Hi Clement,

I attached protocol for measuring antioxidant enzyme activities and I found it more easier and efficient than other methods.

prepare enzyme extract first as follow

1) 200mg of tissue homogenized in 0.1M KPO4 buffer containing 10ul of 0.1M Na2 EDTA.

Centrifuge at max speed for 15 min at 4degree Celsius.

Collect supernatant and use it as source of enzyme

Catalase- reduction of h2o2 at 240nm

Total volume for activity 3ml

1) Enzyme extract-100ul

2) 0.1M KPO4 buffer (pH-7.4) - 2.940mL

3) 14mM H2O2 - 2.5uL

Note- add enzyme extract in last. take reading at 240nm in spectrophotomete time scan of 3 min. note reading after every 30 sec.

from my point of view actually sir any studies on plant tissue or its plant extract , better to do first GC-MS , find the active component in that tissue or extract, then check antimicrobial activity along with minimum inhibitory concentration , and also total antioxidant activity by in vitro studies , after finding that result, you can proceed further for specific antioxidant like both enzymatic ( SOD, CAT , GPX) and non enzymatic (GSH, VIT-C, VIT-E ).

@ ARBIND

HE IS USING PLANT TISSUE. So in plant naturally superoxide radicals forms so the CAT and SOD plays role. So i think there is no need for going GC-MS he can directly do that anti-oxidant assay.

Thank you all- Abdugaffar, Praveen, Arbind for your brilliant contributions. @ Arbind, why did you suggest GC-MS for active compounds first?

because if you take plant tissue, which will be made up of mixture of component , so its better to find the most active component in that mixture , check its activity along with other component , which really help to make that particular compound as patent drugs for antioxidant activity .

take plant tissue homogenize in liquid nitrogen and dissolved in 100 mM sodium phosphate buffer (pH-7.4) containing 0.1mM Na2EDTA, 1% (w/v) PVP and 0.5% (v/v) Triton-X 100. The homogenate was centrifuged at 10,000 rpm for 20 mins at 4ºC. The supernatant was collected for measurement of specific activities of antioxidant enzymes and was stored at -20ºC till use.

for catalase enzyme:In the cuvette 0.2 ml of enzyme extract was mixed with 2 ml of phosphate buffer (pH-7.0) to it 3% H2O2 was added, OD was taken immediately after adding H2O2 at 240nm as initial reading and after 3 mins again OD was taken as final reading. Phosphate buffer was used as a blank for spectrophotometer (Chandlee and Schanalios 1984 ).

for SOD: The activity of superoxide dismutase was determined by the method of Dhindsa and Matowe (1981) by following the photo-reduction of nitroblue tetrazolium (NBT). The reaction mixture contained 50 mM phosphate buffer (pH 7.8), 0 1 mM EDTA, 13 mM methionine, 75 µM nitroblue tetrazolium (NTB), 2 mM riboflavin and 100 µl of the supernatant Riboflavin was added as the last component and the reaction was initiated by placing the tubes under two 15 W fluorescent lamps The reaction was terminated after 10 min by removing the reaction tubes from the light source Non-illuminated and illuminated reactions without supernatant served as calibration standards Reaction products were measured at 560 nm. The volume of the supernatant corresponding to 50% inhibition of the reaction was assigned a value of one enzyme unit.

Following is a useful link in this regard: http://prometheuswiki.publish.csiro.au/tiki-searchresults.php?find=Antioxidant+enzymes&where=wikis&boolean_last=n&search=Search&exact_match=

Aqeel,

Thank you for your interest. It is really appreciated. Could you also send me any detailed protocol in pdf in order to go through also.

Regards

Thank you so much, Aidallal.

Dear Celment,

Here are the two protocols in details:

Dear Hanem

I am so appreciative,. ........thank you so much!

Morespecifically, superoxide dismutase (SOD) and catalase (CAT) activities as antioxidant enzymes, malondialdehyde (MDA) as a sign of lipid peroxidation.The principle of SOD activity assay is based on the inhibition of nitroblue tetrazolium (NBT) reduction. Illumination of riboflavin in the presence of O2 and electron donor like methionine generates superoxide anions and thiscan be  used as the basis of assay of SOD. The reduction of NBT by superoxide radicals to blue coloured formazan is  followed at 560 nm. “One unit of SOD activity is defined as that amount

of enzyme required to inhibit the reduction of NBT by 50% under the specified conditions. The preparation of hemolysate was done by the method of McCord and Fridovich. The catalase activity of the hemolysate is determined by adopting the method of Brannan et al.