Prediction of molecular targets for antidepressant potential of hydroalcoholic extract of Tamarindus indica using network pharmacology approach and evaluating its efficacy in Chronic Unpredictable Mild Stress model in mice

Abstract

The prevalence of psychological disorders has surged since the 1990s, posing a significant global health burden with depressed individuals averaging six lost hours per week and contributing to over 20% of all missed workdays. Current antidepressants, while effective for some, have limited efficacy, dietary restrictions, and adverse effects, including liver damage and hypertension. Natural remedies offer promising therapeutic potential with minimal side effects. Tamarindus indica (TI) is a plant that grows in the shape of a tree. Network pharmacology of TI revealed the key targets MAPK, D1-6, 5HT, DAT, MAO, COMT, PKA, PKC, AKT, and VMAT, which are linked to prominent key pathways such as dopaminergic and serotonergic. The cell viability assays on SH-Sy5y cells indicated a favourable safety profile with an IC50 of 573.99 µg/ml and further, the in vivo efficacy was observed through Chronic Unpredictable Mild Stress (CUMS) model in mice. The hydroalcoholic extract of TI demonstrated antidepressant effects, significantly reducing immobility time in the Tail Suspension Test (TST) and Forced Swim Test (FST). Additionally, locomotor activity, assessed via the Open Field Test (OFT), was significantly increased in the treatment group compared to CUMS mice. Biochemical analyses revealed elevated Brain Derived Neurotropic Factor (BDNF), decreased cortisol levels, and reduced catechol-O-methyltransferase (COMT) concentration in TI-treated (50 mg/kg) groups. These findings underscore the potential of TI as a natural antidepressant, offering a promising avenue for further therapeutic development in depression management. The current study did not evaluate the level of neurotransmitters in the brain, which will be evaluated in future studies.

Introduction

Since the 1990s, the overall prevalence of psychological disorders has been one of the highest reported illnesses of any health condition. Even though the frequency of mental diseases is higher in advanced countries when compared to developing countries, therapy costs are significantly higher in advanced countries than in developing countries, where therapy is often ignored. The number of seriously untreated patients in wealthy nations ranges from 35.5 to 50.3% (Lépine and Briley 2011). This problem is exacerbated in low-income countries, where 76.3–85.4% of major cases go untreated. Due to socio-cultural considerations and health system diversity, evidence-based methods from the advanced world are not applicable to the restrictions of the economic world. The indirect and direct impacts of the depression on the world economy are tremendous and disturbing in every way. Depression, for example, has a significant impact on working productivity. Depressed persons miss approximately 6 h of productive work each week on average, and depression accounts for over 20% of all lost workdays (Sohn et al. 2013).

Antidepressants such as tetracyclic & tricyclic antidepressants, inhibitors of MAO, norepinephrine and serotonin reuptake inhibitors, & mood stabilisers are currently recommended for depression depending on the disease condition and the preferences of the patient (Duval et al. 2006). Tricyclic antidepressants and MAO-inhibiting drugs have been demonstrated to reverse depressive symptoms in the short term. Antidepressants now available have minimal effectiveness and restrictions on dietary practices, & some patients are completely nonresponsive (Fiedorowicz and Swartz 2004). There are also varieties of adverse effects, such as liver damage and hypertension that have been reported while using the antidepressant drugs currently available in the market (Entzeroth and Ratty 2017).

Natural chemicals provide a lot of potential for therapeutic development and discovery for various diseases with minimal side effects. Tamarindus indica (TI) is a plant that grows as a tree. It is a Fabaceae or Leguminosae family member with the Caesalpinioideae subfamily. In Egypt, the TI plant was found to be cultivated first in 400 B.C. Around 600 A.D., the Ayurvedic tradition asserted its medical efficacy. Tamarind was first introduced to America in the sixteenth century, but it is today widely known worldwide (Diallo et al. 2007).

The fruit pulp is mostly used as part of this tree. The pulp of this fruit is used as an ingredient in various Indian food recipes such as Sambar, Rasam, etc. The pulp possesses many proteins, organic acids, aromatic agents, and amino acids. Due to the presence of all these particular nutrients, this plant is used as a food ingredient; this is one of the highly important Ayurvedic medicines for humankind (Bhadoriya et al. 2011). Investigating this herb’s obscure pharmacological characteristics is necessary. The present study uncovered relatively some information on TI CNS effects throughout our literature search. TI leaf extract exhibited significant neuroprotective effects against the neurotoxic impacts of prenatal aluminium chloride (AlCl₃) exposure in neonatal rats (Usman et al. 2022). TI bark extract showed an anti-Parkinsonism effect in haloperidol model in rats (Sarkar et al. 2023). The study reported that different fractions of TI pulp exhibited significant neuroprotective, anti-apoptotic, and anti-amnesic effects against aluminium chloride-induced cerebral damage and cognitive decline, attributed to their antioxidant and anti-acetylcholinesterase properties, making them promising candidates for Alzheimer’s disease therapy (Elmaidomy et al. 2022). These studies comparatively show the antioxidant and neuroprotective effects of TI.

The current study aimed to evaluate TI’s potential as an antidepressant agent. This current study used network pharmacology tools to identify targets and molecular pathways through which Tamarindus indica ameliorates depression using databases and software like Cytoscape. This identification was further validated by performing behavioural analysis in animals and by estimating biochemical parameters like BDNF, cortisol, and neurotransmitter metabolizing enzymes using ELISA.

Materials and methods

Chemicals

TI extract, Escitalopram, Mice Cortisol ELISA kits (Catalogue no: MBS29130, mybiosource.com), COMT ELISA kit (Catalogue no: MBS458496, mybiosource.com), Mice BDNF ELISA Kit (Catalogue no: MBS355435, mybiosource.com), BCA Protein assay kit (Catalogue no: 71285–3, Merk), Catechin (Catalogue no: C0705, TCI Chemicals), CMC (Catalogue no: 59938, SRL Chemical), Gallic Acid (Catalogue no: 13142, SRL Chemicals).

Collection and extract preparation of TI fruit pulp

TI fruit pulp was collected from Market, Hindupur, Andhra Pradesh in the month of May. The hydroalcoholic pulp extract was prepared by following the maceration process. The fruits were separated from the seeds and stored in the refrigerator for 1 month. Afterward, the fruits were cut into small pieces. The pulp was macerated for 7 days with a methanol–water 1:1 ratio (500 ml of methanol in 500 ml of deionised water) while stirring occasionally (Sodde et al. 2015). The components were sifted through a muslin cloth. Rotavapor was used to reduce the amount of the extracted liquid to 40 °C. The lyophilisation process was used in the last drying phase. The rationale behind the utilization of hydroalcoholic extract of the TI is that, in this study, the main focused components of the extract were alkaloids and flavonoids, which were found to be extracted more in hydroalcoholic extraction than in aqueous extract.

Characterisation of plant extract

Total phenolic content

The Folin–Ciocalteau method was used to estimate the total phenolic content. The standard curve was made with gallic acid concentration levels of 25–125 µg/ml. 200 µl (1000 µg/ml) of extract solution or standard solution was added to 1.5 ml of Folin–Ciocalteau reagent. Incubation was done in the dark for five min, and afterwards added 1.5 ml of sodium carbonate buffer (7.5% w/v) (Molole et al. 2022), for 90 min. After determining the absorbance at 760 nm, the extract’s total phenol concentration was calculated using the standard curve (Blainski et al. 2013; Rai et al. 2018).

Total flavonoid content

The aluminium chloride (AlCl₃) method was used to calculate the total flavonoid level. For the investigation, to construct the standard graph, the catechin was used as a standard at varying concentrations from 20 to 100 µg/ml. The reaction mixture was made by adding 0.3 ml sodium nitrite, 4 ml water, and 1 ml catechin/extract solution and incubated for 5 min. After adding 0.3 ml of 10% AlCl₃, the mixture was allowed to stand for 1 min. Then, 2 ml of 1 M NaOH and water were added, increasing the amount to 10 ml. The absorbance was recorded at 510 nm (Chang et al. 2020; Rai et al. 2018).

Network pharmacology

Targets of TI

Targets of various types matched to TI bioactive molecules were gathered from the Therapeutic Target Database, which is frequently utilised in the published literature for monomer study. Therapeutic Target Database offers information on therapeutic protein and DNA targets for the targeted disease (Wang et al. 2020). The species chosen was Homo sapiens.

Targets of depression

A number of sources provided depression-related genes: DisGeNet is a flexible platform that can be used for more than just research; it can be used to analyse the characteristics of disease genes, look into the molecular causes of particular diseases in humans and their complications, and more (Piñero et al. 2017). Consequently, a search was conducted using the keyword “Depression” within this database to identify targets associated with the condition. We then compared these depression-related targets with the potential target genes of TI.

Protein–protein interaction (PPI)

STRING is a comprehensive database focused on proteins, gathering, evaluating, and combining various public sources of data on protein–protein interactions. It enhances this information by incorporating computational forecasts to thoroughly understand protein interactions. After compiling a range of data sources, a worldwide network of protein–protein interactions (PPI) is constructed, encompassing both direct (physical) and indirect (functional) interactions. The organism Homo sapiens is chosen for analysis following the inclusion of the specified target list in the multi-protein search (Szklarczyk et al. 2019).

Active compound-target network

Researchers used the Cytoscape tool to construct a visual network to depict the intricate relationship between active compounds and potential targets (Qin et al. 2020). Nodes within the network represent the active compound and its targets, while edges signify their intermolecular interactions. Cytoscape, an open-source software tool, is employed to analyse and visualise biological networks (Nishida et al. 2014).

Gene ontology (GO) analysis

The gene ontology (GO) project emerged from endeavors to harmonise the functional descriptions of gene products across different databases. GO has established a standardised language, or ontologies, structured around the molecular functions, biological pathways, and cellular components relevant to gene products. The WEB-based GEne SeT AnaLysis Toolkit facilitates the analysis of 324 gene identifiers from diverse platforms and computationally evaluates 150,937 functional categories sourced from public databases (Wang et al. 2017). By opting for overrepresentation enrichment analysis (OSA), 81 UniProt IDs were submitted, with ‘genome’ designated as the reference set.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment

Enrichment analysis using ShinyGO 0.80 was conducted on mutual targets of TI and depression, focusing on the top 20 pathways with significant fold enrichment (Bouter et al. 2023). This analysis aimed to predict the potential mechanisms of action of TI bioactive components in treating depression by gathering information on key targets and associated pathways through online tools.

In-vitro cell viability assay

Cell Viability assay on SH-Sy5Y (Neuroblastoma)-cells was conducted to determine the toxicity associated with TI extract percentage cell viability and hence calculated the IC50 value from the dose–response curve. The 10,000 cells in 100 µl per well were seeded in a 96-well plate and incubated for 24 h. Six concentrations of TI were prepared and kept at −20 °C. After removal of media from inoculated cells, TI extract was added in different concentrations (25, 50, 100, 250, 500, 1000 μg) and incubated for 24 h. A 1 mg/ml MTT solution in 10% PBS and 90% DMEM was prepared. 100 µl of this solution was pipetted to each well after removing the media. The cells were incubated for 1 h at 37 °C. The media was removed from the wells, and 100 µl of DMSO was pipetted into each well. The formazan crystals formed by live cells were pipetted up and down without forming bubbles. The plates were incubated for 5 min, and the absorbance was recorded at 570 nm. Nonlinear regression was used to determine the half-maximal inhibitory concentration (IC50) using the GraphPad Prism program.

In-vivo study

Animals

The male Swiss albino mice, 7–8 weeks old, were 25–30 g and procured from CDRI-Lucknow. Mice were maintained at the animal care facility of NIPER Hajipur, maintained at room temperature with a fixed 12 h of light and dark cycle. The approval of the animal study with the CPCSEA registration No: 2116/GO/Re/S/20/CPCSEA was obtained by the Institutional Animal Ethics Committee of NIPER Hajipur.

Protocols for in vivo experiments

Animal grouping

For the investigation of depression, 30 Swiss albino mice were used. The mice were classified into five groups randomly, with six individuals each, namely normal control, CUMS, Escitalopram (5 mg/kg), TI (50 mg/kg), and TI (100 mg/kg). Except for the normal control group, all of the groups were subjected to stressors. Depressed control animals were included in the Chronic Unpredictable Mild Stress group (CUMS), where only stressors were given without any treatment (TI) compound. Escitalopram (5 mg/kg) was taken as a standard antidepressant drug (Fig. 1).

Fig. 1.

Workflow of animal study

Preparation of escitalopram dose

The escitalopram drug was diluted with 0.25% sodium Carboxy methylcellulose (Na-CMC) and the stock solution was stored in the 4 °C. 5 mg of escitalopram was mixed in 10 ml of 0.25% Na-CMC. A 5 mg/kg dose was administered orally to the animals as per body weight for 6 weeks.

Preparation of TI extract dose

The TI extract was diluted with 0.25% sodium carboxymethylcellulose (Na CMC). A dose of 50 mg/kg and 100 mg/kg body weight was orally given to the animals as per the body weight for 6 weeks.

Disease induction

Swiss albino mice were purchased from an authorised animal vendor and after the arrival of the animals they were acclimatized for 1 week to make the animals familiar with the environment. The CUMS procedure includes daily induction of multiple stressors over a timeline of 6 weeks after acclimatisation. The stressors were assigned randomly in the first week and repeated throughout the 6-weeks experiment (Frisbee et al. 2015), as shown in Table 1.

Table 1.

Schedule of stress induction in the CUMS model

Day	Week 1	Week 2	Week 3	Week 4	Week 5	Week 6
1	No food (24H)	No water	Restraint, tail pinch	No water, tail pinch	Restraint, tail pinch	No water, tail pinch
2	No water (24H)	Forced swim (15 min)	Cold water swimming (for 5 min at 4 °C)	Cage tilt	Cold water swimming (for 5 min at 4 °C)	Cage tilt
3	Cage tilt (24H)	Cage tilt	Tail pinch, no food	No food	Tail pinch, no food	No food
4	Tail pinch (60 s)	Restraint, tail pinch	Cage tilt	Restraint	Cage tilt	Restraint
5	Restraint (4H)	cold water swimming (for 5 min at 4 °C)	No water	Cold water swimming (for 5 min at 4 °C)	No water	Cold water swimming (for 5 min at 4 °C)
6	Cold water swimming (for 5 min at 4 °C)	No food and water	Restraint, tail pinch	Forced swim, no food and water	Restraint, tail pinch	Forced swim, no food and water
7	No food, tail pinch	FST, TST, OFT	Forced swim	FST, TST, OFT	Forced swim	FST, TST, OFT

FST Forced Swim Test, OFT Open Field Test, TST Tail Suspension Test

Schedule for chronic unpredictable mild stress in mice

Cage tilting

The mice housing cage (6 mice in a cage) were inclined horizontally at a 45° angle for 24 h. Restraint: The individual mouse in each group was kept in closed ventilated plastic tubes for 3 h. Tail pinch: A clamp was pinched at a length of about 1 cm from the end part of the tail of the mice for 45–60 s. Food deprivation: The mice were kept fasting for 24 h and coprophagy was prevented by placing steel mesh in their cages. Water deprivation: The mice were kept in cages without water for 24 h. Cold-water swimming: The individual mouse was forced to swim in a glass beaker containing cold water (4 °C) in which the water was filled to 15 cm deep for up to 5 min. The normal control mice were not exposed to any kind of stressors.

Treatment procedure

The experiment was conducted in a normal light–dark cycle. For 6 weeks, all of the mice were given their treatment compound in the morning time between 9:30 a.m. and 10:30 a.m., 1 h before daily exposure to stress. For 6 weeks, Escitalopram 5 mg/kg body weight was given orally to every animal in the standard group. Similar to the standard, TI of 50 mg/kg and 100 mg/kg body weight was given to each animal in the two test groups, respectively, through an oral route once a day for 6 weeks. Animals were fasted for 12 h after 6 weeks of therapy, and blood was withdrawn from the retro-orbital plexus using diethyl ether as an anaesthetic agent. After collecting blood samples, the animals were euthanised by cervical dislocation and collected brain samples of all animals. The brain samples were stored at −80 °C refrigerator for further biochemical tests.

Behavioural parameter testing

Forced Swimming Test (FST)

The mice were placed in a water tank 30 cm in height, 15 cm in diameter, and 20 cm deep with water at 23–25 °C. The mice were unable to jump out from the cylinder or use their tails or hind limbs to reach the bottom of the tank at this position. The first session included 15 min of compelled swimming practice. After an hour of oral dosage, each mouse was placed in the glass tank for a 6-min test session (Tested at the end of the 2nd, 4th, and 6th week). A recording device was used to capture the animal behaviour so that it could subsequently be manually scored. A well-known antidepressant response measurement is the shortening of immobility time. The water in the tank was replenished following each session, and the rats were towel-dried (Kumar et al. 2014).

Tail Suspension Test (TST)

Similar to the FST, the TST uses immobility as a behavioural variable to express behavioural depression. The TST is a straightforward setup in which mice are hung from the ends of horizontal bars while their immobility poses are timed. The TST is the test that utilised the most frequently in rodents. Similar to the FST, the TST has been shown to be responsive to all major drug types of pharmacological antidepressants, such as imipramine, desipramine, SSRIs, such as paroxetine and fluoxetine, and atypical antidepressants (bupropion, citalopram). Both the FSTt and the TST have been under intense scrutiny as behavioural tests for depression in recent years. A prominent source of criticism has been the reality that both experiments mimic an acute context and do not accurately depict the temporal aspects of clinical depression’s development, duration, and treatment effects in real patients (Pollak et al. 2010).

Open Field Test (OFT)

This test is used to investigate rodents’ inquisitive and depressed behaviour. After 60 min per oral dosage, each mouse was kept in the middle square of the OFT Test apparatus and watched for a total of 5 min (1-min acclimatisation and 4 min testing). The animal was taken after 5 min. Before placing the next animal, 70% ethanol was used to clean the apparatus. The animals’ behaviour was monitored using ACTi-track software, which uses infrared beams to record animal movement. The number of line crossings (the frequency with which the body crosses both horizontal and vertical lines) was measured (Walsh and Cummins 1976).

Biochemical estimation through ELISA kit

BDNF estimation

Before homogenisation for this experiment, tissues were thoroughly cleaned in chilled PBS (0.01 mol/l, pH 7.0–7.2) to eliminate any extra blood traces and weighed. The tissue was chopped into tiny chunks and homogenised in 5–10 ml/g of PBS using a tissue homogeniser on packed ice and centrifuged to collect the tissue supernatant. The test was done as per the manufacturer’s protocol.

The BDNF levels in the samples were quantified using a sandwich enzyme-linked immunosorbent assay (ELISA) kit. The assay was based on the principle of a sandwich ELISA, where a specific antibody for BDNF was pre-coated onto the wells of a 96-well plate. The standards, test samples, and a biotin-conjugated detection antibody specific for BDNF were added to the wells sequentially. After incubation and washing, an avidin–biotin-peroxidase complex was added, and unbound conjugates were washed away. The addition of a TMB substrate led to the development of a blue color, which turned yellow upon the addition of an acidic stop solution. The intensity of the yellow color was proportional to the amount of BDNF captured in the wells. Each well’s optical density (OD) was measured at 450 nm using a microplate reader, and the BDNF concentration in the samples was calculated based on the standard curve.

Cortisol estimation

Tissue slices were cleaned in 0.01 M PBS before being mixed in an ice bath with a tissue protein extraction reagent (1 g to 5–10 ml). Once homogenisation was finished. It was centrifuged at 5000–10,000 rpm for 10 min. The supernatant was collected for testing. The absorbance was recorded at 450 nm.

The ELISA kit employed in this study utilized the Double Antibody Sandwich technique for the quantification of mouse cortisol levels. The wells of the ELISA plate were pre-coated with a monoclonal antibody specific for mouse cortisol. A biotinylated polyclonal antibody served as the detection antibody. The samples and biotinylated antibodies were added to the wells and incubated. After washing the wells with PBS or TBS, avidin-peroxidase conjugates were added. TMB substrate was then used for color development after thoroughly washing out the enzyme conjugates from the wells. The peroxidase activity catalyzed the TMB, resulting in a blue product, which turned yellow upon the addition of a stop solution (Color Reagent C). The intensity of the yellow color was directly proportional to the concentration of the target analyte, cortisol, in the sample.

Catechol-O-methyltransferase (COMT) estimation

Tissues were carefully cleansed in Chilled PBS (0.01 mol/L, pH 7.0–7.2) to remove traces of blood and weighed before the homogenisation for this experiment. Using a homogeniser on ice, the tissues were chopped into tiny pieces and homogenised in 10 ml/g of tissue PBS. The suspension was sonicated with an ultrasonicator to break the cell membranes. The homogenates were then centrifuged at 5000g for 5 min. Immediately, the supernatant was tested for COMT estimation.

The assay for measuring COMT levels was conducted using a microtiter plate pre-coated with a specific antibody for COMT. Initially, standards or samples were added to the designated wells along with a biotin-conjugated antibody that targets COMT. Following this, an avidin-Horseradish Peroxidase (HRP) conjugate was introduced to each well, and it was allowed to incubate. After the addition of the TMB substrate solution, color change occurred only in wells containing COMT, the biotin-conjugated antibody, and the enzyme-conjugated avidin. The enzymatic reaction was halted by adding a sulfuric acid solution, and the resulting color change was measured spectrophotometrically at a wavelength of 450 ± 10 nm. The concentration of COMT in the samples was determined by comparing the optical density of the samples to a standard curve.

Statistical analysis

GraphPad Prism (evaluation version 9) and Excel were used to analyse the acquired data statistically. The statistical comparison was conducted using one-way ANOVA and the Tukey multiple comparison test. The p value less than 0.05 was regarded as statistically significant. All values were reported as means with standard deviations.

Results

Extraction and characterisation of extract

The percentage yield from the extract was 20%.

Total phenolic content

The hydroalcoholic extract of TI had a total content of phenolic compounds of 42.988 mg of gallic acid equivalent/g, according to the Folin–Ciocalteau technique.

Total flavonoid content

The aluminium chloride reagent method revealed that 2.088 mg catechin equivalent/g extract was present in the TI extract.

Active compounds of TI

A total of 47 active compounds were identified in TI based on the IMPPAT database and other literature searches and active compound targets were found through the Binding Database.

Disease targets

By screening DisGeNET, 598 depression-related targets were obtained. By comparing the targets of TI and depression, it was found that depression shares 134 targets with the active compounds of TI and 134 targets were correlated with 47 active compounds.

PPI network

The String database was used to perform protein–protein interaction network analysis on the common targets of TI and depression, and the PPI network was formed as shown in Fig. 2. A total of 132 nodes and 1399 edges were obtained from the results, with an average node degree is 21.2.

Fig. 2.

The PPI network of common targets between TI and depression. Each bubble node represents a protein, and the connections between these proteins are illustrated by lines. The width of these lines indicates the level of data validation for the associations. Unconnected nodes were hidden to further minimize the complexity of the network, and the minimum required interaction score was set at 0.700

TI-target network

The network between common targets of TI and depression is shown in Fig. 3. Using Cytoscape, the network was built by mapping 47 active compounds and 134 potential targets. The network embodied 203 nodes and 690 edges, with colorised pink nodes corresponding to the targets and compounds calculated in green from TI. The bioactive compounds with more targets were Hordenine, 3,4-dihydroxyphenylacetate, Gallacetophenone, UNII-0V56HXQ8N5, 1-(2-Furyl)propane-1,2-dione, 2-methyl-1-butanol, 3,5-dimethylphenol, 1.Xi.,6.xi.,7.xi.-Cadina-4,9-diene, 3-Carene, d-Galactose, d-Glucose, Gallacetophenone, l-leucine, l-proline, Phenylalanine, piperidine-2-carboxylic acid, and Xyloglucan Heptasaccharide which corresponds to targets. The result suggested that these active compounds of Tamarindus indica probably served a significant therapeutic role in depression.

Fig. 3.

The TI-target network for potential targets in depression. The green nodes denote bioactive compounds, while the pink nodes indicate the corresponding targets

Gene ontology enrichment analysis

To gain deeper insights into the functions of the 134 target genes, gene ontology (GO) enrichment analysis was conducted using WebGestalt (Fig. 4). This analysis categorises functions into three levels: biological process, cellular component, and molecular function. The top five functional categories identified were response to stimulus (98 out of 99 genes), biological regulation (95 out of 99 genes), cell communication (89 out of 99 genes), multicellular organismal process (88 out of 99 genes), and metabolic process (86 out of 99 genes).

Fig. 4.

Go analysis: The results of the GO analysis: A A bar chart representing the Biological Process categories. B A bar chart illustrating the Cellular Component categories. C A bar chart depicting the Molecular Function categories

KEGG pathway enrichment analysis

To explore the mechanism of TI in the treatment of depression, KEGG pathway analysis was performed for enrichment analysis through the ShinyGO 0.80 database. The top 20 pathways with significant fold enrichment were screened out. Bubble charts were generated to visualise these pathways, showcasing adjusted p values, fold enrichment, and gene counts (Fig. 5).

Fig. 5.

KEGG pathway

Cell viability assay

Using six different TI concentrations, the MTT assay was performed on SH-Sy5y (Neuroblastoma). The IC₅₀ of TI in SH-Sy5y cells was found to be 573.99 µg/ml (Fig. 6).

Fig. 6.

Cell viability analysis: Graph representing the percent of cell viability of SH-Sy5Y (neuroblastoma) cells while treated with different concentrations of the extract

Effect of Tamarindus indica on body weight

The Chronic Unpredictable Mild Stress was used to produce depression. After 4th week, the body weight in CUMS induced mice was less than that of the control group (p < 0.001), suggesting the induction of depression in CUMS group. After 4th week of treatment with Tamarindus indica (50 and 100 mg/kg) and escitalopram, significantly (p < 0.0001) recovered the body weight as compared to the depressed group. The treatment with Tamarindus indica extract improved the body weight from 1 to 3 weeks but dramatically raised body weight from 4 to 6 weeks (Fig. 7).

Fig. 7.

Effect on body weight: Graph representing the effect of TI on body weights of animals at 0–6 weeks. Data were represented as mean ± SD. ^#### p < 0.0001 compared to the control and **** p < 0.0001 were considered significant compared to CUMS

Effect of Tamarindus indica on Forced Swim Test

The induction of stress in CUMS group causes reduced movement and more to be stagnant. This parameter was conducted at different intervals of days to check the induction of stress in mice. In Fig. 8, no significant changes were observed in the 2nd week of FST performance. But in the 4th and 6th week, the immobility duration increased significantly (p < 0.01 to p < 0.001) as compared to the control group. Similarly, treatment of TI (50 mg/kg) reduced the immobility duration more significantly (p < 0.001) in the 4th and 6th week of the experiment and reduced the effect of stress. TI (100 mg/kg) also reduced immobility (p < 0.01, p < 0.05) compared to CUMS. The treatment with a standard drug (Escitalopram) also reversed the immobility duration more significantly (p < 0.001, p < 0.01) in mice in the 4th and 6th week as compared to CUMS group.

Fig. 8.

Effect of TI on Forced Swim Test. CUMS causes increased immobility period when compared with control whereas, treatment with TI and standard drug reverses the action of stress. Data are represented in mean ± SD. ^## p < 0.01, ^### p < 0.001 considered as significant with control group while * p < 0.05, ** p < 0.01, *** p < 0.001 compared to CUMS group

Effect of Tamarindus indica on Tail Suspension Test

Similar to the FST, the Tail Suspension Test is a behavioural parameter to check rodents’ immobile phase duration. In CUMS group, the immobility was more significantly (p < 0.01) observed in the 4th and 6th week of the scheduled test, indicating less movement of mice to escape due to stress induction. The Tail Suspension Test showed that the difference in the immobility time of animals at weeks 2nd,4th and 6th, there was a significant decrease in the immobility time of animals of treatment groups (TI50 and TI100) when compared to the CUMS group. The TI50 was found to be more effective in decreasing immobility time (p < 0.001). In addition, the escitalopram group reduced the immobility period significantly (p < 0.001) since 2nd week of TST (Fig. 9).

Fig. 9.

Effect of TI on Tail Suspension Test. CUMS reduced the mobility action of mice and increased the immobility duration when compared with a control group. In the treatment group, TI and standard drug reduce the immobile phase. Data are represented in mean ± SD. ^# p < 0.05, ^## p < 0.01 considered as significant with control group. Treatment with TI and ESC were considered significant at * p < 0.05, ** p < 0.01, and ***  p < 0.001 compared to CUMS group

Effect of Tamarindus indica on Open Field Test

The open field test showed the difference in the exploratory behaviour of animals at weeks 2nd, 4th and 6th week (Fig. 10). The different stressors created a lesser movement in CUMS group, which was significantly (^#p < 0.05, ^##p < 0.01) reduced as compared to control group indicates the depressed behaviour of mice. Further, there was a significant (***p < 0.001, **p < 0.01) increase in the movement of animals in treatment groups (TI50) when compared to the CUMS group. The TI50 dose was found to be more effective in the number of lines (hypothetical) crossed. The treatment with the standard group increased the locomotory action significantly (***p < 0.001) compared to the control group.

Fig. 10.

Effect on Open Field Test: Graph representing the number of line crossings in OFT at 2, 4 and 6 weeks, respectively. The different stressors create a lesser movement in CUMS group which was significantly (^# p < 0.05, ^## p < 0.01) reduced as compared to the control. In addition, there was a significant (*** p < 0.001, ** p < 0.01) increase in the movement of animals in treatment groups (TI50) when compared to the CUMS group. ESC group significantly (*** p < 0.001) increased the line of crossings compared to control group

TI promoted the neuroprotection and reduction of cortisol and COMT

The reduction of neurogenesis was due to stress which reduced significantly (^##p < 0.01) in CUMS group. The level of BDNF was significantly (***p < 0.001) high in the escitalopram-treated group. Similarly, treatment with TI50 and TI100 significantly (**p < 0.01) and (*p < 0.05) enhanced neuroprotection (Fig. 11). Stress hormones like cortisol played an important role in depressive illnesses. The ELISA showed that there was a significant (p < 0.001) increase in cortisol concentration in CUMS group when compared to the treatment group, which showed depressive behaviour of mice. TI50 was found to be effective in reducing the cortisol levels (Fig. 11). Alteration of COMT might lead to the progression of various psychiatric illnesses. The ELISA showed that there was a significant (^###p < 0.001) increased COMT level in CUMS group when compared to the control group. The treatment with TI and Escitalopram was found to be significantly (p < 0.001) effective in reducing the COMT levels (Fig. 11).

Fig. 11.

Effect on molecular marker of depression: A cortisol, B BDNF, and C catechol-O-methyl transferase (COMT) levels in mice brain tissue. Each statistical data represents the mean ± SD of 6 mice in each group. One-way ANOVA and the Tukey multiple comparison test were used to analyse the data. The data representation ^### p < 0.001, ^## p < 0.01 compared with control group. While *** represents p < 0.001, ** represents p < 0.01, and * represents p<0.05 compared to CUMS group

Discussion

The Chronic Unpredictable Mild Stress (CUMS) induced depression rodent model is an appropriate paradigm for researching depression-like behaviour in mice. In mice, chronic stress causes behavioural and pathological changes that are similar to those seen in sad people. Chronic stress causes changes in central neurotransmitters, such as noradrenaline, serotonin, and dopamine levels and activities, as well as the regulation of particular receptors, destabilisation of the HPA axis, upregulation of several proapoptotic factors, the elevation of cortisol levels, and the production of reactive oxygen species, all of which lead to neuronal damage and an increase in depression symptoms. The goal of this study was to see hydroalcoholic extract of TI had antidepressant properties in a CUMS depression model in mice.

The network pharmacology investigation identified the targets and pathways of TI in depression. And have discovered that TI has 134 potential targets with depression. The PPI network was created to demonstrate the functional interactions between the target proteins. The PPI network analysis revealed that the primary targets of TI in depression are MAPK, D1-6, 5HT, DAT, MAO, COMT, PKA, PKC, AKT, and VMAT. The functional enrichment analysis discovered that these targets were significantly prominent in key pathways such as dopaminergic and serotonergic. The Cell viability assay was performed on SH-Sy5y cells; the MTT assay has shown that the IC50 of TI is 573.99 µg/ml, reflecting that it is safe for the neuronal cells.

The hydroalcoholic extract prepared for this study showed 20% yield, while characterisation for total phenol (42.988 mg of gallic acid equivalent/g of extract) and flavonoid content (2.088 mg catechin equivalent/g of extract), the results were similar to our earlier reports on this extract (Rai et al. 2018). Because of its antioxidant and neuroprotective potential, as well as the flavonoid and phenolic content, which have been found to modulate the Hypothalamus Pituitary Adrenal axis, the results of behavioural and biochemical parameter testing clearly show that hydroalcoholic extract of TI (50 mg/kg) is able to elevate the behavioural and neurochemical effects involved in the etiology of depression.

The FST and TST models are the most extensively used methods for assessing antidepressant activity. To avoid false-positive results, locomotor activity was measured before FST and TST in the current investigation to confirm the antidepressant effect of TI. In mice, the time spent immobilised was dramatically reduced. On the first day of the test, mice treated with TI (50 mg/kg and 100 mg/kg) and escitalopram (5 mg/kg), an SSRI, had significantly shorter immobility times than normal mice, while CUMS mice had significantly longer immobility times than control mice, similar results like other antidepressant study with curcumin (Liao et al. 2020). Another test used to evaluate locomotor activity in mice is the OFT. CUMS mice have been shown in studies to have less line crossings (locomotor activity) (Gencturk and Unal 2024). Compared to the Chronic Unpredictable Mild Stress group, the hydroalcoholic extract of TI substantially increased the number of line crossings.

Given the significance of the BDNF and its corresponding receptors in neural and structural plasticity, as well as the reality that depression and antidepressants have differential effects on BDNF and TrkB expression and capabilities, BDNF signalling appears to be significant in the pathophysiology of depression and the mechanism of antidepressant action (Yang et al. 2020). How a decrease in BDNF expression results in depression is still a mystery. From the studies, it was found that the BDNF was significantly high when treatment groups compared to CUMS group, whereas when compared to the normal control group, these levels were identically decreased in CUMS group. There will be a continuous rise in cortisol levels after stressor exposure in CUMS models, and the results showed that there was a significant rise in the cortisol levels in the brain of CUMS group when compared to the control group, whereas the treatment groups have shown significant decrease when compared to the disease control group. In the metabolism of monoamines, the COMT enzyme is crucial (Dopamine, Norepinephrine). Compared to the disease control group, the concentration of the COMT was significantly lower in the brain samples from the treatment groups (Na et al. 2018). The results of the present study showed that the TI had an effect on neurotransmitter metabolizing enzymes, which helped in increasing neurotransmitter levels. Rise in BDNF levels in treatment groups showed neuroprotection activity which can be correlated with previous studies on plant medicines like Withania somnifera (Prakash et al. 2014) and Mucuna pruriens which is having anti-inflammatory (Rai et al. 2017a), neuromodulatory (Rai et al. 2017b) and antioxidant activity (Yadav et al. 2017; Rai et al. 2020).

Conclusion

According to the current study, the hydroalcoholic extract of the TI pulp was effective in reducing behavioural and neurochemical stimulations involved in the etymology of depression. Further experiments on the molecular mechanism using the network pharmacology, cortisol levels, BDNF level, and COMT level in the brain helped to find the antidepressant activity of the hydroalcoholic extract of TI. Thus, it can be concluded that the hydroalcoholic extract of TI may be an effective alternative for the development of novel therapeutic agents in the treatment of depression.

Declarations

Conflict of interest

We declare no conflict of interest.