The pharmacological mechanism of quercetin as adjuvant therapy of COVID-19

The emergence of novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the global outbreak and major public health concern. After the outbreak human-to-human transmission was confirmed with or without symptoms of upper and lower respiratory tract involvement. Up to date, there has been evidence that COVID-19 is beyond that of a typi-cal pulmonary disease and revealing pathomechanics of COVID-19-associated acute respiratory distress syndrome (CARDS), which include severe inflammation and pulmonary edema leading to impaired alveolar homeostasis, and resulting in an alteration of lung physiology, lung fibrosis, inflammation of endothelium, vascular thrombosis, as well as exaggerated immune response. Concerning this pathophysiology, the use of quercetin as phytotherapeutic may merit in the management of COVID-19 patients. In this review, the authors wish to elaborate on the molecular eﬀ ect of quercetin on SARS-CoV-2 by giving a detailed mechanism of quercetin against the binding of the S-protein of the virus to angiotensin-converting enzyme 2 (ACE2) receptors, the main protease (M pro ) or 3C-like protease (3CL pro ), papain-like protease (PL pro ), and RNA-dependent RNA polymerase (RdRP). Recent clinical evidence supporting the use of quercetin in COVID-19 management is also discussed in this paper.


Introduction
Since the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in Wuhan, Hubei Province, China, in December 2019, it has caused a global outbreak and major public health concern. The human-to-human transmission was confi rmed to elicit both asymptomatic and symptomatic respiratory disease, known as Coronavirus Disease . Numerous eff orts in the medical fi eld have been undertaken to cease the spread of this virus and to determine accurate strategies in treating individuals with confi rmed positive SARS-CoV-2 infection [1]. Globally, the COVID-19 pandemic has aff ected more than 298 million patients and 5.4 million deaths have been reported to the World Health Organization (WHO) during the writing of this review [2]. Ergo, scientists and researchers are racing the clock and breaking records to develop COVID-19 vaccines and repurpose several therapeutic options to treat mild to severe COVID-19. doi: 10.53388/life2022-0205-302 Concerning this pathophysiology, the use of quercetin may merit in the management of COVID-19 through its antiviral, anti-infl ammatory, anti-fi brotic, and immunomodulatory eff ects. In this narrative review, we aim to discuss the evidence related to its antiviral and anti-infl ammatory eff ects in molecular and preclinical studies, and the currently available studies evaluating the use of quercetin as an adjuvant pharmacotherapy of COVID-19.

Molecular Eff ect of Querce n to Coronaviruses
The SARS-CoV-2 pandemic has highlighted the vast multitude replication [14]. SARS-CoV-2 spike protein (S-protein), which constitutes receptor-binding domain (RBD), S1 and S2 subunits, human ACE2 receptors, type II transmembrane serine protease, and cathepsin B/L are the pivotal targets for inhibition of the viral entry [13]. Nevertheless, several compounds and drugs are designed and even repurposed to directly act on SARS-CoV-2 conserved enzymes 3CL pro or main protease (M pro ), PL pro , NSP12, and RNA-dependent RNA polymerase (RdRP) [14]. In this following section, we discuss the antiviral mechanisms of quercetin and its derivatives on coronaviruses of the Coronaviridae family, including SARS-Associated Coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV), and novel SARS-CoV-2, done by but not limited to computational or in silico approaches.

Inhibi on of S-protein interac on to ACE2 receptors
As aforementioned, S-protein can interact with ACE2 receptors in the human body and mediates the viral entry. This S-protein is activated by an enzyme called furin, through its specifi c recognition site toward the enzyme. Milanovic et al. conducted a computational study using an acid-base equilibrium approach and molecular docking simulations to evaluate the inhibitory eff ect of quercetin and its metabolites, compared to chloroquine, hydroxychloroquine, and cinanserin hydrochloride that suppress furin and interfere with the S-protein of SARS-CoV-2. It showed that acid-base forms of quercetin and its oxidative metabolite benzofuranone 2-(3,4-dihydroxy benzoyl)-2,4,6-trihydroxy-3(2H) benzofuranone bind to human furin with a better competitive inhibitory activity than those of chloroquine and hydroxychloroquine at physiological pH. Quercetin and benzofuranone induce alterations in the native conformation of furin and distort its active site. Likewise, their binding affinity toward furin is comparable to those of approved drugs but less comparable in binding to the S-protein. Hence, the approved drugs are still essential for specifi c binding to S-protein [15].
Among natural fl avonoids and synthetic indole chalcones tested computationally by Vijaya Kumar et al., quercetin showed highly potent inhibition against S-protein, dimerization, and catalytic activity of the SARS-CoV-2 M pro [16]. The inhibition of S-protein was shown by tight binding affinity of quercetin to amino residues of SARS-CoV-2, including Spike Receptor Binding Domain (SRBD), consisting of Gly496, Asn501, Tyr505, and Tyr453 with a binding energy of -7.8 kcal/mol. On the other hand, quercetin interacted with amino residues of Glu288, Asp289, Glu290 on M pro dimerization sites and interacted with Leu286 residues on the M pro enzymatic regulatory sites. Moreover, quercetin interacted with β-hairpin residues of NSP12, thus interfering with the stabilization of RdRp of SARS-CoV-2 [16].
Quercetin along with vitamin D and estradiol, namely tripartite combination, were tested by genomic-guided tracing of SARS-CoV-2 targets in human cells [17]. ACE2 and furin play a signifi cant role in SARS-CoV-2 entry to human cells and are expressed in multiple cells and tissues. The tripartite combination from the study was able to regulate the expression of ACE2 and furin by several mechanisms. From the gene expression omnibus (GEO) database, quer-cetin appears to inhibit the expression of several potential SARS-CoV-2 infection-promoting genes like c-FOS in rat and human cells, including Runx1 in rat cells and HNF4a in human cells. C-FOS, Runx1, and HNF4a are reported as activators of the ACE2. Further, the Gene Set Enrichment Analyses (GSEA) demonstrated that quercetin administration renders signifi cant decreases in ACE2 expression during the diff erentiation of intestinal cells in the human model. Intriguingly, quercetin alters approximately 30% of the expression of the genes (98 out of 332), which encode protein targets of SARS-CoV-2 in human cells; hence it is the potential to interfere with 85% (23 out of 27) SARS-CoV-2 proteins. In combination with vitamin D, it may alter the activity of SARS-CoV-2 proteins in human cells, almost 93% (25 out of 27). In the mouse model, quercetin exerts an inhibitory eff ect toward ACE2 by enhancing the activity of GATA5 (repressor) inhibitory activity on surfactant protein-C (SFTPC) gene expression. On the other hand, quercetin upregulates INSIG1 (activator) in the human intestinal cells, allowing inhibition of another activator gene, HIF1a, and ACE2 subsequently [17].
A computational study using traditional Chinese medicine systems pharmacology (TCMSP) Database and Analysis Platform was done to explore quercetin, puerarin, and kaempferol activities in blocking SARS-CoV-2 replication [18]. Among these three compounds, quercetin demonstrated the highest binding affinity to ACE2 with dissociation constant, K D = 4.83x10 -6 M, and was considered to have remarkable value for an unmodifi ed compound. Additionally, it also demonstrated higher binding affi nity to SARS-CoV-2, SRBD with K D = 2.41x10 -8 M; and more notably, it almost suppressed the viral binding to ACE2 receptor identifi ed by Surface Plasmon Resonance (SPR) assay. These dual actions both on SARS-CoV-2 spike protein and ACE2 receptor may generate a better antiviral synergistic eff ect on SARS-CoV-2. Further exploration of quercetin was also performed by Gene Ontology (GO) functional enrichment analysis. It revealed that quercetin was closely linked to infl ammatory response, signal transduction, response to drugs, gene transcription and expression, apoptosis, and oxidation-reduction process. These inhibitory mechanisms are summarized in Figure 1.
In vitro assays to demonstrate quercetin and its metabolites in inhibiting recombinant human ACE2 (rhACE2) activity was done by Liu et al. [19]. Among the polyphenols tested, quercetin was the most potent inhibitor against rhACE2 activity with a half-maximal inhibitory concentration (IC 50 ) of 4.48 μM. The results also showed that by the presence of 5-100 μM quercetin, the reaction rate of 50 ng/mL rhACE2 was concentration-and time-dependent at 37℃. After 2.5 minutes, the rhACE2 IC 50 of quercetin raised from approximately 4.48 μM to 29.5 μM at 10.5 minutes. Since rhACE2 activity is raised with temperature, the inhibition of this enzyme depends on different stages of temperature. Quercetin was also shown to decrease the affinity of rhACE2 to the Mca-APK (Dnp) substrate and lower the rhACE2 catalytic efficiency (K cat /K m ). Consistently, it exerted a dual inhibition action by increasing Km (affinity) and decreasing Vmax of rhACE2. On the other hand, the inhibition of rhACE2 was also demonstrated by quercetin glycosides and quercetin metabolites like isorhamnetin, tamarixetin, 3,4-dihydroxyphenylacetic acid, and quercetin-3-glucuronide, as they decreased the K m and K cat /K m values [19].

Inhibi on of SARS-CoV-2 3CL pro
In this study, a natural compound called quercetin-3-beta-galactoside was identifi ed as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies [20]. The results revealed that Gln189 was pointed to be an important amino residue that plays a role in binding interactions between quercetin-3-β-galactoside with SARS-CoV 3CL pro in wild and mutant type (Q189A). Albeit, SARS-CoV 3CL pro Q189A preserved the same bi-doi: 10.53388/life2022-0202-302 ological activity level as its wild type; in fact, the binding affi nity of quercetin-3-β-galactoside to SARS-CoV 3CL pro Q189A was reduced. Further, the enzymatic activity of these two protease types expressed in E. coli-M15 was similar. Quercetin-3-β-galactoside rendered a significant and dose-dependent increment in SPR response units and demonstrated slow-dissociation curves and characteristics of fast-binding. Also, it acted competitively. The IC 50 value of the quercetin inhibiting the SARS-CoV-3CL pro catalytic activity was calculated to be 42.79 ± 4.97 μM. Whereas, the IC 50 value of the quercetin against the mutant type Q189A, was calculated to be 2-fold greater than the wild-type, 27.89 ± 10.06 μM. Moreover, among eight derivatives of quercetin-3-β-galactoside synthesized in this study, four exhibited a remarkable inhibition percentage at 50μM, more than 50%, designating that these derivatives were potent candidate inhibitors of SARS-CoV-3CL pro [20].
There has been evidence pointing out that the structural basis of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV with a conserved active site are similar based on homology modeling studies [21,22]. Further, it is also highlighted that the binding of lead compounds is similar between the main proteases of SARS-CoV-2 and SARS-CoV, based on the protonation state of the Cys-His catalytic dyad [14]. It is also known that the high sequence identity for main protease 3CL pro between SARS-CoV and MERS-CoV is 95% [23]. In accordance with the previous study, quercetin displayed a good inhibition against recombinant SARS-CoV 3CL pro expressed in Pichia pastoris with an IC 50 value of 73 μM. Along with other flavonoids tested, quercetin also demonstrated greater than 80% inhibition toward the catalytic activity of recombinant 3CL pro and rendered docking energy of -10.2 kcal/mol. Thus, these fi ndings suggest that quercetin has the potential to be an inhibitor candidate for SARS-CoV3CL pro [24]. Among forty fl avonoids tested for inhibition against MERS-CoV 3CL pro catalytic activity, quercetin-3-β-D-glucoside showed prominent inhibition in a dose-dependent manner with an IC 50 value of 37.03 μM. Likewise, the molecular docking results indicated that the hydroxymethyl group of quercetin glucoside was able to form hydrogen bonds with Glu169, hence contributing to a higher affi nity to the S1 side of the MERS-CoV 3CL pro [25].
Explorations of phytochemical potential to tackle SARS-CoV-2 have been done extensively. Jia et al. elucidated the underlying mechanism of Reduning Injection (RDNI), which is a patented traditional Chinese medicine for COVID-19 treatment [26]. They used a combination of modeled Vero E6 cells and computational studies. The results showed that quercetin, among the compounds analyzed from the RDNI, displayed potential in the regulatory mechanism of SARS-CoV-2 3CL pro , ACE2, and PL pro with a binding energy of less than -5 kJ/mol on molecular docking analysis. In the protein microarray analysis, quercetin as a component of RDNI was shown to inhibit cytokine expression, including IL-1α, IL-1β, IL-4, IL-6, IL-8, and TNF-α. These inhibitory mechanisms are summarized in Figure  1. From pathway enrichment analysis, the researchers suggested that RDNI is capable of regulating inflammation through PI3K/AKT, FOXO, MAPK, and T cell receptor signaling pathways. Likewise, RDNI eff ectively suppresses the overexpression of MAPKs, PKC, and p65 based on Western blot analysis [26].
A screening study by computational and biophysical methods was previously done by Abian et al. to identify the inhibitory activity of natural quercetin on SARS-CoV-2 3CLpro. The results revealed that quercetin was able to alter the thermal stability of 3CL pro leading to destabilization in a concentration-dependent manner. Isothermal titration calorimetry (ITC) assay demonstrated an interaction between quercetin and SARS-CoV-2 3CL pro and displayed an inhibition constant of approximately 7 μM, which is considered suffi ciently good in the preclinical studies [27]. Another in silico study revealed that quercetin among other natural products like hispidulin, cirsimaritin, sulfasalazine, artemisinin, and curcumin demonstrated better affi nity against SARS-CoV-2 3CL pro active site, in comparison with hydroxychloroquine. Likewise, quercetin showed better potential inhibitions against SARS-CoV-2 3CL pro and ACE2 than hydroxychloroquine. Albeit, quercetin showed the lowest binding energy to the SARS-CoV-2 3CL pro , as it fi tted quite well to the active pocket of the 3CL pro and its hydroxyl groups were able to form hydrogen bonds with amino acid residues of His163 and Leu141 [28]. Modifying the quercetin scaff old is known to be important for quercetin to bind SARS-CoV-2 3CL pro at a molecular level. Seleno-functionalization of quercetin showed higher selectivity in the binding conformation and favorable affinity toward 3CL pro , as compared to its natural compound by molecular docking. The derivative compounds from the seleno-functionalization approach exhibited no cytotoxic eff ect on Vero cells and exhibited SARS-CoV-2-related cytopathic eff ect under the inverted light microscope. Likewise, at a lower concentration, the seleno-functionalization of quercetin was able to hamper SARS-CoV-2 replication performed by RT-qPCR [29].
Another common glycosylated conjugate of quercetin, namely rutin (quercetin-3-O-rutinose) constantly showed inhibitory activity to SARS-CoV-2 3CL pro . Rutin binds to the catalytic site of 3CL pro thus inhibiting viral replication. It significantly alters the tertiary structure of the protein and/or the aromatic protein environment side chains. The declining enzymatic activity of 3CL pro due to the rutin inhibitory eff ect delineates a concentration-dependent fashion. This eff ect is qualitatively similar to the previous study, utilizing quercetin [27], and stipulating that conjugated moiety of quercetin does not substantially suppress the inhibitory eff ect of the quercetin scaff old [23]. The results of this study were concordant with a previous study that showed allosteric inhibition of quercetin against SARS-CoV-2 3CL pro . Using molecular docking analysis, the binding affinity of 3CL pro for its substrate polypeptide was signifi cantly reduced when complexed with quercetin [30]. Cherrak et al. through their in silico study also revealed that glycosylated fl avonoids were strongly able to inhibit SARS-CoV-2 3CL pro . Their binding against SARS-CoV-2 3CL pro was stronger. The structural and the sugar moieties built in these fl avonoids mainly aff ect the strength of the binding against the 3CL pro active site. The strongest binding is possessed by fl avonoids substituted with mono or disaccharides at position C3 of the fl avonoid sugar. Among the fl avonoids tested, quercetin-3-O-rhamnoside, myricetin 3-rutinoside, and quercetin-3-O-rutinoside (rutin), the latter exhibited the highest score and was stable in the docked 3CL pro complex simulation. Henceforth, rutin is a better candidate to inhibit SARS-CoV-2 3CL pro [31].

Inhibi on of RNA-dependent RNA polymerase (RdRp)
To replicate its genome, SARS-CoV-2 does not rely on host polymerase, rather uses the RdRp. Hence-forth, it can be a potential for drug targeting. The RdRp structure of SARS-CoV-2 incorporates three subdomains and the functionally crucial catalytic sites of the RdRp, which include Asp760, Asp761, Cys622, Cys813, Ser759, and Trp617 [32]. Another in silico study consistently showed that quercetin-3-O-rutinoside (rutin), quercetin-3-O-glucuronide, quercetin-3'-O-sulfate along kaempferol were able to inhibit SARS-CoV-2 3CL pro and RdRp that play a role in viral replication stages. Rutin formed hydrogen bonds with 3CL pro through interaction with some amino residues His41 from the catalytic dyad of 3CL pro , and other residues like Thr25, Cys44, Met165, Gln189, and Thr190. Likewise, the results highlighted key interactions between rutin and binding pockets of RdRp via the formation of hydrogen bonds with Asp623 and Arg624 from motif A, and with the catalytic residue Ser759 and Asp760 from motif C. Other glucuronidated and sulfated quercetin demonstrated hydrogen bonds and π-anion interaction with the catalytic residue from motif C. These fi ndings suggested that glucuronidated and sulfated forms of quercetin and kaempferol are promising doi: 10.53388/life2022-0205-302 candidates for 3CL pro and RdRp inhibitors in SARS-CoV-2 replication [33].
The interaction of quercetin-3-O-sophoroside with SARS-CoV-2 main proteins was done by docking and molecular dynamic studies [32]. The targeted main proteins were E protein ion channel, helicase ADP site, helicase NCB site, N protein NCB site, 3CL pro , Nsp14 ExoN, Nsp14 N7-MTase, Nsp15 endoribonuclease, Nsp 16 2'-O-MTase, PL pro RdRp with or without RNA. The results demonstrated that quercetin-3-O-sophoroside potentially had the highest binding affi nity to the RdRp with RNA, with a considerable value of -9.70 ± 1.58 kcal/mol. It has been shown that quercetin-3-O-sophoroside binds to RdRp with RNA, by forming electrostatic interactions and hydrogen bonding. Likewise, there were hydrophilic interactions between quercetin-3-O-sophoroside with the amino acid residues of Arg555, Asp452, Asp623, Cys622, Lys62, Ser682, Thr556, and RNA G-8 nucleotide (61). Additionally, quercetin-3-O-sophoroside was able to form π-π interactions in the case of RdRp without RNA, and relative to RdRp with RNA, the binding energy was decreased to -8.40 ± 1.12 kcal/mol. These fi ndings suggested that the hydrophilic side chains of the proteins rendered the greatest tendency to interact with quercetin-3-O-sophoroside [32]. These inhibitory mechanisms are summarized in Figure 1.

Inhibi on of respiratory infec ons and infl amma ons
Despite the aforementioned well-established in silico studies regarding the antiviral properties of quercetin, human studies are still lacking. Moreover, the currently available data assessing the eff ectiveness of quercetin in respiratory infections and infl ammatory diseases are confl icting [8], and more randomized-controlled trials (RCTs) addressing this topic are essential. Heinz et al. conducted one of the randomized double-blinded controlled trials in 2010 to assess the infl uence of quercetin on the incidence of Upper Respiratory Tract Infection (URTI) in a large community group [9]. Around 1002 participants were enrolled and a standardized Wisconsin Upper Respiratory Symptom Survey (WURSS) recorded the upper respiratory tract outcomes daily for 12 weeks. After randomly distributed, the participants received either placebo (N = 335), 500 mg quercetin/ day (Q-500, N=334), or 1000 mg/day of quercetin (Q-1000, N=333). Additionally, the intervention arms also received vitamin C (125 or 250 mg/day) and niacin (5-10 mg/day). After a 12-week study period, plasma quercetin was compared to the pretreatment level and demonstrated a signifi cant increase in both Q-500 and Q-1000, compared to the placebo (P<0.001) with no significant adverse effects occurring in the intervention arm. Despite no signifi cant diff erence in URTI symptoms, severity, and symptom scores between intervention and control groups, a separate analysis of subjects > 40 years of age showed signifi cantly better fi tness level (N = 325), lower URTI severity (36% reduction, P = 0.020), and URTI total sick days (31% reduction, P = 0.048) in the Q-1000 group, compared to the placebo. However, adjustment based on gender, age, and body mass index (BMI) demonstrated an insignificant quercetin-related effect on URTI.
There are some other studies evaluating the effi cacy of quercetin in URTI [10] and other lung parenchymal inflammatory diseases, such as sarcoidosis [11]. Those studies have demonstrated some promising results, including a reduction in inflammatory markers, an increase in antioxidant levels, and the incidence of respiratory infections [9][10][11]. Some provide insight to more usefulness of quercetin in a specifi c population such as age more than 40-year-old in reducing the severity of the symptoms [9]. However, due to the lack of available human-interventional studies, more research on this topic is needed to further elucidate the role of quercetin in respiratory infection and infl ammatory diseases.

Querce n supplementa on and its ongoing clinical trials
Several clinical trials are conducted to elucidate the effi cacy of quercetin supplementation in diff erent stages of SARS-CoV-2 infection either in a single or in combination with other interventions. However, among 12 clinical trials data that we retrieved from the clinicaltrials.gov registry presented in Table 1 tionally [6,34,35].

Conclusion and future direc ons
Multiple in silico approaches have extensively been done to elucidate the ability of quercetin and its metabolites in halting SARS-CoV-2 entry and replication. Quercetin and its metabolites showed abilities to inter-fere with furin, an enzyme that is responsible for S-protein activation on the viral surface, thus inhibiting the interaction between SARS-CoV-2 and ACE2 receptors. Quercetin and its metabolites bind to catalytic sites of SARS-CoV-2 3CL pro and interfere with the 3CL pro dimerization by altering the tertiary structure of the protein and/or the aromatic protein environment side chains. Multiple interactions also occur between quercetin and the RdRp structures of SARS-CoV-2, rendering its ability to halt viral replication. Recent clinical trial data support the use of quercetin in terms of prophylaxis and adjuvant therapy of COVID-19. The phytosome formulation is considered a promising ingredient. Hence, we consider quercetin as a good candidate for development and optimization for COVID-19 treatment to a greater extent. Further clinical investigations to assess the safety and effi cacy of quercetin, and its interaction with other drugs in the management of COVID-19, are certainly warranted to provide high-quality clinical data and generalizability.