Introduction

Influenza virus, belonging to the Orthomyxoviridae family, is a single-stranded, negative sense RNA virus and is one of the most significant pathogens threatening to human health. Influenza viruses are classified into four types (A, B, C, and D), of which influenza A virus (IAV) comprised the majority of global seasonal influenza case. Co-circulating with IAV, influenza B virus (IBV) is also responsible for about 25% of all influenza-related hospitalisations, especially cause high morbidity and mortality in children. [1, 2]. Continuing antigenic drift and shift of IAV and IBV results in new variants which facilitates the cross-species transmission and reduced efficacy of current vaccines and antiviral drugs. Thus, identifying new, more effective drugs against both IAV and IBV is of critical importance.

Isoquercitrin, also known as Quercetin-3-O-glucoside, is a kind of flavonol and widely exists in plants, like Houttuynia cordata, Hedyotis diffusa, et al. Currently, besides the direct extraction from plants, the chemical synthesis method was extensively used to prepare isoquercitrin. As a mono-glycoside derivative of quercetin which has diverse biological functions, isoquercitrin has been demonstrated to exhibit anti-tumor [3,4,5], anti-apoptotic [33], but in vivo, 10 mg/kg/day of isoquercitrin had equivalent efficacy to 25 mg/kg/day of oseltamivir phosphate, suggesting this drug is a better in vivo therapeutic drug. Given the continuous emergence of resistance of oseltamivir, perhaps isoquercitrin can become a new treatment option for drug-resistant strains of influenza A virus and influenza B virus.

Isoquercitrin is a flavonoid that can be extracted from natural plants and exhibits antiviral activity against multiple viruses. Isoquercitrin was reported to be the main component of some Traditional Chinese medicine (TCM) that suppressed HSV by inhibiting ROS production and NF-κB activation [9, 10], suppressed IAV (H1N1) replication by inhibiting ROS production [11] and exerted antiviral activity against SARS-CoV-2 [34] and HCoV-229E [13] by blocked Spike protein and Mpro. Isoquercitrin also blocked ZIKV infection through reduced ZIKV NS2B-NS3 protease and NS5 RNA dependent RNA polymerase (RdRp) activity in cells and Ifnar1−/− mice [16, 35]. Taken its inhibitory effect on many RNA viruses and even DNA viruses and results of our researches, isoquercitrin could become a broad-spectrum antiviral candidate.

Cytokine is a kind of peptide or glycoprotein secreted by the cells and perform diverse biological functions, such as immune modulation, involvement in inflammatory responses, etc. Cytokine is a crucial component of antiviral innate immune, but severely viral infections are always accompanied by cytokine dysregulation and amplification, even becomes the main factor for viral pneumonia during the later phase after virus infection [36]. Considering that, reducing cytokine production is a potential treatment strategy. Thus, multiple immunomodulators, such as Janus kinase (JAK) inhibitors [37] and anti‐IL‐6 antibody [38], are developed to control virus-induced “cytokine storm” and mitigate disease progression. In addition to chemical molecules and antibodies, traditional Chinese medicine gradually become an alternative source for anti-influenza virus drugs [39]. Fructuscorni and Radix salviae miltiorrhizae possess resistance to influenza because they can enhance specific and non-specific immunity [40]. As a natural drug, isoquercitrin was reported to reduce the virus-induced ROS production and NF-κB activation [10]. Taken together, isoquercitrin not only has antiviral activity against IAV and IBV, but also represses the expression of pro-inflammatory cytokines by inactiviating NF-κB signal.

The pathogenicity of an influenza virus depends on many factors, such as cell tropism, replicate efficiency, and host immunity. Both IAV and IBV prefer to replicate in respiratory system and innate immune cells are recruited to lung and produce massive cytokines and chemokines after influenza virus infection, inducing pneumonia, even multi-organ failure [41]. Unexpectedly, adolescents and children are more susceptible to IBV and sometimes IBV-associated diseases develop earlier and more severe than IAV [42]. Characterized by inflammatory cell infiltration, alveolar epithelial cells sloughing, alveolar wall hyperemia and thickness, alveolar hemorrhage and damage, interstitial-alveolar edema, etc. the degree of which always becomes a standard for assess the pathogenicity of viruses and the efficacy of therapeutic drugs [43]. Fortunately, in this study, isoquercitrin was found as a treatment drug for viral pneumonia.

The dose and route of administration are main factors affecting the drug efficacy [44]. In other researches, isoquercitrin was demonstrated to exert therapeutic effect at lower doses, so we only explore its dose-dependent relationship. In a murine model, the common route for drug administration include tail-vein injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral gavage and intranasal administration. Compared to tail-vein injection, which can increase the risk of infection and thromboembolic events [45], intraperitoneal injection, intramuscular injection and subcutaneous injection are more safe and long-acting. Oral administration is the predominant method of drug administration of natural products [46], but its bioavailability should be considered. In our study, isoquercitrin treatment via different administration could exert antiviral effect, suggesting that this drug may directly exert the pharmacodynamic effect in target organ, without requirement of metabolic conversion. Additionally, considering the influence of different drug administration on the antiviral effect of isoquercitrin, more experiments should be considered be implemented to identify the optimal dosing regimen.

Conclusion

Above findings suggest that isoquercitrin has potent antiviral activity against influenza A (H1N1) virus and influenza B virus in vitro and in vivo. Lethal infection of influenza virus induced excessive production of cytokines and severe lung damage, but these outcomes were suppressed by isoquercitrin, indicating that this drug could benefit severe patients with influenza virus infection. Additionally, through comparison between different drug delivery methods, the i.m. was proved as a better method for administration of isoquercitrin.