens (Langseth, 1995).
Glutathione reductase (GSSH)
Glutathione reductase is a NADPH-dependent flavoenzyme that converts oxidized glutathione (GSSG) to reduced glutathione (GSH) by the oxidation of NADH to NAD (Schaedle and Bassaham, 1977).
1.10.2. Non-enzymatic antioxidants
The non-enzymatic antioxidants include mainly, low molecular weight antioxidants like glutathione, antioxidant vitamins like vitamin A, C, E and K, minerals like manganese, zinc and copper and phytochemicals like polyphenols, carotenoids, etc. (Patil et al., 2006)
It is substrate for both glutathione peroxidase and transferase. Endogenous glutathione have a significant part in managing endothelial dysfunction and vascular hypo reactivity in response to peroxynitrite and endotoxic shock, as well as in acute oxidative stress and inflammation. Depletion of GSH may enhance the risk of cerebral ischemic injury (KoÅ‚acz et al., 2007).
Alpha-Tocopherol (vitamin E) is a fat-soluble vitamin known to be one of the most potent antioxidant present in biological membranes. Vitamin E protects cells from peroxidation of membrane phospholipids and from oxidative damage of VLDL, LDL, smooth muscle cell proliferation, proteins and DNA and thus provides protection against atherosclerosis, and carcinogenesis. Deficiency of vitamin E induces lipid peroxidation and a reduction in the activities of enzymatic antioxidants causing various disorders. Ascorbic acid (vitamin C), a water-soluble vitamin plays an important antioxidant role in physiological concentrations. Ascorbate prevents lipid hydroperoxide formation in LDL by reducing Î±-tocopherol radicals formed upon reaction with lipid peroxyl radicals, thus preventing atherosclerotic plaque formation. The antioxidant mechanisms of ascorbic acid are due to the donation of electrons to lipid radicals, quenching of singlet oxygen and removal of molecular oxygen. It scavenges superoxide anion radical by forming semi hydro ascorbate radical, which is subsequently reduced by glutathione (Langseth, 1995).
Minerals like copper, zinc, manganese, magnesium, and selenium play an important role in enzyme functions. The enzymatic antioxidants like Cu, Zn-SOD and Mn-SOD requires copper and zinc and manganese respectively for their activities. Hence, dietary deficiency of these minerals significantly decreases the enzyme activities leading to lipid peroxidation and mitochondrial dysfunction (Albion, 2007).
A number of extracts and isolated compounds from plants have been reported to afford protection against free radical-induced oxidative damage in various experimental models. Among them, phenolic and polyphenolic compounds and carotenoids exhibit potent antioxidant activities.
Phenolics found in plants, have been reported to have numerous biological effects, including antioxidant activity. Phenolics show some redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. Phenolics also have significant metal chelating capacity. Phenolics delay oxidative degradation of lipids and improve the quality and nutritional value of food. (Faujan, 2007).
Flavonoids may be coming under the class of polyphenolic compounds. Flavonoids plays an important role in biological effects such as anti-inflammatory, antihepatotoxic, antiulcer, antiallergic, antiviral, anticancer activities. Polyphenolic compounds exert potent antioxidant activity may be because of the prescence of phenolic hydroxyl groups and it also have metal chelating ability.
Carotenoids, a group of tetraterpenoids are potent antioxidants of plant origin. They are most effective singlet oxygen quenchers in biological systems. Carotenoids scavenge superoxide anion radicals by transfer of either hydrogen atoms or electrons to the free radicals. The potent antioxidant activity of carotenoids is helpful in the prevention of free radical-induced diseases like cataract, neurodegeneration, atherosclerosis and multiple sclerosis (Prakash and Gupta, 2007).
1.11. Oxidative stress
Our antioxidant defense system may not always be adequate. The word oxidative stress expresses a shift in the direction of pro-oxidants in the antioxidant and pro-oxidant balance. This imbalance occurs as a result of a raise in oxidative metabolism. Increased oxidative stress at the cellular level can come about as a consequence of many factors including, medications, radiation, exposure to alcohol, cold, poor diet, toxins, trauma, infections, or physical activity. Management of oxidative stress depends on presence of various antioxidants in human body (Mark, 1998).
1.11.1. Oxidative stress and human disease
Oxidative damage to vital biological molecules like DNA, proteins, and other macromolecules has been concerned in the pathogenesis of a broad variety of diseases, most particularly heart disease and cancer (Ames et al., 1993).
184.108.40.206. Heart Disease
Cardio vascular problems are the foremost cause of mortality in the world. While several factors, such as abnormal lipid profile, hypertension, and diabetes are believed to promote atherosclerosis. This hypothesis is explained by a number of epidemiological studies, which connect poor intakes of dietary antioxidants to high incidence of heart disease. Antioxidants may show to prevent LDL oxidation in vitro and delay the development of atherosclerosis in animal models. Several human studies found that vitamin E supplements may increase vitamin E levels in low density lipoprotein; this in turn increases the resistance of low density lipoprotein oxidation, and delay the rate of low density lipoprotein oxidation (Kris-Etherton et al., 2004).
Excluding heart diseases cancer is the second most cause of mortality in the world. It is reported that imbalanced diet may account for all most 36% of all human cancers. Epidemiological support always relates low blood levels of antioxidants with increased cancer threat. Oxidants have a significant influence on stimulating cell division, which is a crucial factor in mutagenesis. When a cell with a damaged DNA strand divides, cell metabolism and duplication becomes deranged. So a mutation can arise as a vital factor in carcinogenesis. It is believed that antioxidants exert their protective effect by decreasing oxidative damage to DNA and by decreasing abnormal increases in cell division (Valko et al., 2006).
220.127.116.11. Pulmonary Disorders
The respiratory tract is one of the major targets for free radical attack. Air pollution can be considered as a major source of ROS. Current studies propose that free radicals may be concerned in the progress of pulmonary disorders. Bronchial inflammation one of the characteristic of asthma may be induced due to the cellular damage caused by free radicals. It has been recommended that increased antioxidant intake may reduce oxidant stress and prevent or diminish the advancement of asthmatic symptoms (Kinnula and Crapo, 2003).
Other major pathologies that may involve free radicals include neurological disorders, hemolysis, atherosclerosis, cataracts, arthritis, diabetes, renal diseases, cataracts and haemodialysis (Mark, 1998).
1.12. Antimicrobial therapy
All the living organisms are victim to infection. Humans are susceptible to diseases caused by viruses, bacteria, protozoa, fungi and helminths. The successful development of such agents, particularly the antibiotic revolution, constitutes one of the most important therapeutic advances in the entire history of medicine (Rang et al., 2005).
1.12.1. Definition and characteristics
Antibiotics are substances obtained from different classes of microorganisms (bacteria, viruses and fungi) that restrain the growth of other microorganisms. Understanding the mechanisms of bacterial replication greatly supports the development of antimicrobials (Tripathi, 2007).
1.12.2. Mechanism of action and classification of antimicrobials
Based on mechanism of action and structures antimicrobials are grouped like follows:
Anti microbial agents that stop the synthesis of bacterial cell walls - Including the ß-lactam class of antibiotics, different agents such as cycloserine, vancomycin, and bacitracin.
Agents that act on the cell membrane of the microorganism - polymyxin, polyene antifungal agents and daptomycin.
Agents that disrupt role of 30S or 50S ribosomal subunits inhibit protein synthesis reversibly -
chloramphenicol, tetracyclines,erythromycin,clindamycin,streptogramins, and linezolid
Agents that connect to the 30S ribosomal subunit and revise protein synthesis -
Agents that affect bacterial nucleic acid metabolism,
Rifamycins - inhibit RNA polymerase.
Quinolones - inhibit topoisomerases.
Trimethoprim and sulfonamides - block essential enzymes of folate metabolism.
Acyclovir or ganciclovir - selectively inhibit viral DNA polymerase.
Zidovudine or lamivudine - inhibit HIV reverse transcriptase.
Non-nucleoside HIV reverse transcriptase inhibitors -
Inhibitors of other essential viral enzymes
Inhibitors of HIV protease or influenza neuraminidase
Fusion inhibitors like enfuvirtide.
Supplementary groups with more complex mechanisms are elucidated. The accurate mechanism of antimicrobial action of some agents is still mysterious (Chambers, 2006).
1.13. Bacterial resistance to antimicrobial agents
Bacterial resistance is a major medical problem, because it seriously limits the usefulness of many antibiotics. Usually some species of microorganisms may be susceptible to some chemotherapeutic agent and resistant to others. But development of strains against the drug, which are effective against the species, is serious (Levy and Marshall, 2004).Bacterial resistance is either natural or acquired.
18.104.22.168. Natural resistance
This type of resistance is genetically determined and depends upon the absence of metabolic process or pathway in the microorganism. Most of the time natural resistance is confined to a particular species (Baquero, 1997).
22.214.171.124. Acquired resistance
Acquired resistance refers to resistance developing in a previously sensitive bacterial species. The development of acquired resistance involves a stable genetic change, heritable from generation to generation. Resistance may acquire to the organism through various mechanisms like mutation, adaptation, transformation, transduction or conjugation (Barar, 2000).
1.14. Test for microbial sensitivity to antimicrobial agents
1.14.1. Bacteria and fungi
Preliminary screening of plants may be performed with pure substances or crude extracts. The screening methods used for both bacteria and fungi are similar. The most commonly used screens to establish antimicrobial susceptibility are the broth dilution assay, the disc diffusion assay and agar well diffusion assay. In some cases, plates or tubes inoculated with microorganisms are exposed to UV light to screen for the existence of light-sensitizing phytochemicals. Antifungal phytochemicals can also be screened by a method known spore germination assay. After initial screening of phytochemicals, more specific media can be used to conduct the specific microorganisms and Minimum inhibitory concentration can be efficiently compared to those of presently used antibiotics (Cowan, 1999).
A number of methods are offered to detect either virucidal or antiviral plant activity. Investigators can assess cytopathic effects, plaque formation, transformation and proliferative effects on cell lines. Viral replication is a measure of antiviral activity. This may be quantified by detection of viral products such as RNA, DNA and polypeptides. Antiviral assays often screen for active substances, those have the capacity to inhibit adsorption of the microorganism to host cells (Ahmad, 2010).
1.14.3. Protozoa and helminths
Compared to the screening of plant extract for their activity against bacteria, fungi, or viruses screening against helminths and protozoa can be more difficult. Culturing the organism is very difficult and very less number of organisms is obtained. Assays are particular for the microorganism (Vital, 2009).
1.15. Choice of antimicrobial agent
Selection from among several drugs depends on host factors that include the following: (1) concomitant disease states (eg, AIDS, severe chronic liver disease); (2) prior adverse drug effects; (3) impaired elimination or detoxification of the drug (may be genetically predetermined but more frequently is associated with impaired renal or hepatic function due to underlying disease); (4) age of the patient; and (5) pregnancy status. Pharmacologic factors include (1) the kinetics of absorption, distribution, and elimination; (2) the ability of the drug to be delivered to the proper site of infection; (3) the potential toxicity of an agent; and (4) pharmacokinetic or pharmacodynamic interactions with other drugs. Finally, increasing consideration is being given to the cost of antimicrobial therapy, especially when multiple agents with comparable efficacy and toxicity are available for a specific infection (Harry and Daniel, 2007).
Use of a combination of antimicrobial agents may be justified (1) for empirical therapy of an infection in which the cause is unknown, (2) for treatment of polymicrobial infections, (3) to enhance antimicrobial activity (i.e., synergism) for a specific infection, or (4) to prevent emergence of resistance. Combination therapy has been advocated for the treatment of infections caused by other gram-negative rods. However, the benefits of using a drug combination over a single, effective agent remain largely unproven (Waterer, 2005).
1.16. Clinical failure of antimicrobial agents
Disadvantages of antimicrobials include super infection, treatment of non responsive infection, and therapy of diseases of unknown origin, improper dosage, inappropriate reliance on chemotherapy alone and lack of adequate bacteriological information (Flammer, 1997).
Superinfection, defined as the appearance of bacteriological and clinical evidence of a new infection during the chemotherapy of a primary one. This phenomenon is relatively common and potentially very dangerous because the microorganisms responsible for the new infection can be misuses of Antibiotics. There are methods to optimize the use of antimicrobial agents to prevent drug resistance and the transmission of infections (Serra et al., 1985).
1.16.2. Treatment of nonresponsive infections
A frequent exploitation of these agents for infections that was proved by experimental and clinical examination to be nonresponsive to treatment with antimicrobial agents. Most of the diseases caused by viruses are self-limited and do not respond to any of the currently available anti-infective compounds.
1.16.3. Therapy of diseases of unknown origin
Fever of undetermined cause may persist for only a few days or a week or for a longer period. Some of these infections may require treatment with antimicrobial agents that are not used commonly for bacterial infections. Others, such as occult abscesses, may require surgical drainage or prolonged courses of pathogen-specific therapy, as in the case of bacterial endocarditis. In appropriately administered antimicrobial therapy may mask an underlying infection, delay the diagnosis, and by rendering cultures negative, prevent identification of the infectious pathogen (Miller, 2008).
1.16.4. Improper dosage
Dosing errors, which can be the wrong frequency of administration or the use of either an excessive or a sub therapeutic dose, are common. Although antimicrobial drugs are among the safest and least toxic of drugs used in medical practice, excessive amounts can result in significant toxicities, including seizures (e.g., penicillin), vestibular damage (e.g., aminoglycosides), and renal failure (e.g., aminoglycosides), especially in patients with impaired drug excretion or metabolism. The use of too low a dose may result in treatment failure (Garcia, 2009).
1.16.5. Inappropriate reliance on chemotherapy alone
Infections complicated by abscess formation, the presence of necrotic tissue, or the presence of a foreign body often cannot be cured by antimicrobial therapy alone. As a general rule, when an appreciable quantity of pus, necrotic tissue, or a foreign body is present, the most effective treatment is an antimicrobial agent given in adequate dose plus a properly performed surgical procedure.
1.16.6. Lack of adequate bacteriological information
Antimicrobial therapy administered to patients too often is given in the absence of supporting microbiological data. Frequent use of drug combinations or drugs with the broadest spectra is a cover for diagnostic imprecision (Chambers, 2006).
1.17. Advantages of herbal products over synthetic products
These days the word natural products are rather frequently understood to refer to herbs, herbal decoctions, dietary supplements, traditional medicine, or alternative medicine. The World Health Organization estimates that more or less 80 percent of the world's population primarily using traditional systems of medicines for their normal health care (Spainhour, 2005).
Herbal drugs have been used since the olden times as a therapy for the treatment of a wide variety of diseases like malaria, jaundice etc. Medicinal plants have significant role in maintaining world health by means of traditional systems of medicines. Regardless of the great improvements observed in modern systems of medicine, plants build an significant role to normal health care. Herbal plants are seen worldwide, but they are most plentiful in tropical countries. Interest in drugs derived from higher plants, has improved expressively. It is calculated that about quarter percentage of all modern medicines are directly or indirectly obtained from medicinal plants (Bozzuto, 1998).
Natural products may be the most creative source of lead compound for the further development of new drugs. More than hundred new products are o the way of clinical investigations, predominantly as anti infectives and anti-cancer agents. Various screening approaches are being utilized to improve and reveal the effectiveness of natural products. It therefore seems advisable to do research on such plants, which have been utilized over the centuries for treatment and curative purposes (Harvey, 2008).