Labsolution software

Total lipids in Caco-2 cells, AC and BLC were extracted, and relative fatty acids methylated as previously described [18 (link)]. Before methylation, pentadecanoic acid was added as internal standard. The qualitative and quantitative content of fatty acid methyl esters (FAMEs) was determined by fast-GC (GC-2030AF; Shimadzu, Kyoto, Japan) using a capillary column (30 mt, 0.2 μm film thickness) with a programed temperature gradient (50–250 °C, 10 °C/min). The gas chromatographic peaks were identified based on their retention time ratios relative to methyl stearate and predetermined by use of authentic samples [19 (link)]. Gas chromatographic traces and quantitative evaluations were obtained using a Lab Solution software (Shimadzu, Kyoto, Japan).

Organic acid produced during the process of Si solubilization was estimated to identify marker organic acids. Microbes with different efficacies of Si and P solubilization along with best Si solubilizers screened with the developed NBRISSM media were used for organic acid profiling using HPLC (Shimadzu, Japan) comprising PDA SPD M 20 A system LC-20AD dual pump system, and SIL-20 AC auto injector (with cooler) furnished with a 20 µL sample loop. Compounds were separated on column with 250 × 4.6 mm, i.d. (inside diameter) and 5 µm pore size, Shimadzu RP-C18 column protected by guard column containing the same packing. The mobile phase of 0.01mol/L sulfuric acid was used for the elution at the flow rate of 0.5 ml/min (Wang et al., 2014 (link)). In brief, the culture supernatant of different microbial cultures grown in NBRISSM medium after 5^th day of inoculation was injected thrice with 20 µl sample loop and run for 25 min. Data was integrated by Shimadzu Lab solution software with the detection of peaks at 510 nm and results were obtained by comparison with standards. Plain mobile phase was used as control for identification of blank peaks.

The HPLC equipment used for the quantitative analysis of the two flavones, kuwanon G and morusin, in M. alba L. was a Shimadzu LC-20A series (Kyoto, Japan) consisting of a pump (LC-20AT), degassing unit (DGU-20A3R), column oven (CTO-20A), auto sampler (SIL-20A), and PDA detector (SPD-M20A). The acquisition and processing of chromatographic data was performed using the LabSolution software (Version 5.53, SP3, Shimadzu, Kyoto, Japan). The column used for the separation of kuwanon G and morusin was the Phenomenex Gemini C18 analytical column (250 mm × 4.6 mm, 5 μm, Torrance, CA, USA), which was maintained at 45 °C. The mobile phase for the efficient separation of analytes was composed of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B), and flowed from the initial 20% B to 90% B for 50 min. The flow rate was kept constant at 1.0 mL/min, the injection volume was 10 μL, and the PDA scanned for quantification was 190–400 nm. For HPLC analysis of the two biomarker compounds in M. alba L., 500.0 mg of the ground raw M. alba L. sample material was dissolved in 20 mL of 70% methanol and extracted for 60 min at 25°C using a Branson ultra-sonicator, 8510E-DTH (Danbury, CT, USA). Then, the extracted solution was filtered through a 0.2 μm membrane filter (Pall Life Sciences, MI, USA), before injection into the HPLC equipment.

The prepared samples were analyzed by ultra-performance liquid chromatography. The Nexera X2UPLC-MS/MS (Shimadzu Corp., Kyoto, Japan) contained two LC-30AD pumps, SiL-30AC autosampler, CTO-20AC column oven, CBM-20A communication bus module, and mass spectrometer LC-MS8050 with electro spray ionization with positive and negative ion mode (Table 1). Chromatographic separation was performed using an analytical column Kinetex 2.6 µm, Phenyl-Hexyl 100 Å 4.6 mm, 150 mm (Phenomenex, Torrance, CA, USA). The gradient program consisted of the following: 7-min sequence of linear gradient flows of solvent B (acetonitrile:methanol 1:1 v/v) balanced with solvent A (water with 0.1% formic acid) at a flow rate of 0.6 mL/min: 50–80% B over 1 min, 80–100% B over 2 min, isocratic 100% B for 3 min, and finally, 100–50% B over 1 min. The injection volume was 1 µL and column temperature was 40 °C.
The main parameters used to identify analytes were their retention times and multiple reaction monitoring (MRM) ratio, which were obtained at 0.25 µg/mL working standard solutions for most standards, and 2.5 µg/mL for AMP, IB, SPEC, STREP, SFC, SNA and TET. Chromatographic data processing was carried out using LabSolution^® software (Shimadzu Corp., Kyoto, Japan).

Chromatographic analysis of the marker components in MHT was performed using the Shimadzu Prominence LC-20A series HPLC system (Kyoto, Japan) equipped with photodiode array (PDA) detector and Lab Solution software (Version 5.54 SP3, Shimadzu, Kyoto, Japan). Waters SunFire C₁₈ analytical column (250 × 4.6 mm; 5 μm, Milford, MA, USA) was used for the separation of the main components as the stationary phase and maintained at 40°C. The mobile phases consisted of 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B) and the gradient elution for chromatographic separation was as follows: 10–60% B for 0–30 min, 60–100% B for 30–40 min, 100% B for 40–45 min, 100–10% B for 45–50 min, and 10% B for 50–60 min. The flow rate was 1.0 mL/min and injection volume was 10 μL.