**1. Introduction**

The uncertainty of the coronavirus disease 2019 (COVID-19) pandemic situation remains elusive, although great success has been accomplished with the rapid development of protective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are currently no effective drugs for the treatment of COVID-19, although clinical trials and case reports involving antiviral and antiparasitic agents have yielded promising results that warrant further investigation [1,2]. Herbal medicines have a long history of vindicated clinical efficacy against infectious diseases. Clinical evidence shows that herbal medicines are effective against viral infections such as influenza, SARS, and SARS-CoV-2 by targeting virus cell entry, viral replication, and host antiviral immune response steps. During the pandemic of COVID-19, several drugs formulated according to principles of Chinese medicine showed therapeutic effects against mild and severe COVID-19. Among the drugs and formulae recommended by the Chinese authority for COVID-19 therapy, the dried root of *Glycyrrhiza*

https://doi.org/10.3390/ molecules26206090 Academic Editors: Muhammad Ilias and Raphaël E. Duval

**Citation:** Li, J.; Xu, D.; Wang, L.; Zhang, M.; Zhang, G.; Li, E.; He, S. Glycyrrhizic Acid Inhibits SARS-CoV-2 Infection by Blocking Spike Protein-Mediated Cell Attachment. *Molecules* **2021**, *26*, 6090.

Received: 10 August 2021 Accepted: 30 September 2021 Published: 9 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

spp. (licorice) is among the most commonly used ingredients in the formulae [3]. The radices of *Glycyrrhiza* spp. have been used as an important ingredient of herbal medicines and also a flavoring agent in traditional formulae since antiquity [4,5]. The use of the dried roots of the plant can be traced back to ancient Assyrian, Egyptian, Chinese, and Indian cultures for symptoms that resemble those of viral respiratory tract infections such as dry cough or hoarse voice [5,6]. Recent reports also suggest that licorice extract may have a potential role in combating COVID-19 and associated conditions [7].

The main chemical component from *Glycyrrhiza* sp. is glycyrrhizic acid (GA), also known as glycyrrhizin, a triterpenoid saponin that can be extracted from the dried roots in high yields [8,9]. Numerous reports show that GA is effective against the infection of enveloped and nonenveloped viruses such as herpes viruses, respiratory syncytial virus, vaccinia virus, and SARS-CoV in cell culture studies [4,10,11]. Cinatl and colleagues found that glycyrrhizin was among the most active compounds tested in inhibiting replication of the SARS-CoV [12]. Thus, licorice and GA could be an old weapon against emerging diseases [13,14].

In this study, we investigated the antiviral effect of GA against SARS-CoV-2 using a pseudotyped lentivirus that has the SARS-CoV-2 S protein on its envelope (Lenti-S). We found that GA inhibited Lenti-S infection through inhibition of virus attachment to host cells. Preincubation of Lenti-S, rather than the host cells, with GA prior to the infection reduced Lenti-S infection, suggesting that GA primarily targets the virus rather than the host cells. GA interacted with the S protein with high affinity and blocked a recombinant S protein binding to the host cells. Thus, this study uncovered a mechanism by which GA blocks SARS-CoV-2 infection by impeding virus and host cell interaction.

### **2. Materials and Methods**
