Ultimate xb c18

The capsule of NXT was completely removed, and the powder was weighed 1g precisely. The powder was treated by ultrasonic wave in 50ml of 75% methanol for 30 min. The supernatant was filtrated and then injected into ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry system for analysis (UFLC-Q-TOF-MS/MS). The column was a Ultimate XB-C18 (4.6×250mm, 5μm, Welch), which is maintained at 30°C. The mobile phases were composed of acetonitrile (A) and water with 0.1% formic acid (B) using a multi-step linear gradient elution of 10% A at 0–5min, 10–25% A at 5–35min, 25–60% A at 35–65min, 60–67% A at 65–70min, 67% A at 70–80min, 67–90% A at 80–95min, 90% A at 95–105min, 90–10% A at 105–110min, and 10% A at 110–120min with the flow rate kept at 1.0 ml/min. Other MS parameters were adopted same as published work (Zeng et al., 2018 (link)). The sample volume injected was set at 20μl. All the acquisition and analysis of data were controlled by the PeakView Software TM V. 1.1 (AB SCIEX, Foster City, CA).

HPLC determinations were performed using an Agilent HPLC 1200 instrument (Agilent Technologies, Palo Alto, CA), equipped with a diode array detector (DAD) detector, an auto sampler, a column heater, and a Welch Ultimate® XB-C18 (250 mm × 4.6 mm, 5 μm) column. The mobile phase consisted of A (acetonitrile) and B (4% glacial acetic acid aqueous) (V/V). Optimum separation was 48% A. The flow rate was 1.0 mL·min⁻¹ and injection volume was 10 μL. The column temperature was set at 30°C and the wavelengths were monitored at 425 nm.

For the analysis, the standard products were purchased from Solarbio Science & Technology Co., Ltd. (HPLC ≥ 98%, Beijing, China). D-(-)-quinic acid, trigonelline, and citric acid were prepared at 5 mg/ml with ultrapure water. Berberine, luteolin, caffeic acid, apocynin, taxifolin, and ferulic acid were prepared at 2.5 mg/ml with 50% methanol aqueous solution. Then take these solutions to make a 1,000 µl mix. The resulting solution was filtered through a 0.22 μm membrane filter before HPLC injection. HPLC was performed using an Agilent 1,260 Infinity Ⅱ system (Agilent Technologies, Santa Clara, CA, United States) equipped with a DAD detector and a Welch Ultimate XB C18 (250 mm × 4.6 mm; 5 µm). The mobile phase included 0.2% phosphoric acid plus 0.4% sodium 1-heptanesulfonate (A) and methanol (B) at a flow speed of 1.0 ml/min in the condition of a column temperature of 30°C. The detection wavelength was set at 214 nm. The reference standard sample injection volume was 10 µl. The gradient elution was as follows: 0–5 min (100–100% A, 0%–0% B), 5–60 min (100–60% A, 0%–40% B), 60–120 min (60–30% A, 40–70% B), 120–125 min (30–30% A, 70–70% B), 125–125.1 min (30–100% A, 70–0% B), 125.1–132 min (100–100% A, 0–0% B). The chromatographic data and peak area scores were collected and analyzed using ChemStation software.

Column chromatographic procedures were performed using silica gel (80–120 mesh and 200–300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China) and Sephadex™ LH-20 gel (40–70 μm; Merck, Darmstadt, Germany), whereas precoated silica gel (GF254, Qingdao Marine Chemical Co. Ltd., Qingdao) plates were used for TLC analyses. Spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH. UV spectra were recorded using a Waters UV-2401A spectrophotometer equipped with a DAD and a cell of 1 cm pathlength. Methanolic samples were scanned from 190 to 400 nm in 1 nm steps. Semipreparative HPLC was performed on an Agilent 1120 apparatus equipped with a UV detector and a reversed-phase C₁₈ column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). 1D (¹H, ¹³C) spectra of all compounds were recorded on Bruker AM-600, AM-500, and AM-400 NMR spectrometers (Bruker, Karlsruhe, Germany), with TMS as the internal reference. |Enzymatic activity experiments were performed using SpectraMax i3x (Molecular Devices, Austria).