Synergi c18 column

Metabolite profiles of test extracts were recorded on a Shimadzu Prominence (Shimadzu Corporation, Kyoto, Japan) equipped with two binary pumps, an autoinjector, a photodiode array (PDA) detector, and an LCMS2020 mass spectrometry detector with a single quadrupole analyzer and electrospray ionization (ESI). Each seed extract was dissolved in absolute ethanol (5 mg/mL) and injected (20 µL) into the HPLC system. The separation system consisted of a Synergi C18 column (Phenomenex, Torrance, CA, USA) (4.6 mm × 150 mm, 4 μm), and a combination of solvent A (1% formic acid in Mili-Q H₂O) and solvent B (1% formic acid in acetonitrile (ACN)). A gradient elution method at 0.7 mL/min was used as follows: 0–2 min 5% B, 2–20 min 5% to 40% B, 20–27 min 40% B, 27–42 min 40% to 90% B, 42–46 min 90%, and 46–50 min 90% to 5% B. The monitoring wavelength was 270 nm. Mass spectra were simultaneously acquired using electrospray ionization in the positive ion mode (scan 100–2000 m/z). The MS parameters involved a voltage detector (1.5 kV), curved desolvation line (CDL) (250 °C), heat block temperature (400 °C), and a nebulization gas flow (1.5 L/min).

Nucleotide hydrolysis reactions were carried out in the SAMHD1 buffer containing 0.5 mM GTP in a 500 μL reaction volume, with various concentrations of dNTPs. Reactions were initiated by the addition of SAMHD1 at a final concentration of 500 nM to the dNTPs solution and incubated in a 37 °C water bath. Reactions were terminated with a 5× dilution into ice-cold buffer containing 10 mM EDTA at various time points. Samples were deproteinized by spinning through an Amicon Ultra 0.5-ml 10-kDa filter (Millipore) for 20 min at 16,000×g. Samples were analyzed by HPLC with a Synergi C18 column 150 × 4.6 mm (Phenomenex). The column was pre-equilibrated in 20 mM ammonium acetate, pH 4.5 (buffer A) and samples were eluted at a flow rate of 1 ml/min with a gradient of methanol (buffer B) over 14 min. UV absorption was recorded at 260 nm.

Metabolic stability was measured by Wuxi AppTech (Shanghai, China). Briefly, peptides (1 μM, 5% MeOH in potassium phosphate buffer) were incubated with human (catalogue no. 452161 from BD Gentest) and mouse (catalogue no. M1000, Xenotech) liver microsomes at 37°C for 10 min. Liver microsomes were at a final assay concentration of 0.7 mg protein/mL. The reaction was started by the addition of 90 μL of NADP cofactor solution and stopped by the addition of 300 μL of stop solution (acetonitrile at 4°C, including 100 ng/mL tolbutamide as an internal standard) after 20 min of incubation. The samples were shaken for 5 min and then centrifuged for 20 min at 1500 g. A 100 μL aliquot of the supernatant was transferred to eight new 96-well plates with 300 μL of HPLC water and centrifuged at 1500 g for LC-MS/MS analysis (Shimadzu LC 10-AD−API 4000). An injection volume of 10 μL was added to a Phenomenex Synergi C18 column eluting with formic acid in water or acetonitrile at a flow rate of 800 μL/min. The percent loss of parent compound was calculated from the peak area ratio of the analyte/internal standard. Compounds and positive controls were tested in duplicate.

Chromatographic profiles of test extracts were recorded on a Shimadzu Prominence (Shimadzu Corporation, Kyoto, Japan) equipped with two binary pumps, autoinjector, and a photodiode array detector. Each botanical extract was then dissolved in absolute ethanol (5 mg/mL) and injected (20 µL) into the HPLC system. The separation system consisted of a Synergi C18 column (Phenomenex, Torrance, CA, USA) (4.6 mm × 150 mm, 4 μm), and a combination of solvent A (1% formic acid in acetonitrile (ACN)) and solvent B (1% formic acid in Mili-Q H₂O). A gradient elution method was used as follows: 0–2 min 0% B, 2–25 min 0% to 50% B, 25–35 min 50% B, 35–50 min 50% to 100% B, 50–55 min 100%, and 55–50 min 100% to 0% B, at 0.7 mL/min. The monitoring wavelength was 270 nm.