**2. Materials and Methods**

The present study was carried out based on the literature review of cinnamic acids with antidepressant activity. The survey, conducted in the Pubmed database, for studies published

from January 2002 to October 2019, used the following keywords: Cinnamic acid, coumaric acid, para-coumaric acid, meta-coumaric acid, ortho-coumaric acid, ferulic acid, caffeic acid, sinapic acid, trimethoxycinnamic acid, methylenedioxycinnamic acid, methoxycinnamic acid, dimethoxycinnamic acid, antidepressant, and depression. The scientific publications were selected from studies published in English language.

#### **3. Antidepressant Activity of Cinnamic Acids**

Based on increasing evidence of the contributions of neuronal pro-inflammatory mediators and oxidative stress in the pathogenesis and development of depression, new therapeutic tools have been experimentally tested in order to improve the current treatment of depressive disorders. In this context, hydroxylated and/or methoxylated aromatic acids have shown promising results as neuroprotective agents [25]. Among these acids, there are phenolic acids, which are divided into hydroxybenzoic acids and hydroxycinnamic acids, based on C6–C1 and C6–C3 skeletons, respectively [26]. According to the literature, phenolic acids have potentially antioxidant properties due to the presence of a phenolic ring that promotes the electron donation and hydrogen atom transfer to free radicals, acting as free-radical scavengers, reducing agents, and quenchers of single oxygen formation [27,28]. It has been reported that phenolic compounds produce a significant reduction of pro-inflammatory cytokines, including TNF-α and IL-1β, and stimulate a concomitant increase of anti-inflammatory cytokines as interleukin-8 (IL-8) in different in vitro and in vivo models of inflammation [29–32]. Here, the antidepressant effect of cinnamic acids was investigated, specifically the trimethoxycinnamic acids, *p*-coumaric acid, caffeic acid, and ferulic acid on animal models and their relevance to the treatment of depression. The last three compounds are classified as phenolic acids.

Fifteen studies were found on cinnamic acids in experimental models of depression; the chemical structures of these acids are shown in Figure 3. Basically, the experimental model used in almost all studies for assessing antidepressant-like activity of cinnamic acids were the forced swim test (FST) and the tail suspension test (TST). Both tests are validated animal models fundamental for understanding the pathogenesis and treatment of mood and anxiety disorders, such as depression. The FST is based on the immersion of rodents in a beaker of water without a possible escape—a compound qualifies as a potential antidepressant if it reverts or delays the initial attempts to escape (active behavior) and promotes a progressive increase in the frequency and duration of episodes of immobile floating (passive behavior). The TST is based on the fact that animals subjected to the short-term, inescapable stress of being suspended by their tail, will develop an immobile posture [33,34]. Other important tests used to evaluate the effect of cinnamic acids on anxiety behaviors were the elevated plus maze (EPM) test, sucrose preference test, and open field test (OFT). In the EPM, the measure for anxiety is calculated by percentage of the total number of arm entries and the period of time spent on the open arms. Open-arm entries indicate security and low anxiety, while closed-arm entries, a sign of need for security. Decreased sucrose preference indicates the loss of the ability to feel pleasure (anhedonia), a common symptom of depression [34,35]. The OFT measures locomotor activity, including ambulation, exploration, latency, escape attempts, exploration, and the aversions of rodents to novel, brightly lit, open environments. In the OFT, reduced locomotor activity suggests anxiety-like behaviors associated with depression [34].

**Figure 3.** Chemical structures of antidepressant cinnamic acids.

In the literature, two studies have reported the effects of trimethoxycinnamic acid (TMCA) on depressive behaviors, showing divergences in results. In Nakazawa et al. (2003), 2,4,5-trimethoxycinnamic acid failed to alter the duration of immobility in FST for Male ddY mice intraperitoneally treated with TMCA, at a dose ranging from 25–200 mg/kg. Differently, Leem and Oh (2015) have shown that Male C57BL/6J mice, orally treated with 3,4,5-trimethoxycinnamicacid (50 mg/kg) for 15 days, showed reduced immobility in the FST and higher time and frequency of visits in the open arms than the control group in the EPM. The data sugges<sup>t</sup> an antidepressant effect of 3,4,5-trimethoxycinnamic acid, which the authors have attributed to increased expression of the ΔFosB protein on the nucleus accumbens observed in TMCA-treated animals. There were differences in some methodological aspects between the two investigative approaches. Thus, it is difficult to conclude about the presence or absence of antidepressant effect of TMCA based on only two experimental studies that used different routes of administration, species of mice, and experimental protocols. In addition, antidepressant activity may be related to the position of the methoxyl group in the aromatic ring, since animals treated with 3,4,5-trimethoxycinnamic acid had antidepressant effects, which did not occur in those treated with 2,4,5-trimethoxycinnamic acid [36,37].

Some studies performed by Takeda and his group have shown that male ICR mice subjected to intraperitoneal treatment with caffeic acid, at a dose of 4 mg/kg, exhibit decreased immobility time in the FST, as well as a reduction in the duration of freezing of mice in the conditioned fear stress test. According to authors, the caffeic acid's ability to reduce depressive behavior might be attributed to, at least in part, an indirect modulation of the α1A-adrenoceptor and α1-adrenoceptor system and regulation of brain-derived neurotrophic factor (BDNF) expression in the frontal cortex caused by caffeic acid [38–40]. These data corroborate a study by Dzitoyeva and colleagues in which caffeic acid demonstrated antidepressant activity, attenuating BDNF mRNA decrease, by forced swimming test, using wild type mice and a 5-lipoxygenase (5-LOX) deficient group—previous studies have shown that caffeic acid inhibits 5-LOX. However, in this study it was observed that attenuation occurred only in wild rats, indicating that this acid can be used as a tool to study the regulation of the 5-LOX pathway of BDNF expression [41]. Therefore, antidepressant activity of caffeic acid may be related to its ability to regulate inflammation, as observed in the study by Huang et al. (2018), in which this acid inhibited dose-dependent increase in inflammatory and inducing cytokines affecting 5-HT, DA, and

NE metabolism, such as tyrosine (Tyr), 3-methoxy-4-hydroxyphenylglycol (MHPG), Tryptophan (Trp), and 5-hydroxyindoleacetic acid (5-HIAA), improving the behavior of depressed rats [42].

The antidepressant activity of ferulic acid is the most investigated among hydroxycinnamic acids [32,43]. According to Zeni and colleagues, ferulic acid exhibits major e ffects on free radical and inflammatory messengers and has the ability to counteract the reduction in reward-seeking behavior that tends to occur with depression. Animals also exhibit decreased immobility time in the FST and TST, using ferulic acid oral treatment ranging from 0.001–80 mg/kg/day. Moreover, several studies show that ferulic acid has no e ffect on locomotor activity in the OFT, indicating its specificity, while others show a reversal of decreased locomotor activity, suggesting ferulic acid's ability to reduce anxiety-like behaviors associated with depression. The antidepressant e ffect of ferulic acid has been attributed to diverse mechanisms, including modulation of serotonergic system by signaling pathway of protein kinase A (PKA), Ca<sup>2</sup>+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK), and phosphoinositide 3-kinases (PI3K) [44,45].

Ferulic acid acts as some of the antidepressant drugs from the pharmaceutical market, as shown by the study by Chen et al. (2015), in which there is an increase on the concentrations of monoamines serotonin and norepinephrine in the hippocampus and frontal cortex through the inhibition of monoamine oxidase A (MAO-A) activity in male ICR mice treated with ferulic acid [46]. However, unlike commercially available selective serotonin reuptake inhibitor (SSRI) drugs, which may cause bowel movement inhibition, ferulic acid exhibited both antidepressant and prokinetic activity. The Zhang et al. study performed the TST on rats, noting a reduction in immobility time, and increased locomotor activity, associated with an increase in gastric emptying speed [47].

In addition, it was noted that oral administration of ferulic acid at a dose of 5 mg/kg in male ICR mice for 7 days decreased TST immobility due to up-regulation of gene expression associated with cell survival and proliferation, energy metabolism, and synthesis of dopamine in the limbic system of the brain of mice [48].

Lenziet al. (2015) have related ferulic acid's antioxidant activity and its e ffects on the central nervous system, evidenced by increasing superoxide dismutase (SOD), catalase (CAT) activities, and low thiobarbituric acid reactive substances (TBARS) levels found in hippocampus of ferulic acid-treated male swiss mice [49]. Furthermore, Li et al. attributed the ferulic acid's ability to reduce depressive-like behaviors, suggested by reducing immobility time in the FST and TST, observed in ferulic acid-treated male ICR mice to anti-inflammatory mechanisms [50]. In addition, a study of Liu et al. (2017) reported that ferulic acid increased the levels of BDNF and synaptic proteins (synapsin I and PSD-95) in the prefrontal cortex and hippocampus, as well as inhibited microglia activation, pro-inflammatory cytokines expression, nuclear factor kappa B (NF-κB) signaling, and decreased PYD domains-containing protein 3 NLRP3 [51].

A similar study by Zheng and colleagues demonstrated the relationship between the antidepressant effect and the anti-inflammatory activity of ferulic acid in prenatally-stressed o ffspring rats. In this work, it was noted that the administration of ferulic acid decreased the time of immobility and total number of crossing, rearing, grooming, and increased sucrose intake in the animals. Furthermore, it was able to reduce the concentration of inflammatory cytokines such as IL-6, IL-1, and TNFα, and increase IL-10. As such, it was concluded that the antidepressant activity of ferulic acid occurs in part due to its anti-inflammatory activity and regulation on hypothalamic-pituitary-adrenal (HPA) axis [52].

In the study of Lee et al., it was observed that the antidepressant e ffects of *p*-coumaric acid may be related to its anti-inflammatory activity, as has also been shown in studies with ca ffeic acid. In this study, *p*-coumaric acid reduced lipopolysaccharide-induced tumor necrosis factorα (LPS)-induced despair-related behavioral symptoms in the FST, TST, and sucrose splash test (SST), preventing the increase of inflammatory cytokines such as cyclooxygenase-2 and lipopolysaccharide-induced tumor necrosis factorα (LPS), as well as inhibiting BDNF reduction [53].

Medicinal plants used to treat behavioral disorders that contain these psychoactive acids may have antidepressant action such as *Eugenia catharinensis* D. Legrand. Ethyl acetate extract of this species had antioxidant and antidepressant-like action in mice treated with corticosterone. Analysis of the extract using HPLC-ESI-MS/MS demonstrated the presence of several phenolic compounds, mainly phenolic acids, such as *p*-coumaric acid, ferulic acid, and caffeic acid. The authors sugges<sup>t</sup> that chemical constituents are the active principles of antidepressant action [54]. Antidepressant action also was demonstrated for butanol fraction of *Olax subscorpioidea* Oliv. and was involved in the monoaminergic mechanism. Analysis of the chemical composition by HPLC indicated caffeic acid as one of the active ingredients of the plant [55]. In another study, ethyl acetate fraction from *Tabernaemontana catharinensis* A. DC. leaves showed antidepressant activity in animal models. Analysis of the chemical composition of this fraction proposes that the pharmacological action may be dependent on the presence of *p*-coumaric acid in the plant [56]. Therefore, the collection of studies discussed in this review (Table 1) show the pharmacological effect of cinnamic acids against the name Table 1 was added in text.

Depressive disorders. Figure 4 summarizes the main effects of cinnamic acids as antidepressants.

**Figure 4.** Antidepressant action of cinnamic acids.




