Introduction

Free-living amoebae of the genus Acanthamoeba constitute an aetiological factor influencing chronic granulomatous amoebic encephalitis (GAE), Acanthamoeba keratitis (AK), amoebic pneumonitis (AP), as well as changes occurring in other human and animal organs. Treatment of Acanthamoeba infections is always very difficult and not always effective. Apart from antibiotics, which prevent further consequences of tissue damage, use is made of compounds that are amoebicidal or amoebistatic. These substances usually used for disinfection are frequently strongly irritating and toxic for the host (Kitagawa et al. 2003; Seal 2003).

The aim of the present study was to investigate the amoebicidal or amoebistatic effect of plant extracts obtained from Rubus chamaemorus, Pueraria lobata, Solidago virgaurea, S. graminifolia, Eryngium maritimum L. and E. planum L. on the growth and development of free-living amoebae of the genus Acanthamoeba.

Materials and methods

The plant material used to isolate pharmacologically active substances comprised the aboveground parts of plants, including flowers of common goldenrod S. virgaurea L. and grass-leaved goldenrod S. graminifolia (L.) Elliott.(Asteraceae), the roots of kudzu P. lobata (Willd.) (Fabaceae) and the leaves of cloudberries R. chamaemorus L. (Rosaceae).

Herbarial specimens of the researched plants are kept at the Department of Pharmaceutical Botany of Poznan University of Medical Sciences.

Each batch of dried and fragmented plant material (approximately 10.0 g) was extracted three times for 1 h in 150 mL of methanol. The combined methanol extracts were filtered off and dried in a vacuum on a water bath at a temperature under 40°C. Research into therapeutic properties was performed on dry extracts of each plant species, which were dissolved in distilled water.

The tested plant extracts underwent a phytochemical analysis aimed at establishing the presence of flavonoids in S. virgaurea and S. graminifolia, ellagic acid in R. chamaemorus and isoflavones in P. lobata.

Phytochemical analysis of extracts was performed using column chromatography, thin-layer preparative chromatography and high-performance liquid chromatography (HPLC; Krawczyk et al. 2003).

The influence of therapeutic substances obtained from plants was tested in vitro on strain 309 Acanthamoeba castellanii (pathogenic for mice, isolated from the environment; Kasprzak and Mazur 1972), and on the “Ograbek” strain Acanthamoeba sp. (pathogenic for humans, isolated for Acanthamoeba keratitis—own, previously undescribed strain).

The amoebae strains were axenically cultured on a liquid medium Bacto-Casitone+horse serum described by Červa (1966, 1969). Plant extracts were added to the axenic culture of amoebae (5 × 104 cells/mL) in the following concentrations: 0.01, 0.05, 0.1, 0.5, 1.0 and 1.5 mg/mL. The increase or decrease in the number of amoebae was checked at 24-h intervals using a Thoma haemocytometric chamber. The control group was a culture of amoebae without extracts. The IC50 coefficient refers to the lowest concentration of the researched substance that halts the growth of amoebae in 50%.

The pathogenic properties of amoebae were tested by infecting 2-week-old white mice of the BALB-c strain using the procedure described by Kasprzak and Mazur (1972) and Mazur (1984).

Research into the therapeutic influence of plant extracts on the progress of the infection and survival time of animals consisted in the daily provision to the intraperitoneally infected mice, over a period of 3 days, of sterile solutions of plant extracts in concentrations ranging from 0.1 to 0.5 mg of dry extract per 1 g of body mass. The extracts were dissolved in normal saline with a small quantity of dimethyl sulfoxide.

All of the experiments were repeated five to seven times. Tests on the animals were repeated five times, using five to ten animals for each test series.

Results

Phytochemical tests of the methanol extract from the flowers and leaves of Solidago sp. have shown the presence of a number of lipophilic flavonoids. Attempts at determining the structure of these compounds are currently in progress.

The main phenolic compound present in the extract from leaves of the species R. chamaemorus L. is ellagic acid, which is most probably responsible for the action of the extract from cloudberry leaves. HPLC was used to designate the quantity of ellagic acid in cloudberry leaves. The content of ellagic acid was 6.996 g/100 g of dry mass.

In addition, thin-layer chromatography was used to determine the presence of gallic acid (apart from ellagic acid) and derivatives of these two acids; the presence of a small quantity of flavones in the extracts was also confirmed.

In the leaves of an Asian species of the kudzu P. lobata (Willd) Ohwi, the main secondary metabolites are compounds belonging to the group of isoflavones: daidzein, genistein and formonentin. In young roots of P. lobata, thin-layer chromatography was used to determine the presence of an isoflavone—puerarin.

Quantitative analysis using the HPLC method for the hydrolysed and non-hydrolysed root methanol extract from P. lobata contained puerarin in a quantity of 5.32 mg/g dry mass and daidzein in a quantity of 5.22 mg/g dry mass in the plant extract.

The coefficients of inhibition of growth of amoebae from the genus Acanthamoeba sp. (IC50) calculated for individual plant extracts and pure substances are presented in Table 1. It was determined that extracts from S. virgauera, P. lobata and R. chamaemorus had chemotherapeutic properties in vitro in concentrations of approximately 0.01–0.05 mg extract/mL.

Table 1 IC50 of plant extracts for the amoebae cultures
Full size table

Table 2 presents the results of designation of therapeutic coefficients ED50 of tested plant extracts for laboratory mice of the BALB-c strain. The results of IC50 and therapeutic index (TI) research for both tested pathogenic strains of Acanthamoeba sp. are nearly identical and statistically significant.

Table 2 Values of the lethal dose (LD50), therapeutic dose (ED50) and TI for the mice
Full size table

Tests concerning the therapeutic action of plant extracts on the experimental infection with Acanthamoeba are presented in Table 3. It was determined that, following the application of the extracts, the animals survived considerably longer (2.5–3 times).

Table 3 The survival time for mice infected with A. castellanii (309) following the application of the plant extracts; n = 5 for five to ten mice
Full size table

Discussion

Free-living amoebae from the genus Acanthamoeba are commonly occurring organisms, and they may be found in the soil, air, in each salty or drinking water reservoir and on every continent, as well as in air-conditioning units, in water mains, showers, sanitary and dental equipment, dialysers, fluids for contact lenses and infected tissue cultures (Visvesvara and Stehr-Green 1990; De Jonckheere 1991; Mergeryan 1991; Szenasi et al. 1998). The first suggestions that amoeba may cause diseases afflicting humans were made in 1958 (Marciano-Cabral and Cabral 2003). Currently, cases of GAE, Acanthamoeba keratitis, amoebic pneumonitis and skin inflammation are observed around the world (Fowler and Carter 1965; Callicott 1968; Jager and Stamm 1972; Willaert et al. 1976; Martinez et al. 1977; Martinez and Visvesvara 1997; Marciano-Cabral et al. 2000).

Clinical symptoms of human GAE include headaches, fever, neurological disorders, such as hallucinations, disorientation and vision disorders, personality changes and coma (Martinez and Visvesvara 1997). Acanthamoeba keratitis, in turn, is characterised by strong ophthalmalgia, photophobia, blue–red vision and blood extravasations. In the lungs, amoebae cause numerous inflammation foci (AP), which is accompanied by the exudation of serous fluid containing trophozoites and cysts. Skin changes are in the form of numerous, more or less extensive ulcerations. All of the infections are typically chronic.

In all cases of Acanthamoeba sp. invasions, chemotherapy poses a serious problem. A majority of invasions end in patient death. Only a few instances of successful chemotherapy have been noted, performed using highly toxic drugs usually used for disinfection, e.g. chlorhexidine derivatives (Kitagawa et al. 2003; Seal 2003). Effective treatment of infections of the central nervous system or eyeballs for immunocompetent persons has been recorded in the event of usage of a combined therapy, commenced at an early stage of the disease. However, in the latter stages of the disease, the majority of therapeutic agents are ineffective (Ficker et al. 1990; Dougherty et al. 1994; Horne et al. 1994; Murdoch et al. 1998).

Regardless of the wide application of therapeutic agents to combat AK, with the value thereof being unquestioned, the majority of drugs display a high toxicity for humans, causing undesirable reactions. For this reason, research is being conducted into alternative methods of treating AK, a disease which oftentimes leads to blindness and even death. Our research focused on plant extracts from S. virgaurea, S. graminifolia (goldenrod), Rubus chamaemors (cloudberries) and P. lobata (kudzu) and the potential application thereof in combating human Acanthamoeba sp. infections (Derda et al. 2004a, b). Drugs of natural origin have already been used to treat other parasitic diseases (Arrieta et al. 2001; Kayser et al. 2003; Said Fernández et al. 2005).

Table 1 presents results which indicate that substances present in methanol extracts from the tested plants are active with respect to both the tested pathogenic strains of amoebae from the genus Acanthamoeba in very low concentrations, i.e. approximately 0.01–0.05 mg extract/mL or approximately 0.35 μg/mL calculated for ellagic acid (cloudberries) and 0.53 μg/mL calculated for puerarin (kudzu), and they may be used in a combined treatment with antibiotics. The TI value is 20 for extracts from R. chamaemors, which is very high; for S. virgaurea, the TI is 7.5. Research has clearly shown that plant extracts may be successfully used both internally and externally in the event of a combined therapy against Acanthamoeba infections. Plant extracts administered to experimentally infected animals considerably lengthened their survival time in comparison with control animals that were not given any treatment. The control animals that were infected usually died after 4 days. The animals which received a plant extract monotherapy survived for a period 2.5–3 times longer. In the case of animals that were not infected, the therapeutic doses of drugs given did not display any toxic activity.

In the case of human infection with Acanthamoeba sp., a combined therapy connected with a standard antibiotic treatment would definitely be more effective than a therapy conducted using a single drug. This is so because numerous drugs have amoebistatic properties, but no amoebicidal properties. Some drugs are deadly to trophozoites, but ineffective in the case of cysts. To date, no effective drug has been found for both instances (cysts and trophozoites). Research into the efficiency of plant extracts in vivo and in vitro in an experimental Acanthamoeba sp. infection is being continued.