Pyrrolizidine Alkaloids from Echium vulgare in

Subscriber access provided by University of Cincinnati Libraries

Article

Pyrrolizidine alkaloids from Echium vulgare in honey originate primarily from floral nectar Matteo A. Lucchetti, Gaétan Glauser, Verena Kilchenmann, Arne Dübecke, Gudrun Beckh, Christophe Praz, and Christina Kast J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02320 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

1

Pyrrolizidine alkaloids from Echium vulgare in honey originate

2

primarily from floral nectar

3

Matteo A. Lucchetti , Gaetan Glauser , Verena Kilchenmann , Arne Dübecke , Gudrun Beckh ,

4

Christophe Praz2, Christina Kast1*

5

1

6

2

7

Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland.

8

3

9

4

1,2

3

1

4

4

Agroscope, Swiss Bee Research Centre, Schwarzenburgstrasse 161, 3003 Bern, Switzerland. Institute of Biology, Laboratory of Fundamental and Applied Research in Chemical Ecology (FARCE), University of Neuchâtel,

Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel, Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland. Quality Services International GmbH (QSI), Flughafendamm 9a, 28199 Bremen, Germany.

10

*Corresponding author: Email: [email protected]

11

Abstract

12

Pyrrolizidine alkaloids (PAs) in honey can be a potential human health risk. So far, it

13

has remained unclear whether PAs in honey originate from pollen or floral nectar. We

14

obtained honey, nectar and plant pollen from two observation sites where Echium

15

vulgare L. was naturally abundant. The PA concentration of honey was determined

16

by targeted analysis using a high pressure liquid chromatography-mass spectrometry

17

system (HPLC-MS/MS), allowing the quantification of six different PAs and PA-N-

18

oxides present in E. vulgare. Echium-type PAs were detected up to 0.153 µg/g in

19

honey. Nectar and plant pollen were analyzed by non-targeted analysis using ultra-

20

high pressure liquid chromatography-high resolution-mass spectrometry (UHPLC-

21

HR-MS), allowing the detection of 10 alkaloids in small size samples. Echium-type

22

PAs were detected between 0.3 - 95.1 µg/g in nectar and 500 - 35000 µg/g in plant

23

pollen. The PA composition in nectar and plant pollen was compared to the

24

composition in honey. Echimidine (+N-oxide) was the main alkaloid detected in

25

honey and nectar samples, while echivulgarine (+N-oxide) was the main PA found in

ACS Paragon Plus Environment

1


Page 2 of 29

26

plant pollen. These results suggest that nectar contributes more significantly to PA

27

contamination in honey than plant pollen.

28

Keywords: Pyrrolizidine alkaloids, Echium vulgare, honey, nectar, plant pollen, high

29

pressure liquid chromatography-mass spectrometry (HPLC-MS/MS), ultrahigh

30

pressure liquid chromatography-high resolution mass spectrometry (UHPLC-HR-MS).

31

1.1

32

Pyrrolizidine Alkaloids (PAs) are toxic compounds produced by plants as a chemical

33

defense against herbivores.1,2 Many unrelated plant species produce PAs, and some

34

of them are particularly abundant in European agro-ecosystems. These PA-

35

containing plants mainly belong to the Asteraceae (Senecioneae and Eupatorieae

36

tribes), Boraginaceae (all genera), and Fabaceae (genus Crotalaria) families.3,4 PAs

37

may get into the food chain when food products are either contaminated with or

38

derived from PA-containing plant tissues.5-7 PAs can occur as N-oxides or as free-

39

base/tertiary forms. These two forms are both hepatotoxic and genotoxic.8,9 In plants,

40

the N-oxides are found in higher concentrations than the corresponding free-bases

41

(tertiary PAs).1,10 Acute poisoning or chronic exposure to PAs mostly affects liver

42

function, since PAs are activated by nucleophilic compounds through the liver’s

43

detoxification enzymes.6,11-13 The chronic intake of low PA-levels in food can lead to

44

liver cirrhosis and cancer.14 Legal limits for PAs in food have not yet been established

45

in the European Union or in Switzerland. However, the German Federal Institute for

46

Risk Assessment (BfR) recommends an intake of not more than 0.007 µg of 1.2-

47

unsaturated PAs per day per kg bodyweight.15

48

Honey is one of the best studied food products with respect to PA contamination.

49

When honeybees collect nectar and plant pollen from PA-containing plants, PAs are

50

transferred into bee products such as honey or bee-collected pollen.16,17 PAs have

Introduction


2

Page 3 of 29


51

been detected in honey samples from various geographical and botanical origins.4,18-

52

27

53

by the BfR have been reported in monofloral honeys from Echium vulgare or E.

54

plantagineum 28-32 and from Senecio jacobaea.31,33,34

55

Honey is mostly composed of concentrated nectar and contains only traces of pollen.

56

Therefore, PAs contained in nectar constitute an important potential source of PAs in

57

honeys. However, the concentration of secondary compounds may be considerably

58

higher in plant pollen than in nectar,35,36,37 and some pollen types contain particularly

59

high amounts of PAs.17 Consequently, it remains unclear whether the PA content in

60

nectar is high enough to substantially contaminate honey,38 and, more generally,

61

whether the PAs in honey predominantly originate from pollen or nectar.39,40

62

Prior research has suggested pollen as the major source of PA contamination in

63

honey.14,17 Contamination of honey could be caused by the liberation of PAs from

64

pollen.38 Lastly, experiments with plant pollen from Senecio vernalis added to PA-free

65

honey have suggested that contamination of honey may occur through diffusion of

66

PAs from pollen into honey.40

67

Unraveling the entry mechanism by which PAs contaminate honey is important for

68

reducing risks associated with PA-containing bee products.

69

Two approaches can be used to examine the pathway from the different plant tissues

70

into bee products. First, quantitative analyses of nectar, pollen, and honey may help

71

determine which plant part is the main contributor to the total PAs found in honey.

72

Second, differences in the PA composition (relative abundance of different PAs)

73

found in nectar can be compared to that found in pollen. This information can be

74

used to determine whether the PA composition in honey more closely matches that

75

of nectar or pollen. In the present study, we performed qualitative and quantitative

76

analysis of the different alkaloids found in Echium nectar and pollen.

PA concentrations of up to two orders of magnitudes over the limits recommended


3


Page 4 of 29

77

We selected Echium vulgare as a model to study the pathway by which PAs are

78

transferred into honey. This plant species is the only species of the genus Echium

79

vulgare regularly found in Switzerland. It is widely distributed in Europe and has been

80

previously described as a major source of PA contamination of European honeys.19,21

81

We chose two observation sites where E. vulgare was blooming during the bee

82

season. These sites were located in two different climatic regions, one located to the

83

north and the other to the south of the Alps.

84

1.2 Material and Methods

85

Chemical reagents

86

The echimidine used in this study was obtained from Phytolab (Vestenbergsgreuth,

87

Germany), while the heliotrine was from Latoxan (Valence, France). For plant

88

extraction, milli-Q water was used. Formic acid and glass beads (Ø 2 mm) were

89

purchased from Sigma-Aldrich (Buchs, Switzerland). HPLC grade methanol was

90

purchased from Merck (Dietikon, Switzerland). Cyclohexane (> 98% purity), sulphuric

91

acid, and ammonia were purchased from Merck Chemicals (Darmstadt, Germany).

92

The solvents and additives used for LC-MS were water, acetonitrile, and ULC-MS

93

grade formic acid acquired from Biosolve (Valkenswaard, Netherlands).

94

Observation sites

95

We selected two observation sites where Echium plants were abundant around bee

96

colonies. The first observation site was located north of the Alps near Basel, close to

97

the border between Switzerland and France (hereafter Basel). The other observation

98

site was located close to Italy, along the southern flank of the Alps in the Verzasca

99

valley (hereafter Verzasca). At the Verzasca site, two beekeepers participated to the


4

Page 5 of 29


100

project (hereafter Verzasca 1 and Verzasca 2). The aerial distance between these

101

two apiaries was approximately 400 meters.

102

Honey and plant sample collection

103

Honeys: In total, four samples of honey were included from Basel and six from

104

Verzasca. In Basel, the honeys were harvested on 8 June and 27 July 2012, as well

105

as on 29 June and 8 August 2013. In Verzasca 1, the honeys were harvested on 1

106

August 2012, 2 August 2013, and 1 August 2014, while in Verzasca 2 they were

107

harvested on 30 July 2012, 2 August 2013, and 1 August 2014. Eight additional

108

honey samples were obtained from apiaries in diverse regions of Switzerland. All of

109

the additional honey samples were produced between 2009 and 2011. Plant

110

material: In Basel, E. vulgare was in blossom during June and July, while in the

111

Verzasca valley E. vulgare was in blossom from June until August. Samples of nectar

112

and plant pollen from E. vulgare were collected at the two observation sites. Samples

113

were only collected under dry weather conditions to avoid wash-out of plant pollen

114

from the anthers and dilution of nectar by the rain. In Basel, the samples were

115

collected on 18 June and 4 July 2013 and on 19 June and 27 June 2014. In

116

Verzasca, the samples were collected on 29 June and 17 July 2013 and on 6 July

117

and 18 July 2014. In total, 20 nectar samples from Basel (n=10 in 2013 and n=10 in

118

2014), 16 nectar samples from Verzasca (n=7 in 2013 and n=9 in 2014), 14 plant

119

pollen samples from Basel (n=5 in 2013 and n=9 in 2014) and 13 plant pollen

120

samples from Verzasca (n=4 in 2013 and n=9 in 2014) were collected. On the day

121

before any given sample collection, plants were tightly bagged with a fine mesh, and

122

a layer of insect glue was spread around the lower part of the stem in order to

123

prevent insect visits. Nectar was collected using a Pasteur pipette previously

124

elongated to a capillary on a flame. The pipette was directly placed into the corolla,


5


Page 6 of 29

125

carefully avoiding any disruption of floral tissues. In 2013, pollen from anthers of

126

Echium flowers (plant pollen) was collected with metal forceps. Plant pollen was

127

carefully removed from the surface of the anthers in order to prevent contamination

128

of the pollen with other flower parts, especially the anthers. This procedure yielded

129

low amounts of plant pollen. In order to facilitate collection and avoid contamination

130

with other plant parts, plant pollen was collected in 2014 by immersing the stamens

131

into cyclohexane.38 The cyclohexane was subsequently evaporated. For comparison,

132

two plant pollen samples were collected from the same plant using both methods

133

(forceps and cyclohexane). Since the two samples gave comparable results (data not

134

shown), we concluded that the cyclohexane did not wash out PAs from the pollen,

135

and hence that both collection methods would yield similar results. All samples were

136

kept on dry ice during collection and subsequently stored at -80°C until extraction.

137

Sample preparation of honey for quantification of PAs with LC-MS/MS

138

Honey samples were prepared as described in Dübecke et al.19 Since PA-N-oxides

139

are polar organic compounds with basic characteristics, they are soluble in polar

140

organic solvents or in mixtures of solvents and acidified water. 10 g of honey,

141

together with 100 ng heliotrine as internal standard and 30 mL of 0.05M sulphuric

142

acid were vigorously shaken for 20 min. Samples were then filtered overnight using a

143

2 mm mesh to remove particles that could block the solid-phase extraction. Clean-up

144

was conducted using SPE-SCX Cartridges (Varian) washed previously with methanol

145

and conditioned with 9 mL of 0.05M sulphuric acid. Samples were loaded onto the

146

column, washed with 9 mL of deionized water, eluted into a glass vial using

147

ammoniated methanol,18 and dried at 40°C in an ambient air stream. Samples were

148

then reconstituted in 1 mL deionized water, shaken vigorously, and filtered into a 2

149

mL glass vial using a 0.45 µm syringe filter.


6

Page 7 of 29


150

The PA concentration was determined by targeted analysis using a HPLC-MS/MS-

151

system as described in Dübecke et al.,19 allowing the detection of six different PAs or

152

PA-N-oxides (echimidine, echimidine-N-oxide, acetylechimidine, acetylechimidine-N-

153

oxide, echivulgarine and echivulgarine-N-oxide) commonly found in E. vulgare.17 The

154

total PA concentration was calculated as the sum of the six different PAs. LC-MS/MS

155

analysis was performed using an HTC PAL autosampler of CTC Analytics AG, a

156

Shimadzu LC-system with a Thermo Hypersil Gold C18 column (50 x 2.1 mm, 1.9 µm

157

particle size) and an Applied Biosystems API4000 QTRAP triple quadrupole mass

158

spectrometer. Concentrations were corrected against the recovery of the internal

159

standard. For quantification, external calibration was performed using echimidine as

160

the standard. The limit of quantitation (LOQ) for echimidine in honey was 1 ng/g. As

161

no further reference standards were available, echimidine-N-oxide, acetylechimidine,

162

acetylechimidine-N-oxide, echivulgarine, and echivulgarine-N-oxide were indirectly

163

quantified using the calibration of echimidine, assuming the same response factor

164

and thus the same LOQ. A linear range was achieved from 0.5 to 100 ng/mL for the

165

echimidine standard. Recovery of echimidine near the LOQ was 97%. Repeatability

166

was 5.4% as determined with six independent sample preparations measured by the

167

same person on the same day.

168

Extraction of PAs from nectar and plant pollen, and UHPLC-HRMS analysis

169

Nectar: 5 µL of nectar was directly transferred into a glass vial containing a 200 µL

170

insert and diluted 10 times with the extraction solvent (70% methanol, 29.5% ultra-

171

pure water and 0.5% formic acid, v/v). Plant pollen: 1 mg of plant pollen was

172

accurately weighed using a microbalance scale (Mettler Toledo), mixed with 100 µL

173

of extraction solvent as described above and transferred into a 2 mL Eppendorf tube.

174

Five glass beads were added and the tube was vigorously shaken at 30 Hz for 4 min


7


Page 8 of 29

175

to disrupt the pollen structure and to extract the PAs. Following centrifugation (18400

176

g, 4 min), 5 µL of the supernatant was transferred into a glass vial containing a 200

177

µL insert and diluted 10 times with the extraction solvent.

178

Non-targeted analysis using the UHPLC-HR-MS system was carried out for the

179

detection and quantification of various alkaloids found in E. vulgare (echimidine,

180

echimidine-N-oxide,

181

echimidine-N-oxide, acetylvulgarine, acetylvulgarine-N-oxide, echivulgarine and

182

echivulgarine-N-oxide17). In brief, separation of the alkaloids was performed on an

183

Acquity BEH C18 column (50 x 2.1 mm i.d., 1.7 µm particle size, Waters), using an

184

Acquity UHPLCTM system (Waters) coupled to a Synapt G2 QTOF mass

185

spectrometer (Waters). A binary gradient was performed at a flow rate of 0.4 mL min-

186

1

187

formic acid in acetonitrile (solvent B). The gradient program was as follows: 0-4 min

188

5-40% B, 4-6 min 40-100% B, 6-9 min 100% B, 9.1-10.5 min 5% B. The temperature

189

of the column was maintained at 30°C and that of the autosampler at 25°C. Injection

190

volume was 1 µL. The QTOF operated in electrospray positive mode over a mass

191

range of 50-600 Da. MS conditions were: Capillary voltage +2800 V, cone voltage

192

+30 V, source temperature 120°C, desolvation gas temperature and flow 350°C and

193

800 L/h, respectively, and scan time 0.4 sec. A leucine-enkephaline solution at 400

194

ng/mL was infused throughout the analysis to ensure high mass accuracy (

Pyrrolizidine Alkaloids from Echium vulgare in

Recommend Documents