The Kinetics of 3-Chymotrypsin-Like Cysteine Protease (3CLpro) Inhibition by <i>Otostegia persica</i> and <i>Otostegia aucheri</i> Extracts and Fractions: An In Vitro Model for Coronavirus Enzyme Inhibition

Background: As of 2025, COVID-19 has caused over 700 million cases globally (WHO, 2025). The COVID-19 epidemic has created a severe global health crisis due to the broad loss of human lives and economic impact. The 3-Chymotrypsin-like cysteine protease (3CLpro, which cleaves viral polyproteins to produce functional proteins essential for replication), performing critical functions in the duplication of severe acute respiratory syndrome (SARS)-CoV, is necessary for its life cycle. Certainly, 3CLpro (as a model for coronavirus protease) is a previously approved drug target for SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. This emphasizes the significant role of 3-chymotrypsin-like cysteine protease in the construction of powerful medicine against COVID-19. Nowadays, finding a novel drug derived from a plant with better treatment is extremely desirable due to the lack of specific treatments for SARS-CoV, MERS-CoV, and SARS-CoV-2. Objectives: The present study aimed to evaluate the efficacy of Otostegia persica and O. aucheri extracts and their fractions in inhibiting COVID-19 marker enzymes, which are called proteases. Methods: Ethanol extracts of these plants were fractionated using petroleum ether (petr), chloroform (chlo), ethyl acetate (eta), and n-butanol (buta) solutions, respectively. For the measurement of shrimp-derived cysteine protease as a 3CLpro model retardation, 200 μL of the enzyme (at 25°C), 400 μL of a substrate, and plant extracts and fractions were used. After determining the percentage of inhibition and IC50, Km and Vmax were also calculated. Results: The results showed that the eta fraction of O. aucheri (with IC50 = 0.002 ± 0.0001 mg/mL) had the greatest inhibitory effect on protease activity compared to iodoacetamide (with IC50 = 0.01 ± 0.01 mg/mL, P = 0.04) as the standard. Also, the chlo fraction of O. aucheri (with an IC50 = 0.09 ± 0.007 mg/mL) and the buta fraction of O. aucheri (with an IC50 = 0.07 ± 0.002 mg/mL) possessed significant inhibitory effects in protease retardation. Otostegia persica fractions had no significant inhibition on protease activity (P > 0.05). The mode of protease inhibition of all samples was uncompetitive, except for the petr fraction of O. aucheri, which was mixed inhibition. Conclusions: The eta, chlo, and buta fractions of O. aucheri had remarkable effects in the inhibition of protease. In vitro results may require in vivo validation.