Lanostane triterpenoids from cultivated fruiting bodies of the wood-rot basidiomycete Ganoderma casuarinicola

Phytochemistry 170 (2020) 112225

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Lanostane triterpenoids from cultivated fruiting bodies of the wood-rot basidiomycete Ganoderma casuarinicola

T

Masahiko Isaka∗, Panida Chinthanom, Pranee Rachtawee, Wilunda Choowong, Rattaket Choeyklin, Tuksaporn Thummarukcharoen National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani, 12120, Thailand

ARTICLE INFO

ABSTRACT

Keywords: Ganoderma casuarinicola Ganodermataceae Lanostane Antimalarial activity

Sixteen previously undescribed lanostane-type triterpenoids (1–16), together with fourteen known compounds, were isolated from cultivated fruiting bodies of the basidiomycete Ganoderma casuarinicola, a recently described species. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Two of these compounds, 9 and 10, showed antimalarial activity with IC50 values of 9.7 and 9.2 μg/ml, respectively.

1. Introduction

2. Results and discussion

Bracket fungi in the genus Ganoderma have been prolific sources of highly oxygenated lanostane-type triterpenoids. Lanostane triterpenoids have been considered as one of the key ingredients of the popular medicinal mushroom lingzhi (G. lucidum or G. lingzhi) especially as potent anti-cancer agents (Ríos et al., 2012). A number of lanostanes and modified lanostanes have been isolated from basidiocarps (natural or cultivated mushroom specimens) and/or mycelial cultures of G. lucidum and several other species (Baby et al., 2015; Xia et al., 2014); however, there are still many minor species that remain chemically uninvestigated or not well studied. As part of our research program on medicinal utilization of fungal sources in Thailand, we have been searching for novel bioactive compounds from basidiomycetes, especially polypores of the family Ganodermataceae. The studies have led to the isolation of antitubercular and antimalarial lanostane triterpenoids (Isaka et al., 2013, 2017, 2019). In a continuation of the research to demonstrate the structural diversity and biological activities of Ganoderma lanostanoids, we have examined fruiting body cultivation and chemical investigation of a Ganoderma species, which was identified afterwards as a recently described species, G. casuarinicola (Xing et al., 2018). The study led to the isolation of a norlanostane, ganocasuarinone A (1), two rearranged lanostanes, ganocasurarins A (2) and B (3), and thirteen intact lanostane triterpenoids, ganocasuarins C–F (4–7) and compounds 8–16, together with a known norlanostane (17) and ten known lanostanes (18–27) (Fig. 1 and Fig. S1). Antimalarial and antitubercular activities of the new compounds were evaluated.

Fruiting bodies of Ganoderma casuarinicola (ca. 1 kg) were successfully produced from the living culture strain BCC 78460 by application of a factory cultivation procedure for G. lucidum (lingzhi medicinal mushroom). The CH2Cl2 and MeOH extracts were fractionated by combination of silica gel column chromatography and preparative HPLC (ODS) to furnish pure compounds. Known lanostane triterpenoids were identified on the basis of their NMR and HRESIMS data, and comparison with literature: 17 (Chen et al., 2017), ganoderol A (18) (Arisawa et al., 1986), (24E)-15α,26-dihydroxy-3-oxo-lanosta-7,9 (11),24-triene (19) (González et al., 2002), ganodermadiol (20) (Arisawa et al., 1986), ganoderal B (21) (Nishitoba et al., 1988), applanoxidic acid A (22) (Chairul et al., 1991), applanoxididic acid F (23) (Chairul and Hayashi, 1994), ganoderone A (24) (Niedermeyer et al., 2005), lucidadiol (25) (González et al., 1999), ganoderal A (26) (Morigiwa et al., 1986), and ganoderic acid T-O (27) (Lin et al., 1988). A fragmented sterol, demethylincisterol A3 (28) (Mansoor et al., 2005), and two meroterpenoids, ganomycins B (29) (Mothana et al., 2000) and I (30) (El Dine et al., 2009), were also isolated from the same extracts (Fig. S1). The molecular formula of ganocasuarinone A (1) was determined by HRESIMS and 13C NMR as C24H32O5, with six carbon atoms lacking from the classic C30 lanostane. The 1H and 13C NMR, DEPT, and HSQC data for 1 supported the presence of three ketones, a trisubstituted olefin, an oxygenated methine (δC 71.7), a trisubstituted epoxide (δC 60.6, δH 4.12; δC 66.1), four quaternary carbons, two methines, four methylenes, and six singlet methyl groups. The planar structure was

∗

Corresponding author. E-mail address: [email protected] (M. Isaka).

https://doi.org/10.1016/j.phytochem.2019.112225 Received 24 July 2019; Received in revised form 15 November 2019; Accepted 8 December 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

Phytochemistry 170 (2020) 112225

M. Isaka, et al.

Fig. 1. Structures of compounds 1–21.

basis of the COSY and HMBC correlations (Fig. 2). The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of the ABCD-ring were similar to those of ganorbiformin A, which was previously isolated from mycelial cultures of G. orbiforme BCC 22324 (Isaka et al., 2013) and Ganoderma sp. BCC 16642 (Isaka et al., 2016). Their structural differences were the functional groups of the C-20–C-27 side-chain. Ganocasuarin A (2) lacked the 22-acetoxy group of ganorbiformin A, and the C-26 carboxyl group was replaced by a hydroxymethyl in 2. The 24E configuration was evident from the intense NOESY correlations of H-24 with H2-26. The NOESY data also suggested the relative configuration of the ABCDring to be the same as ganorbiformin A (Fig. 3). NOESY correlations between Hα-1/H-5, H-5/H3-28, H-5/Hα-6, H3-28/Hα-6, and H3-30/Hα16 indicated their α-orientation. Key NOESY correlations of the β-face protons were H3-19/Hβ-2, Hβ-2/H3-29, H3-29/Hβ-6, H3-18/Hβ-16, and H3-18/H-20. The oxymethine H-7 resonated as a singlet signal with a narrow peak width (small coupling constants with Hα-6 and Hβ-6), which indicated an equatorial (β) orientation of this proton. Significant downfield chemical shifts of Hα-1 (δH 2.19), H-5 (δH 2.52), H-7 (δH 5.25), and H3-30 (δH 1.55) can be reasonably explained by deshielding of these protons by 9α-OH, 9α-OH, 15β-OH, and 7α-OH, respectively. Ganocasuarin B (3) had the molecular formula C30H50O5 as determined by HRESIMS. Its NMR spectroscopic data were similar to those of 2, which suggested a close structure and the same relative configurations. The only structural difference was the replacement of the C-3 ketone in 2 by an equatorial alcohol (δC 78.6; δH 3.33, dd, J = 11.5, 3.5 Hz). The molecular formula of ganocasuarin C (4) was determined by HRESIMS to be C30H42O8. Its planar structure, possessing a classic C30 lanostane carbon skeleton (Tables 1 and 3), was elucidated on the basis

elucidated by analysis of COSY and HMBC spectra (Fig. 2). Presence of an enone was revealed by the HMBC correlations from H-17 and H3-18 to the ketone carbon (δC 200.9, C-12), from H-11 to C-8, C-9, C-10, and C-13, and from Hα-1 and H3-19 to the downfield olefinic carbon (δC 164.4, C-9). An acetyl group was bonded to C-17, which was demonstrated by the HMBC correlations from H2-16, H-17, and H3-21 to the ketone carbon (δC 208.7, C-20). Another ketone located at C-3 was evident from the HMBC correlations from H2-1, H2-2, H3-22, and H3-23 to the ketone carbon (δC 216.1). Location of the epoxide was assigned by the HMBC correlations from H2-6 to C-7 and C-8, from H-11, H-15, and H3-24 to C-8, and from the epoxy proton (H-7) to C-5, C-6, and C-8. A secondary alcohol at C-15 position was revealed by the COSY correlations of the oxymethine proton (H-15) with H2-16, and the HMBC correlations from H3-24 to the oxymethine carbon (C-15). The relative configuration was elucidated on the basis of the NOESY correlations (Fig. 3). Intense NOESY correlations between H3-24/H-17 and H3-18/ H-15 indicated the relative configuration of C-13/C-14/C-15/C-17 with β-orientation of H-15 and CH3-18, and α-orientation of H-17 and CH324. The epoxy proton (H-7) showed intense correlations with H-15, and cross-peak intensities of Hα-6/H-7 and Hβ-6/H-7 were similar. H-7 also showed a weak NOESY correlation with H3-18, while the cross-peak of H-7/H3-30 was absent. These data indicated an α-epoxide (7R,8R) configuration, and they were different from those of the β-epoxide derivatives 4 and 5 (discussed later) and the known norlanostane 17. NOESY spectra of these β-epoxide derivatives showed intense correlations between H-7/H3-30 and H-7/Hα-6, while the cross-peak of H-7/ Hβ-6 was much weaker. The molecular formula of ganocasuarin A (2) was determined by HRESIMS to be C30H48O5. Its planar structure was elucidated on the 2

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Fig. 3. Key NOESY correlations for 1, 2, and 4.

HMBC correlation from H-3 to the carbonyl carbon of the acetyl group. Consequently, ganocasuarin D (5) was identified as the 3-O-acetate derivative of 4. The molecular formula of ganocasuarin E (6) was determined by HRESIMS as C30H40O8. The C-20–C-27 side-chain was proved to be the same as 4 by interpretation of COSY and HMBC data. NMR spectroscopic data for the ABCD-ring were similar to those of ganocasuarinone A (1). The only difference was the presence of additional ketone at C-15 (δC 210.8) replacing the oxymethine (15α-OH) of 1. The α-epoxide (7R,8R) configuration was revealed by the NOESY correlation of H-7/ H3-18 and the absence of the cross-peak for H-7/H3-30. The unusual downfield chemical shift of H-7 (δH 4.70, d, J = 1.8 Hz) can be explained by the deshielding by the oxygen atom of the C-15 ketone. Ganocasuarin F (7) had the molecular formula C30H38O8 as determined by HRESIMS. Its NMR spectroscopic data were similar to those of applanoxidic acid F (23) (Chairul and Hayashi, 1994). The only structural difference was that CH3-29 was hydroxylated in 7. The location of the hydroxymethyl group was determined by the HMBC correlations from H2-29 (δH 3.90, 3.45) to C-3, C-4, C-5, and C-28, and from H-5 and H3-28 to C-29 (δC 66.3), and the NOESY correlations between Ha-29/H3-19, H3-28/H-5, and H3-28/Hα-6. The absolute configuration of C-25 remains unassigned. NMR spectroscopic data of compounds 8–13 suggested that they share the same lanostane carbon skeleton, bearing C-8/C-9 double bond and the side-chain was terminated with an allylic alcohol (Tables 1 and 4). The 24E configuration was confirmed by the NOESY correlations of H-24/H2-26. Compound 8 possessed C-3 ketone (δC 217.7) and 15α-

Fig. 2. COSY and HMBC correlations for 1, 2, and 4.

of the COSY and HMBC correlations (Fig. 2). An axial (α) orientation of H-3 (δH 3.18, dd, J = 11.3, 4.6 Hz) was evident from its coupling constant values and the NOESY correlations between H-3/Hα-1, H-3/H5, and H-3/H3-28. The relative configuration of C-13/C-14/C-17 was confirmed by the intense NOESY correlation of H-17/H3-30. The βepoxide configuration (7S,8S) was deduced from the NOESY correlations of H-7/Hα-6 and H-7/H3-30. The C-20–C-27 side-chain structure is one of the common ones in Ganoderma lanostanoids (Baby et al., 2015; Xia et al., 2014); however, most of the reported compounds with the same side-chain structure have not been assigned the configurations at C-20 and C-25. As for compounds 4–6, we were unable to determine these configurations by NMR spectroscopic analysis. Due to the close chemical shifts for H2-16/H-17/H2-22 and H3-18/H3-21, and the absence of the resonance for 20-OH in CDCl3, the NOESY spectrum was not informative to assign the configuration at C-20. NMR spectroscopic data of ganocasuarin D (5) were similar to those of 4, which suggested a close structure and the same relative configurations. The differences were the presence of an acetyl group and downfield shift of the axial methine proton H-3 (δH 4.43, dd, J = 11.1, 4.2 Hz) in 5. The location of the acetyl group was confirmed by the 3

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Table 1 13 C NMR spectroscopic data for compounds 2–16 (CDCl3). No.

2

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-OAc 3-OAc a,b

31.0 34.6 216.5 47.1 39.8 29.0 66.2 132.6 76.4 41.3 26.3 34.2 45.4 156.0 77.8 47.9 52.6 17.4 16.9 33.5 19.0 35.1 24.0 126.7 134.5 69.0 13.8 25.8 22.0 31.2

3 30.0 27.3 78.6 38.4 38.1 28.2 66.4 132.9 76.6 41.4 26.2 34.2 45.3 155.5 77.7 47.8 52.5 17.3 17.1 33.5 19.0 35.2 24.0 126.6 134.4 68.9 13.7 28.3 15.3 31.1

4 36.6 27.0 78.0 39.2 48.2 20.9 57.3 59.7 163.8 38.0 124.7 203.7 56.0 55.3 210.6 36.3 43.9 18.4 21.8 72.6 27.7 52.6 210.4 47.7 34.2 179.5 16.8 27.7 15.1 20.1

5 a

36.3 23.4 79.5 38.1 48.3 20.7 57.1 59.7 163.3 37.8 124.8 203.4 56.0 55.2 210.6 36.2a 43.8 18.3 21.9 72.6 27.6 52.6 210.4 47.6 34.3 180.4 16.8 27.6 16.2 20.0 170.9 21.2

6

7

8

9

10

11

12

13

14

15

16

36.0 36.0 216.5 45.9 40.5 22.6 58.8 62.7 163.8 40.2 129.3 202.1 57.5 55.9 210.8 33.5 43.0 18.2 24.8 72.8 27.2 52.6 211.0 47.7 34.2 180.0 16.8 28.6 21.6 17.7

35.6 33.9 219.6 49.2 41.3 21.5 58.6 62.4 163.4 39.9 129.9 200.4 57.6 54.9 209.5 38.2 42.8 18.1 25.6 154.6 20.5 126.7 198.6 47.6 34.6 179.9 17.0 23.5 66.3 17.6

35.9 34.5 217.7 47.3 51.0 19.4 26.9 134.5 134.0 37.0 20.9 31.6 45.1 51.9 73.6 39.5 49.0 16.2 18.6 36.1 18.4 35.9 24.4 126.7 134.5 69.0 13.6 26.2 21.3 16.9

35.2 34.3 217.5 46.7 45.0 29.9 66.8 136.6 139.6 37.9 21.0 30.9 45.0 49.7 29.9 28.1 50.6 16.1 17.2 36.3 18.5 35.9 24.5 126.9 134.4 69.0 13.6 26.5 21.3 26.1

35.2 34.3 217.2 46.8 45,4 29.0 66.7 135.5 139.9 37.9 20.7 31.8 45.6 52.2 72.6 38.5 49.8 16.6 17.0 36.1 18.3 35.8 24.4 126.6 134.5 69.0 13.7 26.2 21.3 19.0

35.2 24.1 80.8 37.8 50.3 18.1 27.0 133.5 135.2 37.0 20.8 31.5 45.1 51.7 73.7 39.4 48.9 16.1 19.1 36.1 18.4 35.9 24.4 126.8 134.5 69.0 13.6 27.9 16.5 16.9

34.6 23.9 80.5 37.3 45.2 28.1 67.3 134.7 141.4 38.1 20.6 31.7 45.7 52.2 72.5 38.3 49.7 16.5 17.3 36.1 18.3 35.8 24.4 126.7 134.5 69.0 13.6 27.7 16.6 19.1 171.0 21.3

34.9 27.6 78.6 38.3 45.1 28.3 67.5 134.6b 141.7 38.4 20.7 31.8 45.8 52.3 72.5 38.5 49.8 16.5 17.3 36.1 18.4 35.8 24.4 126.7 134.5b 69.0 13.6 27.8 15.6 19.1

35.4 24.4 80.8 37.6 49.1 22.7 121.1 140.9 145.8 37.3 116.3 38.5 44.3 52.0 74.7 40.1 48.8 15.9 22.9 35.8 18.4 35.8 24.2 126.7 134.5 69.0 13.6 28.1 16.9 17.1 171.0 21.3

35.7 27.8 78.9 38.7 48.9 22.9 121.3 140.8 146.1 37.4 116.0 38.5 44.4 52.0 74.7 40.1 48.8 15.9 22.8 35.8 18.4 35.9 24.4 126/7 134.5 69.0 13.7 28.1 15.8 17.1

35.2 34.3 217.0 46.8 45.4 29.1 66.7 135.4 140.0 37.9 20.7 31.8 45.7 52.3 72.5 38.5 49.8 17.0 17.0 36.2 18.2 34.6 25.9 155.0 139.3 195.3 9.2 26.2 21.3 19.0

The assignment of carbons may be interchanged.

Table 2 1 H NMR spectroscopic data for compounds 2, 3, and 14–16 (CDCl3). No.

2

3

14

15

16

1

α 2.19, dt (14.7, 4.9); β 1.68, m

α 182, m; β 1.40, m

α 2.37, ddd (14.7, 4.9, 3.7); β 2.63, dt (5.9, 14.7)

α 1.70, m; β 1.57, m

α 1.43, dt (4.4, 13.3); β 1.98, m α 1.72, m; β 1.65, m

α 1.68, m; β 1.97, m

2

α 1.52, dt (4.2, 13.2); β 1.98, m α 1.73, m; β 1.69, m 4.50, dd (11.2, 4.6) 1.18, dd (11.6, 3.8) 2.13, ddd (17.5, 6.2, 3.8); 2.06, m 5.84, d (6.2) 5.31, d (6.0) α 2.29, br d (6.0); β 2.06, m 4.27, dd (9.4, 5.4) 1.95, m; 1.73, m

3.25, dd (11.2, 4.4) 1.09, dd (11.3, 3.6) 2.16, ddd (17.2, 6.4, 4.1); 2.07, m 5.85, d (6.4) 5.31, d (6.2) α 2.29, br d (17.6); β 2.05, m 4.28, dd (9.3, 5.3) 1.95, m; 1.72, m

1.65, m 0.60, s 1.00, s 1.35, m 0.89, d (6.4) 1.41, m; 1.08, m 2.05, m; 1.93, m 5.38, t (6.8) 4.00 (2H), s 1.66, s 0.88, s 0.95, s 0.93, s 2.05, s

1.64, m 0.61, s 0.98, s 1.35, m 0.89, d (6.5) 1.44, m; 1.08, m 2.06, m; 1.93, m 5.39, t (7.0) 4.00 (2H), s 1.67, s 1.00, s 0.88, s 0.94, s

3 5 6

2.52, dd (13.3, 3.2) 1.83, m; 1.75, m

7 11 12

5.25, br s 1.85, m; 1.45, m 1.83, m; 1.64, m

15 16

1.98, dd (13.6, 6.4); 1.73, m

17 18 19 20 21 22 23 24 26 27 28 29 30 3-OAc

1.60, m 0.89, s 1.05, s 1.51, m 1.01, d (6.4) 1.43, m; 1.19, m 2.12, m; 1.95, m 5.40, t (7.1) 4.00 (2H), s 1.67, s 1.15, s 1.10, s 1.55, s

3.33, 2.05, 1.87, m 5.20, 1.79, 1.83,

dd (11.5, 3.5) br d (12.4) br d (12.5); 1.64, br s m; 1.40, m m; 1.64, m

1.97, dd (13.7, 6.6); 1.71, m 1.58, m 0.87, s 0.85, s 1.52, m 1.01, d (6.4) 1.42, m; 1.19, m 2.11, m; 1.97, m 5.40, t (6.9) 4.00 (2H), s 1.67, s 1.06, s 0.86, s 1.52, s

hydroxy functional groups. A β-orientation of H-15 and the relative configuration of C-13/C-14/C-15/C-17/C-20 were deduced from the NOESY correlations between H-15/H3-18, H-15/Hβ-16, Hβ-16/H3-18,

α 2.47, ddd (16.0, 7.1, 3.7); β 2.58, ddd (16.0, 10.7, 7.3) 1.90, m 1.89, m; 1.73, m 4.44, br s 2.15–2.10 (2H), m α 1.89, m; β 1.66, m 4.44, dd (9.3, 6.1) 1.92, m; 1.85, m 1.69, 0.68, 1.07, 1.39, 0.93, 1.58, 2.39, 6.48, 9.40, 1.75, 1.14, 1.08, 1.07,

m s s m d (6.4) m; 1.24, m m; 2.29, m t (7.1) s s s s s

H3-18/H-20, H3-18/Hβ-12, Hβ-12/H3-21, H3-30/H-17, and H3-30/Hα12. The trans AB-ring junction was suggested by the NOESY correlations of H3-19 with Hβ-6 (axial) and Hβ-2 (axial). Consequently, it was 4

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Table 3 1 H NMR spectroscopic data for compounds 4–7 (CDCl3). No.

4

5

6

7

1

α 1.49, ddd (13.1, 12.7, 3.6); β 1.86, dt (12.7, 3.1) α 1.75, m; β 1.68, m 3.18, dd (11.3, 4.6) 1.10, dd (13.3, 5.0) 2.14, m; 2.01, m

α 1.57, dt (3.0, 12.8); β 1.86, br d (12.8) α 1.76, m; β 1.68, m 4.43, dd (11.1, 4.2) 1.20, m 2.09, m; 1.99, dd (15.0, 13.5)

α 2.08, ddd (13.9, 10.6, 5.9); β 1.94, ddd (13.9, 9.1, 4.9) α 2.65, m; β 2.46, m

α 2.15, ddd (13.8, 11.3, 5.6); β 1.99, ddd (13.8, 9.3, 4.4) α 2.46, m; β 2.75, m

4.32, 5.92, 2.68, 8.2) 2.58, 1.47, 1.17, 1.40, 2.72, 3.06, 1.24, 1.02, 0.86, 1.12,

d (6.2) s dd (18.4, 9.9); 2.43, dd (18.4,

4.32, d (6.1) 5.92, s 2.67, m; 2.43, dd (18.7, 8.4)

2.74, dd (13.1, 3.2) 2.14, ddd (14.5, 3.8, 3.2); 1.74, dd (14.5, 13.1) 4.70, d (3.8) 6.01, s 2.74, m; 2.49, m

2.90, m 2.20, ddd (14.8, 3.8, 3.4); 1.75, dd (14.8, 13.6) 4.68, d (3.8) 6.06, s 2.67, dd (19.6, 9.5); 2.46, dd (19.6, 9.5)

m s s s d (15.5); 2.55, m dd (13.8, 8.3); 2.51, m d (7.2) s s s

2.55, 1.46, 1.18, 1.40, 2.69, 3.02, 1.22, 0.88, 0.92, 0.93, 2.05,

2.63, m 1.37, s 1.10, s 1.40, s 2.60–2.58 (2H), m 2.98, m; 2.49, m 1.24, d (6,7) 1.11, s 1.09, s 1.22, s

3.51, 1.12, 1.10, 2.27, 6.35, 2.93, 1.23, 1.29, 3.90, 1.30,

2 3 5 6 7 11 16 17 18 19 21 22 24 27 28 29 30 3-OAc

m s s s d (15.7); 2.54, d (15.7) m; 2.50, m d (7.1) s s s s

identified as a new lanostane triterpenoid, (24E)-15α,26-dihydroxy-3oxo-lanosta-8,24-diene. Compound 9 possessed C-3 ketone and 7α-hydroxy groups. H-7 resonated as a broad singlet with narrow peak width, which indicated its equatorial (β) orientation. Accordingly, this compound was identified to be (24E)-7α,26-dihydroxy-3-oxo-lanosta-8,24diene. Compound 10 was shown to possess C-3 ketone and 7α- and 15αhydroxy groups as revealed by interpretation of COSY and HMBC correlations. Thus, it was identified to be (24E)-3-oxo-7α,15α,26-trihydroxylanosta-8,24-diene. Compound 11 possessed 3β-acetoxy and 15αhydroxy groups. An axial (α) orientation of H-3 (δH 4.49, dd, J = 12.7,

t (9,5) s s s s m; 2.59, m d (6.9) s d (11.4); 3.45, d (11.4) s

4.5 Hz) was evident from its coupling constant values. Therefore, this compound was identified to be (24E)-3β-acetoxy-15α,26-dihydroxylanosta-8,24-diene. Compound 12 was structurally close to 11, but it additionally possessed 7α-hydroxy group. Consequently, it was identified to be (24E)-3β-acetoxy-7α,15α,26-trihydroxylanosta-8,24-diene. Inspection of the NMR spectroscopic data of compound 13 suggested that it is the deacetyl derivative of 12. The coupling constant values of H-3 (δH 3.31, dd, J = 11.2, 3.8 Hz) indicated its axial (α) orientation. Thus, compound 13 was identified to be (24E)-3β,7α,15α,26-tetrahydroxylanosta-8,24-diene.

Table 4 1 H NMR spectroscopic data for compounds 8–13 (CDCl3). No.

8

9

10

11

12

13

1

α 1.62, m; β 1.97, m

α 1.68, m; β 1.95, m

α 1.65, m; β 1.98, m

α 1.31, m; β 1.73, m

α 1.25, m; β 1.76, m

2

α 2.41, ddd (15.7, 6.8, 3.5); β 2.58, ddd (15.7, 11.1, 7.2)

α 2.49, ddd (15.8, 8.2, 4.4); β 2.55, m

α 2.47, ddd (15.9, 7.1, 3.6); β 2.57, ddd (15.9, 10.7, 7.3)

α 1.30, ddd (13.6, 12.3, 3.0); β 1.72, m α 1.70, m; β 1.60, m

α 1.73, m; β 1.61, m

α 1.72, m; β 1.63, m

4.49, dd (12.7, 4.5)

4.53, dd (11.8, 4.1)

1.14, br d (11.2) 1.71, m; 1.52, m

1.41, dd (12.0, 2.7) 1.82, m; 1.78, m

3.31, dd (11.2, 3.8) 1.31, m 1.82, m; 1.80, m

2.18, m; 2.11, m 2.03, m; 1.98, m α 1.82, m; β 1.63, m 4.21, dd (9.6, 5.9) 1.93, m; 1.70, m 1.60, m 0.72, s 1.00, s 1.33, m 0.89, d (6.4) 1.41, m; 1.06, m 2.07, m; 1.91, m 5.38, t (6.8) 3.99 (2H), s 1.66, s 0.88, s 0.88, s 0.92, s 2.05, s

4.38, br s 2.07–2.04 (2H), m α 1.85, m; β 1.62, m 4.39, dd (9.5, 6.3) 1.88, m; 1.80, m 1.66, m 0.64, s 0.96, s 1.33, m 0.88, d (6.4) 1.42, m; 1.08, m 2.07, m; 1.91, m 5.38, t (6.6) 3.99 (2H), s 1.65, s 0.91, s 0.89, s 1.03, s 2.05, s

3 5 6

1.60, m 1.64, m; 1.61, m

1.96, m 1.79, m; 1.67, m

7 11 12

2.24–2.17 (2H), m 2.07–2.03 (2H), m α 1.84, m; β 1.67, m 4.22, dd (9.4, 5.9) 1.94, m; 1.72, m 1.60, m 0.75, s 1.11, s 1.36, m 0.90, d (6.4) 1.41, m; 1.07, m 2.07, m; 1.92, m 5.38, t (6.8) 4.00 (2H), s 1.66, s 1.09, s 1.07, s 0.94, s

4.25, br s 2.12–2.08 (2H), m α 1.78, m; β 1.67, m 1.73, m; 1.49, m 1.99, m; 1.38, m 1.54, m 0.63, s 1.05, s 1.39, m 0.92, d (6.3) 1.48, m; 1.09, m 2.10, m; 1.94, m 5.40, t (6.8) 4.00 (2H), s 1.67, s 1.13, s 1.07, s 1.05, s

15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 3-OAc

1.90, m 1.87, m; 1.73, (12.3) 4.43, br s 2.14, m; 2.10, α 1.87, m; β 1.64, m 4.42, m 1.92, m; 1.83, 1.68, m 0.67, s 1.06, s 1.36, m 0.89, d (6.4) 1.43, m; 1.08, 2.09, m; 1.91, 5.39, t (6.8) 4.00 (2H), s 1.66, s 1.13, s 1.07, s 1.06, s

5

br d m

m

m m

4.41, m 2.10, m; 2.07, α 1.86, m; β 1.62, m 4.40, m 1.92, m; 1.84, 1.68, m 0.65, s 0.95, s 1.33, m 0.89, d (6.1) 1.42, m; 1.09, 2.11, m; 1.92, 5.39, t (6.8) 4.00 (2H), s 1.67, s 1.05, s 0.83, s 1.05, s

m

m

m m

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Interpretation of the NMR spectroscopic data of compound 14 (Tables 1 and 2) revealed the same carbon skeleton as the common Ganoderma lanostanoids, ganoderol A (18) and ganodermadiol (20). This compound possessed 3β-acetoxy, 15α-hydroxy, and 26-hydroxy groups. An axial (α) orientation of H-3 (δH 4.50, dd, J = 11.2, 4.6 Hz) was evident from its coupling constant values, which was further supported by the NOESY correlations between H-3/Hα-1 and H-3/H-5. Therefore, compound 8 was identified to be (24E)-3β-acetoxy-15α,26dihydroxylanosta-7,9 (11),24-triene. On the basis of the NMR spectroscopic data, compound 15 was identified to be the deacetyl derivative of 14, (24E)-3β,15α,26-trihydroxylanosta-7,9 (11),24-triene. The NMR spectroscopic data of compound 16 were similar to those of ganoderal B (21), possessing C-3 ketone, 7α-hydroxy, and C-26 formyl groups. This compound additionally possessed 15α-hydroxy group. A β-orientation of H-15 was revealed by its intense NOESY correlation with H3-18. The 24E configuration was confirmed by the NOESY correlations between H-24/H-26 and Ha-23/H3-27. Therefore, compound 8 was identified to be (24E)-7α,15α-dihydroxy-3-oxo-lanosta-8,24-dien-26-al. In our previous studies, several lanostane triterpenoids and modified lanostanes, isolated from Ganodermataceae, were shown to exhibit antimalarial and antitubercular activities (Isaka et al., 2013, 2017, and 2019). New compounds with sufficient sample quantities (12 compounds) were tested for antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain), antitubercular activity against Mycobacterium tuberculosis H37Ra, and cytotoxicity to nonmalignant Vero cells (African green monkey kidney fibroblasts) (Table 5). Lanostanes 9 and 10 exhibited antimalarial activity with IC50 values 9.7 and 9.2 μg/ml, respectively; however, they also showed cytotoxicity. On the other hand, compounds 11 and 12 showed weak antitubercular activity (MIC 25 μg/ml). As for known lanostanes, ganodermadiol (20) and applanoxidic acid F (23) were previously isolated from other sources in our laboratory and were tested for these biological assays. Both compounds were inactive at the tested concentrations (Isaka et al., 2018; Li et al., 2018). In conclusion, the present study demonstrates high structural diversity of lanostane-type triterpenoids in the recently described minor species, G. casuarinicola. Ganocasuarinone A (1) is a side-chain fragmented norlanostane. Ganocasuarins A (2) and B (3) are rearranged lanostanes, sharing the same carbon skeleton only with ganorbiformin

A. It should also be noted that ganorbiformin A was previously isolated from mycelial cultures. The present work has shown that this type of rearranged lanostanes are also produced in fruiting bodies of Ganoderma. 3. Experimental 3.1. General experimental procedures Optical rotations were determined using a JASCO P-1030 digital polarimeter. UV spectra were recorded on an Analytik-jena SPEKOL 1200 spectrophotometer. FTIR spectra were acquired using a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. ESITOF mass spectra were measured using a Bruker micrOTOF mass spectrometer. Merck Silica gel 60H (particle size, 90% < 45 μm) was used for column chromatography. 3.2. Fungal material and cultivation The fungus used in this study was isolated from soil in Dong-Yai forest, Hua Taphan, Amnat Charoen Province, Thailand, on September 22, 2015. The voucher mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 40052, and the living culture is preserved in the BIOTEC Culture Collection as BCC 78460. This fungus was identified as a recently described species, Ganoderma casuarinicola, on the basis of the sequence data of the ITS rDNA (GenBank accession number: MK007289). For fruiting body cultivation, the G. casuarinicola mycelia were grown on PDA agar at 25 °C for 20 days. Then, a 1 × 1 cm fungal colony was cut from the agar plate and transferred into glass bottle containing sterile millet (7 × 17 cm). After incubation at 25 °C for 9 days, spawn was used as inoculum for cultivation on 120 substrate bags. Each plastic bag contained ca. 1 kg of substrate consisting of 92.5% (w/w) sawdust, 4.6% (w/w) rice bran, 1.8% (w/w) calcium sulfate, 0.9% calcium oxide, and 0.2% (w/w) magnesium sulfate. The substrate bags were incubated in cultivation house at 27–30 °C under dark condition for 68 days. The fruiting bodies with suitable size (5–15 cm stalk long) were obtained from all substrate bags and then harvested for further analysis. 3.3. Extraction and isolation Dried fruiting bodies (953 g) were cut into small pieces, and macerated in CH2Cl2 (8.0 l) at room temperature for 7 days. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a dark brown gum. This extraction was repeated to obtain a combined CH2Cl2 extract (20.4 g) as a brown gum. The residue was then extracted with MeOH (5.0 l, 5 days) to obtain a dark brown gum (MeOH extract, 18.0 g). The CH2Cl2 extract was subjected to column chromatography (CC) on silica gel (5.6 × 19 cm, EtOAc/CH2Cl2, step gradient elution 0:100, 20:80, 40:60, 60:40, 80:20, then 100:0, and then with acetone) to obtain 25 fractions (Fr-1–Fr-25). Fr-1–Fr-5 (total 6.58 g) were mainly composed of lipids. Fr-6 (1.75 g) was purified by silica gel CC (5.6 × 16 cm, EtOAc/CH2Cl2 = 5:95) to furnish 20 (805 mg). Fr-7 (322 mg), Fr-8 (1.09 g), Fr-9 (582 mg), Fr-10 (328 mg), Fr-11 (288 mg), Fr-12 (568 mg), and Fr-13 (330 mg) were further fractionated and purified by preparative HPLC using reverse phase column (Dionex SunFire Prep C18 OBD, 19 × 250 mm, 10 μm; mobile phase MeCN/H2O, gradient from 60:40 to 100:0, over 40 min; flow rate 12 ml/min) to furnish 8 (112 mg), 9 (26 mg), 11 (30 mg), 14 (51 mg), 15 (3.1 mg), 16 (20 mg), 19 (5.7 mg), 21 (71 mg), 24 (7.5 mg), 25 (8.3 mg), 26 (5.5 mg), 27 (5.8 mg), 28 (6.0 mg), and 30 (52 mg). Fr-14 (359 mg), Fr-15 (142 mg), Fr-16 (70 mg), Fr-17 (266 mg), Fr-18 (405 mg), Fr-19 (855 mg), Fr-20 (186 mg), Fr-21 (159 mg), Fr-22 (198 mg), Fr-23 (319 mg), and Fr-24 (435 mg) were separately subjected to preparative HPLC (MeCN/H2O, gradient from 40:60 to 80:20,

Table 5 Antiplasmodial, antimycobacterial, and cytotoxic activities of the lanostanes and modified lanostanes from Ganoderma casuarinicola. Compound

1 4 5 6 7 8 9 10 11 12 14 16 Dihydroartemisinina Isoniazidb Ellipticinec a b c

Anti-malaria

Anti-tuberculosis

Cytotoxicity

P. falciparum K1

M. tuberculosis H37Ra

Vero cells

IC50, μg/ml

MIC, μg/ml

IC50, μg/ml

> 10 > 10 > 10 > 10 > 10 > 10 9.7 9.2 > 10 > 10 > 10 > 10 0.00071

> 50 > 50 > 50 > 50 > 50 50 > 50 > 50 25 25 50 > 50

> 50 > 50 > 50 > 50 > 50 5.9 12.0 13.1 6.0 9.0 > 50 16.9

0.047

0.74

Standard antimalarial drug. Standard antituberculosis drug. Standard compound for the cytotoxicity assay. 6

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over 40 min) to furnish pure compounds: 2 (4.9 mg), 3 (4.7 mg), 5 (236 mg), 6 (177 mg), 10 (187 mg), 12 (39 mg), 13 (1.4 mg), 17 (10 mg), 23 (222 mg), and 29 (105 mg). Fr-25 (1.96 g) was purified by silica gel CC (5.6 × 16 cm, EtOAc/CH2Cl2 = 5:95) to furnish 1 (18 mg), 4 (60 mg), 7 (394 mg), 17 (12 mg), and 22 (16 mg). The MeOH extract was also fractionated by silica gel CC (5.6 × 19 cm, EtOAc/CH2Cl2, step gradient elution 0:100, 20:80, 40:60, 60:40, 80:20, then 100:0) and the fractions were further separated by preparative HPLC (MeCN/H2O) to furnish 1 (13 mg), 3 (7.3 mg), 4 (20 mg), 5 (42 mg), 6 (109 mg), 7 (159 mg), 8 (18 mg), 17 (15 mg), 18 (43 mg), 19 (46 mg), 23 (7.3 mg), and 30 (50 mg).

3.3.7. Ganocasuarin F (7) MeOH (nm) Colorless solid; [α]24 D +157 (c 0.31, CHCl3); UV λmax (log ε): 240 (3.85); IR νmax ATR (cm−1): 1743, 1687, 1612; 1H NMR (400 MHz, CDCl3) data, Table 3; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 549.2460 [M + Na]+ (calc. for C30H38O8Na, 549.2459). 3.3.8. (24E)-15α,26-dihydroxy-3-oxo-lanosta-8,24-diene (8) MeOH Colorless solid; [α]24 (nm) D +80 (c 0.54, CHCl3); UV λmax (log ε): 217 (2.93); IR νmax ATR (cm−1): 3408, 1701; 1H NMR (400 MHz, CDCl3) data, Table 4; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 479.3514 [M + Na]+ (calc. for C30H48O3Na, 479.3496).

3.3.1. Ganocasuarinone A (1) MeOH Colorless solid; [α]22 (nm) (log ε): D +245 (c 0.42, CHCl3); UV λmax 241 (3.56), 260 (3.52); IR νmax ATR (cm−1): 3485, 1703, 1684; 1H NMR (400 MHz, CDCl3) δ 6.09 (1H, s, H-11), 4.41 (1H, t, J = 7.7 Hz, H-15), 4.12 (1H, d, J = 3.7 Hz, H-7), 3.59 (1H, dd, J = 10.9, 6.4 Hz, H-17), 2.85 (1H, ddd, J = 14.8, 9.3, 6.4 Hz, Hβ-16), 2.75 (1H, dd, J = 12.7, 2.7 Hz, H5), 2.69 (1H, ddd, J = 15.9, 9.0, 5.9 Hz, Hα-2), 2.48 (1H, ddd, J = 15.9, 10.6, 4.8 Hz, Hβ-2), 2.35 (3H, s, H-21), 2.17 (1H, ddd, J = 14.6, 3.7, 2.7 Hz, Hα-6), 2.13 (1H, m, Hα-1), 1.95 (1H, ddd, J = 13.7, 9.0, 4.8 Hz, Hβ-1), 1.81 (1H, dd, J = 14.6, 12.7 Hz, Hβ-6), 1.56 (1H, ddd, J = 14.8, 10.9, 7.3 Hz, Hβ-16), 1.16 (3H, s, H-24), 1.11 (3H, s, H-22), 1.09 (6H, s, H19 and H-23), 0.97 (3H, s, H-18); 13C NMR (100 MHz, CDCl3) δ 216.1 (C, C-3), 208.7 (C, C-20), 200.9 (C, C-12), 164.4 (C, C-9), 130.4 (CH, C-11), 71.7 (CH, C-15), 66.1 (C, C-8), 60.6 (CH, C-7), 58.2 (C, C-13), 51.0 (C, C14), 49.9 (CH, C-17), 45.9 (C, C-4), 40.6 (CH, C-5), 40.3 (C, C-10), 35.8 (CH2, C-1), 33.5 (CH2, C-2), 31.2 (CH3, C-21), 31.0 (CH2, C-16), 28.7 (CH3, C-22), 24.6 (CH3, C-19), 23.1 (CH2, C-6), 21.6 (CH3, C-23), 17.9 (CH3, C-18), 14.6 (CH3, C-24); HRESIMS (m/z): 423.2138 [M + Na]+ (calc. for C24H32O5Na, 423.2142).

3.3.9. (24E)-7α,26-dihydroxy-3-oxo-lanosta-8,24-diene (9) MeOH Colorless solid; [α]24 (nm) D +89 (c 0.47, CHCl3); UV λmax −1 (log ε): 215 (3.01), 241 (2.84); IR νmax ATR (cm ): 3401, 1699; 1H NMR (500 MHz, CDCl3) data, Table 4; 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 479.3523 [M + Na]+ (calc. for C30H48O3Na, 479.3496). 3.3.10. (24E)-3-oxo-7α,15α,26-trihydroxylanosta-8,24-diene (10) MeOH Colorless solid; [α]24 (nm) D +88 (c 0.33, CHCl3); UV λmax −1 (log ε): 215 (2.93); IR νmax ATR (cm ): 3350, 1700; 1H NMR (400 MHz, CDCl3) data, Table 4; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 495.3446 [M + Na]+ (calc. for C30H48O4Na, 495.3445). 3.3.11. (24E)-3β-acetoxy-15α,26-dihydroxylanosta-8,24-diene (11) MeOH Colorless solid; [α]24 (nm) D +57 (c 0.27, CHCl3); UV λmax (log ε): 216 (3.27), 238 (3.20); IR νmax ATR (cm−1): 1718; 1H NMR (400 MHz, CDCl3) data, Table 4; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 523.3788 [M + Na]+ (calc. for C32H52O4Na, 523.3758).

3.3.2. Ganocasuarin A (2) MeOH Colorless solid; [α]23 (nm) (log ε): D +9 (c 0.21, CHCl3); UV λmax −1 1 215 (3.17); IR νmax ATR (cm ): 3401, 1703; H NMR (500 MHz, CDCl3) data, Table 2; 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 511.3392 [M + Na]+ (calc. for C30H48O5Na, 511.3394).

3.3.12. (24E)-3β-acetoxy-7α,15α,26-trihydroxylanosta-8,24-diene (12) MeOH Colorless solid; [α]23 (nm) D +71 (c 0.25, CHCl3); UV λmax −1 (log ε): 239 (3.44), 238 (3.20); IR νmax ATR (cm ): 3431, 1731; 1H NMR (400 MHz, CDCl3) data, Table 4; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 539.3716 [M + Na]+ (calc. for C32H52O5Na, 539.3707).

3.3.3. Ganocasuarin B (3) MeOH Colorless solid; [α]23 (nm) D −11 (c 0.24, CHCl3); UV λmax (log ε): 217 (3.24), 236 (3.20), 264 (3.22); IR νmax ATR (cm−1): 3402, 1708; 1H NMR (500 MHz, CDCl3) data, Table 2; 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 513.3557 [M + Na]+ (calc. for C30H50O5Na, 513.3550).

3.3.13. (24E)-3β,7α,15α,26-tetrahydroxylanosta-8,24-diene (13) MeOH Colorless solid; [α]23 (nm) D +124 (c 0.07, CHCl3); UV λmax −1 1 (log ε): 239 (3.46); IR νmax ATR (cm ): 3380; H NMR (500 MHz, CDCl3) data, Table 4; 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 497.3613 [M + Na]+ (calc. for C30H50O4Na, 497.3601).

3.3.4. Ganocasuarin C (4) MeOH Colorless solid; [α]22 (nm) D +54 (c 0.27, CHCl3); UV λmax (log ε): 240 (3.69); IR νmax ATR (cm−1): 1741, 1686; 1H NMR (400 MHz, CDCl3) data, Table 3; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 553.2789 [M + Na]+ (calc. for C30H42O8Na, 553.2772).

3.3.14. (24E)-3β-acetoxy-15α,26-dihydroxylanosta-7,9(11),24-triene (14) MeOH Colorless solid; [α]23 (nm) D +89 (c 0.45, CHCl3); UV λmax −1 (log ε): 239 (3.59); IR νmax ATR (cm ): 3410, 1719; 1H NMR (400 MHz, CDCl3) data, Table 2; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 521.3615 [M + Na]+ (calc. for C32H50O4Na, 521.3601).

3.3.5. Ganocasuarin D (5) MeOH Colorless solid; [α]23 (nm) D +57 (c 0.43, CHCl3); UV λmax −1 (log ε): 241 (3.67); IR νmax ATR (cm ): 1733, 1688; 1H NMR (400 MHz, CDCl3) data, Table 3; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 595.2874 [M + Na]+ (calc. for C32H44O9Na, 595.2878).

3.3.15. (24E)-3β,15α,26-trihydroxylanosta-7,9(11),24-triene (15) MeOH Colorless solid; [α]23 (nm) D +105 (c 0.16, CHCl3); UV λmax (log ε): 239 (3.72); IR νmax ATR (cm−1): 3381; 1H NMR (400 MHz, CDCl3) data, Table 2; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 479.3510 [M + Na]+ (calc. for C32H48O3Na, 479.3496).

3.3.6. Ganocasuarin E (6) MeOH Colorless solid; [α]24 (nm) D +209 (c 0.37, CHCl3); UV λmax −1 (log ε): 242 (3.71); IR νmax ATR (cm ): 1741, 1688; 1H NMR (400 MHz, CDCl3) data, Table 3; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 551.2616 [M + Na]+ (calc. for C30H40O8Na, 551.2615).

3.3.16. (24E)-7α,15α-dihydroxy-3-oxo-lanosta-8,24-dien-26-al (16) MeOH Colorless solid; [α]23 (nm) D +120 (c 0.24, CHCl3); UV λmax 7

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(log ε): 239 (3.66); IR νmax ATR (cm−1): 3435, 1704, 1686, 1644; 1H NMR (400 MHz, CDCl3) data, Table 2; 13C NMR (100 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 493.3289 [M + Na]+ (calc. for C30H46O4Na, 493.3288).

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3.4. Biological assays An assay for activity against Plasmodium falciparum (K1, multidrug resistant strain) was performed in duplicate using the microculture radioisotope technique (Desjardins et al., 1979). Antimycobacterial activity against Mycobacterium tuberculosis H37Ra and cytotoxicity to Vero cells were evaluated using the green fluorescent protein microplate assay (Changsen et al., 2003; Hunt et al., 1999). Declaration of competing interest The authors have no competing interests to declare. Acknowledgements Financial support from National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112225. References Arisawa, M., Fujita, A., Saga, M., Fukumura, H., Hayashi, T., Shimizu, M., Morita, N., 1986. Three new lanostanoids from Ganoderma lucidum. J. Nat. Prod. 49, 621–625. Baby, S., Johnson, A.J., Govindan, B., 2015. Secondary metabolites from Ganoderma. Phytochemistry 114, 66–101. Chairul, Tokuyama, T., Hayashi, Y., Nishizawa, M., Tokuda, H., Chairul, S.M., Hayashi, Y., 1991. Applanoxidic acids A, B, C and D, biologically active tetracyclic triterpenes from Ganoderma applanatum. Phytochemistry 30, 4105–4109. Chairul, S.M., Hayashi, Y., 1994. Lanostanoid triterpenes from Ganoderma applanatum. Phytochemistry 35, 1305–1308. Changsen, C., Franzblau, S.G., Palittapongarnpim, P., 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 47, 3682–3687. Chen, X.-Q., Chen, L.-X., Li, S.-P., Zhao, J., 2017. A new nortriterpenoid and an ergostanetype steroid from the fruiting bodies of the fungus Ganoderma resinaceum. J. Asian Nat. Prod. Res. 19, 1239–1244.

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