Lanostane triterpenes from cultures of the Basidiomycete Ganoderma orbiforme BCC 22324

Lanostane triterpenes from cultures of the Basidiomycete Ganoderma orbiforme BCC 22324

Phytochemistry 87 (2013) 133–139 Contents lists available at SciVerse ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytoch...

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Phytochemistry 87 (2013) 133–139

Contents lists available at SciVerse ScienceDirect

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

Lanostane triterpenes from cultures of the Basidiomycete Ganoderma orbiforme BCC 22324 Masahiko Isaka ⇑, Panida Chinthanom, Surisa Kongthong, Kitlada Srichomthong, Rattaket Choeyklin National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand

a r t i c l e

i n f o

Article history: Received 22 February 2012 Received in revised form 18 August 2012 Available online 29 December 2012 Keywords: Plant pathogenic mushroom Ganoderma orbiforme Lanostanes Antimycobacterial activity Antiplasmodial activity

a b s t r a c t Seven lanostane triterpenoids, ganorbiformins A–G, together with twelve known compounds, were isolated from cultures of the mushroom fungus Ganoderma orbiforme BCC 22324. Ganorbiformin A is an unusual rearranged analog, whereas the other compounds share the same lanostane skeleton with known ganoderic acids. The C-3 epimer of ganoderic acid T also exhibited significant antimycobacterial activity against Mycobacterium tuberculosis H37Ra (MIC 1.3 lM). Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

2. Results and discussion

The mushroom Ganoderma lucidum is a well-known Chinese crude drug widely used in Asian countries (Paterson, 2006). This fungus is a prolific source of highly oxygenated lanostane triterpenoids, such as ganoderic acids, that have been isolated both from fruiting bodies and cell cultures (Cole and Schweikert, 2003). Several other species, Ganoderma applanatum (Shim et al., 2004; de Silva et al., 2006; Wang and Liu, 2008), Ganoderma amboinense (Li et al., 2005), Ganoderma colossum (El Dine et al., 2008), and Ganoderma carnosum (Keller et al., 1997) are also known as producers of similar triterpenoids. As part of our research program on utilization of fungal sources in Thailand, the relatively rare species Ganoderma orbiforme, strain BCC 22324, was investigated. A mycelial extract from cell culture of this fungus displayed a complex 1H NMR profile, which suggested the occurrence of many terpenoids. In Thailand, this species has been found in the south area as an oil palm pathogen. To our knowledge, there has been no previous report on the chemical constituents from natural fruiting bodies or cell cultures of this species. Scale-up fermentation and chemical studies of BCC 22324 led to the isolation and characterization of seven new lanostane triterpenoids, ganorbiformins A–G (1–7), along with twelve known ganoderic acid derivatives (8–19) (Fig. 1).

Ganorbiformin A (1) was isolated as a colorless solid, and its molecular formula was determined to be C32H48O8 by HRESIMS. The IR spectrum exhibited a broad absorption band of hydroxy groups at mmax 3418 cm 1 and the intense overlapping carbonyl absorption bands at 1730–1691 cm 1. The 1H and 13C NMR spectroscopic data in CDCl3 suggested that 1 was a triterpenoid bearing one acetoxy group and the skeleton was similar to the known lanostane co-metabolites 8–19. The 1H and 13C NMR, DEPT, and HMQC data for 1 supported the presence of an aliphatic ketone (dC 216.6), two carboxyl or ester groups (dH 172.0 and 171.3), three sp2 quaternary carbons (dC 155.0, 132.9, and 129.8), an sp2 methine (dC 138.5/dH 6.74), two oxygenated quaternary carbons (dC 77.5 and 76.3), two oxymethines (dC 74.6/dH 5.03 and dC 66.2/dH 5.25), three sp3 quaternary carbons, three methines, seven methylenes, and eight methyl groups, respectively (Tables 1 and 2). Structural elucidation of 1 was accomplished by analyses of COSY and HMBC data (Fig. 2). Key HMBC data were the 2J correlations from six singlet-signal methyl groups (H3-18, H3-19, H3-27, H3-28, H3-29, and H3-30) to their attached quaternary sp3 carbons C-13, C-10, C-25, C-4, C-4, and C-15, respectively, and their 3J correlations. The significant structural difference with other lanostane derivatives was the location of CH3-30, which is attached to C-15 in 1. The C-3 ketone (dC 216.6) was assigned on the basis of the HMBC correlations from Hb-2, H3-28 and H3-29 to this carbon. A tetrasubstituted olefin was assigned to C-8/C-14 by the HMBC correlations from Ha-6 and Ha-11 to C-8 (dC 132.9), and the correlations from Hb-16, H3-18, and H3-30 to C-14, respectively. The chemical shifts

⇑ Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707. E-mail address: [email protected] (M. Isaka). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2012.11.022

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Fig. 1. Structures of compounds 1–19.

of protons and carbons for the C-20–C-27 side-chain were very similar to those of known 22-acetoxy-derivatives 10, 12–17, and 19, which strongly suggested the same 20R and 22S configurations and 24E olefinic geometry for 1. The 24E configuration was further supported by the NOESY correlation of H2-23/H3-27. While the NMR spectroscopic data taken in CDCl3 were useful for comparison of the side-chain structure with those of the cometabolites, the 1H NMR spectrum in DMSO-d6 exhibited better peak dispersions and more informative NOESY data was obtained. In addition, a singlet OH signal was observed at dH 4.83, which was assigned to 9-OH on the basis of its HMBC correlations to C-8 and C-11. Analysis of the NOESY correlations from H-5 (axial) to Ha-1 (axial) and 9-OH established their relationship. Significant downfield shifts for Ha-1 (dH 2.00 in DMSO-d6; 2.19 in CDCl3) and H-5 (dH 2.37 in DMSO-d6; 2.52 in CDCl3), when compared with other analogs, can be explained by deshielding of these axial protons by the 9a-OH group. NOESY correlations from H3-19 to Hb-1 (equatorial), Hb-2 (axial), Hb-11 (axial), and the correlations from H3-18 to Hb-11 and Hb-12 (equatorial) were indicative of their being in borientations. The oxymethine H-7 resonated as a singlet signal with a narrow peak width (small coupling constants with Ha-6 and Hb-6), which indicated an equatorial (b) orientation. Intense NOESY cross-peaks for H3-18/H-20 and Hb-12/H3-21 were consistent with the 17R and 20S configurations. NOESY correlations Hb16/H-22 and Ha-16/H3-30 suggested the a-face orientation of CH3-30. The unusual downfield chemical shift of H-7 (dH 5.33 in DMSO-d6; 5.25 in CDCl3) can be explained by deshielding of this proton by the 15b-OH group. The lack of NOESY correlation between H-7 and H3-30 was consistent with a 15R configuration. A b-CH3-30 (15S) would be expected to show a strong NOESY correlation with H-7. A possible mechanism to account for the formation of 1 is the C15 alcohol oxidation of 20 to the ketone 21, whose methyl group (CH3-30) on the a-face migrated to the neighboring ketone carbon

(C-15) under acid catalysis (Scheme 1). In the present study, two compounds, 20 and its 7-O-acetate, were also isolated whose structures were suggested by analysis of their 1H NMR spectra. However, each compound in NMR solvent (CDCl3) was converted to the same 7,9(11)-diene 12. A similar elimination reaction of 7a-OMe ganoderic acid derivatives under acidic conditions was previously reported (Nishitoba et al., 1987a,b,c). Ganorbiformin B (2) was assigned the molecular formula C34H50O7 by HRESIMS. The 1H and 13C NMR spectra displayed similarity to those for known ganoderic acids and suggested the presence of two acetoxy groups and an enone (dC 198.6) functionality. Locations of two acetoxy groups were assigned to C-3 and C-22 positions on the basis of COSY data, and these assignments were further confirmed by the HMBC correlations from the oxymethine protons H-3 and H-22 to the carbonyl carbons of the acetyl group at dC 170.8 and 170.6, respectively. The coupling constant values of H-3 (dd, J = 11.8, 4.3 Hz), including a trans diaxial coupling to Hb-2, indicated an axial (a) orientation. The enone was assigned by the HMBC correlations: from Ha-6 and Hb-6 to the ketone carbon (C7); from H3-30 to C-8; and from H3-19 to C-9. The 1H and 13C NMR spectroscopic data were consistent with those of lucidadiol and lucidal (González et al., 1999) which possess the same ring ABCD structure. The molecular formula of ganorbiformin C (3) was determined by HRESIMS as C30H48O6. The 1H and 13C NMR spectra of 3 exhibited closely related signals to those of 2. The differences were the absence of the resonances for one acetyl group and the upfield shift of H-3 (dH 3.28) when compared to 2 (dH 4.51). Thus, ganorbiformin C (3) was identified as the 3-O-deacetyl analog of 2. Ganorbiformin D (4) had the molecular formula C34H50O8 (HRESIMS). The 1H and 13C NMR spectroscopic data demonstrated that the C-20–C-27 side-chain and ring D structures were the most typical ones for ganoderic acids with two acetoxy groups at C-15 and C-22 positions (Table 3). Other key functional groups were an

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M. Isaka et al. / Phytochemistry 87 (2013) 133–139 Table 1 13 C (125 MHz) NMR spectroscopic data for ganorbiformins A–G (1–7), 8, and 10 in CDCl3.

a–e f–j

No.

1

2

3

4

5

6

7

8

10

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 3-OCOCH3 3-OCOCH3 7-OCH3 15-OCOCH3 15-OCOCH3 22-OCOCH3 22-OCOCH3

31.0 34.6 216.6 47.1 39.7 28.8 66.2 132.9 76.3 41.4 26.2 34.2 45.2 155.0 77.5 47.9 49.7 17.2 17.0 37.8 13.3 74.6 31.6 138.5 129.8 172.0 12.5 25.8 22.0 31.4

34.5 23.8a 79.6 37.8 49.9 36.4 198.6 138.9 164.4 39.6 23.6a 30.1 44.9 47.8 31.9 28.5 45.6 15.6 18.5 39.5 13.1 74.8 31.8 139.4 129.0 171.3 12.3 27.4 16.3 25.1 170.8 21.2

34.8 27.4f 77.9 38.9 49.8 36.6 198.9 138.8 164.6 39.8 23.6 30.1 44.9 47.8 31.9 28.5 45.7 15.6 18.4 39.5 13.1 74.8 31.8 139.4 129.0 171.2 12.3 27.4f 15.3 25.0

35.2 34.2 217.2 46.6 44.7 28.4 66.1 134.6 140.1 38.1 20.7 31.2 45.1 51.2 75.9 36.1 45.9 16.4 17.3 39.9 12.6 74.3 31.9 138.8 129.5 171.9 12.3 26.5 21.2b 20.1

35.3 34.3 217.4 46.7 45.0g 30.0d 66.7 136.4 139.4 37.9 21.3 31.0 45.0g 49.7 29.9d 27.9 47.1 16.0 17.3 39.7 12.8 74.7 31.8 139.6 129.2 172.0 12.3 26.5 21.3 26.1

35.3 34.3 217.4 46.7 45.0 23.3 76.1 135.3 139.5h 37.8 21.0i 31.1 44.9 49.9 30.1 27.8 47.2 16.0 17.4 39.7 12.8 74.7 31.8 139.5h 129.0 171.7 12.3 26.5 21.3 25.4

36.6 34.8 216.9 47.5 50.7 23.7 120.3 142.5 144.6 37.2 116.9 37.8 43.7 50.3 31.4 27.6 47.4 15.5 21.1 39.4 12.7 74.7 31.9 139.5 129.1 171.9 12.3 25.3 22.5 25.5

35.2 34.3 217.2 46.6 44.8 28.4 66.2 134.8 140.1 38.1 20.8 31.2 45.3 51.2 76.4 36.4 49.3 16.6 17.3 36.2 18.2 34.6 25.9 144.9 126.7 171.2 12.1 26.5 21.2e 20.1

36.6j 34.8 216.4 47.4 50.4 23.7 121.3 140.2 144.7 37.3 116.7 38.0 43.9 51.4 76.7 36.6j 45.5 15.8 22.1 39.6 12.7 74.4 31.9 139.0 129.2 171.3 12.3 25.4 22.4 18.3

170.6 21.1e

170.6 21.0

170.5c 21.1b 170.6c 21.0b

171.1 21.4 170.6 21.0

55.8

171.3 21.2

170.6 21.0

170.7 21.1

170.6 21.0i

170.7 21.1

The carbon assignment may be interchanged. The carbon resonances are superimposed.

Table 2 1 H (500 MHz, CDCl3) NMR spectroscopic data for ganorbiformins A–C (1–3). No.

1

2

3

1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OCOCH3 22-OCOCH3

a 2.19, dt (4.4, 13.4); b 1.69, m a 2.37, m; b 2.63, m

a 1.52, m; b 1.83, m a 1.78, m; b 1.69, m

a 1.43, m; b 1.84, m a 1.75, m; b 1.68, m

4.51, dd (11.8, 4.3) 1.71, dd (13.3, 3.9) a 2.38, m; b 2.42, m

3.28, dd (11.6, 4.4) 1.62, dd (12.7, 4.6) a 2.41, m; b 2.43, m

a 2.34, m; b 2.26, m a 1.76, m; b 1.74, m a 2.07, m; b 1.70, m a 2.03, m; b 1.32, m

a 2.33, m; b 2.28, m a 1.77, m; b 1.74, m a 2.05, m; b 1.72, m a 2.03, m; b 1.31, m

1.55, 0.64, 1.18, 1.53, 0.99, 5.09, 2.57, 6.80, 1.86, 0.88, 0.95, 0.89, 2.07, 2.05,

1.54, 0.64, 1.17, 1.54, 0.99, 5.10, 2.57, 6.80, 1.86, 0.98, 0.88, 0.89,

2.52, br d (13.4) a 1.84, m; b 1.84, m 5.25, br s a 1.47, m; b 1.79, a 1.70, m; b 1.81, m

a 2.10, m; b 1.68, m 1.68, 0.89, 1.03, 1.60, 1.07, 5.03, 2.56, 6.74, 1.84, 1.14, 1.09, 1.53,

m s s m d (6.1) m m; 2.34, m m br s s s s

2.05, s

aliphatic ketone (dC 217.2) and an allylic alcohol. The C-3 ketone was assigned by analysis of the HMBC correlations from Ha-1, Hb-1, Hb-2, H3-28, and H3-29 to the carbonyl carbon. The location

m s s m d (6.2) t (7.2) m; 2.35, m dd (7.3, 7.0) br s s s s s s

m s s m d (6.7) t (7.1) m; 2.35, m dd (7.5, 7.0) br s s s s

2.04, s

of the allylic alcohol was determined by HMBC correlations from H-7, Hb-11, H-15, and H3-30 to C-8, and from H-5, H-7, Hb-11, Hb-12, and H3-19 to C-9, respectively. The oxymethine H-7

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Ganorbiformin F (6), possessing a molecular formula C33H50O6 (HRESIMS), was structurally closely related to 5. It was assigned as the 7-O-methyl derivative of 5. An intense HMBC correlation from the methoxy protons (7-OCH3, dH 3.30) to C-7 (dC 66.2) confirmed the location of the methoxy group. Similarly to 4 and 5, H-7 exhibited very small coupling constant values (dH 3.66, br s), which confirmed a 7a-methoxy group. Ganorbiformin F (6) is probably an artifact formed from the 7a-hydroxy or 7a-acetoxy derivative during the mycelial extraction with methanol. Similar 7a-methoxy derivative, 7-O-methyl-ganoderic acid O (9), was previously isolated from the cultured mycelium of Ganoderma lucidae (Hirotani et al., 1987). This original paper also describes that 9 was obtained by methanol extraction, and it was not present in the benzene extract. Ganorbiformin G (7) had the molecular formula C32H46O5 as determined by HRESIMS. The 1H and 13C NMR spectroscopic data were similar to those of the known 7,9(11)-diene-type lanostanes 10–19. Interpretation of the 2D NMR (COSY, HMQC, and HMBC) data indicated that an acetoxy group was attached to C-22, while C-15 was a methylene (dC 31.4). The C-3 ketone (dC 216.9) functionality was assigned by the HMBC correlations from Hb-1, Hb-2, H3-28, and H3-29 to this carbon. Compound 8 was identified on the basis of HRMS and 2D NMR spectroscopic data as ganoderic acid V. Since it was characterized

Fig. 2. COSY and key HMBC correlations for ganorbiformin A (1).

resonated as a broad singlet, which indicated its pseudoequatorial (b) orientation. The NMR spectroscopic data of ganorbiformin E (5) were similar to those of 4, but differed in the ring D moiety. An additional methylene carbon was present, replacing the oxymethine (C-15) in 4. Therefore, it was identified as the 15-deacetoxy analog of 4.

Scheme 1. Plausible biogenetic pathway to 1.

Table 3 1 H (500 MHz, CDCl3) NMR spectroscopic data for ganorbiformins D–G (4–7). No.

4

5

6

7

1

a 1.93, m; b 1.70, m

a 1.96, m; b 1.69, m

a 1.93, m; b 1.70, m

2 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 7-OCH3 15-OCOCH3 22-OCOCH3

a 2.50m; b 2.53, m

a 2.49, m; b 2.54, m

a 2.48, m; b 2.50, m

2.05, m 1.7 3–1.71, m 4.15, br s a 2.08, m; b 2.12, m a 1.89, m; b 1.64, m 5.13, dd (9.5, 6.0) a 1.89, m; b 2.16, m 1.78, m 0.71, s 1.03, s 1.51, m 0.96, d (6.7) 5.02, t (7.0) 2.56, m; 2.33, m 6.77, dd (7.3, 6.8) 1.85, br s 1.11, s 1.06, s 1.12, s

1.97, m a 1.67, m; b 1.79, 4.25, br s 2.11–2.09, m a 1.80, m; b 1.66, a 1.70, m; b 1.52, a 1.38, m; b 2.06, 1.67, m 0.62, s 1.04, s 1.53, m 0.98, d (6.7) 5.09, t (7.1) 2.57, m; 2.38, m 6.80, t (7.0) 1.86, br s 1.12, s 1.07, s 1.03, s

a 1.76, dt (4.5, 13.9) b 2.27, ddd (13.3, 5.3, 3.1) a 2.34, m; b 2.77, dt (5.7, 14.5) 1.53, dd (12.0, 3.6) a 2.05, m; b 2.20, m 5.51, br d (6.6) 5.38, br d (6.1) a 2.21, m; b 2.09, m a 1.64, m; b 1.41, m a 1.33, m; b 2.04, m 1.70, m 0.57, s 1.19, s 1.55, m 0.98, d (6.7) 5.10, t (7.0) 2.57, m; 2.37, m 6.81, t (7.0) 1.86, br s 1.08, s 1.12, s 0.85, s

2.09, s 2.06, s

2.05, s

1.99, dd (13.0, 1.7) m

m m m

a 1.85, m; b 1.48, m 3.66, br s 2.10–2.09, m a 1.81, m; b 1.67, m a 1.68, m; b 1.38, m a 1.37, m; b 2.06, m 1.69, m 0.62, s 1.04, s 1.51, m 0.98, d (6.7) 5.10, t (7.0) 2.57, m; 2.37, m 6.81, t (7.2) 1.87, br s 1.11, s 1.08, s 1.02, s 3.30, s 2.05, s

2.06, s

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M. Isaka et al. / Phytochemistry 87 (2013) 133–139 Table 4 Biological activities of compounds 1, 4–7, 12, 13, 15, 17, and 19, and standard compounds. Compound

Ganorbiformin A (1) Ganorbiformin D (4) Ganorbiformin E (5) Ganorbiformin F (6) Ganorbiformin G (7) 12 13 Ganoderic acid T (15) Ganoderic acid P (17) Ganoderic acid S (19) Doxorubicin hydrochloride Ellipticine Dihydroartemisinin Isoniazid a b

Cytotoxicity (IC50, lM) NCI-H187

MCF-7

KB

Vero

>89 >85 70 44 65 34 13 15 26 39 0.083 – – –

>89 >85 >95 >96 >98 >95 51 78 58 >98 14 – – –

>89 >85 >95 63 65 >95 13 18 40 53 1.0 – – –

>89 >85 >95 36 35 >95 16 28 >88 >98 – 4.1 – –

Anti-malariaa (IC50, lM)

Anti-TBb (MIC, lM)

>18 >17 >19 >19 >20 >19 4.6 5.5 17 >20 – – 0.0021 –

>89 >85 >95 96 >98 >95 1.3 10 >88 98 – – – 0.17–0.34

Antimalarial activity against Plasmodium falciparum K1. Antitubercular activity against Mycobacterium tuberculosis H37Ra.

after conversion to the methyl ester derivative as described in the original report (Toth et al., 1983), the 13C NMR data of the free carboxylic acid form is reported herein (Table 1). The 13C NMR spectroscopic data of 10 are also listed in Table 1, as they were not reported in previous literature (Yang et al., 2002). Other known compounds were identified by comparison of the NMR and MS data with those of literature values; 7-O-methyl-ganoderic acid O (9) (Hirotani et al., 1987), 11 (Lin et al., 1988), 12 (Yang et al., 2002), 13 (Shiao et al., 1988b), 14 (Shiao et al., 1988a), ganoderic acid T (15) (Hirotani et al., 1986), ganoderic acid R (16) (Hirotani et al., 1986), ganoderic acid P (17) (Hirotani et al., 1987), ganoderic acid X (18) (Toth et al., 1983; Li et al., 2005), and ganoderic acid S (19) (Hirotani et al., 1986), respectively. Compounds 1, 4–7, 12, 13, 15, 17, and 19 were subjected to our bioassay protocols: cytotoxicity to three cancer cell-lines (NCIH187, MCF-7 and KB) and nonmalignant Vero cells, antimalarial activity against Plasmodium falciparum K1, and antitubercular activity against Mycobacterium tuberculosis H37Ra (Table 4). Other isolated compounds were not tested due to sample limitation. Compounds (1 and 4–7) and known compounds (12, 17, and 19) were either inactive or exhibited very weak activities in these assays. In contrast, ganoderic acid T (15) and its C-3 epimer 13 exhibited antimalarial and antitubercular activities as well as cytotoxicity. The study of the biological activities of the ganoderic acids have been extensively done (Paterson, 2006; Shi et al., 2010; Xu et al., 2010). Antiplasmodial activity of lanostanes isolated from G. lucidum mushroom was recently reported (Adams et al., 2010). To our knowledge, antitubercular activity of these lanostane triterpenoids from Ganoderma has not been previously reported. 3. Conclusions The present results demonstrate that the minor species G. orbiforme is also a rich source of Ganoderma lanostanoids including a novel rearranged analog, ganorbiformin A (1). The potent antitubercular activity of 13 (MIC 1.3 lM) and its relatively weaker cycotoxicity to noncancerous Vero cells (IC50 16 lM) are noteworthy and deserve further biological evaluation. 4. Experimental 4.1. General procedures Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded

on a GBS Cintra 404 spectrophotometer. FTIR spectra were taken on Bruker VECTOR 22 and ALPHA spectrometers. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. 4.2. Fungal material The fungus used in this study was isolated from a dead oil palm (Elaeis guineensis) trunk in plantation area, Ban Nuea Khlong Village, Krabi Province, Thailand, on May 4, 2006. The mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 19071, and the living culture was deposited in the BIOTEC Culture Collection on July 27, 2006, as BCC 22324. This fungus was identified as G. orbiforme (Fr.) Ryvarden (2000) based on the morphological characteristics: basidiocarps perennial, sessile and broadly attached, corky to woody, upper surface shiny laccate, light brown or deep reddish or chestnut brown, becoming darker by age; pore surface creamy white at first, later ochraceous to pale brown, with rounded pores; hyphal system dimitic, with generative hyphae hyaline, thin-walled, and skeletal hyphae yellowish brown, thick-walled; cuticle cells club-like with irregular lobes, brown, thick-walled, amyloid; basidiospores broadly ellipsoid, truncated, pale brown, finely echinulate,10–12.5  4–5 lm. Finally, this identification was confirmed by the sequence data of the ITS rDNA (GenBank accession number: JX997990). 4.3. Fermentation and isolation The fungus BCC 22324 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into 8  250 ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l). After incubation at 25 °C for 4 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1000 ml Erlenmeyer flask containing 250 ml of the same liquid medium (PDB), and incubated at 25 °C for 4 days on a rotary shaker (200 rpm). The secondary cultures were pooled and each 25 ml portion was transferred into 80  1000 ml Erlenmeyer flasks containing 250 ml of malt extract broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose 1.8 g/l, dextrose 6.0 g/l), and the final fermentation was carried out at 25 °C for 20 days under static conditions. The cultures were filtered to separate broth and mycelia (residue). The broth was extracted with EtOAc (2  15 l) and concentrated under reduced pressure to obtain a brown gum (broth extract, 626 mg). The wet mycelia were macerated in MeOH (4.5 l, 25 °C, 2 days) and filtered.

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Hexanes (4 l) and H2O (300 ml) were added to the filtrate, and the layers were separated. The H2O/MeOH (bottom) layer was partially concentrated by evaporation, and the residue was extracted with EtOAc (2.3 l), which was concentrated under reduced pressure to obtain a brown gum (mycelial extract, 4.35 g). The mycelial extract was passed through a column on Sephadex LH-20 (3.7  58 cm) and eluted with MeOH. The terpenoids-containing fractions were combined (3.45 g) and it was subjected to column chromatography (CC) on silica gel (4.8  18 cm, EtOAc/CH2Cl2, step gradient elution) and the fractions were further fractionated and purified by silica gel CC (EtOAc/hexanes or MeOH/CH2Cl2) and preparative HPLC using a reversed phase column (Phenomenex Luna 10u C18(2) 100A, 21.2  250 mm, 10 lm; mobile phase MeCN/H2O, proportions 50:50–85:15; detection UV 210 and 254 nm) to furnish pure compounds: 1 (23 mg), 2 (4 mg), 3 (8 mg), 4 (102 mg), 5 (17 mg), 6 (29 mg), 7 (51 mg), 8 (12 mg), 10 (8 mg), 11 (2 mg), 12 (41 mg), 13 (14 mg), 14 (5 mg), 15 (49 mg), 17 (30 mg), and 19 (12 mg), respectively. A fraction (20 mg) assigned as 20 by 1H NMR (CDCl3) analysis was converted to 13 during the sample recovery. The broth extract (626 mg) was fractionated by CC on Sephadex LH-20 (MeOH). Analysis of the 1H NMR spectroscopic data of the fractions and the crude extract indicated that the major components of the broth extract were fatty acids and the fractions did not contain triterpenoids. Another fermentation batch (28  250 ml) was also examined. The metabolite profile (1H NMR) of the mycelial extract (1.30 g) was similar to the previous sample, but there were small differences in lanostanoid composition. Chromatographic fractionations using the similar methods as described above gave 9 (6 mg), 16 (1 mg), and 18 (1 mg), which were not obtained from the previous sample, along with 1 (8 mg), 4 (40 mg), 5 (8 mg), 6 (4 mg), 7 (8 mg), 8 (3 mg), 10 (2 mg), 13 (2 mg), 15 (13 mg), 17 (10 mg), and 19 (4 mg). 4.3.1. Ganorbiformin A (1) Colorless solid; [a]26D 4 (c 0.11, CHCl3); IR (ATR) mmax 3418, 2967, 1730 sh, 1700 sh, 1691, 1373, 1237 cm 1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 2 and 1; 1H NMR (400 MHz, DMSO-d6) d 6.52 (1H, dd, J = 9.4, 6.4 Hz, H-24), 5.33 (1H, br s, H-7), 4.89 (1H, t, J = 7.1 Hz, H-22), 4.83 (1H, br s, 9-OH), 2.65 (1H, dt, J = 5.7, 14.2 Hz, Hb-2), 2.45 (1H, m, Ha-23), 2.37 (1H, m, H-5), 2.35 (1H, m, Hb-23), 2.17 (1H, m, Ha-2), 2.00 (1H, m, Ha-1), 1.99 (3H, s, acetyl), 1.92 (1H, m, Hb-16), 1.74 (3H, br s, H-27), 1.72 (1H, m, Hb-11), 1.70 (1H, m, Hb-12), 1.63 (1H, m, Hb-6), 1.60–1.59 (2H, m, Ha-16 and H-17), 1.59–1.58 (2H, m, Hb-1 and H-20), 1.57 (1H, m, Ha-6), 1.46 (1H, m, Ha-12), 1.32 (3H, s, H-30), 1.26 (1H, m, Ha-11), 1.01 (5H, m, H-21 and H-29), 0.99 (3H, s, H-28), 0.97 (3H, s, H-19), 0.84 (3H, s, H-18); 13C NMR (100 MHz, DMSO-d6) d216.2 (C, C-3), 170.7 (qC, acetyl), 169.7 (qC, C-26), 155.3 (qC, C-14), 136.3 (CH, C-24), 131.7 (qC, C-8), 131.1 (qC, C-25), 76.6 (qC-C-9), 76.2 (qC, C-15), 75.0 (CH, C-22), 65.6 (CH, C-7), 50.2 (CH, C-17), 47.5 (CH2, C-16), 47.2 (qC, C-4), 45.2 (qC, C-13), 42.2 (qC, C-10), 40.2 (CH, C-5), 37.3 (CH, C-20), 34.9 (CH2, C-2), 34.6 (CH2, C-12), 31.9 (CH3, C30), 31.6 (CH2, C-1), 31.4 (CH2, C-23), 30.1 (CH2, C-6), 27.2 (CH2, C-11), 26.4 (CH3, C-28), 22.2 (CH3, C-29), 21.6 (CH3, acetyl), 17.6 (CH3, C-18), 17.2 (CH3, C-19), 14.1 (CH3, C-21), 13.2 (CH3, C-27); HRMS (ESI-TOF) m/z 583.3244 [M+Na]+ (calcd. for C32H48O8Na, 583.3241). 4.3.2. Ganorbiformin B (2) Colorless solid; [a]26D +9 (c 0.18, CHCl3); UV (MeOH) kmax (log e) 216 (4.12), 244 sh (3.93), 253 (3.94) nm; IR (ATR) mmax 2967, 1729, 1710 sh, 1690 sh, 1649, 1371, 1237, 1016 cm 1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic

data, see Tables 2 and 1; HRMS (ESI–TOF) m/z 571.3624 [M+H]+ (calcd. for C34H51O7, 571.3629). 4.3.3. Ganorbiformin C (3) Colorless solid; [a]26D +6 (c 0.105, CHCl3); UV (MeOH) kmax (log e) 213 (4.15), 253 (3.95) nm; IR (ATR) mmax 3423, 2925, 1734, 1701, 1645, 1370, 1233, 1015 cm 1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 1; HRMS (ESI–TOF) m/z 529.3530 [M+H]+ (calcd. for C32H49O6, 529.3524). 4.3.4. Garnorformin D (4) Colorless solid; [a]26D +87 (c 0.12, CHCl3); UV (MeOH) kmax (log e) 207 (4.26) nm; IR (KBr disk) mmax 3461, 2971, 1719, 1709, 1377, 1246, 1041 cm 1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 3 and 1; HRMS (ESI–TOF) m/z 609.3396 [M+Na]+ (calcd. for C34H50O8Na, 609.3398). 4.3.5. Ganorbiformin E (5) Colorless solid; [a]26D +52 (c 0.09, CHCl3); UV (MeOH) kmax (log e) 207 (4.18) nm; IR (ATR) mmax 2941, 1730 sh, 1700, 1690 sh, 1372, 1234, 1016 cm 1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 3 and 1; HRMS (ESI–TOF) m/z 551.3347 [M+Na]+ (calcd. for C32H48O6Na, 551.3343). 4.3.6. Ganorbiformin F (6) Colorless solid; [a]26D +52 (c 0.195, CHCl3); UV (MeOH) kmax (log e) 208 sh (4.30) nm; IR (ATR) mmax 2938, 1734, 1702, 1686, 1648, 1372, 1234, 1076 cm 1; for 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 3 and 1; HRMS (ESI–TOF) m/z 565.3502 [M+Na]+ (calcd. for C33H50O6Na, 565.3500). 4.3.7. Ganorbiformin G (7) Colorless solid; [a]26D +25 (c 0.085, CHCl3); UV (MeOH) kmax (log e) 218 (4.25), 234 (4.26), 242 (4.27), 251 (4.08) nm; IR (ATR) mmax 2966, 2934, 1734, 1709, 1686, 1372, 1236 cm 1; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 3 and 1; HRMS (ESI–TOF) m/z 533.3232 [M+Na]+ (calcd. for C32H46O5Na, 533.3237). 4.3.8. Ganoderic acid V (8) Colorless solid; [a]28D +105 (c 0.15, CHCl3); UV (MeOH) kmax (log e) 209 (4.22) nm; IR (ATR) mmax 2937, 1698, 1644, 1376, 1244, 1040 cm 1; for 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HRMS (ESI–TOF) m/z 551.3342 [M+Na]+ (calcd. for C32H48O6Na, 551.3343). 4.3.9. Compound 10 Colorless solid; [a]28D +39 (c 0.12, CHCl3); UV (MeOH) kmax (log e) 216 (4.11), 234 (4.06), 242 (4.07), 251 (3.91) nm; IR (ATR) mmax 2936, 1731, 1712, 1649, 1644, 1375, 1244, 1030, 754 cm 1; 1 H NMR (500 MHz, CDCl3) spectroscopic data were consistent with those reported in the literature (Yang et al., 2002); for 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HRMS (ESI– TOF) m/z 591.3290 [M+Na]+ (calcd. for C34H48O7Na, 591.3292). 4.4. Biological assays Assay for activity against P. falciparum (K1, multi-drug resistant strain) was performed using the microculture radioisotope technique (Desjardins et al., 1979). Anticancer activities against KB cells (oral human epidermoid carcinoma), MCF-7 cells (human breast cancer), and NCI-H187 cells (human small-cell lung cancer),

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were evaluated using the resazurin microplate assay (O’Brien et al., 2000). Cytotoxicity to Vero cells (African green monkey kidney fibroblasts) was performed using the green fluorescent protein microplate assay (Changsen et al., 2003). Acknowledgements Financial support from the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BIOTEC), is gratefully acknowledged. We thank Dr. Sayanh Somrithipol and Ms Sujinda Sommai for description and gene sequence data of the fungus, respectively.

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