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Crocus sativus petals: waste or valuable resource? The answer of HR- and HR-MAS NMR Valeria Righi, Francesca Parenti, Vitaliano Tugnoli, Luisa Schenetti, and Adele Mucci J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03284 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 15, 2015

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Journal of Agricultural and Food Chemistry

1

Crocus sativus petals: waste or valuable resource? The answer of HR- and HR-MAS NMR

2

Valeria Righi,† Francesca Parenti, ‡ Vitaliano Tugnoli,§ Luisa Schenetti,┴Adele Mucci*,‡

3 4 5 6 7 8 9 10 11

†

Dipartimento di Scienze per la Qualità della Vita, Università di Bologna, C.so D'Augusto 237,

47921 Rimini ‡

Dipartimento di Scienze Chimiche e Geologiche, Università di Modena e Reggio Emilia, Via G.

Campi 103, 41125 Modena §

Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Via Belmeloro 8/A,

40123 Bologna ┴

Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Via G. Campi 103,

41125 Modena

12 13 14 15 16

Corresponding author:

17

Prof. A. Mucci

18

Dipartimento di Scienze Chimiche e Geologiche

19

Università di Modena e Reggio Emilia

20

Via G. Campi 103

21

41125 Modena

22

tel: 00390592058636

23

e-mail: [email protected]

24

1 ACS Paragon Plus Environment


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ABSTRACT

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Intact Crocus sativus petals were studied for the first time by HR-MAS NMR spectroscopy,

27

revealing the presence of kinsenoside (2) and goodyeroside A (3), together with 3-hydroxy-γ-

28

butyrolactone (4). These findings were confirmed by HR-NMR analysis of ethanol extract of fresh

29

petals

30

glucopyranosyloxybutanolides occurs during extraction. On the other hand, kaempferol 3-O-

31

sophoroside (1) which is “NMR-silent” in intact petals, is present in extracts.

32

These results suggest to evaluate the utilization of saffron petals for phytopharmaceutical and

33

nutraceutical purposes in order to exploit a waste product of massive production of commercial

34

saffron and point to the application of HR-MAS NMR for monitoring bioactive compounds directly

35

on intact petals avoiding extraction procedure and the consequent hydrolysis reaction.

and

showed

that,

even

though

carried

out

rapidly,

partial

hydrolysis

of

36 37

Keywords: Crocus sativus, Saffron, kinsenoside, goodyeroside A, kaempferol-3-O-sophoroside,

38

HR-MAS NMR

39 40


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INTRODUCTION

42

Crocus sativus (fam. Iridaceae) is cultivated in some part of Italy for the utilization of its stigmas

43

(saffron) in foods and sweets for both their intense colour and strong taste. Moreover, saffron has

44

also been used in preventive medicine: numerous pharmacological tests point to specific therapeutic

45

actions and many studies are focused on it.1

46

Crocus sativus petals represent the main by-product of saffron production. Considering that saffron

47

is one of the most commercialized spices, and that 1 kg of saffron is obtained from more than

48

160000 flowers, it is fair to ask whether it is possible to directly use the petals for

49

phytopharmaceutical or nutraceutical purposes or as raw material to obtain substances with

50

pharmacological activity.2 Very recently, a paper proposing the use of petals of Crocus sativus as

51

source of crocin and and kaempferol, appeared.3

52

A number of studies on hepatoprotective, anti-nociceptive, anti-inflammatory, anti-depressant

53

effects of saffron petal extracts are reported in the literature.3-5 It is also known that petals of Crocus

54

sativus contain kaempferol,6,7 which has been reported to be a tyrosinase inhibitor,7 and its

55

glycosides, especially kaempferol-3-O-sophoroside 1 (Fig. 1), which exhibit antioxidant and anti-

56

infammatory activity.8,9

57

Hence, the properties reported for petal extracts could be simply due to kaempferol derivatives, but

58

they could also be due to other bioactive species. Sprouts of Crocus sativus contain 3-(R)-3-β-D-

59

glucopyranosyloxybutanolide (kinsenoside 2, Fig. 1) and 3-(S)-3-β-D-glucopyranosyloxybutanolide

60

(goodyeroside A 3, Fig. 1),10 that derive from 3-hydroxy-γ-butyrolactone (4, Fig. 1) and have been

61

isolated also from other plants such as Anoectochilus and Goodyera species (Orchidaceae).11-14

62 63

Figure 1 near here

64 65

Both 2 and 3 exhibit hepatoprotective effects;14,15 2 is also antihyperglycemic,12 anti-

66

inflammatory,16 antihyperliposis,17 vascular protective,18 ovariectomy-induced bone loss preventive 3 ACS Paragon Plus Environment


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and osteoclastogenesis suppressing.19 For these reasons efforts towards efficient synthesis of 2 and

68

3 by a chemo-enzymatic approach have recently been reported.20

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In order to gain a deeper insight into this subject, and to establish which are the main metabolites

70

present in Crocus sativus petals, we analysed them by high-resolution magic-angle-spinning NMR

71

(HR-MAS NMR) and their ethanol (95%) extract by high-resolution (HR-) NMR. HR-MAS NMR

72

spectroscopy allows to derive the biochemical profile of intact (human, animal or plant) tissues,21

73

formed by fast moving small metabolites, that give rise to narrow resonances. Through HR-MAS

74

NMR we tried to have a look inside a complex row matrix without disrupting its structure and

75

avoiding hydrolytic processes that can flank extraction. The purpose of this paper is to contribute to

76

the utilization of Crocus Sativus petals directly after the separation from saffron. To the best of our

77

knowledge, this is the first HR-MAS NMR study of petals.

78 79

EXPERIMENTAL SECTION

80 81

Plant Materials

82 83

Crocus sativus flowers were collected in Abruzzo (Italy), in the Aquila Saffron Protected

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Designation of Origin (PDO) status, at the Farm "Vigna di More" located in Tione degli Abruzzi, in

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autumn 2011. The flowers grew at 700 m above sea level. The bulbs were planted in August on a

86

ground prepared a year before (the soil is employed every five years) and the flowers were

87

harvested in October. The bulbs were put at 2-3 cm distance from each other, at least 8 cm depth

88

in rows of two or three, according to the space and land available. Flowers were hand-picked early

89

in the morning, before sunrise before perianth opening, and were placed in traditional wicker

90

baskets. After collection, they were stored at 5 °C and send to the lab, where intact petals were used

91

for HR-MAS NMR measurements within three days after collection. 4 ACS Paragon Plus Environment

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Extraction

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Fresh petals (10 g) were extracted with 95% ethanol (Sigma Aldrich) and the extract vacuum

95

evaporated (150 mg). The whole process was carried out quickly at room temperature (about 1

96

hour). The extracts were analysed in high resolution NMR and in MS.

97 98 99

NMR measurements 1

H HR-MAS NMR spectra were recorded with a Bruker Avance400 (Bruker BioSpin) spectrometer

100

operating at a frequency of 400.13 MHz. The instrument was equipped with a 1H,13C HR-MAS

101

probe for semisolids and with a BBI probe for liquids. Before HR-MAS examination, intact saffron

102

petals were introduced in a MAS zirconia rotor (4 mm OD), 10 µL of D2O were added to provide

103

deuterium for the lock system, fitted with a 50 µl cylindrical insert to increase sample homogeneity,

104

and then transferred into the probe cooled to 5 ˚C. Experiments were performed at a temperature of

105

5˚C controlled by a Bruker cooling unit.

106

Samples were spun at 4000 Hz and one- (1D) and two-dimensional (2D) spectra were acquired by

107

using the sequences implemented in the Bruker software. The same experiments were carried out on

108

D2O extract solution at 25 °C, with a lower number of scans. To record 1H water-presaturated

109

spectrum, a composite pulse sequence (zgcppr) with 2 s water presaturation during the relaxation

110

delay, 8 kHz spectral width, 32k data points, 16-8 scans were used. 2D COSY (COrrelation

111

SpectroscopY) spectra were acquired using a standard pulse sequence (cosygpprqf) and 1 s water

112

presaturation during relaxation delay, 2-4 kHz spectral width, 2 k data points, 4 scans per

113

increment, 128 increments. 2D TOCSY (TOtal Correlation SpectroscopY) spectra were acquired

114

using a standard pulse sequence (mlevgpph19) and 1 s water presaturation during relaxation delay,

115

100 ms mixing (spin-lock) time, 2-4 kHz (spectral width, 4 k data points, 4 scans per increment,

116

512 increments. 2D HSQC (Heteronuclear Single Quantum Coherence) edited spectra were 5 ACS Paragon Plus Environment


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acquired using a standard pulse sequence echo-antiecho phase sensitive (hsqcedetgp) and 0.5 s

118

relaxation delay, 1.725 ms evolution time, 2-4 kHz spectral width in f2, 2 k data points, 16-8 scans

119

per increment, 15 kHz spectral width in f1, 256 increments. 2D HMBC (Heteronuclear Multiple

120

Quantum Coherence) spectra were acquired using a standard pulse sequence (hmbcgplpndqf) and

121

0.5 s relaxation delay, 50 ms evolution time, 2-4 kHz spectral width in f2, 2 k data points, 128-32

122

scans per increment, 22 kHz spectral width in f1, 256 increments.

123

Deconvolution of 1H NMR selected signals was run with MestReNova 9.1 (2014 Mestrelab

124

Research S.L.).

125 126

ESI ion-trap MS measurements

127

The negative ion ESI mass spectra and ESI-MS/MS data (collision energies: 10, 15, 20 or 35 eV)

128

were acquired with a LC-MS(n) Ion Trap 6310A (Agilent Technologies) coupled with a HPLC

129

Agilent Series 1200 equipped with a Zorbax SB-C18 column, 30x2.1mm ID, 3.5 µm particle size

130

(Agilent). Eluents: acetonitrile and H2O, 1% formic acid. Chromatographic runs were performed

131

using a gradient of 1% formic acid in acetonitrile (98 →10) in 1% formic acid in water (2 → 90).

132

The solvent flow rate was 0.2 ml/min, the temperature kept at 25°C and the injector volume

133

selected was 2 µl. Total ion current (TIC) chromatograms were acquired in both positive and

134

negative mode in the mass range between 100 and 1400 m/z. He was used as collision gas in MS2

135

experiments.

136 137

RESULTS & DISCUSSION

138

The water-presaturated 1H HR-MAS NMR spectrum of saffron petals, Fig. 2a, shows the

139

overlapped signals from a number of metabolites, mainly in the carbohydrate region, whereas only

140

broad and weak signals are found in the aromatic region, at about 6.9 and 7.7 ppm, where multiplets

141

from kaempferol derivatives are expected. These two broad resonances are sometimes assigned to 6 ACS Paragon Plus Environment

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polyphenols but they have also been attributed to NH protons.22 On the other hand, some multiplets

143

in the range 3.1-2.5 ppm, outside the common carbohydrate region, are detected and other low

144

signals mainly attributable to free aminoacids and lipids are present at lower ppm.

145

To disentangle the complex spectral pattern it was necessary to use 2D NMR homo- and hetero-

146

correlated experiments, such as COSY, TOCSY, HSQC and HMBC. We were thus able to

147

reconstruct molecular skeletons and to compare the chemical shifts and the correlation patterns to

148

those reported in the literature and in NMR databanks, such as HMDB (http://www.hmdb.ca/).

149

Using this approach, we found that the majority of signals in 1H spectrum are due to sucrose (Suc),

150

glucose (Glc) and fructose (Fru), as can be better seen observing HSQC correlations (Fig. 3). Apart

151

those of sugars, other resonances due to 2-4 are detected and assigned, through 2D experiments, as

152

reported in Table 1. The chemical shifts determined by us on intact petals parallel those reported in

153

pyridine-d5 by Zhang et al.20 Remarkably, even 2D NMR spectra did not highlight signals from

154

kaempferol or its derivatives in the aromatic region, meaning that, in the cell environment of fresh

155

petals, these molecules are not freely tumbling, but are probably associated with rigid structures

156

within the cell. Other minor resonances were confirmed to derive from free aminoacids (mainly

157

alanine, Ala, glutamine, Gln, and valine, Val).

158

The molar ratio of total 2-4 derivatives relative to water signal, estimated through deconvolution of

159

2-H signals of 2-4 at 2.8-2.5 ppm and of H2O at 4.97 ppm in the not-presaturated 1H NMR

160

spectrum, resulted around 0.3 mol %. It can be roughly converted to 0.8 % in weight with respect

161

water and then, considering a 75 % of water in petals, to 0.6 % of 2-4 in intact petals.

162 163

Figure 2 and 3 near here

164 165

Fresh petals were then extracted rapidly with 95 % ethanol, the vacuum evaporated residue was

166

solubilized in D2O and 1D, 2D NMR and ESI MS/MS spectra were acquired.



167

1

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H NMR spectrum of extract is reported in Fig. 2b, and it clearly displays signals in the aromatic

168

region that compare well with those reported by Wolfram et al.23 and were assigned, together with

169

other resonances in the carbohydrate region, to kaempferol 3-O-sophoroside 1 (Table 1), and to a

170

minor derivative that differs mainly for the 2',6'-H signals that are found at 8.09 ppm, 8-H at 6.78

171

(d, 2.2 Hz) and 6-H at 6.50 (d, 2.2 Hz) ppm and 1-HGlc 5.48 ppm (d, 7.6 Hz) and could be a 7-

172

substituted derivative of 1.

173

Apart from differences in the chemical shifts, due to a change from the cell environment to D2O

174

solution, resonances from 2-4 were detected also in the ethanol extract, even though in a ratio

175

different with respect to that observed in intact petals: the (2+3)/4 molar ratio passes from 2.5:1 in

176

intact petals to less that 1:1 in the extracts and at the same time the free glucose signals enhance

177

with respect to those of sucrose (1-HαGlc/1-Hsuc passes from 0.4 to 1.2). Hence, the extraction

178

process, even though carried out rapidly and without acidic conditions, promoted hydrolysis of 2

179

and 3 to 4 and free Glc.

180

ElectroSpray Ionization Ion Trap Mass Spectrometry (ESI-IT MS) was finally employed to confirm

181

the presence of 1 and to clarify the structure of the minor flavonol detected in ethanol extract (Fig.

182

4). The two major peaks detected in negative mode correspond to 609 and 771 m/z [M-H]- pseudo-

183

molecular ions. The collision induced fragmentation (CID) with He gas of 609 (highest) peak gave

184

429 m/z [M-C6H12O6-H]-and 285 m/z [kaempferol-H]- as expected for two subsequent hexose losses

185

starting from 1. On the other hand, CID of 771 m/z gave 609 m/z, corresponding to a loss of

186

C6H10O5 demonstrating that this derivative contains a further hexose unit. Minor peaks,

187

corresponding to pseudomolecular species of 625 m/z (which decomposes to 463 m/z through

188

hexose loss) and 651 m/z (which gives 471 m/z and 285 m/z under CID) were also detected. The

189

former could be due to quercetin sophoroside, the latter is probably an acetylated derivative.

190 191

Figure 4 near here 8 ACS Paragon Plus Environment

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192 193

In conclusion, this work shows that HR-MAS NMR can be employed to monitor bioactive

194

compounds freely tumbling in the cell environment, directly on intact petals. HR-MAS NMR

195

allows detecting the presence of 2, 3 and 4 directly on intact Crocus sativus petals, without the need

196

of extraction processes. Their content was estimated by HR-MAS NMR to be roughly 0.6 %. HR-

197

MAS NMR does not detect instead flavonols, especially 1, that are still contained in petals in a not

198

negligible amount, as demonstrated by 1H NMR analysis of ethanol extract. This implies that

199

flavonol derivatives in saffron petals have low tumbling rates, that could be due to their close

200

association to macromolecular species in cell walls.24

201

The 3-hydroxybutyrolactone derivatives 2-4 contained in Crocus sativus petals are biologically

202

active species that, together with 1, probably contribute to confer hepatoprotective,

203

antihyperglycemic, anti-inflammatory and other effects to petal extracts. The extraction process

204

leads to partial hydrolysis of petal constituents, hence it could be useful to evaluate the direct

205

employment of saffron petals for phytopharmaceutical and nutraceutical purposes, in order to better

206

exploit a waste product of massive production of commercial saffron.

207

The data presented in this study strongly support the idea that petals of Crocus sativus, are not a

208

waste, but they can be used for their biological activity in specific foods, as well as in herbalist

209

products. This can have a major impact for the community of saffron, the cultivation of which

210

represents an opportunity to improve and develop the economy of the agricultural sector in poor

211

European regions. The exploitation not only of stigmas but also of petals as raw material for health

212

enhancing products would further enhance the economical value of this activity and reduce waste

213

production.

214 215 216 9 ACS Paragon Plus Environment


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Acknowledgments

218

We are very grateful to Prof. Dario Iarossi and to "Vigna di More" farm, located in Tione degli

219

Abruzzi, for the supply of Crocus Sativus petals. http://www.valleaterno.it/vignadimore/


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Figure Legends

221

Fig. 1. Structures of identified compounds in Crocus sativus petals and ethanol extract.

222

Fig. 2. Water-presaturated 1H HR-MAS NMR spectrum of fresh saffron petals (a) and 1H NMR

223

spectrum of ethanol (95%) extract of fresh saffron petals in D2O solution (b).

224

Fig. 3. Enlarged carbohydrate (a) and aliphatic (b) regions of HSQC HR-MAS NMR spectrum of

225

fresh saffron petals. ChoCC =choline containing compounds.

226

Fig. 4. ESI-IT MS/MS spectra of 1 (left) and of minor kaempferol derivative (right).

227



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References

229

(1) Winterhalter, P.; Straubinger, M. Saffron: Renewed interest in an ancient spice. Food Rev Int.

230

2000, 16, 39–59.

231

(2) Montoro, P.; Tuberoso, C.I.G.; Maldini, M.; Cabras, P.; Pizza, C. Qualitative Profile and

232

Quantitative Determination of Flavonoids from Crocus sativus L. Petals by LC-MS/MS. Nat. Prod.

233

Commun. 2008, 3, 2013-2016.

234

(3) Zeka, K.; Ruparelia, K.C.; Continenza, M.A.; Stagos, D.; Vegliò, F.; Arroo, R.R.J. Petals of

235

Crocus sativus L. as a potential source of the antioxidants crocin and kaempferol. Fitoterapia 2015.

236

doi 10.1016/j.fitote.2015.05.014.

237

(4) Omidi, A.; Riahinia, N.; Montazer Torbati, M.B.; Behdani, M.A. Hepatoprotective effect of

238

Crocus sativus (saffron) petals extract against acetaminophen toxicity in male Wistar rats. J

239

Phytomed. 2014, 4, 330-336.

240

(5) Vijaya Bhargava, K. Medicinal Uses and Pharmacological Properties of Crocus sativus Linn.

241

(Saffron). Int J. Pharm. Pharm. Sci. 2011, 3, 22-26.

242

(6) Hadizadeh, F.; Khalilia, N.; Hosseinzadeh, H.; Khair-Aldine, R. Kaempferol from Saffron

243

Petals. Iranian J. Pharm. Res. 2003, 251-252.

244

(7) Kubo, I.; Kinst-Hori, I. Flavonols from Saffron Flower: Tyrosinase Inhibitory Activity and

245

Inhibition Mechanism. J. Agric. Food Chem. 1999, 47, 4121-4125.

246

(8) Yoon, H.-R.; Han, H.-G.; Paik, Y.-S. Flavonoid glycosides with antioxidant activity from the

247

petals of Carthamus tinctorius. J. Appl. Biol. Chem. 2007, 50, 175-178.

248

(9) Kim, T. H.; Ku, S.-K.; Lee, I.-C.; Bae, J.-S. Anti-inflammatory effects of kaempferol-3-O-

249

sophoroside in human endothelial cells. Inflammation Res. 2012, 61, 217-224.

250

(10) Gao, W.Y.; Li, Y.M.; Zhu, D.Y. Phenolic glucosides and a γ-lactone glucoside from the

251

sprouts of Crocus sativus. Planta Med. 1999, 65, 425-427.


Page 13 of 22


252

(11) Ito, A.; Kasa, R. Aliphatic and aromatic glucosides from Anoectochilus koshunensis.

253

Phytochemistry 1993, 33, 1133-1137.

254

(12) Du, X.M.; Yoshizawa, T.; Shoyama, Y. Butanoic acid glucoside composition of whole body

255

and in vitro plantlets of Anoectochilus formosanus. Phytochemistry 1998, 49, 1925-1928.

256

(13) Zhang, Y.H.; Cai, J.Y.; Ruan, H.L. Antihyperglycemic activity of kinsenoside, a high yielding

257

constituent from Anoectochilus roxburghii in streptozotocin diabetic rats. J. Ethnopharmacol. 2007,

258

114, 141−145.

259

(14) Du, X.M.; Sun, N.Y.; Chen, Y. Hepatoprotective aliphatic glycosides from three Goodyera

260

species. Biol. Pharm. Bull. 2000, 23, 731-734.

261

(15) Wu, J.B.; Lin, W.L.; Hsieh, C.C. The hepatoprotective activity of kinsenoside from

262

Anoectochilus formosanus. Phytother. Res. 2007, 21, 58-61.

263

(16) Hsiao, H.B.; Wu, J.B.; Lin, H.; Lin, W.C. Kinsenoside isolation from Anoectochilus

264

formosanus suppresses LPS-stimulated inflammatory reactions inmacrophages and endotoxin shock

265

in mice. Shock 2011, 35, 184-190.

266

(17) Du, X.M.; Sun, N.; Tamura, T. Higher yielding isolation of kinsenoside in Anoectochilus and

267

its antihyperliposis effect. Biol. Pharm. Bull. 2001, 24, 65-69.

268

(18) Liu, Z.L.; Liu, Q.; Xiao, B.; Zhou, J. The vascular protective properties of kinsenoside isolated

269

from Anoectochilus roxburghii under high glucose condition. Fitoterapia 2013, 86, 163-170.

270

(19) Hsiao, H.B.; Lin, H.; Wu, J.B.; Lin, W.C. Kinsenoside prevents ovariectomy-induced bone

271

loss and suppresses osteoclastogenesis by regulating classical NF-κB pathways. Osteoporos. Int.

272

2013, 24, 1663-1676.

273

(20) Zhang, Y.; Xia, Y.; Lai, Y.; Tang, F.; Luo, Z.; Xue, Y.; Yao, G.; Zhang Y.; Zhang, J.

274

Antihyperglycemic activity of kinsenoside, a high yielding constituent from Anoectochilus

275

roxburghii in streptozotocin diabetic rats. Molecules 2014, 19, 16950-16958.



Page 14 of 22

276

(21) Righi, V.; Schenetti, L.; Mucci, A. A new NMR technique: HR-MAS applied to animal and

277

vegetable soft materials. In New Trends in Analytical, Environmental and Cultural Heritage

278

Chemistry; Colombini, M.P., Tassi, L. Eds.; Transworld Research Network: Trivandrum, India,

279

2008; 31-42.

280

(22) Marino, G.; Righi, V.; Simoni, A.; Schenetti; L.; Mucci; A., Tugnoli; V., Muzzi, E.; Francioso,

281

O. Effect of a Peat Humic Acid on Morphogenesis in Leaf Explants of Pyrus communis and

282

Cydonia oblonga. Metabolomic Analysis at an Early Stage of Regeneration. J. Agric. Food Chem.

283

2013, 61, 4979-4987.

284

(23) Wolfram, K.; Schmidt, J.; Wray, V.; Milkowski, C.; Schliemann, W.; Strack, D. Profiling of

285

phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus). Phytochemistry 2010,

286

71, 1076-1084.

287

(24) Hutzler, P.; Fischbach, R.; Heller, W.; Jungblut, T.P.; Reuber, S.; Schmitz, R.; Veit, M.;

288

Weissenböck, G.; Schnitzler, J.-P. Tissue localization of phenolic compounds in plants by confocal

289

laser scanning microscopy. J. Exp. Botany 1998, 49, 953-965.

290 291 292 293 294 295 296 297 298 299 300


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Table 1 1 H and 13C NMR data of 2-4 (ppma) in Crocus sativus petals as obtained by HR-MAS NMR and of 1-4 extracted with ethanol (95%) (D2O). 2b

2c

3b 1

C

1

179.7

-

C

1

1

-

182.5

-

180.3

-

-

2

2.99, 38.2 2.71 dd, 18.1, 6.1 Hz; d, 18.1 Hz)

2.87, 37.2 2.65 (dd, 18.0, 6.4 Hz; ddd, 18.0, 1.9, 0.8 Hz)

3.03, 38.5 2.78 dd, 18.6, 6.1 Hz; d, 18.6 Hz)

2.89, 38.1 2.73 (dd, 18.2, 1.4 Hz; dd, 18.2, 6.4 Hz)

2.97, 40.1 2.51 (dd, 18.1, 6.1 Hz; d, 18.1 Hz)

2.83, 39.7 2.37 (dd, 17.7, 5.8 Hz; dt, 17.7, 1.3 Hz)

-

160.3

3

4.86

77.9

4.72

77.2

4.86

77.9

4.73

77.4

4.71 70.0 (t, 4.6 Hz)

4.57 69.5 (ddt, 4.3, 5.8, 1.3 Hz)

-

136.1

4

4.55

77.4

4.54,d 4.49

77.3

4.59

77.9

4.47d

76.4

4.54, 79.8 4.36 (d, 10.3 Hz)

4.42, 78.9 4.22d (dd, 10.0, 4.3 Hz; dt, 10.0, 1.2 Hz)

-

nd

4a

-

106.9

5

-

164.2

6

6.22

101.0

7

-

167.2

8

6.42

95.9

8a

-

159.6

e

C

1

182.5

-

13

H

C

1

13

C

1

180.1

-

183.0

-

1c 13

H

13

4c

13

H

C

4b

1

H

13

3c

H

e

H

H

13

C

1'

4.55

103.9

4.40 104.8 (d, 7.8 Hz)

4.58

103.7

4.38 105.1 (d, 7.8 Hz)

-

123.9

2'

3.27

75.6

3.19

3.27

75.6

3.19

8.05

133.5

75.9

75.9

3'

3.49

78.4

3.36

79.2

3.49

78.4

3.36

79.2

6.92

117.5

4'

f

f

3.29

72.8

f

f

3.29

72.8

-

162.8

5'

f

f

79.2

f

f

3.29

79.2

6.92

117.5

6'

f

f

63.9

f

f

3.67, 3.88

63.9

8.05

133.5

5.43g

102.2

3.29 3.67, 3.88

1”



309 310 311 312 313 314 315 316 317 318 319 320

Page 16 of 22

2”

3.74h

84.3

3”

3.61

79.0

4”

3.36

5”

3.20

79.5

6”

3.49, 3.69

63.7

1”'

4.78

106.0

2”'

3.37

76.8

3”',4”',5”'

3.413.30

6”'

3.69, 3.80

63.5

a

chemical shifts refer to sucrose 1-H at 5.40 and 94.8 ppm, respectively; intact petals; c petals extracted with ethanol (95%); d long range correlation with C=O. e long range correlations at 4.40/77.2 ppm and 4.38/77.4 in HMBC spectrum. f overlapped to β-Glc. g long range correlations with 136.0 ppm carbon in HMBC spectrum h long range correlations with 102.2 and 106.0 ppm carbons in HMBC spectrum. b

321 322 323 324 325 326 327 328 329 330 331 332 333 16 ACS Paragon Plus Environment

Page 17 of 22


334 335 336 337 338

339 340 341

Fig. 1. Structures of identified compounds in Crocus sativus petals and ethanol extract.

342 343 344 345 346 347 348 349



Page 18 of 22

350 351 352 353

354 355

Fig. 2. Water-presaturated 1H HR-MAS NMR spectrum of fresh saffron petals (a) and 1H NMR

356

spectrum of ethanol (95%) extract of fresh saffron petals in D2O solution (b).

357 358 359 360 361 362


Page 19 of 22


363

364 365 366

Fig. 3. Enlarged carbohydrate (a) and aliphatic (b) regions of HSQC HR-MAS NMR spectrum of

367

fresh saffron petals. ChoCC =choline containing compounds.

368 369 19 ACS Paragon Plus Environment


Page 20 of 22

370 371 372

Fig. 4. ESI-IT MS/MS spectra of 1 (left) and of minor kaempferol derivative (right).

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375


TOC image

376



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kinsenoside

goodyeroside A

ACS Paragon Plus Environment

Crocus sativus Petals: Waste or Valuable Resource? The Answer of

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