Bio-Inspired Fabrication of Silver Nanoparticles Using High Altitude Squamulose Lichen Extract and Evaluation of its Antioxidant, Anticandida and Cytotoxic properties

Bio-inspired nanoparticle synthesis has attracted substantial interest among the scientic society owing to its eco-friendly and non-toxic nature. In the present study, Silver nanoparticles (AgNPs) were synthesized using high altitude squamulose lichen – Cladonia subradiata and characterized using different techniques. The antioxidant and anticandida activity of AgNPs were evaluated using multiple in-vitro assays. In-silico molecular docking analysis and in-vitro cytotoxic assay was performed to determine the anti-cancer potential of synthesized AgNPs. The results of the spectroscopic studies revealed the successful synthesis of AgNPs and the presence of different functional groups suggesting the involvement of phytocompounds in the reduction and capping of AgNPs. The average size of the AgNPs was 20 nm and predominantly spherical in shape. AgNPs demonstrated excellent DPPH free radicals scavenging activity with an IC 50 value of 7.51 ± 0.4 µg/mL. C.albicans was identied as the most susceptible strain from the anticandida studies. Usnic acid and Pulvinic acid exhibited low binding energies and showed excellent inhibition interaction with EGFR lung cancer protein. The in-vitro cytotoxic results were impressive with an IC 50 value of 28.75 µg/mL for A549 lung cancer cells treated with AgNPs. Thus, the study demonstrates the effective and non-toxic synthesis of AgNPs using a less explored lichen extract as a promising anticandida and anticancer agent in the eld of nano-medicine.

in uence the synthesis of nanoparticles [11]. Microorganisms and plants are considered as the most promising primal matter for the genesis of MNPs due their ability to produce stable and mono-dispersed particles [12]. Plant-based MNPs synthesis has the advantage of being eco-friendly and economic, while microbes-based MNPs synthesis is regarded effective due to their unique enzyme machinery [13]. In fact, bio-inspired fabrication has become a new trend that is preferred by the scienti c society to produce MNPs that are non-toxic, well-morphed, amicable in size, reproducible and easily scaled-up [14]. MNPs synthesized through biological methods are bio-compatible and reported to have anticancer and antimicrobial properties. Anti-cancer potential of these MNPs are reported in cancer cell lines such as A549 (human lung cancer) [15], MCF 7 (human breast cancer) [16], HCT-116 (human colon cancer) [17] and Hep2 (human hepatic cancer) [18]. The anti-cancer mechanism is elucidated to be dependent on intracellular reactive oxygen species production and apoptosis via mitochondria-dependent and caspasedependent pathways [19]. Application of biogenic MNPs also includes imaging facilitators, theranostics and sensor designing [20,21].
Lichens are complex symbiotic organisms that are formed from the intimate association between a mycobiont (fungi) and a photobiont (algae or cyanobacteria) [22]. They are regarded as the primary colonizers and inhabitants of terrestrial ecosystems with worldwide distribution regardless of the geographical characteristics [23]. Lichens are a part of traditional medicine owing to their rich phytochemical pro le [24]. Unique and novel secondary metabolites have been identi ed from the thallus or the lichenized stroma [25]. Among the several identi ed compounds, metabolites belonging to the class of phenols, depsides, depsones, quinones, furans, dibenzofurans and lactones have gained special interest in the eld of medicine [26,27]. Lichens are regarded as pollution indicators [28] and also their extracts are identi ed to possess vital antibacterial, antifungal, antiviral, antioxidant, antibiotic, antimutagenic, antipyretic and anti-carcinogenic activities [29]. In the present study, Silver nanoparticles (AgNPs) are synthesized in a rapid and affordable approach using the aqueous extract of squamulose lichen -Cladonia subradiata collected from the Kodaikanal hills of Western ghats region, India. For the rst time, in-vitro antioxidant activity, in-silico and in-vitro cytotoxic properties of C. subradiata mediated AgNPs have been reported.

Preparation of extract
The identi ed 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 ne powder using a laboratory mixer grinder. 10g of the ne 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.  [32] ,and Wagner et al., [33] were followed to detect the presence of alkaloids avonoids, phenols, saponins, tannins and glycosides.

GC-MS analysis of C. subradiata extract
Gas chromatography and Mass spectroscopy has been regarded as a "gold standard" for forensic substance identi cation because it is used to perform a 100% speci c test, which positively identi es 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 ow of 1ml / min and an injection volume of 2 ml was employed (split ratio of 10:1

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 con rmed using Ultra-Violet Visible (UV-Vis) spectroscopy (Shimadzu with a range of 200-800nm). Functional groups were identi ed 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).

Determination of Total Phenol Content (TPC) and Total avonoid content (TFC)
The total phenol content and avonoid 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.

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 (H 2 O 2 ) free radical scavenging assay 40 mM H 2 O 2 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: 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].

In-silico molecular docking of C. subradiata compounds
Structure of chemical compounds identi ed 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 le for the preparation of protein in docking simulation. Auto Dock requires pre-calculated grid maps, one for each atom type present in the exible 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].

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% CO 2 , 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% CO 2 , 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.

Identi cation of lichen sample
Kodaikanal hills of the Western Ghats region was a rich repository of several diverse lichen species. Crustose, foliose, fruiticose and squamulose lichens were documented during the survey and most of them belonged to the families Pyrenulaceae, Bacidiaceae, Physciaceae, Parmeliaceae, Cladonniaceae, Arthoniaceae, Graphidaceae, Trochotheliaceae and Ramalinaceae. Western Ghats region of Tamil Nadu has a wide assortment of lichens with more than 657 taxa among which some lichens still remain unexplored and the lichen Parmelia pseudobitteriana was identi ed for the rst time from kodaikanal hills [45]. The lichen sample selected for the study was identi ed as C. subradiata (Fig. 1). The lichen was characterized by squamules of 2x1 mm with whitish graypodetida sparingly branched. The tips were blunt in young poetida and mature tips formed cups. The surface was thinly corticated and the pycnidia were found in young basal squamules.

Phytochemical screening of C. subradiata
Methanol, ethanol, acetone, chloroform, aqueous, petroleum ether and hexane extracts of C.subradiatia were screened for the presence of phytochemicals. Table 1 shows the presence and absence of phytocompounds such as alkaloids, avonoids, phenols, saponins, tannins, glycosides and sterols. Extraction of the phytocompounds was found to be high in mid-polar solvents such as acetone, chloroform and water. Chloroform extract exhibited strong results for avonoids and phenols when compared to other extracts. The extraction quality was poor in petroleum ether and hexane suggesting the minimal quantity of non-polar groups in the phytocompounds. Alkaloids, phenols, avonoids, saponins, glycosides and tannins are the frequently reported phytochemicals in lichen species [46]. Bodicherla et al., [47] has reported the presence of phytochemicals similar to our results in selected macrolichens in methanol, 2-propanol and water extracts. The phytochemicals in biological samples are considered as the major agents that are responsible for the rapid reduction of metal ions to metal nanoparticles in an eco-friendly manner [48].

Identi cation of C. subradiata compounds using GC-MS analysis
The results of the GC-MS analysis con rmed the presence of phytocompounds in the chloroform extract of C.subradiata. 34 phytoconstitents were identi ed from the chromatogram (Fig. 2)

Biogenic synthesis and characterization of AgNPs synthesized from C. subradiata
AgNPs were rapidly synthesized using C.subradiata chloroform extract which was con rmed from the visible colour change in the colloidal solution. The colour change from pale yellow to brown intensi ed with the increase in time (Fig. 3a). The Uv-Visible spectra of the synthesized AgNPs from the lichen extract showed surface plasmon bands between 420 and 450 nm at a time lapse of 5 to 180 minutes.
The intensity of the bands stabilized after 60 minutes and was devoid of red and blue shifts (Fig. 3b). This indicates the stability of the bio-synthesized AgNPs using C.subradiata chloroform extract. Reports of Abdel-Raouf et al., [50] showed that bands were obtained between 400-500 nm for AgNPs synthesized from brown algae extracts and similarly Gudikandulaet al., [51] reported bands between 419-421 nm for AgNPs from white rot fungi. These reports run parallel with our report as lichens are symbionts of algae and fungi. The FT-IR spectrum (Fig. 4) revealed the functional groups of the AgNPs synthesized using C.
subradiata chloroform extract. Peak at 3438 cm − 1 corresponds to O-H stretch of alcohol, the peak at 1416 cm − 1 is attributed to C = C stretch of alkenes and peak at 1107 cm − 1 is designated to C-N for amide or nitro groups. Peaks obtained belonged to the functional groups such as alcohol, nitro compounds, amides and alkenes respectively. The presence of amide and nitro groups showed that other than secondary metabolites, proteins play a major role in the reduction and capping of AgNPs [52]. The X-ray diffraction pattern (Fig. 5) of AgNPs synthesized from C.subradiata chloroform extractcorresponded to (111), (200), (220) and (311) crystallographic planes of face centred cubic (FCC) Silver. Debye-Scherrer formula (D = 0.94 λ/β cos θ) was used to calculate the average size of the AgNPs. The average size of the AgNPs synthesized from C.subradiata chloroform extract was 23 nm. The results were in accordance with the Braggs re ection of silver nanocrystals [53]. The morphological characteristics of the AgNPs were investigated using a TEM instrument. The micrographs (Fig. 6a) revealed that the AgNPs synthesized from lichen extracts were predominantly spherical in shape and their size ranged between 20-50 nm. The crystalline nature of the nanoparticles was evident from the SAED pattern (Fig. 6b) and coincided with the XRD results. Biogenic fabrication of Ag NPs using aqueous-ethanolic extract of Usnea longissima yielded NPs within the size range of 9-11 nm with enhanced antibacterial activity, phytocompounds of the lichen extract were suggested to be the vital reducing and capping agents [54].

Total phenol and avonoids content in C. subradiata chloroform extract and AgNPs
The results of the TPC and TFC exhibited by C. subradiata chloroform extract and AgNPs are given Table 2. It is shown that C. subradiata chloroform extract showed high TPC and TFC. Considerable amounts of phenols and avonoids were detected in the Ag NPs synthesized from the lichen extract. The highest TPC (163.41 ± 0.3 mg GAE/g) and TFC (88.72 ± 0.01 mg GAE/g) were expressed in C. subradiata chloroform extract at a concentration of 100µg/mL. Green synthesized Ag NPs using Tridax procumbens exhibited 68.93 ± 0.36 µg/mg GAE and 64.98 ± 0.46 µg/mg QE as TPC and TFC at 1mg/mL concentration [55]. It is evident from our results that the content of avonoids and phenols increased with the increase in the concentration of the sample, therefore it can be suggested that the TPC and TFC contents of the AgNPs synthesized from chloroform extract of C. subradiata might increase with the increase in the concentration.  Antioxidants play an inevitable role in neutralizing or nullifying the effect of free radicals and management of fatal diseases. Phenols and avonoids in plant extracts are suggested as to be excellent free radical scavenging agents. The potent antioxidant activity exhibited by the AgNPs was due to the phytocompounds that act as reducing and capping agents [35].

In-vitro anticandida activity of C. subradiata extract and AgNPs
Anticandida activity of the Ag NPs synthesized from C. subradiata extract was tested against ve candida strains and the zone of inhibition (ZOI) measured for the strains ranged from 7.3 ± 0.72 mm to 17 ± 1.12 mm. The ZOI observed for C. subradiata chloroform extract ranged from 0.3 ± 1.3 mm to 5.7 ± 0.9 mm. The order of susceptibility of the strains was as follows for AgNPs: C. albicans ≥ C. tropicalis ≥ C. krusei ≥ C. glabrata ≥ C. parapsilosis, whereas the susceptibility order of the lichen extract was observed to be: C. albicans ≥ C. krusei ≥ C. tropicalis ≥ C. parapsilosis ≥ C. glabrata (Fig. 8). C.albicans was highly susceptible to the treatment of Ag NPs and C. subradiata chloroform extract. It was evident from the well diffusion assay that the inhibition pattern was concentration dependent. The ZOIs ranged from 22.3 ± 1.4 mm to 25.7 ± 0.7 mm. The activity of 38 lichen extracts were found to have poor inhibitory effects on planktonic C. albicans yeast and an MIC of 500 µg and above were reported [56]. This suggests that phytocompounds present in lichen extracts were least effective against candida species and was in correspondence with our results. However Ag NPs synthesized from seed extract of Syzygium cumini was reported to have an MIC value of 0.125 mg-0.250 mg/mL against Candida sp. [57]. Similarly the ZOI of Curcumin-Ag NPs was observed as 22.2 ± 0.8 mm, 20.1 ± 0.8 mm, and 16.4 ± 0.7 mm against C. glabrata, C. albicans and C. tropicalis respectively [58]. Results of the anticandida assay suggest that AgNPs synthesized form C. subradiata can be considered as promising anticandida agents.
3.8.In-silico molecular docking of C. subradiata compounds against lung cancer protein In-silico molecular docking was performed using the structure of seven compounds identi ed by GC-MS analysis of C. subradiata chloroform extract using Autodock 4.2. From the analysis, six out of seven compounds showed hydrogen bond interactions. Docked pose of the different compounds with EGFR protein (PDB Id:2GS2). Hydrogen bond interactions, the information about the Binding energies, Number of hydrogen bonds formed and the distance between them is given in Table 3. From the analysis, it is observed that Pulvinic acid and Usnic acid has least binding energy and showed remarkable hydrogen bond interactions with EGFR protein (Fig. 9). Usnic acid suppresses angiogenesis of breast cancer by successfully inhibiting vascular endothelial growth factor receptor (VEGFR) 2 mediated Extracellular signal-regulated protein kinases 1 and 2(ERK1/2) and AKT/P70S6K signaling pathways in endothelial cells [59]. Phytocompounds from the fruticose lichen Rocella montagnei showed impressive docking scores against CDK-10 (Cyclin Dependent Kinase 10) that plays a pivotal role in the pathogenesis of cancer and is attributed for the incessant proliferation of cancer cells [60]. Lichen metabolites are also known to inhibit the activity of cyclooxygenase-2 enzyme which is involved in the in ammation of tissues [61]. Molecular docking results of this study provide a new arena for exploring the anti-cancer potential of lichen metabolites against lung cancer proteins. 3.9. In-vitro cytotoxic activity of C. subradiata extract and AgNPs In-vitro cytotoxic activity of C. subradiata chloroform extract and AgNPs synthesized from the same extract were carried out owing to the encouraging results obtained from in-silico molecular docking study using the structures of C. subradiata extract compounds identi ed by GC-MS analysis. The effects of C. subradiata extract and AgNPson cell viability were analysed and quanti ed by using MTT assay after 24hour treatment with a concentrations of the samples ranging from 3 to 300 g/mL (Fig. 10). Through this assay, cell viability was determined based on the measurement of mitochondrial function, as MTT is transformed into formazan crystals in living cells in which mitochondrial dehydrogenases are functional.
As shown in Fig. 10 The IC 50 value obtained in this study was comparatively less and exhibited potential cytotoxic effects.The mechanism of action of AgNPs dependent cancer cell death is owed to the generation of intracellular reactive oxygen species. Over production of free radicals leads to oxidative stress, thus damaging the DNA of the cancer cells and forcing them to enter apoptosis. Morphology, size, charges of the AgNPs are crucial in determining the cytotoxic effect [64, 65]. Bio-active compound based metal nanoparticles have also showed excellent cytotoxic activity against A549 cells, in a study Palladium nanoparticles synthesized using ascorbic acid by microwave irradiation method showed an IC 50 of 7.2 ± 1.7 ug/mL [66].
However the results of our study suggested that simple and facile synthesis are cost-effective in yielding AgNPs with similar cytotoxic effects.

Conclusion
The present study elucidated a greener, safer, eco-friendly, feasible and rapid alternative approach to synthesize Silver nanoparticles with enhanced biological applications. C. subradiata lichen extract was a successful green resource for the synthesis of AgNPs and are identi ed to have rich secondary metabolite pro le. Lichen extract and synthesized AgNPs showed substantial amounts of phenol and avonoid content with effective free radical scavenging activity and anticandida activity. The in-silico study explicated the effective binding and interaction of C.subradiata compounds with EGFR lung cancer protein.
In-vitro cytotoxicity assay revealed the dose dependent effect of AgNPs and lichen extract on A549 cells with a low IC 50 value. The results present vital evidence that cost-effective synthesis of AgNPs from C. subradiata lichen extract and their anti-cancer properties have potential applications against lung cancer in the eld of nanomedicine.