Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue10.Dec2000:
Biochemical Systematics and Ecology 28 (2000) 949}962
HPLC analysis of the seasonal and diurnal
variation of iridoids in cultivars of Antirrhinum
majus
Birgitte Dr+hse H+gedal, Per M+lgaard*
Department of Medicinal Chemistry, Pharmacognosy Group, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received 17 June 1999; accepted 6 March 2000
Abstract
In this paper we show the seasonal and diurnal variation in the content of the four iridoids
found in cultivars of Antirrhinum majus, antirrhinoside, antirrhide, 5-glucosyl-antirrhinoside
and linarioside. The seasonal variation in total iridoid content showed a marked bimodal
distribution with high total values (around 100 mg/g dry matter) early and late in the season
and a very low content of all iridoids coinciding with the onset of #owering at the beginning of
August. The relative contribution of antirrhinoside was signi"cantly higher before #owering
than after bud break. The relative decrease in antirrhinoside was counteracted by an increase of
antirrhide, which was signi"cantly higher after the onset of #owering than before. This pattern
indicates a change in biosynthesis, although no explanation can be given to the phenomenon.
The diurnal variation showed a variation between 20 and 60 mg/g dry weight, but there was no
relation to light/darkness conditions, temperature patterns or water content. The analyses were
performed by HPLC. The applied method has not previously been used in the quanti"cation of
iridoids, but was developed speci"cally for the analyses of cultivars of Antirrhinum majus. We
have fully validated the method during its development. The limit of detection was calculated to
0.004 mg/ml and the limit of quanti"cation was 0.01 mg/ml. ( 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Antirrhinum majus; Scrophulariaceae; Iridoid glucoside; Antirrhinoside; Seasonal variation;
Diurnal variation
* Corresponding author. Tel.: #45-35-37-67-77; fax: #45-35-30-60-40.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 5 - 4
950
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
1. Introduction
Iridoids are secondary compounds con"ned to a restricted number of sympetalous
plant families. They are found in Corni#orae, Loasi#orae, Lamii#orae and Gentianiflorae and are part of the complex set of characters that bind these superorders
together in the classi"cation of Dahlgren (1989). The value of chemical characters,
including iridoids, in the classi"cation of the Asteridae s.l. has recently been discussed
by Grayer et al. (1999).
There are two di!erent pathways in the biosynthesis of iridoids of which route II
leads to the aucubin-type iridoids common in Lamii#orae (Jensen, 1992). Antirrhinum
majus in the Scrophulariaceae is a member of Lamii#orae and thus produces
aucubin-type iridoids. The cultivars &White Wonder' and &Yellow Monarch' contain
the four major iridoid compounds antirrhinoside, antirrhide, 5-glucosyl-antirrhinoside and linarioside (Fig. 1). Antirrhinoside in Antirrhinum is biosynthetically derived
from 8-epi-iridodial via route II as shown by Breinholt et al. (1992) and Damtoft et al.
(1993, 1995).
The variation in the content of secondary compounds is important for the interaction of plants with pathogens and herbivores as highlighted in general by Coleman
and Jones (1991). In several studies iridoids have been shown to a!ect the feeding
behaviour of herbivores. By a turnover from production of aucubin to catalpol the
seasonal variation could be related to a shift from generalist to specialist herbivores
on Plantago species during the season (Bowers et al., 1992; Bowers and Stamp, 1992;
Bowers, 1996). In general, iridoids act as a defence mechanism against herbivores,
although they may also attract adapted herbivores at certain times according to
composition and concentration (Adler et al., 1995; Bowers and Puttick, 1986; Bowers,
1984; Pereyra and Bowers, 1988).
It is well known that the concentration of phytochemicals varies during the season
(Heeger, 1956), not only iridoids (Bowers et al., 1992), but also other monoterpenoids
(Adams, 1987; Goralka and Langenheim, 1996; Goralka et al., 1996); sesquiterpenes
(Hendriks et al., 1997); triterpenes (Ndamba et al., 1994); phenolics (William and Ellis,
1989; Wilt and Miller, 1992), essential oils (El-Gengaihi and Wahba, 1995), peptides
(Scheer et al., 1992) and cyanide (Boyd et al., 1938). Diurnal variation in plant
secondary chemistry has also been observed, either related to light conditions
Fig. 1. Chemical structures of the four iridoids from Antirrhinum majus: antirrhinoside (1), antirrhide (2),
5-glucosyl-antirrhinoside (3), and linarioside (4).
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
951
(Dickson, 1987; Sporer et al., 1993), to water de"cit (Itenov et al., 1999) or to more
complex interactions (Rosa et al., 1994). Besides, seasonal #uctuations and variations
in secondary plant metabolites may be governed by genotype and in#uenced by
nutrient availability as shown for iridoids by Bowers et al. (1992) and Fajer et al.
(1992), respectively.
This project is part of an ongoing investigation of secondary plant compounds for
their potential use as starting material in the chemical and pharmaceutical industry.
The speci"c interest in iridoids stems from their structure with an inherent chiral
centre, which makes the compounds potential precursors for the semi-synthesis of
drugs with activity against HIV and cancer (Franzyk et al., 1997, 1998a,b).
Although Antirrhinum majus has a long reputation as an ornamental garden plant
with detailed knowledge on the genetics of #ower colour patterns (e.g. Stubbe, 1974),
very little is known on the variation in the content of iridoids. The content of iridoids
may be of importance for the interaction with pathogens and herbivores on A. majus,
and in relation to chemotaxonomy at the generic level. However, not only from a pure
scienti"c point of view but also in relation to A. majus as a potential producer of
starting material for "ne chemicals it is important to "nd out if there is a seasonal or
diurnal variation and turnover of the iridoids. There may be an inherent di!erence
between cultivars, and we designed this project to determine at which state the plants
contain the largest amount of iridoids or the best combination of individual compounds, so that harvesting of raw material for "ne chemicals can be planned accordingly.
Previously, quantitative analyses of iridoids have been based on either gas
chromatography (Bowers, 1996; Bowers and Stamp, 1992), or high-performance
liquid chromatography, HPLC (Baghdikian et al., 1997; Dallenbach-Toelke et al.,
1987; Lenherr et al., 1984; Meier and Sticher, 1977; Miyagoshi et al., 1986). The
present analyses are made by HPLC using a newly developed method. We have
developed this method speci"cally to quantify the iridoids in these cultivars of
A. majus. The method has been fully validated alongside the development.
2. Materials and methods
2.1. Plant material
Two Antirrhinum majus cultivars, White wonder and Yellow monarch, were "eld
grown at the Danish Institute of Agricultural Science, Research Center Flakkebjerg.
The seeds were obtained from Impecta Handels, 640 25 Julita, Sweden. The plants
were grown in open "elds on silty clay soil, with a distance of 45 cm between the rows.
Nitrogen was applied on June 2 at the rate of 50 kg N/ha.
2.1.1. Weather conditions
During the observation period, the weather conditions were monitored for air
temperature (C3), soil temperature (C3), rainfall (mm), evaporation (mm), wind speed
(m/s) and relative humidity (%). Temperature and rainfall are shown in Fig. 4.
952
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
2.1.2. Seasonal variation
Plant samples for chemical analyses were taken weekly between June 17, 1998 and
September 30, 1998, and twice a week during the #owering period (July 19 until
September 4). Seven shoots from separate plants were cut each time, washed, weighed
and pressed. The shoots were dried at 403C for 24 h. After drying the shoots were
weighed and the water content determined in percent. Five of these shoots (the two
extremes were discarded) were selected each sample day, and the leaves were powdered. During the drying process on July 22 and July 29 some of the plants were
damaged and therefore excluded from the study. After September 9 fungi attacked the
plants, and the experiment was terminated. The development of the plants was
observed during the whole period.
2.1.3. Diurnal variation
Samples were taken at the onset of #owering (August 5}6, sunrise 5:22, sunset 21:09,
Danish summer time 2 h ahead of GMT) and in the middle of the #owering period
(August 18}19, sunrise 5:48, sunset 20:38, Danish summertime 2 h ahead of GMT).
Each harvest started at 15:00 h, and "ve plants were sampled every hour over a 24 h
period. No plants were used twice. The lower side branch and the pair of leaves above
were collected. The lower side branch was placed in a plastic bag and frozen on solid
CO to stop instantly any enzymatic activity. The leaves were washed, dried and
2
placed in sample vials, weighed and frozen on solid CO . After 1 h on solid CO both
2
2
samples were moved to a freezer at !203C. The samples were lyophilised on
Drywinner 6-85, Heto Holm & Halby, at !953C under vacuum for 48 h.
2.2. Sample preparation
50 mg of powdered leaf material was weighed out and extracted for 1 h in an
ultra-sonic bath with 10 ml of a 20% methanol solution, containing 0.05 mg/ml of the
internal standard, p-hydroxybenzaldehyde. The extract was "ltered through a Minisart' "lter type RC-0.45 lm and analysed directly on HPLC.
2.3. HPLC analysis
The HPLC analyses were performed on a Shimadzu SCL-6A System controller
with Shimadzu SIL-6A Auto-injector with an injection volume of 10 ll, Shimadzu
CTO-6A Column oven at 403C, Shimadzu SPD-6AV Spectrophotometric detector at
205 nm and two Shimadzu LC-6A pumps with a #ow of 1 ml/min. The data processing
was carried out by a Class-LC10 version 1.41. The column was a Macherey-Nagel
Nucleosil C18-5l, 125]4.6 mm2. The system was an isocratic 3% acetonitrile in
water, with an analysis time of 25 min. The internal standard was p-hydroxybenzaldehyde, and the retention times (Rt) in the system were 6.39 min for antirrhinoside,
6.93 min for antirrhide, 8.44 min for 5-glucosylantirrhinoside, 13.12 min for
linarioside and 19.78 min for p-hydroxybenzaldehyde (IS). A chromatogram of a
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
953
Fig. 2. Chromatogram of a standard solution containing the four iridoids and the internal standard:
antirrhinoside (1), antirrhide (2), 5-glc-antirrhinoside (3), linarioside (4) and p-hydroxybenzaldehyde (5).
Fig. 3. Standard curve for antirrhinoside, using p-hydroxybenzaldehyde as an internal standard.
standard solution with the four iridoids (Fig. 1) and the internal standard p-hydroxybenzaldehyde is shown in Fig. 2.
Calibration curves like the one in Fig. 3 were made, and good linearity
was obtained with the standard solutions of the test compounds with concentrations of 0.1}0.005 mg/ml. The correlation coe$cients were 0.99936, 0.99937, 0.99980
954
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
and 0.99977 for antirrhinoside, antirrhide, 5-glucosylantirrhinosid and linarioside,
respectively.
2.4. Assay validation and recovery
The extraction procedure was decided upon after a range of extractions had been
carried out using a variation in methanol concentrations (0, 10, 20, 40, 60, 80 and
100%) and extraction times (10 and 30 min, 1, 2 and 4 h) in an ultra-sonic bath, prior
to the analyses by HPLC. Based on the most stable results, we decided on extraction
for 1 h with 20% methanol.
The HPLC method was carefully validated. Accuracy was measured on standard
solutions with concentrations lower than those we measured during the season. For
the highest concentration the accuracy for all compounds was between 86 and 100%.
The relative standard deviation was between 0.48 and 2.55% in three replicates of
three standard solutions, which gives a very good repeatability. There was no
di!erence in the result of the standard solutions on di!erent chromatographs or
di!erent analysts, and therefore the method has a good reproducibility. The diodearray scan showed no interference from other compounds in the plant extract, which
gives a good selectivity. The method is very robust to variations in #ow and to change
of column with the same material. The linearity of the calibration curve is shown in
Section 2.3. The limit of detection for antirrhinoside is 0.004 mg/ml, the limit of
quanti"cation 0.01 mg/ml.
3. Results
3.1. Plant analyses
3.1.1. Seasonal variation
The weather was more or less stable during the sampling period. There was
no event of especially warm or cold weather and no period was particularly dry
or wet. Fig. 4 shows the average rainfall and temperature during the period
of sampling. Compared to an average Danish summer this one was a bit cool and
wet (Statistisk As rbog, 1999). The plants developed normally during the season.
After September 9 most of the plants were attacked by fungi, and the experiment
terminated.
Fig. 4 shows the variation in concentration of antirrhinoside and total iridoids for
both cultivars of A. majus. The concentrations of the other three iridoids were very low
and are therefore not shown individually. Table 1 lists the absolute and relative
content of all four iridoids and total during the season, together with the mean values
of iridoid content before (incl. July 29), and during #owering (from August 7). It is
evident that the content of individual iridoids varies during the season (Fig. 4), and in
general the relative amount of antirrhinoside is higher before than after the onset of
the #owering (Table 1).
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
955
Fig. 4. Weather conditions during the sample period: daily rainfall and average temperature (3C).
There is a pronounced bimodal variation in total iridoids during summer, and the
two cultivars show a remarkable uniformity. Coinciding with the onset of #owering,
the content of iridiods in leaves was very low in both cultivars. Some test samples were
made of the spikes and #owers just around the time of #owering. Table 2 shows that
some of the buds and #owers had a higher content of iridoids than the leaves sampled
on the same day.
3.1.2. Diurnal variation
Figs. 5 and 6 show the diurnal variation of antirrhinoside at the beginning of the
#owering and in the middle of the #owering season, respectively. The content of
antirrhinoside varies substantially (between 20 and 60 mg/g) during the day and night,
but no general pattern in relation to day length could be seen, neither for the two
cultivars nor for the two sample days. However, the variation between individual
samples is unquestionable, but presumably not related to light conditions, temperature patterns or humidity, which vary continuously in a symmetric pattern around
midday (cf. Itenov et al., 2000).
4. Discussion
It has not been possible to "nd published observations like those we show in this
study with a clearcut bimodal seasonal variation and a sharp decline in the content of
secondary compounds at the onset of the #owering. Rather, there is a tendency to
956
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Table 1
The seasonal variation in iridoid content, absolute (mg/g dry weight) and relative (%), in the leaves of
cultivars of Antirrhinum majus during the summer of 1998. The season is divided in two periods before
#owering (incl. July 29) and after the onset of #owering (from August 7), respectively. The means of each
period are calculated and listed in the table
White Wonder
Pre-#owering
17/06/98
24/06/98
01/07/98
08/07/98
15/07/98
29/07/98
Mean
s.d.
Total iridoids
Antirrhinoside Antirrhide 5-Glc-antirrhinoside
(mg/g dry weight) relative
relative
relative
Linarioside
relative
n
56.32
74.60
72.04
81.78
102.84
63.00
90.63
90.00
93.98
92.32
90.68
93.33
91.82
1.63
7.03
7.24
3.14
2.57
4.53
3.49
4.67
2.02
1.99
2.09
2.89
4.26
3.83
2.22
2.88
0.97
0.36
0.67
0.00
0.86
0.93
0.95
0.63
0.38
5
5
5
5
5
2
Onset of #owering
31/07/98
9.16
69.87
15.94
14.19
0.00
5
During #owering
04/08/98
07/08/98
12/08/98
14/08/98
21/08/98
25/08/98
28/08/98
01/09/98
04/09/98
09/09/98
Mean
s.d.
80.53
80.71
75.22
83.26
76.19
82.21
86.56
83.56
86.88
85.12
82.02
3.97
12.84
12.86
9.35
8.74
11.98
11.55
8.32
8.88
9.27
8.68
10.25
1.84
5.42
5.72
13.88
6.90
11.42
5.82
4.69
6.66
3.29
5.68
6.95
3.22
1.21
0.71
1.55
1.14
0.40
0.42
0.43
0.89
0.58
0.52
0.79
0.40
5
5
5
5
5
5
5
4
4
2
Yellow Monarch
Pre-#owering
17/06/98
59.54
24/06/98
71.00
01/07/98
96.13
08/07/98
96.82
15/07/98
107.98
29/07/98
92.65
Mean
s.d.
86.29
86.79
93.26
88.25
90.48
91.47
89.42
2.76
10.82
8.62
2.18
3.68
3.67
4.96
5.66
3.34
2.65
3.97
4.40
6.90
5.56
2.97
4.41
1.61
0.24
0.62
0.16
1.18
0.30
0.59
0.52
0.38
5
5
4
5
5
2
Onset of #owering
31/07/98
2.10
45.24
11.90
42.86
0.00
4
During #owering
04/08/98
07/08/98
12/08/98
14/08/98
21/08/98
25/08/98
28/08/98
01/09/98
Mean
s.d.
73.46
75.15
77.91
84.49
82.54
76.77
83.55
82.05
79.49
4.18
16.65
19.84
13.61
10.53
9.05
17.00
12.46
9.95
13.64
3.86
8.21
4.31
5.52
3.96
7.99
6.28
3.79
7.38
5.93
1.81
1.68
0.76
2.97
1.02
0.42
1.39
0.20
0.62
1.13
0.89
5
4
4
5
4
3
3
2
28.04
55.98
76.36
75.38
74.60
62.50
64.44
67.53
60.75
66.85
42.88
36.70
73.35
68.74
96.08
69.60
85.30
64.35
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
957
accumulate secondary compounds during the season as shown for alkaloids
(Williams and Ellis, 1989) and terpenoids (Gershenzon et al., 1993). With the
development of #owering intensity Kite and Smith (1997) showed an increase in the
emission of monoterpenes with day}night variations, and for #avonoids Letchamo
(1996) similarly showed an increase with increasing opening of the #owerheads in
camomile. However, in our experiments, #owering at the main stem of A. majus
is followed by regrowth of lower sidebranches, and as these make up part of
the plant material, the samples subsequently contain more newly developed leaves.
It is well documented that new leaves may have higher content of secondary metabolites, e.g. monoterpenes (Goralka and Langenheim, 1996) and iridoids (Bowers et al.,
1992).
We have not been able to relate this variation to changes in the abiotic conditions
such as temperature, insolation, humidity (cf. Fig. 2), or to nutrient availability, which
could explain the drop in iridoid content in the middle of the season. After September
9 a severe fungal attack to the leaves coincided with the disappearance of iridoids;
however, this gives no explanation to the bimodal seasonal variation as the plants
were attacked only at the end of the season, obviously coinciding with a drop in the
content of iridoids.
The biosynthesis rate of the iridoids must change after the onset of #owering, as
shown by the change in the relative content of antirrhinoside and antirrhide. Translocation of iridoids from the leaves to other parts of the plants, e.g. in#orescenses under
development, may be an explanation to this pattern, with a resultant lack of iridoids in
the leaves during #ower development. We have only a few analyses to justify this
hypothesis (Table 2), and it is noticeable that the iridoid content in some of the plants
from August 4 is substantially higher in the buds than in the leaves. This could be
a result of transport of the iridoids at the onset of #owering, in order to secure
protection of the young tissue. But more detailed analyses are needed to con"rm this
transport to the buds. According to Gowan et al. (1995) antirrhinoside is biosynthesized in the leaf blade and subsequently transported through the petiole to other
parts of the plant. Gupta (1991) states that an attack of Peronospora plantaginiae on
Plantago psyllium appeared at the initiation of spikes. In comparison to our study, this
fungal attack at the onset of #owering in P. psyllium could be due to lack of iridoids in
the leaves.
The diurnal observations show that the content of iridoids varies during the day
(Figs. 5 and 6). Although the variation is substantial, from 20 to 60 mg/g dry matter,
there is no pattern to be read from these graphs. Most of the published results on
diurnal variation in secondary chemistry concerns alkaloids with a rapid change in
concentration, e.g. morphine (Fairbairn and Wassel, 1964) and coniin (Fairbairn and
Suwal, 1961). However, the extremely rapid turnover of alkaloids in the latex of
Papaver somniferum has recently been shown to be an artefact as only the water
content varies (Itenov et al., 1999). To eliminate the e!ect of such variation it is
important to collect the samples at the same time every day. Because of the great
similarity between the two curves for the seasonal variation, we assume that
even though the samples were not taken at exactly the same time, the results are very
valid.
958
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Table 2
The seasonal variation in total iridoid content and antirrhinoside in the leaves and #owersbuds of
Antirrhinum majus in a short period at the onset of #owering. Note the substantial variation between
individuals
Total iridoids
(mg/g dry weight)
Antirrhinoside
(mg/g dry weight)
Leaves
Buds
Leaves
Buds
White Wonder
29/7/98
29/7/98
67.5
63
5.2
111
46.9
58.8
1.6
97.2
31/7/98
31/7/98
31/7/98
31/7/98
31/7/98
4/8/98
4/8/98
4/8/98
4/8/98
4/8/98
2.4
6.1
3
18.5
15.8
10.7
1.9
22.8
63
41.8
0
0.04
0.8
13
8.4
83.6
81.7
64.6
88.6
84.8
1.2
3.9
1
15
10.9
3.3
0.8
16.9
55.1
36.8
0
0.04
0.7
11
6.1
77.3
73.3
55.2
82.3
74.2
102.9
82.4
4.5
1.4
0.5
2
35
34
65.8
29.8
49.8
166.7
n.d.!
1
17.7
1.49
0.5
52.6
26.1
95.2
17.9
99.2
95
74.5
3.3
0
0
0.5
29.7
23.1
51.7
18.1
34.9
145.3
n.d.
0.9
9.9
0.9
0.4
46.3
22.7
68
13.3
87
Yellow Monarch
29/8/98
29/7/98
31/7/98
31/7/98
31/7/98
31/7/98
4/8/98
4/8/98
4/8/98
4/8/98
4/8/98
!n.d.: not determined.
Part of this study was planned to "nd out when it is most appropriate to harvest
these two cultivars of A. majus in order to obtain an optimum yield of iridoids. This
should evidently not be at the onset of #owering. However, this does not "t with the
recommended procedures for medicinal plant production, and Heeger (1956)
suggests that above-ground plant parts should be harvested just before or at
the beginning of the #owering period. The present "ndings show how important it is
to carry out detailed growth experiments, incl. seasonal variation in the content of
active compounds, when introducing new crop plants for the production of "ne
chemicals.
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
959
Fig. 5. Seasonal variation of antirrhinoside and total iridoid content (mg/g leaf dry matter) in the two
cultivars White Wonder (top) and Yellow Monarck of Antirrhinum majus. The total number of #owers is
shown with solid line. The values are means based on samples of "ve individual plants every week and an
additional sample from July 31 when #owering started (compare Tables 1 and 2).
We recommend that cultivars of Antirrhinum majus be harvested 2}4 weeks before
#owering. Under normal conditions this would even give a farmer the opportunity to
harvest two times a year, as the plants will have su$cient time to regrow after the "rst
harvest.
960
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Fig. 6. Diurnal variation in the content of antirrhinoside in two cultivars of Antirrhinum majus, White
Wonder and Yellow Monarch. The values are based on "ve samples sampled every hour during August 5}6
and August 18}19, with sunrise and sunset at 5:22 and 21:09, and 5:48 and 20:38, respectively.
Acknowledgements
We wish to thank Betina S+rensen, Danish Institute of Agricultural Science,
Flakkebjerg, for her patient help with the sampling of the plant material and S+ren
Johnsen for his assistance with the "eldwork of the diurnal variation. Thanks also to
S+ren Rosendal Jensen, Dept. of Organic Chemistry, Danish Technical University,
who kindly supplied the reference material of iridoids and carefully commented on the
manuscript, and to Peter Christensen for skilled help with the HPLC method and the
validation process. An unknown referee gave invaluable comments on the "rst draft of
the manuscript.
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
961
This work was planned in relation to the project &Special chemicals and pharmaca
from plants', which is a cooperation between Danish Institute of Agricultural Science,
The Royal Danish School of Pharmacy and the Danish Technical University, "nancially supported by a grant from the Danish Science Foundation, ref. No. 9501145.
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HPLC analysis of the seasonal and diurnal
variation of iridoids in cultivars of Antirrhinum
majus
Birgitte Dr+hse H+gedal, Per M+lgaard*
Department of Medicinal Chemistry, Pharmacognosy Group, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received 17 June 1999; accepted 6 March 2000
Abstract
In this paper we show the seasonal and diurnal variation in the content of the four iridoids
found in cultivars of Antirrhinum majus, antirrhinoside, antirrhide, 5-glucosyl-antirrhinoside
and linarioside. The seasonal variation in total iridoid content showed a marked bimodal
distribution with high total values (around 100 mg/g dry matter) early and late in the season
and a very low content of all iridoids coinciding with the onset of #owering at the beginning of
August. The relative contribution of antirrhinoside was signi"cantly higher before #owering
than after bud break. The relative decrease in antirrhinoside was counteracted by an increase of
antirrhide, which was signi"cantly higher after the onset of #owering than before. This pattern
indicates a change in biosynthesis, although no explanation can be given to the phenomenon.
The diurnal variation showed a variation between 20 and 60 mg/g dry weight, but there was no
relation to light/darkness conditions, temperature patterns or water content. The analyses were
performed by HPLC. The applied method has not previously been used in the quanti"cation of
iridoids, but was developed speci"cally for the analyses of cultivars of Antirrhinum majus. We
have fully validated the method during its development. The limit of detection was calculated to
0.004 mg/ml and the limit of quanti"cation was 0.01 mg/ml. ( 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Antirrhinum majus; Scrophulariaceae; Iridoid glucoside; Antirrhinoside; Seasonal variation;
Diurnal variation
* Corresponding author. Tel.: #45-35-37-67-77; fax: #45-35-30-60-40.
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 4 5 - 4
950
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
1. Introduction
Iridoids are secondary compounds con"ned to a restricted number of sympetalous
plant families. They are found in Corni#orae, Loasi#orae, Lamii#orae and Gentianiflorae and are part of the complex set of characters that bind these superorders
together in the classi"cation of Dahlgren (1989). The value of chemical characters,
including iridoids, in the classi"cation of the Asteridae s.l. has recently been discussed
by Grayer et al. (1999).
There are two di!erent pathways in the biosynthesis of iridoids of which route II
leads to the aucubin-type iridoids common in Lamii#orae (Jensen, 1992). Antirrhinum
majus in the Scrophulariaceae is a member of Lamii#orae and thus produces
aucubin-type iridoids. The cultivars &White Wonder' and &Yellow Monarch' contain
the four major iridoid compounds antirrhinoside, antirrhide, 5-glucosyl-antirrhinoside and linarioside (Fig. 1). Antirrhinoside in Antirrhinum is biosynthetically derived
from 8-epi-iridodial via route II as shown by Breinholt et al. (1992) and Damtoft et al.
(1993, 1995).
The variation in the content of secondary compounds is important for the interaction of plants with pathogens and herbivores as highlighted in general by Coleman
and Jones (1991). In several studies iridoids have been shown to a!ect the feeding
behaviour of herbivores. By a turnover from production of aucubin to catalpol the
seasonal variation could be related to a shift from generalist to specialist herbivores
on Plantago species during the season (Bowers et al., 1992; Bowers and Stamp, 1992;
Bowers, 1996). In general, iridoids act as a defence mechanism against herbivores,
although they may also attract adapted herbivores at certain times according to
composition and concentration (Adler et al., 1995; Bowers and Puttick, 1986; Bowers,
1984; Pereyra and Bowers, 1988).
It is well known that the concentration of phytochemicals varies during the season
(Heeger, 1956), not only iridoids (Bowers et al., 1992), but also other monoterpenoids
(Adams, 1987; Goralka and Langenheim, 1996; Goralka et al., 1996); sesquiterpenes
(Hendriks et al., 1997); triterpenes (Ndamba et al., 1994); phenolics (William and Ellis,
1989; Wilt and Miller, 1992), essential oils (El-Gengaihi and Wahba, 1995), peptides
(Scheer et al., 1992) and cyanide (Boyd et al., 1938). Diurnal variation in plant
secondary chemistry has also been observed, either related to light conditions
Fig. 1. Chemical structures of the four iridoids from Antirrhinum majus: antirrhinoside (1), antirrhide (2),
5-glucosyl-antirrhinoside (3), and linarioside (4).
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
951
(Dickson, 1987; Sporer et al., 1993), to water de"cit (Itenov et al., 1999) or to more
complex interactions (Rosa et al., 1994). Besides, seasonal #uctuations and variations
in secondary plant metabolites may be governed by genotype and in#uenced by
nutrient availability as shown for iridoids by Bowers et al. (1992) and Fajer et al.
(1992), respectively.
This project is part of an ongoing investigation of secondary plant compounds for
their potential use as starting material in the chemical and pharmaceutical industry.
The speci"c interest in iridoids stems from their structure with an inherent chiral
centre, which makes the compounds potential precursors for the semi-synthesis of
drugs with activity against HIV and cancer (Franzyk et al., 1997, 1998a,b).
Although Antirrhinum majus has a long reputation as an ornamental garden plant
with detailed knowledge on the genetics of #ower colour patterns (e.g. Stubbe, 1974),
very little is known on the variation in the content of iridoids. The content of iridoids
may be of importance for the interaction with pathogens and herbivores on A. majus,
and in relation to chemotaxonomy at the generic level. However, not only from a pure
scienti"c point of view but also in relation to A. majus as a potential producer of
starting material for "ne chemicals it is important to "nd out if there is a seasonal or
diurnal variation and turnover of the iridoids. There may be an inherent di!erence
between cultivars, and we designed this project to determine at which state the plants
contain the largest amount of iridoids or the best combination of individual compounds, so that harvesting of raw material for "ne chemicals can be planned accordingly.
Previously, quantitative analyses of iridoids have been based on either gas
chromatography (Bowers, 1996; Bowers and Stamp, 1992), or high-performance
liquid chromatography, HPLC (Baghdikian et al., 1997; Dallenbach-Toelke et al.,
1987; Lenherr et al., 1984; Meier and Sticher, 1977; Miyagoshi et al., 1986). The
present analyses are made by HPLC using a newly developed method. We have
developed this method speci"cally to quantify the iridoids in these cultivars of
A. majus. The method has been fully validated alongside the development.
2. Materials and methods
2.1. Plant material
Two Antirrhinum majus cultivars, White wonder and Yellow monarch, were "eld
grown at the Danish Institute of Agricultural Science, Research Center Flakkebjerg.
The seeds were obtained from Impecta Handels, 640 25 Julita, Sweden. The plants
were grown in open "elds on silty clay soil, with a distance of 45 cm between the rows.
Nitrogen was applied on June 2 at the rate of 50 kg N/ha.
2.1.1. Weather conditions
During the observation period, the weather conditions were monitored for air
temperature (C3), soil temperature (C3), rainfall (mm), evaporation (mm), wind speed
(m/s) and relative humidity (%). Temperature and rainfall are shown in Fig. 4.
952
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
2.1.2. Seasonal variation
Plant samples for chemical analyses were taken weekly between June 17, 1998 and
September 30, 1998, and twice a week during the #owering period (July 19 until
September 4). Seven shoots from separate plants were cut each time, washed, weighed
and pressed. The shoots were dried at 403C for 24 h. After drying the shoots were
weighed and the water content determined in percent. Five of these shoots (the two
extremes were discarded) were selected each sample day, and the leaves were powdered. During the drying process on July 22 and July 29 some of the plants were
damaged and therefore excluded from the study. After September 9 fungi attacked the
plants, and the experiment was terminated. The development of the plants was
observed during the whole period.
2.1.3. Diurnal variation
Samples were taken at the onset of #owering (August 5}6, sunrise 5:22, sunset 21:09,
Danish summer time 2 h ahead of GMT) and in the middle of the #owering period
(August 18}19, sunrise 5:48, sunset 20:38, Danish summertime 2 h ahead of GMT).
Each harvest started at 15:00 h, and "ve plants were sampled every hour over a 24 h
period. No plants were used twice. The lower side branch and the pair of leaves above
were collected. The lower side branch was placed in a plastic bag and frozen on solid
CO to stop instantly any enzymatic activity. The leaves were washed, dried and
2
placed in sample vials, weighed and frozen on solid CO . After 1 h on solid CO both
2
2
samples were moved to a freezer at !203C. The samples were lyophilised on
Drywinner 6-85, Heto Holm & Halby, at !953C under vacuum for 48 h.
2.2. Sample preparation
50 mg of powdered leaf material was weighed out and extracted for 1 h in an
ultra-sonic bath with 10 ml of a 20% methanol solution, containing 0.05 mg/ml of the
internal standard, p-hydroxybenzaldehyde. The extract was "ltered through a Minisart' "lter type RC-0.45 lm and analysed directly on HPLC.
2.3. HPLC analysis
The HPLC analyses were performed on a Shimadzu SCL-6A System controller
with Shimadzu SIL-6A Auto-injector with an injection volume of 10 ll, Shimadzu
CTO-6A Column oven at 403C, Shimadzu SPD-6AV Spectrophotometric detector at
205 nm and two Shimadzu LC-6A pumps with a #ow of 1 ml/min. The data processing
was carried out by a Class-LC10 version 1.41. The column was a Macherey-Nagel
Nucleosil C18-5l, 125]4.6 mm2. The system was an isocratic 3% acetonitrile in
water, with an analysis time of 25 min. The internal standard was p-hydroxybenzaldehyde, and the retention times (Rt) in the system were 6.39 min for antirrhinoside,
6.93 min for antirrhide, 8.44 min for 5-glucosylantirrhinoside, 13.12 min for
linarioside and 19.78 min for p-hydroxybenzaldehyde (IS). A chromatogram of a
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
953
Fig. 2. Chromatogram of a standard solution containing the four iridoids and the internal standard:
antirrhinoside (1), antirrhide (2), 5-glc-antirrhinoside (3), linarioside (4) and p-hydroxybenzaldehyde (5).
Fig. 3. Standard curve for antirrhinoside, using p-hydroxybenzaldehyde as an internal standard.
standard solution with the four iridoids (Fig. 1) and the internal standard p-hydroxybenzaldehyde is shown in Fig. 2.
Calibration curves like the one in Fig. 3 were made, and good linearity
was obtained with the standard solutions of the test compounds with concentrations of 0.1}0.005 mg/ml. The correlation coe$cients were 0.99936, 0.99937, 0.99980
954
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
and 0.99977 for antirrhinoside, antirrhide, 5-glucosylantirrhinosid and linarioside,
respectively.
2.4. Assay validation and recovery
The extraction procedure was decided upon after a range of extractions had been
carried out using a variation in methanol concentrations (0, 10, 20, 40, 60, 80 and
100%) and extraction times (10 and 30 min, 1, 2 and 4 h) in an ultra-sonic bath, prior
to the analyses by HPLC. Based on the most stable results, we decided on extraction
for 1 h with 20% methanol.
The HPLC method was carefully validated. Accuracy was measured on standard
solutions with concentrations lower than those we measured during the season. For
the highest concentration the accuracy for all compounds was between 86 and 100%.
The relative standard deviation was between 0.48 and 2.55% in three replicates of
three standard solutions, which gives a very good repeatability. There was no
di!erence in the result of the standard solutions on di!erent chromatographs or
di!erent analysts, and therefore the method has a good reproducibility. The diodearray scan showed no interference from other compounds in the plant extract, which
gives a good selectivity. The method is very robust to variations in #ow and to change
of column with the same material. The linearity of the calibration curve is shown in
Section 2.3. The limit of detection for antirrhinoside is 0.004 mg/ml, the limit of
quanti"cation 0.01 mg/ml.
3. Results
3.1. Plant analyses
3.1.1. Seasonal variation
The weather was more or less stable during the sampling period. There was
no event of especially warm or cold weather and no period was particularly dry
or wet. Fig. 4 shows the average rainfall and temperature during the period
of sampling. Compared to an average Danish summer this one was a bit cool and
wet (Statistisk As rbog, 1999). The plants developed normally during the season.
After September 9 most of the plants were attacked by fungi, and the experiment
terminated.
Fig. 4 shows the variation in concentration of antirrhinoside and total iridoids for
both cultivars of A. majus. The concentrations of the other three iridoids were very low
and are therefore not shown individually. Table 1 lists the absolute and relative
content of all four iridoids and total during the season, together with the mean values
of iridoid content before (incl. July 29), and during #owering (from August 7). It is
evident that the content of individual iridoids varies during the season (Fig. 4), and in
general the relative amount of antirrhinoside is higher before than after the onset of
the #owering (Table 1).
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
955
Fig. 4. Weather conditions during the sample period: daily rainfall and average temperature (3C).
There is a pronounced bimodal variation in total iridoids during summer, and the
two cultivars show a remarkable uniformity. Coinciding with the onset of #owering,
the content of iridiods in leaves was very low in both cultivars. Some test samples were
made of the spikes and #owers just around the time of #owering. Table 2 shows that
some of the buds and #owers had a higher content of iridoids than the leaves sampled
on the same day.
3.1.2. Diurnal variation
Figs. 5 and 6 show the diurnal variation of antirrhinoside at the beginning of the
#owering and in the middle of the #owering season, respectively. The content of
antirrhinoside varies substantially (between 20 and 60 mg/g) during the day and night,
but no general pattern in relation to day length could be seen, neither for the two
cultivars nor for the two sample days. However, the variation between individual
samples is unquestionable, but presumably not related to light conditions, temperature patterns or humidity, which vary continuously in a symmetric pattern around
midday (cf. Itenov et al., 2000).
4. Discussion
It has not been possible to "nd published observations like those we show in this
study with a clearcut bimodal seasonal variation and a sharp decline in the content of
secondary compounds at the onset of the #owering. Rather, there is a tendency to
956
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Table 1
The seasonal variation in iridoid content, absolute (mg/g dry weight) and relative (%), in the leaves of
cultivars of Antirrhinum majus during the summer of 1998. The season is divided in two periods before
#owering (incl. July 29) and after the onset of #owering (from August 7), respectively. The means of each
period are calculated and listed in the table
White Wonder
Pre-#owering
17/06/98
24/06/98
01/07/98
08/07/98
15/07/98
29/07/98
Mean
s.d.
Total iridoids
Antirrhinoside Antirrhide 5-Glc-antirrhinoside
(mg/g dry weight) relative
relative
relative
Linarioside
relative
n
56.32
74.60
72.04
81.78
102.84
63.00
90.63
90.00
93.98
92.32
90.68
93.33
91.82
1.63
7.03
7.24
3.14
2.57
4.53
3.49
4.67
2.02
1.99
2.09
2.89
4.26
3.83
2.22
2.88
0.97
0.36
0.67
0.00
0.86
0.93
0.95
0.63
0.38
5
5
5
5
5
2
Onset of #owering
31/07/98
9.16
69.87
15.94
14.19
0.00
5
During #owering
04/08/98
07/08/98
12/08/98
14/08/98
21/08/98
25/08/98
28/08/98
01/09/98
04/09/98
09/09/98
Mean
s.d.
80.53
80.71
75.22
83.26
76.19
82.21
86.56
83.56
86.88
85.12
82.02
3.97
12.84
12.86
9.35
8.74
11.98
11.55
8.32
8.88
9.27
8.68
10.25
1.84
5.42
5.72
13.88
6.90
11.42
5.82
4.69
6.66
3.29
5.68
6.95
3.22
1.21
0.71
1.55
1.14
0.40
0.42
0.43
0.89
0.58
0.52
0.79
0.40
5
5
5
5
5
5
5
4
4
2
Yellow Monarch
Pre-#owering
17/06/98
59.54
24/06/98
71.00
01/07/98
96.13
08/07/98
96.82
15/07/98
107.98
29/07/98
92.65
Mean
s.d.
86.29
86.79
93.26
88.25
90.48
91.47
89.42
2.76
10.82
8.62
2.18
3.68
3.67
4.96
5.66
3.34
2.65
3.97
4.40
6.90
5.56
2.97
4.41
1.61
0.24
0.62
0.16
1.18
0.30
0.59
0.52
0.38
5
5
4
5
5
2
Onset of #owering
31/07/98
2.10
45.24
11.90
42.86
0.00
4
During #owering
04/08/98
07/08/98
12/08/98
14/08/98
21/08/98
25/08/98
28/08/98
01/09/98
Mean
s.d.
73.46
75.15
77.91
84.49
82.54
76.77
83.55
82.05
79.49
4.18
16.65
19.84
13.61
10.53
9.05
17.00
12.46
9.95
13.64
3.86
8.21
4.31
5.52
3.96
7.99
6.28
3.79
7.38
5.93
1.81
1.68
0.76
2.97
1.02
0.42
1.39
0.20
0.62
1.13
0.89
5
4
4
5
4
3
3
2
28.04
55.98
76.36
75.38
74.60
62.50
64.44
67.53
60.75
66.85
42.88
36.70
73.35
68.74
96.08
69.60
85.30
64.35
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
957
accumulate secondary compounds during the season as shown for alkaloids
(Williams and Ellis, 1989) and terpenoids (Gershenzon et al., 1993). With the
development of #owering intensity Kite and Smith (1997) showed an increase in the
emission of monoterpenes with day}night variations, and for #avonoids Letchamo
(1996) similarly showed an increase with increasing opening of the #owerheads in
camomile. However, in our experiments, #owering at the main stem of A. majus
is followed by regrowth of lower sidebranches, and as these make up part of
the plant material, the samples subsequently contain more newly developed leaves.
It is well documented that new leaves may have higher content of secondary metabolites, e.g. monoterpenes (Goralka and Langenheim, 1996) and iridoids (Bowers et al.,
1992).
We have not been able to relate this variation to changes in the abiotic conditions
such as temperature, insolation, humidity (cf. Fig. 2), or to nutrient availability, which
could explain the drop in iridoid content in the middle of the season. After September
9 a severe fungal attack to the leaves coincided with the disappearance of iridoids;
however, this gives no explanation to the bimodal seasonal variation as the plants
were attacked only at the end of the season, obviously coinciding with a drop in the
content of iridoids.
The biosynthesis rate of the iridoids must change after the onset of #owering, as
shown by the change in the relative content of antirrhinoside and antirrhide. Translocation of iridoids from the leaves to other parts of the plants, e.g. in#orescenses under
development, may be an explanation to this pattern, with a resultant lack of iridoids in
the leaves during #ower development. We have only a few analyses to justify this
hypothesis (Table 2), and it is noticeable that the iridoid content in some of the plants
from August 4 is substantially higher in the buds than in the leaves. This could be
a result of transport of the iridoids at the onset of #owering, in order to secure
protection of the young tissue. But more detailed analyses are needed to con"rm this
transport to the buds. According to Gowan et al. (1995) antirrhinoside is biosynthesized in the leaf blade and subsequently transported through the petiole to other
parts of the plant. Gupta (1991) states that an attack of Peronospora plantaginiae on
Plantago psyllium appeared at the initiation of spikes. In comparison to our study, this
fungal attack at the onset of #owering in P. psyllium could be due to lack of iridoids in
the leaves.
The diurnal observations show that the content of iridoids varies during the day
(Figs. 5 and 6). Although the variation is substantial, from 20 to 60 mg/g dry matter,
there is no pattern to be read from these graphs. Most of the published results on
diurnal variation in secondary chemistry concerns alkaloids with a rapid change in
concentration, e.g. morphine (Fairbairn and Wassel, 1964) and coniin (Fairbairn and
Suwal, 1961). However, the extremely rapid turnover of alkaloids in the latex of
Papaver somniferum has recently been shown to be an artefact as only the water
content varies (Itenov et al., 1999). To eliminate the e!ect of such variation it is
important to collect the samples at the same time every day. Because of the great
similarity between the two curves for the seasonal variation, we assume that
even though the samples were not taken at exactly the same time, the results are very
valid.
958
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Table 2
The seasonal variation in total iridoid content and antirrhinoside in the leaves and #owersbuds of
Antirrhinum majus in a short period at the onset of #owering. Note the substantial variation between
individuals
Total iridoids
(mg/g dry weight)
Antirrhinoside
(mg/g dry weight)
Leaves
Buds
Leaves
Buds
White Wonder
29/7/98
29/7/98
67.5
63
5.2
111
46.9
58.8
1.6
97.2
31/7/98
31/7/98
31/7/98
31/7/98
31/7/98
4/8/98
4/8/98
4/8/98
4/8/98
4/8/98
2.4
6.1
3
18.5
15.8
10.7
1.9
22.8
63
41.8
0
0.04
0.8
13
8.4
83.6
81.7
64.6
88.6
84.8
1.2
3.9
1
15
10.9
3.3
0.8
16.9
55.1
36.8
0
0.04
0.7
11
6.1
77.3
73.3
55.2
82.3
74.2
102.9
82.4
4.5
1.4
0.5
2
35
34
65.8
29.8
49.8
166.7
n.d.!
1
17.7
1.49
0.5
52.6
26.1
95.2
17.9
99.2
95
74.5
3.3
0
0
0.5
29.7
23.1
51.7
18.1
34.9
145.3
n.d.
0.9
9.9
0.9
0.4
46.3
22.7
68
13.3
87
Yellow Monarch
29/8/98
29/7/98
31/7/98
31/7/98
31/7/98
31/7/98
4/8/98
4/8/98
4/8/98
4/8/98
4/8/98
!n.d.: not determined.
Part of this study was planned to "nd out when it is most appropriate to harvest
these two cultivars of A. majus in order to obtain an optimum yield of iridoids. This
should evidently not be at the onset of #owering. However, this does not "t with the
recommended procedures for medicinal plant production, and Heeger (1956)
suggests that above-ground plant parts should be harvested just before or at
the beginning of the #owering period. The present "ndings show how important it is
to carry out detailed growth experiments, incl. seasonal variation in the content of
active compounds, when introducing new crop plants for the production of "ne
chemicals.
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
959
Fig. 5. Seasonal variation of antirrhinoside and total iridoid content (mg/g leaf dry matter) in the two
cultivars White Wonder (top) and Yellow Monarck of Antirrhinum majus. The total number of #owers is
shown with solid line. The values are means based on samples of "ve individual plants every week and an
additional sample from July 31 when #owering started (compare Tables 1 and 2).
We recommend that cultivars of Antirrhinum majus be harvested 2}4 weeks before
#owering. Under normal conditions this would even give a farmer the opportunity to
harvest two times a year, as the plants will have su$cient time to regrow after the "rst
harvest.
960
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
Fig. 6. Diurnal variation in the content of antirrhinoside in two cultivars of Antirrhinum majus, White
Wonder and Yellow Monarch. The values are based on "ve samples sampled every hour during August 5}6
and August 18}19, with sunrise and sunset at 5:22 and 21:09, and 5:48 and 20:38, respectively.
Acknowledgements
We wish to thank Betina S+rensen, Danish Institute of Agricultural Science,
Flakkebjerg, for her patient help with the sampling of the plant material and S+ren
Johnsen for his assistance with the "eldwork of the diurnal variation. Thanks also to
S+ren Rosendal Jensen, Dept. of Organic Chemistry, Danish Technical University,
who kindly supplied the reference material of iridoids and carefully commented on the
manuscript, and to Peter Christensen for skilled help with the HPLC method and the
validation process. An unknown referee gave invaluable comments on the "rst draft of
the manuscript.
B. Dr~hse H~gedal, P. M~lgaard / Biochemical Systematics and Ecology 28 (2000) 949}962
961
This work was planned in relation to the project &Special chemicals and pharmaca
from plants', which is a cooperation between Danish Institute of Agricultural Science,
The Royal Danish School of Pharmacy and the Danish Technical University, "nancially supported by a grant from the Danish Science Foundation, ref. No. 9501145.
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