Tea is the most commonly consumed drink in the world after water. Depending on the manufacturing process, tea can be classified into three major classes: non-fermented green tea, semi-fermented oolong tea, and fermented black and red teas (Cabrera et al., 2006). Non-fermented green tea from the plant Camellia sinensis is dried and steamed to prevent oxidation, which is not the case for black and red tea (Cabrera et al., 2006). The natural compound epigallocatechin-3-gallate (EGCG) is an active polyphenolic catechin and accounts for approximately 59% of the total catechins from the leaves of the green tea. Other catechins in green tea include epigallocatechin (EGC) (19%), epicatechin-gallate (ECG) (13.6%) and epicatechin (EC) (6.4%) (McKay and Blumberg, 2002). The functional and structural differences between these catechins are attributed to the number of hydroxyl groups on the B-ring and the presence or absence of a galloyl moiety (Figure 1).
In traditional Chinese medicine, green tea is considered to have beneficial properties for human health including cardioprotective, anti-carcinogenetic and anti-infective effects. Although a detailed molecular understanding as to why green tea has these broad protective effects is lacking, the ability of EGCG to bind many biological molecules and influence the activity of a variety of enzymes and signal transduction pathways at the micromolar and nanomolar level may, at least in part, be responsible for these effects (Lee et al., 2002). EGCG is water soluble and exposure to high temperatures such as boiling water does not greatly influence the stability of the molecule (Wang et al., 2008). Notably, EGCG and various green tea preparations are available as an over the counter remedy in many countries and are inexpensive. The first documented report of an anti-infective effect of tea was made over 100 years ago by the British army surgeon Mc Naught, who showed that tea killed the organism that causes typhoid fever (Salmonella typhi) and brucellosis (Brucella melitensis) (MC Naught, 1906). However, this effect of tea was not studied further until the late 1980s when systematic research on the antimicrobial and antiviral effects of tea was conducted. Today, a literature search at pubmed.gov shows that over 4000 publications on the effects of EGCG and/or green tea have been reported. In this review, we firstly summarize the antiviral effect of EGCG on different viral families (Table 1) with a focus on hepatitis C virus (HCV) and HIV. We then discuss the antibacterial and antifungal activities of EGCG in in vitro and in vivo model systems. EGCG has in general a low bioavailability, therefore, the translation of its anti-infective effects into clinically relevant strategies and the procurement of physiologically concentrations of the molecule at the sites of viral, bacterial and fungal replication are also crucial aspects that need to be considered.
Effect of EGCG against hepatitis C virus
HCV, a positive strand RNA virus of the family Flaviviridae, chronically infects about 160 million individuals (Lavanchy, 2011). These patients are at risk of potentially life-threatening hepatic complications including cirrhosis, liver failure and hepatocellular carcinoma. In fact, chronic HCV infection is associated with about 30% of liver cancers worldwide and is among the leading indications for orthotopic liver transplantation (Brown, 2005). Standard therapy consists of a combination of pegylated interferon-α with ribavirin (PEGIFN-α/RV). However, PEGIFN-α/RV therapy has different success rates dependant on the viral genotype of the infection. The addition of one of two currently licensed viral protease inhibitors, the first generation of direct acting antivirals to current PEGIFN-α/RV combination therapy has substantially increased the treatment success rates for patients infected with the most prevalent genotype 1. However, this triple therapy cannot be used for all viral genotypes and it is associated with a number of side effects that can compromise patient compliance. Therefore, more effective therapies that are applicable for all viral genotypes and with fewer side effects are needed. For instance, in respect of liver transplantation for HCV-associated end-stage liver disease, the ability to block viral cell entry would help to minimize the currently universal re-infection of the donor liver by virions in the blood.
Recently, in the search for new antiviral molecules, three independent groups have identified EGCG as a potent inhibitor of the HCV entry pathway (Ciesek et al., 2011; Calland et al., 2012; Chen et al., 2012). Ciesek and colleagues were initially working on the influence of semen on HCV infection and became interested in EGCG when it was reported that the green tea molecule counteracts semen-mediated enhancement of HIV infection (Hauber et al., 2009). When they performed the first infection experiments with EGCG, a potent inhibition of HCV infection was noted, and the green tea molecule was identified as a novel HCV entry inhibitor (Ciesek et al., 2011). Calland and co-workers became interested in testing EGCG because it was reported to increase lipid droplet formation and to impair lipoprotein secretion in hepatocytes, two cellular functions known to play a role in the life cycle of HCV (Li et al., 2006). In these three studies it was clearly demonstrated that entry of cell culture-derived particles (HCVcc) as well as HCV pseudoparticles (HCVpp) are inhibited by EGCG independent of the HCV genotype (Ciesek et al., 2011; Calland et al., 2012; Chen et al., 2012). This was also the case when primary human hepatocytes, which resemble more closely the natural reservoir for HCV, were used as target cells. Evaluation of each step in the viral life cycle identified EGCG as an entry inhibitor because RNA replication and release of infectious particles were not affected. It had previously been suggested that EGCG inhibits the essential NS3/4A serine protease of HCV (Zuo et al., 2007); however, these assays were performed in a cell-free system and this observation could not be validated in an HCV replication setting (Ciesek et al., 2011; Calland et al., 2012). Chen et al. (2012) reported a slight inhibition (two to threefold) of HCV RNA replication with JFH1 and Con1 constructs in tissue culture, but only at a very high concentration (80 μM) of EGCG. Other catechins, such as EGC, EC and ECG, did not have such a strong inhibitory effect as EGCG, which suggests that inhibition of HCV entry is unique to EGCG and not shared by other green tea catechins (Ciesek et al., 2011).
On testing its effect on other viruses, it was demonstrated that EGCG inhibited Herpes simplex virus (HSV) infection, as described earlier (Isaacs et al., 2008; 2011), but it had no effect on bovine viral diarrhoea virus or yellow fever virus (YFV), which, like HCV, also belong to the family of Flaviviridae, or the on the unrelated Sindbis virus (SINV) (Calland et al., 2012). It has been reported that HCV can be transmitted in cell culture via cell-to-cell spread. This mode of transmission may be particularly relevant in vivo in the context of infected liver tissue. Infection via cell-to-cell spread was found to be refractory to neutralization by E2 monoclonal antibodies and it may occur in a CD81-independent manner (Timpe et al., 2008; Witteveldt et al., 2009). EGCG was able to prevent cell-to-cell transmission when infected cells were overlaid by agarose or incubated with neutralizing antibodies to prevent the extracellular route of infection (Ciesek et al., 2011; Calland et al., 2012). HCV entry is a complex multistep process involving many host factors and is followed by endocytosis and fusion of the viral membrane with the host membrane (Figure 2). To resolve which step in the entry pathway is blocked by EGCG, its antiviral activity was assessed by administration of the molecule at different time points during the early phase of infection. The results from these experiments suggest that EGCG acts on the virus particles and inhibits virus entry by impairing virus binding to the cell surface (Ciesek et al., 2011; Calland et al., 2012) (Figure 2). In line with these results, pretreatment with EGCG had no effect on target cells, but it inhibited the primary attachment of 35S-labelled HCV virions to cells (Ciesek et al., 2011). Importantly, the green tea molecule was also able to clear HCV from cell cultures. At a concentration of 50 μm EGCG led to undetectable levels of infectious virus in the supernatant of human cells after three cell passages (Calland et al., 2012) and clearance of the virus has even been observed after two passages at the same concentration (Chen et al., 2012).
In summary, EGCG potently inhibits HCV entry of all genotypes to hepatoma cell lines and in primary human hepatocytes by preventing viral attachment to target cells. Therefore, EGCG could provide a new approach to prevent HCV infection, especially after liver transplantation of chronically infected patients. The combination of EGCG with other antiviral compounds targeting HCV replication in an interferon-free regimen is possible, as strong and additive inhibition of HCV infection was demonstrated when the molecule was combined with a NS3/4A protease inhibitor or cyclosporine A, which inhibits HCV replication by interfering with the HCV co-factor cyclophilin (Ciesek et al., 2011). Future clinical trials will reveal how effective EGCG is at reducing viraemia in naïve patients with chronic hepatitis C and in preventing graft re-infection in patients undergoing liver transplantation.