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Cytotoxic ergosterols from cultures of the basidiomycete Psathyrella candolleana
Fitoterapia 138 (2019) 104289
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Cytotoxic ergosterols from cultures of the basidiomycete Psathyrella candolleana ⁎
T ⁎
Ya-Pei Liua,c, Chao-Jun Pua,c, Meng Wanga,c, Juan Hea,c, Zheng-Hui Lia,c, Tao Fenga,c, , Jie Xieb, , ⁎ Ji-Kai Liua,c, a
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing 400715, China c National Demonstration Center for Experimental Ethnopharmacology Education, South-Central University for Nationalities, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Psathyrella candolleana Ergosterol Psathergosterols A–C Cytotoxicity
Three newly isolated ergosterols, psathergosterols A–C (1–3), together with two known ones (4 and 5), have been isolated from cultures of the basdiomycete Psathyrella candolleana. Their structures with the absolute configuration were elucidated by means of spectroscopic methods and the single crystal X-ray diffraction. Compounds 2–4 exhibited certain cytotoxicities to five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480).
1. Introduction The fungus Psathyrella candolleana (Psathyrellaceae) is widely distributed in Europe and North America, as well as many Asian areas. It has been demonstrated to be a source for guanacastane-type diterpenoids which attracted great attention of chemists for both organic and biological studies [1–3]. In our previous studies on this fungus, a number of biologically active guanacastane-type diterpenoids, as well as a seco-derivative, have been isolated from the fermentation broths [4,5]. In order to search for more new and bioactive chemical constituents from this fungus, as well as in our ongoing searching for structurally interesting and biologically active natural products from higher fungi [6–11], a continuous study on the cultures of P. candolleana in rice medium has been carried out. As a result, three new ergosterols, psathergosterols A–C (1–3), together with two known ones 4 and 5 (Fig. 1), were obtained from the EtOAc layer. The structures with absolute configurations were identified by means of spectroscopic methods, including MS, NMR, and the single crystal X-ray diffraction. All compounds were evaluated for their cytotoxicities to five human cancer cell lines. Herein, the separation, structural elucidation, and the biological activities of the isolates are discussed. 2. Results and discussion Compound 1 was isolated as colorless crystals. Its molecular formula C28H46O5 was determined on the basis of the positive high
⁎
resolution (HR) ESIMS (measured at m/z 463.34177 [M + H]+; calcd for C28H47O5+, 463.34235), corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3365 cm−1), carbonyl (1653 cm−1), and double bond (1593, 1454 cm−1). The 1H NMR spectrum of 1 recorded in methanol-d4 displayed signals for four tertiary methyl groups as singlets at δH 0.74 (3H, s, Me-18), 0.95 (3H, s, Me-19), 1.12 (3H, s, Me-26), and 1.13 (3H, s, Me-27), two secondary methyl groups as doublets at δH 0.99 (3H, d, J = 6.5 Hz, Me-21), and 0.91 (3H, d, J = 6.8 Hz, Me-28), one oxygenated proton signal at δH 3.50 (1H, tt, J = 11.3, 4.4 Hz, H-3), and one olefinic proton signal at δH 5.83 (1H, s, H-7). All the carbons in compound 1 were detected by the 13 C NMR spectrum, in association with DEPT experiment. A total of 28 carbon signals were assigned as six CH3, nine CH2, six CH, and seven quaternary carbons. These data demonstrated that compound 1 should be an ergosterol derivative. In the 1He1H COSY spectrum, four fragments were readily established as shown in Fig. 2. In which, a hydroxy group at C-3 was identified. Based on these fragments, the HMBC data played an important role for the determination of the main substituents (Fig. 2). In detail, a 7.8-en-6-keto moiety was built by the three carbons with chemical shifts at δC 132.2 (d, C-7), 158.8 (s, C-8), and 201.7 (s, C6), and supported by the HMBC correlation from H-5 to C-6, and C-7, and from H-7 to C-9, and C-14. Three hydroxy groups at C-9, C-14, and C-25 were identified by the HMBC correlations from H-11 to δC 74.4 (s, C-9), from H-18 to δC 85.4 (s, C-14), and from H-26 and H-27 to δC 72.2 (s, C-25), respectively. After detailed analysis of HMBC data, a planar structure of compound 1 was built as shown in Fig. 2. The relative
Corresponding authors at: School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China. E-mail addresses: [email protected] (T. Feng), [email protected] (J. Xie), [email protected] (J.-K. Liu).
https://doi.org/10.1016/j.fitote.2019.104289 Received 12 July 2019; Received in revised form 1 August 2019; Accepted 2 August 2019 Available online 03 August 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved.
Fitoterapia 138 (2019) 104289
Y.-P. Liu, et al.
Compound 2 was isolated as white powder. Its molecular formula C28H46O5, identical to that of 1, was revealed based on its HR-ESIMS data (measured at m/z 463.32571 [M + H]+; calcd for C28H47O5+, 463.34235). By comparison of the 1D and 2D NMR data with those of 1, compound 2 was supposed to be an epimer of 1. The significant difference of the NMR chemical shifts for CH-3 (δH 4.06, m; δC 64.3, d) suggested the different stereoconfiguation of C-3 in 2, with respect to that of 1. In addition, the REOSY correlation between H-3 and H-5 could not be observed any more, while the other 2D NMR data of 2 were the same to those of 1. Compound 2 was, therefore, identified as 3-epi-1, and named as psathergosterol B. Compound 3 was isolated as white powder. Its molecular formula C28H46O4 was determined on the basis of the positive HR-ESIMS (measured at m/z 469.32913 [M + Na]+; calcd for C28H46O4Na, 469.32938), corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3338 cm−1), carbonyl (1698 cm−1) and double bond (1578, 1454 cm−1). The 1H NMR spectrum recorded in methanol-d4 displayed signals for four tertiary methyl groups as singlets at δH 0.82 (3H, s, Me-18), 0.92 (3H, s, Me-19), 1.12 (3H, s, Me-26), and 1.14 (3H, s, Me-27), two secondary methyl groups as doublets at δH 1.02 (3H, d, J = 6.7 Hz, Me-21), and 0.91 (3H, d, J = 7.1 Hz, Me-28), two oxygenated protons resonating at δH 3.56 (1H, m, H-3) and 4.43 (1H, td, J = 7.7, 4.8 Hz, H-16), and one olefinic protons resonating at δH 5.66 (1H, s, H-7). A total of 28 carbon signals were detected in the 13C NMR spectrum and assigned by the DEPT experiment as six methyl, eight methylene, nine methine, and five quaternary carbons. These data demonstrated that compound 3 was also probably an ergosterol derivative. In the 1He1H COSY spectrum, three fragments were also established as shown in Fig. 2. In which, a hydroxy group at C-3 (δC 68.7) and a hydroxy group at C-16 (δC 70.7) were easily identified. The same to that in 1 and 2, a 7,8-en-6-keto moiety was also built by the chemical shifts at δC 120.3 (d, C-7), 166.9 (d, C-8), and 203.2 (s, C-6), as well as the HMBC correlations from H-5 to C-6 and from H-7 to C-9 and C-14 (Fig. 2). In addition, the third hydroxy group at C-25 (δC 72.9, s) was identified by the HMBC correlations from H-24, H-26, and H-27 to C-25. Detailed analysis of HMBC data, a planar structure of 3, as shown in Fig. 2, was figured out. In the ROESY spectrum (Fig. 2), a cross peak between H-19 and H-5 indicated that H-5 should be β oriented, while the cross peak of H-3/H-5 suggested that OH-3 should be α oriented. In addition, the ROESY between H-9 and H-14 indicated that H-9 and H-14 should both be α oriented, consequently the ROESY cross peak of H-16 with H-14 and H-17 indicated that OH-16 should be β oriented. Compound 3 was, therefore, established and named as psathergosterol C. Two known ergosterols were identified as normal ones that usually obtained from fungi as ergosta-5,7,22-trien-3β-ol (4) [11] and 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (5) [12] by comparison their 1D NMR data with the reference samples in the laboratory. All compounds were evaluated for their cytotoxicities against five human cancer cell lines, including human myeloid leukemia cell line HL-60, human hepatocellular carcinoma cell line SMMC-7721, human lung cancer cell line A549, human breast cancercell line MCF-7, and human colon cancer SW-480, using the same method we reported previously [13,14]. As shown in Table 2, compounds 2–4 displayed certain cytotoxicity to several cancer cell lines. In conclusion, five ergoterols including three new ones have been isolated from cultures of the basidiomycete P. candolleana. Their structures were established by extensive spectroscopic methods. Compounds 2–4 showed certain cytotoxicities to several cancer cell lines. To our best known, this is the first time reporting new ergosterols from fungus P. candolleana, while H-5 in compound 3 possessing a β orientation in steroids occurred seldom.
Fig. 1. Psathergosterols A–C (1–3) and two known ergosterols (4 and 5) from Psathyrella candolleana.
Fig. 2. 1He1H COSY, HMBC and ROESY correlations for 1 and 3.
Fig. 3. ORTEP diagram of 1 showing the absolute configuration.
configuration of 1 was mainly elucidated by an ROESY experiment. It seems that H-3 should be β-oriented due to its constant coupling (J = 11.3, 4.4 Hz), while an ROESY cross peak between H-3 and H-5 indicated that H-5 should be α-oriented (Fig. 2). However, the stereoconfigurations of C-9 and C-14 are difficult to be established by the ROESY data. After attempts, a single crystal of 1 was obtained, the Xray diffraction determined not only the planar structure but also the absolute configuration of the molecule (Fig. 3. Flack parameter = 0.03(3)). The structure of compound 1 was, therefore, identified and named as psathergosterol A. 2
Fitoterapia 138 (2019) 104289
Y.-P. Liu, et al.
Table 1 1 H (600 MHz) and No.
13
C (150 MHz) NMR Data of 1–3a in methanol‑d4 (δ in ppm, J in Hz).a
1
2
δH 1
1.96, 1.47, 1.79, 3.50, 2.11, 3.11,
2 3 4 5 6 7 8 9 10 11 12
dd (13.7, 3.7) m m; 1.34, m tt (11.3, 4.4) m; 1.31, m dd (12.3, 3.8)
δC
δH
28.6, CH2
2.36, 1.16, 1.68, 4.06, 1.98, 3.46,
1.79, m; 0.80, m 2.04, m; 1.72, m
29.7, CH2 69.4, CH 29.4, CH2 46.4, CH 201.7, qC 132.2, CH 158.8, qC 74.4, qC 42.0, qC 27.7, CH2 27.4, CH2
13 14 15
1.89, m; 1.63, m
46.3, qC 85.4, qC 30.0, CH2
16
2.06, m; 1.42, m
26.0, CH2
17 18 19 20 21 22 23 24 25 26 27 28
2.00, 0.74, 0.95, 1.46, 0.99, 1.00, 2.14, 1.29,
50.0, 15.1, 15.5, 36.1, 18.3, 34.1, 27.8, 44.9, 72.2, 24.5, 25.7, 13.8,
a
5.83, s
m s s m d (6.5) m; 1.60, m m; 1.72, m m
1.12, s 1.13, s 0.91, d (6.8)
3
td (13.5, 4.1) m m; 1.61, m m m; 1.57, m dd (12.2, 4.0)
5.83, s
2.05, m; 1.42, m 2.14, td (13.2, 4.0) 1.72, m
δH
δC
23.5, CH2
1.86, m; 1.14, m
33.1, CH2
26.9, CH2 64.3, CH 27.1, CH2 41.9, CH 203.5, qC 123.3, CH 158.6, qC 74.6, qC 42.6, qC 26.0, CH2 27.8, CH2
1.50, 3.56, 1.71, 2.02,
29.9, CH2 68.7, CH 34.3, CH2 56.2, CH 203.2, qC 120.3, CH 166.9, qC 37.6, CH 35.9, qC 21.2, CH2 38.9, CH2
46.3, qC 85.5, qC 30.0, CH2
1.90, m; 1.63 m 1.99, m 1.75 dd (8.4, 3.8) 2.00, m 0.77, s 0.94, s 1.46, m 0.98, d (6.5) 1.60, m; 1.00, m 1.80, m; 0.79, m 1.29, m
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
δC
27.2, CH2 49.9, 15.1, 14.8, 36.1, 18.3, 34.9, 27.7, 44.9, 72.7, 24.3, 25.9, 13.8,
1.12, s 1.13, s 0.90, d (6.8)
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
m; 1.79, m m m; 1.32, m dd (13.2, 3.7)
5.66, s 2.72, m 1.79, m; 1.71, m 2.17, dd (9.4, 3.0) 1.48, m 2.13, 2.27, 1.57, 4.43,
dd (12.6, 6.6) m td (12.9, 4.7) td (7.7, 4.7)
1.33, 0.82, 0.92, 1.90, 1.02, 1.71, 1.80, 1.33,
m s s m d (6.7) m; 1.33, m m; 0.88, m m
1.12, s 1.14, s 0.91, d (7.1)
44.8, qC 53.4, CH 35.1, CH2 70.7, CH 61.0, 12.4, 22.8, 30.3, 17.8, 34.2, 27.8, 44.7, 72.9, 24.3, 25.9, 14.0,
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
Data were assigned by HSQC, HMBC, 1He1H COSY and ROESY spectra.
Table 2 Cytotoxicities of 1–5 against five human cancer cell lines (IC50, μM). Sample
HL-60
SMMC-7721
A-549
MCF-7
SW480
1 2 3 4 5 Taxola
> 40 32.3 ± 1.12 > 40 > 40 > 40 < 0.008
> 40 > 40 29.3 ± 1.81 12.3 ± 1.43 > 40 < 0.008
> 40 23.4 ± 0.75 > 40 9.8 ± 1.98 > 40 < 0.008
> 40 28.3 ± 1.15 22.3 ± 1.37 11.9 ± 1.76 > 40 < 0.008
> 40 > 40 > 40 18.6 ± 1.71 > 40 < 0.008
a
Positive control.
9.4 × 150 mm, flowing speed = 10 mL/min).
3. Experimental section 3.1. General experimental procedures
3.2. Fungal material and cultivation conditions Optical rotations were measured on a Rudolph Autopol IV polarimeter. UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer. IR spectra were obtained by using a Shimadu Fourier Transform Infrared spectrometer with KBr pellets. NMR spectra were acquired with a Bruker Avance III 600 instrument. HR-ESIMS were measured on a Thermo Scientific Q Exactive Orbitrap MS system. Silica gel (200–300 mesh and 80–100 mesh, Qingdao Marine Chemical Inc., China), RP-18 gel (40–75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan) and Sephadex LH-20 (Amersham Biosciences, Upssala, Sweden) were used for column chromatography (CC). Fractions were monitored by TLC (Qingdao Marine Chemical Inc., China) and spots were visualized by 10% H2SO4 in methanol, in combination with Agilent 1260 series HPLC system (Zorbax SB-aq-C18 column, 5 μm, 4.9 × 150 mm, flowing speed = 1 mL/min). Preparative HPLC was performed on an Agilent 1260 series with a Zorbax SB-C18 column (5 μm,
Fruiting bodies of P. candolleana were collected at Jingdong, Yunnan Province, China in 2003 and identified by Prof. Zhu-Liang Yang (Kunming Institute of Botany). The voucher specimen (NO.CGBWSHF00118.2) was deposited at School of Pharmaceutical Sciences, South-Central University for Nationalities. Culture medium was composed of glucose (5%), pork pepton (0.15%), yeast (0.5%), KH2PO4 (0.05%) and MgSO4 (0.05%). Initial pH was adjusted to 6.0, the fermentation was first carried out on an erlenmeyer flask for 6 days till the mycelium biomass reached to the maximum. Then transfer it to rice medium for 24 °C in dark culture for 40 days. Rice medium: 50 g of rice, 50 mL of water, placed in a 250 mL Erlenmeyer flask, sterilized at 121 °C for 15 min, a total of 180 bottles.
3
Fitoterapia 138 (2019) 104289
Y.-P. Liu, et al.
1698, 1622, 1578, 1454, 1112, 1033 cm−1. 1H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS at m/z 469.32913 [M + Na]+ (calcd for C28H46O4Na, 469.32938).
3.3. Extraction and isolation The rice culture (9 Kg) was extracted four times with EtOAc. The organic layer was evaporated to give a crude extract (90 g). Then it was subjected to silica gel CC (200–300 mesh) eluted with petroleum ether (PE)-Me2CO gradient system to afford eight fractions A–H. Compound 4 was deposited from fraction B as colorless crystals (120 mg). Fraction E (4 g) was first isolated by the silica gel CC (200–300 mesh) eluted with PE-Me2CO (5/1) to give subfractions E1-G5. Fraction E2 (800 mg) was further isolated by CC using RP-C18 silica gel (MeOH/H2O from 6/4 to 9/1) to give subfractions E2a-E2e. HPLC preparation (MeCN/H2O from 7/3 to 8/2 in 20 mins) on fraction E2c (60 mg) afforded compounds 1 (8.8 mg, retention time (Rt) = 14.6 min) and 2 (4.3 mg, Rt = 13.5 min). Fraction E2e (70 mg) was separated by Sephadex LH-20 (MeOH) to give a mixture, which gave a precipitate when solvent evaporation. After filtrating and washing, white powder for 3 (6 mg) was obtained. Fraction G (8 g) was first isolated by the silica gel CC (200–300 mesh) eluted with PE-Me2CO (2/1) to give subfractions G1-G7. Fraction G3 (120 mg) was further isolated by silica gel CC eluted with PE-Me2CO (5/1) to give a precipitate. After filtrating and washing, white powder for 5 (12 mg) was obtained. Psathergosterol A (1): colorless crystals (MeOH), mp 234–235. [α]18 D = − 2.2 (c 1.8, MeOH). UV (MeOH) λmax (log ε) 225 (2.79) nm. IR (KBr) 3365, 2947, 2833, 1708, 1653, 1593, 1454, 1114, 1031 cm−1. 1 H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS: m/z 463.34177 [M + H]+ (calcd for C28H47O5+, 463.34235). X-Ray crystallographic data for psathergosterol A (1): a light colorless block-like of C28H46O5, M = 462.65, approximate dimensions 0.140 mm × 0.149 mm × 0.163 mm, was used for the X-ray crystallographic analysis on the BRUKER D8 QUEST. The integration of the data using a orthorhombic unit cell yielded a total of 30,921 reflections to a maximum θ angle of 79.19° (0.78 Å resolution), of which 5665 were independent (average redundancy 5.458, completeness = 97.3%, Rint = 3.01%, Rsig = 2.33%) and 5446 (96.13%) were greater than 2σ(F2). The final cell constants of a = 6.7623(7) Å, b = 12.5373(12) Å, c = 31.904(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2704.9(5) Å3, T = 150.(2) K. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P 21 21 21, with Z = 4. The final anisotropic full-matrix least-squares refinement on F2 with 316 variables converged at R1 = 2.90%, for the observed data and wR2 = 8.33% for all data. The goodness-of-fit was 1.084. The absolute configuration was determined by the Flack parameter = 0.03(3), which was determined using 2286 quotients [(I+)-(I)]/[(I+) + (I-)] [15]. The crystallographic data for 1 was deposited in the Cambridge Crystallographic Data Centre (CCDC deposition numbers: 1939957). The data can be obtained freely from the Cambridge Crystallographic Data Centre by visiting sites of www.ccdc.cam.ac.uk/ conts/retrieving.html. Psathergosterol B (2): white powder. [α]20 D = −30.1 (c 0.4, MeOH). UV (MeOH) λmax (log ε): 230 (3.81) nm. IR (KBr) 3338, 2943, 2831, 1702, 1622, 1591, 1452, 1113, 1031 cm−1. 1H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS at m/z 463.32571 [M + H]+ (calcd for C28H47O5+, 463.34235). Psathergosterol C (3): white powder. [α]24 D = + 27.5 (c 0.3, MeOH). UV (MeOH) λmax (log ε): 245 (3.72) nm. IR (KBr): 3338, 2943, 2831,
Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81872762), and the National Key Research and Development Program of China (2017YFC1704007). The authors thank the Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities for the spectra test. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2019.104289. References [1] M.B. Quin, C.M. Flynn, C. Schmidt-Dannert, Traversing the fungal terpenome, Nat. Prod. Rep. 31 (2014) 1449–1473. [2] G.K. Bian, J. Rinkel, Z.Q. Wang, L. Lauterbach, A.W. Hou, Y.J. Yuan, Z.X. Deng, T.G. Liu, J.S. Dickschat, A clade II-D fungal chimeric diterpene synthase from Colletotrichum gloeosporioides produces dolasta-1(15),8-diene, Angew. Chem. Int. Ed. 57 (2018) 15887–15890. [3] D. Tymann, U. Bednarzick, L. Iovkova-Berends, M. Hiersemann, Progress toward the total synthesis of gukulenin a: photochemically triggered two-carbon ring expansion key to α-tropolonic ether synthesis, Org. Lett. 20 (2018) 4072–4076. [4] X. Yin, T. Feng, Z.H. Li, Y. Leng, J.K. Liu, Five new guanacastane-type diterpenes from cultures of the fungus Psathyrella candolleana, Nat. Prod. Bioprospect. 4 (2014) 149–155. [5] Y.P. Liu, Q. Dai, C.J. Pu, M. Wang, Z.H. Li, J.K. Liu, T. Feng, Psathyrellanic acid, a monocyclic diterpenoid from the basidiomycete Psathyrella candolleana, Nat. Prod. Commun. 2019 (2019), https://doi.org/10.1177/1934578X19850958. [6] Z.Y. Zhou, G.Q. Shi, R. Fontaine, K. Wei, T. Feng, F. Wang, G.Q. Wang, T. Qu, Z.H. Li, Z.J. Dong, H.J. Zhu, Z.L. Yang, G. Zeng, J.K. Liu, Evidence for the natural toxins from the mushroom Trogia venenata as a cause of sudden unexpected death in Yunnan province, China, Angew. Chem. Int. Ed. 51 (2012) 2368–2370. [7] X.Y. Yang, T. Feng, Z.H. Li, Y. Sheng, X. Yin, Y. Leng, J.K. Liu, Conosilane A, an unprecedented sesquiterpene from the cultures of basidiomycete Conocybe siliginea, Org. Lett. 14 (2012) 5382–5384. [8] Z.Z. Zhao, H.P. Chen, B. Wu, L. Zhang, Z.H. Li, T. Feng, J.K. Liu, Matsutakone and matsutoic acid, two (nor)steroids with unusual skeletons from the edible mushroom Tricholoma matsutake, J. Organomet. Chem. 82 (2017) 7974–7979. [9] W. Li, J. He, T. Feng, H.X. Yang, H.L. Ai, Z.H. Li, J.K. Liu, Antroalbocin A, an antibacterial sesquiterpenoid from higher fungus Antrodiella albocinnamomea, Org. Lett. 20 (2018) 8019–8021. [10] H.X. Yang, H.L. Ai, T. Feng, W.X. Wang, B. Wu, Y.S. Zheng, H. Sun, J. He, Z.H. Li, J.K. Liu, Trichothecrotocins A−C, antiphytopathogenic agents from potato endophytic fungus Trichothecium crotocinigenum, Org. Lett. 20 (2018) 8069–8072. [11] X.L. Yang, Y.C. Zhu, S.T. Fang, M.Y. Jiang, Chemical constitutes of Termitomyces schimperi collected from Africa, Nat. Prod. Res. Dev. 22 (2010) 972–975. [12] H. Kawagishi, R. Katsumi, T. Sazawa, T. Mizuno, T. Hagiwara, T. Nakamura, Phytochemistry 27 (1988) 2777–2779. [13] S.B. Zhang, Z.H. Li, M. Stadlerd, H.P. Chen, Y. Huang, X.Q. Gan, T. Feng, J.K. Liu, Lanostane triterpenoids from Tricholoma pardinum with NO production inhibitory and cytotoxic activities, Phytochemistry 152 (2018) 105–112. [14] Y. Huang, S.B. Zhang, H.P. Chen, Z.Z. Zhao, Z.Y. Zhou, Z.H. Li, J.K. Liu, New acetylenic acids and derivatives from the edible mushroom Craterellus lutescens (Cantharellaceae), J. Agric. Food Chem. 65 (2017) 3835–3841. [15] S. Parsons, H.D. Flack, T. Wagner, Use of intensity quotients and differences in absolute structure refinement, Acta Cryst B69 (2013) 249–259.
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Cytotoxic ergosterols from cultures of the basidiomycete Psathyrella candolleana ⁎
T ⁎
Ya-Pei Liua,c, Chao-Jun Pua,c, Meng Wanga,c, Juan Hea,c, Zheng-Hui Lia,c, Tao Fenga,c, , Jie Xieb, , ⁎ Ji-Kai Liua,c, a
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing 400715, China c National Demonstration Center for Experimental Ethnopharmacology Education, South-Central University for Nationalities, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Psathyrella candolleana Ergosterol Psathergosterols A–C Cytotoxicity
Three newly isolated ergosterols, psathergosterols A–C (1–3), together with two known ones (4 and 5), have been isolated from cultures of the basdiomycete Psathyrella candolleana. Their structures with the absolute configuration were elucidated by means of spectroscopic methods and the single crystal X-ray diffraction. Compounds 2–4 exhibited certain cytotoxicities to five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480).
1. Introduction The fungus Psathyrella candolleana (Psathyrellaceae) is widely distributed in Europe and North America, as well as many Asian areas. It has been demonstrated to be a source for guanacastane-type diterpenoids which attracted great attention of chemists for both organic and biological studies [1–3]. In our previous studies on this fungus, a number of biologically active guanacastane-type diterpenoids, as well as a seco-derivative, have been isolated from the fermentation broths [4,5]. In order to search for more new and bioactive chemical constituents from this fungus, as well as in our ongoing searching for structurally interesting and biologically active natural products from higher fungi [6–11], a continuous study on the cultures of P. candolleana in rice medium has been carried out. As a result, three new ergosterols, psathergosterols A–C (1–3), together with two known ones 4 and 5 (Fig. 1), were obtained from the EtOAc layer. The structures with absolute configurations were identified by means of spectroscopic methods, including MS, NMR, and the single crystal X-ray diffraction. All compounds were evaluated for their cytotoxicities to five human cancer cell lines. Herein, the separation, structural elucidation, and the biological activities of the isolates are discussed. 2. Results and discussion Compound 1 was isolated as colorless crystals. Its molecular formula C28H46O5 was determined on the basis of the positive high
⁎
resolution (HR) ESIMS (measured at m/z 463.34177 [M + H]+; calcd for C28H47O5+, 463.34235), corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3365 cm−1), carbonyl (1653 cm−1), and double bond (1593, 1454 cm−1). The 1H NMR spectrum of 1 recorded in methanol-d4 displayed signals for four tertiary methyl groups as singlets at δH 0.74 (3H, s, Me-18), 0.95 (3H, s, Me-19), 1.12 (3H, s, Me-26), and 1.13 (3H, s, Me-27), two secondary methyl groups as doublets at δH 0.99 (3H, d, J = 6.5 Hz, Me-21), and 0.91 (3H, d, J = 6.8 Hz, Me-28), one oxygenated proton signal at δH 3.50 (1H, tt, J = 11.3, 4.4 Hz, H-3), and one olefinic proton signal at δH 5.83 (1H, s, H-7). All the carbons in compound 1 were detected by the 13 C NMR spectrum, in association with DEPT experiment. A total of 28 carbon signals were assigned as six CH3, nine CH2, six CH, and seven quaternary carbons. These data demonstrated that compound 1 should be an ergosterol derivative. In the 1He1H COSY spectrum, four fragments were readily established as shown in Fig. 2. In which, a hydroxy group at C-3 was identified. Based on these fragments, the HMBC data played an important role for the determination of the main substituents (Fig. 2). In detail, a 7.8-en-6-keto moiety was built by the three carbons with chemical shifts at δC 132.2 (d, C-7), 158.8 (s, C-8), and 201.7 (s, C6), and supported by the HMBC correlation from H-5 to C-6, and C-7, and from H-7 to C-9, and C-14. Three hydroxy groups at C-9, C-14, and C-25 were identified by the HMBC correlations from H-11 to δC 74.4 (s, C-9), from H-18 to δC 85.4 (s, C-14), and from H-26 and H-27 to δC 72.2 (s, C-25), respectively. After detailed analysis of HMBC data, a planar structure of compound 1 was built as shown in Fig. 2. The relative
Corresponding authors at: School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China. E-mail addresses: [email protected] (T. Feng), [email protected] (J. Xie), [email protected] (J.-K. Liu).
https://doi.org/10.1016/j.fitote.2019.104289 Received 12 July 2019; Received in revised form 1 August 2019; Accepted 2 August 2019 Available online 03 August 2019 0367-326X/ © 2019 Elsevier B.V. All rights reserved.
Fitoterapia 138 (2019) 104289
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Compound 2 was isolated as white powder. Its molecular formula C28H46O5, identical to that of 1, was revealed based on its HR-ESIMS data (measured at m/z 463.32571 [M + H]+; calcd for C28H47O5+, 463.34235). By comparison of the 1D and 2D NMR data with those of 1, compound 2 was supposed to be an epimer of 1. The significant difference of the NMR chemical shifts for CH-3 (δH 4.06, m; δC 64.3, d) suggested the different stereoconfiguation of C-3 in 2, with respect to that of 1. In addition, the REOSY correlation between H-3 and H-5 could not be observed any more, while the other 2D NMR data of 2 were the same to those of 1. Compound 2 was, therefore, identified as 3-epi-1, and named as psathergosterol B. Compound 3 was isolated as white powder. Its molecular formula C28H46O4 was determined on the basis of the positive HR-ESIMS (measured at m/z 469.32913 [M + Na]+; calcd for C28H46O4Na, 469.32938), corresponding to six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3338 cm−1), carbonyl (1698 cm−1) and double bond (1578, 1454 cm−1). The 1H NMR spectrum recorded in methanol-d4 displayed signals for four tertiary methyl groups as singlets at δH 0.82 (3H, s, Me-18), 0.92 (3H, s, Me-19), 1.12 (3H, s, Me-26), and 1.14 (3H, s, Me-27), two secondary methyl groups as doublets at δH 1.02 (3H, d, J = 6.7 Hz, Me-21), and 0.91 (3H, d, J = 7.1 Hz, Me-28), two oxygenated protons resonating at δH 3.56 (1H, m, H-3) and 4.43 (1H, td, J = 7.7, 4.8 Hz, H-16), and one olefinic protons resonating at δH 5.66 (1H, s, H-7). A total of 28 carbon signals were detected in the 13C NMR spectrum and assigned by the DEPT experiment as six methyl, eight methylene, nine methine, and five quaternary carbons. These data demonstrated that compound 3 was also probably an ergosterol derivative. In the 1He1H COSY spectrum, three fragments were also established as shown in Fig. 2. In which, a hydroxy group at C-3 (δC 68.7) and a hydroxy group at C-16 (δC 70.7) were easily identified. The same to that in 1 and 2, a 7,8-en-6-keto moiety was also built by the chemical shifts at δC 120.3 (d, C-7), 166.9 (d, C-8), and 203.2 (s, C-6), as well as the HMBC correlations from H-5 to C-6 and from H-7 to C-9 and C-14 (Fig. 2). In addition, the third hydroxy group at C-25 (δC 72.9, s) was identified by the HMBC correlations from H-24, H-26, and H-27 to C-25. Detailed analysis of HMBC data, a planar structure of 3, as shown in Fig. 2, was figured out. In the ROESY spectrum (Fig. 2), a cross peak between H-19 and H-5 indicated that H-5 should be β oriented, while the cross peak of H-3/H-5 suggested that OH-3 should be α oriented. In addition, the ROESY between H-9 and H-14 indicated that H-9 and H-14 should both be α oriented, consequently the ROESY cross peak of H-16 with H-14 and H-17 indicated that OH-16 should be β oriented. Compound 3 was, therefore, established and named as psathergosterol C. Two known ergosterols were identified as normal ones that usually obtained from fungi as ergosta-5,7,22-trien-3β-ol (4) [11] and 3β,5α,9α-trihydroxyergosta-7,22-dien-6-one (5) [12] by comparison their 1D NMR data with the reference samples in the laboratory. All compounds were evaluated for their cytotoxicities against five human cancer cell lines, including human myeloid leukemia cell line HL-60, human hepatocellular carcinoma cell line SMMC-7721, human lung cancer cell line A549, human breast cancercell line MCF-7, and human colon cancer SW-480, using the same method we reported previously [13,14]. As shown in Table 2, compounds 2–4 displayed certain cytotoxicity to several cancer cell lines. In conclusion, five ergoterols including three new ones have been isolated from cultures of the basidiomycete P. candolleana. Their structures were established by extensive spectroscopic methods. Compounds 2–4 showed certain cytotoxicities to several cancer cell lines. To our best known, this is the first time reporting new ergosterols from fungus P. candolleana, while H-5 in compound 3 possessing a β orientation in steroids occurred seldom.
Fig. 1. Psathergosterols A–C (1–3) and two known ergosterols (4 and 5) from Psathyrella candolleana.
Fig. 2. 1He1H COSY, HMBC and ROESY correlations for 1 and 3.
Fig. 3. ORTEP diagram of 1 showing the absolute configuration.
configuration of 1 was mainly elucidated by an ROESY experiment. It seems that H-3 should be β-oriented due to its constant coupling (J = 11.3, 4.4 Hz), while an ROESY cross peak between H-3 and H-5 indicated that H-5 should be α-oriented (Fig. 2). However, the stereoconfigurations of C-9 and C-14 are difficult to be established by the ROESY data. After attempts, a single crystal of 1 was obtained, the Xray diffraction determined not only the planar structure but also the absolute configuration of the molecule (Fig. 3. Flack parameter = 0.03(3)). The structure of compound 1 was, therefore, identified and named as psathergosterol A. 2
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Y.-P. Liu, et al.
Table 1 1 H (600 MHz) and No.
13
C (150 MHz) NMR Data of 1–3a in methanol‑d4 (δ in ppm, J in Hz).a
1
2
δH 1
1.96, 1.47, 1.79, 3.50, 2.11, 3.11,
2 3 4 5 6 7 8 9 10 11 12
dd (13.7, 3.7) m m; 1.34, m tt (11.3, 4.4) m; 1.31, m dd (12.3, 3.8)
δC
δH
28.6, CH2
2.36, 1.16, 1.68, 4.06, 1.98, 3.46,
1.79, m; 0.80, m 2.04, m; 1.72, m
29.7, CH2 69.4, CH 29.4, CH2 46.4, CH 201.7, qC 132.2, CH 158.8, qC 74.4, qC 42.0, qC 27.7, CH2 27.4, CH2
13 14 15
1.89, m; 1.63, m
46.3, qC 85.4, qC 30.0, CH2
16
2.06, m; 1.42, m
26.0, CH2
17 18 19 20 21 22 23 24 25 26 27 28
2.00, 0.74, 0.95, 1.46, 0.99, 1.00, 2.14, 1.29,
50.0, 15.1, 15.5, 36.1, 18.3, 34.1, 27.8, 44.9, 72.2, 24.5, 25.7, 13.8,
a
5.83, s
m s s m d (6.5) m; 1.60, m m; 1.72, m m
1.12, s 1.13, s 0.91, d (6.8)
3
td (13.5, 4.1) m m; 1.61, m m m; 1.57, m dd (12.2, 4.0)
5.83, s
2.05, m; 1.42, m 2.14, td (13.2, 4.0) 1.72, m
δH
δC
23.5, CH2
1.86, m; 1.14, m
33.1, CH2
26.9, CH2 64.3, CH 27.1, CH2 41.9, CH 203.5, qC 123.3, CH 158.6, qC 74.6, qC 42.6, qC 26.0, CH2 27.8, CH2
1.50, 3.56, 1.71, 2.02,
29.9, CH2 68.7, CH 34.3, CH2 56.2, CH 203.2, qC 120.3, CH 166.9, qC 37.6, CH 35.9, qC 21.2, CH2 38.9, CH2
46.3, qC 85.5, qC 30.0, CH2
1.90, m; 1.63 m 1.99, m 1.75 dd (8.4, 3.8) 2.00, m 0.77, s 0.94, s 1.46, m 0.98, d (6.5) 1.60, m; 1.00, m 1.80, m; 0.79, m 1.29, m
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
δC
27.2, CH2 49.9, 15.1, 14.8, 36.1, 18.3, 34.9, 27.7, 44.9, 72.7, 24.3, 25.9, 13.8,
1.12, s 1.13, s 0.90, d (6.8)
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
m; 1.79, m m m; 1.32, m dd (13.2, 3.7)
5.66, s 2.72, m 1.79, m; 1.71, m 2.17, dd (9.4, 3.0) 1.48, m 2.13, 2.27, 1.57, 4.43,
dd (12.6, 6.6) m td (12.9, 4.7) td (7.7, 4.7)
1.33, 0.82, 0.92, 1.90, 1.02, 1.71, 1.80, 1.33,
m s s m d (6.7) m; 1.33, m m; 0.88, m m
1.12, s 1.14, s 0.91, d (7.1)
44.8, qC 53.4, CH 35.1, CH2 70.7, CH 61.0, 12.4, 22.8, 30.3, 17.8, 34.2, 27.8, 44.7, 72.9, 24.3, 25.9, 14.0,
CH CH3 CH3 CH CH3 CH2 CH2 CH qC CH3 CH3 CH3
Data were assigned by HSQC, HMBC, 1He1H COSY and ROESY spectra.
Table 2 Cytotoxicities of 1–5 against five human cancer cell lines (IC50, μM). Sample
HL-60
SMMC-7721
A-549
MCF-7
SW480
1 2 3 4 5 Taxola
> 40 32.3 ± 1.12 > 40 > 40 > 40 < 0.008
> 40 > 40 29.3 ± 1.81 12.3 ± 1.43 > 40 < 0.008
> 40 23.4 ± 0.75 > 40 9.8 ± 1.98 > 40 < 0.008
> 40 28.3 ± 1.15 22.3 ± 1.37 11.9 ± 1.76 > 40 < 0.008
> 40 > 40 > 40 18.6 ± 1.71 > 40 < 0.008
a
Positive control.
9.4 × 150 mm, flowing speed = 10 mL/min).
3. Experimental section 3.1. General experimental procedures
3.2. Fungal material and cultivation conditions Optical rotations were measured on a Rudolph Autopol IV polarimeter. UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer. IR spectra were obtained by using a Shimadu Fourier Transform Infrared spectrometer with KBr pellets. NMR spectra were acquired with a Bruker Avance III 600 instrument. HR-ESIMS were measured on a Thermo Scientific Q Exactive Orbitrap MS system. Silica gel (200–300 mesh and 80–100 mesh, Qingdao Marine Chemical Inc., China), RP-18 gel (40–75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan) and Sephadex LH-20 (Amersham Biosciences, Upssala, Sweden) were used for column chromatography (CC). Fractions were monitored by TLC (Qingdao Marine Chemical Inc., China) and spots were visualized by 10% H2SO4 in methanol, in combination with Agilent 1260 series HPLC system (Zorbax SB-aq-C18 column, 5 μm, 4.9 × 150 mm, flowing speed = 1 mL/min). Preparative HPLC was performed on an Agilent 1260 series with a Zorbax SB-C18 column (5 μm,
Fruiting bodies of P. candolleana were collected at Jingdong, Yunnan Province, China in 2003 and identified by Prof. Zhu-Liang Yang (Kunming Institute of Botany). The voucher specimen (NO.CGBWSHF00118.2) was deposited at School of Pharmaceutical Sciences, South-Central University for Nationalities. Culture medium was composed of glucose (5%), pork pepton (0.15%), yeast (0.5%), KH2PO4 (0.05%) and MgSO4 (0.05%). Initial pH was adjusted to 6.0, the fermentation was first carried out on an erlenmeyer flask for 6 days till the mycelium biomass reached to the maximum. Then transfer it to rice medium for 24 °C in dark culture for 40 days. Rice medium: 50 g of rice, 50 mL of water, placed in a 250 mL Erlenmeyer flask, sterilized at 121 °C for 15 min, a total of 180 bottles.
3
Fitoterapia 138 (2019) 104289
Y.-P. Liu, et al.
1698, 1622, 1578, 1454, 1112, 1033 cm−1. 1H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS at m/z 469.32913 [M + Na]+ (calcd for C28H46O4Na, 469.32938).
3.3. Extraction and isolation The rice culture (9 Kg) was extracted four times with EtOAc. The organic layer was evaporated to give a crude extract (90 g). Then it was subjected to silica gel CC (200–300 mesh) eluted with petroleum ether (PE)-Me2CO gradient system to afford eight fractions A–H. Compound 4 was deposited from fraction B as colorless crystals (120 mg). Fraction E (4 g) was first isolated by the silica gel CC (200–300 mesh) eluted with PE-Me2CO (5/1) to give subfractions E1-G5. Fraction E2 (800 mg) was further isolated by CC using RP-C18 silica gel (MeOH/H2O from 6/4 to 9/1) to give subfractions E2a-E2e. HPLC preparation (MeCN/H2O from 7/3 to 8/2 in 20 mins) on fraction E2c (60 mg) afforded compounds 1 (8.8 mg, retention time (Rt) = 14.6 min) and 2 (4.3 mg, Rt = 13.5 min). Fraction E2e (70 mg) was separated by Sephadex LH-20 (MeOH) to give a mixture, which gave a precipitate when solvent evaporation. After filtrating and washing, white powder for 3 (6 mg) was obtained. Fraction G (8 g) was first isolated by the silica gel CC (200–300 mesh) eluted with PE-Me2CO (2/1) to give subfractions G1-G7. Fraction G3 (120 mg) was further isolated by silica gel CC eluted with PE-Me2CO (5/1) to give a precipitate. After filtrating and washing, white powder for 5 (12 mg) was obtained. Psathergosterol A (1): colorless crystals (MeOH), mp 234–235. [α]18 D = − 2.2 (c 1.8, MeOH). UV (MeOH) λmax (log ε) 225 (2.79) nm. IR (KBr) 3365, 2947, 2833, 1708, 1653, 1593, 1454, 1114, 1031 cm−1. 1 H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS: m/z 463.34177 [M + H]+ (calcd for C28H47O5+, 463.34235). X-Ray crystallographic data for psathergosterol A (1): a light colorless block-like of C28H46O5, M = 462.65, approximate dimensions 0.140 mm × 0.149 mm × 0.163 mm, was used for the X-ray crystallographic analysis on the BRUKER D8 QUEST. The integration of the data using a orthorhombic unit cell yielded a total of 30,921 reflections to a maximum θ angle of 79.19° (0.78 Å resolution), of which 5665 were independent (average redundancy 5.458, completeness = 97.3%, Rint = 3.01%, Rsig = 2.33%) and 5446 (96.13%) were greater than 2σ(F2). The final cell constants of a = 6.7623(7) Å, b = 12.5373(12) Å, c = 31.904(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2704.9(5) Å3, T = 150.(2) K. Data were corrected for absorption effects using the Multi-Scan method (SADABS). The structure was solved and refined using the Bruker SHELXTL Software Package, using the space group P 21 21 21, with Z = 4. The final anisotropic full-matrix least-squares refinement on F2 with 316 variables converged at R1 = 2.90%, for the observed data and wR2 = 8.33% for all data. The goodness-of-fit was 1.084. The absolute configuration was determined by the Flack parameter = 0.03(3), which was determined using 2286 quotients [(I+)-(I)]/[(I+) + (I-)] [15]. The crystallographic data for 1 was deposited in the Cambridge Crystallographic Data Centre (CCDC deposition numbers: 1939957). The data can be obtained freely from the Cambridge Crystallographic Data Centre by visiting sites of www.ccdc.cam.ac.uk/ conts/retrieving.html. Psathergosterol B (2): white powder. [α]20 D = −30.1 (c 0.4, MeOH). UV (MeOH) λmax (log ε): 230 (3.81) nm. IR (KBr) 3338, 2943, 2831, 1702, 1622, 1591, 1452, 1113, 1031 cm−1. 1H (600 MHz) and 13C NMR (150 MHz) data (methanol-d4): see Table 1; HR-ESIMS at m/z 463.32571 [M + H]+ (calcd for C28H47O5+, 463.34235). Psathergosterol C (3): white powder. [α]24 D = + 27.5 (c 0.3, MeOH). UV (MeOH) λmax (log ε): 245 (3.72) nm. IR (KBr): 3338, 2943, 2831,
Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81872762), and the National Key Research and Development Program of China (2017YFC1704007). The authors thank the Analytical & Measuring Center, School of Pharmaceutical Sciences, South-Central University for Nationalities for the spectra test. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2019.104289. References [1] M.B. Quin, C.M. Flynn, C. Schmidt-Dannert, Traversing the fungal terpenome, Nat. Prod. Rep. 31 (2014) 1449–1473. [2] G.K. Bian, J. Rinkel, Z.Q. Wang, L. Lauterbach, A.W. Hou, Y.J. Yuan, Z.X. Deng, T.G. Liu, J.S. Dickschat, A clade II-D fungal chimeric diterpene synthase from Colletotrichum gloeosporioides produces dolasta-1(15),8-diene, Angew. Chem. Int. Ed. 57 (2018) 15887–15890. [3] D. Tymann, U. Bednarzick, L. Iovkova-Berends, M. Hiersemann, Progress toward the total synthesis of gukulenin a: photochemically triggered two-carbon ring expansion key to α-tropolonic ether synthesis, Org. Lett. 20 (2018) 4072–4076. [4] X. Yin, T. Feng, Z.H. Li, Y. Leng, J.K. Liu, Five new guanacastane-type diterpenes from cultures of the fungus Psathyrella candolleana, Nat. Prod. Bioprospect. 4 (2014) 149–155. [5] Y.P. Liu, Q. Dai, C.J. Pu, M. Wang, Z.H. Li, J.K. Liu, T. Feng, Psathyrellanic acid, a monocyclic diterpenoid from the basidiomycete Psathyrella candolleana, Nat. Prod. Commun. 2019 (2019), https://doi.org/10.1177/1934578X19850958. [6] Z.Y. Zhou, G.Q. Shi, R. Fontaine, K. Wei, T. Feng, F. Wang, G.Q. Wang, T. Qu, Z.H. Li, Z.J. Dong, H.J. Zhu, Z.L. Yang, G. Zeng, J.K. Liu, Evidence for the natural toxins from the mushroom Trogia venenata as a cause of sudden unexpected death in Yunnan province, China, Angew. Chem. Int. Ed. 51 (2012) 2368–2370. [7] X.Y. Yang, T. Feng, Z.H. Li, Y. Sheng, X. Yin, Y. Leng, J.K. Liu, Conosilane A, an unprecedented sesquiterpene from the cultures of basidiomycete Conocybe siliginea, Org. Lett. 14 (2012) 5382–5384. [8] Z.Z. Zhao, H.P. Chen, B. Wu, L. Zhang, Z.H. Li, T. Feng, J.K. Liu, Matsutakone and matsutoic acid, two (nor)steroids with unusual skeletons from the edible mushroom Tricholoma matsutake, J. Organomet. Chem. 82 (2017) 7974–7979. [9] W. Li, J. He, T. Feng, H.X. Yang, H.L. Ai, Z.H. Li, J.K. Liu, Antroalbocin A, an antibacterial sesquiterpenoid from higher fungus Antrodiella albocinnamomea, Org. Lett. 20 (2018) 8019–8021. [10] H.X. Yang, H.L. Ai, T. Feng, W.X. Wang, B. Wu, Y.S. Zheng, H. Sun, J. He, Z.H. Li, J.K. Liu, Trichothecrotocins A−C, antiphytopathogenic agents from potato endophytic fungus Trichothecium crotocinigenum, Org. Lett. 20 (2018) 8069–8072. [11] X.L. Yang, Y.C. Zhu, S.T. Fang, M.Y. Jiang, Chemical constitutes of Termitomyces schimperi collected from Africa, Nat. Prod. Res. Dev. 22 (2010) 972–975. [12] H. Kawagishi, R. Katsumi, T. Sazawa, T. Mizuno, T. Hagiwara, T. Nakamura, Phytochemistry 27 (1988) 2777–2779. [13] S.B. Zhang, Z.H. Li, M. Stadlerd, H.P. Chen, Y. Huang, X.Q. Gan, T. Feng, J.K. Liu, Lanostane triterpenoids from Tricholoma pardinum with NO production inhibitory and cytotoxic activities, Phytochemistry 152 (2018) 105–112. [14] Y. Huang, S.B. Zhang, H.P. Chen, Z.Z. Zhao, Z.Y. Zhou, Z.H. Li, J.K. Liu, New acetylenic acids and derivatives from the edible mushroom Craterellus lutescens (Cantharellaceae), J. Agric. Food Chem. 65 (2017) 3835–3841. [15] S. Parsons, H.D. Flack, T. Wagner, Use of intensity quotients and differences in absolute structure refinement, Acta Cryst B69 (2013) 249–259.
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