**3. Discussion**

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death among women worldwide [28]. Despite the long list of drugs that have been used in the treatment of breast tumors, marked drug multi-resistance during treatment remains one of the main problems facing the clinic today when applying a therapy against breast cancer. Among various mechanisms of chemo-resistance, resistance due to the increased expression of P-gp is the best characterized and it is considered the most important in cancer therapy. The significance of the above mentioned is also supported by the fact that more than 50% of existing anticancer drugs are going to P-gp mediated efflux [29]. Since the results of clinical trials using P-gp inhibitors have not been very promising, the search for new modulators which selectively inhibit P-gp activity without significant negative side effects is currently under high interest and hopes are pinned on the application of some plant polyphenols [11].

P-gp may affect the pharmacokinetic parameters of drugs or other compounds, possibly leading to modifications of their bioavailability as well as of their distribution, metabolism, elimination and toxicity (ADMET) [30]. Together with several CYP450 isoenzymes, P-gp is involved in drug-drug, food-drug and, finally, herb-drug interactions [31]. Herbal medicines may interact with drugs at the intestine, liver, kidneys, and targets of action. Importantly, many of these drugs have very narrow therapeutic indices. Most of them are substrates for P-gp. The underlying mechanisms for most described herb-drug interactions are not entirely understood, and several pharmacokinetic and/or pharmacodynamic interferences are often implicated in these interactions. In particular, enzyme induction and inhibition may play an important role in the occurrence of some herb-drug interactions. Because herb-drug interactions can significantly a ffect circulating levels of drug and, hence, alter the clinical outcome, the identification of herb-drug interactions has important implications [32]. As present results sugges<sup>t</sup> the species *Ageratina havanensis* (Kunth) R. M. King & H. Robinson could be a source of new potential inhibitors of drug e fflux mediated by P-gp; focus on these aspects must be taking into account for future studies.

Today it is known that P-gp function and its regulation can be mediated via reactive oxidative species (ROS) [33–37]. Therefore, the transport of P-gp substrates may be altered by modulation of P-gp expression/activity under conditions of oxidative stress [38]. In this sense, natural antioxidants, such as polyphenols, could influence the function of this membrane transporter. It was reported that polyphenolic compounds, mainly flavonoids or their derivatives, can modulate the main ABC transporters responsible for cancer drug resistance, including P-gp [16,39–41].

The interest in health benefits of polyphenols has increased due to their powerful antioxidant and free radical scavenging activities observed in vitro [42]. Current evidence strongly supports a contribution of polyphenols, in particular flavonoids, to the prevention and therapy of cancer and other chronic diseases [1,43]. Literature data have shown that plant phenolic content and antioxidant activity depend on several factors, mainly environmental conditions [3]. Previous studies show that summer plants (reproductive stage) are richer in phenolic compounds than spring ones (vegetative stage), and consequently exhibit higher antioxidant activities [2,3]. Here, we evaluated the antioxidant potential of the *Ageratina havanensis* extracts by using three in vitro methods of well-established use to strongly predict the antioxidant capacity of several substances [22–24]. But surprisingly, the results of the performed studies showed the opposite, the concentration of flavonoids is higher in the vegetative state. In fact, we observed that *Ageratina havanensis* extracts possess higher reductive capacity in the vegetative period. This finding might be related to the content of sakuranetin and 7-methoxyaromadendrin, which is greater in the vegetative stage. The antioxidant capacity of sakuranetin has been previously demonstrated [44]. Indeed, here we observed that sakuranetin possesses the greatest reductive capacity of the analyzed samples. In order to evaluate the antioxidant capacity in a biological system, we used the lipid peroxidation model in brain homogenates. Rat brain homogenates exposed to oxygen spontaneously exhibit lipid peroxidation by a mechanism which is independent of superoxide and free hydroxyl radical production and whose initiation step may involve and iron-mediated cleavage of lipid hydroperoxides to yield peroxide or alkoxy radicals [45,46]. In this system, the extracts obtained from the flowering stage e ffectively inhibited TBARS generation with estimated values (see Table 1). This result could be explained in term of several mechanisms comprising among others, iron chelation, (at the initiation step) or a synergistic antioxidant activity (at the propagation step). In addition, previous studies demonstrated the metal chelation properties of polyphenols [47,48]. The present results sugges<sup>t</sup> that *Ageratina havanensis* could be represent a source of antioxidants with potential neuroprotective actions. In summary, these results show for first time the antioxidant potential of the extracts obtained from the species *Ageratina havanensis*.
