This article was downloaded by: [Fudan University] On: 16 May 2015, At: 08:07 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast ab
a
cd
Natália N. Saraiva , Bárbara S.F. Rodrigues , Paula C. Jimenez , cd
a
Larissa A. Guimarães , Maria C.M. Torres , Edson Rodriguese
f
f
a
Filho , Ludwig H. Pfenning , Lucas M. Abreu , Jair Mafezoli , a
cd
Marcos C. de Mattos , Letícia V. Costa-Lotufo
Click for updates
Conceição F. de Oliveira
& Maria da
a
a
Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, CE, Brazil b
Departamento de Farmácia, Universidade Federal de Sergipe, Lagarto, SE, Brazil c
Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil d
Instituto de Ciências do Mar, LABOMAR, Universidade Federal do Ceará, Fortaleza, CE, Brazil e
Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil f
Departamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG, Brazil Published online: 23 Dec 2014.
To cite this article: Natália N. Saraiva, Bárbara S.F. Rodrigues, Paula C. Jimenez, Larissa A. Guimarães, Maria C.M. Torres, Edson Rodrigues-Filho, Ludwig H. Pfenning, Lucas M. Abreu, Jair Mafezoli, Marcos C. de Mattos, Letícia V. Costa-Lotufo & Maria da Conceição F. de Oliveira (2014): Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.987772 To link to this article: http://dx.doi.org/10.1080/14786419.2014.987772
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Fudan University] at 08:07 16 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.987772
Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast Nata´lia N. Saraivaab, Ba´rbara S.F. Rodriguesa, Paula C. Jimenezcd, Larissa A. Guimara˜escd, Maria C. M. Torresa, Edson Rodrigues-Filhoe, Ludwig H. Pfenningf, Lucas M. Abreuf, Jair Mafezolia, Marcos C. de Mattosa, Letı´cia V. Costa-Lotufocd* and Maria da Conceic a˜o F. de Oliveiraa*
Downloaded by [Fudan University] at 08:07 16 May 2015
a
Departamento de Quı´mica Orgaˆnica e Inorgaˆnica, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; bDepartamento de Farma´cia, Universidade Federal de Sergipe, Lagarto, SE, Brazil; c Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; d Instituto de Cieˆncias do Mar, LABOMAR, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; e Departamento de Quı´mica, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, SP, Brazil; fDepartamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG, Brazil (Received 14 June 2014; final version received 5 November 2014)
A fungal strain of Aspergillus sp. (BRF 030) was isolated from the sediments collected in the northeast coast of Brazil, and the cytotoxic activity of its secondary metabolites was investigated against HCT-116 tumour cell line. The cytotoxicity-guided fractionation of the extracts from this fungus cultured in potato-dextrose-sea water for 14 days at room temperature yielded the hetero-spirocyclic g-lactams pseurotin A (1), pseurotin D (2) and pseurotin FD-838 (7), the alkaloids fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6), methylsulochrin (4) and bis(dethio)bis (methylthio)gliotoxin (3). Among them, fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6) were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6). Keywords: marine-derived fungi; Aspergillus sp; cytotoxic activity; diketopiperazine; pseurotin
1. Introduction Oceans have been considered a source of potentially useful bioactive compounds, and more than 20,000 secondary metabolites from marine sources have been isolated and characterised since *Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
2
N.N. Saraiva et al.
the early 1960s (Blunt et al. 2012). When compared with that of terrestrial sources, the bioprospection of marine sources in search of novel and bioactive compounds is still incipient, as only 14% of all known natural products are of marine origin (Menna 2009; Blunt et al. 2011). Cytotoxicity in the extracts of organisms from marine environments is one of the most expressive biological activities, and surpasses those of terrestrial origin. When considering the list of the natural compounds under clinical investigation, anticancer chemotherapy is suggested as the stronghold of marine natural products (Menna 2009). On the course of our ongoing research program regarding the identification of new antitumour compounds from the Brazilian marine biodiversity (Montenegro et al. 2012), we report, herein, the bioprospection of cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from sediments from the Northeast coast of Brazil.
Downloaded by [Fudan University] at 08:07 16 May 2015
2. Results and discussion The cytotoxic potential of organic extracts from 35 fungal strains recovered from the sediments collected at Mucuripe Beach, Fortaleza, on the Northeast coast of Brazil was investigated, and strain BRF 030 inhibited cell growth with 95.15% efficiency. This strain was identified as a member of the genus Aspergillus, section Fumigati, based on cultural and micromorphological traits observed under standard conditions (Klich 2002; Samson et al. 2007). Aspergillus is considered an oligotrophic genus found in diverse living environments, including marine sediments. In addition, marine-derived species from this genus have contributed to most of the secondary metabolites from marine fungi (Lee et al. 2013). The crude EtOAc extract from Aspergillus sp. (BRF 030) was fractionated using a cytotoxicity-guided approach, and yielded compounds pseurotin A (1) (Bloch et al. 1976), pseurotin D (2) (Breitenstein et al. 1981), bis(dethio)bis(methylthio)gliotoxin (3) (Yi et al. 2012), methylsulochrin (Turner 1965) (4), fumitremorgin C (5) (Cole et al. 1977), 12,13dihydroxyfumitremorgin C (6) (Abraham & Arfmann 1990) and pseurotin FD-838 (7) (Mizoue et al. 1987) (Figure 1). The elucidation of the structures of all the isolated compounds was established mainly on the basis of their 1D and 2D NMR spectroscopic data, and comparison with the literature data. All compounds were previously isolated from A. fumigatus (Turner 1965; Cole et al. 1977; Mizoue et al. 1987; Abraham & Arfmann 1990; Wink et al. 1991; Wenke et al. 1993; Han et al. 2007; Hayashi et al. 2007; Frisvad et al. 2009; Ge et al. 2009; Mehedi et al. 2010; Rasmussen et al. 2010; Wang et al. 2011; Li et al. 2012) and some congener species (Larsen et al. 2007; Zhang et al. 2008; Zhang et al. 2011; Martı´nez-Luis et al. 2012; Wu et al. 2012). In addition, studies on the cytotoxic activities of these secondary metabolites are reported in the literature. Pseurotins 1, 2 and 7 were assayed against human breast cancer cell line MCF-7, and pseurotin D (2) was the most active compound with IC50 15.6 mM (Martı´nez-Luis et al. 2012). Bis(dethio)bis (methylthio)gliotoxin (3) was inactive when tested for growth inhibition on various cancer cell lines. The same result was observed when methylsulochrin (4) was assayed against K562 tumour cell line. The diketopiperazine 5 was cytotoxic against human leukaemia P-388 (ED50 3.9 mg/ mL), and its analogue 6 showed antiproliferative effect on human leukaemia U-937 (IC50 1.8 mM) and prostate cancer PC-3 (IC50 6.6 mM). In addition, fumitremorgin C (5) was active when tested against multi-drug resistant breast and colon cancer (Bladt et al. 2013). Pseurotin A (1) was isolated from the highly cytotoxic fraction (H2O/MeOH 5:5), which nearly completely inhibited cell growth (Table S1). However, 1 presented an IC50 of 72 mM when tested for its cytotoxicity on HCT-116 cells (Table S2), and this kind of activity does not justify such high potency of the mother-fraction. The neighbouring fraction, H2O/MeOH 6:4, yielded a compound from this same class, pseurotin D (2), which showed a comparable, yet slightly higher, IC50 of 85 mM (Table S2). The fraction MeOH/H2O 7:3, which also completely
Downloaded by [Fudan University] at 08:07 16 May 2015
Natural Product Research
3
Figure 1. Chemical structures of the compounds 1 – 7 isolated from Aspergillus sp.
hindered cell growth (Table S1), yielded five different compounds of which the diketopiperazines fumitremorgin C (5) and 12,13-dihydroxy-fumitremorgin C (6), with respective IC50 of 15.17 and 4.53 mM (Table S2), were the most active compounds. Compounds bis(dethio)bis(methylthio)gliotoxin (3), methylsulochrin (4) and the other pseurotin FD-838 (7) did not show any cytotoxic activity in the range of concentrations tested (IC50 . 120 mM). In order to associate the cytotoxic activity of the extracts with the kinetic production of compounds 1 –7, Aspergillus sp. was grown for 7, 14, 21 and 28 days. The EtOAc extract from each time point was tested for cytotoxicity (Table S3) and analysed by using LC/MS. The extract obtained at day 21 was the most active on the bioassay, with IC50 of 3.54 mg/mL, followed by that of day 14, with IC50 of 13.22 mg/mL. The total ion chromatograms of these most active extracts (Figure S1) showed the presence of compounds 5 and 6 in higher relative concentration, when compared with that from 7 days. The extract from 28 days of fungal growth, which was the least active, did not show the presence of these two compounds. These results might suggest that compounds 5 and 6, which are also among the most active compounds isolated herein, are responsible for the activity of the extracts from Aspergillus sp.
3. Experimental 3.1. General NMR spectra (1H, 13C, DEPT, COSY, HSQC and HMBC) were recorded on Bruker spectrometer DRX 500 (Bruker, Karlsruhe, Germany) with MeOD as solvent and TMS as internal standard. Solvents were purchased from Vetecw and Tediaw (HPLC-grade solvents).
3.2. Fungal strain The fungal strain of Aspergillus sp. (BRF 030) was recovered from sediments collected in Mucuripe Beach (038430 23.0400 S; 388290 27.9800 W) Fortaleza-CE, Brazil, by autonomous diving. The isolation followed the literature procedure (Damare et al. 2006). The strain BRF 030 was three point inoculated on Czapek yeast extract agar (CYA) and malt extract agar, and incubated at 258C, in the dark, for 7 days (Klich 2002). The strain was also incubated at 378C on CYA. The
4
N.N. Saraiva et al.
Downloaded by [Fudan University] at 08:07 16 May 2015
macroscopic features evaluated included colony diameter, colour of the mycelium, conidia and colony reverse. Microscopic preparations were used to evaluate the shape, size, colour and texture of the conidiophores and conidia. 3.3. Fungus culture, extraction and isolation One disc (6.0 mm each), containing the strain BRF 030 previously grown in potato dextrose agar medium in reconstructed sea water for 7 days, was transferred to a 500 mL Erlenmeyer flask with 200 mL of potato dextrose broth. The fungal strain was grown under static conditions for 14 days at room temperature (288C). The mycelium was separated from the liquid medium by vacuum filtration and extracted with EtOAc/MeOH 1:1, yielding extract BRF-030-mycelium (26.26 g). The liquid medium was then extracted with EtOAc and provided extract BRF-030-liquid medium (1.41 g). This extract was dissolved in MeOH 90% and extracted with n-hexane, and yielded fractions BRF-030-liquid medium MeOH 90% (1.36 g) and BRF-030-liquid medium hexane (0.02 g), respectively. Fraction BRF-030 liquid medium MeOH 90% was fractionated on a C18 cartridge (7 cm £ 3 cm; Strata Phenomenex) using MeOH and water as eluent. Eight fractions were collected: H2O 100% (160.50 mg); H2O/MeOH 8:2 (72.10 mg); H2O/MeOH 6:4 (43.70 mg); H2O/MeOH 5:5 (185.60 mg); MeOH/H2O 7:3 (483.50 mg); MeOH/H2O 8:2 (141.20 mg); MeOH/H2O 9:1 (62.10 mg) and MeOH 100% (25.20 mg). Among these, fractions H2O/MeOH 6:4, H2O/MeOH 5:5 and MeOH/H2O 7:3 were, separately, purified by using HPLC (Phenomenex column, ODS 100A 250 mm £ 10.00 mm, 5 mm, 4.7 mL/min flow). Compound 1 (7.7 mg) was isolated from the fraction H2O/MeOH 5:5, by elution with MeOH/H2O 1:1, and identified as pseurotin A. Compound 2 (9.5 mg) was isolated from the fraction H2O/MeOH 6:4 using MeOH/H2O 45:65 as eluent, and identified as pseurotin D. Chromatography of fraction MeOH/H2O 7:3 by elution with MeOH/H2O 55:45 yielded compounds bis(dethio)bis (methylthio)gliotoxin (3, 43.7 mg), methylsulochrin (4, 16.4 mg), fumitremorgin C (5, 9.2 mg), 12,13-dihydroxyfumitremorgin C (6, 13.3 mg) and pseurotin FD-838 (7, 11.2 mg). 3.4. LC – MS analyses of crude extracts from Aspergillus sp. grown in varied time The LC – MS system consisted of UFLC system coupled with IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) equipped with electrospray ionisation (ESI) source operated in positive mode. The spectra were recorded in the range m/z 100 – 600 Da, using a potential of 4.0 kV on the capillary and nitrogen as the desolvation gas. The system was controlled by LCsolution software, which was also used for data analysis. An aliquot (5 mL) of each sample with a concentration of 1 mg/mL was injected into a reversed phase column (C18, 150 nm £ 2.0 nm, Shimadzu), which was maintained at 408C. The mobile phases consisted of the following linear binary gradient of the CH3CN and H2O at a flow rate of 0.2 mL/min: 10 –100% CH3CN for 90 min. At the end of this sequence, the column was equilibrated under initial conditions for 10 min. The column effluent was directly transferred into the ESI interface without splitting. 3.5. Cytotoxic activity Cytotoxic activity of the extracts and pure compounds was evaluated against the tumour cell line HCT-116 (human colon carcinoma), donated from the National Cancer Institute (Bethesda, MD, USA), and followed the literature procedure (Montenegro et al. 2012). 4. Conclusions In summary, we have isolated Aspergillus sp. (strain BRF 030) from the sediments collected in the northeast coast of Brazil. This strain was selected for bioassay-guided fractionation based on
Natural Product Research
5
previous cytotoxic screening assay. Compounds pseurotin A (1), pseurotin D (2), bis(dethio)bis (methylthio)gliotoxin (3), methylsulochrin (4), fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6) and pseurotin FD-838 (7) were isolated. Among them, the indole diketopiperazine alkaloids 5 and 6 were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds 1 – 7. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds 5 and 6.
Supplementary material Figure S1, Tables S1 – S3 and NMR data of the isolated compounds are available online.
Downloaded by [Fudan University] at 08:07 16 May 2015
Acknowledgements The authors thank the Fundac a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Fundac a˜o Cearense de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (FUNCAP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Programa Sisbiota-Brasil and Coordenac a˜o de Aperfeic oamento de Ensino Superior (CAPES) for financial support. E.R.F., M.C.F.O and L.V.C.L. also thank CNPq for their research fellowship.
References Abraham WR, Arfmann HA. 1990. 12,13-Dihydroxy-fumitremorgin C from Aspergillus fumigatus. Phytochem Rev. 29:1025–1026. Bladt TT, Frisvad JC, Knudsen PB, Larsen TO. 2013. Anticancer and antifungal compounds from Aspergillus, Penicillium and other filamentous fungi. Molecules. 18:11338– 11376. Bloch P, Tamm C, Bollinger P, Petcher TJ, Weber HP. 1976. Pseurotin, a new metabolite of Pseudeurotium ovalis STOLK having an unusual hetero-spirocyclic system. Helv Chim Acta. 59:133–137. Blunt J, Munro M, Upjohn M. 2012. The role of databases in marine natural products research. In: Fattorusso E, Gerwick HW, Taglialatela-Scafati O, editors. Handbook of marine natural products. 24th ed. New York: Springer. Blunt JW, Copp BR, Munro MHG, Northcotec PT, Prinsep MR. 2011. Marine natural products. Nat Prod Rep. 28:196–268. Breitenstein W, Chexal KK, Mohr P, Tamm C. 1981. Pseurotin B, C, D, and E. further new metabolites of Pseudeurotium ovalis Stolk. Helv Chim Acta. 64:379–388. Cole RJ, Kirksey JW, Dorner JW, Wilson DM, Johnson Jr JC, Johnson AN, Bedell DM, Springer JP, Chexal KK, Clardy JC, Cox RH. 1977. Mycotoxins produced by Aspergillus fumigatus species isolated from molded silage. J Agric Food Chem. 25:826–830. Damare S, Raghukumar C, Raghukumar S. 2006. Fungi in deep-sea sediments of the central indian basin. Deep-Sea Res Pt I. 53:14–27. Frisvad JC, Rank C, Nielsen KF, Larsen TO. 2009. Metabolomics of Aspergillus fumigatus. Med Mycol. 47:53–71. Ge HM, Yu ZG, Zhang J, Wu JH, Tan RX. 2009. Bioactive alkaloids from endophytic Aspergillus fumigatus. J Nat Prod. 72:753–755. Han X, Xu X, Cui C, Gu Q. 2007. Alkaloidal compounds produced by a marine-derived fungus, Aspergillus fumigatus H1-04, and their antitumor activities. Zhongguo Yaowu Huaxue Zazhi. 17:232–237. Hayashi A, Fujioka S, Nukina M, Kawano T, Shimada A, Kimura Y. 2007. Fumiquinones A and B, nematicidal quinones produced by Aspergillus fumigatus. Biosci Biotechnol Biochem. 71:1697–1702. Klich MA. 2002. Identification of common Aspergillus species. Utrecht: Centraalbureau voor Schimmelcultures. Larsen TO, Smedsgaard J, Nielsen KF, Hansen MAE, Samson RA, Frisvad JC. 2007. Production of mycotoxins by Aspergillus lentulus and other medically important and closely related species in section Fumigati. Med Mycol. 45:225–232. Lee YM, Kim MJ, Li H, Zhang P, Bao B, Lee KJ, Jung JH. 2013. Marine-derived Aspergillus species as source of bioactive secondary metabolites. Mar Biotechnol. 15:499–519. Li X, Zhang Q, An-zhang L, Gao J. 2012. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J Agric Food Chem. 60:3424– 3431. Martı´nez-Luis S, Cherigo L, Arnold E, Spadafora C, Gerwick WH, Cubilla-Rios L. 2012. Antiparasitic and anticancer constituents of the endophytic fungus Aspergillus sp. strain F1544. Nat Prod Commun. 7:165–168.
Downloaded by [Fudan University] at 08:07 16 May 2015
6
N.N. Saraiva et al.
Mehedi MAU, Molla AH, Khondkar P, Sultana S, Islam MA, Rashid MA, Chowdhury R. 2010. Pseurotin A: an antibacterial secondary metabolite from Aspergillus fumigatus. Asian J Chem. 22:2611–2614. Menna M. 2009. Antitumor potential of natural products from Mediterranean ascidians. Phytochem Rev. 8:461–472. Mizoue K, Okazaki T, Hanada K, Amamoto T, Yamagishi M, Omura S. 1987. Physiologically active substance FD-838 and process for preparing the same. European patent application. EP 216607 A2 19870401. Montenegro TGC, Rodrigues FAR, Jimenez PC, Angelim AL, Melo VMM, Rodrigues Filho E, De Oliveira MCF, CostaLotufo LV. 2012. Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei. Chem Biodivers. 9:2203–2209. Rasmussen RR, Rasmussen PH, Larsen TO, Bladt TT, Binderup ML. 2010. In vitro cytotoxicity of fungi spoiling maize silage. Food Chem Toxicol. 49:31–44. Samson RA, Hong S, Peterson SW, Frisvad JC, Varga J. 2007. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol. 59:147–203. Turner WB. 1965. The production of trypacidin and monomethylsulochrin by Aspergillus fumigatus. J Chem Soc Perkin. 1:6658–6659. Wang F, Li D, Zhu T, Zhang M, Gu Q. 2011. Pseurotin A1 and A2, two new 1-oxa-7 azaspiro[4.4]non-2-ene-4,6-diones from the holothurians derived fungus Aspergillus fumigates WFZ-25. Can J Chem. 89:72–76. Wenke J, Anke H, Sternce O. 1993. Pseurotin A and 8-O-demethylpseurotin a from Aspergillus fumigatus and their inhibitory activities on chitin synthase. Biosci Biotechnol Biochem. 57:961– 964. Wink J, Grabley S, Gareis M, Zeeck A, Philipps S. 1991. Biologically active pseurotins A and D, new metabolites from Aspergillus fumigatus, process for their preparation and their use as apomorphine antagonists. European patent application. EP 546475. Wu QX, Jin XJ, Draskovic M, Crews MS, Tenney K, Valeriote FA, Yao XJ, Crews P. 2012. Unraveling the numerous biosynthetic products of the marine sediment-derived fungus, Aspergillus insulicola. Phytochem Lett. 5:114–117. Yi S, Kentaro T, Yasushi T, Minoru Y, Yuichi N, Shigeru O, Shigeki M. 2012. Gliotoxin analogues from a marinederived fungus, Penicillium sp., and their cytotoxic and histone methyltransferase inhibitory activities. J Nat Prod. 75:111–114. Zhang L, Li Z, Bai J, Wu X, Wang Y, Hua H. 2011. Metabolites of Aspergillus sp. HT-2. Zhongguo Yaoxue Zazhi. 46:1154–1158. Zhang M, Wang W, Fang Y, Zhu T, Gu Q, Zhu W. 2008. Cytotoxic alkaloids and antibiotic nordammarane triterpenoids from the marine-derived fungus Aspergillus sydowii. J Nat Prod. 71:985–989.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast ab
a
cd
Natália N. Saraiva , Bárbara S.F. Rodrigues , Paula C. Jimenez , cd
a
Larissa A. Guimarães , Maria C.M. Torres , Edson Rodriguese
f
f
a
Filho , Ludwig H. Pfenning , Lucas M. Abreu , Jair Mafezoli , a
cd
Marcos C. de Mattos , Letícia V. Costa-Lotufo
Click for updates
Conceição F. de Oliveira
& Maria da
a
a
Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Fortaleza, CE, Brazil b
Departamento de Farmácia, Universidade Federal de Sergipe, Lagarto, SE, Brazil c
Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil d
Instituto de Ciências do Mar, LABOMAR, Universidade Federal do Ceará, Fortaleza, CE, Brazil e
Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil f
Departamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG, Brazil Published online: 23 Dec 2014.
To cite this article: Natália N. Saraiva, Bárbara S.F. Rodrigues, Paula C. Jimenez, Larissa A. Guimarães, Maria C.M. Torres, Edson Rodrigues-Filho, Ludwig H. Pfenning, Lucas M. Abreu, Jair Mafezoli, Marcos C. de Mattos, Letícia V. Costa-Lotufo & Maria da Conceição F. de Oliveira (2014): Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.987772 To link to this article: http://dx.doi.org/10.1080/14786419.2014.987772
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Fudan University] at 08:07 16 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.987772
Cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from the sediments of the Brazilian coast Nata´lia N. Saraivaab, Ba´rbara S.F. Rodriguesa, Paula C. Jimenezcd, Larissa A. Guimara˜escd, Maria C. M. Torresa, Edson Rodrigues-Filhoe, Ludwig H. Pfenningf, Lucas M. Abreuf, Jair Mafezolia, Marcos C. de Mattosa, Letı´cia V. Costa-Lotufocd* and Maria da Conceic a˜o F. de Oliveiraa*
Downloaded by [Fudan University] at 08:07 16 May 2015
a
Departamento de Quı´mica Orgaˆnica e Inorgaˆnica, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; bDepartamento de Farma´cia, Universidade Federal de Sergipe, Lagarto, SE, Brazil; c Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; d Instituto de Cieˆncias do Mar, LABOMAR, Universidade Federal do Ceara´, Fortaleza, CE, Brazil; e Departamento de Quı´mica, Universidade Federal de Sa˜o Carlos, Sa˜o Carlos, SP, Brazil; fDepartamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG, Brazil (Received 14 June 2014; final version received 5 November 2014)
A fungal strain of Aspergillus sp. (BRF 030) was isolated from the sediments collected in the northeast coast of Brazil, and the cytotoxic activity of its secondary metabolites was investigated against HCT-116 tumour cell line. The cytotoxicity-guided fractionation of the extracts from this fungus cultured in potato-dextrose-sea water for 14 days at room temperature yielded the hetero-spirocyclic g-lactams pseurotin A (1), pseurotin D (2) and pseurotin FD-838 (7), the alkaloids fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6), methylsulochrin (4) and bis(dethio)bis (methylthio)gliotoxin (3). Among them, fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6) were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds fumitremorgin C (5) and 12,13-dihydroxy fumitremorgin C (6). Keywords: marine-derived fungi; Aspergillus sp; cytotoxic activity; diketopiperazine; pseurotin
1. Introduction Oceans have been considered a source of potentially useful bioactive compounds, and more than 20,000 secondary metabolites from marine sources have been isolated and characterised since *Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
2
N.N. Saraiva et al.
the early 1960s (Blunt et al. 2012). When compared with that of terrestrial sources, the bioprospection of marine sources in search of novel and bioactive compounds is still incipient, as only 14% of all known natural products are of marine origin (Menna 2009; Blunt et al. 2011). Cytotoxicity in the extracts of organisms from marine environments is one of the most expressive biological activities, and surpasses those of terrestrial origin. When considering the list of the natural compounds under clinical investigation, anticancer chemotherapy is suggested as the stronghold of marine natural products (Menna 2009). On the course of our ongoing research program regarding the identification of new antitumour compounds from the Brazilian marine biodiversity (Montenegro et al. 2012), we report, herein, the bioprospection of cytotoxic compounds from the marine-derived fungus Aspergillus sp. recovered from sediments from the Northeast coast of Brazil.
Downloaded by [Fudan University] at 08:07 16 May 2015
2. Results and discussion The cytotoxic potential of organic extracts from 35 fungal strains recovered from the sediments collected at Mucuripe Beach, Fortaleza, on the Northeast coast of Brazil was investigated, and strain BRF 030 inhibited cell growth with 95.15% efficiency. This strain was identified as a member of the genus Aspergillus, section Fumigati, based on cultural and micromorphological traits observed under standard conditions (Klich 2002; Samson et al. 2007). Aspergillus is considered an oligotrophic genus found in diverse living environments, including marine sediments. In addition, marine-derived species from this genus have contributed to most of the secondary metabolites from marine fungi (Lee et al. 2013). The crude EtOAc extract from Aspergillus sp. (BRF 030) was fractionated using a cytotoxicity-guided approach, and yielded compounds pseurotin A (1) (Bloch et al. 1976), pseurotin D (2) (Breitenstein et al. 1981), bis(dethio)bis(methylthio)gliotoxin (3) (Yi et al. 2012), methylsulochrin (Turner 1965) (4), fumitremorgin C (5) (Cole et al. 1977), 12,13dihydroxyfumitremorgin C (6) (Abraham & Arfmann 1990) and pseurotin FD-838 (7) (Mizoue et al. 1987) (Figure 1). The elucidation of the structures of all the isolated compounds was established mainly on the basis of their 1D and 2D NMR spectroscopic data, and comparison with the literature data. All compounds were previously isolated from A. fumigatus (Turner 1965; Cole et al. 1977; Mizoue et al. 1987; Abraham & Arfmann 1990; Wink et al. 1991; Wenke et al. 1993; Han et al. 2007; Hayashi et al. 2007; Frisvad et al. 2009; Ge et al. 2009; Mehedi et al. 2010; Rasmussen et al. 2010; Wang et al. 2011; Li et al. 2012) and some congener species (Larsen et al. 2007; Zhang et al. 2008; Zhang et al. 2011; Martı´nez-Luis et al. 2012; Wu et al. 2012). In addition, studies on the cytotoxic activities of these secondary metabolites are reported in the literature. Pseurotins 1, 2 and 7 were assayed against human breast cancer cell line MCF-7, and pseurotin D (2) was the most active compound with IC50 15.6 mM (Martı´nez-Luis et al. 2012). Bis(dethio)bis (methylthio)gliotoxin (3) was inactive when tested for growth inhibition on various cancer cell lines. The same result was observed when methylsulochrin (4) was assayed against K562 tumour cell line. The diketopiperazine 5 was cytotoxic against human leukaemia P-388 (ED50 3.9 mg/ mL), and its analogue 6 showed antiproliferative effect on human leukaemia U-937 (IC50 1.8 mM) and prostate cancer PC-3 (IC50 6.6 mM). In addition, fumitremorgin C (5) was active when tested against multi-drug resistant breast and colon cancer (Bladt et al. 2013). Pseurotin A (1) was isolated from the highly cytotoxic fraction (H2O/MeOH 5:5), which nearly completely inhibited cell growth (Table S1). However, 1 presented an IC50 of 72 mM when tested for its cytotoxicity on HCT-116 cells (Table S2), and this kind of activity does not justify such high potency of the mother-fraction. The neighbouring fraction, H2O/MeOH 6:4, yielded a compound from this same class, pseurotin D (2), which showed a comparable, yet slightly higher, IC50 of 85 mM (Table S2). The fraction MeOH/H2O 7:3, which also completely
Downloaded by [Fudan University] at 08:07 16 May 2015
Natural Product Research
3
Figure 1. Chemical structures of the compounds 1 – 7 isolated from Aspergillus sp.
hindered cell growth (Table S1), yielded five different compounds of which the diketopiperazines fumitremorgin C (5) and 12,13-dihydroxy-fumitremorgin C (6), with respective IC50 of 15.17 and 4.53 mM (Table S2), were the most active compounds. Compounds bis(dethio)bis(methylthio)gliotoxin (3), methylsulochrin (4) and the other pseurotin FD-838 (7) did not show any cytotoxic activity in the range of concentrations tested (IC50 . 120 mM). In order to associate the cytotoxic activity of the extracts with the kinetic production of compounds 1 –7, Aspergillus sp. was grown for 7, 14, 21 and 28 days. The EtOAc extract from each time point was tested for cytotoxicity (Table S3) and analysed by using LC/MS. The extract obtained at day 21 was the most active on the bioassay, with IC50 of 3.54 mg/mL, followed by that of day 14, with IC50 of 13.22 mg/mL. The total ion chromatograms of these most active extracts (Figure S1) showed the presence of compounds 5 and 6 in higher relative concentration, when compared with that from 7 days. The extract from 28 days of fungal growth, which was the least active, did not show the presence of these two compounds. These results might suggest that compounds 5 and 6, which are also among the most active compounds isolated herein, are responsible for the activity of the extracts from Aspergillus sp.
3. Experimental 3.1. General NMR spectra (1H, 13C, DEPT, COSY, HSQC and HMBC) were recorded on Bruker spectrometer DRX 500 (Bruker, Karlsruhe, Germany) with MeOD as solvent and TMS as internal standard. Solvents were purchased from Vetecw and Tediaw (HPLC-grade solvents).
3.2. Fungal strain The fungal strain of Aspergillus sp. (BRF 030) was recovered from sediments collected in Mucuripe Beach (038430 23.0400 S; 388290 27.9800 W) Fortaleza-CE, Brazil, by autonomous diving. The isolation followed the literature procedure (Damare et al. 2006). The strain BRF 030 was three point inoculated on Czapek yeast extract agar (CYA) and malt extract agar, and incubated at 258C, in the dark, for 7 days (Klich 2002). The strain was also incubated at 378C on CYA. The
4
N.N. Saraiva et al.
Downloaded by [Fudan University] at 08:07 16 May 2015
macroscopic features evaluated included colony diameter, colour of the mycelium, conidia and colony reverse. Microscopic preparations were used to evaluate the shape, size, colour and texture of the conidiophores and conidia. 3.3. Fungus culture, extraction and isolation One disc (6.0 mm each), containing the strain BRF 030 previously grown in potato dextrose agar medium in reconstructed sea water for 7 days, was transferred to a 500 mL Erlenmeyer flask with 200 mL of potato dextrose broth. The fungal strain was grown under static conditions for 14 days at room temperature (288C). The mycelium was separated from the liquid medium by vacuum filtration and extracted with EtOAc/MeOH 1:1, yielding extract BRF-030-mycelium (26.26 g). The liquid medium was then extracted with EtOAc and provided extract BRF-030-liquid medium (1.41 g). This extract was dissolved in MeOH 90% and extracted with n-hexane, and yielded fractions BRF-030-liquid medium MeOH 90% (1.36 g) and BRF-030-liquid medium hexane (0.02 g), respectively. Fraction BRF-030 liquid medium MeOH 90% was fractionated on a C18 cartridge (7 cm £ 3 cm; Strata Phenomenex) using MeOH and water as eluent. Eight fractions were collected: H2O 100% (160.50 mg); H2O/MeOH 8:2 (72.10 mg); H2O/MeOH 6:4 (43.70 mg); H2O/MeOH 5:5 (185.60 mg); MeOH/H2O 7:3 (483.50 mg); MeOH/H2O 8:2 (141.20 mg); MeOH/H2O 9:1 (62.10 mg) and MeOH 100% (25.20 mg). Among these, fractions H2O/MeOH 6:4, H2O/MeOH 5:5 and MeOH/H2O 7:3 were, separately, purified by using HPLC (Phenomenex column, ODS 100A 250 mm £ 10.00 mm, 5 mm, 4.7 mL/min flow). Compound 1 (7.7 mg) was isolated from the fraction H2O/MeOH 5:5, by elution with MeOH/H2O 1:1, and identified as pseurotin A. Compound 2 (9.5 mg) was isolated from the fraction H2O/MeOH 6:4 using MeOH/H2O 45:65 as eluent, and identified as pseurotin D. Chromatography of fraction MeOH/H2O 7:3 by elution with MeOH/H2O 55:45 yielded compounds bis(dethio)bis (methylthio)gliotoxin (3, 43.7 mg), methylsulochrin (4, 16.4 mg), fumitremorgin C (5, 9.2 mg), 12,13-dihydroxyfumitremorgin C (6, 13.3 mg) and pseurotin FD-838 (7, 11.2 mg). 3.4. LC – MS analyses of crude extracts from Aspergillus sp. grown in varied time The LC – MS system consisted of UFLC system coupled with IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) equipped with electrospray ionisation (ESI) source operated in positive mode. The spectra were recorded in the range m/z 100 – 600 Da, using a potential of 4.0 kV on the capillary and nitrogen as the desolvation gas. The system was controlled by LCsolution software, which was also used for data analysis. An aliquot (5 mL) of each sample with a concentration of 1 mg/mL was injected into a reversed phase column (C18, 150 nm £ 2.0 nm, Shimadzu), which was maintained at 408C. The mobile phases consisted of the following linear binary gradient of the CH3CN and H2O at a flow rate of 0.2 mL/min: 10 –100% CH3CN for 90 min. At the end of this sequence, the column was equilibrated under initial conditions for 10 min. The column effluent was directly transferred into the ESI interface without splitting. 3.5. Cytotoxic activity Cytotoxic activity of the extracts and pure compounds was evaluated against the tumour cell line HCT-116 (human colon carcinoma), donated from the National Cancer Institute (Bethesda, MD, USA), and followed the literature procedure (Montenegro et al. 2012). 4. Conclusions In summary, we have isolated Aspergillus sp. (strain BRF 030) from the sediments collected in the northeast coast of Brazil. This strain was selected for bioassay-guided fractionation based on
Natural Product Research
5
previous cytotoxic screening assay. Compounds pseurotin A (1), pseurotin D (2), bis(dethio)bis (methylthio)gliotoxin (3), methylsulochrin (4), fumitremorgin C (5), 12,13-dihydroxy fumitremorgin C (6) and pseurotin FD-838 (7) were isolated. Among them, the indole diketopiperazine alkaloids 5 and 6 were the most active. The cytotoxic activities of the extracts from Aspergillus sp. grown from 7 to 28 days were investigated, and they were associated with the kinetic production of the compounds 1 – 7. The most active extracts (14 and 21 days) were those with the highest relative concentrations of the compounds 5 and 6.
Supplementary material Figure S1, Tables S1 – S3 and NMR data of the isolated compounds are available online.
Downloaded by [Fudan University] at 08:07 16 May 2015
Acknowledgements The authors thank the Fundac a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Fundac a˜o Cearense de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (FUNCAP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Programa Sisbiota-Brasil and Coordenac a˜o de Aperfeic oamento de Ensino Superior (CAPES) for financial support. E.R.F., M.C.F.O and L.V.C.L. also thank CNPq for their research fellowship.
References Abraham WR, Arfmann HA. 1990. 12,13-Dihydroxy-fumitremorgin C from Aspergillus fumigatus. Phytochem Rev. 29:1025–1026. Bladt TT, Frisvad JC, Knudsen PB, Larsen TO. 2013. Anticancer and antifungal compounds from Aspergillus, Penicillium and other filamentous fungi. Molecules. 18:11338– 11376. Bloch P, Tamm C, Bollinger P, Petcher TJ, Weber HP. 1976. Pseurotin, a new metabolite of Pseudeurotium ovalis STOLK having an unusual hetero-spirocyclic system. Helv Chim Acta. 59:133–137. Blunt J, Munro M, Upjohn M. 2012. The role of databases in marine natural products research. In: Fattorusso E, Gerwick HW, Taglialatela-Scafati O, editors. Handbook of marine natural products. 24th ed. New York: Springer. Blunt JW, Copp BR, Munro MHG, Northcotec PT, Prinsep MR. 2011. Marine natural products. Nat Prod Rep. 28:196–268. Breitenstein W, Chexal KK, Mohr P, Tamm C. 1981. Pseurotin B, C, D, and E. further new metabolites of Pseudeurotium ovalis Stolk. Helv Chim Acta. 64:379–388. Cole RJ, Kirksey JW, Dorner JW, Wilson DM, Johnson Jr JC, Johnson AN, Bedell DM, Springer JP, Chexal KK, Clardy JC, Cox RH. 1977. Mycotoxins produced by Aspergillus fumigatus species isolated from molded silage. J Agric Food Chem. 25:826–830. Damare S, Raghukumar C, Raghukumar S. 2006. Fungi in deep-sea sediments of the central indian basin. Deep-Sea Res Pt I. 53:14–27. Frisvad JC, Rank C, Nielsen KF, Larsen TO. 2009. Metabolomics of Aspergillus fumigatus. Med Mycol. 47:53–71. Ge HM, Yu ZG, Zhang J, Wu JH, Tan RX. 2009. Bioactive alkaloids from endophytic Aspergillus fumigatus. J Nat Prod. 72:753–755. Han X, Xu X, Cui C, Gu Q. 2007. Alkaloidal compounds produced by a marine-derived fungus, Aspergillus fumigatus H1-04, and their antitumor activities. Zhongguo Yaowu Huaxue Zazhi. 17:232–237. Hayashi A, Fujioka S, Nukina M, Kawano T, Shimada A, Kimura Y. 2007. Fumiquinones A and B, nematicidal quinones produced by Aspergillus fumigatus. Biosci Biotechnol Biochem. 71:1697–1702. Klich MA. 2002. Identification of common Aspergillus species. Utrecht: Centraalbureau voor Schimmelcultures. Larsen TO, Smedsgaard J, Nielsen KF, Hansen MAE, Samson RA, Frisvad JC. 2007. Production of mycotoxins by Aspergillus lentulus and other medically important and closely related species in section Fumigati. Med Mycol. 45:225–232. Lee YM, Kim MJ, Li H, Zhang P, Bao B, Lee KJ, Jung JH. 2013. Marine-derived Aspergillus species as source of bioactive secondary metabolites. Mar Biotechnol. 15:499–519. Li X, Zhang Q, An-zhang L, Gao J. 2012. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J Agric Food Chem. 60:3424– 3431. Martı´nez-Luis S, Cherigo L, Arnold E, Spadafora C, Gerwick WH, Cubilla-Rios L. 2012. Antiparasitic and anticancer constituents of the endophytic fungus Aspergillus sp. strain F1544. Nat Prod Commun. 7:165–168.
Downloaded by [Fudan University] at 08:07 16 May 2015
6
N.N. Saraiva et al.
Mehedi MAU, Molla AH, Khondkar P, Sultana S, Islam MA, Rashid MA, Chowdhury R. 2010. Pseurotin A: an antibacterial secondary metabolite from Aspergillus fumigatus. Asian J Chem. 22:2611–2614. Menna M. 2009. Antitumor potential of natural products from Mediterranean ascidians. Phytochem Rev. 8:461–472. Mizoue K, Okazaki T, Hanada K, Amamoto T, Yamagishi M, Omura S. 1987. Physiologically active substance FD-838 and process for preparing the same. European patent application. EP 216607 A2 19870401. Montenegro TGC, Rodrigues FAR, Jimenez PC, Angelim AL, Melo VMM, Rodrigues Filho E, De Oliveira MCF, CostaLotufo LV. 2012. Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei. Chem Biodivers. 9:2203–2209. Rasmussen RR, Rasmussen PH, Larsen TO, Bladt TT, Binderup ML. 2010. In vitro cytotoxicity of fungi spoiling maize silage. Food Chem Toxicol. 49:31–44. Samson RA, Hong S, Peterson SW, Frisvad JC, Varga J. 2007. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol. 59:147–203. Turner WB. 1965. The production of trypacidin and monomethylsulochrin by Aspergillus fumigatus. J Chem Soc Perkin. 1:6658–6659. Wang F, Li D, Zhu T, Zhang M, Gu Q. 2011. Pseurotin A1 and A2, two new 1-oxa-7 azaspiro[4.4]non-2-ene-4,6-diones from the holothurians derived fungus Aspergillus fumigates WFZ-25. Can J Chem. 89:72–76. Wenke J, Anke H, Sternce O. 1993. Pseurotin A and 8-O-demethylpseurotin a from Aspergillus fumigatus and their inhibitory activities on chitin synthase. Biosci Biotechnol Biochem. 57:961– 964. Wink J, Grabley S, Gareis M, Zeeck A, Philipps S. 1991. Biologically active pseurotins A and D, new metabolites from Aspergillus fumigatus, process for their preparation and their use as apomorphine antagonists. European patent application. EP 546475. Wu QX, Jin XJ, Draskovic M, Crews MS, Tenney K, Valeriote FA, Yao XJ, Crews P. 2012. Unraveling the numerous biosynthetic products of the marine sediment-derived fungus, Aspergillus insulicola. Phytochem Lett. 5:114–117. Yi S, Kentaro T, Yasushi T, Minoru Y, Yuichi N, Shigeru O, Shigeki M. 2012. Gliotoxin analogues from a marinederived fungus, Penicillium sp., and their cytotoxic and histone methyltransferase inhibitory activities. J Nat Prod. 75:111–114. Zhang L, Li Z, Bai J, Wu X, Wang Y, Hua H. 2011. Metabolites of Aspergillus sp. HT-2. Zhongguo Yaoxue Zazhi. 46:1154–1158. Zhang M, Wang W, Fang Y, Zhu T, Gu Q, Zhu W. 2008. Cytotoxic alkaloids and antibiotic nordammarane triterpenoids from the marine-derived fungus Aspergillus sydowii. J Nat Prod. 71:985–989.
