The effect of Citrus reticulata peel extract containing hesperidin on inhibition of SARS-CoV-2 infection based on pseudovirus entry assays

Orange (Citrus reticulata Blanco) peel contains a flavonoid glycoside hesperidin (HSD) as the primary component. Upon enzymatic hydrolysis, HSD forms a flavonoid aglycone derivative, hesperetin (HST). These two flavonoids have been predicted to have in-silico affinities for human Angiotensin Converting Enzyme 2 (hACE2) and SARS-CoV-2 spike, crucial proteins involved in the SARS-CoV-2 infection mechanisms. However, in vitro antiviral testing of orange peel extract, HSD, and HST has not been reported. This study presents for the first time a pseudovirus entry assay approach to test the anti-SARS-CoV-2 effect of HSD, HST, and orange peel extract prepared by hydrodynamic cavitation (HCV). We used a nonvirulent pseudovirus model as an alternative to the original SARS-CoV-2 wild-type virus to target the entry point and enable research to be conducted in a lower biosafety level laboratory (BSL-2). Based on HPLC analysis of 1,000 μg/mL HCV, we observed that our HCV contained HSD at about 4 w/w. Moreover, HSD 1 and 10 μM, HST 10 μM, and HCV 1 μg/mL showed inhibition of pseudovirus entry in 293/hACE2 cells with percentages inhibition 25.92, 37.40, 27.32, and 38.97, respectively. Despite HCV 1 μg/mL showing about 6 lower inhibitory activity than HSD 1 μM in pseudovirus entry assay, it holds potential as a supplement or source of raw material for HSD as a SARS-CoV-2 antiviral. © (2025), (Open Science Publishers LLP Inc.). All rights reserved.