Capitate Glandular Trichomes of Paragutzlaffia henryi Harbor New

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Capitate Glandular Trichomes of Paragutzlaffia henryi Harbor New Phytotoxic Labdane Diterpenoids Ying Wang, Shi-Hong Luo, Juan Hua, Yan Liu, Shu-Xi Jing, Xiao-Nian Li, and Sheng-Hong Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04113 • Publication Date (Web): 29 Oct 2015 Downloaded from http://pubs.acs.org on November 4, 2015

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Journal of Agricultural and Food Chemistry Submitted to Journal of Agricultural and Food Chemistry

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Capitate Glandular Trichomes of Paragutzlaffia henryi Harbor New Phytotoxic Labdane Diterpenoids Ying Wang,†, ‡ Shi-Hong Luo,† Juan Hua,†, ‡ Yan Liu,† Shu-Xi Jing,† Xiao-Nian Li,† and Sheng-Hong Li*,†

†

State Key Laboratory of Phytochemistry and Plant Resources in West China,

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China ‡

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of

China

Corresponding Author * Tel/Fax: +86-871-65223035. E-mail: [email protected]

ACS Paragon Plus Environment


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1

ABSTRACT

2

The morphology and chemical profile of the capitate glandular trichomes (CGTs) of

3

Paragutzlaffia henryi (Acanthaceae) were investigated. Four new labdane

4

diterpenoids named paraguhenryisins A−D (1−4), together with the known

5

physacoztomatin (5), were localized to the CGTs using laser microdissection coupled

6

with cryogenic 1H NMR and HPLC analyses, and were traced and isolated from the

7

CGTs extract of inflorescences. Their structures were determined by spectroscopic

8

methods and single crystal X-ray diffraction. Bioassays indicated significant

9

inhibitory effect for these diterpenoids on Aradidopsis thaliana seed germination and

10

seedling root elongation. The most potent inhibitor, paraguhenryisin C (3), was

11

interestingly detected in both the rhizosphere soil and water rinsed inflorescences

12

extract of P. henryi but not the roots, with average contents in the rhizosphere soil

13

relevant to its phytotoxic EC50 values. These results suggested that phytotoxic labdane

14

diterpenoids in the CGTs might be released into the environment as a defensive

15

measure for P. henryi against other competitive plants.

16 17

KEYWORDS:

Paragutzlaffia

henryi,

capitate

glandular

18

microdissetion, labdane diterpenoids, phytotoxic activity

19


trichomes,

laser

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INTRODUCTION

21

Plant trichomes (including glandular and non-glandular) are epidermal protuberances

22

on the aerial parts of plant.1 Plant trichomes constitute a key feature in taxonomy but

23

recently also have been gathering the focus of natural product researchers.2 Generally,

24

glandular trichomes (GTs) can be subdivided into capitate glandular trichomes (CGTs)

25

and peltate glandular trichomes (PGTs) based on their respective morphological

26

characteristics. While CGTs typically consist of a basal epidermal cell, few stalk cells,

27

and a few secretory cells at the tip of the stalk, PGTs are comprised of a basal

28

epidermal cell, a short stalk cell and a secretory head, consisting of several secretory

29

cells that are enclosed in a smooth cuticle.3 GTs are involved in the synthesis, storage,

30

and emission of a wide range of secondary metabolites such as terpenoids, alkaloids

31

and phenolics which often increase the plant resistance against insect herbivores,

32

pathogenic bacteria, fungi and other natural enemies.4

33

GTs are present in many important plant families such as Lamiaceae, Asteraceae or

34

Solanaceae.1 For example, the Lamiaceae sage Salvia divinorum produced an array of

35

clerodane diterpenoids in its PGTs, the most abundant of which is salvinorin A.3

36

Peppermint (Mentha piperita, Lamiaceae) accumulated large quantities of

37

monoterpenoids in the subcuticular space of the PGTs.5 The GTs of Tanacetum

38

cinerariifolium (Asteraceae) were found to secrete pyrethrins in a basipetal direction

39

which can reach distant tissues, and protect seedlings against insects and fungi.6 The

40

important antimalarial drug artemisinin was synthesized and stored in the GTs of

41

Artemisia annua which belongs to the Asteraceae family.7-8 High levels of




42

calamenene-type sesquiterpenoids have been isolated from the GTs of Heterolheca

43

subaxillaris (Asteraceae), and reported strong plant growth inhibition of these

44

compounds,9 however the bioactivity of the sesquiterpenoids in the plant rhizosphere

45

soil or surrounding environment remains unknown. Moreover, acyl sugars are stored

46

in GTs of many Solanaceae, including Solanum, Nicotiana, Datura,10 which stick to

47

arthropod cuticles and thereby immobilize or suffocate arthropods.11 Previous studies

48

by our research group on native plants in Yunnan have demonstrated that the PGTs of

49

Colquhounia coccinea var. mollis (Lamiaceae) harbor new classes of unique

50

defensive sesterterpenoids colquhounoids A−C.12 Based on the variety of secondary

51

metabolites produced in GTs and their diverse functions, the investigation of plant

52

families and genera which so far have been scarcely analyzed is an intriguing research

53

area.

54

The Acanthaceae family comprises about 221 genera and 4000 species widely

55

distributed in pantropical and subtropical regions in the world.13 Sesquiterpenoid

56

glycoside has been isolated from GTs exudates of Brillantaisia owariensis

57

(Acanthaceae),14 but the exact type of the GTs and the bioactivity of this secondary

58

metabolite is still unknown. Paragutzlaffia henryi (Figure 1A), also named as

59

Strobilanthes henryi, belongs to the Acanthaceae family. This plant occurs as

60

subshrubs, up to 70 cm tall, which are mainly distributed in southwest of China at

61

altitudes from 1000 to 2800 m, and generally growing on mountain slopes.15 In our

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continuing systematic studies of secondary metabolites from plant GTs, we found that

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P. henryi thrived in many sites with few other plants growing nearby, and the


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inflorescences felt very sticky (Figure 1A). By stereo microscopic analysis, numerous

65

CGTs (Figure 1B and 1C) were for the first time observed to densely cover the

66

inflorescences of P. henryi, while no CGTs were presented on the leaves and stems of

67

the plant.

68

Herein the phytochemical investigation of CGTs of P. henryi were described and

69

the isolation of five labdane diterpenoids (1–5), including four new ones,

70

paraguhenryisins A−D (1–4) were described. The CGTs were precisely collected by

71

LMD, and the main compounds were isolated by extraction of CGTs of inflorescences

72

and classical phytochemical techniques. Bioassay results disclosed significant

73

phytotoxic activies of these compounds against A. thaliana seed germination and root

74

elongation, and compound 3 was the strongest phytotoxin among them. Interestingly,

75

3 was also detected in the rhizosphere soil and the water rinsed inflorescences of P.

76

henryi, but was not present in the roots.

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MATERIALS AND METHODS

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General Experimental Procedures. Optical rotation values were measured on

79

Jasco P-1020 spectropolarimeter (Jasco, Tokyo, Japan). UV spectral data were

80

obtained on a Shimadzu-210A double-beam spectrophotometer (Shimadzu, Tokyo,

81

Japan). CD spectra were obtained on a Chirascan spectropolarimeter (Applied

82

photophysics, Surrey, UK). IR spectra were recorded on a Bruker-Tensor-27

83

spectrometer (Bruker Optics, Ettlingen, Germany) with KBr pellets. NMR

84

experiments were carried out on a Bruker Avance III 600 spectrometer (Bruker,

85

Karlsruhe, Germany) with tetramethyl silane (TMS) as internal standard. Mass spectra




86

were recorded on a Waters Autospec Premier P776 mass spectrometer (Waters Corp.,

87

Massachusetts, USA). Semipreparative HPLC was performed on an Agilent 1200

88

system (Agilent, CA, USA) equipped with a Zorbax SB-C18 (9.4 × 250 mm) column.

89

X-ray diffraction data collection was performed on a Bruker SMART APEX CCD

90

(Bruker, Karlsruhe, Germany) crystallography system. Column chromatography was

91

performed on either 200-300 mesh silica gel (Qingdao Marine Chemical Factory, P. R.

92

China) or Sephadex LH-20 (GE Healthcare Bio-Xciences AB). TLC spots were

93

visualized under UV light and by dipping into 10% H2SO4 in ethanol followed by

94

heating.

95

Plant Material. Leaves, inflorescences, stems, and rhizosphere soil of P. henryi for

96

microscopic study, LMD, and metabolic analysis were collected from the plants (ca.

97

60 cm in height) grown in Kunming Botanical Garden, in July 2013. Inflorescences

98

and roots for HPLC analysis were collected from the same place in October 2015. A.

99

thaliana seeds were Columbia wild ecotype maintained in our laboratory.

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Microscopy. Fresh leaves and inflorescences of P. henryi were examined under a

101

Leica S8 APO light stereo microscope (Leica Microsystems, Wetzlar, Germany) with

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bright-field optics. For scanning electron microscopy, samples were processed

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according to our previously reported protocols,12, 16 and the specimens were observed

104

using a Hitachi S-4800 scanning electron microscope (SEM) (Hitachi Ltd., Tokyo,

105

Japan) at 10.0 KeV accelerating potential. Magnifications ranged from 30× to 500×.

106 107

Laser microdissection of CGTs and metabolic analysis by cryogenic NMR and HPLC.

CGTs

were

microdissected

and

collected


from

freshly-harvested

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inflorescences of P. henryi with a Leica LMD 7000 system (Leica Microsystems,

109

Wetzlar, Germany) using a procedure similar to that we previously described. 12, 16

110

Samples were collected in two microtubes, respectively, each containing

111

approximately 600 CGTs. Both were centrifuged at 4 °C (12,000 g, 10 min) to settle

112

the contents. One sample was directly extracted with acetone-d6 (500 µL,

113

99.9%+0.03% TMS, Cambridge Isotope Laboratories, Inc., USA) by ultrasonication

114

for 10 min. Extract was transferred to a 5 mm NMR tube for 1H NMR measurement

115

on a Bruker Avance III 600 spectrometer equipped with a TCI cryoprobe (5 mm). 1H

116

NMR spectrum was recorded with 128 scans at 276 K and referenced to internal

117

TMS. The residual HDO signal was suppressed by using the PURGE sequence. The

118

other sample was extracted with MeOH (300 µL) by ultrasonication for 10 min.

119

Extract was directly analyzed with HPLC (column: ZORBAX SB-C18, 5 µm, 4.6 ×

120

250 mm; mobile phase: acetonitrile in H2O, gradient 5–95% in 35 min; detection: 202

121

nm; injection volume: 40 µL; column temperature: 30 °C; flow rate: 1 mL/min).

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Fractionation and purification of CGTs metabolites. CGTs were wiped from the

123

fresh inflorescences of P. henryi with acetone-soaked cotton swabs, which were then

124

washed with fresh acetone. The extract were combined and evaporated to dryness to

125

yield a brownish oily residue (750 mg). The residue was subjected to silica gel

126

column chromatography eluting with petroleum ether/acetone (15:1, v/v) to afford

127

two fractions. Fraction A (65 mg) mainly containing compounds 1 and 2 was further

128

chromatographed on a Sephadex LH-20 column eluting with acetone, and was finally

129

purified by reversed-phase semi-preparative HPLC (mobile phase: 70% MeOH in




130

water; flow rate: 3 mL/min, column: ZORBAX SB-C18, 5 µm, 9.4 × 250 mm,

131

detection: UV 202 nm) to yield 1 (t = 16.4 min, 3.1 mg) and 2 (t = 21.5 min, 2.0 mg),

132

respectively. Fraction B (420 mg) mainly containing compounds 3–5 was further

133

separated by silica gel column chromatographies (petroleum ether/EtOAc 8:1, v/v)

134

and then purified on a Sephadex LH-20 column (acetone) to yield 3 (4.5 mg), 4 (7.6

135

mg) and 5 (5.2 mg), respectively.

136

Paraguhenryisin A (1). colorless crystals; [α]D20 −2.4 (c 0.10, MeOH); UV (MeOH)

137

λmax (log ε): 202 (2.18) nm; IR (KBr) νmax: 3441, 2964, 2921, 2852, 1631, 1446, 1384,

138

1112, 836, 562 cm-1; For 1H and

139

EI-MS m/z (%): 323 (35) [M+H]+, 322 (32) [M]+, 307 (37), 304 (38), 204 (93), 161

140

(39), 119 (57), 109 (100), 93 (59), 81 (89), 69 (80), 54 (85); HR-EI-MS: m/z 322.2513

141

[M]+ (m/zcalcd [C20H34O3]+ = 322.2508). Crystal data for 1: C20H34O3, M = 322.47,

142

triclinic, size 1.30 × 0.70 × 0.02 mm3, a = 7.656(4) Å, b = 8.170(5) Å, c = 17.060(10)

143

Å, α = 90.276(9)°, β = 97.280(9)°, γ = 115.602(8)°, V = 952.4(10) Å3, T = 100 (2) K,

144

space group P1, Z = 2, µ(MoKα) = 0.073 mm-1, 12024 reflections measured,

145

measured in the range 1.21° ≤ θ ≤ 28.71°, completeness θmax = 97.3%, 8805

146

independent reflections (Rint = 0.0863). The final R1 values were 0.0985 (I > 2σ(I)).

147

The final wR(F2) values were 0.1958 (I > 2σ(I)). The final R1 values were 0.1961 (all

148

data). The final wR (F2) values were 0.2445 (all data). The goodness of fit on F2 was

149

1.027. Flack parameter = 0.2(19). CCDC 1419393 (1) contains the supplementary

150

crystallographic data for this paper. These data can be obtained free of charge from

151

The

Cambridge

13

C NMR spectroscopic data see Tables 1 and 2;

Crystallographic

Data


Centre

via

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www.ccdc.cam.ac.uk/data_request/cif. The crystal structure of 1 was solved by direct

153

methods with SHELXS-9717 and refined by full-matrix least-squares refinements

154

based on F2 with SHELXL-97.

155

Paraguhenryisin B (2). colorless oil; [α]D18 +2.9 (c 0.30, MeOH); UV (MeOH) λmax

156

(log ε): 202 (2.85) nm; IR (KBr) νmax: 3420, 2935, 2868, 1741, 1632, 1460, 1445,

157

1388, 1372, 1237, 1125, 1084, 1010, 966, 937, 604 cm-1; For 1H and

158

spectroscopic data see Tables 1 and 2; EI-MS m/z (%): 367 (3) [M+H]+, 366 (10)

159

[M]+, 348 (65), 315 (23), 191 (83), 177 (100), 123 (71), 54 (59); HR-EI-MS: m/z

160

366.2763 [M]+ (m/zcalcd [C22H38O4]+ = 366.2770).

13

C NMR

161

Paraguhenryisin C (3). colorless oil; [α]D17 −3.1 (c 0.05, MeOH); UV (MeOH) λmax

162

(log ε ): 202 (3.06) nm; IR (KBr) νmax: 3439, 3434, 2946, 2926, 2868, 1729, 1631,

163

1458, 1422, 1386, 1375, 1247, 1229, 1125, 1041, 1023, 967, 936, 668 cm-1; For 1H

164

and

165

348 (20), 333 (27), 315 (19), 191 (61), 177 (85), 109 (62), 69 (100), 54 (85);

166

HR-EI-MS: m/z 366.2764 [M]+ (m/zcalcd [C22H38O4]+ = 366.2770).

13

C NMR spectroscopic data see Tables 1 and 2; EI-MS m/z (%): 366 (5) [M]+,

167

Paraguhenryisin D (4). colorless oil; [α]D21 +1.7 (c 0.10, MeOH); UV (MeOH) λmax

168

(log ε): 202 (2.46) nm; IR (KBr) νmax: 3441, 2955, 2925, 2853, 1735, 1715, 1631,

169

1458, 1443, 1385, 1367, 1264, 1239, 1096, 1030, 545 cm-1; For 1H and

170

spectroscopic data see Tables 1 and 2; EI-MS m/z (%): 348 (11) [M]+, 149 (31), 134

171

(78), 121 (100), 88 (91), 86 (95), 61 (31); HR-EI-MS: m/z 348.2655 [M]+ (m/zcalcd

172

[C22H36O3]+ = 348.2664).

173

Phytotoxicity Bioassay.


13

C NMR



174

Seed germination bioassay. A. thaliana seeds were washed with ethanol (70%

175

v/v) for 2 min, and then surface sterilized using sodium hypochlorite (0.5% v/v) for 2

176

min, followed by washing with sterile distilled water for three times. The sterilized

177

seeds were placed in a refrigerator at 4 °C for 3 days before use. The CGTs extracts of

178

P. henryi and compounds 3–5 were assayed at 12.5, 25, 50 and 100 µg/mL,

179

respectively. To avoid toxic effects of the organic solvent, the final concentration of

180

MeOH was not exceeding 0.2%. Fifty sterilized seeds were equidistantly sown on

181

Murashige and Skoog medium [0.8% agar (w/v), pH 5.8] supplemented with the test

182

sample at varying concentrations in each Petri dish (6.0 cm diameter). The same

183

volume of MeOH was used as a blank control. Three duplicates of each treatment

184

were carried out. Seeds were allowed to germinate under 16 h light and 8 h dark at 23

185

°C (day) and 18 °C (night). The light intensity in the growth chamber was 100 µmol

186

m-2 s-1. Emergence of the radicle (≥1 mm) was used as an index of germination and

187

the number of germinated seeds were checked daily until most seeds (≥95%) in the

188

control Petri dishes had germinated. Germination counts were performed over a

189

period of four days. The seed germination inhibition rate (IG) was evaluated by the

190

following equation:

191

IG% = (1 −

) × 100

192

NT: Number of germinated seeds at each treatment for the last time measurement;

193

N: Number of seeds used in the bioassay. The effective dose to give 50% inhibitions

194

(EC50) was then determined by probit analysis using SPSS.


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Root elongation bioassay. A. thaliana seeds were pretreated as described above.

196

The CGTs extracts of P. henryi and compounds 3–5 were also assayed at 12.5, 25, 50

197

and 100 µg/mL, respectively, and three replicates were carried out for each

198

concentration. Ten seeds were distributed in a horizontal line on each Petri dish (6.0

199

cm diameter). The Petri dishes were vertically placed in a growth chamber at 16 h

200

light and 8 h dark at 23 °C (day) and 18 °C (night). The light intensity in the growth

201

chamber was 100 µmol m-2 s-1. The length of the seedling roots were measured using

202

a calliper after seven days. The percentage of growth inhibition of root lengths (IR)

203

was calculated from the following equation:

204

IR% = (1 − )×100

205

T: the average length of treatment (cm); C: the average length of control (cm). The

206

EC50 was determined by probit analysis using SPSS.

207

Detection of GTs compounds in the inflorescences and water rinsed

208

inflorescences extract. Freshly harvested inflorescences of P. henryi (3.0 g) were

209

immediately frozen with liquid nitrogen and ground in a mortar to powder, which was

210

then extracted with MeOH (3 × 15 mL) by ultrasonication for 30 min at room

211

temperature, followed by centrifugation for 5 min at 12,000 rpm. The supernatant was

212

filtered and concentrated in vacuo, which was analyzed by HPLC equipped with a

213

diode array detector (mobile phase: 0–35 min: linear gradient of 5−95% of acetonitrile;

214

column: ZORBAX SB-C18, 5 µm, 4.6 × 250 mm; injection: 10 µL; flow rate: 1

215

mL/min; detection: 202 nm). To explore the allelochemicals released from the plant

216

into surroundings by rain, aqueous rinsed solution of P. henryi inflorescences was




217

also analyzed. Ten inflorescences of P. henryi were collected and rinsed with distilled

218

water (100 mL) for 30 s. The extract was filtered and concentrated in vacuo, and was

219

then dissolved in MeOH (1 mL) and analyzed by the same HPLC method as described

220

above.

221

Detection of GTs compounds in rhizosphere soil and roots of P. henryi. The

222

rhizosphere soil of P. henryi (15.0 g) was randomly collected at 0−10 cm depths.

223

Plant residues were carefully removed with a sieve (30 mesh) and the soil samples

224

were then extracted with MeOH (3 × 20 mL) by ultrasonication for 30 min at room

225

temperature, followed by centrifugation for 5 min at 12,000 rpm. The supernatant was

226

filtered and concentrated in vacuo, which was analyzed by the same HPLC method as

227

described above. The roots of P. henryi (0.5 g) were collected and gently washed in

228

tap water to remove the adhering soil particles. Roots samples were immediately

229

frozen with liquid nitrogen and ground in a mortar to powder, which was then

230

extracted with MeOH (3 × 10 mL). The extract was worked up and analyzed by the

231

same HPLC method as mentioned above.

232

Quantification of paraguhenryisin C (3) in rhizosphere soil of P. henryi.

233

Quantification of 3 in the rhizosphere soil of P. henryi was carried out using the same

234

HPLC method, with the isolated authentic sample as external standard. The

235

rhizosphere soil of P. henryi (15.0 g) was collected in triplicate a day for three

236

consecutive days with occasional rain. Samples were also prepared in the same way as

237

described above. For quantification, calibration curve for 3 was prepared. Triplicate

238

injections were carried out at eight concentrations (1, 5, 10, 20, 50, 100, 200 and 500


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µg/mL), followed by plotting the peak area versus concentration to obtain linear

240

calibration curves. The equation and correlation coefficient obtained from the

241

linearity study for 3 was y = 0.0872x−20.841 (R2=0.9945).

242

Statistical analysis. Each treatment was conducted with three replicates, the data

243

for inhibition of seed germination and root elongation were expressed as means ±

244

standard deviation (SD). The statistical analysis of experimental data utilized the

245

SPSS Student's t-test. P < 0.01 was considered significant.

246

RESULTS AND DISCUSSION

247

Morphology and distribution of trichomes. To study the morphology and

248

distribution of the trichomes on P. henryi, slices of the leaves, inflorescences and

249

stems were analyzed with a stereo microscope and SEM. The nonglandular trichomes

250

appear as unicellular pyramids with 100–200 µm length on the leaves, inflorescences

251

and young stems (Figure 1D), and are particularly abundant on the latter. Moreover,

252

both CGTs and PGTs have been found in this plant. The CGTs feature a globular-like

253

storage cavity (20–25 µm in diameter) atop a long stalk (0.25–1.00 mm) (Figure 1E

254

and 1F), and occur abundantly on the inflorescences, but are entirely absent from the

255

young leaves and stems. The PGTs have a storage cavity ( 15–20 µm in diameter),

256

and are embedded into the epidermal cells (Figure 1E and 1F), which are distributed

257

on the inflorescences and are not present on the leaves and stems.

258

LMD of CGTs and secondary metabolites analysis by cryogenic NMR and

259

HPLC. For analyzing secondary metabolites, intact CGTs were carefully selected

260

under a microscope and dissected by using LMD,16 as illustrated in Figure 1G−1I.




261

Thus, two samples were prepared in the caps of 0.5 mL Eppendorf microcentrifuge

262

tubes, with each one containing approximately 600 CGTs. To avoid degradation of

263

secondary metabolites, the dissected samples were stored at − 80 °C immediately after

264

LMD.

265

One of the CGTs samples was analyzed by cryo-1H NMR (600 MHz) in acetone-d6,

266

and the results showed the presence of methyl groups in the high field region between

267

δH 0.84 and 2.06, and olefinic and oxygenated functionalities between δH 3.85 and

268

5.62 (Figure 3A). The other CGTs sample was extracted with 300 µL MeOH by

269

ultrasonication for 10 min. After centrifugation, the supernatant was directly analyzed

270

by reversed-phase HPLC, equipped with a diode array detector recording at 202 nm.

271

Five major peaks (I, II, V, III, IV) with retention times of 22.45, 24.18, 24.74, 26.34

272

and 32.42 min, respectively, were detected in the chromatogram (Figure 2A).

273

Structural elucidation of secondary metabolites. The amount of CGTs collected

274

by LMD was insufficient for direct isolation and identification of these compounds

275

appearing as peaks Ι–V in the chromatograms in Figure 2A. Therefore CGTs extracts

276

were obtained by wiping fresh inflorescences with acetone-soaked cotton swabs. The

277

swabs were gently brushed over the epidermis without impairment of the epidermal

278

surface. Examination of the inflorescences under a microscope ensured that the CGTs

279

had been extracted by this method. The extracts were subjected to silica gel, Sephadex

280

LH-20 column chromatographies and semi-preparative HPLC to afford five labdane

281

diterpenoids (1–5). Through HPLC analysis and comparison of their retention times

282

and UV spectra, the identities of peaks Ι–V in the CGTs of P. henryi were finally


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283

validated and assigned to compounds 1, 2, 5, 3 and 4 in Figure 2A. Meanwhile, the 1H

284

NMR signals of olefinic protons between δH 3.85 and 5.62 in compounds 1‒5 were

285

also found in the cryo-1H NMR spectrum of P. henryi CGTs (Figure 3).

286

Compound 1 was obtained as colorless crystal, and a molecular formula of 13

287

C20H34O3 was deduced from its

C NMR spectroscopic data and (HR) EI-MS (m/z

288

322.2513 [M]+, calcd 322.2508), indicating the presence of four degrees of

289

unsaturation. The IR spectrum showed characteristic absorptions for hydroxyl (3441

290

cm-1) and olefinic (1631 cm-1) groups. In the 1H NMR spectrum (Table 1), five

291

tertiary methyl groups at δH 0.75, 0.85, 0.88, 1.25 and 1.65 (all singlets) were clearly

292

visible. Six protons at δH 2.93 (1H, t, J = 5.4 Hz), 3.29 (1H, ddd, J = 11.4, 5.4, 1.8 Hz),

293

3.61 (1H, m), 3.69 (1H, m), 3.81 (1H, dd, J = 5.3, 1.2 Hz) and 3.90 (1H, d, J = 6.0 Hz)

294

were resonated from either oxygenated methines or oxygenated methylenes or free

295

hydroxyl groups. An olefinic proton at δH 5.37 (1H, br s) indicated the presence of a

296

trisubstituted double bond. Other overlapping proton resonances occurred between

297

1.10 and 2.08 ppm, resulting from either methine or methylene protons. The 13C NMR

298

spectrum (Table 2) indicated 20 carbon resonances, which were further classified by

299

DEPT experiments as five methyls, six methylenes including one oxymethylene (δC

300

61.1), five methines including two oxymethines (δC 61.6 and 77.2) and one olefinic

301

methine (δC 123.0), and four quaternary carbons including one olefinic (δC 136.1) and

302

one oxygenated (δC 64.0). These data were consistent with the resonances observed in

303

the 1H NMR spectrum and suggested the structure of 1 to be an oxygenated labdane

304

diterpenoid18.




305

The 1H NMR spectrum in conjunction with the HSQC spectrum indicated the

306

presence of two hydroxyl groups at δH 3.81 (15-OH) and 3.90 (12-OH), respectively.

307

The 1H-1H COSY correlations demonstrated the former to be at C-15 (δC 61.1) the

308

latter to be at C-12 (δC 77.2) which was also confirmed by the HMBC correlations

309

(Figure 5A) from δH 3.90 to the carbons at δC 31.5 (C-11), 64.0 (C-13) and δC 77.2

310

(C-12). Furthermore, the HMBC correlations from Me-17 (δH 1.65) to the olefinic

311

carbons at δC 123.0 (C-7) and 136.1 (C-8) and the methine carbon at δC 50.6 (C-9),

312

suggested the presence of a double bond between C-7 and C-8. The molecular

313

formula of 1, corresponding to four degrees of unsaturation, together with the

314

consideration of the remaining oxygen atom in the molecular formula and the

315

presence of an oxymethine carbon at δC 64.0 (C-13) and an oxygenated quaternary

316

carbon at δC 61.6 (C-14), implied the existence of an oxirane moiety. The HMBC

317

correlations from H-12 (δH 3.29), H2-15 (δH 3.61 and 3.69) and Me-16 (δH 1.25) to the

318

carbons at δC 64.0 and 61.6 further indicated the occurrence of an oxirane between

319

C-13 and C-14. The planar structure of 1 was thus identified as shown in Figure 4.

320

In the ROESY spectrum of 1, the correlations of H-9 with H-5 and H-12, of Me-20

321

with Me-19, and of Me-18 with H-5, indicated that Me-18, H-12, H-9 and H-5 were

322

α-oriented while Me-20 and Me-19 were β-oriented, as expected with the labdane

323

skeleton.18 However, the configurations at C-12, C-13 and C-14 were difficult to

324

determine with ROESY method due to the free rotation of the C-9 side chain and lack

325

of any proton at C-13. Therefore, a single crystal of 1 was obtained by crystallization

326

from a mixture of MeOH/water (5:1), and X-ray crystallographic analysis with


Page 17 of 40


page 17 of 40

327

molybdenum radiation was carried out. The result unambiguously established the

328

complete structure of 1 including the configurations of C-12, C-13 and C-14.

329

Accordingly, compound 1 was characterized as shown in Figure 5B, and was named

330

paraguhenryisin A.

331

Compound 2 was obtained as colorless oil. According to the (HR) EI-MS (m/z

332

366.2763 [M]+, calcd 366.2770) and

13

333

molecular formula of C22H38O4 was deduced. Comparison of the 1H and

334

spectral data of 2 with those of 1 (Tables 1 and 2) clearly revealed that the structure of

335

2 was also a labdane diterpenoid, similar to that of 1. All proton signals, except for the

336

ones at δH 2.94 and 3.61 could be unambiguously assigned to their respective carbons

337

by analysis of the HSQC spectrum, suggesting that these two signals were ascribed to

338

free hydroxyl groups. The HMBC correlations from the former to four carbons at δC

339

45.3 (C-7), 73.5 (C-8), 62.2 (C-9) and 24.4 (C-17), and from the latter to two carbons

340

at δC 130.6 (C-14) and 58.5 (C-15), indicated that the two hydroxy groups occurred at

341

C-8 and C-15, respectively. The HMBC correlations from the signal at δH 5.59 (H-14)

342

to the carbons at δC 39.2 (C-12) and 62.1 (C-16) indicated the presence of a double

343

bond between C-13 and C-14. In addition, the simultaneous HMBC correlations from

344

the methyl protons at δH 1.98 and the oxymethylene protons at δH 4.61 and 4.63 to the

345

carbonyl carbon at δC 170.9 indicated the existence of an acetoxy group that was

346

connected to C-16 (Fig. S13).

C NMR and DEPT spectra (Table 2) a 13

C NMR

347

The ROESY spectrum of 2 showed a similar correlation pattern as that of 1,

348

suggesting that the relative configurations of the chiral centers in 2 remain unchanged.




349

The ROE correlations of Me-17 with Me-19 and Me-20 indicated that Me-17 was

350

β-oriented and 8-OH α-oriented. Correlations between H2-12 and H-14, and between

351

H2-15 and H2-16 indicated a Z-configuration for the double bond between C-13 and

352

C-14. Accordingly, compound 2 was characterized as shown in Figure 4, and was

353

named paraguhenryisin B. Compound 3 was isolated as colorless oil, and the same molecular formula

354

13

355

(C22H38O4) as 2 was deduced from its

356

366.2764 [M]+, calcd 366.2770). The 1D and 2D NMR spectra of 3 revealed the same

357

substituents on the same skeleton as paraguhenryisin B (2). The two diterpenoids

358

differed only in the nature of their substituents at C-15 and C-16. The acetoxyl group

359

at C-16 and hydroxyl group at C-15 in 2 were transposed in 3. The HMBC observed

360

correlations from H2-15 (δH 4.64) to C-13 (δC 146.9), C-14 (δC 121.2) and the acetyl

361

carbonyl carbon at δC 171.0 (Fig. S13). The ROESY correlations also indicated a

362

Z-configuration for the double bond between C-13 and C-14. Consequently,

363

compound 3 was characterized as shown in Figure 4, and was named paraguhenryisin

364

C.

365

C NMR and (HR) EI-MS analysis (m/z

Compound 4 was obtained as colorless oil. Its molecular formula was established 13

366

as C22H36O3 based on analysis of its

C NMR data and (HR) EI-MS (m/z 348.2655

367

[M]+, calcd 348.2664), which indicated five degrees of unsaturation. The 1H and 13C

368

spectra of 4 were quite similar to those of 2. The only difference between them was

369

the loss of the hydroxyl group at C-8, and the appearance of an additional double

370

bond (δH 5.38 and 5.62) between C-7 and C-8 in 4. This was supported by the HMBC


Page 19 of 40


page 19 of 40

371

correlations from Me-17 (δH 1.70) to δC 55.3 (C-9), the olefinic carbons at δC 122.9

372

(C-7) and 135.8 (C-8). ROESY spectrum of 4 suggested that the relative configuration

373

of 4 were the same as in 2 and 3. Thus, compound 4 was identified as shown in Figure

374

4, and was named paraguhenryisin D.

375 376

Compound 5 was identified as physacoztomatin by comparison of its spectral data with those of literature values. 19

377

Seed germination bioassay. The phytotoxic effect of CGTs extracts of P. henryi

378

inflorescences and three major compounds 3–5 on A. thaliana seed germination were

379

investigated. While inhibitory effect was observed to some extent for all tested

380

samples, clear differences were found at higher concentrations. The CGTs extracts

381

and compound 3 showed significant inhibition, with EC50 values of 42.2 ± 2.1 and

382

37.3 ± 0.6 µg/mL respectively (Figure 6), while compounds 4 and 5 (EC50 = 66.9 ±

383

0.8 and 64.7 ± 0.8 µg/mL, respectively) only retarded germination at the highest

384

concentration 100 µg/mL. The results suggested that paraguhenryisin C (3) is an

385

efficient seed germination inhibitor against A. thaliana and its phytotoxic activity

386

seems to be structure specific.

387

Root elongation bioassay. The CGTs extracts of P. henryi inflorescences and

388

compounds 3−5 were also tested for their phytotoxic effect on the root elongation of A.

389

thaliana seedlings. As shown in the exemplified Figure 7, all tested samples showed

390

significant inhibitory activity in a dose dependant manner. The EC50 values were 28.4

391

± 2.4, 29.1 ± 2.2, 44.4 ± 2.9 and 37.6 ± 2.1 µg/mL for CGTs extracts and compounds

392

3−5, respectively. At the maximum concentration 100 µg/mL, 80−90% reduction of




393

root elongation was observed for all samples (Figure 8), indicating that all samples are

394

phytotoxic substances.

395

Detection of GTs compounds in the inflorescences and aqueous rinsed

396

inflorescences extract. It has been well known that secondary metabolites could be

397

released into the environment as allelochemicals. In the fields we observed breakdown

398

of approximately 30-50% CGTs of P. henryi by moderate rain. It was therefore

399

interesting to find out the fate of the above identifed diterpenoids in CGTs of P. henryi.

400

The chemical profile of the inflorescences of P. henryi with rich CGTs was first

401

analyzed. Four major peaks (I, II, V, III) with retention times of 22.40, 24.23, 24.82

402

and 26.25 min, respectively, were detected in the HPLC chromatogram of the MeOH

403

inflorescences extract (Figure 9A), which were in accordance with compounds 1, 2, 5

404

and 4 (Figure 2B−2E) by comparison of their retention times and UV spectra with

405

those of isolated authentic samples. Then the inflorescences of P. henryi were

406

collected and rinsed with distilled water to mimic rain leaching, and the extracts were

407

analyzed by HPLC-UV method, and compared with the retention times and UV

408

spectra of separate injections of the authentic samples. As shown in Figure 9B, the

409

most potent phytotoxic diterpenoid, paraguhenryisin C (3), was clearly detected in the

410

aqueous rinsed solution.

411

Detection of GTs compounds in rhizosphere soil and roots. To find out if CGTs

412

compounds also accumulated in the surrounding soil of P. henryi, rhizosphere soil

413

(15.0 g) was collected in triplicate for three consecutive days with occasional rain,

414

which was extracted with MeOH and analyzed in the same way as described above.


Page 21 of 40


page 21 of 40

415

The results indicated that paraguhenryisin C (3) was also readily detected in the

416

rhizosphere soil (Figure 9C). A quantitative analysis was carried out, which disclosed

417

that the average content of 3 in rhizosphere soil was between 19.4 ± 4.4 and 27.3 ±

418

3.3 µg/g (Figure 10), which were close to its phytotoxic EC50 values. In addition, the

419

chemical profile of the roots of P. henryi was also investigated. It was found that

420

physacoztomatin (5) was the predominant constituent, while paraguhenryisin C (3)

421

was almost not detectable, which excluded the possibility that paraguhenryisin C (3)

422

in the rhizosphere soil of P. henryi was from the roots exudates. These findings

423

suggested that the labdane diterpenoids accumulated in the CGTs could be released

424

into environment by rain and act as allelochemical defense for P. henryi.

425

In conclusion, we have identified five labdane diterpenoids including four new ones

426

precisely from the CGTs of P. henryi through LMD coupled with cryogenic NMR and

427

HPLC methods. Significant phytotoxic effect for these diterpenoids on Aradidopsis

428

thaliana seed germination and seedling root elongation were observed. The most

429

abundant and also most potent diterpenoid, paraguhenryisin C (3), was detected in

430

both the rhizosphere soil of P. henryi and the aqueous rinsed inflorescences extract of

431

P. henryi in similar concentrations, suggesting that this compound could be released

432

into the environment possibly by rain as allelochemical of P. henryi against other

433

neighboring competitive plant species, which thus provided evidence for a new

434

defense function for the plant GTs. Further investigation are required to confirm its

435

role in mediating ecological interactions and to explore the biosynthetic pathway of

436

these special GTs metabolites. To our knowledge, phytotoxic activity for natural




437

labdane diterpenoids has been rarely reported. The unusual phytotoxic activity of

438

these GTs secondary metabolites heralds their potential as promising phytotoxins for

439

development of novel herbicides based on natural products that are useful for weed

440

management in organic farming systems nowadays.

441

Abbreviations Used

442

Glandular trichomes, GTs; capitate glandular trichomes, CGTs; peltate glandular

443

trichomes, PGTs; the effective dose to give 50% inhibitions, EC50; laser

444

microdissection, LMD; scanning electron microscope, SEM; nuclear magnetic

445

resonance, NMR; high performance liquid chromatography, HPLC; tetramethyl silane,

446

TMS.

447

ACKNOWLEDGMENT

448

We thank the members of the analytical center of Kunming Institute of Botany,

449

Chinese Academy of Sciences, for measurements of the NMR, MS, IR, and UV

450

spectral data. This research was supported financially by the National Science Fund

451

for Distinguished Young Scholars (31525005), the National Basic Research Program

452

of China (973 Program) on Biological Control of Key Crop Pathogenic Nematodes

453

(2013CB127505), the NSFC-Yunnan Joint Fund (U1202263), and the “Hundred

454

Talents Program” of the Chinese Academy of Sciences (awarded to S.-H. Li).

455

SUPPORTING INFORMATION

456

1

457

key HMBC correlations of compounds 2 and 3. This information is available free of

458

charge via the Internet at http://pubs.acs.org.

H and 13C NMR spectra of compounds 1-5. CD spectra of compounds 1−4. Selected


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page 23 of 40

459

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460

1.

461

J. 2012, 70, 51-68.

462

2.

463

trichomes. Plant Biotechnol. 2014, 31, 353-361.

464

3.

465

trichomes of the psychoactive sage, Salvia divinorum. Ann. Bot. 2004, 93, 763-771.

466

4.

467

Press: Israel, 2000; Vol. 31, pp 1-35.

468

5.

469

trichomes of peppermint. Plant Physiol. 2000, 124, 665-680.

470

6.

471

M.; Jongsma, M. A. Bidirectional secretions from glandular trichomes of pyrethrum

472

enable immunization of seedlings. Plant Cell 2012, 24, 4252-4265.

473

7.

474

of isoprenoids in glanded and glandless Artemisia annua L. Phytochemistry 1999, 52,

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1035-1040.

476

8.

477

Escherichia coli for production of functionalized terpenoids using plant P450s. Nat.

478

Chem. Biol. 2007, 3, 274-277.

479

9.

480

constituents of glandular trichomes and the leaf surface of camphorweed, Heterotheca

Tissier, A. Glandular trichomes: what comes after expressed sequence tags? Plant

Wang, G. D. Recent progress in secondary metabolism of plant glandular

Siebert, D. J. Localization of salvinorin A and related compounds in glandular

Werker, E. Trichome diversity and development. In Adv. Bot. Res., Academic

Turner, G. W.; Gershenzon, J.; Croteau, R. B. Development of peltate glandular

Ramirez, A. M.; Stoopen, G.; Menzel, T. R.; Gols, R.; Bouwmeester, H. J.; Dicke,

Tellez, M. R.; Canel, C.; Rimando, A. M.; Duke, S. O. Differential accumulation

Chang, M. C. Y.; Eachus, R. A.; Trieu, W.; Ro, D.-K.; Keasling, J. D. Engineering

Morimoto, M.; Cantrell, C. L.; Libous-Bailey, L.; Duke, S. O. Phytotoxicity of




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subaxillaris. Phytochemistry 2009, 70, 69-74.

482

10. Kroumova, A. B.; Wagner, G. J. Different elongation pathways in the

483

biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous

484

plants. Planta 2003, 216, 1013-1021.

485

11. Wagner, G. J.; Wang, E.; Shepherd, R. W. New approaches for studying and

486

exploiting an old protuberance, the plant trichome. Ann. Bot. 2004, 93, 3-11.

487

12. Li, C. H.; Jing, S. X.; Luo, S. H.; Shi, W.; Hua, J.; Liu, Y.; Li, X. N.; Schneider,

488

B.; Gershenzon, J.; Li, S. H. Peltate Glandular trichomes of Colquhounia coccinea var.

489

mollis harbor a new class of defensive sesterterpenoids. Org. Lett. 2013, 15,

490

1694-1697.

491

13. Scotland, R. W.; Vollesen, K. Classification of Acanthaceae. In Kew Bulletin,

492

Springer: Germany, 2000; Vol. 55, pp 513-589.

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14. Asai, T.; Hirayama, Y.; Fujimoto, Y. Epi-α-bisabolol 6-deoxy-β-D-gulopyranoside

494

from the glandular trichome exudate of Brillantaisia owariensis. Phytochem. Lett.

495

2012, 5, 376-378.

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15. Hu, J. Q.; Deng, Y. F.; Wood, J. R. I. Acanthaceae. In Flora of China, Science

497

Press: Beijing, 2011; Vol. 19, p 398.

498

16. Li, C. H.; Liu, Y.; Hua, J.; Luo, S. H.; Li, S. H. Peltate glandular trichomes of

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Colquhounia seguinii harbor new defensive clerodane diterpenoids. J. Integr. Plant

500

Biol. 2014, 56, 928-940.

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17. Sheldrick, G. M. SHELXS97 and SHELXL97; University of Göttingen, Germany,

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18. Abad, A.; Agullo, C.; Arno, M.; Cunat, A. C.; Zaragoza, R. J., Synthesis of

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(-)-borjatriol. J. Org. Chem. 1992, 57, 50-54.

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19. Perez-Castorena, A. L.; Oropeza, R. F.; Vazquez, A. R.; Martinez, M.; Maldonado,

506

E. Labdanes and withanolides from Physalis coztomatl. J. Nat. Prod. 2006, 69,

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1029-1033.




Figure captions Figure 1. Morphology and distribution of trichomes of P. henryi and LMD of CGTs. (A) P. henryi in flowering. (B and C) CGTs on the inflorescences. Scale bar: 100 µm. (D) Unicellular pyramid nonglandular trichomes on the leaves surface. Scale bar: 200 µm. (E) CGTs and PGTs on the inflorescences. Scale bar: 1.00 mm. (F) CGTs and PGTs on the inflorescences. Scale bar: 100 µm. (G) Intact CGTs before dissection. Scale bar: 400 µm. (H) The remaining tissue after dissection. Scale bar: 400 µm.

(I)

Collected CGTs. Scale bar: 200 µm. Figure 2. HPLC analysis and comparison of secondary metabolites in microdissected CGTs (202 nm). (A) Total HPLC chromatogram of the MeOH extract of laser microdissected CGTs. (B−F) HPLC chromatograms of the isolated authentic compounds 1 (B), 2 (C), 5 (D), 3 (E) and 4 (F) from the CGTs extracts of inflorescences of P. henryi. Figure 3. 1H NMR (600 MHz in acetone-d6) analysis of P. henryi CGTs and comparison of secondary metabolites isolated from CGTs extracts. (A) Cryo-1H NMR spectrum of the extract of P. henryi CGTs. (B−F) 1H NMR spectra of the isolated compounds 1 (B), 2 (C), 5 (D), 3 (E) and 4 (F) from the CGTs extracts of inflorescences of P. henryi. Figure 4. Compounds isolated from CGTs of P. henryi inflorescences. Figure 5. (A): Selected key HMBC (from H to C) of paraguhenryisin A. (B): X-ray crystallographic structure of paraguhenryisin A. Figure 6. Effects of CGTs extracts (A) and compounds 3−5 (B−D) on A.thaliana seed


Page 27 of 40


page 27 of 40

germination. Asterisks indicate significant differences between treatments and control as determined by Student’s t-test: *P < 0.01. Figure 7. Inhibition of the root elongation of A. thaliana by different concentrations (12.5, 25, 50 and 100 µg/mL) of CGTs extracts of P. henryi, photographs were taken after growth for seven days. Figure 8. Inhibition of A. thaliana root elongation by CGTs extracts (A) and compounds 3–5 (B–D) at different concentrations. Asterisks indicate significant differences between treatments and control as determined by Student’s t-test: *P < 0.01. Figure 9. HPLC chromatograms (202 nm) of different extracts of P. henryi (A-C and E) and authentic isolated compound 3 (D). (A) MeOH of the iInflorescences. (B) Aqueous rinsed extract of the inflorescences. (C) MeOH extract of rhizosphere soil. (E) MeOH extract of the roots. Figure 10. Content of paraguhenryisin C (3) in the rhizosphere soil of P. henryi.




Table 1. 1H NMR (600 MHz) spectroscopic data of compounds 1–4 in acetone-d6 (δH [ppm], J [Hz])[a]. No.

1

2

3

4

1a 1b

1.07 m 1.85 m

0.99 m 1.69 m

0.99 m 1.69 m

0.99 m 1.87 m

2a 2b

1.43 m 1.53 m

1.42 m 1.63 m

1.42 m 1.63 m

1.43 m 1.56 m

3a 3b

1.17 m 1.38 m

1.17 m 1.35 m

1.18 m 1.35 m

1.17 m 1.40 m

5

1.22 dd (12.6, 4.8)

0.95 dd (12.0, 1.8)

0.95 dd (12.0, 1.8)

1.18 dd (12.6, 4.8)

6a 6b

1.89 m 1.97 m

1.30 m 1.62 m

1.30 m 1.62 m

1.89 m 1.96 m

7a 7b

5.37 br s

1.44 m 1.79 m

1.44 m 1.79 m

5.38 br s

9

2.05 m

1.11 m

1.15 m

1.63 m

11a 11b

1.35 m 1.52 m

1.32 m 1.60 m

1.38 m 1.63 m

1.30 m 1.61 m

2.12 m 2.24 m

2.21 m 2.27 m

2.01 m 2.33 m

12a 12b

3.29 ddd (11.4, 5.4, 1.8)

14

2.93 t (5.4)

5.59 t (6.5)

5.37 t (6.6)

5.62 t (6.5)

15a 15b

3.61 m 3.69 m

4.14 t (2H, 6.1)

4.64 d (2H, 6.6)

4.15 t (2H, 6.1)

16a 16b

1.25 (3H, s)

4.61 d (12.5) 4.63 d (12.5)

4.15 (2H, m)

4.61 d (12.5) 4.64 d (12.5)

17

1.65 (3H, s)

1.10 (3H, s)

1.10 (3H, s)

1.70 (3H, s)

18

0.85 (3H, s)

0.86 (3H, s)

0.86 (3H, s)

0.85 (3H, s)

19

0.88 (3H, s)

0.80 (3H, s)

0.80 (3H, s)

0.88 (3H, s)

20

0.75 (3H, s)

0.82 (3H, s)

0.82 (3H, s)

0.77 (3H, s)

[a] Signals for hydroxy groups: 1: δH 3.90 (d, 6.0, 12-OH), 3.81 (dd, 5.3, 1.2, 15-OH); 2: δH 2.94 (s, 8-OH); δH 3.61 (t, 5.4, 15-OH); 3: δH 3.09 (s, 8-OH); δH 3.83 (t, 5.7, 16-OH); 4: δH 3.67 (t, 5.5, 15-OH); Signals for acetoxy groups 2: δH 1.98 (s, 3H, 16-OCOCH3); 3: δH 1.97 (s, 3H, 15-OCOCH3 ); 4: δH 2.00 (s, 3H, 16-OCOCH3).


Page 29 of 40


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Table 2. 13C NMR (150 MHz) spectroscopic data of compounds 1–4 in acetone-d6 (δC [ppm])[a]. No.

1

2

3

4

1

39.7 t

40.5 t

40.6 t

39.7

2

19.4 t

19.1 t

19.1 t

19.4 t

3

43.1 t

42.8 t

42.8 t

42.9 t

4

33.6 s

33.8 s

33.8 s

33.5 s

5

51.0 d

57.0 d

57.0 d

50.9 d

6

24.5 t

21.2 t

21.2 t

24.4 t

7

123.0 d

45.3 t

45.2 t

122.9 d

8

136.1 s

73.5 s

73.7 s

135.8 s

9

50.6 d

62.2 d

61.9 d

55.3 d

10

37.1 s

39.8 s

39.8 s

37.5 s

11

31.5 t

25.0 t

24.7 t

26.7 t

12

77.2 d

39.2 t

39.3 t

38.1 t

13

64.0 s

137.7 s

146.9 s

136.7 s

14

61.6 d

130.6 d

121.2 d

131.3 d

15

61.1 t

58.5 t

61.2 t

58.5 t

16

12.0 q

62.1 t

60.4 t

61.9 t

17

22.7 q

24.4 q

24.4 q

22.3 q

18

33.5 q

33.8 q

33.8 q

33.5 q

19

22.1 q

21.8 q

21.8 q

22.1 q

20

14.0 q

15.9 q

16.0 q

13.9 q

[a] Signals for acetoxy groups 2:, δC 170.9 s, 20.8 q (16-OCOCH3); 3:, δC 171.0 s, 20.9 q (15-OCOCH3); 4: δC 170.9 s, 20.8 q (16-OCOCH3).



Figure 1.



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Figure 2.


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Figure 3.



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Figure 4.

508


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Figure 5.

OH HO O

H

A



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Figure 6.

509


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510

Figure 7.



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Figure 8.


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Figure 9.

511



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Figure 10.

512


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Capitate Glandular Trichomes of Paragutzlaffia henryi Harbor New

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