Assessment of antibacterial potential of different solvent extract of foliose lichens against human pathogenic bacteria

In vitro antibacterial activity of acetone, methanol, petroleum ether, and diethyl ether extracts of three foliose lichen species Dirinaria picta, Dirinaria Papillulifera, and Dirinaria applanata have been investigated against two clinically significant human pathogenic bacteria (Gram-positive Staphylococcus aureus, MTCC 737 and Gramnegative Escherichia coli, MTCC 443) using agar cup diffusion method. Commercial drug streptomycin (1 mg/ml) was taken as a standard positive and 5% dimethyl sulphoxide (DMSO) as a negative control. All the extracts were found effective by showing the solvent dependent zone of inhibition. Largest zone of inhibition against E. coli by D. picta, D. papillulifera, and D. applanata have been recorded for acetone (37.1 ± 0.2 mm), methanol (23.5 ± 0.6 mm), and acetone (30.6 ± 0.4 mm) extracts, respectively. On the other hand, S. aureus was inhibited with maximum zone by methanol (23.6 ± 0.4 mm), diethyl ether (24.2 ± 0.4 mm), and acetone (21.6 ± 0.5 mm) extracts for D. picta, D. papillulifera, and D. applanata, respectively. The principal component analysis concluded that negative control (5% DMSO) does not affect the growth of test pathogens and most of the lichen extracts were more effective than the positive control.


INTRODUCTION
Despite notable achievement in drug discovery by human being infectious diseases caused by pathogenic microorganisms pose a serious threat to health. Among them, pathogenic bacteria are the causal organism for a variety of diseases. The centuryold practice of antibiotics has led to the rise of numerous drugresistant pathogenic strains. Due to this reason, the achievement of biologically active compounds has been paid more attention by scientist all over the World. Such compounds obtained from plants have been the source of pharmaceutical science for a long time (Chauhan and Abraham, 2013).
Since ancient times lichens and their products have been conventionally used as a remedy for various disorders throughout the world. Up until now, many investigators have evaluated the bioactivity of lichen secondary metabolites against serious infectious organisms (Fournet et al., 1997). Such metabolites unique to lichens possess manifold biological activities, including antiviral, antibiotic, antitumor, and antiherbivore abilities (Dayan and Romagni, 2001;Huneck, 2001;Müller, 2001).
India, the world's 12th mega-diversity region, comprises great numbers of flora and fauna (Ali and Adithyakumari, 2015). Although numerous taxonomic works followed by Awasthi (2007) on lichen has been carried out in different states of India, studies on lichen metabolites are very few, particularly from the state of Assam. Therefore, the current study was designed to explore the in vitro analysis of the antibacterial potential of three foliose lichen species against two clinically significant human pathogenic bacteria.

Collection and identification of lichen samples
The lichen samples for this study were collected from Chapanalla region of Nagaon district of Assam State, India which is located 25 km in the direction of the east from District headquarters Nagaon. It lies between 26°19′9.22″ and 26°19′45.12″ North latitude and 92°54′29.80″ to 92°54′32.48″ east longitude in the foothills of Karbi Anglong and the elevation of the region ranges between 87 and 136 m. The samples were collected from the bark of trees. The morphological and anatomical identification was done using Leica S9i stereomicroscope and Leica DM 2500 optical microscope. For chemical identification color spot test (Nylander, 1866) and microcrystallography (Asahina, 1938;Orange et al., 2001) was used. The specimens were identified with the help of relevant literatures (Awasthi, 2007) and confirmed by comparing them to the specimens available in the lichen herbarium of Nowgong College, Assam, authenticated by CSIR-National Botanical Research Institute, Lucknow (LWG). The voucher specimens were submitted at the herbarium of Nowgong College, Assam, India.

Extraction of lichen samples in different solvent
Carefully cleaned lichen thalli were washed and dried by spreading on the paper sheet at room temperature in the laboratory. The dried lichen specimens were powdered using an electric grinder. Four different solvent systems, viz., acetone, methanol, petroleum ether, and diethyl ether were used for the extraction. Around 10 g of powdered sample wrapped in Whatman No. 1 filter paper was extracted in 100 ml of solvent using Soxhlet extractor (Soxhlet, 1879). Furthermore, the metabolites were obtained through the removal of solvents using Rotary evaporator. The crude extract was subsequently dissolved in dimethyl sulphoxide (DMSO) for antimicrobial bioassay. The extraction process was executed by maintaining the boiling temperature of the solvents (acetone 56°C, methanol 65°C, Petroleum ether 62°C, and Diethyl ether 35°C).

Test pathogens and media
From the Institute of Microbial Technology, Chandigarh, India, clinically significant human pathogenic bacterial strains have been obtained. The test pathogens include one Gram-positive bacterium: Staphylococcus aureus (MTCC 737) and one Gramnegative bacterium: Escherichia coli (MTCC 443). Bacterial cultures were maintained on Nutrient Agar (NA) Media.

Screening of antibacterial potential
Antibacterial potential of test pathogens was carried out using the agar cup diffusion method (Tayung et al., 2011). Nutrient agar was poured into the Petri plates. Inoculation of 12-14 hours broth was done by streaking the plates with a sterile cotton swab. Even distribution of inoculum was ensured by rotating the plates during swabbing. The plates were allowed to dry for 15 minutes in Bio-safety cabinet after inoculation. Wells were made in agar plates using a cork borer (7 mm diameter) and 100 μl of the extract was added into each well. The plates were incubated at 37°C and after 24 hours the zone of inhibition was recorded. The diameter of the inhibition zones was measured in millimeter and expressed as the mean value. Streptomycin (1 mg/ml) was taken as a standard positive and 5% DMSO as a negative control (Prabhu and Sudha, 2015;Tomović et al., 2017). All the experiments were carried out in triplicates.

Identified lichen species
In the current investigation, the collected lichen species were identified as D. picta, D. papillulifera, and D. applanata (Fig. 1).

Antibacterial potential of lichen extracts
In the present study, the antibacterial activity of acetone, methanol, petroleum ether, and diethyl ether extracts of three foliose lichen species D. picta, D. papillulifera, and D. applanata have been investigated against two clinically significant human pathogenic bacteria using agar cup diffusion method (Ganesan et.al., 2017;Joshi and Sati, 2011;Pavithra et al., 2013;Plaza et al., 2018). It has been found that most of the lichen species in all solvent system showed potent antibacterial activities in terms of inhibition zone as compared to commercial drug streptomycin (Tables 1-3) which was similar to results obtained by (Srivastava   (Fig. 2) which is similar to work carried out by Gunasekaran et al. (2016), Pavithra et al. (2013), and Ranković et al. (2011).

Principal component analysis
The PCA biplots revealed that two extracts (methanol and diethyl ether) of D. picta and the positive control streptomycin were more effective against S. aureus, while the two other extracts (acetone and petroleum ether) were more effective against E. coli. For the species of D. papillulifera, three extracts (acetone, diethyl ether, and petroleum ether) were more effective against S. aureus as compared to E. coli, which is highly inhibited by methanol extract and the positive control streptomycin. On the other hand, biplot for D. applanata showed high activity of two extracts (methanol and petroleum ether) along with the positive control against S. aureus, while the remaining two extracts   (acetone and diethyl ether) were more effective against E. coli with almost similar potency. In both the cases of D. papillulifera and D. applanata, the extract of petroleum ether was found to be less effective than the positive control. The negative control (DMSO) was unable to show any antagonistic activity against the human test pathogens. Thus, PCA biplots concluded that all the lichen extracts were effective against the pathogens in a solvent dependent manner and most of the extracts were more effective than the commercial drug streptomycin (Fig. 2). The same result to evaluate the effectiveness of lichen extracts has been carried out by Ganesan et al. (2015).
The discovery of different antibiotics has revolutionized the human history of medicine. However, multidrug-resistant pathogens have been emerging out continuously due to rapid uses of antibiotics discovered so far. Presently, scientists all over the world pay attention to lichen secondary metabolites due to their promising effectiveness over traditionally used compounds (Huneck, 1999). Similar to the present work acetone, chloroform, diethyl ether, methanol, and petroleum ether extracts of Parmelia sulcata comprising salazinic acid showed potent antibacterial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Yersinia enterocolitica, S. aureus, Streptococcus faecalis, Candida albicans, and Candida glabrata (Candan et al., 2007). Diethyl ether, acetone, and ethanol extracts of Cetraria aculeate comprising protolichesterinic acid have been investigated for antibacterial activity against different bacterial strains belonging to Gram-positive and Gram-negative groups which are also similar to the present investigation (Türk et al., 2003). The literature revealed that antibacterial activity of lichen extracts was mostly tested on Bacillus, Pseudomonas, E. coli, S. aureus, Kleibsiella, Salmonella, Yersinia, and Proteus sp. (Ingólfsdóttir et al., 1985;Karagöz et al., 2009;Karthikaidevi et al., 2009;Manojlovic et al., 2010;Martins et al., 2010;Yilmaz et al., 2004).

CONCLUSION
The present bioassay of comparative effectiveness of acetone, methanol, petroleum ether, and diethyl ether extracts of D. picta, D. papillulifera, and D. applanata against Gram-positive and Gram-negative bacteria show solvent dependent inhibition activity. Except few instances, most of the crude extracts showed far better inhibition activity than commercial streptomycin. Therefore, it can be concluded that lichen extracts can be used as an alternative to commercially available antibacterial drugs. Thus, further investigations on purification, characterization, and identification of antimicrobial compound from lichen is needed.