Article pubs.acs.org/JAFC

Isolation and Characterization of Sesquiterpenes from Celastrus orbiculatus and Their Antifungal Activities against Phytopathogenic Fungi Meicheng Wang,†,‡ Qiang Zhang,§ Quanhui Ren,†,‡ Xianglei Kong,⊥ Lizhong Wang,⊥ Hao Wang,†,‡ Jing Xu,*,†,‡ and Yuanqiang Guo†,‡ †

College of Pharmacy; ‡Tianjin Key Laboratory of Molecular Drug Research; and ⊥State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People’s Republic of China § College of Science, Northwest A&F University, Yangling 712100, People’s Republic of China S Supporting Information *

ABSTRACT: Celastrus orbiculatus is an insecticidal plant belonging to the Celastraceae family. In this survey on the secondary metabolites of plants for obtaining bioactive substances to serve agriculture, the chemical constituents of the fruits of C. orbiculatus were investigated. This phytochemical investigation resulted in the isolation of nine new and one known sesquiterpene. Their structures, especially the complicated stereochemical features, were elucidated on the basis of extensive NMR spectroscopic data analyses, time-dependent density functional theory CD calculations, and the CD exciton chirality method. Biological screenings disclosed that these sesquiterpenes showed antifungal activities against six phytopathogenic fungi. The results of our phytochemical investigation further disclosed the chemical components of C. orbiculatus, and biological screening implied that it may be potentially useful to protect crops against phytopathogenic fungi and the bioactive compounds may be regarded as candidate agents of antifungal agrochemicals for crop protection products. KEYWORDS: insecticidal plant, Celastrus orbiculatus, sesquiterpenes, antifungal activities, agrochemicals

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INTRODUCTION Celastrus orbiculatus Thunb., belonging to the Celastraceae family, is a deciduous vine distributed throughout China. This plant plays an important role in China and has been used extensively in the fields of agriculture and medicine.1 The plant possesses insecticidal activity and has been used as a natural botanical insecticide.2 Previous phytochemical surveys on C. orbiculatus disclosed the main presence of terpenoids, especially sesquiterpenes, which displayed a broad spectrum of biological effects, such as neuroprotective, cytotoxic, NO inhibitory, and anti-inflammatory activities and the modulation of multidrug resistance.3−10 However, to the best of our knowledge, although the chemical constituents and their biological activities were investigated, studies on the role of this plant in the agriculture field are limited, and there have been no reports on the antifungal activities against phytopathogenic fungi of the extract or the chemical constituents from this plant. As a continuation of our survey on the secondary metabolites of plants for obtaining bioactive substances to serve agriculture, the insecticidal and medicinal plant C. orbiculatus, possessing multiple functions in the medicine and agriculture field, evoked our interest, and a phytochemical investigation of the fruits of this plant was carried out. This procedure resulted in the isolation of nine new and one known sesquiterpenes. Their structures, especially the complicated stereo features, were elucidated on the basis of the NMR spectroscopic data analyses, the time-dependent density functional theory (TDDFT) CD calculations, and the CD exciton chirality method. The compounds were tested for antifungal activity against six phytopathogenic fungi. © XXXX American Chemical Society

MATERIALS AND METHODS

General. The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). The IR spectra were taken on a Bruker Tensor 27 FT-IR spectrometer with KBr disks (Bruker, Germany). ECD spectra were obtained on a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., UK). The ESIMS spectra were acquired on an LCQ-Advantage mass spectrometer (Finnigan Co., Ltd., San Jose, CA, USA). HR-ESIMS spectra were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA, USA). 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (Bruker, Switzerland) with TMS as an internal standard. HPLC separations were performed on a CXTH system, equipped with a UV3000 detector at 210 nm (Beijing Chuangxintongheng Instruments Co. Ltd., China), and a YMC-pack ODS-AM (250 × 20 mm i.d.) column (YMC Co. Ltd., Japan). Silica gel was used for column chromatography (200−300 mesh) (Qingdao Marine Chemical Group Co. Ltd., China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Chemical Reagent Co., China. Biological reagents were from Sigma Co. Plant Material. The fruits of C. orbiculatus were purchased in July 2010 from Shenyang Materia Medica Market, Liaoning province, China. A voucher specimen (no. 20100713) was identified by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China) and deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China. Received: August 3, 2014 Revised: October 11, 2014 Accepted: October 21, 2014

A

dx.doi.org/10.1021/jf503735t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 1. 13C NMR Data (δC) of Compounds 1−9 (100 MHz)a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

a

1

2

3

4

5

6

7

8

9

79.2 23.3 26.5 33.5 91.0 74.7 53.3 71.4 73.3 51.1 81.1 14.9 60.8 24.2 30.1

75.4 22.1 26.8 39.8 86.9 36.7 49.5 72.6 70.8 52.8 82.2 16.8 62.5 25.1 31.4

73.3 22.2 26.7 39.7 86.4 36.8 48.2 71.5 68.3 51.5 82.3 17.2 64.8 25.0 31.0

76.7 73.4 31.5 33.4 90.5 75.3 52.8 73.5 74.8 48.3 81.4 18.7 12.2 24.0 30.9

79.0 23.5 26.8 39.8 88.3 36.6 47.3 78.2 76.3 51.1 81.7 17.0 62.7 24.7 30.9

76.4 69.4 31.1 39.3 88.1 35.5 47.3 76.3 78.1 52.6 81.5 18.8 60.4 24.6 30.6

76.3 69.3 31.1 39.2 87.9 36.1 49.1 75.8 80.5 50.5 82.0 18.4 61.9 24.8 31.0

73.7 21.3 26.8 33.8 90.6 75.5 55.5 74.7 80.6 49.5 81.6 17.3 19.1 25.7 31.0

74.6 22.2 26.8 39.8 86.9 36.4 48.1 72.0 68.6 52.9 82.0 17.0 62.2 25.1 31.2

169.8 20.8

169.2 20.8

169.6 20.7

130.4 129.7 128.5 133.1 166.4

169.7 21.3

169.8 21.4

170.0 21.4

169.3 20.8

169.8 20.6

169.7 20.4

1-OR

1 2/6 3/5 4 7

2-OR

1 2/6 3/5 4 7

6-OR

1 2

169.8 20.5

8-OR

1 2/6 3/5 4 7

130.6 130.1 128.8 133.5 166.2

9-OR

1 2/6 3/5 4 7

130.0 129.8 128.7 133.5 165.2

13-OR

1 2 3 4 5 6

170.5 21.2

130.0 129.8 128.6 133.2 167.4 169.9 21.3 169.9 20.8

129.4 130.3 128.3 133.4 167.7

130.0 129.7 128.5 133.2 166.3

170.0 20.8 130.0 129.6 128.4 133.1 166.1

129.3 130.2 128.3 133.3 165.9

170.5 21.0

129.8 129.4 128.8 133.3 165.4

125.6 137.5 123.6 153.8 151.2 165.3

125.9 137.2 123.6 153.7 151.1 165.4

169.9 20.9

129.7 129.7 128.7 133.4 167.4

129.0 130.1 128.4 133.5 165.7

129.7 130.2 128.3 133.1 166.0

170.2 21.5

Compound 1 was recorded in pyridine-d5, and the others were in CDCl3. (F1−F7) according to the TLC analyses. Fraction F3 (eluted by petroleum ether/acetone, 100:4 and 100:7) was subjected to mediumpressure liquid chromatography (MPLC) over octadecylsilyl (ODS) eluting with a step gradient from 68 to 87% MeOH in H2O to give seven subfractions (F3‑1−F3‑7). Subfraction F3‑4 was further purified by preparative HPLC (YMC-pack ODS-AM, 250 × 20 mm i.d., 85% MeOH in H2O) to afford compound 1 (tR = 29 min, 14.7 mg). Fraction F5 (eluted by petroleum ether/acetone, 100:10 and 100:15), using the above MPLC (67−87% MeOH in H2O), provided 12 subfractions F5‑1−F5‑12, and the following purification of F5‑1 by the

Extraction and Isolation. The air-dried fruits of C. orbiculatus (9.0 kg) were powdered and extracted with methanol three times (3 × 54 L) under reflux. The organic solvent was evaporated to afford a crude extract (1.2 kg). The extract was suspended in H2O (1.2 L) and then partitioned with petroleum ether (5 × 1.2 L) and ethyl acetate (5 × 1.2 L) successively. The ethyl acetate soluble part (190 g) was fractionated by silica gel column chromatography (silica gel, 1.2 kg; column, 9 × 70 cm), using a gradient solvent system of petroleum ether/acetone (100:0, 100:2, 100:4, 100:7, 100:10, 100:15, 100:20, and 100:28, 21 L for each gradient elution), to afford seven fractions B

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Table 2. 1H NMR Data (δH) of Compounds 1−4 (400 MHz)a position

1

1 2α 2β 3α 3β 4 6α β 7 8 9 12 13 14 15

a

5.87 1.80 1.90 2.27 1.41 2.25

dd (11.7, 4.7) m m m m m

7.07 2.66 6.13 6.20 1.06 5.24 5.06 1.57 1.48

s d (3.7) dd (5.6, 3.7) d (5.6) d (7.0) d (13.2) d (13.2) s s

1-OR

2/6

2.17 s

2-OR

2/6 3/5 4

6-OR

2

1.59 s

8-OR

2/6 3/5 4

8.34 d (7.9) 7.47 t (7.9) 7.56 t (7.9)

9-OR

2/6 3/5 4

8.27 d (7.9) 7.35 t (7.9) 7.48 t (7.9)

13-OR

2 3 4 5

2.18 s

2 5.60 1.63 1.88 2.25 1.56 1.86 2.18 2.23 2.26 4.43 6.03 1.04 4.30 3.79 1.56 1.22

dd (12.0, 4.0) m m m m m overlapped d (13.2) overlapped dd (6.2, 3.0) d (6.2) d (7.6) d (12.0) d (12.0) s s

1.64 s

3 5.60 1.69 1.77 2.30 1.47 1.94 2.22 2.26 2.28 5.67 5.94 1.16 4.86 4.80 1.57 1.24

dd (12.2, 4.1) m m m m m overlapped overlapped overlapped dd (6.4, 2.9) d (6.4) d (7.8) d (12.4) d (12.4) s s

4 4.31 d (3.6) 5.52 dd (6.5, 3.6) 2.36 m 2.00 m 2.32 m 6.14 2.60 5.68 4.37 1.31 1.59

s d (4.7) t (4.7) d (4.7) d (7.6) s

1.48 s 1.44 s

1.56 s 8.15 d (7.9) 7.48 t (7.9) 7.58 t (7.9) 2.09 s 1.92 s

8.11 d (8.0) 7.44 t (8.0) 7.56 t (8.0)

8.02 d (7.9) 7.45 t (7.9) 7.58 t (7.9)

8.11 d (7.2) 7.46 t (7.6) 7.59 t (7.4) 8.56 7.48 8.83 9.44

dt (8.0,1.7) dd (8.0,4.6) d (4.0) s

Compound 1 was recorded in pyridine-d5, and the others were in CDCl3. CDCl3) data, Tables 1 and 2, respectively; ESIMS m/z 455 [M + Na]+; HR-ESIMS m/z 455.2042 [M + Na]+, calcd for C24H32NaO7, 455.2046. Celaspene C (3): colorless oil; [α]22 D +23.0 (c 0.20, CH2Cl2); IR (KBr) νmax 2918, 2850, 1729, 1540, 1452, 1367, 1273, 1228, 1150, and 1025 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 2, respectively; ESIMS m/z 602 [M + Na]+; HR-ESIMS m/z 602.2366 [M + Na]+, calcd for C32H37NNaO9, 602.2366. Celaspene D (4): colorless oil; [α]21 D +2.7 (c 0.15, CH2Cl2); IR (KBr) νmax 3446, 2917, 2849, 1714, 1452, 1273, 1229, 1095, and 1032 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 2, respectively; ESIMS m/z 575 [M + Na]+; HR-ESIMS m/z 575.2246 [M + Na]+, calcd for C31H36NaO9, 575.2257. Celaspene E (5): Colorless oil; [α]22 D +1.9 (c 0.21, CH2Cl2); IR (KBr) νmax 3431, 2917, 2849, 1705, 1463, 1276, 1082, and 1039 cm−1; 13 C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 3, respectively); ESIMS m/z 622 [M + Na]+; HR-ESIMS m/z 622.2417 [M + Na]+, calcd for C35H37NNaO8, 622.2417. Celaspene F (6): colorless oil; [α]22 D +1.6 (c 0.25, CH2Cl2); IR (KBr) νmax 3448, 2917, 2849, 1741, 1709, 1463, 1367, 1264, 1108, and 1071 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz,

same HPLC system (72% MeOH in H2O) yielded compounds 2 (tR = 28 min, 13.5 mg) and 7 (tR = 31 min, 12.7 mg). By the same HPLC system, compound 3 (tR = 29 min, 40.6 mg) was obtained from subfraction F5‑5 (82% MeOH in H2O). The further purification of subfractions F5‑2 (78% MeOH in H2O) and F5‑10 (88% MeOH in H2O) yielded compounds 4 (tR = 12 min, 8.7 mg) and 6 (tR = 48 min, 5.0 mg), respectively. Compounds 5 (tR = 23 min, 6.0 mg) and 10 (tR = 25 min, 6.9 mg) were acquired from the subfraction F5‑12 (91% MeOH in H2O) with the same HPLC system. Compounds 8 (tR = 47 min, 4.9 mg) and 9 (tR = 45 min, 6.6 mg) were isolated from subfraction F4‑2 (72% MeOH in H2O), which was obtained from F4 (eluted by petroleum ether/acetone, 100:7 and 100:10) by the above MPLC (67−87% MeOH in H2O). Celaspene A (1): white powder; [α]21 D −21.4 (c 0.19, CH2Cl2); CD (CH3CN) 198 (Δε −19.3), 222 (Δε +9.4), 239 (Δε −13.9) nm; IR (KBr) νmax 2955, 2917, 2849, 1736, 1602, 1369, 1279, 1229, 1114, 1069, and 1026 cm−1; 13C NMR (100 MHz, pyridine-d5) and 1H NMR (400 MHz, pyridine-d5) data, Tables 1 and 2, respectively; ESIMS m/z 659 [M + Na]+; HR-ESIMS m/z 659.2466 [M + Na]+, calcd for C35H40NaO11, 659.2468. Celaspene B (2): colorless oil; [α]21 D +8.2 (c 0.15, CH2Cl2); IR (KBr) νmax 3432, 2917, 2849, 1734, 1654, 1374, 1277, 1236, and 1024 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, C

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Table 3. 1H NMR Data (δH) of Compounds 5−9 (CDCl3, 400 MHz) position

5

1 2α 2β 3α 3β 4 6α β 7 8 9 12 13 14 15 1-OR

2-OR 6-OR 8-OR

9-OR

13-OR

2/6 3/5 4 2 2 2/6 3/5 4 2/6 3/5 4 2 3 4 5

5.80 1.63 1.87 1.60 1.55 1.91 2.14 2.68 2.47 5.70 4.70 1.21 5.15 5.07 1.64 1.22 8.03 7.41 7.54

dd (10.8, 5.9) m m m m m dd (12.7, 4.8) d (12.7) t (4.8) dd (9.5, 4.8) dd (9.5) d (7.7) d (12.6) d (12.6) s s d (7.0) t (7.0) t (7.0)

6

7

5.46 d (3.2) 5.38 dd (6.3, 3.2)

5.51 d (3.3) 5.37 dd (6.3, 3.3)

2.35 1.70 1.91 2.12 3.04 2.40 5.58 5.97 1.36 4.45 4.20 1.54 1.20 2.01

2.39 1.72 1.91 2.09 2.64 2.38 4.32 5.80 1.28 4.87 4.80 1.63 1.23 2.08

m m m dd (12.7, 4.9) d (12.7) t (4.9) dd (9.5, 4.9) d (9.5) d (8.0) d (13.0) d (13.0) s s s

1.56 s 8.01 d (7.1) 7.40 t (7.1) 7.52 t (7.1)

m m m dd (12.6, 4.2) d (12.6) t (4.2) dd (9.2, 4.2) d (9.2) d (8.0) d (13.0) d (13.0) s s s

dd (11.8, 4.1) m m m m m

5.91 2.41 4.31 4.89 1.03 1.48

s d(3.2) s s d (7.3) s

9

1.35 s 1.42 s 1.58 s

5.55 1.64 1.88 2.27 1.65 1.87 2.15 2.52 2.26 5.52 5.94 1.09 4.25 3.92 1.53 1.21 1.65

dd (12.3, 4.0) m m m m m dd (12.8, 4.2) d (12.8) t (4.2) dd (6.4, 4.2) d (6.4) d (7.8) d (11.7) d (11.7) s s s

2.12 s

1.89 s

8.05 d (7.2) 7.45 t (7.2) 7.57 t (7.2)

8.11 d (7.3) 7.46 t (7.3) 7.57 t (7.3)

1.43 s

1.89 s

7.95 d (7.2) 7.48 t (7.2) 7.59 t (7.2) 8.50 7.50 8.84 9.41

8 5.43 1.66 1.89 2.26 1.48 2.27

7.99 7.46 7.57 2.21

dt (7.9, 1.8) dd (7.9, 4.0) d (4.0) s

CDCl3) data, Tables 1 and 3, respectively; ESIMS m/z 555 [M + Na]+; HR-ESIMS m/z 555.2207 [M + Na]+, calcd for C28H36NaO10, 555.2206. Celaspene G (7): colorless oil; [α]21 D −13.2 (c 0.17, CH2Cl2); IR (KBr) νmax 3450, 2917, 2849, 1743, 1721, 1464, 1367, 1279, and 1026 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 3, respectively; ESIMS m/z 555 [M + Na]+; HR-ESIMS m/z 555.2205 [M + Na]+, calcd for C28H36NaO10, 555.2206. Celaspene H (8): colorless oil; [α]22 D +3.2 (c 0.25, CH2Cl2); IR (KBr) νmax 3444, 2917, 2849, 1714, 1464, 1367, 1275, 1234, and 1096 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 3, respectively; ESIMS m/z 497 [M + Na]+; HR-ESIMS m/z 497.2144 [M + Na]+, calcd for C26H34NaO8, 497.2151. Celaspene I (9): colorless oil; [α]22 D −11.6 (c 0.21, CH2Cl2); IR (KBr) νmax 3499, 2918, 2850, 1737, 1711, 1462, 1367, 1283, 1265, and 1025 cm−1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, Tables 1 and 3, respectively; ESIMS m/z 497 [M + Na]+; HR-ESIMS m/z 497.2153 [M + Na]+, calcd for C26H34NaO8, 497.2151. Computational Methods. Details have been previously reported.11,12 Antifungal Activity Assay. The antifungal activities of the isolated compounds were tested against six pathogenic fungi (Gibberella zeae, Physalospora piricola, Alternaria solani, Cercospora arachidicola, Cladosporium cucumerinum, and Phytophthora capsici) using a mycelium growth inhibition method.13−17 The test solution (1 mL, 50 μg/mL) was poured into sterile culture plates (9 cm diameter), and agar culture medium (9 mL) was added. Sterile water (1 mL) and agar culture medium (9 mL) were used as controls. The inocula, 4 mm

d (7.5) t (7.5) t (7.5) s

in diameter, were removed from the margins of actively growing colonies of mycelium, placed in the centers of the above plates, and incubated at 24 °C. After an incubation of 72 h, the diameter of the mycelium was measured. Each treatment was performed in two replicates. The inhibitory rate was used to describe the antifungal activities of compounds: inhibition rate (%) = (av hyphal diam in control − av hyphal diam in treatment)/av hyphal diam in control

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RESULTS AND DISCUSSION The ethyl acetate-soluble part of the methanol extract of the fruits of C. orbiculatus was subjected to silica gel column chromatography and further purified by HPLC to acquire nine new (1−9) and one known (10) compound (Figure 1). The known compound was identified by comparison of spectroscopic data with those reported in the literature as 6α-acetoxy1β,2β,9β-tribenzoyloxy-8β-nicotinoyloxy-β-dihydroagarofuran, 10.18 Compound 1 was isolated as a white powder. Its molecular formula was determined as C35H40O11 on the basis of HR-ESIMS spectroscopic data, which were consistent with the NMR data. The 1H NMR spectrum of 1 exhibited six methyl groups [δH 2.18 (3H, s, COCH3-13), 2.17 (3H, s, COCH3-1), 1.59 (3H, s, COCH3-6), 1.57 (3H, s, H3-14), 1.48 (3H, s, H3-15), and 1.06 (3H, d, J = 7.0 Hz, H3-12)], four oxygenated methine protons [δH 5.87 (1H, dd, J = 11.7, 4.7 Hz, H-1), 7.07 (1H, s, H-6), 6.13 (1H, dd, J = 5.6, 3.7 Hz, H-8), and 6.20 (1H, d, J = D

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at C-6 and C-13, respectively. The two benzoyloxy groups located at C-8 and C-9 were verified by the HMBC correlations of H-8 and H-9 to the corresponding carbonyl carbons of two benzoyloxy groups. Further analyses of the 2D NMR spectra (Figure 2) led to the assignments of all the proton and carbon signals, and the planar structure for 1 was thus established.

Figure 2. Selected HMBC and compound 1.

1

H−1H COSY correlations of

The relative configuration of compound 1 was established on the basis of the NOESY spectrum and Chem3D modeling (Figure 3). NOESY correlations observed for H3-12/H2-13, H312/H-2β, H2-13/H-2β, H2-13/H-6, H3-12/H-6, H-1/H-9, H9/H3-14, H-8/H3-14, and H-1/H-3α (Figure 3), but not for H2-13/H-1, H2-13/H-9, and H3-12/H-1, and the Chem3D modeling indicated a conformation for compound 1 as shown in Figure 3, where the two six-membered rings B and A were trans-fused and existed in a twist-chair and chair conformation, and the furan ring C, almost perpendicular to the six-membered ring B, was located below ring B. The arrangement of the three rings and the NOESY correlations required that the C-12 methyl group and the C-13 methylene group were both βaxially oriented, the C-1 acetoxy group and the C-9 benzoyloxy group were both β-equatorially oriented, the C-6 acetoxy group was in an α-equatorial position, and the C-8 benzoyloxy group was in a β-axial position.5,19 Thus, the relative configuration of 1 was designated as shown in Figure 3. The absolute configuration of 1 was tentatively established by the TDDFT CD calculations20,21 and substantiated by the CD exciton chirality method.22 Through the systematic conformational search and optimization and TDDFT CD calculations, the calculated ECD spectra were obtained. The calculated ECD spectrum of 1a (Figure 4) is agreement with the experimental result, which suggested an absolute configuration of 1S,4R,5S,6R,7R,8R,9S,10S for compound 1, whereas the experimental CD spectrum of 1 showed a characteristic Davidoff-type split curve with a first negative Cotton effect at 239 nm (Δε −13.9) and a second positive effect at 222 nm (Δε +9.4), due to the couplings of the two benzoyloxy chromophores at C-8β and C-9β, which confirmed the absolute configuration of 1S,4R,5S,6R,7R,8R,9S,10S unequivocally. On the basis of the above evidence, the structure of 1 was established as (1S,4R,5S,6R,7R,8R,9S,10S)-1β,6α,13-triacetoxy8β,9β-dibenzoyloxy-β-dihydroagarofuran, which has been named celaspene A. Compound 2, a colorless oil, had a molecular formula of C24H32O7 as deduced from the HR-ESIMS. From the 1H NMR spectrum of 2, four methyl groups, five aromatic protons, three oxygenated methine protons, and a pair of methylene protons were displayed (Table 2). The 13C NMR spectrum of 2 exhibited 24 carbon resonances. From the 1H and 13C NMR spectra, one acetyl group was apparent from the methyl singlet

Figure 1. Structures of compounds 1−10 from C. orbiculatus.

5.6 Hz, H-9)], and a pair of methylene protons [δH 5.24 and 5.06 (each 1H, d, J = 13.2 Hz, H2-13)]. Additionally, 10 aromatic protons were also revealed by the 1H NMR spectrum. The 13C NMR spectrum of 1 showed 35 carbon resonances (Table 1). From the 1H and 13C NMR spectra, three acetyl groups and two benzoyl groups were deduced and defined from the above proton signals and the observation of the corresponding carbon signals (Tables 1 and 2).5 In addition to the above 20 signals for the acyl groups, there are additional 15 resonances displayed for the parent skeleton in the 13C NMR spectrum. These skeletal carbon signals comprised three methyls [δC 14.9 (C-12), 24.2 (C-14), and 30.1 (C-15)], two methylenes [δC 23.3 (C-2), and 26.5 (C-3)], six methines [δC 79.2 (C-1), 33.5 (C-4), 74.7 (C-6), 53.3 (C-7), 71.4 (C-8), and 73.3 (C-9)], and four quaternary carbons [δC 91.0 (C-5), 51.1 (C-10), and 81.1 (C-11)] on the basis of the HMQC and DEPT spectra. The above spectroscopic features and the 15 skeletal carbons displayed in the 13C NMR spectrum suggested that compound 1 is a sesquiterpene having five acyloxy groups (three acetoxy and two benzoyloxy groups).7−10 When the chemical shifts of skeletal carbons of 1 were compared with those of sesquiterpenes reported in the literature,5,19 the presence of β-dihydroagarofuran skeleton for 1 was obvious. To corroborate the above deductions and confirm the βdihydroagarofuran sesquiterpene skeleton, the HMBC and 1 H−1H COSY experiments were performed. By interpretation of the HMQC, HMBC, and 1H−1H COSY spectra, the βdihydroagarofuran sesquiterpene skeleton for 1 was elucidated, where the oxygenated methine and methylene carbon signals at δC 79.2, 74.7, 71.4, 73.3, and 60.8 were assigned to C-1, C-6, C8, C-9, and C-13, respectively. The locations of the five acyloxy groups were determined via HMBC correlations. The longrange correlation of the proton H-1 (δH 5.87) with the carbonyl signal at δC 169.8 (CO of the acetoxy moiety) revealed the presence of the acetoxy group at C-1. Similarly, the long-range couplings of the protons H-6 (δH 7.07) and H2-13 (δH 5.24 and 5.06), with the carbonyl carbon signals at δC 169.8 and 170.5 evidenced that the remaining two acetoxy groups were attached E

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Figure 4. Calculated ECD spectra of 1a (1S,4R,5S,6R,7R,8R,9S,10S)and 1b (1R,4S,5R,6S,7S,8S,9R,10R)-isomers and the experimental ECD spectrum of 1 in acetonitrile.

and 167.7 demonstrated that the acetoxy group was attached at C-1 and the benzoyloxy group was at C-9, respectively. There were no additional acyloxy groups in compound 2, so the substituent groups at C-8 and C-13 could only be hydroxy groups, which was confirmed by the HR-ESIMS data. Further analyses of the HMQC, HMBC, and 1 H− 1 H COSY spectroscopic data resulted in the assignments of all the proton and carbon signals. The planar structure with a β-dihydroagrofuran sesquiterpene skeleton for 2 was therefore disclosed. The relative configuration was elucidated on the basis of the NOESY spectrum and the coupling constants. NOESY correlations observed for H3-12/H2-13, H3-12/H-2β, H2-13/ H-2β, H2-13/H-6β, H3-12/H-6β, H2-13/H-9, H-8/H-6β, and H-1/H-3α (Figure 3), but not for H-1/H-9, H2-13/H-1, H314/H-8, H-9/H3-14, and H3-12/H-1, and the Chem3D modeling revealed a conformation for compound 2 as shown in Figure 3, where the C-1 acetoxy group was in a β-equatorial position and the C-9 benzoyloxy group was in an α-axial position. The C-8 hydroxy group was determined as αequatorially oriented, which was supported by the coupling constant between H-9 and H-8 (J8,9 = 6.2 Hz).7,19 Thus, the relative configuration of 2 was characterized as in Figure 3. Considering the biosynthetic origin, the conformations of three skeletal rings, and the same relative configurations for C-4, C-5, and C-10 in compounds 2 and 1, the absolute configurations of C-4, C-5, and C-10 were established to be 4R, 5S, and 10S and the absolute configurations of C-1, C-8, and C-9, consequently, were determined as 1S, 8S, and 9R.5 Thus, compound 2 was characterized as (1S,4R,5S,7S,8S,9R,10S)-1β-acetoxy-9α-benzoyloxy-8α,13-dihydroxy-β-dihydroagarofuran, which was named celaspene B. Compound 3 possessed the molecular formula of C32H37NO9 on the basis of the HR-ESIMS. The 1H and 13C NMR spectra implied that 3 had the same 1,8,9,13tetrasubstituted-β-dihydroagrofuran sesquiterpene skeleton as compound 2 and possessed four acyl groups.5−10,19 Besides one benzoyl and two acetyl groups, an additional nicotinoyl group was deduced and defined on the basis of the proton signals and the typical carbon resonances (Tables 1 and 2).19,23 The following 2D NMR experiments validated the above deductions. The locations of the acyl groups were elucidated by interpretation of the HMBC data as in the case of 2. HMBC correlations of the protons at H-1 (δH 5.60), H-8 (δH 5.67), H-

Figure 3. Key NOESY correlations of compounds 1, 2, 4−6, 8, and 9.

and the corresponding carbon signals (Tables 1 and 2). In addition, one benzoyloxy group was also deduced and defined from the observation of the aromatic proton signals (Table 2) and the typical aromatic carbon signals (Table 1).5−10 Apart from the above nine resonances for the substituent groups, the remaining 15 skeletal carbon resonances indicated in the 13C NMR spectrum form a characteristic β-dihydroagrofuran skeleton.5 To elucidate the skeleton and assign the substituent groups, the following HMQC, HMBC, and 1H−1H COSY experiments were performed. By interpretation of the 2D NMR spectra, the characteristic 1,8,9,13-tetrasubstituted-β-dihydroagrofuran sesquiterpene skeleton was confirmed, where the oxygenated methylene and methine carbons at δC 75.4, 72.6, 70.8, and 62.5 were ascribed to C-1, C-8, C-9, and C-13, respectively. The HMBC correlations of the protons H-1 (δH 5.60) and H-9 (δH 6.03) with the carbonyl carbons at δC 169.2 F

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ESIMS data. As in the case of 1−4, the acyloxy groups were positioned by interpretation of the HMBC spectrum. The HMBC correlations of the protons H-1, H-8, and H2-13 with the carbonyl carbons at δ C 166.4, 166.1, and 165.4 demonstrated that the two benzoyloxy groups were located at C-1 and C-8, and the nicotinoyloxy group was at C-13, respectively. Consequently, the substituent group at C-9 could only be hydroxy group. The coupling constants and the NOESY spectrum allowed the relative configuration of 5 to be assigned. The coupling constant (J8,9 = 9.5 Hz) between H-9 and H-8 suggested that both H-8 and H-9 had an axial orientation, with a nearly 180° dihedral angle between H-8 and H-9.7,19 The NOESY spectrum displayed the correlations of H3-12/H2-13, H3-12/H-6β, H2-13/H-6β, H-6β/H-8, H3-12/H2β, H2-13/H-2β, H2-13/H-8, H-1/H-9, H-9/H3-14, and H-1/ H-3α (Figure 3), but not for H2-13/H-1, H2-13/H-9, and H312/H-1. According to the above evidence, the C-1 benzoyloxy group, the C-9 hydroxy group, and the C-8 benzoyloxy group were characterized as β-equatorial, β-equatorial, and αequatorial, respectively. The relative stereochemistry of 5 was therefore elucidated, where the orientation of the C-8 substituent group was different from those in compounds 1 and 4. Also on the basis of the conformations of three skeletal rings and the biosynthetic origin, the absolute configuration of 5 was established as 1S,4R,5S,7S,8S,9S,10R.5 Thus, compound 5 was elucidated as (1S,4R,5S,7S,8S,9S,10R)-1β,8α-dibenzoyloxy-9β-hydroxy-13-nicotinoyloxy-β-dihydroagarofuran and named celaspene F. Compounds 6 and 7 had the same molecular formula, C28H36O10, on the basis of their HR-ESIMS data. Analyses of the 13C and 1H NMR spectra (Tables 1 and 3) of the two compounds revealed that compounds 6 and 7 had the same 1,2,8,9,13-pentasubstituted-β-dihydroagarofuran skeleton and the same substituent groups (one hydroxy, one benzoyloxy, and three acetoxy groups).5,19 The difference was that the substituent groups in the two compounds were in different positions. Through the same 2D NMR experiments as those for compounds 1−5, the positions of the hydrxoy and acyloxy groups in compounds 6 and 7 were determined. For compound 6, the three acetoxy groups and the benzoyloxy group were attributed to C-1, C-2, and C-8, and C-9 by the HMBC correlations of H-1, H-2, H-8, and H-9 to the corresponding carbonyl carbons, and the remaining one hydroxy group could only be located at C-13. The following NOESY experiment and the coupling constants led to the elucidation of the relative configuration of 6. The NOESY correlations observed for H312/H2-13, H3-12/H-6β, H2-13/H-6β, H-6β/H-8, H2-13/H-8, H-1/H-9, H-9/H3-14, and H-1/H-3α (Figure 3), but not for H2-13/H-1(2), H2-13/H-9, and H3-12/H-1(2), the coupling constant of H-9 and H-8 (J9,8 = 9.5 Hz), and the Chem3D modeling suggested a conformation for 6 as shown in Figure 3, where the C-1 acetoxy group and the C-9 benzoyloxy group were both in β-positions with an equatorial orientation, the C-2 acetoxy group was in a β-position with an axial orientation, and the C-8 acetoxy group was in an α-position with an equatorial orientation. Compound 7 had the same substituent groups as compound 6, and their relative locations were assigned by the HMBC spectrum, which demonstrated the three acetoxy groups and the benzoyloxy group were attached at C-1, C-2, C-13, and C-9, respectively. The only hydroxy group supported by the HR-ESIMS data was verified at C-8 by interpretation of the 1H−1H COSY spectrum. The same relative configuration for compounds 7 and 6 was inferred on the basis of the analyses

9 (δH 5.94), and H2-13 (δH 4.86 and 4.80) with the corresponding carbonyl carbons at δC 169.6, 169.9, 165.9, and 165.3 indicated that two acetoxy, one benzoyloxy, and one nicotinoyloxy group were attached at C-1, C-8, C-9, and C-13, respectively. By further analyzing the 2D NMR spectra, the planar structure of 3 was established. The relative configuration of compound 3 was elucidated on the basis of the NOESY spectrum and Chem3D modeling as that of compound 2. The NOESY correlations observed for H3-12/H2-13, H3-12/H-2β, H2-13/H-2β, H2-13/H-6β, H3-12/H-6β, H2-13/H-9, H-8/H6β, and H-1/H-3α (Figure 3), but not for H2-13/H-1, H-1/H9, H-9/H3-14, and H3-12/H-1, and the Chem3D modeling disclosed a molecular conformation for compound 3 as depicted in Figure 3, where all of the chiral carbons had the same relative configurations as those of compound 2. On the basis of the above spectroscopic evidence and the biosynthetic standpoint, compound 3 was assigned as (1S,4R,5S,7S,8S,9R,10S)-1β,8α-diacetoxy-9α-benzoyloxy-13nicotinoyloxy-β-dihydroagarofuran and named celaspene C, which is the C-8 isomer of the known compound 1β,8αdiacetoxy-9β-benzoyloxy-13-nicotinoyloxy-β-dihydroagarofuran.24,25 Analyses of the 13C and 1H NMR data (Tables 1 and 2) of compound 4 revealed that 4 had the characteristic 1,2,6,8,9pentasubstituted-β-dihydroagarofuran sesquiterpene skeketon.5−10 The same acyl groups as those appearing in compounds 1−3 were apparent, which were deduced and determined as one acetyl and two benzoyl groups according to its 13C and 1H NMR spectra.19,23 The residual substituent groups could only be hydroxy groups, which were validated by the HR-ESIMS data. Through the same 2D NMR experiments, especially HMBC and 1H−1H COSY experiments, as for compounds 1−3, the positions of the substituent groups (one acetoxy and two benzoyloxy groups) of 4 were determined, which suggested that the two benzoyloxy groups were attached at C-2 and C-8, the two hydroxy groups were ascribed to C-1 and C-9, and the acetoxy group was at C-6, respectively. The NOESY spectrum and the coupling constants allowed the relative configuration of 4 to be assigned. The NOESY correlations observed for H3-12/H3-13, H3-13/H-6, H3-12/H6, H-1/H-9, H-9/H3-14, H-8/H3-14, and H-1/H-3α (Figure 3), but not for H3-13/H-1, H3-13/H-9, H3-12/H-1, and H312(13)/H-2, and Chem 3D revealed that the two hydroxy groups attributed to C-1 and C-9 were both β-equatorially oriented, the two benzoyloxy groups ascribed to C-2 and C-8 were both β-axially oriented, and the acetoxy attached at C-6 was α-equatorially oriented.5,19 The coupling constants between H-9 and H-8 (J8,9 = 4.7 Hz) and H-1 and H-2 (J1,2 = 3.6 Hz) also confirmed that the protons H-1 and H-9 were in α-axial positions and H-2 and H-8 were in α-equatorial positions, respectively. According to the above spectroscopic evidence and the biosynthetic standpoint, the structure of compound 4 was therefore assigned as (1R,2S,4R,5S,6R,7R,8R,9S,10S)-6α-acetoxy-2β,8β-dibenzoyloxy-1β,9β-dihydroxy-β-dihydroagarofuran,5 which has been named celaspene D. The HR-ESIMS provided a molecular formula of C35H37NO8 for compound 5. The 13C and 1H NMR spectra (Tables 1 and 3) suggested a 1,8,9,13-tetrasubstituted-β-dihydroagarofuran sesquiterpene skeketon for 5.5−10 The four substituent groups were deduced as two benzoyloxy groups, one nicotinoyloxy group,19,23 and one hydroxy group according to its 13C and 1H NMR spectra, which was supported by the 2D NMR and HRG

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7S,8S,9R,10S)-1β,8α-diacetoxy-9α-benzoyloxy-13-hydroxy-β-dihydroagarofuran and named celaspene I. To explore the potential biological effects of these new sesquiterpenes to serve agriculture, these isolated compounds from C. orbiculatus were preliminarily investigated for their antifungal activities against six pathogenic fungi (G. zeae, P. piricola, A. solani, C. arachidicola, C. cucumerinum, and P. capsici), which commonly infect crops, leading to severe crop yield reduction and dramatic economic losses in agriculture.30 Chlorothalonil and carbendazol were used as the corresponding positive controls. At a concentration of 50 μg/mL, all of the evaluated compounds exhibited antifungal activities. Their preliminary antifungal activities are shown in Figure 5

and the careful comparison of the NOESY spectra of 7 and 6. On the basis of the same conformations of three skeletal rings in compounds 1−7 and the biosynthetic origin, compounds 6 and 7 were characterized as (1R,2S,4R,5S,7S,8S,9S,10S)1β,2β,8α-triacetoxy-9β-benzoyloxy-13-hydroxy-β-dihydroagarofuran and (1R,2S,4R,5S,7S,8S,9S,10R)-1β,2β,13-triacetoxy-9βbenzoyloxy-8α-hydroxy-β-dihydroagarofuran, which have been named celaspenes G and H, respectively. Compound 8 possessed the molecular formula of C26H34O8 as determined by the HR-ESIMS. Its 13C and 1H NMR spectra (Tables 1 and 3) revealed that compound 8 was a 1,6,8,9tetrasubstituted-β-dihydroagarofuran sesquiterpene with two acetyl, one benzoyl, and one hydroxy group, which was supported and confirmed by 2D NMR spectra. The further interpretation of 2D NMR spectra led to the determination of the relative positions of these substituent groups, which verified that the two acetoxy groups were located at C-1 and C-6, the benzoyloxy group was at C-9, and the hydroxy group was at C8, respectively. By further analyzing the HMQC, HMBC, and 1 H−1H COSY spectra, all of the proton and carbon signals were assigned unambiguously. On the basis of these assignments, the proton signals for H-8 and H-9 presented in the form of singlet, which implied the different configurations for C-8 and C-9 in 8. The NOESY correlations of H3-12/H3-13, H3-12/H-6, H3-13/H-6, H3-13/H-9, H3-13/H-2β, H3-12/H-2β, and H-1/H-3α (Figure 3), but not for H-6/H-8, H-1/H-9, H9/H3-14, H3-13/H-1, and H3-12/H-1, the coupling constant of H-9 and H-8 (J9,8 = 0 Hz), and Chem3D modeling suggested that H-8 was α-equatorially oriented, H-9 was β-equatorially oriented, and H-1 was α-axially oriented.5,19 Thus, the relative configuration of 8 was elucidated. According to the same conformations of three skeletal rings of compounds 1−7, the absolute configuration of 8 was assigned as 1S,4R,5S,6R,7R,8R,9R,10S.5,19 Compound 8 was therefore characterized as (1S,4R,5S,6R,7R,8R,9R,10S)-1β,6α-diacetoxy9α-benzoyloxy-8β-hydroxy-β-dihydroagarofuran and named celaspene H, which is the C-9 isomer of the known compound 1β,6α-diacetoxy-9β-benzoyloxy-8β-hydroxy-β-dihydroagarofuran.26−29 Compound 9 possessed a molecular formula of C26H34O8 from its HR-ESIMS. The molecular formula and its 13C and 1H NMR spectra (Tables 1 and 3) suggested that compound 9 was a 1,8,9,13-tetrasubstituted-β-dihydroagarofuran sesquiterpene with two acetoxy, one benzoyloxy, and one hydroxy group. To achieve the assignments of all the proton and carbon signals, the 2D NMR spectra were recorded. By interpretation and analyses of 2D NMR spectra, the planar structure of a 1,8,9,13-tetrasubstituted-β-dihydroagarofuran sesquiterpene was established, where the two acetoxy groups and one benzoyloxy group were ascribed to C-1, C-8, and C-9, and the residual hydroxy group was securely assigned to C-13. The coupling constant of H-9 and H-8 (J9,8 = 6.4 Hz), Chem3D modeling, and the NOESY correlations of H3-12/H2-13, H312/H-6β, H2-13/H-6β, H2-13/H-9, H2-13/H-8, H-6β/H-8, H213/H-2β, H3-12/H-2β, and H-1/H-3α (Figure 3), but not for H-1/H-9, H-9/H3-14, H2-13/H-1, and H3-12/H-1, revealed a molecular conformation for 9 as shown in Figure 3.5 The proton H-1 was in an α-position with an axial orientation, H-8 was in a β-position with an axial orientation, and H-9 was in a β-position with an equatorial orientation. Similarly, from a biosynthetic standpoint, the absolute configuration was also assigned on the basis of the same conformations of the three skeletal rings.5 Thus, compound 9 was elucidated as (1S,4R,5S,

Figure 5. Antifungal effects of compounds 1−4 and 6−10 against six pathogenic fungi. Six common pathogenic fungi (G. zeae, P. piricola, A. solani, C. arachidicola, C. cucumerinum, and P. capsici) were selected to evaluate the antifungal activities of compounds 1−4 and 6−10. The biological data presented are the mean scores for each treatment across replicates. The symbols ▲ and ▼ indicate the positive controls chlorothalonil and carbendazol, respectively.

(compound 5 was not assayed for the antifungal effects because of inadequate residual amount). For the pathogenic fungi A. solani and P. capsici, all of the compounds showed weak or moderate antifungal effects. For the fungus G. zeae, compound 4 showed a strong activity with an inhibitory rate of 62%. For the fungus C. cucumerinum, compound 7 showed a strong activity with an inhibitory rate of 62%. For the fungus C. arachidicola, compound 6 had a stronger activity with an inhibitory rate of 69% than other compounds. Compounds 1, 3, 6, 8, and 9 showed promising antifungal activities against the fungus P. piricola with inhibitory rates of 84, 84, 70, 66, and 71%, respectively. From the above antifungal data, there seem to be no obvious correlations among these activities against the six pathogenic fungi and the selectivity or sensitivity of each pathogenic fungus toward the same compound is different, which implies that the antifungal activity against different pathogenic fungus may have different mechanisms. In conclusion, nine new and one known sesquiterpene were obtained from the fruits of the plant C. orbiculatus. Their structures, especially the stereochemical features, were elucidated by NMR data analyses, TDDFT CD calculations, and the CD exciton chirality method. The subsequent biological screenings exhibited that these isolated compounds showed antifungal activities against six phytopathogenic fungi. The results of our phytochemical investigation further revealed the chemical components of C. orbiculatus as an insecticidal and H

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Agrochemicals and Plant Protection; Wiley: Surrey, UK, 1998; pp 109−148. (14) Zhao, H.; Liu, Y.; Cui, Z.; Beattie, D.; Gu, Y.; Wang, Q. Design, synthesis, and biological activities of arylmethylamine substituted chlorotriazine and methylthiotriazine compounds. J. Agric. Food Chem. 2011, 59, 11711−11717. (15) Li, X. J.; Zhang, Q.; Zhang, A. L.; Gao, J. M. Metabolites from Aspergillus f umigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J. Agric. Food Chem. 2012, 60, 3424−3431. (16) Zhang, Y.; Wang, J. S.; Wang, X. B.; Gu, Y. C.; Wei, D. D.; Guo, C.; Yang, M. H.; Kong, L. Y. Limonoids from the fruits of Aphanamixis polystachya (Meliaceae) and their biological activities. J. Agric. Food Chem. 2013, 61, 2171−2182. (17) Wang, D. M.; Zhang, C. C.; Zhang, Q.; Shafiq, N.; Pescitelli, G.; Li, D. W.; Gao, J. M. Wightianines A−E, dihydro-β-agarofuran sesquiterpenes from Parnassia wightiana, and their antifungal and insecticidal activities. J. Agric. Food Chem. 2014, 62, 6669−6676. (18) Takaishi, Y.; Tokura, K.; Noguchi, H.; Nakano, K.; Murakami, K.; Tomimatsu, T. Sesquiterpene esters from Tripterygium wilfordii. Phytochemistry 1991, 30, 1561−1566. (19) Kennedy, M. L.; Cortés-Selva, F.; Pérez-Victoria, J. M.; Jiménez, I. A.; González, A. G.; Muñoz, O. M.; Gamarro, F.; Castanys, S.; Ravelo, A. G. Chemosensitization of a multidrug-resistant Leishmania tropica line by new sesquiterpenes from Maytenus magellanica and Maytenus chubutensis. J. Med. Chem. 2001, 44, 4668−4476. (20) Conflex software, version 6.7; Conflex Corp., Tokyo, Japan, 2010. (21) Gaussian 09, revision B.01; Gaussian Inc.: Pittsburgh, PA, USA, 2010. (22) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton Coupling in Organic Stereochemistry; University Science Books: Mill Valley, CA, USA, 1983. (23) Xu, J.; Jin, D. Q.; Guo, Y.; Xie, C.; Ma, Y.; Yamakuni, T.; Ohizumi, Y. New myrsinol diterpenes from Euphorbia prolifera and their inhibitory activities on LPS-induced NO production. Bioorg. Med. Chem. Lett. 2012, 22, 3612−3618. (24) Wang, Y.; Yang, L.; Tu, Y.; Zhang, K.; Chen, Y.; Fan, J. Two new sesquiterpenoids from the seeds of Celastrus angulatus. J. Nat. Prod. 1998, 61, 942−944. (25) Yang, L.; Wang, Y.; Tu, Y.; Chen, Y. Two sesquiterpenoids from Celastrus angulatus. Zhongshan Daxue Xuebao, Ziran Kexueban 1997, 36, 123−124. (26) Guo, Y. Q.; Li, X.; Ma, Z. J.; Cui, W. S.; Wang, J. H.; Ju, Z. X. A new sesquiterpene ester from the fruits of Celastrus orbiculatus. J. Asian Nat. Prod. Res. 2005, 7, 157−160. (27) Tu, Y.; Wang, Y. Complete proton and carbon-13 chemical shift assignments and the relationship between carbon-13 chemical shifts and stereochemistry for β-dihydroagarofuran sesquiterpene. Bopuxue Zazhi 1992, 9, 329−335. (28) Tu, Y.; Wu, D.; Zhang, X.; Hao, X. Bioactive sesquiterpenoids from Celastrus paniculatus. Gaodeng Xuexiao Huaxue Xuebao 1992, 13, 1548−1550. (29) Tu, Y. Q.; Chen, Y. Z.; Wu, D. G.; Zhang, X. M.; Hao, X. J. Sesquiterpenoids from Celastrus paniculatus. J. Nat. Prod. 1993, 56, 122−125. (30) Savary, S.; Teng, P. S.; Willocquet, L.; Nutter, F. W., Jr. Quantification and modeling of crop losses: a review of purposes. Annu. Rev. Phytopathol. 2006, 44, 89−112.

medicinal plant, and the biological screenings implied that C. orbiculatus may be potentially useful to protect crops against phytopathogenic fungi and that the bioactive compounds may probably be considered as candidate agents of antifungal agrochemicals for crop protection products.

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR and HR-MS spectra of compounds 1−9. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.X.) Phone/fax: +86-22-23502595. E-mail: xujing611@ nankai.edu.cn. Funding

This work was supported in part by the National Natural Science Foundation of China (No. 21372125). Notes

The authors declare no competing financial interest.

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REFERENCES

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