Chemical and pharmacological evaluation of manglicolous lichen Roccella montagnei Bel em. D. D. Awasthi

Abstract

Background

Lichen is a composite organism composed of fungus in association with algae or cyanobacteria. Particularly, lichen betide to mangroves are named as manglicolous lichens. From the folklore, lichen extracts were used in the management of many infections and diseases.

Results

The chemical investigation of acetone extract of manglicolous lichen Roccella montagnei (RM-Ac) yielded nine known metabolites namely divarinolmonomethylether (1), ethyl divaricatinate (2), divarinol (3), orcinol (4), methyl 2,6-dihydroxy-4-methylbenzoate (5), haematommic acid (6), atranol (7), ethyl haematommate (8) and ethyl orsellinate (9). Except 4, all are for the first reported from this species. The RM-Ac and its metabolites (1–9) were screened for antioxidant (DPPH, ABTS and superoxide free radical assays), anti-inflammatory, anticancer (SRB assay using A549, DLD-1, FADU, HeLa and MCF-7) and acute toxicity studies. The pharmacological results showed that compounds 6 and 8 depicted potent inhibitory profile against ABTS free radical (with IC₅₀ value of 40.0 and 40.5 μg/mL, respectively) and protein denaturation (with IC₅₀ value of 435 and 403 μg/mL, respectively). LD₅₀ of RM-Ac was found to be above 2 g/Kg body weight. Moreover, the RM-Ac showed prominent inhibition of formalin-hind albino rat paw oedema at both the tested doses, i.e., 100 and 200 mg/Kg b.w than that of the standard drug (indomethacin). Furthermore, the compounds 6 and 8 exhibited significant degree of specificity against HeLa, FADU and A549; besides, they showed very little degree of specificity against NHME cell line specifying less toxicity to normal cells.

Conclusion

It can be concluded that the manglicolous lichen R. montagnei has an aptitude to act against free radicals, inflammation and cancer, and the main metabolites responsible for its biological activity are 6 and 8.

Background

Lichen (a symbiotic organism) belongs to bryophytes which have an aptitude to persevere on any geographical region or any substratum [1, 2]. Due to their unique survival and mutualistic characteristics, lichens and their secondary metabolites are used for the treatment of several infections and diseases [2, 3]. The lichens particularly associated with the mangroves/mangals are termed as manglicolous lichens [2, 3]. As mangals persist in stressful environment such as high concentration of moisture and salt and low and high tidal water, lichens habituated on these groups of plants are also exposed to the aforementioned stressed conditions. As a result, they show a difference in phytochemical constituents than normal lichens due to stressed physiological adaptations [2,3,4]. Besides, there are very few chemical and pharmacological reports existing on manglicolous lichens due to their slow growth (1 cm/year) and the difficulty to collect a good amount of specimen from mangrove regions.

In recent times, the Roccella genus has been reviewed, reporting 24 main species which are constrained to coastal habitats. Among them, Roccella montagnei Bel em. D. D. Awasthi is one having a fructose growth form which commonly persists in mangroves of India [4, 5]. In 1940, Rao and Seshadri reported novel metabolites namely erythrin, erythritol, lecanoric acid, orcinol and roccellic acid from R. montagnei [6, 7]. In 1942, occurrence of active montagnetol in R. montagnei was also reported by Seshadri’s group [8]. In 1958 and 1959, Murty and Subramanian’s group reported the carotene content of R. montagnei and also isolated carotene and ergosterol from R. montagnei [9,10,11].

In 2006, a biological report regarding the in vitro antimicrobial assay of R. montagnei from the mangrove tree, Rhizophora sp. in Pichavaram mangroves, Tamil Nadu, India, has been determined and found that methanolic extract of R. montagnei were active against microbes (bacterial and fungal) [12]. In 2016, the phytochemical and in vitro antioxidant assay of R. montagnei was quantified and it was reported that the species contains alkaloids, saponins, phytosterols, phenols, tannins, flavonoids and good inhibitory profile against DPPH and ferric radicals [13]. Furthermore, Mallavadhani and Sudhakar reported roccellatol along with nine known metabolites from R. montagnei [14]. In recent times, isomer of methyl-γ-orsellinate and roccellatol which were isolated from R. montagnei were screened for radical scavenging potential, anti-inflammatory and anti-arthritic activities [15], as well as, isolation of cytotoxic compounds along with docking evidences has been reported by Mishra’s group [16]. Earlier, our group has established and reported the phytochemical analysis, antimicrobial, antimycobacterial, antioxidant and cytotoxicity activities of various extracts of R. montagnei [4]. In continuation of our research work to identify bioactive metabolites from manglicolous lichen R. montagnei, the present study was performed to identify the bioactive constituents present in manglicolous lichen R. montagnei.

Methods

Collection

From the twigs of mangrove plant Excoecaria agallocha, the specimens of manglicolous Roccella montagnei Bel em. D. D. Awasthi was collected from Godavari estuary, Andhra Pradesh, India, in December, 2016. The species was determined by Dr. D. K. Upreti, and a voucher specimen (14–027172) was deposited at Lucknow Lichen herbarium, National Botanical Research Institute, Lucknow, India [1].

Extraction

The collected manglicolous lichen Roccella montagnei was shade dried, and about 250 g of dried lichen material was exhaustively extracted with acetone. The attained acetone extract of R. montagnei (RM-Ac; 5.45 g, 2.18%, percentage based on total lichen material) was exposed to column chromatography (CC) (#230–400) by n-hexane in ethyl acetate (EA) (increasing polarity) as eluent, which eventually resulted in three fractions. Fraction I was re-treated with CC (#230–400) by n-hexane in dichloromethane (DCM) (increasing polarity) yielded 1 (30 mg, 0.012%, percentage based on total lichen material) as pale yellow oil and 2 (18 mg, 0.007%, percentage based on total lichen material) as sharp colorless needles. Fraction II was re-treated with CC (#230–400) by n-hexane in EA (increasing polarity) as eluent, yielded 3 (12 mg, 0.005%, percentage based on total lichen material) as pinkish powder, 4 (15 mg, 0.006%, percentage based on total lichen material) as colorless needles, 5 (675 mg, 0.027%, percentage based on total lichen material) as pale yellow crystals and 6 (35 mg, 0.014%, percentage based on total lichen material) as pale yellow needles. Fraction III was re-treated with CC (#230–400) by DCM in EA (increasing polarity) as eluent, yielded 7 (12 mg, 0.005%, percentage based on total lichen material) as yellowish powder, 8 (15 mg, 0.006%, percentage based on total lichen material) as greenish solid and 9 (35 mg, 0.014%, percentage based on total lichen material) as pale yellow solid.

Antioxidant activity

DPPH assay

The metabolites (1–9) and RM-Ac was subjected to 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay in triplicate, and results are reported as percentage of inhibition of DPPH free radicals [17]. Initially, to the known concentrations of the sample added 0.004% DPPH dissolved in methanol and incubated for half-an-hour at 37 °C. By using UV-Visible spectrophotometry (Spectra MAX plus 384, USA), all the samples were noted with absorbance at 517 nm against blank. IC₅₀ values of the metabolites (1–9) and RM-Ac were determined by plotting concentrations against their percentage inhibition.

ABTS radical scavenging assay

The metabolites (1–9) and RM-Ac was exposed to 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay in triplicate, and results are reported as percentage of inhibition of ABTS free radicals [18]. To 7 mM ABTS⁺, added potassium persulfate (2.45 mM) in water at room temperature and standardized. Then, to know the concentrations of the sample, added 1 mL of above standardized solution and incubated for half-an-hour. In later incubation, all the samples were observed to have absorbance at 750 nm against blank. IC₅₀ values of the metabolites (1–9) and RM-Ac were determined by plotting concentrations against their percentage inhibition.

Superoxide radical scavenging assay

The metabolites (1–9) and RM-Ac was exposed to superoxide radical scavenging assay in triplicate, and results are reported as percentage of inhibition of superoxide free radicals [19]. To NADH (73 μM), added 15 μM of PMS and NBT (50 μM) in 20 mM phosphate buffer (pH 7.4) and standardized. Then, to know the concentrations of the sample, added 1 mL of above standardized solution and incubated for half-an-hour. After incubation, all the samples were observed to have absorbance at 562 nm against blank. IC₅₀ values of the metabolites (1–9) and RM-Ac were determined by plotting concentrations against their percentage inhibition.

In vitro anti-inflammatory activity

The in vitro anti-inflammatory activity for metabolites (1–9) and RM-Ac were determined by using protein denaturation method [20] in triplicate, and results are reported as percentage of inhibition of protein denaturation against blank. Bovine serum albumin protein (1%) was solubilized in sodium phosphate buffer (50 mM, pH 6.4). Then, to know the concentrations of the sample, added 0.2 mL of above protein solution and make up to 5 mL with sodium phosphate buffer and incubated for 20 min at 37 °C. Later, all the samples were boiled for 20 min in steam bath at 95 °C and set to room temperature. After incubation, all the samples were observed to have absorbance at 562 nm against blank. IC₅₀ values of the metabolites (1–9) and RM-Ac were determined by plotting concentrations against their percentage inhibition.

Animals

Albino rats (either sex) weighing 190–200 g were utilized for the current experimental study, and the experimental procedure were performed as per OECD regulations (Regd No. 516/01/A/CPCSEA).

Acute toxicity studies

A week before the acute toxicity study, five male albino rats were kept on standard diet under room temperature. Orally, RM-Ac at 2000 mg/kg body weight (b.w) were administered to the selected albino male rats and kept under observed for 24 h [21]. The mortality number triggered by the RM-Ac within this time duration was observed, from which log dose response plots were calibrated and median lethal dose (LD₅₀) of the sample was determined.

In vivo anti-inflammatory activity

The RM-Ac was subject to in vivo anti-inflammatory assay by plethysmographic measurement of formalin-induced hind albino rat (either sex; n = 6) paw oedema [2, 20]. Group I is for normal control (dosed with 0.5% CMC), group II for indomethacin (Indo, 100 mg/Kg b.w) and group III and group IV assisted for RM-Ac at 100 and 200 mg/Kg b.w, respectively. After 30 min of samples administration intraperitoneally to albino rats, 1% w/v formalin (0.1 mL) was dosed in the plantar area of albino rat left paw and paw volume was measured at 2 and 4 h by using right paw (non-inflammated) as a reference. The % variation in albino rat paw oedema was calculated using below formula.

$$ \mathrm{Percentage}\kern0.17em \mathrm{reduction}=\frac{\left(\mathrm{Volume}\kern0.17em \mathrm{of}\;\mathrm{paw}\;\mathrm{oedema}\kern0.17em \mathrm{in}\kern0.17em \mathrm{controlled}\kern0.17em \mathrm{animals}\hbox{-} \mathrm{Volume}\kern0.17em \mathrm{of}\ \mathrm{paw}\;\mathrm{oedema}\kern0.17em \mathrm{in}\kern0.17em \mathrm{treated}\kern0.17em \mathrm{animals}\right)\;}{\mathrm{Volume}\kern0.17em \mathrm{of}\;\mathrm{paw}\;\mathrm{oedema}\kern0.17em \mathrm{in}\kern0.17em \mathrm{controlled}\kern0.17em \mathrm{animals}}\times 100 $$

Statistical analysis

The outcomes are stated as mean ± SEM values of three and six independent tests of in vitro and in vivo assays, respectively. The results of in vivo anti-inflammatory assay were deliberate by one-way ANOVA followed by Dunnett’s test. The p < 0.05 value between the experimental groups is statistical significance.

Anticancer activity

Cancer cell lines

Lung (A549), colon (DLD-1), cervical (HeLa), breast (MCF-7), head & neck (FADU), cancer cells and normal human mammary epithelial (NHME) cell lines were attained from National Centre for Cell Science, Pune. All the cancer cell lines were preserved in minimal essential medium MEM media, which contain 5% mixture of streptomycin (100 μg/mL) and penicillin (100 units), and fetal calf serum (10%), in presence of CO₂ (5%) incubator with humidity (90%) for 72 h at 37 °C.

Cell growth medium

Three days earlier to assay, selected cancer cell lines were maintained in MEM and grown on 10% FBS supplemented with trypsin (0.25%). In a sterilized polypropylene tube, final suspension of cancer cells were taken, and concentration of the cells in each well was calculated by using 0.4% trypan blue solution using hematocytochameter chamber under a microscope. The minimal concentration of 1 × 10⁴ cells per well was noted as nominal seed density.

Sample preparation

The samples were dissolved in DMSO, which is used as a control and doxorubicin as a standard. Primary screening of the samples were performed at 100 μg/mL for RM-Ac, 30 μg/mL for compounds 1–9 and 10 μg/mL for standard drug, i.e. doxorubicin. Active samples were further screened at different concentrations for RM-Ac (25, 50, 75 and 100 μg/mL), compounds 1–9 (5, 10, 20 and 30 μg/mL) and doxorubicin (2.5, 5.0, 7.5 and 10 μg/mL) against particular cancer cells.

Sulforhodamine B (SRB) colorimetric assay

The anticancer activity of RM-Ac and compounds 1–9 were performed by SRB assay in triplicate (n = 3) [3]. In 96-well plate, added 190 μL screened ideal cancer cells suspension and test samples and incubate with relative humidity (90%), 5% CO₂ for 3 h at 37 °C. Formerly, to each well, 100 μL cold TCA was added and incubated for 1 h at 4 °C. Thereafter, 96-well plate was gently washed with water, air-dried at 25 °C. Next, to each well, SRB solution (100 μL of 0.057%) was added, incubated for half an hour and stained with CH₃COOH (1%). Then, Tris base (200 μL of 10 mM at pH 10.5) solution was added to each well, agitated for few minutes and observed the optical density at 510 nm. The control contains only cancer cells, whereas blank contains only MEM medium. The % growth inhibition was determined by

$$ \%\mathrm{Growth}\kern0.34em \mathrm{inhibition}=100\hbox{-} \left[\mathrm{Absorbance}\kern0.17em \mathrm{of}\kern0.17em \mathrm{Sample}/\mathrm{Absorbance}\kern0.17em \mathrm{of}\kern0.17em \mathrm{Control}\right]\times 100 $$

IC₅₀ values of the active samples were determined by plotting concentrations against their percentage inhibition.

Results

Chemistry

Chemical investigation of acetone extract from Roccella montagnei (RM-Ac) yielded compounds 1–9, which are illustrated in Fig. 1. The chemical structures of metabolites 1–9 were characterized by using elemental analysis, ¹H & ¹³C NMR, and mass spectral data and correlating with the existing literature data [22].

Fig. 1

Structures of secondary metabolites isolated from Roccella montagnei

Divarinolmonomethylether

Pale yellow oil, R_f: 0.6 (Hex:DCM, 9:1), UV (λ_max): 273 in methanol, Mol. For.: C₁₀H₁₄O₂; ¹H NMR (400 MHz, DMSO-d₆): δ 0.74–0.82 (m, 3H), 1.41–1.52 (m, 2H), 2.66–2.74 (m, 2H), 3.76 (brs, 1H), 4.39 (brs, 3H), 6.24 (d, 1H, J = 2.1 Hz), 6.49 (s, 1H), 6.61 (d, 1H, J = 3 Hz); ¹³C NMR (400 MHz, DMSO-d₆): δ 12.40 (C-10), 23.11 (C-9), 35.21 (C-8), 52.81 (C-7), 101.49 (C-2), 108.59 (C-6), 111.23 (C-4), 141.81 (C-5), 155.76 (C-3), 162.12 (C-1). Elemental analysis found C-72.33, H-8.42(%), calcd. C, 72.26, H, 8.49(%). ESI-MS: m/z 166.9 ([M-H⁺], 100%).

Ethyl divaricatinate

Sharp colorless needles, R_f: 0.4 (Hex:DCM, 9:1), m.p: 43–44 °C, UV (λ_max): 201 in methanol, Mol. For.: C₁₃H₁₈O₄; ¹H NMR (400 MHz, DMSO-d₆): δ 0.97–1.05 (m, 6H), 1.70–1.72 (m, 2H), 2.38–2.39 (m, 2H), 2.98–3.04 (m, 2H), 3.87 (s, 3H), 6.67 (d, 1H, J = 2.3 Hz), 6.79 (d, 1H, J = 2.8 Hz), 11.36 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 14.50 (C-12), 14.60 (C-9), 24.87 (C-11), 31.17 (C-10), 55.75 (C-13), 56.53 (C-8), 99.46 (C-5), 106.21 (C-3), 108.59 (C-1), 144.12 (C-2), 162.92 (C-6), 166.74 (C-4), 172.65 (C-7). Elemental analysis found C-65.16, H-7.26(%), calcd. C, 65.53, H, 7.61(%). ESI-MS: m/z 239.0 ([M-H⁺], 100%).

Divarinol

Pinkish powder, R_f: 0.8 (Hex:EA, 1:1), m.p: 51–52 °C, UV (λ_max): 274 in methanol, Mol. For.: C₉H₁₂O₂; ¹H NMR (400 MHz, DMSO-d₆): δ 0.97–1.04 (m, 3H), 1.65–1.76 (m, 2H), 2.95–3.04 (m, 2H), 3.53 (brs, 2H), 6.18 (s, 1H), 6.40–6.42 (dd, 1H, J = 8, 9.2 Hz), 6.66 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 12.40 (C-9), 23.11 (C-8), 35.21 (C-7), 101.49 (C-2), 108.59 (C-4/C-6), 141.81 (C-5), 155.76 (C-1/C-3). Elemental analysis found C-71.87, H-7.82(%), calcd. C, 71.03, H, 7.95(%). ESI-MS: m/z 153.0 ([M-H⁺], 18.75%).

Orcinol

Colorless needles, R_f: 0.7 (Hex:EA, 1:1), m.p: 108–109 °C, UV (λ_max): 215.5 in methanol, Mol. For.: C₇H₈O₂; ¹H NMR (400 MHz, DMSO-d₆): δ 2.46 (s, 3H), 3.34 (s, 2H), 6.23 (s, 2H), 6.30 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 27.93 (C-7), 98.36 (C-4/C-6), 109.34 (C-2), 155.22 (C-5), 163.90 (C-3), 157.52 (C-1). Elemental analysis found C-67.59, H-6.52(%), calcd. C, 67.73, H, 6.50(%). ESI-MS: m/z 125.1 ([M-H⁺], 28.88%).

Methyl-2,6-dihydroxy-4-methylbenzoate

Pale yellow crystals, R_f: 0.6 (Hex:EA, 1:1), m.p: 138–139 °C, UV (λ_max): 219.5 in methanol, Mol. For.: C₉H₁₀O₄; ¹H NMR (400 MHz, DMSO-d₆): δ 2.23 (s, 3H), 3.75 (s, 3H), 6.12 (d, 2H, J = 1.2 Hz), 9.93 (s, 1H), 10.65 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 22.46 (C-9), 52.16 (C-8), 100.83 (C-1), 107.93 (C-5), 110.58 (C-3), 141.15 (C-4), 161.46 (C-2/C-6), 170.59 (C-7). Elemental analysis: found C-59.66, H-5.62(%), calcd. C, 59.34, H, 5.53(%). ESI-MS: m/z 183.0 ([M-H⁺], 68.81%).

Haematommic acid

Pale yellow needles, R_f: 0.4 (Hex:EA, 1:1), m.p: 172–173 °C, UV (λ_max): 219.5 in ethanol, Mol. For.: C₉H₈O₅; ¹H NMR (400 MHz, DMSO-d₆): δ 2.54 (s, 3H), 6.42 (s, 1H), 9.68 (s, 1H), 10.59 (s, 1H), 11.46 (s, 1H), 13.75 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 17.10 (C-9), 105.25 (C-1), 109.34 (C-3/C-5), 155.22 (C-6), 163.90 (C-4), 167.04 (C-2), 173.42 (C-7), 191.73 (C-8). Elemental analysis: found C-55.64, H-4.52(%), calcd. C-55.11, H-4.11(%). ESI-MS: m/z 198.3 ([M-H⁺], 5.64%).

Atranol

Yellowish powder, R_f: 0.8 (DCM:EA, 7:3), m.p: 125–126 °C, UV (λ_max): 225 in methanol, Mol. For.: C₈H₈O₃; ¹H NMR (400 MHz, DMSO-d₆): δ 2.23 (s, 3H), 6.12 (s, 2H), 9.94 (s, 1H), 10.66 (s, 1H), 11.29 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 22.46 (C-8), 100.83 (C-1), 107.93 (C-5), 110.58 (C-3), 141.15 (C-4), 161.46 (C-2/C-6), 192.87 (C-7). Elemental analysis: found C-63.50, H-5.52(%), calcd. C-63.15, H-5.30(%). ESI-MS: m/z 153.1 ([M-H⁺], 6.91%).

Ethyl haematommate

Greenish solid, R_f: 0.6 (DCM:EA, 7:3), m.p: 112–113 °C, UV (λ_max): 209.5 in methanol, Mol. For.: C₁₁H₁₂O₅; ¹H NMR (400 MHz, DMSO-d₆): δ 0.93–0.97 (t, 3H), 1.59–1.65 (m, 2H), 2.54 (s, 3H), 6.42 (s, 1H), 9.68 (s, 1H), 10.59 (s, 1H), 11.46 (s, 1H), 13.75 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 14.28 (C-11), 20.14 (C-9), 68.63 (C-8), 106.38 (C-1), 109.34 (C-5), 114.78 (C-3), 155.22 (C-6), 163.90 (C-4), 167.04 (C-2), 173.42 (C-7), 191.73 (C-8). Elemental analysis found C-58.64, H-5.52(%), calcd. C-58.93, H-5.39(%). ESI-MS: m/z 224.9 ([M-H⁺], 31.36%).

Ethyl orsellinate

Pale yellow solid, R_f: 0.4 (DCM:EA, 7:3), m.p: 131–132 °C, UV (λ_max): 219 in methanol, Mol. For.: C₁₀H₁₂O₄; ¹H NMR (400 MHz, DMSO-d₆): δ 0.80–0.84 (m, 3H), 1.46–1.52 (m, 2H), 2.54 (s, 3H), 6.42 (s, 2H), 9.68 (s, 1H), 10.59 (s, 1H); ¹³C NMR (400 MHz, DMSO-d₆): δ 14.28 (C-9), 20.14 (C-10), 68.63 (C-8), 105.25 (C-5), 109.34 (C-1), 114.78 (C-3), 155.22 (C-2), 163.90 (C-4/C-6), 173.42 (C-7). Elemental analysis found C-61.26, H-6.55(%), calcd. C-61.22, H-6.17(%). ESI-MS: m/z 197.1 ([M-H⁺], 100%).

Antioxidant activity

In DPPH assay, reduction of the DPPH free radicals to a DPPH-H (non-radical) form by antioxidant capable substance takes place [17, 19]. As shown in Fig. 2, IC₅₀ value of ascorbic acid on DPPH free radicals was found to be 27.0 μg/mL. Furthermore, the IC₅₀ values of 1, 2, 3, 4, 5, 6, 7, 8, 9 and RM-Ac were determined to be 88.5, 58.0, 99.5, 84.0, 40.0, 53.0, 45.0, 50.5, 56.25 and 95.0 μg/mL, respectively.

Fig. 2

IC₅₀ values of acetone extract and all compounds of Roccella montagnei against DPPH, ABTS and Superoxide free radicals

In ABTS radical assay, radical cation ABTS^·+ is decoyed [18]. As shown in Fig. 2, the IC₅₀ value of ascorbic acid on ABTS free radicals was found to be 41.0 μg/mL. Among all samples, 6 and 8 showed better IC₅₀ than that of the standard. The IC₅₀ values of Roccella montagnei samples on ABTS radical were in the order 6 (40.0 μg/mL) > 8 (40.5 μg/mL) > 5 (43.5 μg/mL) > 7 (48.25 μg/mL) > 9 (65.0 μg/mL) > 2 (67.0 μg/mL) > RM-Ac (87.0 μg/mL) > 1 (91.0 μg/mL).

Generally, the superoxide free radicals ascend from biological metabolisms interrelate with chemical species, i.e. substrates in occurrence of metallic or enzymatic catalyzed routes to produce ¹O₂ and OH radical [17, 19]. These superoxide radicals influence oxidative impairment in lipids, DNA, and, proteins. The superoxide free radical assay of all the prepared lichen samples was tabulated in Additional file 1: Table S3. As shown in Fig. 2, the concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9 and RM-Ac required for 50% reticence of superoxide free radicals were found to be 81.5, 60.0, 98.0, 99.75, 40.0, 37.25, 38.0, 36.0, 62.5 and 99.75 μg/mL, respectively, whereas standard was 35.5 μg/mL.

In vitro anti-inflammatory activity

The route cause for inflammation is biological protein denaturation, which occurs by alkaline/acidic/radiation reactions and heat treatment, etc. [2, 3]. Therefore, in the current work, the acetone extract (RM-Ac) and isolates (1–9) from Roccella montagnei were experimented for reticence of albumin protein denaturation persuaded by heat. The results of in vitro anti-inflammatory assay were tabulated in Additional file 1: Table S4, which specified that almost all isolates showed significant anti-inflammatory activity. The IC₅₀ values of 2, 5, 6, 7, 8, 9 and RM-Ac on protein denaturation were determined to be 529, 664, 435, 531, 403, 533 and 330 μg/mL, respectively, whereas standard drug (Indo) with 110 μg/mL (Fig. 3).

Fig. 3

IC₅₀ values of acetone extract and all compounds of Roccella montagnei against albumin protein denaturation

Acute toxicity studies

Acute toxicity studies of RM-Ac gave LD₅₀ as above 2 g/Kg, and the low and high dose of RM-Ac was determined to be 100 and 200 mg/Kg b.w, respectively.

In vivo anti-inflammatory activity

Based on the in vitro bioassay and toxicological studies of RM-Ac, the RM-Ac at low and high dosage, i.e. 100 and 200 mg/Kg b.w, respectively, were exposed to formalin-induced albino rat paw oedema assay against Indo at single dose, i.e. 100 mg/Kg b.w. From the outcomes of in vivo assay, it is noticed that RM-Ac showed dosage-reliant reduction of albino rat paw oedema.

The albino rats treated with lower dosage of RM-Ac showed 34.87 and 41.89% reduction of albino rat paw oedema at both intervals of time, i.e. 2 and 4 h, respectively, whereas prominent result of 40.91 and 47.12%, respectively, reduction of albino rat paw oedema was noticed in albino rats administered with higher dosage of RM-Ac (Fig. 4). From the results, it can be assumed that RM-Ac are very effective in reducing albino rat paw oedema than that of the Indo.

Fig. 4

In vivo anti-inflammatory assay of acetone extract of R. montagnei on formalin-induced hind rat paw oedema method

Anticancer activity

In general, chronic inflammation is route cause for numerous lethal disorders and diseases including cancer. Additionally, compounds (1–9) displayed better anti-inflammatory activities, so we further evaluated 1–9 and acetone extract of R. montagnei (RM-Ac) for their anti-cancer activity. Primary screening of the samples were performed at 100 μg/mL for RM-Ac, 30 μg/mL for compounds 1–9 and 10 μg/mL for standard drug, i.e. doxorubicin against MCF-7, DLD-1, HeLa, FADU and A549 cancer cells and NHME cell lines, by using SRB assay, and the % cell growth inhibition was represented in Additional file 1: Table S4. Samples that are active against cancer cell lines were further screened at different concentrations for RM-Ac (25, 50, 75 and 100 μg/mL), compounds 1–9 (5, 10, 20 and 30 μg/mL) and doxorubicin (2.5, 5.0, 7.5 and 10 μg/mL). The outcomes of percentage cell growth inhibition against concentrations are plotted to attain IC₅₀ values. The poorer IC₅₀ value directs improved inhibitory activity against cancer cells.

From the primary evaluation, RM-Ac (72.23 ± 1.75) at 100 μg/mL concentration showed equivalent inhibitory profile against DLD-1 as that of the standard drug doxorubicin (10 μg/mL concentration, 72.67 ± 0.21). Among the metabolites of RM-Ac, only 2, 6, 8 and 9 displayed reasonable degree of specificity towards experimented series of cancer cells. Moreover, RM-Ac and its metabolites showed very little degree of specificity against normal cell lines, i.e. NHME indicates non-toxic (Additional file 1: Table S4).

From Fig. 5, it was clearly evident that the RM-Ac showed more pronounced degree of specificity against DLD-1, HeLa, FADU and A549 with IC₅₀ values of 61.0, 74.5, 62.5 and 64.9 μg/mL, respectively. Further screening of the isolates obtained from this extract showed significant inhibitory profile against all the experimented cancer cells. Among all isolates, only 2 showed better IC₅₀ value of 28.20 μg/mL on MCF-7, whereas standard with 5.5 μg/mL, 2 and 9 depicted IC₅₀ value of 18.5 and 26.5 μg/mL on DLD-1, respectively, while standard with 5.4 μg/mL; 2, 6 and 8 revealed IC₅₀ value of 25.5, 27.0 and 26.5 μg/mL on HeLa, respectively, whereas standard with 4.5 μg/mL; 6 and 8 showed IC₅₀ value of 20.0 and 25.5 μg/mL on FADU and 22.5 and 27.5 μg/mL on A549, respectively, while standard with 3.8 and 6.3 μg/mL, respectively.

Fig. 5

IC₅₀ values of acetone extract and all compounds of Roccella montagnei against panel of cancer cell lines

Discussion

In present study, the chromatographic examination of acetone extract of Roccella montagnei (RM-Ac) yielded nine monoaromatic compounds (1–9) substituted with hydroxyl group(s), which are confirmed by UV, NMR, mass spectral and elemental analysis (Fig. 1). All the isolated metabolites and RM-Ac were subjected to antioxidant activity using DPPH, superoxide and ABTS free radicals, and in vitro & in vivo anti-inflammatory by using protein denaturation method and formalin-induced albino rat paw oedema bioassay, and anticancer activity using SRB assay. In addition, the acute toxicity of RM-Ac was performed on male albino rats [20].

From the antioxidant and in vitro anti-inflammatory outcomes, it can be determined that RM-Ac and samples showed prominent inhibitory capabilities against DPPH, superoxide and ABTS free radicals, and albumin protein denaturation is due to the existence of freely available oxygenated substances namely phenolics, carboxylic acids, etc. in their chemical structure (Figs. 2 and 3).

The LD₅₀ of RM-Ac was found to be above 2 g/Kg and low and high dosage i.e. 100 and 200 mg/Kg b.w, respectively RM-Ac was determined. As RM-Ac was active against in vitro anti-inflammatory assay, we prolonged the anti-inflammatory study for its in vivo model. The outcomes are denoted as the % reduction of albino rat paw oedema calculated to basal rat paw size (Fig. 4). From outcomes of in vivo assay, it is evident that RM-Ac showed dose-reliant reduction of albino rat paw oedema. In addition, higher dose of RM-Ac are extremely effective in reducing paw oedema in albino rat than that of the Indo (Fig. 4).

The illustrations of the current research proposed that the RM-Ac has pronounced anti-inflammatory capability against prolonged models of inflammation like formalin-induced assay [20], which is also used to measure anti-arthritic activity. Therefore, outcomes of current research explained the application of above lichen extract, i.e. RM-Ac in folklore support for the management of both types of inflammation, i.e. acute and chronic, and in predominantly allied with arthritis. Besides, the dominance consequence of the RM-Ac may be due to the blockade of the biosynthesis of thromboxane (TXA2), prostanoids (PGE2, PGF2α, PGD2, PGI2), and Interluekin-8 [2, 3, 20].

As chronic inflammation is route cause for several deadly diseases including cancer. Moreover, as the RM-Ac and isolates depicted better anti-inflammatory properties, we evaluated metabolites (1–9) and acetone extract of R. montagnei (RM-Ac) for their anti-cancer activity on MCF-7, DLD-1, HeLa, FADU and A549 cancer cells and NHME cells. Form the SRB study it can be determined that key agents accountable for RM-Ac were 2, 6, 8 and 9. In addition, the redox reactions (free radical production) and chronic inflammation are the key elements that cause deadly diseases like cancer (Fig. 5). By comparing the antioxidant and anti-inflammatory bioassay results, it is evident that the anticancer activity of these compounds were may be due to the presence of their antioxidant and anti-inflammatory capabilities. Moreover, RM-Ac and its metabolites showed very little degree of specificity against normal cells states non-toxic (Additional file 1: Table S4). Therefore, metabolites (1–9) can be a benchmarks for designing powerful anti-cancer agents.

Conclusion

This is a primitive study of chemical and biological evaluation of manglicolous lichen R. montagnei. Chemical investigation of acetone extract of R. montagnei results in the isolation of nine known metabolites namely divarinolmonomethylether (1), ethyl divaricatinate (2), divarinol (3), orcinol (4), methyl 2,6-dihydroxy-4-methylbenzoate (5), haematommic acid (6), atranol (7), ethyl haematommate (8) and ethyl orsellinate (9) which were confirmed by spectral data. The pharmacological evaluation revealed the inhibitory capabilities of R. montagnei against DPPH free radicals, superoxide free radicals, ABTS free radicals, albumin protein denaturation and rat paw oedema, MCF-7, HeLa, FADU, DLD-1 and A549. Moreover, key agents responsible for pharmacological activity of R. montagnei were also identified as 2, 6, 8 and 9.