is that the high content of sugar in the extract may act in the TBARS assay? because i tried to evaluate the antioxidant activity of date fruit using this assay but it doesn't work.
TBARS means Thiobarbituric Acid Reactive Substances. This assay allows to evaluate lipid peroxidation and it is widely used to assess the antioxidant activity of plant material
yes. aldehydes interfere with TBARS assay, thus sugar effect the result of TBARS assay
Yes! But this is not a measure of antioxidant activity, but a marker of the accumulated oxidative stress/damage.
Relative high contentes of sugars, phenols and proteins might interfere the reading of the adduct at 532nm. Try Hodges modification of the method that consideres interfering compounds.
Hodges, D.M. et al., 1999. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207(4), pp.604–611.
[Edited to note that is a mesure of oxidative stress/damage on the lipidic phase]
Dear Eimad,
Yes, sugars do interfere with TBARS assay. Unfortunately, majority of researchers overlook the same as they believe that this assay is specific for quantification of MDA (which is a cytotoxic product released during lipid peroxidation) and accordingly degree of lipid peroxidation. It is unfortunate that scientific world (in particular Editors/Chief-Editors and Reviewers) believes blindly on published protocols and directs researchers to follow the same.
MDA level (or let us say lipid peroxidation) gives an indication regarding the degree of oxidative stress (i.e. quantity/degree of ROS being generated) and not the antioxidant activity. If you wish to determine antioxidant activity, you may use DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. To the best of my knowledge sugars do not interfere with this assay.
Dear Eimad,
yes! aldehydes interfere with TBARS assay as MDA is an aldehyde, thus sugars effect the result of TBARS assay. But this is a measure of the capacity of a plant extract to inhibit lipid peroxidation induced in vitro. If a plant extract inhibits this oxidation, it has antioxidant activity. To eliminate this interference, you should set a blank test tube containing all the ingredients solutions and plant extract except the substrat of MDA. Then substract this contribution from other tubes.
Dear Njayou,
Please consider the following points:
(i) MDA in living systems is generally a product of lipid peroxidation;
(ii) Lipids peroxidation is (generally) directly proportional to reactive oxygen species (ROS);
(iii) ROS are generated due to oxidative stress (to the best of my knowledge all abiotic stresses lead to oxidative stress);
In light of above, lipid peroxidation is a measure of oxidative stress.
Yes, I certainly agree that anything (be it be plant extract) that inhibits/reduces lipid peroxidation is an antioxidant,
We can certainly, plot a perfect standard curve by making different levels of MDA (if available) react with TBA. But, if you want to measure the potential of plant extract to inhibit lipid peroxidation (that results in MDA production), how can one get rid of MDA?
Point that we need to address is the way we can get rid of sugars from date palm fruit extract or can we have a protocol alternate to TBARS assay?
thank you Dear Sradhi for the light. I used to induce lipid peroxidation in vitro using rat liver homogenate. I suggest to Eimad to consult the following article Too
Comparison of two methods used to analyse lipid peroxidation from
Vaccinium myrtillus(L.) during snow removal, reacclimation and cold acclimation
Erja Taulavuori, Eeva-Kaisa Hellström, Kari Taulavuori and Kari Laine
ournal of Experimental Botany, Vol. 52, No. 365, pp. 2375–2380, December 2001
Dear Njayou
i have tested my extracts of date fruit using rat liver homogenate to evaluate the ability of these extracts to inhibe lipide peroxidation as test of antioxidant activity but i found that these extract induced lipide peroxidation. when i use an other assay such as FRAP, DPPH or ABTS i observe that these extract possessed an antioxidant activity.
hence i think that other compound excested in extract especially sugar may react with TBA and absorbe at 532nm
I agree with all the above. Also the level of fat and therefore lipid oxidation by-products should be very low and may below the range of the assay.
Dear Eimad,
FRAP, DPPH or ABTS tests measure iron chelator or antiradicalar properties of your extract. I think you used a suitable model for evaluating antioxidant activity. My earlier proposition to eliminate interference would be useful.
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