Plant may contain a wide variety of secondary metabolites, such as phenolic compounds (e.g. phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, and tannins), nitrogen compounds (alkaloids, amines, and betalains), vitamins, terpenoids and some other endogenous metabolites, which are rich in medicinal activity. The intake of natural products has been associated with reduced risks of cancer, cardiovascular disease, diabetes, and other diseases associated with ageing. Herbal drugs are hastily becoming accepted in recent years as an optional therapy (Agarval et al., 2008).
In nature there are wide variety of naturally occurring compounds which are different in their composition, physical and chemical property, mechanism and site of action (Gupta et al., 2006). For example flavonoids are widely distributed in plant foods, and constitute the most common phenolic compounds in plants. Flavonoids have been described to modulate certain immune processes and anti oxidant property (Oteiza et al., 2005). The Phytochemical screening of the four ficus bark extract showed classes of useful chemical compounds such as steroids, flavonoids, glycosides, saponins, tannins, phenols and triterpenoids. To the best of our knowledge, there is no previous reported work on anti hemolytic activity of methanolic bark extract of four ficus variety (Ficus bengalensis, Ficus racemosa, Ficus microcarpa and Ficus religosa).
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Hemolysis is the rupture of red blood cells membrane, causing the liberation of hemoglobin and other components into the extra cellular fluid. (Arzoumanian, 2003). The present study examined the inhibitory effects of methanolic extract of ficus bark on human erythrocyte hemolysis .The production of free radical is a normal physiological process and free radicals act on lipids to cause lipid per oxidation (Sekiya et al.,2005).
Red blood cell membranes contain lipids rich in unsaturated fatty acids. RBCs are more frequently exposed to oxygen than other body tissue and are more susceptible to oxidative damage. Invasion of the RBC membrane by peroxidant may lead to cell hemolysis. Lipid peroxidation can also be catalysed by the hemoglobin which is released from the hemolysed RBC. Other than lipid peroxidation, oxidants influence vital -SH groups of proteins which are extremely active and possibly under attack in oxidative stress. Decreased glutathione amount leads to a decline in -SH groups. Diminished levels of glutathione bring about oxidization of membrane -SH groups and loss of membrane constancy. Protein -SH groups have a fundamental role in keeping cell membrane strength. In the time of oxidative stress, -SH groups guard cellular structures in opposition to free radicals by undergoing oxidization and creating disulfide bonds. If antioxidant compounds found important in preserving -SH groups in opposition to oxidization, they are probable to enhance cellular resistance to oxidative stress (Asgary et al., 2005). To evaluate the ability of the extracts to prevent damage to human blood constituents, three in vitro methods were utilised.
Free radical initiators such as 2, 2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH) have been used to generate free radicals by thermal decomposition in the aqueous phase to produce carbon radicals which react with oxygen rapidly to give peroxyl radicals, which attack the erythrocyte membranes. Where lysis occurs by two events, lipid peroxidation and redistribution of oxidized band within the cell membrane. It is considered that AAPH attack the membrane to induce the chain oxidation of lipids and proteins leading to the change of spectrin, a membrane protein by oxidative cross linking which may ultimately result in hemolysis (Zhu et al., 2002).
Water-soluble radical scavengers scavenge the peroxyl radicals derived from AAPH in the aqueous phase efficiently before the radicals attack the erythrocyte membranes and protect membranes from the oxidative damage. On the other hand, lipid-soluble scavenger residing in the membranes scavenges radicals predominantly within the membranes and suppresses the oxidative damage of erythrocytes (Niki et al., 1988).
Band 3 proteins of erythrocytes membrane play an important role in a rapid exchange of HCO3 - and Cl- across the membrane. The plant extract may block the hemolysis by inhibiting the formation of hemolytic holes in erythrocyte cell membrane by blocking oxidation and redistribution of band 3 proteins. Inhibition of hemolysis in the presence of test sample was determined by measuring the absorbance of the supernatant fraction of the reaction mixture (amount of hemoglobin) at 540 nm (Lavhale and Mishra et al., 2007). Previous studies have established that AAPH-produced hemolysis in RBCs is efficiently repressed by natural antioxidants. In our study the FBBE showed significant anti hemolytic activity compared to other extract.
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The name viridans is a group of bacteria which can produce alpha-hemolysis in the presence of an agent known alpha-hemolysin. The study done by Bernard indicates that the alpha-hemolysin is hydrogen peroxide and this peroxide plays an important role in pathogenesis. It has been reported that concentrations of H2O2 produced by viridans are sufficient to cause lethal damage to human cells. Acidic conditions dramatically enhance the effects of H2O2 on erythrocytes because reduced pH may increase cell permeability, inhibit antioxidant defences, or supply protons needed as reagents in the oxidation of heme iron. Here we have induced the hemolysis by using hydrogen peroxide. The concentration of H2O2 in the reaction mixture was adjusted so as to bring about 90% hemolysis of blood cells (Bernard et al., 1996).
Hydrogen peroxide acts rapidly to generate free radicals, which then cause chain reaction-type lipid peroxidation (Younkin et al., 1971). Hydrogen peroxide may produce hemolysis through a mechanism which was dependent on the oxygenated state of hemoglobin. It is suggested that hemolysis is due to interaction of H2O2 with intracellular hemoglobin and some product of such interaction is the lytic agent (Falcioni et al., 2003). It was reported that flavonols and their glycosides are effective antioxidants which can protect human red blood cells from free radical induced oxidative hemolysis (Dai et al ., 2006). FBBE showed a strong dose-dependent protection toward the hemolysis of red blood cells in vitro. The inhibitory effect of the FBBE was almost similar to ascorbic acid, which has been shown to act as an antioxidant against human low-density lipoprotein oxidation. Other extracts also showed potential antihemolytic activity at very low concentrations.
In the third assay hemolysis was induced by osmotic stress. Hypotonic PBS was used to induce hemolysis. This method is used to study structural modification of the red blood cell membrane in the occurrence of osmotic strain. Sodium chloride which is present in the hypotonic PBS will induce protein oxidation in erythrocyte cell membrane as well as decrease sulfhydryl content (Alanazi., 2010). The increased fragility of erythrocytes membrane may be due to increased oxidative stress. Moreover, excess of free radicals can overcome the ability of antioxidants enzymes to preserve and sustain the membrane integrity (Tsuchiya et al., 2002). In the current study, the vulnerability of erythrocytes to hemolysis in presence of ficus extract was lower than that of control. This discovery was comparable to the earlier study reported that presence of flavonoids present in the ficus extract critically inhibits the hemolysis by binding of to the red blood cell membranes significantly inhibits lipid peroxidation and at the same time enhances their integrity against lysis(Chaudhuri et al., 2007).
In all the three assays extend of hemolysis was determined corresponding to liberation of haemoglobin by measurement of the absorbance at 540 nm (Ebrahimzadeh et al., 2010). Vitamin C is a most important antioxidant in plasma and cells. It also interact with the plasma membrane was used as standard. Ascorbic acid can act as antioxidant both inside the cell as well as across the plasma membrane. Transformation of α-tocopherol by ascorbate and ascorbate-dependent oxido reductase activity helps to protect membrane lipids from peroxidation (May, 1999).
The natural wealth of both potential and recognized antioxidants are huge. Some antioxidant compounds are derived from simply accessible sources, for example agricultural and horticultural crops, medicinal plants etc. Generally defined, an antioxidant is a compound that prevent or delays the oxidation of substrates even though the compound is present in a extensively lower concentration than the oxidized substrate (Hodzic et al., 2009). The scavenging of reactive oxygen species (ROS) is one among the probable method of action. Others comprise the avoidance of ROS development by metal binding or enzyme inhibition. Chain breaking antioxidants avoid injure by interfering with the free radical transmission cascades (Hepsibha et al., 2010).
The antioxidant activity of plant derived metabolites has been generally recognized by in vitro method and involves a number of of the above-mentioned mechanisms of action. In the present study we are trying to reveal the anti oxidant property of methanolic extract of bark of four ficus variety by using in vitro techniques.
There are a number of plain, a little more complex, and fairly complicated methods for antioxidant testing. The antioxidant testing can disclose a variety of mechanisms of action, based on description of the specific assay (Matkowski et al., 2008). Here we have done around ten in vitro methods to evaluate the anti oxidant activity of the plant.
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Phosphomolybdenum method can be used to find out the total antioxidant capacity of the extract. It was considered based on the development of phosphomolybdenum compound which was measured spectrometrically at 695 nm (Rakesh et al 2010). This assay is based on the conversion of Mo (VI) to Mo (V)(reduction) in presence of the antioxidant compounds and the consequent development of a green phosphate/Mo (V) complex at lower pH (Kumar et al., 2008). All the extracts possessed antioxidant activity. The phosphomolybdate method to measure the antioxidant capacity is quantitative assay, so the antioxidant capacity is expressed as α-tocopherol equivalents (Umamaheswari and Chatterjee, 2007). FBBE and FMBE have high antioxidant capacity compared to others and a extremely constructive association among total phenols and antioxidant activity expressed in our study.
Fe 2+ forms complexes with ferrozine quantitatively. But in the presence of ion chelating agents, the complex development is disturbed, following a reduction in the red colour of the complex. The iron (II) chelating properties of the antioxidant extract may be attributed to their endogenous chelating agents, mainly phenolics. Some phenolic compounds have correctly positioned functional groups, which can chelate metal ions (Senevirathne et al., 2006). Measurement of this colour change consequently allows for the estimation of metal chelating ability of the synchronized chelator. Plant extract having metal chelating ability will disrupt the formation of Fe2+-ferrozine complex indicating that plant extract chelate the iron. Metal chelating agents diminish the concentration of transition metal which catalyses the lipid peroxidation by forming sigma bonds. This in turn reduces the redox potential and stables the oxidized form of the metal ion (Kumar et al., 2007). It was studied that the chelating agents who make bond with metals, are valuable antioxidants derivative. Since they decrease the redox potential thus stabilising the oxidised form of the metal ion (Duh et al., 1999).The findings of our study expressed that the extracts have valuable ability for iron binding, suggesting its antioxidant potential. Moreover, the metal chelating capacity of the fractions established that they decrease the concentration of the catalysing transition metal concerned in the peroxidation of lipids.
Peroxynitrite (ONOO−) has a potent oxidative activity toward -SH and other reduced moieties. Peroxy nitrous acid (ONOOH), protonated form of ONOO− can evoke lipid peroxidation (Beckman et al., 1994). One way for disintegration of this molecule was considered to be homolytic cleavage to generate hydroxyl radicals and nitrogen dioxide (Yang et al., 2001). An azo dye initiator, AAPH, was used to generate peroxyl radicals, and the scavenging activity of the extracts was determined via the spectrophotometric examination of 2,7- dichlorofluorescin-diacetate (Cui.,2005). Carbon radicals generated from AAPH quickly bind with oxygen to create peroxyl radicals in an AAPH concentration based manner. Dichlorofluorescin after being oxidized by various oxidants, will become 2, 7-dichlorofluorescin-diacetate and absorbance was measured at 490nm (Wang and Joseph, 1999). All the four extracts showed dose-dependent peroxy radical scavenging activity. In particular, the FBBE extract presented the strongest effect, although its scavenging effect was lower than that of ascorbic acid.
Flavonoids are a group of effective antioxidants which are present abundantly throughout the plant kingdom. Flavonoid and related compound are effective in scavenging hydroxyl radical and in metal-chelating capacity. Flavonoids are found to exhibit numerous biological activities like vasodilatory, anticarcinogenic, antiinflammatory, antibacterial, immune-stimulating, antiallergic, and antiviral effects (Hepsibha et al., 2010). In our study the total flavonoid content was estimated spectrophotometrically by the aluminium chloride method based on the formation of complex flavonoid aluminu. In the present study the FBBE and FMBE was found to possess significant amount, when compared to other extracts. The total flavonoid content assay is quantitative, since it is expressed as quercetin equivalents.
Phosphomolybdic acid and phosphotungstic acid present in the Folin- Ciocalteu reagent, react with the phenolic compounds and experience a complex redox reaction (Tyagi et al., 2010).The total phenolic compound estimation by Folin- Ciocalteu assay was not definite to polyphenols but to any other substance that can be oxidised by the Folin reagent .However, it should be prominent that some chemical group of organic acids, amino acids, sugars, aromatic amines and proteins. could react with the reagent(Wong et al., 2004). In this investigation, bark was dried before extraction while ascorbic acid was lost during drying process and amino acids and proteins can be removed from the extraction solvents. Thus, interference from ascorbic acid and other compounds should be very little. The amount of total phenolic constituent present in the various extracts was determined as microgram of pyrocatechol equivalent. FBBE and FMBE were found to possess significant amount of phenols.
Nitric oxide is an essential bio regulatory molecule required for several physiological processes like neural signal transmission, immune response, control vasodilatation and control of blood pressure etc. The elevation of the NO can leads to several pathological conditions, including cancer (Balakrishnan et al., 2009). In our study nitric oxide is formed in physiological pH from sodium nitroprusside in aqueous medium. Nitric oxide then reacts with oxygen to generate nitrite ions and it may be measured by means of Greiss reagent. The nitric oxide scavenger competes with oxygen leads to deceased generation of nitric oxide (Jagetia et al., 2004). Nitric oxide radical inhibition was measured by using the principles of Griess Illosvoy reaction. The Nitric oxide radical was produced from the sodium nitropruside and measured by the Greiss reduction (Chakraborthy et al., 2009). The absorbance was calculated at 546 nm in the presence of plant extract. In the present study all the four extracts reduced the quantity of nitrite formed from the breakdown of sodium nitroprusside. This might be because of the antioxidant constituents in the extract which participate with oxygen to react with NO· thus inhibiting the production of nitrite.
In biochemical coordination, superoxide radical and H2O2 react mutually to generate singlet oxygen and hydroxyl radical and this is one of the most reactive oxygen species among all ROS (Philips et al., 2010). In vitro, (OH.) were generated by a mixture of Fe3+ - EDTA, H2O2 and ascorbic acid and were assessed by monitoring the degraded fragments of deoxyribose, by reaction with thio barbituric acid(Fenton reaction). If any drug scavenges the hydroxyl radical, they may either scavenge the radical or may chelate the Fe2+ ion making them unavailable for the Fentons reaction (Hepsibha et al., 2010). Lipid oxidation and enormous biological damage are induced by hydroxyl radicals. On Ph 7.4, ferric-EDTA complex was incubate with ascorbic acid and hydrogen peroxide and leads to the formation of hydroxyl radicals in free solution and measured by their capability to degrade 2-deoxy-2-ribose into fragments. These fragments by heating with thiobarbituric acid at low pH appears a pink colour (Gupta et al., 2004).In our studies, all the extracts exhibit concentration depended scavenging activity against OH radical generated in Fenton system. This can be due to highly vigorous hydrogen giver capacity of OH substitution or its chelating power of phenolic group present in the extract. This in vitro model showed that our plant extract can be used to diminish the adverse effect of OH radical, to stop lipid peroxidation and DNA destruction.
It is well recognized that superoxide anions injure biomolecules by generating H2O2, .OH, singlet oxygen and peroxy nitrite during aging and pathological events such as ischemic reperfusion injury. Superoxide has also been reported for its capacity to start lipid peroxidation. Superoxide anions can be generated by both enzymatic and non-enzymatic system (Krishnaraju et al., 2009). In this study we adopted the non-enzymatic method to generate superoxide anion radical. In this assay PMS /NADH -NBT system was used to generate and estimate super oxide anions. Super oxides obtained from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The reduced absorbance with antioxidants indicates consumption of super oxide anion in the reaction mixture (Basniwal et al., 2009). The decline in absorbance at 560 nm with acetone, ethanol and antioxidants shows the utilization of superoxide anion in the reaction blend. The degree of discoloration indicates the scavenging potential of the plant extracts. As expected, the lower the IC50 values the higher the percentage of NBT radical inhibition of the samples (Salar and Dhall, 2010). All the four extracts when added to the reaction mixture, scavenge the hydroxyl radicals by preventing the decomposition of deoxyribose.
Even though hydrogen peroxide itself is not extremely reactive, it can occasionally produce cytotoxicity by generating hydroxyl radicals in the cell. Therefore disappearing, H2O2 is very vital all through the food scheme (Ebrahimzadeh et al., 2010). H2O2 was considered poorly reactive because of its weaker oxidizing and reducing capabilities. Biologically, it acts as a toxicant to the cell by converting itself into hydroxyl radical in the presence of metal ions and superoxide anion and also produces singlet oxygen through reaction with superoxide anion or with hypochlorous acid (HOCl) or chloramines in living systems (Saumya and Basha,2011). Transition metal ion supporting hydroxyl radical mediated oxidative DNA injures may occur because of the accumulation of H2O2 to cells in cultures. Levels of H2O2 around 20-50 mg appears to have partial or limited cytotoxicity to a lot of cell types (Sahgal et al., 2009). Therefore, scavenging of H2O2 can be used as a gauge of the antioxidant activity of the plant extract. All the extracts of ficus scavenged hydrogen peroxide which may be credited due to the occurrence of phenolic constituents that might contribute electrons to hydrogen peroxide, thus neutralising it into water.
The seek out for compounds with antimicrobial activity has got rising significance due to worldwide concern about the shocking increase in the rate of infection by antibiotic-resistance microorganisms. Many of the plants have been known to synthesize active secondary metabolites with established potent insecticidal and antimicrobial activities, which indeed have formed the basis for their applications in some pharmaceuticals, alternative medicines and natural therapies (Devi et al., 2009).
Numerous of the complex relations between the invading microorganism and its host are implicated by reactive oxidant species. Oxidants demonstrate a wide variety of toxic effects to biomolecules that are crucial for cell endurance. Generation of these oxidants by microorganisms make them to have a survival benefit in their surroundings. Host oxidant creation, particularly by phagocytes, is a counteractive mechanism meant at microbial killing. However, this mechanism may donate a harmful significance of oxidant contact. Both the host and the microorganism have produced difficult adaptive mechanisms to prevent oxidant-mediated damage, together with enzymatic and nonenzymatic oxidant-scavenging systems (Miller and Britigan, 1997).
Plants are significant source of potentially valuable structures for the growth of new chemotherapeutic compounds. The primary move on the way to this goal is the in vitro antibacterial activity assay. A lot of information's are obtainable on the antiviral, antifungal, anthelmintic, antibacterial, antimolluscal and anti-inflammatory properties of plants. Some of these interpretations have helped in identifying the active principle accountable for such actions and in the development of drugs for the therapeutic use in human beings (Tona et al., 1998).
In the present study, the methanolic extract of four ficus species showed the activity against S. aureus, P.aeruginosa and E. coli. Plant based products have been effectively proven for their utilization as source for antimicrobial compounds. Ciprofloxacin was approved by the U.S. Food and Drug Administration as a broad spectrum antibiotic that is active against both Gram positive and Gram-negative bacteria were used as standard It has been used for the treatment of 14 types of infection including respiratory and urinary tract infections, skin, and other gastro-intestinal infections(Riyazullah et al., 2010).
The antimicrobial importance of medicinal plants has been explained by the chemical structure of active substances (Ushimaru et al., 2007). Utilization of crude plant extracts and its phytochemicals, with predictable antimicrobial property, can be of enormous implication in curative treatments. In the most recent years, several studies have been take place in different countries to establish such effectiveness. A lot of plants have been used as of their antimicrobial qualities, which are because of compounds synthesized in the derivative metabolism of the plant. These plant species are recognized by their active constituents. For instance, the phenolic compounds which are derivatives of the oils as well as tannins (Mahesh and Satish, 2008).
The Ficus bark extracts contains tannins and phenolics. Our study showed that ficus extract inhibited both gram positive and gram negative bacteria. Flavonoids are established to be valuable antimicrobial substances against extensive variety of micro organism, possibly because of their capacity to complex with bacterial cell wall; more lipophilic flavonoids may disturb microbial membrane (Bylka et al., 2004).
Antibacterial activity of tannins may be connected to their capacity to inactivate microbial bond enzymes and cell wall envelope transport proteins, they also complex with polysacchrides( Scalbert,2001).The presence of tannins and flavonoids present in the bark of ficus implied that which may be responsible for in vitro anti bacterial activity in this study.
Results of the current study showed a direct correlation between the concentration of the tested plant extract and its antihemolytic effect, antioxidant effect and antimicrobial effect. Specified that these effects were also observed at satisfactory levels at much lower concentrations, it may be probable to use them in the clinical setting with negligible side effects (once in vivo and toxicology tests have been performed).In radiance of the results of the present and parallel studies on plant extracts, these substances may give beneficial effects in inhibiting oxidative damage to membranes. They may also be useful in preventing disease conditions in which lipid peroxidation have an important role. Further studies are required assess their effects and mechanisms of actions.