Lanostane triterpenes from the mushroom Ganoderma resinaceum and their inhibitory activities against α-glucosidase

Phytochemistry 149 (2018) 103e115

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Lanostane triterpenes from the mushroom Ganoderma resinaceum and their inhibitory activities against a-glucosidase Xian-Qiang Chen, Jing Zhao*, Ling-Xiao Chen, Shen-Fei Wang, Ying Wang**, Shao-Ping Li*** State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 July 2017 Received in revised form 29 December 2017 Accepted 12 January 2018 Available online 25 February 2018

Eighteen previously undescribed lanostane triterpenes and thirty known analogues were obtained from the fruiting bodies of Ganoderma resinaceum. Resinacein C was isolated from a natural source for the first time. The structures of all the above compounds were elucidated by extensive spectroscopic analysis and comparisons of their spectroscopic data with those reported in the literature. Furthermore, in an in vitro assay, Resinacein C, ganoderic acid Y, lucialdehyde C, 7-oxo-ganoderic acid Z3, 7-oxo-ganoderic acid Z, and lucidadiol showed strong inhibitory effects against a-glucosidase compared with the positive control drug acarbose. The structure-activity relationships of ganoderma triterpenes on a-glucosidase inhibition showed that the C-24/C-25 double bond is necessary for a-glucosidase inhibitory activity. Moreover, the carboxylic acid group at C-26 and the hydroxy group at C-15 play important roles in enhancing inhibitory effects of these triterpenes. © 2018 Published by Elsevier Ltd.

Keywords: Ganoderma resinaceum Ganodermataceae Lanostane triterpenes a-Glucosidase inhibitor

1. Introduction The genus Ganoderma is a group of common mushrooms belonging to the family Ganodermataceae that are widely distributed in tropical and temperate regions worldwide (Richter et al., 2015). In China, Japan, and Korea, Ganoderma has been regarded as one of the most important medicinal fungi for preventing and treating various human diseases (Paterson, 2006). More than 430 small molecule compounds have been reported from various Ganoderma species (Baby et al., 2015). Chemical investigations have revealed that Ganoderma species contain a substantial number of biologically active constituents, including triterpenes, polysaccharides, steroids, fatty acids, and alkaloids (Paterson, 2006; Wang et al., 2015). Among the compounds present in the genus Ganoderma, triterpenes and polysaccharides are the main bioactive constituents. Lanostane-type triterpenes isolated from Ganoderma species have attracted considerable attention due to their potentially significant pharmacological activities and structural diversity.

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected], [email protected] (J. Zhao), [email protected] (Y. Wang), [email protected], [email protected] (S.-P. Li). https://doi.org/10.1016/j.phytochem.2018.01.007 0031-9422/© 2018 Published by Elsevier Ltd.

These compounds have shown cancer cell cytotoxic (Rios et al., 2012), hepatoprotective (Peng et al., 2014), antimicrobial (Isaka et al., 2013), antiviral (Niedermeyer et al., 2005), and antiinflammatory activities (Kleinwaechter et al., 2001) as well as inhibitory effects against a-glucosidase (Zhao et al., 2015), aldose reductase (Fatmawati et al., 2011), 5a-reductase (Liu et al., 2006), neuraminidase (Zhu et al., 2015), and HMG-CoA reductase (Wang et al., 2015). Ganoderma resinaceum Boud. (Ganodermataceae) has been used as an enthnomedicines for lowering blood sugar (Oyetayo, 2011); however, the chemical composition of G. resinaceum and the biological activities of its components have not been fully determined. Previous studies have reported a few lanostane-type triterpenes, phenolic compounds, and polysaccharides from the fruiting bodies and cultured mycelia of G. resinaceum (Amaral et al., 2008; Peng et al., 2013; Zengin et al., 2015). A recent study revealed that the methanol extract of G. resinaceum shows inhibitory activity against a-glucosidase (Zengin et al., 2015). To investigate the biologically active constituents of G. resinaceum, we carried out a phytochemical investigation on G. resinaceum. As a result, forty-eight triterpenes including 18 previously undescribed compounds (1e2 and 4e19) and 30 known compounds (3 and 20e48) were obtained from its ethanol extract. All compounds isolated from G. resinaceum were identified by extensive spectroscopic analysis (MS, IR, UV, and NMR) and a

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comparison of their spectroscopic data with those reported in the literature. Among the components isolated, compound 3 was isolated from a natural source for the first time. The presence of 3 in Ganoderma lucidum had been presumed based on the analysis of MS fragmentation (Cheng et al., 2011); however, its NMR, IR, and UV spectroscopic data have not been reported, and its structure was not fully established. Compounds 2e4, 18e22, 25e27, 29, 33e36, 39, 43, and 45 were evaluated for their a-glucosidase inhibitory effects. Compounds 3, 21, 22, 26, 27, and 29 had measurable IC50 values and showed much stronger inhibitory activities against aglucosidase than the positive control drug acarbose. 2. Results and discussion 2.1. Structural elucidation The ethanol extract of G. resinaceum was partitioned successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc and nBuOH extracts were combined and repeatedly subjected to column chromatography including silica gel, ODS, MCI, Sephadex LH-20, and semi-preparative HPLC. Finally, eighteen previously undescribed triterpenes (1e2 and 4e19) (Fig. 1) and 30 known analogues (3 and 20e48) were obtained from the fruiting bodies of G. resinaceum.

Compound 1 was isolated as a white powder. Its molecular formula was determined to be C30H46O4 due to the HRESIMS ion peak at m/z 469.3322 [M
H]
(calcd. for C30H45O4, 469.3318). Its 1 H NMR, 13C NMR (Table 1), and HSQC spectra showed the presences of six methyl singlets [dH 0.83 (6H, s), 1.14 (3H, s), 1.02 (3H, s), 1.13 (3H, s), and 1.84 (3H, s)], one methyl doublet (dH 0.92), nine methylenes, four methines including an oxygenated methine at dH 3.25 and dC 78.7, four sp3 quaternary carbons, two pairs of olefinic carbons (dC 127.0, 145.1, 139.5, and 164.3), one ketone carbon (dC 199.3), and one carboxyl group (dC 172.9). The above NMR data suggested 1 was a lanostane triterpenoid similar to 7-oxo-ganoderic acid Z2 (Peng et al., 2013) except 1 is missing a ketone group. The a,b-unsaturated ketone at C-8/C-9/C-11 was defined on the basis of the HMBC correlations from H-19 (dH 1.14) to C-8 (dC 164.3), H-7 (dH 2.26 and 2.36) to C-8 (dC 164.3) and C-9 (dC 139.5), and H-12 (dH 2.67 and 2.48) to C-11 (dC 199.3), and the assignment was also supported by the UV absorption at 258 nm and the IR (KBr) absorption band at vmax 1651 cm
1. The carboxylic acid group at C-26, which is part of an a,b-unsaturated ketone system at C-24/C-25/C26, was further confirmed by the HMBC correlations from both H27 (dH 1.84) and H-24 (dH 6.78) to C-26 (dC 172.9). Finally, the planar structure of 1 was confirmed by the 1H-1H COSY and HMBC experiments (Fig. 2). The ROESY correlations (Fig. 3) of H-27 (dH 1.84) with H-23 (dH 2.26 and 2.11) suggested the D24 double bond to be E-

Fig. 1. Structures of compounds 1e19.

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115 Table 1 1 H (600 MHz, CDCl3) and

13

C (150 MHz, CDCl3) NMR Spectroscopic Data for 1e5 (d in ppm, J in Hz).

No 1

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 a b

105

2

dC

dH

34.3, CH2 27.9, CH2 78.7, CH 39.0, C 51.8, CH 17.3, CH2 30.0, CH2 164.3, C 139.5, C 37.6, C 199.3, C 51.7, CH2 47.2, C 51.6, C 31.0, CH2 27.1, CH2 50.1, CH 16.7, CH3 19.0, CH3 36.1, CH 18.2, CH3 34.6, CH2 25.7, CH2 145.1, CH 127.0, C 172.9, C 12.0, CH3 28.3, CH3 15.7, CH3 25.8, CH3

3.04, dt (13.8, 3.0); 1.07, dd (13.8, 3.6) 1.69, m; 1.65, m

dC

34.3, CH2 28.0, CH2 3.25, dd (11.4, 4.8) 78.7, CH 39.0, C 0.92, m 51.9, CH 1.77 m; 1.48, m 17.3, CH2 2.36, dd (19.8, 6.0); 2.27, 30.0, m CH2 164.4, C 139.5, C 37.6, C 199.4, C 2.67, d (16.2); 2.48, 51.8, d (16.2) CH2 47.2, C 51.6, C 1.77 m; 1.38, m 31.0, CH2 1.98, m; 1.37, m 27.0, CH2 1.74, m 50.1, CH 0.83, s 16.7, CH3 1.14, s 19.0, CH3 1.45, m 36.0, CH 0.92, d (6.6) 18.3, CH3 1.57, m; 1.19, m 35.7, CH2 2.26, m; 2.11, m 24.4, CH2 6.87, br s 126.6, CH 134.5, C 69.0, CH2 1.84, s 13.7, CH3 1.02, s 28.3, CH3 0.83, s 15.7, CH3 1.13, s 25.8, CH3

4a

3

dH

dC

3.04, dt (13.8, 3.0); 34.3, 1.09, m CH2 1.70, m; 1.66, m 27.9, CH2 3.25, dd (11.4, 4.8) 78.6, CH 38.7, C 0.92, m 51.7, CH 1.76 m; 1.47 m 17.0, CH2 2.36 dd (19.8, 6.0); 30.4, 2.26 m CH2 163.5, C 139.8, C 37.8, C 198.7, C 2.67, d (16.4); 2.48, 52.2, d (16.4) CH2 47.2, C 53.4, C 1.77, m; 1.36, m 73.1, CH 2.00, m; 1.39, m 39.0, CH2 1.73, m 48.8, CH 0.85, s 17.3, CH3 1.15, s 19.0, CH3 1.45, m 35.9, CH 0.92, d (6.0) 18.0, CH3 1.48, m; 1.08, m 34.5, CH2 2.11, m 1.95, m 25.7, CH2 5.40, t (6.9) 144.7, CH 127.0, C 4.01, s 171.9, C 1.69, s 12.1, CH3 1.05, s 28.3, CH3 0.85, s 15.8, CH3 1.14, s 18.9, CH3

5b

dH

dC

dH

dC

dH

3.00, (dt 13.8, 3.6); 1.08, (dt 13.8, 3.0) 1.68, m; 1.64, m

34.6, CH2 28.3, CH2 77.1, CH 39.5, C 50.8, CH 37.4, CH2 200.2, C 140.7, C 162.0, C 40.8, C 64.1, CH 45.5, CH2 48.4, C 47.9, C 33.4, CH2 28.2, CH2 50.2, CH 17.1, CH3 19.8, CH3 36.3, CH 18.3, CH3 35.0, CH2 25.7, CH2 142.2, CH 128.7, C 170.4, C 12.6, CH3 27.7, CH3 15.8, CH3 25.1, CH3

2.56, m; 2.18, m

35.4, CH2 28.2, CH2 78.2, CH 39.9, C 51.1, CH 37.3, CH2 207.4, C 150.7, C 155.9, C 41.4, C 203.1, C 53.8, CH2 49.8, C 54.3, C 73.4, CH 31.1, CH2 51.5, CH 19.4, CH3 17.7, CH3 75.0, C

2.81, dt (13.8, 3.0); 1.28, dt (13.8, 3.6) 1.73, m; 1.68, m

25.7, CH3 43.2, CH2 24.5, CH2 143.7, CH 129.0, C 171.8, C 12.4, CH3 28.3, CH3 16.0, CH3 20.7, CH3

1.24, s

3.25, dd (12.0, 4.2)

0.91, m 1.78, m; 1.45, m 2.48, m; 2.45, m

2.74, d (16.2); 2.45, d (16.2)

4.36, dd (9.6, 6.0) 1.98, m; 1.76, m 1.83, m 0.87, s 1.12, s 1.83, m 0.89, d (6.6) 1.51, m; 1.18, m 2.25, m; 2.12, m 6.86, t (7.2)

1.84, s 1.02, s 0.83, s 1.17, s

2.01, m 3.49, dd (9.6, 6.6)

2.00, m 2.71, m

4.79, dd (4.8, 4.2) 2.59, dd (13.2, 4.2); 2.31, dd (13.2, 4.8)

2.51, m; 1.97, m 2.01, m; 1.33, m 1.69, m 0.79, s 1.32, s 1.45, m 1.01, d (6.6) 1.61, m; 1.18, m 2.31, m; 2.17, m 7.24, t (7.2)

2.12, s 1.13, s 1.14, s 1.57, s

3.22, dd (11.4, 4.8)

1.58, dd (15.0, 2.4) 2.71, t (15.6); 2.53, dd (16.2, 2.4)

2.98, d (16.8); 2.60, d (16.8)

4.42, dd (10.2, 6.0) 2.42, dt (13.8, 9.6); 1.67, m 2.20, m 1.04, s 1.30, s

1.55, m; 1.49, m 2.23, m 6.77, dd (7.2, 6.0)

1.82, s 1.00, s 0.88, s 1.16, s

Recorded in Pyridine-d5. Recorded in CD3OD.

configuration. The 3-OH group was assigned as b-oriented based on its large coupling constant (dH 3.25, dd, J ¼ 11.4, 4.8 Hz) and the ROESY correlation of H-3 (dH 3.25) with H-28 (dH 1.02). The ROESY correlation of H-17 (dH 1.74) with H-21 (dH 0.92) and H-30 (dH 1.13), and H-18 (dH 0.83) with H-20 (dH 1.45) suggested methyl-21 and H20 were a-oriented and b-oriented, respectively. Therefore, compound 1 was elucidated as (R,E)-6-((3S,5R,10S,13R,14R,17R)-3hydroxy-4,4,10,13,14-pentamethyl-11-oxo2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-17-yl)-2-methylhept-2-enoic acid, named resinacein

A. Compounds 2 and 3 were both obtained as a white powder. The molecular formulas of 2 and 3 were established as C30H48O3 and C30H46O5 based on their HRESIMS ion peaks at m/z 455.3509 [M
H]
(calcd. for C30H47O3, 455.3525) and m/z 485.3272 [M
H]
(calcd. for C30H45O5, 485.3267), respectively. Comparison of their NMR spectroscopic data (Table 1) with those of 1 suggested their structures are similar. The differences were the presence of a carbinol group at C-26 in 2 instead of the carboxylic acid group present in 1, and the presence of an additional oxygenated methine at C-15 in 3. The HMBC

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X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

Fig. 2. Key COSY, HMBC, and ROESY correlations of 1, 8, 11, and 15.

Fig. 3. Key ROESY correlations of 1, 8, 11, and 15.

correlations from H-27 (dH 1.69) to C-24 (dC 126.6), C-25 (dC 134.5), and C-26 (dC 69.0), and H-26 (dH 4.01) to C-24 (dC 126.6), C-25 (dC 134.5), and C-27 (dC 13.7) confirmed the presence of a C-24/C-25 double bond and a carbniol group at C-26 in the side chain of 2. The 15a-OH in 3 was assigned based on the HMBC correlations from H-15

(dH 4.36) to C-30 (dC 18.9), the 1H-1H COSY correlations of H-15 (dH 4.36) with H-16 (dH 1.76 and 1.98), and the ROESY correlation of H-15 (dH 4.36) with H-18 (dH 0.87). The relative configurations of 3-OH and the D24 double bond were determined based on the results of the ROESY experiment. Therefore, compounds 2 and 3 were determined

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

to be (3S,5R,10S,13R,14R,17R)-3-hydroxy-17-((R,E)-7-hydroxy-6methylhept-5-en-2-yl)-4,4,10,13,14-pentamethyl-1,2,3,4,5,6,7,10, 12,13,14,15,16,17-tetradecahydro-11H-cyclopenta[a]phenanthren11-one (resinacein B) and (R,E)-6-((3S,5R,10S,13R,14R,15S,17R)-3,15dihydroxy-4,4,10,13,14-pentamethyl-11-oxo-2,3,4,5,6,7,10,11,12, 13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)2-methylhept-2-enoic acid (resinacein C), respectively. Compound 4 was obtained as a white powder. Its molecular formula, C30H46O5, was deduced from the HRESIMS ion peak at m/z 485.3269 [M
H]
(calcd. for C30H45O5, 485.3267). The 1H NMR, 13 C NMR (Table 1), and HSQC spectra of 4 indicated that the structure of 4 was highly similar to that of 7-oxo-ganoderic acid Z (27); however, 4 showed the presence of an additional oxygenated methine at C-11. The 11-OH moiety was confirmed by key HMBC correlations from H-11 (dH 4.79) to C-9 (dC 162.0) and C-8 (dC 140.7), and H-12 (dH 2.31 and 2.59) to C-11 (dC 64.1), together with 1H-1H COSY correlations of H-12 (dH 2.31 and 2.59) with H-11 (dH 4.79). The ROESY correlation of H-11 (dH 4.79) with H-19 (dH 1.32) indicated that 11-OH was a-oriented. Based on above analyses, compound 4 was elucidated as (R,E)-6-((3S,5R,10S,11R,13R,14R,17R)3,11-dihydroxy-4,4,10,13,14-pentamethyl-7-oxo-2,3,4,5,6,7,10,11,12, 13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17yl)-2-methylhept-2-enoic acid, named resinacein D. Compound 5 was obtained as a pale-yellow powder. Its molecular formula was assigned as C30H44O7 based on the HRESIMS ion peak at m/z 515.2999 [M
H]- (calcd. for C30H43O7, 515.3009). Comparison of its 1D NMR spectroscopic data (Table 1) with those of 3 showed their structures were similar except for an additional ketone group and one oxygenated sp3 quaternary carbon in 5 instead of one methylene and one methine in 3. Seven singlet methyl groups were observed in the 1H NMR spectrum of 5, suggesting the presence of hydroxy group at C-20. The hydroxy group at C-20 was supported by the HMBC correlations from H-21 (dH 1.24), H-17 (dH 2.20), H-16 (dH 2.42 and 1.67), and H-22 (dH 1.55 and 1.49) to C-20 (dC 75.0). A comparison of the 13C NMR data of 5 with those of 3 revealed the chemical shifts of the C-8/C-9 double bond were significantly different, indicating the presence of a ketone group at C-7 that is part of the C-7/C-8/C-9/C-11 conjugate system. The key HMBC correlations from H-5 (dH 1.58) and H-6 (dH 2.53 and 2.71) to C-7 (dC 207.4) confirmed the position of the C-7 ketone group. The relative configurations of the hydroxy groups at C-3 and C-15 were established based on the ROESY experiment. Accordingly, compound 5 was elucidated as (E)-6-((3S,5R,10S,13R,14R,15S,17S)-3,15dihydroxy-4,4,10,13,14-pentamethyl-7,11-dioxo-2,3,4,5,6,7,10,11,12, 13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17yl)-6-hydroxy-2-methylhept-2-enoic acid, named resinacein E. Compound 6 was obtained as a white powder. Its molecular formula was determined to be C31H42O7 based on the HRESIMS ion peak at m/z 525.2852 [M
H]
(calcd. for C31H41O7, 525.2852). Comparison of its spectroscopic data with those of 25 suggested their structure were similar except for the presence of a ketone group instead of the oxymethine group present in 25. The chemical shift of C-8 was almost the same as that of C-9 in the 13C NMR spectrum, indicating a ketone group was located at C-7; this assignment was further confirmed by HMBC correlations from H-5 (dH 1.72) and H-6 (dH 2.72 and 2.53) to C-7 (dC 198.2). The methyl-21 and H-20 were assigned as a-oriented and b-oriented based on the ROESY correlation of H-12a (dH 3.02) with H-21 (dH 1.11) and H-18 (dH 1.09) with H-20 (dH 2.99), respectively. Thus, compound 6 was determined to be methyl (6R)-6-((3S,5R,10S,13R,14R)-3-hydroxy4,4,10,13,14-pentamethyl-7,11,15-trioxo-2,3,4,5,6,7,10,11,12,13,14,15dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methyl-4oxoheptanoate, named resinacein F. Compound 7 was obtained as a white powder and has a molecular formula of C30H40O8 based on the HRESIMS ion peak at m/z

107

527.2643 [M
H]
(calcd. for C30H39O8, 527.2645). Comparison of its 1H and 13C NMR spectroscopic data (Table 2) with those of 6 revealed that the structure of 7 closely resembled that of 6 except for the presence of a hydroxy group at C-12 and the disappearance of the methoxy group that was present in 6. Additionally, the combination of the downfield carboxyl group and the HMBC correlations indicated 7 was a ganoderic acid. The 12-OH moiety was established by the HMBC correlations of H-12 (dH 4.70) with C-11 (dC 203.0), C-13 (dC 57.7), C-17 (dC 187.2), and C-18 (dC 25.3). The ROESY correlation of H-12 (dH 4.70) with H-30 (dH 1.51) indicated the 12-OH moiety was in the b-orientation. Therefore, compound 7 was elucidated as (6R)-6-((3S,5R,10S,12S,13R,14R)-3,12-dihydroxy4,4,10,13,14-pentamethyl-7,11,15-trioxo-2,3,4,5,6,7,10,11,12,13,14,15dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methyl-4oxoheptanoic acid, named resinacein G. Compound 8 was obtained as a white powder. Its molecular formula was established as C30H42O8 based on the HRESIMS ion peak m/z 530.2855 [M]
(calcd. for C30H42O8, 530.2880) and its 1D NMR and HSQC spectra. The 1H NMR, 13C NMR (Table 2), and HSQC spectra suggested 8 was a lanostane-type ganoderic acid similar to 25. However, a comparison of their NMR data revealed 8 possessed an oxygenated sp3 quaternary carbon but was missing the methoxy group present in 25. It was speculated that the hydroxy group was located at C-20 due to the presences of six singlet methyl signals and oxygenated quaternary carbon at dC 77.4. This hypothesis was confirmed by the key HMBC correlations from H-16 (dH 5.75), H-21 (dH 1.37), and H-22 (dH 2.85 and 3.05) to C-20 (dC 74.4). The carboxylic acid group at C-26 was assigned based on the HMBC correlations (Fig. 2) and its downfield chemical shift (dC 180.0). Thus, compound 8 was determined to be 6-((3S,5R,7S,10S,13R,14R)-3,7dihydroxy-4,4,10,13,14-pentamethyl-11,15-dioxo-2,3,4,5,6,7,10,11, 12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)-6hydroxy-2-methyl-4-oxoheptanoic acid, named resinacein H. Compound 9 was isolated as a white powder. The HRESIMS ion peak at m/z 529.3137 [M
H]
(calcd. for C31H45O7, 529.3165) suggested its molecular formula was C31H46O7. Interpretation of its NMR data revealed that 9 was very similar to the known compound 3b,7b,15b-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (Hu et al., 2013), but there were the major chemical differences in the 1H NMR shifts of H-15 and H-30 compared with those of 3b,7b,15b-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester. Accordingly, it was hypothesized that the 15OH group was a-oriented unlike that of 3b,7b,15b-trihydroxy11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester. The hypothesis was confirmed by the ROESY correlation of H-15 (dH 5.35) with H-18 (dH 1.02). Thus, compound 9 was determined to be methyl (6R)-2-methyl-4-oxo-6-((3S,5R,7S,10S,13R,14R,15S)-3,7,15trihydroxy-4,4,10,13,14-pentamethyl-11-oxo-2,3,4,5,6,7,10,11,12,13, 14,15-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)heptanoate, named resinacein I. Compound 10 was obtained as a white powder. Its molecular formula was established as C30H44O8 by the HRESIMS ion peak at m/ z 531.2949 [M
H]
(calcd. for C30H43O8, 531.2958). The 1H and 13C NMR (Table 2) and HSQC spectra suggested 10 was a ganoderic acid with a structure similar to that of 8 except that one of carbonyl groups of 8 was reduced to an oxygenated methine at C-15 in 10. The presence of the 15a-OH group was supported by the key HMBC correlations from H-15 (dH 5.45) to C-16 (dC 127.9), C-17 (dC 156.2), and C-30 (dC 23.5), the 1H-1H COSY correlation of H-15 (dH 5.45) with H-16 (dH 5.38), and the ROESY correlation of H-15 (dH 5.45) with H-18 (dH 1.17). Therefore, compound 10 was assigned as 6hydroxy-2-methyl-4-oxo-6-((3S,5R,7S,10S,13S,14R,15S)-3,7,15trihydroxy-4,4,10,13,14-pentamethyl-11-oxo2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl)heptanoic acid, named resinacein J.

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Table 2 1 H NMR (600 MHz, CD3OD) and No

6

a

dC 1

13

C NMR (150 MHz, CD3OD) Spectroscopic Data for 6e10 (d in ppm, J in Hz). 7

dH

dC

dH

2 3

34.0, CH2 2.95, m; 1.43, dd (13.2, 3.6) 34.6, CH2 2.86, m; 1.26, dt (13.8, 3.6) 27.4, CH2 1.78, m; 1.71, m 28.1, CH2 1.67, m; 1.62, m 77.7, CH 3.32, dd (11.4, 4.2) 78.4, CH 3.17, dd (11.7, 4.4)

4 5 6

39.0, C 49.1, CH 1.72, 35.2, CH2 2.72,

7

198.2, C

8 9 10 11 12

150.6, C 152.4, C 40.8, C 199.7, C 44.7, CH2 3.02,

13 14 15 16 17 18 19 20 21 22

51.6, C 55.1, C 203.6, C 123.2, CH 183.1, C 29.8, CH3 17.4, CH3 28.7, CH 20.0, CH3 47.9, CH2

23 24

206.4, C 46.6, CH2 2.42,

25 26 27 28 29 30 26-OMe

34.7, CH 176.3, C 17.3, CH3 27.8, CH3 15.3, CH3 33.2, CH3 52.2, CH3

a

5.65, 1.09, 1.14, 2.99, 1.11, 2.81,

2.94, 1.19, 1.04, 0.90, 1.46, 3.67,

9a

8

dC

dH

36.0, CH2 2.77, m; 1.00, dd (13.2, 3.6) 28.4, CH2 1.61, m; 1.54, m 79.0, CH 3.09, dd (12.0, 4.8) 40.1, C 39.9, C dd (14.4, 4.2) 51.2, CH 1.68, m 50.6, CH 0.93, m m; 2.53, dd (18.0,14.4) 37.5, CH2 2.53, dd (10.2, 8.4) 27.6, CH2 2.08, dd (12.6, 8.4); 1.47, m 200.2, C 68.4, CH 4.75, dd (9.6, 8.4) 152.0, C 159.5, C 153.5, C 143.9, C 41.7, C 40.4, C 203.0, C 200.4, C d (16.8); 2.57, d (16.8) 76.8, CH 4.70, s 47.1, CH2 3.15, d (16.8); 2.85, d (16.8) 57.7, C 52.8, C 40.4, C 60.8, C 206.0, C 211.9, C s 123.5, CH 5.61, s 125.0, CH 5.75, s 187.2, C 188.7, C s 25.3, CH3 0.93, s 32.3, CH3 1.26, s s 17.6, CH3 1.14, s 19.1, CH3 1.13, s m 30.9, CH 3.33, m 74.4, C d (7.2) 19.7, CH3 1.12, d (6.6) 29.1, CH3 1.37, s dd (17.4, 9.8); 2.71, m 48.3, CH2 2.89, dd (17.4, 8.4); 53.7, CH2 3.05, d (16.2); 2.66, dd (17.4, 6.0) 2.85, d (16.2) 209.2, C 210.4, C dd (10.8, 3.0); 2.94, m 47.3, CH2 2.80, m; 2.46, dd 48.7, CH2 2.86, dd (18.0, (13.2, 3.6) 8.4); 2.54, dd (18.0, 4.8) m 36.8, CH 2.77, m 36.1, CH 2.75, m 179.3, C 180.0, C d (6.6) 17.8, CH3 1.08, d (6.6) 17.7, CH3 1.08, d (7.2) s 28.3, CH3 1.08, s 28.9, CH3 0.96, s s 16.0, CH3 0.95, s 16.5, CH3 0.77, s s 33.0, CH3 1.51, s 33.7, CH3 1.48, s s

dC

10

dH

dC

dH

34.6, CH2 2.85, m; 0.98, m

36.0, CH2 2.81, dt (13.2, 3.6); 0.99, m 28.3, CH2 1.64, m 28.5, CH2 1.66, m; 1.61, m 78.2, CH 3.22, dd (11.4, 4.8) 79.1, CH 3.15, dd (11.4, 4.8) 38.9, C 40.0, C 49.5, CH 0.98, m 50.8, CH 0.96, d (12.0) 27.9, CH2 2.14, m; 1.63, m 29.2, CH2 2.13 dd (12.0 7.8); 1.59, m 69.9, CH 4.53, br s 70.2, CH 4.47, dd (9.6, 7.8) 158.8, C 162.1, C 141.3, C 143.0, C 39.0, C 39.9, C 199.8, C 202.5, C 47.9, CH2 2.94, d (14.4); 2.37, 50.2, CH2 3.11, d (15.6); d (14.4) 2.66, d (15.6) 51.9, C 53.2, C 56.3, C 59.0, C 77.7, CH 5.35, s 77.8, CH 5.45, s 123.9, CH 5.23, s 127.9, CH 5.38, s 155.2, C 156.2, C 22.0, CH3 1.02, s 24.2, CH3 1.17, s 19.7, CH3 1.23, s 19.7, CH3 1.23, s 27.7, CH 2.66, m 73.7, C 20.2, CH3 1.03, s 28.9, CH3 1.34, s 48.6, CH2 2.67, m; 2.50, dd 53.6, CH2 3.02, d (16.2); (18.6, 9.6) 2.67, d (16.2) 207.5, C 211.9, C 46.6, CH2 2.88, m; 2.43, dd 48.7, CH2 2.93, dd (18.0, (17.4, 2.4) 8.4); 2.60, dd (18.0, 4.8) 34.7, CH 2.93, m 36.2, CH 2.81, m 176.5, C 180.3, C 17.3, CH3 1.17, d (7.2) 17.8, CH3 1.15, d (7.2) 28.4, CH3 1.03, s 29.0, CH3 1.01, s 16.0, CH3 0.85, s 16.6, CH3 0.84, s 22.4, CH3 1.30, s 23.5, CH3 1.29, s 52.2, CH3 3.67, s

Recorded in CDCl3.

Compound 11 was obtained as a white powder. Its molecular formula was determined to be C30H40O7 based on the HRESIMS ion peak at m/z 511.2692 [M
H]
(calcd. for C30H39O7, 511.2696). The 1 H NMR, 13C NMR (Table 3), and HSQC spectroscopic data revealed that the structure of 11 was almost identical to that of ganoderenic acid H (35). However, the 13C NMR spectrum of 11 showed that the signal of C-17 was shifted upfield approximately 10 ppm in comparison with that of ganoderenic acid H, and the chemical shifts at C-20, C-21, C-22, and C-23 in 11 were shifted from dC 155.5 to 156.5, 21.6 to 24.7, 126.1 to 129.2, and 201.1 to 200.3, respectively. Furthermore, it was hypothesized that the relative configuration of the C-20/C-22 double bond was different from that of ganoderenic acid H. The ROESY correlation of H-21 (dH 1.90) with H-22 (dH 6.37) confirmed the C-20/C-22 double bond was in the Z-configuration. Therefore, compound 11 was elucidated as (Z)-6((3S,5R,10S,13R,14R,17R)-3-hydroxy-4,4,10,13,14-pentamethyl7,11,15-trioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-17-yl)-2-methyl-4-oxohept-5-enoic acid, named resinacein K. Compounds 12 and 13 were both obtained as a white powder. The molecular formulas of 12 and 13 were determined to be C30H40O8 and C30H42O8 based on the HRESIMS ion peak at m/z 596.2560 [M þ Na þ HCOOH
H]
(calcd. for C31H41O10Na, 596.2569), and m/z 529.2788 [M
H]
(calcd. for C30H41O8, 529.2801), respectively. Their 1H NMR, 13C NMR (Table 3), and HSQC

spectroscopic data indicated that the structures of 12 and 13 were similar to that of 11. The differences among them were that the presence of an additional hydroxy group at C-12 in 12 was observed while two oxygenated methines in 13 replaced one ketone group and one methylene in 11. The HMBC correlations from H-12 (dH 4.63) to C-11 (dC 202.7), C-13 (dC 52.0) and C-18 (dC 12.5) combined with the ROESY correlation of H-12 (dH 4.63) with H-30 (dH 1.66) confirmed the presence of a 12b-OH functionality in 12. The chemical shifts of C-8 and C-9 in 13 were dramatically different from those of the corresponding peaks of 11, suggesting that the hydroxy group was located at C-7 or C-11. Ultimately, the location of the 7b-OH in 13 was confirmed based on the key HMBC correlations from H-7 (dH 4.88) to C-6 (dC 28.1), C-8 (dC 159.0), and C-9 (dC 144.0), the 1H-1H COSY correlations of H-6 (dH 1.54 and 2.18) with H-7 (dH 4.88), as well as the ROESY correlations of H-7 (dH 4.88) with H-5 (dH 0.95) and H-30 (dH 1.42). The presence of a hydroxy group at C24 in 13 was confirmed by the HMBC correlations from H-24 (dH 4.55) to C-23 (dC 201.7), C-25 (dC 43.6), C-26 (dC 177.6), and C-27 (dC 11.0) and the 1H-1H COSY correlation of H-24 (dH 4.55) with H-25 (dH 2.88). In their 13NMR spectra, the C-22 signals of 12 and 13 were shifted downfield compared with that of 11, implying relative configurations of their C-20/C-22 double bonds were different from that of 11. The relative configurations of their C-20/C-22 double bonds were assigned as E based on the ROESY correlations of H-22 with H-17 and H-16. Thus, compounds 12 and 13 were determined

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115 Table 3 1 H (600 MHz, CD3OD) and

13

C (150 MHz, CD3OD) NMR Spectroscopic Data for 11e14 (d in ppm, J in Hz).

No 11

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

109

12

dC

dH

34.8, CH2 28.2, CH2 78.3, CH 40.3, C 52.6, CH

2.72, dt (13.2, 3.6); 1.16, dd (13.2, 34.5, 4.2) CH2 1.64, m; 1.58, m 28.2, CH2 3.15, dd (11.4, 4.2) 78.2, CH 40.3, C 1.54, dd (15.0, 2.4) 52.8, CH

37.3, CH2 201.7, C 147.0, C 154.0, C 41.9, C 201.9, C 48.6, CH2 49.3, C 49.3, C 209.9, C 37.5, CH2 40.2, CH 19.0, CH3 18.1, CH3 156.5, C 24.7, CH3 129.2, CH 200.3, C 48.8, CH2 36.4, CH 180.2, C 17.8, CH3 28.5, CH3 16.3, CH3 22.0, CH3

2.62, d (15.0); 2.42, m

3.07, d (15.6); 2.03, d (15.6)

dC

37.7, CH2 201.3, C 147.4, C 153.5, C 41.8, C 202.7, C 78.9, CH

52.0, C 59.3, C 208.6, C 2.58, dd (18.6, 9.0); 2.43, dd (18.6, 39.5, 9.0) CH2 5.00, t (9.0) 50.0, CH 0.68, s 12.5, CH3 1.23, s 18.4, CH3 157.4, C 1.90, s 23.1, CH3 6.37, s 126.8, CH 201.8, C 2.87, dd (17.4, 8.4); 2.52, dd (17.4, 49.1, 4.8) CH2 2.78, m 37.9, CH 182.0, C 1.12, d (7.2) 18.1, CH3 0.94, s 28.5, CH3 0.81, s 16.3, CH3 1.59, s 20.9, CH3

13

dH 2.68, m; 1.15, dd (13.2, 3.6)

dC

35.9, CH2 1.68, m; 1.60, m 28.2, CH2 3.15, dd (12.0, 4.2) 78.9, CH 39.7, C 1.54, m 50.34, CH 2.66, m; 2.44, m 28.1, CH2 67.7, CH 159.0, C 144.0, C 40.0, C 199.8, C 4.63, s 50.32, CH2 47.6, C 59.4, C 216.7, C 2.74, dd (18.6, 9.6); 2.30, dd (18.6, 38.8, 9.0) CH2 3.47, t (9.0) 51.1, CH 0.60, s 19.4, CH3 1.30, s 18.7, CH3 158.9, C 2.21, s 21.7, CH3 6.25, s 122.4, CH 201.7, C 2.83, dd (17.4, 8.4); 2.43, dd (17.4, 78.9, CH 9.0) 2.73, m 43.6, CH 177.6, C 1.07, d (6.6) 11.0, CH3 0.94, s 28.7, CH3 0.83, s 16.3, CH3 1.66, s 25.3, CH3

to be (E)-6-((3S,5R,10S,12S,13R,14R,17R)-3,12-dihydroxy-4,4,10, 13,14-pentamethyl-7,11,15-trioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methyl-4oxohept-5-enoic acid (resinacein L) and (E)-6-((3S,5R, 7S,10S,13R,14R,17R)-3,7-dihydroxy-4,4,10,13,14-pentamethyl-11,15dioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-3-hydroxy-2-methyl-4-oxohept-5enoic acid (resinacein M), respectively. Compound 14 was obtained as a white powder. Its molecular formula was determined to be C30H44O8 based on the HRESIMS ion peak at m/z 531.2947 [M
H]
(calcd. for C30H43O8, 531.2958). Comparison of its spectroscopic data with those of 13 revealed their structures were similar except for the presence of an oxygenated methine at C-15 in 14 instead of the ketone group in 13. The 15-OH group was confirmed by the key HMBC correlations from H-15 (dH 4.90) to C-30 (dC 20.5) and C-14 (dC 55.0), and H-30 (dH 1.32) to C-15 (dH 73.6), as well as the 1H-1H COSY correlations of H-15 (dH 4.90) with H-16 (dH 1.73 and 2.49). The ROESY correlation of H-15 (dH 4.90) with H-18 (dH 0.78) suggested the 15-OH moiety was in the aorientation. The ROESY correlation of H-22 (dH 6.47) with H-17 (dH 3.04) confirmed the double bond at C-20 was in the E-

14

dH

dC

dH

2.81, m; 1.04, m

36.0, CH2 28.5, CH2 79.0, CH 39.8, C 50.6, CH

2.71, m; 0.96, m

1.66, m; 1.57, m 3.15, dd (12.0, 4.8) 0.95, d (13.2)

2.18, dd (13.2, 9.0); 1.54, dd (13.2, 29.2, 9.0) CH2 4.88 t (9.0) 70.3, CH 161.0, C 143.2, C 39.9, C 201.5, C 3.07, d (16.8); 2.47, d (16.8) 52.2, CH2 49.9, C 55.0, C 73.6, CH 2.82, dd (19.2, 9.6); 2.62, dd (19.2, 33.0, 8.4) CH2 3.29, dd overlapped 54.0, CH 0.86, s 19.6, CH3 1.20, s 19.9, CH3 161.4, C 2.20, s 22.5, CH3 6.46, s 122.0, CH 202.4, C 4.55, d (4.2) 80.0, CH 2.88, m 1.03, d (9.0) 1.02 s 0.83, s 1.42, s

45.7, CH 182.7, C 11.9, CH3 28.8, CH3 16.6, CH3 20.5, CH3

1.66, m; 1.58, m 3.15, dd (12.0, 4.8) 0.95, m 2.11, dd (12.0, 7.2); 1.60, m 4.55, dd (10.2, 7.2)

2.98, d (15.0); 2.22, d (15.0)

4.90, dd (9.6, 7.2) 2.49, m; 1.73, m 3.04, dd (9.6, 6.0) 0.78, s 1.24, s

2.16, s 6.47, s

4.58, d (3.0) 2.70, m 0.98, d (7.2) 1.02, s 0.83, s 1.32, s

configuration. Therefore, compound 14 was elucidated as (E)-3hydroxy-2-methyl-4-oxo-6-((3S,5R,7S,10S,13R,14R,15S,17R)-3,7,15trihydroxy-4,4,10,13,14-pentamethyl-11-oxo2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-17-yl)hept-5-enoic acid, named resinacein N. Compound 15 was isolated as a white powder. Its molecular formula was determined to be C31H45O8 based on the HRESIMS ion peak at m/z 545.3113 [M
H]
(calcd. for C31H45O8, 545.3114). The 1 H NMR, 13C NMR (Table 4), and HSQC spectra revealed that 15 was a methylated derivative of ganoderic acid similar to the known compound 24. The only difference between 15 and 24 was the presence of an additional hydroxy group attached to C-24 in 15. The HMBC correlations from H-24 (dH 4.57) to C-23 (dC 209.4), C-25 (dC 41.9), C-26 (dC 174.2), and C-27 (dC 10.0) in conjunction with the 1 H-1H COSY correlation of H-24 (dH 4.57) with H-25 (dH 2.97) confirmed the presence of 24-OH. Thus, compound 15 was elucidated as methyl (6R)-6-((3S,5R,7S,10S,13R,14R,17R)-3,7-dihydroxy4,4,10,13,14-pentamethyl-11,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15, 16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-3hydroxy-2-methyl-4-oxoheptanoate, named resinacein O. Compound 16 was obtained as a white powder. The molecular

110

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

Table 4 1 H (600 MHz, CDCl3) and No

13

C (150 MHz, CDCl3) NMR Spectroscopic Data for 15e19 (d in ppm, J in Hz).

15

16

dC

dH

1

34.8, CH2

2 3

27.7, CH2 78.3, CH

2.85, 1.00, 1.69, 3.23,

4 5

38.6, C 49.1, CH

0.90, m

39.1, C 50.7, CH

6

26.6, CH2

2.22, m; 1.64, m

36.2, CH2

7 8 9 10 11 12

66.8, CH 156.9, C 142.7, C 38.8, C 197.7, C 50.2, CH2

4.82, m

199.3, C 146.8, C 151.3, C 40.4, C 200.0, C 49.9, CH2

13 14 15 16

45.3, C 59.4, C 217.2, C 40.9, CH2

17

45.0, CH

2.52, m

48.5, CH

18 19 20 21 22

17.4, 18.4, 31.5, 19.8, 45.6,

1.04, 1.24, 2.24, 1.02, 2.52,

17.8, 17.7, 73.1, 26.9, 52.3,

23 24

209.4, C 77.1, CH

4.57, m

210.8, C 47.6, CH2

25

41.9, CH

2.97, qd (7.2, 3.6)

26 27 28 29 30 26-OMe

174.2, C 10.0, CH3 28.2, CH3 15.5, CH3 24.4, CH3 52.4, CH3

1.09, 1.05, 0.87, 1.37, 3.78,

a

CH3 CH3 CH CH3 CH2

dt (13.8, 3.6); dd (13.8, 4.2) m; 1.65, m dd (11.4, 4.8)

2.80, d (16.8); 2.73, d (16.8)

2.70, dd (19.2, 8.4); 2.06, dd (19.2, 9.6)

s s m d (6.6) m; 2.17, m

d (7.2) s s s s

18a

17

19a

dC

dH

dC

dH

dC

dH

33.6, CH2

2.85, dt (13.2, 3.6); 1.27, m 1.75, m; 1.69, m 3.27, dd (12.0, 4.8)

33.6, CH2

2.85, dt (13.2, 3.6); 1.25, m 1.73, m; 1.66, m 3.28, dd (12.0, 4.8)

36.0, CH2 28.5, CH2 79.1, CH

2.64, 0.88, 1.59, 3.08,

39.8, C 50.5, CH

27.4, CH2 77.5, CH

44.8, C 57.2, C 207.8, C 35.2, CH2

CH3 CH3 C CH3 CH2

1.58, dd (13.8, 3.6) 2.64, d (15.0); 2.59, dd (15.0, 3.6)

2.88, d (16.2); 2.81, d (16.2)

2.68, dd (18.0, 9.6); 2.50, dd (18.0, 8.4) 2.30, t (9.6) 1.01, s 1.25, s 1.38, s 2.64, d (16.8); 2.50, d (16.8)

27.4, CH2 77.5, CH 39.1, C 50.8, CH 36.2, CH2

199.4, C 146.7, C 151.6, C 40.1, C 199.7, C 49.4, CH2 44.3, C 57.0, C 207.3, C 40.4, CH2

44.9, CH 16.2, 17.8, 31.6, 19.8, 45.0,

CH3 CH3 CH CH3 CH2

209.4, C 77.1, CH

34.4, CH

2.89, dd (17.4, 3.6); 2.46, dd (17.4, 4.2) 2.96, m

41.9, CH

176.0, C 17.0, CH3 27.8, CH3 15.5, CH3 22.4, CH3 52.1, CH3

1.20, 1.03, 0.89, 1.52, 3.69,

174.1, C 9.9, CH3 27.8, CH3 15.5, CH3 21.8, CH3 52.4, CH3

d (7.2) s s s s

1.59, dd (13.8, 3.0) 2.63, d (15.0); 2.59, dd (15.0, 3.0)

2.87, d (15.6); 2.69, d (15.6)

dC

dH

36.0, CH2 28.3, CH2 79.0, CH

2.78, 1.03, 1.66, 3.15,

0.87, d (13.2)

39.7, C 50.3, CH

0.93, d (13.2)

29.2, CH2

2.04, m; 1.52, m

28.0, CH2

2.17, m; 1.56, dd (13.2, 9.0)

70.3, CH 161.4, C 143.2, C 39.8, C 202.4, C 53.3, CH2

4.45, dd (10.2, 9.8)

67.9, CH 158.9, C 144.1, C 39.9, C 200.4, C 51.5, CH2

4.84, t (9.0)

48.5, 55.5, 73.4, 37.2,

C C CH CH2

dt (13.2, 3.6); m m; 1.53, m dd (12.0, 4.8)

2.80, d (15.0); 2.33, d (15.0)

dt (13.2, 3.6); m m; 1.58, m dd (12.0, 4.8)

2.93, d (16.8); 2.62, d (16.8)

4.70, dd (9.6, 7.2) 1.76, m; 1.71, m

46.7, C 60.5, C 218.4, C 41.9, CH2

49.3, CH

1.85, m

46.9, CH

2.22, m

17.7, 19.9, 33.5, 20.3, 46.8,

0.91, 1.19, 1.98, 0.81, 2.55, 2.42,

17.8, 18.9, 32.6, 20.2, 46.4,

1.00, 1.21, 2.18, 0.99, 2.61,

2.72, dd (18.0, 8.4); 1.91, dd (18.0, 8.4) 2.24, dd (10.2, 8.4) 0.88, s 1.27, s 2.19, m 0.99, d (6.0) 2.50, m

4.56, m

213.0, C 78.9, CH

4.30, d (4.8)

212.5, C 78.8, CH

4.38, br d (4.8)

43.5, CH

2.86, qd (7.2, 5.4)

43.4, CH

2.87, m

176.2, C 11.5, CH3 28.8, CH3 16.6, CH3 20.2, CH3 52.6, CH3

1.01, 0.95, 0.77, 1.18, 3.62,

177.9, C 11.8, CH3 28.7, CH3 16.2, CH3 24.9, CH3

1.10, 1.02, 0.83, 1.37,

2.95, qd (7.2, 3.0) 1.07, 1.03, 0.89, 1.54, 3.76,

d (7.2) s s s s

CH3 CH3 CH CH3 CH2

s s m d (6.6) dd (18.0, 2.4); dd (18.0, 9.6)

d (7.2) s s s s

CH3 CH3 CH CH3 CH2

2.74, dd (19.2, 7.8); 2.11, dd (19.2, 9.6)

s s m d (7.2) m; 2.55, m

d (7.2) s s s

Recorded in CD3OD.

formula, C31H44O8, was confirmed by the HRESIMS ion peak at m/z 543.2960 [M
H]
(calcd. for C31H43O8, 543.2958). Comparison of its 1H and 13C NMR (Table 4) spectroscopic data with those of methyl ganoderate I showed most of the signals in 16 were the same as those of methyl ganoderate I (Kikuchi et al., 1985) except for the presence of a ketone at C-7 instead of the oxygenated methine seen in methyl ganoderate I. The ketone group at C-7 was assigned by the key HMBC correlations of H-6 (dH 2.59 and 2.64) with C-7 (dC 199.3) and was also supported by comparison of the chemical shifts of C-8 and C-9 with those of the corresponding carbons of methyl ganoderate I. Therefore, compound 16 was determined to be methyl 6-hydroxy-6-((3S,5R,10S,13R,14R,17S)-3hydroxy-4,4,10,13,14-pentamethyl-7,11,15-trioxo-2,3,4,5,6,7,10,11, 12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren17-yl)-2-methyl-4-oxoheptanoate, named resinacein P. Compounds 17e19 were isolated as a white powder. The molecular formulas of 17, 18, and 19 were established as C31H43O8, C31H48O8, and C30H44O8 based on the HRESIMS ion peaks at m/z 543.2963 [M
H]
(calcd. for C31H43O8, 543.2958), 571.3281 [M þ Na]þ (calcd. for C31H48O8Na, 571.3247), and 531.2919 [M
H]
(calcd. for C30H43O8, 531.2958), respectively. The similarities in the

NMR data (Table 4) of 17e19 and 15 revealed that they are analogues. The differences among them were that the ketone or hydroxy groups were located at C-7 and/or C-15 and the methylation of the carboxylic group at C-26. A comparison of the NMR data of 17 with those of 15 showed that the chemical shifts of a tetrasubstituted olefinic group (dC 146.7 and 151.6) were significantly different from those of 15, indicating that the ketone in 17 may be located at C-7. The ketone at C-7 in 17 was assigned by the HMBC correlations from H-6 (dH 2.59 and 2.63) to C-7 (dC 199.4). A detailed comparison of the NMR data of 15 and 18 indicated the presence of an oxygenated methine at C-15 in 18 instead of the ketone in 15. The 15a-OH in 18 was established by the key HMBC correlation from H-15 (dH 4.70) to C-30 (dC 20.2), the 1H-1H COSY correlations of H-15 (dH 4.70) with H-16 (dH 1.71 and 1.76), and the ROESY correlation of H-15 (dH 4.70) with H-18 (dH 1.19). A detailed comparison of the NMR data of 19 with those of 15 showed their structures were similar except for the absence of a methoxy group and the presence of downfield carboxylic group at dC 177.9 in 19, suggesting 15 was a methylated derivative of 19. The carboxylic acid group at C-26 in 19 was ultimately confirmed by the HMBC experiment. Accordingly, compounds 17, 18, and 19 were

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

determined to be methyl (6R)-3-hydroxy-6((3S,5R,10S,13R,14R,17R)-3-hydroxy-4,4,10,13,14-pentamethyl7,11,15-trioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-17-yl)-2-methyl-4-oxoheptanoate (resinacein Q), methyl (6R)-3-hydroxy-2-methyl-4-oxo-6-((3S,5R,7S, 10S,13R,14R,15S,17R)-3,7,15-trihydroxy-4,4,10,13,14-pentamethyl11-oxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)heptanoate (resinacein R), and (6R)-6((3S,5R,7S,10S,13R,14R,17R)-3,7-dihydroxy-4,4,10,13,14-pentame thyl-11,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro1H-cyclopenta[a]phenanthren-17-yl)-3-hydroxy-2-methyl-4-oxo heptanoic acid (resinacein S), respectively. The known compounds were identified as ganodermadiol (20) (Arisawa et al., 1986), ganoderic acid Y (21) (Toth et al., 1983), lucialdehyde C (22) (Gao et al., 2002), ganodermanondiol (23) (Fujita et al., 1986), methyl ganoderate B (24) (Kubota et al., 1982), 3b,7b-dihydroxy-11,15,23-trioxo-lanost-8,16-dien-26-oic acid methyl ester (25) (Guan et al., 2007), 7-oxo-ganoderic acid Z3 (26) (Peng et al., 2013), 7-oxo-ganoderic acid Z (27) (Li et al., 2006), lucidumol B (28) (Min et al., 1998), lucidadiol (29) (Gonzalez et al., 1999), ganodermatriol (30) (Arisawa et al., 1986), ganodermic acid Jb (31) (Shiao et al., 1988), astraodoric acid C (32) (Arpha et al., 2012), ganoderenic acid B (33) (Komoda et al., 1985), ganoderic acid B (34) (Chen and Yu, 1993; Kubota et al., 1982), ganoderenic acid H (35) (Nishitoba et al., 1989), ganoderenic acid AM1 (36) (Liu et al., 2014), methyl ganoderate C6 (37) (Kikuchi et al., 1986), 20(Z)ganoderenic acid I (38) (Nishitoba et al., 1989), ganoderic acid I (39) (Kikuchi et al., 1985), 20-hydroxy-ganoderic acid AM1 (40) (Liu et al., 2014), ganoderic acid C6 (41) (Cheng et al., 2012; Kikuchi et al., 1986), methyl ganoderate I (42) (Kikuchi et al., 1985), 3b,7b,15b-trihydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid methyl ester (43) (Hu et al., 2013), ganoderenic acid C (44) (Komoda et al., 1985), ganolucidate F (45) (Liu et al., 2012), ganoderic acid L (46) (Nishitoba et al., 1986), ganodermic acid XL1 (47) (Liu et al., 2014), and ganodermic acid XL2 (48) (Liu et al., 2014) by comparing their spectroscopic data with those reported in the literature, respectively. Some methods including single-crystal Xray diffraction, electronic circular dichroisms, and Mosher's method were used to determine the absolute configuration at C-24 of 13e15 and 17e19; however, these methods failed. At present, the chirality at C-24 is unidentified in 13e15 and 17e19. 2.2. a-Glucosidase inhibitory activity

111

against a-glucosidase than the positive control drug acarbose (Table 5). Compound 26 showed the strongest a-glucosidase inhibition and with an IC50 value 26 times lower than that of acarbose, suggesting that 26 is a potentially strong a-glucosidase inhibitor. In addition, the IC50 values of 21 and 27 were almost 14 times and 24 times lower than that of acarbose, respectively. The compounds with measurable IC50 values all have C-24/C-25 double bonds in their side chains, suggesting that the C-24/C-25 double bond at the branch is key factor affecting inhibitory activity against a-glucosidase. Structures without this double bond showed no significant inhibitory activity and their IC50 values could not be determined. However, the presence of C-24/C-25 double bond in the side chain was not the “magic bullet” guaranteeing inhibitory activity; the hydroxy and ketone groups at some positions also play important roles in inhibitory effects. For instance, the structure of 4 was highly similar to that of 27 with the only difference being that 4 contained a hydroxy group at C-11 where 27 did not. This difference resulted in an IC50 value of 27 of 0.114 mM while 4 exhibited much weaker inhibitory activity (30.3% inhibition at 2.0 mM). For this reason, the hydroxy group at C-11 may decrease the a-glucosidase inhibitory effect. Although 1, 3, and 45 showed many structural similarities, compound 3 showed strong a-glucosidase inhibitory activity, 1 displayed weaker inhibitory effects (50.3% inhibition at 2.5 mM), and 45 showed no inhibitory activity against a-glucosidase. The differences among these compounds were the presence of hydroxy groups at the position of C-15 and C-23. Compound 3 contained a 15-hydroxy substituent and 1 did not, which suggests the 15-OH may play an important role in increasing the a-glucosidase inhibitory activity. This hypothesis was also supported by comparing the structure and activity of 26 with those of 27. Compound 45 showed the presence of 15-hydroxy group, but it displayed no significant inhibitory activity. The poor activity of 45 may be because the 23-OH substituent in 45 negatively impacted 45's ability to inhibit a-glucosidase. The IC50 value of 27 was 5 times and 15 times lower than those of 22 and 29, respectively. The only difference among these compounds was that 27, 22, and 29 possessed carboxylic acid, aldehyde, and hydroxy groups at C-26, respectively. These results suggested that the carboxylic acid group at C-26 is essential for the inhibitory effects. The same trend was also observed between 20 and 21. None of the compounds with 11- and/or 23-ketone groups showed any significant inhibitory activity. The presence of 11- and 23-ketone groups is therefore believed to decrease a-glucosidase inhibitory

The inhibitory effects against a-glucosidase were examined using p-NPG as the substrate and acarbose as the positive control drug. Compounds 1, 3, 4, 18e22, 25e27, 29, 33e36, 39, 43, and 45 were chosen as representatives of different types of common lanostane triterpenoids, and their a-glucosidase inhibitory effects were evaluated. Compounds 3, 21, 22, 26, 27, and 29 presented measurable IC50 value and showed stronger inhibitory activities Table 5 Inhibitory effects of yeast a-glucosidase. Compounds

IC50 (mM)a

3 21 22 26 27 29 Acarboseb

1.049 ± 0.062 0.170 ± 0.015 0.635 ± 0.04 0.106 ± 0.002 0.114 ± 0.019 1.735 ± 0.11 2.761 ± 0.029

a Inhibitory activity was expressed as the mean ± SD of IC50 in triplicates determination. b Positive control.

Fig. 4. Summary of structure-activity relationships of triterpenes from Ganoderma resinaceum on a-glucosidase inhibition.

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X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

activity. For example, compound 3 possessed an 11-ketone group and its IC50 value was approximately 10 times higher than that of 26 and 27, indicating that the presence of a ketone at C-11 might decrease a-glucosidase inhibition within this series of compounds. The structure-activity relationships of the triterpenes isolated from G. resinaceum on a-glucosidase inhibitory activity are summarized in Fig. 4. 3. Conclusions In summary, forty-eight lanostane-type triterpenes including 18 previously undescribed compounds were isolated from the fruiting bodies of G. resinaceum. Compound 3 was isolated from a natural source for the first time. Compounds 1, 3, 4, 18e22, 25e27, 29, 33e36, 39, 43, and 45 were evaluated for their a-glucosidase inhibitory effects. Compounds 3, 21, 22, 26, 27, and 29 showed stronger inhibitory activity against a-glucosidase than the positive control acarbose. Analysis of the structure-activity relationships on a-glucosidase inhibitory effects of these compounds evaluated showed that the C-24/C-25 double bond is necessary for the aglucosidase inhibitory activity. The carboxylic acid group at C-26 and the hydroxy group at C-15 play important roles in enhancing the a-glucosidase inhibitory effect. Hydroxy and ketone groups at the position of C-11 or/and C-23 decrease the inhibitory activity. These results provide an approach to understanding the structural requirements for lanostane-type triterpenes from G. resinaceum to inhibit a-glucosidase activity. This understanding is necessary to develop new types of a-glucosidase inhibitors. Our bioactivity study also showed that ganoderma triterpenes, such as compounds 3, 21, 22, 26, 27, and 29, may play important roles in the antidiabetic effects of G. resinaceum extracts. 4. Experimental 4.1. General experimental procedures NMR spectra were recorded on a Bruker Ascend 600 spectrometer with TMS used as a reference. Optical rotations were measured on a PerkinElmer Model 341 polarimeter. UV spectra were acquired using a HACH DR6000 UVevisible spectrophotometer. IR spectra were recorded as KBr disks on PerkinElmer Spectrum 100 Series FT-IR spectrometers. HRESIMS data were obtained on an LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap FT-MS spectrometer. TLC was carried out on silica gel GF254 plates (Yantai Institute of Chemical Industry, Yantai, China) and spots were visualized by UV light (254 and/or 365 nm) and spraying with 10% H2SO4 followed by heating. Column chromatography was carried out using silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI gel (75e150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan), ODS (35e70 mm, Grace, Maryland, USA), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) as packing materials. Semi-preparative HPLC separations were performed on a Shimadzu instrument coupled to a CBM-20A system controller, two LC-20AP pumps, an SPD-M20A Photodiode Array Detector, and an SIL-10AP autosampler, and the system was equipped with a Shimadzu PRC-ODS column (250 mm  20 mm i.d., 15 m m). Absorbance was measured at 405 nm using a Flexstation 3 multi-mode microplate reader (Molecular Devices, USA). 4.2. Fungal material Fruiting bodies of Ganoderma resinaceum Boud. (Ganodermataceae) were purchased in Dec. 2014 from Haikou Ruizhitang Wild Lingzhi Co., Ltd., and the sample has been collected in Changjiang (N 19.0066 , E 109.1811 ), Hainan province, China. The mushroom

was identified as Ganoderma resinaceum Boud. (Ganodermataceae) by Professor Shaoping Li, a corresponding author. A voucher specimen (No. ICMS-SQC-20141201) has been deposited with the Institute of Chinese Medical Sciences, University of Macau. 4.3. Extraction and isolation The dried fruiting bodies of G. resinaceum (48 kg) were powdered and extracted with 95% EtOH (600 L  2 h  2) under reflux. The organic solvent was removed under vacuum to afford the crude extract (2.6 kg). The extract was dispersed in water and sequentially extracted with petroleum ether, EtOAc, and n-BuOH, successively. The HPLC and TLC profiling of the EtOAc and n-BuOH extracts showed they contained similar constituents; therefore, the EtOAc and n-BuOH extracts were combined. The mixture of the EtOAc and n-BuOH extracts was subjected to silica gel column chromatography (CC) eluted with a gradient of CHCl3-MeOH (100:0e0:100, v/v) to obtain three fractions (WE1eWE3). WE1 (358.0 g) was separated using silica gel CC eluted with petroleum ether-acetone (100:0e1:100, v/v) to obtain four subfractions (WE11eWE14). WE11 (72.0 g) was further purified over silica gel CC eluted with petroleum ether-EtOAc (10:1e0:1, v/v) to yield WE11A and WE11B. WE11B (24.0 g) was subjected to silica gel CC eluted with CHCl3-MeOH (100:0e20:1, v/v) to obtain five fractions (WE11B1-WE11B5). WE11B1 was chromatographed over silica gel CC eluted with petroleum ether-EtOAc (8:1e1:1, v/v) to yield three fractions (WE11B11eWE11B13). WE11B12 was purified over silica gel CC eluted with CHCl3-EtOAc (30:1, v/v) to afford 20 (100.1 mg) and 21 (238.0 mg). WE11B13 was chromatographed over ODS CC eluted with MeOH-H2O (80:20e100:0, v/v) to afford 22 (601.3 mg) and 23 (8 mg). WE12 (135.0 g) was separated using silica gel CC eluted with a gradient of petroleum ether-EtOAc (10:1e0:1, v/v) to yield fractions WE12A and WE12B. WE12B was further subjected to MCI gel CC eluted with MeOH-H2O (70:30e100:0, v/v) to obtain seven fractions (WE12B1eWB12B7). WE12B5 was chromatographed over ODS CC eluted with MeOH-H2O (50:50e100:0, v/v) to afford four fractions (WE12B51eWE12B54). WE12B51 was purified over silica gel CC eluted with petroleum ether-EtOAc (2:1, v/v) to yield five fractions (WE12B51AeWE12B51E). Compound 25 (60.8 mg) was isolated from WE12B51B by semi-preparative HPLC using an isocratic MeCN-H2O (40:60, v/v) mobile phase. WE12B51D was purified using semi-preparative HPLC eluted with MeCN-H2O (45:55, v/ v) to afford 24 (16.4 mg). WE12B53 was separated on Sephadex LH20 CC eluted with methanol and then purified over silica gel CC eluted with CHCl3-EtOAc (8:1, v/v) to afford 3 (450.5 mg). WE12B54 was further purified over silica gel CC eluted with CHCl3-EtOAc (8:1, v/v) to obtain 26 (380.2 mg). WE12B6 was fractionated using silica gel CC eluted with CHCl3-acetone (15:1e8:1, v/v) to obtain four fractions (WE12B61eWE12B64). WE12B61 was separated by silica gel CC to yield two fractions (WE12B61A and WE12B61B). Compounds 2 (10.2 mg), 28 (14.2 mg), and 29 (380.5 mg) were obtained from WE12B61A by silica gel CC eluted with petroleum ether-EtOAc (2:1, v/v). WE12B61B was purified over silica gel CC and then semipreparative HPLC using an isocratic MeCN-H2O (65:35, v/v) solvent system to afford 1 (110.3 mg). WE12B62 was purified over Sephadex LH-20 gel CC eluted with CHCl3-MeOH (1:1, v/v) to afford 27 (1.6 g). Compound 30 (5.1 mg) was isolated from WE12B63 by silica gel CC eluted with petroleum ether-EtOAc (2:1, v/v) and then semipreparative HPLC using MeCN-H2O (65:35, v/v) as the mobile phase. WE12B64 was subjected to silica gel CC eluted with petroleum ether-EtOAc (2:1, v/v) and then semi-preparative HPLC eluted with MeCN-H2O (70:30, v/v) to afford 31 (40.3 mg) and 32 (8.1 mg). WE13 (40 g) was subjected to silica gel CC eluted with CHCl3acetone (10:1e0:1, v/v) to obtain three fractions (WE13AeWE13C).

X.-Q. Chen et al. / Phytochemistry 149 (2018) 103e115

WE13A was separated on MCI gel CC eluted with MeOH-H2O (50:50e100:0, v/v) to afford three fractions (WE13A1eWE13A3). WE13A2 was further separated using silica gel CC eluted with CHCl3-acetone (10:1e8:1, v/v) to obtain three fractions (WE13A21eWE13A23). WE13A22 was purified over Sephadex LH20 gel CC eluted with MeOH and then semi-preparative HPLC using MeCN-H2O (70:30, v/v) as the mobile phase to yield 6 (14.2 mg) and 37 (6.1 mg). WE13A23 was further purified using semi-preparative HPLC eluted with MeCN-H2O (40:60, v/v) to afford 38 (28.3 mg). WE13B was separated using ODS CC eluted with MeOH-H2O (50:50e80:20, v/v) to afford four fractions (WE13B1eWE12B4). WE13B2 was subjected to silica gel CC eluted with petroleum etherEtOAc (1:2e1:4, v/v) to yield three fractions (WE13B21eWE13B23) all of which were purified over Sephadex LH-20 gel CC followed by semi-preparative HPLC. Consequently, compound 36 (45.2 mg) was isolated from WE13B21, and compounds 33 (190.2 mg), 34 (1.0 g), and 35 (1.2 g) were obtained from WE13B22. WB13B4 was also purified over Sephadex LH-20 gel CC followed by semi-preparative HPLC to afford 4 (80.6 mg). WE13C was fractionated using MCI gel CC eluted with MeOH-H2O (40:60e80:20, v/v) to obtain two fractions (WE13C1 and WE13C2). WE13C1 was separated using ODS CC eluted with MeOH-H2O (50:50e80:20, v/v) to yield five fractions (WE13C11eWE13C15). WE13C11, WE13C12, and WE13C15 were purified over Sephadex LH-20 gel CC and then semi-preparative HPLC. Compounds 39 (210.2 mg) and 7 (20.1 mg) were obtained from WE13C11, and compounds 40 (220.6 mg) and 11 (26.3 mg) were isolated from WE13C12 and WE13C15, respectively. WE13C14 was further separated on ODS CC eluted with MeOH-H2O (50:50, v/ v) and then semi-preparative HPLC using an isocratic MeCN-H2O (28:72, v/v) solvent system to obtain 8 (11.0 mg) 12 (3.2 mg), and 41 (6.0 mg). WE2 (390.0 g) was separated on silica gel CC eluted with CHCl3acetone (6:1e0:1, v/v) to obtain two fractions (WE21 and WE22). WE21 was subjected to ODS CC eluted with MeOH-H2O (40:60e70:20, v/v) to obtain four fractions (WE21AeWE21D). WE21B was separated over silica gel CC eluted with CHCl3-acetone (6:1e0:1, v/v) to obtain four fractions (WE21B1eWE21B4). WE21B1 was purified over Sephadex LH-20 gel CC and semipreparative HPLC to obtain 15 (8.1 mg), 42 (7.0 mg), and 9 (13.1 mg). WE21B2 was chromatographed over ODS CC eluted with MeOH-H2O (50:50, v/v) to obtain three fractions (WE21B21eWE21B23) all of which were purified using Sephadex LH-20 gel CC and semi-preparative HPLC. Compounds 16 (5.1 mg) and 17 (5.0 mg) were obtained from WE21B21. Compounds 18 (120.1 mg) and 43 (117.4 mg) were isolated from WE21B22 and WE21B23, respectively. WE21B3 was separated on ODS CC eluted with MeOH-H2O (50:50, v/v) to afford fractions WE21B31 and WE21B32 which were both subjected to semi-preparative HPLC. Finally, compound 19 (94.1 mg) was isolated from WE21B31, and 44 (22.0 mg) was isolated from WE21B32. WE21B4 was purified using Sephadex LH-20 gel CC and then semi-preparative HPLC to obtain 45 (46.0 mg). WE21C was chromatographed over ODS CC eluted with MeOH-H2O (40:60, v/v) to obtain three fractions (WE21C1eWE21C3). WE21C1 was purified over Sephadex LH-20 gel CC to obtain 13 (5.0 mg). WE21D was separated on silica gel CC eluted with CHCl3-acetone (6:1e0:1, v/v) to obtain fractions WE21D1 and WE21D2. WE21D1 was purified using Sephadex LH20 gel CC and then semi-preparative HPLC to afford 5 (15.3 mg). WE22 (90.0 g) was subjected to MCI CC eluted with MeOH-H2O (50:50e80:20, v/v) to obtain WE22A and WE22B. WE22A was further separated on silica gel CC eluted with CHCl3-acetone (2:1, v/ v) to obtain three fractions (WE22A1eWE22A3). WE22A1 was purified using Sephadex LH-20 gel CC and then semi-preparative HPLC to obtain 46 (35.0 mg), 10 (11.1 mg), 14 (10.0 mg), and 47 (18.3 mg). WE22B was purified using silica gel CC eluted with

113

CHCl3-acetone (2:1, v/v) and then semi-preparative HPLC eluted with MeCN-H2O (30:70, v/v) to obtain 48 (17.1 mg). The spectroscopic data of known compounds (20e48) are shown in the supporting information (Spectroscopic data of known compounds 20e48). The physical properties and spectroscopic data of compounds 1e19 are as follows. 4.3.1. Resinacein A (1) White powder; [a]20 D þ97.0 (c 0.43, MeOH); UV (MeOH) lmax (log 3 ) 216 (4.09), 258 (3.84) nm; IR (KBr) vmax 3420, 2959, 2931, 2869, 1703, 1651, 1587, 1457, 1418, 1377 cm
1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 469.3322 [M
H]
(calcd. for C30H45O4, 469.3318), 939.6718[2M
H]
(calcd. for C60H91O8, 939.6714). 4.3.2. Resinacein B (2) White powder; [a]20 D þ146.7 (c 0.11, MeOH); UV (MeOH) lmax (log 3 ) 201 (3.76), 258 (3.78) nm; IR (KBr) vmax 3422, 2954, 2928, 2867, 1651, 1586, 1459, 1383 cm
1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 455.3509 [M
H]
(calcd. for C30H47O3, 455.3525). 4.3.3. Resinacein C (3) White powder; [a]20 D þ144.0 (c 0.18, MeOH); UV (MeOH) lmax (log 3 ) 205 (3.37), 213 (3.39), 258 (3.14) nm; IR (KBr) vmax 3438, 2934, 2872, 1688, 1654, 1539, 1456, 1413, 1379 cm
1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 485.3272 [M
H]
(calcd. for C30H45O5, 485.3267), 971.6617 [2M
H]
(calcd. for C60H91O10, 971.6612). 4.3.4. Resinacein D (4) White powder; [a]20 D þ44.64 (c 0.22, MeOH); UV (MeOH) lmax (log 3 ) 217 (4.13), 251(3.87) nm; IR (KBr) vmax 3360, 3283, 2955, 2879, 1714, 1669, 1461, 1404, 1380 cm
1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 485.3269 [M
H]
(calcd. for C30H45O5, 485.3267), 971.6616 [2M
H]
(calcd. for C60H91O10, 971.6612). 4.3.5. Resinacein E (5) Pale yellow powder; [a]20 D þ124.9 (c 0.17, MeOH); UV (MeOH) lmax (log 3 ) 215 (4.16), 272(3.81) nm; IR (KBr) vmax 3462, 2962, 2929, 2869, 1672, 1459, 1386 cm
1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 515.2985 [M
H]
(calcd. for C30H43O7, 515.3009). 4.3.6. Resinacein F (6) White powder; [a]20 D þ106.5 (c 0.12, MeOH); UV (MeOH) lmax (log 3 ) 234 (4.02), 271(3.80) nm; IR (KBr) vmax 3424, 2965, 2933, 2866, 1729, 1709, 1678, 1600, 1457,1378, 1341 cm
1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 525.2852 [M
H]
(calcd. for C31H41O7, 525.2852). 4.3.7. Resinacein G (7) White powder; [a]20 D þ83.4 (c 0.34, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.73), 238 (3.97) nm; IR (KBr) vmax 3443, 2974, 2935, 2866, 1713, 1678, 1597, 1459, 1382 cm
1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 527.2643 [M
H]
(calcd. for C30H39O8, 527.2645). 4.3.8. Resinacein H (8) White powder; [a]20 D þ87.8 (c 0.76, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.60), 244 (3.90) nm; IR (KBr) vmax 3443, 2972, 2933, 2874, 1696, 1655, 1460, 1379 cm
1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 530.2855 [M]
(calcd. for C30H42O8, 530.2880), 598.2731 [M þ Na þ HCOOH
H]
(calcd. for C31H43O10Na, 598.2754).

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4.3.9. Resinacein I (9) White powder; [a]20 D þ57.5 (c 1.54, MeOH); UV (MeOH) lmax (log 3 ) 203 (3.69), 254 (3.72) nm; IR (KBr) vmax 3433, 2965, 2929, 2876, 1717, 1659, 1459, 1377, 1281 cm
1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 529.3137 [M
H]
(calcd. for C31H45O7, 529.3165). 4.3.10. Resinacein J (10) White powder; [a]20 D þ54.2 (c 0.35, MeOH); UV (MeOH) lmax (log 3 ) 203 (3.76), 255 (3.67) nm; IR (KBr) vmax 3429, 2968, 2929, 2872, 1705, 1653, 1566, 1459, 1378 cm
1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 531.2949 [M
H]
(calcd. for C30H43O8, 531.2958). 4.3.11. Resinacein K (11) White powder; [a]20 D
68.4 (c 0.19, MeOH); UV (MeOH) lmax (log 3 ) 203 (3.75), 247 (4.03) nm; IR (KBr) vmax 3440, 2953, 2925, 2863, 1748, 1680, 1626, 1459, 1382 cm
1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 511.2692 [M
H]
(calcd. for C30H39O7, 511.2696). 4.3.12. Resinacein L (12) White powder; [a]20 D þ38.5 (c 0.07, MeOH); UV (MeOH) lmax (log 3 ) 201 (3.81), 245 (4.03) nm; IR (KBr) vmax 3435, 2956, 2924, 2851, 1745, 1683, 1608, 1461, 1383 cm
1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 528.2694 [M]
(calcd. for C30H40O8, 528.2723), 596.2560 [M þ Na þ HCOOH
H]
(calcd. for C31H41O10Na, 596.2569). 4.3.13. Resinacein M (13) White powder; [a]20 D þ79.7 (c 0.17, MeOH); UV (MeOH) lmax (log 3 ) 249 (4.24) nm; IR (KBr) vmax 3438, 2962, 2927, 2869, 1729, 1659, 1611, 1458, 1382 cm
1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 529.2788 [M
H]
(calcd. for C30H41O8, 529.2801). 4.3.14. Resinacein N (14) White powder; [a]20 D þ45.6 (c 0.89, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.63) nm, 251 (4.01); IR (KBr) vmax 3427, 2965, 2926, 2866, 1656, 1583, 1459, 1389 cm
1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 531.2947 [M
H]
(calcd. for C30H43O8, 531.2958). 4.3.15. Resinacein O (15) White powder; [a]20 D þ107.5 (c 0.24, MeOH); UV (MeOH) lmax (log 3 ) 201 (3.47), 254 (3.77) nm; IR (KBr) vmax 3441, 2962, 2926, 2872, 1723, 1655, 1461, 1380 cm
1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 545.3113 [M
H]
(calcd. for C31H45O8, 545.3114). 4.3.16. Resinacein P (16) White powder; [a]20 D þ82.9 (c 0.51, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.61), 260(3.72) nm; IR (KBr) vmax 3456, 2956, 2925, 2866, 1742, 1679, 1460, 1379 cm
1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 543.2960 [M
H]
(calcd. for C31H43O8, 543.2958). 4.3.17. Resinacein Q (17) White powder; [a]20 D þ102.4 (c 0.17, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.60), 259(3.74) nm; IR (KBr) vmax 3430, 2951, 2920, 2860, 1738, 1685, 1461, 1433, 1382 cm
1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 543.2963 [M
H]
(calcd. for C31H43O8, 543.2958). 4.3.18. Resinacein R (18) White powder; [a]20 D þ81.6 (c 0.36, MeOH); UV (MeOH) lmax

(log 3 ) 202 (3.40), 254 (3.87) nm; IR (KBr) vmax 3436, 2971, 2932, 2877, 1719, 1653, 1563, 1461, 1380 cm
1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 571.3281 [M þ Na]þ (calcd. for C31H48O8Na, 571.3247). 4.3.19. Resinacein S (19) White powder; [a]20 D þ118.0 (c 0.49, MeOH); UV (MeOH) lmax (log 3 ) 202 (3.55), 254 (3.86) nm; IR (KBr) vmax 3444, 2969, 2929, 2872, 1720, 1650, 1458, 1380 cm
1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 531.2919 [M
H]
(calcd. for C30H43O8, 531.2958). 4.4. Inhibition assay against a-glucosidase The a-glucosidase inhibitory assay was carried out according to a method previously reported by Li et al. (2010). Acknowledgements This research program was financially supported by grants from the National Natural Science Foundation of China (No. 81673389 and No. 81603069), the Science and Technology Development Fund of Macau (040/2016/A), and the Research Fund of University of Macau (MYRG2015-00122). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at https://doi.org/10.1016/j.phytochem.2018.01. 007. These data include MOL files and InChiKeys of the most important compounds described in this article. References Amaral, A.E., Carbonero, E.R., Simao, R.d.C.G., Kadowaki, M.K., Sassaki, G.L., Osaku, C.A., Gorin, P.A.J., Iacomini, M., 2008. An unusual water-soluble betaglucan from the basidiocarp of the fungus Ganoderma resinaceum. Carbohydr. Polym. 72, 473e478. Arisawa, M., Fujita, A., Saga, M., Fukumura, H., Hayashi, T., Shimizu, M., Morita, N., 1986. Three new lanostanoids from Ganoderma lucidum. J. Nat. Prod. 49, 621e625. Arpha, K., Phosri, C., Suwannasai, N., Mongkolthanaruk, W., Sodngam, S., 2012. Astraodoric acids AeD: new lanostane triterpenes from edible mushroom Astraeus odoratus and their Anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity. J. Agric. Food Chem. 60, 9834e9841. Baby, S., Johnson, A.J., Govindan, B., 2015. Secondary metabolites from Ganoderma. Phytochemistry 114, 66e101. Chen, R., Yu, D., 1993. Studies on the triterpenoid constituents of the spores from Ganoderma lucidum Karst. J. Chin. Pharm. Sci. 2, 91e96. Cheng, C.-R., Li, Y.-F., Xu, P.-P., Feng, R.-H., Yang, M., Guan, S.-H., Guo, D.-A., 2012. Preparative isolation of triterpenoids from Ganoderma lucidum by countercurrent chromatography combined with pH-zone-refining. Food Chem. 130, 1010e1016. Cheng, C.-R., Yang, M., Wu, Z.-Y., Wang, Y., Zeng, F., Wu, W.-Y., Guan, S.-H., Guo, D.-A., 2011. Fragmentation pathways of oxygenated tetracyclic triterpenoids and their application in the qualitative analysis of Ganoderma lucidum by multistage tandem mass spectrometry. Rapid Commun. Mass Spectrom. 25, 1323e1335. Fatmawati, S., Shimizu, K., Kondo, R., 2011. Structure-activity relationships of ganoderma acids from Ganoderma lucidum as aldose reductase inhibitors. Bioorg. Med. Chem. Lett. 21, 7295e7297. Fujita, A., Arisawa, M., Saga, M., Hayashi, T., Morita, N., 1986. Two new lanostanoids from Ganoderma lucidum. J. Nat. Prod. 49, 1122e1125. Gao, J.J., Min, B.S., Ahn, E.M., Nakamura, N., Lee, H.K., Hattori, M., 2002. New triterpene aldehydes, lucialdehydes A-C, from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells. Chem. Pharm. Bull. 50, 837e840. Gonzalez, A.G., Leon, F., Rivera, A., Munoz, C.M., Bermejo, J., 1999. Lanostanoid triterpenes from Ganoderma lucidum. J. Nat. Prod. 62, 1700e1701. Guan, S.-H., Yang, M., Wang, X.-M., Xia, J.-M., Zhang, Z.-M., Liu, X., Guo, D.-A., 2007. Spectral assignments and reference data structure elucidation and complete NMR spectral assignments of three new lanostanoid triterpenes with unprecedented D16,17 double bond from Ganoderma lucidum. Magn. Reson. Chem. 45, 789e791. Hu, L.-L., Ma, Q.-Y., Huang, S.-Z., Guo, Z.-K., Ma, H.-X., Guo, J.-C., Dai, H.-F., Zhao, Y.-

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