Lc 20at prominence

Fifty microliters of A. sculptum saliva, collected after 7–9 days of host attachment, were diluted in 450 μL of PBS, and filtrated through a 3-kDa molecular weight cutoff microfilter (Vivaspin 500, Sartorius Biolab Products, Goettingen, NI, Germany), separating the saliva into a low molecular weight (LMW; < 3 kDa) and a high molecular weight (HMW; >3 kDa) fractions. The HMW fraction was diluted again in PBS and filtrated a second time. The resulting HMW and LMW fractions were pooled, sterilized through a 0.22 μm membrane (Millipore Corporation, Billerica, MA, United States), and used in DC cultures described later.
Two hundred microliters of the LMW fraction of A. sculptum saliva were applied onto analytical reversed phase C₁₈ column (Shim-pack Shimadzu VP-ODS, size 250 mm × 4.6 mm, 5 μm), coupled to a ultra fast liquid chromatography (UFLC) system (LC-20AT Prominence, Shimadzu, Japan) previously equilibrated with 2% acetonitrile (ACN) in acidified water (0.05% trifluoroacetic acid). Salivary molecules were eluted with a linear gradient of 2–60% ACN in acidified water over 60 min, at a flow rate of 1 mL/min. Fractions were individually used in DC cultures described later.

TEGDMA, Bis-GMA, bisphenol A, UDMA, and HEMA monomers were investigated by HPLC. HPLC was performed on an isocratic HPLC instrument (Shimadzu LC-20AT Prominence) with an LC-20AT pump, a manual injector with a loop volume of 20 µL, and a programmable variable wavelength Shimadzu SPD-20A detector. Separation was carried out with a Supelcosil LC-18 reverse-phase column (4.6 × 250 mm, 5 µm particle size). The mobile phase was 65% acetonitrile (gradient grade, Sigma-Aldrich, St. Louis, MO, USA) and 35% water (Direct-Q 3 UV system, Millipore, Burlington, MA, USA). The flow rate of the mobile phase and the run time were set to 1 mL/min and 12 min, respectively. The column temperature was 25 °C. The detection wavelength was 205 nm, and the chromatograms were analyzed using LC Solution (Shimadzu, Kyoto, Japan) software.
The qualitative and quantitative evaluations of monomer release were carried out according to the report of Pelka et al. [26 (link)]. A mixture of 10 mg of each of the monomers (Table 2) in a 4 mL acetonitrile/water mixture (1:1) was used as standard. Standard HPLC spectra were obtained by injecting this mixture into the device at appropriate rates. The retention times and peak values of the monomers were recorded, and the concentrations were determined in μmol/L based on the calculation of the area under the peaks obtained from the standard solutions.

HQD was prepared by grinding 90 g of skullcap, 60 g of licorice, 60 g of peony, and 490 g of jujube, then boiling at 100°C for 30 min in a 10X volume of distilled water. After filtration, the residue was extracted using an 8X volume of distilled water. The filtrates were combined in a container and stored at 4°C for subsequent animal experiments.
The HQD extract was analyzed using a Shimadzu LC-20AT Prominence high-performance liquid chromatography (HPLC) system equipped with a diode array detector. Chromatographic separation was performed on Diamonsil C18 (250 mm × 4.6 mm, 5 μm) maintained at 30°C. The mobile phase flow rate was 1 ml/min. The mobile phases were 0.1% (v/v) formic acid (A) and acetonitrile (B). The gradient elution program was as follows: 0–15 min, 95–95% A (v/v), 5–5% B (v/v); 15–30 min, 95–85% A, 5–15% B; 30–60 min, 85–77% A, 15–23% B; 60–90 min, 77–55% A, 23–45% B; 90–110 min, 55–40% A, 45–60% B; 110–115 min, 40–90% A, 60–5% B; 115–120 min, 40–95% A, 60–5% B. The injection volume was 10 μl and the detection wavelength was 280 nm (Yang et al., 2017 (link)).