Coniferin dimerisation in lignan biosynthesis in flax cells

Article abstract of DOI:10.1016/j.phytochem.2007.09.016

[¹³C₂]-Coniferin was provided to a flax (Linum usitatissimum L.) cell suspension to monitor subsequent dimerisation by MS and NMR. The label was mainly incorporated into a 8-8′-linked lignan, lariciresinol diglucoside, a 8-5′-linked

Full text of DOI:10.1016/j.phytochem.2007.09.016

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 68 (2007) 2744–2752
www.elsevier.com/locate/phytochem
Coniferin dimerisation in lignan biosynthesis in flax cells
a,b
a
c
d
b
´
Vickram Beejmohun , Ophelie Fliniaux , Christophe Hano , Serge Pilard , Eric Grand ,
b
d
c
c
b
´ ´
´
´
David Lesur , Dominique Cailleu , Frederic Lamblin , Eric Laine , Jose Kovensky ,
a
a,
*
´
Marc-Andre Fliniaux , Franc¸ois Mesnard
a
Laboratoire de Phytotechnologie EA 3900, Faculte´ de Pharmacie, Universite´ de Picardie Jules Verne, 1 Rue des Louvels, 80037 Amiens, France
Laboratoire des Glucides CNRS UMR 6219, Faculte´ des Sciences, Universite´ de Picardie Jules Verne, 33 Rue Saint-Leu, 80039 Amiens, France
b
c
Laboratoire de Biologie des Ligneux et des Grandes Cultures UPRES EA 1207, Antenne Scientifique Universitaire de Chartres, 21 Rue de Loigny la
Bataille, 28000 Chartres, France
d
Plate-forme Analytique, Faculte´ des Sciences, Universite´ de Picardie Jules Verne, 33 Rue Saint-Leu, 80039 Amiens, France
Received 6 April 2007; received in revised form 15 September 2007
Available online 7 November 2007
Abstract
[13C2]-Coniferin was provided to a flax (Linum usitatissimum L.) cell suspension to monitor subsequent dimerisation by MS and
NMR. The label was mainly incorporated into a 8–80-linked lignan, lariciresinol diglucoside, a 8–50-linked neolignan, dehydrodiconiferyl
alcohol glucoside and a diastereoisomeric mixture of a 8-O-40-linked neolignan, guaiacylglycerol-b-coniferyl alcohol ether glucoside. This
latter compound is reported for the first time in flax. The strong and transient increase in these compounds in fed cells was concomitant
with the observed peak in coniferin content. These results suggest (i) a rapid metabolisation of coniferin into lignans and neolignans and
indicate the capacity of flax cells to operate different types of couplings, and (ii) a continuous synthesis and subsequent metabolisation of
coniferin-derived dimers all over the culture period.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Linum usitatissimum; Linaceae; Flax; LC-MS; MS/MS; NMR; Labelled coniferin; Lignan; Neolignan
1. Introduction
lites. When this dimerisation involves an oxidative linkage
through the C-8 of the propenyl side chains of two conife-
ryl alcohol moieties, forming 8–80 bonds, the resulting
metabolites are called lignans. The term neolignan is used
to define all the other types of linkage (Moss, 2000). Many
of the dimers present in vascular plants have linkages other
than 8–80 and, of these, the bulk of the biochemical work
has thus far been directed towards elucidating mechanisms
of 8–50, 8-O-40 and 8–20-linked neolignan formation (Davin
and Lewis, 2003).
Lignans and neolignans, found in a wide range of plant
species, display numerous pharmacological activities (Pool-
Zobel et al., 2000; Arroo et al., 2002). The major lignans
from flaxseed (Linum usitatissimum), secoisolariciresinol
diglucoside and matairesinol, are converted into the ‘‘mam-
malian lignans’’ enterodiol and enterolactone by intestinal
bacteria (Wang et al., 2000). The beneficial effects of these
compounds on human health are well recognized (Westcott
Monolignols are derived from cinnamic acids and sup-
ply precursors for various phenylpropanoid compounds
such as lignins and lignans. The current view of the mono-
lignol biosynthetic pathway envisages a metabolic grid
leading to G and S units, through which the successive
hydroxylation and O-methylation reactions may occur at
different levels of side chain oxidation (Dixon et al.,
2001). This general monolignol biosynthetic pathway
occurring in most angiosperms is presented in Fig. 1
(adapted from Hoffmann et al., 2004 and from Shadle
et al., 2007). The oxidative dimerisation of two coniferyl
alcohol units leads to a great variety of secondary metabo-
*
Corresponding author. Tel.: +33 3 22 82 74 91; fax: +33 3 22 82 74 69.
E-mail address: francois.mesnard@u-picardie.fr (F. Mesnard).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.09.016

V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
2745
Fig. 1. Monolignol-derived products investigated in this study. (a) Monolignol biosynthetic pathway occurring in most angiosperms (from Hoffmann
et al., 2004 and from Shadle et al., 2007). PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate: CoA ligase; HCT:
hydroxycinnamoyltransferase; C3H: p-coumarate 3-hydroxylase; CCoAOMT: caffeoyl-CoA O-methyltransferase; COMT: caffeic acid O-methyltrans-
ferase; CCR, cinnamoyl-CoA reductase; F5H: ferulate 5-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; SAD, sinapyl alcohol dehydrogenase. (b)
Coniferyl alcohol-derived products: pinoresinol (8-80-linked lignan) guaicylglycerol-b-coniferyl ether (8-O-40-linked neolignan) and dehydrodiconiferyl
alcohol (8-50-linked phenylcoumaran lignan) resulting from simple dimerisation of two coniferyl alcohols.
and Muir, 2003; McCann et al., 2005) and in vitro studies
confirm the observed in vivo effect (Bylund et al., 2005).
In particular, they were shown to reduce the incidence of
breast and prostate cancers by modulating steroidal hor-
mone synthesis (Adlercreutz and Mazur, 1997). 8-O-40 neo-
lignans are used as lead compounds for antifungal agents
(Apers et al., 2003).
In view of the important pharmacological properties,
numerous studies have been carried out in an attempt to
get a better knowledge of the biological events linked to
the biosynthesis and the accumulation of lignans and neo-
lignans (Seidel et al., 2002; Sicilia et al., 2003). In flax,
coniferyl alcohol was clearly evidenced to be the monolig-
nol involved in lignan biosynthesis (Ford et al., 2001). Flax
suspension cells are also known to accumulate neolignans
(Attoumbre et al., 2006).
was used in feeding experiments with 13C-labelled coniferin
to investigate monolignol dimerisation into both lignans
and neolignans. In order to visualise its dimerisation by
NMR and MS, doubly labelled (8,9-13C2)-coniferin was
synthesised to offer easier detection (Beejmohun et al.,
2006). Coniferin, considered as the putative storage form
of coniferyl alcohol (Gross, 1985) was used to facilitate
the solubilisation into aqueous culture media (Van Uden
et al., 1995).
2. Results and discussion
2.1. Preliminary experiments
2.1.1. Growth kinetics
In an effort to study the biosynthesis and the accumula-
tion of secondary metabolites, in vitro cultures have been
established as a very useful tool because this system allows
uniformity, accessibility and reduced complexity (Facchini,
2001; Mesnard et al., 2002; Verpoorte et al., 2002). In the
present study, a recently established flax (L. usitatissimum)
cell suspension (Hano et al., 2006; Attoumbre et al., 2006)
For the feeding experiments, a minimum concentration
of 0.5 g/l (1.46 mM) of labelled coniferin was required
due to the sensitivity of the analytical methods used. Such
a concentration of an exogeneous precursor might be toxic
for the flax suspension cells (Edahiro et al., 2005). Prior to
experiments with labelled coniferin, the effect of unlabelled
coniferin supplementation on cell growth was therefore

2746
V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
evaluated under the same conditions as in the feeding
experiments (see Section 4.3).
Fig. 2 presents the L. usitatissimum cell growth kinetics
in presence or absence of coniferin. Over the cultivation
period of 14 days, the dry weight increased progressively
until a maximum value measured at day 8 for both condi-
tions. After one week of cultivation, the weight stabilised.
In spite of a slight difference during the exponential growth
phase (day 2 and day 4), both curves showed large similar-
ities indicating that coniferin supplementation at 0.5 g/l in
the culture medium did not affect cell growth.
2.1.2. Evolution of coniferin content in medium and cells
In order to verify that coniferin was incorporated into
the cells, the evolution of coniferin amount in the culture
media and inside the cells was evaluated by HPLC analysis.
Two control experiments were performed by following the
evolution of coniferin (i) in culture medium supplemented
with coniferin but without cells and (ii) in cells (and their
culture medium) untreated with coniferin.
As shown in Fig. 3a, the amount of coniferin remained
stable in culture medium supplemented with coniferin with-
out suspension cells over the 14 days period indicating that
coniferin was not spontaneously degraded in the medium.
The coniferin content rapidly decreased in coniferin supple-
mented culture media in presence of flax cells and was no
longer detectable from day 2 (Fig. 3a). From the 2.5 mg
coniferin added in the media in each well at the beginning
of the feeding experiments, only 0.2 mg was remaining at
day 1, suggesting that 2.3 mg were taken up by the cells
or cleaved into coniferyl alcohol. Indeed coniferin could
have been metabolised in coniferyl alcohol under the effect
of external glucosidases. The occurrence of coniferyl alco-
hol was therefore sought in the media, but was not detected
by HPLC, whatever the time of culture, which suggests that
coniferin was rapidly incorporated into the cells, as con-
firmed in Fig. 3b. This graph shows that the flax cells fed
with coniferin accumulated more coniferin in the first two
Fig. 3. Coniferin content in culture medium (a) and in L. usitatissimum
cells extracts (b). Experiments were done in triplicate.
days of the feeding experiment as compared to untreated
cells. Then, the coniferin content from day 2 evolved in
the same manner with or without supplementation. At
day 1, in treated cells, the coniferin concentration is
19.7 mg/g dry wt (Fig. 3b) which, for a total dry weight
of ca 25 mg, corresponds to a coniferin amount of
0.5 mg. At the same time of culture, the coniferin amount
in untreated cells was only 0.1 mg. Compared to untreated
cells, the total intracellular coniferin amount increased
therefore by 0.4 mg in treated cells, and it appears that
an unrecovered fraction of 1.9 mg (2.3–0.4) of the supple-
mented coniferin was metabolised. Only traces of coniferyl
alcohol were found in cell extracts showing that this com-
pound is not accumulated in these flax cells. However,
these data do not exclude a rapid conversion of coniferin
into coniferyl alcohol before a subsequent metabolisation
into other compounds, potentially lignans.
2.2. Metabolisation of coniferin
2.2.1. Identification of the labelled compounds
Feeding experiments were carried out with labelled and
unlabelled coniferin. Since the aim of the study was the
Fig. 2. Growth curves of cell suspensions of L. usitatissimum cultured with
or without coniferin. Experiments were done in triplicate.

V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
2747
investigation of (neo)lignan synthesis in flax cells, a classi-
cal methanolic extraction was performed before applying
appropriate chromatographic analysis (Hano et al., 2006).
By comparison with feeding experiments with unlabelled
coniferin, LC-MS analysis of the flax cells methanolic
extracts revealed the incorporation of the label mainly into
five compounds: peak 1 at M+2 and peaks 2, 3a, 3b, 4 at
M+2 and at M+4 (Figs. 4 and 5), whatever the time of har-
vest. It is noteworthy that these five compounds were also
accumulated in untreated cells, indicating that their biosyn-
thesis is not specifically related to the supplementation.
Peak 1 with a retention time of 21.3 min was easily
attributed to coniferin by comparison with a reference
compound. This observation confirmed the incorporation
of exogenous labelled coniferin into the intracellular conif-
erin pool before it is metabolised (Fig. 3b).
By comparison to a reference compound, peak 2 (Rt =
24.6 min) was assigned to lariciresinol diglucoside (Beejmo-
hun, 2006). This tetrahydrofuran lignan is obtained from
the reduction of pinoresinol – known to be formed by the
dimerisation of two coniferyl alcohol moities through an
8–80 bond (Davin and Lewis, 2003) – catalysed by pinores-
inol-lariciresinol reductase. The same enzyme catalyses the
formation of secoisolariciresinol from lariciresinol.
Pinoresinol diglucoside was found labelled in trace amount
(data not shown) but no secoisolariciresinol or secoisolaric-
iresinol diglucoside was detected. This result is somewhat
surprising but could be explained by a metabolisation of
secoisolariciresinol at a rate higher than its biosynthesis
from lariciresinol, thus leading to the accumulation of this
intermediate product. The presence of the aglycone laric-
iresinol has already been reported at low amounts in a cell
suspension of another Linum species (L. album, Smollny
et al., 1998).
[M+Na]+ m/z 561, found 561.1931 for C26O12H34Na
requires 561.1948). The structure of these compounds
was elucidated by MS/MS and NMR experiments. They
are the erythro (a) and threo (b) forms of the glucoside of
1-(4-hydroxy-3-methoxyphenyl)-2-(4-(3-hydroxy-(E)-prop-
1-enyl)-2-methoxyphenoxy)propane-1,3-diol, also called
guaiacylglycerol-b-coniferyl alcohol ether glucoside
(GGCG). Several glucosides formed from the same agly-
cone have been already described in several plants: Arum
italicum (Della Greca et al., 1994), Echinacea purpurea (Li
and Barz, 2006), Citrus limon, as citrusin A (Matsubara
et al., 1991), Glehnia littoralis, as citrusin A (Yuan et al.,
2002), Angelica furcijuga as hyuganoside III (Morikawa
et al., 2004). However, they have not yet been observed
in Linaceae. The MS/MS spectrum of these compounds
isolated from the cell extracts cultured with unlabelled
coniferin, obtained in the positive ion mode from the
CID (collision induced dissociation, collision energy
37 V) process of the [M+Na]+ ion (m/z 561), showed a
fragmentation pattern similar to that described in the liter-
ature (Li and Barz, 2006). The identification of compounds
3a and 3b was confirmed by 1H NMR data which were in
accordance with previous data reported in the literature
(Takara et al., 2002; Morikawa et al., 2004).
Peak 4 was eluted at 33.0 min. By comparison with an
authentic standard, it was attributed to a 8–50 neolignan
(dehydrodiconiferyl alcohol-4-b-D-glucoside) which has
already been characterised in L. usitatissimum cell cultures
(Attoumbre et al., 2006). It was shown to accumulate at
high amounts which is in agreement with the concentration
values (5–30 mg/g dry wt) found during the experiments
reported here. These labelling experiments showed that this
compound was not released from the cell-wall but was pro-
duced directly from coniferin/coniferyl alcohol. This result
is in agreement with previous labelling experiments
reported by Orr and Lynn (1992) who showed the labelling
of dehydrodiconyferyl alcohol when incubating Vinca cells
with [3H]-coniferyl alcohol. They proposed a direct biosyn-
thetic pathway involving the 8–50 coupling of two coniferyl
alcohol moities. In our experiments, the M+4 enrichment
of this compound revealing the incorporation of two dou-
bly-labelled coniferin molecules might confirm this hypoth-
esis but nothing is known about which carbons
incorporated the label and consequently the mechanism
of coupling. Fig. 6a represents the NMR spectrum of a
methanolic extract of flax cells 24 h after feeding with
(8,9-13C2)-coniferin. By comparison with a control per-
formed in the same conditions with unlabelled coniferin
(Fig. 6b), several doublets are clearly visible at
d = 55.5 ppm (m = 38.1 Hz), d = 64.0 ppm (m = 47.7 Hz),
d = 65.1 ppm (m = 38.1 Hz), and d = 127.8 ppm (m =
47.7 Hz). They respectively correspond to the C80, C9,
C90 and C8 (coupled with their labelled neighbour carbon)
of DCG, unambiguously confirming the biosynthetic path-
way described above.
Peaks 3a and b showed similar positive and negative
ESI-MS spectra ([M+Na]+ m/z 561 and [MꢀH]ꢀ m/z
537) corresponding to the same molecular weight of 538.
A formula of C26O12H34 was proposed following accurate
mass measurements (3a: [M+Na]+ m/z 561, found
561.1960 for C26O12H34Na requires 561.1948, 3b:
The biosynthesis of these four labelled molecules is
highly likely to involve the same enzymatic systems as
Fig. 4. HPLC chromatogram of an extract of L. usitatissimum cell
suspension fed with coniferin for 8 h.

2748
V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
Fig. 5. ESI-MS spectra of the compounds 1, 2, 3 and 4 from the extract of a L. usitatissimum cell suspension cultured with 13C2-coniferin for 48 h.
Fig. 6. NMR spectra of a flax cell extract after feeding of the cells with labelled (a) or unlabelled (b) coniferin for 24 h.

V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
2749
those involved in untreated cells since the coniferin supple-
mentation did not induce the formation of any new com-
pound (fed-cell extracts show the same HPLC profile as
untreated cell extracts). Note that even isomeric forms of
these labelled molecules would have been detected. Indeed,
these molecules being glucosylated, enantiomers cannot be
found (since it would involve the biosynthesis of L-glucose
molecules) but only diastereoisomers which are detectable
by LC-MS and NMR.
2.2.2. Kinetics of (neo)lignan biosynthesis
The kinetics of the accumulation of coniferin derived
lignans and neolignans reported in Fig. 7 indicated a rapid
increase in the first two days of culture after addition of
coniferin, reaching a maximum amount at day 1 (from 3-
to 6-fold increase when compared to untreated cells val-
ues). These increases (by 0.7 mg DCG, 0.1 mg LDG,
0.2 mg erythro-GGCG, 0.2 mg threo-GGCG) were con-
comitant with the observed increase in coniferin content
in fed-cells; they correspond to the use of ca 80% of the
1.9 mg unrecovered coniferin (calculated in Section 2.1.2).
After 2 days, lignan and neolignan contents in coniferin
fed-cells progressively returned to that observed in
untreated cells. These results suggest a rapid metabolisa-
tion of coniferin into lignans and neolignans, which would
be quickly further transformed. This hypothesis is sup-
ported by the ratio of labelled versus unlabelled com-
pounds (Table 1). Indeed labelled dimers were mainly
accumulated at 48 h (maximum ratios labelled vs unla-
belled), following the maximum ratio of labelled coniferin
observed at day 1. Then these ratios decreased, suggesting
a continuous synthesis and subsequent metabolisation of
coniferin and coniferin-derived dimers all over the culture
period. Coniferin and/or coniferin-derived dimers might
be incorporated into higher polymers such as lignin; which
might correspond to the remaining 20% of the unrecovered
coniferin, at day 1.
Indeed, it is generally assumed that coniferin is a stor-
age form of lignin and/or lignan precursors because this
glycosylated form of coniferyl alcohol has a markedly
higher water solubility and stability towards oxidation
(Whetten et al., 1998). Our results seem to indicate the
existence of a mechanism regulating the intracellular
steady state levels of coniferin and coniferin-derived
dimers in flax cells keeping them constant in both
untreated and coniferin fed-cells.
Lignins and lignans share monolignols as common pre-
cursors and are both potentially involved in plant defence
against pathogens (Gang et al., 1999). Elicitation of cell
cultures with Fusarium oxysporum extracts triggered a
strong incorporation of monolignols in the non condensed
labile ether-linked lignin fraction concomitantly with a
decrease in 8–80 and 8–50 lignan accumulation (Hano
et al., 2006). One hypothesis to explain such a decrease in
lignan and neolignan accumulation in elicited cell cultures
was their possible incorporation in the lignin polymer to
contribute to cell wall reinforcement. Such metabolisation
Fig. 7. Kinetics of accumulation of compounds 2 (LDG), 3 (GGCG) and
4 (DCG) over the 14-day experiment, in untreated cells and in cells fed
with coniferin. Both forms (erythro and threo) of GGCG show similar
accumulation kinetics. The kinetics of the erythro form is presented here.
Experiments were done in triplicate.
of lignan and neolignan compounds has been already
observed during tracheary element differentiation of iso-
lated Zinnia elegans mesophyll cells (Tokunaga et al.,
2005). These authors also reported the incorporation of
pinoresinol, erythro- and threo-guaicylglycerol-b-coniferyl
ether and dehydrodiconiferyl alcohol into the lignin
polymer.

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V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
Table 1
syl bromide and silver oxide in quinoline. The
monolignol glucoside (8,9-13C2)-coniferin was then syn-
thesised by condensation of the corresponding aldehyde
with (1,2,3-13C3)-malonic acid as previously described
(Beejmohun et al., 2006). The carboxylic acid was con-
verted to the acyl chloride prior to its reduction to alco-
hol. Pure double labelled lignan precursors could only
be obtained after separation from their contaminating
Z-isomers and dihydro by-products by high-performance
liquid chromatography.
Kinetics of label incorporation in compounds 1, 2, 3, and 4
Cultivation time (h)
8
24
48
96
192
336
Coniferin
Unlabelled
Labelled
LDG
86
14
38
62
45
55
75
25
84
16
84
16
Unlabelled
Labelled
GGCG
87
13
60
40
36
74
29
71
52
48
41
59
Unlabelled
Labelled
DCG
93
7
47
53
20
80
26
74
42
58
44
56
4.3. Feeding experiments
Unlabelled
Labelled
61
39
34
66
18
82
26
74
51
49
45
55
All feeding experiments were performed in 6-well sterile
plates. To each well was added 300 mg fresh weight of cells
in exponential growth-phase and 5 ml of sterile culture
medium containing labelled or unlabelled coniferin at
0.5 mg/ml. At 8, 24, 48, 96, 192 and 336 h following precur-
sor addition, the cells were harvested, separated from the
medium by filtration, washed with 5 ml water, frozen and
lyophilised. Culture media and washings were pooled, fro-
zen and lyophilised. Standard experiments were carried out
under the same conditions but without coniferin addition
in culture media.
Both forms (erythro and threo) of GGCG show similar incorporation
kinetics. The kinetics of the erythro form is presented here. Results are
given in percent
3. Concluding remarks
Four major labelled-dilignols were identified in conifer-
in-fed cell suspensions: lariciresinol diglucoside (8–80-
linked lignan), dehydrodiconiferyl alcohol glucoside (8–
50-linked neolignan) and finally the erythro and threo forms
of guaicylglycerol-b-coniferyl ether glucoside (8-O-40-
linked neolignan). To our knowledge, this latter compound
was evidenced for the first time in flax.
Limited information is available regarding lignan and
neolignan biosynthesis accumulation and regulation. The
system presented herein will be of interest in order to con-
firm or infirm the hypothesis of an incorporation of
(neo)lignans into lignin under developmental or stress con-
ditions. Indeed it will be possible to purify different 13C-
labelled (neo)lignans which will be provided to cell cultures
to see whether the label is incorporated into lignin.
4.4. Extraction
Each sample of dried cells was extracted twice with
20 ml MeOH 70% (v/v) with gentle shaking, at room tem-
perature, for 24 h. The extracts were filtered and reduced to
dryness under vacuum. Residues were resuspended in
500 ll MeOD for NMR analysis. 20 ll of o-coumaric acid
solution (0.25 g/l) was added to 80 ll of the methanolic
extract for LC analysis.
Lyophilised culture media were resuspended in 1100 ll
of MeOH and 20 ll of o-coumaric acid solution (1 g/l) were
added to 180 ll of the methanolic extract for LC analysis.
4. Experimental
4.5. NMR analysis
4.1. Plant material
13C NMR spectra were recorded at 300 K on a Bruker
Avance DMX 300 spectrometer, operating at 75.47 MHz
using a 5 mm broad-band probe head. Spectra were accumu-
lated using a 90ꢁ pulse angle, a recycle time of 4 s and an
acquisition time of 1.08 s, for a spectral width of 15 kHz
for 32 K data points. Before Fourier transformation, a zero
filling to 64 K was applied, and a line broadening of 5 Hz was
used to improve the spectral signal-to-noise ratio.
Spectra were acquired for 10,900 scans (meaning
15h 30 experiment time). All 13C resonances were
assigned through comparison with the chemical shifts
of authentic reference compounds and with the literature
data.
The 2-1B cell suspension culture of L. usitatissimum L. cv
Barbara was established from hypocotyl-derived calli in
MS-derived medium (Murashige and Skoog, 1962) supple-
mented with 3% (w/v) sucrose, 8.88 lM benzylaminopurine
and 2.68 lM a-naphthalene acetic acid. All suspension cul-
tures were incubated on a gyratory shaker at 120 rpm, with a
5 cm orbital size, in darkness at 25 ꢁC and subcultured every
14 days. Usually, 100 ml MS-derived medium in a 250 ml
Erlenmeyer flask were inoculated with 5 g of cells. Growth
was evaluated by measuring dry weight of lyophilised cells.
4.2. Synthesis of [8,9-13C2]-coniferin
1H NMR spectra of compounds 3 were recorded at
300 K on a Bruker Avance DPX 300 spectrometer, operat-
ing at 300.13 MHz using a 5 mm selective probe head.
Spectra were accumulated using a 30ꢁ pulse angle, a recycle
Conversion of vanillin to its corresponding b-gluco-
side was performed with tetra-O-acetyl-a-D-glucopyrano-

V. Beejmohun et al. / Phytochemistry 68 (2007) 2744–2752
2751
time of 1 s and an acquisition time of 2 s, for a spectral
width of 3 kHz for 32 K data points.
The source and desolvation temperatures were kept at
80 and 150 ꢁC, respectively. Nitrogen was used as the dry-
ing and nebulizing gas at flow rates of 350 and 50 l hꢀ1
,
4.6. HPLC and LC-MS analysis
respectively. The capillary voltage was 3.5 kV, the cone
voltage 50 V and the RF lens 1 energy 20 V ( ESI).
For collision-induced dissociation (CID) experiments,
argon was used as collision gas at an indicated analyser
pressure of 5 · 10ꢀ5 Torr and the collision energy was opti-
mised for each parent ion (10 to 60 V). Lock mass correc-
tion, using appropriate cluster ions of an ortho-phosphoric
acid solution (0.1% in H2O/CH3CN 50/50, v/v), was
applied for accurate mass measurements. The mass range
was typically 50–1000 Da and spectra were recorded at
1 s/scan in the profile mode at a resolution of 10,000
(FWMH). Data acquisition and processing were performed
with MassLynx 4.0 software.
4.6.1. HPLC
A Merck-Hitachi HPLC system (Fontenay sous Bois,
France) equipped with a L7100 pump and a L7400 UV–
Vis detector was used. The separation was performed at
room temperature on a KROMASILꢂ (Macherey Nagel,
Hoerdt, France) C18 column (250 mm · 4.6 mm i.d.;
5 lm). The mobile phase consisted of 0.2% acetic acid in
water (solvent A) and acetonitrile (solvent B). The compo-
sition of the mobile phase varied during the run according
to a nonlinear gradient as follows: A–B: 0–10 min (100:0,
v/v), 10–30 min (82:18, v/v), 30–55 min (64:36, v/v) at a
flow rate of 0.8 ml/min. Detection was performed at
280 nm. Lignan and monolignol glucosides were identified
by comparison of their retention times to those of authentic
standards and by mass spectrometry measurements.
Acknowledgements
This work was carried out with the financial contribu-
´
ˆ
tion of the Conseil Regional de Picardie (Pole IBFBio).
4.6.2. LC-MS
´
V.B. thanks the Conseil Regional de Picardie for a
LC-MS analyses were performed on a Waters 2695 Alli-
ance coupled with a single quadrupole mass spectrometer
ZQ (Waters-Micromass, Manchester, UK) equipped with
an electrospray ion source (ESI-MS).
fellowship.
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