Sampling and study area

MSCBU campus positioned at 21.9303° N 86.7636° E, Odisha, India (Fig. 1). Seasonal field visits (Spring, Rainy and Winter) and collection (Dates: 07/03/2021, 22/08/2021 and 21/01/2022) of lichens were performed regularly, and all collected lichens were sampled by random sampling technique. Collected lichens are wrapped in white paper bags with zippers, with its microhabitat data (latitude, longitude, temperature and humidity) of each bisect.

Fig. 1

Study area: MSCBU campus, shadow area showing coverage and distribution of lichens

Diversity index of collected samples

This study explored a number of epiphytic lichen species under 12 genera in 8 families from the MSCBU campus, which is situated near the Similipal Biosphere Reserve (SBR). The preceding study was carried out by studying the epiphytic lichen species on twenty dominant trees/phorophytes. The lichen family Graphidaceae has the maximum distribution and is represented by SWI (Shannon-Weiner Index) as H = 2.053, with a SIV (species index value) of 206.72, and both the lichen species with SIV as in Pyrenula nitida- OQ146904 (55.96) and Graphis scripta-OP861477 (63.46) were distributed within vegetation.

Morphological identification of lichens

Morphological identification was done using a stereo-zoom microscope (Stemi-305, 40 × magnifications) and scanning electron microscope (Model-S3400N, magnification 10X to 300,000 X Max) observation. Morphological identifying characteristics are studied using the lichen identifying key book [12].

Molecular identificationDNA isolation from lichens

For the isolation of DNA from Lichen thallus, 10 mg of scraped lichen thallus was used and put into a micro-vial, deepen in liquid nitrogen with three or four 2.5-mm sterile glass beads for 30 s and disrupted with a Mini-Beadbeater-24. The sample was stirred vertically with 300 µL KCl extraction buffer, 300 µL chloroform and kept reversed. The sample mixture was centrifuged at 12,000 rpm for 1 min. The supernatant was transferred to a microcentrifuge vial with the addition of 180 µL of chilled isopropanol and centrifuge at 12,000 rpm for 1 min; the supernatant was descanted. The remaining pellet was poured out with 300 µL chilled ethanol (70%). The pellet was dried at 55 °C in oven, then 100 µL of TE buffer was added, and purified DNA was stored at 4 °C [13].

PCR and sequencing

A partial sequence of DNA for analysis (28s, and 18s rDNA) was isolated from the upper cortex of both lichens. Amplification of rDNA performed using PCR primers for the 28s larger sub-unit of P. nitida strain PNSB04 used forward primer used was PN1- 5′-aacaggggggtgagatgtcaga-3′ (25 nm STD, GC: 54.5%, Tm: 60 °C, ΔG: − 41.7 kcal/mol), and reverse primer PN2-5′-ctagtacgatacattcaaatgt-3′ (25 nm STD, GC: 31.8%, Tm: 47.8 °C, ΔG: -34.33 kcal/mol) for G. scripta strain SPB25 rDNA, Forward primer-GS1: 5′-ttgtaatttggagaaggtgttt-3′ (25 nm STD, GC: 31.8%, Tm: 50.4 ΔG: − 38.42 kcal/mol), and reverse primer-GS2: 5′-catcctagcttttgcgcggacc-3′ was used, following the protocol of White et al. [14]. PCR products were visualized on a 1.5% agarose gel analysis for 25 min with content 120 V, to authenticate the presence of amplicons size, then exonuclease-I and recombinant shrimp alkaline phosphatase used for molecular purification and sequenced bidirectionally by genetic analyzer (ABI 3730). Proofreads (forward and reverse) were assembled in MEGA and Sequencher v. 5, with GenBank submission having accession number for G. scripta (OP861477) and P. nitida (OQ146904).

Antioxidant activity of LichensPreparation of the lichen extracts for non-enzymatic antioxidant activity

Collected lichen samples were dried in a hot air oven and then crushed to a fine powder, and 10 g of dry powder was poured into a Borosil Soxhlet at 45–50 °C for 24 h using methanol, acetone, benzene and diethyl ether. After 6–8 cycles, the solutions are collected and further evaporated in oven at 42–45 °C. After evaporation, the dry extract of each solvent was taken for further analysis.

DPPH radicals scavenging assay

DPPH (1,1-diphenyl-2-picryl-hydrazil) is a complex radical mixture used in an H+ Ion transformed-based scavenging non-enzymatic antioxidant assay [15]. An earlier prepared 1 ml DPPH solution (0.1 mM) was added to 3 ml of different progressive concentrations (100, 250, 500, 750 and 1000 μg/ml) of lichen extract and instantly incubated in dark conditions for 30 min at room temperature. After incubation, the absorbance was taken at 517 nm using a Janway-119 spectrophotometer. The radical scavenging activity (RSA) was measured in percentage (%) by using the formula Activity (%) = [(control Absorbance (A0)—sample Absorbance (A1))/control Absorbance (A0)] × 100, where A0 is the absorbance of the − ve control, and A1 is the absorbance of the sample added reaction mixture or standards, i.e., butylated hydroxytoluene. Based on the percentage of radical scavenging activity, the IC-50 value was calculated according to the increase in percentage of radical scavenging. For comparative analysis, the natural antioxidant Ascorbate was used as a + ve control against the sample.

Ferric-reducing assay

ET (electron transfer)-based antioxidant assay determined the ferric ion-reducing activity of both lichens by the method of Oyaizu [16]. The concentrations of a standard range of lichen extracts (100–1000 μg/ml) were mixed with 2.5 ml of PO4 buffer of pH 6.6 (0.2 M) with 1% potassium ferricyanide and incubated for 25 min at 50 °C and subsequently mixed with 10% trichloroacetic acid, followed by centrifugation at 3000 rpm for 20 min. The collected supernatant was vigorously mixed with 2.5 ml of distilled water while simultaneously adding 0.5 ml of 0.1% FeCl3, and absorbance was measured at 700 nm. For the comparative observation, BHT (butylated hydroxytoluene) was taken as the positive control.

Determination of antioxidant enzymatic assayPreparation of cellular extract

For analysis of superoxide dismutase and catalase activity, 0.5 g of lichen extract was prepared by using ethylene diamine-tetra acetate (50 mM), sodium phosphate buffer of pH 7.4 (50 mM), polyvinyl pyrrolidone 10% (w/v) and phenyl methyl sulfonyl fluoride (2 mM) in a frozen condition with a pestle in the dark. The well-gelatinous ground material was centrifuged at 14,000 rpm at 4 °C for 20 min in a collapsing centrifuge. After centrifugation, the supernatant was evaluated for SOD and CAT activities.

Superoxide dismutase (SOD) activity

The activity and quantity measures of SOD were evaluated by the standard procedure of Das et al. [17]. This activity measured the superoxide-driven nitrite formation inhibition from hydroxylamine hydrochloride. The reaction cocktail was prepared in dark conditions by adding 1.11 ml of phosphate buffer of 50 mM (pH 7.8), 0.075 ml of 10 mM hydroxylamine hydrochloride, 0.04 ml of 1% Triton X-100 (v/v), 0.075 ml of L-methionine (20 mM), 0.1 ml of 50 mM EDTA and 80 µl of 50 mM, riboflavin to the mixture. After preparation of the reaction mixture, 10 min of light exposure is required to produce the appearance of white fluorescence and absorbance measured at 543 nm with the addition of Griess reagent. A single unit of enzyme activity represented the amount of SOD, which prevented 50% of nitrite formation. The enzymatic activity was measured using the formula V0/V−1, where V0 (control absorbance) and V (sample absorbance). The activity and total enzyme were expressed in units nkat (nanokatal per liter)/mg or U (Unit per liter)/ml.

Catalase (CAT) activities

The catalase activity of two tested lichens is evaluated by the method of Aebi [18]. The preparative chemical mixture for catalase analysis contained 2 ml of 0.1, potassium phosphate buffer (pH 6.8), an enzyme extract of 500 μL and H2O2 of 500 μL that reached a final volume of 3 ml. The catalase activity was measured over a 3-min time interval at 240 nm against the blank by the rate of H2O2 consumption.

Native-PAGE analysis for SOD and CAT enzyme

For the analysis and estimation of total SOD and catalase, native-PAGE analysis was performed. The SOD and catalase staining was performed by Beauchamp and Fridovich [19]. Enzymatic analysis through native-PAGE is done by early preparation of 10% resolving and 5% stacking gels and then loaded the sample solution at 4 °C with a constant current of 40 V for 12 h. For gel support, 10% glycerol was added [20]. The gel was stained in dark for 30 min by using solutions (i.e., 50 mM sodium phosphate buffer of pH 7.8), tetramethylethylenediamine (28 mM), riboflavin (0.003 mM), NBT(0.25 mM) and EDTA (0.1 mM), and the gel after 30 min of light exposure, resulting in the visible protein bands. Similarly, the catalase staining was done in a dark condition. The gel was washed with water (ddH2O) and stained with 0.003% H2O2 for 10 min. Thereafter, a mixture of 2% potassium ferric cyanide, 2% ferric chloride and 1% HCl was added for the catalase isoforms to appear dark green in the background of the gel. The activity and total enzyme were expressed in units nkat (nanokatal per liter)/mg or U (Unit per liter)/ml.

Fourier transform infrared spectroscopy (FTIR)

In FTIR analysis, the IR spectra are equipped with a Thermo Nicolet iS10 FTIR spectrometer (Thermo Scientific, USA) with the Smart iTR attenuated total refraction (ATR) accessory. Soxhlated dry lichen extract is placed on a horizontal ATR crystal of the spectrometer under constant pressure. The sample is 32 scans across the range of (ν4000 to 400 cm−1) with a resolution of 4.0 cm−1, an absolute threshold of 96.395 and a sensitivity of 50. For analysis of spectral data, essential FTIR and prism software (version 8.0.1) was used, and all sample analysis was carried out in triplicate.

Gas chromatography–mass spectrometry analysis (GC–MS)

For GC–MS analysis, methanol is used as a reliable solvent for extraction. GC–MS analyses were performed on an Agilent Capillary-column 60.0 m × 250 μm × 25 μm. Injection in autosampler was used 1.5 μl of the sample split to 10:1. Oven: Initial temp 60 °C, ramp (temperature regulation) 7 C/min to 200 °C, (hold for 3 min), ramp 10 °C /min to 300 °C (hold 5 min), inject autosampler = 280 °C, Volume = 1.5 µl, split = 10:1, Helium as carrier gas, Solvent delay = 7.00 min, Transfer temp = 160 °C, Source temp = 150 °C, scan = 50 to 600 Da and column 60.0 m × 250 μm. The GC peak regions were used to calculate the percentage of extract composition. PubChem and the NIST Chemistry web-book used for analysis.

Determination of total phenolic

The estimation of total phenols includes total aromatic and polyphenols and is expressed against the gallic acid standard calibration curve as GAE mg/g of total DE (dry extract). The Folin–Ciocalteu reagent method is used for estimating total phenol [21]. Lichen extract of 100 μl was taken and mixed with 2 ml of 2% sodium carbonate, and in a 10-min interval, 500 μl of Folin’s reagent was added and incubated for 20 min. Absorbance was measured by Jenway-119 spectrometer at 650 nm.

Estimation of total flavonoid

Total flavonoid estimation of both lichen species was carried out by method of Zhishen et al. [22] and expressed as quercetin equivalent, i.e., μg QE/g dry extract. One milliliter of Lichen extract was mixed with 10% of aluminum chloride (300 μl), 500 μl of sodium nitrite and 1 ml of 1 M hydroxide, and final volume was made up to 5 ml with dd H2O. After a period of incubation absorbance was measured at 510 nm.

Estimation of total tannin

The total tannin content of two lichens was estimated, expressed as tannic acid equivalent (TAE/mg/g DE) of dry extract and calibrated in a tannic acid calibration curve using the method of Oyaizu [16]. The lichen extract of 200 µg/ml was taken and added to 7 ml of distilled water, followed by 0.5 ml of Folin phenol reagent and 1 ml of a 35% sodium carbonate solution and volume adjusted with added dd H2O and up to 10 ml. The mixture was shaken and incubated for 30 min, and absorbance was taken at 725 nm.

Estimation of total terpene

The quantitative estimation of total terpene in both lichens was carried out by the standard procedure of Ghorai et al. [23], and total terpene was plotted against the linalool equivalent (a monoterpene) calibration curve and expressed as LE mg/g DW. Lichen extract of 1 ml was mixed with conc. 1 ml, conc. H2SO4 and 1 ml chloroform and then shaked gently, to take absorbance at 538 nm.