*2.4. Structural Characterization of Chalcones and Coumarines*

To find the regulators for tyrosinase and the indicator component, the acetone extract was dissolved in acetone and coated in silica gel (1.5 g) and was then subjected to medium-pressure liquid chromatography (MPLC) and semi-preparative HPLC purification using the LC/MS-guided isolation approach. The process resulted in the successful isolation of six compounds, **1**, **3**, **6**, **7**, **16** and **17** (Figure 5). By using extensive NMR spectroscopic methods (1D and 2D-NMR), and LC/MS analysis to compare our results with previously reported spectroscopic values, the isolated compounds were determined to be xanthoangelol (**1**) [23], 4-hydroxyderricin (**7**) [24], xanthoangelol D (**3**) [25], xanthoangelol H (**6**) [26], laserpitin (**16**) [23] and isolaserpitin (**17**) [23] (Figure 5). These types of chalcones and coumarins are common amongst the *Angelica* species. In order to further confirm the effectiveness of the screening system, purified compounds, which in the S-plot are different between the blank and test groups, like the compounds **1**, **7** and **16**, were subjected to tyrosinase inhibition assay using kojic acid as the positive control. Results showed that both compounds **1** and **7** inhibited tyrosinase activity with IC50 values of 15.87 ± 1.21 μM and 60.14 ± 2.29 μM, respectively, whereas compound 16 had no inhibitory effect (IC50 > 100 μM) on tyrosinase. These findings corroborated the results found earlier via the screening system (Tables 1–3).

**Figure 5.** The chemical structure of 17 compounds in *Angelica keiskei* Koidzumi, xanthoangelol (**1**), B (**2**), D (**3**), E (**4**), G (**5**), H (**6**), 4-hydroxyderricin (**7**), xanthokeismin B (**8**), (2*E*)-1-[4-hydroxy-2-(2-hydroxy-2-propanyl)-2,3-dihydro-1-benzofuran-7-yl]-3-(4-hydroxyphenyl)-2-propen-1-one (**9**), umbelliferone (**10**), selinidin (**11**), isopimpinellin (**12**), phellopterin (**13**), xanthyletin (**14**), ashitabaol A (**15**), laserpitin (**16**) and isolaserpitin (**17**).


**Table 1.** Identification of the different amount of compounds from *A. keiskei* between the blank and test groups by UPLC-MS/MS in positive ion mode.

**Table 2.** Identification of the different amount of compounds from *A. keiskei* between the blank and test groups by UPLC-MS/MS in negative ion mode.



**Table 3.** Effect of kojic acid, 4-hydroderricin, xanthoangelol and laserpitin on mushroom tyrosinase activity.

<sup>1</sup> Relative inhibitory activity, <sup>2</sup> Positive control.

#### **3. Materials and Methods**

#### *3.1. Reagents and Materials*

The fresh *Angelica keiskei* was collected from Ali Mountain, Chiayi County of Taiwan, and authenticated by Dr. Yu-Hsin Chen (Taichung District Agricultural Research and Extension Station, Taichung, Taiwan). The voucher specimens (TMU-LCK-77) were deposited at the School of Pharmacy, Taipei Medical University, Taipei, Taiwan.

All the reagents including phosphate buffer (sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4)), kojic acid (positive control for the enzyme activity assay), dimethyl sulfoxide (DMSO) and acetone were purchased from Sigma Aldrich (St. Louis, MO, USA). Methanol and acetonitrile (all MS grade), on the other hand, were purchased from Macron Fine Chemicals™ (Radnor, PA, USA). The ultra-pure water was prepared with the Millipore-Q water purification system (Bedford, MA, USA).

#### *3.2. Sample Preparation*

The fresh *A. keiskei* was weighed, ground and precisely cut into thin blocks. The root (425 g) and leaf (490 g) specimens of *A. keiskei* were then purified and extracted three times using acetone in a 1:5 ratio to obtain 20.58 g and 18.11 g of crude extract, respectively.
