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The potential bioactive compounds predicted by virtual screening were experimentally analysed to confirm their inhibitory action against sars-cov-2 3CLpro and PLpro. The inhibitory activity was assessed using the fluorescence resonance energy transfer (FRET) protease assay.  The preliminary screening was conducted at 10 μM and 100 μM. Fig5A and Fig5B presents the SARS-CoV-2 3CLpro and PLpro inhibition values (%) of 10 tested compounds TCMID-05973, TCMID-15972, TCMID-16012, TCMID-22246, TCMID-22314, TCMID-26509, TCMID-27300, TCMID-27476, TCMID-27543 and TCMID-28905. At 100 μM, TCMID-27300 (isobavachalcone) exhibited a inhibition percentage of 98.3% on Mpro and 43.6% on PLpro , while TCMID-27476 (glabrol) exhibited  a inhibition percentage of 93.4% on Mpro and TCMID-15972 (licoflavone B) exhibited  a inhibition percentage of 43.3% on PLpro. These results verfied the antivial activity of bioactive compounds derived from HSBD. 

Following the preliminary screening results, the compounds mentioned above which shows best inhibition activity on Mpro and PLpro were selected for further testing to determine their IC50 values, with nirmatrelvir (Mpro) and GRL-0617 (PLpro) used as a positive control. As shown in Fig5C and Fig5D, TCMID-27300 (isobavachalcone) displayed inhibitory activity against both Mpro and PLpro, with IC50 values of  6.27 μM and 99.93 μM. TCMID-27476 (glabrol) displayed inhibitory activity against Mpro with IC50 values of  3.38 μM while TCMID-15972 (licoflavone B) displayed inhibitory activity against PLpro with IC50 values of 84.35 μM. 

The IC50 value of the positive control was consistent with the activity data reported in the literature which proves the reliability of experimental data. These results suggested that while these 3 natural products derived from HSBD exhibit promising inhibitory effects on SARS-CoV-2 Mpro or PLpro, they are less potent than the known inhibitor nirmatrelvir and GRL-0617. Further optimization and structural modifications may enhance their efficacy, providing a foundation for developing effective antiviral therapies.

isobavachalcone (TCMID‑27300) 

 Glabrol (TCMID‑27476) 

  licoflavone B (TCMID‑15972)