Edited by: Zhendong Jin, The University of Iowa, United States
Reviewed by: Zhen Liu, Heinrich Heine Universität Düsseldorf, Germany; Xiuping Lin, Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology (CAS), China; Fernando Reyes, Fundación MEDINA, Spain
This article was submitted to Organic Chemistry, a section of the journal Frontiers in Chemistry
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Plant pathogenic fungi can produce toxic substances, known as phytotoxins, that adversely affect the host plants, and might play a role in the development of plant disease (Strange,
As part of our continuing interest in the secondary metabolites, especially the phytotoxins, of
Optical rotations were recorded on a Rudolph Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, New Jersey). Ultraviolet (UV) spectra were recorded on a TU-1810 UV/vis spectrophotometer (Beijing Persee General Instrument Co., Ltd., Beijing, China). Circular dichroism (CD) spectra were recorded on a JASCO J-815 CD spectrometer (JASCO Corp., Tokyo, Japan). Infrared (IR) spectra were measured on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Scientific Instrument Crop., Madison, Wisconsin). High-resolution electrospray ionization mass spectrometry (HRESIMS) spectra were recorded on a LC 1260/Q-TOF-MS 6520 instrument (Agilent Technologies, Santa Clara, CA). 1H, 13C, and 2D NMR (1H-1H COSY, HSQC/HMQC, HMBC, and NOESY) spectra were measured on Bruker Avance 600 or 400 NMR spectrometers (Bruker BioSpin, Zürich, Switzerland). 1H and 13C NMR chemical shifts were expressed in δ (ppm) referring to the inner standard tetramethylsilane (TMS), and coupling constants in Hertz. HPLC-DAD analysis of the EtOAc extracts was performed on a Shimadzu LC-20A instrument equipping with a SPD-M20A photodiode array detector (Shimadzu Corp., Tokyo, Japan) using an analytical C18 column (250 × 4.6 mm i.d., 5 μm; Phenomenex Inc., Torrance, California). Semipreparative HPLC separation was carried out on a Lumtech instrument (Lumiere Tech. Ltd., Beijing, China) equipped with a K-501 pump (flow rate: 3 mL/min) and a K-2501 UV detector (detection was set at 370 nm) using a Luna-C18 column (250 × 10 mm i.d., 5 μm, Phenomenex Inc., Torrance, California) eluting with a mixture of MeOH and water (containing 0.02% TFA).
The fungal strain
The strain
The SF (60 g) was subjected to vacuum liquid chromatograph over silica gel (200~300 mesh, i.d. 8 × 16 cm), eluting with a gradient of CH2Cl2/MeOH (100:0~0:100), to yield six fractions (Frs. A~F).
Fr. B was further fractionated by medium pressure liquid chromatography over silica gel (i.d. 4.0 × 50 cm) to obtain ten subfractions (Frs. B1~B10). Compound
Fr. C was subjected to column chromatography (i.d. 3.0 × 60 cm) over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to yield seven subfractions (Frs. C1~C7), among which Fr. C5 was purified by semi-preparative HPLC (85% MeOH/H2O) to give oxosorbicillinol (
Fr. D was fractionated by column chromatography (i.d. 4.0 × 40 cm) on RP-18 eluting with MeOH/H2O (gradually increased from 55:45 to 100:0) to obtain 12 subfractions (Frs. D1~D12). Fr. D7 was further chromatographed (i.d. 3.0 × 60 cm) over Sephadex LH-20 (CH2Cl2/MeOH, 1:1) to afford six fractions (Frs. D7.1~D7.6). Compounds
Fr. E was chromatographed (i.d. 4.0 × 40 cm) over ODS eluting with a gradient increase of MeOH in water (from 30:70 to 100:0) to obtain six subfractions (Frs. E1~E6). The 2-pyrones,
Ustisorbicillinol A (
1H and 13C NMR data of
1 | 33.1, CH2 | 2.55, d (15.2)2.26, d (15.2) | 34.2, CH2 | 2.48, d (16.0)2.30, d (16.0) | 55.4, CH | 3.12, s | 54.9, CH | 3.12, s |
2 | 74.5, C | 74.2, C | 79.0, C | 79.2, C | ||||
3 | 107.6, C | 107.7, C | 105.3, C | 104.3, C | ||||
4 | 57.7, C | 57.8, C | 55.7, CH | 3.00, d (12.0) | 56.0, CH | 3.04, d (12.2) | ||
5 | 172.1, C | 171.6, C | 170.8, C | 172.3, C | ||||
6 | 110.2, C | 110.2, C | 109.1, C | 109.8, C | ||||
7 | 193.8, C | 194.3, C | 188.3, C | 188.7, C | ||||
8 | 41.1, CH2 | 2.44, dd (17.0, 13.0)2.33, dd (17.0, 4.0) | 42.3, CH2 | 2.23, dd (16.9, 3.6)2.12, dd (16.9, 14.7) | 41.1, CH2 | 2.58, dd (16.7, 4.8)2.18, dd (16.7, 8.7) | 41.7, CH2 | 2.56, dd (16.8, 13.4)2.29, dd (16.8, 3.5) |
9 | 79.3, CH | 4.26, ddd (13.0, 5.9, 4.0) | 80.6, CH | 4.56, m | 80.2, CH | 4.93, m | 81.5, CH | 4.38, ddd (13.4, 7.0, 3.5) |
10 | 128.6, CH | 5.47, dd (15.5, 5.9) | 129.2, CH | 5.45, dd (15.5, 6.3) | 128.3, CH | 5.43, ddq (15.2, 7.3, 1.7) | 128.7, CH | 5.57, ddq (15.0, 7.0, 1.7) |
11 | 131.7, CH | 5.80, m | 130.7, CH | 5.76, dq (15.5, 6.5) | 131.4, CH | 5.76, dqd (15.2, 6.6, 1.0) | 132.0, CH | 5.73, dqd (15.0, 6.6, 1.0) |
12 | 18.0, CH3 | 1.72, d (6.3) | 18.0, CH3 | 1.73, d (6.5) | 17.9, CH3 | 1.65, ddd (6.6, 1.7, 0.7) | 17.8, CH3 | 1.66, ddd (6.6, 1.7, 0.7) |
13 | 22.3, CH3 | 1.11, s | 22.6, CH3 | 1.17, s | 20.9, CH3 | 1.27, s | 20.9, CH3 | 1.25, s |
14 | 19.7, CH3 | 1.32, s | 19.1, CH3 | 1.29, s | ||||
1′ | 53.9, CH | 3.69, s | 54.0, CH | 3.69, s | 48.6, CH | 3.67, d (12.0) | 48.5, CH | 3.67, d (12.2) |
2′ | 80.2, C | 80.2, C | 81.3, C | 81.1, C | ||||
3′ | 171.2, C | 170.9, C | 104.9, C | 105.3, C | ||||
4′ | 108.8, C | 108.7, C | 60.4, C | 60.7, C | ||||
5′ | 192.7, C | 193.3, C | 202.2, C | 202.2, C | ||||
6′ | 102.8, C | 102.7, C | 105.1, C | 105.3, C | ||||
7′ | 168.3, C | 168.3, C | 174.2, C | 174.0, C | ||||
8′ | 122.0, CH | 6.45, d (14.8) | 121.9, CH | 6.45, d (14.8) | 120.7, CH | 6.56, d (14.8) | 120.6, CH | 6.53, d (14.8) |
9′ | 139.4, CH | 7.16, dd (14.8, 10.9) | 139.3, CH | 7.17, dd (14.8, 10.8) | 142.6, CH | 7.28, dd (14.8, 10.9) | 142.6, CH | 7.29, dd (14.8, 10.9) |
10′ | 132.5, CH | 6.33, dd (15.1, 10.9) | 132.5, CH | 6.32, dd (15.1, 10.8) | 132.0, CH | 6.38, m | 132.0, CH | 6.36, m |
11′ | 137.4, CH | 6.10, dq (15.1, 6.8) | 137.3, CH | 6.10, dq (15.1, 6.8) | 139.5, CH | 6.25, dq (15.1, 6.8) | 139.6, CH | 6.26, dq (15.0, 6.8) |
12′ | 18.8, CH3 | 1.86, d (6.8) | 18.8, CH3 | 1.86, d (6.8) | 18.8, CH3 | 1.87, dd (6.8, 1.6) | 18.8, CH3 | 1.87, dd (6.8, 1.6) |
13′ | 26.6, CH3 | 1.46, s | 26.6, CH3 | 1.46, s | 21.5, CH3 | 1.35, s | 21.5, CH3 | 1.36, s |
14′ | 7.8, CH3 | 1.62, s | 7.9, CH3 | 1.59, s | 20.1, CH3 | 1.31, s | 19.9, CH3 | 1.31, s |
Ustisorbicillinol B (
Ustisorbicillinol C (
Ustisorbicillinol D (
Ustisorbicillinol E (
1H (600MHz) and 13C (150MHz) NMR data of
1 | 45.5, CH | 3.60, d (3.5) |
2 | 74.2, C | |
3 | 209.2, C | |
4 | 62.5, C | |
5 | 194.9, C | |
6 | 104.7, C | |
7 | 172.3, C | |
8 | 117.2, CH | 6.12, d (14.9) |
9 | 144.6, CH | 7.38, dd (14.8, 10.2) |
10 | 130.8, CH | 6.29, dd (15.3, 10.2) |
11 | 141.4, CH | 6.24, m |
12 | 19.0, CH3 | 1.91, d (6.0) |
13 | 24.4, CH3 | 1.28, s |
14 | 10.4, CH3 | 1.23, s |
1′ | 76.3, CH | 5.44, dd (8.9, 3.5) |
2′ | 41.4, CH | 2.89, ddd (11.5, 8.9, 5.5) |
3′ | 30.2, CH2 | 2.70, dd (19.2, 11.6) |
4′ | 174.8, C | |
7-OH | 14.32, s |
Ustisorbicillinol F (
1H (600MHz) and 13C (150MHz) NMR data of
1 | 109.7, C | |
2 | 169.2, C | |
3 | 73.7, C | |
4 | 196.9, C | |
5 | 106.1, C | |
6 | 165.5, C | |
7 | 180.5, C | |
8 | 111.7, CH | 6.31, s |
9 | 163.9, C | |
10 | 118.6, CH | 6.12, d (15.4) |
11 | 140.1, CH | 7.24, ov. |
12 | 130.1, CH | 6.26, ov. |
13 | 140.6, CH | 6.27, ov. |
14 | 18.9, CH3 | 1.92, br. d (4.9) |
15 | 30.0, CH3 | 1.64, s |
16 | 6.8, CH3 | 1.88, s |
6-OH | 13.43, br. s |
Ustilopyrone A (
1H (400 MHz) and 13C (100 MHz) NMR data of
2 | 167.5, C |
167.03, C |
168.0, C | |||
3 | 100.6, C | 102.3, C | 100.8, C | |||
4 | 167.4, C |
166.95, C |
167.6, C | |||
5 | 110.5, C | 113.7, C | 102.3, CH | 6.07, s | ||
6 | 153.7, C | 152.2, C | 158.0, C | |||
7 | 120.7, CH | 6.53, d (15.1) | 128.2, CH | 6.97, d (15.0) | 123.2, CH | 6.18, d (15.3) |
8 | 135.4, CH | 7.06, dd (15.1, 11.0) | 132.2, CH | 7.13, dd (15.0, 11.2) | 135.6, CH | 7.05, dd (15.3, 11.0) |
9 | 130.5, CH | 6.49, dd (15.0, 11.0) | 144.6, CH | 7.44, dd (15.2, 11.2) | 129.8, CH | 6.43, dd (15.2, 11.0) |
10 | 139.2, CH | 6.12, dt (15.0, 5.3) | 126.0, CH | 6.12, d (15.2) | 140.0, CH | 6.15, dt (15.2, 5.2) |
11 | 63.1, CH2 | 4.19, d (5.3) | 170.0, C | 63.0, CH2 | 4.18, d (5.2) | |
3-Me | 9.2, CH3 | 1.94, s | 9.4, CH3 | 1.96, s | 8.6, CH3 | 1.89, s |
5-Me | 9.6, CH3 | 2.04, s | 9.9, CH3 | 2.09, s |
Ustilopyrone B (
5-Demethylustilopyrone A (
The Molecular Merck force field (MMFF) conformational search, geometry optimization and frequency calculations at the B3LYP 6-31G(d) level
For the calculations of 13C NMR chemical shifts, B3LYP/6-31G(d,p) method was used to optimize the selected conformers. For all optimized structures, vibrational spectra were calculated to ensure that no imaginary frequencies for energy minimum were obtained. NMR calculations were performed at the level of mPW1PW91/6-31G(d,p) with the gauge-independent atomic orbital (GIAO) method (Forsyth and Sebag,
The b3lyp/6-31g(d,p)-optimized conformers of (
The major compounds (
Cytotoxicities of the isolated compounds were tested against human carcinoma cells using the microculture tetrazolium (MTT) assay as described previously (Sun et al.,
The isolated compounds were evaluated for antibacterial activities against six human/plant pathogenic bacteria, including
The antifungal activities of compounds
The crude EtOAC extract was repeatedly fractionated over sephadex LH-20, silica gel, and RP-18, and purified by semi-preparative HPLC to give 12 bisorbicillinoids, and five sorbicillinoid-like metabolites (
Structures of the isolated sorbicillinoids.
Compound
A long spin system was easily recognized by anlysis of the coupling constants and 1H-1H COSY spectrum, in which cross-peaks were observed between CH3-12′ (δH 1.86, d)/H-11′ (δH 6.10, dq), H-11′/H-10′ (δH 6.33, dd), H-10′/H-9′ (δH 7.16, dd), and H-9′/H-8′ (δH 6.45, d). The HMBC correlations from H-9′ to C-7′ (δC 168.3), as well as from H-8′ to C-6′ (δC 102.8) and C-7′ established an enol-sorbyl chain (box A,
Selected 1H-1H COSY (bold) and HMBC (H→ C, arrow) correlations of two monomeric units (
The second monomeric unit (box B,
The linkage of the two units was deduced by analysis of the HMBC spectrum. A key HMBC correlation from CH3-14 to C-1′ indicated a direct sigma bond between C-1′ and C-4. Considering the molecular formula and the required degrees of unsaturation, one additional ether ring had to be formed. Then, a C-2′-O-C-3 ether was deduced by analysis of the chemical shifts of C-2′ (δC 80.2) and C-3 (δC 107.6), which was consistent with those of the reported bisvertinols (Trifonov et al.,
The stereochemistry of
Selected NOESY correlations of
Structurally,
Experimental ECD spectra of
Ustisorbicillinol B (
Ustisorbicillinol C (
ECD spectra of
Ustisorbicillinol D (
Ustisorbicillinol E (
Selected 1H-1H COSY, and HMBC
The relative configuration of
Experimental ECD spectrum of
Ustisorbicillinol F (
Selected HMBC correlations of
Ustilopyrone A (
Additionally, a new analog of ustilopyrone A (
The other isolated compounds were identified by comparing the spectroscopic data with those published in the literature, and included 5-demethylustilopyrone A (also named saturnispol H,
The major bisorbicillinoids
Phytotoxicity of the isolated compounds against the radicle and germ elongation of rice and lettuce seeds.
50 | 65.37 ± 3.54c | 20.33 ± 6.74c | 8.31 ± 1.06ij | 14.73 ± 3.07hi | |
100 | 88.42 ± 1.79b | 29.81 ± 7.01c | 16.06 ± 1.77fg | 15.68 ± 1.35hi | |
200 | 98.33 ± 3.33a | 51.92 ± 8.85b | 58.30 ± 3.91e | 33.58 ± 3.32d | |
400 | 98.86 ± 1.41a | 64.84 ± 6.65a | 100.00 ± 0.00a | 100.00 ± 0.00a | |
50 | 9.79 ± 5.90c | 14.56 ± 7.93a | 7.82 ± 1.53j | 8.55 ± 2.02j | |
100 | 23.49 ± 9.38b | 12.91 ± 3.52a | 11.74 ± 2.82hi | 13.64 ± 2.64i | |
200 | 35.07 ± 8.80b | 14.70 ± 5.63a | 18.85 ± 2.97f | 16.85 ± 3.35ghi | |
400 | 68.47 ± 5.31a | 18.41 ± 7.18a | 100.00 ± 0.00a | 100.00 ± 0.00a | |
50 | 7.49 ± 3.66b | 3.57 ± 7.14c | 3.95 ± 0.57k | 6.00 ± 0.27j | |
100 | 5.37 ± 5.35b | 16.62 ± 3.25b | 11.72 ± 2.59hi | 21.06 ± 2.73fg | |
200 | 4.38 ± 7.19b | 18.41 ± 6.80b | 15.95 ± 2.47fg | 26.87 ± 4.15e | |
400 | 45.89 ± 6.87a | 36.95 ± 5.06a | 60.60 ± 1.31e | 50.87 ± 4.34c | |
50 | 18.25 ± 6.04b | 12.64 ± 10.63bc | 9.96 ± 1.28hij | 8.97 ± 2.04 |
|
100 | 23.24 ± 8.05b | 7.55 ± 6.29b | 13.03 ± 1.92gh | 17.21 ± 3.28fghi | |
200 | 43.83 ± 1.28a | 22.25 ± 8.62ab | 18.86 ± 2.08f | 21.31 ± 3.68f | |
400 | 51.49 ± 5.09a | 31.46 ± 3.02a | 100.00 ± 0.00a | 100.00 ± 0.00a | |
Glyphosate |
50 | 84.51 ± 3.29c | 59.34 ± 8.94c | 75.92 ± 2.80d | 18.51 ± 0.77 |
100 | 93.93 ± 2.24b | 74.18 ± 3.17ab | 77.41 ± 3.59cd | 27.63 ± 0.95e | |
200 | 95.80 ± 1.30b | 72.12 ± 4.43b | 80.23 ± 1.77bc | 28.80 ± 2.43e | |
400 | 99.62 ± 0.76a | 81.46 ± 4.36a | 82.97 ± 1.53b | 56.81 ± 3.65b |
Inhibitory activity of
When compared the inhibitory activity of each tested compound at each tested concentration against both type of seeds, a general potency of
The isolated compounds were also evaluated for their cytotoxicities against nine human carcinoma cell lines including NCI-H460, BGC823, Daoy, HepG2, HCT116, A375, A549, MCF-7, and Capan2 (
Cytotoxicity of the isolated compounds (IC50, μM).
8.83 | 38.2 | 21.2 | 25.8 | 12.9 | – | – | – | – | |
– | – | – | – | 28.7 | 52.4 | 25.4 | 38.3 | >50.0 | |
– | – | – | – | >50.0 | 41.3 | 45.0 | 74.7 | >50.0 | |
– | – | – | – | 31.7 | 45.0 | 60.5 | 48.8 | >50.0 | |
Taxol |
<0.008 | <0.008 | 0.00504 | 0.0752 | 0.0019 | 0.0220 | 0.0232 | <0.008 | 0.0167 |
The antibacterial activities of the isolated compounds were also tested (
Antibacterial activity of the isolated compounds.
MIC | 64 | 64 | 128 | 8 | 32 | 5 | |
IC50 | 35.43 | 75.68 | 38.76 | 3.36 | 22.72 | 1.41 | |
MIC | 64 | 128 | 128 | 8 | 32 | 5 | |
IC50 | 41.92 | 67.26 | 43.23 | 4.01 | 24.23 | 1.37 | |
MIC | 32 | 64 | 64 | 8 | 32 | 7.5 | |
IC50 | 37.15 | 73.55 | 16.71 | 3.87 | 12.87 | 2.34 | |
MIC | 64 | 64 | 64 | 4 | 24 | 5 | |
IC50 | 80.66 | 22.29 | 29.53 | 3.67 | 9.58 | 1.21 | |
MIC | 64 | 128 | 128 | 4 | 32 | 5 | |
IC50 | 14.78 | 35.20 | 49.80 | 2.02 | 15.34 | 1.4 | |
MIC | 64 | 64 | 64 | 8 | 24 | 10 | |
IC50 | 25.66 | 41.41 | 19.59 | 4.73 | 10.13 | 2.59 |
Additionally, compounds
In the present study, we isolated 17 sorbicillinoids including eight new compounds from the pathogenic fungus
The biosynthesis of sorbicillinol has been reported in
Proposed biosynthetic route for the monomeric sorbicillinoid and related metabolites (
The biosynthetic pathway for bisorbicillinols
Compounds
Sorbicillinoids were reported to have diverse biological activities, such as cytotoxic, antimicrobial, and antioxidant activity (Harned and Volp,
In conclusion, 17 sorbicillinoids were isolated from the fermentation of
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
LZ, DL, and JM conceived and designed the experiments. JM was responsible for the isolation of compounds. DL and JM elucidated the structures. JD tested cytotoxicity of the compounds. JM, GG, PD, XZ, and WW performed the experiments of phytotoxic and antimicrobial activities. DL, LZ, and JM interpreted the data and wrote the paper. YL and JD revised the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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