Attenuation of Protective Effect on DNA and Antioxidant Efficacy of Extracts from Terminalia chebula Prepared by Sequential Method

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1 Advances in Biological Research 6 (6): , 212 ISSN IDOSI Publications, 212 DOI: /idosi.abr Attenuation of Protective Effect on DNA and Antioxidant Efficacy of Extracts from Terminalia chebula Prepared by Sequential Method Harpreet Walia, Subodh Kumar and Saroj Arora 1 Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar-1435, Punjab, India 2 Department of Chemistry, Guru Nanak Dev University, Amritsar-1435, Punjab, India Abstract: The aim of this work was then to assess the total phenolics content and the antioxidant properties of methanol, ethyl acetate and n-butanol extract of fruits of Terminalia chebula prepared by sequential method. Differences in the hydroxylation and glycosylation of various phytochemicals are known to contribute to differing results especially when the activity is evaluated by methods that have different end-point measurements of oxidation. The use of a variety of methods that differ in either source of free radical generation or specific quantification has been shown to be useful in comprehensively evaluating antioxidant activities of plant materials. This approach was taken in the present study to characterize the antioxidant activity of the extracts prepared by sequential method. Key words: DNA Damage Phytochemicals TBARS Hydroxyl Radicals Phenolic Content INTRODUCTION belongs to family combretaceae is a plant native to India and Southeast Asia, where it is extensively cultivated and "Free radicals" and "anti-oxidants" are the latest is known to be rich in polyphenolic compounds. In this buzzwords of the health industry. The role of free-radical study the fruits of this plant are used for evaluation of its reactions in biology has become an area of intense antioxidant potential. It is assumed that the fruits are interest. It is increasingly recognized that reactive oxygen mainly rich in phytochemicals. This plant in India is species play an imperative role as mediators in the commonly known as harar and its dried ripe fruit has physiopathology of various diseases, so it aroused a been traditionally used to treat various ailments in ancient considerable interest among the scientists [1]. Although Indian medicinal system [9-11]. In India, it is one of the organisms are bestowed with antioxidant and repair three components of a herbal preparation popularly systems that have evolved to protect them against known as triphala i.e. three fruits known as Wonder oxidative damage, yet these systems are insufficient to Drug used routinely in the ayurvedic system of medicine prevent the damage totally [2, 3]. Hence, antioxidants in as laxative and ophthalmic problems [12-15]. It has been diet are important as possible protective agents to help reported to exhibit a variety of biological activities the human body to reduce oxidative damage. including, antidiabetic, antimutagenic, antibacterial, Plants are the potent source of many bioactive antifungal and antiviral activities [16-2]. compounds such as phenolics, flavonoides, which have Keeping in mind the immense medicinal importance antioxidative properties and promoted as Magic Bullets of this plant and polyphenols, the present work was for optimum health [4-8]. Although these compounds planned to check the antioxidative potential of extracts of have enormous structural diversity and activity, only a fruits Terminalia chebula prepared by sequential method small portion of that diversity has been explored for its in in vitro assays viz. DPPH assay, Deoxyribose (Site pharmacological potential so far and there is therefore specific and Non-site specific), Reducing power, little reason to believe that this potential has now run dry. Chelating Power, Lipid Peroxidation assay and DNA Among important medicinal plants Terminalia chebula Nicking assay. The most widely used methods for Corresponding Author: Harpreet Walia, Department of Botanical & Environmental Sciences, Guru Nanak Dev University, Amritsar-1435, Punjab, India. 231

2 measuring antioxidant activity are those that involve the properly. After some time the two layers were formed generation of radical species, where the presence of which were separated to get ethyl acetate filtrate and antioxidants determines the disappearance of radicals. It 2% aqueous methanol stock. The three filtrates of is pertinent to use different assays in an efficient ethyl acetate were collected and dried at room extraction medium, instead of relying on a single assay to temperature in petri plates to get ethyl acetate extract. assess and compare the antioxidant capacity. Our results After this n-butanol was added serially in 2% aqueous enable us to proceed towards more detailed chemical and methanol to get n-butanol extract (Flow chart 1). pharmacological understanding of this plant material and show the interest of natural antioxidants for the Chemical Analysis prevention of free radical-mediated pathology. Determination of Total Phenolic Content: The total phenolic content of the extracts was determined using MATERIALS AND METHODS Folin-Ciocalteu method [21]. To 1µl of extract 9µl of water was added. To this mixture 5µl of FC reagent was Extraction Procedure: 1 kilogram of fruit powder of added. This was followed by the addition of 1.5ml of 2% T. chebula was suspended in pure methanol (15ml) and Sodium carbonate. The mixture was shaken thoroughly the mixture was kept for 24hrs on shaker at room and allowed to stand for 2 hours. The volume of mixture temperature. The residue was filtered through Whatman was made up to 1ml with distilled water and absorbance no.1 filter paper and filtrate was collected. The residue was observed at 765nm. The phenolic content was was again suspended in methanol (1ml) twice in similar calculated as gallic acid (mg/g) equivalents. manner. The filtrate were combined and dried at room temperature by putting in petri plates to get methanol Spectroscopic Analysis of Extracts: The extracts were extract. 1g of methanol extract was dissolved in 2% analyzed by UV spectroscopy. For this the solution of aqueous methanol (1ml) and put into the separatory extracts was prepared in spectroscopic grade methanol in funnel. The hexane and chloroform was added to remove the concentration of 1mg/1ml, diluted four times and a the fatty compounds from the methanol layer. Then ethyl spectrum was recorded on UV-Visible spectrophotometer acetate was added in separatory funnel and mixed (Shimadzu-161). Flow Chart 1: Extraction procedure. 232

3 Antioxidant Testing phosphate buffer (.2M, ph 6.6) and 2.5 ml of 1% DPPH Assay: The H-donor activity of the extracts was potassium ferricyanide. The mixture was incubated at 5 C measured by 1, 1 diphenyl picrylhydrazyl (DPPH) for 2 min. 2.5 ml of 1% tricholroacetic acid was then method [22]. The reaction mixture contained 2µl of added to the mixture and centrifuged at 3 rpm for 1 different extract/fractions concentrations (5-3µg/ml) min. 1ml of aliquot of supernatant was mixed with 2.5ml of and 2 ml of DPPH (.1 mm in methonolic solution). The distilled water and.5 ml of FeCl 3 (.1%) and absorbance reaction mixture was observed at 517nm against a blank, was measured at 7nm. Increase in absorbance was which did not contain extract. The % age inhibition was interpreted as increased reducing activity. calculated as: Lipid Peroxidation Assay: TBA reacts with = B-B 1/Bx1 malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected Where, B is the absorbance of control, B 1is the spectrophotometrically at 532 nm [26]. Normal albino rats absorbance of reaction mixture. of the Wistar strain were used for the preparation of liver homogenate. The perfused liver was isolated and 1% Deoxyribose Assay: This was done by non-site specific (w/v) homogenate was prepared using a homogenizer at and site-specific methods [23]. Stock solution of EDTA -4 C with.15 M KCl. The homogenate was centrifuged (1mM), FeCl 3(1mM), ascorbic acid (1mM), H2O 2 (1mM) at 3 rpm for 15 min and clear cell-free supernatant was and deoxyribose (1mM) were prepared in distilled water. used for the study of in vitro lipid peroxidation. Different In non- site specific assay,.1ml of EDTA,.1ml of FeCl3, concentrations of extracts mixed with 1ml of.15 M KCl.1 ml of H2O 2,.36 ml of deoxyribose, 1ml of extract and.5 ml of rat liver homogenates were added to the test concentrations (1-1 µg/ml),.33 ml of phosphate buffer tubes. Peroxidation was initiated by adding 1 µl of.2 (5mM, ph 7.4) and.1 ml of ascorbic acid were added in mm ferric chloride. After incubation at 37 C for 3 min, the sequence. The mixture was incubated at 37 C for 1 hour. reaction was stopped by adding 2 ml of ice-cold HCl 1ml of the incubated mixture was mixed with 1 ml of 1% (.25 N) containing 15% trichloroacetic acid (TCA),.38% trichloroacetic acid and 1 ml of thiobarbituric acid thiobarbituric acid (TBA) and.5% butylated (.25M NaOH) and heated for one hour on water bath at hydroxytoluene (BHT). The reaction mixtures were heated 8 C and pink chromogen developed, which was at 8 C for 6 min. The samples were cooled and measured at 532 nm. In site-specific assay EDTA was centrifuged and the absorbance of the supernatants was replaced with phosphate buffer. The hydroxyl radical measured at 532 nm. An identical experiment was scavenging activity of extract is reported as % inhibition performed to determine the amount of lipid peroxidation of deoxyribose. The % age inhibition was calculated as in obtained in the presence of inducing agents without any given above assay. extract. The % age inhibition was calculated as give above. Chelating Power Assay: The chelating activity of extracts was measured as given by Dinis with little modifications DNA Nicking Assay: A DNA nicking assay was [24]. 1 ml of extract with different concentrations was performed using supercoiled pbr322 plasmid DNA [27]. mixed with 3.5ml of methanol and then the mixture was Plasmid DNA (.5 µg) was added to Fenton reagent mixed with ferrous chloride (2mM,.1 ml) and ferrozine (3 mm H2O 2, 5 µm ascorbic acid and 8 µm FeCl 3) (1mM,.2ml) for 1 min at room temperature. The containing different concentrations of the absorbance was measured at 562 nm against a blank in extract/fractions and the final volume of the mixture was which the extract was not added. The % age inhibition brought up to 2 µl. The mixture was then incubated for was calculated as given in DPPH assay. 3 min at 37 C. After incubation bromophenol blue dye (.25% in 5% glycerol) was added. This was followed by Reducing Power Assay: The relative reducing activity of electrophoresis using 1% agrose gel. The gel was extracts was determined according to the method of dissolved in TBE buffer (Tris base: 1.8g, boric acid: 5.5g, Oyaizu [25]. 1ml of extract/fractions (5-3µg/ml) was EDTA:.75g) and heated until the agrose dissolved prepared in distilled water and mixed with 2.5 ml of completely. After cooling it to bearable to touch, ethidium 233

4 bromide (.5µg/ml) solution was added. The agrose gel the extracts was between 1-15 minutes. Although the was casted in the tray and solidified. After solidification antioxidant capacities are influenced by many factors, the comb was removed and the wells were loaded with which cannot be fully described by a single method, the reaction mixture (2µl) and electrophoresis was carried out DPPH radical scavenging activity is the most commonly for 2.5 hrs at 5mV. The DNA bands were analyzed on gel used method for assessment of the antioxidant properties documentation system. of natural products [31,32]. The assay is suitable for solvent extracts and as a rapid assay, it can be applied for RESULTS AND DISCUSSION monitoring the activity of numerous samples over a limited period of time. Moreover, it is reproducible and Plant phenolics exhibit an array of solubility in strongly correlated with phenolic compounds [33]. solvents with different polarity and they constitute one of. The hydroxyl radical ( OH) constitutes the chemically the major groups of compounds acting as primary most reactive species of activated oxygen formed by antioxidants of free radical terminators [28]. For the successive monovalent reduction of dioxygen (O 2) in cell determination of total phenolics content, in the present metabolism and it can inflict damage to biomolecules in its study Folin Ciocalteu s method was used, which vicinity [34]. Hydroxyl radicals are known to be produced response depending on the chemical structure of by Fenton s reaction in the presence of H2O 2and free iron phenolics (i.e. the higher the number of functional OH [35,36]. The free iron promotes Fenton s reaction by group the higher the total phenolics content). The liberating hydroxyl radicals spontaneously from H2O 2. phenolic content of methanol extract was 875mg/g of The produced hydroxyl radical attach on deoxyribose and GAE, which was maximum. Ethyl acetate and n-butanol produced malondialdehyde that react with thiobarbituric exhibited phenolic content of 41 and 63mg/g of GAE acid to yield pink chromogen detectable at 532nm respectively. For further analysis of presence of phenolic [37]. In the presence of extract hydroxyl radicals are content the UV analysis of the extracts was done. The UV scavenged and did not get the chance to react with spectrum of methanol, ethyl acetate and n-butanol deoxyribose, thus enabling the antioxidant activity of extracts were exhibited maximum absorption maxat 364nm, extracts. Insets of Figures 3 and 4 shows the inhibition 361nm and 365nm respectively and strong end absorption plot for the scavenging of hydroxyl radicals in non-site at around 2nm, which pointed towards phenolic and specific and site-specific deoxyribose assay respetively. polyphenolic compounds (Figure 1). The order of inhibition of extracts in non-site specific DPPH assay is often used to evaluate the ability of assay was n-butanol (93.1%)> ethyl acetate (91.6%) > antioxidant to scavenge free radicals, which are known to methanol (87.2%) at 2µg/ml concentration (Figure 3). be a major factor in biological damages caused by On the other hand in site-specific assay the order of oxidative stress. This assay is known to give reliable inhibition was ethyl acetate > methanol > n-butanol with information concerning the antioxidant ability of the values of 9.7%, 86.8% and 85.6% respectively at tested compounds [29]. This assay measures the ability of 3µg/ml concentration (Figure 4). By means of compounds to transfer labile H-atoms to radicals, is the site-specific and non-site specific deoxyribose assay, the commonest method of antioxidant activity evaluations hydroxyl radical scavenging ability as well as chelating [3]. The abstraction of hydrogen by this stable free power of extracts were determined. Deoxyribose has radical is known to lead to the bleaching of the absorption 2+ affinity for the Fe and in the case of site specific assay maxima at 517 nm and can easily be monitored 2+ the Fe is present in free form that directly attack on spectrophotometrically. Based on the results obtained. deoxyribose before the generation of OH radical via from this established assay system, it appears that Fenton reaction. However in non-site specific assay the extracts showed a potent and concentration dependent 2+. EDTA decrease the binding of Fe to deoxyribose and OH free radical scavenging effect. The maximum effect was radicals formed freely in the solution. So, by site-specific observed in methanol which was 77.3% at 5µg/ml deoxyribose assay we can calculate the chelating as well concentration. Ethyl acetate and n-butanol extract as hydroxyl scavenging activity and by non-site specific exhibited 72.1% and 74.7 % at the highest concentration assay we can calculate only the hydroxyl radical tested (Figure 2). The extracts significantly fade the purple scavenging activity. High activity of extracts in site color of DPPH to pale yellow color and reaction time for specific assay denotes that the chelating potential of 234

5 Fig 1: The UV-visible spectra of extracts of T. chebula acquired by sequential method Ethyl acetate n- butanol Methanol Ethyl acetate n-butanol Methanol Fig. 2: Effect of different concentrations of extracts of T. Fig. 4: Effect of different concentrations of extracts chebula in DPPH assay. of T. chebula in site-specific deoxyribose assay. ferrozine test that provided information regarding the 6 reactivity of extracts with iron and it was found that the 4 extracts were interfered with the formation of ferrousferrozine 2 complex, suggesting that they have marked iron chelating activities and capture ferrous ion before the formation of ferrozine [38]. The high chelating potential was observed with methanol extract which was Ethyl acetate n-butanol Methanol 75.3% at 5µg/ml of dose. The ethyl acetate and n- Fig. 3: Effect of different concentrations of extracts of butanol extract exhibited 69.9% and 71.1% chelating T.chebula in non-site specific deoxyribose assay. potential at the same dose (Figure 5). It is possible that the antioxidant effect of extracts may at least in part result. extracts surpass the OH scavenging activity. The activity from its iron chelating activities. The general chelating of extracts in DPPH assay further support the hydrogen abilities of the extracts is probably related to the high donating ability, which scavenge the hydroxyl radicals. nucleophilic chelation of the aromatic ring of polyphenolic To clarify the possibility of extract as good chelators, compounds rather than to specific chelation groups with we examined the iron chelating effect of extracts in the the molecules [39]. 235

6 Absorbanc Ethyl aceate n-butanol Methanol Fig. 5: Effect of different concentrations of extracts of T.chebula in chelating power assay Ethyl acetate n-butanol Methanol Fig. 7: Effect of different concentrations of extracts of T.chebula in lipid peroxidation assay. 1.5 ability. The high amount of phenolic compounds within 1 plant extracts can be responsible for its antioxidant activity [43]..5 Lipid peroxidation consists of a series of free radical mediated chain reaction processes and is associated with several types of biological damage. Lipid is not only a major component of biomembrane system, but also an important ingredient of foods. Lipid decomposition Ethyl acetate n-butanol Methanol through peroxidation results in injury of cells and tissues Fig. 6: Effect of different concentrations of extracts of in living system and decreases the safety and nutritional T.chebula in reducing power assay. value in oil food products [44]. Lipid peroxidation has been broadly defined as the oxidative deterioration of The reducing power of extracts of T. chebula is polyunsaturated lipids [45]. Inhibition of lipid peroxidation shown in Figure 6. The reducing power of all extracts by any external agent is often used to evaluate its increased with the increase in concentration. The antioxidant capacity. In order to determine, whether the reducing power of methanol, ethyl acetate and n-butanol extracts are capable of reducing in vitro oxidative stress, was 1.229, and.917 respectively at 5µg/ml the traditional lipid peroxidation assay that determines the concentration. It is pertinent to mention that the extracts production of malondialdehyde and related lipid peroxides showed dose response curve up to 3µg/ml in living system was carried out. In the present study the concentration. extracts showed strong activity in lipid peroxidation assay The reducing capacity of a compound may serve as and dose response relationship was found up to a significant indicator of its potential antioxidant activity 1µg/ml. The maximum activity was found in methanol [4]. Literature reports have shown that the reducing extract which posses 8.9% inhibition at 1µg/ml dose. power of bioactive compounds is associated with The ethyl acetate and n-butanol showed 75.3% and 72.3% antioxidant activity [41]. The antioxidants can take part inhibition at the same dose (Figure 7). into oxidation-reduction potential and termed as In order to study the protective effects of extracts on reductones. These reductones have been shown to exert hydroxyl radical-mediated DNA strand breaks, a free antioxidant action by breaking the free radical chain by radical induced plasmid pbr322 DNA breaks system in donating a hydrogen atom [42]. The extracts convert vitro was used in the present assay. Attacked by ferricyanide to ferrocyanide ions, which on reaction with hydroxyl radical generated from the Fenton reaction, 2+ Fe yield green colour and can be detected by increase in supercoiled plasmid DNA was broken into three forms, absorbance. More is the absorbance means, more including supercoiled, open circular and linear form. As reducing ability of the compound to reduce ferricyanide shown in Figure 8, the incubation of DNA with Fenton ions. From the results it is apparent that among the reagent results in increasing formation of open circular extracts, the methanol extract revealed strong reducing and linear forms of DNA. The role of the extract in altering 236

7 the radiation induced strand break in plasmid pbr Halliwell, B. and J.M.C. Gutteridge, Free DNA was investigated. In this assay when pbr322 plasmid DNA was exposed to Fenton reaction, it caused a change in DNA band from Form I (Native plasmid DNA) to Form II (single-stranded, nicked circular plasmid DNA) or to Form III (Linear plasmid DNA). It is clear from the 3. st Radicals in Biology and Medicine. 1 ed. Oxford: Clarendon Press, pp: Halliwell, B. and J.M.C. Gutteridge, Free radicals. results that extracts scavenges the OH radicals and protects the pbr322 plasmid DNA (Figure 8). The different concentrations were tried but at the concentration of 6µg/ml the extracts showed the reduction in forms II and III and increased form I which is a normal DNA. The extracts showed comparable effect to rutin (Lane 3). It is clear from the results that the methanol extract (Lane 4), ethyl acetate extract (Lane 5) and n-butanol extract (Lane 6) exhibited a notable protection against hydroxyl radical induced by Fenton reaction. The extract may have phenolics compounds, which bear free hydroxyl for donation. The UV analysis of methanol extract showed max in the region 365nm indicating the presence of phenolic compounds (Figure1). Overall, scavenging activity of polyphenolic compounds might be due to their active hydrogen donating ability. Since phenolic compounds present in the extract are good electron donor, they may accelerate the conversion of H2O 2 into H2O. Polyphenols can also chelate the active sites of iron and make them inert consequently they do not take part in Fenton reaction. So, the polyphenolic compounds present in the Terminalia chebula extracts/ fractions might be responsible for inhibiting the DNA nicking. CONCLUSION From the results it is clear that the extracts prepared by sequential method showed good activity in all the assays. From chemical investigations it is clear that the extracts are rich in polyphenolic compounds. Presumably, the different antioxidant activity among these extracts could be ascribed to the different qualitative/quantitative chemical profiles. The major antioxidant components might selectively be extracted into methanol extract as it shows strong activity in all the assays. REFERENCES 1. Canakci, C.F., Y. Cicek and V. Canakci, 25. Reactive Oxygen Species and Human Inflammatory Periodontal Diseases. Biochemistry, 7: rd in biology and medicine. 3 ed. Oxford: Oxford University Press, pp: Dhir, H., A. Kumar and A. Sharma, Relative efficiency of Phyllanthus emblica fruit extract and ascorbic acid in modifying lead and aluminium induced sister chromatid exchanges in mouse bone marrow. Environ. Mol. Mutag., 21: Shahidi, F. and M. Naczk, Antioxidant st Properties of Food Phenolics. 1 ed. Basel: Technomic Publishing Co. Inc. Lancester, pp: Cozzi, R., R. Ricordy, T. Aglitti, V. Gatta, P. Perticone and R. De Salvia, Ascorbic acid and b-carotene as modulators of oxidative damage. Carcinogenesis, 18: Velioglu, Y.S., G. Mazza, L. Gao and B.D. Oomah, Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J. Agric. Food Chem., 46: Cai, Y.Z., M. Sun and H. Corke, 23. Antioxidant activity of betalains from plants of the Amaranthaceae. J Agric Food Chem., 51: Hartwell, J.L., Plants used against cancer. A survey. USA: Quarterman Publications, Lawrence, MA. 1. Singh, C., a-hydroxymicromeric acid, a pentacyclic triterpene from Terminalia chebula. Phytochemistry, 29: Barthakur, N.N. and N.P. Arnold, Nutritive value of the chebulinic myrobalan (Terminalia chebula Retz.) and its potential as a food source. Food Chem., 4: Singh, P.K., 23. Mycotoxin elaboration in Triphala and its constituents. Indian Phytopathol., 56: Jagetia, G.C., K. J. Malagi, M. S. Baliga, P. Venkatesh and R.R. Veruva, 24. Triphala, an ayurvedic rasayana drug, protects mice against radiationinduced lethality by free-radical scavenging. J Altern Complement Med., 1: Kaur, S., H. Michael, S. Arora, P. L. Harkonen and S. Kumar, 25. The in vitro cytotoxic and apoptotic activity of Triphala - an Indian herbal drug. J. Ethnopharmacol., 97:

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