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Metabolomic Differentiation of Cannabis s ativa Cultivars Using 1 H NMR Spectroscopy and Principal Component Analysis

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  • Hazekamp Herbal Consulting

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The metabolomic analysis of 12 Cannabis sativa cultivars was carried out by 1H NMR spectroscopy and multivariate analysis techniques. Principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by principal component 1 (PC1) and principal component 3 (PC3) in cannabinoid fraction. The loading plot of PC value obtained from all 1)H NMR signals shows that Delta9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are important metabolites to differentiate the cultivars from each other. The discrimination of the cultivars could also be obtained from a water extract containing carbohydrates and amino acids. The level of sucrose, glucose, asparagine, and glutamic acid are found to be major discriminating metabolites of these cultivars. This method allows an efficient differentiation between cannabis cultivars without any prepurification steps.
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NMR ASSIGNMENTS OF CANNABINOIDS AND CANNABIFLAVONOIDS 345
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345–354 (2004)
PHYTOCHEMICAL ANALYSIS
Phytochem. Anal. 15, 345–354 (2004)
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002.pca.787
Copyright © 2004 John Wiley & Sons, Ltd.
Received 8 August 2003
Revised 10 October 2003
Accepted 10 October 2003
NMR Assignments of the Major Cannabinoids
and Cannabiflavonoids Isolated from Flowers of
Cannabis sativa
Young Hae Choi,1 Arno Hazekamp,1 Anja M. G. Peltenburg-Looman,1 Michel Frédérich,1,2
Cornelis Erkelens,3 Alfons W. M. Lefeber3 and Robert Verpoorte1,*
1Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
2Laboratory of Pharmacognosy, Natural and Synthetic Drug Research Center, University of Liège, Liège, Belgium
3Division of NMR, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
The complete 1H- and 13C-NMR assignments of the major Cannabis constituents,
9-tetrahydrocannabinol,
tetrahydrocannabinolic acid,
8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid,
cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra
including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the
cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical
shift of H-1
of the C5 side chain and 2
-OH. It was also observed that carboxylic acid substitution reduces
intermolecular hydrogen bonding resulting in a sharpening of the H-5
signal in cannabinolic acid in deuterated
chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to
show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the
other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3
and C-4
of cannflavin A
and B were determined by HMBC spectra. Copyright © 2004 John Wiley & Sons, Ltd.
Keywords: NMR assignment; cannabinoids; cannabiflavonoids; Cannabis sativa.
* Correspondence to: R. Verpoorte, Division of Pharmacognosy, Section
Metabolomics, Institute of Biology, Leiden University, PO Box 9502, 2300 RA
Leiden, The Netherlands.
Email: verpoort@chem.leidenuniv.nl
Contract/grant sponsor: van Leersumfonds (KNAW).
INTRODUCTION
Cannabis preparations, derived from Cannabis sativa
L., are considered to be amongst the most dangerous
of illicit drugs because of their narcotic and addictive
properties. Nevertheless, their promising therapeutic
potential has driven researchers to consider in detail
their clinical uses (Formukong et al., 1989), for example
in the treatment of menstrual cramps and convulsions,
inflamed tonsils, migraine and headaches (Russo,
1998), glaucoma (Helper and Frank, 1971) and asthma
(Abdoud and Sanders, 1976), and in pain relief (Iversen
and Chapman, 2000). The active constituents belong to
a group of compounds classified as cannabinoids, includ-
ing 9-tetrahydrocannabinol, 8-tetrahydrocannabinol,
cannabigerol and cannabinol. In addition to these
major cannabinoids, approximately 60 further cannabin-
oids including metabolites have been isolated so far
(Mechoulam et al., 1976; Turner et al., 1981). How-
ever, the skeletons of the cannabinoids do not differ
greatly one from another, and modification of the struc-
tures are limited to changes in the C5 side chain, sub-
stitution of a carboxylic acid and a hydroxyl group, or
an additional cyclisation. For the easy identification
of the minor cannabinoids and metabolites, complete
assignments of the 1H- and 13C-NMR spectra of the
major cannabinoids are necessary. However, most of
the previous work has been performed mostly using low
field NMR, which resulted in ambiguous assignments
of the cannabinoids.
In a continuation of our investigation on the meta-
bolomic profiling of the constituents of C. sativa, we here
report the isolation and identification, together with
NMR assignment, of the major cannabinoids, includ-
ing 9-tetrahydrocannabinol (1), tetrahydrocannabinolic
acid (2), 8-tetrahydrocannabinol (3), cannabigerol (4),
cannabinol (5), cannabidiol (6) and cannabidiolic acid
(7), and of the major flavonoids, namely cannflavin B (8),
and cannflavin A (9), using one- and two-dimensional
NMR spectra, such as 1H- and 13C-NMR, 1H-1H-COSY,
HMQC and HMBC, and mass spectrometry.
EXPERIMENTAL
Spectroscopic analyses. 1H-NMR (400 MHz) and 13C-
NMR (100 MHz) spectra were recorded on a Bruker
model AV-400 FT-NMR spectrometer (Karlsruhe,
Germany) with reference to TMS as standard. EI-MS
spectra were obtained using a Finnigan MAT 700 instru-
ment (San Jose, CA, USA).
Plant material. Flowers of Cannabis sativa L. (cultivar
SIMM 04 and Kompolti) harvested in October 2002
were obtained from Stichting Institute for Medicinal
Marijuana (SIMM; Rotterdam, The Netherlands). Plant
materials were air-dried in the dark at ambient tempera-
ture for 2 weeks.
346 Y. H. CHOI ET AL.
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345 –354 (2004)
Extraction and isolation. Air-dried flowers (200 g) of
C. sativa (cultivar SIMM 04) were extracted with
chloroform:methanol (1:1) which, on removal of solvent
in vacuo, afforded the initial extract (47.0 g). A suspen-
sion of this extract in 90% methanol was partitioned with
n-hexane (30.3 g), leaving a residual 90% methanol ex-
tract (15.5 g). The n-hexane extract was dissolved in
ethanol and subjected to CC over HP-20 resin (213 g;
Mitsubishi Chemicals, Tokyo, Japan) using a stepwise
gradient of 90% ethanol, 100% ethanol, 70% ethanol
in acetone and 30% ethanol in acetone. A total of 22
fractions (500 mL) were collected. Fractions showing
similar TLC profiles (silica gel 60F254 layers) using
CHCl3-MeOH (19:1) were pooled to afford five com-
bined fractions. Fraction 2 was separated by CC over
silica gel eluted with a gradient of chloroform:methanol
from 100:1 to 10:1, and the third fraction of the 18 frac-
tions collected was further purified over Sephadex LH-
20 (40 g) with dichloromethane:methanol (1:1). The first
fraction of the five fractions obtained from the Sephadex
LH-20 column was purified by CC over silica gel (30 g)
eluted with a gradient of n-hexane:diethyl ether from
10:1 to 1:1. From this separation, compounds 1 (36 mg),
2 (64 mg), and 4 (132 mg) were isolated. Fraction 4 from
the first CC over silica gel was further purified by
CC Sephadex LH-20 using methanol as eluent and
afforded 30 fractions from which 5 (5.0 mg) and 6
(4.3 mg) were obtained from the 28th and 29th, respect-
ively: cannabiflavonoids 8 (12.8 mg) and 9 (3.8 mg) were
isolated from the 18th fraction by further CC over
Sephadex LH-20 (30 g) using methanol as mobile phase.
Cannabidiolic acid (7) had been previously isolated
from the n-hexane (1.5 L × 2) extracts of fibre (100 g)
of C. sativa (cultivar Kompoli; unpublished data). The
n-hexane extracts were filtered over a glass filter, evapo-
rated under reduced pressure and then subjected to
centrifugal partition chromatography using a Sanki
(Kyoto, Japan) model LLB-M instrument. A two-phase
NMR ASSIGNMENTS OF CANNABINOIDS AND CANNABIFLAVONOIDS 347
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345–354 (2004)
system composed of n-hexane:methanol:water (5:3:2)
acidified with 25 mm formic acid was employed, in which
the upper phase was the stationary phase and the lower
phase the mobile phase. The flow rate, rotational speed
and fraction size were 3.5 mL/min, 500 rpm, and 11.5 mL,
respectively. 8-Tetrahydrocannabinol (3) was purchased
from Sigma (St Louis, MO, USA).
RESULTS AND DISCUSSION
The EI-MS spectrum of 9-tetrahydrocannabinol (1) ex-
hibited peaks due to [M]+, [M-CH3]+ and [M-C3H7]+ at
m/z values of 314, 299 and 271, respectively. The 1H-
NMR spectrum was in accordance with that of a
cannabinoid and showed signals due to two angular me-
thyl groups [3H each, s at
δ
1.41, C8-methyl (
β
) and
δ
1.09, C9-methyl (
α
)], one tertiary methyl group (3H, t, J
= 7.0 Hz at
δ
0.87, H-5), and three aromatic and olefinic
protons (1H, q, J = 1.6 Hz, at
δ
6.31, H-2; 1H, d, J =
1.6 Hz at
δ
6.27, H-5; 1H, d, J = 1.6 Hz at
δ
6.14, H-3).
The H-2 signal was a quintet because it was coupled not
only to the adjacent H-1 but also long-range coupled to
H-4 and H-6, which was confirmed by the 1H-1H-COSY
spectrum. Compound 1 from C. sativa was reported to
have a trans configuration for H-1 and H-6, in contrast
to the synthetic compound (Crombie et al., 1988; Evans
et al., 1997; Razdan et al., 1974). The coupling constant
of H-1, 10.9 Hz [Fig. 1(A)], indicated that 1 had a trans
configuration for H-1 and H-6 by comparison with the J
values calculated through dihedral angle and Karplus
equation, which were 11.9 and 3.9 Hz for the trans and
cis configurations, respectively (Karplus, 1959). The 13C-
NMR spectrum of the compound was compared with
that previously reported (Archer and Johnson, 1977). All
1H- and 13C-NMR assignments of 1 are shown in Table 1
and the HMBC correlations in Fig. 2.
Tetrahydrocannabinolic acid (2) did not show an [M]+
peak by virtue of easy decarboxylation at C-3. In the 1H-
NMR a significant difference was observed for H-2 and
H-1 compared with the spectrum of 1. The splitting
pattern of H-2 was found to be a broad singlet, which
was different from that for 1, in which H-2 occured as a
quintet (J = 1.6 Hz). This may be because the carboxyl
group changes the stereochemistry between H-2 and the
adjacent protons (H-1 and H-4). While the two protons
H-1 of 1 were detected at
δ
2.42, apparently as a triplet
through coupling with the two H-2, those of 2 were
separate at
δ
2.94 (1H, m) and 2.78 (1H, m) as shown in
Fig. 1(B); the carboxylic acid group at C-3 affected the
protons differently. Moreover, the signal of 2-OH in 1
was observed at
δ
4.87 (1H, s) whilst that of 2 was at
δ
12.19 (1H, s). This assignment was based on the correla-
tion with H-2. The shift was probably occasioned by
intra-molecular hydrogen bonding between the OH and
the ortho-COOH. In the 13C-NMR spectrum, an upfield
shift of 5.0 ppm was found for C-3, but the other aro-
matic carbons, C-1, C-2, C-4, C-5, and C-6, showed
downfield shifts of 1.1, 10.0, 4.1, 2.5 and 5.6 ppm, respect-
ively. The chemical shifts of C-1, C-2 and C-3 of the
side chain were also shifted downfield by 0.5–1.0 ppm
when compared with those of 1. The complete 1H- and
13C-NMR assignments are shown in Table 1 and HMBC
correlations in Fig. 2. They are in accordance with a
previous report (Fellemeier et al., 2001).
The EI-MS spectrum of 8-tetrahydrocannabinol (3)
was similar to that of 1 and showed peaks due to [M]+,
Figure 1. 1H-NMR spectra in the range
δ
3.3– 2.3 of 9-
tetrahydrocannabinol (A) and tetrahydrocannabinolic acid (B) measured
in deuterochloroform.
348 Y. H. CHOI ET AL.
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345 –354 (2004)
Table 1.
1
H- and
13
C-NMR assignments for
9
-tetrahydrocannabinol (1)
9
-tetrahydrocannabinolic acid (2) and
8
-tetrahydrocannabinol (3) measured in deuterochloroform
9
-Tetrahydrocannabinol (1)
9
-Tetrahydrocannabinolic acid (2)
8
-Tetrahydrocannabinol (3)
Position
1
H-NMR
a,b 13
C-NMR
a,c 1
H-NMR
a,b 13
C-NMR
a,c 1
H-NMR
a,b 13
C-NMR
a,c
13.20 (1H,
dm
, 10.9 Hz) 33.6 3.23 (1H,
dm
, 7.0 Hz) 33.5 2.70 (1H,
td
, 10.8 Hz, 4.8 Hz) 31.6
26.31 (1H,
q
, 1.6 Hz) 123.7 6.39 (1H,
brs
)123.6 3.24 (2H,
dd
, 16.5 Hz, 3.7 Hz), 1.80 (
m
)
f
36.0
3134.3 133.8 134.7
3-Me 1.68 (3H,
s
)23.4 1.68 (3H,
s
)23.3 1.70 (3H,
s
)23.5
42.16 (2H,
m
)31.2 2.17 (2H,
m
)31.2 5.43 (1H,
brd
, 4.8 Hz) 119.3
51.90 (1H,
m
), 1.40 (
m
)25.0 1.92 (1H,
m
), 1.35 (
m
)25.0 2.13 (1H,
m
), 1.64 (1H,
s
)27.9
61.69 (
m
)45.8 1.67 (
m
)45.6 1.80 (
m
)
f
44.8
776.7 78.8 76.7
81.41 (3H,
s
)27.6 1.44 (3H,
s
)27.4 1.38 (3H,
s
)27.6
91.09 (3H,
s
)19.3 1.11 (3H,
s
)19.6 1.10 (3H,
s
)18.5
1110.8 109.9 110.5
2154.7 164.7 154.8
36.14 (1H,
d
, 1.6 Hz) 107.5 102.3 6.11 (1H,
d
, 1.6 Hz) 107.6
4142.8 146.9 142.7
56.27 (1H,
d
, 1.6 Hz) 110.1 6.26 (1H,
s
)112.6 6.27 (1H,
d
, 1.5 Hz) 110.1
6154.2 159.8 154.8
12.42 (2H,
td
, 7.3 Hz, 1.6 Hz) 35.5 2.94 (1H,
m
), 2.78 (1H,
m
)36.5 2.44 (2H,
td
, 8.3 Hz, 2.1 Hz) 35.4
21.55 (2H,
q
, 7.8 Hz) 30.6 1.57 (2H,
m
)31.3 1.56 (2H,
q
, 7.6 Hz) 30.6
31.29 (
m
)
d
31.5 1.35 (
m
)
e
32.0 1.32 (
m
)
g
31.1
41.29 (
m
)
d
22.5 1.35 (
m
)
e
22.5 1.32 (
m
)
g
22.5
50.87 (3H,
t
,
7.0 Hz) 14.0 0.90 (3H,
t
, 6.9 Hz) 14.1 0.88 (3H,
t
, 7.1 Hz) 14.0
2-OH 4.87 (1H,
s
)12.19 (1H,
s
)4.82 (1H, s)
COOH 176.2
a
Chemical shifts (in ppm) were determined with reference to TMS.
b
Spectra determined at 400 MHz.
c
Spectra determined at 100 MHz.
d–g
Chemical shifts bearing the same symbol overlap.
NMR ASSIGNMENTS OF CANNABINOIDS AND CANNABIFLAVONOIDS 349
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345–354 (2004)
Figure 2. HMBC correlations in 9-tetrahydrocannabinol (1), tetrahydrocannabinolic acid (2), 8-
tetrahydrocannabinol (3), cannabigerol (4), cannabinol (5), cannabidiol (6) and cannabidiolic acid (7).
[M-CH3]+ and [M-C3H7]+ at m/z values of 314, 299 and
271, respectively, together with further fragments at m/z
258, 243 and 231. The intensity of each peak, however,
was quite different from that of the corresponding peak
in 1, for example the intensity of the fragment ion at m/z
299 was found to be 100% for 1 but only 9.3% (with
respect to the base peak) for 3. This was due to the
different position of the vinyl group which affects the
elimination of the methyl group in MS fragmentation;
possible fragmentation is shown in Fig. 3. The 1H- and
13C-NMR spectrum of 3 indicated a cannabinoid struc-
ture similar to 1. The H-4 vinyl proton was moved upfield
to
δ
5.43 (dm, J = 4.84 Hz) compared with
δ
6.31
(1H, tm, 1.60 Hz) for 1 because the OH group had no
deshielding effect on this proton. The coupling constant
of H-1 in 3 was 10.8 Hz, indicating a trans configuration
of H-1 and H-6, the same as in 1. The assignments of the
1H- and 13C-NMR spectra of 3 were made on the basis of
two-dimensional NMR spectra and are shown in Table 1
whilst the correlations in HMBC are shown in Fig. 2. The
assignments of the 13C-NMR spectrum as reported by
Archer and Johnson (1977) are confirmed.
Cannabigerol (4) exhibited EI-MS peaks due to [M]+,
[M-C6H13]+ and [M-C9H15]+ at m/z values of 316, 231 and
193, respectively. In the 1H-NMR spectrum, the four
methyl peaks at
δ
1.82 (3H, s, H-3),
δ
1.69 (3H, s, H-9),
δ
1.60 (3H, s, H-8) and
δ
0.90 (3H, t, J = 6.9 Hz, H-3)
confirmed that 4 possessed a cannabinoid structure. A
sharp singlet at
δ
6.26 (2H) was ascribed to the two
identical aromatic protons (H-4 and H-6) of 4.
The EI-MS spectrum of cannabinol (5) exhibited
peaks due to [M]+ and [M-CH3]+ at m/z values of 310 and
350 Y. H. CHOI ET AL.
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345 –354 (2004)
Figure 3. EI-MS fragmentations of 9-tetrahydrocannabinol (A), 8-tetrahydrocannabinol (B) and cannabidiol (C).
δ
2.38 (3H, s, C-3 methyl). The chemical shifts of the two
methyls at C-7 of 1 and of 3 were different because of
different orientations of those groups, however, the
methyls of 5 had identical chemical shifts (
δ
1.60) since
the additional aromatic ring results in a flat molecule in
295 (base peak), respectively. In the 1H-NMR spectrum
of 5, the extra aromatic moiety resulted in additional
peaks due to aromatic protons at
δ
8.16 (1H, s, H-2),
δ
7.07 (1H, d, J = 7.9 Hz, H-4), and
δ
7.14 (1H, d, J =
7.9 Hz, H-5), and a methyl attached to a benzene ring at
NMR ASSIGNMENTS OF CANNABINOIDS AND CANNABIFLAVONOIDS 351
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345–354 (2004)
Table 2. 1H- and 13C-NMR assignments for cannabigerol (4) and cannabinol (5) measured in
deuterochloroform
Cannabigerol (4)Cannabinol (5)
Position 1H-NMRa,b 13C-NMRa,c Position 1H-NMRa,b 13C-NMRa,c
1154.8 1 108.7
2110.7 2 8.16 (1H,
s
)126.3
3154.8 3 136.9
46.26 (2H,
s
)d108.4 3-Me 2.38 (3H,
s
)21.5
5— 142.7 4 7.07 (1H,
d
, 7.9 Hz) 127.6
66. (2H,
s
)d108.4 5 7.14 (1H,
d
, 7.9 Hz) 122.6
13.41 (2H,
d
, 7.0 Hz) 22.5 6 136.9
25.29 (1H,
m
)121.8 7 77.5
3138.0 8 1.60 (6H,
s
)f27.1
3-Me 1.82 (3H,
s
)16.1 9 1.60 (6H,
s
) f27.1
42.09 (4H,
m
)e39.7 1110.8
52.09 (4H,
m
)e26.4 2154.7
65.07 (1H,
m
)123.8 36.29 (1H,
d
, 1.1 Hz) 109.8
7132.0 4144.5
81.60 (3H,
s
)17.6 56.44 (1H,
d
, 1.1 Hz) 110.8
91.69 (3H,
s
)25.6 6153.0
12.45 (2H,
t
, 7.5 Hz) 35.5 12.50 (2H,
t
, 7.5 Hz) 35.6
21.56 (2H,
q
, 7.8 Hz) 30.8 21.63 (
m
)30.4
31.33 (4H,
m
)31.5 31.32 (
m
)g31.5
41.33 (4H,
m
)22.2 41.32 (
m
)g22.5
50.90 (3H,
t
,
6.9 Hz) 14.0 50.89 (3H,
t
,
6.8 Hz) 14.0
OH 5.36 (2H,
s
)2-OH 5.13 (1H,
s
)
a Chemical shifts (in ppm) were determined with reference to TMS.
b Spectra determined at 400 MHz.
c Spectra determined at 100 MHz.
d–g Chemical shifts bearing the same symbol overlap.
which the two methyl groups have similar positions
above and below the plane of the aromatic moiety. The
assignments of 1H- and 13C-NMR spectra of 4 and 5
are shown in Table 2 and the HMBC correlations are
depicted in Fig. 2.
The EI-MS spectrum of cannabidiol (6) exhibited
peaks due to [M]+ and [M-C6H11]+ at m/z values of 314
and 231 (base peak), respectively. These peaks are com-
patible with a cannabinoid structure (Fig. 3). In the 1H-
NMR spectrum of 6 measured in deuterochloroform,
broad peaks were observed. These signals were not
affected by the addition of deuterium oxide, which re-
veals that these are aromatic hydrogens rather than
hydroxyl groups [Fig. 4(A)]. Previously, it was shown
that the shifts of these aromatic protons are temperature-
dependent and the broadening is likely to be due to
restricted rotation (Mechoulam et al., 1976). This
broadening hampered the assignments of the signals; for
example, there were no correlations observed in the
HMQC and HMBC for these signals. The problem could
be solved using a protic solvent such as deuteromethanol
[Fig. 4(B)]; such a solvent breaks up the inter-molecular
hydrogen bonding and allows the terpenoid moiety in 5
to rotate freely resulting in sharp singlets for the aromatic
protons. Further, this solvent-dependent conformation
change was confirmed by the shift of the signal of H-6.
This proton was at
δ
2.40 in deuterochlorofom and over-
lapped with H-1 but was well separated from H-1 at
δ
2.89 (1H, td, 15.1 Hz, 5.05 Hz) in deuteromethanol. The
free rotation of the terpenoid group in protic solvent
allowed H-6 and OH to be closer when compared with
the conformations in deuterochloroform solution. A
Figure 4. 1H-NMR spectra of cannabidiol (6) in deuterochloro-
form (A) and deuteromethanol (B), and of cannabidiolic acid
(7) in deuterochloroform (C) and deuteromethanol (D).
352 Y. H. CHOI ET AL.
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345 –354 (2004)
Table 3.
1
H- and
13
C-NMR assignments for cannabidiol (6) and cannabidiolic acid (7) measured in deuterochloroform and deuteromethanol
Cannabidiol (6)Cannabidiolic acid (7)
Position
1
H-NMR
a,b
in CDCl
3
1
H-NMR
a,b
in CD
3
OD
13
C-NMR
a,c
in CD
3
OD
1
H-NMR
a,b
in CDCl
3
1
H-NMR
a,b
in CD
3
OD
13
C-NMR
a,c
in CD
3
OD
13.90 (1H,
dm
, 11.8 Hz) 3.93 (1H,
dm
, 11.1 Hz) 37.5 4.09 (1H,
m
)3.97 (1H,
dm
, 8.9 Hz) 37.3
25.57 (1H,
s
)5.28 (1H,
s
)127.3 5.56 (1H,
s
)5.28 (1H,
s
)126.9
3134.2 133.0
42.21 (1H,
m
), 2.09 (1H,
m
)2.18 (1H,
m
), 1.99 (1H,
m
)30.7 2.20 (1H,
m
), 2.10 (1H,
m
)2.18 (1H, m), 1.98 (1H,
m
)31.7
51.84 (
m
)1.74 (2H,
m
)31.7 1.86 (
m
)1.79 (2H,
m
)30.8
62.40 (
m
)2.89 (1H,
td
, 15.1 Hz, 5.05 Hz) 46.4 2.40 (1H,
m
)3.02 (1H,
m
)45.7
71.79 (3H,
s
)1.67 (3H,
s
)23.7 1.80 (3H,
s
)1.65 (3H,
s
)23.6
8—150.3
g
150.4
k
94.64 (
trans
, 1H,
m
), 4.46 (
trans
, 1H,
m
), 110.5 4.55 (
trans
, 1H,
m
), 4.45 (
trans
, 1H,
m
), 110.5
4.54 (
cis
, 1H,
m
)4.42 (
cis
, 1H,
m
)4.40 (
cis
, 1H,
m
)4.42 (
cis
, 1H,
m
)
10 1.66 (3H,
s
)1.63 (3H,
s
)19.5 1.72 (3H,
s
)1.62 (3H,
s
)19.3
1115.9 116.5
2157.5 161.8
36.26 (1H,
brs
)6.07 (2H,
s
)
e
108.3
h
104.2
4142.7 147.0
56.16 (1H,
brs
)6.07 (2H,
s
)
e
108.3
h
6.26 (1H,
s
)6.13 (1H,
s
)111.3
6150.3
g
150.4
k
12.43 (2H,
t
, 7.5 Hz) 2.37 (2H,
t
, 7.46 Hz) 36.6 2.92 (1H,
m
), 2.82 (1H,
m
)2.81 (2H,
t
, 7.5 Hz) 37.6
21.55 (2H,
q
, 7.6 Hz) 1.53 (2H,
q
, 7.34 Hz) 32.0 1.57 (2H,
m
)1.57 (2H,
m
)32.8
31.29 (
m
)
d
1.29 (4H,
m
)
f
32.6 1.33 (4H,
m
)
i
1.31 (4H,
m
)
i
33.2
41.29 (
m
)
d
1.29 (4H,
m
)
f
23.6 1.33 (4H,
m
)
i
1.31 (4H,
m
)
i
23.5
50.88 (3H,
t
, 6.8 Hz) 0.89 (3H,
t
, 7.13 Hz) 14.4 0.89 (3H,
t
, 6.9 Hz) 0.89 (3H,
t
, 7.2 Hz) 14.4
2-OH 5.99 (1H, s) 11.93 (1H,
s
)
6-OH 5.02 (1H,
s
)6.63 (1H,
s
)
COOH 175.6
a
Chemical shifts (in ppm) were determined with reference to TMS.
b
Spectra determined at 400 MHz.
c
Spectra determined at 100 MHz.
d–k
Chemical shifts bearing the same symbol overlap.
NMR ASSIGNMENTS OF CANNABINOIDS AND CANNABIFLAVONOIDS 353
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345–354 (2004)
large coupling constant of H-6 (15.1 Hz) also indicated
that it had a trans conformation. The 1H- and 13C-
NMR assignments deduced from two-dimensional NMR
spectra such as 1H-1H-COSY, HMQC, and HMBC are
shown in Table 3 and HMBC correlations are shown
in Fig. 2.
In contrast to the above, the broad signals in the 1H-
NMR measured in deuterochloroform were not detected
in cannabidiolic acid (7) because of intra-hydrogen bond-
ing between the carboxylic acid at C-3 and the hydroxyl
group at C-2, which permits the terpenoid moiety to
rotate freely [Fig. 4(C)]. The two protons at C-1,
similar to those in 2, were affected differently by the
carboxylic acid at C-3 and were observed at
δ
2.20 (1H,
m) and 2.10 (1H, m), respectively. The hydroxyl group
at C-2 was shifted to
δ
11.9 by the hydrogen bonding of
the hydroxyl and the carboxyl acid groups. In the 13C-
NMR spectrum of 7, the signals of C-1, C-2, C-4, C-5
and C-6 showed downfield shifts of 0.5–5 ppm. The 1H-
and 13C-NMR assignments are shown in Table 3 and the
HMBC correlations in Fig. 2.
In addition to cannabinoids, prenylated flavonoids
having a prenyl group on C-6, for example cannflavin B
and A (8 and 9, respectively), have been reported as con-
stituents of C. sativa (Barrett et al., 1986). The 1H-NMR
spectrum confirmed that 8 was a methoxyflavonoid
showing signals due to five aromatic protons at
δ
7.60
(1H, d, 1.9 Hz, H-2),
δ
7.58 (1H, dd, 8.2 Hz, 1.9 Hz,
H-6),
δ
7.00 (1H, d, 8.2 Hz, H-5),
δ
6.85 (1H, s, H-3),
δ
6.63 (1H, s, H-8) and methoxy peaks at
δ
3.99 (3H, s).
The additional prenyl peaks, including
δ
3.36 (2H, d,
7.12 Hz, H-1),
δ
5.29 (1H, tt, 7.24 Hz, 1.52 Hz, H-2),
δ
1.80 (3H, s, H-4) and
δ
1.65 (3H, s, H-5), indicated
that this compound has a prenyl moiety, the position of
which was confirmed to be C-6 by HMBC. The position
of the methoxy group could be assigned to be C-3 from
the NOESY spectrum in which H-2 correlated with
the methoxy group. The assignments of C-3 and C-4
were obtained using HMQC and HMBC data: H-6
was not correlated with C-3 but with C-4 in the HMBC
spectrum (Fig. 5). The assignment given here is different
from that previously reported (Agrawal, 1989).
The 1H-NMR spectrum of cannflavin A (9) showed
additional signals, including
δ
1.93 (2H, t, 10.0 Hz, H-4),
δ
2.03 (2H, t, 7.21 Hz, H-5),
δ
5.04 (1H, t, 7.08 Hz, H-6)
and
δ
1.54 (3H, s, H-8) when compared to those of 8.
The 1H- and 13C-NMR assignments of the flavonoids are
shown in Table 4.
Acknowledgements
The Post-doctoral Fellowship Program of Korea Science and
Engineering Foundation (KOSEF) is gratefully acknowledged
for the support of Y. H. Choi. The authors also thank Professor
J. De Graeve and Mr. J.-C. van Heugen at the University of
Liège, Liège, Belgium for the measurements of ESI-MS spectra.
Financial support of the van Leersumfonds (KNAW) is gratefully
acknowledged.
Table 4. 1H- and 13C-NMR assignments for cannflavin B (6) and cannflavin A (7) measured in deuteroacetone
Cannflavin B (6)Cannflavin A (7)
Position 1H-NMRa,b 13C-NMRa,c 1H-NMRa,b 13C-NMRa,c
2—164.7 — 164.7
36.68 (1H,
s
)104.4 6.69 (1H,
s
)104.4
4—183.1 — 183.1
5—162.5 — 162.4
6—112.3 — 112.4
7—160.1 — 160.2
86.63 (1H,
s
)94.2 6.63 (1H,
s
)94.1
9—156.6 — 156.6
10 — 105.1 — 105.2
1123.7 — 123.7
27.60 (1H,
d
, 1.88 Hz) 110.4 7.61 (1H,
d
, 1.88 Hz) 110.5
3148.8 — 148.8
4151.3 — 151.3
57.00 (1H,
d
, 8.28 Hz) 116.3 7.02 (1H,
d
, 8.28 Hz) 116.3
67.58 (1H,
dd
, 8.24 Hz, 1.90 Hz) 121.2 7.58 (1H,
dd
, 8.24 Hz, 1.90 Hz) 121.2
13.36 (2H,
d
, 7.12 Hz) 22.0 3.36 (2H,
d
, 7.20 Hz) 22.0
25.29 (1H,
tt
, 7.24 Hz, 1.52 Hz) 123.2 5.28 (1H,
tt
, 7.24 Hz, 1.52 Hz) 123.2
3131.6 — 131.6
41.80 (3H, s) 25.8 1.93 (2H,
t
, 10.0 Hz) 40.5
51.65 (3H,
s
)17.9 2.03 (2H, t, 7.21 Hz) 27.4
6——5.04 (1H, t, 7.08 Hz) 125.2
7———131.6
8——1.54 (3H,
s
)16.3
9——1.78 (3H, s) 25.8
10——1.65 (3H,
s
)17.7
OMe 3.99 (3H,
s
)56.7 3.99 (3H,
s
)56.6
5-OH 13.30 (1H,
s
)— 13.3 (1H, s)
a Chemical shifts (in ppm) were determined with reference to TMS.
b Spectra determined at 400 MHz.
c Spectra determined at 100 MHz.
354 Y. H. CHOI ET AL.
Copyright © 2004 John Wiley & Sons, Ltd. Phytochem. Anal. 15: 345 –354 (2004)
Figure 5. HMBC spectrum of cannflavin B (8) in deuteroacetone.
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Examination of the toluene-p-sulphonic acid-catalysed reaction of (1S,2S,3R,6R)-(+)-trans-car-2-ene epoxide with olivetol shows that, inconsistently with the accepted mechanism, (3R,4R)-(-)-o- and -p-cannabidiols are produced as well as (3R,4R)-(-)-Δ 1-and Δ 6-tetrahydrocannabinols. Evidence is now presented that, as in Petrzilka's reaction employing chiral p-mentha-2,8-dien-1-ols, the reacting species is the delocalised (4R)-p-mentha-2,8-dien-1-yl cation (9). Similar terpenylation using (1S,3S,4R,6R)-(+)-trans-car-3-ene epoxide shows that besides the reported (-)-Δ 6-THC, o- and p-cannabidiols, Δ 4.8-THC and Δ 4.8-iso-THC can also be produced. The nature of the products, the chirality, and the characteristics of the reaction implicate again the delocalised cation (9). Its formation via Kropp-type rearrangement is excluded and a pathway leading to (4/7)-/)-mentha-2,6,8-triene, which on protonation gives (9), is proposed. Protonated on C-8, the triene can be trapped and isolated as (4/7)-p-mentha-2,6-dien-8-ol. The latter, made in (±)-form from citral, proved to be an excellent terpenylating agent for producing cannabinoids. Terpenylation of olivetol by the pinanes (1S,4S,5S)-(-)-cis-verbenol and (1R,5S,7R)-(+)-cis-chrysanthenol is compared. A major drawback of the latter is partial racemisation which occurs in the verbenone-chrysanthenone isomerisation during its photochemical preparation. Whilst Δ 1-THC cannot be directly obtained from verbenol, its tertiary allylic cation permits a much higher yielding terpenylation than the secondary cation from chrysanthenol.
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Chemical characterization of Italian red wines from different geographical locations in the Apulia, region of southern Italy, have been performed by means of chromatographic, analyses routine analyses (density, alcohol content, acidity, dry extract and ash content), inductively coupled plasma-atomic emission spectrometric measurements and nuclear magnetic resonance (1HNMR) spectrometric determinations. Multivariate statistical methods were applied separately to the analytical and NMR data. The results showed that Apulian red wines are divided in three groups according to their geographical origin. . Keywords: Italian red wines; NMR; Multivariate statistical analysis; Geographical origin; ICP-AES
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The valence‐bond theory for the contact electron‐spin coupling of nuclear magnetic moments is used to calculate the proton‐proton, proton‐fluorine, and fluorine‐fluorine coupling constants in ethanic and ethylenic molecules. A considerable simplification is introduced into the theory by approximations which reduce the problem to one involving only a small number of electrons and canonical structures. The agreement between calculated and experimental values is such as to demonstrate that the mechanism considered is the one of primary importance for the nuclear coupling in the compounds studied. Of particular interest is the theoretical confirmation of the observation that in ethylenic compounds the trans coupling between nuclei (HH, HF, FF) is considerably larger than cis coupling.