Article pubs.acs.org/jnp
Ganoboninketals A−C, Antiplasmodial 3,4-seco-27-Norlanostane Triterpenes from Ganoderma boninense Pat. Ke Ma,†,‡,⊥ Jinwei Ren,†,⊥ Junjie Han,† Li Bao,† Li Li,§ Yijian Yao,† Chen Sun,∥ Bing Zhou,∥ and Hongwei Liu*,† †
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China § Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China ∥ State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, 100084, People’s Republic of China S Supporting Information *
ABSTRACT: Three new nortriterpenes, ganoboninketals A−C (1−3), featuring rearranged 3,4-seco-27-norlanostane skeletons and highly complex polycyclic systems were isolated from the medicinal mushroom Ganoderma boninense. The structures of the new metabolites were established by spectroscopic methods. The absolute configurations in 1−3 were assigned by electronic circular dichroism (ECD) calculations. Compounds 1−3 showed antiplasmodial activity against Plasmodium falciparum with IC50 values of 4.0, 7.9, and 1.7 μM, respectively. Compounds 1 and 3 also displayed weak cytotoxicity against A549 cell line with IC50 values of 47.6 and 35.8 μM, respectively. Compound 2 showed weak cytotoxicity toward HeLa cell line with an IC50 value of 65.5 μM. Compounds 1−3 also presented NO inhibitory activity in the LPS-induced macrophages with IC50 values of 98.3, 24.3, and 60.9 μM, respectively.
(one oxymethlene), three methines (one oxymethine), five quaternary carbons including one doubly oxygenated sp3 quaternary carbon (δC 110.4), four olefinic carbons including one terminal olefinic carbon, two carboxylic carbons (δC 174.8 and 170.8), and one carbonyl carbon (δC 211.0). Five isolated spin systems were established by 1H−1H COSY and HSQC correlations as illustrated in Figure 2. In the HMBC spectrum of 1 (Figure 2), the correlations of H-7/C-6, C-8, C-9, and C10, H3-19/C-1, C-5, C-9, and C-10, H3-30/C-8, C-13, C-14, and C-15, and H2-12/C-9, C-11, C-13, C-14, C-17, and C-18 furnished a 6-6-5 ring system. HMBC correlations from H-1 to C-2, C-3, C-5, C-9, C-10, and C-19, with the correlation of H331 to C-3 not only confirmed the presence of methyl propionate moiety but also connected C-1 with C-10. HMBC correlations from H2-28 (H3-29) to C-4 and C-5 located the propenyl group at C-5. Cross-peaks from H-7 to C-32, and from H3-33 to C-32 located the acetoxyl group at C-7. Furthermore, HMBC correlations from H3-21 to C-17, C-20, and C-22, and from H2-25 to C-23, C-24, and C-26 established the chain of C21−C26 (via C-20), meanwhile attached C-20 to C-17. Finally, considering the chemical shift of C-18 (δC 66.4), C-20 (δC 87.7), and C-24 (δC 110.4), the two C-24 bonded
T
he genus of Ganoderma includes more than 200 species distributed throughout the world. For centuries, Ganoderma mushrooms have been widely used as herbal medicines for the treatment of various diseases in China.1 G. lucidum is the most widely used medicinal mushroom and its extracts have been shown to have antiproliferative, immuno-modulatory, anti-inflammatory, and antimalarial activities.1,2 Chemical investigations of Ganoderma species have resulted in the identification of over 200 lanostane triterpenoids.3−5 In our search for new bioactive agents from medicinal mushrooms, the fruiting bodies of G. boninense collected from the Hainan province of China (Qinlan Nature Reserve) were chemically investigated. Fractionation of an EtOAc extract of the fruiting bodies afforded ganoboninketals A−C, three new 3,4-seco-27norlanostane-triterpenes with two types of new chemical skeletons. Details of the structural elucidation, bioactivities, and the plausible biogenesis of ganoboninketals A−C (1−3) are reported herein (Figure 1)..
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RESULTS AND DISCUSSION Ganoboninketal A (1) was isolated as a yellow oil. The molecular formula of 1 was determined as C32H46O7 (10 degrees of unsaturation) on the basis of the molecular ion using HRTOFMS. Analysis of the 1H and 13C NMR data (Table 1) revealed seven methyl groups (one methoxy), ten methylenes © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 31, 2014
A
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corresponding HMBC correlations (Figure S7, SI). The full assignments of the 1H and 13C NMR signals were achieved by detailed interpretations of 2D NMR data including 1H−1H COSY, HSQC, and HMBC. The relative stereochemistry of 2 was assigned unambiguously by selected NOEs of H3-19 with H-28(29), and H-18 (δH 3.76), H3-30 with H-12 (δH 2.85), and H-17, H3-26 with H-12 (δH 3.80), H-18 (δH 3.84) with H-16 (δH 1.87), and H-22 (δH 2.37) (Figure S9, SI). Accordingly, the structure of 2 was determined as shown. The absolute configuration of 2 was deduced as 5S,10S,13S,14R,17S,20S,24R by comparison of the experimental and simulated ECD spectra using the procedures described for 1 (Figure S10, SI). Ganoboninketal C (3) was isolated as colorless oil. The HRTOFMS spectrum indicated a molecular formula of C32H46O8. Analysis of the 1H, 13C,1H−1H COSY, HSQC, and HMBC spectra revealed the same 6-6-5 rings system as that of 1 (Figure 2). As described for 1, the HMBC correlations observed from H2-1, H-7, H3-29, H3-31, and H3-33 confirmed the location of a methyl propionate moiety at C-10, a propenyl group at C-5, and an acetoxyl group at C-7, respectively. The HMBC correlation from H2-18 to C-8, together with the oxygen-bearing charactersitics of C-8 (δC 87.8) and C-18 (δC 73.5) indicated the linkage of C-18 with C-8 via an oxygen atom. Futher HMBC correlations from H3-21 to C-17, C-20, and C-22, and from H2-25 to C-23, C-24, and C-26, together with the 1H−1H COSY correlations of H-22 with H-23, and H25 with H-26, confirmed the fragment of C-21-C-26 via C-20 and the connection of C-20 with C-17. Considering the doubly oxygenated nature of C-24 (δC 110.4), and the chemical shifts for C-17 (δC 92.9) and C-20 (δC 88.3), and the unsaturation requirement of 3, the two C-24 bonded oxygen atoms were linked with C-17 and C-20, respectively, to complete the planar structure of 3. In selective NOE experiments, NOEs of H3-19/ H-18 (δH 3.75), H-28 (δH 4.87), and H3-29, H-18 (δH 3.75)/ H2-21, H-30/H-7, and H-9, H-12 (δH 2.70)/H-22 (δH 1.69) and H2-23 indicated their spatial proximity (Figure 2). As described for 1 and 2, the absolute configuration of 3 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by timedependent density functional theory at the B3LYP/6-31+G(d, p) lever (Figure 4). The absolute configuration of 3 was assigned as 5S,7S,8S,9R,10S,13S,14R,17R,20S,24S. In order to verify compounds that 1−3 are natural, the deposited fruiting bodies were extracted with acetonitrile, and the extract was subjected to RP-HPLC analysis. Ganoboninketals A−C were all detected on the HPLC chromatogram of the acetonitrile extract (Figure S17, SI). Since ganoderic aldehyde TR from G. lucidum and ganoderic acids T and P from G. orbiforme were reported to possess good antimalarial activity,10,11 ganoboninketals A−C (1−3) were evaluated against the 3D7 strain of P. falciparum in vitro using the method described by Corbett.12,13 Compounds 1−3 showed antiplasmodial activity with the IC50 values of 4.0, 7.9, and 1.7 μM, respectively. We also tested cytotoxicity and NO inhibitory activity of ganoboninketals A−C (Table 2). Compounds 1 and 3 displayed weak activity against the A549 cell line with IC50’s of 47.6 and 35.8 μM, respectively, and compound 2 showed weak activity against HeLa cell line with IC50 of 65.5 μM. In the NO inhibition assay, ganoboninketal B displayed more potent NO inhibitory activity with an IC50 value of 24.3 μM in the LPS-induced macrophages than ganoboninketals A (IC50, 98.3 μM) and C (IC50, 60.9 μM).
Figure 1. Structures of ganoboninketals A (1), B (2), and C (3).
oxygen atoms were attached to C-18 and C-20, respectively, to provide the complete structure and satisfy the unsaturation requirement. The relative configuration of 1 was established by selected NOE experiments (Figure 2 and Figure 3). NOEs from H3-19 to H-18 (δH 3.93), H-28 (δH 4.82), and H3-29 indicated these protons were on the same face of the 6-6-5 rings system, whereas NOEs of H-7 with H-5 and H3-30, and H3-30 with H-17 and H-12 (δH 2.84) placed them on the opposite side of the rings. In addition, NOEs of H-12 (δH 3.79) with H18 (δH 3.93), and H3-26, and those of H-18 (δH 4.07) with H22 (δH 2.43), H2-23, and H-15 (δH 2.29) revealed their spatial proximity. Consequently, the relative stereochemistry of 1 was established as 5S*,7S*,10S*,13S*,14R*,17S*,20S*,24R*. The absolute configuration of 1 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) curves generated by time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d) lever.6−9 Considering the relative configuration determined above, one of the two isomers, (5S,7S,10S,13S,14R,17S,20S,24R)-1 or (5R,7R,10R,13R,14S,17R,20R,24S)-1 should represent the actual absolute configuration of 1. Considering the complicated conformation of the flexible fragments at C-10 and C-24 and their insignificant effect on the CD spectrum, compound 1 was simplified as structure 4 for CD calculation. A systematic conformational analysis was performed for 4a and 4b by the molecular operating environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at B3LYP/6-31G(d) basis set level afforded six lowest-energy conformers for enantiomers 4a and 4b, respectively. The calculated ECD spectra were generated by Boltzmann-weighting of the conformers. Comparison of the experimental and calculated ECD curves of 4a and 4b (Figure 4) allowed the unambiguous assignment of the absolute configuration of 1 as 5S,7S,10S,13S,14R,17S,20S,24R. The molecular formula of ganoboninketal B (2) was established as C30H42O6, determined by HRTOFMS. The 1H and 13C NMR spectra of 2 are very similar to those of 1, except for the absence of the acetyl group and the presence of an extra carbonyl carbon that was assigned to C-7 (δC 201.4) by B
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Table 1. NMR Spectroscopic Data for 1−3 in Pyridine-d5 1 No.
δCa
1
32.5
2
30.5
3 4 5 6
44.6 30.9
7 8 9 10 11 12
70.7 159.8 141.2 41.5 200.0 48.5
13 14 15
54.5 51.4 33.5
16
26.6
17 18
55.3 66.4
19 20 21 22
22.6 87.7 33.0 35.7
23 24 25 26 28
35.4 112.1 32.6 9.6 116.7
29 30 31 32 33
23.4 29.6 51.9 170.7 21.7
2
δH (mult., J in Hz)b 3.08 m 2.09 m 2.46c 2.07 m
2.46c 2.20 m 1.95 m 5.95 dd (9.3, 7.1)
3.79 d (16.7) 2.84 d (16.7)
2.29 m 1.46 m 1.84 m 1.56 m 2.41d 4.07 d (13.2) 3.93 d (13.2) 1.31 s 1.32 2.43 1.62 1.94 1.94
s md m m m
1.77 1.02 4.99 4.82 1.71 1.35 3.59
q (7.5) t (7.5) s s s s s
δC
3
δH (mult., J in Hz)
32.3
2.97 2.27 2.50 2.20
30.4 174.4 145.7 47.2 41.4
m m me m
2.86f 2.86f 2.50e
201.4 153.4 148.5 42.4 202.7 48.4
3.80 d (15.7) 2.85 d (15.7)
54.2 48.4 34.0
2.50e 1.85 m 1.87 m 1.57 m 2.41g 3.84 d (13.2) 3.76 d (13.2) 1.27 s
26.2 54.8 66.4 21.4 87.6 33.0 35.7
35.4 112.1 32.4 9.5 117.6 23.4 28.3 51.9
2.15 s
a
1.31 2.39 1.61 1.97 1.97
s mg m m m
1.77 1.01 5.02 4.85 1.71 1.35 3.61
q (7.4) t (7.4) s s s s s
δC
δH (mult., J in Hz)
35.1
2.87 m 1.99 m 2.63 m 2.57 m 174.8 146.1 2.47h 2.47h 1.81 m 5.35 dd (11.0, 4.6)
29.7 174.2 144.9 47.8 31.8 71.0 87.8 56.1 42.0 211.0 48.8
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3.27 dd (18.6, 3.0) 2.70 d (18.6)
56.4 57.9 32.9
2.37 1.41 2.57 1.99
39.5 92.9 73.5
m m m m
4.10 dd (8.6, 3.0) 3.75 d (8.6) 1.31 s
20.6 88.3 19.6 34.0 36.7 36.7 110.4 26.8 8.9 115.8 23.9 16.9 51.9 170.8 22.0
Recorded at 500 MHz. bRecorded at 125 MHz. c These signals are overlapped. dThese signals are overlapped. These signals are overlapped. g These signals are overlapped. h These signals are overlapped.
27-Norlanostane triterpenes are rare in nature. Examples included eight 27-norlanostane saponins from Muscari paradoxam, 14 and ten norlanostane saponins from Scilla scilloides.15−17 Ganoboninketals A (1) and B (2) share a new skeleton featuring a 2,9-dioxabicyclo(4.2.1)nonane moiety fused with a 6-6-5 ring system. Ganoboninketal C (3) was characterized by a new skeleton of decahydro-2′,7′-dioxaspiro(9a,3a-(epoxymethano)cyclopenta(a)naphthalene-3,3′bicyclo(2.2.1)heptane. Compounds 1−3 are new members of 27-norlanostane triterpenes. The possible biosynthetic pathway for 1−3 is proposed in the SI (Figure S18). In conclusion, we presented three lanostane-type nortriterpenes possessing new chemical skeletons and antiplasmodial activity from the mushroom G. boninense.
2.43 s
1.47 1.69 1.49 1.63 1.63
s m m m m
1.86 1.04 4.99 4.87 1.76 1.23 3.64
q (7.5) t (7.4) s s s s s
2.15 s e
These signals are overlapped.
f
EXPERIMENTAL SECTION
General Experimental Procedures. Solvents used for extraction and chromatographic separation were analytical grade. TLC was carried out on silica gel HSGF254 and the compounds were visualized by spraying with 10% H2SO4 and heating. Silica gel (Qingdao Haiyang Chemical Co., Ltd., People’s Republic of China and ODS (Merck) were used for column chromatography. HPLC separation was performed on an Agilent 1200 HPLC system using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm; detector: UV) with a flow rate of 2.5 mL/min. The optical rotations were measured on a PerkinElmer 241 polarimeter and UV spectra were determined on a ThermoGenesys-10S UV−vis spectrophotometer. IR data were measured using a Nicolet IS5FT-IR spectrophotometer. CD spectra were recorded on a JASCO J-815 Spectropolarimeter. 1H and 13C NMR data were acquired with a Bruker Avance-500 spectrometer. The HMQC and C
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of MeOH−H2O (20%−100%) yielded four fractions (A-D). Fraction C was further separated by chromatography over silica gel (3 × 35 cm) with a stepwise gradient elution of CH2Cl2−MeOH (100:1 to 0:1) to afford six subfractions C1−C6. Subfraction C2 was purified by reverse phased HPLC (RP-HPLC) using 68% acetonitrile in water to afford compound 2 (12.0 mg, tR 34.5 min) and 3 (110.0 mg,tR 30 min). Compound 1 (13.0 mg, tR 18 min) was obtained by RP-HPLC using 65% acetonitrile in water from subfraction C3. Ganoboninketal A (1). Yellow oil (methanol); [α]25D −52.0 (c 1.5, methanol); CD (c 1.5 × 10−3 M, methanol) λmax (Δε): 225 (−4.09), 258 (+8.34), 349 (−1.75); UV (methanol) λmax nm (log ε): 260 (4.42); IR (neat) νmax: 2968, 1735, 1663, 1233, 1198, 1028, 895 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HR-ESI-MS: m/ z [M + H]+ 543.3319 (calcd for C32H47O7 543.3316). Ganoboninketal B (2). Yellow oil (methanol); [α]25D 43.5 (c 1.2, methanol); CD (c 1.2 × 10−3 M, methanol) λmax (Δε): 227 (+8.02), 264 (+3.31), 414 (−1.02); UV (methanol) λmax nm (log ε): 272 (4.20); IR (neat) νmax: 2969, 1735, 1695, 1286, 1195, 1072, 895 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HR-ESI-MS: m/ z [M + H]+ 499.3059 (calcd for C30H43O6 499.3054). Ganoboninketal C (3). Colorless oil (methanol); [α]25D 11.4 (c 1.5, methanol); CD (c 1.5 × 10−3 M, methanol) λmax (Δε): 223 (−0.39), 304 (0.81); UV (methanol) λmax nm (log ε): 200 (4.69); IR (neat) νmax: 2974, 2948, 2882, 1734, 1701, 1220, 1196, 1038, 986, 905 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HRESIMS: m/z [M + H]+ 559.3272 (calcd for C32H47O8, 559.3265). Computational Details. Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software package was used for systematic conformational analyses for compounds 1−3 with the MMFF94 molecular mechanics force field. Subsequently, The MMFF94 conformational analyses were optimized using DFT at B3LYP/6-31G(d) basis set level using Gaussian03 package. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 30 lowest electronic transitions were calculated using the TDDFT methodology at the B3LYP/6-
Figure 2. Selected key HMBC and NOESY correlations of 1 and 3. The molecular models of 1 and 3 in minimal energy were obtained by Conflex calculations in MMFF94s force field. HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESI-MS data were acquired using an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument. Fungal Material. Fruiting bodies of Ganoderma boninense were collected from Hainan province (China) in 2010, and identified by Prof. Yijian Yao (one of coauthors) on the basis of morphological characteristics. Extraction and Isolation. Sixty g of dry fruiting body of G. boninense were extracted with ethyl acetate (3 × 1 L), and 8 g crude extract was obtained by evaporating solvent under vacuum. Separation by ODS column chromatography (column 5 × 50 cm) with a gradient
Figure 3. Selected excitation NOE of ganoboninketal A (1) in pyridine (500 MHz). D
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Figure 4. Experimental CD spectra of 1 and 3 in MeOH and the calculated ECD spectra of 4a, 4b, 6a, and 6b. Structures 4a, 4b, 6a, and 6b represent possible stereoisomers for 1 and 3, respectively. 39.9 min), and 3 (tR 38.8 min) were all detected on the HPLC chromatogram of the crude extract. NO Inhibition and Cytotoxicity Assays. Mouse monocyte/ macrophages RAW 264.7 were maintained in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 mg/ mL), and 10% heat-inactivated fetal bovine serum at 37 °C with 5% CO2 and 95% air; 200 μL of cells (5 × 105 cells/mL) were added in a 96-well plate. After 1 h incubation, cells were treated with 1 mg/mL of LPS and various concentrations of test compounds (DMSO as solvent) for 24 h. The NO level in the supernatant of RAW 264.7 cells was determined by the Griess reaction.18 Cytotoxic activity against A549 and HeLa cell lines was assayed with the MTT assay.19 Cells were incubated with tested compounds (DMSO as solvent) at 37 °C in a humidified atmosphere of 5% CO2 95% air for 72 h. Into each well was added 50 μL of MTT/medium solution (0.5 mg/mL), and cells were incubated for another 4 h. After removing the MTT/medium, 100 μL of DMSO was added to each well. The plate was shaken to dissolve the precipitate, and activity was measured at 540 nm using a microplate reader. The inhibition rates were calculated and plotted versus test concentrations to afford the IC50 (±SD) for three independent experiments, each carried out in triplicate. Hydrocortisone (IC50, 53.7 ± 3.9 μM) was used as a positive control for the NO inhibition assay. Cisplatin was used as the reference substance that showed cytotoxicity against A549 and HeLa cell lines with IC50 values of 19.2 ± 1.1 and 15.9 ± 1.5 μM, respectively. Antimalarial Assay..12,13 The 3D7 strain of Plasmodium falciparum was maintained in RPMI 1640 medium (Gibco-BRL Laboratories) supplemented with 2.5 g/L NaHCO3, 0.3 g/L Lglutamine, 3.96 g/L HEPES, 25 mg/L penicillin, 4 g/L Albumax II, 0.07 g/L streptomycin, and 0.02 g/L hypoxanthine. Parasites were
Table 2. Antiplasmoidial, NO Inhibitory and Cytotoxic Activities of 1−3 (IC50, μM) antiplasmodial effect
antiinflammation
cmpds
P. falciparum
NO inhibitiona
A549
HeLa
1 2 3 positive control
4.0 ± 0.7 7.9 ± 0.9 1.7 ± 0.5 artemisinin 30.4 ± 0.6 nM
98.3 ± 8.3 24.3 ± 1.9 60.9 ± 5.5 hydrocortisone 53.7 ± 3.9
47.6 ± 3.6 >100 35.8 ± 2.8 cisplatin 19.2 ± 1.1
>100 65.5 ± 3.7 >100 cisplatin 15.9 ± 1.5
cytotoxicity
a
The growth of macrophage cells were not influenced by compounds 1−3 at the concentration of 200 μM.
31G(d) or B3LYP/6-31+G(d, p) lever. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.34 eV. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correlation. HPLC Analysis of the Extract of G. boninense. The deposited fruiting bodies were extracted with acetonitrile (3 × 100 mL). The acetonitrile extract was dissolved in acetonitrile (10 mg/mL) and subjected to RP-HPLC analysis. The detection wavelength was set to 210 nm. The mobile phase consisted of MeCN (A) and H2O (B) using a gradient elution of 15−40% A at 0−10 min, 40−60% A at 10− 25 min, 60−100% A at 25−45 min, and 100% A at 45−55 min. The flow rate was kept at 1 mL/min. Compounds 1 (tR 39.4 min), 2 (tR E
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maintained in human red blood cells (hRBC) at a hematocrit of 5% at 37 °C. Cultures were under an atmosphere of 5% CO2, 5% O2 and 90% N2. The parasites were diluted to a final hematocrit of 2% and a parasitemia of 1% by using medium and hRBC. The dilution of parasitemia and compounds (DMSO as solvent) were added to a 96-well plate and cultured in a 37 °C incubator for approximately 48 h. Fifty μL of fluorochrome mixture consisting of PicoGreen (Molecular Probes, Inc.), 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE buffer), and 2% Triton X-100 diluted with double distilled water, was then added to release and label the parasitic DNA. The plates were then incubated for 30 min in the dark. The fluorescence signal, measured as relative fluorescence units (RFU), was determined with a fluorescence microplate reader (Fluoroskan Ascent) with excitation at 485 nm and emission at 538 nm. Simultaneously, the RFU from positive and negative control samples were obtained, stored, and analyzed. IC50 (±SD) values were calculated using linear interpolation of inhibition curves for three independent experiments, each carried out in triplicate. Artemisinin was used as positive control with IC50 of 30.4 ± 0.6 nM.
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(15) Ono, M.; Ochiai, T.; Yasuda, S.; Nishida, Y.; Tanaka, T.; Okawa, M.; Kinjo, J.; Yoshimitsu, H.; Nohara, T. Chem. Pharm. Bull. 2013, 61, 592−598. (16) Ono, M.; Takatsu, Y.; Ochiai, T.; Yasuda, S.; Nishida, Y.; Tanaka, T.; Okawa, M.; Kinjo, J.; Yoshimitsu, H.; Nohara, T. Chem. Pharm. Bull. 2012, 60, 1314−1319. (17) Ono, M.; Toyohisa, D.; Morishita, T.; Horita, H.; Yasuda, S.; Nishida, Y.; Tanaka, T.; Okawa, M.; Kinjo, J.; Yoshimitsu, H.; Nohara, T. Chem. Pharm. Bull. 2011, 59, 1348−1354. (18) Zhang, F.; Liu, S.; Lu, X.; Guo, L.; Zhang, H.; Che, Y. S. J. Nat. Prod. 2009, 72, 1782−1785. (19) Qiu, L.; Zhao, F.; Liu, H. W.; Chen, L. X.; Jiang, J. H.; Liu, H. X.; Wang, N. L.; Yao, X. S.; Qiu, F. Chem. Biodivers. 2008, 5, 758−763.
ASSOCIATED CONTENT
* Supporting Information S
NMR data of 1-3, CD data and calculations for 2. This material is available free of charge via the Internet at http://pubs.acs.org
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 10 62566577. E-mail: [email protected] (H.-W.L.). Author Contributions ⊥
K.M. and J.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the National Program on Key Basic Research Project (973 program 2014CB138304), the National Natural Science Foundation of China (21072219).
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REFERENCES
(1) Paterson, R. R. M. Phytochemistry 2006, 67, 1985−2001. (2) Oluba, O. M.; Olusola, A. O.; Fagbohunka, B. S.; Onyeneke, E. C. Int. J. Med. Mushrooms 2012, 14, 459−466. (3) Cole, R. J.; Schweikert, M. A. Handbook of Secondary Fungal Metabolites; Academic Press: New York, 2003; Vol. 2, pp 271−430. (4) Yan, Y. M.; Ai, J.; Zhou, L. L.; Chung, A. C. K.; Li, R.; Nie, J.; Fang, P.; Wang, X.-L.; Luo, J.; Hu, Q.; Hou, F. F.; Cheng, Y. X. Org. Lett. 2013, 15, 5488−5491. (5) Wang, C. F.; Liu, J. Q.; Yan, Y. X.; Chen, J. C.; Lu, Y.; Guo, Y. H.; Qiu, M. H. Org. Lett. 2010, 12, 1656−1659. (6) Diedrich, C.; Grimme, S. J. Phys. Chem. A 2003, 107, 2524−2539. (7) Crawford, T. D.; Tam, M. C.; Abrams, M. L. J. Phys. Chem. A 2007, 111, 12057−12068. (8) Stephens, P. J.; Devlin, F. J.; Gasparrini, F.; Ciogli, A.; Spinelli, D.; Cosimelli, B. J. Org. Chem. 2007, 72, 4707−4715. (9) Bringmann, G.; Bruhn, T.; Maksimenka, K.; Hemberger, Y. Eur. J. Org. Chem. 2009, 17, 2717−2727. (10) Isaka, M.; Chinthanom, P.; Kongthong, S.; Srichomthong, K.; Choeyklin, R. Phytochemistry 2013, 87, 133−139. (11) Adams, M.; Christen, M.; Plitzko, I.; Zimmermann, S.; Brun, R.; Kaiser, M.; Hamburger, M. J. Nat. Prod. 2010, 73, 897−900. (12) Corbett, Y.; Herrera, L.; Gonzalez, J.; Cubilla, L.; Capson, T. L.; Coley, P. D.; Kursar, T. A.; Romero, L. I.; Ortega-Barria, E. Am. J. Trop. Med. Hyg. 2004, 70, 119−124. (13) Trager, W.; Jensen, J. B. Science 1976, 193, 673−675. (14) Kuroda, M.; Mimaki, Y.; Ori, K.; Sakagami, H.; Sashida, Y. J. Nat. Prod. 2004, 67, 2099−2103. F
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Ganoboninketals A−C, Antiplasmodial 3,4-seco-27-Norlanostane Triterpenes from Ganoderma boninense Pat. Ke Ma,†,‡,⊥ Jinwei Ren,†,⊥ Junjie Han,† Li Bao,† Li Li,§ Yijian Yao,† Chen Sun,∥ Bing Zhou,∥ and Hongwei Liu*,† †
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China § Department of Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China ∥ State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, 100084, People’s Republic of China S Supporting Information *
ABSTRACT: Three new nortriterpenes, ganoboninketals A−C (1−3), featuring rearranged 3,4-seco-27-norlanostane skeletons and highly complex polycyclic systems were isolated from the medicinal mushroom Ganoderma boninense. The structures of the new metabolites were established by spectroscopic methods. The absolute configurations in 1−3 were assigned by electronic circular dichroism (ECD) calculations. Compounds 1−3 showed antiplasmodial activity against Plasmodium falciparum with IC50 values of 4.0, 7.9, and 1.7 μM, respectively. Compounds 1 and 3 also displayed weak cytotoxicity against A549 cell line with IC50 values of 47.6 and 35.8 μM, respectively. Compound 2 showed weak cytotoxicity toward HeLa cell line with an IC50 value of 65.5 μM. Compounds 1−3 also presented NO inhibitory activity in the LPS-induced macrophages with IC50 values of 98.3, 24.3, and 60.9 μM, respectively.
(one oxymethlene), three methines (one oxymethine), five quaternary carbons including one doubly oxygenated sp3 quaternary carbon (δC 110.4), four olefinic carbons including one terminal olefinic carbon, two carboxylic carbons (δC 174.8 and 170.8), and one carbonyl carbon (δC 211.0). Five isolated spin systems were established by 1H−1H COSY and HSQC correlations as illustrated in Figure 2. In the HMBC spectrum of 1 (Figure 2), the correlations of H-7/C-6, C-8, C-9, and C10, H3-19/C-1, C-5, C-9, and C-10, H3-30/C-8, C-13, C-14, and C-15, and H2-12/C-9, C-11, C-13, C-14, C-17, and C-18 furnished a 6-6-5 ring system. HMBC correlations from H-1 to C-2, C-3, C-5, C-9, C-10, and C-19, with the correlation of H331 to C-3 not only confirmed the presence of methyl propionate moiety but also connected C-1 with C-10. HMBC correlations from H2-28 (H3-29) to C-4 and C-5 located the propenyl group at C-5. Cross-peaks from H-7 to C-32, and from H3-33 to C-32 located the acetoxyl group at C-7. Furthermore, HMBC correlations from H3-21 to C-17, C-20, and C-22, and from H2-25 to C-23, C-24, and C-26 established the chain of C21−C26 (via C-20), meanwhile attached C-20 to C-17. Finally, considering the chemical shift of C-18 (δC 66.4), C-20 (δC 87.7), and C-24 (δC 110.4), the two C-24 bonded
T
he genus of Ganoderma includes more than 200 species distributed throughout the world. For centuries, Ganoderma mushrooms have been widely used as herbal medicines for the treatment of various diseases in China.1 G. lucidum is the most widely used medicinal mushroom and its extracts have been shown to have antiproliferative, immuno-modulatory, anti-inflammatory, and antimalarial activities.1,2 Chemical investigations of Ganoderma species have resulted in the identification of over 200 lanostane triterpenoids.3−5 In our search for new bioactive agents from medicinal mushrooms, the fruiting bodies of G. boninense collected from the Hainan province of China (Qinlan Nature Reserve) were chemically investigated. Fractionation of an EtOAc extract of the fruiting bodies afforded ganoboninketals A−C, three new 3,4-seco-27norlanostane-triterpenes with two types of new chemical skeletons. Details of the structural elucidation, bioactivities, and the plausible biogenesis of ganoboninketals A−C (1−3) are reported herein (Figure 1)..
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RESULTS AND DISCUSSION Ganoboninketal A (1) was isolated as a yellow oil. The molecular formula of 1 was determined as C32H46O7 (10 degrees of unsaturation) on the basis of the molecular ion using HRTOFMS. Analysis of the 1H and 13C NMR data (Table 1) revealed seven methyl groups (one methoxy), ten methylenes © XXXX American Chemical Society and American Society of Pharmacognosy
Received: March 31, 2014
A
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corresponding HMBC correlations (Figure S7, SI). The full assignments of the 1H and 13C NMR signals were achieved by detailed interpretations of 2D NMR data including 1H−1H COSY, HSQC, and HMBC. The relative stereochemistry of 2 was assigned unambiguously by selected NOEs of H3-19 with H-28(29), and H-18 (δH 3.76), H3-30 with H-12 (δH 2.85), and H-17, H3-26 with H-12 (δH 3.80), H-18 (δH 3.84) with H-16 (δH 1.87), and H-22 (δH 2.37) (Figure S9, SI). Accordingly, the structure of 2 was determined as shown. The absolute configuration of 2 was deduced as 5S,10S,13S,14R,17S,20S,24R by comparison of the experimental and simulated ECD spectra using the procedures described for 1 (Figure S10, SI). Ganoboninketal C (3) was isolated as colorless oil. The HRTOFMS spectrum indicated a molecular formula of C32H46O8. Analysis of the 1H, 13C,1H−1H COSY, HSQC, and HMBC spectra revealed the same 6-6-5 rings system as that of 1 (Figure 2). As described for 1, the HMBC correlations observed from H2-1, H-7, H3-29, H3-31, and H3-33 confirmed the location of a methyl propionate moiety at C-10, a propenyl group at C-5, and an acetoxyl group at C-7, respectively. The HMBC correlation from H2-18 to C-8, together with the oxygen-bearing charactersitics of C-8 (δC 87.8) and C-18 (δC 73.5) indicated the linkage of C-18 with C-8 via an oxygen atom. Futher HMBC correlations from H3-21 to C-17, C-20, and C-22, and from H2-25 to C-23, C-24, and C-26, together with the 1H−1H COSY correlations of H-22 with H-23, and H25 with H-26, confirmed the fragment of C-21-C-26 via C-20 and the connection of C-20 with C-17. Considering the doubly oxygenated nature of C-24 (δC 110.4), and the chemical shifts for C-17 (δC 92.9) and C-20 (δC 88.3), and the unsaturation requirement of 3, the two C-24 bonded oxygen atoms were linked with C-17 and C-20, respectively, to complete the planar structure of 3. In selective NOE experiments, NOEs of H3-19/ H-18 (δH 3.75), H-28 (δH 4.87), and H3-29, H-18 (δH 3.75)/ H2-21, H-30/H-7, and H-9, H-12 (δH 2.70)/H-22 (δH 1.69) and H2-23 indicated their spatial proximity (Figure 2). As described for 1 and 2, the absolute configuration of 3 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by timedependent density functional theory at the B3LYP/6-31+G(d, p) lever (Figure 4). The absolute configuration of 3 was assigned as 5S,7S,8S,9R,10S,13S,14R,17R,20S,24S. In order to verify compounds that 1−3 are natural, the deposited fruiting bodies were extracted with acetonitrile, and the extract was subjected to RP-HPLC analysis. Ganoboninketals A−C were all detected on the HPLC chromatogram of the acetonitrile extract (Figure S17, SI). Since ganoderic aldehyde TR from G. lucidum and ganoderic acids T and P from G. orbiforme were reported to possess good antimalarial activity,10,11 ganoboninketals A−C (1−3) were evaluated against the 3D7 strain of P. falciparum in vitro using the method described by Corbett.12,13 Compounds 1−3 showed antiplasmodial activity with the IC50 values of 4.0, 7.9, and 1.7 μM, respectively. We also tested cytotoxicity and NO inhibitory activity of ganoboninketals A−C (Table 2). Compounds 1 and 3 displayed weak activity against the A549 cell line with IC50’s of 47.6 and 35.8 μM, respectively, and compound 2 showed weak activity against HeLa cell line with IC50 of 65.5 μM. In the NO inhibition assay, ganoboninketal B displayed more potent NO inhibitory activity with an IC50 value of 24.3 μM in the LPS-induced macrophages than ganoboninketals A (IC50, 98.3 μM) and C (IC50, 60.9 μM).
Figure 1. Structures of ganoboninketals A (1), B (2), and C (3).
oxygen atoms were attached to C-18 and C-20, respectively, to provide the complete structure and satisfy the unsaturation requirement. The relative configuration of 1 was established by selected NOE experiments (Figure 2 and Figure 3). NOEs from H3-19 to H-18 (δH 3.93), H-28 (δH 4.82), and H3-29 indicated these protons were on the same face of the 6-6-5 rings system, whereas NOEs of H-7 with H-5 and H3-30, and H3-30 with H-17 and H-12 (δH 2.84) placed them on the opposite side of the rings. In addition, NOEs of H-12 (δH 3.79) with H18 (δH 3.93), and H3-26, and those of H-18 (δH 4.07) with H22 (δH 2.43), H2-23, and H-15 (δH 2.29) revealed their spatial proximity. Consequently, the relative stereochemistry of 1 was established as 5S*,7S*,10S*,13S*,14R*,17S*,20S*,24R*. The absolute configuration of 1 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) curves generated by time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d) lever.6−9 Considering the relative configuration determined above, one of the two isomers, (5S,7S,10S,13S,14R,17S,20S,24R)-1 or (5R,7R,10R,13R,14S,17R,20R,24S)-1 should represent the actual absolute configuration of 1. Considering the complicated conformation of the flexible fragments at C-10 and C-24 and their insignificant effect on the CD spectrum, compound 1 was simplified as structure 4 for CD calculation. A systematic conformational analysis was performed for 4a and 4b by the molecular operating environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at B3LYP/6-31G(d) basis set level afforded six lowest-energy conformers for enantiomers 4a and 4b, respectively. The calculated ECD spectra were generated by Boltzmann-weighting of the conformers. Comparison of the experimental and calculated ECD curves of 4a and 4b (Figure 4) allowed the unambiguous assignment of the absolute configuration of 1 as 5S,7S,10S,13S,14R,17S,20S,24R. The molecular formula of ganoboninketal B (2) was established as C30H42O6, determined by HRTOFMS. The 1H and 13C NMR spectra of 2 are very similar to those of 1, except for the absence of the acetyl group and the presence of an extra carbonyl carbon that was assigned to C-7 (δC 201.4) by B
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Table 1. NMR Spectroscopic Data for 1−3 in Pyridine-d5 1 No.
δCa
1
32.5
2
30.5
3 4 5 6
44.6 30.9
7 8 9 10 11 12
70.7 159.8 141.2 41.5 200.0 48.5
13 14 15
54.5 51.4 33.5
16
26.6
17 18
55.3 66.4
19 20 21 22
22.6 87.7 33.0 35.7
23 24 25 26 28
35.4 112.1 32.6 9.6 116.7
29 30 31 32 33
23.4 29.6 51.9 170.7 21.7
2
δH (mult., J in Hz)b 3.08 m 2.09 m 2.46c 2.07 m
2.46c 2.20 m 1.95 m 5.95 dd (9.3, 7.1)
3.79 d (16.7) 2.84 d (16.7)
2.29 m 1.46 m 1.84 m 1.56 m 2.41d 4.07 d (13.2) 3.93 d (13.2) 1.31 s 1.32 2.43 1.62 1.94 1.94
s md m m m
1.77 1.02 4.99 4.82 1.71 1.35 3.59
q (7.5) t (7.5) s s s s s
δC
3
δH (mult., J in Hz)
32.3
2.97 2.27 2.50 2.20
30.4 174.4 145.7 47.2 41.4
m m me m
2.86f 2.86f 2.50e
201.4 153.4 148.5 42.4 202.7 48.4
3.80 d (15.7) 2.85 d (15.7)
54.2 48.4 34.0
2.50e 1.85 m 1.87 m 1.57 m 2.41g 3.84 d (13.2) 3.76 d (13.2) 1.27 s
26.2 54.8 66.4 21.4 87.6 33.0 35.7
35.4 112.1 32.4 9.5 117.6 23.4 28.3 51.9
2.15 s
a
1.31 2.39 1.61 1.97 1.97
s mg m m m
1.77 1.01 5.02 4.85 1.71 1.35 3.61
q (7.4) t (7.4) s s s s s
δC
δH (mult., J in Hz)
35.1
2.87 m 1.99 m 2.63 m 2.57 m 174.8 146.1 2.47h 2.47h 1.81 m 5.35 dd (11.0, 4.6)
29.7 174.2 144.9 47.8 31.8 71.0 87.8 56.1 42.0 211.0 48.8
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3.27 dd (18.6, 3.0) 2.70 d (18.6)
56.4 57.9 32.9
2.37 1.41 2.57 1.99
39.5 92.9 73.5
m m m m
4.10 dd (8.6, 3.0) 3.75 d (8.6) 1.31 s
20.6 88.3 19.6 34.0 36.7 36.7 110.4 26.8 8.9 115.8 23.9 16.9 51.9 170.8 22.0
Recorded at 500 MHz. bRecorded at 125 MHz. c These signals are overlapped. dThese signals are overlapped. These signals are overlapped. g These signals are overlapped. h These signals are overlapped.
27-Norlanostane triterpenes are rare in nature. Examples included eight 27-norlanostane saponins from Muscari paradoxam, 14 and ten norlanostane saponins from Scilla scilloides.15−17 Ganoboninketals A (1) and B (2) share a new skeleton featuring a 2,9-dioxabicyclo(4.2.1)nonane moiety fused with a 6-6-5 ring system. Ganoboninketal C (3) was characterized by a new skeleton of decahydro-2′,7′-dioxaspiro(9a,3a-(epoxymethano)cyclopenta(a)naphthalene-3,3′bicyclo(2.2.1)heptane. Compounds 1−3 are new members of 27-norlanostane triterpenes. The possible biosynthetic pathway for 1−3 is proposed in the SI (Figure S18). In conclusion, we presented three lanostane-type nortriterpenes possessing new chemical skeletons and antiplasmodial activity from the mushroom G. boninense.
2.43 s
1.47 1.69 1.49 1.63 1.63
s m m m m
1.86 1.04 4.99 4.87 1.76 1.23 3.64
q (7.5) t (7.4) s s s s s
2.15 s e
These signals are overlapped.
f
EXPERIMENTAL SECTION
General Experimental Procedures. Solvents used for extraction and chromatographic separation were analytical grade. TLC was carried out on silica gel HSGF254 and the compounds were visualized by spraying with 10% H2SO4 and heating. Silica gel (Qingdao Haiyang Chemical Co., Ltd., People’s Republic of China and ODS (Merck) were used for column chromatography. HPLC separation was performed on an Agilent 1200 HPLC system using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm; detector: UV) with a flow rate of 2.5 mL/min. The optical rotations were measured on a PerkinElmer 241 polarimeter and UV spectra were determined on a ThermoGenesys-10S UV−vis spectrophotometer. IR data were measured using a Nicolet IS5FT-IR spectrophotometer. CD spectra were recorded on a JASCO J-815 Spectropolarimeter. 1H and 13C NMR data were acquired with a Bruker Avance-500 spectrometer. The HMQC and C
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of MeOH−H2O (20%−100%) yielded four fractions (A-D). Fraction C was further separated by chromatography over silica gel (3 × 35 cm) with a stepwise gradient elution of CH2Cl2−MeOH (100:1 to 0:1) to afford six subfractions C1−C6. Subfraction C2 was purified by reverse phased HPLC (RP-HPLC) using 68% acetonitrile in water to afford compound 2 (12.0 mg, tR 34.5 min) and 3 (110.0 mg,tR 30 min). Compound 1 (13.0 mg, tR 18 min) was obtained by RP-HPLC using 65% acetonitrile in water from subfraction C3. Ganoboninketal A (1). Yellow oil (methanol); [α]25D −52.0 (c 1.5, methanol); CD (c 1.5 × 10−3 M, methanol) λmax (Δε): 225 (−4.09), 258 (+8.34), 349 (−1.75); UV (methanol) λmax nm (log ε): 260 (4.42); IR (neat) νmax: 2968, 1735, 1663, 1233, 1198, 1028, 895 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HR-ESI-MS: m/ z [M + H]+ 543.3319 (calcd for C32H47O7 543.3316). Ganoboninketal B (2). Yellow oil (methanol); [α]25D 43.5 (c 1.2, methanol); CD (c 1.2 × 10−3 M, methanol) λmax (Δε): 227 (+8.02), 264 (+3.31), 414 (−1.02); UV (methanol) λmax nm (log ε): 272 (4.20); IR (neat) νmax: 2969, 1735, 1695, 1286, 1195, 1072, 895 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HR-ESI-MS: m/ z [M + H]+ 499.3059 (calcd for C30H43O6 499.3054). Ganoboninketal C (3). Colorless oil (methanol); [α]25D 11.4 (c 1.5, methanol); CD (c 1.5 × 10−3 M, methanol) λmax (Δε): 223 (−0.39), 304 (0.81); UV (methanol) λmax nm (log ε): 200 (4.69); IR (neat) νmax: 2974, 2948, 2882, 1734, 1701, 1220, 1196, 1038, 986, 905 cm−1; for 1H NMR and 13C NMR data see Table 1; Positive HRESIMS: m/z [M + H]+ 559.3272 (calcd for C32H47O8, 559.3265). Computational Details. Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software package was used for systematic conformational analyses for compounds 1−3 with the MMFF94 molecular mechanics force field. Subsequently, The MMFF94 conformational analyses were optimized using DFT at B3LYP/6-31G(d) basis set level using Gaussian03 package. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 30 lowest electronic transitions were calculated using the TDDFT methodology at the B3LYP/6-
Figure 2. Selected key HMBC and NOESY correlations of 1 and 3. The molecular models of 1 and 3 in minimal energy were obtained by Conflex calculations in MMFF94s force field. HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HR-ESI-MS data were acquired using an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument. Fungal Material. Fruiting bodies of Ganoderma boninense were collected from Hainan province (China) in 2010, and identified by Prof. Yijian Yao (one of coauthors) on the basis of morphological characteristics. Extraction and Isolation. Sixty g of dry fruiting body of G. boninense were extracted with ethyl acetate (3 × 1 L), and 8 g crude extract was obtained by evaporating solvent under vacuum. Separation by ODS column chromatography (column 5 × 50 cm) with a gradient
Figure 3. Selected excitation NOE of ganoboninketal A (1) in pyridine (500 MHz). D
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Figure 4. Experimental CD spectra of 1 and 3 in MeOH and the calculated ECD spectra of 4a, 4b, 6a, and 6b. Structures 4a, 4b, 6a, and 6b represent possible stereoisomers for 1 and 3, respectively. 39.9 min), and 3 (tR 38.8 min) were all detected on the HPLC chromatogram of the crude extract. NO Inhibition and Cytotoxicity Assays. Mouse monocyte/ macrophages RAW 264.7 were maintained in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 mg/ mL), and 10% heat-inactivated fetal bovine serum at 37 °C with 5% CO2 and 95% air; 200 μL of cells (5 × 105 cells/mL) were added in a 96-well plate. After 1 h incubation, cells were treated with 1 mg/mL of LPS and various concentrations of test compounds (DMSO as solvent) for 24 h. The NO level in the supernatant of RAW 264.7 cells was determined by the Griess reaction.18 Cytotoxic activity against A549 and HeLa cell lines was assayed with the MTT assay.19 Cells were incubated with tested compounds (DMSO as solvent) at 37 °C in a humidified atmosphere of 5% CO2 95% air for 72 h. Into each well was added 50 μL of MTT/medium solution (0.5 mg/mL), and cells were incubated for another 4 h. After removing the MTT/medium, 100 μL of DMSO was added to each well. The plate was shaken to dissolve the precipitate, and activity was measured at 540 nm using a microplate reader. The inhibition rates were calculated and plotted versus test concentrations to afford the IC50 (±SD) for three independent experiments, each carried out in triplicate. Hydrocortisone (IC50, 53.7 ± 3.9 μM) was used as a positive control for the NO inhibition assay. Cisplatin was used as the reference substance that showed cytotoxicity against A549 and HeLa cell lines with IC50 values of 19.2 ± 1.1 and 15.9 ± 1.5 μM, respectively. Antimalarial Assay..12,13 The 3D7 strain of Plasmodium falciparum was maintained in RPMI 1640 medium (Gibco-BRL Laboratories) supplemented with 2.5 g/L NaHCO3, 0.3 g/L Lglutamine, 3.96 g/L HEPES, 25 mg/L penicillin, 4 g/L Albumax II, 0.07 g/L streptomycin, and 0.02 g/L hypoxanthine. Parasites were
Table 2. Antiplasmoidial, NO Inhibitory and Cytotoxic Activities of 1−3 (IC50, μM) antiplasmodial effect
antiinflammation
cmpds
P. falciparum
NO inhibitiona
A549
HeLa
1 2 3 positive control
4.0 ± 0.7 7.9 ± 0.9 1.7 ± 0.5 artemisinin 30.4 ± 0.6 nM
98.3 ± 8.3 24.3 ± 1.9 60.9 ± 5.5 hydrocortisone 53.7 ± 3.9
47.6 ± 3.6 >100 35.8 ± 2.8 cisplatin 19.2 ± 1.1
>100 65.5 ± 3.7 >100 cisplatin 15.9 ± 1.5
cytotoxicity
a
The growth of macrophage cells were not influenced by compounds 1−3 at the concentration of 200 μM.
31G(d) or B3LYP/6-31+G(d, p) lever. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.34 eV. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correlation. HPLC Analysis of the Extract of G. boninense. The deposited fruiting bodies were extracted with acetonitrile (3 × 100 mL). The acetonitrile extract was dissolved in acetonitrile (10 mg/mL) and subjected to RP-HPLC analysis. The detection wavelength was set to 210 nm. The mobile phase consisted of MeCN (A) and H2O (B) using a gradient elution of 15−40% A at 0−10 min, 40−60% A at 10− 25 min, 60−100% A at 25−45 min, and 100% A at 45−55 min. The flow rate was kept at 1 mL/min. Compounds 1 (tR 39.4 min), 2 (tR E
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maintained in human red blood cells (hRBC) at a hematocrit of 5% at 37 °C. Cultures were under an atmosphere of 5% CO2, 5% O2 and 90% N2. The parasites were diluted to a final hematocrit of 2% and a parasitemia of 1% by using medium and hRBC. The dilution of parasitemia and compounds (DMSO as solvent) were added to a 96-well plate and cultured in a 37 °C incubator for approximately 48 h. Fifty μL of fluorochrome mixture consisting of PicoGreen (Molecular Probes, Inc.), 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE buffer), and 2% Triton X-100 diluted with double distilled water, was then added to release and label the parasitic DNA. The plates were then incubated for 30 min in the dark. The fluorescence signal, measured as relative fluorescence units (RFU), was determined with a fluorescence microplate reader (Fluoroskan Ascent) with excitation at 485 nm and emission at 538 nm. Simultaneously, the RFU from positive and negative control samples were obtained, stored, and analyzed. IC50 (±SD) values were calculated using linear interpolation of inhibition curves for three independent experiments, each carried out in triplicate. Artemisinin was used as positive control with IC50 of 30.4 ± 0.6 nM.
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ASSOCIATED CONTENT
* Supporting Information S
NMR data of 1-3, CD data and calculations for 2. This material is available free of charge via the Internet at http://pubs.acs.org
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86 10 62566577. E-mail: [email protected] (H.-W.L.). Author Contributions ⊥
K.M. and J.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the National Program on Key Basic Research Project (973 program 2014CB138304), the National Natural Science Foundation of China (21072219).
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