Arch. Pharm. Res. DOI 10.1007/s12272-015-0620-9
RESEARCH ARTICLE
A new 9,11-secosterol with a 1,4-quinone from a Korean marine sponge Ircinia sp. Inho Yang1 • Hyukjae Choi2 • Sang-Jip Nam3 • Heonjoong Kang1,4
Received: 28 January 2015 / Accepted: 26 May 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract An intensive investigation of chemical components for Ircinia sp. led to the isolation of a new 9,11secosterol. The chemical structure of this compound was elucidated based on the interpretation of NMR and MS spectroscopic data. The isolated compound exhibited antibacterial activities against Staphylococcus epidermidis and Bacillus subtilis with MIC values of 6.3 and 25 lg ml-1, respectively. Keywords Secosterol Sponge Ircinia Antibacterial Marine natural product
Inho Yang and Hyukjae Choi have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-015-0620-9) contains supplementary material, which is available to authorized users. & Sang-Jip Nam [email protected] & Heonjoong Kang [email protected] 1
Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Korea
2
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea
3
Department of Chemistry and Nano Science, Global Top5 Program, Ewha Womans University, Seoul 120-750, Korea
4
Research Institute of Oceanography, Seoul National University, NS-80, Seoul 151-747, Korea
Introduction A seco-steroid is a structural class of modified steroid with a cleavage on one of the four rings in the basic steroid structure. Several structure subtypes of seco-steroids such as the 9,10-seco-steroid, 13,14-seco-steroid, and 9,11-secosteroid have been identified in nature. Vitamin D is a representative of 9,10-seco-steroid that is frequently found in diverse living organisms including mammals (USDA 2011), fish (USDA 2011), and mushrooms (Mattila et al. 1994). They have been reported to have physiological functions on bone growth and remodeling (Matsumoto et al. 2014), cancer (Davis 2008), cardiovascular disease (Norman and Powell 2014), and inflammation (Yin and Agrawal 2014). Likewise, 13,14-secosteroids have been isolated from terrestrial plants (Bhandari et al. 2006; Kirson et al. 1976; Qiu et al. 2008) and some of which have been demonstrated to possess anti-inflammatory activities (Qiu et al. 2008). 9,11-Secosterols, the third group of secosteroids, are very rare in terrestrial organisms, and are instead of found primarily in marine invertebrates, mainly corals (Huang et al. 2012; Cheng et al. 2011; Chen et al. 2011a, b; Ioannou et al. 2009; Minh et al. 2007; Epifanio et al. 2007; Su et al. 2006; Anta et al. 2002; Aknin et al. 1998; Morris et al. 1998; He et al. 1995; Lopp et al. 1994; Koljak et al. 1993; Ochi et al. 1991; Fusetani et al. 1989; Bonini et al. 1983) and sponges (Van Altena et al. 1999; De Rosa et al. 1999; Rueda et al. 1998; Lu and Faulkner 1997; Reddy et al. 1997; Adinolfi et al. 1994; Li et al. 1994; Migliuolo et al. 1992, Migliuolo et al. 1991; Capon and Faulkner 1985). In addition, 9,11-secosterols have been reported to display diverse bioactivities including cytotoxicity against cancer cell lines (Huang et al. 2012; Cheng et al. 2011; Chen et al. 2011a, b; Su et al. 2006; Morris et al. 1998; Rueda et al. 1998), anti-proliferative effects
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against cancer cells (Ioannou et al. 2009; He et al. 1995; Lopp et al. 1994; Koljak et al. 1993), stimulation of bone formation (Minh et al. 2007), fish feeding deterrence for coral and coral-associated algae (Epifanio et al. 2007), protein kinase C inhibition (He et al. 1995), anti-inflammatory activity (Huang et al. 2012; He et al. 1995), brine shrimp toxicity (Ochi et al. 1991; De Rosa et al. 1999), inhibition of starfish embryo cell division (Fusetani et al. 1989), antifungal activity (Li et al. 1994), and ichthyotoxicity (Capon and Faulkner 1985). During the chemical and pharmacological investigation on the extract of Korean marine invertebrates, anti-bacterial activity was observed from an extract of Ircinia sp., of which a 9,11-secosterol was identified as an anti-bacterial component (Yang et al. 2014). An intensive chemical investigation of the residual extract led us to the purification of a new 9,11-secosterol. Herein, we report the isolation, structure elucidation and evaluation of the anti-bacterial activities of a new 9,11-secosterol with a 1,4-quinone moiety from the Korean marine sponge Ircinia sp.
Materials and methods General experimental procedures Optical rotation was measured using a Rudolph Research Autopol III polarimeter with a 5 cm cell. The UV spectrum was recorded in a Scinco UVS-2100 with a path length of 1 cm. Infrared spectra were recorded on a Thermo Electron Corporation spectrometer. NMR spectral spectroscopic data were obtained using Bruker Avance 600 MHz spectrometer [CDCl3 (dH 7.26; dC 77.0) was used as an internal standard]. HRFAB-MS data were measured on a JEOL, JMS-AX505WA mass spectrometer. Animal material The genus Ircinia sp. sponge was collected by SCUBA off the-shore of Yeongdeok-gun in the East Sea, and immediately frozen on dry ice. Extraction and isolation The wet animal tissue (3 kg) was extracted three times with 50 % methanol (MeOH) in dichloromethane. These extracts were concentrated and partitioned three times between n-hexane and MeOH. Next, the MeOH-soluble layer was partitioned three times between ethylacetate (EtOAc) and water. The EtOAc-soluble layer (7.0 g) was subjected to silica flash column chromatography using step-gradient elution of EtOAc in hexanes (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 %) to afford eleven fractions (Fr 1–Fr 11).
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Fr 10 (78.1 mg) was further purified by reversed-phase ˚ , UV detecHPLC (Polar-RP, 250 9 10 mm, 5 lm, 80 A tion = 210 nm, flow rate 2.0 ml min-1), eluting with 70 % acetonitrile in H2O to afford compound 1 (1.7 mg), as a colorless oil. Compound 1 Colorless oil; [a]21 D = -5.0° (0.002, CHCl3); UV (MeOH) kmax (log e) 274 (3.96) nm; IR (film) mmax 3422, 2951, 2851, 1718, 1681, 1464 cm-1; 1H NMR data, see Table 1; 13 C NMR data, see Table 1; LRFABMS m/z 461 [M?H]?; HRFABMS m/z 461.3280 [M?H]? (calcd for C28H45O5, 461.3267). Antibacterial assay A total of seven bacterial strains were used: four Grampositive strains (Staphylococcus epidermidis ATCC 12228, Micrococcus lutes ATCC 9341, Bacillus subtilis ATCC 6633, S. aureus ATCC 65381) and three Gram-negative strains (Escherichia coli ATCC 11,775, Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 4352). These bacteria were inoculated in Mueller–Hinton agar media for 24 h at 37 °C. The bacterial colony was cultivated into a 15 ml round tube containing 6 ml of Mueller–Hinton broth media at 37 °C and 225 rpm for 24 h. Test compound (1) and a positive control (gentamicin) dissolved in DMSO were added to a final volume of 100 ll each in a 96-well microtiter plate containing 50 ll of Mueller– Hinton broth. The samples were serially diluted and 50 ll of bacterial Mueller–Hinton broth media was adjusted to the concentration of 1/100 diluted McFarland 0.5 % standard. The 96-well was then incubated for 24 h at 37 °C. Then, the minimum inhibitory concentration (MIC) values were determined as the concentration of compounds at transparent well, which inhibits the growth of bacteria.
Results and discussion The molecular formula of 1 was deduced as C28H44O5 from the HRFABMS data (a pseudomolecular ion peak at m/ z 461.3280 [M?H]?) and from the interpretation of 13C NMR data. The 1H NMR spectrum of 1 showed an oxygenated methine proton [dH 4.04 (m)], three olefinic proton [dH 6.49 (br s), 5.22 (dd, J = 6.9, 1.2), 5.20 (dd, J = 6.9, 1.2)], and two protons in one downfielded methylenes [dH 3.86 (m). 3.71 (m)]. The 1H NMR spectrum also displayed two methyl singlets (dH 1.22, 0.70) and four methyl doublets [dH 1.03 (d, J = 6.8 Hz), 0.91 (d, J = 6.8 Hz), 0.83 (d, J = 6.8 Hz), 0.81 (d, J = 6.8 Hz)]. The 13C NMR and HSQC spectroscopic data analysis revealed six methyls,
A new 9,11-secosterol with a 1,4-quinone from a Korean marine sponge Ircinia sp Table 1 NMR spectroscopic data of 1a (CDCl3)
No.
dC, mb
1
25.9, t
dH, m, J (Hz)
COSY
HMBC (10 Hz)
1.77 m
2
3, 5, 10, 19
1, 3
–
4.04 m
2, 4
–
1.77 dd (11.2, 11.1)
3
2, 5, 10
2.20 dt (11.2, 3.3) 2
29.9, t
1.55 m 2.00 m
3
66.6, d
4
35.7, t
2.16 dd (11.2, 3.3) 5
80.5, s
–
–
–
6
197.5c, s
–
–
–
7
134.4, d
6.49, s
–
5, 6, 8, 9, 14
8
152.1, s
–
–
–
9
203.1c, s
–
–
–
10
52.1, s
–
–
–
11
59.9, t
3.71 m
12
–
12
41.1, t
3.86 m 1.15 m
11
–
13
47.4, s
–
–
–
14
44.0, d
3.52 dd (11.0, 8.5)
15
7, 8, 9, 12, 13, 15, 18
15
25.9, t
1.75 m
14, 16
–
15, 17
–
1.73 m
1.84 m 16
26.6, t
1.68 m 1.75 m
17
50.2, d
1.73 m
16, 20
–
18
18.8, q
0.70 s
–
12, 13, 14, 17
19
20.6, q
1.22 s
–
1, 5, 9, 10
20
36.6, d
1.41 m
17, 21, 22
–
21
17.8, q
1.03 d (6.8)
20
17, 20, 22
22
133.3, d
5.22, dd (6.9, 1.2)
20, 23
24
23
134.2, d
5.20, dd (6.9, 1.2)
22, 24
–
24 25
43.0, d 33.1, d
1.81 m 1.51 m
23, 25, 28 24, 26, 27
– 23
26
19.6, q
0.81 d (6.8)
25
24, 25, 27,
27
20.0, q
0.83 d (6.8)
25
24, 25, 26
28
17.8, q
0.91 d (6.8)
24
23, 24, 25
a
1
600 MHz for H NMR and 150 MHz for
b
Multiplicity was determined by the interpretation of 2D NMR spectroscopic data
c
Chemical shifts were assigned from the interpretation of HMBC correlations
seven methylenes, eight methines, and six fully-substituted carbons. Analysis of 2D NMR spectroscopic data allowed the structure assignment of 1 to be established as shown in Fig. 1. COSY crosspeaks on two sets of spin systems, [H-1/ H-2/H-3/H-4] and [H-11/H-12], established two fragments with a four-carbon unit (a) and a two-carbon unit (b), respectively. Additional COSY corrections on [H-14/H-15/ H-16/H-17/H-20], [H-21/H-20/H-22/H-23/H-24/H-28], and [H-24/H-25/H-26, H-27] provided a fragment with a thirteen-carbon unit (c) as shown in Fig. 2. The establishment of the 1,4-quinone moiety was determined by the HMBC
13
C NMR
correlations from an olefinic proton (H-7) to fully substituted carbons (C-6, C-8, and C-9) and by the carbon chemical shifts of C-6 (dC 197.5), C-7 (dC 134.4), C-8 (dC 152.1), and C-9 (dC 203.1). The connectivity of the three fragments (a, b, and c) and the 1,4-quinone moiety was constructed from HMBC correlations. The long-range HMBC correlations from H-19 to C-1, C-5, C-9, and C-10, and from H-4 to C-2, C-5, and C-10, and from H-7 to C-5 allowed the connection of A/B ring with the fragment (a) and the 1,4-quinone moiety. The connection of the two-carbon unit (b) and the thirteen-
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HO
21
11
O 12
19 1
HO
10 5
3
18
8
H
6
HO
O
13 14
HO
22
21
11
20 24
17 28
19
25 1
15
HO
5
3
HO
1
18
O 12
10
8 6
22
13 14
H
O
20 17
15
2
Fig. 1 Chemical structure of compound 1 and known derivative 2
HO b O
Table 2 Antibacterial activity of 1
c
HO
O
COSY HMBC
Fig. 2 COSY (bold lines) and key HMBC (arrows) correlations of 1
carbon unit (c) were also secured from the interpretation of HMBC correlations. A two-bond HMBC correlation from a methyl singlet proton H-18 to a carbon C-13, and threebond HMBC correlations from H-18 to carbons C-12, C-17 allowed the connectivity of C-12/C-13/C-17. Lastly, threebond HMBC correlations from the olefinic proton H-7 to a carbon C-14, and from the methyl singlet proton H-18 to a carbon C-14 allowed the connectivity of C-8/C-14, which completed structure assignment of 1. The relative stereochemistry of the side chain and rings of 1 was established from comparison with NMR data of previously reported secosterols and from the interpretation of NOESY correlations (Reddy et al. 1997; Lu and Faulkner 1997). NOESY correlations [H-7/H-14, H-14/H12, H-12/H-21, H-18/H-20] were well matched to the NOE correlations of reported secosterols (Anta et al. 2002). The a-configuration of the 5-hydroxy group at C-5 was proposed from the comparison of the 13C data to those of a synthetic analog (dC 80.3 for 5a-carbon, dC 81.9 for 5bcarbon, Kovganko and Chernov 2001) and previously reported steroids (dC 80.8–80.9, Rho et al. 2000). The bconfiguration of the 3-hydroxy group at C-3 was also defined from the coupling constants of H-4a [dH 2.16 (dd, J = 11.2, 3.3 Hz)] and NOESY correlations [H3/H4a, H4b/H19]. The geometry of the double bond at C-22 was determined as Z based on the coupling constants of H-22 and H-23 (J = 6.9 Hz), however, we were unable to establish the configuration of C-24.
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Gentamicina
(MIC, lg ml-1)
a HO
1a
Strain
Staphylococcus epidermidis ATCC 12228
6.3
Micrococcus lutes ATCC 9341
100
0.2 3.1
Bacillus subtilis ATCC 6633
25
0.2
S. aureus ATCC 65381
[200
0.2
Escherichia coli ATCC 11775
[200
0.8
Salmonella typhimurium ATCC 14028
[200
1.6
Klebsiella pneumonia ATCC 4352
[200
0.8
a
Each experiment was replicated three times
We previously isolated a secosterol (2) that was structurally related to compound 1 (Yang et al. 2014). As the previously identified steroid (2) exhibited antibacterial activities, compound 1 was also investigated for its antibacterial activity against seven pathogens (Table 2). Compound 1 was found to display antibacterial activities on Staphylococcus epidermidis ATCC 12228 and Bacillus subtilis ATCC 6633 with MIC values of 6.3 and 25 lg ml-1, respectively. Interestingly, 1 exhibited weak activity against Micrococcus lutes ATCC 9341, whereas 2 displayed potent activity. Acknowledgments This work was supported by Suncheon Research Center for Natural Medicines. I. Yang was in part supported by the BK21 program, Ministry of Education, Korea. Conflict of interest of interest.
The authors declare that they have no conflict
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A new 9,11-secosterol with a 1,4-quinone from a Korean marine sponge Ircinia sp Aknin, M., V. Costantino, A. Mangoni, E. Fattorusso, and E.M. Gaydou. 1998. New 9,11-secosterols from gorgonian Subergorgia suberosa of the Indian Ocean. Steroids 63: 575–578. Anta, C., N. Gonza´lez, J. Rodrı´guez, and C. Jime´nez. 2002. A new secosterol from the Indonesian octocoral Pachyclavularia violacea. Journal of Natural Products 65(9): 1357–1359. Bhandari, P., N. Kumar, B. Singh, and V.K. Kaul. 2006. Bacosterol glycoside, a new 13,14-seco-steroid glycoside from Bacopa monnieri. Chemical & Pharmaceutical Bulletin 54(2): 240–241. Bonini, C., C.B. Cooper, R. Kazlauskas, R.J. Wells, and C. Djerassi. 1983. Minor and trace sterols in marine invertebrates. 41. Structure and stereochemistry of naturally occurring 9,11-seco sterols. Journal of Organic Chemistry 48: 2108–2111. Capon, R.J., and D.J. Faulkner. 1985. Herbasterol, an ichthyotoxic 9,11-secosterol from the sponge Dysidea herbacea. Journal of Organic Chemistry 58: 4771–4773. Chen, B.-W., S.-M. Chang, C.-Y. Huang, J.-H. Su, Z.-H. Wen, Y.-C. Wu, and J.-H. Sheu. 2011a. Hirsutosterols A–G, polyoxygenated steroids from a Formosan soft coral Cladiella hirsuta. Organic & Biomolecular Chemistry 9: 3272–3278. Cheng, S.-Y., H.-P. Chen, S.-K. Wang, and C.-Y. Duh. 2011b. Three new 9,11-secosterols from the Formosan soft coral Sinularia leptoclados. Bulletin of the Chemical Society Japan 84(6): 648–652. Davis, C.D. 2008. Vitamin D and cancer: current dilemmas and future research needs. The American Journal of Clinical Nutrition 88: 565S–569S. De Rosa, S., A. De Giulio, A. Crisping, C. Iodice, and G. Tommonaro. 1999. New 9,11-secosterol from the Tyrrhenian sponge Fasciospongia cavernosa. Natural Product Letters 13(1): 15–20. Epifanio, R., L. Maia, J. Pawlik, and W. Fenical. 2007. Antipredatory secosterols from the octocoral Pseudopterogorgia americana. Marine Ecology Progress Series 329: 307–310. Fusetani, N., H. Nagata, H. Hirota, and T. Tsuyuki. 1989. Astrogorgiadiol and astrogorgin, inhibitors of cell division in fertilized starfish eggs, from a gorgonian Astrogorgia sp. Tetrahedron Letters 30(50): 7079–7082. He, H., P. Kulanthaivel, B.J. Baker, K. Kalter, J. Darges, D. Cofield, L. Wolff, and L. Adams. 1995. New antiproliferative and antiinflammatory 9,11-secosterols from the gorgonian Pseudopterogorgia sp. Tetrahedron 51(1): 51–58. Huang, C.-Y., J.-H. Su, C.-Y. Duh, B.-W. Chen, Z.-H. Wen, Y.-H. Kuo, and J.-H. Sheu. 2012. A new 9,11-secosterol from the soft coral Sinularia granosa. Bioorganic & Medicinal Chemistry Letters 22(13): 4373–4376. Ioannou, E., A.F. Abdel-Razik, X. Alexi, C. Vagias, M.N. Alexis, and V. Roussis. 2009. 9,11-secosterols with antiproliferative activity from the gorgonian Eunicella cavolini. Bioorganic & Medicinal Chemistry 17(13): 4537–4541. Kirson, I., Z. Zaretskii, and E. Glotter. 1976. Withaphysalin C, a naturally occurring 13,14-seco-steroid. Journal of the Chemical Society, Perkin Transactions 1: 1244–1247. Koljak, R., T. Pehk, I. Jarving, M. Liiv, A. Lopp, K. Varvas, A. Vahemets, U. Lille, and N. Samel. 1993. New antiproliferative 9,11-secosterol from soft coral Gersemia fruticosa. Tetrahedron Letters 34(12): 1985–1986. Kovganko, N., and Y. Chernov. 2001. 13C NMR spectra of 6-hydroximinosteroids of the stigmastane series. Chemistry of Natural Compounds 37(4): 351–355. Li, H., S. Matsunaga, and N. Fusetani. 1994. A new 9,11-secosterol, stellettasterol from a marine sponge Stelletta sp. Experientia 50: 771–773. Lopp, A., A. Pihlak, H. Paves, K. Samuel, and R. Koljak. 1994. The effect of 9,11-secosterol, a newly discovered compound from the soft coral Gersemia fruticosa, on the growth and cell cycle
progression of various tumor cells in culture. Steroids 59: 274–281. Lu, Q., and D.J. Faulkner. 1997. Two new sesterterpenoids and a new 9,11-secosterol from Spongia matamata. Journal of Natural Products 60: 195–198. Matsumoto, T., T. Takano, H. Saito, and F. Takahashi. 2014. Vitamin D analogs and bone: Preclinical and clinical studies with eldecalcitol. BoneKEy Reports 3: 513. Mattila, P.H., V.I. Piironen, E.J. Uusi-rauva, and P.E. Koivistoinen. 1994. Vitamin D contents in edible mushrooms. Journal of Agriculture and Food Chemistry 42: 2449–2453. Migliuolo, A., V. Piccialli, and D. Sica. 1991. Structure elucidation and synthesis of 3b,6a-dihydroxy-9-oxo-9,11-seco-5a-cholest-7en-11-al, a novel 9,11-secosterol from the sponge Spongia officinalis. Tetrahedron 47(37): 7937–7950. Migliuolo, A., V. Piccialli, and D. Sica. 1992. Two new 9,11secosterols from the marine sponge Spongia oficinalis. Synthesis of 9,11-seco-3b,6a,11-trihydroxy-5a-cholest-7-en-9-one. Steroids 57: 344–347. Minh, C.V., N.X. Cuong, T.A. Tuan, E.M. Choi, Y.H. Kim, and P.V. Kiem. 2007. A new 9,11-secosterol from the Vietnamese Sea soft coral, Sarcophyton mililatensis, increases the function of osteoblastic MC3T3-E1 cells. Natural Product Communications 2(11): 1095–1100. Morris, L.A., E.M. Christie, M. Jaspars, and L.P. Van Ofwegen. 1998. A bioactive secosterol with an unusual A- and B-ring oxygenation pattern isolated from an Indonesian soft coral Lobophytum sp. Journal of Natural Products 61: 538–541. Norman, P.E., and J.T. Powell. 2014. Vitamin D and cardiovascular disease. Circulation Research 114(2): 379–393. Ochi, M., K. Yamada, H. Kotsuki, and K. Shibata. 1991. Calicoferols A and B, two novel secosterols possessing brine-shrimp lethality from the gorgonian Calicogorgia sp. Chemistry Letters 20(3): 427–430. Qiu, L., F. Zhao, Z.-H. Jiang, L.-X. Chen, Q. Zhao, H.-X. Liu, X.-S. Yao, and F. Qiu. 2008. Steroids and flavonoids from Physalis alkekengi var. franchetii and their inhibitory effects on nitric oxide production. Journal of Natural Products 71: 642–646. Reddy, M.V., M.K. Harper, and D.J. Faulkner. 1997. Luffasterols A– C, 9,11-secosterols from the Palauan sponge Luffariella sp. Journal of Natural Products 60(1): 41–43. Rho, J.-R., H.-S. Lee, Y. Seo, K.W. Cho, and J. Shing. 2000. New bioactive steroids from the gorgonian Acalycigorgia inermis. Bulletin of the Korean Chemical Society 21: 518–520. Rueda, A., E. Zubia, M.J. Ortega, J.L. Carballo, and J. Salva. 1998. New metabolites from the sponge Spongia agaricina. Journal of Natural Products 61: 258–261. Su, J.-H., Y.-J. Tseng, H.-H. Huang, A.F. Ahmed, C.-K. Lu, Y.-C. Wu, and J.-H. Sheu. 2006. 9,11-Secosterols from the soft corals Sinularia lochmodes and Sinularia leptoclados. Journal of Natural Products 69: 850–852. USDA (U.S. Department of Agriculture), Agricultural Research Service. 2011. USDA National Nutrient Database for Standard Reference, Release 24. Nutrient Data Laboratory home page. http://www.ars.usda.gov/ba/bhnrc/ndl. Accessed 5 Jan 2015. Van Altena, I.A., A.J. Butler, and S.J. Dunne. 1999. A new cyclized 9,11-secosterol enol-ether from the Australian sponge Euryspongia arenaria. Journal of Natural Products 62: 1154–1157. Yang, I., H. Choi, D.H. Won, S.-J. Nam, and H. Kang. 2014. An antibacterial 9,11-secosterol from a marine sponge Ircinia sp. Bulletin of the Korean Chemical Society 35(11): 3360–3362. Yin, K., and D.K. Agrawal. 2014. Vitamin D and inflammatory diseases. Journal of Inflammation Research 2014: 69–87.
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RESEARCH ARTICLE
A new 9,11-secosterol with a 1,4-quinone from a Korean marine sponge Ircinia sp. Inho Yang1 • Hyukjae Choi2 • Sang-Jip Nam3 • Heonjoong Kang1,4
Received: 28 January 2015 / Accepted: 26 May 2015 Ó The Pharmaceutical Society of Korea 2015
Abstract An intensive investigation of chemical components for Ircinia sp. led to the isolation of a new 9,11secosterol. The chemical structure of this compound was elucidated based on the interpretation of NMR and MS spectroscopic data. The isolated compound exhibited antibacterial activities against Staphylococcus epidermidis and Bacillus subtilis with MIC values of 6.3 and 25 lg ml-1, respectively. Keywords Secosterol Sponge Ircinia Antibacterial Marine natural product
Inho Yang and Hyukjae Choi have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12272-015-0620-9) contains supplementary material, which is available to authorized users. & Sang-Jip Nam [email protected] & Heonjoong Kang [email protected] 1
Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University, NS-80, Seoul 151-747, Korea
2
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Korea
3
Department of Chemistry and Nano Science, Global Top5 Program, Ewha Womans University, Seoul 120-750, Korea
4
Research Institute of Oceanography, Seoul National University, NS-80, Seoul 151-747, Korea
Introduction A seco-steroid is a structural class of modified steroid with a cleavage on one of the four rings in the basic steroid structure. Several structure subtypes of seco-steroids such as the 9,10-seco-steroid, 13,14-seco-steroid, and 9,11-secosteroid have been identified in nature. Vitamin D is a representative of 9,10-seco-steroid that is frequently found in diverse living organisms including mammals (USDA 2011), fish (USDA 2011), and mushrooms (Mattila et al. 1994). They have been reported to have physiological functions on bone growth and remodeling (Matsumoto et al. 2014), cancer (Davis 2008), cardiovascular disease (Norman and Powell 2014), and inflammation (Yin and Agrawal 2014). Likewise, 13,14-secosteroids have been isolated from terrestrial plants (Bhandari et al. 2006; Kirson et al. 1976; Qiu et al. 2008) and some of which have been demonstrated to possess anti-inflammatory activities (Qiu et al. 2008). 9,11-Secosterols, the third group of secosteroids, are very rare in terrestrial organisms, and are instead of found primarily in marine invertebrates, mainly corals (Huang et al. 2012; Cheng et al. 2011; Chen et al. 2011a, b; Ioannou et al. 2009; Minh et al. 2007; Epifanio et al. 2007; Su et al. 2006; Anta et al. 2002; Aknin et al. 1998; Morris et al. 1998; He et al. 1995; Lopp et al. 1994; Koljak et al. 1993; Ochi et al. 1991; Fusetani et al. 1989; Bonini et al. 1983) and sponges (Van Altena et al. 1999; De Rosa et al. 1999; Rueda et al. 1998; Lu and Faulkner 1997; Reddy et al. 1997; Adinolfi et al. 1994; Li et al. 1994; Migliuolo et al. 1992, Migliuolo et al. 1991; Capon and Faulkner 1985). In addition, 9,11-secosterols have been reported to display diverse bioactivities including cytotoxicity against cancer cell lines (Huang et al. 2012; Cheng et al. 2011; Chen et al. 2011a, b; Su et al. 2006; Morris et al. 1998; Rueda et al. 1998), anti-proliferative effects
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against cancer cells (Ioannou et al. 2009; He et al. 1995; Lopp et al. 1994; Koljak et al. 1993), stimulation of bone formation (Minh et al. 2007), fish feeding deterrence for coral and coral-associated algae (Epifanio et al. 2007), protein kinase C inhibition (He et al. 1995), anti-inflammatory activity (Huang et al. 2012; He et al. 1995), brine shrimp toxicity (Ochi et al. 1991; De Rosa et al. 1999), inhibition of starfish embryo cell division (Fusetani et al. 1989), antifungal activity (Li et al. 1994), and ichthyotoxicity (Capon and Faulkner 1985). During the chemical and pharmacological investigation on the extract of Korean marine invertebrates, anti-bacterial activity was observed from an extract of Ircinia sp., of which a 9,11-secosterol was identified as an anti-bacterial component (Yang et al. 2014). An intensive chemical investigation of the residual extract led us to the purification of a new 9,11-secosterol. Herein, we report the isolation, structure elucidation and evaluation of the anti-bacterial activities of a new 9,11-secosterol with a 1,4-quinone moiety from the Korean marine sponge Ircinia sp.
Materials and methods General experimental procedures Optical rotation was measured using a Rudolph Research Autopol III polarimeter with a 5 cm cell. The UV spectrum was recorded in a Scinco UVS-2100 with a path length of 1 cm. Infrared spectra were recorded on a Thermo Electron Corporation spectrometer. NMR spectral spectroscopic data were obtained using Bruker Avance 600 MHz spectrometer [CDCl3 (dH 7.26; dC 77.0) was used as an internal standard]. HRFAB-MS data were measured on a JEOL, JMS-AX505WA mass spectrometer. Animal material The genus Ircinia sp. sponge was collected by SCUBA off the-shore of Yeongdeok-gun in the East Sea, and immediately frozen on dry ice. Extraction and isolation The wet animal tissue (3 kg) was extracted three times with 50 % methanol (MeOH) in dichloromethane. These extracts were concentrated and partitioned three times between n-hexane and MeOH. Next, the MeOH-soluble layer was partitioned three times between ethylacetate (EtOAc) and water. The EtOAc-soluble layer (7.0 g) was subjected to silica flash column chromatography using step-gradient elution of EtOAc in hexanes (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 %) to afford eleven fractions (Fr 1–Fr 11).
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Fr 10 (78.1 mg) was further purified by reversed-phase ˚ , UV detecHPLC (Polar-RP, 250 9 10 mm, 5 lm, 80 A tion = 210 nm, flow rate 2.0 ml min-1), eluting with 70 % acetonitrile in H2O to afford compound 1 (1.7 mg), as a colorless oil. Compound 1 Colorless oil; [a]21 D = -5.0° (0.002, CHCl3); UV (MeOH) kmax (log e) 274 (3.96) nm; IR (film) mmax 3422, 2951, 2851, 1718, 1681, 1464 cm-1; 1H NMR data, see Table 1; 13 C NMR data, see Table 1; LRFABMS m/z 461 [M?H]?; HRFABMS m/z 461.3280 [M?H]? (calcd for C28H45O5, 461.3267). Antibacterial assay A total of seven bacterial strains were used: four Grampositive strains (Staphylococcus epidermidis ATCC 12228, Micrococcus lutes ATCC 9341, Bacillus subtilis ATCC 6633, S. aureus ATCC 65381) and three Gram-negative strains (Escherichia coli ATCC 11,775, Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 4352). These bacteria were inoculated in Mueller–Hinton agar media for 24 h at 37 °C. The bacterial colony was cultivated into a 15 ml round tube containing 6 ml of Mueller–Hinton broth media at 37 °C and 225 rpm for 24 h. Test compound (1) and a positive control (gentamicin) dissolved in DMSO were added to a final volume of 100 ll each in a 96-well microtiter plate containing 50 ll of Mueller– Hinton broth. The samples were serially diluted and 50 ll of bacterial Mueller–Hinton broth media was adjusted to the concentration of 1/100 diluted McFarland 0.5 % standard. The 96-well was then incubated for 24 h at 37 °C. Then, the minimum inhibitory concentration (MIC) values were determined as the concentration of compounds at transparent well, which inhibits the growth of bacteria.
Results and discussion The molecular formula of 1 was deduced as C28H44O5 from the HRFABMS data (a pseudomolecular ion peak at m/ z 461.3280 [M?H]?) and from the interpretation of 13C NMR data. The 1H NMR spectrum of 1 showed an oxygenated methine proton [dH 4.04 (m)], three olefinic proton [dH 6.49 (br s), 5.22 (dd, J = 6.9, 1.2), 5.20 (dd, J = 6.9, 1.2)], and two protons in one downfielded methylenes [dH 3.86 (m). 3.71 (m)]. The 1H NMR spectrum also displayed two methyl singlets (dH 1.22, 0.70) and four methyl doublets [dH 1.03 (d, J = 6.8 Hz), 0.91 (d, J = 6.8 Hz), 0.83 (d, J = 6.8 Hz), 0.81 (d, J = 6.8 Hz)]. The 13C NMR and HSQC spectroscopic data analysis revealed six methyls,
A new 9,11-secosterol with a 1,4-quinone from a Korean marine sponge Ircinia sp Table 1 NMR spectroscopic data of 1a (CDCl3)
No.
dC, mb
1
25.9, t
dH, m, J (Hz)
COSY
HMBC (10 Hz)
1.77 m
2
3, 5, 10, 19
1, 3
–
4.04 m
2, 4
–
1.77 dd (11.2, 11.1)
3
2, 5, 10
2.20 dt (11.2, 3.3) 2
29.9, t
1.55 m 2.00 m
3
66.6, d
4
35.7, t
2.16 dd (11.2, 3.3) 5
80.5, s
–
–
–
6
197.5c, s
–
–
–
7
134.4, d
6.49, s
–
5, 6, 8, 9, 14
8
152.1, s
–
–
–
9
203.1c, s
–
–
–
10
52.1, s
–
–
–
11
59.9, t
3.71 m
12
–
12
41.1, t
3.86 m 1.15 m
11
–
13
47.4, s
–
–
–
14
44.0, d
3.52 dd (11.0, 8.5)
15
7, 8, 9, 12, 13, 15, 18
15
25.9, t
1.75 m
14, 16
–
15, 17
–
1.73 m
1.84 m 16
26.6, t
1.68 m 1.75 m
17
50.2, d
1.73 m
16, 20
–
18
18.8, q
0.70 s
–
12, 13, 14, 17
19
20.6, q
1.22 s
–
1, 5, 9, 10
20
36.6, d
1.41 m
17, 21, 22
–
21
17.8, q
1.03 d (6.8)
20
17, 20, 22
22
133.3, d
5.22, dd (6.9, 1.2)
20, 23
24
23
134.2, d
5.20, dd (6.9, 1.2)
22, 24
–
24 25
43.0, d 33.1, d
1.81 m 1.51 m
23, 25, 28 24, 26, 27
– 23
26
19.6, q
0.81 d (6.8)
25
24, 25, 27,
27
20.0, q
0.83 d (6.8)
25
24, 25, 26
28
17.8, q
0.91 d (6.8)
24
23, 24, 25
a
1
600 MHz for H NMR and 150 MHz for
b
Multiplicity was determined by the interpretation of 2D NMR spectroscopic data
c
Chemical shifts were assigned from the interpretation of HMBC correlations
seven methylenes, eight methines, and six fully-substituted carbons. Analysis of 2D NMR spectroscopic data allowed the structure assignment of 1 to be established as shown in Fig. 1. COSY crosspeaks on two sets of spin systems, [H-1/ H-2/H-3/H-4] and [H-11/H-12], established two fragments with a four-carbon unit (a) and a two-carbon unit (b), respectively. Additional COSY corrections on [H-14/H-15/ H-16/H-17/H-20], [H-21/H-20/H-22/H-23/H-24/H-28], and [H-24/H-25/H-26, H-27] provided a fragment with a thirteen-carbon unit (c) as shown in Fig. 2. The establishment of the 1,4-quinone moiety was determined by the HMBC
13
C NMR
correlations from an olefinic proton (H-7) to fully substituted carbons (C-6, C-8, and C-9) and by the carbon chemical shifts of C-6 (dC 197.5), C-7 (dC 134.4), C-8 (dC 152.1), and C-9 (dC 203.1). The connectivity of the three fragments (a, b, and c) and the 1,4-quinone moiety was constructed from HMBC correlations. The long-range HMBC correlations from H-19 to C-1, C-5, C-9, and C-10, and from H-4 to C-2, C-5, and C-10, and from H-7 to C-5 allowed the connection of A/B ring with the fragment (a) and the 1,4-quinone moiety. The connection of the two-carbon unit (b) and the thirteen-
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HO
21
11
O 12
19 1
HO
10 5
3
18
8
H
6
HO
O
13 14
HO
22
21
11
20 24
17 28
19
25 1
15
HO
5
3
HO
1
18
O 12
10
8 6
22
13 14
H
O
20 17
15
2
Fig. 1 Chemical structure of compound 1 and known derivative 2
HO b O
Table 2 Antibacterial activity of 1
c
HO
O
COSY HMBC
Fig. 2 COSY (bold lines) and key HMBC (arrows) correlations of 1
carbon unit (c) were also secured from the interpretation of HMBC correlations. A two-bond HMBC correlation from a methyl singlet proton H-18 to a carbon C-13, and threebond HMBC correlations from H-18 to carbons C-12, C-17 allowed the connectivity of C-12/C-13/C-17. Lastly, threebond HMBC correlations from the olefinic proton H-7 to a carbon C-14, and from the methyl singlet proton H-18 to a carbon C-14 allowed the connectivity of C-8/C-14, which completed structure assignment of 1. The relative stereochemistry of the side chain and rings of 1 was established from comparison with NMR data of previously reported secosterols and from the interpretation of NOESY correlations (Reddy et al. 1997; Lu and Faulkner 1997). NOESY correlations [H-7/H-14, H-14/H12, H-12/H-21, H-18/H-20] were well matched to the NOE correlations of reported secosterols (Anta et al. 2002). The a-configuration of the 5-hydroxy group at C-5 was proposed from the comparison of the 13C data to those of a synthetic analog (dC 80.3 for 5a-carbon, dC 81.9 for 5bcarbon, Kovganko and Chernov 2001) and previously reported steroids (dC 80.8–80.9, Rho et al. 2000). The bconfiguration of the 3-hydroxy group at C-3 was also defined from the coupling constants of H-4a [dH 2.16 (dd, J = 11.2, 3.3 Hz)] and NOESY correlations [H3/H4a, H4b/H19]. The geometry of the double bond at C-22 was determined as Z based on the coupling constants of H-22 and H-23 (J = 6.9 Hz), however, we were unable to establish the configuration of C-24.
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Gentamicina
(MIC, lg ml-1)
a HO
1a
Strain
Staphylococcus epidermidis ATCC 12228
6.3
Micrococcus lutes ATCC 9341
100
0.2 3.1
Bacillus subtilis ATCC 6633
25
0.2
S. aureus ATCC 65381
[200
0.2
Escherichia coli ATCC 11775
[200
0.8
Salmonella typhimurium ATCC 14028
[200
1.6
Klebsiella pneumonia ATCC 4352
[200
0.8
a
Each experiment was replicated three times
We previously isolated a secosterol (2) that was structurally related to compound 1 (Yang et al. 2014). As the previously identified steroid (2) exhibited antibacterial activities, compound 1 was also investigated for its antibacterial activity against seven pathogens (Table 2). Compound 1 was found to display antibacterial activities on Staphylococcus epidermidis ATCC 12228 and Bacillus subtilis ATCC 6633 with MIC values of 6.3 and 25 lg ml-1, respectively. Interestingly, 1 exhibited weak activity against Micrococcus lutes ATCC 9341, whereas 2 displayed potent activity. Acknowledgments This work was supported by Suncheon Research Center for Natural Medicines. I. Yang was in part supported by the BK21 program, Ministry of Education, Korea. Conflict of interest of interest.
The authors declare that they have no conflict
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