Metabolic analysis
Tissues of rosemary leaves, stems and roots (three biological
replicates) were used for RNA extraction and metabolite analyses. High
performance liquid chromatography (Waters e2695) and high-performance
gas chromatography (Agilent Technologies 7890B-5977B) were used for
metabolite analysis of S. rosmarinus . The rosemary samples were
oven-dried to a constant weight at 60°C, crushed with a grinder, and
sieved through a 60-mesh screen. An aliquot of the test sample powder
(0.02g) was added to 1 mL of 70% methanol, ultrasonically extracted for
45 minutes, centrifuged at 8,000 rpm/min for 10 minutes, and the
supernatant was collected and passed through a 0.22 µm filter membrane
to obtain the test solution, each group of extracts (3 replicates) was
placed in a 4 ℃ refrigerator for later use.
A Waters e2695 high performance liquid chromatography system was used
for analysis, the detector model was a Waters2998 ultraviolet light
detector, the chromatographic column model was a Waters sunfire C18
reversed-phase chromatographic column, and the chromatographic
acquisition software was Empoeder 2. The chromatographic conditions were
as follows: flow rate 1 mL/min, column temperature 30°C, sample loading
volume 20 µL. The sample running time was 96 minutes, 0.02 percent
phosphoric acid water and acetonitrile, utilizing gradient elution.
Qualitative and quantitative analysis of volatile components in
different parts of the rosemary plant was carried out using GC-MS.
Precisely weighed 1 g of fresh sample of rosemary was placed in a 20 mL
headspace bottle, sealed, and then the sample was injected directly into
the headspace sample tray. The chromatographic and headspace conditions
were as follows: chromatography initial temperature 35°C, held for 2
min; 3°C/min to 130°C, 25°C/min to 250°C, held for 3 minutes; cooling to
35°C. Headspace conditions: Equilibrium: 80℃ for 30 min, oven
temperature: 80℃, loop temperature: 90℃, transfer line temperature:
100℃, GC cycle time: 70 min, split ratio: 1:5, flow rate: 1mL/min, M/Z:
35-600, EI: 70eV, MS ion source temperature: 230°C, detector
temperature: 260°C.