Diosgenin

To date, the burden of alcohol-related seizures is increasing, with an unexplored etiological complex, and the psychopharmacological interplay remains significantly scarce. In this study, we developed an experimental approach to investigate the contrasting impact of alcohol on pentylenetetrazol-induced seizures and the effects of diosgenin, a phytosteroid agent with neuroprotective effects. After 7 days of binge alcoholism with ethanol (2 g/kg, oral gavage) in male mice, they were subjected to maximum and sub-convulsive pentylenetetrazol-induced seizures concomitantly with diosgenin (25 and 50 mg/kg, p.o.) or diazepam (3 mg/kg, p.o) treatments from days 8-14. The interaction between ethanol and pentylenetetrazol-induced seizures was investigated, along with behavioral comorbidities, hypothalamic-adrenal-pituitary-axis (HPA-axis), neurochemical and neurotrophic dysfunctions, oxidative stress, and neuroinflammation in the hippocampus, prefrontal cortex, and striatum. Ethanol-exacerbated pentylenetetrazol-induced seizure and frequency, characterized by rearing with myoclonic jerks, and clonic-tonic convulsions. It increased anxiety, depressive behavior and impaired spatial working memory, influenced by heightened alcohol preference and corticosterone levels, which were normalized by diosgenin. Concomitant ethanol administration exacerbated reductions in GABAergic-dependent glutamic acid decarboxylase and increased glutamate levels associated with pentylenetetrazol-induced seizures, alongside depletions of serotonin and brain-derived neurotrophic factor in the hippocampus, prefrontal cortex, and striatum. Among others, diosgenin, compared to ethanol-pentylenetetrazol exacerbation, reduced levels of myeloperoxidase, TNF-α, and IL-6, nitrite and malondialdehyde in the hippocampus, prefrontal cortex, and striatum while increasing IL-10 cytokine and antioxidant system (superoxide-dismutase, glutathione, and glutathione-transferase). These findings suggest that alcoholism exacerbates seizures across brain regions, involving neurochemical imbalance, HPA-axis dysfunction, oxidative stress, and neuroinflammation, which are reversible by diosgenin.

Diosgenin is a key precursor for steroidal pharmaceuticals; however, traditional acid hydrolysis produces significant environmental pollution, whereas enzymatic conversion presents a more environmentally sustainable alternative. Herein, four saponinases were identified from Aspergillus tubingensis HG57: the β-glucosidases ATGH I and ATGH II, which hydrolyzed residual β-d-glucosyl moieties, and the rhamnosidases ATRH I and ATRH II, which cleaved terminal α-L-(1 → 2) rhamnosyl linkages in steroidal saponins. ATGH I (7.46 U mg^-1) and ATRH I (285.31 U mg^-1) demonstrated some of the highest catalytic efficiencies documented to date. Both enzymes displayed broad pH tolerance and thermostability (≤50 °C), with ATRH I maintaining over 90 % activity in 2 M rhamnose. Structure-guided engineering of ATGH I led to the development of an improved variant, ATGH M, which displayed a two-fold improvement in catalytic efficacy (14.99 U mg^-1). High-density fermentation produced enzyme-rich supernatants (ATGH M: 98.52 U mL^-1; ATRH I: 2564.36 U mL^-1) that facilitated gram-scale, one-pot enzymatic hydrolysis to produce diosgenin. This bioprocess achieved 138 % of the diosgenin yield obtained by traditional acid hydrolysis while markedly reducing wastewater pollutants, lowering chemical oxygen demand by 76 %, sulfate by 99 %, and acidity by nearly 100 %. The integration of efficient saponinase discovery, protein engineering, and high-density fermentation established a green and scalable enzymatic platform for diosgenin production, offering a sustainable alternative to conventional acid hydrolysis.