2.4.6 Total Soluble Sugar and Proline Content
Dry samples of S. hieraciifolia (0.1 g) were homogenized with 5 ml 70% ethanol, the homogenate was boiled at 80 °C for 3 min and centrifuged at 10.000 g for 5 min. For spectrophotometric measurement, 900 µl of distilled water was added to 100 µl of the supernatant and diluted. 1 ml of 5% phenol was added to this mixture and 5 ml of 96% sulfuric acid was added and then the mixture was homogenized by vortex. The mixture was cooled to room temperature and the absorbance of the mixture was recorded at 490 nm (Dubois, 1956).
Proline content of S. hieraciifolia was measured according to Bates et al. (1973). Oven dried shoots (0.1 g) were homogenized with 3% sulfosalicylic acid (5ml) and filtrated. One ml of filtrate, equal one ml of glacial acetic acid and ninhydrin reagent were mixed and subsequently incubated for 1 h at 100°C. After that, the test tubes were placed in an ice bath in order to stop reaction. Toluene (3 ml) were then added to the samples and shaken by a vortex. The UV-VIS spectrophotometer (Nicolet Evolution 100, Thermo) recorded absorbance of the toluene phase at 520 nm. A standard curve was used to determine the proline concentration.