To assess the presence and absence of mycoplasma contamination in cell culture, Fourier transform infrared (FTIR) microspectroscopy, coupled with multivariate analysis, was deployed to determine the biomolecular changes in hepatocellular carcinoma cells, HepG2, before and after mycoplasma contamination. The contaminated HepG2 cells were treated with antibiotic BM-Cyclin to decontaminate the mycoplasma, and polymerase chain reaction (PCR) was then performed to confirm the presence or the absence of mycoplasma contamination. The contaminated and decontaminated HepG2 cells were analyzed by FTIR microspectroscopy with principal component analysis (PCA) and peak integral area analysis. The results showed that the FTIR spectra of contaminated HepG2 cells demonstrated the alteration in the IR spectra corresponding to the lipid, protein, and nucleic acid regions. PCA analysis distinguished the spectral differences between the groups of mycoplasma-contaminated and -decontaminated cells. The PCA loading plots suggest that lipid and protein are the main contributed molecules for the difference between these two cell groups. Peak integral area analysis illustrated the increase of lipid and nucleic acid and the decrease of protein contents in the contaminated HepG2 cells. FTIR microspectroscopy is, therefore, proven to be a potential tool for assessing mycoplasma removal by monitoring biomolecular alterations in cell culture.

Background: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. Methods: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Molecular docking analyses of these compounds against the main protease (M^pro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. Results: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC₅₀ of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 M^pro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-β-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ − 9 kcal/mol. The stability of each ligand–protein complex of these compounds with the M^pro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.