Integrating In Silico and In Vitro Approaches to Screen the Antidiabetic Properties from Tabernaemontana divaricata (Jasmine) Flowers

The purpose of this study was to assess different in vitro biological activities such as phytochemical constituents, enzymatic antioxidant status, cytotoxicity through hemolytic activity, and antidiabetic potential of plant methanolic extract through glucose uptake by yeast cells. Further, using in silico approach by the SwissADME technique the drug-likeness rules for bioactive components were characterized, while potential interactions were identified via molecular docking of a ligand with target proteins by GOLD 5.3.0. The results showed that T. divaricata was rich in TPC and TFC, i.e., 62.32 ± 4.02 and 24.53 ± 0.61, respectively, and the cytotoxic potential was 10% towards human RBCs, while protein estimation revealed the presence of protein in the extract, which was 22.82 ± 4.6. DPPH assay in comparison with ascorbic acid and several enzymatic assays, such as CAT, SOD, and POD, showed maximum antioxidant potential, i.e.,15.9 ± 2.33%, 65.57 ± 13.4%, 3.02 ± 3.4, 15.87 ± 0.5, and 0.74 ± 0.2, respectively. Glucose uptake by yeast cells, i.e., α-amylase and α-glucosidase, showed a maximum antidiabetic potential such as 75.11 ± 1.44%, 41.81 ± 3.75%, and 35.9 ± 1.24%, respectively. Our results indicate that the methanolic extract of T. divaricata has antioxidant potential and inhibits α-amylase and α-glucosidase activity and possesses maximum antidiabetic potential. The results provide scientific proof that the medicinal plant being studied is a powerful source of natural antioxidant, antidiabetic, and medicinally significant substances. In silico study, using a molecular docking, unveiled that two compounds showed good interactions with 5kzw protein with considerable binding affinities and fulfilled docking parameters. It may conclude that T. divaricata is an important vegetable with a potent source of natural antioxidants and antidiabetic activity justifying its traditional use in green therapeutics.


Introduction
Diabetes is a metabolic disorder characterized by a high level of glucose in the bloodstream triggered by inadequate insulin output or insulin activity [1]. Diabetes is a dynamic chronic disease needing ongoing medical care with harm reduction approaches within glycemic control [2]. Polyuria, polydipsia, dry mouth, itchy skin, blurry vision, nausea, and exhaustion are the typical physical symptoms of DM [3]. Diabetes accounts for over 3.8 million deaths annually and is therefore the fifth leading cause of mortality [4]. is disease is becoming disastrously underdeveloped (low-income) countries [5]. Pakistan is amongst the ten countries that are expected to have high figures of people with diabetes by 2030 [6].
Diabetes increases the risk of several health problems and has a severe effect on the eye, kidney, foot, blood pressure, etc. [2]. α-Amylase involves the starch hydrolysis into the small scraps of sugar [7]. During diabetes, ROS are induced and cause β-cell glucose toxicity [8]. Cytotoxic T lymphocytes (CTLs) are one of the types of T cells, and CTLs are triggered to clear cells associated with the virus [9].
Oxidative stress is caused by an increase in reactive oxygen species, which is the primary cause of diabetes and can have serious consequences. Enzymes including superoxide dismutase, glutathione, peroxidase, and catalase are involved in enzymatic defense systems [10,11]. e peroxidases are a family of enzymes with the ability to oxidize different substrates using H 2 O 2 [12]. Antioxidants (e.g., superoxide dismutase (SOD), reduced glutathione (GSH), and other antioxidant enzymes) are generally providing tools to investigate the stress-related diabetes [13]. Several natural antioxidants are present in plants, and usually, these are vitamins C and E, tannins, and flavonoids. ese antioxidants have the competency to sustain or uphold β-cell administration, and in this way, they could diminish the glucose level in the blood. It has been found that medicinal plants, e.g., Coriandrum sativum, Fraxinus excelsior, Casearia esculenta, Caesalpinia bonducella, and Biophytum sensitivum, are more cost-effective, have fewer side effects, and are more persuasive in curing diabetes mellitus than conventional drugs [14].
Due to the high cost of pharmaceuticals, typical and traditional plants could be used to cure different diseases as 70 to 80% of the developing world depends on them [15]. For this purpose, 800 plants could be considered for their antidiabetic potential [16]. A plant species known as Tabernaemontana divaricata (TD) belongs to the family Apocynaceae, locally acknowledged as Tagar/Chandni/Crepe Jasmine. In contrast to Staphylococcus aureus and Escherichia coli, Tabernaemontana divaricata flower extract has strong antibacterial activity [17].
In silico approaches to the drug design has the benefit of reducing the time and expense of developing new targets. In silico methods that can describe interacting molecules and predict three-dimensional (3D) structures have been used to solve several biological problems. In this context, this study aimed to evaluate the phyto-components, total phenolic content (TPC), total flavonoid content (TFC), and in vitro biological properties such as DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging, enzymatic antioxidant activities, hemolysis, and antidiabetic potentials of methanolic extract of T. divaricate flower. Further, to develop effective inhibitors, in silico analysis was performed to determine the interaction of identified antidiabetic compounds with the target protein. (TPC and TFC). By following the protocol of Singleton et al. [18], the TPC of methanolic extract of T. divaricata was evaluated. e absorbance for the mixtures of solutions was measured at 760 nm with gallic acid as a standard, and TPC results were expressed as milligram gallic acid equivalent per gram dry weight (mg GAE/g dw) in triplicates. e procedure of Bao et al. [19] was followed to calculate the total flavonoid content (TFC) of T. divaricata. Eventually, the absorbance of the mixtures was measured against a reagent blank at 510 nm and TFC was noted in triplicate samples and presented as milligram quercetin per gram dry weight (mg QE/g dw).

Antioxidant Activity Assays
2.2.1. DPPH Free Radical Scavenging Assay. By following the protocol of Gyamfi et al. [20], the free radical scavenging capability of T. divaricata flower methanolic extract was evaluated against free radicals of DPPH. Ascorbic acid was used as a standard. e following formula was used to calculate the percentage inhibition of plant extract against DPPH free radicals: where AB is the absorbance of blank and AS is the absorbance of the sample.

Enzymatic Antioxidant Activities.
e method proposed by Mohebbi et al. [20] was performed to measure the catalase (CAT) activity. In this method, 0.1 mL of T. divaricata extract was added in 0.05 M K 2 HPO 4 and 1.4 mL hydrogen peroxide (H 2 O 2 ), which was taken as a substrate, and catalase enzymes were added for the decomposition of H 2 O 2. e decomposition was detected using a UV spectrophotometer (UV-1601, Shimadzu, Germany) by calculating the reduction in the absorbance for 5 min at 240 nm. e results of this activity are denoted as μM of consumed H 2 O 2 /min/mg of protein. Similarly, peroxidase (POD) activity was checked in which guaiacol was used as hydrogen. e experiment was carried out by measuring the difference at 470 nm for 1 minute. e enzymatic activity was described as a unit (one activity unit defined as absorbance at 470 nm changes 0.001 per min) [21]. e rate of inhibition in the photoreduction of nitroblue tetrazolium (NBT) by the mean of superoxide dismutase (SOD) enzymes was calculated to determine SOD operation. e reaction mixture used in this activity has 50 mM sodium phosphate buffer of pH 7.6, 50 mM sodium carbonate, 0.1 mM EDTA, 50 μM NBT, 12 mM·L-methionine, 10 μL riboflavin, and 100 μL crude extract within an exact having 3.0 mL volume. On the other hand, the control reaction was preceded in the absence of extract. e SOD behavior was measured by exposing the following reaction mixture to white light at room temperature for 15 minutes. After incubation for 15 min, the absorbance was noted at 560 nm with the help of a spectrophotometer [22].

Cytotoxicity through Hemolytic Activity.
To estimate the hemolytic activity of plant extract against human red blood cells (RBCs), the method of Powell et al. [23] was followed. PBS was used as the negative control, while 0.1% Triton X-100 was served as a positive control. e absorbance at 576 nm was noted.  [24]. Various concentrations of extract were incubated in phosphate buffer of 0.1 mol/L at pH 6.8 and 2 U/mL porcine pancreatic amylase 500 μL for 20 minutes at 37°C. 1% starch of 200 μL was mixed in phosphate buffer of 0.1 mol/L with pH 6.8 and added to mixtures of extract. After incubation at 37°C for 1 h, 1 mL of color reagent (3,5dinitrosalicylic acid) was added to the mixtures, and finally, the absorbance was recorded at 540 nm after boiling this mixture for 10 minutes. Control was without inhibitor, and the percentage of inhibitory efficacies of the extract was measured using the following formula:

Intestinal α-Glucosidase Inhibitory
Assay. e inhibitory capability of α-glucosidase was evaluated by following the procedure of Ademiluyi and Oboh [25] with some modifications. Different concentrations of the extract were mixed with a 100 μl solution of 1.0 U/mL α-glucosidase and phosphate buffer whose pH was adjusted to 6.8, and the mixtures were incubated for 15 minutes at 37°C. After this, 5 mmol/L solutions of PNPG with the amount of 50 μL in phosphate buffer 0.1 mol/L (pH 6.8) were poured into the solutions and incubated again for 20 minutes at 37°C. e absorbance of the control and sample was measured at 405 nm, and the percentage inhibitory activity of glucosidase was calculated using the following formula: where A control is the absorbance of the mixture without extract and A sample is the absorbance of the mixture with the extract.

Glucose Uptake in Yeast
Cells. e protocol of Cirillo [26] was used to perform glucose uptake in yeast cells. Suspension of yeast (1%) was centrifuged for 5 min at 4200 rpm. By taking supernatant 10% v/v yeast cells, the suspension was prepared. 2 mg of an extract was dissolved in 1 mL of DMSO. ese mixtures were then added in the different molar concentrations of 1 mL solution of glucose and at 37°C incubated for 10 min. For starting the reaction, 100 μL of suspension was added to this mixture, then vortexed, and incubated again at 37°C for 60 min. e mixture was then centrifuged at 3800 rpm for 5 min, and for the estimation of glucose, the absorbance was taken at 520 nm. e percent increase in glucose uptake was calculated using the following formula: Increase in percentage of glucose uptake e control consisted of all the reagents except plant extract, and metronidazole was used as a standard drug.

In Silico Study
2.5.1. SwissADME Analysis. Fourteen important bioactive compounds in the methanolic extract of T. divaricata flowers were selected due to their bioactive potential towards antitumor, antibacterial, and anti-inflammatory properties and to give better pharmacological activities [27][28][29]. ese compounds, enlisted in Table 1, are subjected to a theoretical in silico ADME prediction study using the Web tool, Swis-sADME (http://www.sib.swiss). To predict suitable properties, 2D structural models of analyzed compounds were drawn in SDF format and transferred to the simplified molecular-input line-entry system (SMILES) format. e SwissADME server tool was used to measure physicochemical properties of the compounds such as molecular weight, number of hydrogen bond acceptors, number of hydrogen bond donors, number of rotatable bonds, molar refractivity, lipophilicity (ALogP), and topological polar surface area (TPSA). e drug-likeness efficiency of the selected compounds was examined for firstround screening using Lipinski, Ghose, and Veber rules based on their physicochemical properties [30][31][32], and then, pharmacokinetics and medicinal properties were also examined.
Drug-likeness, bioavailability, and pharmacokinetic abilities are important parameters to predict the active potential molecules for the purpose of drug development. In the clinical trials, many potent medicines fail in the drug development phase because of their weak abilities. To ensure that the bioactive compounds of Tabernaemontana divaricata have drug-like characteristics, all of the compounds in this study were subjected to ADME and prediction experiments before molecular docking.

Molecular Docking Analysis.
e compounds that went through the filter were docked with the target protein, resulting in improved docking studies that predicted the potential binding of inhibitor to the protein. According to the results of SwissADME, 9 of 26 compounds were chosen to describe the major compounds contained in the extracts. e three-dimensional structures of compounds and target protein were calculated using the Protein Data Bank (PDB) database. GOLD version 5.3.0 and BIOVIA Discovery Studio were used to perform docking calculations (http:// www.3dsbiovia.com/) for designing and visualization [33].

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(1) Protein Preparation for Dockings. For the molecular docking studies of the synthesized compounds, GOLD docking software version 5.3.0 was used. Protein Data Bank was used to obtain the coordinated crystal structure of the 5kzw protein (http://www.rcsb.org/pdb/home/home.do) [34] and saved in PDB format. Essential hydrogen atoms were added with the aid of GOLD. GOLD score is a function that mimics a molecular mechanism and has been optimized for calculating ligand-binding positions [35]. Affinity (grid) maps of 10Å and grid points with 0.75Å spacing were generated [36].
(2) Ligand and Energy Minimization. For ligand's energy minimization, the ChemDraw Ultra 12.0 and Chem3D Pro were used. e ligand atoms were given the Gasteiger partial charges. Rotatable bonds were described by merging nonpolar hydrogen atoms. e ligands were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov) [37]. GOLD takes into account the degree of freedom in the binding site that corresponds to the reorientation of hydrogen bonds of donor and acceptor groups. Despite accounting for a small fraction of the total conformational space available, this degree of freedom accounts for a significant difference in binding energy values [38].
(3) Ligand-Protein Docking. For predicting the binding affinities of a variety of ligands, molecular docking protocols are commonly used [39]. e parameters of molecular docking software were used to conduct the experiments (https://www.ccdc.cam.ac.uk/). e Lamarckian genetic algorithm (LGA) and the Solis and Wets local search method were used to simulate docking [40]. e initial positions, orientations, and torsions of the ligand molecules were chosen at random. Each docking experiment was divided into ten separate runs, each of which was set to end after a maximum of 1.5 energy evaluations.

Statistical Analysis.
e experimental results were performed in triplicate, and the data were expressed as mean ± S.E. One-way analysis of variance (ANOVA) was done for statistical analysis of data employing SPSS version 22.0. For statistical significance, p < 0.05 was considered. Using GOLD version 5.3.0 and BIOVIA Discovery studio visualizer (http://www.3dsbiovia.com), docking calculations were carried out.

Phytochemical Assays.
e estimated total phenolic content of T. divaricata methanolic extract ranged from 35.62 ± 5.31 to 62.32 ± 4.02. e estimated total flavonoid content of T. divaricata flower extract ranged from 13.95 ± 1.33 to 24.53 ± 0.61. e maximum quantity of total phenol and flavonoid was 1 mg, while the minimum was 31 μg as shown in Table 2.

Antioxidant Activities.
Extract antioxidant activity was measured in comparison with ascorbic acid. e measured antioxidant potential ranges from 1.49 ± 0.33 to 15.9 ± 2.33, while ascorbic acid was 1.15 ± 0.58 to 65.57 ± 13.4. e antioxidant potential of the extract was less than ascorbic Evidence-Based Complementary and Alternative Medicine acid at maximum concentration; however, the potential was improved at lower concentration in comparison with ascorbic acid as shown in Table 2.

Enzymatic Antioxidant Activities.
Enzymatic antioxidant activities by T. divaricata such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), as shown in Table 3, were 3.99, 1.27, and 23.80 U/mg protein, respectively. e free radical scavenging, along with an increase in physiological antioxidants (e.g., CAT, SOD, and POD), was possibly due to the presence of the phytochemicals of this plant.

Cytotoxicity through Hemolytic Activity.
Hemolytic activity of any compound is a sign of common cytotoxicity towards normal healthy cells. In this research, the extract exhibited 10% cytotoxicity towards human erythrocytes as shown in Table 3. Such findings demonstrated that if medicinal formulations from this plant are used at low concentrations, the parameters of the RBC membrane will not change. Further research is essential on this medicinal edible plant in the context of drug development and discovery.

α-Amylase and α-Glucosidase Inhibitory.
e estimated α-amylase concentration range of T. divaricata flower extract was 16.76 ± 3.97 to 41.81 ± 3.75. e average values of α-glucosidase inhibitory activity ranged from 17.88 ± 1.40 to 35.9 ± 1.24. e inhibition potential of both enzymes was maximum at 200 ug/mL and minimum at 50 ug/mL as shown in Table 4.

Glucose Uptake by Yeast Cells.
e uptake of glucose was evaluated by incubation of yeast cells in different molar concentrations of glucose and T. divaricata extract. At 5, 10, and 25 mM glucose concentrations with 1, 2, and 3 mg/mL concentration of T. divaricata extract, the results showed that by rising the extract concentration the uptake of glucose percentage was increased, while it showed an inverse relation with the glucose molar concentration as described in detail in Figure 1, which shows the maximum absorbance at 5 mM of glucose and 3 mg/mL concentration of extract.

3.5.
In Silico SwissADME Analysis. Due to weak drug-likeliness and pharmacokinetic characteristics, many potent drugs fail in clinical trials or later stages of drug discovery.
All of the compounds in this sample were subjected to druglikeness and ADME prediction tests before molecular docking to ensure that they had drug-like characteristics.

Physicochemical Properties.
A drug's physicochemical properties have a significant effect on its metabolic destiny in the body. e results from Table 1 showed that the molecular weight of all compounds met the criterion (which should be ≤ 500 g/mol) except D-glucopyranoside (MW 504.44 g/ mol) in accordance with one of the criteria laid down in the Lipinski rule of five. Of all the studied compounds, 17 compounds had less than 10 rotatable bonds, and others had more than 10, which is not acceptable. Further, the molar refractivity of all compounds was within the acceptable range (40 and 130) except squalene, 1-heptatriacotanol, and methyl ester, and these three compounds satisfy the criteria for oral bioavailability.
TPSA is another key parameter correlated with the drug's bioavailability. High oral bioavailability for passively absorbed compounds has TPSA < 140Å2. Table 1 reveals that all the selected compounds were found to be polar with TPSA values ranging from 0.00Å2 to 80.48Å2 except D-glucopyranoside, desulphosinigrin, and lactose, which had the highest TPSA (>140Å2). High solubility can facilitate complete absorption of the administered through oral administration, while low solubility limits the drug absorption in the gastrointestinal tract [41]. Table 1 shows that all of the tested 26 compounds have good to moderate water solubility, with a log S value between −0.18 and −5.6, which may promote good oral adsorption.

Pharmacokinetic Properties.
Interestingly, except for three compounds, as shown in Table 5, all were observed with high intestinal absorption and thus could penetrate very easily through the intestinal lining and be accessible to the cell membrane. To meet their molecular target, drugs that function in the central nervous system (CNS) need to move through the blood-brain barrier (BBB). However, for  Evidence-Based Complementary and Alternative Medicine drug molecules with a peripheral target, little to no BBB permeation may be needed to prevent side effects on the central nervous system [42]. e blood-brain barrier (BBB) permeation expresses the relative affinity of the drug for the blood or brain tissue. Table 5 indicates that 15 compounds are estimated to have no penetration of the blood-brain barrier, and thus, the chance of CNS side effects is expected to be absent. P glycoprotein (P-gp) plays a significant role in protecting the central nervous system from xenobiotics [43]. e predicted outcome shows that only 9 of 26 compounds are P-gp substrates and do not cause phospholipidosis. e other 17 compounds on the other hand are non-substrates of P-gp and are therefore required to induce phospholipidosis on the phenyl ring. e cytochrome P450 (CYP) superfamily is critical in drug removal through metabolic biotransformation [44]. e less skin permeant the molecule is, the lower the log Kp (in cm/s) is. It is found that log Kp measurements of all the compounds evaluated are within the limits (−8.0 to −1.0) except D-glucopyranoside, desulphosinigrin, and lactose [45].

Lipophilicity and Drug-Likeness.
e result of Table 6 showed that the log P values of all the compounds except benzene dicarboxylic acid, methylnonadecane, phthalic acid, 1-heptatriacotanol, and cyclopropane tetradecanoic acid. e selected compounds were found to be within the limits, i.e., between 0.7 and + 5.0, indicating that they should have strong permeability and oral absorption. Drug-likeness qualitatively tests a molecule's likelihood of being an oral drug candidate for bioavailability [45].

Rule of Five by Lipinski.
As per Lipinski's rule of five [31], the drug is likely to be produced as a prospective oral drug if the applicant violates none or less than one of the following four conditions. e Abbot bioavailability score (BAS) is a rule-based semi-quantitative score that relies on a total charge, TPSA, and the Lipinski filter violation that distinguishes four compound groups. All of the selected molecules have a bioavailability score of 0.55, except for tetrazole, octadecadienoic acid, n-hexadecanoic acid, and lactose, indicating a chance of becoming the oral drug candidates [45].
In this investigation, 17 of 26 of the selected compounds did not pass ADME screening, leaving 9 candidate compounds for the molecular docking analysis (Table 6), thus demonstrating the potential usefulness of the series for druglike compound growth.

Medicinal Chemistry.
Pan-assay interference compounds or promiscuous compounds (PAINS) are substances with substructures that display a false reaction, irrespective of the protein receptor, with biologically potent output [46]. Table 6 shows that all of the compounds return no PAINS alert. For all the candidates in the library, the synthetic accessibility (SA) scores were found to be less than 5 except for the 6 compounds that had scores more than 5, as shown in Table 6. e score of SA is normalized between 1 (easy synthesis) and 10 (very difficult synthesis). For most candidates in the library, the SA scores were found to be less than 5 and thus have strong synthesis feasibility. e lead-like rule-based approach lets the medicinal chemist define the required molecule to start lead optimization. Interestingly, Table 6 shows that 10 compounds of 26 have one violation of lead-likeness, and these molecules are therefore considered appropriate for initiating lead optimization. Furthermore, as is evident from the radar depictions, 8 compounds with two violations are also found to meet the requirement for oral bioavailability.   e drug-likeness of a molecule can be quickly determined using the bioavailability radar. e pink-colored region is the required physiochemical space for oral bioavailability, and the molecule's radar plot must fall entirely into the field to be considered drug-like [47].
e SwissADME prediction output revealed that 9 compounds had the optimal range of all six properties, which indicates that they have competent chemotherapeutic potential (Figure 2).

Molecular Docking.
e molecular docking technique is used to calculate ligand-binding affinities and energies, which is essential in the structure-based drug design process. 3D protonation, energy minimization, and prediction of the active site for ligands were used to prepare the protein for molecular docking, with the parameters left at their defaults. en, using GOLD version 5.3.0 software, ligands were docked with the target protein (5 kzw). In our study, 9 secondary metabolites from T. divaricata (Jasmine) flower were docked with α-glucosidase protein (5 kzw), which is the important protein for the key regulatory enzymes that are important in the diabetes management. To prevent sugar digestion and postprandial hyperglycemia, glucosidase, a digestive enzyme involved in carbohydrate digestion, must be inhibited. Table 7 contains a list of the compounds, which are downloaded from PubChem (https://pubchem.ncbi.nlm. nih.gov).
Following the selection of these nine compounds based on SwissADME findings, ChemDraw Ultra 12.0 and Chem3D Pro were used in GOLD docking for energy minimization of ligands. e coordinate crystal structure of the 5kzw protein was obtained from the Protein Data Bank and loaded into GOLD suite version 5.3.0 with a resolution of 0. GOLD 5.3.0 version was used to screen various docked complexes based on docking fitness and GOLD ratings. e GOLD program identified the most effective compound for interacting with the receptor of 2.70. e binding compatibility, i.e., docking score and fitness, was used to test the results. e best drug was selected with the highest binding affinity with the receptor molecule. As the ligand molecule, the number of hydrogen bonds formed and the bond distance between the active site and inhibited atomic coordinates were used to decide the final docked conformation for various chemicals. e GOLD docking scores for the phytochemicals are given in Table 8.  For each ligand, each docking routine returned the top ten rated docked poses. Compounds having maximum ligand-receptor binding energy and interactions with the receptor (<6Å bond lengths) were predicted to be most effective. Cyclopropane tetradecanoic acid, 2-octyl-, methyl ester, 4-(4-methyl-2-biphenylyloxy) phthalonitrile showed the best interaction with α-glucosidase (PDB ID � 5kzw), and the protein having Gold fitness (39.28, 37.21) and GOLD docking score is -7.51and -6.

Discussion
Ethnobotanicals are widely used for the treatment of diabetic and oxidative stress-related conditions [48]; however, it still requires rigorous scientific validation. e amount of postprandial blood glucose is the key factor that must be controlled in T2D management [49]. e drug intervention often has inexorable consequences, mostly hypoglycemia, gastrointestinal damage, and weight gain [50]. Several studies have verified the antihyperglycemic properties of plant extracts and herbal formulations that could be used as antidiabetic tonics. Herbal medications are often thought to have fewer adverse side effects than prescription drugs [51]. e correlation was found between hypoglycemic events of medicinal plants to the presence of phenol and flavonoids [52]. According to previous studies, flavonoids have an antidiabetic property and are a source of glucose uptake in tissue with relevance to oxidative stress during diabetic conditions [53]. Antidiabetic activity is due to the co-adjuvant effect of bioactive compounds present in T. divaricata.
Plants have a phenolic compound that shows antioxidant activity to prevent tissue damage. Rauter et al. [53] reported phenolic content as antioxidants in selected Nigerian medicinal plants, such as A. platyneuron and B. nitida, in which the value of phenolic content ranged from 82.33 ± 0.30 to 11.67 ± 0.09 mg GAE/g, while Agbo et al. [54] reported that the phenolic content ranged from 97.77 ± 0.77 mg GAE/g. In our results, T. divaricate showed maximum phenolic content of 62.32 ± 4.02. Flavonoid is another parameter that is used for the determination of antioxidant activity in medicinal plants. In this study, the average range of flavonoid ranged from  [55] reported the value of 59.6 ± 1.5 for methanolic plant extract of T. leptophylla. Previous research has shown that diabetic beta cells produce reactive oxygen species (ROS), which are counteracted by the overexpression of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX). Antioxidants protect beta cells from oxidative damage by scavenging the free radicals produced, thus preventing diabetes from developing [27][28][29]. In transgenic mice, overexpression of SOD has been shown to prevent the development of diabetic complications. Since oxidative stress is linked to the development of diabetic complications and inflammation, this study aimed to assess Tabernaemontana divaricata's antioxidant activity.
Biological properties, such as the antioxidant property, are considered as an estimation of the nutritional and medicinal value of plants [56]. e antioxidant activity of methanolic extract of T. divaricata flowers was estimated by DPPH assay. DPPH is a neutral, free radical that helps a stable molecule to tolerate an electron or hydrogen radical. e ability of natural antioxidants to reduce the DPPH free radical is assessed by a decrease in absorbance at 520 nm. In comparison with regular ascorbic acid, the extract showed strong scavenging activity. e lower the absorbance, the more the scavenging operation there was. As a result, the bioactive compounds in this plant can serve as antioxidants and aid in the treatment of a variety of diseases, including diabetes. Antioxidant properties of natural compounds are said to correlate with antidiabetic properties or vice versa. In this study, the given antioxidant activity of the T. divaricata flower extract could be connected to their good amount of TFC and TPC, which function as metal chelators, reduction agents, hydrogen donors, and singlet oxygen quenchers and free radical scavenger [57].

Evidence-Based Complementary and Alternative Medicine
Toxicity is an important factor in the design of pharmaceutical drugs and an important starting point for hemolytic actions, providing principal knowledge on the interaction at the cellular level between biological entities and molecules. Hemolytic activity of any compound is a sign of common cytotoxicity towards normal healthy cells. In this research, MEAE exhibited 10% cytotoxicity toward human erythrocytes, as shown in Table 3. Such findings demonstrate that if medicinal formulations from this plant are used at low concentrations, parameters of the RBC membrane will not change. Reports on the toxic effects of A. esculentus are insufficient. A previous study conducted with aqueous and methanolic extracts of the A. esculentus fruit confirmed that there were no deaths up to a dose of 2000 mg/kg (p.o.) in Swiss mice (n � 6) for 7 days and no signs of toxicity [58], which may be one of the significant issues for considering it as an important vegetable for human use. Further research is essential on this medicinal edible plant in the context of drug development and discovery.
Dietary phenols and flavonoids, in accumulation to their antioxidant effects, have been noticed to exert antihyperglycemic effects by binding to the transporters of glucose [49] and spirited digestive enzyme inhibition [59]. e carbohydrate-digesting enzymes, α-glucosidase and α-amylase, digest dietary starch and produce glucose, resulting in surge of postprandial glucose. So, the inhibition of α-glucosidase and α-amylase activities is one of the primary approaches to managing hyperglycemic T2D patients.
T. divaricata has antidiabetic activity, according to alpha-amylase inhibitory tests. e percentage inhibitory activity of plant methanolic extract against alpha-amylase enzyme increased in a dose-dependent manner. e plant extract concentration of 50 g/ml showed a percentage inhibition of 28%, while 200 g/ml showed a percentage inhibition of 61%. At a high concentration of 200 g/ml, the extract inhibited the alpha-amylase enzyme by 50%. As a result of the current inhibitory studies, it has been discovered that the T. divaricata plant extract is successful in inhibiting the alpha-amylase enzyme, which helps to postpone the breakdown of starch into glucose and thus maintain glucose levels in diabetic patients. Dastjerdi et al. [48] observed α-amylase inhibition activity of some plant extracts of T. eucrium species values ranged from 41.59 ± 0.64 to 77.07 ± 0.49 at 1.56 mg/mL to 25 mg/mL. e α-amylase  Evidence-Based Complementary and Alternative Medicine inhibitory activity of methanol plant extract is most likely by polar compounds that should be investigated further by isolating pure active compounds. At 50-200 g/ml concentrations of T. divaricata extract, the percentage inhibition increased in a dose-dependent manner. For the highest and lowest concentrations, respectively, the percentage inhibition ranged from 60% to 31% (Table 4). us, by inhibiting the alpha-glucosidase enzyme, the extract of T. divaricata, which contains various natural bioactive compounds, helps to reduce the rate of carbohydrate digestion, lowering blood glucose levels and maintaining diabetic conditions.
Glucose uptake is one of the in vitro methods used to explore the antihyperglycemic effect of different compounds [60]. e average values of glucose uptake by yeast cells in this study were 13.05 ± 1.3% to 69.5 ± 4.78%. Shehzadi et al. [60] reported the evaluation of antihyperglycemic activity through in vitro assays (0.2 to 26.3%) at 2 mg/mL, which was below the current research values. e results indicated all used concentrations under examination are capable of increasing the utilization and uptake of glucose in yeast cells. e in silico prediction of molecular physicochemical parameters, bioavailability, and the pharmacokinetics have become more relevant for the investigation of productive potential drug molecules from a drug discovery standpoint [47]. eoretical experiments play a critical role in presenting accurate data in a timely and comfortable manner. Many free online platforms have recently been established for faster screening, reducing the time and expense of drug testing (no animal testing) [46]. SwissADME is a modern comprehensive method developed by the Swiss Institute of Bioinformatics (SIB) that allows drug candidates to have their absorption, delivery, metabolism, and excretion (ADME) parameters estimated [45]. At the outset of the drug development process, ADME properties, which determine either the potential drug's access to the target or its removal by the organism, are the required properties that must be evaluated for the drug to be selected. In silico experiments based on measured physicochemical requirements can be used to check these parameters. SwissADME also provides information on gastrointestinal absorption (GIA) and blood-brain barrier (BBB) permeability in humans. From twenty-six selected compounds, only eight compounds fulfilled the criteria of  drug-likeness parameters. So, all of these nine compounds were further processed for molecular docking by GOLD.
To predict the affinity and behavior of small molecules and drug candidates, computational molecular docking is commonly used to predict the binding orientation to their protein targets [61]. We used molecular docking to model various bioactive compounds isolated from the Jasmine plant that is known to inhibit glucosidase. Because of their low price and comparatively greater protection, with a low frequency of severe gastrointestinal side effects, plants or plant-based substances may be a suitable source of glucosidase inhibitors [62]. Hydrogen bonds play a crucial role in the structure and interaction of protein-protein or ligandreceptor complexes. Hydrogen bonds are important in drug design to ensure that the drug is unique to the protein target. To back up the findings of this research, a molecular docking study was performed (cyclopropane tetradecanoic acid, 2octyl-, methyl ester, 4-(4-methyl-2-biphenylyloxy) phthalonitrile), which showed excellent interaction with 5kzw protein having Gold fitness (39.28, 37.21) and GOLD docking score of -7.51 and -6.53 including the formation of hydrogen bond with amino acids residues (1,6,10-dodecatriene, 7,11-dimethyl-3-methylene, 2-phenylthiolane, and cyclohexane propanoic acid, 3-oxo-, methyl ester). ese molecules showed exceptionally good interaction with α-glucosidase protein and can be considered as potential molecules that may prove to be beneficial in antiviral activity through their direct action on new castle disease [63]. ese compounds showed moderate binding affinity of 32.69, 29.14, and 28.29 and GOLD docking scores of -4.03, 0.205, and 0.93, respectively. N-methylallylamine, propanamide, cyclohexene, and 3-ethenyl have the least interaction in the range of 20.00, 19.16, and 23.83 and docking scores of -0.16, -0.59, and 0.14, while 1H-tetrazol-5-amine compound showed the least affinity towards 5kzw binding site.

Conclusion
Since diabetes mellitus is a global epidemic, effective drugs with less or no toxicity must be developed and one such way is the use of herbal medicines having no side effects. is study investigated the antidiabetic and antioxidant properties of T. divaricata extract and found promising inhibitors of two carbohydrate-related enzymes. T. divaricata has rich phenolic and flavonoid contents. As a flavonoid, it has great antidiabetic potential and also causes active uptake of glucose. e methanolic extract of T. divaricate has great potential for stimulation of α-amylase and α-glucosidase, so that it can be used to treat diabetes. In silico docking studies of phytoactive compounds from T. divaricata have been proven to have potential drug capabilities in terms of their pharmacokinetic and drug-likeness. e molecular docking analysis described some compounds that inhibited the targets related to diabetes mellitus and showed encouraging results with prominent inhibitory activity. is study shows that T. divaricata extract, which is rich in important bioactive compounds, can be used for diabetes. It is suggested to carry out long-term research work to recognize and isolate the active moieties responsible for antidiabetic property and to understand the mechanisms involved in glucose-lowering properties of the plant.

Data Availability
Data are available from authors on reasonable demand.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this study.