Introduction

Depression is one of the most important psychological conditions associated with life-threatening behavioral symptoms (Leonard 2010). It is widely accepted that stressful life events often precede a major role in depressive disorders. Chronic mild stress (CMS) is generally acceptable animal model to test depression-like behavior, which employs a series of stressors to imitate the depressive behavior (Shang et al. 2017). Recent studies have suggested that hyperactivity of HPA (hypothalamic-pituitary-adrenal) axis and glucocorticoids over secretion are the major indicators in depression-like disorders (Wang et al. 2015). An alteration in monoamine oxidases (MAO) is responsible in pathophysiology of neuropsychiatric and neurodegenerative disorders via regulation of neurotransmitters, such as serotonin (5-HT), dopamine (DA), epinephrine, and nor-epinephrine (NE) (Duncan et al. 2012). BDNF (brain-derived neurotrophic factor) plays a major role in the physiology of depression by activating series of pathways such as, PI3K-Akt, MEK/ERK, and PLCγ-Ca2+ (Numakawa et al. 2013).

Chronic mild stress (CMS) model is a commonly used rodent model to understand the mechanism of depression. Additionally, this model modulates neurotransmitter levels in brain, corticosterone and corticotropin-releasing factor (CRF), expression of tumor necrosis factor-α, BDNF, CREB etc. (Grippo et al. 2005). Recent drug research had recognized that reversible or competitive inhibition of brain monoamine oxidase (MAO) might be more important in depressive patients. Moreover, MAO inhibitors were proven to be beneficiary antidepressant effects by improving brain neurotransmitter levels and reducing MAO activities (Asnis and Henderson 2014).

In recent years, medicinal plants have been given importance as alternative agents to treat depression-like behavior (Kumar et al. 2014). Terminalia bellirica (Gaertn.) Roxb., Combretaceae, is well known in Ayurveda for having medicinal properties like anti-spasmodic, anti-asthmatic, anti-tussive, and expectorant (Trivedi et al. 1979). Terminalia bellirica contains several triterpenoids and polyphenols such as chebulagic acid, ellagic acid, gallic acid, phyllembin, and ethyl gallate (Kadian and Parle 2015). Keeping in view of several mechanisms involved in pathophysiology of depression-like disorders, the present study was undertaken to examine the association of neurotransmitters, monoamine oxidases, and antioxidant enzymes in the antidepressant-like activity of T. bellirica. Furthermore, we investigated the ability of T. bellirica in modulation/regulation of hyperactivity of HPA-axis, oxidative stress, and stress-induced apoptosis.

Materials and Methods

Plant Material and Preparation of Gallic Acid Rich Extract

Terminalia bellirica (Gaertn.) Roxb., Combretaceae, fruits were obtained from local market of Mysore, India, and was authenticated by the Department of Botany, University of Mysore, Mysore, India (voucher specimen no. AND4680). The shade dried fruits were extracted by using a Soxhlet apparatus with alcohol (70%; v/v) for 72 h and lyophilized. Further, the lyophilized powder was subjected to column chromatography (Sephadex LH-20; equilibrated with 95% ethanol). Fraction I (low molecular weight phenolic compounds) was obtained by using mobile phase ethanol. Fraction II (gallic acid rich fraction) was eluted by 50% (v/v) acetone. Gallic acid rich fraction was lyophilized and used for the study, which contained the major component gallic acid and other components in smaller quantities.

HPLC Conditions

The separation of phenolic compounds was performed by using HPLC (JASCO) with UV-VIS detector. The compounds were separated by using RP-C18 column (waters; 250 mm × 4 mm). The mobile phase used for the study was methanol, water, and formic acid (80:19:1) with a flow rate of 0.8 ml min−1 in isocratic mode (Adiyaman et al. 2016). Injection volume was 20 μl with a run time of 60 min and detection wavelength was 280 nm.

LC–MS Analysis

Terminalia bellirica extract was analyzed by using Agilent 1260 LC–MS and the chromatographic conditions were DL temperature of 250 °C, scan range of 100–1500 m/z, 2 Hz scan speed, 0.8 kV detector voltages, and mobilizing gas flow of 7 l/min was used for MS analysis of the samples.

Animals and Experimental Design

Animals studies were conducted as per CPCSEA (committee for the purpose of control and supervision of experiments on animals) with approval no. IAEC-2016/AN/11. Forty-two female Balb/c mice (weighing 25–35 g) were divided into six experimental groups viz., control group (CON), chronic mild stress group (CMS), imipramine group (IMP10 mg/kg; p.o.), and treatment groups (TB-25, 50 and 100 mg/kg bwt; p.o.) (Fig. S1, Supplementary material). CMS protocol was performed as mentioned in our previous study (Chandrasekhar et al. 2017). Various kinds of stressors were applied individually and continuously as mentioned in Fig. S1, Supplementary material. Control mice were housed in a separate room and had no contact with the CMS group. Body weight was recorded on weekly basis and the animals were submitted to the behavioral tests after 24 h of the last stressor.

Behavioral Observations

Behavioral studies, such as sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST) were conducted in accordance with previous studies (Chandrasekhar et al. 2018; Kim et al. 2018). Before conducting the SPT, animals were allowed to drink sucrose solution (1%; w/v) to avoid neophobia. Amount of water or sucrose consumed by the mice in a period of 1 h was recorded and calculated in percentage. In TST, mice were individually placed approximately 50 cm above the floor with the help of tape from the tip of the tail. Immobile time was recorded on the basis of mice movements by ANY-maze software. FST was conducted in a transparent glass tank, which is filled with 30-cm high water (25 ± 0.5 °C). The test was conducted for a period of 6 min and the cumulative immobility time during the last 4 min was recorded. The immobile time was considered when the mice remained floating without struggling.

Collection of Hippocampus and Serum

Mice were sacrificed after the behavioral studies under the mild anesthetic condition. Mice brain was immediately dissected and followed by further dissection of hippocampus in a chilled condition. Blood samples were collected into the serum separator and centrifuged for 10 min at 3000×g at 4 °C. All the samples were frozen at − 80 °C for further analysis.

Estimation of Serum Acetylcholine Esterase and Corticosterone by ELISA

Serum acetylcholine esterase (AChE) and corticosterone (CORT) were estimated by using ELISA kits as per the manufacturer’s protocol (Abcam, USA). The AChE and CORT concentrations were expressed in nanogram per liter and gram per liter, respectively.

Estimation of Antioxidant Activities

Brain samples were minced and homogenized with 50-mM phosphate buffer (pH 7.4) and centrifuged at 1200×g for 10 min at 4 °C. The supernatants were then subjected to the measurement of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidises (GPx), and glutathione reductase (GR) levels according to the assay kit manual (BioVisoin Pvt. Ltd., USA).

Estimation of Monoamine Oxidase

Estimation of MAO was carried according to the previously described procedure (Dhingra and Goyal 2008). The hippocampal tissue was homogenized with isolation buffer (0.25 M sucrose, 10 mM Tris, 0.5 mM EDTA, and pH 7.4), then mitochondrial fraction was obtained by centrifugation at 18,500×g for 20 min. MAO-A and MAO-B were measured at 280 nm and 242 nm, respectively.

Monoamine Neurotransmitter Levels

Monoamine neurotransmitters such as dopamine (DA), 5-hydroxytryptamine (5-HT), epinephrine, and nor-epinephrine (NE) are measured by using HPLC-ECD. The hippocampal tissue was homogenized in ice cold perchloric acid and HPLC conditions were followed as described in our previous study (Chandrasekhar et al. 2017).

Analysis of Quantitative RT-PCR

qRT-PCR studies were followed as per the manufacturer’s instruction (SsoFast Eva Green, Bio-Rad). qRT-PCR results were normalized against β-actin (control) and calculated 2ΔΔCT values. Primer sequence and amplification conditions of targeted genes are placed in Table 1.

Table 1 Primer sequences of mice for RT-PCR and amplification conditions
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Western Blotting

Hippocampal tissue samples were homogenized in HEPES lysis buffer and the supernatant of the lysates were separated by SDS–PAGE mini gels and transferred onto polyvinylidene fluoride (PVDF) membrane. The membranes were subsequently probed via incubation with Gapdh, Bax, Bcl2, Caspase-3, Creb, and Bdnf (Santa Cruz, USA; 1:1000). Incubation, blocking, and detection of immune reactivity were followed as mentioned in our previous study (Chandrasekhar et al. 2017).

Statistical Analysis

The results were expressed as mean ± standard deviation (SD). Data were analyzed by using one-way ANOVA followed by Tukey’s test and Dunnett’s multiple comparisons using GraphPad Prism version 6.03. Differences were considered significant when p < 0.05.

Results

Gallic Acid Estimation from Terminalia Bellirica by using HPLC and LC–MS

Total tannin content in the T. bellirica was 477 mg/g, which is 47% dry weight. HPLC analysis revealed that T. bellirica contains gallic acid (36.38%), corilagin (2.14%), chebulinic acid (0.82%), and ellagic acid (3.26%) (Fig. S2, Supplementary Material). Further, LC–MS study was conducted for its confirmation and results are shown in Fig. S2, Supplementary Material.

Effect of Terminalia Bellirica on CMS-induced Body Weight Loss

Percentage of body weight gain/loss during 7-week stress protocol is shown in Fig. 1a. CMS exposure induced a significant body weight loss when compared with control mice (p < 0.05; Fig. 1a). However, TB-25, TB-50, and TB-100 showed a dose-dependent body weight gain. Treatment with T. bellirica and IMP significantly reversed (p < 0.05) the CMS-induced body weight loss.

Fig. 1
figure 1

Effects of Terminalia bellirica on body weight (a) and sucrose preference (b). Control (CON) and chronic mild stress group (CMS) received saline water, imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o. and 100 mg/kg., p.o., respectively). Data were presented as mean ± SD (n = 7). For statistical significance: #, p < 0.05 vs CON; *, p < 0.05; **, p < 0.01 vs CMS

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Effect of Terminalia Bellirica on Sucrose Preference Test

Chronic mild stress exposure for a period of 7 weeks resulted in reduced sucrose consumption in CMS group as compared with control mice (p < 0.05). However, treatment of T. bellirica (25, 50, 100 mg/kg) enhanced the uptake of sucrose solution when compared with CMS mice (p < 0.05; Fig. 1b).

Effect of Terminalia Bellirica on CMS-induced on TST and FST

The effect of T. bellirica treatment on immobility time of mice subjected to TST and FST is shown in Fig. 2a and b, respectively. There was a significant increase in immobility time by CMS exposure in mice when compared with control animals (p < 0.05). Mice treated with T. bellirica along with stress protocol showed a reduced immobility time in TST and FST as compared with CMS group (p < 0.05).

Fig. 2
figure 2

Effects of Terminalia bellirica on tail suspension test. a Forced swim test. b In CMS-induced mice. Control (CON) and chronic mild stress group (CMS) received saline water, imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o. and 100 mg/kg., p.o., respectively).Data were presented as mean ± SD (n = 7). For statistical significance: #, p < 0.05 vs CON; *, p < 0.05 vs CMS

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Effect of Terminalia Bellirica on CMS-induced Serum CORT and AChE Levels

The results indicated that continuous stress protocol of the study significantly elevated the levels of serum CORT and serum AChE (56% and 14%, respectively) as compared with the control mice (p < 0.05). However, treatment with T. bellirica significantly reduced these serum CORT and AChE levels (p < 0.05; Fig. 3a, b).

Fig. 3
figure 3

Effects of Terminalia bellirica on serum corticosterone levels (a) and acetylcholine esterase levels (b) in CMS-treated mice. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively). Data were presented as mean ± SD (n = 6). For statistical significance: #, p < 0.05 vs CON; *, p < 0.05 vs CMS

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Effect of Terminalia Bellirica on CMS-induced Oxidative Stress

Continuous stress protocol of the study significantly downregulated the levels of antioxidant enzymes such as SOD (11.05 ± 0.8 to 4.68 ± 0.38 unit g/protein), CAT (66.28 ± 1.6 to 37.29 ± 1.92 unit g/protein), GPx (15.11 ± 1.06 to 5.67 ± 0.94 unit g/protein), and GR (18.26 ± 1.24 to 6.73 ± 0.86 unit g/protein) as compared with control animals (p < 0.05). In contrast, T. bellirica treatment significantly increased these antioxidant enzymes (Fig. 4; p < 0.05).

Fig. 4
figure 4

Effects of Terminalia bellirica on antioxidant levels in CMS-induced mice. ASOD, superoxidase dismutase; bCAT, catalase; cGPx, glutathione peroxidase; dGR, glutathione reductase. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively).Data were presented as mean ± SD (n = 3). For statistical significance: #, p < 0.05 vs CON; *, p < 0.05 vs CMS

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Effect of Terminalia Bellirica on CMS-induced Monoamine Oxidase Activities

CMS exposure significantly increased the hippocampal MAO-A by 2.5-fold (Fig. 5a) and MAO-B by 4-fold (Fig. 5b) as compared with unstressed animals (p < 0.05). In contrast, these CMS-induced MAO activities were significantly reduced by the treatment of T. bellirica in a dose-dependent manner from p < 0.05.

Fig. 5
figure 5

Effects of Terminalia bellirica on the levels of MAO-A and MAO-B. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively). Data were presented as mean ± SD (n = 3). For statistical significance: #, p < 0.05 vs CON; *, p < 0.05 vs CMS

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Effect of Terminalia Bellirica on CMS-induced Monoamine Levels

Figure 6 revealed that the CMS procedure was able to induce a significant decrease on epinephrine, nor-epinephrine, dopamine, and serotonin levels when compared with control mice (p < 0.01). However, these monoamine levels, such as nor-epinephrine (108.5 ± 2.9 and 225.9 ± 2.8 ng/100 mg), epinephrine (78.2 ± 1.0 and 92.3 ± 3.4 ng/100 mg), dopamine (83.6 ± 1.2 and 101.5 ± 1.5 ng/100 mg), and serotonin (56.4 ± 3.8 and 74.4 ± 1.2 ng/100 mg) were significantly increased by TB-50 and TB-100 treatments, respectively. In contrast, CMS-induced monoamine levels were not affected by low dose of T. bellirica, i.e., TB-25.

Fig. 6
figure 6

Effects of Terminalia bellirica on the levels of monoamine neurotransmitters in hippocampus of CMS-stressed mice. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively). Data were presented as mean ± SD (n = 6). For statistical significance:*, p < 0.05; **, p < 0.01 vs CMS; #, p < 0.01 vs CON

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Effect of Terminalia Bellirica on CMS-induced Bcl2 and Bax Expression

Chronic mild stress induced apoptotic cell death by increasing Bax/Bcl2 ratio in mice hippocampus (35:1). CMS-induced Bax transcript levels were downregulated by the T. bellirica treatment (Fig. 7). Mice groups treated with IMP and TB-100 could significantly increase the transcript levels of Bcl2 by 86% and 72%, respectively, when compared with CMS (10%) exposed mice. Western blot analysis further confirmed the protective effect of T. bellirica on CMS-induced cell death by downregulated Bax levels and upregulated Bcl2 levels (Fig. 8a, b).

Fig. 7
figure 7

Effects of Terminalia bellirica on the mRNA expression of CREB, BDNF, Bcl2, and Bax in hippocampus of CMS-stressed mice. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively). Data were presented as mean ± SD (n = 6). For statistical significance: *, p < 0.05; **, p < 0.01 vs CMS; #, p < 0.01 vs CON

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Fig. 8
figure 8

Effect of Terminalia bellirica on protein expression levels. Bax (a), Bcl2 (b), caspase-3 (c), CREB (d), BDNF (e), and western blot images (f) in the hippocampus of CMS-treated mice. Control (CON) and chronic mild stress group (CMS) received saline water; imipramine (IMP; 10 mg/kg., p.o.), TB-25, TB-50, and TB-100 (extract of T. bellirica, 25 mg/kg., p.o., 50 mg/kg., p.o., and 100 mg/kg., p.o., respectively). Data are represented as the mean ± SD (n = 6). #, p < 0.05 vs CON; *, p < 0.05, **, p < 0.01 vs CMS

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Effect of Terminalia Bellirica on CMS-induced BDNF, CREB, and Caspase-3 Expression

Figure 8c illustrated that CMS significantly induces upregulation of caspase-3 expression by twofold when compared with control animals (p < 0.05). In contrast, treatment with T. bellirica and IMP normalized the CMS-induced caspase-3 expression. RT-PCR and western blot analysis (Figs. 7 and 8, respectively) illustrated that CMS exposure significantly downregulated BDNF and CREB expressions in hippocampus (p < 0.05). CMS-induced BDNF downregulation and increased CREB phosphorylation were significantly reverted by T. bellirica and IMP treatments (p < 0.05; Fig. 8d, e).

Discussion

Chronic mild stress plays a key role in the pathophysiology of depression, in which mice were exposed to various stressors for certain period to mimic chronic stressful life events of human (Auriacombe et al. 1997). There are multiple mechanisms that have been associated in the pathogenesis of depressive disorder, such as change in neurotransmitter systems, over activation of monoamine oxidases, oxidative stress, dysregulation of HPA-axis, inflammatory cytokines, and apoptotic pathway (Messay et al. 2012).

The sucrose preference is an index of anhedonia-like behavioral changes. In the present study, mice exposed to CMS showed reduced sucrose preference as compared with control animals at week 7. T. bellirica treatment significantly improved the sucrose preference, which showed the antidepressant-like action of T. bellirica. FST and TST have been widely used as behavioral parameters to assess the locomotor activity in depression models. CMS exposure significantly increased the immobility time of the mice in the TST and FST; these results are in consistent with previous studies by Mao et al. (2009). However, T. bellirica treatment in the present study reversed the CMS-induced behavioral despair by reducing the immobility time.

In depression-like disorders, BDNF plays a crucial role in regulating synaptic plasticity and neuronal survival (Popova and Naumenko 2019). As mentioned in the previous studies, BDNF transcription was downregulated in the pathogenesis of depression and lead to hippocampal atrophy, and the same was reversed by antidepressant drug treatment (Mao et al. 2012). Present RT-PCR and western blot studies confirmed that CMS exposure induced a downregulated hippocampal BDNF expression, while T. bellirica treatment significantly reversed it. In addition, BDNF has been proved to have neuroprotective property by activating serotonergic and dopaminergic neurotransmission (Huang and Reichardt 2001). Based on these results, we can speculate that gallic acid from T. bellirica can increase the levels of neurotransmitters and signaling to promote the expression of BDNF.

It has been postulated that over production of glucocorticoids may eventually lead to atrophy and decreased hippocampal neurogenesis, which results in neuro-anatomical, molecular and behavioral changes (Lanfumey et al. 2008). It has been reported that release of serum CORT activates glutamate receptor; it may lead to open Ca2+ channel by NMDA receptor (N-methyl-D-aspartate) binding activity, resulting an overload of calcium influx, induces neuronal cell death (Roozendaal 2002). In the present study, T. bellirica treatment suppressed the CMS-induced serum CORT levels, indicating the antidepressant-like effects of T. bellirica. Furthermore, overload of calcium influx may affect the activity of CREB expression (Hardingham and Bading 2002), which directly affects the regulation of BDNF. Present study confirmed that treatment with T. bellirica regulates hyperactivity of HPA-axis via modulating overload of calcium influx and its consequent upregulation in the hippocampal BDNF and CREB expressions. Over production of AchE dysregulates cholinergic neurotransmission in the nervous system (Dang et al. 2009). The present study suggested that CMS induced AChE levels in mice. Moreover, T. bellirica treatment significantly reversed the stress-dependent AChE levels.

Previous neurobehavioral studies proved the serotonin, dopamine, epinephrine, and nor-epinephrine involvement in many neurological and psychiatric diseases (Naughton et al. 2000). In the present study, neurochemical analysis revealed that treatment with T. bellirica elevated the levels of neurotransmitters. Previous reports on antidepressant drugs like serotonin reuptake inhibitors (SRI’s) and nor-epinephrine reuptake inhibitors (NRI’s) are in line with the present study, which were found to have effects on the treatment of depression by elevating monoamine levels through the inhibition of monoamine reuptake (Piñeyro and Blier, 1999).

Monoamino oxidases (MAO-A and MAO-B) are responsible for metabolic degradation of catecholamines and monoamines. MAO-A catalyzes nor-epinephrine, dopamine, and serotonin, whereas MAO-B catalyzes dopamine more specifically (Bortolato et al. 2008). Gallic acid has been reported with MAO’s inhibition and hippocampal 5-HT receptor elevation (Chhillar and Dhingra 2013). In the present study, treatment with T. bellirica has shown a significant inhibition of CMS-induced hippocampal MAO-A and MAO-B levels. Moreover, the study demonstrated that T. bellirica treatment has an ameliorative effect on CMS-induced metabolic degradation of catecholamines and monoamines.

Previous studies have demonstrated that depression was accompanied by oxidative stress (Liu et al. 2015). Present study suggested that CMS not only induced depressive-like behavior but also stimulated oxidative stress and decreased antioxidant enzyme activities. Further findings of the study demonstrated that T. bellirica treatment demolishes CMS-induced oxidative stress via upregulation of antioxidant enzymes viz., SOD, CAT, GPx, and GR.

Neuroprotective effect of gallic acid has been previously studied and demonstrated that gallic acid can reduce the neuronal apoptosis via regulating the Bax/Bcl2 ratio (Chhillar and Dhingra 2013). In the present investigation, CMS exposure elevated Bax expression levels and induced the levels of Bcl2. Further, the study confirms that T. bellirica treatment reversed CMS-induced neuronal apoptosis via regulating the Bax/Bcl2 ratio. Therefore, T. bellirica can be considered anti-apoptotic agent in controlling depressive-like behavior.

Conclusion

The present study showed that CMS induced behavioral, biochemical, and neurochemical changes. These findings of the study demonstrated that gallic acid from T. bellirica fruits exhibited antidepressant-like effects as evidenced by increased sucrose preference and decreased immobility time in behavioral observations. Also, T. bellirica ameliorated CMS-induced depression in mice through reduced hyperactivity of HPA-axis, enhancement of antioxidant defense system, normalization of neurotransmitter disturbances, and modulating proteins at molecular levels by which reduced hippocampal neuronal apoptosis. Thus, T. bellirica could be considered to be an alternative medical therapy to treat depressant-like disorders. However, additional studies involving its toxicity and clinical studies are necessary to prove the potential application of T. bellirica as a therapeutic agent.