Tq8040

Hormone levels were quantified from leaf no. 8. Five leaves were pooled at random to produce four replicates per developmental stage. After harvest, leaves were flash-frozen in liquid nitrogen and stored at −80 °C. The samples were ground twice for 0.5 min at 30 Hz with a Retsch Mixermill and were cooled with liquid nitrogen before and between milling steps. They were then extracted in 2 × 750 µl (total 1500 µl) ethyl acetate with 0.1% formic acid, containing the internal standard (3-hydroxybutyrate 60 ng, 9,10-dihydrojasmonic acid 80 ng, and 3-indole pyruvic acid 50 ng per ml), for 10 min in an ultrasonic bath. After centrifugation, the supernatant was used to quantify soluble hormones, whereas the pellet was hydrolysed to release any bound compounds. After removal of the solvent from the supernatant by using an Eppendorf vacuum concentrator (mode HV) at 30 mbar, 70 µl of a fresh 1:1 mix of methanol and trimethylsilyldiazomethane, Sigma-Aldrich) was added to the dry samples. GC-MS (Shimadzu TQ8040) in the splitless MRM mode was used for sample analyses. Hydrolysis of the dry pellet was performed using 200 µl of 3 M HCl and 200 µl of 3 M NH₃.

The chemical constituents of EOs isolated from the aerial part of Thymus sp. were determined by using a gas chromatograph (Shimadzu 2010, Kyoto, Japan) coupled with a triple quadrupole mass spectrometer (TQ 8040, Shimadzu, Kyoto, Japan). The used column was an optima 1MS + WAX column (30 m × 0.25 mm i.d., 0.25 µm film thickness, Macherey–Nagel, Duren, Germany) using helium as carrier gas at 1 mL min⁻¹ flow. The oven temperature was initiated at +70 °C for 11 min and raised to +190 °C at a rate of 5 °C min⁻¹ and then to +240 °C at a rate of +20 °C min⁻¹, where it was kept for 5 min. Injector and MS source temperatures were set to +250 °C and +200 °C, respectively. The samples were diluted before injection (1:25, v:v), and the injection volume was 1 µL, with a split ratio of 10:1.
The EO constituents were identified based on the comparison of their retention indices (abbreviated RI^a), determined relative to the t_R values of n-alkanes (C10–C35) on both capillary columns with those in the literature [59 ] and their mass spectra with those of standard compounds (α-pinene; sabinene; β-pinene; β-myrcene; α-phellandrene; 3-carene; D-limonene; cis-β-ocimene; trans-β-ocimene; carvacrol; caryophyllene) available in our laboratories or those listed in the NIST 14 and Wiley 09 mass spectral libraries (abbreviated RI ^b).

The MESF was analyzed in a mass spectrometer (TQ 8040, Shimadzu Corporation, Kyoto, Japan) using the electron impact ionization (EI) method and a gas chromatograph (GC-17A, Shimadzu Corporation) with a fused silica capillary column (Rxi-5 ms; 0.25 m film, 30 m long and internal diameter 0.32 mm) coated with DB-1 (J&W). The oven temperature was set at 70 °C (0 min); 10 °C, 150 °C (5 min); 12 C, 200 °C (15 min); 12 °C, 220 °C (5 min), with a hold time of 10 min. The inlet temperature was 260 °C. The flow rate of the column was 0.6 mL/min helium gas at constant pressure (90 kPa). The GC to MS interface temperature was 280 °C. The MS was used in scanning mode, with a scanning range of 40–350 amu. The ionization mode was electron ionization (EI), and the mass range was 50–550 m/z. One microliter of the sample was injected in the split-less injection mode. The total GC-MS run time was 50 min. The compounds in the peak areas were identified by comparison with the national institute of standards and technology (NIST) GC-MS library version 08-S.

The pH of the fermentation system was measured with a standard pH meter (DELTA320, Mettler Toledo Co., Ltd. Shanghai, China). SCFAs were extracted by absolute ether following the method of Bai et al. (2021) (link) and were analyzed by gas chromatography–mass spectrometry (GC–MS)-TQ8040 (Shimadzu, Kyoto, Japan) equipped with SH-Rtx®-WAX column (30 m × 0.25 mm i. d.; film thickness 0.25 μm). In brief, the fermented solution was centrifuged at 6000 g for 10 min. The 20 μl of 10% H₂SO₄ was added to acidify 500 μl of supernatant, and 500 μl of absolute ether was used to extract SCFAs. The mixtures were then centrifuged at 8000 g for 10 min at 4°C, and the phases were separated. The supernatant was taken and filtered using a 0.20 μm filter into a sample injection bottle. The temperature increased raised to 140°C at 7.5°C/min and held for 4 min, which then raised to 200°C at 60°C/min. Carrier gas helium was employed, the flow rate was 2.0 ml/min, the full scan mode in the m/z range was 20.0–300.0, and the injection volume was 1 μl. SCFA concentration was determined by the external standard method with corresponding standards.

Table 1 details the origin of the 19 EOs samples used in this study. Industrial-scale extractions by dragging water vapor were applied to obtain the EOs, which were selected based on their commercial availability. All samples were standardized according to the methods and quality indicators described in the Brazilian Pharmacopoeia (≥ 90% purity).
The chemical composition of the EOs was assessed by gas chromatography/mass spectrum (GC/MS) performed with a Shimadzu 2030 equipment coupled to a sequential Shimadzu TQ8040 mass detector, using an HP-5MS column (30 m × 0.25 mm × 0.25 μm). Analytical conditions were established at 250 and 260 °C injector and transfer line temperatures, respectively; the oven temperature was programmed from 60 to 240 °C, at a rate of 3 °C min⁻¹ and maintained at 240 °C for 10 min; helium gas at 1.0 mL.min⁻¹; 0.1 μL injection volume (5% HPLC grade n-hexane solution); and a 1:30 split ratio.
The identification of the components was performed by comparing the mass spectra with those of commercial libraries [55 (link)], and by their linear retention rates [56 (link)] after injection of a homologous series of alkanes (C₈–C₂₆) under the same experimental conditions, and compared with data from the literature [57 ].