2.1. Collection and identification of lichen sample
A brief survey of lichen distribution was carried out at different altitudes covering various agro-climatic zones of Kodaikanal located in Western Ghats of Tamil Nadu, India. Diverse living and non-living substrata were collected along with lichen thalli to examine the nature of attachment. The collected samples were cautiously packed without disturbing the thalli and the substrata of the sample material and transported to the laboratory at the Department of Biotechnology, Mother Teresa Women’s University, Kodaikanal. The sample was identified by Lichenolgist Dr.SanjeevNayaka (CSIR-NBRI),Lucknow,India.
2.2. Preparation of extract
The identified lichen sample was washed gently washed under running tap water and rinsed with double distilled water. The sample was shade dried for 7 days and grinded to fine powder using a laboratory mixer grinder. 10g of the fine sample powder was weighed and dissolved in 100 mL of various solvents (polar to non-polar). The mixture was agitated using an orbital shaker at room temperature for 72 hours. The mixture was later filtered, evaporated and stored for further use at 4°C [30].
2.3. Phytochemical screening of C. subradiata extract
Methanol, ethanol, acetone, chloroform, aqueous, petroleum ether and hexane extracts were screened for the presence of phytochemicals. The methods of Harborne [31],Brunton [32] ,and Wagner et al., [33] were followed to detect the presence of alkaloids flavonoids, phenols, saponins, tannins and glycosides.
2.3. GC-MS analysis of C. subradiata extract
Gas chromatography and Mass spectroscopy has been regarded as a "gold standard" for forensic substance identification because it is used to perform a 100% specific test, which positively identifies the presence of a particular substance. Aqueous extract of C. subradiata dissolve in DMSO was analyzed using a GC Clarus 500 PerkinElmer system and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions. The column Elite-1 was fused silica capillary column, operating in electron impact mode at 70eV. Helium (99.999%) was used as carrier gas at a constant flow of 1ml / min and an injection volume of 2 ml was employed (split ratio of 10:1). Injector temperature was 250°C. Ion source temperature was 280°C. The oven temperature was programmed from 110°C (isothermal for 2 min) with an increase of 10°C / min, to 200°C then 5°C / min, to 280°C, ending with a 9 min ISO thermal at 280°C. Mass spectrum was taken at 70eV with a scan interval of 0.5 seconds and fragments from 45 to 450 Da. The Total GC running time was 36 minutes and the relative percentage of each component was calculated by comparing its average peak area to the total area in the NIST library [34].
2.3. Biogenic fabrication of Ag NPs using C. subradiata extract
15 mL of aqueous lichen extract was added to 285 mL of 1mM Silver nitrate (Ag NO3) solution. The extract and salt combination was mixed well and allowed to react. The resultant solution was centrifuged at 10,000g for 10 minutes. The supernatant was discarded and the pellet was dispersed in double distilled water and washed thrice to remove the unreacted Ag NO3 and extract constituents [35]. The pellet was air dried and stored at 4°C for further experiments.
2.3.1. Characterization of Silver nanoparticles
The pellet, either directly or as redisposed in distilled water was used for the characterization procedures. The fabrication of the nanoparticles was confirmed using Ultra-Violet Visible (UV-Vis) spectroscopy (Shimadzu with a range of 200-800nm). Functional groups were identified using Fourier Transform Infrared (FTIR) spectrophotometer (Perkin Elmer, range 4000 to 500 cm− 1). Powder X-ray Diffractrometer (XRD) (X’ Pert Pro – PANalytic) was used for particle nature analysis. Morphology and average size of the particles were visualized and calculated from the Transmission electron microscope images (Joel/Jem 2100 HR-TEM operating at a voltage of 200kv).
2.4. Determination of Total Phenol Content (TPC) and Total flavonoid content (TFC)
The total phenol content and flavonoid content of C.subradiata aqueous extract and AgNPs green synthesized using the same extract were evaluated by different spectroscopic methods. Different concentrations of the lichen extract (20, 40, 60, 80 and 100 µg/mL) and AgNPs (2, 4, 6, 8 and 10 µg/mL) were used to perform the analysis. The TPC in the samples were determined using Folin-ciocalteau method [36] and TFC was determined using Aluminium chloride method [37]. The samples were measured for absorbance using a Shimadzu spectrophotometer (200–800 nm) in the wavelength 725 nm and 430 nm for the assays respectively.
2.5. In-vitro antioxidant activity of C. subradiata extract and AgNPs
The antioxidant activity of C. subradiata extract and AgNPs were determined using different in-vitro assays. Different concentrations of C. subradiata extract (20, 40, 60, 80 and 100 µg/mL) and AgNPs (2, 4, 6, 8 and 10 µg/mL) were used. 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was performed by adding 2 ml of DPPH solution in methanol to the aliquots of the samples. The mixture was allowed to react in dark for 30 minutes and the absorbance was measured at 517 nm [38]. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free radical scavenging assay was done by adding diluted ABTS which was incubated in dark for 14 hours to the samples and incubated for 15 minutes. The absorbance of the samples was measured at 734 nm [39]. To perform the Hydrogen peroxide (H2O2) free radical scavenging assay 40 mM H2O2 was added to the different aliquots of samples and incubated for 10 minutes. The absorbance of the samples was recorded at 230 nm [40]. Ascorbic acid was used as the positive control for all the assays and the free radical scavenging percentage of the samples were calculated as follows:
% inhibition = [(AC- AS) / AC] x 100
Where AC is the absorbance of the control; AS is the absorbance of the sample
2.6. In-vitro anticandida activity of C. subradiata extract and AgNPs
In-vitro anticandida activities of C. subradiata extract and synthesized AgNPs were evaluated by agar well-diffusion method. The samples were tested at different concentrations (15–100 µg). Candida strains were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India (C. albicans -MTCC 183, C. tropicalis -MTCC 184, C.glabrata -MTCC 3019, C.parapsilosis MTCC − 7043 and C. krusei -MTCC 9215). The assay was performed using Potato Dextrose Agar (PDA) as the culture medium and 0.1 mL of each inoculum was swabbed on individual petriplates and allowed to dry in order to assure the absorbance of the inoculum by PDA. Wells were cut using a cork borer and different concentrations of the samples were added to each well. Distilled water served as the negative control and standard antifungal agent Amphotericin B was used as the positive control. The plates were incubated at 37°C for 24 hours after which the zones of inhibition was observed and measured in millimetres [41].
2.7. In- silico molecular docking of C. subradiata compounds
Structure of chemical compounds identified using GC-MS analysis were obtained from the Pubchem database and the three-dimensional protein structure of EGFR protein involved in lung cancer pathogenesis was downloaded from RCSCB protein data bank (PDB) [42]. The Graphical User Interface program "AutoDock Tools" was used to prepare, run, and analyze the docking simulations. Koll man united atom charges, solvation parameters and polar hydrogen’s were added into the receptor PDB file for the preparation of protein in docking simulation. Auto Dock requires pre-calculated grid maps, one for each atom type present in the flexible molecules being docked and its stores the potential energy arising from the interaction with rigid macromolecules. This grid must surround the region of interest in the rigid macromolecule. The grid box size was set at 126, 126 and 126A° (x, y, and z) to include all the amino acid residues that present in rigid macromolecules. AutoGrid 4.2 Program, supplied with AutoDock 4.2 was used to produce grid maps. The spacing between grid points was 0.375 angstroms. The Lamarckian Genetic Algorithm (LGA) was chosen search for the best conformers. During the docking process, a maximum of 10 conformers was considered. The population size was set to 150 and the individuals were initialized randomly. Maximum number of energy evaluation was set to 25,00,000, maximum number of generations 27,000, maximum number of top individual that automatically survived set to 1, mutation rate of 0.02, crossover rate of 0.8, Step sizes were 0.2 A for translations, 5.0° for quaternions and 5.0° for torsions. Cluster tolerance 0.5A, external grid energy 1,000.0, max-initial energy 0.0, max number of retries 10,000 and 10 LGA runs was performed. The best ligand-receptor structure from the docked structures was chosen based on the lowest energy and minimal solvent accessibility of the ligand. Docking results of each calculation were clustered on the basis of root mean square deviation (RMSD) between the Cartesian coordinates of ligands and were ranked according to binding energy [43].
2.8. In- vitro cytotoxic activity of C. subradiata extract and AgNPs
The human lung cancer cell line (A 549) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 37°C, 5% CO2, 95% air and 100% relative humidity. Maintenance of the cultures was done by weekly passaging and the culture medium was changed twice a week. 100 µL per well of cell suspension were seeded into 96-well plates. After 24 h the cells were treated with serial concentrations of AgNPs. Following sample addition, the plates were incubated for an additional 48 h at 37°C, 5% CO2, 95% air and 100% relative humidity [44]. The medium without samples served as control and the experiments were performed in triplicate.
Statistical analysis
All the assays were performed in triplicates and expressed as mean ± standard error. Origin 8 pro and Excel 2010 softwares were used for plotting the graphs.