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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new α-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp. a
a
a
b
Yoshihito Shiono , Fumiaki Shibuya , Takuya Koseki , Harizon , b
c
c
Unang Supratman , Shota Uesugi & Ken-ichi Kimura a
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan b
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang 45363, Indonesia c
Laboratory of Chemical Biology, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan Published online: 30 Oct 2014.
To cite this article: Yoshihito Shiono, Fumiaki Shibuya, Takuya Koseki, Harizon, Unang Supratman, Shota Uesugi & Ken-ichi Kimura (2014): A new α-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp., Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.961919 To link to this article: http://dx.doi.org/10.1080/10286020.2014.961919
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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.961919
A new a-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp.
Downloaded by [University of Colorado - Health Science Library] at 11:29 14 December 2014
Yoshihito Shionoa*, Fumiaki Shibuyaa, Takuya Kosekia, Harizonb, Unang Supratmanb, Shota Uesugic and Ken-ichi Kimurac a
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan; bDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang 45363, Indonesia; cLaboratory of Chemical Biology, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan (Received 30 May 2014; final version received 31 August 2014) A new a-pyrone derivative, compound 2, and a known one, cladobotrin V, were isolated from the culture broth of the mangrove endophyte Fusarium sp. IM-37. Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 2 restored the growth inhibition caused by hyperactivated Ca2þ-signaling in mutant yeast. Keywords: mangrove; endophyte; Fusarium sp; cladobotrin V; Ca2þ-signaling
1.
Introduction
Marine-derived microorganisms have become rich sources of novel and bioactive secondary metabolites for drug discovery. In fact, structurally unique and biologically active compounds have been isolated from marine-derived microorganisms [1]. In our search for new metabolites from mangrove endophytic fungi from the Java Sea of Indonesia, we have isolated a new bioactive metabolite, 12-O-deacetylphomoxanthone A from the Phomopsis sp. IM 41-1, which showed antimicrobial activity against plant pathogenic fungi [2]. Generally, endophytes inhabit the inner tissues of plants as their specific microbial biota. Ecological interaction of the mangrove microenvironment has not only evolved the host plants but also endophytes, which share a common substrate. The habitat of mangroves demands a frequent variation in salinity, temperature, and moisture; hence, microorganisms isolated from unexplored habitats like
mangroves have a unique and proven capacity to produce novel compounds [3]. This paper reports on the isolation, structural elucidation, and biological activities of cladobotrin V (1), and one new compound 2 from the mangrove endophytic fungus Fusarium sp. IM-37 isolated from Rhizophora mucronata (Figure 1). 2.
Results and discussion
Fusarium sp. IM-37 was isolated from R. mucronata, and the producing strain IM37 was cultivated on sterilized, unpolished rice (18 g/Petri dish £ 50; total of 900 g) at 258C for 4 weeks. A methanol extract from the fermented unpolished rice was concentrated and then partitioned between ethyl acetate (EtOAc) and water. The purification of the EtOAc layer was guided by the characteristically intense blue coloration with a vanillin –sulfuric acid solution on the TLC plates. The EtOAc layer was chromatographed on a silica gel
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structures of compounds 1 –4.
column using a stepwise elution of nhexane/EtOAc. Further chromatographic separation using silica and ODS gel yielded two compounds: cladobotrin V (1, 25 mg) and the new related derivative 2 (12 mg). Cladobotrin V (1, 5-hydroxymethyl-4methoxy-6-(E-propenyl)-2-pyrone) was identified on the basis of its 1H and 13C NMR, 1H – 1H COSY, HMQC, and HMBC data (Table 1) [4].
Compound 2 was assigned the molecular formula C10H10O6 on the basis of the [M 2 H]2 peak in the HR-ESI-TOFMS data. The UV spectrum of 2 exhibited absorption maxima at 235 and 341 nm, suggesting the presence of a conjugated enone system in its structure. The IR spectral data showed absorption bands at 3300, 1720, 1700, and 1675 cm21, indicating the presence of a hydroxyl and an unsaturated ester group, respectively.
Journal of Asian Natural Products Research Table 1.
1
H, 13C NMR, and HMBC data for 1 and 2 (d in ppm, J in Hz).a 1b
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3
No.
dC
1 2 3 4 5 6 7 8 9 10
170.2 s 88.6 d 163.3 s 109.3 s 157.0 s 137.0 d 119.0 d 18.7 q 56.3 q 54.1 t
dH
2c
dC
HMBC
5.48 (1H, s)
1, 3
6.81 (1H, 6.43 (1H, 1.94 (3H, 3.86 (3H, 4.52 (2H,
4, 8 5, 8 6, 7 3 3, 4, 5
d, 16.0) m) d, 6.7) s) s)
171.7 s 91.6 d 164.9 s 117.1 s 156.1 s 130.4 d 129.6 d 169.9 s 57.5 q 49.4 t
dH
HMBC
5.76 (1H, s)
3, 4
7.57 (1H, d, 16.1) 6.76 (1H, d, 16.1)
4, 8 5, 8
3.93 (3H, s) 4.55 (2H, s)
3 3, 5
a 13
C NMR spectrum was recorded at 100 MHz and 1H NMR spectrum at 400 MHz. Measured in CDCl3. c Measured in CD3OD. b
The 13C NMR spectrum (Table 1) showed the presence of 10 carbon signals, and further analysis of the DEPT experiment revealed that the 13C NMR signals consisted of two sp3, six sp2, and two carbonyl carbon atoms. The signals of six olefinic carbon atoms indicated the presence of three double bonds in 2. These carbon atoms along with two carbonyl carbon atoms accounted for five degrees of unsaturation; therefore, the remaining one degree of unsaturation should be due to the presence of one ring in the molecule. In the 1 H NMR spectrum, one methoxy, one oxymethylene, and three olefinic methines were observed. Analysis of 1H – 1H COSY data showed one spin network of the couplings between H-6 and H-7 (Figure 2). The lactone linkage required by the molecular formula was shown to be
Figure 2. Key HMBC (arrows) correlations observed for 2.
between C-1 and C-5 by the highly similar 13 C shift values of 2 compared with those of the a-pyrone [5,6]. In the HMBC spectrum, HMBC correlations from H-6 to C-4 and C-8, and from H-7 to C-5 showed the presence of a propenoic acid moiety [dH 6.76 (1H, d, J ¼ 16.1 Hz, H-7), 7.57 (1H, d, J ¼ 16.1 Hz, H-6), dC 171.7 (C-1), 130.4 (C-6), 129.6 (C-7)] at C-5 of the pyrone. Observations of HMBC correlations from H-10 to C-3 and C-5, and from H-2 to C-1 and C-4, together with those from OMe-9 to C-3 established the planar structure of 2 as shown in Figure 2. These observations indicate that 2 is a new derivative of 1. The microbial metabolites closest in similarity to 1, which possess the unique basic structural units 5-hydroxymethyl-4methoxy-6-(E-propenyl or propenoic acid)-2-pyrone, were allantopyrone A (3) and islandic acid-II methyl ester (4) that were isolated from the endophyte Allantophomopsis lycopodina KS-97 [6]. Compounds 3 and 4 showed a 50% cell growth inhibition of HL60 cells at a concentration of 0.32 and 6.55 mM, respectively. Assessment of cytotoxicity of 1 and 2 against HL60 cells by a MTT assay showed no cytotoxic activity at a concentration of 100 mM. The lower activity of 1 and 2 compared with that of
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dose-dependent growth-restoring activity in mutant yeast involving Ca2þ-signal transduction (from 25 to 1.56 mg/spot) (Figure 3). However, 1 exhibited no activity even at a dose of 25 mg/spot (data not shown). Further pharmacological studies of 1 and 2 are currently underway. 3. Experimental 3.1 General experimental procedures Figure 3. Growth-restored activity of 2 against the mutant strain, S. cerevisiae YNS17. Growth-restored activities of 2 against S. cerevisiae YNS17 (zds1D erg3D pdr1D pdr3D) in the presence of 0.3 M CaCl2. 1:25 mg/spot, 2: 12.5 mg/spot, 3: 6.25 mg/spot, 4. 3.13 mg/spot, 5: 1.56 mg/spot, 6: 0.78 mg/ spot, 7: 2.5 mg/spot (FK506).
3 and 4 confirmed that the substitution at C-2 of the 2-pyrone seems to enhance cytotoxic activity. In addition, two isolated compounds were tested for their inhibitory effect on Ca2þ-signaling using the mutant yeast strain Saccharomyces cerevisiae (zds1D erg3D pdr1D pdr3D: YNS17 strain). This mutant strain is not able to grow at high CaCl2 concentrations because of blockage of the cell-cycle progression of the G2 period by hyperactivation of the Ca2þsignaling pathway [7,8]. Cell-cycle regulation by high Ca2þ levels is affected by two parallel pathways, the calcineurin and the Mpk1 MAPK cascade. These pathways coordinately activate Swe1 at the transcriptional and post-translational level. In turn, Swe1 inhibits the cell cycle in the G2 phase by the phosphorylation of Cdc28-Clb at Tyr19. This assay system was able to detect inhibitors of Ca2þsignal transduction by their ability to stimulate cell growth as a growth zone around a paper disc containing the active compound [9,10]. In fact, the calcineurin inhibitor FK506 was detected in this mutant yeast by restoration of growth. In this screening assay system, 2 showed a
IR and UV spectra were recorded with FT710 (HORIBA, Kyoto, Japan) and Shimadzu UV mini-1240 (Shimadzu, Kyoto, Japan) spectrophotometer, respectively. Mass spectra were obtained using a waters Synapt G2 instrument (Waters, Milford, MA, USA), and 1H and 13C NMR spectra were acquired with a JEOL EX-400 spectrometer (JEOL, Tokyo, Japan). Chemical shifts are given on a d (ppm) scale with TMS as an internal standard. Semipreparative HPLC was carried out with Shimadzu pump and UV LC-10A detector (set at 210 nm, Shimadzu) on Mightysil ODS column (5 mm, 150 mm £ 6.0 mm; Kanto Chemical Co., Inc., Tokyo, Japan) at the flow rate of 2.0 ml/ min. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc.) and ODS (Fuji Silysia, Kasugai, Aichi, Japan). TLC was carried out on Merck precoated silica gel plates (silica gel 60 F254, Darmstadt, Germany), and spots were detected by spraying with 10% vanillin in H2SO4 followed by heating or by UV irradiation. 3.2 The producing strain The fungal strain Fusarium sp. IM-37 was isolated from a mangrove plant collected from a forest in Muara Angke, Jakarta, Indonesia in October 2012. The branch samples were surface-sterilized successively with 70% EtOH for 1 min, 5% sodium hypochlorite for 5 min, and 70% EtOH for 1 min, then rinsed in sterile water for two times. The sterilized samples were
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Journal of Asian Natural Products Research dried on sterilized paper and cut into 1-cm pieces. The pieces were placed on plates of potato-dextrose-agar (PDA) containing chloramphenicol (100 mg/l). After incubation at 258C for 7 days, the hyphal tips of the fungi on the plates were removed from the agar plates and transferred to PDA plates (slant). The strain IM-37 was isolated and grew on slants of PDA as white colored culture. This strain was identified to be Fusarium sp. by BEX. Co. LTD, Tokyo Japan, using a DNA analysis of the 18S rDNA regions. This fungus has been deposited at our laboratory in the Faculty of Agriculture of Yamagata University. 3.3 Fermentation, extraction, and isolation The fungal strain IM-37 was cultivated on sterilized unpolished rice (total 900 g, 18 g/Petri dish £ 50) at 258C for 4 weeks. The moldy unpolished rice was extracted with MeOH (2 l), and MeOH extract was concentrated. The resulting aqueous concentrate was partitioned into n-hexane layer (0.5 l), EtOAc layer (1.5 l), and aqueous layer (0.5 l). Purifications of eluates were monitored by the characteristic intense blue coloration with 10% vanillin in H2SO4 on TLC plates. The EtOAc layer (2.5 g) was chromatographed on a silica gel column with stepwise elution of n-hexane – EtOAc (100:0 – 0:100) and EtOAc – MeOH (50:50, 0:100), respectively, to afford fractions 11 to 1-13. Fraction 1-12 (EtOAc– MeOH, 50:50, 445 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0 –0:100) to afford fractions 2-1 to 213. Fractions 2-6 and 2-7 (CHCl3 –EtOAc, 50:50, 40:60, 210 mg) were combined and further separated by semipreparative ODS HPLC (MeOH – H2O, 60:40) to give compound 1 (25.0 mg, tR 11.9 min). Fraction 2-11 (CHCl3 – EtOAc, 0:100, 110 mg) was subjected to semipreparative
5
ODS HPLC (MeOH – H2O, 80:20) to give compound 2 (12.0 mg, tR 9.7 min). 3.3.1
Compound 2
White amorphous powder; UV (MeOH) lmax (nm): 235, 341; IR (KBr) nmax (cm21): 3300, 1720, 1700, 1675, and 1050; 1 H (400 MHz) and 13C NMR (100 MHz) spectral data, see Table 1; Negative HR-ESI-TOF-MS: m/z 225.0419 [M 2 H]2 (calcd for C10H9O6, 225.0399). 3.4 Growth-restored activity of samples against YNS17 strain Screening was performed according to the previously described method [11]. Each sample was dissolved in MeOH and twofold dilutions of them were used. Difcow yeast –peptone – dextrose (YPD) broth and YPD agar were purchased from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA). The mutant yeast, YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1:: hisG-URA3-hisG pdr3::hisG) yeast strain was derivative of strain W303-1A [10]. A 5-ml aliquot of samples was spotted on a YPD agar medium containing YNS17 strain and 0.3 M CaCl2. After 3 days of incubation at 288C, the intensity of the growth spot was observed as the result of inhibition of Ca2þ-signal transduction. FK506 (2.5 ng/spot) was used as a positive control. FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (the present Astellas Pharma, Inc., Tokyo, Japan). References [1] K. Duarte, T.A.P. Rocha-Santos, A. C. Freitas, and A.C. Duarte, TrAC Trents Anal. Chem. 34, 97 (2012). [2] Y. Shiono, T. Sasaki, F. Shibuya, Y. Yasuda, T. Koseki, and U. Supratman, Nat. Prod. Commun. 12, 1735 (2013). [3] Z. Huang, X. Cai, C. Shao, Z. She, X. Xia, Y. Chen, J. Yan, S. Zhou, and Y. Lin, Phytochemistry 69, 1604 (2008).
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[4] Y. Tezuka, Q. Huang, T. Kikuchi, A. Nishi, and K. Tsubaki, Chem. Pharm. Bull. 42, 2612 (1994). [5] R.A. Hussain and P.G. Waterman, Phytochemistry 21, 1393 (1982). [6] Y. Shiono, M. Yokoi, T. Koseki, T. Murayama, N. Aburai, and K. Kimura, J. Antibiot. 63, 251 (2010). [7] M. Mizunuma, D. Hirata, K. Miyahara, E. Tsuchiya, and T. Miyakawa, Nature 392, 303 (1998). [8] T. Miyakawa and M. Mizunuma, Biosci. Biotechnol. Biochem. 71, 633 (2007).
[9] A. Shitamukai, M. Mizunuma, D. Hirata, H. Takahashi, and T. Miyakawa, Biosci. Biotechnol. Biochem. 64, 1942 (2000). [10] R. Chanklan, E. Aihara, S. Koga, H. Takahashi, M. Mizunuma, and T. Miyakawa, Biosci. Biotechnol. Biochem. 72, 132 (2008). [11] K. Kimura, Y. Minamikawa, Y. Ogasawara, J. Yoshida, K. Saitoh, H. Shinden, Y.Q. Ye, S. Takahashi, T. Miyakawa, and H. Koshino, Fitoterapia 83, 907 (2012).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new α-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp. a
a
a
b
Yoshihito Shiono , Fumiaki Shibuya , Takuya Koseki , Harizon , b
c
c
Unang Supratman , Shota Uesugi & Ken-ichi Kimura a
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan b
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang 45363, Indonesia c
Laboratory of Chemical Biology, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan Published online: 30 Oct 2014.
To cite this article: Yoshihito Shiono, Fumiaki Shibuya, Takuya Koseki, Harizon, Unang Supratman, Shota Uesugi & Ken-ichi Kimura (2014): A new α-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp., Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.961919 To link to this article: http://dx.doi.org/10.1080/10286020.2014.961919
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.
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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
Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.961919
A new a-pyrone metabolite from a mangrove plant endophytic fungus, Fusarium sp.
Downloaded by [University of Colorado - Health Science Library] at 11:29 14 December 2014
Yoshihito Shionoa*, Fumiaki Shibuyaa, Takuya Kosekia, Harizonb, Unang Supratmanb, Shota Uesugic and Ken-ichi Kimurac a
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan; bDepartment of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang 45363, Indonesia; cLaboratory of Chemical Biology, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan (Received 30 May 2014; final version received 31 August 2014) A new a-pyrone derivative, compound 2, and a known one, cladobotrin V, were isolated from the culture broth of the mangrove endophyte Fusarium sp. IM-37. Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 2 restored the growth inhibition caused by hyperactivated Ca2þ-signaling in mutant yeast. Keywords: mangrove; endophyte; Fusarium sp; cladobotrin V; Ca2þ-signaling
1.
Introduction
Marine-derived microorganisms have become rich sources of novel and bioactive secondary metabolites for drug discovery. In fact, structurally unique and biologically active compounds have been isolated from marine-derived microorganisms [1]. In our search for new metabolites from mangrove endophytic fungi from the Java Sea of Indonesia, we have isolated a new bioactive metabolite, 12-O-deacetylphomoxanthone A from the Phomopsis sp. IM 41-1, which showed antimicrobial activity against plant pathogenic fungi [2]. Generally, endophytes inhabit the inner tissues of plants as their specific microbial biota. Ecological interaction of the mangrove microenvironment has not only evolved the host plants but also endophytes, which share a common substrate. The habitat of mangroves demands a frequent variation in salinity, temperature, and moisture; hence, microorganisms isolated from unexplored habitats like
mangroves have a unique and proven capacity to produce novel compounds [3]. This paper reports on the isolation, structural elucidation, and biological activities of cladobotrin V (1), and one new compound 2 from the mangrove endophytic fungus Fusarium sp. IM-37 isolated from Rhizophora mucronata (Figure 1). 2.
Results and discussion
Fusarium sp. IM-37 was isolated from R. mucronata, and the producing strain IM37 was cultivated on sterilized, unpolished rice (18 g/Petri dish £ 50; total of 900 g) at 258C for 4 weeks. A methanol extract from the fermented unpolished rice was concentrated and then partitioned between ethyl acetate (EtOAc) and water. The purification of the EtOAc layer was guided by the characteristically intense blue coloration with a vanillin –sulfuric acid solution on the TLC plates. The EtOAc layer was chromatographed on a silica gel
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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Figure 1. Structures of compounds 1 –4.
column using a stepwise elution of nhexane/EtOAc. Further chromatographic separation using silica and ODS gel yielded two compounds: cladobotrin V (1, 25 mg) and the new related derivative 2 (12 mg). Cladobotrin V (1, 5-hydroxymethyl-4methoxy-6-(E-propenyl)-2-pyrone) was identified on the basis of its 1H and 13C NMR, 1H – 1H COSY, HMQC, and HMBC data (Table 1) [4].
Compound 2 was assigned the molecular formula C10H10O6 on the basis of the [M 2 H]2 peak in the HR-ESI-TOFMS data. The UV spectrum of 2 exhibited absorption maxima at 235 and 341 nm, suggesting the presence of a conjugated enone system in its structure. The IR spectral data showed absorption bands at 3300, 1720, 1700, and 1675 cm21, indicating the presence of a hydroxyl and an unsaturated ester group, respectively.
Journal of Asian Natural Products Research Table 1.
1
H, 13C NMR, and HMBC data for 1 and 2 (d in ppm, J in Hz).a 1b
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3
No.
dC
1 2 3 4 5 6 7 8 9 10
170.2 s 88.6 d 163.3 s 109.3 s 157.0 s 137.0 d 119.0 d 18.7 q 56.3 q 54.1 t
dH
2c
dC
HMBC
5.48 (1H, s)
1, 3
6.81 (1H, 6.43 (1H, 1.94 (3H, 3.86 (3H, 4.52 (2H,
4, 8 5, 8 6, 7 3 3, 4, 5
d, 16.0) m) d, 6.7) s) s)
171.7 s 91.6 d 164.9 s 117.1 s 156.1 s 130.4 d 129.6 d 169.9 s 57.5 q 49.4 t
dH
HMBC
5.76 (1H, s)
3, 4
7.57 (1H, d, 16.1) 6.76 (1H, d, 16.1)
4, 8 5, 8
3.93 (3H, s) 4.55 (2H, s)
3 3, 5
a 13
C NMR spectrum was recorded at 100 MHz and 1H NMR spectrum at 400 MHz. Measured in CDCl3. c Measured in CD3OD. b
The 13C NMR spectrum (Table 1) showed the presence of 10 carbon signals, and further analysis of the DEPT experiment revealed that the 13C NMR signals consisted of two sp3, six sp2, and two carbonyl carbon atoms. The signals of six olefinic carbon atoms indicated the presence of three double bonds in 2. These carbon atoms along with two carbonyl carbon atoms accounted for five degrees of unsaturation; therefore, the remaining one degree of unsaturation should be due to the presence of one ring in the molecule. In the 1 H NMR spectrum, one methoxy, one oxymethylene, and three olefinic methines were observed. Analysis of 1H – 1H COSY data showed one spin network of the couplings between H-6 and H-7 (Figure 2). The lactone linkage required by the molecular formula was shown to be
Figure 2. Key HMBC (arrows) correlations observed for 2.
between C-1 and C-5 by the highly similar 13 C shift values of 2 compared with those of the a-pyrone [5,6]. In the HMBC spectrum, HMBC correlations from H-6 to C-4 and C-8, and from H-7 to C-5 showed the presence of a propenoic acid moiety [dH 6.76 (1H, d, J ¼ 16.1 Hz, H-7), 7.57 (1H, d, J ¼ 16.1 Hz, H-6), dC 171.7 (C-1), 130.4 (C-6), 129.6 (C-7)] at C-5 of the pyrone. Observations of HMBC correlations from H-10 to C-3 and C-5, and from H-2 to C-1 and C-4, together with those from OMe-9 to C-3 established the planar structure of 2 as shown in Figure 2. These observations indicate that 2 is a new derivative of 1. The microbial metabolites closest in similarity to 1, which possess the unique basic structural units 5-hydroxymethyl-4methoxy-6-(E-propenyl or propenoic acid)-2-pyrone, were allantopyrone A (3) and islandic acid-II methyl ester (4) that were isolated from the endophyte Allantophomopsis lycopodina KS-97 [6]. Compounds 3 and 4 showed a 50% cell growth inhibition of HL60 cells at a concentration of 0.32 and 6.55 mM, respectively. Assessment of cytotoxicity of 1 and 2 against HL60 cells by a MTT assay showed no cytotoxic activity at a concentration of 100 mM. The lower activity of 1 and 2 compared with that of
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dose-dependent growth-restoring activity in mutant yeast involving Ca2þ-signal transduction (from 25 to 1.56 mg/spot) (Figure 3). However, 1 exhibited no activity even at a dose of 25 mg/spot (data not shown). Further pharmacological studies of 1 and 2 are currently underway. 3. Experimental 3.1 General experimental procedures Figure 3. Growth-restored activity of 2 against the mutant strain, S. cerevisiae YNS17. Growth-restored activities of 2 against S. cerevisiae YNS17 (zds1D erg3D pdr1D pdr3D) in the presence of 0.3 M CaCl2. 1:25 mg/spot, 2: 12.5 mg/spot, 3: 6.25 mg/spot, 4. 3.13 mg/spot, 5: 1.56 mg/spot, 6: 0.78 mg/ spot, 7: 2.5 mg/spot (FK506).
3 and 4 confirmed that the substitution at C-2 of the 2-pyrone seems to enhance cytotoxic activity. In addition, two isolated compounds were tested for their inhibitory effect on Ca2þ-signaling using the mutant yeast strain Saccharomyces cerevisiae (zds1D erg3D pdr1D pdr3D: YNS17 strain). This mutant strain is not able to grow at high CaCl2 concentrations because of blockage of the cell-cycle progression of the G2 period by hyperactivation of the Ca2þsignaling pathway [7,8]. Cell-cycle regulation by high Ca2þ levels is affected by two parallel pathways, the calcineurin and the Mpk1 MAPK cascade. These pathways coordinately activate Swe1 at the transcriptional and post-translational level. In turn, Swe1 inhibits the cell cycle in the G2 phase by the phosphorylation of Cdc28-Clb at Tyr19. This assay system was able to detect inhibitors of Ca2þsignal transduction by their ability to stimulate cell growth as a growth zone around a paper disc containing the active compound [9,10]. In fact, the calcineurin inhibitor FK506 was detected in this mutant yeast by restoration of growth. In this screening assay system, 2 showed a
IR and UV spectra were recorded with FT710 (HORIBA, Kyoto, Japan) and Shimadzu UV mini-1240 (Shimadzu, Kyoto, Japan) spectrophotometer, respectively. Mass spectra were obtained using a waters Synapt G2 instrument (Waters, Milford, MA, USA), and 1H and 13C NMR spectra were acquired with a JEOL EX-400 spectrometer (JEOL, Tokyo, Japan). Chemical shifts are given on a d (ppm) scale with TMS as an internal standard. Semipreparative HPLC was carried out with Shimadzu pump and UV LC-10A detector (set at 210 nm, Shimadzu) on Mightysil ODS column (5 mm, 150 mm £ 6.0 mm; Kanto Chemical Co., Inc., Tokyo, Japan) at the flow rate of 2.0 ml/ min. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc.) and ODS (Fuji Silysia, Kasugai, Aichi, Japan). TLC was carried out on Merck precoated silica gel plates (silica gel 60 F254, Darmstadt, Germany), and spots were detected by spraying with 10% vanillin in H2SO4 followed by heating or by UV irradiation. 3.2 The producing strain The fungal strain Fusarium sp. IM-37 was isolated from a mangrove plant collected from a forest in Muara Angke, Jakarta, Indonesia in October 2012. The branch samples were surface-sterilized successively with 70% EtOH for 1 min, 5% sodium hypochlorite for 5 min, and 70% EtOH for 1 min, then rinsed in sterile water for two times. The sterilized samples were
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Journal of Asian Natural Products Research dried on sterilized paper and cut into 1-cm pieces. The pieces were placed on plates of potato-dextrose-agar (PDA) containing chloramphenicol (100 mg/l). After incubation at 258C for 7 days, the hyphal tips of the fungi on the plates were removed from the agar plates and transferred to PDA plates (slant). The strain IM-37 was isolated and grew on slants of PDA as white colored culture. This strain was identified to be Fusarium sp. by BEX. Co. LTD, Tokyo Japan, using a DNA analysis of the 18S rDNA regions. This fungus has been deposited at our laboratory in the Faculty of Agriculture of Yamagata University. 3.3 Fermentation, extraction, and isolation The fungal strain IM-37 was cultivated on sterilized unpolished rice (total 900 g, 18 g/Petri dish £ 50) at 258C for 4 weeks. The moldy unpolished rice was extracted with MeOH (2 l), and MeOH extract was concentrated. The resulting aqueous concentrate was partitioned into n-hexane layer (0.5 l), EtOAc layer (1.5 l), and aqueous layer (0.5 l). Purifications of eluates were monitored by the characteristic intense blue coloration with 10% vanillin in H2SO4 on TLC plates. The EtOAc layer (2.5 g) was chromatographed on a silica gel column with stepwise elution of n-hexane – EtOAc (100:0 – 0:100) and EtOAc – MeOH (50:50, 0:100), respectively, to afford fractions 11 to 1-13. Fraction 1-12 (EtOAc– MeOH, 50:50, 445 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0 –0:100) to afford fractions 2-1 to 213. Fractions 2-6 and 2-7 (CHCl3 –EtOAc, 50:50, 40:60, 210 mg) were combined and further separated by semipreparative ODS HPLC (MeOH – H2O, 60:40) to give compound 1 (25.0 mg, tR 11.9 min). Fraction 2-11 (CHCl3 – EtOAc, 0:100, 110 mg) was subjected to semipreparative
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ODS HPLC (MeOH – H2O, 80:20) to give compound 2 (12.0 mg, tR 9.7 min). 3.3.1
Compound 2
White amorphous powder; UV (MeOH) lmax (nm): 235, 341; IR (KBr) nmax (cm21): 3300, 1720, 1700, 1675, and 1050; 1 H (400 MHz) and 13C NMR (100 MHz) spectral data, see Table 1; Negative HR-ESI-TOF-MS: m/z 225.0419 [M 2 H]2 (calcd for C10H9O6, 225.0399). 3.4 Growth-restored activity of samples against YNS17 strain Screening was performed according to the previously described method [11]. Each sample was dissolved in MeOH and twofold dilutions of them were used. Difcow yeast –peptone – dextrose (YPD) broth and YPD agar were purchased from Becton Dickinson Biosciences (Franklin Lakes, NJ, USA). The mutant yeast, YNS17 (MATa zds1::TRP1 erg3::HIS3 pdr1:: hisG-URA3-hisG pdr3::hisG) yeast strain was derivative of strain W303-1A [10]. A 5-ml aliquot of samples was spotted on a YPD agar medium containing YNS17 strain and 0.3 M CaCl2. After 3 days of incubation at 288C, the intensity of the growth spot was observed as the result of inhibition of Ca2þ-signal transduction. FK506 (2.5 ng/spot) was used as a positive control. FK506 was kindly provided by Fujisawa Pharmaceutical Co., Ltd. (the present Astellas Pharma, Inc., Tokyo, Japan). References [1] K. Duarte, T.A.P. Rocha-Santos, A. C. Freitas, and A.C. Duarte, TrAC Trents Anal. Chem. 34, 97 (2012). [2] Y. Shiono, T. Sasaki, F. Shibuya, Y. Yasuda, T. Koseki, and U. Supratman, Nat. Prod. Commun. 12, 1735 (2013). [3] Z. Huang, X. Cai, C. Shao, Z. She, X. Xia, Y. Chen, J. Yan, S. Zhou, and Y. Lin, Phytochemistry 69, 1604 (2008).
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[4] Y. Tezuka, Q. Huang, T. Kikuchi, A. Nishi, and K. Tsubaki, Chem. Pharm. Bull. 42, 2612 (1994). [5] R.A. Hussain and P.G. Waterman, Phytochemistry 21, 1393 (1982). [6] Y. Shiono, M. Yokoi, T. Koseki, T. Murayama, N. Aburai, and K. Kimura, J. Antibiot. 63, 251 (2010). [7] M. Mizunuma, D. Hirata, K. Miyahara, E. Tsuchiya, and T. Miyakawa, Nature 392, 303 (1998). [8] T. Miyakawa and M. Mizunuma, Biosci. Biotechnol. Biochem. 71, 633 (2007).
[9] A. Shitamukai, M. Mizunuma, D. Hirata, H. Takahashi, and T. Miyakawa, Biosci. Biotechnol. Biochem. 64, 1942 (2000). [10] R. Chanklan, E. Aihara, S. Koga, H. Takahashi, M. Mizunuma, and T. Miyakawa, Biosci. Biotechnol. Biochem. 72, 132 (2008). [11] K. Kimura, Y. Minamikawa, Y. Ogasawara, J. Yoshida, K. Saitoh, H. Shinden, Y.Q. Ye, S. Takahashi, T. Miyakawa, and H. Koshino, Fitoterapia 83, 907 (2012).
