INTRODUCTION:

Lichens and lichen products have been used in traditional medicines for centuries and still hold considerable interest as alternative treatments in various parts of the world¹. They produce characteristic secondary metabolites that are unique with respect to those of higher plants^2,3. In various systems of traditional medicine worldwide, including the Indian system of medicine, these lichen species are said to effectively cure dyspepsia, bleeding piles, bronchitis, scabies, stomach disorders, and many disorders of blood and heart^4-7. Numerous lichens were screened for antibacterial activity in the beginning of the antibiotic era in the 1950s⁸.

Several lichen metabolites were found to be active against Gram-positive organisms⁹. The anti-mycobacterial activity of lichen compounds was reported against nontubercular species of Mycobacterium¹⁰. Parmotrema pseudotinctorum (des. Abb.) Hale (Parmeliaceae) is a foliose lichen with thallus loosely adnate to the substratum, corticolous, up to 6.0 cm across, margin entire to crenate, and eciliate. Ramalina hossei H. Magn and G. Awasthi (Ramalinaceae) is a tufted, errect, fruticose lichen with thallus corticolous, yellowish grey in color and branched¹¹. Silver has been used in many applications in pure free metal or compound form because it possesses antimicrobial activity against pathogens but is nontoxic to humans^12,13. Silver ions are very reactive, leading to inhibition of microbial respiration and metabolism as well as physical damage^14,15. Moreover, it has been suggested that silver ions intercalate into bacterial DNA once entering the cell, which prevents further proliferation of the pathogen¹⁶. Recently, nanotechnology has amplified the effectiveness of silver particles as antimicrobial agents. In the present study, the antibacterial efficacy of methanolic extracts of lichens namely Parmotrema pseudotinctorum (des. Abb.) Hale and Ramalina hossei H. Magn and G. Awasthi and silver nanoparticles singly and the combination of silver nanoparticles and lichen extracts was studied. The objective of the investigation was to evaluate synergistic antibacterial effect of lichen extracts and silver nanoparticles, if any, against bacteria causing food poisoning.

MATERIALS AND METHODS:

Collection and Identification of lichen:

The lichens, Parmotrema pseudotinctorum (des. Abb.) Hale (Voucher No. KSV/KU001130) and Ramalina hossei H. Magn and G. Awasthi (Voucher No. KSV/KU00905) were collected from forest area of Bhadra wildlife sanctuary. The collected lichen specimens were dried and identified by using standard manua^l1 and also by morphological, anatomical, chemical tests. The color tests were performed with the usual reagent, i.e., K (5% Potassium hydroxide), C (aqueous solution of Calcium hypochlorite) and PD (Paraphenylene diamine). Thin layer chromatography (TLC) in solvent A (180 toluene: 60 1-4, dioxine: 8 acetic acid) was carried using technique of Culberson and Walker and James^17,18. The lichen specimens were preserved in Department of Applied Botany, Kuvempu University, Karnataka, India for future reference.

Extraction of powdered lichen material using solvents:

For extraction, 20g of air-dried and powdered lichen sample was taken and added to 100 ml of solvent methanol. The mixtures were sonicated for 30 min, and then left at room temperature overnight. The extracts were filtered over Whatman No. 1 filter paper, and the filtrates were concentrated under reduced pressure to pasty mass. The condensed extracts were used for antibacterial assay alone and in combination with silver nanoparticles¹⁹. The extracts were subjected to detect secondary metabolites²⁰.

Silver nanoparticles:

The silver nanoparticles of distorted circular shape and approximately 100nm in size as determined by TEM and X-Ray diffraction analysis were obtained from Dept. of Industrial Chemistry, School of Chemical Sciences, Jnanasahyadri, Shankaraghatta-577451, Karnataka ²¹.

Antibacterial susceptibility testing:

Four species of bacteria, known to be the causative agents of food poisoning, namely Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhi were screened for their susceptibility towards lichen extracts and silver nanoparticles alone and a combination of lichen extracts and nanoparticles by Agar well diffusion method²². Bacteria strains were inoculated onto Muller-Hinton agar plate (10⁸ cells/ml) by swabbing the broth culture of test bacteria. Then wells of 6mm diameter were bored in the inoculated plates and the lichen extracts (10mg/ml in DMSO), Silver nanoparticles (1mg/ml in DMSO), Standard (Chloramphenicol, 1mg/ml), combination of lichen extract and silver nanoparticles (1:1 ratio) and Control (DMSO) were added into respectively labeled wells. The plates were allowed to stand for about half an hour and then incubated at 37^oC for 24 hours in upright position and the zone of inhibition was recorded. The experiment was repeated twice and average reading was taken.

RESULTS AND DISCUSSION:

The TLC in solvent A shows the presence of Atranorin and Lecanoric acid in the methanol extract of P. pseudotinctorum. Usnic acid and Sekikaic acid were detected in the extract of R. hossei (Table-1). Table-2 shows the presence of secondary metabolites in methanol extracts of selected lichens. Tannins and Steroids were detected in P. pseudotinctorum while extract of R. hossei shows the presence of tannins and terpenoids.

Table-1: Metabolites identified by TLC in lichen

Metabolite	P. pseudotinctorum	*R. hossei*
Atranorin	+	-
Lecanoric acid	+	-
Usnic acid	-	+
Sekikaic acid	-	+

‘+’Detected; ‘-‘Not detected

The result of antibacterial activity of lichen extracts, silver nanoparticles and their combination is given in Table-3. In case of P. pseudotinctorum extract, marked inhibition of B. cereus was observed which is followed by S. aureus, S. typhi and E. coli. The extract of R. hossei was more effective than P. pseudotinctorum as it caused wider inhibition zone. The methanol extract of R. hossei inhibited B. cereus to more extent (ZOI 1.9 cm) followed by S. aureus, E. coli and S. typhi. The inhibition produced by silver nanoparticles was found to be lesser than the extracts in most trials. The silver nanoparticles were found to affect Gram negative bacteria to more extent than Gram positive bacteria. Marked inhibition of test bacteria was observed by combining the lichen extracts and the silver nanoparticles. In both cases, more inhibition of test bacteria was observed as compared to extract or the nanoparticles alone. In case of combination of silver nanoparticles and P. pseudotinctorum combination, more inhibition of S. typhi (ZOI 2.8 cm) was observed followed by E. coli, B. cereus and S. aureus. Marked inhibition of Gram negative bacteria was observed in combination trial involving the methanol extract of R. hossei. E. coli was found to be inhibited to maximum extent (ZOI 2.7 cm) which is followed by S. typhi, B. cereus and S. aureus. The standard antibiotic has shown high inhibition of test bacteria among all the trails except in case of inhibition of E. coli and S. tyhpi by lichen extract and nanoparticles combination where inhibition of bacteria was more when compared to standard antibiotic . The inhibition of E. coli and S. typhi by combination of R. hossei and silver nanoparticles was higher than that of the standard antibiotic Chloramphenicol. Of all the bacteria tested, in trials involving the lichen extracts, the gram positive bacteria were found to be greatly affected than gram negative bacteria. In case of combination of lichen extracts and silver nanoparticles, more inhibition of gram negative bacteria was observed.

Table-2: Secondary metabolites in methanol extracts of selected lichens

Metabolite	P. pseudotinctorum	*R. hossei*
Tannins	+	+
Terpenoids	-	+
Alkaloids	-	-
Steroids	+	-
Flavonoids	-	-
Saponins	-	-

‘+’Detected; ‘-‘Not detected

India is a rich center of lichen diversity, contributing nearly 15% of the 13,500 species of lichens so far recorded in the world. Lichen is one of the most widely distributed eukaryotic organisms in the world. There are about 20,000 known lichen species, which account for approximately 25% of all the fungi described²³. Lichens produce a wide range of organic compounds that can be grouped as primary metabolites and secondary metabolites²⁴. Lichen compounds are also known to show some biological activities against microorganisms. Usnic acid is one of the most common and investigated lichen compounds. Its antimicrobial, antiprotozoal, antiviral, antiproliferative, anti-inflammatory, analgesic, antipyretic, and anti-tumour activities as well as some other properties such as UV protection, allergen, toxicity have been summarized ^25,26. The antibacterial activity of usnic acid against Streptococcus mutans has been examined²⁷. In vitro activities of (+)-usnic acid, (-)-usnic acid, and vulpinic acid against aerobic and anaerobic microorganisms²⁸. Both forms of usnic acid inhibited the growth of Mycobacterium tuberculosis and Mycobacterium tufu in vitro at a relatively low concentration²⁹. The antimicrobial activity of the chloroform, diethyl ether, acetone, petroleum ether, and ethanol extracts of the lichen Cladonia foliacea and its (-)-usnic acid, atranorin, and fumarprotocetraric acid constituents against 9 bacteria and fungi has been investigated¹⁹. The antibacterial activity of extracts of selected lichens could be mainly due to the presence of secondary metabolites.

Table-3: Antibacterial activity of lichen extracts, Ag nanoparticles alone and combination of lichen extracts and Ag nanoparticles

Treatment	Zone of inhibition (ZOI) in cm
Treatment	*S. aureus*	*B. cereus*	*E. coli*	*S. typhi*
Control (DMSO)	-	-	-	-
Ag nanoparticle	1.0	1.1	1.2	1.2
P. pseudotinctorum	1.6	1.7	1.2	1.5
P. pseudotinctorum + Ag nanoparticle	1.9	2.1	2.6	2.8
R. hossei	1.8	1.9	1.7	1.7
R. hossei + Ag nanoparticle	2.2	2.4	2.7	2.6
Standard	2.6	2.8	2.4	2.5

Results are average of three trials

Among the inorganic antibacterial agents, silver (Ag) has been known most extensively since ancient times to fight infections and control spoilage. The antibacterial and antiviral actions of Ag, Ag+ and Ag compounds have been thoroughly investigated^30-32. It is well known that silver ion and silver-based compounds are highly toxic to microorganisms³³, showing strong biocidal effect against as many as 16 species of bacteria, including Escherichia coli³⁴. Silver nanoparticles are highly reactive because they generate Ag+ ions, whereas metallic silver is relatively unreactive³⁵. It has also been shown that nanoparticles efficiently penetrate microbial cells, suggesting that lower concentrations of nanosized silver particles would be sufficient for microbial control. This approach could be more efficient than existing treatments, especially for certain organisms that are less sensitive to antibiotics because of their resistance to cell penetration³⁶.

The higher resistance of Gram-negative bacteria to plant extracts has previously been documented and related to thick murein layer in their outer membrane, which prevents the entry of inhibitor substances into the cell³⁷. Gram negative bacteria contain a lipopolysaccharide layer at the exterior, followed underneath by a thin layer of peptidoglycan³⁸. Lipopolysaccharides contain negative charge³⁹ and attract the weak, positively charged Ag nanoparticles⁴⁰. On the other hand, Gram-positive bacteria are principally composed of a thick layer of peptidoglycan, consisting of linear polysaccharide chains cross-linked by short peptides to form a three dimensional rigid structure⁴¹. The rigid and extended cross-linking not only endows the cell wall with fewer anchoring sites for the Ag nanoparticle, but also makes it difficult to penetrate. This may account for less effect of the Ag nanoparticles on Gram positive bacteria when compared to Gram negative bacteria in this study. Ag nanoparticle was shown to cause antibacterial effect by rupturing the membrane of bacterial cells at low concentration. The concentration of nanoparticles was responsible for biocidal effect along with the treatment time. TEM image showed the particle binding and damage in cell wall of bacteria. Synergistic effect of nanoparticles along with ultrasonic treatment showed an enhanced antibacterial effect ⁴².

CONCLUSION:

Both lichen extracts and silver nanoparticles have exhibited marked antibacterial activity. From the study, it is arrived that the lichen extracts and silver nanoparticles have synergistic action against test bacteria as individually the activity was found to be lesser. Further experiments are to be conducted to isolate the active principle from extract and to evaluate the in vivo potential of extracts singly and in combination with silver nanoparticles.

ACKNOWLEDGEMENT:

Authors express their sincere thanks to the Head of the dept. of Microbiology and Principal, S.R.N.M.N College of Applied Sciences, Shivamogga-577201 for the facilities provided. Authors also thank the N.E.S for the moral support to conduct the work.

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Received on 30.08.2009 Modified on 19. 10.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 67-70