Dual-Action Compounds for Glycemic Control: N-Benzyl-2,5-Dioxopyrrolidin-3-yl-Methanesulfonamide and 2-Benzylsuccinimide mimic α-Amylase and α-Glucosidase activities to regulate blood glucose levels

Dual-Action Compounds for Glycemic Control: N-Benzyl-2,5-Dioxopyrrolidin-3-yl-Methanesulfonamide and 2-Benzylsuccinimide mimic α-Amylase and α-Glucosidase activities to regulate blood glucose levels | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual-Action Compounds for Glycemic Control: N-Benzyl-2,5-Dioxopyrrolidin-3-yl-Methanesulfonamide and 2-Benzylsuccinimide mimic α-Amylase and α-Glucosidase activities to regulate blood glucose levels Zakir Ullah, Adnan Nadir, Muhammad Junaid, Umar Nishan, Muhammad Saeed Jan, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7306006/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Diabetes mellitus is one of the most alarming metabolic syndromes caused by high blood sugar levels. Herein, we report the identification of two potent antidiabetic agents: N-benzyl-2,5-dioxopyrrolidin-3-yl-methanesulfonamide (NDMS) and 2-benzylsuccinimide (2-BS). These molecules were evaluated for their antidiabetic potential using in vitro, in vivo, and in silico assays against their potential targets, α-amylase and α-glucosidase. For in vivo studies, an alloxan-induced diabetic rat model was used. Our results show that NDMS and 2-BS exhibit good in vitro enzyme inhibition compared to the standard acarbose drug. In in vivo assays, the acute toxicity profiles and IC50 values showed that NDMS at doses of 5 and 10 mg/kg/b. w. resulted in hypoglycemia with 247.9,178 mmol/L and 246.4,171 mmol/L. Similarly, 2-BS at the same dose resulted in a hypoglycemic effect by lowering blood glucose levels from 251.6 and 199 mmol/L to 244.8 and 181 mmol/L, respectively. Furthermore, their antihyperlipidemic effects revealed that these compounds also reduced total cholesterol, low-density lipoprotein (LDL), and triglyceride (TG) levels, while increasing high-density lipoprotein (HDL) levels. Moreover, these molecules significantly enhanced the number of pancreatic β-cells and restored normal liver morphology and serum creatinine level. Additionally, the in-silico results showed that both NDMS and 2-BS have remarkable interactions with α-amylase and α-glucosidase compared to those of the standard acarbose drug. Thus, our findings indicate that these molecules have good hypoglycemic and hypolipidemic effects and can be used as lead pharmacophores in the development of new antidiabetic drugs. Diabetes α-amylase α-glucosidase N-benzyl-2 5-dioxopyrrolidin-3-yl-methanesulfonamide 2-benzylsuccinimide alloxan Figures Figure 1 Figure 2 Figure 3 Figure 4 1) Introduction Diabetes mellitus is a group of metabolic complications characterized by hyperglycemia, predominantly caused by insulin synthesis or sensitization deficiency 1 . Insulin and glucagon hormone levels are crucial for maintaining blood sugar levels. Relative or absolute insulin deficiency or large-scale glucagon secretion causes blood glucose abnormalities. Diabetes is generally characterized by urination, extreme thirst, significant weight loss, high blood sugar levels, and a decline in the metabolism of biomolecules, including carbohydrates, proteins, and lipids 2 – 4 . Owing to autoimmune destruction of β-cells, absolute insulin deficiency leads to type 1 diabetes, whereas relative insulin secretion deficiency or its resistance to receptors, or both, cause type 2 diabetes 5 – 8 . Major macrovascular complications include myocardial infarction and peripheral arterial disease, whereas microvascular complications include nephropathy, neuropathy, retinopathy, and diabetic feet. In 2019, approximately 463 million patients with diabetes were recorded. It is projected that by 2035, the diabetic mortality figure will cross 592 million. Current antidiabetic drugs include insulin therapy, biguanides, sulfonylureas, thiazolidinediones, meglitinides, DPP-4 inhibitors, glucagon-like peptide receptor agonists, and sodium-glucose transporter inhibitors 9 . One clinical approach to treating diabetes involves the inhibition of carbohydrate-metabolizing enzymes. These enzymes have a direct impact on blood glucose levels because they are crucial for the breakdown of carbohydrates into glucose. Clusters of α-amylase and α-glucosidase, α-glycosidic O -linkage-hydrolases, a common exo-type, release D-glucose from the substrate at the non-reducing end. Hydrolytic processes are caused by the breaking of the bond between the anomeric carbon of the glucosyl residue and the glycosidic oxygen (C1-O). Since they exclusively release D-glucose, α-glucosidase and α-amylase do not appear to differ from one another. However, the anomer type of product—α-glucosidase produces α-glucose and α-amylase, β-glucose—essentially distinguishes the two enzymes 10 , 11 . The enzyme α-amylase is found in plants, animals, and microbes 12 . These metalloenzymes require a minimum of one Ca2 + ion per molecule for complex formation and retention of morphology 13 . The amino acid 496 residues that make up porcine pancreatic α-amylase are 83% similar to those of human pancreatic amylase 14 . Pancreatic juice and saliva contain the enzyme α-amylase 15 . The brush-border membrane of enterocytes in the intestine secretes α-glucosidase, also known as maltase, which hydrolyzes α-1,4-glucoside-bonded polysaccharides into monosaccharides that are ready for assimilation. Pancreatic α-amylase hydrolyzes the amylose α-1,4-glycosidic linkages of a starch 8 , 16 – 19 . α-Glucosidase inhibitors (acarbose, which neutralizes α-amylase, sucrose, and maltase; miglitol, which works on glucoamylase and isomaltose; and voglibose) are a new group of antidiabetic The activity of glucosidases is essential for various biological reactions, including (i) the breakdown of dietary polysaccharides to yield monosaccharide units that the body can metabolize and use, (ii) the processing of glycoproteins and catabolism of lysosome glycoconjugates, and (iii) the biosynthesis of oligosaccharide units in glycolipids or glycoproteins 20 . At present, miglitol (1) (Glyset), acarbose (2) (Precose), and N-butyl-1-deoxynojirimycin (3) (Zavescae) are the three drugs relied on commercially as anti-glucosidases. Drugs 1 and 2 are prescribed to treat non-insulin-dependent type II diabetes. Drug 3 is used to treat Gaucher's disease, which is associated with disrupted lysosomal storage. In keeping with the growing interest in both natural and synthetic glucosidase inhibitors as potential beneficial medicines and crucial strategies for mastering biochemical processes 19 , 21 – 23 . Thus, the inhibition of α-amylase and α-glucosidase is necessary to control blood sugar levels. 2) Methodology 2.1. Drugs, solvents, and equipment NDMS and 2-BS compounds were synthesized with alloxan monohydrate 98% (Chai 10G, Sigma, CAS 2244-11-3), acarbose (Alfa Aesar), yeast α-glucosidase (CAS 9001-42-7), porcine α-amylase (CAS 9000-90-2), Tween 80 (CAS 9005-65-6) (Sigma Aldrich), normal saline (Medipak Limited), creatinine Jafee (Kit Lot CF 11231JB-CentronicGmbH/Germany), GPT/ALT-LQ- (Lot-LIQ-1259 LABKIT, China), ACCU-CHEK glucose measuring kits (Roche), ethanol strips, and pricking needles. All chemicals and solvents were of analytical grade and used without further purification. 2.2. Animal Healthy, 8–12 weeks Swiss albino mice were purchased from the Directorate of Veterinary Research Institute, Peshawar, and housed in sanitized polypropylene cages. Mice were housed in the animal house of the Department of Pharmacy, University of Swabi, Pakistan, and nourished under standard conditions (12 h light/dark) at 25 ± 2°C temperature 34 and 50 ± 15% relative humidity and were fed standard food (pellet diet) and water ad libitum. Upon reaching an average body weight of 35 g, the animals were inspected for in vivo experimentation. The randomized animals were adapted to the laboratory environment for ten days prior to the start of the study. The experimental protocol was approved by the Ethical Committee (UOS/Pharm11), Department of Pharmacy, University of Swabi, Khyber Pakhtunkhwa, Pakistan. The Advance Academic and Research Board (ASRB), University of Swabi, approved this study (study permission no. F. NO. DA/UOS/2023/1766 dated August 01, 2023). Following the experiment, the animals were euthanized in compliance with AVMA regulations 35 . 2.3. Compounds chemistry NDMS and 2-BS were synthesized by M.S. Jan et a l . (2020) and further evaluated for their antidiabetic potential. The synthesis of the N-benzene sulfonamide derivative of pyrrolidine-2,5-dione shown in Scheme 2 (see S. Figure 1 ) 36 . The analyzed compounds were synthesized by Michael's addition of ketones to N-substituted maleimides. A mixture of ethyl-2-oxy-cyclohexanecarboxylate (2.0 equivalents) and a catalyst (20 mol% of either 8-hydroxyquinoline or a combination of creatinine and KOH) was mixed in 1 M dichloromethane (DCM) and magnetically agitated for 3–4 min, followed by N-cyclohexylmaleimide additions to synthesize the desired molecules. Thin-layer chromatography (TLC) plates were used to track the reaction progress (see S. Figure 2 ). After the reaction, the mixture was cooled and diluted with 15 mL of water. Dichloromethane (3 × 15 mL) was then used to extract the material, which was filtered and concentrated using a low-pressure rotary evaporator. Column chromatography was used to purify the crude mixture. The in-depth summary begins with the combination of N-substituted maleimides with different cyclic alkyl/alkyl ketones to create ketone derivatives of pyrrolidine-2,5-dione. All reactions were straightforward, single-step processes that were completed quickly and produced good isolated yields. To the best of our knowledge, this is the first report of a tricomponent non-covalent organocatalytic system for these reactions. N-sulfamoyl-phenylmaleic acid ([(4-sulfamoylphenyl) carbamoyl] prop-2-enoic acid) intermediate was produced in the first stage by reacting maleic anhydride with sulfanilamide in diethyl ether to form an N-substituted sulfonamide derivative. The sulfanilamide derivative was obtained by cyclizing the intermediate in acetic anhydride with sodium acetate. After reacting with the N-sulfonamide derivative, cyclohexanone and cycloheptanone were produced in the reaction mixture. Biologically screened compounds were > 95% pure, as determined by HPLC 36 . The physical and chemical properties of NDMS include the following: Molecular Formula: C₁₂H₁₄N₂O₄S, Molecular Weight, 282.32 g/mol; Melting Point, 160°C; Solubility: Soluble in methanol, ethanol, and DMSO Low solubility in hexane and chloroform; moderate water solubility; Appearance: White to off-white solid; and Boiling Point, 360°C. 2-Benzylsuccinimide molecular formula of C₁₁H₁₁NO₂, a Molecular Weight of 189.21 g/mol, and a Melting Point of 102°C. solubility in water is likely low, as is common for succinimide derivatives, owing to their relatively nonpolar nature. 2-BS is more soluble in polar organic solvents such as acetone, ethanol, methanol, and dimethyl sulfoxide (DMSO) because of the ability of the succinimide and benzyl groups to interact with these solvents. Appearance: white or off-white crystalline solid; boiling point: 325°C. 2.4. Toxicity Before the administration of the compounds to lab animals, 5-2000 mg/kg/b. w., comprising 8 doses, was determined by the results of preliminary range-finding experiments from LD0-100. For every eight doses, three replicates were examined using both the positive and negative controls. Oral routes were followed for each group of five mice, and after implementing the predetermined operations, the animals were closely monitored for allergic reactions, behavioral changes, and mortality rates for up to three days. The comprehensive dosage schedules and animal requirements are provided in Tables 36 , 37 . 2.5. Invitro α-amylase and α-glucosidase inhibition 2.5.1. α-amylase Inhibition assay The α-amylase inhibition test was performed by measuring the amount of reducing sugar (maltose) generated during the assay. A decline in the amount of maltose released was used to indicate the inhibitory action of the enzyme. The maltose equivalent was calculated using a modified dinitrosalicylic acid (DNS) technique. To prepare the sample solution, alpha amylase was mixed with the solvent (phosphate buffer), and various concentrations of the pre-synthesized compounds (500, 250, 125, 62.5, and 31.25 µg/ml) were mixed in this solution. A 1% w/v starch solution was then mixed after the compounds were pre-incubated with 1 U/mL α-amylase. The above solution was incubated for approximately 30 min at 37°C. After incubation, the reactant mixture was kept in a water bath for 5 min at 100°C. Instead of the amylase enzyme, equal amounts of buffer (20 mM sodium phosphate buffer with 6.7 mM sodium chloride, pH 6.9 at 20°C) were used as a blank. After that, 1 mL of DNS reagent (12.0 g of sodium potassium tartrate tetrahydrate in 8 mL of 2 M NaOH and 96 mM 3, 5-dinitrosalicylic acid solution) was added to stop the reaction 38 . The color intensity was measured using a microplate reader at 656 nm. The percentage of inhibition was calculated using the following formula: % inhibition = \(\:\frac{Abs\:control-abs\:sample}{Abs\:control}\) x100 2.5.2. α-glucosidase inhibition assay The α-glucosidase chromogenic inhibitory assay of NDMS and 2-BS was performed using standard protocols 39 , 40 . Briefly, approximately 20 µL of the prepared α-glucosidase solution (0.5 U/mL) was combined with 120 µL of pH 6.9 phosphate buffer. The ultimate concentration of the enzymatic solution was 20 µL of α-glucosidase (0.5 U/mL). The substrate para-nitrophenyl–D-glucopyranoside (5 mM) was manufactured in the same buffer (pH 6.9). After preparing test samples with different concentrations ranging from 62.5 to 1000 µg/mL, they were combined with the enzyme solution and incubated for 15 min at 37°C. Finally, 80 µL of 0.2 M sodium carbonate solution was added to complete the reaction. Acarbose blank solution served as the positive control, and the system devoid of α-glucosidase served as the blank. The absorbance was recorded at 405 nm using a UV spectrophotometer (Shimadzu, Japan). The experiment was repeated in triplicate, and the inhibitory activity was determined using the following equation: % inhibition = \(\:\frac{Abs\:control-abs\:sample}{Abs\:control}\) x100 2.6. Alloxan-induced model and blood glucose profiling Diabetes was induced by administering intraperitoneal alloxan at 120 mg/kg/bw. w. to mice 41 . Not only was the symptomatic assessment performed for polyuria and polydipsia, but the model was also evaluated by fasting blood glucose levels above 200 mg/dL for diabetes induction. The animals were grouped into normal control (nondiabetic, saline nourished), diabetic control (alloxan administered), group 3 was standardly treated with glibenclamide (0.5 mg/kg), and groups 4 and 5 were treated with oral NDMS at doses of 5 and 10 mg/kg, and 2-BS at the same dose of NDMS was administered orally to groups 6 and 7. Glucose levels were recorded regularly for up to 15 days using an Accu-Check glucometer. 2.7. Lipid profiling After 15 days, blood samples were collected in heparin-coated sample tubes. Serum was isolated and processed for determination of total cholesterol, LDL, HDL, and TGs 17 , 42 before centrifugation at room temperature for 30 minutes at 15000 rpm. For triglyceride (TG), the separated serum was analyzed using a calorimetric machine (MicroLab 300; Q-Line Biotech Pvt. Ltd.). Yellow gel tubes (clot activators) were used for TG quantification. Approximately 1 ml of TG reagent was added to the tube and mixed with 10 µL of serum, followed by incubation for 10 min at 37°C. The tube was then processed using a machine. Similarly, for total cholesterol, the same process was repeated, but the serum was mixed with a cholesterol reagent and placed in a water bath for 10 min at 37°C. The prepared tube was placed in a MicroLab 300 (Q-Line Biotech Pvt. Ltd.) for analysis, and the readings were recorded. The levels of total cholesterol, total triglycerides, low-density lipoproteins, and high-density lipoproteins in the plasma were measured. 2.8. Liver functions profile The same isolated plasma was used for the analysis of liver enzymes (SGPT, SGOT, and ALP). Briefly, for SGPT, SGOT, and ALP profiling, the previously separated serum (10–50 µL) was mixed in a tube with an ALT reagent (Thermo Fisher Scientific Company) and subjected to calorimetric analysis. For creatinine analysis, mice blood samples (1.5 ml) in a heparin-coated sample tube already placed in the tube rack were used using the standard kit method. A blank solution was tested to check the machine function, followed by validation that the creatinine reagent fell within the acceptable reading range of ≤ 2 mg/dL. The desired blood sample was then aspirated after mixing with the reagent and waiting for 10 s using a stopwatch, followed by machine readings for further statistical analysis. 2.9. Histopathological Examination To check the recovery of the pancreas, approximately 3–4 pancreas samples from each group were collected from the sacrificed mice for histopathological examination of the pancreas at the end of the anti-diabetic study. The study was continued for up to 4 weeks after the administration of various dosages to the respective groups. A standard histological protocol was used (IETIE). Briefly, pancreatic sections were preserved in formalin saline. Subsequently, ethanol was used to dehydrate the tissues, followed by xylene tissue cleansing at increasing concentrations. Before embedding in paraffin blocks, the tissues were impregnated with molten paraffin. Fine sectioning was followed by hematoxylin and eosin (H and E) staining, and the samples were examined under a light microscope. For histological examination of the liver, three to four mice from each group that had the greatest change in blood sugar levels were sacrificed. This was performed using a standard histological method. The liver was immediately preserved in 10% formalin for 24 h at room temperature after euthanasia. The tissues were subsequently divided into sections, placed on glass microscope slides, and fixed in paraffin. The slices (4 mm) were stained with hematoxylin and eosin and viewed under a light microscope. 2.10. Molecular docking A molecular docking study was carried out using MOE (2016) software (CCG, 2016). The 3D structures of α-amylase and α-glycosidase with Protein Data Bank (PDB) ID 3BAJ and 5NN5, respectively, were obtained from the PDB database 43 , 44 . All water molecules were eliminated before energy minimization. Energy minimization was performed using the MMFF94 force field with a convergence criterion of 0.05. The 2D structural coordinates of the synthesized compounds, NDMS and 2-BS, were constructed and saved in a new MOE database, and the 2D structure of acarbose was obtained from the PubChem database (PubChem CID-41774). The substrate-binding site residues of α-amylase and α-glucosidase were defined for molecular docking studies. Flexibility was applied to all ligand atoms during docking to obtain the lowest-energy ligand-protein complex 45. The compounds were ranked using the GBVI/WSA dG scoring system. Finally, the protein-ligand interactions were analyzed using PyMOL v.1.7. 2.11. Statistical analysis SigmaPlot@17 was used for all graphical and statistical analyses. A P-value < 0.05 was considered to indicate a significant statistical difference. 3) Results 3.1. In-vitro α-amylase and α-glucosidase inhibition NDMS and 2-BS directly blocked α-amylase and α-glucosidase enzymes via an enzyme inhibition assay experiment using acarbose as the reference drug (Fig. 1). NDMS showed significant inhibition of α-amylase in a dose-dependent manner of 85.49 ± 0.60, 76.28 ± 1.94, 71.08 ± 1.04, 61.37 ± 0.56, and 54.12 ± 0.52 at a concentration range of 62.5–1000 µg/mL, with an IC50 value of 49.05 µM. The 2-BS displayed percent inhibition of 77.58 ± 0.63, 716.61 ± 0.43, 60.93 ± 0.67, 53.65 ± 0.91, and 45.12 ± 0.12 at the same concentration, with an IC50 value of 95.05 µM. In comparison, acarbose had an IC50 value of 22.61 µM (Fig. 1B) 46–48 . The reduction in the activity of α-glucosidase by NDMS was 87.30 ± 1.42, 82.80 ± 0.41, 69.51 ± 0.59, 65.90 ± 0.32, and 61.56 ± 0.52 at a concentration range of 62.5–1000 µg/ml with an IC50 value of 30.93 µM, whereas 2-BS displayed an inhibition of 76.32 ± 0.87, 69.33 ± 0.66, 56.40 ± 0.82, 51.90 ± 0.45, and 42.34 ± 0.42 at the same dose, respectively, with an IC50 value of 120.3 µM (Fig. 1C). In comparison, acarbose has an IC50 of 23.78 µM 49 . 3.2 In-vivo study of NDMS and 2-BS in alloxan-induced diabetic mice The body weights of treated and nontreated mice were measured at different time intervals ranging from days 1 to 15, and the percent change in body weight was calculated, as shown in Fig. 2A. The data demonstrated that weight gain in diabetic mice (23.59%) was less than that in normal control mice (41.7%). Furthermore, diabetic mice treated with glibenclamide (0.5 mg/kg/b. w.) resulted in almost equivalent weight gain to that of normal control mice (47.8%). The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice have shown a good weight gain of 50–52.3% compared to glibenclamide 41, 50 . While the 2-BS (5 and 10 mg/kg/b. w.)-treated animals had a minimum effect on controlling weight loss of approximately 29–36% in diabetic mice. Thus, 2-BS is less effective in controlling weight loss in diabetic models than NDMS and glibenclamide (Fig. 2A). Similarly, the antihyperglycemic effects of NDMS and 2-BS (5 and 10 mg/kg/b. w.) with the reference drug, glibenclamide (0.5 mg/kg/b.w.), via oral administration considerably reduced fasting blood glucose levels from days 1–15. The NDMS (5 and 10 mg/kg/b.w.)-treated Blood glucose levels (BGL) of C. sinensis-treated mice were noted as 247.9 ± 0.9, 236.1 ± 1.7, 208 ± 1.9, 187 ± 1.4, and 178 ± 1.3, respectively. While the 2-BS (5 and 10 mg/kg/b. w.)-treated mice exhibited a decreased BGL of 251.6 ± 1.4, 232.4 ± 1.5, 223 ± 1.1, 208 ± 1.7, and 199 ± 1.6 mg/dL, respectively. The noted BGL decline was significantly less than that in the diabetic control but was still higher than that in glibenclamide-treated mice (Fig. 2B). Furthermore, liver function analysis was performed by assessing the key enzymes, including serum pyruvic transaminase (SGPT; 61.4 ± 1.5), serum glutamic oxaloacetic transaminase (SGOT; 46.9 ± 0.8), alkaline phosphatase (ALP; 275 ± 1.1), and serum creatinine (2.8 ± 0.7) in diabetic mice. The reference drug, glibenclamide treatment, halted the liver enzymes SGPT (25.8 ± 1.8), SGOT (23.9 ± 1.5), ALP (188 + 2.5), and serum creatinine (0.5 ± 0.8), respectively. The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice reduced the same markers up to SGPT (24–26.8 ± 1.4), SGOT (23–27.8 ± 1.5), ALP (193–182 + 1.8), and serum creatinine (0.7–0.9 ± 0.2), respectively. The reduction was significant at both doses compared to that in the diabetic control group. Similarly, 2-BS (5 and 10 mg/kg/b. w.)-treated diabetic mice reduced the SGPT (22-25.8 ± 1.2), SGOT (21-22.9 ± 1.4), ALP (187-191.4 + 1.17), and serum creatinine (0.6–0.8 ± 0.15) by respectively 47, 51–53 . The reduction was remarkable at both doses compared to that in the diabetic control group (Fig. 2C and E). Lipid profiling studies on mice treated with NDMS and 2-BS for 15 days showed considerably reduced levels of total cholesterol, triglycerides (TGs), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). This outcome was proportionate to that obtained with the recommended dosage of the standard drug glibenclamide (0.5 mg/kg/b.w.) 3, 7, 8, 16 . HDL cholesterol levels were also remarkably higher in mice treated with NDMS and 2-BS (Fig. 2D). 3.3. Histopathological Findings The effects of various combinations of treatments on the structure of pancreatic β-cells were compared with those of the alloxan-induced diabetic model (control) and reference drug (positive control) groups. The cell morphology of islets in alloxan-induced diabetic control mice was altered by healing and fibrosis, which showed β-cell destruction, causing the cells to contract and deviate from their normal histological appearance (Fig. 3Ac). In the NMDS-treated (5 and 10 mg/kg/b. w.) group, a good trend of pancreatic β-cell recovery was noted compared to the reference molecules, as indicated by the red arrow in Fig. 3A1. Similarly, the diabetic model group treated with 2-BS (5 and 10 mg/kg/b. w.) also shows a better trend of pancreatic β-cell recovery, as depicted in Fig. 3A2 16 . The NDMS- and 2-BS-treated groups showed significantly improved cell mass, islet cell shape, and total islet cell volume compared to the reference group (Fig. 3A). The livers of diabetic animals after receiving alloxan injections displayed several abnormalities, including hazy edema, lymphocyte infiltration, severe congestion, necrotic foci, and hydropic changes (Fig. 3B). The hepatic portal vein was also congested in the alloxan (Diabetic Control) section from the liver of a diabetic mouse, showing hydropic degeneration (HD) and severe congestion (CO) (Fig. 3Bc) 8, 50 . The NMDS and 2-BS-treated diabetic mice livers (5 and 10 mg/kg/b. w.) show a nearly similar morphology of hepatic architecture with an apparently normal appearance and good recovery (Fig. 3B1-2). 3.4. Molecular docking To investigate the specific sites of interaction responsible for these biological activities, a docking study was performed on these keys’ pharmacophores. Docking analyses of NDMS and 2-BS revealed several possible binding interactions with the target enzymes. NDMS established a significant interaction with the binding site residues of amylase, with a dock score of 5.6607 kcal/mol, while 2-BS had a lower amylase binding affinity, with a docking score of -4.3213 kcal/mol. The docking scores of NDMS and 2-BS to α-glucosidase were 5.8803 and − 4.9448 kcal/mol 52, 54 . Overall, the docking simulations of the synthetic compounds demonstrated the significant inhibitory activities of α-amylase (Table 1) and α-glucosidase (Table 2), both of which showed significant interactions with varying binding affinities toward the target proteins (Fig. 4AB). 4) Discussion Up until now, the FDA has approved ≥ 10 classes of diabetic drugs for diabetic therapies, including biguanides, which reduce gluconeogenesis and improve insulin peripheral sensitization 55 ; sulfonylureas, which increase insulin synthesis from β-cells 46 ; sodium-glucose co-transporters and 2 inhibitors, which enhance renal glucose elimination from the body 6 . This suggests that nearly all diabetes treatments are rationalized by the post-assimilation of monosaccharides. Therefore, pre-assimilatory inhibition of glucose-based strategies must be developed. This can be overcome by changing dietary habits, such as consuming foods high in fiber and less glucose and sugar sweetener substitutes 4 , 56 . Intestinal glucose absorption proceeds via the conversion of long-chain polysaccharides to glucose by the glucosidase enzyme. The most prominent monosaccharide-creating enzymes in the GIT are α-amylase (salivary and pancreatic) and α-glucosidase. Salivary α-amylase hydrolyzes starch at the α-(1,4) glycosidic linkages to form small oligomers. Pancreatic α-amylase reduces the size of oligomers, resulting in smaller oligosaccharides such as maltose and dextrin 16 . These oligomers are transformed into dextrose by α-glucosidases, such as maltase, sucrose, and isomaltose, which are readily absorbed by the intestines. α-amylase/glucosidase inhibitors hinder gastric carbohydrate-hydrolyzing enzymes to control postprandial hyperglycemia 8 . Pharmacologic treatments require the development of new compounds to inhibit polysaccharide-hydrolyzing enzymes 51 . The one-drug, one-disease ideology is antiquated, and scientists are struggling to develop multifaceted compounds 57 , 58 . Pyrrolidinedione, commonly called succinimide, is reported to have pharmacologic activities 59 covering inflammation, tumors, microbes, and analgesia 60 , 61 and has a close resemblance to FDA-approved antidiabetic-class thiazolidinediones 62 , 63 . Pyrrolidinedione and its chiral derivatives have been studied for pancreas, heart, and liver diseases 54 , 61 . Structural activity relationships show rational derivatives of existing compounds with more potency 64 . The current study aimed to investigate the antidiabetic and antilipidomic activities of sulfonamide and succinimide derivatives. Both NDMS and 2-BS positively inhibit the target enzymes, α-amylase and glucosidase 65 . A toxicity profile was determined for these synthetic compounds before testing on an animal model, and they were found to be safe up to 1000 mg/kg/b. w., with acute toxicity above it. Therefore, a 1000 mg concentration was selected as the optimal dose for all assays. These compounds showed a good response in both in vitro (Fig. 1 ) and in vivo (Figs. 2 – 3 ) α-amylase/glucosidase inhibition assays using acarbose as a standard drug (Fig. 1 D). NDMS exhibited significantly better activity, whereas 2-BS had comparable antidiabetic activity to that of the reference drug (PC), as shown in Fig. 1 – 3 . Molecular docking (MOE) interactions of NDMS and 2-BS with target α-amylase and glucosidase enzymes also support these biological and in silico activities (Figs. 1 – 3 ). 5) Conclusion In search of finding good antidiabetic molecules, our current findings show that NDMS and 2-BS compounds significantly inhibit the activities of the target α-amylase/glucosidase enzymes in vitro and show good antihyperglycemic and antihyperlipidemic effects in diabetic models in vivo. It significantly reduced blood glucose levels and diabetes-associated markers, including LFTs (SGPT, SGOT, and ALP) and serum creatinine levels. Docking of NDMS and 2-BS with the target α-amylase/glucosidase enzymes showed good and stable interactions. Thus, this finding provides good evidence to justify the pharmacological use of NDMS and 2-BS compounds in the treatment and management of various diabetic disorders. Declarations - Ethical Approval: This study was approved by the Ethical Committee of Animal Study at the University of Swabi (study permission No. F. NO. DA/UOS/2023/1766 dated August 01, 2023) - Consent to Participate : Not applicable - Consent to Publish : Not applicable -Author’s Contribution: Z. U. and A. N.: data curation and original draft writing. M. J.: Methodology and Data analysis. U. N.: Supervision, Reviewing, and Editing. M. S. J., Z. U. H., S. U. A. H., A. H., and O. M.A. D.: Writing, Data Visualization, Data draft preparation, and I.B. & Z. W. Reviewing and Editing: Z. W.: Investigation, Reviewing, and Editing. K.F.F: Funding acquisition, S.A: Funding acquisition, Investigation, Conceptualization, Reviewing, Editing and original draft finalization. -Funding: The authors express their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for supporting this work through a research group program under grant number RGP-2/630/45. Additionally, this study was supported by the National Natural Science Foundation of China (NSFC) Grant No. 31850410479. -Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study. - Availability of data and materials All data generated and analyzed are included in the submitted manuscript and Supplementary Materials. Acknowledgement The authors express their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for this work through a research group program under grant number RGP-2/630/45. References Issue Information. Diabetes/Metabolism Research and Reviews 2019, 35 . de Oliveira Neto, X. A.; Barssotti, L.; Fiori-Duarte, A. T.; de Lima Barbosa, H. C.; Kawano, D. F., Entering the sugar rush era: revisiting the antihyperglycemic activities of biguanides after a century of metformin discovery. 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13:51:25","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30091,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/88f59c8e6332bc87adadc59f.png"},{"id":93691468,"identity":"66dbb83d-e846-4785-b47d-eaf7a30f8a84","added_by":"auto","created_at":"2025-10-16 14:07:25","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":285480,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/bbd97b62a48f71d55d91a8fb.png"},{"id":93689168,"identity":"bcaf0c00-aa60-41b0-a742-6ba53dbba438","added_by":"auto","created_at":"2025-10-16 13:51:25","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":437343,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/517673f313373eb9c4ccb85e.png"},{"id":93689170,"identity":"03b32c09-4b85-44fd-8f64-9d9b7af08312","added_by":"auto","created_at":"2025-10-16 13:51:25","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97759,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/8a3d2f2efd9ca50325a10247.png"},{"id":93689173,"identity":"e6c771d0-3aa7-4c4b-a964-d75a9e6e16ad","added_by":"auto","created_at":"2025-10-16 13:51:25","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166978,"visible":true,"origin":"","legend":"","description":"","filename":"42af6f2e7fab48c09da21d3932c8da401structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/2a1617c277ecf41dd40d6065.xml"},{"id":93689174,"identity":"c96a6a3e-79e0-4e0a-927b-0519113e2799","added_by":"auto","created_at":"2025-10-16 13:51:26","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183111,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/5bc7c03b50b8ac733f1b6088.html"},{"id":93690712,"identity":"ec517d89-24d9-45e3-bde5-9e843d6123d9","added_by":"auto","created_at":"2025-10-16 13:59:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn-vitro enzyme inhibitory effect of NMDS and 2-BS compounds:\u003c/strong\u003e The inhibitory influence of NMDS and 2-BS compounds on α-amylase and α-glucosidase enzyme responses was determined by measuring % inhibition after 0.25 h of incubation with test molecules using acarbose as a PC (positive control). A) Shows enzyme inhibition % at a fixed 1000 µg/mL concentration. B-C) Represent dose- response curves for α-amylase (B) and α-glucosidase (C) enzyme inhibition. D) Show the chemical structure of the respective NMDS, 2-BS, and acarbose molecules. Data represent the means ± SD of three replicate experiments.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/c1bd8df770e377f90dfaeb65.png"},{"id":93689156,"identity":"65a4ac24-2b04-45bb-9cce-64200a7c5c3b","added_by":"auto","created_at":"2025-10-16 13:51:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":890140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn-vivoeffect profiling of NMDS and 2-BS compounds on biochemical parameters. \u003c/strong\u003eA) The graph shows the change in body weights in the respective groups. B) Represent the serum glucose level taken at different time intervals. The graph shows liver function tests for key enzymes, including serum pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), and alkaline phosphatase (ALP) profiling. D) shows the lipid profiling for cholesterol, triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoproteins (HDL)\u003cstrong\u003e. \u003c/strong\u003eE) highlights the creatinine contents. Data represent the mean (n = 6) ± standard error of the mean in triplicate.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/68e7ff0bd55355542deb99ea.png"},{"id":93690713,"identity":"ccdfa770-001b-446d-b5c5-e4d4605ecec3","added_by":"auto","created_at":"2025-10-16 13:59:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1172230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathology and hepatoprotective activities of NDMS and 2-BS compounds. A) Shows overall histopathological examination of pancreas. \u003c/strong\u003e(C) Shows alloxan-induced diabetic control mice beta cell destruction as indicated by the red arrow; (PC) shows an alloxan-induced diabetic rat treated with glibenclamide (0.5 mg/kg/b. w.) as a reference drug. (1-2) Shows an alloxan-induced diabetic rat treated with (5 mg/kg/b. w.) test samples (1) NMDS \u0026amp; (2) 2-BS, respectively; recovery of pancreatic beta cells compared to reference as indicated by white dashed lines and circles with red arrows. \u003cstrong\u003eB) Shows overall hepatoprotective activities of NDMS and 2-BS compounds.\u003c/strong\u003e The hepatoprotective activities of the desired compounds (NDMS \u0026amp; 2-BS) were investigated by using rat model liver histopathology HE techniques. (C). Shows Alloxan-induced diabetic control rat liver sections with hydropic degeneration and severe congestion (PC). Shows alloxan-glibenclamide-treated rat liver sections. (1-2) Shows Alloxan-induced diabetic rat livers treated with (5 mg/kg/b.w.) test samples (1) NMDS \u0026amp; (2) 2-BS. The apparently normal appearance of hepatic strands to referenced as indicated by white dished lines and circles with red arrows. Images of the HE-stained slides were taken at 40X.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/8985390e5de007ebb12882cf.png"},{"id":93690715,"identity":"db855373-57ce-4eb4-b281-dc5afca7d54b","added_by":"auto","created_at":"2025-10-16 13:59:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":435613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking of NDMS and 2-BS compounds with respective enzymes \u003c/strong\u003eA) Represent the predictive docking poses of NDMS (a), 2-BS (b), and acarbose (c) inside the active site of the respective enzyme, α-amylase. B) Represent the predictive docking poses of NDMS (d), 2-BS (e), and acarbose (f) inside the active site of the respective enzyme, α-glucosidase. Hydrogen bonds (green) were represented in dashed lines.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/6c5bb3e1df46cbb9528f10fe.png"},{"id":98428968,"identity":"839a046c-14a6-4a24-98dd-6afb2f48c803","added_by":"auto","created_at":"2025-12-17 16:42:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3916539,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/28023b9f-2e3a-49f8-9a9f-e03e8c4df9f0.pdf"},{"id":93689158,"identity":"c1202faa-89b6-453f-9f3d-186266d589c3","added_by":"auto","created_at":"2025-10-16 13:51:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1749598,"visible":true,"origin":"","legend":"","description":"","filename":"4.Suplimentrymaterials04.08.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7306006/v1/4e0e35737eec1a43658c1506.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDual-Action Compounds for Glycemic Control: N-Benzyl-2,5-Dioxopyrrolidin-3-yl-Methanesulfonamide and 2-Benzylsuccinimide mimic α-Amylase and α-Glucosidase activities to regulate blood glucose levels\u003c/p\u003e","fulltext":[{"header":"1) Introduction","content":"\u003cp\u003eDiabetes mellitus is a group of metabolic complications characterized by hyperglycemia, predominantly caused by insulin synthesis or sensitization deficiency \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Insulin and glucagon hormone levels are crucial for maintaining blood sugar levels. Relative or absolute insulin deficiency or large-scale glucagon secretion causes blood glucose abnormalities. Diabetes is generally characterized by urination, extreme thirst, significant weight loss, high blood sugar levels, and a decline in the metabolism of biomolecules, including carbohydrates, proteins, and lipids \u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Owing to autoimmune destruction of β-cells, absolute insulin deficiency leads to type 1 diabetes, whereas relative insulin secretion deficiency or its resistance to receptors, or both, cause type 2 diabetes\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Major macrovascular complications include myocardial infarction and peripheral arterial disease, whereas microvascular complications include nephropathy, neuropathy, retinopathy, and diabetic feet. In 2019, approximately 463\u0026nbsp;million patients with diabetes were recorded. It is projected that by 2035, the diabetic mortality figure will cross 592\u0026nbsp;million. Current antidiabetic drugs include insulin therapy, biguanides, sulfonylureas, thiazolidinediones, meglitinides, DPP-4 inhibitors, glucagon-like peptide receptor agonists, and sodium-glucose transporter inhibitors \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOne clinical approach to treating diabetes involves the inhibition of carbohydrate-metabolizing enzymes. These enzymes have a direct impact on blood glucose levels because they are crucial for the breakdown of carbohydrates into glucose. Clusters of α-amylase and α-glucosidase, α-glycosidic \u003cem\u003eO\u003c/em\u003e-linkage-hydrolases, a common exo-type, release D-glucose from the substrate at the non-reducing end. Hydrolytic processes are caused by the breaking of the bond between the anomeric carbon of the glucosyl residue and the glycosidic oxygen (C1-O). Since they exclusively release D-glucose, α-glucosidase and α-amylase do not appear to differ from one another. However, the anomer type of product\u0026mdash;α-glucosidase produces α-glucose and α-amylase, β-glucose\u0026mdash;essentially distinguishes the two enzymes \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe enzyme α-amylase is found in plants, animals, and microbes \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These metalloenzymes require a minimum of one Ca2\u0026thinsp;+\u0026thinsp;ion per molecule for complex formation and retention of morphology \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The amino acid 496 residues that make up porcine pancreatic α-amylase are 83% similar to those of human pancreatic amylase \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Pancreatic juice and saliva contain the enzyme α-amylase \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The brush-border membrane of enterocytes in the intestine secretes α-glucosidase, also known as maltase, which hydrolyzes α-1,4-glucoside-bonded polysaccharides into monosaccharides that are ready for assimilation. Pancreatic α-amylase hydrolyzes the amylose α-1,4-glycosidic linkages of a starch \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eα-Glucosidase inhibitors (acarbose, which neutralizes α-amylase, sucrose, and maltase; miglitol, which works on glucoamylase and isomaltose; and voglibose) are a new group of antidiabetic The activity of glucosidases is essential for various biological reactions, including (i) the breakdown of dietary polysaccharides to yield monosaccharide units that the body can metabolize and use, (ii) the processing of glycoproteins and catabolism of lysosome glycoconjugates, and (iii) the biosynthesis of oligosaccharide units in glycolipids or glycoproteins \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. At present, miglitol (1) (Glyset), acarbose (2) (Precose), and N-butyl-1-deoxynojirimycin (3) (Zavescae) are the three drugs relied on commercially as anti-glucosidases. Drugs 1 and 2 are prescribed to treat non-insulin-dependent type II diabetes. Drug 3 is used to treat Gaucher's disease, which is associated with disrupted lysosomal storage. In keeping with the growing interest in both natural and synthetic glucosidase inhibitors as potential beneficial medicines and crucial strategies for mastering biochemical processes \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Thus, the inhibition of α-amylase and α-glucosidase is necessary to control blood sugar levels.\u003c/p\u003e"},{"header":"2) Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Drugs, solvents, and equipment\u003c/h2\u003e\u003cp\u003eNDMS and 2-BS compounds were synthesized with alloxan monohydrate 98% (Chai 10G, Sigma, CAS 2244-11-3), acarbose (Alfa Aesar), yeast α-glucosidase (CAS 9001-42-7), porcine α-amylase (CAS 9000-90-2), Tween 80 (CAS 9005-65-6) (Sigma Aldrich), normal saline (Medipak Limited), creatinine Jafee (Kit Lot CF 11231JB-CentronicGmbH/Germany), GPT/ALT-LQ- (Lot-LIQ-1259 LABKIT, China), ACCU-CHEK glucose measuring kits (Roche), ethanol strips, and pricking needles. All chemicals and solvents were of analytical grade and used without further purification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Animal\u003c/h2\u003e\u003cp\u003eHealthy, 8\u0026ndash;12 weeks Swiss albino mice were purchased from the Directorate of Veterinary Research Institute, Peshawar, and housed in sanitized polypropylene cages. Mice were housed in the animal house of the Department of Pharmacy, University of Swabi, Pakistan, and nourished under standard conditions (12 h light/dark) at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C temperature 34 and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;15% relative humidity and were fed standard food (pellet diet) and water ad libitum. Upon reaching an average body weight of 35 g, the animals were inspected for in vivo experimentation. The randomized animals were adapted to the laboratory environment for ten days prior to the start of the study. The experimental protocol was approved by the Ethical Committee (UOS/Pharm11), Department of Pharmacy, University of Swabi, Khyber Pakhtunkhwa, Pakistan. The Advance Academic and Research Board (ASRB), University of Swabi, approved this study (study permission no. F. NO. DA/UOS/2023/1766 dated August 01, 2023). Following the experiment, the animals were euthanized in compliance with AVMA regulations\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Compounds chemistry\u003c/h2\u003e\u003cp\u003eNDMS and 2-BS were synthesized by M.S. Jan et a\u003cem\u003el\u003c/em\u003e. (2020) and further evaluated for their antidiabetic potential. The synthesis of the N-benzene sulfonamide derivative of pyrrolidine-2,5-dione shown in \u003cb\u003eScheme 2 (see S.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The analyzed compounds were synthesized by Michael's addition of ketones to N-substituted maleimides. A mixture of ethyl-2-oxy-cyclohexanecarboxylate (2.0 equivalents) and a catalyst (20 mol% of either 8-hydroxyquinoline or a combination of creatinine and KOH) was mixed in 1 M dichloromethane (DCM) and magnetically agitated for 3\u0026ndash;4 min, followed by N-cyclohexylmaleimide additions to synthesize the desired molecules. Thin-layer chromatography (TLC) plates were used to track the reaction progress (see S. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After the reaction, the mixture was cooled and diluted with 15 mL of water. Dichloromethane (3 \u0026times; 15 mL) was then used to extract the material, which was filtered and concentrated using a low-pressure rotary evaporator. Column chromatography was used to purify the crude mixture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe in-depth summary begins with the combination of N-substituted maleimides with different cyclic alkyl/alkyl ketones to create ketone derivatives of pyrrolidine-2,5-dione. All reactions were straightforward, single-step processes that were completed quickly and produced good isolated yields. To the best of our knowledge, this is the first report of a tricomponent non-covalent organocatalytic system for these reactions. N-sulfamoyl-phenylmaleic acid ([(4-sulfamoylphenyl) carbamoyl] prop-2-enoic acid) intermediate was produced in the first stage by reacting maleic anhydride with sulfanilamide in diethyl ether to form an N-substituted sulfonamide derivative. The sulfanilamide derivative was obtained by cyclizing the intermediate in acetic anhydride with sodium acetate. After reacting with the N-sulfonamide derivative, cyclohexanone and cycloheptanone were produced in the reaction mixture. Biologically screened compounds were \u0026gt;\u0026thinsp;95% pure, as determined by HPLC\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The physical and chemical properties of NDMS include the following: Molecular Formula: C₁₂H₁₄N₂O₄S, Molecular Weight, 282.32 g/mol; Melting Point, 160\u0026deg;C; Solubility: Soluble in methanol, ethanol, and DMSO Low solubility in hexane and chloroform; moderate water solubility; Appearance: White to off-white solid; and Boiling Point, 360\u0026deg;C. 2-Benzylsuccinimide molecular formula of C₁₁H₁₁NO₂, a Molecular Weight of 189.21 g/mol, and a Melting Point of 102\u0026deg;C. solubility in water is likely low, as is common for succinimide derivatives, owing to their relatively nonpolar nature. 2-BS is more soluble in polar organic solvents such as acetone, ethanol, methanol, and dimethyl sulfoxide (DMSO) because of the ability of the succinimide and benzyl groups to interact with these solvents. Appearance: white or off-white crystalline solid; boiling point: 325\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Toxicity\u003c/h2\u003e\u003cp\u003eBefore the administration of the compounds to lab animals, 5-2000 mg/kg/b. w., comprising 8 doses, was determined by the results of preliminary range-finding experiments from LD0-100. For every eight doses, three replicates were examined using both the positive and negative controls. Oral routes were followed for each group of five mice, and after implementing the predetermined operations, the animals were closely monitored for allergic reactions, behavioral changes, and mortality rates for up to three days. The comprehensive dosage schedules and animal requirements are provided in Tables\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.5. Invitro\u003c/em\u003eα-amylase and α-glucosidase inhibition\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. α-amylase Inhibition assay\u003c/h2\u003e\u003cp\u003eThe α-amylase inhibition test was performed by measuring the amount of reducing sugar (maltose) generated during the assay. A decline in the amount of maltose released was used to indicate the inhibitory action of the enzyme. The maltose equivalent was calculated using a modified dinitrosalicylic acid (DNS) technique. To prepare the sample solution, alpha amylase was mixed with the solvent (phosphate buffer), and various concentrations of the pre-synthesized compounds (500, 250, 125, 62.5, and 31.25 \u0026micro;g/ml) were mixed in this solution. A 1% w/v starch solution was then mixed after the compounds were pre-incubated with 1 U/mL α-amylase. The above solution was incubated for approximately 30 min at 37\u0026deg;C. After incubation, the reactant mixture was kept in a water bath for 5 min at 100\u0026deg;C. Instead of the amylase enzyme, equal amounts of buffer (20 mM sodium phosphate buffer with 6.7 mM sodium chloride, pH 6.9 at 20\u0026deg;C) were used as a blank. After that, 1 mL of DNS reagent (12.0 g of sodium potassium tartrate tetrahydrate in 8 mL of 2 M NaOH and 96 mM 3, 5-dinitrosalicylic acid solution) was added to stop the reaction \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The color intensity was measured using a microplate reader at 656 nm. The percentage of inhibition was calculated using the following formula:\u003c/p\u003e\u003cp\u003e\u003cem\u003e% inhibition =\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Abs\\:control-abs\\:sample}{Abs\\:control}\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003ex100\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. α-glucosidase inhibition assay\u003c/h2\u003e\u003cp\u003eThe α-glucosidase chromogenic inhibitory assay of NDMS and 2-BS was performed using standard protocols \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Briefly, approximately 20 \u0026micro;L of the prepared α-glucosidase solution (0.5 U/mL) was combined with 120 \u0026micro;L of pH 6.9 phosphate buffer. The ultimate concentration of the enzymatic solution was 20 \u0026micro;L of α-glucosidase (0.5 U/mL). The substrate para-nitrophenyl\u0026ndash;D-glucopyranoside (5 mM) was manufactured in the same buffer (pH 6.9). After preparing test samples with different concentrations ranging from 62.5 to 1000 \u0026micro;g/mL, they were combined with the enzyme solution and incubated for 15 min at 37\u0026deg;C. Finally, 80 \u0026micro;L of 0.2 M sodium carbonate solution was added to complete the reaction. Acarbose blank solution served as the positive control, and the system devoid of α-glucosidase served as the blank. The absorbance was recorded at 405 nm using a UV spectrophotometer (Shimadzu, Japan). The experiment was repeated in triplicate, and the inhibitory activity was determined using the following equation:\u003c/p\u003e\u003cp\u003e\u003cem\u003e% inhibition =\u003c/em\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Abs\\:control-abs\\:sample}{Abs\\:control}\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003ex100\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Alloxan-induced model and blood glucose profiling\u003c/h2\u003e\u003cp\u003eDiabetes was induced by administering intraperitoneal alloxan at 120 mg/kg/bw. w. to mice \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Not only was the symptomatic assessment performed for polyuria and polydipsia, but the model was also evaluated by fasting blood glucose levels above 200 mg/dL for diabetes induction. The animals were grouped into normal control (nondiabetic, saline nourished), diabetic control (alloxan administered), group 3 was standardly treated with glibenclamide (0.5 mg/kg), and groups 4 and 5 were treated with oral NDMS at doses of 5 and 10 mg/kg, and 2-BS at the same dose of NDMS was administered orally to groups 6 and 7. Glucose levels were recorded regularly for up to 15 days using an Accu-Check glucometer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Lipid profiling\u003c/h2\u003e\u003cp\u003eAfter 15 days, blood samples were collected in heparin-coated sample tubes. Serum was isolated and processed for determination of total cholesterol, LDL, HDL, and TGs \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e before centrifugation at room temperature for 30 minutes at 15000 rpm. For triglyceride (TG), the separated serum was analyzed using a calorimetric machine (MicroLab 300; Q-Line Biotech Pvt. Ltd.). Yellow gel tubes (clot activators) were used for TG quantification. Approximately 1 ml of TG reagent was added to the tube and mixed with 10 \u0026micro;L of serum, followed by incubation for 10 min at 37\u0026deg;C. The tube was then processed using a machine. Similarly, for total cholesterol, the same process was repeated, but the serum was mixed with a cholesterol reagent and placed in a water bath for 10 min at 37\u0026deg;C. The prepared tube was placed in a MicroLab 300 (Q-Line Biotech Pvt. Ltd.) for analysis, and the readings were recorded. The levels of total cholesterol, total triglycerides, low-density lipoproteins, and high-density lipoproteins in the plasma were measured.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Liver functions profile\u003c/h2\u003e\u003cp\u003eThe same isolated plasma was used for the analysis of liver enzymes (SGPT, SGOT, and ALP). Briefly, for SGPT, SGOT, and ALP profiling, the previously separated serum (10\u0026ndash;50 \u0026micro;L) was mixed in a tube with an ALT reagent (Thermo Fisher Scientific Company) and subjected to calorimetric analysis. For creatinine analysis, mice blood samples (1.5 ml) in a heparin-coated sample tube already placed in the tube rack were used using the standard kit method. A blank solution was tested to check the machine function, followed by validation that the creatinine reagent fell within the acceptable reading range of \u0026le;\u0026thinsp;2 mg/dL. The desired blood sample was then aspirated after mixing with the reagent and waiting for 10 s using a stopwatch, followed by machine readings for further statistical analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Histopathological Examination\u003c/h2\u003e\u003cp\u003eTo check the recovery of the pancreas, approximately 3\u0026ndash;4 pancreas samples from each group were collected from the sacrificed mice for histopathological examination of the pancreas at the end of the anti-diabetic study. The study was continued for up to 4 weeks after the administration of various dosages to the respective groups. A standard histological protocol was used (IETIE). Briefly, pancreatic sections were preserved in formalin saline. Subsequently, ethanol was used to dehydrate the tissues, followed by xylene tissue cleansing at increasing concentrations. Before embedding in paraffin blocks, the tissues were impregnated with molten paraffin. Fine sectioning was followed by hematoxylin and eosin (H and E) staining, and the samples were examined under a light microscope. For histological examination of the liver, three to four mice from each group that had the greatest change in blood sugar levels were sacrificed. This was performed using a standard histological method. The liver was immediately preserved in 10% formalin for 24 h at room temperature after euthanasia. The tissues were subsequently divided into sections, placed on glass microscope slides, and fixed in paraffin. The slices (4 mm) were stained with hematoxylin and eosin and viewed under a light microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10. Molecular docking\u003c/h2\u003e\n \u003cp\u003eA molecular docking study was carried out using MOE (2016) software (CCG, 2016). The 3D structures of \u0026alpha;-amylase and \u0026alpha;-glycosidase with Protein Data Bank (PDB) ID 3BAJ and 5NN5, respectively, were obtained from the PDB database \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. All water molecules were eliminated before energy minimization. Energy minimization was performed using the MMFF94 force field with a convergence criterion of 0.05. The 2D structural coordinates of the synthesized compounds, NDMS and 2-BS, were constructed and saved in a new MOE database, and the 2D structure of acarbose was obtained from the PubChem database (PubChem CID-41774). The substrate-binding site residues of \u0026alpha;-amylase and \u0026alpha;-glucosidase were defined for molecular docking studies. Flexibility was applied to all ligand atoms during docking to obtain the lowest-energy ligand-protein complex 45. The compounds were ranked using the GBVI/WSA dG scoring system. Finally, the protein-ligand interactions were analyzed using PyMOL v.1.7.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eSigmaPlot@17 was used for all graphical and statistical analyses. A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a significant statistical difference.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3) Results","content":"\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e3.1. In-vitro α-amylase and α-glucosidase inhibition\u003c/h2\u003e\n \u003cp\u003eNDMS and 2-BS directly blocked α-amylase and α-glucosidase enzymes via an enzyme inhibition assay experiment using acarbose as the reference drug (Fig.\u0026nbsp;1). NDMS showed significant inhibition of α-amylase in a dose-dependent manner of 85.49 ± 0.60, 76.28 ± 1.94, 71.08 ± 1.04, 61.37 ± 0.56, and 54.12 ± 0.52 at a concentration range of 62.5–1000 µg/mL, with an IC50 value of 49.05 µM. The 2-BS displayed percent inhibition of 77.58 ± 0.63, 716.61 ± 0.43, 60.93 ± 0.67, 53.65 ± 0.91, and 45.12 ± 0.12 at the same concentration, with an IC50 value of 95.05 µM. In comparison, acarbose had an IC50 value of 22.61 µM (Fig.\u0026nbsp;1B) \u003csup\u003e46–48\u003c/sup\u003e. The reduction in the activity of α-glucosidase by NDMS was 87.30 ± 1.42, 82.80 ± 0.41, 69.51 ± 0.59, 65.90 ± 0.32, and 61.56 ± 0.52 at a concentration range of 62.5–1000 µg/ml with an IC50 value of 30.93 µM, whereas 2-BS displayed an inhibition of 76.32 ± 0.87, 69.33 ± 0.66, 56.40 ± 0.82, 51.90 ± 0.45, and 42.34 ± 0.42 at the same dose, respectively, with an IC50 value of 120.3 µM (Fig.\u0026nbsp;1C). In comparison, acarbose has an IC50 of 23.78 µM \u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.2 In-vivo study of NDMS and 2-BS in alloxan-induced diabetic mice\u003c/h2\u003e\n \u003cp\u003eThe body weights of treated and nontreated mice were measured at different time intervals ranging from days 1 to 15, and the percent change in body weight was calculated, as shown in Fig.\u0026nbsp;2A. The data demonstrated that weight gain in diabetic mice (23.59%) was less than that in normal control mice (41.7%). Furthermore, diabetic mice treated with glibenclamide (0.5 mg/kg/b. w.) resulted in almost equivalent weight gain to that of normal control mice (47.8%). The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice have shown a good weight gain of 50–52.3% compared to glibenclamide \u003csup\u003e41, 50\u003c/sup\u003e. While the 2-BS (5 and 10 mg/kg/b. w.)-treated animals had a minimum effect on controlling weight loss of approximately 29–36% in diabetic mice. Thus, 2-BS is less effective in controlling weight loss in diabetic models than NDMS and glibenclamide (Fig.\u0026nbsp;2A).\u003c/p\u003e\n \u003cp\u003eSimilarly, the antihyperglycemic effects of NDMS and 2-BS (5 and 10 mg/kg/b. w.) with the reference drug, glibenclamide (0.5 mg/kg/b.w.), via oral administration considerably reduced fasting blood glucose levels from days 1–15. The NDMS (5 and 10 mg/kg/b.w.)-treated Blood glucose levels (BGL) of C. sinensis-treated mice were noted as 247.9 ± 0.9, 236.1 ± 1.7, 208 ± 1.9, 187 ± 1.4, and 178 ± 1.3, respectively. While the 2-BS (5 and 10 mg/kg/b. w.)-treated mice exhibited a decreased BGL of 251.6 ± 1.4, 232.4 ± 1.5, 223 ± 1.1, 208 ± 1.7, and 199 ± 1.6 mg/dL, respectively. The noted BGL decline was significantly less than that in the diabetic control but was still higher than that in glibenclamide-treated mice (Fig.\u0026nbsp;2B).\u003c/p\u003e\n \u003cp\u003eFurthermore, liver function analysis was performed by assessing the key enzymes, including serum pyruvic transaminase (SGPT; 61.4 ± 1.5), serum glutamic oxaloacetic transaminase (SGOT; 46.9 ± 0.8), alkaline phosphatase (ALP; 275 ± 1.1), and serum creatinine (2.8 ± 0.7) in diabetic mice. The reference drug, glibenclamide treatment, halted the liver enzymes SGPT (25.8 ± 1.8), SGOT (23.9 ± 1.5), ALP (188 + 2.5), and serum creatinine (0.5 ± 0.8), respectively. The NDMS (5 and 10 mg/kg/b.w.)-treated diabetic mice reduced the same markers up to SGPT (24–26.8 ± 1.4), SGOT (23–27.8 ± 1.5), ALP (193–182 + 1.8), and serum creatinine (0.7–0.9 ± 0.2), respectively. The reduction was significant at both doses compared to that in the diabetic control group. Similarly, 2-BS (5 and 10 mg/kg/b. w.)-treated diabetic mice reduced the SGPT (22-25.8 ± 1.2), SGOT (21-22.9 ± 1.4), ALP (187-191.4 + 1.17), and serum creatinine (0.6–0.8 ± 0.15) by respectively \u003csup\u003e47, 51–53\u003c/sup\u003e. The reduction was remarkable at both doses compared to that in the diabetic control group (Fig.\u0026nbsp;2C and E).\u003c/p\u003e\n \u003cp\u003eLipid profiling studies on mice treated with NDMS and 2-BS for 15 days showed considerably reduced levels of total cholesterol, triglycerides (TGs), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). This outcome was proportionate to that obtained with the recommended dosage of the standard drug glibenclamide (0.5 mg/kg/b.w.) \u003csup\u003e3, 7, 8, 16\u003c/sup\u003e. HDL cholesterol levels were also remarkably higher in mice treated with NDMS and 2-BS (Fig.\u0026nbsp;2D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.3. Histopathological Findings\u003c/h2\u003e\n \u003cp\u003eThe effects of various combinations of treatments on the structure of pancreatic β-cells were compared with those of the alloxan-induced diabetic model (control) and reference drug (positive control) groups. The cell morphology of islets in alloxan-induced diabetic control mice was altered by healing and fibrosis, which showed β-cell destruction, causing the cells to contract and deviate from their normal histological appearance (Fig.\u0026nbsp;3Ac). In the NMDS-treated (5 and 10 mg/kg/b. w.) group, a good trend of pancreatic β-cell recovery was noted compared to the reference molecules, as indicated by the red arrow in Fig.\u0026nbsp;3A1. Similarly, the diabetic model group treated with 2-BS (5 and 10 mg/kg/b. w.) also shows a better trend of pancreatic β-cell recovery, as depicted in Fig.\u0026nbsp;3A2 \u003csup\u003e16\u003c/sup\u003e. The NDMS- and 2-BS-treated groups showed significantly improved cell mass, islet cell shape, and total islet cell volume compared to the reference group (Fig.\u0026nbsp;3A).\u003c/p\u003e\n \u003cp\u003eThe livers of diabetic animals after receiving alloxan injections displayed several abnormalities, including hazy edema, lymphocyte infiltration, severe congestion, necrotic foci, and hydropic changes (Fig.\u0026nbsp;3B). The hepatic portal vein was also congested in the alloxan (Diabetic Control) section from the liver of a diabetic mouse, showing hydropic degeneration (HD) and severe congestion (CO) (Fig.\u0026nbsp;3Bc) \u003csup\u003e8, 50\u003c/sup\u003e. The NMDS and 2-BS-treated diabetic mice livers (5 and 10 mg/kg/b. w.) show a nearly similar morphology of hepatic architecture with an apparently normal appearance and good recovery (Fig.\u0026nbsp;3B1-2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.4. Molecular docking\u003c/h2\u003e\n \u003cp\u003eTo investigate the specific sites of interaction responsible for these biological activities, a docking study was performed on these keys’ pharmacophores. Docking analyses of NDMS and 2-BS revealed several possible binding interactions with the target enzymes. NDMS established a significant interaction with the binding site residues of amylase, with a dock score of 5.6607 kcal/mol, while 2-BS had a lower amylase binding affinity, with a docking score of -4.3213 kcal/mol. The docking scores of NDMS and 2-BS to α-glucosidase were 5.8803 and − 4.9448 kcal/mol \u003csup\u003e52, 54\u003c/sup\u003e. Overall, the docking simulations of the synthetic compounds demonstrated the significant inhibitory activities of α-amylase (Table\u0026nbsp;1) and α-glucosidase (Table\u0026nbsp;2), both of which showed significant interactions with varying binding affinities toward the target proteins (Fig.\u0026nbsp;4AB).\u003c/p\u003e\n \u003cp\u003e\u003cimg 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vCBYJwsrZMwGcPLkycmJEyfSdoQTFTBTlqkhzpy1z4DQNnnK1IYpCjQLx0XzS5yBR3/VS6Ys/mVCUbpYprbj1WIsk6uxdaB2U49cBtRfFz/4Uz9tKw3EemtbYVFGyhea+ofyUXKvXr1KflFeoLyIp+1VHNiwTXKJZcmR7zK4cOFC+kfB5uM9tl9+eVsgtkfxcNqP4ZI1ILcYR8hPsl4XtE30qb/GVfTXPmnUPslNYXJ/9md/Nt0mrspcd7vnIY4XiHLCsS9K/SuiTP7yL/9yus04jDLXdpRLDI/xIqdPn07/6BqlRSlL78TxHvNuqnc8FrStNE3jYO1UVzJF2oLv37+fwqsrrdqnmUqI0zj8sw+7u7spvcrQNv7V1U/arq7gUpj2CQO2lafCQHUiHS7G419hQD3YJ43S7e3tpbCmdMSZB9K0oTKpv2Afl8uAeqm+gnrFOqp+Sqd8SBfbF/MF9Y/kRVz1jfKHWD75sK18CMP1QXmU2Fa5xLKUD/9NKL8Sqit5CeWp8qgj+9SD+Dj545e3Re0kntoh2anu5B3jxTD8ySuWr7z5V16LQP5tqA5yqptoq7/kIJmo/vLHafsPf/hD+ie/khzUTv2vA8ptgzoSp2288B/7S/5qV96/Mc+SvCkDSMM+/gojXS5DtlWHHI1JOaVRHuSrNhE31jv6g+qOI4z6KY7iq52rgrKaWHiGfOzYsfTfZq7jKqNq4PRKhn/249VHJbh66/sVEOYO8fXr13rrO0ePHk3/VaelK3tgLYR0oDq9ePEi/fchN2upbqrzjRs30v+6yGUAuemHq1far9kN/7npsRp8KR/6hzZWfT159uxZkhcg29g/OOIQV1ekXVQDeyqnVbNNctE4/vz5c/pfBsorzhDE9evXp2uGWK6oW94WtZO4sjp8+vQp/Yt79+7VWz948uRJ+ie9zPd9+mEVIHf6rURT/Z8+fZr+GV/Xrl1L6ePskPMT4wXZHDlypPb9cX5QXJ1HJI+hE8dL23m6qX8jyJSxFWVWQjIqQR+U9ImWQjl+gTKZGetcz7lf/UTcWG8dbxznkXgu6BoH62JhhawDulRxKbYxka9VDIH8oiTChQkDEKclgiboH06M58+fX9naC+NgnYxNLpyMqBfKDsd21wlqHqTwZIqM6GTGyZf2lk40mPg4weqk10afk1XfflgmnIAxZwJ1LNUz96efVFcpphzkiILG1I8cIbaLMPJBjleuXKl9h0nbeJkHpY/LBH3gXIucq9loUq7IE9m9fv26jvEDFK/0icYyLPOCVvQZB6tkYYVMZTW7vXjxYvoXDEhAeFxxaPDzzz7+iyCFyRU3BzvEq2J1UNPBQJo+qG4aGKvo9IOC7JEjbZJsJfMmHj58mP65iIoDXm2NV7jzHlhDYgxy0cG+TAXFCYs20/YmJU/diUOZxHn37l0d8gNOeOTBCXZ/f7/27UbHvtboKGveflgV1K3rpCqFopkW9Vedm0CRqQ9xEcmeMrvK3ST5eGk7Tzf1b+TMmTPp/Et+ixwj3KPTRwlyERvrB5SNA57uAcYx7bt582baJ670BtafEvOOg5VRCaFIR3CiEmaKF111hV2H/hzOPkS/SqlOt6uBMLMNCo9hygeiP/Z/EfOtFPj0v7qQmImvbRxQf+2XyuxCeTWhushRRqxrrJNkEOtMPXK5Ql5vEdNGWUJTPqA6Ei9Pj5/2Y10J64J4JbZZLvk4k38ThJWIZUQX84plUV/CcOzH9se2xPYqLk7rpjjlpX3KiflEv5L8FqEtfSw7d4TFesxTf+3jyAPyNuGiPIH84/lvHVCPEn3GC+RtU5shl09+POGn/fPnz0+3cfEYifFwEPejf0RlxraQr4j5UlcR6634pXMBNI2DVUEZTZR7sqKUaN3oIFr3QD8IQ5Hd0DjMcqHtuYsnPoG/+cGQ5BFP2nLx5B0VxboYknzmJV4gyW1Chuum1GcHeg7ZGNMPTGDViYajcOpgyKZNMwumUIh9WCnoyR/90R+lNdAx3jezSZAXS0VRnpWCnt6cexgZ/Nee6DTd9QnVjGIUJ7EhyG6IHFa55OMY9vb2puvVEY+dWYYkD+oS4SKLdVj5b6KeYx4v3GjIOV2gkHVT3jZT6jN/fnFFWHbNWC7dWEazWB7tWD7jo9RnNlkbY4wxA8AK2RhjjBkAVsjGGGPMALBCNsYYYwaAFbIZJPErMMt6nITHVpRfzN8YY4aAFbIZJDxKwiMQfdGr/fpC/idPfv8oiTHGDAErZDN6UMZ9viYUvwZjjDFDwwp5Q/CyfZlM5ZZlmh0zbaZk5KMwvcie/3k/tK83LkViGl5WYIwx68YKeQPoayu7u7vTT93xxp/DPnND4aJc9YpJvrYU4U1XMYz4fDoReIUh/ihe8sDcLdnyBRfe7kbaEnw/lf5oeljfGGPWgRXyhtFHz/OPwR9G9Nk/vcs2riFrVhu/RUv8RT603wSfa+PD5My4Sx9JN8aYVWKFvAFYy0TZoAC4sQh3GN7fugw0Q8bp+7M5LAf0/dC+IC/yZOZtk7UxZhNYIW8ITKtSLJ6RfUcfG+ej5RA/EK6ZMKZl0KNLOfizHDDvh/ZRwpTHi+5xsWxjjFkLlUIo0hFsWmiTHd91Jjw6vrN6GGiTC1Qz4KlM9EFyfUi860PkxOP7wopzK3xrNX5oHxfLIR5O5bG9SaiD+YHl0Y7lMz5KfeavPa2ILtnlnx2D0uf4tgmPqW4OIqO/+r/+jfT/X//Lv5v8tVPH0/bY8Zhpx/IZH6U+s8l6A2BqvXjxYuoQOdaRdYOXMYvw395/mvzzf/rnk//vf/4/kxf/+mXta4wZC1bIG+DKlSvp5iGukuS4CYk7hI3pC1YW1swFM+J/+fzfpFnylT+9XPt+v0Ndz21vM8iDdsbjCsf9AMgp95uHmD7KHNlyE+EyUP5csJNnLGcZWD7Dxwp5A+jRnOi489qYvnBy5VGtuMTBDPnjf/+StqO5WmNrWSfGISJ58LSC7q7H6sSxxfGGnHiyQcfbvBe/Sp/DDYg3btyo9w4Gz8GLly9fpsf29LjfQbF8xoEVsjEjg1kOJ9j8sS/M1P/2X3+/C/1fPPs36V9wIubu8207gUFJHhFmVnqJzDLB0rWqi2kUEI9GHhTLZzxYIRszIjAlcpJrmnX8j89f08z4/7jxDyf/8T/959r3B8xg7t69W+9tB23yAMJRNsz6csWgMDmZaXOzbgkubpBpzCe+ghWTKlYJtslTlMrNUX2bHu/rC3lbPuPBCtmYEfHu3bv0f/To0fQvMFf/vb/7t9P2P7ry9yf/7Om/StuR48ePb90z1iV5AG3FLAtNlgFurOSVq3ragVesoiRQYLyARibSklUBcyyzSsy7ei0rz8krP0yqXABRBnkKlYsS5J/9EtS/z4dTSlg+48IK2ZgR8fnz53prFszV//s/+S3d0HXxH/zj5JebrfVK0q9fv6b/baAkD+BkrfVSTJvxQgTFgmJAWeA4+WPSZS2UbWQlc2ipDJRIPqvk5kyBMiE/QfmxXOCfffybwNR8kBcHWT7jwgrZmC2Bx53kMFtzx/VhhxO+ZmeaDXbBSZ7ZXbyJKAcFgUIZO5bPsLBCNmZEaJYbYSb8d07/rXrvO5it//1/+E/JlC00k9mm592b5JHDzE53AOtOc83MmMVpZsg6JuZXZmSYWlE6JZ49e7bQ3cOUi+JTmfyzH2eKEWZ/KMBFsXxGxrcWOoJNC5ZdM5ZLN20yqk6GKbyanaT9f/J//l/f/sr/8teT+3//239PfvDX/ub/NvX/vx88Tn7VSXf6GtIxMY88qtle2o9OcaIjXu4Pe+G1tshL2/HVq8iQf5HnE9MpLq6aMTbGZx9KftS1DeKUUFmHWT5DhHo34VdnrgjLrhnLpZsuGTFTYebAs5jzwEyCNbx8XW/orEoeQwezLzc+dR0vls/4zielPrPJ2piRwTPFmOrmedyDkzIKeWzKuA+LyGMMoGza1mn7YvmMBytkY0YIJ1ge9yjdfRrRYynbNkOKzCOPMcBaLndAL+sCyvIZBzZZrwjLrhnLpRvLaBbLox3LZ3yU+swzZGOMMWYAWCEbY4wxA8AK2RhjjBkAVsjGGGPMALBC3gA8fsKifpNb9C5I8tRbdiLKtynMGGPMcLBC3gA8gnCrflUdt+5ztx3/wLN1y+TDh+9fVjHGGDNsrJAHgt7hCnqP6zyg5Lf5OVNjjNl2rJAHAqZqZrN8IUWfHkMx5ybn6IcjHW9hYju+ZF3hDx8+rH2+I3/e2tOUTn44mc+jn03fxhizGqyQNwwmahQdX0bBdB1nuXyYG9M2/syAeeMSHwlHaeMnszevxotIaRKHj9JH4qvm8nTkz9dduDAgnr7Wgh8mdZu/jTFmdVghbxgUHQoWZcdMVGjGjDJEYbPN586uXLkyefXqVfJDoTZ9loxwmb+J35enT5+mf9Ly8XHKZEaO4ufCgYsBm8WNMWY1WCEPACk5lK/eOyw0Q8ahgDFna3aMUl70ruw2UMQqk/IoF7+dnR2brI0xZkVYIQ8E3WXNzJRZafxAOODHDJq1XxxKkpn1/v5+Co9oxk2ar1+/1r4/kyvzCxcupH9mwkB5WrNGMVNHZt/GGGNWQDULKtIRbFpok138KDdOH9iOHw9nu1KA033SKE41O576Q9xXXtqPZekj5fKrFPc0TOmin+I35b8o5GHasYxmsTzasXzGR6nP/LWnFWHZNWO5dGMZzWJ5tGP5jI9Sn9lkbYwxxgwAK2RjjDFmAFghG2OMMQPACtkYY4wZAFbIxhhjzACwQjbGGGMGgBWyMcYYMwCskI0xxpgBYIVsjDHGDAArZGOMMWYAWCEbY4wxA8AK2RhjjBkAVsjGGGPMALBCNsYYYwZA5+cXjTHGGLNcmlSvv4e8Iiy7MpZNO5bPLJZHO5bP+Cj1mU3WxhhjzACwQjbGGGMGwMIK+fLly2najbt9+3btO5mcOnVq6v+nf/qn0+3oiCN+//33mbDnz5/XIT+IeaqsWP7Hjx9n6jAW8raz/+DBgxRGe2JYTh6ODHL/JlnOg+Qe+wsoq62M2K55iX1NHwMykR9ObT2sIHPJotTH+diKcfukHzs6P2gMtaFjZoznkEWJx1SXjEryiecBHZNx3LEt4vkal59TTM23FjqCv926dSvFwe3u7ta+3/0F/oTfv38/7e/t7U3TfPjwIfmB/C5dulT7fCfGVxnkdfLkybQNbMf9IUB925BcaJ+g7TEdcXJ55OR5QFeaPtCH6h/yU/8BZSoMucd+1JiIfjmxjU2QRywPaOPQ+hjiWF8WbfJBripTx0YT8XgE9Wff9ENi3jrG8cN/PpYi5L2M42WTLNKHbTKJlORDesLiuYexFeuibfzzeKs4bsZEqc9ae7KUSCBUKRYcgpa/UHgcAOrM2NFs5/kAcRWmkwz55yfnoR1U1LeN2J5ILruudkm+AnlF+S1K3gc6oPI6EaY6E9ZHaXbJhvzIN0L5Q1PIq6pTm3zyvi2Njzye+qhv+iHRNV5yiK928l9KT9vzcTZG5pUPY4E0XWO3JB/GfVOZ8VwAOo7zMcd5Iirow0ipzw68hnz16tVJJfi0ffHixfTfxZkzZ9L/q1ev0j9cuHBhUg2QtP3ixYv0X+L8+fOTqpOT6UNmlJcvX6b/MSAz4enTp9N/5NGjR/VWP5B/deAkOchsdOLEifR/EJSH8jx79mz6f/v27Uz+x44dm8a5e/duGgMyS0WT1arJTbQimtWimSya7LRMANG0pnYhW+LInM4+5Z07d246DtmPZcX0hJFWect/EaLsqUdp3Md4lH/8+PG03Tf9WKGtoHbqP5c58Tj/fPr0KfXJYTKh/vrrr2iD6bHaRJt87t27l875+ON0Pnvz5s10nAHbpI9jDp4+fTo9n5hZlnJTF0oEZcrJqc+azZEjR+qt2QPl5s2b6f/Jkyfpn46+cuVK2o6ghKorr7T9+PHjmUExBj5//pz+85OmBjhOJ5Y+oAiRAyfYO3fu1L4HByVEv+7s7MzINx50gn6k/7lY4mCnf1BYi0KZUR5deXGSqK68U9lcoKi+Gpf4A/44Th74VVfqqSxAflwY4k9enLCQAbIlzuvXr1Ne7ANpyZ/4nGCIz7bSE4ZcpLjpJ8LyE9QiIBPq0UeR7O/v/3QczZN+bCD3LpAJSkXjFej/w4DGH+dtZBAvSEWbfFDUnAM0zq9duzY9j3OB3sUyxv+2srS7rN+/f5/+6SydsEp8/fq13prtHCkTTl4oJBRXqfOIy4CQYo6DYuho0Mb6ckLXwOdEP88VJHE5eFAmy0QyRsGhTNqgTzkRcrEE6stF+4R+pWw5ZNIGMz3KRtHI8sIYou4aQ4xR4jDLv3HjRvKLckfh6kKA8cQ4pB3IlvqQD459TlgRyorKTZYL5CLFPU+fdiGZUMeui1EuPvLjaJ702wgzN5SKxisXdGM5fywTZsvIIqckH8lIxzfhjO14Tm+DsYaSN80s9bGnrpOm0MmME1YOJztgAJSutuKVLAND5fYdFJtGpuqHDx+m/6ETzegcTJzgBRdNXAhg9eDkvg5yawInCcYE2yiaOK50oZgjK0UOY4k85OahVNaq0IVYG8imdKHWJ/3QYXanccAFEW1iHEpx8I/CyC9IZE7ddnL5NNFk8SrJR3KUfCOMMy52Belz5Uu4lLxpoDrpFOkInlnAF9VMYiYdC/js4w/VwZL2cWwLhVcnxGm4qE6waZ+8gHKrgyxtg8oYEl31kZzUboEfMgDaRdsjhOVpAJk0+S8D8o15U0f1XdymrorXVHfRJZumttDu2Oc5eT00VvDXNpBvPl7YJ20+rjS++Y9tUVl5nfKyFNZV95xYty664tJejacm5ilrU8xbxzh+msYS0H/k2zRmxsZB+pB2SwaRNvkgUx0bcWwrTb4dicfRYabUZ6092dbRdALhuPyAl9DpNMWJLp6cSCt/peNfHR7T4TQY1OFybSedTUCduohtl+uSHU4HiYh9sawBH8trUo4Ky09i8m9TQISXaGoL5csPl7cforyUB/XMZay0MX6UGdvyjyf16K82xzGIXz4m8zh9+0Zpm8hlESH/vIx8vy39UFmknhoDsf30EX4aA9rH5WN8TMwrH7UZF8+b88gnHqeRmCbmDYThTLnP/C7rFWHZlRmbbDCHY8LTutmq8diZxfJox/IZH6U+W+oasjHGGGMWwzPkFWHZlRmTbLgpRo9F7e7uruWGFI+dWSyPdiyf8VHqMyvkFWHZlbFs2rF8ZrE82rF8xkepz2yyNsYYYwaAFbIxxhgzAKyQjTHGmAFghWyMMcYMACvkDcAr7FjUx+ljHPEVd7jffvttZl8fm9AXg8iD19fFF8PH+Pmr7VRm0wv0CYv5bJI+sml6bZ8xxowdK+QNwDuPb9UfLNDn7/RO7pMnv3896M///M+n7+hmn3f0oph4lyz7fAiBuALlhT+OvOOnMAnj3eCEQfyYAArww5reQd2HPrLJ30u8SZoucIwxZhGskEeEvsACKKWoSON3bfmCi8KYTfL1Iz0/e/369ZkvN6EALzV85MN0g9WCCyNjjFkGVsgjgi+n8JIKmXJRyqXXOWr2/O7du5mZ9Dq/yrRO8i9ACS5I5Ic1QEQzeDTXa0kAJ9M4s2DiRLM/5ek7x/ixH8uK6QkjrfKWvzHGzPCthY5g00KX7OLHDaKrlGcd48dHHCLyw5FHE/El7vzHF+xXCuSnPAlf58v18/Jz+sgmhzaozXGbdLQZSC/ZSCZRxpQrORCH+OyrfPKR/EiHi3WK26RXGqUnfh+Ia35gebRj+YyPUp95hrxBWCet+mDqqhN2HVKGtWTiVif8yePHj6ez5cg2fHN0XtlgsqfNzEAx0QMz00rxTtecMc8TB/ncuHEj+UmegPkZCwR5XLt2Lc1+sUBoTZt8cOzrm95Cs2BBOZTNN7qpe6WsU1nGGFPCCnlEYCqVuZMTPgpDykegFB49elTvTSanT5+eiYOCQFGMGZSfzMIyASMbtlGusX0o4SY+f/5cb82C8owXAvNQKssYY/pghTwy4l29+fows2XdZISS0l3ZKCjdWc3d1podjhXNauVoI9YCLlAixMMv3lWOTLQWLwhHXtyZHmUTZf3mzZt6a5LKunLlSr33naayUNCeFRtjelOd0Ip0BJsW2mTHWiPhuEpZJr+4VomrFMbMPuuPrHHyH/1F9JOrFEQd+iOcPCKUrzDqsA4oq0Qf2cR2CdqlcOWBrHJ5KW2Mr3KgSR7Ejf6sDwN5Rb+4j8vjxHLaUFrzHcujHctnfJT6zF97WhGWXZmxyYaZMo+ble5oXzYeO7NYHu1YPuOj1Gc2WRtjjDEDwArZmBZYc2bNmDXnuD48FFj75mobF9e8S+jmN8WdN/0YoQ/VxjYkm22WhRk41bS5SEewacGyK2PZtDOPfOK6NOlYMy9BeL6OPU/6TXGQ8cLavtpI20rr+Kz1696AsXEQ+ZjNUOozz5CNGTHxEbdK2RTv6uYOfJ6ljq9Yhb7px8rTp09nnjnnzncem8thRsyz503P9RuzLqyQjRkxeukJ5vT4jvIICohn0XkXOubY+AKTPunHDO0+evRovff9Of0vX77Uez/gwqSaoKTtKB9j1okVsjEjh1kdszverc2acA5vFbt161Z6/lpKJ66RdqUfO7y/vQtdmGBBQCEP8X4Bs/1YIRszclAiUrQPHz5M/xF9JUyvU+XlMFHxdqU/bGApKL3JzZhVYoVszJbA+82bQBmjlLsopR8LTa9UZV2cN9oJ1pB5nWwXx44dq7eMWR9WyMZsCdzAxPeuc3jNJ49uaVYcb3SKlNKPBW7aYqYvhxmadtIuwAyNOVrm6RKkkTXBmHVihWzMSMlnhJhadZe0nr0FFBCzX957jt+FCxeSwmlLvy3QTtpP+1gn113majv/XKhEOeh98MasG786c0VYdmUsm3Ysn1ksj3Ysn/FR6jPPkI0xxpgBYIVsjDHGDAArZGOMMWYAWCEbY4wxA8AK2RhjjBkAVsjGGGPMALBCNsYYYwaAFfIG4G1BPIeG0+fe4kfUcb/99tvMPi8wAOKzTx680IB0XcS88w+vt328nbB1f/mmj2zie5iNMWZr+NZCR7BpoUt2t27d+nb//v167zt8QP3kyZP13vf9mA/xSQd8UJ2wPI8c8lAcpdGH2PkvfbydNMSN9VkWy5DNUFB/LJMu+Rw2LI92LJ/xUeozz5BHhL7aA7wOsFKwabsNXoV4586dtE0aPsOnL9m0fbydNGP/2MCqQVZ+zaIxZllYIY8Ivme7s7MzNeWiYKVs50Ffsun78fYxoHcTy4n4nuJofo9mcLaFlgRwMo1j5pf5Hn/2KY/vB3NRhB/7+TuRlZ4w0ipvm9yNMY3UM+VGOoJNC12yw9RJnNy1maxBfrhFzKXRLE0emLEFYTJnA9ubMlmrjdG11SXWPW6TTm0kPf44ySHKmHJlKlfbZbpXPji2SZeb0eM26ZVG6YnfB+KaH1ge7Vg+46PUZ54hb5DqZE+vTF11wq5DyugTc9UJP31ST7PlPjDLe/ToUb03bOaVDV/x4cs+zECZ+QMz00rxJksCYJInztu3b2dM9eQPmJ+xQJAHXwaqlGmyQGDmpz7kI7P//v5+SiM0CxaUQ9lfv35Nda+U9dZ9SckYs1yskEcEplKZOznhozCkfLrgW7Ag5QQojEU+3r5pUH4yC8sEjGzYRrnSLkGbmtA6eg7KM14IzEOpLGOM6YMV8siIjy2hTJl5dYECY1ao9WblscjH24eAZrVy1BlrARcoEeLhp4sRwEqgtXhBOEr94sWL05kzRFm/efOm3pqksvjof6SpLBS0Z8XGmN5UJ7QiHcGmhTbZsdZIOE5rmXGtElcpjJl91h9Z4+Q/+neR54sjH8G2/CNaA8XFtdFlkJcV6SObSvEl/0hsh/JAVrm8lDbGVznAtvwpF4gb/ZENkFf0i/u4PE4spw2lNd+xPNqxfMZHqc9+4acKbEQmQDM/ll2ZscmGmTKPmy1yR/sieOzMYnm0Y/mMj1Kf2WRtzMjQejkuPrKVE+OxbBHBtI7/tsNShGTQp73EjzfnGbNOrJCNaQGFx5oxa85xfXhTUAfWwLm65gY06pUrW5CiVjyemdYNgeTBXeSHAS5KkAHu0qVLnU8lxPsGjFk3NlmvCMuujGXTzjzyQcFwIxp33Ufwv3v37vSmMvYvXLgwNbujxFHSY+iHZY0X2oysSnfDc6HC3fdPnjwZ1R3zPp7GR6nPPEM2ZuTEt60JFEp869oY7p5fB23maJ5EOHPmTL1nzPqxQjZm5DQ9WsUjXHqkzXyHl7lgNWgCU/VYXppjthcrZGNGCkqEN5Q1gXLhpTGYxnCsgx/m2Z/Wz5suXjBlsy5vzKaxQjZmhOgFJ22wRoXjNavc0LTtLylBseoCBCclDFgMSo+tPXv2LN3kRpr4wZCY3ph1YIVszMjQ3d66kavtzmAUN2ba0kx6m+CCQxchOK2bs26sm7RQ2vnd8lgTlIY70nn7XUxvzLqwQjZmRKB8mc3pIxg4fSObO6n1WA//hPEN7fyOYd1hDcTZVpjh0j7NeHG0mwsZzab5N2Yo+LGnFWHZlbFs2rF8ZrE82rF8xkepzzxDNsYYYwaAFbIxxhgzAKyQjTHGmAFghWyMMcYMACtkY4wxZgBYIRtjjDEDwArZGGOMGQBWyMYYY8wAsEI2xhhjBoAVsjHGGDMArJCNMcaYAWCFbIwxxgwAK2RjjDFmAFghG2OMMQOg8/OLxhhjjFkuTarX30NeEZZdGcumHctnFsujHctnfJT6zCZrY4wxZgBYIRtjjDED4EAK+fnz52nqjbt9+3bt+zOKk7vLly/XMSaTjx8/zviz/+DBgzp0u6BdUQ7REXbq1KnpPtsi+v/xH//xdDs65DYP6kOVE/sBt25iGzU+cnnN28ZtIx53bHchmSpulDEuHofbAm3q07Yoiz6y3DZod4l43P3++++17yRty1/njQi6IJd7TLOt5/Wl8K2FtuC9vb0UHt3u7m4d+jOXLl1KcT58+DCzf+vWrbR/8uTJb/fv30/bylv7Y4T6l6DNkgPbsd25DCKkkbyAuHFfMu1LU3z8BNtxf1l01THKRCAPxsjQiPJfFm3yiWOgaYxEmo4j/DT2gP1c1kOja7zkxPHDf6l9nK+iLMbKvPIRyKmUNo4L5BSPvXhOwD8eA8ozP29oH3kTvg1yPwgluS88Q3727NmkEiq5Tiph1779uXHjRvrXbIe8xNmzZyfVgKj3to9Hjx5NTpw4Ue/9gHbfuXNnul0N7pkrzYcPH6a0Je7evZv++8wgdZVK/0VevnxZb33P7/379/WeyeGq//Xr1/Xe+tAYYIy0HXvnzp1Lx5HGFBw5cmRm7O3v70+uXLlS720Hjx8/nraJ/52dnbSdc+3atUmlUA7ljI2xe/z48XrvZxgnGjenT5+eXLx4MW1DPEfcu3dv5nzD2KwUeb33HcKVhrHHmG06/5kDmKyjUpFypeP6IuWhtHQSB47MI1E5HVaQ8atXr5IpDXf9+vU6pBlk2XewI2viyYxUUuJNJqkhEk1iOEG75BfbEs1x8YQsUydOMsEERxyZN9mnPBQeF5L4sR/LiukJI63yLsm6D7FvqUc8OUaoL2OBMUGZWlLKx8abN296jZexgKxBbdJ/LnP2uRjFcSxIPocFJlRtF2JxTHRNBLrGTwxnXLbldeipBmSRjuApbWYhUZ0cUn7RYRaJYP6I4WOmb/2jea0JwnKzkCAsyiuXZwnikaeWGMib/Rzy75vnPHTJRmav3DXVUTC+1J64TTqZx9RmHHGA9hEHYl8Qh/hRxuSDY5t0kqOI26RXGqXvK0vidqE8SzKhfbQn1lkyEfirvUOmjzxE3idAetraRp84Q2Ue+QB9HsdFCYXjdLzkaIxFyL8pPv2i/A47JRks5S7rT58+9Z7NVp1HTZIpjRlGNMliHiWs6ri0n98YcBiRXEuz4+qAmMrsy5cvtW87xGPGdvXq1bT/66+/ziwZgGYUWCo2QXVQp3bJMV7aYKZIe5gNYlUAZkvViWF6hc74Is7bt2+nlhnaR/6A+VlWGsyZyAT5I2PqQz449jH1RjQLFpRD2V+/fk11p3+WKUvJhDo23YxEWxkzsc6fP3+uQ7/z4sWLyZkzZ+q9ww39S19tOzqudUy0QRzGWXUhl44pWR8E++fPn++VFzAmdZ6JVinzgwMrZJSmTBCcGOgkTmhypTsXdXLSyTOajOg4TiD8m36gTFAifcyhR48e7ZQta0ZDXTLIxxhtZvywzQkERShK7cyVk0DJkYfcPKx7vHIMcZw0wcVB1wUaF9KbuuBaFpzYNQ5oM+3hpK/jgH8uhvooDdZNt41cPlyE6aJTEx+2c2Ub0cVlBLmyfqyL+r7QD1z8mGYOpJA5CaJQ1eHMPDgg4gmt1GG6QtKg4EaMeNVEh8cbCUw7DHSuZCXPNnQS1sUSB2k8sXPgSrlwoJYuqjZFPsZoO+Mnn+Xr5Bzrzxjjqp6TkiBc400zZ4gXiay1injTkGgqCxlK1quCujQdY7RD92lAXmcprLHDRaPGgcYsY5kxDax/3rx5M22XQBbbtpYucvnEfR0vbLeNU+STj2WOFd2/wJhvU+g5T548aV2/PtRUnVGkLZg1AsKjY+2giTyeXKU86hg/1u+awsYIbeiCNqq9yDOnOmCm4bgo3yj/KCv5d8kv5q2y8/Lklk1bnk0yieMCRz1zGD8KVx7VbDc5+eOUNsaPso9ylbyJG/21FhvlhV8uvzxOLKcNpW0il0WE/GMZsY3IIUI+ud9QydvZB42BKA/6CD/6RNu4rmNl6CwiH9DYFFE++fEk8uMpD4/jU7KPssaR/2EHOTThd1mvCMuuzNhkw0yZR0TWZcL32JnF8mjH8hkfpT5byk1dxhhjjDkYniGvCMuuzJhkw5qz1pt3d3fnvollETx2ZrE82rF8xkepz6yQV4RlV8ayacfymcXyaMfyGR+lPrPJ2hhjjBkAVsjGGGPMALBCNsYYYwaAFbIxxhgzAKyQjTHGmAFghWyMMcYMACtkY4wxZgBYIRtjjDEDwArZGGOMGQBWyMYYY8wAsEI2xhhjBoAVsjHGGDMAOj8uYYwxxpjl0qR6/bWnFWHZlbFs2rF8ZrE82rF8xkepz2yyNsYYYwaAFbIxxhgzAJaikJl+y12+fDn53b59O/1vgufPn0/r8/Hjx9p3OJw6deoneT148GDqh/vtt99m9n///fcUj/jskwdtI10TMT+lFcpDZUeUpindOugjm64+JbxPvLHCsSVZlPofYrzYl1HGuKZxMHbaxriIMsBt8py1bmK7S8eJZIjjnCry4xFHHjruotP4ZPzlfqaBby10BH/b29tLcWI8+V26dKn2WZz79+/XW/ND+dTjw4cPtc966ZLdrVu3fmofsjt58mS990OWgvikA9pFWJOMSCf/3d3dmTxjufzH9E15rYJlyKYN4m2y7w9Km3zoTxxofPCfg/w0VhQPebAd5cL+uvp9UbrGS07bGBeSoYhyHRvzyodzY5RP07kaf40r5KLxo/2I0uf+MQ/F0XlrrMfmsij12YFmyOfOnUv/lXDTP5w9e5aS6r3F4SrqzZs39Z6BT58+TY4fP562T5w4MSP3yJEjRyZ37txJ26dPn55cvHgxbcPjx48nV65cSdv87+zspG2ubtnmCjZeDY+R169f11vbx9WrV5MDjrXqRDf58uVL2o9w7Fy/fj1tK96LFy/S2GDsiP39/el42BZKYzzCcRF5+/btVK6HgWPHjs3853D+YNwAcqkucidfv35N+1F2nDcuXLiQtnOZcr4iD+K8fPky+TH2GItxDJofLKyQddKmo5qEqw7IzRjsg/aj+UN5YjriIHr16lXyj+YObWN2U1yFbbvJ6fz580kuMsMhdyneSOyPhw8fTh49epS2ZbZUuP7VJ1xIoeSvXbs2SrOSTGy0ORKXMDRugPFCOzWG4vgpjSvKaIq/SY4ePVpv/eD9+/czijrvc4Hizv3GTNcYF3mb8/Bt5saNG+kYh6dPn07PD11wMQdRdlzknTlzJm1H/yjP6M/x1re8Q0maJxdoC8YcQbhMESUwHyoO/zI7ygyiMmJeCot5Y4ZSfMLJR/Ewi+AUBqRlf1OmEcpuI7YnOskH1KaI/HAySTZBuxVPciRtzB8Iz2WktKuiK+8+ssnJzZTEpx24fByxrzg4xkyUDfvKC/BnX/WK+a0CyuhLqS5qp4jyEcgm9xsi88ij7xiPkEbnjTEyj3yExj9t7wLZMX6aKPnrmIrQL5S5SH23jZIMFp4hy9TBlXgJrlarzpxeIfHPvq5ioRoY9dYPctNHpOrkZEKhXK7ugCs3Xb3JbwzQ9qoPpo62daElgWrAJ9Nc6aYVZK14WBqizLsgbXWgbXTWMK9scjOl4ApeJjXg6hx5YFmgjZQjUyVjExhDuuoH9plJkrZScmmGMQSYocsSlaN2apaPfGKbIM5uDjPPnj1rPedsGzquOaZYduxaoopWtkjb+YFjRiZvwTlbx9gYLXDrYGGFrAGMgJs6Zh4FINqUu/l+ApasUSLInpNuG8RDiQAHSOwv/qur1hmTUqTkv2kYW1I0uLYTA7CWNS9N67JDghMaSxht6GKGizLGQH6C1BrfmEEOGgcsR8w7xsVQx/pByeUD3FPChRiyQinLfN0E5xzdi5Dz7t27YljbOaVpEma+s7BCjoKNNw2BZm10OAdDfnAs6ySgmQo3G+iGg6HMXlZFXLfkgECebSBzLnQkc2aFzIyAK9+bN2+m7cgiF1PrhLbE2TNjETlo7Ri5gE48zA41BmkbMmiDGfXdu3frve83Pg1pXGlGo5l921o2J2Taks+kJY+xg6VD40AX9H3GuGA86EbJbaRJPsCYFqVziC76dO7Ix1nTLBgYn20Xi0+ePNm6GwmXRtVRRTqCE9XV6HRdQK666qpDfw5nH7RfDYbpegaObWD9S+HkF+NHqoNvGsY2sH4hv7iOtk4ouwRtyOsXZYDb2dmZ2UcGtC/KAtdElEkuL1D5UTax/FXLjDJK9JGNxlAkjjPSxXhN4yEfc9qWvKIMCc/94hhfNuRfItZBTvWjbWqfZED8JkizyjYsE9oxL01jXOMgjh/k0zSexsS88onHSpRHlI/GT3SEC+KUxlaUOcTjDzd2eS8D5NCE32W9Iiy7MpZNO5bPLJZHO5bP+Cj12YGeQzbGGGPMcrBCNsYYYwaAFbIxxhgzAKyQjTHGmAFghWyMMcYMACtkY4wxZgBYIRtjjDEDYCkKmVey5W934q0upfcsH5T46sRVvFWKPFdVd2OMMaaJAytklDGvpstfocbrCnnP8ipe0UdZtzpef3gQyJ/XJ8ZP9RljjDGr5EAKmVkwSiv/Ji/vQJXC1Dtlx4baRFuMMcaYVbOwQmbmyyy49NJ9fQWEF4nnoMRlctaL8uNH5KMpGtMx+/zryyXxc2H37t2bpomz8aYyoqk7xle+MS4w89/Z2an3jDHGmNWxsELWF3WOHj2a/gVKT5/2OnnyZPoUWlznZVaNH+/xJFzfL+YTYJcuXUphwDdKQd/h1GcGSRPBtKx3guqrU7GM+/fvp7xRvuTJzB1/ygLqhtLd29v76VNk+uZzrL8xxhizChZWyJ8/f663ZkHp8dFrZpu5cgVm1VKqfA5Mn4VDSaJ8FZavPaNAMSOTRp+dA31gPSrzWIaUKqZzZu2EMXumXD7bp7pRZxxIAetiY+jfxjXGGDN+DnxTVw7fFkW54qKCzBVsDrNaZrhKswqYtVMvmbNjnZgdq975DWrGGGPMqllYIWvmGUGpxg9PMwPVzV36eLxmslqrJQ2KEaWNQpYpfF4waass/qXYNZOnXpRFucyOmUFTlj6krZky68maIWtmnJvljTHGmKVTzQiLtAVXCi+F66PVlRJM+zh9+Dz64diHShn+FDfG0TZ5Vwp8Zl8QT3XAES8Sy4h1VP6Ei1imPvYObOO3CIumOwxYNu1YPrNYHu1YPuOj1Ge/8FMFNoJZtyV4OrvVOvC2wZ3d3DSWP9bVhy7ZHWYsm3Ysn1ksj3Ysn/FR6rMDKWRgPZbHgxZRWkMG0zWPbHET2SL4IClj2bRj+cxiebRj+YyPUp8d+KYuFNabN2+26tEg2kKbFlXG80LnxOefI4ThtP5N3eJz09F13Tg3L7r5jf8I5ajMvN7xufFFUJkxj7y9Xe1U/ZYtj6HAWJAskE2JGC8en2zLnzjbBmNS7eszDiWnbZSFGRmVli7SEWxa6Cs71rdZz87XwAE/rX/zT55ac+c/roNrrX1ZaI0eyDuurVOOwqiDtkmjePzHNJGuesZ8RN7eNogX6zg22uTDONCYQCbE1ZiIID/kCIqHPHAxf2RV6qeh0CaPHNqndgNp29pHeNOxNybmkY8ZBqU+W/pjT2Y+3r59m16O0vTeb2bousObZ6+rE3HabuLu3bvpf1mWil9//TXdJQ+so+t5b2Yf1QlsGsZyhe6g50553WXP/6becvb69et6a/tgHOg5fB7Poy+anpPHwqO35Skez+LzZEGlsJI/8Ka7prfpjRXGpV4mBJUynnz69Knem4XZM+Hbeg+MGR9WyBsEBcxz23qrWf7IF4+B8bISmSU5EZeekdZjW8t6hloKVxcJypcLCIUBj78RRxcCCsvTrwOZy3WBIKIJM5rfMVEi2yaTpfxyf8poir9Jmh7L42IuKmr1B48Bxj45DI/0cYzlMF65CEZZ5+PCmE1hhbxBUMCaUTLT1GtEBVf6XMEz0+Skka/XfvjwYao0mJ3u7e3VIcsBZcWFAuXHsptOcEDcZaE2y+ktaiVQjnqNaqwfyge54o/jIgelStuQGeXwLDqy08yatsYX3OAvxS1LBv5xJrZJmi7CaGc+ngBLR5M1ZlvBUtB0w+n+/n6yFND39CUM5QLLHGKqwVikI9i00Ed2rF0RL7rSuifrYoRrPYx1Qdb/1gH1VFnUI67JsZ5JeFN9Su3Bv428DOhqbyyLf+2TT56Xyo/lkL/81R7BNn7Av9ZwV0WXfAT1b4N8olObNJbkuvLZNNRxEein2I+RfIxpHI+RReVjNkepzzxD3hDMUPhSVtUHU8cMM36uMt4hymyMNWSu+NdNnAkyo4h1wATKzJRZWqUApzMv/mlPNG8vi3iXMK5rtldaQ2xj6O8vZ8aut8yV0Lhi3FTKZjqTpj8VBtwvMFaQg8ZBNDszRlheKS3hYAFZZFwYs0qskDcE65y6OUdgto432LAOyAlH5Ou364KLBOoG1DmaPDH5yuxezTqmFxS0T2mWDSdZKRQcMkH5a+1Ya/GYbTHRxnepc6Kmnm1wgaGb5ADzZukzo5tAywcaP22mVsYPbclvXEIeKLFqBrmRMbUsMEdrHOgxRdrGzWq6kEQG+UUbYzaOC8z7Q+pjc0ipBnKRjmDTQpvsML0SHk2wMrPKYWqTWU1+MqlFP5xMtcsk5p+bfKmbwnLzrdrWZv4jvITSxzz6tDfKj3QxHnWMYRDzjNvqE2Qfw3O/kil0GZB/iVgHOdWPtql9kgHxI1EWY2Geusb2yUkmCmsaF5LhGKH+ZlyU+uzAb+oyzVh2ZSybdiyfWSyPdiyf8VHqM5usjTHGmAFghWyMMcYMACtkY4wxZgBYIRtjjDEDwArZGGOMGQBWyMYYY8wAsEI2xhhjBoAV8gbJXwHJvt7MxduXYlhOHq43DkX//GMU88KrCMkn/xKO3vJUKiO2yxhjTE++tdARbFrokp3eEhTf+KS3Kwni6C1DJfI8oCtNH3jDk95oRH7xTUaUqTDebKVt0Jukol9ObKP5GctnFsujHctnfJT6zDPkDXHt2rX00v/48nveN1wptHqvH+QRP03IDHsZnwXkgwN6xzHvduad0MCMuFLQ0zDeV613SBPGpwqrcTXq9yMbY8wmsELeADLznj59Ov1H5lWmfGAABYmpWmbrZShD5aE8deGQf+Di2LFj0zh8xIAPOshcjenaGGNMP6yQNwCfLISo2JrWk/uCIuTLNSjlpo+xLwqz7ZMnT6YvOsW1Yj5dl4NS/vDhw/SD7/fv35+ZuRtjjGnHCnkDMKsEzSxBnxSEvb294ndcmyAupm5My8tEn7ZjBh4/R9jE169fk/LWJwF1YRDbaIwxpowV8gaQqVprr0MnmtGZAb9586be+z7b50LgyJEjaYZsjDFmMayQNwCmaky6mJn1mFNf4qNR6+LFixfp5i1gBvzq1avpzBdzNh97p03MpFW3/OYvY4wxHXxroSPYtNBHdvFD/3KVEkthenyoyeWPFDV91P+gxPLyj7fHevNoVkT++tB/E4SbMpbPLJZHO5bP+Cj12S/8VIGNcHNRS7BpwbIrY9m0Y/nMYnm0Y/mMj1Kf2WRtBoXeDoa7fPly8sMMLj/cYb9RjOUAySLe/d4EMuTu+8g86ccKY4T2dT2twNjK30RnzKawQjaD4v379+mOcdbYeVEKcMc2d55zFzdXlUNal86V3apB0fAsOHJAJrxgpgTKmPX+yDzpxwptZKx0gTLmHghjhoIVsjELwuyLN5OtG931zuNu3DhXovTmt77pxwoXbH1MuFzo8aY7Y4aCFbLZCvIXqwiZLnHRNBnN4LozHJhVyp+0wCxYpk382ac8XnzCo174sR/LiukJI63ylv8iROsA9ZAVoS8HTW+MWSHVlWSRjmDTgmVXpks2pTvM2+7c5u5y3fEdt0mnu9JJjz9Od6PrjnGgXN1RThzis6/yyQfHNulwsU5xm/RKo/TE7wNxu1CebTIB2oTL6Zt+CPSRRxOk65K5+nnMLCofszlKfeYZshkkrCFX43PqqhNrHdIMMz2ekWYGqnVTZqaYZDUrZH2aOKyh3rhxI/nFN6RhfmZNkTxYW2X2i1kTsy/1IR8c+/v7+ymN0CxY6B3jvMGMulcn/bnevtaFZEIdF7kx66DpjTHLxwrZjA6Un8zCMgFjfmUbRYMiFCjhJvQ+8RyUFHnIzUOprFWhV6YuykHTD4G49OC7pc3YsULeILliYV/rmVIwcjl5eFzvlN9BZz5aM81PdHGttKmM2K5VoFmtHLNW3nrGbC9CvHwGiHx5/We8u5Zw2sSXqjRzBmQp4utCKYu3k0WaykJB479KqIveH74IB02/afS+ddy6L4iMWTrVQC7SEWxa6JKd1hirGVnt833tM6aLa50l8jygK00fWHesFEzaJj+trQJlKoz1N20D6WJ4E7GNOeRHOE7tiOu4uKa8VS5OeSAXnPxxShvjR3mxLX+1mbjRn34B8op+cR+Xx4nltKG0TeSyiJB/LCO2kW1oSz9UFqlnbKOODx1zGgPax415HZn6m3FR6rPWnnRHL06X7AjXiT2iEycQ3nUS10lFcMLVCecgxDzIM57UYp0IU50J63NiG9u4on20c134uJvF8mjH8hkfpT6zyXoDyKyprz5F4peV+qCbhzCvymwdH21ZFOWhPGV65YaomD+fklQcPtGI2VfmakzXxhhj+mGFvAF0Q1FUbE3ryX1BEbIWiFLWd4iXAeut1Yw3rbfGtdHjx4/XWz9AKVez6rQ+W13opbuSeU537CADZJvLwBhjlo0V8gZgVgmaWYJuVIK9vb25bgYi7q1bt9J3iZeJbphhBo7Sb4PHe1DeukFIFwaxjWMk3jQ05pufjDHDxwp5A8hU/fDhw/Q/dKIZnRlwvOOY2T4XAkeOHPnpLmdjjDH9sULeAJiqMeliCtVjTn2Jj0atixcvXkxu3ryZtpkl8uINzXwx5fIIEG1iJq26Yd5lP5rljTHGtPCthY5g00If2eWP5OB0BzN39uZhcvld1HrMB6f0ByWWl99hHOud3yku/7a7rQk3ZSyfWSyPdiyf8VHqs1/4qQIb4eailmDTgmVXxrJpx/KZxfJox/IZH6U+s8naDAq9HQzH15EAM7j8cGO/UcwYY5rwDHlFWHZlumTD41s8WhUf4WLtnNdaDu31iNR13mfHu/DYmcXyaMfyGR+lPvMM2ZgF4SKBL0QZY8wysEI2W0H+YhWBeVt+8SMZ0Qwe71rHTC5/mcaZBRNH5nT2KY8Xn/CoF37sx7JiesJIq7xtcjfGNFJNm4t0BJsWLLsyXbIp3WHeduc2d5frju+4TTrdlU56/HG6G113jEN8ZzVxiM++yicfHNukw8U6xW3SK43SE78PxDU/sDzasXzGR6nPPEM2g4TntKvxOXWVMqtDmnn58mV6RpoZKM9JAzPTSvFOn4Vm/Zk4vI9bn1mMb0jD/Mxz1eRx7dq1NPtlHZu3oFEf8sGxv7+/n9IIzYIF5VA2bzCj7pWynuvta8aYw4cVshkdKD+ZhWUCxozMNsoVRShKN4HpfeI5KM94ITAP/h6vMeYgWCFvgD6P9vz2228z+ygh0DokeaCI4vpnJK6F5h9FkH+c0UGsw5A/pKBZrRyzVt56xow2Qjz8YltoI6//ZCYsCEeWfKlKM2dAyYv4ulDK4u1kkaayUNCeFRtjelOd0Ip0BJsWumQX1ytFvibJfsyH+KQDrU3meQB+pIW4lgnkrzDy0lpqXFdV3oq3bNpkQ/0Ix6k+tEd+OLUlQlsUrjyov2Qop7QxvsoBtuUv2UpO8kdWIDnJL+7j8jixnDaU1nzH8mjH8hkfpT7zc8grokt2zL66nrVlnzt5lU+ehlkd75mOeTTBTPjp06fpAxDMApU/6VnbrJRG+tAFM0d90Uizw2U/YwtjG1dNfbVKfNzNYnm0Y/mMj1Kf2WQ9ImRqlZkbU21fJYEy5gYjlK/QzU6Aco7rqk3fPDbGGLM6rJA3iO7olev6oD+z1729vXQXMfHjGmcJrY2ifLWe2bQ+zCcUnzx5Uu8ZwZoza8b01VDW1amHxkxbnRQHxzgQ8R4GnC7wtgndQ9HWtijH0r0YxqyVatpcpCPYtNAlO9Yl8/Vf1jvb1pAjWhvuWpeknIjSycXy4votTmuly4a8TZk2+bAmrT5tGx9xXBBf/Uwa8hDsN92HMCTmHS/x2OK/qX35sbbK8b5qfDyNj1KftfakO3pxumS3iEImTTyZst1WDvHJowQn7aaTUF6PZeNx1U6bfGL/Q58bxeI4ydMzBnO/oTHveCG+2hTbHskVNdt9b7obGvPKx2yeUp/ZZD0yopn63bt36aasJvR4j8zUuXkbsyVmat3EJTDjYTr3M7XDJK7706e8EKUPGicxPfA4V+43ZvR4oNqk/2iyh0+fPiUnjh07Vm8Zs0FqxdxIR7BpoU12zD4Jx+mqnCt0+eF2dnZm9pm1asYb/ZsgzxgHx0xYswVcPnMmb/zXMUugHFOmj3zUj30sGfR9kyWE8RBniUNlnvHSZN0hfW4FQB4xX/Y9QzbrotRnrT3pjl4cy66MZdNOX/no4qxJ2Ua42GoCZZxfmA2RecZLX4UM+YXrGC5OmphHPmYYlPrMJmtjRgrLEZWyrfeaYWmi9Cw5Jtuxv0ksvl2OttKeSvlOTdT8Y65vMstj7q/OgdNHAdf1nLkxJayQjRkxPJKV3wcgeORH32tGMcVHe/I11bGCEkWp4nTfAxcpvDAHeOHNzZs303YTrDmjsKWUjdkkVsjGjIg4I8ShiAQKWM/dEsbz6igbtvmP799GYV2/fr3e2y6wCPBMPe3mwkMzXz13rIsTtnkzHjLcphvbzHjxqzNXhGVXxrJpx/KZxfJox/IZH6U+8wzZGGOMGQBWyMYYY8wAsEI2xhhjBoAVsjHGGDMArJCNMcaYAWCFbIwxxgwAK2RjjDFmAFghm0ERP56vl1zkL8PYlrdMGWNMxC8GWRGWXZku2fBZwePHj8+8W5hXHPJWpaF9FpK6lt4VvSgeO7NYHu1YPuOj1GeeIRuzIFwk6F3RxhhzUKyQzVaAcuSqU05g3pYf5nARzeDxowuYyeUv0zizYOLInM4+5Z07dy59lAA/9mNZMT1hpFXeNrkbYxqpps1FOoJNC5ZdmS7Z8P1e4uQu/85thG/b6rvAcZt0+hYu6fGPH6PXN4WBcvVNXOIQn32VTz44tkmXf3s3bpNeaZS+77eHiWt+YHm0Y/mMj1KfeYZsBkmlCBmxU1cpszqkGb5ty2cImYHylSNgZlop3umXfFh/Js7bt2/TejTw/VzyB8zPOzs7KY9r166l2S/r2HzOj/qQD479/f39lEZoFiwoh7K/fv2a6l4p69F/e9gYs1qskM3oQPnJLCwTMGZktlGuKEJRugns8+fP9dYsKM94ITAPQ7vhzBgzLqyQzejQrFaOWSsf6mdGGyEefnwHV7AWfP78+TQTFoSj1C9evDidOQNKXrx586bemqSy4reFoaksFLRnxcaY3lQntCIdwaYFy65Mm2xYhyUcp3XeuI6LY102J647Kw/WbHHyxyltjK9ygG35az2ZuNFf69PkFf3iPi6PE8tpQ2nNdyyPdiyf8VHqMz+HvCIsuzJjkw0z5fy56FXisTOL5dGO5TM+Sn1mk7UxxhgzAKyQjWmBNWfWjFlzjuvDm0Q3sOHiM9Q5MR43wol4U1y8M3yb0DPhsd05ioMbSt+aw40VsjEtYKbGtITjUaZNg+LgpjTqwx3hXCg0KR0pasXjJSZ6IQnbupschdym1McI7eQxsy6I8+HDhySHu3fvTuVjzKawQjZmRHBRoAsD7uC+dOnS5MuXL2k/wl3h169fT9uK9+LFi7QPR44cSf96RnuboE0o2Ta4sEEmav/NmzcnDx8+TNvGbAorZGNGztGjR+utH/DIVVTUUfHyYhM90sXLUNZ1s9qQ4OUwUSbHjh3zDNlsHCtkY0ZO07POPFP99OnTem8WfZ2KtdPD/DIT7pw3ZkhYIRszUpjl8srQJlC6vEJUNy1xY9qZM2dSGOZa1kx5HShhnhkaMwyskI0ZIXrjWBuso+J2d3fTeikzaZQv7+lmG1M1Slnm6zGCHHTRMc8d48guvn2NV6leuHCh3jNmM1ghGzMy9IiObu5qU6goLGbD+Uw63pk95hu74l3w85jfkR0WBFkHuFs9fx2qMevGCtmYEYHyZYarr1LhtBbK95Zx2ibs06dPM4oK5cuMmUefCH/y5Ml0TXmboG1AO3XxwYUM/lLCPPrFo0/4IZMxX5iY7cCvzlwRll0Zy6Ydy2cWy6Mdy2d8lPrMM2RjjDFmAFghG2OMMQPACtkYY4wZAFbIxhhjzACwQjbGGGMGgBWyMcYYMwCskI0xxpgBYIVsjDHGDAArZGOMMWYAWCEbY4wxA8AK2RhjjBkAVsjGGGPMALBCNsYYYwZA59eejDHGGLNcmlRvq0I2xhhjzHqwydoYY4wZAFbIxhhjzACwQjbGGGM2zmTy/wPzHC9IXNIhWgAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n 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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \n\u003c/div\u003e"},{"header":"4) Discussion","content":"\u003cp\u003eUp until now, the FDA has approved\u0026thinsp;\u0026ge;\u0026thinsp;10 classes of diabetic drugs for diabetic therapies, including biguanides, which reduce gluconeogenesis and improve insulin peripheral sensitization \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e; sulfonylureas, which increase insulin synthesis from β-cells \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e; sodium-glucose co-transporters and 2 inhibitors, which enhance renal glucose elimination from the body \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This suggests that nearly all diabetes treatments are rationalized by the post-assimilation of monosaccharides. Therefore, pre-assimilatory inhibition of glucose-based strategies must be developed. This can be overcome by changing dietary habits, such as consuming foods high in fiber and less glucose and sugar sweetener substitutes\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Intestinal glucose absorption proceeds via the conversion of long-chain polysaccharides to glucose by the glucosidase enzyme. The most prominent monosaccharide-creating enzymes in the GIT are α-amylase (salivary and pancreatic) and α-glucosidase. Salivary α-amylase hydrolyzes starch at the α-(1,4) glycosidic linkages to form small oligomers. Pancreatic α-amylase reduces the size of oligomers, resulting in smaller oligosaccharides such as maltose and dextrin \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These oligomers are transformed into dextrose by α-glucosidases, such as maltase, sucrose, and isomaltose, which are readily absorbed by the intestines. α-amylase/glucosidase inhibitors hinder gastric carbohydrate-hydrolyzing enzymes to control postprandial hyperglycemia \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Pharmacologic treatments require the development of new compounds to inhibit polysaccharide-hydrolyzing enzymes \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe one-drug, one-disease ideology is antiquated, and scientists are struggling to develop multifaceted compounds \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Pyrrolidinedione, commonly called succinimide, is reported to have pharmacologic activities \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e covering inflammation, tumors, microbes, and analgesia \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003eand has a close resemblance to FDA-approved antidiabetic-class thiazolidinediones \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Pyrrolidinedione and its chiral derivatives have been studied for pancreas, heart, and liver diseases \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Structural activity relationships show rational derivatives of existing compounds with more potency \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The current study aimed to investigate the antidiabetic and antilipidomic activities of sulfonamide and succinimide derivatives. Both NDMS and 2-BS positively inhibit the target enzymes, α-amylase and glucosidase \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. A toxicity profile was determined for these synthetic compounds before testing on an animal model, and they were found to be safe up to 1000 mg/kg/b. w., with acute toxicity above it. Therefore, a 1000 mg concentration was selected as the optimal dose for all assays. These compounds showed a good response in both in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) α-amylase/glucosidase inhibition assays using acarbose as a standard drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). NDMS exhibited significantly better activity, whereas 2-BS had comparable antidiabetic activity to that of the reference drug (PC), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Molecular docking (MOE) interactions of NDMS and 2-BS with target α-amylase and glucosidase enzymes also support these biological and in silico activities (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e"},{"header":"5) Conclusion","content":"\u003cp\u003eIn search of finding good antidiabetic molecules, our current findings show that NDMS and 2-BS compounds significantly inhibit the activities of the target α-amylase/glucosidase enzymes in vitro and show good antihyperglycemic and antihyperlipidemic effects in diabetic models in vivo. It significantly reduced blood glucose levels and diabetes-associated markers, including LFTs (SGPT, SGOT, and ALP) and serum creatinine levels. Docking of NDMS and 2-BS with the target α-amylase/glucosidase enzymes showed good and stable interactions. Thus, this finding provides good evidence to justify the pharmacological use of NDMS and 2-BS compounds in the treatment and management of various diabetic disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e-\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Ethical Committee of Animal Study at the University of Swabi (study permission No. F. NO. DA/UOS/2023/1766 dated August 01, 2023)\u003c/p\u003e\n\u003cp\u003e-\u003cstrong\u003eConsent to Participate\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e-\u003cstrong\u003eConsent to Publish\u003c/strong\u003e: \u0026nbsp;Not applicable\u003c/p\u003e\n\u003cp\u003e-Author\u0026rsquo;s Contribution:\u0026nbsp;Z. U. and A. N.: data curation and original draft writing. M. J.: Methodology and Data analysis. U. N.: Supervision, Reviewing, and Editing. M. S. J., Z. U. H., S. U. A. H., A. H., and O. M.A. D.: Writing, Data Visualization, Data draft preparation, and I.B. \u0026amp; Z. W. Reviewing and Editing: Z. W.: Investigation, Reviewing, and Editing. K.F.F: Funding acquisition, S.A: Funding acquisition, Investigation, Conceptualization, Reviewing, Editing and original draft finalization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Funding:\u0026nbsp;\u003c/strong\u003eThe authors express their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for supporting this work through a research group program under grant number RGP-2/630/45. Additionally, this study was supported by the National Natural Science Foundation of China (NSFC) Grant No. 31850410479.\u003c/p\u003e\n\u003cp\u003e-Competing Interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.\u003c/p\u003e\n\u003cp\u003e-\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed are included in the submitted manuscript and Supplementary Materials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia, for this work through a research group program under grant number RGP-2/630/45.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIssue Information. \u003cem\u003eDiabetes/Metabolism Research and Reviews \u003c/em\u003e\u003cstrong\u003e2019,\u003c/strong\u003e\u003cem\u003e35\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003ede Oliveira Neto, X. 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B., Towards the sustainable discovery and development of new antibiotics. \u003cem\u003eNature Reviews Chemistry \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e\u003cem\u003e5\u003c/em\u003e (10), 726-749.\u003c/li\u003e\n\u003cli\u003eGenovese, M.; Nesi, I.; Caselli, A.; Paoli, P., Natural \u0026alpha;-glucosidase and protein tyrosine phosphatase 1B inhibitors: A source of scaffold molecules for synthesis of new multitarget antidiabetic drugs. \u003cem\u003eMolecules \u003c/em\u003e\u003cstrong\u003e2021,\u003c/strong\u003e\u003cem\u003e26\u003c/em\u003e (16), 4818.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 2","content":"\u003cp\u003eScheme 2 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetes, α-amylase, α-glucosidase, N-benzyl-2,5-dioxopyrrolidin-3-yl-methanesulfonamide, 2-benzylsuccinimide, alloxan","lastPublishedDoi":"10.21203/rs.3.rs-7306006/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7306006/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetes mellitus is one of the most alarming metabolic syndromes caused by high blood sugar levels. Herein, we report the identification of two potent antidiabetic agents: N-benzyl-2,5-dioxopyrrolidin-3-yl-methanesulfonamide (NDMS) and 2-benzylsuccinimide (2-BS). These molecules were evaluated for their antidiabetic potential using in vitro, in vivo, and in silico assays against their potential targets, α-amylase and α-glucosidase. For in vivo studies, an alloxan-induced diabetic rat model was used. Our results show that NDMS and 2-BS exhibit good in vitro enzyme inhibition compared to the standard acarbose drug. In in vivo assays, the acute toxicity profiles and IC50 values showed that NDMS at doses of 5 and 10 mg/kg/b. w. resulted in hypoglycemia with 247.9,178 mmol/L and 246.4,171 mmol/L. Similarly, 2-BS at the same dose resulted in a hypoglycemic effect by lowering blood glucose levels from 251.6 and 199 mmol/L to 244.8 and 181 mmol/L, respectively. Furthermore, their antihyperlipidemic effects revealed that these compounds also reduced total cholesterol, low-density lipoprotein (LDL), and triglyceride (TG) levels, while increasing high-density lipoprotein (HDL) levels. Moreover, these molecules significantly enhanced the number of pancreatic β-cells and restored normal liver morphology and serum creatinine level. Additionally, the in-silico results showed that both NDMS and 2-BS have remarkable interactions with α-amylase and α-glucosidase compared to those of the standard acarbose drug. Thus, our findings indicate that these molecules have good hypoglycemic and hypolipidemic effects and can be used as lead pharmacophores in the development of new antidiabetic drugs.\u003c/p\u003e","manuscriptTitle":"Dual-Action Compounds for Glycemic Control: N-Benzyl-2,5-Dioxopyrrolidin-3-yl-Methanesulfonamide and 2-Benzylsuccinimide mimic α-Amylase and α-Glucosidase activities to regulate blood glucose levels","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 13:51:20","doi":"10.21203/rs.3.rs-7306006/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d673fe84-4ddf-4346-b1a3-3ddf5765f0ed","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T16:09:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 13:51:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7306006","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7306006","identity":"rs-7306006","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}