Bioactive Meroterpenoids and Isocoumarins from the Mangrove

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Bioactive Meroterpenoids and Isocoumarins from the MangroveDerived Fungus Penicillium sp. TGM112 Meng Bai,†,‡,# Cai-Juan Zheng,*,†,‡,# Guo-Lei Huang,†,‡ Rong-Qing Mei,†,‡ Bin Wang,†,‡ You-Ping Luo,†,‡ Chao Zheng,†,‡ Zhi-Gang Niu,†,‡ and Guang-Ying Chen*,†,‡ †

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Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, Hainan 571158, People’s Republic of China ‡ Key Laboratory of Tropical Medicinal Plant Chemistry of Hainan Province, Hainan Normal University, Haikou, Hainan 571158, People’s Republic of China S Supporting Information *

ABSTRACT: Two new meroterpenoids, penicianstinoids A and B (1 and 2), and eight new isocoumarins, peniciisocoumarins A−H (3−10), together with 10 known analogues (11−20) were obtained from the mangrove-derived fungus Penicillium sp. TGM112. The structures and absolute configurations of 1−10 were determined by interpretation of detailed NMR, MS spectroscopic data, X-ray diffraction analyses, modified Mosher’s method, and calculated electronic circular dichroism data. Compounds 1−4, 7, 8, 10, 12, 13, and 16 showed growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner with IC50 values ranging from 50 to 200 μg/mL, respectively. Compounds 1, 2, and 11−15 displayed activity against Caenorhabditis elegans with EC50 values ranging from 9.4 (± 1.0) to 38.2 (± 0.6) μg/mL, respectively. Compound 1 represents an austinoid-like meroterpenoid that is reported here for the second time, in which a carbon−carbon double bond was oxidized to a carbonyl group at C-1′−C-2′.

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ungi from the genus Penicillum produce a variety of novel bioactive metabolites, including the antiosteoporotic citrofulvicin,1 anti-inflammatory chrysogenester,2 α-glycosidase inhibitory chrysines B and C,3 cytotoxic penicimenolides B−D and penitalarins A−C,4,5 antibacterial brocapyrrozin A,6 and the antiviral simpterpenoid A.7 These results have increasingly attracted the attention of both pharmaceutical and natural product chemists, due to the novel structures and notable biological activities reported. Mangrove-derived fungi have been shown to be useful in the search for medicinal lead compounds.8 In our continuing investigation into bioactive metabolites of mangrove-derived fungi,9−14 the mangrovederived fungi Penicillium sp. TGM112 isolated from the medicinal mangrove Bruguiera sexangula var. rhynchopetala, showed significant activity against newly hatched larvae of Helicoverpa armigera Hubner. A chemical investigation of this fungus led to the identification of two new meroterpenoids penicianstinoids A and B (1 and 2), and eight new isocoumarins, peniciisocoumarins A−H (3−10), together with 10 known analogues (11−20). Herein, the isolation, structure elucidation, and bioactivities of these compounds are described.

of HR-ESIMS, implying 14 degrees of unsaturation. Analysis of the 1H and 13C NMR data (Table 1) indicated that 1 had six carbonyl carbons and four olefinic carbons, suggesting that 1 was a hexacyclic compound and had a meroterpenoid skeleton. In addition, the 1H NMR data (Table 1) displayed one olefinic proton signal at δH 6.78 (q, J = 5.6 Hz, H-20), one terminal double-bond group at δH 5.83 (dd, J = 24.0, 1.6 Hz, H-13), three oxymethines at δH 5.93 (m, H-11), 5.47 (dd, J = 10.0, 5.6 Hz, H-7), and 5.19 (q, J = 6.8 Hz, H-5′), and three methylenes at δH 2.61 (m, H-2), 2.53 (m, H-1a) and 2.33 (m, H-1b), and 1.86 (d, J = 5.2 Hz, H-6a) and 1.83 (d, J = 5.2 Hz, H-6b). The 1 H and 13C NMR spectroscopic data of 1 (Table 1) were similar to the known compound 1,2-dehydroterredehydroaustin.15 The obvious differences were the lack of two olefinic carbons (δC 116.7 and 137.0) at C-1′ and C-2′ in 1 and the presence of one carbonyl group (δC 191.7) at C-2′, indicating that the carbon−carbon double bond at C-1′−C-2′ in 1,2dehydroterredehydroaustin was oxidized to a carbonyl group at C-2′ in 1. In addition, the HR-ESIMS spectrum revealed that 1 contained one more oxygen atom than 1,2-dehydroterredehydroaustin. These were confirmed by the HMBC correlations of 9′-Me to C-2′/C-3′/C-11 and of H-11 to C-2′. The whole structure was further determined by the 2D NMR

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals, and the molecular formula was deduced to be C31H36O12 on the basis © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 17, 2018

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DOI: 10.1021/acs.jnatprod.8b00866 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

which a carbon−carbon double bond was oxidized to a carbonyl group at C-1′−C-2′.16 Compound 2 was obtained as colorless crystals. The molecular formula was deduced to be C25H28O9 on the basis of HR-ESIMS, implying 12 degrees of unsaturation. The 1D NMR data (Table 1) of 2 closely resembled those of dehydroaustinol,17 with the main difference being the replacement of a methylene group at δH 1.70 (m) and δC 27.7 (CH2) for C-7 in dehydroaustinol with a methine group [δH 4.32 (dd, J = 12.0, 4.8 Hz) and δC 64.7 (CH)] for C-7 in 2. These results were confirmed by the 1H−1H COSY correlation of H-6 to H-7 and the HMBC correlations of H-7 to C-5/C-8/ C-9 (Figure 1). The absolute configuration of 2 was determined by interpretation of the NOESY data and the Xray crystal diffraction (Figures 3 and 4). The absolute configuration could be determined as 5S,7R,8S,9R,11R,3′S,5′R,6′R,7′S by comparing experimental and calculated ECD spectra using TDDFT (Figure 5). Thus, the structure of 2 was determined and named penicianstinoid B. Compound 3 was isolated as a colorless, amorphous powder, with the molecular formula of C14H22O5 (four degrees of unsaturation) on the basis of its HR-ESIMS data. In the 1H NMR data of 3 (Table 2), three oxymethine protons at δH 4.44 (m, H-3), 4.25 (m, H-6), and 3.75 (m, H-4′), one methine proton at δH 2.89 (m, H-4a), six methylene groups at δH 2.64 (d, J = 19.6 Hz, H-7a)/2.34 (d, J = 19.6 Hz, H-7b), 1.96 (m, H-5a)/1.31 (m, H-5b), 1.75 (m, H-1′), 1.70 (m, H-4), and 1.69 (m, H-3′a)/1.48 (m, H-3′b) and 1.65 (m, H-2′), and one

data (Figure 1). Additionally, a 2-methylcrotonate unit could be determined by the HMBC correlations from H-22 to C-18/ C-19/C-20. On the basis of these results, the planar structure of 1 was elucidated. The relative configuration of 1 was based on the NOESY correlations as indicated in Figure 3. The NOESY correlations of H-11 to 15-Me and H-7, H-7 to 10′-Me, and 9′-Me to H-5′ and 12-Me indicated that 9′-Me to H-5′ and 12-Me were on the opposite side of the H-7, H-11, 15-Me, and 10′-Me. To support the above deduction and determine the absolute configuration of 1, an X-ray crystal structure with a Flack parameter of −0.03(6) was obtained (Figure 4). The absolute configuration of 1 were also determined by comparing experimental and calculated electronic circular dichroism (ECD) spectra for the truncated model (5S,7R,8S,9R,11S,3′R,5′R,6′R,7′S)-1a and the truncated model (5R,7S,8R,9S,11R,3′S,5′S,6′S,7′R)-1b using time-dependent density-functional theory (TDDFT). The DFT reoptimization of the initial Merck molecular force field (MMFF) minima was performed at the B3LYP/6-31+G(d,p) level using a conductorlike polarizable continuum model (CPCM). The theoretical spectrum of 1 showed an excellent fit with the experimental plot recorded in MeOH (Figure S1), which supported the absolute configuration to be 5S,7R,8S,9R,11S,3′R,5′R,6′R,7′S. Thus, the structure of 1 was determined and named penicianstinoid A. Compound 1 represents an austinoid-like meroterpenoid that is reported here for the second time, in B



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Table 1. 1H NMR and 13C NMR Data (δ) for 1 and 2 (400 MHz) (δ in ppm, J in Hz) 1a

2b

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1

29.7, CH2

154.8, CH

7.07, d (10.0)

2 3 4 5 6

25.8, CH2 170.5, C 87.7, C 43.0, C 35.6, CH2

2.33, m 2.53, m 2.61, m

116.2, CH 166.2, C 88.4, C 47.5, C 37.3, CH2

5.88, d (10.0)

position

1.83, d (5.2) 1.86, d (5.2)

7

67.4, CH

8 9 10 11 12 13

61.4, C 93.8, C 137.1, C 75.9, CH 12.0, CH3 125.2, CH2

14 15 16 17 18 19 20 21 22 1′

23.7, CH3 27.2, CH3 168.5, C 20.9, CH3 166.8, C 127.3, C 140.8, CH 14.1, CH3 12.4, CH3

2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 7-OH 11-OH

191.7, C 83.6, C 163.8, C 76.9, CH 85.9, C 65.5, C 164.4, C 16.1, CH3 13.9, CH3

carbonyl (δC 173.8), one double bond (δC 173.3 and 97.9), one methyl (δC 23.5), six sp3 methylenes, and four sp3 methines (three oxygenated at δC 82.2, 68.3, and 64.4). The 1 H−1H COSY spectrum displayed two fragments due to the observed correlations of H-4 to H-4a/H-5 and sequentially from H-6 to H-5/H-7. The above data combined with the HMBC correlations from H-4a to C-1/C-8/C-8a suggested the presence of an isocoumarin structure (Figure 2). The NMR spectroscopic data of 3 were very similar to those of 6hydroxyramulosin.18 The obvious difference was that a methyl unit at C-3 in 6-hydroxyramulosin had been replaced by a 2pentanol unit at C-3 in 3. The 1H−1H COSY correlation of H3 to H-1′ and the HMBC correlation of H-2′ to C-3 further confirmed that the 2-pentanol unit was connected at C-3. The 1 H−1H COSY, HMQC, and HMBC spectra allowed the complete assignment for 3. The relative configuration of 3 was determined by NOESY correlation from H-3 to H-4a and the lack of NOESY correlation between H-4a and H-6, which indicated that H-6 was on the opposite side of H-3 and H-4a. The absolute configuration of 3 was determined by the modified Mosher’s method.19 The differences in 1H NMR chemical shifts between (S)- and (R)-MTPA esters (Δδ = δS − δR) (Figure 6) were calculated to assign the absolute configuration of C-6 and C-4′ to be the same (R). The absolute configuration of 3 was also resolved by comparing experimental and calculated ECD spectra using TDDFT (Figure 5).20 Thus, the absolute configuration of 3 was established as 3R,4aS,6R,4′R and named peniciisocoumarin A. Compound 4 was isolated as a colorless, amorphous powder, possessing the molecular formula C14H20O5 (five degrees of unsaturation) determined by HR-ESIMS. The 1H and 13C NMR data (Tables 2 and 3) revealed that 4 was an isocoumarin and suggested a close structural relationship to 3. The obvious differences were the presence of a carbonyl carbon at δC (211.5) for C-4′ in 4 and the presence of one oxygenated methine carbon at δC (68.3) for C-4′ in 3. The HMBC correlations (Figure 2) from the oxymethylene H-3′ to C-1′/C-4′ and the methyl H-5′ to C-3′/C-4′ established an isocoumarin derivative with a carbonyl group at C-4′. The 1 H−1H COSY, HMQC, and HMBC spectra allowed the complete assignment for 4. The relative configuration of 4 was the same as 3 according to the NOESY correlation of H-3 to H-4a. The absolute configuration of 4 was confirmed by comparing experimental and calculated ECD spectra using TDDFT (Figure 5).20 Thus, the absolute configuration of 4 was established as 3R,4aS,6R and named peniciisocoumarin B. Compound 5 was isolated as a colorless oil. The molecular formula was determined as C13H16O5 (six degrees of unsaturation) on the basis of its HR-ESIMS data. In the 1H NMR data of 5 (Table 2), the proton signals and the coupling constants at δH 7.17 (d, J = 8.0 Hz, H-6) and 6.75 (d, J = 8.0 Hz, H-5) indicated the presence of a 1,2,3,4-tetrasubstituted phenyl. In addition, the 1H NMR data (Table 2) also displayed signals for one oxymethine proton at δH 4.63 (m, H-3), one oxymethylene proton at δH 3.62 (m, H-3′), three methylene protons at δH 2.95 (m, H-4), 1.82 (m, H-1′), and 1.69 (m, H2′), and one methoxy group at δH 3.85 (s, 7-OMe). The combination of 13C NMR and HMQC spectra exhibited 13 carbon signals, including six aromatic carbons at δC (132.2, 118.5, 119.5, 148.6, 153.4, and 109.6), one lactone carbonyl at δC (171.8), one oxymethylene group at δC (62.5), one oxymethine group at δC (81.9), three methylene groups at

5.47, dd (10.0, 5.6)

5.93, m 1.46, s 5.83, dd (24.0,1.6) 1.39, s 1.42, s

64.7, CH 58.0, C 93.9, C 142.0, C 76.9, CH 11.2, CH3 125.1, CH2

1.73, dd (12.0, 4.8) 1.86, dd (12.0, 2.0) 4.32, dd (12.0, 4.8)

26.1, CH3 24.2, CH3

1.24, s 5.79, brd (1.6) 6.11, d (1.6) 1.51, s 1.54, s

115.3, CH2

5.72, s 5.99, s

2.08, s

6.78, q (5.6) 1.83, s 1.83, s

5.19, q (6.8)

1.46, s 1.67, d (6.8)

140.4, C 86.0, C 170.3, C 77.1, CH 85.9, C 63.1, C 171.9, C 20.7, CH3 14.1, CH3

5.19, m

1.62, s 1.64, d (6.8)

a

CDCl3. bCD3OD.

Figure 1. 1H−1H COSY correlations and key HMBC correlations for compounds 1 and 2.

methyl group at δH 1.16 (d, J = 6.4 Hz, H-5′) were deduced. The 13C NMR data (Table 3) displayed 14 signals, which were classified by DEPT and HMQC spectra as one lactone C



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Figure 2. 1H−1H COSY correlations and key HMBC correlations for compounds 3−10.

Figure 3. Key partial structures of compounds 1 and 2 from NOESY data.

δC (33.2, 32.3, and 28.9), and one methoxy carbon at δC (56.9). Detailed inspection of the 1H and 13C NMR data (Tables 2 and 3) revealed that 5 belongs to the isocoumarin class. The HMBC correlations from 7-OMe to C-7, H-5 to C4/C-7/C-8a, and H-3′ to C-1′/C-2′ (Figure 2) established an isocoumarin derivative with a methoxy group at C-7 and a hydroxy group at C-3′. The 1H−1H COSY, HMQC, and HMBC spectra allowed the complete assignment for 5. The absolute configuration of C-3 was determined by circular dichroism (CD) spectroscopy (Figure 5). The negative circular dichroism at 258 nm suggested the R configuration at C-3, by comparison with data for dihydroisocoumarins described in the literature (Figure 5).20 Thus, the absolute configuration of 5 was defined as 3R, and the compound named peniciisocoumarin C. Compound 6 was isolated as a colorless oil. The molecular formula was determined as C15H18O5 (seven degrees of unsaturation) by HR-ESIMS. Detailed inspection of the 1H and 13C NMR data (Tables 2 and 3) revealed that 6 was an isocoumarin. In addition, these spectroscopic features suggested that 6 closely resembled those of the known compound penicimarin C (19).27 The 13C NMR data of C-4′ was shifted from δC 68.0 (CH) in penicimarin C to δC 211.5 (C) in 6, suggesting the oxygenated methine group for C-4′ in penicimarin C was replaced by a carbonyl group in 6. Furthermore, in the HMBC correlations (Figure 2) from the oxymethylene H-2′ to C-4′, from H-3′ to C-1′, C-4′, and C-5′, and from the methyl H-5′ to C-3′ and C-4′ established an isocoumarin derivative with a carbonyl unit at C-4′. The 1 H−1H-COSY, HMQC, and HMBC spectra allowed the complete assignment for 6 (Figure 2). The absolute configuration of C-3 was determined as R by CD spectroscopy (Figure 5),20 and 6 was named peniciisocoumarin D.

Compound 7 was isolated as a colorless oil. Its molecular formula was determined as C14H16O6 (seven degrees of unsaturation) on the basis of its HR-ESIMS data. The 1H and 13 C NMR data (Tables 2 and 3) of 7 were structurally similar to those of penicimarin I,13 except for the absence of one aromatic proton at δH 6.70 (d, J = 7.6 Hz) for C-7 in penicimarin I and the presence of one methoxy group at δH 3.84 (s) and δC 61.8 for C-8 in 7. These results indicated that the aromatic proton (H-7) in penicimarin I was replaced by a hydroxy group (7-OH) in 7, and 8-OH in penicimarin I was replaced by a methoxy group in 7, which was supported by HMBC correlations from H-5 to C-7 and C-8a, from H-6 to C8 and C-4a, and from 8-OMe to C-8 (Figure 2). The absolute configuration of C-3 was determined as R by CD spectroscopy comparison with the known dihydroisocoumarins (Figure 5),20 and 7 was named peniciisocoumarin E. Compound 8 was isolated as a colorless oil, with the molecular formula C14H18O5, determined by HR-ESIMS. The 1 H and 13C NMR data (Tables 2 and 3) revealed that 8 closely resembled those of the known compound penicimarin G13 with an isocoumarin structure, but 8 lacked a methoxy signal at C-5. These indicated that the methoxy (5-OMe) in penicimarin G was replaced by a hydroxy group (5-OH) in 7, which was supported by a decrease of 14 mass units in the HR-ESIMS analysis and HMBC correlations from the aromatic protons H-6 to C-8 and C-4a and from H-7 to C-5 and C-8a (Figure 2). The absolute configuration of C-3 was defined as R by CD spectroscopy comparison with the known dihydroisocoumarins (Figure 5).20 The absolute configuration of C-4′ at the side chain was defined as R by the modified Mosher’s method (Figure 6).19 Thus, the absolute configuration of 8 was established as 3R,4′R, and this metabolite named peniciisocoumarin F. D



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Figure 4. X-ray of compounds 1, 2, 11, and 13.

The large coupling constant of J3‑4 (7.6 Hz) indicated that they should be placed on opposite sides. The absolute configurations of C-4 and C-4′ were defined as S and R by the modified Mosher’s method (Figure 6),19 which were also confirmed by comparing experimental and calculated ECD spectra using TDDFT (Figure 5). Thus, on the basis of these results the absolute configuration of 10 was defined as 3R,4S,4′R, and this compound named peniciisocoumarin H. By comparing physical and spectroscopic data with literature values, 10 known compounds were identified as furanoaustinol (11),23 austinol (12),24 1,2-dihydro-7-hydroxydehydroaustin (13),25 7-hydroxydehydroaustin (14),25 dehydroaustinol (15),26 austin (16),25 11β-acetoxyisoaustinone (17),17 (R)-3(3-hydroxypropyl)-8-hydroxy-3,4-dihydroisocoumarin (18),22 penicimarin C (19),27 and aspergillumarin A (20).21 The relative configuration of 11 was confirmed by the X-ray crystal diffraction (Figure 4), and the absolute configuration of 11 was determined as 1S,5S,8S,11S,3′S,5′R,6′R,7′R by comparing experimental and calculated ECD spectra using TDDFT (Figure 5) for the first time. The structure and absolute configuration of 13 were confirmed by X-ray crystal analysis for the first time. Compounds 1−4, 7, 8, 10, 12, 13, and 16 showed inhibition of growth activity against newly hatched larvae of H. armigera Hubner with IC50 values of 200, 200, 200, 200, 200, 100, 100, 100, 50, and 50 μg/mL, respectively. Azadirachtin was used as positive control with an IC50 value of 25 μg/mL.

Compound 9 was isolated as a colorless oil. Its molecular formula was determined as C14H16O5 by HR-ESIMS. The 1H and 13C NMR data (Tables 2 and 3) revealed that 9 and (R)3-(3-hydroxypropyl)-8-hydroxy-3,4-dihydroisocoumarin (18)22 showed a close structural relationship, except for the presence of a singlet methyl signal at δH 2.02 (s) of 9 in the 1H NMR spectrum. The 13C NMR spectrum revealed a carbonyl group at C-4′ (δC 170.4) and a methyl group at C-5′ (δC 20.7) in 9. These spectroscopic features indicated that the hydroxy group at C-4′ was acetylated in 9. The location of the acetoxyl group at C-4′ was further confirmed by the HMBC correlations from H-3′ and H-5′ to C-4 (Figure 2). The absolute configuration of C-3 was determined as R by CD spectroscopy (Figure 5),20 and 9 was named peniciisocoumarin G. Compound 10 was assigned the molecular formula C 14 H 18 O 5 , as deduced from the positive HR-ESIMS. Comparison of the 1H and 13C NMR spectra data of 10 (Tables 2 and 3) with that of aspergillumarin A (20)21 showed a similar structure. The major difference in the 1D NMR spectra was that one oxymethylene carbon at δC 68.3 (C-4) appeared in 10 instead of a methylene carbon at δC 43.8 (C-4) in 20, and the carbonyl carbon (δC 211.5) for C-4′ in 20 was replaced by one oxygenated methine carbon at δH 3.74 (m) and δC 68.1 (CH) for C-4′ in 10. These were confirmed by the 2D NMR spectra (Figure 2). The relative configuration of 10 was determined by the large coupling constant of H-3 and H-4. E



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Figure 5. Experimental ECD spectra of 2−11 in MeOH and the calculated ECD spectra of 2−4, 10, and 11 at the B3LYP/6-311+G(d,p) level.

The above results suggest that anstinoid-like moreterpenoids and isocoumarins could potentially be developed as new biopesticides, with good efficiency and low toxicity.

Compounds 1, 2, and 11−15 displayed insecticidal activity against C. elegans with EC50 values of 9.4 (± 1.0), 9.9 (± 0), 19.1 (± 0.6), 19.5 (± 1.0), 20.5 (± 0.6), 20.6 (± 2.1), and 38.2 (±0.6) μg/mL, respectively. The positive control was levamisole, with an EC50 value of 4.8 (± 0.6) μg/mL. No activity was observed against human A549, HeLa, and HepG2 cell lines, Gram positive and Gram negative bacteria, or the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced RAW 246.7 mouse macrophages.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on an X-6 micromelting point apparatus and were uncorrected. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer. 1D and 2D NMR spectra were measured on a Bruker AV-400 spectrometer with tetramethylsilane as the internal standard. HR-ESIMS spectra were obtained on a F



Article

Table 2. 1H NMR Data (400 MHz, δ in ppm, J in Hz) for 3−10 in CD3OD and DMSO-d6 3a

4a

5a

6a

7a

8a

9b

10a

3 4

4.44, m 1.70, m

4.43, m 1.96, m

4.63, m 2.95, m

4.43, m 2.85, m

4.47, m 2.88, m

4.60, m 2.66, dd (17.2, 11.6) 3.18, dd (17.2, 2.8)

4.71, m 2.99, m

4.46, m 4.64, d (7.6)

4a 5

2.89, m 1.31, m 1.96, m 4.25, m 2.34, d (19.6) 2.64, d (19.6)

2.88, m 1.32, m

6.75, d (8.0)

6.93, d (8.0)

6.93, d (8.0)

6.84, d (8.0)

7.01, d (8.0)

7.17, d (8.0)

7.10, d (8.0)

7.08, d (8.0)

7.03, d (8.8) 6.75, d (8.8)

7.50, dd (8.0, 8.0) 6.84, d (8.0)

7.41, dd (8.0, 8.0) 6.93, d (8.0)

3.86, s 1.74, m 1.72, m 2.57, m

3.84, s 2.57, m 2.05, m

1.83, m 1.56, m 1.50, m

1.77, m 1.73, m 4.05, m

1.68, m 1.60, m 1.49, m

3.76, m 1.18, d (6.0)

2.02, s

3.74, m 1.16, d (6.0)

position

6 7 7-OMe 8-OMe 1′ 2′ 3′

4.24, m 2.31, d (19.6) 2.64, d (19.6)

3.85, s 1.75, m 1.65, m 1.69, m 1.48, m 3.75, m 1.16, d (6.4)

4′ 5′ 3′-OMe

1.75, m 1.65, m 2.56, t (6.0)

1.82, m 1.69, m 3.62, m

2.14, s

2.14, s 3.69, s

a

CD3OD. bDMSO-d6.

Table 3.

a

13

C NMR Data (100 MHz, δ in ppm) for 3−10 in CD3OD and DMSO-d6

position

3a

4a

5a

6a

7a

8a

9b

10a

1 2 3 4 4a 5 6 7 8 8a 7-OMe 8-OMe 1′ 2′ 3′ 4′ 5′ 3′-OMe

173.8, C

173.8, C

171.8, C

165.1, C

164.8, C

171.7, C

169.2, C

170.2, C

82.2, CH 36.7, CH2 27.6, CH 36.1, CH2 64.4, CH 37.9, CH2 173.3, C 97.9, C

81.9, CH 36.7, CH2 27.6, CH 36.1, CH2 64.4, CH 37.9, CH2 173.0, C 97.8, C

81.9, CH 33.2, CH2 132.2, C 118.5, CH 119.5, CH 148.6, C 153.4, C 109.6, C 56.9, CH3

80.2, CH 34.2, CH2 133.1, C 124.3, CH 123.1, CH 151.6, C 150.5, C 118.7, C

79.4, CH 34.1, CH2 132.8, C 124.3, CH 123.1, CH 151.7, C 150.8, C 119.3, C

81.3, CH 27.8, CH2 125.9, C 147.1, C 125.2, CH 116.5, CH 156.4, C 109.4, C

79.1, CH 31.9, CH2 140.4, C 118.4, CH 136.3, CH 115.4, CH 160.9, C 108.4, C

85.3, CH 68.3, CH 144.2, C 117.9, CH 137.8, CH 117.8, CH 162.9, C 108.0, C

36.9, CH2 22.2, CH2 39.9, CH2 68.3, CH 23.5, CH3

36.1, CH2 20.2, CH2 43.7, CH2 211.5, C 29.8, CH3

32.3, CH2 28.9, CH2 62.5, CH2

61.8, CH3 34.9, CH2 20.3, CH2 43.7, CH2 211.5, C 29.8, CH3

61.8, CH3 30.3, CH2 30.8, CH2 175.1, CH

36.0, CH2 22.3, CH2 39.8, CH2 68.4, CH 23.5, CH3

30.5, CH2 23.8, CH2 63.4, CH2 170.4, CH2 20.7, CH3

32.8, CH2 22.3, CH2 39.8, CH2 68.1, CH 23.5, CH3

52.2, CH3 b

CD3OD. DMSO-d6.

Figure 6. Δδ (= δS − δR) values for (S)- and (R)-MTPA esters of 3, 8, and 10. chromatography (CC). All solvents were purchased from Xilong Chemical Reagent Factory (Guangzhou, China). Fungal Materials. The fungal strain Penicillium sp. TGM112 was isolated from the mangrove B. sexangula var. rhynchopetala, collected in the South China Sea in August 2014. The strain was deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, China. The fungus was identified according to its morphological characteristics and by

Bruker Daltonics Apex-Ultra 7.0 T and a Q-TOF Ultima Global GAA076 LC mass spectrometer. Single-crystal data were measured by an Agilent Gemini Ultra X-ray single-crystal diffractometer (Cu Kα radiation). Preparative HPLC was done using an Agilent 1260 prepHPLC system with an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 7 μm). Sephadex LH-20 (Pharmacia Co. Ltd., Sandwich, UK) and silica gel (200−300 and 300−400 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column G



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(1.55) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 225 (−7.0), 259 (−3.0); IR (KBr) νmax 3513, 1714, 1607, 1277, 1255, 1232 cm−1; 1 H and 13C NMR Tables 2 and 3; HR-ESIMS m/z 281.1021 [M + H]+ (calcd for C14H17O6, 281.1020). Peniciisocoumarin F (8): colorless oil; [α]24 D −17.0 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 220 (2.48), 245 (2.55), 310 (1.65) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 265 (−3.5); IR (KBr) νmax 3511, 1714, 1606, 1271, 1251, 1224, 1109, 789 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 267.1229 [M + H]+ (calcd for C14H19O5, 267.1227). Peniciisocoumarin G (9): colorless oil; [α]24 D −24.4 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 219 (2.26), 245 (2.35), 311 (1.45) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 221 (3.0), 261 (6.5); IR (KBr) νmax 3517, 1711, 1603, 1275, 1251, 1231 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 265.1073 [M + H]+ (calcd for C14H17O5, 265.1071). Peniciisocoumarin H (10): colorless oil; [α]24 D −21.0 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 220 (2.56), 246 (2.66), 310 (1.75) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 250 (12.5); IR (KBr) νmax 3513, 1715, 1603, 1274, 1252, 1232, 1114, 799 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 267.1229 [M + H]+ (calcd for C14H19O5, 267.1227). X-ray Crystallographic Analysis of 1, 2, 11, and 13. Colorless crystals of 1, 2, 11, and 13 were obtained from MeOH. Single-crystal X-ray diffraction data were collected on an Xcalibur, Atlas, Geminiultra diffractometer with Cu Kα radiation (λ = 1.541 80 Å) at 120.01(10) K, respectively. The structure was solved by direct methods (ShelXS) and refined with the ShelXL refinement package using least squares minimization. All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined relatively isotropically with a riding model. Crystallographic data of 1, 2, 11, and 13 have been deposited in the Cambridge Crystallographic Data Centre with the deposition numbers CCDC 1836068, 1836079, 1836084, and 1836089, respectively. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: +44-(0)1223-336033, or e-mail: deposit@ccdc. cam.ac.uk]. Crystal data for 1: C31H36O12, Mr = 600.60, orthorhombic, a = 10.25981(15) Å, b = 16.0292(3) Å, c = 19.2493(3) Å, α = 90°, β = 90°, γ = 90°, V = 3165.67(9) Å3, space group P212121, Z = 4, Dx = 1.260 mg/mm3, μ(Cu Kα) = 0.816 mm−1, and F(000) = 1272. Independent reflections: 5654 (Rint = 0.0380). The final R1 values were 0.0373, wR2 = 0.0902 (I >2σ(I)). Flack parameter = −0.03(6). Crystal data for 2: C25H28O0, Mr = 472.47, orthorhombic, a = 8.88932(10) Å, b = 10.03059(13) Å, c = 25.1654(3) Å, α = 90°, β = 90°, γ = 90°, V = 2243.87(5) Å3, space group P212121, Z = 4, Dx = 1.399 mg/mm3, μ(Cu Kα) = 0.892 mm−1, and F(000) = 1000. Independent reflections: 4005 (Rint = 0.0365). The final R1 values were 0.0340, wR2 = 0.0877 (I >2σ(I)). Flack parameter = 0.10(7). Crystal data for 11: C25H30O9, Mr = 474.49, trigonal, a = 11.6668(4) Å, b = 11.6668(4) (3) Å, c = 31.6316(13) Å, α = 90°, β = 90°, γ = 120°, V = 3728.7(3) Å3, space group P3221, Z = 6, Dx = 1.268 mg/mm3, μ(Cu Kα) = 0.805 mm−1, and F(000) = 1512. Independent reflections: 3383 (Rint = 0.0579). The final R1 values were 0.0579, wR2 = 0.1502 (I >2σ(I)). Flack parameter = 0.6(2). Crystal data for 13: C27H32O9, Mr = 500.53, orthorhombic, a = 7.76421(14) Å, b = 15.0679(3) Å, c = 21.2025(4) Å, α = 90°, β = 90°, γ = 90°, V = 2480.49(8) Å3, space group P212121, Z = 4, Dx = 1.340 mg/mm3, μ(Cu Kα) = 0.836 mm−1, and F(000) = 1064. Independent reflections: 4385 (Rint = 0.0616). The final R1 values were 0.0350, wR2 = 0.0994 (I >2σ(I)). Flack parameter = 0.07(6). Preparation of (S)- and (R)-MTPA Ester Derivatives of Compounds 3, 8, and 10. Preparation of (S)- and (R)-MTPA ester derivatives of 3, 8, and 10 was performed as described previously.19 (S)-MTPA ester of 3 (3a): 1H NMR (CDCl3, 400 MHz) δH 5.52 (1H, m, H-6), 5.14 (1H, m, H-4′), 4.23 (1H, m, H-3), 2.70 (1H, m, H-7a), 2.56 (1H, m, H-4a), 2.55 (1H, m, H-7b), 2.13 (1H, m, H-5a), 1.72 (2H, m, H-4), 1.61 (2H, m, H-2′), 1.44 (1H, m, H-5b), 1.71

comparison of the ITS sequence amplification, primer pair ITS1 and ITS4, and sequencing of the internal transcribed spacer (ITS) region. The sequence data have been submitted to GenBank, with accession number MK028136, and the fungal strain was identified as Penicillium. The fungal strain was cultivated in 20 L of potato glucose liquid medium (15 g of glucose and 30 g of sea salt in 1 L of potato infusion, in 1 L Erlenmeyer flasks each containing 300 mL of culture broth) at 25 °C without shaking for 4 weeks. Extraction and Isolation. The fungal cultures were filtered through cheese cloth, and the filtrate was extracted with EtOAc (3 × 20 L, 24 h each). The organic extracts were concentrated in vacuo to yield an oily residue (20.6 g), which was subjected to silica gel CC (petroleum ether, EtOAc v/v, gradient 100:0−0:100) to generate five fractions (Fr. 1−Fr. 5). Fr. 4 (8 g) was separated by silica gel CC and eluted with petroleum ether−EtOAc (from 5:1 to 1:1) to afford four subfractions (4a−4d). Subfractions 4a−4d were further separated by semipreparative HPLC (CH3CN−H2O, 45:55 for subfraction 4a, 35:65 for subfraction 4b, 30:70 for subfraction 4c, and 25:75 for subfraction 4d, v/v) to obtain 1 (3 mg), 11 (3.5 mg), and 14 (5 mg) from subfraction 4a; 3 (5 mg), 7 (4.6 mg), 9 (5.3 mg), and 12 (5 mg) from subfraction 4b; 6 (3.8 mg), 8 (3.5 mg), 13 (6.7 mg), and 15 (4 mg) from subfraction 4c; and 19 (3.7 mg) and 20 (4.2 mg) from subfraction 4d. Fr. 5 (5 g) was separated by silica gel CC and eluted with petroleum ether−EtOAc (from 4:1 to 1:1) to afford three subfractions (5a−5c). Subfraction 5b was isolated by CC on silica gel eluting with CHCl3−MeOH (30:1, v/v) and then separated by Sephadex LH-20 CC eluting with CHCl3−MeOH (2:1, v/v) and further purified by using ODS eluting with MeOH−H2O (45:55, v/v) to obtain 4 (8.5 mg), 10 (5.5 mg), and 18 (4.4 mg). Subfraction 5c was further separated by semi-preparative HPLC (CH3CN−H2O, 35:65, v/v) to obtain 2 (5.5 mg), 5 (4 mg), 16 (7 mg), and 17 (4.5 mg). Penicianstinoids A (1): colorless crystals; [α]24 D +30.7 (c 0.10, CHCl3); mp 298.8−301°C; UV (MeOH) λmax (log ε) 212 (3.02) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 191 (−12.3), 209 (10.1) nm; IR (KBr) νmax 3501, 1787, 1747, 1740, 1710, 1250, 1230, 1112, 1082, 1064, 798 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 601.2282 [M + H]+ (calcd for C31H37O12, 601.2280). Penicianstinoids B (2): colorless crystals; [α]24 D +34.5 (c 0.30, CHCl3); mp 298−301 °C; UV (MeOH) λmax (log ε) 232 (4.20) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 201 (−104.9), 239 (107.2) nm; IR (KBr) νmax 3518, 1787, 1747, 1740, 1710, 1250, 1230, 1112, 1082, 1064, 798 cm−1; 1H and 13C NMR see Tables 1; HRESIMS m/z 473.1811 [M + H]+ (calcd for C25H29O9, 473.1806). Peniciisocoumarin A (3): colorless, amorphous powder; [α]D24 +20.8 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 202 (2.12), 263 (2.50) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 260 (9.0) nm; IR (KBr) νmax 3564, 1736 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 271.1543 [M + H]+ (calcd for C14H23O5, 271.1540). Peniciisocoumarin B (4): colorless, amorphous powder; [α]D24 +16.0 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 200 (3.12), 265 (3.50) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 260 (25.0) nm; IR (KBr) νmax 3564, 1736 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 269.1384 [M + H]+ (calcd for C14H21O5, 269.1383). Peniciisocoumarin C (5): colorless oil; [α]24 D −25.4 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 219 (2.40), 247 (2.50), 313 (1.65) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε), 225 (−6.0), 258 (−2.75); IR (KBr) νmax 3518, 1712, 1602, 1272, 1250, 1230, 1112, 1082 cm−1; 1H and 13C NMR see Tables 2 and 3; HR-ESIMS m/z 253.1076 [M + H]+ (calcd for C13H17O5, 253.1071). Peniciisocoumarin D (6): colorless oil; [α]24 D −20.0 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 220 (2.34), 246 (2.35), 312 (1.52) nm; CD (c 2 × 10−4 mol/L, MeOH) λmax (Δε) 212 (−10.0), 261 (−2.75); IR (KBr) νmax 3510, 1717, 1610, 1251, 1232, 1102, 1064 cm−1; 1H and 13C NMR Tables 2 and 3; HR-ESIMS m/z 279.1232 [M + H]+ (calcd for C15H19O5, 279.1227). Peniciisocoumarin E (7): colorless oil; [α]24 D −18.8 (c 0.25, MeOH); UV (MeOH) λmax (log ε) 218 (2.30), 245 (2.40), 312 H



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(2H, m, H-3′), 1.25 (2H, m, H-1′), 1.28 (3H, d, J = 5.6 Hz, H-5′); ESIMS m/z 725 [M + Na]+. (R)-MTPA ester of 3 (3b): 1H NMR (CDCl3, 400 MHz) δH 5.51 (1H, m, H-6), 5.13 (1H, m, H-4′), 4.03 (1H, m, H-3), 2.72 (1H, m, H-7a), 2.56 (1H, d, m, H-7b), 2.54 (1H, m, H-4a), 2.08 (1H, m, H5a), 1.62 (2H, m, H-4), 1.61 (2H, m, H-3′), 1.60 (2H, m, H-2′), 1.32 (1H, m, H-5b), 1.35 (3H, d, J = 5.6 Hz, H-5′), 1.24 (2H, m, H-1′); ESIMS 725 [M + Na]+. (S)-MTPA ester of 8 (8a): 1H NMR (CDCl3, 400 MHz) δH 7.04 (1H, d, J = 8.8 Hz, H-6), 6.78 (1H, d, J = 8.8 Hz, H-7), 5.15 (1H, m, H-4′), 4.49 (1H, m, H-3), 2.61 (2H, m, H-4), 1.95 (2H, m, H-1′), 1.79 (2H, m, H-3′), 1.54 (2H, m, H-2′), 1.29 (3H, d, J = 6.0 Hz, H5′); ESIMS m/z 697 [M − H]−. (R)-MTPA ester of 8 (8b): 1H NMR (CDCl3, 400 MHz) δH 7.45 (1H, d, J = 8.4 Hz, H-6), 7.00 (1H, d, J = 8.4 Hz, H-7), 5.13 (1H, m, H-4′), 4.30 (1H, m, H-3), 2.53 (2H, m, H-4), 1.86 (2H, m, H-1′), 1.67 (2H, m, H-3′), 1.48 (2H, m, H-2′), 1.35 (3H, d, J = 6.0 Hz, H5′); ESIMS m/z 697 [M − H]−. (S)-MTPA ester of 10 (10a): 1H NMR (CDCl3, 400 MHz) δH 7.63 (1H, dd, J = 8.4, 7.6 Hz, H-6), 7.46 (1H, d, J = 7.6 Hz, H-5), 7.15 (1H, d, J = 8.4 Hz, H-7), 5.08 (1H, m, H-4′), 4.65 (1H, m, H-4), 4.58 (1H, m, H-3), 1.64 (2H, m, H-2′), 1.50 (2H, m, H-1′), 1.49 (2H, m, H-3′), 1.31 (3H, d, J = 6.0 Hz, H-5′); ESIMS m/z 697 [M − H]−. (R)-MTPA ester of 10 (10b): 1H NMR (CDCl3, 400 MHz) δH 6.92 (1H, dd, J = 8.4, 7.6 Hz, H-6), 7.56 (1H, d, J = 7.6 Hz, H-5), 7.08 (1H, d, J = 8.4 Hz, H-7), 5.09 (1H, m, H-4′), 4.63 (1H, m, H-4), 4.53 (1H, m, H-3), 1.48 (2H, m, H-1′), 1.53 (2H, m, H-2′), 1.37 (2H, m, H-3′), 1.32 (2H, d, J = 6.0 Hz, H-5′); ESIMS m/z 697 [M − H]−. Computational Section. Monte Carlo conformational searches were carried out by means of the Spartan 10 software using the MMFF. The conformers with Boltzmann populations of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at the B3LYP/6-31+G(d,p) level in MeOH using the CPCM polarizable conductor calculation model.27 The theoretical calculation of ECD was conducted in MeOH using TDDFT at the B3LYP/6-31+G(d,p) level for all conformers of compounds.28 ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussaian band shapes with sigma = 0.3 eV. Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G (d) energies in the gas-phase calculations and from the B97D/TZVP energies in the PCM model ones. The Spartan 10 software package was used for visualization of the results. Cell Culture and Inhibition of NO Production. RAW 246.7 cells were cultured as previously described. Cells were seeded in 96well plates at a density of 50 000 cells/well for 24 h, pretreated with the tested compounds for 30 min at 37 °C, and co-incubated with LPS (100 ng/mL) for 24 h. NO production was analyzed through the Griess reaction. Briefly, cell culture supernatant (50 μL) and Griess reagent (50 μL) were mixed for 10 min, and absorptions were then monitored at 540 nm using a plate reader. All experiments were performed in triplicate. The IC50 values were calculated using SPSS 20 software. All the tested compounds were prepared as stock solutions with a concentration of 10 mM in DMSO (final DMSO concentration should not exceed 0.1% in each well).29 Dexamethasone was used as the positive control. Insecticidal Activities. Insecticidal activities against newly hatched larvae of H. armigera Hubne were assessed. In the test, there were three groups, each containing three neonate larvae of H. armigera Hubner, and the tested compounds were dissolved in DMSO at the concentration of 1 mg/mL. The insecticidal activity was investigated by adding serial dilutions of the isolated compounds and the positive control azadirachtin at 200, 100, 50, 25, and 12.5 μL/well with 3 replicates per treatment to the artificial diet for the newly hatched larvae, and the bioassay diet was placed into six-well plates. Newly hatched larvae were incubated at 25 ± 1 °C and a relative humidity of 80%. DMSO was used as the negative control, azadirachtin was used as positive control, and artificial diet was

used as the blank control. The number of dead larvae was recorded on the 2nd, 4th, 6th, and 8th day after treatment, respectively.30 C. elegans Motility Inhibition Assay. Filter-sterilized M-9 medium was used as a measurement media and diluent for the compounds tested. The assay was performed in a 48-well plate. Each well contained 40 μL of Escherichia coli OP50 in S medium, 5 μL of L1 worms in M9 medium (30−40 worms total), 1.2 μL of chloramphenicol, isolated compounds, and the positive control levamisole in a range of dilutions at 40, 20, 10, 5, 1, and 0 μg/mL and S medium to bring the total volume to 400 μL, with 3 replicates per treatment. The negative control was M-9 medium, and the positive control was 1 mg/mL of levamisole. The 48-well plates were sealed with Parafilm to avoid evaporation and incubated at 24 °C for 60 h in the dark. The number of dead larvae was recorded on the 12th, 24th, 36th, 48th, and 60th day after treatment, respectively.31,32 Cytotoxic Activities. Cytotoxic activities of all compounds against human A549, HeLa, and HepG2 cell lines were evaluated by the MTT method.33 5-Fluorouracil and adriamycin were used as positive controls. Antibacterial Activities. Antibacterial activity was determined against four pathogenic bacteria, E. coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Methicillin-resistant S. aureus MRSA (ATCC 33591), and Bacillus cereus (ATCC 11778) and two marine pathogenic bacteria, Vibrio parahaemolyticus (ATCC 17802) and V. alginolyticus (ATCC 17749), by the microplate assay method.34 Ciprofloxacin was used as the positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00866. 1

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H NMR, 13C NMR, DEPT, HMQC, COSY, HMBC, NOESY, and HR-ESIMS spectra of compounds 1−10 (PDF) X-ray crystallographic data of compounds 1, 2, 11, and 13 (ZIP)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-898-65889422. Fax: +86-898-65889422. E-mail: [email protected] (C.-J. Zheng). *Tel: +86-898-65889422. Fax: +86-898-65889422. E-mail: [email protected] (G.-Y. Chen). ORCID

Meng Bai: 0000-0002-1024-3458 Cai-Juan Zheng: 0000-0003-1779-8736 Author Contributions #

M. Bai and C.-J. Zheng contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21462015 and 31760093), National Natural Science Foundation of Hainan Province (218MS045), Program for Innovative Research Team in University (IRT16R19), Hainan Province Education Department ProjectChina (No. HnKy2015-21), and Hainan Province Natural Science Foundation of Innovative Research Team Project (No. 2016CXTD007) and Special Projects of the Central Government for the Development of Local Science and Technology (No. ZY2018HN07). I



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