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].
Divarinolmonomethylether
Pale yellow oil, Rf: 0.6 (Hex:DCM, 9:1), UV (λmax): 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, Rf: 0.4 (Hex:DCM, 9:1), m.p: 43–44 °C, UV (λmax): 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, Rf: 0.8 (Hex:EA, 1:1), m.p: 51–52 °C, UV (λmax): 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, Rf: 0.7 (Hex:EA, 1:1), m.p: 108–109 °C, UV (λmax): 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, Rf: 0.6 (Hex:EA, 1:1), m.p: 138–139 °C, UV (λmax): 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, Rf: 0.4 (Hex:EA, 1:1), m.p: 172–173 °C, UV (λmax): 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, Rf: 0.8 (DCM:EA, 7:3), m.p: 125–126 °C, UV (λmax): 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, Rf: 0.6 (DCM:EA, 7:3), m.p: 112–113 °C, UV (λmax): 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, Rf: 0.4 (DCM:EA, 7:3), m.p: 131–132 °C, UV (λmax): 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.
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).
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.
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.