(TMV) Quassinoids from Ailanthus altissima (Mill.) Swingle

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Bioactive Constituents, Metabolites, and Functions

Anti-Tobacco mosaic virus (TMV) 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01280 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Journal of Agricultural and Food Chemistry

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Anti-Tobacco mosaic virus (TMV) Quassinoids from

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Ailanthus altissima (Mill.) Swingle

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Qing-Wei Tan,*,#,†,‡ Jian-Cheng Ni,#,‡ Lu-Ping Zheng,†,‡ Pei-Hua Fang,‡ Jian-Ting

4

Shi,‡ Qi-Jian Chen*,†,‡

5

†

6

Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, P. R. China

7

‡

8

Agriculture and Forestry University, Fuzhou 350002, P. R. China

9

#

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant

Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian

Qing-Wei Tan and Jian-Cheng Ni contribute equally to this article.

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Supporting Information

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ABSTRACT: Quassinoids are bitter constituents characteristic of the family Simaroubaceae.

12

Eighteen C20 quassinoids, including nine new quassinoid glycosides, named as chuglycosides

13

A–I (1–6 and 8–10), were identified from the samara of Ailanthus altissima (Mill.) Swingle. All

14

of the quassinoids showed potent anti-Tobacco mosaic virus (TMV) activity. A preliminary

15

structure–anti-TMV activity relationship of quassinoids was discussed. The effects of three

16

quassinoids, including chaparrinone (12), glaucarubinone (15), and ailanthone (16), on the

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accumulation of TMV coat protein (CP) were studied by Western blot analysis. And ailanthone

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(16) was further investigated for its influence on TMV spread in Nicotiana benthamiana plant.

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KEYWORDS: Tobacco mosaic virus (TMV), Ailanthus altissima (Mill.) Swingle, quassinoids,

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structure-activity relationship (SAR)

ACS Paragon Plus Environment


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INTRODUCTION The genus Ailanthus (Simaroubaceae) is reputed for its medical values, and more than 200

23

compounds of diverse structural patterns have been characterized from Ailanthus plants.1,2

24

Ailanthus altissima (Mill.) Swingle [syn. Ailanthus glandulosa (Desf)], native to China and

25

naturalized in many temperate regions, is one of the most well studied Ailanthus deciduous

26

tree.3,4 Phytochemical investigations into this species have revealed the presence of quassinoids,

27

alkaloids, terpenoids, coumarins, sterols, and phenolics, etc.3–4 In addition, we have recently

28

reported the identification of phenylpropionamides and piperidine derivatives from its methanol

29

samara extract.5 Pharmacological and clinical investigations revealed that natural products from

30

Ailanthus plants, especially quassinoids, the characteristic bitter constituents of Simaroubaceae,

31

are very promising for their medical use. So far, more than 40 quassinoids have been

32

characterized from A. altissima, including ailanthone,6–11 dihydroailanthone,7 amarolide,7

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11-acetyl amarolide,7 ailantholide,10 shinjulactones A–O,12–20 shinjudilactone,21 shinjuglycosides

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A–F,22,23 ailantinols A–H,24–27 altissinols A and B,3 ∆13(18)-dehydroglaucarubinone,24

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∆13(18)-dehydroglaucarubolone,24 chapparin,26 and chapparinone,10 etc. We herein reported the

36

isolation and structure elucidation of eighteen quassnioids, including nine new quassinoid

37

glycosides (1–6 and 8–10), from the samara of A. altissima. All of the quassinoids showed potent

38

inhibitory effects against the replication of Tobacco mosaic virus (TMV).

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MATERIALS AND METHODS


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General Experimental Procedure. Melting points were determined on an INESA SGW X-4

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microscopic melting point apparatus (uncorrected), and optical rotations were measured with an

42

INESA SGW-532 polarimeter (Shanghai INESA Physico-Optical Instrument Co., Ltd, China).

43

IR spectra (KBr) were recorded using a Nicolet iS50 FT-IR spectrometer (Thermo Scientific,

44

USA), HR-ESI-MS data were acquired on an Agilent 6520 Q-TOF mass spectrometer (Agilent

45

Technologies, USA), and NMR spectra were obtained on a Bruker AVANCE III 500

46

spectrometer (Bruker BioSpin, Switzerland) with TMS as the internal standard at the Test Center

47

of Fuzhou University (Fuzhou, China). The absorbance were read at 405 nm with a Multiskan

48

MK3 microplate reader (Thermo Fisher Scientific (China) Co. Ltd, China). Silica gel (200–300

49

and 300–400 mesh, and silica gel H, Qingdao Oceanic Chemical Co., China), Sephadex LH-20

50

(25–100 µm, Pharmacia Fine Chemical Co., Ltd., Sweden), Lichroprep RP-18 gel (40–63 µm,

51

Merck, Germany), and MCI gel CHP-20P (75−150 µm, Mitsubishi Chemical Co., Japan) were

52

used for column chromatography. Thin-layer chromatography (TLC) were carried out with silica

53

gel H and visualized by heating after spraying 5% H2SO4 in EtOH. All solvents used for

54

extraction and separation were of analytical reagent grade.

55

Extraction and Isolation of Quassinoids. The A. altissima samara, collected in Muyang City

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(Jiangsu, China) in 2013 and identified by associate Professor Chun-Mei Huang (College of Life

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Sciences, Fujian Agriculture and Forestry University), was milled and extracted with MeOH to

58

give a crude extract. The MeOH extract was concentrated in vacuo and resuspended in distilled

59

H2O and then partitioned with organic solvents to yield n-hexane, CHCl3, n-BuOH, and



60

water-soluble fractions. From the CHCl3- and n-BuOH-soluble fractions, eighteen quassinoids

61

were isolated by column chromatography using silica gel, Sephadex LH-20, RP-C18 and MCI

62

gel (Figure 1).

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Chuglycoside A (1). Colorless crystal; mp 220–222 ºC; [α]20 +109.1 (c 0.1, MeOH); IR (KBr) D

64

υmax: 3419, 2876, 1727, 1660, 1503, 1436, 1374, 1316, 1237, 1167, 1034, 910 cm–1；HR-ESI-MS

65

m/z 623.2333 [M + Na]+ (calcd for C28H40O14Na 623.2316); 13C NMR (Table 1) and 1H NMR

66

(Table 2).

67

Chuglycoside B (2). White needle crystal; mp 213–215 ºC; [α]20 +116.5 (c 0.1, MeOH); IR D

68

(KBr) υmax: 3411, 2963, 2925, 2884, 1747, 1702, 1648, 1453, 1387, 1254, 1192, 1134, 1080,

69

1034, 955 cm–1；HR-ESI-MS m/z 663.2647 [M + Na]+ (calcd for C31H44O14Na 663.2629); 13C

70

NMR (Table 1) and 1H NMR (Table 2).

71

Chuglycoside C (3). Colorless crystal; mp 218–219 ºC; [α]20 +141.4 (c 0.1, MeOH); IR (KBr) D

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υmax: 3406, 2958, 2888, 1710, 1602, 1453, 1382, 1283, 1254, 1171, 1063, 1134, 1026, 993, 955

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cm–1；HR-ESI-MS m/z 685.2483 [M + Na]+ (calcd for C33H42O14Na 685.2472); 13C NMR (Table

74

1) and 1H NMR (Table 2).

75

Chuglycoside D (4). Colorless crystal; mp 208–210 ºC; [α]20 +62.6 (c 0.1, MeOH); IR (KBr) D

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υmax: 3406, 2967, 2938, 2880, 1739, 1635, 1457, 1378, 1320, 1229, 1192, 1154, 1076, 1051, 914

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cm–1；HR-ESI-MS m/z 651.2642 [M + Na]+ (calcd for C30H44O14Na 651.2629); 13C NMR (Table

78

1) and 1H NMR (Table 2).


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Chuglycoside E (5). White needle crystal; mp 232–233 ºC; [α]20 +58.3 (c 0.1, MeOH); IR D

79 80

(KBr) υmax: 3498, 3423, 3257, 2950, 2876, 2747, 1760, 1731, 1669, 1457, 1378, 1233, 1084,

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1042, 988, 918 cm–1；HR-ESI-MS m/z 665.2807 [M + Na]+ (calcd for C31H46O14Na 665.2785);

82

13

83

C NMR (Table 1) and 1H NMR (Table 2). Chuglycoside F (6). White needle crystal; mp 218–219 ºC; [α]20 +75.0 (c 0.1, MeOH); IR D

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(KBr) υmax: 3402, 2921, 2884, 1731, 1640, 1598, 1382, 1349, 1316, 1233, 1183, 1107, 1076,

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1046, 959 cm–1；HR-ESI-MS m/z 685.2493 [M + Na]+ (calcd for C33H42O14Na 685.2472); 13C

86

NMR (Table 1) and 1H NMR (Table 2).

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Chuglycoside G (8). White needle crystal; mp 216–218 ºC; [α]20 +100.6 (c 0.1, MeOH); IR D

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(KBr) υmax: 3406, 2929, 2888, 1706, 1644, 1441, 1378, 1250, 1192, 1129, 1076, 918 cm–1；

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HR-ESI-MS m/z 661.2491 [M + Na]+ (calcd for C31H42O14Na 661.2472); 13C NMR (Table 1)

90

and 1H NMR (Table 3).

91

Chuglycoside H (9). Colorless crystal; mp 211–213 ºC; [α]20 +110.0 (c 0.01, MeOH); IR D

92

(KBr) υmax: 3406, 2963, 2929, 2892, 1714, 1656, 1449, 1316, 1283, 1246, 1163, 1109, 1063,

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1026 cm–1；HR-ESI-MS m/z 683.2323 [M + Na]+ (calcd for C33H40O14Na 683.2316); 13C NMR

94

(Table 1) and 1H NMR (Table 3).

95

Chuglycoside I (10). White needle crystal; mp 230–232 ºC; [α]20 +43.9 (c 0.1, MeOH); IR D

96

(KBr) υmax: 3411, 2967, 2938, 2884, 1735, 1652, 1457, 1179, 1150, 1051, 914 cm–1；HR-ESI-MS

97

m/z 663.2646 [M + Na]+ (calcd for C31H44O14Na 663.2629); 13C NMR (Table 1) and 1H NMR

98

(Table 3).



99

Anti-Tobacco mosaic virus Assay. Tobacco (N. tabacum cv. K326, N. benthamiana, and N.

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glutinosa L.) with 5−6 fully expanded true leaves were used for experiments. TMV U1 strain

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was available from the Institute of Plant Virology, Fujian Agriculture and Forestry University,

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and the virus concentration was determined as described.5,28,29 Virus infection was achieved by

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rubbing leaves pre-dusted with silicon carbide (400 mesh) with a solution of TMV (15 µg/mL) in

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0.01 M phosphate buffer saline (PBS). The quassinoids were dissolved to 10 mM in DMSO and

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diluted to the required concentration with 0.01 M PBS before use. Two commercial agents used

106

as control were ningnanmycin and ribavirin.

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Local-lesion Method. Three of the top fully expanded leaves of N. glutinosa L. were used for

108

TMV infection. A mixture of quassinoid and TMV was mechanically inoculated on one half of

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the leaves, while the other half was used as control, which was treated with a mixture of TMV

110

and 1% DMSO. Local lesions formed were counted 3 or 4 days after inoculation. The anti-TMV

111

activity of quassinoids were evaluated through their inhibition rate, which were calculated as

112

follows: inhibition rate (%) = [1 − (lesions number of treatment)/(lesions number of positive

113

control)] × 100.

114

Leaf-disk Method. The whole fully expanded N. tabacum cv. K326 leaf was inoculated and

115

infected with TMV. Six hours later, leaf-disks (1 cm diameter) were punched excluding the main

116

vein. Six leaf-disks were random selected and floated on the quassinoids solution in Petri dish

117

and placed in a climate chamber at 25 ºC. Leaf-disks from the healthy or TMV infected tobacco

118

leaves, which were treated with a solution of 1% DMSO in 0.01 M PBS was used as negative


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and positive control. After 48 h, the leaf-disks were used for indirect enzyme-linked

120

immunosorbent assay (ELISA) or Western blot analysis.5,28,29

121

Indirect ELISA. Leaf disks were ground in carbonate coating buffer (0.01 M, pH 9.6, 500 µL

122

per leaf-disk) and centrifuged. The supernatants were used for indirect ELISA to determine the

123

OD405 values.30,31 Meanwhile, a series of purified TMV with five different diluted concentrations

124

varying from 0.01 to 0.1 µg/mL were prepared for indirect ELISA, and a standard curve was

125

conducted to indicated the relationship between the virus concentration and corresponding OD405

126

values. The virus concentrations in the TMV infected leaf disks were calculated using the

127

standard curve and inhibition rate of quassinoids against TMV were calculated as follows:

128

inhibition rate (%) = [1 − (virus concentration of the treatment)/(virus concentration of the

129

positive control)] × 100.

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SDS-PAGE and Western Blot Analysis. TMV infected leaf-disks treated with different

131

concentrations of selected quassinoids were extracted for total proteins, which were applied to

132

Western blot immunoassay after SDS-PAGE. SDS-PAGE and Western blot were performed as

133

described by Sambrook et al.32,33

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Observe of TMV Spread in N. benthamiana. An Agrobacterium carrying an TMV-based

135

expression vector 30B:GFP, available from the Institute of Plant Virology, Fujian Agriculture

136

and Forestry University, was used to investigate the influence of quassinoids on virus spread in

137

host plant. The engineered Agrobacterium (Agro35S-30B:GFP) was constructed as described.33

138

Bacterial Agrobacterium (Agro35S-30B:GFP) suspensions mixed with or without different



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concentrations of ailanthone (16) were infiltrated into leaves of N. benthamiana. Green

140

fluorescence visualized under UV light (365 nm, UVP B100AP, Analytikjena, USA) were

141

indicative of the expression of green fluorescence protein (GFP) and the distribution of TMV in

142

plants.

143

RESULTS AND DISCUSSION

144

Bioassay Guided Isolation. The crude MeOH extract of the samara of A. altissima, which

145

showed potent anti-TMV activity (IC50 80.7 ± 3.5 µg/mL) as tested using leaf-disk method, was

146

suspended in H2O and extracted with n-hexane, chloroform, and n-BuOH, successively. The

147

CHCl3- and n-BuOH-soluble extracts exhibited significant antiviral activity with IC50 values of

148

3.3 ± 0.1 and 28.9 ± 1.1 µg/mL, respectively. However, the n-hexane- and water-soluble extracts

149

were inactive (IC50 > 100 µg/mL). From the CHCl3- and n-BuOH-soluble fractions, eighteen

150

quassinoids, including nine new quassinoid glycosides (1–6 and 8–10) (Figure 2), were isolated

151

using a combination of column chromatography with silica gel, RP-18, MCI, and Sephadex

152

LH-20 gel. The structures of nine known quassinoids (Figure 2) were identified by comparing

153

their spectroscopic data with those reported in the literatures as shinjuglycoside A (7),22,34

154

shinjuglycoside B (11),1,22 chaparrinone (12),3,10,11 glaucarubolone (13),34,35 ailanthinone (14),35–

155

37

156

dehydroailanthinone (18).7,38

glaucarubinone

(15),11,36,37

ailanthone

(16),6–11

∆13(18)-glaucarubolone

(17),38

and

157

Structure Elucidation of New Quassinoids. Compound 1 was isolated as a colorless crystal,

158

which molecular formula was established as C28H40O14 by HR-ESI-MS (m/z 623.2333 [M + Na]+,


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159

calcd for C28H40O14Na 623.2316). Its IR spectrum (Figure S1) displayed absorption bands

160

indicating the presence of hydroxyl (3419 cm-1), δ-lactone (1727 cm-1), and double bond (1660

161

cm-1). The 1H NMR (Figure S3) and HSQC (Figure S7) spectra of 1 displayed signals of an

162

olefinic proton [δH 5.77 (1H, td, J = 3.1, 1.5 Hz, H-3)], five oxygenated methines [δH 5.44 (1H,

163

dd, J = 12.3, 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

164

Hz, H-1), 3.44 (1H, d, J = 4.2 Hz, H-12)], one oxygenated methylene [δH 4.16 and 3.80 (each 1H,

165

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

166

(1H, td, J = 6.9, 4.2 Hz, H-13), 2.07 (1H, dt, J = 12.8, 6.2 Hz, H-14)], one methylene [δH 2.90

167

(1H, dd, J = 19.1, 13.5 Hz, H-15) and 2.64 (1H, dd, J = 19.1, 5.8 Hz, H-15)], four methyl groups

168

[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)],

169

as well as signals for a characteristic glucopranosyl moiety [δH 4.55 (1H, d, J = 7.8 Hz, H-1′),

170

3.91 (1H, dd, J = 11.8, 1.8 Hz, H-6′), 3.72 (1H, m, H-6′), 3.42–3.37 (1H, overlap, H-3′), 3.37–

171

3.30 (2H, overlap, H-4′ and H-5′), 3.27 (1H, dd, J = 9.1, 7.8 Hz, H-2′)]. The 13C NMR (Figure

172

S4) and DEPT (Figure S5) spectra exhibited 28 carbon signals including two carbonyls, two

173

olefinic carbons, one hemiketal carbon, four methyls, one methylene, nine methines, and two

174

quaternary carbons, as well as six saccharide-type carbons. The above data were similar to those

175

of shinjuglycoside A (7),22 which suggested that 1 is an analogue of C20 quassinoid glycoside

176

derivative. The carbon signals observed at δC 171.9 and 21.4 suggested the existence of an

177

additional acetoxy group in 1, which was established by HMBC correlations (Figure 3) between

178

H-2′′ (δH 2.14, 3H, s) and C-1′′ (δC 171.9). And the observed HMBC correlations between H-6



179

(δH 5.44, 1H, dd, J = 12.3, 2.6 Hz) and C-1′′, as well as correlations between H-2′′ and C-6 (δC

180

69.4) indicated that the acetoxy group was attached at C-6 position. The HMBC correlations

181

between the anomeric proton H-1′ and C-2 (δC 83.8), and between H-2 and C-1′ (δC 105.4)

182

confirmed that the glucopyranosyl unit was attached at the C-2 position, and it must be a

183

β-anomer as suggested by the coupling constant of the anomeric proton. The NOESY

184

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,

185

H-13/Hb-20, and H-14/Hb-20 indicated that H-2, H-6, H-7, H-13, H-14, and Me-19 were cofacial

186

and assigned as β-orientations. While the NOESY correlations of H-1/H-5, H-1/H-9, and

187

H-5/H-9 showed that these protons were α-oriented. The coupling constant of H-12 at δH 3.44 (d,

188

J = 4.2 Hz, 1H) indicated that the hydroxyl group at C-12 was also α-oriented. Thus, the

189

structure of 1 was characterized as 6α-acetoxyl-chaparrin-2-O-β-D-glucopyranoside, and named

190

as chuglycoside A.

191

Compound 2 was determined to have a molecular formula of C31H44O14 by the [M + Na]+ ion

192

peak appearing at m/z 663.2647 (calcd for C31H44O14Na 663.2629) in its HR-ESI-MS. The 1H

193

and

194

substituted group connected at C-6. The side chain attached at C-6 in 2 was established as a

195

tigloyloxy moiety, which could be deduced from the key HMBC correlations (Figure 3) observed

196

from H3-4′′ to C-2′′ and C-3′′, and correlations from H3-5′′ to C-1′′. Meanwhile, the HMBC

197

correlations observed from H-6 [5.58 (1H, dd, J = 12.3, 2.6 Hz)] to C-1′′ (δC 168.5) confirmed

198

that the tigloyloxy moiety was attached at C-6. The planar structure of 2 was confirmed by the

13

C NMR spectra of 2 were consistent with those of 1 except for signals assigned to the


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199

key COSY and HMBC correlations observed as shown in Figure 3, while the relative

200

configuration was determined based on NOESY experiment (Figure 4). Compounds 2 was

201

established as 6α-tigloyloxy-chaparrin-2-O-β-D-glucopyranoside, and named as chuglycoside B.

202

Compound 3 was found to possess a molecular formula of C33H42O14 based on the [M + Na]+

203

ion peak at m/z 685.2483 (calcd for C33H42O14Na 685.2472) in the HR-ESI-MS. Comparison of

204

the NMR spectra of 3 with those of 1 and 2 revealed that it was also a

205

chaparrin-2-O-β-D-glucopyranoside bearing a substitute group at C-6. The 1H NMR resonances

206

at δH 8.16–8.13, (2H, overlap), 7.65 (1H, m), and 7.56–7.50 (2H, overlap) indicated the presence

207

of a benzoate moiety in 3, and it was located at C-6, since the C-6 methine proton at δH 5.77 (1H,

208

dd, J = 12.2, 2.6 Hz)] was observed correlated with the carbonyl carbon at C-7′′ (δC 167.2) in its

209

HMBC spectra (Figure 3). NOESY experiment (Figure 4) was conducted to determine the

210

relative

211

6α-benzoyloxy-chaparrin-2-O-β-D-glucopyranoside, and named as chuglycoside C.

configuration.

Compound

3

was

then

identified

as

212

Compound 4 was obtained as a colorless crystal, and its molecular formula was determined as

213

C30H44O14 based on the HR-ESI-MS ([M + Na]+ m/z 651.2642, calcd for C30H44O14Na 651.2629).

214

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

215

1.20 (3H, d, J = 7.0 Hz) observed in the 1H NMR spectra of 4 suggested the presence of an

216

isobutyrate group, which was confirmed by HMBC correlations (Figure 3) from H-3′′ to C-1′′,

217

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

218

comparison of the 13C NMR and DEPT data of 4 with those of shinjuglycoside A (7) showed that



Page 12 of 38

219

the resonance of a methylene carbon appearing at δC 30.6 (C-15) in 7 was not observed in 4.

220

Instead, a methine carbon signal was observed at δC 70.9. These findings indicated 4 was a

221

quassinoid glycoside with an isobutyrate group attached at C-15, which was confirmed by

222

HMBC correlations (Figure 3) observed from the methine proton at δH 5.73 (1H, d, J = 11.3 Hz,

223

H-15)

224

15β-isobutyryloxy-chaparrin-2-O-β-D-glucopyranoside, and named as chuglycoside D.

to

C-1′′

(δC

177.4).

Thus,

compound

4

was

determined

as

225

Compound 5 was assigned the molecular formula of C31H46O14 based on HR-ESI-MS ([M +

226

Na]+ m/z 665.2807, calcd for 665.2785). Its 1H NMR spectrum displayed signals of one terminal

227

[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′′)]

228

protons, which was assigned to a 2-methylbutanoyl based on the HMBC correlations (Figure 3)

229

from H-3′′ to C-1′′, C-2′′, C-4′′, and C-5′′, from H-4′′ to C-2′′ and C-3′′, as well as from H-5′′ to

230

C-1′′, C-2′′, and C-3′′. And it was attached at C-15 as confirmed by HMBC correlations (Figure

231

3) from H-15 to C-1′′. The 1H and 13C NMR data of 5 except for those attributed to substitute

232

group connected to C-15 were in consistent with those of 4. Thus, the structure of 5 was

233

determined as 15β-(2-methylbutanoyloxy)-chaparrin-2-O-β-D-glucopyranoside, and named as

234

chuglycoside E.

235

Compound 6, yield as a white needle crystal, had a molecular formula C33H42O14 as deduced

236

from HR-ESI-MS ([M + Na]+ m /z 685.2493, calcd for C33H42O14Na 685.2472). It has the same

237

molecular formula with that of 3, and resonances for a benzoate moiety [δH 8.10–8.06 (2H,

238

overlap), 7.67 (1H, m), and 7.56–7.51 (2H, overlap)] were also observed in its 1H NMR spectra.


Page 13 of 38


239

However, the HMBC correlations (Figure 3) observed between δH 5.95 (1H, d, J = 12.1 Hz,

240

H-15) and C-1′′ (δC 166.9) revealed that a benzoate group was attached at C-15. The structure of

241

6 was thus presented as 15β-benzoyloxy-chaparrin-2-O-β-D-glucopyranoside, and named as

242

chuglycoside F.

243

Compound 8 had a molecular formula of C31H42O14, as revealed by its HR-ESI-MS data (m/z

244

661.2491 [M + Na]+, calcd for 661.2472). Its NMR data were similar to those of 2, except that

245

signals indicating of a terminal double bond [δH 5.24 and 5.22 (each 1H, d, J = 1.5 Hz, H2-21);

246

δC 146.6 (C-13) and 120.1 (C-21)] were observed in the 1H and 13C NMR of 8. And HMBC

247

correlations observed from H2-21 to C-12 and C-14, from H-12 to C-13 and C-21, and from

248

H-14 to C-13 and C-21 confirmed that a terminal double bond was located at C-13. HMBC and

249

NOESY correlations observed as shown in Figure 3 and 4 confirmed the structure and relative

250

configuration of 8, which was established as 6α-tigloyloxy-shinjulactone

251

A-2-O-β-D-glucopyranoside, and named as chuglycoside G.

252

Comparison of the NMR data of compounds 9–11 with those of 8 indicated that they were all

253

quassinoid derivatives with the same skeleton as that of 8. Compound 9 possessed a molecular

254

formula of C33H40O14, as determined by the HR-ESI-MS ([M + Na] + m /z 683.2323, calcd for

255

C33H40O14Na 683.2316). Signals for a benzoate group [δH 8.17–8.13 (2H, overlap), 7.66 (1H, m),

256

and 7.56–7.51 (2H, overlap); δC 167.2, 134.8, 131.2, 130.9, 129.8] were observed in 1H and 13C

257

NMR spectra of 9, and HMBC correlations (Figure 3) observed from H-6 (δH 5.77, 1H, dd, J =

258

12.1, 2.6 Hz) to C-1′′ (δC 167.2) indicated that it was connected to C-6. The structure of 9 was



259

then established as 6α-benzoyloxy-shinjulactone A-2-O-β-D-glucopyranoside, and named as

260

chuglycoside H.

261

Compound 10 was determined to have the molecular formula C31H44O14 on the basis of

262

HR-ESI-MS ([M + Na]+ m /z 663.2646, calcd for C31H44O14Na 663.2629). One terminal methyl

263

triplet [δH 0.98 (3H, t, J = 7.4 Hz, H-4′′)] and one secondary methyl doublet [δH 1.18 (3H, d, J =

264

7.0 Hz, H-5′′)] were observed in the 1H NMR spectra of 10, which were attributed to a

265

2-methylbutanoyl attached to C-15. These findings were confirmed by COSY correlations

266

(Figure 3) observed between H-2′′/H-3′′, H-3′′/H-4′′, and between H-2′′/H-5′′, as well as HMBC

267

correlations (Figure 3) observed from H3-4′′ to C-2′′ and C-3′′, and correlations from H3-5′′ to

268

C-1′′ and C-2′′. Thus, compound 10 was elucidated as 15β-(2-methylbutanoyloxy)-shinjulactone

269

A-2-O-β-D-glucopyranoside, and named as chuglycoside I.

270

Antiviral Properties of Quassinoids against TMV. Leaf-disk method was employed to test

271

the inhibitory effect of quassinoids against TMV. All of the eighteen quassinoids exhibited

272

significant antiviral activities with IC50 < 100 µM, and were all more effective than the

273

commercial antiviral agents, ninanmycin and ribavirin (Table 4). Six quassinoids including 12–

274

16 and 18 showed significant antiviral activities with IC50 values ranging from 0.19 to 5.74 µM.

275

Among the quassinoids obtained, ailanthone (16) exhibited the best antiviral activity, followed

276

by chaparrinone (12), both of which were quassinoids bearing a 3-en-2-one unit. The increased

277

IC50 values from 0.19 (16) to 0.93 µM (12) suggested that the saturation of the 13,21-double

278

bond could result in the decline of bioactivity. Compounds 13–15 were all quassinoids sharing


Page 14 of 38

Page 15 of 38


279

the same skeleton with that of chaparrinone (12), while 17 and 18 both had the same skeleton

280

with that of ailanthone (16). A comparison of the IC50 values of 13–15 with that of 12, as well as

281

a comparison of the IC50 values of 17 and 18 with that of 16, respectively, indicated that the

282

introduction of substitute groups at C-15 resulted in the decrease of antiviral activity. The nine

283

new compounds (chuglycosides A–I, 1–6 and 8–10), as well as 7 and 11 (IC50 values ranging

284

from 18.37 to 92.04 µM) were all quassinoids bearing the same O-β-D-glucose unit at C-2. The

285

declined IC50 values from 91.10 (7) to 0.93 (12), from 35.01 (5) to 5.04 (14), from 21.90 (11) to

286

0.19 (16), and from 18.37 (10) to 10.38 (18) µM, respectively, indicated that the replacement of

287

2-keto group with O-β-D-glucose led to considerable loss of antiviral activities. Meanwhile, the

288

antiviral properties of the quassinoids were also investigated by an alternative half-leaf method.

289

The results (Table 4) revealed that seven quassinoids including 12–18 exhibited potent antiviral

290

activities with IC50 values ranging from 5.65 to 26.54 µM, while the antiviral activities of

291

quassinoid 2-O-β-D-glucosides (1–11) declined dramatically with IC50 values >100 µM. The

292

results from leaf-disk and half-leaf methods together proved that the more active quassinoids had

293

a ketone group and one double bond within ring A, and the addition of a glycosidic linkage at

294

C-2 decreased their bioactivities.

295

Western blot analysis of the TMV coat protein (CP) expression in systemic infection host N.

296

tabacum cv. K326 in the presence of three quassinoids, chaparrinone (12), glaucarubinone (15),

297

and ailanthone (16), of different concentrations were carried out to further investigate their

298

antiviral property. The results indicated that the accumulation of TMV CP decreased along with



299

the presence of increasing concentrations of ailanthone (16, Figure 5a), chaparrinone (12, Figure

300

5b), and glaucarubinone (15, Figure 5c), respectively, which indicated the dose-dependent

301

inhibition of quassinoids on the accumulation of TMV CP.

302

The Agro35S-30B:GFP suspension containing different concentrations (0.156, 0.313, and

303

0.625 µM) of ailanthone (16) were used to infect N. benthamiana, and green fluorescence were

304

observed under UV light at 14 days postinfection. When the plants were injected with an

305

Agro35S-30B:GFP suspension containing 0.156 µM ailanthone, green fluorescence were visible

306

both in inoculated leaves and the upper uninoculated leaves (Figure 6a), which appeared similar

307

to those of control. When the concentration of ailanthone in the suspension was increased to

308

0.313 µM, green fluorescence was still visible (Figure 6b), however, the spots area around the

309

inoculated sites were much smaller, and green fluorescence was visible but weak in the upper

310

uninoculated leaves and appeared mainly along the leaf vein. And when the concentration of

311

ailanthone in the Agro35S-30B:GFP suspension was increased to 0.625 µM, green fluorescence

312

could hardly be observed under UV light in either the inoculated leaves or the upper

313

uninoculated leaves (Figure 6c).

314

In summary, eighteen C20 quassinoids were characterized from the samara of A. altissima. All

315

of the quassinoids obtained were more effective as inhibitors against the replication of TMV than

316

commercial antiviral agents, ningnanmycin and ribavirin. Among these quassinoids, ailanthone

317

(16) and chapparinone (12) showed the best antiviral activity. And quassinoids could inhibit the

318

TMV CP expression and the virus systemic spread in tobacco. The present work revealed that the


Page 16 of 38

Page 17 of 38


319

samara of A. altissima is a natural origin containing an abundance of bioactive quassinoids. And,

320

quassinoids from A. altissima, especially ailanthone (16) and chapparinone (12), could be

321

considered as lead structures for antiviral agent design and development.

322

Supporting Information

323

IR, HR-ESI-MS, and NMR spectra of 1–6 and 8–11 (PDF)

324

Corresponding Author

325

*Phone: +86 591 83789365. E-mail: [email protected] or [email protected].

326

Funding

327

This work was supported by the National Natural Science Foundation of China (grant numbers

328

31501687 and 31371987) and the Innovation Foundation of Fujian Agricultural and Forestry

329

University (KF2015061).

330

Notes

331

The authors declare no competing financial interest.

332

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333

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Page 24 of 38

Table 1. 13C NMR Data for Compounds 1–11 in CD3OD (125 MHz) Position 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''

1 82.6, CH 83.8, CH 127.9, CH 137.1, C 44.9, CH 69.4, CH 80.6, CH 47.8, C 43.5, CH 45.5, C 110.9, C 80.3, CH 32.3, CH 42.8, CH 30.6, CH2 172.8, C 24.6, CH3 11.3, CH3 71.6, CH2 12.9, CH3 105.4, CH 75.5, CH 78.0, CH 71.5, CH 77.9, CH 62.7, CH2 171.9, C 21.4, CH3 – – – – –

2 82.7, CH 83.9, CH 127.7, CH 137.3, C 45.0, CH 69.2, CH 80.9, CH 47.9, C 43.6, CH 45.6, C 110.9, C 80.3, CH 32.3, CH 42.8, CH 30.6, CH2 172.9, C 24.5, CH3 11.4, CH3 71.7, CH2 12.9, CH3 105.4, CH 75.5, CH 78.1, CH 71.5, CH 78.0, CH 62.7, CH2 168.5, C 129.7, C 140.6, CH 14.5, CH3 12.1, CH3 – –

3 82.7, CH 83.8, CH 127.9, CH 137.1, C 45.1, CH 69.8, CH 80.9, CH 48.0, C 43.6, CH 45.7, C 110.9, C 80.3, CH 32.3, CH 42.8, CH 30.6, CH2 172.9, C 24.6, CH3 11.4, CH3 71.7, CH2 12.9, CH3 105.4, CH 75.6, CH 78.1, CH 71.5, CH 78.0, CH 62.7, CH2 167.2, C 131.2, C 130.9, CH 129.7, CH 134.7, CH 129.7, CH 130.9, CH

4 82.6, CH 84.2, CH 125.0, CH 137.4, C 42.0, CH 26.3, CH2 80.0, CH 48.8, C 45.8, CH 42.5, C 110.8, C 80.4, CH 33.5, CH 46.4, CH 70.9, CH 170.1, C 21.3, CH3 10.7, CH3 72.3, CH2 15.4, CH3 105.7, CH 75.6, CH 78.0, CH 71.5, CH 77.9, CH 62.7, CH2 177.4, C 35.3, CH 19.2, CH3 18.9, CH3 – – –

5 82.6, CH 84.2, CH 125.0, CH 137.3, C 42.0, CH 26.3, CH2 80.0, CH 48.8, C 45.8, CH 42.5, C 110.8, C 80.3, CH 33.5, CH 46.5, CH 70.8, CH 170.0, C 21.3, CH3 10.7, CH3 72.3, CH2 15.4, CH3 105.7, CH 75.6, CH 78.0, CH 71.5, CH 77.9, CH 62.7, CH2 177.1, C 42.3, CH 27.8, CH2 11.8, CH3 16.5, CH3 – –

6 82.6, CH 84.2, CH 125.0, CH 137.4, C 42.0, CH 26.3, CH2 80.2, CH 48.9, C 45.9, CH 42.6, C 110.8, C 80.4, CH 33.5, CH 46.4, CH 71.5, CH 170.0, C 21.3, CH3 10.7, CH3 72.3, CH2 15.4, CH3 105.7, CH 75.6, CH 78.0, CH 71.6, CH 78.0, CH 62.7, CH2 166.9, C 130.9, C 130.8, CH 129.7, CH 134.7, CH 129.7, CH 130.8, CH

7 82.6, CH 84.2, CH 125.0, CH 137.4, C 42.3, CH 26.5, CH2 80.5, CH 47.1, C 43.0, CH 45.0, C 110.7, C 80.1, CH 32.4, CH 42.3, CH 30.6, CH2 173.8, C 21.4, CH3 10.4, CH3 72.4, CH2 13.0, CH3 105.6, CH 75.6, CH 78.0, CH 71.5, CH 77.9, CH 62.7, CH2 – – – – – – –

435


8 82.6, CH 83.7, CH 127.8, CH 137.1, C 45.0, CH 69.1, CH 80.7, CH 47.3, C 43.8, CH 45.7, C 110.5, C 81.2, CH 146.6, C 47.9, CH 35.2, CH2 171.7, C 24.6, CH3 11.3, CH3 72.4, CH2 120.1, CH2 105.3, CH 75.6, CH 78.1, CH 71.5, CH 78.0, CH 62.7, CH2 168.4, C 129.7, C 140.6, CH 14.6, CH3 12.1, CH3 – –

9 82.6, CH 83.7, CH 128.0, CH 136.9, C 45.1, CH 69.7, CH 80.7, CH 47.3, C 43.8, CH 45.7, C 110.5, C 81.2, CH 146.6, C 47.9, CH 35.2, CH2 171.7, C 24.7, CH3 11.4, CH3 72.4, CH2 120.1, CH2 105.3, CH 75.6, CH 78.1, CH 71.5, CH 78.0, CH 62.7, CH2 167.2, C 131.2, C 130.9, CH 129.8, CH 134.8, CH 129.8, CH 130.9, CH

10 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 – –

11 82.5, CH 84.0, CH 125.0, CH 137.3, C 42.3, CH 26.4, CH2 80.4, CH 48.0, C 46.5, CH 42.4, C 110.2, C 80.4, CH 146.9, C 48.0, CH 35.4, CH2 172.6, C 21.4, CH3 10.3, CH3 73.2, CH2 120.3, CH2 105.6, CH 75.6, CH 78.0, CH 71.5, CH 78.0, CH 62.7, CH2 – – – – – – –

Page 25 of 38

436


Table 2. 1H NMR Data for Compounds 1–7 in CD3OD (500 MHz) Position 1 2 3 4 5 6

1 3.78, d (7.9) 4.18, m 5.77, td (3.1, 1.5) – 2.91–2.85, overlap 5.44, dd (12.3, 2.6)

2 3.80, d (7.8) 4.19, m 5.76, td (3.1, 1.5) – 2.94, br d (12.3) 5.58, dd (12.3, 2.6)

3 3.82, d (7.9) 4.20, m 5.78–5.75, overlap – 3.06, br d (12.2) 5.77, dd (12.2, 2.6)

7 8 9 10 11 12 13 14 15

1'' 2'' 3''

4.59, d (2.6) – 2.56, s – – 3.44, d (4.2) 2.30, td (6.9, 4.2) 2.07, dt (12.8, 6.2) 2.89, dd (19.1, 13.5) 2.64, dd (19.1, 5.8) – 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 – 2.14, s –

4.54, d (2.6) – 2.58 (s) – – 3.46, d (4.2) 2.31, td (7.0, 4.2) 2.08, dt (12.9, 6.1) 2.91, dd (19.2, 13.5) 2.65, dd (19.2, 5.7) – 1.78, s 1.47, s 4.19, d (8.8) 3.81, d (8.8) 1.04, d (7.1) 4.56, d (7.8) 3.28, dd (9.0, 7.8) 3.43–3.38, overlap 3.37–3.31, overlap 3.37–3.31, overlap 3.92, dd (11.8, 1.8) 3.73, m – – 7.06, m

4'' 5'' 6'' 7''

– – – –

1.87, d (7.1) 1.91, s – –

16 18 19 20a 20b 21 Glc–1' 2' 3' 4' 5' 6'

4.68, d (2.6) – 2.61, s – – 3.46, d (4.2) 2.32, td (7.0, 4.2) 2.10, dt (12.8, 6.1) 2.93, dd (19.1, 13.5) 2.66, dd (19.1, 5.7) – 1.78, s 1.50, s 4.25, d (8.8) 3.84, d (8.8) 1.04, d (7.1) 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 3.90, dd (11.8, 1.6) 3.71, m – – 8.16–8.13, overlap

4 3.66, d (7.9) 4.17, m 5.70, m – 2.43, br d (12.9) 2.06, dt (14.9, 2.8) 1.99, ddd (14.9, 12.9, 2.3) 4.64, t (2.8) – 2.56, s – – 3.43–3.40, overlap 2.40–2.30, overlap 2.40–2.30, overlap 5.73, d, (11.3)

5 3.67, d (7.9) 4.18, m 5.70, td (2.9, 1.5) – 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) – 2.56, s – – 3.43–3.40, overlap 2.39–2.32, overlap 2.39–2.32, overlap 5.75, d (11.2)

6 3.72, d (8.1) 4.20, m 5.72, td (2.9, 1.5) – 2.50, br d (13.1) 2.11, dt (14.9, 3.0) 2.03, ddd (14.9, 13.1, 2.3) 4.73, t (3.0) – 2.66, s – – 3.44, d (3.4) 2.39, m 2.56, dd (12.1, 6.2) 5.95, d (12.1)

– 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 – 2.61, hep (7.0) 1.21, d (7.0)

– 1.72, s 1.31, s 4.04, d (8.9) 3.73, d (8.9) 1.08, d (6.4) 4.54, d (7.8) 3.25, dd (9.1, 7.8) 3.43–3.36, overlap 3.35–3.29, overlap 3.35–3.29, overlap 3.90, dd (11.9, 1.6) 3.73–3.68, 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 – – 8.10–8.06, overlap

7 3.65, d (8.0) 4.18, m 5.71, td (2.8, 1.5) – 2.35–2.26, overlap 2.07, dt (14.7, 2.9) 2.02–1.94, overlap 4.54, overlap – 2.51, s – – 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) – 1.73, s 1.30, s 4.06, d (8.6) 3.69, d (8.6) 1.03, d (7.2) 4.55, d (7.8) 3.27, dd (9.1, 7.8) 3.42–3.36, overlap 3.36–3.30, overlap 3.36–3.30, overlap 3.90, dd (11.9, 1.4) 3.74–3.69, overlap – – –

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

1.20, d (7.0) – – –

7.56–7.51, overlap 7.67, m 7.54, overlap 7.56–7.51, overlap

– – – –

437


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) – –


438

Table 3. 1H NMR Data for Compounds 8–11 in CD3OD (500 MHz) Position 1 2 3 4 5 6

8 3.82, d (7.8) 4.20, m 5.76, td (3.1, 1.6) – 2.99, br d (12.2) 5.57, dd (12.2, 2.6)

9 3.86, d (7.8) 4.21, m 5.78–5.75, overlap – 3.10, br d (12.1) 5.77, dd (12.1, 2.6)

7 8 9 10 11 12 13 14 15

1'' 2'' 3''

4.59, d (2.6) – 2.77, s – – 3.93, s – 2.85, dd (13.1, 6.0) 3.13, dd (18.8, 13.1) 2.71, dd (18.8, 6.0) – 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 – – 7.07, m

4.73, d (2.6) – 2.81, s – – 3.94, s – 2.87, dd (13.1, 6.0) 3.15, dd (18.8, 13.1) 2.72, dd (18.8, 6.0) – 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) –

4'' 5'' 6'' 7''

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'

439

Page 26 of 38

8.17–8.13, overlap

10 3.75, d (8.0) 4.19, m 5.72, td (2.8, 1.5) – 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) – 2.83, s – – 3.90, s – 3.03, br d (12.6) 5.75–5.66, overlap

11 3.71, d (8.0) 4.21, m 5.73, td (2.8, 1.5) – 2.39, br d (13.0) 2.11, dt (14.9, 2.8) 2.00, m 4.60, t (2.8) – 2.71, s – – 3.92, s – 2.83, dd (13.6, 5.4) 3.06, dd (18.7, 13.6) 2.68, dd (18.7, 5.4) – 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 – – –

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

– – – –

Table 4. Inhibitory activities of compounds 1–18 towards the replication of TMV on N. tabacum cv. K326 IC50 ± SD (µM)

IC50 ± SD (µM) Compounds

Compounds Leaf-disk method

Half-leaf method

Leaf-disk method

Half-leaf method

1

78.48 ± 3.22

> 100

10

18.37 ± 1.25

> 100

2

23.10 ± 1.58

> 100

11

21.90 ± 1.51

> 100

3

44.46 ± 1.86

> 100

12

0.93 ± 0.12

7.35 ± 1.15

4

25.32 ± 1.52

> 100

13

5.74 ± 0.68

9.19 ± 1.73

5

35.01 ± 2.26

> 100

14

5.04 ± 0.45

8.68 ± 1.23

6

92.04 ± 3.81

> 100

15

2.91 ± 0.33

7.92 ± 1.30

7

91.10 ± 3.15

> 100

16

0.19 ± 0.06

5.65 ± 0.94

8

22.47 ± 0.82

> 100

17

10.38 ± 0.75

26.54 ± 3.21

9

34.12 ± 1.73

> 100

18

2.99 ± 0.23

7.71 ± 1.32

Ningnanmycin

183.31 ± 4.26

> 100

Ribavirin

255.19 ± 4.57

> 100


Page 27 of 38


440 441

Figure 1. Extraction and isolation of compounds 1−18 from A. altissima samara.

442 443

Figure 2. Chemical structures of compounds 1–18.



444 445

Figure 3. Selected HMBC (→) and 1H-1H COSY (

) correlations of 1–6 and 8–10.

446 447

Figure 4. Key NOESY correlations of 1–6 and 8–10.


Page 28 of 38

Page 29 of 38


448 449

Figure 5. Effects of ailanthone (16), chaparrinone (12), and glaucarubinone (15) on accumulation of TMV CP in N.

450

tabacum K326. Total protein was extracted from leaf-disks infected with TMV pretreated with different

451

concentrations of ailanthone (1.25–0.078 µM, Figure 5a), chaparrinone (5–0.313 µM, Figure 5b), and glaucarubinone

452

(20–1.25 µM, Figure 5c), respectively. Leaf-disks from plants inoculated with 2% DMSO in water were used as

453

negative control (CK−), while leaf-disks from plants infected with TMV were used as positive control (CK+). An

454

equal amount of total protein was electrophoresed in each lane for Western blot analysis.



455 456

Figure 6. Effects of ailanthone (16) on the spread of TMV in N. benthamiana. a) Plant inoculated with

457

Agro35S-30B:GFP containing 0.156 µM ailanthone. b) Plant inoculated with Agro35S-30B:GFP containing 0.313

458

µM ailanthone. c) Plant inoculated with Agro35S-30B:GFP containing 0.625 µM ailanthone. All the plants were

459

photographed under long-wave UV light at 14 days postinjection. Inoculation sites were marked with arrows. Plant

460

inoculated with Agro35S-30B:GFP were used as positive control (CK+), while plants injected with 2% DMSO in

461

water were used as negative control (CK−).

462


Page 30 of 38

Page 31 of 38


463 464

TOC Graphic



Page 32 of 38

Dried A. altissima samara (7.5 kg) Extracted with 25 L MeOH for three times

MeOH extract (390.0 g) Suspended in distilled H2O (2 L) and partitioned with n-hexane, CHCl3, and n-BuOH, successively

n-Hexane-soluble (168.0 g)

CHCl3-soluble (30.0 g)

n-BuOH-soluble (90.0 g)

Water-soluble (102.0 g)

Silica gel (200−300 mesh) column chromatography, eluted with a gradient of MeOH in CHCl3 (0−100%) to give fractions T1−T11

Fraction T6 (972.0 mg)

Fraction T8 (990.0 mg)

14 (30.0 mg) 18 (14.0 mg)

15 (47.0 mg)

Fraction B4 (5.4 g)

Column chromatography 1. MCI gel, eluted with a gradient of MeOH in H2O (15%−100%) 2. RP-C18, eluted with MeOH in H2O (25%)

13 (15.0 mg)

12 (87.0 mg) 16 (181.0 mg)

Fraction B6 (9.3 g)

Column chromatography 1. MCI gel, eluted with a gradient of MeOH in H2O (15%−100%) 2. RP-C18, eluted with MeOH in H2O (30%) 3. Silica gel H, eluted with CHCl3−MeOH (v/v 95:5)

17 (6.5 mg)

Silica gel (300−400 mesh) column chromatography, eluted with CHCl3−MeOH−H2O (v/v/v, 95:5:0, 90:10:0, 80:20:2, 70:30:5, 60:40:10, and 0:100:0) to give fractions B1−B14

Column chromatography 1. RP-C18, eluted with a gradient of MeOH in H2O (35%−100%) 2. Sephadex LH-20, eluted with CHCl3−MeOH (v/v 1:1) 3. Silica gel H, eluted with CHCl3−MeOH (v/v 98:2)

Column chromatography 1. RP-C18, eluted with a gradient of MeOH in H2O (50%−100%) 2. Sephadex LH-20, eluted with CHCl3−MeOH (v/v 1:1) 3. Silica gel H, eluted with CHCl3−MeOH (v/v 98:2)

Column chromatography 1. Sephadex LH-20, eluted with CHCl3−MeOH (v/v 1:1) 2. RP-C18, eluted with a gradient of MeOH in H2O (60%−100%)

Fraction B3 (3.5 g)

Fraction T10 (1.9 g)

Fraction B7 (6.3 g)

Column chromatography 1. MCI gel, eluted with a gradient of MeOH in H2O (15%−100%) 2. RP-C18, eluted with a gradient of MeOH in H2O (30%−60%)

4 (70.2 mg) 5 (1017.0 mg) 10 (226.0 mg)


Fraction B8 (10.1 g)

Column chromatography 1. MCI gel, eluted with a gradient of MeOH in H2O (15%−100%) 2. Silica gel H, eluted with CHCl3−MeOH (v/v 90:10)

1 (20.3 mg) 2 (96.3 mg) 3 (167.1 mg) 6 (16.4 mg) 7 (1019.6 mg) 8 (7.2 mg) 9 (4.6 mg)

Column chromatography 1. MCI gel, eluted with a gradient of MeOH in H2O (5%−100%) 2. RP-C18, eluted with MeOH in H2O (30%)

11 (1031.1mg)

Page 33 of 38


Figure 2. Chemical structures of compounds 1–18. 84x39mm (300 x 300 DPI)



Figure 3. Selected HMBC (→) and 1H-1H COSY (-) correlations of 1–6 and 8–10. 346x201mm (300 x 300 DPI)


Page 34 of 38

Page 35 of 38


Figure 4. Key NOESY correlations of 1–6 and 8–10. 304x151mm (300 x 300 DPI)



Figure 5. Effects of ailanthone (16), chaparrinone (12), and glaucarubinone (15) on accumulation of TMV CP in N. tabacum K326. Total protein was extracted from leaf-disks infected with TMV pretreated with different concentrations of ailanthone (1.25–0.078 µM, Figure 5a), chaparrinone (5–0.313 µM, Figure 5b), and glaucarubinone (20–1.25 µM, Figure 5c), respectively. Leaf-disks from plants inoculated with 2% DMSO in water were used as negative control (CK−), while leaf-disks from plants infected with TMV were used as positive control (CK+). An equal amount of total protein was electrophoresed in each lane for Western blot analysis. 247x120mm (300 x 300 DPI)


Page 36 of 38

Page 37 of 38


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. c) Plant inoculated with Agro35S-30B:GFP containing 0.625 µM ailanthone. All the plants were photographed under long-wave UV light at 14 days postinjection. Inoculation sites were marked with arrows. Plant inoculated with Agro35S-30B:GFP were used as positive control (CK+), while plants injected with 2% DMSO in water were used as negative control (CK−). 251x168mm (300 x 300 DPI)



TOC Graphic. 84x47mm (300 x 300 DPI)


Page 38 of 38

(TMV) Quassinoids from Ailanthus altissima (Mill.) Swingle

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