Yes, Guilherme is right, b-carotene bleaching inhibition may be the best way to test this, or even TBARS assay, for it's sensibility...

Not related at all, TPTZ is the component of FRAP reagent only.

If you are using Trolox as standard, the FRAP value is the conc of Trolox equivalent.

What do you mean by TPTZ?

TPTZ means 2,4,6-Tri(2-pyridyl)-s-triazine

Yes, results are expressed in uM Trolox equivalents/g of essential oil. So for calculations I don't need to put into the equation the amount of FRAP reagent?

Usually, FRAP method is not good method for antioxidant activity determination of essential oils due to low pH in system (reduction of Fe3+ to Fe2+ at pH 3,6) or water as medium (lipophilic caracteristic of essential oils- usually very small number of polar substances- CHO fraction).

Generally:

TPTZ is just a indicator an it is responsible for blue color of final product (Fe2+-TPTZ complex).

You are actually measuring the ability of your sample to reduce Fe3+ to Fe2+ form. Bioactive compounds are able to do that reduction (electron transfer (ET) characteristic of antioxidants). You need to create the calibration curve using pure Fe2+ (usually FeSO4 or FeCl2) by testing this compound (standard) in different concentrations. From the equation of the calibration line you calculate the ability of your sample to reduce iron. The results are usually expressed in equivalents of mg Fe2+/per liter or in some other units that you used when you prepared standard solution.

I am sorry, I didn't read your question carefully, but again I think that the easiest way is to create calibration curve (x-concentration of the standard; y- absorbance) for standard and from its equation calculate the FRAP value.

Don't forget to take in account the dilution factor because great number of samples must be previously diluted.

Also every instrument (spectrophotometer, microplate reader) give different values so you can not use data obtained on some other instrument.

Follow the answer of Ivana Generalic

i have used same methodology

ANTIOXIDANT CAPACITY AND PHENOLIC CONTENT OF THE AQUEOUS EXTRACT OF COMMONLY CONSUMED CUCURBITS

Comparative Analysis of the Antioxidant Activity of Cassia fistula Extracts

You can download from research gate

Thanks Ivana. I made a standard curve of trolox solution in methanol. Different concentrations of essential oils were diluted in methanol as well. I can't agree with you that FRAP method isn't an appropriate method for EOs antioxidant activity determination because in many articles this method is used.

I know that you can find this method used in lot of articles, as well as DPPH (dissolved in EtOH), but the logic is that if you mix something polar and non-polar (like most of terpens present in essential oils) you get emulsion and you have problems with results obtained on spectrophotometer (reproducibility of the results). Also it depends on chemical profile of oils.

According to my opinion It is better to fractionate oils in CH and CHO fraction (polar). Then you will also see that CHO fraction is mostly responsible for antioxidant activity (done that with sage oil).

But don't worry, probably you will not have problems with the publication of the results.

:-)

P.S. I think that standard curve obtained by Trolox is identical as one you get when you use Vitamin C. Fe2+ curve have 2-fold lower slope.

You're right ;)

Dear Agnieszka , I agree with Ivana , His advise is complete but i think that for getting the accurate results you should survey antioxidant activity of the EO by two methods of (DPPH and FRAP) and finally by comparing antioxidant activity of standard compounds such as Ascorbic Acid and Vit E ( alpha tocopherol) decide which one is better and present the results as EC50.

Witam, niestety nie pracuje z metoda FRAP wiec nie moge pomoc.

Dear Majid, thank you for advice.

Dear All Colleagues,

Very useful and valuable answers but I think b-carotene bleaching test is the more appropriate to this intent

Yes, Guilherme is right, b-carotene bleaching inhibition may be the best way to test this, or even TBARS assay, for it's sensibility...

Aqueous ascorbic acid solutions at 100, 250, 500 and 1000 micromol (equivalent to 200, 500, 1000 and 2000 micromol FRAP) can be used. For details please see Parihar MS et al., (2004). Susceptibility of hippocampus and cerebral cortex to oxidative damage in streptozotocin treated mice: prevention by extracts of Withania somnifera and Aloe vera. J Clin Neurosci. 2004 May;11(4):397-402. (PMID: 15080956)

Reducing power assay (Iron reducing activity)

Different concentrations of Ceratonia siliqua leaves extracts (0-1 mg) in 1 ml of distilled water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml. 0.1%) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid, tannic acid and gallic acid were used as standards. Phosphate buffer (pH 6.6) was used as blank solution. All analyses were run in triplicate and results averaged.

Hopefully you can find the answer in details in this article

The FRAP assay measures the ability of the antioxidants to reduce a ferric-

TPTZ (Fe3+-TPTZ) complex to the blue-coloured ferrous form

(Fe2+), which absorbs light at 593 nm. Briefly, 0.06 ml of the standard

(ferrous sulfate, FeSO4.7H2O) or sample was mixed with 2 ml of

FRAP reagent (prepared by mixing 300 mM acetate buffer [pH

3.6], 20 mM ferric chloride, and 10 mM TPTZ in 40 mM HCl in a

10:1:1 [v/v/v] ratio) in a cuvette (Fisher Scientific), which was

incubated at 37°C for the reaction. After 4 min, absorbance readings

were taken at 593 nm with a Lambda Bio UV Spectrophotometer

(PerkinElmer Ltd.). The FRAP results were expressed as μg of

ascorbic acid eq./g fresh-frozen weight. Concentrations beyond

the highest point (1 mM) of the linear range of the standard curve

were diluted before final analysis.