Flaviviruses are (+)ssRNA viruses that include important human pathogens such as hepatitis C virus (HCV) and dengue virus (DENV). HCV is known to cause chronic infection (hepatitis C) that can lead to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC) [6
]. DENV, on the other hand, is the etiologic agent of dengue fever (DF) and the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are fatal illnesses that could lead to death in young children [7
]. Currently, there are no effective vaccines against these viruses. Although the treatments for HCV infection has remarkably improved with the advent of direct-acting antivirals (DAAs), important issues such as cost, selection of drug-resistant mutants, and challenges in the difficult-to-treat populations have limited the widespread use of these drugs. DENV infection on the other hand, has no available antiviral treatments. These circumstances necessitate the search for novel/alternative forms of therapy to complement the existing treatment options.
2.1.1. Hepatitis C Virus
The effect of silymarin on HCV has been extensively studied and the antiviral activity of the drug against HCV in vitro is well documented. Using a standardized silymarin extract (MK-001), Polyak et al. demonstrated that MK-001 not only inhibited the genotype 2a HCV strain JFH-1 infection in both the pretreatment and post-infection analysis, but also blocked TNF-α and NFκB transcriptional activity in peripheral blood mononuclear cells (PBMCs) and hepatoma Huh-7 cells, respectively, suggesting that the extract possesses both antiviral and anti-inflammatory bioactivities [8
]. Further mechanistic studies demonstrated that although MK-001 treatment alone only modestly affected the interferon (IFN) JAK-STAT pathway, the combination of MK-001 with IFN-α augmented the antiviral efficacy of exogenously added IFN, leading to the conclusion that the antiviral effect of MK-001 is mediated by potentiating the JAK-STAT antiviral signaling pathway which, in turn, inhibits HCV replication.
Following the above discovery, the same authors in two independent studies demonstrated that silymarin treatment blocked different steps of the HCV (JFH-1) life cycle, including entry/fusion, replication, and virion production in the host cells [9
]. Furthermore, silymarin and its derived pure compounds exhibited potent hepatoprotective functions by inhibiting the HCV-induced oxidative stress, NFκB-dependent transcription, and T-cell receptor (TCR)-mediated proliferation [9
]. Interestingly, both studies observed an impact of silymarin on HCV NS5B RNA-dependent RNA polymerase (RdRp) activity, albeit at concentrations higher than that required for its anti-HCV effect. Consistent with the inhibition of the RdRp activity, Belkacem et al. employing silybin A, silybin B, and Legalon®
SIL demonstrated that all three forms of silymarin components inhibited HCV replication by targeting the HCV RdRp activity of NS5B, with an IC50 value ranging between 75–100 μM [11
In contrast, Blaising et al. in 2013 showed that the major bioactive component silibinin exerts antiviral effect against HCV by blocking clathrin-mediated endocytosis [12
]. Another study in 2013 indicated that silibinin impedes HCV infection by targeting the HCV NS4B protein [13
], which is known to mediate the membranous web formation where HCV RNA replication occurs [14
] and hence affecting the morphogenesis of the viral replication sites. More recently, we developed silibinin nanoparticles (SB-NPs) and showed that both the SB-NPs and the conventional silibinin inhibited HCV infection by blocking viral cell-to-cell spread [15
]. However, in our hand, the drug had minimal impact on other steps of the viral life cycle (entry, replication, and virion production) or in modulating the type I IFN response.
In 2016, DebRoy et al. investigated the antiviral effect of intravenous Legalon®
SIL monotherapy on uPA-SCID chimeric mice with humanized livers [16
]. Mice chronically infected with HCV were treated with different intravenous doses of Legalon®
SIL (469, 265 or 61.5 mg/kg) for 14 days before analyzing serum HCV, human albumin, and liver HCV RNA levels. The results demonstrated that Legalon®
SIL monotherapy led to a biphasic serum viral decline without affecting the human albumin levels, suggesting that the antiviral effect observed was not due to a decline in the human hepatocytes. Furthermore, administration of Legalon®
SIL induced anti-inflammatory and anti-proliferative gene expressions that were demonstrated by a decrease in TNF-α and NFκB-associated transcriptional activations. Interestingly, the microarray analysis showed that Legalon®
SIL treatment inhibited the expression of genes such as interleukin 8 (IL-8), nicotamidine N-methyltransferase (NNMT), and osteopontin/secreted phosphoprotein 1 (SPP1), which are known to facilitate HCV replication, consistent with the inhibition of HCV production. These results suggest that Legalon®
SIL could efficiently inhibit HCV production in mice in the absence of adaptive immunity in vivo.
Together, the results discussed above support the robust anti-HCV activity of silymarin and its derivatives, although the underlying anti-HCV mechanism varies substantially from study to study. Possible explanations include discrepancy in the source or type of drug used (e.g., silymarin component, purity, and extraction method), variation in experimental design such as concentration and treatment protocol, and the specific model systems employed. This is supported by Wagoner et al.’s work demonstrating that the oral and intravenous (i.e., Legalon®
SIL) formulations of silibinin exert different effects on HCV life cycle, inflammation, and antiviral signaling in vitro [17
]. Thus, concerted efforts should be made to delineate the main mechanism of action of this promising drug. Nonetheless, given that silymarin and its constituents are bioactive against several cell physiological processes, it is likely that silymarin and its major active components may exhibit impact on multiple steps of the virus life cycle, either directly or indirectly.