Plant may incorporate a broad assortment of secondary metabolites, such as phenolic compounds ( e.g. phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, and tannic acids ) , nitrogen compounds ( alkaloids, aminoalkanes, and betalains ) , vitamins, terpenoids and some other endogenous metabolites, which are rich in medicative activity. The consumption of natural merchandises has been associated with decreased hazards of malignant neoplastic disease, cardiovascular disease, diabetes, and other diseases associated with ageing. Herbal drugs are hurriedly going accepted in recent old ages as an optional therapy ( Agarval et al. , 2008 ) .
In nature there are broad assortment of of course happening compounds which are different in their composing, physical and chemical belongings, mechanism and site of action ( Gupta et al. , 2006 ) . For illustration flavonoids are widely distributed in works nutrients, and represent the most common phenolic compounds in workss. Flavonoids have been described to modulate certain immune procedures and anti oxidant belongings ( Oteiza et al. , 2005 ) . The Phytochemical showing of the four Ficus bark infusion showed categories of utile chemical compounds such as steroids, flavonoids, glycosides, saponins, tannic acids, phenols and triterpenoids. To the best of our cognition, there is no old reported work on anti haemolytic activity of methanolic bark infusion of four Ficus assortment ( Ficus bengalensis, Ficus racemosa, Ficus microcarpa and Ficus religosa ) .
Hemolysis is the rupture of ruddy blood cells membrane, doing the release of haemoglobin and other constituents into the excess cellular fluid. ( Arzoumanian, 2003 ) . The present survey examined the repressive effects of methanolic infusion of Ficus bark on human erythrocyte haemolysis.The production of free group is a normal physiological procedure and free groups act on lipoids to do lipid per oxidization ( Sekiya et al.,2005 ) .
Red blood cell membranes contain lipoids rich in unsaturated fatty acids. RBCs are more often exposed to oxygen than other organic structure tissue and are more susceptible to oxidative harm. Invasion of the RBC membrane by peroxidant may take to cell haemolysis. Lipid peroxidation can besides be catalysed by the haemoglobin which is released from the hemolysed RBC. Other than lipid peroxidation, oxidizers influence critical -SH groups of proteins which are highly active and perchance under onslaught in oxidative emphasis. Decreased glutathione sum leads to a diminution in -SH groups. Diminished degrees of glutathione bring about oxidation of membrane -SH groups and loss of membrane stability. Protein -SH groups have a cardinal function in maintaining cell membrane strength. In the clip of oxidative emphasis, -SH groups guard cellular constructions in resistance to free groups by undergoing oxidation and making disulfide bonds. If antioxidant compounds found of import in continuing -SH groups in resistance to oxidation, they are likely to heighten cellular opposition to oxidative emphasis ( Asgary et al. , 2005 ) . To measure the ability of the infusions to forestall harm to human blood components, three in vitro methods were utilized.
Free extremist instigators such as 2, 2′-azo-bis ( 2-amidinopropane ) dihydrochloride ( AAPH ) have been used to bring forth free groups by thermic decomposition in the aqueous stage to bring forth C groups which react with O quickly to give peroxyl groups, which attack the red blood cell membranes. Where lysis occurs by two events, lipid peroxidation and redistribution of oxidised set within the cell membrane. It is considered that AAPH attack the membrane to bring on the concatenation oxidization of lipoids and proteins taking to the alteration of spectrin, a membrane protein by oxidative cross associating which may finally ensue in haemolysis ( Zhu et al. , 2002 ) .
Water-soluble extremist scavengers scavenge the peroxyl groups derived from AAPH in the aqueous stage expeditiously before the groups attack the red blood cell membranes and protect membranes from the oxidative harm. On the other manus, lipid-soluble scavenger shacking in the membranes scavenges groups preponderantly within the membranes and suppresses the oxidative harm of red blood cells ( Niki et al. , 1988 ) .
Band 3 proteins of red blood cells membrane play an of import function in a rapid exchange of HCO3 – and Cl- across the membrane. The works infusion may barricade the haemolysis by suppressing the formation of haemolytic holes in red blood cell cell membrane by barricading oxidization and redistribution of set 3 proteins. Inhibition of haemolysis in the presence of trial sample was determined by mensurating the optical density of the supernatant fraction of the reaction mixture ( sum of haemoglobin ) at 540 nanometer ( Lavhale and Mishra et al. , 2007 ) . Previous surveies have established that AAPH-produced haemolysis in RBCs is expeditiously repressed by natural antioxidants. In our survey the FBBE showed important anti haemolytic activity compared to other infusion.
The name viridans is a group of bacteriums which can bring forth alpha-hemolysis in the presence of an agent known alpha-hemolysin. The survey done by Bernard indicates that the alpha-hemolysin is hydrogen peroxide and this peroxide plays an of import function in pathogenesis. It has been reported that concentrations of H2O2 produced by viridans are sufficient to do deadly harm to human cells. Acidic conditions dramatically heighten the effects of H2O2 on red blood cells because reduced pH may increase cell permeableness, inhibit antioxidant defense mechanisms, or supply protons needed as reagents in the oxidization of haem Fe. Here we have induced the haemolysis by utilizing H peroxide. The concentration of H2O2 in the reaction mixture was adjusted so as to convey approximately 90 % haemolysis of blood cells ( Bernard et al. , 1996 ) .
Hydrogen peroxide acts quickly to bring forth free groups, which so cause concatenation reaction-type lipid peroxidation ( Younkin et al. , 1971 ) . Hydrogen peroxide may bring forth haemolysis through a mechanism which was dependant on the oxygenated province of haemoglobin. It is suggested that haemolysis is due to interaction of H2O2 with intracellular haemoglobin and some merchandise of such interaction is the lytic agent ( Falcioni et al. , 2003 ) . It was reported that flavonols and their glycosides are effectual antioxidants which can protect human ruddy blood cells from free group induced oxidative haemolysis ( Dai et al. , 2006 ) . FBBE showed a strong dose-dependent protection toward the haemolysis of ruddy blood cells in vitro. The repressive consequence of the FBBE was about similar to ascorbic acid, which has been shown to move as an antioxidant against human low-density lipoprotein oxidization. Other infusions besides showed possible antihemolytic activity at really low concentrations.
In the 3rd assay haemolysis was induced by osmotic emphasis. Hypotonic PBS was used to bring on haemolysis. This method is used to analyze structural alteration of the ruddy blood cell membrane in the happening of osmotic strain. Sodium chloride which is present in the hypotonic PBS will bring on protein oxidization in red blood cell cell membrane every bit good as lessening sulfhydryl content ( Alanazi. , 2010 ) . The increased breakability of red blood cells membrane may be due to increased oxidative emphasis. Furthermore, surplus of free groups can get the better of the ability of antioxidants enzymes to continue and prolong the membrane unity ( Tsuchiya et al. , 2002 ) . In the current survey, the exposure of red blood cells to hemolysis in presence of Ficus infusion was lower than that of control. This find was comparable to the earlier survey reported that presence of flavonoids present in the Ficus infusion critically inhibits the haemolysis by adhering of to the ruddy blood cell membranes significantly inhibits lipid peroxidation and at the same clip enhances their unity against lysis ( Chaudhuri et al. , 2007 ) .
In all the three checks extend of haemolysis was determined matching to release of hemoglobin by measuring of the optical density at 540 nanometer ( Ebrahimzadeh et al. , 2010 ) . Vitamin C is a most of import antioxidant in plasma and cells. It besides interact with the plasma membrane was used as standard. Ascorbic acid can move as antioxidant both inside the cell every bit good as across the plasma membrane. Transformation of ?-tocopherol by ascorbate and ascorbate-dependent oxido reductase activity helps to protect membrane lipoids from peroxidation ( May, 1999 ) .
The natural wealth of both possible and recognized antioxidants are immense. Some antioxidant compounds are derived from merely accessible beginnings, for illustration agricultural and horticultural harvests, medicative workss etc. By and large defined, an antioxidant is a compound that prevent or delays the oxidization of substrates even though the compound is present in a extensively lower concentration than the oxidised substrate ( Hodzic et al. , 2009 ) . The scavenging of reactive O species ( ROS ) is one among the likely method of action. Others comprise the turning away of ROS development by metal binding or enzyme suppression. Chain interrupting antioxidants avoid injure by interfering with the free extremist transmittal Cascadess ( Hepsibha et al. , 2010 ) .
The antioxidant activity of works derived metabolites has been by and large recognized by in vitro method and involves a figure of of the above-named mechanisms of action. In the present survey we are seeking to uncover the anti oxidant belongings of methanolic infusion of bark of four Ficus assortment by utilizing in vitro techniques.
There are a figure of field, a little more complex, and reasonably complicated methods for antioxidant testing. The antioxidant testing can unwrap a assortment of mechanisms of action, based on description of the specific check ( Matkowski et al. , 2008 ) . Here we have done around 10s in vitro methods to measure the anti oxidant activity of the works.
Phosphomolybdenum method can be used to happen out the entire antioxidant capacity of the infusion. It was considered based on the development of phosphomolybdenum compound which was measured spectrometrically at 695 nanometer ( Rakesh et al 2010 ) . This check is based on the transition of Mo ( VI ) to Mo ( V ) ( decrease ) in presence of the antioxidant compounds and the consequent development of a green phosphate/Mo ( V ) composite at lower pH ( Kumar et al. , 2008 ) . All the infusions possessed antioxidant activity. The phosphomolybdate method to mensurate the antioxidant capacity is quantitative check, so the antioxidant capacity is expressed as ?-tocopherol equivalents ( Umamaheswari and Chatterjee, 2007 ) . FBBE and FMBE have high antioxidant capacity compared to others and a highly constructive association among entire phenols and antioxidant activity expressed in our survey.
Fe 2+ signifiers composites with ferrozine quantitatively. But in the presence of ion chelating agents, the complex development is disturbed, following a decrease in the ruddy coloring material of the composite. The Fe ( II ) chelating belongingss of the antioxidant infusion may be attributed to their endogenous chelating agents, chiefly phenoplasts. Some phenolic compounds have right positioned functional groups, which can chelate metal ions ( Senevirathne et al. , 2006 ) . Measurement of this coloring material alteration accordingly allows for the appraisal of metal chelating ability of the synchronised chelator. Plant extract holding metal chelating ability will interrupt the formation of Fe2+-ferrozine complex indicating that works infusion chelate the Fe. Metal chelating agents decrease the concentration of passage metal which catalyses the lipid peroxidation by organizing sigma bonds. This in bend reduces the redox potency and stables the oxidised signifier 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 possible therefore bracing the oxidized signifier of the metal ion ( Duh et al. , 1999 ) .The findings of our survey expressed that the infusions have valuable ability for Fe binding, proposing its antioxidant potency. Furthermore, the metal chelating capacity of the fractions established that they decrease the concentration of the catalyzing passage metal concerned in the peroxidation of lipoids.
Peroxynitrite ( ONOO? ) has a powerful oxidative activity toward -SH and other decreased medieties. Peroxy azotic acid ( ONOOH ) , protonated signifier of ONOO? can arouse lipid peroxidation ( Beckman et al. , 1994 ) . One manner for decomposition of this molecule was considered to be homolytic cleavage to bring forth hydroxyl groups and N dioxide ( Yang et al. , 2001 ) . An azo dye instigator, AAPH, was used to bring forth peroxyl groups, and the scavenging activity of the infusions was determined via the spectrophotometric scrutiny of 2,7- dichlorofluorescin-diacetate ( Cui.,2005 ) . Carbon groups generated from AAPH rapidly bind with O to make peroxyl groups in an AAPH concentration based mode. Dichlorofluorescin after being oxidized by assorted oxidizers, will go 2, 7-dichlorofluorescin-diacetate and optical density was measured at 490nm ( Wang and Joseph, 1999 ) . All the four infusions showed dose-dependent peroxy extremist scavenging activity. In peculiar, the FBBE infusion presented the strongest consequence, although its scavenging consequence was lower than that of ascorbic acid.
Flavonoids are a group of effectual antioxidants which are present copiously throughout the works land. Flavonoid and related compound are effectual in scavenging hydroxyl extremist and in metal-chelating capacity. Flavonoids are found to exhibit legion biological activities like vasodilatory, anticarcinogenic, antiinflammatory, antibacterial, immune-stimulating, antiallergic, and antiviral effects ( Hepsibha et al. , 2010 ) . In our survey the entire flavonoid content was estimated spectrophotometrically by the aluminum chloride method based on the formation of complex flavonoid aluminu. In the present survey the FBBE and FMBE was found to possess important sum, when compared to other infusions. The entire flavonoid content check is quantitative, since it is expressed as quercetin equivalents.
Phosphomolybdic acid and phosphotungstic acid nowadays in the Folin- Ciocalteu reagent, react with the phenolic compounds and see a complex oxidation-reduction reaction ( Tyagi et al. , 2010 ) .The entire phenolic compound appraisal by Folin- Ciocalteu check was non definite to polyphenols but to any other substance that can be oxidised by the Folin reagent.However, it should be outstanding that some chemical group of organic acids, aminic acids, sugars, aromatic aminoalkanes and proteins. could respond with the reagent ( Wong et al. , 2004 ) . In this probe, bark was dried before extraction while ascorbic acid was lost during drying procedure and amino acids and proteins can be removed from the extraction dissolvers. Therefore, intervention from ascorbic acid and other compounds should be really small. The sum of entire phenolic component nowadays in the assorted infusions was determined as mcg of pyrocatechol equivalent. FBBE and FMBE were found to possess important sum of phenols.
Nitric oxide is an indispensable bio regulative molecule required for several physiological procedures like nervous signal transmittal, immune response, control vasodilatation and control of blood force per unit area etc. The lift of the NO can take to several pathological conditions, including malignant neoplastic disease ( Balakrishnan et al. , 2009 ) . In our survey azotic oxide is formed in physiological pH from Na nitroprusside in aqueous medium. Nitric oxide so reacts with O to bring forth nitrite ions and it may be measured by agencies of Greiss reagent. The azotic oxide scavenger competes with O leads to asleep coevals of azotic oxide ( Jagetia et al. , 2004 ) . Nitric oxide extremist suppression was measured by utilizing the rules of Griess Illosvoy reaction. The Nitric oxide radical was produced from the Na nitropruside and measured by the Greiss decrease ( Chakraborthy et al. , 2009 ) . The optical density was calculated at 546 nanometer in the presence of works infusion. In the present survey all the four infusions reduced the measure of nitrite formed from the dislocation of Na nitroprusside. This might be because of the antioxidant components in the infusion which participate with O to respond with NO· therefore suppressing the production of nitrite.
In biochemical coordination, superoxide extremist and H2O2 respond reciprocally to bring forth vest O and hydroxyl extremist and this is one of the most reactive O species among all ROS ( Philips et al. , 2010 ) . In vitro, ( OH. ) were generated by a mixture of Fe3+ – Ethylenediaminetetraacetic acid, H2O2 and ascorbic acid and were assessed by supervising the debauched fragments of deoxyribose, by reaction with thio barbituric acid ( Fenton reaction ) . If any drug scavenges the hydroxyl extremist, they may either scavenge the extremist or may chelate the Fe2+ ion doing them unavailable for the Fentons reaction ( Hepsibha et al. , 2010 ) . Lipid oxidization and tremendous biological harm are induced by hydroxyl groups. On Ph 7.4, ferric-EDTA composite was incubate with ascorbic acid and H peroxide and leads to the formation of hydroxyl groups in free solution and measured by their capableness to degrade 2-deoxy-2-ribose into fragments. These fragments by heating with thiobarbituric acid at low pH appears a pink coloring material ( Gupta et al. , 2004 ) .In our surveies, all the infusions exhibit concentration depended scavenging activity against OH extremist generated in Fenton system. This can be due to extremely vigorous H giver capacity of OH permutation or its chelating power of phenolic group nowadays in the infusion. This in vitro theoretical account showed that our works infusion can be used to decrease the inauspicious consequence of OH extremist, to halt lipid peroxidation and DNA devastation.
It is good recognized that superoxide anions injure biomolecules by bring forthing H2O2, .OH, vest O and peroxy nitrite during aging and pathological events such as ischaemic reperfusion hurt. Superoxide has besides been reported for its capacity to get down lipid peroxidation. Superoxide anions can be generated by both enzymatic and non-enzymatic system ( Krishnaraju et al. , 2009 ) . In this survey we adopted the non-enzymatic method to bring forth superoxide anion group. In this check PMS /NADH -NBT system was used to bring forth and gauge ace oxide anions. Super oxides obtained from dissolved O by PMS/NADH matching reaction reduces NBT. The decreased optical density with antioxidants indicates ingestion of ace oxide anion in the reaction mixture ( Basniwal et al. , 2009 ) . The diminution in optical density at 560 nanometer with propanone, ethyl alcohol and antioxidants shows the use of superoxide anion in the reaction blend. The grade of stain indicates the scavenging potency of the works extracts. As expected, the lower the IC50 values the higher the per centum of NBT extremist suppression of the samples ( Salar and Dhall, 2010 ) . All the four infusions when added to the reaction mixture, scavenge the hydroxyl groups by forestalling the decomposition of deoxyribose.
Even though H peroxide itself is non highly reactive, it can on occasion bring forth cytotoxicity by bring forthing hydroxyl groups in the cell. Therefore vanishing, H2O2 is really critical all through the nutrient strategy ( Ebrahimzadeh et al. , 2010 ) . H2O2 was considered ill reactive because of its weaker oxidizing and cut downing capablenesss. Biologically, it acts as a poison to the cell by change overing itself into hydroxyl extremist in the presence of metal ions and superoxide anion and besides produces vest O through reaction with superoxide anion or with hypochlorous acid ( HOCl ) or chloramines in populating systems ( Saumya and Basha,2011 ) . Transition metal ion back uping hydroxyl extremist mediated oxidative DNA injures may happen because of the accretion of H2O2 to cells in civilizations. Degrees of H2O2 around 20-50 milligram appears to hold partial or limited cytotoxicity to a batch of cell types ( Sahgal et al. , 2009 ) . Therefore, scavenging of H2O2 can be used as a gage of the antioxidant activity of the works infusion. All the infusions of Ficus scavenged H peroxide which may be credited due to the happening of phenolic components that might lend negatrons to hydrogen peroxide, therefore neutralizing it into H2O.
The seek out for compounds with antimicrobic activity has got lifting significance due to worldwide concern about the flooring addition in the rate of infection by antibiotic-resistance micro-organisms. Many of the workss have been known to synthesise active secondary metabolites with established potent insecticidal and antimicrobic activities, which so have formed the footing for their applications in some pharmaceuticals, alternate medical specialties and natural therapies ( Devi et al. , 2009 ) .
Numerous of the complex dealingss between the occupying micro-organism and its host are implicated by reactive oxidant species. Oxidants demonstrate a broad assortment of toxic effects to biomolecules that are important for cell endurance. Coevals of these oxidizers by micro-organisms make them to hold a survival benefit in their milieus. Host oxidant creative activity, peculiarly by scavenger cells, is a counteractive mechanism meant at microbic violent death. However, this mechanism may donate a harmful significance of oxidant contact. Both the host and the micro-organism have produced hard adaptative mechanisms to forestall oxidant-mediated harm, together with enzymatic and nonenzymatic oxidant-scavenging systems ( Miller and Britigan, 1997 ) .
Plants are important beginning of potentially valuable constructions for the growing of new chemotherapeutic compounds. The primary move on the manner to this end is the in vitro antibacterial activity assay. A batch of information ‘s are gettable on the antiviral, fungicidal, anthelminthic, antibacterial, antimolluscal and anti-inflammatory belongingss of workss. Some of these readings have helped in placing the active rule accountable for such actions and in the development of drugs for the curative usage in human existences ( Tona et al. , 1998 ) .
In the present survey, the methanolic infusion of four Ficus species showed the activity against S. aureus, P.aeruginosa and E. coli. Plant based merchandises have been efficaciously proven for their use as beginning for antimicrobic compounds. Ciprofloxacin was approved by the U.S. Food and Drug Administration as a wide spectrum antibiotic that is active against both Gram positive and Gram-negative bacteriums were used as criterion It has been used for the intervention of 14 types of infection including respiratory and urinary piece of land infections, tegument, and other gastro-intestinal infections ( Riyazullah et al. , 2010 ) .
The antimicrobic importance of medicative workss has been explained by the chemical construction of active substances ( Ushimaru et al. , 2007 ) . Use of petroleum works infusions and its phytochemicals, with predictable antimicrobic belongings, can be of tremendous deduction in healing interventions. In the most recent old ages, several surveies have been take topographic point in different states to set up such effectivity. A batch of workss have been used as of their antimicrobic qualities, which are because of compounds synthesized in the derivative metamorphosis of the works. These works species are recognized by their active components. For case, the phenoplast compounds which are derived functions of the oils every bit good as tannic acids ( Mahesh and Satish, 2008 ) .
The Ficus bark extracts contains tannic acids and phenoplasts. Our survey showed that Ficus infusion inhibited both gram positive and gram negative bacteriums. Flavonoids are established to be valuable antimicrobic substances against extended assortment of micro being, perchance because of their capacity to complex with bacterial cell wall ; more lipotropic flavonoids may upset microbic membrane ( Bylka et al. , 2004 ) .
Antibacterial activity of tannic acids may be connected to their capacity to demobilize microbic bond enzymes and cell wall envelope conveyance proteins, they besides complex with polysacchrides ( Scalbert,2001 ) .The presence of tannic acids and flavonoids nowadays in the bark of Ficus implied that which may be responsible for in vitro anti bacterial activity in this survey.
Consequences of the current survey showed a direct correlativity between the concentration of the tried works infusion and its antihemolytic consequence, antioxidant consequence and antimicrobic consequence. Specified that these effects were besides observed at satisfactory degrees at much lower concentrations, it may be likely to utilize them in the clinical scene with negligible side effects ( one time in vivo and toxicology trials have been performed ) .In glow of the consequences of the present and parallel surveies on works infusions, these substances may give good effects in suppressing oxidative harm to membranes. They may besides be utile in forestalling disease conditions in which lipid peroxidation have an of import function. Further surveies are required assess their effects and mechanisms of actions.