Hi Kaneria

There is no the best method for the evaluation of antioxidant activity of certain samples. I think, the method of choices are depended on solubility of your sample. If your sample dissolve in organic solvent only (chloroform, acetone, methanol or ethanol), DPPH method in methanol is your first choice. By contrast, the sample only dissolved water, ABTS and FRAP method are more likely fit your need.

Other methods like complex lipid peroxidation, hydroxyl radical and superoxide anion scavenging assays are used in case of you want to investigate which type of reactive species scavenging mechanisms.

My previous projects, I use just only 2-5 mg of extract or pure compounds to evaluate the antioxidant activity. Anyway, you should have level of cutoff concentration to judge them 'active' or 'not active'. I usually use 10 micromolar for this purpose.

Feel free ta ask me.

Good luck with you work.

Dear Mital, Kindly follow the following question of Zongo Cheikna,

Can anyone suggest the best technique to estimate the level of antioxidant compounds in a plant extract?

You will get useful discussions of peers there.

There is no technique considered as best for determination of antioxidant potential of plant extracts. researchers have used collection of techniques to show this activity in plants both in vitro and in vivo.

Kit based assays such as "http://www.bmglabtech.com/media/35216/1044022.pdf" might be helpful for "sensitivity issues", esp. for your smaller fractions. But none of the methods are complete, but are merely indicative and rely on different mechanisms.

Depending on what you are doing- a PhD or MS Thesis, you can judge what you want as the outcome- some data for a degree and, if NOT, but a good research investigation leading to a PhD- then you just can not say, that you do not have enough plant materials, fractions, or extracts. Simply, you need to redo the entire exercise (using bulk starting materials) to be able to perform all these assays.

Also depends the resourcefulness of you department/ PI,  and hence judicious use of these reagents, than merely repeating them.

Be reminded that anything in vitro is JUST in vitro, and is least significant in terms of publication value, meaningfulness- rather, what the world believes and is helpful to mankind is IN VIVO- like genetic mice models, mutants and so on. Or else, if you look at the literature there have been thousands of plants, with millions of assays and zillions of molecules and correlations between assays and activities, but people are still repeating antioxidant activities of lemon, ginger, garlic- which we already know (from thousands of years of use by humanity!)- but no therapeutic/ medicinal commodities have developed from these as on-the-counter/ prescription drugs !

Best of luck for your research though.

You'd better work with at least two different methods, then I suggest DPPH and FRAP for presenting good correlation.

Good luck.

Thank you all for your valuable suggestions...

Hi Kaneria

There is no the best method for the evaluation of antioxidant activity of certain samples. I think, the method of choices are depended on solubility of your sample. If your sample dissolve in organic solvent only (chloroform, acetone, methanol or ethanol), DPPH method in methanol is your first choice. By contrast, the sample only dissolved water, ABTS and FRAP method are more likely fit your need.

Other methods like complex lipid peroxidation, hydroxyl radical and superoxide anion scavenging assays are used in case of you want to investigate which type of reactive species scavenging mechanisms.

My previous projects, I use just only 2-5 mg of extract or pure compounds to evaluate the antioxidant activity. Anyway, you should have level of cutoff concentration to judge them 'active' or 'not active'. I usually use 10 micromolar for this purpose.

Feel free ta ask me.

Good luck with you work.

Each assay is based on very complex chemistry of free radicals behind the scene. You have to understand well what you are measuring by each assay. Might be helpfull to have a look at these pubs:

DOI: 10.1016/S0076-6879(02)59199-0

DOI: 10.1023/A:1021249717025

It is good to use assay combination to asses antioxidant capacity. Certain antioxidant tests do have the limitation and involve complex mechanisms. You can use a combination of assays for scavenging electron or free radical (i.e DPPH, FRAP) with the assays associated with lipid peroxidation (B-carotene bleaching, TBA). Combination of methods will tell us the antioxidant properties of the sample in more detail. 

Good luck

Dear you cannot ascertain antioxidant potential of and sample with one method. To evaluate the antioxidant potential of any sample you have to use different method viz. DPPH, FRAP, ABTS, Mo reducing assay, NO scavenging assay and Bita-carotenoid linoleic acid assay. Because every method have its own importance, also keep one thing in your mind that method like DPPH, FRAP, Mo reducing assay have not any biological relevance. where as NO scavenging assay, Bita-carotenoid linoleic acid assay, H2O2 reducing assay have biological relevance.

I agree with Serm Surapinit that there is no best method for the evaluation of antioxidant activity of samples especially plant extracts. It depends on the solubility of your sample, that will determine the method to use. 

DPPH is easy and reliable  

If you want to get rapid results to be published without any relevance to the "real world" the best option is obviously DPPH in methanol. However, if you want to obtain results that are representative in some extents to natural and biological systems, you have to pay attention to two points when chosing an antioxidant activity measurement protocol:

1. Be sure to use an assay that is mainly constituted of water as solvent and not organic solvent. Otherwise, you will get results that are not directly applicable in aqueous systems that represent 99 % of the known systems on earth (be careful with DPPH using methanol). From this point of view, ORAC is a good option but you will need a microplate reader.

2. An assay with a lipid/water interface is ideal for this purpose, since homogenous solution is an artifact that is only encountered in laboratories ; world as we know it is full of heterogenous and compartmentalized systems with interfaces, mostly lipid/water ones (micelles, emulsions, membranes, living cells, etc.). Conjugated autoxidized triene assay is a rapid method performed in an emulsified system that can be done using a microplate reader. You can also use all the lipid oxidation measurements. To do so you have to mix an oil with water plus emulsifier to form a stable emulsion, afterward you measure oxidation of this oil in presence or not of your antioxidant extract. The only problem is that it is very long and tedious! Good luck...

see the paper 

Evaluation of antioxidant properties of some Indian vegetable and fruit peels by decoction extraction method

S Chanda, N Amrutiya, K Rakholiya

Am J Food Technol 8, 173-82

If u have small samples..you can try on beta carotene bleaching or ORAC using microplate methods..only use in ug amount..I like beta carotene because it also tell us the antioxidant or prooxidant activities..you can check my papers

Best of luck

cheers

What is the best method and technique to measure antioxidant activity in water?

I socked grounded moringa seeds in greywater, and a week (being sure of coagualtion). I suppose to analyse for antibacterial, antioxidant, antibiotics and anti fungi properties.

There are several methods that can be used to evaluate antioxidant activity but a rapid, simple and inexpensive method to measure antioxidant capacity of food is DPPH which involves the use of the free radical, 2, 2-Diphenyl-1- picrylhydrazyl (DPPH) which is widely used to test the ability of compounds to act as free radical scavengers or hydrogen donors and to evaluate antioxidant activity.