Conceptualization, M.J.S.; methodology, A.S.L.; software, A.S.L.; validation, A.S.L.; formal analysis, A.S.L.; investigation, A.S.L.; resources, A.S.L.; data curation, A.S.L.; writing original draft preparation, A.S.L.; writing review and editing, A.S.L.; visualization, A.S.L.; supervision, A.S.L.; project administration, M.J.S.; funding acquisition, M.J.S. All authors have read and agreed to the published version of the manuscript.

Figure 1. (A) Chrysanthemum coronarium L. (CC) extract reduces the senescent phenotype in H2O2 (100 μmol/L)-stimulated HUVECs, as shown by β-gal staining. The magnification was set to 40×. Scale bar = 50 μm. (B) Quantitative analysis of positive SA-β-gal stained cells. Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). (C) CC reduced the expression of p21, p53, and PAI-1 induced by H2O2. (D–F) The relative ratios from comparison to β-actin in duplicate blots are shown in a graph chart. Control cells (CB) received the vehicle alone. The T Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). **, p < 0.01, ***, p < 0.001, versus CB; #, p < 0.05, ###, p < 0.001, vs. H2O2.

Figure 1. (A) Chrysanthemum coronarium L. (CC) extract reduces the senescent phenotype in H2O2 (100 μmol/L)-stimulated HUVECs, as shown by β-gal staining. The magnification was set to 40×. Scale bar = 50 μm. (B) Quantitative analysis of positive SA-β-gal stained cells. Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). (C) CC reduced the expression of p21, p53, and PAI-1 induced by H2O2. (D–F) The relative ratios from comparison to β-actin in duplicate blots are shown in a graph chart. Control cells (CB) received the vehicle alone. The T Data shown are derived from three independent experiments and expressed as the mean ± SEM (n = 6 per group). **, p < 0.01, ***, p < 0.001, versus CB; #, p < 0.05, ###, p < 0.001, vs. H2O2.

Figure 2. Chrysanthemum coronarium L. (CC) extract reduces endothelial senescence in HUVECs induced by ROS. (A) Fluorescent staining images illustrating ROS production in HUVECs via DCF-DA staining. The relative ratios of DCF-DA fluorescence intensities indicate the degree of endothelial ROS formation in HUVECs. The original magnification was set to 20 ×. Scale bar = 50 μm. (B) The fluorescence sensitivity of DCF-DA was standardized for the total number of cells in each dish. (C–D) Expression of phospho-ERK, total ERK, and β-actin in H2O2-treated cells. (E) NADPH was measured using a colorimetric assay. Control cells (CB) received the vehicle alone. Bars represent the mean ± SEM from three dishes per group. ***, p < 0.001, versus CB; #, p < 0.05, ##, p < 0.01, ###, p < 0.001, vs. H2O2.

Figure 2. Chrysanthemum coronarium L. (CC) extract reduces endothelial senescence in HUVECs induced by ROS. (A) Fluorescent staining images illustrating ROS production in HUVECs via DCF-DA staining. The relative ratios of DCF-DA fluorescence intensities indicate the degree of endothelial ROS formation in HUVECs. The original magnification was set to 20 ×. Scale bar = 50 μm. (B) The fluorescence sensitivity of DCF-DA was standardized for the total number of cells in each dish. (C–D) Expression of phospho-ERK, total ERK, and β-actin in H2O2-treated cells. (E) NADPH was measured using a colorimetric assay. Control cells (CB) received the vehicle alone. Bars represent the mean ± SEM from three dishes per group. ***, p < 0.001, versus CB; #, p < 0.05, ##, p < 0.01, ###, p < 0.001, vs. H2O2.

Figure 3. Chrysanthemum coronarium L. (CC) extract improves endothelial NO formation to combat senescence in HUVECs. (A) CC reversed the H2O2 (100 μmol/L)-induced decrease of eNOS expression in H2O2-, L-NAME- (100 μM), and NAM- (5 mM) treated HUVECs. (B) Expression of Sirt1 in H2O2 and H2O2 + CC HUVECs (C) The relative ratios of DAF-FM fluorescence intensities indicate the degrees of endothelial NO formation in the H2O2-, L-NAME- (100 μM), and NAM- (5 mM) treated HUVECs. ***, p < 0.001, versus CB; #, p < 0.05, ###, p < 0.001, versus H2O2; ‡, p < 0.05, vs. H2O2 + CC.

Figure 3. Chrysanthemum coronarium L. (CC) extract improves endothelial NO formation to combat senescence in HUVECs. (A) CC reversed the H2O2 (100 μmol/L)-induced decrease of eNOS expression in H2O2-, L-NAME- (100 μM), and NAM- (5 mM) treated HUVECs. (B) Expression of Sirt1 in H2O2 and H2O2 + CC HUVECs (C) The relative ratios of DAF-FM fluorescence intensities indicate the degrees of endothelial NO formation in the H2O2-, L-NAME- (100 μM), and NAM- (5 mM) treated HUVECs. ***, p < 0.001, versus CB; #, p < 0.05, ###, p < 0.001, versus H2O2; ‡, p < 0.05, vs. H2O2 + CC.

Figure 4. (A) Representative chromatogram of Chrysanthemum coronarium L. extract (B) and spectra of major compounds. The extract was analyzed using UPLC-Q-TOF MS and its major compounds were tentatively identified using the online databases connected to the UNIFI software (Waters).

Figure 4. (A) Representative chromatogram of Chrysanthemum coronarium L. extract (B) and spectra of major compounds. The extract was analyzed using UPLC-Q-TOF MS and its major compounds were tentatively identified using the online databases connected to the UNIFI software (Waters).