- Email: [email protected]
The isolation of two new lanostane triterpenoid derivatives from the edible mushroom Astraeus asiaticus
Phytochemistry Letters 14 (2015) 79–83
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
The isolation of two new lanostane triterpenoid derivatives from the edible mushroom Astraeus asiaticus Preeyanuch Pimjuka , Cherdchai Phosrib , Tsuyoshi Waukec, Sirirath McCloskeya,* a Natural Products Research Unit, Centre of Excellence for Innovation in Chemistry (PERCH-CIC), Department of Chemistry, Faculty of Science, Khon Kaen University, Thailand b Division of Science, Faculty of Liberal Arts and Science, Nakhon Phanom University, Nakhon Phanom, 48000, Thailand c Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, Okinawa, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 July 2015 Received in revised form 3 September 2015 Accepted 17 September 2015 Available online xxx
Two lanostane triterpenoid derivatives, astrasiaone (2), (22S, 25R, 26R)-26-methoxy-22-26-epoxylanost8-en-3-one, and astrasiate (3), (3a, 22S, 25R)-3, 22-dihydroxylanost-8-en-26 -oate, together with six known compounds, astraodorol (1), artabotryol B (4), artabotryol C1 (5), 6-dehydrocerevisterol (6), ergosterol (7) and hypaphorine (8) were isolated from the edible mushroom Astraeus asiaticus. 3 and 4 exhibited weak cytotoxicity against KB and NCI-H187 cancer cell lines. A comparison of the structures of 2 and 3 to that of 1,4 and 5 suggest that these two new compounds could be the intermediate form that occurs during biogenesis. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Astraeus asiaticus Lanostane triterpenoid Cytotoxic activity Biogenesis
1. Introduction Astraeus are common ectomycorrhizal fungi that belong to the family Diplocystaceae (Astraeaceae), which can been found throughout temperate to tropical ecosystems (Fangfuk et al., 2010; Andrew et al., 2012; Phosri et al., 2013). To date, nine species have been recognized, of which Astraeus odoratus, Astraeus asiaticus and Astraeus sirindhorniae have been studied in Thailand (Phosri et al., 2004, 2007, 2014). The six remaining species are Astraeus pteridis, Astraeus koreanus, Astraeus morganii, Astraeus telleriae, Astraeus smithii and Astraeus hygrometricus. In Thailand Astraeus are a popular but expensive edible mushroom, however, information on their chemical constituents and their potential uses in other areas such as medicines have not been widely studied. The most abundant chemical constituents found so far in these genera are triterpenoids, which exhibit various biological activities such as, antifungal, antibacterial, antituberculosis and cytotoxicity against cancer cell lines (Kope et al., 1991; Stanikunaite et al., 2008; Tapan et al., 2012). Our studies into A. odoratus also isolated four new lanostane triterpenoid astraodoric acids A–D with astraodoric acids A and B exhibiting antituberculosis and anticancer activities (Arpha et al., 2012). A modified astraodorol
* Corresponding author at: Department of Chemistry, Faculty of Science, Khon Kaen University, 40002, Thailand. E-mail address: [email protected] (S. McCloskey).
also exhibited the antimalarial activity that had been reported by Nasomjai et al., 2014. Further research of the bioactive chemical constituents in these genera has led to the isolation of two lanostane triterpenoids derivatives (2-3) and six known compounds including astraodorol (Arpha et al., 2012), artabotryol B (Gupta et al., 2010), artabotryol C1 (Gupta et al., 2010), 6dehydrocerevisterol (Lee et al., 2010), ergosterol (Arpha et al., 2012) and hypaphorine (Arpha et al., 2012) from the edible mushroom A. asiaticus, which are described below. 2. Results and discussion Crude extracts of A. asiaticus were purified by chromatographic methods to yield a total of eight compounds (1–8). Their structures were determined on the basis of IR, 1H and 13C NMR and 2D NMR spectral data and also compared to those reported in literature. Compound 1 was obtained as a white amorphous solid. Its IR spectrum showed absorption bands at 3562, 1725, 1650, and 1242 cm1, which corresponded to OH, C¼O, C¼C and C O. The 13 C NMR spectral data of 1 suggested a terpenoid structure containing 30 carbons (Table 1). As lanostane triterpenoids have been isolated from A. odoratus previously, the two signals of quaternary olefinic carbons at dC 133.9 (C-8) and 134.8 (C-9) and a hydroxyl group signal at dC 76.0 (C-3), which are associated with dH 3.42 (1H, brs, H-3), supported that 1 possessed a lanostane skeleton. The carbonyl carbon signal at dC 174.8 (C-26) and oxygenated carbon signal at dC 84.1 (C-22), which are associated
http://dx.doi.org/10.1016/j.phytol.2015.09.009 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
80
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
Table 1 1 H and 13C NMR data (400 MHz; CDCl3) of compounds 1–3. No.
1
2 a
dC
dH
1 2
30.9 25.7
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
76.0 37.6 44.2 18.2 27.6 133.9 134.8 36.9 20.9 26.0 44.5 49.9 30.8 30.0 46.0 15.6 19.0 41.4 12.8 84.1
1.57 m 1.79 m, 1.62 m 3.42 brs
23 24 25 26 27 28 29 30 OCH3 a
3 a
dC
dH
36.1 34.6
2.01 m 2.59 m
1.21 (t, J = 9.6) 1.43 m 1.98 m 0.69 s 0.98 s 1.53 m 0.95 s 4.40 (dd, J = 11.6, 2.4)
217.8 47.4 51.2 19.4 26.3 133.3 135.2 36.9 21.1 31.0 44.3 50.0 30.8 28.4 46.5 15.5 18.7 41.4 13.0 69.6
27.2 28.7 36.4
1.65 m 1.98 m 2.39 (ddq, J = 12.8, 6.0, 6.8)
29.1 26.7 34.8
1.82 m 1.39 m 2.03 m 0.72 s 1.11 s 1.37 m 0.93 (d, J = 6.4) 3.77 (d, J = 11.2) 1.29 m 1.51 m 1.69 m
174.8 17.5 24.3 28.0 22.2
1.29 (d, J = 7.2) 0.90 s 0.96 s 0.86 s
102.2 16.8 24.4 26.2 21.3 55.0
4.46 (d, J = 2.8) 0.85 (d, J = 6.4) 0.91 s 1.09 s 1.06 s 3.38 s
1.50 m 1.46 m 1.32 m
1.93 m 2.02 m
1.63 m 1.61 m 2.10 m
2.06 m 1.24 m
dC
dH a
30.1 25.7
1.47 m 1.59 m
76.0 37.6 44.2 18.2 26.0 134.0 134.8 36.9 20.9 31.0 44.4 49.9 30.8 27.6 46.7 15.7 19.0 41.0 11.6 73.5
3.42 brs
33.0 30.5 39.3
1.53 m 1.82 m 2.47 (sext, J = 6.8)
177.1 17.0 24.3 28.0 22.2 51.5
1.51 m 1.57 m 2.04 m
2.11 m 1.66 m
1.62 m 1.37 m 1.87 m 0.70 s 0.99 s 1.43 m 0.85 s 3.65 m
1.17 (d, J = 6.8) 0.91 s 0.97 s 0.87 s 3.67 s
Chemical shift values are in ppm, and J values are in Hz.
with dH 4.40 (1H, dd, J = 11.6, 2.4 Hz, H-22), suggested lactone moiety. The 1H and 13C NMR spectral data were then carefully compared to the data from previous work and the result showed that it was identical to that of astraodorol isolated from A. odoratus. Therefore, 1 was a known astraodorol. Compound 2 was obtained as a white amorphous solid. The molecular formula C31H50O3 was deduced from the [M + Na]+ peak at m/z 493.3314 in the HR-ESI–MS. The IR absorption bands at 1707 cm1 indicated a CO group, and 1111 and 1038 cm1 indicated a C-O group. Except for the presence of a carbonyl ketone signal at dC 217.8 and there being no oxygenated carbon at C-3 (HC–OH) the 1H and 13C NMR spectra of 2 suggested a lanostane skeleton similar to that of 1 (Table 1). The 1H NMR spectrum showed signals of acetal (O–CH–O) at dH 4.46 (1H, d, J = 2.8 Hz, H26), CH–O–C–O at dH 3.77 (1H, d, J = 11.2 Hz, H-22) and also an
oxymethyl proton at dH 3.38(CH3 O). These data suggested that 2 had a cyclic acetal rather than a lactone side chain. This was confirmed by the correlations of H-21 to C-13, C-17, C-20 and C-22; H-27 to C-24, C-25 and C-26 and HC-OMe to C-26 in the HMBC spectrum (Fig. 1). The analysis of the above data and a comparison of the 1H and 13C NMR spectral data with literature, showed that compound 2 was a C-3 oxidative of the known artabotryol C1, isolated from the seeds of Artabotrys odoratissimus (Gupta et al., 2010). The correlation between HC-OMe and H-20 in NOESY suggested an R configuration at C-26. Therefore 2 was considered as a new lanostane-type derivative found for the first time in A. asiaticus. Compound 3 was obtained as a white amorphous solid. The molecular formula C31H52O4 was indicated from the [M + Na]+ peak at m/z 511.3842 in the HR–ESI–MS. The IR spectrum showed broad
Fig. 1. Key HMBC correlations of 2–3.
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
absorption bands at 3469, 1718 and 1067 cm1, which indicated the OH, C¼O and C O. The 1H and 13C NMR spectral data of 3 showed a similar lanostane skeleton to that of 1 (Table 1). The signal of an oxymethine proton at dH 3.42 (brs), associated with dC 76.0 carbon, confirmed the hydroxylation at C-3. The 1H NMR spectrum showed another group of oxymethine protons at dH 3.65 (1H, m, H-22) and a methoxy proton at dH 3.67 (3H, s). The 13C NMR data at dC 177.1 (C26) of an ester carbonyl carbon and a methoxy carbon signal at dC 51.5 suggested a side chain of ester rather than lactone. This was supported by the correlations of H-21 to C-20; H-22 to C-21; H24 to C-20; H-25 to C-23, C-24, C-26 and C-27; H-27 to C-24, C-25, C-26; and HC-OMe to C-26 in the HMBC spectrum (Fig. 1). Spectroscopic data suggested that 3 was a new methylation form of artabotryol D (Gupta et al., 2010). The stereochemistry at C-3, C-20, C-22 and C-25 were assigned as 3a, 22S, 25R, according to its identical 1H and 13C NMR data to that of artabotryol D. The spectral data of 4 and 5 were carefully compared to some of the compounds found by Gupta et al., (2010) and the result showed that 4 was artabotryol B, which appeared as an epimers mixture. This was confirmed by selecting the relevant 1H and 13C NMR data, i.e, the signal that appeared at dc 101.8 and 95.2 for C26, and dc 78.6 and 69.8 for C-22. This data were carefully measured in a ca. 2.4:1 ratio of 26R:26S. Compound 5 was artabotryol C1.The rest of the known compounds were 6dehydrocerevisterol (6), ergosterol (7) and D-hypaphorine (8). With regards to compounds 1–5 found in this study, their biogenesis could be proposed via the mevalonate pathway in which the cyclization of long chain squalene triterpenes occurred via the intermediate of 2, 3-oxidosqualene, which was produced in
81
a reaction catalyzed by squalene epoxidase, thus creating lanosterol (Dewick, 2009). Lanosterol could be transformed by several reactions such as oxidation, reduction, and cyclisation to the products found in this work. Astraodorol (1, 2.86 g) may have undergone lactone hydrolysis to form a side chain containing carboxylic acid, which then methylated at the hydroxyl group to form the new compound 3, (22.1 mg). The reduction at C-26 of 1 yielded the hemiacetal epimers product of Artabotryol B (4, 1.04 g). The methylation at the hydroxyl group (C-26) of 4 formed 5 (2.19 g) which further oxidised at the hydroxyl group (C-3) to yield 2 (6.5 mg) (Fig. 2). To ensure that 2 and 3 are naturally occurring compounds, 1 and 5 were individually stirred overnight together with silica gel in various solvent systems; the result showed no change. Compounds 1–8 were evaluated for cytotoxicity activities. 3 and 4 were slightly toxic against KB and NCI-H187 cancer cell lines with IC50 values of 46.49 and 36.94 mg/mL, and 38.78 and 19.54 mg/ mL respectively; 4 was also toxic to normal Vero cells with an IC50 of 17.95 mg/mL. In this work, eight chemical constituents isolated from the edible mushroom A. asiaticus were reported. These ectomycorrhizal fungi are thought to supplement healthy eating, used in folk remedies and also play an important role of growth associated with several pine tree species. Although most of the isolated compounds showed very weak biological activity when tested, the information found in this study could benefit further related research in areas such as alternative biological activities evaluation, the more specific biological activity evaluation for its growth association, the chemical modification to enhance the biological activity of the
Fig. 2. Proposed biogenesis pathways and chemical structures of 1–5.
82
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
isolated compounds, biosynthesis and molecular studies for improving the production of the bioactive compounds.
(33.3 mg, 0.0008%) was purified from M13 by preparative layer chromatography (PLC) and developed twice using t-BuOH: MeOH: CH2Cl2 (3:5:2) as an eluent.
3. Experimental 3.1. General Melting points were determined on a Gallenkamp SANYO MPU250BM 3.5 melting point apparatus. Optical rotations were measured on a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer. UV spectra were recorded on an Agilent 8453 UV–vis spectrophotometer. 1H and 13C NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer, with TMS as an internal standard. Electrospray ionization (ESI) mass spectra were measured on a micrOTOF Bruker mass spectrometer. Flash column chromatography (FCC) was performed on silica gel 60 (0.063–0.200 mm and 0.040–0.063 mm, Merck, Germany). Thin layer chromatography (TLC) was performed using silica gel 60 F254 precoated sheets (Merck, Germany). All solvents used in the chromatographic separations were commercial grade and were distilled before being used. Analytical grade solvents were used for crystallization. 3.2. Fungal material The fruiting bodies of A. asiaticus mushroom were collected and identified from Phu Khieo wildlife sanctuary, Thailand by Dr. Cherdchai Phosri, Division of Biology, Faculty of Science, Nakhon Phanom University, Nakhon Phanom, Thailand. The sample specimen was deposited at MycoBank (no. MB510459). 3.3. Extraction and isolation The air-dried A. asiaticus (4.4 kg) were ground into powder using a blender and then extracted 3 times at room temperature with hexane, ethyl acetate (EtOAc) and methanol (MeOH), respectively. Removal of the solvents from each extract under reduced pressure gave crude hexane (144.08 g, 3.27%), crude EtOAc (201.83 g, 4.59 %) and crude MeOH (186.70 g, 4.24 %) extracts. All crude extracts were repeatedly separated by column chromatography (silica gel; 0.040–0.063 mm and less than 0.063 mm mesh, Merck) with various stepwise gradient elutions and recrystallizations to yield eight compounds (1–8). Compound 4 (1.04 g, 0.024%) was isolated from the crude hexane when the crude extract was subjected to FCC and eluted with EtOAc:hexane (1:9). Compound 5 was obtained when the hexane extract was purified and eluted with EtOAc:CH2Cl2:hexane (1:4:5) and was additionally isolated from crude EtOAc and MeOH extracts eluted by the same solvent system to a total of 2.19 g (0.05%). Crude EtOAc extract was subjected to silica gel column chromatography (CC), eluted with a gradient system of EtOAc:hexane (0:10–10:0) and MeOH:EtOAc (0:10–10:0) for 400 mL each. The collected solutions were combined by TLC analysis into 6 fractions (E1–E6). When E5 was further purified by FCC, compounds 1 and 6 were isolated when eluted with CH2Cl2 (1.70 g, 0.039%) and MeOH:CH2Cl2 (0.5:9.5) (6.2 mg, 0.0001%) respectively. The crude MeOH extract was subjected to silica gel column chromatography (CC) eluted with a gradient system of CH2Cl2:hexane (5:5–10:0) and MeOH:CH2Cl2 (0:10–10:0) to give 14 fractions (M1–M14). Compound 1 (1.12 g, 0.026%) was additionally obtained when M8 was repeatedly purified and eluted with CH2Cl2 to 2.86 g (0.065%) in total. Compounds 2 (6.5 mg, 0.0002%) and 7 (64.6 mg, 0.0015%) were isolated when M9 was eluted with CH2Cl2:hexane (3:7) and EtOAc: CH2Cl2:hexane (1:4:5), respectively. The new compound 3 (22.1 mg, 0.0005%) was obtained when M10 was separated by FCC, eluted with MeOH:EtOAc:CH2Cl2 (1:1:8). Compound 8
3.3.1. Astrasiaone (2) A white amorphous solid (6.5 mg, 0.0002 %); Rf = (5:5CH2Cl2: hexane); [a]D28.1 = +250.4 (c = 1.0, CHCl3); UV (CHCl3): lmax (log e) nm: 252 (3.65); IR (CH2Cl2) vmax: 2926, 2855, 1707, and 1038 cm1; 1 H and 13C NMR data are given in Table 1; HRESIMS m/z 493.3314 [M + Na]+ (calc. 493.3658 for C31H50O3). 3.3.2. Astrasiate (3) A white amorphous solid (22.1 mg, 0.0005 %); Rf = 0.61 (5:5 EtOAc: hexane); m.p. 159–161 ; [a]D27.3 = +222.9 (c = 1.0, CHCl3); UV (CHCl3): lmax (log e) nm: 243 (4.81); IR vmax: 3469, 2940, 2875, 2833, 1718, 1451, 1372 and 1067 cm1; 1H and 13C NMR data are given in Table 1; HRESIMS m/z 511.3842 [M + Na]+ (calc. 511.3763 for C31H52O4). 3.4. Biological screening 3.4.1. Cytotoxicity Cytotoxicity of 1–8 against human epidermoid carcinoma (KB), human small cell lung cancer (NCI-H187), and human breast cancer cells (MCF7) were carried out at the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, employing the Resazurin Microplate assay (REMA) as described by Brien et al., 2000. The positive control of KB and NCI-H187 were ellipticine (IC50 values of 0.769 and 0.268 mg/mL) and doxorubicin (IC50 values of 0.779 and 0.110 mg/mL). Tamoxifen and doxorubicin were used as a positive control drugs against MCF-7 with IC50 values of 5.83 and 9.61 mg/mL, respectively. Acknowledgements This work was supported by the Development and Promotion of Science and Technology Talents Project (DPST), an office of the National Research Council of Thailand (NRCT) and the Centre of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the Higher Education Commission, Ministry of Education. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2015.09.009. References Andrew, W.W., Manfred, B., David, S.H., 2012. Diversity and evolution of ectomycorrhizal host associations in the Sclerodermatineae (Boletales, Basidiomycota). New Phytol. 194, 1079–1095. Arpha, K., Phosri, C., Suwannasai, N., Mongkolthanaruk, W., Sodngam, S., 2012. Astraodoric acids A–D: new lanostane triterpenes from edible mushroom Astraeus odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity. J. Agric. Food Chem. 60, 9834–9841. Brien, J.O., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Dewick, P.M., 2009. Medicinal Natural Products: A Biosynthetic Approach, 3rd edition John Wiley & Sons, Ltd., Chichester, UK. Fangfuk, W., Petchang, R., To-aanun, C., Fukuda, M., Yamada, A., 2010. Identification of Japanese Astraeus, based on morphological and phylogenetic analyses. Mycoscience 51, 291–299. Gupta, C., Prasad, S., Sahai, M., Asai, T., Hara, N., Fujimoto, Y., 2010. Artabotryols A–E, new lanostane triterpenes from the seeds of Artabotrys odoratissimus. Helv. Chim. Acta 93, 1925–1932. Kope, H.H., Tsantrizos, Y.S., Fortin, J.A., Ogilvie, K.K., 1991. p-Hydroxybenzoylformic acid and (R)-()-p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolithus arhizus. Can. J. Microbiol. 37, 258–264.
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83 Lee, S.Y., Kim, J.S., Lee, S., S, Kang, 2010. Polyoxygenated ergostane-type sterols from the liquid culture of Ganoderma applanatum. Nat. Prod. Res. 25, 1304–1311. Nasomjai, P., Arpha, K., Sodngam, S., Brandt, S.D., 2014. Potential antimalarial derivatives from astraodorol. Arch. Pharm. Res. 37, 1538–1545. Phosri, C., Watling, R., Martin, P.M., Whalley, A.J.S., 2004. The genus Astraeus in Thailand. Mycotaxon 89, 453–463. Phosri, C., Watling, R., Suwannasai, N., Wilson, A., Martin, P.M., 2014. A new representative of star-shaped fungi: Astraeus sirindhorniae sp. nov. from Thailand. PLoS One 9, 1–10. Phosri, C., Martin, P.M., Sihanonth, P., Whalley, A.J.S., Watling, R., 2007. Molecular study of the genus Astraeus. Mycol. Res. 111, 275–286.
83
Phosri, C., Martin, P.M., Watling, R., 2013. Astraeus: hidden dimensions. IMA Fungus 4, 347–356. Stanikunaite, R., Radwan, M.M., Trappe, J.M., Fronczek, F., Ross, S.A., 2008. Lanostane-type triterpenes from the mushroom Astraeus pteridis with antituberculosis activity. J. Nat. Prod. 71, 2077–2079. Tapan, K.L., Gunjan, B., Soumya, C., Aritri, D., Chiranjib, P., Julie, B., Nattamai, B., Joseph, H.R., Krishnendu, A., 2012. Leishmanicidal and anticandidal activity of constituents of Indian edible mushroom Astraeus hygrometricus. Chem. Biodivers. 9, 1517–1524.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
The isolation of two new lanostane triterpenoid derivatives from the edible mushroom Astraeus asiaticus Preeyanuch Pimjuka , Cherdchai Phosrib , Tsuyoshi Waukec, Sirirath McCloskeya,* a Natural Products Research Unit, Centre of Excellence for Innovation in Chemistry (PERCH-CIC), Department of Chemistry, Faculty of Science, Khon Kaen University, Thailand b Division of Science, Faculty of Liberal Arts and Science, Nakhon Phanom University, Nakhon Phanom, 48000, Thailand c Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, Okinawa, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 July 2015 Received in revised form 3 September 2015 Accepted 17 September 2015 Available online xxx
Two lanostane triterpenoid derivatives, astrasiaone (2), (22S, 25R, 26R)-26-methoxy-22-26-epoxylanost8-en-3-one, and astrasiate (3), (3a, 22S, 25R)-3, 22-dihydroxylanost-8-en-26 -oate, together with six known compounds, astraodorol (1), artabotryol B (4), artabotryol C1 (5), 6-dehydrocerevisterol (6), ergosterol (7) and hypaphorine (8) were isolated from the edible mushroom Astraeus asiaticus. 3 and 4 exhibited weak cytotoxicity against KB and NCI-H187 cancer cell lines. A comparison of the structures of 2 and 3 to that of 1,4 and 5 suggest that these two new compounds could be the intermediate form that occurs during biogenesis. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Astraeus asiaticus Lanostane triterpenoid Cytotoxic activity Biogenesis
1. Introduction Astraeus are common ectomycorrhizal fungi that belong to the family Diplocystaceae (Astraeaceae), which can been found throughout temperate to tropical ecosystems (Fangfuk et al., 2010; Andrew et al., 2012; Phosri et al., 2013). To date, nine species have been recognized, of which Astraeus odoratus, Astraeus asiaticus and Astraeus sirindhorniae have been studied in Thailand (Phosri et al., 2004, 2007, 2014). The six remaining species are Astraeus pteridis, Astraeus koreanus, Astraeus morganii, Astraeus telleriae, Astraeus smithii and Astraeus hygrometricus. In Thailand Astraeus are a popular but expensive edible mushroom, however, information on their chemical constituents and their potential uses in other areas such as medicines have not been widely studied. The most abundant chemical constituents found so far in these genera are triterpenoids, which exhibit various biological activities such as, antifungal, antibacterial, antituberculosis and cytotoxicity against cancer cell lines (Kope et al., 1991; Stanikunaite et al., 2008; Tapan et al., 2012). Our studies into A. odoratus also isolated four new lanostane triterpenoid astraodoric acids A–D with astraodoric acids A and B exhibiting antituberculosis and anticancer activities (Arpha et al., 2012). A modified astraodorol
* Corresponding author at: Department of Chemistry, Faculty of Science, Khon Kaen University, 40002, Thailand. E-mail address: [email protected] (S. McCloskey).
also exhibited the antimalarial activity that had been reported by Nasomjai et al., 2014. Further research of the bioactive chemical constituents in these genera has led to the isolation of two lanostane triterpenoids derivatives (2-3) and six known compounds including astraodorol (Arpha et al., 2012), artabotryol B (Gupta et al., 2010), artabotryol C1 (Gupta et al., 2010), 6dehydrocerevisterol (Lee et al., 2010), ergosterol (Arpha et al., 2012) and hypaphorine (Arpha et al., 2012) from the edible mushroom A. asiaticus, which are described below. 2. Results and discussion Crude extracts of A. asiaticus were purified by chromatographic methods to yield a total of eight compounds (1–8). Their structures were determined on the basis of IR, 1H and 13C NMR and 2D NMR spectral data and also compared to those reported in literature. Compound 1 was obtained as a white amorphous solid. Its IR spectrum showed absorption bands at 3562, 1725, 1650, and 1242 cm1, which corresponded to OH, C¼O, C¼C and C O. The 13 C NMR spectral data of 1 suggested a terpenoid structure containing 30 carbons (Table 1). As lanostane triterpenoids have been isolated from A. odoratus previously, the two signals of quaternary olefinic carbons at dC 133.9 (C-8) and 134.8 (C-9) and a hydroxyl group signal at dC 76.0 (C-3), which are associated with dH 3.42 (1H, brs, H-3), supported that 1 possessed a lanostane skeleton. The carbonyl carbon signal at dC 174.8 (C-26) and oxygenated carbon signal at dC 84.1 (C-22), which are associated
http://dx.doi.org/10.1016/j.phytol.2015.09.009 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
80
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
Table 1 1 H and 13C NMR data (400 MHz; CDCl3) of compounds 1–3. No.
1
2 a
dC
dH
1 2
30.9 25.7
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
76.0 37.6 44.2 18.2 27.6 133.9 134.8 36.9 20.9 26.0 44.5 49.9 30.8 30.0 46.0 15.6 19.0 41.4 12.8 84.1
1.57 m 1.79 m, 1.62 m 3.42 brs
23 24 25 26 27 28 29 30 OCH3 a
3 a
dC
dH
36.1 34.6
2.01 m 2.59 m
1.21 (t, J = 9.6) 1.43 m 1.98 m 0.69 s 0.98 s 1.53 m 0.95 s 4.40 (dd, J = 11.6, 2.4)
217.8 47.4 51.2 19.4 26.3 133.3 135.2 36.9 21.1 31.0 44.3 50.0 30.8 28.4 46.5 15.5 18.7 41.4 13.0 69.6
27.2 28.7 36.4
1.65 m 1.98 m 2.39 (ddq, J = 12.8, 6.0, 6.8)
29.1 26.7 34.8
1.82 m 1.39 m 2.03 m 0.72 s 1.11 s 1.37 m 0.93 (d, J = 6.4) 3.77 (d, J = 11.2) 1.29 m 1.51 m 1.69 m
174.8 17.5 24.3 28.0 22.2
1.29 (d, J = 7.2) 0.90 s 0.96 s 0.86 s
102.2 16.8 24.4 26.2 21.3 55.0
4.46 (d, J = 2.8) 0.85 (d, J = 6.4) 0.91 s 1.09 s 1.06 s 3.38 s
1.50 m 1.46 m 1.32 m
1.93 m 2.02 m
1.63 m 1.61 m 2.10 m
2.06 m 1.24 m
dC
dH a
30.1 25.7
1.47 m 1.59 m
76.0 37.6 44.2 18.2 26.0 134.0 134.8 36.9 20.9 31.0 44.4 49.9 30.8 27.6 46.7 15.7 19.0 41.0 11.6 73.5
3.42 brs
33.0 30.5 39.3
1.53 m 1.82 m 2.47 (sext, J = 6.8)
177.1 17.0 24.3 28.0 22.2 51.5
1.51 m 1.57 m 2.04 m
2.11 m 1.66 m
1.62 m 1.37 m 1.87 m 0.70 s 0.99 s 1.43 m 0.85 s 3.65 m
1.17 (d, J = 6.8) 0.91 s 0.97 s 0.87 s 3.67 s
Chemical shift values are in ppm, and J values are in Hz.
with dH 4.40 (1H, dd, J = 11.6, 2.4 Hz, H-22), suggested lactone moiety. The 1H and 13C NMR spectral data were then carefully compared to the data from previous work and the result showed that it was identical to that of astraodorol isolated from A. odoratus. Therefore, 1 was a known astraodorol. Compound 2 was obtained as a white amorphous solid. The molecular formula C31H50O3 was deduced from the [M + Na]+ peak at m/z 493.3314 in the HR-ESI–MS. The IR absorption bands at 1707 cm1 indicated a CO group, and 1111 and 1038 cm1 indicated a C-O group. Except for the presence of a carbonyl ketone signal at dC 217.8 and there being no oxygenated carbon at C-3 (HC–OH) the 1H and 13C NMR spectra of 2 suggested a lanostane skeleton similar to that of 1 (Table 1). The 1H NMR spectrum showed signals of acetal (O–CH–O) at dH 4.46 (1H, d, J = 2.8 Hz, H26), CH–O–C–O at dH 3.77 (1H, d, J = 11.2 Hz, H-22) and also an
oxymethyl proton at dH 3.38(CH3 O). These data suggested that 2 had a cyclic acetal rather than a lactone side chain. This was confirmed by the correlations of H-21 to C-13, C-17, C-20 and C-22; H-27 to C-24, C-25 and C-26 and HC-OMe to C-26 in the HMBC spectrum (Fig. 1). The analysis of the above data and a comparison of the 1H and 13C NMR spectral data with literature, showed that compound 2 was a C-3 oxidative of the known artabotryol C1, isolated from the seeds of Artabotrys odoratissimus (Gupta et al., 2010). The correlation between HC-OMe and H-20 in NOESY suggested an R configuration at C-26. Therefore 2 was considered as a new lanostane-type derivative found for the first time in A. asiaticus. Compound 3 was obtained as a white amorphous solid. The molecular formula C31H52O4 was indicated from the [M + Na]+ peak at m/z 511.3842 in the HR–ESI–MS. The IR spectrum showed broad
Fig. 1. Key HMBC correlations of 2–3.
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
absorption bands at 3469, 1718 and 1067 cm1, which indicated the OH, C¼O and C O. The 1H and 13C NMR spectral data of 3 showed a similar lanostane skeleton to that of 1 (Table 1). The signal of an oxymethine proton at dH 3.42 (brs), associated with dC 76.0 carbon, confirmed the hydroxylation at C-3. The 1H NMR spectrum showed another group of oxymethine protons at dH 3.65 (1H, m, H-22) and a methoxy proton at dH 3.67 (3H, s). The 13C NMR data at dC 177.1 (C26) of an ester carbonyl carbon and a methoxy carbon signal at dC 51.5 suggested a side chain of ester rather than lactone. This was supported by the correlations of H-21 to C-20; H-22 to C-21; H24 to C-20; H-25 to C-23, C-24, C-26 and C-27; H-27 to C-24, C-25, C-26; and HC-OMe to C-26 in the HMBC spectrum (Fig. 1). Spectroscopic data suggested that 3 was a new methylation form of artabotryol D (Gupta et al., 2010). The stereochemistry at C-3, C-20, C-22 and C-25 were assigned as 3a, 22S, 25R, according to its identical 1H and 13C NMR data to that of artabotryol D. The spectral data of 4 and 5 were carefully compared to some of the compounds found by Gupta et al., (2010) and the result showed that 4 was artabotryol B, which appeared as an epimers mixture. This was confirmed by selecting the relevant 1H and 13C NMR data, i.e, the signal that appeared at dc 101.8 and 95.2 for C26, and dc 78.6 and 69.8 for C-22. This data were carefully measured in a ca. 2.4:1 ratio of 26R:26S. Compound 5 was artabotryol C1.The rest of the known compounds were 6dehydrocerevisterol (6), ergosterol (7) and D-hypaphorine (8). With regards to compounds 1–5 found in this study, their biogenesis could be proposed via the mevalonate pathway in which the cyclization of long chain squalene triterpenes occurred via the intermediate of 2, 3-oxidosqualene, which was produced in
81
a reaction catalyzed by squalene epoxidase, thus creating lanosterol (Dewick, 2009). Lanosterol could be transformed by several reactions such as oxidation, reduction, and cyclisation to the products found in this work. Astraodorol (1, 2.86 g) may have undergone lactone hydrolysis to form a side chain containing carboxylic acid, which then methylated at the hydroxyl group to form the new compound 3, (22.1 mg). The reduction at C-26 of 1 yielded the hemiacetal epimers product of Artabotryol B (4, 1.04 g). The methylation at the hydroxyl group (C-26) of 4 formed 5 (2.19 g) which further oxidised at the hydroxyl group (C-3) to yield 2 (6.5 mg) (Fig. 2). To ensure that 2 and 3 are naturally occurring compounds, 1 and 5 were individually stirred overnight together with silica gel in various solvent systems; the result showed no change. Compounds 1–8 were evaluated for cytotoxicity activities. 3 and 4 were slightly toxic against KB and NCI-H187 cancer cell lines with IC50 values of 46.49 and 36.94 mg/mL, and 38.78 and 19.54 mg/ mL respectively; 4 was also toxic to normal Vero cells with an IC50 of 17.95 mg/mL. In this work, eight chemical constituents isolated from the edible mushroom A. asiaticus were reported. These ectomycorrhizal fungi are thought to supplement healthy eating, used in folk remedies and also play an important role of growth associated with several pine tree species. Although most of the isolated compounds showed very weak biological activity when tested, the information found in this study could benefit further related research in areas such as alternative biological activities evaluation, the more specific biological activity evaluation for its growth association, the chemical modification to enhance the biological activity of the
Fig. 2. Proposed biogenesis pathways and chemical structures of 1–5.
82
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83
isolated compounds, biosynthesis and molecular studies for improving the production of the bioactive compounds.
(33.3 mg, 0.0008%) was purified from M13 by preparative layer chromatography (PLC) and developed twice using t-BuOH: MeOH: CH2Cl2 (3:5:2) as an eluent.
3. Experimental 3.1. General Melting points were determined on a Gallenkamp SANYO MPU250BM 3.5 melting point apparatus. Optical rotations were measured on a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer. UV spectra were recorded on an Agilent 8453 UV–vis spectrophotometer. 1H and 13C NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer, with TMS as an internal standard. Electrospray ionization (ESI) mass spectra were measured on a micrOTOF Bruker mass spectrometer. Flash column chromatography (FCC) was performed on silica gel 60 (0.063–0.200 mm and 0.040–0.063 mm, Merck, Germany). Thin layer chromatography (TLC) was performed using silica gel 60 F254 precoated sheets (Merck, Germany). All solvents used in the chromatographic separations were commercial grade and were distilled before being used. Analytical grade solvents were used for crystallization. 3.2. Fungal material The fruiting bodies of A. asiaticus mushroom were collected and identified from Phu Khieo wildlife sanctuary, Thailand by Dr. Cherdchai Phosri, Division of Biology, Faculty of Science, Nakhon Phanom University, Nakhon Phanom, Thailand. The sample specimen was deposited at MycoBank (no. MB510459). 3.3. Extraction and isolation The air-dried A. asiaticus (4.4 kg) were ground into powder using a blender and then extracted 3 times at room temperature with hexane, ethyl acetate (EtOAc) and methanol (MeOH), respectively. Removal of the solvents from each extract under reduced pressure gave crude hexane (144.08 g, 3.27%), crude EtOAc (201.83 g, 4.59 %) and crude MeOH (186.70 g, 4.24 %) extracts. All crude extracts were repeatedly separated by column chromatography (silica gel; 0.040–0.063 mm and less than 0.063 mm mesh, Merck) with various stepwise gradient elutions and recrystallizations to yield eight compounds (1–8). Compound 4 (1.04 g, 0.024%) was isolated from the crude hexane when the crude extract was subjected to FCC and eluted with EtOAc:hexane (1:9). Compound 5 was obtained when the hexane extract was purified and eluted with EtOAc:CH2Cl2:hexane (1:4:5) and was additionally isolated from crude EtOAc and MeOH extracts eluted by the same solvent system to a total of 2.19 g (0.05%). Crude EtOAc extract was subjected to silica gel column chromatography (CC), eluted with a gradient system of EtOAc:hexane (0:10–10:0) and MeOH:EtOAc (0:10–10:0) for 400 mL each. The collected solutions were combined by TLC analysis into 6 fractions (E1–E6). When E5 was further purified by FCC, compounds 1 and 6 were isolated when eluted with CH2Cl2 (1.70 g, 0.039%) and MeOH:CH2Cl2 (0.5:9.5) (6.2 mg, 0.0001%) respectively. The crude MeOH extract was subjected to silica gel column chromatography (CC) eluted with a gradient system of CH2Cl2:hexane (5:5–10:0) and MeOH:CH2Cl2 (0:10–10:0) to give 14 fractions (M1–M14). Compound 1 (1.12 g, 0.026%) was additionally obtained when M8 was repeatedly purified and eluted with CH2Cl2 to 2.86 g (0.065%) in total. Compounds 2 (6.5 mg, 0.0002%) and 7 (64.6 mg, 0.0015%) were isolated when M9 was eluted with CH2Cl2:hexane (3:7) and EtOAc: CH2Cl2:hexane (1:4:5), respectively. The new compound 3 (22.1 mg, 0.0005%) was obtained when M10 was separated by FCC, eluted with MeOH:EtOAc:CH2Cl2 (1:1:8). Compound 8
3.3.1. Astrasiaone (2) A white amorphous solid (6.5 mg, 0.0002 %); Rf = (5:5CH2Cl2: hexane); [a]D28.1 = +250.4 (c = 1.0, CHCl3); UV (CHCl3): lmax (log e) nm: 252 (3.65); IR (CH2Cl2) vmax: 2926, 2855, 1707, and 1038 cm1; 1 H and 13C NMR data are given in Table 1; HRESIMS m/z 493.3314 [M + Na]+ (calc. 493.3658 for C31H50O3). 3.3.2. Astrasiate (3) A white amorphous solid (22.1 mg, 0.0005 %); Rf = 0.61 (5:5 EtOAc: hexane); m.p. 159–161 ; [a]D27.3 = +222.9 (c = 1.0, CHCl3); UV (CHCl3): lmax (log e) nm: 243 (4.81); IR vmax: 3469, 2940, 2875, 2833, 1718, 1451, 1372 and 1067 cm1; 1H and 13C NMR data are given in Table 1; HRESIMS m/z 511.3842 [M + Na]+ (calc. 511.3763 for C31H52O4). 3.4. Biological screening 3.4.1. Cytotoxicity Cytotoxicity of 1–8 against human epidermoid carcinoma (KB), human small cell lung cancer (NCI-H187), and human breast cancer cells (MCF7) were carried out at the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, employing the Resazurin Microplate assay (REMA) as described by Brien et al., 2000. The positive control of KB and NCI-H187 were ellipticine (IC50 values of 0.769 and 0.268 mg/mL) and doxorubicin (IC50 values of 0.779 and 0.110 mg/mL). Tamoxifen and doxorubicin were used as a positive control drugs against MCF-7 with IC50 values of 5.83 and 9.61 mg/mL, respectively. Acknowledgements This work was supported by the Development and Promotion of Science and Technology Talents Project (DPST), an office of the National Research Council of Thailand (NRCT) and the Centre of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the Higher Education Commission, Ministry of Education. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2015.09.009. References Andrew, W.W., Manfred, B., David, S.H., 2012. Diversity and evolution of ectomycorrhizal host associations in the Sclerodermatineae (Boletales, Basidiomycota). New Phytol. 194, 1079–1095. Arpha, K., Phosri, C., Suwannasai, N., Mongkolthanaruk, W., Sodngam, S., 2012. Astraodoric acids A–D: new lanostane triterpenes from edible mushroom Astraeus odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity. J. Agric. Food Chem. 60, 9834–9841. Brien, J.O., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Dewick, P.M., 2009. Medicinal Natural Products: A Biosynthetic Approach, 3rd edition John Wiley & Sons, Ltd., Chichester, UK. Fangfuk, W., Petchang, R., To-aanun, C., Fukuda, M., Yamada, A., 2010. Identification of Japanese Astraeus, based on morphological and phylogenetic analyses. Mycoscience 51, 291–299. Gupta, C., Prasad, S., Sahai, M., Asai, T., Hara, N., Fujimoto, Y., 2010. Artabotryols A–E, new lanostane triterpenes from the seeds of Artabotrys odoratissimus. Helv. Chim. Acta 93, 1925–1932. Kope, H.H., Tsantrizos, Y.S., Fortin, J.A., Ogilvie, K.K., 1991. p-Hydroxybenzoylformic acid and (R)-()-p-hydroxymandelic acid, two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolithus arhizus. Can. J. Microbiol. 37, 258–264.
P. Pimjuk et al. / Phytochemistry Letters 14 (2015) 79–83 Lee, S.Y., Kim, J.S., Lee, S., S, Kang, 2010. Polyoxygenated ergostane-type sterols from the liquid culture of Ganoderma applanatum. Nat. Prod. Res. 25, 1304–1311. Nasomjai, P., Arpha, K., Sodngam, S., Brandt, S.D., 2014. Potential antimalarial derivatives from astraodorol. Arch. Pharm. Res. 37, 1538–1545. Phosri, C., Watling, R., Martin, P.M., Whalley, A.J.S., 2004. The genus Astraeus in Thailand. Mycotaxon 89, 453–463. Phosri, C., Watling, R., Suwannasai, N., Wilson, A., Martin, P.M., 2014. A new representative of star-shaped fungi: Astraeus sirindhorniae sp. nov. from Thailand. PLoS One 9, 1–10. Phosri, C., Martin, P.M., Sihanonth, P., Whalley, A.J.S., Watling, R., 2007. Molecular study of the genus Astraeus. Mycol. Res. 111, 275–286.
83
Phosri, C., Martin, P.M., Watling, R., 2013. Astraeus: hidden dimensions. IMA Fungus 4, 347–356. Stanikunaite, R., Radwan, M.M., Trappe, J.M., Fronczek, F., Ross, S.A., 2008. Lanostane-type triterpenes from the mushroom Astraeus pteridis with antituberculosis activity. J. Nat. Prod. 71, 2077–2079. Tapan, K.L., Gunjan, B., Soumya, C., Aritri, D., Chiranjib, P., Julie, B., Nattamai, B., Joseph, H.R., Krishnendu, A., 2012. Leishmanicidal and anticandidal activity of constituents of Indian edible mushroom Astraeus hygrometricus. Chem. Biodivers. 9, 1517–1524.