Alkaline phosphatase (AP, EC 3.1.3.1) is a key metalloenzyme and has been widely used as a reliable marker for milk pasteurization. Milk containing antibiotic residues tends to inhibit AP function, which can result in faulty pasteurization determination. AP from bovine milk was purified by a purification strategy involving butanol homogenization, rennet precipitation, and Sephadex G-100 gel filtration chromatography. The enzyme was purified 53.43-fold with a specific activity of 7516.40 EU/mg. Kinetic characterization showed a KM value of 2.27 mM (Vmax value: 555.56 EU/min); substrate was p-nitrophenyl phosphate. Electrophoretic studies verified the purity and molecular nature of the product enzyme. To functionally validate the identity of the purified enzyme, tetramisole hydrochloride was evaluated as a standard reference inhibitor, yielding a potent IC₅₀ value of 0.045 mM. Fifteen veterinary drugs and antibiotics were used to test the inhibitory effects of purified milk AP in vitro. Among the antibiotic compounds tested, Danofloxacin Mesylate induced the strongest antibiotic inhibition (IC50: 3.36 mM). Gentamycin Sulfate, Metamizole Sodium, Enrofloxacin, Kanamycin Sulfate, Sulfadimidine Sodium, and Neomycin Sulfate had IC50 values ranging between 22.89 and 312.13 mM of inhibition in AP, while Dexamethasone, Oxytetracycline Hydrochloride, Marbofloxacin, Tilmicosin, Doxycycline Monohydrate, Tylosin, Florfenicol and Flunixin Meglumine made no detected inhibition in the presence of 0.5 mM p-nitrophenyl phosphate. The molecular docking studies were additionally performed that the density functional theory (DFT), Hartree–Fock (HF), and M06-2X calculations were used to validate the experimental results with the 6-31++G(d,p) basis set. The theoretical results also reported that Oxytetracycline Hydrochloride, Tilmicosin, and Doxycycline Monohydrate displayed superior electronic reactivity, lower energy gaps between HOMO–LUMO, and more electrophilic character compared with the other compounds. These results are the first complete assessment of the impact of veterinary antibiotics in milk AP and underscore the possible role of antibiotic residues in establishing the reliability of pasteurization control measures.
Plant protease inhibitors have been suggested as one of the pest control strategies to control Helicoverpa armigera. To isolate protease inhibitors that are effective against H. armigera gut proteases, plants were screened for the presence of potent protease inhibitors after segregating them into host and non-host depending on whether H. armigera was reported to feed on it or not. A carboxypeptidase inhibitor and a cysteine protease inhibitor were isolated from non-host plants Oxalis corniculata (OcPI) and Withania somnifera (WsPI) respectively. Both inhibitors have Ki in the micromolar range, active between pH 2.6 to pH 10.6, and are stable up to 75 °C. At 3 inhibitor units per gram of diet (IU/g) each, OcPI caused higher mortality (24
Leech saliva contains a diverse array of bioactive compounds, most notably hirudin, a potent natural thrombin inhibitor. Despite the clinical utility of hirudotherapy, inherent risks such as localized infection, uncontrolled hemorrhage, and leech migration limit its widespread application. To overcome these limitations, this study presents a refined extraction, partial purification, and stabilization protocol for hirudin-containing salivary extract derived from the Iranian leech, Hirudo orientalis. Saliva was harvested using 0.001 M arginine in 0.15 M saline under controlled temperatures (23 °C vs. 37–40 °C) and subsequently stored at 4 °C. The crude extract underwent fractionation/purification via centrifugation, followed by stabilization with sucrose, fructose, and mannitol, and final lyophilization (freeze-drying). Optimal extraction efficiency was observed at 37–40 °C. The short-term stability (44 days at 4 °C) and antibacterial efficacy of the resulting lyophilizate were evaluated using High-Performance Liquid Chromatography (HPLC) against authentic reference parameters and Brain Heart Infusion (BHI) broth assays. The findings demonstrate that this stabilization matrix preserves the short-term chromatographic profile and in-vitro biological activity of the salivary protein extract, providing a preliminary foundation for future in-vivo evaluation and development of standardized leech-derived biotherapeutics.
Plaque psoriasis is a long-term skin disease driven by the immune system, impacting around 1–3
A series of thiadiazol–hydrazine derivatives (3a–3d) were rationally designed, synthesized, and characterized to explore their antimicrobial potential and possible interactions with dihydrofolate reductase (DHFR). The antimicrobial activity of the synthesized compounds was evaluated against representative Gram-positive bacteria (S. aureus and S. pyogenes), Gram-negative bacteria (E. coli and P. aeruginosa), and fungal strains (C. albicans, A. niger, and A. clavatus) using the minimum inhibitory concentration (MIC) method. Among the synthesized derivatives, compound 3b exhibited the most favorable overall antimicrobial profile, particularly against the tested bacterial strains, whereas the antifungal activity of the derivatives was comparatively limited. To investigate the possible molecular basis of the observed antimicrobial activity, molecular docking studies were performed against dihydrofolate reductase (DHFR; PDB ID: 1AOE). The synthesized derivatives exhibited favorable predicted binding within the DHFR active-site region, with compound 3b showing the most favorable docking score among the investigated compounds. The predicted binding mode of 3b was characterized by hydrogen-bonding and hydrophobic interactions with residues lining the DHFR binding pocket. Molecular dynamics simulations and interaction-energy analyses were subsequently employed to examine the stability and dynamic behavior of the predicted protein–ligand complexes. The simulation results supported the persistence of the predicted interactions and provided additional computational evidence for the favorable accommodation of 3b within the DHFR binding site. Density functional theory (DFT) calculations at the B3LYP-D3BJ/def2-TZVP level were performed to investigate the electronic properties of the synthesized derivatives. The calculated frontier molecular orbital characteristics and related electronic descriptors provided complementary insights into the influence of structural variation on molecular reactivity and the observed biological activity. Furthermore, haemolytic toxicity studies demonstrated low erythrocyte membrane disruption (< 3
