Characterisation of phenolic compounds of the ethyl acetate fraction from Tabernaemontana catharinensis and its potential antidepressant-like effect

Abstract This study evaluates the antidepressant-like effect and analysed the qualitative and quantitative 74 phenolic standards of ethyl acetate fraction from Tabernaemontana catharinensis leaves. Acute administration of fraction in mice reduced the immobility time in forced swimming and tail suspension tests confirming its antidepressant-like activity. The anti-immobility effect elicited by this fraction was prevented by the pretreatment of mice with PCPA (100 mg kg−1), ketanserin (5 mg kg−1), SCH 23,390 (0.05 mg kg−1) or yohimbine (1 mg kg−1). A sub effective dose of the fraction produced a synergistic effect with fluoxetine (5 mg kg−1). Chromatographic analysis identified 4-hydroxybenzoic and p-coumaric acids in the ethyl acetate fraction from T. catharinensis. Capillary electrophoresis presented 7.34 ± 0.02 mg g−1 of p-coumaric acid concentration in the fraction. Therefore, it is possible that antidepressant-like effect elicited by ethyl acetate fraction from T. catharinensis be dependent on the p-coumaric acid.


Introduction
Major depressive disorder is a heterogeneous syndrome, unleashed by cultural factors, genetic predisposition and emotional experience (Nestler et al. 2002). Depressive disorders are characterised by sadness, loss of pleasure and can lead to suicide. More than 320 million people live with depression, which has increased its rate by 18.4% in ten years (WHO 2017).
Plants are important raw materials for biologically active natural products that are used for the development of drugs (Riaz et al. 2016). Brazil is privileged due to its rich plant genetic diversity, where Tabernaemontana catharinensis is an angiosperm of the Apocynaceae family that is largely found in several regions of Brazil. Pharmacological activities were demonstrated in plants of the same family, such as Tabernaemontana divaricata, which presented antioxidant (Jain et al. 2010a) and anti-fertility activity (Jain et al. 2010b). Studies on the T. catharinensis have demonstrated anticholinesterase, antioxidant (Nicola et al. 2013) and antinociceptive activities (Da Silva et al. 2016) and antidepressant-like effects (Cardoso et al. 2015). Most T. catharinensis activities have been associated with indole alkaloids; however, Belke et al. (2013) demonstrated a considerable presence of phenolic compounds in the branches of this plant, which can be extracted from the ethyl acetate fraction of this plant. This study aimed to identify these compounds in the ethyl acetate fraction of T. catharinensis (EAF) and investigate its antidepressant-like effect in mice using the forced swimming test (FST) and tail suspension test (TST).

Results and discussion
Data depicted in Figures S1(a) and S1(b) show that treatment of mice with EAF (250 mg kg −1 and 500 mg kg −1 ) significantly reduced the immobility time of mice in the FST and TST. There were no significant changes in the numbers of crossing of treated animals, compared to the control group ( Figure S1(c)) in the open field test (OFT). The present study shows, for the first time, that EAF causes a significant reduction in the immobility time of mice in the FST and TST, indicating an antidepressant-like activity in this experimental model, as described by Porsolt et al. (1977).Other plants of the Apocynaceae family, such as Apocynum venetum and Hedranthera barteri, have also been shown to induce reductions in immobility times in the FST and TST, respectively (Butterweck et al. 2001;Onasanwo et al. 2010). EAF, at a sub effective dose (25 mg kg −1 ), potentiated the action of fluoxetine (5 mg kg −1 ) in the FST ( Figure  S2(a)) and TST ( Figure S2(b)), suggesting that EAF and fluoxetine may act through similar mechanisms to promote antidepressant-like effects. It also suggests an involvement of the serotonergic system in the antidepressant-like effect caused by EAF. The deficiency of endogenous monoamines, especially serotonin and noradrenaline, is considered the main cause of depression (Lopez-Muñoz and Alamo 2009).
The pretreatment of mice with SCH 23,390 ( Figure S3(a)), ketanserin ( Figure S3(b)), yohimbine ( Figure S3(c)) and PCPA (p-chlorophenylalanine methyl ester) ( Figure S3(d)) were able to block the decrease in the immobility in the FST caused by EAF. Data suggest the involvement of the endogenous monoamines in the antidepressant-like effect of EAF. Major antidepressants used in clinical practice act by modulating the monoaminergic system and the serotoninergic receptors, such as subtype 5-HT 2A (Ansorge et al. 2007).
Qualitative analysis by HPLC-ESI-MS/MS led to the identification of two phenolic acids, p-coumaric acid and 4-hydroxybenzoic acid (Table S1); the chromatogram obtained is depicted in Figure S4. The p-coumaric acid concentration in EAF was 7.34 ± 0.02 mg g −1 , as quantified by capillary electrophoresis (CE). Another study identified the presence of phenolic compounds in the ethyl acetate fraction of T. catharinensis, including p-coumaric acid, chlorogenic acid and caffeic acid (Da Silva et al. 2016). The fact that p-coumaric acid can be metabolised into other phenolic acids, such as caffeic acid and chlorogenic acid (El-Seed et al. 2012), suggests that the mature state of the leaves could have contributed to these divergent results.
Reports in the literature suggest that the antidepressant-like activity presented by EAF may be dependent on phenolic compounds, such as the extract of Aloysia gratissima, where its analysis showed quantities of phenolic acids, such as ferulic and chlorogenic acids (Zeni et al. 2013). Some phenolic acids, such as caffeic (Takeda et al. 2002) and ferulic acids (Li et al. 2015) showed antidepressant-like effects in mice. Since the chemical structure of p-coumaric acid is similar to those of caffeic and ferulic acid, it is possible that this phenolic acid is responsible for the antidepressant-like effects demonstrated by EAF.

Conclusions
Our results demonstrate, for the first time, that EAF promotes an antidepressant-like effect in the FST and TST, which is dependent on the serotoninergic, noradrenergic and dopaminergic systems. Furthermore, EAF's antidepressant-like activities may be mediated by pcoumaric acid; further studies should be conducted to confirm this relationship.

Supplementary material
Experimental details relating to this paper are available online, alongside with Figures S1-S4 and  Table S1.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the CNPq; the Universidade Regional de Blumenau.