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  Cheminformatics bioprospection and experimental validation of corn silk for interventive type 2 diabetes therapeutics

  (2025-05) Akoonjee, Ayesha; Sabiu, Saheed

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  Diabetes mellitus (DM) is one of the oldest known human diseases, with type 2 diabetes mellitus (T2DM) being the most prevalent form. Type 2 diabetes mellitus (T2DM) is characterized by elevated blood glucose levels due to defective insulin production and/or resistance to insulin. If left untreated, it can lead to severe complications affecting various body systems. While synthetic medications are commonly used to treat T2DM, their associated drawbacks, such as high cost, inaccessibility and side effects, mitigate their application in managing T2DM. Consequently, there has been a growing interest in natural products with antidiabetic potential. Natural products, including medicinal plants and plantderived products, have been used for centuries, and their active compounds continue to be explored for therapeutic applications. For example, corn silk (CS), a waste material of corn cultivation, possesses several therapeutic properties, including antidiabetic potential. Although, studies reporting the promising hypoglycaemic potentials of CS exist, its exact mechanism of action remains incompletely elucidated, a research gap that was fulfilled in this study through metabolomics, cheminformatics bioprospection and in vitro experimental validation. To identify the constituents in CS, ultra-performance liquid chromatography-mass spectrometry analysis and principal component analysis was performed on its three extracts (aqueous, hydro-ethanolic and ethanolic) at two developmental growth stages (premature and mature). A library consisting of 128 metabolites was generated from all the samples of CS with qualitative and quantitative variations observed between the two growth stages of CS and the type of solvent used for extraction. Specifically, the mature CS had a higher abundance of most metabolites, with the hydro-ethanolic extract of CS being the most metabolites-rich compared to the aqueous and ethanolic extracts of CS. These metabolites were thereafter subjected to bioprospection against the therapeutic targets, such as enzymes and genes implicated in the pathogenesis of T2DM using computational techniques. The modulatory role of CS metabolites on six enzymes implicated in the pathogenesis of T2DM and its secondary complication, particularly alpha-amylase (AA), alpha-glucosidase (AG), aldose reductase (AR), dipeptidyl peptidase-4 (DPP-4), protein tyrosine phosphatase 1B (PTP1B) and sorbitol dehydrogenase (SDH), was analysed using molecular docking complemented with molecular dynamics (MD) simulation. Molecular docking analysis identified aesculin (-8.1 kcal/mol), austricin (-7.8 kcal/mol), (6E)-1-(4-hydroxyphenyl)-7- phenylhepta-4,6-dien-3-one (-9.9 kcal/mol), (-)-11-hydroxy-9,10-dihydrojasmonic acid 11-beta-D-glucoside (-8.6 kcal/mol), phaseic acid (-6.0 kcal/mol), and erythronolide B (- 9.2 kcal/mol) as compounds with the most negative scores against AA, AG, AR, DPP-4, PTP1B and SDH, respectively. However, a further insight into the binding free energy (ΔGbind) calculations of the putative leads against each enzyme over a 150-ns simulation period revealed that R-7-butyl-6,8-dihydroxy-3-[(3e)-pent-3-en-1-yl]-3,4- dihydroisochromen-1-one (BHP), 1-O-vanilloyl-beta-D-glucose (VBJ), (-)-11-hydroxy9,10-dihydrojasmonic acid 11-beta-D-glucoside (HDJ), p-coumaroyl malic acid (CMA), 2- hydroxydecanedioic acid (HDA), and (-)-11-hydroxy-9,10-dihydrojasmonic acid 11-beta-D-glucoside (HDJ) hold remarkable therapeutic promise as modulators of AA, AG, AR, DPP-4, PTP1B, and SDH, respectively. The post-MD dynamic simulation analysis and interaction plots in each case revealed the formation of thermodynamically stable complexes suggestive of the putative leads as potential modulators of the respective investigated enzymes and their possible applications in the management of T2DM and its secondary complications. Density functional theory (DFT) analysis was used to determine the molecular characteristics of the top ranked CS metabolites identified to modulate the investigated enzyme targets. Although the lower energy gaps, higher softness and lower chemical hardness of the metabolites did not correlate with their high negative binding free energy (potentially due to the observed relative residue fluctuations and increased surface area of the targets upon ligand binding), their electrophilicity indices which were above 1.5 electron volt (eV) alluded to their strong electrophilic potential. This highlights their ability to interact with amino acids with nucleophilic side chains of the target enzymes that is indicative of enhanced specificity and binding to the enzymes. Subsequently, a network pharmacology study was conducted to elucidate the relationship between CS constituents and signaling pathways implicated in T2DM. The analysis identified the cAMP pathway as the central signaling pathway, with adenosine receptor A1 (ADORA1), hydroxycarboxylic acid receptor 2 (HCAR2) and gamma-aminobutyric acid type B subunit 1 (GABBR1) as key therapeutic targets. Gallicynoic acid (-48.74 kcal/mol), dodecanedioc acid (-34.53 kcal/mol), and tetradecanedioc acid (-36.80 kcal/mol) interacted effectively with ADORA1, HCAR2, and GABBR1, respectively, relative to the reference standards (metformin and resveratrol) and formed thermodynamically stable complexes, as indicated by post-MD analysis results. These findings suggest the compounds as potential drug candidates for T2DM through modulation of cAMP pathway genes. The cAMP pathway is implicated in the pathogenesis of T2DM through various levels including glucagon and epinephrine-stimulated cAMP production, increased glucose release from the liver, modulation of insulin signaling, insulin resistance and the regulation of gut hormone secretion, including glucagon-like peptide-1. To complement and validate the results obtained through network pharmacology as a further way of elucidating the mechanism of antidiabetic action of CS, experimental validation employing the use of HepG2 cells was performed. The effect of different CS formulations on HepG2 cells was firstly assessed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) viability and glucose consumption assays, followed by real-time polymerase chain reaction (RT-qPCR) to understand the effect of CS on the expression of ADORA1 and GABBR1, the top two target genes modulated by the CS metabolites as identified by the network pharmacology study. For the MTT assay, CS extracts at concentrations 75 – 100 µg/mL promoted viability of HepG2 cells, with the ethanolic extract of the mature CS being the most viable relative to the controls (insulinand metformin-treated) and the untreated cells. Generally, higher HepG2 cell viability and glucose uptake were observed following treatment with mature CS extracts compared to premature CS. Specifically, the most significant and enhanced glucose uptake level was observed with both normal and insulin-resistant HepG2 cells following treatment with the aqueous extracts of mature CS extract compared to the controls. Furthermore, compared to the untreated cells, as well as insulin- and metformin-administered cells, treatment with CS extracts remarkably inhibited the expression of ADORA1 and GABBR1 in insulin-resistant HepG2 cells with the most prominent effect observed with the aqueous extract of premature CS. These observation with the CS aqueous extracts may be attributed to their relatively higher abundance of the profiled metabolites such as gallicynoic acid B and tetradecanedioc acid, which were more than 40% each by composition in both the mature and premature extracts. These findings regarding the high concentrations of gallicynoic acid B and tetradecanedioc acid in CS aqueous extracts are not only significant in modulating the expression of ADORA1 and GABBR1, resulting in increased glucose uptake in the treated cells but consistent with the results of MD simulation that profiled the two compounds as putative leads against the two most significant therapeutic targets in the cAMP signalling pathway associated with T2DM. Overall, the findings from this study have contributed to the elucidation of the mechanisms of antidiabetic action of CS metabolites which would be vital in the development of CS as a therapeutic agent for the management of T2DM and its associated secondary complications.

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  Development of a microalgae-based consortium for the bioremediation of sugar mill effluent

  (2025-05) Sibisi, Siphelele; Rawat, Ismail; Bux, Faizal; Mogany, Trisha

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  Industrial and agricultural activities have increased exponentially to meet the rising demands for food. The sugar production process is water-intensive and requires high volumes of freshwater that are subsequently discharged as effluent. An average of 1000 L of wastewater is produced per ton of sugarcane processed. The sugarcane industry wastewater is characterized by high chemical oxygen demand (COD: 1752 - 8339 mg L-1), and biochemical oxygen demand (BOD:1052 - 4641 mg L-1) but remains low in nutrients and other minerals. Discharging untreated wastewater into the environment might have negative consequences, thus reusing and treating wastewater is essential. Conventional physiochemical treatment methods including sedimentation, filtration, and coagulation-flocculation have shown limited efficacy. The sugar industry wastewater exhibits high biodegradability, thus biological treatment techniques are preferred due to environmental friendliness and sustainability. In recent years, the co-culturing approach has gained interest as a strategy to improve the biotechnological productivity of microalgae in wastewater treatment. This study aims to develop stable microalgae-based consortia using native, microalgal, bacterial and yeast strains. The steps adopted for achieving the above-stated aim were: (1) isolation, identification, and characterization of indigenous microorganisms from wastewater, and (2) assembling microbial consortia to identify the most effective and compatible strains. Indigenous microalgal, bacterial, and yeast strains were isolated and screened for growth in synthetic wastewater. The strains exhibiting significant growth were further characterized in real wastewater from the sugar industry to evaluate their performance efficiency based on COD removal efficiencies. To attain higher wastewater treatment efficiencies, different primary and secondary combinations of consortia were constructed from the pool of previously screened and selected microbial strains guided by the bottom-up approach. The microalgal, bacterial, and yeast microbial strains present in the final consortia were identified using polymerase chain reaction and sequencing. The final microalgal-based consortia were characterised in real effluent to assess wastewater treatment efficiency and elucidate their basic interactions for better application. Two microalgal, seven bacterial, and four yeast strains were isolated from the sugar industry wastewater. In the primary screening procedure in synthetic wastewater, two microalgal strains (A7 and C12), and four bacterial (B003, B009, B010, and B013) strains were found to grow substantially and thus selected for further studies and subsequent microalgae-consortia development. The two microalgal strains showed high removal efficiency for NO3--N (98–100%), NH4+-N (62-65%), and PO43--P (75-80%), however, the removal rate for COD was mainly observed in bacterial strains ranging between 1–73%. Also, the yeast strain (Y2) reduced COD from 22660 to 11690 mg L-1 (48% removal rate) within 168 h of cultivation. The co-culturing of microalgae with bacteria and yeast could improve the treatment of high-strength wastewater. In this regard, four primary, and three secondary consortia were considered. In all the primary consortia (B009A7, B010A7, B013A7, and Y2A7), improvement in removal efficiency for Total Nitrogen (TN) and Total phosphorus (TP) was recorded in ranges between 75-80% and 84-94%, respectively. However, a significantly lower removal rate (4-7%) was observed for COD. Furthermore, three secondary (B009B010A7, B009B013A7, and B010B013A7) and one tertiary consortia (B009B010B013A7) were considered. All secondary consortia exhibited a prolonged lag phase with reduced COD removal efficiency. In contrast, the tertiary consortia showed improved COD, TN, and TP removal efficiency corresponding to 26%, 85% & 73% (respectively) in synthetic wastewater. The microalgal, bacterial, and yeast strains in the final consortia were identified as Chlorella sorokiniana A7, Rhodococcus sp B009, Bacillus sp B010, Bacillus sp B013, and Saccharomyces cerevisiae Y2. The wastewater treatment efficiency of the consortia (MBC and MYC) was further evaluated in real sugar industry wastewater. The COD removal efficiency was found to be 86% and 71% after 4 days of cultivation for MBC and MYC, respectively. In addition, the co-culture with S. cerevisiae Y2 markedly increased chlorophyll-a content, photosynthesis, and respiration in microalgae. The microalgal-based consortia exhibited physical and biochemical interactions, with improved yield parameters and metabolite production between microalgae, bacteria, and yeast. The co-cultivation of Chlorella sorokiniana A7 and Saccharomyces cerevisiae Y2 was observed to have the highest COD removal efficiency from wastewater (100% within 4 d of cultivation). Microalgae and yeast mutually benefited from each other in the MYC system with synergistic cross-feeding between specific parameters such as CO2/O2, and organic acids. In addition, indole-3-acetic acid (IAA) was selected as a marker for evaluating the plant growth-promoting effects of co-cultured partners and determining the communication intensity. All co-cultured strains were found to produce and secrete indole acetic acid (IAA), suggesting plant growth-promoting effects. All the co-culture partners produced different concentrations of IAA under tryptophan ranging between 2 to 129 mg L-1. Meanwhile, IAA production was highest within 24 hr of cultivation in the MBC system, while the MYC exhibited a steady increase in IAA production, with the highest production observed after 72 h. The IAA signals are suggested to facilitate the establishment of mutualistic associations between microalgae and yeast/bacteria under varying environmental conditions. This indicates that yeast/bacteria may promote the growth of the co-existing microalgae through secretion of IAA, and microalgae would selectively enhance IAA secretion, thus, shaping the physiology and ecology of the partners in the microbial consortia. The study demonstrated the efficacy of microalgae-based consortia that have potential use in treating high-strength COD wastewater. The results could help improve the performance of the current treatment methods by introducing low-cost and sustainable biological technologies. The study demonstrated that microalgal-based co-cltivation is a promising bioremediation tool for high-strength biodegradable wastewater and presents environmental value in the design of low-energy, small-scale biological treatment systems. An insight into mechanisms of interactions between microalgae and co-cultured microbes still requires further study through integrated omics, studying the ecology and diversity of microbial communities could improve their application in environmental monitoring and bioremediation.

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  Metabolomic profiling, computational and experimental validation of sunflower seeds as therapeutics against type-2diabetes mellitus

  (2025-05) Rampadarath, Athika; Sabiu, Saheed; Makunga, Nokwanda

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  Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by impaired glucose metabolism due to insufficient insulin secretion or insulin resistance. This global health crisis is projected to affect an estimated 7079 individuals per 100,000 by 2030. While medications like metformin are effective, accessibility and affordability are issues consistent with low-income populations alongside potential side effects like hypoglycaemia, nausea and gastrointestinal issues that have limited their use in clinical practice. More importantly, uncontrolled T2DM can lead to serious complications like retinopathy, nephropathy, neuropathy, and delayed wound healing. Therefore, this prompts the search for alternative management options that are safer, easily accessible, affordable and with minimal side effects. Plants and their products are becoming increasingly important due to their relative ease of accessibility, affordability and potential health benefits. Sunflower seed, a popular dietary snack, has rich nutritional profile and has found significant health benefits as an antiinflammatory, antioxidant, anticancer, antimicrobial, and antidiabetic agent. While the antidiabetic potential of sunflower seeds has been explored, there remains a lack of understanding on its mechanism of action. This study addressed this knowledge gap by establishing the comprehensive metabolite profiles and investigating the antidiabetic efficacy of sunflower seed extracts through a two-pronged approach: targeted enzyme inhibition and network pharmacology analyses complemented with experimental validation in vitro. Metabolomic profiling of six cultivars of sunflower seeds commonly consumed in South Africa, namely, AGSUN 8251, 5270, 5101 CLP, 5103 CLP, 5106 CLP and 5108 CLP was performed using Liquid chromatography – mass spectrometry (LC-MS) and Gas chromatography – mass spectrometry (GC-MS) techniques. A total of 94 metabolites were identified, with LC-MS analysis revealing 44 phenolic compounds across the six cultivars with a minor variance of 39.7%, while GC-MS analysis revealed the presence of volatile compounds such as organic acids, alkanes, alcohols, terpenes, heterocyclic compounds and hydrocarbons in all the cultivars in similar abundance. Noteworthily, 84 of the 94 metabolites profiled passed Lipinski’s rule of five and were selected for further analysis. For the enzyme inhibition study, molecular docking analysis was initially used to screen the profiled metabolites against the key enzymes [α-amylase (AAMY), α-glucosidase (AGLU), aldose reductase (AR), sorbitol dehydrogenase (SDH), dipeptidyl peptidase 4 (DPP-4) and protein tyrosine phosphatase 1B (PTP1B)] implicated in T2DM pathogenesis and its secondary complications. The top-ranked metabolites against each enzyme were further subjected to molecular dynamics (MD) simulation to identify putative leads with the strongest binding affinity, and unperturbed structural integrity through evaluation of their stability, compactness and intermolecular interactions. This aspect of the study identified sonchuside I (SON I) - AAMY (–47.26 kcal/mol), sacranoside A (SAC A) - α-glucosidase (–40.10 kcal/mol), pelatoside A (PLT) - AR (–58.84 kcal/mol), sacranoside A (SAC A) - SDH (–48.03 kcal/mol), 4α,6S,7α)-6α-[6-O-(4-Hydroxybenzoyl)-β-D-glucopyranosyloxy]-7βmethyloctahydrocyclopenta[c]pyran-1-one) (PYR) -DPP-4 (–37.93 kcal/mol) and chlorogenic acid (CGA)-PTP1B (–24.32 kcal/mol) as potential lead inhibitors of the respective enzyme relative to their respective reference standards. This was further supported by their improved thermodynamic properties and favourable post-dynamic simulation parameters such as improved stability and compactness of their resulting complexes. These observations are suggestive of multiple mechanisms by which sunflower seed may exert its antidiabetic effects such as anti-hyperglycaemia (α-amylase and α-glucosidase), prevention and management of diabetic complications (AR and SDH), increasing insulin signalling (DPP-4) and sensitivity (PTP1B) by the respective putative leads. For network pharmacology analysis, the filtered sunflower seed metabolites were used to create a gene-compound library that was subsequently used to identify genes commonly associated with both the metabolites and T2DM. Thereafter, Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to identify the most significantly enriched pathways with key target genes for molecular docking and MD simulations to identify lead metabolites. Finally, the antidiabetic activity of sunflower seed extracts and the findings from the network pharmacology analysis were validated using insulin-resistant HepG2 cells where glucose consumption assay and gene expression analysis were performed. The network pharmacology analysis revealed a total of 87 genes common to sunflower seeds metabolites and T2DM, whereas KEGG enrichment analysis highlighted 35 signalling pathways potentially influenced by the metabolites. Of these, the Peroxisome proliferator-activated receptor (PPAR) signalling pathway and its hub receptors, Matrix metalloproteinase-1(MMPI) and peroxisome proliferator-activated receptor alpha (PPAR) were selected as the most significant. These receptors interacted mostly with the identified metabolites, with CGA (– 43.74 kcal/mol), GPA (–41.62 kcal/mol), and CFG (–45.36 kcal/mol) having lower binding free energy than both reference standards, rosiglitazone (ROS) and metformin (MET) against MMP1 after 100 000 ps MD simulation. In contrast, ROS (–46.98 kcal/mol) had better affinity against PPARA compared to the top-hits derived from sunflower seeds. However, against both genes, the top-hits had significant thermodynamic stability, flexibility, and compactness, which are attributable to their bond interactions and molecular orbital properties. These findings are suggestive of the essential role of the top-hits in the antidiabetic potential of sunflower seeds through activation of the PPAR signalling pathway and most especially MMP1. In this regard, the modulation of MMP1 and PPARA genes by the identified metabolites of sunflower seeds may enhance insulin sensitivity and glucose homeostasis in the management of T2DM. Finally, the in vitro validation using insulin-resistant HepG2 cells revealed cultivar-specific effects on cell viability, with each cultivar having a unique optimal concentration. Overall, all cultivars demonstrated the ability to stimulate glucose consumption, suggesting their potential antihyperglycemic activity. Among the cultivars, AGSUN 5103 CLP (14.4 mmol/L), 8251 (14.6 mmol/L), and 5101 CLP (13.7 mmol/L) exhibited the most pronounced glucose lowering action compared to the untreated cells (23.3 mmol/L) after 24 h, highlighting their promising antidiabetic effects. These three cultivars also modulate the PPAR signalling pathway, as evidenced by the upregulation of MMP1 and PPARA expression. Specifically, AGSUN 5101 CLP emerged as a particularly promising candidate based on its superior glucose lowering potential and higher fold increase expression of MMP1 (1.88) and PPARA (4.59) compared to the effect observed with the untreated cells (1.00). In conclusion, this study provides compelling evidence for the antidiabetic potential of sunflower seeds. The observed effects on enzyme inhibition, activation of the PPAR signalling pathway, and stimulation of glucose uptake in HepG2 cells suggest a multifaceted approach by the seeds in regulating blood sugar levels. The identification of cultivar-specific effects and promising lead compounds warrants further investigation to explore the therapeutic potential of sunflower seeds in managing T2DM.

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  Application of cold plasma for inactivation of waterborne pathogens

  (2025-05) Rampersad, Amelia; Swalaha, Feroz Mahomed; Reddy, Kevin P.

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  The inactivation of harmful pathogenic microorganisms during water treatment is essential for ensuring safe and clean water for human consumption. Current treatment technologies exhibit limitations in effectively eliminating pathogenic contaminants, necessitating the exploration of advanced disinfection technologies. This study investigates the application of atmospheric cold plasma (ACP) as a novel disinfection method for water contaminated with various pathogens, optimising key treatment parameters such as electrode distance and gas type (oxygen, argon and air) at voltages of 9.56 kV- 13.53 kV over treatment durations of 0.5-2.5 minutes. The study assessed ACP’s efficacy against chlorine-resistant and non-chlorineresistant bacteria, examining direct ACP treatment and effects after 24 h storage post-treatment. Bacterial suspensions at initial concentrations of 1 x 107 CFU/mL were exposed to varying treatment conditions, with bacterial inactivation analysed via colony counts and statistical analyses (two-way ANOVA with Tukey’s post-hoc). The results indicated significant log reductions in bacterial populations, with ACP achieving up to 4-log reductions, particularly against chlorine-resistant, Gram-negative bacteria. Among the gases tested, argon plasma had the highest bacterial inactivation rates, outperforming oxygen and air plasma, particularly against chlorine–resistant bacteria. Environmental water samples treated with ACP showed 90-100% bacterial inactivation, corresponding to log reductions of 3-5 logs, consistent across both direct and 24 h storage post-treatment samples. Argon and oxygen plasma showed high efficacy, with oxygen plasma having the highest inactivation in the uMhlanga Lagoon sample. The sustained antibacterial effect of ACP after 24 h storage post-treatment was attributed to its disruption of bacterial cellular functions, effectively inhibiting regrowth and ensuring long-term water safety. These findings confirm the potential of ACP as a highly effective and adaptable disinfection technology for water purification, particularly in targeting bacteria with carrying resistance profiles. While this study highlights the efficacy of ACP in inactivating pathogenic bacteria, further research is needed to ensure the safety of treated water for human consumption and to evaluate its effectiveness in removing chemical contaminants. This study demonstrates that ACP is a highly effective disinfection technology for water treatment, with consistent success in inactivating a wide range of pathogenic bacteria.

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  Multicomponent synthesis, characterization and antimicrobial evaluation of pyrazole derivatives

  (2025-05) Mntambo, Bahle Luyanda Dave-Junior; Makhanya, Talent Raymond; Gengan, Robert Moonsamy

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  People have been ailing regularly nowadays and conventional antibiotics have become less potent due to drug resistance. The molecular architecture of the antibiotics issued to patients has only marginally changed since their discovery, thus this has caused microorganisms to adapt or survive the repeated administration of the same drugs over time, causing medication to be less effective, and people becoming sicker because the lack of new, different and potent antibiotic structures has enabled bacteria to mutate. This presented the pressing need to develop pyrazole compounds because they suit the category of being structurally different and have been reported to exhibit excellent antibacterial activities. To obtain pyrazole derivatives, Microwave-Assisted Organic Synthesis (MAOS) was utilized to synthesize ten novel compounds of (4Z)-4-arylidene-4,5-dihydro-3-methyl-5-oxopyrazole-1-carbothioamide derivatives (4a-4j) by a one-pot multicomponent reaction (MCR) methodology that is ecofriendly and adhering to green chemistry principles, occurring by catalyst-free in a waterethanol solvent system. The methodology to access novel pyrazoles (4a-4j) in this study is superior to conventional pyrazole synthesis because it requires harsh reaction conditions and expensive catalysts, which deviates from green chemistry. All synthesized compounds (4a-4j) were fully characterized and confirmed by 1H NMR, 13C NMR, 2D NMR, FTIR and TOF-MS. Antimicrobial study was performed against two Gram-negative and two Gram-positive strains viz. Escherichia Coli (E.Coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus Aureus (S. Aureus), and Streptococcus pneumoniae (S. pneumoniae), respectively, and amoxicillin was used as the reference drug. It was found that only 4c and 4j exhibited activity against E. coli and their Minimum Inhibitory Concentration ( MIC) values were 1.38 and 2.50 mg/mL, respectively. In contrast, Amoxicillin displayed a lower MIC value of 0.0306 mg/mL, thereby suggesting that amoxicillin is more effective than the synthesized pyrazoles against E. coli. Pyrazoles 4a, 4d, and 4g showed no antibacterial activity against P. aeruginosa. Compounds 4b, 4c, 4e, 4f, 4h, 4i, and 4j displayed varying MIC values ranging from 0.212 to 0.625 mg/mL against S. aureus, 4i being 0.212 mg/mL the lowest MIC value. Amoxicillin displayed a lower MIC value than all the pyrazoles against P. aeruginosa. Against S. pneumoniae, 4j demonstrated an excellent antibacterial potential with a lower MIC value of 0.0156 mg/mL compared to that of amoxicillin (0.0306 mg/mL). The other pyrazoles displayed marginally higher MIC values than amoxicillin; 4g showed no activity for S. aureus. However, 4i displayed the lowest MIC value, suggesting its potential as an antibacterial agent. It was percieved that the unsubstituted phenyl ring in 4i contributed to its enhanced potency against test bacteria. Molecular docking studies were performed to predict the binding sites and affinities of the pyrazoles, revealing that they primarily target Penicillin Binding Proteins (PBPs) in the test bacteria. Docking scores for E. coli ranged from -6.7 to -7.9 kcal/mol and compound 4f exclusively exhibited the best docking score of -8.1 kcal/mol against E. coli better than amoxicillin’s docked score of -7.0 kcal/mol. Docking scores for P. aeruginosa ranged from - 6.8 to -7.7 kcal/mol. Compounds 4g and 4j displayed the highest negative docking scores of - 7.7 kcal/mol, outperforming amoxicillin thereby suggesting their potential as inhibitors, however 4b and 4f were comparable. Furthermore, in silico simulation, Molecular Dynamics (MD) studies were conducted for 4g and 4j. Docking scores for S. pneumoniae range from - 7.2 to -8.1 kcal/mol. Compound 4j displayed the highest negative docking score. Agreement between docking and in vitro results reinforces 4j as a potential inhibitor. The pyrazoles exhibited docking scores between -5.5 and -6.6 kcal/mol for S. aureus with 4j showing potential as an inhibitor. While lower than other PBPs, some pyrazoles are comparable to amoxicillin. Furthermor, the pyrazoles exhibited LogP (Lipophilicity) values oscillating between 1 and 2.5, indicating better bioavailability. Amoxicillin, partitioned close to an aqueous phase, showed by a negative LogP value of -0.29. Amoxicillin’s LD50 (lethal dose for killing 50% of the bacteria) is 15 g/kg, suggesting non-toxicity orally. Among pyrazoles, only compound 4c shares this safety profile as amoxicillin, qualifying 4c to be an orally administered antibacterial agent. Other pyrazoles fall into toxicity class 4, necessitating alternative administration routes, such as IV (Intravenous) or IM (Intramuscular). All studied compounds showed no affinity for BBB (Blood-Brain Barrier) permeability or P-glycoprotein binding, indicating they can traverse the system without hindrance from nonspecific enzymes or tissues. Compound 4d does not inhibit any CYP (Cytochrome P450) isoenzymes, similar to amoxicillin. Compound 4e selectively inhibits CYP 3A4, relevant for drug metabolism.

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