Antihemolytic And Free Radical Scavenging Activity Biology Essay

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In this study, antihemolytic and free radical scavenging activities of methanolic bark extract of Ficus benghalensis Linn. were by various in vitro assays. The extract exhibited its antihemolytic effect in concentration dependent manner on 2, 2 azo (2-asmidino propane) hydrochloride (AAPH), hydrogen peroxide and osmotic stress induced hemolysis with IC 50 values 171.10µg/mL, 84.60µg/mL and 164.10 µg/mL respectively. The extract was also studied for free radical scavenging activity in a series of assays and IC50 values were determined. Considerable activity was observed in all the test systems (peroxy radical trapping potential IC 50 166.70 µg/mL, hydroxyl radical scavenging activity IC 50 36.70 µg/mL, nitric oxide radical scavenging activity IC 50 105.10 µg/mL, hydrogen peroxide scavenging assay IC 50 86.87 µg/mL and superoxide radical scavenging assay IC 50 138.20 µg/mL) and it is comparable to that of standards. The results obtained in the present study indicate that Ficus benghalensis Linn. bark extract may be used as a major therapeutic agent in future.

Keywords: antihemolytic activity; free radical scavenging activity; Ficus benghalensis Linn.; 2, 2 azo (2-asmidino propane) hydrochloride; peroxy radical trapping potential; hydrogen peroxide scavenging activity


Plants have been the foremost source of drugs in antique systems of medicine in the world. Herbalism is a conventional medicine or folk medicine practice based on the exploitation of plants and plant extracts (Kamboj, 2000). The genus Ficus includes around 750 species of plants distributed in most tropical and subtropical forests throughout the world. The genus is significant for the huge distinction in the habits of its species. Many plants of this genus are used in medicine for the treatment of skin diseases, liver enlargement, diarrhoea, diabetes, respiratory disorder, leucorrhoea, cardiovascular diseases, piles, ulcers and rheumatism (Kirtikar and Basu, 1933).

Ficus bengalensis is commonly known as Banyan tree. This tree is considered to be sacred tree in India. The plant is a large evergreen tree with widely spreading branches bearing many aerial roots. Different part of this tree are used as haemostatic, anti-septic, anti-inflammatory, and anticancer agent and also in the treatment of diarrhoea, skin diseases, ulcers, vaginal disorders, leucorrhoea, menorrhagia and deficient lactation. In the traditional system of medicine, the plant is used for various health problems and diseases (Hosamani et al., 2003).

Leucopelargonidin -3-0-α-L rhamnoside and leuco cynidin 3-0-α-D galactosyl cellobioside, glucoside, β-glucoside, pentatriacontan-5-one, β- sitosterol α- D glucose were isolated from bark of Ficus benghalensis. The leaves contain, protein, fibres, phosphorous, rutin, friedelin, taraxsterol, lupeol and β-amyrin. The latex contains chemical constituents like caoytchoue, resin, albumin, cerin, sugar, and malic acid (Thakare et al., 2010).

Hemolysis is the rupture of red blood cells membrane, causing the liberation of hemoglobin and other components into the surrounding fluid. Hemolysis is visually detected by viewing crimson to red shade in serum or plasma (Lena, 2003).Hemolysis may be associated with numerous disorders like hemolytic anemia, glucose 6-phosphate dehydrogenase deficiency etc. The main objective of the present work was, therefore, to estimate the levels of in vitro anti hemolytic activity and free radical scavenging activity of methanol extract of Ficus benghalensis bark , by various assays.

Materials and Methods


All solvents used in this investigation were of analytical grade: Distilled, deionized water was obtained from purification system (Millipore S.A., Molsheim, France). ascorbic acid , 2, 2 azo (2-asmidino propane) hydrochloride (AAPH), deoxy-D-ribose, thiobarbituric acid, 2, 7-dichlorofluresecin-diacetate, butylated hydroxytoluene , nicotinamide adenine dinucleotide , nitroblue tetrazolium , butylated hydroxyl anisole were purchased from Sigma Chemical Co, USA. Phenazine methosulphate was procured from Himedia, Mumbai. All the other chemicals were also of analytical grade.

Plant material and extraction procedure

The dried bark of F. benghalensis was collected from Coimbatore, Tamilnadu India in July 2009, identified and authenticated by Dr.G.V.S.Murthy Director, Botanical Survey of India, Tamilnadu Agriculture University, Coimbatore and the voucher specimen (BSI/SRC/5/23/10-11/Tech. 597) has been preserved in our division for upcoming reference. The extraction was carried out by cold maceration. The solvent used was 100%v/v methanol. About 50 g of powder was soaked with 250mL of solvent then macerated at room temperature for 72 h. The extract was filtered off and the marc was again soaked with the same volume of solvent for 72 h .The extract was filtered off and the marc was again soaked with the same volume for 72 h and filtered. The filtrates were then combined concentrated to dryness under controlled temperature between 60-700c (Thakare et al., 2010). The percentage yield of the Ficus bengalensis bark extract (FBBE) was 11.5%w/w.

In vitro antihemolytic activity

Preparation of erythrocyte suspensions

Heparinized human blood was obtained and RBCs were separated from plasma by centrifugation at 1500 g for 10 min. Crude RBC was then washed three times with 5 volumes of PBS (pH 7.4). Finally re-suspended using the same buffer to hematocrit level of 5% (Benedetti et al., 2004).

Hemolysis induced by AAPH.

The inhibitory activity of AAPH induced red blood cell damage was evaluated as described previously (Sekiya et al., 2005). One milliliter of RBC suspension was mixed with 1mL of PBS solution containing various amounts of FBBE. One milliliter of RBC suspension mixed with 1 mL of PBS solution alone was used as control. One milliliter of 200mM AAPH in PBS solution was then added to the mixture. The incubation mixture was shaken gently in a water bath at 37 °C for 3 h. After incubation, 4mL of PBS solution was added to the reaction mixture, and this was followed by centrifugation at 1000 g for 10 min. The absorbance of the supernatant at 540 nm was recorded with UV-visible spectrophotometer (UV-265FS, Shimadzu, Kyoto, Japan).We used the L-ascorbic acid as a reference antioxidant compound for comparison purpose. The values are presented as the means of triplicate analysis.

Hemolysis induced by Hydrogen peroxide.

The inhibitory activity of H2O2 induced red blood cell damage was evaluated by slight modification in the method described by Ebrahimzadech (Ebrahimzadech et al., 2009). To 100µl of 5% (v/v) suspensions of erythrocytes in PBS, 50 µl of extract with different concentrations (25-400µg in PBS pH 7.4), was added. To this, 100 µl of 100 µ M H2O2 (in PBS pH 7.4) was added. The reaction mixture was shaken gently while being incubated at 370c for 3h. The reaction mixture was diluted with 8 mL of PBS and centrifuged at 2000 x g for 10minutes.The absorbance of the resulting supernatant was measured at 540 nm by spectrophotometer to determine hemolysis. Likewise erythrocytes were treated with 100 µM H2O2 and without inhibitors to obtain complete hemolysis. The absorbance of the supernatant was measured at same condition. The inhibitory effect of extract was compared with standard L-ascorbic acid. The values are presented as the means of triplicate analysis.

Osmotic stress induced hemolysis

The inhibitory activity of osmotic stress induced red blood cell damage was evaluated by slight modification of previously described method (Tsuchiya et al., 2002). Fresh human erythrocytes 20 µl were mixed with 50 µl of extract with different concentrations (25-400µg in PBS pH 7.4). Add 2 mL of hypotonic PBS solution, and incubated in a shaking water bath at 370C for 30 min. After incubation, the samples were centrifuged at 750x g for 10 min at 40C, and the extent of hemolysis was determined spectrophotometrically at 540 nm. We used the L-ascorbic acid again as a reference antioxidant compound for comparison purpose. The values are presented as the means of triplicate analysis.

In vitro - anti oxidant activity

Total peroxyl radical trapping potential

A water soluble azo initiator 2, 2' azo bis (2-amidino propane) dihydrochloride (AAPH) produced the peroxyl radicals while a spectrophotometric analysis of 2, 7-dichlorofluresecin-diacetate (DCF) monitored the scavenging activity of the plant extracts (Asokkumar et al., 2009). A 350 μl of 1 mM stock of extract in ethanol was mixed with 1.75 mL of 0. 01 N sodium hydroxide and allowed to stand for 20 min before the addition of 17.9 mL of 25 mM sodium phosphate buffer (pH 7.2). The reaction mixture contained 0.5 mL of various concentrations of the fractions of plant extracts in ethanol, 150 µl of activated DCF solution and 25 µl of AAPH (56 mM). The reaction was initiated with the addition of the AAPH. Absorbance was read at 490 nm. Ascorbic acid was used as standard and all the determination was done in triplicate.

Nitric oxide scavenging assay

Nitric oxide (NO) was generated from sodiumnitroprusside (SNP) and was measured by the Griess reagent by using a previously described method (Balakrishnan et al., 2009). SNP in aqueous solution at physiological pH spontaneously generates NO which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess Reagent. Scavengers of NO compete with oxygen leading to reduced production of NO. SNP (10mM) in phosphate buffer saline (PBS) was mixed with different concentration of extract of the drug dissolved in ethanol and water and incubated at 25°C for 180 minutes. The samples from the above were reacted with Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine dichloride and 3% phosphoric acid). The absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylenediamine dichloride was read at 546 nm and referred to the absorbance of ascorbic acid, used as a positive control treated in the same way with Griess reagent. All the determination was done in triplicate.

Hydroxyl radical scavenging (Deoxy ribose degradation) assay

The hydroxyl radical scavenging activity was determined by Deoxy ribose degradation assay (Subhadradevi et al., 2010).The assay mixture for Deoxy ribose degradation assay contains a final volume of 1 mL. It consists of 100 µl of 2-deoxy 2-ribose dissolved in phosphate buffer (28 mM, pH 7.4), 500 µl of the plant extract of various concentrations in buffer, 200µ l of 20 mM ferric chloride (1:1 v\v) and 1.04 µM EDTA and 100 µl of 1.0 µM hydrogen peroxide and 100 µl of 1.0 µM ascorbic acid. After incubation of the test sample at 370 C for 1 h the level of free radical injure forced on the substrate deoxyribose was calculated using thiobarbituric acid test. For the thiobarbituric acid test, about 1 mL of thiobarbituric acid in 50 mM sodium hydroxide and 1 mL of 2.8% w/v trichloroacetic acid was added to the test tubes and heated at 1000 C for 20 min. Subsequent to cooling absorbance was measured at 532 nm against a blank. Percentage inhibition of deoxyribose degradation was calculated. Quercetin was used as standard and all the determination was done in triplicate.

Super oxide anion radical scavenging (NBT reduction) assay

The scavenging activity of the plant extracts towards superoxide anion radicals was measured using formerly described method (Sannigrahi et al., 2010).Superoxide anions were generated in a non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS, NADH, and oxygen. It was assayed by the reduction of nitro blue tetrazolium (NBT). In these experiments the superoxide anion was generated in 3 mL of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 mL of NBT (300 μM) solution, 0.75 mL of NADH (936 μM) solution and 0.3 mL of different concentrations of the extract. The reaction was initiated by adding 0.75 mL of PMS (120 μM) to the mixture. After 5 min of incubation at room temperature, the absorbance at 560 nm was measured in spectrophotometer. Butylated hydroxy toluene was used as reference compounds. All the determination was done in triplicate

Hydrogen peroxide scavenging assay

The ability of extracts to scavenge hydrogen peroxide was determined (Asokkumar et al., 2009). The solution of hydrogen peroxide (100mM) was prepared in 40mM phosphate buffer saline of (PH7.4), at various concentration of plant extracts were added to hydrogen peroxide solution (2 mL). Absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. In case of control takes absorbance of hydrogen peroxide at 230 nm without sample extracts. Gallic acid was used as standard. The values are presented as the means of triplicate analysis.

Calculation of 50% Inhibitory Concentration (IC50)

The concentration of the extracts that was essential to scavenge 50% of the radicals was calculated by means of the percentage scavenging activities at five different concentrations of the extract.

Percentage inhibition was calculated using the formula,

I % = (Ac-As) x 100


Here Ac is the absorbance of the control and As is the absorbance of the sample.

Results and Discussion

In vitro antihemolytic activity

The peroxyl radical generated by AAPH on addition to erythrocyte suspension and its consequent scavenging action produced by graded higher concentration of the extract are specified in Table 1.The results of the study were compared with the standard ascorbic acid and an increase in inhibition was noticed for the extract tested. FBBE exhibited significant IC 50 value (171.10µg/mL).Free radical initiators such as 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. 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). Earlier studies have demonstrated that AAPH-induced hemolysis in RBCs is effectively inhibited by natural antioxidants. In this study the FBBE showed significant (P<0.01) anti hemolytic activity compared to other extract.

Lipid oxidation of erythrocyte membrane mediated by H2O2 induces membrane damage and subsequently hemolysis. The antihemolytic activity of the FBBE on erythrocytes is presented in Table 1. The sample extract was found to have significant (P<0.01) antihemolytic activity.The name viridans is a group of bacteria which can produce alpha-hemolysis in the presence of an agent known alpha-hemolysin. The alpha-hemolysin is hydrogen peroxide and this peroxide plays an important role in pathogenesis. The concentrations of H2O2 produced by viridans are sufficient to cause lethal damage to human cells. 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 (Snyder et al., 1985). 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.

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 FBBE was lower than that of control. This discovery was comparable to the earlier study reported that presence of flavonoids present in the FBBE critically inhibits the hemolysis by binding of to the red blood cell membranes potentially inhibits lipid peroxidation and at the same time enhances their integrity against lysis. The FBBE produced significant (P<0.01) and strong antihemolytic activity at very low concentrations.

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 the 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).

In vitro free radical scavenging activity

The peroxy radical trapping potential was determined for FBBE and results were compared using L ascorbic acid. Addition of increasing concentration of extractss to the solution containing AAPH decreased the luminescence produced by DCF and absorbance decreased in a linear fashion. Only FBBM exhibited significant activity with IC 50 of 166.70µg/ mL. 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. One route for decomposition of this molecule is thought to be homolytic cleavage to produce hydroxyl radicals and nitrogen dioxide (Yang et al., 2001). An azo initiator, AAPH, was used to produce peroxyl radicals, and the scavenging activity of the extracts was monitored via the spectrophotometric analysis of 2, 7- dichlorofluorescin-diacetate (Cui., 2005). The FBBE extract showed the significant (P<0.01) peroxyl radical scavenging activity

Prepare solutions of sodium nitroprusside in phosphate buffered saline and incubate at 25 0c for 150 min resulted in production of nitric oxide. The extract is effectively reduced the generation of nitric oxide radicals. The FBBE exhibited nitric oxide scavenging activity action with IC 50 105.10 µg/mL (Table 2). 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 this 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). In the present study FBBE reduced the quantity of nitrite formed from the breakdown of sodium nitroprusside. FBBE expressed strongest and significant (P<0.01) effect. 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.

The degradation of deoxyribose by Fe 3+ - ascorbate - ethylene diamine tetra acetic acid (EDTA) - hydrogen peroxide (H2O2) system was markedly decreased by the FBBE tested (Table 2) indicating the significant (P<0.01) hydroxyl radical scavenging ability. In biochemical system, superoxide radical and H2O2 react together to form singlet oxygen and hydroxyl radical, this is the most reactive oxygen species among all reactive oxygen species (Philips et al., 2010). In vitro, hydroxyl radical (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). In this study FBBE exhibit concentration depended and significant (P<0.01) scavenging activity against OH radical. This can be due to high active hydrogen donor ability of OH substitution or its chelating power of phenolic group present in the extract.

The FBBE was established as scavenger of superoxide anion in NBT reduction system (Table 2). The extracts showed significant (P<0.01) superoxide inhibiting activity at a concentration range of 25-400 µg/ml. The IC 50 of FBBE, was found to be 138.20 µg/ml, 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). This study 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 in the presence of antioxidants shows the utilization of superoxide anion in the reaction blend. The degree of discoloration indicates the scavenging potential of the plant extracts (Salar and Dhall, 2010). FBBE when added to the reaction mixture, scavenge the hydroxyl radicals by preventing the decomposition of deoxyribose. FBBE produced significant (P<0.01) effect.

The methanolic extracts FBBE were scavenged hydrogen peroxide in a concentration dependent manner which could be seen by its graded increase in percentage inhibition (Table 2). The IC 50 of FBBE was found to be 86.87 µg/mL This indicating the significant (P<0.01) hydrogen peroxide radical scavenging ability. Even though hydrogen peroxide itself is not extremely reactive, it can occasionally produce cytotoxicity by generating hydroxyl radicals in the cell. Therefore disappearing of H2O2 is very vital all through the food scheme (Ebrahimzadeh et al., 2010). 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 dependent hydroxyl radical mediated oxidative DNA damage may occur due to 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. The extracts FBBE 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. FBBE showed significant (P<0.01) effect against hydrogen peroxide scavenging activity.

Results of the present study showed a direct relationship between the concentration of the tested plant extract and its antihemolytic effect and Free radical scavenging effect. Given that these effects were also observed at acceptable levels at much lower concentrations, it may be possible to use them in the clinical setting with minimal side effects (once in vivo and toxicology tests have been performed). In light of the results of the present and similar studies on plant extracts, these substances may afford beneficial effects in preventing oxidative damage to membranes. They may also be useful in preventing or treating other disease conditions in which lipid peroxidation plays a role. Further studies are required to assess their effects and mechanisms of actions.

Conflict of Interest

The authors have declared that there is no conflict of interest