Then, 5mL of reagent C was added. To this, 0.5mL of reagent D was added and was allowed to incubated in dark for 30 minutes, and the absorbance was determined at 660nm using spectrophotometer.2.5.3. Determination of Total Carbohydrates The carbohydrate was estimated as described by Sadasivam and Manikam [18] using Glucose as different a standard. 100mg of sample powder was ground with 10mL of 80% acetone in mortar and pestle. Then, the filtrate was centrifuged at 5000rpm for 5 minutes. The supernatant was used for further analysis. 400mg of anthrone reagent was dissolved in 190mL of ice-cold concentrated sulphuric acid with 10mL of distilled water. Glucose was used as a standard. 10mg of glucose was dissolved in 100mL of distilled water. A 0.5 to 1mL of diluted supernatant (10?1) was taken in the test tubes.
It was made up to 1mL with distilled water. A 4mL of anthrone reagent was added. The tubes were treated over a boiling water bath for 10 minutes and then cooled down to room temperature. The absorbance of a blue green solution was measured at 630nm using spectrophotometer and compared with a standard curve preparation with known amounts of glucose. The amount of total carbohydrate present in each sample was calculated and the results were tabulated.2.5.4. Estimation of Amino Acids Amino acids in leaves were determined according to the procedure of Ishida et al. [19]. Extracted samples were filtered through a 0.45��m membrane, filter, and 20��L of the filtrate was injected in to a HPLC (model LC 10 AS, Shimadzu, Mount holly, New Jersey) equipped with a cation exchange column packed with a strongly acidic cation exchange resin, that is, styrene divinylbenzene copolymer with sulphonic group.
The amino acid analysis was with the nonswitching flow method and fluorescence detection after postcolumn derivatization with o-phthalaldehyde. Amino acid standards were used to calculate amino acid concentrations in samples.2.5.5. Mineral Quantification For the determination of mineral contents in the sample, digestion mixture was prepared following standard method. For digestion, 0.5g of dried sample was mixed with 5mL digestion mixture and kept in digestion unit at 300��C. The process was allowed to continue till the mixture turns colourless. Desired volume of distilled water is added to the digested and cooled samples. Solution was filtered and mixed well till all sediments got dissolved.
Subsequently, minerals were determined as follows: nitrogen Entinostat (N) through micro-Kjeldahl method; phosphorus (P) by treating the digested samples with ammonium molybdate and freshly prepared ascorbic acid and analyzed by spectrophotometer (Hitachi U-2001 Japan); potassium (K), sodium (Na), and calcium (Ca) were determined by Flame Photometer by the method of Allen [20]. The microelements (Fe, CO, Cu, Mg, Mn, and Zn) were determined through atomic absorption spectrophotometer.2.5.6.