FRAP - Science topic

the results are converted as mg/100 mL (expressed as dry weight soluble solids fraction by 100 ml in the vol of sample used.

Usually when you want to compare the effect of two different techniques, you must standardize by a parameter that is constant in the samples, for example the protein concentration in the tissue.

Biological activities of plant extracts that some can tested are many. In addition to the one cited above, you also have : anti-tuberculosis a antitumor, anticancer, antimalarial, , anti-inflammatory, anti-aging, anti-proliferative, hypoglycemic, hypocholesterolemic, antihypertensive, induction of resistance activities...................

The orientation of the objectives of your project should guide you, on which biologocal activity you can evaluate

Hello Duri Eun

The molecular weight of FeCl3.6H2O is 270.2.

So, add 0.0541g of Iron (III) chloride hexahydrate to 10ml ultrapure water.

You may weigh 0.0541g of Iron (III) chloride hexahydrate and transfer the Iron (III) chloride hexahydrate to a beaker of appropriate size. Add 5 mL of ultrapure water to the beaker to dissolve the salt. Transfer your solution to a 10 mL volumetric flask. Using ultrapure water, complete the solution's volume to reach 10 mL. You may prepare this solution on the day of the experiment.

It is better to prepare a fresh solution. This solution is sensitive to light.

Best.

Yes. You can use anhydrous ferric chloride. The key point is the equal molar amount of iron.

IC50 is used for DPPH method, as Weihao Meng mentioned , the reason is the absorbency because the relation in DPPH between conc. and abs. is not linear its (S shape ) line which don't allow to calculate the conc. .

In FRAP we preface the use calculation the concentration using standard as the relation is linear.

I'm wondering why do you use some methods without reading the descriptions of these methods. You must do a minimal home work yourself before asking for the help.

Hello everyone,

Sorry for late response, it's been some busy two weeks with other more pressing matters on hand. I'll do my best to answer the questions that came up tomorrow when I have access to my data again. (This is just a quick PSA so you dont think I didn't bother to check here again after getting an answer.)

Have a nice sunday and until then

Official definitions by IUPAC

The terms “antioxidant activity” and “antioxidant capacity” have different meanings: antioxidantactivity deals with the kinetics of a reaction between an antioxidant and the prooxidant or radical itreduces or scavenges, whereas antioxidant capacity measures the thermodynamic conversion efficiencyof an oxidant probe upon reaction with an antioxidant.

"Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report)

The performance antioxidant activity of essential oil (EOs) is the result of a complex interplay between components and the oxidizable tested material. ABTS and DPPH are antioxidant assays to measure the activity of the compounds to scavenge free radicals. The essential oil especially terpenoid groups containing phenolic skeleton can react rapidly with peroxyl radical, therefore could protect oxidation by those radicals. Most of the phenolic compounds capable of scavenging free radicals by donating their electrons or H (from a hydroxyl (—OH) in the benzene ring) to the radicals. On the other hand, non-phenolic terpenoids can not protect lipid from oxidation due to (for example alpha-pinene or its similar compounds) undergo auto-oxidation. Furthermore, not all EOs have the ability to reduce reduces Fe³⁺ to Fe²⁺ (as measured in FRAP assay), even the phenolic structure with ortho-dihydroxy moiety (ex. hydroxytyrosol) could chelate Fe²⁺ whereas the tyrasol that has only one OH does not show that ability.

Hi! First, you should know the photophysics of you dye. For example, may be in FRAP your observed the fast reverse photobleacing recovery.

Second, experimental geometry. FLIP is more sensitive to border proximity. May be the observed fluorecence decrease (logarithmic, in first approximation) strongly interferes with total dye decrease in all area. In FRAP, it will result to appearance of so called immobile fraction, but smaller effect on recovery rate

Dear Harto Widodo,

please see in

https://www.researchgate.net/publication/315552642.

Best regards,

I. Popov

Thank you so much Yasmine for your correction. Go a head.

Dear Chong!

Please You look at following publication:

Figure 1-3

Hi Kajanthan,

The blank in any antioxidant assay must contain all the chemicals involved in the reaction except the plant extract or the standard molecule (antioxidant).

You are wellcome Paolo Fagherazzi , I hope it helps you with your research. Good luck!

Best!

We used μmol Fe2+.g-1 TP while observing FRAP in semen plasma.

As LAM was found to disrupt microdomains in artificial membrane (doi: 10.1529/biophysj.107.104075) and potentially enriched in cell membrane nanodomains (doi: 10.1128/IAI.01549-07), a guess would be that if domains are smaller, disrupted, or even in fewer number, you would have more "rigid" lipids from rafts such as NBD-DPPE now in "fluid" membranes. So you would see an increase of mobile fraction from NBD-DPPE, and a decrease with nRhDHPE due to an enrichment of "rigid" lipids in fluid membranes. You might want to try with cholesterol probes

Do the conversion as follows: 1 microgramme per mL X 1 mMole per 55.84g (molecular weight of Fe2+)=0.0179, appropriately 0.018 micromole of Fe2+ is equaled to 1 microgramme per mL of the same Fe2+. So you can use the conversion factor to convert all your EC50 in vitro values of the results.. I wish you the best of luck!

Total Antioxidant Determination by FRAP Method

FRAP assay was carried out according to the previous study with some modification.[16] FRAP reagent was prepared from acetate buffer (1.6 g sodium acetate and 8 mL acetic acid makeup to 500 mL; (pH 3.6), 10 mM TPTZ solution in 40 mM HCL and 20 mM iron (III) chloride solution in proportion of 10:1:1(v/v), respectively. The FRAP reagent was prepared fresh daily and was warmed to 37°C in oven prior to use. A total of 200 μL of samples extract were added to 4 mL of the FRAP reagent and mixed well. The absorbance was measured at 593 nm using UV-VIS spectrophotometer (UV-VIS specord 205). Samples were measured in three replicates. Standard curve of ascorbic acid (125, 250, 500, 750, and 1000 μmol) and GA were prepared using a similar procedure.

like

follow

This paper would be helpful: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3763360/

Thank you very much! do you have any references for the said statements?

Maher H.S. Al-Mohammad Duried Alwazeer Thank you so much.

Hello Manju Tembhre, thanks for the answer, but can you tell me what is the blank to read the absorbance only for the FRAP solution?, without the analyte, because I am working with a methodology that requires the absorbance of only the initial reagents (FRAP solution). Get my kind regards.

Melissa García Reyes

What is the nature of extract.(i.e. Polar or non polar).

If polar you can dilute the samples in deionized water and also prepare Stock ABTS and pottasium per sulphate in same water.

If sample mid polar, after preparation of stock reagents u dissolved in ethanol or acetone.

I agree with Dr. María Antonieta López Hernández

Ascorbic acid is a standard antioxidant and soluble in water.

You can go ahead with AA

Adam B Shapiro agree with

Why do you want to measure antioxidan tpropoerties (using the usual colorimetric tests I suppose), it is just not relevant.

Many intervention trials showed that pure antioxidants or phenol-rich extracts cannot prevent radical associated diseases (cancer, cardio-vascular, aging …). Considering recent studies, it appears advisable not to interfere with the delicate internal redox homeostasis of the cell by non-physiological exogenous dosages of phenolic antioxidants:

- The use of total antioxidant capacity as surrogate marker for food quality and its effect on health is to be discouraged, Pompella, A. et al., Nutrition (New York, NY, United States) (2014), 30(7-8), 791-793.

- No correlation is found for vegetables between antioxidant capacity and potential benefits in improving antioxidant function in aged rats, Ji, Linlin et al., Journal of Clinical Biochemistry and Nutrition (2014), 54(3), 198-203;

- Free radicals and antioxidants: myths, facts and mysteries; Lone, Abid A. et al., African Journal of Pure and Applied Chemistry (2013), 7(3), 91-113.).

The debate on the advantages of an additional dietary uptake of antioxidants is long-standing: "Although total antioxidant capacity (TAC) may be helpful in comparing different food items, the extrapolation to their contribution of antioxidant defense in vivo and to health issues should be discouraged, with possible exception of the gastrointestinal tract. This is of particular importance because dietary phytochemicals and other small molecules have other nonantioxidant activities."(Total antioxidant capacity: appraisal of a concept, Sies, H. Journal of Nutrition (2007), 137(6), 1493-1495.)

The EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) more recently inclined to a critical position. The current "Guidance for the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health" https://doi.org/10.2903/j.efsa.2018.5136) not even contains the words "phenol" or "ferulic acid".

There is no gape the constituents of the plant and the biological activities are differ according to its location even in the same species. The different methods of estimation also affected the amounts determined. Just write your methods and results and compare with others

Javier Casas Yes,I think this is more probable than the converse if the DAG-lipid

interaction is favorable and also because of the larger effective mass of the complex.Did you actually observe slower diffusion?

Hey, try this method

1. Add 0.9ml of HPLC grade water to 0.1ml of sample solution.

2. Then add 2ml of FRAP reagent to it and mix well.

3. Incubate it for 30mins at 34 degree Celsius

4. Read the Abs. at 593nm

*FRAP reagent prepared by mixing the following solutions accordingly in the ratio of 10:1:1

300mM Acetate Buffer

10mM TPTZ in 40mM HCl

20mM ferric chloride hexahydrate

Dear read the protocol for the kit you are using.

Make 10mg/ml stock of your sample and dilute them as per your working concentrations and please see the reference article I think it is useful to you

Ok I'll try that I will tell you about the result

First, I don't use these assays. Second, what is the obvious answer?

Third, I would not recommend FRAP assay. This test is performed under much lower pH, ~2, than physiological. Fe(II) is a very active PRO-OXIDANT (https://en.wikipedia.org/wiki/Pro-oxidant). Chelating can reduce this activity.. FRAP measures the amount of Fe(III) reduced by antioxidant. Chelating activity is measured by the amount of Fe(II) bound by a standard chelator. In other words, FRAP measured the antioxidant activity, while other the pro-oxidant activity of Fe(II)

You can use DMSO to dilute the extracts

Chelation ability is best showing using Ferrous-ion chelating activity (FICA) assay. I used it during my PhD to test the ferrous chelating activity of seaweed extracts.

I used the method described by Wang et al. (2009). In a 96-well plate 100 µl of each sample extract (1 mg/ml) were mixed with 150 µl distilled water and 5 µl FeCl2 (2 mM). The reaction was initiated by the addition of 5 µl ferrozine (5 mM). Following 10 mins incubation at room temperature the absorbance was measured at 562 nm using a plate reader . The assay positive control contained distilled water and all assay reagents. The colour blank (containing sample extracts and all reagents apart from ferrozine) was used to correct the colour of the seaweed extracts. The % ferrous ion-chelating activity (FICA) was calculated as follows: FICA, % = (1 - ((Abs562nm Seaweed extract - Abs562nm Colour blank) /Abs562nm Control)) × 100

Olusegun Abayomi Olalere Ahmed Mostafa Thank you for answering. Your input is much appreciated

this link is useful

https://www.sciencedirect.com/.../fluorescence-recovery-after-photobl... -

regards

I agree with Dr. @Sabry Abdallah

Thank you Syed Mukhtar Ahmed, this assay is called FRAP assay (ferric reducing anti-oxidant power).

Thanks to all of you.

But one more enquiry, the concentration will be same as for trolox standard or it will be different? and if its different please guide how should i calculate?

Antioxidant activity must be evaluated under physiological conditions. This means either in water at neutral pH or in nonpolar organic solvents (mimicking lipids) such as alkanes. There is no sense to measure antioxidant activity of compounds dissolved in chloroform. First, make your extract soluble in water or in heptane/octane

You say you have 5 levels by factor and a value of alpha of 1.41 (square root of 2). So, I suppose you realized a central composite design with 2 factors. Am I wight?

In such a case, during the analysis of results, removing non statistically significant effects and minimizing the PRESS (maximizing the prediction R²) will give you a quite stable model. If your response vary very much (max/min is about 100 or 1000...) it could be interesting to use a transformation of your response (Box-Cox transformation) or model the log of your response or inverse (these 2 last cases are particular cases of Box-Cox transformation).

With data, it would be more simple to answer.

Hope this can help you.

Pierre Chagnon

Hi Dr Chibuike Udenigwe,

Can you help me for the calculation part. I am stucked..

For example, my sample has a FRAP value og 1.53 mM FE.. Then, I want to express it in mM Fe/g DW.. So how?

Thank you.

First of all, you should choose the way in which you are going to express your results.

For example, you could measure the absorbance change of a standard antioxidant sush us trolox, gallic acid, ascorbic acid or else you can use the IC50 value.

In my opinion, make a literature review about frap calculation in the sample you work on. There are plenty of published works.

Free radical is defined by the occurrence of an unpaired electron in an organic molecule or metal. This potentially unstable electronic configuration can react either to acquire or to lose an electron and form a more stable product. Free radicals have the potential to serve as either effective oxidants or reductants while Free radical quenching can be defined as any reaction in which the free radical forms products that are not free radicals.

I hope this information is helpful.

Plz, don't spam the RG forum. Combine your three similar questions about ABTS, DPPH, and FRAP assays into a single question.

Antioxidant action for different assays (regardless of mechanism) is similar in the sense of quenching/reducing reactive species/radicals or oxidant probes, but kinetics, potential for side reactions, and dependence on reaction conditions may differ.

no single “universally accepted” assay is adequate in itself to precisely and quantitatively detect/determine all actions of a putative antioxidant.

For more details, see the links below

Noted. Thank you all for your valuable input.

Dear Sir. Concerning your issue about the calculation of FRAP value of plant extract. The ability to reduce ferric ions was measured using the method described by Benzie and Strain. The FRAP reagent was generated by mixing 300 mM sodium acetate buffer (pH 3.6), 10.0 mM (tripyridyl triazine) TPTZ solution and 20.0 mM FeCl3.6H2O solution in a ratio of 10:1:1 in volume. Samples at different concentrations (100,200,300,400 and 500 μg/ml) was then added to 3 ml of FRAP reagent and the reaction mixture was incubated at 37 °C for 30 min. The increase in absorbance at 593 nm was measured. Fresh working solutions of FeSO4 were used for calibration. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as mmol FeSO4 equivalents per gram of sample (DW). I think the following below links and the attached file may help you in your analysis:

Thanks

Dear Nazma

If the slope of absorbance vs concentration graph of standard is also positive as that of sample then there may be chance of error in reagent preparation or in method. Take care in reagent preparation and procedure 

FRAP ASSAY

Assay: Experiments were done according to [Benzie I.F., Strain J.J.: The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem., 1996; 239: 70-76] with modifications. FRAP working solution was prepared freshly each time: 0.3 M acetate buffer (pH=3.6), 0.01 M TPTZ (2,4,6-tripyridyl-s-triazine) in 0.04 M HCl and 0.02 M FeCl3 6H2O were mixed in 10:1:1 (v/v/v) and kept away from light. 0.075 ml of phenols (final concentration 0.0001-0.01 mg/ml) or FeSO4 7H2O (final concentration 0-1.8 µmol Fe2+/ml) solution were added to 2.25 ml FRAP working solution and 0.225 ml of deionized water. The mixture was vortexed and incubated at 37°C for 30 min

away from light. Absorbance was measured at 593 nm using the spectrophotometer. FRAP working solution with deionized water instead of a sample was used as a blank.

Johannes, as Peter mentioned , please give sufficient time for incorporation of NR in hydrophobic lipid domain and then performed the experiment.

You may refer this paper- http://journal.medsys-site.com/JISANH/article/view/1320

Perv, I always measure at 593nm. I blank/ zero it using FRAP reagent+water.

I would choose the standard depending on the matrix: re human nutrition (food, beverages, blood, bile, ...) it would be the best to use Trolox (vitamin E derivative), for antioxidants in plants per se I would use gallic acid.

If money is an object, than use gallic acid for all analyses.

dear elek,

just try it out and play with the parameter - and then think also what it means to change the parameter, you know you should have a result in the end which is independent of certain parameters, or better you should always have an effect, clearer or not so clear, but if you get only a result because of a very special setting, then the question is: do you measure a real effect or just an effect because of...

consequently, you should have a logical explanation why a certain parameter set works better or worse than another one...

Thank you!

If  50mW , nearly perfect TEM00, all solidstate, CW at 473 nm is enough, I would recommend http://www.spectra-physics.com/products/cw-lasers/excelsior#specs . 

Udenigwe is correct. Have a look at the attached protocol for details.

you are welcome 

You can use the 505nm emission filter if it is long pass filter, or if it is a band pass filter extending beyond 510nm, as this is your dichroic cut-off. How it affects the counts you obtain will depend on what the two filter specs are (the 520 and 505), and where the emission maximum of your sample lies.

Whether it is essential to have an emission filter as well as the dichroic depends on how high contrast the sample you are looking at is, but since dichroics are often only 90% efficient, it is usually a good idea to use an emission filter.

hello Mr. Palani, i know what do you describe and it is a problem! First, ensure that the acetic buffer is on room temperature. Then put 1 ml of TPTZ solution in 10 ml buffer and then add 1 ml of FeCl solution. This working solution takes a straw-brown color in 37o C.

i had this blue-purple color when my acetic buffer was cold or had been made time ago.

Good luck!

Thank you!

I use tocopherol in the test of  phenols,,catechins and vitamins.

Both of them are spectrophotometric methods, which are used to investigate the in vitro antioxidant capacity of foods and biological samples. They are based on electron transfer reactions, which visually results on the reduction of a coloured oxidant (DPPH or FRAP as oxidant). Therefore, usually the results obtained from these methods present excellent correlation.

I believe the main difference is that DPPH is a stable organic nitrogen radical. The reaction progress between the radical and the antioxidant is monitored for aproximatelly 30-40 minutes at 515-517 nm, or until stability. So, we must be careful with antioxidant compouns such as some anthocyanins that absorb at similar wavelenghts and could interfere in the final results.

In the case of FRAP, there are no free radicals. What is monitored is the reduction of ferric iron to ferrous iron in the presence of the antioxidant. This is usually done every 15s until 4 minutes, at 597nm. For some polyphenols, FRAP values cannot be accurately obtained after this time.

The file article attached (The chemistry behind antioxidant capacity assays.) may be helpful.

Hope it could be helpful.

The mechanism of FRAP assay and DPPH scavenging assay is similar i.e., these methods measure the electron transfer ability of antioxidants. Therefore, higher correlation has been reported by many researchers. Moreover, FRAP assay gives excellent result for polyphenol rich compounds (antioxidants). It is better to use at least one alternate method based on hydrogen atom transfer ability. You can select either Oxygen Radical Absorbance Capacity (ORAC) or Low-Density Lipoprotein (LDL) Oxidation or Total Radical-Trapping Antioxidant Parameter (TRAP) or other assay methods.   

For methodology of FRAP assay please go through link below

Dear Vikas,

I have copied the experimental section of FRAP assay from a publication entitled " ANTIOXIDANT ACTIVITY OF ETHANOLIC EXTRACT OF Maranta arundinacea .L TUBEROUS RHIZOMES" Published in Asian Journal of Pharmaceutical and Clinical Research Vol 5, Issue 4, 2012 (for full paper see attached file) for quick view. You should make a curve such as that illustrated in Figure 6 of the paper.

FRAP Assay

Frap assay measures the reducing potential of an antioxidant reacting with a ferric tripyridyltriazine [Fe3+-TPTZ] complex and producing a coloured ferrous tripyridyltriazine [Fe2+-TPTZ]11. Generally, the reducing properties are associated with the presence of compounds which exert their action by breaking the free radical chain by donating a hydrogen atom26.Frap assay treats the antioxidants in the sample as a reductant in a redox-linked colorimetric reaction27. In the present study, the trend for ferric ion reducing activities of M.arundinacea and BHT are shown in Fig 6. The absorbance of M.arundinacea clearly increased, due to the

formation of the Fe2+-TPTZ complex with increasing concentration. The water and ethanol extracts of sumac (Rhus.coriaria L.) showed increased ferric reducing power with the increased concentration as standard antioxidants28. Hence they should be able to donate electrons to free radicals stable in the actual biological and food system.

Fig 6:FRAP Assay

The ethanolic extract of M.arundinacea was found to be an effective scavenger of ABTS, DPPH, H2O2, and NO and also possessed a good reducing power and Frap activity. Earlier reports on the antioxidant activity of M.arundinacea are very rare in the literature. Therefore, it is very difficult to compare our results with that of previous studies. The high antioxidant activity of M.arundinaacea enhanced the potential interest in these under-exploited tubers for improving the efficacy of different products as nutraceutical and pharmacological agents. The consumption of the arrowroot may play a role in preventing human diseases in which free radicals are involved, such as cancer, cardiovascular disease, and aging. We conclude that, the results presented indicate that M.arundinacea extract attenuated oxidative stress via its antioxidant properties. However, further investigations on phenols,  flavonoids, active principle, their in vivo antioxidant activity, and the different

antioxidant mechanism are warranted.

Hoping this will be helpful,

Rafik

Colour controls are OK, but I guess the question is how to discriminate whether or not the observation of high FRAP activity in the sample is real,  or there is an artefact. As redox reactions are involved, we should be sure there is not pH change among the assays of the different sanples when FRAP reagents is used.

what equation did you get?

Hi Ashlesha. You will need to do a standard curve using FeSO4 and also use a standard concentration of ascorbic acid. Have a look at my attached FRAP protocol. if you need help just ask.

A highly photobleacheable fluorophore with a high brightness... This would be the best obviously!

I would go for NBD, not because of its photophysics properties, but because of its chemical structure... I expect it to less perturbates the BLM than the Cy5 will.

i did not check, but biotin label is not good for live study. Simple you do not know, what you see... The label is not specific. If specificity is not an issue,  you can use it, i think.

You have to prepare a calibration curve of the selected standar in micromolar (X axis) and O.D (Y axis). With the linear equation you can interpolate your O.D. results. Then you have an amount of 0.1 mg of sample in the 7.5 uL of analysis (0.0001 g). So, if you divide the micromolar obtained in the 0.0001 g of sample you get your results in micromolar/g. Now it depends if you need to express in grams of extract or grams of plant/frui/ etc, you have to relate this result with the yield of extraction.

I hope this can be helpful. Please find attached a publication were we used the FRAP assay for the antioxidant activity of a fruit extract.

Best regards, 

Dear Bernard,