Do you have some Optical density for exemple ? You know the abs of ur sample before adding the dpph ?

Well, if technique has been properly performed (methanol / 515 nm / kinetic measurement during 120 min), based on your details I would think that the sample absorbs (carotene composition) at the wave length of 515 nm or causes an increase in turbidity (check it by measuring at 700 nm the mixture reaction without DPPH). I would not believe the samples would have a pro-oxidant activity.

Well, if technique has been properly performed (methanol / 517 nm with pink color indications)

Dear Margarida,

The most important parameter is the absorbance (A). Does the absorbance increase or decrease? If you observed only increase of the absorbance it is high possibility that this method (with DPPH) is not proper for measurement of oil antioxidant capacity. For evaluation the antioxidant activity of oil the best methods are: method for determining the ability of reducing copper (II) (Copper Reduction Assay, CRA) and a variation of this method: CUPRAC (Cupric Reducting Antioxidant Capacity) which is based on the ability to reduce Cu (II) to Cu (I). The Cu (II) ion occurs as a complex with neocurpine.

Kind regards,

Anna

If the free radicals are increasing only then the absorbance will increase. Please see what is the error. The absorbance in DPPH is because of the presence of free radicals.

If the incresing of absorbance appears at the beginning, you could make a blank with oil and without DPPH in order to verify if the oil has an absorbance at 515nm ?

I ve had similar experience with essential oils of Stachys species. Use ABTS radical instead.

Thanks for all the answers.

I am using this method:

Oil samples (1.5 ml) (diluted in methanol, soluble fraccion) were added to 1.5 ml of 0.15 mM in 95% methanol. The mixture was mixed vigorously and allowed to stand at room temperature in the dark for 30 min. The absorbance of the resulting solution was measured at 517 nm The sample blank was prepared in the same manner except that methanol was used instead of sample solution. A standard curve was prepared using Trolox in the range of 10–60 lM. The activity was calculated after the sample blank substraction and expressed as lmol Trolox equivalents/ 100g sample.

The sample that gives me a negative value is the residue fracction of molecular distillation (at 200 °C). The inlet samples give me a positive value and the distillate fraccion gives me a positive value (higher than the inlet sample value). When the molecular distillation is at lower temperatures than 200°C the residual fraccion gives me a positive value (lower than the inlet fraccion).

Maybe in some of your samples there is a molecule which, at the conditions of the antioxidant protocol, produces a chromophore with characteristic absorption at the same wavelength as the DPPH radical. Did you check what is going on if running the antioxidant measurement with DPPH radical omitted?

No! I didn´t, I will try it.

How stable is your oil sample? sometimes if your oil is not from the same batch can give you problems with the auto-oxidation. And if you're making the extraction of oil for much worse. I would advise that besides the DPPH test, you tried other methodological alternative.

Best wishes

Looking at only one wavelength will give you a cyclopean image of your problem. To understand why adding your oil leads to an increase of the 515 nm-signal, you should scan from 300 to 600 nm to have the whole spectrum of 'DPPH + methanol' ; 'DPPH + methanol + oil'; and 'methanol + oil'. It is possible that your oil sample contains minor compounds absorbing close to 515 nm. But even in the case your oil absorbs at, say 400 nm, the shoulder of this band may artificially increase absorbance at 515 nm. It is also possible that your oil is not 100 % solubilized in methanol and that a sort of "emulsion" is forming, but this is unlikely since methanol and oil are usually quite soluble.

I agree with all the above comments. The oil that resulted in increasing the DPPH purple colour could be having pro-oxidant activity (increasing the radicals in the solution) which is the opposite of antioxidant activity

The basic concept of DPPH and antioxidant stands on decoloration potency of test agent. I think a blank from oil could help you. on the other hand it could be also possible that oil at the used concentration does have oxidant activity.

Taking account of my predecessor (Use DPPH in the blank), the radical DPPH is purple colour and in presence of an antixidant the intensity of the coloration is reduce. Taken for yellow coloration. Because radical DPPH obtain Hydorgen. If your your extracts are antioxidants, the radical DPPH loss progressively the purple coloration. Thank you

Decolourization is stoichiometric with respect to degree of reduction.

For me, you have problems with solubility. Oil samples doesn’t dissolve good in alcohols, in some cases mixture of oil and methanol gives emulsion, which increase absorbance. But it doesn’t increasing the absorbance, but turbidity, which makes detector higher signal.

DPPH assay is not good method of measure antioxidant (antiradical) potential for oil samples.

I agree with Katarzyna that your oil samples may have low solubility in methanol. Absorbance at 517nm increased due to the emulsion. DPPH assay is not appropriate for your oil samples.

Before, must evaluate:

What is the concentration of DPPH in the analysis?

What is the concentration of oil? (maybe You should dilute the oil)

What is the chemical grade of oil? (due to chromophore production)

Discrepancy in results were observed in our DPPH assay also. We observed that absorbance was increased with increasing concentration. It might be the pro-oxidant effect and for sure the solubility issue cannot be ruled out.

Well there can be two reasons 1: the whole chemical soup is having pro-oxidant activity or 2: the test sample possesses some substance having absorption at 517 nm.

I agree with Mr. Mickaël Laguerre comments that there may be some constituents in your sample which absorb light at the same wave length that of DPPH methanolic solution. For that you should have to do some experiments suggested by Mr. Mickaël Laguerre. One more thing is that what is the colour of your oil. Because ANTHRAQUINONES absorbs light in the range about 540nm and they gives purble colour absorption. So please check it out.

http://onlinelibrary.wiley.com/doi/10.1002/1097-0010(20000901)80:11%3C1686::AID-JSFA694%3E3.0.CO;2-Y/full

Not only quinones, this problem already occurs in a lot of std solutions, just care when you work with natural products and more over when you warm that much

I think u should dilute sample in this case and then check antioxidant activity

I also want to align with the comments made by Mr. Mickaël Laguerre. However, in carrying out further experiments, I will suggest making a serial dilution of the test sample oil in MeOH up to 1: 512 and run the DPPH experiment again using appropriately the respective sample blanks. Perhaps this effort may throw some light as to where the problem lies. You may also try the ABTS assay. that works better in both lipophilic and hydrophilic milieu

It is unusual that you are observing an increase in absorbance of DPPH with your sample. Two things can be expected in your condition. Firstly, your sample has internal candidate(s) who absorbs nearly at 515 nm range. This point has extremely been taken care off by previous discussions. The second and minor point could be DPPH is not fully dissolved and in time it get dissolved with your sample. This can be avoided by testing the saturation time of dissolve of DPPH in methanol. Other controls such as only sample, sample + DPPH, DPPH in methanol etc. can be incubated to find out the above saturation time.

DPPH in methanol or ethanol medium forms DPPH radicals which have absorption maxima at 515 nm. Antioxidants scavenge this radical thereby reduce the absorption at 515 nm. Since DPPH is already in the radical form whatever pro-oxidants present in your sample will not increase the radicals further. Therefore only answer for getting the increased absorption with some of the samples is due to the absorption by certain compounds present in the oil at 515 nm. This can be eliminated by taking one more reading with only methanol and oil at 515 nm and subtract it with the sample reading.

I totally agree with Mr Shivashankara comment that adding whatever pro-oxidant cannot lead to an increase of the absorbance at 515 nm.

Most likely they are observations of Mr Laguerre'a and Mr Shivashankara confirmed also our research. Try to measure the absorbance at a time.

please correlate with negative control. please check absorption of oil separately and correlate with the (DPPH + test solution) by taking the same concentration of the sample. then also if you are not finding any conclusion, i think, the oil in interfering with DPPH not in defined mechanism.

There cant be a relation between the oxidant or pro-oxidant nature of the sample on DPPH radical coloration.

DPPH is a coloremetric method depend on change of the color (spectrophotmetrically)

You can try to perform a color correction for the oil with methanol and negate that value from your DPPH+oil reading.

DPPH is artificial Free radical. To the best of my knowledge, it is not detected from any living entity and therefore, there is no chance to have it in extract of plant under Study. Taking into account your observation, Addition of plant extract never increases the concentration of DPPH but, it reduces the concentration of DPPH by reaction of DPPH radical with those of any antioxidant(s) compound if it is containing.

For dissolving oil, petroleum ether of chloroform are best solvents, though methanol is universal extractant for most of the phytochemicals. Increased absorbance is may be the result of formation emulsion (slight turbid solution) formed by oil as, it might be partially soluble. Therefore, this emulsified solution is not absorbing the light, but it acts as barrier to light to pass through the solution. This obstacle, increases the absorbance by the test sample, which reflects the high antioxidant capacity of an oil sample as observed by you, but it is not actually so.

Dear, Keep in mind, the absorbance of the standered solution of DPPH should be always Higher than the test sample.

It is not possible that any oil will increase color because then it is helping in generation of free radicals. If it will generate free radicals then it will be very toxic living cells. May be this type of oils can be used in generation of mutants in plants.

Did you test sample dilution before analysis in order to see if that still happens?

It seems that your sample is absorbing at 515 nm.

DPPH is a stable organic proton free radical in its crystalline form, but in aqueous solution it becomes reactive. It contains an odd electron which is responsible for the absorbance at 515-517nm and also for a visible deep purple colour. It accepts an electron or hydrogenatom to become a stable diamagnetic molecule causing a colour change from purple to yellow and the solution losses color with number of electrons taken up. Remaining DPPH measured after a certain period of time is inversely proportional to the activity of the antioxidant i.e. maximum yellow color indicate high antioxidant activity of plant extract with less DPPH molecules remained in performed assay. I think it is enough to clear difference between coloration and decoloration.

when you leav the solution of DPPH for long time the color changed ,, that is the reson for making blank every time.

It's quite usual in case of oils to arrive at the results which you have obtained. The probable reason which could be thought of includes phase separation while mixing oil and methanol, and presence of minor components in oil which absorbs at 517 nm. In my suggestion, a modified DPPH method can be tested where methanol is replaced by iso-octane. Iso-octane has better oil solubility and its fairly compatible with DPPH reagent. You may wish to test it and adopt, if it works.

There should be color interference. Test your sample for any color interference without DPPH.