*2.5. LC/UVD Quantitative Analysis of Coumarin-Based Compounds*

Quantitative analysis of coumarin-based compounds was performed by using LC/UVD with various solvents. An UltiMate 3000 HPLC system (Thermo Fisher Scientific Lin., San Jose, CA, USA) coupled with a UV detector (Thermo Fisher Scientific, San Jose, CA, USA) was applied for quantitative analysis of coumarin-based compounds. The separation of each compound was achieved by a column (5 μm, 250 mm × 4.6 mm, Bischoff Analysentechnik und-geräte GmbH., Leonberg, Garmany). The mobile phase comprised water (A) and acetonitrile (B). The gradient elution conditions were: 0–5 min, 80% A; 5–60 min, 80–0% A; 60–70 min, 0% A; 70–71 min, 0–80% A; 71–80 min, 80% A. The temperature of the column oven was maintained at 30 ◦C. A flow rate of 1.0 mL/min was used and the injection volume was 10 μL. The chromatograms of the compounds were acquired at 203 nm. Standard chemical compounds such as scopoletin, umbelliferone, phellopterin, and imperatorin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Stock standard chemicals (1000 ng/mL) were prepared in 100% methanol. The stock solutions were maintained at 4 ◦C and used to construct calibration curves after appropriate dilution. Amounts of different quantified compounds were calculated as mean values from HPLC analyses based on the calibration

curves of the corresponding standard compounds. The regression equation of the standard compounds were as follows.

$$\begin{aligned} \text{Scopoletin}\_{\prime} \text{ y = } &0.482 \text{x} + 0.077 \text{, R}^2 = 0.999 \\\\ \text{Umbelliferoone}\_{\prime} \text{ y = } &1.005 \text{x} + 0.027 \text{, R}^2 = 0.999 \\\\ \text{Imperatorin}\_{\prime} \text{ y = } &0.996 \text{x} + 0.017 \text{, R}^2 = 0.999 \\\\ \text{Phellopeterin}\_{\prime} \text{ y = } &0.993 \text{x} + 0.010 \text{, R}^2 = 0.999 \end{aligned}$$
