Chemical and Pharmacological Profiling of Wrightia coccinea (Roxb. Ex Hornem.) Sims Focusing Antioxidant, Cytotoxic, Antidiarrheal, Hypoglycemic, and Analgesic Properties

The aim of the study was to conduct phytochemical and pharmacological investigations of Wrightia coccinea (Roxb. ex Hornem.) Sims via several in vitro, in vivo, and in silico models. A total of four compounds were identified and isolated from the methanol extract of the bark and the methanol extract of the seed pulp of W. coccinea through successive chromatographic techniques and were characterized as 3β-acetyloxy-olean-12-en-28-ol (1), wrightiadione (2), 22β-hydroxylupeol (3), and β-sitosterol (4) by spectroscopic analysis. The aqueous fraction of the bark and chloroform fraction of the fruits provided the most potent antioxidant capacity (IC50 = 7.22 and 4.5 µg/mL, respectively) in DPPH free radical scavenging assay compared with the standard ascorbic acid (IC50 = 17.45 µg/mL). The methanol bark extract and the methanol fruit coat extract exerted anti-diarrheal activity by inhibiting 74.55 ± 0.67% and 77.78 ± 1.5% (mean ± SEM) of the diarrheal episode in mice, respectively, after four hours of loading the samples. In the hypoglycemic test, the methanol bark extract and the methanol fruit coat extract (400 mg/kg) produced a significant (p < 0.05) reduction in the blood glucose level in mice. Both doses of the plant extracts (200 mg/kg and 400 mg/kg) used in the study induced a significant (p < 0.05) increase in pain reaction time. The in vitro and in vivo findings were supported by the computational studies. The isolated compounds exhibited higher binding affinity compared with the standard drugs towards the active binding sites of glutathione reductase, epidermal growth factor receptor (EGFR), kappa opioid receptor, glucose transporter 3 (GLUT 3), Mu opioid receptor, and cyclooxygenase 2 (COX-2) proteins due to their potent antioxidant, cytotoxic, anti-diarrheal, hypoglycemic, and central and peripheral analgesic properties, respectively. The current findings concluded that W. coccinea might be a potential natural source for managing oxidative stress, diarrhea, hyperglycemia, and pain. Further studies are warranted for extensively phytochemical screening and establishing exact mechanisms of action.


Extraction and Partitioning Process
The cleaned bark and fruit coat of the plant were sun-dried for 14 days while the seeds contained in the fruit generated cotton-like pulp. The dried samples were ground separately, and the pulverized samples of the bark, fruit coat, and seed pulp were extracted using Soxhlet apparatus [22]. The finely ground materials were packed in a 'thimble' made with filter paper and placed in a thimble chamber. The solvent (methanol) placed in the bottom chamber of the Soxhlet apparatus was then vaporized and allowed to be condensed and dripped on the thimble. The condensed solvent was collected in the bottom chamber containing the extracted compounds from the powdered material residing in the porous bag. Three different samples (bark, fruit coat, and seed pulp) were subjected to the process separately. The filtrate was condensed at reduced temperature and pressure using a rotary evaporator (Heidolph, Germany). The resulting % yield of crude methanol extract of the bark, fruit coat, and seed pulp was 17.83%, 15.38%, and 16.93%, respectively. Vacuum liquid chromatography (VLC) was employed for the fractionation of the plant extracts [23]. The column was packed with VLC grade silica and was washed with petroleum ether to ensure the compact packing. Approximately 15 g extracts of the bark and seed pulp were prepared by dissolving them into methanol and were mixed with silica, then dried. The dried sample of the bark extract (MEB) and seed pulp extract (MES) were applied separately to the top of the column, and elution was commenced starting with petroleum ether. The polarity of the eluting solvent was gradually increased by adding more polar solvents including ethyl acetate and methanol (Table S1). The fractions 4-8 of VLC run were mixed together due to their identical characteristics and subjected to preparative TLC (stationary phase-silica gel PF 254 , mobile phase-ethyl acetate: petroleum ether = 5:95). The fractions of SEC were also mixed together due to their identical characteristics and subjected to preparative TLC. These compounds along with their sample ID are stated in Table S2.
Both MEB and MEF were subjected to modified Kupchan partitioning into petroleum ether (PE), dichloromethane (DCM), chloroform (CF), and aqueous (AQ) soluble fractions [24]. All the plant samples were subjected to pharmacological evaluation.

Isolation of Chemical Compounds
The selected VLC fractions of the bark methanol extract were analyzed by gel permeation chromatography over lipophilic Sephadex LH-20 and PTLC over silica gel (F 254 ). Following TLC screening of the chromatographic fractions and the subsequent PTLC analysis of the fractions, compounds 1-3 were isolated from the bark methanol extract (Figure 1). The chromatographic column separation of the seed pulp methanol extract and the subsequent PTLC of column fractions using ethyl acetate and toluene yielded compound 4 ( Figure 1). PTLC was performed over silica gel 60 F254 coated with glass plates, and 1% vanillin-sulfuric acid reagents were used to detect compounds. The NMR spectra of the isolated compounds were recorded in CDCl 3 on Bruker 400 NMR machine.

In Vitro Antioxidant Activity: DPPH Assay
DPPH assay was used to estimate the antioxidant potential of a variety of plant samples using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical [25][26][27]. It is noted that 2.0 mL solution of the plant sample at different concentrations (500.0 to 0.977 µg/mL) was mixed with 3.0 mL of DPPH solution in methanol, rendering the concentration of DPPH working solution of 20 µg/mL [28,29]. After 30 min incubation in the dark, the absorbance of each reaction mixture was recorded at 517 nm using a UV-visible spectrophotometer. The DPPH radical quenching capacity of W. coccinea was measured according to the following equation: where A = absorbance for each group at 517 nm. The IC50 value (50% inhibition) for each tested sample was calculated from a plot of % inhibition of DPPH free radical vs. concentration (µg/mL) of the test materials.

In Vitro Cytotoxicity
Cytotoxic activity of the bark and fruit coat extracts was performed using brine shrimp lethality assay with vincristine sulfate (VS) as the positive standard [29,30]. A serial dilution of each tested sample (4 mg) in 99% DMSO (dimethyl sulfoxide) was prepared to obtain variable concentrations (400.0-0.781 µg/mL) of the tested solutions. Simulated seawater containing approximately ten live brine shrimp nauplii was added to each concentration of the tested solutions. After 24 h, the surviving nauplii were inspected by visual inspection with the help of a magnifying glass. For the individual concentration of the tested sample, the level of toxicity towards the shrimp was estimated by determining the LC50 value. LC50 value of the tested sample was determined from a plot of percentage of non-viable shrimps against the log concentration of plant extract using the standard curve of vincristine.

In Vitro Antioxidant Activity: DPPH Assay
DPPH assay was used to estimate the antioxidant potential of a variety of plant samples using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a free radical [25][26][27]. It is noted that 2.0 mL solution of the plant sample at different concentrations (500.0 to 0.977 µg/mL) was mixed with 3.0 mL of DPPH solution in methanol, rendering the concentration of DPPH working solution of 20 µg/mL [28,29]. After 30 min incubation in the dark, the absorbance of each reaction mixture was recorded at 517 nm using a UV-visible spectrophotometer. The DPPH radical quenching capacity of W. coccinea was measured according to the following equation: where A = absorbance for each group at 517 nm. The IC 50 value (50% inhibition) for each tested sample was calculated from a plot of % inhibition of DPPH free radical vs. concentration (µg/mL) of the test materials.

In Vitro Cytotoxicity
Cytotoxic activity of the bark and fruit coat extracts was performed using brine shrimp lethality assay with vincristine sulfate (VS) as the positive standard [29,30]. A serial dilution of each tested sample (4 mg) in 99% DMSO (dimethyl sulfoxide) was prepared to obtain variable concentrations (400.0-0.781 µg/mL) of the tested solutions. Simulated seawater containing approximately ten live brine shrimp nauplii was added to each concentration of the tested solutions. After 24 h, the surviving nauplii were inspected by visual inspection with the help of a magnifying glass. For the individual concentration of the tested sample, the level of toxicity towards the shrimp was estimated by determining the LC 50 value. LC 50 value of the tested sample was determined from a plot of percentage of non-viable shrimps against the log concentration of plant extract using the standard curve of vincristine.

Experimental Animals
Swiss-albino mice were used in the study. The mice of both sexes were collected from the Animal Resource Branch of the International Centre for Diarrheal Diseases and Research, Bangladesh (ICDDR,B). The mice weighed between 20-25 g and aged 4-5 weeks during the experiment. The standard polypropylene cages were utilized during housing the mice, and the temperature was controlled at 24 ± 2 • C with relative humidity 60-70% in the animal house, where a 12 h light-dark cycle was maintained. Because of the high susceptibility of the animals to environmental variation, all the acquired mice were kept in the laboratory environment for at least 3-4 days for acclimation. Standard guidelines for the care and use of laboratory animals were adopted while conducting the experiments. The Institutional Ethical Review Committee (SUB-IERC), State University of Bangladesh, has critically reviewed and approved the ethical issues. All detailed procedures and protocols of the study were approved, and an approval number (2018-08-13-SUB/A-ERC/007) was provided.

Preparation of the Loading Doses for In Vivo Study
For the in vivo model, the tested animals were given bark extract and fruit coat extract at doses of 200 and 400 mg/kg body weight. To prepare these loading doses, the accurately weighed samples were measured (24 mg on average for 200 mg/kg and 48 mg on average for 400 mg/kg) and were triturated in a unidirectional technique by mixing a small amount of 1% Tween 80 in normal saline. The volume of the suspension was made up to 3.0 mL.

In Vivo Study Design
Mice were divided into the following groups, with four mice in each group. The negative and positive control group received 1% Tween 80 in normal saline (10 mL/kg) and standard drug, respectively. Group I and II were given 200 and 400 mg/kg of methanol extract of the bark, while Group III and IV received methanol extract of the fruit coat at 200 and 400 mg/kg, respectively.

Anti-Diarrheal Assay
The bark and fruit coat extracts were subjected to castor oil-induced diarrhea model in mice [31,32]. The positive control group received the standard loperamide and the negative control group received only 1% Tween 80 solutions. After half an hour of administering the sample and standard doses, 0.5 mL castor oil was administered to each animal orally with the help of a feeding needle. Each animal was separated into an individual cage. The floor was covered with a blotting paper that can hold clear stains of feces-they were observed for 4 h to monitor the anti-diarrheal effect of the tested samples. Data were collected every hour after castor oil administration. The percentage of inhibition of the defecation by the plant extracts was determined using the following formula where D = mean number of diarrheal episodes in each group.

Hypoglycemic Assay
The blood-glucose-lowering effect of the plant extract (200 and 400 mg/kg) was evaluated by oral glucose tolerance test according to the method described by Peungvicha et al. [33]. Six subgroups of animals were given sugar syrup orally with the help of a feeding needle. After 5 min, glibenclamide, water, 24 mg tested sample, and 48 mg tested sample were given to each animal of the positive control, negative control, Group-I, Group-II, Group-III, and Group-IV, respectively. Blood sugar was checked with diabetic strips every half an hour for 3 h. The % reduction in blood glucose level was calculated by the equation below: where BG = average blood glucose level for each group.

Central Analgesic Activity
Tail immersion assay is a thermal process that was employed to assess the central analgesic activity of W. coccinea extracts [34]. The standard morphine (2 mg/kg, subcutaneous) solution was prepared by diluting the supplied morphine (15 mg/mL) with saline water [26]. The tested materials were orally administered utilizing a feeding needle to the mice. For the test, the mouse tail was immersed in hot water at 55 • C. The pain reaction time (PRT) or latency period for each mouse to flick its tail from the warm water was measured before (0 min) and at 0, 30, 60, and 90 min following the loading of the tested samples.

Peripheral Analgesic Activity
Acetic acid-induced writhing method was employed to assess the peripheral analgesic activity of W. coccinea extracts [35]. The acetic acid-induced writhing method was applied to investigate the activity of the crude extract. Glacial acetic acid was used as a pain inducer to the animals of each subgroup. After an oral administration of the acid, aspirin and 24 and 48 mg sample was given to the positive control group, Group-I, Group-II, Group-III, and Group-IV, respectively, whereas the negative control group received Tween 80 solution alongside acetic acid. Writhing happens because of acetic acid. The number of writhes was measured for ten min after intraperitoneal injection of acetic acid. The percent inhibition of writhing was determined as follows where N = mean number of abdominal writhing for each group.

Molecular Docking Study
Molecular docking analysis was performed to interpret the interactive profile of the four isolated compounds from W. coccinea with their target proteins. The widely used popular software packages, including PyRx, PyMoL 2.3, and BIOVA Discovery Studio version 4.5, were utilized during for the in silico study of the isolated compounds from W. coccinea according to the semiflexible procedures described in several studies [36][37][38][39][40][41].

Target Protein Selection
Computational docking was conducted to reveal the potential bioactivities of the identified compounds including antioxidant, cytotoxicity, antidiarrheal, hypoglycemic, and analgesic properties. To run the molecular interaction and determine the radical scavenging capacity, cytotoxicity, antidiarrheal, hypoglycemic, and central and peripheral analgesic properties, glutathione reductase (PDB ID: 3GRS), epidermal growth factor receptor (PDB ID: 1XKK), kappa opioid receptor (PDB ID: 6VI4), glucose transporter 3 [GLUT 3] (PDB ID: 4ZWB), Mu-opioid receptor (PDB ID: 5C1M), and cyclooxygenase 2 (COX-2) (PDB ID: 1CX2) proteins were selected based on the biochemical mechanisms and the current evidence [36,[42][43][44][45][46]. The targeted proteins' three-dimensional (3D) crystal structures were retrieved from the RCSB protein data bank (https://www.rcsb.org; accessed on 1 January 2022) and were saved in PDB format. All collected biomolecules were opened in the PyMoL 2.3 software to delete water molecules and any unwanted residue from the proteins. Then the cleaned proteins were arranged by adding non-polar hydrogen atoms and were converted into the lowest energy state by applying Swiss PDB viewer for an energy minimization program. Finally, the cleaned and optimized proteins were saved in PDB format for further analysis.

Ligand Protein Interaction
Molecular docking was conducted to assume the potential binding profiles of the isolated phytoconstituents with their binding affinities towards the target macromolecules [47]. A widely used advanced software PyRxAutoDock Vina was applied for the drug-protein interaction, where a semiflexible modeling approach was adopted during the computer-aided docking process. The targeted protein was loaded and was selected as a macromolecule. The literature-based amino acids with their three-letter IDs were chosen for ascertaining the site-specific ligand-protein interaction. A total of eleven amino acids, VAL 102, LYS 127, ASN 129, VAL 130, GLN 131, LYS 143, SER 145, SER 147, GLY 148, ASP 183, and THR 185 were selected in glutathione reductase enzyme (PDB ID: 3GRS) for conducting active site-specific docking to predict the antioxidant effects of these isolated phytoconstituents [37].  [43]. The active sites of the GLUT3 (PDB ID: 4ZWB), Mu-opioid receptor (PDB ID: 5C1M), and cyclooxygenase 2 (COX-2) (PDB ID: 1CX2) proteins were selected based on the literature [44][45][46]. All the 3D conformers of the ligands (SDF format) were imported into the PyRx software and were run for the energy minimization of the ligands. All ligands were converted into pdbqt format in the PyRxAutoDock Vina software by utilizing Open Bable tool to equip the most suitable optimal hit. Then the grid box originated and the active binding sites of the proteins were kept within the center of the box, where the grid box mapping was as for center (X, Y, Z): (15.

Statistical Analysis
Experimental data found from the in vitro and in vivo assays were reported as mean ± standard error of mean (SEM), whenever possible. Data were evaluated by Student's t-test using GraphPad Software, USA. p values less than 0.05 were considered statistically significant. The molecular docking was conducted in triplicate, and the mean docking scores were presented in which the standard errors for all docking times were less than 1%.

Phytochemical Studies
We isolated four compounds including 3β-acetyloxy-olean-12-en-28-ol, wrightiadione, 22β-hydroxylupeol, and β-sitosterol ( Figure 1). The structures of these compounds were determined and were confirmed comparing their NMR spectra with the reported data for similar compounds. 20. An extra oxymethine signal was also present, indicating that compound 3 is a hydroxylated lupeol derivative [50]. The spectroscopic data were identical to the published values [51]. The 1 H NMR (400 MHz, CDCl 3 ) of compound 4 ( Figure 1) agreed with the published report of β-sitosterol and the spectrum was superimposable to the 1 H NMR spectrum acquired for an actual sample formerly isolated in the laboratory [51]. The 1 H NMR spectrum of 4 was provided in the Supplementary Materials ( Figure S6).

DPPH Free Radical Scavenging Activity
The tested samples showed quenching activities against DPPH radical in a concentrationdependent manner in the antioxidant assay. The IC 50 values (50% inhibition) of the DPPH radical quenching capacity of the plant extracts are presented in Table 1. The smaller the IC 50 values, and the higher the antioxidant potential of the tested sample. Among all tested samples, the chloroform fraction of the fruit coat extract and the aqueous fraction and dichloromethane fraction of the bark extract was the most potent DPPH radical scavenger, with IC 50 values of 4.55, 7.22, and 10.91 µg/mL, respectively, in comparison with BHT (4.3 µg/mL) as the positive control. From the analysis of Table 1, we can conclude that the scavenging effect shown by different solvent fractions of the bark extracts on DPPH radicals were more potent than the fruit coat extracts of W. coccinea.

Cytotoxic Activity
All the tested samples exhibited significant brine shrimp larvicidal activity in terms of LD 50 , which was comparable to the standard vincristine sulphate (LC 50 = 0.451 µg/mL) ( Table 1). The petroleum ether fraction of the fruit coat extract and the bark extract of W. coccinea displayed the maximum larvicidal activity with lethality concentrations (LC 50 ) of 10.67 and 32.65 µg/mL, respectively. The second highest cytotoxic extracts were the dichloromethane and chloroform fraction of the bark extract and fruit coat extract of W. coccinea.

Anti-Diarrheal Property
The tested fractions and standard loperamide demonstrated a significant (p < 0.05) and dose-dependent anti-diarrheal property in the animal study (Table 2). Castor oil-induced diarrhea persisted up to 4 h in the control group. This effect was diminished by loperamide (80.40 ± 0.61%) and by both bark extract and fruit coat extract of W. coccinea at dose 400 mg/kg with the maximum inhibitory effect of 74.55 ± 0.67% and 77.78 ± 1.5%, respectively.

Hypoglycemic Property
The hypoglycemic effects of the bark extract and fruit coat extract of W. coccinea in mice were summarized in Table 3. The tested samples of W. coccinea demonstrated significant (p < 0.05) and concentration-dependent glucose-lowering activity, which continued up to three hours following the loading dose. Both the bark extract and fruit coat extract exhibited promising hypoglycemic effects (74.7 ± 0.19% and 70.6 ± 0.30%, respectively) after 3 h while the standard glibenclamide produced 66.7 ± 0.61% reduction.

Central Analgesic Activity
The results of the central analgesic effect of the bark extract and fruit coat extract in the tail immersion method are shown in Table 4. All the tested samples showed a potent (p < 0.05) increase in the pain reaction time in comparison with the reference drug morphine (Table 4). Both the bark extract and fruit coat extract at a dose of 400 mg/kg body weight caused a significant increase in PRT, up to 8.50 ± 0.28 and 8.57 ± 0.19, respectively, compared with the standard morphine (12.06 ± 0.53) at 90 min after loading the plant sample. These extracts exhibited a potential analgesic effect in the experimental mice.

Peripheral Analgesic Activity
The results of the peripheral analgesic effect of the bark extract and fruit coat extract (200 and 400 mg/kg body weight) in mice are summarized in Table 5. The plant extracts showed noticeable (p < 0.05) and concentration-dependent activity in reducing acetic acidinduced abdominal writhing in mice. The bark extract and fruit coat extract at the 400 mg/kg body weight dose exhibited 66.07 ± 0.88% and 54.39 ± 1.20% writhing inhibition in mice, respectively, compared with the standard diclofenac sodium (76.79 ± 0.33% inhibition).

In Silico Study
To understand the pharmacological activities of the extracts and different solvent fractions prepared from W. coccinea, molecular docking of the plant's derived compounds against the corresponding molecular receptors was conducted by applying several suitable computer-based tools. All the docking scores collected from PyRx were tabulated in Table 6. The amino acid responsible for the interactions with the atom of the ligands, including the bond distance, bond type, and nature of the interaction, were tabulated in Tables S1-S6 (Supplementary Materials). The lower the binding affinity (kcal/mol), the higher the binding strength. The extrapolated binding affinity having a null RMSD (root mean square deviation) value indicated the best docking prediction [37]. The enzyme/receptor inhibitory capacity of these isolated compounds was described as follows.

Inhibition of Glutathione Reductase Enzyme: Antioxidant Activity
Glutathione reductase enzyme is associated with the regulation, modulation, and maintenance of redox homeostasis and oxidative stress [52]. All isolated compounds exerted good binding affinity (−8.4 to −9.3 kcal/mol; Table 6) towards the glutathione reductase enzyme compared with the standard antioxidant drug butylated hydroxytoluene (BHT) (−5.8 kcal/mol). The binding affinity order of the compounds were 22β-hydroxylupeol (compound 3) > 3β-acetyloxyolean-12-en-28-ol (compound 1) > wrightiadione (compound 2) = β-sitosterol (compound 4) > BHT. The active binding sites of the glutathione reductase enzyme while interacting with the isolated compounds are summarized in Figure 2. A total of 13 hydrophobic interactions were noticed during the molecular docking of 22β-hydroxylupeol, where the number of alkyl and pi-alkyl interaction was eight and five, respectively. All the bonding sites including their corresponding distances were stated in   (Table S3). All the active binding sites of the compounds 3, 4, and standard drug BHT are summarized in Figure 2.    4) 3β-acetyloxy-olean-12en-28-ol, Wrightiadione, 22β-hydroxylupeol, β-sitosterol, and the standard BHT, respectively).

Inhibition of GLUT 3: Hypoglycemic Activity
The molecular docking scores of the four compounds (1-4) were found as −8.6, −9.5, −8.6, and −9.4 kcal/mol, respectively, in the interaction with the GLUT ASN 413 in A chain were found as the active binding sites while interacting with compound 2 (wrightiadione) which showed the most hypoglycemic property among the isolated phytoconstituents. All the active binding sites of the protein during docking with the reported compounds are summarized in Figure 5.

Inhibition of Mu-Opioid Receptor and COX-2 Proteins: Analgesic Activity
Molecular docking of the isolated compounds with the Mu-opioid receptor and COX-2 protein was performed to reveal the molecular mechanism of analgesic activity of the W. coccinea extracts. All compounds exerted higher binding affinity towards COX 2 enzyme (−8.5 to −9.6 kcal/mol) compared with the standard analgesic agent, diclofenac (−7.0 kcal/mol). The order of the docking scores of the compounds against COX-2 proteins were β-sitosterol > wrightiadione > 3β-acetyloxy-olean-12-en-28-ol > 22β-hydroxylupeol > diclofenac (Table 6). Wrightiadione and β-sitosterol showed interaction with Mu-opioid receptor (−9.1 kcal/mol and −9.7 kcal/mol, respectively) than the standard drug mor-   Figure 4.    ASN 413 in A chain were found as the active binding sites while interacting with compound 2 (wrightiadione) which showed the most hypoglycemic property among the isolated phytoconstituents. All the active binding sites of the protein during docking with the reported compounds are summarized in Figure 5.

Discussion
Medicinal plants are gifted sources of bioactive phytochemicals, which possess various pharmacological properties. The isolation and characterization of phytochemicals is an established way of ascertaining the bioactive profile of a medicinal plant. In phytochemical analysis, various chromatographic techniques such as CC, TLC, HPLC, GC, and PTLC can be used to isolate and purify bioactive compounds from plant extracts. In our study, we extracted W. coccinea with methanol and followed gel permeation chromatographic technique, TLC screening, and subsequent PTLC analysis. Several pure compounds (Figure 1) were obtained from the bark and seed pulp extracts of W. coccinea.
Several pharmacological activities of the plant extracts were evaluated to search for new therapeutic potentials of the plant and validate its folk medicinal use. The antioxidant property of W. coccinea was investigated in the search for safe and effective antioxidant candidates from natural resources. The antioxidant capacity of this plant species was assayed in terms of estimation the total phenol content and the DPPH radical quenching activity. W. coccinea bark and fruit coat extracts exhibited promising antioxidant potential compared with the reference antioxidant BHT and ascorbic acid. A moderate level of the phenolic content was also observed in different solvent fractions of the bark and fruit coat extracts. The plant-derived phenolic compounds serve as antioxidants through many possible pathways [56][57][58][59][60]. The hydroxyl group of the polyphenols scavenge reactive free radicals and thereby protect the biological system from free radical-induced oxidative stress [61]. All isolated compounds revealed higher affinities towards the glutathione reductase enzyme than the standard compound BHT. Among these molecules, 22β-hydroxylupeol showed the maximum free radical scavenging property in the molecular docking study. The compound exerted direct DPPH scavenging and membrane permeability, revealing strong antioxidant properties [62]. All the hydrophobic interactions of the compound with the glutathione reductase enzyme through alkyl and pi-alkyl interactions might be responsible for such actions. Sudhahar et al. [63] demonstrated that lupeol decreased lipid peroxidation (LPO) and showed enzymatic and nonenzymatic antioxidant capacity in animal models by reducing oxidative cellular damage. This might be due to the higher hydrophobic interactions with the glutathione reductase enzyme (Table S3). β-Sitosterol is also responsible for minimizing the detrimental effect of free radicals like peroxynitrite and inhibiting the LPO and NO generation [64,65].
As toxicity is a major concern for a crude drug, brine shrimp (Artemia salina) lethality bioassay, a cost-effective and reliable technique for preliminary screening of cytotoxicity, was conducted to predict the toxicity of the plant extracts [66]. According to the study performed by Logarto Parra et al. [67], there is a good correlation (r = 0.85; p < 0.05) between the 50% lethal concentration (LC 50 ) found in brine shrimp assay and 50% lethal dose (LD 50 ) obtained from an animal study, suggesting the brine shrimp test can be used as an alternative model. Meyer et al. [68] reported that the cutoff point for bioactive phytochemicals is LC 50 value less than 1000 µg/mL. The current study revealed that the measured LC 50 values in the brine shrimp lethality bioassay were below 1000 µg/mL. No crude extract and fraction derived from the plant might be considered severely toxic or lethal, endorsed by the findings of the previous studies (LC 50 > 10 µg/mL) [66,69]. All the isolated compounds from the plant were molecularly docked with the EGFR protein, which has a significant role in cellular signal transduction and cell survival [70]. The overproduction of EGFR is strongly associated with several types of cancer progression like ovarian cancer, breast cancer, and colon cancer [71]. Thus, EGFR inhibition might be an exciting target for developing selective anticancer molecules [42,72]. All the isolated phytoconstituents exerted more potent cytotoxic potential and safety than the standard vincristine. All the plant-derived compounds exhibited 1 to 3 conventional H-bond formation (except β-sitosterol) and 11 to 20 hydrophobic interactions and showed better fitting to the active sites of EGFR (Table S4). Notably, compound 2 (wrightiadione) exhibited the highest potent EGFR protein inhibition (binding affinity = −9.4 kcal/mol), exerted as a promising candidate for anticancer drug development. The abundance of hydrophobic interactions of the compound with the protein through pi-alkyl and pi-sigma might be responsible for the actions. Recently, scientists have reported that wrightidione and its derivatives showed promising anticancer potentillas through inhibiting tropomyosin-related kinases (Trks) [73,74].
Since ancient times, medicinal plants have been used to manage several gastrointestinal (GI) related disorders, including diarrhea. However, the safety and efficacy profiles of the major part of these plants have not been investigated. Therefore, this study was designed to appraise the safety and efficiency of W. coccinea as an anti-diarrheal potential option in the management of diarrheal illness in mice. Ricinoleic acid, the chief constituent of castor oil, is reported to irritate the gut wall, which can bring out peristaltic movement and finally induces diarrhea [75]. The both crude extracts prepared from the bark and fruit coat of W. coccinea showed notable antidiarrheal properties in a dose-dependent manner. In the case of fruit coat methanolic extract, the lower dose extract showed no significant antidiarrheal effect because the lower dose had not enough capacity to prolong the onset of diarrhea [75]. This study demonstrated that the antidiarrheal activity of the bark and fruit coat extracts of W. coccinea might be attributed to the bioactive phytochemicals responsible for the antidiarrheal effect [76]. All compounds isolated from the plant extracts showed comparable binding affinity towards kappa opioid receptor (−7.0 to −7.7 kcal/mol), supporting the antidiarrheal effect of the plant extracts. β-Sitosterol, an isolated secondary metabolite from W. coccinea, might play a significant role in reducing the peristalsis in the GI system and thus prevents GI motility [75,77].
Diabetes mellitus, a metabolic disorder, is now prevailing as a universal public health problem. Therefore, the demand for safe and effective antidiabetic molecules from natural sources intensifies day by day. Due to the low toxicity, affordable price, and availability of plants, herbal medicines can be widely used to manage hyperglycemia. Several reports were published explaining the blood-glucose-lowering effect of the plant extracts. Therefore, the present work is concerned with the preliminary assessment of the hypoglycemic effect of W. coccinea in animals. Our study revealed that the soluble fractions of the bark and fruit coat extracts of W. coccinea exhibited significant lowering of blood glucose after glucoseinduced hyperglycemia in mice. Therefore, the significant hypoglycemic effect of W. coccinea produced in this analysis could be attributed to the presence of phytochemicals [78]. All the isolated compounds exhibited a notable binding affinity towards GLUT 3 in the computational docking study. This is a novel investigation of the plant extract and thus there are no previous data regarding the hypoglycemic property of the plant. However, the petroleum ether extract (400 mg/kg) of W. tinctoria L. showed almost 75% glucose-lowering capacity after 14 days in alloxan-induced diabetic rats [79]. The secondary metabolites, β-sitosterol and lupeol or its derivatives, isolated from the W. coccinea, showed a significant antidiabetic activity in several studies [80,81]. Many studies reported that oxidative stress is directly associated with the etiology and pathogenesis of several disorders like cancer and diabetes mellitus [82,83]. As all the compounds showed higher binding affinity (−8.4 to −9.3 kcal/mol) towards the glutathione reductase enzyme than the standard BHT (−5.8 kcal/mol) and comparable inhibition property of glucose transporter 3 (GLUT 3) (−8.6 to 9.4 kcal/mol), these four compounds (compounds 1 to 4) could be potentially considered the responsible molecules isolated from W. coccinea having glucose-lowering property [64,84].
Pain-relieving agents are being searched from natural sources as alternatives to synthetic drugs because they have fewer adverse effects [85]. In our study, the analgesic activity of methanol extract of the bark and methanol extracts of the fruit coat was evaluated by tail immersion method and writhing method in mice. In our experimental condition, the plant samples significantly reduced both heat-induced and acetic acid-induced pain sensation in mice. Our study suggested that the bark and fruit coat extracts of W. coccinea may contain phytochemicals that can reduce pain sensation by inhibiting prostaglandin synthesis [86,87]. Furthermore, the compounds found in the plant extract exhibited more potent affinities towards the COX 2 protein than the standard drug diclofenac in the computational study supporting the in vivo findings. Furthermore, the β-sitosterol and wrightiadione exerted higher binding affinity than the standard morphine through conventional hydrogen bond and hydrophobic interactions. Nirmal et al. [88] reported that the isolated compound β-sitosterol exhibited analgesic activity in hot plate and acetic acid-induced assays by inhibiting central opioid receptors or facilitating the discharge of endogenous opioid peptides and blocking the production of prostaglandins and bradykinins.

Conclusions
The phytochemical analysis of bark extract and seed pulp extract of W. coccinea afforded four compounds characterized as 3β-acetyloxy-olean-12-en-28-ol, wrightiadione, 22β-hydroxylupeol, and β-sitosterol by spectroscopic techniques. The isolated plant metabolites may be functional for medicinal purposes. In biological screening, the chloroform soluble fraction of fruit coat extract of W. coccinea showed maximum DPPH radical quenching capability as an antioxidant agent with an IC 50 value of 4.55 µg/mL compared to the standard ascorbic acid (IC 50 = 17.45 µg/mL). The in vivo assay results demonstrated that the bark extract and fruit coat extract of the plant species possess outstanding antidiarrheal and analgesic properties. The bark extract and fruit coat extract exerted promising glucose-lowering capacity (74.7 ± 0.19% and 70.6 ± 0.30% of blood glucose level reduction, respectively) after three hours, while the standard drug glibenclamide produced 66.7 ± 0.61% of blood glucose level reduction. Furthermore, the in-silico investigations supported the in vitro and in vivo results as most of the isolated compounds exhibited promising binding affinities towards the corresponding receptors. Thus, the present pharmacological study unveiled the effectiveness of W. coccinea as a hopeful source of potential bioactive molecules that may be considered for novel drug discovery and therapeutic progress. Nonetheless, further studies are imperative to discover the bioactive compounds from W. coccinea extracts accountable for these bioactivities.

Informed Consent Statement: Not applicable.
Data Availability Statement: All the raw data of this research can be obtained from the corresponding authors upon reasonable request.