key: cord-1001354-5ptqe51w
authors: Khazeei Tabari, Mohammad Amin; Iranpanah, Amin; Bahramsoltani, Roodabeh; Rahimi, Roja
title: Flavonoids as Promising Antiviral Agents against SARS-CoV-2 Infection: A Mechanistic Review
date: 2021-06-25
journal: Molecules
DOI: 10.3390/molecules26133900
sha: 99edeecce1a1c3539e11dea0cef08f7fd44a9009
doc_id: 1001354
cord_uid: 5ptqe51w

A newly diagnosed coronavirus in 2019 (COVID-19) has affected all human activities since its discovery. Flavonoids commonly found in the human diet have attracted a lot of attention due to their remarkable biological activities. This paper provides a comprehensive review of the benefits of flavonoids in COVID-19 disease. Previously-reported effects of flavonoids on five RNA viruses with similar clinical manifestations and/or pharmacological treatments, including influenza, human immunodeficiency virus (HIV), severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Ebola, were considered. Flavonoids act via direct antiviral properties, where they inhibit different stages of the virus infective cycle and indirect effects when they modulate host responses to viral infection and subsequent complications. Flavonoids have shown antiviral activity via inhibition of viral protease, RNA polymerase, and mRNA, virus replication, and infectivity. The compounds were also effective for the regulation of interferons, pro-inflammatory cytokines, and sub-cellular inflammatory pathways such as nuclear factor-κB and Jun N-terminal kinases. Baicalin, quercetin and its derivatives, hesperidin, and catechins are the most studied flavonoids in this regard. In conclusion, dietary flavonoids are promising treatment options against COVID-19 infection; however, future investigations are recommended to assess the antiviral properties of these compounds on this disease.

By the end of 2019, an unusual pneumonia was reported from China which was further diagnosed as a novel coronavirus (CoV) causing severe acute respiratory syndrome (SARS) and was called COVID-19 [1, 2] . Later, the virus (SARS-CoV-2) spread to other countries and was declared a pandemic by WHO on 11 March 2020 [3] . The virus is transmitted mostly by respiratory droplets, and the severity ranges from mild to severe lethal symptoms. The asymptomatic cases in the incubation period are thought to be an important source of contagion [4] . In most cases, mild symptoms take 1-2 weeks to resolve, whereas severe cases can lead to death [5] .

SARS-CoV-2 affects the respiratory system, causing fever and dry cough [4] ; however, the virus can cause organ failure, mainly in the heart and kidneys, as well as causing cytokine storms, which further increase mortality. The viral life cycle of SARS-CoV-2 includes 3.10 µg/mL to 8.19 µg/mL. Oseltamivir also showed a weaker activity against influenza B than influenza A; whereas Q7G demonstrated strong activity against all influenza viruses. Additionally, quantitative PCR assays reported a higher decrease in viral RNA synthesis after Q7G treatment compared with oseltamivir, indicating the inhibitory effect of Q7G on viral RNA polymerase. Molecular docking analysis revealed this interaction to be due to the attachment of Q7G to the PB2 subunit of viral RNA polymerase [50] .

Oroxylin A (OA) is a flavonoid found in Oroxylum indicum (L.) Kurz. It has been shown that OA can inhibit several influenza A strains in MDCK cells in a dose-dependent manner. Oral treatment of mice infected with influenza virus H1N1 with OA also decreased virus-induced death, bodyweight loss, and lung injury, with a survival rate of 60.0% at 100 mg/kg daily dose. Antiviral effects of OA were reported to be due to downregulation of H1N1 matrix 1 (M1) mRNA transcription and protein synthesis (Jin et al. 2018 ). The M1 protein is a protein within the viral envelope that binds to the viral RNA and can mediate encapsidation of RNA nucleoprotein cores into the membrane envelope [51] . Although OA could inhibit protein synthesis, it could not block viral entry to host cell or nucleoprotein (NP) entrance to host cell nucleus [52] . Baicalin and biochanin A could inhibit influenza H5N1 infection in A549 cells with the IC50 values of 18.79 and 8.92 µM, respectively. This effect was mediated by suppressing nuclear viral ribonucleoprotein (RNP) export [53] . Other studies have also shown that baicalin can downregulate influenza M1 protein expression [54, 55] .

Host cdc2-like kinase 1 (CLK1) has a key role in the splicing of the H1N1 influenza virus M2 gene and is an important anti-influenza target. M2 is a proton channel in the viral envelope of the influenza A virus [56, 57] . It was demonstrated that gallocatechin-7-gallate isolated from Pithecellobium clypearia is an inhibitor of host cdc2-like kinase 1 (CLK1), an anti-influenza target due to its role in viral M2 mRNA alternative splicing. Investigations on the effect of gallocatechin-7-gallate at the daily dose of 30 mg/kg on ICR mice infected with H1N1 virus showed a significantly higher survival up to 8 days. It also inhibited virus-induced acute lung injury and weight loss. Additionally, assessments on H1N1-infected A549 cells demonstrated a significant downregulation of viral NP and M2 mRNAs. Moreover, the phosphorylation of splicing factors SF2/ASF and SC35, key factors for virus M2 gene alternative splicing, was significantly decreased after treatment with gallocatechin-7-gallate [58] . Cirsimaritin (CST), a flavonoid from Artemisia scoparia Waldst. and Kitam was assessed regarding its in vitro antiviral effects on MDCK and THP-1 cells infected with three influenza virus strains which showed IC50 values ranging from 5.8 to 11.1 µg/mL, compared with 3.4 to 8.9 µg/mL for ribavirin. Data demonstrated that CST could effectively reduce influenza M2 and protein expression in a dose-dependent manner so that the potency of CST at 20 µg/mL was higher than 10 µM of the standard antiviral ribavirin [59] . Luteolin is another flavonoid with an inhibitory effect on M2 mRNA expression. In MDCK cells infected with different influenza strains, 15 µM of luteolin was more effective than 10 µM of oseltamivir in both H1N1 and H2N3 infected cells. Luteolin also downregulated influenza virus coat protein I (COPI) expression, mediating virus entry and endocytic pathway, in infected cells [60] . Santin is a flavonoid extracted from Artemisia rupestris L., which was also suggested to have anti-influenza virus effects through suppression of M2 mRNA expression in a dose-dependent manner [61] .

It was indicated that quercetin could be a probable therapeutic agent against influenza infection at the early stages of infection so that it can be used for influenza virus prophylaxis. Investigations on the effects of quercetin on MDCK and A549 cells infected with influenza virus A strains revealed that it could inhibit viral NP mRNA in a dose-dependent manner, with the highest activity at 50 µM concentration [62] .

Research has shown that tricin (4 ,5,7-trihydroxy-3 ,5 -dimethoxyflavone) exhibits antiviral activities against influenza A and B strains. RT-PCR tests indicated that tricin could suppress M protein mRNA synthesis in MDCK cells infected with influenza virus; with no significant effects on neuraminidase and hemagglutinin biosynthesis. The 50% effective concentration of tricin, which could inhibit viral mRNA synthesis, was 3.4-10 µM Understanding these host cell necessities not only informs us of the molecular pathways used by the virus, but also presents additional targets for drug development [64] .

An in vitro study assessed the effect of quercetin and isorhamnetin on SARS-CoV-2 entry to ACE2h cells. ACE2 expressed on lung cells is a co-receptor of viral spike protein and, thus, is a main target of antiviral agents against SARS-CoV-2. It was observed that these two flavonoids have a high binding affinity to ACE2 and subsequently decrease viral entry via the inhibition of spike protein attachment to this receptor [65] . Another study assessed the effect of baicalein on SARS-CoV-2 infection in Vero E6 cells and hACE2 transgenic mice. A significant reduction was observed in in vitro and in vivo viral replication, as well as body weight loss and lung injury of animals [66] . Dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone and myricetin-3 ,5 -dimethyl ether 3-O-β-D-galactopyranoside are flavonoids derived from Cleistocalyx operculatus (Roxb.) Merr. and L.M. Perry. Cytopathic effect (CPE) reduction assay showed that these flavonoids inhibit viral replication of influenza virus H1N1 in MDCK cells. Structure-activity relationship (SAR) studies indicated that OH groups at C-7 and C-4, a double bond between C-2 and C-3, and especially a carbonyl group at the C-4 position, are critical functional groups that significantly improve the antiviral properties of flavonoids [67] . 3-deoxysappanchalcone (3DSC) isolated from Caesalpinia sappan L. could inhibit influenza virus replication in high concentrations via inhibition of viral NP expression in MDCK cells infected with the H1N1 virus. At an equal concentration (30 µM), both ribavirin and 3DSC showed significant inhibition of NP expression, though ribavirin had a stronger effect [68] . Studies have demonstrated that biochanin A and baicalein inhibited caspase-3 activation, an enzyme involved in viral replication [53, 69] . These compounds could also inhibit the nuclear export of viral RNP complexes, which is critical in viral replication [53] . Biochanin A showed an inhibitory effect against 1 2 mitogen-activated p38 and NF-κB, which were shown to be involved in viral replication. NF κB and p38 are activated due to oxidative stress and are known to affect influenza A virus replication and pathology [53, 70] . Investigations on cell cultures of MDCK cells and A549 cells infected with influenza virus showed that baicalein could inhibit viral replication at 20-80 µg/mL concentrations. Interestingly, baicalin showed similar antiviral activity to ribavirin and oseltamivir at concentrations of 40 µg/mL and 60 µg/mL, respectively. Baicalin also inhibited viral replication in the lungs of mice in vivo [71] . CST was shown to downregulate NF-κB protein and NF-κB phosphorylation in the nucleus [59] . It is already known that NF-κB has an important role in inflammation, oxidative stress, and host immunity suppression [72] . The downregulation of NF-κB also inhibits replication in various types of viruses, including influenza virus [73] .

In Vero E6 cells infected with SARS-CoV-2, naringenin could inhibit CPE in a timeand concentration-dependent manner. This effect was mediated through inhibition of endolysosomal Two-Pore Channels (TPCs), a pathway involves in infectivity of SARS-CoV-2, Ebola, and MERS via facilitating viral entry [74] . EGCG has shown a dose-dependent inhibitory effect (25, 50 µM) on HIV replication in T-cells; however, inhibition of viral replication was not directly affected by RT inhibition. Fassina et al. analyzed the p24 enzyme, which is involved in packaging viral particles. The results showed a downregulation of p24 concentration and RT activity in HIV-infected T lymphoblasts. Based on the following results, it was noted that EGCG inhibited viral replication through the downregulation of viral infection. There is yet no certainty about the exact effect of EGCG on viral infection [75] . gp120 signaling is commonly associated with increased HIV-1 replication in previously infected cells [75] . Studies show the inhibitory effect of genistein on gp120 and, subsequently, HIV-1 viral replication. There was no change in viral replication after admin- istration of genistein with a concentration of 1-2.5 µg/mL, but in a range of 5-10 µg/mL, genistein could suppress viral replication [76] . Herbacitrin is a flavonoid derived from Drosera peltata Thunb. and was previously known as an antiviral agent. It was shown that herbacitrin inhibits both RT and integrase in HIV-1 infected MT-4 and MT-2 cell cultures, resulting in viral replication blockade at different stages. At a concentration of 21.5 µg/M, herbacitrin could suppress RT activity, while it could inhibit integrase at a lower concentration, 2.15 µM [77] . Scutellarin purified from Erigeron breviscapus is a flavonoid with anti-HIV-1 activity. This flavonoid inhibited HIV-1 RT activity and cell fusion as major participants of viral replication [78] . Hesperidin and linarin are flavonoids with rutinose at the A ring and methoxy (-OCH3) substitution at the B ring. Isoquercetin has been shown to inhibit influenza A and B virus replication in infected MDCK cells. The combination of isoquercetin with amantadine also showed a synergistic effect on viral replication in MDCK cells infected with influenza A virus only in low doses (0.5 µM for isoquercetin and 1 µM for amantadine). Virus titer values after administration of isoquercetin and amantadine were about 7.5; while increasing isoquercetin and amantadine concentrations lowered the synergistic effect on virus titers to the value of 5 [79] . Q3R derived from Houttuynia cordata exerts anti-influenza virus effects. The effects of Q3R on MDCK cells infected with influenza virus A were compared to oseltamivir. Pulmonary lesions and edema were inhibited by Q3R more than oseltamivir. Q3R also had a higher efficacy compared to oseltamivir. The inhibitory effect of Q3R on influenza virus replication was indirect and through interaction with viral particles. Oseltamivir demonstrated moderate antiviral activity, about 58% against influenza A virus, and weak antiviral activity less than 49% with doses under 10 µg/mL; while Q3R showed 86% viral inhibition at 100 µg/mL and 66% inhibition in 10 µg/mL concentrations [80] . Quercetin 3-β-O-D-Glucoside (Q3G) was shown to prevent Ebola virus replication in vitro. Prophylactic administration of Q3G 30 min before the infection showed significant prevention of the Ebola virus. Q3G could also inhibit viral entry at the early stages. So Q3G could be an effective flavonoid for Ebola virus prophylaxis [81] .

Interferons (IFNs) comprise a group of proteins produced by several immune cells in response to many pathogens like viruses, parasites, bacteria, and tumor cells. There are three major classes of IFNs, including type I or acid-stable interferons (IFN-α subtypes, IFN-β, IFN-κ, IFN-, IFN-ω, and IFN-τ), type II (IFN-γ), and type III IFNs that known as IFN-λ [82] [83] [84] . They show a wide range of biological activities like activation of the innate immune response, increasing the expression of major histocompatibility complex (MHC) molecules, suppressing angiogenesis. Their most important role is to interfere with viral infections [84, 85] .

In the early phases of viral infection, IFNs activate the innate immune system. Recent studies have reported a decrease in the type I and type II IFN induction and signaling in COVID-19 patients [86] . These types of IFN have demonstrated antiviral effects by decreasing neutrophils immigration to the inflammation site, increasing antigen presentation, suppression of mononuclear macrophage-mediated pro-inflammation, and activating the acquired immunity for the progression of antigen-specific B and T cell responses [86] [87] [88] [89] [90] . Thus, IFNs usage at the early phase of the disease could decrease symptoms of the COVID-19 by reducing viral replication. Researchers also reported that IFN-γ levels could increase in COVID-19 patients with ARDS. The rapid rise in IFNs levels could invite proinflammatory cytokines into the alveolar tissue and resulting in pulmonary inflammation and lung injury [90, 91] . Therefore, it seems that either upregulation or dysregulation of IFNs and other pro-inflammatory cytokines responses or both could exert a significant role in the progression and pathological features of SARS-CoV-2.

Li et al. investigated the anti-influenza effects of baicalin, a glycosyloxyflavone that is the 7-O-glucuronide of baicalein, in the in vitro and in vivo model of influenza A virus infection. TNF receptor-associated factor 6 (TRAF6) is an effective mediator in the IFN production signaling pathway. Overexpression of TRAF6 leads to increased production of type I IFN [54] . MicroRNAs (miRNAs) are small molecules that control gene regulation post-transcriptionally [92] . miR-146a has been shown to have a regulatory role in inflammation [93] . miR-146a could enhance the replication of H1N1 and H3N2 through the downregulation of TRAF6. Baicalin (20 µg/mL) indicated a significant reduction in the miR-146a expression, viral NP, M1 protein levels, viral titer, and also increased mice survival rate [54] . In another study, Nayak and colleagues represented anti-influenza virus (H1N1-pdm09) activity of baicalin through regulating viral protein NS1, resulting in up-regulation of interferon regulatory factor 3 (IRF-3), IFN-γ, and IFN-β. This IFN up-regulation decreased viral replication that could reduce viral transcripts and pro-inflammatory cytokines expression, including IL-8 and TNF-α [55] .

Ding et al. designed a study to investigate the effects of hesperidin, a flavanone glycoside, in the influenza A virus (H1N1)-induced lung injury in male rats. The results showed that hesperidin attenuated lung injury via decreasing pro-inflammatory cytokine production, including IFN-α, TNF-α, and IL-6, through suppressing MAPK signaling pathways. Hesperidin also decreased IFN-α in the H1N1 infected pulmonary microvascular endothelial cells [94] . In another study by Kim et al. isoquercetin effectively attenuated lung injury induced by the H1N1 virus in mice via reducing IFN-γ, iNOS, RANTES, virus titers, viral bronchitis, and bronchiolitis [79] .

Oroxylin A (OA) from Oroxylum indicum (L.) Kurz prevented the lung injury induced by influenza A H1N1 virus in mice via up-regulation of IFN-β and IFN-γ [52] . Wogonin, another flavonoid isolated from Scutellaria baicalensis Georgi, exhibited a significant antiinfluenza activity by regulation of AMPK pathways. Wogonin also increased the regulation of IFN-β, IFN-λ1, and IFN downstream molecules, including myxovirus resistance gene A (MxA) and 2-5 oligoadenylate synthetase (OAS), in MDCK and A549 infected cells [95] .

CoVs contain some open reading frames which encode a few accessory proteins. These accessory proteins have been shown to modulate inflammatory pathways such as IFN signaling and pro-inflammatory cytokines [96] . It has been elucidated that the prognosis of COVID-19 could be worsened by the secretion of pro-inflammatory cytokines, including interleukins, IFNγ, and TNF-α [97] . Blanco-Mello et al. indicated that inappropriate immune response might help virus replication and complications due to severe types of COVID-19 [98] . Ruan et al. also showed that an elevation in inflammatory cytokines such as IL-6 is associated with ARDS, respiratory failure, and adverse clinical outcomes [99] . Respiratory failure caused by lung damage is a result of the overproduction of pro-inflammatory cytokines after the infiltration of immune cells into the lung [100] . Cytokine storm is a systemic inflammatory response associated with a broad range of factors like infections and certain drugs. Several studies showed a significant connection between the cytokine storm, severe inflammation, and multiple organ failure in COVID-19 patients [101] [102] [103] . SARS-CoV-2 virus recognition with innate and adaptive immune systems could result in the activation and production of inflammatory cytokines. According to recent studies, plasma levels of pro-inflammatory cytokines are enhanced in COVID-19 patients. These inflammatory cytokines like TNF-α, IL 6, IL 2, IL-1β, IL 7, IL 10, and IL-18, as well as monocyte chemoattractant protein-1 (MCP-1), have pivotal roles in pathological progression and severity of COVID-19 through an increase in viral load, pneumonia, lung damage, neurological disorders, and mortality [97, 101] .

These events could lead to multi-organ failure and lung injury as the main complication of SARS-CoV-2; therefore, modulation of pro-inflammatory cytokines can be considered as a reasonable treatment goal in COVID-19. In addition, significant anti-inflammatory effects of flavonoids have been demonstrated in many studies; thus, they may be promising compounds in combating inflammation-related complications of COVID-19 [97] .

Yang et al. proved the protective effect 3-deoxysappanchalcone (30 µM) on in vitro influenza H1N1 virus-induced inflammation and apoptosis by decreasing IL-1β and IL-6 levels [68] . Baicalein, a flavone, and biochanin A, an O-methylated isoflavone, reduced pro-inflammatory cytokine expression in A549 cells and primary human monocyte-derived macrophages (MDM) infected with influenza H5N1 virus strains, that could prevent inflammatory pathway activation and tissue damages [53] . In influenza A-infected A549 and MDCK cells, baicalin, the glycosylated form of baicalein (baicalein-7-glucuronide), could increase IFN levels, resulting in a reduction of pro-inflammatory cytokines production. Thus, IL-8 and TNF-α were significantly lower in baicalin-treated cells compared with the untreated control cells [55] .

An in vitro study has shown that 2.5, 5, and 10 µg/mL concentrations of CST, a dimethoxyflavone, has a significant effect on the attenuation of NF-κB signal transduction pathway in THP-1 cells infected with influenza A (H1N1) virus. Following NF-κB inhibition, the production of pro-inflammatory cytokines including IL-1β, IL-8, IL-10, and TNF-α, as well as the inflammation-related protein COX-2, were suppressed by CST in a dose-dependent manner [59] . In an in vitro study by Yonekawa et al. on the antiviral properties of hesperidin and linarin, these flavonoid glycosides inhibited R5-HIV-1-NL(AD8) viral replication in CD4+ NKT cells by increasing the production of anti-inflammatory cytokines including IL-2, IL-5, and IL-13. It was observed that the stimulatory effect of these two flavonoids are critically dependent on the sugar moiety as the aglycones (hesperetin and acacetin) failed to show such activity. Furthermore, methoxy (-OCH3) substitution at the B ring is essential for the stimulatory activity of hesperidin and linarin on CD4+ NKT cells. They could also induce RANTES, MIP-1α, and MIP-1β secretion from Vδ1+ expressing T cell receptors which subsequently suppressed viral replication in CD4+ NKT cells [104] . Kang suppressed TNF-α production as one of the important inflammatory mediators causing fever and triggering NF-κB pro-inflammatory pathway, further worsening the condition of patients [106] . The trimethoxyflavone santin has demonstrated anti-influenza activity in THP-1 and MDCK cells in a 60 µM concentration. Influenza A (H3N2) virus induces pro-inflammatory cytokine production in THP-1 cells that results in lung inflammation and injury [61] . Anti-inflammatory cytokines might also be altered during influenza virus infection. IL-10 is an anti-inflammatory cytokine that can be induced by influenza virus. IL-10 inhibits invariant natural killer T cells by downregulating the production of IL-12 by pulmonary monocyte-derived dendritic cells [107] . The levels of IL-6, IL-8, IL-10, IL-1β, and TNF-α were significantly decreased in the santin-treated group through downregulation of MAPKs and NF-κB signaling pathways [61] .

In addition to the above, gallocatechin-7-gallate, genistein and theaflavins are other flavonoids with modulating effects on the production of pro-inflammatory cytokines [58, 76, 108] ; thus, these molecules seem to have a desirable anti-inflammatory effect, helpful in controlling viral infection-related inflammation.

When a virus enters a host cell, the host cell recognizes its replication via pattern recognition receptors (PRRs) [109] . Virus RNA structure is involved in oligomerization of PRRs and activation of downstream transcription factors, in particular, interferon regulatory factors (IRF) and NF-κB. Activation of NF-κB and IRFs leads to engagement of Molecules 2021, 26, 3900 9 of 36 cellular antiviral defense by the induction of type I and III interferons and chemokine secretion [110] .

Chiou et al. investigated the effects of 8-prenylkaempferol (8-PK) in A549 cells infected with the influenza A (H1N1) virus. Results showed that interfering with the PI3K-Akt pathway is the main mechanism of 8-PK lead to protective effects against the influenza A virus. 8-PK decreased NF-κB and IRF-3 nuclear translocation through attenuation of Akt phosphorylation and PI3K activity. Finally, reduced production of regulated activation, normal T cell expressed and secreted (RANTES) through H1N1-infected A549 cells [111] . Zhu et al. represented that influenza A (H3N2) virus-induced autophagy in the A549 and Ana-1 infected cells via suppressing the mTOR signaling pathway. Baicalin could increase mTOR phosphorylation and rescued H3N2 virus effects in a dose-dependent manner [112] . In another study, baicalin was found to exert anti-influenza virus (H1N1-pdm09) activity by downregulation of the PI3k/Akt pathway caused through modulating viral protein NS1 expression [55] . Besides, biochanin A, an O-methylated isoflavone, indicated protective effects on H5N1 influenza A virus-infected cells via decreasing AKT, ERK1/2, JNK, and p38 phosphorylation. It could also modulate cellular signaling pathways, decrease IL-6, IL-8, CXCL10 (IP-10), TNF-α, and improved IκB levels [53] .

CST represented inhibitory effects on the in vitro model of influenza A virus infection through inhibition of the NF-κB/p65 signal pathway, resulting in the downregulation of pro-inflammatory cytokines. CST also decreased phospho-p38 MAPK and phospho-JNK levels [59] . In another study by Ding et al., administration of hesperidin at the daily doses of 200 and 500 mg/kg for five days could inhibit pulmonary inflammation in influenza A virus (H1N1)-induced lung injury in rats. This effect was mediated via attenuating pro-inflammatory cytokine production, including IL-6 and TNF-α. Hesperidin also decreased IL-6 and TNF-α expression in H1N1 infected pulmonary microvascular endothelial cells through inhibition of MAPK signaling pathways [94] . Further, studies suggested ERK signaling pathway as a main modulator of the MAPK signaling pathway. Isorhamnetin (50 µM), a monomethoxyflavone, decreased ERK phosphorylation in MDCK cells after influenza A (H1N1) virus infection [113] . Jeong and colleagues investigated the cytotoxic effects of oroxylin A and tectorigenin in the CHME5 cells and primary human macrophages infected with HIV-1-D3. These flavonoids exert their effects via reducing the phosphorylation of PI3K, Akt, m-TOR, PDK1, GSK-3β, and Bad in the lipopolysaccharide/cycloheximide treated cells santin suppressed influenza A virus replication in the MDCK and THP-1 infected cells [114] . At the concentration of 60 µM, santin attenuated phosphorylation of p38 MAPK, ERK, JNK/SAPK, and NF-κB [61] .

Flavonoids as a class of safe and abundant phytoconstituents have attracted a lot of attention regarding their beneficial effects in COVID-19, and several attempts have been made to assess the structure-activity relationship of these compounds against SARS-CoV-2 proteins [115, 116] . This paper reviewed the potential antiviral mechanisms of flavonoids based on the in vitro and in vivo studies on different viruses that follow the same pathogenic mechanisms as SARS-CoV-2, including HIV, influenza virus, ebola virus, SARS, and MERS. Available data on all virus and host targets were included in this study. Figures 1 and 2 provide an overview of the direct and indirect mechanisms of flavonoids.

2 proteins [115, 116] . This paper reviewed the potential antiviral mechanisms of flavonoids based on the in vitro and in vivo studies on different viruses that follow the same pathogenic mechanisms as SARS-CoV-2, including HIV, influenza virus, ebola virus, SARS, and MERS. Available data on all virus and host targets were included in this study. Amongst direct antiviral mechanisms, inhibition of viral proteases are the most frequently reported property of flavonoids. Due to the high similarity of SARS-CoV-2 proteases to those of SARS, flavonoids with inhibitory effects on these enzymes, such as isoliquiritigenin, kaempferol, and its derivatives, quercetin and its derivatives, theaflavins, flavonoids derived from Angelica keiskei (Miq.) Koidz. and Broussonetia papyrifera (L.) L'Hér. ex Vent. can be considered as candidates for future antiviral assessments against SARS-CoV-2 (Table 1) . On the other hand, modulation of inflammatory host responses to Amongst direct antiviral mechanisms, inhibition of viral proteases are the most frequently reported property of flavonoids. Due to the high similarity of SARS-CoV-2 proteases to those of SARS, flavonoids with inhibitory effects on these enzymes, such as isoliquiritigenin, kaempferol, and its derivatives, quercetin and its derivatives, theaflavins, flavonoids derived from Angelica keiskei (Miq.) Koidz. and Broussonetia papyrifera (L.) L'Hér. ex Vent. can be considered as candidates for future antiviral assessments against SARS-CoV-2 (Table 1) . On the other hand, modulation of inflammatory host responses to the viral infections by the flavonoids seems to be the most important mechanism by which the complications of viral infection are managed. Baicalin and baicalein, biochanin A, cirsimaritin, gallocatechin-7-gallate, and hesperidin are flavonoids with modulating effects on both TNF-α and ILs and thus, can regulate severe conditions due to malfunction of host immune system such as cytokine storm.

According to the current literature, theaflavins, quercetin, luteolin, myricetin, kaempferol, catechins, hesperidin, and baicalin were the most promising flavonoids against the aforementioned viruses. Regarding the herbal sources of flavonoids, the most studied plants were Camellia sinensis (L.) Kuntze (tea) and Scutellaria baicalensis Georgi (skullcap). Green tea is a rich source of catechins, whereas black tea mostly contains theaflavins. Flavonoids from both types of tea have shown direct antiviral properties. Since tea is a popular drink in the human diet, it can be suggested as a safe dietary intervention for COVID-19 patients with mild to moderate symptoms. Due to its acceptable safety profile, tea can also be introduced as a suitable candidate for investigation in future clinical trials. Skullcap is a medicinal plant mostly used in Chinese medicine and is the natural source of baicalin, baicalein, oroxylin A, and wogonin. These flavonoids have demonstrated significant effects on the immune response of infected cells and animals via modulation of IFNs, endogenous antioxidant defense mechanisms, and inflammatory responses, as well as direct antiviral properties.

Some of the flavonoids reviewed in this study, such as cirsimaritin were shown to have antiviral activity higher than standard chemically synthesized drugs like ribavirin [59] . It should be mentioned that the results of in vitro antiviral studies do not necessarily guarantee the same potency and efficacy in clinical settings; though, they can be considered as a screening method to select the most effective compounds amongst numerous candidates for further in vivo and mechanistic evaluations. As previously mentioned, oseltamivir which is an anti-influenza agent has been designed and synthesized using shikimic acid, a plant-derived compound; thus, the introduced flavonoids in this review can be used as molecular backbones for the design and development of novel semisynthetic medicines with better bioavailability and clinical efficacy.

Despite hundreds of flavonoids evaluated against SARS-CoV-2 through virtual screenings, the experimental evidence on the in vitro or in vivo antiviral effect of these compounds against this exact type of virus is limited. Amongst the included flavonoids in our review, only four compounds, including baicalin, baicalein, quercetin, and isorhamnetin, were experimentally assessed in SARS-CoV-2-infected cells or animals.

Previous in silico studies and molecular analysis of different CoVs showed the potential antiviral effects of phytochemicals at different stages of viral biogenesis, including binding to ACE2, surface gangliosides, RdRp, viral spike protein, and viral protease in host cells and paved the way for more clinical and experimental studies [9, [117] [118] [119] [120] [121] [122] [123] . Nevertheless, it should be considered that an acceptable antiviral activity in virtual screenings does not necessarily guarantee in vivo antiviral activity, and that is why an overview of flavonoids with antiviral properties in experimental studies is a further step toward the selection of natural antiviral agents. On the other hand, several of the mechanisms suggested for antiviral flavonoids in virtual screenings are not yet experimentally evaluated. In vitro and in vivo evidence discussed in this review, together with the results of virtual screenings, provides a better overview of the proper compounds for further investigations.

Additionally, there are some recently-published review articles that have focused on the effect of flavonoids on one specific target (e.g., ACE-2) or clinical manifestation (cytokine storms or lung injury) of SARS-CoV-2 infection [124] [125] [126] [127] . Such points of view can put a focus on the development of natural medicines against one specific viral target; however, we preferred a more general approach in our study. We considered no limitation for antiviral/symptoms relieving mechanisms of flavonoids, and all experimental evidence of flavonoids on the above-mentioned viruses were included.

In conclusion, flavonoids can be considered as promising plant-derived compounds to manage SARS-CoV-2 infection via direct antiviral properties or management of host immune response to viral infection. Future experimental mechanistic and clinical studies are needed to further clarify the role of these compounds in primary and secondary prevention of SARS-CoV-2 infection.

Electronic databases, including PubMed, Scopus, and Web of Science, were searched from inception until April 2021 with the following formula: (COVID-19 OR SARS OR MERS OR corona OR HIV OR ebola OR influenza (title/abstract)) AND (plant OR extract OR herb OR phytochemical OR flavonoid (all fields)). As a supplementary search, the names of popular flavonoids including catechin, quercetin, rutin, hesperidin, hesperetin, naringenin, naringin, baicalin, bailaein, and epigallocatechin gallate (EGCG) were also individually searched in order to collect all related papers. After excluding duplicates, primary retrieved results were screened by two independent investigators based on the title and abstract. Selected papers were then checked based on their full text. Inclusion criteria were any in vitro or in vivo study in which the antiviral effect and mechanism of a flavonoid were evaluated. Studies on phytochemicals other than flavonoids, antiviral assessments of flavonoids without clarifying the mechanisms, and studies with non-English full-texts were excluded from our review. In silico studies were excluded unless coupled with an in vitro/in vivo experiment. We also did not discuss antiviral mechanisms such as inhibition of hemagglutinin and neuraminidase of influenza virus since these proteins are not mutual with SARS-CoV-2 and cannot be extrapolated to this virus. Those studies included in the final article are summarized in Table 1 . 

Review on the potential action mechanisms of Chinese medicines in treating Coronavirus Disease 2019 (COVID-19)

Country Quarantine During COVID-19: Critical or Not?

Epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus-2 (SARS-CoV-2

Coronavirus Disease-19)

A review of coronavirus disease-2019 (COVID-19)

The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China

The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex

Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter

Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding

Applying computer simulations in battling with COVID-19, using pre-analyzed molecular and chemical data to face the pandemic

Genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Compassionate Use of Remdesivir for Patients with Severe Covid-19

Experimental treatment with favipiravir for Ebola virus disease (the JIKI Trial): A historically controlled, single-arm proof-of-concept trial in Guinea

Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase

Pharmacokinetics and Concerns about Clinical Trials for 2019-nCoV Infection

Remdesivir, lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro

A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19

Antiviral therapy in management of COVID-19: A systematic review on current evidence

The preventive and therapeutic potential of natural polyphenols on influenza

An Evaluation of Traditional Persian Medicine for the Management of SARS-CoV-2

How Can Persian Medicine (Traditional Iranian Medicine) Be Effective to Control COVID-19? Tradit

Clinically Improvement in a Case of COVID-19 with Gastrointestinal Manifestations through Using Natural Therapy based on Persian Medicine: A Case Report

COVID 19: Natural Products and Traditional Medicines; Opportunity or Threat? Tradit

Current therapeutic options for coronavirus disease 2019 (COVID-19)-lessons learned from severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) therapy: A systematic review protocol

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 M(pro) enzyme through in silico approach

Identification of phytochemical inhibitors against main protease of COVID-19 using molecular modeling approaches

Docking study of flavonoid derivatives as potent inhibitors of influenza H1N1 virus neuraminidase

Chemistry and biological activities of flavonoids: An overview

Kaempferol: An encouraging flavonoid for COVID-19

Natural Bioactive Compounds from Fungi as Potential Candidates for Protease Inhibitors and Immunomodulators to Apply for Coronaviruses

Global landscape of HIV-human protein complexes

An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy

Characterization and inhibition of SARS-coronavirus main protease

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti-COVID-19 drug design

Improving Viral Protease Inhibitors to Counter Drug Resistance

In silico and in vitro evaluation of kaempferol as a potential inhibitor of the SARS-CoV-2 main protease (3CLpro)

Kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus

Evaluation of polyphenols from Broussonetia papyrifera as coronavirus protease inhibitors

Molecular mechanisms of action of epigallocatechin gallate in cancer: Recent trends and advancement

Antibacterial and antifungal activities of new acylated derivatives of epigallocatechin gallate

Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)

Phytochemical and Pharmacological Role of Liquiritigenin and Isoliquiritigenin From Radix Glycyrrhizae in Human Health and Disease Models. Front

Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins

Inhibition of SARS-CoV 3C-like protease activity by theaflavin-3,3 -digallate (TF3)

Prenylisoflavonoids from Erythrina senegalensis as novel HIV-1 protease inhibitors

Inhibitory activity of flavonoids and tannins against HIV-1 protease

A Comprehensive Superposition of Viral Polymerase Structures

Tentative identification of RNA-dependent RNA polymerases of dsRNA viruses and their relationship to positive strand RNA viral polymerases

Tinkering with translation: Protein synthesis in virus-infected cells

Baicalein and Baicalin Inhibit SARS-CoV-2 RNA-Dependent-RNA Polymerase. Microorganisms

Probing the impact of quercetin-7-O-glucoside on influenza virus replication influence

Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1

Oroxylin A suppresses influenza A virus replication correlating with neuraminidase inhibition and induction of IFNs

Differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin A and baicalein in H5N1 influenza A virus-infected cells

Baicalin inhibits influenza virus A replication via activation of type I IFN signaling by reducing miR-146a

Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses

Drug Discovery of Host CLK1 Inhibitors for Influenza Treatment

Influenza M2 proton channels

Anti-influenza effect and action mechanisms of the chemical constituent gallocatechin-7-gallate from Pithecellobium clypearia Benth

Cirsimaritin inhibits influenza A virus replication by downregulating the NF-kappa B signal transduction pathway

Luteolin decreases the yield of influenza A virus in vitro by interfering with the coat protein I complex expression

Santin inhibits influenza A virus replication through regulating MAPKs and NF-kappaB pathways

Quercetin as an Antiviral Agent Inhibits Influenza A Virus (IAV) Entry. Viruses

Anti-influenza virus activity of tricin, 4',5,7-trihydroxy-3',5'-dimethoxyflavone

Human host factors required for influenza virus replication

Potential antiviral activity of isorhamnetin against SARS-CoV-2 spike pseudotyped virus in vitro

The comprehensive study on the therapeutic effects of baicalein for the treatment of COVID-19 in vivo and in vitro

Antiviral phenolics from the leaves of Cleistocalyx operculatus

The protective effect of 3-deoxysappanchalcone on in vitro influenza virus-induced apoptosis and inflammation

Caspase 3 activation is essential for efficient influenza virus propagation

N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus

Antiviral activity of baicalin against influenza A (H1N1/H3N2) virus in cell culture and in mice and its inhibition of neuraminidase

NF-kappaB and virus infection: Who controls whom

NF-kappaB signaling differentially regulates influenza virus RNA synthesis

Naringenin is a powerful inhibitor of SARS-CoV-2 infection in vitro

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent

The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages

Flavonol 7-O-Glucoside Herbacitrin Inhibits HIV-1 Replication through Simultaneous Integrase and Reverse Transcriptase Inhibition

Sesquiterpenes and butenolides, natural anti-HIV constituents from Litsea verticillata

Inhibition of influenza virus replication by plant-derived isoquercetin

Inhibitory effects of quercetin 3-rhamnoside on influenza A virus replication

Prophylactic Efficacy of Quercetin 3-beta-O-D-Glucoside against Ebola Virus Infection

Dynamics of Immune Activation in Viral Diseases

Interferon-inducible antiviral effectors

Molecular mechanisms of IFN-γ to up-regulate MHC class I antigen processing and presentation

Potential role of interferons in treating COVID-19 patients

Interferons: Mechanisms of action and clinical applications

Current treatment options and the role of peptides as potential therapeutic components for Middle East respiratory syndrome (MERS): A review

Ribavirin and interferon-β synergistically inhibit SARSassociated coronavirus replication in animal and human cell lines

Human type I interferon antiviral effects in respiratory and reemerging viral infections

Dysregulation of type I interferon responses in COVID-19

Chromosomes; Cancer, Functional mechanisms of miR-192 family in cancer

MicroRNAs in the anticancer effects of celecoxib: A systematic review

Hesperidin attenuates influenza A virus (H1N1) induced lung injury in rats through its antiinflammatory effect

a flavonoid isolated from Scutellaria baicalensis, has anti-viral activities against influenza infection via modulation of AMPK pathways

Accessory proteins of SARS-CoV and other coronaviruses

SARS-CoV-2 infection: The role of cytokines in COVID-19 disease

Imbalanced host response to SARS-CoV-2 drives development of COVID-19

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China

COVID-19 cytokine storm: The interplay between inflammation and coagulation

Targeting cytokine storm to manage patients with COVID-19: A mini-review

Cytokine storm and COVID-19: A chronicle of pro-inflammatory cytokines

COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death

Suppression of R5-type of HIV-1 in CD4(+) NKT cells by Vdelta1(+) T cells activated by flavonoid glycosides, hesperidin and linarin

Studies on the bioactive flavonoids isolated from Pithecellobium clypearia Benth. Molecules

Immunomodulatory properties of quercetin-3-O-alpha-L-rhamnopyranoside from Rapanea melanophloeos against influenza a virus

Influenza A virus-induced release of interleukin-10 inhibits the anti-microbial activities of invariant natural killer T cells during invasive pneumococcal superinfection

In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives

Innate immune recognition

Double-stranded RNA sensors and modulators in innate immunity

8-Prenylkaempferol Suppresses Influenza A Virus-Induced RANTES Production in A549 Cells via Blocking PI3K-Mediated Transcriptional Activation of NF-kappaB and IRF3

Baicalin inhibits autophagy induced by influenza A virus H3N2

Antiviral effect of methylated flavonol isorhamnetin against influenza

7-Dihydroxy-6-Methoxy-Flavonoids Eliminate HIV-1 D3-transfected Cytoprotective Macrophages by Inhibiting the PI3K/Akt Signaling Pathway

Flavonoids: A complementary approach to conventional therapy of COVID-19?

Roles of flavonoids against coronavirus infection

Inhibition of SARS-CoV 3CL protease by flavonoids

Characteristics of flavonoids as potent MERS-CoV 3C-like protease inhibitors

Evaluation of flavonoids as 2019-nCoV cell entry inhibitor through molecular docking and pharmacological analysis

In silico study of some natural flavonoids as potential agents against COVID-19: Preliminary results

Lead Finding from Selected Flavonoids with Antiviral (SARS-CoV-2) Potentials against COVID-19: An in-silico Evaluation

In silico evaluation of flavonoids as effective antiviral agents on the spike glycoprotein of SARS-CoV-2

Investigation of the inhibitory activity of some dietary bioactive flavonoids against SARS-CoV-2 using molecular dynamics simulations and MM-PBSA calculations

Flavonoids as potential phytotherapeutics to combat cytokine storm in SARS-CoV-2

Flavonoids against the SARS-CoV-2 induced inflammatory storm

Natural flavonoids as potential angiotensinconverting enzyme 2 inhibitors for anti-SARS-CoV-2

New perspectives on natural flavonoids on COVID-19-induced lung injuries

Dual HIV-1 reverse transcriptase and integrase inhibitors from Limonium morisianum Arrigoni, an endemic species of Sardinia (Italy)

In vitro anti-influenza viral activities of constituents from Caesalpinia sappan

Anti-influenza virus activity of biflavonoids

Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase

a Biflavonoid from Anacardium occidentale L. Inhibits Influenza Virus Neuraminidase

A new anti-HIV flavonoid from glucoronide from Chrysanthemum marifolium

Flavonoid baicalin inhibits HIV-1 infection at the level of viral entry

Catechin inhibiting the H1N1 influenza virus associated with the regulation of autophagy

Antiviral effect of catechins in green tea on influenza virus

Catechin inhibition of influenza neuraminidase and its molecular basis with mass spectrometry

The evaluation of catechins that contain a galloyl moiety as potential HIV-1 integrase inhibitors

Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase

Inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate

Preclinical development of the green tea catechin, epigallocatechin gallate, as an HIV-1 therapy

Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy

Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding

C-Methylated Flavonoid Glycosides from Pentarhizidium orientale Rhizomes and Their Inhibitory Effects on the H1N1 Influenza Virus

Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris

Genistein as Antiviral Drug against HIV Ion Channel

Methoxyflavones from Marcetia taxifolia as HIV-1 Reverse Transcriptase Inhibitors

Antiviral activities of compounds from aerial parts of Salvia plebeia R. Br

A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate-and Co-Receptor-Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

A flavonoid, luteolin, cripples HIV-1 by abrogation of tat function

HIV-1 integrase inhibitory substances from Coleus parvifolius

Anti-HIV-1 activity of flavonoid myricetin on HIV-1 infection in a dual-chamber in vitro model

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro

Glycosylated Flavonoids from Psidium guineense as Major Inhibitors of HIV-1 Replication in vitro

Pongamone A-E, five flavonoids from the stems of a mangrove plant, Pongamia pinnata

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Flavanone and flavonol glycosides from the leaves of Thevetia peruviana and their HIV-1 reverse transcriptase and HIV-1 integrase inhibitory activities

Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora

Structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities

Anti-influenza virus activities of flavonoids from the medicinal plant Elsholtzia rugulosa

Anti HIV-1 flavonoid glycosides from Ochna integerrima

Antiviral Effect of Flavonol Glycosides Isolated from the Leaf of Zanthoxylum piperitum on Influenza Virus

Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities

Anti-cholinergic, cytotoxic and anti-HIV-1 activities of sesquiterpenes and a flavonoid glycoside from the aerial parts of Polygonum viscosum

The anti-HIV-1 effect of scutellarin

Funding: This research received no external funding.Institutional Review Board Statement: Not applicable.

Data Availability Statement: Not applicable.

The authors declare no conflict of interest.