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Hopane triterpenes from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276
Phytochemistry Letters 5 (2012) 734–737
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Hopane triterpenes from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276 Masahiko Isaka *, Panida Chinthanom, Sumalee Supothina, Suchada Mongkolsamrit 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
A B S T R A C T
Article history: Received 8 May 2012 Received in revised form 27 July 2012 Accepted 1 August 2012 Available online 13 August 2012
Two new hopane-type triterpenenes, 6a,15a,22-trihydroxyhopane (4) and 3b-acetoxy-6a,22-dihydroxyhopane (5), together with the known 6a,22-dihydroxyhopane (1, zeorin), 15a,22-dihydroxyhopane (2, dustanin), and 3b-acetoxy-15a,22-dihydroxyhopane (3) were isolated from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Aschersonia calendulina Entomopathogenic fungi Hopanoid
1. Introduction Aschersonia is a genus of entomopathogenic fungi that specifically attacks scale insects. Species in this genus have been sources of bioactive secondary metabolites such as hopane-type triterpenoids from A. aleyrodis (van Eijk et al., 1986), A. tubulata BCC 1785 (Boonphong et al., 2001) and A. paraphysata BCC 11964 (Isaka et al., 2010), tetrahydroxanthone dimers (ascherxanthones A and B) from Aschersonia sp. BCC 8401 (Isaka et al., 2005) and A. luteola BCC 8774 (Chutrakul et al., 2009), binaphthopyrones from A. paraphysata BCC 11964 (Isaka et al., 2010), and cyclodepsipeptides (destruxins A4 and A5) from an Aschersonia species (Krasnoff et al., 1996). Recently we conducted a broad survey of Aschersonia species and their Hypocrella sensu lato teleomorphs (Moelleriella and Hypocrella species) for their secondary metabolites, which resulted in the conclusion that this group of fungi commonly produce three hopane triterpenes, 6a,22-dihydroxyhopane (1, zeorin), 15a,22-dihydroxyhopane (2, dustanin), and 3b-acetoxy15a,22-dihydroxyhopane (3) (Isaka et al., 2009). In our further study of this group of fungi, we have investigated a later described species, Aschersonia calendulina (strain BCC 23276) (Mongkolsamrit et al., 2009), which led to the isolation of two new hopanoids, 6a,15a,22-trihydroxyhopane (4) and 3b-acetoxy-6a,22-dihydroxyhopane (5) in addition to 1–3.
* Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707. E-mail address: [email protected] (M. Isaka).
2. Results and discussion Aschersonia calendulina BCC 23276 was fermented in a bioreactor using 7 l of potato dextrose broth (PDB) medium. Two new compounds (4 and 5) were isolated by fractionation of the extracts from the fermentation broth (see Section 3) by column chromatography on Sephadex LH-20 and silica gel. Three known hopanoids, 1 (Tsuda et al., 1967), 2 (Tsuda and Isobe, 1965; Tsuda et al., 1967), and 3 (Boonphong et al., 2001), ergosterol, and mevalonolactone were also isolated from the same extracts. Compound 4 was isolated as a colorless solid, and the molecular formula was determined as C30H52O3, by the sodiated quasi-molecular ion peak at m/z 483.3807 (calc. 483.3809) in the
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.08.002
M. Isaka et al. / Phytochemistry Letters 5 (2012) 734–737
HR–ESI-MS. The 1H and 13C NMR spectroscopic data of 4 were similar to those of the co-metabolites 1 and 2. Interpretation of the 1 H and 13C NMR, DEPT135, and HMQC data for 4 revealed the presence of an oxygenated quaternary carbon (dC 73.7), two oxygenated methines (dC 69.0 and 74.8), five quaternary carbons, five methines, nine methylenes, and eight methyl groups (Table 1). The planar structure was deduced by analyses of COSY and HMBC data. Key HMBC correlations were those from eight methyl groups (H3-23, H3-24, H3-25, H3-26, H3-27, H3-28, H3-29, and H3-30) to their attached quaternary carbons (2J), C-4, C-4, C-10, C-8, C-14, C18, C-22, and C-22, respectively, and their 3J correlations. HMBC correlations from H-5 to C-4, C-6, C-7, C-9, C-10, C-23, C-24, and C25 were employed for the assignments of protons and carbons of the ring AB moiety. The upfield methine H-5 (dH 0.83, d, J = 10.7) exhibited 1,2-diaxial 1H–1H coupling to a secondary alcohol methine (dH 3.93, dt, J = 3.9, 10.7 Hz, H-6), which indicated the 6ahydroxy group. The 1H and 13C chemical shifts in the ring AB region showed close resemblance to those of zeorin (1). The location of the other secondary alcohol was achieved by the HMBC correlations from H-15 (dH 3.84, dd, J = 10.0, 5.4 Hz) to C-27, and from H327 (dH 1.00, s) to C-15 (dC 73.1). The axial orientation of this oxymethine proton was evident from the observed coupling constants involving an 1,2-diaxial 1H–1H coupling (J = 10.0 Hz). The axial (b) orientations of H-5 and H-15 were further supported by the NOESY correlations of these protons with H3-26. Chemical shifts of protons and carbons of the ring CDE moiety were similar to those of dustanin (2). The tertiary alcohol functionality was located at C-22 on the basis of the HMBC correlations from H-17, H-21, H329, and H3-30 to the oxygenated quaternary carbon (dC 73.7). Compound 4 was therefore assigned as a new hopane-type triterpene, 6a,15a,22-trihydroxyhopane.
735
The molecular formula of compound 5 was determined as C32H54O4 by HRESIMS. The 1H and 13C NMR spectra suggested the presence of a tertiary alcohol (dC 73.7), and two oxymethines at dC 68.8 and dC 80.8, and an acetoxy group (dC 171.1, 21.3; dH 2.06, 3H, s). An intense sharp absorption band at nmax 1735 cm 1 in the IR spectrum further confirmed the presence of an acetoxy group, which was located at C-3 (dC 80.8) based on the HMBC correlation from the attached proton (H-3) to the ester carbonyl (dC 171.1) and the correlations from H3-23 and H3-24 to the oxymethine carbon (C-3). The axial (a) orientation of H-3 was evident from its coupling constant values (dH 4.44, dd, J = 11.5, 5.1 Hz). The secondary alcohol was assigned as 6-OH on the basis of the COSY correlations of the oxymethine proton (H-6, dH 4.03) to H-5 and H2-7. The coupling constants for H-6 (dt, J = 3.9, 10.6 Hz) indicated its axial (b) orientation. The C-22 tertiary alcohol was confirmed by the HMBC correlations from H3-29 and H3-30 to this quaternary carbon. The planar structure was established by interpretation of COSY and HMBC (Table 1) data. The 1H and 13C NMR spectroscopic data for the ring A moiety were similar to those of 3, whereas the data for all others (BCDE rings) showed very close resemblance to those of zeorin (1). The following NOESY correlations (Fig. 1) were consistent with the hopane-type relative configuration: H-5/H325, H-5/H3-26, H3-26/H-13, H3-26/Hb-15, H-17/H-21, H-1/H-3, H3/H-5, H-9/H3-27, H3-27/Ha-12, and Ha-16/H3-28. Compound 5 was therefore identified as 3b-acetoxy-6a,22-dihydroxyhopane. Hopanoids 1–5, isolated from BCC 23276, were tested for antitubercular activity against Mycobacterium tuberculosis H37Ra, since the known 1–3 were previously shown to exhibit the same MIC value of 12.5 mg/ml (Boonphong et al., 2001; Isaka et al., 2009). Unexpectedly, all compounds (1–5) were inactive at a concentration of 50 mg/ml in our present assay. The reason of the
Table 1 NMR data for 4 and 5 in CDCl3. No.
4
5
dC, mult.
dH, mult. (J in Hz)
1 2 3 4 5 6 7
40.5, 18.5, 43.7, 33.6, 60.8, 69.0, 48.8,
a 0.80, m; b 1.65, m a 1.37, m; b 1.54, m a 1.17, m; b 1.32, m
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
44.1,a qC 50.1, CH 39.2, qC 21.0, CH2 24.0, CH2 48.6, CH 47.1, qC 74.8, CH 32.8, CH2 50.5,b CH 44.2,a qC 40.9, CH2 26.9, CH2 50.6,a CH 73.7, qC 36.7, CH3 22.1, CH3 17.2, CH3 18.8, CH3 11.8, CH3 15.7, CH3 28.6, CH3 31.0, CH3
a–c
CH2 CH2 CH2 qC CH CH CH2
HMBC
0.83, d (10.7) 3.93, dt (3.9, 10.7) a 1.70, m b 1.87, dd (12.5, 3.9)
4, 6, 7, 10, 23, 24, 25
1.17, m
25
a 1.59, m; b 1.21, m a 1.57, m; b 1.41, m
13, 14
6, 8, 26 5, 6, 8, 9, 26
1.27, m 3.84, dd (10.0, 5.2) a 1.65, m; b 2.23, m 1.44, m
27 14, 18 22, 28
a 1.52, m; b 0.90, m a 1.47, m; b1.76, m 2.21, m
13, 18, 20 22 17, 20, 22
1.16, 1.00, 0.87, 1.12, 1.02, 0.75, 1.16, 1.18,
3, 4, 5, 24 3, 4, 5, 23 1, 5, 9, 10 7, 8, 9, 14 8, 13, 14, 15 13, 17, 18, 19 21, 22, 30 21, 22, 29
s s s s s s s s
The carbon assignment may be interchanged.
dC, mult.
dH, mult. (J in Hz)
38.2, 23.3, 80.8, 38.1, 60.5, 68.8, 45.9,
a 1.03, m; b 1.68, m a 1.65, m; b 1.60, m
CH2 CH2 CH qC CH CH CH2
43.0,c qC 49.7, CH 39.2, qC 21.0, CH2 23.9, CH2 49.4, CH 41.9,c qC 34.4, CH2 21.9, CH2 53.9, CH 44.0, qC 41.2, CH2 26.6, CH2 51.1, CH 73.9, qC 30.8, CH3 16.6, CH3 16.9, CH3 18.3, CH3 17.0, CH3 16.1, CH3 28.8, CH3 30.9, CH3 171.1, qC 21.3, CH3
4.44, dd (11.5, 5.1) 0.92, d (10.6) 4.03, dt (3.9, 10.6) a 1.45, m; b 1.57, m
HMBC 10 3-OCOCH3 4, 6, 7, 9, 10, 24, 25 6, 26
1.23, m
a 1.57, m; b 1.24, m a 1.54, m; b 1.42, m 1.34, m
15
a 1.22, m; b 1.42, m a 1.60, m; b 1.96, m
17 15
1.43, m
a 1.52, m; b 0.93, m a 1.49, m; b 1.76, m 2.22, m
21, 22 19
1.17, 1.05, 0.89, 1.04, 0.96, 0.76, 1.18, 1.20,
3, 4, 5, 24 3, 4, 5, 23 1, 5, 9, 10 7, 8, 9, 14 8, 13, 14, 15 13, 17, 18, 19 21, 22, 30 21, 22, 29
s s s s s s s s
2.06, s
3-OCOCH3
M. Isaka et al. / Phytochemistry Letters 5 (2012) 734–737
736
Fig. 1. Key NOESY correlations for 5.
inconsistent assay result for 1–3 is uncertain; however, it may be related to the change of the assay protocol from the visial microplate Alamar Blue (resazurin) assay (Collins and Franzblau, 1997) to the currently employing green fluorescent protein microplate assay (Changsen et al., 2003). 3. Experimental 3.1. General experimental procedures Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. IR spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on a Bruker AV500D spectrometer. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. Merck Silica gel 60H (particle size, 90% <45 mM) was used for column chromatography. 3.2. Fungal material Aschersonia calendulina was collected from a scale insect (Hemiptera) in Doi Inthanon National Park, Chiang Mai Province, Thailand, by one of the authors (S.M.). This fungus was deposited in the BIOTEC Culture Collection (BCC) on October 11, 2006 as BCC 23276. The identification is based on the morphology of the fungus and macro morphology on the scale insect (Mongkolsamrit et al., 2009).
was fractionated by Sephadex LH-20 column chromatography (3.6 cm 54 cm; MeOH) to obtain five pooled fractions. The second fraction (560 mg) was subjected to column chromatography (CC) on silica gel (3.0 cm 14 cm, step gradient elution with 0–100% EtOAc in CH2Cl2) to afford 2 (10 mg), 3 (10 mg), mevalonolactone (14 mg), and a fraction containing 4 (12 mg). The last fraction was purified by CC on silica gel (1.2 cm 10 cm, EtOAc/hexane = 70:30) to furnish 4 (4.8 mg). Extract B was fractionated by CC on silica gel (3.2 cm 11 cm, step gradient elution with 0–100% EtOAc in CH2Cl2) to obtain ergosterol (30 mg), 1 (20 mg), and a fraction containing 5 (3 mg). The last fraction was purified by CC on silica gel (EtOAc/hexane = 30:70) to afford 5 (1.0 mg). 3.3.1. 6a,15a,22-Trihydroxyhopane (4) Colorless solid; mp 268–269 8C; [a]26D +28 (c 0.10, MeOH); IR nmax ATR (cm 1): 3375, 2934, 1464, 1387, 1016; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 483.3807 [M+Na]+ (calc. for C30H52O3Na, 483.3809). 3.3.2. 3b-Acetoxy-6a,22-dihydroxyhopane (5) Colorless powder; [a]25D +25 (c 0.08, MeOH); IR nmax ATR (cm 1): 3350, 2943, 1735, 1458, 1368, 1247, 1031; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 525.3904 [M+Na]+ (calc. for C32H54O4Na, 525.3914). 3.4. Antimycobacterial assay Growth inhibitory activity against Mycobacterium tuberculosis H37Ra was performed using the green fluorescent protein microplate assay (Changsen et al., 2003). The MIC values of the standard antituberculosis drugs (positive control), rifampicin, streptomycin, isoniazid, ofloxacin, and ethambutol were 0.025, 0.625, 0.094, 0.781, and 1.88 mg/ml, respectively.
Acknowledgement Financial support from the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BIOTEC), is gratefully acknowledged.
3.3. Fermentation, extraction, and isolation The fungus BCC 23276 was maintained on potato dextrose agar at 25 8C. The agar plugs (1 cm 1 cm) were cut into small pieces and inoculated into 3 250 ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l) and incubated at 25 8C for 5 days on a rotary shaker (200 rpm). Each primary seed culture was transferred into a 1 l Erlenmeyer flask containing 250 ml of PDB, and incubated at 25 8C for 5 days on a rotary shaker (200 rpm). The seed cultures were combined and a 700 ml portion was transferred into a 10 l bioreactor containing 6.3 l of PDB, and the final fermentation was carried out at 25 8C for 7 days under agitation at 200 rpm and aeration 0.5 vvm. The culture was diluted with acetone (4 l) and the mixture was stood for 3 days, and then filtered. The filtrate was partially evaporated, and the residual aqueous solution (ca. 3 l) was extracted with EtOAc (3 800 ml). The combined EtOAc phase was concentrated under reduced pressure to obtain a brown gum (1.52 g, extract A). The residual mycelial cakes were macerated in MeOH (1.2 l) for 4 days and then filtered. The filtrate was evaporated and the residue was diluted with H2O (200 ml) which was then extracted with EtOAc (2 l). The EtOAc layer was concentrated to give a brown gum (1.11 g, extract B). Extract A
References Boonphong, S., Kittakoop, P., Isaka, M., Palittapongarnpim, P., Jaturapat, A., Danwisetkhanjana, K., Tanticharoen, M., Thebtaranonth, Y., 2001. A new antimycobacterial, 3b-acetoxy-15a,22-dihydroxyhopane, from the insect pathogenic fungus Aschersonia tubulata. Planta Med. 67, 279–281. 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. Chutrakul, C., Boonruangprapa, T., Suvannakad, R., Isaka, M., Sirithunya, P., Toojinda, T., Kirtikara, K., 2009. Ascherxanthone B from Aschersonia luteola, a new antifungal compound active against rice blast pathogen Magnaporthe grisea. J. Appl. Microbiol. 107 1634-1631. Collins, L.A., Franzblau, S.G., 1997. Microplate Alamar Blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 41, 1004–1009. Isaka, M., Palasarn, S., Kocharin, K., Saenboonrueng, J., 2005. A cytotoxic xanthone dimer from the entomopathogenic fungus Aschersonia sp. BCC 8401. J. Nat. Prod. 68, 945–946. Isaka, M., Hywel-Jones, N.L., Sappan, M., Mongkolsamrit, S., Saidaengkham, S., 2009. Hopane triterpenes as chemotaxonomic markers for the scale insect pathogens Hypocrella s. lat. and Aschersonia. Mycol. Res. 113, 491–497. Isaka, M., Yangchum, A., Rachtawee, P., Komwijit, S., Lutthisungneon, A., 2010. Hopane-type triterpenes and binaphthopyrones from the scale insect pathogenic fungus Aschersonia paraphysata BCC 11964. J. Nat. Prod. 73, 688–692.
M. Isaka et al. / Phytochemistry Letters 5 (2012) 734–737 Krasnoff, S.B., Gibson, D.M., Belofsky, G.N., Gloer, K.B., Gloer, J.B., 1996. New destruxins from the entomopathogenic fungus Aschersonia sp. J. Nat. Prod. 59, 485–489. Mongkolsamrit, S., Luangsa-ard, J.J., Spatafora, J.W., Sung, G.-H., Hywel-Jones, N.L., 2009. A combined ITS rDNA and b-tubulin phylogeny of Thai species of Hypocrella with non-fragmenting ascospores. Mycol. Res. 113, 684–699.
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Tsuda, Y., Isobe, K., 1965. Dustanin: a new pentacyclic triterpenoid as a fungal metabolite. Tetrahedron Lett. 6, 3337–3343. Tsuda, Y., Isobe, K., Fukushima, S., Ageta, H., Iwata, K., 1967. Final clarification of the saturated hydrocarbons derived from hydroxyhopane, diploptene, zeorin, and dustanin. Tetrahedron Lett. 8, 23–28. van Eijk, G.W., Roeijmans, H.J., Seykens, D., 1986. Hopanoids from the entomogenous fungus Aschersonia aleyrodis. Tetrahedron Lett. 27, 2533–2534.
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Hopane triterpenes from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276 Masahiko Isaka *, Panida Chinthanom, Sumalee Supothina, Suchada Mongkolsamrit 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
A B S T R A C T
Article history: Received 8 May 2012 Received in revised form 27 July 2012 Accepted 1 August 2012 Available online 13 August 2012
Two new hopane-type triterpenenes, 6a,15a,22-trihydroxyhopane (4) and 3b-acetoxy-6a,22-dihydroxyhopane (5), together with the known 6a,22-dihydroxyhopane (1, zeorin), 15a,22-dihydroxyhopane (2, dustanin), and 3b-acetoxy-15a,22-dihydroxyhopane (3) were isolated from the scale insect pathogenic fungus Aschersonia calendulina BCC 23276. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Aschersonia calendulina Entomopathogenic fungi Hopanoid
1. Introduction Aschersonia is a genus of entomopathogenic fungi that specifically attacks scale insects. Species in this genus have been sources of bioactive secondary metabolites such as hopane-type triterpenoids from A. aleyrodis (van Eijk et al., 1986), A. tubulata BCC 1785 (Boonphong et al., 2001) and A. paraphysata BCC 11964 (Isaka et al., 2010), tetrahydroxanthone dimers (ascherxanthones A and B) from Aschersonia sp. BCC 8401 (Isaka et al., 2005) and A. luteola BCC 8774 (Chutrakul et al., 2009), binaphthopyrones from A. paraphysata BCC 11964 (Isaka et al., 2010), and cyclodepsipeptides (destruxins A4 and A5) from an Aschersonia species (Krasnoff et al., 1996). Recently we conducted a broad survey of Aschersonia species and their Hypocrella sensu lato teleomorphs (Moelleriella and Hypocrella species) for their secondary metabolites, which resulted in the conclusion that this group of fungi commonly produce three hopane triterpenes, 6a,22-dihydroxyhopane (1, zeorin), 15a,22-dihydroxyhopane (2, dustanin), and 3b-acetoxy15a,22-dihydroxyhopane (3) (Isaka et al., 2009). In our further study of this group of fungi, we have investigated a later described species, Aschersonia calendulina (strain BCC 23276) (Mongkolsamrit et al., 2009), which led to the isolation of two new hopanoids, 6a,15a,22-trihydroxyhopane (4) and 3b-acetoxy-6a,22-dihydroxyhopane (5) in addition to 1–3.
* Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707. E-mail address: [email protected] (M. Isaka).
2. Results and discussion Aschersonia calendulina BCC 23276 was fermented in a bioreactor using 7 l of potato dextrose broth (PDB) medium. Two new compounds (4 and 5) were isolated by fractionation of the extracts from the fermentation broth (see Section 3) by column chromatography on Sephadex LH-20 and silica gel. Three known hopanoids, 1 (Tsuda et al., 1967), 2 (Tsuda and Isobe, 1965; Tsuda et al., 1967), and 3 (Boonphong et al., 2001), ergosterol, and mevalonolactone were also isolated from the same extracts. Compound 4 was isolated as a colorless solid, and the molecular formula was determined as C30H52O3, by the sodiated quasi-molecular ion peak at m/z 483.3807 (calc. 483.3809) in the
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.08.002
M. Isaka et al. / Phytochemistry Letters 5 (2012) 734–737
HR–ESI-MS. The 1H and 13C NMR spectroscopic data of 4 were similar to those of the co-metabolites 1 and 2. Interpretation of the 1 H and 13C NMR, DEPT135, and HMQC data for 4 revealed the presence of an oxygenated quaternary carbon (dC 73.7), two oxygenated methines (dC 69.0 and 74.8), five quaternary carbons, five methines, nine methylenes, and eight methyl groups (Table 1). The planar structure was deduced by analyses of COSY and HMBC data. Key HMBC correlations were those from eight methyl groups (H3-23, H3-24, H3-25, H3-26, H3-27, H3-28, H3-29, and H3-30) to their attached quaternary carbons (2J), C-4, C-4, C-10, C-8, C-14, C18, C-22, and C-22, respectively, and their 3J correlations. HMBC correlations from H-5 to C-4, C-6, C-7, C-9, C-10, C-23, C-24, and C25 were employed for the assignments of protons and carbons of the ring AB moiety. The upfield methine H-5 (dH 0.83, d, J = 10.7) exhibited 1,2-diaxial 1H–1H coupling to a secondary alcohol methine (dH 3.93, dt, J = 3.9, 10.7 Hz, H-6), which indicated the 6ahydroxy group. The 1H and 13C chemical shifts in the ring AB region showed close resemblance to those of zeorin (1). The location of the other secondary alcohol was achieved by the HMBC correlations from H-15 (dH 3.84, dd, J = 10.0, 5.4 Hz) to C-27, and from H327 (dH 1.00, s) to C-15 (dC 73.1). The axial orientation of this oxymethine proton was evident from the observed coupling constants involving an 1,2-diaxial 1H–1H coupling (J = 10.0 Hz). The axial (b) orientations of H-5 and H-15 were further supported by the NOESY correlations of these protons with H3-26. Chemical shifts of protons and carbons of the ring CDE moiety were similar to those of dustanin (2). The tertiary alcohol functionality was located at C-22 on the basis of the HMBC correlations from H-17, H-21, H329, and H3-30 to the oxygenated quaternary carbon (dC 73.7). Compound 4 was therefore assigned as a new hopane-type triterpene, 6a,15a,22-trihydroxyhopane.
735
The molecular formula of compound 5 was determined as C32H54O4 by HRESIMS. The 1H and 13C NMR spectra suggested the presence of a tertiary alcohol (dC 73.7), and two oxymethines at dC 68.8 and dC 80.8, and an acetoxy group (dC 171.1, 21.3; dH 2.06, 3H, s). An intense sharp absorption band at nmax 1735 cm 1 in the IR spectrum further confirmed the presence of an acetoxy group, which was located at C-3 (dC 80.8) based on the HMBC correlation from the attached proton (H-3) to the ester carbonyl (dC 171.1) and the correlations from H3-23 and H3-24 to the oxymethine carbon (C-3). The axial (a) orientation of H-3 was evident from its coupling constant values (dH 4.44, dd, J = 11.5, 5.1 Hz). The secondary alcohol was assigned as 6-OH on the basis of the COSY correlations of the oxymethine proton (H-6, dH 4.03) to H-5 and H2-7. The coupling constants for H-6 (dt, J = 3.9, 10.6 Hz) indicated its axial (b) orientation. The C-22 tertiary alcohol was confirmed by the HMBC correlations from H3-29 and H3-30 to this quaternary carbon. The planar structure was established by interpretation of COSY and HMBC (Table 1) data. The 1H and 13C NMR spectroscopic data for the ring A moiety were similar to those of 3, whereas the data for all others (BCDE rings) showed very close resemblance to those of zeorin (1). The following NOESY correlations (Fig. 1) were consistent with the hopane-type relative configuration: H-5/H325, H-5/H3-26, H3-26/H-13, H3-26/Hb-15, H-17/H-21, H-1/H-3, H3/H-5, H-9/H3-27, H3-27/Ha-12, and Ha-16/H3-28. Compound 5 was therefore identified as 3b-acetoxy-6a,22-dihydroxyhopane. Hopanoids 1–5, isolated from BCC 23276, were tested for antitubercular activity against Mycobacterium tuberculosis H37Ra, since the known 1–3 were previously shown to exhibit the same MIC value of 12.5 mg/ml (Boonphong et al., 2001; Isaka et al., 2009). Unexpectedly, all compounds (1–5) were inactive at a concentration of 50 mg/ml in our present assay. The reason of the
Table 1 NMR data for 4 and 5 in CDCl3. No.
4
5
dC, mult.
dH, mult. (J in Hz)
1 2 3 4 5 6 7
40.5, 18.5, 43.7, 33.6, 60.8, 69.0, 48.8,
a 0.80, m; b 1.65, m a 1.37, m; b 1.54, m a 1.17, m; b 1.32, m
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
44.1,a qC 50.1, CH 39.2, qC 21.0, CH2 24.0, CH2 48.6, CH 47.1, qC 74.8, CH 32.8, CH2 50.5,b CH 44.2,a qC 40.9, CH2 26.9, CH2 50.6,a CH 73.7, qC 36.7, CH3 22.1, CH3 17.2, CH3 18.8, CH3 11.8, CH3 15.7, CH3 28.6, CH3 31.0, CH3
a–c
CH2 CH2 CH2 qC CH CH CH2
HMBC
0.83, d (10.7) 3.93, dt (3.9, 10.7) a 1.70, m b 1.87, dd (12.5, 3.9)
4, 6, 7, 10, 23, 24, 25
1.17, m
25
a 1.59, m; b 1.21, m a 1.57, m; b 1.41, m
13, 14
6, 8, 26 5, 6, 8, 9, 26
1.27, m 3.84, dd (10.0, 5.2) a 1.65, m; b 2.23, m 1.44, m
27 14, 18 22, 28
a 1.52, m; b 0.90, m a 1.47, m; b1.76, m 2.21, m
13, 18, 20 22 17, 20, 22
1.16, 1.00, 0.87, 1.12, 1.02, 0.75, 1.16, 1.18,
3, 4, 5, 24 3, 4, 5, 23 1, 5, 9, 10 7, 8, 9, 14 8, 13, 14, 15 13, 17, 18, 19 21, 22, 30 21, 22, 29
s s s s s s s s
The carbon assignment may be interchanged.
dC, mult.
dH, mult. (J in Hz)
38.2, 23.3, 80.8, 38.1, 60.5, 68.8, 45.9,
a 1.03, m; b 1.68, m a 1.65, m; b 1.60, m
CH2 CH2 CH qC CH CH CH2
43.0,c qC 49.7, CH 39.2, qC 21.0, CH2 23.9, CH2 49.4, CH 41.9,c qC 34.4, CH2 21.9, CH2 53.9, CH 44.0, qC 41.2, CH2 26.6, CH2 51.1, CH 73.9, qC 30.8, CH3 16.6, CH3 16.9, CH3 18.3, CH3 17.0, CH3 16.1, CH3 28.8, CH3 30.9, CH3 171.1, qC 21.3, CH3
4.44, dd (11.5, 5.1) 0.92, d (10.6) 4.03, dt (3.9, 10.6) a 1.45, m; b 1.57, m
HMBC 10 3-OCOCH3 4, 6, 7, 9, 10, 24, 25 6, 26
1.23, m
a 1.57, m; b 1.24, m a 1.54, m; b 1.42, m 1.34, m
15
a 1.22, m; b 1.42, m a 1.60, m; b 1.96, m
17 15
1.43, m
a 1.52, m; b 0.93, m a 1.49, m; b 1.76, m 2.22, m
21, 22 19
1.17, 1.05, 0.89, 1.04, 0.96, 0.76, 1.18, 1.20,
3, 4, 5, 24 3, 4, 5, 23 1, 5, 9, 10 7, 8, 9, 14 8, 13, 14, 15 13, 17, 18, 19 21, 22, 30 21, 22, 29
s s s s s s s s
2.06, s
3-OCOCH3
M. Isaka et al. / Phytochemistry Letters 5 (2012) 734–737
736
Fig. 1. Key NOESY correlations for 5.
inconsistent assay result for 1–3 is uncertain; however, it may be related to the change of the assay protocol from the visial microplate Alamar Blue (resazurin) assay (Collins and Franzblau, 1997) to the currently employing green fluorescent protein microplate assay (Changsen et al., 2003). 3. Experimental 3.1. General experimental procedures Melting points were measured with an Electrothermal IA9100 digital melting point apparatus. Optical rotations were measured with a JASCO P-1030 digital polarimeter. IR spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on a Bruker AV500D spectrometer. ESITOF mass spectra were measured with a Bruker micrOTOF mass spectrometer. Merck Silica gel 60H (particle size, 90% <45 mM) was used for column chromatography. 3.2. Fungal material Aschersonia calendulina was collected from a scale insect (Hemiptera) in Doi Inthanon National Park, Chiang Mai Province, Thailand, by one of the authors (S.M.). This fungus was deposited in the BIOTEC Culture Collection (BCC) on October 11, 2006 as BCC 23276. The identification is based on the morphology of the fungus and macro morphology on the scale insect (Mongkolsamrit et al., 2009).
was fractionated by Sephadex LH-20 column chromatography (3.6 cm 54 cm; MeOH) to obtain five pooled fractions. The second fraction (560 mg) was subjected to column chromatography (CC) on silica gel (3.0 cm 14 cm, step gradient elution with 0–100% EtOAc in CH2Cl2) to afford 2 (10 mg), 3 (10 mg), mevalonolactone (14 mg), and a fraction containing 4 (12 mg). The last fraction was purified by CC on silica gel (1.2 cm 10 cm, EtOAc/hexane = 70:30) to furnish 4 (4.8 mg). Extract B was fractionated by CC on silica gel (3.2 cm 11 cm, step gradient elution with 0–100% EtOAc in CH2Cl2) to obtain ergosterol (30 mg), 1 (20 mg), and a fraction containing 5 (3 mg). The last fraction was purified by CC on silica gel (EtOAc/hexane = 30:70) to afford 5 (1.0 mg). 3.3.1. 6a,15a,22-Trihydroxyhopane (4) Colorless solid; mp 268–269 8C; [a]26D +28 (c 0.10, MeOH); IR nmax ATR (cm 1): 3375, 2934, 1464, 1387, 1016; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 483.3807 [M+Na]+ (calc. for C30H52O3Na, 483.3809). 3.3.2. 3b-Acetoxy-6a,22-dihydroxyhopane (5) Colorless powder; [a]25D +25 (c 0.08, MeOH); IR nmax ATR (cm 1): 3350, 2943, 1735, 1458, 1368, 1247, 1031; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS (m/z): 525.3904 [M+Na]+ (calc. for C32H54O4Na, 525.3914). 3.4. Antimycobacterial assay Growth inhibitory activity against Mycobacterium tuberculosis H37Ra was performed using the green fluorescent protein microplate assay (Changsen et al., 2003). The MIC values of the standard antituberculosis drugs (positive control), rifampicin, streptomycin, isoniazid, ofloxacin, and ethambutol were 0.025, 0.625, 0.094, 0.781, and 1.88 mg/ml, respectively.
Acknowledgement Financial support from the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BIOTEC), is gratefully acknowledged.
3.3. Fermentation, extraction, and isolation The fungus BCC 23276 was maintained on potato dextrose agar at 25 8C. The agar plugs (1 cm 1 cm) were cut into small pieces and inoculated into 3 250 ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l) and incubated at 25 8C for 5 days on a rotary shaker (200 rpm). Each primary seed culture was transferred into a 1 l Erlenmeyer flask containing 250 ml of PDB, and incubated at 25 8C for 5 days on a rotary shaker (200 rpm). The seed cultures were combined and a 700 ml portion was transferred into a 10 l bioreactor containing 6.3 l of PDB, and the final fermentation was carried out at 25 8C for 7 days under agitation at 200 rpm and aeration 0.5 vvm. The culture was diluted with acetone (4 l) and the mixture was stood for 3 days, and then filtered. The filtrate was partially evaporated, and the residual aqueous solution (ca. 3 l) was extracted with EtOAc (3 800 ml). The combined EtOAc phase was concentrated under reduced pressure to obtain a brown gum (1.52 g, extract A). The residual mycelial cakes were macerated in MeOH (1.2 l) for 4 days and then filtered. The filtrate was evaporated and the residue was diluted with H2O (200 ml) which was then extracted with EtOAc (2 l). The EtOAc layer was concentrated to give a brown gum (1.11 g, extract B). Extract A
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