Natural Product Research Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New cytotoxic constituents from the Red Sea soft coral Nephthea sp. Mohamed-Elamir F. Hegazy, Amira M. Gamal-Eldeen, Tarik A. Mohamed, Montaser A. Alhammady, Abuzeid A. Hassanien, Mohamed A. Shreadah, Ibrahim I. Abdelgawad, Eman M. Elkady & Paul W. Paré To cite this article: Mohamed-Elamir F. Hegazy, Amira M. Gamal-Eldeen, Tarik A. Mohamed, Montaser A. Alhammady, Abuzeid A. Hassanien, Mohamed A. Shreadah, Ibrahim I. Abdelgawad, Eman M. Elkady & Paul W. Paré (2016) New cytotoxic constituents from the Red Sea soft coral Nephthea sp., Natural Product Research, 30:11, 1266-1272, DOI: 10.1080/14786419.2015.1055266 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1055266
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Published online: 13 Jul 2015.
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Date: 07 December 2016, At: 10:23
Natural Product Research, 2016 Vol. 30, No. 11, 1266–1272, http://dx.doi.org/10.1080/14786419.2015.1055266
New cytotoxic constituents from the Red Sea soft coral Nephthea sp. Mohamed-Elamir F. Hegazya*, Amira M. Gamal-Eldeenb, Tarik A. Mohameda, Montaser A. Alhammadyc, Abuzeid A. Hassaniend, Mohamed A. Shreadahe, Ibrahim I. Abdelgawadd, Eman M. Elkadye and Paul W. Pare´f a Phytochemistry Department and Center of Excellence for Advanced Sciences, National Research Centre, 33 El Bohouth st. (Former El Tahrir st.) Dokki, Giza, P.O. Box 12622, Egypt; bCancer Biology Lab, Center of Excellence for Advanced Sciences, and Biochemistry Department, National Research Centre, 33 El Bohouth st. (Former El Tahrir st.) Dokki, Giza, P.O. Box 12622, Egypt; cNational Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada 84511, Egypt; dDepartment of Chemistry, Faculty of Science, Suez University, Egypt; eNational Institute of Oceanography & Fisheries, Alexandria, Egypt; fDepartment of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
(Received 20 March 2015; final version received 20 May 2015)
Nephthea are soft coral species rich in sesquiterpenoids and steroids. An organic extract of Nephthea sp. resulted in the isolation of a new steroid (1), as well as several previously reported metabolites (2 – 9). Structures were elucidated by employing NMR and HR-EI-MS analyses. The total extract, fractions and purified compounds exhibited differential cytotoxicity against the breast cancer MCF-7 cell line. Keywords: Nephthea sp.; terpenes; cytotoxicity; breast cancer cell line MCF-7
1. Introduction The Red Sea which is arguably the world’s warmest (up to 358C in summer) and most saline marine habitat (ca. 40 psu in the northern Red Sea) contains extensive reef formation (Edwards & Head 1987). Despite the sea’s size and diversity of reef-associated inhabitants, marine invertebrates associated with this ecosystem remain poorly studied compared to other coral reef systems around the world. The genus Nephthea (Acyonaceae, subfamily Nephtheidae) is present in the Red Sea and is a rich source of sesquiterpenoids and steroids. In addition, the genus produces metabolites that have been shown to possess diverse biological properties including cytotoxic (Duh et al. 1999; Januar et al. 2010; Liang et al. 2010), antimicrobial and antiinflammatory activities (Cheng et al. 2009). As part of the project to chemically and biologically
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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characterise metabolites from the Red Sea soft corals, steroids (1– 6), sesquiterpenes (7 and 8) and a ceramide (9) were isolated and identified from an organic extract of Nephthea sp. with isolated compounds chemically characterised by spectroscopic methods. Compound purification was guided by a breast cancer toxicity assay using cell line MCF-7 since a previous study has shown Nephthea compound efficacy against this cancer line. 2. Results and discussion A dichloromethane –methanol (1:1) extract of freshly collected Nephthea sp. was subjected to normal and reversed phase chromatography to afford nine metabolites with 1 being a newly reported natural product (Figure 1). Compound 1 was obtained as a colourless oil with an optical rotation ([a ]25 D þ 25.5 in MeOH). HR-ESI-FT-MS analysis showed a molecular ion peak at m/z 469.3649 [M þ Na]þ corresponding to a molecular formula of C29H50O3Na (calcd. 469.3651). The 1H NMR spectrum showed six methyl signals: one tertiary methyl dH 0.98 (3H, s), five secondary methyls dH 1.00 (3H, d, J ¼ 6.2 Hz), 0.80 (3H, d, J ¼ 6.2 Hz), 0.81 (3H, d, J ¼ 6.8 Hz), 0.90 (3H, d, J ¼ 6.8 Hz) and 0.93 (3H, d, J ¼ 6.2 Hz), two olefinic protons at dH 5.08 (1H, dd, J ¼ 15.1, 8.3 Hz) and 5.18 (1H, dd, J ¼ 15.1, 8.3 Hz), one oxymethine proton at dH 2.95 (1H, m) and one oxymethylene at dH 3.54 (1H, d, J ¼ 11.7 Hz) and 3.66 (1H, d, J ¼ 11.7 Hz). The 13C NMR and DEPT spectra also exhibited six methyl at dC 12.6(q), 19.9(q), 18.8(q), 19.1(q), 16.8(q) and 14.4(q); two olefinic at at dC 135.4(d), and 132.1(d), and three OH bearing carbon at dC 76.0(d), 71.5(s), and 62.1(t), which was confirmed by HR-ESI-FT-MS analysis. NMR comparisons with nebrosteroid-C, reported from Nephthea chabroli (Huang et al. 2008), indicated compound similarities with 1 except for a disappearance of an acetate group as well as a combined keto group loss/exomethylene to an endo-olefin shift. A loss of downfield H-18 signals originally observed in nebrosteroid-C occurred with a concurrent appearance of oxygenated methylene protons at dH 3.54 (d, J ¼ 11.7 Hz) and 3.66 (d, J ¼ 11.7 Hz); these methylene protons correlated with dC 38.6 (C12), 46.5 (C-13) and 57.0 (C-17) in heteronuclear multiple bond coherence (HMBC), indicating an acetate/hydroxyl group substitution in 1. The endo-olefin position was based on mutiplet signals at dH 1.80 (H-20) and 1.92 (H-24) that correlate with two olefinic protons at dH 5.08 (1H, dd, J ¼ 15.1, 8.3 Hz), 5.18 (1H, dd, J ¼ 15.1, 8.3 Hz), methyl signals at dH 0.80 (d, 6.8, 3H) and 1.00 (d, 6.8, 3H) in DQF-COSY spectrum. HMBC correlation of two methyl doublets at dH 0.80 (J ¼ 6.2 Hz, H-26) and 1.00 (J ¼ 6.2 Hz, H-21) with olefinic signals at dC 132.1 and 135.4 confirmed the double bond assignment at C-22/C-23. Postion of the secondary hydroxyl group was based on HMBC correlation between doublet signal at dH 0.93 (J ¼ 6.2 Hz, H-29) and the oxygenated carbon at dC 76.0, allowing for the assignment of C-3. Postion of the third hydroxyl group was based on correlation of multiplet signals dH 1.20 and 1.80 (H2-7) with quaternary oxygenated signal at dC 71.5, allowing for the assignment of C-8. Complete NMR assignments (Table S1) were determined by analyses of DQF-COSY, HMQC and HMBC data (Figure S1), establishing the structure of 1. The relative stereochemistry of 1 was deduced from NOESY data in which identified alpha protons as 1 and 5 correlated with H-3 allowing for the beta hydroxyl assignment at C-3. In addition, correlations between H3-19/H-4 and H3-19/H-6b indicated the b configuration of H319 and a configuration of H3-29. The coupling constant between H-22 and H-23 (J ¼ 15.1 Hz) suggested the double bond to have E configuration. From all available data, 1 was deduced to be 4a,24-dimethyl-5a-cholest-8b,18-dihydroxy, 22E-en-3b-ol (1). Compound 2 was obtained as a colourless oil with an optical rotation ([a ]25 D þ 44.0 in MeOH). HR-ESI-FT-MS analysis showed a molecular ion peak at m/z 444.3635 [M]þ corresponding to a molecular formula of C29H50O3 (calcd. 444.3539).
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3
21 28
HO
20
18
11
10
2 3
HO
4
26
16 27
15
9
O
24 25
13 14
1
23
17
12 19
22
OH
8 7
5
OH
HO
6
2
1
29
HO
HO
R
HO
3 R = OH 4 R = OAc
5 H
R
HO H HO
7 R = OH 8 R = OCH3
6 O HN
10 8
OH
OH
9
Figure 1. Structures of metabolites 1 – 9.
The 13C NMR and DEPT spectra revealed the presence of 6 methyls, 10 methylenes (one of them olefinic), 8 methines (one of them oxygenated, dC 76.0) and 5 quaternary carbons (one of them oxygenated, dC 72.7, one olefinic dC 155.9, one keto dC 203.3). NMR comparison of 2 with those of nebrosteroid-C, published by Huang et al. (2008), indicated that the disappearance of downfield oxygenated methylene protons of nebrosteroid-C and the appearance of additional methyle group in 2. HMBC correlations were observed between the carbonyl carbon signal C-23 (dC 203.3) and dH 2.00 (H-20), 2.45 (H-22), 5.95 (H-28b) and 5.71 (H-28a). In addition, H-28 signals showed long range coupling with dC 155.9 (C-24) and 27.8 (C-25). These correlations led to the
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establishment of connectivity between C23 and C25. The DQF-COSY displayed correlations between signal at dH 2.00 (H-20) and 2.45 (H-22), as well as between H-20 and dH 0.84 (H-21), suggesting a spin system containing H22 –H20 – H21. On the other hand, location of CH3-29 group was assaigned to be at C-4 which was supported by HMBC correlations between dH 0.92 (H3-29) and dC 76.0 (C-3)/51.9 (C-5). Correlation between H3-18 and H3-19 with signal at dC 72.7 allowed the assignment quaternary hydroxyl group to be at C-8. From all the above data, 2 was deduced to be 4a-methyl-3b,8b-dihydroxy-5a-ergost-24(28)-en-23-one with 13C NMR data recorded here for the first time (Bortolotto et al. 1976). In addition to compounds 1 and 2, seven known metabolites were identified as 24-methylcholesta-5,24(28)-diene-3b,7b,19-triol (3) (Bortolotto et al. 1976), 7b-acetoxy-24-methylcholesta-5,24(28)-diene-3b,19-diol (4) (Faheem et al. 2012), 24-methyl-cholesta-5,24(28)diene-3b-ol (5) (Ahmed et al. 2006), 4a,24R-dimethyl-5a-cholest-22-en-3b-ol (6) (Kokke et al. 1981), alismoxide (7) (Jin et al. 2012), 10-O-methyl alismoxide (8) (Jin et al. 2012) and erythroN-dodecanoyl-docosasphinga-(4E,8E)-dienine (9) (Bortolotto et al. 1976) by comparing NMR and MS data with literature reports. From previous studies about cytotoxicity against breast cancer MCF-7 cell line have been studies only for purified diterpenes by Januar et al. ( 2010), with a non-selective activity towards the MCF-7 cell line (GI50 . 100 mM), which encourage us to study the total extract, fractions and purified compounds against breast cancer MCF-7 cell line. As shown in Figure 2, fraction N4 produced the most potent cytotoxicity against MCF-7 cell recording the lowest IC50 ¼ 37 mg/mL among other tested fractions/compounds. The order of the activity of the rest of the samples is 3 . 4 . 8 . 2 . 1 (Table S1). It can be concluded that compounds 3 and 4 isolated from fraction N-6 have anatagonitic effect when present together as a mixture in fraction N-6, as their individual activities against the MCF-7 cell viability are more potent than the activity displayed by the N-6 fraction. It can also be concluded that compound 8 may synergise with other non-isolated compound(s) in fraction N-4. Similar weak effects (higher IC50 values) on MCF-7 cytotoxicity were produced by the rest of the tested fractions/compounds (Table S1). 3. Experimental 3.1. General experimental procedures H and 13C NMR spectra were recorded in CDCl3 or CD3OD on a JEOL ECA-600 spectrometer (600 MHz for 1H and 150 MHz for 13C). All chemical shifts (d) are given in ppm units with reference to TMS as an internal standard and coupling constants (J) are reported in Hz. HR-ESIFT-MS experiments were performed on HR-ESI-FT-MS: Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer in m/z. Purification was run on a Agilent HPLC system equipped with an Agilent-G1314 variable wavelength UV detector at 254 nm and compound separation was performed on YMC-Pack ODS-A (250 £ 4.6 mm i.d.) and (250 £ 20 mm i.d.) columns for analytical and preparative separation, respectively. Normal-phase chromatography separation included silica gel 60 (230 –400 mesh, Merck, Darmstadt, Germany). Pre-coated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F254, 0.25 mm) were used for TLC analyses. Spots were visualised by heating after spraying with 10% H2SO4.
1
3.2. Animal material Soft coral Nephthea sp. was collected from the Egyptian Red Sea off the coast of Hurghada in March 2013.The soft coral was identified by one of the authors Alhammady and a voucher specimen (03RS38) has been deposited in the National Institute of Oceanography and Fisheries, Marine Biological Station, Hurghada, Egypt.
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Figure 2. The comparable cytotoxic effect of different Nephthea products (calcutated IC50, mg/mL) on the growth of MCF-7 using MTT assay. The data represent the IC50 values calculated by means of %viability versus dose (n ¼ 4) as explained in ‘Statistical analysis’.
3.3. Extraction and separation Frozen soft coral (6.0 kg, total wet weight) was chopped into small pieces and extracted with dichloromethane – methanol (1:1) at room temperature (4 L £ 5). The combined dichloromethane– methanol extracts were concentrated in vacuo to a brown gum. The dried material (350 g) was subjected to gravity chromatography in a silica gel column (6 £ 120 cm) eluting with n-hexane (4 L) followed by a gradient of n-hexane – CH2Cl2 up to 100% CH2Cl2 and CH2Cl2 – MeOH up to 50% MeOH (4 L each of the solvent mixture) to afford nine major fractions (N-1 to N-9). The n-hexane – CH2Cl2 (1:3) fraction (N-4) (4.5 g) eluted with n-hexane – EtOAc (9:1) was subjected to silica gel column separation (100 cm £ 3 cm) to afford compounds 5 (80 mg) and 6 (70 mg). The n-hexane – CH2Cl2 (1:2) (N-3) fraction (3.0 g) eluted with n-hexane –EtOAc (8:1) was subjected to silica gel column separation (100 cm £ 3 cm) to afford 8 (45 mg). The n-hexane – CH2Cl2 (1:2) (N-6) fraction (5.5 g) eluted with n-hexane –EtOAc (6:1) was subjected to silica gel column separation (100 cm £ 3 cm) sub-fractions were collected from which five pure were obtained 2 (55 mg), 3 (35 mg) 4 (40 mg) and 9 (60 mg) and re-purified on reverse phase HPLC using MeOH – H2O (7:3 v/v) to generate compounds 1 (30 mg) and 7 (20 mg).
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3.3.1. 4a, 24-Dimethyl-5a-cholest-8b,18-dihydroxy,22E-en-3b-ol (1) Amorphous powder, 1H NMR [CD3OD] dH 1.44 (2H, m, H-6), 0.90 (3H, d, J ¼ 6.8 Hz, Me-28) 0.98 (3H, s, Me-19), 1.20, 1.80 (1H each, m, H-7), 2.95 (1H, m, H-3), 0.80 (3H, d, J ¼ 6.2 Hz, Me-26), 0.81 (3H, d, J ¼ 6.8 Hz, Me-27), 1.00 (3H, d, J ¼ 6.2, Me-21), 3.54, 3.66 (1H each, d, J ¼ 11.7 Hz, H-18). 13C NMR [CD3OD] dC 38.0 (CH2, C-1), 30.1 (CH, C-2), 76.0 (CH, C-3), 38.3 (CH2, C-4), 51.9 (C-5), 20.4 (CH, C-6), 37.5 (CH, C-7), 71.5 (CH, C-8), 56.1 (CH, C-9), 36.2 (C-10), 18.5 (CH2, C-11), 38.6 (CH2, C-12), 46.5 (C-13), 61.0 (C-14), 20.4 (CH2, C-15), 28.5 (CH2, C-16), 57.0 (CH, C-17), 62.1 (CH3, C-18), 12.6 (CH2, C-19), 40.0 (CH, C-20), 19.9 (CH3, C-21), 135.4 (CH2, C-22), 132.1 (CH2, C-23), 42.9 (C-24), 33.0 (CH, C-25), 18.8 (CH3, C26), 19.1 (CH3, C-27), 16.8 (CH2, C-28)
3.3.2. 4a-Methyl-3b, 8b-dihydroxy-5a-ergost-24(28)-en-23-one (2) Amorphous powder, 1H NMR [CD3OD] dH 1.47, 1.60 (1H each, m, H-6), 5.71, 5.98 (1H each, s, H-28), 0.98 (3H, s, Me-19), 1.70 (1H each, m, H-7), 3.02 (1H, m, H-3), 1.00 (3H, d, J ¼ 6.5 Hz, Me-26), 1.10 (3H, d, J ¼ 6.5 Hz, Me-27), 0.84 (3H, d, J ¼ 6.5, Me-21), 0.96 (3H each, s, Me18). 13C NMR [CD3OD] dC 37.7 (CH2, C-1), 30.2 (CH, C-2), 76.0 (CH, C-3), 38.3 (CH2, C-4), 51.9 (C-5), 18.8 (CH, C-6), 39.4 (CH, C-7), 72.7 (CH, C-8), 56.7 (CH, C-9), 36.2 (C-10), 18.3 (CH2, C-11), 41.4 (CH2, C-12), 43.2 (C-13), 59.9 (C-14), 20.0 (CH2, C-15), 27.8 (CH2, C-16), 57.1 (CH, C-17), 13.0 (CH3, C-18), 12.9 (CH2, C-19), 33.0 (CH, C-20), 18.8 (CH3, C-21), 44.9 (CH2, C-22), 203.3 (CH2, C-23), 155.9 (C-24), 27.8 (CH, C-25), 21.2 (CH3, C-26), 21.3 (CH3, C27),14.6 (CH2, C-28)
3.4. Anti-tumour activity Compound cytotoxicity against MCF-7 cell lines was assayed via a 3-[4,5-dimethylthiazole-2yl]-2,5-diphenyl tetrazolium bromide (MTT) spectrophotometric cell-viability assay. The MTT assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium ring of the yellow MTT to form dark-blue insoluble formazan crystals that are largely impermeable to cell membranes, resulting in its accumulation within healthy cells. With loss of cell viability, formazan crystals leak into the medium. Cell viability is monitored and quantified spectrophotometrically from formazan crystals extracted from the cells (Hansen et al. 1989). In brief, cells are diluted in serum-free media to 5.0 £ 104 cells/well based on turbidity measurements, plated into flat-bottomed 96-well microplates, and incubated for 48 h with 20 mL of test sample. After incubation, the medium is removed and 40 mL MTT (0.5 mg MTT/mL 0.9% aqueous NaCl) is added into each well and then incubated for an additional 4 h. MTT crystals are solubilised by adding 180 mL of acidified isopropanol to each well and plates are agitated at room temperature, followed by absorbance measurements at 570 nm using a microplate ELISA reader (BMG LABTECH, Ortenberg, Germany). Triplicate repeats were performed for each concentration and the average was calculated. Data are expressed as the percentage of relative viability compared with the untreated cells and with the vehicle control. Cytotoxicity was reported as a relative viability compared with the untreated control. Percentare relative viability was determined based on cell viability: (absorbance of treated cells/absorbance of control cells) £ 100.
3.5. Statistical analysis Half-maximal inhibitory concentrations (IC50) of the compounds, fractions and extracts were calculated using regression analysis from the corresponding mean of %viability versus
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concentration. r 2 values (goodness of fit) of the regression fit are presented in supplementary data. 4. Conclusion Investigation of Red Sea Nephthea sp. led to the isolation of metabolites (1– 9): a new steroid, 4a,24-dimethyl-5a-cholest-8b,18-dihydroxy,22E-en-3b-ol (1), known steroids (2– 6), known sesquiterpenoids (7 and 8) and a known ceramide (9). Supplementary material Supplementary material for this article is available at http://dx.doi.org/10.1080/14786419. 2015.1055266. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This project was supported financially by the Science and Technology Development Fund (STDF), Egypt [grant number 1102].
References Ahmed AF, Hsieh YT, Wen ZH, Wu YC, Sheu JH. 2006. Polyoxygenated sterols from the formosan soft coral Sinularia gibberosa. J Nat Prod. 69:1275 –1279. doi:10.1021/np0601509. Bortolotto M, Braekman JC, Daloze D, Losman D, Tursch. B. 1976. Chemical studies of marine invertebrates. XXIII. A novel polyhydroxylated sterol from the soft coral Litophyton viridis (Coelenterata, Octocorallia, Alcyonacea). Steroids. 28:461–466. doi:10.1016/0039-128X(76)90015-5. Cheng SY, Huang YC, Wen ZH, Hsu CH, Wang SK, Dai CF, Duh CY. 2009. New 19-oxygenated and 4-methylated steroids from the formosan soft coral Nephthea chabroli. Steroids. 74:543–547. doi:10.1016/j.steroids.2009.02. 004. Duh CY, Wang SK, Weng YL, Chiang MY, Dai CF. 1999. Cytotoxic terpenoids from the formosan soft coral Nephthea brassica. J Nat Prod. 62:1518– 1521. doi:10.1021/np990212d. Edwards AJ, Head SM. 1987. Key Environments –Red Sea. Oxford: Pergamon Press. Faheem A, Yen C, Yam WS. 2012. Chemical constituents and biological properties of the marine soft coral Nephthea sp. Trop J Pharma Res. 11:485–498. Hansen MB, Nielsen SE, Berg K. 1989. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods. 119:203 –210. doi:10.1016/0022-1759(89)90397-9. Huang YC, Wen ZH, Wang SK, Hsu CH, Duh CY. 2008. New anti-inflammatory 4-methylated steroids from the formosan soft coral Nephthea chabroli. Steroids. 73:1181–1186. doi:10.1016/j.steroids.2008.05.007. Januar HI, Chasanah E, Motti CA, Tapiolas DM, Liptrot CH, D. wright AD. 2010. Cytotoxic cembranes from Indonesian specimens of the soft coral Nephthea sp. Mar Drugs. 8:2142– 2152. doi:10.3390/md8072142. Jin H-G, Jin Q, Ryun kim AR, Choi H, Lee JH, Kim YS, Lee DG, Woo E-R. 2012. A new triterpenoid from Alisma orientale and their antibacterial effect. Arch Pharm Res. 35:1919–1926. doi:10.1007/s12272-012-1108-5. Kokke WCMC, Fenical L, Djerassi W, Djerassi C. 1981. Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates. Phytochemistry. 20:127–134. doi:10.1016/0031-9422(81)85231-4. Liang CH, Chou TH, Yang CC, Hung WJ, Chang LC, Cheng DL, Wang GH. 2010. Cytotoxic effect of Discosoma sp., Isis hippuris and Nephthea chabrolii on human oral SCC25 cells. J Taiwan Inst Chem Eng. 41:333– 337. doi:10. 1016/j.jtice.2009.09.006.
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New cytotoxic constituents from the Red Sea soft coral Nephthea sp. Mohamed-Elamir F. Hegazy, Amira M. Gamal-Eldeen, Tarik A. Mohamed, Montaser A. Alhammady, Abuzeid A. Hassanien, Mohamed A. Shreadah, Ibrahim I. Abdelgawad, Eman M. Elkady & Paul W. Paré To cite this article: Mohamed-Elamir F. Hegazy, Amira M. Gamal-Eldeen, Tarik A. Mohamed, Montaser A. Alhammady, Abuzeid A. Hassanien, Mohamed A. Shreadah, Ibrahim I. Abdelgawad, Eman M. Elkady & Paul W. Paré (2016) New cytotoxic constituents from the Red Sea soft coral Nephthea sp., Natural Product Research, 30:11, 1266-1272, DOI: 10.1080/14786419.2015.1055266 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1055266
View supplementary material
Published online: 13 Jul 2015.
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Article views: 132
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View Crossmark data
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Download by: [University of Warwick]
Date: 07 December 2016, At: 10:23
Natural Product Research, 2016 Vol. 30, No. 11, 1266–1272, http://dx.doi.org/10.1080/14786419.2015.1055266
New cytotoxic constituents from the Red Sea soft coral Nephthea sp. Mohamed-Elamir F. Hegazya*, Amira M. Gamal-Eldeenb, Tarik A. Mohameda, Montaser A. Alhammadyc, Abuzeid A. Hassaniend, Mohamed A. Shreadahe, Ibrahim I. Abdelgawadd, Eman M. Elkadye and Paul W. Pare´f a Phytochemistry Department and Center of Excellence for Advanced Sciences, National Research Centre, 33 El Bohouth st. (Former El Tahrir st.) Dokki, Giza, P.O. Box 12622, Egypt; bCancer Biology Lab, Center of Excellence for Advanced Sciences, and Biochemistry Department, National Research Centre, 33 El Bohouth st. (Former El Tahrir st.) Dokki, Giza, P.O. Box 12622, Egypt; cNational Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada 84511, Egypt; dDepartment of Chemistry, Faculty of Science, Suez University, Egypt; eNational Institute of Oceanography & Fisheries, Alexandria, Egypt; fDepartment of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
(Received 20 March 2015; final version received 20 May 2015)
Nephthea are soft coral species rich in sesquiterpenoids and steroids. An organic extract of Nephthea sp. resulted in the isolation of a new steroid (1), as well as several previously reported metabolites (2 – 9). Structures were elucidated by employing NMR and HR-EI-MS analyses. The total extract, fractions and purified compounds exhibited differential cytotoxicity against the breast cancer MCF-7 cell line. Keywords: Nephthea sp.; terpenes; cytotoxicity; breast cancer cell line MCF-7
1. Introduction The Red Sea which is arguably the world’s warmest (up to 358C in summer) and most saline marine habitat (ca. 40 psu in the northern Red Sea) contains extensive reef formation (Edwards & Head 1987). Despite the sea’s size and diversity of reef-associated inhabitants, marine invertebrates associated with this ecosystem remain poorly studied compared to other coral reef systems around the world. The genus Nephthea (Acyonaceae, subfamily Nephtheidae) is present in the Red Sea and is a rich source of sesquiterpenoids and steroids. In addition, the genus produces metabolites that have been shown to possess diverse biological properties including cytotoxic (Duh et al. 1999; Januar et al. 2010; Liang et al. 2010), antimicrobial and antiinflammatory activities (Cheng et al. 2009). As part of the project to chemically and biologically
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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characterise metabolites from the Red Sea soft corals, steroids (1– 6), sesquiterpenes (7 and 8) and a ceramide (9) were isolated and identified from an organic extract of Nephthea sp. with isolated compounds chemically characterised by spectroscopic methods. Compound purification was guided by a breast cancer toxicity assay using cell line MCF-7 since a previous study has shown Nephthea compound efficacy against this cancer line. 2. Results and discussion A dichloromethane –methanol (1:1) extract of freshly collected Nephthea sp. was subjected to normal and reversed phase chromatography to afford nine metabolites with 1 being a newly reported natural product (Figure 1). Compound 1 was obtained as a colourless oil with an optical rotation ([a ]25 D þ 25.5 in MeOH). HR-ESI-FT-MS analysis showed a molecular ion peak at m/z 469.3649 [M þ Na]þ corresponding to a molecular formula of C29H50O3Na (calcd. 469.3651). The 1H NMR spectrum showed six methyl signals: one tertiary methyl dH 0.98 (3H, s), five secondary methyls dH 1.00 (3H, d, J ¼ 6.2 Hz), 0.80 (3H, d, J ¼ 6.2 Hz), 0.81 (3H, d, J ¼ 6.8 Hz), 0.90 (3H, d, J ¼ 6.8 Hz) and 0.93 (3H, d, J ¼ 6.2 Hz), two olefinic protons at dH 5.08 (1H, dd, J ¼ 15.1, 8.3 Hz) and 5.18 (1H, dd, J ¼ 15.1, 8.3 Hz), one oxymethine proton at dH 2.95 (1H, m) and one oxymethylene at dH 3.54 (1H, d, J ¼ 11.7 Hz) and 3.66 (1H, d, J ¼ 11.7 Hz). The 13C NMR and DEPT spectra also exhibited six methyl at dC 12.6(q), 19.9(q), 18.8(q), 19.1(q), 16.8(q) and 14.4(q); two olefinic at at dC 135.4(d), and 132.1(d), and three OH bearing carbon at dC 76.0(d), 71.5(s), and 62.1(t), which was confirmed by HR-ESI-FT-MS analysis. NMR comparisons with nebrosteroid-C, reported from Nephthea chabroli (Huang et al. 2008), indicated compound similarities with 1 except for a disappearance of an acetate group as well as a combined keto group loss/exomethylene to an endo-olefin shift. A loss of downfield H-18 signals originally observed in nebrosteroid-C occurred with a concurrent appearance of oxygenated methylene protons at dH 3.54 (d, J ¼ 11.7 Hz) and 3.66 (d, J ¼ 11.7 Hz); these methylene protons correlated with dC 38.6 (C12), 46.5 (C-13) and 57.0 (C-17) in heteronuclear multiple bond coherence (HMBC), indicating an acetate/hydroxyl group substitution in 1. The endo-olefin position was based on mutiplet signals at dH 1.80 (H-20) and 1.92 (H-24) that correlate with two olefinic protons at dH 5.08 (1H, dd, J ¼ 15.1, 8.3 Hz), 5.18 (1H, dd, J ¼ 15.1, 8.3 Hz), methyl signals at dH 0.80 (d, 6.8, 3H) and 1.00 (d, 6.8, 3H) in DQF-COSY spectrum. HMBC correlation of two methyl doublets at dH 0.80 (J ¼ 6.2 Hz, H-26) and 1.00 (J ¼ 6.2 Hz, H-21) with olefinic signals at dC 132.1 and 135.4 confirmed the double bond assignment at C-22/C-23. Postion of the secondary hydroxyl group was based on HMBC correlation between doublet signal at dH 0.93 (J ¼ 6.2 Hz, H-29) and the oxygenated carbon at dC 76.0, allowing for the assignment of C-3. Postion of the third hydroxyl group was based on correlation of multiplet signals dH 1.20 and 1.80 (H2-7) with quaternary oxygenated signal at dC 71.5, allowing for the assignment of C-8. Complete NMR assignments (Table S1) were determined by analyses of DQF-COSY, HMQC and HMBC data (Figure S1), establishing the structure of 1. The relative stereochemistry of 1 was deduced from NOESY data in which identified alpha protons as 1 and 5 correlated with H-3 allowing for the beta hydroxyl assignment at C-3. In addition, correlations between H3-19/H-4 and H3-19/H-6b indicated the b configuration of H319 and a configuration of H3-29. The coupling constant between H-22 and H-23 (J ¼ 15.1 Hz) suggested the double bond to have E configuration. From all available data, 1 was deduced to be 4a,24-dimethyl-5a-cholest-8b,18-dihydroxy, 22E-en-3b-ol (1). Compound 2 was obtained as a colourless oil with an optical rotation ([a ]25 D þ 44.0 in MeOH). HR-ESI-FT-MS analysis showed a molecular ion peak at m/z 444.3635 [M]þ corresponding to a molecular formula of C29H50O3 (calcd. 444.3539).
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3
21 28
HO
20
18
11
10
2 3
HO
4
26
16 27
15
9
O
24 25
13 14
1
23
17
12 19
22
OH
8 7
5
OH
HO
6
2
1
29
HO
HO
R
HO
3 R = OH 4 R = OAc
5 H
R
HO H HO
7 R = OH 8 R = OCH3
6 O HN
10 8
OH
OH
9
Figure 1. Structures of metabolites 1 – 9.
The 13C NMR and DEPT spectra revealed the presence of 6 methyls, 10 methylenes (one of them olefinic), 8 methines (one of them oxygenated, dC 76.0) and 5 quaternary carbons (one of them oxygenated, dC 72.7, one olefinic dC 155.9, one keto dC 203.3). NMR comparison of 2 with those of nebrosteroid-C, published by Huang et al. (2008), indicated that the disappearance of downfield oxygenated methylene protons of nebrosteroid-C and the appearance of additional methyle group in 2. HMBC correlations were observed between the carbonyl carbon signal C-23 (dC 203.3) and dH 2.00 (H-20), 2.45 (H-22), 5.95 (H-28b) and 5.71 (H-28a). In addition, H-28 signals showed long range coupling with dC 155.9 (C-24) and 27.8 (C-25). These correlations led to the
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establishment of connectivity between C23 and C25. The DQF-COSY displayed correlations between signal at dH 2.00 (H-20) and 2.45 (H-22), as well as between H-20 and dH 0.84 (H-21), suggesting a spin system containing H22 –H20 – H21. On the other hand, location of CH3-29 group was assaigned to be at C-4 which was supported by HMBC correlations between dH 0.92 (H3-29) and dC 76.0 (C-3)/51.9 (C-5). Correlation between H3-18 and H3-19 with signal at dC 72.7 allowed the assignment quaternary hydroxyl group to be at C-8. From all the above data, 2 was deduced to be 4a-methyl-3b,8b-dihydroxy-5a-ergost-24(28)-en-23-one with 13C NMR data recorded here for the first time (Bortolotto et al. 1976). In addition to compounds 1 and 2, seven known metabolites were identified as 24-methylcholesta-5,24(28)-diene-3b,7b,19-triol (3) (Bortolotto et al. 1976), 7b-acetoxy-24-methylcholesta-5,24(28)-diene-3b,19-diol (4) (Faheem et al. 2012), 24-methyl-cholesta-5,24(28)diene-3b-ol (5) (Ahmed et al. 2006), 4a,24R-dimethyl-5a-cholest-22-en-3b-ol (6) (Kokke et al. 1981), alismoxide (7) (Jin et al. 2012), 10-O-methyl alismoxide (8) (Jin et al. 2012) and erythroN-dodecanoyl-docosasphinga-(4E,8E)-dienine (9) (Bortolotto et al. 1976) by comparing NMR and MS data with literature reports. From previous studies about cytotoxicity against breast cancer MCF-7 cell line have been studies only for purified diterpenes by Januar et al. ( 2010), with a non-selective activity towards the MCF-7 cell line (GI50 . 100 mM), which encourage us to study the total extract, fractions and purified compounds against breast cancer MCF-7 cell line. As shown in Figure 2, fraction N4 produced the most potent cytotoxicity against MCF-7 cell recording the lowest IC50 ¼ 37 mg/mL among other tested fractions/compounds. The order of the activity of the rest of the samples is 3 . 4 . 8 . 2 . 1 (Table S1). It can be concluded that compounds 3 and 4 isolated from fraction N-6 have anatagonitic effect when present together as a mixture in fraction N-6, as their individual activities against the MCF-7 cell viability are more potent than the activity displayed by the N-6 fraction. It can also be concluded that compound 8 may synergise with other non-isolated compound(s) in fraction N-4. Similar weak effects (higher IC50 values) on MCF-7 cytotoxicity were produced by the rest of the tested fractions/compounds (Table S1). 3. Experimental 3.1. General experimental procedures H and 13C NMR spectra were recorded in CDCl3 or CD3OD on a JEOL ECA-600 spectrometer (600 MHz for 1H and 150 MHz for 13C). All chemical shifts (d) are given in ppm units with reference to TMS as an internal standard and coupling constants (J) are reported in Hz. HR-ESIFT-MS experiments were performed on HR-ESI-FT-MS: Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer in m/z. Purification was run on a Agilent HPLC system equipped with an Agilent-G1314 variable wavelength UV detector at 254 nm and compound separation was performed on YMC-Pack ODS-A (250 £ 4.6 mm i.d.) and (250 £ 20 mm i.d.) columns for analytical and preparative separation, respectively. Normal-phase chromatography separation included silica gel 60 (230 –400 mesh, Merck, Darmstadt, Germany). Pre-coated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F254, 0.25 mm) were used for TLC analyses. Spots were visualised by heating after spraying with 10% H2SO4.
1
3.2. Animal material Soft coral Nephthea sp. was collected from the Egyptian Red Sea off the coast of Hurghada in March 2013.The soft coral was identified by one of the authors Alhammady and a voucher specimen (03RS38) has been deposited in the National Institute of Oceanography and Fisheries, Marine Biological Station, Hurghada, Egypt.
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Figure 2. The comparable cytotoxic effect of different Nephthea products (calcutated IC50, mg/mL) on the growth of MCF-7 using MTT assay. The data represent the IC50 values calculated by means of %viability versus dose (n ¼ 4) as explained in ‘Statistical analysis’.
3.3. Extraction and separation Frozen soft coral (6.0 kg, total wet weight) was chopped into small pieces and extracted with dichloromethane – methanol (1:1) at room temperature (4 L £ 5). The combined dichloromethane– methanol extracts were concentrated in vacuo to a brown gum. The dried material (350 g) was subjected to gravity chromatography in a silica gel column (6 £ 120 cm) eluting with n-hexane (4 L) followed by a gradient of n-hexane – CH2Cl2 up to 100% CH2Cl2 and CH2Cl2 – MeOH up to 50% MeOH (4 L each of the solvent mixture) to afford nine major fractions (N-1 to N-9). The n-hexane – CH2Cl2 (1:3) fraction (N-4) (4.5 g) eluted with n-hexane – EtOAc (9:1) was subjected to silica gel column separation (100 cm £ 3 cm) to afford compounds 5 (80 mg) and 6 (70 mg). The n-hexane – CH2Cl2 (1:2) (N-3) fraction (3.0 g) eluted with n-hexane –EtOAc (8:1) was subjected to silica gel column separation (100 cm £ 3 cm) to afford 8 (45 mg). The n-hexane – CH2Cl2 (1:2) (N-6) fraction (5.5 g) eluted with n-hexane –EtOAc (6:1) was subjected to silica gel column separation (100 cm £ 3 cm) sub-fractions were collected from which five pure were obtained 2 (55 mg), 3 (35 mg) 4 (40 mg) and 9 (60 mg) and re-purified on reverse phase HPLC using MeOH – H2O (7:3 v/v) to generate compounds 1 (30 mg) and 7 (20 mg).
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3.3.1. 4a, 24-Dimethyl-5a-cholest-8b,18-dihydroxy,22E-en-3b-ol (1) Amorphous powder, 1H NMR [CD3OD] dH 1.44 (2H, m, H-6), 0.90 (3H, d, J ¼ 6.8 Hz, Me-28) 0.98 (3H, s, Me-19), 1.20, 1.80 (1H each, m, H-7), 2.95 (1H, m, H-3), 0.80 (3H, d, J ¼ 6.2 Hz, Me-26), 0.81 (3H, d, J ¼ 6.8 Hz, Me-27), 1.00 (3H, d, J ¼ 6.2, Me-21), 3.54, 3.66 (1H each, d, J ¼ 11.7 Hz, H-18). 13C NMR [CD3OD] dC 38.0 (CH2, C-1), 30.1 (CH, C-2), 76.0 (CH, C-3), 38.3 (CH2, C-4), 51.9 (C-5), 20.4 (CH, C-6), 37.5 (CH, C-7), 71.5 (CH, C-8), 56.1 (CH, C-9), 36.2 (C-10), 18.5 (CH2, C-11), 38.6 (CH2, C-12), 46.5 (C-13), 61.0 (C-14), 20.4 (CH2, C-15), 28.5 (CH2, C-16), 57.0 (CH, C-17), 62.1 (CH3, C-18), 12.6 (CH2, C-19), 40.0 (CH, C-20), 19.9 (CH3, C-21), 135.4 (CH2, C-22), 132.1 (CH2, C-23), 42.9 (C-24), 33.0 (CH, C-25), 18.8 (CH3, C26), 19.1 (CH3, C-27), 16.8 (CH2, C-28)
3.3.2. 4a-Methyl-3b, 8b-dihydroxy-5a-ergost-24(28)-en-23-one (2) Amorphous powder, 1H NMR [CD3OD] dH 1.47, 1.60 (1H each, m, H-6), 5.71, 5.98 (1H each, s, H-28), 0.98 (3H, s, Me-19), 1.70 (1H each, m, H-7), 3.02 (1H, m, H-3), 1.00 (3H, d, J ¼ 6.5 Hz, Me-26), 1.10 (3H, d, J ¼ 6.5 Hz, Me-27), 0.84 (3H, d, J ¼ 6.5, Me-21), 0.96 (3H each, s, Me18). 13C NMR [CD3OD] dC 37.7 (CH2, C-1), 30.2 (CH, C-2), 76.0 (CH, C-3), 38.3 (CH2, C-4), 51.9 (C-5), 18.8 (CH, C-6), 39.4 (CH, C-7), 72.7 (CH, C-8), 56.7 (CH, C-9), 36.2 (C-10), 18.3 (CH2, C-11), 41.4 (CH2, C-12), 43.2 (C-13), 59.9 (C-14), 20.0 (CH2, C-15), 27.8 (CH2, C-16), 57.1 (CH, C-17), 13.0 (CH3, C-18), 12.9 (CH2, C-19), 33.0 (CH, C-20), 18.8 (CH3, C-21), 44.9 (CH2, C-22), 203.3 (CH2, C-23), 155.9 (C-24), 27.8 (CH, C-25), 21.2 (CH3, C-26), 21.3 (CH3, C27),14.6 (CH2, C-28)
3.4. Anti-tumour activity Compound cytotoxicity against MCF-7 cell lines was assayed via a 3-[4,5-dimethylthiazole-2yl]-2,5-diphenyl tetrazolium bromide (MTT) spectrophotometric cell-viability assay. The MTT assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium ring of the yellow MTT to form dark-blue insoluble formazan crystals that are largely impermeable to cell membranes, resulting in its accumulation within healthy cells. With loss of cell viability, formazan crystals leak into the medium. Cell viability is monitored and quantified spectrophotometrically from formazan crystals extracted from the cells (Hansen et al. 1989). In brief, cells are diluted in serum-free media to 5.0 £ 104 cells/well based on turbidity measurements, plated into flat-bottomed 96-well microplates, and incubated for 48 h with 20 mL of test sample. After incubation, the medium is removed and 40 mL MTT (0.5 mg MTT/mL 0.9% aqueous NaCl) is added into each well and then incubated for an additional 4 h. MTT crystals are solubilised by adding 180 mL of acidified isopropanol to each well and plates are agitated at room temperature, followed by absorbance measurements at 570 nm using a microplate ELISA reader (BMG LABTECH, Ortenberg, Germany). Triplicate repeats were performed for each concentration and the average was calculated. Data are expressed as the percentage of relative viability compared with the untreated cells and with the vehicle control. Cytotoxicity was reported as a relative viability compared with the untreated control. Percentare relative viability was determined based on cell viability: (absorbance of treated cells/absorbance of control cells) £ 100.
3.5. Statistical analysis Half-maximal inhibitory concentrations (IC50) of the compounds, fractions and extracts were calculated using regression analysis from the corresponding mean of %viability versus
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concentration. r 2 values (goodness of fit) of the regression fit are presented in supplementary data. 4. Conclusion Investigation of Red Sea Nephthea sp. led to the isolation of metabolites (1– 9): a new steroid, 4a,24-dimethyl-5a-cholest-8b,18-dihydroxy,22E-en-3b-ol (1), known steroids (2– 6), known sesquiterpenoids (7 and 8) and a known ceramide (9). Supplementary material Supplementary material for this article is available at http://dx.doi.org/10.1080/14786419. 2015.1055266. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This project was supported financially by the Science and Technology Development Fund (STDF), Egypt [grant number 1102].
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