982
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
New Triterpenoids from the Fruiting Bodies of Ganoderma lucidum and Their Bioactivities by Dong-Ze Liu* a ), Yi-Qun Zhu b ), Xiao-Fei Li b ), Wei-Gang Shan b ), and Peng-Fei Gao b ) a
) Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China (e-mail: [email protected]) b ) Sichuan Agricultural University, Yaan 625014, P. R. China
Phytochemical investigation of the AcOEt extract of G. Lucidum has led to the isolation of two new triterpenoids, 1 and 2, together with five known ones, 3 – 7. The structures of the new compounds were identified as 12b-acetoxy-3b,7b-dihydroxy-11,15,23-trioxolanost-8-en-26-oic acid butyl ester (1) and 12bacetoxy-3,7,11,15,23-pentaoxolanost-8-en-26-oic acid butyl ester (2) on the basis of detailed spectroscopic analysis (mass spectrometry, and 1D- and 2D-NMR experiments). The antimicrobial activities of 1 and 2 were also evaluated.
Introduction. – Ganoderma lucidum (Fr.) Karst, a species of basidiomycetes that belongs to the family Polyporaceae, has been widely used as a medical remedy to promote health and longevity in China, Japan, Korea, and other Asian countries for centuries. In addition, it has been used for the treatment of a wide range of ailments and chronic diseases, including migraine, hypertension, arthritis, bronchitis, hemorrhoids, diabetes, hypercholesterolemia, nephritis, etc. [1] [2]. Because of the potential medicinal value and wide applicability of G. lucidum, much attention has been paid to search for the pharmacologically significant compounds from this mushroom. Triterpenoids are typical chemical constituents in G. lucidum, and have an important role in the pharmacological effects described above. Since the first triterpenoid of ganoderic acid A had been reported [3], more than 150 triterpenes including highly oxygenated lanostane derivatives were isolated from the fruiting bodies, cultured mycelia, and spores of G. Lucidum over the past 30 years [4], and some of them possess biological activities of interest [5 – 10]. Even today, the number of new triterpenoids identified from it seemes to continously increase. In our screening for naturally occurring bioactive metabolites, chemical studies of the fruiting bodies of G. lucidum led to the isolation and characterization of two new triterpenoids, 1 and 2, together with five known ones, 3 – 7 (Fig.). Herein, we describe the isolation, structure elucidation, and biological activity of these compounds. Results and Discussion. – Compound 1 was assigned the molecular formula C36H54O9 by positive-ion-mode HR-ESI-MS (m/z 653.3663 ([M þ Na] þ )), corresponding to ten degrees of unsaturation. The UV absorption maximum at 250 nm (log e 3.25) and the IR band at 1708 cm 1 suggested the presence of an a,b-unsaturated C¼O system. The 13C- and DEPT-NMR spectra (Table) exhibited 36 signals attributed to five C¼O groups (d(C) 170.4, 176.1, 192.1, 207.3, and 216.3), two sp2 quaternary C-atoms 2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
983
Figure. Structures of compounds 1 – 7
(d(C) 155.8 and 142.9), three O-bearing CH groups (d(C) 78.2 and 66.1), one Obearing CH2 group (d(C) 64.8), as well as four sp3 quaternary C-atoms (d(C) 38.5, 38.6, 49.5, and 60.6), four CH groups (d(C) 49.1, 45.2, 28.2, and 35.1), eight CH2 groups (d(C) 34.4, 27.4, 36.7, 37.8, 47.9, 46.8, 30.9, and 19.3), and nine Me groups (d(C) 12.1, 18.6, 28.1, 15.4, 21.9, 17.3, 23.9, 13.9, and 20.9), of which 30 were assigned to a triterpene skeleton. Comparison of these spectroscopic data with those of butyl ganoderate B [11], suggested that the skeleton and side-chain moiety of 1 should be the same except for the H-atom (d(H) 5.60 (s), 2.25 (s)) and C-atom (d(C) 79.6, 170.4, and 20.9) resonances which were characteristic for an AcO group at C(12). This assignment was supported by cross-peaks between HC(12) and C¼O C-atom of the AcO group in the HMBC spectrum. The relative configuration of 1 was determined by analyzing the NOESY spectrum. Key correlations HC(3)/HC(5), HC(7)/HC(5) and Me(30), and HC(12)/Me(30) were observed, indicating that HC(3) was a-orientated as were HC(5), HC(7), HC(12), and Me(30). Thus, the data above conclusively allowed elucidation of structure for 1, as depicted in the Figure. Compound 2 was isolated as white powder. It possessed the molecular formula C36H50O9 (m/z 626.3452 ([M þ Na] þ )), as deduced from HR-EI-MS. Its UV absorption maximum at 252 nm was characteristic of an a,b-unsaturated C¼O group. The 13C- and DEPT-NMR spectra (Table) revealed the presence of 36 C-atoms, including seven C¼O groups, two sp2 quaternary C-atoms, one O-bearing CH group, one O-bearing CH2 group, as well as four sp3 quaternary C-atoms, four CH groups, and eight CH2 , and nine Me groups. Comparing the 1H- and 13C-NMR spectra with known compounds, compound 2 had almost the same chemical shifts as those of ganoderic acid F [12], except for additional signals consistent with the presence of an O-Bu moiety (d(H) 4.08 (t, J ¼ 6.6), 1.57 – 1.62 (m), 1.36 – 1.41 (m), 0.94 (t, J ¼ 7.2); d(C) 13.8, 19.2, 30.9, and 64.8), indicating that 2 is a butyl ester derivative of ganoderic acid F. In addition to the two new compounds described above, five known ones were identified by comparing their physicochemical and spectral data with those in the literature as lucidumol B (3) [13], ganodermanondiol (4) [13], ganodermanontriol (5) [13], lucidenic acid N (6) [14], and lucidenic acid A (7) [15].
984
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table. 1H- and 13C-NMR (500 and 125 MHz, resp.; CDCl3 ) Data for 1 and 2. d in ppm, J in Hz. Position
1 d( H )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Bu MeCO
0.87 – 0.93 (m), 2.62 (ddd, J ¼ 3.6, 7.2, 13.6) 1.62 – 1.68 (m) 3.19 (dd, J ¼ 5.2, 10.8) 0.87 – 0.93 (m) 2.21 – 2.27 (m), 1.62 – 1.68 (m) 4.80 (t, J ¼ 8.8)
5.60 (s)
2.10 (dd, J ¼ 9.6, 19.6), 2.67 (dd, J ¼ 8.0, 19.2) 2.41 – 2.49 (m) 0.95 (s) 1.27 (s) 2.41 – 2.49 (m) 0.96 (d, J ¼ 5.6) 2.29 (dd, J ¼ 8.4, 16.0), 2.41 – 2.49 (m) 2.41 – 2.49 (m), 2.83 (dd, J ¼ 8.7, 17.5) 2.91 – 2.97 (m) 1.17 (d, J ¼ 7.2) 1.02 (s) 0.84 (s) 1.48 (s) 4.08 (t, J ¼ 6.6), 1.57 – 1.63 (m), 1.36 – 1.40 (m), 0.94 (t, J ¼ 7.2) 2.25 (s)
2 d(C ) 34.4 27.4 78.2 38.5 49.1 36.7 66.1 155.8 142.9 38.6 192.1 79.6 49.5 60.6 216.3 37.8 45.2 12.1 18.6 28.2 21.9 47.9 207.3 46.8 35.1 176.1 17.3 28.1 15.4 23.9 64.8, 30.9, 19.3, 13.9 170.4, 20.9
d( H ) 1.72 (ddd, J ¼ 5.6, 8.8, 14.8), 2.73 (ddd, J ¼ 4.0, 9.6, 14.4) 2.44 – 2.49 (m) 3.19 (dd, J ¼ 5.2, 10.8) 2.28 – 2.35 (m) 2.76 – 2.80 (m), 2.45 – 2.53 (m) 4.80 (t, J ¼ 8.8)
5.66 (s)
1.90 (dd, J ¼ 8.0, 18.0), 2.79 (dd, J ¼ 8.4, 18.8) 2.55 – 2.60 (m) 0.85 (s) 1.32 (s) 2.28 – 2.32 (m) 0.98 (d, J ¼ 6.0) 2.28 – 2.33 (m), 2.38 – 2.44 (m) 2.41 – 2.49 (m), 2.83 (dd, J ¼ 8.7, 17.5) 2.90 – 2.96 (m) 1.17 (d, J ¼ 7.2) 1.13 (s) 1.11 (s) 1.80 (s) 4.08 (t, J ¼ 6.6), 1.57 – 1.62 (m), 1.36 – 1.41 (m), 0.94 (t, J ¼ 7.2)
d(C ) 33.9 33.6 215.1 46.8 50.8 37.4 198.6 149.8 145.8 39.1 194.2 78.9 47.6 58.7 205.6 37.8 44.3 12.1 18.6 29.4 21.9 48.4 207.3 46.8 35.1 175.9 17.3 27.6 20.3 20.7 64.8, 30.9, 19.2, 13.8
New metabolites were tested for their antibacterial activities against a panel of bacteria. Compound 1 displayed significant antimicrobial activities against the Grampositive bacteria Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC6633), with MIC values of 68.5 and 123.8 mm, respectively (the positive control ampicillin showed MIC values of 4.1 and 19.3 mm, resp.). However, compound 2 did not show any noticeable in vitro antibacterial activities against the above-mentioned organisms.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
985
Experimental Part General. Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Qingdao Marine Chemicals, Qingdao, P. R. China) or Sephadex LH-20 (GE Healthcare, SE-Uppsala). Semiprep. HPLC: Agilent 1200 HPLC system with an ODS column (RP-8, 250 10 mm, YMC Pak, 5 mm; detector: UV); flow rate, 2.0 ml/min. TLC: GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, P. R. China; visualization by spraying with 5% H2SO4 in EtOH, followed by heating. Optical rotation: Horiba SEPA-300 polarimeter (Horiba, Kyoto, Japan). UV Spectra: UV/VIS Evolution 300 spectrometer (Thermo Scientific, USA). IR Spectra: Bruker Tensor 27 spectrometer (Bruker Optics, DE-Ettlingen); KBr pellets. NMR Spectra: Bruker Avance III 500 MHz spectrometer (Bruker BioSpin, DE-Rheinstetten) with TMS as internal standard. ESI-MS: Thermo Fisher LTQ Fleet spectrometer (Thermo Scientific, USA). HR-ESIMS: Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, USA). Plant Matarial. The fruiting bodies of G. lucidum (purchased in Tibet, P. R. China) were supplied and identified by one of the authors. The voucher specimen (200543715) was deposited with the Herbarium of Sichuan Agricultural University. Extraction and Isolation. The air-dried and powdered fruit bodies of G. lucidum (2.5 kg) were extracted with 95% EtOH (3 10 l) at r.t. for 6 d. The EtOH extract was next partitioned between AcOEt and H2O, and the org. layer was evaporated in vacuo to afford the AcOEt extract (45 g). The latter was subjected to CC (SiO2 ; CHCl3/MeOH from 100 : 0 to 0 : 100 (v/v)) to afford Frs. A – H. The fractions eluted with CHCl3/MeOH 100 : 0 and 99 : 1 were combined and separated by CC (Sephadex LH20; CHCl3/MeOH 1 : 1 (v/v)) to give Subfrs. B1 – B4. Subfr. B2 was repeatedly subjected to CC (SiO2 ; hexane/AcOEt 15 : 1 to 8 : 1) to yield compounds 2 (4.2 mg) and 4 (8.9 mg). Subfrs. B3 and B4 were further purified by semiprep. reversed-phase HPLC (45% MeOH in H2O over 5 min, 45 – 80% over 35 min) to give 7 (13.5 mg). The fractions eluted with CHCl3/MeOH 98 : 2 and 95 : 5 were combined and separated by CC (Sephadex LH-20; MeOH) to yield three fractions, Frs. D1 – D3, one of which, Fr. D2, was further purified by CC (SiO2 ; hexane/Me2CO 30 : 1 to 10 : 1) to afford compound 1 (5.8 mg). Fr. D3 was further separated repeatedly by CC (Sephadex LH-20; MeOH), followed by semiprep. reversedphase HPLC (30% MeOH in H2O over 5 min, 30 – 90% over 30 min) to yield compounds 3 (3.7 mg), 5 (4.1 mg), and 6 (8.7 mg). Butyl (3b,7b,12b)-12-(Acetyloxy)-3,7-dihydroxy-11,15,23-trioxolanost-8-en-26-oate (1). Colorless powder. [a] 25 D ¼ þ 22.5 (c ¼ 0.45, CHCl3 ). UV (CHCl3 ): 250 (3.25). IR (KBr): 3540, 2970, 1740, 1660. 1 H- and 13C-NMR (CDCl3 , 500 MHz): see the Table. HR-ESI-MS: 653.3663 ([M þ Na] þ , C36H54NaO þ9 ; calc. 653.3666). Butyl (12b)-12-(Acetyloxy)-3,7,11,15,23-pentaoxolanost-8-en-26-oate (2). Colorless powder. [a] 25 D ¼ þ 135.9 (c ¼ 0.12, CHCl3 ). UV (MeOH): 252 (2.15). IR (KBr): 3435, 2970, 2935, 1751, 1701, 1462, 1175. 1 H- and 13C-NMR (CDCl3 , 500 MHz): see the Table. HR-EI-MS: 626.3452 (M þ , C36H50O þ9 ; calc. 626.3455). Antibacterial Assay. Compounds 1 and 2 were tested for their antibacterial activities using a diskdiffusion assay [16]. Seed cultures of three bacteria, Staphylococcus aureus ATCC29213, Bacillus thuringiensis ATCC39765, and Bacillus subtilis ATCC6633, were cultivated in LB (lysogeny broth) medium by incubating the organisms for 10 h at 378. Then, the seed cultures were added to LB agar medium. Sterile filter disks (6-mm diameter) infused with 5 ml of test soln. (2 mg/ml DMSO) and positive control (5 mg/ml DMSO ampicillin) were added to the plates. The plates were placed in an incubator at 378. After 12 h, the diameters of the zones of growth inhibition around each disk were recorded. This work was supported by the National Natural Science Foundation of China for Young Scholars (31100012).
REFERENCES [1] S. C. Jong, J. M. Birmingham, Adv. Appl. Microbiol. 1992, 37, 101. [2] C. Hobbs, Medicinal Mushrooms: An exploration of tradition, healing and culture, Botanica Press, Inc., Santa Cruz, CA, 1995.
986
[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
T. Kubota, Y. Asaka, I. Miura, H. Mori, Helv. Chim. Acta 1982, 65, 611. B. Boh, M. Berovic, J. Zhang, Z.-B. Lin, Biotechnol. Ann. Rev. 2007, 13, 265. J. O. Toth, B. Luu, G. Ourison, Tetrahedron Lett. 1983, 24, 1081. C.-N. Lin, W.-P. Tome, S.-J. Won, J. Nat. Prod. 1991, 54, 998. A. Morigiwa, K. Kitabatake, Y. Fujimoto, N. Ikekawa, Chem. Pharm. Bull. 1986, 34, 3025. D.-H. Kim, S.-B. Shin, N.-J. Kim, I.-S. Jang, Biol. Pharm. Bull. 1999, 22, 162. S. Lee, S. Park, J.-W. Oh, C.-H.Yang, Planta Med. 1998, 64, 303. B.-S. Min, N. Nakamura, H. Miyashiro, K.-W. Bae, M. Hattori, Chem. Pharm. Bull. 1998, 46, 1607. I. Lee, J. Seo, J. Kim, H. Kim, U. Youn, J. Lee, H. Jung, M. Na, M. Hattori, B. Min, K. Bae, J. Nat. Prod. 2010, 73, 172. Y. Komoda, H. Nakamura, S. Ishihara, M. Uchida, H. Kohda, K. Yamasaki, Chem. Pharm. Bull. 1985, 33, 4829. A. Fujita, M. Arisawa, M. Saga, T. Hayashi, N. Morita, J. Nat. Prod. 1986, 49, 1122. T.-S.Wu, L.-S. Shi, S.-C. Kuo, J. Nat. Prod. 2001, 64, 1121. T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, S. Sakamura, Agric. Biol. Chem. 1985, 49, 1793. X. Wang, J. G. Sena Filho, A. R. Hoover, J. B. King, T. K. Ellis, D. R. Powell, R. H. Cichewicz, J. Nat. Prod. 2010, 73, 942. Received January 2, 2014
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
New Triterpenoids from the Fruiting Bodies of Ganoderma lucidum and Their Bioactivities by Dong-Ze Liu* a ), Yi-Qun Zhu b ), Xiao-Fei Li b ), Wei-Gang Shan b ), and Peng-Fei Gao b ) a
) Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China (e-mail: [email protected]) b ) Sichuan Agricultural University, Yaan 625014, P. R. China
Phytochemical investigation of the AcOEt extract of G. Lucidum has led to the isolation of two new triterpenoids, 1 and 2, together with five known ones, 3 – 7. The structures of the new compounds were identified as 12b-acetoxy-3b,7b-dihydroxy-11,15,23-trioxolanost-8-en-26-oic acid butyl ester (1) and 12bacetoxy-3,7,11,15,23-pentaoxolanost-8-en-26-oic acid butyl ester (2) on the basis of detailed spectroscopic analysis (mass spectrometry, and 1D- and 2D-NMR experiments). The antimicrobial activities of 1 and 2 were also evaluated.
Introduction. – Ganoderma lucidum (Fr.) Karst, a species of basidiomycetes that belongs to the family Polyporaceae, has been widely used as a medical remedy to promote health and longevity in China, Japan, Korea, and other Asian countries for centuries. In addition, it has been used for the treatment of a wide range of ailments and chronic diseases, including migraine, hypertension, arthritis, bronchitis, hemorrhoids, diabetes, hypercholesterolemia, nephritis, etc. [1] [2]. Because of the potential medicinal value and wide applicability of G. lucidum, much attention has been paid to search for the pharmacologically significant compounds from this mushroom. Triterpenoids are typical chemical constituents in G. lucidum, and have an important role in the pharmacological effects described above. Since the first triterpenoid of ganoderic acid A had been reported [3], more than 150 triterpenes including highly oxygenated lanostane derivatives were isolated from the fruiting bodies, cultured mycelia, and spores of G. Lucidum over the past 30 years [4], and some of them possess biological activities of interest [5 – 10]. Even today, the number of new triterpenoids identified from it seemes to continously increase. In our screening for naturally occurring bioactive metabolites, chemical studies of the fruiting bodies of G. lucidum led to the isolation and characterization of two new triterpenoids, 1 and 2, together with five known ones, 3 – 7 (Fig.). Herein, we describe the isolation, structure elucidation, and biological activity of these compounds. Results and Discussion. – Compound 1 was assigned the molecular formula C36H54O9 by positive-ion-mode HR-ESI-MS (m/z 653.3663 ([M þ Na] þ )), corresponding to ten degrees of unsaturation. The UV absorption maximum at 250 nm (log e 3.25) and the IR band at 1708 cm 1 suggested the presence of an a,b-unsaturated C¼O system. The 13C- and DEPT-NMR spectra (Table) exhibited 36 signals attributed to five C¼O groups (d(C) 170.4, 176.1, 192.1, 207.3, and 216.3), two sp2 quaternary C-atoms 2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
983
Figure. Structures of compounds 1 – 7
(d(C) 155.8 and 142.9), three O-bearing CH groups (d(C) 78.2 and 66.1), one Obearing CH2 group (d(C) 64.8), as well as four sp3 quaternary C-atoms (d(C) 38.5, 38.6, 49.5, and 60.6), four CH groups (d(C) 49.1, 45.2, 28.2, and 35.1), eight CH2 groups (d(C) 34.4, 27.4, 36.7, 37.8, 47.9, 46.8, 30.9, and 19.3), and nine Me groups (d(C) 12.1, 18.6, 28.1, 15.4, 21.9, 17.3, 23.9, 13.9, and 20.9), of which 30 were assigned to a triterpene skeleton. Comparison of these spectroscopic data with those of butyl ganoderate B [11], suggested that the skeleton and side-chain moiety of 1 should be the same except for the H-atom (d(H) 5.60 (s), 2.25 (s)) and C-atom (d(C) 79.6, 170.4, and 20.9) resonances which were characteristic for an AcO group at C(12). This assignment was supported by cross-peaks between HC(12) and C¼O C-atom of the AcO group in the HMBC spectrum. The relative configuration of 1 was determined by analyzing the NOESY spectrum. Key correlations HC(3)/HC(5), HC(7)/HC(5) and Me(30), and HC(12)/Me(30) were observed, indicating that HC(3) was a-orientated as were HC(5), HC(7), HC(12), and Me(30). Thus, the data above conclusively allowed elucidation of structure for 1, as depicted in the Figure. Compound 2 was isolated as white powder. It possessed the molecular formula C36H50O9 (m/z 626.3452 ([M þ Na] þ )), as deduced from HR-EI-MS. Its UV absorption maximum at 252 nm was characteristic of an a,b-unsaturated C¼O group. The 13C- and DEPT-NMR spectra (Table) revealed the presence of 36 C-atoms, including seven C¼O groups, two sp2 quaternary C-atoms, one O-bearing CH group, one O-bearing CH2 group, as well as four sp3 quaternary C-atoms, four CH groups, and eight CH2 , and nine Me groups. Comparing the 1H- and 13C-NMR spectra with known compounds, compound 2 had almost the same chemical shifts as those of ganoderic acid F [12], except for additional signals consistent with the presence of an O-Bu moiety (d(H) 4.08 (t, J ¼ 6.6), 1.57 – 1.62 (m), 1.36 – 1.41 (m), 0.94 (t, J ¼ 7.2); d(C) 13.8, 19.2, 30.9, and 64.8), indicating that 2 is a butyl ester derivative of ganoderic acid F. In addition to the two new compounds described above, five known ones were identified by comparing their physicochemical and spectral data with those in the literature as lucidumol B (3) [13], ganodermanondiol (4) [13], ganodermanontriol (5) [13], lucidenic acid N (6) [14], and lucidenic acid A (7) [15].
984
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table. 1H- and 13C-NMR (500 and 125 MHz, resp.; CDCl3 ) Data for 1 and 2. d in ppm, J in Hz. Position
1 d( H )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Bu MeCO
0.87 – 0.93 (m), 2.62 (ddd, J ¼ 3.6, 7.2, 13.6) 1.62 – 1.68 (m) 3.19 (dd, J ¼ 5.2, 10.8) 0.87 – 0.93 (m) 2.21 – 2.27 (m), 1.62 – 1.68 (m) 4.80 (t, J ¼ 8.8)
5.60 (s)
2.10 (dd, J ¼ 9.6, 19.6), 2.67 (dd, J ¼ 8.0, 19.2) 2.41 – 2.49 (m) 0.95 (s) 1.27 (s) 2.41 – 2.49 (m) 0.96 (d, J ¼ 5.6) 2.29 (dd, J ¼ 8.4, 16.0), 2.41 – 2.49 (m) 2.41 – 2.49 (m), 2.83 (dd, J ¼ 8.7, 17.5) 2.91 – 2.97 (m) 1.17 (d, J ¼ 7.2) 1.02 (s) 0.84 (s) 1.48 (s) 4.08 (t, J ¼ 6.6), 1.57 – 1.63 (m), 1.36 – 1.40 (m), 0.94 (t, J ¼ 7.2) 2.25 (s)
2 d(C ) 34.4 27.4 78.2 38.5 49.1 36.7 66.1 155.8 142.9 38.6 192.1 79.6 49.5 60.6 216.3 37.8 45.2 12.1 18.6 28.2 21.9 47.9 207.3 46.8 35.1 176.1 17.3 28.1 15.4 23.9 64.8, 30.9, 19.3, 13.9 170.4, 20.9
d( H ) 1.72 (ddd, J ¼ 5.6, 8.8, 14.8), 2.73 (ddd, J ¼ 4.0, 9.6, 14.4) 2.44 – 2.49 (m) 3.19 (dd, J ¼ 5.2, 10.8) 2.28 – 2.35 (m) 2.76 – 2.80 (m), 2.45 – 2.53 (m) 4.80 (t, J ¼ 8.8)
5.66 (s)
1.90 (dd, J ¼ 8.0, 18.0), 2.79 (dd, J ¼ 8.4, 18.8) 2.55 – 2.60 (m) 0.85 (s) 1.32 (s) 2.28 – 2.32 (m) 0.98 (d, J ¼ 6.0) 2.28 – 2.33 (m), 2.38 – 2.44 (m) 2.41 – 2.49 (m), 2.83 (dd, J ¼ 8.7, 17.5) 2.90 – 2.96 (m) 1.17 (d, J ¼ 7.2) 1.13 (s) 1.11 (s) 1.80 (s) 4.08 (t, J ¼ 6.6), 1.57 – 1.62 (m), 1.36 – 1.41 (m), 0.94 (t, J ¼ 7.2)
d(C ) 33.9 33.6 215.1 46.8 50.8 37.4 198.6 149.8 145.8 39.1 194.2 78.9 47.6 58.7 205.6 37.8 44.3 12.1 18.6 29.4 21.9 48.4 207.3 46.8 35.1 175.9 17.3 27.6 20.3 20.7 64.8, 30.9, 19.2, 13.8
New metabolites were tested for their antibacterial activities against a panel of bacteria. Compound 1 displayed significant antimicrobial activities against the Grampositive bacteria Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC6633), with MIC values of 68.5 and 123.8 mm, respectively (the positive control ampicillin showed MIC values of 4.1 and 19.3 mm, resp.). However, compound 2 did not show any noticeable in vitro antibacterial activities against the above-mentioned organisms.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
985
Experimental Part General. Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Qingdao Marine Chemicals, Qingdao, P. R. China) or Sephadex LH-20 (GE Healthcare, SE-Uppsala). Semiprep. HPLC: Agilent 1200 HPLC system with an ODS column (RP-8, 250 10 mm, YMC Pak, 5 mm; detector: UV); flow rate, 2.0 ml/min. TLC: GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, P. R. China; visualization by spraying with 5% H2SO4 in EtOH, followed by heating. Optical rotation: Horiba SEPA-300 polarimeter (Horiba, Kyoto, Japan). UV Spectra: UV/VIS Evolution 300 spectrometer (Thermo Scientific, USA). IR Spectra: Bruker Tensor 27 spectrometer (Bruker Optics, DE-Ettlingen); KBr pellets. NMR Spectra: Bruker Avance III 500 MHz spectrometer (Bruker BioSpin, DE-Rheinstetten) with TMS as internal standard. ESI-MS: Thermo Fisher LTQ Fleet spectrometer (Thermo Scientific, USA). HR-ESIMS: Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, USA). Plant Matarial. The fruiting bodies of G. lucidum (purchased in Tibet, P. R. China) were supplied and identified by one of the authors. The voucher specimen (200543715) was deposited with the Herbarium of Sichuan Agricultural University. Extraction and Isolation. The air-dried and powdered fruit bodies of G. lucidum (2.5 kg) were extracted with 95% EtOH (3 10 l) at r.t. for 6 d. The EtOH extract was next partitioned between AcOEt and H2O, and the org. layer was evaporated in vacuo to afford the AcOEt extract (45 g). The latter was subjected to CC (SiO2 ; CHCl3/MeOH from 100 : 0 to 0 : 100 (v/v)) to afford Frs. A – H. The fractions eluted with CHCl3/MeOH 100 : 0 and 99 : 1 were combined and separated by CC (Sephadex LH20; CHCl3/MeOH 1 : 1 (v/v)) to give Subfrs. B1 – B4. Subfr. B2 was repeatedly subjected to CC (SiO2 ; hexane/AcOEt 15 : 1 to 8 : 1) to yield compounds 2 (4.2 mg) and 4 (8.9 mg). Subfrs. B3 and B4 were further purified by semiprep. reversed-phase HPLC (45% MeOH in H2O over 5 min, 45 – 80% over 35 min) to give 7 (13.5 mg). The fractions eluted with CHCl3/MeOH 98 : 2 and 95 : 5 were combined and separated by CC (Sephadex LH-20; MeOH) to yield three fractions, Frs. D1 – D3, one of which, Fr. D2, was further purified by CC (SiO2 ; hexane/Me2CO 30 : 1 to 10 : 1) to afford compound 1 (5.8 mg). Fr. D3 was further separated repeatedly by CC (Sephadex LH-20; MeOH), followed by semiprep. reversedphase HPLC (30% MeOH in H2O over 5 min, 30 – 90% over 30 min) to yield compounds 3 (3.7 mg), 5 (4.1 mg), and 6 (8.7 mg). Butyl (3b,7b,12b)-12-(Acetyloxy)-3,7-dihydroxy-11,15,23-trioxolanost-8-en-26-oate (1). Colorless powder. [a] 25 D ¼ þ 22.5 (c ¼ 0.45, CHCl3 ). UV (CHCl3 ): 250 (3.25). IR (KBr): 3540, 2970, 1740, 1660. 1 H- and 13C-NMR (CDCl3 , 500 MHz): see the Table. HR-ESI-MS: 653.3663 ([M þ Na] þ , C36H54NaO þ9 ; calc. 653.3666). Butyl (12b)-12-(Acetyloxy)-3,7,11,15,23-pentaoxolanost-8-en-26-oate (2). Colorless powder. [a] 25 D ¼ þ 135.9 (c ¼ 0.12, CHCl3 ). UV (MeOH): 252 (2.15). IR (KBr): 3435, 2970, 2935, 1751, 1701, 1462, 1175. 1 H- and 13C-NMR (CDCl3 , 500 MHz): see the Table. HR-EI-MS: 626.3452 (M þ , C36H50O þ9 ; calc. 626.3455). Antibacterial Assay. Compounds 1 and 2 were tested for their antibacterial activities using a diskdiffusion assay [16]. Seed cultures of three bacteria, Staphylococcus aureus ATCC29213, Bacillus thuringiensis ATCC39765, and Bacillus subtilis ATCC6633, were cultivated in LB (lysogeny broth) medium by incubating the organisms for 10 h at 378. Then, the seed cultures were added to LB agar medium. Sterile filter disks (6-mm diameter) infused with 5 ml of test soln. (2 mg/ml DMSO) and positive control (5 mg/ml DMSO ampicillin) were added to the plates. The plates were placed in an incubator at 378. After 12 h, the diameters of the zones of growth inhibition around each disk were recorded. This work was supported by the National Natural Science Foundation of China for Young Scholars (31100012).
REFERENCES [1] S. C. Jong, J. M. Birmingham, Adv. Appl. Microbiol. 1992, 37, 101. [2] C. Hobbs, Medicinal Mushrooms: An exploration of tradition, healing and culture, Botanica Press, Inc., Santa Cruz, CA, 1995.
986
[3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
T. Kubota, Y. Asaka, I. Miura, H. Mori, Helv. Chim. Acta 1982, 65, 611. B. Boh, M. Berovic, J. Zhang, Z.-B. Lin, Biotechnol. Ann. Rev. 2007, 13, 265. J. O. Toth, B. Luu, G. Ourison, Tetrahedron Lett. 1983, 24, 1081. C.-N. Lin, W.-P. Tome, S.-J. Won, J. Nat. Prod. 1991, 54, 998. A. Morigiwa, K. Kitabatake, Y. Fujimoto, N. Ikekawa, Chem. Pharm. Bull. 1986, 34, 3025. D.-H. Kim, S.-B. Shin, N.-J. Kim, I.-S. Jang, Biol. Pharm. Bull. 1999, 22, 162. S. Lee, S. Park, J.-W. Oh, C.-H.Yang, Planta Med. 1998, 64, 303. B.-S. Min, N. Nakamura, H. Miyashiro, K.-W. Bae, M. Hattori, Chem. Pharm. Bull. 1998, 46, 1607. I. Lee, J. Seo, J. Kim, H. Kim, U. Youn, J. Lee, H. Jung, M. Na, M. Hattori, B. Min, K. Bae, J. Nat. Prod. 2010, 73, 172. Y. Komoda, H. Nakamura, S. Ishihara, M. Uchida, H. Kohda, K. Yamasaki, Chem. Pharm. Bull. 1985, 33, 4829. A. Fujita, M. Arisawa, M. Saga, T. Hayashi, N. Morita, J. Nat. Prod. 1986, 49, 1122. T.-S.Wu, L.-S. Shi, S.-C. Kuo, J. Nat. Prod. 2001, 64, 1121. T. Nishitoba, H. Sato, T. Kasai, H. Kawagishi, S. Sakamura, Agric. Biol. Chem. 1985, 49, 1793. X. Wang, J. G. Sena Filho, A. R. Hoover, J. B. King, T. K. Ellis, D. R. Powell, R. H. Cichewicz, J. Nat. Prod. 2010, 73, 942. Received January 2, 2014
