*3.5. Mass Fragmentation Analyses and Compound Identifications*

The MassLynx Mass Spectrometry software version 2.0.w.15 (Waters Corporation, Milford, MA, USA) was used for analyses. We extracted the mass fragment spectrum of each candidate in positive and negate mode, separately, and then transferred the mass fragment spectrum to the mass list. In the last step, via the MassFragment software in the MarkerLynx XS, structures were assigned to our

observed fragment ions of small molecule compounds and, further, the reported structures were cross referenced with the literature on *A. keiskei.*

#### *3.6. Statistics*

The raw data were aligned and extracted using the SIEVE v2.2 application software from Thermo Fisher Scientific. The experimental target was metabolomics and the minimum intensity for the base peak was 1,000,000. The frames and threshold values were defined as 481,351 for the positive mode and 674,992 for the negative mode. SIMCA-P 13.0.3 software from MKS Umetrics (Umeå, Sweden) was then used to obtain OPLS-DA. The S-plots were also utilized for finding different candidates between blank and test groups.

#### **4. Conclusions**

Besides xanthoangelols (**1**), B (**2**), D (**3**), E (**4**), G (**5**), H (**6**), 4-hydroxyderricin (**7**), xanthokeismin B (**8**) and (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**) and ashitabaol A (15) in the positive and negative modes of UPLC-MS/MS, along with the differences between untreated and treated, revealed that there were nearly 15 different tyrosinase modulators found in these two groups. In addition to umbelliferone (**10**) and isopimpinellin (**12**), which are already known to inhibit tyrosinase activity, we also identified compounds **1** and **7** as active components with similar effects. This is the first research to investigate the tyrosinase-modifying effects of *A. keiskei*. We compared the compounds with the published literature and found xanthoangelols (**1**) [20], 4-hydroxyderricin (**7**) [20], umbelliferone (**10**) [27], isopimpinellin (**12**) [28] and phellopterin (**13**) [28] had been reported to have tyrosinase inhibition activity. The compounds from its roots demonstrate promising potential for treating hyperpigmentation and related disorders. Future research can focus on elucidating the in vivo effect and the optimal dosage.

With the aim of evaluating the effectiveness of the novel screening system, we examined the purified compounds to check the candidates from the base peak chromatograms and the tyrosinase inhibitory assay and found out that the results from the two experiments support each other. We therefore conclude this novel screening is highly accurate and effective. Furthermore, in comparison to the traditional protocols, our method reduces the sample and solvent volumes required for UPLC-MS/MS analysis. In addition, it allows researchers to quickly analyze and screen for more candidates with tyrosinase modulatory effects. This strategy can also be used for the rapid development of applications in other screening platforms.

**Author Contributions:** Investigation, J.-H.L. performed the experiments, data analysis and wrote the manuscript. Resources, Y.-H.C. executed the experiments and identified the plant. Conceptualization, C.-K.L. designed the research experiments and provided the study outline. Methodology, T.-H.L. created models and suggestion of direction. Writing—original draft, I.-C.K. and writing—review and editing, H.-C.M. All authors edited and approved the final manuscript.

**Funding:** This research was funded by the National Science Council (Taiwan), grant number NSC104-2320- 038-020-MY3.

**Acknowledgments:** We are grateful to S.-H.W. and S.-Y.S. of the Instrumentation Center of Taipei Medical University and the Instrumentation Center of the College of Science, National Taiwan University, respectively, for the NMR and MS data acquisition.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

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*Article*
