Stevia Genotypes

MATERIALS AND METHODS

Stevia genotypes (SRB-512, SRB-123, SRB-128 and Madhupatra) obtained from different locations in India were used in the present study. The plants were raised at the research farm attached to the Department of Genetics and Plant Breeding while the laboratory works were carried out at the Department of Bio-Chemistry, Ch. Charan Singh University Campus Meerut. The clean and fresh leaves and stems were collected separately from growing plants of Stevia and dried at room temperature. The dry leaves/stems (in small pieses) of Stevia plant were added in boiling water. After some time, the materials (leaves and stems) were re-boiled for short time and kept at room temperature for one to two hours. After that, Stevia leaves/stems

The spectra were recorded by making dried KBr pellet. A total scans of 16 scans per sample at resolutions were obtained over mid IR region of 400 to 4000 cm-1. The percentages of the diterpene glycosides were determined by High Pressure Liquid Chromatography (HPLC). For mobile phase, HPLC grade acetonitrile and water (80:20) were mixed (pH of 3.0) and filtered through 0.22 µm Millipore filter. Accurately weighed 50 mg (dried at 1000C, 3h) of stevioside standard was taken in a 100 ml volumetric flask and diluted to volume with mobile phase. Then, accurately weighed 70 - 120 mg of the sample was taken in 100 ml volumetric flask and diluted with the mobile phase. Supelcosil LC-NH2 (length: 15 - 30 cm; inner diameter: 3.9 - 4.6 mm) column was used. The flow rate was adjusted so that the retention time of stevioside is about 10

A broad band at 3510 - 3490 cm-1 may be attributed to O-H stretching of alcohol group. The maximum absorption for O-H stretching depends upon concentration, nature of the solvent and temperature. In case, an alcohol is branched heavily in the α-position, the absorption band corresponds to the free O-H group. In intermolecular hydrogen bonded molecules, absorption shifts are concentration dependent. The O-H stretching absorption band appears at 3570 - 3450 cm-1. The presence of a band at 2985-3000 cm-1 is attributed to C-H stretching of sp2 hybridized while the band at 2790-2865 cm-1 may be attributed to C-H stretching of sp3 hybridized carbon present in diterpene glycoside. Bands at 1724 cm-1 and 1685 cm-1 are assigned to the carbonyl group and hydrogen bonded carbonyl groups, respectively. The band at 1600 - 1585 cm-1 indicates the presence of C=C stretching of alkene group while bands at 1085 - 1020 cm-1 in FTIR spectrum are attributed to C-O stretching of ether group. The bands present at wave lengths 980 - 1000 cm-1 are attributed to C-O stretching of ethers group. Band in the range 850 - 835 cm-1 are assigned to C-C stretching in diterpene glycoside extract from Stevia leaves and