Abstract
Antibacterial and antifungal activity of methanol extracts of the mosses Pleurozium schreberi (Willd. Ex Brid.) Mitt. (Hylocomiaceae), Palustriella commutata (Hedw.) Ochyra (Amblystegiaceae), Homalothecium philippeanum (Spruce) Schimp. (Brachytheciaceae), Anomodon attenuatus (Hedw.) Huebener (Anomodontaceae), Rhytidium rugosum (Hedw.) Kindb. (Rhytidiaceae), Hylocomium splendens (Hedw.) Schimp. (Hylocomiaceae), Dicranum scoparium Hedw. (Dicranaceae), and Leucobryum glaucum (Hedw.) Ångstr. (Leucobryaceae), were tested against six bacterial and seven fungal species by microdilution and disc diffusion methods. The extract of A. attenuatus possessed the highest antibacterial activity (MIC of 1.25–5.0 mg/ml and MBC of 2.5–5.0 mg/ml), while L. glaucum extract showed the lowest activity (MIC of 20.0–25.0 mg/ml and MBC of 25.0 mg/ml). The best antifungal activity was obtained from P. schreberi extract (MIC of 0.5 mg/ml and MFC of 2.5–5.0 mg/ml, while the lowest antifungal potential was obtained from A. attenuatus (MIC 2.5–5 mg/ml and MFC 10 mg/ml). The extracts proved to be more active against Gram (+) bacteria than Gram (−) and showed strong antifungal activity.
Introduction
The Bryopsida (Musci, genuine mosses) is a large group of non-vascular plants, consisting of about 14,500 species. It is interesting that only some birds and insects use mosses in feeding and that other groups of organisms avoid them (CitationSaxena & Harinder, 2004). The investigations of secondary metabolites, such as flavonoids, of bryophytes are still insufficient. It has been shown that mosses rich with flavonoids possess strong antimicrobial activity. CitationMarkham and Given (1987) showed that species of genus Bryum Hedw. (Bryaceae) are rich with flavonoid glycosides (apigenin and luteolin glycosides and their 6′′ malonyl esters, and 8-hydroxyapigenin-7-O-glucoside and 8-hydroxyluteolin-7-O-glucoside). Dicranin isolated from CH2Cl2 extract of Dicranum scoparium Hedw. (Dicranaceae) showed antimicrobial activity (CitationBorel et al., 1993). An acetone extract of Rhynchostegium riparioides (Hedw.) Cardot. (Brachytheciaceae) exhibited antibacterial activity against Gram (−) bacteria (CitationBasile et al., 1998). Methanol and acetone extracts of Palustriella commutata were active against Gram (+) and Gram (−) bacteria (CitationIlhan et al., 2006).
This study analysed the antimicrobial activity of methanol extracts of Pleurozium schreberi (Willd. Ex Brid.) Mitt. (Hylocomiaceae), Palustriella commutata (Hedw.) Ochyra (Amblystegiaceae), Homalothecium philippeanum (Spruce) Schimp. (Brachytheciaceae), Anomodon attenuatus (Hedw.) Huebener (Anomodontaceae), Rhytidium rugosum (Hedw.) Kindb. (Rhytidiaceae), Hylocomium splendens (Hedw.) Schimp. (Hylocomiaceae), Dicranum scoparium Hedw. (Dicranaceae), and Leucobryum glaucum (Hedw.) Ångstr. (Leucobryaceae) against plant, animal, and human pathogenic bacteria and fungi.
Materials and Methods
Plant material and extract preparation
All mosses tested were collected from the natural habitats. Leucobryum glaucum was collected in Ražanj, Serbia, during May 1998 (voucher no. 16122). Pleurozium schreberi (no. 16118), Rhytidium rugosum (no. 16120), Dicranum scoparium (no. 16121) and Homalothecium philippeanum (no. 16123) were collected from Zlatar mountain, Serbia, in September 2000. Hylocomium splendens (no. 16117) was collected from Zlatar mountain, Serbia, in October 2001, Palustriella commutata (no. 16119) from the canyon of the Moraèa river, Montenegro in May 2004, and Anomodon attenuatus (no. 16124) from Vrdnik, Serbia, in June 2005. All moss taxa have been identified by the first author (M.V.).
Samples of each moss (10 g) were dried by airflow at room temperature. They were then finely ground with a hammer mill and extracted separately with 80% methanol (100 × 2 ml) in H2O for 24 h at 40°C (CitationIlhan et al., 2006). Analytical grade methanol was purchased from Zorka Pharma Šabac. Extracts were filtered with celullose-acetate membrane (0.45 μ m). Filtrates were evaporated to dryness with a rotary evaporator and 80 mg dry extracts were dissolved with 1 ml of dimethyl sulfoxide (DMSO).
Masses of dried extracts were as follows: H. philippeanum (0.431 g), P. schreberi (0.360 g), R. rugosum (0.243 g), P. commutata (0.168 g), A. attenuatus (0.186 g), H. splendens (0.198 g), D. scoparium (0.155 g), and L. glaucum (0.191 g) were dissolved in DMSO to obtain stock solution 1 mg/ml.
Tests for antibacterial activity
The following bacterial species were used: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Micrococcus flavus (ATCC 9341), Bacillus subtilis (ATCC 10707), Escherichia coli (ATCC 25922), Enterobacter cloacae (human isolate), Salmonella typhimurium (ATCC 13311).
Bacterial species were cultured overnight at 37°C in LB medium. Inoculum suspensions containing ∼ 106 cells/ml were used for experiments.
The antibacterial assays were carried out by the modified disc-diffusion method (CitationVerpoorte et al., 1983) and microdilution method (CitationHanel & Raether, 1988; CitationDaouk et al., 1995).
Disc diffusion method
In Petri dishes (diameter 90 mm) filled with the Mueller-Hinton agar and seeded with 0.3 ml of the test organism, a sterile filter disc (diameter 4 mm, Whatman paper no. 3) was placed. The disc was impregnated with test concentrations (0.05–2 mg/disc) of compounds investigated dissolved in DMSO. The zones of growth inhibition around the discs were measured after 24 h of incubation at 37°C.
Each microorganism was tested in triplicate and the solvent (DMSO) was used as a control, while streptomycin was used as a positive control.
Microdilution method
In order to obtain quantitative data for compounds investigated, the modified microdilution technique was used (CitationHanel & Raether, 1988; CitationDaouk et al., 1995). Bacterial species were cultured overnight at 37°C in LB medium. The inoculum suspension used for the experiment contained ∼ 106 cells/ml.
The inoculum suspension was adjusted with sterile saline to a concentration of approximately 1.0 × 105 in a final volume of 100 μ l per well. The inocula were stored at 4°C for further use. Dilutions of the inocula were cultured on solid MH medium to verify the absence of contamination and to check the validity of the inoculum.
Minimum inhibitory concentrations (MICs) determination was performed by a serial dilution technique using 96-well microtiter plates. Compounds investigated were dissolved in broth medium with inoculum to achieve desired concentrations (0.05–20 mg/ml). The microplates were incubated for 72 h at 28°C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations which completely inhibited bacterial growth (MICs). The minimum bactericidal concentrations (MBCs) were determined by serial subcultivation of a 2 μ l into microtiter plates containing 100 μ l of broth per well and further incubation for 72 h at 28°C. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the original inoculum.
DMSO was used as a control, while streptomycin was used as a positive control.
Tests for antifungal activity
The following fungi were used: Aspergillus niger (ATCC 6275), A. ochraceus (ATCC 12066), A. versicolor (ATCC 11730), A. flavus (ATCC 9170), Penicillium funiculosum (ATCC 10509), Trichoderma viride (IAM5061) and Candida albicans human isolate.
Disc diffusion method
In order to test antifungal activity of extracts of mosses against Candida albicans, the disc diffusion method was used. In Petri dishes (diameter 90 mm) filled with Sabouraud dextrose agar and seeded with 0.3 ml of C. albicans inoculum (containing ∼ 106 cells/ml), a sterile filter disc (diameter 4 mm, Whatman paper No. 3) was placed. The disc was impregnated with test concentrations (0.5–2 mg/disc) of compounds investigated dissolved in dimethyl sulfoxide (DMSO). The zones of growth inhibition around the discs were measured after 24 h of incubation at 37°C.
Each microorganism was tested in triplicate and the solvent DMSO was used as a control, while bifonazole was used as a positive control (0.1–2 mg/disc).
Microdilution method
In order to investigate the antifungal activity of extracts the microdilution technique was used. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0 × 105 in a final volume of 100 μ l/ml. The inoculates were stored at 4°C for further use. Dilutions of the inocula were cultured on solid MA to verify the absence of contamination and to check the validity of the inoculum.
Determination of minimum inhibitory concentrations (MICs) was performed by a dilution technique using 96-well microtiter plates. The extracts were added in broth medium with fungal inoculum to achieve required concentrations (0.5–10 mg/ml). Commercial fungicide, bifonazole, was used as a control, 0.1, 0.5, and 1 mg/ml. The microplates were incubated for 72 h at 28°C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations which completely inhibited fungal growth (MICs).
Results and Discussion
Summary results of antibacterial activity of moss methanol extracts are presented in and . As a preliminary screening of the antibacterial potency, the obtained results are presented in . The disc-diffusion assay is a qualitative non-standardized method that is useful only for the detection, but not for the comparison, of antimicrobial properties of different samples. The comparison of the size of inhibition halos of different extracts cannot be used for the determination of the relative antimicrobial potency since a more diffusible but less active extract could give a bigger diameter than a non-diffusible but more active extract.
Table 1. The antibacterial activity of methanol extracts (disc-diffusion method).
Table 2. The antibacterial activity of methanol extracts (microdilution method).
For the comparison of antimicrobial activity of different samples, the microdilution method was used. The results from the microdilution method showed that the extract of A. attenuatus possessed the highest antibacterial activity with a MIC of 1.25–5.0 mg/ml and a MBC of 2.5–5.0 mg/ml. The extract of L. glaucum showed the lowest activity with a MIC of 20.0–25.0 mg/ml and a MBC of 25.0 mg/ml. The most resistant bacteria was as in the previous method—E. cloacae. Only extracts of P. commutata and A. attenuatus showed bactericidal activity at a lower concentration (5.0 mg/ml). Streptomycin showed better antibacterial activity than extracts from the investigated mosses ().
The results of antifungal activity of methanol extracts of mosses are presented in and . All the extracts tested showed great antifungal activity. The antifungal potential of the extracts can be present in this order: P. schreberi, P. commutata, D. scoparium, L. glaucum. H. philippeanum, R. rugosum, H. splendens and A. attenuatus.
Table 3. The antifungal activity of methanol extracts (microdilution method).
Table 4. The antifungal activity of methanol extracts (disc-diffusion method).
The best antifungal activity was obtained for P. schreberi extract with a MIC of 0.5 mg/ml, and a MFC of 2.5–5.0 mg/ml. The antifungal effect of this extract is greater than the activity which was obtained for bifonazole against T. viride, while against P. funiculosum and A. Ochraceus, this extract showed the same effect as bifonazole. The lowest antifungal potential was obtained for A. attenuatus, MIC 2.5–5 mg/ml, and MFC 10 mg/ml. However, this extract showed better activity than bifonazole against T. viride. The most sensitive microfungi was T. viride (MIC for all the extracts tested were 0.5 mg/ml, and MFC 2.5 mg/ml, while MIC and MFC for bifonazole were 1 mg/ml). The most resistant fungus was A. niger, MIC for all the extracts tested were 2.5 mg/ml, and MFC 5.0 mg/ml (except for A. attenuatus MIC 2.5 mg/ml and MFC 10.0 mg/ml). Bifonazole showed better activity against this species, MIC and MFC 0.1 mg/ml.
The antifungal activity of methanol extracts of mosses was also tested against yeast Candida albicans by the disc-diffusion method (). The antifungal effect was obtained for P. schreberi, P. commutata and H. splendens, with inhibition zones of 11.5, 8.5, and 8.0 mm, respectively. The rest of the extracts did not show activity against C. albicans even at higher concentrations (1 and 2 mg/disc). Bifonazole showed antifungal activity against yeast at 0.2 mg/disc with inhibition zone 30 mm.
Methanol extracts tested in this work showed antimicrobial activity but it was observed that they possessed greater antifungal than antibacterial activity. Extracts showed better antibacterial activity against Gram (+) bacteria than against Gram (−).
The growth of tested microorganisms responded differently to the investigated extracts, which indicated that may have different modes of action or that the metabolism of some fungi was able to better overcome the effect of the compound tested or adapt to it.
The literature data concerning antimicrobial activity of mosses are poor and need more attention. The methanol extract of H. sericeum (30 mg/ml) inhibited growth of C. albicans in our work. The previous data indicated the presence of flavonoids in the extract which could explain the activity (CitationDulger et al., 2005). In different species of the genus Bryum flavonoid glycosides are present (apigenin and luteolin glycosides and their 6″ malonyl esters, and 8-hydroxapigenin-7-O-glucosides and 8-hydroxiluteolin-7-O-glucosides (CitationMarkham & Given, 1987). The antimicrobial activity of ethanol extracts of Bryum argenteum was tested against bacteria (S. aureus, M. luteus, B. subtilis, and E. coli), and fungi (A. niger, P. ochrochloron, C. albicans and T. mentagrophyes) and showed activity against all the organisms tested (CitationSaboljević et al., 2006).
Methanol extracts of H. cupressiforme and M. undulatum (30 mg/ml) was inhibited Gram (−) and Gram (+) bacterial species and C. albicans. The previous data showed that in H. cupressiforme methanol extract polycyclic aromatic hydrocarbon, hipnogenol, biflavonoids and hydroxiflavonoids were present, while in extract of M. undulatum flavonoids glycosides and other type of flavonoids were detected (CitationDulger et al., 2005).
The previous investigation of antifungal activity of other secondary metaboiltes showed that T. viride is the most resistant fungal species even with fungicides (CitationSoković et al., 2002). It is important to notice that extracts tested in this paper showed the strongest antifungal effects against this species (MIC 0.5 mg/ml, MFC 5.0 mg/ml) and that these extracts showed higher activity than commercial fungicide—bifonazole.
These results clearly indicate that extracts investigated should find a practical application in the prevention and protection of fungal infections of plants, animals, and humans. Essential oils could be safely used as preservative materials on foods to protect it from fungal infection, since they are natural and non-toxic to humans.
Acknowledgements
The authors are grateful to the Ministry of Sceince for financial support (grants 143049 and 143041).
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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