**2. Results**

#### *2.1. P-gp Modulation by Extracts Obtained from Ageratina havanensis*

The first step of this study was to investigate if the extracts obtained from *Ageratina havanensis* could inhibit P-gp activity under no cytotoxicity conditions. In order to mimic the chemo-resistance in humans, the cells chosen for this research were the well-characterized mouse mammary carcinoma 4T1 cells that express multi-resistance phenotype after exposure to di fferent anticancer drugs mediated by P-gp.

Firstly, to determine the cytotoxic e ffects of the eleven extracts obtained from *Ageratina havanensis* on 4T1 cells, the MTT assay was employed. Table 1 reports the IC50 values calculated after exposure of the cells to the *Ageratina havanensis* extracts for 24 h. As shown, the treatments reduced cell viability showing only slight di fferences between the products. In all the cases, significant di fferences were observed in comparison with control cells for values above 250 μg/mL. Thus, a range of concentrations under IC50 values was selected for evaluating e ffects of the extracts on P-gp function.

Rho-123 is a fluorescent compound, which enters the cells passively and concentrates in mitochondria. As P-gp substrate, the intracellular loading of this probe is inversely proportional to P-gp activity. Intracellular fluorescence increased in a dose-dependent fashion in 4T1 cells exposed to Lf-EtOH until F-EtOH extract for 1 h, but important di fferences between the extracts were found. The percentage of the inhibitory e ffect on the activity of the transporter produced at the highest concentration tested (200 μg/mL) are showed in Table 1. As reported, all ethanolic extracts (Lf-EtOH, Lv-EtOH, Sf-EtOH and F-EtOH) showed promissory inhibitory e ffects. More in details, the inhibitory activity was above 50% for both Sf-EtOH and Sv-EtOH, the two extracts obtained from the stem bark, compared to controls. On the contrary, the F-EtOAc and F-*n*-BuOH extracts were not able to inhibit the function of the transporter under the same experimental conditions.


**Table 1.** Cytotoxicity and inhibitory effects on P-gp function of *Ageratina havanensis* (Kunth) R. M. King & H. Robinson extracts on breast cancer 4T1 cells.

IC50 is defined, as the concentration required achieving 50% inhibition over control cells, values are shown as mean ± SEM. %: represents the percentage of inhibition P-gp activity at the higher concentration tested (200 μg/mL) respect to control cells (untreated cells) after 1 h of exposure. Verapamil used as control positive was included, showing values of inhibition in the order of 300% to the activity exhibit by control cells. Values showed are from two independent experiments with three replicas.

#### *2.2. Antioxidant E*ff*ects of Extracts Obtained from Ageratina havanensis*

The screening of the antioxidant activity of the substances may require a combination of di fferent methods to describe the background about the antioxidant properties of the samples. Here, the antioxidant potential of the *Ageratina havanensis* extracts was determined by using three in vitro methods, which previously have been used to predict the antioxidant capacity of several substances (DPPH free radical scavenging assay, FRAP assay and the determination of lipid peroxidation in brain rat homogenates) [22–24].

The model of scavenging the stable DPPH radical has been used method to evaluate the free radical scavenging ability of substances [23,24]. In this case, the antioxidant e ffect of the analyzed sample on DPPH radical scavenging may be due to their hydrogen donating ability and it reduce the stable violet DPPH radical to the yellow DPPH-H. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers [25]. On the other hands, FRAP assay is based on the ability of antioxidant to reduce Fe3<sup>+</sup> to Fe2<sup>+</sup> in the presence of tripyridyltriazine (TPTZ), forming the intense blue Fe2<sup>+</sup>–TPTZ complex with an absorption maximum at 593 nm; the absorbance increase is proportional to the antioxidant content [22]. As shown in Table 2, the radical scavenging activity of the eleven *Ageratina havanensis* extracts evaluated was significantly (*p* < 0.05) higher in the flowering compared to the vegetative season, meanwhile, the reductive capacity was significantly (*p* < 0.05) higher in vegetative state.

During the last years, lipid peroxidation has received renewed attention from the viewpoints of nutrition and medicine. Lipid peroxidation is implicated in the underlying mechanisms of several disorders and diseases such as cardiovascular diseases, cancer, neurodegenerative diseases, and even aging [26]. It is the accumulated result of reactive oxygen species and a chain reaction that causes the dysfunction of biological systems [27]. Furthermore, the extracts from the flowering season showed a significant (*p* < 0.05) inhibition of lipid peroxidation against brain phospholipid peroxidation compared with the extracts from the vegetative stage (Table 2).


**Table 2.** In vitro antioxidant capacity of *Ageratina havanensis* (Kunth) R. M. King & H. Robinson extracts.

Values represent the mean ± SEM of the antioxidant activity of *A. havanensis* extracts. IC50 values were calculated as the extract concentration required to scavenge 50% of DPPH•, Ascorbic acid was employed as standard for DPPH• assay and Trolox-C for lipid peroxidation assay. Different letters in the same column represent statistical differences (ANOVA, Dunnet post-hoc test; *p* < 0.05). Comparisons were carried out between extracts according to the seasonal stage (leaves, stems or flowers) or according to the solvent (EtOH, EtOAc or *n*-BuOH). Three independent experiments were done and samples were analyzed by triplicate.

#### *2.3. Quantification of Sakuranetin and 7-Methoxyromadendrin in the Extracts of Ageratina havanensis*

The major flavonoids sakuranetin and 7-methoxyaromadendrin only differ for a hydroxyl group (Figure 1) and thus elute with very close retention times. Because of this, it was necessary to develop a rapid, sensitive and accurate method that would allow for their quantification in *Ageratina havanensis* extracts. UPLC-ESI-MS/MS system provides high separation capacity, high analytical speed and high analytical sensitivity. The addition of 0.1% formic acid to the mobile phase helped achieve satisfactory peak symmetry, good resolution, and significantly enhanced sensitivity. In this case, this method was useful for quantifying sakuranetin and 7-methoxyaromadendrin in the extracts of *Ageratina havanensis* collected in both seasons.

**Figure 1.** Chemical structures of: (**a**) sakuranetin; (**b**) 7-methoxyromadendrin.
