Progress in Antiviral Research of Glycyrrhizic Acid

Glycyrrhizic acid (GA), one of the most important active ingredients in licorice, has a chemical structure as shown in the following figure. Modern research indicates that glycyrrhizic acid possesses extensive antiviral pharmacological activities.

1.Anti-Respiratory Virus Activity

Research has shown that intragastric administration of glycyrrhizic acid monoammonium salt to mice can significantly inhibit the pneumonia lesions caused by H9N2 subtype influenza virus. Mice infected with lethal doses of A2 influenza virus (H2N2) and treated with intraperitoneal injection of glycyrrhizic acid exhibited significantly prolonged mean survival time, with lower degrees of lung parenchymal consolidation and lower virus titers compared to the control group. Further studies have revealed that high mobility group box protein 1 (HMGB 1) plays a crucial role in the replication of influenza virus in the nucleus. By inhibiting the binding of HMGB 1 to the influenza virus nucleoprotein, which promotes virus growth and enhances the activity of the virus polymerase, glycyrrhizic acid is able to reduce the activity of the influenza virus polymerase and thus inhibit the replication of the influenza virus.

Glycyrrhizic acid also has inhibitory effects on SARS-CoV (SARS-coronavirus). According to research reports, 1g·L-1 of GA can reduce the expression of viral antigens in the culture medium, effectively inhibiting the replication of SARS-CoV in cells. At a concentration of 4g·L-1, GA can completely block the replication of SARS-CoV. Antiviral activity tests on two coronaviruses, FFM-1 and FFM-2, using 6-azauridine, ribavirin, mycophenolic acid, pyrazomycin, and glycyrrhizic acid, showed that GA was the most significant compound among these compounds in inhibiting the replication of SARS-CoV. Clinical studies have also indicated that the combination of lopinavir/ritonavir and diammonium glycyrrhizinate for the treatment of COVID-19 patients resulted in significantly improved total effective rate, cure rate, and marked effectiveness in the combined treatment group.

2.Anti-Hepatitis Virus Activity

Glycyrrhizic acid possesses direct antiviral activity against hepatitis B virus (HBV) without being toxic to host cells. Research has shown that glycyrrhizic acid can inhibit the secretion of hepatitis B surface antigen (HBsAg) from HBV-infected cells to the outside of the cell, thereby inhibiting the destruction of liver cells and improving liver dysfunction in patients with chronic hepatitis B, thus improving the immune status against HBV. Clinically, glycyrrhizic acid has shown significant effects in reducing indicators such as glutamyl transpeptidase and hepatitis B virus. The clinical application of diammonium glycyrrhizinate has also achieved good results, demonstrating superior activity with strong anti-inflammatory, protective effects on hepatocyte membranes, and improvement of liver function. Glycyrrhizic acid also has a strong antiviral effect against hepatitis C virus (HCV). When used in non-toxic doses in hepatocytes infected with HCV, the expression levels of HCV core gene at both mRNA and protein levels decrease in a dose-dependent manner, and it has a synergistic effect with interferon. Formulations containing glycyrrhizic acid, such as SNMC (stronger neo-minophagen C), can protect mitochondria from oxidative stress damage induced by HCV proteins, effectively reducing the risk of cirrhosis in patients with hepatitis C.

3.Anti-Herpes Simplex Virus Activity

Research indicates that water extracts from licorice roots possess antiviral properties against herpes simplex virus 1 (HSV-1), likely due to their strong anti-adhesive properties that can directly inhibit cell-to-cell adhesion during HSV-1 virus infection. One of its main components, glycyrrhizic acid, not only inhibits virus growth but also inactivates virus particles. Glycyrrhetinic acid derivatives, such as carbenoxolone (CBX) and cicloxolone (CCX), exhibit antiviral effects against HSV-1 and HSV-2. They significantly inhibit the replication of HSV-1 and HSV-2, reducing the number of infectious virus particles by 10,000 to 100,000 times. Glycyrrhizic acid also has inhibitory activity against the replication of Epstein-Barr virus (EBV) in a dose-dependent manner, with an IC50 value of 0.04 mmol·L-1. Glycyrrhizic acid mainly exerts its effects in the early stages of the EBV replication cycle, without affecting virus adsorption or inactivating EBV particles. Additionally, glycyrrhizic acid inhibits the lytic replication of Kaposi's sarcoma-associated herpesvirus-8 and can effectively clear the virus during latent infection.

Mechanism of Antiviral Action of Glycyrrhizic Acid (GA)

GA exerts its antiviral effects directly by inhibiting virus attachment and penetration, suppressing virus replication, and inhibiting the expression of virus-related proteins. It can also indirectly exert antiviral effects by modulating the function of the human immune system.

1.Inhibition of Virus Attachment and Penetration

Virus particles enter host cells by binding to specific receptors on the host cell membrane. GA can inhibit the attachment and penetration processes of viruses, thus directly inhibiting viral invasion. GA can reduce cell membrane fluidity and inhibit cell-to-cell fusion induced by HIV-1. GA possesses strong anti-adhesive properties that can directly inhibit cell adhesion during HSV-1 virus infection, preventing viral invasion. GA blocks respiratory syncytial virus (RSV) from attaching to cells, inhibiting RSV attachment and penetration. Furthermore, GA inhibits the interaction between influenza A virus and the cell membrane, leading to reduced endocytic activity and decreased viral invasion.

2.Inhibition of Viral DNA Replication

The massive replication of viral DNA is one of the critical causes of host cell necrosis and severe inflammation. Numerous studies have shown that GA can effectively inhibit viral DNA replication, thus exerting its antiviral effects. Research has found that GA can inhibit the protease SARS-CoV-2 (Mpro) and thereby suppress virus replication. Network pharmacology and molecular docking studies have discovered that GA may be an inhibitor of the 3CL protease of SARS-CoV-2. GA does not directly inactivate H1N1 or affect its adsorption and entry into MDCK cells, but it inhibits the replication of H1N1 virus that has entered the cells in a concentration-dependent manner. GA can also inhibit high-mobility group protein B1, blocking virus replication by inhibiting the polymerase activity of influenza virus. Studies have shown that GA can counteract the expression of chemokine ligands 10, 5, and IL-6 induced by influenza virus H5N1 in human macrophages, effectively inhibiting the replication of influenza virus. Research on the antiviral mechanism of GA against HIV suggests that GA may reduce the activity of protein kinase C, induce the production of β-chemokines including chemokine ligands 4 and 5, and effectively inhibit HIV replication. Experiments using monocyte-derived macrophages have also confirmed that GA can inhibit the replication of R5 HIV virus dependent on polymorphonuclear neutrophils by inhibiting the production of CCL2 or interleukin-10. GA can alter the expression of hepatitis B-related antigens and inhibit the sialylation of hepatitis B surface antigen, suppressing its transport through the Golgi apparatus of cells and thus inhibiting hepatitis B virus replication. Studies have shown that glycyrrhizin can inhibit the synthesis of KSHV latent proteins, induce apoptosis of infected cells, and block the replication of latent viruses. GA inhibits early viral replication cycles, exerting an antiviral effect against herpesviruses.

3.Inhibition of Virus-Related Protein Expression

Research has revealed that GA can upregulate the expression of viral cell cycle regulatory proteins by downregulating the expression of LA-NA antigen, thereby inhibiting the transcription and translation of latent KSHV-related proteins in B lymphocytes. GA can also downregulate the expression of KSHV-related nuclear antigens during latent infection and upregulate the expression of viral cyclins, leading to a reduction in infection. Additionally, GA inhibits the expression of full-length hepatitis C virus particles and core genes in a dose-dependent manner, and exhibits a synergistic effect with interferon. Experiments using non-toxic doses of GA on hepatocytes infected with hepatitis C virus have shown that the expression levels of hepatitis C core genes at both mRNA and protein levels decrease in a dose-dependent manner, and there is a synergistic effect with interferon. Furthermore, GA inhibits SARS-CoV-2 by suppressing the synthesis of spike protein, disrupting the interaction between the binding domain of the SARS-CoV-2 receptor and ACE2, thereby exerting an inhibitory effect on SARS-CoV-2.

4.Indirect Antiviral Activity Mechanisms

Both in vitro and in vivo experiments have found that GA can exert antiviral activity against influenza virus by stimulating T cells to produce γ-interferon. Further research on the antiviral mechanism of GA against influenza virus has shown that GA can upregulate the expression level of IFN-γ mRNA and downregulate the expression level of inflammatory factor TNF-α mRNA. Through its immunomodulatory function, GA can resist influenza virus infection. It has also been reported that GA may play a role in innate immunity by inducing cellular synthesis of nitric oxide (NO), a host defense molecule, thus inhibiting coronavirus replication. In terms of the antiviral mechanism of GA against hepatitis, it has been found that GA can inhibit the migration of macrophages and NK cells to damaged hepatocytes by suppressing the expression of MCP-2, and may enhance the antiviral efficacy by regulating ENA-78. Additionally, GA can act on multiple immune-related signaling pathways in the body, such as blocking the IL-17/STAT3 pathway, inhibiting NF-κB and MAPK signaling pathways, regulating the expression of inflammatory cytokines and chemokines-2, and suppressing intracellular levels of proinflammatory factors like COX-2 and IL-6. Through its anti-inflammatory and immunomodulatory effects, GA improves the antiviral activity of the body.

References

[1] Yang Xiuwei. Antiviral Effects of Glycyrrhizic Acid [J]. Modern Chinese Medicine Materials, 2020, 22(04): 533-541.

[2] Song Yanling, Ren Meng, Zhou Lingzhu, et al. Research Progress in Antiviral Effects of Glycyrrhizic Acid and Its Derivatives [J]. Journal of Shenyang Pharmaceutical University, 2023, 40(12): 1703-1712.

Author Introduction

Xiaomichong, a pharmaceutical quality researcher, has long been committed to the research of drug quality and the validation of drug analysis methods. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection, analysis, and analytical method validation.