A Review on in-vitro Antioxidant Methods Comparisions Correlations and Considerations

Saudi Pharm J. 2013 Apr; 21(2): 143–152.

Review on in vivo and in vitro methods evaluation of antioxidant activity

Received 2012 Mar 14; Accepted 2012 May ane.

Abstract

A good number of abstracts and research articles (in total 74) published, and then far, for evaluating antioxidant activity of diverse samples of research interest were gone through where 407 methods were come up beyond, which were repeated from 29 different methods. These were classified every bit in vitro and in vivo methods. And those are described and discussed beneath in this review commodity. In the later part of this review article, frequency of in vitro as well as in vivo methods is analyzed with a bar diagram. Solvents are important for extracting antioxidants from natural sources. Frequency of solvents used for extraction is also portrayed and the results are discussed in this article. As per this review there are 19 in vitro methods and ten in vivo methods that are being used for the evaluation of antioxidant activity of the sample of interest. DPPH method was plant to be used more often than not for the in vitro antioxidant activity evaluation purpose while LPO was found as mostly used in vivo antioxidant analysis. Ethanol was with the highest frequency equally solvent for extraction purpose.

Keywords: Antioxidant, In vivo methods, In vitro methods

ane. Introduction

The human body has a complex organization of natural enzymatic and non-enzymatic antioxidant defenses which annul the harmful furnishings of complimentary radicals and other oxidants. Free radicals are responsible for causing a large number of diseases including cancer (Kinnula and Crapo, 2004), cardiovascular affliction (Singh and Jialal, 2006), neural disorders (Sas et al., 2007), Alzheimer's disease (Smith et al., 2000), mild cerebral impairment (Guidi et al., 2006), Parkinson's disease (Bolton et al., 2000), booze induced liver affliction (Arteel, 2003), ulcerative colitis (Ramakrishna et al., 1997), crumbling (Hyun et al., 2006) and atherosclerosis (Upston et al., 2003). Protection against free radicals can exist enhanced past ample intake of dietary antioxidants. Substantial bear witness indicates that foods containing antioxidants and possibly in detail the antioxidant nutrients may be of major importance in disease prevention. At that place is, however, a growing consensus amid scientists that a combination of antioxidants, rather than unmarried entities, may exist more constructive over the long term. Antioxidants may be of nifty do good in improving the quality of life past preventing or postponing the onset of degenerative diseases. In add-on, they accept a potential for substantial savings in the cost of wellness care delivery.

Diverse methods are used to investigate the antioxidant holding of samples (diets, plant extracts, commercial antioxidants etc.). The objective of this review commodity is to accumulate all probable methods that are used to evaluate the antioxidant holding of various samples. A compiled description of all available in vitro and in vivo antioxidant models would provide prolific advantages to the researchers of this loonshit by reducing their time for literature review and method development. Two review articles have been published before (Chanda and Dave, 2009 and Badarinath et al., 2010) on in vitro evaluation of antioxidant activity. In this article, attempts have been taken to include in vivo as well and to analyze the frequency of the utilize of different methods.

2. Methods

Internet browsing from Google Scholar database was used to identify and to download abstracts and research papers related to antioxidant activity study using suitable keywords (antioxidant + plant extract +in vitro +in vivo) in the calendar month of August 2011. In the first thirty-4 pages, a total of three hundred and forty articles appeared and those were subjected to preliminary screening. The basis of the selection of the manufactures was (i) antioxidant activity of plant extracts and (ii) description of antioxidant test procedures. A full of seventy-iv papers and abstracts were identified and reviewed for in vivo and in vitro methods related to antioxidant evaluation. Solvents used for extraction purpose are also reviewed from the downloaded scientific records.

iii.In vitro methods

Antioxidant activity should not be concluded based on a single antioxidant exam model. And in practice several in vitro test procedures are carried out for evaluating antioxidant activities with the samples of interest. Another attribute is that antioxidant test models vary in different respects. Therefore, it is difficult to compare fully one method to other one. To some extent comparison amidst dissimilar in vitro methods has been done by Badarinath et al. (2010), while we discussed the methods in terms of grouping in the present manuscript. Researcher has to critically verify methods of analysis before adopting that one for his/her enquiry purpose. Generally in vitro antioxidant tests using free radical traps are relatively straightforward to perform. Among free radical scavenging methods, DPPH method is furthermore rapid, simple (i.e. not involved with many steps and reagents) and cheap in comparison to other test models. On the other paw ABTS decolorization assay is applicable for both hydrophilic and lipophilic antioxidants. In this article all in vitro methods are described and it is important to notation that 1 may optimize logically the respective method to serve his/her experimental objective as no one method is absolute in nature rather than an example.

3.1. DPPH scavenging action

The molecule i, 1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) is characterized every bit a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, and so that the molecule does not dimerize, as would be the example with almost other free radicals. The delocalization of electron as well gives rise to the deep violet color, characterized by an absorption band in ethanol solution centered at about 517 nm. When a solution of DPPH is mixed with that of a substrate (AH) that can donate a hydrogen atom, then this gives ascent to the reduced course with the loss of this violet color.

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In order to evaluate the antioxidant potential through free radical scavenging by the test samples, the modify in optical density of DPPH radicals is monitored. According to Manzocco et al., 1998 the sample excerpt (0.2 mL) is diluted with methanol and two mL of DPPH solution (0.5 mM) is added. Afterward 30 min, the absorbance is measured at 517 nm. The percentage of the DPPH radical scavenging is calculated using the equation every bit given beneath:

% inhibition of DPPH radical = ([A _br -A _ar]/A _br) × 100

where A _br is the absorbance before reaction and A _ar is the absorbance afterward reaction has taken identify.

three.2. Hydrogen peroxide scavenging (H_iiO_two) assay

Human beings are exposed to H_twoO₂ indirectly via the surround well-nigh almost 0.28 mg/kg/solar day with intake mostly from foliage crops. Hydrogen peroxide may enter into the man body through inhalation of vapor or mist and through eye or skin contact. H₂O₂ is rapidly decomposed into oxygen and water and this may produce hydroxyl radicals (OH^•) that can initiate lipid peroxidation and crusade Deoxyribonucleic acid damage in the torso.

The ability of plant extracts to scavenge hydrogen peroxide can exist estimated according to the method of Ruch et al. (1989). A solution of hydrogen peroxide (40 mM) is prepared in phosphate buffer (l mM pH 7.4). The concentration of hydrogen peroxide is determined by absorption at 230 nm using a spectrophotometer. Extract (20–60 μg/mL) in distilled water is added to hydrogen peroxide and absorbance at 230 nm is determined after x min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage of hydrogen peroxide scavenging is calculated equally follows:

% scavenged (H₂O₂) = [(A _i -A _t)/A _i] × 100

where A _i is the absorbance of command and A _t is the absorbance of test.

iii.3. Nitric oxide scavenging activeness

NO^• is generated in biological tissues by specific nitric oxide synthases, which metabolizes arginine to citrulline with the formation of NO^• via a 5 electron oxidative reaction (David, 1999; Ghafourifar and Cadenas, 2005; Marletta, 1989; Moncada et al., 1989; and Virginia et al., 2003). The compound sodium nitroprusside is known to decompose in aqueous solution at physiological pH (seven.2) producing NO^•. Under aerobic conditions, NO^• reacts with oxygen to produce stable products (nitrate and nitrite), the quantities of which can be adamant using Griess reagent (Marcocci et al., 1994). Two (2) mL of 10 mM sodium nitroprusside dissolved in 0.5 mL phosphate buffer saline (pH vii.4) is mixed with 0.five mL of sample at diverse concentrations (0.2–0.8 mg/mL). The mixture is so incubated at 25 °C. Later on 150 min of incubation, 0.5 mL of the incubated solution is withdrawn and mixed with 0.5 mL of Griess reagent [(1.0 mL sulfanilic acrid reagent (0.33% in 20% glacial acetic acrid at room temperature for 5 min with one mL of naphthylethylenediamine dichloride (0.1% west/v)]. The mixture is then incubated at room temperature for xxx min and its absorbance pouring into a cuvette is measured at 546 nm. The amount of nitric oxide radical inhibition is calculated following this equation:

% inhibition of NO radical = [A ₀ -A ₁]/A ₀ × 100

where A ₀ is the absorbance before reaction and A ₁ is the absorbance after reaction has taken place with Griess reagent.

three.4. Peroxynitrite radical scavenging activity

Peroxynitrite (ONOO^•) is a cytotoxicant with strong oxidizing backdrop toward various cellular constituents, including sulfhydryls, lipids, amino acids and nucleotides and can cause cell death, lipid peroxidation, carcinogenesis and aging. It is generated in vivo by endothelial cells, Kupffer cells, neutrophils and macrophages. Peroxynitrite radical is a relatively stable species compared with other free radicals but once protonated gives highly reactive peroxynitrous acid (ONOOH), decomposing with a very brusk one-half-life (1.9 s) at 37 °C to form various cytotoxicants and that can induce the oxidation of thiol (−SH) groups on proteins, nitration of tyrosine, lipid peroxidation and too nitrosation reactions, affecting jail cell metabolism and signal transduction. It can ultimately contribute to cellular and tissue injury with Dna strand breakage and apoptotic cell death, e.g. in thymocytes, cortical cells and HL-60 leukemia cells. Its excessive germination may likewise be involved in several homo diseases such as Alzheimer's disease, rheumatoid arthritis, cancer and atherosclerosis. Due to the lack of endogenous enzymes responsible for ONOO^• inactivation, developing specific ONOO^• scavengers is of considerable importance. The method described by Kooy et al., 1994 involves the use of a stock solution of dihydroxyrhodamine 123 (DHR 123, 5 mM) in dimethylformamide that is purged with nitrogen and stored at −80 °C. Working solution with DHR 123 (terminal concentration 5 μM) is diluted from the stock solution and is placed on water ice in the dark immediately prior to the experiment. Buffer solution, 50 mM sodium phosphate (pH vii.4), containing xc mM sodium chloride and v mM potassium chloride with 100 μM diethylenetriaminepentaacetic acrid (DTPA) are purged with nitrogen and placed on water ice before utilise. Scavenging action of ONOO^• past the oxidation of DHR 123 is measured on a microplate fluorescence spectrophotometer with excitation and emission wavelengths of 485 nm and 530 nm at room temperature, respectively. The background and final fluorescent intensities are measured five min after treatment without 3-morpholino- sydnonimine (SIN-1) or accurate (ONOO^•). Oxidation of DHR 123 by decomposition of SIN-1 gradually increased whereas authentic ONOO^• rapidly oxidized DHR 123 with its final fluorescent intensity being stable over time.

3.v. Trolox equivalent antioxidant capacity (TEAC) method/ABTS radical cation decolorization assay

This method, uses a diode-array spectrophotometer to measure the loss of color when an antioxidant is added to the blueish–greenish chromophore ABTS· + (2,two-azino-bis(iii-ethylbenzthiazoline-vi-sulfonic acid)). The antioxidant reduces ABTS·+ to ABTS and decolorize it. ABTS·+ is a stable radical not found in the human torso. Antioxidant action can be measured equally described past Seeram et al. (2006). ABTS radical cations are prepared past adding solid manganese dioxide (80 mg) to a v mM aqueous stock solution of ABTS (20 mL using a 75 mM Na/G buffer of pH 7). Trolox (six-hydroxy-2,v,7,viii-tetramethylchroman-2-carboxylic acid), a water-soluble analog of vitamin Eastward, can be used as an antioxidant standard. A standard calibration curve is synthetic for Trolox at 0, fifty, 100, 150, 200, 250, 300, and 350 μM concentrations. Samples are diluted appropriately co-ordinate to antioxidant activity in Na/K buffer pH, 7. Diluted samples are mixed with 200 μL of ABTS^•+ radical cation solution in 96-well plates, and absorbance is read (at 750 nm) subsequently 5 min in a microplate reader. TEAC values tin exist calculated from the Trolox standard curve and expressed every bit Trolox equivalents (in mM).

3.6. Total radical-trapping antioxidant parameter (TRAP) method

This method is based on the protection provided by antioxidants on the fluorescence decay of R-phycoerythrin (R-PE) during a controlled peroxidation reaction. The fluorescence of R-Phycoerythrin is quenched by ABAP (2,2′-azo–bis(2-amidino-propane)hydrochloride) as a radical generator. This quenching reaction is measured in the presence of antioxidants. The antioxidative potential is evaluated past measuring the decay in decoloration. According to Ghiselli et al. (1995) 120 μL of diluted sample is added to 2.iv mL of phosphate buffer (pH 7.4), 375 μL of bidistilled water, 30 μL of diluted R-PE and 75 μL of ABAP; the reaction kinetics at 38 °C is recorded for 45 min by a luminescence spectrometer. TRAP values are calculated from the length of the lag-phase due to the sample compared with standard.

3.vii. Ferric reducing-antioxidant ability (FRAP) analysis

This method measures the power of antioxidants to reduce ferric fe. Information technology is based on the reduction of the complex of ferric fe and 2,3,five-triphenyl-i,3,4-triaza-2-azoniacyclopenta-one,4-diene chloride (TPTZ) to the ferrous form at low pH. This reduction is monitored by measuring the change in assimilation at 593 nm, using a diode-assortment spectrophotometer. Antioxidant analysis can be conducted by the method adult by Benzie and Strain (1999). Iii milliliter of prepared FRAP reagent is mixed with 100 μL of diluted sample; the absorbance at 593 nm is recorded after a 30 min incubation at 37 °C. FRAP values can exist obtained past comparison the absorption change in the test mixture with those obtained from increasing concentrations of Fe3+ and expressed as mM of Fe2+ equivalents per kg (solid food) or per L (beverages) of sample.

3.8. Superoxide radical scavenging activity (SOD)

Although superoxide anion is a weak oxidant, information technology ultimately produces powerful and dangerous hydroxyl radicals besides as singlet oxygen, both of which contribute to oxidative stress (Meyer and Isaksen, 1995). The superoxide anion scavenging activeness can be measured as described by Robak and Gryglewski (1988). The superoxide anion radicals are generated in 3.0 mL of Tris–HCl buffer (16 mM, pH 8.0), containing 0.5 mL of nitroblue tetrazolium (NBT) (0.3 mM), 0.5 mL NADH (0.936 mM) solution, 1.0 mL extract and 0.5 mL Tris–HCl buffer (16 mM, pH 8.0). The reaction is initiated past calculation 0.v mL phenazine methosulfate (PMS) solution (0.12 mM) to the mixture, incubated at 25 °C for v min and then the absorbance is measured at 560 nm confronting a bare sample.

3.ix. Hydroxyl radical scavenging activity

Hydroxyl radical is one of the strong reactive oxygen species in the biological system that reacts with polyunsaturated fat acid moieties of jail cell membrane phospholipids and causes harm to cell. The scavenging power of hydroxyl radicals is measured past the method of Kunchandy and Rao (1990). The reaction mixture (1.0 mL) consist of 100 μL of ii-deoxy-Dribose (28 mM in twenty mM KH₂PO_four-KOH buffer, pH 7.4), 500 μL of the extract, 200 μL EDTA (1.04 mM) and 200 μM FeCl_three (i:ane v/v), 100 μL of H₂O₂ (1.0 mM) and 100 μL ascorbic acrid (one.0 mM) which is incubated at 37 °C for 1 h. 1 milliliter of thiobarbituric acid (ane%) and 1.0 mL of trichloroacetic acid (2.8%) are added and incubated at 100 °C for 20 min. After cooling, absorbance is measured at 532 nm, confronting a blank sample.

3.x. Hydroxyl radical averting capacity (HORAC) method

The HORAC analysis described by Ou et al. (2002) measures the metallic-chelating action of antioxidants in the weather condition of Fenton-similar reactions employing a Co (II) circuitous and hence the protecting power confronting formation of hydroxyl radical. Hydrogen peroxide solution of 0.55 M is prepared in distilled water and 4.six mM Co (Ii) is prepared past dissolving 15.7 mg of CoF₂·4H₂O and 20 mg of picolinic acid in 20 mL of distilled water. Fluorescein – 170 μL (threescore nM, final concentration) and ten μL of sample are incubated at 37 °C for 10 min. directly in the plate reader. After incubation 10 μL H₂O₂ (27.5 mM, terminal concentration) and 10 μL of Co (2) (230 μM concluding concentration) solutions are added subsequently. The initial fluorescence is measured afterwards which the readings are taken every min. after shaking. For the bare sample, phosphate buffer solution is used. 100, 200, 600, 800 and 1000 μM standard antioxidant solutions (in phosphate buffer 75 mM, pH seven.4) are used for building the standard curve. The final HORAC values are calculated using a regression equation between the standard antioxidant concentration and the net area under the curve. One HORAC unit is assigned to the cyberspace protection area provided by i μM standard antioxidant and the activity of the sample is expressed as μM standard antioxidant equivalents per gram of fresh weight of the samples. Gallic acid can be used equally standard antioxidant.

3.11. Oxygen radical absorbance chapters (ORAC) Method

ORAC is an exciting and revolutionary new test tube analysis that can be utilized to test "Antioxidant Power" of foods and other chemic substances. This test tin can exist washed using either β-phycoerythrin (β-PE) or fluorescein as target molecule. While reviewing, β-PE is not encountered in the publications later than 2005. Thus it seems fluorescein is replacing the β-PE as target molecule in ORAC assay. The test is performed using Trolox (a water-soluble analog of Vitamin E) equally a standard to determine the Trolox Equivalent (TE). The ORAC value is then calculated from the Trolox Equivalent and expressed equally ORAC units or value. The college the ORAC value, the greater the "Antioxidant Power".

This assay is based on generation of gratuitous radical using AAPH (2,two-azobis two-amidopropane dihydrochloride) and measurement of decrease in fluorescence in the presence of free radical scavengers. Prior et al. (2003) accept reported an automated ORAC assay. In this assay β-phycoerythrin (β-PE) was used as target free radical impairment, AAPH every bit a peroxy radical generator and Trolox as a standard control. Later addition of AAPH to the examination solution, the fluorescence is recorded and the antioxidant activity is expressed every bit trolox equivalent (Cao et al., 1993; Frei et al., 1990).

The analysis can be carried out co-ordinate to Prior et al. (2003) in 96-well polypropylene fluorescence plates with a final volume of 200 μL. Assays are conducted at pH 7.0 with Trolox (6.25, 12.5, 25, and 50 μmol/50 for lipophilic assays; 12.5, 25, fifty and 100 μmol/L hydrophilic assays) as the standard and 75 mM/50 phosphate buffer as the blank. After the add-on of AAPH, the plate is placed immediately in a multilabel counter preheated to 37 °C. The plate is shaken in an orbital manner for 10 south and the fluorescence is read at 1 min intervals for 35 min at the excitation wavelength of 485 nm and emission wavelength of 520 nm. Surface area-under-the-curve is calculated for each sample using Wallac Conditioning 1.5 software. Concluding ciphering of results is fabricated by taking the difference of areas-under-the-decay curves between blank and sample and/or standard (Trolox) and expressing this in μM of Trolox equivalents (TE) per g dry out weight of sample (μM TE/g).

iii.12. Reducing power method (RP)

This method is based on the principle of increase in the absorbance of the reaction mixtures. Increase in the absorbance indicates an increase in the antioxidant activity. In this method, antioxidant compound forms a colored complex with potassium ferricyanide, trichloro acerb acid and ferric chloride, which is measured at 700 nm. Increase in absorbance of the reaction mixture indicates the reducing power of the samples (Jayaprakash et al., 2001). In the method described past Oyaizu (1986) 2.5 mL of 0.2 K phosphate buffer (pH six.6) and 2.5 mL of Grand₃Fe (CN)_half-dozen (1% due west/5) are added to i.0 mL of sample dissolved in distilled water. The resulting mixture is incubated at 50 °C for 20 min, followed past the addition of 2.5 mL of Trichloro acerb acid (x% w/5). The mixture is centrifuged at 3000 rpm for ten min to collect the upper layer of the solution (2.v mL), mixed with distilled water (2.5 mL) and 0.5 mL of FeCl₃ (0.1%, w/five). The absorbance is then measured at 700 nm against blank sample.

3.13. Phosphomolybdenum method

Total antioxidant capacity analysis is a spectroscopic method for the quantitative conclusion of antioxidant capacity, through the formation of phosphomolybdenum complex. The assay is based on the reduction of Mo (Half dozen) to Mo (V) past the sample analyte and subsequent formation of a light-green phosphate Mo (V) circuitous at acidic pH. Total antioxidant chapters can be calculated past the method described by Prieto et al. (1999). 0.1 mL of sample (100 μg) solution is combined with 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tube is capped and incubated in a boiling water bath at 95 °C for 90 min. Later on cooling the sample to room temperature, the absorbance of the aqueous solution is measured at 695 nm against bare in UV spectrophotometer. A typical blank solution contained 1 mL of reagent solution and the appropriate volume of the aforementioned solvent used for the sample and information technology is incubated under same atmospheric condition as residual of the sample. For samples of unknown limerick, antioxidant capacity tin exist expressed as equivalents of α-tocopherol.

3.fourteen. Ferric thiocyanate (FTC) method

This method tin be exploited to determine the antioxidant activity equally illustrated by Kikuzaki et al. (1991). A mixture of iv mg of sample (final concentration of 0.02% w/v) in 4 mL ethanol, four.i mL of two.51% linoleic acid in ethanol, 8.0 mL of 0.02 Grand phosphate buffer (pH 7.0) and 3.nine mL of distilled h2o contained in spiral cap vial is placed in an oven at forty °C in the nighttime. 0.1 mL of the reaction mixture is transferred to a exam tube and, to it; 9.7 mL of 75% (v/v) aqueous ethanol, followed by 0.1 mL of 30% aqueous ammonium thiocyanate and 0.1 mL of 0.02 Thou ferrous chloride in three.5% hydrochloric acid are added. Three minutes after the improver of ferrous chloride to the reaction mixture, the absorbance of the resulting mixture (red color) is measured at 500 nm every 24 h until the absorbance of the command reached its maximum. Standard antioxidant (last concentration of 0.02% w/v) is used as positive control, and the mixture without the sample is used as the negative control.

3.15. Thiobarbituric acid (TBA) method

TBA method, described by Ottolenghi (1959) is as follows: The concluding sample concentration of 0.02% w/v was used in this method. 2 mL of 20% trichloroacetic acid and 2 mL of 0.67% of thiobarbituric acid were added to 1 mL of sample solution. The mixture was placed in a boiling water bath for 10 min and then centrifuged afterward cooling at 3000 rpm for twenty min. The absorbance activity of the supernatant was measured at 552 nm and recorded after it has reached its maximum.

three.xvi. DMPD (North,Northward-dimethyl-p-phenylene diamine dihydrochloride) method

DMPD radical cation decolorization method has been adult for the measurement of the antioxidant activeness in food and biological samples. This analysis is based on the reduction of buffered solution of colored DMPD in acetate buffer and ferric chloride. The procedure involves measurement of decrease in absorbance of DMPD in the presence of scavengers at its absorption maximum of 505 nm. The activeness was expressed every bit percentage reduction of DMPD.

Fogliano et al. (1999) obtained the radical by mixing one mL of DMPD solution (200 mM), 0.4 mL of ferric chloride (III) (0.05 Chiliad), and 100 mL of sodium acetate buffer solution at 0.i M, modifying the pH to 5.25. The reactive mixture has to be kept in darkness, under refrigeration, and at a low temperature (four–5 °C) .The reaction takes identify when l μL of the sample (a dilution of 1:10 in water) is added to 950 μL of the DMPD·+ solution. Absorbance is measured after 10 min of continuous stirring, which is the time taken to attain constant decolorization values. The results are quantified in mM Trolox on the relevant calibration curve.

iii.17. β-carotene linoleic acrid method/conjugated diene assay

This is one of the rapid method to screen antioxidants, which is mainly based on the principle that linoleic acid, which is an unsaturated fatty acid, gets oxidized by "Reactive Oxygen Species" (ROS) produced by oxygenated h2o. The products formed volition initiate the β-carotene oxidation, which volition lead to discoloration. Antioxidants subtract the extent of discoloration, which is measured at 434 nm and the activity is measured.

The method as described by Kabouche et al. (2007): β-carotene (0.5 mg) in 1 mL of chloroform is added to 25 μL of linoleic acid and 200 mg of tween-80 emulsified mixture. Chloroform is evaporated at twoscore °C, 100 mL of distilled water saturated with oxygen is slowly added to the rest and the solution is vigorously agitated to course a stable emulsion. Four mL of this mixture is added into the test tubes containing 200 μL of sample prepared in methanol at final concentrations (25, 50, 100, 200 and 400 μg/mL). Equally soon as the emulsified solution is added to the tubes, cipher time absorbance is measured at 470 nm. The tubes are incubated for ii h at 50 °C. Vitamin C can be used equally standard. Antioxidant activity is calculated as percentage of inhibition (I%) relative to the control using the following equation:

I% = [1 - (As - As120)/Air-conditioning - Ac120)]

where As was initial absorbance, As120 was the absorbance of the sample at 120 min, Ac was initial absorbance of negative control and Ac120 was the absorbance of the negative control at 120 min.

3.18. Xanthine oxidase method

The xanthine oxidase activity with xanthine as sub-substrate can be measured spectrophotometrically, by the method of Noro et al. (1983). The extract (500 μL of 0.1 mg/mL) and allopurinol (100 μg/mL) (in methanol) are mixed with 1.3 mL phosphate buffer (0.05 K, pH 7.5) and 0.2 mL of 0.ii units/mL xanthine oxidase solution. After 10 min of incubation at room temperature (25 °C), i.5 mL of 0.xv M xanthine substrate solution is added to this mixture. The mixture is again incubated for thirty min at room temperature (25 °C) and and so the absorbance is measured at 293 nm using a spectrophotometer confronting blank (0.5 mL methanol, 1.3 mL phosphate buffer, 0.two mL xanthine oxidase). The solution of 0.5 mL methanol, 1.3 mL phosphate buffer, 0.two mL xanthine oxidase and 1.5 mL xanthine substrate is used as a control. Percent of inhibition is calculated using the formula:

Percentage of inhibition = [1 - (As/Ac)] × 100

where As and Ac are the absorbance values of the examination sample and control, respectively.

three.nineteen. Cupric ion reducing antioxidant capacity (CUPRAC) method

The chromogenic oxidizing reagent of the developed CUPRAC method, that is, bis(neocuproine)copper(II) chloride [Cu(Ii)-Nc], reacts with polyphenols [Ar(OH)n] in the manner.

$ii n Cu {(Nc)}_{2}^{two +} + Ar (OH) n = two n Cu {(Nc)}^{ii +} + Ar (= O) n + ii n H^{+}$

where the liberated protons may be buffered with the relatively concentrated ammonium acetate buffer solution. In this reaction, the reactive Ar-OH groups of polyphenols are oxidized to the respective quinones and Cu (Ii)-Nc is reduced to the highly colored Cu (I)-Nc chelate showing maximum absorption at 450 nm.

According to Apak et al. (2008), ane mL of 10⁻² M of CuCl₂, 1 mL of 7.5 × 10⁻³ M neocuproine and 1 One thousand NH_ivCH_threeCOO solution are added into the glass test tube. And then, 400 μL of freshly prepared standard solution is added and diluted to the terminal volume of 4.i mL with deionized water. This procedure is repeated for 400 μL, 300 μL, 200 μL, 100 μL and 50 μL additions of freshly prepared solutions of the sample. The prepared solutions are mixed and incubated at room temperature for xxx min. The absorbance at 450 nm is adamant against a reagent blank past spectrometer. The calculation of antioxidant chapters of compounds as Trolox equivalents (TEAC values) by the CUPRAC method has been reported.

three.twenty. Metallic chelating activity

Ferrozine tin course a circuitous with a red colour by forming chelates with Feⁱⁱ⁺. This reaction is restricted in the presence of other chelating agents and results in a subtract of the crimson color of the ferrozine-Feⁱⁱ⁺ complexes. Measurement of the color reduction determines the chelating activity to compete with ferrozine for the ferrous ions (Soler-Rivas et al., 2000). The chelation of ferrous ions is estimated using the method of Dinis et al. (1994). 0.1 mL of the extract is added to a solution of 0.v mL ferrous chloride (0.two mM). The reaction is started by the add-on of 0.2 mL of ferrozine (5 mM) and incubated at room temperature for 10 min and then the absorbance is measured at 562 nm. EDTA or citric acid (Dinis et al., 1994) tin be used as a positive control.

iv.In vivo models

For all in vivo methods the samples that are to be tested are usually administered to the testing animals (mice, rats, etc.) at a definite dosage regimen every bit described by the respective method.

After a specified period of time, the animals are usually sacrificed and blood or tissues are used for the assay.

four.1. Ferric reducing ability of plasma

It is one of the most rapid test and very useful for routine assay (Umesh et al., 2010). The antioxidative activity is estimated by measuring the increase in absorbance caused past the germination of ferrous ions from FRAP reagent containing TPTZ (ii,four,6-tripyridyl-s-triazine) and FeCl_two·6H₂O. The absorbance is measured spectrophotometrically at 593 nm.

The method illustrated by Benzie and Strain (1996) involves the use of claret samples that are collected from the rat retro-orbital venous plexus into heparinized glass tubes at 0, 7 and 14 days of treatment. Three mL of freshly prepared and warm (37 °C) FRAP reagent [1 mL (10 mM) of 2,iv,6 tripyridyl-s-triazine (TPTZ) solution in xl mM HCl, 1 mL xx mM FeCl₂·6H₂O, 10 mL of 0.3 G acetate buffer (pH 3.half-dozen)] is mixed with 0.375 mL distilled water and 0.025 mL of test samples. The absorbance of developed color in organic layer is measured at 593 nm. The temperature is maintained at 37 °C. The readings at 180 s are selected for the calculation of FRAP values.

4.2. Reduced glutathione (GSH) estimation

GSH is an intra-cellular reductant and plays major part in catalysis, metabolism and transport. It protects cells confronting free radicals, peroxides and other toxic compounds (Sapakal et al., 2008) Deficiency of GSH in the lens leads to cataract formation. Glutathione besides plays an important role in the kidney and takes part in a transport system involved in the reabsorption of amino acids. The method illustrated by Ellman (1959) can be used for determination of antioxidant activity. The tissue homogenate (in 0.1 K phosphate buffer pH 7.4) is taken and added with equal volume of 20% trichloroacetic acid (TCA) containing i mM EDTA to precipitate the tissue proteins. The mixture is allowed to stand for five min prior to centrifugation for ten min at 2000 rpm. The supernatant (200 μL) is so transferred to a new set of examination tubes and added with ane.viii mL of the Ellman's reagent (5,5′-dithiobis-2-nitrobenzoic acid (0.1 mM) prepared in 0.3 Yard phosphate buffer with ane% of sodium citrate solution). So all the exam tubes are made up to the volume of two mL. After completion of the full reaction, solutions are measured at 412 nm against blank. Absorbance values were compared with a standard curve generated from known GSH.

4.3. Glutathione peroxidase (GSHPx) interpretation

GSHP_X is a seleno-enzyme two third of which (in liver) is nowadays in the cytosol and one third in the mitochondria. It catalyzes the reaction of hydroperoxides with reduced glutathione to grade glutathione disulfide (GSSG) and the reduction product of hydroperoxide. GSHPx is found throughout the tissues, being present equally four different isoenzymes, cellular glutathione peroxidase, extracellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase and gastrointestinal glutathione peroxidase. GSHPx measurement is considered in detail with patients who are under oxidative stress for whatever reason; low activity of this enzyme is ane of the early on consequences of a disturbance of the prooxidant/antioxidant balance (Paglia and Valentin, 1967; Yang et al., 1984).

According to Wood (1970), Cytosolic GPx is assayed via a three-mL cuvette containing 2.0 mL of 75 mM/Fifty phosphate buffer, pH 7.0. The following solutions are so added: 50 μL of threescore mM/Fifty glutathione reductase solution (30 U/mL), fifty μL of 0.12 M/L NaN_three, 0.x of 0.15 mM/L Na₂EDTA, 100 μL of three.0 mM/L NADPH, and 100 μL of cytosolic fraction obtained after centrifugation at 20,000 g for 25 min. Water is added to make a total volume of 2.9 mL. The reaction is started by the addition of 100 μL of vii.five mM/Fifty H₂O₂, and the conversion of NADPH to NADP is monitored past a continuous recording of the change of absorbance at 340 nm at 1 min interval for 5 min. Enzyme activity of GSHPx was expressed in terms of mg of proteins.

four.4. Glutathione-Southward-transferase (GSt)

Glutathione-S-transferase is thought to play a physiological role in initiating the detoxication of potential alkylating agents, including pharmacologically agile compounds. These enzymes catalyze the reaction of such compounds with the -SH group of glutathione, thereby neutralizing their electrophilic sites and rendering the products more than water-soluble. The method tin can be used as described by Jocelyn (1972). The reaction mixture (1 mL) consisted of 0.1 N potassium phosphate (pH vi.5), ane nM/L GSt, 1 Yard/Fifty 50-chloro-ii, 4-dinitrobenzene equally substrate and a suitable amount of cytosol (half-dozen mg protein/mL). The reaction mixture is incubated at 37 °C for 5 min and the reaction is initiated by the addition of the substrate. The increase in absorbance at 340 nm was measured spectrophotometrically.

4.5. Superoxide dismutase (SOD) method

This method is well described by Mccord and Fridovich (1969) and tin can be applied for determination of antioxidant activeness of a sample. It is estimated in the erythrocyte lysate prepared from the v% RBC suspension. To 50 μL of the lysate, 75 mM of Tris–HCl buffer (pH eight.2), 30 mM EDTA and two mM of pyrogallol are added. An increase in absorbance is recorded at 420 nm for iii min past spectrophotometer. I unit of enzyme activity is 50% inhibition of the rate of autooxidation of pyrogallol as determined by change in absorbance/min at 420 nm. The activity of SOD is expressed every bit units/mg protein.

iv.vi. Catalase (CAT)

Catalase activity tin can exist determined in erythrocyte lysate using Aebi's method (Aebi, 1984). Fifty microliter of the lysate is added to a cuvette containing 2 mL of phosphate buffer (pH seven.0) and one mL of 30 mM H_twoO₂. Catalase activeness is measured at 240 nm for 1 min using spectrophotometer. The tooth extinction coefficient of H₂O₂, 43.6 M cm⁻¹ was used to decide the catalase activity. I unit of activity is equal to 1 mmol of H_twoO_ii degraded per minute and is expressed as units per milligram of protein.

four.7. γ-Glutamyl transpeptidase activeness (GGT) assay

According to Singhal et al. (1982), the serum sample is added to a substrate solution containing glycylglycine, MgCl₂ and thou-Glutamyl-p-nitroanilide in 0.05 M tris (free base), pH eight.2. The mixture is incubated at 37 °C for 1 min and the absorbance read at 405 nm at i m interval for 5 yard. The activity of GGT is calculated from the absorbance values.

4.8. Glutathione reductase (GR) assay

The ubiquitous tripeptide glutathione (GSH), which is the most abundant low molecular weight thiol in nigh all cells, is involved in a wide range of enzymatic reactions. A major function of GSH is to serve as a reductant in oxidation–reduction processes; a function resulting in the germination of glutathione disulfide (GSSG). A heat labile system capable of reducing GSSG was discovered in liver. The enzyme straight involved in reduction of GSSG.

The method illustrated past Kakkar et al. (1984) is as follows: Livers (about 400 yard) are obtained from killed rats (200–250 one thousand). The livers are cut into pocket-sized pieces and homogenized in ix mL of 0.25 Chiliad water ice-cold sucrose per one thousand of rat liver in a blender. The homogenate is centrifuged for 45 min at xiv,000 rpm. The pellets are suspended in a small volume of 0.25 G sucrose and centrifuged. The supernatants are combined with the previous centrifugate. The pooled material is adjusted to pH five.5 with cold 0.2 M acetic acid and centrifuged once again for 45 min at xiv,000 rpm. The rate of oxidation of NADPH by GSSG at 30 °C is used as a standard measure of enzymatic activity. The reaction system of 1 mL independent: ane.0 mM GSSG, 0.1 mM NADPH, 0.5 mM EDTA, 0.10 M sodium phosphate buffer (pH 7.6), and a suitable corporeality of the glutathione reductase sample to give a modify in absorbance of 0.05–0.03/min. The oxidation of 1 μM of NADPH/min under these conditions is used as a unit of measurement of glutathione reductase activity. The specific activity is expressed as units per mg of protein.

4.ix. Lipid peroxidation (LPO) assay

LPO is an autocatalytic procedure, which is a common consequence of prison cell death. This process may cause peroxidative tissue harm in inflammation, cancer and toxicity of xenobiotics and aging. Malondialdehyde (MDA) is 1 of the end products in the lipid peroxidation process. Malondialdehyde (MDA) is formed during oxidative degeneration as a product of free oxygen radicals, which is accepted as an indicator of lipid peroxidation.

This method described by Ohkawa et al. (1979) is as follows: The tissues are homogenized in 0.1 M buffer pH 7.4 with a Teflon-drinking glass homogenizer. LPO in this homogenate is determined by measuring the amounts of malondialdehyde (MDA) produced primarily. Tissue homogenate (0.2 mL), 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), i.5 mL of twenty% acetic acid and i.5 mL of 8% TBA are added. The volume of the mixture is fabricated up to four mL with distilled water and and so heated at 95 °C on a h2o bath for sixty min using glass assurance as condenser. Subsequently incubation the tubes are cooled to room temperature and final book was fabricated to five mL in each tube. Five mL of butanol: pyridine (15:1) mixture is added and the contents are vortexed thoroughly for 2 min. Later centrifugation at 3000 rpm for x min, the upper organic layer is taken and its OD is taken at 532 nm against an appropriate blank without the sample. The levels of lipid peroxides can exist expressed equally northward moles of thiobarbituric acid reactive substances (TBARS)/mg poly peptide using an extinction coefficient of i.56 × ten⁵ ML cm⁻¹.

4.10. LDL assay

The isolated LDL is washed and dialyzed against 150 mmol/L NaCl and 1 mmol/L Na_iiEDTA (pH 7.4) at iv °C. The LDL is then sterilized by filtration (0.45 μM), kept under nitrogen in the nighttime at four °C. LDL (100 μg of protein/mL) is incubated for 10 min at room temperature with samples. Then, five μmol/L of CuSO4 is added, and the tubes are incubated for 2 h at 37 °C. Cu²⁺-induced oxidation is terminated by the addition of butylated hydroxytoluene (BHT, 10 μM). At the finish of the incubation, the extent of LDL oxidation is determined by measuring the generated amount of lipid peroxides and also by the thiobarbituric acrid reactive substances (TBARS) analysis at 532 nm, using malondialdehyde (MDA) for the standard bend equally described past Buege and Aust, 1978; El-Saadani et al., 1989.

5. Results and give-and-take

In improver to compilation of diverse methods related to evaluation of antioxidant activity, it was our involvement to see the frequency of each method of a given number of citations beingness used. The results of the said frequency assay for in vitro and in vivo methods are shown in Figs. 1 and 2, respectively. It is clear from Fig. 1 that four in vitro methods were virtually frequently used and these were in order of decreasing frequency: DPPH > Hydroxyl radical scavenging > SOD > β-carotene linolate. Out of all the in vitro methods, DPPH is the most easy, simple and reasonably costly method and hence it might have been used generally for the antioxidant activity evaluation of a sample. If one looks into Fig. 2, it immediately appears that the frequency of use is higher for LPO assay and it was followed by CAT and GSHPx. Lipid is a major component of cell membrane and thus its peroxidation about straight co-relates peroxidative damage of cell in vivo and hence it might have been found to accept the highest frequency in vivo antioxidant action assay.

An external file that holds a picture, illustration, etc. Object name is gr1.jpg

Frequency of unremarkably used In vitro methods.

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Frequency of usually used In vivo methods.

Fig. 3 represents the frequency of utilise of solvent for the extraction of a material to evaluate its antioxidant property. Information technology is axiomatic from the figure that four solvents are prominently being used for the extraction purpose in relation to the stated experiment. These solvents are ethanol, water, methanol and aqueous ethanol. Ethanol, methanol and water have expert polarity and hence are used favorably to extract polar compounds such every bit phenolic compounds and flavonoids which are believed to exist effective antioxidants. Ethanol being organic and nontoxic might have the highest frequency of use for extraction purpose. H2o needs a dissimilar footstep of freeze drying to remove it from the extract after extraction. Toxicity of methanol limits its use in some extraction and subsequent experiment. Not-polar solvents such every bit ether and low polarity solvents such every bit chloroform, ester, acetone etc. have been used in specific cases and their availability as well limits their use in the experiment and hence their frequency of use was found very low.

An external file that holds a picture, illustration, etc. Object name is gr3.jpg

Commonly used extracting solvents for antioxidant written report.

vi. Conclusion

This review article is focused on in vitro and in vivo methods of antioxidant evaluation. It was prepared based on plenty literature search. Presently, 19 in vitro and 10 in vivo methods are being used for antioxidant evaluation purpose. DPPH method is the most oftentimes used one for in vitro antioxidant activity evaluation while LPO was found equally the by and large used in vivo antioxidant assay. Ethanol excerpt was found with the highest frequency for antioxidant study. This article will be a comprehensive set up reference for those who are interested on antioxidant report.

Footnotes

Peer review under responsibility of King Saud University

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References

Aebi H. Catalase. Methods Enzymol. 1984;105:121–126. [PubMed] [Google Scholar]
Apak R., Güçlü K., Özyürek Yard., Karademir S.E. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchim. Acta. 2008;160:413–419. [Google Scholar]
Arteel Thou.E. Oxidants and antioxidants in alcohol induced liver disease. Gastroenterol. 2003;124:778–790. [PubMed] [Google Scholar]
Badarinath A.V., RAo K.M., Chetty C.Chiliad.S., Ramkanth V., Rajan T.Five.S., Gnanaprakash Thousand. A review on in-vitro antioxidant methods: comparisons, correlations and considerations. Int. J. PharmTech Res. 2010;2(2):1276–1285. [Google Scholar]
Benzie I.F.F., Strain J.J. The ferric reducing ability of plasma (FRAP) equally a measure of 'antioxidant power': The FRAP analysis. Anal. Biochem. 1996;239:seventy–76. [PubMed] [Google Scholar]
Benzie I.F.F., Strain J.J. Ferric reducing antioxidant power assay: direct measure of total antioxidant action of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 1999;299:xv–27. [PubMed] [Google Scholar]
Bolton J.L., Trush M.A., Penning T.M., Dryhurst G., Monks T.J. Part of quinones in toxicology. Chem. Res. Toxicol. 2000;13:135–160. [PubMed] [Google Scholar]
Buege J.A., Aust S.D. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302–310. [PubMed] [Google Scholar]
Cao K., Alessio H.One thousand., Culter R.G. Oxygen radical absorbance capacity assay for antioxidant free radicals. Biol. Med. 1993;fourteen:303–311. [PubMed] [Google Scholar]
Chanda Southward., Dave R. In vitro models for antioxidant activeness evaluation and some medicinal plants possessing antioxidant properties: an overview. Afr. J. Microbiol. Res. 2009;3(thirteen):981–996. [Google Scholar]
David S.B. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Gratis Radic. Res. 1999;31(vi):577–596. [PubMed] [Google Scholar]
Dinis T.C.P., Madeira V.M.C., Almeida 50.Yard. Activity of phenolic derivatives (acetaminophen, salicylate and five-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxy radical scavengers. Curvation. Biochem. Biophy. 1994;315:161–169. [PubMed] [Google Scholar]
Ellman K.Fifty. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959;82:seventy–77. [PubMed] [Google Scholar]
El-Saadani M., Esterbauer H., El-Sayed M., Goher M., Nassar A.Y., Juergens 1000.A. Spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J. Lipid Res. 1989;xxx:627–630. [PubMed] [Google Scholar]
Fogliano V., Verde V., Randazzo G., Ritieni A. Method for measuring antioxidant activity and its awarding to monitoring antioxidant capacity of wines. J. Agric. Food Chem. 1999;47:1035–1040. [PubMed] [Google Scholar]
Frei B., Stocker R., England L., Ames B.N. Ascorbate the most effective antioxidant in human blood plasma. Adv. Med. Exp. Biol. 1990;264:155–163. [PubMed] [Google Scholar]
Ghafourifar P., Cadenas E. Mitochondrial nitric oxide synthase. Trends Pharmacol. Sci. 2005;26:190–195. [PubMed] [Google Scholar]
Ghiselli A., Serafini K., Maiani Chiliad., Azzini E., Ferro-Luzzi A. A fluorescence-based method for measuring total plasma antioxidant capability. Free Radic. Biol. Med. 1995;xviii:29–36. [PubMed] [Google Scholar]
Guidi I., Galimberti D., Lonati S., Novembrino C., Bamonti F., Tiriticco M., Fenoglio C., Venturelli Eastward., Baron P., Bresolin N. Oxidative imbalance in patients with mild cognitive harm and Alzheimer'south disease. Neurobiol. Crumbling. 2006;27:262–269. [PubMed] [Google Scholar]
Hyun D.H., Hernandez J.O., Mattson M.P., de Cabo R. The plasma membrane redox system in aging. Aging Res. Rev. 2006;5:209–220. [PubMed] [Google Scholar]
Jayaprakash Thousand.K., Singh R.P., Sakariah Thousand.K. Antioxidant activity of grape seed extracts on peroxidation models in-vitro. J. Agric. Food Chem. 2001;55:1018–1022. [Google Scholar]
Jocelyn P.C. Academic Press; London: 1972. Biochemistry of the SH Group. pp. 10. [Google Scholar]
Kakkar P., Das B., Viswanathan P.North. A modified spectrophotometric assay of superoxide dismutase. Ind. J. Biochem. Biophys. 1984;21:131–132. [PubMed] [Google Scholar]
Kabouche A., Kabouche Z., Ôzturk 1000., Kolal U., Topçu K. Antioxidant abietane diterpenoids from Salvia barrelieri. Nutrient. Chem. 2007;102:1281–1287. [Google Scholar]
Kikuzaki H., Usuguchi J., Nakatani Due north. Constituents of Zingiberaceae I. Diarylheptanoid from the rhizomes of ginger (Zingiber officinale Roscoe) Chem. Pharm. Bull. 1991;39:120. [Google Scholar]
Kinnula V.Fifty., Crapo J.D. Superoxide dismutases in malignant cells and man tumors. Free Radic. Biol. Med. 2004;36:718–744. [PubMed] [Google Scholar]
Kooy Due north.Westward., Royall J.A., Ischiropoulos H., Beckman J.S. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic. Biol. Med. 1994;16:149–156. [PubMed] [Google Scholar]
Kunchandy E., Rao Chiliad.N.A. Oxygen radical scavenging action of curcumin. Int. J. Pharm. 1990;58:237–240. [Google Scholar]
Manzocco Fifty., Anese G., Nicoli K.C. Antioxidant properties of tea extracts as affected by processing. Lebens-mittel-Wissenschaft Und-Technologie. 1998;31(7–8):694–698. [Google Scholar]
Marcocci I., Marguire J.J., Droy-lefaiz M.T., Packer L. The nitric oxide scavenging backdrop of Ginkgo biloba extract. Biochem. Biophys. Res. Commun. 1994;201:748–755. [PubMed] [Google Scholar]
Marletta Chiliad.A. Nitric oxide: biosynthesis and biological significance. Trends Biochem. Sci. 1989;14(12):488–492. [PubMed] [Google Scholar]
McCord J., Fridovich I. Superoxide dismutase, an enzymic office for erythrocuprin. J. Biol. Chem. 1969;244:6049–6055. [PubMed] [Google Scholar]
Moncada S., Palmer R.1000., Jr, Higgs Eastward.A. Biosynthesis of nitric oxide from l-arginine: a pathway for the regulation of prison cell office and communication. Biochem. Pharmacol. 1989;38:1709–1715. [PubMed] [Google Scholar]
Meyer A.Due south., Isaksen A. Application of enzymes as nutrient antioxidants. Trends Food Sci. Technol. 1995;half-dozen:300–304. [Google Scholar]
Noro T., Oda Y., Miyase T., Ueno A., Fukushima S. Inhibitors of xanthine oxidase from the flowers and buds of Daphne genkwa. Chem. Pharm. Balderdash. 1983;31:3984–3987. [PubMed] [Google Scholar]
Ohkawa H., Onishi N., Yagi K. Assay for lipid peroxidation in creature tissue by thiobarbituric acid reaction. Anal. Biochem. 1979;95:351–358. [PubMed] [Google Scholar]
Ottolenghi A. Interaction of ascorbic acid and mitochondria lipids. Arch. Biochem. Biophys. 1959;79:355. [Google Scholar]
Ou B.Ten., Huang D.J., Hampsch-Woodill Grand., Flanagan J.A., Deemer E.K. Analysis of antioxidant activities of mutual vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative written report. J. Agric. Nutrient Chem. 2002;fifty(11):3122–3128. [PubMed] [Google Scholar]
Oyaizu G. Studies on products of browning reactions: antioxidant activities of products of browning reaction prepared from glucosamine. J. Nutrit. 1986;44:307–315. [Google Scholar]
Paglia, D.E., Valentin, W.North., l967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. seventy, 158–169. [PubMed]
Prieto P., Pineda 1000., Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the decision of vitamin E. Anal. Biochem. 1999;269:337–341. [PubMed] [Google Scholar]
Prior R.L., Hoang H., Gu L. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC (FL)) of plasma and other biological and food samples. J. Agric. Food Chem. 2003;51:3273–3279. [PubMed] [Google Scholar]
Ramakrishna B.S., Varghese R., Jayakumar Southward., Mathan 1000., Balasubramanian K.A. Circulating antioxidants in ulcerative colitis and their relationship to disease severity and action. J. Gastroenterol. Hepatol. 1997;12:490–494. [PubMed] [Google Scholar]
Robak J., Gryglewski R.J. Flavonoids are scavengers of superoxide anions. Biochem. Pharmacol. 1988;37:837–841. [PubMed] [Google Scholar]
Ruch R.J., Cheng S.J., Klaunig J.E. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogen. 1989;10:1003–1008. [PubMed] [Google Scholar]
Sapakal Five.D., Shikalgar T.S., Ghadge R.V., Adnaik R.S., Naikwade N.S., Magdum C.S. In vivo screening of antioxidant profile: a review. J. Herbal Med. Toxicol. 2008;ii(2):ane–viii. [Google Scholar]
Sas Thou., Robotka H., Toldi J., Vecsei L. Mitochondrial, metabolic disturbances, oxidative stress and kynurenine system, with focus on neurodegenerative disorders. J. Neurol. Sci. 2007;257:221–239. [PubMed] [Google Scholar]
Seeram N.P., Henning S.Thou., Lee R., Niu Y., Scheuller H.S., Heber D. Catechin and caffeine contents of green tea dietary supplements and correlation with antioxidant activeness. J. Agric. Food Chem. 2006;54:1599–1603. [PubMed] [Google Scholar]
Singh U., Jialal I. Oxidative stress and atherosclerosis. Pathophysiology. 2006;xiii:129–142. [PubMed] [Google Scholar]
Singhal P.K., Kapur North., Dhillon K.South., Beamish R.E., Dhalla Due north.S. Role of complimentary radicals in catecholamine cardiomyopathy. Canadian J. Physiol. Pharmacol. 1982;60:1390. [PubMed] [Google Scholar]
Smith M.A., Rottkamp C.A., Nunomura A., Raina A.K., Perry Thousand. Oxidative stress in Alzheimer's disease. Biochim. Biophys. Acta. 2000;1502:139–144. [PubMed] [Google Scholar]
Soler-Rivas C., Espin J.C., Wichers H.J. An easy and fast test to compare total gratuitous radical scavenger capacity of foodstuffs. Phytochem. Anal. 2000;11:330–338. [Google Scholar]
Umesh B., Jagtap S.Due north., Panaskar, Bapat V.A. Evaluation of antioxidant capacity and phenol content in jackfruit (Artocarpus heterophyllus Lam.) fruit pulp. Plant Foods Hum. Nutr. 2010;65:99–104. [PubMed] [Google Scholar]
Upston J.M., Kritharides Fifty., Stocker R. The function of vitamin E in atherosclerosis. Prog. Lipid Res. 2003;42:405–422. [PubMed] [Google Scholar]
Virginia H., Sarah Fifty.E., Rachel J.Due south., Nathaniel T., Joseph S., Adam Eastward., Cecilia G. Mitochondrial nitric-oxide synthase: role in pathophysiology. IUBMB Life. 2003;55(10 &11):599–603. [PubMed] [Google Scholar]
Wood J.50. In: Fishman W.H., editor. vol. 2. Bookish Printing; New York: 1970. pp. 61–299. (Metabolic Conjugation and Metabolic Hydrolysis). [Google Scholar]
Yang G., Chen J., Wen Z., Ge K., Zhu L., Chen X. The function of selenium in Keshan affliction. Adv. Nutr. Res. 1984;6:203–220. [PubMed] [Google Scholar]

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