Antioxidant Assays in Pharmacological Research

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Asian J. Pharm. Tech. 2011; Vol. 1: Issue 4, Pg 99-103

ISSN- 2231–5705 (Print) www.asianpharmaonline.org ISSN- 2231–5713 (Online) 0974-3618 REVIEW ARTICLE

Antioxidant Assays in Pharmacological Research Selvakumar K.*, Madhan R., Srinivasan G. and Baskar V. Department of Applied Biosciences, BioLim Centre for Life Science, Chennai, Tamil nadu, India *Corresponding Author E-mail: [email protected]

ABSTRACT:

Antioxidants research has become active to a greater extent in many fields. The publications on antioxidants’ potential, their availability from vitamins, flavonoids, polyphenols etc has increased too many folds since past decade. Several reviews have also been published on validated and specific assays. This review summarises the versatility of antioxidants against the free radicals, their dual mechanism of action and the chemical principles of many antioxidant assays. The antioxidants assay is a complex topic due to the unavailabilty of a standard assay. This review intends to be comprehensive to cover almost all the reported assays which have few influence and applications. Choosing an adequate assay is critical to investigate the antioxidant activity of foods and biological samples. Two general types such as; lipid peroxidation associated assays and electron or radical scavenging assays are widely used for different antioxidant studies have been discussed here. The former includes, -carotene bleaching assay, anti-lipid peroxidation assay involving TCA-TBA solution and the latter includes ABTS (2, 2’-azino-bis3-ethylbenzthiazoline-6-sulfonic) radical cation decolorisation assay, DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) radical scavenging assay, ferric reducing antioxidant power assay, superoxide anion scavenging activity assay, ferrous ion-chelating assay etc. The chemistry behind all the above mentioned assays has been reviewed here emphasising the need of discovery of a convenient method for the quick quantitation of antioxidants.

KEYWORDS: Free radicals, ROS, antioxidants, assays, lipid peroxidation, radical scavenging INTRODUCTION:

Free radicals, particularly reactive oxygen species (ROS) have a greater impact on humans both from within the body and the environment. During metabolism, ROS such as superoxide (O2-), hydroxyl (OH) and hydrogen peroxide (H2O2) can arise normally or sometimes the immune cells create them purposefully to neutralise the foreign bodies. Moreover, environmental factors such as pollution, radiation, cigarette smoke and herbicides can also generate free radicals. These ROS can damage essential proteins, DNA and lipids and cause various human diseases like atherosclerosis1, cancer, liver injury, cardiovascular disease2, neurodegenerative disorders and rheumatism3 as a result of ‘oxidative stress’. Although, the body possesses defence mechanisms as enzymes and antioxidant nutrients, which arrest the damaging properties of ROS4, 5, continuous exposure to chemicals and contaminants may increase the amount of free radicals in the body beyond its ability to control and cause irreversible oxidative damages6. Received on 03.11.2011 Accepted on 29.11.2011 © Asian Pharma Press All Right Reserved Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 40-48

Therefore, antioxidants with free radical scavenging activities may be relevant in the prevention and therapeutics of diseases where free radicals are implicated7. WHO has recommended the use of natural antioxidants that can delay or inhibit the lipids or other molecules oxidation by inhibiting the initiation or propagation of oxidative chain reactions8. Antioxidants are substances that when present at low concentrations, compared to those of the oxidisable substrate significantly delays or inhibits the oxidation of the substrate9. An important role of antioxidants is to suppress free radical−mediated oxidation by inhibiting the formation of free radicals by scavenging radicals. Radical scavenging action is dependent on both the reactivity and concentration of the antioxidant. The research on the role of antioxidants in biology focused earlier on their use in preventing the oxidation of unsaturated fats, which is the cause of rancidity. However, it was the identification of vitamins A, C, and E as antioxidants that revolutionised the field and led to the realisation of the importance of antioxidants in the biochemistry of living organisms. Antioxidants are found in varying amounts in foods such as vegetables, fruits, and a variety of other foods10 naturally. Besides, many pharmaceutical companies are also offering the antioxidant

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capsules from natural substances to boost the metabolism and the immune system of the body. In pharmacological research, many assays are involved for the measurement of antioxidants. It is not a straightforward process, as this is a diverse group of compounds with different reactivities to different reactive oxygen species. The current industry standard for assessing antioxidant strength of whole foods, juices and food additives is improving with many new assays being involved. Some of the measurement tests include the Folin-Ciocalteu reagent, the Trolox equivalent antioxidant capacity assay11, the oxygen radical absorbance capacity (ORAC)12,13 etc. However, more detailed analytical information on the constituents mediating the observed biological effects is needed prior the promotion or development of effective and safe foods or food supplements, e.g. nutraceuticals, for human consumption. Hence, this article intends to offer a critical evaluation of existing antioxidant assays by emphasising the need of developing more refined, rapid, simple and low cost assays.

adjusted to 0.700 ± 0.02 at 734 nm with distilled water and used for the assay purposes. Assay: 1 ml of ABTS reagent is added to 10 l of different concentrations of sample and the absorbance is measured at 734 nm at 3 min interval. Trolox is used as standard. Percentage inhibition of the sample is calculated by the following equation, % Inhibition = [(A0 – A1)/A0] × 100 A0- absorbance of control A1- absorbance of the tested sample The ABTS radical anion scavenging assay is expressed as Trolox equivalent antioxidant capacity (TEAC) and defined as mg of trolox equivalents per 1 g of sample16.

DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) radical scavenging assay: DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) is a stable free radical with red colour. On scavenging, these free radicals turn to yellow. This common principle has been utilised in Mechanism of action: The redox properties of antioxidants play an important role this assay. in absorbing and neutralising free radicals, quenching singlet and triplet oxygen, or decomposing peroxides14. In Assay: 1.2 ml of test sample is added to 0.1 ml of 1 M Trisdoing so, the antioxidants themselves become oxidised. HCl buffer (pH 7.9) and mixed with 1.2 ml of 5 mM DPPH This urges the constant need of antioxidants to replenish in methanol. The reaction mixture is then kept in dark at them. The mechanism of antioxidants work has two room temperature for 30 min. The absorbance of the functions; the first function is that they act as the giver of resulting solution is measured at 517 nm. Phenolic organic the hydrogen atom, which is a main function. Antioxidants, acids can be used as standard (e.g. gallic acid). The which have such main functions, are referred to as primary decrease of the absorbance at 517 nm is calculated as the antioxidants. They can provide hydrogen atoms at a faster percentage of inhibition by the following equation, rate to the lipid radical (R*, Roo*) or change it to a more % Inhibition = [(A0 – A1)/A0] × 100 stable form. It is a chain breaking step. The second function A0 - absorbance of control is a secondary one, which is a preventive step. It reduces the A1 - absorbance of the tested sample rate of auto-oxidation with a variety of mechanisms beyond the auto-oxidation mechanism of chain termination by DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) radical scavenging radical conversion of lipids to form more stable15 i.e., by assay is expressed as Standard phenolic acid equivalent and scavenging initiating radicals, such antioxidants can thwart defined as mg of gallic acid equivalents per 1 g of sample16. an oxidation chain from ever setting in motion. The effectiveness of an antioxidant in the body depends on Ferric reducing antioxidant power assay: which free radical is involved, how and where it is The FRAP assay is simple, inexpensive, robust and fast generated, and where the target of damage is present. assay which uses anitoxidants as reductants in a redox linked colorimetric method to test the total antioxidant ABTS (2, 2’-azino-bis3-ethylbenzthiazoline-6-sulfonic) power directly. radical cation decolourisation assay: ABTS assay can be used to determine the antioxidant Preparation: FRAP reagent is prepared by mixing the activity of biological fluids, cells, tissues, natural and other reagents such as; 0.1 M acetate buffer (pH 3.6), 10 mM synthetic therapeutical compounds. The assay measures 2,4,6-tris(2-pyridyl)- s-triazine (TPTZ) and 20 mM ferric ABTS+ radical cation formation induced by metmyoglobin chloride in the ratio 10:1:1 (v/v/v). and hydrogen peroxide. A water soluble form of vitamin E called Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2- Assay: 10 ìl of testing sample (250 ìl /ml) is mixed with carboxylic acid], is used as a positive control for inhibiting 300 ml of FRAP reagent, incubated at room temperature for 15 min and the absorbance is read at 593 nm. The assay the formation of the radical cation in the assay. involves FeSO4.7H2O as a standard reference and with Preparation: The ABTS reagent is prepared by mixing 5 different concentrations of which the standard curve was ml of 14 mM ABTS with 5 ml of 4.9 mM potassium plotted. The FRAP values are expressed in ìmole Fe2+/mg persulphate (K2S2O8) and the mixture is kept in dark at dry weight of the test sample17. room temperature for 16 h. The reagent absorbance is then

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562 nm. Methanol without test sample is used as a control Reducing power assay: It is another form of reducing assay, which is found to be and methanol without ferrozine solution is used as a sample simple and effective for analysing the antioxidants. blank. EDTA is used as a reference standard for the assay. A lower absorbance value indicates a better ferrous ionAssay: 25 l of test sample (250 g/ml) is mixed with 50 l chelating ability of the test sample18. The ferrous ionof 50 M phosphate buffer (pH 6.6), 50 l of 0.1% (w/v) chelating ability can be calculated using the following potassium ferricyanide and incubated in a water bath at formula, 50°C for 20 min. 100 l of 1% (w/v) trichloroacetic acid %Ferrous ion-chelating ability = [1- (Abss-Absb)/Absc] × 100 solution is added to the mixture and centrifuged at 3000 rpm for 10 min. 175 l of the upper layer is carefully Abss - Absorbance value of test sample removed and combined with 25 l of 5 mM ferric chloride Absb - Absorbance value of blank and then the absorbance of the reaction mixture is measured Absc - Absorbance value of control at 700 nm. Ascorbic acid diluted in methanol is used as a standard material. The reducing power is expressed as g of BSA oxidative damage assay: ascorbate equivalent per mg dry weight of the test sample17. This method is used to evaluate the tendency of the antioxidants to inhibit protein oxidation. Bovine serum albumin (BSA) is oxidised in phosphate buffer with pH 7.4. -Carotene/LA assay: Preparation: 0.5 mg of -carotene is dissolved in 1 ml of 50 l of BSA (20.0 mg/ml), 50 l of FeCl2/citric acid chloroform and added with 25 l of linoleic acid (LA) and (4.0/4.0 mM), 50 l of H2O2 (4.0 mM) and 50 l of a 200 mg of Tween 20. The chloroform is evaporated under sample (concentration range 62.5 to 250 g/ml) are taken in vacuum and the residue is added with 100 ml of distilled a 1.5 ml reaction tube and incubated in water bath at 37°C for 60 min. Butylated hydroxytoluene (BHT) is used as a water. positive control agent for comparison. The carbonyl content Assay: 500 l of testing sample is taken in a test tube and of oxidised BSA is then determined in following steps; 0.5 added with 5 ml of the stock solution. BHT (90 g/ml) is ml of 2, 4-dinitrophenylhydrazine (2,4-DNPH) (10 mM in 2 employed as a positive control agent. The absorbance of the N HCl) is added to the samples and allowed to react for 60 mixture is noted at 470 nm. The reaction mixture is then min at room temperature, with vortexing every 15 min. The incubated for 2 h at 50 ºC. After incubation the absorbance protein is then precipitated by adding 0.5 ml of is measured again at 470 nm (t = 120 min)18. The trichloroacetic acid (TCA) (20%) to reaction samples and antioxidant activity is calculated as percentage inhibition of followed by centrifugation for 5 min. The supernatant is discarded and the precipitate is washed three times with 1 oxidation using the following equation, % inhibition = [1 – (Abss0– Abss120)/ (Absc0– Absc120)] ×100 ml of EtOH/EtOAc (1:1). Each wash is followed by centrifugation and discarding of the supernatant. The washed precipitate is dissolved in 0.6 ml of guanidine (6 M Superoxide anion scavenging activity assay: The superoxide anion radicals are produced in 2 ml of in phosphate buffer, adjusted to pH 2.3 with trifluoroacetic phosphate buffer (100 mM, pH 7.4) with 78 M - acid (TFA), incubated at 37°C for 15 to 20 min, centrifuged nicotinamide adenine dinucleotide (NADH), 50 M nitro and then the absorbance is read at 390 nm. Results are blue tetrazoliumchloride (NBT) and test samples at expressed as % inhibition in carbonyl formation, relative to 18 different concentrations. The reaction mixture is kept for control . incubation at room temperature for 5 min. It is then added with 5-methylphenazinium methosulphate (PMS) (10 M) Phosphomolybdenum assay: to initiate the reaction and incubated for 5 min at room The phosphomolybdenum assay used for determining the temperature. The colour reaction between superoxide anion antioxidant capacity is based on the reduction of Mo (VI)– radical and NBT is read at 560 nm. Gallic acid is used as a Mo (V) by the antioxidants and subsequent formation of a positive control agent for comparative analysis. The green phosphate/Mo (V) complex at acid pH. reaction mixture without test sample is used as control and without PMS is used as blank18. The scavenging activity is Assay: 0.3 ml of test sample is taken in a tube and mixed with 3 ml of reagent solution containing 0.6 M sulphuric calculated as follows, acid, 28 mM sodium phosphate and 4 mM ammonium % Scavenging activity = [(Absc – Abss)/Absc] × 100 molybdate and incubated at 95°C for 90 min. Ascorbic acid is utilised as a reference standard. The absorbance of the Ferrous ion-chelating assay: The ferrous ion chelating (FIC) activity can be used to mixture is then measured at 695 nm with methanol blank. assay the antioxidants and it is measured by the decrease in The antioxidant activity is expressed as the number of gram 20 the absorbance at 562 nm of the iron (II) and ferrozine equivalents of ascorbic acid . 19 complex . 1 ml of test sample (concentration range 50 to 200 g/ml) is mixed with 3.7 ml of methanol and 0.1 ml of Cupric ions chelation assay: 2 mM FeCl2 and the reaction is initiated by the addition of Test sample is diluted 10 times with hexamine–HCl buffer 0.2 ml of 5 mM ferrozine. The mixture is incubated at room containing 10 mM KCl at pH 5.0. 1ml of prepared test temperature for 10 min and the absorbance is determined at sample is then mixed with 1 ml of 400 µM of CuSO4 prepared using hexamine–HCl buffer. 100 µl of 2 mM

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tetramethylmurexide ammonium salt (TMM) solution is added to this mixture subsequently and the absorbance of the final reaction mixture is recorded at 460 and 530 nm and the ratio of the absorbance at 460 and 530 nm is calculated. Free cupric ion concentration is determined using a standard curve of absorbance ratio against concentration of free cupric ion. The value of free cupric ion concentration is then subtracted from the total amount of cupric ions and there by the total concentration of chelated cupric ions is identified and converted to percentage21.

CONCLUSION:

The interest in natural antioxidants has grown in the recent years with the awareness of several deadliest diseases. The result of which, is the development of several assays seen above for the measurement of total antioxidant capacity of a biological sample or any food. Indeed, these assays are being utilised in medical field as well as food industries. Due to the complexity of the composition of foods or other biological sample, studying each antioxidant individually is costly and inefficient. Therefore, it is very appealing to researchers to have a convenient method for the quick quantitation of antioxidants. However, such methods are yet Hydroxyl radical scavenging activity assay: to be developed. Hence, this review besides discussing The scavenging activity for hydroxyl radicals can be various methodologies, it emphasises on improvisation of determined using Fenton reaction. the existing assays and its future research on innovating better methods to comprehensively study different aspects Assay: 60 l of 1.0 mM FeCl2, 90 l of 1mM 1,10- of antioxidants. phenanthroline, 2.4 ml of 0.2 M phosphate buffer (pH 7.8), 150 l of 0.17 M H2O2 and 1.5 ml of test solution with REFERENCES: various concentrations are mixed together. H2O2 is added to 1. Wu Y, Hong C, Lin S, Wu P and Shiao M. Increase of vitamin the reaction mixture in order to initiate the reaction and the Econtent in LDL and reduction of atherosclerosis in cholesterolfedrabbits by a water-soluble antioxidant-rich fraction of mixture is kept for incubation at room temperature for 5 Salviamiltiorrhiza. Arterioscler. Thomb. Vasc. Biol. 18; 1998: min. After incubation the absorbance of mixture is read at 481–486. 560 nm using a spectrophotometer and the hydroxyl 2. Liao KL and Yin MC. Individual and combined antioxidant 22 radicals scavenging activity is calculated . effects of seven phenolic agents in humanerythrocyte membrane

Anti-lipid peroxidation assay: Anti-lipid peroxidation assay is a standard method which can be performed with the help of goat liver homogenate. 2.8 ml of 10% goat liver homogenate, 0.1 ml of 50 mM ferrous sulphate and 0.1 ml of test sample are added and the reaction mixture is incubated at 37°C for 30 min. The reaction is then inhibited by TCA-TBA solution. 2ml of 10% TCA-0.67% TBA made in 50% acetic acid is added to 1 ml of the reaction mixture and boiled for 1 hour at 100°C, followed by centrifugation for 5 min at 10,000 rpm. The supernatant is observed for absorbance at 535 nm against blank. Induced vitamin E is used as a standard. Reaction mixture without test sample and FeSO4 is used as control23. Anti-lipid peroxidation percentage is calculated using the following formula,

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%ALP = Abs of Fe2+ induced peroxidation - abs of sample X 100 Abs of Fe2+ induced peroxidation - abs of control

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Hydrogen peroxide scavenging activity assay: Hydrogen peroxide scavenging activity of the extract can be determined using replacement titration methodology. 1.0 ml of 0.1 mM H2O2 and 1.0 ml of various concentrations of test sample are mixed together. 2 drops of 3% ammonium molybdate, 10 ml of 2 M sulphuric acid and 7.0 ml of 1.8 M potassium iodide are added to the reaction mixture. The mixed solution is then titrated with 5.09 mM NaS2O3. Appearance of yellow colour is marked as the end point of the reaction. The reaction mixture without test sample is used as control22. Percentage of scavenging of hydrogen peroxide is calculated as follows % Inhibition = (V0 - V1) / V0 × 100 V0 - volume of NaS2O3 solution used to titrate the control V1 - volume of NaS2O3 solution used in titrate the test mixture

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