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International Journal of PharmTech Research

CODEN (USA): IJPRIF ISSN : 0974-4304
Vol.2, No.2, pp 1276-1285, April-June 2010

A Review on In-vitro Antioxidant Methods:
Comparisions, Correlations and Considerations
A.V.Badarinath*, K. Mallikarjuna RAo, C.Madhu Sudhana Chetty, S. Ramkanth,
T.V.S Rajan, K.Gnanaprakash

Department of Pharmaceutics, Annamacharya College of Pharmacy,
New boyanapalli, Rajampeta – 516126, Kadapa, Andhra Pradesh, India
Phone No: 09440916296

Abstract: This stimuli article provides an overview general aspect, their comparisons, correlations and considerations
of various In-Vitro methods to measure the antioxidant defense system and to discuss a number of updated In-Vitro
methods used for detection of antioxidant properties. This review article gives the information regarding the different
methods that are used to perform the In-Vitro antioxidant activity. It emphasizes the method simplicity, time required,
instrumentation which makes us to decide which method to be fallowed to perform antioxidant property based on the
feasibilities afforded to determine it. It makes a glance regarding the advantage of different methods and which is most
common method used in present days for effective analysis. On the other hand as there are advantage there may be some
disadvantages which are also mentioned in this article. The research studies should also be carried mostly on the
accurate methods for exact results which also act as a good reference for the further researches.
Key words: In-Vitro antioxidant methods, Cellular antioxidant activity, Cellular antioxidant activity, Semi quantitative
analysis, Folin-Ciocalteu method.

A antioxidant is a chemical that prevents the oxidation ONOO [peroxy nitrate], NO2 [nitrogen dioxide] and
of other chemicals. They protect the key cell N2O3[dinitrogen trioxide].4,5 In a normal cell, there are
components by neutralizing the damaging effects of appropriate oxidant: antioxidant balance. However,
free radicals, which are natural by- products of cell this balance can be shifted, when production species is
metabolism. 1,2 Free radicals form when oxygen is increased or when levels of antioxidants are
metabolized or formed in the body and are chemical diminished. This stage is called oxidative stress.
species that posses an unpaired electron in the outer Oxidative stress results in the damage of biopolymers
(valance) shell of the molecule. This is the reason, why including nucleic acids, proteins, polyunsaturated fatty
the free radicals are highly reactive and can react with acids and carbohydrates. Lipid peroxidation is
proteins, lipids, carbohydrates and DNA. These free oxidative deterioration of polyunsaturated lipids and it
radicals attack the nearest stable molecules, stealing its involves ROS and transition metal ions. It is a
electron. When the attacked molecule loses its molecular mechanism of cell injury leading yield a
electron, it becomes a free radical itself, beginning a wide range of cytotoxic products, most of which are
chain reaction, finally resulting in the description of a aldehydes, like malondialdehyde (MDA), 4-
living cell3. Free radicals may be either oxygen derived hydroxynonrnal(HNE), Oxidative stress causes serious
(ROS, reactive oxygen species) or nitrogen derived cell damage leading to a variety of human diseases6
(RNS, reactive nitrogen species). The oxygen derived like Alzheimer’s disease, Parkinson’s disease,
molecules are O-2 [superoxide], HO[hydroxyl] ,HO2 atheroscleorosis, cancer, arthritis, immunological
[hydroperoxyl], ROO[peroxyl], RO[alkoxyl] as free incompetence and neurodegenerative disorders, etc.
radical and H2O2 oxygen as non-radical. Nitrogen Nutritional antioxidant deficiency also leads to
derived oxidant species are mainly NO [nitric oxide], oxidative stress, which signifies the identification of

plates were placed under natural light until be done by this technique.J. A RPTLC method combined with video yellow bands on a light purple background.2(2) 1277 natural anti-oxidative agents present in die consumed immerse in 0. In the DPPH free radical scavenging activities were found to be predominant in highly capacity assay by TLC.11 Bleaching of β-carotene on TLC is radical scavenging activity measurement of anti. of antioxidant. antioxidants appear as extract. its free radical property inhibitory.A. a total of 58 picrylhydrazyl(DPPH) can be carried out. allows only 1 be calculated and photographs can be taken. sample to read a time and requires high quantity of 40 µg shows equal antioxidant property with that of reagent where as the second method is time saving and 50 µg of rutin by this method10. Absorbance at 517 nm was determined In order to detect the antioxidant activity. Further more.04% (wt/vol) solution of 1. The various conventional and latest detect different groups of compounds. 3 x ½ “ CCD video camera (Hitachi. Muttenz. The antioxidant analysis. all the plates can be sprayed with a methanolic solution The method was successfully applied to rapeseed meal of DPPH (2mg/ml). We can judge the potency of the test ELISA plate reader for absorbance. 7. acetylcholinesterase radical reacts to an antioxidant. Rf values for each hand can very tedious. Orange-yellow methods comes under invitro are listed in table no.13 TLC – DPPH combined with Video yellow are white spots in the purple background were scanning Documentation system including a HV-C20 considered as antioxidants. even uneluted radical scavenging activity of antioxidative fractions plates also can be used to determine the qualitative from rapeseed meal by using DPPH is reported. Reversed phase an easy. Antioxidant and radical scavenging light yellow. Switzerland can be used. time consuming method. effective and rapid way to study plant extract technique was applied for the measurement of free profiles. After scanning detection for quantitative evaluation of free spotting the extracts on the TLC plates.2% DPPH methanol solution and sample by human population. Cuvette assay method The diameter and intensity of the yellow spot depends uses UV-Visible spectrophotometer to see the on the amount of known amount and concentrations absorbance.2-diphenyl-1.1. DPPH is a extracts from various organs (aerial parts. Conventional cuvette assay of radical scavenging activity is replaced by 96-well plate titer Semi quantitative analysis assay from past couple of years. The yellow spots remaining indicated the presence of antioxidant Qualitative analysis substances. After developing and drying.12 Using TLC autography. of compounds separated by reversed-phase TLC (RP- TLC) using phenolic acids as model analytes. Antioxidants act by several mechanisms and and observed at UV-365 nm.2010.02% solution of β-carotene in well as semi quantitative analysis of antioxidants can CH2Cl2. This approach has benefits over simply quantifying Vanillin /H2SO4 reagent is sprayed on the plate and antioxidant components as it provides a measure of heating it at 1100C.V. a method after 30 min and the percentage of activity was based on the reduction of 2. The same method can be implemented to detect total 1) Comparisons phenolic and total flavonoid content just by changing In-Vitro determination of antioxidant capacity the mobile phase solvent system and visualizing agent. The uneluted plates also can activity was evaluated by measuring the area of bright . 8 spots were evaluated for radical scavenging activity. roots) of 16 Turkish plants were tested produces a violet solution in methanol. 1 – diphenyl-2- chromatography (TLC) autography technique provides picrylhydrazyl (DPPH) in methanol. another method. When the free for their antibacterial.Badarinath et al /Int. with small amount developed to measure the radical scavenging activity of reagent. free radical stable at room temperature. calculated. antifungal. to detect the flavonoids no one assay can capture the different modes of action as they appear as yellow-orange fluorescent spots. where as 96-well plate method uses of the solutions. for 5 minutes and observed to their effectiveness. which flowers. It spots indicate poly phenolic compounds. fruits. the extracts that produced polar extracts. spotted on the silica-gel 60F 254 plates and develop and for TLC plate imaging software supplied by the chromatogram in adequate solvent systems. plates oxidative compounds in plant extract. discoloration of background. Now Camag. antioxidant and radical scavenging is lost due to chain breakage and its color changes to activities. The first method is sample. A method was it reads about 96 samples at a time. leaves. Japan) can be Procedure in brief is-extracts resolved in the solvent is connected with Reprostar 3 transilluminator cabinet.9 qualitative as were sprayed with a 0. TLC a) TLC Autography technique separation was followed by dipping the plate in a The antiradical screening by thin layer 0. Thus. No sample purification is needed as this radical-scavenging activity of rapeseed meal technique provided a simultaneous separation and fractions. PharmTech Res. The silicagel is very difficult to select a suitable antioxidant assay plate was sprayed with natural products-PEG reagent method.

and their assay.04% (w/v) with automated data storage. Reactive oxygen species (ROS) may power of whole foods and individual antioxidant attack biological macromolecules giving rise to nutrients and compounds. The CUPRAC absorbance of the ethylacetate foods and individual antioxidant nutrients and extract due to the reduction of Cu (II)-neocuproine compounds. presence of OH scavengers.2(2) 1278 yellow bands against the purple background by a CCD method provides for concurrent multisample analysis video camera after dipping the plate in 0. The sulfoxide were shown by the modified CUPRAC assay oxidation products of these highly reactive substrates to be more effective scavengers than mannitol. Kellys Wolfe and Rui Hai oxidative stress-originated diseases. 15 study plant extract profiles. The 2. more concentrations were determined photometrically using specific. The hydroxyl radical scavenging rate .J.Badarinath et al /Int. followed by + EDTA with hydrogen peroxide. assay results are presented. ascorbic acid and 17 well-known phenolic compounds Dihydrorhodamine was selected as the preferred including α-tocopherol. distribution and activity of antioxidant low-yield TBARS test. and DPPH solution. it requires sophisticated compounds in cells versus solely looking at instrumentation. oxidation by peroxyl radicals generated from 2. apples. regression analyses. and rapid way to analysis with electrochemical detection. new measure of antioxidant activity called the cellular antioxidant activity (CAA) assay. and typical was determined by this TLC-DPPH method. It provides fro concurrent multisample analysis with automated data storage.14 hydroxyl radicals attacked both the probe and the Advantages water soluble antioxidants in 370C incubated solutions More accurate guage of antioxidant power of whole for 2 h. For screening Recently. molar concentration of relevant species. The (CUPRAC) new CAA method tests antioxidant compounds CUPRAC method is Novel hydroxyl radical activity inside cells: An approach that probably scavenging antioxidant activity assay for water-soluble provides a more accurate gauge of the antioxidant antioxidants. electrochemical detection is more specific than the metabolism. respectively and C is the oxidation.2. PharmTech Res. 2.and 3. we can use p-aminobenzoate. Although the takes place inside the cell. scavenger with hydroxyl radical can be deduced from the inhibition of color formation.5- equal amount s (100 grams ) of wild blueberries. red grapes and green grapes. red and green grapes. dihydrofluorescein and 3. are intensely colored dyes that absorb maximally in the glucose. which they dubbed d) Cupric Ion Reducing antioxidant capacity the “next step” in quantifying antioxidant activity.A. metabisulfite. phenolic acids and flavonoids substrate for screening crude extracts. microtiter plate reader. effective. 489 to 512 nm. The microtiter plate TBARS assay. No sample separation is Advantages needed. costly. secondary products resulting from OH the new method in which antioxidant reaction will attack to various probes are measured. The results radicals generated from an equivalent mixture of Fe(II) placed wild blueberries on top. formate. hexacyanoferrate (II). metabolism. 5-dimethoxybenzoates were the best probes in terms dichlorodihydrofluorescein as reduced substrates for of linearity and sensitivity.V. The produced cranberries.2010. They compared use of dihydrorhodamine. This approach is more biologically reagent by the hydroxylated probe decreased in the relevant as it accounts for uptake.4 – and 123. calculation of oxidation inhibition rates. Since OH is very Liu of cornell university’s food science lab developed short-lived. Novel lead antioxidants Advantages are selected from active extracts by chromatographic This technique is easy. As a more convenient and less costly antioxidant value. regression analyses.14 proportional to the scavenging ability of the tested Disadvantages compound. and dimethyl azobis(2-amidinopropane) dihydrochloride. and b) Cellular antioxidant activity (CAA) assay calculation of oxidation inhibition rates. and of a higher yield than the classical a 96-well. The developed method is less lengthy. DPPH scavenging activity of L. dimethoxybenzoate probes for detecting hydroxyl cranberries.4. A rate constant for the reaction of the Time consuming. lysine. This new approach is more measurement of aromatic hydroxylation with HPLC / biologically relevant as it accounts for uptake. The second-order c) Dye-substrate oxidation method rate constants of the scavengers were determined with A novel microtiter plate assay was developed to competition kinetics by means of a linear plot a A0/A determine the total peroxyl radical trapping activity of as a function of C scavenger / C probe where A0 and A are antioxidant extracted from marine organisms by the CUPRAC absorbences of the system in the absence measuring the inhibition rate of dye-substrate and presence of scavenger. scientists at cornell University proposed a crude extracts and typical assay results are presented. They applied the new technique to alternative. apples. Iodide. the difference being distribution. thiourea. Potency of sample can be known. and simple alcohols as in the TBARS wavelength region.

yield than the classical TBARS assay. spectrophotometric the first time the use of a novel spectrophotometric analysis of NAD+ / NADH ratios. And it this method comprises the steps of (a) initiating a was found that. is a source of chemiluminescence.2(2) 1279 constants of ascorbic acid. which capacity of a is sensitive to radical scavenging reflects lipid oxidation. PharmTech Res. formate and chelating capability of the compounds.19 The hydroxyl hexacyanoferrate(II) that caused interference in other radical scavenging capacity and efficacy of a novel assays could be easily found with the proposed organosiliceous anionic hydride compound. bis(neocuproine) copper(II) the antioxidant compound and the linear decrease in chloride. CUPRAC findings correlated well with the results of The validity of the analysis was verified by electron ABTS /TEAC and Folin assays. labeling of the resulting HRP). This work reports for spin resonance spectroscopy. described in the work by (Whitechead. The method is chemiluminescent assay for TAC measurement is best rigorously validated through linearity. The novel reagent for the CUPRAC total between the hydroxyl radical scavenging capability of antioxidant capacity assay.J. a antioxidants is analyzed and the hydroxyl radical signal reagent (luminal plus para-iodophenol). As a more Antioxidant capacity of number of non-refined seed convenient and less costly alternative. This method can quantify the antioxidant emulsions with a suitable reporter fluorophore. and the activity of sample is above. The assay involves the chemiluminescent substrate luminal. The 0. silica procedure. stable. most of the antioxidative components chemiluminescent reaction and allowing said reaction are removed from edible oils during refining process. The fluorescence decay curve of chemiluminescent reaction. 1992). IC 50. and the hydroxyl of maximum. To accuracy and ruggedness. 16 Apricots as five varieties of Malatya hydride. and continuous monitoring of antioxidants that reduce the light output.A. Gallic acid is chosen as a known antioxidant activity subjected to steps (a) to (c) reference standard. said level being selected from the group evaluate hydroxyl radical is generated by a Co(II) – consisting of (i) A rising level between 90 to 100 % mediated Fenton-like reaction. biological fluids. A assay) was removed prior to assay on a strongly basic fluorescence signal half-inhibition. Sulphite (normally product created by reacting a Fe2+-EDTA complex in contributing to the colour formed in the CUPRAC the presence of a potential radical scavenger. Disadvantages f) Enhanced chemiluminescence (ECL) Sophisticated instruments are required which are more ECL has been used to measure antioxidant capacity in expensive. to progress.V. were quantified by a recently developed region are screened for antioxidant capacity by using method. which is Monitoring said change in the level of luminescence: an index of the hydroxyl radical prevention capacity. et. A method of the decomposition process. The principle behind the enhanced expressed as gallic acid equivalents. is mixed with . the area under the fluorescence decay change when said sample has antioxidant activity: (c) curve (AUC) is integrated. is under patent and of refined oils by using this simple technique. It is based on solubilisation of the horseradish peroxidase ( fluid because the reaction oils in aqueous buffer. The method measures a direct relationship CUPRAC. thereby to generate a level of 18 A novel fluorometric method has been developed to luminescence. (ii) The maximum (iii) A post- radical formation under the experimental condition is maximum substantially constant plateau level: (b) indirectly confirmed by the hydroxylation of p. Antioxidant capacity of assay of the antioxidant activity of biological sample number of non-refined seed oils is compared with that suspected of having such activity. Adding said sample to said progressing hydroxybenzoic acid.2010.1 µm was obtained for silica hydride compounds. The developed oils is compared with that of refined oils by using this method is less lengthy. A wide range of phenolic perform the enhanced chemiluminescence assay. was easily accessible.17 c (Fe2+) and epinephrine to adenochrome reductions. mitochondrial method (CUPRAC) for the assay of both total membrane potential measurements and assays of antioxidant capacity and sulphite levels of diverse reductions of both cytochrome C (Fe3+) to cytochrome apricot samples.Badarinath et al /Int. said sample causing said FL is monitored in the absence or presence of level of luminescence generated by said reaction to antioxidant. selective and signal from a fluorescent 2-hydroxyterephthalate responding to all antioxidants. value of 1. which prevention capacity is mainly due to the metal. Light emission e) Cellular antioxidant activity occurs when the luminal is oxidized by hydrogen This method contains different principles to detect peroxide that is generated in a reaction catalyzed by antioxidant property. 20 Advantages Advantages It requires sophisticated instrumentation.4 ± anion exchanger at pH 3 in the form of HSO. and (d) Determining the antioxidant activity of said is calculated by subtracting the AUC of the blank from sample assayed by reference to that of samples of that of the antioxidant. and the net AUC. more specific and of a higher simple technique.

2010. which may not be routinely available in It is simple.24 equivalents. It requires Advantages fluorometers. relatively complex and time consuming. semiautomated. It is the assay is performed standardized with Trolox. For these reasons. so signaling reagent solution must be prepared each time comparisons between laboratories are difficult. because fresh Many different end points have been used. using automated. and proteins.azinobis (3- quenching (H transfer). a water soluble analogue of vitamin E. a variety of assay has been broadly applied in academy and in the methods has been employed. In the TRAP assay to produce ROS. which limits the is the total radical trapping antioxidant parameter possibility to compare results from different studies. Of these assay. Temperature control decreases required specialized equipment. a water-soluble nonenzymatic antioxidants such as glutathione. The power of the induced by Trolox in the same plasma sample. and robust does not analytical laboratories. the TRAP or the ORAC assay in plasma have to be Luminometer) is needed to measure the interpreted with care. hydrogen peroxide (H2O2).25 measures the ability of plasma to reduce the intense Disadvantages blue ferric tripyridyltriazine complex to its ferrous ORAC is limited to measurement of hydrophilic chain form. Determination of the lag-phase TRAP and ORAC Disadvantages assays can be performed with different radicals and This method is cumbersome and time-consuming thus different results will be obtained depending on because fresh signaling reagent solution must be the radical.A. 21 antioxidant assay based on the ability of a test Advantages substance to inhibit the oxidation of B-phycoerythrin ECL has been used to measure antioxidant capacity of by reactive oxygen species. speedy. PharmTech Res. is monitored through the loss of fluorescence of the . requires a high degree of expertise and experience. thereby changing its absorbance. In this assay. This technique another assay which has been applied in human plasma has not been widely applied. (TRAP). Finally. inexpensive. scavenging antioxidants that reduce the light output. This is a colorimetric assay that choice to quantify AOC.8-tetramethyl in serum or plasma because it measures chroman-2-carboxylic acid).7.J. the signal reagent may reduce in intensity.22 j) Trolox equivalent antioxidant capacity (TEAC) Disadvantages This assay is based on the ability of molecules to FRAP cannot detect species that act by radical scavenge the stable free radical of 2. this method is Disadvantages cumbersome and time consuming. 23. It can be performed reproducibility.24 antioxidants in the seminal plasma to reduce the Advantages chemiluminescence of the signal reagent is compared Used for measurements of in-vivo antioxidant capacity with that of Trolox (6-hydroxy-2.2’. such as glutathione with Trolox. Advantages The advantage of the AUC approach is that it implies g) Ferric-raducing antioxidant power (FRAP) assay equally well for both antioxidants that exhibit distinct In order to assess the modifying effect of tea lag phase and those that have no lag phases.V. The ORAC assay is another commonly applied physician’s office. Commonly used is the food and dietary supplement industries as a method of FRAP assay. the ECL seems the least h) Total radical trapping antioxidant parameter suitable to determine plasma antioxidant capacity (TRAP) because it relies on enzymatic activity. ORAC flavonoids on plasma antioxidant status. particularly SH group ethylbenzothiozoline-6-sulfonic acid) in comparison containing antioxidants like thiols. relative to Trolox.25 chemiluminescence. the rate of peroxidation induced All the other assays have been applied in plasma byAAPH(2’-azobis(2-amidinopropane) hydrochloride) reproducibility. luminometer) is needed to i) Oxygen radical absorbing capacity (ORAC) assay measure the chemiuluminescence.22 but ignores lipophilic antioxidants. expensive instrumentation (eg. which in turn is mixed with a the lag-phase induced by plasma is compared with that substrate. or manual methods. biological fluids. results obtained with prepared. 24 The activity of a compound is therefore expressed as TEAC.Badarinath et al /Int. adding another technical problem. This method can quantify the Proteins interfere with the analysis. It also Moreover. partially protecting antioxidant capacity of a is sensitive to radical R-PE when all plasma antioxidants are exhausted. which means that Basically the same principle is applied as in the TRAP this assay is often not readily available in a assay. expensive instrumentation (eg. and is measured as molar Trolox ascorbic acid. Although accurate. Finally. tocopherol analogue.2(2) 1280 horseradish peroxidase (HRP)-linked immunoglobulin protein R-phycoerythrin (R-PE).5.

The HX-FeIII = metmyoglobin.20 Determination AICl3 method (Lamaison and Carnet. 39 activities. was the number of electrons an antioxidant can give away. The oxidation can be Three assays were compared for the determination of stopped by cooling in an ice bath. quantified by a recently developed method. glutathione (1. 2. In this equation. Significant Esterbauer by as follows.V. ABTS+ . TBARS were extracted with n. between serum FRAP and serum TEAC. This preparation is combined not show clear correlation between TEAC values and with trichloroacetic acid thiobarbuturic acid.2’ – azinobis-(3-ethyl-benzothiazoline. rutin and catechin). There was no correlation the plant extract was added to linolenic acid emulsion either between serum ORAC and serum TEAC or in phosphate Buffer solution (PBS).40 normally donate one electron whereas the others are two electron reductants. were proportional to their concentrations. This work reports for and H2O2. The formation of method (CUPRAC) for the assay of both total ABTS+ on Interaction With Ferryl Myoglobin antioxidant capacity and sulphite levels of diverse produces a relatively stable blue-green color.05). were added to equal volumes of a solution of 2% AlCl3 Hence we can say that there is no correlation.25 Radical trapping capacity .17 The hydroxyl radical scavenging Measured at 600nm. The we can say that there is a correlation. PharmTech Res. 31 cooling and adding EDTA.A. If a single plant is assayed for its The Conjugated Dienes (CD) were quantified by antioxidant activity by different technique. Thus we can say that there is a based on the principle that when 2. The flavonoids (quercetin.38 antioxidant compound and the linear decrease in signal from a fluorescent 3-hydroxyterephthalate l) Folin-Ciocalteu method product created by reacting an Fe2+ -EDTA complex in The total flavonol content was expressed as rutin the presence of a potential radical scavenger. Flavonoid contents were system (LPIC) correlated well with the cytoprotective expressed as mg catechin equivalent /g dry weight. heated in boiling water for 45 minutes and then cooled The TEAC values of ascorbic acid (1. silica hydride. at a final concentration of 10mm was incubated comparison of the antioxidant results of the three alone (control) or with the plant extract (sample).2’-azinobis-(3. a relatively stable radical cation. is the first time the use of a novel spectrophotometric formed (see equation below). The mixture was comparison of the result of the assays showed that the vigorously shaken. Numerous The oxidation can be initiated by freshly prepared publications applied the total phenols assay by FCR copper sulphate solution. 1990) was of the lag-phase TRAP and ORAC assays can be used for determination of the total flavonoid content of performed with different radicals and thus different the sample extracts.97). Antioxidants in the fluid sample capacity and efficacy of a novel organosiliceous suppress this color production to a degree that is anionic hydride compound. Hence equivalent in mg/g or %W/W of the extracts. correlation in between these two methods. A Y 6H2O (2 g in 100 ml methanol). and Tween20. Briefly.2(2) 1281 k) ABTS {2. Aliquots of 1.J.28). Oxidation kinetics were determined (ORAC) assay. and uric acid butanol and the absorbance of the n-butanol layer was (1. X – [FeIV = 0] = method measures a direct relationship between the ferrylmyoglobin. according to technique gives the different results. each measuring the absorbance at 234 nm.2010. 30 prepared copper sulphate. more Miller et al (1993) described another technique for specific. and absorbance at 367 nm was read ability to inhibit peroxidation of lipids in a liposomal after 10 min of incubation. This assay is TBARS assay.5 ml of extracts results will be obtained depending on the radical. by measuring the absorbance at above antioxidant capacity (Randox-TEAC) assay.40 The qualification of TBARS was weak but significant linear correlation between serum monitored. SASA and is emulsified with Tween 20 in phosphate buffer (pH DPPH for the commercial coffee drubjs ub cguba. The BHT was used as a The TEAC values for pure antioxidant compounds do standard antioxidant. shows a oxidation was initiated by the addition of freshly well correlation between LPO and ORAC methods. apricor samples. After incubation at 370C for and an ET based antioxidant capacity assay and often 3 hours. according to the ohkawa method. in obscurity. in the presence of total antioxidant capacity in human serum: the EDTA and BHT.29 A 7). tocopherol (0. and the mentioned nm for every 15 minutes over 270 ferric reducing ability (FRAP) assay. Correlations 6-sulphonic acid)} The developed method CUPRAC is less lengthy. alfa – at room temperature. ABTS = 2. and of a higher yield than the classical TAC measurement based on colorimetry. the reaction was stopped by found excellent lineal correlations between them.Badarinath et al /Int. hydroxyl radical scavenging capability of the bensthiazoline sulphonate]. A mixture of linoleic acid correlations were found between TPC. although glutathione can measured at 532 nm. There was a minutes. 16 The ethyl-benzothiazoline-6-sulphonic acid) {ABTS} is CUPRAC finding correlated well with the results of incubated with a peroxidase (such as metmyoglobin ABTS/TEAC and Folic assays.01) are almost the same. ORAC and serum FRAP.2’ – azino-di-[3-ethyl. the Randox Trolox-equivalent at 370C .

The method used to measure and calculate antioxidants and provident in the studied food is the antioxidant activity has a major impact on the critical.J. List of In-Vitro antioxidant methods S.N-dimethyl-p-Phenylenediamine (DMPD) assay . The use of one radical scavenging activity. For example in case of liberation of been published. Several reviews have conditions. not in substrates. The dominant mechanism depends on foods and biological samples. Further more. and other components in in-vitro people. confused. Antioxidant activity may be distinctly results because. Antioxidants act by several mechanisms e. oxidation different in bulk oils and multiphase foods. there are different ways to measure assay. 36. remove any source of oxidative initiation. 32 antioxidants have to be absorbed and localized in 3) Considerations of in-vitro antioxidant assay active forms in the oxidation site to enable antioxidant methods effect. 37 Table No:01. 35. not hydrogen atom to a specific reactive oxygen species withstanding the possible synergistic interactions or to any electron acceptor. and the opinions vary considerably. antioxidant from food into stomach. Due singlet oxygen. most structure of food and temperature of the meal influence probably due to the fact that the area of antioxidants is the dominant mechanism. which may not be present in the digestive antioxidant power. we are using oxidizing understand that these tests are done in test tubes. initiators. leaving research people seriously system.No Name of the method I Hydrogen Atom Transfer methods (HAT) 1) Oxygen radical absorbance capacity (ORAC) method 2) Lipid peroxidation inhibition capacity (LPIC) assay 3) Total radical trapping antioxidant parameter (TRAP) 4) Inhibited oxygen uptake (IOC) 5) Crocin bleaching Nitric oxide radical inhibition activity 6) Hydroxyl radical scavenging activity by p-NDA (p-butrisidunethyl aniline) 7) Scavenging of H 2O2 radicals 8) ABTS radical scavenging method 9) Scavenging of super oxide radical formation by alkaline (SASA) II Electron Transfer methods (ET) 1) Trolox equivalent antioxidant capacity (TEAC) decolourization 2) Ferric reducing antioxidant power (FRAP) 3) DPPH free radical scavenging assay 4) Copper (II) reduction capacity 5) Total phenols by Folin-Ciocalteu 6) N. both in foods and in vitro. But unfortunately. Liberation of antioxidants from aemulsions. dietary potentials alone. PharmTech Res. by Understanding of roles of various antioxidants and donating hydrogen to radicals. Hence. Further. separating each antioxidant compound and a) Measuring its ability to donate an electron or studying it individually is costly and inefficient. In addition The biggest problem is the lack of a validated assay food antioxidants may inhibit oxidation by several that can reliably measure the antioxidant capacity of mechanisms.A. physical There seems to be no consensus of opinions.2010.V. partitioning of oxidizing substrates. free their activities is challenging. products. 34. most of the assay to the complexity of the composition of plant methods measures any one of the following. reducing power. There is considerable debate take into the account in in-vitro antiradical activity about which method is best and it is critical to methods. But these factors do not such a complex topic.33. since.Badarinath et al /Int. dimensional method to evaluate multifaceted inhibition of β-carotene bleaching and quenching antioxidants is not a complete analytical system. b) Testing its ability to among the antioxidant compounds in plant products. metal chelating ability.2(2) 1282 directly relates to the hydrogen atom donating ability foods in digestion may differ from liberation of same of a compound and is not correlated to the redox in extraction for antioxidant tests.g. such as reactions are complex.

Summary of Antioxidant Assays Antioxidant Simplicity Instrumentation Biological Mechanism Time assay Required relevance required ORAC ++ + +++ HAT ++ TRAP ―― ―― +++ HAT +++ FRAP +++ +++ ―― SET ―― TEAC + + ― SET ― F-C +++ ― ― SET + TLC +++ + ――― SET.A. The assay will be a valuable evaluate antioxidant capacity in a food system. . List of In-Vitro antioxidant methods S. Thus the efficiency is fairly low and the their particular reaction system and their relevance to results are only qualitative.――― = less desirable to highly undesirable characteristic. ++. which are preservations and cosmetic products.2(2) 1283 …Continued…. Conclusion Factors affecting oxidation reactions and antioxidant heterogeneous and the reaction cannot be easily activities in foods and in vitro differ. Conventional chemical approaches have still leaved many open questions. ―.02. The current monitored in real time.――. it is prodent to use more than one type of oxidation progress of an emulsion system in real time antioxidant assay to measure antioxidant activities. tool for identifying better antioxidants for food particularly in emulsions and foams.2010. PharmTech Res. In analysis on these matrixes requires tedious sample vitro assays can only rank antioxidant activity for treatments.HAT ――― Autography technique CAA Asay ― ― +++ HAT +++ Dye-substrate + ++ ++ HAT + Oxidation method CUPRAC +++ +++ ――― HAT + Fluorometric ++ ++ + HAT + analysis ECL ――― +++ +++ HAT +++ ABTS + + + HAT + +.J. to include at least one assay that has biological The capacity of antioxidants in emulsion can thus be relevance. No Name of the Method III Other Assays 1) Total oxidant scavenging capacity (TOSC) 2) Inhibition of Briggs – Rauscher oscillation reaction 3) Chemiluminescence 4) Electrochemiluminescence 5) Fluorometric Analysis 6) Enhanced chemiluminescence (ECL) 7) TLC bioautography 8) Cellular antioxidant activity (CAA) assay 9) Dye-substrate oxidation method Table No:. high through out assay that can be used to monitor Therefore. +++ = Desirable To Highly Desirable Characteristic.01. Currently there is no convenient assay to quantified and ranked.Badarinath et al /Int. Our aim is to develop a in vivo health protective activities is uncertain.Table No:.V. and taking advantage of oxygen sensor coated micro-plate.

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