COVID-19: Quercetin has high binding affinity for the spike protein helping to inhibit SARS-CoV-2


Numerous studies have been conducted showing its efficacy not only as an antiviral agent but also as a therapeutic supplement that has  anti-inflammatory and immunomodulatory properties along with tissue and organ protective and repair properties.

One study conducted by scientists from Tianjin Medical University that has been published in the peer reviewed Computational and Structural Biotechnology Journal indicates that Quercetin is one of the many phytochemicals in TCM (Traditional Chinese Medicine) herbs that may inhibit SARS-CoV-2 Virus.

Using computational methods, the study team identified three phytochemicals in traditional Chinese medicine that could be used against SARS-CoV-2: Quercetin, Puerarin and Kaempferol​. 

Significantly of the three compounds, Quercetin showed the highest binding affinity to both the ACE2 receptor and the receptor-binding domain of the SARS-CoV-2 spike protein, and could thus provide a dual synergistic effect.  

It has been known that the SARS-CoV-2 coronavirus infects human hosts by binding with the human angiotensin-converting enzyme 2 (ACE2) receptor on their cells, notably the epithelium lining the respiratory tract. The receptor-binding domain (RBD) of the coronavirus spike protein binds to ACE2 followed by membrane fusion to the host cell, thus allowing the virus to infiltrate the cell and commence replication.

TCM or Traditional Chinese Medicine, widely used for many diseases, showed therapeutic effects during the 2003 SARS-CoV epidemic.

The RBD or receptor-binding domain of the SARS-CoV-2 has significant structural homology with SARS-CoV. Although the use of Chinese herbs with modern medicine has shown benefits in COVID-19 patients, several components are present in the herbs and have complex interactions, making it challenging to uncover the molecular mechanisms responsible for its therapeutic effects.

Many computational studies have helped predict active compounds in the medicinal herbs with the potential to accelerate traditional medicine-based drug discovery.

The Chinese study team used computational analysis to discover potential molecule candidates against SARS-CoV-2 infection. Utilizing the Traditional Chinese Medicine Pharmacology database, they screened for molecules that could target ACE2.

The study team identified the compound puerarin that could target ACE2. Then, they screened for Chinese herbs that have this compound in the database and found five.

Furthermore, sinc e it is thought that compounds in the same herbal medicine have synergistic properties, they expanded their search to include all the compounds in the five herbs to arrive at 41 compounds.

Upon analyzing which compounds were present in the maximum number of herbs however, they found puerarin was present in all the five herbs, and quercetin and kaempferol were present in three herbs.

The team then predicted potential drug targets of the selected compounds using the database, leading to 240 possible targets. Upon further analysis, they selected puerarin, quercetin, and kaempferol for further study.

The researchers next performed molecular docking analysis to determine potential binding sites and binding affinity to ACE2. All the three compounds could bind on the same region of ACE2, which is located some distance from the binding position of SARS-CoV-2. It is likely the compounds are causing changes in conformations rather than competing with the spike protein to bind to ACE2.

Importantly, it was found that Quercetin had the highest binding affinity, forming both strong and weak hydrogen bonds.

The study team also experimentally determined the binding of the three compounds to ACE2 using surface plasmon resonance. Similar to the theoretical analysis, they found quercetin had higher binding affinity to ACE2 than puerarin.

They also observed that puerarin affected the binding of spike protein to ACE2, and quercetin almost completely disrupted the spike protein binding to ACE2.

Quercetin could bind to RBD domain of S-protein with a high binding affinity. (A) Hydrophilic-hydrophobic interaction between (i) quercetin and SARS-CoV-2 Spike in candidate protein binding pocket, and (ii) quercetin and relative amino acids. (B) The KD of the SARS-CoV-2 Spike RBD protein with a series of concentrations of quercetin was calculated by SPR.

Importantly molecular docking analysis showed that quercetin has high binding affinity to the spike protein.

Utilizing pathway enrichment analysis for the COVID-19-related genes, the study team found quercetin affected the immune-modulation and viral infection activities.

It should be noted that Quercetin has been found in all the 26 Chinese herbal medicines advised by traditional Chinese medicine practitioners to combat COVID-19.

The phytochemical Quercetin showed a higher binding affinity to both ACE2 and the RBD of the spike protein. The dual binding effect of quercetin could therefore be synergistic and provide a strong antiviral effect against SARS-CoV-2.

Also since analysis suggested that quercetin could affect immunomodulation, and because studies have shown patients with severe COVID-19 disease tend to experience cytokine storms, quercetin as one of the any COVID-19 Supplements could help alleviate symptoms in such cases.

Two separate randomized clinical trials led by Italian researchers but supported by multinational teams of scientists found that Quercetin not only prevented disease severity and mortality in infected COVID-19 patients, but also resolved clinical symptoms faster and lead to faster viral clearance times.

Another study conducted by researchers from Iran showed the mechanics by which quercetin could function as an anti-inflammatory compound and prevent cytokine storms in those infected by the SARS-CoV-2.

Studies have also emerged that quercetin can help in the treatment of acute kidney injury as a result of COVID-19, hence showing its potent role for Long COVID as well.

Studies are also currently underway with regards to using quercetin as a prophylaxis against SARS-CoV-2 infections.

Quercetin is a plant flavonol from the flavonoid group of polyphenols. It is found in many fruits, vegetables, leaves, seeds, and grains; red onions and kale are common foods containing appreciable amounts of quercetin.

Quercetin supplements are relative safe to take. In preliminary human studies, oral intake of quercetin in doses up to one gram per day over three months did not cause adverse effects.

However readers have to be careful when buying quercetin supplements as many supplement brands take advantage of lax regulatory controls over supplements and also weak labeling laws.

Flavonoids in COVID-19

Flavonoids are defined as important naturally occurring compounds with a phenolic structure [7]. Chemically, flavonoids are composed of fifteen-carbon skeleton that consists of two benzene rings connected through a pyrane ring [35]. Flavonoids are classified by their chemical structure, level of oxidation, and the pattern of the substitution of their heterocyclic pyrane ring also known as C ring.

The classification of individual compounds within each class is based on the substitution of benzene rings (A and B rings) [7]. Table 2 provides an overview of the classification and food sources of flavonoids [7], [8], [9], [36], [37], [38], [39], [40], [41].

Table 2
Classification and food sources of flavonoids [7], [8], [9], [36], [37], [38], [39], [40], [41].

Flavonoid classificationMembers of the flavonoids subgroupFood sources
FlavanonesHesperidin, naringenin, naringin, taxifolin, eriodictyol, naringeninOranges, lemons, oregano, grapes, medicinal plants
FlavonolsKaempferol, quercetin, fisetin, myricetin, morin, rutinOnion, apples, tomatoes, kale, grapes, berries, lettuce, tea, red wine, olive oil, medicinal plants
FlavanolsCatechin, epicatechin, epigallocatechin-3-gallateGreen tea, apples, bananas, blueberries, cacao beans, peaches, pears, medicinal plants
FlavonesApigenin, luteolin, hispidulin, wogonin, oroxylin, scutellarin, rhamnocitrin baicalein, chrysin, morusin, tangeretin, pectolinarigenin, scutellarinChamomile, mint, celery, parsley, Ginkgo biloba, tomatoes, fruit skin, red wine, medicinal plants
IsoflavonoidsGenistein, glycitein, daidzeinSoya, medicinal plants
ChalconesPhloretin, xanthohumol, isoliquiritigenin, velutone FStrawberries, apples, medicinal plants
AnthocyanidinsCyanidin, delphinidin, apigenidin, malvidinBlack/cran/rasp/straw/blue-berries, grapes, cherries, blackcurrants, nuts, medicinal plants

Flavonoids are secondary plant metabolites responsible for their color, flavor, and are also related to plants’ pharmacological activities. Flavonoids possess significant anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and immunomodulatory abilities [7], [8], [42].

In addition, flavonoids exert a strong anti-viral capacity in numerous pathologies [43], [44], [45], [46]. More importantly, flavonoids demonstrated anti-viral and immunomodulatory activities against coronaviruses [47]. Therefore, the anti-viral properties of flavonoids might be applicable also in the current COVID-19 pandemic.

The potentially beneficial role of flavonoids or flavonoid-rich whole plants in COVID-19 pandemic is currently a widely discussed topic [48], [49], [50], [51]. One of the suggested targets of SARS-CoV-2 therapies is the ACE-2 receptor [52]. The applicability of flavonoids is associated with the SARS-CoV-2 spike-protein that engages ACE-2 receptors to entry into host cells as was demonstrated by bioinformatics and molecular docking in case of tea flavonoids, especially epigallocatechin-3-gallate (EGCG) and theaflavin gallate [53], fisetin, quercetin, kaempferol [54], quercetin, luteolin, or naringenin [55]. Moreover, the biological activity of flavonoids predetermines them to be effective also in terms of the modulation of inflammatory and immune pathways of SARS-CoV-2-associated pathology.

Flavonoids as potential inflammatory modulators in COVID-19

The anti-inflammatory and immunomodulatory properties of flavonoids are well-described [7], [8], [9], [10], [11], [12]. Thus, flavonoids could potentially be useful in the modulation of COVID-19-related inflammatory processes and immune responses.

Due to the excessive immune responses that trigger cytokine release and can result in the overproduction of proinflammatory cytokines, the modulation of systemic immune responses and reversion of hyper-inflammation are suggested to possess a potential role in the management of COVID-19 patients [23].

Generally speaking, flavonoids can modulate the production of inflammatory mediators. Luteolin inhibited IL-1β-induced inflammation in rat chondrocytes [56]. Similarly, apigetrin, a glucoside conjugate of apigenin [57], reduced inflammatory factors including IL-lβ, TNF-α, IL-6, and VEGF in mice with acute otitis media [58].

Also, Smilax campestris aqueous extract, which contains catechin and glycosylated derivatives of quercetin (quercetin-3-O-glucoside, quercetin-3-O-galactoside, rutin, and quercetin-3-rhamnoside) as its main constituents, reduced the production of proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and MCP-1 in lipopolysaccharide (LPS)-activated macrophages derived from the monocytic cell line THP-1 [59].

In addition, apigenin alleviated inflammation demonstrated through reduced plasma levels of IL-6, TNF-α, and interferon-γ (IFN-γ) in vivo [60]. Furthermore, flavonoids can modulate the switch of macrophages from proinflammatory to anti-inflammatory phenotype [61]. As was demonstrated by et al, flavonoids (quercetin, naringenin and naringin) induced metabolic variations opposite to proinflammatory metabolic reprogramming elicited by LPS and IFN-γ stimulation in in vitro cultured human macrophages [62].

Also, flavonoids regulate the immune cell functions through the enhancement of the activity of NK cells and cytotoxic T lymphocytes and also through the macrophage functions via modulation of lysosomal activity and the release of nitric oxide [63].

Moreover, potent modulatory properties of hesperidin on systemic immunity (demonstrated by enhanced NK cell cytotoxicity and proportion of phagocytic monocytes, amelioration of the secretion of cytokines stimulated by macrophages, or increased T helper cells) were observed in rats following an intensive training and exhausting exercise [64]. Therefore, a significant benefit of flavonoids is associated with their potent immunomodulatory properties [11], [62], [65].

Based on the immunomodulatory properties of flavonoids discussed above, we can assume their significant effects on the production of proinflammatory cytokines also within COVID-19. Despite the high affinity to the spike protein, helicase and protease sites on ACE-2 demonstrated in in silico studies, the flavonoid-based phytomedicine caflanone also exhibited the ability to inhibit the production of cytokines including IL-1β, IL-6, IL-8, Mip-1α, TNF-α [47].

Moreover, Bellavite and Donzelli [13] recently discussed the nutraceutical properties of citrus fruits, primarily focusing on its flavonoid component hesperidin as a potential substance against SARS-CoV-2 due to its anti-viral, anti-oxidant, and inflammation-modulatory activities [13]. Hesperidin ameliorated altered level of inflammatory mediators in ischemia/reperfusion-induced kidney injury in rats [66] and also triggered anti-inflammatory responses resulting in a decreased level of IL-33 and TNF-α in mice co-treated with hesperidin and LPS [67]. Nevertheless, the hypothetical role of citrus flavonoid hesperidin against COVID-19 requires corroboration in further pre-clinical, epidemiological, and clinical studies [13].

Moreover, vascular endothelial activation has a crucial role in the excessive cytokine production leading to the cytokine storm and severe pathologies in infectious diseases such as SARS or COVID-19. Therefore, rhamnocitrin, a flavonoid extracted from Nervilia fordii, which has been identified as a potent inhibitor of endothelial activation, might be suggested as a potential modulator of cytokine storm in the management of these diseases [68].

Notably, the traditional Chinese medicine (TCM) with its main active ingredients including flavonoids (such as quercetin, kaempferol, luteolin, baicalein, naringenin, and wogonin) could also exert beneficial effects in the management of COVID-19 via the inhibition of viral adsorption and replication as well as the regulation of inflammatory mediators, anti-inflammatory, and immune-regulatory effects to prevent cytokine storm and to protect the target organs [69].

Indeed, Niu et al. [70] recently evaluated chemical constituents of three TCM formulas that were proven to be effective in COVID-19. Eventually, the network pharmacology research revealed decreased IL-6 through several TCM compounds, including but not restricted to quercetin, luteolin, and rutin. These observations suggest the positive association between TCM efficiency in the prevention and rehabilitation of at-risk COVID-19 [70]. Similarly, other TCM formula, which includes quercetin among others, could inhibit COVID-19 through ACE2 downregulation [71]. Moreover, Table 3 [72], [73], [74], [75] briefly summarizes the applicability of other TCM formulas with defined core compounds (represented mostly by flavonoids among others) in COVID-19 through the modulation of inflammatory/immune pathways, based on studies of network pharmacology and molecular docking.

Table 3

Effects of TCM (mostly flavonoids among core compounds) in COVID-19 evaluated through network pharmacology and molecular docking.

TCMPotential effects in COVID-19Reference
TCM prescription DayuanyinSuppression of the inflammatory storm and regulation of immune function.[72]
Observed affinity between the core compounds of Dayuanyin (kaempferol, quercetin, 7-Methoxy-2-methyl isoflavone, naringenin, formononetin) and its target genes such as IL-6, IL1β, and CCL2.
Maxingyigan decoctionRecognized and verified gene targets (including IL-6) and three components of Maxingyigan (quercetin, formononetin, luteolin).[73]
The potential role of Maxingyigan in the prevention and treatment of COVID-19 could be based on its anti-inflammatory and immunity-based actions including the activation of T-cells, lymphocytes, leukocytes, cytokine-cytokine-receptor, and chemokine signaling pathways.
Toujie Quwen granuleThe potential role of Toujie Quwen granule and its key active ingredients (including quercetin, kaempferol, luteolin, and oroxylin A, among others) in the treatment of COVID-19 associated with the mechanisms that elevate immunity, suppress inflammatory stress, and regulate inflammatory responses among others.[74]
Qing-Fei-Pai-Du decoctionObserved immuno-regulatory, anti-inflammatory and multi-organ protective abilities (attributed to four compounds including also flavonoids baicalin and hesperidin and its targets) that could be applicable in COVID-19 management (thrombin and TLR signaling suggested as essential pathways of its anti-inflammatory effects).[75]

Abbreviations: CCL2, monocyte chemo-attractant protein-1; IL, interleukin; TCM, Traditional Chinese medicine; TLR, Toll-like receptor.

Thus, flavonoids are currently a widely discussed source of agents potentially applicable in the management of COVID-19, as demonstrated by network pharmacology and molecular docking experiments.

The potential of flavonoids to modulate specific inflammatory pathways deregulated in SARS-CoV-2 infection

Apart from the above-discussed effects of flavonoids against the SARS-CoV-2, research databases offer a wide range of results that could indirectly point to flavonoids’ role in targeting inflammatory mediators that are altered in COVID-19. Based on the above-described inflammatory processes associated with COVID-19 and with diverse biological effects of the flavonoids taken into account, it is possible to hypothesize their significant effects on SARS-CoV-2-associated pathways such as the modulation of NLRP3 inflammasome, TLRs, or BRD4, and activation of Nrf2, or the effects on ACE-2 ( Fig. 3) [1], [16], [23], [32], [76], [77], [78].

Fig. 3
Fig. 3
Inflammatory pathways associated with SARS-CoV-2 that can be potentially targeted by flavonoids. Abbreviations: AngII, angiotensin II; ACE, angiotensin-converting enzyme; ACE-2, angiotensin-converting enzyme 2; Ang1-7, angiotensin 1-7; AngII, angiotensin II; AT1R, angiotensin II receptor type 1; BRD4, bromodomain-containing protein 4; CRP, c-reactive protein; IL, interleukin; Mas, mitochondrial assembly receptor; NF-κB, nuclear factor kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; RAAS, renin-angiotensin-aldosterone system; TLRs, toll-like receptors. Explanatory notes: (A) An essential determinant of the inflammatory response is the cleavage and secretion of pro-IL-1β and pro-IL-18 into bioactive cytokines activated by the NLRP3 inflammasome [23]. The NLRP3 inflammasome is activated in response to AngII stimulation [32]. (B) TLR activation followed by viral infection can induce the production of IL-6 by macrophages and monocytes. TLRs, TNFα, and IL-1β are considered as the most important stimulators of IL-6. IL-6 is the main regulator of T-cells and can modulate the function of Th17 cells to serve as proinflammatory self-reactive T-cells. IL-6 can also induce the production of acute phase proteins such as CRP [23]. (C) The recruitment of BRD4 by NF-κB leads to the activation of NF-κB-mediated proinflammatory signaling while BRD4 inhibitors decrease the recruitment of macrophages and infiltration of T-cells. The transmembrane E protein of SARS-CoV-2 has been recently demonstrated to bind to BRD4 [1]. (D) The activity of Nrf2 is associated with the modulation of execution and resolution of inflammation through the repression of proinflammatory signals such as IL-6 or IL-1β [76]. (E) Despite the crucial role for viral entry, ACE-2 paradoxically exerts protective effects via conversing AngII to Ang1-7 [77]. SARS-CoV-2 spike protein attachment to ACE-2 leads to ACE-2 downregulation (increase in the level of AngII and augmentation of AngII/AT1R axis activation that are associated with proinflammatory responses). RAAS activation can promote proinflammatory responses through AT1R in kidney and vascular system [16]. The ACE-2-cleaved protein Ang1-7 bind to Mas that is followed by a decrease in proinflammatory cytokine production (TNF-α, IL-6) [78]. Therefore, the binding of SARS-CoV-2 to ACE-2 prevents the production of anti-inflammatory Ang1-7 and leads to the accumulation of proinflammatory AngII [16].

Flavonoids potentially targeting NLRP3 inflammasomes and TLRs in COVID-19

Flavonoids demonstrated reasonable effects in the modulation of inflammatory mediators or signaling cascades including TLRs and NLRP3 inflammasomes ( Table 4) [41], [79], [80], [81], [82], [83], [84], [85], [86], [87]. Altogether, these results highlight flavonoids’ capacity to target inflammatory processes associated with TLRs and NLRP3 inflammasome, the deregulation of which is discussed also in terms of SARS-CoV-2 pathology.

Therefore, it can be assumed that flavonoids exert significant anti-viral and immunomodulatory effects mediated through TLRs or NLRP3 inflammasomes in COVID-19 patients while these effects need to be precisely evaluated in well-defined pre-clinical and clinical studies.

Table 4

Effects of flavonoids on inflammatory cascades TLRs and NLRP3 inflammasomes.

Target of inflammatory pathwayFlavonoidAim of the studyEffectsReference
TLREpigallocatechin-3-gallateBALB/C mice (lipopolysaccharide-induced acute lung injury)Ameliorated lipopolysaccharide-induced acute lung injury by suppression of TLR4/NF-κB signaling.[79]
Decreased proinflammatory cytokines TNF-α, IL-1β, and IL-6 in lung, serum, and bronchoalveolar lavage fluid.
LuteolinC57BL/6J mice (inflammation-mediated metabolic diseases)TLR signaling modulation.[80]
Reduction of macrophage infiltration and modulation of the inflammatory response.
NobiletinProstate cancer cells (anti-inflammatory activities)Anti-inflammatory effects (inhibition of TLR4 and TL9-dependent signaling).[81]
Pycnogenol® (extract of French maritime pine bark rich in flavonoids)TLR-dependent immunomodulatory activitiesTLRs inhibition (after gastrointestinal metabolization).[82]
Flavonoids from Houttuynia cordataEffects and mechanism of flavonoid glycosides from H. cordata on influenza A virus-induced acute lung injury in miceAttenuation of H1N1-induced acute lung injury (inhibition of TLR signaling).[83]
NLRP3 inflammasomesApigeninEffects on NLRP3 inflammasome pathways – measurement of active IL-1β (differentiated THP-1 cells)Inhibition of IL-1β.[84]
ScutellarinEffects on NLRP4 inflammasome activation (macrophages)Suppression of NLRP3 inflammasome activation in macrophages.[85]
MyricetinEffects on NLRP3-driven inflammatory diseasesInhibition of NLRP3 inflammasome assembly.[86]
BaicalinEffects on neuroinflammation (amyloid beta precursor protein/presenilin-1 mice)Protection of neurons from microglia-mediated neuroinflammation via suppression of NLRP3 inflammasomes and the TLR4/NF-κB signaling pathway.[87]
Flavonoids isolated from Millettiavelutina (velutone F)Effects on NLRP3 inflammasome activation (THP1 cells)Suppression of NLRP3 inflammasome activation and serum IL-1β release.[41]
Abbreviations: IL, interleukin; NF-κB, nuclear factor kappa B; TLRs, Toll-like receptors; TNFα, tumor necrosis factor.

Flavonoids potentially targeting BRD4 in COVID-19
As mentioned above, the transmembrane E protein of SARS-CoV-2 has been recently demonstrated to bind to BRD4 while its recruitment by NF-κB activates proinflammatory signaling [1]. Importantly, fisetin and amentoflavone are putative ligands of BRD4 and amentoflavone can establish contacts with non-canonical residues for BET inhibition [88]. Moreover, Yokoyama [89] recently discussed the structural and thermodynamic characteristics of isoliquiritigenin interactions with BRD4 and suggested it as a novel template for BDR inhibitors [89]. Hence, BRD4 inhibition represents another approach potentially applicable to overcome COVID-19 associated inflammatory chaos.

Flavonoids potentially targeting Nrf2 in COVID-19

The Nrf2-response has been recently demonstrated to be suppressed in COVID-19 patient biopsies while the Nrf2 agonists 4-octyl-itaconate and the clinically approved dimethyl fumarate inhibited SARS-CoV-2 replication across cell lines in vitro [90]. The poor reproducibility of COVID-19 in animal models limits the effectiveness of the development of therapies against SARS-CoV-2. Nevertheless, genetic or pharmacological activation of Nrf2 demonstrated anti-inflammatory and anti-viral effects in other pathologies while the most relevant mechanisms of its action are associated with targeting specific cysteine receptors within KEAP1 [76].

Although the evaluation of Nrf2 inducers for the reduction of oxidative stress and inflammation in SARS-CoV-2 infections has not been yet performed, a wide range of compounds including flavonoids can activate Nrf2 within other pathologies (7-O-methylbiochanin A, biochanin A, flavonoids of Abelmoschus esculentus L. flowers, cyanidin chloride) [91], [92], [93], [94], [95]. Moreover, xanthohumol protected LPS-induced acute lung injury against inflammatory damage and oxidative stress via induction of the AMPK/GSK3β-Nrf2 signaling axis in vivo and in vitro [96].

Also, EGCG was observed to protect endothelial cells against inflammation induced by environmental pollutants while the mechanisms of action included the induction of Nrf2-regulated genes [97]. Moreover, Crateva nurvala Buch. Ham extracts containing flavonoids as the major class of bioactive phytochemicals activated Nrf2 and decreased proinflammatory TNF-α, NF-κβ, and IL-6 in vivo [98].

Besides, flavonoids from Apios americana Medikus leaves reduced inflammatory cytokines and activated Nrf2-KEAP1 pathways in RAW264.7 cells that are, when induced by LPS, accepted as a classic inflammatory model [99]. Furthermore, a study evaluating flavonoids’ role in the inhibition of Influenza A viral replication demonstrated that 6-demethoxy-4′-O-methylcapillarisin, a flavonoid derivative of Artemisia rupestris L., activated Nrf2/heme oxygenase pathway [100]. It can therefore be hypothesized that flavonoids could decrease the severity of SARS-CoV-2 via the activation of Nrf2 and subsequent modulation of inflammatory and immune processes [25].

Flavonoids potentially targeting ACE-associated pathways in COVID-19

The dual impact of ACE-2 in COVID-19 is associated with its ability to convert AngII to Ang1-7, thus counteracting the inflammatory action of AngII [77]. The attachment of SARS-CoV-2 spike protein to ACE-2 is followed by down-regulation of ACE-2 through its intracellular binding site and then an increase in the level of AngII and augmentation of AngII/AT1R axis activation. Increased production of AngII and activation of angiotensin II receptor type 1 (AT1R) are processes associated with proinflammatory response.

The activation of NF-κB by AngII also leads to the production of TNF-α, IL-6, IL-1β. AT1R activation is followed by the regulation of mitogen-activated protein kinase (MAPK) by AngII, which also affects the release of cytokines (TNF-α, IL-1). Furthermore, the activation of the renin-angiotensin-aldosterone system (RAAS) can be associated with proinflammatory responses through AT1R in the kidney and vascular system.

In fact, increased circulatory levels of AngII have been observed in COVID-19 patients with the association of its plasma level and lung injury [16]. However, the ACE-2-cleaved protein Ang1-7 is hypothesized to possess beneficial effects on immune regulation and its low expression during SARS-CoV-2 infection can be associated with COVID-19 severity.

The antagonist effects of AngII-derived pathway is associated with the binding of Ang1-7 to the mitochondrial assembly (Mas) receptor and consequent decrease in proinflammatory cytokine production (TNF-α, IL-6) [78]. Therefore, the binding of SARS-CoV-2 to ACE-2 decreases its anti-inflammatory Ang1-7 production and promotes the accumulation of proinflammatory AngII [16].

In 1982, Agarwal demonstrated anti-inflammatory effects of a flavonoid nepitrin that could be mediated through its anti-angiotensin action [101]. More recently, in hypertensive rats models, high levels of AngII were attenuated by hesperidin [102] and RAS activation was inhibited by tangeretin [103]. Similarly, kaempferol exerted inflammation-inhibitory effects mediated by a decrease in AngII-induced collagen accumulation in cardiac fibroblasts [104]. A precise analysis of the effects of ACE-2 on the overall course of SARS-CoV-2 pathogenesis may contribute to the identification of key agents, potentially even among flavonoids, targeting the discussed ACE-2 and related signaling mechanisms.

Other potential mechanisms of flavonoids targeting inflammation as a strategy against COVID-19

Other mechanisms that could potentially be modulated by flavonoids in COVID-19 include the inhibition of DPP4, the neutralization of 3CLpro, or the effects on gut microbiota.

Dipeptidyl peptidase 4

In addition to ACE-2, recently suggested interaction between SARS-CoV-2 and dipeptidyl peptidase 4 (DPP4) as a co-receptor could lead to the development of novel therapeutic COVID-19 strategies. DPP4 is a ubiquitous membrane-bound aminopeptidase with multiple roles in metabolism, nutrition, and the endocrine and immune system [105]. A large interface was observed between the SARS-CoV-2 spike glycoprotein and DPP4 using a docked complex model.

The capacity of DPP4 to cleave numerous substrates, such as chemokines or growth factors, is associated with its ability to regulate numerous physiological processes [18] and diseases of the immune system. DPP4 is expressed on epithelia and endothelia of the systemic vasculature, lung, small intestine, kidney, and heart.

Accordingly, DPP4 distribution may contribute to the virus’s entry through the respiratory tract and may also facilitate the development of cytokine storm and immune-pathologies associated with fatal COVID-19 consequences [19]. Therefore, some investigators suggest the inhibition of DPP4 as a therapeutic strategy to slow the progression of COVID-19 or to hamper cytokine storm and inflammation [18], [19].

DPP-4 inhibitors, generally known as anti-diabetic drugs, may possess immunomodulatory functions and could be beneficial in inflammatory diseases [106], [107] and could act beneficially also in COVID-19 patients through the reduction of inflammation [108]. Natural compounds, including flavonoids such as EGCG also target DPP4 [109], [110], [111], [112]. Indeed, citrus flavonoids [113], epicatechin [114], and chrysin [115] were demonstrated to be potent DPP4 inhibitors. Overall, flavonoids may represent a source of DPP4 inhibitors with potential efficacy against COVID-19.

3-Chymotrypsin-like protease

3-Chymotrypsin-like protease (3CLpro) is a non-structural protein of coronaviruses. The 3CLpro of SARS-CoV-2 shares 96.1% of its sequence with other SARS-CoV family members such as SARS-CoV or MERS-CoV. 3CLpro cleaves polyproteins into viral replication-related proteins, a process essential for viral replication and maturation. Another crucial function of 3CLpro is to cleave host proteins related to innate immune responsiveness such as the signal transducer and activator of transcription 2 (STAT-2) and NF-κB transcription factor.

Therefore, the neutralization of 3CLpro can avert viral maturation and restore the natural immune response [18]. As in the case of SARS-CoV [116], [117] and MERS-CoV [118], the promising role of flavonoids has been recently suggested also in SARS-CoV-2 pathology. Interestingly, quercetin has been demonstrated as a potent inhibitor of SARS-CoV-2 3CLpro in vitro and can be considered a proper candidate for further optimization and development [119].

Similarly, the docking study revealed quercetin, scutellarin, and myricetin as potent candidates to target 3CLpro [120]. Despite quercetin’s promising efficiency in COVID-19 targeting 3CLpro, its anti-viral properties may be challenged by its poor oral bioavailability. Therefore, Di Pierro et al. have recently demonstrated an increased bioavailability of quercetin’s phospholipid complex (Quercetin Phytosome®) in humans [121].

Furthermore, a 3CLpro inhibitory activity of quercetin-3-O-rutinoside (rutin), kaempferol-3-O-rutinoside (nicotiflorin), and their human metabolites has been demonstrated using molecular docking approach [122]. Thus, the neutralization of 3CLpro by flavonoids to restore immune response represents another potential strategy against COVID-19.

Gut microbiota

Despite that SARS-CoV-2 is associated with acute respiratory syndrome, there are also gastrointestinal manifestations that may precede respiratory events. Therefore, due to the capacity of normal gut microbiota restoration to maintain immune response to viral diseases and improve respiratory symptoms, the prebiotic properties of polyphenolic compounds may represent a therapeutic strategy for COVID-19, primarily due to the possibility to affect the gut microbiota of patients [18].

Flavonoids can modulate intestinal immune function through the modulation of T-cell differentiation, alteration of gut microbiota as well as the regulation of cytokines [123], [124]. Also, Estruel-Amades [125] demonstrated the immunomodulatory activity of hesperidin on gut-associated lymphoid tissue and reinforced its prebiotic role [125]. Hence, the restoration of the immune responses through gut microbiota may potentially support the organism to overcome COVID-19.

Above all, flavonoids modulate the synthesis or activity of a plethora of inflammatory mediators and immunomodulatory signals [51], [74], [113], [114], [118], [121], [123], [126], [127]. However, pre-clinical evidence might be limited by the utilization of non-physiological concentrations in in vitro models while flavonoids are extensively metabolized in vivo [126]. Low bioavailability of flavonoids can limit or hinder their activity.

Generally speaking, the bioavailability of flavonoids is affected by several factors including molecular weight, glycosylation, or metabolic conversion [128]. The metabolism of flavonoids occurs in small and large intestine, and liver. Also, the absorption, distribution, and metabolism of flavonoids and their circulating concentrations, elimination, and tissue exposure are also affected by age, sex, genotype, habitual diet, prescribed medicine, and gut microbiome [7].

Gut microbiota plays an essential role in the metabolism of flavonoids [129]. However, some metabolites were observed to paradoxically exert more robust physiological functions than their precursors [7]. Nevertheless, the increase in flavonoids bioavailability as well as the safety evaluation of their application are the goals of ongoing research [7], [9], [130], [131], [132]. Furthermore, human studies are necessary to clarify the anti-inflammatory properties of flavonoids [126].

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