Table 1.

Various in vitro and in vivo antioxidant assays, their principles, advantages, and limitations.

Mechanism (Category)	Assay	Technique/Principle	Advantages	Limitations	Ref.
	In vitro
Electron transfer (Total Antioxidant Capacity)	CUPRAC (Cupric ion reducing antioxidant capacity method)	In this assay, phenolic groups in the polyphenols are oxidized to quinones, whereas Cu(II) is reduced to Cu(I), which is measured at 450 nm.	(+) Copper reaction rates are faster than that of ferric ions, and it is more specific for antioxidants	(−) it ignores the reaction kinetics	[36]
	DMPD (N,N-dimethyl-p-phenylene diamine dihydrochloride) method	In the presence TROLOX, reduction of DMPD radical cation by antioxidants, the absorbance at 505 nm is decreased.	(+) easy, cheaper, and reproducible	(−) it ignores the reaction kinetics, and the DMPD radical is a non-physiological radical	[62]
	FRAP (Ferric reducing-antioxidant power assay)	Antioxidants at low pH reduce ferric-tripyridyltriazine (FeIII-TPTZ)to FeII form which is measured at 593 nm.	(+) a good representation of electron transfer mechanism (+) the is inexpensive, easy to prepare reagents, reproducibility, and speedy and a straight forward procedure	(−) it ignores the reaction kinetics and non-specific to antioxidants	[51]
	Follin-Ciocalteu reducing capacity	In this assay, phenols are oxidized in a basic medium by a mixture of tungstate and molybdate (Folin-Ciocalteu reagent), with the consequent formation of colored molybdenum ions, MoO4+ (750 nm).	(+) easy and reproducible	(−) it ignores the reaction kinetics and non-specific to antioxidants	[14]
	Trolox equivalent antioxidant capacity (TEAC) method (ABTS radical cation decolorization assay)	Upon reaction with an antioxidant (Trolox), ABTS (2,2-azo-bis(3-ethylbenz-thiozoline-6-sulfonic acid)) radical cation, which is a blue-green chromophore, reduces and decolorized. This assay uses a diode-array spectrophotometer at 750 nm.	(+) it can screen both hydrophilic and lipophilic antioxidants, easy and reproducible	(−) it ignores the reaction kinetics, and the ABTS is a non-physiological radical (−) The assay is not suitable for the determination of proteins antioxidant activity	[63]
Hydrogen Atom Transfer (Antioxidant Activity)	ORAC (Oxygen radical absorbance capacity) method	AAPH (2,2-azobis-2-aminopropane dihydrochloride) decomposition induces peroxyl radicals, and radical scavengers are used to measure the decrease in fluorescence. AAPH is used as a radical generator and Trolox as the antioxidant control. 485 nm is used as the excitation wavelength, and 520 nm is used as the emission wavelength.	(+) physiologically resemble method, and it takes initiation and propagation into account	(−) lack of consistency and the possible underestimation of antioxidant activity as B-PE can interact with phenolic acids (−) The method has been reported to fail determining both hydrophilic and lipophilic antioxidants	[64]
	TRAP (Total radical-trapping antioxidant parameter method), both in vivo and in vitro	In this method, the antioxidant potential is assessed by measuring the decay in decoloration. ABAP (2,2′-azo-bis(2-ami-dino-propane)hydrochloride) is a radical initiator that quenches the fluorescence of R-Phycoerythrin (R-PE).	(+) peroxyl radical is a common and physiologically representative radical	(−) detection probe(oxygen) that is not stable and may cause issues in measurements	[65,67,68]
ROS/RNS scavenging activity/lipid oxidation	β-carotene linoleic acid method/conjugated diene assay	The ROS oxidizes linoleic acid, and the resulting products initiate β-carotene oxidation, which leads to discoloration. In the presence of antioxidants, the discoloration will be delayed and measured at 434 nm.	(+) shows a strong correlation with the total phenolics measured by the F-C method.	(−) lack of reproducibility and crude kinetic treatment	[14,68,69]
	Ferric thiocyanate (FTC) method	During linoleic acid peroxidation, peroxides were formed, which oxidize Fe(II) to Fe(III). The Fe(III) reacts with thiocyanate to form a red color complex, which is measured at 500 nm.	(+) used to measure peroxide amount at the starting phase of peroxidation	(−) lack of specificity	[46,70,71]
	Thiobarbituric acid (TBA) method	In this assay, TBA and trichloroacetic acid are mixed with the sample solution, placed in the hot water bath for 10 min, centrifuged in the solution, and supernatant absorbance activity is measured at 552 nm.	(+) used to measure the concentration of free radicals present at the end of peroxide oxidation	(−) Not specific	[14]
	Hydrogen peroxide scavenging (H2O2) assay	Antioxidants reduce hydrogen peroxide concentration, which is measured at 230 nm using a spectrophotometer.		(−) most plant and food samples also absorb at this wavelength, which can compromise both the precision and accuracy of the method	[72]
	Hydroxyl radical scavenging activity	In the presence of antioxidant, the degraded product of deoxyribose (TBARS) measured colorimetrically at 532 nm.	(+) Useful for ketone containing antioxidants	(−) Higher concentration of antioxidants required	[73]
	Nitric oxide scavenging activity	Under aerobic conditions, nitric oxide reacts with oxygen to form nitrate and nitrite, which can be quantified using Griess reagent, and the absorbance is measured at 546 nm.	(+) relatively simple experimentation and physiologically relevant	(−) detection technique is not easily available and has a long reaction time	[18,74]
	Peroxynitrile radical scavenging activity	ONOO.scavenging activity is measured by the oxidation of dihydroxyrhodamine to rhodamine fluorescence spectrophotometer with an excitation wavelength of 485 nm and emission wavelength of 530 nm.	(+) Peroxynitrile is a good oxidizing agent for dihydroxyrhodamine	(−) Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine	[75]
	Superoxide radical scavenging activity (SRSA/SOD)	This assay is based on the removal rate of superoxide radical (O2−) using antioxidants, which is measured by nitro blue tetrazolium (NBT) at 560 nm.	(+) peroxyl radical is a common and physiologically representative radical	(−) irreproducibility due to the water insolubility issue of diformazan, the end product of NBT reduction	[76,77]
	Xanthine oxidase inhibition assay	Xanthine is a substrate in XOD- catalyzed reaction, which yields uric acid as a product. Allopurinol is used as a xanthine oxidase inhibitor, measured at 293 nm.	(+) Possible to get kinetics	(−) Enzyme collection is tricky	[8,78]
ET/HAT_mixed	DPPH scavenging activity	1-diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl; DPPH) is a stable free radical due to electron delocalization, which prevents its dimerization. DPPH reacts with antioxidants, which diminishes its deep violet color, which is measured at 517 nm (515–518 nm).	(+) easy and reproducible	(−), difficult to get the reaction kinetics, and the DPPH radical is a non-physiological radical	[66]
Metal chelation	Ferrous ion chelating activity assay/Ferrozine assay-Fe(II)	TAC assay obtained via reduction of Fe(III) to Fe(II), and formed Fe(II) is determined with ferrozine using spectrophotometric absorbance measurement at 562 nm.	(+) High sensitivity, correlated with structure-activity relationships, higher molar absorptivity, relatively lower interference from foreign ions, wide pH tolerance, complex stability constant, water solubility, and low viscosity	(−) Not correlated with FRAP, DPPH, and TPC	[87,88]
	Cuprous ion chelating activity/Pyrocatechol violet-Cu(II)	Free Cu(II) that is not complexed with antioxidants is bound to Pyrocatechol, which is assessed at 632 nm.	(+) good repeatability and reproducibility, Cu2+ chelating ability is significantly and positively correlated to DPPH, FRAP, and total phenolic content		[88]
	In vivo
Hydrogen atom transfer	Catalase (CAT)	The catalase activity is measured in an erythrocyte lysate as the difference in absorbance (λ240) per unit as the H2O2 maximum absorption wavelength is 240 nm. Catalase activity is used both in vivo and in vitro.	(+) a good representation of physiological conditions		[78]
Electron transfer/reducing power (Total Antioxidant Capacity)	Ferric reducing ability of plasma (FRAP)	This assay is primarily based on the principle that, at low pH, ferric-tripyridyltriazine (FeIII-TPTZ) is reduced to Fe(II). The antioxidant capacity is measured using the increased FeII, which is measured spectrophotometrically at 593 nm.	(+) most simple, rapid, inexpensive tests and very useful for routine analysis, a good representation of electron transfer mechanism	(−) it ignores the reaction kinetics and non-specific to antioxidants	[51,79]
	γ-glutamyl transpeptidase (GGT)	GGT transfers the γ-glutamyl group from the L-γ-Glutamyl-p-nitroanilide and liberates the chromogen p-nitroanilide (pNA, 418 nm) proportional to the GGT present.	(+) a good representation of physiological conditions		[80]
Lipid peroxidation inhibition	Glutathione peroxidase (GSHPx) estimation	GSHPx is a seleno-enzyme that catalyzes the reaction of hydroperoxides with GSH to form GSSG and reduction of hydrogen peroxide.	(+) a good representation of physiological conditions		[13,85]
	Glutathione reductase (GR) assay	GR catalyzes the reduction of GSSG to GSH. GR activity is determined at 340 nm and 412 nm. One may expect a decrease of activity at 340 nm as a result of the oxidation of NADPH or an increase at 412 nm caused by the reduction of dithiobis (2-nitrobenzoic acid) DTNB.	(+) a good representation of physiological conditions		[13,81]
	Glutathion-S-transferase (GSt)	This assay utilizes 1-Chloro-2,4-dinitrobenzene (CDNB). Potassium phosphate, GSt, and CDNB mixture are incubated at 37 C, pH 6.5 for 5 min, followed by adding substrate. 340 nm absorbance is used for monitoring the assay.	(+) a good representation of physiological conditions		[13]
	LDL assay	The extent of low-density lipoprotein (LDL) oxidation is determined by the amount of lipid peroxides, also by using a thiobarbituric acid reactive substances (TBARS) assay determined at 532 nm.	(+) LDL is a true representation of physiologically	(−) limitations in the isolation of LDL from the blood, and it is difficult to monitor the lag phase	[13,83,84,85]
Lipid peroxidation inhibition	Lipid peroxidation (LPO) assay	Malondialdehyde (MDA) is one of the end products of lipid peroxidation, which is used for the LPO assay measured at 586 nm.	(+) a good representation of physiological conditions		[84,85]
	Superoxide dismutase (SOD) method	The SOD assay works based on the absorbance change at 420 nm related to pyrogallol.	(+) a good representation of physiological conditions		[86]