Zb 5ht

Polysaccharide samples (0.5 mg) were hydrolyzed with 10 M HCl at 80 °C for 25 min and evaporated under a stream of N₂. The resulting monosaccharides were converted into alditol acetates, according to Sawardeker, Sloneker, and Jeanes (1956) [53 (link)]. For methylation analysis, the polysaccharide samples were permethylated according to the method described by Ciukanu and Kerek (1984) [54 (link)]. The product was purified by water-chloroform extraction. The methylated polysaccharides were hydrolyzed in 2 M TFA at 120 °C for 2 h and evaporated under N₂. Finally, methylated monosaccharides were reduced with NaBD₄ and acetylated for gas-liquid chromatography-mass spectrometry (GLC-MS) analysis using the same conditions as for the sugar analysis and butyl glycosides as described below. The absolute configurations of the sugars were determined by GLC-MS of their acetylated glycosides using (S)-/+2-butanol essentially as described by Gerwig, Kamerling, and Vliegenthart (1979) [55 (link)]. Alditol acetates and acetylated 2-butyl glycosides were analyzed by GLC-MS using an ITQ 700 Thermo Scientific system equipped with a ZB-5HT (Phenomenex) capillary column with a temperature gradient from 150 to 270 °C at 8 °C/min.

EOs were analyzed with the same method and instrument as in our previous work [18 (link)]. We used a Hewlett Packard gas chromatograph (HP 6890 series GC) with a Hewlett Packard 5973 mass detector. The instrument was equipped with a high-temperature ZB-5HT (5% diphenyl- and 95% dimethyl-polysiloxane) 30 m long capillary column with an inner diameter of 0.32 mm and a film thickness of 0.25 μm (Phenomenex Inc., Torrance, CA, USA). EOs (20 µL) were diluted with dichloromethane (Sigma-Aldrich ACS Grade, Merck, Darmstadt, Germany) and directly analyzed. We injected 1 μL of a sample using the a split injection technique with a split ratio of 20:1 and an injector temperature of 250 °C. Helium was used as the carrier gas, and its flow rate was 2 mL/min. The temperature oven program was 40–180 °C with a heating rate of 5 °C/min and 180–280 °C with a heating rate of 10 °C/min. The solvent delay was three minutes. Peaks were identified by comparing the mass spectra data with the NIST 11 Library and by comparing their retention indices with literature values. In some cases, comparisons with standards were necessary. Retention index mixture, the terpineol mixture of isomers, and sabinene hydrate analytical standards were obtained from Merck. Amounts of the detected components represent % abundance (area percent, solvent peak excluded).

The volatile compounds of the S. montana L. herb were identified by comparing the mass spectra data with spectrometer database of the NIST 11 Library and by comparison of their retention index calculated against n-alkanes (C₉–C₂₀). Each chromatographic analysis was repeated three times. The average value of the relative composition of the essential oil percentage was calculated from the peak areas. The Hewlett Packard HP 6890 series GC system chromatograph (Hewlett Packard, WALDBRONN, Germany) was used for the study, which was coupled with the Hewlett Packard 5973 mass selective detector (Hewlett Packard, Waldbronn, Germany). The chromatograph was equipped with the non-polar, high-temperature ZB-5HT (5% diphenyl- and 95% dimethylpolysiloxane) capillary column of length of 30 m, inner diameter of 0.32 mm, film thickness of 0.25 μm (Phenomenex Inc., Torrance, CA, USA). The gas chromatograph was equipped with a split injector; the split ratio was 20:1 and 1 μm of a sample was introduced. Helium served as the carrier gas, and its flow rate was 2 mL/min. Analyses were performed at the temperature range of 40–280 °C and the heating rate was 10 °C/min. Injector temperature was 250 °C.