Characterizing Tyrosinase Modulators from the Roots of Angelica keiskei Using Tyrosinase Inhibition Assay and UPLC-MS/MS as the Combinatorial Novel Approach

Abstract

In this study, an in vitro tyrosinase inhibition assay in combination with ultra performance liquid chromatography-orbitrap mass spectrometry (UPLC-orbitrap-MS) was developed for the rapid screening and identification of tyrosinase modulators from roots of Angelica keiskei. Of the 15 candidates considered, nine chalcones, xanthoangelols (1), B (2), D (3), E (4), G (5), H (6), 4-hydroxyderricin (7), xanthokeismin B (8) and (2E)-1-[4-hydroxy-2-(2-hydroxy-2-propanyl)-2,3-dihydro-1-benzofuran-7-yl]-3-(4-hydroxyphenyl)-2-propen-1-one (9), five coumarins, umbelliferone (10), selinidin (11), isopimpinellin (12), phellopterin (13) and xanthyletin (14), and one other compound, ashitabaol A (15), were distinguished between the test samples and the controls with statistical significance, and the structure of each compound was determined by comparing with in-house standards and the literature. Among these, six compounds, xanthoangelol (1), xanthoangelol D (3), xanthoangelol H (6), 4-hydroxyderricin (7), laserpitin (16) and isolaserpitin (17), were isolated from roots of A. keiskei. Of the compounds isolated, compounds 1, 7 and 16 were subjected to tyrosinase inhibitory assay, and the IC₅₀ values were 15.87 ± 1.21, 60.14 ± 2.29 and >100 μM, respectively. The present study indicated that the combination of in vitro tyrosinase inhibition assay coupled with UPLC-MS/MS could be widely applied to the rapid screening of active substances from various natural resources.

Keywords: Angelica keiskei; UPLC-MS/MS; chalcone; coumarins; tyrosinase.

Figure 1

The comparative ratio of tyrosinase activity of the A. keiskei Leaf (AK. Leaf) at 500 μg/mL and roots (AK. Root) at 200, 500, 750 and 1000 μg/mL concentrations using kojic acid (10 and 20 μM) as the positive control.

Figure 2

The base peak chromatograms (BPC) of untreated (A) and tyrosinase-treated samples (B) of A. keiskei in positive ion mode.

Figure 3

The base peak chromatograms (BPC) of untreated (A) and tyrosinase-treated samples (B) of A. keiskei in negative ion mode.

Figure 4

OPLS-DA score plot (A) and S-plot (B) of positive mode, OPLS-DA score plot (C) and S-plot (D) of negative mode, mass spectra obtained from untreated (blank) and tyrosinase-treated (test) groups.

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), (2E)-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).