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57
A Peer-reviewed Official International Journal of Wollega University, Ethiopia
DOI: http://dx.doi.org/10.4314/star.v3i3.10
ISSN: 2226-7522(Print) and 2305-3372 (Online)
Science, Technology and Arts Research Journal
Sci. Technol. Arts Res. J., July-Sep 2014, 3(3): 57-62
Journal Homepage: http://www.starjournal.org/
Antimicrobial Activity of Ramalina conduplicans Vain. (Ramalinaceae)
Yashoda Kambar, Vivek M.N, Manasa M, Prashith Kekuda T.R* and Onkarappa R
Post Graduate Department of Studies and Research in Microbiology, Sahyadri Science College
(Autonomous), Kuvempu University, Shivamogga-577203, Karnataka, India
Abstract
Article Information
The members of the genus Ramalina (Ramalinaceae) are fruticose lichens growing on
various types of substrata. The present study was conducted with an aim of determining
antimicrobial activity of Ramalina conduplicans Vain. The powdered lichen material was
extracted sequentially using petroleum ether, ethyl acetate and ethanol. The solvent extracts
were screened for antibacterial activity by Agar well diffusion assay against 15 bacteria which
included reference strains and isolates from burn, dental caries and urinary tract infections.
Poisoned food technique was performed to determine antifungal effect of solvent extracts
against fungal isolates from anthracnose of chilli, foot rot of finger millet and mouldy grains of
sorghum. Usnic acid, Salazinic acid and Sekikaic acid were detected in the lichen. The
solvent extracts were shown to exhibit dose dependent inhibitory activity against test bacteria.
Ethyl acetate extract inhibited reference bacterial strains to higher extent. Among solvent
extracts, only ethanol extract inhibited all urinary tract bacteria. S. aureus isolates from burn
were inhibited to higher extent when compared to S. mutans isolates from dental caries. The
solvent extracts exhibited varied inhibitory activity against test fungi. Ethyl acetate extract
inhibited Alternaria sp., Aspergillus flavus and Sclerotium rolfsii to higher extent while
Colletotrichum capsici and Helminthosporium sp. were inhibited to higher extent by petroleum
ether and ethanol extract respectively. The observed inhibitory potential of solvent extracts of
R. conduplicans could be ascribed to the presence of secondary metabolites. The lichen can
be used in the treatment of bacterial infections and to manage plant pathogenic fungi.
Article History:
Received : 14-05-2014
Revised : 19-08-2014
Accepted : 27-08-2014
Keywords:
Ramalina conduplicans
Antimicrobial
Agar well diffusion
Poisoned food technique
*Corresponding Author:
Prashith Kekuda T.R
E-mail:
p.kekuda@gmail.com
Copyright@2014 STAR Journal. All Rights Reserved.
INTRODUCTION
Lichens represent self-supporting symbiotic
association comprising of a Photobiont (algae or blue-
green algae) and a Mycobiont (fungi). The lichens grow
on rocks, roofs, tree trunks, leaves etc. and occur in
different growth forms namely crustose, foliose and
fruticose. They lack specialized organs such as roots,
leaves etc., which allows them to survive in harsh
environmental conditions. Lichens are considered as the
primary colonizers of terrestrial ecosystem. Lichens are
the indicators of air pollution and are valuable resources
of medicine, food, fodder, perfume, spices and dyes. They
are consumed in certain parts of the world as food
especially during famine. Several lichen species are often
used as spice and flavoring agents in certain foods.
Lichens are traditionally used worldwide to treat various
ailments. Lichens produce a number of secondary
metabolites called lichen substances which seldom occur
in other organisms. Most of these metabolites are of
fungal origin. These metabolites are useful in the lichen
taxonomy and have diverse bioactivities such as
antimicrobial, antioxidant, enzyme inhibitory, cytotoxic,
antiherbivore, phytotoxic, analgesic, wound healing,
antitermite, antiinflammatory etc. (Perry et al., 1999;
Kumar et al., 2011; Ul Haq et al., 2012; Pavithra et al.,
2013; Shukla et al., 2014 and Vivek et al., 2014).
The members of the genus Ramalina Ach. are
fruticose lichens belonging to the family Ramalinaceae,
order Lecanorales, class Ascomycetes. The genus was
first described by Acharius and comprises approximately
200 species. These lichens occur in diverse vegetation
types and on diverse substrates such as rocks, wood,
bark, peaty soil etc. The thallus is attached to the
substrate by a basal holdfast. The thallus may be
dichotomously or irregularly branched. The fresh thallus is
usually gray, greenish-gray to yellowish gray and become
yellowish brown to dark-brown on drying.
Pseudocyphellae are found with a solid thallus (Fu et al.,
2008; Lin, 2009). R. conduplicans is an edible lichen
species being commonly used in central and southeastern
Asian countries. In Yunnan province of southwestern
China, it is used to prepare a traditional cold dish served
at marriage banquets and in a stir-fried pork dish (Wang
et al., 2001). The Rai and Limbu communities of East
Nepal use R. conduplicans traditionally for preparation of
food (Bhattarai et al., 1999). In many places of India, the
lichen is used as a spice (Upreti et al., 2005). The
proximate composition of R. conduplicans collected at
Bhadra wildlife sanctuary, Karnataka has been reported
(Vinayaka et al., 2009). Besides, R. conduplicans is
shown to exhibit bioactivities such as antifungal (Wei et
al., 2008), antioxidant (Luo et al., 2010; Vinayaka et al.,
Original Research
Yashoda
et al
., Sci. Technol. Arts Res. J., July-Sep 2014, 3(3): 57-62
58
2009), insecticidal (Kumar et al., 2010a), anthelmintic
(Vinayaka et al., 2009) and amylase inhibitory activity
(Vinayaka et al., 2013). The present study was conducted
with an aim of determining antimicrobial activity of solvent
extracts of R. conduplicans.
MATERIALS AND METHODS
Collection and Identification of Lichen
The fruticose lichen R. conduplicans found growing on
barks of areca trees (corticolous) was collected at Hosalli,
Shivamogga, Karnataka. The lichen was identified by
morphological, anatomical and color tests. The color tests
were done on cortex as well as medulla using 10%
potassium hydroxide (K), Steiner’s stable para-
phenylenediamine solution (P) and calcium hypochlorite
solution (C). Secondary metabolites were detected by thin
layer chromatography using solvent system A (Benzene:
1,4-Dioxane:Acetic acid in the ratio 90:25:4) (Awasthi,
2000; Culberson and Kristinsson, 1970; Culberson, 1972).
Extraction
A known quantity (50g) of powdered lichen material
was added to a clean flask and subjected to sequential
extraction using solvents viz., petroleum ether, ethyl
acetate and ethanol. The lichen material was left in each
solvent for 48 hours and stirred occasionally. The
contents were filtered through Whatman No. 1. The
solvents were evaporated to dryness (Vinayaka et al.,
2009).
Antibacterial Activity of Solvent Extracts of R.
conduplicans
A panel of 15 bacteria (which included 6 reference
bacteria (2 Gram positive and 4 Gram negative), 5 drug
resistant uropathogens (2 Gram positive and 3 Gram
negative), 2 isolates of Staphylococcus aureus from burn
and 2 isolates of Streptococcus mutans from dental
caries) were tested for their susceptibility to extracts of R.
conduplicans by Agar well diffusion assay. The test
bacteria were seeded into sterile Nutrient broth (HiMedia,
Mumbai) tubes and incubated overnight at 37oC. The
broth cultures of test bacteria were swab inoculated on
sterile Nutrient agar (HiMedia, Mumbai) plates. Using
sterile cork borer, wells of 6mm were punched in the
inoculated plates. 100µl of lichen extracts (10 and
20mg/ml of 25% dimethyl sulfoxide [DMSO; HiMedia,
Mumbai]), reference antibiotic (Chloramphenicol, 1mg/ml
of sterile distilled water) and DMSO (25%, in sterile
distilled water) were added into labeled wells. The plates
were incubated at 37oC for 24 hours in upright position.
Using a ruler, the zones of inhibition formed around the
wells were measured (Kekuda et al., 2013).
Antifungal Activity of Solvent Extracts of R.
conduplicans
Colletotrichum capsici (isolate from anthracnose of
chilli), Sclerotium rolfsii (isolate from foot rot of
fingermillet), and Aspergillus flavus, Helminthosporium
sp., and Alternaria sp. (isolates from moldy grains of
sorghum) were screened for their susceptibility to solvent
extracts of R. conduplicans by Poisoned food technique
(Dileep et al., 2013). The test fungi were inoculated at the
centre of control (without solvent extract) and poisoned
plates (1mg of solvent extract/ml of Potato dextrose agar).
The plates were incubated at 28oC for 5 days. The colony
diameter of test fungi (on control and poisoned plates)
was measured in mutual perpendicular directions using a
ruler. The antifungal effect of solvent extracts in terms of
reduction in mycelial growth of test fungi on poisoned
plates was determined using the formula:
Inhibition of mycelial growth (%) = (C – T / C) x 100,
where C is the colony diameter on control plate and T is
the colony diameter on poisoned plates.
RESULTS
Characteristics of R. conduplicans
Table 1 shows the morphological characteristics of the
thallus and result of color tests and secondary metabolites
detected by TLC.
Table 1: Characteristics of R. conduplicans of this study
Thallus
Fruticose thallus 3-5cm long, corticolous,
pendulous, flattened, greenish grey,
branched; upper side smooth, scarcely
pseudocyphellate; lower side rugose, with
raised, round, prominent pseudocyphellae;
soredia and isidia absent, chondroid tissue
present and uneven in thickness, distinctly
cracked into hyphal bundles; medulla
solid, white; pith filled
Colour test
Cortex K-; Medulla K-, C-, KC -, Pd+
yellow
Secondary
metabolites
Usnic acid, Salazinic acid, Sekikaic acid
Colour and Yield of Solvent Extracts
The sequential extraction of the lichen resulted in high
yield in case of ethyl acetate (2.37%) followed by ethanol
(2.03%) and petroleum ether (0.45%). The color of ethyl
acetate, petroleum ether and ethanol extracts obtained
was brownish green, green and brown respectively.
Antibacterial Activity of Solvent Extracts against
Reference Bacteria
The result of antibacterial activity of solvent extracts of
R. conduplicans against reference bacteria is shown in
Table 2. All solvent extracts displayed dose dependent
inhibitory activity against reference bacteria. Among
bacteria, high and least susceptibility was shown by S.
flexneri and S. typhi respectively. B. cereus and S. flexneri
inhibited to high extent among Gram positive and Gram
negative bacteria respectively. Among solvent extracts,
ethyl acetate extract was shown to be more potent and
inhibited bacteria to high extent. Least inhibitory activity
was shown by ethanol extract. Reference antibiotic
inhibited bacteria to high extent when compared to solvent
extracts. DMSO did not cause inhibition of any bacteria.
Antibacterial activity of Solvent Extracts against
Urinary Tract Bacteria
The result of antibacterial activity of extracts of R.
conduplicans against uropathogens is shown in Table 3.
The inhibitory potential of solvent extracts was dose
dependent. Among bacteria, high and least susceptibility
was recorded in case of E. faecalis and K. pneumoniae
respectively. E. faecalis and P. aeruginosa were inhibited
to high extent among Gram positive and Gram negative
bacteria respectively. Among solvent extracts, ethanol
extract inhibited all test bacteria. Petroleum ether and
ethyl acetate extract was not effective against K.
pneumoniae and E. coli respectively. Reference antibiotic
showed high inhibitory efficacy when compared to lichen
extracts. DMSO did not cause inhibition of any bacteria.
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59
Table 2: Antibacterial activity of R. conduplicans extracts against reference bacteria
Test Bacteria
Zone of Inhibition in cm
Ethyl acetate
Petroleum ether
Ethanol
Antibiotic
20mg/ml
10mg/ml
20mg/ml
10mg/ml
20mg/ml
10mg/ml
S. aureus
2.0
1.9
1.9
1.7
1.8
1.5
2.2
B. cereus
2.4
2.0
2.2
2.0
2.2
2.0
3.0
K. pneumoniae
2.5
2.4
2.4
2.2
2.0
1.8
2.7
E. aerogenes
2.5
2.3
2.3
2.1
2.0
1.8
2.9
S. typhi
1.9
1.6
1.6
1.0
1.4
1.2
2.6
S. flexneri
2.6
2.5
2.4
2.2
2.2
2.0
2.6
Table 3: Antibacterial activity of R. conduplicans against uropathogens
Test Bacteria
Zone of Inhibition in cm
Ethyl Acetate
Petroleum Ether
Ethanol
Antibiotic
20mg/ml
10mg/ml
20mg/ml
10mg/ml
20mg/ml
10mg/ml
S. aureus
1.5
1.3
1.3
1.1
1.1
1.1
2.6
E. faecalis
1.8
1.7
1.8
1.7
2.2
2.1
3.8
E. coli
0.0
0.0
1.3
0.8
1.1
0.8
1.7
K. pneumoniae
0.8
0.0
0.0
0.0
1.0
0.8
1.6
P. aeruginosa
1.8
1.6
1.7
1.5
1.7
1.6
2.8
Antibacterial Activity of Solvent Extracts against Burn
Isolates
Antibacterial activity of solvent extracts of R.
conduplicans against isolates of S. aureus (from burn) is
depicted in Table 4. The extracts exhibited concentration
dependent inhibitory activity. Among isolates, Sa-02 was
inhibited to higher extent than Sa-01. Ethyl acetate extract
was found to inhibit isolates to higher extent followed by
petroleum ether and ethanol extracts. Reference antibiotic
inhibited isolates to higher extent when compared to
solvent extracts. DMSO was not effective in inhibiting
isolates.
Table 4: Antibacterial activity of R. conduplicans against
S. aureus isolates
Solvent
Extract
Concentration
(mg/ml)
Zone of Inhibition in cm
Sa-01
Sa-02
Ethyl acetate
10
2.0
2.3
20
2.1
2.5
Petroleum
ether
10
1.9
2.3
20
2.0
2.3
Ethanol
10
1.6
2.0
20
1.8
2.0
Antibiotic
1
2.8
2.6
Antibacterial Activity of Solvent Extracts against
Dental Caries Isolates
Table 5 depicts the result of inhibitory efficacy of
solvent extracts of R. conduplicans against S. mutans
isolates. Extracts have shown the dose dependent
antibacterial activity. Ethyl acetate extract inhibited isolate
Sm-02 to high extent than isolate Sm-01. Other extracts
inhibited isolates to more or less similar extent. Inhibitory
activity of reference antibiotic was higher than that of
solvent extracts. DMSO did not cause inhibition of
isolates.
Table 5: Antibacterial activity of R. conduplicans against
S. mutans isolates
Solvent
Extract
Concentration
(mg/ml)
Zone of Inhibition in cm
Sm-01
Sm-02
Ethyl acetate
10
1.7
2.0
20
1.8
2.1
Petroleum
ether
10
1.6
1.6
20
1.7
1.7
Ethanol
10
1.6
1.5
20
1.7
1.6
Antibiotic
1
3.7
3.5
Antifungal Activity of Solvent Extracts
The result of antifungal potential of solvent extracts of
R. conduplicans is shown in the Table 6 and Figure 1. All
solvent extracts inhibited test fungi to a varied extent (12
to 100% inhibition). Ethyl acetate extract inhibited
Alternaria sp., A. flavus and S. rolfsii to higher extent
when compared to other extracts. C. capsici was inhibited
by petroleum ether extract to higher extent. Ethanol
extract was more effective against Helminthosporium sp.
when compared to other solvent extracts. Ethyl acetate
extract completely suppressed the growth of Alternaria sp.
Overall, marked antifungal effect was exhibited by Ethyl
acetate extract.
Table 6: Antifungal effect of solvent extracts of R. conduplicans
Treatment
Colony Diameter in cm
C. capsici
Helminthosporium sp.
Alternaria sp.
A.flavus
S. rolfsii
Control
3.4
5.5
3.1
3.5
5.0
Ethyl acetate
1.7
3.7
0.0
1.7
0.5
Petroleum ether
1.1
4.2
2.2
2.3
4.4
Ethanol
1.2
2.9
1.4
2.0
1.8
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Figure 1: Inhibition of test fungi (%) by solvent extracts of R. conduplicans (EtOAc- ethyl acetae; Pet. Ether- petroleum
ether)
DISCUSSION
Discovery of antibiotics is one of the major milestones
in the field of chemotherapy. The use of these antibiotics
prevented and controlled millions of deaths by life
threatening infections caused by a range of pathogenic
microorganisms. However, continuous and uncontrolled
use of these wonder drugs resulted in the development of
resistance in pathogens. Besides, long term use of such
drugs often causes several side effects. This situation
intensified search for finding lead compounds having
inhibitory activity from natural origin. Lichens are known to
be one of the best sources of bioactive agents with activity
against a range of human pathogens. The lichen extracts
and the bioactive principles from lichens are shown to
exhibit inhibitory activity against wide variety of human
pathogens including clinical strains (Smith and Coast,
2002; Verma et al., 2011; Javeria et al., 2013 and Kosanić
et al., 2014). In the present study, we evaluated
antibacterial activity of solvent extracts of R. conduplicans
against reference bacterial strains and clinical isolates of
burn, dental caries and urinary tract infections. It was
observed that the extracts exhibited marked inhibitory
effect against clinical isolates and the effect was dose
dependent. The species of the lichen genus Ramalina
were shown to exhibit inhibitory effect against bacteria
including clinical isolates. The aqueous extract of R.
farinacea was shown to exhibit inhibitory effect against S.
aureus, E. coli and B. subtilis (Karagöz et al., 2009). The
extract of R. hossei was shown to exhibit inhibitory activity
against S. aureus, E. coli and P. aeruginosa (Kekuda et
al., 2009). Hoskeri et al. (2010) reported inhibitory
potential of solvent extracts of R. pacifica against clinical
isolates of P. aeruginosa, K. pneumoniae, Salmonella
typhi, Salmonella paratyphi, E. coli and S. aureus. The
study of Santigo et al. (2010) revealed potent inhibitory
effect of extracts of R. dendriscoides against Gram
positive bacteria. Agboke and Esimone (2011) observed
antibacterial activity of methanol extract of R. farinacea
against clinical strains of S. aureus. It has been shown
that acetone extract of R. menziesii exhibited marked
activity against reference strains of bacteria and
methicillin resistant S. aureus when compared to
methanol extract (Shrestha and St. Clair, 2013).
Plants are vulnerable to infections caused by various
pathogens such as bacteria, viruses and fungi. Among
these, fungi are considered to be the major pathogens of
plants causing huge number of diseases in agricultural
and horticultural crops. The fungal diseases of plants
results in drastic loss of yield and deterioration of its
quality. The pre- and post-harvest crop losses are higher
in developing countries. The fungal infection of plants
leads to quality problems such as aspect, nutritional
value, organoleptic characteristics, and limited shelf life.
Besides, some fungi produce toxins in food commodities
which cause adverse health effects on consumption. The
management of fungal infections of agricultural
commodities is mainly by the use of chemical fungicides.
However, the use of these fungicides suffers from
drawbacks such as resistance development in fungi, high
cost and deleterious effects on non-target organisms
including humans. Hence, search for alternative strategies
for prevention and control of mycotic infections of plants is
of immense interest. Natural products including lichens
are shown to be promising alternates for synthetic
fungicides (Park et al., 2008; Kumar et al., 2010b;
Dellavalle et al., 2011; Shukla et al., 2011; Panea et al.,
2013 and Vinayaka et al., 2014). In this study, we
evaluated antifungal effect of solvent extracts of R.
conduplicans against 5 fungi collected from different
sources namely anthracnose of chilli, foot rot of finger
millet and mouldly grains of sorghum. The extracts were
effective against test fungi but to a varied extent. When
compared to other extracts, Ethyl acetate extract was
more effective against Alternaria sp., A. flavus and S.
rolfsii. C. capsici and Helminthosporium sp. were inhibited
to higher extent by petroleum ether and ethanol extracts
respectively. Earlier studies have shown that Ramalina
species exhibit antifungal activity against a range of fungi.
In a previous study, Wei et al. (2008) reported antifungal
activity of lichen forming fungi from R. conduplicans
against Colletotrichum acutatum, causal agent of
anthracnose of hot pepper. The solvent extracts of R.
hossei were shown to exhibit varied inhibitory effect
Yashoda
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against Asperigllus niger and A. fumigatus (Kumar et al.,
2010b). Shukla et al. (2011) showed the antifungal effect
of aqueous and acetone extracts of Ramalina sp. against
phytopathogenic fungi. It has been shown that extracts of
R. roesleri exhibit inhibitory activity against Rhizoctonia
bataticola and other soil borne fungi (Goel et al., 2011).
CONCLUSIONS
The solvent extracts of R. conduplicans were shown to
exhibit marked antimicrobial activity. The extracts
exhibited inhibitory effect against reference strains of
bacteria and clinical isolates from burn, dental caries and
urinary tract infections. The extracts inhibited fungal
isolates from anthracnose of chilli, foot rot of finger millet
and mouldy grains of sorghum. The observed bioactivities
could be ascribed to the presence of secondary
metabolites in the lichen. The lichen R. conduplicans can
be used in the treatment of bacterial infections and to
manage fungal diseases of plants.
ACKNOWLEDGEMENTS
Authors are thankful to Dr. N. Mallikarjun, Associate
Professor and Chairman, P.G Department of Studies and
Research in Microbiology and Principal, Sahyadri Science
College (Autonomous), Kuvempu University for providing
all facilities and moral support to conduct work. Authors
also thank Dr. Vinayaka K.S for helping in identification of
lichen.
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