Extract from Arrabidaea chica (Fridericia chica) leaves show preventive action for the mitigation of doxorubicin-induced cardiotoxicity

Doxorubicin (dox) is used for treatment of several types of cancer in humans and dogs; however, it shows important off-target cardiotoxic effects, which appear even months after completing the treatment in both species (Almeida et al., 2015; Hallman et al., 2019). Thus, it is imperative to find a treatment that reduces dox cardiotoxicity maintaining its anticancer efficacy. Considering the oxidative stress induced by dox, an antioxidant agent is an option. Green tea from Camellia sinensis (L.) Kuntze has been studied and demonstrated cardioprotective potential against dox toxicity (Aboulwafa et al., 2019; Saeed et al., 2015). In this scenario, a plant extract which has high flavonoid content, such as Arrabidaea chica (Bonpl.) Verl. [Fridericia chica (Bonpl.) L.G.Lohman] of the Bignoniaceae family, should be considered to prevent dox-induced oxidative stress in the myocardium. Due to its high flavonoid content (Barbosa et al., 2008), we hypothesized that A. chica has a previously uninvestigated cardioprotective role. Thus, the present study aimed to evaluate the cardioprotective effect of A. chica, compared with C. sinensis (a known positive control), and the maintenance of dox anticancer activity in the presence of both extracts.

Ventricular cardiomyocyte isolation from neonatal Wistar rats was performed as described earlier (Ott et al., 2008) and human mammary adenocarcinoma cell line MDA-MB-231 was used. Both cell cultures were maintained in a humidified incubator with 5% CO2 at 37°C. Cell viability was assessed through the MTT assay (Mosmann, 1983) and cellular death was evaluated by the propidium iodide (PI) nuclear staining (Riccardi & Nicoletti, 2006)  or 2.6x10 5 cells/cm 2 and MDA-MB-231 cells were plated at 1.3x10 4 cells/cm 2 in 24-well plates. For PI, 6x10 5 cardiomyocytes and 1x10 5 MDA-MB-231 cells were plated in 6-well plates.
Data of MTT assay demonstrated that AC and CS were not toxic to the cells at a concentration rate from 12.5 up to 50μg/ml, as cell viability was not different from control (P>0.05).
However, extracts at higher concentrations of 100 and 200μg/ml were toxic (lower cell viability compared to control, P<0.05) and, therefore, excluded from further investigations. These findings are explained byhormesis, a doseresponse relationship that is characterized by low-dose stimulation and high-dose inhibition. Such pro-oxidative property was also observed with resveratrol (Plauth et al., 2016). Flavonoids are considered antioxidants, but they can become pro-oxidative as described here for high doses. This paradoxical effect highlights the misleading concept of considering antioxidant agents always beneficial (Lei et al., 2016).
The percentage of death cardiomyocytes significantly increased after incubation with dox, compared to control (p=0.015), as expected. However, cellular death was lower for cardiomyocytes incubated with dox+plant extracts (P<0.05 for all dosage tested), compared to dox only ( Figure 1A), indicating a protective effect of AC and CS against dox over cardiomyocytes. Such protection was not observed for MDA-MB-231 cells, which is desirable, because the anti-neoplastic property of dox must be kept over cancer cells ( Figure 1B). In fact, the association of Dox with AC at 12.5μg/ml and Dox with CS at 25 and 50μg/ml resulted in more cell death to breast cancer cells, compared to Dox only (P<0.05), indicating a potentializing action of dox by these extracts in cancer cells. All data obtained for PI assay were confirmed by MTT assay.

Extract from Arrabidaea chica…
The CS used had high concentration of epigallocatechin (> 45%), which was proved to be cardioprotective against dox (Saeed et al., 2015), explaining the preservation of cardiomyocyte viability. Mass spectrometry analyses of the AC identified the presence of kaempferol. In ischemia-reperfusion injury in the heart, kaempferol decreased levels of inflammatory markers (TNF-alpha and IL-6) and proapoptotic proteins (Bax and caspase-3), and increased expression of the antiapoptotic protein Bcl-2 (Suchal et al., 2016). In dox-induced toxicity, kaempferol protected heart cells by inhibiting the p53-mediated mitochondriadependent intrinsic apoptotic signaling (Xiao et al., 2012). While dox promotes apoptosis by increasing Bax and decreasing Bcl-2, kaempferol inhibits apoptosis by showing an opposite action (Xiao et al., 2012). In this context, at least part of AC effectiveness demonstrated here is due to the presence of kampferol.
Despite the potential therapeutic properties of A. chica, described with antineoplastic, antiinflammatory and antimicrobial properties and useful for treating osteoarthritis (Barbosa et al., 2008;Michel et al., 2015;Vasconcelos et al., 2019), there is no previous study evaluating its effect against oxidative injury to the heart. Present data demonstrated a novel antioxidant activity of A. chica on dox-induced cardiotoxicity.   Vet. Intern. Med., v.33, p.783-791, 2019.