In-vitro antimicrobial activity of lichen Ramalina ...

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Research Article

In-vitro antimicrobial activity of lichen Ramalina conduplicans Vain.

collected from Eastern Ghats, India

Anjali Devi B.1, Satish Mohabe

1, Sanjeeva Nayaka

2 and A. Madhusudhana Reddy

1*

1 Lichenology Laboratory, Department of Botany, Yogi Vemana University, Vemanapuram, Kadapa -

516003, Andhra Pradesh, India 2Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow - 226

001, Uttar Pradesh, India

*[email protected]

Article Info

Abstract

Received: 02-08-2016

Revised: 26-09-2016

Accepted: 29-09-2016

The research work investigates the in-vitro antimicrobial efficacy of lichen

Ramalina conduplicans Vain. (Ramalinaceae). The 2-Propanol, methanol,

acetone and petroleum ether extract of the species were tested each against

eleven bacterial and fungal pathogens by using Kirby-Bauer disc diffusion

method with 15 μL extract per disc. It is observed that the extracts are effective

against bacteria in than to the fungi. Among various extracts methanol was

much effective against most of the bacteria and diameter of inhibition zone

ranged from 12.66±0.66 to 16.66±1.6 mm. In case of antifungal activity the

extract formed zone of inhibition against only six pathogens. The R.

conduplicans can be a potential source of bioactive agents against disease

causing microbes of humans and plants.

Keywords:

Antifungal, antibacterial,

Kirby-Bauer disc diffusion

assay, bioprospecting,

lichenized fungi.

INTRODUCTION

The lichens are symbiotic organisms

composed of a fungal partner (mycobiont) and an

algal partner (photobiont), which may be either a

green algae or cyanobacteria (Nash, 1996). The alga

provides nutrients by photosynthesis to the

mycobiont, and the fungus helps in absorption of

water and nutrients from surroundings to the

photobiont. Lichens usually grow on the surface of

rocks, on non-fertile ground, as well as on the barks

of trees and leaves as epiphytic lichens. A variety of

secondary compounds synthesized from lichens are

unique and called as “lichen compounds”. These

secondary metabolites differ in the chemical

structure as well as in their biological activities. The

lichen metabolites are found be effective anti-biotic,

anti-viral, anti-microbial, analgesic, anti-

inflammatory, antipyretic, antioxidant, anticancer,

and antiproliferative antifungal agents (Wei et al.,

2008). Since the times of the Chinese and Egyptians

civilizations lichens are used as food materials and

also in traditional medicine (Palo, 1993; Bernasconi

et al., 2000; Turk et al., 2006; Vinayaka et al., 2009;

Behera et al., 2009; Temina et al., 2010; Nayaka et

al., 2010; Kowalski et al., 2011; Verma et al.,

2012).

The genus Ramalina Ach. is a fruticose

lichens belonging to the family Ramalinaceae, order

Lecanorales, class Ascomycetes. The genus was first

described by Acharius (1810) and comprises

approximately 200 species known from the world in

which 22 species reported from India (Singh &

Sinha, 2010). R. conduplicans occur in diverse

vegetation types and on different substrates such as

rocks, wood, bark, peaty soil etc. The thallus is

attached to the substratum by the basal holdfast. The

thallus may be long, erect to decumbent greenish

grey to yellowish brown, branched, branches

uniformly wide upper side smooth with scarcely

pseudocyphellate, lower side of the thallus is rugose,

with raised, round to oblong, pseudocyphellate

prominent, sorediate, chondroid tissue uneven


Anjali Devi et al.,

in the thickness, with distinctly cracked hyphal

bundles, medulla solid, apothecia lecanorine,

ascospores straight or curved and thallus contains

usnic acids, salazinic acid and sekikaic acid in three

chemical strains (Awasthi, 2007).

Ramalina conduplicans is an edible lichen

species in Central and South Eastern Asian

countries. It is used to prepare a traditional cold dish

served at marriage banquets in Yunnan province of

south western China, and in a stir-fried pork dish

(Wang et al. 2001). R. conduplicans is used in the

preparation of traditional food by the Limbu and Rai

communities of East Nepal (Bhattarai et al., 1999).

Vinayaka et al. (2009) reported the proximate

composition of R. conduplicans collected at Bhadra

Wildlife Sanctuary, Karnataka which has high

protein (5.95%) and carbohydrates (79.08%). Upreti

et al., (2005) reported that the tribals of Himachal

Pradesh use R. conduplicans as food materials and

are being sold in the markets for commercial

purposes. The fruticose lichen R. conduplicans

exhibits antioxidant, insecticidal antihelmentic (Luo

et al., 2010; Vinayaka et al., 2009; Kumar et al.,

2010a). India exhibits a rich diversity of lichens,

found in all phytogeographical regions of the

country. A total of 20,000 species of lichens are

known in the world, more than 2,450 species

(Awasthi, 2007) of lichens are reported from Indian

Subcontinent and India alone has 2,300 species

(Singh and Sinha, 2010) and more than 500 species

were reported from South India but a few data on

the biological screening of lichens are available.

Recently, Kambar et al., (2014) studied the

antimicrobial activity of R. conduplicans against six

bacterial and five fungal pathogens with ethyl

acetate, petroleum ether and ethanol extracts in

which petroleum ether extracts showed effective

results against the bacterial pathogens while all the

three solvents showed effective results against

fungal pathogen. Therefore in search of a new

molecules of natural origin for the production of

biosafety products lichens can play a dynamic role

and the present study on In-vitro antimicrobial

efficacy R. conduplicans against the bacterial and

fungi pathogens was initiated with this aim.

MATERIAL AND METHODS

Collection and identification

The thalli of R. conduplicans (Fig. 1A) were

collected from Mangifera indica orchards near

Talakona Jungle Thrills, Chittoor District of Andhra

Pradesh at an altitude of N 14°28.698’ E

078°42.749’ alt. 539 m. The specimen identification

was carried out by following the standard

procedures given by Nayaka (2014). The literature

of Awasthi, (2007) and Mohabe, (2016) was referred

for taxonomic characters and Orange et al., (2001)

was followed for chemical analysis. The voucher

specimens (ADB & SM 3734, 3760, 3772) were

deposited at the Lichen Herbarium, Department of

Botany, Yogi Vemana University (YVUH), Kadapa,

Andhra Pradesh, India.

Extraction of bioactive compounds The freshly collected lichen material was

cleaned, shade dried and powdered mechanically.

About 10g of the powdered samples was wrapped in

Whatman No.1 filter paper and placed inside the

extractor tube of Soxhlet apparatus. The extraction

was carried out in 250 ml of solvents of different

polarity such as 2-Propanol, petroleum ether,

acetone, methanol and water (Soxhlet, 1879;

Harwood and Moody, 1989) for 48 h at room

temperature at the specific boiling temperature of

the solvents (2-propanol: 82.3ºC, Petroleum ether:

60ºC, acetone: 56ºC and methanol: 65ºC). The

lichen extracts obtained were filtered and

concentrated to dryness in vacuum under reduced

pressure at 40°C using a Heidolph rota-vapour. Few

drops of the extract were used to identify the major

secondary compounds for Thin Layer

Chromatography (TLC). Extracts were preserved in

a Deep Freezer at -80°C until they are used for

further assays.

Microorganisms and media

The microorganisms used in the present

study were procured form National Collection of

Industrial Microorganisms (NCIM), Pune. Eleven

bacterial pathogens viz. Bacillus cereus, B. subtilis,

Corynebacterium rubrum, Staphylococcus aureus

Streptococcus pyogenes (Gram +ve) Enterobacter

cloacae, Escherichia coli, Klebsiella pneumonia,

Pseudomonas aeruginosa, Salmonella abony, S.

typhimurium (Gram –ve) were selected and

maintained on Muller Hington Agar (MHA) media

at 37°C. Among all the bacterial pathogens a single

pathogen E. cloacae is plant pathogen rest of all

human pathogens. Similarly, eleven fungal

pathogens viz. Aspergillus niger, Colletotrichum

falcatum, Fusarium solani, Rhizoctonia bataticola,

Trichoderma lignorum, Yeast sp. (plant pathogens)

Fusarium moniliformae, P. notatum Mucor sp.

(human pathogens) Aspergillus flavus and Fusarium

oxysporum (plant and human pathogens) were

selected and maintained on Potato Dextrose Agar

(PDA) at 27°C.



Screening of antimicrobial activity The antimicrobial efficacy of crude lichen

extracts was studied by Kirby-Bauer disc diffusion

method using selected bacterial and fungal micro-

organisms (Bauer et al., 1966). Bacterial cultures

(1.5 × 108

CFU/ml) were seeded onto the Muller

Hinton Agar plates with the help of L – spreader

while the fungal mat of 2–3 days old cultures were

seeded onto PDA plates. On the seeded plates the

sterile filter paper discs of 5 mm soaked with 15 μL

of lichen extract concentration were placed. The

inoculated plates with bacteria were incubated at

37°C for 24 hrs while fungal plates were incubated

at 27°C for 2–3 days. Amphicillin (30μg/ml) and

Nystanin (10μg/ml) were taken as a standard

positive control for bacteria and fungus respectively,

while solvent alone used as negative control. All the

experiments were carried out in triplicates. The

diameters of the zone of inhibitions were measured

in millimeter (including size of paper disc) and the

mean and standard error was calculated.

RESULTS

Among the different solvents 2-propanol

extract showed maximum zone of inhibitions of

7.33±0.33 mm against B. cereus, E. cloacae, S.

abony and S. typhimurium while it showed

minimum inhibition zone of 2.33±2.33 mm was

against C. rubrum. However,2-propanol did not

have effect against the E. coli. Petroleum ether

extract exhibited maximum and minimum zone of

12.66±0.33 mm and 7.33±3.17mm against P.

aeruginosa and C. rubrum respectively. Methanolic

extract showed highest and lowest inhibition zones

of 16.66±1.6 mm and 12.33±1.45 mm against S.

typhimurium and B. subtilis respectively. Acetone

extract presented a highest inhibition zone of

11.66±0.33 mm against S. typhimurium and lowest

inhibition zone of 7.66±0.66 mm against S. abony.

The standard Amphicillin showed an inhibition zone

of 18.0±0.57 mm and E. cloacae and lowest

inhibition zone of 13.33±0.33 mm was observed

against P. aeruginosa (Plate-1). In case of

antifungal activity the extracts showed zone of

inhibition against only six fungal pathogens only.

The 2-propanol extract showed maximum and

minimum inhibition zones of 10.0±0.57 and

8.66±4.66 mm against C. falcatum and and T.

lignorum respectively. Petroleum ether extract

exhibited a highest zone of inhibition of 19.0±3.0

mm against F. moniliformae and lowest inhibition

zone of 9.33±9.33 mm against T. lignorum. The

methanolic extracts formed maximum and minimum

inhibition zone of17.33±4.09 mm and.0±0.57 mm

against F. moniliformae and C. falcatum

respectively.

DISCUSSION The present investigation evaluated antimicrobial

efficacy of R. conduplicans against diverse human

and plant fungal pathogens in different solvents viz.

2-propanol, petroleum ether, acetone and methanol

extract. R. conduplicans consists of a yellowish

cortical pigment and occurs in two enantiomeric

forms differing in the direction of the methyl group

located in the stereo genic centre at the 9b position.

It is noticeable that inhibitory effects of the species

against the microbes exhibited in very low

concentration (15 μL) especially against bacteria.

The lichens and their extracts containing usnic acid

have been used for several purposes such as

medicinal, perfumery, cosmetic etc. As a pure

substance, usnic acid has been formulated in the

preparations of toothpaste, mouthwash, creams,

sunscreen lotions and deodorants products. Kilpiö

(1952) studied that the Topical formulations of an

usnic acid salt (“Usno”) have been successfully used

clinically against bacterial infections. Cocchietto et

al., (2002) reported that the usnic acid has effective

result against Tinea pedis and furthermore reports on

usnic acid showed that the acid has been used

topically as an adjuvant against papilloma virus

infection. Usnic acid is being used in antifeedants by

Durazo et al., (2004). It exhibits several important

biological and pharmacological properties (Yilmaz

et al., 2004; Ribeiro-Costa et al., 2004; Behera et

al., 2009). Sati et al., (2011) used Parmotrema

nilgherrense extracts of Chloroform, Ethanol and

Methanol and recorded maximum zone of inhibition

against Bacillus subtilis and Escherichia coli by

using 200μL of the extract while in the present

study, R. conduplicans extracts inhibited the same

bacterial pathogens with a very less quantity (15 μL)

of the bioactive compound. Pavithra et al., (2013)

enumerated that the usnic acid compound from U.

pictoides exhibited a noticeable zone of inhibition

against the Gram +ve bacteria i.e. S. aureus than the

Gram ˗ve bacteria P. aeruginosa. Whereas in the

present study with R. conduplicans the bioactive

compound exhibited highest zone of inhibition

against the P. aeruginosa with the petroleum ether

extract and S. aureus showed less zone of inhibition

with the petroleum ether extract. The studies of

Kambar et al., (2014) assessed antifungal effect of

solvent extracts of R. conduplicans against five


Anjali Devi et al.,

Plate–1.Antibacterial efficacy of R. conduplicans A. Habit of the thallus, B. Bacillus cereus, C. B. subtilis,

D. Corynebacterium rubrum E.Enterobacter cloacae, F. Escherichia coli, G. Klebsiella pneumonia, H.

Pseudomonas. aeruginosa, I. Salmonella abony, J. S. typhimurium, K. Staphylococcus aureus,L.

Streptococcus pyogenes; Abbreviations: Am=Amphicillin (control), Extract used: 2P=2-Propanol,

Ac=Acetone, M=Methanol, PE=Petroleum ether, W=Water; Scale bars: B–L = 10 mm.



Table-1: Antibacterial efficacy of R. conduplicans Vain.

S.No. Bacterial

pathogens

Amphicillin Diameter of zone of inhibition

2pol Methanol Acetone PE

1 B. cereus 13.66±0.33 7.33±0.33 12.66±0.66 10.0±0.0 11.0±0.57

2 B. subtilis 14.66±0.33 6.66±0.33 12.33±1.45 10.0±0.33 10.0±0.66

3 C. rubrum 13.33±2.02 2.33±2.33 15.66±1.45 11.0±1.0 7.33±3.17

4 E. cloacae 18.0±0.57 7.33±0.33 13.33±4.37 8.33±0.33 8.66±0.33

5 E. coli 13.66±0.88 0.0±0.0 13.0±1.15 9.66±0.66 11.33±1.45

6 K. pneumonia 13.66±1.20 7.0±0.0 13.33±0.6 9.33±0.33 12.33±0.66

7 P. aeruginosa 13.33±0.33 4.66±2.33 16.0±1.15 9.66±0.66 12.66±0.33

8 S. abony 14.33±1.33 7.33±0.33 12.66±4.66 7.66±0.66 8.0±0.57

9 S. typhimurium 14.66±0.66 7.33±0.33 16.66±1.6 11.66±0.33 11.66±0.33

10 Sta. aureus 13.33±1.76 4.66±2.33 14.66±1.45 11.0±0.57 9.33±0.33

11 Str. pyogenes 15.33±1.33 2.66±2.66 15.66±2.02 9.33±0.33 11.33±1.76

(*values are in mean ± standard error)

collected from different sources namely

anthracnose of chilli, foot rot of finger millet and

mouldy grains of sorghum. Whereas, the present

work with different solvents of R. conduplicans

resulted in the activity against six fungal pathogens

in which most of them are plant and human disease

causing fungal microbes. Tiwari et al., (2014)

reported that the acetone, methanol and chloroform

extracts of Flavoparmelia caperata exhibited

effective results against Aspergillus flavus, A. niger,

Fusarium solani and Fusarium oxysporum. The

bioactive compounds of R. conduplicans have not at

all having any activity against the same fungal

pathogens.

Fig.1. Graph showing zone of inhibitions of R. conduplicans extract in different solvents against bacterial

pathogens; 2Pol = 2-Propanol, PE= Petroleum ether


Anjali Devi et al.,

Acetone extract revealed highest and lowest inhibition zones of 13.66±1.85 mm and 8.66±0.88 mm against

F. moniliformae and R. bataticola respectively. The standard Nystanin showed effective results against P.

chrysogenum with an inhibition zone of 31.0±2.08 mm and lowest activity against T. lignorum with an

inhibition zone of 7.33±0.33 mm (Plate-2). Bacterial and fungal pathogens taken for the study has no

activity with the water extracts. The mean and standard values of R. conduplicans were shown in (Table-1

& 2)

Plate-2. Antifungal efficacy of R. conduplicans A. Fusarium oxysporum, B. F. solani, C. Mucor sp., D.

Penicillium chrysogenum, E P. notatum, F. Rhizoctonia bataticola, G. Trichoderma lignorum, H.

Yeast sp.; Abbreviations: NS=Nystanin (control), Extract used: 2P=2-Propanol, Ac=Acetone,

M=Methanol, Pe=Petroleum ether, W=Water; Scale bars: A–I = 10 mm.



Table-2: Antifungal efficacy of R. conduplicans Vain.

S.No Fungal pathogens Diameter of zone of inhibition

Nystanin 2pol Methanol Acetone PE

1 A. flavus 25.66±0.66 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0

2 A. niger 26.33±0.88 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0

3 C. falcatum 24.33±1.33 10.0±0.57 8.0±0.57 11.66±1.76 0.0 ±0.0

4 F. moniliformae 2.33±2.33 0.0 ±0.0 17.33±4.09 13.66±1.85 19.0±3.05

5 F. solani 21.0±3.51 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0

6 Mucor sp. 14.0±1.52 0.0 ±0.0 0.0 ±0.0 10.0±1.15 0.0 ±0.0

7 P. chrysogenum 31.0±2.08 10.0±0.88 11.0±1.52 9.0±1.15 9.66±1.20

8 P. notatum 29.0±1.05 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0 0.0 ±0.0

9 R. bataticola 17.0±1.15 0.0 ±0.0 10.0±1.73 8.66±0.88 0.0 ±0.0

10 T. lignorum 7.33±0.33 8.66±4.66 6.0±6.0 0.0±0.0 9.33±9.33

11 Yeast sp. 11.33±2.40 0.0 ±0.0 12.33±3.38 9.33±1.85 10.0±2.51

(*values are in mean ± standard error)

Fig. 2 Graph showing zone of inhibitions of R. conduplicans extract in different solvents against fungal

pathogens. 2Pol = 2-Propanol, PE= Petroleum ether


Anjali Devi et al.,

The usnic acid, a low molecular weight

dibenzofurans derivative formed by the fungal

partner, is one of the important lichen substances

found in plenty of genera especially in some

Alectoria, Cladonia, Evernia, Ramalina, Usnea and

Lecanora. In this connection the major bioactive

compound like usnic and salazinic acid occurred in

R. conduplicans exhibited effective results against

eleven bacterial pathogens and seven fungal

pathogens with 2-propanol, petroleum ether, acetone

and methanolic extracts.

CONCLUSION

Lichens have been documented as potent

antimicrobial agents since ancient times and many

studies conducted all over the world showed the

potential of lichen extracts and purified metabolites

to inhibit a wide range of bacterial and fungal

pathogens (Yilmaz et al., 2004; Turk et al., 2006;

Canarasan et al., 2006, Candan et al., 2006;

Vinayaka et al., 2009; Kekuda et al., 2011; Kambar

et al., 2014; Prabhu & Sudha 2015). Hence the

present study has created an enthusiasm to workout

with different solvents for R. conduplicans against

diverse human and plant disease causing bacterial

and fungal pathogens. The present species used for

microbial assay has screened out as potential lichen

especially from Sheshachalm Biosphere Reserve of

Andhra Pradesh in Eastern Ghats of India. The study

also includes three different solvents viz. 2-

propanol, methanol and acetone whereas the use of

these solvents with R. conduplicans were not yet

reported from the study area (Anjali et al., 2014,

2015a, b). Now it became clear that the bioactive

compound present in R. conduplicans attributed the

best results against the bacterial pathogens instead of

fungal pathogens with the standard controls. The

study encourages to explore the novel antimicrobial

bio-molecules within lichen biodiversity. The

availability of R. conduplicans in Eastern Ghats of

India may also yield potential antimicrobial

compound in some other organic solvent systems. It

is very useful for the pharmacological industries for

further development of novel drug discovery.

ACKNOWLEDGMENTS

The authors are thankful to the Department

of Science and Technology (INSPIRE) and Director

and Dr. D.K. Upreti, Chief Scientist, CSIR-National

Botanical Research Institute, Lucknow for their kind

permission to utilize the infrastructure facilities of

Lichenology Laboratory for the identification of

lichens. The authors are also thankful to the Forest

Official of Andhra Pradesh for their help.

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How to Cite this Article:

Anjali Devi B, Satish Mohabe, Sanjeeva Nayaka and A. Madhusudhana Reddy, 2016. In-vitro

antimicrobial activity of lichen Ramalina conduplicans Vain. collected from Eastern Ghats, India. Science

Research Reporter, 6(2):99-108.

In-vitro antimicrobial activity of lichen Ramalina ...

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