Anticoronavirus and Immunomodulatory Phenolic Compounds: Opportunities and Pharmacotherapeutic Perspectives

In 2019, COVID-19 emerged as a severe respiratory disease that is caused by the novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). The disease has been associated with high mortality rate, especially in patients with comorbidities such as diabetes, cardiovascular and kidney diseases. This could be attributed to dysregulated immune responses and severe systemic inflammation in COVID-19 patients. The use of effective antiviral drugs against SARS-CoV-2 and modulation of the immune responses could be a potential therapeutic strategy for COVID-19. Studies have shown that natural phenolic compounds have several pharmacological properties, including anticoronavirus and immunomodulatory activities. Therefore, this review discusses the dual action of these natural products from the perspective of applicability at COVID-19.


Introduction
Coronaviruses (CoVs) are positive single-stranded (+ss) RNA viruses belonging to family Coronaviridae [1]. A large number of CoVs have been discovered as the causative agents of diseases in animals and humans [2]. Seven human CoVs (HCoVs) were discovered to date and they have all been linked to respiratory diseases. Four HCoVs cause mild diseases; whereas three HCoVs are the causative agents of severe respiratory diseases [3,4]. Of those three HCoVs, Severe Acute Respiratory Syndrome-CoV (SARS-CoV) was the first discovered in 2002-2003, followed by Middle East Respiratory Syndrome-CoV (MERS-CoV) in 2012, and finally the causative agent of the current COVID-19 pandemic; SARS-CoV-2 in 2019 [3,4]. SARS-CoV-2 was first discovered in patients that were linked to Huanan Seafood Market in Wuhan, China [4]. Since its emergence according to World Health Organization (WHO), millions of COVID-19 cases have been reported worldwide with over 4 million deaths. The severity of the diseases associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 and the high fatality rates have prompted several research groups to develop effective antivirals against coronaviruses. Natural products have shown antiviral activities against several viruses including coronaviruses [5]. Of these natural products, phenolic compounds have shown a wide range of pharmacological activities [6].
Phenolic compounds are chemically characterized by having at least one aromatic rings attached to one or more hydroxyl substituent, and more than 8000 phenolic compounds have already been identified in plants [7]. Several plant families contain phenolic

Flavonoids and Other Natural Phenolic Compounds as Inhibitors of SARS-CoV-2
Since the emergence of SARS-CoV-2 in December 2019, several studies have focused on repurposing drugs that have been used for other health conditions including drugs with reported anti-SARS-CoV activity. In line with that, flavonoids that inhibited SARS-CoV were tested against SARS-CoV-2. In one study, quercetin and EGCG were shown to interact with and inhibit SARS-CoV-2 3CL pro activity in a FRET-based enzymatic assay [34,35]. Moreover, EGCG inhibited the entry of SARS-CoV-2-pseudotyped virus and live SARS-CoV-2 into HEK293T-hACE2 and Vero cells respectively [36]. Another study has demonstrated that EGCG inhibited the endoribonuclease enzymatic activity of SARS-CoV-2 nonstructural protein-15 (Nsp15) with an IC 50 of 1.62 µM, while blocking viral replication in Vero cells with an IC 50 , 0.2 µM [37]. The low IC 50 of ECGC indicates its potency and warrants its further development as a potential SARS-CoV-2 antiviral. GCG was also found to inhibit the binding of SARS-CoV-2 N protein to viral RNA inhibiting viral replication in A549-hACE2 with an IC 50 , 44.4 µM [38]. As shown previously with SARS-CoV 3CL pro , herbacetin and pectolinarin inhibited SARS-CoV-2 3CL pro with an IC 50 of 53.90 and 51.64 µM, respectively [28,39]. However, rhoifolin exhibited weaker inhibition, whereas baicalin showed stronger inhibition of SARS-CoV-2 3CL pro than that observed for SARS-CoV 3CL pro . These differences in inhibition of 3CL pro by rhoifolin and baicalin may be attributed to the slight differences in the amino acid sequence since the two 3CL pro have 96% sequence identity. A study has also demonstrated potent inhibition of recombinant SARS-CoV-2 3CL pro by myricetin, which suggests that myricetin could be further tested and developed as a potential SARS-CoV-2 antiviral [40].
A recent study that screened for inhibitors of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, identified the flavonoids rutin, quercetin, and tamarixetin as inhibitors of ACE2 activity [41]. However, the most potent of all flavonoids tested was quercetin with an IC 50 of 4.48 µM.

Immune Response to SARS-CoV-2
Immune responses to viral infections are essential to control viral replication, kill infected cells and induce protective immunity against virus [65,66]. Following infection, viral nucleic acid and viral proteins are detected by patter recognition receptors (PRRs), such as Toll-like receptors (TLRs) on immune cells and other cells [65]. The recognition of viral proteins and nucleic acid results in production of inflammatory cytokines, chemokines and adhesion molecules by immune tissue resident cells, such as macrophages [67]. Although appropriate levels of proinflammatory cytokines are required to activate immune cells involved in viral control, extremely high levels of IL-1β, IL-10, G-CSF, GM-CSF, IFN-γ and TNF-α were detected in COVID-19 patients [68]. Moreover, disease severity positively correlated with increased IL-6 levels [69].
Type I interferon (IFN) is required to activate cellular antiviral mechanisms to suppress viral replication and virion assembly [70]. Severe COVID-19 patients demonstrated decreased type I IFN response and exacerbated inflammation [71]. Indeed, autoantibodies against type I IFN were detected in severe COVID-19 pneumoniae [72] and mutations in genes related to type I IFN immunity were also detected in critical patients [73]. Besides the host defects in type I IFN immunity, a study showed that SARS-CoV-2 ORF6 protein inhibited type I interferon production and signaling pathway [74]. In addition, natural killer (NK) cell numbers were reduced in blood of severe COVID-19 patients [75]. Therefore, impaired innate immune responses and increased production of proinflammatory cytokines may contribute to disease severity and worse outcomes in COVID-19 patients.
Natural products with immunomodulatory activities are worth investigation as promising therapeutics for COVID- 19. In addition to antiviral activity, many natural products have antifungal and antibacterial activities, which are interesting since coinfections have been reported in severe SARS-CoV-2 pneumonia patients [84]. Anti-inflammatory, antiapoptotic, antioxidant, and immunomodulatory activities have been described for natural compounds [85][86][87][88]. Indeed, natural compounds capable of reducing inflammation without compromising host immunity would be beneficial for treatment of severe COVID-19 [80]. Herein, we review the immunomodulatory activities of natural phenolic compounds, including flavonoids, that possess anti-SARS-CoV, anti-MERS-CoV, and anti-SARS-CoV-2 activities.

Immunostimulatory Activities of Natural Phenolic Compounds
A variety of natural compounds have shown anti-inflammatory and antioxidant activities in addition to immunomodulatory activities that are reported in different experimental models [87][88][89] (Table 2). Apigenin and luteolin at 10 µM induced activation of NK and CD8 + T cells (CTLs) in vitro, and enhanced the proliferation of splenocytes stimulated with lipopolysaccharide (LPS) [90].
EGCG enhanced the antiviral state in Huh7 cells, a hepatoma cell line, infected with hepatitis C virus (HCV) [91]. Treatment of HCV-infected Huh7 cells with 10 µM of EGCG enhanced polyinosinic-polycytidylic acid (Poly I:C) induced expression of IFN-stimulated genes (ISGs), increased TLR3 and IFN-λ1 expression, and decreased viral replication [91]. Indeed, pretreatment of Huh7 cells with 10 µM epigallocatechin gallate followed by HCV dsRNAs enhanced antiviral defense that is mediated by interferon-λ1 (IFN-λ1), TLR3, RNA-sensing retinoic acid-inducible gene I (RIG-I) and IFN-stimulated gene (ISG) expression [92]. In a murine leukemia model, oral treatment with 87.26 µmol/kg of EGCG induced T and B cell proliferation and NK cell activity [93]. Furthermore, EGCG (50 µM) increased macrophage receptor with collagenous structure (MARCO) expression and improved macrophage phagocytosis of Streptococcus pneumoniae [94]. Ex vivo experiments using cells from mice orally treated, every day for 6 weeks, with 1000 mg/kg of EGCG fraction of green tea extract demonstrated enhanced innate and adaptive immune responses such as NK cytolysis, peritoneal cells phagocytosis, splenocyte proliferation, and IL-2 and IFN-γ production [95].
Resveratrol at 25 µM inhibited influenza virus replication through activation of TLR9/MyD88/IRF7 pathway in A549 infected cells, and enhancing IFN-β production [97]. Also, pre-treatment of RAW 264.7 cells with 100 µg/mL of aqueous extract of Eupatorium fortune demonstrated antiviral activity against influenza A virus infection by enhancing production of type I IFN. Indeed, quercetin was identified as one of the active antiviral and immunomodulatory compounds of the extract [100]. This was confirmed by a study which showed that pre-treatment with 3.0 µg/mL quercetin inhibited influenza virus replication in RAW 264.7 cells and increased IFN-β production [101]. Moreover, quercetin dose dependently decreased nontypeable Haemophilus influenzae (NTHi) bacterial viability in vitro, reduced production of proinflammatory markers in the lungs of infected mice that were pre-treated with 60 mg/kg for 8 days and for 24h postinfection, and decreased mortality of NTHi-infected zebrafish that were intraperitoneally treated with 0.3 mg/g of quercetin at 29 and 53h postinfection [102].
Hesperetin is another flavonoid with antioxidant, anti-inflammatory, anticancer and antimicrobial activity [103,104]. It has been shown that hesperetin (25 µM) activated host cellular and humoral responses [105], enhanced LPS-mediated in vitro proliferation of splenocytes, and potentiated killing activity of NK and CTLs [105]. Moreover, hesperetin activated antigen presenting cells (APCs), enhanced CTL response, and antitumor immunity when used as an adjuvant at 2.65 µmol/mouse in combination with inactivated B16F10 melanoma cells vaccine which prolonged the survival of tumor-bearing mice [106].

Effects of Natural Phenolic Compounds on NF-κB Pathway and Inflammation
It has been established that activated nuclear factor-κB (NF-κB) translocates to the nucleus and induces the transcription of genes involved in inflammation, apoptosis, cell proliferation, survival, and differentiation [107]. Since NF-κB drives the expression of cytokines and others inflammatory mediators involved in COVID-19 hyperinflammatory state, targeting NF-κB pathway has been proposed to ameliorate severe inflammation in COVID-19 [108,109].
Monocyte-derived macrophages are involved in lung and multiorgan inflammation observed in severe COVID-19 patients [110][111][112] which necessitate the investigation of potential natural phenolic compounds that could reduce NF-κB activation and inhibit the production of proinflammatory cytokines and chemokines by macrophages.
Also, THP-1 macrophages that were pre-treated with 40 µM catechin, before infection with Porphyromonas gingivalis, showed downregulation of NF-κB activation, and reduced IL-1β and TNF-α production with no effect on bacterial growth [124].
Studies showed that resveratrol impaired NF-κB activation in different cells including myeloid cells, HeLa, and Jurkat cells that were stimulated with phorbol myristate acetate (PMA), LPS, H 2 O 2 , okadaic acid or ceramides [131]. Indeed, human and murine macrophages stimulated with TNF-α or LPS in the presence of 25 µM of resveratrol showed reduced production of proinflammatory cytokine and chemokine [132]. In human epithelial cells, high concentration of resveratrol (300 µM) inhibited rhinovirus replication and ICAM-1 expression, and decreased basal levels of IL-6 and RANTES in uninfected human epithelia [133].
Studies showed that helichrysetin possessed anti-inflammatory, anti-oxidant and antitumor activities in different cell lines [141,142]. Helichrysetin (50 µM) impaired NF-κB activation in mouse pancreatic β-MIN-6 cells [141], HeLa, and T98G cells [142]. Rheumatoid arthritis fibroblast-like synoviocytes treated for 48h with 10 or 20 µM of pectolinarin showed decreased activation of the phosphatidylinositol 3 kinase/protein kinase B pathway, reduced cell proliferation and decreased production of IL-6, IL-18, NO and PGE 2 [143]. However, in LPS-stimulated RAW 264.7 macrophages, pectolinarin at 1, 10, 25 or 50 µM did not affect COX-2 expression and PGE 2 synthesis [144]. Table 3 summarizes the antiinflammatory activities of phenolic compounds discussed in this study.                                                 LPS-stimulated proliferation of TNF-α, IL-1β, IL-6 and NO production [140] The compounds in the table are in the order in which the compounds are presented in the section Effects of Natural Phenolic Compounds on NF-κB Pathway and Inflammation.

Inhibitory Effects of Natural Phenolic Compounds on NLRP3 Inflammasome
Viral nucleic acids are recognized by PRRs, such as TLR 3,7,8 in the endosomes [145]. Recognition of viral proteins and nucleic acid by PRRs triggers myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling pathways culminating in the activation of interferon-regulatory factor 3/7 (IRF) and NF-κB transcription factors resulting in expression of pro-IL1β and pro-IL-18 [107]. Moreover, activation of cytosolic NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome by pathogens, including viruses, results in activation of caspase-1, and consequently the processing of pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 [146,147]. It is noteworthy to mention that the activation of inflammatory caspases can induce a type of cell death called pyroptosis, which may be involved in exacerbated production of inflammatory cytokines during acute phase of COVID-19 [68,148]. Those events are important in the defense against infectious diseases but could promote inflammation, death and tissue injury.
A study has shown that quercetin, at 100 µM, inhibited caspase-recruitment domain (ASC) oligomerization and NLRP3 inflammasome activation resulting in decreased IL-1β production by in vitro-stimulated macrophages [152]. Moreover, in a Kawasaki disease experimental model, treatment of mice with 100 mg/kg of quercetin prevented vascular inflammation and IL-1β production [152]. It was also found that treatment of macrophages with 25 µM apigenin blocked caspase-1 activation by targeting ASC and impairing NLRP3 inflammasome assembly [123]. Endoplasmic reticulum (ER) stress induced by palmitate in EA.hy926 cells, a hybridoma line derived from human endothelium and A549/8 cells, led to NLRP3 activation, IL-1β production and endothelial cell dysfunction. However, treatment of EA.hy-926 cells with 10 µM of quercetin, luteolin or epigallocatechin gallate reduced reactive oxygen species (ROS) production and thioredoxin-interacting protein (TXNIP) and NLRP3 inflammasome activation, resulting in lower IL-1β expression [126]. Moreover, EGCG (25 µM) reduced nucleus pulposus cell inflammation and cell death, induced by H 2 O 2, by interfering with cGAS/Sting/NLRP3 pathway [157].

Natural Phenolic Compounds in Sepsis and Lung Injury
Sepsis manifestations including cytokine storm, endothelial cell dysfunction, intravascular coagulation, pulmonary, cardiovascular, and renal complications have all been reported in COVID-19 patients [164,165]. Dysregulated immune response and cytokine storm [166], with elevated levels of IL-6, IL-10, and TNF-α, and lymphopenia, correlated with worse outcomes in COVID-19 patients [167]. Therefore, anti-inflammatory and anti-coagulant drugs could be considered to reduce hyperinflammation and incidence of thrombosis, multiple organ failure and death [168].
Experimental models to study sepsis include, for example, cecum ligation and puncture (CLP) and LPS lethal dose, since LPS binds to TLR4 which activates NF-κB and IRF3 pathways inducing the production of proinflammatory cytokines and cellular activation [169,170].
EGCG is among the natural phenolic compounds that inhibit TLR signaling. EGCG inhibited TLR4 signaling and ameliorated acute lung injury in mice infected with H9N2 influenza virus [173]. Resveratrol was also found to impairs TLR4 and TLR3 pathways independent of MyD88 signaling [174]. In addition, resveratrol suppressed RSV replication, IL-6 secretion and TRIF-TBK1 pathway in 9HTEo cells; human epithelium tracheal cells, that are infected and treated with 100 µM of resveratrol [175]. In vivo, resveratrol (30 mg/kg) reduced RSV titer in the lungs of infected mice, and impaired TLR3-TRIF signaling pathway, alleviating airway hyperresponsiveness and inflammation [176].
Amentoflavone and apigenin were shown to reduce inflammation in sepsis models. In CLP-induced sepsis, amentoflavone treatment (50 mg/kg) protected rats from acute lung injury by decreasing TNF-α and IL-1β levels, impairing NF-κB activity and reducing oxidative stress in the lung tissue [177]. Mice treated with 50 mg/kg of apigenin, 3h before receiving a lethal dose of LPS, showed enhanced survival with decreased lung cell death, and reduced TNF-α production and neutrophil infiltration into the lung tissue. In addition, cardiac function and heart mitochondrial complex I activity were restored in these mice [178].
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are also described in COVID-19 patients, and correlated with worse outcome and higher mortality [179]. Hesperetin demonstrated the ability to suppress inflammatory cytokines production, inflammatory cell infiltration into the lung tissue, and reduced myeloperoxidase and LDH activities in different models of ALI [180][181][182].
Besides the importance of neutrophils in early responses to infections, they can damage tissues and are also involved in sepsis-induced tissue injury [183]. It has been shown that neutrophils accumulate in lungs of severe COVID-19 patients [184]. Neutrophilextracellular traps (NETs) were detected in high levels in the plasma and lung tissues of COVID-19 patients [185], indicating that neutrophils activation is detrimental in COVID-19 patients. A study has shown that luteolin (30 µM) inhibited oxidative stress, and reduced NETs formation in human neutrophils that were activated with PMA [186]. Amentoflavone impaired oxidative burst in human neutrophils stimulated with PMA and protected human erythrocytes from oxidative hemolysis. These effects were explained by the ability of amentoflavone to inhibit NADPH oxidase and ROS production in human neutrophils and to prevent membrane damage and lipid peroxidation in human erythrocytes [187]. However, more studies are needed to further understand the mechanism by which amentoflavone inhibit neutrophil oxidative burst and erythrocyte lysis.

Natural Phenolic Compounds in Extrapulmonary Complications of COVID-19
Neurologic symptoms have been described in COVID-19 patients, including anosmia, ageusia, encephalopathy, seizures, encephalitis, stroke, and cognitive disturbance [188,189]. SARS-CoV-2 have been shown to infect neurons and damage the central nervous system (CNS) [190,191]. The detection of low or no viral copies in the brain tissue has been described in a number of COVID-19 cases with neurologic complications [192], and it remains unclear whether the CNS complications are caused by direct infection or inflammation. Seizures are among the neurologic complications that have been reported during and after recovery from SARS-CoV-2 infection [193][194][195]. Brain inflammation, genetic factors, developmental dysfunction, environmental risk and neurological insults are involved in epileptogenesis and seizures susceptibility [196]. Amentoflavone has been described as neuroprotective in experimental models of epilepsy. Amentoflavone suppressed NF-κB activation, decreased production of NO, PGE 2 , IL-1β, and IL-6, prevented hippocampus neurons apoptosis, and decreased epileptic seizures in pilocarpine-treated mice [197]. Moreover, amentoflavone blocked apoptosis, impaired NLRP3 inflammasome activation, and decreased production of IL-18, IL-1β, and TNF-α in brains of pentylenetetrazole-induced kindling mice [151].
The anti-inflammatory, anti-oxidant and anti-apoptotic effects of hesperetin have been related to its ability to protect neuronal [198,199], cardiac [200] and renal tissues [201] in different injury models. Hesperetin ameliorated neuroinflammation, memory, and impaired neuronal apoptosis in vivo [198]. Hesperetin interfered with the TLR4-NF-κB signaling pathway. Accordingly, mice treated with LPS and hesperetin (50 mg/kg) showed decreased brain levels of p-NF-κB, IL-1β and TNF-α compared to mice that received only LPS. The anti-inflammatory and cytoprotective effects of hesperetin were also confirmed in vitro using BV-2, and HT-22 mouse hippocampal neuronal cell line [198].
Acute myocarditis is one of the extrapulmonary complications in COVID-19 patients [202], and is associated with inflammatory cell infiltration into the heart tissue [203]. It has been shown that apigenin prevented myocarditis in an experimental model of autoimmune myocarditis. Treatment with 200 mg/Kg (gavage) of apigenin reduced inflammatory cell infiltration into the heart, decreased TNF-α, IL-2 and IFN-γ, and ameliorated cardiac dysfunction compared to untreated mice [204]. Anti-apoptotic effects of 25 µM of hesperetin was also demonstrated in in vitro LPS-treated H9C2 cardiomyocytes [205], and in a myocardial infarction (MI) model in vivo. Indeed MI mice, treated with 30 mg/kg/day of hesperetin for 8 weeks, showed impaired NF-κB activation, reduced cardiac fibrosis and inflammation compared to untreated MI-mice [200].
Kawasaki-like disease (KD) was also described in COVID-19 pediatric patients [206]. Proinflammatory cytokines are related to hyperinflammation, vasculitis and coronary artery damage in KD patients. Increased TNF-α and IL-1β levels in KD patients result in endothelial cell activation and expression of adhesion molecules which leads to leukocyte adherence and endothelial injury, promoting vasculitis and coronary artery aneurysms [207]. Human coronary arterial endothelial cells, activated with 10 ng/mL of TNF-α, showed enhanced VCAM-1 and ICAM-1 expression, oxidative stress and proinflammatory cytokines production. However, in the presence of 10 µM of resveratrol, expression of ICAM-1, iNOS, and IL-1β were reduced which indicate that resveratrol has anti-inflammatory actions on coronary arterial cells and could be promising in treatment of KD patients [208]. Moreover, quercetin treatment (50 mg/kg) prevented cardiac injury, inflammation and oxidative stress in the heart of streptozocin (STZ) and nicotinamide-induced diabetic rats [209]. Luteolin (10 µM) protected H9C2 cardiomyocytes from inflammation and oxidative stress induced by high glucose concentration. Additionally, reduced inflammation was observed in the heart of STZ-diabetic mice that were treated with 20 mg/kg of luteolin for 15 weeks [210]. Figure 3 illustrates the main mechanisms of the immunomodulatory actions of phenolic compounds discussed in this study. Most of the compounds discussed in this review are found in foods and beverages of natural origin, such as resveratrol, which is commonly present in wine. However, it is not possible to conclude that a diet based on these foods will result in prevention or improvement of the clinical conditions of affected people by COVID-19. Discussion of this possibility requires carrying out comprehensive studies in populations that have an appropriate diet. For example, the high consumption of wine in France may contribute to the low frequency of coronary heart disease, possibly due to the presence of resveratrol in this drink. This evidence represents the French paradox [211]. However, countries with high consumption of wine, such as France and Italy, had a high number of deaths caused by COVID-19 [212,213]. Therefore, studies using standardized methods with these phytoconstituents are needed to advance the knowledge of their therapeutic potential against COVID-19.

Conclusions
Among natural phenolic compounds discussed, we highlighted the antiviral effects of quercetin, luteolin, resveratrol, and amentoflavone against coronaviruses as well as their ability to modulate immune response and inflammatory status in a variety of in vitro and in vivo models. Despite the structural complexity of some bioactive compounds, there are perspectives for the development of synthetic analogues with an anticoronavirus and immunomodulator profile, but structurally simpler and easier to obtain using the phytoconstituents in this review as prototypes. In addition, it is possible to manufacture plant products containing a significant amount of these phenolic compounds and use them as potentially therapeutic agents against COVID-19. So, further experimental studies focusing on anti-SARS-CoV-2 and immunomodulatory activities of these compounds are needed.

Conflicts of Interest:
The authors declare no conflict of interest.