Journal of Complementary and Integrative Medicine

Journal of Complementary and Integrative Medicine Volume 7, Issue 1

2010

Article 5

The Antioxidant, Iron Chelating and DNA Protective Properties of 70% Methanolic Extract of ‘Katha’ (Heartwood extract of Acacia catechu) Bibhabasu Hazra∗

Rhitajit Sarkar†

Santanu Biswas‡

Nripendranath Mandal∗∗

∗

Bose Institute, Kolkata, [email protected] Bose Institute, Kolkata, [email protected] ‡ Bose Institute, Kolkata, [email protected] ∗∗ Bose Institute, Kolkata, [email protected] †

c Copyright 2010 The Berkeley Electronic Press. All rights reserved.

The Antioxidant, Iron Chelating and DNA Protective Properties of 70% Methanolic Extract of ‘Katha’ (Heartwood extract of Acacia catechu)∗ Bibhabasu Hazra, Rhitajit Sarkar, Santanu Biswas, and Nripendranath Mandal

Abstract The present study was carried out to determine the antioxidant and iron chelating property of 70% methanolic extract of ‘katha’ (heartwood extract of Acacia catechu). The extract was found to be an antioxidant with a TEAC value of 0.72 ± 0.02. The extract has shown its scavenging activity for different radical and 17.8 ± 0.63 µg/ml, 39.55 ± 1.36 µg/ml, 55.31 ± 7.12 µg/ml, 746.85 ± 30.46 µg/ml, 44.2 ± 4.48 mg/ml, 57.5 ± 4.23 µg/ml, 155.48 ± 3.78 µg/ml were determined as IC50 value for DPPH, superoxide, nitric oxide, peroxynitrite, hydrogen peroxide, singlet oxygen and hypochlorous acid radicals, respectively. The plant was found to inhibit lipid peroxidation with an IC50 of 12.35 ± 0.43 µg/ml. The extract also has the ability to chelate iron with an IC50 of 810.8 ± 20.74 µg/ml as well as DNA protective property with [P]50 of 60.44 ± 5.73 µg/ml. It was also observed that the plant extract (100 mg) has 97.13 ± 0.006 mg/ml gallic acid equivalent phenolic and 383.66 ± 0.014 mg/ml quercetin equivalent flavonoid content. In a word, the present results provide evidence that 70% methanol extract of ‘katha’ acts as an antioxidant, iron chelator and DNA protector which is partly due to the phenolic and flavonoid compounds present in it. KEYWORDS: Acacia catechu, TEAC, hydroxyl radical, iron chelation, DNA protection, phenolic content

∗

The authors are immensely grateful to Dr. Rajagopal Chattopadhyay, Biochemistry Department, Bose Institute for allowing to use the rotary evaporator. The authors would also like to acknowledge Mr. Ranjit Das for his assistance in the study.

Hazra et al.: Antioxidant & DNA Protective Property of A. catechu

INTRODUCTION Reactive oxygen species (ROS) play an important role in several pathological situations (Valko et al. 2007). Exogenous chemical and endogenous metabolic processes in the human body produce ROS which are capable of oxidizing biomolecules and generate cellular oxidative stress (Halliwell 1991). The endogenous antioxidant system can reduce oxidative stress by reacting with free radicals, chelating catalytic metals, and also by acting as oxygen scavengers (Niki et al. 1994). But excessive generation of ROS, beyond the capacity of antioxidant system to control them, leads the development of many diseases such as cancer, diabetes, liver injury, atherosclerosis, neurodegenerative disorders and cardiovascular diseases (Stadtman 1992; Maxwell 1995; Braca et al. 2002). In treatment of these diseases, antioxidant compounds have gained an immense importance. Although some synthetic antioxidants have been developed in the past few years, their wide application has been restricted due to negative side effects. Thus, the interest in natural antioxidants of plant origin has increased considerably. The antioxidant activity of several plant materials has recently been described (Jovanovic and Simic 2000; Gupta et al. 2009; Jadeja et al. 2009). Acacia catechu (L.f.) wild (Family-Leguminoseae), a moderate size deciduous tree with dark grayish or brown rough bark, is native to Southern Asia and widely distributed in India, especially in the Deccan. It is commonly known as “khair” in India and various parts of this plant have been used since ancient times in ayurvedic medicine (Chopra et al. 1996). The bark and roots of “khair” are used in treating sore mouth, bronchial asthma, piles, eczema and certain forms of leprosy. The mucilaginous gum exudates from the tree also reported to have antioxidant activity (Surveswaran et al. 2007). Several natural products such as (+)-catechin, (-)-epicatechin, (-)-epicatechin-3-O-gallate, epigallocatechin-3-Ogallate, quercetin, (+)-cyanidanol (Sharma et al. 1997; Shen et al. 2006; Hye et al. 2009) have been isolated from heartwood, bark, roots, leaves and stem of Acacia catechu. The heartwood extract of Acacia catechu, called pale catechu or “katha” in Hindi, is a traditional ayurvedic medicine used in the treatment of cough, dysentery, throat infections, chronic ulcers and wounds (Chopra et al. 1996). Catechu is an indispensable ingredient of “pan” which is beetle leaf preparation chewed in India. Catechu is also reported to possess antimicrobial (Patel et al. 2009), anti-inflammatory (Burnett et al. 2007), antipyretic, antidiarrhoeal, hypoglycemic and hepatoprotective (Jayasekhar et al. 1997; Ray et al. 2006) property. In the present study, 70% methanolic extract of “katha” has been taken up to evaluate its antioxidant, iron chelating and DNA protective property.

Published by The Berkeley Electronic Press, 2010

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Journal of Complementary and Integrative Medicine, Vol. 7 [2010], Iss. 1, Art. 5

MATERIALS AND METHODS CHEMICALS 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtained from Roche diagnostics, Mannheim, Germany. 6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox) was obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), 2-deoxy-2-ribose, mannitol, sodium nitroprusside (SNP), lipoic acid, quercetin and ferrozine were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Folin-ciocalteu reagent, xylenol orange, butylated hydroxytoluene (BHT) and N,N-dimethyl-4-nitrosoaniline were obtained from Merck, Mumbai, India. 1,1-diphenyl-2-picrylhydrazyl (DPPH), Gallic acid and curcumin were obtained from MP Biomedicals, France. Catalase was obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Evans blue was purchased from BDH, England. Diethylene-triamine-pentaacetic acid (DTPA) was obtained from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India. CRUDE EXTRACT PREPARATION OF “KATHA” The pale catechu was purchased from local market and finely powdered. The powder (100 g) was mixed with 500 ml mixture of methanol:water (7:3) and centrifuged at 2850 x g. The supernatant was decanted and the process was repeated again by adding the solvent with the precipitated pellet. The collected supernatants were concentrated in a rotary evaporator and then lyophilized. The dried extract was stored at -20°C until use. TOTAL ANTIOXIDANT ACTIVITY Antioxidant capacity was evaluated by an improved ABTS.+ radical cation decolorisation assay in comparison to trolox standard (Hazra et al. 2008). The ABTS.+ radical cation was generated from the overnight mixture of ABTS solution with potassium persulfate. Then 10 µl extract was added into 1 ml ABTS.+ solution and the decrease in absorbance was measured at 734 nm. All experiments were repeated six times. The trolox equivalent antioxidant concentration (TEAC) was determined by plotting the percentage inhibition of absorbance as a function of concentration of standard and sample.

http://www.bepress.com/jcim/vol7/iss1/5 DOI: 10.2202/1553-3840.1335

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DPPH RADICAL SCAVENGING ASSAY The free radical scavenging activity of the extract was evaluated by 1,1-diphenyl2-picrylhydrazyl (DPPH) using a standard method (Mahakunakorn et al. 2004). Briefly the reaction mixture containing 0.05 ml of 1mM DPPH solution, 0.5 ml of 99% ethanol and 0.45 ml of sample extract and standard ascorbic acid at different concentrations. The solution was rapidly mixed and the reduction of DPPH was measured by reading the decrease in absorbance at 517nm. All tests performed six times. Ascorbic acid was used as a reference compound. HYDROXYL RADICAL SCAVENGING ASSAY The Fenton inhibition assay was carried out to measure the hydroxyl radical scavenging activity of the extract (Hazra et al. 2008). Hydroxyl radical was generated by a Fe3+-ascorbate-EDTA-H2O2 system. The assay quantifies the 2deoxyribose degradation product, by its condensation with TBA. All tests were carried out six times. Mannitol, a classical .OH scavenger, was used as a positive control. Percent inhibition was calculated from the results of the test and blank solution. SUPEROXIDE RADICAL SCAVENGING ASSAY The activity was measured according to a previously reported method (Hazra et al. 2008). The non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system generates superoxide radicals. These radicals reduce nitro blue tetrazolium (NBT) into a purple colored formazan which was measured spectrophotometrically at 562 nm. Quercetin was used as positive control. All tests were performed six times. NITRIC OXIDE RADICAL SCAVENGING ASSAY Nitric oxide generated from sodium nitroprusside (SNP) interacts with oxygen and produce nitrite ions which forms a pink colored chromophore by Griess Illosvoy reaction (Hazra et al. 2008). The chromophore generated was spectrophotometrically measured at 540 nm against blank sample. All tests were performed six times. Curcumin was used as a standard. PEROXYNITRITE SCAVENGING ASSAY Evans blue bleaching assay was used to measure the peroxynitrite scavenging activity (Mandal et al. 2009). Peroxynitrite (ONOO-) was synthesised 12 hrs


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before the assay; according to a standard method (Beckman et al. 1994). The percentage of scavenging of ONOO- was calculated by comparing the results of the test and blank sample. All tests were performed six times. Gallic acid was used as reference compound. HYDROGEN PEROXIDE SCAVENGING ASSAY As previously described, this activity was determined by FOX-reagent method using sodium pyruvate as a reference compound (Mandal et al. 2009). The absorbance of the ferric-xylenol orange complex was measured at 560 nm. All tests were performed six times. SINGLET OXYGEN SCAVENGING ASSAY Singlet oxygen (1O2) generated by a reaction between NaOCl and H2O2 can bleach N,N-dimethyl-4-nitrosoaniline (RNO) which was measured at 440 nm. The scavenging activity of the sample and the reference compound lipoic acid can be assessed by bleaching RNO using a previously depicted method (Mandal et al. 2009). All tests were carried out six times. HYPOCHLOROUS ACID SCAVENGING ASSAY Hypochlorous acid (HOCl) was prepared just before the experiment according to a previously depicted method (Mandal et al. 2009). HOCl degrades the prosthetic group of catalase resulting decrease in the absorbance of catalase. The scavenging activities of the plant extract and the standard, ascorbic acid, a potent HOCl scavenger was evaluated by measuring the increase in the absorbance of catalase at 404 nm. All tests were performed six times. FE2+ ION CHELATING ACTIVITY To evaluate test sample-iron interaction further, the ferrozine test was performed according to an earlier depicted method (Hazra et al. 2008). The chelating of Fe2+ with ferrozine was interrupted in presence of extract and standard EDTA and decrease in absorbance was read at 562 nm. All tests were performed six times. REDUCING POWER ASSAY The Fe3+-reducing power of the extract was determined by a standard method (Mandal et al. 2009). A higher absorbance of the reaction mixture at 700 nm indicated greater reducing power. All tests were performed six times. BHT was used as a reference compound.


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LIPID PEROXIDATION INHIBITION ASSAY The homogenate was prepared by centrifuging Swiss Albino mice brain (20 ± 2 gm) with 50 mM phosphate buffer (pH 7.4) and 120 mM KCl, at 3000 rpm for 10 min. The inhibition of lipid peroxidation activity of extract was determined by quantification of thiobarbituric acid-reactive substances (TBARS) using a previously reported method (Sarkar et al. 2009). The TBARS generated, were spetrophotometrically measured at 532 nm using trolox as a standard. All tests were repeated six times. DNA STRAND SCISSION ASSAY The DNA scission assay was performed using supercoiled pUC18 plasmid DNA according to a previously described method (Hermes-Lima et al. 1998). The reaction mixture containing pUC18 DNA (0.5 mg/ml) in a 100 mM Hepes buffer, pH 7.2, 750 µM FeSO4, varying concentrations of plant extract (30-300 µg/ml) and 7.5 mM. H2O2. After 10 min of incubation, the reaction was terminated by the addition of desferal followed by loading buffer and loaded in 1% agarose gel. After migration, the gel was stained with ethidium bromide and visualized in a UV transilluminator. The DNA bands were quantified through densitometry and the following formulae were used to calculate the percentage of protection. % SC = [1.4 X SC/(OC+(1.4 X SC))] X 100 where, SC = supercoiled; OC = open circular; 1.4 = correction factor % protection = 100 X [(control SC – chelator SC)/( control SC – no chelator SC) – 1] The ability of the plant extracts to protect the DNA supercoil can be expressed by the concentration of sample required for 50% protection, designated as the [P]50 value. ESTIMATION OF PHENOLIC AND FLAVONOID CONTENTS As previously described (Hazra et al. 2008), the Folin-Ciocalteu (FC) reagent was used to determine total phenolics present in the extract. The phenolic content was evaluated from gallic acid standard curve. The amount of total flavonoids was determined with aluminium chloride (AlCl3) according to a known method (Hazra et al. 2008). The flavonoid content was calculated from quercetin standard curve.


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STATISTICAL ANALYSIS All data were reported as the mean ± SD of six measurements. The statistical analysis was performed by KyPlot version 2.0 beta 15 (32 bit). The IC50 values were calculated by the formula, Y = 100*A1/(X + A1) where A1 = IC50, Y = response (Y = 100% when X = 0), X = inhibitory concentration. The IC50 values were compared by paired t test. p < 0.05 was considered significant.

RESULTS TOTAL ANTIOXIDANT ACTIVITY The total antioxidant capacity of the extract was evaluated based on the decolorization of ABTS.+, which was measured spectrophotometrically at 734 nm. The interaction of the ABTS.+ with the extract or standard trolox results in suppression of this cation and plotted as percentage inhibition of absorbance, as shown in Figure 1. The TEAC value of the extract was 0.72 ± 0.02.

Fig. 1: Total Antioxidant Activity Total antioxidant activity of plant extract and trolox. Effect of Acacia catechu extract and reference compound trolox on ABTS radical cation decolorization assay. The percentage of inhibition was plotted against concentration of sample. All data are expressed as mean ± S.D. (n=6).


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DPPH RADICAL SCAVENGING The results shown in Figure 2 is a comparison of the DPPH radical scavenging activity of the plant extract to the standard ascorbic acid where the IC50 values (Table 1) were found to be 17.8 ± 0.63 µg/ml and 5.27 ± 0.27 µg/ml, respectively.

Fig. 2: DPPH Radical Scavenging DPPH radical scavenging activities of the plant extract and the reference compound ascorbic acid. The data represent the percentage of scavenging of DPPH radical. The results are mean ± S.D. of six parallel measurements. ***p < 0.001 vs. 0 µg/ml.

HYDROXYL RADICAL SCAVENGING A. catechu extract did not show any noteworthy result in scavenging hydroxyl radical, in comparison to the standard mannitol (IC50 = 571.45 ± 20.12 µg/ml). So, the results and the figures are not provided. SUPEROXIDE RADICAL SCAVENGING The decrease of the absorbance at 560 nm with the plant extract and the reference compound quercetin indicates their ability to quench superoxide radicals from reaction mixture. As shown in Figure 3, the IC50 values (Table 1) of the plant extract and quercetin were 39.55 ± 1.36 µg/ml and 32.29 ± 1.66 µg/ml, respectively.


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Fig. 3: Superoxide Radical Scavenging Scavenging effect of Acacia catechu plant extract and standard quercetin on superoxide radical. The data represents the percentage of superoxide radical inhibition. All data are expressed as mean ± S.D. (n=6). *p < 0.05 and ***p < 0.0001 vs 0 µg/ml.

NITRIC OXIDE RADICAL SCAVENGING A. catechu extract also inhibits nitric oxide in dose dependent manner (figure 4) with the IC50 (Table 1) value being 55.31 ± 7.12 µg/ml. Curcumin was used as a reference compound and 90.82 ± 4.75 µg/ml curcumin was needed for 50% inhibition.

Fig. 4: Nitric oxide Radical Scavenging Nitric oxide radical scavenging activities of A. catechu plant extract and standard curcumin. The data represent the % of nitric oxide inhibition. Each value represents mean ± S.D. (n=6). ***p < 0.001 vs. 0 µg/ml.


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PEROXYNITRITE SCAVENGING Figure 5 shows the peroxynitrite scavenging activity of the plant extract in a concentration dependent manner. The calculated IC50 was 746.85 ± 30.46 µg/ml which was lower than that of the reference compound gallic acid (IC50 = 876.24 ± 56.96 µg/ml) (Table 1) indicating that the sample is more potent scavenger of peroxynitrite than gallic acid.

Fig. 5: Peroxynitrite Anion Scavenging The peroxynitrite anion scavenging activity of Acacia catechu plant extract and standard gallic acid. Each value represents mean ± S.D. (n=6). ***p < 0.0001 vs 0 µg/ml.

HYDROGEN PEROXIDE SCAVENGING The hydrogen peroxide scavenging assay of the extract and standard by the FOX reagent method was shown in Figure 6. The IC50 value (Table 1) of the plant extract was 44.2 ± 4.48 mg/ml whereas that of standard sodium pyruvate was 3.24 ± 0.3 mg/ml.


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Fig. 6: H2O2 Scavenging Activity Effect of Acacia catechu plant extract and standard sodium pyruvate on the scavenging of H2O2. The data represents the percentage of H2O2 scavenging. All data are expressed as mean ± S.D. (n=6). **p < 0.001 and ***p < 0.0001 vs 0 mg/ml.

SINGLET OXYGEN SCAVENGING A. catechu extract was an effective scavenger of singlet oxygen (Fig 7) and this activity was comparable to that of lipoic acid. The IC50 value (Table 1) of the test sample was found to be 57.5 ± 4.23 µg/ml, in comparison of that of lipoic acid which was found to be 46.15 ± 1.16 µg/ml.

Fig. 7: Singlet Oxygen Scavenging Effect of Acacia catechu extract and standard lipoic acid on the scavenging of singlet oxygen. The results are mean ± S.D. of six parallel measurements. ***p < 0.001 vs 0 µg/ml.


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HYPOCHLOROUS ACID SCAVENGING Figure 8 shows the dose-dependent hypochlorous acid scavenging activity of A. catechu extract compared to ascorbic acid. The obtained results indicate that the extract scavenges (IC50 = 155.48 ± 3.78 µg/ml) hypochlorous acid more efficiently than ascorbic acid (IC50 = 235.95 ± 5.75 µg/ml) (Table 1).

Fig. 8: HOCl Scavenging Hypochlorous acid scavenging activity of Acacia catechu plant extract and standard ascorbic acid. All data are expressed as mean ± S.D. (n=6). ***p < 0.0001 vs 0 µg/ml.

FE2+ CHELATION Ferrozine makes a violet colored complex with Fe2+ ion. The complex formation is interrupted in presence of chelating agent and as a result the violet color of the complex is decreased. The results [figures 9(a) and (b)] demonstrated that the formation of ferrozine-Fe2+ complex is inhibited in the presence of test and reference compound. The IC50 values (Table 1) of the plant extract and EDTA were 810.8 ± 20.74 µg/ml and 1.27 ± 0.05 µg/ml, respectively.


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Fig. 9: Iron Chelation Effect of (a) Acacia catechu plant extract and (b) standard EDTA on ferrozine-Fe2+ complex formation. The data expressed as % inhibition of chromogen formation. The results are mean ± S.D. of six parallel measurements. ***p < 0.001 vs 0 µg/ml.

REDUCING POWER As illustrated in Figure 10, reduction of Fe3+ to Fe2+ was facilitated in the presence of A. catechu extract and the reference compound BHT. At the concentration of 0.05 mg/ml, the absorbance of the plant extract and BHT was 0.25 and 0.006 respectively while at the concentration of 0.4 mg/ml, the absorbances of both extract and BHT were almost same. This result indicates that, at a low dose (0.05 mg/ml), the extract showed better activity than BHT.

Fig. 10: Reducing Power The reductive ability of Acacia catechu extract and standard BHT. The absorbance (A700) was plotted against concentration of sample. Each value represents mean ± S.D. (n=6). ***p < 0.0001 vs 0 mg/ml.


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LIPID PEROXIDATION As shown in Figure 11, the plant extract and the reference compound trolox exhibited a concentration dependant suppressive effect on the TBARS formation in mice brain homogenate with the IC50 value (Table 1) 12.35 ± 0.43 µg/ml and 26.88 ± 0.65 µg/ml, respectively

Fig. 11: Inhibition of Lipid Peroxidation Lipid peroxidation inhibiting capacity of the A. catechu extract and the standard trolox. The data is expressed as the % of lipid peroxidation inhibition of brain homogenate, induced by Fe2+/ascorbic acid. Each value represents mean ± S.D. (n=6). *** p < 0.001 vs. 0 µg/ml.

DNA STRAND SCISSION The protective effect of A. catechu extract against Fe2+-H2O2 mediated DNA breakdown was demonstrated in Figure 12. pUC18 supercoiled DNA was used as control (lane 1). The treatment of supercoiled DNA with Fenton’s reagent led to the conversion of DNA to open circular form (lane 2). The addition of different concentrations of plant extracts resulting in the restoration of DNA in the supercoiled form (lane 3-9). The results had showed the dose dependant protective effect of extract. The [P]50 value of the extract was found to be 60.44 ± 5.73 µg/ml.


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Fig. 12: DNA Strand Scission Assay Protection against oxidative damage to pUC18 by Acacia catechu extract. Picture of agarose gel of pUC18 DNA showing bands of supercoiled (SC) and open ciecular (OC) forms. Lanes on the gel represent: (Lane 1) control DNA (no H2O2 or Fe2+); (Lane 2) reaction mixture without extract; (Lane 3-9) reaction mixture with extract of increasing concentration (30-300 µg/ml).

DETERMINATION OF PHENOLIC AND FLAVONOID CONTENTS The calculated total phenolic and flavonoid contents of 70% methanolic extract of pale catechu were found to be 97.13 ± 0.006 mg/ml equivalent gallic acid and 383.66 ± 0.014 mg/ml equivalent quercetin per 100 mg plant extract, respectively.


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TABLE 1: Reactive oxygen species scavenging, iron chelating activity and lipid peroxidation inhibition (IC50 values) of Acacia catechu and reference compounds Activity Extract/Reference IC50 (#) Acacia catechu 17.8 ± 0.63 (6) DPPH radical scavenging Ascorbic acid 5.27 ± 0.27 (6)*** Acacia catechu 39.55 ± 1.36 (6) Superoxide anion (O2.-) scavenging Quercetin 32.29 ± 1.66 (6)** Nitric oxide radical (NO) Acacia catechu 55.31 ± 7.12 (6) scavenging Curcumin 90.82 ± 4.75 (6)*** Acacia catechu 746.85 ± 30.46 (6) Peroxynitrite (ONOO-) scavenging Gallic acid 876.24 ± 56.96 (6)*** Hydrogen Peroxide (H2O2) Acacia catechu 44.2 ± 4.48 (6) scavenging Sodium pyruvate 3.24 ± 0.3 (6)*** Acacia catechu 57.5 ± 4.23 (6) Singlet oxygen (1O2) scavenging Lipoic acid 46.15 ± 1.16 (6)** Hypochlorous acid (HOCl) Acacia catechu 155.48 ± 3.78 (6) scavenging Ascorbic acid 235.95 ± 5.75 (6)*** Acacia catechu 810.8 ± 20.74 (6) Iron Chelating Activity EDTA 1.27 ± 0.05 (6)*** Acacia catechu 12.35 ± 0.43 (6) Lipid Peroxidation Inhibition Trolox 26.88 ± 0.65 (6)*** # Unit of IC50 of all activities are µg/ml, except H2O2 scavenging, where units are mg/ml. Data expressed as mean ± S.D. Data in parenthesis indicate number of independent assays. EDTA, Ethylene diamine tetra acetic acid. DPPH, 1,1-diphenyl-2-picrylhydrazyl. ** p< 0.01. *** p< 0.001 vs. Acacia catechu.

DISCUSSION In the living system, free radicals are constantly generated and they can cause extensive damage of tissues and biomolecules leading to create various disease conditions, especially degenerative diseases (Finkel & Holbrook 2000) and extensive lysis (Halliwell & Gutteridge 1998). Due to the adverse side effects of synthetic drugs, natural antioxidants from plant origin have drawn much attention in recent years. It is already reported that the aqueous extract of Acacia catechu was found to content phenolic antioxidants (Naik et al. 2003). The present study


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is focused on the evaluation of antioxidant and free radical scavenging activity of 70% methanol extract of “katha” (pale catechu). The reaction between ABTS and potassium persulfate results in the production of a blue colored chromophore, ABTS.+. Addition of the plant extract to this pre-formed radical cation reduces it to ABTS on a concentration dependant manner. Compared to trolox and the TEAC, the methanolic extract is a potent antioxidant. The bleaching of DPPH indicates the free radical scavenging capacity of 70% methanolic extract of catechu but to a lower extent than the standard ascorbic acid. Superoxide anion is a harmful ROS with detrimental effect on the cellular components in biological systems (Aruoma et al. 1987). As shown in Figure 3, the plant methanolic extract is a better scavenger of superoxide radical than the standard quercetin. Nitric oxide, a diffusible free radical, plays an important role as an effector molecule in diverse biological systems including neuronal messenger, vasodialation and antimicrobial and antitumor activities. But chronic emergence of nitric oxide radical is linked with various carcinomas and inflammatory conditions including juvenile diabetes, multiple sclerosis, arthritis and ulcerative colitis (Tylor et al. 1997). The reaction of NO with superoxide radical generates the highly reactive peroxynitrite anion (ONOO-) implicated in the pathogenesis of diseases such as heart disease, Alzheimer’s disease, and atherosclerosis (Ischiropoulos et al. 1995). The obtained results showed that the methanolic plant extract is more potent nitric oxide and peroxynitrite scavenger than the standards curcumin and gallic acid, respectively (Figures 4 & 5). Hydrogen peroxide, a weak oxidizing agent and cross cell membrane rapidly, reacts with Fe2+ and possibly Cu2+ ions to form the damaging toxic hydroxyl radical. The scavenging activity of the methanolic plant extract was found to be poor to the standard sodium pyruvate (Figure 6). Singlet oxygen, a high energy form of oxygen, is generated in the skin upon UV-irradiation. It induces hyperoxidation, oxygen cytotxicity and decreases the antioxidant activity (Kochevar and Redmond 2000). The scavenging activity of methanolic plant extract is as good as the standard lipoic acid (Figure 7). Hypochlorous (HOCl), another harmful ROS, is produced through the oxidation of Cl- ions by the neutrophil myeloperoxidase at the sites of inflammation (Aruoma et al. 1989). HOCl inactivates the antioxidant enzyme catalase by breaking down its heme prosthetic group. The HOCl scavenging activity is illustrated by the inhibition of catalase deactivation. The obtained results (Figure 8) indicate that the plant extract is a much better scavenger of HOCl than the standard ascorbic acid Although iron is significant for various biological reactions, its excess leads to the production of highly reactive hydroxyl radical (OH.), by the Haber-


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Weiss reaction, that causes severe injury to membranes, proteins and DNA. The metal chelating capacity is significant since it reduces the concentration of the catalyzing transition metal in lipid peroxidation (Chang et al. 2002). The methanolic plant extract was found to be a poor iron chelator compared to the standard EDTA as shown in Figure 9. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. As illustrated in Figure 10, at 0.05 mg/ml, the absorbances of the plant extract and BHT are 0.25 and 0.006, respectively, while the absorbance of the sample and standard BHT is almost the same at 0.4 mg/ml. This result indicates that the activity of the extract, at low doses, is higher than the standard BHT (Figure 10). Lipid peroxidation most often affects polyunsaturated fatty acids present in cell membrane resulting in the cell damage. It is initiated through iron catalysed generation of ferry-perferryl complex or hydroxyl radicals. The highly reactive hydroxyl radical can damage biological molecules, eventually yielding carbonyl products like malondialdehyde (MDA), which generate a pink chromogen with TBA. As shown in Figure 11, the plant methanolic extract inhibits lipid peroxidation, in a dose dependent manner, more efficiently than the standard trolox. The effect of plant extract on Fe2+-dependent hydroxyl radical induced DNA nicking of pUC18 plasmid was studied. The addition of plant extract to the reaction mixture substantially decreased the DNA strand scission, in a dose dependent manner, leading to the protection of DNA under oxidative stress (Figure 12). The significant reduction in the formation of nicked DNA and increase in supercoiled DNA in the presence of the extract reveal its excellent DNA protector effect. The phytochemical analysis showed that A. catechu methanolic plant extract is rich in flavonoids and phenols. Flavonoids exhibit their antioxidative action through scavenging or chelating process (Kessler et al. 2003) whereas phenolic compounds, important plant constituent (Rice-Evans et al. 1995), exercise their scavenging activity via their hydroxyl groups. The presence of both these compounds in human diet and their effects on human nutrition and health are considerable.

CONCLUSIONS Based on the results of this study, it may be concluded that 70% methanolic extract of “katha” possess significant free radical scavenging and antioxidant as well as iron chelating properties in vitro. The extract has also shown its DNA protective property against free radical damage. These activities might be due to


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the presence of phenolic and flavonoid contents. Further studies are in progress to evaluate the in vivo iron chelating and antioxidant potential. The active principles are also essential to identify the compounds responsible for the bioactivity of this plant.

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