Have you tried the DPPH method? I used and found it to be a friendly method to assay for AA

Yes , I  have  tried  for the   DPPH  method  and  I  have already  standerized the protocol.

I have  tried  for the  Hydroxyl  radical  scavenging  method, but till I am  not get the success.

I also get a problem like that and my professor suggest me to try phosphomolybdic acid assay...

Dear Sumana

We estimated Hydroxyl Radical Scavenging Activity for Kalonchoe pinnata. The procedure is given below: 

Hydroxyl radical  scavenging activity was measured comparing mannitol  and plant extract for hydroxyl radical generated by Fe3+ -ascorbate-EDTA-H2O2  system (Fentons’s reaction) using the method of Kunchandy Rao. The reaction  mixture containing 100µl 2-deoxy-2-ribose(23mM in  28mM phosphate buffer, pH 7.4), 500µl of each  fraction of the plant extract or the reference  compound in phosphate buffer (28mM, pH 7.4), 200µl  1.04mM EDTA and 200µm FeCl3 (1:1 v/v), 100µl 1.0mM H2O2 and 100 µl 1.0mm ascorbic acid ,was incubated at 37oC for an hour. The 1ml of the 1% thiobarbituric acid and 1.0ml of 2.8% trichloro acetic acid were added and incubated at 100oC for 20 minutes.

Ref:Kunchandy E, Rao MNA: Oxygen radical scavenging activity of

curcuminoid. Int. J. Pharmacogn. 1990; 58: 235-242.

Regards

Chandra Mohan

Please recommend mine latest work in Biomedicine and Preventive Nutrition from mine publication list.

Dear Sumana, 

Deoxyribose assay is used to determine the hydroxyl radical scavenging activity in an aqueous medium. The reaction mixture containing FeCl3 (100 µM), EDTA (104 µM), H2O2 (1 mM) and 2-deoxy- D-ribose (2.8 mM) are mixed with or without the test sample (plant extract/fraction/compound) at various concentrations (10-250 µg) in 1 ml final reaction volume made with potassium phosphate buffer (20 mM, pH 7.4) and incubated for 1 hr at 37 0C. The mixture is then heated at 950C on water bath for 15 min followed by the addition of 1 ml each of Trichloroacetic acid (TCA) (2.8%) and Thiobarbituric acid (TBA) [0.5% TBA in 0.025 M NaOH containing 0.02% 2-Deoxy-D-ribose, butylated hydroxyanisole (BHA)]. Finally the reaction mixture is cooled on ice and centrifuged at 5000 rpm for 15 min. Absorbance of supernatant is measured at 532 nm. All readings are corrected for any interference from brown colour of the extract or antioxidant by including appropriate controls. The negative control without any antioxidant or sample is considered 100% deoxyribose oxidation. The % hydroxyl radical scavenging activity of test sample is determined accordingly in comparison with negative control. Ascorbic acid is taken as the positive control. 

Hello, Refer this,

Hydroxyl radical scavenging activity

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell et al (1987). Stock solutions of EDTA (1 mM), FeCl3 (10 mM), Ascorbic Acid (1 mM), H2O2 (10 mM) and Deoxyribose (10 mM), were prepared in distilled deionized water.

The assay was performed by adding 0.1 mL EDTA , 0.01 mL of FeCl3,0.1 mL H2O2, 0.36 mL of deoxyribose, 1.0 mL of the extract of different concentration (50, 100, 200, 400 & 800 μg/mL) dissolved in distilled water, 0.33 mL of phosphate buffer (50 mM , pH 7.9), 0.1 mL of ascorbic acid in sequence. The mixture was then incubated at 370C for 1 hour. 1.0 mL portion of the incubated mixture was mixed with 1.0 mL of 10% TCA and 1.0 mL of 0.5% TBA (in 0.025 M NaOH containing 0.025% BHA) to develop the pink chromogen measured at 532nm. The percentage inhibition was calculated by comparing the results of the test with those of the control using the formula.

% Inhibition = {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged

Reference

 Halliwell B, Gutteridge JMC, Aruoma OI. The deoxyribose method: a simple test to be assay for determination of rate constants for reaction of hydroxyl radicals, Ana Biochem. 1987; 65: 215-219.

several  methods are avaible

The ability to scavenge hydrogen peroxide evaluated according to the method of (Ruch et al., 1989).

A solution of H2O2 (2mmole) was prepared in phosphate buffer (pH 7.5). Plant extracts (50-250µg/ml) were added to the hydrogen peroxide solution (0.6ml). Absorbance of hydrogen peroxide at 230nm was determined after 15 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. BHT and α- tocopherol were taken as known standards. The scavenging activity of the plant extracts on H2O2 was expressed as:

% scavenged [H2O2] = [(A0-A1)/A0] x 100

Where A0 is the absorbance of the control and A1 is absorbance in the presence of plant extracts and known standards.

I hope this can be of some assistance

Please refer the attached file. still u r having doubt means i will give working protocol too.. Best of luck

DPPH activity . According to method given by Wang et. al.,  1998.

Hydroxyl radical scavenging assay can be performed by Klein et al and Halliwell methods. 

Hydroxyl  radical  scavenging  activity  may be  assayed  by 

the method of Smirnoff and Cumbes. The reaction mixture 3.0 ml contained 1.0 ml of 1.5 mM  FeSO4, 0.7  ml  of  6  mM  hydrogen  peroxide,  0.3  ml  of  20  mM sodium  salicylate  and  varied  concentrations  of  the  extracts.  After incubation for  1 hour at 37°C,  the  absorbance  of  the  hydroxylated salicylate complex was measured at 562 nm. The scavenging activity 

of  hydroxyl  radical  effect  was  calculated  as  follows  :

[1‐(A1-A2)/A0] x 100,

 where A0 is absorbance of the control (without extract) and A1 is the absorbance in the presence of the extract, A2  is the absorbance

without sodium salicylate. 

Good luck

I am  also following  the  method  of  Smirnoff  and  Cumbes. But the problem arising during the  calculation.  Actually, the percentage of  hydroxyl radical scavenging activity  decreases with the increasing concentration of ascorbic acid. I am  facing this problem again and again.  Is the  colour of the  samples increases with the  increasing concentration of  extracts/ ascorbic acid.

Respected Madam,

                                    Kindly refer the following methodology.

Hydroxyl Radical Scavenging activity

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell et al. (1987). Stock solutions of EDTA (1 mM), FeCl3 (10 mM), Ascorbic Acid (1 mM), H2O2 (10 mM) and Deoxyribose (10 mM), were prepared in distilled deionized water. The assay was performed by adding 0.1 ml EDTA, 0.01 ml of FeCl3, 0.1 ml H2O2, 0.36 ml of deoxyribose, 1.0 ml of the extract of different concentration (125, 250, 500 & 1000 μg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM , pH 7.9), 0.1ml of ascorbic acid in sequence. The mixture was then incubated at 37ºC for 1 hour. 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10 % TCA and 1.0 ml of 0.5 % TBA (in 0.025 M NaOH containing 0.025 % BHA) to develop the pink chromogen measured at 532 nm. The hydroxyl radical scavenging activity of the extract is reported as % inhibition of deoxyribose degradation is calculated by using the following equation

Hydroxyl radical scavenging activity= {(A0 –A1)/A0)*100}

Where, A0 is the absorbance of the control reaction, and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged.

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

Good Luck

I am also experiancing the same problem posed by Sumana Datta. I also followed the Smirnoff and Cumbes protocol When I am increasing the concentration of extract the absorbance is also increasing. I refered the oroginal paper published in 1989 by Smirnoff and Cumbes. Their graphs represent salicylate hydroxylation, % control(downward trend). But the recent paper indicate them as % inhibition(upward trend). I would like to know the difference between. 

To Dr. Tresina Soris P mam,

I had followed the protocol with ascorbic acid as standard (0.05 mg/ml-0.25 mg/ml), however the absorbance increases with concentration and are higher than absorbance of control.

kindly suggest what can i do.?

You have follow the method of ,Choi et al., 2002 for DPPH activity

I did not try any method yet but the Smirnov method seems to be much easier than the others - in some way it seems logical that the OH. activity decreases with the increase of Vit C since this molecule would catch these radicals (?)

You have follow the method of ,Choi et al., 2002 for DPPH activity

i have followed so many methods still i m facing the same problem as the concentration of std/extract increases the absorbance also increases as well, please suggest me what should i do.

which method you followed and did u success or not for this assay please guide me .

Perhaps this could be useful

https://www.researchgate.net/publication/321330966_Antioxidant_Potential_and_Total_Phenolic_Changes_in_Malawian_Herbal_Formulations_Stored_In_Clay_Pots_and_Plastic_Bottles

can anyone suggest the method of Hydroxyl radical estimation using Terepthalic acid (TA) by in-vitro?

The main issue with this determination is the highly reactive nature of OH radicals which are readily dismutase into hydrogen peroxide due to SOD activity in living system. So, I believe there is no method to determine the accurate calculation in the literature except ESR. Moreover, the homogenization of sample should be done in liquid nitrogen to minimize the interconversion of OH radicals which will reduce SOD activity. If some has any standardized method please share.