Thesinine-4'-O-beta-D-glucoside the first glycosylated plant pyrrolizidine alkaloid from Borago officinalis.

Article abstract of DOI:10.1016/S0031-9422(02)00069-9

The glycosylated pyrrolizidine alkaloid, thesinine-4'-O-beta-D-glucoside, has been isolated from the aqueous methanol extract of dried, defatted seeds of Borago officinalis (Boraginaceae). The structure was established by means of spectroscopic and chemic

Full text of DOI:10.1016/S0031-9422(02)00069-9

Phytochemistry 60 (2002) 399–402
www.elsevier.com/locate/phytochem
Thesinine-40-O-b-d-glucoside the first glycosylated plant
pyrrolizidine alkaloid from Borago officinalis
Martina Herrmann*, Holger Joppe, Gerhard Schmaus
Corporate Research Division, DRAGOCO Gerberding & Co. AG, D-37601 Holzminden, Germany
Received 19 November 2001; received in revised form 7 February 2002
Abstract
The glycosylated pyrrolizidine alkaloid, thesinine-40-O-b-d-glucoside, has been isolated from the aqueous methanol extract of
dried, defatted seeds of Borago officinalis (Boraginaceae). The structure was established by means of spectroscopic and chemical
analysis. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Borago officinalis; Boraginaceae; Borage; Glycosylated pyrrolizidine alkaloid; Thesinine-40-O-b-d-glucoside
1. Introduction
In the present study we report on the isolation and
structure elucidation of the first glycosylated pyrrolizi-
dine alkaloid, thesinine-40-O-b-d-glucoside (1), obtained
from the aqueous methanol extract of borage seeds.
Borage (Borago officinalis L., Boraginaceae) is an
annual herb originating from the Mediterranean area,
now cultivated not only in the northern hemisphere but
also in Australia, New Zealand, South Africa and South
America for the production of its seed oil. Borage seed
oil is a rich source of the essential fatty acid g-linolenic
acid (18–25%) and is, therefore, used in nutritional,
cosmetical and medical applications (Roeder et al.,
2001).
However, members of the Boraginaceae family are
also known for their content of pyrrolizidine alkaloids
(PAs). Especially unsaturated PAs have been shown
to be hepatotoxic and carcinogenic. So far seven PAs
were identified in leaves, flowers and seeds of borage
with thesinine, a saturated and therefore probably non-
toxic necine base described as a major alkaloid. Besides
thesinine six unsaturated PAs, amabiline, supinine,
lycopsamine, intermedine, acetyllycopsamine and acetyl-
intermedine, were identified as minor constituents
(Langer and Franz, 1997). The total alkaloid amount of
the plant was determined as less than 0.001% (Roeder,
1995) whereas mature seeds yielded about 0.03% crude
alkaloids (Dodson and Stermitz, 1986).
2. Results and discussion
Compound 1 was isolated as a colourless, amorphous
solid from the aqueous methanol extract of defatted,
dried borage seeds after partitioning between aqueous
HCl and ethyl acetate followed by RP-18 chromato-
graphy of the concentrated aqueous phase. The mole-
cular formula of 1 was determined as C23H31NO8 by
HR–DCIMS, which exhibited a [M+H]+ ion peak at
m/z 450.2143 (calc. 450.2128). Three structure units
could be identified by spectroscopic means. From the
UV spectral data with absorption maxima at 305, 225
and 204 nm the presence of a p-hydroxy cinnamoyl
group was deduced (Lee, 2000), which was supported by
a set of AA0XX0 doublets at ꢀ 7.51 (2 H, d, J=8.6 Hz)
and 7.08 (2 H, d, J=8.6 Hz) and two trans-configurated
olefinic protons at ꢀ 7.66 (1 H, d, J=16.0 Hz) and 6.34
(1H, d, J=16.0 Hz) in the 1H NMRspectrum. A further
unit was established from the chemical shifts of the six
methylene and two methine groups C-1 to C-9 in the 1H
and 13C NMRspectra and their correlations in the
1H–1H COSY NMRspectrum as 1-hydroxymethyl
pyrrolizidine residue (Table 1). Both units together
indicate the structural relation to the known thesinine.
* Corresponding author. Tel.: +49-5531-971414; fax: +49-5531-
971158.
E-mail address: herrmann.martina@eu.dragoco.com (M. Herrmann).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00069-9

400
M. Herrmann et al. / Phytochemistry 60 (2002) 399–402
Unfortunately, no confirming HMBC correlation was
observed between H-9 and the carbonyl carbon C-90, even
when HMBC data were recorded under a variety of
different experimental conditions. Further signals in the
1H and 13C NMRspectra of 1 could be assigned to a
sugar unit, as evident from an anomeric proton signal at
ꢀ 4.99 (1H, d, J=6.8 Hz) and carbon signals at ꢀ 100.6,
76.7 (2 carbon atoms), 73.5, 70.1 and 61.7. The con-
nectivity of the sugar unit was established from the
HMBC spectrum showing a correlation between H-100
and C-40.
Full structure elucidation was achieved by acidic and
alkaline hydrolysis of 1 (Fig. 1). Acidic hydrolysis gave
the hexopyranose and the PA aglycone. The latter was
confirmed to be thesinine (2) by LC–MS (ESI) spectro-
scopy, giving the expected pseudomolecular ion peak at
m/z 288 for [M+H]+, and by comparison of the 1H and
13C NMRdata with those published by Dodson and
Stermitz (1986). The complete assignments of the pro-
ton and carbon shifts of 1 and 2 aided by DEPT, 1H–1H
COSY and C,H correlated 2D NMRspectra are given
in Table 1 and the experimental section, respectively.
The sugar residue was determined as glucose by TLC
and HPLC comparison with an authentic sample. From
the large coupling constant of the anomeric proton H-100
(J=6.8 Hz) the b-configuration of the hexopyranose
was deduced. Alkaline hydrolysis yielded p-coumaric
acid-4-O-b-d-glucoside (3), which was identified by
NMRspectroscopy (Foo et al., 2000). Comparison of
the optical rotation data of 3 with those published in
literature (Chapman & Hall Chemical Databases, 2001)
established the D-configuration of C-100 of the glucose
moiety. Thus, the structure of compound 1 was eluci-
dated as thesinine-40-O-b-d-glucoside.
In 1999 a PA glycoside, most likely senecionine-O-
glucoside, was identified by LC-MS as detoxification
product in PA sequestering chrysomelid leaf beetles
(Hartmann et al., 1999). However, to our knowledge
thesinine-40-O-b-d-glucoside (1) is the first glycosylated
plant PA described in literature. With a content of
about 0.5% in dried borage seeds and 0.02% in fresh
aerials, 1 is occurring in much higher amounts than
the non glycosylated PAs earlier described for Borago
officinalis. Presumably because of its high polarity it has
not been isolated before. Applying the usual alkaloid
extracting procedure including acidic-basic liquid-liquid
partition (e. g. Parvais et al., 1994), 1 is not found in the
organic alkaloid phase but remains completely in the
aqueous layer.
Structurally related to 1 are several lupin alkaloid
glycosides, e. g. (trans-40-glucosyloxycinnamyl)lupinine
(4), isolated from Lupinus luteus (Leguminosae) seed-
Table 1
0
1H and 13C NMRspectral data of thesinine-4 -O-b-d-glucoside (1)a
Position ꢀC
ꢀH
1H–1H COSY
1
2
44.5 2.40 (m)
29.5 2.23 (m)
1.91 (m)
H-2, 8, 9
H-1, 3
H-1, 3
H-2
3
5
54.1 3.90 (m)
2.88 (ddd, J=6.0, 10.9 and 10.9 Hz) H-2
54.3 3.52 (m) H-6
2.99 (ddd, J=5.8, 5.8 and 11.5 Hz) H-6
6
7
25.1 2.10 (m)
30.7 2.23 (m)
H-5, 6
H-6, 8
H-6, 8
H-1, 7
H-1
1.91 (m)
69.3 4.01 (m)
8
9
64.7 4.33 (dd, J=5.9 and 11.4 Hz)
4.23 (dd, J=7.1 and 11.4 Hz)
128.6
H-1
10
20/60
30/50
40
130.1 7.51 (d, J=8.6 Hz)
117.0 7.08 (d, J=8.6 Hz)
159.5
H-30, 50
H-20, 60
70
145.6 7.65 (d, J=16.0 Hz)
115.4 6.34 (d, J=16.0 Hz)
167.4
H-80
H-70
800
900
100
200
3
100.6 4.99 (d, J=6.8 Hz)
73.5 3.52 (m)
76.7 3.52 (m)
H-200
400
500
600
70.1 3.52 (m)
76.7 3.52 (m)
61.7 3.79 (m)
H-500
a
Fig. 1. Thesinine-40-O-b-d-glucoside (1) and reaction products
obtained from acidic and alkaline hydrolysis.
Measured in CDCl3 with CD3OD added to improve solubility at
300 (1H NMR) and 75 MHz (13C NMR ).

M. Herrmann et al. / Phytochemistry 60 (2002) 399–402
401
lmax nm (log "): 305 (4.29), 297 (4.28), 225 (4.06), 204
(4.15); IR(KBr) nmax cmꢂ1: 3410, 1711, 1604, 1510,
1240, 1171, 1073; 1H NMR(see Table 1); 13C NMR(see
Table 1); DCIMS (NH3), 120 eV, m/z=450 [M+H]+,
HR–DCIMS (NH3), 120 eV, calc. for C23H32NO8
[M+H]+ 450.2128, found: 450.2143.
Fig. 2. (trans-40-Glucosyloxycinnamyl)lupinine (4).
3.4. Acidic hydrolysis of 1
Forty-five milligrams of 1 were dissolved in 6 ml of
MeOH and 25 ml of 3% aq. HCl and hydrolysed under
reflux (1 h). After concentration in vacuo, aq. NaHCO3
was added and the alkaline solution was extracted with
EtOAc. The organic phase was evaporated to dryness
and separated by preparative RP18-HPLC (column:
Eurospher 80 C18, 10 mm, 250ꢀ16 mm; eluent: MeOH/
H2O 50:50+0.5 ml HOAc/l, flow rate: 10 ml/min, l: 330
nm) to give 5 mg of thesinine (2).
lings by Murakoshi et al. (1977, 1979). In contrast to 1
they consist of quinolizidine 6-rings instead of the pyr-
rolizidine 5-rings (Fig. 2).
3. Experimental
3.1. General
The aqueous layer was concentrated in vacuo and
glucose was identified by Co-TLC [aluminium sheets
silica gel 60 F254, eluent: CHCl3/Me2CO/MeOH/H2O
3:3:3:1, detection: anisaldehyde/H2SO4, 120 C, Rf (glu-
cose) 0.42] and HPLC [column: Phenomenex REZEX
The following instruments were used: OR: polari-
meter 241 (Perkin-Elmer), IR: IFS25 FTIR- spectro-
meter (Bruker), UV: U3300 spectrophotometer
(Hitachi), 1H and 13C NMR: Varian VXR-300 (300
and 75.5 MHz, respectively with TMS as internal stan-
dard), MS: MAT-95 mass spectrometer (Finnigan).
Analytical HPLC: HP1100, column YMC ODS-AQ,
5 mm, 150ꢀ3 mm with 10ꢀ3 mm guard column, flow
rate 0.6 ml/min, l 330 nm, temp. 40 ꢁC, gradient:
A=water+0.1% HCO2H, B=CH3CN, 0–10 min/2.5–
25% B, 10–15 min/25–40% B, HPLC–MS: HP1100
with Bruker Esquire LC, ion source: ESI, nebulizer gas
ꢁ
RPM Monosaccaride, 300ꢀ7 mm, eluent: H2O, temp.
ꢁ
80 C, flow rate 0.6 ml/min, Rt (glucose) 12.1 min] with
MS detection (ESI, positive ion mode, m/z 203 for
[M+Na]+) with an authentic sample.
3.4.1. Thesinine (2)
Analytical HPLC: Rt=12.6 min; 1H NMR(300 MHz,
CDCl3) ꢀ 7.56 (1H, d, J=16.0 Hz, H-70), 7.35 (2 H, d,
J=8.5 Hz, H-20 and H-60), 6.89 (2 H, d, J=8.5 Hz, H-30
and H-50), 6.13 (1 H, d, J=16.0 Hz, H-80), 4.32 (1 H, dd,
J=5.3 and 11.3 Hz, H-9A), 4.13 (1 H, dd, J=7.4 and
11.3 Hz, H-9B), 4.07 (1 H, m, H-8), 3.81 (1H, m, H-3A),
3.53 (1 H, m, H-5A), 2.88 (1 H, ddd, J=6.1, 6.1 and 12.0
Hz, H-5B), 2.78 (1 H, ddd, J=6.4, 10.9 and 10.9 Hz, H-
3B), 2.33 ( 1H, m, H-1), 2.18 (2 H, m, H-2A and H-7A),
1.98 (3 H, m, H-2B and H-6), 1.81 (1 H, m, H-7B); 13C
NMR(CDCl 3) ꢀ 167.0 (s, C-90), 160.4 (s, C-40), 146.0 (d,
C-70), 130.2 (d, C-20 and 60), 125.4 (s, C-10), 116.2 (d, C-
30 and C-50), 113.1 (d, C-80), 68.7 (d, C-8), 63.9 (t, C-9),
54.2 (t, C-5), 54.0 (t, C-3), 44.3 (d, C-1), 30.7 (t, C-7),
29.3 (t, C-2), 25.2 (t, C-6); HPLC–MS (ESI, positive ion
mode) m/z 288 for [M+H]+.
ꢁ
30 PSI, dry gas 10 l/min, dry temp. 300 C.
3.2. Plant material
Borage seeds (Boraginis sem. tot.) were purchased
from Alfred Galke GmbH, D-37534 Gittelde, Germany.
Locally grown borage (1999) was used for the extraction
of fresh aerials.
3.3. Extraction of borage seeds and isolation of 1
Fifty grams of milled borage seeds were defatted by
hexane extraction (2ꢀ100 g, 1 h under reflux each). The
residue was refluxed with 200 g of MeOH/H2O 4:1 (w/
w) for 1 h. After evaporation, the MeOH/H2O extract
(1.8 g) was dissolved in aq. HCl (pH 2–3) and extracted
twice with EtOAc. The aqueous phase was neutralized
with Na2CO3, concentrated to dryness and separated by
RP18-MPLC (YMC ODS-AQ, 10/20 mm, 120 C, 470 ꢀ
40 mm, MeOH/H2O 20:80+0.5 ml HCO2H/l, l 220 nm,
flow rate 15 ml/min) to give 85 mg of 1.
3.5. Alkaline hydrolysis of 1
Twenty milligrams of 1 were dissolved in 5 ml 2.5%
aq. NaOH and stirred for 10 min at room temp. H2SO4
was added and the acidic solution was extracted twice
with EtOAc. The organic phase was evaporated to dry-
ness and separated by preparative RP18-HPLC (col-
umn: Eurospher 80 C18, 10 mm, 250ꢀ16 mm; eluent:
MeOH/H2O 50:50+0.5 ml HOAc/l, flow rate: 10 ml/
min, l: 330 nm) to give 6 mg of 3.
3.3.1. Thesinine-40-O-ꢁ-d-glucoside (1)
A Colourless amorphous solid; analytical HPLC:
Rt=9.5 min; [a]2D0ꢂ22.8ꢁ (DMSO, c 1.99); UV (MeOH)

402
M. Herrmann et al. / Phytochemistry 60 (2002) 399–402
3.5.1. trans-p-Coumaric acid 4-O-ꢁ-d-glucoside (3)
Analytical HPLC: Rt=7.4 min; [ꢂ]2D4 ꢂ69.5ꢁ (MeOH,
c 0.7) Lit. [ꢂ]2D4 ꢂ72.6ꢁ (Chapman & Hall Chemical
Databases, 2001); HPLC–MS (ESI, negative ion mode)
m/z 325 for [M–H]ꢂ.
Foo, L.Y., Lu, Y., Molan, A.L., Woodfield, D.R., McNabb, W.C.,
2000. The phenols and prodelphinidins of white clover flowers.
Phytochemistry 54, 539–548.
Hartmann, T., Theuring, C., Schmidt, J., Rahier, M., Pasteels, J.M.,
1999. Biochemical strategy of sequestration of pyrrolizidine alka-
loids by adults and larvae of chrysomelid leaf beetles. J. Insect
Physiol. 45, 1085–1095.
Langer, T., Franz, C., 1997. Pyrrolizidine alkaloids in commercial
samples of borage seed oil products by GC–MS. Sci. Pharm. 65,
321–328.
Acknowledgements
Lee, H.S., 2000. HPLC analysis of phenolic compounds. In: Nollet,
L.M.L. (Ed.), Food Analysis by HPLC, 2nd ed. Marcel Dekker Inc.
Basel, New York, pp. 775–824.
We thank Professor L.-F. Tietze (Institut fur Orga-
¨
¨
nische Chemie, Universitat Gottingen) for providing the
¨
Murakoshi, I., Kakegawa, F., Toriizuka, K., Haginiwa, J., Ohmiya,
S., Otomasu, H., 1977. (ꢂ)-(trans-40-Rhamnosyloxycinnamyl)-
lupinine, a new lupin alkaloid in Lupinus luteus. Phytochemistry 16,
2046–2047.
optical rotation data, M. Degener for recording the UV
and IRspectra, S. Seilwind for NMRmeasurements
(both DRAGOCO Gerberding & Co. AG, Holzminden)
and R. Christ (GBF, Braunschweig) for measuring the
MS spectra.
Murakoshi, I., Toriizuka, K., Haginiwa, J., Ohmiya, S., Otomasu, H.,
1979. (ꢂ)-trans-40-b-d-Glucopyranosyloxycinnamoyl)lupinine,
a
new lupin alkaloid in Lupinus seedlings. Chem. Pharm. Bull. 27,
144–146.
Parvais, O., Vander Stricht, B., Vanhaelen-Fastre, R., Vanhaelen,
M., 1994. TLC detection of pyrrolizidine alkaloids in oil extracted
from the seeds of Borago officinalis. J. Planar Chromatogr. 7,
80–82.
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