Apigenin’s Therapeutic Potential Against Viral Infection

Several antiviral drugs are clinically approved to treat influenza that is a highly prevalent acute respiratory disease. However, emerging drug-resistant virus strains undermine treatment efficacy, highlighting the exigency for novel antiviral drugs to counter these drug-resistant strains. Plants and their derivates have been historically utilized as medicinal remedies, and extensive studies have evidenced the antiviral potential of phytochemicals. Notably, apigenin is a predominant flavonoid with minimal toxicity and substantial therapeutic effects in various disease models. Despite its many anti-inflammatory, anti-oxidant, anti-cancer, anti-bacterial, and other beneficial bioactivities, existing reviews have yet to focus on apigenin’s antiviral effects. Therefore, this review elucidates apigenin’s therapeutic and antiviral properties in vitro and in vivo , discussing its mode of action and future prospects. Apigenin’sremarkable inhibition by modulating multiple mechanisms against viruses has promising potential for novel plant-derived antiviral drugs and further clinical study developments.


Introduction
Influenza is a viral acute respiratory disease, and a prominent recurrent threat to human health.Several influenza pandemics have transpired in recent history, such as the Spanish flu in 1918, the Hong Kong flu in 1968, and the Swine flu in 2009 [1].Approximately 500 million people were infected, and 50 million died during the most severe 1918 Spanish pandemic [2].Although anti-influenza drugs and vaccines have significantly prevented and treated influenza viral infection, they remain limited due to the virus' high mutation rate and subsequent subtype generation [3].Thus, influenza vaccines must be updated annually to stay ahead of rapidly mutating viruses [4].Discordance between influenza vaccine strains and circulating viruses may engender a more severe endemic or pandemic due to reduced vaccine efficacy [5].Furthermore, emerging drug-resistant influenza strains threaten current antiviral drug applications [6], emphasizing the urgency for new antiviral drugs.
Nearly 90 drugs have been approved for nine human viral disease treatments [7].Plants have been used as traditional remedies for thousands of years, and many studies have revealed plant derivative and metabolite therapeutic potentials against diverse diseases.An estimated 34% of approved small molecule drugs in the past four decades are natural products or their derivatives, and 25% are from plants exclusively [8,9].Due to recent recurrent viral pandemics, phytochemicals have garnered attention as promising antiviral drug agents [10].Plant derivatives or phytochemicals maintain significant structural complexity and can target viral and host proteins in various ways [11].
Apigenin is a natural plant flavonoid found in fruits, herbs, and various plants.As part of the flavone class, apigenin maintains a flavone structure with hydroxyl group substitutions at positions 4 ′ ,5, and 7, as indicated by its chemical name (4 ′ ,5,7,-trihydroxyflavone).Apigenin has low solubility in water and high permeability, notable Biopharmaceutics Classification System Class II drug features [12].Many studies have verified apigenin's antiinflammatory, antioxidant, and anti-apoptotic activities [13][14][15], showcasing its therapeutic potential for diverse human diseases, such as cancer, cardiometabolic disorders, and skin inflammatory conditions [16][17][18].This review details apigenin's therapeutic effects by focusing on its antiinfluenza properties and mode of action.

Enterovirus 71
Apigenin's antiviral properties were also effective against Enterovirus 71 (EV71).Apigenin from dried Paulownia tomentosa flowers inhibited EV71 infection with an 11.0 µM EC 50 by disrupting the internal ribosome EV71 entry site and heterogenous nuclear ribonucleoprotein (hnRNPs) interaction required for viral translation [35].The antiviral effect against EV71 was more efficient with high dose of apigenin, indicating apigenin inhibits EV71 in a dose-dependent manner [36][37][38].Of note, the underlying mechanism of anti-EV71 activity of apigenin has been established.Pre-treatment of host cells with apigenin significantly reduced EV71 infection, indicating that apigenin targeted the host cells for its anti-EV71 effect [35].Further studies confirmed that apigenin targeted heterogenous nuclear ribonucleoprotein (hnRNPs).HnRNP A1 and A2 are known to interact with internal ribosome entry site, required for viral translation, thus considered as essential factors for initiation of EV71 polypeptide translation [39].Apigenin treatment resulted in significantly reduced EV71 RNA in hnRNP A1 and A2 by disrupting viral RNA association with hnRNPs while not affecting the redistribution of those proteins [35,36].The antiviral effect in vitro was corroborated in vivo showing 88.89% of survival protection against lethal dose of EV71 in newborn mouse, suggesting apigenin as promising antiviral agents [38].

Hepatitis C Virus
Similarly, several studies have observed apigenin's effect on HCV.As an Eclipta alba component, apigenin substantially interfered with HCV non-structure protein (NS) 5B's RNA-dependent-RNA polymerase (RdRp) activity, exhibiting a 175.5 µM IC 50 .Concomitantly, apigenin treatment reduced HCV replication by 90% in a cell culture system.Interestingly, apigenin directly binds to HCV NS5B, inhibiting its enzymatic activity [40].Likewise, in vitro system using Huh7 cells expressing HCV replicons, apigenin treatment with very low concentrations (0.1 µM) significantly reduced the HCV RNA copy number up to 40% [41].Furthermore, Shibata et al. [42] reported that apigenin impeded HCV replication by depleting mature miR122 expression levels, which are essential for HCV RNA propagation and stability [69].Inhibiting mature miR122 ex-pression levels with apigenin is likely due to inhibiting TAR RNA binding protein (TRBP) phosphorylation.In previous reports by Ohno et al. [43], apigenin suppressed TRMP phosphorylation, an integral factor for the maturation of miRNA subsets by binding to Dicer [70], modulating ERK activity, and impairing miRNA subset maturation.

Dengue Virus
Apigenin is also known to inhibit the DENV protein NS5.In a cell culture system antagonized by DENV's NS5 during infection [44], apigenin obstructed DENV2 replication by restoring STAT2 phosphorylation.Notably, apigenin stimulated STAT2 even without infection by promoting STAT2 Tyr 689 phosphorylation and activation, indicating a low probability for viral escape mutations affecting apigenin [45].Concomitant to the inhibitory activity of apigenin against DENV protein, apigenin treatment reduced DENV titer in vitro.Apigenin inhibited DENV-3 infection in macrophage (U937-DC-SIGN) up to 50% at 40 µM [46].Also, apigenin inhibited DENV2 infectivity with EC 50 value of 10.55 ± 3.36 µM.Interestingly, by using alkyne-tagged apigenin to visualize the compound localization inside the cells, they found that apigenin was localized at the perinuclear region and colocalized with a dengue protein in the early phase of DENV infection, suggesting further insight for underlying mechanisms of anti-DENV activity of apigenin [47].
These findings substantiate that apigenin has immense potential for antiviral therapeutics against diverse viruses such as HSV, EV71, HCV, and DNV (Table 2, Ref. ).

Apigenin's Anti-Influenza Viral Activity during Late-Stage Infection
Viral mRNA translation, protein packaging, egress, and other critical propagation processes occur during late- stage viral replication (4-8 hpi).The influenza virus forms a complete structure by assembling its progeny particles and releasing them from the host cell for propagation [57,58].Notably, apigenin also inhibited late-stage IAV replication.When cells were treated with apigenin 5 hpi, (+)-strand viral RNA synthesis was attenuated, supporting apigenin's potential for IAV RNA-dependent-RNA polymerase (RdRP) inhibition [54].Similarly, viral particle production was considerably reduced when apigenin was administered 4-8 hpi [59][60][61].Apigenin treatment after virus infection decreased the level of viral NP after 8 hpi with inhibited NA activity, indicating the inhibitory role of apigenin in virus assembly and release stages [41].Thus, apigenin suppresses IAV replication by obstructing multiple IAV replication stages (Fig. 1).

The Regulation of Influenza Virus-Induced Signaling Pathways by Apigenin
IAV infection activates various signaling pathways within the host cell, such as mitogen-activated protein kinase (MAPK) [63,64] and retinoic acid-inducible gene-I (RIG-I) pathways [65].Assessing alterations in signaling pathways up-regulated by IAV provides invaluable insights for antiviral drug developments targeting these components.Joo et al. [54] revealed that apigenin effectively suppressed IAV infection-induced MAPK signaling pathway activations that are critical for virus replication RIG-I is a prominent innate RNA sensor that recognizes influenza viral RNA and activates antiviral host responses, including cytokine cascade [65].However, uncontrolled overproduction of cytokines such as interferons (IFNs) is a major factor that exacerbates immunopathologic lung injury upon IAV infection [66][67][68].Therefore, regulating cytokine production induced by RIG-I pathway activation is vital for managing IAV infection outcomes.Xu et al. [59] revealed that apigenin treatment significantly suppressed IAV-induced RIG-I activation and proinflammatory cytokines such as IFNs.Interestingly, apigenin-mediated suppression of RIG-I was due to ubiquitin-mediated RIG-I protein degradation, leading to disruption of interactions between RIG-I and heat shock protein 90α.In addition, apigenin's RIG-I inhibition was RIG-I pathway-specific as it minimally affected other PRRs, such as melanoma differentiation-associated gene 5 (MDA5), cyclic GAM-AMP synthase (cGAS), and Toll-like receptors (TLRs).

In Vivo Anti-Influenza Viral Potential of Apigenin
Lastly, apigenin's anti-IAV activity strongly suggests its potential as an antiviral drug in vitro, inspiring many in vivo studies.Intranasally administered apigenin significantly reduced viral loads in mouse lungs infected with the IAV H1N1 strain (A/California/07/2009), corroborating apigenin's IAV suppression in vitro.Furthermore, oral administration of an aqueous Agrimonia pilosa and Galla rhois extracts mixture (APRG64), where apigenin is the main antiviral component, considerably curtailed IAVinfected mice morbidity and mortality.In addition, proinflammatory cytokine expressions, such as IFN-γ, TNF-α, and IL-6, in IAV-infected mice lungs and LPS-stimulated splenocytes were notably suppressed by APRG64 treatment.The observed therapeutic effects regarding IAV's mortality and APRG immunopathogenesis were comparable to oseltamivir phosphate (Tamiflu) [54].However, the oral administration of 30 mg/kg apigenin in another study failed to exhibit anti-IAV (A/PR/8/34) efficacy in mice [62].Therefore, despite apigenin's promising anti-IAV activity, the administration route and dose must be carefully considered and require further investigation for new herbal antiviral drug development.

Concluding Remarks
Influenza has continuously threatened human health, invoking unpredictable and recurrent pandemics; four influenza A pandemics have occurred within the last century.Although most influenza A and B strains are susceptible to oseltamivir or zanamivir (neuraminidase inhibitors), resistant strains were observed frequently since the 2009 H1N1 influenza A pandemic.Therefore, developing novel antiinfluenza drugs that effectively target current drug-resistant strains is urgently needed.Apigenin is a plant flavonoid component with substantial antiviral effects against various viruses, including influenza virus, evidenced through numerous in vitro and in vivo experiments.As summarized in the present review, apigenin exerts anti-viral activity against HSV, HCV, EV71, DENV, SARS-CoV, and influenza by disrupting virus particles, interfering with multiple viral replication stages by inhibiting viral proteins, regulating virus-induced signaling pathways, and interrupting virus interactions with host factors, such as RdRP, the ACE-2 receptor, and TMPRSS2.
Apigenin directly inhibits influenza virus and host factor interactions while alleviating pathogenic inflammatory responses caused by infection, similar to clinically used anti-IAV drugs.Furthermore, apigenin has also proven effective against drug-resistant influenza strains.These results indicated that apigenin is a promising candidate for anti-influenza therapy, as it can directly inhibit influenza infectivity and replication while reducing clinical symptoms associated with robust inflammatory responses.However, despite its potential as an anti-influenza drug, further studies using in vivo and nonhuman primate models are needed to reveal apigenin's intricate anti-influenza mechanisms for clinical use, as current findings are limited to in vitro and in vivo mouse experiments.
Various studies have studied apigenin's pharmacological features, reporting its nontoxic and nonmutagenic nature with various biological benefits, including antiinflammatory, anti-cancer, anti-oxidant, anti-bacterial, and anti-depressant.However, high apigenin doses may induce slight side effects [71][72][73].Apigenin exerts biological effects through numerous signaling molecule interactions, including ERK, c-Jun N-terminal kinase (JNK), p38, enzymes like cytochrome P450s, and the benzodiazepine receptor [73][74][75].Based on their remarkable beneficial effects, apigenin's pharmacokinetics has been investigated.Researchers found that oral apigenin administration in a rat model was absorbed, resulting in deficient blood levels [76,77].Apigenin absorption primarily undergoes Phase I to produce luteolin, scutellarein, and iso-scutellarein by utilizing Phase I enzymes and Phase II metabolism through glucuronidation and sulfation of apigenin in enteric and enterohepatic cycling [72,[77][78][79][80]. Up to 63% of apigenin was excreted through urinary and fecal routes, as corroborated in clinical trials [77,81].Also, apigenin elimination was reported as considerably slow; 24.8% of radiolabeled apigenin was observed in the body ten days postadministration [77].Apigenin's higher absorption and accumulation extent than other dietary flavonoids, such as luteolin and quercetin, indicate better oral bioavailability and pharmacokinetics [77,79,82,83].However, apigenin's clinical developments are severely limited due to its low solubility in water and organic solvents.Therefore, new technologies or formulations must be developed to improve apigenin's bioavailability.
Similar to other plant extracts, apigenin's slow production rate or inefficient processing techniques could hamper its clinical applications, particularly during an influenza pandemic.Therefore, a systemic mass-production manufacturing system must be developed.