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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new compounds from a marinederived fungus Penicilliumoxalicum ab
b
ab
c
Pei-Le Wang , Dan-Yi Li , Lei-Rui Xie , Xin Wu , Hui-Ming Hua Zhan-Lin Li
ab
&
ab
a
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China c
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China Published online: 21 Nov 2013.
To cite this article: Pei-Le Wang, Dan-Yi Li, Lei-Rui Xie, Xin Wu, Hui-Ming Hua & Zhan-Lin Li (2014) Two new compounds from a marine-derived fungus Penicilliumoxalicum, Natural Product Research: Formerly Natural Product Letters, 28:5, 290-293, DOI: 10.1080/14786419.2013.856906 To link to this article: http://dx.doi.org/10.1080/14786419.2013.856906
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. 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 &
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Natural Product Research, 2014 Vol. 28, No. 5, 290–293, http://dx.doi.org/10.1080/14786419.2013.856906
Two new compounds from a marine-derived fungus Penicillium oxalicum Pei-Le Wanga,b, Dan-Yi Lib, Lei-Rui Xiea,b, Xin Wuc, Hui-Ming Huaa,b* and Zhan-Lin Lia,b*
Downloaded by [University of Cambridge] at 06:03 16 October 2014
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; bSchool of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; cSchool of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China
(Received 29 May 2013; final version received 15 October 2013) Two new anthraquinones, emodin-3-O-sulphate (1) and citreorosein-3-O-sulphate (2), as well as five known anthraquinones, were isolated from a marine-derived fungus Penicillium oxalicum. The structures of these compounds were determined by spectroscopic methods (1D and 2D NMR, HR-ESI-MS). Keywords: marine-derived fungus; Penicillium oxalicum; anthraquinone sulphate
1. Introduction Natural products from marine sources, especially from marine-derived microorganisms, are still of great interest for their distinctive scaffold and diverse bioactivities (Blunt et al. 2012, 2013). In this investigation into the antitumour products from marine-derived fungi, two new anthraquinones, emodin-3-O-sulphate (1) and citreorosein-3-O-sulphate (2), and five known compounds including emodin (3, Choi et al. 1996), citreorosein (4, Ren et al. 2006), isorhodoptilometrin (5, Ren et al. 2006), chrysophanol (6, Kang et al. 2008) and aloe-emodin (7, Kang et al. 2008) (Figure 1) were obtained from a fungus Penicillium oxalicum 2HL-M-6, which was isolated from the sea mud sample collected in Bohai Bay, China. This paper reports the isolation and structural elucidation of compounds 1 and 2.
2. Results and discussion Compound 1 was isolated as a yellow powder. Its molecular formula of C15H10O8S (12 degrees of unsaturation) was determined by HR-ESI-MS (m/z 349.0001 [M 2 H]2). A fragment ion peak at m/z 269.0446 [M-H-SO3]2, calcd for C15H9O5, was also observed from the HR-ESI-MS spectrum, indicative of a sulphate group. The UV spectrum with maximum absorption at 220, 253 and 436 nm was suggestive of an anthraquinone derivative (Lee & Kim 1995). The 1H NMR spectrum of compound 1 revealed two chelated hydroxyl groups at d 12.01 (1H, s) and 11.97 (1H, s), two sets of meta-coupled aromatic protons at d 7.55 (1H, br. s), 7.54 (1H, d, J ¼ 2.4 Hz), 7.20 (1H, br. s) and 7.07 (1H, d, J ¼ 2.4 Hz), along with a methyl protons at d 2.43 (3H, s). Fifteen carbon signals, including two carbonyl carbons at dC 190.3 and 181.2, three oxygenated sp2 carbons at dC 163.3, 161.5 and 160.9, nine aromatic carbons signals and a methyl carbon at dC 21.5, were observed in the 13C NMR spectrum. The above data suggested that 1 was an anthraquinone derivative composing of a sulphate group, a methyl group and two a-hydroxyl groups, based on the downfield shifting of dC 190.3 (Berger & Castonguay 1978). *Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
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Figure 1. Structures of compounds 1 – 7.
HMBC correlations established the structures of two aromatic rings. One of the metacoupled aromatic protons at d 7.55 showed long-range correlations with dC 181.2, 124.0, 113.6 and 21.5, suggesting that dH 7.55 and the aromatic proton at d 7.20, coupling with it, were signals for H-5 and H-7, respectively. The methyl group at d 2.43 was assigned to be attached to C-6, which was supported by its long-range correlations with dC 148.6, 124.0 and 120.6. The hydroxyl group at d 11.97 showing long-range correlations with the carbons at dC 161.5, 124.0 and 113.6 was placed at C-8. As for the other aromatic ring, the meta-coupled aromatic protons at d 7.54 displayed long-range correlations with dC 160.9 and 111.1, and proton at d 7.07 was correlated with dC 160.9 and 111.2, indicating that dH 7.54 and dH 7.07 were assigned to H-4 and H-2, respectively. The long-range correlations between the hydroxyl group at d 12.01 and the carbon signals at d 163.3 and 112.2 suggested that this hydroxyl group was placed at C-1. Comparison of the 13C NMR data of 1 with those of emodin (3) showed that the signal for C-3 of 1 at d 160.9 shifted up-field from that of 3 at d 165.5, while those for C-2, C-4 and C-9a of 1 at d 111.1, 111.2 and 112.2 shifted down-field from those of 3 at d 107.8, 108.7 and 108.7, respectively, indicating that the sulphate unit was located at C-3 (Lee & Kim 1995), supported by the down-field shifting of H-2 and H-4 compared with 3. Consequently, compound 1 was identified as 1,8-dihydroxy-5-methylanthraquinone-3-O-sulphate (emodin-3-O-sulphate). Compound 2 was obtained as a yellow powder. Its molecular formula was determined to be C15H10O9S (12 degrees of unsaturation) by HR-ESI-MS (m/z 364.9974 [M 2 H]2), and a fragment ion peak at m/z 285.0403 [M-H-SO3]2 was indicative of the existence of a sulphate unit. Comparison of the 1H and 13C NMR data of 2 with those of 1 showed that the signals for CH3-11 in 1 were replaced by a hydroxymethyl group [dH 4.60 (2H, d, J ¼ 4.5 Hz); dC 62.1], which showed long-range correlations with d 153.2, 120.9 and 117.8 in the HMBC experiment. Hence, compound 2 was assigned as 1,8-dihydroxy-5-hydroxymethylanthraquinone-3-Osulphate (citreorosein-3-O-sulphate). 3. Experimental 3.1. General experimental procedures IR spectra were recorded on a Bruker IFS-55 spectrometer (KBr Pellet; Bruker Optics, Ettlingen, Germany). UV spectra were run on a Shimadzu UV-1700 spectrometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra were recorded on a Bruker ARX-300 or AV-600 NMR spectrometer (Bruker BioSpin, Fallanden, Switzerand), with tetramethyl silane as an internal standard. HR-ESI-MS spectra were recorded on a Bruker micrOTOF-Q mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Chromatographic silica gel (200 –300 mesh) was produced by Qingdao Haiyang Chemical Co. Ltd, Qingdao, China and Sephadex LH-20 was
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P.-L. Wang et al.
purchased from GE Healthcare (Little Chalfont, Bucks, UK). Octadecylsilyl silica gel (50 mm) was produced by YMC Co. Ltd, Kyoto, Japan.
3.2. Microorganism material The strain, Penicillium sp. (2HL-M-6), was isolated from the mud sample collected from Bohai Bay, Liaoning Province, China, and identified as P. oxalicum by the morphological evaluation and ITS1-5.8S-ITS2 of rRNA gene sequence (accession number in GenBank: KC515450).
Downloaded by [University of Cambridge] at 06:03 16 October 2014
3.3. Fermentation and extraction This fungus was stationary cultivated in a 75 L scale using Erlenmeyer flasks containing fermentation medium (D -mannitol 2%, D -glucose 2%, yeast extract 0.5%, peptone 1%, KH2PO4 0.05%, MgSO4 0.03%, corn syrup 0.1%, sea salt 3.3%) at 288C for 30 days. The fermented broth was filtered by cheesecloth to be separated into the supernatant and the mycelia. The supernatant was partitioned with EtOAc and the mycelia were extracted by ultrasonicator with acetone.
3.4. Separation and purification The acetone extract (100 g) of mycelia was chromatographed over silica gel using a gradient system of petroleum ether (PE) (60 –908C) –acetone (100:0 to 0:100). The collected fractions were combined on basis of their thin-layer chromatography characteristics to yield eight fractions (Fr. A – Fr. H). Fr. A was separated by Sephadex LH-20 CC eluted with MeOH to yield three fractions (Fr. A1 –Fr. A3), and Fr. A1 was recrystallised repeatedly in PE –acetone to afford 6 (15.6 mg), so did Fr. A2 and Fr. A3 to afford 3 (14.7 mg) and 7 (20.2 mg), respectively. Fr. H (15 g) was chromatographed on silica gel eluted with CH2Cl2 – MeOH from 10:1 to 3:1 to afford three fractions (H1 –H3). Fr. H1 was separated by Sephadex LH-20 CC eluted with MeOH to yield three subfractions (Fr. H1A –Fr. H1C), and compound 1 (3.0 mg) was obtained by repeated recrystallisation in PE –acetone of Fr. H1A. Fr. H1C was purified on Sephadex LH20 CC eluted with MeOH to yield 2 (2.6 mg). The EtOAc extract (30 g) was subjected to silica gel CC eluted with PE – acetone to give seven fractions Fr. A0 to Fr. G0 . Compound 4 (11.2 mg) and compound 5 (2.2 mg) were purified by Sephadex LH-20 CC eluted with MeOH from Fr. E0 . Emodin-3-O-sulphate (1): yellow powder; UV (MeOH) lmax: 220, 253, 436 nm; IR (KBr): 3417, 2949, 1675, 1384, 1261, 1216, 1171 cm21; ESI-MS: m/z 348.9 [M 2 H]2; HR-ESI-MS m/ z: 349.0001 [M 2 H]2 (calcd for C15H9O8S 349.0018). 1H NMR (600 MHz, DMSO-d6): d 7.07 (1H, d, J ¼ 2.4 Hz, H-2), 7.54 (1H, d, J ¼ 2.4 Hz, H-4), 7.55 (1H, br. s, H-5), 7.20 (1H, br. s, H7), 2.43 (3H, s, H-11), 11.97 (1H, s, 8-OH), 12.01 (1H, s, 1-OH). 13C NMR (150 MHz, DMSOd6): d 163.3 (C-1), 111.1 (C-2), 160.9 (C-3), 111.2 (C-4), 120.6 (C-5), 148.6 (C-6), 124.0 (C-7), 161.5 (C-8), 190.3 (C-9), 181.2 (C-10), 21.5 (C-11), 134.5 (C-4a), 113.6 (C-8a), 112.2 (C-9a), 133.0 (C-10a). Citreorosein-3-O-sulphate (2): yellow powder; UV (MeOH) lmax: 223, 254, 436 nm; IR (KBr): 3421, 2923, 1651, 1384, 1272, 1220, 1110 cm21; ESI-MS: m/z 364.9 [M 2 H]2; HRESI-MS m/z: 364.9974 [M 2 H]2 (calcd for C15H9O9S 364.9967). 1H NMR (600 MHz, DMSOd6): d 7.06 (1H, d, J ¼ 2.4 Hz, H-2), 7.54 (1H, d, J ¼ 2.4 Hz, H-4), 7.68 (1H, br. s, H-5), 7.26 (1H, br. s, H-7), 4.60 (2H, d, J ¼ 4.5 Hz, H-11), 12.02 (2H, s, 1 or 8-OH), 5.54 (1H, br. s, 11OH). 13C NMR (150 MHz, DMSO-d6): d 163.5 (C-1), 111.1 (C-2), 160.7 (C-3), 111.6 (C-4), 117.8 (C-5), 153.2 (C-6), 120.9 (C-7), 161.6 (C-8), 190.3 (C-9), 181.5 (C-10), 62.1 (C-11), 134.7 (C-4a), 114.5 (C-8a), 112.4 (C-9a), 133.2 (C-10a).
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4. Conclusion The isolation of emodin and citreorosein, together with their sulphates in this work, prompted us to synthesise the sulphates of phenolic natural products such as anthraquinones and flavonoids which are more hydrophilic and convenient to be prepared as injections, and to investigate the possibility that P. oxalicum (2HL-M-6) could be used for the bio-sulphation of phenols. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S9.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21002064 & 20872094) and Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University.
References Berger Y, Castonguay A. 1978. The carbon-13 nuclear magnetic resonance spectra of anthraquinone, eight polyhydroxyanthraquinones and eight polymethoxyanthraquinones. Org Magn Reson. 11:375–377. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR. 2012. Marine natural products. Nat Prod Rep. 29:144–222. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR. 2013. Marine natural products. Nat Prod Rep. 30:237–323. Choi JS, Jung JH, Lee HJ, Kang SS. 1996. The NMR assignments of anthraquinones from Cassia tora. Arch Pharm Res. 19:302–306. Kang SC, Lee CM, Choung ES, Bak JP, Bae JJ, Yoo HS, Kwak JH, Zee OP. 2008. Anti-proliferative effects of estrogen receptor-modulating compounds isolated from Rheum palmatum. Arch Pharm Res. 31:722–726. Lee NK, Kim YH. 1995. New cytotoxic anthraquinones from the crinoid ptilometra: 10 -deoxyrhodoptilometrin-6-Osulfate and rhodoptilometrin-6-O-sulfate. Bull Korean Chem Soc. 16:1011–1013. Ren H, Tian L, Gu QQ, Zhu WM. 2006. Secalonic acid D: a cytotoxic constituent from marine lichen-derived fungus Gliocladium sp. T31. Arch Pharm Res. 29:59–63.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new compounds from a marinederived fungus Penicilliumoxalicum ab
b
ab
c
Pei-Le Wang , Dan-Yi Li , Lei-Rui Xie , Xin Wu , Hui-Ming Hua Zhan-Lin Li
ab
&
ab
a
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China b
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China c
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China Published online: 21 Nov 2013.
To cite this article: Pei-Le Wang, Dan-Yi Li, Lei-Rui Xie, Xin Wu, Hui-Ming Hua & Zhan-Lin Li (2014) Two new compounds from a marine-derived fungus Penicilliumoxalicum, Natural Product Research: Formerly Natural Product Letters, 28:5, 290-293, DOI: 10.1080/14786419.2013.856906 To link to this article: http://dx.doi.org/10.1080/14786419.2013.856906
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. 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 &
Downloaded by [University of Cambridge] at 06:03 16 October 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 5, 290–293, http://dx.doi.org/10.1080/14786419.2013.856906
Two new compounds from a marine-derived fungus Penicillium oxalicum Pei-Le Wanga,b, Dan-Yi Lib, Lei-Rui Xiea,b, Xin Wuc, Hui-Ming Huaa,b* and Zhan-Lin Lia,b*
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a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; bSchool of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; cSchool of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China
(Received 29 May 2013; final version received 15 October 2013) Two new anthraquinones, emodin-3-O-sulphate (1) and citreorosein-3-O-sulphate (2), as well as five known anthraquinones, were isolated from a marine-derived fungus Penicillium oxalicum. The structures of these compounds were determined by spectroscopic methods (1D and 2D NMR, HR-ESI-MS). Keywords: marine-derived fungus; Penicillium oxalicum; anthraquinone sulphate
1. Introduction Natural products from marine sources, especially from marine-derived microorganisms, are still of great interest for their distinctive scaffold and diverse bioactivities (Blunt et al. 2012, 2013). In this investigation into the antitumour products from marine-derived fungi, two new anthraquinones, emodin-3-O-sulphate (1) and citreorosein-3-O-sulphate (2), and five known compounds including emodin (3, Choi et al. 1996), citreorosein (4, Ren et al. 2006), isorhodoptilometrin (5, Ren et al. 2006), chrysophanol (6, Kang et al. 2008) and aloe-emodin (7, Kang et al. 2008) (Figure 1) were obtained from a fungus Penicillium oxalicum 2HL-M-6, which was isolated from the sea mud sample collected in Bohai Bay, China. This paper reports the isolation and structural elucidation of compounds 1 and 2.
2. Results and discussion Compound 1 was isolated as a yellow powder. Its molecular formula of C15H10O8S (12 degrees of unsaturation) was determined by HR-ESI-MS (m/z 349.0001 [M 2 H]2). A fragment ion peak at m/z 269.0446 [M-H-SO3]2, calcd for C15H9O5, was also observed from the HR-ESI-MS spectrum, indicative of a sulphate group. The UV spectrum with maximum absorption at 220, 253 and 436 nm was suggestive of an anthraquinone derivative (Lee & Kim 1995). The 1H NMR spectrum of compound 1 revealed two chelated hydroxyl groups at d 12.01 (1H, s) and 11.97 (1H, s), two sets of meta-coupled aromatic protons at d 7.55 (1H, br. s), 7.54 (1H, d, J ¼ 2.4 Hz), 7.20 (1H, br. s) and 7.07 (1H, d, J ¼ 2.4 Hz), along with a methyl protons at d 2.43 (3H, s). Fifteen carbon signals, including two carbonyl carbons at dC 190.3 and 181.2, three oxygenated sp2 carbons at dC 163.3, 161.5 and 160.9, nine aromatic carbons signals and a methyl carbon at dC 21.5, were observed in the 13C NMR spectrum. The above data suggested that 1 was an anthraquinone derivative composing of a sulphate group, a methyl group and two a-hydroxyl groups, based on the downfield shifting of dC 190.3 (Berger & Castonguay 1978). *Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
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Figure 1. Structures of compounds 1 – 7.
HMBC correlations established the structures of two aromatic rings. One of the metacoupled aromatic protons at d 7.55 showed long-range correlations with dC 181.2, 124.0, 113.6 and 21.5, suggesting that dH 7.55 and the aromatic proton at d 7.20, coupling with it, were signals for H-5 and H-7, respectively. The methyl group at d 2.43 was assigned to be attached to C-6, which was supported by its long-range correlations with dC 148.6, 124.0 and 120.6. The hydroxyl group at d 11.97 showing long-range correlations with the carbons at dC 161.5, 124.0 and 113.6 was placed at C-8. As for the other aromatic ring, the meta-coupled aromatic protons at d 7.54 displayed long-range correlations with dC 160.9 and 111.1, and proton at d 7.07 was correlated with dC 160.9 and 111.2, indicating that dH 7.54 and dH 7.07 were assigned to H-4 and H-2, respectively. The long-range correlations between the hydroxyl group at d 12.01 and the carbon signals at d 163.3 and 112.2 suggested that this hydroxyl group was placed at C-1. Comparison of the 13C NMR data of 1 with those of emodin (3) showed that the signal for C-3 of 1 at d 160.9 shifted up-field from that of 3 at d 165.5, while those for C-2, C-4 and C-9a of 1 at d 111.1, 111.2 and 112.2 shifted down-field from those of 3 at d 107.8, 108.7 and 108.7, respectively, indicating that the sulphate unit was located at C-3 (Lee & Kim 1995), supported by the down-field shifting of H-2 and H-4 compared with 3. Consequently, compound 1 was identified as 1,8-dihydroxy-5-methylanthraquinone-3-O-sulphate (emodin-3-O-sulphate). Compound 2 was obtained as a yellow powder. Its molecular formula was determined to be C15H10O9S (12 degrees of unsaturation) by HR-ESI-MS (m/z 364.9974 [M 2 H]2), and a fragment ion peak at m/z 285.0403 [M-H-SO3]2 was indicative of the existence of a sulphate unit. Comparison of the 1H and 13C NMR data of 2 with those of 1 showed that the signals for CH3-11 in 1 were replaced by a hydroxymethyl group [dH 4.60 (2H, d, J ¼ 4.5 Hz); dC 62.1], which showed long-range correlations with d 153.2, 120.9 and 117.8 in the HMBC experiment. Hence, compound 2 was assigned as 1,8-dihydroxy-5-hydroxymethylanthraquinone-3-Osulphate (citreorosein-3-O-sulphate). 3. Experimental 3.1. General experimental procedures IR spectra were recorded on a Bruker IFS-55 spectrometer (KBr Pellet; Bruker Optics, Ettlingen, Germany). UV spectra were run on a Shimadzu UV-1700 spectrometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra were recorded on a Bruker ARX-300 or AV-600 NMR spectrometer (Bruker BioSpin, Fallanden, Switzerand), with tetramethyl silane as an internal standard. HR-ESI-MS spectra were recorded on a Bruker micrOTOF-Q mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Chromatographic silica gel (200 –300 mesh) was produced by Qingdao Haiyang Chemical Co. Ltd, Qingdao, China and Sephadex LH-20 was
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P.-L. Wang et al.
purchased from GE Healthcare (Little Chalfont, Bucks, UK). Octadecylsilyl silica gel (50 mm) was produced by YMC Co. Ltd, Kyoto, Japan.
3.2. Microorganism material The strain, Penicillium sp. (2HL-M-6), was isolated from the mud sample collected from Bohai Bay, Liaoning Province, China, and identified as P. oxalicum by the morphological evaluation and ITS1-5.8S-ITS2 of rRNA gene sequence (accession number in GenBank: KC515450).
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3.3. Fermentation and extraction This fungus was stationary cultivated in a 75 L scale using Erlenmeyer flasks containing fermentation medium (D -mannitol 2%, D -glucose 2%, yeast extract 0.5%, peptone 1%, KH2PO4 0.05%, MgSO4 0.03%, corn syrup 0.1%, sea salt 3.3%) at 288C for 30 days. The fermented broth was filtered by cheesecloth to be separated into the supernatant and the mycelia. The supernatant was partitioned with EtOAc and the mycelia were extracted by ultrasonicator with acetone.
3.4. Separation and purification The acetone extract (100 g) of mycelia was chromatographed over silica gel using a gradient system of petroleum ether (PE) (60 –908C) –acetone (100:0 to 0:100). The collected fractions were combined on basis of their thin-layer chromatography characteristics to yield eight fractions (Fr. A – Fr. H). Fr. A was separated by Sephadex LH-20 CC eluted with MeOH to yield three fractions (Fr. A1 –Fr. A3), and Fr. A1 was recrystallised repeatedly in PE –acetone to afford 6 (15.6 mg), so did Fr. A2 and Fr. A3 to afford 3 (14.7 mg) and 7 (20.2 mg), respectively. Fr. H (15 g) was chromatographed on silica gel eluted with CH2Cl2 – MeOH from 10:1 to 3:1 to afford three fractions (H1 –H3). Fr. H1 was separated by Sephadex LH-20 CC eluted with MeOH to yield three subfractions (Fr. H1A –Fr. H1C), and compound 1 (3.0 mg) was obtained by repeated recrystallisation in PE –acetone of Fr. H1A. Fr. H1C was purified on Sephadex LH20 CC eluted with MeOH to yield 2 (2.6 mg). The EtOAc extract (30 g) was subjected to silica gel CC eluted with PE – acetone to give seven fractions Fr. A0 to Fr. G0 . Compound 4 (11.2 mg) and compound 5 (2.2 mg) were purified by Sephadex LH-20 CC eluted with MeOH from Fr. E0 . Emodin-3-O-sulphate (1): yellow powder; UV (MeOH) lmax: 220, 253, 436 nm; IR (KBr): 3417, 2949, 1675, 1384, 1261, 1216, 1171 cm21; ESI-MS: m/z 348.9 [M 2 H]2; HR-ESI-MS m/ z: 349.0001 [M 2 H]2 (calcd for C15H9O8S 349.0018). 1H NMR (600 MHz, DMSO-d6): d 7.07 (1H, d, J ¼ 2.4 Hz, H-2), 7.54 (1H, d, J ¼ 2.4 Hz, H-4), 7.55 (1H, br. s, H-5), 7.20 (1H, br. s, H7), 2.43 (3H, s, H-11), 11.97 (1H, s, 8-OH), 12.01 (1H, s, 1-OH). 13C NMR (150 MHz, DMSOd6): d 163.3 (C-1), 111.1 (C-2), 160.9 (C-3), 111.2 (C-4), 120.6 (C-5), 148.6 (C-6), 124.0 (C-7), 161.5 (C-8), 190.3 (C-9), 181.2 (C-10), 21.5 (C-11), 134.5 (C-4a), 113.6 (C-8a), 112.2 (C-9a), 133.0 (C-10a). Citreorosein-3-O-sulphate (2): yellow powder; UV (MeOH) lmax: 223, 254, 436 nm; IR (KBr): 3421, 2923, 1651, 1384, 1272, 1220, 1110 cm21; ESI-MS: m/z 364.9 [M 2 H]2; HRESI-MS m/z: 364.9974 [M 2 H]2 (calcd for C15H9O9S 364.9967). 1H NMR (600 MHz, DMSOd6): d 7.06 (1H, d, J ¼ 2.4 Hz, H-2), 7.54 (1H, d, J ¼ 2.4 Hz, H-4), 7.68 (1H, br. s, H-5), 7.26 (1H, br. s, H-7), 4.60 (2H, d, J ¼ 4.5 Hz, H-11), 12.02 (2H, s, 1 or 8-OH), 5.54 (1H, br. s, 11OH). 13C NMR (150 MHz, DMSO-d6): d 163.5 (C-1), 111.1 (C-2), 160.7 (C-3), 111.6 (C-4), 117.8 (C-5), 153.2 (C-6), 120.9 (C-7), 161.6 (C-8), 190.3 (C-9), 181.5 (C-10), 62.1 (C-11), 134.7 (C-4a), 114.5 (C-8a), 112.4 (C-9a), 133.2 (C-10a).
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4. Conclusion The isolation of emodin and citreorosein, together with their sulphates in this work, prompted us to synthesise the sulphates of phenolic natural products such as anthraquinones and flavonoids which are more hydrophilic and convenient to be prepared as injections, and to investigate the possibility that P. oxalicum (2HL-M-6) could be used for the bio-sulphation of phenols. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S9.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21002064 & 20872094) and Program for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University.
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