Deciphering the Pharmacological Potentials of Methanol Extract of Sterculia foetida Seeds Using Experimental and Computational Approaches

The edible herb Sterculia foetida L. has potential nutraceutical and medicinal effects. The present study is performed to assess the possible antidiabetic, neuropharmacological, and antidiarrheal activity of the methanolic extract of S. foetida seeds (MESF) through in vitro, in vivo, and in silico approaches. When compared to standard acarbose, the results of the antidiabetic study provided strong proof that the glucose level in the MESF was gradually decreased by inhibiting the function of α-amylase enzymes. The sedative potential of MESF (200 and 400 mg/kg) was determined by employing open field, hole cross, and thiopental sodium-induced sleeping time tests, which revealed significant reductions in locomotor performance and increased sleep duration following MESF treatment. In addition, mice treated with MESF exhibited superior exploration during elevated plus maze and hole board tests. MESF also showed good antidiarrheal activity in castor oil-induced diarrhea and intestinal motility tests. Previously isolated compounds (captan, 1-azuleneethanol, acetate, and tetraconazole) exhibited good binding affinity in docking studies and drug-likeliness properties in absorption, distribution, metabolism, excretion/toxicity (ADME/T), and toxicological studies. Collectively, these results indicate the bioactivity of S. foetida, which represents a potential candidate in the food and pharmaceutical industries.


Introduction
Plants have long been used by humans to cure various diseases; however, modern medicines have trended away from the utilization of plants to prioritize the utilization of laboratory-produced agents. However, components derived from plants tend to display distinct structural diversity and reduced toxicity and are more readily obtainable than synthetic chemicals, and plant-based substances display a variety of mechanisms of action, leading researchers to increasingly explore the developmental potential of components derived from plants for drug design during recent decades [1][2][3]. Psychological and behavioral disorders have become significant consequential factors associated with disability, disrupting people's emotions and moods [4]. Neuropsychiatric disorders have been identified as the third leading cause of disability, particularly among women, according to the Global Burden of Disease (GBD), Injuries, and Risk Factors Study Highlights (2017) [5]. Moreover, the World Health Organization has identified depressive disorder, which is considered the largest contributor to worldwide nonfatal health issues, with anxiety disorders representing the sixth most common contributor [6].
Depression is characterized as a multifactorial disorder that affects broad swathes of the population and is associated with physical dysfunction and increased morbidity and mortality rates, and anxiety represents another type of chronic psychiatric disorder [7]. In both developed and developing countries, gastrointestinal disorders, which are commonly associated with bacteria and parasites, represent common health issues. Enteric infections that cause dysentery-like chronic diarrhea are commonly associated with infection by numerous etiological agents, including Salmonella, Campylobacter, Escherichia, Shigella, Yersinia enterocolitica, parasites, and viruses [8]. Because of the development of multiple resistance against accessible drugs, current infectious diseases cannot always be readily treated; therefore, assessing plants for the presence of potential new therapeutically functional agents may represent an alternative and effective approach to the development of novel treatments. e medicinal herb Sterculia foetida, which belongs to the family Malvaceae, is commonly referred to as "Jangli badam" in the Hindi and Bengali languages and as "Gorapu badam" in the Tamil language. is herb can be found in temperate and tropical regions, such as Bangladesh, North-East Australia, Malaysia, and India [9,10]. e S. foetida tree yields edible seeds, which contain 30-36% fat and 11.4% protein, and its bark and leaves can be utilized for aperient, diuretic, and diaphoretic purposes and have also been used to treat obesity, rheumatism, gonorrhea, dropsy, and skin disease [11]. is plant also contains a wide range of bioactive components that have been isolated previously, including acetamiprid, α-BHC, tetradecanoic acid, methyl ester, terbufos, benfuresate, dichlofluanid, DEP (trichlorfon), 1-azuleneethanol, acetate, captan, etridiazole, diethofencarb, etobenzanid, cyfluthrin, cypermethrin, pendimethalin, kresoxim-methyl, tetraconazole, and pyributicarb [12]. Although S. foetida has broad traditional applications, several of the pharmacological characteristics of this plant remain to be explored.
us, the purpose of the present study was to assess the in vitro antidiabetic, in vivo neuropharmacological, and antidiarrheal activities of a methanolic extract of S. foetida (MESF). In addition, computer-aided studies, including molecular docking; absorption, disruption, metabolism, and excretion/toxicity (ADME/T); and toxicological properties of the compounds isolated from this herb were evaluated to explore their biological activities. ese studies contributed to the identification of bioactive compounds present in MESF and provide insights regarding potential targets for development as lead compounds for therapeutic purposes.

Plant Material Collection and Extraction.
e plant material collection and detailed extraction process used to obtain the methanolic extract of S. foetida were described in detail in a previous publication [12]. e extract was stored at 4°C for additional use.

Ethical Statement and Experimental
Animals. Swiss albino mice of both sexes, with an average weight of 20-30 grams, were obtained from Jahangirnagar University, Dhaka, Bangladesh, and were maintained under standard laboratory circumstances (room temperature: 25 ± 2°C; relative humidity: 55%-60%), with access to food and water. All experiments were performed in an isolated and soundless environment.
e research was conducted in compliance with the guidelines of e Planning and Development (P&D) Committee of the International Islamic University Chittagong, Department of Pharmacy, according to current government guidelines [12].

α-Amylase Inhibitory Activity.
e α-amylase inhibitory activity method was described in [13] with some modifications. A mixture of 1 mL of plant extract and 1 mL of α-amylase was incubated in a test tube at 37°C for 10 min. After preincubation, 1 mL of 1% (v/v) starch solution was added to each tube and incubated for 15 min at 37°C. After that, 0.4 mL aliquots of the incubate were transferred to sample tubes containing 3 mL starch and 2 mL phosphate buffer (pH 6.5), and 0.1 mL of the combination was removed before reincubation to measure absorbance at 565 nm; the mixture was then reincubated for 45 minutes. After mixing, 0.1 mL of the reaction mixture was removed from each tube and discharged into 10 mL of iodine solution at the conclusion of the reincubation time. e absorbance (At) was measured immediately at 565 nm after the solutions were completely mixed. e % inhibition of α-amylase by each plant extract can be calculated using the following formula: where A0 and At are the absorbance values at zero time and at the end of the incubation, respectively. e open field apparatus was employed to assess locomotor activity [14]. e treatments were applied to the four groups of animals as described above in Section 2.4. e mice were placed in a box (40 cm × 40 cm × 30 cm) separated into 25 black and white squares by a series of lines on the floor. In brief, movement was recorded as the number of lines crossed by each individual mouse during a 3-minute period starting at 0, 30, 60, 90, and 120 minutes.

Hole Cross Test (HCT).
An HCT apparatus was prepared, consisting of a cage (30 cm × 20 cm × 14 cm) separated into two chambers using a 7.5 cm tall divider featuring a hole (three centimeters in length) in the center [15]. e treatments were applied to four groups of animals, as described above in Section 2.4. e mice were placed on one side of the divider, and the number of times the mice passed through the hole during a 3-minute period was recorded, starting at 0, 30, 60, 90, and 120 minutes.

2.7.3.
iopental Sodium-Induced Sleeping Test. Treatments were applied to four groups of mice as described above in Section 2.4, and after 30 minutes, all mice were treated with thiopental sodium (40 mg/kg) to induce sleep. e latency between the administration of thiopental and the loss of the correction reflex, sleep onset, and sleep duration was recorded [16].

Elevated plus Maze (EPM).
e EPM utilized an apparatus situated 40 cm above the ground, featuring two open arms and two closed arms, to evaluate the anxiolytic effects of MESF in mice [17]. e arms open to a center platform, such that the maze resembles a plus sign. e treatments were applied to four groups of mice as described above in Section 2.4, and 30 minutes after treatment, each mouse was placed on the center platform, and the time spent in both the open and closed arms was recorded over a 5minute period.

Hole Board Test (HBT).
A device containing 16 holes (3 cm in diameter) across a flat platform was utilized in the experiment, and the apparatus was placed 25 cm above the floor [18]. e treatments were applied to four groups of mice as described above in Section 2.4, and after 30 minutes, the experimental mouse was placed on the center of the HBT device and allowed to move freely. e number of times the mouse dipped its head through the holes was recorded over a 5-minute period.

Antidepressant Activity
e glass cylinder (25 cm × 15 cm × 25 cm) was filled with water at a constant temperature of 25 ± 1°C to a height of 15 cm. e treatments were applied to four groups of animals as described above in Section 2.4, and the mice were forced to swim freely in the cylinder. e FST lasted for a total of 6 minutes, and the duration of immobility was recorded during the last 4 minutes [19].

Tail Suspension Test (TST).
e TST test was used to assess the antidepressant activities of MESF. e treatments were applied to four groups of animals as described above in Section 2.4, after which the mice were hung by their tails to induce immobility (depression), and the time spent immobilized was recorded for six minutes [20].

Castor Oil-Induced Diarrhea.
Castor oil-induced diarrhea was performed as previously described [21]. Treatments were applied to four groups of animals described above in Section 2.4, and after 1 hour, 0.5 mL castor oil was administered orally. Each mouse was then placed in a box containing absorbent paper. en, the fecal production of the mice was monitored for 4 hours for each mouse, and the absorbent paper was replaced every h. e defecation inhibition percentage was measured using the following equation: where A � average feces number of the control group and B � average feces number of the test group.

Charcoal-Induced Intestinal Motility
Test. e test was conducted as described previously [22]. e treatments were applied to four groups of animals as described above in Section 2.4, and after 1 hour, 0.5 mL castor oil was administered orally, followed by 1 mL of charcoal marker (10% charcoal + 5% gum acacia), 1 hour after the castor oil treatment. e animals were sacrificed 1 hour after charcoal treatment. e distance between the charcoal meal and the Evidence-Based Complementary and Alternative Medicine pylorus caecum was measured, and the overall length of the intestine (percentage) was estimated in cm. e following equation was used to determine the peristalsis index and inhibition percentage: 2.11. Compounds Investigated. Based on the results of a previously described gas chromatography-mass spectrometry study, acetamiprid, α-BHC, tetradecanoic acid, methyl ester, terbufos, benfuresate, dichlofluanid, DEP (trichlorfon), 1-azuleneethanol, acetate, captan, etridiazole, diethofencarb, etobenzanid, cyfluthrin, cypermethrin, pendimethalin, kresoxim-methyl, tetraconazole, and pyributicarb were selected [12], and the chemical structures were obtained from the PubChem database.
2.14. Statistical Analysis. All values (in vivo) are described as the mean ± standard error mean (SEM). Using SPSS v 20.0 software, Dunnett's test was used to compare values between groups. At p < 0.05, 0.01, and 0.001, values were considered significant.

α-Amylase Inhibitory Activity.
In this test, S. foetida seeds and standard acarbose are represented as percentage of inhibition where the highest 80.28% of inhibition was achieved by 500 μg/ml of MESF (Figure 1).

Open Field Test (OFT).
e present study utilized the OFT to assess the neuropharmacological activity of MESF in mice. Each mouse in the test group was treated with MESF (200 and 400 mg/kg; b.w.), which resulted in a notable decrease in the numbers of squares crossed (Figure 2). At alltime points measured in this experiment, the mice treated with the extract displayed significant decreases in locomotor activity compared with the control group.

Hole Cross Test (HCT).
e time required for each mouse in the control group to cross from one chamber to the next ranged from 30 to 120 minutes. In this study, the groups treated with MESF demonstrated decreased locomotion at 0-120 minutes from its initial value. However, the maximal inhibition of locomotor activity was observed for both MESF doses tested (p < 0.001), referring to diazepam (reference drug; Figure 3).

iopental Sodium-Induced Sleeping Time Test.
In a dose-dependent manner, MESF (200 and 400 mg/kg) treatment resulted in a significant (p < 0.001) reduction in the sleep onset time, similar to that of the standard drug. In addition, the duration of sleep induced by thiopental sodium in MESF-treated animals increased compared with that of the control group (Table 1).  Figure 4).

Forced Swimming Test (FST).
e animals treated with MESF (200 and 400 mg/kg) and imipramine (standard drug) revealed significant (p < 0.001) reduction in the period of immobility, which were measured at 120.37, 70.03, and 48.28 seconds, respectively ( Figure 6) compared with the control group.  motility in mice are presented in Figure 9 as the percentage inhibition of intestinal transit. MESF (p < 0.001) significantly reduced the peristalsis index relative to that of the negative control group for both tested doses (p < 0.001). e 400 mg/kg dose had the greatest suppressive effect on intestinal motility (36.75%), similar to that of the standard drug loperamide (48.01%).

Molecular Docking Analysis for Sedative and Anxiolytic
Properties. e 18 primary compounds identified in S. foetida were docked with the human gamma-aminobutyric acid receptor (PDB : 4COF) to test sedative effects and displayed scores between +2.296 and − 6.33 kcal/mol. e results show that captan (− 6.33 kcal/mol) displayed the strongest binding score with the target receptor, which was stronger than the docking score of the standard drug diazepam (− 5.961 kcal/mol). e docking score ranking order is shown as follows: captan > cypermethrin > 1-azuleneethanol, acetate > dichlofluanid > DEP (trichlorfon)   > dichlofluanid did not display any interactions with the target receptor. e results of the molecular docking analysis are shown in Table 2, and the ligand-target interactions are shown in Figures 10(a) and 10(b).

Molecular Docking Study for Antidepressant Activity.
In the antidepressant docking analysis, our investigation revealed that tetraconazole and tetradecanoic acid, methyl ester have the strongest and weakest binding affinities for the human serotonin receptor (PDB : 5I6X), with docking scores of − 4.842 kcal/mol and +0.842 kcal/mol, respectively. e   docking score rank order for the antidepressant effect is as follows: tetraconazole > kresoxim-methyl > cyfluthrin > pyributicarb > dichlofluanid > 1-azuleneethanol, acetate > etridiazole > cypermethrin > captan > acetamiprid > diethofencarb > α-BHC > terbufos > pendimethalin > tetradecanoic acid, methyl ester. However, two compounds, benfuresate and DEP (trichlorfon), did not show any interactions with the target receptor. e reference standard drug imipramine displayed a docking score of − 5.35 kcal/mol against the human serotonin receptor (PDB : 5I6X). ese scores are shown in Table 2, and the binding interactions of the bestdocked compounds are displayed in Figure 11.

ADME/T and Toxicological Properties Prediction.
As specified by Lipinski's rule of five, a compound may possess drug-like properties if it does not fail more than one of the following principles: MW < 500 amu; HBA sites < 10; HBD sites < 5; and lipophilicity value (LogP) ≤ 5. In addition, Veber et al. recommended that a compound should have a number of rotatable bonds ≤10 and a total polar surface area value ≤ 140Å to provide optimal molecular flexibility, which might facilitate the transport of medications through membranes.
Our study found that all tested compounds satisfied both Lipinski's rule of five and Veber's rule, which indicated that each compound could represent a good starting point for the development of new drugs (Table 3). e admetSAR online tool was used to predict the toxic properties of the compounds, and the results are presented in Table 4. None of the chemicals studied exhibited Ames toxicity, carcinogenicity, acute oral toxicity, or rat acute toxicity.

Discussion
e seeds of S. foetida were examined to assess α-amylase inhibitory and its behavioral effects through the application of various experimental tests, including the OFT, HCT, HBT, EPM, FST, TST, and thiopental sodium-induced sleep test, which were conducted on available and approved animal models of CNS activity.
Previous preclinical evaluation of antidiabetic study concluded that the methanol leaf extract [29], ethanol, and aqueous flower extracts of S. foetida revealed a significant reduction in diabetic rats' blood glucose levels [30]. Seeds of S. foetida are rich in secondary metabolites such as alkaloids, flavonoids, and saponins and have antidiabetic properties [12]. According to the previous study, the bioactive molecules found in the plant S. foetida play a significant role in antidiabetic properties. e current study of the antidiabetic screening test found that the methanol seed extract of S. foetida has the better antidiabetic activity.
Both the OFT and HCT were conducted to examine the effects of MESF on locomotor activity. Sedative agents will decrease locomotion, which can be assessed as a lack of inquisitive behavior when a mouse is introduced to a new environment [31]. Locomotor activity is considered to represent a mental state indicator of consciousness and awareness, and decreased locomotion serves as an indicator of sedation and a general lack of agitation, which can be interpreted as diminished CNS excitability [32]. e administration of MESF to mice reduced the number of square blocks crossed in the OFT and the number of holes crossed in the HCT starting at 30 minutes after treatment, indicating the sedative activity effects on reduced locomotion. e effects of MESF in mice were also examined using the thiopental-induced sleep test. In addition to extending the duration of sleeping time, the latent period of drowsing off is reduced and the onset of sleep in mice increased. iopental acts on CNS activity through the stimulation of the GABAergic system repression, and plant extracts derived from S. foetida have been shown to produce hypnosis at large dosages, such as 200 and 400 mg/kg. e extract exhibited anxiolytic-like behavior in mice during the HBT and EPM. During the HBT, anxiogenic conditions of the animals are indicated by a decrease in head dipping activity, whereas an anxiolytic state is indicated by an increase in head dipping behavior [33]. We observed that when the MESF dose increased from 200 to 400 mg/kg, the number of head dips increased, revealing the extract's anxiolytic effect. e duration spent in the open arm of the EPM test further supports the extract's anxiolytic effects.
Generally, corticotrophin-releasing factor (CRF) is produced when the hypothalamic-pituitary-adrenal (HPA) axis is overstimulated, whereas dysfunction in this system can result in the display of depressive symptoms [34]. However, potent antidepressant management suppresses the stimulation of the HPA axis caused by stress, followed by the reinstallation of CRF's rational expression and function [35]. In our study, the FST and TST were conducted following MESF administration to inspect antidepressant-like activity and the underlying mechanism of depression.
ese two experiments involve very stressful environments in which the mice are subjected to a state of behavioral distress, manifested as immobility, in the absence of an antidepressant agent. e administration of MESF (200 and 400 mg/kg) to mice resulted in key behaviors, including reduced immobility in the FST and an increased tendency to struggle in the TST. is antidepressant activity of MESF might be due to the inhibition of monoamine reuptake or the stimulation of HPA axis.
Diarrhea is associated with diverse recognized mechanisms, including unstable intestinal motility (motility diarrhea), infected or inflated-induced mucosal damage (exudative diarrhea), and increased water and electrolyte output (secretory diarrhea) [36]. e nitric acid found in castor oil triggers the stimulation of diarrheal activity, whereas the ricinoleic acid in castor oil triggers diarrhea via a hypersecretory response and may represent the most potent castor oil constituent [37]. In the lumen of the intestine, the castor oil containing ricinoleic acid produces ricin oleate salts with Na + and K + , which then suppresses N + /K + ATPase activity and consequently enhances the penetrability of the intestinal epithelium and increases water and electrolyte secretion [38,39]. Both models for antidiarrheal activity showed the significant suppression of diarrhea activity in our study investigation. MESF (200 and 400 mg/kg) significantly suppressed defecation in the castor oil-induced diarrhea model (p < 0.001) and significantly inhibited intestinal motility in the charcoal-induced intestinal motility model (p < 0.001).
Molecular docking represents an important approach for defining ligand-target relations because it can demonstrate the conduct of minor molecules at the active sites of target proteins and can help researchers to better understand the mechanisms underlying several pharmacological reactions [22,40]. erefore, molecular docking was conducted to better recognize the molecular processes underlying these observed pharmacological reactions.
Our findings demonstrated that three key compounds, captan, 1-azuleneethanol, acetate, and tetraconazole, docked against the target enzymes for the described pharmacological actions. e findings of our docking investigation suggested that the investigated compounds may be responsible for MESF's biological actions (sedative, anxiolytic, antidepressant, and antidiarrheal) through interactions with the target proteins.
In addition, the ADME/T analysis demonstrated that captan, 1-azuleneethanol, acetate, and tetraconazole meet Lipinski's rule of five, and these compounds did not possess any toxic properties, including Ames toxicity, carcinogenicity, acute oral, or rat acute toxicity (Tables 3 and 4). Drugs or compounds that violate these rules are not considered to demonstrate good oral bioavailability [41,42].

Conclusions
In summary, various phytochemicals, which may induce synergistic or individual effects, are responsible for such pharmacological activities. ese chemical compounds may offer such pharmacological activities as the molecular docking study assessed. In addition, the in silico docking analysis indicated that captan, 1-azuleneethanol, acetate, and tetraconazole might represent suitable candidates for sedative, anxiolytic, antidepressant, and antidiarrheal activities. However, further widespread research remains necessary to inspect the complete array of biological reactions and mechanisms in animal models. Additional advanced studies remain to identify the potential compounds responsible for pharmacological activities.  Category-III (500 mg/kg < LD 50 < 5,000 mg/kg).