Pharmaceutical Biology2008, Vol. 46, No. 12, pp. 871–875

Antimicrobial Activity of Methanol Extracts of Mosses

from Serbia

Milan Veljic,1 Maja Tarbuk,1 Petar D. Marin,1 Ana Ciric,2 Marina Sokovic,2 and Marija Marin1

1Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, University of Belgrade, Belgrade, Serbia;2Institute for Biological Research “Sinisa Stankovic” Belgrade, Serbia

Abstract

Antibacterial and antifungal activity of methanol extractsof the mosses Pleurozium schreberi (Willd. Ex Brid.)Mitt. (Hylocomiaceae), Palustriella commutata (Hedw.)Ochyra (Amblystegiaceae), Homalothecium philip-peanum (Spruce) Schimp. (Brachytheciaceae), Anomodonattenuatus (Hedw.) Huebener (Anomodontaceae), Rhytid-ium rugosum (Hedw.) Kindb. (Rhytidiaceae), Hylocomiumsplendens (Hedw.) Schimp. (Hylocomiaceae), Dicranumscoparium Hedw. (Dicranaceae), and Leucobryum glaucum(Hedw.) Angstr. (Leucobryaceae), were tested against sixbacterial and seven fungal species by microdilution anddisc diffusion methods. The extract of A. attenuatus pos-sessed the highest antibacterial activity (MIC of 1.25–5.0mg/ml and MBC of 2.5–5.0 mg/ml), while L. glaucumextract showed the lowest activity (MIC of 20.0–25.0mg/ml and MBC of 25.0 mg/ml). The best antifungalactivity was obtained from P. schreberi extract (MIC of0.5 mg/ml and MFC of 2.5–5.0 mg/ml, while the lowestantifungal potential was obtained from A. attenuatus (MIC2.5–5 mg/ml and MFC 10 mg/ml). The extracts proved tobe more active against Gram (+) bacteria than Gram (−)and showed strong antifungal activity.

Keywords: Mosses, methanol extracts, antibacterial activ-ity, antifungal activity.

Introduction

The Bryopsida (Musci, genuine mosses) is a large group ofnon-vascular plants, consisting of about 14,500 species.It is interesting that only some birds and insects usemosses in feeding and that other groups of organisms

Accepted: April 17, 2008

Address correspondence to: Petar D. Marin, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Studentski trg 16,University of Belgrade, 11000 Belgrade, Serbia. Tel: +381 11 3342 114; Fax: +381 11 3243 603. E-mail address: pdmarin@bfbot.bg.ac.yu

avoid them (Saxena & Harinder, 2004). The investiga-tions of secondary metabolites, such as flavonoids, ofbryophytes are still insufficient. It has been shown thatmosses rich with flavonoids possess strong antimicrobialactivity. Markham and Given (1987) showed that speciesof genus Bryum Hedw. (Bryaceae) are rich with flavonoidglycosides (apigenin and luteolin glycosides and their6′′ malonyl esters, and 8-hydroxyapigenin-7-O-glucosideand 8-hydroxyluteolin-7-O-glucoside). Dicranin isolatedfrom CH2Cl2extract of Dicranum scoparium Hedw. (Di-cranaceae) showed antimicrobial activity (Borel et al.,1993). An acetone extract of Rhynchostegium riparioides(Hedw.) Cardot. (Brachytheciaceae) exhibited antibacterialactivity against Gram (−) bacteria (Basile et al., 1998).Methanol and acetone extracts of Palustriella commutatawere active against Gram (+) and Gram (−) bacteria (Ilhanet al., 2006).

This study analysed the antimicrobial activity ofmethanol extracts of Pleurozium schreberi (Willd. Ex Brid.)Mitt. (Hylocomiaceae), Palustriella commutata (Hedw.)Ochyra (Amblystegiaceae), Homalothecium philip-peanum (Spruce) Schimp. (Brachytheciaceae), Anomodonattenuatus (Hedw.) Huebener (Anomodontaceae), Rhytid-ium rugosum (Hedw.) Kindb. (Rhytidiaceae), Hylocomiumsplendens (Hedw.) Schimp. (Hylocomiaceae), Dicranumscoparium Hedw. (Dicranaceae), and Leucobryum glaucum(Hedw.) Angstr. (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 Razanj, Serbia, dur-ing May 1998 (voucher no. 16122). Pleurozium schreberi

DOI: 10.1080/13880200802367502 C© 2008 Informa UK Ltd.

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(no. 16118), Rhytidium rugosum (no. 16120), Dicranumscoparium (no. 16121) and Homalothecium philippeanum(no. 16123) were collected from Zlatar mountain, Serbia, inSeptember 2000. Hylocomium splendens (no. 16117) wascollected from Zlatar mountain, Serbia, in October 2001,Palustriella commutata (no. 16119) from the canyon of theMoraŁa river, Montenegro in May 2004, and Anomodonattenuatus (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 atroom temperature. They were then finely ground with ahammer mill and extracted separately with 80% methanol(100 × 2 ml) in H2O for 24 h at 40◦C (Ilhan et al., 2006). An-alytical grade methanol was purchased from Zorka PharmaSabac. Extracts were filtered with celullose-acetate mem-brane (0.45 µm). Filtrates were evaporated to dryness witha rotary evaporator and 80 mg dry extracts were dissolvedwith 1 ml of dimethyl sulfoxide (DMSO).

Masses of dried extracts were as follows: H. philip-peanum (0.431 g), P. schreberi (0.360 g), R. rugosum (0.243g), P. commutata (0.168 g), A. attenuatus (0.186 g), H.splendens (0.198 g), D. scoparium (0.155 g), and L. glau-cum (0.191 g) were dissolved in DMSO to obtain stocksolution 1 mg/ml.

Tests for antibacterial activity

The following bacterial species were used: Staphylococcusaureus (ATCC 25923), Staphylococcus epidermidis (ATCC12228), Micrococcus flavus (ATCC 9341), Bacillus subtilis(ATCC 10707), Escherichia coli (ATCC 25922), Enter-obacter cloacae (human isolate), Salmonella typhimurium(ATCC 13311).

Bacterial species were cultured overnight at 37◦C in LBmedium. Inoculum suspensions containing ∼106cells/mlwere used for experiments.

The antibacterial assays were carried out by the modifieddisc-diffusion method (Verpoorte et al., 1983) and microdi-lution method (Hanel & Raether, 1988; Daouk 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, asterile filter disc (diameter 4 mm, Whatman paper no. 3) wasplaced. The disc was impregnated with test concentrations(0.05–2 mg/disc) of compounds investigated dissolved inDMSO. The zones of growth inhibition around the discswere measured after 24 h of incubation at 37◦C.

Each microorganism was tested in triplicate and the sol-vent (DMSO) was used as a control, while streptomycinwas used as a positive control.

Microdilution method

In order to obtain quantitative data for compounds inves-tigated, the modified microdilution technique was used

(Hanel & Raether, 1988; Daouk et al., 1995). Bacterialspecies 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 salineto a concentration of approximately 1.0 × 105 in a finalvolume of 100 µl per well. The inocula were stored at 4◦Cfor further use. Dilutions of the inocula were cultured onsolid MH medium to verify the absence of contaminationand to check the validity of the inoculum.

Minimum inhibitory concentrations (MICs) determina-tion was performed by a serial dilution technique using 96-well microtiter plates. Compounds investigated were dis-solved in broth medium with inoculum to achieve desiredconcentrations (0.05–20 mg/ml). The microplates were in-cubated for 72 h at 28◦C. The lowest concentrations withoutvisible growth (at the binocular microscope) were defined asconcentrations which completely inhibited bacterial growth(MICs). The minimum bactericidal concentrations (MBCs)were determined by serial subcultivation of a 2 µl into mi-crotiter plates containing 100 µl of broth per well and fur-ther incubation for 72 h at 28◦C. The lowest concentrationwith no visible growth was defined as the MFC, indicating99.5% killing of the original inoculum.

DMSO was used as a control, while streptomycin wasused as a positive control.

Tests for antifungal activity

The following fungi were used: Aspergillus niger (ATCC6275), A. ochraceus (ATCC 12066), A. versicolor (ATCC11730), A. flavus (ATCC 9170), Penicillium funiculosum(ATCC 10509), Trichoderma viride (IAM5061) and Can-dida albicans human isolate.

Disc diffusion method

In order to test antifungal activity of extracts of mossesagainst Candida albicans, the disc diffusion methodwas used. In Petri dishes (diameter 90 mm) filled withSabouraud dextrose agar and seeded with 0.3 ml of C. al-bicans inoculum (containing ∼106 cells/ml), a sterile filterdisc (diameter 4 mm, Whatman paper No. 3) was placed.The disc was impregnated with test concentrations (0.5–2mg/disc) of compounds investigated dissolved in dimethylsulfoxide (DMSO). The zones of growth inhibition aroundthe discs were measured after 24 h of incubation at 37◦C.

Each microorganism was tested in triplicate and the sol-vent DMSO was used as a control, while bifonazole wasused as a positive control (0.1–2 mg/disc).

Microdilution method

In order to investigate the antifungal activity of extracts themicrodilution technique was used. The fungal spores werewashed from the surface of agar plates with sterile 0.85%

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Table 1. The antibacterial activity of methanol extracts (disc-diffusion method).

Zone of inhibition (mm)Concentration of extracts 2 mg/disc

Pleurozium Palustriella Homalothecium Anomodon Rhytidium Hylocomium Dicranum LeucobryumBacteria schreberi commutata philippeanum attenuatus rugosum splendens scoparium glaucum Streptomycin

S. aureus 12.3 — 13.3 15.0 — — — — 40.0S. epidermidis — — — — 14.7 12.7 29.3 18.3 28.6M. flavus — — — — — — — 12.7 —E. coli — — 15.0 — — — — — 26.3E. cloacae — — — — — — — — —S. typhimurium — 16.7 — — — — — — 35.3

—, absence of inhibition; streptomycin 0.02 mg/disc.

saline containing 0.1% Tween 80 (v/v). The spore suspen-sion was adjusted with sterile saline to a concentration ofapproximately 1.0 × 105 in a final volume of 100 µl/ml.The inoculates were stored at 4◦C for further use. Dilu-tions of the inocula were cultured on solid MA to verify theabsence of contamination and to check the validity of theinoculum.

Determination of minimum inhibitory concentrations(MICs) was performed by a dilution technique using 96-well microtiter plates. The extracts were added in brothmedium with fungal inoculum to achieve required con-centrations (0.5–10 mg/ml). Commercial fungicide, bifon-azole, was used as a control, 0.1, 0.5, and 1 mg/ml. Themicroplates were incubated for 72 h at 28◦C. The lowestconcentrations without visible growth (at the binocular mi-croscope) were defined as concentrations which completelyinhibited fungal growth (MICs).

Results and Discussion

Summary results of antibacterial activity of moss methanolextracts are presented in Tables 1 and 2. As a preliminaryscreening of the antibacterial potency, the obtained resultsare presented in Table 1. The disc-diffusion assay is a qual-itative non-standardized method that is useful only for the

Table 2. The antibacterial activity of methanol extracts (microdilution method).

Concentration of extracts and streptomycin (mg/ml)

Pleurozium Palustriella Homalothecium Anomodon Rhytidium Hylocomium Dicranum Leucobryumschreberi commutata philippeanum attenuatus rugosum splendens scoparium glaucum Streptomycin

Bacteria MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

S. aureus 25.0 25.0 5.0 5.0 5.0 10.0 5.0 5.0 5.0 10.0 5.0 5.0 25.0 25.0 20.0 25.0 1.0 1.0M. flavus 25.0 25.0 5.0 5.0 5.0 10.0 1.25 2.5 5.0 10.0 5.0 5.0 25.0 25.0 25.0 25.0 0.5 1.0B. cereus 10.0 20.0 5.0 5.0 10.0 20.0 2.5 2.5 20.0 20.0 2.5 5.0 5.0 5.0 20.0 25.0 1.0 1.0E. cloacae 25.0 25.0 5.0 5.0 10.0 10.0 5.0 5.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 1.0 1.5S. typhimurium 10.0 20.0 25.0 25.0 5.0 10.0 5.0 5.0 25.0 25.0 5.0 5.0 25.0 25.0 25.0 25.0 0.1 0.1

detection, but not for the comparison, of antimicrobial prop-erties of different samples. The comparison of the size ofinhibition halos of different extracts cannot be used for thedetermination of the relative antimicrobial potency since amore diffusible but less active extract could give a biggerdiameter than a non-diffusible but more active extract.

For the comparison of antimicrobial activity of differentsamples, the microdilution method was used. The resultsfrom the microdilution method showed that the extract of A.attenuatus possessed the highest antibacterial activity witha 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 witha MIC of 20.0–25.0 mg/ml and a MBC of 25.0 mg/ml. Themost resistant bacteria was as in the previous method—E.cloacae. Only extracts of P. commutata and A. attenua-tus showed bactericidal activity at a lower concentration(5.0 mg/ml). Streptomycin showed better antibacterial ac-tivity than extracts from the investigated mosses (Table 2).

The results of antifungal activity of methanol extractsof mosses are presented in Tables 3 and 4. All the ex-tracts tested showed great antifungal activity. The antifun-gal 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.

The best antifungal activity was obtained for P. schre-beri extract with a MIC of 0.5 mg/ml, and a MFC of

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Table 3. The antifungal activity of methanol extracts (microdilution method).

Concentration of extracts and bifonazole (mg/ml)

Pleurozium Palustriella Homalothecim Anomodon Rhytidium Hylocomium Dicranum Leucobryumschreberi commutata philippeanum attenuatus rugosum splendens scoparium glaucum Bifonazole

Fungi MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC

T. viride 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 1.0 1.0A. flavus 0.5 2.5 2.5 10.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 0.1 0.1A. versicolor 2.5 10.0 0.5 0.5 2.5 10.0 2.5 10.0 2.5 10.0 2.5 10.0 0.5 2.5 0.5 0.5 0.1 0.1P. funiculosum 0.5 2.5 0.5 2.5 0.5 2.5 5.0 10.0 0.5 2.5 0.5 2.5 0.5 2.5 2.5 10.0 0.5 1.0A. ochraceus 0.5 5.0 2.5 10.0 2.5 10.0 2.5 10.0 2.5 10.0 2.5 10.0 2.5 10.0 0.5 5.0 0.5 1.0A. niger 2.5 5.0 2.5 5.0 2.5 5.0 2.5 10.0 2.5 5.0 2.5 5.0 2.5 5.0 2.5 5.0 0.1 0.1

2.5–5.0 mg/ml. The antifungal effect of this extract isgreater than the activity which was obtained for bifona-zole against T. viride, while against P. funiculosum and A.Ochraceus, this extract showed the same effect as bifona-zole. The lowest antifungal potential was obtained for A. at-tenuatus, 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 forall the extracts tested were 0.5 mg/ml, and MFC 2.5 mg/ml,while MIC and MFC for bifonazole were 1 mg/ml). Themost resistant fungus was A. niger, MIC for all the extractstested were 2.5 mg/ml, and MFC 5.0 mg/ml (except forA. attenuatus MIC 2.5 mg/ml and MFC 10.0 mg/ml). Bi-fonazole showed better activity against this species, MICand MFC 0.1 mg/ml.

The antifungal activity of methanol extracts of mosseswas also tested against yeast Candida albicans by the disc-diffusion method (Table 4). The antifungal effect was ob-tained for P. schreberi, P. commutata and H. splendens, withinhibition zones of 11.5, 8.5, and 8.0 mm, respectively. Therest of the extracts did not show activity against C. albicanseven at higher concentrations (1 and 2 mg/disc). Bifonazoleshowed antifungal activity against yeast at 0.2 mg/disc withinhibition zone 30 mm.

Methanol extracts tested in this work showed antimicro-bial activity but it was observed that they possessed greaterantifungal than antibacterial activity. Extracts showed betterantibacterial activity against Gram (+) bacteria than againstGram (−).

The growth of tested microorganisms responded differ-ently to the investigated extracts, which indicated that may

Table 4. The antifungal activity of methanol extracts (disc-diffusion method).

Zone of inhibition (mm)

Pleurozium Palustriella Homalothecim Anomodon Rhytidium Hylocomium Dicranum LeucobryumC.albicans schreberi commutata philippeanum attenuatus rugosum splendens scoparium glaucum Bifonazole

1 mg/disc 11.5 8.5 — — — 8.0 — — 30.02 mg/disc — — — — — — — —

—, absence of inhibition; bifonazole 0.2 mg/disc.

have different modes of action or that the metabolism ofsome fungi was able to better overcome the effect of thecompound tested or adapt to it.

The literature data concerning antimicrobial activity ofmosses are poor and need more attention. The methanolextract of H. sericeum (30 mg/ml) inhibited growth of C.albicans in our work. The previous data indicated the pres-ence of flavonoids in the extract which could explain theactivity (Dulger et al., 2005). In different species of thegenus Bryum flavonoid glycosides are present (apigeninand luteolin glycosides and their 6′′malonyl esters, and8-hydroxapigenin-7-O-glucosides and 8-hydroxiluteolin-7-O-glucosides (Markham & Given, 1987). The antimicro-bial activity of ethanol extracts of Bryum argenteum wastested against bacteria (S. aureus, M. luteus, B. subtilis, andE. coli), and fungi (A. niger, P. ochrochloron, C. albicansand T. mentagrophyes) and showed activity against all theorganisms tested (Saboljevic et al., 2006).

Methanol extracts of H. cupressiforme and M. undulatum(30 mg/ml) was inhibited Gram (−) and Gram (+) bacterialspecies and C. albicans. The previous data showed that inH. cupressiforme methanol extract polycyclic aromatic hy-drocarbon, hipnogenol, biflavonoids and hydroxiflavonoidswere present, while in extract of M. undulatum flavonoidsglycosides and other type of flavonoids were detected(Dulger et al., 2005).

The previous investigation of antifungal activity of othersecondary metaboiltes showed that T. viride is the mostresistant fungal species even with fungicides (Sokovicet al., 2002). It is important to notice that extracts testedin this paper showed the strongest antifungal effects against

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this species (MIC 0.5 mg/ml, MFC 5.0 mg/ml) and thatthese extracts showed higher activity than commercialfungicide—bifonazole.

These results clearly indicate that extracts investigatedshould find a practical application in the prevention andprotection of fungal infections of plants, animals, and hu-mans. Essential oils could be safely used as preservativematerials on foods to protect it from fungal infection, sincethey are natural and non-toxic to humans.

Acknowledgements

The authors are grateful to the Ministry of Sceince forfinancial support (grants 143049 and 143041).

Declaration of interest: The authors report no conflicts ofinterest. The authors alone are responsible for the contentand writing of the paper.

References

Basile A, Vuotto L, Ielpo T (1998): Antibacterial activity in Rhyn-chostegium riparioides (Hedw.) Card. extract (Bryophyta).Phytother Res 12: 146–148.

Borel C, Welthy H, Fernandez I, Colmenares M (1993): Di-cranin, an antimicrobial and 15-lipoxygenase inhibitor from

the moss Dicranum scoparium. J Nat Prod 56: 1071–1077.

Daouk D, Dagher S, Sattout E (1995): Antifungal activity of theessential oil Origanum syriacum L. J Food Protect 58: 1147–1149.

Dulger B, Yayintas T, Gonuz A (2005): Antimicrobial activity ofsome mosses from Turkey. Fitoterapia 76: 730–732.

Hanel H, Raether W (1988): A more sophisticated method ofdetermining the fungicidal effect of water-insoluble prepara-tions with a cell harvester, using miconazoleans an example.Mycoses 31: 148–154.

Ilhan S, Savaroglu F, Colak F, Iscen C, Erdemgil F (2006): Antimi-crobial activity of Palustriella commutata (Hedw.) Ochyraextracts (Bryophyta). Turkish J Biol 30: 149–152.

Markham KR, Given R (1987): The major flavonoids of an antarc-tic Bryum Phytochemistry 27: 2843–2845.

Saboljevic A, Sokovic M, Saboljevic M, Grubisic D (2006): An-timicrobial activity of Bryum argenteum. Fitoterapia 77:144–145.

Saxena K, Harinder S (2004): Uses of Bryophytes. Resonance9(6): 56–65.

Sokovic M, Tzakou O, Pitarokili D, Couladis M (2002): Antifungalactivities of selected aromatic plants growing wild in Greece.Nahrung/Food 5: 317–320.

Verpoorte R, Van Beek TA, Thomassen PHAM, Aandeweil J,Baerheim-Svendsen A (1983): Screening of antimicrobal ac-tivity of some plants belonging to the Apocynanceae andLoganinaceae. J Ethnopharmacol 8: 287–302.

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