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Free Radical Scavengers - Science topic

Free Radical Scavengers are substances that influence the course of a chemical reaction by ready combination with free radicals. Among other effects, this combining activity protects pancreatic islets against damage by cytokines and prevents myocardial and pulmonary perfusion injuries.
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Do not need it anymore. The question has already been solved.
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Dear Chunhua Wang,
Radical scavengers are the species which can interfere with the effect of Radical species in a reaction. Scavengers can react very fast and specifically to the radical and can quench the effect of the radical in a reaction. Mostly these scavenging agents are electron-accepting or electron-donating species or have a particular site where they can specifically react with the radical ion.
This paper might help you with a better understanding .
Thank you.
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I have quantified the free radical scavenging activity of plant extracts.
With excel calculate the EC50 value from the linear graph(y = mx + c) (%inhibition Vs. Concentration) where i substitute the y value with 50, and solve for x.
With Graphpad prism, i first transform the experimental data to logarithmic values. Thereafter, i normalise the data set (whereby the highest value was taken as 100 and lowest value as 0).
I then use the normalised data set to graph a non-linear regression curve fit, from were the IC50 is then generated by the program.
These two methods give me significantly different EC50 values. Which is the accurate approach to calculating the EC50?. Please help.
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[1]. Use linear regression (LR) to determine IC50 for radical scavenging. This is by far the most common method with the DPPH assay. The use of NLR is more common for extracting the pharmacological IC50 from drug-dose response curves.[2] The spectrophotometric antioxidant assays are all based on Beer Lamberts Law, which is a linear equation. [3] Please fit you data to Y = mx. The intercept is always zero as in zero % inhibition for zero antioxidant concentration. see this articlel,[ [Molyneux, P., 2004. The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity. Songklanakarin J. sci. technol, 26(2), pp.211-219.]] I hope this reply is not too late.
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Need help regarding protocols
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The following links may be useful for measurement of melanin content:
Quantitative analysis of melanin content in a three-dimensional melanoma cell culture. Scientific Reports volume 9, Article number: 780 (2019).
Methodology for evaluation of melanin content and production of pigment cells in vitro. .Photochem Photobiol May-Jun 2008;84(3):645-9.
For free radical scavenging assay:
Evaluation of free radical scavenging activity of various extracts of leaves from Kedrostis foetidissima (Jacq.) Cogn. Food Science and Human Wellness
Volume 4, Issue 1, March 2015, Pages 42-46.
Evaluation of the free-radical scavenging and antioxidant activities of Chilauni, Schima wallichii Korth in vitro. Future Sci OA. 2018 Feb; 4(2): FSO272.
Evaluation of Antioxidant, Free Radical Scavenging, and Antimicrobial Activity of Quercus incana Roxb. Front. Pharmacol., 23 November 2015 | https://doi.org/10.3389/fphar.2015.00277
In vitro antioxidant and free radical scavenging activity of different parts of Tabebuia pallida growing in Bangladesh. BMC Research Notes volume 8, Article number: 621 (2015).
There are several published research papers that gives protocol for the analysis of free radical scavenging activity assay (as mentioned above). As per your lab set up and research objectives, you can go through aforesaid links and conduct the experiments.
Good luck
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I am performing DPPH test. My crude plant extract is showing higher percentage of free radical scavenging activity than standard BHT for each time. Is it okay or is there any problem? I'm not understanding it.
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this means that your extract has excellent antiradical power and this is very good. several articles have found that their extracts have shown results like yours. good luck
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I am looking for alkali stable radical scavengers.
I have a glycol solution of 80:20 ethyl diglycol:butyl glycol.
And want to add 10% KOH (so a waterless alkaline).
I have tried spiking the glycol solution with up to 2% BHT (butylated hydroxy toluene) before adding the KOH. As BHT is commonly used as antioxidant (radical scavenger)
However, it still seems unstable.
I was wondering if there are any Alkali stable radical scavengers or anything to supress discoloration and further (aldol) condensation of the present glycols.
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I'm performing the antioxidant activity of essential oil by DPPH manner, and I was wondering about the value of IC50 it seems so high because that of reference which is ascorbic acid is around 0.018 mg/ml, whereas that of my sample is 2 mg/ml. There is a big difference!
When we can consider an essential oil as an antioxidant agent? Is there any intervale of IC50?
Is there any interest to say that this essential oil having an antioxidant activity?
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Harto Widodo The definition of antioxidant can't not be based on the mechanism. There area thousands of molecules which fit your definition, but they are not antioxidants. The concept of antioxidants belongs to life-sciences. In the field of chemistry dealing with oxidation by dioxygen the term "inhibitor" is commonly used. I think that the best definition of an antioxidant was given by Halliwell and Gutteridge: "an antioxidant is any substance that delays, prevents or removes oxidative damage to a target molecule”
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I am looking for the reagent which can scavenge superoxide in living cells. Most of ROS scavenger, like tempol, inhibit not only superoxide accumulation but also reactive nitrogen spices (RNS). But I am actually looking at the effect of RNS in oxidative stress but not superoxide. Dose anyone know the cell permeable superoxide scavenger?
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Hey Kazushi,
Tiron (1,2-dihydroxybenzene-3,5-disulfonate) is a vitamin E analog, cell-permeable
and acts as a global superoxide scavenger as an alternative for TEMPOL.
If you like you can have a look at our recent review:
"Functions of ROS in macrophages and antimicrobial immunity".
In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).
Functions of ROS in Macrophages and Antimicrobial Immunity
February 2021, Antioxidants 10(2):313
All the best and stay healthy,
Marc
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Please look at the image. It is showing DPPH free radical scavenging activity of something...
According to the image, what would be the IC50 value of Ascorbic acid used as a standard for that study?
Formula-
IC50 = (50-b)/a
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You can use ED50 Plus v1.0 software.
This is very precise software to calculate EC50.
You can find it for free online via google search with "ED50 Plus v1.0 software".
With kind regards
Christophe
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Specifically am asking for antioxidant analysis( TAC, TFC and TPC) and free radical scavenging activity assays ( ADDH, FRAP AND BTS). it would be great if the reason could also be explained?
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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).
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please i hope to now if the usage of diffuse basis function in calculs, would change the free radical scavenging capacities of biological molecules?
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There are many essays to assess free radical scavenging activity in plant based preparations. How do I select the best method? What are the factors to be considered in selecting the most suitable essay?
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DPPH, ABTS, DMPD, and many more methods are available
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I have determined the % Inhibition of DPPH free radical scavenging capacity. I have the absorbance value at 517 nm of my samples. Can I determine the Trolox Equivalent Antioxidant Capacity (TEAC) by plotting these same absorbance values in the standard curve prepared from different concentrations of Trolox?
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You can try to find the curve for the Trolox from the equation. For y, we substitute the EC50 absorbance from the extract and we obtain x, the desired equivalent in mM, i.e. TEAC for short.
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A extract of a compound herbal drug was found to contain alkaloids, flavonoids, steroids, tannin and phenolic compounds and cyanogenic glycosides. It also contain considerable amount of antioxidants. DPPH assay revealed that the compound has potent free radical scavenging activity. Therefore, may it be efficacious on minimizing free radical induced tissue damage in normal ageing process?
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Which compound you mean? Please, can you explain your question in clear details?
Regards
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I am currently working on prospects on molecular structure modification and tweaking of readily available compounds that are well-known good antioxidants in various in vitro assays. The goal is to modify the structures, synthesize them, and then assess the activities of the derivatives. Now, I am trying to find some understudied yet promising compounds that exhibit superior antioxidant capacity and has wide structural variability . I was wondering if anyone can suggest any understudied compounds and derivatives I can start with? Some literature and supporting articles would be a great help. Thanks
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Good question
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Vitamin C (ascorbic acid) is a water-soluble vitamin that is thought to have beneficial effects in patients with severe and critical illnesses. It is an antioxidant and free radical scavenger that has anti-inflammatory properties, influences cellular immunity and vascular integrity, and serves as a cofactor in the generation of endogenous catecholamines.1,2Because humans may require more vitamin C in states of oxidative stress, vitamin C supplementation has been evaluated in numerous disease states, including serious infections and sepsis. Because serious COVID-19 may cause sepsis and acute respiratory distress syndrome (ARDS), the potential role of high doses of vitamin C in ameliorating inflammation and vascular injury in patients with COVID-19 is being studied.
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Vitamin C increases the body's immunity, so it is definitely beneficial against Covid-19.
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for example what is the difference between N-acetylcistein and BHT(BUTYLATE HYDROXY TOLOUEN)? 
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Hi Elah,
all the answers given so far are very useful, but are all given from the point of (cell)biologists. You always have to consider also the chemistry and not only the cellular point of view.
In general, substances, which have a preference to give electrons are reducing components (reductants) and substances, which have a preference of taking electrons are oxidzing components (also called oxidants). Not all but many radicals are oxidizing compounds (because they are lacking one or more electrons and want to fill that gap to become stable). Of course there, are also strong oxidants that are not radicals.
A cell, as a stable biological system, wants to avoid a lot of radialcs (or strong oxidizing or reducing subtances) going around, because they react with biomolecules and therefore damage the cell. "Antioxidant" is a general term for all chemical substances, which can reduce an oxidizing substance (not only in the cell).
Reactive oxygen species (ROS) are just one example of oxidizing substances in biological systems. In this group you have to distinguish between radicals (Superoxide anion, Hydroxyl radical, Peroxyradical) and non-radicals (Hydrogen peroxide, hypocchloride, singelt oxygen, ozone). If a substance you add to your cell can react with at least one of these substances (it does not matter if radical or not) this substance is a "ROS-Scavenger". How the substance is doing the job depends on its chemistry and the ROS subspecies it reacts with. NAC for example can directly, as stated above correctly, exepct eletrons from radicals. It also refills the gluthathion pool of cells, which is necessary to decompose hydrogen peroxide. So NAC can (directly and indirectly) "scavenge" different types of ROS (not only radicals). From the cellular pooint of view it is an ROS-Scavenger, from the chemical point of view it reduces and oxdizing comopnent, so it is an antioxidant.
I am sorry this answer is a little long, but I hope this helps you.
All the best,
Marc
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Antioxidants are chemical substances that can neutralize and scavenge the free radicals
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please Mr. Elham ,, i am also not understanding what you want to say !!! please be more accurate and precise in your choosing the words for a question?
Mr. Prof.Yurii V Geleti has a right when he didn't understand your question
with regard
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Please be kind enough to clarify me what is the difference between assessing antioxidant activity and at the same time assessing free radical scavenging activity!
Looking forward for your kind response!
Thank You!
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there are myriad of antioxidant assays. some assays use free radical scavenging activity to measure antioxidant activity of crude extract or biological sample (DPPH assay). However there are many other ways to determine the antioxidant activity. some assays use reducing activity (FRAP assay).
To measure total antioxidant activity one assay such as DPPh is absolutely not enough.
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I have extracted methanol, ethanol, chloroform and water extracts of fenugreek and I have done DPPH and ABTS assays to determine free radical scavenging activity. However, I got different DPPH and ABTS results (inhibition percentages) for same concentration in same extract. Is that possible??
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Yes the data are different because of the different radicals but the trend of IC50 change by all extracts must be the same. DPPH and ABTS data correlate very well.
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Hello, I am looking for a compound which could selectively quench reactive oxygen species while not quenching carbon centered radicals (for a polymerization reaction). Thanks a lot.
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Melanin is a polyphenol.
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I am working with some oils which are not soluble in methanol, but soluble in IPA, etc. Can anyone help me the complete procedure regarding the use of IPA for DPPh assay to be done in microplate reader? also please help me with the complete protocol to be followed.
Thanks in advance.
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Sure, absolute ethanol works better than methanol
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For doing DPPH free radical scavenging assay, I have used ascorbic acid as the standard. and took different concentrations (5,10,15,20,25 and 30 microgram/ml) for both standard as well as for plant extract. Above 30 microgram/ ml, the OD value for ascorbic acid remains to be same for all concentrations. For standard (Ascorbic acid) the % inhibition values are 24.04, 54.06, 86.68, 96.63, 96.78 and 96.93.And that for sample, values are 9.63, 17.34, 27.50, 33.51, 41.68 and 46.47. I plotted the scattered plot using % inhibition versus concentation (microgram/ml). Do I need to modify anything? Please help me!!
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My dear you have to prepare higher concentration of the plant extract because the 30 microgram/ ml sample is not able to scavenge the DPPH radical and to make such concentrations until is reached higher than 90 % inhibition. Obviously the plant extract has lower radical scavenging activity than ascorbic acid.
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What is the meaning of IC 50 value in case of DPPH free radical scavenging method and how it is related with the antioxidant activity???
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IC50 value is inversely proportional to the free radical scavenging property/ antioxidant property of the sample.
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Hello all
I have an agent that increase the level of reactive oxygen species, I would like to do co-treatment with free radical scavengers (N-Acetyl-L-cysteine) to see if this will rescue cells from death. MY cells is at 5 x 10⁵ cells/ml.
For my agent I prepared working solution (20.4 µM) and then I to culture my cells in final concentration 0.4 µM, I add 20 µl of this working solution to my cells (1 ml). Now I need to do co- treatment with 50mM or 10mM of N-Acetyl-L-cysteine…my questions is how can I prepare that and how much do I need to add to my cells to get these concentration
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thanks all
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If I follow this protocol: The antioxidant activity of the extracts was  tested using DPPH as previously described with some modifications (Zhang et al., 2011). Briefly, the DPPH free radical scavenging activity of grain extracts was determined using a 2 × 10−4 M DPPH solution. Each sample (0.5 ml) was mixed with 4 ml 2 × 10−4 M DPPH in ethanol. The mixture was shaken, and then left to stand for 60 min in the dark. The absorbance was measured at 517 nm in a spectrophotometer. The absorbance of the control was obtained by replacing the sample with 80% methanol. The DPPH radical scavenging activity of the sample was calculated as follows:
% scavenged= [1- abs of sample/abs of control]x 100
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I think that the use of EXCEL method is the most prevalent and easiest method for determination of IC50, as it depends only on the plot of your data as concentration vs inhibition %%, and then use the equation of obtained line.
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I have done catalase, peroxidase, ascorbic acid and reduced gluthathione tests in antioxidant assays. I have done superoxide scavenging activity, hydroxyl radical scavenging activity and TBA test. I have calculated control, test and standard for all. How to calcuate them and plot them in graph?
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Please have a look at enclosed PDF..
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Dear everyone,
Do you happen to know any carbon free radical scavenger stable at high temperature (~ 300 oC)? I am currently trying to determining whether a reaction is using radical mechanism. The reaction breaks a C-Cl bond and forms the C-H bond. I suspected the reaction undergoes the Carbon-chloride homolysis to form the radical, then the radical abstract a hydrogen from the solvent. To test it, I could think of using either a radical scavenger, or deuteride donor. Unfortunately, the reaction occurs only at high temperature. So I need to find something stable at that temperature. Anyone have any suggestions? Thank you so much
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You should use activated charcoal. It´s an excellent radical scavenger at elevated temperatre.
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Dear All
I am studying the quenching effect in a system containing SO4 and OH radicals. I want to scavenge Oxygen using a liquid quencher since It will be sophisticated to pump N2 as an oxygen quencher. I have read that hydrazine can quench oxygen, but I am inquiring if it can also scavenge SO4 and OH radicals or not. If it can quench them, Is there any other alternative oxygen quencher that can scavenge oxygen only or what is the reaction rate?
Thanks in advance
Regards,
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Hello,
You can performed your reaction under purged N2 or Ar.
Regards,
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I'm looking for a solid OH radical scavenger, not liquid.
Are there any effective solid OH radical scavenger??
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The " effective solid OH radical scavenger" means that this material scavenges radicals faster than any other possible reactants in the solution. HO(.) react with almost all organic molecules with a rate constant >(1e+9)M-1s-1. If a solution contains about 0.1 M of organics, the life-time of HO(.) is shorter than (1e-8) s. A radical scavenger should significantly decrease this life time, say to (1e-9) s. In order to achieve this, the ratio of surface area of scavenger to the reaction volume must be very high. It's possible if you use nanoparticles. For simplicity, we can assume that all nanoparticles are the same size, say 5 nm. Such nanoparticle contains about 100 atoms and can be considered as a molecule of average size. Therefore, the reaction kinetics in such solution can be described by the same way as in homogeneous systems. In order to have the life-time of HO(.) shorter than (1e-9) s, you need to add about 10-100 times more of your solid material by weight compared with that of already present in solution. Based on this, you can answer your question yourself.
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I am embarking on an exploratory research project on secondary metabolites of marine organisms particularly on sea stars and brittle stars. What natural products should I consider first? Im looking for a list of natural products and various methods I can use to identify and quantify them. I am planning to do zoochemical analyses, in vitro antioxidant activity, and cytotoxicity.
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I hv carried out dpph assay on sargassum extract and found out following result..can someone please tell me ic50 value of it and whether it is correct or should i have to repeat the expt again..? Thankyou
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Both options are fine. But i strongly go for indicating that ic50 is >1000, clearly explaining in your thesis that 1000 was the highest concentration tested in your study. The other problem with testing higher concentrations of crude extracts is the issue of masking. Highly concentrated crude extracts have the ability to mask molecules being tested then you end up with false positives; thinking that your sample is inhibiting while it's actually just masking radicals or proteins in the case of enzymes, or even cells in the case of cytotoxicity testing. 
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Dear All
I want to measure sulfate radicals concentration using dimethyl sulfoxide. Please, can you provide me with a reference for measurement of sulfate radicals using dimethylsulfoxide.
Regards, 
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Hey Amal Elsonbaty, I also observe oxidation of DMSO by HO2-radikals to DMSO2 in solution. I think there are many possibilities to oxidize DMSO. 
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We have ROS such as O2•−, OH, H2O2,and 1O2.  The ROS scavengers are NADPH, Uric acid, Vitamin A, Vitamin C, Vitamin E, Glutathione, Beta-carotene and polyphenols, etc.  In these, which is more powerful scavengers to ROS.  Is there any selectivity?  what is the order of scavenging ability of above bio-molecules?
Please share your knowledge and Thank you in advance.
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Dear
Antioxidants have two known mechanism of action first is chain breaker and the second is the elements blockers. However H2O2 is not a free radical. All mentioned bioactive compounds have high antioxidant properties but they are more effective in different parts of the body, for example vitamin C is effective in polar parts but vitamin E is more effective in fat witch can be absorbed well. However the intake of these secondary antioxidant have a great impact on health as they help the primary antioxidant extract e.g. glutathione peroxidase and catalase.
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the results I got are oppose to each others ... hydrogen peroxide scavenging decreased and the inhibition activity of DPPH increased .. Is there a relationship between these methods?
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What are your reaction conditions ? Often hydroperoxide scavenging assays are done in a more lipophilic medium. Howver what do you expect during the hydroperoxide assay: Either scavenging hydroxyl radicals, or peroxyl radicals, or (in some assays) Lipid = alkyl radicals....from the chemical structure they´are not even close to the structure of DPPH...further you have not said a word about your scavenging samples. can be also that some radicals are sterically hindered while others are not....and all of the mentioned radicals have quite short half-life times compared to DPPH. So, it seems to be obvious that there might be differences. This is what studying antioxidant activity is about: "Expect the unexpected" !!!
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I am planning to immobilize anti-oxidants like Gallic acid in a nanocarrier with targeting moiety decorated on the surface of the nanocarrier. If i link the anti-oxidant with a covalent bond is it still possible for the nanocarrier to exert free radical scavenging properties once it reaches the target site or is it essential to release the anti-oxidant cargo to exert free radical scavenging properties at the target site.
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Dear Yaswanth
Antioxidants usually reacts themselves when exerting their function and they are consumed in the process. As such, I can not see the application of inmobilizing in the surface. In such case, the drug will be probably consumed before it reaches its target. In the interlayer they will be protected, but they will have to be released in order to perform its job.
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AAI/AAU is used for calculation of DPPH free radical scavenging assay. Its a new method for standardizing DPPH results.
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Antioxidant activity measured in terms of IC50 values, lower IC50 values better antioxidant activity. It is negative correlation between antioxidant activity and IC50 value
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Please share you experiences about the best method to determine radical scavenging on natural product samples.
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Dear Ahmad,
The following describes all radical scavenging assay methods:
Review: Methods Used to Evaluate the Free Radical Scavenging Activity in Foods and Biological Systems
C. Sánchez-Moreno
Abstract
Free radical generation is directly related with oxidation in foods and biological systems. Therefore, the search for methods to determine free radical scavenging is important. In this work are described the methods used for this purpose in both substrates as well as in specific cases of their application. The main methods comprise superoxide radicals scavenging (O2 ·-); hydrogen peroxide scavenging (H2O2); hypochlorous acid scavenging (HOCl); hydroxyl radical scavenging (HO.); peroxyl radical scavenging (ROO.), among them are the methods that use azo-compounds to generate peroxyl radicals, such as the ``TRAP'' method (Total Radical-Trapping Antioxidant Parameter) and the ``ORAC'' method (Oxygen-Radical Absorbance Capacity); the scavenging of radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) or the ABTS or the ``TEAC'' method (Trolox Equivalent Antioxidant Capacity); the scavenging of stable radical 2,2-diphenyl-1-picrylhydrazyl or DPPH . method and the scavenging of radical cation N,N-dimethyl-p-phenylenediamine or DMPD method. At present, in spite of the diversity of methods, there is a great need to standardize measurements of antioxidant activity. The search for more specific assays, giving us chemical information that could be related directly to oxidative deterioration of foods and biological systems could be the objective of future research.
The most used method is based on reducing DPPH.
Hoping this will be helpful,
Rafik
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The way to determine DPPH radical scavenging activity has been described in so many papers, but I need a protocol with more details.
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Mahmood, I believe Neha sent you the correct information. The second file has the protocol you are looking for.
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I performed hydroxyl radical scavenging by the method of Halliwell et al 1987. According to literature, TBA makes a pink color with the reaction product (MDA) after degradation of 2- Deoxy ribose. But I obtained a yellow color after the completion of the test and therefore I am unable to calculate radical scavenging activity. Please suggest solutions for the problem.
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Uma Kant, sorry my English is poor.
Now I also get the slimier result in my TBA assay. I want to make sure your conclusion. you mean that maybe other chemicals would effect this assay, and too much MDA product would cause product color become yellow?
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The ABTS•+ radical cation was produced by reacting 7.4 mM ABTS and 2.6 mM potassium persulfate solutions (1:1) and  stored in the dark at room temperature for 12 h and then diluting  with  methanol to get an absorbance of 1.1 ± 0.02 at 734 nm. Plant extracts (150µl) at 200µg/ml was mixed with ABTS•+ solution (2850 µl) and then incubated at dark for 2 hr. Absorbance was found to be 0.218 at 734nm. Trolox standard is prepared from 50 to 600µM. The equation of trolox is y = -0.0010x + 0.6713, R2 = 0.9968. Most of the paper followed in the Re et al (1999) was expressed in % inhibition or IC50 or trolox equivalent and its ABTS•+ solution was diluted to 0.7 absorbance. But I don’t find any paper expressed in % inhibition for the protocol which I have followed. Can it express in % inhibition using the same protocol or it should express as µM TE/g? If it should express in trolox equaivalent, then how can it calculate?  Please help me 
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Hi jag,
For the ABTS assay, the Re et al. method is the best. For the first time I saw this protocol. If you are getting the good calibration curve then its ok, you can use following equation to calculate % scavenging
ABTS cation radical scavenging activity (%)  =  [(A blank -  A sample) / A blank] x 100
It is always good to show activity in terms of IC50 value. even for protocol you have used, you can calculate activity in terms of IC50. For that you have to prepare series of various conc. of your sample solutions and evaluate them all for the ABTS assay. Further you need to find value at 50% inhibition and that will be your IC50 value.
Sending herewith one of my article, you can go through it.
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Good day. I am using the DPPH free radical scavenging assay to determine antioxidant activity in tomatoes. I have carried out the extraction, read the absorbance and calculated the scavenging activity in percentage (%). I have also read the absorbance of various concentration of of ascorbic acid and plotted the percentage scavenging effect against concentration. With what formular do I convert or express my answer in mg AA/100g?
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 Dear Johnson Esua,
First of all you have to draw a standard curve with different concentration of ascorbic acid and find out the straight line equation of that curve (eg. y=mx+c). In the equation you will get a value for m and also for c. Ignore the value c and consider the equation as y=mx. Now put your OD value of unknown concentration in place of y and equate thereafter. 
FOR EXAMPLE- If I have 200 mg of sample and my OD value of DPPH is 0.617 and my total reaction mixture (RM) is 10 ml and the straight line equation for standard curve is y=0.008x+0.0429. then,
0.617=0.008x
x=617/8=77.125
therefore, 10 ML RM contains 77.125 µg/ml or 771.25 µg.
again, 200 mg or 0.2 gm of sample contains 771.25 µg or 0.77125mg
therefore, 100 gm of sample contains 385 mg AAE (ASCORBIC ACID EQUIVALENT)/100 gm dry weight.
THANKING YOU,
SANDIPAN RAY (UGC-SRF)
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Different antioxidant assay have different mechanism as well give different activity. How can we decide which method is suitable for free radical scavengers?
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Attached file would help you to get an insight on antioxidant activity and the methods used for analysis. 
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What will happen on IC50 if I'll prefer to use 0.002% of DPPH instead of 0.004 % of DPPH as the free radical scavenging method? Will IC50 of 0.002 % DPPH be decrease with comparison to the IC50 of  0.004  % of DPPH ?
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DPPH is one of the standard method for determination of antioxidant activity in natural plant extracts. So, you can use DPPH method for your studies.
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what is the mechanism of superoxide anion free radical scavenging assay of plant extract?
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the mechanism for  non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system which generates superoxide radicals,  reduce nitro blue tetrazolium (NBT) to a purple formazan. The active compounds in ur extracts inhibits the reduction of NBT by quenching the superoxide radicals..
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i want to know the scavengers of hydroperoxide radical. Who knows about that?
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Recently I’m working on hydroxyl radicals detection and I use pCBA as a probe. But here is the problem, I can’t dissolve pCBA in water! I understand that pCBA is insoluble in water but there were many research work have been done before but the author didn’t mention how to solve the the dissolve problem. I tried to raise the temperature to 80℃ but the pCBA didn’t dissolve, and I put NaOH to react with pCBA hoping to form soluble salt but it didn’t work at all! I really confused why the Na- pCBA can’t dissolve in water! In short, I’m confused about how to dissolve pCBA in water, is there anyone facing the same problem? Welcome to share! Thanks in advance!
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I assume that you are referring to p-chloro-benzoic acid as p-CBA. But for hydroxyl radical detection, you can also use benzoic acid directly instead of p-CBA. The reaction between OH radical and benzoic acid gives 3 isomeric hydroxylated products. The para isomer (p-hydroxy benzoic acid or p-HBA) is easily soluble in water and is widely used for quantitative estimation of OH radical. This para isomer also has different HPLC retention time from the other isomers and parent benzoic acid. So it is easy to quantify.
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What is the meaning of IC value in case of DPPH free radical scavenging method. Say for the IC50 value of the _ _ _ extract and and ascorbic acid were _ _ _ microgram/mL. Then, what is IC here ? and how can I calculate it ?
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I agree with all the answers with the meaning of IC50, but I want to add that this value it is not correct to compare antioxidant activity abilities between different compounds, extracts or pure compounds from different researchers, just in case both have been done with the same DPPH method. And as you all know, DPPH method is not standarized as other analytical methods, so evry method can have different DPPH initial concentration. So, in my opinion is much more useful to express your results as Trolox equivalents/g compound(fruit, leave, or whatever), otherwise we will never be able to compare results as we do in polyphenolic content. 
And it is extremely easy making a calibrated curve with %Inhibition and Troilox concentration ;-)
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what is the standard DPPH free radical scavenging activity for plant  extracts? i am working withmedicinal plants for the estimation of their antioxidant properties and i have read many research papers for various methods for antioxidant estimation. ther are many different methods for the DPPH method. Can anyone help me to solve this problem?
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Hi Urvashi,
Then, antioxidant determination by DPPH assay there are different ways to express your data. Your data can be expressed in IC50, % inhibition, and umol TE/g, the your choice will depend of literature used to compare your results.
Best regards
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Can anyone explain the importance of using a positive control (eg quercetin) and a negative control (methanol) while calculating the DPPH free radical scavenging activity?
The formula that I am using for my extracts is as follows:
% Inhibition = [(A0-A1)/A0] × 100
Where A0 is the absorbance of the control; A1 is the absorbance of test samples.
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DPPH radical scavenging activity is expressed as mg of standard ( Gallic acid/ Quercetin/ Catechin/ Torolox) per g of sample or fresh weight. That's why we need standard or positive control , what ever you termed. Negative control is solvent plus DPPH to take absorbance with out sample which gives the absorbance of DPPH before scavenging by extract of sample. 
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We are preparing chitosan encapsulated naringenin. For that we need to know the mode of action of naringenin. It would be greatly helpful if anyone tells the answer with reference.
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Naringenin inhibits the assembly and long-term production of infectious hepatitis C virus particles through a PPAR-mediated mechanism. Naringenin is a non-toxic assembly inhibitor of HCV and that other PPARα agonists play a similar role in blocking viral production. The combination of naringenin with STAT-C agents could potentially bring a rapid reduction in HCV levels during the early treatment phase, an outcome associated with sustained virological response. Contact poisoning is possible in living animals.
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I am calculating DPPH inhibition data. I have graphed my DPPH inhibition against concentration and I am trying to calculate the IC50 for my samples. However, when I calculate the IC50, using the equation fo the line (intercept=0), I find that compounds with highest %DPPH inhibition have highest IC50 values. Surely this is incorrect? Does anybody know what I am doing wrong?
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You are just saying theoretical DPPH activity, go for a log scale graph and plot real values to find an intercept at its maximum by deducting values of control-test/control+testX 100, you will get a figure that can tell you about the value less than IC50, when you plot the same you may a value of higher % of DPPH activity that will not touch IC 50 value and remain below. 
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We have different medicinal plant extracts. So please suggest the recent trends used for free radical scavenger activity.
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Through the DPPH assay.
Take a look at this article: Influence of the drying method in the antioxidant potential and chemical composition of four shrubby flowering plants from the tribe Genisteae (Fabaceae)
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There are quite a number of methods to evaluate the in vitro antioxidant activities. Can you please suggest me the best and the most effective method?
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What role does solvent play in extracting antioxidants from natural sources? Please suggest. 
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Yes, Arvind, solvent have a crucail importance! There are some antioxidants (tocopherol) what require non-polar solvent. Moreover, solvent is cruail alos for stabiluty of some antioxuants. For exmap0le, for ascorbate and gluthatione racid pH (3 of 6% MPA) is require to keep stabilty and prevent oxidation. Some of summary you can find here: Kothari, V., Gupta, A., & Naraniwal, M. (2012). Comparative study of various methods for extraction of antioxidant and antibacterial compounds from plant seeds. Journal of Natural Remedies, 12(2), 162-173.
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I am working with a phenolic compound that may act as a free-radical scavenger within macrophages, reducing the initiation of apoptosis.  I have seen reports that test the free radical scavenging capacity of food products, but I don't that these will be useful in my case, since I am interested in the intracellular compartment.   Thanks!
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Dear Reid,
I usually see works that uses the DPPH antioxidant assay. If I'm not wrong, this test is based on spectrophotometric measurements trough the color changes of DPPH when reduced.
I guess these two articles attached can help.
Best regards and hope that helps!
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Antioxidant property of polyphenols could prevent brains from free radical attack to cells and scavenge them.
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hi this may be useful to you
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Can anyone list the in vitro radical scavenging assays with same substrates (involved in the reaction) so that i can avoid those non-substrate specific assays?
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hi this may useful to you
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Can anyone tell me about the IC50 standard for NO that our sample (plant extract) is active as a radical scavenger (see link below)
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hi
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Dear all,
Can anybody explain to me the exact difference between FRAP analysis and Free Radical Scavenging analysis? Do they measure the same thing? What's the difference between them? Thank you so much.
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Hi,
the both methods are quite different. FRAP fives you information about the ability of the antioxidants (AO) to reduce Fe in very low pH (3.6) and is based on the single electron transfer mechanism. But if you read more literature you will see that this method has very disadvantages because of the "not realistic" pH. The other methods give you info about the ability of the AO to quench free radicals, and these methods are generally based on H-transfer mechanism, Here are ORAC, DPPH, ABTS which differ on the radical used and the conditions of the reaction.
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In many papers, the researchers use both of DPPH and OH radical scavenging. But why should we use both of them?
What is difference between the principle of 2 methods? 
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The molecule DPPH is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecule does not dimerize and also gives rise to the deep violet color. After accepting the hydrogen atom from donor it gives rise to the reduced form with the loss of this violet color. Hydroxyl radical is one of the potent reactive oxygen species in the biological system that reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes damage to cell. Out of all the in vitro methods, DPPH is the most easy, simple and reasonably costly method and hence it might have been used mostly for the antioxidant activity evaluation of a sample.
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The IC50 value of ascorbic acid reported in research papers differs significantly. I have used DPPH assay, galivonoxyl free radical scavenging assay and ferric ion scavenging assay for evaluating in vitro antioxidant activity of plant extracts and used ascorbic acid as a standard for the same. Could you kindly help me to know the exact IC50 value of this compound in all the above mentioned assays?
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Ascorbic acid has a strong antioxidant activity. i have used it as a positive control and the IC50 varied depending on the experiment conditions. I would say from 1.5-5 μg/m 
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 Is it possible to understand using PetroOXY whether antioxidants are free radical terminators or oxygen scavengers?
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Hi Ali,
Thanks for papers but I can't open these paper. its damaged.
could you please try once more.
Regards,
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DPPH, a free radical, is used to measure potential to scavenge free radical. Is there any effect of pH as well as temperature on the experiment?
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Temp. can be an imp. factor in degrading DPPH activity. The absorbance of DPPH was found to decrease almost by 35% at 25 °C under light. Studies show that the evaluation of antioxidant activity by the changes of DPPH absorbance should be carefully interpreted since the absorbance of DPPH at 517 nm is decreased by light, oxygen, pH and type of solvent in addition to the antioxidant.
Refer:
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see above
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Respected Sir,
You can proceed your analysis using Superoxide radical scavenging activity, Hydroxyl radical scavenging activity and ABTS radical cation scavenging activity. Kindly go through the attachment.
Good Luck
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This question was raised by a reviewer. Please help me to answer this in a scientific way,
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I believe there is plenty of evidence around that flavonoids are strong antioxidants. However, i think this is not what the reviewer intended with his question. Probably you need to answer the question whether there are no other antioxidants present in the sample. One way to prove that phenolics are most important antioxidants is by selectively removing flavonoids from the sample, f.i. By adsorption on PVPP
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Is there a default value because I found the absorption rate sometimes fluctuates.
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Thank you very much Prof. Sharma for your valuable list
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Please share your experiences about the advantages and disadvantages of radical scavenging methods from medicinal plants.
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The antioxidant capacity was determined by chemiluminiscence using the “PHOTOCHEM” dedicated device produced by Jena Analytic, Germany.
Eight 2 mL aliquots from the working solution are frozen at −10°C. By successively defreezing these aliquots, the antioxidant capacity is measured every 3 hours. The determination of antioxidant capacity is based upon the photochemical generation of the superoxide anion radical initiated by a 185–240 nm UV beam.
The determination of the antioxidant capacity is based upon photochemical generation initiated by an UV radiation in the 185–240 nm domains, of the anion (O– 2◦) superoxide radical. The sequence presented in Schemes 1 and 2 includes the optical excitation of the free radical generator (damnacantal) thus obtaining the singlet state (1 s) of the molecule (process [A]). By an “inter system crossing”-type process (process [B]), the molecule goes into the triplet state which, due to known molecular spectroscopy selection rules, is stable enough to react either with oxygen in the normal triplet state (generating the singlet oxygen reactive species) (process [C]), or with a reducing (also called a reductant or reducer) agent (mono-electronic reduction process) (process [D]) with the generation of an anion radical in doublet state (2S) and the formation of the superoxide anion (process [E]). The latter, by a series of processes ([A]–[H]), transforms into the amino orthophthalic acid dianion, in the singlet state, which, by reversal process to the fundamental state, emits a light beam in the 425–450 nm spectral domain. Antioxidants from the captured samples yield superoxide ions and reduce the radiation intensity generated by excited luminol (luminol reaction inhibiting-blank). Standard soluble compounds are TROLOX (derivate of α–tocopherol), and for the water–soluble, ascorbic acid. Process [A]–obtaining the singlet state (1 s) of the molecule; process [B]–process the molecule goes into the triplet; process [C]–generating the singlet oxygen reactive species; process [D]–the generation of an anion radical in doublet state (2S).
Process [E], [F] and [G]– the formation of the superoxide anion; processes [H]– transforms into the acid dianion. For both it performs a calibration curve and estimate the advance between the integral under the curve of the blank (solution without antioxidant) and sample (standard solution or extract with antioxidant-plant) and distribute by the standard integral. These estimates are done automatically by the software unit. The intensity of the light signal, measured by a photomultiplier, depends on the speed of the processes [F] and [G]. If the system does not include a compound capable to bond free radicals, the entire amount of generated anion superoxide is consumed by the light supplying agent “luminol”, and the intensity of the emitted light is maximal. If the system contains an amount of free radical binding agents (antioxinants) a competition between luminol and the free radical binding agents occurs for the superoxide anion radical. In this case the light signal detected by the photomultiplier has a lower intensity.
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I was wondering if anyone could point me to literature or maybe some knowledge with regards to how I should prepare my samples prior to running a DPPH assay for measuring antioxidant activity?
I have Joshanda tea, green tea and lyons tea, I'm just unsure on how to begin preparing my samples and have been hitting a wall with regards to searching online.
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Dear Shahbaz,
DPPH is a stable radical frequently used to measure hydropilic antioxidant activity. You are talking abou tea, thus you could make the assay on a polyphenoli extract to check for polyphenols activity (mainly) or on a whole sample water extract. The main porblem is to stay within specific absorbance values. You have to start with a maximum absorbance of 0.8 -0.9 otherwise the risk is to be in excess of radical. If you decide to use a water extract you have to dissolve a certain amount of your sample in a certain volume of water and then homogenize and centrifuge (I use 9000xg for 15 minutes at 4°C). You can start by diluting your sample tenfold, as an example, centrifuge, than fllter the surnatant with filtering paper and filtering again with a 0.45 micron filter. Then you will use a small aliquot (50 microliters for example) or 3 mL of your freshly prepared DPPH solution, in order to have a decrease in absorbance not more of 0.5-0.6 points (to have a final abs of 0.3-0.4). If your sample reduce too much the absorbance dilute more or use a smaller amount (you are in excess of sample), if the decrease is too small reduce the dilution or increase the sample (you are in excess radical). I hope this will help you
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The oscillations in the Bray-Liebhafsky reaction (hydrogen peroxide decomposition catalyzed by iodate and iodine in acidic solutions) are strongly perturbed by oxygen, a product of the reaction. We (G. Schmitz & S. Furrow) believe that the oscillations are explained by non-radical reactions but that the perturbing effect of oxygen is the result of radical reaction. In order to obtain "clean" oscillations, we are looking for a radical scavenger that does not react with the iodine compounds involved in the oscillations, IO3, IO2H, IOH and I2. Who has a suggestion?
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Nitrogen does not eliminate the effect of the oxygen because this is a reaction product and, if nitrogen is bubbled through the solution, it removes iodine which is a key intermediate.
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What is the effect or the mechanisms of activities of ferrocene compounds,(ferrocenium) free radical on breast cancer cells and on normal cells?
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Bleomycin binds ferrous iron and oxygen and after reduction in vivo produces an activated intermediate, a Fe3+ hydroperoxide [BLM-Fe(III)-OOH, ferric peroxide complex] that cleaves DNA by hydrogen abstraction. Bleomycin destroys malignant cells based on a metal-dependent prooxidant free radical mechanism leading to DNA fragmentation.
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Can the repetition of assays of same substrates be avoided?
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Thank you Juliana and Bilal for your reply....