Plants contain various chemical compounds. For assessment of antioxidant potential of endogenous compounds, single assay methods is not sufficient. Because different antioxidant assays differs in terms of assay principle and experimental conditions. Also antioxidant are of two main types,

1- Polar (phenolics, flavanoids etc)

2- non-polar (vitamin E )

Therefore for assessment of different antioxidants we need different assays.

The DPPH and ABTS free radical scavenging assay are the commonly used methods generally used evaluation of proton donating antioxidants (phenolics or polyphenols) from plants.

thank you Ajay.

What about FRAP & CUPRAC?

Frap assay only measure reducing activity by deactivating free radicals via single electron transfer mechanism. Also you can try Folin-Ciocalteu (Total phenolics) method. Frap and Folin-Ciocalteu methods have the same antioxidant mechanism. On the other hand a complementary method which has hydrogen atom transfer mechanism such as ORAC can be used to obtain complementary data about antioxidant potential of the extratcs. I recommend to read the articles of;

1) Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry 53, 4290-4302.

2) Huang, D., Ou, B., Prior, R.L., 2005. The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry 53, 1841-1856.

Please have a look at the attached paper which describes in detail the methods currently used to assess antioxidant activity. Hope this will help you to arrive at a solution.

Regards

Normally, combination of few antioxidants activity assay are conducted, especially those involved different mechanisms on how antioxidant takes place. Common assays like FRAP, DPPH and TEAC can be considered.

DPPH is the most common assay method for antioxidant activity

How about CUPRAC? Have you heard it before???

FRAP & CUPRAC both are also employed for assessment of antioxidant potential

In these assay methods the reduction of Ferric (Fe3+) to Ferrous (Fe2+) in case of FRAP and cupric to cuprous in case of CUPRAC is measured in presence of antioxidant compounds.

The antioxidants has the tendency to donate electrons. The electron from antioxidant compounds are gained by Ferric ions and they convert in to ferrous ions. This will cause change in colour of reaction which is recorded with the help of spectrophotometer.

The antioxidant capacity was determined by chemiluminiscence using the “PHOTOCHEM” dedicated device produced by Jena Analytic, Germany.

Eight 2 mL aliquots from the working solution are frozen at −10°C. By successively defreezing these aliquots, the antioxidant capacity is measured every 3 hours. The determination of antioxidant capacity is based upon the photochemical generation of the superoxide anion radical initiated by a 185–240 nm UV beam.

The determination of the antioxidant capacity is based upon photochemical generation initiated by an UV radiation in the 185–240 nm domains, of the anion (O– 2◦) superoxide radical. The sequence presented in Schemes 1 and 2 includes the optical excitation of the free radical generator (damnacantal) thus obtaining the singlet state (1 s) of the molecule (process [A]). By an “inter system crossing”-type process (process [B]), the molecule goes into the triplet state which, due to known molecular spectroscopy selection rules, is stable enough to react either with oxygen in the normal triplet state (generating the singlet oxygen reactive species) (process [C]), or with a reducing (also called a reductant or reducer) agent (mono-electronic reduction process) (process [D]) with the generation of an anion radical in doublet state (2S) and the formation of the superoxide anion (process [E]). The latter, by a series of processes ([A]–[H]), transforms into the amino orthophthalic acid dianion, in the singlet state, which, by reversal process to the fundamental state, emits a light beam in the 425–450 nm spectral domain. Antioxidants from the captured samples yield superoxide ions and reduce the radiation intensity generated by excited luminol (luminol reaction inhibiting-blank). Standard soluble compounds are TROLOX (derivate of α–tocopherol), and for the water–soluble, ascorbic acid. Process [A]–obtaining the singlet state (1 s) of the molecule; process [B]–process the molecule goes into the triplet; process [C]–generating the singlet oxygen reactive species; process [D]–the generation of an anion radical in doublet state (2S).

Process [E], [F] and [G]– the formation of the superoxide anion; processes [H]– transforms into the acid dianion. For both it performs a calibration curve and estimate the advance between the integral under the curve of the blank (solution without antioxidant) and sample (standard solution or extract with antioxidant-plant) and distribute by the standard integral. These estimates are done automatically by the software unit. The intensity of the light signal, measured by a photomultiplier, depends on the speed of the processes [F] and [G]. If the system does not include a compound capable to bond free radicals, the entire amount of generated anion superoxide is consumed by the light supplying agent “luminol”, and the intensity of the emitted light is maximal. If the system contains an amount of free radical binding agents (antioxinants) a competition between luminol and the free radical binding agents occurs for the superoxide anion radical. In this case the light signal detected by the photomultiplier has a lower intensity.

I think Dr.Dalar has answered your question very appropriately.

So there aren't correlation between in vitro and in vivo assay Dr. Haenen?

Thank you for the explanation Dr. Haenen

I would go for DPPH too

I measured the capacity of aqueous extracts from Aloysia triphylla to scavenge superoxide and hydrogen peroxide. Both are simple spectrophotometric techniques and they are very common in papers that involve natural antioxidants.

I used DPPH and TBARS methods...they are simple and quickly.....

Here are a few ways to do this: measure activities of those enzymes involved in scavenging reactive oxygen species, like superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase - the higher the activity the higher is the antioxidative function; determine levels of antioxidants like reduced ascorbate and reduced glutathione - higher levels of antioxidants give better protection against damage due to oxidative stress; quantify damage of oxidative stress by the levels of TBARS - like malondialdehyde and the higher the level, the higher is the damage.

for radical scaveniging assay, normally i use DPPH method as it is easy and simple

You can use chemical procedures or kits for determine malondialdehyde, glutathion peroxidase, superoxide dismutase, catalase, tyrosine and total antioxidant activity which were very efficient indicators for general antioxidant status in the body.

Respected Sir,

I use to follow the following assay

DPPH radical scavenging activity

The free radical scavenging activity of methanol extract was measured by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) using the method of Blois (1958). 0.1 mM solution of DPPH in methanol was prepared and I ml of this solution was added to 3 ml of various concentrations of methanol extract and reference compound (125, 250, 500 and 1000 µg/ml). After 30min, absorbance was measured at 517 nm. BHT was used as a reference material. All the tests were performed in triplicate and the graph was plotted with mean value. The percentage of inhibition was calculated by comparing the absorbance values of reference compound (control) and samples.

% of inhibition = [(A blank/A sample)/A blank] x 100

Where ‘A blank’ is the absorbance of the control reaction (containing all reagents except the test sample) and ‘A sample’ is the absorbance of the extracts/standard.

Hydroxyl Radical Scavenging activity

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell et al. (1987). Stock solutions of EDTA (1 mM), FeCl3 (10 mM), Ascorbic Acid (1 mM), H2O2 (10 mM) and Deoxyribose (10 mM), were prepared in distilled deionized water.

The assay was performed by adding 0.1 ml EDTA, 0.01 ml of FeCl3, 0.1 ml H2O2, 0.36 ml of deoxyribose, 1.0 ml of the extract of different concentration (125, 250, 500 & 1000 μg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM , pH 7.9), 0.1ml of ascorbic acid in sequence. The mixture was then incubated at 37ºC for 1 hour. 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10 % TCA and 1.0 ml of 0.5 % TBA (in 0.025 M NaOH containing 0.025 % BHA) to develop the pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation is calculated by using the following equation

Hydroxyl radical scavenging activity= {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged.

Superoxide radical scavenging activity

Superoxide anion scavenging activity was measured according to the method of Robak and Gryglewski (1988) with some modifications. All the solutions were prepared in 100 mM phosphate buffer (pH 7.4) 1ml of reduced Nicotinamide adenine dinucleotide (NADH, 468 µm) 3ml of plant extract of different concentration (125, 250, 500 & 1000 μg/ml) were mixed. The reaction was initiated by adding 100 ml of phenanzine methosulphate (PMS, 60µm). The reaction mixture was incubated at 25oC for 5 min, followed by measurement of absorbance at 560 nm. The percentage inhibition was calculated by using the following equation.

Superoxide radical scavenging activity =

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged.

Antioxidant activity by ABTS assay

The radical scavenging activity of the extract was also analyzed by the 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay (TEAC). The total antioxidant activity of the samples was measured by the ABTS radical cation decolourization assay according to the method of Re et al. (1999). ABTS was produced by reacting 7mM ABTS aqueous solution with 2.4 mM potassium persulfate. This mixture was kept at ambient temperature for 12-16 hours. Prior to assay, this solution was diluted in ethanol (about 1.89 v/v) and equilibrated at 30ºC to give an absorbance at 734 nm of 0.700±0.02.

After the addition of 1ml of diluted ABTS solution to 10 µl of sample or Trolox standard (final concentration 0-15 µM) in ethanol, absorbance was measured at 30ºC exactly 30 minutes after initial mixing. Appropriate solvent blank was also run. Triplicate analyses were made at each dilution of the standard and the percentage inhibition was evaluated at 734 nm. The percentage inhibition was plotted against Trolox concentration.

Reducing power

Reducing power was estimated according to the method of Oyaizu (1986). An aliquot of sample and standard solution were prepared. MeOH (250 µl) that was mixed with 250 µl of sodium phosphate buffer (0.2 M, pH 6.6) and 250 µl of 1% K3Fe (CN)6 which was incubated at 50ºC for 20 min. After adding 250 µl of 10% trichloroacetic acid, the mixture was centrifuged at 3750 rpm for 10 min. The supernatant (100 µl) was then taken out and immediately mixed with 100 µl of MeOH and 25 µl of 0.1% ferric chloride. After 10 min incubation, the absorbance against blank was taken at 700 nm. Ascorbic acid was taken as the standard.

I am measuring   chelating activity of microbial extract using ferrozine-Fe2+ complex formation.  According to literature, control should be devoid of microbial extract (which has colour).   Due to this, my control OD is less than test OD.  How to solve the problem Please help.   

Measuring total thiol is the significant way of calculating total antioxidant capacity. 

I also use DPPH assay. 

I Think Dr. Tresina has rightly answered your question and that too in a very elaborative way.

Please refer the attached file as an additional information

Article Antioxidative and radioprotective potential of rutin and que...

The assays employed were ferric reducing antioxidant power, trolox equivalent antioxidant capacity and scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl free radical. ... Fruits, vegetables, grains and medicinal plants are known to contain number of phenolic compounds with strong antioxidant activity.

Please have a look at these useful links.

https://journals.sagepub.com/doi/abs/10.1106/108201302026770

Article Free radical scavenging capacity and antioxidant activity of...

There is no one method preferred over the others. it is recommended to use more than one method for more reliable evaluation of antioxidant level in your samples.