Lanostane triterpenoids and sterols from Antrodia camphorata

Lanostane triterpenoids and sterols from Antrodia camphorata

Phytochemistry 84 (2012) 177–183 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytoch...

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Phytochemistry 84 (2012) 177–183

Contents lists available at SciVerse ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Lanostane triterpenoids and sterols from Antrodia camphorata Hui-Chi Huang a, Chih-Chuang Liaw b, Hsin-Ling Yang c, You-Cheng Hseu d, Hsiou-Ting Kuo e, Yao-Ching Tsai e, Shih-Chang Chien f, Sakae Amagaya g, Yu-Chang Chen a,⇑, Yueh-Hsiung Kuo a,e,h,i,⇑ a

Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404, Taiwan Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan c Department of Nutrition, China Medical University, Taichung 404, Taiwan d Department of Cosmeceutics and Graduate Institute Cosmeceutics, China Medical University, Taichung 404, Taiwan e Department of Chemistry, National Taiwan University, Taipei 106, Taiwan f The Experimental Forest Management Office, National Chung-Hsing University, Taichung 402, Taiwan g Department of Kampo Pharmaceutical Sciences, School of Pharmacy, Nihon Pharmaceutical University, Saitama 362-0806, Japan h Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung 404, Taiwan i Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan b

a r t i c l e

i n f o

Article history: Received in revised form 27 March 2012 Accepted 13 August 2012 Available online 18 September 2012 Keywords: Antrodia camphorata Polyporales Fomitopsidaceae Cinnamomun kanehirai Hay Lauraceae Chinese herb Lanostane-type triterpenoids Fungal sterols Camphosterol A

a b s t r a c t Four lanostane triterpenes, 3,7,11-trioxo-5a-lanosta-8,24(E)-dien-26-oic acid, methyl 11a-3,7-dioxo-5alanosta-8,24(E)-dien-26-oate, methyl 3,7,11,12,15,23-hexaoxo-5a-lanost-8-en-26-oate, and ethyl 3,7,11,12,15,23-hexaoxo-5a-lanost-8-en-26-oate, two sterols, (14a,22E)-14-hydroxyergosta-7,22diene-3,6-dione and a steroid named as camphosterol A were isolated from a mixture of fruiting bodies and mycelia of solid cultures of Antrodia camphorata. The 1H and 13C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, HMQC, HMBC and NOESY sequences. Six compounds were evaluated for cytotoxicity against several human tumor cell lines, all of which has moderate activity. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Antrodia camphorata (Polyporaceae, Aphyllophorales, Fomitopsidaceae) is a fungus parasitic on the inner wall of heartwood of the endemic and endangered species Cinnamomun kanehirai Hay (Lauraceae) in Taiwan. The fruiting bodies of A. camphorata, which are called ‘‘jang-jy’’ or ‘‘niu-chang-chih’’ in Taiwan, are highly valued in folk medicine and are rare in the wild (Wu et al., 1997). The fruiting bodies are used in traditional Chinese medicine to treat abdominal pain, diarrhea, food and drug intoxication, hypertension, liver cancer, and pruritus (Tsai and Liaw, 1982). The pharmacological effects and phytochemical aspects of the fruiting body, solid-state cultivated mycelia, and mycelia from submerged cultivations of A. camphorata have been studied (Geethangili and Tzeng, 2009; Yeh et al., 2009; Lien et al., 2009). Previous chemical studies ⇑ Corresponding authors. Tel.: +886 4 22053366x5209 (Y.-C. Chen), tel.: +886 4 22053366x5701; fax: +886 4 22071693 (Y.-H. Kuo). Address: Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung 404, Taiwan. E-mail addresses: [email protected] (Y.-C. Chen), [email protected] edu.tw (Y.-H. Kuo). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.08.011

of the mixture of fruiting bodies and the mycelia from solid cultures of A. camphorata have reported the presence of several components, including fatty acids, lignans, benzenoids, benzoquinones, sesquiterpenes, steroids, and triterpenoids (Chien et al., 2008). Generally, the fruiting bodies of A. camphorata are composed of abundant triterpenoids including those with ergostane and lanostane skeleta (Geethangili and Tzeng, 2009). Additionally, some triterpenoids isolated from fruiting bodies show cytotoxic (Yeh et al., 2009), anti-Helicobacter pylori (Geethangili et al., 2010), antiinflammatory (Shen et al., 2004), and immuno-modulating (Shen et al., 2003) activities. Herein, reported an isolation and structural elucidation of four lanostane triterpenes 3,7,11-trioxo-5alanosta-8,24(E)-dien-26-oic acid (1), methyl 11a-hydroxy-3, 7-dioxo-5a-lanosta-8,24(E)-dien-26-oate (2), methyl 3,7,11,12,15, 23-hexaoxo-5a-lanost-8-en-26-oate (3), and ethyl 3,7,11,12,15, 23-hexaoxo-5a-lanost-8-en-26-oate (4), and two sterols, (14a,22E)-14-hydroxyergosta-7,22-diene-3,6-dione (5) and a degraded steroid camphosterol A (6) from a mixture of fruiting bodies and the mycelia of A. camphorata. The biological evaluation of the isolated compounds (1–6) against a panel of cancer cell lines is also described.

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2. Results and discussion A MeOH extract of the mixture of fruiting bodies and mycelia of A. camphorata was suspended in H2O and successively partitioned with EtOAc and n-butanol, successively. After evaporation of the solvent, the EtOAc layer was subjected to successive column chromatography on silica gel and Sephadex LH-20, and then separated by HPLC to afford 1–6 and seven known compounds. Compound 1 was obtained as colorless needles with the molecular formula C30H42O5, as established by a [M]+ ion peak at m/z 482.3038 in its HREIMS spectrum. The IR spectrum of 1 confirmed the presence of isolated carbonyl (1712 cm1), carboxylic acid (3300–2500, 1690 cm1), and a,b-unsaturated carbonyl (1681 cm1) groups. The 1H NMR spectrum indicated presence of six tertiary methyl groups at dH 1.82 (s), 1.26 (s), 1.20 (s), 1.10 (s), 1.08 (s), and 0.80 (s) (Table 1), a secondary methyl group at dH 0.91 (d, J = 6.5 Hz), and an olefinic methine at dH 6.85 (t, J = 7.4 Hz). The olefinic proton (H-24) was downfield-shifted to dH 6.85 due to the conjugation with the carboxylic acid in a cis configuration, in comparing with 3a-acetoxy-5a-lanosta-8,24-dien21-oic acid (Lin et al., 1997; Min et al., 2000). Thus, compound 1 had a lanostane skeleton with a conjugated carboxylic acid located at the terminal of a side-chain. Analysis of UV absorption data and four signals from the 13C NMR spectrum (dC 201.9, 201.3, 151.6 and 149.5) (Table 2) suggested the presence of a 1,4-enedione system in this compound. The two methylene proton resonances at dH 2.72 and 2.60 with larger AB geminal coupling (J = 16.7 Hz), which

exhibited a HMBC (Fig. 2) correlation with dC 201.9 and 151.6, may be assigned as H2-12, in an a-position to one of the carbonyl groups (dC 201.9). The other methylene group resonances (H2-6, dH 2.51 and 2.37) with ABX patterns were neighbors to another carbonyl group at dC 201.3 based on the HMBC correlation with dC 201.3 and 149.5. From the above arguments, the 1,4-enedione functionality presented at C-7, C-8, C-9 and C-11 between the two assigned methylene groups was deduced as shown (Fig. 2). The 13C NMR, DEPT, and HMQC experiments had signals for seven methyl groups, eight methylene groups, four methine groups including an olefinic carbon at dC 144.1, eleven quaternary carbons, including four carbonyl carbons at dC 215.7, 201.9, 201.3, and 172.4, and three olefinic carbons at dC 151.6, 149.5, and 126.9. The carbon resonances were similar to those of lanostanoid, 3,7-dioxo-5a-lanosta-8,24(E)-diene-26-oic acid (Wang et al., 1997), except for additional signals due to presence of one carbonyl group instead of a methylene group. According to the above mentioned discussion, the additional oxo functionality was unambiguously assigned at C-11. The configuration of the double bond at C-24 was assigned as E based on the NOESY correlations between H327 and H2-23. The complete 1H and 13C NMR spectroscopic assignments were made by a combination of COSY, NOESY, HMQC and HMBC data, and the key COSY and HMBC correlations are shown in Fig. 2. Structure 1 was ultimately identified as 3,7,11-trioxo5a-lanosta-8,24(E)-dien-26-oic acid. Compound 2 has the molecular formula C31H46O5 based on its HREIMS data (m/z 498.3360 [M]+) and 13C NMR spectroscopic data

Table 1 1 H NMR spectroscopic data of compounds 1–5 (CDCl3, d in ppm, J in Hz, 400 MHz). Position

1

1

2.93 2.11 2.59 2.48

2

2 ddd (13.9, 5.6, 2.3) m m m

2.28 2.18 2.70 2.45

3 m m m m

2.87 1.75 2.61 2.48

4 m m m m

2.86 1.75 2.61 2.50

5 m m m m

4 5 6

2.22a 2.51a 2.37 dd (14.3, 2.4)

7 9 11 12 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCH3

a

2.24 dd (14.5, 3.0) 2.58a 2.39a

2.36a 2.75a 2.52a

2.36a 2.74a 2.53a

4.50 dd (9.0, 5.0) 2.72 2.60 2.14 1.64 1.98 1.66 1.74 0.80 1.26 1.41 0.91 1.57 1.10 2.25 2.08 6.85

1.82 1.20 1.10 1.08

d (16.7) d (16.7) m m m m m s s m d (6.5) m m m m t (7.4)

s s s s

2.50a 1.84a 2.06 m 1.59 m 1.96 m 1.28 m 1.55 m 0.66 s 1.38 s 1.38 m 0.92 d (6.5) 1.54 m 1.17 m 2.20 m 2.06 m 6.72 t (7.5)

1.81 1.12 1.09 1.08 3.71

s s s s s

2.82 m 1.90 m 2.78 m 1.16 s 1.33 s 1.97 m 0.87 d (6.4) 2.40a 2.33a

2.82 m 1.89 m 2.78 m 1.17 s 1.34 s 1.97 m 0.88 d (6.4) 2.39a 2.35a

5.93 2.80 1.78 1.64 2.05 1.72 1.97 1.46 1.90 1.40 1.95 0.70 1.05 2.08 1.01 5.17

d (2.5) tdd (10.1, 7.0, 2.5) m m m m m m m m m s s m d (6.5) dd (15.2, 8.3)

5.26 dd (15.2, 7.6) 2.84a 2.43a 2.92 m

2.84a 2.41a 2.93 m

1.15 1.53 1.11 1.13 3.65

1.15 1.53 1.12 1.13

d (6.8) s s s s

d (6.4) s s s

OCH2CH3

1.22 t (7.2)

OCH2CH3

4.10 q (7.2)

Assignments were confirmed by 1H–1H COSY and HMQC.

2.15 m 1.74 m 2.38 m 2.32 m 2.15a 1.74a 2.65a

1.85 m 1.46 0.82 0.81 0.91

m d (7.0) d (7.0) d (7.0)

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H.-C. Huang et al. / Phytochemistry 84 (2012) 177–183 Table 2 13 C NMR spectroscopic data for compounds 1–5 (CDCl3, d in ppm, 100 MHz). Position

1

2

3

4

5

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 OCH3

34.9 t 34.0 t 215.7 s 46.7 s 49.7 d 37.0 t 201.3 s 149.5 s 151.6 s 38.8 s 201.9 s 51.2 t 46.8 s 48.7 s 31.9 t 27.5 t 49.1 d 16.9 q 18.3 q 36.0 d 17.9 s 34.5 t 25.8 t 144.1 d 126.9 s 172.4 s 12.0 q 25.8 q 20.3 q 27.3 q

34.8 t 34.7 t 214.4 s 47.5 s 50.8 d 37.5 t 199.3 s 142.1 s 158.7 s 40.1 s 65.8 s 44.6 t 47.6 s 48.1 s 32.6 s 27.9 t 49.7 d 17.0 q 19.2 q 36.0 d 18.4 q 34.6 t 25.6 s 142.8 t 127.3 d 168.7 s 12.3 q 25.3 q 21.6 q 25.1 q 51.7 q

33.6 t 33.5 t 214.6 s 47.0 s 50.9 d 37.2 t 198.3 s 149.5 s 150.0 s 39.2 s 192.6 s 197.1 s 61.1 s 59.0 s 203.9 s 38.9 t 38.4 d 12.4 q 18.6 q 32.2 d 19.4 q 48.7 t 207.6 s 46.9 t 34.6 d 176.1 s 17.1 q 23.3 q 20.3 q 27.5 q 51.9 q

33.6 t 33.5 t 214.6 s 47.0 s 50.9 d 37.2 t 198.3 s 149.5 s 150.0 s 39.2 s 192.6 s 197.1 s 61.1 s 59.0 s 203.8 s 38.9 t 38.4 d 12.4 q 18.6 q 32.2 d 19.4 q 48.7 t 207.6 s 46.9 t 34.6 d 175.5 s 17.1 q 23.3 q 20.2 q 27.5 q

38.1 t 37.2 t 210.7 s 38.1 t 54.7 d 198.9 s 122.5 d 163.3 s 45.6 d 38.6 s 20.6 t 30.5 t 46.4 s 85.1 s 32.0 t 26.5 t 50.3 d 16.1 q 12.6 q 39.9 d 21.2 q 135.2 d 132.6 d 42.8 d 33.0 d 19.9 q 19.6 q 17.5 q

OCH2CH3

14.2 q

OCH2CH3

60.6 t O

21 24

18 R1

O

26

O

R2

11

R

O

19

O

27

3

30 7

O

29

(Table 2). The IR absorptions of 2 at 3492, 1711, and 1667 cm1 suggested the presence of hydroxyl, conjugated ester, and a,bunsaturated cyclohexanone groups, respectively. Based on a comparison of NMR spectroscopic data, 2 had the same lanostane skeleton as 1 with a carbinol group at C-11 (dC 65.8 in 2) instead of a ketone group (dC 201.9 in 1) (Tables 1 and 2). The location of the carbinol group at C-11 was confirmed by HMBC correlations between the proton signal at dH 4.50 (dd, J = 9.0, 5.0 Hz) and the carbon resonances at dC 142.1 (C-8) and 158.7 (C-9) (Fig. 2). H-11 exhibited NOESY correlations with H3-18 and H3-19, which indicated that H-11 is in a b-axial orientation. The HMBC correlation between the signals dH 3.71 and dC 168.7 confirmed the presence of a methyl carboxylate at the side-chain terminal (Fig. 1). Like 1, the olefinic proton resonance of H-24 was downfield-shifted to dH 6.72 in 2 due to its conjugation with the methyl ester in a cis configuration. The HMBC correlation from dH 1.12 (H3-28) and 1.09 (H3-29) to dC 214.4 identified the third carbonyl group at the C-3 position. Complete 1H and 13C NMR spectroscopic assignments and functional group location were determined from a combination of COSY, NOESY, HMQC and HMBC spectroscopic analyses (Fig. 2). Therefore, compound 2 was identified as methyl 11a-hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-26-oate. Compound 3 was isolated as a colorless solid. Its HREIMS spectrum exhibited an ion peak at m/z 540.2733 [M]+, consistent with a molecular composition of C31H40O8. Analysis of the IR absorptions at 1750, 1735, 1709 and 1689 cm1 indicated the presence of cyclopentanone, ester carbonyl, isolated carbonyl, and conjugated carbonyl groups, respectively. Compound 3 was identified as a lanostane by analyses of its 1H and 13C NMR spectra (Tables 1 and 2) and from observations of connectivities in the 1H–1H COSY, HMQC, and HMBC NMR spectra (Fig. 2). The 1H NMR spectrum indicated

O

O

O

O

28

α

21 14

15

R2

12 R1 O

1

OH

O

O

13

4

16

17

H

8

O

R Fig. 1. Structures of compounds 1–6, 6a and 6b isolated from A. camphorata.

18 20

11 3

19

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H.-C. Huang et al. / Phytochemistry 84 (2012) 177–183

3

2

3

3

1

13

1

1

Fig. 2. Key COSY and HMBC correlations for compounds 1–6.

the presence of five tertiary methyl signals at dH 1.53 (s), 1.33 (s), 1.16 (s), 1.13 (s) and 1.11 (s), and two secondary methyls at dH 1.15 (d, J = 6.8 Hz) and 0.87 (d, J = 6.4 Hz). The 13C NMR spectrum and DEPT data for 3 suggested that the molecule contained 31 carbons: eight methyls (including one methoxy carbon), six methylenes, four methines, seven carbonyls, and six quaternary carbons. Among the seven carbonyl resonances, six carbonyls belong to ketone functionalities at dC 214.6, 207.6, 203.9, 198.3, 197.1 and 192.6, and one belongs to an ester at dC 176.1. The 1H and 13C NMR spectroscopic characteristics are very similar to those of methyl ganoderate E (methyl 3,7,11,15,23-pentaoxo-5a-lanost-8en-26-oate) (Hirotani et al., 1985). A comparison of the NMR spectra indicated that an extra oxo functionality (dC 197.1) was present in 3 instead of a methylene (C-12) as in methyl ganoderate E. The additional ketone group was assigned at C-12 by the HMBC correlation of H-17 (dH 2.78, m) and H3-18 (dH 1.16, s) to C-12 (dC 197.1). The methyl proton signals for H3-18 shift from dH 1.16 in 3 to dH 0.80 in 1, due to deshielding from the C-12 carbonyl group, further supported this assignment. Since C-25 is a to the C-26 carbonyl group, this chiral center will be present as a mixture of two epimers, spontaneously (Chen et al., 2010). Based on the above evidence, 3 was established as methyl 3,7,11,12,15,23-hexaoxo-5alanost-8-en-26-oate. The molecular formula for 4 is C32H42O8 (m/z 554.2878 [M]+) based on HREIMS and 13C NMR spectroscopic data. The IR spectrum of 4 showed the presence of cyclopentanone (1745 cm1), ester carbonyl (1731 cm1), isolated carbonyl (1712 cm1), and conjugated carbonyl (1688 cm1) groups. Analysis of the 1H and 13 C NMR spectra indicated that its structure was similar to that of 3, the only difference being that 4 has an ethyl ester [dH 4.10 (q, J = 7.2 Hz) and 1.22 (t, J = 7.2 Hz)] instead of the methyl ester in 3. These findings suggested that this functionality is located at

the side-chain terminal. The HMBC (Fig. 2) correlations, CH3 (dH 1.22)/–OCH2– (dC 60.6); H3-27 (dH 1.15)/C-26 (dC 175.5), confirmed the carboxylate at C-26. C-25 is also present as a mixture of two epimers, the reason being the same as in compound 3. Consequently, compound 4 was identified as ethyl 3,7,11,12,15,23-hexaoxo-5a-lanost-8-en-26-oate. The molecular formula of 5 was determined to be C28H42O3 (m/z 426.3142 [M]+) by HR-EIMS. The IR spectrum showed absorption bands for a hydroxyl group at 3493 cm1, an isolated cyclohexanone at 1718 cm1, and an a,b-unsaturated cyclohexanone at 1662 cm1. Signals for two tertiary methyls at dH 1.05 (s, H-19) and 0.70 (s, H-18) and four secondary methyls at dH 1.01 (d, J = 6.5 Hz, H-21), 0.91 (d, J = 7.0 Hz, H-28), 0.82 (d, J = 7.0 Hz, H26) and 0.81 (d, J = 7.0 Hz, H-27) were observed in the 1H NMR spectrum (Table 1). The 13C NMR (Table 2) and DEPT spectra indicated 28 carbons, composed of four olefinic carbons, six methyls, seven methylenes, six methines, and five quaternary carbons (Table 2), suggesting that 5 was an ergostane-type steroid. In addition, the three olefinic proton resonances at dH 5.93 (d, J = 2.5 Hz), 5.26 (dd, J = 15.2, 7.6 Hz, H-23), and 5.17 (dd, J = 15.2, 8.3 Hz, H-22), the three tertiary carbon signals at dC 135.2 (C-22), 132.6 (C-23), and 122.5 (C-7), the quaternary olefinic carbon resonances at dC 163.3, and the two carbonyl carbon signals at dC 210.7 and 198.9 were similar to those of ergosta-7,22-diene-3,6-dione (Kawahara et al., 1994), except for presence of an additional oxygen-bearing carbon at dC 85.1 in compound 5. The complete assignments of the 1H and 13C NMR spectroscopic data were made by a combination of COSY, NOESY, HMQC and HMBC spectra. Detailed inspection of the key COSY and HMBC correlations between the proton and carbon pairs are shown in Fig. 2. The double bond units were located at D7 and D22 based on the HMBC correlations, H-7 (dH 5.93)/C-5, C-8, C-14 (dC 85.1), H-22/C-20, -21, -23, -24, and H-23/

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C-20, -22, -24, -25, respectively. The geometry of D22 was assigned as E according to the coupling constant between H-22 and H-23 with J = 15.2 Hz. One hydroxyl group was located at C-14 (dC 85.1) on the basis of the HMBC correlation: H-7, -12, -15, -16, 18/C-14. The higher chemical shift of H3-18 at dH 0.70 and H3-19 at dH 1.05 indicated that the junctions of rings A/B and C/D should be trans (Kawahara et al., 1995). On the basis of the above spectroscopic data, compound 5 was assigned as (14a, 22E)-14-hydroxyergosta-7,22-diene-3,6-dione. The molecular formula (C21H32O3, m/z 332.2360 [M]+), the UV (kmax 216 and 256 nm) data, and the IR (3415 and 1746 cm1) data of compound 6 suggested that it contained hydroxyl and a,bunsaturated c-lactone groups. The 1H NMR spectrum (Table 3) displayed signals for three secondary methyl at dH 1.02 (d, J = 6.5 Hz), 0.83 (d, J = 7.0 Hz) and 0.97 (d, J = 7.0 Hz), one tertiary methyl at dH 0.58 (s), and an olefinic proton at dH 5.61 (s), together with resonances for a trans-disubstituted double bond at dH 5.26 (dd, J = 15.5, 7.0 Hz), and 5.21 (dd, J = 15.5, 7.0 Hz). The 13C NMR (Table 3) and DEPT spectra exhibited signals for four methyl groups, five methylene signals including one hydroxymethyl group at dC 66.7, nine methine resonances (including one oxygenated methine at dC 81.7, three olefinic carbons at dC 135.7, 131.3 and 111.2), and three quaternary carbons, including one carbonyl carbon at dC 173.8 and one olefinic carbon at dC 172.6. The 1H and 13C NMR spectra (Table 3) of 6 were similar to those of demethylincisterol A3 (6a), a degraded C21 steroid (Riccardis et al., 1995; Mansoor et al., 2005), except for an additional hydroxyl group at C-19 [dC 66.7 and dH 3.43 (dd, J = 10.8, 6.3 Hz) and 3.54 (dd, J = 10.8, 6.3 Hz)] and a C-4 proton instead of a hydroxyl group. In the HMBC spectrum (Fig. 2), the proton signals of the hydroxymethyl group (dH 3.43 and 3.54) were correlated to the carbon resonances at C17, 18 and 20 and also showed COSY (Fig. 2) correlation to H-18. This evidence suggested that the hydroxyl group is linked to C19 (Fig. 1). By comparison with the 1H NMR spectroscopic data of camphoratin I, the relative configuration at C-17 and C-18 in 6 may be assigned as erythro, (Wu et al., 2010) which was further verified as 17S,18S due to the down-field shift of H2-19 (dH 4.15) in its (R)-MTPA derivative. The 1H NMR signal at dH 5.61 (s, H-2) and the 13C NMR resonances at dC 173.8 (C-1), 172.6 (C-3), and 111.2 (C-2) suggested the presence of an a,b-unsaturated c-lactone, and the UV absorption at kmax 216 nm is additional proof.

Table 3 1 H NMR and 13C NMR spectroscopic data for compound 6 (CDCl3, d in ppm, J in Hz, 400 MHz for 1H NMR, 100 MHz for 13C NMR). Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1

H NMR

5.61 s 4.64 dd (9.4, 6.8) 2.37 m, 1.56 m 2.03 m, 1.40 m 2.30 1.71 1.85 1.41 0.58 2.04 1.02 5.21 5.26 2.20 1.57 3.43 0.83 0.97

dd (12.0, 7.0) m, 1.50 m m, 1.42 m m s m d (6.5) dd (15.5, 7.0) dd (15.5, 7.0) m m dd (10.8, 6.3) 3.54 dd (10.8, 6.3) d (7.0) d (7.0)

13

C NMR

173.8 s 111.2 d 172.6 s 81.7 d 30.7 t 35.6 t 48.0 s 52.1 d 21.5 t 28.9 t 55.2 d 12.2 q 40.1 d 21.0 q 135.7 d 131.3 d 38.0 d 40.7 d 66.7 t 12.8 q 18.4 q

The signal at dC 81.7 was assigned at C-4 based on the observed HMBC correlations, H-4 (dH 4.64)/C-2, -3; H-2 (dH 5.61)/C-1, -4, 8. The relative configuration of the degraded sterol nucleus was determined based on its NOESY correlations, which suggested key correlations depicted in Fig. 3. The H-4 was a-oriented, based on NOESY correlations from H-4 to the a-axial H-8 and from H-4 to H-5a and H-6a. Furthermore, the UV spectrum showed kmax (MeOH) values at 216 and 256 nm, while its CD spectrum exhibited a strong positive Cotton effect at kmax 252 nm, indicating that compound 6 had the S configuration at C-4 (Mansoor et al., 2005). The NOESY correlation of H-11/H-6a, -8 established that the side-chain was in a b-orientation and the correlation of H-13/H3-12 and the chemical shift of H3-14 (dH 1.02) also supported its 13R configuration (Mansoor et al., 2005). Further proof for the trans-fusion of the B and C rings was given by the following NOESY correlations; H312/H-5b, -9b and H-8/H-6a, -11. The double bond between C-2 and C-3 and the trans fusion of the C and D rings can cause a shift of H-12 up to dH 0.58, as seen in 5a-stigmasta-7-en-3b-ol (Akihisa et al., 2007). The phenomenon is additional evidence for the double bond location and the trans-junction of C and D rings. Based on these considerations, 6 was shown as the structure which was name camphosterol A. Seven known compounds were identified by comparison of their spectroscopic data with those reported in the literature as: methyl antcinate G (Cherng et al., 1996), ganoderic acid Sz (Li et al., 2005), ganoderic acid S (Morigiwa et al., 1986), lanosta8,24-diene-3,21-diol (Su et al., 2000), ganodermadiol (Arisawa et al., 1986), 5a,8a-epidioxy-6,9(11),22E-ergostatrien-3b-ol (Leslie et al., 1981), and ergosterol peroxide (Leslie et al., 1981). Compounds 1–6 were evaluated for cytotoxicity against several human tumor cell lines [Huh-7 (hepatocarcinoma), MCF-7 (human breast adenocarcinoma), A-2058 (human melanoma), HSC3 (human oral squamous cell carcinoma), B16F10 (melanoma) and SKOV3 (ovarian carcinoma)] (Table 4). The bioassay data showed that compounds 1, 2, 4, 5, and 6 have moderate cytotoxicity, whereas 3 was inactive (IC50 > 100 lM) in the tumor cell lines tested. 2.1. Concluding remarks In conclusion 13 compounds, including four lanostane triterpenes (1–4), one sterol (5), and one degraded sterol (6), were separated from the fruiting bodies and mycelia of a solid culture of A. camphorata. Compounds 1–4 and 6 had a bitter taste, but have not yet been evaluated quantitatively. Compounds 1–6 were assayed for their cytotoxicity against seven cancer cell lines. Although activities were moderate, the bioassay data obtained showed that a C-26 with an ethyl ester group in 4 exhibited greater available cytotoxicity, whereas that of 3, which possessed a C-26 with a methyl ester group, was inactive. These results suggest that the presence of an ethyl ester group at C-26 plays a role in mediating cytotoxicity. This topic is under further investigation.

2

12

3

9 10

8

7 13

5 11

4

6

3 Fig. 3. Key NOESY correlations (

) for compound 6.

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H.-C. Huang et al. / Phytochemistry 84 (2012) 177–183

Table 4 Cytotoxicity data for compounds 1–6 against tumor cells. Compounds

1 2 3 4 5 6 Mitomycin Cb a b

IC50 (lM) Huh-7

MCF-7

A-2058

HSC-3

B16F10

B16F1

SKOV3

()a () () 37.00 38.66 43.03 20.01

() 40.25 () 35.02 () 77.59 7.74

() 20.43 () 26.91 29.01 31.10 4.63

() 29.62 () () 29.94 70.15 7.17

70.83 20.35 () 33.31 27.90 26.69 11.37

() 44.16 () 43.58 () () 6.02

() () () () () () 34.07

(): Inactive, IC50 > 100 lM. Positive control.

3. Experimental 3.1. General experimental procedures Melting points were determined with a Yanagimoto micromelting point apparatus and are uncorrected. UV spectra and specific optical rotations were determined using a Hitachi S-3200 spectrometer and a JASCO DIP-180 digital polarimeter, respectively. IR spectra were recorded on a Perkin-Elmer 983 G spectrometer. CD spectra were obtained using a JASCO J-810 spectropolarimeter. 1 H and 13C NMR spectra were recorded on a Bruker DMX-400 spectrometer. EIMS and HREIMS were measured with JEOL Finnigan TSQ-46C and JEOL SX-102A mass spectrometers. Extracts were purified by silica gel column chromatography (CC) (Merck 70– 230 mesh and 230–400 mesh) and purified on a semi-preparative reversed-phase HPLC column [250  10 mm, Cosmosil 5C18-AR II, pore size: 5 lm] monitored with a RI detector, LCD Refracto Monitor III. 3.2. Plant material The solid cultural fruiting bodies and mycelia of A. camphorata were provided from Well Shine Biotechnology Development and identified by Prof. Sheng-Yang Wang (Department of Forestry, National Chung-Hsing University, Taichung, Taiwan). A voucher specimen (No. CMU-AC200706) was deposited at School of Chinese Pharmaceutical Sciences of Chinese Medicine Resources, China Medical University, Taichung, Taiwan. 3.3. Extraction and isolation The dried powder of fruiting bodies and mycelia from a solid culture of A. camphorata (3.0 kg) were extracted with MeOH (12 L) at room temperature (5 days  2), and the MeOH extracts were then evaporated to give a residue (288.0 g). The latter was successively partitioned between H2O, EtOAc, and n-BuOH. The EtOAc extract (161.6 g) was then subjected to silica gel CC (10  70 cm, Merk 70–230 mesh), eluting with n-hexane/EtOAc in a gradient to give 10 fractions. Fraction 3 [n-hexane–EtOAc (5:1, v/v)] was subjected to Sephadex LH-20 CC (10  70 cm) eluting with MeOH to yield six subfractions. Subfraction 4 of the EtOAc fraction 3 was subjected to semi-preparative reversed-phase HPLC, eluting with isocratic MeOH–H2O (80:20) to yield 2 (3.8 mg), 5 (1.7 mg), and methyl antcinate G (2.2 mg). Subfraction 5 of fraction 3 was further purified by semi-preparative reversed-phase HPLC with isocratic MeOH–H2O (70:30) to yield ganoderic acid Sz (2.4 mg), ganoderic acid S (4.6 mg) and ganodermadiol (3.2 mg). Fraction 4 [n-hexane–EtOAc (7:3, v/v)] was further separated by Sephadex LH-20 (10  70 cm) CC with MeOH to yield six subfractions. Subfraction 4 of fraction 4 was further purified using semipreparative reversed-phase HPLC eluted with MeOH–H2O (70:30)

to yield 1 (2.4 mg), 3 (6.0 mg), 4 (3.6 mg), and 6 (3.5 mg). Using semi-preparative reversed-phase HPLC with MeOH–H2O (65:35), lanosta-8,24-diene-3,21-diol (8.4 mg), 5a,8a-epidioxy6,9(11),22E-ergostatrien-3b-ol (4.6 mg), and ergosterol peroxide (2.1 mg) were obtained from subfraction 5 of fraction 4. 3.3.1. 3,7,11-Trioxo-5a-lanosta-8,24(E)-dien-26-oic acid (1) Colorless needle; ½a25 D +13.8 (c 0.04, MeOH); mp: 96–98 °C; UV (MeOH) kmax (log e) 216 (4.16) and 268 (3.96) nm; IR (KBr) mmax 3300–2500, 2970, 2882, 1712, 1690 and 1681 cm1; CD (c 5.0  105 M, CH3OH) De252 +1.30; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS m/z 482.3038 [M]+ (calcd for C30H42O5, 482.3032). 3.3.2. Methyl 11a-hydroxy-3,7-dioxo-5a-lanosta-8,24(E)-dien-26oate (2) Colorless needle; ½a25 D +2.3 (c 0.09, MeOH); mp: 97–99 °C; UV (MeOH) kmax (log e) 220 (4.45) and 248 (4.25) nm; IR (KBr) mmax 3492, 2955, 2881, 1711 and 1667 cm1; CD (c 5.0  105 M, CH3OH) De252 +0.49; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS m/z 498.3360 [M]+ (calcd for C31H46O5, 498.3345). 3.3.3. Methyl 3,7,11,12,15,23-hexaoxo-5a-lanost-8-en-26-oate (3) Yellow needles; ½a25 D +4.7 (c 0.06, MeOH); mp: 172–174 °C; UV (MeOH) kmax (log e) 206 (3.80), 224 (3.74) and 258 (3.77) nm; IR (KBr) mmax 2977, 2950, 1750, 1735, 1709 and 1689 cm1; CD (c 5.0  105 M, CH3OH) De252 +0.15; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS m/z 540.2733 [M]+ (calcd for C31H40O8, 540.2723). 3.3.4. Ethyl 3,7,11,12,15,23-hexaoxo-5a-lanost-8-en-26-oate (4) Pale yellow solid; ½a25 D +4.4 (c 0.03, MeOH); mp: 96–98 °C; UV (MeOH) kmax (log e) 204 (3.81), 224 (3.75) and 261 (3.77) nm; IR (KBr) mmax 2978, 2938, 1745, 1731, 1712 and 1688 cm1; CD (c 5.0  105 M, CH3OH) De252 +0.78; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS m/z 554.2878 [M]+ (calcd for C32H42O8, 554.2880). 3.3.5. (14a,22E)-14-Hydroxyergosta-7,22-diene-3,6-dione (5) Colorless needle; ½a25 D +4.8 (c 0.02, MeOH); mp: 215–217 °C; UV (MeOH) kmax (log e) 240 (4.05), 266 (3.41), and 292 (2.83) nm; IR (KBr) mmax 3493, 2956, 2868, 1718 and 1662 cm1; CD (c 5.0  105 M, CH3OH) De252 +1.40; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS m/z 426.3142 [M]+ (calcd for C28H42O3, 426.3136). 3.3.6. Camphosterol A (6) Colorless gum; ½a25 D +17.1 (c 0.02, MeOH); UV (MeOH) kmax (log e) 216 (4.26) and 256 (3.57) nm; IR (KBr) mmax 3415, 2960, 2876, 1746, 1650 cm1; CD (c 1.20  104 M, CH3OH) De252

H.-C. Huang et al. / Phytochemistry 84 (2012) 177–183

+5.00; see Tables 3 for 1H and 13C NMR spectroscopic data; HREIMS m/z 332.2360 [M]+ (calcd for C21H32O3, 332.2351). 3.3.7. Preparation of (R)-MTPA ester (6b) of 6 To a solution of 6 (0.5 mg) in dry pyridine (0.5 mL) was added (S)-MTPA chloride (15 lL), and the solution was allowed to stand at room temperature for 3 h. After the reaction mixture was concentrated under a stream of N2, the residue was partition between CHCl3 and H2O. The CHCl3 layer was subjected to silical gel CC using n-hexane–EtOAc (3:1) as eluent to yield the (R)-MTPA ester, 6b (0.6 mg). Selective 1H NMR (CDCl3, 500 MHz) data of 6b: dH 7.30–7.48 (5H, m, Ph), 5.20 (1H, dd, J = 15.0, 7.0 Hz, H-16), 5.22 (1H, dd, J = 15.0, 7.0 Hz, H-15), 4.15 (2H, br d, J = 6.5 Hz, H-19), 3.55 (3H, s, OCH3), 1.02 (3H, d, J = 6.5 Hz, H-21), 0.82 (3H, d, J = 6.5 Hz, H-20), and 0.56 (3H, s, H-12). 3.4. Cytotocixity The human oral squamous cell carcinoma (HSC-3), murine melanoma (B16F1 and B16F10), hepatocarcinoma (Huh-7), ovarian carcinoma (SKOV3), human breast adenocarcinoma (MCF-7), and human melanoma (A-2058) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS, 2 mM of glutamine, and 1% penicillin–streptomycin-neomycin at 37 °C in a humidified incubator with 5% CO2. Cultures were harvested and monitored for changes in cell number by counting cell suspensions using a haemocytometer with phase-contrast microscopy. Cell viability was monitored by the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) colorimetric assay. Briefly, cells (5  105 cells/24-well dish) were treated with up to 30 lM samples or standard agent (mytomycin C) for 24 h before 400 lL of 0.5 mg/mL MTT in PBS was added to each well. After incubation at 37 °C for 2 h, an equal cell culture volume of 10% SDS (400 lL) was added to dissolve the MTT formazan, and the absorbance was measured at 570 nm (A570). The cell viability (%) was calculated as: (A570 of treated cells/A570 of untreated cells)  100. Acknowledgements This research was supported by the China Medical University (CMU97-217 CMU-222), the National Science Council of the Republic of China, and in part by the Taiwan Department of Health, Clinical Trial (DOH 100-TD-B-111-004 and DOH 101-TD-B-111004). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2012. 08.011. References Akihisa, T., Nakamura, Y., Tagata, M., Tokuda, H., Yasukawa, K., Uchiyama, E., Suzuki, T., Kimura, Y., 2007. Anti-inflammatory and anti-tumor-promoting of triterpene

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