Anti-Tobacco Mosaic Virus Quassinoids from Ailanthus altissima (Mill

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Cite This: J. Agric. Food Chem. 2018, 66, 7347−7357

Anti-Tobacco Mosaic Virus Quassinoids from Ailanthus altissima (Mill.) Swingle Qing-Wei Tan,*,†,‡,§ Jian-Cheng Ni,†,§ Lu-Ping Zheng,‡,§ Pei-Hua Fang,§ Jian-Ting Shi,§ and Qi-Jian Chen*,‡,§ State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection and §Key Laboratory of Biopesticide and Chemistry Biology, Ministry of Education, Fujian Agriculture and Forestry University, 15 Shangxiadian Road, Cangshan, Fuzhou, Fujian 350002, People’s Republic of China

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‡

S Supporting Information *

ABSTRACT: Quassinoids are bitter constituents characteristic of the family Simaroubaceae. A total of 18 C20 quassinoids, including nine new quassinoid glycosides, named chuglycosides A−I (1−6 and 8−10), were identified from the samara of Ailanthus altissima (Mill.) Swingle. All of the quassinoids showed potent anti-tobacco mosaic virus (TMV) activity. A preliminary structure−anti-TMV activity relationship of quassinoids was discussed. The effects of three quassinoids, including chaparrinone (12), glaucarubinone (15), and ailanthone (16), on the accumulation of TMV coat protein (CP) were studied by western blot analysis. Ailanthone (16) was further investigated for its influence on TMV spread in the Nicotiana benthamiana plant. KEYWORDS: tobacco mosaic virus (TMV), Ailanthus altissima (Mill.) Swingle, quassinoids, structure−activity relationship (SAR)

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recorded using a Nicolet iS50 FTIR spectrometer (Thermo Scientific, Waltham, MA, U.S.A.). High-resolution electrospray ionization mass spectrometry (HR−ESI−MS) data were acquired on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, U.S.A.). Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker AVANCE III 500 spectrometer (Bruker BioSpin, Switzerland), with tetramethylsilane (TMS) as the internal standard, at the Test Center of Fuzhou University (Fuzhou, China). The absorbance were read at 405 nm with a Multiskan MK3 microplate reader [Thermo Fisher Scientific (China) Co., Ltd., China]. Silica gel (200−300 and 300−400 mesh, silica gel H, Qingdao Oceanic Chemical Co., China), Sephadex LH-20 gel (25−100 μm, Pharmacia Fine Chemical Co., Ltd., Sweden), Lichroprep RP-18 gel (40−63 μm, Merck, Germany), and MCI gel CHP-20P (75−150 μm, Mitsubishi Chemical Co., Japan) were used for column chromatography. Thinlayer chromatography (TLC) were carried out with silica gel H and visualized by heating after spraying 5% H2SO4 in EtOH. All solvents used for extraction and separation were of analytical reagent grade. Extraction and Isolation of Quassinoids. The A. altissima samara, collected in Muyang (Jiangsu, China) in 2013 and identified by Associate Professor Chun-Mei Huang (College of Life Sciences, Fujian Agriculture and Forestry University), was milled and extracted with MeOH to give a crude extract. The MeOH extract was concentrated in vacuo, resuspended in distilled H2O, and then partitioned with organic solvents to yield n-hexane, CHCl3, n-BuOH, and water-soluble fractions. From the CHCl3- and n-BuOH-soluble fractions, 18 quassinoids were isolated by column chromatography using silica, Sephadex LH-20, RP-C18, and MCI gels (Figure 1). Chuglycoside A (1). Colorless crystal; melting point (mp), 220− 222 °C; [α]20 D , +109.1 (c 0.1, MeOH); IR (KBr) υmax, 3419, 2876, 1727, 1660, 1503, 1436, 1374, 1316, 1237, 1167, 1034, 910 cm−1;

INTRODUCTION The genus Ailanthus (Simaroubaceae) is reputed for its medical values, and more than 200 compounds of diverse structural patterns have been characterized from Ailanthus plants.1,2 Ailanthus altissima (Mill.) Swingle [syn. Ailanthus glandulosa (Desf.)], native to China and naturalized in many temperate regions, is one of the most well-studied Ailanthus deciduous trees.3,4 Phytochemical investigations into this species have revealed the presence of quassinoids, alkaloids, terpenoids, coumarins, sterols, phenolics, etc.3,4 In addition, we have recently reported the identification of phenylpropionamides and piperidine derivatives from its methanol samara extract.5 Pharmacological and clinical investigations revealed that natural products from Ailanthus plants, especially quassinoids, the characteristic bitter constituents of Simaroubaceae, are very promising for their medical use. Thus far, more than 40 quassinoids have been characterized from A. altissima, including ailanthone,6−11 dihydroailanthone,7 amarolide,7 11-acetyl amarolide,7 ailantholide,10 shinjulactones A− O,12−20 shinjudilactone,21 shinjuglycosides A−F,22,23 ailantinols A−H,24−27 altissinols A and B,3 Δ13(18)-dehydroglaucarubinone,24 Δ13(18)-dehydroglaucarubolone,24 chapparin,26 chapparinone,10 etc. We herein reported the isolation and structure elucidation of 18 quassnioids, including nine new quassinoid glycosides (1−6 and 8−10), from the samara of A. altissima. All of the quassinoids showed potent inhibitory effects against the replication of tobacco mosaic virus (TMV).

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MATERIALS AND METHODS

General Experimental Procedure. Melting points were determined on an INESA SGW X-4 microscopic melting point apparatus (uncorrected), and optical rotations were measured with an INESA SGW-532 polarimeter (Shanghai INESA Physico-Optical Instrument Co., Ltd., China). Infrared (IR) spectra (KBr) were © 2018 American Chemical Society

Received: Revised: Accepted: Published: 7347

March 18, 2018 June 18, 2018 June 28, 2018 June 28, 2018 DOI: 10.1021/acs.jafc.8b01280 J. Agric. Food Chem. 2018, 66, 7347−7357

Article

Journal of Agricultural and Food Chemistry

Figure 1. Extraction and isolation of compounds 1−18 from A. altissima samara. Chuglycoside H (9). Colorless crystal; mp, 211−213 °C; [α]20 D, +110.0 (c 0.01, MeOH); IR (KBr) υmax, 3406, 2963, 2929, 2892, 1714, 1656, 1449, 1316, 1283, 1246, 1163, 1109, 1063, 1026 cm−1; HR−ESI−MS m/z, 683.2323 [M + Na]+ (calcd for C33H40O14Na, 683.2316); and 13C NMR (Table 1) and 1H NMR (Table 3). Chuglycoside I (10). White needle crystal; mp, 230−232 °C; [α]20 D, +43.9 (c 0.1, MeOH); IR (KBr) υmax, 3411, 2967, 2938, 2884, 1735, 1652, 1457, 1179, 1150, 1051, 914 cm−1; HR−ESI−MS m/z, 663.2646 [M + Na]+ (calcd for C31H44O14Na, 663.2629); and 13C NMR (Table 1) and 1H NMR (Table 3). Anti-TMV Assay. Tobacco (Nicotiana tabacum cv. K326, Nicotiana benthamiana, and Nicotiana glutinosa L.) with 5−6 fully expanded true leaves were used for experiments. TMV U1 strain was available from the Institute of Plant Virology, Fujian Agriculture and Forestry University, and the virus concentration was determined as described.5,28,29 Virus infection was achieved by rubbing leaves predusted with silicon carbide (400 mesh) with a solution of TMV (15 μg/mL) in 0.01 M phosphate-buffered saline (PBS). The quassinoids were dissolved to 10 mM in dimethyl sulfoxide (DMSO) and diluted to the required concentration with 0.01 M PBS before use. Two commercial agents used as the control were ningnanmycin and ribavirin. Local-Lesion Method. Three of the top fully expanded leaves of N. glutinosa L. were used for TMV infection. A mixture of quassinoid and TMV was mechanically inoculated on one-half of the leaves, while the other half was used as the control, which was treated with a mixture of TMV and 1% DMSO. Local lesions formed were counted 3 or 4 days after inoculation. The anti-TMV activity of quassinoids was evaluated through their inhibition rate, which was calculated as follows: inhibition rate (%) = [1 − (lesion number of treatment)/(lesion number of positive control)] × 100. Leaf-Disk Method. The whole fully expanded N. tabacum cv. K326 leaf was inoculated and infected with TMV. After 6 h, leaf disks (1 cm

HR−ESI−MS m/z, 623.2333 [M + Na]+ (calcd for C28H40O14Na, 623.2316); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside B (2). White needle crystal; mp, 213−215 °C; [α]20 D, +116.5 (c 0.1, MeOH); IR (KBr) υmax, 3411, 2963, 2925, 2884, 1747, 1702, 1648, 1453, 1387, 1254, 1192, 1134, 1080, 1034, 955 cm−1; HR−ESI−MS m/z, 663.2647 [M + Na]+ (calcd for C31H44O14Na, 663.2629); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside C (3). Colorless crystal; mp, 218−219 °C; [α]20 D, +141.4 (c 0.1, MeOH); IR (KBr) υmax, 3406, 2958, 2888, 1710, 1602, 1453, 1382, 1283, 1254, 1171, 1063, 1134, 1026, 993, 955 cm−1; HR−ESI−MS m/z, 685.2483 [M + Na]+ (calcd for C33H42O14Na, 685.2472); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside D (4). Colorless crystal; mp, 208−210 °C; [α]20 D, +62.6 (c 0.1, MeOH); IR (KBr) υmax, 3406, 2967, 2938, 2880, 1739, 1635, 1457, 1378, 1320, 1229, 1192, 1154, 1076, 1051, 914 cm−1; HR−ESI−MS m/z, 651.2642 [M + Na]+ (calcd for C30H44O14Na, 651.2629); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside E (5). White needle crystal; mp, 232−233 °C; [α]20 D, +58.3 (c 0.1, MeOH); IR (KBr) υmax, 3498, 3423, 3257, 2950, 2876, 2747, 1760, 1731, 1669, 1457, 1378, 1233, 1084, 1042, 988, 918 cm−1; HR−ESI−MS m/z, 665.2807 [M + Na]+ (calcd for C31H46O14Na, 665.2785); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside F (6). White needle crystal; mp, 218−219 °C; [α]20 D, +75.0 (c 0.1, MeOH); IR (KBr) υmax, 3402, 2921, 2884, 1731, 1640, 1598, 1382, 1349, 1316, 1233, 1183, 1107, 1076, 1046, 959 cm−1; HR−ESI−MS m/z, 685.2493 [M + Na]+ (calcd for C33H42O14Na, 685.2472); and 13C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside G (8). White needle crystal; mp, 216−218 °C; [α]20 D, +100.6 (c 0.1, MeOH); IR (KBr) υmax, 3406, 2929, 2888, 1706, 1644, 1441, 1378, 1250, 1192, 1129, 1076, 918 cm−1; HR−ESI−MS m/z, 661.2491 [M + Na]+ (calcd for C31H42O14Na, 661.2472); and 13C NMR (Table 1) and 1H NMR (Table 3). 7348

DOI: 10.1021/acs.jafc.8b01280 J. Agric. Food Chem. 2018, 66, 7347−7357

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

position

82.6, 83.8, 127.9, 137.1, 44.9, 69.4, 80.6, 47.8, 43.5, 45.5, 110.9, 80.3, 32.3, 42.8, 30.6, 172.8, 24.6, 11.3, 71.6, 12.9, 105.4, 75.5, 78.0, 71.5, 77.9, 62.7, 171.9, 21.4,

1

CH CH CH C CH CH CH C CH C C CH CH CH CH2 C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C CH3

82.7, 83.9, 127.7, 137.3, 45.0, 69.2, 80.9, 47.9, 43.6, 45.6, 110.9, 80.3, 32.3, 42.8, 30.6, 172.9, 24.5, 11.4, 71.7, 12.9, 105.4, 75.5, 78.1, 71.5, 78.0, 62.7, 168.5, 129.7, 140.6, 14.5, 12.1,

2

CH CH CH C CH CH CH C CH C C CH CH CH CH2 C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C C CH CH3 CH3

82.7, 83.8, 127.9, 137.1, 45.1, 69.8, 80.9, 48.0, 43.6, 45.7, 110.9, 80.3, 32.3, 42.8, 30.6, 172.9, 24.6, 11.4, 71.7, 12.9, 105.4, 75.6, 78.1, 71.5, 78.0, 62.7, 167.2, 131.2, 130.9, 129.7, 134.7, 129.7, 130.9,

3 CH CH CH C CH CH CH C CH C C CH CH CH CH2 C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C C CH CH CH CH CH

82.6, 84.2, 125.0, 137.4, 42.0, 26.3, 80.0, 48.8, 45.8, 42.5, 110.8, 80.4, 33.5, 46.4, 70.9, 170.1, 21.3, 10.7, 72.3, 15.4, 105.7, 75.6, 78.0, 71.5, 77.9, 62.7, 177.4, 35.3, 19.2, 18.9,

4 CH CH CH C CH CH2 CH C CH C C CH CH CH CH C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C CH CH3 CH3

Table 1. 13C NMR Data for Compounds 1−11 in CD3OD (125 MHz) 82.6, 84.2, 125.0, 137.3, 42.0, 26.3, 80.0, 48.8, 45.8, 42.5, 110.8, 80.3, 33.5, 46.5, 70.8, 170.0, 21.3, 10.7, 72.3, 15.4, 105.7, 75.6, 78.0, 71.5, 77.9, 62.7, 177.1, 42.3, 27.8, 11.8, 16.5,

5 CH CH CH C CH CH2 CH C CH C C CH CH CH CH C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C CH CH2 CH3 CH3

82.6, 84.2, 125.0, 137.4, 42.0, 26.3, 80.2, 48.9, 45.9, 42.6, 110.8, 80.4, 33.5, 46.4, 71.5, 170.0, 21.3, 10.7, 72.3, 15.4, 105.7, 75.6, 78.0, 71.6, 78.0, 62.7, 166.9, 130.9, 130.8, 129.7, 134.7, 129.7, 130.8,

6 CH CH CH C CH CH2 CH C CH C C CH CH CH CH C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2 C C CH CH CH CH CH

82.6, 84.2, 125.0, 137.4, 42.3, 26.5, 80.5, 47.1, 43.0, 45.0, 110.7, 80.1, 32.4, 42.3, 30.6, 173.8, 21.4, 10.4, 72.4, 13.0, 105.6, 75.6, 78.0, 71.5, 77.9, 62.7,

7 CH CH CH C CH CH2 CH C CH C C CH CH CH CH2 C CH3 CH3 CH2 CH3 CH CH CH CH CH CH2

82.6, 83.7, 127.8, 137.1, 45.0, 69.1, 80.7, 47.3, 43.8, 45.7, 110.5, 81.2, 146.6, 47.9, 35.2, 171.7, 24.6, 11.3, 72.4, 120.1, 105.3, 75.6, 78.1, 71.5, 78.0, 62.7, 168.4, 129.7, 140.6, 14.6, 12.1,

8 CH CH CH C CH CH CH C CH C C CH C CH CH2 C CH3 CH3 CH2 CH2 CH CH CH CH CH CH2 C C CH CH3 CH3

82.6, 83.7, 128.0, 136.9, 45.1, 69.7, 80.7, 47.3, 43.8, 45.7, 110.5, 81.2, 146.6, 47.9, 35.2, 171.7, 24.7, 11.4, 72.4, 120.1, 105.3, 75.6, 78.1, 71.5, 78.0, 62.7, 167.2, 131.2, 130.9, 129.8, 134.8, 129.8, 130.9,

9 CH CH CH C CH CH CH C CH C C CH C CH CH2 C CH3 CH3 CH2 CH2 CH CH CH CH CH CH2 C C CH CH CH CH CH

82.3, CH 84.0, CH 125.1, CH 137.1, C 42.1, CH 26.1, CH2 80.4, CH 48.5, C 46.2, CH 42.6, C 110.2, C 80.8, CH 143.4, C 52.4, CH 69.4, CH 169.2, C 21.4, CH3 10.4, CH3 73.1, CH2 122.4, CH2 105.7, CH 75.60, CH 78.0, CH 71.5, CH 77.9, CH 62.7, CH2 177.1, CH3 42.3, CH 27.8, CH2 11.9, CH3 17.0, CH3

10 82.5, 84.0, 125.0, 137.3, 42.3, 26.4, 80.4, 48.0, 46.5, 42.4, 110.2, 80.4, 146.9, 48.0, 35.4, 172.6, 21.4, 10.3, 73.2, 120.3, 105.6, 75.6, 78.0, 71.5, 78.0, 62.7,

CH CH CH C CH CH2 CH C CH C C CH C CH CH2 C CH3 CH3 CH2 CH2 CH CH CH CH CH CH2

11

Journal of Agricultural and Food Chemistry Article

7349


7350

4″ 5″ 6″ 7″

1″ 2″ 3″

16 18 19 20a 20b 21 Glc-1′ 2′ 3′ 4′ 5′ 6′

7 8 9 10 11 12 13 14 15

1 2 3 4 5 6

position

3.46, 2.31, 2.08, 2.91, 2.65,

3.44, 2.30, 2.07, 2.89, 2.64,

2.14, s

1.84, s 1.44, s 4.16, d (8.8) 3.80, d (8.8) 1.04, d (7.2) 4.55, d (7.8) 3.27, dd (9.1, 7.8) 3.42−3.37, overlap 3.37−3.30, overlap 3.37−3.30, overlap 3.91, dd (11.8, 1.8) 3.72, m

1.87, d (7.1) 1.91, s

7.06, m


d (4.2) td (7.0, 4.2) dt (12.9, 6.1) dd (19.2, 13.5) dd (19.2, 5.7)

2.58, s

2.56, s

d (4.2) td (6.9, 4.2) dt (12.8, 6.2) dd (19.1, 13.5) dd (19.1, 5.8)

4.54, d (2.6)

2.94, br d (12.3) 5.58, dd (12.3, 2.6)

2.91−2.85, overlap 5.44, dd (12.3, 2.6)

4.59, d (2.6)

3.80, d (7.8) 4.19, m 5.76, td (3.1, 1.5)

2

3.78, d (7.9) 4.18, m 5.77, td (3.1, 1.5)

1

d (4.2) td (7.0, 4.2) dt (12.8, 6.1) dd (19.1, 13.5) dd (19.1, 5.7)

7.56−7.50, overlap 7.66, m 7.56−7.50, overlap 8.16−8.13, overlap

8.16−8.13, overlap


3.46, 2.32, 2.10, 2.93, 2.66,

2.61, s

4.68, d (2.6)

3.06, br d (12.2) 5.77, dd (12.2, 2.6)

3.82, d (7.9) 4.20, m 5.78−5.75, overlap

3

Table 2. 1H NMR Data for Compounds 1−7 in CD3OD (500 MHz)

br d (12.9) dt (14.9, 2.8) ddd (14.9, 12.9, 2.3) t (2.8)

1.20, d (7.0)

2.61, hep (7.0) 1.21, d (7.0)

1.71, s 1.30, s 4.04, d (9.0) 3.72, d (9.0) 1.07, d (6.6) 4.53, d (7.8) 3.25, dd (9.0, 7.8) 3.43−3.35, overlap 3.35−3.28, overlap 3.35−3.28, overlap 3.89, dd (11.8, 1.7) 3.74−3.68, overlap

3.43−3.40, overlap 2.40−2.30, overlap 2.40−2.30, overlap 5.73, d, (11.3)

2.56, s

2.43, 2.06, 1.99, 4.64,

3.66, d (7.9) 4.17, m 5.70, m

4

2.48−2.40, overlap 1.74, tt (14.6, 7.4) 1.53, tt (13.6, 7.4) 0.98, t (7.4) 1.18, d (7.0)


3.43−3.40, overlap 2.39−2.32, overlap 2.39−2.32, overlap 5.75, d (11.2)

2.56, s

2.48−2.40, overlap 2.07, dt (14.8, 3.1) 1.99, ddd (14.8, 13.0, 2.3) 4.65, t (3.1)

3.67, d (7.9) 4.18, m 5.70, td (2.9, 1.5)

5

br d (13.1) dt (14.9, 3.0) ddd (14.9, 13.1, 2.3) t (3.0)

d (3.4) m dd (12.1, 6.2) d (12.1)

7.56−7.51, overlap 7.67, m 7.54, overlap 7.56−7.51, overlap

8.10−8.06, overlap

1.74, br s 1.33, s 4.08, d (9.0) 3.77, d (9.0) 1.07, d (7.4) 4.56, d (7.8) 3.27, dd (9.1, 7.8) 3.43−3.37, overlap 3.36−3.30, overlap 3.36−3.30, overlap 3.91, dd (11.9, 1.6) 3.75−3.69, overlap

3.44, 2.39, 2.56, 5.95,

2.66, s

2.50, 2.11, 2.03, 4.73,

3.72, d (8.1) 4.20, m 5.72, td (2.9, 1.5)

6


3.43, d (4.3) 2.35−2.26, overlap 2.07−1.99, overlap 2.78, dd (19.0, 13.8) 2.62, dd (19.0, 5.3)

2.51, s

2.35−2.26, overlap 2.07, dt (14.7, 2.9) 2.02−1.94, overlap 4.54, overlap

3.65, d (8.0) 4.18, m 5.71, td (2.8, 1.5)

7

Journal of Agricultural and Food Chemistry Article


Article

Journal of Agricultural and Food Chemistry Table 3. 1H NMR Data for Compounds 8−11 in CD3OD (500 MHz) position

8

9

10

11

1 2 3 4 5 6

3.82, d (7.8) 4.20, m 5.76, td (3.1, 1.6)

3.86, d (7.8) 4.21, m 5.78−5.75, overlap

3.75, d (8.0) 4.19, m 5.72, td (2.8, 1.5)

3.71, d (8.0) 4.21, m 5.73, td (2.8, 1.5)

2.99, br d (12.2) 5.57, dd (12.2, 2.6)

3.10, br d (12.1) 5.77, dd (12.1, 2.6)

7 8 9 10 11 12 13 14 15

4.59, d (2.6)

4.73, d (2.6)

2.53−2.43, overlap 2.11, dt (15.0, 2.9) 1.99, ddd (15.0, 13.3, 2.5) 4.66, t (2.9)

2.39, 2.11, 2.00, 4.60,

2.77, s

2.81, s

2.83, s

2.71, s

3.93, s

3.94, s

3.90, s

3.92, s

2.85, dd (13.1, 6.0) 3.13, dd (18.8, 13.1) 2.71, dd (18.8, 6.0)

2.87, dd (13.1, 6.0) 3.15, dd (18.8, 13.1) 2.72, dd (18.8, 6.0)

3.03, br d (12.6) 5.75−5.66, overlap

2.83, dd (13.6, 5.4) 3.06, dd (18.7, 13.6) 2.68, dd (18.7, 5.4)

1.78, s 1.45, s 4.15, d (8.6, Ha) 3.60, d (8.6, Hb) 5.24, d (1.5, Ha) 5.22, d (1.5, Hb) 4.56, d (7.8) 3.27, dd (9.1, 7.8) 3.43−3.37, overlap 3.36−3.31, overlap 3.36−3.31, overlap 3.91, dd (12.1, 1.7) 3.72, m

1.78, s 1.49, s 4.21, d (8.6) 3.64, d (8.6) 5.24, d (1.5) 5.22, d (1.5) 4.56, d (7.8) 3.26, dd (9.1, 7.8) 3.42−3.36, overlap 3.36−3.30, overlap 3.36−3.30, overlap 3.90, dd (11.8, 1.6) 3.70, dd (11.9, 5.4)

1.73, s 1.29, s 4.01, d (8.6) 3.54, d (8.6) 5.28, d (1.8) 5.18, d (1.8) 4.55, d (7.8) 3.26, dd (9.1, 7.8) 3.42−3.36, overlap 3.36−3.29, overlap 3.36−3.29, overlap 3.90, dd (11.8, 1.5) 3.71, dd (11.8, 5.3)

1.75, s 1.31, s 4.03, d (8.4) 3.52, d (8.4) 5.26−5.24, overlap 5.26−5.24, overlap 4.56, d (7.8) 3.28, dd (9.1, 7.8) 3.44−3.37, overlap 3.37−3.32, overlap 3.37−3.32, overlap 3.94−3.89, overlap 3.75−3.69, overlap

7.07, m

8.17−8.13, overlap

1.87, dd (7.1, 1.3) 1.91, s

7.56−7.51, overlap 7.66, m 7.56−7.51, overlap 8.17−8.13, overlap

16 18 19 20a 20b 21 Glc-1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

diameter) were punched, excluding the main vein. Six leaf disks were randomly selected, floated on the quassinoid solution in a Petri dish, and placed in a climate chamber at 25 °C. Leaf disks from the healthy or TMV-infected tobacco leaves, which were treated with a solution of 1% DMSO in 0.01 M PBS, were used as negative and positive controls, respectively. After 48 h, the leaf disks were used for the indirect enzyme-linked immunosorbent assay (ELISA) or western blot analysis.5,28,29 Indirect ELISA. Leaf disks were ground in carbonate coating buffer (0.01 M, pH 9.6, 500 μL per leaf disk) and centrifuged. The supernatants were used for indirect ELISA to determine the OD405 values.30,31 Meanwhile, a series of purified TMVs with five different diluted concentrations varying from 0.01 to 0.1 μg/mL were prepared for indirect ELISA, and a standard curve was conducted to indicate the relationship between the virus concentration and corresponding OD405 values. The virus concentrations in the TMV-infected leaf disks were calculated using the standard curve, and the inhibition rate of quassinoids against TMV was calculated as follows: inhibition rate (%) = [1 − (virus concentration of the treatment)/(virus concentration of the positive control)] × 100. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS−PAGE) and Western Blot Analysis. TMV-infected leaf disks treated with different concentrations of selected quassinoids were

br d (13.0) dt (14.9, 2.8) m t (2.8)

2.53−2.43, overlap 1.80−1.68, overlap 1.53, m 0.98, t (7.4) 1.18, d (7.0)

extracted for total proteins, which were applied to the western blot immunoassay after SDS−PAGE. SDS−PAGE and western blot were performed as described by Sambrook et al.32,33 Observation of TMV Spread in N. benthamiana. An Agrobacterium carrying a TMV-based expression vector 30B:GFP, available from the Institute of Plant Virology, Fujian Agriculture and Forestry University, was used to investigate the influence of quassinoids on virus spread in the host plant. The engineered Agrobacterium (Agro35S-30B:GFP) was constructed as described.33 Bacterial Agrobacterium (Agro35S-30B:GFP) suspensions mixed with or without different concentrations of ailanthone (16) were infiltrated into leaves of N. benthamiana. Green fluorescence visualized under ultraviolet (UV) light (365 nm, UVP B100AP, Analytik Jena, Upland, CA, U.S.A.) was indicative of the expression of the green fluorescence protein (GFP) and the distribution of TMV in plants.

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RESULTS AND DISCUSSION Bioassay-Guided Isolation. The crude MeOH extract of the samara of A. altissima, which showed potent anti-TMV activity (IC50 of 80.7 ± 3.5 μg/mL) as tested using the leafdisk method, was suspended in H2O and extracted with nhexane, chloroform, and n-BuOH, successively. The CHCl37351


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Figure 2. Chemical structures of compounds 1−18.

Figure 3. Selected HMBCs (arrows) and 1H−1H COSY (bold lines) of compounds 1−6 and 8−10.

and n-BuOH-soluble extracts exhibited significant antiviral activity, with IC50 values of 3.3 ± 0.1 and 28.9 ± 1.1 μg/mL, respectively. However, the n-hexane- and water-soluble extracts were inactive (IC50 > 100 μg/mL). From the CHCl3- and nBuOH-soluble fractions, 18 quassinoids, including nine new quassinoid glycosides (1−6 and 8−10) (Figure 2), were isolated using a combination of column chromatography with silica, RP-18, MCI, and Sephadex LH-20 gels. The structures of nine known quassinoids (Figure 2) were identified by comparing their spectroscopic data to those reported in the literature as shinjuglycoside A (7),22,34 shinjuglycoside B (11),1,22 chaparrinone (12),3,10,11 glaucarubolone (13),34,35 ailanthinone (14),35−37 glaucarubinone (15),11,36,37 ailanthone (16),6−11 Δ13(18)-glaucarubolone (17),38 and dehydroailanthinone (18).7,38 Structure Elucidation of New Quassinoids. Compound 1 was isolated as a colorless crystal, whose molecular formula

was established as C28H40O14 by HR−ESI−MS (m/z, 623.2333 [M + Na]+; calcd for C28H40O14Na, 623.2316). Its IR spectrum (Figure S1 of the Supporting Information) displayed absorption bands, indicating the presence of a hydroxyl (3419 cm−1), δ-lactone (1727 cm−1), and double bond (1660 cm−1). The 1H NMR (Figure S3 of the Supporting Information) and heteronuclear single quantum correlation (HSQC) (Figure S7 of the Supporting Information) spectra of compound 1 displayed signals of an olefinic proton [δH 5.77 (1H, td, J = 3.1 and 1.5 Hz, H-3)], five oxygenated methines [δH 5.44 (1H, dd, J = 12.3 and 2.6 Hz, H-6), 4.59 (1H, d, J = 2.6 Hz, H-7), 4.18 (1H, m, H-2), 3.78 (1H, d, J = 7.9 Hz, H1), and 3.44 (1H, d, J = 4.2 Hz, H-12)], one oxygenated methylene [δH 4.16 and 3.80 (each 1H, d, J = 8.8 Hz, H2-20)], four methines [δH 2.91−2.85 (1H, overlap, H-5), 2.56 (1H, s, H-9), 2.30 (1H, td, J = 6.9 and 4.2 Hz, H-13), 2.07 (1H, dt, J = 12.8 and 6.2 Hz, H-14)], one methylene [δH 2.90 (1H, dd, J = 7352


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Figure 4. Key NOESY of compounds 1−6 and 8−10.

19.1 and 13.5 Hz, H-15) and 2.64 (1H, dd, J = 19.1 and 5.8 Hz, H-15)], four methyl groups [δH 2.14 (3H, s, H3-2″), 1.84 (3H, s, H3-18), 1.44 (3H, s, H3-19), 1.04 (3H, d, J = 7.2 Hz, H3-21)], and signals for a characteristic glucopranosyl moiety [δH 4.55 (1H, d, J = 7.8 Hz, H-1′), 3.91 (1H, dd, J = 11.8 and 1.8 Hz, H-6′), 3.72 (1H, m, H-6′), 3.42−3.37 (1H, overlap, H3′), 3.37−3.30 (2H, overlap, H-4′ and H-5′), 3.27 (1H, dd, J = 9.1 and 7.8 Hz, H-2′)]. The 13C NMR (Figure S4 of the Supporting Information) and distortionless enhancement by polarization transfer (DEPT) (Figure S5 of the Supporting Information) spectra exhibited 28 carbon signals, including two carbonyls, two olefinic carbons, one hemiketal carbon, four methyls, one methylene, nine methines, two quaternary carbons, and six saccharide-type carbons. The above data were similar to those of shinjuglycoside A (7),22 which suggested that compound 1 is an analogue of the C20 quassinoid glycoside derivative. The carbon signals observed at δC 171.9 and 21.4 suggested the existence of an additional acetoxy group in compound 1, which was established by heteronuclear multiple bond correlations (HMBCs) (Figure 3) between H-2″ (δH 2.14, 3H, s) and C-1″ (δC 171.9). The observed HMBCs between H-6 (δH 5.44, 1H, dd, J = 12.3 and 2.6 Hz) and C-1″ as well as HMBCs between H-2″ and C-6 (δC 69.4) indicated that the acetoxy group was attached at the C-6 position. The HMBCs between the anomeric proton H-1′ and C-2 (δC 83.8) and between H-2 and C-1′ (δC 105.4) confirmed that the glucopyranosyl unit was attached at the C-2 position and must be a β-anomer, as suggested by the coupling constant of the anomeric proton. The nuclear Overhauser effect spectroscopy (NOESY) cross peaks (Figure 4) of H-2/ H3-19, H-6/H3-19, H-7/H-14, H-7/Ha-20, H3-19/Ha-20, H13/Hb-20, and H-14/Hb-20 indicated that H-2, H-6, H-7, H13, H-14, and Me-19 were cofacial and assigned as β orientations, while the NOESY of H-1/H-5, H-1/H-9, and H-5/H-9 showed that these protons were α-oriented. The coupling constant of H-12 at δH 3.44 (d, J = 4.2 Hz, 1H) indicated that the hydroxyl group at C-12 was also α-oriented.

Thus, the structure of compound 1 was characterized as 6αacetoxyl-chaparrin-2-O-β-D-glucopyranoside and named as chuglycoside A. Compound 2 was determined to have a molecular formula of C31H44O14 by the [M + Na]+ ion peak appearing at m/z 663.2647 (calcd for C31H44O14Na, 663.2629) in its HR−ESI− MS. The 1H and 13C NMR spectra of compound 2 were consistent with those of compound 1, except for signals assigned to the substituted group connected at C-6. The side chain attached at C-6 in compound 2 was established as a tigloyloxy moiety, which could be deduced from the key HMBCs (Figure 3) observed from H3-4″ to C-2″ and C-3″ and HMBCs from H3-5″ to C-1″. Meanwhile, the HMBCs observed from H-6 [5.58 (1H, dd, J = 12.3 and 2.6 Hz)] to C1″ (δC 168.5) confirmed that the tigloyloxy moiety was attached at C-6. The planar structure of compound 2 was confirmed by the key correlation spectroscopy (COSY) and HMBCs observed, as shown in Figure 3, while the relative configuration was determined on the basis of the NOESY experiment (Figure 4). Compound 2 was established as 6αtigloyloxy-chaparrin-2-O-β-D-glucopyranoside and named as chuglycoside B. Compound 3 was found to possess a molecular formula of C33H42O14 based on the [M + Na]+ ion peak at m/z 685.2483 (calcd for C33H42O14Na, 685.2472) in its HR−ESI−MS. A comparison of the NMR spectra of compound 3 to those of compounds 1 and 2 revealed that it was also a chaparrin-2-Oβ-D-glucopyranoside bearing a substitute group at C-6. The 1H NMR resonances at δH 8.16−8.13 (2H, overlap), 7.65 (1H, m), and 7.56−7.50 (2H, overlap) indicated the presence of a benzoate moiety in compound 3, and it was located at C-6, because the C-6 methine proton at δH 5.77 (1H, dd, J = 12.2 and 2.6 Hz)] was observed to correlate with the carbonyl carbon at C-7″ (δC 167.2) in its HMBC spectra (Figure 3). The NOESY experiment (Figure 4) was conducted to determine the relative configuration. Compound 3 was then 7353


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Journal of Agricultural and Food Chemistry Table 4. Inhibitory Activities of Compounds 1−18 toward the Replication of TMV on N. tabacum cv. K326 IC50 ± SD (μM) compound 1 2 3 4 5 6 7 8 9 ningnanmycin

leaf-disk method 78.48 23.10 44.46 25.32 35.01 92.04 91.10 22.47 34.12 183.31

± ± ± ± ± ± ± ± ± ±

3.22 1.58 1.86 1.52 2.26 3.81 3.15 0.82 1.73 4.26

IC50 ± SD (μM)

half-leaf method

compound

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100

10 11 12 13 14 15 16 17 18 ribavirin

leaf-disk method 18.37 21.90 0.93 5.74 5.04 2.91 0.19 10.38 2.99 255.19

± ± ± ± ± ± ± ± ± ±

1.25 1.51 0.12 0.68 0.45 0.33 0.06 0.75 0.23 4.57

half-leaf method >100 >100 7.35 9.19 8.68 7.92 5.65 26.54 7.71 >100

± ± ± ± ± ± ±

1.15 1.73 1.23 1.30 0.94 3.21 1.32

thus presented as 15β-benzoyloxy-chaparrin-2-O-β-D-glucopyranoside and named as chuglycoside F. Compound 8 had a molecular formula of C31H42O14, as revealed by its HR−ESI−MS data (m/z, 661.2491 [M + Na]+; calcd, 661.2472). Its NMR data were similar to those of compound 2, except that signals indicating a terminal double bond [δH 5.24 and 5.22 (each 1H, d, J = 1.5 Hz, H2-21); δC 146.6 (C-13) and 120.1 (C-21)] were observed in the 1H and 13 C NMR of compound 8. HMBCs observed from H2-21 to C12 and C-14, from H-12 to C-13 and C-21, and from H-14 to C-13 and C-21 confirmed that a terminal double bond was located at C-13. HMBCs and NOESY observed as shown in Figures 3 and 4 confirmed the structure and relative configuration of compound 8, which was established as 6αtigloyloxy-shinjulactone A-2-O-β- D -glucopyranoside and named as chuglycoside G. A comparison of the NMR data of compounds 9−11 to those of compound 8 indicated that they were all quassinoid derivatives with the same skeleton as that of compound 8. Compound 9 possessed a molecular formula of C33H40O14, as determined by its HR−ESI−MS ([M + Na] + m/z, 683.2323; calcd for C33H40O14Na, 683.2316). Signals for a benzoate group [δH 8.17−8.13 (2H, overlap), 7.66 (1H, m), and 7.56− 7.51 (2H, overlap); δC 167.2, 134.8, 131.2, 130.9, 129.8] were observed in 1H and 13C NMR spectra of compound 9, and HMBCs (Figure 3) observed from H-6 (δH 5.77, 1H, dd, J = 12.1 and 2.6 Hz) to C-1″ (δC 167.2) indicated that it was connected to C-6. The structure of compound 9 was then established as 6α-benzoyloxy-shinjulactone A-2-O-β-D-glucopyranoside and named as chuglycoside H. Compound 10 was determined to have the molecular formula C31H44O14 on the basis of HR−ESI−MS ([M + Na]+ m/z, 663.2646; calcd for C31H44O14Na, 663.2629). One terminal methyl triplet [δH 0.98 (3H, t, J = 7.4 Hz, H-4″)] and one secondary methyl doublet [δH 1.18 (3H, d, J = 7.0 Hz, H-5″)] were observed in the 1H NMR spectra of compound 10, which were attributed to a 2-methylbutanoyl attached to C15. These findings were confirmed by COSY (Figure 3) observed between H-2″/H-3″, H-3″/H-4″, and H-2″/H-5″ and HMBCs (Figure 3) observed from H3-4″ to C-2″ and C3″ and HMBCs from H3-5″ to C-1″ and C-2″. Thus, compound 10 was elucidated as 15β-(2-methylbutanoyloxy)shinjulactone A-2-O-β-D-glucopyranoside and named as chuglycoside I. Antiviral Properties of Quassinoids against TMV. The leaf-disk method was employed to test the inhibitory effect of quassinoids against TMV. All of the 18 quassinoids exhibited significant antiviral activities with IC50 < 100 μM and were all

identified as 6α-benzoyloxy-chaparrin-2-O-β-D-glucopyranoside and named as chuglycoside C. Compound 4 was obtained as a colorless crystal, and its molecular formula was determined as C30H44O14 based on its HR−ESI−MS ([M + Na]+ m/z, 651.2642; calcd for C30H44O14Na, 651.2629). A septet at δH 2.62 (1H, hep, J = 7.0 Hz) and two doublets at δH 1.21 (3H, d, J = 7.0 Hz) and 1.20 (3H, d, J = 7.0 Hz) observed in the 1H NMR spectra of compound 4 suggested the presence of an isobutyrate group, which was confirmed by HMBCs (Figure 3) from H-3″ to C1″, C-2″, and C-4″, from H-4″ to C-1″, C-2″, and C-3″, and from H-2″ to C-1″, C-3″, and C-4″. A comparison of the 13C NMR and DEPT data of compound 4 to those of shinjuglycoside A (7) showed that the resonance of a methylene carbon appearing at δC 30.6 (C-15) in compound 7 was not observed in compound 4. Instead, a methine carbon signal was observed at δC 70.9. These findings indicated that compound 4 was a quassinoid glycoside with an isobutyrate group attached at C-15, which was confirmed by HMBCs (Figure 3) observed from the methine proton at δH 5.73 (1H, d, J = 11.3 Hz, H-15) to C-1″ (δC 177.4). Thus, compound 4 was determined as 15β-isobutyryloxy-chaparrin-2-O-β-D-glucopyranoside and named as chuglycoside D. Compound 5 was assigned the molecular formula of C31H46O14 based on HR−ESI−MS ([M + Na]+ m/z, 665.2807; calcd, 665.2785). Its 1H NMR spectrum displayed signals of one terminal [δH 0.98 (3H, t, J = 7.4 Hz, H-4″)] and one secondary methyl [δH 1.18 (3H, d, J = 7.0 Hz, H-5″)] protons, which were assigned to a 2-methylbutanoyl based on the HMBCs (Figure 3) from H-3″ to C-1″, C-2″, C-4″, and C5″, from H-4″ to C-2″ and C-3″, and from H-5″ to C-1″, C-2″, and C-3″. It was attached at C-15, as confirmed by HMBCs (Figure 3) from H-15 to C-1″. The 1H and 13C NMR data of compound 5, except for those attributed to the substitute group connected to C-15, were consistent with those of compound 4. Thus, the structure of compound 5 was determined as 15β-(2-methylbutanoyloxy)-chaparrin-2-O-β-Dglucopyranoside and named as chuglycoside E. Compound 6, with a yield as a white needle crystal, had a molecular formula of C33H42O14 as deduced from HR−ESI− MS ([M + Na]+ m/z, 685.2493; calcd for C33H42O14Na, 685.2472). It has the same molecular formula as that of compound 3, and resonances for a benzoate moiety [δH 8.10− 8.06 (2H, overlap), 7.67 (1H, m), and 7.56−7.51 (2H, overlap)] were also observed in its 1H NMR spectra. However, the HMBCs (Figure 3) observed between δH 5.95 (1H, d, J = 12.1 Hz, H-15) and C-1″ (δC 166.9) revealed that a benzoate group was attached at C-15. The structure of compound 6 was 7354


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Journal of Agricultural and Food Chemistry more effective than the commercial antiviral agents, ninanmycin and ribavirin (Table 4). Six quassinoids, including 12−16 and 18, showed significant antiviral activities, with IC50 values ranging from 0.19 to 5.74 μM. Among the quassinoids obtained, ailanthone (16) exhibited the best antiviral activity, followed by chaparrinone (12), both of which were quassinoids bearing a 3-en-2-one unit. The increased IC50 values from 0.19 μM (16) to 0.93 μM (12) suggested that the saturation of the 13,21 double bond could result in the decline of bioactivity. Compounds 13−15 were all quassinoids sharing the same skeleton as that of chaparrinone (12), while compounds 17 and 18 both had the same skeleton as that of ailanthone (16). A comparison of the IC50 values of compounds 13−15 to that of compound 12 and a comparison of the IC50 values of compounds 17 and 18 to that of compound 16 indicated that the introduction of substitute groups at C-15 resulted in the decrease of antiviral activity. The nine new compounds (chuglycosides A−I, compounds 1− 6 and 8−10) and compounds 7 and 11 (IC50 values ranging from 18.37 to 92.04 μM) were all quassinoids bearing the same O-β-D-glucose unit at C-2. The declined IC50 values from 91.10 μM (7) to 0.93 μM (12), from 35.01 μM (5) to 5.04 μM (14), from 21.90 μM (11) to 0.19 μM (16), and from 18.37 μM (10) to 10.38 μM (18) indicated that the replacement of the 2-keto group with O-β-D-glucose led to a considerable loss of antiviral activities. Meanwhile, the antiviral properties of the quassinoids were also investigated by an alternative half-leaf method. The results (Table 4) revealed that seven quassinoids, including compounds 12−18, exhibited potent antiviral activities, with IC50 values ranging from 5.65 to 26.54 μM, while the antiviral activities of quassinoid 2-O-β-D-glucosides (1−11) declined dramatically, with IC50 values of >100 μM. The results from leaf-disk and half-leaf methods together proved that the more active quassinoids had a ketone group and one double bond within ring A and the addition of a glycosidic linkage at C-2 decreased their bioactivities. Western blot analysis of the TMV coat protein (CP) expression in systemic infection host N. tabacum cv. K326 in the presence of three quassinoids, chaparrinone (12), glaucarubinone (15), and ailanthone (16), of different concentrations were carried out to further investigate their antiviral properties. The results indicated that the accumulation of TMV CP decreased along with the presence of increasing concentrations of ailanthone (16; Figure 5a), chaparrinone (12; Figure 5b), and glaucarubinone (15; Figure 5c), which indicated the dose-dependent inhibition of quassinoids on the accumulation of TMV CP. The Agro35S-30B:GFP suspension containing different concentrations (0.156, 0.313, and 0.625 μM) of ailanthone (16) were used to infect N. benthamiana, and green fluorescence were observed under UV light at 14 days postinfection. When the plants were injected with an Agro35S30B:GFP suspension containing 0.156 μM ailanthone, green fluorescence were visible in both inoculated leaves and upper uninoculated leaves (Figure 6a), which appeared similar to those of the control. When the concentration of ailanthone in the suspension was increased to 0.313 μM, green fluorescence was still visible (Figure 6b); however, the spot area around the inoculated sites was much smaller, and green fluorescence was visible but weak in the upper uninoculated leaves and appeared mainly along the leaf vein. When the concentration of ailanthone in the Agro35S-30B:GFP suspension was increased to 0.625 μM, green fluorescence could hardly be observed

Figure 5. Effects of ailanthone (16), chaparrinone (12), and glaucarubinone (15) on the accumulation of TMV CP in N. tabacum K326. The total protein was extracted from leaf disks infected with TMV pretreated with different concentrations of ailanthone (a, 1.25− 0.078 μM), chaparrinone (b, 5−0.313 μM), and glaucarubinone (c, 20−1.25 μM). Leaf disks from plants inoculated with 2% DMSO in water were used as the negative control (CK−), while leaf disks from plants infected with TMV were used as the positive control (CK+). An equal amount of total protein was electrophoresed in each lane for western blot analysis.

Figure 6. Effects of ailanthone (16) on the spread of TMV in N. benthamiana: (a) plant inoculated with Agro35S-30B:GFP containing 0.156 μM ailanthone, (b) plant inoculated with Agro35S-30B:GFP containing 0.313 μM ailanthone, and (c) plant inoculated with Agro35S-30B:GFP containing 0.625 μM ailanthone. All of the plants were photographed under long-wave UV light at 14 days postinjection. Inoculation sites were marked with arrows. Plants inoculated with Agro35S-30B:GFP were used as the positive control (CK+), while plants injected with 2% DMSO in water were used as the negative control (CK−).

under UV light in either the inoculated leaves or upper uninoculated leaves (Figure 6c). In summary, 18 C20 quassinoids were characterized from the samara of A. altissima. All of the quassinoids obtained were more effective as inhibitors against the replication of TMV than commercial antiviral agents, ningnanmycin and ribavirin. Among these quassinoids, ailanthone (16) and chapparinone (12) showed the best antiviral activity. Quassinoids could inhibit the TMV CP expression and the virus systemic spread in tobacco. The present work revealed that the samara of A. altissima is a natural origin containing an abundance of bioactive quassinoids. Quassinoids from A. altissima, especially ailanthone (16) and chapparinone (12), could be considered as lead structures for antiviral agent design and development. 7355


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(13) Ishibashi, M.; Murae, T.; Hiroshi, H.; Tsuyuji, T.; Takahashi, T.; Itai, A.; Iitaka, Y. Shinjulactone C, a new quassinoid with a 1α,12α:5α,13α-dicyclo-9βH-picrasane skeleton from Ailanthus altissima Swingle. Tetrahedron Lett. 1982, 23, 1205−1206. (14) Furuno, T.; Ishibashi, M.; Naora, H.; Murae, T.; Hirota, H.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y. Structure determination of bitter principles of Ailanthus altissima. Structures of shinjulactones B, D, and E. Bull. Chem. Soc. Jpn. 1984, 57, 2484−2489. (15) Ishibashi, M.; Yoshimura, S.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y.; Matsushita, K. Shinjulactone F, a new bitter principle with a 5βH-picrasane skeleton from Ailanthus altissima Swingle. Chem. Lett. 1984, 13, 555−556. (16) Ishibashi, M.; Yoshimura, S.; Tsuyuki, T.; Takahashi, T.; Matsushita, K. Shinjulactones G and H, new bitter principles of Ailanthus altissima Swingle. Bull. Chem. Soc. Jpn. 1984, 57, 2013− 2014. (17) Ishibashi, M.; Yoshimura, S.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y. Structure determination of bitter principles of Ailanthus altissima. Structures of shinjulactones F, I, J, and K. Bull. Chem. Soc. Jpn. 1984, 57, 2885−2892. (18) Ishibashi, M.; Tsuyuki, T.; Takahashi, T. Structure determination of a new bitter principle, shinjulactone L, from Ailanthus altissima. Bull. Chem. Soc. Jpn. 1985, 58, 2723−2724. (19) Niimi, Y.; Tsuyuki, T.; Takahashi, T.; Matsushita, K. Structure determination of shinjulactones M and N, new bitter principles from Ailanthus altissima Swingle. Bull. Chem. Soc. Jpn. 1986, 59, 1638− 1640. (20) Yang, X. L.; Yuan, Y. L.; Zhang, D. M.; Li, F.; Ye, W. C. Shinjulactone O, a new quassinoid from the root bark of Ailanthus altissima. Nat. Prod. Res. 2014, 28, 1432−1437. (21) Ishibashi, M.; Murae, T.; Hirota, H.; Naora, H.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y. Shinjudilactone, a new bitter principle from Ailanthus altissima Swingle. Chem. Lett. 1981, 10, 1597−1598. (22) Yoshimura, S.; Ishibashi, M.; Tsuyuki, T.; Takahashi, T.; Matsushita, K. Constituents of seeds of Ailanthus altissima Swingle. Isolation and structures of shinjuglycosides A, B, C, and D. Bull. Chem. Soc. Jpn. 1984, 57, 2496−2501. (23) Niimi, Y.; Tsuyuki, T.; Takahashi, T.; Matsushita, K. Bitter principles of Ailanthus altissima Swingle. Structure determination of shinjuglycosides E and F. Chem. Pharm. Bull. 1987, 35, 4302−4306. (24) Kubota, K.; Fukamiya, N.; Hamada, T.; Okano, M.; Tagahara, K.; Lee, K. H. Two new quassinoids, ailantinols A and B, and related compounds from Ailanthus altissima. J. Nat. Prod. 1996, 59, 683−686. (25) Kubota, K.; Fukamiya, N.; Okano, M.; Tagahara, K.; Lee, K. H. Two new quassinoids, ailantinols C and D, from Ailanthus altissima. Bull. Chem. Soc. Jpn. 1996, 69, 3613−3617. (26) Tamura, S.; Fukamiya, N.; Okano, M.; Koyama, J.; Koike, K.; Tokuda, H.; Aoi, W.; Takayasu, J.; Kuchide, M.; Nishino, H. Three new quassinoids, ailantinol E, F, and G, from Ailanthus altissima. Chem. Pharm. Bull. 2003, 51, 385−389. (27) Tamura, S.; Fukamiya, N.; Okano, M.; Koyama, J.; Koike, K. A new quassinoid, ailantinol H, from Ailanthus altissima. Nat. Prod. Res. 2006, 20, 1211−1215. (28) Yan, X. H.; Chen, J.; Di, Y. T.; Fang, X.; Dong, J. H.; Sang, P.; Wang, Y. H.; He, H. P.; Zhang, Z. K.; Hao, X. J. Anti-Tobacco mosaic virus (TMV) quassinoids from Brucea javanica (L.) Merr. J. Agric. Food Chem. 2010, 58, 1572−1577. (29) Chen, J.; Yan, X. H.; Dong, J. H.; Sang, P.; Fang, X.; Di, Y. T.; Zhang, Z. K.; Hao, X. J. Tobacco mosaic virus (TMV) inhibitors from Picrasma quassinoids Benn. J. Agric. Food Chem. 2009, 57, 6590−6595. (30) Shen, J. G.; Zhang, Z. K.; Wu, Z. J.; Ouyang, M. A.; Xie, L. H.; Lin, Q. Y. Antiphytoviral activity of bruceine-D from Brucea javanica seeds. Pest Manage. Sci. 2008, 64, 191−196. (31) Wu, Z. J.; Ouyang, M. A.; Wang, C. Z.; Zhang, Z. K.; Shen, J. G. Anti-Tobacco mosaic virus (TMV) triterpenoid saponins from the leaves of Ilex oblonga. J. Agric. Food Chem. 2007, 55, 1712−1717. (32) Sambrook, J.; Fritsch, E.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b01280. IR, HR−ESI−MS, and NMR spectra of compounds 1−6 and 8−11 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +86-591-83789150. E-mail: tanqingwei@fafu. edu.cn. *Telephone: +86-591-83789365. E-mail: fafuchenqijian@163. com. Author Contributions †

Qing-Wei Tan and Jian-Cheng Ni contributed equally to this work. Funding

This work was supported by the National Natural Science Foundation of China (Grants 31501687 and 31371987) and the Innovation Foundation of Fujian Agricultural and Forestry University (KF2015061). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Kim, H. M.; Kim, S. J.; Kim, H. Y.; Ryu, B.; Kwak, H.; Hur, J.; Choi, J. H.; Jang, D. S. Constituents of the stem barks of Ailanthus altissima and their potential to inhibit LPS-induced nitric oxide production. Bioorg. Med. Chem. Lett. 2015, 25, 1017−1020. (2) Kundu, P.; Laskar, S. A brief resume on the genus Ailanthus: Chemical and pharmacological aspects. Phytochem. Rev. 2010, 9, 379− 412. (3) Wang, Y.; Wang, W. J.; Su, C.; Zhang, D. M.; Xu, L. P.; He, R. R.; Wang, L.; Zhang, J.; Zhang, X. Q.; Ye, W. C. Cytotoxic quassinoids from Ailanthus altissima. Bioorg. Med. Chem. Lett. 2013, 23, 654−657. (4) Dao, T. T.; Tran, T. L.; Kim, J.; Nguyen, P. H.; Lee, E. H.; Park, J.; Jang, I. S.; Oh, W. K. Terpenylated coumarins as SIRT1 activators isolated from Ailanthus altissima. J. Nat. Prod. 2012, 75, 1332−1338. (5) Ni, J. C.; Shi, J. T.; Tan, Q. W.; Chen, Q. J. Phenylpropionamides, piperidine, and phenolic derivatives from the fruit of Ailanthus altissima. Molecules 2017, 22, 2107. (6) Casinovi, C. G.; Ceccherelli, P.; Grandolini, G.; Bellavita, V. On the structure of ailanthone. Tetrahedron Lett. 1964, 5, 3991−3997. (7) Casinovi, C. G.; Ceccherelli, P.; Fardella, G.; Grandolini, G. Isolation and structure of a quassinoid from Ailanthus glandulosa. Phytochemistry 1983, 22, 2871−2873. (8) Naora, H.; Furuno, T.; Ishibashi, M.; Tsuyuki, T.; Takahashi, T.; Itai, A.; Iitaka, Y.; Polonsky, J. On the structure of ailanthone, a bitter principle from Ailanthus altissima. Chem. Lett. 1982, 11, 661−662. (9) Naora, H.; Ishibashi, M.; Furuno, T.; Tsuyuki, T.; Murae, T.; Hirota, H.; Takahashi, T.; Itai, A.; Iitaka, Y. Structure determination of bitter principles in Ailanthus altissima. Structure of shinjulactone A and revised structure of ailanthone. Bull. Chem. Soc. Jpn. 1983, 56, 3694−3698. (10) Polonsky, J.; Fourrey, J. L. Constituents of Ailanthus altissima seeds. Structure of ailanthone. Tetrahedron Lett. 1964, 5, 3983−3990. (11) Polonsky, J.; Baskévitch, Z.; Gottlieb, H. E.; Hagaman, E. W.; Wenkert, E. Carbon-13 nuclear magnetic resonance spectral analysis of quassinoid bitter principles. J. Org. Chem. 1975, 40, 2499−2504. (12) Naora, H.; Ishibashi, M.; Furuno, T.; Tsuyuki, T.; Murae, T.; Hirota, H.; Takahashi, T.; Itai, A.; Iitaka, Y. Structure determination of bitter principles in Ailanthus altissima. Structure of shinjulactone A and revised structure of ailanthone. Bull. Chem. Soc. Jpn. 1983, 56, 3694−3698. 7356


Article

Journal of Agricultural and Food Chemistry (33) Li, Y.; Wang, L.; Li, S.; Chen, X.; Shen, Y.; Zhang, Z.; He, H.; Xu, W.; Shu, Y.; Liang, G.; Fang, R.; Hao, X. Seco-pregnane steroids target the subgenomic RNA of alphavirus-like viruses. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 8083−8088. (34) Chaudhuri, S. K.; Kubo, I. Two quassinoid glucosides from Castela tortuosa. Phytochemistry 1992, 31, 3961−3964. (35) Joshi, B. C.; Pandey, A.; Sharma, R. P.; Khare, A. Quassinoids from Ailanthus excelsa. Phytochemistry 2003, 62, 579−584. (36) Ogura, M.; Cordell, G. A.; Kinghorn, A. D.; Fransworth, N. R. Potential anticancer agents VI. Constituents of A. excelsa. Lloydia 1977, 40, 579−584. (37) Usami, Y.; Nakagawa-Goto, K.; Lang, J. Y.; Kim, Y.; Lai, C. Y.; Goto, M.; Sakurai, N.; Taniguchi, M.; Akiyama, T.; Morris-Natschke, S. L.; Bastow, K. F.; Cragg, G.; Newman, D. J.; Fujitake, M.; Takeya, K.; Hung, M. C.; Lee, E. Y. H. P.; Lee, K. H. Antitumor agent. 282. 2′(R)-O-acetylglaucarubinone, a quassinoid from Odyendyea gabonensis as a potential anti-breast and anti-ovarian cancer agent. J. Nat. Prod. 2010, 73, 1553−1558. (38) Kupchan, S. M.; Lacadie, J. A. Dehydroailanthinone, a new antileukemic quassinoid from Pierreodendron kerstingii. J. Org. Chem. 1975, 40, 654−656.

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Anti-Tobacco Mosaic Virus Quassinoids from Ailanthus altissima (Mill

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