Ultra high-Performance Liquid Chromatography (UHPLC) method development and validation for the identification of oxidized product of Epigallocatechin-3-Gallate (EGCG).

OBJECTIVE: In the present study we reported oxidation of epigallocatechin-3-gallate (EGCG) and validation of oxidized product by a validated ultra high-performance liquid chromatography (UHPLC) method. METHODS: Two hundred milligrams of EGCG was oxidized in 5 mL of hydrogen peroxide (H2O2) and was identified by a validated UHPLC method with precision and robustness. Confirmation of parameters like C-H stretching and mass was carried out using infrared spectroscopy and mass spectroscopy, respectively. Identification of oxidized EGCG (O-EGCG) was done by UHPLC. RESULTS: The infrared spectroscopy chromatograms observed less intensity C-H stretching as compared to O-EGCG. The mass of EGCG and O-EGCG were 459.09 and 915.16, respectively. Structure elucidation revealed a loss of one proton in O-EGCG as compared to EGCG. Validation of the developed method was specific, with linear correlation coefficient 0.9981 and 0.9917, respectively for EGCG and O-EGCG, the accuracy rate of 95.2%-99.6% for EGCG, and 99.18%-101.5% for O-EGCG. CONCLUSION: Together, the results of this study demonstrate the formation of a dimer also the UHPLC method developed for identification of both EGCG and O-EGCG is validated as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.

A novel RP-HPLC method for quantification of cholinesterase activity in human blood: An application for assessing organophosphate and carbamate insecticide exposure.

Several methods have been reported to estimate Acetylcholinesterase (AChE) enzyme activity in blood samples. The Ellman assay is the most important among all but with several shortcomings, and there is a need to develop a method which is accurate, sensitive and quick for analyzing AChE. Therefore, we have developed an assay utilizing RP-HPLC with UV detection for the determination of AChE activity. This method measured the conversion of 1-naphthol acetate to 1-naphthol to estimate AChE activity in blood samples. Performance was judged on the basis of reproducibility, sensitivity, accuracy, and the ability to screen enzyme activity within 20minutes. A series of experiments were performed, varying the concentration of blood and substrate, with optimal sensitivity using 50 µM substrate and 10µL blood. The validation parameters such as linearity (R2 of ≥ 0.9842 for 1-naphthol and ≥ 0.9897 for 1-naphthol acetate), precision (94.21-96.41%), accuracy (85.2%-99.6% and 82.6%-99.3% for 1-naphthol and 1-naphthol acetate respectively), and robustness were validated according to International Conference on Harmonization (ICH) guidelines. Blood samples were collected from healthy people, farmers exposed to spraying of pesticides, and suicidal patients who ingested pesticides and were hospitalized and were analyzed by the developed method. The AChE level was approximately 21 units/mL compared to 24units/mL in controls, whereas suicidal patients showed the least AChE levels of 1 unit/mL. The employment of this method is recommended for estimating AChE level on various matrices.

Assessment of Antioxidant, Immunomodulatory Activity of Oxidised Epigallocatechin-3-Gallate (Green Tea Polyphenol) and Its Action on the Main Protease of SARS-CoV-2-An In Vitro and In Silico Approach.

Owing to the instability of Epigallocatechin Gallate (EGCG), it may undergo auto-oxidation and form oxidised products or dimers. In the present study, we aimed to evaluate the therapeutic effects, including antioxidation and immunomodulatory action, of the Oxidised Epigallocatechin Gallate (O-EGCG) as compared to native EGCG and the action of these compounds on main protease (Mpro) docking against SARS-CoV-2. HCT-116 (Human Colon Cancer) cell lines were used to estimate the total antioxidant capacity and lipid peroxidation levels and pro-inflammatory markers (human IL-6, IL-1ß, TNF-α). Further, molecular docking analysis was performed by AutoDock and visualised in Discovery studio. Improved antioxidant capacity of O-EGCG was observed, and there was a significant decrease in the inflammatory markers (IL-1ß, IL-6, and TNF-α) when O-EGCG was applied as compared to EGCG. The O-EGCG was shown to be strongly associated with the highest docking score and active site residues of IL-1, IL-6, and TNF- α, as well as the Mpro of SARS-CoV-2, according to in silico approach. The in vitro and in silico analyses indicate an improved therapeutic action of the oxidised form of EGCG. The effective inhibitory action of O-EGCG against SARS-CoV-2 suggests further exploration of the compound against COVID-19 and its efficacy. However, in vivo studies and understanding of the mechanism of action of O-EGCG may yield a better opinion on the use of O-EGCG and future human clinical trials.

Toxicokinetic analysis of commonly used pesticides using data on acute poisoning cases from Hyderabad, South India.

Organophosphorus pesticides (OPs) interfere with the activity of acetylcholinesterase (AChE), a vital enzyme that regulates the functioning of the nervous system, resulting in acetylcholine (Ach) accumulation at the synapses and myoneural junctions. It remains unknown whether the commonly used OPs in South India also interfere with the AChE activity and their toxicokinetics in humans remains poorly understood. We collected peripheral blood samples from OP-associated suicide cases (hospitalised) and analysed the pesticide concentration and AChE activity, and the toxicokinetics of six commonly used pesticides. LC-MS/MS was used for the estimation of pesticide concentration. Based on a comparison of six pesticide kinetic profiles and toxicokinetic parameters, we concluded that chlorpyrifos ingestion resulted in the highest concentration of chlopyrifos among the identified pesticides, followed by acephate, triazophos, propanil, while dimethoate exhibited the lowest concentration. Based on a time-course analysis, we observed a faster elimination phase for monocrotophos and dimethoate. We observed that there was a significant decrease in the mean concentration of monocrotophos (64 ng/mL) (P = 0.015), while the mean value of AChE (1.08 unit/mL) increased over time. While monocrotophos and dimethoate elimination phases were remarkable in human subjects, the other pesticides did notdemonstrate similar elimination phases owing to their low rate of metabolism and high stability.