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Studies on Anthriscus sylvestris L. (Hoffm.)Hendrawati, Oktavia

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ChapterIdentification of lignans and related compounds in Anthriscus sylvestris

by LC-ESI-MS/MS and LC-SPE-NMR

Oktavia Hendrawati Herman J. Woerdenbag

Paul J.A. Michiels Herald G. Aantjes

Annie van Dam Oliver Kayser

Phytochemistry 2011; in press

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oktavia Hendrawati1, Herman J. Woerdenbag2, paul J.A. michiels3, Herald g. Aantjes3,

Annie van dam4, oliver Kayser1,5

1Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands; 2Department of Pharmaceutical Technology and Biopharmacy, University of Groningen Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands; 3Spinnovation Analytical BV, Nijmegen, the Netherlands; 4Mass Spectrometry Core Facility, University of Groningen, Groningen, The Netherlands; 5Technical University Dortmund, Technical Biochemistry, Emil-Figge-Strasse 66, 44227 Dortmund, Germany

Phytochemistry 2011; in press

AbStrACt

The aryltetralin lignan deoxypodophyllotoxin is much more widespread in the plant kingdom than podophyllotoxin. The latter serves as a starting compound for the production of cytostatic drugs like etoposide. A better insight into the occurrence of deoxypodophyllotoxin combined with detailed knowledge of its biosynthestic pathway(s) may help to develop alternative sources for podophyllotoxin. Using HPLC combined with electrospray tandem mass spectrometry and NMR spectroscopy techniques, we found lignans and related structures in roots of Anthriscus sylvestris (L.) Hoffm. (Apiaceae), a common wild plant in temperate regions of the world. Podophyllotoxone, deoxypodophyllotoxin, yatein, anhydropodorhizol, 1-(3’-methoxy-4’,5’-methylenedioxyphenyl)1-ξ-methoxy-2-propene, and 2-butenoic acid, 2-methyl-4-[[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1-yl ester, (2Z)- were the major compounds. a-Peltatin, podophyllotoxin, b-peltatin, isopicropodophyllone, b-peltatin-a-methylether, (Z)-2-angeloyloxymethyl-2-butenoic acid, anthriscinol methylether, and anthriscrusin were present in lower concentrations. a-Peltatin, b-peltatin, isopicropodophyllone, podophyllotoxone, and b-peltatin-a-methylether have not been previously reported to be present in A. sylvestris. Based on our findings we propose a hypothetical biosynthetic pathway of aryltetralin lignans in A. sylvestris.

Keywords: Anthriscus sylvestris L. (Hoffm.), Apiaceae, cow parsley, wild chervil, LC-ESI-MS/MS, LC-SPE-NMR, aryltetralin lignans, biosynthetic pathway, deoxypodophyllotoxin

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Anthriscus sylvestris L. (Hoffm.) (Apiaceae) is a perennial herb that grows in Europe and in parts of North America, Africa, Asia and New Zealand.1-3 The aryltetralin lignan deoxypodophyllotoxin (6) is an interesting constituent of the plant because it can be used as a precursor for the production of podophyllotoxin (2).4 Podophyllotoxin (2) is important as a semi-synthetic precursor for the anticancer drugs etoposide, teniposide, and etopophos.5

To date, 2 is obtained by isolation from Podophyllum species. In the future, the availability of 2 from this source is likely to become a major bottleneck. Podophyllum species have been listed on the endangered species list in India, proving that the continuous demand of 2 is a serious threat for their existence.6 An alternative source of 2 may be found by the (biotechnological) hydroxylation of 6 at the C7 position.7 Compound 6 is much more widespread in the plant kingdom than 2. 1-4 A better insight into the occurrence of 6 combined with profound knowledge of its biosynthetic pathway(s) may help to develop alternative sources for the desired lignans.

In addition to 6, several other compounds have been reported to occur in A. sylvestris, including monoterpenes, anthricinol, angeloyl butenoid acid, anthriscusin (13), anthricin, isoanthricin and crocactone.8-10 The objective of the present study was to further investigate the occurrence of lignans and related structures in A. sylvestris. A better insight into the phytochemistry of A. sylvestris will increase our understanding of possible biosynthetic pathways for podophyllotoxin and related lignans and contribute to the development of alternative sources for the desired compounds. LC-SPE-NMR and LC-ESI-MS/MS techniques were used for the identification and structure elucidation.

reSuLtS And diSCuSSion

A typical HPLC chromatogram of the aerial and the root parts of A. sylvestris is shown in figure 1. Because the underground parts contained more compounds and at higher concentrations compared to the aerial parts, we used the root extract for the separation, isolation and identification of lignans and related structures, 14 in total.

Compounds 1, 2, 3, and 6-9 were identified based on comparison with pure reference compounds using HPLC, molecular weights and fragment ions by LC-MS/MS. Compound 4, 5, and 10-14 were identified based on molecular weights, fragment ions, 13C NMR and 1H NMR spectra. The chemical structures are shown in figure 2. The molecular weights and the fragment ions are summarized in table 1. The comprehensive and detailed assignment of the multiplicity and coupling constants of 13C NMR and 1H NMR spectra are summarized in table 2.

We are the first to report on the presence of a-peltatin (1), b-peltatin (3), isopicropodophyllone (4), podophyllotoxone (5) and b-peltatin-a-methylether (9) in A. sylvestris.

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Figure 1. UV chromatograms obtained at 240 nm of aerial parts and root extract of Anthriscus sylvestris.

Figure 1. UV chromatograms obtained at 240 nm of aerial parts and root extract of

Anthriscus sylvestris

Compounds 1, 2, 3, and 6-9 were identified based on comparison with pure

reference compounds using HPLC, molecular weights and fragment ions by LC-

MS/MS. Compound 4, 5, and 10-14 were identified based on molecular weights,

fragment ions, 13C NMR and 1H NMR spectra. The chemical structures are shown

in figure 2. The molecular weights and the fragment ions are summarized in

table 1. The comprehensive and detailed assignment of the multiplicity and

coupling constants of 13C NMR and 1H NMR spectra are summarized in table 2.

Compound 1 (mw: 400), 2 (mw: 414), 3 (mw: 414), and b-peltatin-a-methylether (9, mw: 428) are aryltetralin lignans. The general fragmentation ions were produced by loss of the pendant phenyl substituent [B+H]+ and subsequent loss of CO2 from the lactone ring [B-CO2+H]+.11 The latter fragment is either not observed or very weak. [B+H]+ shows the dehydration product [B-H2O+H]+ with somewhat higher intensity.11

The fragment ions of 1 are 247 [B+H]+, 229 [B+H-H2O]+, and 185 [A]+. The fragment ions of 2 are 313 [A+H]+, 247 [B+H]+, 229 [B+H-H2O]+, and 185 [B+H-H2O-CO2]

+. Compound 3 has the same fragmentation as 1 with additional 203 [B-CO2+H]+. The fragment ions of compond 9 are 261 [B+H]+ and 217 [B-CO2+H]+. The concentration of these peltatins in A. sylvestris were low and hardly detectable by HPLC. However using combined LC-MS/MS, a technique more sensitive than HPLC-UV alone, we could confirm the identity of compounds 1, 2, 3 and 9, also in comparison to the literature.11

Compounds 4 and 5 have the same molecular weight of 412 and the same fragment ion patterns, 245 [B+H]+ and 201 [B-CO2]

+. We further identified the stereochemistry by 1H-NMR and 13C-NMR and compared it to the literature.12, 13 The coupling constant and multiplicity of especially H-7’, H-8’, H-8 and H-9 determine the stereochemistry. The signals of H-7’ of compound 4 and 5 appear as doublets at δ 4.72 (J = 6.3 Hz) and δ 4.84 (J = 4.6 Hz), respectively. These results are not in agreement with picropodophyllone13 which has a singlet for this proton resonance. The multiplicity of H-8’ of compound 4 is multiplet at δ 3.77 whereas this signal in compound 5 appears

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as a doublet of doublets (dd) at δ 3.77 (J = 15.6, 4.6 Hz). The multiplicity of H-8 of compound 4 is multiplet at δ 3.83 whereas this signal in compound 5 appears as a doublet of doublets (ddd) at δ 3.59 (J = 15.6 and 4.7 Hz). The multiplicity of H-9 of compound 4 is multiplet at δ 3.87 and triplet at δ 4.50 (J= 7.9 Hz) whereas this signal in compound 5 appears as a doublet of doublets (dd) at δ 4.31 (J=10.3 and 8.4 Hz) and triplet at δ 4.49 (J=7.9 Hz). The 13C-NMR spectra also showed characteristic values distinguishing these stereochemistry at C-3, C-7, C-8, C-9, C-8’, and C-9’. These values are similar with the reported data.12, 13

The main lignan in A. sylvestris is deoxypodophyllotoxin (6). The identity of this compound was based on comparison of the retention time of reference compound by HPLC, the molecular weight (398), the fragment ions, and the 13C NMR and 1H NMR spectra. Our data are in agreement with literature.14

Table 1. LC-ESI-MS and LC-ESI-MS/MS data of the compounds identified in root extract of Anthriscus sylvestris.

No Compound MW Molecular Ions of [M+NH4]

+ or [M+H]+ion

Fragment ions

1 a-Peltatin 400 418 247, 229, 1852 Podophyllotoxin 414 432 313, 247, 229, 1853 b-Peltatin 414 432 247, 229, 203, 1854 Isopicropodophyllone 412 430 245, 2015 Podophyllotoxone 412 430 245, 2016 Deoxypodophyllotoxin 398 416 231, 1877 Yatein 400 418 223, 1818 Anhydropodorhizol 398 416 231, 1359 b-Peltatin-a-methylether 428 446 261, 21710 (Z)-2-angeloyloxymethyl-2-butenoic acid 198 199 18111 Anthriscinol methyl ether 222 223 191, 16112 1-(3’-methoxy-4’,5’-

methylenedioxyphenyl)1-ς-methoxy-2-propene

222 229d 191, 161

13 Anthriscrusin 388 406 19114 2-butenoic acid, 2-methyl-4-[[(2Z)-

2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1yl ester, (2Z)-

388 406 191

a Unknown 428b Unknown 416c Unknown 392

d[M+Li]+

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Figure 2. Chemical structures of compounds in the root extract of Anthriscus sylvestris

Table 1. LC-ESI-MS and LC-ESI-MS/MS data of the compounds identified in root extract

of Anthriscus sylvestris

No Compound MW Molecular Ions of [M+NH4]+ or [M+H]+ion

Fragment ions

1 α-Peltatin 400 418 247, 229, 185

Figure 2. Chemical structures of compounds in the root extract of Anthriscus sylvestris.

We identified compound 10 as (Z)-2-angeloyloxymethyl-2-butenoic acid, an acyloxycarbocyclic acid, based on the molecular weight (198), and 1H-NMR and 13C-NMR spectra. Our proton NMR data (table 2) are in agreement with those published by Kozawa who isolated this compound from A. sylvestris.8

Compound 11 was identified as anthriscinol methyl ether, a phenylpropanoid derivative, based on the molecular weight (222), the 1H-NMR and 13C-NMR spectra. The NMR spectra were confirmed with reported literature.15 Compound 11 has been reported to display weak insecticidal activity.16

The molecular weight of compound 12 is 222. The 13C NMR and 1H NMR data compared with the literature13 confirmed that compound 12 is 1-(3’-methoxy-4’,5’-methylenedioxyphenyl)-1ξ-methoxy-2-propene.15 The isolation and uses of compound 12 in A. sylvestris are for prevention or treatment of cancer or condyloma.17

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3The molecular weight of compounds 13 and 14 is 388 and showed similar fragment

ions in LC-MS data. The NMR data and compared with data from the literature9, 15 suggested that compound 13 is O-[(Z)-2-angeloyloxymethyl-2-butenoyl]-3-methoxy-4,5-methylendioxyamyl alcohol (anthriscusin). It is a phenylpropanoid ester derived from anthriscinol and compound 10.16 Based on our intepretation of 13C NMR and 1H NMR data, we propose that compound 14 is 2-butenoic acid, 2-methyl-4-[[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1-yl ester, (2Z)-. Selective 1D NOE experiments were carried out to identify the NOE long range coupling for the 4’’ CH to the 3’’ CH3 group. The NOE was difficult to identify in the 2D NOESY experiment because it overlapped with the large NOE between 3’’’ and 4’’’. The weak NOE between CH2 and the CH3 could be explained by spin-diffusion because it is very far away through space. The biological study, isolation and uses of compound 14 in A. sylvestris for prevention or treatment of cancer or condyloma were patented.17 However there was no NMR or MS data reported in the patent.

The major compounds in the roots were compounds 5-8, 12 and 14. Compound 1 and 3 were isolated from P. hexandrum for the first time.18 In A. sylvestris, we now report and structure elucidated these compounds for the first time. Trace amounts of compound 2 (< 0.01 µg/mg d.w.) have been reported in A. sylvestris.19 Compounds 10 and 13 have been isolated earlier from the roots of A. sylvestris.8, 9 Compound 6 was isolated for the first time from the roots of A. sylvestris.10 Compound 7, 11, 12 have been isolated from the roots of A. sylvestris.15 Compound 7 and 12 were also isolated from the fruit of A. sylvestris.20

The low concentration of 1 and 3 might be due to the fact that A. sylvestris was collected after having flowered in May 2007. A decrease of these compounds after flowering has also been shown in Podophyllum species.21 The loss of 1 and 3 during growth might due to metabolic turnover.21

The lignans isolated in this study (1-9) may be involved in the late biosynthesis pathway of aryltetralin lignans in A. sylvestris. Federolf et al.22 has recently proposed hypothetical pathway of 2 in Linum album. Combined with our findings, we propose a biosynthetic pathway of 6 and related lignans in A. sylvestris (figure 3).

Matairesinol was incorporated effectively into 1, 2, 3, 4’-demethylpodophyllotoxin, and 4’-demethyldeoxypodophyllotoxin in P. hexandrum root, P. peltatum leaves and Diphylleia cymosa leaves.23 In A. sylvestris, matairesinol may be incorporated into 7, 8, 4’-demethylyatein (putative intermediate) and other compounds24 which are further metabolized into 6, 2, 5, 1, 3, and 9 (figure 3).

Feeding experiments with labelled 6 and 4’-demethyldeoxypodophyllotoxin in P. hexandrum showed that these compounds are the likely precursors of 1 and 3 respectively, by hydroxylation in the aromatic ring.21 The transformation of compound 6 into 2 and 3 has been shown in a feeding experiment in P. hexandrum.21

In P. hexandrum, it has been shown that 2 is derived from 6 by hydroxylation. Further oxidation yielded 5, the later reaction being reversible.18, 25 Compound 5 may be also reduced in vivo into 2.25 In our studies with A. sylvestris, 2 was detected in low amount together with its oxidation products, 5 and its diasteoisomer, 4 were present.

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3 Table 2. NMR spectroscopic data (500MHz, d6-DMSO) for metabolites identified in Anthriscus sylvestris root extracts by LC-SPE-NMR.

Position2 4 5 6

δ C δ H (J in Hz)

δ C δ H (J in Hz)

δ C δ H (J in Hz)

δ C δ H (J in Hz)

1 166.9 128.0 127.9 131.0 6.49, dt (15.8)2 127.3 140.0 141.6 124.9 6.25, dt

(15.8, 5.8, 5.8)3 137.1 6.10, qq 108.0 6.86, s 109.4 6.85, s 71.9 4.00, dd

(6.0, 1.8)4 15.2 1.88, dq (7.4, 1.8,

1.6, 1.5)152.7 152.4

5 20.0 1.81, q (1.45, 1.57, 1.62, 1.46)

147.6 147.6

6 104.7 7.31, s 104.4 7.40, s7 195.0 192.48 44.2 3.83, m 42.8 3.59, ddd

(15.6, 4.7)9 68.5 3.87, m, 4.50,

t (7.9) 66.4 4.31, dd

(10.3, 8.4) and 4.49, t (7.9)

1’ 64.7 4.71, s 135.1 133.2 131.32’ 128.5 106.3 6.35, s 107.5 6.37, s 106.7 6.72, d (1.6)3’ 141.1 6.41, q (7.5, 7.2) 152.6 152.4 143.44’ 15.2 2.00, d (7.2) 136.8 136.8 134.55’ 167.2 152.6 152.4 148.86’ 12.67, s 106.3 6.35, s 107.5 6.37, s 99.4 6.77, d (1.1)7’ 42.7 4.72, d (6.3) 43.6 4.84, d (4.6)8’ 43.5 3.77, m 44.8 3.77, dd (15.6, 4.6)9’ 175.4 173.6

OCH2O 102.6 6.13, d (0.8), 6.13, d (0.8)

102.9 6.16, d (0.8), 6.16, d (0.8)

101.0 5.97, s

C-3’, OCH3

55.5 3.63, s 55.6 3.65, s 56.0 3.83, s

C-4’, OCH3

60.1 3.62, s 59.8 3.63, s

C-5’, OCH3

55.5 3.63, s 55.6 3.65, s

C-3, OCH3

57.0 3.27, s

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3Position

7 8 12 13δ C δ H

(J in Hz)δ C δ H

(J in Hz)δ C δ H

(J in Hz)δ C δ H

(J in Hz)

1 130.6 83.1 4.58, d (6.6) 133.2 6.57, dt (15.9, < 1) 133.1 6.61, dt (15.9, < 1)

2 129.1 138.7 5.86, ddd (17.1, 10.3,

6.6)

122.0 6.26, dt (15.9, 6.2) 122.1 6.31, dt (15.9, 6.3)

3 119.6 6.51, s 115.3 5.14 ddd (10.3, 1.8,

1.1); 5.25, ddd (17.2, 1.8, 1.3)

64.5 4.77, m (nd) 64.7 4.78, dd (6.2, 1.2)

4 145.9

5 145.9

6 108.2 6.81, s

7 31.8 2.75, dd (15.4, 11.7); 3.03, dd (16.4, 5.1)

8 32.3 2.62, m

9 71.3 3.96, dd (10.6, 8.2) ; 4.42, t (7.6)

1’ 137.1 135.7 131.0 130.8

2’ 108.0 6.30, s 106.1 6.58, d (1.3) 106.9 6.71, d (1.4) 107.1 6.76, d (1.4)

3’ 152.1 143.1 142.9 143.2

4’ 136.3 134.1 134.9 134.9

5’ 152.1 148.4 148.8 148.8

6’ 108.0 6.30, s 100.2 6.51, d (1.3) 99.7 6.75, d (1.3) 99.7 6.80, d (1.4)

7’ 42.8 4.52, d (5.3)

8’ 45.9 2.99, dd, (13.5, 5.2)

OCH2O 99.8 5.94, d (0.9)5.97, d (0.9)

100.0 5.95, s 101.0, CH2

5.96, s 100.6 5.96, s

C-3’, OCH3

55.6 3.64, s 56.0 3.79, s 56.1 3.83, s 56.1 3.84, s

C-4’, OCH3

59.7 3.62, s

C-5’, OCH3

55.6 3.64, s

C-1, OCH3

55.3 3.20, s

1’’ 165.3 166.2

2’’ 127.2 128.4

3’’ 143.1 6.53, qt (7.2, < 1) 19.3 1.93, tq (5.3, 1.6nd)

4’’ 15.6 2.03, dt (7.2, < 1) 138.1 6.18, tq (5.3, 16)

5’’ 64.6 4.78, m (nd) 61.8 5.02, dq (5.3, 1.8)

1’’’ 166.8 167.0

2’’’ 127.2 127.2

3’’’ 137.5 6.07, qq (7.2, 1.5) 137.8 6.14, qq (7.2, 1.5)

4’’’ 15.3 1.85, dq (7.2, 1.5) 15.5 1.92, dq (nd, 1.5)

5’’’ 20.1 1.78 , p (1.5) 20.1 1.85, p (1.5)

nd: not determined due to overlap

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Compound 4 was first isolated in nature from P. pleianthum.26 Compounds 4 and 5 have been reported to be present in P. pleianthum, P. hexandrum and P. peltatum.27 Since 5 is readily isomerised to 4 by heat, the latter compound may well be an artefact from 5 in Podophyllum extracts.26 Compound 4 is quite likely to be an artefact formed from 5.

Compound 7 has been reported to be an earlier precursor of deoxypodophyllo-toxin,25 while compound 3 is the product of the metabolization of 6 according to the hypothetical biosynthetic pathway of lignans which has been reported previously.18, 22 Broomhead et al.23 proposed 4’-demethylyatein and 4’-demethyldeoxypodophyllotoxin as putative intermediates between matairesinol and 1 in P. hexandrum root, P. peltatum leaf and Diphylleia cymosa leaf. Although 4’-demethylyatein and 4’-demethyldeoxypodophyllotoxin were not detected in the root extract of

present. Compound 4 was first isolated in nature from P. pleianthum.26

Compounds 4 and 5 have been reported to be present in P. pleianthum, P.

hexandrum and P. peltatum.27 Since 5 is readily isomerised to 4 by heat, the latter

compound may well be an artefact from 5 in Podophyllum extracts.26 Compound 4

is quite likely to be an artefact formed from 5.

Figure 3. Proposed biosynthetic pathway of deoxypodophyllotoxin and related lignans in Anthriscus sylvestris (additional compounds: 1-5, 8, and 9, nd = not detectable).

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3A.  syvlestris, these compounds may be putative intermediates between matairesinol and 1 (figure 3). However, we could not trace these earlier precursors such as matairesinol and later products of lignan biosynthesis detected in other plants, such as 6-methoxypodophyllotoxin.22

In the late biosynthetic pathway of podophyllotoxin,18, 22 we suggest a role of other (or: additional) compounds related to the formation of podophyllotoxin in A. sylvestris (figure 3). Our results add to what is known about the biosynthetic pathway of lignans in A. sylvestris. Additional experiments to further unravel this biosynthetic pathway of lignans in A. sylvestris are needed.

In summary, the combination of the liquid chromatography, electrospray ionization mass spectrometry (LC-ESI-MS/MS), and LC-SPE-NMR techniques are proven to be powerful tools to elucidate the structures unknown compounds in A. sylvestris extracts. In total, we identified 14 compounds in the root extract of A. syvlestris which may be involved in the late biosynthesis pathway of aryltetralin lignans as shown in figure 3. We identified the 6-methoxy lignans (1 and 3) as new side pathway, which previously was not known.

mAteriAL And metHodS

ChemicalsDeoxypodophyllotoxin was a gift from Dr. M. Angeles Castro (Salamanca University, Salamanca, Spain). Anhydropodorhizol, yatein, a-peltatin, b-peltatin and b-peltatin-a-methylether were kindly provided from Prof. M. Medarde (Salamanca University, Salamanca, Spain). Podophyllotoxin was from Sigma Aldrich (Zwijndrecht, the Netherlands). The identity and purity of these reference compounds were checked using HPLC. Acetonitrile and methanol were HPLC grade from Biosolve, Valkenswaard, the Netherlands. Dichloromethane was from Fisher Scientific, Landsmeer, the Netherlands. Formic acid and ammonium formate were from Sigma Aldrich, Zwijndrecht, the Netherlands. Deuterated DMSO (99.96 %) was from Eurisotop, Saint Aubin Cedex, France.

plant materialAnthriscus sylvestris L. Hoffm. (Apiaceae) plants at the flowering stage were collected in Groningen, the Netherlands (53o 11’ 34” N and 06o 37’ 04” E) in May 2007. Voucher specimen have been deposited in our department, encoded Asylv2007-4. The roots were separated from the aerial parts and the material was dried at room temperature after harvesting and prior to extraction.

Sample preparationThe extraction method was as described by Koulman et al.19 Shortly, 100 mg dried plant material were weighed into a Sovirel tube. A 2.0 mL portion of 80% methanol was added and the mixture was sonicated during 1 hour. Subsequently 4.0 mL of dichloromethane

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3and 4.0 mL H2O were added. The mixture was vortexed and centrifuged at 1,000 g for 5 minutes. The aqueous layer was discarded and 2.0 mL of organic layer were transferred into a 2 mL Eppendorf tube. The organic layer was left in the fume hood until dried. The residue was redissolved in 2.0 mL methanol and filtered over a 0.45 µm HPLC syringe filter (nylon). The samples were submitted to HPLC analysis. For LC-MS/MS and LC-SPE-NMR analysis 1 g dried root material was used and the aliquot residue was reconstituted in methanol at a concentration corresponding to 1 g dried plant material.

HpLC-uV analysisThe HPLC analysis was as described by Vasilev et al.7 with some modifications. A Shimadzu-VP system was used, consisting of an LC-10AT pump, a Kontron 360 auto sampler, an SPD M10A DAD detector, an FCV-10AL low pressure gradient mixer, an SCL-10A system controller, an FIAtron system CH-30 column heater, operated with LC Solution software, version 1.2. The column used was Zorbax Eclipse C18 (150 x 4.6 mm, 5 µm), together with Phenomenex guard cartidge C18 (4 x 3 mm) Phenomenex, Bester, The Netherlands. The detection wavelength was 240 nm. Mobile phase A [H2O: ACN (95:5)] and B [ACN: H2O (95:5)], both in 0.1% formic acid and 2 mM ammonium formate. The injection volume was 5 µL with a flow rate of 1 mL/min using a gradient program of 30 min consisting of 1 min of B 30%, followed by a linear gradient until 15 min B 90%, at 20 min B 90%, at 25 min B 30% and at 30 min B 30%.

LC-eSi-mS/mS analysis HPLC was performed using a Shimadzu LC system, consisting of 2 LC-20AD gradient pumps and a SIL-20AC autosampler. The LC system was coupled to an API 3000 triple-quadrupole mass spectrometer (Applied Biosystems/MDS Sciex) via a TurboIonSpray source. Data were collected and analyzed by Analyst 1.4.2 data acquisition software (Applied Biosystems/MDS Sciex). Chromatographic separation was achieved on an Alltima C18 (Grace Davison) narrow-bore guard column (2.1 x 150 mm, 5 µm). The mobile phase and the gradient system were the same as used for the HPLC analysis. The flow rate was 0.2 mL/min. The injection volume was dependent on the expected analyte concentration. The ionization was performed by electrospray in the positive mode, which resulted in the formation of (M+H)+ and/or (M+NH4)

+ adduct ions. The source temperature was set to 450°C. The instrument was operated with an ionspray voltage of 5.2 kV. Nitrogen was used both for curtain gas and nebulizing gas. Full scan mass spectra were acquired at a scan rate of 1 scan/sec with a scan range of 100-1100 amu and a step size of 1 amu. Defined product ion scans were acquired in specified time windows.

LC-Spe isolationHPLC was applied for the isolation of the individual compounds in pure form. Mobile phase A consisted of 0.1% formic acid in H2O, mobile phase B consisted of 0.1% formic acid in acetonitrile. The following method with gradient was applied with a flow-rate of 1 mL/min: 5 min isocratic at 32% B, followed by a linear gradient until 15 min B

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386%, quick increase in 0.1 min to 95% B, where kept constant for 5 min and followed by a quick descent in 1 min to 32% B, where kept for 5 min of equilibration prior to the next analysis. The purification was carried out on an Agilent 1200 system (Agilent Technology, SantaClara, USA) equipped with a Prospekt 2 (Spark Holland), using an Agilent column; Zorbax Eclipse XDB-C18, (150 x 4.6 mm, 5 µm). Compounds were trapped on general purpose (GP) cartridges (Spark Holland) and dried for at least 40 min under nitrogen air flow. Elution was performed using 600 mL of acetonitril, the purified fraction was dried under nitrogen air flow and dissolved in deuterated DMSO prior to NMR analyses.

nmr spectroscopyNMR analysis was performed on a Bruker Avance III 500MHz spectrometer equipped with a 5-mm CPTCI cryo-probe (1H-13C/15N/2H + Z-gradients) operating at 303 K. The structure identification of the purified compounds was based on a selection of 1D 1H, 1D selective NOE, 1H-1H-DQF-COSY, 1H-1H-TOCSY, 1H-13C-HSQC, 1H-13C-HMQC, and 1H-1H-NOESY spectra based on complexity or quantity of the sample. The proton and carbon chemical shifts were referenced to the internal reference TMS (proton, d = 0.00 ppm; carbon, d = 0.00 ppm). The data were processed using Topspin 2.1 pl5 and analysed with Mnova 6.2.1.

ACKnoWLedgementS

Financial support by the Ubbo Emmius Fund, University of Groningen, and the Müggenburg Foundation is greatly acknowledged.

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