Hi! I hope this article can help you on your research.

http://pac.iupac.org/publications/pac/pdf/2013/pdf/8505x0957.pdf

What do you mean by "level of antioxidant compounds"? Do yo want to estimate the net concentration of compounds with antioxidant properties in the extract?

Sorry but we studed only in human blood and different tissue extract

There is a number of assays you may conduct if you want to measure the antioxidant levels in an extract. Those that I am familiar with is ORAC, FRAP and TEAC. You should be able to get ample litterature on these assays. Hope this helps.

Do you need to estimate the total content of antioxidant compounds or individual compounds?

There is not any technique showed the best for estimation of the antioxidant activity in plant extract. I think it should be evaluated from more than one technique because the antioxidant activity of the extract might be act as a chain breaking antioxidant or a preventive antioxidant.

I think the answer could help you.

Good Luck..

To evaluate the antioxidant power of the extract, you can proceed with reducing power assay model and total antioxidant activity model in vitro. Further more, to evaluate the antioxidant activity of the crude extract, you can choose any one or all of the in vitro assays such as DPPH radical scavenging activity/ ABTS radical scavenging activity/ Hydroxyl radical scavenging activity/ Hydrogen peroxide radical scavenging activity/ Nitric oxide scavenging activity/ Superoxide radical scavenging activity / Metal chelating activity etc. Then you can go for in vivo studies.

You can fractionate the extract with different solvents and evaluate each fraction in the above mentioned models. The fraction with stronger antioxidant activity can be selected for column chromatography for isolation of responsible antioxidant compound/s.

Plant extracts contain several phytoconstituents. Normally phenolic and flavanoid compounds are thought to be responsible for antioxidant activity. Accordingly you can estimate the total phenolic content as well as total flavanoid content in the crude extract. These are preliminary assays for evaluation of antioxidant power of a plant extract. Please refer to published articles for details.

Dear Zongo Cheinka,

hi, actually there is no such as best technique! you can either perform easy assays e.g. Folin, Dpph, Teac, Frap to test total phenols, antioxidant capacity and reducing power of plant extracts. then if wanted you can perform HPLC-DAD or LC-MS analysis to determine specific antioxidant compounds in your extracts

http://www.sciencedirect.com/science?_ob=ArticleListURL&_method=list&_ArticleListID=-307637359&_sort=r&_st=13&view=c&_acct=C000228598&_version=1&_urlVersion=0&_userid=10&md5=29156fa539c0da840d8ce81d8f07e4c4&searchtype=a

Hi Zongo,

The relevant question, as other have pointed out is: what do you need to estimate the level of antioxidants in the extract... is it to test purification steps? is it to propose it for an antioxidant action? is it to compare its antioxidant compounds against other extracts? the answer to these questions is extremely relevant since the method will vary (imo) for each case. There is no consensus regarding which antioxidant molecules or organic functions are more significant for the antioxidant activity: there are water-soluble molecules with antioxidant functions as well as lipid-soluble molecules. Organic functions vary from phenols to thiols to antocyanins to even mucins, all of which exert their antioxidant action through different (yet often complementary and synergistic fashion). Therefore, quite frequently, the overall antioxidant capacity of an extract is not just the sum of the antioxidant capacity of the main molecules with antioxidant properties occurring in such an extract. Finally, you can measure and compare general antioxidant properties using ORAC, DPPH, APPH (and many other nitrogen centered free radicals, which differ in reactivity and stability with those oxygen-centered radicals) all you want but until you don't measure the protection towards oxidative stress in a biological system (that is in the presence of biological molecules), it is extremely difficult to predict what will happen in vivo.

So, my advice is to first establish what is the underlying goal for measuring the antioxidant activity of an extract and then figure out what is the best way to assess that.

Best wishes! Paula

There are good answers you can follow viz Mr. Jovale, Mr. Dario, Mr. Manjir, Mr. paula.

As there are different mechanisms of in vitro antioxidant action, and if you are interesting in an estimation of the overall antioxidant effect of the extract, you are obliged to use at least one method that is seisitive to each of the possible types of AOA. If it is me, I will use two methods for each possible type of AOA, to be on the safe side:) And bare in mind that in vitro and in vivo AOA may significantly differ. There are compounds that do net exhibit AOA in vitro, but are good in vivo antioxidants...

Good luck:)

The simplest and most widely used, first level assay is DPPH. scavenging study with your extract at different concentarions say 100-1000mug/ml. See how the % radical scavenging activity varies.

Hi，zongo. There is a new assay to identify antioxidant ability by HpeG2 Cell,named Cellular Antioxidant Activity (CAA) Assay. You can find more detail in the following links. Good luck. http://www.ncbi.nlm.nih.gov/pubmed/17902627

I agree with all the above statements and suggest you to select the method on the basis of mechanism of antioxidant action you want to assess relating to the particular activity and the related disease. If you want to calculate for 'some medicinal plants' as your question says, I believe you are interested in comparison among different plants you have. In such a case, any method you use, you will find a comparative result. But as different plants have different modes of anitoxidant activity, you can find the results variable. Means in one method eg DPPS, you can get a good activity for sample A but in another method like NBT, it might not be the same. I hope you understand my point. I am attaching a file containing detailed procedure for antioxidant activity using different methods available.

I am attaching another file in which antioxidant activity has been done on 32 different plants. This should also help you.

Pls find the attached paper wherein antioxidant activity of various tea samples has been assessed. Hope will help your cause.

I'd suggest the 2,2´-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) / Horseradish peroxidase (HRP) decoloration method (Arnao et al., 2000; Cano et al., 2000). We modified this method to evaluate the antioxidant activity of essential oils of thyme (Blanco et al. 2010, Blanco et al 2012)

You may consider adding free radical ( superoxide,hydroxyl nitric oxide)scavenging assays and inhibition of hydrogen peroxide-induced lipid peroxidation in brain homogenates. these are all useful assays to determine antioxidant potentials of plant extracts.

moreover,

You could also think about the FRAP (Ferric reducing Antioxidant Power)test. It allows you to test the ability of a crude extract to reduce Iron. Thus, you plant extract can be consider as a good electrons donor.

It is a simple test that you can easily perform.

Dear Zongo,

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.

Kindly find the papers on the same.

Respected Sir,

                           Before estimating the antioxidant properties in a plant extract, first analyse the total phenolic and flavonoid content in your sample and then proceed. The following methods may be useful for you to estimate antioxidant activity.

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.

Halliwell, B., Gutteridge, J. et al. 1987. The deoxiribose method: a simple test to be assay for determination of rate constants for reaction of hydroxyl radicals. Ana. Biochem.165: 215-219.

Oyaizu, M. 1986. Studies on product of browning reaction prepared from glucose amine, Jap. J. Food Chem. 44: 307-315.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, E. 1999. Antioxidant activity applying an improved ABTS radical cation decolonization assay. Free Rad. Bio. Med. 26: 1231-1237.

Robak, J and Gryglewski, R.J. 1988. Flavonoids are scavenges of superoxide anions. Biochem. Pharmacol. 37: 837-841.

Blois, M.S. 1958. Antioxidant determination by the use of a stable free radical. Nat. 181: 1199-1200.

Good Luck

Dr. Tresina is right and may be followed

Hi.. I want to ask few questions regarding DPPH assay. How do I plot the graph for my DPPH result? Im quite confused with the calculation. How should I expressed my result in Trolox equivalent? Thank you.

You have to do total phenolic content first and you have to extract dry powder than do DPPH, FRAP, metal ion & SOSA.

Abts and FRAP test may also be suitable to assess anti oxidant activity of plant extracts. They are easy to implement.

there are a lot of stable free radicals such as ( ABTS, DPPH, FRAP ) available to evaluate the potential of natural or synthetic antioxidants.

Total Polyphenol Content is the first and foremost steps and than you can go for individual texts like DPPH, FRAP, SOSA etc.