The Eulophia macrobulbon (EM) ethanolic extract and its active component, 1-(4´-hydroxy-benzyl)-4,8-dimethoxyphenanthrene-2,7-diol, have shown potential therapeutic benefits in the treatment of erectile dysfunction and cardiovascular diseases. However, their safety in relation to herb-drug interactions remains unclear. This study aimed to evaluate the potential for herb-drug interactions mediated by cytochrome P450 (CYP) modulation. CYP1A, CYP2B, CYP2C, and CYP3A activities were assessed in primary rat hepatocytes exposed to EM extract or phenanthrene, and in rat liver microsomes following 21-day oral administration. In vitro, EM extract decreased CYP2C activity and increased CYP1A and CYP3A activities, whereas phenanthrene showed no significant effects. In vivo, EM extract inhibited CYP2C and CYP3A activities at low doses (15 mg/kg) and induced CYP1A activity at higher doses (450 mg/kg), with no significant changes at 1,000 mg/kg. Phenanthrene decreased CYP1A activity at 750 µg/kg. Overall, CYP modulation was approximately 2-fold compared with the control. These findings indicate that EM extract can modulate key CYP enzymes and may pose a risk of herb-drug interactions, particularly through inhibition of CYP2C and CYP3A at low in vivo doses. The discrepancies between in vitro and in vivo results suggest that metabolites may contribute to CYP modulation. HIGHLIGHTS Cytotoxicity of EM extract and its phenanthrene was evaluated in primary rat hepatocytes. CYP1A, 2B, 2C, and 3A activities were assessed using in vitro and in vivo models. The EM extract and its phenanthrene modulated CYP isoforms, indicating a potential risk of herb-drug interactions. GRAPHICAL ABSTRACT
This research investigated the efficacy of plasma-activated media (PAM) as a tool for inducing stable skin color variants in Nile tilapia (Oreochromis niloticus). PAM was generated using a transient corona discharge system (20 W, 6 - 10 kV) in atmospheric air, resulting in the time-dependent accumulation of hydrogen peroxide, nitrite, and nitrate. A 2-factorial experimental design was employed to evaluate the interaction between PAM activation duration (0–20 min) and developmental stage (Stages 1 - 5). Survival analysis at 14 days post-immersion identified Stage 2 (eyed egg) as a critical window of sensitivity, showing significant mortality and phenotypic shifts at the 20-minute activation level. In contrast, Stage 3 (pre-hatched embryo) and later larval stages demonstrated high tolerance to PAM exposure. PAM immersion successfully induced 4 distinct color phenotypes: Natural, Whole Pink, White, and Black Blotch, with an induction rate of 28.5% in the most intense treatment group. Notably, a chemically simulated control group matching the PAM’s RONS concentrations failed to produce any variants, suggesting that short-lived species or physical plasma factors are the primary drivers of the phenotypic shift. The induced phenotypes were 100% stable from the larval stage through the 39-week adult harvest. Quantitative CIELAB analysis and microscopic quantification revealed that Whole Pink and White phenotypes were characterized by a significant reduction in melanophore density (5 - 12 cells/mm2) compared to the Natural control (145 cells/mm2). Long-term monitoring demonstrated that PAM-treated groups exhibited significantly higher body weights and Average Daily Gain (ADG) by Week 39, consistent with a hermetic response. Texture Profile Analysis (TPA) confirmed that PAM exposure did not negatively affect flesh firmness across anatomical regions. These results indicate that PAM immersion is a safe, stable, and non-genetic method for inducing commercially desirable color variants and enhancing growth performance in Nile tilapia. HIGHLIGHTS Plasma-activated media induced stable skin color variants in Nile tilapia. Stage 2 eyed eggs were the most sensitive window for color induction. Whole Pink and White phenotypes remained stable until 39 weeks of growth. PAM treatment enhanced growth performance without reducing flesh firmness. Short-lived plasma species likely contributed to pigmentation changes. GRAPHICAL ABSTRACT
Pseudomonas aeruginosa is a highly resilient bacterial pathogen intrinsically resistant to multiple antibiotics due to its low outer membrane permeability and the overexpression of multidrug efflux pumps, particularly MexAB-OprM. Targeting efflux activity through efflux pumps inhibitors (EPIs) represents a promising strategy to restore antibiotic efficacy. This in silico study explores caffeic acid and its structurally diverse derivatives (benzyl caffeate, caffeoylmalic acid, caftaric acid, caffeic acid phenethyl amide (CAPA), caffeic acid phenethyl ester (CAPE), dihydrocaffeic acid, ethyl caffeate, methyl caffeate, octyl caffeate, prenyl caffeate, and rosmarinic acid) as potential EPIs targeting P. aeruginosa, specifically the MexB protein of the MexAB-OprM system. Molecular docking simulations were performed using AutoDock 4.2 to determine binding affinities and interaction profiles of the selected compounds within the MexB distal binding pocket. Docking analysis revealed that all derivatives preferentially localized to the distal binding pocket of the MexB transporter, interacting extensively with hydrophobic and aromatic residues enriched in the PN2 and PC1 subdomains of the T monomer. These regions are enriched with phenylalanine residues (Phe136, Phe178, Phe610, Phe615 and Phe628), which facilitate with π–π stacking and hydrophobic interactions and hydrogen bond interaction. Among the tested compounds, CAPE and CAPA showed the most favorable docking energies and interaction patterns, with binding conformations closely mimicking the binding behavior of those established EPI, ABI-PP. Although their binding energies were moderately higher than ABI-PP, CAPA and CAPE demonstrated strong structural alignment and interaction with key residues, suggesting their potential as novel efflux pump inhibitors to combat multidrug resistance in P. aeruginosa. Overall, these findings suggest that CAPA and CAPE possess promising structural features for efflux pump inhibition and may serve as lead compounds for the development of novel adjuvant therapies against multidrug‑resistant P. aeruginosa. Further experimental validation is warranted to confirm their inhibitory potential and assess their clinical relevance. HIGHLIGHTS The study targets the MexB efflux pump of Pseudomonas aeruginosa, a key contributor to multidrug resistance. Twelve caffeic acid derivatives were docked in silico, showing preferential binding near the PN2 and PC1 subdomains of MexB. Stable interactions involved key phenylalanine residues (Phe136, Phe178, Phe610, Phe615 and Phe628) with CAPE and CAPA identified as the most promising inhibitors. GRAPHICAL ABSTRACT
Guazuma ulmifolia leaves have traditionally been used for managing diabetes, weight control, and cholesterol reduction. This study evaluated metabolite profiles of G. ulmifolia leaf extracts prepared with different solvents and assessed their antidiabetic and antihypercholesterolemic activities. The leaves were extracted using water, water:ethanol mixtures, and ethanol, followed by 1H NMR-based metabolomics and multivariate analysis. Total flavonoid content was determined, and biological activities were evaluated using alpha-glucosidase inhibitory, DPPH, FRAP, and HMG-CoA reductase inhibitory assays. Approximately 31 compounds were putatively identified, including tiliroside, quercitrin, quercetin, rutin, catechin, kaempferol, luteolin, apigenin, chlorogenic acid, and primary metabolites. PCA score plot successfully classified the samples based on the extraction solvents. The evaluation of the total flavonoid content suggested that the flavonoid content increases with a higher ethanol composition in the extraction solvent. The extracts of G. ulmifolia leaves showed low alpha-glucosidase inhibitory activity with IC50 values ranging from 125.99 ± 5.25 mg/mL to 176.43 ± 10.43 mg/mL. Meanwhile, the ethanol extract exhibited the highest flavonoid content, strongest antioxidant activity, and greatest HMG-CoA reductase inhibition, with 82.79 ± 3.04% inhibition. These findings suggest that ethanol is the most effective solvent for extracting flavonoid-rich G. ulmifolia leaf metabolites associated with antioxidants and HMG-CoA reductase inhibitory activities, supporting its potential for further development as a natural cholesterol-lowering candidate. HIGHLIGHTS The ethanol extract of Guazuma ulmifolia shows potential as a cholesterol-lowering agent. The polarity of extraction solvents influences the ulmifolia bioactivities. Higher ethanol composition in the extraction solvent increases the flavonoid content. HMG-CoA inhibitory activity is correlated positively with the flavonoid contents. GRAPHICAL ABSTRACT
In this study, serratiopeptidase was covalently immobilized on polyaldehyde dextran (PAD) to improve handling stability and obtain a more reproducible proteolytic response under defined biochemical assay conditions. PAD was selected as a hydrophilic, water-soluble polysaccharide carrier because, unlike many particulate silica or inorganic supports, it permits mild aqueous Schiff-base coupling while providing a flexible hydrated microenvironment that may reduce enzyme self-association. PAD was prepared by periodate oxidation of dextran to an oxidation degree of approximately 25% - 30%, determined by hydroxylamine hydrochloride titration. Immobilization was performed at 20 - 25 °C for 6 - 24 h using enzyme-to-carrier ratios of 0.5 - 4.0 mg serratiopeptidase per 0.5 g PAD and coupling pH values of 4.0 - 10.0. Quantitative comparisons were performed as mean +/− SD from triplicate measurements and analyzed by one-way ANOVA with Tukey post hoc testing. The best functional balance was obtained at an enzyme-to-carrier ratio of 1:3 and pH 6.5 - 7.5, where apparent immobilization efficiency reached approximately 85% - 90% while proteolytic activity was retained. FTIR spectroscopy supported PAD formation and enzyme attachment through changes in aldehyde- and imine-associated bands, although these data are interpreted as supportive rather than conclusive structural proof. Casein digestion assays showed that PAD-immobilized serratiopeptidase produced a more proportional concentration-activity response and an apparent activity enhancement of 2.1 - 3.1-fold compared with the free enzyme. Apparent kinetic analysis gave Km values of approximately 0.52 mM for free serratiopeptidase and 0.84 mM for the immobilized preparation, with an apparent Vmax of about 0.96 umol/min for the immobilized form; kcat was not calculated because the catalytically accessible immobilized enzyme concentration was not directly determined. The immobilized preparation retained > 80% activity at pH 6.0 - 8.0, about 70% activity after 1 h at 50 °C, and about 85% activity after 30 days at 4 °C. These findings suggest that PAD is a useful polysaccharide carrier for stabilized serratiopeptidase systems, while future work should address direct leaching, reusability, substrate specificity, and application-specific validation. HIGHLIGHTS Serratiopeptidase was covalently immobilized on polyaldehyde dextran via Schiff base Immobilization parameters were optimized to balance binding efficiency and catalytic The immobilized preparation showed a proportional concentration-activity response, with an apparent activity enhancement of 2.1 - 3.1-fold compared with the free enzyme under the tested assay conditions. The immobilized preparation retained > 80% activity at pH 6.0 - 8.0, about 70% activity after 1 h at 50 °C, and about 85% activity after 30 days at 4 °C. Polyaldehyde dextran provided a mild hydrophilic matrix for experimentally controllable serratiopeptidase stabilization and dosing. GRAPHICAL ABSTRACT
