*2.2. The Structure-Activity Relationships (SAR)*

The structure-activity relationships were analyzed basing on the MTT results, and we found that, in the core structure of 10-oxyderivatives of oleoside secoiridoids, **1**–**4** all had a hydroxyl substituent at the 10 position and only differed at the 7 and 11 positions. **1** had a hydroxyl group at the 7 position and a methyl group at the 11 position, **2** had methyl groups at the 7 and 11 positions, and **3** had a *p*-hydroxy-phenylethyl group at the 7 position and a methyl group at the 11 position, while **4** (multifloroside) had an *o*-hydroxy-*p*-hydroxy-phenylethyl group at both the 7 and 11 positions (Figure 1), which indicated that the *o*-hydroxy-*p*-hydroxy-phenylethyl group may contribute to the anti-proliferative activity of multifloroside against A431 cells.

#### *2.3. Multifloroside Inhibits Tumor Cell Colony Formation*

Encouraged by the above results, we further investigated the bioactivity of multifloroside. First, the inhibitory effect of multifloroside on the proliferative ability of A431 cells was determined via colony formation assay. Herein, A431 cells were seeded into a 6-well cell culture plate at a density of 400 cells/well and treated with multifloroside at several concentrations (200, 100, 50, and 25 μM), gefitinib (25 μM) or 0.5% DMSO for 12 days, and then stained with crystal violet. As shown in Figure 3, multifloroside resulted in a significant suppression of cell colony formation. When the cells were incubated with multifloroside (200 and 100 μM) or gefitinib (25 μM), colony formation were completely suppressed. In addition, the cells incubated with multifloroside (50 and 25 μM) formed fewer and smaller colonies in a concentration-dependent manner compared to the control group. There were 337 viable colonies when A431 cells were treated with 0.5% DMSO, but only 183 and 76 colonies were observed when the cells were exposed to multifloroside at concentrations of 50 and 25 μM, respectively. Therefore, the plating efficiency (PE) showed a significant decrease compared to the control (*p* < 0.001). The PEs were 84%, 46%, and 24% for the control, and the 25 μM and 50 μM multifloroside treatments, respectively, and were 0 for the other groups. These results demonstrate that multifloroside inhibits the growth of A431 cells and that the inhibitory effect of multifloroside persists for a significant period of time.

**Figure 3.** Colony formation of A431 cells inhibited by multifloroside. (**A**) A431 cells were incubated with the indicated concentrations of multifloroside or gefitinib and fixed with 4% paraformaldehyde and stained with 0.2% crystal violet 12 days after cell treatment. (**B**) Bar chart showing the decrease in the number of colonies after incubation with multifloroside. (**C**) Micrographs showing differences between the cell colonies. Images were taken of stained single colonies observed under a microscope. A single colony was defined to be an aggregate of >50 cells. Data are shown as mean ± SEM (*n* = 3), \*\*\* *p* < 0.001 indicates a significant difference compared with the control.

#### *2.4. E*ff*ect of Multifloroside on Cell Apoptosis in A431 Cells*

After treatment with multifloroside for 48 h, apoptosis of A431 cells was measured by flow cytometry using annexin V-FITC and PI labeling. As shown in Figure 4, 96.30% of A431 cells were in their normal state in the untreated control groups. When cells were treated with multifloroside (200 μM) or gefitinib (25 μM), the numbers of early apoptotic cells were significantly higher than in the control groups (*p* < 0.001). However, there were no significant differences in the numbers of early apoptotic cells when cells exposed to lower concentrations of multifloroside (25, 50 and 100 μM). In addition, there were no significant differences in the numbers of late apoptotic cells and necrotic cells when cells were treated with multifloroside at 25, 50, 100, and 200 μM and gefitinib at 25 μM. These

results indicate not only that early apoptosis of A431 cells was not significantly influenced by treatment with low concentrations of multifloroside but also that late apoptosis was not significantly affected by multifloroside at the tested concentrations (25–200 μM).

**Figure 4.** Effect of multifloroside on cell apoptosis in A431 cells. (**A**) Representative histograms of apoptosis in the cells treated with multifloroside for 48 h, (**B**) Percentages of apoptotic cells in each group from (**A**). All values are expressed as mean ± SEM (*n* = 3). \*\*\* *p* < 0.001 indicates a significant differences compared with the control at the same group.

#### *2.5. S-Phase Cell Cycle Arrest Induced by Multifloroside*

In order to study the relationship between the anti-proliferative activity of multifloroside and cell cycle arrest, A431 cells were treated with multifloroside or gefitinib for 48 h, after which the cells were stained with PI and examined using flow cytometry. As shown in Figure 5, when cells were treated with multifloroside at four different concentrations (25, 50, 100 and 200 μM), the number of A431 cells in S phase were significantly increased (*p* < 0.001), from 28.55% to 31.78%, 49.29%, and 55.30%, respectively, accompanied by a decrease in the number of cells in G0/G1 and G2/M. In the gefitinib (25 μM) treated cells, the percentage of cells in G0/G1, S, and G2/M were 77.54%, 16.17%, and 6.29%, respectively. These results indicate, unlike gefitinib which arrests the A431 cells in the G0/G1-phase, multifloroside can arrest the cell cycle of A431 cells in the S-phase and showed concentration-dependent activity.

**Figure 5.** Effect of multifloroside on the cell cycle phase distribution in A431 cells. (**A**) Representative histograms of DNA content in the cells treated with multifloroside for 48 h, and (**B**) percentages of cell populations in the G0/G1, S and G2/M phases from (**A**). All values are expressed as mean ± SEM (*n* = 3). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 indicate significant differences compared with the control at the same phase.

#### *2.6. Intracellular ROS Production Induced by Multifloroside*

Intracellular ROS generation was evaluated via MitoSox red reagen<sup>t</sup> staining and flow cytometry analysis. As shown in Figure 6, treatment with gefitinib (25 μM) significantly increased the fluorescence intensity (*p* < 0.001), and treatment with multifloroside at high concentrations (50 and 100 μM) also significantly increased the fluorescence intensity (*p* < 0.001) in a dose-dependent manner. Treatment with multifloroside at a low concentration (25 μM) also increased the fluorescence intensity, but the difference was not statistically significant (*p* > 0.05). These results indicate that ROS production can be induced by multifloroside at the tested concentrations.

**Figure 6.** Effect of multifloroside on ROS production. A431 cells were treated with different concentrations of multifloroside for 48 h, then MitoSox red reagen<sup>t</sup> (5 μM) was loaded and the cells were analyzed by flow cytometry for the quantification of multifloroside-induced oxidative stress in A431 cells. The fluorescence intensity of MitoSox Red reagen<sup>t</sup> in cells was obtained by FACS (**A**) and the data was analyzed using GraphPad Prism 5 (**B**). The values are presented as mean ± SEM (*n* = 3). \*\*\* *p* < 0.001 indicates a significant differences compared with the control.

#### *2.7. E*ff*ect of Multifloroside on the MMP*

MMP of A431 cells stained with JC-10 was assayed by flow cytometry. As shown in Figure 7, the JC-10 fluorescence ratio (% of J-aggregates with Red Fluorescence divided by the % of J-monomers with Green Fluorescence) decreased but was not significantly different (*p* > 0.05) when cells were treated with gefitinib (25 μM), while the ratios increased when cells were treated with multifloroside (25, 50, and 100 μM). Moreover, the differences in the JC-10 fluorescence ratios were not significant when cells were treated with multifloroside at 25 and 100 μM, and they were significant when cells were treated with 50 μM multifloroside (*p* < 0.01). The above results indicate the MMP did not decrease, but rather increased, with multifloroside treatment

**Figure 7.** Effect of multifloroside on the MMP of A431 cells. A431 cells were treated with different concentrations of multifloroside for 48 h and analyzed by flow cytometry after JC-10 staining. The fluorescent intensity of JC-10 in cells was obtained by FACS (**A**) and the data was analyzed by GraphPad Prism 5 (**B**). The percentage of cells with JC-10 red fluorescence is indicated. JC-10 fluorescence ratio (%) equals the red/green fluorescence intensity ratio. The values are presented as mean ± SEM (*n* = 3). \*\* *p* < 0.01 indicates a significant differences compared with the control.
