**Multifloroside Suppressing Proliferation and Colony Formation, Inducing S Cell Cycle Arrest, ROS Production, and Increasing MMP in Human Epidermoid Carcinoma Cell Lines A431**

**Xin Zhang 1,2,**†**, Yamei Li 1,2,**†**, Zhengping Feng 1,2, Yaling Zhang 1,2,\*, Ye Gong 1,2, Huanhuan Song 1,2, Xiaoli Ding 1,2 and Yaping Yan 1,2,\***


Academic Editors: Ra ffaele Pezzani and Sara Vitalini Received: 23 November 2019; Accepted: 16 December 2019; Published: 18 December 2019

**Abstract:** Multifloroside (**4**), together with 10-hydroxyoleoside 11-methyl ester (**1**), 10-hydroxyoleoside dimethyl ester (**2**), and 10-hydroxyligustroside (**3**), are all secoiridoids, which are naturally occurring compounds that possess a wide range of biological and pharmacological activities. However, the anti-cancer activity of **1**–**4** has not been evaluated yet. The objective of this work was to study the anti-cancer activities of **1**–**4** in the human epidermoid carcinoma cell lines A431 and the human non-small cell lung cancer (NSCLC) cell lines A549. The results indicate that **1**–**4** di ffer in potency in their ability to inhibit the proliferation of human A431 and A549 cells, and multifloroside (**4**) display the highest inhibitory activity against A431 cells. The structure-activity relationships sugges<sup>t</sup> that the *o*-hydroxy-*p*-hydroxy-phenylethyl group may contribute to the anti-cancer activity against A431 cells. Multifloroside treatment can also inhibit cell colony formation, arrest the cell cycle in the S-phase, increase the levels of reactive-oxygen-species (ROS), and mitochondrial membrane potential (MMP), but it did not significantly induce cell apoptosis at low concentrations. The findings indicated that multifloroside (**4**) has the tendency to show selective anti-cancer e ffects in A431 cells, along with suppressing the colony formation, inducing S cell cycle arrest, ROS production, and increasing MMP.

**Keywords:** anti-cancer activities; multifloroside; 10-oxyderivatives of oleoside secoiridoids; structure-activity relationship; flow cytometry
