Natural Antioxidants: A Review of Studies on Human and Animal Coronavirus

The outbreaks of viruses with wide spread and mortality in the world population have motivated the research for new therapeutic approaches. There are several viruses that cause a biochemical imbalance in the infected cell resulting in oxidative stress. These effects may be associated with the development of pathologies and worsening of symptoms. Therefore, this review is aimed at discussing natural compounds with both antioxidant and antiviral activities, specifically against coronavirus infection, in an attempt to contribute to global researches for discovering effective therapeutic agents in the treatment of coronavirus infection and its severe clinical complications. The contribution of the possible action of these compounds on metabolic modulation associated with antiviral properties, in addition to other mechanisms of action, is presented.


Introduction
Coronaviruses (CoVs) belong to a family of enveloped viruses with a positive sense, single-stranded RNA genome. CoVs cause illness ranging from upper respiratory tract infections (URTIs) resembling the common cold to lower respiratory tract infections (LRTIs) such as bronchitis, pneumonia, and even severe acute respiratory syndrome (SARS) with most serious disease outcomes in the elderly, immunocompromised patients, and infants [1,2]. HCoV-OC43 (OC43), HCoV-229E (229E), HCoV-NL63 (NL63), and HCoV-HKU1 (HKU1) were the first documented human CoVs (HCoVs), which usually cause URTIs and less frequently are associated with LTRI diseases [3]. In the last decades, two human coronaviruses created great concern for the world medical community due to significant disease and mortality [4,5]. In 2003, severe acute respiratory syndrome-coronavirus (SARS-CoV) was characterized by acute atypical pneumonia and diffuse alveolar damage (DAD) in roughly 8000 patients and with almost 800 deaths, representing a nearly 10% mortality rate [6]. More recently, in 2012, a new human coronavirus, designated as Middle East respiratory syndrome-coronavirus (MERS-CoV), was identified, and the global ongoing outbreak of MERS with over 2519 official cases and 866 deaths represented approximately 34% case fatality rate to date in humans [7].
Over the last few months, a new strain of human coronavirus, SARS-CoV-2 (also known as 2019-nCoV), has caught the world's seven continents' attention with its rapid global spread, affecting at least 200 countries and territories, infecting more than 3,000,000 and claiming more than 202,597 lives worldwide [8]. The coronavirus pandemic has promoted isolation and uncertainly fear and panic worldwide. In addition, it will likely lead to changes in political and economic power in ways that can be determined only later [9].
It is important to note that there are many similarities among different coronavirus species, but not in all aspects.
Depending on the molecular mechanism of viral inhibition promoted by an antiviral agent, the analysis of the data and comparison between animal and human CoVs must be done very carefully. In fact, it is important to note that there are differences between human and animal CoV receptors, which will likely result in different affinities, or unlikely interactions, of an antiviral agent with the different CoV receptors. However, if the antiviral agent interferes with the replication and/or assembly of the CoVs, there is a higher probability of obtaining similar antiviral activity results in human CoV tests [1,2,10,11]. Following this line, our search in specialized literature was focused, mainly, on studies that investigated the anticoronavirus effects of natural antioxidants by inhibiting proteases for viral replication.

Materials and Methods
The present study was carried out based on a search of the literature of natural antioxidants and coronavirus. The search, performed in the PubMed database, included studies published until March 2020 and used the following keywords: coronavirus, antioxidants, flavonoids, oxidative stress, MERS-CoV; SARS-CoV, 229E, NL63, OC43, HKU1, MERS-CoV virus infection; and Middle East Respiratory Syndrome Virus. The scientific publications were selected from studies published in the English language.

Pathogenic Mechanism of Coronavirus-Induced Cell Damage
The high mortality rate associated with the three pathogenic HCoVs has been mainly attributed to the development of digestive and respiratory tract injuries observed following infection. Acute atypical pneumonia and diffuse alveolar damage that progress to deposition of fibrous tissue, denuded airways, haemorrhage, and elevated macrophage infiltration are sometimes accompanied by watery diarrhoea, dehydration, and vomiting [2,12,13]. Despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidated and still unclear, studies have suggested that late-term disease progression is unrelated to viremia. It is now believed more likely to be associated with the immunopathological mechanism [14,15]. Viral clearance and subsequent recovery from infection require activation of an effective host immune response; however, many immune effector cells may also cause injury to host tissues [16]. Together with inflammatory and immune response signaling, the presence of oxidative compounds, such as reactive oxygen species (ROS), plays important roles in the pathogenic mechanism of cell damage induced by CoVs through oxidative stress [17].
Oxidative stress is defined as an interruption and/or deregulation of the signaling and redox system that can be caused by an imbalance in the production of oxidant and antioxidant species [18]. Among the main oxidant agents, ROS and reactive nitrogen species (RNS) stand out. In order to counterbalance the oxidant species, there is an antioxidant system formed by enzymes and nonenzymatic molecules [19,20]. However, during pathological events, such as viral infec-tions, there may be an increase in the production of oxidant species not neutralized by the antioxidant system, resulting in oxidative stress that promotes cellular damage through protein denaturation, changes in the functions of nucleic acids, lipid peroxidation, and cell death [21][22][23].
In addition, during viral infection, oxidative stress contributes to viral pathogenesis through stimulating inflammation, loss of immune function, and increased viral replication that may occur due to the activation of the nuclear factor kappa B (NF-κB) transcription pathway [24][25][26]. Current evidence suggests that cytokine dysregulation-also called cytokine storm-contributes to severe disease caused by the pathogenic CoVs [27,28]. The exact mechanisms are not clear yet, but research on influenza A virus shows that infection causes a rapid influx of inflammatory cells. This is followed by an increase in reactive oxygen species production and cytokine expression and release, which ultimately leads to acute lung injury [29]. In general, RNA viruses promote changes in the body's antioxidant defense system, affecting enzymes such as superoxide dismutase (SOD) and catalase (CAT), in addition to reducing the levels of antioxidant molecules such as ascorbic acid, carotenoids, and reduced glutathione (GSH) [30][31][32]. Wu et al. reported that glucose-6-phosphate dehydrogenase-(an important antioxidant enzyme that produces NADPH) knockdown cells were more susceptible to infection by HCoV-229E than normal cells [33]. Interestingly, Ye and colleagues have reported that the inhibition of ROS production alleviates inflammation caused by influenza A virus infections [29].
In an experimental model of SARS-induced acute lung injury in mice, it was noted that phospholipid oxidation, due to oxidative stress, is one of the main triggering factors of acute lung injury. This happens through the activation of the innate immune response, culminating in the activation of pulmonary macrophages via TLR4-TRIF-TRAF6-NF-κB signaling [17]. Furthermore, hypoxia caused by acute lung injury can cause myocardial injury due to the production of ROS, aggravating infections caused by coronavirus disease 2019 (COVID-19) [34].
Mitochondria have an essential function in energy generation, and for this reason, their function and integrity are strictly regulated in order to respond to varying energy requirements and environmental conditions [35]. Mitochondria are known to function as the control point in apoptotic pathways, releasing proapoptotic factors, mainly ROS, which function as a signaling molecule that may result in cell death [36,37]. Some studies have shown a relationship between coronavirus infection and dysfunctional or damaged mitochondria, leading to the release of ROS and other proapoptotic substances [38,39]. In a recent study, Xu et al. reported that ROS and p53 play key roles in regulating many kinds of the cell process during coronavirus infection in Vero cells. According to the authors, coronavirus infection appears to induce a time-dependent ROS accumulation, which in turn is linked to regulatory mechanisms of p53 activation and apoptosis in infected cells [40].
Antioxidant substances promote improvement in cases of disease caused by coronaviruses, such as apolipoprotein D-a lipocalin that promoted a neuroprotective effect against 2 Oxidative Medicine and Cellular Longevity encephalitis induced by human coronavirus OC43-in mice. This protective effect occurred through the reduction of oxidative stress, cerebral lipid peroxidation, and regulation of inflammation [41,42]. Also, the treatment with antioxidants, such as pyrrolidine dithiocarbamate or N-acetylcysteine, significantly inhibits coronavirus-induced apoptosis [43]. Moreover, melatonin promotes downregulation of acute lung oxidative injury due to its anti-inflammatory and antioxidant actions, making it a possible compound in the treatment of COVID-19 [44]. Based on these studies, compounds that have antioxidant actions can be helpful in the treatment of infections promoted by coronavirus.
In general, antioxidant properties of polyphenolic compounds, such as some flavonoids, have been associated with the presence of aromatic phenolic rings that promote the electron donation and hydrogen atom transfer to free radicals, acting as free radical scavengers, reducing agents, and quenchers of single oxygen formation [45]. Thus, the aim of this study was to investigate the antioxidant capacity and antiviral activity of natural antioxidants against coronavirus. The compounds are illustrated in Figure 1.

Occurrence and Antioxidant Properties of Anticoronavirus Compounds
Quercetin can be found in plants such as Rubus fruticosus L. and Lagerstroemia speciosa (L.) Pers. [46,47]. Also, quercetin shows antioxidant activity at a concentration of 10 μmol/L in HepG2 cells, inhibiting oxidative stress promoted by H 2 O 2 [48], promotes an increase in SOD, CAT, and glutathione peroxidase (GPx), and reduces lipid peroxidation in rats with chronic prostatitis/chronic pelvic pain syndrome [49]. Moreover, quercetin improves sepsis-induced acute lung injury in rats, by reducing lipid peroxidation and inflammation and increasing SOD and CAT levels [50]. In addition, quercetin glycosides with antioxidant activity, such as quercetin 3-β-glucoside, have already been isolated from plants such as Passiflora subpeltata Ortega and Chamomilla suaveolens (Pursh) Rydb. [51,52]. The administration of quercetin 3-β-glucoside (40 mg/kg p.o.) in streptozotocin-induced diabetic rats promotes an increase in the levels of antioxidant enzymes (SOD, CAT, and GPx) and nonenzymatic antioxidants (vitamins C and E and GSH) and a reduction of lipid peroxidation [53]. Quercetin 3-β-galactoside (hyperoside) is found mainly in plants of the Hypericum genus such as Hypericum perforatum L. [54,55]. Moreover, it showed cardioprotective activity in high glucose-induced injury of myocardial cells through decreased apoptosis and ROS production and increased SOD levels [56]. Quercetin 7-ramnoside is also found in plants of the Hypericum genus such as Hypericum japonicum Thunb. ex Murray. This flavonoid shows hepatoprotective activity against carbon tetrachloride in mice by decreasing lipid peroxidation and increasing CAT and GSH levels, in addition to presenting values of 118.75 μM and 128.47 μM in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 ′ -azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays, respectively [57].
Epigallocatechin gallate is present in Parkia roxburghii G. Don and is one of the main metabolites found in green tea and Liubao tea (Camellia sinensis LO Kuntze). Also, gallocatechin gallate can be found in this plant [58][59][60][61]. Literature data reveal that the administration (i.p.) of 2.5 mg/100 g of epigallocatechin gallate in rats, with streptozotocin-induced diabetes mellitus, promotes a reduction in oxidative stress through reductions in parameters such as indirect nitric oxide synthesis and status total oxidative, as well as an increase in levels of CAT and total antioxidant capacity of plasma [62]. Furthermore, it promotes cardioprotection by antioxidant mechanisms [63].
Luteolin is present in foods such as carrot, cabbage, tea, and apple and is found in Ugni molinae Turcz. [73,74]. Data show that luteolin (50 μg/mL) increases the levels of GSH, the expression of GSH synthetase, and the activity of SOD and CAT in human colon cancer cells (HT-29) [75]. Furthermore, luteolin attenuates the sepsis-induced acute lung injury in mice by reducing lipid peroxidation and increasing SOD and CAT activity, in addition to suppressing the NF-κB pathway [76].
Herbacetin is ubiquitous in plants of the genus Rhodiola, such as Rhodiola rosea L. [77]. Herbacetin glycosides are also present in the roots of R. sachalinensis A. Bor and show antioxidant activity [78]. Veeramani Figure 1: Chemical structures of bioactive antioxidants against coronavirus. 4 Oxidative Medicine and Cellular Longevity Catechin Scavenging of free radicals [86] 5 Oxidative Medicine and Cellular Longevity injury induced by D-galactosamine by increasing levels of SOD, GSH, glutathione reductase, and glutathione Stransferase [80,81].
Psoralidin is a prenylated coumestan, which is found in plants of the Fabaceae family, such as Psoralea corylifolia L. Xiao and collaborators, investigating the antioxidant potential of compounds isolated from P. corylifolia, observed that psoralidin shows the best antioxidant activity by the method of electron spin resonance spectroscopy with an IC 50 value of 44.7 μM [87].
The compound isobavachalcone has been isolated from plants of the Fabaceae and Moraceae families [88,89]. Isobavachalcone shows a strong antioxidant activity in DPPH SC 50 , FRAP, and ABTS SC 50 assays with values of 250.8 μM, 0:4 ± 0:05 mM equivalent to FeSO 4 ·7H 2 O, and 510.1 mM, respectively. In addition, the compound has been reported to inhibit the NF-κB pathway in Sephadex-induced lung injury in rats [88,90].
Helichrysetin is a chalcone that is found in plants of the Helichrysum genus such as Helichrysum odoratissimum L. [91]. In a study investigating the antioxidant activity of natural and prenylated chalcones, Vogel et al. found that helichrysetin is the substance that shows the highest antioxidant activity in the ORAC test with values of 4:4 ± 0:6 Trolox equivalents [92].
Resveratrol is found in grapes, peanuts, and blueberries and can be isolated from Veratrum grandiflorum O. Loes [100]. Literature shows that resveratrol has good antioxidant activity with DPPH SC 50 /r 2 values of 26:37/0:849 μmol/dm. Moreover, it is able to reduce the production of ROS by inhibiting the activity and expression of NADPH oxidases, by eliminating oxidant agents, including radical hydroxyl, superoxide, hydrogen peroxide, and peroxynitrite [101,102]. The treatment of resveratrol (50 mg/kg p.o.) in rats reduces oxidative stress in obstructive lung disease by increasing SOD activity and reducing MDA levels, indicating a decrease in lipid peroxidation [103]. Table 1 shows the main actions of natural antioxidants discussed in this study, and Figure 2 illustrates these activities.

Effect of Natural Antioxidants in Coronavirus Infections
This review focused on studies reporting on the anticoronavirus activity of natural antioxidants. Based on exclusion criteria, data from nineteen compounds were discussed. The oxidative stress pathway could potentially be a key element in coronavirus-induced apoptosis and pathogenesis [104]. For this reason, it is interesting to investigate the use of antioxidants as potential therapeutic tools-either as an alternative or as an adjuvant to conventional therapies-in the treatment of coronavirus infections. Among the antioxidant compounds evaluated as for coronavirus infections are the flavonoids, which are compounds widely found in fruits, vegetables, and certain beverages. In fact, research groups have reported that antioxidant flavonoids, including (+)-catechin, luteolin, apigenin, quercetin, and quercetin 7rhamnoside, inhibit ROS accumulation and apoptosis of cells infected with different coronavirus, including porcine epidemic diarrhoea coronavirus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) [105][106][107].
As shown with the recent COVID-19 pandemic, the search for alternative or new antiviral therapies for the treatment of coronavirus diseases remains important. Based on the literature, antioxidant therapies offer an attractive option.

Inhibition of SARS-CoV replication
Myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity [116] 8 Oxidative Medicine and Cellular Longevity SARS-CoV with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory results, mainly because corticosteroids exert immunosuppressor effects on the humoral and cellular immune systems [108,109]. Other drugs such as pentoxifylline were considered for the treatment of SARS due to its interesting therapeutic properties that include anti-inflammatory, antiviral, immunomodulatory, and bronchodilatory effects. However, it too was not successful in the clinical treatment of SARS-CoV infection [110]. Many antioxidant compounds show antiviral activity against SARS-CoV. The antiviral activity has been mainly attributed to the inhibition of the 3C-like protease (3CL pro ) of SARS-CoV, a vital enzyme for SARS-CoV replication [111]. As an example, multiples studies have reported that quercetin and quercetin-derived compounds, such as quercetin 3-β-galactoside, display potent 3CL pro inhibitory e5ffect and consequent reduction of SARS-CoV replication [112]. Other antioxidants, such as epigallocatechin gallate, gallocatechin gallate, amentoflavone, apigenin, luteolin, herbacetin, rhoifolin, and pectolinarin, are also found to efficiently block the enzymatic activity of SARS-CoV 3CL pro [111,113,114].
Moreover, some natural antioxidants exhibit promising antiviral activity against SARS-CoV infection by interfering with different targets involved in SARS-CoV replication, in particular the SARS-CoV papain-like protease (PL pro ) and SARS-CoV helicase protein. Kim et al. reported that isobavachalcone and psoralidin inhibit PL pro in a dose-dependent manner with IC 50 ranging between 4.2 and 38.4 μM [115]. Previously, Yu et al. reported that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity [116].
MERS-CoV is another zoonotic coronavirus transmitted between animals and human beings that causes severe morbidity and mortality. No antiviral medicines with satisfactory efficacy for the treatment of MERS-CoV-infected patients have been identified to date. Similar to SARS-CoV, natural antioxidant libraries have been probed for potential inhibitory compounds against MERS-CoV 3Clike protease. Jo et al. showed that herbacetin, isobavachalcone, quercetin 3-β-d-glucoside, and helichrysetin, four compounds with recognized antioxidant activity, can block the enzymatic activity of MERS-CoV 3CL pro using a tryptophan-based fluorescence method. Furthermore, the experimental and computational studies show that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of MERS-CoV 3CL pro [117].
In a study performed by Lin et al., the antiviral activities of resveratrol were investigated in MERS-infected Vero E6 cells. The authors reported a significant inhibition of MERS-CoV infection and prolonged host cell survival after virus infection, which they speculate was promoted by resveratrol. In addition, they also found that the expression of the nucleocapsid (N) protein, which is essential for MERS-CoV replication, is decreased after resveratrol treatment [118]. It is important to mention that in vitro models of coronavirus infection also show antiviral activity of flavonoids extracted from flowering cherry cultivars and black tea [119,120]. Finally, antioxidants, such as resveratrol, also are able to block infection produced by herpesvirus [121,122]. The discovery of antiviral compounds from a bioactive compound against other viruses is an interesting strategy for obtaining new antiviral drugs. Table 2 shows the main actions of the natural antioxidants against the coronavirus, and Figure 3 summarizes these activities.   Oxidative Medicine and Cellular Longevity

Conclusions
In conclusion, this review shows that antioxidant compounds, prominently flavonoids, exhibit antiviral action in models of coronavirus infections. In general, the antiviral activity might be attributed, at least in part, to the inhibitory effect on the enzymatic activity of targets involved in coronavirus replication, including SARS-CoV 3CL pro , SARS-CoV papain-like protease (PL pro ), SARS-CoV helicase protein, and MERS-CoV 3CL pro . In addition, some studies provide evidence that the reduction of ROS accumulation retards the coronavirus-activated apoptotic signaling. Therefore, the mechanisms of oxidative stress could be the key element to be studied in coronavirus infections, including those related to inflammatory processes arising from the action of this virus. Obviously, further investigations are needed to elucidate other pharmacological mechanisms by which natural antioxidants play an antiviral effect. Despite the findings reported in this review, they cannot be generalized to COVID-19. However, the data provided support to the investigation of natural antioxidants as a potential therapeutic approach in the treatment for COVID-19 and its severe clinical complications, either as an alternative or as an adjuvant to conventional therapies, and contribute to the search for new prototypes in the development of drugs against coronavirus infections.

Disclosure
Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors, and therefore, the NRF does not accept any liability in regard thereto.

Conflicts of Interest
No potential conflict of interest was reported by the authors.