THERAPEUTIC PROFILE OF Glycyrrhiza glabra : A RAY OF HOPE IN TREATING COVID-19

Glycyrrhiza glabra Linn (licorice) has been widely studied because of the possible ethnopharmacological benefit. It was among the essential Ayurvedic medicines studied for immunomodulatory, antiviral, antibacterial, antioxidant, hepato-protective activities. Nowadays, licorice’s root parts have been evaluated to contain numerous bioactive components responsible for the treatment of respiratory tract infections and influenza-like illnesses. Most importantly, several investigations have now assessed the multiple mechanisms by which these biomolecules could attack the essential proteins and enzymes responsible for SARS-CoV-2 cellular entry and interaction. Our findings gathered that the metabolites of Glycyrrhiza glabra could interfere with COVID-19 cellular entrance and replication by blocking all five key proteins and enzymes essential for the survival and attachment of the virus. In addition to their ability to neutralise, the inflammatory cytokines storm reaction following the COVID-19 viral infection. This review was designed to digest the potential pharmacological importance of Glycyrrhiza glabra and its likeness in combating the pandemic COVID-19.


INTRODUCTION
Coronavirus is a pandemic disease outbreak at the Wuhan market of China in December 2019.The pandemic COVID-19 has now infected more than 163 million people and claimed more than 3.3 million lives across 220 countries.The highest prevalence of infection was reported in the USA followed by India, Spain and Italy.At present, there is no licensed drug marketed for the treatment of SARS-CoV-2 infection (Rabaan et al. 2020).In the Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 Ibrahim Aminu Shehu and Aparna Datta 148 meantime, eight vaccines were approved for emergency use that was distributed globally for shooting amidst the pandemic.WHO reported that more than 1.5 billion vaccine doses were administered globally, in which about 369 million people were fully vaccinated (Holder 2021).
The COVID-19 vaccines are under the emergency authorisation of the FDA and the shooting is helping both asymptomatic patients and pre-infected people.However, the vaccines suffer from several limitations like lack of sufficient dose to supply the world population, therefore, only the people at the front-line risk of infection are prioritised during the vaccination initially.Although the shooting is underway with a massive record of safety, some vaccines have been reported to cause serious adverse effects and consequently, the shooting was suspended in some countries.For instance, the shooting of the AstraZeneca vaccine was suspended in some European countries (Estonia, Latvia, Lithuania and Luxembourg) over serious blood clot formation (Anon 2021a).Two severe anaphylactic reactions were reported in the UK over the use of the Pfizer vaccine (Cherry 2020).Besides, the available vaccines might be less effective against the new strength of coronavirus, as the mutation in SARS-CoV-2 spike protein (E484Q and L452R) has been reported in India (Hodgson et al. 2021).Moderna has announced the development of a novel form of its vaccines that can neutralise the new COVID-19 variant of South Africa (Grady, Mandavilli and Thomas 2021).The current shortage in COVID-19 vaccines supply was reported in more than 60 low-income countries amidst the pandemic.Importantly, India has reported the largest causality in the second wave of the pandemic, for being the first country to report more than 400,000 cases a day but suffered the major disruption in the vaccine supplychain (Anon 2021b).
According to the WHO report, about three-quarters of the world populace relies solely on herbal medicines as the primary source of the remedy, because of their safety, affordability and a broad spectrum of activities (Amawi et al., 2020;Rastogi, Pandey and Singh 2020).The different bioactive constituents that are traditionally known as immunemodulators and antivirals have been considered as an alternative measure of curving COVID-19 infection.
Glycyrrhiza glabra Linn (licorice) is an 'essential herbal medicinal plant' used to cure various ailments for more than 4,000 years (Dastagir and Rizvi 2016).Hippocrates had prescribed it in 400 BC for the treatment of ulcer and thirst-quenching (Lakshmi and Geetha 2011).It belongs to the family Papilionaceae found in abundance in tropical and subtropical regions of the world (Damle 2014).The two Greek words Glycyrrhiza glabra, mean sweet root.Binocularly, it was called 'licorice' in English, Jothi-madhu in Hindi, Jeshthmadh in Marathi and Yashtimadhu in Sanskrit (Damle 2014).Currently, more than 80% of the world population relies on traditionally based medicines (Anagha et al. 2014) and the number of traditional medicines users is alarmingly increasing.Consequent to the SARS-CoV-2 pandemic outbreak and the lack of allopathically approved medication or vaccine, the world relied on potential herbs known to provide potent immunomodulatory and antiviral antiinflammatory activities (Rastogi, Pandey and Singh 2020;Amawi et al. 2020).Different bioactive constituents extracted from licorice were found to have shown hepato-protective, anti-inflammatory and antiviral effects, in addition to their effectiveness on the management of metabolic syndromes and oxidative stress.Hence, they have shown high tolerance and safety profile (Li, Sun and Liu 2019).The root extracts show significant potency in treating many viral infections, including SARS-CoV-2 viruses and other respiratory infectious diseases (Roshan et al. 2012).The exact mechanism of the antiviral activity of glycyrrhizinic acid (the main Glycyrrhiza glabra component) has not yet been established.Hoever et al. (2005) investigated the antiviral effect of glycyrrhizinic acid and found it to have doubled that Licorice in Handling COVID-19 and Allied Infection Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 of zidovudine and twice lower than that of acyclovir.Therefore, the author's finding could be a considerable evidence of using glycyrrhizinic acid for treating COVID-19 infection.
Thus, it could be essential to benefit licorice's therapeutic variance in combating SARS-CoV-2 infections.It could be crucial to investigate the current literature on how Glycyrrhiza glabra could be beneficial in combating SARS-CoV-2 infections thereby exploiting the bioactive constituents responsible for the activities and the possible distinct mechanisms of action.This study focuses on finding the potential of Glycyrrhiza glabra, its important pharmacological activities relevant to its likeness to fight COVID-19 pandemic infection.The latest literature for the period the past 30 years to date was extracted from the official scientific database such as PubMed, Google Scholar, Research Gate and Science Direct.

Morphology of Glycyrrhiza glabra
Glycyrrhiza glabra is a stout herb with imparipinnate and multifoliolated leaves.Flowers are papilionaceous present in axillary spikes which are violet in colour.The reniform seeds are compressed in pods and the hardy rootstock makes numerous amounts of perennial roots.The drug (licorice) can be accumulated either in dried, peeled or unpeeled underground stems or in roots.The image of plants' leaves has been presented schematically in Figure 1.Licorice in Handling COVID-19 and Allied Infection Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167

Hepato-protective activity
Several studies have proved the hepato-protective effect of licorice.It was evident that glycyrrhizinic acid exerts hepato-protective activity by modulating the permeability of hepatocytes (Nakamura, Fujii and Ichihara 1985).Al-Jawad et al. (2012) evaluated the effect of aqueous root extract of licorice on acute liver injured rats by administering a daily dose of 2 gm/kg body weight orally for 1 week.The study found licorice to have shown a significant hepato-protective effect by restoring the injured rats' hepatic tissues (Rafi, Al-Jawad and Jinan 2012)).Laylani (2016) administered both aqueous and ethanolic extracts of Glycyrrhiza glabra at 250 mg/kg and 500 mg/kg to hepato-toxicity-induced rats.
Histopathological parameters taken after 24 h injection revealed a significant reduction in liver enzymes level, thus indicating the potential hepato-protective effect of licorice (Laylani 2016).Jeong et al. (2002) accessed the hepato-protective activities of licorice.At the end of the study, it was found that 18ß-glycorrhizinic acid is a principally active agent behind J Pharm Sci, Vol. 20, No. 2 (2022): 147-167

Malay
Ibrahim Aminu Shehu and Aparna Datta 152 the therapeutic effect against licorice against liver injury, and it works by the mechanism of competitive agonism to specific glycine arginine rich (GAR) hepatic proteins.

Antioxidant activity
The flavonoids of licorice are 100 times more potent in antioxidant activities than vitamin E. It has a potential free radical cleaving effect that can be used for skin and hair protective cosmetics products (Kiso et al. 1984;Demizu et al. 1988).Numerous scientists have shown that the extract of Glycyrrhiza glabra has shown potential antioxidant and anti-candidate properties and thus interferes with oxidative stress induced by HIV in the HIV/AIDS treatment, it could therefore serve as a potent adjuvant agent (Aluckal et al. 2017).Gaitry Chopra et al. (2013) demonstrated the antioxidant activity of methanol extract of licorice roots at a dose of 500 μg/m, using the DPPH (1,1-diphenyl-2-picrylhydrazile) model and found that there was substantial antioxidant activity.

Anti-inflammatory
Several studies suggest that many phyto constituents of Glycyrrhiza glabra are potent COX-2 and phospholipase A2 inhibitors, and therefore, they might be potent anti-inflammatory agents (Pastorino et al. 2018;Kaur et al. 2010).Alcoholic extract of licorice of 200 mg/kg exerts equivalent anti-inflammatory effect to 10 mg/kg indomethacin as demonstrated on carrageenan-induced oedema rats (Gupta, Maheshwari and Khandelwal 2013).Moreover, it also illustrated an anti-inflammatory effect similar to that of hydrocortisone.Glycyrrhizinic acid is known to exhibit anti-inflammatory activity in vivo and work through a distinct mechanism of inhibiting pro-inflammatory cytokines (Okimasu, Moromizato and Watanabe 1983;Račková et al. 2007).Therefore, this could contribute to the prevention of cytokine storms caused by the SARS Cov-2 infection.An in vivo study performed by Matsumoto et al. (2013) has shown the anti-apoptotic and anti-inflammatory activity of glycyrrhizin on titanium dioxide intoxicated rats.

Respiratory diseases treatment
Glycyrrhiza glabra has shown the bronco-relaxant effect better than prednisolone and Boswellia carterii (Olibanum), following the trial conducted by Shi et al. (1998) on 54 patients, and therefore, it has been found to reduce the asthmatic attack on the tested group significantly.However, it was reported to have been a potent expectorant and demulcent agent; it stimulated tracheal mucus' secretions.The extracts and powders formulations of Glycyrrhiza glabra have been used in traditional settings as an antitussive and expectorant (Al-Jawad et al. 2012;Tsay et al. 2016).Azman et al. (2021) has proved the effectiveness of licorice in the treatment of upper respiratory tract infection, where the study indicated the 'phenyl groups' to have been a bioactive principle responsible for the antibacterial action of Glycyrrhiza glabra.According to the findings of a study performed by Tanaka et al. (2001), licorice is a potent nasal decongestant, efficacious in treating upper respiratory tract infections.Nazari, Rameshrad and Hosseinzadeh (2017) found that glycyrrhizin of 200 mg/kg enhanced the activities of respiratory tract enzymes like superoxide dismutase, tetrahydrocannabinol (THC), phenoloxidase (PO) and glycyrrhizin have shown to inhibit the respiratory syncytial virus (RSV) in vitro (Chen et al. 2010).

Licorice in Handling COVID-19 and Allied Infection
Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 Anticoagulant Wang et al. (2006) have assessed the anticoagulant activity of glycyrrhizinic acid using in vivo model by orally administering glycyrrhizinic acid 200 mg/kg, 300 mg/kg and 400 mg/ kg, respectively to the rats 90 min before the tail cutting.The bleeding time was noted to have significantly reduced after immersing the wounded tail at physiological saline.Finally, GA was proved a competitive inhibitor of FXa (Factor Xa, enzyme belonging to the serine protease family which plays a vital role in hemostasis) in a dose-dependent manner (Wang et al. 2006).Jiang et al. (2020) virtually screened 1,571 compounds for FXa inhibitory activity, docking, pharmacophore modeling and 3D-QSAR studies performed the result obtained predicted the licorice, trifolium and olive to have been the best FXa inhibitors.
Following the in vitro study on the different extracts of Glycyrrhiza glabra, the ethyl acetate extract at 3 mg/mL exhibited the highest anti-FXa activity (Jiang et al., 2014;Ibrahim et al. 2020).Mendes-Silva et. al. (2003) have demonstrated the first known in vivo study on the antithrombotic effect of glycyrrhizin and the study result proved the compound to have possesses a promising antithrombotic impact.

Immuno stimulatory effect
Glycyrrhiza glabra extract has shown a drastic increase in the phagocytic activity of neutrophils.In contrast, the root extract was identified to elicit no immunological effect on broiler chickens (Vikhe et al. 2013;Moradi 2014).The mechanism of anti-inflammatory activity Glycyrrhiza glabra is by radical scavenging effect (Arivoli et al. 2012).Jassim and Naji (2003) gathered that sulfur fumigation is the ancient Chinese method of herbal drug formulation.Several investigations prove its harmful effect on the liver after long-term consumption was found to compromise the immune-modulatory effect of glycyrrhizae radix rhizome glycopeptide resistant enterococci (GRER).Furthermore, the study revealed that sulfur-fumigated-GRER caused liver damage.However, the reduction of liquiritin potency in GRER following sulfur fumigation is possibly due to the depletion of it is the immunomodulatory effect as demonstrated on cyclophosphamide immune-suppression induced mice (Jiang et al. 2020).The Clinical Education (2020) investigated the immunological activities of Glycyrrhiza glabra (Revitonil tabs) and Echinacea purpurea in both in vitro and in vivo models.The result obtained through in vitro experiments revealed that Revitonil tabs at the dose of 10 μg/mol stimulate human granulocytes 44%-53% while Echinacea purpurea enhanced by 15%-40% at a concentration of 100 μg/mol.Upon in vivo bioassay, 2.2 mg/30 mg and 1.1 mg/30mg application of Revitonil tabs and Echinacea purpurea was orally administered in mice, respectively.The study result indicated Revitonil tabs to be the most potent immune-modulator when compared with Glycyrrhiza glabra and Echinacea purpurea mono-extract (Clinical Education 2020).

Antiviral Activities
The extracts of Glycyrrhiza glabra was found to inhibit different types of viruses and the available literature indicated the two active components of Glycyrrhiza glabra to have been responsible for such antiviral activities, namely; 18β-glycyrrhetinic acid and glycyrrhizin acid (Yang et al. 2017).Omer et al. (2014) investigated the antiviral effect of licorice and Ribavirin against Newcastle disease, the author found that the minimal concentration of 60 mg/100 mL Glycyrrhiza glabra extract inhibit the viral replication processes without causing any Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 Ibrahim Aminu Shehu and Aparna Datta 154 toxic effect.On the other hand, the concentration of 20 μg/mL of Ribavirin is the maximum safe dose for antiviral activity (Omer et al. 2014).

Human Immunodeficiency Virus (HIV)
Simone et al. (2001), prepared HIV-infected culture media of giant cells and tested the effectiveness of cochalcone A, glycocoumarin and licopyranocoumarin, the result indicated the significant inhibition of HIV growth on the giant cell.Batiha (2020), conducted an in vitro experiment and synthesised amides, glycopeptides and salts from derivatives of glycyrrhizin glycopeptides and biological evaluation confirmed their potency in inhibition of HIV-1 and HIV-2.Furthermore, the 'Penta-O-nicotinate' of glycyrrhizin was clinically evaluated to have anti-HIV as well as immune-modulatory activities.The anti-HIV activity of Glycyrrhiza lepidota (wild licorice) where they identified the diprenylated bibenzyl to have been the principal compound responsible for the anti-HIV activity and in contrast, glepidotin A and glepidotin B proved to be inactive against viruses (Batiha 2020).

Activity on influenza virus
Glycyrrhizinic acid and its derivatives could be a promising source of developing novel antiinfluenza virus's agents.Low dose of glycyrrhizinic acid was found to promote the recovery of embrocated hen eggs infected from influenza and Newcastle disease in cell monolayer cultures.The inhibitory mechanism of glycyrrhizinic acid involves the interference with the late replication stage of the viruses rather than viral viability and virions' haemagglutination, as highlighted in Figure 3 (Ashraf et al. 2017;Grienke et al. 2014).Kim et al. (2011) examined the anti-H5N1 effect of injection Neo-Minophagen ® approved glycyrrhizin acid derived drug.
Upon evaluation, the study found that glycyrrhizin acid product inhibited influenza A virus H5N1 and its associated inflammatory induced effect without compromising natural killer (NK) cells' activities.In continuation of the earlier experiment, the author discovered the drug to inhibit inflammatory responses elicited by H5N1 influenza viruses (Kim et al. 2011).Therefore, glycyrrhizin acid could be a promising compound for the treatment of H5N1 disease.Significant immune-stimulatory activity of the extract of saponin from Glycyrrhiza glabra L (Glabilox) demonstrated that it can serve as an adjunct for vaccination against the H7N1 influenza virus.The research compares the immune-stimulatory activity of Glabilox and Quil-A against H7N1 induced mice, and the mice were immunised subcutaneously by the formulation of both extracts.Following an animal assessment after one month of immunisation, Glabilox immunised mice showed a high level of H7N1 influenza virusspecific antibodies with no haemolytic and toxicity side-effects compared to Quil-A (Alexyuk et al. 2019).Moreover, Seniya (2012), virtually screened 657 phytochemical compounds from 191 antiviral plants, out of which the author identified three combinations of Glycyrrhiza glabra namely, licofalvone B, 2,2,4' trihydroxychalcone and davidigenin to have followed the Lipinski rule of five and excellently docked in inhibition of the active targeted site of influenza A viruses.However, the study has lacked support from animal bioassay, both in vitro and or in vivo (Oh, Barr and Hurt 2015).

Effect on human ortho-pneumovirus (HRSV)
Feng Yeh et. al. (2013) evaluated the anti-HRSV activity of the aqueous extract of G. glabra, the study result suggested that both 'Radix Glycyrrhizae' and '18β-glycyrrhetinic' increase the level of interferons and inhibit the viral cellular fusion mechanism.

Effect on hepatitis C (HCV)
According to the findings of Ashfaq et al. (2011), glycyrrhizin acid is very useful in targeting HCV, while showing more effectiveness in synergy with interferons.
Effect on herpes simplex virus Ghannad et al. (2014) conducted an in vivo experiment on an animal model, and the study revealed the antiviral effectiveness of Glycyrrhiza glabra with specificity on 'harps simplex viral' infection through which it can be taken as a prophylactic drug.The in vitro study conducted by Lin (2003), revealed that 0.04 mM-0.08 mM of glycyrrhizinic acid inhibited the replication of type 4 herpes virus.

Effect on SARS-CoV-2 (COVID-19)
There are some important drug targeted sites in the SARS-CoV-2 life cycle including the cellular entry mechanism, involving angiotensin converting enzyme 2 (ACE2); transmembrane protease, serine 2 (TMPRSS2) and COVID-19 interaction.RNA transcription and replication mediated by helicase and RdRp.The means of translation and proteolytic effect of viral proteins mediate by chymotrypsin-like and papain-like proteases, in addition to virion assembling and exocytosis (Shehu et al. 2021).These targetable proteins are essential for curing COVID-19 with existing drugs that have therapeutic potency against these specific proteins and enzymes (Kiplin Guy et al. 2020).

ACE2 receptors
It has been confirmed that SARS-CoV-2 enters cells via the ACE2 receptor.This implies that the blockage or modulation of ACE2 could be a potential means to halt the cellular entry of SARS-CoV-2.The mechanism of blockage involves either inhibiting the virus ACE2-RBD interaction, directly inhibiting ACE2-receptors or using recombinant human ACE2 (rhACE2); APN01 to disrupt virus endocytosis (Heinrich, Martina and Prakash 2020).

Affecting TMPRSS2
Human TMPRSS2 more powerfully activates the COVID-19 spike protein and facilitates its flush in across the host cell membrane via ACE2 than endosomal cathepsin and other proteases enzymes.As a result, targeting TMPRSS2 may be critical for developing anti-SARS-CoV-2 molecules.Some potent TMPRSS2 inhibitors, such as camostate and bromhexine, have been shown to be repurposed against COVID-19.Stopsack K investigated the anti-TMPRSS2 activity of androgens and other corticosteroids and demonstrated their potential for repurposing in the treatment of COVID-19.Furthermore, determining the crystal structure of the TMPRSS2 protein could aid in the development of COVID-19 (Iwata-Yoshikawa et al. 2019).

Affecting furin
The SARS-CoV-2 S-protein possessed a furin-like cleavage site (FCS), which is responsible for the S-structural protein's form, electrostatic interaction and furin binding affinity.The study in Zhejiang province examined the effect of FCS mutation (F1-2).Hasan et al. (2021) Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 Ibrahim Aminu Shehu and Aparna Datta 156 demonstrated the impact of furin-ACE2 enzyme interaction and its pathogenic role in COVID-19 infection, determining its relative proclivity to influence the structure of S-protein and the interaction between FCS and Furin.

Non-structural protein
Coronavirus possessed 16 non-structural protein (nsp) rendering varying functions, although, the specific functions elicited by nsp remain unclear.The crystal structure of nsp has been established aiding the development of novel molecules to block its action (Dong et al. 2020;Kandeel et al. 2020).

RNA-dependent RNA polymerase (RdRp)
RdRp plays a crucial role in facilitating the process of genome replication and transcription in the COVID-19 life cycle (Chan et al. 2020).The development of the RdRp structure in complex with its cofactors (nsp7 and nsp8) is encouraging.Therefore, interfering with the function of these enzymes would help the drug design for the treatment of COVID-19 infection (McDonald 2013).
The study conducted by Luo, Liu and Li (2020) concluded that at optimum dose, glycyrrhizin acid could elicit a desirable anti-COVID-19 effect and its analogs were predicted to have been more effective.Besides, the dosing of glycyrrhizin acid needs to be with caution in geriatric and in patients with heart disease (Luo Liu and Li 2020).Yonesi and Rezazadeh (2020) surveyed the available literature and confirmed the effectiveness of glycyrrhizin acid against SARS-CoV-2.However, the claim needs in vivo experimental support (Yonesi and Rezazadeh 2020).Chen and Du (2020) discovered glycyrrhizin acid to have worked via the inhibition of viral cellular entrance and interfere with SARS-CoV-2 receptors binding domain at minimal toxicity.The study found that molecular analogs of glycyrrhizin acid produce molecules with better antiviral potency than glycyrrhizin acid itself.However, unfortunately, found to have toxicity in the level profile (Chen and Du 2020).The molecular docking study conducted by Maurya (2020) indicated that the use of licorice may prevent SARS-CoV-2 infection by reducing the effect of cytokine storm in infected patients and boosting the body immune system.However, the findings need support of in vitro and in vivo experiments.Alternatively, Yashtimadhu (Telugu name of licorice) could be administered as an immunity booster as well as a prophylactic agent for mitigation and treatment of COVID-19 (Maurya 2020).Vardhan and Sahoo (2020) screened out 154 phyto-compounds from different plant sources based on a molecular docking study against five active binding sites of SARS-CoV-2.When compared with that of the FDA, the score-approved drugs, arbidol, remdesivir and hydroxychloroquine.The best 47 ligands were further screened based on drug-likeness and ADMET using in silico evaluation.However, the best 15 molecules were further processed to 'protein-ligand interaction' evaluations, upon which seven compounds were found to have succeeded and in overall two molecules, including glycyrrhizinic acid limuloids by having the highest binding scores of -9.9 kcal/mol and -8.2 kcal/mol, respectively.Hence, these molecules were also found to have obeyed the Lipinski rule of five; therefore, they were predicted to have higher drug-likeness properties for targeting COVID-19 (Vardhan and Sahoo 2020).Maurya (2020) evaluated the druggability of licorice's phytochemicals in terms of their binding affinities to spike protein, ACE2 enzyme, Mpro, RdRp, and furin residue of COVID-19.The docking study predicted that glycyrrhetic acid possessed the highest binding affinity to spike protein -8.3 kcal/mol, followed by shinflavanone with the binding energy of -7.8 kcal/mol and glycyrrhizin acid that has -7.2 kcal/mol, respectively.However, Licorice in Handling COVID-19 and Allied Infection Malay J Pharm Sci, Vol. 20, No. 2 (2022): 147-167 the relative binding affinities of shinpterocarpin, apioside, glabridin and glycyrrhetc acid toward ACE2 enzymes are predicted to be -8.2 kcal/mol, -7.8 kcal/mol, -7.7 kcal/mol and -7.6 kcal/mol, respectively.Meanwhile, the result of binding to furin protease shows that shinpterocarpin, apioside, and glycyrrhizin acid have the highest affinity with the relative binding energies of -8.6 kcal/mol, -8.5 kcal/mol and -8.5 kcal/mol, respectively.On the other hand, glabrin B, shinflavanone and glycyrrhizic acid found to have higher binding affinities to COVID-19.Main protease (Mpro) following binding affinities; -8.9 kcal/mol, -8.4 kcal/mol and -8.2kcal/mol, respectively.Lastly, the molecular binding to 'RNA-dependent RNA polymerase' revealed that liquiritin, shinflavanone, glycyrrhizin acid and glycyrrhizic acid scored the binding affinities of -8.8 kcal/mol, -8.5 kcal/mol, -8.4 kcal/mol and -8.4 kcal/mol, respectively (Maurya 2020).Therefore, phyto-compounds of Glycyrrhiza glabra can target all the five active sites of SARS CoV-2, as illustrated in Figure 2. Therefore, it could be a great source for the active ingredient in the drug designing of COVID-19.Sinha et. al. (2020) screened 20 bioactive compounds of licorice based on inhibition of COVID-19 non-structural protein (Nsp-15) and spike protein.After 'pharmacoinformatics' evaluation, glycyrrhizin acid was found to have scored the highest binding affinity to spike glycoprotein and ACE2.In contrast, glyasperin A has emerged as the molecule with the highest binding affinity to Nsp15.Thus, synergistically both these compounds can block the binding as well as the replication process of COVID-19.The author isolated numerous phyto-compounds of Glycyrrhiza glabra and screened them for in silico examination against COVID-19 inhibitory efficiency.The study was conducted by predicting the relative binding affinities of such molecules to spike glycoprotein and Nsp15 endoribonuclease of SARS-CoV-2 and comparing it with that of FDA-approved candidates Lopinavir and Ribavirin.The docking study result revealed that glyasperin A found a high affinity toward Nsp15 endoribonuclease blockage and a viral replication process.Simultaneously, glycyrrhizic acid is predicted to have a high affinity to block the COVID-19-ACE-2 receptor attachment process and thus could combat the cellular entry mechanism.However, these findings need biological validation (Sinha et al. 2020).Sci, Vol. 20, No. 2 (2022): 147-167 Ibrahim Aminu Shehu and Aparna Datta 158

SARS-CoV-2 inhibitory mechanism of Glycyrrhiza glabra
Glycyrrhizin acid, a principal active compound of Glycyrrhiza glabra was reported to prevent the replication and cellular penetration of SARS-CoV-2 via indistinct mechanisms.Although, investigation shows that the nitrous oxide produced by glycyrrhizin could be responsible for inhibiting the replication of COVID-19.The pharmacological effect exerted by glycyrrhizin acid was minimal due to the rapid metabolism of the compound and this can be enhanced by the structural modification of the compound using amino acid conjugate and amides formation in the ring (Bailly and Vergoten 2020).The

CONTRAINDICATIONS
Glycyrrhiza glabra has an abortifacient effect in pregnancy and thus was contraindicated in pregnancy and compromised kidney or heart, failure patients (Ramalingam et al. 2018).
It is contraindicated to take licorice extracts along with oral contraceptives and steroids, primarily prednisolone and hydrocortisone (Batiha et al. 2020).Daily administration of more than 15 g may lead to a surge in blood pressure, mineralocorticoid and hyperkalemia (Kaur et al. 2010;Nahidi et al. 2012).To date, there is no clinically established dosage limit of different Glycyrrhiza constituents, efficiently alleviating the inherently associated side effects.Therefore, advanced research need to prove the optimal safety dose of licorice for other medical conditions and patient categories.

CONCLUSION
From the surveyed literature, we concluded that Glycyrrhiza glabra is a rich source of numerous bioactive compounds that elicit different pharmacological activities against the treatment of infections and metabolic disorders.Interestingly, it has been exploited as potent antivirals, impotently, influenza, HIV and SARS-CoV-2 related viruses, some in silico and few in vivo studies have predicted the licorice metabolites to have promising potential in targeting all the five key proteins and enzymes responsible for COVID-19 cellular entrance and replication as evident from Figure 2, even though research lacked in vivo support and validation.Besides, the body immune system response to COVID-19 infection in immunecompromised people results in intensive inflammatory reactions, which caused 'cytokines storm' the mechanism to lead to multi-organ damages and subsequent death.Glycyrrhiza glabra has the potentiality to inhibit these mechanisms because of its inherent immunemodulatory, antioxidant, anti-inflammatory, anticoagulant and other desirable therapeutic benefits.Therefore, there is a ray of hope for licorice to provide effective medications for COVID-19 infection.

Future Perspectives
The research on licorice and its bioactive constituents should be conducted extensively on animal models, both, in vivo and in vitro.Meanwhile, the clinical and pharmacokinetic validation of such ligands will pave the way for inventing novel drugs effective against COVID-19.

Figure 2 :
Figure 2: Different mechanism of action of Glycyrrhiza glabra metabolites.
in vitro study conducted byVan de Sand et al. (2021) demonstrated the potential of glycyrrhizin acid to inhibit the main protease 'Mpro' (a vital enzyme responsible for SARS-CoV-2 virus replication) and halt the replication process.Glycyrrhizin acid could also prevent the cytokine storm by inhibiting the accumulation of reactive oxygen species (ROS) intracellularly.As illustrated in Figure3, it was found to be more effective than glucocorticoids in the management of SARS-CoV infections (Van de Sand 2021).Tan et al. (2022) have investigated the ability of glycyrrhizin acid to bind with the ACE2 and prevent the cellular entry of SARS CoV2 clinically.The author treated the COVID-19 infected patient with diammonium glycyrrhizinate and yielded a positive prognosis(Tan et al. 2022).

Table 1 :
Licorice metabolites and their pharmacological activates