EGCG against fungi
Over 600 different fungi have been reported to infect humans, ranging from common to fatal infections (Brown et al., 2012). They infect billions of people every year and due to the use of more modern and interventional medicine and an increased number of immunosuppressed patients, the incidence of invasive fungal infections is rising. The antifungal effects of EGCG were mainly studied against yeasts such as Candida spp. and moulds such as dermatophytes. Currently, data relating to aspergilli or other human-pathogenic fungi as zygomycetes are lacking.
Yeasts such as Candida spp. are generally considered as commensals of the skin, mucosa and gut flora. Superficial infections by Candida spp. are commonly present in cases of deferment of bacterial flora or dysfunction of the local defence system. Candidaemia is the fourth most common source of bloodstream infection in the US and is associated with high morbidity and mortality (Rangel-Frausto, 1999; Pappas et al., 2009).
The dermatophytes are a distinct group of fungi, which have the ability to utilize keratin as a nutrition source. These fungi cause superficial infections of the skin, hair and nails of humans and animals.
The problem with the most currently available antifungals is not the existing antimycotic activity; it is more the potential side effects of the different classes of drugs as most of them are nephro- or hepatotoxic. Thus, developing and testing compounds from nature with less toxic effects is desirable.
The fungicidal activities of EGCG against Trichophyton mentagrophytes, T. rubrum, Cryptococcus neoformans and C. albicans were first analysed in 1991 (Okubo et al., 1991). Low concentrations of EGCG (2.5 mg mL−1) showed no antifungal effects against C. albicans and C. neoformans in vitro. However, the tea extract with EGCG inhibited the growth of Trichphyton in a dose- and contact time-dependent manner. Using scanning and transmission electron microscopy to study the mode of action, the same research group examined the effects of EGCG against T. mentagrophytes (Toyoshima et al., 1994). EGCG was shown to inhibit the germination of conidia and subsequent hyphal growth. After 3 days of EGCG treatment, the morphological characteristics of the conidia were changed in terms of deformation and swelling and after 5 days, most of the ungerminated conidia were broken down. In addition, the hyphal cell walls were exfoliated. It was concluded that EGCG can cause lysis of the conidia and hyphae suggesting that it has an antidermatophytic effect against T. mentagrophytes.
It was over 15 years later before the in vitro activity of EGCG against clinical isolates of dermatophytes was investigated (Park et al., 2011). The susceptibility of 35 dermatophytes to a wide range of EGCG concentrations was tested using the standard protocol (M38-A2) from the Clinical and Laboratory Standards Institute (CLSI). The MIC50 and MIC90 of EGCG were 2–4 and 4–8 μg mL−1, respectively. Interestingly, T. rubrum was more susceptible than T. mentagrophytes and Microsporum canis. However, more in vivo and ex vivo experiments need to be performed to verify a potential effect of EGCG.
While infections with dermatophytes only sometimes present therapeutic challenges, yeasts like Candida spp. possess a substantially higher medical relevance in terms of associated morbidity and mortality.
A study testing the susceptibility of C. albicans to catechins as single agents and in combination with antifungal agents by a broth microdilution method showed that EGCG had pH-dependent anti-C. albicans effects (Hirasawa and Takada, 2004). At a pH of 7.0, the MIC90 of EGCG ranged between 15.6 and 250 μg mL−1. The combination of EGCG with antifungal agents (amphotericin B, fluconazole) inhibited the growth of different reference strains indicating additive or synergistic effects. The results from another investigation evaluating the antifungal activity of EGCG (CLSI M27-A) on 21 clinical isolates of seven Candida species in vitro was mainly in agreement with those obtained previously (Park et al., 2006). The MIC90 of EGCG against C. albicans was >16 μg mL−1 whereas C. glabrata, C. guilliemondii and C. parapsilosis exhibited the highest susceptibility (MIC90; 1–16 μg mL−1). As expected, most antifungals revealed lower MIC values against Candida spp. than EGCG. Hence, it has been suggested that EGCG could be used as an agent or adjuvant for antifungal therapy in candidiasis. However, the mechanism of the antifungal effect of EGCG has not been defined and in vivo experiments are currently lacking. So far three studies have been performed to try and address these issues.
In an in vitro study, it was shown that EGCG, EGC and ECG cause metabolic instability of C. albicans cultures even at physiological polyphenol concentrations found in green tea (Evensen and Braun, 2009). Of the three catechins, EGCG was found to be the most potent at retarding the formation and maintenance of Candida biofilm and to disrupt a preformed biofilm. It was demonstrated that higher EGCG concentrations inhibited C. albicans proteasomol chymotrypsin-like activity in vivo suggesting that the impairment of proteasol activity contributes to the cellular metabolic and structural disruptions of this yeast.
A study by Navarro-Martínez and colleagues explored the mechanism of the inhibitory effect of tea cathechins on C. albicans (Navarro-Martinez et al., 2006). They found nearly the same MICs of EGCG against C. albicans as previously shown by Hirasawa et al. (2004). In addition, they demonstrated that the inhibitory effect of EGCG on the C. albicans DHFR (Ki = 0.7 μM), a key enzyme in the biosynthesis of purines, pyrimidines and several amino acids, was pH-independent. When EGCG was combined with azole antifungals (ketonazole and itraconazole) or inhibitors of the ergosterol biosynthesis pathway it mainly had a synergistic effect. EGCG was shown to inhibit ergosterol production by disturbing the folate metabolism in C. albicans cells. In addition, EGCG was also shown to have activity against an azole-resistant isolate and it was proposed that EGCG might be an alternative treatment for C. albicans infections. Hence, the results from this investigation provided new information as to the mode of action of EGCG: EGCG not only indirectly disrupts the ergosterol synthesis pathway through disruption of the folate cycle, but also inhibits ergosterol biosynthesis by reducing the cellular pools of the methyl donor S-adenosyl-methionine.
As the results from in vitro experiments suggested that EGCG could be an effective treatment for Candida spp., Han conducted the first in vivo investigation of the anticandidal effects of EGCG alone and combined with amphotericin B in a murine model of disseminated candidiasis (Han, 2007). It was found that when EGCG, 1–4 mg kg−1 (i.p.), was administered alone to BALB/c mice before an i.v. inoculation of 5 × 105 C. albicans cells it had a dose-dependent inhibitory effect on the survival of the cells: the mean survival time was 29.0 days with 4 mg kg−1 compared with 11.0 days with 1 mg kg−1. In addition, the combination treatment of EGCG, 2 mg kg−1, and amphotericin B, 0.5 mg kg−1, enhanced the resistance of these inoculated mice for up to 42.1 days compared with the survival rates of the untreated control (10.9 days). These results demonstrate that EGCG has anticandidal activity in vivo, and further show that it has a synergist effect when combined with amphotericin B in a murine model of disseminated candidiasis.
In summary, most of the in vitro and in vivo data on the antifungal activity of EGCG were obtained against Candida and indicate that EGCG could be used as an additional or alternative therapeutic agent against disseminated candidiasis. However, future work is needed to determine its in vivo efficacy in different settings.