Synergistic effect of chlorogenic acid and vitamin D3 (cholecalciferol) on in-vitro glycation may assist in prevention of Polycystic Ovarian Syndrome (PCOS) progression - A biophysical, biochemical and in-silico study.

Advanced glycation end products (AGEs) are formed through non-enzymatic glycation, that have been linked to various diseases, including polycystic ovarian syndrome (PCOS) playing a critical role leading to secondary comorbidities such as diabetes-related problems, cardiovascular complications, infertility, etc. As a result, there has been a lot of research into AGE-inhibiting phytochemicals for the remediation and obstruct progression of glycation-related illnesses. The current study is based on in-vitro protein model, in which human serum albumin have been used to investigate the cumulative effect of chlorogenic acid (CGA) and cholecalciferol (vitamin D) on glycation and evaluate their inhibitory impact on AGEs production in the presence of methylglyoxal. Through the application of several biochemical and biophysical techniques, we were able to examine the synergistic impact of both the compounds on albumin structure and its biochemical properties during different stages of glycation. According to Nitro-blue tetrazolium assay results indicate that CGA and vitamin D inhibited fructosamine (early glycation product) production. Moreover, free thiol and lysine residues were significantly increased whereas protein carbonyl levels were significantly decreased. Additive effect of CGA and vitamin D were associated with reduced AGEs fluorescence and increased tryptophan and tyrosine fluorescence. Amadori-albumin after treatment showed some evidence of regaining its alpha-helicity as measured by far-UV CD spectrum. Furthermore, secondary structural alterations were confirmed by Fourier transform infrared spectroscopy (FTIR). ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence spectra also displayed less revelation of hydrophobic patches. Bilirubin binding capacity was also restored which showed functional recovery of HSA. The electrophoretic mobility was also restored which is portrayed by SDS-PAGE. Additionally, to predict the anti-aggregation potential of CGA and vitamin D, congo red assay and ThT fluorescence was performed which reveal low aggregate formation after treatment. These results corroborated with scanning electron microscopy and confocal microscopy. Docking and simulation results also reveal spontaneous binding of CGA and vitamin D on subdomain IIA of HSA favoring their binding thermodynamically. All the findings suggest that chlorogenic acid and cholecalciferol given in combination might help in prevention of PCOS progression and its related complications.

In silico and in vitro studies on the inhibition of laccase activity by Ellagic acid: Implications in drug designing for the treatment of Cryptococcal infections.

In recent years, the increased frequency of drug-resistant strains of Cryptococcus neoformans has depleted our antifungal armory. In the present study, we investigated the inhibitory potential of ellagic acid (EA) against C. neoformans laccase through in silico and in vitro studies. For the first time, a homology modelling was established to model laccase and modelled protein served as a receptor for docking EA. Thermodynamic stability of the docked complex was ascertained by molecular dynamics simulation (MD). The analysis of root mean square deviation and fluctuation of alpha carbons of protein justifies the stability of the bound EA in the binding pocket of laccase. Frontier molecular orbitals of the EA was studied by density functional theory-based optimization by using the Lee-Yang-Parr correlation functional (B3LYP) approach. Negative values of the highest occupied/unoccupied molecular orbitals (HOMO/LUMO) indicated that laccase with EA forms a stable complex. Interestingly, EA inhibited laccase activity both in vitro and in yeast cells of C. neoformans. Moreover, EA treatment remarkably inhibited the proliferation of C. neoformans inside macrophages. The findings of the present study unveil the molecular basis of the interactions of laccase with EA, which may prove to be beneficial for designing laccase inhibitors as potential anti-cryptococcal agents.

Catalytic roles, immobilization and management of recalcitrant environmental pollutants by laccases: Significance in sustainable green chemistry.

We are facing a high risk of exposure to emerging contaminants and increasing environmental pollution with the concomitant growth of industries. Persistence of these pollutants is a major concern to the ecosystem. Laccases, also known as "green catalysts" are multi-copper oxidases which offers an eco-friendly solution for the degradation of these hazardous pollutants to less or non-toxic compounds. Although various other biological methods exist for the treatment of pollutants, the fact that laccases catalyze the oxidation of broad range of substrates in the presence of molecular oxygen without any additional cofactor and releases water as the by-product makes them exceptional. They have a good possibility of utilization in various industries, especially for the purpose of bioremediation. Besides this, they have also been used in medical/health care, food industry, bio-bleaching, wine stabilization, organic synthesis and biosensors. This review covers the catalytic behaviour of laccases, their immobilization strategies, potential applications in bioremediation of recalcitrant environmental pollutants and their engineering. It provides a comprehensive summary of most factors to consider while working with laccases in an industrial setting. It compares the benefits and drawbacks of the current techniques. Immobilization and mediators, two of the most significant aspects in working with laccases, have been meticulously discussed.

Liposomal Ellagic Acid Alleviates Cyclophosphamide-Induced Toxicity and Eliminates the Systemic Cryptococcus neoformans Infection in Leukopenic Mice.

Cryptococcus neoformans infections rose sharply due to rapid increase in the numbers of immunocompromised individuals in recent years. Treatment of Cryptococcosis in immunocompromised persons is largely very challenging and hopeless. Hence, this study aimed to determine the activity of ellagic acid (EA) in the treatment of C. neoformans in cyclophosphamide injected leukopenic mice. A liposomal formulation of ellagic acid (Lip-EA) was prepared and characterized, and its antifungal activity was assessed in comparison to fluconazole (FLZ). The efficacy of the drug treatment was tested by assessing survival rate, fungal burden, and histological analysis in lung tissues. The safety of the drug formulations was tested by investigating hepatic, renal function, and antioxidant levels. The results of the present work demonstrated that Lip-EA, not FLZ, effectively eliminated C. neoformans infection in the leukopenic mice. Mice treated with Lip-EA (40 mg/kg) showed 70% survival rate and highly reduced fungal burden in their lung tissues, whereas the mice treated with FLZ (40 mg/kg) had 20% survival rate and greater fungal load in their lungs. Noteworthy, Lip-EA treatment alleviated cyclophosphamide-induced toxicity and restored hepatic and renal function parameters. Moreover, Lip-EA treatment restored the levels of superoxide dismutase and reduced glutathione and catalase in the lung tissues. The effect of FLZ or EA or Lip-EA against C. neoformans infection was assessed by the histological analysis of lung tissues. Lip-EA effectively reduced influx of inflammatory cells, thickening of alveolar walls, congestion, and hemorrhage. The findings of the present study suggest that Lip-EA may prove to be a promising therapeutic formulation against C. neoformans in immunocompromised persons.

Immobilization of laccase on Sepharose-linked antibody support for decolourization of phenol red.

Laccases which are considered as "green tools" in biotechnology have potential to degrade toxic contaminants/synthetic dyes present in industrial effluents. The loss in activity and stability of laccases are key challenges faced in their potential industrial applications. Here, laccase from Trametes versicolor (polypore mushroom) was immobilized on Sepharose-linked antibody support to carry out the decolourization of phenol red. This support was prepared by covalent linking of anti-laccase antibodies to CNBr activated Sepharose at pH 8.5, and then laccase was immobilized on this affinity support at pH 5.0. The amount of laccase immobilized was approximately 33 mg per gram of the affinity support, giving an immobilization yield of 83.4%. The immobilized enzyme displayed an activity of 3.88 U with an effectiveness factor (η) of 0.90. Immobilization of laccase led to significant enhancement in thermal and storage stability. The immobilized enzyme retained 44% of its activity after 10 cycles of continuous use. The decolourization of phenol red dye obtained by immobilized and soluble laccase after 6 h of incubation at 50 °C was 80 and 56%, respectively. Thus, immobilization of laccase on Sepharose-linked antibody support leads to remarkable improvement in its various properties, making it more versatile for industrial applications.