*5.6. Nitric Oxide (NO) Assay*

NO analysis was performed to evaluate inflammatory response and to measure NO release by macrophages. RAW 264.7 cells (1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) were seeded in 96-well cell culture plates and allowed to adhere for 12 h. The cells were incubated on swertiamarin and loganic acid (0, 5, 10, 20, 40, 80, and 100 μM), or swertiamarin and vitexin (0, 5, 10, 20, 40, and 50 μM), respectively, with stimulation by LPS (1 μg/mL) for 24 h [34]. NO secretion by LPS-stimulated macrophages was determined by Griess reagents (Beyotime, Shanghai, China) according to the instructions of manufacturer [35]. Absorbance was measured at 540 nm and NO concentration was determined using sodium nitrite as a standard. Three replicates were carried out for each of the different treatments.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/24/4478/s1, Figure S1: the mass spectra of loganin and secologanol in positive ion mode by UPLC-Q Exactive mass spectrometer ([2M + Na]+). Figure S2. the mass spectra of loganin and secologanol in negative ion mode by UPLC-Q Exactive mass spectrometer ([2M + HCOOH-H]−). Figure S3. Effects of loganic acid, swertiamarin, and vitexin on LPS-induced NO production in RAW 264.7 cells.

**Author Contributions:** Z.P. and G.-Y.Z. conceived and designed the study, F.X., Y.-L.C., Z.-W.C. and G.-G.W. performed the detailed experiments; Z.P., G.-Y.Z. and W.-F.C.; Funding acquisition, F.X. and Y.Z., samples investigation, Z.P., G.-G.W., and Y.-L.C. wrote the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant number 81973567), and the Chong Qing Science Foundation Project (grant number CSTC2016jcyj A0288 & 2019jcyj-msxmX0180), and the Development Project of Qinghai Provincial Key Laboratory (grant number 2017-ZJ-Y10).

**Conflicts of Interest:** The authors declare no conflicts of interest associated with this manuscript.
