Laurencia is a common genus of marine red algae, taxonomically classified as Rhodophyta, Rhodophyceae, Ceramiales, Rhodomelaceae. Laurencia obtusa is one of the most widely investigated marine species by natural product chemists. It provides a unique source of halogenated secondary metabolites such as C15-acetogenises , diterpenes, and sesquiterpenoid skeletons [1, 2, 3, 4, 5, 6, 7, 8, 9]. Many of these compounds are biologically active, showing antioxidant, antimalarial, antimicrobial, and cytotoxic activity [3,6,10].
To find new bioactive compounds in marine macroorganisms, Laurencia obtusa Lamouroux was collected from the Saudi Red Sea and its organic fraction extracted to provide four new sesquiterpenoids (Figure 1). The antimicrobial and anticandidal activities of the new compounds were also investigated.
Column chromatography was performed with aluminum oxide Fluka, neutral type 507C. Fractions were analyzed by? TLC on silica gel 60 F254. Preparative TLC was performed on glass plates (20 cm × 20 cm) coated with silica gel (250μm thickness). Spots were visualized using UV light (254 nm), then p-anisaldehyde-sulfuric acid spray reagent. High resolution electron impact mass spectra (HREIMS) were recorded on a Krators EIMS-25 instrument at an ionizing voltage of 70 eV. 1D and 2D NMR spectra were recorded on a Bruker 850 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) using the solvent residual signal as the internal standard (CDCl3: δ 7.26 for 1H and δ 77.0 for 13C).
2.2 Extraction and Isolation
Laurencia obtusa Lamouroux was collected in May 2016 from Salman Gulf, north of Jeddah, Saudi Arabia (21°51′39.8″ N; 38°58′42.7″ E). Reference standard (JAD 03060) was stored at the Faculty of Marine Sciences, King Abdulaziz University (Jeddah, Saudi Arabia). L. obtusa was dried, then extracted with equal volumes of dichloromethane and methanol. The residue (6 g) was purified first by column chromatography on Sephadex LH-20 (MeOH/CHCl3 = 9.5:0.5), then by column chromatography on neutral aluminum oxide using gradient elution (n-hexane/diethyl ether to n-hexane/EtOAc). Fractions containing the product were combined and dried under reduced pressure. Final purification by PTLC afforded the pure product.
2.3 Chemical characterization
purified by column chromatography (eluent: 30% diethyl ether) followed by PTLC (eluent: 30% diethyl ether). The violet band (p-anisaldehyde-sulfuric acid reagent) was collected to provide 1 as a colorless liquid (1.4 mg, 0.0007% yield). Rf 0.30; [α]D +54.0 (c 0.014, CH2Cl2); IR ν (cm−1) 3424, 2924, 2854, 1712, 1640, 1455, 1377, 1261, 1172, 1074, 904; EI-MS m/z 236; HREIMS m/z 236.1764 (Calcd. 236.1776, C15H24O2); 1H and 13C NMR (Tables 1 and 2, respectively).
purified by column chromatography (eluent: 15% diethyl ether) followed by PTLC (eluent: 10% diethyl ether). The brown band (p-anisaldehyde-sulfuric acid reagent) was collected to provide 2 as a pale yellow liquid (4.5 mg, 0.0023% yield). Rf 0.53; [α]D +79.0 (c 0.023, CH2,Cl2); UV (MeOH) λmax 252, 259 and 290 nm; IR ν (cm−1) 3274, 2972, 2927, 1701, 1606, 1461, 1392, 1369, 1223, 1178, 1075, 854; EI-MS m/z 232; HREIMS m/z 232.1451 (Calcd. 232.1463, C15H20O2); 1H and 13C NMR (Tables 1 and 2, respectively).
purified by column chromatography (eluent: 40% diethyl ether) followed by PTLC (eluent: 30% diethyl ether). The light blue band (p-anisaldehyde-sulfuric acid reagent) was collected to provide 3 as a colorless liquid (0.7 mg, 0.0004% yield). Rf 0.4; [α]D +61.8 (c 0.007, CH2Cl2); UV (MeOH) λmax 230 and 280 nm; IR ν (cm−1) 3383, 2921, 2851, 1736, 1605, 1463, 1377, 1286, 1239, 1183, 1075, 832; EI-MS m/z 220; HREIMS m/z 220.1451 (Calcd. 220.1463, C14H20O2); 1H and 13C NMR (Tables 1 and 2, respectively).
purified by column chromatography (eluent: 40% diethyl ether) followed by PTLC (eluent: 30% diethyl ether). The light blue band (p-anisaldehyde-sulfuric acid reagent) was collected to provide 4 as a colorless liquid (0.7 mg, 0.0004% yield). Rf 0.4; [α]D +50.4 (c 0.007, CH2,Cl2); UV (MeOH) λmax 230 and 280 nm; IR ν (cm−1) 3383, 2921, 2851, 1736, 1605, 1463, 1377, 1286, 1239, 1183, 1075, 832, 557; HREIMS m/z 298.0556 and 300.0536 (1:1) (Calcd. 298.0568 and 300.0548 for C14H1979BrO2 and C14H1981BrO2, respectively); 1H and 13C NMR (Tables 1 and 2, respectively).
2.4 Biological evaluation
2.4.1 Antibacterial activity
The antibacterial activity was tested on Muller-Hinton agar using agar diffusion well test . The minimum inhibitory concentration (MIC) was determined by ELISA using 96-well plates and fluorescein diacetate (FDA, 5 μl of a 0.25% w/w in acetone) as an indicator. The green color due to FDA hydrolysis was estimated at λmax 490 nm using the ELISA tray reader [13,14].
Compound toxicity was assessed using Artemisia salina as test creature and dimethylsulfoxide (DMSO) as a negative control .
3 Results and discussion
Four new sesquiterpenoids (1–4) were isolated from Laurencia obtusa Lamouroux after extraction and purification of its organic fraction. Spectroscopic analysis of the four compounds afforded the following results.
Compound 1 was isolated as a liquid with specific rotation [α]D +54 (c 0.014, CH2Cl2). HREIMS analysis of the molecular ion peak at m/z 236.1764 provided the molecular formula C15H24O2, which requires four degrees of unsaturation. The lack of absorption in the UV spectrum revealed the absence of conjugation. Hydroxyl group (ν 3424 cm−1) and exocyclic C=C bond (ν 1640 cm−1) were assigned from the IR spectrum. The 13C NMR spectrum displayed 15 signals, which were categorized by DEPT into 5 quaternary, 8 methylene and 2 methyl carbons. HSQC experiment indicated the presence of two tertiary methyls (δH/δC = 1.82/18.9 and 0.93/22.2), two oxygenated quaternary carbons (δC 78.3 and 75.0), two olefinic quaternary carbons (δC 151.6 and 150.9), and two exocyclic methylene groups (δH/δC 5.06 and 4.84/108.2; δH/δC 5.04 and 4.83/109.6; Tables 1 and 2). The 1H─1H correlation experiment highlighted three sequences: three correlated methylene groups, two correlated methylene groups, and an isolated methylene group (δH 1.50 and 2.25). Overall, these results suggest that compound 1 is an eudesmane-type sesquiterpenoid with two C=C bonds fulfilling four degrees of unsaturation. HMBC experiment revealed additional correlations: the methyl protons at δH 0.93 (Me-14) correlate to carbons at δC 78.3, 34.2 and 31.6, confirming that Me-14 is an angular methyl; methyl protons at δH 1.82 (Me-13) correlate to carbons at δC 150.9, 109.6 and 75.0, confirming that Me-13 is part of the isopropylidene group (Figure 2). The relative configuration of 1 was assigned by NOESY experiment as well as by analogy with known compounds. The absence of correlation between Me-13 and Me-14 in NOESY spectrum suggests the two groups are not co-facially oriented. As in natural eudesmanes Me-14 is β-oriented,7 Me-14 and OH group at C-5 must be trans correlated. Based on structure, 1 was named eudesma-4(15),11-diene-5,7-diol.
Compound 2 was isolated as a liquid with specific rotation [α]D +79.2 (c 0.025, CH2Cl2). HREIMS analysis of the molecular ion peak (m/z 232) provided the molecular formula C15H20O2, which requires six degrees of unsaturation. The UV absorptions at 252, 259 and 290 nm indicated a substituted benzene ring. The IR spectrum showed the characteristic bands of OH, C=O and gem dimethyl groups (ν 3274, 1701 and 1369 cm−1, respectively). The presence of the OH group was confirmed by a peak at m/z 214 [M–H2O] in the EI-MS spectrum. The 13C NMR spectrum of 2 showed 15 signals, categorized by DEPT into 4 quaternary, 6 methine, 2 methylene and 3 methyl carbons. The 1H─13C HSQC experiment revealed the presence of the following groups (Tables 1 and 2): three aliphatic methyl groups (singlets at δH/δC 1.48/25.1, 1.13/21.1 and 0.62/22.6), a formyl group (singlet at δH/δC 9.99/191.8), and a p-disubstituted benzene ring (two doublets in the aromatic region at δH/δC 7.79 [J = 8.5 Hz, 2H]/128.9 and δH 7.39 [J = 8.5 Hz, 2H]/128.2). In addition, the proton-proton correlation spectrum revealed two proton sequences: the aromatic system and a CH2─CH2─CH fragment. The aforementioned analysis points to a cuparane-type sesquiterpenoid structure containing a cyclopentane ring connected to a benzaldehyde skeleton. The location of the aldehyde group in the p-position of the benzene ring was confirmed by HMBC correlation of the aldehyde proton (H-15, δH 9.99) to C-3 (δC 134.4), C-2 (δC 128.9), and C-4 (δC 128.9). Correlations of H-14 (δH 1.48) to C-7 (δC 48.8), C-11 (δC 49.6), C-8 (δC 36.5), and C-6 (δC 154.5) are also in agreement with the structure of 2.
Comparison of the spectral data of 2 with those reported for cuparane  showed similarity, except for the presence of an aldehyde group in 2 in the place of a methyl group in cuparane. The increase in chemical shift value of Me-14 in compound 2 (δH 1.48) with respect to cuparane (δH 1.23) is likely explained in terms of anisotropy created by the aromatic ring as well as the electron withdrawing effect of the aldehyde group. The relative configuration of 2 was determined by NOESY experiment, which showed the correlation of Me-14 (δH 1.48) to Me-12 (δH 1.13), and of Me-13 (δH 0.62) to H-10 (δH 4.01). These correlations indicate that Me-14, Me-12 and the hydroxyl group on C-10 have the same orientation. Consequently, the relative configurations of C-7 and C-10 were assigned as 7R* and 10R*, respectively. Compound 2 was given the trivial name 10-hydroxycuparaldehyde.
Metabolite 3 was isolated as a liquid with specific rotation [α]D +61.8 (c 0.007, CH2Cl2). Its molecular formula was determined as C14H20O2 (with five degrees of unsaturation) by HREIMS. The UV absorptions at 230 and 280 nm indicated a substituted benzene ring. The IR spectrum revealed the presence of OH and gem dimethyl groups (ν 3383 and 1377 cm−1, respectively). The 13C NMR spectrum of 3 showed 14 signals, categorized by DEPT into 4 quaternary, 5 methine, 2 methylene and 3 methyl carbons. The 1H─13C HSQC spectrum allowed to identify three tertiary methyls (singlets at δH/δC 1.41/25.2, 1.08/20.9 and 0.61/22.5) and a p-disubstituted benzene ring (two doublets in the aromatic region at δH/δC 6.74 [2H, J = 8.5 Hz]/114.2 and at δH/δC 7.07 [2H, J = 8.5 Hz]/128.7; Tables 1 and 2). Two OH groups are located on C-3 (δC 153.6) and C-10 δH/δC 4.04/62.0). In addition, the proton-proton correlation spectrum revealed two proton sequences: the aromatic system and the CH2─CH2─CH fragment. With five degrees of unsaturation, the carbon skeleton of compound 3 must contain a (p-OH)C6H4 group attached to a 1,2,2-trimethylcyclopentan-3-ol moiety. The position of the hydroxyl groups was confirmed by HMBC correlation of the phenolic carbon C-3 (δC 153.6) to the aromatic protons H-2 and H-4 (δH 6.74), and that of H-10 (δH 4.04) to C-11 (δC 48.3) and C-9 (δC 33.1).
The abovementioned analysis indicates that compound 3 is a nor-cuparane-type sesquiterpenoid. Compound 3 was named 3-hydroxy-15-nor-cuparan-10β-ol.
Compound 4 (isolated as a liquid with specific rotation [α]D +50.4 [c 0.007, CH2Cl2]) was identified by comparison with the spectroscopic data of 3 (Tables 1 and 2). Their IR, UV and NMR spectra were similar, with a difference in the aromatic region of the 1H NMR spectrum. The 1H─13C HSQC spectrum of 4 exhibited three methine carbons in the aromatic region δC/δC 7.14 [d, J = 2.6 Hz]/127.9, 6.92 [d, J = 8.5 Hz]/115.1 and 7.02 [dd, J = 8.5 Hz, J = 2.6 Hz]/127.6) and three quaternary carbons (δC 140.6, 119.1, and 149.4) indicating a 1,2,4-trisubstituted benzene ring. The molecular formula of 4 was assigned as C14H19BrO2 by HREIMS. The parent peaks at m/z 298 and 300 in the EI-MS spectrum, with relative intensities in the 1:1 ratio, alongside the absorption band at ν 557 cm−1 in the IR spectrum, clearly indicate the presence of a bromine atom.
The analysis indicates that compound 4 is a nor-cuparane bearing two hydroxyl groups and a bromine atom. The position of bromine was assigned to C-2 (δC 119.1). 4 was named 2-bromo-3-hydroxy-15-nor-cuparan-10β-ol.
The antibacterial activity of 1 and 2 was tested against several strains of multidrug-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus). 1 displayed weak activity against all tested bacteria (results not shown), whereas 2 showed an inhibition zone of 10.0-15.5 mm diameter on Muller-Hinton agar (Table 3).
The MICs of 2 were in the range of 0.08–0.15 mM for all tested bacteria. Noteworthy, 2 was more active against Gram-positive bacteria (Table 3). All compounds showed no toxicity (LD50 >0.5 mM) against Artemia salina as test creature.
Compound 1 displayed anticandidal activity against Candida albicans and Candida tropicalis (MICs = 8.27 and 10.13 μM, respectively. This activity is relatively high if compared to amphotericin B (positive control; MICs = 4.63 and 5.27 μM, respectively).
Four new compounds were isolated from the organic extract of the red alga L. obtusa Lamouroux. These compounds belong to two major classes of sesquiterpenoids: eudesmanes [eudesma-4(15),11-diene-5,7-diol (1)] and cuparanes [10-hydroxycuparaldehyde (2), 3-hydroxy-15-nor-cuparan-10β-ol (3), and 2-bromo-3-hydroxy-15-nor-cuparan-10β-ol (4)]. Compounds 1 and 2 displayed relatively high antimicrobial activities, whereas 3 and 4 belong to a specific group of compounds (nor-cuparane sesquiterpenoids) that are rarely isolated from the marine environment.
The authors are indebted to Prof. Mohsen El-Sherbiny, Faculty of Marine Sciences (King Abdulaziz University) for the collection and identification of the alga sample. They also thank King Fahd Center for Medical Research for giving them the opportunity to work in its central laboratory.
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About the article
Published Online: 2017-10-22
Conflict of interest: Authors declare no conflict of interest.
Citation Information: Open Chemistry, Volume 15, Issue 1, Pages 219–224, ISSN (Online) 2391-5420, DOI: https://doi.org/10.1515/chem-2017-0025.
© 2017 Nahed O. Bawakid et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0