Non-volatile floral oils of Diascia spp. (Scrophulariaceae)

Article abstract of DOI:10.1016/j.phytochem.2007.12.012

The floral oils of Diascia purpurea, Diascia vigilis, Diascia cordata, Diascia megathura, Diascia integerrima and Diascia barberae (Scrophulariaceae) were selectively collected from trichome elaiophores. The derivatized floral oils were analyzed by gas chromatography-mass spectrometry (GC-MS), whilst the underivatized samples were analysed by electrospray ionization mass spectrometry (ESI-MS) and Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). The most common constituents of the floral oils investigated are partially acetylated acylglycerols of (3R)-acetoxy fatty acids (C₁₄, C₁₆, and C₁₈), as was proven with non-racemic synthetic reference samples. The importance of these oils for Rediviva bees is discussed in a co-evolutionary context.

Full text of DOI:10.1016/j.phytochem.2007.12.012

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1372–1383
www.elsevier.com/locate/phytochem
Non-volatile floral oils of Diascia spp. (Scrophulariaceae)
a
a
a
b
c
Kanchana Dumri , Lars Seipold , J u¨ rgen Schmidt , G u¨ nter Gerlach , Stefan D o¨ tterl ,
d
a,
*
Allan G. Ellis , Ludger A. Wessjohann
a
Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany
b
Botanical Garden Munich, Nymphenburg, Menzinger Straße 65, D-80638 M u¨ nchen, Germany
Department of Plant Systematics, Universit a¨ tsstraße 30, University of Bayreuth, D-95447 Bayreuth, Germany
Department of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville PMB 3209, South Africa
c
d
Received 17 October 2007; received in revised form 17 December 2007
Available online 7 March 2008
Abstract
The floral oils of Diascia purpurea, Diascia vigilis, Diascia cordata, Diascia megathura, Diascia integerrima and Diascia barberae
Scrophulariaceae) were selectively collected from trichome elaiophores. The derivatized floral oils were analyzed by gas chromatogra-
(
phy–mass spectrometry (GC–MS), whilst the underivatized samples were analysed by electrospray ionization mass spectrometry (ESI-
MS) and Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS). The most common constituents of the floral oils
investigated are partially acetylated acylglycerols of (3R)-acetoxy fatty acids (C14, C16, and C18), as was proven with non-racemic syn-
thetic reference samples. The importance of these oils for Rediviva bees is discussed in a co-evolutionary context.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Diascia; Scrophulariaceae; Floral oils; Fatty acid profiling; Gas chromatography–mass spectrometry (GC–MS); Electrospray Fourier-trans-
form ion cyclotron resonance mass spectrometry (ESI-FTICR-MS); Oxidized fatty acids-asymmetric derivatization
1
. Introduction
tube-like petal appendages. The bees use the lipid secre-
tions (mostly instead of nectar) along with pollen as provi-
sion for their larvae and likely also for water-resistant cell
lining of the soil nests (Simpson and Neff, 1981; Buch-
mann, 1987).
The main literature reports are focused on the floral
morphology, ecology, taxonomy and evolution of several
oil-secreting flowers (Vogel, 1974, 1986, 1990; Simpson
et al., 1977, 1979; Seigler et al., 1978; Buchmann, 1987;
Cane et al., 1983; Vinson et al., 1997; Seipold et al.,
2004). The morphology and anatomy of elaiophores in
the families Malpighiaceae, Krameriaceae, Scrophularia-
ceae, Iridaceae and Orchidaceae were initially described
by Vogel (1974). He reported the diacylglycerol of acetic
acid and b-acetoxy palmitic acid as a major component
of Calceolaria pavonii (Scrophulariaceae) trichome
exudates.
Flowering plants have evolved a wide range of floral
rewards to achieve pollination by insects. Nectar and/or
pollen, containing carbohydrates, amino acids and proteins
have long been recognized as the most common recom-
pense for pollinating animals. Various specialized pollina-
tors are, however, attracted to other substances, including
feeding tissue, resins and gums, or floral scent compounds
(
Simpson and Neff, 1983). Non-volatile (fatty) oils are less
well known floral rewards, despite of being significant for
many plants and their pollinating bees, especially in the
neotropics (Vogel, 1969; Simpson and Neff, 1981, 1983).
Oil-collecting bees have special adaptations to gather the
lipids, which are offered in so called elaiophores, e.g., in
*
Corresponding author. Tel.: +49 (0)345 5582 1301; fax: +49 (0)345
In most oil-secreting flowers studied, free b-acetoxy fatty
acids, free fatty acids and unique acylglycerols containing
5582 1309.
E-mail address: wessjohann@ipb-halle.de (L.A. Wessjohann).
0
031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.12.012

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1373
b-acetoxy fatty acids were found as main lipid components
Vogel, 1969, 1974, 1986, 1990; Simpson et al., 1977, 1979;
Seigler et al., 1978; Cane et al., 1983; Simpson and Neff,
983; Buchmann, 1987; Vinson et al., 1997). The structure
2. Results and discussion
(
2.1. FAME profiling of the Diascia species
1
of a novel diacylglycerol of Ornithophora radicans was elu-
cidated from spectroscopic data (Reis et al., 2000, 2003).
Our group (Seipold et al., 2004) was first to report partially
acetylated 3,7-dihydroxy fatty acids as a new type of con-
stituent in Malpighia coccigera (Malpighiaceae) oil; and
to discuss a possible relationship between floral oil and
plant epicuticular wax biosynthesis. Recently, Reis et al.
Diascia oils from trichome elaiophores are yellowish by
nature. Table 1 shows the fatty acid methyl ester (FAME)
profiles of the six Diascia spp., and as an example, Fig. 3
illustrates the total ion chromatogram (TIC) of the FAME
profiling of Diascia vigilis. Unbranched fatty acids and
(3R)-hydroxy fatty acids with even-numbered chain length
ranging from C14 to C18 represent the main compounds of
the lipids. In all cases, there were no remainders of acylgly-
cerols due to the complete transesterification reaction. The
main compound of all derivatized Diascia oil samples was
(3R)-hydroxypalmitic acid in a range of 55–75% (Table 1).
Non-oxidized fatty acids could not be detected in Diascia
barberae oil. EI mass spectra of trimethylsilyl (TMSi)-
derivatives of 3-hydroxy fatty acid methyl esters exhibit a
predominant peak at m/z 175 attributed to a cleavage at
(
2007) proved R configuration for both positions 3 and 7.
The genus Diascia comprises about 70 species, 20 of
which are found in the eastern parts of Southern Africa
Fig. 1). Most of the Diascia spp., perennial or annual,
(
are characterized by twin floral sacs (Steiner and White-
head, 1988). The trichome elaiophores are located within
the tips of paired spurs. The females of Rediviva bees have
specially adapted elongated forelegs that allow only them
to exploit the floral oils from the spurs of Diascia flowers.
The bees transfer oil to their hind legs and then carry it to
the nest, where it is used as larval food and presumably
also for nest cell lining and construction (Steiner and
Whitehead, 1990, 1991). The lipid-based association of
Diascia and Rediviva has both historical and evolutionary
implications beyond pollination ecology. Vogel (1974)
assumed that Rediviva bees and Diascia hosts might have
a co-evolutionary association. The morphological data
strongly indicated that the Rediviva leg length is correlated
to the length of the Diascia spurs (Steiner and Whitehead,
+
the C /C -linkage as well as a strong [MꢀCH ] ion (May-
3
4
3
berry, 1980; Mielniczuk et al., 1993). Experiments to deter-
mine the absolute configuration of the target compounds
proved that the hydroxyl at C-3 shows (R)-configuration.
Results are based on GC-comparison between the (2S)-
phenylpropionyl derivative of a chiral standard and the
samples (Hammarstr o¨ m, 1975; Weil et al., 2002).
2.2. EI-MS analysis of the acylglycerols of Diascia oils
A GC/EI-MS study of TMSi-derivatives of Diascia oils
yielded diacylglycerols (DAGs) and monoacylglycerols
(MAGs) along with small amounts of triacylglycerols
(TAGs) as main constituents. According to the results
obtained from the TMSi-derivatives, the detected acylgly-
cerols of Diascia spp. contain one or two acetyl groups
and an (3R)-acetoxy fatty acid attached to the glycerol
backbone. Furthermore, the ESI-FTICR-MS profiling
analysis of underivatized Diascia oils confirmed the (3R)-
acetoxy fatty acids as long-chain moiety of the acylglyce-
rols (see below). The acetylation of the 3-hydroxy acids
may be related to the export of the floral oils out of the
1
990, 1991).
In the present study, we have extensively investigated
the chemical composition of the floral oils of six Diascia
spp. (Scrophulariaceae) by different mass spectrometric
methods (GC/EI-MS, ESI-MS and ESI-FTICR-MS). The
identification of acylglycerols of Diascia spp. oil is
described in detail including the stereochemistry (see
Fig. 2). The application of high resolution FTICR-MS of
underivatized Diascia oils is presented. The co-evolution-
ary relation between Diascia flowers and Rediviva oil-col-
lecting bees is discussed.
Table 1
a
FAME profiling of TMSi-derivatives of Diascia spp.
b
Number Compound
t
R
Relative composition (%)
t
R
(
min)
b
(min)
D. purpurea D. vigilis D. cordata D. megathura D. integerrima D. barberae
1
2
Myristic acid (C14:0)
(3R)-Hydroxymyristic
acid (C14:0)
13.24
14.12
1.3
0.2
1.1
6.6
0.3
24.5
1.7
3.7
0.9
9.2
–
3.1
–
13.63
3
4
Palmitic acid (C16:0)
(3R)-Hydroxypalmitic
acid (C16:0)
15.69
16.71
6.7
56.5
5.0
72.1
2.0
65.0
11.3
55.8
8.4
61.1
–
75.6
–
16.07
5
6
Stearic acid (C18:0)
(3R)-Hydroxystearic
acid (C18:0)
18.48
19.54
5.3
30.1
3.4
11.9
1.4
6.8
7.8
19.7
6.4
14
–
18.0
–
18.77
a
GC/EI-MS analysis obtained from TMSi-derivatives of Diascia spp.
D. barberae floral oil was measured on an Agilent 6890 series gas chromatograph equipped with a Micromass, GCT mass-sensitive reflectron time-of-
b
flight (TOF) detector (DB-5 MS, 30 m ꢁ 0.25 mm i.d., 0.25 lm film thickness). Non-oxidized fatty acids were not detected.

1374
K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
+
cells. It has been reported that a hydroxy group in fatty
acids reduces the lipid transporter affinity compared to
unfunctionalized fatty acids (Zachowski et al., 1998).
Therefore, the acetylation could be crucial for an improved
transport (Seipold et al., 2004).
Fig. 4 shows the total ion chromatogram of the TMSi-
derivatives of D. vigilis. The mass spectral data of the iden-
tified compounds are presented in Table 2. The two main
components of D. vigilis were 2-[(3R)-acetoxypalmi-
toyl]glycerol (14) and 2-[(3R)-acetoxypalmitoyl]-1-acetyl-
glycerol (16, Table 3).
Our results prove that in case of 3-acetoxy fatty acids
the 1-monoacyl and 2-monoacyl glycerols (MAGs) can
be distinguished by the EI-MS data of their bis-TMSi-
derivatives. Fig. 5 shows a comparison of the EI mass spec-
tra of the 1-monoacyl and 2-monoacyl isomer of (3R)-acet-
oxypalmitoylglycerol. The molecular weight of MAGs of
the fragment [MꢀCH ] , formed by loss of a methyl radi-
3
cal from the trimethylsilyl group, represents the peak of
highest mass (Curstedt, 1974; Wood, 1980). Scheme 1
shows the characteristic fragmentation of bis-TMSi-deriva-
tives of 2-[(3R)-acetoxypalmitoyl]glycerol (14) and 1-[(3R)-
acetoxypalmitoyl]glycerol (15). An important key fragment
of the 2-MAG isomer is the ion of type e at m/z 218 (com-
pounds 7, 14 and 20), while the ion of the type (bꢀHOAc)
is a significant ion for 1-MAGs (compounds 8, 15 and 21).
As suggested by Johnson and Holman (1966) the 2-MAGs
display a significant signal at m/z 218 which is formed by
+
loss of the 3-acetoxy fatty acid from [M] . Rearrangement
of a TMSi group from the acylglycerol backbone to the
carboxy group of the fatty acids leads to a c-type ion at
m/z 311 (after loss of a HOAc unit). The ion of type (d-
HOAc) corresponds to the acylium ion after loss of the
3-acetoxy group. The fragment ion at m/z 203 (eꢀMe) is
produced by both 1- and 2-monoacyl isomers. The most
significant evidence of the 1-MAGs of (3R)-acetoxy fatty
acid is the formation of an ion at m/z 369 (bꢀHOAc) as
(
3R)-acetoxy fatty acids in Diascia spp. can be determined
by the appearance of significant ions of type a,
+
[
MꢀMeꢀHOAc] (Seipold, 2004). In case of the MAGs,
Table 2
GC/EI-MS data of the identified compounds from Diascia spp. (as TMSi-derivatives)
a
Number
t
R
Compound
Characteristic fragmentation ions (m/z (relative intensity, %))
(min)
+
7
8
9
22.96
23.41
23.93
24.22
24.64
24.79
25.18
25.67
26.14
26.66
26.96
27.39
27.92
28.28
28.73
29.27
29.56
30.00
2-[(3R)-Acetoxymyristoyl]glycerol
1-[(3R)-Acetoxymyristoyl]glycerol
504 (M , ꢀ), 429 (a, 15), 373 (9), 283 (c, 35), 209 (d-HOAc, 60), 218 (e, 85), 203 (e-Me,
3
9), 147 (66), 129 (100), 73 (55)
+
504 (M , ꢀ), 429 (a, 10), 401 (b, 2), 341 (b-HOAc, 88), 283 (c, 5), 209 (d-HOAc, 58),
2
05 (f, 15), 203 (e-Me, 22), 147 (56), 129 (28), 73 (100)
+
2-[(3R)-Acetoxymyristoyl]-1-
acetylglycerol
1-[(3R)-Acetoxymyristoyl]-3-
acetylglycerol
2-[(3R)-Acetoxymyristoyl]-1,3-
diacetylglycerol
Not identified [isomer of 16: an
474 (M , ꢀ), 399 (a, 12), 283 (c, 48), 209 (d-HOAc, 95), 189 (g, 50), 188 (e, 23), 146
(10), 145 (k, 72), 129 (79), 117 (96), 73 (75), 43 (100)
+
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
a
474 (M , ꢀ), 399 (a, 14), 341 (b-HOAc, 27), 283 (c, 5), 209 (d-HOAc, 44), 189 (g, 30),
1 1 2
188 (e , 3), 175 (h, 100), 146 (e -CH CO, 12), 117 (33), 43 (82)
+
444 (M , ꢀ), 324 (2), 269 (d, 5), 209 (d-HOAc, 48), 159 (88), 117 (11), 43 (100)
+
502 (M , ꢀ), 427 (a, 6), 311 (c, 56), 237 (d-HOAc, 72), 189 (g, 7), 188 (e, 31), 146 (12),
(
acetoxypalmitoyl)-acetylglycerol?]
Not identified [isomer of 17: an
acetoxypalmitoyl)-acetylglycerol?]
145 (k, 100), 129 (55), 117 (72), 73 (77), 43 (68)
+
502 (M , ꢀ), 427 (a, 5), 369 (b-HOAc, 10), 311 (c, 2), 237 (d-HOAc, 24), 189 (g, 2), 188
(
1 1 2
(e , 2), 175 (h, 100), 146 (e -CH CO, 12), 117 (20)
+
2-[(3R)-Acetoxypalmitoyl]glycerol
532 (M , ꢀ), 457 (a, 5), 401 (4), 311 (c, 15), 237 (d-HOAc, 36), 218 (e, 60), 203 (e-Me,
4), 147 (55), 129 (100), 73 (84)
2
+
1-[(3R)-Acetoxypalmitoyl]glycerol
532 (M , ꢀ),457 (a, 6), 429 (b, 2), 369 (b-HOAc, 92), 311 (c, 4), 237 (d-HOAc, 48), 205
(
f, 17), 203 (e-Me, 24), 147 (60), 129 (32), 73 (100)
+
2-[(3R)-Acetoxypalmitoyl]-1-
acetylglycerol
1-[(3R)-Acetoxypalmitoyl]-3-
acetylglycerol
2-[(3R)-Acetoxypalmitoyl]-1,3-
diacetylglycerol
Not identified [isomer of 23: an
502 (M , ꢀ), 427 (a, 10), 311 (c, 43), 237 (d-HOAc, 83), 189 (g, 66), 188 (e, 26), 146 (9),
145 (k, 74), 129 (96), 117 (80), 73 (77), 43 (100)
+
502 (M , ꢀ), 427 (a, 12 ), 369 (b-HOAc, 22), 311 (c, 5), 237 (d-HOAc, 33), 189 (g, 40),
1 1 2
188 (e , 2), 175 (h, 100), 146 (e -CH CO, 11), 117 (44), 43 (91)
+
472 (M , ꢀ), 352 (1), 297 (d, 4), 237 (d-HOAc, 37), 159 (96), 117 (10), 43 (100)
+
530 (M , ꢀ), 455 (a, 5), 397 (b-HOAc, 9), 339 (c, 1), 265 (d-HOAc, 13), 189 (g, 2), 188
(acetoxystearoyl)-acetylglycerol?]
1 1 2
(e , 1), 175 (h, 100), 146 (e -CH CO, 10), 117 (14)
+
2-[(3R)-Acetoxystearoyl]glycerol
560 (M , ꢀ), 485 (a, 4), 429 (3), 339 (c, 13), 265 (d-HOAc, 28), 218 (e, 60), 203 (e-Me,
3), 147 (54), 129 (100), 73 (85)
2
+
1-[(3R)-Acetoxystearoyl]glycerol
560 (M , ꢀ), 485 (a, 5), 457 (b, 1), 397 (b-HOAc, 60), 339 (c, 5), 265 (d-HOAc, 40), 205
(
f, 15), 203 (e-Me, 19), 147 (48), 129 (40), 73 (100)
+
2-[(3R)-Acetoxystearoyl]-1-
acetylglycerol
1-[(3R)-Acetoxystearoyl]-3-
acetylglycerol
2-[(3R)-Acetoxystearoyl]-1,3-
diacetylglycerol
530 (M , ꢀ), 455 (a, 8), 339 (c, 34), 265 (d-HOAc, 60), 189 (g, 67), 188 (e, 25), 146 (9),
145 (h, 70), 129 (98), 117 (88), 73 (95), 43 (100)
+
530 (M , ꢀ), 455 (a, 7), 397 (b-HOAc, 17), 339 (c, 2), 265 (d-HOAc, 21), 189 (g, 28),
1 1 2
188 (e , 2), 175 (h, 100), 146 (e -CH CO, 12), 117 (44), 43 (89)
+
500 (M , ꢀ), 380 (ꢀ), 325 (d, 4), 265 (d-HOAc, 27), 159 (88), 117 (20), 43 (100)
The retention time and relative abundance data were obtained from the mass spectrum of TMSi-derivatives of D. vigilis.

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1375
Table 3
Relative composition of the acylglycerols in Diascia spp. (obtained from the GC/EI-MS data of their TMSi-derivatives)
a
Number
t
R
(min)
Relative composition (%)
t
R
(min)
a
D. purpurea
D. vigilis
D. cordata
D. megathura
D. integerrima
D. barberae
7
8
9
22.96
23.41
23.93
24.22
24.64
24.79
25.18
25.67
26.14
26.66
26.96
27.39
27.92
28.28
28.73
29.27
29.56
30.00
1.2
0.3
0.3
0.1
0.9
17.4
6.5
16.2
9.2
17.6
6.8
3.2
2.0
8.2
5.8
2.5
1.8
–
1.3
0.2
5.8
0.2
0.6
2.5
1.1
35.6
3.6
35.7
2.0
0.1
0.3
4.8
1.2
4.4
0.4
0.1
1.7
9.5
1.2
–
–
–
39.8
8.7
4.2
25.8
1.5
–
–
1.7
–
3.1
–
6.6
–
–
40.2
5.6
17.9
1.7
9.2
–
8.9
2.8
2.4
0.1
0.4
–
–
1.4
–
0.9
–
–
–
0.8
1.6
4.7
0.2
–
23.00
–
23.70
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
b
b
–
–
–
13.6
12.5
24.9
19.6
5.5
–
2.1
4.0
5.0
5.0
0.6
8.0
0.5
80.6
0.3
1.4
–
0.6
–
6.3
–
24.70
25.15
25.72
25.98
26.40
–
27.27
–
28.23
–
b
–
0.3
9.1
–
–
–
0.1
28.97
a
D. barberae floral oil was measured on an Agilent 6890 series gas chromatograph equipped with a Micromass, GCT mass-sensitive reflectron time-of-
flight (TOF) detector (DB-5, 30 m ꢁ 0.25 mm, i.d., 0.25 lm film thickness).
b
Unidentified.
Scheme 1. Mass spectral fragmentation of TMSi-derivatives of the acylglycerols 2-[(3R)-acetoxypalmitoyl]glycerol (14) and 1-[(3R)-acetoxypalm-
itoyl]glycerol (15) (n.d. = not detected).

1376
K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
an unique peak, including the f-type ion, m/z 205. In most
structure is k at m/z 145 (Curstedt, 1974) which is not
observed in the TMSi-derivatives of 1,3-diacylglycerols
(compounds 10, 17 and 23). It should be pointed out that
the signals at m/z 175 (type h) and m/z 146 (e -CH CO)
+
cases of 1-MAGs, the fragment of type b ([Mꢀ103] ) cor-
responding to the loss of CH OSi(CH ) forms the base
2
3 3
peak (Fig. 5) (Johnson and Holman, 1966; Curstedt,
1
2
1
974; Myher et al., 1974; Wood, 1980).
Fig. 6 demonstrates the comparison of the EI mass spec-
are only present in the mass spectra of TMSi-derivatives
of 1,3-diacylglycerols (Scheme 2 and Table 2; Seipold,
2004).
tra of TMSi-derivatives of the 1,2- and 1,3-diacylglycerols
DAGs) of (3R)-acetoxypalmitic acid and an acetic acid
(
The TAGs of D. vigilis flowers (compounds 11, 18 and
24) consist of one (3R)-acetoxy fatty acid (C , C , or
moiety, also displaying significant differences. Generally,
the mass spectral behaviour of DAGs containing (3R)-acet-
oxy fatty acids and an acetyl moiety is similar to that of the
MAGs. A signal at m/z 189 (g) in both the TMSi-deriva-
tives of 1,2- and 1,3-DAGs can be explained as a cyclic
structure (Curstedt, 1974) as shown in Scheme 2. The
TMSi-derivatives of 1,2-diacylglycerols (compounds 9, 16
and 22) show an analogous signal at m/z 188 (type e) with
1
4
16
C ) at C-2 and two acetyl moieties at C-1 and C-3 of the
1
8
glycerol backbone. They were detected in amounts lower
than 1% (Table 2). Some of these compounds were also
reported from Oncidium oil (Reis et al., 2000). EI mass
spectra of 1-acyl-2,3-diacetyl-glycerols were also previously
described (Reis et al., 2003).
The unidentified compounds 12, 13, and 19 appear to be
isomers of 2-[(3R)-acetoxypalmitoyl]-1-acetylglycerol (16),
3-[(3R)-acetoxypalmitoyl]-1-acetylglycerol (17) and 3-[(3R)-
acetoxystearoyl]-1-acetylglycerol (23), respectively (Table
2), but no unequivocal assignment is possible due to the
limitations of the MS-techniques with the amounts available.
moderate intensity (Table 2). On the other hand, e (m/z
1
1
88), in 1,3-DAGs is of low abundance. This ion can be
a first hint indicating that the (3R)-acetoxy fatty acid is
attached to the secondary hydroxy group of the glycerol
backbone. A further key ion confirming the 1,2-DAG
Scheme 2. Mass spectral fragmentation of TMSi-derivatives of the acylglycerols 2-[(3R)-acetoxypalmitoyl]-1-acetylglycerol (16) and 1-[(3R)-
acetoxypalmitoyl]-3-acetylglycerol (17) (n.s. = not significant; n.d. = not detected).

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1377
Table 4
Positive-ion ESI-FTICR mass spectral data of the floral oils of the Diascia spp.
a
Number
Elemental
composition
m/z
([M+Na] )
Error
(ppm)
M
r
Relative abundance (%)
D. purpurea D. vigilis D. cordata D. megathura D. integerrima
+
a
+
+
+
+
+
+
+
+
+
7
9
1
1
1
1
1
2
2
, 8
, 10
1
C
C
C
C
C
C
C
C
C
19
H
21
H
23
H
23
H
21
H
25
H
25
H
23
H
27
H
36
38
40
42
40
44
46
44
48
O
6
O
7
O
8
O
7
O
6
O
8
O
7
O
6
O
8
Na
Na
Na
Na
Na
Na
Na
Na
Na
383.29800
425.25130
467.26179
453.28256
411.27216
495.29381
481.31404
439.30342
523.32383
+0.2
+0.8
+0.8
+0.8
+1.1
+0.7
+1.4
+1.0
ꢀ0.6
360
402
444
430
388
472
458
416
500
1.3
4.8
7.0
0.3
10.5
4.0
100
8.4
3.5
28.0
2.6
–
2.5
38.3
22.7
100
16.0
8.6
15.6
2.2
–
1.8
38.9
25.4
2.9
45.9
22.1
100
16.1
6.8
13.5
2.2
–
2, 13, 16, 17
4, 15
8
100
100
28.2
38.9
62.7
17.9
13.8
12.0
10.1
16.5
2.2
9, 22, 23
0, 21
4
1.4
a
+
The exemplary m/z ([M+Na] ) and error (ppm) data were obtained from D. integerrima (exception: compound 24 from D. purpurea).
+
+
+
An isomerization via an acyl-migration might occur during
the storage or during the analytical procedure (Lyubachevs-
kaya and Boyle-Roden, 2000; Seipold, 2004; Christie,
detected, namely [M+H] , [M+NH ] , [M+Na] and
4
+
+
[M+K] . The ion type [M+NH ] represented the predom-
inant adduct. The DAG of (3R)-acetoxypalmitic acid rep-
4
2
006). Therefore, compounds 12, 13, and 19 also may be
artefacts.
Table 3 shows the relative composition of acylglycerols
resents the compound with the highest abundance as
+
shown by ions at m/z 448 [M+NH ] and m/z 453
4
+
[M+Na] . The results indicated the DAGs and MAGs as
of the Diascia flower oils investigated. In most of the cases,
the six Diascia spp. exhibited a similar pattern with respect
to their MAG, DAG and TAG distribution. Exceptionally,
in Diascia cordata no TAG could be detected.
the predominant constituents along with a small amount
of TAGs and were in good agreement with EI-MS
experiments.
In conclusion, positive-ion ESI-FTICR mass spectra of
the non-derivatized floral oils of Diascia spp. exposed
essential results with respect to the acylglycerol profile.
The determination of the elemental composition can yield
a faster and simple look at the lipid pattern of the oil-
secreting Diascia flowers. With respect to the relative com-
position of the floral oils the most prominent components
are shown both in the FTICR-MS or ESI-MS and the
GC–MS data (Tables 3 and 4).
By TMSi-derivatization of the floral oils of Diascia spp.,
fatty acids could not be detected. DAGs were the most
abundant class (60–80%) along with MAGs (20–30%)
and TAGs (<15%). MAGs and DAGs as well as a small
amount of TAGs were also described as the main oil com-
ponents in Byrsonima crassifolia (Malpighiaceae) elaio-
phores (Vinson et al., 1997). The dominance of MAGs
and DAGs could be related to the insects’ digestive system.
It could be shown that both MAGs and DAGs are better
digestible than TAGs (Vinson et al., 1997).
3. Conclusions
2
.3. Analysis of acylglycerols of underivatized Diascia oils
Floral oils represent the vital floral reward of Diascia
The underivatized floral oils of the Diascia spp. were
species. The variation in foreleg lengths of Rediviva could
be explained as an evolutionary response to Diascia floral
spur lengths (Steiner and Whitehead, 1990, 1991). The flo-
ral oils play an important role in larval provision and have
also been suggested to be used in nest construction (Cane
et al., 1983; Buchmann, 1987). Some hydroxylated fatty
acids are reported to possess antibiotic properties (Valcavi
et al., 1989; Weil et al., 2002). The prevalence of such chem-
ical species in the Diascia flower oils instead of simple fatty
acid oils – may be necessary to keep the larval food free of
microbial decomposition, or for the nest cell lining. The
additional acetylation may either be required by the plant
for excretion (Seipold et al., 2004) or to reduce the oils
water content, thereby avoiding, e.g., fungal decomposi-
tion. Also, there is no detailed report on the chemistry of
Rediviva bee nest cell lining. Therefore, some further inves-
tigations have to be carried out to verify the chemical nat-
ure of the association between Diascia flower oil and
Rediviva bee cell lining. Future questions concern the
investigated by ESI-FTICR-MS to obtain high resolution
mass data of the lipid compounds allowing a rapid profil-
ing of the various oils. All measurements were performed
in the positive-ion mode. In these cases the electrospray
mass spectra of the investigated oils display sodium
+
adducts ([M+Na] ) of the corresponding compounds
(
Table 4). The positive ESI-FTICR mass spectrum of Dias-
cia integerrima displays a DAG signal (base peak) compris-
ing compounds 12, 13, 16 and 17 (m/z 453.28256) (Fig. 7).
It should be noted that the ESI-FTICR-MS of D. cordata
indicates the presence of TAGs with (3R)-acetoxy fatty
acids with chain lengths of C14 and C , whereas the EI-
16
MS data yield no evidence of such TAGs. However, all
the significant acylglycerol components could be detected
with both methods.
The underivatized D. barberae oil was investigated by
positive ESI-MS experiments using ammonium acetate
(
Fig. 8). In that case, four types of adduct ions were

1378
K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
natural variation of flower oil compositions within a spe-
cies or with flower age, the absolute configuration of glyce-
rols with a chiral center at the sn-2 position, and if
stereochemistry has any relevance in the biological context.
the twin spurs. The amounts of the lipids per flower varied
from 1 to 5 ll. All samples were dissolved into 450 ll of
t-butylmethylether (MTBE)–MeOH (2:1, v/v) and stored
under N at ꢀ18 °C (Seipold, 2004; Seipold et al., 2004;
2
Neff and Simpson, 2005).
4
. Experimental
4.3. Derivatization of floral oils
4
.1. Oil-secreting flowers
Fatty acid methyl ester (FAME) profiling was carried
out according to the previously described procedure by
using the transesterification followed by trimethylsilylation
(Seipold et al., 2004). Both the crude Diascia oils and the
methanol transesterified oils were then converted to the tri-
methylsilyl (TMSi)-derivatives by using 2,2,2-trifluoro-
N-methyl-N-(trimethylsilyl) acetamide (MSTFA) as
reagent (2 h at room temperature, see Morrison and Smith,
1964; Seipold, 2004; Seipold et al., 2004). Diluted deriva-
tized samples were analyzed by GC–MS as described
below.
Different specimens of Diascia purpurea, D. vigilis, D.
cordata, Diascia megathura, D. integerrima (Scrophularia-
cece) were collected in the Drakensberg area of Southern
Africa. D. barberae was cultivated in a green house of the
Botanical Garden Munich (Germany). All samples were
collected during the blooming stage between January and
February 2006. Samples of each species were collected at
single locations from 1 to 4 individuals with 2–10 flowers
each, except for D. purpurea, of which two collections
(
S.D., Bayreuth: SA3, SA9) were made. In order to gather
enough material for this first survey, all samples of a spe-
cies were united, thereby averaging the composition.
4.4. Synthesis of (3R)-hydroxypalmitic acid methyl ester
(Fig. 2)
4
.2. Gathering of floral oils
In a 50 ml two neck-flask, equipped with a drop funnel
with pressure balance and reflux cooler, 1.44 g calcium
chloride (144.13 g/mol, 10 mmol) were added to 1.38 g
Meldrum’s acid (0.983 g/ml, 79.10 g/mol, 17.4 mmol) in
12 ml CH Cl and 1.4 ml pyridine at 0 °C. 2.9 ml or
2.64 g myristic acid chloride (246.82 g/mol, 0.91 g/ml,
10.7 mmol) were added dropwise. The solution was stirred
for 1 h at 0 °C and additional 4 h at room temperature. The
Most Diascia species have twin spurs containing oil-
secreting glands located within their tip. Fig. 1 shows D.
megathura (Scrophulariaceae) as an example. Fresh flowers
were cut and floral oils were collected from the trichome
elaiophores in the field. Tiny pieces of filter papers were
carefully manipulated to adsorb the oil from the inside of
2
2
Fig. 1. Diascia megathura (Scrophulariaceae): (a) inflorescence showing spurs and (b) spur longitudinally split, showing the elaiophores with free oil.
Arrows show the spurs (Photos: G. Gerlach).

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1379
Fig. 2. Synthetic route to (3R)-hydroxypalmitic acid methyl ester.
orange solution was washed twice with 2 N HCl and then
300 °C for 20 min. The mass spectrometer was operated
with an electron impact (EI) 70 eV, ion source temperature
180 °C and mass range 40–800 a.m.u.
with 5% aq. NaHCO solution. The upper layer was sepa-
3
rated and the solvent evaporated under vacuum. The
orange residue was dissolved in 25 ml MeOH and refluxed
for 3 h. The crude 3-ketopalmitic acid methyl ester was
purified by chromatography on silica gel 60 (particle size
4.5. Synthesis of (2S)-phenylpropionyl chloride
Ninety milligrams (+)-(2S)-phenylpropionic acid
0
.040–0.063 mm (230–400 mesh), Merck, Darmstadt, Ger-
many) (hexane–EtOAc (6:1, v/v)) to give an overall yield of
1% (1.61 g) (Valcavi et al., 1989; Oikawa et al., 1978; Sei-
(164.08 g/mol, 0.55 mmol) and 120 ll thionyl chloride were
mixed at 0 °C and kept at 70 °C for 30 min. Dried benzene
was added, and the mixture was evaporated to dryness.
This step was repeated to remove traces of thionyl chloride.
The residue was dissolved in 1 ml dry benzene and kept at
6
1
pold, 2004). H NMR (300 MHz, CDCl ): d 0. 88 (3H, t,
3
J = 6.8 Hz), 1.23–1.30 (22H, m), 1.61 (2H, m), 2.53 (2H,
t, J = 7.31 Hz), 3.45 (2H, s); 3.74 (3H, s) (Seipold, 2004).
Five hundred milligrams of 3-ketopalmitic acid methyl
ester (284.44 g/mol, 1.76 mmol) in 15 ml of MeOH/CH Cl
4
°C in a sealed flask (Hammarstr o¨ m, 1975; Seipold, 2004).
2
2
4.6. Determination of the absolute configuration
(
96:4 v/v) was degassed by triple freezing and
thawing under vacuum. Under argon, 10 mg of
dichloro[(R)(+)2,2 bis(diphenylphosphino)1,1 binaphthyl]-
0
0
The procedures were carried out using GC–MS tech-
niques, due to the limited amount of floral oil samples. A
ruthenium(II) ([RuCl (C H P )] , 794.67 g/mol, 0.013
2
44 32 2 x
mmol, Strem Chemicals, USA) was added. The reduction
was carried out in a pressure reactor under a hydrogen
atmosphere of 34.5 bar (500 psi) for 24 h at 80 °C (Heiser
et al., 1991). The solvent was evaporated under vacuum.
The residue was dissolved in a mixture of benzene–EtOAc
(
4:1, v/v) and filtered through 2 g silica gel to eliminate the
catalyst (Heiser et al., 1991). (3R)-Hydroxypalmitic acid
methyl ester was obtained in a purity >99% (GC). The
optical rotation was ꢀ14.2° at 24 °C (k 589 nm) (literature
ꢀ
14.3°, Tulloch and Spencer, 1964). The (2S)-phenylpropi-
onate derivative of (3R)-hydroxypalmitic acid methyl ester
revealed in 93% ee (GC).
(
3R)-Hydroxypalmitic acid methyl ester was obtained in
9
1
7% yield (m.p. 82 °C, literature: 83–85 °C, Valcavi et al.,
1
989). H NMR (300 MHz, CDCl ): d 0.88 (3H, t,
3
J = 6.9 Hz), 1.23–1.30 (22H, m), 1.44 (2H, m), 2.41 (1H,
dd, J1 = 16.5 Hz, J2 = 8.8 Hz), 2.52 (1H, dd,
J = 16.5 Hz, J = 3.3 Hz), 3.71 (3H, s), 4.0 (1H, m) (Sei-
1
2
pold, 2004). The purity (%) and ee (%) measurements were
obtained with a GC 8000 series (Fisons Instruments)
gas chromatograph with MS-detector (DB-5 MS, 20 m ꢁ
Fig. 3. Partial total ion chromatogram (TIC) of the FAME profiling of D.
vigilis oil. (For the identified compounds of interest see Table 1; the non-
labeled peaks correspond to fatty acid TMSi-derivatives, saturated
0
.18 mm, i.d., 0.18 lm film thickness) (J&W Scientific,
hydrocarbons, artefacts, and not identified compounds:
t
R
13.56
Folsom, CA, USA). The column temperature was
(
min) = trace of non-silylated hydroxy fatty acid methyl ester (C14),
programmed for 1 min at 60 °C, increased 15 °C/min from
1
4.60 = dibutyl phthalate, 14.66 = not identified, 17.23 = octadecanol,
6
0 to 200 °C, and 5 °C/min to 300 °C and completed at
17.37 = not identified, 18.01 = saturated hydrocarbon.)

1380
K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
comparison of retention times between the (2S)-phenylpro-
pionyl derivatives of Diascia oils and of (3R)-hydroxypal-
mitic acid methyl ester as chiral standard were performed
complete separation of the R- and S-derivatives
(a = 1.01) under the GC–MS conditions described below
(Hammarstr o¨ m, 1975; Seipold, 2004).
(
Hammarstr o¨ m, 1975; Wollenweber et al., 1985; Grad-
owska and Larsson, 1994). Racemic mixtures were
obtained directly from the Diascia oils through oxidation
with potassiumdichromate (K CrO ) and backward reduc-
4.7. GC/EI-MS analysis of natural samples
The GC–MS investigations were performed with two
GC–MS systems. Five floral oils (D. purpurea, D. vigilis,
D. cordata, D. megathura and D. integerrima) were
2
4
tion with sodiumborohydride (NaBH ) (Fabritius et al.,
4
1
996). (2S)-Phenylpropionyl derivatives of samples gave a
Fig. 4. Total ion chromatogram (TIC) of TMSi-derivatives of D. vigilis oil. (For the corresponding identified compounds see Table 2.)
Fig. 5. Mass spectra of TMSi-derivatives of monoacylglycerols (MAGs): (a) 2-[(3R)-acetoxypalmitoyl]glycerol (14); and (b) 1-[(3R)-acetoxypalm-
itoyl]glycerol (15). The significant fragment ions are described in Scheme 1.

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1381
Fig. 6. Mass spectra of TMSi-derivatives of diacylglycerols (DAGs): (a) 2-[(3R)-acetoxypalmitoyl]-1-acetylglycerol (16); and (b) 1-[(3R)-acetoxypalm-
itoyl]-3-acetylglycerol (17). The significant fragment ions are described in Schemes 1 and 2.
Fig. 7. Positive-ion ESI-FTICR mass spectrum of the acylglycerol profile of D. integerrima. Peak heights are scaled relative to the highest magnitude peak.
(For the corresponding identified compounds see Table 4.)
separated using a Finnigan Voyager GC/MS system
electron energy, ion source temperature 200 °C and a mass
range of 40–800 a.m.u.
mounted with capillary column (DB-5 MS, 30 m ꢁ
0
.25 mm i.d., 0.25 lm film thickness) (J&W Scientific,
The investigation of the floral oil of D. barberae was car-
ried out on an Agilent 6890 series (Wilmington, USA) gas
chromatograph equipped with a Micromass (Manchester,
UK) GCT mass sensitive reflectron time-of-flight (TOF)
detector (DB-5 MS, 30 m ꢁ 0.25 mm, i.d., 0.25 lm film
thickness) (J&W Scientific, Folsom, CA, USA) (see above
for the conditions).
Folsom, CA, USA). The following temperature program
was used: from 60 to 200 °C with a rate of 10 °C/min, fol-
lowed by 5 °C/min to 300 °C and a hold at 300 °C for addi-
tional 20 min. Helium was used as the GC carrier gas at a
constant flow of 1 ml/min. The mass spectrometer was
operated with an electron impact (EI) ion source at 70 eV

1382
K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
Fig. 8. Positive-ion ESI mass spectrum of the acylglycerol profile of underivatized D. barberae oil. (For the corresponding identified compounds see Tables
2
and 3.)
4
.8. ESI-FTICR-MS analysis
Kuhnt (IPB Halle) and Jutta Babczinsky (Botanical Gar-
den Munich) for helpful technical assistance. K. Dumri
thanks the German Academic Exchange Service (DAAD)
for a Leibniz-DAAD scholarship.
The positive-ion high resolution ESI mass spectra of oils
of all species except D. barbarae were obtained from a Bru-
ker Apex III Fourier-transform ion cyclotron resonance
(
FTICR) mass spectrometer (Bruker Daltonics, Billerica,
TM
USA) equipped with an Infinity cell, a 7.0 T supercon-
ducting magnet (Bruker, Karlsruhe, Germany), an RF-
only hexapole ion guide and an APOLLO electrospray
ion source (Agilent, off axis spray, voltages: endplate,
References
Buchmann, S.L., 1987. The ecology of oil flowers and their bees. Annu.
Rev. Econ. Syst. 18, 343–369.
Cane, J.H., Eickwort, G.C., Wesley, F.R., Spielholz, J., 1983. Foraging,
grooming and mate-seeking behaviors of Macropis nuda (Hymenop-
tera, Melittidae) and use of Lysimachia ciliata (Primulaceae) oils in
larval provisions and cell linings. Am. Midl. Nat. 110, 257–264.
Christie, W.W., 2006. The Lipid Library. Online available at: www.lipi-
dlibrary.co.uk.
ꢀ
3.700 V; capillary, ꢀ4.200 V; capillary exit, 100 V; skim-
mer 1, 15.0 V; skimmer 2, 10.0 V). N was used as drying
2
gas at 150 °C. 0.1 mg of Diascia oil in MeOH was intro-
duced continuously via a syringe pump with a flow rate
of 120 ll h . All data were acquired with 512k data points
and zero filled to 2048k by averaging 32 scans.
ꢀ1
2
Curstedt, T., 1974. Mass spectra of trimethylsilyl ethers of H-labelled
mono- and diacylglycerides. Biochim. Biophys. Acta 360, 12–23.
Fabritius, D., Sch a¨ fer, H.J., Steinb u¨ chel, A., 1996. Identification and
production of 3-hydroxy-9-cis-1,18-octadecenedioic acid by mutants of
Candida tropicalis. Appl. Microbiol. Biotechnol. 45, 342–348.
Gradowska, W., Larsson, L., 1994. Determination of absolute configu-
rations of 2- and 3-hydroxy fatty acids in organic dust by gas-
chromatography–mass spectrometry. J. Microbiol. Method 20, 55–67.
Hammarstr o¨ m, S., 1975. Microdetermination of stereoisomers of
2-hydroxy and 3-hydroxy fatty acids. Method Enzymol. 35, 326–334.
Heiser, B., Broger, E.A., Crameri, Y., 1991. New efficient methods for the
synthesis and in situ preparation of ruthenium(II) complexes of
atropisomeric diphosphines and their application in asymmetric
catalytic hydrogenations. Tetrahedron: Asymm. 2, 51–62.
4
.9. ESI-MS analysis
Positive-ion ESI mass spectra of D. barberae were
obtained by using a Finnigan TSQ 7000 instrument. Fifty
microlitres of D. barberae oil was dried in a constant
stream of N . The oil residue was dissolved in 1 ml of
5
The sample was injected using a syringe pump at a constant
flow 5 ll/min. The electrospray voltage was 4.5 kV, the
capillary temperature 200 °C. N was used as a sheath
gas (Seipold, 2004; Seipold et al., 2004).
2
mM ammonium acetate in CHCl –MeOH (70:30, v/v).
3
2
Johnson, C.B., Holman, R.T., 1966. Mass spectrometry of lipids. II:
monoglycerides, their diacetyl derivatives and their trimethylsilyl
ethers. Lipids 1, 371–380.
Lyubachevskaya, G., Boyle-Roden, E., 2000. Kinetics of 2-monoacyl-
glycerol acyl migration in model chylomicra. Lipids 35, 1353–1358.
Mayberry, W.R., 1980. Hydroxy fatty acids in Bacteroides species: D-(ꢀ)-
Acknowledgements
3
1
-hydroxy-15-methylhexadecanoate and its homologs. J. Bacteriol.
43, 582–587.
The authors are grateful to the Deutsche Forschungs-
gemeinschaft (DFG) for financial support, and to Christine
Mielniczuk, Z., Mielniczuk, E., Larsson, L., 1993. Gas chromatography–
mass spectrometry methods for analysis of 2- and 3-hydroxylated fatty

K. Dumri et al. / Phytochemistry 69 (2008) 1372–1383
1383
acids: application for endotoxin measurement. J. Microbiol. Method
7, 91–102.
Simpson, B.B., Seigler, D., Neff, J.L., 1979. Lipid from the floral glands of
Krameria. Biochem. Syst. Ecol. 7, 193–194.
1
Morrison, W.R., Smith, L.M., 1964. Preparation of fatty acid methyl
esters and dimethylacetals from lipids with boron fluoride–methanol.
J. Lipid Res. 5, 600–608.
Myher, J.J., Marai, L., Kuksis, A., 1974. Identification of monoacyl- and
monoalkylglycerols by gas–liquid chromatography–mass spectrometry
using polar siloxane liquid phases. J. Lipid Res. 15, 586–592.
Neff, J.L., Simpson, B.B., 2005. Other rewards: oils, resins, and gums. In:
Dafni, A., Keven, P.G., Husband, B.C. (Eds.), Practical Pollination
Biology. Enviroquest Ltd., Cambridge, pp. 314–328.
Steiner, K.E., Whitehead, V.B., 1988. The association between oil-
producing flowers and oil-collecting bees in the Drakensberg of
Southern Africa. Monogr. Syst. Bot. Missouri Bot. Gard. 25,
259–277.
Steiner, K.E., Whitehead, V.B., 1990. Pollinator adaptation to oil-
secreting flowers Diascia and Rediviva. Evolution 44, 1701–1707.
Steiner, K.E., Whitehead, V.B., 1991. Oil flowers and oil bees: further
evidence for pollinator adaptation. Evolution 45, 1493–1501.
Tulloch, A.P., Spencer, J.F.T., 1964. Extracellular glycolipids of Rhodo-
torula species. Can. J. Chem. 42, 166–173.
Valcavi, U., Albertoni, C., Brandt, A., Corsi, G.B., Farima, P., Foresta,
P., Pascucci, G., Ramacci, M.T., 1989. New potential immuno
enhancing compounds. Synthesis and pharmacological evaluation of
new long-chain 2-amido-2-deoxy-D-glucose derivatives. Arzneim. For-
sch./Drug Res. 39, 1190–1195.
Oikawa, Y., Sugano, K., Yonemitsu, O., 1978. Meldrum’s acid in organic
synthesis. 2: a general and versatile synthesis of b-keto esters. J. Org.
Chem. 43, 2087–2088.
Reis, M.G., Faria, A.D., Bittrich, V., Amaral, M.C.E., Marsaioli, A.J.,
2000. The chemistry of flower rewards – Oncidium (Orchidaceae). J.
Braz. Chem. Soc. 11, 600–608.
Reis, M.G., Faria, A.D., Bittrich, V., Amaral, M.C.E., Marsaioli, A.J.,
Vinson, S.B., Williams, H.J., Frankie, G.W., Shrum, G., 1997. Floral lipid
chemistry of Byrsonima crassifolia (Malpighiaceae) and a use of floral
lipids by Centris bees (Hymenoptera: Apidae). Biotropica 29,
76–83.
Vogel, S., 1969. Flowers offering fatty oil instead of nectar. In:
Proceedings of the XI International Botanical Congress, 24 August–
2 September 1969 and International Wood Chemistry Symposium, 2–4
September 1969, Seattle, WA, USA. Abstract No. 229.
2003. Oncidinol – a novel diacylglycerol from Ornithophora radicans
Barb. Rodr. (Orchidaceae) floral oil. Tetrahedron Lett. 44, 8519–8523,
Please note that the given structure of oncidinol is misassigned as 3,6-
diacetoxy fatty acid, but actually is a 3,7-diacetoxy derivative.
Reis, M.G., de Faria, A.D., dos Santos, I.A., Amaral, M.d.C.E.,
Marsaioli, A.J., 2007. Byrsonic acid – the clue to floral mimicry
involving oil-producing flowers and oil-collecting bees. J. Chem. Ecol.
¨
33, 1421–1429.
Vogel, S., 1974. Olblumen und o¨ lsammelnde Bienen. Akademie der
Seigler, D., Simpson, B.B., Martin, C., Neff, J.L., 1978. Free 3-acetoxy
Wissenschaften und der Literatur, Mainz.
¨
fatty acids in floral glands of Krameria species. Phytochemistry 7, 995–
Vogel, S., 1986. Olblumen und o¨ lsammelnde Bienen. Akademie der
996.
Wissenschaften und der Literatur, Mainz.
¨
Seipold, L., 2004. Bl u¨ ten o¨ le – Chemische Analyse, Biosynthese und
Vogel, S., 1990. Olblumen und o¨ lsammelnde Bienen. Akademie der
¨
Betrachtungen zur Entstehung von Olblumen. Ph.D. Thesis, Leibniz
Wissenschaften und der Literatur, Mainz.
Institute of Plant Biochemistry and Martin-Luther-Universit a¨ t Halle-
Wittenberg, Halle.
Weil, K., Humpf, H.-U., Schwab, W., Schreier, P., 2002. Absolute
configuration of 3-hydroxy acids formed by Stenotrophomonas malto-
philia: application of multidimensional gas chromatography and
circular dichroism spectroscopy. Chirality 14, 51–58.
Wollenweber, H-W., Schramek, S., Moll, H., Rietschel, E.T., 1985.
Nature and linkage type of fatty acids present in lipopolysaccharides of
phase I and II Coxiella burnetii. Arch. Microbiol. 142, 6–11.
Wood, G.W., 1980. Complex lipids. In: Waller, G.R., Dermer, O.C.
(Eds.), Biochemical Applications of Mass Spectrometry, First supple-
mentary vol. John Wiley & Sons, New York, pp. 173–209.
Zachowski, A., Guerbette, F., Grobois, M., Jolliot-Croquin, A., Kader,
J.C., 1998. Characterisation of acyl binding by a plant lipid transfer
protein. Eur. J. Biochem. 257, 443–448.
Seipold, L., Gerlach, G., Wessjohann, L., 2004. A new type of floral oil
from Malpighia coccigera (Malpighiaceae) and chemical consider-
ations on the evolution of oil flowers. Chem. Biodivers. 1, 1519–1528.
Simpson, B.B., Neff, J.L., 1981. Floral rewards: alternatives to pollen and
nectar. Ann. Mo. Bot. Gard. 68, 301–322.
Simpson, B.B., Neff, J.L., 1983. Evolution and diversity of floral rewards.
In: Jones, C.E., Little, R. (Eds.), Handbook of Experimental Pollina-
tion Biology. Scientific and Academic Editions, New York, pp. 142–
157.
Simpson, B.B., Neff, J.L., Seigler, D., 1977. Krameria, free fatty acids and
oil-collecting bees. Nature 267, 150–151.