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Advances in Microbiology, 2012, 2, 345-353
http://dx.doi.org/10.4236/aim.2012.23042 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Antibacterial Activity of Extracts from Some Bryophytes
Vizma Nikolajeva1*, Ligita Liepina1, Zaiga Petrina1, Guntra Krumina1,
Mara Grube2, Indrikis Muiznieks1
1Faculty of Biology, University of Latvia, Riga, Latvia
2Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia
Email: *[email protected]
Received June 25, 2012; revised July 23, 2012; accepted August 4, 2012
ABSTRACT
The antimicrobial activity of aqueous and ethanolic extracts of 11 Bryophyta species and 9 Marchantiophyta species
collected in Latvia was tested against Staphylococcus aureus, Escherichia coli and Bacillus cereus. The extract of Lo-
phocolea heterophylla inhibited the growth of B. cereus, but none of the tested extracts inhibited the growth of E. coli.
70% of bryophyte species demonstrated certain activity in relation to S. aureus. In general, 73% of ethanolic extracts
and 39% of aqueous extracts exhibited antibacterial activity against S. aureus. The highest degree of antibacterial ac-
tivity against S. aureus was shown by the ethanolic extract of Dicranum scoparium and aqueous extracts of Atrichum
undulatum and Rhytidiadelphus squarrosus. The bactericidal action was not ascertained. For the first time antimicrobial
activity has been proved for three moss species—Eurhynchium angustirete, Rhytidiadelphus squarrosus and Rhodo-
bryum roseum, and for two liverwort species Frullania dilatata and Lophocolea heterophylla. Qualitative and quantita-
tive differences of plant extracts were evaluated by FT-IR spectra.
Keywords: Bryophyta; Marchantiophyta; Antimicrobial Activity
1. Introduction
Bryophytes, including liverworts (Marchantiophyta), ho-
rnworts (Anthocerotophyta), and mosses (Bryophyta), are
a diverse group of land plants that usually colonize habi-
tats with moist or extremely variable conditions. Tradi-
tionally, because of their antimicrobial activity, mosses
were used as a natural medicine in the Indian culture [1]
and as natural diapers [2]. Today, mosses and liverworts
are interesting for biotechnological use in medicine, ag-
riculture, and pharmacology [1,3,4]. Liverworts have
been proposed as ideal models for genetic studies and
biotechnological applications [3].
The search for plants with antimicrobial activity has
grown in importance in recent years, due to a growing
concern about increase in the rate of infection caused by
antibiotic-resistant microorganisms. Asakawa [5,6] has
analyzed approximately 1000 bryophyte species from the
world total of 27,000. However, few studies have been
carried out about the antimicrobial properties of Euro-
pean bryophytes. In literature, reports have been found
about antibacterial activity of 23 bryophyte species [7-15]
that are common in Latvia [16] and other European
countries [17-20].
In presented paper, the antimicrobial activity of 20
bryophyte species collected in Latvia was evaluated.
2. Materials and Methods
2.1. Plant Material
Samples of all tested plants were collected from their
native habitats in Salaspils, Kemeri, Iecava and Ropazi
(Latvia) in August and September and the specimens
were identified. Taxonomic references used were Abolina
[16] and Smith [21-23] for liverworts and mosses, and
Ignatov and Ignatova [24,25] for mosses. Once harvested,
the plant material was maintained in the refrigerator
(+4˚C) and processed in five days to obtain extracts.
2.2. Preparation of the Extracts
At first, plants were washed with sterile water to remove
attached litter, dead material and fragments of epiphytic
hosts. One gram of plant material per repetition was
finely ground with a pestle and mortar, and then extract
was made using 10 ml of sterile water or 50% ethanol.
The suspensions were kept in refrigerator for 18 hours
and then centrifuged (Eppendorf, 4000 rpm, 4˚C, 30 min).
Autoclaved (121˚C, 15 min) aqueous extracts were used.
2.3. Antimicrobial Activity
Antimicrobial assays were performed on three species of
microorganisms maintained in the Microbial Strain Col-
lection of Latvia (MSCL). Following strains were used:
*Corresponding author.
C
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V. NIKOLAJEVA ET AL.
346
Bacillus cereus MSCL 330, Escherichia coli MSCL
332 and Staphylococcus aureus MSCL 334. Gentamicin
(KRKA, Slovenia) 10 mg/ml was used as a positive con-
trol. For the evaluation of antimicrobial activity two
methods were used: the agar-well diffusion method [26]
and broth microdilution assay [27]. The tests were per-
formed in triplicates for each microorganism evaluated.
The final results were presented as the arithmetic aver-
ages.
2.3.1. A ga r-Well Dif fusion Met h o d
Agar diffusion test was performed on Müller-Hinton
Agar (Oxoid). Fresh inoculum approximately 106 CFU
(colony-forming units)/ml of tested microorganisms was
used. Aliquots of 70 μl of each test-sample solution and
control (distilled water and 50% ethanol) were applied
into 6.0 mm diameter wells. After incubation at 37˚C
1˚C for 18 hours the inhibition zone corresponding to the
halo formed from well edge to the beginning of the zone
of microbial growth was measured.
2.3.2. Broth Microdilution Assay
Müller-Hinton Broth (BD DifcoTM) was used. Test
strains were suspended in broth to obtain a final density
of approximately 106 CFU/ml. To confirm the initial
bacterial counts, serially diluted bacterial cultures were
plated on the Müller-Hinton Agar plates and enumerated.
The test was performed using three concentrations of
each extract (3%, 17%, and 33%, v/v) in test tubes, in-
cluding growth (in water or ethanol dilutions) and steril-
ity controls. Tubes were incubated at 37˚C 1˚C for 48 h.
After incubation, the mixtures were subjected to succes-
sive 10-fold serial dilutions, mixed with a vortex shaker
to ensure dispersion and quantitatively cultured in dupli-
cate onto agar plates to determine the number of viable
bacteria. Viable cell counts were expressed as CFU/ml
and if applicable the minimal inhibitory concentration
(MIC80) according to Qaiyumi [28] was evaluated.
2.4. Identification of Chemical Constituents by
FT-IR Spectroscopy
FT-IR absorption spectra of bryophyte extracts were reg-
istered on a microplate reader HTS-XT (Bruker, Ger-
many). 50 - 330 µl of each sample were dried on a 96-
place silicon plate at <50˚C. Spectra were collected over
the wave-number range of 4000 - 600 cm–1, 32 scans,
resolution 4 cm–1. Data were processed with OPUS 6.0
(Bruker, Germany) software. Spectra were Vector nor-
malized and baseline corrected by the rubber-band me-
thod.
2.5. Statistics
Statistical analysis was done by analysis of variance and
by Chi-square (
2) significance test. P < 0.05 was con-
sidered statistically significant.
3. Results
The agar-well diffusion method did not show any anti-
bacterial effect of the tested extracts against the investi-
gated microorganisms, and for that reason in subsequent
experiments we used the method of broth microdilution
assay. Using this method, in many cases significant in-
fluence of bryophytes on the growth of microorganisms
was found (Table 1). None of the tested bryophyte spe-
cies (Lophocolea heterophylla, Nowellia curvifolia, Po-
lytrichum commune, Rhodobryum roseum) had a signifi-
cant influence (P > 0.05) on the growth of E. coli. The
growth of Bacillus cereus was inhibited by the aqueous
extracts of L. heterophylla (MIC80 27%), and P. com-
mune (MIC80 was not achieved) but was not inhibited by
the aqueous extracts of N. curvifolia and R. roseum. The
most comprehensive researches have been made about
the influence of bryophyte extracts on the growth of
Staphylococcus aureus.
3.1. Antibacterial Activity of Mosses against
Staphylococcus aureus
Of the 12 species, 7 species (58%) exhibited antimicro-
bial activity against S. aureus (Figure 1). Antibacterial
activity against S. aureus was not established for Fis-
sidens taxifolius, Hypnum cupressiforme, Plagiomnium
undulatum, Pleurozium schreberi and Sphagnum girgen-
sohnii aqueous extracts. On the contrary, H. cupressi-
forme and S. girgensohnii extracts stimulated the growth
of S. aureus (F i g ure 2).
3.2. Antibacterial Activity of Liverworts against
Staphylococcus aureus
Of the 8 species, 3 species (38%) exhibited antimicrobial
activity against S. aureus (Figure 3). Antibacterial activ-
ity against S. aureus was not established for Lepidozia
reptans, Marchantia polymorpha, Nowellia curvifolia,
Plagiochila asplenioides and Radula complanata aque-
ous extracts. The extract of N. curvifolia stimulated the
growth of S. aureus (Figure 2).
3.3. Main Constituents of Bryophytes Extracts
FT-IR spectra of extracts showed intensive band of C=C
group at 1600 cm–1 and characteristic stretching bands of
C=O in 1300 - 1720 cm–1 region indicating the carbonyl
groups of phenolic esters [29], and C-O stretching bands
of esters and phenols at 1240 and 1052 cm–1 [30]. The
intensities of absorption bands in 1500 - 1720 cm–1 re-
gion varied in different samples thus indicating qualita-
ive and quantitative differences of the biochemical com- t
Copyright © 2012 SciRes. AiM
V. NIKOLAJEVA ET AL.
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347
Table 1. Minimal inhibitory concentration (MIC80) of plant extracts (in %) against Staphylococcus aureus.
Taxonomic position Extract
Division Class Species Water Ethanol
Dicranum scoparium Hedw. 30 3
Eurhynchium angustirete (Broth.) T.J. Kop. - 13 - 30a
Fissidens taxifolius Hedw. - N.T.
Hylocomium splendens (Hedw.) B., S. et G. 27 >33
Hypnum cuppressiforme Hedw. - N.T.
Plagiomnium undulatum (Hedw.) T. Kop. - -
Pleurozium schreberi (Brid.) Mitt. N.T. -
Rhodobryum roseum (Hedw.) Limpr. 24 13
Bryopsida
Rhytidiadelphus squarrosus (Hedw.) Warnst. - 7
Atrichum undulatum (Hedw.) P. Beauv. - 7
Polytrichopsida Polytrichum commune Hedw. >33 30
Bryophyta
Sphagnopsida Sphagnum girgensohnii Russ. - N.T.
Frullania dilatata (L.) Dum. 13 33
Lepidozia reptans (L.) Dum. - N.T.
Lophocolea heterophylla (Schrad.) Dum. 30 10
Nowellia curvifolia (Dicks.) Mitt. - N.T.
Plagiochila asplenioides (L. emend. Tayl.) - N.T.
Ptilidium pulcherrimum (G. Web.) Vainio 27 N.T.
Jungermannosida
Radula complanata (L.) Dum. - N.T.
Machantiophyta
Marchantiopsida Marchantia polymorpha L. - N.T.
-Did not have MIC80; aOnly in the indicated interval of concentration; N.T. not tested.
position of bryophyte extracts depending on the extrac-
tion method and species (Figure 4). Thus the ethanolic
extract of Frullania dilatata showed considerably higher
concentration of phenolics (1600 cm–1) while that of
Dicranum scoparium much higher concentration of es-
ters (1712 cm–1). In aqueous extracts higher concentra-
tion of phenolics was found in extracts of Marchantia
polymorpha, Lophocolea heterophylla and Nowellia cur-
vifolia. In general, the concentration of phenolics was
higher in ethanolic extracts.
In all extracts, the proportion of carbohydrates was
higher than that of other determined compounds, except
for four liverwort species (Frullania dilatata, Lopho-
colea heterophylla, Marchantia polymorpha and Nowe l -
lia curvifolia); in these species, the proportion of phenols
was the highest. In other species, phenols have been
proved to be the second largest group of substances; the
only exception was Plagiochila asplenioides, for which
the second largest group was made up by amides (Table
2). Dicranum scoparium and Atrichum undulatum ex-
tracts differed by significant ester content. In other ex-
tracts ester bands were weak and in many samples even
missed.
4. Discussion
In Latvia, folk medicine and ethnopharmacological tradi-
tions of using bryophytes virtually do not exist; never-
theless more than 550 species of bryophytes have been
found growing in this country [16]. In our study, extracts
of 20 bryophytes collected in Latvia were screened for
antibacterial activity. Microbiological tests indicated that
different bryophytes possess different influence on the
V. NIKOLAJEVA ET AL.
348
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
031
conc entration, %
log CFU/ ml
733
*
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
031733
co n cent ration, %
l og CFU/ m
*
*
*
l
* *
* * *
(a) (b)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
031
concentration, %
l og CFU/ m
733
l
*
*
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
031733
co ncent ration, %
l og CFU/ m
l
***
(c) (d)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
031
concentration, %
log CFU/ml
733
*
*
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
0317 33
concentration, %
log CFU/ml
* *
* *
* *
(e) (f)
6.0
6.5
7.0
7.5
8.0
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10.0
03 17
concentration, %
log CFU/ml
33
*
*
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
030.533
concentration, %
l og CFU/ m
*
*
*
l
*
(g) (h)
Figure 1. Inhibition of Staphylococcus aureus growth by mosses in dependence of the conce ntration of their aqueous or etha-
nolic extract. (a) Eurhynchium angustirete; (b) Rhytidiadelphus squarrosus; (c) Polytrichum commune; (d) Atrichum undula-
tum; (e) Dicranum scoparium; (f) Rhodobryum roseum; (g) Hylocomium splendens; (h) Fissidens taxifolius;  aqueous
extract; ethanolic extract; *P < 0.05 in comparison with control, without extract.
Copyright © 2012 SciRes. AiM
V. NIKOLAJEVA ET AL. 349
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
0317 33
co ncentration, %
log CFU/ml
Sphagnum girgensohnii
Novellia curvifolia
Hypn um cupressifo rme
**
*
* *
Figure 2. Stimulation of Staphylococcus aureus growth by three species of bryophy tes in dependenc e of concentration of their
aqueous extracts. *P < 0.05 in comparison with control, without extract.
growth of microorganisms, from inhibition to stimula-
tion.
70% of bryophyte species demonstrated certain activ-
ity in relation to Staphylococcus aureus. 55% of the spe-
cies showed a more or less pronounced antibacterial ac-
tivity. MIC80 was achieved and therefore could be esti-
mated for 10 extracts (Table 1). Bactericidal effect has
not been found in any case.
Chi-square test showed that significantly more moss
species had antibacterial properties in comparison to liv-
erwort species (Table 2, P < 0.001). Probably it was due
to the fact that only for two liverwort species ethanolic
extracts were studied instead of aqueous extracts. On the
other hand, ethanolic extracts were studied for nine moss
species.
In general, 73% of ethanolic extracts and 39% of aque-
ous extracts exhibited antibacterial activity against S.
aureus. Ethanolic extracts exhibited also a higher degree
of antimicrobial activity as compared with aqueous ex-
tracts with exception of Frullania dilatata and Hyloco-
mium splendens, the aqueous extracts of these species
showed higher antimicrobial activity than the ethanolic
extracts (Table 1). In our experiments, the aqueous ex-
tract of Marchantia polymorpha did not influence the
growth of S. aureus, although in literature data can be
found about the antibacterial influence of this liverwort
species on gram-positive bacteria among others [31].
This is also explainable by the type of extract. The etha-
nolic extracts of M. palmata have been described as hav-
ing slightly higher antibacterial activity in comparison
with the aqueous extracts [32]. It is known that one of
characteristic features of Marchantiophyta, in difference
from Bryophyta and Anthocerotophyta, is the presence of
cellular oil bodies and production of a number of lipo-
philic terpenoids, aromatic compounds and acetogenins,
several of which show biological activity including anti-
bacterial and antifungal activities [4]. These oil bodies
can be extracted with organic solvents.
The highest antimicrobial activity against S. aureus
was shown by the ethanolic extract of Dicranum sco-
parium (MIC80 3%) and aqueous extracts of Atrichum
undulatum and Rhytidiadelphus squarrosus (MIC80 of
7% for both species) (Table 1). The antibiotically active
substances of Atrichum and Dicranum spp. are consid-
ered to be polyphenolic compounds [33]. In particular,
flavonoids, including phenolic acids, are the main group
of phenols obtained from mosses [34]. It is important that
the antibacterial activity of aqueous extracts in our ex-
periments was heat stable. This fact can lighten the work
in case if any of the extracts would be introduced in fu-
ture practice.
The interconnection between the antimicrobial activity
and content of phenolics, esters, amides and/or carbohy-
drates was not estimated.
To the best of our knowledge, the antimicrobial activ-
ity of five of above mentioned bryophyte extracts has not
been previously reported. For the first time, antimicrobial
activity has been found for three moss species, Eu-
rhynchium angustirete, Rhytidiadelphus squarrosus and
Rhodobryum roseum, and for two liverwort species,
Frullania dilatata and Lophocolea heterophylla, al-
though in the genus Frullania, antifungal activity had
been previously reported for the species F. muscicola [35]
and antibacterial activity—for species F. nisquallens is
[36]. Previously, the chemical composition of L. hetero-
phylla had been investigated. Asakawa [5] believes it is
unique, because this liverwort species contains monoter-
pene 2-methylisoborneol together with calamenene-type
sesquiterpenes and eudesmanolides. Further research is
needed to obtain information about correlation between
chemical composition and antimicrobial activity of bryo-
phyte species.
Copyright © 2012 SciRes. AiM
V. NIKOLAJEVA ET AL.
350
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
03 17
co ncen t rat ion, %
l og CF U/m
33
l
*
(a)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
0317
c oncentration, %
log CFU/ml
33
**
*
*
(b)
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
03 17
c oncentration, %
log CFU/ ml
33
**
*
*
(c)
Figure 3. Inhibition of Staphylococcus aureus growth by liverworts in dependence of the concentration of their aqueous or
ethanolic extract. (a) Ptilidium pulcherrinum; (b) Frullania dilatata; (c) Lophocolea heterophylla;  aqueous extract;
ethanolic extract; *P < 0.05 in comparison with control, without extract.
Copyright © 2012 SciRes. AiM
V. NIKOLAJEVA ET AL. 351
3500 3000 2500 2000 1500 1000
Absorbance Units
Wavenumber cm
–1
–0.01 0.00 0.01 0.02 0.03 0.04
ic r anum scoparium
P
olytrichum commune
1712
Figure 4. FT-IR absorption spectra of Dicranum scoparium and Polytrichum commune ethanolic extracts.
Table 2. Features of chemical composition of bryophytes extracts, expressed in relative units (FT-IR data).
Amides Esters Phenolics Carbohydrates
Species
aqueous ethanolic aqueous ethanolicaqueousethanolic aqueous ethanolic
Bryophyta
Atrichum undulatum N.T. 0.024 N.T. 0.013 N.T. 0.042 N.T. 0.069
Dicranum scoparium N.T. 0.022 N.T. 0.018 N.T. 0.030 N.T. 0.065
Eurhynchium angustirete N.T. 0.027 N.T. 0.007 N.T. 0.040 N.T. 0.081
Fissidens taxifolius 0.016 N.T. 0.000 N.T. 0.024 N.T. 0.037 N.T.
Hylocomium splendens 0.016 N.T. 0.000 N.T. 0.024 N.T. 0.043 N.T.
Hypnum cupressiforme 0.015 N.T. 0.004 N.T. 0.028 N.T. 0.035 N.T.
Plagiomnium undulatum N.T. 0.031 N.T. 0.000 N.T. 0.051 N.T. 0.069
Polytrichum commune N.T. 0.025 N.T. 0.003 N.T. 0.038 N.T. 0.050
Rhodobryum roseum 0.014 0.030 0.000 0.000 0.015 0.026 0.045 0.045
Rhytidiadelphus squarrosus N.T. 0.024 N.T. 0.005 N.T. 0.035 N.T. 0.066
Sphagnum girgensohnii 0.014 N.T. 0.000 N.T. 0.024 N.T. 0.041 N.T.
Marchantiophyta
Frullania dilatata N.T. 0.034 N.T. 0.000 N.T. 0.067 N.T. 0.036
Lepidozia reptans 0.015 N.T. 0.000 N.T. 0.023 N.T. 0.033 N.T.
Lophocolea heterophylla 0.023 0.040 0.000 0.000 0.041 0.068 0.031 0.057
Marchantia polymorpha 0.026 N.T. 0.000 N.T. 0.055 N.T. 0.028 N.T.
Nowellia curvifolia 0.017 N.T. 0.000 N.T. 0.032 N.T. 0.030 N.T.
Plagiochila asplenioides 0.014 N.T. 0.000 N.T. 0.013 N.T. 0.039 N.T.
Ptilidium pulcherrimum 0.017 N.T. 0.000 N.T. 0.026 N.T. 0.040 N.T.
Radula complanata 0.019 N.T. 0.000 N.T. 0.026 N.T. 0.040 N.T.
N.T.: not tested.
Copyright © 2012 SciRes. AiM
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352
5. Acknowledgements
This study was supported by the European Regional De-
velopment Fund (ERDF) (2010/0295/2DP/2.1.1.1.0/10/
APIA/VIAA/134).
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