*2.3. Pro-oxidant Activity of ROS Generation and Copper Reduction*

Oxyresveratrol was found to generate ROS in the presence of copper(II) in a dose-dependent manner and in a higher amount compared to resveratrol and trans-stilbene (Figure 4C,D). The three extracts also exhibited ROS formation consistent with oxyresveratrol content (data not shown). Investigating the ability of oxyresveratrol to reduce Cu2<sup>+</sup> ions, we report the amount of Cu<sup>+</sup> produced by oxyresveratrol was dose-dependent (Figure 4E). Oxyresveratrol was also found to produce significantly higher amounts of copper (I) than resveratrol (Figure 4F). Trans-stilbene showed no copper-reducing activity; the ability of oxyresveratrol to generate ROS is consequently reported to be specifically induced by copper ions. Speculation exists as to the mechanism responsible although it has been previously noted that copper–oxo complex or copper–peroxide complex have the capacity to cause DNA damage following the reactions shown in Scheme 1 [6,14,15]. However, the exact structural feature of the complexes that caused damage to the DNA is still unclear. Copper to some certain extent is well-known to be one of the transition metals playing an important role in biological functions. However, It has been reported that significantly elevated levels of copper have been found in both serum and tissue of cancer patients [16]. Moreover, elevated copper levels have been documented in breast, cervical, ovarian, lung, prostate, stomach cancers, and leukemia. Serum copper concentration has also been found to correlate with tumour incidence and burden; malignant progression in Hodgkin's lymphoma; and leukemic, sarcoma, brain, breast, cervical, liver, and lung cancer [17]. The generation of ROS by stilbenoids in the presence of copper may be one of the key mechanisms by which they (stilbenoids) may be useful in the treatment of cancer.

**Scheme 1.** The assumed mechanism of oxyresveratrol for ROS production. The compound reduces cupric (II) into cuprous (I), then the cuprous reacting with O2 generates superoxide radical (•O2 −); the radical causes direct oxidative stress or reacts to hydrogen ions presenting hydrogen peroxide (H2O2). Lastly, the cuprous ion and hydrogen peroxide in the reaction generates a copper–peroxide complex or copper oxo-species complex.

**Figure 4.** Effect of oxyresveratrol on DNA, ROS generation and copper reduction in cell-free assays. (**A**) DNA damage induced by oxyresveratrol at six concentrations of Cu(OAc)2. (**B**) Comparison of DNA damage induced by oxyresveratrol, resveratrol, and *trans*-stilbene. (**C**) Generation of ROS by oxyresveratrol in the presence or absence of Cu(II). (**D**) Comparison of generation of ROS by oxyresveratrol, resveratrol, and *trans*-stilbene. (**E**) Reduction of Cu(II) by oxyresveratrol. (**F**) Comparison of copper-reducing ability of the three stilbenoids. Values express mean ± SD (*n* = 3).
