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Research Journal of Botany

Year: 2007  |  Volume: 2  |  Issue: 1  |  Page No.: 54 - 59

In vitro Antimicrobial Activity of Parmotrema praesorediosum Thallus Extracts

P. Balaji and G.N. Hariharan

Abstract

Hexane, dichloromethane, ethyl acetate, acetone and methanol extracts of the thalli of Parmotrema praesorediosum (Nyl.) Hale was tested for their antimicrobial activity against eleven human pathogenic microorganisms using standard disc diffusion method. The potential secondary metabolite constituents of ethyl acetate and dichloromethane extracts were active against the microorganisms viz., Bacillus cereus, Corynebacterium diptheriae, Shigella flexnerii, Staphylococcus aureus, Vibrio cholerae and Candida albicans. Solvents treated discs were used as negative control and discs treated with standard antibiotics served as positive controls in the experiment. The dichloromethane extract exhibited potential antibacterial and antifungal activity.

Table 1). The statistical analysis was carried out using Student’s t-test. The sterile discs soaked with respective solvents served as negative control, while standard antibiotic discs, Tetracycline-30 μg/disc, Chloramphenicol-50 μg/disc, Erythromycin-15 μg/disc, Streptomycin- 10 μg/disc and Kanamycin 30 μg disc (Himedia, Mumbai) were used as positive control.

RESULTS

Disc diffusion assays of crude extracts of P. praesorediosum indicated that dichloromethane (DCM), ethyl acetate, acetone and methanol showed antimicrobial activity against various pathogens tested, while crude hexane extract exhibited no activity (Table 1). Among the crude extracts, ethyl acetate showed activity against maximum number (6) of pathogens (Bacillus cereus, C. diptheriae, Staphylococcus aureus, Shigella flexnerii, Vibrio cholerae and Candida albicans) followed by DCM extract against 4 pathogens (Bacillus cereus, Corynebacterium diptheriae, Staphylococcus aureus and Candida albicans), Acetone extract against 3 pathogens (Staphylococcus aureus, Shigella flexnerii and Candida albicans) and Methanol extract against 3 pathogens (Proteus mirabilis, Salmonella typhi and Shigella flexnerii). The antibacterial assays revealed that both DCM and ethyl acetate extracts inhibited the growth of Bacillus cereus, Corynebacterium diptheriae, Shigella flexnerii, Staphylococcus aureus, Vibrio cholerae and Candida albicans in a concentration dependent manner. The inhibition was detected at 0.5-5% for both DCM and ethyl acetate extracts. The DCM extract of P. praesorediosum had a larger inhibition zone (14.1±0.2 mm) when compared to ethyl acetate extract (5.7±0.3 mm) against C. albicans at 5% concentration. However, the growth of microbial pathogens such as Proteus vulgaris, P. mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Streptococcus pyogenes were not inhibited by both DCM and ethyl acetate extracts.


Table 1: In vitro antimicrobial activity of the extractives from Parmotrema praesorediosum
#Mean of triplicate, Zones represents diameter in mm, + ve Controls: T*-Tetracycline-30 μg/disc, C*-Chloramphenicol-50 μg/disc, E*-Erythromycin-15 μg/disc, S*- Streptomycin-10 μg/disc and K* - Kanamycin 30 μg/disc, Tr- Activity in trace; (-) no inhibition, Organisms coded as 1- Bacillus cereus, 2 - Corynebacterium diptheriae, 3 - Staphylococcus aureus, 4 - Streptococcus pyogenes, 5 - Proteus mirabilis, 6- Proteus vulgaris, 7 - Pseudomonas aeruginosa, 8 - Salmonella typhi, 9 - Shigella flexnerii, 10 - Vibrio cholerae and 11 - Candida albicans

There was a trace antibacterial activity of crude acetone extract against Streptococcus pyogenes, Proteus vulgaris and Pseudomonas aeruginosa and methanol extracts against few pathogens only. The methanol extract of P. praesorediosum was able to inhibit the growth of both Proteus mirabilis and Salmonella typhi. However except methanol extracts none of the extracts (DCM, ethyl acetate and acetone) were not able to inhibit the growth of P. mirabilis and S. typhi. The comparison of different concentrations of DCM extract against pathogens growth inhibition showed greater zone of inhibition 8, 11 and 14 mm in 0.5, 2 and 5% concentration respectively for C. albicans (Table 1). The inhibition zone (14.1±0.2 mm) for DCM extract of P. praesorediosum against the growth of C. albicans at 5% concentration was closer to the inhibition zone of commercially available antibiotic drug Chloramphenicol (15 mm) (50 μg/disc), where as the antibacterial properties of DCM extract of P. praesorediosum was not as affective as the commercial antibiotics like Tetracycline, Chloramphenicol, Erythromycin, Streptomycin and Kanamycin. Among the antimicrobial activities of various extracts of P. praesorediosum, the DCM extract showed both antibacterial and antifungal activity against C. albicans at various concentrations. The inhibitory effect of lichen extracts is obviously due to the presence of lichen secondary compounds.

DISCUSSION

The search for novel bioactive natural compounds to improve pharmaceutical, cosmetic and agriculture applications is an ancient one and currently it is regaining its importance and lichen compounds are not an exception. In the past, many lichen compounds including usnic acid were very much in use as a remedy for bacterial infections and it was the main ingredient of the antibiotic cream Usno (Richardson, 1988). Currently the interest on the lichen secondary compounds is again increasing because of previously reliable drug becoming ineffective and since lichens are the repositories of pharmacologically relevant unique polyketide compounds (Huneck, 1999; Müller, 2001). In the natural product research, drug-prospecting odds have been much better around 1% when polyketides were screened compared to other compounds. The polyketides are small, cyclized molecules produced by sharing biosynthetic pathways that produce a common ketone structure and between 5000 and 10,000 are known and about 1% of them possess bioactivity and lichens possess a rich array of these compounds (Miao et al., 2001; Balunas and Kinghorn, 2005).

The present bioassay with crude extracts of P. praesorediosum (Table 1) against Gram-positive and Gram-negative bacteria and a fungus are comparable to Dülger et al. (1998) where in the crude extracts of ethyl acetate, acetone, chloroform and ethanol of Cetraria islandica showed activity against gram-positive bacteria. The various solvent extracts of Usnea florida loaded on sterile paper discs showed antimicrobial activity against Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus var. mycoides, Bacillus sphaericus, Bacillus megaterium and Mycobacterium smegmatis (Dülger et al., 1997). Similarly the ethanol extract of Parmelia kamstchandalis tested using disc diffusion technique showed antibacterial activity (Mazid et al., 1999). The acetone extract of Ramalina farinacea showed activity against Bacillus subtilis, L. monocytogenes, Proteus vulgaris, Staphylococcus aureus, Yersinia enterocolitica, Candida albicans and C. glabrata (Tay et al., 2004). Ylmaz et al. (2004) reported that the chloroform, diethyl ether, acetone and petroleum extracts of C. foliacea showed activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Proteus vulgaris, Listeria monocytogenes, Aeromonas hydrophila, Candida albicans and C. glabrata. Behera et al. (2005) reported that the acetone, methanol and light Petroleum extracts of Usnea ghattensis showed activity against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus. Ray et al. (2003) reported that ethanol extracts of Usnea articulate, Ramalina jamesii and Parmotrema tinctorum showed activity against gram-positive and gram-negative bacteria. Ozturk and Guvenc (1995) reported that the chloroform extracts of Pseudevernia furfuracea (L.) Zopf. var furfuracea and Ramalina farinacea (Esimone and Adikwu, 1999) showed activity against Bacillus subtilis and Staphylococcus aureus. We found that 0.5% concentration of ethyl acetate; acetone and methanol crude extracts were adequate to inhibit the growth of Shigella flexnerii while 200 μg/disc of ethyl haematommate was adequate to inhibit the growth of S. flexnerii. The ethanolic extract of Parmelia kamstchandalis was active against Salmonella typhi at 200 μg/disc (Mazid et al., 1999) while methanol extract of P. praesorediosum was active at 5% concentration. The Dichloromethane extract found to be broad spectrum with higher zone of inhibition compared to ethyl acetate extracts. It is generally known that, the anti-bacterial action of metabolites isolated from lichenized fungi involves interference within the ATP machinery of the cell wall. Lichen derived scabrosin esters have been reported for their ATP synthase inhibition activity and direct influence on the production of ATP within the mitochondrial apparatus. When the mitochondrial ATP synthase is inhibited then the mitochondrial membrane becomes hyperpolarized and finally apoptotic cell death occurs (Gardiner et al., 2005). The above studies including ours on P. praesorediosum indicate the antimicrobial potentials of these extracts and that lichens are the potential source of novel bioactive molecules with high drug prospecting odds.

Natural products provide a starting point for new synthetic compounds with diverse structures and often with multiple stereocentres that can be challenged synthetically. Hence the lead molecule exhibiting antimicrobial activity has to be further characterized and further assays involving its derivative molecules to target sites through high throughput screening and drug discovery have to be carried out to increase its drug prospecting odds (Balunas and Kinghorn, 2005).

The antifungal compounds present in P. praesorediosum extracted with DCM is a non-polar compound. Hence this extract is currently subjected to further fractionation to identify the potential antimicrobial compounds. Therefore the leads that were described in this paper will provide the basis for future studies for the discovery of potential compounds from P. praesorediosum.

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