key: cord-0932089-qn7yv95p
authors: Brito, Júlio César Moreira; Lima, William Gustavo; Cordeiro, Lídia Pereira B.; da Cruz Nizer, Waleska Stephanie
title: Effectiveness of supplementation with quercetin‐type flavonols for treatment of viral lower respiratory tract infections: Systematic review and meta‐analysis of preclinical studies
date: 2021-04-17
journal: Phytother Res
DOI: 10.1002/ptr.7122
sha: e80fc0cd92fd44cf7e10e2e55f884675020442a8
doc_id: 932089
cord_uid: qn7yv95p

Viral infections of the lower respiratory tract are considered a public health problem. They affect millions of people worldwide, causing thousands of deaths, and are treated with expensive medicines, such as antivirals or palliative measures. In this study, we conducted a systematic review to describe the use of quercetin‐type flavonols against lower respiratory tract viruses and discussed the preclinical impact of this approach on different signs and clinical mechanisms of infection. The systematic review was performed in PubMed/MEDLINE, Scopus, Scielo, and Biblioteca Virtual de Saúde (BVS). After the database search, 11 relevant studies were identified as eligible. The analysis of these studies showed evidence of antiviral activity of quercetin‐type flavonols with significantly reduced mortality rate (M‐H = 0.19, 95% CI: 0.05 to 0.65, p‐value = 0.008) of infected animals and a reduction in the average viral load (IV = −1.93, 95% CI: −3.54 to −0.31, p‐value = 0.02). Additionally, quercetin and its derivatives reduced the amount of proinflammatory cytokines, chemokines, reactive oxygen species, mucus production, and airway resistance in animals infected with a respiratory virus. Overall, supplementation with quercetin‐type flavonols is a promising strategy for treating viral‐induced lower respiratory tract infections.

(e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2) (Batiha et al., 2020; Biancatelli et al., 2020; Kaul, Middleton, & Ogra, 1985) . In these studies, quercetin induced a concentration-dependent reduction in the infectivity of these viruses. Quercetin also affects multiple viral virulence steps essential for the infectious process in lung cells (i.e., viral entry, replication, release, maturation, and protein assembly) (Kaul et al., 1985) .

Moreover, the in vivo antiviral activity of quercetin and its derivatives is also related to their modulatory effect on immune response mechanisms. For instance, quercetin-like flavonols are known to upregulate the interferon (IFN)-γ from T helper-1 (T H 1) lymphocyte, increasing cell-mediated immunological activity and downregulate IL-4 release by T helper-2 lymphocyte (T H 2) (Chen et al., 2012; Nair et al., 2002) .

However, the evidence of the antiviral activity of quercetin and its derivatives against viral infections of lower airways remains decentralized. In this context, little effort has been made to determine the antiviral activity of these compounds, and the therapeutic effect of supplementation with quercetin-like compounds in vivo needs to be clarified. Therefore, in this study, we aim to summarize the available data on the antiviral effect of quercetin and its derivatives against viral lower respiratory tract infections. For this, we quantified the magnitude of the in vivo therapeutic effect of these compounds present in many apitherapy products through a systematic review and meta-analysis.

The therapeutic effects of quercetin and its derivatives on virusinduced lower respiratory tract infections in experimental models were evaluated by a systematic review and meta-analysis performed according to the principles described in the Cochrane Handbook (Higgins & Green, 2011) . The search, selection of studies, extraction, analysis, and interpretation of data of interest were conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (Liberati et al., 2009) . To identify studies of interest, we applied the PICOS strategy (Eriksen & Frandsen, 2018) as follows: Population-rodents with virus-induced lower respiratory tract infections; Intervention-treatment with quercetin or its derivatives; Control-treatment with placebo; Outcomesmortality and lung viral load; and Study design-in vivo studies conducted in rodents (mice, rats, hamster, or rabbits).

Initially, the systematic search was performed in four databases (PubMed/MEDLINE, Scopus, Biblioteca Virtual em Saúde, and SciELO) using the Medical Subject Heading (MeSH) term "Quercetin*" combined with at least two of the following descriptors: "Virus," "Viruses," "Respiratory System," "Respiratory Tract," "Lung," "Low Respiratory Tract." These descriptors were connected using the connector "AND" between them as in the following example: "Quercetin" AND "Virus" AND "Lower Respiratory Tract." The search was conducted until April 20th, 2020, restricted to studies written in English, and no date limit was established. A gray literature search of dissertations and thesis was also performed in the databases Thesis and Dissertation Catalog of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Digital Library of Thesis and Dissertations of the Universidade Federal de Minas Gerais (UFMG) and Universidade de São Paulo (USP). In addition, the reference lists of all included articles and relevant narrative reviews were evaluated for any relevant studies.

Subsequently, review articles, notes, correspondences, editorials, and letters were excluded. Furthermore, other studies were excluded based on the following criteria: (a) in vivo studies of virus-induced upper respiratory tract infections; (b) studies that do not identify the microorganism used in the lower respiratory tract infection; and (c) studies in which none of the primary outcomes (i.e., lung viral load and mortality) was the subject of the study. In cases where the study was in accordance with the inclusion criteria, but the full text was not available, the corresponding author was contacted by e-mail three times (with 14-day intervals between them). The articles were included if the authors provided the full paper. The details of the search strategy for each database are shown in Data S1. We did not use any type of research limit on the bases selected. Thus, the usage of the search equations provided in Data S1 guarantees the reproducibility of searches.

In the first phase of study selection, two independent researchers (J.C.M.B. and L.P.B.C.) searched the databases. Duplicated records were deleted using the software EndNote X9 version 19.0.0.12062 (Clarivate Analytics, Sydney, Australia), and titles and abstracts of the selected studies were screened according to the PICOS eligibility criteria. Thereby, studies that evaluated the therapeutic effects of quercetin and its derivatives in rodent models of virus-induced lower respiratory tract infection were selected and then evaluated by fulltext review. Any discrepancies were resolved by discussion with a third investigator (W.G.L), and the Kappa coefficient (performed with 95% confidence interval) was used to analyze the degree of agreement between the evaluators (Landis & Koch, 1977) .

After a full analytical reading, all data of interest were summarized in Table 1 for further analysis and interpretation. Furthermore, the data regarding mortality rate and viral load in the lung of mice with virusinduced lower respiratory tract infection treated or not with quercetin and its derivatives were pooled to estimate the mean difference and the confidence intervals by meta-analysis using Review Manager (RevMan)® 5.3 software (Lima et al., 2020b) . All pooled data were estimated using a random effect model (Lima, Silva Alves, Sanches, Antunes Fernandes, & de Paiva, 2019; Lima, Souza, Fernandes, Cardoso, & God oi, 2019) . Heterogeneity of the primary data was analyzed using the I 2 statistic, in which I 2 > 50% was considered to have substantial heterogeneity (de Carvalho, Lima, Coelho, Cardoso, & Fernandes, 2020; Lima et al., 2020c) . In all procedures, the significance level was 5%. To assess the robustness of our findings, we conducted a sensitivity analysis considering only studies that used the A/PR/8/34 strain of influenza virus H1N1 to induce viral pneumonia in mice. After excluding duplicated studies, the titles and abstracts of 124 records were screened, resulting in 15 studies that met the inclusion criteria. Therefore, the full text of 15 articles were assessed for eligibility, and 4 of them were excluded for the following reasons:

(a) different study design (n = 3) or (b) incomplete text (n = 1) ( Figure 1) . Finally, 11 studies were selected for the qualitative analysis (Choi, Song, & Kwon, 2012; Davis, Murphy, McClellan, Carmichael, & Gangemi, 2008; Dayem, Choi, Kim, & Cho, 2015; Fan et al., 2011; Farazuddin et al., 2018; Ganesan et al., 2012; Y. Kim, Narayanan, & Chang, 2010; Kumar et al., 2005; Kumar, Sharma, Khanna, & Raj, 2003; Raju, Lakshmi, Anand, Rao, & Sharma, 2000; Savov et al., 2006) .

Of these, 4 were included in the quantitative studies and used in the meta-analysis for the outcomes of interest (i.e., mortality [Choi et al., 2012; Davis et al., 2008; Dayem et al., 2015] and lung viral load [Choi et al., 2012; Dayem et al., 2015; Y. Kim et al., 2010] ). The degree of agreement between the two researchers was considered substantial (Kappa coefficient of 0.613).

The viral infection of the lower respiratory tract in the majority of the studies was induced by Influenza virus (IV) (9/11; 81.8%) (Choi et al., 2012; Davis et al., 2008; Dayem et al., 2015; Fan et al., 2011; Y. Kim et al., 2010; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006) , using the variants H1N1 (5/9; 55.6%) (Choi et al., 2012; Davis et al., 2008; Dayem et al., 2015; Fan et al., 2011; Y. Kim et al., 2010) or H3N2 (4/9; 44.4%) (Kumar et al., 2003 (Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006) . Only two studies induced lung infection with noninfluenza viruses, both of which employed RV (2/11; 18.2%) (Farazuddin et al., 2018; Ganesan et al., 2012) . In all studies included, viral infection was performed by intranasal instillation of a viral suspension in phosphate-buffered saline (PBS) or 0.9% saline (11/11; 100%) (Choi et al., 2012; Davis et al., 2008; Dayem et al., 2015; Fan et al., 2011; Farazuddin et al., 2018; Ganesan et al., 2012; Y. Kim et al., 2010; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006) .

Regarding the animals infected with IV, the average viral load employed was 10 4 plaque-forming units (PFU), which is equivalent to 1-1.5 times the lethal dose for 50% of mice (LD 50 ). The two studies that used RV as the infectious agent employed a viral load of 5 × 10 6 PFU. Mice were used in all experimental models of viral pneumonia.

The strains employed were: BALB/c (4/11; 36.4%) (Choi et al., 2012;  Y. Kim et al., 2010; Kumar et al., 2003 Kumar et al., , 2005 , C57BL/6 (3/11; 27.3%) (Dayem et al., 2015; Farazuddin et al., 2018; Ganesan et al., 2012) , ICR (2/11; 18.2%) (Davis et al., 2008; Savov et al., 2006) , Kunming (1/11; 9.1%) (Fan et al., 2011) , or Swiss (1/11; 9.1%) (Raju et al., 2000) . Male animals (6/11; 54.5%) (Choi et al., 2012; Davis et al., 2008; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006) were preferred over female animals (4/11; 36.4%) (Dayem et al., 2015; Farazuddin et al., 2018; Ganesan et al., 2012; Y. Kim et al., 2010) , and a study did not identify the sex of the animals (1/11; 9.1%) (Fan et al., 2011) .

Most of the included studies used quercetin as a therapeutic or prophylactic agent against viral pneumonia (7/11; 63.6%) (Davis et al., 2008; Farazuddin et al., 2018; Ganesan et al., 2012; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006 (Choi et al., 2012) , or quercetin-3-O-β-D-glucuronide (Fan et al., 2011) . One study used a nonglycosylated analog of quercetin (1/11; 9%) called isorhamnetin (Dayem et al., 2015) , and another one used a combination of quercetin and rutin (Savov et al., 2006) . Among the studies that evaluated the antiviral effect of quercetin, six used this flavonoid orally (6/7; 85.7%) at doses ranging from 1 to 12.5 mg/Kg (Davis et al., 2008; Farazuddin et al., 2018; Ganesan et al., 2012; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000) , and one used it intraperitoneally (1/7; 14.3%) at a dosage of 20 mg/Kg alone or combined with rutin (Savov et al., 2006 Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000) or as a diet containing 0.1% quercetin and offered ad libitum (1/6; 16.7%) (Farazuddin et al., 2018) . The quercetin derivatives quercitrin and quercetin-3-O-β-Dglucuronide were used orally at doses of 6.25 mg/Kg (Choi et al., 2012 ) and 3 or 6 mg/Kg (Fan et al., 2011) , respectively. Isoquercetin was administered to infected animals at doses of 2 or 10 mg/Kg intraperitoneally (Y. Kim et al., 2010) , and the studies on the therapeutic effects of isorhamnetin utilized a local use (intranasal) of this aglycone at 1 mg/Kg (Dayem et al., 2015) . Ganesan et al., 2012; Kumar et al., 2003 Kumar et al., , 2005 Raju et al., 2000) , two before infection (prophylaxis) (2/11; 18.2%) (Davis et al., 2008; Farazuddin et al., 2018) , and three used the compounds in a combined schedule of pre-and posttreatment (3/11; 27.3%) (Choi et al., 2012; Y. Kim et al., 2010; Savov et al., 2006) . The average time for studies using pretreatment was 8.5 days, while for those that used posttreatment was 5.2 days. In the schemes that combined pre-and posttreatment, the compounds were administered on an average of 2.5 days before infection and maintained for up to 6 days after the start of the treatment.

Three (Choi et al., 2012; Davis et al., 2008; Dayem et al., 2015) 

Among the included studies, 5 described the effect of quercetin and its derivatives on the pulmonary viral load of mice infected with respiratory viruses (Choi et al., 2012; Dayem et al., 2015; Farazuddin et al., 2018; Ganesan et al., 2012; Y. Kim et al., 2010) .

Two of these studies used RV to induce infection and assessed viral load indirectly by quantifying copies of viral RNA by RT-PCR and, therefore, were excluded from the meta-analysis (Farazuddin et al., 2018; Ganesan et al., 2012) . However, in both studies, quercetin was administered orally and reduced the viral lung load of animals infected with RV, suggesting that it possesses antiviral activity against this RNA virus. The remaining 3 studies evaluated the effect of quercetin derivatives on pulmonary viral load in IV H1N1 models.

These studies included 26 animals, 5 of which received quercitrin, 3 isorhamnetin, 5 isoquercetin, and 13 received placebo. Figure 3 shows that animals infected with IV and treated with quercetin derivatives had a significantly lower pulmonary viral load than ani- 

Typically, viral infections of the lower respiratory tract induce significant changes in airway dynamics, which can be measured by the reduction in expiratory flow rates, an increase in airway resistance, or disturbances in dynamic lung compliance (Holtzman et al., 2005) . Two of the included studies investigated the therapeutic benefits of quercetin's oral use on airway resistance induced by RV. According to 

Four studies (Kumar et al., 2003 (Kumar et al., , 2005 Raju et al., 2000; Savov et al., 2006) investigated the effect of quercetin on ROS-producing and ROS-scavenging enzymes induced by IV (H3N2). Studies have shown that the use of quercetin orally or intraperitoneally (combined or not with rutin) reduced the pulmonary oxidative damage induced by the IV (H3N2), as suggested by the reduction in the levels of lipid peroxidation markers (i.e., the reactive substances to thiobarbituric acid

[TBARS]) (Raju et al., 2000; Savov et al., 2006) . Quercetin has been shown to affect superoxide dismutase production in pulmonary macrophages, reducing ROS generation by immune lung cells (Raju et al., 2000; Savov et al., 2006) . The antioxidant effect of this flavonoid was also associated with the modulation of antioxidant enzyme activity. In this context, quercetin increased the catalytic efficiency of catalase and superoxide dismutase in infected BALB/c mice (Kumar et al., 2005) , but not in Swiss mice (Raju et al., 2000) . in liver tissue (Savov et al., 2006) . However, the levels of natural antioxidants that are often reduced due to viral infection, such as vitamin E, were not re-established after oral administration of quercetin (Kumar et al., 2005) . vegetables, seeds, buckwheat, nuts, flowers, barks, broccoli, olive oil, apples, onions, green tea, red grapes, red wine, dark cherries, and berries, such as blueberries and cranberries (Anand David et al., 2016; Jan et al., 2010) . Furthermore, these flavonoids are the most abundant phenolic compounds in apitherapy products, being recognized as the active component of honey, propolis, royal jelly, beeswax, and pollen (Fratellone et al., 2016; Lima et al., 2020a) .

Among the main clinical applications of bee products, therapeutic and prophylactic use against respiratory diseases of viral etiology stands out. In this context, a study showed that the frequency of flu during an influenza outbreak was significantly lower (3.7% incidence) among patients who used an apicomplex composed of honey, royal jelly (2%), pollen (3%), and propolis (1%) in comparison to untreated patients (38% incidence) (Bratko & Miha, 1976) . Several studies report that quercetin and its derivatives are the main active ingredients in these bee products responsible for their antiviral activity (Lima et al., 2020a; Mohamed, Hassan, Hammad, Amer, & Riad, 2015; Schnitzler et al., 2010; Watanabe, Rahmasari, Matsunaga, Haruyama, & Kobayashi, 2014) . Moreover, many in vitro assays confirmed the activity of flavonol against respiratory viruses of medical importance (Jan et al., 2010; Kaul et al., 1985) . showed that the use of quercetin or its derivatives prevented lung damage, reduced inflammation, and oxidative stress, and minimized viral infection effects on respiratory dynamics ( Figure 5 ).

The antiviral activity of quercetin and its derivatives is mainly due to the inhibition of virus entry into the host cell, a crucial stage during viral infections (Biancatelli et al., 2020; Ganesan et al., 2012; Wu et al., 2015) . Some studies have demonstrated that quercetin effectively inhibits IV (Wu et al., 2015) and RV (Ganesan et al., 2012) cells, quercetin reduced the cytopathic effect when administered during viral entry, which was dependent on its binding to hemagglutinin proteins (HA) (Wu et al., 2015) . The binding of IV HA to sialic acids presented by cellular receptors triggers virus cell entry by clathrinmediated endocytosis (Benton et al., 2018) . Thus, the interaction of quercetin with this molecular target justifies its suggested mechanism of action.

Moreover, an in silico study suggested that quercetin interacts with neuraminidase (NA) and acts in the late stages of the viral life cycle. The binding of quercetin to the active site of the H1N1 NA crystallographic structure was more stable (binding energy of −6.8

Kcal/mol) than that observed for oseltamivir (binding energy of −6.8

Kcal/mol), a potent and selective inhibitor of IV NA enzymes (Sadati, Gheibi, Ranjbar, & Hashemzadeh, 2019) . Immunomodulatory properties are also well reported for quercetin and contribute to its therapeutic effect in animals with viral pneumonia. For instance, a study showed that the antiviral activity of TNF against vesicular stomatitis virus and encephalomyocarditis virus is greatly enhanced by quercetin in WISH cells. However, polyclonal antibodies to interferon completely blocked this effect, suggesting that the antiviral activity of quercetin may be mediated by interferon induction (Ohnishi & Bannai, 1993) . Nair et al. (2002) confirmed that the antiviral activity of quercetin is mediated by IFN-γ production. In this study, quercetin at a concentration of 5 μM, similar to plasma levels, stimulated T-helper cells to produce and secrete (Th-1)-derived IFN-γ, enhancing cell-mediated antiviral immunity (Nair et al., 2002) .

Dietary intake of quercetin has also been shown to increase antiviral immune tone. Mice feed with a diet rich in polyphenols (gallic acid, catechin, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, sinapic acid, ferulic acid, quercetin, and rutin) for 5 weeks showed an increase in macrophage chemotaxis, phagocytosis, microbicidal activity, interleukin-2 release, natural killer activity, and lymphoproliferative response to concanavalin A and lipopolysaccharide compared to F I G U R E 5 Schematic summary of the clinical activity of quercetin-type flavonols in animals with virus-induced lower respiratory tract infections animals that received control diet ( Alvarez et al., 2006) . Thus, the combination of direct (i.e., inhibition of the entry and release of viral particles) and indirect antiviral effects (i.e., increased antiviral immune tone) of quercetin contribute to the reduction of the pulmonary viral load observed in the meta-analysis. Due to the direct relationship between viral load and disease lethality (Ngaosuwankul et al., 2010) , these data also support the decrease in mortality found among animals that received supplementation with quercetin or its glycosylated derivatives.

Respiratory virus infections change the neural control of the airway's smooth muscle and are associated with an increase in air flux resistance (Farazuddin et al., 2018; Fryer & Jocoby, 1991; Rynko, Fryer, & Jacoby, 2014) . Physiologically, parasympathetic nerves release acetylcholine (ACh) onto M 3 muscarinic receptors on airway smooth muscle, causing muscle contraction, and bronchoconstriction.

ACh also activates M 2 muscarinic receptors on postganglionic nerves, inhibiting further ACh release, and limiting bronchoconstriction (Fryer & Jacoby, 1998) . However, in viral infections of airways, the M 2 receptors in parasympathetic nerves are dysfunctional, which cause loss of M 2 receptor-mediated negative feedback and increase

ACh release onto airway smooth muscle, producing intense bronchoconstriction (Farazuddin et al., 2018; Fryer & Jocoby, 1991; Rynko et al., 2014) . Parainfluenza virus, for example, decreases M 2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals (Rynko et al., 2014) and impairs the bronchodilatation effect of pilocarpine, an agent that stimulates inhibitory M 2 muscarinic receptors on parasympathetic nerves (Fryer & Jocoby, 1991) . Quercetin and its derivatives are known to reduce airway resistance induced by viral infections by reducing virus-induced M 2 receptor dysfunction (Farazuddin et al., 2018; Ganesan et al., 2012) . A study showed that the treatment with quercetin reverts the (Li et al., 2016) . On the other hand, quercetin can also inhibit the proliferation of cells that synthesize IL-4, IL-5, IL-6, IL-10, and IL-13 by inducing the production of Th-1-derived IFN-γ (Li et al., 2016; Nair et al., 2002) . Another characteristic of quercetin is the negative regulation of the vascular cell adhesion molecule 1 (VCAM-1) and the expression of CD80 . VCAM-1 is usually expressed on the membrane of lung endothelial cells and is involved in the adhesion and migration of monocytes, lymphocytes, eosinophils, and basophils to the pulmonary tissue (Muller, 2011) . Furthermore, quercetin also inhibits the production of inflammation-producing enzymes, such as cyclooxygenase-2 and lipoxygenase, by blocking the activation of PI3K. Thus, it compromises the synthesis of inflammatory mediator prostaglandin E2 (Li et al., 2016; Xiao et al., 2011) .

Markers of redox misbalance in blood and lung are often taken into account in viral pneumonia. In general, lung infections caused by respiratory viruses are associated with cytokine production, inflammation, cell death, and other pathological processes, which could be triggered by enhanced reactive oxygen or nitrogen species (ROS and RNS) production. Quercetin has been shown to act against lung oxidative stress caused by the virus through various mechanisms, such as reducing the generation of ROS (Raju et al., 2000; Savov et al., 2006) , increasing the expression of antioxidant enzymes (Kumar et al., 2005) , and inducing the metabolism of compounds with oxidative proprieties (Savov et al., 2006) . Interestingly, Raju et al. (2000) showed that supplementation with quercetin alone in healthy mice did not significantly affect lipid peroxidation. However, after the viral infection, it showed a significant decrease in the lipid peroxide level. Together, these studies demonstrate that supplementation with quercetin alleviates the toxic effects of free radicals induced during viral infection by influencing signal transduction pathways that modulate the antioxidant properties of organisms, thereby preventing disease development.

Mucus is a viscoelastic fluid produced by mucous membranes composed of glycoproteins and proteoglycans with a crucial protective function (Williams, Sharafkhaneh, Kim, Dickey, & Evans, 2006) .

However, in respiratory infectious disease, the production and secretion of mucus are markedly upregulated, and this excess can reach the back of the throat and lungs and enter the trachea. Therefore, it is important to control and reduce this exacerbated mucus production in respiratory tract infections because it is associated with airway obstruction and mucociliary clearance impairment, which results in the development of debilitating airflow limitation and particulate/ pathogen retention, respectively. Mucus hypersecretion may result from an increase in the steady-state levels of mucin production, mucin exocytosis, or both. Mucin is produced by the Gob5 (or mCLCA3) protein, a member of the calcium-dependent chloride channel 1 (CLCA1)

family of proteins considered a new therapeutic target to treat hypersecretory airway diseases. Quercetin downregulated the Gob5 gene in mice infected with IV, justifying the reduction in mucus production in these animals. It is also in accordance with the improvement of airway resistance observed by some authors. Thus, these studies show that quercetin reduces mucus production by decreasing the synthesis at the gene level. However, the effect of these flavonoids on the exocytosis of mucus-containing vesicles must be determined in future studies.

This meta-analysis has some limitations. First, multiple variables influence the overall therapeutic effect of the studied compounds, such as the wide variety of animals, scheme of treatment used (dose, period, administration via), and type of derivative used. Second, the included studies evaluated the effect of quercetin and its derivatives mostly against IV and HR, which limits the extrapolation of the associations shown here for other respiratory viruses, such as human respiratory syncytial virus, human metapneumovirus, parainfluenza, and coronavirus (e.g., SARS-CoV, MERS-CoV, and SARS-CoV-2). Third, none of the studies identified the limitations of the experimental design employed, which may compromise the observed correlations.

Fourth, the heterogeneity of the studies analyzed in this paper is considerably high, which tends to weaken the robustness of our findings.

Fifth, almost all articles included represented the various factors evaluated graphically (e.g., cytokine levels, airway resistance rate, markers of oxidative damage, and mucus content), which restricted our metaanalysis only to the lethality curve and viral lung load. Sixth, the protocol of this review was not preregistered; consequently, it is not easily available for access, which may introduce a potential additional bias in the study. Finally, it is worth emphasizing that the correlation found in the meta-analysis does not imply any causation, and there is always the possibility of residual confounding in the included studies.

Supplementation with quercetin and its glycosylated or aglycone derivatives by oral, local, or parenteral routes reduces the lethality and pulmonary viral load of mice infected with IV. Moreover, these flavonols reduce the inflammatory process, oxidative damage, airway resistance, mucus hypersecretion, and tissue necrosis associated with the respiratory virus. These findings support the potential of quercetin and its derivatives as a curative agent against viral pneumonia. Furthermore, due to their lack of severe side effects and low cost, quercetin may be developed as a safer and cheaper option for the prophylaxis of seasonal or emerging respiratory viral infections, helping to control outbreaks, epidemics, and/or pandemics.

W.G.L. is grateful to Coordenação de Aperfeiçoamento de Pessoal do Nível Superior (CAPES) for a PhD fellowship.

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The authors declare that they have no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.

The data that support the findings of this study are available from the corresponding author upon reasonable request.