The antimicrobial activity of the methanol extract from the liverwort, Ptilidium pulcherrimum was evaluated against five bacterial and six fungal species. In vitro antibacterial activity was assessed by disc diffusion and microdilution methods. The extract showed a stronger effect against tested Gram (+) than Gram (-) bacteria. The antifungal activity of the methanol extract was tested using a microdilution method. The methanol extract showed strong antifungal activity. The best antifungal activity was achieved against Trichoderma viride compared to the synthetic fungicide bifonazol.Antibakterijsko i antifungalno dejsto metanolnog ekstrakta jetrenjače Ptilidium pulcherrimum ispitivano je na 5 vrsta bakterija i 6 mikromiceta. Za ispitivanje in vitro antimikrobnog dejstva ekstrakta korišćena je metoda mikrodilucije na mikrotitracionim pločama i metoda difuzije sa filter diskova. Ekstrakt je pokazao jači efekat na testirane Gram (+), nego na Gram (-) bakterije. Antifungalna aktivnost metanolnog ekstrakta ispitana je mikrodilucionom metodom. Testirani ekstrakt pokazao je jak antifungalni potencijal na Trichoderma viride, Bifonazol je pokazao skoro identičnu fungistatičku aktivnost, dok je njegova fungicidna aktivnost bila nešto viša u poređenju sa metanolnim ekstraktom analizirane jetrenjače.Projekat ministarstva br. 143049 i 14304

Janaćković, P.

Marin, P.D.

Soković, Marina

Veljić, M.

Ćirić, Ana

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Biol. Sci., Belgrade, 62 (2), 381-395, 2010 DOI:10.2298/ABS1002381V 
381 
ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF THE LIVERWORT  
(PTILIDIUM PULCHERRIMUM) METHANOL EXTRACT 
M. VELJIĆ1*, ANA ĆIRIĆ2, MARINA SOKOVIĆ2, P. JANAĆKOVIĆ1 and P. D. MARIN1 
1 University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, 
11000 Belgrade, Serbia 
2 University of Belgrade, Institute for Biological Research "Siniša Stanković", 11060 Belgrade, Serbia 
Abstract - The antimicrobial activity of the methanol extract from the liverwort, Ptilidium pulcherrimum was evaluated 
against five bacterial and six fungal species.  In vitro antibacterial activity was assessed by disc diffusion and micro-
dilution methods. The extract showed a stronger effect against tested Gram (+) than Gram (-) bacteria. The antifungal 
activity of the methanol extract was tested using a microdilution method. The methanol extract showed strong 
antifungal activity. The best antifungal activity was achieved against Trichoderma viride compared to the synthetic 
fungicide bifonazol. 
Keywords: Ptilidium pulcherrimum, methanol extract, antibacterial activity, antifungal activity, microdilution method, 
disc diffusion method 
UDC 582.321:615.281/.282 
INTRODUCTION 
The liverwort Ptilidium pulcherrimum (Weber) 
Vainio is distributed in Fennoscandia and N. Rus-
sia, Iceland, the Caucasus, Siberia, Shensi and 
North America. It grows on the branches, trunks 
and exposed roots of deciduous shrubs or trees, 
rotting logs and rarely on rocks (Smith, 1999). This 
species is recorded in Serbia on the mountains 
Zlatar, Kopaonik, Crni Vrh and Tara.  
Bryophytes have been used as medicinal 
plants in traditional medicine for a long time. 
They are used for treating various skin diseases 
and injuries, since they are believed to protect 
skin and open wounds from pathogenic microor-
ganisms (Flowers, 1957).  
The chemical constituents of liverworts have 
been investigated because this group contains 
cellular oil bodies which are composed of lipophilic 
terpenoids and aromatic compounds, in contrast to 
genuine mosses and hornworts. The phytochemis-
try of liverworts has been investigated from the 
aspects of chemotaxonomic and biological activity 
(Asakawa, 2001, 2004). A number of the isolated 
and identified compounds possess significant phar-
macological activity.  
Various kinds of biological activities of the 
compounds from bryophytes have been reported so 
far. Even in a single species, multiple types of activity 
have been reported. Thus, the liverworts Marchantia 
tosana and Plagiochasma japonica exhibited antifun-
gal, antibacterial and antitumor activity, inhibition of 
superoxide release, inhibition of thrombin activity, 
and muscle relaxation (Lahlou et al., 2000). It has been 
shown that the ethanol extracts of Plagiochila 
stephensoniana were active against Trichophyton 
mentagrophyes and Candida albicans, and Bacillus 
subtilis, but not Gram (-) bacterium E. coli (Lorimers 
and Perry, 1993). A bioassay-guided separation of the 
antifungal constituents of Marchantia polymorpha led 
to the isolation of seven bis[bibenzyl]-type macro-
cycles (Niu et al., 2006).  
No evidence of the use of Ptilidium pulcherrimum 
or related species in traditional medicine has been 
382 M. VELJIĆ ET AL. 
Table 1. Antibacterial activity of the Ptilidium pulcherrimum
methanol extract assessed by  the disc diffusion method. 
Zone of inhibition (mm) 
Bacteriа P. pulcherrimum 
(2 mg/disc) 
Streptomycin 
(0.02 mg/disc) 
Staphylococcus aureus 12.3 40.0 
Micrococcus flavus 13.3 55.0 
Escherichia coli - 26.3 
Enterobacter cloacae - - 
Salmonella 
typhimurium 
- 35.3 
found. Since literature data indicate that liverworts 
possess various kinds of pharmacological activities, the 
goal of our work was to evaluate the effect of an extract 
from this species against bacteria and microfungi.  
MATERIAL AND METHODS 
Plant material was collected from its native habitat 
(mountain Zlatar, Serbia) in September 2000. A 
voucher specimen of this species is kept in the 
Herbarium of the Institute of Botany and Botanical 
Garden "Jevremovac", Faculty of Biology, Univer-
sity of Belgrade (BEOU - 16116). 
A sample (10 g) was dried by airflow at room 
temperature, powdered and extracted with 80% 
methanol (100 x 2 ml) for 24 h at 40˚C. The extract 
was filtered with a cellulose-acetate membrane 
(0.45 μm). The filtrate was evaporated until dry 
with a rotary evaporator and 80 mg of dry extract 
was dissolved with 1 ml dimethyl sulfoxide 
(DMSO) (Ilhan et al., 2006). 
Tests for antibacterial and antifungal activity 
The following bacterial species were used: Staphylo-
coccus aureus (ATCC 25923), Micrococcus flavus 
(ATCC 9341), Echerichia coli (ATCC 25922), 
Enterobacter cloacae (human isolate), Salmonella 
typhimurium (ATCC 13311). The bacterial species 
were cultured overnight at 37ºC in LB medium. 
Inoculum suspensions containing ~106 cells/ml were 
used for the experiments. The antibacterial assays were 
carried out by the modified disc-diffusion method 
(Verpoorte et al., 1983) and microdilution method 
(Hanel and Raether, 1988; Daouk et al., 1995). 
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), Tricho-
derma viride (IAM5061). 
Disc diffusion method 
A sterile filter disc (diameter 4 mm, Whatman 
paper No. 3) was placed in Petri dishes (diameter 
90 mm) filled with Mueller-Hinton agar and 
seeded with 0.3 ml of the test organism. The disc 
was impregnated with test concentrations (0.05-2 
mg/disc) of the compounds investigated 
dissolved in DMSO. The zones of growth 
inhibition around the discs were measured after 
24h of incubation at 37ºC. 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 
The modified microdilution technique was used to 
obtain quantitative data for the compounds 
investigated, (Hanel and Raether, 1988; Daouk  et 
al., 1995). Bacterial species were cultured overnight 
at 37ºC in LB medium. The inoculum suspension 
was adjusted with sterile saline to a concentration of 
approximately 1.0 x 105 in a final volume of 100 μl 
per well. The fungal spores were washed from the 
surface of agar plates with sterile 0.85% saline 
containing 0.1% Tween 80 (vol/vol) and adjusted 
with sterile saline to a concentration of 1.0 x 105 in a 
final volume of 100 μl/ml. The inocula were stored 
at +4° C for further use. Dilutions of the inocula 
were cultured on solid MH for bacteria and MA for 
fungi to verify the absence of contamination and to 
check the validity of the inoculum. 
 ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF THE LIVERWORT (PTILIDIUM PULCHERRIMUM)  383 
Table 2. Antibacterial activity of the Ptilidium pulcherrimum 
methanol extract assessed by the microdilution method. 
 P. pulcherrimum 
(mg/ml) 
Streptomycin 
(mg/ml) 
Bacteria MIC MBC MIC MBC 
Staphylococcus 
aureus 
10 20 0.05 0.1 
Micrococcus flavus 10 20 0.05 0.1 
Escherichia coli 20 >20 0.1 0.2 
Enterobacter 
cloacae 
10 20 0.1 0.3 
Salmonella  
typhimurium 
- - 0.1 0.4 
Table 3. Antifungal activity of the Ptilidium pulcherrimum
methanol extract assessed by the microdilution method. 
                              Concentration of extract and bifonazol 
(mg/ml) 
 P. pulcherrimum Bifonazol 
Fungi MIC MFC MIC MFC 
Trichoderma 
viride 
0.5 2.5 1.0 1.0 
Aspergillus 
flavus 
0.5 2.5 0.1 0.1 
Aspergillus 
versicolor 
2.5 5.0 0.1 0.1 
Aspergillus  
ochraceus 
2.5 5.0 0.5 1.0 
Aspergillus  
niger 
2.5 5.0 0.1 0.1 
Penicillium 
funiculosum 
0.5 2.5 0.5 1.0 
Minimum inhibitory concentration (MIC) 
determination was performed by a serial dilution 
technique using 96-well microtiter plates. The 
investigated compounds were dissolved in broth 
medium with inoculum to achieve the desired 
concentrations (0.05-20 mg/ml). Microplates were 
incubated for 48 h at 37° C for bacteria and 72 h at 
28° C for fungi. The lowest concentrations without 
visible growth (a binocular microscope was used) 
were defined as concentrations which completely 
inhibited bacterial or fungal growth (MICs). The 
minimum bactericidal concentrations (MBCs) and 
minimum fungicidal concentrations (MFCs) were 
determined by serial subcultivation of 2 μl into 
microtiter plates containing 100 μl of broth per well 
and further incubation for 24/72 h at 28/37° C. The 
lowest concentration with no visible growth was 
defined as MBC/MFC, indicating = 99.5% killing of 
the original inoculum. DMSO was used as a 
control, while streptomycin and bifonazole were 
used as a positive control. 
RESULTS AND DISCUSSION 
In this work, the antibacterial and antifungal 
activity of the methanol extract of Ptilidium pul-
cherrimum was tested against Gram (+) and Gram 
(-) bacteria and 6 micromycetes. For comparison of 
the antimicrobial activity a synthetic antibiotic 
Streptomycin was used, while for the antifungal 
activity a synthetic fungicide Bifonazol was used. 
Using the disc diffusion method and a concen-
tration of 2 mg per disc, the results differed to a 
degree. The extract showed activity against Staphy-
lococcus aureus and Micrococcus flavus, while the 
other organisms were resistant (Tab. 1). It is also 
evident that the antibacterial activity of Streptomy-
cin was higher than the tested extract.  
According to the obtained results it is evident 
that the extract of Ptilidium pulcherrimum at a 
concentration of 20 mg/ml has high antibacterial 
activity. Using the microdilution method MBC was 
observed against most of the investigated species, 
while inhibitory activity against Escherichia coli was 
observed. Only Salmonella typhimurium was resis-
tant at the concentration tested (Tab. 2).  
Antifungal activity of the methanol extract 
of Ptilidium pulcherrimum was tested using a 
microdilution method. The activity was tested 
against 6 micromycetes (Table 3). Ptilidium 
pulcherrimum had a fungicidal effect on one half 
384 M. VELJIĆ ET AL. 
of tested organisms. Regarding Trichoderma 
viride, this concentration showed a better 
inhibitory effect than the synthetic fungicide 
Bifonazol. Similar results were obtained with 
Penicillium funiculosum. The concentration of 
2.5 mg/ml showed a significant fungicidal effect 
against one half of the tested organisms, while 
against the other half of the species a higher 
(double) concentration was needed (Tab. 3).  
From various investigations it can be seen 
that the extracts of liverworts show considerable 
antimicrobial effect against micromycetes and G 
(+) bacteria, while their activity against G (-) 
bacteria is weak. A similar effect is given in our 
work. Thus, the methanol extract of Ptilidium 
pulcherrimum showed a moderate antibacterial 
and strong antifungal activity. The results 
indicate that the methanol extract of this species 
(20 mg/ml) have bacteriostatic activity against 
Escherichia coli, while against Staphylococcus 
aureus, Micrococcus flavus and Enterobacter 
cloacae the extract showed bactericidal activity. 
There is no activity against Salmonella typhi-
murium. The micromycetes tested were more 
sensitive than the bacterial species (MIC 0.5-2.5 
mg/ml and MFC 2.5-5.0 mg/ml). Moreover, 
Trichoderma viride, which is known as a 
resistant species, was also very sensitive to this 
extract. Bifonazol showed almost the same 
fungistatic activity, while its fungicidal activity 
was slightly higher than the methanol extract of 
the analyzed liverwort.  
Acknowledgment - The authors are grateful to Ministry of 
Science and Technological Development of Republic of 
Serbia for financial support (Grants No. 143049 and 
143041). 
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Asakawa, Y. (2004). Chemosystematics of the Hepaticae. 
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Daouk, D., Dagher, S., and E. Sattout, (1995). Antifungal ac-
tivity of the essential oil Origanum syriacum L.  J Food 
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Flowers, P. (1957). Ethnobryology OF the Gosuite Indians OF 
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Hanel, H., and W. Raether, (1988). A more sophisticated 
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Ilhan, S., Savaroğlu, F., Çolak, F., Işçen, C., and F. Erdemgil, 
(2006). Antimicrobial Activity of Palustriella commu-
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56, 1444-1450. 
Niu, C., Qu, J. B., and H. X. Lou, (2006). Antifungal 
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Verpoorte, R., Van Beek, T. A., Thomassen, P. H. A. M., 
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 ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF THE LIVERWORT (PTILIDIUM PULCHERRIMUM)  385 
АНТИБАКТЕРИЈСКА И АНТИФУНГАЛНА АКТИВНОСТ МЕТАНОЛНОГ ЕКСТРАКТА 
ЈЕТРЕЊАЧЕ PTILIDIUM PULCHERRIMUM 
М. ВЕЉИЋ1*, МАРИНА СОКОВИЋ2, АНА ЋИРИЋ2, П. ЈАНАЋКОВИЋ1, и П. Д. МАРИН1 
1Универзитет у Београду – Биолошки факултет, Институт за ботанику 
и Ботаничка башта  "Јевремовац", 11000 Београд, Србија 
2Универзитет у Београду - Институт за биолошка истраживања "Синиша Станковић", 
11000 Београд, Србија 
Антибактеријско и антифунгално дејсто мета-
нолног екстракта јетрењаче Ptilidium pulcher-
rimum испитивано је на 5 врста бактерија и 6 
микромицета. За испитивање in vitro антимик-
робног дејства екстракта коришћена је метода 
микродилуције на микротитрационим плочама и 
метода дифузије са филтер дискова. Екстракт је 
показао јачи ефекат на тестиране Грам (+), него 
на Грам (-) бактерије. Антифунгална активност 
метанолног екстракта испитана је микро-
дилуционом методом. Тестирани екстракт пока-
зао је јак антифунгални потенцијал на Trichoder-
ma viride, Бифоназол је показао скоро идентичну 
фунгистатичку активност, док је његова фунги-
цидна активност била нешто виша у поређењу са 
метанолним екстрактом анализиране јетрењаче. 
 
 


Antibacterial and antifungal activity of the liverwort (Ptilidium pulcherrimum) methanol extract

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Antibacterial and antifungal activity of the liverwort (Ptilidium pulcherrimum) methanol extract

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