The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a huge public health emergency.
Although several clinical trials have been conducted to identify effective drugs for the treatment of COVID-19, many promising drugs such as hydroxychloroquine, remdesivir and lopinavir have been shown to have little effect on hospitalized COVID-19 patients.
A popular traditional Chinese medicine (TCM), Glycyrrhiza uralensis Fisch (licorice, Gan-Cao) has shown antiviral activity against H1N1 influenza and other SARS-CoV viruses, but information on its use against SARS-CoV-2 is sparse.
Examination: Natural triterpenoids from licorice strongly inhibit SARS-CoV-2 infection. Image credit: marilyn barbone / shutterstock
I silico study of licorice products against SARS-CoV-2
In a study recently published in Journal of Advanced Research, researchers from China conducted an experiment to determine the potency of licorice and related TCM products against SARS-CoV-2. The main objective of the work was to discover small molecule inhibitors that can target SARS-CoV-2 spike protein receptor-binding domain (RBD) from licorice.
The researchers performed virtual screening of 125 small molecules obtained after optimization of Glycyrrhiza uralensis Fisch against the ligand structure RBD of SARS-CoV-2 spike protein (S) and non-structural protein – 7 (nsp7). Surprisingly, glycyrrhetinic acid (GA), 3-O-β-D-glucuronosyl-glycyrrhetinic acid (GA-g) and licorice saponin A3 (A3) showed remarkably high affinity compared to glycyrrhizinic acid (GA-gg), which was previously reported as a SARS-CoV-2 inhibitor. Further evaluation of the potential hits identified from virtual screening was performed using studies in Vero E6 cells and a pharmacokinetic study.
Licorice Vero Cell Study against SARS-CoV-2
The study of the inhibitory activities of licorice triterpenoids against SARS-CoV-2 spike protein by enzyme-linked immunoassay (ELISA) showed that at 10 μM concentration of GA, GA-g and A3, they showed inhibition rates of 51.9%, 50 .2% and 45.1%, respectively, which was significantly higher than other licorice compounds such as GA-gg. Six non-licorice triterpenoids were also evaluated, among which ursolic acid and betulinic acid exhibited observable inhibitory activities. The half-maximal inhibitory concentration values (IC 50) for GA, GA-g, A3, betulinic acid and uric acid were found to be 10.9, 14.1, 8.3, 15.1 and 9.0 μM, respectively.
Similar results were observed when GA, GA-g and A3 were tested in Vero E6 cells infected with SARS-CoV-2 pseudovirus. Although the non-licorice compounds ursolic acid and betulinic acid showed appreciable inhibitory activity, they were not further evaluated due to significant cytotoxicity.
The inhibitory activities of GA and A3 against SARS-CoV-2 were assessed in Vero E6 cells using remdesivir as a positive control, and it revealed that GA and A3 both have significant inhibitory activity against SARS-CoV-2 at 3 μM. Viral RNA levels decreased in a dose-dependent manner with a half-maximal effective concentration (EC50) at 3.17 μM for GA and 75 nM for A3.
Since RNA-dependent RNA polymerase (RdRp) is one of the main targets of SARS-CoV-2, and the proteins nsp12, nsp7 and nsp8 are its essential components, the researchers assessed the binding affinities of A3 and GA with nsp12, nsp7, and nsp8 through surface plasmon resonance ( SPR) analysis. The results revealed a higher binding affinity for A3 to nsp7 compared to the other two proteins, suggesting that nsp7 may be the desirable target for A3.
Pharmacokinetic study of licorice against SARS-CoV-2
The results of the pharmacokinetics study (PK) showed that both GA and GA-gg were easily absorbed into the circulation and were inter-convertible. Even the abundant amount of GA-gg exhibits low antiviral activity and may reduce ACE2 expression in the lungs; it could be easily metabolized to GA, which is an active inhibitor of SARS-CoV-2.
The elimination rate of GA was low and thus its plasma concentration was between 8-24 hours high. GA was also obtained from lung samples as the lungs are the main target organ for SARS-CoV-2. When administered intravenously to rats, A3 showed first-order elimination with a slow elimination rate and plasma concentration of 48.9 μM after one hour, which decreased to 27.3 μM after 24 hours.
Pharmacokinetic data show the highest plasma concentration of GA as 7.7 μM after administration of 200 mg / kg licorice extract with EC50 of GA (3.17 μM) supports the use of licorice as a potent drug for the prevention and treatment of COVID-19 after oral administration.
The researchers concluded that licorice triterpenoids could be promising candidates for the development of anti-SARS-CoV-2 drugs, as confirmed by Vero cell models and PK studies. Licorice compounds A3 and GA inhibit SARS-CoV-2 by targeting different proteins, ie. nsp7 and S-RBD protein, respectively.
The researchers recommended further evaluation of the effects of GA-gg against COVID-19 as a pro-drug of GA in both clinical and animal experiments.
The mode of action of the multicomponents and multi-targets is a unique feature of herbal medicines and contributes to the overall clinical effects of licorice in the treatment of COVID-19. “