Biochemical estimations were carried out for all the samples. Estimation of ascorbic acid done by 2–4 dinitrophenylhydrazine method. The Principle of this method is that Dehydroascorbic acid was coupled with 2, 4 dinitrophenylhydrazine and the resulting derivative is treated with sulphuric acid to produce a newly observed color which is measured at 545 nm.[13 (link)] Iron estimation was done by Ferrozine method and the principle of transferrin bound iron breaks into free ferric ions in an acidic medium. These ferric ions react with Hydroxylamine Hydrochloride reduced into ferrous ions which react with Ferrozine to form a violet colored complex measured at 560 nm. The difference before and after the addition of ferrozine is proportional to iron concentration reaction in the specimen.[14 ]