Asteraceae plant extracts, from which apigenin is abundantly isolated, were found to have an antiviral effect against HSV-1 in a previous report [30]. Similarly, chamomile extract is one of the richest natural apigenin sources, exhibiting anti-HSV-1 and -HSV-2 activity in vitro. Moreover, chamomile extract inhibits HSV-1 and HSV-2 during or after viral absorption; the most effective viral inhibition stage was when the extract was provided during viral absorption. Chamomile extract also presented the most substantial virucidal activity on both HSV-1 and HSV-2 particles than other plant extracts, completely inactivating viral particles within 3 and 1 hours against HSV-1 and HSV-2, respectively, indicating apigenin’s potent antiviral activity against HSV [31]. Also, Rittà et al. [32] discovered that Arisaema tortuosum leaf extract exhibited anti-HSV-2 activity even against acyclovir-resistant HSV-1 and HSV-2. Among extract components, apigenin produced the most inhibitory activity against HSV-1 (EC${}_{50}$ = 7.04 µg/mL) and HSV-2 (EC${}_{50}$ = 0.05 $\pm$ 0.02 µg/mL). Through this study, the authors demonstrated that apigenin inhibited the post-entry step of the virus replication cycle, interfering with its proliferation and transmittance [32]. Likewise, the anti-HSV-1 activity of apigenin was reported by other investigators (EC${}_{50}$ = 5 µg/mL) [33]. Interestingly, vitexin, an apigenin flavone glucoside, also exhibited anti-HSV-1 activity with EC${}_{50}$ value of 18 $\pm$ 3.3 µg/mL [34], corroborating the inhibitory activity of apigenin against HSV.

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].

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 expression 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.

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 $\pm$ 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].

SARS-CoV has recently emerged as a predominantly threatening virus, prompting researchers to seek novel antiviral agents. Therefore, as a prominent plant-derived antiviral material, apigenin’s antiviral effect against SARS-CoVs has been increasingly investigated. Chaves and colleagues revealed apigenin’s antiviral activity against SARS-CoV-2 B.1 lineage in Calu-3 cells with a 5.11 $\pm$ 0.26 µM EC${}_{50}$. Moreover, apigenin treatment reduced inflammatory cytokine TNF-alpha production in SARS-CoV-2 infected Calu-3 cells [48]. Thus, an enzyme inhibition assay was performed in silico and in vitro to ascertain how apigenin inhibits coronavirus replication. In silico docking analysis revealed that apigenin potentially interferes with the main SARS-CoV protease (Mpro), essential for SARS-CoV replication [49]. Molecular docking analysis evidenced potent binding energies of –7.2 Kcal/mol (PDB: 6WTT) [50] and –6.7 Kcal/mol (PDB: 6LU7) [51]. However, in vitro enzyme inhibition activity was not as efficient, resulting in a 280.8 $\pm$ 21.4 IC${}_{50}$ [52]. Apigenin also interacts with host factors SARS-CoVs target, such as the angiotensin-converting enzyme (ACE)-2 receptor and transmembrane protease serine subtype 2 (TMRPSS2). The in silico molecular docking assay demonstrated that apigenin has a high binding energy to ACE-2 receptor (–8.5 Kcal/mol) and TMPRSS2 (–7.7 Kcal/mol) [51]. Apigenin’s high-binding affinity to host factors may explain its antiviral effect on SARS-CoV replication. Interestingly, rhoifolin, an apigenin glycoside, inhibited Mpro activity at 20 µM with a high binding affinity (–9.565 Kcal/mol) [53].

These findings substantiate that apigenin has immense potential for antiviral therapeutics against diverse viruses such as HSV, EV71, HCV, and DNV (Table 2, Ref. [30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]).

Upon replication onset (0–4 hours post-infection, hpi), influenza viruses attach to host cells and enter by endocytosis [57, 58]. Apigenin treatment before viral infection (cell protection assay) did not significantly reduce IAV mRNA expression [54] or cytopathic effect [59, 60], whereas apigenin pre-incubation with IAV did [54, 60]. These results demonstrate apigenin’s disruptive activity against IAV and provide insight into IAV particle and apigenin interactions. Moreover, apigenin inhibited replication when cells were simultaneously treated with apigenin and the influenza virus. Similarly, apigenin post-treatment (2 and 4 hpi) effectively reduced virus-induced cytopathic effects and IAV mRNA expression in host cells. Therefore, these results following apigenin administration 0–4 hours post-infection (hpi) indicate interference with the influenza A virus’ attachment and penetration. Apigenin’s anti-influenza viral activity during early-stage infection was also observed for other IAV strains, including H1N1 (A/California/07/2009, A/PR/8/34) and H3N2 (A/Jinan/15/90, A/Minfang/151/2000) [54, 56, 59, 60, 61, 62]. Collectively, these results prove that apigenin can inhibit early-stage IAV replication (Fig. 1).

Fig. 1.

The inhibitory activity of apigenin against IAV on multiple stages of infection. (+), positive strand; (-), negative strand. Created with https://www.biorender.com/.

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).

Apigenin’s antiviral effect on drug-resistant IAV strains was also investigated. Morimoto et al. [61] noted that apigenin exhibited antiviral effects on oseltamivir-resistant strains (Osaka/2024/2009 and Osaka/71/2011) with 23.3 $\pm$ 7.4 and 76.7 $\pm$ 7.8 µM (selectivity index = 40 and 12, respectively) IC${}_{50}$ values. Moreover, this effect was not strain-specific, as it was also observed in A/HebeiXinhua/SWL1106/2017 (oseltamivir & amantadine-resistant H1N1) and A/FujianXinluo/SWL2457/2014 (Amantadine-resistant H1N1) strains [60]. Apigenin’s striking antiviral effects on drug-resistant IAV strains may be due to its many inhibitory methods compared to existing antiviral drugs. Thus, apigenin could substantially improve treatments against novel mutated IAVs that threaten severe pandemics worldwide.

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 and cytokine production. Importantly, apigenin severely suppressed the elevation of extracellular signal-regulated (ERK) and stress-activated (SAPK) protein kinases phosphorylation upon IAV infection, subsequently reducing viral 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 90$\alpha$. 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). The authors also proved that apigenin inhibited viral replication, virus-induced cell death, and A/PR/8/34 (H1N1) virus NA activity in vitro, independently from RIG-I suppression [59]. Therefore, apigenin inhibits signaling pathway activation induced by IAV infection, further illuminating apigenin’s underlying mechanisms against viruses (Fig. 2).

Fig. 2.

Regulation of apigenin in IAV-induced signaling pathways. Created with BioRender.com.

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 IAV-infected mice morbidity and mortality. In addition, proinflammatory cytokine expressions, such as IFN-$\gamma$, TNF-$\alpha$, 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.