Pharmacological Significance of Hesperidin and Hesperetin,Two Citrus Flavonoids,as Promising Antiviral Compounds for Prophylaxis Against and Combating COVID-19

Abstract

Hesperidin and hesperetin are flavonoids that are abundantly present as constituents of citrus fruits. These compounds have attracted attention as several computational methods, mostly docking studies, have shown that hesperidin may bind to multiple regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (spike protein, angiotensin-converting enzyme 2, and proteases). Hesperidin has a low binding energy, both with the SARS-CoV-2 “spike” protein responsible for internalization, and also with the “PL^pro” and “M^pro” responsible for transforming the early proteins of the virus into the complex responsible for viral replication. This suggests that these flavonoids could act as prophylactic agents by blocking several mechanisms of viral infection and replication, and thus helping the host cell to resist viral attack.

Keywords

citrus flavonoids hesperidin hesperetin prophylaxis antiviral COVID-19

Introduction

The coronavirus disease 2019 (COVID-19) epidemic that originated at the beginning of 2020 in Wuhan, China has now spread worldwide and has become an international pandemic. It was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new member of the coronavirus family.^1,2 Despite the development of vaccines, zoonotic SARS-CoV continues to be a major threat to humans, and most research groups do not exclude the possibility of the continual reemergence of severe acute respiratory syndrome (SARS).

Structure of SARS-CoV-2

The genome of SARS-CoV-2 is a positive-sense, single-stranded ribonucleic acid of approximately 30 000 nucleotides in length, encapsulated within a membrane envelope. It has 4 structural proteins, known as S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope (Figure 1).

Figure 1.

Schematic diagram of SARS-COV-2 reproduced from open-source CDC and anatomy of a Killer. Credit: https://www.economist.com/briefing/2020/03/12/understanding-sars-cov-2-and-the-drugs-that-might-lessen-its-power.

The S protein on the virion surface is primarily responsible for the establishment of host-protein receptor recognition for binding to the surface of a host cell through a cell membrane protein receptor called angiotensin-converting enzyme 2 (ACE2).² The transmembrane protease, serine-2, helps in priming of the S protein and subsequent membrane fusion.⁶ The S protein is cleaved into S1 and S2 subunits during viral infection.⁸ The S1 subunit contains the receptor-binding domain (RBD), which directly binds to the peptidase domain (PD) of ACE2,¹¹ while the S2 subunit is responsible for membrane fusion. ACE2 is expressed in several organs such as lungs, gastrointestinal tract, kidney, endothelium, heart, and, to some extent, in pancreas.¹³ ACE2 has been demonstrated to be a functional receptor for SARS-CoV.¹⁸ It has also been shown that SARS-CoV-2 RBD was capable of entering cells expressing human ACE2, but not any other receptors.¹³

The viral gene essential for the replication of the virus is expressed as a polyprotein that must be broken into functional subunits for replication and transcription activity.¹⁹ The viral genome encodes more than 20 proteins, among which are 2 virally encoded cysteine proteases that are vital for virus replication, SARS-CoV-2 papain-like protease (PL^pro) and SARS-CoV-2 main protease, M^pro, also referred to as the 3C-like protease (3CL^pro); they cleave the 2 translated polyproteins (pp1a and pp1ab) into individual 16 functional nonstructural proteins (NSPs).²⁰ Each of these NSPs has its own specific function in replication and transcription.^27,28 These NSPs, with the help of host machinery, translate the RNA coding for the viral spike, envelope, nucleocapsid, and membrane proteins and thus are involved in replication events and transcription of the genome.²⁹ These NSPs then enter the endoplasmic reticulum—Golgi apparatus and are involved in viral assembly and packaging. The SARS-CoV-2 S protein contains a protease cleavage site and is likely processed by these intracellular proteases during exit from the cell.³² The new viral particles spread into the environment and/or infect other cells and organs in the body, in a chain expansion.

Targeted Inhibition Sites for Virus Infection

Based on the above concise description, enzymes/proteins from either the host or the virus could be targeted for either inhibiting or reducing viral infection. These involve a number of potentially targetable steps, including (a) endocytic entry of the virus into host cells, that is, preventing viral entry by either interfering with the binding of viral S-proteins to human ACE2 receptors or by inhibiting the function of the ACE2 receptor, (b) inhibiting viral replication by either blocking RNA dependent RNA polymerase (RdRp) or proteases (involving M^pro- and/or PL^pro), as these are responsible for viral replication after entry into cells for new virus particle assembly, (c) helping the cell to resist viral attack, (d) blocking the spread of the virus in the body, and (e) modulating the inflammation when starting as an innate defensive mechanism, it becomes offensive and cytotoxic.^10,33,34

The search for naturally occurring bioactive compounds is part of the general vision that perceives that the boost to the individual immune system, and effective inhibition of the infection by SARS-CoV-2 as the most effective shield against the onset and serious progress of COVID-19.³⁵

Hesperidin and Hesperetin

Chemically, hesperidin (1) is a glycoside having rutinose (α-L-rhamnopyranosyl-[1→6]-β-D-glucopyranose) linked to OH-7 of hesperetin (2) [(3´,5,7-trihydroxy-4´-methoxy-flavanone), or (S)-5-hydroxy-2-(3-hydroxy-4-methoxy-phenyl)-2,3-dihydro-4H-chromen-4-one], as shown in Figure 2.

Figure 2.

Structure of hesperidin (1) and hesperetin (2).

Citrus fruits, such as bergamots, grapefruit, lemons, limes, mandarins, oranges, and pomelos, are part of our daily food due to their nutritional and pharmacological significance. These represent an important source of dietary flavonoids, including hesperidin, hesperetin, taxifolin, naringin, naringenin, diosmin, quercetin, rutin, nobiletin, tangeretin, and others.³⁸ Studies have demonstrated antiviral activities of various flavonoids,^42,43 including their use for respiratory diseases and SARS-CoV-1.⁴⁴ Among these, hesperidin is the predominant flavonoid in citrus fruits,⁴⁷ primarily in sweet orange (in young immature oranges it accounts for up to 14% of the fresh weight of the fruit).⁴⁸

The consumption of orange juice-rich food has been reported to exert beneficial effects on some intermediate-risk factors for cardiovascular diseases (CVDs), such as low-density lipoprotein cholesterol, blood pressure, and endothelial function.⁴⁹ A dietary intake history of over 10 000 Finnish men and women suggested that individuals with high polyphenols (flavonoids) intake in terms of raw fruits/vegetables and/or their juices had a lower incidence of cardiovascular disease and bronchial asthma.⁵²

Although citrus fruits and juices are widely consumed worldwide, little information has been published on flavanone bioavailability in humans. Low micromolar levels of hesperidin (around 1 µM range) have been detected in blood serum for 5 to 7 h after the intake of citrus juice,^53,54 probably high enough to exert its health-promoting activities in the human body. Hesperidin itself is absorbed from the intestine intact as a glycoside. Its aglycone, hesperetin, appears in plasma 3 h after ingestion, reaching a peak between 5 and 7 h. The circulating forms of hesperetin are glucuronides (87%) and sulfoglucuronides (13%). For hesperidin, urinary excretion is nearly complete 24 h after the orange juice ingestion and does not depend on the dose.⁵⁵

It is worthwhile to note that the presence of sugar moieties usually increases the bioavailability, but the nature of the substituted sugar moieties affects the digestibility and the absorbability. Indeed, flavonoids with glucose moieties are absorbed more rapidly than flavonoids with rhamnoside, rutinoside, and neohesperidoside substitutions.⁵⁶ However, the lack of α-L-rhamnosidase and rutinosidase in human cells makes the bioavailability of flavonoid rutinosides largely dependent on their hydrolysis by intestinal bacteria.⁵⁷ Furthermore, few intestinal bacterial strains can achieve cleavage of these types of glycosidic bonds,⁵⁸ but the tested flavonoid could be active because the bioavailability of the molecules is due to the aglycone part.^59,60

Toxicity studies have confirmed the high safety profile of hesperidin after oral intake of more than 2 g/kg.⁶¹ Daflon 500 mg, a tablet containing a micronized flavonoid mixture (50 mg of hesperidin + 450 mg of diosmin), is used as a vasoprotective venotonic agent.⁶¹ Similarly, intragastrically given Daflon, at a daily dose of 100 mg, did not show either signs or symptoms of side effects in rats.⁶² Likewise, oral administration of diets containing either methyl hesperidin or hesperidin did not show either signs or symptoms of side effects in mice and rats.^63,64 Moreover, no adverse events were observed in mice followed daily intraperitoneal injections of phosphorylated hesperidin at a dose of 20 mg/kg body weight for over 4 weeks,⁶⁵ although one study showed that orally given Dafon-500 mg twice-daily for 60 days caused minor, temporal side effects such as headache and faintness;⁶⁶ other studies showed that oral Dafon-500 mg is safe in humans.^67,68 According to an oral toxicity study, it can be concluded that hesperidin can be safely used in herbal formulations with its LD₅₀ value of more than 2000 mg/kg.⁶⁹ Hesperidin has a long history as a herbal medication.⁷⁰

Clinical trials involving more than 2850 patients treated with a hesperidin mixture for a period of 6 weeks to 1 year showed normal hematological parameters, and hepatic and renal functions, with no signs of toxicity.⁷¹ No CVD risk factor was observed in participants (mean age: 60.6 ± 1.4 years) receiving either 767 mL orange juice or a hesperidin supplement (both providing 320 mg hesperidin and 439 mg vitamin C).⁷²

Pharmacological Significance of Hesperidin and Hesperetin

Hesperidin and its aglycone hesperetin, 2 flavonoids found primarily in sweet oranges and lemons,^38,73 have been documented to possess a wide range of pharmacological properties including anticancer, antihypertensive, antioxidant, antidiabetic, hepatoprotective, neuroprotective, wound healing, cardiovascular, anti-inflammatory, anti-obesity, hypoglycemic, lipid-lowering and beneficial effects on bone and Alzheimer's disease.^40,53,73

Overwhelmingly, the pharmacological effects of hesperidin are related to its antioxidant activity, arising through its ability to scavenge free radicals.¹¹¹ Furthermore, hesperidin can alleviate diabetic neuropathy¹¹² and nephropathy.¹¹³ Studies also suggested that hesperidin had antidepressant-like effects.¹¹⁴ The effects of hesperidin on ameliorating the depression- and anxiety-like behaviors of diabetic rats, which are mediated by the enhancement of glyoxalase 1 (Glo-1), may be due to the activation of the Nrf2/ARE pathway,¹¹⁴ immunomodulatory targets. The binding energy of hesperidin for TNF-α was −6.96 kcal/mol; some important interactions were observed with Ser69, Leu120, and Tyr151; with IL-1β the binding energy was −6.64 kcal/mol, with binding to Glu37 and Lys65 from the A chain. In the case of IL-6, the binding energy was found to be −7.07 kcal/mol, with interaction at Met67, Glu172, and Arg179 (B chain).^115,116

Hesperidin has been found to have protective effects in mice infected with encephalomyocarditis virus and Staphylococcus aureus when given before either single or combined viral-bacterial infections.¹¹⁷ Hesperidin was found to be effective against human rotavirus, which is the causative agent of diarrhea in infants and young children,¹¹⁸ and also inhibited replication of influenza virus in vitro, as well as decreasing the number of infected cells.¹¹⁹ Hesperidin was also found to inhibit various sexually transmitted pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus-2, and human immunodeficiency virus, but had no toxic effects either on the host cells or on the growth of normal vaginal lactobacilli.¹²⁰ Hesperidin was evaluated in vitro against canine distemper virus and found to be potentially active.¹²¹

Cell culture and molecular docking studies suggested that hesperidin had moderate (41%) efficacies against hepatitis B virus replication.¹²² Hesperidin displayed antiviral activity against simian rotavirus strain SA-11 at a concentration of 1200 μg/mL.¹²³ Hesperidin up-regulated P38 and JNK expression and activation, thus resulting in the enhanced cell-autonomous immunity to defend against influenza A virus infection.¹²⁴ Hesperidin is also known to possess antiviral activity by altering the immune system, mainly by regulating interferons in the influenza A virus.¹²⁵ In another study, hesperidin was noticed to attenuate influenza A virus H1N1 induced lung injury through its anti-inflammatory effect.¹²⁶

Hesperidin and hesperetin have shown promising results in the suppression of various types of cancer (colon, prostate, hepatic, bladder, and lung cancer), as either a drug or as a pro-drug and co-adjuvant.¹²⁷ It has been extensively reported that hesperetin exerts neuroprotective effects in experimental models of neurodegenerative diseases.¹²⁸

Hesperetin displays marked inhibitory activity against replication of several diverse viruses. SARS-CoV, herpes simplex virus type-1, influenza A 14 virus, parainfluenza virus type-3, respiratory syncytial virus, and poliovirus type-1 have been inhibited by hesperetin in in vitro conditions.¹²⁹ Hesperitin also exhibited antiviral action on the replication of the 17D strain of yellow fever virus.¹³³ Hesperetin, quercetin, sinigrin, and other flavonoids have been shown to exhibit antiviral effects against SARS-CoV-2 3CL^pro.¹³⁴ Hesperetin was shown to be a highly potent inhibitor of SARS-CoV-2 3CL^pro (IC₅₀ = 8.3 µM) when tested in a cell-based cleavage assay,^131,135 and was also active against Sindbis neurovirulent strain infection.¹³⁵

Recently, it has been reported that flavonoids from citrus, such as hesperidin and hesperetin, may stimulate antiviral pathways by upregulation of expression of the transcription factor interferon regulatory factor 7 gene, as well as due to their ability to activate the interferon-stimulated response element.¹³⁶ Thus, hesperidin and hesperetin have gained interest due to their health-promoting properties, including antitumor, antibacterial, antifungal, and also antiviral actions.

Computational/Docking Studies

Computational-based methods offer considerable promise for screening drugs and other molecules that may have favorable effects on any relevant SARS-CoV-2 virus protein targets by predicting their binding affinities. Better inhibition is usually reflected by low binding energy (the lower the energy required, the stronger and more specific the binding is). This technique, called “in silico,” is currently used to help the design of specific inhibitors of SARS-CoV-2, and significantly enhances the quality of compounds selected for in vitro and in vivo bioassays.¹³⁷ However, it is important to mention that in silico analyses only provide a preliminary theoretical view on the ligand–protein interactions and hence require experimental validation of the molecular targets.

Therapies That May Act on SARS-CoV-2

The therapies that may act on the coronavirus can be divided into several categories based on the specific pathways: (i) acting on structural proteins of the virus, thus either blocking the virus from binding to human cell receptors or inhibiting the virus's self-assembly process, that is, inhibiting virus entry; (ii) acting on either enzymes or functional proteins that are critical to the virus, that is, preventing virus RNA synthesis and replication; (iii) reducing the virulence factor to restore the host's innate immunity; (iv) acting on the host's specific receptors or enzymes, thus preventing the virus from entering the host's cells.

Blocking Initiation Process of Virus Infection

The S glycoprotein of SARS-CoV-2 contains the RBD that recognizes the target receptor leading to the splicing of the trimeric S protein into subunits S1 and S2, facilitating membrane fusion; virus infection then occurs through endocytosis.¹⁴² The receptor ACE2 is a preferable receptor for the S glycoprotein.¹⁴³ The drug candidates may target binding with the RBD of the S protein,^142,146 whereas the ACE2 ligand binding site is recognized as a protease domain (PD) that plays a role in the cleavage of the trimeric structure of S glycoprotein as the important step in virus infection.^142,147,148 Therefore, the RBD of S glycoprotein and ACE2 are preferable candidates as drug targets to inhibit the initiation process of virus infection.

Blocking Virus Replication

As significant functional proteins of coronaviruses, NSPs are involved in RNA transcription, translation, protein synthesis, processing and modification, virus replication, and infection of the host. Among them, the SARS-CoV-2 M^pro/NSP5,¹⁴⁹ PL^pro/NSP3,¹⁵⁰ RdRp/NSP12,¹⁵¹ and helicase/NTPase (NSP13)¹⁵² are considered essential to the viral cycle.^28,153 Therefore, these have also been targeted for docking and/or for the development of small-molecule inhibitors due to their clear biological functions.¹⁵⁸

Computational/Docking Studies Related to Hesperidin/Hesperitin for Their Possible COVID-19 Significance

Several in silico molecular dynamics (MD) docking studies for drug repurposing have reported that flavonoids were high on their hit list. Chen et al¹⁶¹ reported results based on docking 1500 drugs with SARS-CoV 3CL^pro (protein data bank [PDB]: 2DUC) and found that hesperidin and diosmin were the 2 top candidates. Adem et al¹⁶² reported that after evaluating 80 flavonoids for MD docking with the SARS-CoV-2 protease 3CL_pro (PDB:6LU7), hesperidin, rutin, and diosmin were the top 3 candidates for docking at the active site. Peterson investigated 72 flavonoids for their potential to bind with the active catalytic site of 3CL^pro (PDB:6LU7) and identified hesperidin as one of the top 14 flavonoids.¹⁶³

Inhibiting Virus Invasion

Several computational methods, independently applied by different researchers, showed that hesperidin can bind to the SARS-COV-2 S protein and, in doing so, prevent its initial interaction with ACE2 receptors.¹⁶⁴ This may interfere with viral entry into host cells. In a preferential binding affinity study of selected natural compounds, hesperidin exhibited high affinity (−8.1 kcal/mol) for binding to SARS-CoV-2 S glycoprotein (PDB: 6VYB).¹⁶⁷ Hesperidin was found to have positioning at Phe456 and Phe490 in the target protein when the interactions were visualized.¹⁶⁷ Hesperidin has been cited to be better than nelfinavir, with a docking from −8.3 to −13.51 kcal/mol for S protein.¹⁶⁸

The RBD region of SARS-CoV-2 S protein interacts with the host cell ACE2 receptors to form the SARS-CoV-2-RBDACE2 complex, which is responsible for mediation of virus invasion. The RBD of the S glycoprotein (RBD-S) can bind to the ACE2 receptor at the PD of the host cell, thereby leading to viral infection. The docking results show that hesperidin has the lowest docking score for binding with RBD-S (PDB ID:6LXT) and PD-ACE2 (PDB ID:6VW1), indicating that it has potential for inhibiting viral infection.¹⁶⁹

Target-based virtual ligand screening for binding to S protein was undertaken for 1066 antiviral natural substances, plus 78 known antiviral drugs. Hesperidin was found to be the most suitable for targeting the binding interface between viral S protein and ACE2 human receptors. Hesperidin was predicted to lie on the middle shallow pit of the surface of the RBD of the S protein, with the aglycone part parallel to the b-6 sheet of the RBD. The sugar part was inserted into the shallow pit in the direction away from ACE2, where a few hydrophobic amino acids, including Tyr436, Try440, Leu442, Phe443, Phe476, Try475, Try481, and Tyr49 form a relatively hydrophobic shallow pocket to contain the hesperidin. By superimposing the ACE2-RBD complex on the hesperidin-RBD complex, a distinct overlap of hesperidin with the interface of ACE2 could be observed, suggesting that hesperidin may disrupt the interaction of ACE2 with RBD. This suggested that hesperidin, by blocking the interface of ACE2 and RBD-S binding, could probably be used for treating SARS-CoV-2.^171,172 The molecular docking results showed that hesperidin had a binding affinity of −7.5 kcal/mol with the S protein active site.¹⁷³

A computational and experimental study found that multiple flavonoids abundant in citrus peels cooperate to prevent SARS-CoV-2 infection. In particular, simulated molecular docking showed that hesperidin, hesperetin, and naringin have a strong binding affinity for the ACE2 receptor.^172,174 The molecular docking studies of hesperidin with the ACE2 enzyme demonstrated that hesperidin could bind to ACE2 with a predicted ΔG of –8.3 kcal/mol, with binding sites at Tyr613, Ser611, Arg482, and Glu479.^169,175 In another molecular docking study, hesperidin was found to have a binding affinity of −8.0 kcal/mol with ACE2 protein.¹⁷³ Hesperidin showed a high docking affinity for the ACE2 receptor protein model 1R4L (docking score −11.4 kcal/mol), exhibiting key binding with Cy3344, His345, Asp368, Arg514, Tyr515, and Arg518.¹⁷³ In another study, hesperidin has been reported to form H-bonds with residues Asp405, Lys417, Ser494, and Tyr505 of RBD with a binding energy of −8.6 kcal/mol.¹⁷⁶

Considering that the fusion of the RBD region with the host cell membrane needs a big conformational change in the S protein part, any small molecule bound to the S protein at this time may interfere with its re-folding, therefore inhibiting the viral infection process. Based on virtual screening, hesperidin may disrupt the interaction of ACE2 with the RBD of SARS-CoV-2¹⁵⁸ and, therefore, hesperidin has promise as a prophylactic agent against COVID-19 infection.^165,171

Haggaga et al¹⁶⁵ studied the S glycoprotein inhibition activity of hesperidin and other natural products in comparison with nelfinavir (an antiretroviral drug), chloroquine, and hydroxychloroquine sulfate (antimalarial drugs recommended by the FDA as emergency drugs), and the results showed that hesperidin has better poses than the other 3 as S glycoprotein inhibitors. Thus, hesperidin binds to 2 key protein targets: RBD-S and PD-ACE2, thereby preventing binding of the RBD-S to PD-ACE2 of the host cell, thus inhibiting the viral infection.^169,172

Molecular docking of hesperetin to the ACE2 enzyme showed that hesperetin has the potential to bind to ACE2 with an estimated ΔG of −8.3 kcal/mol, with binding sites at Tyr613, Ser611, Arg482, and Glu479, and thus suggesting that hesperetin may block the infection.¹⁷⁵ In silico studies show that hesperetin can bind (binding energy −9.1 kcal/mol) with high affinity to the S protein, helicase, and protease sites on the ACE2 receptor causing conformational change to inhibit viral entry.¹⁷⁷

Inhibiting Virus Replication

Binding With SARS-CoV-2 M^pro

M^pro, also known as 3CL^pro (NSP5), is one of the most attractive targets against SARS-CoV-2 since it plays a key role in viral transcription and replication. M^pro is first automatically cleaved from poly-proteins by autolytic cleavage between NSP4 and NSP6¹⁷⁷ to produce mature enzymes, and then further cleaves downstream viral polyprotein at 11 sites to release functional units NSP6-NSP16 for virus replication and packaging within the host cells.^164,180 M^pro directly mediates the maturation of NSPs, which is essential to the life cycle of the virus. Detailed investigation of the structure and catalytic mechanism of M^pro has revealed it as an attractive target for anti-coronavirus drug development since it plays a key role in viral transcription and replication, and no human proteases are known with the same substrate specificity.^{140,149,158,180}

The M^pro structure is composed of 3 domains; the catalytic dyad is located in the cleft between domains I and II,^19,149,183 and domain III is responsible for the enzyme dimerization, enabling the active form of the macromolecule.¹⁸⁴ M^pro is a cysteine protease with a catalytic Cys145 and His41 dyad at its active site.¹⁸⁷

Adem et al¹⁶² performed molecular docking investigations by using Molegro Virtual Docker 7 to analyze the inhibition probability of flavonoids against SARS-CoV-2 M^pro (PDB:6LU7). According to the obtained results, hesperidin, rutin, diosmin, and apiin were among other phenolic compounds found to be more effective on COVID-19 than nelfinavir. Hesperidin exhibited the lowest binding energy at the active site of M^pro as it formed 14 hydrogen bond interactions with Thr26, Glu166, Arg188, Gln189, Met49, Asp187, Tyr54, His163, Leu141, and Ser144. According to the Moldock binding score, the potent flavonoids can be ranked as follows by affinity hesperidin > rutin > diosmin > apiin.¹⁶² In a study related to virtual screening of drugs on the active sites of SARS-CoV-2 3CL^pro, Chen et al¹⁶¹ revealed the binding energy for hesperidin as −10.1 kcal/mol. In a molecular docking study by Kralevska et al,¹⁷³ hesperidin was found to have a binding affinity of −6.5 kcal/mol with M^pro.

In a comparative M^pro inhibition study, hesperidin showed better binding free energy compared to nelfinavir, chloroquine, and hydroxychloroquine sulfate, which so far are recommended for the treatment of COVID-19. Hesperidin forms 4 hydrogen bonds with M^pro at the amino acid residues Phe140, Glu166, Cys145, and Ser144.¹⁷⁰ In a docking screening study against the viral main protease (M^pro), of the major components of 38 Chinese Patent Drugs that are commonly used in respiratory diseases, hesperidin was identified as one of the top hits having good binding affinity (−8.5 kcal/mol) against M^pro (PDB:6LU7) targets. The key residues Gly143, Ser144, Cys145, and Glu166 were identified for potential inhibitor binding.^171,190

Tomic et al¹⁶⁷ showed that hesperidin had the highest affinity (−5.8 kcal/mol) for SARS-CoV-2 main protease (3CL^pro; PDB:6Y84). The other results show that hesperidin has the best docking score for SARS-CoV-2 main protease (PDB:6LU7).¹⁶⁹ Hesperidin interacts through hydrogen bonding with Thr24, Thr25, Thr45, His4, Ser46, and Cys145, amide-π stacked interaction with Thr45, and π-alkyl interactions with Met49 and Cys145. In a comparison of the ΔG value, which gives the estimated free energy of binding, the binding affinity of hesperidin towards SARS-CoV-2 main protease was found to be −9.02 kcal/mol.¹⁹¹

A recent study indicated that 2 of the compounds present in ginger (Zingiber officinale), namely chlorogenic acid and hesperidin, had high binding affinities for M^pro, with predicted binding energies of −7.5 and −8.3 kcal/mol. The 2D and 3D interactions further showed that hesperidin interacts with Phe140, Asn142, Cys145 (1 of the 2 amino acids forming the catalytic dyad), His163, and Met165. The combination of hydrophobic bonding with Phe140 and Met165, along with interaction with Cys145, would create a favorable situation for blocking the entry of any other compound to the catalytic site and possibly accounts for the predicted higher binding affinity observed for hesperidin.¹⁹²

Binding With PL^pro

Another essential protease for the cleavage of the viral polyproteins is the PL^pro, a cysteine protease with a classical Cys-His-Asp catalytic triad (Cys112, His273, Asp287), which cleaves the viral polyprotein, releasing NSP1, NSP2, and NSP3.^192,193 Computational approaches have also been used to predict potential SARS-CoV-2 PL^pro inhibitors. In a study for screening the preferential binding affinity of artemisinin, hesperidin, and chloroquine, Tomic et al¹⁶⁷ found that hesperidin had the highest binding score (−10.08 kcal/mol for PL^pro; PDB:6W9C).

Binding With SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp)

RdRp catalyzes SARS-CoV-2 RNA replication and, hence, is an obvious target for antiviral drug design. Hesperidin was successfully docked in the catalytic pocket of RdRp with a binding energy of −8.8 kcal/mol, suggesting that it can strongly bind to the catalytic site of the SARS-CoV-2 RdRp. It forms 13 hydrogen bonds with Tyr619, Asp618, Lys798, Ser795, Met794, Pro793, Asp164, Val166, Pro620, Lys621, Asp623, Arg555, and Tyr455, inhibiting the RdRp activity, thus blocking replication and preventing viral transcription.¹⁹⁴

Binding With Non-Structural Protein-15 Endoribonuclease (NSP15)

The docking results for the NSP15 endoribonuclease of SARS-CoV-2 revealed that the flavonoids baicalin, rutin, ilexgenin A, and hesperidin have the best docking scores in the range −8.168 to −5.29 kcal/mol; while the docking score for the standard drug remdesivir is −5.636 kcal/mol. The key residues at the binding site belong to the C-terminal catalytic domain catalytic triad His235, His250, Lys290, Thr341, Tyr343, and Ser294.¹⁹⁵ The carbonyl oxygen and a 5-hydroxyl group of hesperidin form hydrogen bonds with Lys290 and protonated His250 residues, respectively, while the hydroxyl groups on the sugar moieties form hydrogen bonds with Glu340 and Asp240 residues.

Thus, multiple computational methods, independently applied by different researchers, showed that hesperidin has low binding energy both with the coronavirus S protein, responsible for binding with host receptors, and with other proteases such as M^pro, PL^pro, RdRp, and NSP15 endoribonuclease (Figure 3). Based on these predictive results, it is likely that, because of its binding affinity to these 6 main targets, hesperidin would fight the viral infection by inhibiting either virus binding to ACE2 or virus replication in cells.

Figure 3.

Hesperidin can inhibit severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) entry, and its lifecycle, by targeting replication processes.

Mouth Rinses Containing Citrox

Citrox, which is derived from citrus fruits, is composed of soluble bioflavonoids and hydroxylated phenolic structures produced by plants. Although no in vitro or in vivo studies have been published on Citrox mouth rinse, in silico studies based on computer virtual screening predicted that citrus flavonoids, such as hesperidin and rutin, can act as possible components having antiviral action against SARS-CoV-2. Based on these predictive results, it is likely that mouth rinses having citrus flavonoids could help to fight against COVID-19.¹⁹⁶

Nasal Rinse and Nasal Delivery of Hesperidin

An Ayurvedic herbal formulation of Citrus medica and Zingiber officinale is recommended as a nasal rinse in the management of contagious fevers. Molecular docking studies of the constituents of these plants reflect that hesperidin is one of the components that could inhibit the receptor binding of SARS-CoV-2 S protein, as well as ACE2, thus reducing viral load and shedding of SARS-CoV-2 in the nasal passages.¹⁹⁷

Nasal and inhaled drug delivery methods represent a promising strategy for the treatment of inflammatory lung disease as a result of their ability to improve drug delivery to lungs. Recently, chitosan nanoparticles loaded with hesperidin were developed for nasal delivery of the anti-inflammatory hesperidin to treat inflamed lungs. It is worth mentioning that the hesperidin dose that had protective effects on cytokine storm syndrome-induced acute lung injury was also used in the treatment of acute respiratory distress syndrome (ARDS).¹⁹⁸

Hesperidin in Management of Inflammatory Mediators

Cytokine storm is a major cause of ARDS as the body releases various immune-active molecules, such as interferons (eg, IFNγ), interleukins (eg, IL-1β, IL-2, IL-6), chemokines, and tumor necrosis factor-alpha (TNF-α). Hesperidin/hesperetin has been found to modulate inflammatory mediators, such as IL-6, IL-1β, and TNF-α, in the heart, lungs, and central nervous system in multiple animal models.¹⁹⁹ Hesperidin, with its high anti-inflammatory activity, inhibited the secretion of pro-inflammatory cytokines such as IFN-γ and IL-2.²⁰⁵ Besides, hesperidin inhibited IL-1β-stimulated inflammatory responses by inhibiting the activation of the NF-κB signaling cascade.²⁰⁶ It also played a major role in suppressing the release of inflammatory markers such as TNFα and IL-6 in type 2 diabetic patients.²⁰⁷ Activation of coagulation pathways following the immune response to COVID-19 infection promotes clot formation. A prophylactic dose of heparin (with low molecular weight) is recommended for protection against venous thromboembolism.²⁰⁸ Therefore, it can be used as adjuvant therapy to control the severe inflammatory reaction against COVID-19.

COVID-19 Diets

Interestingly, among the many approaches to COVID-19 prevention, the possible role of diet has so far been somewhat marginal. Diets incorporating citrus fruits, and especially of the orange (Citrus sinensis), could be recommended. These fruits are well known for their vitamin content, as well as being a rich source of flavonoids, especially hesperidin and hesperetin, which, as reported earlier, are known for their antiviral action, and may be important in modulating the immune system and for defending cells from the oxidative stress associated with infection.^{10,34,40,45,47,53,73,95,164,209}

Concluding Remarks

The above-mentioned studies suggest the ability of citrus flavonoids, such as hesperidin and hesperetin, to prevent the SARS-CoV-2 virus from binding to the ACE2 enzyme of the host cell and inhibit virus replication after its penetration of the host cell, as well as either restraining or counteracting the proinflammatory overreaction of the immune system. Thus, hesperidin supplementation may be useful as a prophylactic agent against SARS-CoV-2 infection and as a complementary treatment of COVID-infected patients. The biological actions of the flavonoid may counteract infection by SARS-CoV-2 and modulate the immune system's response to the disease. Further preclinical, epidemiological, and clinical studies are needed to corroborate this hypothesis.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Pawan K. Agrawal

Abbreviations

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Pharmacological Significance of Hesperidin and Hesperetin,Two Citrus Flavonoids,as Promising Antiviral Compounds for Prophylaxis Against and Combating COVID-19

Abstract

Keywords

Introduction

Structure of SARS-CoV-2

Targeted Inhibition Sites for Virus Infection

Hesperidin and Hesperetin

Pharmacological Significance of Hesperidin and Hesperetin

Computational/Docking Studies

Therapies That May Act on SARS-CoV-2

Blocking Initiation Process of Virus Infection

Blocking Virus Replication

Computational/Docking Studies Related to Hesperidin/Hesperitin for Their Possible COVID-19 Significance

Inhibiting Virus Invasion

Inhibiting Virus Replication

Binding With SARS-CoV-2 Mpro

Binding With PLpro

Binding With SARS-CoV-2 RNA-Dependent RNA Polymerase (RdRp)

Binding With Non-Structural Protein-15 Endoribonuclease (NSP15)

Mouth Rinses Containing Citrox

Nasal Rinse and Nasal Delivery of Hesperidin

Hesperidin in Management of Inflammatory Mediators

COVID-19 Diets

Concluding Remarks

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

Abbreviations

References

Binding With SARS-CoV-2 M^pro

Binding With PL^pro