DPP-4 inhibition mediated antidiabetic potential of phytoconstituents of an aqueous fruit extract of Withania coagulans (Stocks) Dunal: in-silico, in-vitro and in-vivo assessments

Abstract The DPP-4 inhibition is an interesting target for the development of antidiabetic agents which promotes the longevity of GPL-1(Glucagon-like peptide 1). The current study was intended to assess DPP-4(Dipeptidyl Peptidase-4) inhibition mediated antidiabetic effect of phytocompounds of an aqueous fruit extract of Withania coagulans (Stocks) Dunal by in-vitro, in-silico and in-vivo approaches. The phytoconstituents screening was executed by LCMS (Liquid Chromatography with tandem mass spectrometry). The in-vitro and in-vivo, DPP-4 assays were performed by using available kits. The in-vitro DPP-4 activity was inhibited up to 68.3% by the test extract. Accordingly, in-silico determinations of molecular docking, molecular dynamics and pharmacokinetics were performed between the target enzyme DPP-4 and leading phytocompounds. The molecular dynamics authenticated the molecular docking data by crucial parameters of cytosolic milieu by the potential energy, RSMD (Root Mean Square Deviation), RSMF (Root Mean Square Fluctuation), system density, NVT (Number of particles at fixed volume, ensemble) and NPT (Number of particles at fixed pressure, ensemble). Accordingly, ADMET predictions assessed the druggability profile. Subsequently, the course of the test extract and the sitagliptin (positive control), instigated significant (p ≤ 0.001) ameliorations in HOMA indices and the equal of antioxidants in nicotinamide-streptozotocin induced type 2 diabetic animal model. Compassionately, the histopathology represented increased pancreatic cellular mass which caused in restoration of histoarchitectures. It has been concluded that phytoconstituents in W. coagulans aqueous fruit extract can regulate DPP-4, resulting in improved glucose homeostasis and enhanced endocrinal pancreatic cellular mass. Communicated by Ramaswamy H. Sarma


Introduction
Type 2 diabetes is a complex metabolic disease characterized by altered insulin sensitivity and impaired insulin secretion (Halban et al., 2014). Several targets have been identified to manage diabetes mellitus, includingdipeptidyl peptidase 4 (DPP-4), sodium-glucose cotransporter (SGLT2), GLP-1, a-amylase, and a-glucosidase (Lankatillake et al., 2019). Existing antidiabetic drugs, however, have associated side-effects such as hypoglycemia, weight loss, dizziness, pancreatitis, and adverse hepatocyte interactions, that can potentially be lethal (Nanjan et al., 2018). Therefore, there is significant interest in identifying natural phytoconstituents present in food and herbs that are currently used in traditional Indian, Ayurvedic medicine, that can be used to specifically treat diabetes and/or other metabolic syndromes without any debilitating or toxic side effects. Indigenous people in many countries rely on natural herbal medicines for treating different diseases, including diabetes (Giovannini et al., 2016;Patwardhan & Mashelkar, 2009;Sharma et al., 2022). In this regard, several studies have reported that many secondary metabolites of plants as act as therapeutic agents due to their free-radical scavenging potential and ability to inhibit the activity of many enzymes associated with metabolism (Sharma et al., 2022;Yeshi et al., 2022). Beside this, the exiting antidiabetic except gliptins (DPP-4 inhibitors) targeting the carbohydrate metabolic enzymes whereas the DPP-4 inhibitors promoting the longevity of incretin hormone which stimulate the pancreatic insulin secretion as well as b-cell functions (Campbell & Drucker, 2013;Kumar, 2016;Newsholme et al., 2022). In similar context, there are several studies reported that numerous phytocompounds have potential to inhibit DPP-4 and shown protective activity in pancreatic tissues (Ram et al., 2019;Singh et al., 2017). Accordingly, earlier study reported that an aqueous fruit extract of Withania coagulans (Stocks) Dunal fruit can inhibit DPP-4 and thus alter glucose homeostasis, however, the effect of the extract on the histology of the pancreas in diabetic rats was not analysed and a definitive mechanism was not presented (Bharti et al., 2012;Hemalatha et al., 2004;Shukla, Dikshit, Tyagi, et al., 2012). Therefore, the present study was conducted to fill in the gaps of information existing in the previous study. W. coagulans (Family: Solanaceae), is a stiff, grey undershrub that grows natively in the Thar desert and surrounding regions in India, and areas of the Hindukush Mountain range. Several parts of this plant are used as therapeutic agents to treat different ailments (Ashutosh et al., 2018;Goyal, 2015;Ram et al., 2021).In this regard, several studies were conducted to evaluate the efficacy of the W. coagulans fruit in the treatment of diabetes using a chemically-induced diabetic animal model. The recent study assessed the efficacy of an aqueous fruit extract of W. coagulans on glucose homeostasis and the histopathology of the pancreatic tissues through a combination of in-silico, in-vitro, and in-vivo studies. The phytocompounds exhibited in the ethanolic fruit extract were also recognized by LCMS.

Extract, drug, and essential substances
The fruit of Withania coagulans (Stocks) were precured from the Ayurvedic store and herbal material store of panchariya, Jodhpur which was further authenticated by the expert of botany (BSI/AZRC/1.12012/Tech./2020-21(PI.Id)). A Soxhlet apparatus was used to render an aqueous fruit extract of Withania coagulans (Stocks) Dunal as per the standard protocol. The resulting extract was then vacuum dried. The standard diabetic drug, sitagliptin (Januvia) was purchased as 50 mg doses from a confined pharmacy in Jodhpur, Rajasthan, India. According to the existing literature for invivo research, the dosing regime of test extract was estimated as per body weight. (Bharti et al., 2012). All chemical and substances were obtained from the confined supplier of Loba chemie Pvt. Ltd. The biochemical analytical kits for blood serum were purchased from Erba, Pvt. Ltd and the DPP-4 inhibition assay kit was purchased from Sigma Aldrich.

LC-MS analysis of the fruit extract
The LC-MS data was analysed with Mass Hunter (Agilent) software. Peaks produced above !3000 ionization calculations in both positive and negative manners of ionization were measured with peak layout tolerance of 0.0080 m/z to obtain reasonable resolution of the chromatograms (Keskes et al., 2017;Zhu et al., 2013). A standardised methodology was used to run samples from each experimental group in triplicate. A UPLC C18 systematic column (dimensions: 2.1 mm 100 mm, i.d. 1.7 m) was used to separate the phytoconstituents in the fruit extract at a temperature of 20 C. For the MS/MS, a 150 V fragmentor voltage was used. Positive mode mass spectra were obtained for mass-tocharge ratios (m/z) ranging from 100 to 1800. As a buffer, a solution of water containing 0.01 percent THF was used. For the movable phase, acetonitrile (A) and methanol (B) were employed with an optimised linear gradient elution as follows: 0-5 minutes, 20% A; 6-15 minutes, 40% A; 15-20 minutes, 75% A; 21-24 minutes, 100% A; 25-30 minutes, 10% A. The study was performed with a 3 ll injection volume and a flow rate of 0.4 mL/min.

Animal experimentation
The animal experiments were incorporated two comparative sets i.e., standard pathological diabetic assessments in comparison to vehicle control and second set incorporated test extract and standard drug treatments which access comparative antidiabetic efficacy through DPP-4 inhibition in type 2 diabetes in rats. The diabetes was encouraged by ensuing the modified standard practices of the studies. The overnight fasted animals were sacrificed by cervical dislocations through standard norms and recommendations of AVMA Guidelines for the Euthanasia of Animals 2020 (Iung et al., 2020). The experimental procedures were endorsed by the IAEC (Institutional Animal Ethical committee), Jai Narain Vyas University, Jodhpur conducted on 22.05.2017 as per CPCSEA norms (Registration No.1646/GO/a/12/CPCSEA valid up to 27.03.23).

Development of type-2 diabetic animal model
Wistar male albino rats (Rattus norvegicus) were divided in two groups with each group consisting of seven animals. Group I was administered a normal diet and oral doses of normal saline (vehicle), while the animals of group II were intraperitonially administered by streptozotocin (60 mg/kg) in pre-treated nicotinamide (200 mg/kg) for development of type-2 diabetes. The induction of type-2 diabetes was confirmed by evaluation of insulin and glucose levels in the test rats (Furman, 2021;Masiello et al., 1998;Singh et al., 2022).

Effect of fruit extracts on DPP-4 activity and hyperglycemia
This portion of the study encompassed two treatment groups i.e., treatment of the test of fruit extract and the treatment of standard drug, sitagliptin. Group III was dispensed to assess effect of DPP-4 inhibition activity of the test extract in type 2-diabetic rats (400 mg/kg BW per day) whereas the group IV was treated with sitagliptin (5 mg/kg body weight of rat, which is correspondent to a 50 mg clinical oral dose) (Kagal et al., 2017). The supervision of drug and vehicle was executed by oral cannulation between 10 and 11 AM to circumvent conceivable disparities in response to circadian rhythms.

DPP-4 inhibition assay
The DPP-4 inhibition through in-vitro and in-vivo (serum) were accomplished by ensuing the standard protocols form the test extract and serum. The DPP-4 inhibition assay is based on Gly-Pro-p-nitroanilide being broken down by DPP-4, resulting in the generation of a constant chromophore. The ability of the fruit extract and serum sample to inhibit DPP-4 was assessed by conniving the production of 4-nitroaniline after an examine assortment of comprising 0.1 M Tris-HCl (pH 8.0) and 2 m MGly-Pro p-nitroanilide (substrate). The buildup of sodium acetate buffer made the response stationary later in gestation at 37 C (pH 4.5) and the transmission density of the test solution was recorded by using a UV-VIS Spectrophotometer microplate reader at k ex ¼ 360 nm and k em ¼ 460 nm at 37 C (Al-Masri et al., 2009;Chakrabarti et al., 2011). The percentage inhibition was considered by persecution of the subsequent equation.
% inhibition ¼ Absorbance of controlÀAbsorbance of inhibitor Absorbance of Control x 100

Serum biochemistry
The serum biochemistry was performed by following the assessments of basic parameters, antioxidant determination and HOMA indices calculations.

Homeostatic model assessment (HOMA) analysis
The concentrations of fasting glucose and serum insulin were used to determine (HOMA-IR and HOMA-) scores and sensitivity of insulin. The HOMA scores were computed conferring to the formula of Matthews et al. (1985), which has been validated in animal models (Guyot et al., 2017;Mart ınez et al., 2016;van Dijk et al., 2013). The formulas used are as follows: The HOMA indices were considered by appropriate units (Where conversion made as pmol/L to uIU/mL for insulin and mg/dL to mmol/L for glucose).

Histology of pancreatic tissues
Samples of pancreatic tissues were prepared for observations of pancreatic histopathology by using standard methods. After exsanguination, a pancreas tissue portion was cleaned with ice relaxed phosphate buffer (0.1 M, pH 7.4) and fixed for 24 hours (Lal et al., 2017). The tissues were dehydrated in ethanol with increasing concentrations, cleaned in xylene, and then implanted in liquefied paraffin wax. The cutting of sections was made at thickness of 5 mm which further proceeded for staining of hematoxylin-eosin and examinations performed under clinical microscope (Leica-DM RA, Research Microscope, Germany). The camera attached to the microscope was used to photograph the sections.

In-silico assessments
The in-silico assessments i.e., molecular docking, molecular dynamics simulations, and ADMET predictions were performed by following the standard protocols.

Molecular docking
The molecular docking was performed against the screened phytoconstituents and target protein DPP-4 to assess interactions capabilities by using the PyMol and Autodock (Rudnitskaya et al., 2010). The catalytic residues of amino acid of DPP-4 encompasses Glu205, Glu206, and Tyr226 and hydrophobic central contained of 10 amino acid residues (Ser209, Val207, Arg358, Phe357, Tyr547, Ser630, Tyr667, Asn710, Val711, and His740). The 3 D structure of DPP-4 having ID 5y7k, retrieved from the PDB (Protein data bank). PyMol was used to eliminate co-crystallized ligand inhibitor along with water molecules, thereafter accurate chain incorporation was done. The 3 D structures of screened phytocompounds and standard drug (sitagliptin) were obtained using pubchem database. PyMol was used to process retrieved phytocompounds (ligands). The procedures of docking were authenticated by execution of the re-docking against prepared ligands and target protein and plots were produced. Re-docking authenticate the docking of phytoconstituent with DPP-4 (Kaur et al., 2018). Binding energies, molecular interactions and ligand conformations were recorded.

Molecular dynamic (MD) studies
GROMACS was used to determine conformational dynamics of docked complexes with DPP4 employing molecular dynamics simulations. The MD simulations of Withacoagin with DPP4, Withanolide with DPP4 and Withasomnine with DPP4 complexes were executed. The CHARMM36 force field was used (Huang et al., 2017). DPP4 topology was generated using GROMACS module while the topology of ligand was generated by using PRODRG 2.5 server (Sch€ uttelkopf & Van Aalten, 2004). Dodecahedron box was made for complex solvation, and the complex was put at least 1.0 nm away from the box's edge. After making system electroneutral, energy minimization was accomplished. Energy minimization was done considering max force 1000.0 KJ/mol/nm with 50,000 steps cut-off. NVT equilibration was performed at 310 K for 100ps, with complex coordinates being recorded every 10ps. NPT equilibrium was carried out for 100ps. Finally, the system was subjected to a 1 ns molecular dynamics simulation to assess stability of Withacoagin and DPP4, Withanolide and DPP4 and Withasomnine and DPP4 complexes. Structural analysis (Radius of Gyration, RMSD, and RMSF) was carried out, and graphs were created using xm grace.

ADMET assessment
To examine the druggability characteristics of the screened phytoconstituents, an ADMET analysis was performed using Drulito software (Venkatesan et al., 2012). The phytoconstituents were assessed using the Lipinski rule and their capacity to pass the blood-brain barrier (BBB). According to the Lipinski rule, a perfect therapeutic molecule should have a mass of less than 500 g/mol, H bond donors 5 and H bond acceptors 10 along with a separation coefficient f 5. As a result, phytoconstituents with these qualities could cross the BBB if the number of hydrogen bonds in the molecule is between 8 to 10, and the molecule contains no acidic groups. According to Weber's rule, the total polar surface area (TPSA) describes the medicinal molecule's bioavailability. The TPSA 140Å specifies decent oral bioavailability.

Statistical analysis
The data from serum and other biochemical calculations are expressed as a mean ± standard error of mean (S.E.M.). Mean separations between treatment means were determined by a post hoc Dunnett'st test (p 0.05) expending SPSS 22 trial version for windows (Assaad et al., 2014).

Results
The treatments of test extract and sitagliptin caused significant alterations in type 2 diabetic rats such as glucose homeostasis (HOMA), the lipid profile, free radical scavenging capabilities, and the pancreatic histopathology. The DPP-4 inhibition potential and evidence of molecular docking were also obtained from in-vitro and in-silico analyses.

Phytocompounds screening from test extract by LCMS
The LC-MS/MS data combined with an analysis using Mass Hunter software indicated the presence of the three major bioactive compounds, withacoagin, withasomnine, and withanolide E in the test extract (fruit aqueous extract of Withania coagulans), were identified from chromatogram peaks based on MS-MS fragmentation ( Figure 1A and 1B).
Mass Bank software along with published data were used to determine and identify the phytoconstituents (Table 1A and 1B).

DPP-4 inhibition assay of the test extract
The in-vitro analysis of the fruit extract of the Withania coagulans revealed a concentration-dependent inhibition of DPP-4 activity, with a maximum level of 68.4% inhibition at 60 mg/ml. In comparison, the maximum level of inhibition of DPP-4 activity by sitagliptin was 90.1% (Figure 2A and 2B).

DPP-4 inhibition assay from serum
The course of the test extract and standard drug (sitagliptin) caused significantly (p 0.001) changes in DPP-4 levels as equated to the control groups diabetes and intact. The significant elevated level of DPP-4 shown in diabetic control in comparison to vehicle control (Figure 3).

Serum assessments
The assigned assessments of serum i.e., basic parameters of metabolism, HOMA indices, and antioxidant assays were shown significant alterations after the course of the study.

Basic parameters of metabolism
The total cholesterol and consequent parameters such as LDL-cholesterol, triglyceride and VLDL-cholesterol were considerably elevated in the diabetic rats. The fruit extract was given to diabetic rats, and it lowered overall cholesterol levels, LDL-cholesterol, triglyceride, and VLDL-cholesterol by a considerable (P 0.001) amount ( Figure 5).

Antioxidant alterations
Accordingly, the GSH, catalase, and SOD levels were relatively low in untreated, diabetic rats, which also exhibited increased levels of lipid peroxidation (LPO), though no significant impact was observed in total protein. In contrast, course of the fruit extract caused in noteworthy (P 0.001) ameliorations in GSH, LPO, catalase, and SOD ( Figure 6).

Histology of the pancreas
The histoarchitecture of pancreatic islet (endocrinal tissue) samples in the vehicle control group exhibited fully differentiated cells and appropriate vasculature. The islets of the Langerhans were composed of a lightly stained, diverse cellular mass ( Figure 7A). The development of diabetes through the course of nicotinamide-streptozotocin caused degenerative changes in the islet of the Langerhans tissue at a cellular and nucleoplasmic level ( Figure 7B). Aside from that, test extract and sitagliptin treatments resulted in considerable

In-silico determinations
The in-silico assessments or data obtained from the molecular docking of protein-ligand interactions, molecular dynamics, and pharmacokinetics prediction by ADMET which shown significant interpretations as followings descriptions.

Molecular docking
DPP-4 has an active triad containing of three amino acid residues Tyr226, Glu206 and Glu205. The specific phytoconstituents found in the fruit extract would impede DPP-4 enzyme function by forming strong H bonds to the catalytic site amino acids residues, as per the molecular interactions explored using Auto Dock. Withacoagin was found to have a lower binding energy than the positive control, sitagliptin, indicating that it would strongly inhibit DPP4. Notably, withacoagin interacted via hydrogen bonds formation with the hydrophobic core (Tyr547) as did sitagliptin (Ser630); however, unlike sitagliptin, withacoagin did not interact with the catalytic triad ( Figure 8A-C). Withasomnine and withanolide E both formed hydrogen bonds with the catalytic triad residue Glu206. The identified compounds were found to have strong binding energies with the main catalytic site residues indicating they would inhibit the protein irreversibly (Table 2). These leading phytocompounds such as withasomnine, withacoagulin and withanolide have potent functional groups and reactivity capabilties (8D).

Molecular dynamic studies
By adding 12 sodium ions to each system, the target protein DDP-4 and ligand complexes (withacoagin, withanolide, and withasomnine) were made electro neutral. The potential energy (P.E.) graph showed a decline in the system's potential energy in the first few ps, but thereafter stabilised. P.E. minimization of the Withacoagin and DPP4 system achieved at 1843 EM steps, 1772 EM steps for Withanolide and DPP4 Data are means ± S.E.M. (n ¼ 5); a P 0.05; b P 0.01; c P 0.001; and d non-significant as compared to the respective control values. e P 0.05; g P 0.001; and h non-significant as compared to the respective values of the diabetic control group. and 1663 EM steps for Withasomnine and DPP4, representing that the structure Withasomnine and DPP4 equilibrised fastest between all three ( Figure 9A). The systems were in a dynamic condition at 300 K and sustained pressure after NVT and NPT equilibration ( Figure 9B and 9C). The density of the systems ranged from 1025 to 1035 kg/m 3 , with the average hovering around 1030 kg/m 3 . The density of all three systems remained constant over time, showing that they were pressure and density-equilibrated ( Figure 9D). All three Systems were put to MD simulations for 1 ns each. To determine the instabilities in the 3 D structure of proteins over time, the RMSD of systems was examined. It revealed that proteins undergo minute changes during simulations, resulting in a modest rise in the Root-Mean-Square Distance (RMSD). The RMSD value of the Withanolide equilibrated system was the highest (9E). Data are means ± S.E.M. (n ¼ 5); a P 0.05; b P 0.01; c P 0.001; and d non-significant as compared to the respective control values. e P 0.05; g P 0.001; and h non-significant as compared to the respective values of the diabetic control group.
Radius of gyration value of Withanolide System detected little more as related to other two systems. The complexes of withacoagin and withasomnine were initiate little more condensed as equated to withanolide System ( Figure 9F). RMSF (Root-Mean-Square-Fluctuation) per residues were considered which demonstrates fluctuation over all the course of study of all residues of all three protein. Peak demonstrations show the protein area that fluctuates the most during the simulation. Almost equal RMSF values were observed for all three Systems ( Figure 9G).

ADMET pharmacokinetics predictions
Pharmacokinetic (ADMET) examinations of the major screened phytoconstituents of the test extract shown that withacoagin, Withasomnine, and withanolide-E had an ideal drug profile, conforming to the Lipinski rule of five along with having good potential to cross the BBB (Blood brain barriers) (Table 3).

Discussion
The traditional medicines are working based on exhibiting phytocompounds, nature of phytocompounds and formulations. Accordingly, the antidiabetic extract and formulation are functioning by following the polarity, solubility and structural characteristics of the exhibiting phytocompounds by following the well-established mechanisms of action (Prabhakar & Doble, 2011;Yeshi et al., 2022). The current was Data are means ± S.E.M. (n ¼ 5); a P 0.05; b P 0.01; c P 0.001; and d non-significant as compared to the respective control values. e P 0.05; g P 0.001; and h non-significant as compared to the respective values of the diabetic control group. evaluated the potential use of an aqueous fruit extract of Withania coagulans, with conveyed antidiabetic properties, as therapeutics for the curing of type 2-diabetic rats. The induction of type 2-diabetes in rats was carried out by the course of nicotinamide-streptozotocin along with high sucrose diet which resulted in increased insulin resistance and degenerative changes in the histology of pancreatic tissues. Similar kind of results were reported in several previous studies which validate the protocol (Masiello et al.,1998;Premilovac et al., 2017).
The course of the type 2-diabetic rats with the fruit extract caused in significant improvement in glucose and insulin levels, as well as in the lipid profile. The results were shown consistence with the previous studies which demonstrated that a variety of other phytoconstituents can alter glucose metabolism, as well as lipid metabolism, resulting in reduced hyperglycemia and improvements in lipid profiles (Bharti et al., 2012;Brahmachari et al., 2017;Hoda et al., 2010). Subsequently, there were significant alterations performed in insulin resistance, b-cell function, insulin sensitivity which may following through insulin receptor modulation and an indirect interference in carbohydrate metabolism as reported by previous studies ( Bharti et al., 2012;Srinivasan & Muruganathan, 2016 ). Accordingly, the 68.4% inhibition of DPP-4 activity was performed by in-vitro examination as well as treatments caused  significant reductions in serum DPP-4 activities which could be attributed to the interactions between DPP-4 and the three leading phytoconstituents of extract as determined by in-silico molecular docking analysis. The molecular docking analysis indicated that all three of the major constituents of the fruit extract, withacoagin, withasomnine, and withanolide E, could bind to the catalytic region of DPP-4 and inhibit its activity as reported by earlier studies (Ashutosh et al., 2018;Bharti et al., 2012;Maher et al., 2020). The further, investigations were validated by potential energies levels of interactions, RSMD, NVT, NPT, system density, gyration and RMSF by molecular dynamics studies. The molecular dynamics simulations displaying the flexibility, geometry fitness, equilibrium and accuracy between the protein and small molecule phytoconstituents which acceptable for a comprehensive judgment of the structures and confirmed the differences that cannot be confirmed in a wet lab (Ishak et al., 2017;Salmaso & Moro, 2018). The bioavailability, bioreactivity and Molecular docking: Figure 8A. Molecular interactions of withacoagin with DPP-4 studied by docking analysis. Figure 8B. Molecular interactions of withanolide E with DPP-4 studied through docking analysis. Figure 8C. Molecular interactions of withasomnine with DPP-4 examined by docking analysis. druggability of phytoconstituents were conformed by the pharmacokinetic filters based on the Lipinski rule of five, indicating that they represent ideal drug candidates as reported by similar findings (Mohammadhassan et al., 2020;Walters, 2012). Beside this, diabetes induction resulted by degenerative changes by indicating the an excess production of free radicals which demonstrating by abnormal alterations in antioxidants (Asmat et al., 2016). Following treatment of the diabetic rats with the fruit extract or sitagliptin, however, resulted in significant alterations in the level of catalase, total antioxidant capacity, lipid peroxidation, as well as SOD (Superoxide dismutase) and GSH (Glutathione) levels; indicating the free-radical-scavenging potential of the phytoconstituents and sitagliptin Figure 9A. Potential energy minimization of Withacoagin and DPP4 system achieved at 1843 P.E. steps, Withanolide and DPP4 system achieved at 1772 P.E steps, Withasomnine and DPP4 system achieved at 1663 P.E steps and comparative potential energy minimization of all three systems.
(Al- Rowais, 2002;Ceriello, 2000;Majeed et al., 2020). Subsequently, diabetic rats treated with either the fruit extract and sitagliptin exhibited significant improvements in the morphology of islet of Langerhans cells where normal histology has been restored. These improvements may be attributed to reduced levels of b-cell apoptosis brought about by the small-molecule phytoconstituents present in the fruit extract, as evidenced in this study and previous studies (Monika et al., 2009;Song et al., 2015). In this regard, the present study demonstrated the significant beneficial effect of an aqueous fruit extract of Withania coagulans in the treatment of type 2-diabetes using a hyperglycemic animal model. Figure 9B. Withacoagin system temperature graph after temperature minimization for 100 picoseconds, Withanolide system temperature graph after temperature minimization for 100 picoseconds, Withasomnine system temperature graph after temperature minimization for 100 picoseconds, and all three-system comparative NVT graph after temperature minimization for 100 picoseconds. Figure 9C. Withacoagin system pressure graph accounted after NPT equilibration, Withanolide system pressure graph accounted after NPT equilibration for 100ps, Withasomnine system pressure graph accounted after NPT equilibration for 100ps, and all three-systems pressure graph accounted after NPT equilibration for 100ps. Figure 9D. Withacoagin system density graph accounted after equilibration for 100ps, Withanolide system density graph accounted after equilibration for 100ps, Withasomnine system density graph accounted after equilibration for 100ps and all three-systems density graph accounted after equilibration for 100ps. Figure 9E. Withacoagin system RMSD crystal backbone (black) equilibrated structure backbone, Withanolide system RMSD crystal backbone (black) equilibrated structure backbone (red), Withasomnine system RMSD crystal backbone (black) equilibrated structure backbone (red) and all three systems comparative RMSD accounted during MD run for 1 ns. Figure 9F. Radius of gyration for Withacoagin system accounted after 1000 ps., radius of gyration for Withanolide system accounted after 1000 ps., radius of gyration for Withasomnine system accounted after 1000 ps., and comparative radius of gyration for all three Systems accounted after 1000 ps. Figure 9G. Withacoagin system RMSF accounted during the 1 ns of MD simulations run., Withanolide system RMSF accounted during the 1 ns of MD simulations run., Withasomnine system RMSF accounted during the 1 ns of MD simulations run and all three systems comparative RMSF accounted during the 1 ns of MD simulations run.

Conclusion
It can be illustrated that phytoconstituents of aqueous extract of W. coagulans fruit has the capability to restore glucose homeostasis and reduce insulin resistance, as well as restoration in histoarchitecture of pancreas in type 2-diabetic rats, through its ability to inhibit DPP-4 activity and free radicals scavenging activity. The interactions between the DPP-4 and leading phytoconstituents systematically supported by in-vitro, in-silico and in-vivo determinations. Therefore, it can be claimed that the phytoconstituents of the fruit extract of W. coagulans (withacoagin, withasomnine and withanolide E) might play a crucial role in the inhibition of DPP-4 activity which resulted in antidiabetic activity.