COVID-19 Herbs: German In Vitro Study Shows That The Phytochemical Glycyrrhizin Extracted From Licorice Root Is Able To Inhibit SARS-CoV-2

COVID-19 Herbs: Researchers from the Medical University Duisburg-Essen-Germany, Ruhr University Bochum-Germany and the Institute for Virology at the University Hospital Essen-Germany have in a new vitro study demonstrated that the phytochemical Glycyrrhizin extracted from licorice root is able to inhibit the SARS-CoV-2 coronavirus.


 
The SARS-CoV-2 coronavirus, the causative pathogen that causes the COVID-19 disease so far does not have an effective antiviral or therapeutic drug that can work against it.
 
Traditional herbal medicine substances were discussed as promising candidates for the complementary treatment of viral diseases and recently suggested for the treatment of COVID-19.
 
In this study, the scientist investigated the aqueous licorice root extract for its neutralizing activity against SARS-CoV-2 in vitro.
 
The study team identified the active compound glycyrrhizin and uncovered the respective mechanism of viral neutralization. The study team demonstrated that glycyrrhizin, the primary active ingredient of the licorice root, potently neutralizes SARS-CoV2 by inhibiting the viral main protease. The study experiments highlight glycyrrhizin as a potential antiviral compound that should be further investigated for the treatment of COVID-19.
 
The research findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.12.18.423104v1
 
Thailand Medical News had proposed studying using the licorice root to treat COVID-19 as early as January this year, during the initial stages of the COVID-19 breakout in China. https://www.thailandmedical.news/news/further-research-into-the-phytochemicals-contained-in-licorice-root-might-yield-a-treatment-for-the-new-coronavirus-strain-in-china
 
Previous in-silico simulation study proposed an antiviral activity of glycyrrhizin against SARS-CoV-2, but this hypothesis remained experimentally unproven till now. https://onlinelibrary.wiley.com/doi/full/10.1002/ptr.6852
 
Licorice root had been studied and used as a therapeutic and prophylaxis during the earlier SARS virus outbreak in 2002.
 
It is also one of the key components in various Traditional Chinese Medicine (TCM) Herbal formulations to treat COVID-19 in China.
 
For the study dried licorice roots were brewed at a concentration of 8 mg/ml (w/v) and the fluid was subsequently sterile filtered to obtain an aqueous licorice root extract.
 
The virus neutralization capacity of licorice root extract was determined in cell culture by endpoint dilution. For this purpose, serial dilutions of the licorice root extract (0.004 mg/ml – 4 mg/ml) were pre-incubated with 100 TCID50 of SARS-CoV-2 for 1 hour at 37 °C and subsequently incubated on confluent Vero E6 cells grown in 96-well microtiter plates (pre-entry approach). After 48 hours, the cells were stained with cry stal violet and analysed for plaque formation.
 
Cytotoxicity was determined at four distinct time points (5 minutes, 12 hours, 24 hours and 4 hours).
 
The aqueous licorice root extract showed neutralizing effects even at a subtoxic concentration of 2 mg/ml. This concentration is lower than the normal consuming dilution e.g. in tea (12.5 mg/ml). Although licorice root tea may represent a good candidate for complementary use, the identification and characterization of the active compound is of great importance for a potential clinical application.
 
Based on this results, the study team further investigated the antiviral activity of glycyrrhizin acid against a clinical SARS-CoV-2 isolate and subsequently examined the underlying mechanism of viral neutralization.
 
The neutralizing activity of glycyrrhizin against a clinical SARS-CoV-2 isolate was investigated in cell culture.
 
Glycyrrhizin acid ammonium-nitrate was dissolved in DMEM containing 2% (v/v) FCS and 1% penicillin–streptomycin at 37 °C and adjusted to pH 7. A potential cytotoxic effect of glycyrrhizin was investigated as described above.
 
No cytotoxic effect could be observed even at a concentration of 4 mg/ml. The neutralization capacity of glycyrrhizin was determined by endpoint dilution.
 
The antiviral activity against SARS-CoV-2 was analyzed under pre- and post-entry conditions. Descending glycyrrhizin concentrations (0.002 – 4 mg/ml) were pre-incubated with 100 TCID50 SARS-CoV-2 for 1 hour at 37°C (pre-entry condition) and subsequently added to confluent Vero E6 cells in 96-well microtiter plates for 48 hours. In a second approach, Vero E6 cells were inoculated with 100 TCID50 SARSCoV-2 for 4 hours before the glycyrrhizin-containing inoculation medium with various glycyrrhizin concentrations (0.002 – 4 mg/ml end-concentration) was added (post-entry conditions). Complete virus neutralization was achieved at sub-toxic concentrations of 0.5 mg/ml under pre- and 1 mg/ml under post-entry conditions.
 
The study team determined the half-maximal effective concentration (EC50) sufficient to neutralize the virus. Confluent Vero E6 cells grown in 6-well plates were infected with 1000 TCID50 SARS-CoV-2 and at the same time treated with various concentrations of glycyrrhizin ranging from 0.0625 to 4 mg/ml. After 48 hours of incubation, the supernatants were harvested and the viral loads were determined by endpoint dilution. The experiment was performed in triplicates.
 
The EC50 was calculated with 0.44 mg/ml, uncovering glycyrrhizin as a potent compound effective against SARS-CoV-2 (Figure 1C). The initial finding was supported by quantifying the SARS-CoV-2 RNA from the supernatants of SARSCoV-2 infected cells treated with glycyrrhizin. Confluent Vero E6 cells grown in 24-well plates were infected with 500 TCID50 and simultaneously treated with 1 mg/ml of glycyrrhizin. Supernatants were collected at seven different time points (0 hours, 4 hours, 8 hours, 24 hours, 28 hours, 32 hours and 48 hours) post-infection.
 
The study findings showed that glycyrrhizin treatment significantly reduced the genomic SARS-CoV-2 RNA levels. Taken together, the study team demonstrated that glycyrrhizin exhibited a high antiviral activity against SARS-CoV-2.
 
The study team also investigated the underlying mechanism how glycyrrhizin may interfere with the virus replication.
 
The team focused on the SARS-CoV-2 main protease (Mpro) as a potential target for glycyrrhizin. Mpro is essential for processing the viral polyproteins that are translated from the viral RNA and thus, for virus replication .
 
Glycyrrhizin was suggested as a possible inhibitor of Mpro by in silico analysis, but this hypothesis has never been experimentally proven. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556787/
 
The study team provide evidence that glycyrrhizin potently inhibits Mpro activity in vitro. Mpro were incubated with two different concentrations of glycyrrhizin (30 µM and 2000 µM, dissolved in water). As control, the protease inhibitor GC376 was used. The enzyme-sample solution was incubated at room temperature for 30 minutes. The enzyme activity was measured at 360 nm excitation and 460 nm emission after overnight incubation of the inhibitor-Mpro mixtures with substrate (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH2) at room temperature. Glycyrrhizin completely inhibited Mpro activity at a concentration of 2000 µM (1.6 mg/ml) and reduced its activity by 70.3% at a concentration of 30 µM (0.024 mg/ml).
 
Glycyrrhizin was clinically evaluated in the context of a clinical trial and described to be a safe and well-tolerated compound. https://pubmed.ncbi.nlm.nih.gov/11125713/
 
The pharmacological effects include antioxidative and antiinflammatory, corticosteroid-like activities. The potent antiviral activity as well as antiinflammatory properties highlight glycyrrhizin as an excellent candidate for further clinical investigations in COVID-19 treatment. https://pubmed.ncbi.nlm.nih.gov/31874059/
 
More significantly, a case report described compassionate use of glycyrrhizin among other potential antivirals for the treatment of COVID-1910. Although the patient recovered from disease, further controlled studies are needed to prove the therapeutic effects of glycyrrhizin in COVID-19. https://pubmed.ncbi.nlm.nih.gov/32458502/
 
The study team concluded, “We demonstrated that glycyrrhizin, the primary active ingredient of the licorice root, potently neutralizes SARS-CoV-2 by inhibiting the viral main protease. Our experiments highlight glycyrrhizin as a potential antiviral compound that should be further investigated for the treatment of COVID-19.”

Licorice Root and the phytochemical Glycyrrhizin are both key components in Thailand Medical New’s Prophylactic and Therapeutic Teas For COVID-19. https://www.thailandmedical.news/news/new-therapeutic-teas-

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