Thermo dionex ultimate 3000 hplc system

The methylglyoxal trapping assay followed a published method with some modifications [5 (link),8 (link)]. Methylglyoxal (10 mM), o-phenylenediamine (50 mM) were freshly prepared in phosphate buffer (50 mM, pH 7.4). The final concentration of test samples (the five fractions, compounds 1–3 and kaempferol) and aminoguanidine was 5 mg/mL. Three mL methylglyoxal were incubated with 0.75 mL test samples or phosphate buffer in pH 7.4 at 37 °C water bath for 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h. After incubation, 200 μL of o-phenylenediamine were added with 200 μL of each test solution. Before HPLC analysis, the mixtures were kept in dark at room temperature for 1 h. The remaining methylglyoxal was detected on an Thermo Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific Inc., Massachusetts, USA) equipped with a diode array detector, and a Thermo Syncronis-C18 column (250 mm × 4.6 mm i.d., 5 μm; Thermo Fisher Scientific Inc., Massachusetts, USA). Mobile phases were composed of 0.1% formic acid in water (A) and methanol (B). The elution started at 50% B and then increased to 55% in 10 min, and then increased to 100% in 11 min and lasted for 5 min, then returned to 50% in 16.5 min and lasted for 5 min. The flow rate was 1 mL/min and the injection volume was 20 μL. Detection wavelength was monitored at 315 nm.

SND herbs (150 g) were consisted of 30 g Lonicerae japonicae Flos (Jin Yin Hua), 30 g Angelicae sinensis Radix (Dang Gui), 30 g Lonicerae japonicae Caulis (Ren Dong Teng), 30 g Spatholobi Caulis (Ji Xue Teng), and 30 g Glycyrrhizae Radix et Rhizoma (Gan Cao). All of them were purchased from the Zhejiang Chinese Medical University (Zhejiang, China), and were further authenticated by pharmacologist, Professor Jingxia Wang. A total of 150 g SND herbs were decocted twice with eightfold of water for 1 h. The extracts were combined, filtered, and then concentrated to a volume of 200 mL at 100 °C. The extracts were named as SND. SND used in this study were analyzed by a validated reversed-phase HPLC system (Thermo Dionex Ultimate 3000 HPLC system, Thermo Fisher Scientific, Waltham, MA, USA) using an Agilent Zorbax SB C18 column (4.6×150 mm, 5µm). A representative HPLC chromatogram of the SND was shown in Figure 1.Figure 1

HPLC analysis of the Shouzu Ning Decoction (SND)

Administration method: The patients were treated with the SND twice a day. For each treatments, 200 mL SND was diluted with water to a final volume of 1000 mL. Temperature was kept at 35 °C to 40 °C in a thermostatic bath. Then the hands and feet of the patients were soaked in the bath for 20 minutes.