Article pubs.acs.org/jnp
Antitubercular Activity of Mycelium-Associated Ganoderma Lanostanoids Masahiko Isaka,*,† Panida Chinthanom,† Malipan Sappan,† Sumalee Supothina,† Vanicha Vichai,† Kannawat Danwisetkanjana,† Thitiya Boonpratuang,† Kevin D. Hyde,‡ and Rattaket Choeyklin§ †
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand ‡ Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand § Biodiversity-Based Economy Development Office, The Government Complex, Chaeng Wattana Road, Bangkok 10210, Thailand S Supporting Information *
ABSTRACT: In a continuation of our research into antitubercular lanostane triterpenoids from submerged cultures of Ganoderma species, three strains, Ganoderma orbiforme BCC 22325, Ganoderma sp. BCC 60695, and Ganoderma australe BCC 22314, have been investigated. Fourteen new lanostane triterpenoids, together with 35 known compounds, were isolated. Antitubercular activities of these myceliumassociated Ganoderma lanostanoids against Mycobacterium tuberculosis H37Ra were evaluated. Taken together with the assay data of previously isolated compounds, structure−activity relationships of the antitubercular activity are proposed. Most importantly, 3β- and 15α-acetoxy groups were shown to be critical for antimycobacterial activity. The most potent compound was (24E)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (35). TB lanostanoids.8 These studies led to a preliminary proposal of structure−activity relationship (SAR), suggesting the significance of the 3β-acetoxy- and 15α-acetoxy groups.8 Considering the urgent need to discover a new chemical class of TB drug lead compounds, caused by the increased incidence of multidrug-resistant TB and extensively drug resistant TB,9,10 we have been continuing this search for anti-TB lanostanoids. Three other Thai strains, G. orbiforme BCC 22325, Ganoderma sp. BCC 60695, and G. australe BCC 22314, have been investigated, since mycelial extracts from small-scale fermentations of these strains exhibited relatively higher anti-TB activity, with MIC values of 6.25, 3.13, and 0.781 μg/mL, respectively, among the 15 tested Ganoderma strains. We report here the isolation of 14 new lanostanoids (1−14) together with 35 known compounds (15−49) (Figure 1). On the basis of the anti-TB activities of the new and several known compounds, taken together with the previously reported data,7,8 a conclusive SAR is proposed in this paper.
Ganoderma has long been regarded as one of the most significant medicinal mushrooms worldwide, and laccate species of Ganoderma, i.e., the G. lucidum species complex, have been used in traditional Chinese medicine for over two millennia. Many health products are marketed with Ganoderma as an ingredient, especially in East Asia and the USA. This is because they have putative anticancer, antiaging, and antimicrobial/viral functions, among others.1,2 Lanostane-type triterpenoids, such as ganoderic acids, have been known as one of the key constituents of the medicinal mushroom Lingzhi (G. lucidum or G. lingzhi).2,3 Many related species such as G. applanatum, G. amboinense, G. colossum, and G. carnosum are also known as producers of similar triterpenoids.4,5 More than 250 lanostanetype triterpenoids have been isolated from basidiocarps (natural or cultivated mushroom specimens) and/or mycelial cultures of Ganoderma.4 These lanostanoids have been shown to possess various biological activities. Most importantly, some of the lanostanoids have been considered as potent anticancer agents, showing cytotoxic activities against tumor cell lines.6 As part of our research on the utilization of fungal resources in Thailand, we recently reported the isolation of lanostanoids from mycelial cultures of the relatively rare species G. orbiforme, strain BCC 22324, and their antituberculosis (anti-TB) activity and relatively weaker cytotoxicity. It was the first report of the significant anti-TB activity of Ganoderma lanostanoids.7 This work was followed by the investigation of Ganoderma sp. BCC 16642, which resulted in the isolation of additional potent anti© 2017 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Ganoderma orbiforme BCC 22325 was subjected to scale-up fermentation, extraction, and chromatographic separation of lanostanoids, which led to the isolation of five new compounds Received: October 22, 2016 Published: May 15, 2017 1361
DOI: 10.1021/acs.jnatprod.6b00973 J. Nat. Prod. 2017, 80, 1361−1369
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Article
Ganoderma sp. BCC 16642, are also depicted.The structural variations of the mycelium-associated Ganoderma lanostanoids were the functional groups at C-3, C-7, C-15, and C-22. Compounds 1−59 were divided into six structural groups (Figure 1) based on the C-7 functionality: 7α-acetoxy, 7αhydroxy, 7-oxo, 7-unsubstituted (methylene), 7α-methoxy, and 7,9(11)-diene. The isolated compounds were initially identified by 1H NMR spectroscopy (CDCl3) and ESIMS. Structures of the new compounds were further confirmed by analysis of 2D NMR spectroscopic data. Known compounds were identified by comparison of the spectroscopic data with those reported in the literature. These are 15,8 16,8 17,8 18,8 ganorbiformin E (19),7 ganoderic acid V (20),7,12 ganorbiformin D (21),7 ganorbiformin B (22),7 ganorbiformin C (23),7 24,8 25,8 2613 27,8 astraodoric acid A (28),14 ganoderic acid Md (29),15 7-Omethylganoderic acid O (30),11 31,8 ganorbiformin F (32),7 33,8 34,8 35,16 36,17 ganoderic acid T-O (37),18 ganoderic acid Y (38),12 39,8 40,19 ganoderic acid R (41),20 ganoderic acid T (42),20 ganoderic acid S (43),20 ganoderic acid X (44),12,21 ganoderic acid P (45),11 ganorbiformin G (46),7 47,19 48,22 and 49.22 Compound 1 was isolated as a colorless solid, and the molecular formula was determined to be C34H52O7 by HRESIMS. The 1H and 13C NMR spectroscopic data suggested that 1 had a lanostane skeleton similar to the known lanostane co-metabolites and bearing two acetoxy groups. The 1H and 13 C NMR, DEPT, and HSQC data for 1 supported the presence of three carboxyl or ester groups (δC 172.4, 172.0, and
Figure 1. COSY and key HMBC correlations for 1.
(9, 10, 12, 13, and 14) and 19 known compounds. As mentioned in previous reports,7,8,11 the 7α-methoxy congeners, 12−14 and 29−32, should be isolation artifacts due to the use of MeOH for mycelial extraction. Indeed these congeners were not present when strain BCC 22325 was refermented in small scale and extracted with acetone−EtOAc or EtOH−EtOAc as checked by 1H NMR spectroscopic analysis of crude extracts. Non-MeOH extraction/isolation procedures (see Experimental Section) were employed for Ganoderma sp. BCC 60695 and G. australe BCC 22314. Eleven new compounds (1−11) and 22 known compounds (15−19, 22, 23, 25−28, 33−40, 46, 48, and 49) were isolated from the mycelial extract of BCC 60695, and four new compounds (1, 3, 4, and 6) and 10 known compounds (15−20, 26, and 35−37) from BCC 22314. In total, 14 new lanostanoids (1−14, overlapped production of several compounds among the three strains) were isolated in the present study. Structures of 10 known compounds (50− 59), previously isolated from G. orbiforme BCC 22324 and/or Chart 1
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DOI: 10.1021/acs.jnatprod.6b00973 J. Nat. Prod. 2017, 80, 1361−1369
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Table 1. 13C NMR Spectroscopic Data for Compounds 1−14 in CDCl3 no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
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-OCOCH3 7-OCOCH3 7-OCH3 15-OCOCH3 15-OCOCH3 22-OCOCH3 22-OCOCH3
34.6a 24.0 80.2 37.2 45.2 25.8e 70.2 131.3 144.6 38.2 21.2 31.1 45.6 51.9 72.4 39.6 48.7 16.3 17.7b 36.1 18.3 34.6a 25.7e 145.1 126.8 172.4 12.0 27.5 16.7 17.7b 171.0 21.3 172.0 21.7
34.6 24.0 80.2 37.2 45.2 25.8 70.1 131.2 144.5 38.2 21.1 31.1 45.4c 51.9 72.2 39.2 45.4c 16.2 17.77f 39.3 12.8 74.5 31.6 139.1 129.2 171.6 12.3 27.5 16.7 17.84f 171.0 21.6
34.7 24.0 80.6 37.3 45.2 27.9c 67.3 135.9 140.9 38.2 20.98 31.0 45.0 49.7 29.8 28.9 47.1 15.9 17.5 39.7 12.8 74.8 31.8 138.9 129.0 171.5 12.4 27.9c 16.8 26.3 170.8g 21.3h
34.7d 24.0 80.5 37.3 45.2 28.2 67.3 134.7 141.5 38.1 21.3 31.8 45.8 52.3 72.4 38.3 49.8 16.6 17.4 36.2 18.2 34.7d 25.8 145.0 126.8 171.9 12.1 27.8 16.7 19.1 171.0 21.3
34.7 24.0 80.6 37.3 45.2 28.0 67.0 134.7 141.1 38.1 20.6 31.8 45.6 52.3 72.2 38.1 46.4 16.4 17.4 39.4 12.7 74.8 31.7 138.6 129.6 171.5 12.4 27.7 16.6 19.1 171.0 21.3i
35.3 34.3 217.3 46.7 45.1 29.9j 66.9 136.6 139.6 38.0 21.1 31.0 45.1 49.7 30.0j 28.2 50.6 16.2 17.2 36.4 18.4 34.8 25.9 145.4 126.7 172.1 12.0 26.5 21.3 26.1
35.3 34.3 217.2 46.8 45.3 28.8 66.4 135.5 139.5 37.9 20.9 31.8k 45.4 52.3 72.4 38.1 46.5 16.5 17.4 39.5 12.7 74.7 31.7k 138.5 129.6 171.4 12.4 26.3 21.3 19.0
34.4l 24.0m 79.4 37.7 49.5 36.3 201.3 138.8 169.3 40.1 23.8m 30.7 45.8 51.3 72.0 37.1 48.2 16.2 18.3 36.0 18.5 34.6l 25.7 144.9 126.8 171.3 12.1 27.3 16.4 18.7 170.8 21.2
29.3 25.6 75.2 37.8 44.1 36.7 199.3 138.9 165.8 40.0 23.7 30.2 45.1 48.1 32.2 28.7 45.8 15.8 18.6 39.7 13.3 75.0 32.0 139.7 129.2 171.6 12.5 27.5 22.1 25.3
35.5 27.3 78.9 38.9 50.2 18.18 26.5 132.9 135.6 37.1 20.8 31.2 44.7 51.0 76.1 36.5 49.1 16.1 19.1 36.2 18.20n 34.7 25.9 145.1 126.6 171.8 12.0 28.0 15.4 18.3n
36.0 34.7 217.5 47.4 51.1 19.4 26.3 133.8 134.3 37.0 20.9 31.1 44.7 51.2 76.0 36.5 49.1 16.2 18.6 36.2 18.2o 34.5 25.9 145.0 126.6 171.2 12.1 26.2 21.3p 18.3o
34.6q 24.0 80.3 37.3 44.3 21.4r 76.1 133.2 142.2 38.7 21.1r 30.8 45.7 51.6 75.7 37.4 48.6 16.2 17.7 36.2 18.2 34.5q 25.9 145.1 126.7 172.0 12.0 27.8 17.1 18.9 171.3 21.7
30.2 23.4 77.4 36.5 39.9 21.4 76.2 133.1 143.0 39.0 21.7 31.1 46.1 51.9 75.9 37.7 48.9 16.6 17.8 36.4 18.4 34.8 26.1 145.3 126.9 172.2 12.3 27.5 22.5 19.2 171.5 21.9s
35.0 34.2 217.4 46.5 44.2 22.5 75.4t 133.8 140.9 38.3 21.0 30.8 45.5 51.7 75.3t 37.0 45.3 16.3 17.7 39.9 12.6 74.4 31.9 138.8 129.4 171.7 12.3 27.0 21.2 18.9
171.1 21.4
171.0 21.4p
55.2 170.8 21.3
55.5 171.3 21.8s
55.4 171.2 21.7 170.6 20.9
a−d
Overlapping signals.
170.5 21.3 e−t
170.9g 21.0h
170.8 21.0i
170.9 21.0
170.8 21.3
The carbon assignment may be interchanged.
171.0), four olefinic carbons (including a methine, δC 145.1/δH 6.86), three oxymethines, four sp3 quaternary carbons, three methines, eight methylenes, and nine methyl groups (Tables 1 and 2). The structure of the tetracyclic system (ABCD) was determined by analysis of COSY and HMBC data (Figure 1). Key HMBC data were the 2J correlations from the six methyl group singlets (H3-18, H3-19, H3-28, H3-29, and H3-30) to their attached carbons C-13, C-10, C-4, C-4, and C-14, respectively, and their 3J correlations. A tetrasubstituted olefin was assigned to C-8/C-9 by the HMBC correlations from H-7, H-11, H-15, and H3-30 to C-8 (δC 131.3) and from H-5, H-7, H-11, and H319 to C-9 (δC 144.6). The presence of two acetoxy groups at C3 and C-7 was confirmed by the HMBC correlations from H-3 and H-7 to the acetyl carbonyl carbons (δC 171.0 and 172.0, respectively). The chemical shifts of protons and carbons and the 1H−1H coupling constant values for the C-20−C-27 sidechain were very similar to those of known co-metabolites. The 24E configuration was confirmed by the intense NOESY correlations of Hb-23 (δH 2.09)/H3-27. The same relative
configuration as lanosterol (normal relative configuration) was suggested by the NOESY correlations (Figure 2). NOESY correlations Hα-1/H-3, H-3/H-5, and H-5/Hα-1 indicated their axial orientations. The axial (α) orientation of H-3 (δH 4.56, dd, J = 11.6, 4.2 Hz) was evident from its coupling constant values. The equatorial (β) orientation of H-7 (δH 5.42, br s) was suggested by its small coupling constants (resonated as a broad singlet with narrow peak width). The β-orientation of H-15 was revealed by the intense NOESY correlations H-15/H3-18 and H-15/H-7. NOESY correlations H3-30/H-17 and H3-30/Hα-12 indicated their α-orientations. Consequently, compound 1 was identified as a new lanostanoid, (24E)-3β,7α-diacetoxy-15αhydroxylanosta-8,24-dien-26-oic acid. Compound 2 was assigned the molecular formula C36H54O9 by HRESIMS, which suggested the presence of three acetoxy groups. The 1H and 13C NMR spectra displayed similarity to those of 1. The significant difference was the presence of an additional acetoxy group attached to C-22 (δC 76.8; δH 5.00, t, J = 6.9 Hz). Location of this oxymethine was confirmed by the 1363
DOI: 10.1021/acs.jnatprod.6b00973 J. Nat. Prod. 2017, 80, 1361−1369
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Table 2. 1H NMR Spectroscopic Data for Compounds 1, 2, 8, and 9 in CDCl3 no.
1
2
1 2 3 5 6
α 1.38, m; β 1.76, m α 1.73, m; β 1.64, m 4.56, dd (11.6, 4.2) 1.56, dd (11.2, 2.9) 1.74−1.72 (2H), m
α 1.38, m; β 1.76, m α 1.70, m; β 1.62, m 4.56, dd (11.5, 3.8) 1.55, m 1.74−1.72 (2H), m
8
7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OAc 7-OAc 22-OAc
5.42, br s 2.12−2.05 (2H), m 1.84, m; 1.62, m 4.24, dd (9.3, 5.7) 1.90, m; 1.74, m 1.64, m 0.65, s 1.00, s 1.34 m 0.90, d (6.4) 1.50, m; 1.15, m 2.22, m; 2.09, m 6.86, t (6.9) 1.82, s 0.83, s 0.87, s 1.03, s 2.05, s 2.01, s
5.41, br s 2.15−2.07 (2H), m 1.84, m; 1.60, m 4.23, dd (9.6, 4.3) 1.91, m; 1.81, m 1.78, m 0.64, s 1.00, s 1.46, m 0.95, d (6.6) 5.00, t (6.9) 2.55, m; 2.35, m 6.78, t (6.7) 1.85, s 0.83, s 0.88, s 1.02, s 2.06, s 2.00, s 2.04, s
9
α 1.53, m; β 1.86, m α 1.82, m; β 1.71, m 4.51, dd (11.9, 4.3) 1.72, dd (13.7, 4.0) α 2.47, dd (17.0, 4.0) β 2.53, dd (17.0, 13.7)
α 1.80, m; β 1.55, m α 1.70, m; β 1.99, m 3.49, s 2.15, dd (14.3, 3.2) α 2.31, m; β 2.41, m
2.40, 1.87, 4.27, 1.92, 1.60, 0.69, 1.19, 1.39, 0.92, 1.54, 2.24, 6.86, 1.83, 0.90, 0.96, 0.91, 2.07,
2.38−2.26 (2H), m 1.81, m; 1.77, m 2.05, m; 1.71, m 2.02, m; 1.32, m 1.54, m 0.65, s 1.17, s 1.55, m 1.00, d (6.1) 5.10, t (7.0) 2.56, m; 2.36, m 6.80, t (7.2) 1.86, s 0.96, s 0.94, s 0.89, s
dd (11.3, 8.5); 2.29, m m; 1.73, m dd (9.3, 6.6) m; 1.88, m m s s m d (6.5) m; 1.19, m m; 2.11, m t (7.0) s s s s s
2.05, s
Hβ-6, Hα-15, and Hβ-15. Consequently, compound 3 was identified as a new lanostanoid, (22S,24E)-7α-hydroxylanosta8,24-dien-26-oic acid. Compounds 4−7 were also identified as 7α-hydroxy derivatives with an α,β-unsaturated carboxylic acid (C-24−C27). Compound 4 possessed additionally 3β-acetoxy (δH 4.53, dd, J = 11.6, 3.7 Hz, H-3) and 15α-hydroxy (δH 4.42, m, H-15) functional groups. Compound 5 was a 22S-acetoxy (δH 5.01, t, J = 6.8 Hz, H-22) derivative of 4. Compound 6 possessed a C-3 ketone (δC 217.3) functional group. Compound 7 had C-3 ketone (δC 217.2), 15α-hydroxy (δH 4.42, m, H-15), and 22Sacetoxy (δH 5.02, t, J = 7.0 Hz, H-22) groups. The molecular formula of compound 8 was determined by HRESIMS as C32H48O6. The 1H and 13C NMR spectroscopic data demonstrated the presence of an α,β-unsaturated ketone, similar to the known 7-oxo derivatives 22−25. The location of the tetrasubstituted olefin (C-8/C-9) was confirmed by the HMBC correlations from H3-30 to C-8 (δC 138.8) and from H3-19 to C-9 (δC 169.3). The C-7 ketone (δC 201.3) was evident from the HMBC correlations from H-6α and Hβ-6 to this carbon and the downfield chemical shift of C-9. Accordingly, compound 8 was identified as a new lanostanoid, (24E)-3β-acetoxy-15α-hydroxy-7-oxolanosta-8,24-dien-26-oic acid. Compound 9 had the molecular formula C32H48O6 (HRESIMS), and its NMR spectroscopic data suggested that it was also a 7-oxo derivative. Other functionalities of 9 were 3α-hydroxy (δH 3.49, s, H-3) and 22S-acetoxy (δH 5.10, t, J = 7.0 Hz, H-22) groups. The molecular formula of compound 10 was assigned by HRESIMS as C32H50O5. The 1H and 13C NMR spectroscopic data suggested the absence of any functional group at C-7 and the presence of a hydroxy and an acetoxy group (Tables 1 and 4). The 3β-hydroxy group was assigned based on the coupling
Figure 2. Key NOESY correlations for 1.
HMBC correlation from H-22 and an acetyl methyl (δH 2.04, 3H, s) to an ester carbonyl carbon at δC 170.5. The chemical shifts of protons and carbons and the 1H−1H coupling constant values for the C-20−C-27 side-chain were very similar to those of known (22S,24E)-22-acetoxy derivatives. Therefore, compound 2 was identified as (22S,24E)-15α-hydroxy-3β,7α,22triacetoxylanosta-8,24-dien-26-oic acid. The molecular formula of compound 3 was determined by HRESIMS as C34H52O7. The 1H and 13C NMR spectra of 3 suggested the same side-chain structure as 2 and several other known co-metabolites, with a 22S-acetoxy group and 24Egeometry of the α,β-unsaturated carboxylic acid (Tables 1 and 3). Other functional groups in the tetracyclic core were a tetrasubsituted olefin (C-8/C-9), a 3β-acetoxy group, and a 7αhydroxy group. An axial (α) orientation of H-3 (δH 4.54, dd, J = 11.9, 4.1 Hz) was evident from its coupling constant values. It was further supported by the NOESY correlations from H-3 to Hα-1, Hα-2, H-5, and H3-28. An equatorial (β) orientation of H-7 (δH 4.19, br s) was indicated by its small coupling constants and NOESY correlations from this proton to Hα-6, 1364
DOI: 10.1021/acs.jnatprod.6b00973 J. Nat. Prod. 2017, 80, 1361−1369
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Table 3. 1H NMR Spectroscopic Data for Compounds 3−7 in CDCl3
a
no.
3
4
5
6
7
1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OAc 22-OAc
α 1.32, m; β 175, m α 1.71, m; β 1.63, m 4.54, dd (11.9, 4.1) 1.42, m 1.75, m; 1.72, m 4.19, br s 2.08−2.03 (2H), m 1.76, m; 1.63, m 1.68, m; 1.52, m 2.03, m; 1.36, m 1.65, m 0.59, s 0.97, s 1.51, m 0.98, d (6.3) 5.09, t (7.2) 2.55, m; 2.35, m 6.78, t (6.8) 1.85, s 0.91, s 0.89, s 1.01, s 2.06,a s 2.05,a s
α 1.32, m; β 1.74, m α 1.72, m; β 1.62, m 4.53, dd (11.6, 3.7) 1.41, m 1.81, m; 1.77, m 4.41, m 2.09−2.02 (2H), m 1.83, m; 1.64, m 4.42, m 1.89, m; 1.78, m 1.67, m 0.64, s 0.96, s 1.35, m 0.90, m 1.52, m; 1.18, m 2.23, m; 2.12, m 6.86, t (7.0) 1.83, s 0.92, s 0.89, s 1.04, s 2.05, s
α 1.32, m; β 1.73, m α 1.72, m; β 1.62, m
α 1.68, m; β 1.98, m α 2.49, m; β 2.56, m
α 1.67, m; β 1.96, m α 2.47, ddd (12.5, 7.3, 3.9); β 2.56, m
1.44, 1.80, 4.39, 2.10, 1.86, 4.40, 1.90, 1.79, 0.64, 0.96, 1.47, 0.94, 5.01, 2.53, 6.75, 1.84, 0.92, 0.89, 1.03, 2.05, 2.05,
1.95, m 1.79, m; 1.68, 4.26, br s 2.12−2.09, m 1.78, m; 1.70, 1.75, m; 1.53, 2.01, m; 1.38, 1.54, m 0.63, s 1.05, s 1.41, m 0.94, d (6.4) 1.56, m; 1.19, 2.26, m; 2.12, 6.88, t (7.0) 1.84, s 1.13, s 1.07, s 1.05, s
1.96, m 1.93, m; 1.73, m 4.43, m 2.14−2.09 (2H), m 1.88, m; 1.62, m 4.42, m 1.91, m; 1.83, m 1.79, m 0.66, s 1.05, s 1.49, m 0.95, d (6.7) 5.02, t (7.0) 2.54, m; 2.35, m 6.75, t (7.5) 1.85, s 1.13, s 1.07, s 1.04, s
dd (12.1, 3.5) m; 1.78, m m m; 2.03, m m; 1.60, m m m; 1.77, m m s s m d (6.8) t (6.8) m; 2.35, m t (6.8) s s s s s s
m
m m m
m m
2.05, s
The assignments may be interchanged.
Table 4. 1H NMR Spectroscopic Data for Compounds 10−14 in CDCl3 no. 1 2 3 5 6 7 11 12 15 16 17 18 19 20 21 22 23 24 27 28 29 30 3-OAc 7-OCH3 15-OAc 22-OAc
10 α 1.23, dt (3.0, 13.1) β 1.72, m α 1.67, m; β 1.61, m
12
13
14
α 1.62, m; β 1.98, m
11
α 1.34, m; β 1.72, m
α 1.88, m; β 1.48, m
α 1.76, m; β 1.91, m
α 2.40, ddd (15.7, 6.7, 3.6) β 2.59, ddd (15.7, 11.4, 7.0)
α 1.75, m; β 1.62, m
α 1.64, m; β 1.85, m
α 2.49, m; β 2.51, m
4.53, 1.58, 1.95, 3.57, 2.08, 1.87, 5.09, 2.14, 1.70, 0.69, 0.99, 1.38, 0.91, 1.49, 2.23, 6.85, 1.82, 0.90, 0.90, 1.17, 2.09, 3.18, 2.04,
4.68, br s 1.90, m 1.70, m; 1.37, m 3.57, br s 2.10−2.05 (2H), m 1.88, m; 1.64, m 5.11, dd (10.0, 4.8) 2.16, m; 1.70, m 1.71, m 0.70, s 0.99, s 1.39, m 0.92, d (6.5) 1.51, m; 1.15, m 2.22, m; 2.19, m 6.86, t (7.4) 1.84, s 0.89, s 0.94, s 1.21, s 2.10, s 3.19, s 2.08, s
3.23, dd (11.7, 4.4) 1.01, m 1.69, m; 1.48, m 2.03, m; 1.87, m 2.06−2.01 (2H), m 1.84, m; 1.67, m 5.03, dd (9.6, 5.4) 2.07, m; 1.68, m 1.66, m 0.77, s 0.97, s 1.40, m 0.92, d (6.5) 1.49, m; 1.13, m 2.23, m; 2.09, m 6.85, t (7.0) 1.82, s 1.00, s 0.80, s 1.00, s
1.56, m 1.72, m; 1.58, m 2.07, m; 1.95, m 2.10−2.08 (2H), m 1.86, m; 1.66, m 5.05, dd (9.3, 5.0) 2.10, m; 1.70, m 1.67, m 0.80, s 1.12, s 1.40, m 0.92, d (6.5) 1.50, m; 1.14, m 2.22, m; 2.11, m 6.84, tq (7.3, 1.3) 1.83, br s 1.09, s 1.06, s 1.01, s
2.04, s
2.04, s
constants of H-3 (δH 3.23, dd, J = 11.7, 4.4 Hz) and NOESY correlations H-3/Hα-1, H-3/Hα-2, and H-3/H-5. The 15α-
dd (11.8, 4.0) br d (12.5) m; 1.36, m br s m; 2.05, m m; 1.62, m dd (10.5, 5.0) m; 1.71, m m s s m d (7.0) m; 1.15, m m; 2.20, m t (7.0) s s s s s s s
2.12, m 1.90, m; 1.39, m 3.59, br s 2.11−2.09 (2H), m 1.88, m; 1.64, m 5.10, dd (10.0, 4.5) 2.14, m; 1.83, m 1.81, m 0.71, s 1.03, s 1.50, m 0.96, d (6.7) 5.01, t (7.0) 2.56, m; 2.33, m 6.76, t (7.3) 1.86, s 1.10, s 1.08, s 1.17, s 3.21, s 2.09, s 2.06, s
acetoxy group was assigned by the HMBC correlations from H3-30 to C-15 (δC 76.1) and from H-15 (δH 5.03, dd, J = 9.6, 1365
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Table 5. Biological Activities of the Lanostane Triterpenoids from Cultures of Ganoderma spp. compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16d 17d 18d 19c 20 21c 22 23 24d 25d 26d 27d 28d 29 30 31
anti-TBa
cytotoxicityb
MIC, μg/mL
IC50, μM
25 12.5 25 50 12.5 3.13 >50 50 50 12.5 50 3.13 50 >50 25 25 1.56 3.13 >50 50 >50 6.25 1.56 >50 50 0.781 3.13 >50 50 12.5 25
compound c
31 77 84 39 83 103 >92 92 95 >96 >98 30 >85 >83 >95 72 32 28 >95 >95 >85 32 87 33 91 22 15 93 38 26 >103
32 33 34d 35 36c 37 38 39 40 41 42c 43c 44 45c 46c 47 48 49c 50 51d 52 53d 54d 55d 56d 57 58d 59d isoniazidf ellipticineg
anti-TBa
cytotoxicityb
MIC, μg/mL
IC50, μM
50 >50 12.5 0.391 0.781 6.25 >50 6.25 >50 50 6.25 50 25 >50 >50 12.5 25 >50 12.5 50 50 >50 25 12.5 50 50 >50 50 0.094
36 >101 32 32 16 13 >110 95 33 32 28 >98 94 >88 35 >98 87 >95 28 30 105 86 28 26 32 87 89 >107 3.3
a
Growth inhibitory activity against Mycobacterium tuberculosis H37Ra. bCytotoxic activity against Vero cells (African green monkey kidney fibroblasts). cPreviously reported data of the compounds isolated from G. orbiforme BCC 22324.6 dPreviously reported data of the compounds isolated from Ganoderma sp. BCC 16642.7 ePreviously isolated from G. orbiforme BCC 22324 and/or Ganoderma sp. BCC 16642, whose biological activities were newly tested in the present study. fPositive control for the anti-TB assay. gPositive control for the cytotoxicity assay.
5.4 Hz) to an ester carbonyl carbon at δC 171.1 and the presence of intense NOESY correlations H-15/H3-18 and H15/Hβ-7. Consequently, compound 10 was identified as a new lanostanoid, (24E)-15α-acetoxy-3β-hydroxylanosta-8,24-dien26-oic acid. Compound 11 was also assigned as a 7unsubstituted (methylene) derivative. It had 3-oxo (δC 217.5, C-3) and 15α-acetoxy (δH 5.05, dd, J = 9.3, 5.0 Hz, H-15) functional groups. The molecular formula of compound 12 was determined by HRESIMS as C35H54O7. Interpretation of its 1D and 2D NMR spectroscopic data revealed the presence of a 7α-methoxy (δH 3.18, 3H, s; δC 55.2) and 3β- and 15α-acetoxy groups in addition to the C-8/C-9 double bond and an E-configured α,βunsaturated carboxylic acid. HMBC correlations from the methoxy protons, H-5, and Hα-6 to the oxymethine carbon (δC 76.1, C-7) and from the oxymethine proton (δH 3.57, s, H-7) to C-5, C-8, and C-9 confirmed the location of the methoxy group. Similar to the known 7α-methoxy derivatives, H-7 exhibited very small coupling constants, which confirmed its equatorial (β) orientation. Intense NOESY correlations H-15/ H-7 and H-15/H3-18 confirmed the β-orientations of H-7 and H-15. Accordingly, compound 12 was identified as a new Ganoderma lanostanoid, (24E)-3β,15α-diacetoxy-7α-methoxy-
lanosta-8,24-dien-26-oic acid. Compound 13 was identified as the C-3 epimer of 12. An equatorial (β) orientation of H-3 (δH 4.68, br s) was evident from its small coupling constants. Compound 14 was also identified as a 7α-methoxy derivative. Other key functional groups of 14 were C-3 ketone (δC 217.4) and 15α- and 22S-acetoxy groups (δH 5.10, dd, J = 10.0, 4.5 Hz, H-15; δH 5.01, t, J = 7.0 Hz, H-22). All new compounds (1−14) were subjected to our bioassay protocols: anti-TB activity against Mycobacterium tuberculosis H37Ra and cytotoxicity against nonmalignant Vero cells (Table 5). Activities of several known compounds, which have not been previously tested in our research group, were also evaluated. For convenience of the SAR discussion, our previously reported data for the lanostanoids isolated from G. orbiforme BCC 22324 and/or Ganoderma sp. BCC 16642 are also listed in Table 5.7,8 (24E)-3β,15α-Diacetoxylanosta-7,9(11),24-trien-26-oic acid (35) exhibited the highest anti-TB activity, with an MIC value of 0.391 μg/mL, while compounds 26 and 36 were the second highest activity group, with an MIC of 0.781 μg/mL. These compounds possessed both 3β-acetoxy and 15α-acetoxy groups. Comparison of the anti-TB MIC values of 36, 40, 42, 45, and 48, which share the same structure (7,9(11)-diene and 1366
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Figure 3. Structure−activity relationship for the anti-TB activities of mycelium-associated Ganoderma lanostanoids.
15α-acetoxy and 22S-acetoxy groups), but differ only in the C-3 functional group, revealed that the 3β-acetoxy group (36) is crucial for the anti-TB activity. The significance of the 15αacetoxy group was suggested by the comparison of the anti-TB activities of 33, 35, and 37 and also by comparison of 4, 16, and 17. Comparisons of the MIC values between 35 and 36 and between 26 and 27 suggested that the 22-acetoxy group slightly reduces anti-TB activity. The MIC values of the 3β,15αdiacetoxy derivatives, 17, 26, 12, and 35, which differ only in the functional groups at C-7, revealed that the 7,9(11)-diene isomer (35) showed higher activity than the 8-ene congeners. In addition, the bulky 7α-methoxy group (12) was shown to reduce the activity. The overall SAR is proposed in Figure 3. This SAR follows our preliminary report,8 but in the present study it is further supported by additional data. In particular, the optimal anti-TB lanostane triterpenoid 35 was isolated and evaluated. This compound was originally isolated from cultures of G. lucidum by Shiao and co-workers and named ganoderic acid S.16 However, it should also be noted that this trivial name had been assigned for 43 in an earlier published paper by Shiro and co-workers;20 therefore, in this paper we avoid using this name for compound 35. It is not certain if the SAR rule is applicable to the 7α-acetoxy-8-ene derivatives, because of the lack of data for 3β,15α-diacetoxy congeners in this group. However, all the isolated 7α-acetoxy derivatives (1, 2, and 15) exhibited weak anti-TB activity, and these activities were similar to corresponding 7α-hydroxy derivatives (4 and 5). As an exception, 7-oxo derivatives (8, 9, and 22−25) are not applicable to the proposed SAR rule. In particular, compound 23, a 3β-hydroxy-7-oxo derivative, exhibited significant and higher anti-TB activity (MIC 1.56 μg/mL) than the 3β-acetoxy7-oxo derivative 22 (MIC 6.25 μg/mL). Cytotoxic activities of the Ganoderma lanostanoids were relatively weaker when compared with their anti-TB activities, and there was no clear correlation in the inhibitory potential between these biological activities. The mechanism of action of the anti-TB activity for these mycelium-associated Ganoderma lanostanoids is currently unknown. The present results, taken together with our previous reports, demonstrate that Ganoderma is a rich source of lanostane triterpenoids. Many of these mycelium-associated lanostanoids possess significant anti-TB activity. The SAR for anti-TB activity has been proposed, wherein (24E)-3β,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (35) is the optimal
lanostanoid. We are currently planning chemical modification of this TB drug lead compound.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a JASCO P-1030 digital polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. FTIR spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on Bruker DRX400 and AV500D spectrometers. NMR solvent (CDCl3) was passed through a short column on aluminum oxide (Merck, aluminum oxide 90 active basic) prior to sample preparation in order to remove the contamination of acid.8 ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. Fungal Material. Ganoderma orbiforme was isolated from a dead oil palm (Elaeis guineensis) trunk in a plantation area, Klong Thom, Krabi Province, Thailand, on May 4, 2006. The mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 19068, and the living culture was deposited in the BIOTEC Culture Collection as BCC 22325. Identification of this fungus is based on the morphology and ITS rDNA sequence data (GenBank accession number KX421867). Ganoderma australe was also isolated from a dead oil palm (Elaeis guineensis) trunk in the plantation area, Klong Thom, Krabi Province, Thailand, on May 4, 2006. The mushroom specimen was deposited in the BIOTEC Bangkok Herbarium as BBH 19074, and the living culture was deposited in the BIOTEC Culture Collection as BCC 22314. Identification of this fungus is based on the morphology and ITS rDNA sequence data (GenBank accession number KX421866). Ganoderma sp. was isolated from an unidentified wood sample from Doi Suthep-pui National Park, Chiang Mai Province, Thailand, on October 7, 2012. The mushroom specimen was deposited in the Herbarium of Mae Fah Luang University as MFLU 13-0534. The living culture was deposited both in the Mae Fah Luang University Culture Collection as MFLUCC 12-0527 and in the BIOTEC Culture Collection as BCC 60695, and the latter biological material was used for the present chemical study. Identification of this fungus is based on the morphology and ITS rDNA sequence data (GenBank accession number KP142173). A BLAST search showed 99% identity to Ganoderma gibbosum strain T 10 and Ganoderma sp. 4 YD-2015 voucher PDD 101331. Fermentation, Extraction, and Isolation: Ganoderma orbiforme BCC 22325. The fungus BCC 22325 was maintained on potato dextrose agar at 25 °C. The agar was cut into small plugs and inoculated into 6 × 250 mL Erlenmeyer flasks containing 25 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). After incubation at 25 °C for 7 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 (MEB) and incubated at 25 °C for 7 days on a 1367
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λmax (log ε) 219 (3.89) nm; IR (ATR) νmax 1719, 1687, 1374, 1246, 1026 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 595.3606 [M + Na]+ (calcd for C34H52O7Na, 595.3605). (22S,24E)-15α-Hydroxy-3β,7α,22-triacetoxylanosta-8,24-dien-26oic acid (2): colorless solid; [α]25D +20 (c 0.365, CHCl3); UV (MeOH) λmax (log ε) 218 (3.73) nm; IR (ATR) νmax 1730, 1375, 1242, 1024 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 653.3662 [M + Na]+ (calcd for C36H54O9Na, 653.3660). (22S,24E)-3β,22-Diacetoxy-7α-hydroxylanosta-8,24-dien-26-oic acid (3): colorless solid; [α]24D +23 (c 0.145, CHCl3); UV (MeOH) λmax (log ε) 215 (3.63) nm; IR (ATR) νmax 1735, 1718, 1374, 1242 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESITOF) m/z 595.3615 [M + Na]+ (calcd for C34H52O7Na, 595.3605). (24E)-3β-Acetoxy-7α,15α-dihydroxylanosta-8,24-dien-26-oic acid (4): colorless solid; [α]25D +53 (c 0.175, CHCl3); UV (MeOH) λmax (log ε) 217 (3.91) nm; IR (ATR) νmax 3323, 1732, 1713, 1686, 1375, 1247 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESI-TOF) m/z 553.3493 [M + Na]+ (calcd for C32H50O6Na, 553.3500). (22S,24E)-3β,22-Diacetoxy-7α,15α-dihydroxylanosta-8,24-dien26-oic acid (5): colorless solid; [α]25D +41 (c 0.355, CHCl3); UV (MeOH) λmax (log ε) 218 (3.79) nm; IR (ATR) νmax 1732, 1714, 1375, 1243, 1027 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESITOF) m/z 611.3560 [M + Na]+ (calcd for C34H52O8Na, 611.3554). (24E)-7α-Hydroxy-3-oxolanosta-8,24-dien-26-oic acid (6): colorless solid; [α]25D +89 (c 0.18, CHCl3); UV (MeOH) λmax (log ε) 218 (3.83) nm; IR (ATR) νmax 1689, 1379, 1282 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESI-TOF) m/z 493.3282 [M + Na]+ (calcd for C30H48O4Na, 493.3288). (22S,24E)-22-Acetoxy-7α,15α-dihydroxy-3-oxoanosta-8,24-dien26-oic acid (7): colorless solid; [α]25D +55 (c 0.285, CHCl3); UV (MeOH) λmax (log ε) 218 (3.71) nm; IR (ATR) νmax 3370, 1733, 1704, 1377, 1240, 1061 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 3; HRMS (ESI-TOF) m/z 567.3290 [M + Na]+ (calcd for C32H48O7Na, 567.3292). (24E)-3β-Acetoxy-15α-hydroxy-7-oxolanosta-8,24-dien-26-oic acid (8): colorless solid; [α]25D +43 (c 0.175, CHCl3); UV (MeOH) λmax (log ε) 220 (3.86), 254 (3.80) nm; IR (ATR) νmax 1732, 1686, 1643, 1372, 1245 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESITOF) m/z 551.3342 [M + Na]+ (calcd for C32H48O6Na, 551.3343). (22S,24E)-22-Acetoxyl-3α-hydroxy-7-oxolanosta-8,24-dien-26oic acid (9): colorless solid; [α]25D +9 (c 0.215, CHCl3); UV (MeOH) λmax (log ε) 218 (3.90), 252 (3.84) nm; IR (ATR) νmax 1733, 1712, 1375, 1241 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 2; HRMS (ESI-TOF) m/z 551.3340 [M + Na]+ (calcd for C32H48O6Na, 551.3343). (24E)-15α-Acetoxy-3β-hydroxylanosta-8,24-dien-26-oic acid (10): colorless solid; [α]28D +52 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 219 (3.93), 294 sh (3.46) nm; IR (ATR) νmax 1735, 1725, 1374, 1249, 1031 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESITOF) m/z 537.3555 [M + Na]+ (calcd for C32H50O5Na, 537.3550). (24E)-15α-Acetoxy-3-oxolanosta-8,24-dien-26-oic acid (11): colorless solid; [α]25D +77 (c 0.095, CHCl3); UV (MeOH) λmax (log ε) 218 (3.85) nm; IR (ATR) νmax 1733, 1706, 1376, 1248, 1042 cm−1; for 1 H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESI-TOF) m/z 534.3389 [M + Na]+ (calcd for C32H48O5Na, 535.3394). (24E)-3β,15α-Diacetoxy-7α-methoxylanosta-8,24-dien-26-oic acid (12): colorless solid; [α]28D +36 (c 0.335, CHCl3); UV (MeOH) λmax (log ε) 217 (3.89) nm; IR (ATR) νmax 1729, 1688, 1375, 1248 cm−1; for 1H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESITOF) m/z 609.3751 [M + Na]+ (calcd for C35H54O7Na, 609.3762).
rotary shaker (200 rpm). The secondary cultures were pooled, and each 25 mL portion was transferred into 60 × 1000 mL Erlenmeyer flasks containing 250 mL of MEB. The final fermentation was carried out at 25 °C for 20 days under static conditions. The cultures were filtered, and the residual wet mycelia were macerated in MeOH (2.9 L, 25 °C, 2 days) and filtered. Hexane (2.9 L) was added to the filtrate, and the layers were separated. The MeOH (bottom) layer was partially concentrated by evaporation, and the residue was extracted with EtOAc (4.7 L), which was concentrated under reduced pressure to obtain a brown gum (extract A1, 3.24 g). The hexane layer was concentrated under reduced pressure to leave a pale brown gum (extract B1, 760 mg). The residual mycelial cakes were extracted once again using the same procedure to give a MeOH layer (extract A2, 1.73 g) and hexane layer (extract B2, 765 mg). Extracts A1 and A2 were subjected to column chromatography (CC) on silica gel (EtOAc− CH2Cl2, step gradient elution), and the fractions were further fractionated and purified by preparative HPLC using reversed-phase columns (Phenomenex Luna 10u C18 100A, 21.2 × 250 mm, 10 μm, or Grace Grom-Sil 120 ODS-4 HE, 20 × 150 mm, 5 μm; mobile phase MeCN−H2O, proportions 55:45−80:20; flow rate 8 mL/min) to furnish pure compounds 9 (9.1 mg), 10 (6.6 mg), 14 (48 mg), 18 (14 mg), 19 (24 mg), 20 (175 mg), 21 (243 mg), 22 (10 mg), 24 (3.0 mg), 25 (15 mg), 30 (28 mg), 32 (49 mg), 36 (59 mg), 40 (5.0 mg), 42 (202 mg), 43 (19 mg), 44 (11 mg), 45 (131 mg), 46 (72 mg), 47 (45 mg), 48 (54 mg), and 49 (2.6 mg). Extracts B1 and B2 were also fractionated using the same chromatographic procedures to furnish pure compounds 12 (6.8 mg), 13 (8.9 mg), 29 (3.8 mg), 31 (1.0 mg), 32 (3.5 mg), 34 (1.8 mg), 35 (5.9 mg), 36 (30 mg), 41 (10 mg), 42 (64 mg), 43 (9.0 mg), 44 (3.4 mg), and 46 (18 mg). Fermentation, Extraction, and Isolation (Non-MeOH Procedure): Ganoderma sp. BCC 60695. The fermentation of the fungus BCC 60695 was conducted using a similar procedure to that for BCC 22325. Final fermentation was carried out using 40 × 1000 mL Erlenmeyer flasks (media, MEB) at 25 °C for 28 days under static conditions. The cultures were filtered, and the residual wet mycelia were macerated in acetone (4.5 L, 25 °C, 2 days) and filtered. The filtrate was concentrated under reduced pressure, and the residue was extracted with EtOAc (2.3 L × 3) and concentrated under reduced pressure to obtain a brown gum (mycelial extract, 3.64 g). The mycelial extraction was repeated again (2.67 g). The combined mycelial extract was subjected to CC on silica gel (4.8 × 15 cm; EtOAc−CH2Cl2, step gradient elution from 0:100 to 100:0, then with acetone−CH2Cl2 20:80, 50:50, and 100:0). The fractions were further fractionated and purified by a combination of CC on silica gel (acetone−hexane or EtOAc−hexane) and preparative HPLC (MeCN−H2O) to furnish pure compounds 1 (152 mg), 2 (63 mg), 3 (14 mg), 4 (154 mg), 5 (95 mg), 6 (8.0 mg), 7 (64 mg), 8 (2.9 mg), 9 (14 mg), 10 (11 mg), 11 (2.0 mg), 15 (39 mg), 16 (12 mg), 17 (144 mg), 18 (21 mg), 19 (25 mg), 22 (38 mg), 23 (2.0 mg), 25 (2.2 mg), 26 (123 mg), 27 (8.7 mg), 28 6.9 mg), 33 (1.7 mg), 34 (21 mg), 35 (131 mg), 36 (10 mg), 37 (88 mg), 38 (2.8 mg), 39 (18 mg), 40 (5.0 mg), 46 (20 mg), 48 (11 mg), and 49 (2.6 mg). Fermentation, Extraction, and Isolation (Non-MeOH Procedure): Ganoderma australe BCC 22314. The fermentation of the fungus BCC 22314 was conducted using a similar procedure to that for BCC 22325. Final fermentation was carried out using 83 × 1000 mL Erlenmeyer flasks (media, MEB) at 25 °C for 96 days under static conditions. The cultures were filtered, and the residual wet mycelia were macerated in acetone (2.5 L, 25 °C, 2 days) and filtered. The filtrate was concentrated under reduced pressure, and the residue was extracted with EtOAc (1.6 L × 3) and concentrated under reduced pressure to obtain a brown gum (mycelial extract, 1.72 g). The mycelial extraction was repeated again (2.32 g). The combined mycelial extract was fractionated by a combination of silica gel CC and preparative HPLC (MeCN−H2O) to furnish pure compounds 1 (304 mg), 3 (3.0 mg), 4 (134 mg), 6 (24 mg), 15 (30 mg), 16 (53 mg), 17 (76 mg), 18 (6.0 mg), 19 (5.0 mg), 20 (8.0 mg), 26 (165 mg), 35 (141 mg), 36 (8.0 mg), and 37 (78 mg). (24E)-3β,7α-Diacetoxy-15α-hydroxylanosta-8,24-dien-26-oic acid (1): colorless solid; [α]25D +29 (c 0.265, CHCl3); UV (MeOH) 1368
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(24E)-3α,15α-Diacetoxy-7α-methoxylanosta-8,24-dien-26-oic acid (13): colorless solid; [α]27D +16 (c 0.29, CHCl3); UV (MeOH) λmax (log ε) 217 (3.75) nm; IR (ATR) νmax 1726, 1375, 1249 cm−1; for 1 H NMR (400 MHz) and 13C NMR (100 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESI-TOF) m/z 609.3760 [M + Na]+ (calcd for C35H54O7Na, 609.3762). (22S,24E)-15α,22-Diacetoxy-7α-methoxy-3-oxolanosta-8,24dien-26-oic acid (14): colorless solid; [α]28D +49 (c 0.41, CHCl3); UV (MeOH) λmax (log ε) 217 (4.03), 298 sh (3.51) nm; IR (ATR) νmax 1728, 1375, 1244 cm−1; for 1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data in CDCl3, see Tables 1 and 4; HRMS (ESITOF) m/z 623.3560 [M + Na]+ (calcd for C35H52O8Na, 623.3554). Biological Assays. Antimycobacterial activity against Mycobacterium tuberculosis H37Ra and cytotoxicity to Vero cells (African green monkey kidney fibroblasts) were evaluated using the green fluorescent protein (GFP)-based microplate assay.23,24
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(14) Arpha, K.; Phosri, C.; Suwannasai, N.; Mongkolthanaruk, W.; Sodngam, S. J. Agric. Food Chem. 2012, 60, 9834−9841. (15) Nishitoba, T.; Sato, H.; Shirasu, S.; Sakamura, S. Agric. Biol. Chem. 1987, 51, 619−622. (16) Shiao, M.-S.; Lin, L.-J. J. Nat. Prod. 1987, 50, 886−890. (17) Shiao, M.-S.; Lin, L.-J.; Yeh, S.-F. Phytochemistry 1988, 27, 2911−2914. (18) Lin, L. J.; Shao, M. S.; Yeh, S. F. Phytochemistry 1988, 27, 2269− 2271. (19) Shiao, M.-S.; Lin, L.-J.; Yeh, S.-F. Phytochemistry 1988, 27, 873− 875. (20) Hirotani, M.; Ino, C.; Furuya, T.; Shiro, M. Chem. Pharm. Bull. 1986, 34, 2282−2285. (21) Li, C.-H.; Chen, P.-Y.; Chang, U.-M.; Kan, L.-S.; Fang, W.-H.; Tsai, K.-S.; Lin, S.-B. Life Sci. 2005, 77, 252−265. (22) Yang, P. G.-M.; Wang, H.-H.; Cheng, T.-L.; Liou, J.-J. Rept. Taiwan Sugar Res. Inst. 2002, 175−176, 21−31. (23) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Chemother. 2003, 47, 3682−3687. (24) Hunt, L.; Jordan, M.; De Jesus, M.; Wurm, F. M. Biotechnol. Bioeng. 1999, 65, 201−205.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00973. NMR spectra of compounds 1−14 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +66-25646700, ext 3554. Fax: +66-25646707. E-mail:
[email protected]. ORCID
Masahiko Isaka: 0000-0002-9229-3394 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support from the Thailand Research Fund (Grant No. DBG5980002) is gratefully acknowledged. T. Luangharn is thanked for collection and isolation of the Ganoderma sp. from Chiang Mai.
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