# HSBDF-Derived Bioactive Components Broadly Inhibit Enteroviruses by Targeting 3C Protease and Attenuating Inflammatory Responses

## TL;DR

A Chinese herbal formula and its flavonoid compounds show broad-spectrum antiviral activity against enteroviruses by inhibiting a key viral enzyme and reducing inflammation.

## Contribution

Identification of three flavonoids in Huashi Baidu Formula as potent broad-spectrum anti-enteroviral agents with anti-inflammatory effects.

## Key findings

- HSBDF and its compound velutin inhibit enterovirus replication in mice.
- Three flavonoids suppress 3C protease activity and reduce inflammatory markers like IL-6 and TNF-α.
- HSBDF modulates MAPK and NF-κB pathways to attenuate inflammation.

## Abstract

Human enteroviruses are important pathogens of hand-foot-and-mouth disease, poliomyelitis, and encephalitis, etc., and there are currently no medicines that specifically treat them. Although traditional Chinese herbal medicines showed some antiviral activity during the COVID-19 pandemic, their efficacy and pharmacodynamic material basis against enteroviruses remains underexplored. In this work, we characterized a Chinese herbal mixture, Huashi Baidu Formula, which has already been approved for COVID-19, and found that it shows broad-spectrum anti-enterovirus activity. We identified 152 chemical compounds in Huashi Baidu Formula, among which three flavonoids—velutin, isorhamnetin, and (−)-epicatechin gallate—exhibited potent pan-enteroviral inhibition activity. These compounds block a key viral enzyme and also calm the body’s inflammatory response. Tests in young mice showed that the herbal formula and the compound velutin greatly reduced viral growth. The study demonstrates that this herbal formula and its flavonoid components could become safe, affordable treatments for enterovirus infections, helping to lower the global health burden of these diseases.

Human enteroviruses are important pathogens of hand-foot-and-mouth disease, poliomyelitis, and encephalitis, etc., posing substantial global health burdens with no specific approved therapeutics. While traditional Chinese medicine (TCM) has demonstrated antiviral potential during the COVID-19 pandemic, its efficacy and pharmacodynamic material basis against enteroviruses remains underexplored. Here, we systematically characterized the broad-spectrum anti-enterovirus activity of Huashi Baidu Formula (HSBDF), a clinically approved TCM for COVID-19, and identified three flavonoid compounds as its active components responsible for this antiviral effect. Transcriptomics analysis revealed that HSBDF attenuated CV-A9-induced inflammation by modulating MAPK and NF-κB signaling pathways. High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) analysis identified 152 chemical compounds in HSBDF, among which three flavonoids—velutin, isorhamnetin, and (−)-epicatechin gallate—exhibited potent pan-enteroviral inhibition. Mechanistically, these compounds suppressed the activity of 3C proteases in enteroviruses, while concurrently attenuating CV-A9-induced upregulation of IL-6, TNF-α, MCP-1, and COX-2. Utilizing a BALB/c young mouse model, it was demonstrated that the HSBDF and its compound velutin effectively suppressed viral replication in vivo. Collectively, this study advances TCM-based strategies for enterovirus therapy exemplified by HSBDF and highlights flavonoid scaffolds as promising candidates for developing broad-spectrum anti-enteroviral agents.