A dual in silico–in vitro assessment of nine glimepiride apis: efficacy and oxidative stress protection in type 2 diabetes

Abstract

Type 2 Diabetes Mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and impaired glucose metabolism. Glimepiride, a sulfonylurea drug, is commonly used to manage blood glucose levels in T2DM patients. However, the efficacy of various active pharmaceutical ingredients (APIs) of Glimepiride has not been thoroughly compared. Here, we systematically evaluate nine Glimepiride APIs for their modulation of oxidative stress pathways via computational and laboratory models. Molecular docking simulations were performed to investigate the binding affinity between Glimepiride and aldose reductase (AR), revealing a strong binding interaction. Furthermore, High-Performance Liquid Chromatography analysis was conducted to assess the purity of the APIs, showing consistent results with all samples exceeding 99.5% purity. In vitro experiments were performed using liver enzymes extracted from diabetic rats to assess the effects of the nine APIs of Glimepiride on AR and glutathione reductase (GR) activity. Significant variations in AR inhibition and GR activation were observed among the APIs. In particular, compound G1 exhibited potent AR inhibition and a marked reduction in MDA levels, highlighting its strong antioxidant potential. Meanwhile, compound I2 was the most effective at activating GR, suggesting a potential role in enhancing the natural antioxidant defences. These results support the therapeutic potential of Glimepiride in mitigating oxidative stress and its beneficial effects on key metabolic enzymes in T2DM. The observed differences in the efficacy of the Glimepiride APIs were likely due to factors such as variations in bioavailability, formulation composition, and pharmacokinetic properties. The findings highlight the importance of assessing these factors when evaluating generic formulations for therapeutic use in T2DM management.

Graphical Abstract

Highlights

Molecular docking studies have shown a strong binding affinity between glimepiride and aldose reductase.

In vitro assays revealed that Glimepiride Active Pharmaceutical Ingredients exhibited a global therapeutic effect by inhibiting aldose reductase, activating glutathione reductase and significantly reducing MDA levels.

Differences in therapeutic efficacy between Active Pharmaceutical Ingredients are related to bioavailability, formulation and pharmacokinetics.

Introduction

Diabetes mellitus is a long-term metabolic condition characterised by elevated blood glucose levels, primarily due to impairments in insulin production, insulin responsiveness, or a combination of both [1]. The incidence of this disease has shown a continuous upward trend globally. According to recent data from the International Diabetes Federation (IDF), over 530 million individuals are currently affected, and this number is expected to grow substantially in the near future [2]. This upward trajectory represents a significant public health challenge, not only because of the disease’s chronic nature but also due to its associated healthcare costs and serious long-term complications.

Several interrelated risk factors contribute to the onset and progression of diabetes, including genetic susceptibility, unhealthy diet, physical inactivity, obesity and advancing age [3]. If there is no adequate medical care, chronic hyperglycaemia can lead to a number of serious health complications. These include microvascular disease such as diabetic retinopathy, nephropathy and neuropathy, and macrovascular disease such as cardiovascular disease and stroke, which significantly increase morbidity and mortality in people with diabetes [4].

One of the key pathological mechanisms involved in these complications is oxidative stress, which is increasingly recognised as a major contributor to diabetes-induced cell damage. Oxidative stress occurs when the production of reactive oxygen species (ROS) overwhelms the body’s antioxidant defences, leading to the degradation of essential biomolecules such as lipids, proteins and DNA [5].

One of the most reliable indicators of oxidative stress is malondialdehyde (MDA), a by-product of lipid peroxidation. Elevated levels of MDA are commonly observed in people with diabetes and reflect oxidative damage to cell membranes [6]. In addition, two enzymes - glutathione reductase (GR) and aldose reductase (AR) - play contrasting roles in this oxidative pathway. GR is a key antioxidant enzyme that restores the reduced form of glutathione (GSH), a vital intracellular antioxidant, helping to protect cells from oxidative damage [7]. Conversely, AR, a metabolic enzyme active in the polyol pathway, is overexpressed under hyperglycaemic conditions and mainly contributes to complications such as retinopathy [8].

The management of diabetes typically involves pharmacological approaches based on the type of disease. While insulin therapy is essential for type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) is often treated with oral hypoglycaemic agents [9]. Glimepiride (GLM), a sulfonylurea of the third generation, is one of the most widely used oral antidiabetic agents for the treatment of T2DM. Glimepiride acts primarily by stimulating insulin secretion from pancreatic β-cells through the inhibition of ATP-sensitive potassium channels. This inhibition causes cell depolarization, which triggers calcium influx and leads to insulin release [10]. In addition to this pancreatic effect, Glimepiride also helps reduce oxidative stress, exhibits antioxidant properties, and enhances insulin sensitivity through extra-pancreatic mechanisms [11].

The present study aims to evaluate the therapeutic efficacy of various active pharmaceutical ingredients (APIs) of Glimepiride, used in the manufacture of multiple generic samples, through both in silico and in vitro experiments. In the in silico part, molecular docking was performed to investigate the interaction between Glimepiride and AR, with a focus on identifying key binding residues. The in vitro evaluation assessed the effects of the different generic samples on AR activity, GR activity, and MDA levels in the context of T2DM. These biochemical markers provide valuable insights into the antioxidant and protective potential of generic Glimepiride against diabetes-related oxidative stress. A comparative analysis among the generics was also conducted to explore variability in their biological effectiveness.

Materials and methods

Molecular docking

AutoDock Tools (ADT) (version 1.5.6) was used to analyze the conformations of the Glimepiride. In this study, we used crystal 1IEI from the Protein Data Bank, bound to the potent inhibitor zenarestat. This crystal allowed to delineate the binding pocket of the enzyme, thus indicating the part of the molecule in which the simulated binding was performed. The antagonist was manually extracted with the use of a text editor and binding of the main constituents of CEO was simulated. Ligand structures were obtained from the PubChem database in SDF file and then converted to pdbqt format via Open Babel GUI. The crystal structure of human AR (accession id: 1IEI; Resolution 2.5 Å) was downloaded from Protein Data Bank (PDB) (https://www.rcsb.org/structure/1IEI) in PDB format. A grid map was prepared using a grid box, the grid box size was set to 88 × 88 × 110 (x, y, and z points) with a grid spacing of 0.375 Å and the grid center was designated at −5.611, 1.861 and 0.083 (x, y, and z). The docking program was analyzed by Lamarckian Genetic Algorithm to search the optimal conformation. The Discovery Studio 2021 program was used to visualize the protein-ligand interactions.

Glimepiride APIs preparation

Nine samples (I₁, I₂, P₁, P₂, G₁, G₃, G₄, G₅, G₆) of the active pharmaceutical ingredient of Glimepiride (APIs), in accordance with pharmacopeial standards, were generously provided to us by the Moroccan National Drug Control Laboratory. Each sample was prepared at a concentration of 0.1 mg/mL in a phosphate buffer at pH 7.8.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV)

Prior to analysis, all samples were checked for related substances by HPLC-UV in accordance with the European Pharmacopoeia [12], to ensure that they had not degraded or oxidised and were free from impurities that could affect the results.

Chemicals and standards

The chemical reference standards used for the glimepiride assay were obtained from EDQM (European Directorate for the Quality of Medicines & Health Care), phosphoric acid and sodium dihydrogen phosphate analytical grade were purchased from Sigma Aldrich^®, Germany. Acetonitrile, HPLC grade, was purchased from Merck^®, Germany.

Solution preparations

A test solution was prepared by dissolving 20 mg of the substance to be examined in a 1:4 (v: v) solvent mixture of water and acetonitrile for chromatography, and diluting to 100 mL with the same mixture. The reference solution for the assay was prepared in the same way, by dissolving 20 mg of glimepiride in the solvent mixture and diluting it to 100 mL. For the reference solution for related substances, 1 mL of the test solution is diluted to 100 mL with the solvent mixture. Then, 1 mL of this dilution is further diluted to 10 mL using the same solvent mixture.

Instrument and chromatographic conditions

Prior to analysis, all samples were assayed for related substances by an Agilent^® 1200 Infinity HPLC-UV system according to the European Pharmacopoeia, to ensure that they were not subject to degradation or oxidation, and that they did not contain impurities that could compromise the results. The chromatographic conditions used for the purity of the drug substance are shown below.

Chromatographic separation was performed using a Merck^® Superspher^® 100 RP-18 end-capped column (L = 0.25 m, Ø = 4 μm) at 35 °C, with a constant flow rate of 1.5 mL/min. The mobile phase was a sodium dihydrogen phosphate buffer solution (pH 2.5)/acetonitrile mixture in a ratio of 1:1 (v: v). All analytes were monitored at 228 nm using UV detection and the injection volume was set at 20 µL.

Animal husbandry

Sprague-Dawley rats, aged 4–6 weeks, were obtained from the Faculty of Sciences, Ain Chock, Hassan II University, Casablanca, and used in the experiments. Sprague-Dawley rats were selected for their well-documented physiology and widespread use in toxicological and pharmacological studies, which facilitates comparison with existing literature. Moreover, the use of male rats was chosen to avoid hormonal fluctuations related to the estrous cycle that could introduce variability. The animals were housed in standard cages in a ventilated room maintained at 25 ± 2 °C with a 12-hour light/dark cycle. They were allowed to acclimatise to the laboratory environment for at least one week prior to the start of the experiments and were provided with standard rodent chow and water ad libitum. The number of animals used was minimized in accordance with the 3Rs principle (Replacement, Reduction, and Refinement) to reduce animal use while maintaining sufficient statistical power for valid conclusions. The study complied with the Animal Experimentation Act: Reporting of In Vivo Experiments (ARRIVE) guidelines, which enhance the clarity and transparency of animal research by requiring comprehensive details of study design, methodology and ethical considerations. This ensured that all relevant aspects of animal welfare, housing conditions and experimental procedures were carefully documented, promoting ethical research practices and allowing the results to be evaluated and reproduced by other researchers [13]. A timeline diagram illustrating the treatment schedule and induction procedures is provided in Fig. 1 to clarify the experimental design.

Fig. 1.

A timeline diagram illustrating the treatment schedule and induction procedures of type II diabetes

Induction of type 2 diabetes mellitus

Male Sprague-Dawley rats were fed an ad libitum cafeteria diet for 5 weeks, with free access to water, to induce T2DM through obesity, a major contributing factor to the disease. The diet included 28% protein, 57% carbohydrates, and 15% fat, with each rat receiving a daily mixture of salami, biscuits, cheese, sausages, crisps, chocolate, and almonds in a 2:2:2:1:1:1:1:1 ratio [14]. During the 5-week period, all rats showed significant weight gain, with body weights ranging from 400 to 550 g. Fasting blood glucose levels ranged from 300 to 600 mg/dL, confirming the development of T2DM.

Following induction, the rats were euthanized to collect liver samples, which were used to isolate enzymes associated with diabetes. These enzymes were then assessed in vitro to evaluate the effects of Glimepiride samples on AR, GR, and MDA levels. The study involved two groups of five rats each (n = 5): The first group was composed of normal rats receiving a standard rodent diet ad libitum with free access to water, whereas the second group included obese rats fed with cafeteria diet.

Enzyme suspension preparation

To assess the effect of Glimepiride on enzymatic activities and markers of oxidative stress, an enzyme suspension was prepared from rat liver. 1 g of liver tissue was homogenised in 3 mL of a buffer solution (pH 7.4) consisting of 0.25 M sucrose, 10 mM 4-(2-hydroxyethyl)−1-piperazine ethane sulfonic acid (HEPES), 2 mM phenylmethylsulphonyl fluoride (PMSF) and 1 mM ethylenediaminetetraacetic acid (EDTA) using a homogeniser (Ultra-Turrax^®, Germany). The homogenate was then centrifuged at 28,200 × g for 1 h at 4 °C and the resulting supernatant containing the enzyme-rich fraction was collected for in vitro analysis (Thermo Scientific^®, France). Protein concentration was determined according to the method of Bradford, using bovine serum albumin as standard [15]. All enzyme specific activity measurements were normalized to total protein.

In vitro effect of glimepiride on aldose reductase

Aldose reductase (AR) is an enzyme that catalyses the reduction of D-glyceraldehyde to sorbitol using reduced nicotinamide adenine dinucleotide phosphate (NADPH, H⁺) as a cofactor. Excessive AR activity has been linked to the development of several diabetic complications, mainly due to the accumulation of sorbitol [16].

In the present study, the activity of AR was assessed according to the method of Hmimid F. et al. (2021) [17], which measures the oxidation of NADPH, H⁺ by monitoring its absorbance at 340 nm. This assay is based on the reduction of D-glyceraldehyde to sorbitol, with NADPH, H⁺ as a cofactor. The reaction mixture consisted of 50 µL of liver enzyme suspension, 10 mM D-glyceraldehyde, 50 µL of the Glimepiride sample, and 10 µM NADPH, H⁺ in a 50 mM phosphate buffer (pH 6.2). The reaction was initiated by the addition of the cofactor, and the decrease in absorbance at 340 nm was measured every 5 s during 1 min using a spectrophotometer with cuvettes (BIOMATE^® 3, USA). The molar extinction coefficient (ε) of NADPH, H⁺ at 340 nm (6,220 M⁻¹·cm⁻¹) was used to calculate the enzymatic activity based on the observed changes in absorbance [17].

The results were presented as specific activity, enabling comparison of enzymatic activity relative to the protein content in the sample. Enzymatic activity was expressed in international units (IU), representing the quantity of enzyme that catalyses the conversion of 1 µmol of substrate per minute under optimal temperature and pH conditions.

The enzymatic activity was determined using the following equation:

1

Δ _Absorbance represents the difference in absorbance measured during the reaction.

Specific activity was determined as follows:

2

C _protein represents the protein concentration in the sample, expressed in mg/mL.

In vitro effect of glimepiride on glutathione reductase

Glutathione reductase (GR) is an enzyme that catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH), using NADPH, H⁺ as an electron donor. It plays a crucial role in maintaining cellular redox balance and protecting cells from oxidative damage [7].

The activity of the enzyme was assessed following the method described by Di Ilio et al. (1983) [18], which is based on the reduction of GSSG to GSH. The reaction mixture consisted of 50 mM phosphate buffer (pH 7.4), 25 µM GSSG, 1 mM EDTA, 50 µL of liver enzyme homogenate, and 50 µL of the sample. After pre-incubating at 30 °C for 2 min, the reaction was initiated by the addition of 1.6 µM NADPH, H⁺. Absorbance was measured at 340 nm every 5 s during 1 min [18].

The results were expressed using the previously mentioned Eqs. 1 and 2, allowing the determination of specific enzymatic activity, reflecting the reduction of GSSG to GSH in the presence of NADPH, H⁺.

In vitro effect of glimepiride on malondialdehyde levels

Malondialdehyde (MDA) is a by-product of lipid peroxidation and serves as a reliable biomarker of oxidative stress and tissue damage. The reaction between MDA and thiobarbituric acid (TBA) forms a stable MDA-TBA adduct, which can be quantitatively assessed by spectrophotometry [6].

The assay was performed according to the protocol described by Samokyszyn and Marnett (1990) [19]. Briefly, 100 µL of liver homogenate extract and 100 uL of the sample were mixed with 900 µL of an acidic solution (0.25 M of hydrochloric acid) containing 0.375% thiobarbituric acid (TBARS) and 15% trichloroacetic acid (TCA). The reaction was initiated by heating the tubes at 100 °C for 15 min in a water bath, followed immediately by cooling in an ice bath to allow the MDA-TBA complex to form. To separate the proteins from the supernatant, the mixture was centrifuged at 1000 g. The absorbance of the supernatant was then measured at 535 nm using a spectrophotometer. The molar extinction coefficient for MDA at this wavelength is ε = 1.56 × 10⁵ M-¹ cm-¹, which was used to quantify MDA in mol/L in the samples. The concentration of polyunsaturated fatty acid degradation products was determined based on Beer’s law.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0.2 for Windows (GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA was applied to compare differences between multiple groups, with statistical significance defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Each experiment consisted of three independent replicates.

Results and discussion

Glimepiride bind to the aldose reductase active center: In silico evidence

The docking results revealed that Glimepiride fits in the active site of crystal structure of human AR (Fig. 2). Two hydrophobic interactions mediated by Tyrosine²⁰⁹ and Valine⁴⁷ tend to form contact with Glimepiride with an energy change of −11.0 kcal/mol (Table 1). Additionally, 16 amino acids were attached by van der waals energy bonds, which are crucial for the formation of protein-ligand complex (Fig. 2). Glimepiride had the best binding affinity with AR (−11.0 kcal/mol), comapred to Zenarestat (−6.7 kcal/mol), respectively (Fig. 2). The stronger binding affinity of Glimepiride compared to Zenarestat, a well-established AR inhibitor, indicates a promising capacity to modulate AR activity. This interaction may contribute to reducing the accumulation of sorbitol and limiting oxidative damage in hyperglycemic conditions.

Fig. 2.

Molecular interaction of ligands with AR protein. (a) Interaction of Glimepiride with AR protein and its associated amino acid residues. (b) Interaction of Zenarestat with AR protein and its associated amino acid residues

Table 1.

Molecular Docking analysis of glimepiride

Enzyme	Ligand	Binding energy (kcal/mol)
AR (PDB id: 1IEI)	Glimepiride	−11.0
	Zenarestat	−6.7

HPLC evaluation of apis glimepiride

The assay method provided optimal chromatographic performance with a consistent retention time of 7.5 ± 0.2 min (Fig. 3).

Fig. 3.

Typical chromatogram of the active pharmaceutical ingredient of Glimepiride

Quantitative analysis showed that the purity of all nine Glimepiride samples exceeded 99.5%. These results indicate that the APIs tested are of high quality and meet the required pharmaceutical standards.

Evaluation of glimepiride apis on aldose reductase activity

The specific activity of AR was assessed in different experimental groups treated in vitro with different generic samples of Glimepiride. The results, shown in Fig. 4, highlight the levels of enzyme specific activity expressed in IU per mg of protein.

Fig. 4.

Specific activity of aldose reductase in IU/mg of proteins between the different groups treated in vitro with APIs Glimepiride, compared with the untreated diabetic group (*** highly significant). Values were expressed as mean ± standard deviation, n = 5 animals/group, p ˂ 0.001 (two-factor analysis of variance (ANOVA))

Analysis of AR activity revealed significant differences among the experimental groups (Fig. 4). In the control group, AR activity was relatively low at 3.23 ± 0.20 U/mg of proteins. In contrast, untreated diabetic rats exhibited markedly higher AR activity, measuring 12.55 ± 0.50 U/mg, indicative of the enzyme’s up-regulation in diabetic conditions. This increase is attributed to the accumulation of glucose and its metabolites, which activate the polyol pathway, leading to elevated AR activity [20].

Treatment with various APIs Glimepiride resulted in notable variability in AR activity (Fig. 4). Samples I₁, P₁, G₁, and G₆ exhibited elevated AR activity compared to the control group. Notably, G₁ showed an activity of 10.75 ± 0.10 U/mg, indicating a limited inhibitory effect on the enzyme. Conversely, samples I₂, P₂, G₄, and G₅ demonstrated moderate AR activity levels, suggesting a more pronounced inhibitory effect. Remarkably, G₃ exhibited a significantly reduced AR activity of 2.03 ± 0.05 U/mg, even lower than the control group, indicating a strong inhibitory effect on the enzyme.​.

These variations in AR activity among different APIs may result from factors such as differences in bioavailability, composition, and pharmacokinetic properties. The influence of excipients and the release rates of these samples also likely play roles in modulating AR activity. Collectively, the findings suggest that both proprietary and generic Glimepiride samples can influence AR activity, with effects varying according to their specific samples and the pharmacodynamics of the active ingredient [21].

Evaluation of glimepiride apis on glutathione reductase activity

The impact of APIs Glimepiride on GR activity was assessed and the corresponding results were presented in Fig. 5.

Fig. 5.

Specific activity of glutathione reductase in IU/mg of proteins between the different groups treated in vitro with APIs Glimepiride generics, compared with the untreated diabetic group (*** highly significant). Values were expressed as mean ± standard deviation, n = 5 animals/group, p ˂ 0.001 (two-factor analysis of variance (ANOVA))

In the control group (Fig. 5), GR activity was notably high at 33.5 ± 0.5 U/mg of proteins, reflecting a robust antioxidant defence system. Conversely, the untreated diabetic group exhibited a significant reduction in GR activity, measuring 3.6 ± 0.01 U/mg, indicating compromised antioxidant capacity due to hyperglycaemia-induced oxidative stress. This decrease in GR activity under diabetic conditions has been documented in previous studies, highlighting the detrimental effects of elevated glucose levels on antioxidant enzyme functions [5].

Among the treated groups (Fig. 5), samples I₂, P₁, and G₅ demonstrated sufficient results, with GR activity exceeding that of the control group. Notably, I₂ achieved a GR activity of 36.5 ± 0.03 U/mg, suggesting an enhanced restoration of antioxidant defence mechanisms. Additionally, samples I₁, P₂, G₃, and G₆ also increased GR activity, although to a lesser extent.​ In contrast, samples G₁ and G₄ did not effectively restore GR activity, indicating a limited impact on enhancing antioxidant defences.

Interestingly, these results differ from those observed for AR activity (Fig. 4), where patterns of enzyme modulation varied between samples. Such differences underscore that the response to treatments can vary between enzymes, reflecting the complex interplay between drug samples and specific enzymatic pathways. Overall, the study highlights the importance of considering individual enzyme responses when evaluating the efficacy of pharmaceutical samples, as modulation of one antioxidant enzyme does not necessarily predict similar effects on others.

Evaluation of apis glimepiride generics on lipid peroxidation

Lipid peroxidation levels were measured by quantifying MDA concentrations in the different experimental groups (Fig. 6).

Fig. 6.

Lipid peroxidation evaluation in µM/100 g of proteins between the different groups treated in vitro with APIs Glimepiride generics, compared with the untreated diabetic group (*** highly significant). Values were expressed as mean ± standard deviation, n = 5 animals/group, p ˂ 0.001 (two-factor analysis of variance (ANOVA))

MDA levels, as shown in Fig. 6, were found to be significantly elevated in untreated diabetic group at 0.09 ± 0.001 µM/100 g of proteins, while the control group exhibited much lower levels at 0.02 ± 0.001 µM/100 g protein. This marked increase in MDA reflected an intensification of oxidative stress, a common feature of uncontrolled diabetes, in which chronic hyperglycaemia had promoted excessive production of ROS, consequently leading to lipid peroxidation of cellular membranes [6].

However, the groups treated with APIs Glimepiride (Fig. 6), showed a considerable amelioration of this parameter, as Glimepiride exerted a remarkable antioxidant effect, significantly reducing MDA levels in all treated groups. These levels were very close to those observed in the control group, and the generics G₁ and I₂ exhibited the most significant results, with the lowest levels recorded at 0.015 ± 0.001 µM/100 g protein, suggesting a strong efficacy in attenuating diabetes-induced oxidative stress.

In the context of T2DM, the in vitro treatment with Glimepiride generics effectively reduced oxidative stress markers such as MDA, demonstrating significant antioxidant properties of the drug. Notably, the samples G₁ and I₂ exhibited the most remarkable effects, with the lowest MDA levels observed in these groups. The sample G₃ displayed the strongest inhibitory effect on AR activity, confirming its potential in modulating this enzyme. As for GR activity, almost all samples were successful in restoring its activity to levels comparable with the control group, except for G₄, which had a limited effect.

Conclusion

In conclusion, this study has highlighted the significant therapeutic potential of glimepiride in modulating key enzymes, such as AR and GR, while effectively reducing oxidative stress, as demonstrated by decreased MDA levels. The findings have underscored the importance of optimising the quality of APIs Glimepiride and formulations to maximise these beneficial effects, particularly in alleviating oxidative stress associated with T2DM. The results suggest that glimepiride’s modulation of antioxidant mechanisms may have played a crucial role in preventing or mitigating complications related to diabetes, such as cardiovascular diseases, nephropathy, and neuropathy. By reducing oxidative stress, glimepiride may help slow the progression of these complications, which are common in individuals with poorly managed diabetes. Future research should focus on refining the pharmacokinetic and pharmacodynamic properties of these formulations to enhance their clinical application in the management of T2DM and its associated complications.

However, a limitation of this study is the very small number of animals employed in this study. To confirm these results for glimepiride’s therapeutic effects, larger sample sizes are needed in future research. Also, future studies should also improve these formulations pharmacokinetic and pharmacodynamic characteristics in order to improve their clinical use in the treatment of type 2 diabetes and its related complications.

Author contributions

Author contribution: Amine HSSAINE: Writing – Original Draft Preparation, Conceptualization, Formal Analysis. Fatima Ez-zahra OUSAID: Writing – Original Draft Preparation, Conceptualization, Formal Analysis. Imane Nait Irahal: Investigation, Conceptualization, Docking. Fatima Zahra KHADDACH: Conceptualization. Miloud EL KARBANE: Investigation, HPLC. Mohamed AZOUGAGH: Validation. Noureddine BOURHIM: Writing, Review, editing and Validation. Brahim BENAJI: Project Administration, Writing - Review and Editing.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics declaration

Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978).

Competing interests

The authors declare no competing interests.