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.