Lanostane Triterpenes Isolated from Antrodia heteromorpha and Their

Jun 7, 2016 - Two new spiro-lanostane triterpenoids, antrolactones A and B (1 and 2), along with polyporenic acid C (3), were isolated from an EtOAc-s...
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Lanostane Triterpenes Isolated from Antrodia heteromorpha and Their Inhibitory Effects on RANKL-Induced Osteoclastogenesis Jaeyoung Kwon,† Hyaemin Lee,† Yeo Dae Yoon,‡ Bang Yeon Hwang,§ Yuanqiang Guo,⊥ Jong Soon Kang,‡ Jae-Jin Kim,∥ and Dongho Lee*,† †

Department of Biosystems and Biotechnology and ∥Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea ‡ Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea § College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea ⊥ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300353, People’s Republic of China S Supporting Information *

ABSTRACT: Two new spiro-lanostane triterpenoids, antrolactones A and B (1 and 2), along with polyporenic acid C (3), were isolated from an EtOAc-soluble extract of Antrodia heteromorpha culture medium, and the chemical structures of the new compounds were elucidated by application of NMR, MS, and ECD spectroscopic techniques. All isolated compounds exhibited inhibitory effects on receptor activator of nuclear factor-kappaB ligand-induced osteoclastogenesis. one homeostasis, or so-called “bone remodeling”, is regulated by the balance of two coupled processes. Osteoclastic bone resorption is followed by osteoblastic bone formation and consequently resorbed lacunae by osteoblasts.1,2 Osteoclasts, which play a critical role in bone remodeling, are large multinucleated cells derived from their mononuclear precursor as monocytes and macrophages through a differentiation process induced by receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colonystimulating factor (M-CSF).3,4 However, excessive osteoclastinduced bone resorption in comparison with osteoblastic bone formation has been observed in several skeletal disorders such as osteoporosis, rheumatoid arthritis, and periodontal disease.4,5 Therefore, inhibition of RANKL-induced osteoclast differentiation can be a therapeutic strategy for preventing osteoclastogenesis.6 There have been reports that several natural-product-derived molecules exhibit inhibitory activities against osteoclastogenesis,7,8 and fungi-derived molecules may be significant sources owing to their structural diversity and biological activity. Antrodia heteromorpha (Fr.) Donk, belonging to the family Fomitopsidaceae, is a common wood-rotting basidiomycete fungus characterized by a dimitic hyphal structure with clamped

B

© XXXX American Chemical Society and American Society of Pharmacognosy

generative hyphae and a white or pale cream pore surface.9 Although there have not been previous chemical investigation of this species, Antrodia sp. has been found to possess structurally unique and bioactive secondary metabolites.10−12 In an attempt to discover biologically active constituents, two new spiro-lanostane triterpenoids, antrolactones A and B (1 and 2), together with polyporenic acid C (3),13 were isolated from an EtOAc-soluble extract of A. heteromorpha culture medium, and their chemical structures were determined by the application of spectroscopic techniques. All isolated compounds exhibited inhibitory effects on RANKL-induced differentiation of bone marrow macrophages (BMMs) into osteoclasts, as evidenced by the use of tartrate-resistant acid phosphatase (TRAP) as a primary marker. Described herein are the isolation, structural determination, and biological evaluation of these compounds. Compound 1 was obtained as a white and amorphous solid, and its elemental formula was determined to be C31H42O7 by HRESIMS analysis, suggesting 11 degrees of unsaturation. The IR spectrum exhibited absorptions due to hydroxy (3465 cm−1) Received: March 8, 2016

A

DOI: 10.1021/acs.jnatprod.6b00207 J. Nat. Prod. XXXX, XXX, XXX−XXX

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8.5 Hz, H-17), 1.54 (1H, dd, J = 12.0, 3.5 Hz, H-5)]. The 13C NMR data (Table 1) exhibited 31 carbon signals assigned to eight methyl carbons, four methylene carbons, seven methine carbons including three oxymethines, two carbonyl carbons, one dioxygenated secondary carbon, one oxygenated tertiary carbon, and eight additional quaternary carbons. Detailed analysis of the 1H and 13C NMR data revealed that 1 is a triterpene spirolactone derivative similar to fomlactone C,14 which possesses a lanostane triterpene skeleton. The differences between them were that 1 has three hydroxy groups at C-2, C15, and C-20, as indicated by the HMBC cross-peaks of H-1/C2 (δC 69.1), H-30/C-15 (δC 74.4), and H-21/C-20 (δC 74.0) and three double bonds at C-7 and C-8, C-9 and C-11, and C24 and C-25, as evidenced by the HMBC cross-peaks of H-19/ C-9 (δC 146.0), H-12/C-11 (δC 118.8), H-6/C-7 (δC 123.0), H-30/C-8 (δC 139.5), H-27/C-25 (δC 121.5), and H-31/C-24 (δC 161.7), respectively. Furthermore, the HMBC cross-peaks of H-22a and H-31/C-23 (δC 107.4) suggested the connectivity of a spiro center at C-23, and the HMBC cross-peaks of H-18/ C-12 (δC 79.1) and H-21 and H-22b/C-17 (δC 53.0) demonstrated that this spirolactone ring is connected at C-12

and carbonyl (1759 and 1709 cm−1) groups. The 1H NMR data (Table 1) showed the presence of eight methyl groups [δH 1.97 (3H, d, J = 1.0 Hz, H-31), 1.77 (3H, d, J = 1.0 Hz, H-27), 1.32 (3H, s, H-19), 1.23 (3H, s, H-21), 1.19 (3H, s, H-28), 1.16 (3H, s, H-29), 1.03 (3H, s, H-30), and 0.70 (3H, s, H-18)], four methylene groups [δH 2.64 (1H, dd, J = 12.5, 6.0 Hz, H1a), 2.51 (1H, m, H-16a), 2.45 (1H, d, J = 16.0 Hz, H-22a), 2.25 (1H, dd, J = 18.0, 12.0 Hz, H-6a), 2.14 (1H, ddd, J = 18.0, 7.0, 3.5 Hz, H-6b), 1.96 (1H, d, J = 15.5 Hz, H-22b), 1.81 (1H, m, H-16b), and 1.54 (1H, overlapped, H-1b)], and seven methine groups [δH 6.04 (1H, d, J = 6.5 Hz, H-7), 5.18 (1H, s, H-11), 4.73 (1H, s, H-12), 4.65 (1H, dd, J = 12.5, 6.0 Hz, H-2), and 4.38 (1H, dd, J = 9.5, 6.5 Hz, H-15), 2.79 (1H, dd, J = 10.5,

Table 1. NMR Spectroscopic Data of Compounds 1 and 2 in CDCl3 1 δC

position

a

δH

1

46.3,

CH2

2 3 4 5 6

69.1, 215.7, 46.9, 51.1, 23.6,

CH C C CH CH2

7 8 9 10 11 12 13 14 15 16

123.0, 139.5, 146.0, 37.9, 118.8, 79.1, 47.6, 52.9, 74.4, 34.6,

CH C C C CH CH C C CH CH2

17 18 19 20 21 22

53.0, 11.3, 22.4, 74.0, 27.8, 49.4,

CH CH3 CH3 C CH3 CH2

23 24 25 26 27 28 29 30 31

107.4, 161.7, 121.5, 171.9, 8.3, 22.4, 24.7, 17.1, 10.8,

C C C C CH3 CH3 CH3 CH3 CH3

2.64, dd (12.5, 6.0) 1.54, overlapped 4.65, dd (12.5, 6.0)

1.54, 2.25, 2.14, 6.04,

dd (12.0, 3.5) dd (18.0, 12.0) ddd (18.0, 7.0, 3.5) d (6.5)

2 2, 3, 5, 10, 19 2, 10, 19

6 7, 8 7, 8, 10 5, 9, 14

5.18, br s 4.73, br s

8, 10, 13 9, 11, 14, 18

4.38, 2.51, 1.81, 2.79, 0.70, 1.32,

30 15 13, 15 13, 15, 16, 18, 20 12, 13, 14, 17 1, 5, 9, 10

dd (9.5, 6.5) m m dd (10.5, 8.5) s s

1.23, s 2.45, d (16.0) 1.96, d (15.5)

1.77, 1.19, 1.16, 1.03, 1.97,

d (1.0) s s s d (1.0)

δC

HMBCa

17, 20, 22 21, 23 17, 20

24, 25, 26 3, 4, 5, 29 3, 4, 5, 28 8, 13, 14, 15 23, 24, 25

δH

50.1,

CH2

210.4, 82.2, 44.7, 47.6, 23.3,

C CH C CH CH2

123.7, 139.3 146.7, 43.5, 118.3, 79.0, 47.6, 52.9, 74.3, 34.6,

CH C C C CH CH C C CH CH2

53.0, 11.2, 23.5, 74.1, 27.8, 49.3,

CH CH3 CH3 C CH3 CH2

107.4, 161.3, 121.5, 172.0, 8.3, 16.6, 28.6, 17.1, 10.8,

C C C C CH3 CH3 CH3 CH3 CH3

HMBCa

2.80, d (12.5) 2.54, d (12.5)

2, 3, 5, 10, 19 2, 10, 19

3.96, s

2, 4, 28, 29

1.77, 2.33, 2.14, 6.04,

4 7, 8, 10 7, 8 5, 9, 14

dd (12.0, 3.5) ddd (18.0, 7.0, 4.0) dd (18.0, 12.0) d (6.5)

5.03, br s 4.75, br s

8, 10, 13 9, 11, 14, 18

4.38, 2.52, 1.83, 2.81, 0.66, 1.01,

30

dd (10.0, 6.5) m m m s s

13, 15 15, 16, 18, 20 12, 13, 14, 17 1, 5, 9, 10

1.24, s 2.45, d (15.5) 1.96, d (15.5)

17, 20, 22 21, 23 17, 20

1.82, 0.76, 1.19, 1.09, 1.98,

24, 25, 26 3, 4, 5, 29 3, 4, 5, 28 8, 13, 14, 15 23, 24, 25

d (1.0) s s s d (1.0)

HMBC correlations, optimized for 8 Hz. B

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30 indicated that they are on the opposite face of the system. However, the relative configuration of the spiro center at C-23 was not determined despite the NOESY correlations of H-31 with H-22. The relative configuration of compound 2 was similar to that of 1 (Figure 2), except for C-3, which was confirmed by NOESY correlations of H-5 with H-3 and H-29. The absolute configurations of compounds 1 and 2 were established by comparison of their experimental electronic circular dichroism (ECD) spectra with those calculated using the time-dependent density functional theory (TDDFT) method, and 23R and 23S models were calculated to determine the absolute configurations of C-23 while the configurations of the remaining positions were fixed. Conformational searches were performed using the MMFF force field in Spartan’14 software,17 and geometry optimization for selected conformers was carried out at the B3LYP/6-31+G(d,p) level in Gaussian 09 software.18 Following optimization, the theoretical ECD spectra were calculated at the CAM-B3LYP/SVP level with a CPCM solvent model in MeCN. The experimental ECD spectrum of 1 exhibited a negative Cotton effect (CE) at 281 nm (Δε −1.6), a positive CE at 238 nm (Δε +9.9), and a negative CE at 211 nm (Δε −8.6), and the ECD spectrum of 2 showed almost the same pattern [Δε −7.5 (210), +11.7 (238), −3.1 (288)]. Among the two candidates, the calculated ECD spectra of 23R models were in agreement with the experimental spectra despite a slight difference at 260−290 nm (Figure 3), suggesting the absolute configuration of 1 as 2R, 5R, 10S, 12R, 13R, 14S, 15S, 17S, 20S, and 23R and that of 2 as 3R, 5R, 10S, 12R, 13R, 14S, 15S, 17S, 20S, and 23R. Three isolated compounds (1−3) were evaluated for their inhibitory effects on RANKL-induced osteoclast differentiation in mouse bone marrow macrophages with TRAP as a primary marker, as it is highly expressed in osteoclasts. BMMs were differentiated into TRAP-positive multinucleated osteoclasts in the presence of RANKL and M-CSF, whereas treatment with compounds 1−3 showed dose-dependent suppressive activities on RANKL-induced BMM differentiation into osteoclasts, as evidenced by a TRAP assay (Figure 4A) and staining (Figure 4B). Furthermore, the IC50 values of compounds 1−3 were 7.4, 4.5, and 4.9 μM, respectively. The cell viability assessed by an XTT assay suggested that these compounds had no significant cytotoxic effects on BMMs at their effective concentrations for

and C-17. The positions of the remaining substituents were deduced from additional 2D NMR studies (Figure 1). Accordingly, the new compound 1 was elucidated as shown and has been given the trivial name antrolactone A.

Figure 1. HMBC and COSY correlations for compounds 1 and 2.

The molecular formula of compound 2 was C31H42O7 based on HRESIMS analysis. The 1H and 13C NMR spectroscopic data were similar to those of 1, indicating that both compounds share the same triterpene framework, except that the functional group patterns of C-2 and C-3 were different, which was confirmed by the change in 1H NMR data [δH 4.65 (1H, dd, J = 12.5, 6.0 Hz, H-2), 2.64 (1H, dd, J = 12.5, 6.0 Hz, H-1a), and 1.54 (1H, overlapped, H-1b) in 1 and δH 3.96 (1H, s, H-3), 2.80 (1H, d, J = 12.5 Hz, H-1a), and 2.54 (1H, d, J = 12.5 Hz, H-1b) in 2], the disappearance of COSY cross-peaks of H-1/H2, and the HMBC cross-peaks of H-28 and H-29/C-3 (δC 82.2). The gross structure of 2 was confirmed from 2D NMR data (Figure 1). Thus, the structure of the new compound 2 (antrolactone B) was assigned as shown. The relative configuration of compound 1 was determined by comparison of 1D NMR chemical shifts and NOESY correlations with those of previously reported lanostane triterpene spirolactones, austrolactone and fomefficinol B (Figure 2).15,16 The NOESY correlations of H-2 with H-19, H-18 with H-15, H-19, and H-21, and H-19 with H-28 allowed them to be located on the same face of the ring system. Those of H-5 with H-29, H-12 with H-17 and H-30, and H-17 with H-

Figure 2. Key NOESY correlations for compounds 1 and 2. C

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Figure 3. Calculated and experimental ECD spectra of compounds 1 (A) and 2 (B).

Figure 4. Inhibitory effects of compounds 1−3 on RANKL-induced osteoclast differentiation. BMMs were treated with vehicle or indicated concentrations of 1−3 in the presence of RANKL and M-CSF for 4 days. (A) Supernatants mixed with a chromogenic substrate containing TRAP, with their optical density at 540 nm measured. (B) Cells fixed with 10% formalin, stained with a chromogenic substrate containing TRAP, and visualized by light microphotography. IR spectrometer (Varian, Palo Alto, CA, USA). ECD spectra were recorded on a JASCO J-1100 spectropolarimeter. NMR spectra were recorded at room temperature on a Varian 500 MHz NMR spectrometer with tetramethylsilane as an internal standard. ESIMS were measured on a Waters Q-TOF Micromass spectrometer (Waters, Milford, MA, USA). MPLC was performed by a Biotage Isolera One system with a SNAP cartridge KP-Sil 100 g (Biotage AB, Uppsala, Sweden). HPLC was performed by a Waters system comprising a 515 pump and a 2996 PDA detector with a YMC Pack ODS-A column (5 μm, 250 × 20 mm i.d., YMC, Kyoto, Japan). TLC was performed on precoated plates with silica gel (0.25 mm, Merck, Darmstadt, Germany). Fungal Material. A. heteromorpha was collected in November 2008 from Gapyeong, Gyeonggi-do, Korea, and authenticated by Professor Jae-Jin Kim (Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea). A voucher specimen (KUC9196) was deposited at the Division of Environmental Science and Ecological Engineering, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea. A. heteromorpha was cultivated on potato

the inhibition of differentiation of osteoclasts (Figure S16, Supporting Information). These biological data indicate that further research including mechanism studies is needed. It has been previously reported that several types of triterpenes such as oleanolic acid acetate inhibit osteoclastogenesis, and further research including mechanism studies has also been carried out.19−21 The present study describes the isolation and characterization of lanostane triterpenes from A. heteromorpha, with two new constituents elucidated, and all isolated compounds exhibited inhibitory effects against osteoclastogenesis. This indicates that the lanostane triterpene derivatives may have a significant role in the inhibition of osteoclast differentiation.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 (JASCO, Tokyo, Japan). UV spectra were recorded on a Mecasys Optizen Pop spectrometer (Mecasys, Daejeon, Korea), and IR spectra were obtained with a Varian 640 FTD

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dextrose agar medium on Petri dishes (150 mm × 2 cm × 70 plates) at 25 °C for 18 days. Extraction and Isolation. The fermented materials were extracted with MeOH (3 × 2.0 L) at room temperature and evaporated in vacuo at 35 °C. The residue was suspended in H2O (1.0 L) and partitioned with EtOAc (3 × 1.0 L) to provide an EtOAc-soluble extract (700.0 mg). The EtOAc extract was fractionated on silica gel by MPLC (CHCl3−acetone, 1:0 to 0:1, 35.0 mL/min) to afford 11 fractions (Fr 1 to Fr 11). Fr 6 (92.7 mg) was purified by preparative HPLC (MeCN−H2O, 1:1 to 1:0, 8 mL/min) to obtain polyporenic acid C (3, 7.0 mg). Fr 8 (58.8 mg) was purified by preparative HPLC (MeCN− H2O, 1:4 to 1:0, 8 mL/min) to give antrolactone A (1, 4.1 mg) and antrolactone B (2, 4.0 mg). Antrolactone A (1): colorless, amorphous solid; [α]27D +21.3 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 231 (4.94), 239 (4.98), 246 (5.00), 255 (4.85) nm; IR νmax (ATR) 3465, 2977, 1759, 1709, 1446, 1378, 1248, 1156, 1049 cm−1; ECD (c 0.5 mM, MeCN) Δε −8.6 (211), +9.9 (238), −1.6 (281); 1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 1; ESIMS (negative) m/z 571 [M + HCOO]−; HRESIMS m/z 571.2906 [M + HCOO]− (calcd for C32H43O9, 571.2907). Antrolactone B (2): colorless, amorphous solid; [α]27D +15.4 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 231 (4.98), 239 (5.02), 247 (5.03), 255 (4.89) nm; IR νmax (ATR) 3479, 2978, 1780, 1712, 1437, 1377, 1249, 1169, 1048 cm−1; ECD (c 0.5 mM, MeOH) Δε −7.5 (210), +11.7 (238), −3.1 (288); 1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 1; ESIMS (negative) m/z 571 [M + HCOO]−; HRESIMS m/z 571.2913 [M + HCOO]− (calcd for C32H43O9, 571.2907). Computational Methods. 3D models of 1 and 2 were built from NOESY spectra and Chem3D modeling, and conformational analysis was performed by the MMFF force field as implemented in Spartan’14 software.17 The selected conformers were optimized at the B3LYP/631+G(d) level, and TDDFT ECD calculations were performed at the CAM-B3LYP/SVP level with a CPCM solvent model in MeCN using Gaussian 09 software.18 The calculated ECD spectra were simulated with a half-bandwidth of 0.4 eV. The ECD curves were extracted by SpecDis 1.64 software22 and weighted by Boltzmann distribution after UV correction. Osteoclastogenesis Assay. Mouse bone marrow cells were isolated from the femurs and tibiae of 6- to 8-week-old female C57BL/ 6 mice (Koatech, Pyungtaek, Gyeonggi, Korea). After lysing red blood cells, the cells were incubated in α-minimal essential medium (Gibco BRL, MD, USA) supplemented with 10% fetal bovine serum, 100 U/ mL penicillin, and 100 μg/mL streptomycin in the presence of M-CSF (50 ng/mL) for 3 days. Bone marrow macrophages were obtained by removing floating cells. For osteoclast differentiation, BMMs (4 × 104 cells/well) were cultured in the presence of M-CSF (50 ng/mL) and RANKL (100 ng/mL) in 96-well plates with or without compounds 1−3. After 4 days, cells were fixed with 10% formalin for 5 min, and staining of TRAP-positive cells and quantitation of TRAP activity in culture supernatants were performed using a TRAP staining kit (Kamiya Biomedical Company) according to the manufacturer’s instructions.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the National Research Foundation of Korea (NRF2015R1D1A1A01060321) and the KRIBB Research Initiative Program.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00207. 1 H and 13C NMR as well as MS spectra of compounds 1 and 2 and 1H NMR data of compound 3 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: [email protected] (D. Lee). E

DOI: 10.1021/acs.jnatprod.6b00207 J. Nat. Prod. XXXX, XXX, XXX−XXX