Introduction

Diabetes is a chronic condition that affects the body’s metabolism marked by elevated circulating blood sugar. The increasing incidence of diabetes and the complications linked to it put significant pressure on healthcare systems. Ongoing high blood sugar levels can result in small blood vessel complications, including damage to the eyes (retinopathy), kidneys (nephropathy), and nerves (neuropathy), collectively accounting for approximately 1.5 million deaths worldwide annually1,2,3. Moreover, people living with diabetes are at a markedly higher risk of developing macrovascular complications, including cardiovascular disorders, cerebrovascular accidents, and peripheral arterial disease4. Present-day approaches to diabetes management emphasize achieving optimal blood sugar regulation through a combination of pharmacological treatments, lifestyle modifications, and nutritional interventions. Various traditional antidiabetic drugs are available, functioning by augmenting insulin levels, improving insulin responsiveness, stimulating pancreatic insulin release, and facilitating glucose absorption into cells. Interventions. Given these challenges, there are various strategies available that can provide effective management solutions for diabetes. From a physiological standpoint, blood glucose levels can also be regulated by restricting the amount of glucose that enters the bloodstream. This strategy mainly targets blocking key enzymes involved in carbohydrate breakdown, notably α-amylase and α-glucosidase. One of the major consequences of chronic hyperglycemia is the non-enzymatic glycation of endogenous proteins, which results in the irreversible generation of advanced glycation end products (AGEs)5,6. This process happens naturally in the body, where reducing sugars form stable covalent links with protein amino groups via their reactive aldehyde or ketone structures, leading to the production of AGE compounds characterized by their yellow-brown pigmentation and intrinsic fluorescence. Short-term an excess of glucose in the blood induces oxidative stress, which is reversible, and activates monocytes in circulation. According to Natarajan and collaborators7,8, elevated glucose concentrations and the accumulation of AGEs can stimulate the release of inflammation-related cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), along with chemokines such as MCP-1 and IP-10.These inflammatory responses have been associated by altering signaling molecules like protein kinase C, p47phox, and MAPKs through both oxidant stress-dependent and independent pathways. These signaling pathways lead to the activation of the NF-κB transcription factor, which subsequently regulates the expression and synthesis of various downstream pro-inflammatory mediators. Research has consistently shown a complex and significant relationship between protein glycation and inflammation, identifying glycation as a key trigger of inflammatory responses. Standard therapies for these disorders are frequently associated accompanied by negative side effects, highlighting the urgent demand for alternative treatment strategies. Medicinal plants represent a plentiful source of bioactive compounds that include notable antiglycation potential, and recent studies have confirmed their efficacy in modulating inflammatory responses9. Solanum obtusifolium Dunal., a member of the Solanaceae family, is predominantly distributed across tropical and subtropical zones, notably in regions of Africa, Asia, and Central America10. This species is traditionally utilized for its therapeutic benefits in the management of conditions such as inflammation, fever, and skin injuries. Preliminary research indicates that it may exhibit antioxidant, anti-inflammatory, and antimicrobial properties; however, further investigation is required to validate and clarify its pharmacological potential10. These biological effects are believed to be linked, in part, owing to its abundance of quercetin, astragalin, and kaempferol 8-C-D-galactoside10,11.

The purpose of this research is to comprehensively analyze the phytochemical profile of the hydroethanolic extract of Solanum obtusifolium Dunal. using HPLC in conjunction with a diode-array detector (DAD). The antiglycation activity on hemoglobin and albumin at various stages, as well as the ability to combat inflammation of the extract, will be assessed using in vitro experiments. Furthermore, molecular docking studies, a well-established tool for exploring interactions between proteins and ligands, will be employed to investigate the potential molecular targets and mechanisms underlying the observed bioactivities.

Materials and methods

Plant collection

Fruits of Solanum obtusifolium Dunal was collected at the maturation phase in late March 2022 in the region of Fez, Morocco (34°04′04.2 N, 5°01′26.4 W). Professor Amina Bari identified the plant under the reference E17/1405 before being deposited in the herbarium of the Faculty of Sciences, Dhar El Mahraz, University Sidi Mohamed Ben Abdellah, Fez 30,050, Morocco.

Preparation of the extract

The extraction process was performed using an ultrasonic water bath and a direct sonication technique with slight modifications12. Sonication was conducted at a frequency of 50 kHz, with a power output of 120 W, and the temperature maintained at 35 °C. Five grams of Solanum obtusifolium Dunal fruit were subjected to the experimental conditions defined by each run of the Box-Behnken Design (BBD). The resulting mixtures were subsequently passed through Whatman filter paper and reduced under vacuum at 40 °C using a rotary evaporator to eliminate the solvent. The dried extracts were then transferred into dark vials Then kept at − 20 °C prior to subsequent analysis.

Identification of phenolic compound

HPLC-DAD analysis

Sample and standard solutions were prepared at 30 mg/mL and filtered through 0.4 μm membranes. Chromatographic separation employed an HPLC-DAD system using a method adapted from earlier reported studies13,14 featuring a Kinetex C18 column (250 × 4.6 mm, 2.6 μm) and a binary gradient of 0.1% acetic acid in water (A) and methanol (B). The gradient program was: 0–3 min (5→25% B), 3–6 min (25% B isocratic), 6–9 min (25→37% B), 9–13 min (37% B isocratic), 13–18 min (37→54% B), 18–22 min (54% B isocratic), 22–26 min (54→95% B), 26–29 min (95% B isocratic), 29–35 min (95→5% B), 35–45 min (5% B isocratic). The system operated at 1 mL/min flow rate, 30 °C column temperature, and a 10 µL injection volume for both samples and standards. Detection spanned 200–400 nm, with analysis performed at 280 nm, and compounds were identified by matching retention times to authentic standards.

Antiglycation activity

Determination of haemoglobin antiglycation activity assay

The assessment of antiglycation potential was carried out following the framework established by Abdnim et al.15. Briefly, 25 µL of SEFR (80 mg/mL) was combined 1 mL of hemoglobin solution (5%.). To prevent microbial growth, 5 µL of gentamicin (40 mg/mL) was introduced into each reaction tube. Next, 1 mL of a glucose solution (4 mg/mL) was added to the mixture. The resulting solution was allowed to incubate in the dark for 72 h. For comparison, gallic acid served as a positive control. After incubation, absorbance was recorded at 443 nm, and the percentage of anti-glycation efficiency was determined using the following equation:

$$\% {\text{ of inhibition }}={\text{ }}\left[ {1{\text{ }} - {\text{ }}\left( {{\text{Abs blank }} - {\text{ }}\left( {\left( {{\text{Abs Control }} - {\text{ Abs Sample}}} \right){\text{ }}/{\text{ AbsControl}}} \right)} \right)} \right] \times 100$$

The subsequent involved recording the following measurements:

Abs blank:

The absorbance of hemoglobin alone, without any added sample or glucose.

Abs control:

The absorbance of hemoglobin combined with glucose.

Abs sample:

The absorbance of hemoglobin mixed with both glucose and the test sample.

Determination of albumin antiglycation activity assay

The glycation of albumin was carried out based on the method described by Abdnim et al. (2024)16? with subtle tweaks. Briefly, a solution containing 10 mg/mL BSA was prepared in 100 mM potassium phosphate buffer (pH 7.4), supplemented with 0.2% sodium azide to prevent microbial contamination. Subsequently, 1 mL of the BSA solution was combined with a 500 mM glucose solution. SEFR and aminoguanidine (AG), both dissolved in phosphate-buffered saline (PBS), were incorporated into the mixture, which was then incubated at 37 °C in the dark for one week. The formation of advanced glycation end products (AGEs) was evaluated using a spectrofluorometer, with excitation and emission wavelengths set to 355 nm and 460 nm, respectively. Aminoguanidine acted as the positive control.

Estimation of Fructosamine

The formation of fructosamine was evaluated following the guidelines set by Ravan et al. (2016)17, using the TBA test. In this method, glycated BSA was incubated with 0.05 M TBA solution. After 20 minutes’ exposure to a heated water bath, the samples Were left to cool to ambient temperature, and absorbance was measured at 443 nm. The percentage of fructosamine inhibition was then Calculated according to the following expression.

$${\text{Inhibitory activity }}\left( \% \right){\text{ }}={\text{ }}\left[ {\left( {{\text{A}}0 - {\text{ A}}1} \right)/{\text{A}}0} \right] \times 100$$

A0: absorbance assessement from the positive control group, A1: absorbance following the addition of the extract sample.

Estimation of carbonyl groups

The quantification of albumin carbonyl groups was carried out using the strategy presented by Uchida et al.18, with subtle tweaks. Briefly, equal volumes (0.5 mL) of the glycated specimen and 10 mM DNPH made up in 2.5 M HCl were mixed in glass tubes and allowed to stand at room temperature for one hour. The absorbance was assessed afterward measured at 365 nm. Carbonyl content was calculated using the molar extinction coefficient of DNPH (365 nm = 21 mM/cm). the rate level of inhibition was determined using the following formula:

$${\text{Inhibitory activity }}\left( \% \right){\text{ }}={\text{ }}\left[ {1 - {\text{ }}\left( {{\text{A}}0 - {\text{ A}}1} \right)/{\text{A}}0} \right] \times 100$$

A0: absorbance assessement from the positive control group, A1: absorbance following the addition of the extract sample.

Estimation of amyloid β-structures

The assessment of Congo red affinity for β-amyloid aggregates was conducted by monitoring absorbance at 530 nm, As per the procedure specified by Rashmi S. Tupe et al. (2015)19. In this assay, 100 µL of Congo red reagent (100 mM prepared in PBS containing 10% ethanol) was mixed with 0.5 mL of glycated albumin. The blend was left at room temperature to settle for 20 min. Subsequently, the optical density was recorded at 530 nm. The formula outlined below was used to calculate the inhibition percentage:

$${\text{Inhibitory activity }}\left( \% \right){\text{ }}={\text{ }}\left[ {1 - {\text{ }}\left( {{\text{A}}0 - {\text{ A}}1} \right)/{\text{A}}0} \right] \times 100$$

A0 represents the absorbance measurement from the positive control group, while A1 represents the absorbance after the inclusion of the extract sample.

Anti-hyperglycemic activities

In vitro α-amylase Inhibition assay

The suppression of α-amylase activity was evaluated following The process explained by Abdnim et al.20. A reaction mixture was prepared by 100 µL of SEFR or acarboe was combined with 100 µL of α-amylase enzyme solution (13 IU) and 100 µL of 0.2 M phosphate buffer (pH 6.9) at different concentrations. This blend was pre-incubated at 37 °C for 10 min. Following pre-incubation, 100 µL (1% starch solution) was added, and the blend was incubated at 37 °C for a further 20 min. To cease the reaction, DNSA reagent Was poured in, and the blend was incubated at 100 °C for 8 min to allow for color development. Afterward, the samples were chilled in a cold water bath for 5 min, diluted incorporating 1 mL of distilled water, and the α-amylase inhibitory activity was measured spectrophotometrically at 540 nm. The inhibitory activity of α-amylase value was calculated according to the formula:

$$\% {\text{ Inhibition of }}\alpha - {\text{amylase}}={\text{ }}[({\text{Ab}}{{\text{s}}_{\text{C}}} - {\text{Ab}}{{\text{s}}_{\text{S}}})] / \left( {{\text{Ab}}{{\text{s}}_{\text{C}}}} \right) \times 100$$

Where: AbsC denotes the absorbance of the control reaction, and AbsS denotes the absorbance of the sample reaction.

In vitro α-glucosidase Inhibition test

A 0.1 U/mL α-glucosidase solution was formulated in a 100 mM phosphate buffer with pH 7.5, and 50 mM sucrose (0.1 mL) was inserted as the substrate. Then, 20 µL of SEFR at different concentrations was introduced to the blend, which was subjected to incubation at 37 °C for 20 min. After incubation, the reaction was terminated by warming the mixture at 100 °C for 5 min. The absorbance was subsequently recorded at 500 nm by means of a spectrophotometer20. The glucose produced throughout the reaction was quantified through the use of a commercial glucose oxidase kit. The inhibition percentage was computed using the given formula:

$$\% {\text{ Inhibition of }}\alpha - {\text{glucosidase}}=\left[ {\left( {{{\text{A}}_{\text{C}}} - {{\text{A}}_{\text{S}}}} \right)/{{\text{A}}_{\text{C}}}} \right] \times 100$$

AC: Absorbance indicating enzymatic activity in the absence of an inhibitor.

AS: Absorbance representing enzymatic activity in the presence of an extract or Acarbose.

Pancreatic lipase Inhibition assay

The inhibition of pancreatic lipase activity was tested with subtle tweaks, following the method portrayed by Jaradat et al. in 201921. In this assay, pNPP was Acting as the substrate. A series of SEFR dilutions were prepared at different concentrations. To perform the inhibition experiment, 0.1 mL of a 1 mg/mL lipase solution was blended with different volumes of the extract (0.2 mL), and 0.7 mL of Tris-HCl buffer (pH 8) was added afterward. the solution was properly homogenized and incubated at 37 °C for 15 min. After this incubation, volume of 0.1 mL of pNPP was incorporated to the blend, which was then incubated at 37 °C for an additional 30 min. The absorbance was monitored at 410 nm with a UV-Vis spectrophotometer. Orlistat was applied as the reference standard. The percentage of inhibition was computed using the following equation:

$${\text{Inhibitory activity }}\left( \% \right){\text{ }}={\text{ }}[({\text{A}}0-{\text{A}}1){\text{ }}/{\text{A}}0]{\text{ }} \times {\text{ }}100$$

Anti-inflammatory

In vitro, Inhibition of albumin denaturation- Heat-induced haemolysis

The anti-inflammatory activity of the extract was reviewed in vitro through the use of a distinct approach, such as the albumin denaturation assay., with modifications based on previously established methods13. In short, 0.5 mL of a 0.2% w/v BSA solution in Tris buffer (pH 6.8) was incubated at 37 °C for 15 min with varying concentrations of the plant extract SEFR or diclofenac sodium, the latter serving as the standard reference. The blend was then warmed at 72 °C for 5 min to trigger protein denaturation. Absorbance was measured at 660 nm to evaluate the degree of protein aggregation.

Red blood cell (RBC) membrane stabilization assay

Red blood cells were selected from human blood samples and were selected as a model for membrane stabilization assessment because their membranes are similar to lysosomal membranes. Through the enhancement of erythrocyte membrane stability, the lysosomal bilayer is also stabilized, it serves as pivotal for reducing inflammation. This is achieved by restraining the secretion of damaging proteases and enzymes produced upon activation neutrophils, which can trigger inflammation and lead to tissue damage22,23.

Preparation of phosphate buffer saline (PBS)

A buffering solution was prepared by dissolving 100 g of sodium chloride (NaCl), 2.5 g of potassium chloride (KCl), 18 g of disodium hydrogen phosphate dihydrate (Na₂HPO₄·2 H₂O), and 8 g of potassium dihydrogen phosphate (KH₂PO₄) in 1 L of purified water.

Preparation of a suspension of erythrocytes

Blood aliquots (7 mL) were sourced from healthy human participants aged 20 to 30 years, with no prior use of anti-inflammatory drugs in the month leading up to the experiments. The blood samples were drawn into heparin-coated tubes and centrifuged at 2,500 rpm for 5 min to isolate the erythrocytes. The erythrocyte pellet was subsequently rinsed three times with sterile saline (0.9% w/v NaCl) and resuspended in phosphate-buffered saline (pH 7.4) to prepare a 40% hematocrit suspension24,25.

Heat-induced hemolysis

Heat-triggered hemolysis was evaluated using the procedure outlined by Gunathilake et al.26. In this process, 0.05 mL of blood cell suspension was combined with 0.05 mL of extracts at varying concentrations and 2.95 mL of phosphate buffer (pH 7.4) in three sets. The blend was subsequently incubated at 54 °C for 20 min in a shaking water bath. After incubation, the mixture was spun at 2,500 rpm for 3 min. The supernatant’s absorbance was quantified at 540 nm using a UV/VIS spectrophotometer. Phosphate buffer solution was employed as the baseline, and aspirin was the standard for comparison. Hemolysis suppression was determined using the following equation:

$$\% {\text{ Inhibition of Hemolysis}}={\text{ }}[({\text{Ab}}{{\text{s}}_{{\text{Control}}}} - {\text{Ab}}{{\text{s}}_{{\text{test}}}})] / \left( {{\text{Ab}}{{\text{s}}_{{\text{control}}}}} \right) \times 100$$

In silico studies

Molecular docking

Preparation of ligands

The compounds identified in the hydroethanolic extract of Solanum obtusifolium Dunal, along with Acarbose (CID: 41774), Orlistat (CID: 3034010), and Warfarin (CID: 54678486)27, used as reference inhibitors, were acquired from the PubChem database in 3D SDF format (https://pubchem.ncbi.nlm.nih.gov/)28 and used as reference inhibitors.Their antagonistic potential toward human α-glucosidase and α-amylase was evaluated using molecular docking simulations. PyMOL (version 2.5.3) was used to convert the ligands into PDB format initiallyand then to PDBQT format with AutoDockTools (ADT; version 1.5.7, The Scripps Research Institute)29. Binding affinities and molecular interactions concerning to the reference inhibitors were analyzed and visualized using Discovery Studio Visualizer (Biovia, 2021), which also facilitated the creation of graphical representations29.

Preparation of proteins

The crystallographic structures of human α-glucosidase (PDB ID: 5NN8), human α-amylase (PDB ID: 1B2Y), bovine serum albumin (BSA) (PDB ID: 4OR0)28 and used as reference inhibitors.Their antagonistic potential toward human α-glucosidase and α-amylase was evaluated using molecular docking simulations. PyMOL (version 2.5.3) was used to convert the ligands into PDB format initiallyand then to PDBQT format with AutoDockTools (ADT; version 1.5.7, The Scripps Research Institute)29. Binding affinities and molecular interactions concerning to the reference inhibitors were analyzed and visualized using Discovery Studio Visualizer (Biovia, 2021), which also facilitated the creation of graphical representations29.

Preparation of proteins

The crystallographic structures of human α-glucosidase (PDB ID: 5NN8), human α-amylase (PDB ID: 1B2Y), bovine serum albumin (BSA) (PDB ID: 4OR0)30, and human pancreatic lipase (PDB ID: 1LPB) were downloaded from the Protein Data Bank (PDB) (www.rcsb.org)31. Each structure was processed by eliminating water molecules and incorporating polar hydrogens, and applying Kollman charges using AutoDockTools (ADT; version 1.5.7). For the docking simulations, a grid with a spacing of 0.375 Å and dimensions of 40 × 40 × 40 was set up, positioned at specific x, y, and z coordinates to encompass the catalytic sites and surrounding areas. the prepared macromolecules were subsequently stored in PDB format for subsequent docking analysis29.

ADME studies

With the advancement of computational tools, predicting the pharmacokinetic characteristics of compounds, encompassing absorption, distribution, metabolism, and elimination (ADME), has become increasingly essential32. These properties determine a compound’s journey from administration to elimination, influencing its effectiveness and safety in the body33. ADME analysis evaluates the capability of a molecule to pass through cell membranes, its interactions with the transporters and enzymes responsible for absorption and elimination, along with its metabolic stability. In this study, we utilized the SwissADME platform (available at www.swissadme.ch, accessed on February 22, 2025)29,34 to comprehensively analyze the phytochemicals present in the hydroethanolic extract of Solanum obtusifolium Dunal. This approach enabled a detailed assessment of their pharmacokinetic properties and potential therapeutic applications.

In-silico prediction of toxicity

Early evaluation of the toxicological properties of chemical structures is a critical step in the drug discovery process35. In silico toxicity models are designed to predict the potential toxic effects of chemical compounds, thereby minimizing time, animal testing, and associated costs35. In this study, the toxicity of the compounds previously identified in the hydroethanolic extract of Solanum obtusifolium Dunal36 was assessed using the online platform ProTox-II37. This tool employs a statistical algorithm that compares the chemical structure of a substance to a database of known toxic compounds. It also provides information on LD₅₀ values, toxicity classification, and several toxicological endpoints, including hepatotoxicity, nephrotoxicity, cytotoxicity, and immunotoxicity. The predicted results are presented categorically as either active or inactive38.

Statistical analysis

The results are presented as mean ± standard error of the mean (SEM) (n = 3). Statistical analysis was conducted using One-way ANOVA, with a significance level set at p < 0.05 for differences between mean values. All statistical calculations were carried out using GraphPad 5 (GraphPad Software, San Diego, CA, USA). IC50 values were determined through nonlinear regression analysis, also using the same software.

Results

Phytochemical composition of SEFR

Our results are based on a detailed HPLC-DAD analysis, which revealed the polyphenolic composition of The hydroethanolic extract from Solanum Obtusifolium Dunal fruit (SEFR). This study emphasizes the significant presence of quercetin, p-coumaric acid, catechin, and gallic acid. These compounds demonstrate a remarkable diversity of flavonoids and phenolic acids, which constitute the dominant molecular families in this extract. These findings are backed by the empirical data in Table 1; Fig. 1, highlighting the vital role of the aqueous extract in maintaining and enhancing these compounds.

Fig. 1
Fig. 1
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HPLC-DAD chromatogram of the hydroethanolic extract from Solanum Obtusifolium Dunal fruit and standards.

Table 1 Phytochemical composition of SEFR detected by HPLC-DAD.
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Antiglycation activity

In vitro, hemoglobin antiglycation activity of SEFR

The antiglycation activity of Solanum Obtusifolium Dunal fruit hydroethanolic extract (SEFR) was evaluated across a spectrum of concentration 0.06 to 1 mg/mL. The observations demonstrated that SEFR significantly suppressed hemoglobin glycation compared to the untreated group, revealing a substantial significant statistical distinction (p < 0.0001). At an amont of 1 mg/mL, SEFR displayed its greatest activity, with a percentage inhibition of 73.76 ± 0.002% (Fig. 2). The IC50 value of SEFR, calculated as 0.55 ± 0.003 mg/mL, highlights its significant antiglycation activity. Gallic acid demonstrated the highest antiglycation activity, with a percentage inhibition of 92.12 ± 5.46% at a concentration of 1 mg/mL (p < 0.001), and its IC50 measurement was recorded as 0.41 ± 0.003 mg/mL (Table 2). These insights underscore the potential antiglycation activity of SEFR, using gallic acid serving as a benchmark standard exhibiting superior activity.

Table 2 IC50 value (mg/mL) of hemoglobin antiglycation by hydroethanolic extract from Solanum obtusifolium Dunal fruit (SEFR) and Gallic acid.
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Fig. 2
Fig. 2
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Inhibitory effect of hydroethanolic extract from Solanum Obtusifolium Dunal fruit (SEFR) and Gallic acid on hemoglobin antiglycation activity. Results are shown as mean ± SEM (n = 3). ***p < 0.001 as function of the control group.

Antiglycation effect of albumin at different stages
Effect of hydroethanolic extract from Solanum obtusifolium Dunal fruit (SEFR) on multiple stages of albumin glycation

The findings presented in Table 3 reveals that SEFR significantly inhibits albumin glycation at Each of the three stages: Fructosamine, an early glycation product, was used as a typical glycation compound (TBA), Was observed to be suppressed by SEFR with an IC50 value of 0.46 ± 0.002 mg/mL, contrasted to 0.45 ± 0.006 mg/mL for AG. For protein carbonyl compounds, serving as indicators of the intermediate stage (DNPH), inhibition was observed with IC50 values of 0.49 ± 0.006 mg/mL for SEFR and 0.46 ± 0.003 mg/mL for AG, respectively. Fluorescence assessment of advanced glycation end-products (AGEs) in the later stages indicated that SEFR inhibits their formation, with IC50 values of 0.47 ± 0.005 mg/mL for SEFR and 0.45 ± 0.005 mg/mL for AG, respectively. The inhibition followed a dose-dependent pattern, with the most effective dose being 1 mg/mL for both extracts (Fig. 3A-C).

Effect of hydroethanolic extract from Solanum obtusifolium Dunal fruit (SEFR) on β-aggregation during glycation

Glycation serves as a fundamental mechanism that triggers changes in protein structure by increasing the abundance of amyloid cross-β structures. Such structures are crucial in protein aggregation. We executed a study to evaluate how SEFR is able to prevent The accumulation of glycated albumin, a mechanism associated with amyloidosis, using Congo red amyloid markers. Figure 2C illustrates that the addition of SEFR showed significant inhibition in conjunction with the amyloid signals. The IC50 values shown in Table 3 demonstrate significant suppression of amyloid cross-β structures by SEFR and AG, with values of 0.47 ± 0.008 mg/mL and 0.46 ± 0.006 mg/mL, respectively.

Table 3 IC50 values for the Inhibition of albumin glycation determined through assessments using TBA, DNPH, and congo red tests, along with AGE formation analysis.
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Fig. 3
Fig. 3
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Impact of hydroethanolic extract from Solanum Obtusifolium Dunal fruit (SEFR) and AG (1 mg/mL) (A) inhibition of fructosamine, (B) inhibition of protein carbonyls, and (C) on the alteration of albumin glycation, concerning β aggegation inhibition.

Anti-hyperglycemic actvities

In vitro α-amylase and α-glucosidase Inhibition

We investigated the in vitro blocking action of SEFR on pancreatic α-amylase and intestinal α-glucosidase enzymes at various concentrations, comparing it to the reference molecule. SEFR demonstrated a significant proportional to dosage suppression of pancreatic α-amylase activity (p < 0.0001) as shown in Fig. 4A. At a dosage of 1 mg/mL, SEFR exhibited an inhibition of 70.33%, which was close to the inhibition percentage of acarbose at the same concentration, which was 96.71%. The IC50 value for the SEFR extract was approximately 0.54 ± 0.002 mg/mL. Additionally, its influence on pancreatic α-amylase was comparable to that of acarbose, which had an IC50 value of 0.38 ± 0.003 mg/mL (Table 4).

The extract SEFR also exhibited a significant effect (p < 0.0001) on intestinal α-glucosidase enzyme inhibition, achieving a 72% inhibition, while acarbose demonstrated the highest effect with a suppression rate of 96.71% (Fig. 4B). These findings were consistent with the IC50 values recorded for the SEFR extract and acarbose, which were 0.53 ± 0.005 mg/mL and 0.40 ± 0.002 mg/mL, respectively (Table 4).

Table 4 IC50 value (mg/mL) of Inhibition of α-amylase and α-glucosidase by hydroethanolic extract from Solanum obtusifolium Dunal fruit (SEFR) and acarbose.
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Fig. 4
Fig. 4
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Inhibitory effect of α-amylase (A) and α-glucosidase (B) enzymes through hydroethanolic extract from Solanum Obtusifolium Dunal fruit and acarbose in vitro. The values are the means ± SEM (n = 3). *** p < 0.001 as function of the control group.

In vitro, pancreatic lipase Inhibition

In this study, SEFR extract was evaluated for its activity as an inhibitor of Porcine-derived pancreatic lipase. The findings, condensed in Fig. 5, demonstrate that SEFR and orlistat Strongly suppress (p < 0.001) pancreatic lipase across different concentrations, with IC50 measurements of 0.59 ± 0.004 mg/mL for SEFR and 0.48 ± 0.005 mg/mL for orlistat, respectively (Table 5).

Table 5 IC50 value (mg/mL) of Inhibition of lipase enzyme by hydroethanolic extract from Solanum obtusifolium Dunal fruit (SEFR) and orlistat.
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Fig. 5
Fig. 5
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Inhibitory effect on porcine pancreatic lipase of hydroethanolic extract from Solanum Obtusifolium Dunal fruit (SEFR) and Orlistat. Data were presented as mean ± SEM (n = 3). *** p < 0.001 as function of the control group.

Anti-inflammatory activity

Inhibition of albumin denaturation

The current study assessed the inhibitory effects on protein denaturation in vitro of hydroethanolic extract from Solanum Obtusifolium Dunal fruit (SEFR). Table 6 presents the IC50 values obtained for the inhibition of BSA denaturation. SEFR demonstrated stronger protein protection compared to diclofenac sodium (control), with IC50 values of 197.31 ± 6.89 and 76.46 ± 1.94 µg/mL (Fig. 6).

Fig. 6
Fig. 6
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The effect of Solanum Obtusifolium Dunal on inhibiting albumin denaturation is presented as the mean value ± SD, n = 3, **p < 0.01.

Membrane stabilization tests

Figure 7. shows a concentration-dependent protective impact of SEFR in preventing hemolysis. SEFR demonstrated superior efficacy, with IC50 values of 196.81 ± 4.59 µg/mL for hemolysis induced by heat, outperforming aspirin with IC50 values of 74.81 ± 3.01 µg/mL (Table 6).

Table 6 Membrane stabilizing effect of SEFR on heat-induced hemolysis test.
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Fig. 7
Fig. 7
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Impact of SEFR and aspirin on preventing hemolysis in the heat-induced assay.

Molecular Docking

Exploring antidiabetic potential: molecular interactions with α-Glucosidase (PDB ID: 5NN8) and α-Amylase (PDB ID: 1B2Y)

α-glucosidase and α-Amylase are two essential enzymes in carbohydrate metabolism, playing a fundamental role in digestion and blood sugar regulation. α-Glucosidase, located at the brush border of the small intestine, hydrolyzes oligosaccharides into glucose and other monosaccharides, thereby facilitating their absorption into the bloodstream39. This process is preceded by the action of α-amylase, which initiates starch breakdown in the oral cavity through its salivary form and continues its hydrolytic activity in the intestine via pancreatic α-amylase, producing simpler oligomers40. Beyond their digestive function, these enzymes have garnered increasing interest in the therapeutic field, particularly in diabetes management. α-Glucosidase is considered a promising pharmacological target, as its inhibition reduces glucose absorption and thereby contributes to better glycemic control41. Similarly, inhibiting α-amylase could slow down starch digestion and help mitigate postprandial blood sugar spikes42. Thus, in addition to their physiological role, these two enzymes play a key role in the development of new therapeutic strategies for diabetes, highlighting their significance in medicine and pharmacology43. In this study, the results obtained from the molecular docking of ligands with α-glucosidase (PDB: 5NN8) and α-amylase (PDB: 1B2Y) revealed that all molecules exhibit binding affinities lower than or comparable to those of acarbose (Table 7), except for ursolic acid which is distinguished by binding affinities greater than those of acarbose.In the α-amylase - ursolic acid complex (1B2Y), the dominant interactions include hydrogen bonds with GLY334, ARG252, and PRO332, as well as alkyl interactions with PRO4. These contacts suggest that ursolic acid inserts deeply into the catalytic pocket, stabilizing the interaction with key residues involved in substrate recognition. This binding may potentially disrupt enzymatic activity by preventing substrate access.In the α-glucosidase - ursolic acid complex (5NN8), the inhibitor interacts with HIS584, HIS717, TYR360, PRO595, and LEU865 through hydrophobic interactions and hydrogen bonds (Fig. 8). The presence of histidine and tyrosine residues in the binding site suggests an interaction stabilized by hydrophobic and π-alkyl forces, enhancing the ligand’s affinity for the enzyme. This molecule, along with other compounds present in the hydroethanolic extract of Solanum elaeagnifolium, plays a key role in the extract’s biological activity. The presence of these molecules, combined with their strong affinity for these enzymes, explains the inhibitory effect observed in vitro. This molecular interaction could be responsible for the enzymatic modulation activity of the extract, further highlighting its potential as a natural therapeutic agent.

Table 7 Summary of molecular docking studies on compounds identified in the hydroethanolic extract of Solanum obtusifolium Dunal against α-amylase, α-glucosidase, lipase, and BSA proteins.
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Fig. 8
Fig. 8
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2D and 3D Binding Interactions of Ursolic acid with α-Glucosidase (PDB:5NN8) and α-Amylase (PDB:1B2Y). (Generated in Discovery Studio Visualizer)

Exploring anti-obesity potential: molecular interactions with lipase (PDB ID: 1LPB)

Lipase plays a crucial role in lipid metabolism, playing a crucial role in fat digestion and absorption. Secreted primarily in its pancreatic form, it acts in the small intestine, where it Facilitates the breakdown of triglycerides into monoglycerides and free fatty acids through hydrolysis, facilitating their uptake by enterocytes44. This process is tightly regulated and influenced by colipase, a cofactor protein that stabilizes the enzyme and optimizes its activity in the presence of bile salts. Beyond its digestive function, lipase has garnered increasing interest in the therapeutic field, particularly in relation to diabetes and associated metabolic disorders. Excessive lipase activity promotes fat absorption and may contribute to obesity, a key contributor to the development of type 2 diabetes. Thus, lipase inhibition represents a promising pharmacological approach to reducing dietary lipid absorption, thereby limiting excessive weight gain and improving insulin sensitivity45. In this study, the findings from the molecular docking analysis of ligands with Lipase (PDB ID: 1LPB) indicated that all compounds exhibit binding affinities lower than or comparable to that of Orlistat (Table 7), except for ursolic acid and quercetin, which stand out due to their higher binding affinities compared to Orlistat. In the Lipase-ursolic acid complex (1LPB), the molecule is stabilized by hydrogen bonds and alkyl interactions. Conventional hydrogen bonds and carbon-hydrogen interactions are formed with LEU41, ALA43, and ARG44, while alkyl interactions are observed with LYS60 and BOG452 (Fig. 8). These interactions suggest that ursolic acid binds effectively to the active site of lipase, which may contribute to its inhibitory effect on the enzyme. The presence of hydrogen bonding interactions implies that ursolic acid may interfere with the enzymatic hydrolysis of lipids, potentially leading to an antilipase activity relevant for anti-obesity or metabolic regulation applications. The presence of these molecules, combined with their strong affinity for this enzyme, explains the inhibitory effect observed in vitro. This molecular interaction could underlie the enzymatic modulation activity of the extract.

Exploring anti-glycation potential: molecular interactions with bovine serum albumin (PDB ID: 4OR0)

Bovine Serum Albumin (BSA) is a widely used protein model in glycation studies, a non-enzymatic process implicated in various pathologies, including diabetes and its complications. Glycation occurs through the gradual attachment of reactive sugars binding to lysine and arginineresidues in proteins, resulting in the production of advanced glycation end-products (AGEs). These AGEs contribute to oxidative stress and inflammation, exacerbating cellular damage and metabolic dysfunctions observed in diabetes46. In this context, BSA is commonly used as a laboratory model to assess the effects of anti-glycation agents. Preventing AGE formation is a promising strategy to mitigate diabetes-related complications, including cardiovascular diseases, diabetic nephropathy, and retinopathy46. Several natural and synthetic compounds have demonstrated the ability to inhibit BSA glycation, thereby reducing AGE accumulation and its harmful effects. Thus, studying BSA in the context of anti-glycation research plays a fundamental role in shaping the development of new therapeutic strategies for diabetes and its complications, highlighting its significance in medicine and pharmacology47. In this study, the findings from the molecular docking analysis of ligands with Bovine Serum Albumin (BSA) (PDB ID: 4OR0) indicated that all molecules demonstrate binding affinities lower than or comparable to that of warfarin (Table 6), except for ursolic acid and quercetin, which stand out due to their higher binding affinities than warfarin. In the ursolic acid–BSA complex (PDB: 4OR0), docking results indicate that ursolic acid primarily interacts through π-alkyl interactions with HIS584, HIS717, TYR360, and LEU865 (Fig. 9). Histidine and tyrosine residues play a crucial role in ligand stabilization via hydrophobic and π-alkyl forces. These interactions suggest a strong binding affinity between ursolic acid and BSA, indicating its high potential for plasma protein binding. The high affinity of the molecules present in the extract for BSA could play a key role in regulating coagulation mechanisms, thereby suggesting the extract’s therapeutic potential as a natural anticoagulant agent.

Fig. 9
Fig. 9
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2D and 3D Binding Interactions of Ursolic acid with Lipase (PDB:1LPB) and Bovine serum albumin (BSA)(PDB:4ORO). (Generated in Discovery Studio Visualizer)

ADME analysis

In the early stages of drug development, in silico models are essential for evaluating pharmacokinetic properties, enabling the rapid identification of promising candidates while improving the overall efficiency of the process48. For oral administration, compounds are generally required to comply with Lipinski’s Rule of Five, with one exception. These criteria include: (1) a molecular weight ≤ 500 Da, (2) no more than five hydrogen bond donors, (3) no more than ten hydrogen bond acceptors (oxygen or nitrogen atoms), (4) an octanol/water partition coefficient (MLogP) ≤ 5, and (5) a total polar surface area (PSA) ≤ 140 Ų (54). In our study, all phytochemicals detected in the hydroethanolic extract of Solanum obtusifolium Dunal met Lipinski’s criteria. Moreover, the majority of compounds exhibited high intestinal absorption (Table 7), except for ursolic acid. All compounds, except catechin (7), were classified as non‑substrates of P‑glycoprotein (PGP) (Fig. 9). Regarding brain permeability, our results indicated that p-coumaric acid (TPSA = 57.5 Ų; WLogP = 1.3) is able to cross the blood–brain barrier (BBB) (Fig. 10). Another critical factor in drug safety assessment is metabolism, largely influenced by cytochrome P450 enzymes, which play a major role in detoxification processes, especially in the liver. Our analysis revealed that none of the identified compounds act as inhibitors or substrates of the main CYP450 isoforms, except quercetin, which was identified as a CYP1A2 inhibitor (Table 8). Collectively, these findings highlight the potential of the phytochemicals present in the hydroethanolic extract of Solanum obtusifolium Dunal as promising candidates for the development of orally administered drugs.

Table 8 Assessment of the Pharmacokinetic properties (ADME) of the phytochemicals detected in the hydroethanolic extract of Solanum obtusifolium Dunal (in silico).
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Fig. 10
Fig. 10
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BOILED-Egg Model of the GI absorption and BBB permeability of phytochemicals identified in the hydroethanolic extract of Solanum Obtusifolium Dunal. (1) Ursolic acid., (2) p-Coumaric Acid, (3) Syringic acid, (4) Caffeic acid, (5) 3-4-dihydroxybenzoic acid, (6) Gallic acid, (7) Catechin, and (8) Quercitin. PGP+: P-glycoprotein substrate, PGP-: non-substrate of P-glycoprotein.

Prediction of compounds toxicity

An in silico toxicological assessment was performed on major phytochemicals identified in the hydroethanolic extract of Solanum obtusifolium Dunal. The evaluation focused on hepatotoxicity, immunotoxicity, cytotoxicity, and acute toxicity (LD50) predictions. All tested compounds were predicted to be inactive for hepatotoxicity, with probability values ranging from 0.51 to 0.72. This suggests a favorable hepatic safety profile for the constituents. Additionally, none of the compounds exhibited immunotoxic or cytotoxic potential, with high probabilities for inactivity (P ≥ 0.84), indicating a low likelihood of causing immune or general cellular toxicity. The acute toxicity profiles, expressed as LD50 values, varied considerably. Catechin showed the highest safety margin with an LD50 of 10,000 mg/kg (toxicity class 6), while quercetin displayed the greatest predicted toxicity, with a low LD50 of 159 mg/kg (toxicity class 3). Most other compounds, such as gallic acid, 3,4-dihydroxybenzoic acid, ursolic acid, and syringic acid, belonged to toxicity class 4, suggesting moderate toxicity. Caffeic acid and p-coumaric acid fell into class 5, reflecting low toxicity. Overall, these predictions indicate that the main constituents of the extract have a good safety profile, particularly in terms of liver, immune, and cellular toxicity. However, the relatively high predicted acute toxicity of quercetin suggests that its presence should be considered when assessing the safety of the entire extract (Table 9).

Table 9 In silico prediction of organ toxicity and toxicological endpoints of phytochemicals from the hydroethanolic extract of Solanum obtusifolium Dunal.
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Discussion

This research assessed the effects of a hydro-ethanolic extract from Solanum obtusifolium Dunal (SEFR) on antiglycation, antihyperglycemic, and anti-inflammatory activities. We performed a screening of SEFR to discover potential phytochemicals with the results summarized in Table 1. The hydroethanolic extract Indicated the existence of the following compounds: Quercetin, P-comaric acid, catechin, gallic acid. The results were nearly similar to those of Bouslamti et al.49, who found that the analysis of Solanum Obtusifolium Dunal extracts demonstrated a high concentration of bioactive compounds, including flavonoids, polyphenols, and phenolic acids. The fruit extract (SEFR) was found to contain catechin, vanillin, quercetin 3-O-β-D-glucoside, rutin, quercetin, and kaempferol.

Chronic hyperglycemia is now recognized as the leading cause of most diabetic complications48,50. Glucose influences cell function through a variety of transient and reversible effects, as well as irreversible changes that can lead to progressive and cumulative dysfunction51,52. This indicates that chronic metabolic alterations, rather than acute changes, are essential in the onset and advancement of diabetic complications. One of the irreversible changes directly caused by hyperglycemia involves the production of advanced glycation end products (AGEs) via the Maillard reaction. AGEs exert diverse chemical, cellular, and tissue effects, serving ss central mediators not only in diabetic complications but also in broader aging-related changes53. Glycation is an automatic, enzyme-independent reaction in which free reducing sugars interact with unbound amino groups in proteins, DNA, and lipids, leading to the formation of Amadori products. These products subsequently undergo a series of irreversible dehydration and molecular rearrangements, ultimately resulting in the formation of AGEs. Louis-Camille Maillard first described this process in 1912, laying the foundation for our understanding of the chemical interactions between sugars and amino acids that drive glycation54. Glycation results in protein loss functionality and decreased elasticity in various tissues, such as tendons, skin, and blood vessels55,56,57. Since no enzymes exist to eliminate glycated compounds from the human body, glycation aligns with the hypothesis that the buildup of metabolic by products contributes to the aging process. Therefore, our study seeks to assess the inhibitory effects of SEFR on protein glycation, with a specific focus on albumin and hemoglobin. Our findings suggest that SEFR effectively inhibits hemoglobin glycation and reduces the formation of fructosamine, carbonyl compounds, and AGEs at various stages. The most abundant compound in this extract is quercetin, as previous studies have shown Quercetin significantly inhibited post-Amadori glycation, as shown based on the findings of the HSA-glucose and HSA-MG assays, indicating that quercetin targets nearly all stages of AGE formation58. Also the study by Li et al.59. Demonstrated that quercetin can quickly capture MGO and subsequently inhibit AGEs formation by generating mono- and di-MGO adducts formed under neutral and alkaline conditions in vitro. A previous study showed quercetin and catechin were found to inhibit early glycation, with an inhibitory concentration value of 15.58 mM and 35.01 mM, respectively60. The proposed mechanism suggests that catechin interacts with G-BSA and digestive enzymes, reducing enzyme activity and modifying the conformation of G-BSA. Catechin decreased the β-sheet content of G-BSA while preserving its helical structure. Additionally, catechin boosted the antioxidant capacity of G-BSA, potentially reducing oxidative stress in the gastrointestinal tract induced after meals by AGE release. This study improves our insight into the nutritional and health aspects impacts of catechin’s impact on dietary AGEs during gastrointestinal digestion61. However, the administration of Gallic acid (GA) alongside AGE infusion inhibited AGE-induced fibrosis. Moreover, GA treatment effectively inhibited the AGE-induced upregulation of pro-fibrotic genes and ECM proteins, including the expression of TNF-α, TGF-β, and MMP-2 and − 962.

Another therapeutic strategy for managing the initial phase of diabetes is also to reduce postprandial hyperglycemia. This is achieved by slowing down glucose absorption by inhibiting carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase, within the gastrointestinal tract. This enzymes: α-amylase, found in both salivary and pancreatic secretions, which breaks down α-(1,4) -glycosidic bonds in starch molecules to produce oligosaccharides or disaccharides. The next step involves α-glucosidase, located on the apical surface of enterocytes, which hydrolyzes the non-reducing α-(1,4) bonds in oligosaccharides or disaccharides, ultimately releasing individual glucose molecules. Consequently, inhibitors targeting these enzymes reduce the rate of glucose absorption, thereby reducing the postprandial increase in plasma glucose levels63,64. The SEFR demonstrates significant suppressive effects on the activity of both α-amylase and α-glucosidase enzymes in vitro. Flavonols stand out as the most common type of flavonoids found in plants. The inhibitory effect of myricetin and quercetin on pancreatic α-amylase from Hovenia dulcis was also investigated in the study of Meng et al.65. As shown by Tadera et al.66, myricetin demonstrated slightly stronger inhibitory activity than quercetin. Both compounds were identified as competitive inhibitors, a finding consistent with the work of Li et al.67, who also observed that quercetin acts through competitive inhibition. Therefore, the presence of an -OH group at the 5-position of the A ring appears to enhance the effectiveness of flavonoids to inhibiting α-amylase. In the other investigation of Solanum obtusifolium Dunal68, the enzymatic analysis of α-amylase and α-glucosidase, the IC50 values for ethanol extracts were found to be 17.78 ± 2.38 µg/ml for α-amylase and 27.90 ± 5.02 µg/ml for α-glucosidase, followed by acetone extracts with values of 17.96 ± 6.05 µg/ml for α-amylase and 36.44 ± 3.30 µg/ml for α-glucosidase. The antidiabetic activity of Solanum obtusifolium Dunal was assessed through the enzymes α-amylase and α-glucosidase by Bouslamti et al.69. Notably, SEFr demonstrated significant anti-α-amylase activity, with an IC50 value of 40.31 ± 2.04 µg/mL. The results also revealed that SEFr exhibited strong anti-α-glucosidase activity, with an IC50 value of 20.53 ± 0.37 µg/mL.

The lipase enzyme is also involved in the mechanism of uncontrolled hyperglycemia. Therefore, lipase does not directly regulate blood sugar levels. However, factors such as diet composition, especially the amount and variety of fats ingested, can indirectly affect blood glucose levels management. A diet rich in saturated fats may promote insulin resistance, potentially raising blood sugar levels. Lipase plays a role in triglyceride decomposition, a form of lipid found in the bloodstream. Increased blood lipid levels may be linked to conditions such as hyperglycemia or diabetes. In this research, the hydroethanolic extract from Solanum obtusifolium Dunal fruit was evaluated for its potential to inhibit porcine pancreatic lipase. Pancreatic lipase, an essential enzyme in lipid digestion, breaks down triglycerides into free fatty acids and glycerol through hydrolysis. Inhibiting lipase remains an effective strategy for managing conditions related to lipid imbalances, particularly obesity70. Orlistat is a widely used medication for lipase inhibition, decreasing the absorption of dietary fats by approximately 30%. However, it could lead to adverse effects such as oily stools, diarrhea, abdominal cramps, fecal urgency, and gas71,72. The study by Zhou et al.73 demonstrated that quercetin demonstrated an inhibitory effect on pancreatic lipase, with an IC50 value of 70 µg/mL. In vivo studies revealed that pre-administration of quercetin at doses of 5 and 10 mg/kg body weight significantly reduced fat absorption in rats. Additionally, the inhibition of lipase by quercetin was observed to last for at least 2 h in vivo. The computational study by Ahmed et al.74 found that the selected compound, catechin, adhered to Lipinski’s Rule of 5 and met other drug-like properties. Furthermore, catechin exhibited favorable docking scores, superior fit and molecular interactions, as well as enhanced structural stability and flexibility compared to Orlistat.

The hyperglycemia contributes to diabetes complications primarily by activating inflammatory pathways, which exacerbates cellular and tissue damage, thereby increasing the risk of chronic complications. This is why this study highlighted the anti-inflammatory activity of SEFR to investigate this correlation. Inflammation is a defensive response triggered within tissues to eradicate harmful pathogens and promote restoration of tissue following injury or infection75. In this investigation, the anti-inflammatory effects of SEFR were demonstrated by inhibiting protein denaturation and the stabilization of cell membranes. Protein denaturation is closely associated with inflammation and plays a role in various inflammatory conditions76. Therefore, the ability of a substance (SEFR) to inhibit protein denaturation suggests potential anti-inflammatory properties. SEFR exhibited greater protein protection. Kürbitz et al.77 demonstrated that catechins, particularly ECG, inhibited TNFα-induced activation of NF-κB, resulting in decreased secretion of pro-inflammatory and invasion-promoting proteins, such as IL-8 and uPA. Their findings further revealed that the green tea catechins ECG and CG demonstrate significantly stronger anti-proliferative and anti-inflammatory effects on PDAC cells than the extensively studied catechin EGCG. When RBCs are exposed to extreme conditions, such as hypotonic solutions or high temperatures, their membranes disintegrate, leading to the release of hemoglobin followed by its oxidation78. Because human RBC membranes share structural similarities with lysosomal components, inhibiting hemolysis under these conditions may indicate the extract’s potential anti-inflammatory mechanism. A possible explanation for the membrane-stabilizing effect is the extracts’ capability to suppress the release of lytic enzymes and inflammatory mediators, thereby preventing protein denaturation79. The anti-inflammatory potential of SEFR may stem from the presence of bioactive compounds such as quercetin, gallic acid, p-coumaric acid, and catechin, or from the synergistic interactions among these components.

Molecular docking studies demonstrated that the major bioactive compounds present in the extract, particularly flavonoids such as quercetin, exhibited strong binding affinities toward key enzymes involved in carbohydrate metabolism and glycation. Quercetin showed excellent docking scores against α-amylase and α-glucosidase, with multiple hydrogen bonds and hydrophobic interactions stabilizing its binding within the active sites of these enzymes. These interactions suggest a potential inhibitory mechanism that aligns with the observed in vitro enzyme inhibition. Additionally, quercetin demonstrated favorable binding to glycation-related targets, supporting its antiglycation activity. Overall, the docking results reinforce the hypothesis that the phenolic compounds in the extract contribute significantly to its antidiabetic and antiglycation potential through effective molecular interactions.

The in silico toxicological evaluation of the main phytochemicals from the hydroethanolic extract of Solanum obtusifolium Dunal revealed an overall favorable safety profile. None of the tested compounds showed predicted hepatotoxicity, immunotoxicity, or cytotoxicity, which is consistent with prior reports on the safety of polyphenolic compounds commonly found in medicinal plants. For instance, catechin and gallic acid, both predicted as non-toxic in our study, have previously been shown to exhibit minimal toxicity in animal models at therapeutic doses80. A study demonstrated that catechin-rich green tea extracts did not induce liver or kidney toxicity in rats even at high doses81, which supports our in silico prediction of catechin’s safety (LD50: 10,000 mg/kg; class 6)82. Similarly, gallic acid has been widely reported to be safe in vivo, although it may cause mild toxicity at extremely high concentrations80. In contrast, quercetin exhibited a relatively low LD50 (159 mg/kg; class 3) in our prediction model, indicating a potential for acute toxicityThis aligns with studies showing that, despite its beneficial pharmacological effects, quercetin can induce cytotoxicity and pro-oxidant effects at high doses or prolonged exposure83. Regarding p-coumaric and caffeic acids, their predicted low toxicity (class 5) also correlates with findings from several toxicological studies. Caffeic acid, in particular, has been shown to have a wide margin of safety in subchronic toxicity tests in rodents84. These findings reinforce the predictive accuracy of the in silico models employed. The absence of predicted hepatotoxic and immunotoxic effects for all compounds is particularly encouraging, as these are common limiting factors in the development of phytomedicines. Our ADME study reveals that the phytochemical compounds detected in the hydroethanolic extract of Solanum obtusifolium Dunal comply with Lipinski’s rule of five, suggesting their potential as candidates for oral drug formulation. Most of these molecules also exhibit high intestinal absorption, with the exception of ursolic acid. This parameter is a key determinant of drug efficacy, as good intestinal absorption enhances bioavailability, therapeutic efficiency, ease of administration, and overall tolerability85. Only catechin was classified as a non-substrate of P-glycoprotein (PGP-), indicating a lower likelihood of early elimination via P-gp. This could contribute to better retention and prolonged therapeutic stability within the body86. Moreover, p-coumaric acid (TPSA = 57.5 Ų; WLogP = 1.3) demonstrated the ability to cross the blood-brain barrier (BBB), highlighting its potential activity on the central nervous system and making it a promising candidate for neurological disorder therapies29. Another critical factor in drug safety evaluation is metabolism, mainly regulated by cytochrome P450 enzymes, which play a major role in hepatic detoxification87. Our analysis revealed that none of the identified compounds, except for quercetin, act as inhibitors or substrates of major CYP450 isoforms, particularly CYP1A2. This suggests a reduced risk of metabolic interference, thereby enhancing the safety profile of these phytochemicals88. Taken together, these findings highlight the potential of the compounds present in the hydroethanolic extract of Solanum obtusifolium Dunal as promising candidates for the development of orally administered drugs.

The use of the ProTox-II platform for toxicity prediction is justified by its reliability and its wide application in the field of computational toxicology. This approach provides a preliminary estimation of the safety of the identified compounds by classifying them into different toxicity levels and predicting their LD5033. Unlike many studies that focus solely on pharmacological efficacy, the integration of ProTox-II offers a more comprehensive perspective, combining therapeutic potential with safety profiling. This methodological choice therefore enhances the scientific value of the work by addressing an essential requirement for the future development of these compounds as therapeutic agents85.

Conclusion

This research highlighted that SEFR displayed substantial activity, potentially aiding in the prevention of diabetes complications through its antiglycation activity and the reduction of hyperglycemia. These results indicate that SEFR holds promise as a valuable agent for inhibiting glycation, counteracting hyperglycemia, and exhibiting anti-inflammatory activity. HPLC-DAD analysis suggested that these effects could be linked to various chemical compounds, including flavonoids and phenolic acids. These components exhibited antiglycation, antihyperglycemic, and anti-inflammatory properties, further reinforcing the study’s findings. Additionally, ADMET analysis confirmed that the major compounds identified in SEFR are non-toxic, supporting their potential safety for therapeutic use.