Questions related to Antioxidant Activity
Hello everyone. I need help regarding my DPPH assay.
I'm currently trying to measure the Antioxidant Activity of my sample, Water Kefir infused with Plant extract (Turmeric and Green Tea) using DPPH method.
My DPPH Assay protocols are as follows :
A DPPH stock solution was prepared by mixing 24 mg DPPH in 100 mL Methanol. The working solution was obtained by mixing 10 mL stock solution in 45 mL Methanol to obtain an absorbance of 1.1 0.05 units at 515 nm using ELISA plate reader (Bio-tek Instruments, USA). 50 µL of sample was allowed to react with 250 µL DPPH working solution in dark for 30 minutes. The absorbance was then read at 515 nm using ELISA plate reader (Bio-tek Instruments, USA). Trolox solution was used as positive control in this experiment.
However, I noticed that there is precipitate/sediment formed after I incubated my samples for 30 minutes.
Does anyone know what might be the possible cause and how can I minimize the occurrence of this precipitate/sediment?
Also, if I aliquot the supernatant to a new plate and get the absorbance reading, does it affect my result somehow?
I also attached this photo of my plate to give you a better idea on how does the precipitate/sediment looks like
Note : I serially diluted my samples (2 fold each, 6 times). I noticed that the higher the concentration, the more likely the precipitate/sediment to form (A = 1mL, B = 0.5 mL, C = 0.25 mL, D = 0.125 mL etc)
I intend to measure the antioxidant activity of plant extracts and oils using two devices (96 well plate reader and a double beam ultraviolet-visible (UV / vis) spectrophotometer). Which equation should I use to calculate?
I was determining flavonoid concentration using AlCl3 method from natural product extract (based on Estimation of Total Phenols and Flavonoids in Extracts of
Actaea spicata Roots and Antioxidant Activity Studies; by Madan, Banshal, Kumar, Sharma; 2011) and used Sodium acetate instead of Potassium acetate (Evaluation of Antioxidant Activity, Total Flavonoids, Tannins
and Phenolic Compounds in Psychotria Leaf Extracts; Formagio et al, 2014).
I have prepared a calibration curve (With R^2=0.9918); by preparing different concentration of Quercetin (SRL chemicals) yellow powder in methanol. I did not notice any 'perceivable' colour change after I add reagents into Quercetin aliquot. However I prepared a conventional calibration curve.
It gave me a strange result. Very high flavonoid content in the test extracts; even greater than Total phenol content (by FCR method) of the test extracts
Based on this ( https://chemistry.stackexchange.com/questions/35389/does-the-aluminium-chloride-assay-ordonez-et-al-2006-for-total-flavonoid-conte ) stackexchange post; I prepared a control set for the yellowish background correction (for each concentration of quercetin) where only water was added to quercetin aliquot (instead of same volume of AlCl3 reagent). This second experiment gave me weird result; the ODs increase with quercetin aliquot but OD(Reaction) - OD(Control) keeps fluctuating (positive and negative values at random).
Where I might be doing the mistake? Anybody else encountered similar problems?
I want to use the Hydroxyl Radical Scavenging method for a series of antioxidant tests.
In the written protocol, I should use iron sulfate, salicylic acid and 0.1% hydrogen peroxide.
Does 0.1% hydrogen peroxide mean by weight or by volume?
and What is the solvent of ferrous sulfate in this protocol? Is the Distilled Water?
In the tests related to Hydroxyl Radical Scavenging, two different methods were used.
In some articles, EDTA, deoxyribose, etc. have been used, but in other articles, these two substances have not been used at all and salicylic acid has been used (Hydrogen peroxide and iron chloride are common in both methods).
What is the reason for this difference?
And what is the difference between these two methods? Are both valid?
Thanks a lot
I wish you good health
I have a question:
In different articles, different volumetric ratios have been used for DPPH testing. For example, one paper used 0.5 cc of sample and 2.5 cc of DPPH solution, and another article used 1 cc of sample (ascorbic acid) and 1 cc of DPPH solution.
What is the reason for these different ratios and how do I know which method is correct?
Thanks for your response.
I'm performing antiplatelet aggregation induced by collagen, and I have only this manner, I can not test it by other methods such as ADP or TRAP-6 ...
And using different manners helps to confirm finding results and making a correlation. How can we deal with this kind of circumstances?
Can we give a little bit whether an explanation or interpretation of the potential played by essential oil against free radicals for example without saying only the synergistic effect?
Is there any in-depth way or theoretical study to confirm which compounds, in particular, are responsible for the activity?
I prepared a dose of 1200 ug/ml from my crude extract and doing a serial dilution 600,300,150,75 ug/ml then I poured 1 ml each dose in a 3 ml DPPH solution and final volume of solution was 4 ml. Finally my dose was 300,150,75, 37.5 and 18.75 ug/ml. Now, which doses will I consider, final one or initial one? Most of the authors do not clarify it in their antioxidant article.
As we know, when we increase the concentration, the percentage of inhibition gets increased until reaching the maximum point. But, is it necessary to reach 100% ? Because, sometimes even if we increase the concentration over and over, the value remains stable!
What leads this phenomenon and how can we interpret it?
C1= 1 mg gives 60 % of inhibition
C2= 1.5 mg gives 86 % of inhibition
C3= 3 mg gives 86.2 % of inhibition
C4= 10 mg gives 86.5 % of inhibition
Even increasing the concentration, inhibitions percentage started to look stagnated, no more progress!
Having a molecule containing double bonds (e.g Limonene), or a molecule containing a functional group (e.g citronellal). Which one of those are you expecting will have a good antioxidant activity? and why?
Limonene: containing 2 double bonds
Citronellal: containing 1 double bond + functional group ( aldehyde)
We determine the antioxidant activity through either donation H or donation of an electron.
Could you give deeply more details about this explanation?
As we know, an essential oil is a mixture of components playing a pivot role in various domains.
I'm carrying out the antioxidant activity using in vitro tests.
Before doing this task, can we predict the activity of my essential oil only from the compounds found in my sample?
Is there any software to make a theoretical study?
In order to make a good comparison between your present work and the previous reported studies, we need to compare with results obtained below the same conditions.
Sometimes, we don't find works realised under the conditions that we used !
How can we valorize our work?
Are we allowed to compare even if the conditions are not alike?
Recently, FRAP (Ferric Reducing Antioxidant Power Assay) is not accepted by some journals for the investigation of the antioxidant power of a sample like essential oil!
I don't know the reason behind this refusal!
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?
We tested some new compounds and I would like to write a discussion and compare these with other similar compounds... but we used as standard trolox and in study I would like to use they used AA. Thanks for any answer!
I do flavonoid test for biscuit incorporated with muntingia fruit powder.
Sample extracted with metanol (1:10 ,w/v), macerated 24h, sentrifuged and filtered. Supernatan used for analysis.
I have done dpph, phenol earlier with the supernatan from the same extracts. Dpph done well. Phenol had white precipitation.
And now flavonoid have white cloudy like solution.
An aliquot supernatan, add with 0,15 ml nano2 5%, then 0,15 ml alcl3 10% and 1 ml naoh. Then read abs at 510 nm. Quercetin as standard.
But i have cloudy solution which make it difficult to read in spectro.
Do anyone could help me for these? Thanks before
I will be thankful if anybody answer my question and send me a clear protocol to measure ABTS Radical scavenging activity of protein samples.I want to measure antioxidant capacity of my samples by ABTS assay.
I made ice cream using red pitaya peel and i need to measure the DPPH inhibition percentage for both red pitaya peel and ice cream. As we know, red pitaya peel has such strong red purplish color meanwhile my ice cream is slightly pink color.
Can I use different sample volume for my DPPH assay? I've tried using 1,5 ml ice cream extract+2,5 ml DPPH solutions and 0,5 ml red pitaya peel extract+3,5 ml DPPH solution
But the ice cream gives bigger DPPH inhibition percentage than my red pitaya peel
Is it because of different sample's volume? I can't use 1,5 ml red pitaya peel extract like ice cream one because the color is too strong so it doesn't turn to yellow after incubation
Is there any solution? Because I already performed Phenolic content assay and my ice cream's phenolic lower than red pitaya peel's
I read this paper - Introduction of Purple and Deep Purple F1 Carrot Hybrids to Egypt Showed High Antioxidant Activity and High Content of Total Flavonoids and Phenols
It mentions that carrots grow in Egypt. I am trying to find out whether the ancient Egyptians cultivated and used carrots. If so is there documentary or graphical evidence (e.g. tomb paintings) I can refer to.
I believe, without proper evidence, that they used the leaves and seeds for medicinal purposes.
Some history books refer to tomb paintings but never give detail.
Does one use only one concentration of ascorbic acid and measure over time to get the linear equation? Or use several concentrations? Is it advisable to use dilutions of blank DPPH solutions as a standard curve and ascorbic acid as a positive control?
In the determination of Antioxidant capacity of fruit samples, I used DPPH assay and have a few questions regarding the DPPH assay
1) why is it necessary to vortex and incubate in dark the sample and DPPH solution? Is it so the sample and the DPPH can mix and allow the redox reaction to take place?
2) Why must DPPH be mixed with methanol to form methanolic DPPH? What is the purpose of methanol?
3) How did the equation %Inhibition= [(A0-A1)/A0] × 100 come about? Why is it not using beer-lambert law of calibration curve instead?
Please help! Thank you(:
I am finding a way to determine the all the possible derivatives of a certain compound (say derivatives of curcumin). And, I am having a hard time pointing out possible pathways and for novel product derivatives. Can anyone suggest how I can do it systematically or any technique at all, is there a software for this? Thanks
Carried 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS), Cupric Reducing Antioxidant Capacity (CUPRAC), Total phenolic content (TPC), Reducing Power (RP), Hydroxyl radical scavenging assay (HRSA), Total Antioxidant Activity (TAA) in standard and sample. In DPPH, TPC, CUPRAC and HRSA standard was superior than sample. What may be the reason for this result?
I'm looking for laboratories working with oxidative stress to test nitrocellulose redox permanganometry (NRP). NRP is a simple method for reductive capacity assessment based on the analysis of the MnO2 precipitate following redox reaction between KMnO4 and biological samples fixed onto the nitrocellulose. This method estimates how well the sample can give up its electrons to KMnO4, so it estimates the amount of chemical antioxidants. In other words this method can be used to measure total chemical antioxidant capacity of biological samples. Advantages of the method are: great precision and accuracy; the possibility to measure a lot of samples simultaneously; simplicity; cost (you only need a piece of nitrocellulose membrane and KMnO4). Moreover, the method can be used to obtain information on the spatial distribution of antioxidant capacity in tissue sections (both cryosections and FFPE can be used), providing unique information on the redox status of the tissue.
As the method is new, and we proposed it just recently, I'm looking for laboratories that are working with oxidative stress to test the method in their samples. We conducted thorough analyses and the method seems to be very robust. Nevertheless, I believe additional independent testing is always a good idea. Also, I believe comments of researchers closely working with oxidative stress would be beneficial for further development of this simple method.
The original paper can be found here:
A step by step protocol can be found here:
I am trying to measure the antioxidant activity in food samples. Which is the best way to express Antioxidant capacity - %DPPH inactivation or Trolox Equivalent Antioxidant Capacity? How do they differ? I see the usage of two ways of representations in a same article.
what are the best plant extract dilution concentrations used in Antioxidant Activities?any well known dilution procedure and concentration for Antioxidant activities?
What is the criteria should I follow to study the antioxidant effect of an extract of marine algae?
I mean determination of the concentration that recommended to be administrated in water.
Is there a standard method should I follow?
Should I perform a toxicity test first?
Could I use the concentration to the its effect on amino acids and fatty acids metabolism of adult Zebrafish?
I want to measure the antioxidant capacity of cosmetic creams and lotions with the DPPH and
ABTS methods, but I am not sure how to prepare the samples. Will dissolving them in a mixture of methanol or ethanol and water work?
Thank you in advance for your collaboration
Can anybody suggest me one/more plant names for doing project either in alone or combination therapy of diabetic mellitus, wound healing and anti oxidant property?
antioxidants studies in C. quinoa in different parts of the plant
i use ethyl acetat and ethanol as solvent for extraction fruit of mangrove Avicennia marina. I have a problem with crude extract from ethanol solvent contain liquids that are very similar like oil. I did DPPH test from the extract of both solvents, and the results for ethanol have very weak activity IC50 = almost 2000 µg/mL. Does the liquid like oil affect the value of the activity obtained?
How can I calculate and express total phenolic content as mg GAE/g DE? Below are the details: 1. I dissolved 10mg dry extract in 1ml methanol 2. 200 micro was taken for folin ciocalteu assay 3. The absorbances for one sample were: (0.915, 0.917 & 0.919) 4. i used (0, 10, 30, 50, 70 & 90 micro g/ml) and got the equation (y = 0.0193x + 0.047)
Hi, I am trying to estimate total flavonoid content in crude extract, can you please provide any protocol to prepare catechin standard curve.
Thank you in advance.
The systemic antioxidant therapy is one of the therapeutic options for oral mucosal lesions. In Localized delivery of antioxidant molecules to the oral mucosal lesion, will the oral mucosal cells uptake/absorb the antioxidant molecules? Will the antioxidant molecules perform their action when delivered locally? Which route of delivery (systemic/local) of antioxidant molecules can have better activity?
Dear Sir/Madam, I invited as a guest editor from high quality journals to handle special issues. If anyone can prepare a review similar to my review papers, particularly about a natural product in cancer prevention with focus on the structure activity relationship and mechanism of action, please kindly let me know to send an official letter. At this stage you should just send the title, authors and affiliation and abstract. Please kindly let me know as soon as you can. The suggested deadline for sending review is about 3 month. Best wishes, Suggeted topic: Genotoxicity of different agents and possble protection. Reducing side effects of radiotherapy and chomotherapy. Next generation of cancer therapy; Natural products. Natural products as novel therapeutic compounds. Radiation protection.
Based on my research journals say that fermentation can increase antioxidant activity because the acid lactid bacteria will form primary and secondary metabolites which the secondary metabolite is polyphenol, can someone explain how that happen? Thankyou
I've a question regarding lipid oxidation - there are secondary products with truncated chains (like PoxnoPC and PazePC). The question is if they are stable, since I can't find any publication regarding the problem.
In seedlings it is important to verify that ozone does not cause damage, because plants are more sensitive to ozone in juvenile stages (Timonen et al., 2004).
Ozone, applied diluted in water and sprinkled on seedlings of ornamental perennial species such as Salix integra, Hydrangea paniculata or Spiraea japonica, has proven to be a very effective treatment against fungal foliar diseases, ultimately causing a saving of chemical products. This fact in a world that increasingly requires more ecological treatments, and in which the demands for reducing the residues of fungicides and other pesticides are increased not only in fruits but also in foods processed with them (Edder et al., 2009 ).
Ozone is considered as a possible organic biocide because apart from not leaving chemical residues, it acts in two ways, a direct and an indirect way through the ROS species (Zotti et al., 2008). Toxic doses in sprinkler irrigation were observed from 1.5 ppm on the mentioned perennial seedlings, in applications of 7.5 minutes of irrigation / day (approx 1L per plant / day) for 6 weeks. The non-toxic dose was observed in concentrations below 1.5 ppm, such as 0.5 ppm. At these levels, an elimination of algae and biofilm is ensured, without showing phytotoxic effects (Graham et al., 2009).
In hydroponic or semi-hydroponic systems, water (30%) and fertilizer (40%) were saved by recycling leached or closed-circuit water (Runia, 1988, 1993, 1994; McDonald, 2007).
In hydroponic tomato cultivation with nutritive solution + ozone, larger tomato plants were obtained than in nutritive solution - ozone. It was demonstrated in this work that the concentration of macro and micronutrients of the nutrient solution is not modified: there is no variation in the levels of Calcium, Potassium, Magnesium, Phosphate, Ammonium, Nitrate, Copper, Molybdenum or Zinc depending on the dose increasing ozone up to 10 ppm. There is a drastic decrease in Manganese (from 0.5 ppm) and Iron (from 5 ppm). To avoid that, Terazoe (2001) proposed the ozonation of the water in a separate tank before adding the nutrients or separating the irrigation with ozone and the irrigation of nutrients. Despite this drastic decrease in the irrigation solution, there are no differences in the content of these nutrients in the leaves, which is not negatively affecting their normal development. There is a notable decrease in the incidence of root rot, typical of hydroponic cultivation (Ohashi-Kaneko et al., 2009).
So, everything seems quite good in that way, but many questions comes to my mind:
1) Given the potential of this tool, why is ozone not an essential tool in agriculture today?
2) Are we still in a time of transition to tools such as ozone in agriculture? Or, on the other hand, do people distrust this technology due to unethical actions carried out by certain companies in the manufacturing sector of ozone generators and other innovative technologies?
3) Are there few studies in agriculture with ozone application? Or perhaps the studies show very contradictory results among them?
Feel free to discuss and propose your own questions/answers,
Edder, P., Ortelli, D., Viret, O., Cognard, E., Montmollin, A. D., & Zali, O. (2009). Control strategies against grey mould (Botrytis cinerea Pers.: Fr) and corresponding fungicide residues in grapes and wines. Food Additives and Contaminants, 26(5), 719-725.
Graham, T., Zhang, P., Zheng, Y., & Dixon, M. A. (2009). Phytotoxicity of aqueous ozone on five container-grown nursery species. HortScience, 44(3), 774-780.
Ohashi-Kaneko, K., Yoshii, M., Isobe, T., Park, J. S., Kurata, K., & Fujiwara, K. (2009). Nutrient solution prepared with ozonated water does not damage early growth of hydroponically grown tomatoes. Ozone: Science & Engineering, 31(1), 21-27.
Timonen, U., Huttunen, S., & Manninen, S. (2004). Ozone sensitivity of wild field layer plant species of northern Europe. A review. Plant Ecology, 172(1), 27-39.
Zotti, M., Porro, R., Vizzini, A., & Mariotti, M. G. (2008). Inactivation of Aspergillus spp. by ozone treatment. Ozone: Science and Engineering, 30(6), 423-430.
I am read this sentence in artical paper >
(( Successive extraction with petroleum ether (6×50 mL), ethyl acetate (3×100 mL), and n-butanol (3×50 mL) afforded fractions))
I am using the dpph test for some plant extracts, the extracts are colored and they interfere with the resulted absorbance and I get an absorbance above the negative control....if any one could give me the protocol I'll be very thankful
Coffee is a simple beverage, but it’s full of complex compounds with health benefits. It contains hundreds of bioactive components including vitamins, minerals, and anti-inflammatory polyphenols such as flavonoids. The amount of caffeine in a cup of coffee can vary, depending on factors ranging from the type of bean used. So, What about the healthy daily amount of caffeine was recommended to be healthy with no risk?
I need the full protocol for analysis Chlorophyll a and b, and Anthocyanin for hydroponics lettuce and the ideal solvent for analysis such as acetone for cholophyll and methanol for anthocyanin.
Estoy evaluando actividad antioxidante de un extracto de hojas de ortiga para ello quiero determinar el IC50 por lo cual prepare diferentes concentraciones del extracto para lo cual tuve que disolverlo por lo que las concentraciones están expresadas en mg/mL por lo tanto al calcular el IC50 este tendrá las mismas unidades (mg/mL) sin embargo revisando un articulo científico encuentro que el IC50 esta expresado en mg/g como logro esto? y como se interpreta estos resultados.
is it possible to measure 8-OHDG biomarker molecule in frozen sperm sample (epididymal) or it has to be a fresh sample necessarily?
I want to determine some extracts' antioxidant activity but I am looking for methods which do not need to spectrometery.
Please let me know if you have any idea. Thanks.
I am conducting a experiment for antioxidant activity. For example let a sample X( Containing alpha-Tocopherol and ascorbyl palmitate) giving 250 (mM/g)(TEAC) for ABTS and 450((mM/g) ) for DPPH for a single sample. But most of research publication shows DPPH values have always less than ABTS for a single sample. Is there possible reason behind my result being high in terms of DPPH than ABTS.
As far as I know, ascorbate peroxidase enzyme (APX) is the major enzyme involved in H2O2 scavenging. It is specific to plants and algae.
Recently, I was shocked when I came across an article in which APX and guaiacol peroxidase (GPOX) from fungi were unusually assayed !
In fact, GPOX activity monitoring in fungal cells is not surprising since the famous class II peroxidases such as lignin peroxidase that is found in fungi can use guaiacol as electron donor. However, APX assay is well conducted and APX activity was even higher than catalase that is well reported in fungi !
Although ascorbate (or erythroascorbate) could be a natrural substrate for many peroxidases, what do you think about the presence of APX enzyme in non-photosynthetic organisms and especially in fungi ?
Your contributions will be welcomed.
I'm using "Use of a Free Radical Method to Evaluate Antioxidant
Activity W. Brand-Williams, M. E. Cuvelier and C. Berset" as reference.
I' m trying to measure antioxidant activity on garlic extract but something react with metoh so the solution isn't clear and can't be read on spectrophotometer .
thanks a lot
I want to conduct a theoretical study in which the antioxidant activity and capacity of a phenolic chemical compound is supposed to be evaluated. The number of atoms of the compound is less than 40. I am absolutely in need of a computational method to use in the molecular modeling. Moreover, for doing the calculation, I need an open-source computational software, preferably a windows version. I thank you in advance for your valuable suggestions.
Some polymer additives are added during polymerization reactions in petrochemical plants while masterbatchs are added to the final polymer out of the plent.
Is the above sentence right?
Actually i cannot get the proper list of polymer additives added in petrochemical plants.
I google it but there is not any specific term yet i confuse with masterbatch and additives.
Suggest some best journals which offer speedy publication of research articles covering physio-chemical, sensory and antioxidant analysis of a developed food product. The journals should be impacted.
Thanks in anticipation
I'm a total newbie on antioxidant assay. Trying to determine antioxidant activity of an encapsulated polyphenolic compound
So I was looking at some journals for ABTS Assay procedures, and got quite confused as below:
This is the confusion part for me:
For (1), 7mM ABTS solution mixed with K2S2O8 solution. In that mixture, the concentration of K2S2O8 is 2.45mM.
Whereas for (2) equal amount of 7mM ABTS solution is mixed 2.45mM K2S2O8 solution. Doesnt that mean the final concentration of K2S2O8 in that mixture is 1.225mM.
Is (2) correct. Are they trying to get final concentration of 1.225mM. Or am I reading it incorrectly?
Actually, I'm trying to extract Spirulina microalgae using ethyl acetate to evaluate antioxidant activity. I want the ethyl acetate to be 50% diluted, meaning that I'm planning to add 50 ml water into 50ml of ethyl acetate. Is this applicable, because ethyl acetate has a low solubility in Water? Will this work?
I have a query regarding ''the possible reasons behind the higher phenolic content but lower antioxidant activity''. I used the raw bamboo shoots and it showed good amount of phenolic/flavonoid content and antioxidant activity but when I prepare candies from it without adding any other antioxidant rich ingredient, processing decline the phenolic content but antioxidant activity increase significant higher. Kindly suggest what could be the reason behind it.
LED treated plants gave lower concentrations on APX, SOD and CAT antioxidant activities than fluorescent light...what does it tell from the concentration of the enzymes? which is better?
I am trying to measure a snapshot of the hydrogen peroxide levels in plasma using a quick assay that consists of Horse Radish peroxidase (HRP).
Whenever I spike the plasma samples to be tested, with a specific concentration of hydrogen peroxide, the hydrogen peroxide is undetectable when measured 15 minutes later. This suggests that any endogenous hydrogen peroxide in the plasma will decompose just as fast as soon as the plasma samples are brought to room temperature.
Is there a way to stop hydrogen peroxide levels in my plasma samples from decomposing that wouldn't affect HRP activity of the assay?
I am performing DPPH assay test of citrate and ammonium based compounds. While performing DPPH assay test color of solution changes from violet to brown after incubation. Peak at 417 nm disappears and shifted 425 nm while increasing overall intensity at 417 nm. now colour change is happening BUT why intensity is increasing after adding my samples to DPPH solution. Any particular reason?
P.S using absolute EtOH as solvent. and incubation time is 60 min.
I use quercentin as a standard and reacting the sample with NaOH, AlCl3 and NaNO2
I saw some journal about antioxidant activity with extract in aquades, but i'm still confused about that.