Novel Properties of Old Propranolol—Assessment of Antiglycation Activity through In Vitro and In Silico Approaches

Hypertension has earned the “silent killer” nickname since it may lead to a number of comorbidities, including diabetes and cardiovascular diseases. Oxidative stress and protein glycation play vital roles in the pathogenesis of hypertension. Several studies have shown that they profoundly account for vascular dysfunction, endothelial damage, and disruption of blood pressure regulatory mechanisms. Of particular note are advanced glycation end products (AGEs). AGEs alter vascular tissues’ functional and mechanical properties by binding to receptors for advanced glycation end products (RAGE), stimulating inflammation and free radical-mediated pathways. Propranolol, a nonselective beta-adrenergic receptor antagonist, is one of the most commonly used drugs to treat hypertension and cardiovascular diseases. Our study is the first to analyze propranolol’s effects on protein glycoxidation through in vitro and in silico approaches. Bovine serum albumin (BSA) was utilized to evaluate glycoxidation inhibition by propranolol. Propranolol (1 mM) and BSA (0.09 mM) were incubated with different glycating (0.5 M glucose, fructose, and galactose for 6 days and 2.5 mM glyoxal and methylglyoxal for 12 h) or oxidizing agents (chloramine T for 1 h). Biomarkers of protein glycation (Amadori products (APs), β-amyloid (βA), and advanced glycation end products (AGEs)), protein glycoxidation (dityrosine (DT), kynurenine (KYN), and N-formylkynurenine (NFK)), protein oxidation (protein carbonyls (PCs), and advanced oxidation protein products (AOPPs)) were measured by means of colorimetric and fluorimetric methods. The scavenging of reactive oxygen species (hydrogen peroxide, hydroxyl radical, and nitric oxide) and the antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl radical and ferrous ion chelating (FIC) assays)) of propranolol were also evaluated. Additionally, in silico docking was performed to showcase propranolol’s interaction with BSA, glycosides, and AGE/RAGE pathway proteins. The products of protein glycation (↓APs, ↓βA, ↓AGEs), glycoxidation (↓DT, ↓KYN, ↓NFK), and oxidation (↓PCs, ↓AOPPs) prominently decreased in the BSA samples with both glycating/oxidizing factors and propranolol. The antiglycoxidant properties of propranolol were similar to those of aminoguanidine, a known protein oxidation inhibitor, and captopril, which is an established antioxidant. Propranolol showed a potent antioxidant activity in the FIC and H2O2 scavenging assays, comparable to aminoguanidine and captopril. In silico analysis indicated propranolol’s antiglycative properties during its interaction with BSA, glycosidases, and AGE/RAGE pathway proteins. Our results confirm that propranolol may decrease protein oxidation and glycoxidation in vitro. Additional studies on human and animal models are vital for in vivo verification of propranolol’s antiglycation activity, as this discovery might hold the key to the prevention of diabetic complications among cardiology-burdened patients.


INTRODUCTION
−4 Consequently, hypertension is a civilizational disease.It is diagnosed more often in males than in females for individuals below 65 years of age. 5 Hypertension is a significant risk factor for cardiovascular diseases, strokes, kidney diseases, and hypertensive retinopathy.The prevalence of hypertension has risen 2-fold to reach 1.28 billion individuals since 1990. 6Treatment of hypertension is considered the most common reason for visits to general practitioners and for the prescription of chronic medications. 7he most common hypertensive drugs are beta-blockers, thiazide and thiazide-like diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), and alpha-blockers. 8ropranolol (C 16 H 21 NO 2 ; 1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol; Figure 1) is a nonselective betaadrenergic antagonist used for treating hypertension, angina pectoris, myocardial infarction, migraine, pheochromocytoma, cardiac arrhythmias, and hypertrophic cardiomyopathy. 9It competitively blocks beta 1 -and beta 2 -adrenergic stimulation, which decreases the blood pressure, heart rate, myocardial contractility, and myocardial oxygen demand.Propranolol also reduces portal pressure by producing splanchnic vasoconstriction (beta 2 effect).After oral administration, the drug is absorbed rapidly and completely. 10The onset of actions is 1 to 2 h after administration, and the peak effect is usually seen within a few days to several weeks.The main action of propranolol is mainly due to its parent compound.Propranolol is metabolized by cytochrome P450 in the liver; however, propranolol's metabolites exhibit significantly lower activity than the parent compound. 11rotein glycation, particularly the formation of advanced glycation end products (AGEs), plays a vital role in the evolution and advancement of cardiovascular diseases.Protein glycation profoundly impacts endothelial cell function, as it compromises the inner lining of blood vessels.It leads to reduced vasodilation, increased vascular permeability, and cellular inflammation, leading to atherosclerosis. 12AGEs also play a crucial role in myocardial damage by being accumulated in the heart tissues and thus affecting the structure and function of cardiac proteins. 13AGE accumulation may impact myocardial fibrosis and contractility impairment and increase the likelihood of arrhythmia. 14On the molecular level, AGEs activate specific receptors, mainly for advanced glycation end products (RAGE), thereby triggering pro-inflammatory and oxidative stress pathways. 15rugs with antiglycoxidative effects are particularly favored in cardiology.Nevertheless, little is known about the antioxidant and antiglycation properties of propranolol.The studied literature/current data are inconclusive, and thus, we are the first to investigate propranolol for its antiglycoxidative activity using various in vitro and in silico models.We have also conducted a systematic literature review on the antiglycation properties of propranolol.1.

MATERIALS AND METHODS
At first, initial data was investigated by analyzing titles and abstracts of publications independently by two researchers (K.K.L, M.N.).Then, two other researchers inspected all of the previously extracted manuscripts (M.N., D.T.).Only the papers compliant with the inclusion and exclusion criteria were employed for the final analysis.The Cohen's kappa coefficient (κ) was calculated to measure the level of the researcher's reliability.The result was κ = 0.94.Every article was evaluated methodologically, with the following elements undergoing the analyses: authors, publication year, study design, experiment population size, inclusion and exclusion criteria, length of research, and end points (Figure 2).

Reagents and Equipment.
All of the analytical grade reactants were obtained from Sigma-Aldrich (Numbrecht, Germany/St.Louis, Missouri, USA).0.2 mm membrane filters were used for sterilizing all chemical solutions promptly before utilization.An M200 PRO multimode microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland) assayed the absorbance and fluorescence.

SCAVENGING OF REACTIVE OXYGEN SPECIES (ROS)
3.1.Hydrogen Peroxide (H 2 O 2 ) Scavenging Capacity.The method recommended by Kwon et al. was implemented to assess the H 2 O 2 scavenging activity. 16First of all, 87.3 mg of butylated hydroxytoluene (BHT), 10 μL of sulfuric acid (H 2 SO 4 ), 7.6 mg of xylenol orange, and 10 mg of ferrous ammonium sulfate were amalgamated in 100 mL of 90% methanol−water solution to acquire a solution of ferrous ion oxidation-xylenol orange (FOX).Afterward, H 2 O 2 (50 mM) as well as the samples (final concentration: 1 mM) were mixed (1:1 and v/v) and next incubated for 30 min in room temperature conditions.Later, high-performance liquid chromatography (HPLC)-grade methanol (10 μL) was added to the sample solution (90 μL) in H 2 O 2 .Next, FOX reagent (0.9 mL) was mixed with the produced mixture, , where A 0 represents the control absorbance (without added drugs), A 1 represents the absorbance following the addition of the drugs, and A 2 represents the absorbance without the addition of FOX reagent. 16.2.Hydroxyl Radical (HO•) Scavenging Capacity.The assay modified by Su et al. was implemented to measure the scavenging activity of HO•. 17 0.25 mL of ferrous sulfate (FeSO 4 ), 0.4 mL of hydrogen peroxide (H 2 O 2 ) (6 mM), 0.25 mL of distilled water (H 2 O), 0.5 mL of the samples (final concentration: 1 mM), and 0.2 mL of sodium salicylate (C 7 H 5 NaO 3 ) (20 mM) were all mixed to be subsequently incubated for 1 h at 37 °C. 17At the 562 nm wavelength, the reaction mixture's absorbance was measured.Next, the following formula was employed to count the scavenged HO• (%) by means of the formula [1 − f(A 1 − A 2 )/A 0 ] × 100%, where A 0 represents the control absorbance (without added drugs), A 1 represents the absorbance following the addition of the drugs, and A 2 represents the absorbance without the addition of sodium salicylate. 17 16 Summarily, the diluted sample (30 μL) was mixed with the DPPH• solution (0.13 mg/mL) (180 μL), after which the solution was adjusted to a final volume of 210 μL by adding methanol.The DPPH solution assisted as a control.Then, the reaction mixture was incubated for 20 min at room temperature and the absorbance of the reaction mixture was measured at a 518 nm wavelength using a spectrophotometer.The inhibition rate (%) was calculated using the formula [(A blank − A sample )/A blank ] × 100%, where A blank represents the absorbance of the blank DPPH solution and A sample represents the DPPH solution after the addition of the sample. 16.4.Nitric Oxide (NO•) Scavenging.First, 100 μL of phosphate-buffered saline (PBS) containing 5 mM sodium nitroprusside (SNP) was added to each 50 μL sample.The mixture was next incubated at 25 °C for 150 min.Next, 155 μL of Griess reagent containing 1% of sulfanilamide, 2% of phosphoric acid (H 3 PO 4 ), and 0.1% of N-(1-naphthyl) ethylenediamine were added to the reaction mixture.A chromophore was released through nitrite diazotization of sulfanilamide with its conjugation with N-(1-naphthyl) ethylenediamine.The absorbance of the reaction product was determined spectrophotometrically at a 546 nm wavelength.The scavenged NO• (%) was calculated using the formula [1 − (A 1 /A 2 )] × 100%, where A 1 represents the sample absorbance (with the addition of drugs) and A 2 represents the absorbance without Griess reagent. 18.5.Ferrous Ion Chelation (FIC).FIC activity was determined by measuring the decrease in the formation of the Fe 2+ −ferrozine complex.FeCl 2 (18 μL, 0.06 mM) and CH 3 OH (16 μL) were mixed together with the samples (90 μL, terminal strength of 1 mM) or the BHT control.Immediately afterward, a ferrozine solution (18 μL, 5 mM) was added in order for the mixture to be incubated for an extra 5 min at room temperature.A spectrophotometer was used for measuring the absorbance at a wavelength of 562 nm.The percentage decrease in absorbance as compared to the control was then calculated to determine the ferrous ion chelating (FIC) activity. 16gure 2. Flowchart in accordance with PRISMA guidelines: the systematic review methodology.

BOVINE SERUM ALBUMIN (BSA) MODEL
Glycation/oxidation of BSA was performed based on previously applied methods. 19−25 BSA (98% purity) was immediately dissolved in 0.1 M sodium phosphate buffer (pH 7.4) that contained a preservative of 0.02% sodium azide.The following glycation agents were used: glucose (Glc), fructose (Fru), and galactose (Gal).In addition, aldehydes, glyoxal (GO), and methylglyoxal (MGO) were utilized.BSA was incubated with propranolol (1 mM) as well as 0.5 M of Glc, Fru, and Gal for 6 days or GO and MGO (2.5 mM) for a period of 12 h. 20−22,24−27 GO and MGO were utilized within a month after delivery, and working solutions were assembled briefly before assessment.In order to study the antioxidant properties of propranolol, BSA with propranolol was incubated with 20 mM of chloramine T (ChT) for 1 h. 28All the samples were subjected to strict incubation conditions, including incubation in darkness, sealed vials, and continuous shaking at a speed of 50 rpm and at a temperature of 37 °C. 19,20,26All of the incubation mixtures achieved a final concentration of 0.09 mM BSA.
In order to differentiate the results obtained for propranolol, aminoguanidine as a known protein oxidation inhibitor and captopril as an established antioxidant were utilized.−25 Despite this, the concentrations of oxidants, sugars, and aldehydes were substantially higher than the physiological reference values.They are instrumental in modeling the physiological processes that occur in the human body over weeks or even months in a notably short time. 19,20,26−25 The study was conducted in three series, each duplicated.
Products of Protein Glycation.2.4.1.Amadori Products (APs).A colorimetric nitroblue tetrazolium (NBT) assay was conducted to determine the total levels of APs.The monoformazan extinction coefficient, determined at 12,640 M −1 cm −1 , allowed us to calculate the absorbance at a wavelength of 525 nm. 32.4.2.β-Amyloid (βA).The fluorescence emitted during the binding of amyloid fibrils/oligomers to thioflavin T was analyzed.First, 10 μL of thioflavin T and 90 μL of samples were mixed on a microplate.The fluorescence was measured at a wavelength of 385/485 nm.33 2.4.3.Advanced Glycation End Products (AGEs).The content of AGEs was measured spectrofluorimetrically at a wavelength of 440/370 nm in a 96-well microplate reader.34,35 Before the study, H 2 SO 4 (0.1 M, 1:5, v/v) had been used for diluting the samples.36 2.4.4. Pructs of Glycoxidation.Dityrosine (DT), Nformylkynurenine (NFK), and kynurenine (KN) were assayed spectrofluorimetrically at the excitation/emission wavelengths of 365/480, 325/434, and 330/415 nm, respectively.Before the study, all the samples had been diluted with H 2 SO 4 (0.1 M, 1:5, v/v).36 All the results were standardized to the fluorescence of quinine sulfate solution (0.1 mg/mL) in 0.1 M H 2 SO 4 .37 Products of Protein Oxidation.2.4.5. Prote Carbonyls  (PCs).In order to determine the concentration of PCs, 2,4dinitrophenylhydrazine (2,4-DNPH) and carbonyls reacted in proteins damaged by oxidation.The absorbance of the reaction product was determined colorimetrically at a wavelength of 355 nm.The absorbance coefficient for 2,4-DNPH was used as a standard (22 000 M −1 cm −1 ).38 2.4.6. Adnced Oxidation Protein Products (AOPPs).A spectrophotometric assay was conducted to evaluate the concentration of the AOPPs.First, PBS was used for diluting the assayed samples (200 μL) in a 1:5 (v/v) ratio.The mixture and 0−100 μmol/L standard and blank PBS solutions (200 μL) were placed on a 96-well microplate.Next, 1.16 M potassium iodide (10 μL) (KI) as well as acetic acid (20 μL) (chem formula) was put into the wells.At a wavelength of 340 nm, the absorbance was calculated instantaneously in the microplate reader and compared with the blank solution (PBS (200 μL), potassium iodide (10 μL), acetic acid (20 μL)).The linear absorbance was represented (range: 0−100 μmol/L) by the ChT solutions.34 Molecular Docking.Molecular docking is a clear in silico method of predicting the best-preferred position of a ligand postbinding with a macromolecule (commonly a protein).We examined the possible interaction of BSA, glycosidases (αamylase (αA), α-glucosidase (αG), and sucrase-isomaltase (SI)), and AGE pathway proteins (RAGE, signal transducer and activator of transcription (STAT), Janus kinase 2 (JAK2), cAMP response element-binding protein (CREB), activating transcription factor 4 (ATF4), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p38 mitogen-activated protein kinase (P38 MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), phosphoinositide-3-kinase (PI3-K), protein kinase beta (PKB/Akt2), C/ EBP homologous protein (CHOP), nuclear factor-kB (NF-kB), RAF, rapidly accelerated fibrosarcoma 1 (RAF1), RASrelated 2 protein (RAS), and mechanic target of rapamycin (MTOR) with the propranolol molecule.The Protein Data Bank (PDB) Web site (https://www.rcsb.org/)was accessed to download a 3D crystal structure of BSA (ID: 4F5S) 39 in the.pdb format.The protein structure was determined by means of X-ray diffraction at a resolution value of 2.47 Å. 39 The National Library of Medicine Web site (https://pubchem.ncbi.nlm. ni.gov/) provided the 3D structure of propranolol (ID: 6882).40 In the beginning, AutoDock MGLTools 41 allowed to remove all the water molecules and replace them with polar hydrogen and Kollman's partial charges to minimize energy input.Next, the prepared protein structure was saved as a.pdbqt file.AutoDock Vina 42 (grid size of 40 × 40 × 40, with 0.375 Å spacing, located at coordinates 34.885, 23.976, and 98.792) simulated the possible molecular docking.The exhaustiveness parameter was set at a level of 8. Finally, PyMOL 2.5 allowed us to visualize the possible molecular docking.23,43−46,47−49 Statistical Analysis.GraphPad Prism 9.000 (GraphPad Software, San Diego, California, USA) allowed the statistical analysis to be completed. Thesults were conveyed in terms of the percentage share of the respective control values (BSA with glycation agents).The one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons allowed to determine the differences between the groups, and p < 0.05 was found to be statistically significant.Furthermore, a multiplicity-adjusted p-value was also determined.

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In vivo studies 2-month-old APPswe/PS 1dE9 mice served the purpose of the analysis whether the B-adrenoreceptor antagonist, propranolol, would impact fear memory persistence.
the intra-CA1 infusion of propranolol impaired long-term fear memory only when administered immediately before conditioning in their wild-type counterparts.propranolol was shown to diminish cognitive deficits, amyloid and Tau pathology in Alzheimer's transgenic mice.

Systematic Review.
The systematic review of the bibliography allowed for 279 publications to be recognized from the Medline (PubMed) database, including 223 that were omitted due to their title.Tihrty-three out of 56 abstracts were in line with the inclusion and exclusion criteria.Out of the eligible papers, 15 were found not to be connected to the topic of our research.Nevertheless, 18 papers were finally included (Figure 2).The final results of our systematic review are listed in Table 2. Propranolol scavenged H 2 O 2 at a rate of 5% in the assay.There were no meaningful differences in the inhibition rate of H 2 O 2 scavenging in comparison to that of propranolol (Figure 3A).
The level of APs was relevantly enhanced in GO+aminoguanidine (+50%) as compared to GO.On the other hand, the concentration of APs effectively increased in GO+aminoguanidine (+79%) as compared to BSA (Figure 5D).
The content effectively diminished in GO+captopril (−27%) versus GO.That parameter was alleviated more effectively in GO+aminoguanidine (−60%) than in the case of GO.The fluorescence of AGEs was enhanced in GO +aminoguanidine (+43%) as compared to that in BSA.That parameter was significantly elevated in GO (+255%), GO +propranolol (+246%), and GO+captopril (+160%) as compared to that in BSA (Figure 5N).
The PC level was improved in propranolol (+33%) as compared to that in GO (Figure 7D).
Binding Affinity Analysis.The ability of propranolol to impede protein glycoxidation was also assessed in in silico studies.The molecular docking simulation between BSA and propranolol revealed its binding solid affinity, 7.8 kcal/mol.Just two docking sites had root-mean-square deviations of atomic positions (RMSD) below 3 (Table 3); however, only mode 2 revealed a polar contact with TYR-160 of the BSA particle.Mode 2 is presented in Table 3.
The molecular docking for AGE pathway proteins indicated propranolol to have antiglycative properties in vivo, as it demonstrated the drug's ability to bind to all the tested ligands, from −5.2 kcal/mol for cyclin-dependent kinase inhibitor 1 (P21).Outstandingly high binding affinity was highlighted for NF-kB, PI3-K, and MTOR (−7.4,−7.2, and −7.2 kcal/mol, respectively) (Table 5 and Figure 9).Molecular docking analysis did not exhibit any polar contact for CREB (4NYX), P38 MAPK (2FSL), or CHOP (3T92).Propranolol's ability to bind to the AGE pathway may prevent adverse complications such as insulin resistance, inflammation, or the production of ROS, which aggravate protein glycation (Table 5 and Figure 9).

■ DISCUSSION
Propranolol is a nonselective beta-adrenergic blocker that competitively blocks the response to beta 1 -and beta 2adrenergic stimulation.It causes a reduction in heart rate, blood pressure, myocardial contractility, and myocardial oxygen demand, making propranolol widely used in hypertension, tremor, angina, arrhythmia, and other cardiac or circulatory disorders. 9−78 Our study is the first to comprehensively evaluate the antiglycation properties of propranolol using a glycated albumin model.Albumin, a major plasma protein, plays a vital role as a transport and buffering molecule. 79Nevertheless, albumin also has other biological properties.It can bind transition metal ions and endogenous and exogenous ligands such as hormones, inflammatory mediators, drugs, and pollutants.It also has a vigorous antioxidant activity. 80Not surprisingly, BSA is often used as a model protein because of its structural and functional similarities to human serum albumin. 81Within the framework of our study, several glycation factors (Glc, Fru, Gal, GO, and MGO) have been applied to induce BSA glycation.Various glycation factors have been found to be necessary to mimic physiological conditions in the human body.−86 The wide variety of glycation products formed under those conditions mimics (to some extent) the complexity of glycation processes observed in vivo.
The first step of protein glycation is to form a Schiff base, the preliminary reaction between the carbonyl group of a reducing sugar/aldehyde and the amino group of a protein to form a reversible Schiff base. 87The Schiff base undergoes an irreversible rearrangement to produce an early glycation product known as the APs. 88Oxidation, dehydration, and rearrangement reactions transform APs into AGEs that are very stable protein structures. 72Both dicarbonyl compounds, methylglyoxal (MGO) and glyoxal (GO), are highly toxic due to their glycating solid abilities. 89MGO acts as a potent glycation agent by rapidly reacting with lysine and arginine residues in proteins to produce AGEs. 90Similarly, GO facilitates the glycation process by reacting with amino groups and initiating the formation of AGEs. 85It has been proven that the concentration of MGO and GO increases inside  mitochondria under hyperglycemic conductions, which profoundly affects mitochondrial respiration. 91,92he contents of protein glycation (↑APs, ↑βA, ↑AGEs), glycoxidation (↑DT, ↑KYN, ↑NFK), and oxidation products (↑PCs, ↑AOPPs) were immensely increased in the BSA samples with the addition of all the glycation agents, in comparison to BSA without additives (Figures 3−7).The specific incubation conditions and reagent concentrations were chosen based on kinetic studies, further demonstrating the feasibility of sugars and aldehydes in an in vitro BSA model.
In all of the glycation models, the contents of protein glycation (↓APs, ↓βA, ↓AGEs), glycoxidation (↓DT, ↓KYN, and ↓NFK), and oxidation (↓PCs, ↓AOPPs) products were significantly lower under the influence of propranolol (Figures 3 −7).Propranolol often restored them to the initial BSA levels and, sometimes (βA in Gal), even more effectively than the baseline.Only APs, βA, and AGEs in GO were not significantly altered by the drug.The antiglycation effect of propranolol is also supported by the results of a systematic literature review (Table 2).Although few clinical studies have been conducted so far, in vitro and animal studies show that the drug's antiglycation properties are mainly due to the reduced production/modification of Aβ.Protein glycoxidation inhibition is the key to the prevention of cardiovascular complications.nonenzymatic glycation plays a vital role in the development of diabetes and micro-and macrovascular disorders. 93,94In atherosclerosis, the accumulation of AGEs in blood vessels contributes to endothelial dysfunction and promotes the adhesion of inflammatory cells and lipids to the vessel walls. 95AGEs may also modify lipoproteins, which increases their susceptibility to oxidation. 96ong-term hyperglycemia significantly increases the rate of the AGE formation and accumulation. 97That impairs the mechanism of the AGE removal from the body by reducing the activity of glyoxalase and other proteolytic enzymes. 98nhibition of BSA glycation by propranolol is also confirmed by in silico analyses.Foremost, the molecular docking between propranolol and BSA was conducted.The purpose was to assess propranolol's affinity in respect of albumin's binding sites and its ability to compete with or displace another substance.The simulation of propranolol manifested its very weak affinity with the BSA particle with a score of −8 kcal/ mol.Next, the molecular docking was conducted between propranolol and selected glycosidases.−101 Propranolol proved to have low binding energy for all hydrolases above (−6.8,−6.3, and −6.0 kcal/mol) (Table 4 and Figure 8).It is a widely understood concept that as the energy of the ligand−receptor decreases, the docking improves and affinity increases. 102The positive affinity of propranolol with the enzymes indicates the potential for inhibiting their activity.Therefore, propranolol's ability to decrease the sugars may be connected to its antiglycative properties.The αA particle's docking site consisted of two interactions with propranolol through aspartate (ASP)-300 and histidine (HIS)-299.The αG′s particle's docking site had one polar interaction with propranolol through N-acetylglucosamine (NAG)-1, and the sucrase-isomaltase (SI) docking site had one interaction with propranolol through asparagine (ASN)-43.
Finally, in silico docking was conducted for all of the AGE pathway proteins (Table 5 and Figure 9).The role of the AGEs/RAGE signaling in the modulation of gene transcription is closely associated with the development of type 2 diabetes and its related complications. 103Propranolol portrayed perfect binding affinity (not lower than −5.2 kcal/mol) for all 21 proteins.The drug bound most prominently to NF-kB (−7.4 kcal/mol; four polar contacts) as well as PI3-K (−7.2 kcal/ mol; two polar contacts) and MTOR (−7.2 kcal/mol; one polar contact).The NF-kB overactivation may increase protein glycation, stimulate vascular cell adhesion molecule-1 (VCAM-1), and activate inflammatory cell adhesion to the vascular endothelium.Propranolol proved to have four polar contacts with NF-kB through deoxyadenosine (DA)-608, (DA)-609, LYS-22, and LYS-25.Propranolol's interactions with various cellular regulators such as transcription factors, MAPKs, and cell cycle proteins may contribute to the preservation of the pancreatic β-cell function and insulin sensitivity and regulation of cellular responses to oxidative stress and abnormal protein synthesis induced by AGEs.In patients with diabetes, there is an increased expression of the AGE−RAGE signaling, directly contributing to the development of metabolic complications associated with diabetes. 94ell-established protein glycation inhibitors and antioxidants were utilized to compare propranolol's potential to protect against carbonyl stress.The antiglycation effect of propranolol was comparable to the protein glycation (aminoguanidine) and oxidation (captopril) inhibitors, which successfully prevented the changes induced by glycating/ oxidizing agents.Aminoguanidine prevents glycation due to a guanidinium group competing or scavenging dicarbonyls. 104It also exhibits a direct antioxidant activity.Captopril shows antioxidant properties by scavenging free radicals and increasing the activity of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase.Captopril may also inhibit the formation of AGEs by interfering with MGO and GO, which are essential precursors to the AGE formation.
Propranolol is a propanolamine in which propran-2-ol is switched by a propran-2-ylamino group at position 1 and a napthalen-1-yloxy group at position 3. 105 Therefore, propranolol contains an aryloxypropanolamine structure.In combination with propranolamine, the aryloxy ring promotes its binding to beta-adrenergic receptors, resulting in a competitive blockade of epinephrine and norepinephrine. 106In contrast, propranolol's hydroxyl and secondary amino groups promote water solubility and metabolite formation during conjugation reactions in the liver. 107The OH group also promotes interactions with biological targets, such as beta-adrenergic receptors. 108That chemical structure may also account for propranolol's antioxidant activity, thus explaining the drug's antiglycation effect.The potential antioxidant properties of propranolol confirm the results of our study.Although propranolol poorly scavenges the DPPH or HO radical, the ability to neutralize hydrogen peroxide or bind iron ions in the FIC assay is comparable to captopril and aminoguanidine.Gomes et al. demonstrated that certain β-blockers, including propranolol, acted as effective ROS (O 2 − , H 2 O 2 , HO•, HOCl, and ROO•) and/or RNS (•NO and ONOO − ) scavengers.They proposed that those effects could prevent cardiovascular complications, including hypertension and other comorbidities.The HO• scavenging activity for propranolol was significantly lower than that for labetalol and pindolol but higher than that for sotalol, timolol, atenolol, and metoprolol.Similarly, the results of the •NO scavenging assay proved that atenolol and pindolol had a higher scavenging capacity than propranolol and carvedilol, both of which did not reach 50% effect at the maximum tested concentrations (5000 and 50 μm, respectively).All the assayed compounds, except for timolol and labetalol, were able to scavenge peroxynitrite (ONOO − ); however, propranolol and atenolol showed IC 50 (half-maximal inhibitory concentration) of 1112 ± 232 and 2415 ± 278 μM (mean ± standard error of the mean (SEM)), respectively.In a concentration-dependent fashion, propranolol was able to delay the loss of fluorescence due to the ROO• dependent fluorescein oxidation. 109he available related literature also confirms the antioxidant effects of propranolol in vivo.The D-isomer of propranolol has been shown to reduce cardiac Fe uptake and inflammation and protect against oxidative stress and progressive cardiac dysfunction in rats overloaded with iron. 110,111Propranolol may also act as a "chain-breaking" antioxidant to protect cardiac membrane lipids from peroxidative damage, in addition to simple (reversible) xanthine oxidase (XOD) inhibition. 112n the other hand, chronic propranolol treatment strengthens the antioxidant barrier and protects against ischemiareperfusion injury in isolated hearts of animals without βblockade. 76Therefore, propranolol's antioxidant properties may be due to its beta-blocking effects.By reduction of the activity of the sympathetic nervous system, propranolol mitigates oxidative stress by inhibiting catecholamines that generate free radicals.Ranasinghe et al. assessed the impact of propranolol on nitrosative stress and antioxidant potential in patients suffering from resistant hypertension.Serum nitrate and nitrite levels were significantly lower after 90 days of propranolol treatment.The serum total antioxidant capacity (AOC) also increased in the study group as compared to the placebo group. 75ur study has shown that propranolol has antiglycation properties in in vitro and in silico models.The additional effect of propranolol is comparable to that of known inhibitors of protein glycoxidation.Although further studies are required, the drug may be particularly indicated for people with cardiovascular disease and diabetes.Propranolol is a wellknown drug with an established safety profile.The drug is toxic at plasma concentrations of more than 2 μg/mL, and mortality occurs at doses of more than 3 μg/mL. 113,114It should be remembered that propranolol is a very lipophilic beta blocker.It may easily cross the lipid cell membrane/blood−brain barrier and cause seizures in overdose cases.In diabetic patients, propranolol may also mask some of the symptoms of hypoglycemia, e.g., tachycardia and tremors. 113,114t should be mentioned that our study has a possible limitations.The BSA glycoxidation model simplifies the complex molecular interactions between proteins in vivo, which creates difficulties in transferring the results to more complex physiological models.Additional studies of animal and human models are needed for further analysis.Thus, our work provides a starting point for further research.

Data Availability Statement
The supporting data of this study is available from the corresponding author upon a reasonable request.

. 1 .
Systematic Review.The literature review was performed between 1995 and 2023 on the Medline (PubMed) database.The available bibliography was studied by using the following keywords: [propranolol and antiglycoxidative properties], [propranolol and antiglycation properties], [propranolol and antioxidative properties], [propranolol and oxidative stress], [propranolol and carbonyl stress], [propranolol and protein glycation], [propranolol and nitrosative stress], and [propranolol and ROS scavenging].Inclusion and exclusion criteria are demonstrated in Table

53 [
an enantioselective analytical technique was used for assessing the interaction of propranolol with α1-acid-glycoprotein (AGP) at the microliquid−liquid interface.AGP was added to aqueous solutions of propranolol hydrochloride both cyclic voltammetry and differential pulse voltammetry current responses decreased.Enantioselective binding by AGP of (S)-propranolol (2polypeptide (residues 121−231; PrP121−231)�only autonomous folding unit of PrP with a defined 3D structure.Utilizing a PrP-immobilized biosensor chip, mouse embryonic fibroblasts were isolated from a higher response value was expressed by diazepam, promethazine, and propranolol than by quinine hydrochloride, a well-established effective antiprion compound.Propranolol was identified as a new antiprion compound.51QC13.5-day-oldembryos of angiotensin II type 1 receptor (AT1aR)-deficient mice.It was done to analyze the interaction between the angiotensin receptor and the β-adrenergic receptor and its impact on the production of amyloid β-protein(βA).thewell-known increase in βA production after treatment with Telm actually decreased following the addition of propranolol in a dose-dependent manner.Therefore, the interaction between AT1R and the β-adrenergic receptor (β-AR) may play a role in the pathway that stimulates βA production caused by Telm.52 assessing the βA-induced reduction of soluble amyloid precursor protein α (sAPPα) in SH-SY5Y neuroblastoma cells.50 μM of propranolol ameliorated the βA-induced reduction of sAPPα secretion; therefore, proposing diacylglycerol (DAG) may account for the βA-induced reduction of sAPPα.3H] myristic acid-prelabeled LA-N-2 cells were exposed to various concentrations of amyloid beta protein 25−35 ranging from 20 to 250 μg/mL, and the activation of phospholipases A and D was assessed.Various substances such as propranolol, 7-chlorokyneurenic acid, metabotropic amino acid antagonist, and [Tyr 4 -D-Phe 12 ] bombesin were assessed for βA protein stimulation of the phospholipase C activity.propranolol, 7-chlorokyneurenic acid, metabotropic amino acid antagonist, and [Tyr 4 -D-Phe 12 ] bombesin were determined to decrease the βA protein stimulation of the phospholipase C activity inside the [ 3 H]inositol-prelabeled LA-N-2 cells.Therefore, the βA protein activation of phospholipase C may be receptor-mediated.
Total Oxidant Properties.ROS are products of enzymatic and nonenzymatic reactions of oxidative metabolism.Those chemically active molecules, despite low concentrations, participate in many physiological processes.Increased ROS concentrations lead to oxidative alterations of the cellular biomolecules.Antioxidant properties of the test sample may be determined by studying the scavenging capacity of hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO•), and 2,2-diphenyl-1-picrylhydrazyl (DPPH•). 68,693.2.1.Scavenging of H 2 O 2 .

Table 1 .
Inclusion and Exclusion Criteria of the Examined Publications inclusion criteria exclusion criteria publications written in English publications written in other languages manuscripts relevant to human/animal in vivo and in vitro experiments review papers, surveys, and case descriptions articles on antiglycooxidative activity of propranolol articles not describing the antiglycooxidative activity of propranolol vortexed, and incubated at room temperature for 30 min.The reaction product (ferric ion-xylenol orange) was assayed spectrophotometrically at a wavelength of 560 nm.The scavenging of H 2 O 2 (%) was quantified using the formula [1

Table 3 .
Results of Molecular Docking Simulations between Propranolol and BSA a

Table 4 .
Results of Molecular Docking Simulations Conducted between Propranolol and Glycosidases a

Table 5 .
Results of Molecular Docking Simulations between Propranolol and Advanced Glycation End Product (AGE) Pathway Proteins a Department of Hygiene, Epidemiology and Ergonomics, Medical University of Bialystok, Bialystok 15-233, Poland; Email: mat.maciejczyk@gmail.com