Biological activities
Of the mesopelagic organisms screened, two species showed strong anticancer and antibacterial activities, the fish M. punctatum (Family Myctophidae) and the krill M. norvegica (Family Euphausiidae) (Fig. 1 shows the flowchart of the experimental procedure).
Flowchart of the experimental procedure.
Regarding the anticancer assay, M. punctatum was more active against A549 (lung) and MCF7 (breast) cells (Fig. 2a), while M. norvegica was more active against HepG2 (liver) cells (Fig. 2b). Table 2 reports values of the minimum concentration inhibiting at least half cell viability (IC50). For M. punctatum, IC50 for A549 was 13.77–23.26 while IC50 for MCF7 was 25.34–29.62 μg/mL. For M. norvegica, IC50 for HepG2 cells was between 3.81 and 7.51 μg/mL.
Percentage cell viability inhibition of A549 (lung), A2058 (skin), HepG2 (liver), MCF7 (breast) and MiaPaca-2 (pancreas) cancer cell lines after incubation for 72 h with 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 µg/mL of total extract of (a) M. punctatum and (b) M. norvegica.
Regarding antibacterial assays, both M. punctatum and M. norvegica extracts were able to inhibit the growth of methicillin resistant Staphylococcus aureus (MRSA), methicillin sensitive Staphylococcus aureus (MSSA) and Mycobacterium tuberculosis depending on the tested concentrations (Fig. 3). M. punctatum extract was able to inhibit 100% MRSA viability at extract concentrations between 40 and 320 µg/mL (Fig. 3a). At lower concentrations, the activity decreased. M. punctatum extract was also active against MSSA and M. tuberculosis, but only at the highest concentrations tested (320 and 160 µg/mL). M. norvegica extract was active against MRSA from 80–320 µg/mL, while it was active against the other bacteria only at the highest concentrations (Fig. 3b). No activity was observed against Gram-negative bacteria (i.e. Escherichia coli and Klebsiella pneumoniae).
M. punctatum (a) and M. norvegica (b) extracts tested in triplicates against methicillin resistant S. aureus (MRSA), methicillin sensitive S. aureus (MSSA) and M. tuberculosis at different concentrations (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL).
Dereplication results
Since isolation and characterisation of new compounds is a very time consuming and costly process19, dereplication by LC-UV-HRMS was performed to identify possible known compounds at an early stage using the platform available at MEDINA20. The anticancer and antibacterial activities were tested for 7 different mesopelagic fish species but only M. punctatum (Mp) and M. norvegica (Mn) displayed interesting anticancer and antibacterial activities. For this reason, the bioactive extracts selected for dereplication were Mp and Mn. A simple fractionation step using semipreparative HPLC-DAD was performed; 8 different fractions (F1 to F8) were obtained from each fish extract and each fraction was injected in the LC-UV-HRMS system.
For Mp, the most abundant components were found in fractions F4, F5 and F6 (Fig. 4). F4 had a peak with an assigned molecular formula of C20H30O2. Dereplication using the dictionary of natural product database21 (DNP) indicated that the compound had a molecular formula coincident with that of eicosapentaenoic acid (EPA, Fig. 5a). This is a known compound with an accurate mass of 302.2246, which is found in fish oil22. EPA, a well-known omega-3 fatty acid, has been reported to possess antibacterial activity against Bacillus cereus and Staphylococcus aureus with minimum inhibitory concentrations (MIC) of 64 µg/mL and 128 µg/mL, respectively23. It also displayed anticancer activity. In particular, after 72 h of incubation with EPA, lung human A549 cancer cells showed a significant reduction in cell viability24. EPA inhibited 50% of proliferation of A549 cells at 6.05 μM. Ogo et al.25 also showed a significant synergic effect when EPA was combined with paclitaxel or docetaxel on a human esophageal cancer cell line (TE-1), enhancing its antiproliferative effect.
Myctophum punctatum (Mp) LC-UV spectrum. Fractions 1 to 8 (F1 to F8) are indicated, as well as the most abundant components detected by HPLC-TOF-HRMS.
EPA (a), DHA (b) and ETA (c) structures.
F5 was a peak corresponding to the formula C22H32O2. Dereplication using the DNP database indicated that the compound had the same molecular formula as docosahexaenoic acid (DHA, Fig. 5b), an omega-3 fatty acid with an accurate mass of 328.2402. DHA is found in fish oil from several species as widely reported in the literature22. DHA has been reported to be active against breast (MDA-MB-231, MCF-7), pancreatic (MiaPaca-2) and colorectal (CaCo-2, SW-620) cancer cell lines at a range of concentrations between 10 and 100 µM26.
A molecular formula of C20H32O2 was assigned to F6, which corresponds to the compound 8,11,14,17-eicosatetraenoic acid (ETA, Fig. 5c). This is a known compound closely related to EPA (one less double bond at position 5) with an accurate mass of 304.2402. ETA is an ω-3 fatty acid naturally present in fish oils at levels of around 1–2%27. No biological activities have been reported for this compound.
F2 and F3 also contained some minor components with molecular formulae C25H38O4 (acc. mass 402.2765) and C29H40O6 (acc. mass 484.2817), most likely corresponding to polyunsaturated fatty acids (PUFAs) due to their retention time and formulae. F7 contained traces of linoleic acid (C18H32O2, acc. mass 280.2402) and 4,8,12,15,19-docosapentaenoic acid (C22H34O2, acc. mass 330.2556). Linoleic acid is an ω-6 fatty acid present in various fish species28 whose anticancer activity has been studied29,30. However, previous studies did not conclude whether or not linoleic acid is active against the cancer cells tested (including colorectal, breast and lung cancer cell lines). 4,8,12,15,19-Docosapentaenoic acid is an ω-3 fatty acid with an unusual distribution of its double bonds. It was isolated for the first time in the Japanese sardine Clupanodon melanostica31. The biological activity of this fatty acid is not reported in the literature. F1 and F8 did not show the presence of any significant components when analysed.
For Mn, the most abundant components were found in fractions 3 and 4 (Fig. 6). F3 corresponded to a peak assigned to the molecular formula C20H30O2, identical to the fraction F4 of the sample Mp and, hence, EPA. The main component of F4 was assigned the molecular formula C22H32O2, and identified as DHA, the same major component that was also present in F5 of Mp extract. F1 also contained a minor peak with a molecular formula of C24H36O4 (acc. mass 402.2765), most likely to be a PUFA due to its retention time and formula. F5 and F6 contained traces of linoleic acid (C18H32O2, acc. mass 280.2402), 4,8,12,15,19-docosapentaenoic acid (C22H34O2, acc. mass 330.2556) and 8,11,14,17-eicosatetraenoic acid (C20H32O2, acc. mass 304.2402). F2, F7 and F8 did not show any significant components by LC/MS.
M. norvegica (Mn) LC-UV spectrum. Fractions 1 to 8 (F1 to F8) are indicated, as well as the components detected by HPLC-TOF-HRMS.
Even if similar, also the combination of the various components present in the extracts of the two species may be responsible for the biological activities observed in this study. Forthcoming studies will concern the sampling of more individuals of the two active species and further chemical fractionation in order to isolate and screen each extract component. Some of the detected compounds have already displayed anticancer and/or antibacterial activity in studies reported in the literature23,24,25. Major compounds such as EPA, DHA and ETA are especially interesting for the food and supplement industries32. This is the first time that such activities have been found for these mesopelagic species and we here propose them as new abundant sources of compounds useful for human health.
Source: Ecology - nature.com
