ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF THE LIVERWORT ( PTILIDIUM PULCHERRIMUM ) METHANOL EXTRACT

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.


INTRODUCTION
The liverwort Ptilidium pulcherrimum (Weber) Vainio is distributed in Fennoscandia and N. Russia, 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 microorganisms (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 phytochemistry of liverworts has been investigated from the aspects of chemotaxonomic and biological activity (Asakawa, 2001(Asakawa, , 2004)).A number of the isolated and identified compounds possess significant pharmacological 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 antifungal, 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 macrocycles (Niu et al., 2006).
No evidence of the use of Ptilidium pulcherrimum or related species in traditional medicine has been 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).

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 10 5 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 10 5 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.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 pulcherrimum 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 concentration of 2 mg per disc, the results differed to a degree.The extract showed activity against Staphylococcus aureus and Micrococcus flavus, while the other organisms were resistant (Tab.1).It is also evident that the antibacterial activity of Streptomycin 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 resistant 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 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 typhimurium.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.

Table 1 .
Antibacterial activity of the Ptilidium pulcherrimum methanol extract assessed by the disc diffusion method.

Table 2 .
Antibacterial activity of the Ptilidium pulcherrimum methanol extract assessed by the microdilution method.

Table 3 .
Antifungal activity of the Ptilidium pulcherrimum methanol extract assessed by the microdilution method.