The influenza virus causes severe respiratory diseases that remains as a leading source of annual morbidity . The discovery and development of novel anti-influenza compounds, preferably of natural origin, are required to prevent and treat potential influenza pandemics. Existing therapeutic antiviral agents have limited clinical efficiency with many toxic side effects, but antiviral compounds of natural origin are more easily available and are mostly nontoxic [9, 10].
It has been shown that an influenza infection triggers the induction of pro-inflammatory cytokines which activate GTPase proteins by isoprenylation and begins the process of recognizing ssRNA  to express the target genes for cytokines, such as IL-1, IL-6, TNF-α and IFN-γ. However, the increased level of pro-inflammatory cytokines after an influenza virus infection, which is caused by an over-responsive immune system, sometimes causes hypercytokinemia. Hypercytokinemia is the systemic expression of a strong immune system and is a potentially lethal reaction which consists of a positive feedback between cytokines and immune cells that, in turn, causes lung inflammation [7, 8, 12, 13]. The most important pro-inflammatory cytokines that are responsible for these reactions are IL-6 and TNF-α. These cytokines are pleiotropic inflammatory cytokines that play important roles in metabolism, apoptosis, massive systemic effects and inflammation, which are hallmarks of influenza [14–16].
HESA-A is a natural compound of herbal-marine origin which contains inorganic, organic and aqueous fractions with a wide range of therapeutic applications [4, 17, 18]. In this study, the interaction between HESA-A and influenza virus A/H1N1 was evaluated. HESA-A was not toxic on MDCK cells at up to 0.05 mg/ml. As calculated from the MTT results by two-way ANOVA test, the EC50 of this compound was obtained at 0.025 mg/ml, with no cytotoxic effect on the cells. The optical densities obtained from the MTT assay showed that co-penetration and pre-penetration treatments had 86.92% ± 9.79 and 69.17% ± 11.8 of protection, respectively. These treatments were more protective (p ≤ 0.05) against viral cytopathic effects in comparison with the other exposures, even with different Amantadine treatments. Meanwhile, HA results showed a significant decrease in HA titre in all combination treatments compared to the positive control of a virus-treated sample. From these results, it is postulated that HESA-A may interfere with viral membrane fusion by inhibiting penetration or adsorption through HA glycoprotein interference.
The antiviral effects of HESA-A on influenza viral load and cytokine levels in MDCK cell cultures were analyzed using quantitative real-time PCR assay. This technique, which provides absolute copy numbers of the template, is a reliable and more accurate tool compared to relative quantification [19, 20]. In this assay, the quantity of targeted genes can be evaluated with reasonable accuracy by using a reliable standard . A standard curve, constructed from standard concentrations (data not shown), was used to determine the copy numbers of target genes related to the Ct value. Significant increments in the cycle thresholds (Cts) of M2 PCR products were observed once HESA-A was applied in all types of combination treatments. The log10 copy numbers, which were calculated from the concentrations against mean Ct values, confirmed these significant differences, especially when HESA-A was applied in the co-penetration treatment. In HESA-A and virus combination treatments, TNF-α and IL-6 copy numbers, which were calculated from the standard curves, showed some decrements. The standard curves were obtained by plotting concentrations against mean Ct values. Significantly low expression in TNF-α, but not IL-6, was related to the co-penetration treatment.
The quantification of cytokines by qRT-PCR was also analyzed and confirmed using ELISA. It was found that infection by the virus causes high expression levels of these pro-inflammatory cytokines, while in all combination treatments, the expression of these proteins significantly decreased, especially in co-penetration treatments, which showed 96.51% and 90.77% decrements in TNF-α and IL-6 protein expression, respectively.
In conclusion, it is possible to limit immune system over-expression and turn off lung inflammation caused by influenza infection if proper treatment with HESA-A is applied. Clinical control can prevent this intense cytokine response through early diagnosis and efficient treatment. Further research on the interaction of HESA-A active compounds with different parts of cellular and viral genes are currently underway.