Deeper Insights on Cnesmone javanica Blume Leaves Extract: Chemical Profiles, Biological Attributes, Network Pharmacology and Molecular Docking

This study assessed the anxiolytic and antidepressant activities of a methanol leaves extract of Cnesmone javanica (CV) in Swiss albino mice. The study found a significant increase in the percentage of time spent in the open arms of an elevated plus maze and in the incidence of head dipping in hole-board tests following the administration of 400 mg/kg of CV or 1 mg/kg diazepam. Moreover, a significant (p < 0.001) dose-dependent reduction was observed in the immobility time following CV (200 and 400 mg/kg) and fluoxetine (20 mg/kg) administration for forced swimming and tail suspension tests. Gas chromatography–mass spectroscopy (GC–MS) analysis identified 62 compounds in CV, consisting primarily of phenols, terpenoids, esters, and other organic compounds. A molecular docking study was performed to assess the anxiolytic and antidepressant effects of 45 selected compounds against human serotonin transporter and potassium channels receptors. Network pharmacology was performed to predict the pathways involved in these neuropharmacological effects. Overall, CV demonstrated significant and dose-dependent anxiolytic and antidepressant effects due to the presence of several bioactive phytoconstituents, which should be further explored using more advanced and in-depth mechanistic research.


Introduction
Medical plant use has achieved growing interest in developing countries, representing first therapeutic approach in 80% of developing countries. A significant proportion of the world's population (87.5%) uses herbal remedies to treat health problems [1]. Many plants' medicinal properties, their effects on the human body, and their treatment methods have been known since the 18th century, but their active compounds were unknown [2].
Mental disorders represent a large public health threat, affecting nearly 450 million people globally, and representing 12.3% of all diseases worldwide [3,4]. Mental disorders are among the world's most alarming health problems, and the two most common mental

Effects of CV on Anxiolytic and Antidepressant Activity
Rodent exploratory behavior has recently stimulated the interest of researchers in several behavioral pharmacology studies [16]. The behavioral tests used in this study are known to measure anxiolytic and antidepressant activities, and most have been previously validated [17,18]. The EPM and HBT represent the two most commonly used methods for screening anxiolytic effects [19]. The elevated plus maze (EPM) exposes the animal to elevated open and closed arms, and time spend in the open arm is an indication of reduced anxiety [18,20]. In behavioral pharmacology, the hole-board test (HBT) is a common test of anxiolytic activity, in which the increased frequency of head dipping through the holes is considered an indication of reduced anxiety behavior in mice [16,21]. The tail suspension test (TST) and forced swimming test (FST) are the most commonly used methods to assess antidepressant-like activity in rodents [22]. The immobility behavior expressed by mice during these tests is considered to represent a depressive state [22,23]. Animal immobility induced by the FST and TST have been reported to model human depression and can be reversed with the application of antidepressant drugs [23]. The findings for the EPM, shown in Figure 2, demonstrated that the percentages of time spent in the open arms were 21.52% and 33.48% in mice treated with CV doses of 200 (p < 0.01) or 400 mg/kg (p < 0.001), respectively, which were significantly increased compared with that of the control group (11.25%). However, the intraperitoneal (IP) administration of the positive control diazepam (1 mg/kg) significantly (p < 0.001) increased the percentage of time spent in the open arm (69.33%). The results of the HBT, shown in Figure 3, indicated that the oral administration of CV at 400 mg/kg and the IP administration of 1 mg/kg diazepam significantly increased the frequency of head dipping (p < 0.01 and p < 0.001, respectively) compared with mice in the control group. However, no significant difference was identified between the control and 200 mg/kg CV treatment. The antidepressant effects of CV were assessed using the FST. As shown in Figure 4, the FST demonstrated that the immobility index of mice was reduced by 43.74%, 49.82%, and 80.53% at doses of 200 and 400 mg/kg CV and 20 mg/kg fluoxetine (p < 0.001), respectively, compared with that of the control group. A similar observation was found for the TST, as shown in Figure 5. Compared with the control group, CV (200 and 400 mg/kg) and fluoxetine (20 mg/kg) treatment significantly (p < 0.001) reduced the immobility index in the TST by 36.38%, 53.74%, and 82.02%, respectively.    Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter (NT) found in the central nervous system (CNS) [24]. GABA receptor dysfunction has been associated with an increased risk of both anxiety and depression in both pharmacological and genetic studies [25]. Recent studies have reported that the inactivation of the γ-2 subunit gene, which results in a partial decrease in GABA A receptor function in the mouse brain, resulted in an increased risk of anxiety and depressive behaviors in adults [24,26]. Similar to clinical outcomes in humans, mice with γ-2 subunit deficiencies showed reduced anxiety behaviors when treated with diazepam or with the chronic use of fluoxetine [24,26,27]. Furthermore, in antidepressant-sensitive tests, such as FST and TST, the γ-2 subunit-deficient mice also exhibited significantly improved depression-like responses [24,[27][28][29]. These studies indicate that the GABAergic deficiency that has been reported among patients with anxiety and depression may represent a causative factor for both disorders. Therefore, targeting the GABA system can provide a novel avenue for therapeutic strategies to treat both disorders, such as the use of selective GABA A receptor agonists [24]. In the tentative identification of phytoconstituents in CV, the GC-MS analysis revealed several compounds that could potentially act on the GABA system.

Molecular Docking
Molecular docking has become an excellent tool for the discovery of novel drugs based on structural molecular biology [30,31]. Molecular docking has proven to be a very efficient strategy for identifying small molecules that bind to protein targets, and the modeling of protein-ligand docking is often used by the pharmaceutical industry to screen potentially active compounds [32]. In our present study, the tentative compounds identified in CV were used for the molecular docking study, as presented in Table 2.
To examine antidepressant activity, a molecular docking simulation study was used to explore the interactions between selected compounds identified in CV and human serotonin receptor (PDB ID: 5I6X). The serotonin receptor has a significant dominance in depression, while 15 documented serotonin receptors were involved in depression-like behavior [37]. Interestingly, all of the phytochemicals identified in CV interacted with human serotonin receptor. Among these phytochemicals, some compounds possessed higher docking scores with the receptor than others. 2H-1-benzopyran-6-ol, 3,4-dihydro-2,8- Asn177 and hydrophobic interactions with Tyr95, Ala169, Ile172, Ala173, Tyr176, Phe335, Phe341, Gly442, Leu443, and Val501. 2H-1-benzopyran-6-ol, 3,4-dihydro-2,8-dimethyl-2-(4,8,12-trimethyltridecyl)-, [2R-[2R*(4R*,8R*)]]-exerted an even higher binding affinity than paroxetine (−8.978 kcal/mol), the positive control used in this study. Paroxetine interacted with the target receptor through the formation of hydrogen bonds with Tyr95 and Ala96, and hydrophobic bonds with Tyr95, Asp98, Ala169, Ile172, Ala173, Tyr176, Ser336, Phe341, Ser438, and Ser439 (Table 4 and Figure 7).    Additionally, network pharmacology was used to explore the interactions among the 14 best-docked compounds using the STITCH platform. Interactions were identified for β-tocopherol, γ-tocopherol, DL-α-tocopherol (vitamin E), campesterol, and β-sitosterol with several genes. In the KEGG analysis, six different pathways were identified as being directly correlated with the neuropharmacology of CV (Table 5 and Table 6, and the molecular functions are listed in Table 7. Future research remains necessary to analyze the compounds identified in the extract and to validate the existing results using individual biological models. (PRKACA, PRKCA), and Parkinson's disease (CASP3, PRKACA). The biological process associated with the identified phytoconstituents is presented in Table 6, and the molecular functions are listed in Table 7. Future research remains necessary to analyze the compounds identified in the extract and to validate the existing results using individual biological models.

Preparation and Optimization of the Methanol Extract
The leaves of C. javanica were collected from the Lohagara Hill area in Chittagong, Bangladesh, in February 2020, which was identified by a renowned taxonomist from Bangladesh Forest Research Institute, Chittagong, Bangladesh. The leaves were air-dried and ground into a coarse powder. The crude extraction procedure was optimized using an established procedure [38]. Briefly, at a ratio of 1:4, the powder was macerated in methanol for seven days, after which the extract was filtrated by Whatman filter paper (#1), and the filtrate was evaporated at 40-45 • C, resulting in a black, semisolid extract. The CV extract was stored at 4 • C until use.

GC-MS Analysis
An Agilent GC 7890A (Agilent Technologies Inc., Wilmington, DE, USA), combined with a triple-axis detector 5975 C single-quadrupole mass spectrometer, was used for GC-MS analysis. The chromatographic column was an Agilent HP 5MS column (30 m × 0.25 mm × 0.25 µm film thickness), with high-purity helium as the gas carrier, at a flow rate of 1 mL per min. The injector temperature was 230 • C, and the sample was injected using a splitless injector at 20:1. The temperature was set primarily to 40 • C (held for 1 min), and then raised to 150 • C at a rate of 5 • C per min (held for 2 min), before being increased to 300 • C at a rate of 5 • C per min (held for 10 min). The temperature of the MS ion source was set to 150 • C, and the temperature of the inlet line was set to 280 • C. The scan range was set between 50-550 mass ranges, with 70 eV electron energy and a 4-min solvent delay. Finally, by comparing the spectra against the NIST 2008 database (National Institute of Standard and Technology library), tentative compounds were identified. The total analysis time required for the sample was 65 min.

Animals and Experimental Design
Swiss albino mice (20-25 g), six weeks old and of either sex, were randomly used for all tests. Mice were acclimated to the laboratory conditions for seven days, at a temperature of 25 ± 2 • C with free access to food and water. The institutional animal ethical committee approved this study, which followed the ARRIVE guidelines, in accordance with government regulations [39,40].
The mice were randomly distributed into four groups (n = 5) for each experiment. Two doses (200 and 400 mg/kg) of CV were used in this study, which were administered orally through gavage. Diazepam (1 mg/kg) was administered intraperitoneally (IP) for the elevated plus maze (EPM) and hole-board test (HBT). Fluoxetine was also administrated intraperitoneally for the forced swimming test (FST) and tail suspension test (TST). As a negative control, 1% Tween 80 in water (10 mL/kg) was administered orally.

Elevated Plus Maze (EPM) Test
The EPM was performed to evaluate the anxiolytic-like activity of CV using a previously described method [6]. The apparatus consisted of two open arms and two closed arms (50 × 10 × 40 cm 3 ), made of wood. The apparatus was elevated from the surface of the floor at a height of 40 cm. The treatments administered to each group were described in The HBT apparatus was elevated at a height of 25 cm and consisted of 16 evenly distributed holes. The treatments administered to each group were described in Section 3.3 (Animals and Experimental Design). Each mouse was placed in the apparatus 30 min after treatment, and the number of times they dipped their heads in the holes was counted over a 5-min period [41].

Antidepressant Tests
The antidepressant effects of CV were assessed by the FST [42]. A transparent cylindrical glass apparatus (15 × 25 cm 2 ) containing freshwater (24 ± 2 • C) at a 10 cm depth was used for this experiment. The treatments administered to each group were described in Section 3.3 (Animals and Experimental Design). Each mouse was forced to swim in the apparatus for 6 min, 60 min after treatment. The last four min were considered the immobile period, whereas the first two minutes were considered the initial adjustment period. The results were reported as the percentage of inhibition, determined as follows: Immobile period of control group − Immobile period of test group Immobile period of control group (2)

Tail Suspension Test (TST)
The antidepressant activity of CV was evaluated using a method previously described by Steru et al. [43]. Each group of mice was treated as described in Section 3.3 (Animals and Experimental Design). Each mouse was suspended from the rim of the apparatus using adhesive tape placed on the tail tip (1 cm). The observations and calculations for the TST were performed as described for the FST.

Statistical Analysis
The differences between groups were (* p < 0.01 and ** p < 0.001) compared using one-way analysis of variance (ANOVA, followed by Dunnett's test) in GraphPad Prism version 8.4 (GraphPad Software, San Diego, CA, USA). The results are presented as the mean ± standard error of the mean (SEM).

Protein Preparation
Three-dimensional (3D) crystal structures of human serotonin transporter (PDB id: 5I6X) [44] and potassium channels receptor (PDB ID: 4UUJ) [45] were downloaded from the Protein Data Bank (www.rcsb.org/pdb) in pdb format. The Protein Preparation Wizard in Schrödinger-Maestro v10.1 was used to prepare and refine the structures. The other parameters were set to the system default. Minimization was performed using the force field OPLS 2005, with a root-mean-square deviation (RMSD) value of 0.30 Å.

Ligand Preparation
For the molecular docking analysis, we selected 45 isolated compounds from C. javanica based on the GC-MS analysis results. The targeted compounds were downloaded from the PubChem database in sdf format. Additionally, we used paroxetine (compound CID: 43815) bound to human serotonin transporter (PDB ID: 5I6X) and diazepam (compound CID: 3016) bound to potassium channels receptor (PDB ID: 4UUJ) as positive controls for this study. The selected compounds' three-dimensional (3D) structures were generated using Ligprep in Schrödinger suite 2015 and the OPLS_2005 force field. Using Epik 2.2 in the Schrödinger suite, we generated the ionization states of the compounds at pH 7.0 ± 2.0. For each ligand, up to 32 stereoisomers were retained.

Receptor Grid Generation and Molecular Docking
Glide (Schrödinger Suite-Maestro version 10.1) (Schrödinger, LLC, New York, NY, USA), was used to generate receptor grids and to run molecular docking simulations [46,47]. In glide, the grids were produced using the default system and the OPLS_2005 force field. The receptor was given a cubic box of a particular size, oriented on the center of the active site residues, and a bounding box (14 Å × 14 Å × 14 Å). Glide's standard precision (SP) scoring function was used to conduct all docking experiments, and the lowest glide score was recorded for each ligand.

Network Pharmacology and Pathway Analysis
The network pharmacology analysis was assessed using the STITCH platform [48,49]; multiple compound targets were analyzed by considering the 14 best-docked compounds identified from C. javanica leaves. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was utilized to identify pathways and genes involved in neuropharmacology. Moreover, the biological processes and molecular functions of these compounds were also analyzed using the STITCH platform.

Conclusions
Our findings demonstrated that in several animal models, CV had significant and dose-dependent anxiolytic and antidepressant effects, likely due to the presence of several bioactive phytoconstituents, which were identified by GC-MS analysis. The molecular docking study between the tentatively identified compounds and two receptors associated with anxiety and depression indicated good interaction scores between these two receptors and several of the identified compounds. Overall, these findings provide valuable preliminary outcomes regarding the potential anxiolytic and antidepressant activity of C. javanica, which required future advanced and in-depth mechanistic studies to verify the potential therapeutic applications of these bioactive compounds.