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 ROS^4,
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.

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 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.

Mechanism of action:

The redox properties of antioxidants play an important role in absorbing and neutralising free radicals, quenching singlet and triplet oxygen, or decomposing peroxides¹⁴. In doing so, the antioxidants themselves become oxidised. This urges the constant need of antioxidants to replenish them. The mechanism of antioxidants work has two functions; the first function is that they act as the giver of the hydrogen atom, which is a main function. Antioxidants, which have such main functions, are referred to as primary antioxidants. They can provide hydrogen atoms at a faster rate to the lipid radical (R^*, Roo^*) or change it to a more stable form. It is a chain breaking step. The second function is a secondary one, which is a preventive step. It reduces the rate of auto-oxidation with a variety of mechanisms beyond the auto-oxidation mechanism of chain termination by radical conversion of lipids to form more stable¹⁵ i.e., by scavenging initiating radicals, such antioxidants can thwart an oxidation chain from ever setting in motion. The effectiveness of an antioxidant in the body depends on which free radical is involved, how and where it is generated, and where the target of damage is present.

ABTS (2, 2’-azino-bis3-ethylbenzthiazoline-6-sulfonic) radical cation decolourisation assay:

ABTS assay can be used to determine the antioxidant activity of biological fluids, cells, tissues, natural and other synthetic therapeutical compounds. The assay measures ABTS⁺radical cation formation induced by metmyoglobin and hydrogen peroxide. A water soluble form of vitamin E called Trolox [6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], is used as a positive control for inhibiting the formation of the radical cation in the assay.

Preparation: The ABTS reagent is prepared by mixing 5 ml of 14 mM ABTS with 5 ml of 4.9 mM potassium persulphate (K₂S₂O₈) and the mixture is kept in dark at room temperature for 16 h. The reagent absorbance is then 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 μl 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 this assay.

Assay: 1.2 ml of test sample is added to 0.1 ml of 1 M Tris-HCl buffer (pH 7.9) and mixed with 1.2 ml of 5 mM DPPH in methanol. The reaction mixture is then kept in dark at room temperature for 30 min. The absorbance of the resulting solution is measured at 517 nm. Phenolic organic acids can be used as standard (e.g. gallic acid). The decrease of the absorbance at 517 nm is calculated as the percentage of inhibition by the following equation,

% Inhibition = [(A₀ – A₁)/A₀] × 100

A₀ - absorbance of control

A₁ - absorbance of the tested sample

DPPH (1, 1-Diphenyl-2-picryl-hydrazyl) radical scavenging assay is expressed as Standard phenolic acid equivalent and defined as mg of gallic acid equivalents per 1 g of sample¹⁶.

Ferric reducing antioxidant power assay:

The FRAP assay is simple, inexpensive, robust and fast assay which uses anitoxidants as reductants in a redox linked colorimetric method to test the total antioxidant power directly.

Preparation: FRAP reagent is prepared by mixing the reagents such as; 0.1 M acetate buffer (pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)- s-triazine (TPTZ) and 20 mM ferric chloride in the ratio 10:1:1 (v/v/v).

Assay: 10 ìl of testing sample (250 ìl /ml) is mixed with 300 ml of FRAP reagent, incubated at room temperature for 15 min and the absorbance is read at 593 nm. The assay involves FeSO₄.7H₂O as a standard reference and with different concentrations of which the standard curve was plotted. The FRAP values are expressed in ìmole Fe²⁺/mg dry weight of the test sample¹⁷.

Reducing power assay:

It is another form of reducing assay, which is found to be simple and effective for analysing the antioxidants.

Assay: 25 μl of test sample (250 μg/ml) is mixed with 50 μl of 50 μM phosphate buffer (pH 6.6), 50 μl of 0.1% (w/v) potassium ferricyanide and incubated in a water bath at 50°C for 20 min. 100 μl of 1% (w/v) trichloroacetic acid solution is added to the mixture and centrifuged at 3000 rpm for 10 min. 175 μl of the upper layer is carefully removed and combined with 25 μl of 5 mM ferric chloride and then the absorbance of the reaction mixture is measured at 700 nm. Ascorbic acid diluted in methanol is used as a standard material. The reducing power is expressed as μg of ascorbate equivalent per mg dry weight of the test sample¹⁷.

β-Carotene/LA assay:

Preparation: 0.5 mg of β-carotene is dissolved in 1 ml of chloroform and added with 25 μl of linoleic acid (LA) and 200 mg of Tween 20. The chloroform is evaporated under vacuum and the residue is added with 100 ml of distilled water.

Assay: 500 μl of testing sample is taken in a test tube and added with 5 ml of the stock solution. BHT (90 μg/ml) is employed as a positive control agent. The absorbance of the mixture is noted at 470 nm. The reaction mixture is then incubated for 2 h at 50 ºC. After incubation the absorbance is measured again at 470 nm (t = 120 min)¹⁸. The antioxidant activity is calculated as percentage inhibition of oxidation using the following equation,

% inhibition = [1 – (Abss₀– Abss₁₂₀)/ (Absc₀– Absc₁₂₀)] ×100

Superoxide anion scavenging activity assay:

The superoxide anion radicals are produced in 2 ml of phosphate buffer (100 mM, pH 7.4) with 78 μM β-nicotinamide adenine dinucleotide (NADH), 50 μM nitro blue tetrazoliumchloride (NBT) and test samples at different concentrations. The reaction mixture is kept for incubation at room temperature for 5 min. It is then added with 5-methylphenazinium methosulphate (PMS) (10 μM) to initiate the reaction and incubated for 5 min at room temperature. The colour reaction between superoxide anion radical and NBT is read at 560 nm. Gallic acid is used as a positive control agent for comparative analysis. The reaction mixture without test sample is used as control and without PMS is used as blank¹⁸. The scavenging activity is calculated as follows,

% Scavenging activity = [(Abs_c – Abs_s)/Abs_c] × 100

Ferrous ion-chelating assay:

The ferrous ion chelating (FIC) activity can be used to assay the antioxidants and it is measured by the decrease in the absorbance at 562 nm of the iron (II) and ferrozine complex¹⁹. 1 ml of test sample (concentration range 50 to 200 μg/ml) is mixed with 3.7 ml of methanol and 0.1 ml of 2 mM FeCl₂ and the reaction is initiated by the addition of 0.2 ml of 5 mM ferrozine. The mixture is incubated at room temperature for 10 min and the absorbance is determined at 562 nm. Methanol without test sample is used as a control and methanol without ferrozine solution is used as a sample blank. EDTA is used as a reference standard for the assay. A lower absorbance value indicates a better ferrous ion-chelating ability of the test sample¹⁸. The ferrous ion-chelating ability can be calculated using the following formula,

%Ferrous ion-chelating ability = [1- (Abs_s-Abs_b)/Abs_c] × 100

Abs_s- Absorbance value of test sample

Abs_b- Absorbance value of blank

Abs_c - Absorbance value of control

BSA oxidative damage assay:

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. 50 μl of BSA (20.0 mg/ml), 50 μl of FeCl₂/citric acid (4.0/4.0 mM), 50 μl of H₂O₂ (4.0 mM) and 50 μl of a sample (concentration range 62.5 to 250 μg/ml) are taken in a 1.5 ml reaction tube and incubated in water bath at 37°C for 60 min. Butylated hydroxytoluene (BHT) is used as a positive control agent for comparison. The carbonyl content of oxidised BSA is then determined in following steps; 0.5 ml of 2, 4-dinitrophenylhydrazine (2,4-DNPH) (10 mM in 2 N HCl) is added to the samples and allowed to react for 60 min at room temperature, with vortexing every 15 min. The protein is then precipitated by adding 0.5 ml of trichloroacetic acid (TCA) (20%) to reaction samples and followed by centrifugation for 5 min. The supernatant is discarded and the precipitate is washed three times with 1 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 in phosphate buffer, adjusted to pH 2.3 with trifluoroacetic acid (TFA), incubated at 37°C for 15 to 20 min, centrifuged and then the absorbance is read at 390 nm. Results are expressed as % inhibition in carbonyl formation, relative to control¹⁸.

Phosphomolybdenum assay:

The phosphomolybdenum assay used for determining the antioxidant capacity is based on the reduction of Mo (VI)–Mo (V) by the antioxidants and subsequent formation of a green phosphate/Mo (V) complex at acid pH.

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 acid, 28 mM sodium phosphate and 4 mM ammonium molybdate and incubated at 95°C for 90 min. Ascorbic acid is utilised as a reference standard. The absorbance of the mixture is then measured at 695 nm with methanol blank. The antioxidant activity is expressed as the number of gram equivalents of ascorbic acid²⁰.

Cupric ions chelation assay:

Test sample is diluted 10 times with hexamine–HCl buffer containing 10 mM KCl at pH 5.0. 1ml of prepared test sample is then mixed with 1 ml of 400 µM of CuSO₄ prepared using hexamine–HCl buffer. 100 µl of 2 mM 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.

Hydroxyl radical scavenging activity assay:

The scavenging activity for hydroxyl radicals can be determined using Fenton reaction.

Assay: 60 μl of 1.0 mM FeCl₂, 90 μl of 1mM 1,10-phenanthroline, 2.4 ml of 0.2 M phosphate buffer (pH 7.8), 150 μl of 0.17 M H₂O₂ and 1.5 ml of test solution with various concentrations are mixed together. H₂O₂ is added to the reaction mixture in order to initiate the reaction and the mixture is kept for incubation at room temperature for 5 min. After incubation the absorbance of mixture is read at 560 nm using a spectrophotometer and the hydroxyl radicals scavenging activity is calculated²².

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 FeSO₄is used as control²³. Anti-lipid peroxidation percentage is calculated using the following formula,

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

Abs of Fe₂+ induced peroxidation - abs of control

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

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 to be developed. Hence, this review besides discussing various methodologies, it emphasises on improvisation of the existing assays and its future research on innovating better methods to comprehensively study different aspects of antioxidants.

REFERENCES:

1. Wu Y, Hong C, Lin S, Wu P and Shiao M. Increase of vitamin Econtent in LDL and reduction of atherosclerosis in cholesterol-fedrabbits by a water-soluble antioxidant-rich fraction of Salviamiltiorrhiza. Arterioscler. Thomb. Vasc. Biol. 18; 1998: 481–486.

2. Liao KL and Yin MC. Individual and combined antioxidant effects of seven phenolic agents in humanerythrocyte membrane ghosts and phosphatidylcholine liposome systems: Importance of the partition coefficient. Journal of agricultural and food chemistry. 48; 2000: 2266-2270.

3. Halliwell B. Establishing the significance and optimal intake ofdietary antioxidants: The biomarker concept. Nutr. Rev. 57(4); 1999: 104-113.

4. Halliwell B, Aeschbach R, Löliger J and Aruoma OI. The characterization of antioxidants. Food Chemistry and Toxicology. 33; 1995: 601-617.

5. Sies H. Strategies of antioxidant defense. European Journal of Biochemistry. 215; 1993: 213–219.

6. Tseng TH, Kao ES, Chu CY, Chou FP, Lin Wu HW and Wang CJ. Protective Effects of Dried Flower Extracts of Hibiscus sabdariffa L. against Oxidative Stress in Rat Primary Hepatocytes. Food Chemistry and Toxicology. 35; 1997:1159-1164.

7. Soares JR, Dinis TCP, Cunha AP and Almeida LM (1997). Antioxidant Activities of some Extracts of Thymus zygis. Free Radical Research. 26; 1997: 469-478.

8. Velioglu Y, Mazza G, Gao L, Oomah B. Antioxidant activity andtotal phenolics in selected fruits, vegetables, and grain products. J.Agric. Food Chem. 46(10); 1998: 4113-4117.

9. Gutteridge JMC. Free Radicals and Aging. Rev. Clin. Gerontol. 4; 1994: 279-288.

10. Moon J and Shibamoto T. Antioxidant assays for plant and foodcomponents. J. Agric. Food Chem. 57; 2009: 1655–1666

11. Prior R, Wu X and Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 53 (10); 2005: 4290–302. doi:10.1021/jf0502698. PMID 15884874.

12. Cao G, Alessio H, Cutler R. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med. 14 (3); 1993: 303–11. doi:10.1016/0891-5849(93)90027-R. PMID 8458588.

13. Ou B, Hampsch-Woodill M, Prior R. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem. 49 (10); 2001: 4619–26. doi:10.1021/jf010586o. PMID 11599998.

14. Osawa T. Novel natural antioxidants for utilization in food and biological systems. In Post harvest biochemistry of plant food-materials in the tropics, Edited by Uritani I, Garcia VV, Mendoza EM. Japan Scientific Societies Press, Japan. 1994; pp. 241-251.

15. Gordon MH. The mechanism of antioxidant action in vitro. In Food antioxidants, Edited by Hudson BJF, Elsevier Applied Science, London. 1990; pp. 1–18.

16. Boonchum W, Peerapornpisal Y, Vacharapiyasophon P, Pekkoh J, Pumas C, Jamjai U, Amornlerdpison D, Noiraksar T and Kanjanapothi D. Antioxidant activity of some seaweed from the gulf of Thailand. Int. J. Agric. Biol.13; 2011: 95–99.

17. Jen-Chieh Tsai, Guan-Jhong Huang, Tai-Hui Chiu, Shyh-Shyun Huang, Shun-Chieh Huang, Tai-Hung Huang, Shang-Chih Lai and Chao-Ying Lee. Antioxidant activities of phenolic components from various plants of Desmodium species. African Journal of Pharmacy and Pharmacology. 5(4); 2011: 468-476.

18. Nuno Rainha, Elisabete Lima, José Baptista and Carolina Rodrigues. Antioxidant properties, total phenolic, total carotenoid and chlorophyll content of anatomical parts of Hypericum foliosum. Journal of Medicinal Plants Research. 5(10); 2011: 1930-1940.

19. Lim YY, Lim TT and Tee JJ. Antioxidant properties of several tropical fruits: A comparative study. Food Chemistry. 103; 2007: 1003–1008.

20. Sahaa MR, Hasana SMR, Aktera R, Hossaina MM, Alamb MS, Alam MA, and Mazumder MEH. In vitro free radical scavenging activity of methanol extract of the leaves of Mimusops elengi Linn. Bangl. J. Vet. Med. 6 (2); 2008: 197–202.

21. Shih Peng Wong, Lai Peng Leong and Jen Hoe William Koh. Antioxidant activities of aqueous extracts of selected plants. Food Chemistry. 99(4); 2006: 775-783.

22. Nagulendran KR, Velavan S, Mahesh R and Haseena Begum V. In Vitro Antioxidant Activity and Total Polyphenolic Content of Cyperus rotundus Rhizomes. E-Journal of Chemistry. 4(3); 2007: 440-449.

23. Mandal P, Misra TK and Ghosal M. Free-radical scavenging activity and phytochemical analysis in the leaf and stem of Drymaria diandra Blume. International Journal of Integrative Biology. 7(2); 2009: 80-84.

Received on 03.11.2011 Accepted on 29.11.2011

Asian J. Pharm. Tech. 1(4): Oct. - Dec. 2011; Page 99-103