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Antimicrobial Activity of Ramalina conduplicans Vain. (Ramalinaceae)

Authors:
  • S.R.N.M.N College of Applied Sciences, Shimoga, Karnataka, India

Abstract and Figures

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.
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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.
Yashoda
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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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61
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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... Lichens contain around 53-79% carbohydrates, 5-16% crude fiber, 6-16% protein, 1.3-6.5% lipidic fraction and 4-12% ash (up to 17.2 g Ca/kg, 5.5 g K/ kg, 1 g P/kg, 0.56-0.88 g Mg/kg, and lower amounts of Na, Fe, Cu and Zn) (Kumar and Pandey 2010;Kambar et al. 2014;Meli et al. 2018;Zhao et al. 2020). ...
... In order to powder the sample, the mechanical pre-treatment usually includes milling (Esimone et al. 2007;Brovko et al. 2017;Ivanov et al. 2019) and grinding (Tarasov et al. 2009;Zugic et al. 2016a;Rashid et al. 2019). The relative importance of the internal mass transfer compared to the external one can be visualized by the fact that the strirring conditions do not become limiting, since some maceration studies have been performed for prolonged times with only occasional stirring (Kambar et al. 2014). ...
... For analytical and characterization purposes of a lichen, a mixture of species or lichen products, most methods involve the extraction with organic solvents, among them acetone, chloroform, dichloromethane, methanol, carbon tetrachloride, hexane, petroleum ether, their mixtures or a sequence of them (Yilmaz et al. 2004;Bomfim et al. 2009;Behera et al. 2012;Kambar et al. 2014;Brovko et al. 2017;Hawryl et al. 2020;Staples et al. 2020;Kerboua et al. 2021), followed by evaporation of the solvent and further purification with solvent extraction (Kerboua et al. 2021), or with column chromatography (Manojlovic et al. 2020;Maulidiyah et al. 2021;Nguyen et al. 2021), further elution with organic solvents, solvent separation by distillation, and precipitation and crystallization of the product (Polovinka et al. 2006;Gao et al. 2020). As mentioned for polysaccharides, crude fractions could in occasions exhibit higher activity than the purer fractions, i.e. the antioxidants activity of crude acetone extracts was higher than for some purified compounds (Maulidiyah et al. 2021). ...
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... Lichens growing on the barks of tress are known as corticolous lichens. In India, >70% lichens are ., 2007; Kambar et al., 2014a). Barks of a number of trees support the growth of epiphytic ., 2012; Kambar et al., ...
... The study of Vivek et al. (2014a) showed the inhibitory effect of three Parmotrema species against Gram positive and Gram negative bacteria. Similarly, studies by Karthikaidevi et al. (2009), Karagöz et al. (2009), Ranković et al. (2010, Paudel et al. (2012), Pavithra et al. (2013), Kekuda (2014), Kambar et al. (2014a) and Anjali et al. (2015) showed the antimicrobial efficacy of lichens. ...
... The use of these chemicals is associated with certain drawbacks such as resistance development in fungi, high cost and adverse effects on non-target organisms (including humans). Natural products including extracts and purified metabolites from lichens are considered to be alternative strategies for prevention and control of fungal diseases (Wei et al., 2008;Park et al., 2008;Shukla et al., 2011;Goel et al., 2011;Kambar et al., 2014a). ...
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The present study was conducted to screen antimicrobial activity of three foliose macrolichens viz ., Parmotrema reticulatum (Taylor) M. Choisy (Parmeliaceae), Heterodermia obscurata (Nyl.) Trevis (Physciaceae) and Dirinaria consimilis (Stirt.) D.D. Awasthi (Physciaceae) and two fruticose macrolichens viz ., Ramalina pacifica Asahina (Ramalinaceae), and Ramalina hossei H. Magn & G. Awasthi (Ramalinaceae) growing on barks of areca trees. The lichens were identified on the basis of morphological, anatomical and color tests and the secondary metabolites (lichen substances) were identified by thin layer chromatography. Antibacterial activity of lichen extracts was evaluated against three Gram positive and five Gram negative bacteria by Agar well diffusion assay. The lichen extracts were effective in inhibiting all test bacteria. Overall, extract of D. consimilis and P. reticulatum displayed marked and least antibacterial efficacy. Marked inhibitory activity was observed against Klebsiella pneumoniae . Antifungal effect of lichen extracts was determined against four molds by Poisoned food technique. Among fungi, the growth of Colletrotrichum capsici was suppressed to higher extent by lichen extracts. The observed inhibitory activity of lichens could be ascribed to the presence of lichen substances. K ey w o r d s: Lichens, Antimicrobial, Agar well diffusion, Poisoned food technique
... Lichen extracts also have powerful antifungal and antimicrobial activities, yet many lichen secondary metabolites have not been characterized, and their modes of action, interactions and toxicities have not been defined. Beyond the known lichen metabolites, it is likely that many as-yetunidentified compounds remain to be extracted from lichens (Tiwari 2011;Kambar et al. 2014). ...
... yeast growth, with the most effective being a C. flavoruscens extract. To identify functional interactions between gene products and the C. flavoruscens extracts(Kambar et al. 2014), we used chemical-genetic and chemogenomic approaches. HIP-HOP assays revealed that strains lacking Rsc8, Pro1 and Toa2 were by guest on August 10, 2016 http://femsle.oxfordjournals.org/ ...
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Usnea longissima is traditional medicine in Asia and several polyketide synthase (PKS) genes responding bioactive compound were isolated from U. longissima. However, the intron splicing of PKS gene from U. longissima is not clear in Aspergilli. In this study, a PKS of U. longissima (UlPKS6) was ligated into vector pNQ-ArgB which contained the A. nidulans niiA promoter, the Neurospora crassaqa4 terminator, took the ampicillin resistance gene and arginine as a selectable marker, first and then UlPKS6 was transferred into Aspergillus nidulans by polyethylene glycol mediated protoplasts transformation method. The integrity and copy number of UlPKS6 in transformants were detected by long and accurate polymerase chain reaction (LA-PCR) and Southern blot respectively; Reverse transcription-polymerase chain reaction (RT-PCR) and sequence alignment were used to detect gene expression and intron splicing of UlPKS6 in transformants. Nine transformants were obtained with single full UlPKS6 gene DNA from 106 protoplast. Intron splicing of UlPKS6 in transformants is same with in lichen forming fungi. UlPKS6 was transferred into A. nidulans and expressed in A. nidluans successfully. The DNA from U. longissima or other lichen can be used in heterologous expression in A. nidulans directly.
... Lichen extracts also have powerful antifungal and antimicrobial activities, yet many lichen secondary metabolites have not been characterized, and their modes of action, interactions and toxicities have not been defined. Beyond the known lichen metabolites, it is likely that many as-yetunidentified compounds remain to be extracted from lichens (Tiwari 2011;Kambar et al. 2014). ...
... ited yeast growth, with the most effective being a C. flavoruscens extract. To identify functional interactions between gene products and the C. flavoruscens extracts (Kambar et al. 2014), we used chemical-genetic and chemogenomic approaches. HIP-HOP assays revealed that strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations of the C. flavoruscens extract. ...
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Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and thirty-nine extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide Haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking RSC8, PRO1, and TOA2 were sensitive to three concentrations (IC25.5, IC25, and IC50, respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling.
... Hence, alternatives to several synthetic fungicides currently in use are needed. It has been shown that lichen and lichen forming fungi exhibit marked antifungal activity against a wide range of phytopathogenic fungi (Shahi et al., 2001;Oh et al., 2006;Wei et al., 2008;Goel et al., 2011;Tiwari et al., 2011;Kambar et al., 2014a). In the present study, we screened the potential of extract of P. cristiferum and D. applanata to inhibit fungi. ...
... Strong inhibitory activity against mycelial growth of fungi was observed in case of P. praesorediosum when compared to other two lichens. More recently, Kambar et al. (2014a) showed antifungal efficacy of solvent extracts of Ramalina conduplicans against fungi from moldy sorghum grains. ...
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Lichens are self-supporting symbiotic association of mycobiont and photobiont. The present study was conducted to investigate antimicrobial, insecticidal and radical scavenging potential of methanol extract of two macrolichens viz. Parmotrema cristiferum (Taylor) Hale and Dirinaria applanata (Fée) D.D. Awasthi. Antibacterial activity was tested against a panel of 5 bacteria by Agar well diffusion assay. Minimum inhibitory concentration was determined against 2 bacteria by broth dilution method. Antifungal efficacy was determined against 5 molds by Poisoned food technique. Free radical scavenging activity was screened by DPPH radical scavenging assay. Total phenolic content was estimated by Folin-Ciocalteau reagent method. Insecticidal activity was determined against 2nd instar larvae of Aedes aegypti Among lichen extracts, D. applanata exhibited stronger inhibition of bacteria as evidenced by wider zones of inhibition and low MIC values. Extract of D. applanata suppressed the mycelial growth of test fungi to higher extent when compared to extract of P. cristiferum The scavenging of DPPH radicals was also marked in case of D. applanta when compared to P. cristiferum. The content of total phenolics was also higher in D. applanata than that of P. cristiferum. Lichen extracts showed concentration dependent larvicidal effect. D. applanata exhibited stronger larvicidal effect than P. cristiferum. Overall, D. applanata displayed marked bioactivities when compared to P. cristiferum. This could be mainly due to high phenolic content. The macrolichens of this study appear to be promising sources of bioactive agents.
... Next to R. pacifica, R. conduplicans was found to exhibit marked antibacterial activity against Gram positive and Gram negative bacteria. In an earlier study, Kambar et al.14 showed concentration dependent inhibitory activity of solvent extracts of R. conduplicans against Gram positive and Gram negative bacteria. The study of Devi et al. 43 showed that methanol extract of R. showed the potential of R. hossei, R. conduplicans and R. pacifica to inhibit clinical isolates of Streptococcus mutans. ...
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A systematic review of literature data on the antifungal potential of extracted lichen compounds and individual secondary metabolites against mold species of the genus Aspergillus is provided. Crude extracts from 49 epiphytic, 16 epigeic and 22 epilithic species of lichens and 44 secondary metabolites against 10 species, Aspergillus candidus, A. favus, A. fumigatus, A. nidulans, A. niger, A. ochraceus, A. parasiticus, A. restrictus, A. stellatus and A. ustus, were analysed. Several measuring techniques were employed for such analyses. Lichen substances were extracted with alcoholic and other organic solvents mainly using the Soxhlet apparatus. Among the three most-studied mold species, the results showed that the crude extracts from the thalli of the lichens Cladonia foliacea, Hypotrachyna cirrhata, Leucodermia leucomelos, Platismatia glauca and Pseudevernia furfuracea against Aspergillus favus, from C. foliacea, Nephroma arcticum and Parmelia sulcata against A. fumigatus and from Evernia prunastri, Hypogymnia physodes, Umbilicaria cylindrica and Variospora dolomiticola against A. niger have the greatest antifungal potential. The lichen secondary metabolites showed a higher inhibitory potential, e.g. protolichesterinic acid against A. favus, lecanoric acid against A. fumigatus and orsellinic acid against A. niger; the other seven species of Aspergillus have been poorly studied and require further investigation. A comparison of the inhibitory potential of the tested mixtures of lichen substances and their secondary metabolites shows that they can compete with commonly used antifungal substances, such as ketoconazole and clotrimazole against A. favus, A. nidulans, A. niger and A. parasiticus and fuconazole in the case of A. fumigatus.
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The filtrate from Bacillus subtilis AAF2 fermentation was reported to have high anti-phytopathogenic activity in the corn steep liquor modified medium with glucose as its carbon source. Besides media and carbon sources, the nitrogen source also affects these activities. This study aims to obtain the best nitrogen source on anti-phytopathogenic activities fermented filtrate of B. subtilis AAF2. The nitrogen sources used were yeast extract (15 G L-1), peptone (15 G L-1), NH4NO3 (1.71 G L-1), and (NH4)2SO4 (2.83 G L-1), with phytopathogen test were Fusarium oxysporum and Sclerotium rolfsii. The parameters measured were pH fluctuations, the number of B. subtilis AAF2 cells, and the efficacy of phytopathogen growth inhibition (measured every 24 hours for 3 x 24 hours). The results showed pH ranged from 5.7 to 7.1; the number of cells ranges from 2.1X106 -1.00X1015 CFU ml-1; the efficacy against F. oxysporum ranged from 7.10 -71.40% and against S. rolfsii ranged from 17.70 -71.80%. The best nitrogen source in the fermentation of anti-phytopathogenic compounds by B. subtilis AAF2 was peptone.
Chapter
Lichens are composite organisms formed upon symbiotic association of fungi and algae doing photosynthesis (blue green algae). Lichenized fungi produce unique secondary metabolites which bears a wide spectrum in biological activities viz., anti-HIV, production of antibiotics, anti-protozoan, anticancer etc. This review focuses primarily on the antibacterial activity of lichen’s secondary chemicals related to major antibacterial activity and highlights of each study. The literature published clearly demonstrates that the lichen extracts and their constituent compounds have potential of significant inhibitory activity against various pathogenic bacteria viz., Bacillus cereus, Bacillus subtilis, Chrysobacterium indolthecium, Clavibacter michiganensis subsp. Michiganensis, Enterococcus faecalis, Erwinia carotovora sub sp. carotovora, Escherichia coli, Methicillin-resistant S. aureus, Pseudomonas aeruginosa. Pseudomonas cichorii, Pseudomonas savastoni pv. Fraxinus, Pseudomonas syringae pv. syringae, Pseudomonas vesicularis, Staphylococcus aureus, Staphylococcus aureus, Streptococcus pneumoniae, Xanthomonas axanopodis pv. Malvecearum, Xanthomonas hortanum pv. pelargonii, Xanthomonas phaseoli, Yersiniapseudotuberculosis, even at low concentrations. The literature records however shows no studies reported anything on the specific mechanism of action against pathogenic bacteria. Lichens harbours many biologically active compounds in which less numbers have been experimentally tested. It needs very deep studies for search of active compounds. It needs to do more clinical trials and search of mechanism of action for potent compounds in lichens.
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The aim of the study was to evaluate the antifungal activity of extracts of 10 plant species used in traditional Uruguayan medicine against the phytopathogenic fungus Alternaria spp. The plants were selected on the basis of their reported ethnobotanical uses. Aqueous, saline buffer and acid extracts of different plant species were screened in vitro for their antifungal activity against Alternaria spp. For the antifungal evaluation we used a microspectrophotometric assay. Minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the extracts were determined. Three solvents were assayed on different tissues of the plants and among the 29 evaluated extracts, 31% of the extracts inhibited growth, similar to the effects of a chemical fungicide. Acid extracts of the plants were more effective than the aqueous or buffer extracts against Alternaria spp. The MIC values of the extracts were determined ranging between 1.25 and 25 μg mL-1. The MFC values of the extracts ranged between 1.25 μg mL-1 (Rosmarinus officinalis L.) and 10 μg mL-1 (Cynara scolymus L.). MICs and MFCs values obtained from leaves (Salvia officinalis L. and R. officinalis) and seeds extracts (Salvia sclarea L.) were quite comparable to values obtained with the conventional fungicide captan (2.5 μg mL-1). The extracts of Salvia sclarea, S. officinalis and R. officinalis could be considered as potential sources of antifungal compounds for treating diseases in plants. These extracts showed maximum activity, even at very low concentrations, and the same fungicide effects as chemical fungicide. We conclude from this that these extracts exhibit amazing fungicidal properties that support their traditional use as antiseptics.
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So far 279 lichen species belonging to 79 genera and 33 families have been reported from the state of Jammu & Kasmir. The taxa under lichen families Parmeliaceae and Physiaceae dominates the state. Genera such as Xanthoria, Cladonia, Lecanora and Caloplaca showed the maximum diversity of species. The paper presents occurrence and probable utilization of lichens for bioprospection in the state. [Manzoor Ul Haq, Zafar A Reshi, D. K. Upreti and M.A. Sheikh. Lichen wealth of Jammu and Kashmir- A promising plant source for Bioprospection. Life Sci J 2012;9(4):926-929] (ISSN:1097-8135). http://www.lifesciencesite.com. 144
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In the present investigation the methanol extracts of the lichens Lecanora muralis, Parmelia saxatilis, Parmeliopsis ambigua, Umbilicaria crustulosa and Umbilicaria polyphylla were tested for antioxidant, antimicrobial and anticancer potential. Antioxidant activity was evaluated by five separate methods: free radical scavenging, superoxide anion radical scavenging, reducing power, determination of total phenolic compounds and determination of total flavonoid content. Of the lichens tested, Umbilicaria polyphylla had largest free radical scavenging activity (90.08% inhibition). Moreover, the tested extracts had effective reducing power and superoxide anion radical scavenging. Total content of phenol and flavonoid in extracts were determined as pyrocatechol equivalent, and as rutin equivalent, respectively. The strong relationships between total phenolic and flavonoid contents and the antioxidant effect of tested extracts were observed. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method against six species of bacteria and ten species of fungi. The most active was extract of Umbilicaria polyphylla with minimum inhibitory concentration values ranging from 0.78 to 1.56 mg/mL. Cytotoxic activity was tested against colon cancer adenocarcinoma cell line HTC-116 using MTT method. All extracts were found to be strong cytotoxic activity toward cell line with IC50 values ranging from 14.77 to 497μg/mL. The present study shows that tested lichen extracts demonstrated a strong antioxidant, antimicrobial and cytotoxic effects. That suggest that lichens may be used as possible natural antioxidant, antimicrobial and anticancer agents and also, could be of significance for different applications in the food industry and to control various diseases.
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Lichens are used as traditional food by Rai and Limbu communities of East Nepal. Lichens are cooked mixed with various foods. These lichens are locally called as Yangben. Three lichen species belonging to Yangben: Parmelia nepalensis, Ramalina farinacea, and R. conduplicans were chemically analyzed to assess their food value. The lichens were found rich in carbohydrate, fat, crude fibre, and minerals. Their carbohydrate and protein contents were comparable to that of rice. If cooked mixed with other food, these lichens will provide various minerals in sufficient amount and add carbohydrate, protein, fat, and fibre.
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Western Ghats are one of the biodiversity hotspots in the world. The present study was conducted to determine antibacterial and radical scavenging potential of three Parmotrema species viz., P. tinctorum, P. grayanum and P. praesorediosum from Maragalale and Guliguli Shankara, Western Ghats of Karnataka, India. The powdered lichen materials were extracted using methanol. Antibacterial activity of lichen extracts was tested against three Gram positive and five Gram negative bacteria by Agar well diffusion assay. Radical scavenging activity of lichen extracts was determined by DPPH free radical scavenging assay. Total phenolic content of lichen extracts was estimated by Folin-Ciocalteau reagent method. The lichen extracts showed dose dependent antibacterial activity. Overall, the lichen extracts were more inhibitory to Gram positive bacteria than Gram negative bacteria. P. grayanum displayed high inhibitory activity against test bacteria. Scavenging of DPPH radicals by lichen extracts was concentration dependent. Among the lichen species, P. grayanum showed higher scavenging potential as indicated by lower IC50 value. Total phenolic content was also high in P. grayanum. Thin layer chromatogram revealed the presence of Lecanoric acid, Orsellinic acid, Protolichesterinic acid, Chloroatranorin, Protopraesorediosic acid and Praesorediosic acid in lichen samples. The observed bioactivities of lichens could be ascribed to the presence of secondary metabolites. These lichens can be considered as suitable candidates for development of bioactive agents active against pathogenic microbes and oxidative damage.
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In the present study, bactericidal potential of some lichen secondary compounds has been presented. Three lichen species namely Cladonia ochrochlora, Parmotrema nilgherrensis and Parmotrema sancti-angelii were successively fractionated in various organic solvents n-hexane, ethyl acetate, acetone and methanol. The ethyl acetate fraction (10 μg/ml) of these lichen species showed promised bactericidal activity and the order of the activity of lichen species were found as P. nilgherrensis > P. sanctiangelii > C. ochrochlora. Seven lichen secondary compounds alectoronic acid, atranorin, α-collatolic acid, fumarprotocetraric acid, hypoprotocetraric acid, lecanoric acid and protocetraric acid isolated from ethyl acetate fraction of above lichen species demonstrated moderate to strong bactericidal activity with low MIC value; alectoronic acid showed MIC 21.9 μg to 162.1 μg/ml, atranorin (5 μg -70.7 μg/ml), α-collatolic acid (8.6 μg - 245 μg/ml), fumarprotocetraric acid (24 μg - 227.3 μg/ml), hypoprotocetraric acid (12.2 μg - 278.5 μg/ml), lecanoric acid (24.6 μg - 591.7 μg/ml), protocetraric acid (18.7 μg-740.7 μg/ml). These secondary compounds were found most effective to kill microorganisms within a period of 0 hrs to 6 hrs incubation as evident from the kinetic time kill assay study. As far as the stability of the lichen compounds for the observed activity, it was found that these compounds were stable at 4 oC without loosing their bactericidal potential.
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Bactericidal activity of crude extracts from lichen Ramalina pacifica were screened against 20 clinical pathogenic strains isolated from different infectious sources which belong to Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhi, Salmonella paratyphi, Echerichia coli, and Staphylococcus aureus. The minimal inhibitory concentration of petroleum ether extract and ethanol extract was determined against AmericanTypeCellCulture and MicrobialTypeCellCulture strains. Both the extracts exhibited predominant antibacterial activity against all the multi-resistant strains isolated from infected patient's sample with significant zone of inhibition at MIC=100 The bactericidal activity was assessed comparatively with the reference ATCC strains (Pseudomonas aeruginosa-ATCC-20852; Staphylococcus aureus-ATCC 29737), (Salmonella typhi – ATCC-19430), (Salmonella paratyphi – ATCC-9150), (E. coli – ATCC-25922) and MTCC strains (Klebsiella pneumoniae – MTCC-618) respectively. Ciproflaxin at the concentration 50 was used as standard. Ethanolic extract exhibited significant zone of inhibition against the clinical strains of S. aureus (16.67±1.05mm) and E. coli (17.00±1.24mm) isolated form the abscess and hospital effluent respectively. The results were promising and supported the traditional use of lichens for the treatment of respiratory infections, urinary tract infections and pneumonia. [Researcher. 2010;2(3):81-85]. (ISSN: 1553-9865).
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Ampicillin is a member of the group of antibiotics called penicillin otherwise known as B-lactam drugs; it is a selective inhibitor of bacterial cell wall synthesis and therefore is active against growing bacteria. Ampicillin is one of the most widely prescribed antibiotics. It is considered as penicillin and is a close relative of amoxicillin. Unlike penicillin, ampicillin and amoxicillin can penetrate and prevent the growth of certain types of bacteria, called gram-negative bacteria. Ampicillin is used mainly to treat infections of the middle ear, sinuses, bladder, kidney, and uncomplicated gonorrhea. It is used intravenously to treat meningitis and other serious infections. The activities of this antibiotic were in some cases hindered by the β β β β-lactamase producing resistant strains of Staphylococcus aureus. Antimicrobial interaction screening of the methanol extract with ampicillin, reveled that most of the combination ratio of methanol extract and ampicillin shows synergism while few were additive and no indifference and antagonism. This shows that in the treatment of infections of S. aureus the combination of the methanol extract of Lichen, Ramalina Farinacea and ampicilin can be used together to enhance potency of the ampicilin in some cases of infection by S. aureus.
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Thirteen species of the lichen genus Ramalina are reported from Northeastern China. Descriptions, comments and illustrations of two species new to China, R. conduplicans and R. yasudea are provided. The earlier reports of R. fraxinea and R. polymorpha are found to be misidentified, and the correct names of them are R. sinensis and R. sekika respectively.