Evaluation As Antioxidant Agents Of 1,2,4-triazole .
Journal of Applied Pharmaceutical Science Vol. 5 (06), pp. 120-126, June, 2015Available online at http://www.japsonline.comDOI: 10.7324/JAPS.2015.50620ISSN 2231-3354Evaluation as antioxidant agents of 1,2,4-triazole derivatives: effectsof essential functional groupsAhmet Cetin, Ibrahim Halil GeçibeslerDepartment of Chemistry, Faculty of Science and Art, Bingol University, 12000, Turkey.ARTICLE INFOABSTRACTArticle history:Received on: 27/03/2015Revised on: 08/04/2015Accepted on: 03/05/2015Available online: 27/06/2015A series of 1,2,4-triazole derivative compounds substituted with groups of phenol and pyridine were synthesizedin high yields and screened against several antioxidant activity parameters such as DPPH, ABTS, metalchelating, reducing power and the total antioxidant activity. The compounds showed better than expectedantioxidant activity between the studied biological activity parameters. Among these, compound G(2-(5mercapto-4H-1,2,4-triazol-3-yl)phenol) had a high total antioxidant activity potential with value of 232.12 6.89mmol/ml. Also showed fairly good ABTS cation radical and DPPH radical scavenging activity with values ofIC50 4.59 4.19 and IC50 7.12 2.32µg/mL respectively. Further, the antioxidant potential of heterocycliccompounds that 1,2,4-triazole derivatives containing different functional groups were compared with varioustests performed and has been shown to increase the activity of electron donating groups. Therefore, the presentstudy demonstrates that phenol and pyridine substituted 1,2,4-triazole compounds would be a better prospectivein the development of antioxidant agent.Key words:1,2,4-triazole, antioxidantactivity, 2-(5-mercapto-4H1,2,4-triazol-3-yl), phenol.INTRODUCTIONIn recent times there is increasing interest in thenitrogen-containing such as triazole heterocyclic compounds asutilized in medicinal chemistry. Currently the drugs used fortreatment such as Ribavirin (antiviral agent), Rizatriptan(antimigraine agent), Alprazolam (anxiolytic agent), Fluconazoleand Itraconazole (antifungal agents) are the best examples forpotent molecules possessing triazole nucleus. (Knabe et al., 1983;Lopes et al., 2004). The 1,2,4-Triazoles is another heterocyclicring having value as human therapeutic agents. Biologicalactivity studies related with 1,2,4-triazole ring system promotes itas an interesting class of heterocyclic compounds with a broadspectrum of pharmacological activities which include antifungal(Karabasanagouda et al., 2007), anti-inflammatory, analgesic(Chawla et al., 2012), antitumor (Ibrahim, 2009), anthelmintic(El-Khawass et al., 1989), antibacterial (Holla et al., 1996),antihypertensive, antidepressant, anticonvulsant and antiviral(Kritsanida et al., 2002). Free radicals are chemical speciescontaining one or more unpaired electrons, most of them beingunstable and capable of abstracting electrons from othermolecules.* Corresponding AuthorEmail: ibrahimgecibesler@gmail.comThe predominant reactive oxygen species generated bycell metabolism or by exogenous factors include hydrogenperoxide (H2O2), the hydroxyl radical (HO), the superoxide anionradical (O2 ˉ). These free radicals have essential roles in cellsignaling, apoptosis and gene expression. On the other hand,excessive free radical attack can damage DNA, proteins and lipids,resulting in diseases like cancer, neurological degeneration andarthritis, as well as the process of aging (Halliwell and Gutteridge,1990; Halliwell et al., 1992). Therefore, considerable speculationhas been directed towards the identification of antioxidants for usein preventive medicine. The radical-mediated oxidation of DNA isrelated to many diseases (Jomova et al., 2010). In addition, theradical-scavenging capacities of antioxidants can be estimated byreacting with ationic radical (ABTS) (Re et al., 1999)and 2,2-diphenyl-1picrylhydrazyl radical (DPPH)(Foti et al., 2011). During theinflammatory processes many reactive species are produced,among them reactive oxygen species (ROS) such as, superoxideradical (O2 ˉ), hydrogen peroxide (H2O2), hypochlorous acid(HOCl), singlet oxygen (1O2) and peroxyl radical (ROO ), as wellas reactive nitrogen species (RNS), like nitric oxide ( NO) andperoxynitrite anion (ONOOˉ). Indeed, ROS and RNS are alsoproduced by the endothelial cells, Kupffer cells, neutrophils and. 2015 Ahmet Cetin and Ibrahim Halil Geçibesler. This is an open access article distributed under the terms of the Creative Commons Attribution License NonCommercial-ShareAlikeUnported License ).
Cetin and Geçibesler / Journal of Applied Pharmaceutical Science 5 (06); 2015: 120-126macrophages as mechanism of defense against foreign infectiouspathogens (Vapaatalo, 1986; Halliwell et al., 1988; MouithysMickalad et al., 2000; Nikolic and Breemen, 2001).To date, the prevention of oxidative stress relateddiseases has been tentatively achieved by the development ofantioxidant compounds that are able to scavenge ROS and RNS,and thus avoid radical-induced oxidation damage. When avalidated scaffold has been identified, the study of structurereactivity relationship (SAR) may provide useful information forthe understanding of its mode of action and to the improvement ofthe antioxidant activity of future generated compounds.In the present study, due to a wide range of applicationsto find their possible antioxidant activity, some 1,2,4-triazolederivatives A–M, functionalized with phenolic, thiol, 2-hydroxybenzene, and pyridine groups were evaluated by using bis(3ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS)scavenging, reducing power, 1,1-diphenyl-2-picryl-hydrazyl(DPPH) free radical scavenging, total antioxidant and metalchelating activities. The search and evaluation of 1,2,4-triazolecompounds and their derivatives with a specific pharmacologicalactivity is a demanding task in the drug discovery process. In thiscontext, the study of structure reactivity relationship (SAR) is avaluable tool for the planning of potential novel drugs and, in alater stage, for the development of novel treatments.MATERIALS AND METHODSMaterialsThe Ferrous chloride, α-tocopherol (Vit E), 1,1-diphenyl2-picryl-hydrazyl (DPPH), iazine (ferrozine), butylatedhydroxyanisole (BHA),ethylene-diaminetetraacetic acid (EDTA), trichloroacetic acid(TCA) Diammonium salt of 2,2̍-azinobis(3-ethylbenzothiazoline6-sulfonate) (ABTS salt), were purchased from Merck, SigmaAldrich and Fluka. Other agents and solvents were of analyticalgrade and used without further purification directly.Synthesis of CompoundsThe required compounds were prepared as reported inliterature (Genc et al., 2004;Dincer et al; 2005; Cansiz et al.,2012). Thus, l-5-pyridin-4-yl4H-1,2,4-triazole-3-thiol (C)4 - 3-thiol l(F)2 - (5-mercapto-4H-1,2,4-triazol-3-yl) phenol (G)2 - (4-Ethyl5-mercapto-4H-1,2,4-triazol-3-yl) phenol (H)2 - (5-Mercapto-4phenyl-4H-1,2,4-triazol-3-yl) phenol (I)2 - l] phenol (K)2 - (4-Benzyl-5mercapto-4H-1,2,4-triazol-3-yl) phenol (L)2 - wereprepared:structures are listed in Table 1.121DPPH free radical-scavenging activityThe samples were tested with DPPH free radicalaccording to the method previously defined by Zovko Koncic etal., (2010)with some modifications. Shortly, 0.5 ml of synthesizedcompounds and synthetic antioxidant compound (BHA) preparedat different concentrations (12,5–400 µg/ml) were taken into testtubes and stirred with 2.5 ml of 2 mM DPPH solution. The mixturewas stirred thoroughly and incubated for 30 min in dark laboratoryconditions. The absorbance at a wavelength of 517 nm wasmeasured by UV spectrophotometry. To determine DPPH freeradical scavenging activity (FRSA) was used in the followingequation.FRSA(%) [(A0-A1)/A0]x100A0 is the absorbance values without specimen and A1 theabsorbance in the presence of specimen. As opposed to increasingconcentration of specimens decline of absorbance is an indicationthat destroyed DPPH radical. Antioxidant activity results areexpressed as IC50 value (µg extract/mL) that reduces by half theeffective concentration of DPPH radicals and was calculated byinterpolation from linear regression analysis.Ferrous chelating capacityThe ferrous chelating capacity of samples and thereference compound (EDTA) was conducted following the methodused by Decker and Welch (1990) with slight modifications. 2 mlof extract, sub-fractions and reference compound (EDTA) ofconcentration labeling between 25 and 250 µg/mL were added to50 μl of 2 mM FeCl2 and well mixed. The reaction mixture wasincubated in laboratory conditions. The reaction occurred by theaddition of 100 µl 5 mM ferrozine. After 10 min of incubationperiod, the absorbance of the solution was measured at 562 nmusing a UV–visible spectrophotometer. For the ferrous-chelatingactivity, IC50 values were calculated using the equation asdescribed above was used for DPPH free radical scavengingactivity.Assay of Reducing PowerThe reducing power was determined according to themethod of Oyaizu (1986) with slight modifications variousconcentrations of triazole derivatives (2.5 ml) were mixed with 2.5ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1%potassium ferricyanide.The mixture was incubated at 50 ºC for 20 min. After 2.5ml of 10% trichloroacetic acid (w/v) were added, the mixture wascentrifuged at 1000 rpm for 8 min. The upper layer (5 ml) wasmixed with 5 ml of deionized water and 1 ml of 0.1% of ferricchloride, and the absorbance was measured spectrophotometricallyat 700 nm. The assays were carried out in triplicate and the resultsexpressed as mean values standard deviations. BHA, BHT andα-tocopherol were used as standards. It was indicated that highabsorbance of the sample was good reducing power in the reactionconditions.
122Cetin and Geçibesler / Journal of Applied Pharmaceutical Science 5 (06); 2015: 120-126Evaluation of total antioxidant capacity byphosphomolybdenum methodRESULTS AND DISCUSSIONThe total antioxidant capacity of samples was evaluatedby the method of Prieto, Pineda, and Aguilar(1999) with slightmodifications. The antioxidant capacity of the extracts wasmeasured spectrophotometrically using a phosphomolybdenummethod, based on the reduction of Mo(VI) to Mo(V) by the sampleanalyte and the subsequent formation of specific greenphosphate/Mo(V) compounds. An aliquot of 0.1 mL of samplesolution (100 μg/mL) was combined with 1 mL of reagent solution(0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mMammonium molybdate). The tubes were capped and incubated in aboiling water bath at 95 C for 90 min. After the samples hadcooled to room temperature, the absorbance of the aqueoussolution of each was measured at 695 nm against blank. A typicalblank solution contained 1 mL of reagent solution and theappropriate volume of the same solvent used for the sample and itwas incubated under same conditions as rest of the sample. Stocksolutions of α-tocopherol were prepared in methanol. The totalantioxidant activity was expressed as equivalents of α-tocopherol(mmol α-tocopherol/ml).ABTScation radical scavenging activityTo determine the antioxidant activity of compounds,ABTS cation radical-scavenging activity was employed in thisstudy with slight modifications (Re et al., 1999). The ABTS cationradical cation was generated by mixing ABTS stock solution (7mM in water) with 2.45 mM potassium persulfate. This mixturewas kept at ambient temperature for 24 h until the reaction wascomplete and the absorbance was stable. Briefly, 1.9 ml of anABTS cation radical was added to 0.1 ml of differentconcentrations of standard (trolox) or samples. After 10 min, anabsorbance was read at 734 nm, and distilled water was used as ablank (each measured in triplicate). The IC50 is the concentrationof an antioxidant that is required to quench 50% of the initialABTS cation radicals under the experimental conditions given.Trolox, a well-known antioxidant, was used as a positive control.Table. 1:Chemical structure of 1,2,4-triazole derivative compounds.NNNNNNNNNNNNSHSHSHCH3HDPPH radical scavenging activityThe DPPH radical scavenging activity 1,2,4-triazolederivatives containing phenol groups (H-M) have showncomparable antioxidant potential with the BHA. whereas thecompounds (A-F) showed a moderate DPPH radical scavengingactivity. In compounds (A-F) pyridine rings are present at triazolering while in compounds (G-M) the phenol ring are present attriazole ring which different contributed in the radical scavengingability. It is clear from the results that the antioxidant potential ofcompounds is associated with the substituents on mentioned 1,2,4triazole rings. The inhibition percentage of compounds and Vit-Eat different concentrations are given in Fig.1A.Compound Gshowed highest DPPH scavenging activity with percent inhibitionof 93.751 0.47 at concentration of 100μg/ml, when compared withthe other compounds. This increased activity may be due toexistence of the –SH, -C6H5OH, and –NH groups. The presence ofphenolic group was inadequate for antioxidant activity.DPPH radical scavenging is a widely used method toevaluate antioxidant activities in a relatively short time comparedwith other methods. DPPH radical scavenging activities ofsynthesized compounds (A–M) were tested using ethanolicsolution of the stable free radical DPPH. The freshly preparedDPPH solution exhibits a deep purple color generally convert themto a colorless/ bleached product when an antioxidant is present inthe medium. Thus, antioxidant compounds can quench DPPH freeradical by providing hydrogen atoms or by electron donationresulting in absorbance at 517nm and the more rapidly theabsorbance decreases, the more potent the antioxidant activity ofthe compounds. The 1,2,4-triazole derivatives compounds wasfound to be significantly active against DPPH radical. On DPPHassay, IC50 values were obtained for compounds and BHA, so thata lower value of IC50 indicated a higher antioxidant activity andvice versa. Table 1 shows the statistical analysis of DPPH radicalscavenging activity and value of IC50 obtained from antioxidantactivity of compounds and BHA (as a reference compound). Thecompound G with an IC50 value 7.12 2.32 μg/mL was found to bemore active than the standard antioxidant BHA.NNNNNNSHNSHNNNNSHNH2CCH3ANNOH GHIKLMNSH
Cetin and Geçibesler / Journal of Applied Pharmaceutical Science 5 (06); 2015: 120-126123Fig. 1: Free radical-scavenging activities of compounds measured using the DPPH radical(A), Ferrous ion chelating activities of compounds at differentconcentrations (B), Reducing power of compounds at different concentrations by spectrophotometric detection of the Fe3 -Fe2 transformation (C), Highabsorbance at 700 nm indicates high reducing power. Concentration-response curves for inhibition of the absorbance of ABTS cation radical at 734 nm forcompounds (D). Results are means SD (n 3).Fig. 2 Total antioxidant activity of compounds measured by phosphomolybdenum reduction assay. Each value represents means SD (n 3).
124Cetin and Geçibesler / Journal of Applied Pharmaceutical Science 5 (06); 2015: 120-126Ferrous ion chelating activityFerrous ion chelating activities of the compounds andEDTA are shown in Table 1 IC50 for Fe2 chelating abilitywere60.16 2.65, 66.76 1.14 and 79.29 2.33 µg/ ml forcompounds G, M and L, respectively. Furthermore, the percentageinhibition of compounds obtained from Fig.1Breveal that the metalchelating effects of the compounds D were concentrationdependent the other compounds were not. Thus the compounds Ddemonstrate a marked capacity for iron chelating, suggesting thattheir action as peroxidation protectors may be related to their ironchelating capacity. However, the ferrous ion chelating activities ofcompounds were lower than the EDTA (IC50 28.48 0.65µg/ ml)Itwas reported that the compounds with structures containing two ormore of the functional groups, such as -OH. -SH.-COOH.-PO3H2.C configuration should have chelation activity (Lindsay,1996; Yuan et al., 2005; Gulcin, 2006). In this respect,compound G may chelate the ferrous ions with hydroxyl and thiolgroups.Transition metals have a pivotal role in the generation ofoxygen free radicals in living organism. The production of theseradicals may lead to lipid peroxidation, protein modification andDNA damage. Chelating agents may inactivate these metal ionsand potentially inhibit the metal-dependent processes (Finefrock etal., 2003). Compounds with metal chelating activity have ability toconvert metal ions into insoluble metal complexes or generatesteric hindrance, which can prevent the interactions betweenmetals. In the presence of chelating agents the complex formationis disrupted with the result that the red colour of the complex isdecreased.Measurement of colour reduction therefore allowsestimation of the chelating activity of the coexisting chelators(Yamaguchi et al., 2000). The ferrous state of iron accelerateslipid oxidation by breaking down the hydrogen and lipid peroxidesto reactive free radicals. The ferric iron (Fe3 ) is the relativelybiologically inactive form of iron. Fe3 but ion produces radicalsfrom peroxides, even though the rate is tenfold less than that ofFe2 ion, which is the most powerful pro-oxidant among thevarious types of metal ions (Calıs et al., 1993). Chelating agentsmay not activate metal ions and potentially inhibit the metaldependent processes (Finefrock et al., 2003). Free iron is known tohave low solubility and a chelated iron complex has greatersolubility in solution, which can be contributed solely by theligand. Furthermore, the compound–iron complex may also beactive, since it can participate in iron-catalyzed reactions.Reducing power capacityThe capacity of reducing power of 1,2,4-triazolederivatives was almost comparable with the synthetic antioxidant,Vitamine E (Vit-E). The reducing power of test compoundsincreases with increase in concentration. Compounds C–D and Lexhibited very low absorption, whereas compounds A, I, M and Gwere found to have higher absorption at a high concentration of200 μg/ml. The actively mentioned 1,2,4-triazole derivatives couldreduce the most Fe3 ions, which had a higher reductive activitythan the reference standard of Vit-E. Reducing power of thecompounds mostly depended on the groups where the compoundswere substituted. The presence of pyridine, thiol and phenolfunctions in the 1,2,4-triazole compounds may play an importantrole to act as a better electron donor which may enhance reducingpower ability of active compounds. The reducing power of theinvestigated the 1,2,4-triazole derivatives differed according tocontaining phenol and pyridine groups. This may be due to thesubstitution of electron-releasing groups at 1,2,4-triazolecompounds, which may be helping for stabilization of the freeradical form after donating electron and thus to leadingmaximum reducing ability compared to other less activecompounds.The reducing capacity of a compound may serve as asignificant indicator of its potential antioxidant activity. Reducingpower is a measure of reductive ability of antioxidants and isevaluated by the transformation of Fe3 to Fe2 in the presence ofcompounds. The reducing ability of a compound generallydepends on the presence of reductants (Duh et al., 1999), whichhave been exhibited antioxidative potential by breaking the freeradical chain, donating a hydrogen atom (Gordon, 1990).
antioxidant methodologies such e) cationic radical (ABTS) scavenging, reducing power, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, total antioxidant and metal chelating activities. The search and evaluation of 1,2,4-triazole
The antioxidant parameters including Superoxide dismutase (SOD), glutathione peroxidase and glutathione levels were evaluated [16,17]. Then the animals were sacrificed under anaesthesia using diethyl ether and the tissue samples of liver, kidney and heart were collected for evaluation of antioxidant levels in tissues. Antioxidant Assays
antioxidant, which accounts for the scavenging of free radicals and protective effect on antioxidant enzymes (Ravi et al., 2004). Total phenolics of the seeds said have an important antioxidant activity (Bajpai et al., 2005). The fruit is rich in sugar, mineral salts, vitamins C. Fruit of Syzygium cumini contain malic acid and a small quantity of
Antioxidant property In this experiment, methanol extract of Aporosa wallichii Hook.f. leaves were tested properly through DPPH assay and TPC to determine the antioxidant property of this plant ( Pavithra et al., 2009). Antioxidant activity is very important in preventing free radical reactions because they can neutralize free radical by their
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Theoretical Evaluation of Antioxidant Activity of Tea Catechins N.S. Labidi1*, L. Guerguer 1, A. Kacemi 1 1. Institut des Sciences, Département des Sciences de la Matière, Centre Universitaire de Tamanrasset, BP (10034) Sersouf- Tamanrasset (11000)-Algeria Abstract To quickly evaluate the antioxidant activity of tea catechins epicatechin (EC), -
evaluation of the free radical scavenging activity of the drug sample. All eight marketed hepatic formulation showed the capacity of scavenging of free radical. The lowest antioxidant activity is shown by Udiliv i.e. 8.97% and silybon shows the highest antioxidant activity i.e. 60.86%.
Evaluation of antioxidant activities of flower extract (fresh and dried) . eliminate free radicals and other reactive oxygen and nitrogen species, and these . Table.1 Phytochemical and antioxidant activity analysis of water and ethanol extract of fresh flowers of Saraca indica
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