**3. Discussion**

Cancer incidence and mortality are rising rapidly worldwide. Throughout the development of human society, nature has catered to the basic needs of humans, not the least of which is the

provision of medicines for the treatment of a wide spectrum of diseases [22,23]. There is growing evidence that specific bioactive compounds present in plants used as spices, fruits, vegetables, and nuts can be effective against some cancers. Secoiridoids, isolated from *Oleaceae* family, which are now widely used in the fields of food and medicine, have been extensively investigated in recent years and have been shown to possess a variety of pharmacological effects [24–29], such as anti-diabetic [30,31], anti-oxidant [32,33], anti-inflammatory [34–36], immunosuppressive [37], neuroprotective [36,38,39], and anti-cancer [1,40–42]. Multifloroside (**4**), together with 10-hydroxyoleoside 11-methyl ester (**1**), 10-hydroxyoleoside dimethyl ester (**2**), and 10-hydroxyligustroside (**3**), are all 10-oxyderivatives of oleoside secoiridoids.

**1**–**3,** together with other eight secoiridoid glycosides, were isolated from the bark of *Osmanthus asiaticus Nakai in 1993* [15]; **2** was isolated from *Jasminum lanceolarium* Roxb in 1997 and 2007 [16,17]; **1** was also isolated from *Jasminum lanceolarium* Roxb in 2007 [16]; **1**, **3**, and **4** were isolated from the water soluble fraction of *Jasminum multiflorum* extract [18]; **1** and **4**, with the other 11 types of iridoid glycosides and 15 other compounds, were identified by UPLC-MS in extracts of *Jasminum elongatum* (Bergius) Willd in 2016 [19]. However, by now, only **1** and **4** have been found to possess coronary dilating and cardiotropic activities [18]. Therefore, we aimed to investigate the in vitro anti-cancer activities of **1**–**4** in this study. Cancer is characterized by uncontrolled cell growth, invasiveness, and formation of metastasis, and the cells of most of malignant tumors are proliferate intensively [14]. Thus, we first evaluated the anti-proliferative activities of **1**–**4** against A431 human epidermoid carcinoma cells and A549 human NSCLC cells. The results showed that **1**–**4** possess different anti-proliferative activities against A549 and A431 cells, and that **4** (multifloroside) had the most potent inhibitory activity against A431 cells (Figure 2). As reported, it appears that the best way to prevent cancer is through rational dietary habits and behaviors, and consumption of sufficient amounts of antioxidants and bioactive plant-derived compounds that demonstrate protective effects against carcinogenesis in pre-clinical and clinical studies [43], **4** (multifloroside) and its derivatives may be the way to prevent epidermoid carcinoma cancer. The SAR were analyzed, and we found that the *o*-hydroxy-*p*-hydroxy-phenylethyl group may contribute to the anti-proliferative activity of multifloroside against A431 cells. In a future study, it may be advantageous to study other oleoside secoiridoids with *o*-hydroxy-*p*-hydroxy-phenylethyl substituents in order to identify the most potent compounds in the *Oleaceae* that can protect against the epidermoid carcinomas.

Second, a colony formation assay was performed to detect the inhibitory effect of multifloroside on the proliferative ability of A431 cells. The assay is now widely used to examine the effect of agents with potential clinical applications [44], and it shows the ability of a cancer cell to produce a viable colony after drug treatment; thus, the results obtained may help to predict the efficacy of agents in vivo [45]. Similarly to other reported plant anti-cancer molecules and their derivatives, such as panaxatriol [46], cycloartenol [47], and oridonin derivatives [48], the number and the size of tumor cell colonies were significantly decreased when A431 cell were treated by 200, 100, 50, and 25 μM multifloroside (Figure 3). These results all illustrate that multifloroside can significantly inhibit the growth of A431 cells, which is consistent with the results of MTT assay.

As reported, most of malignant tumors whose cells proliferate intensively, represent very dynamic structures that create numerous mutations resulting in new tumor cell lines with different genotypes and phenotypes within the tumor mass. In such malignancies, a highly variable sensitivity to therapeutics can be observed, and some of cell lines develop resistance to the treatment [14,49]. Therefore, the biological effects of combining various plant molecules (phytochemicals) with proven cytotoxic effects administered together with conventional therapy to target a markedly wider range of signaling pathways in cancer cells should be superior compared to single compound in cancer treatment and thus may delay the development of drug resistance in cancer [43]. So, further urgen<sup>t</sup> research is needed for the identification of new molecules (including plant-derived compounds) with well-validated anticancer properties within combinational clinical approach in oncology. Further studies were then done to elucidate the mechanism of action of multifloroside on A431 cells. In general, drug-induced apoptosis is one major mechanism of action for the treatment of cancer, and various signaling pathways are involved in the process [50]. However, the apoptotic assay suggested multifloroside at high concentration (200 μM) induced cell apoptosis but that at lower concentrations (100, 50, and 25 μM) could not (Figure 4). Cell cycle regulating is a key method in controlling tumor propagation [50]. As reported, most antitumor compounds inhibit cell proliferation by inducing cell cycle arrest [48]. Cell-cycle analyses indicated the cell-cycle distribution was significantly changed, and was also di fferent from that resulting from gefitinib, where the cells were arrested in the G0/G1-phase, multifloroside-treated cells were arrested cells in the S-phase (Figure 5). Mitochondria are the main source of cellular ROS, which have a double-edged sword role in cytotoxicity in cancer cells [51,52]. Herein, ROS levels were significantly increased after cells were treated with multifloroside at 50 and 100 μM for 48 h (Figure 6). Similarly, the MMP was increased when cells were treated with multifloroside at the tested concentrations compared with the control group (Figure 7). JC-10 forms "J-aggregates" displaying red fluorescence in healthy cells, in contrast, as the membrane potential decreases, JC-10 monomers are generated, resulting in a shift to green fluorescence [52]. These results all sugges<sup>t</sup> that multifloroside may exert its anti-cancer e ffect through cell-cycle arrest and an increase in the ROS and MMP in A431 cells. The above gives a well-validated anticancer properties for multifloroside and suggests multifloroside may be the potential one in applying to anti-epidermoid carcinoma cancer.
