INTRODUCTION

Giardia duodenalis is a flagellated protozoan parasite that colonizes and reproduces in the human intestine, causing giardiasis, of which the most common symptom is serious diarrhoea. Effective drug treatment for G. duodenalis is available, using orally administered metronidazole, but there is evidence of resistance (e.g. Harris et al. ²⁰⁰¹; Upcroft et al. ²⁰⁰⁶). In addition, side effects such as nausea and headaches combined with an unpalatable metallic taste reduce patient compliance. In poorer countries where giardiasis is endemic, this condition affects both child development and mortality and chemotherapy may be minimally effective due to continual re-infection from the environment and therefore novel drugs and treatments are sought (Rossignol, ²⁰¹⁰).

Plants and plant products have been used as traditional remedies for various ailments in numerous countries, with little or no side effects (Jones, ¹⁹⁹⁶). Wild blueberries (Vaccinium myrtillus) and wild strawberries (Fragaria vesca) have been historically used to treat ‘fluxes’ (or florid diarrhoea) in the UK (see Anthony et al.²⁰⁰⁷). The true causes of such diarrhoea/fluxes cannot be confirmed but certainly could be caused by enteropathogens (Cox, ²⁰⁰²) such as Giardia and Cryptosporidium.

The anti-microbial properties of plant products have been recognized for numerous years, and recently there has been renewed interest in berries and their phenolic constituents such as anthocyanins and ellagitannins (e.g. Nohynek et al.²⁰⁰⁶). Polyphenol-rich berry extracts inhibited the growth of enteropathogenic bacteria such as Salmonella, Escherichia, Staphylococcus, Helicobacter, Bacillus, Clostridium and Campylobacter species, and were bacteriostatic for 3 Staphylococcus aureus strains (Puupponen-Pimia et al.²⁰⁰⁵).

Trophozoites from infective cysts infect the duodenum, attaching onto enterocytes via a combination of hydrodynamic forces and their ventral ‘sucking’ disc and through lectins specific for D-glucosyl and D-mannose residues (Inge et al. ¹⁹⁸⁸). Berry extracts may inhibit the adhesion and colonization of Giardia to human enterocytes. Proanthocyanidins found in cranberry juice (Vaccinium macrocarpon, and in other Vaccinium species) have potent bacterial anti-adhesion activity (Howell et al. ²⁰⁰⁵) and the proanthocyanidin, Geranin D, from Geranium niveum has potent anti-giardial activity (Calzada et al.²⁰⁰¹) Plant products have been demonstrated to possess anti-protozoan effects, but relatively little is known. Garlic extract inhibits G. duodenalis trophozoite growth in vitro (Lun et al. ¹⁹⁹⁴) and treatment with garlic extract can reduce clinical signs and symptoms more rapidly than metronidazole, the current drug of choice (Soffar and Mokhtar, ¹⁹⁹¹). Whole garlic extract co-cultured with trophozoites causes internalization of flagella and ventral disc fragmentation (Harris et al. ²⁰⁰⁰; Anthony et al. ²⁰⁰⁵). Isoflavones, especially formononetin from Dalbergia frutescens, have potent anti-giardial activity in vitro (Khan et al.²⁰⁰⁰) with a possible mode of action being the detachment of Giardia trophozoites in vitro and ex vivo through the inhibition of flagellar motility (Lauwaet et al.²⁰¹⁰).

Previous work investigated a pressed extract of blueberries, a commercial drink made from this extract and a polyphenolic-rich extract for their ability to influence G. duodenalis trophozoite viability in vitro and confirmed the effectiveness of the polyphenolic components (Anthony et al.²⁰⁰⁷). This study examines the effects of polyphenol-rich extracts from a range of berry species with disparate composition on Giardia viability in vitro.

MATERIALS AND METHODS

RESULTS

Strawberry, blackberry, cloudberry and arctic bramble were as effective in killing Giardia as the positive control, metronidazole, at 50 μg GAE/well (Fig. 1). These effects were not due to changes in pH as the polyphenol-enriched extracts were effectively devoid of organic acids and did not alter the pH of the medium. There was no correlation between anthocyanin content and effectiveness as both cloudberry (<0.1%) and blackberry (58.0%) anthocyanin content; (see Supplementary data, Table S1, Online version only) caused complete trophozoite killing at 50 μg GAE well⁻¹.

The most effective berry extracts were re-assayed at lower concentrations to assess dose-response effects (Fig. 2). This revealed an order of effectiveness of blueberry < strawberry < blackberry=arctic bramble < cloudberry. Cloudberry extracts were most effective and caused high levels of killing at 20 μg GAE well⁻¹ when the other extracts had lost activity.

LC-MS analysis (Fig. 3) confirmed that ellagitannins were the predominant polyphenolic components in the cloudberry extracts (Table 1) but also identified significant amounts of benzoic acid and p-coumaric acid. As an example, the mass spectral properties leading to the identification of peak 6 as Sanguiin H6 are discussed in more detail in supplementary data (Supplementary Fig. S1, Online version only). Smaller amounts of flavonol derivatives (e.g. quercetin-3-O-glucuronide) could also be identified. The ellagitannin content of the cloudberry extract was estimated at approximately 72.0±1.4% compared to 44.1±0.8% for the less effective extract from the related arctic bramble. Although this value is only an estimate (calculated from a ratio of the peak area due to ellagitannins and ellagic acid divided by the total peak area of all identified phenolic components), it certainly reflects the higher content of non-ellagitannin contents such as anthocyanins in arctic bramble (see Supplementary data, Table S1).

Indeed, the high ellagitannin content of cloudberry has been noted previously in this laboratory (McDougall et al. ²⁰⁰⁸) and in previous work (Kähkönen et al.²⁰⁰¹; Kopenen et al.²⁰⁰⁷). For example, Kähkönen et al. (²⁰⁰¹) estimated the ET content in different cloudberry samples that ranged from 65 to 88% of total phenol content. However, the ellagitannin content of arctic bramble has not been published previously.

DISCUSSION

Berry extracts have long been known to interfere with human microbial pathogens. One of the most widely known is the inhibitory effect of proanthocyanidins from cranberry (Howell et al.²⁰⁰⁵), and lingonberry (Kontiokari et al.²⁰⁰¹), on bacterial urinary tract infections. Berry extracts rich in ellagitannins have also been noted as being particularly effective against human pathogenic micro-organisms (Heinonen et al. ²⁰⁰⁷) with cloudberry being the most effective against the widest range of bacteria and yeasts (Nohynek et al. ²⁰⁰⁶). This builds on work that outlined anti-microbial activity of ellagitannins from other plant sources (Latte and Kolodziej, ²⁰⁰⁰; Asres et al. ²⁰⁰¹; Machado et al. ²⁰⁰²; Parashar et al. ²⁰⁰⁹, and reviewed by Yoshida et al. ²⁰⁰⁹).

Indeed, ellagitannin-rich extracts and purified ellagitannins have been reported to have effects on human protozoan parasites such as Trypanosoma (Shuaibu et al. ²⁰⁰⁸), Plasmodium species (Dell'Agli et al. ²⁰⁰⁹) and Leishmania species (Kolodziej and Kiderlen, ²⁰⁰⁵). Ellagic acid, which can be formed by ellagitannin degradation, has also been shown to be effective against Plasmodium species in vivo and in vitro (Soh et al. ²⁰⁰⁹). Ellagic acid was also identified as an active anti-giardial agent in Rubus coriifolius (Alanis et al. ²⁰⁰³). In addition, other studies have shown toxicity of ellagitannins against the nematode, C. elegans (Yamasaki et al. ²⁰⁰²).

Here we have shown that the polyphenol-rich extracts of strawberry, blackberry, cloudberry and arctic bramble were as effective as metronidazole in the in vitro killing of G. duodenalis trophozoites and that cloudberry proved to be the most effective. The minimum concentration of cloudberry phenolics found to be effective against Giardia trophozoites in this study was between 10 and 20 μg GAE per well (which equates to 33–66 μg ml⁻¹). Considering that cloudberries can provide around 150 mg GAE/100 g fruit (approx. 1500 μg ml⁻¹: Kähkönen et al. ²⁰⁰¹), the amount of phenolic material entering the small intestine could easily reach levels shown to be effective in vitro, even allowing for partial degradation in the stomach (Clifford and Scalbert, ²⁰⁰⁰). In studies with ileostomy volunteers, Gonzalez-Barrio et al. (²⁰¹⁰) have shown that a large proportion of ingested raspberry polyphenols were recovered in ileal fluid and, moreover, about a quarter of the main ellagitannin component, Sanguiin H6, survived digestion in the upper gastrointestinal tract.

As well as having a high percentage of ellagitannins, cloudberries are notable for having substantial levels of unconjugated p-coumaric acid and benzoic acid (Maatta-Riihinen et al. ²⁰⁰⁴). p-Coumaric acid has been shown to have a weak effect on Giardia trophozoites (Calzada and Alanis, ²⁰⁰⁷) and the coumaric acid derivative (melilotoside) was identified as an active anti-giardial agent from Teloxys graveolens (Calzada et al. ²⁰⁰³). The presence of p-coumaric and benzoic acid may potentiate the effects on Giardia caused by the ellagitannins but perhaps by a different and synergistic mechanism (Fyfe et al. ¹⁹⁹⁸; Puupponen-Pimiä et al. ²⁰⁰¹). In addition, quercetin derivatives, also identified in cloudberry, may also contribute to anti-giardial activity (Amaral et al. ²⁰⁰⁶; Calzada and Alanis ²⁰⁰⁷).

In conclusion, this study has confirmed that berry polyphenols can influence Giardia survival in vitro and suggests that ellagitannins are most effective. Tannin-rich preparations may also have efficacious effects on diarrhoeal symptoms (Palombo, ²⁰⁰⁶) often associated with Giardia infection. Unlike many plant sources of anti-giardial agents (e.g. Amaral et al. ²⁰⁰⁶), berries are a natural and palatable foodstuff and therefore have few issues with toxicity, side effects or acceptance. However, further work is required to uncover whether these in vitro effects described here can be transferred to the in vivo situation and contribute to giardiasis treatment.

ACKNOWLEDGEMENTS

SCRI receives grant-in-aid from the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD). In memoriam of Huw Vaughn Smith who sadly passed away in October 2010 and contributed greatly to parasitology and to the analysis presented in this manuscript.

[TableWrap ID: tab01] Table 1 

Putative identification of ellagitannin peaks in cloudberry extract

  —(All MS data are from negative mode ionization. The major signal is given in bold. Putative identifications are based on previous work on cloudberry (McDougall et al. ²⁰⁰⁸, ²⁰⁰⁹) and previous literature (Mullen et al.²⁰⁰³; Gasperotti et al.²⁰¹⁰; Hager et al.²⁰⁰⁸). More detailed information about the identification of peak 6 as sanguiin H6 is shown in the Supplementary data (Fig. S1), Online version only.).