Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC-MS/MS, LC-NMR, and LC-CD analyses

Article abstract of DOI:10.1021/np8001496

The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification p

Full text of DOI:10.1021/np8001496

1376
J. Nat. Prod. 2008, 71, 1376–1385
Online Structural Elucidation of Alkaloids and Other Constituents in Crude Extracts and
Cultured Cells of Nandina domestica by Combination of LC-MS/MS, LC-NMR, and LC-CD
Analyses
Kinuko Iwasa,*,† Teturo Takahashi,† Yumi Nishiyama,† Masataka Moriyasu,† Makiko Sugiura,† Atsuko Takeuchi,†
Chisato Tode,† Harukuni Tokuda,‡ and Kazuyoshi Takeda§
Kobe Pharmaceutical UniVersity, 4-19-1 Motoyamakita, Higashinada-ku, Kobe-shi 658-8558, Japan, Department of Biochemistry and
Molecular Biology, Kyoto Prefectural UniVersity of Medicine, Kawaramachi-dori, Kamigyo-ku, Kyoto 602-0841, Japan, and Yokohama College
of Pharmacy, 601 Matanocyo, Hodogayaku, Yokohama-shi 245-0066, Japan
ReceiVed March 10, 2008
The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration
of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation
and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR
and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were
identified from N. domestica. O-Methylpallidine and (E,E)-, (E,Z)-, and (Z,Z)-terrestribisamide were also characterized
for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E,E)-,
(E,Z)-, and (Z,Z)-terrestribisamide were identified in the callus of N. domestica.
Nandina domestica Thumb. grows wild in Japan and China.1 In
Japan, the fruits of this plant have long been used to treat asthma,
whooping cough, pharyngeal tumors, and uterine bleeding.2 In
China, other parts of the plant, e.g., stems and leaves, have also
been used as medicines.3 Shoji et al.2 reported that the alkaloid
nantenine is a serotonergic receptor antagonist. The fruits, seeds,
roots, and stem bark contain aporphine-, promorphine-, protober-
berine-, and protopine-type alkaloids.4-18 Twelve protoberberines
and magnoflorine have been identified from tissue cultures of N.
domestica.19-22
Application of LC-NMR combined with LC-MS to drug
metabolism, identification of natural products in crude plant extracts
and the characterization of isomeric mixtures prepared by chemical
reactions have been summarized.23-25 The coupling of HPLC to a
CD detector is one of the most powerful hyphenated techniques
for stereochemical investigation.26 The combination of three online
coupling methods, LC-NMR, LC-MS, and LC-CD, permitted
determination of the structures including absolute configuration of
the natural products in crude plant extracts without the necessity
of isolation and purification.27,28
NOESY spectroscopic data (Figure 2) also supported this structure.
Similarly, the compounds associated with peaks b1 and c1 were
identified as sinoacutine and domesticine, respectively (Figure 2,
1
B and C), on the basis of their stopped-flow H NMR [peak b1
displayed two aromatic proton doublets (J ) 8.0 Hz) at δ 6.89 and
6.71, two olefinic proton singlets at δ 7.78 and 6.46, two methoxy
groups at δ 3.76 and 3.66, an N-methyl group at δ 2.70; c1 showed
three aromatic proton singlets at δ 7.85, 6.84, and 6.76, a
methylenedioxy group at δ 5.93, a methoxy group at δ 3.81, and
an N-methyl group at δ 2.95] and NOESY data (Figure 2). The
stopped-flow 1H NMR spectrum of peak d1 displayed three aromatic
proton singlets at δ 7.77, 6.86, and 6.81, a methylenedioxy group
at δ 5.96, two methoxy groups at δ 3.81 and 3.58, and an N-methyl
group at δ 2.87. The compound corresponding to peak d1 was
identified as nantenine by comparison of its LC-NMR spectrum
(Figure 2, D) with that of domesticine (Figure 2, C). Isoboldine,
sinoacutine, domesticine, and nantenine have been isolated previ-
ously from N. domestica.4-8,10-18
The absolute configuration at C-6a of the isolated aporphine
alkaloids (isoboldine, domesticine, and nantenine) were determined
from LC-CD analysis. Initially, CD spectra of commercially
available (S)-boldine and (S)-isocorydine in the stopped-flow mode
were recorded at 220-420 nm to find a suitable wavelength for
the measurement of LC-CD spectra. (S)-Boldine and (S)-isocorydine
showed a positive CD sign near 240 nm (Figure 3). A wavelength
of 236 nm was used in subsequent LC-CD measurements. In an
LC-CD spectrum of Plant Fr. E measured under the same
conditions, peaks a1, c1, and d1 corresponding to isoboldine,
domesticine, and nantenine, respectively, had a positive CD sign
(Figure 4). Therefore, the absolute configuration at C-6a of these
alkaloids was identified as S. The proaporphine-type alkaloid
sinoacutine (peak b1) showed a negative CD sign.
In this report, we describe the characterization of components,
particularly alkaloids, in crude extracts of intact plants and tissue
cultures of N. domestica using LC-MS-MS, LC-NMR, and LC-
CD analyses.
Results and Discussion
The ground parts of N. domestica were extracted to give Plant
Fr. E and Plant Fr. C (see Experimental Section), which were
subjected to LC-NMR, LC-MS, and LC-CD analyses. In the LC-
MS chromatogram of Plant Fr. E (Figure 1), peaks a1, b1, c1, and
d1 showed protonated molecular ions [M + 1]+ at m/z 328 (a1 and
b1), 326 (c1), and 340 (d1), respectively. The stopped-flow 1H NMR
spectrum of peak a1 showed three aromatic proton singlets at δ
8.02, 6.83, and 6.77, two methoxy groups at δ 3.85 and 3.82, and
an N-methyl group at δ 2.99. From these data, the compound
associated with peak a1 was identified as isoboldine (Figure 2, A).
LC peaks a2-e2 of Plant Fr. C (Figure 5) exhibited protonated
molecular ions [M + 1]+ or molecular ions [M]+ of m/z 356 (a2),
m/z 342 (b2), m/z 328 (c2), m/z 441 (d2), and m/z 336 (e2). The
1
stopped-flow H NMR spectrum of peak a2 contained a singlet
aromatic proton at δ 7.02, two aromatic proton doublets (J ) 8.0
Hz) at δ 7.04 and 6.99, three methoxy groups at δ 3.86, 3.80, and
3.60, and two N-methyl groups at δ 3.28 and 2.89 (Figure 6, A).
The compound associated with peak a2 was identified as menisper-
mine by comparison of LC-NMR data with those of magnoflorine.
NOESY data (Figure 6) supported this structure.
* Corresponding author. E-mail: k-iwasa@kobepharma-u.ac.jp. Tel: 081-
78-441-7544. Fax: 081-78-441-7544.
† Kobe Pharmaceutical University.
‡ Kyoto Prefectural University of Medicine.
§ Yokohama College of Pharmacy.
10.1021/np8001496 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/01/2008

Alkaloids in Crude Extracts of Nandina domestica
Journal of Natural Products, 2008, Vol. 71, No. 8 1377
Figure 1. LC data of the alkaloid fraction (Plant Fr. E) obtained from N. domestica. Column: Cosmosil 5C -AR-II (4.6 × 150 mm). Eluent:
A: 0.1 M NH4OAc/D2O (0.05% TFA); B: MeCN (0.05% TFA). Gradient A/B: initial 90/10, 5 min 75/25, 15 min 75/25, 30 min 0/100.
Flow rate: 1.0 mL/min. UV detector: 280 nm.
Figure 2. Stopped-flow LC-1H NMR spectra and NOESY data of major components in the alkaloid fraction (Plant Fr. E) obtained from
1
1
1
N. domestica. (A) H NMR spectrum of peak a1 (isoboldine). (B) H NMR spectrum of peak b1 (sinoacutine). (C) H NMR spectrum of
1
peak c1 (domesticine). (D) H NMR spectrum of peak d1 (nantenine).
The LC-NMR spectrum of peak b2 exhibited two aromatic proton
singlets at δ 7.07 and 6.83, two singlet olefinic protons at δ 6.93
and 6.50, three methoxy groups at δ 3.80, 3.79, and 3.67, and one
N-methyl group at δ 2.74 (Figure 6, C). The compound associated
with peak b2 was recognized as O-methylpallidine by comparison
of LC-NMR data with those of sinoacutine. NOESY data (Figure
6) supported this structure.
doublets (J ) 16.0 Hz) at δ 7.35 and 6.41, a methoxy group at δ
3.80, and two methylene proton singlets at δ 3.23 and 1.53 (Figure
7, Table 1). After preparative HPLC of Plant Fr. C, HR-SIMS
analysis of the compound corresponding to peak d2 gave a molecular
formula of C24H28N2O6 with a molecular ion at m/z 440 and
fragment ions at m/z 265 and 177. 13C NMR and DEPT spectra
showed only 12 carbons: a methyl at δ 56.36, two methylenes at δ
40.14 and 27.92, five methines at δ 142.02, 123.15, 118.73, 116.46,
and 111.53, three quaternary carbons at δ 149.82, 149.27, and
128.25, and a carbonyl at δ 169.23 (Figure 8 and Table 2). Thus,
on the basis of HR-SIMS and 13C NMR data, this compound is
Peak d2 displayed a protonated molecular ion at m/z 441 and
1
product ion at m/z 177. The stopped-flow H NMR spectrum of
peak d2 showed an aromatic proton singlet at δ 7.12, two aromatic
proton doublets (J ) 8.0 Hz) at δ 7.04 and 6.82, two olefinic proton

1378 Journal of Natural Products, 2008, Vol. 71, No. 8
Iwasa et al.
Figure 3. Stopped-flow LC-CD spectra and LC-CD data of (S)-boldine and (S)-isocorydine. Column: Cosmosil 5C18-AR-II (6.0 × 150
mm). Gradient: A: 0.1 M NH4OAc (0.05% TFA); B: MeCN (0.05% TFA). A/B: initial 100/0, 40 min 0/100. Flow rate: 2.0 mL/min. Temp:
40 °C.
Figure 4. LC-CD data of the alkaloid fraction (Plant Fr. E) obtained from N. domestica. Column: Cosmosil 5C18-AR-II (6.0 × 150 mm).
Gradient: A: 0.1 M NH4OAc (0.05% TFA); B: MeCN (0.05% TFA). A/B: initial 100/0, 40 min 0/100. Flow rate: 2.0 mL/min. Temp: 40
°C. UV detector: 236 nm.
dimeric. The structure corresponding to peak d2 was identified as
terrestribisamide. Compositions of C14H21N2O3 and C10H9O3 for
HR-SIMS fragment ions m/z 265 and 177, respectively, supported
this structure (Scheme 1). The coupling constant (16 Hz) of the

Alkaloids in Crude Extracts of Nandina domestica
Journal of Natural Products, 2008, Vol. 71, No. 8 1379
Figure 5. LC data of the alkaloid fraction (Plant Fr. C) obtained from N. domestica. Column: Cosmosil 5C18-AR-II (4.6 × 150 mm).
Eluent: A: 0.1 M NH4OAc/D2O (0.05% TFA); B: MeCN (0.05% TFA). Gradient A/B: initial 90/10, 5 min 75/25, 15 min 75/25, 30 min
0/100. Flow rate: 1.0 mL/min. UV detector: 280 nm.
Figure 6. Stopped-flow LC-1H NMR spectra of peaks a2 and b2 in LC of the alkaloid fraction (Plant Fr. C) obtained from N. domestica and
magnoflorine. (A) 1H NMR spectra of peak a2 (menispermine). (B) 1H NMR spectra of standard magnoflorine. (C) 1H NMR spectra of peak
b2 (O-methylpallidine).
olefinic protons indicated that both double bonds have an E
configuration. HMBC data (Table 2) were also consistent with
identification of this compound (peak d2) as (E,E)-terrestribisamide.
The LC-NMR spectrum of peak e2 exhibited four aromatic proton
singlets at δ 9.60, 8.55, 7.54, and 6.94, two aromatic proton doublets
(J ) 9.0 Hz) at δ 8.03 and 7.95, a methylendioxy group at δ 6.06,
and two methoxy groups at δ 4.06 and 4.04. The compound
associated with peak e2 was determined to be berberine (identical
weeks were extracted to give Callus Frs. E and C (see Experimental
Section), which were subjected to LC-NMR, LC-MS, and LC-CD
analyses. In the LC of Callus Fr. E (Figure 9), peaks a3, b3, c3, and
d3 exhibited protonated molecular ions at m/z 222 (a3) and 441 (b3,
c3, and d3). The stopped-flow 1H NMR spectrum of peak a3 showed
the presence of two aromatic proton singlets at δ 6.82 and 6.76,
two methoxy groups at δ 3.77 (6H, s), and one methyl doublet (J
) 6.0 Hz) at δ 1.56. The compound associated with peak a3 was
determined to be carnegine by comparison of its LC-NMR data
with those of an authentic sample (Figure 10). The absolute
configuration at C-1 of carnegine was predicted from LC-CD
analysis. (R)- and (S)-Carnegine were resolved with L-(+)- and
D-(-)-tartaric acid and showed negative and positive CD signs,
respectively, in LC-CD spectra on Chiralcel OD-RH (Figure 11).
An LC-CD spectrum of Callus Fr. E was measured under the same
conditions. The peak corresponding to carnegine had a negative
CD sign (Figure 11); therefore, the absolute configuration at C-1
of the isolated alkaloid was determined to be R.
1
with the H NMR spectrum of C in Figure 15) by comparison of
LC-NMR data with those of an authentic sample.
By using LC-NMR, LC-MS/MS, and LC-CD techniques, we
have identified four known alkaloids, (S)-isoboldine, (S)-domesti-
cine, (S)-nantenine, sinoacutine, and menispermine, from N. do-
mestica. O-Methylpallidine and (E,E)-, (E,Z)-, and (Z,Z)-terrestri-
bisamide were also characterized for the first time from this plant.
Calli of N. domestica were derived from the stems of wild plants
grown in Kobe (Japan) and cultured in the dark on Murashige and
Skoog’s medium. Methanol extracts of callus cultured for 5-6

1380 Journal of Natural Products, 2008, Vol. 71, No. 8
Iwasa et al.
Figure 7. Stopped-flow LC-1H NMR spectrum of peak d2 in LC of the alkaloid fraction (Plant Fr. C) obtained from N. domestica.
1
Table 1. Stopped-Flow 1H NMR and HMBC Data of (E,E)-
and (Z,Z)-Terrestribisamide
identified as (Z,Z)-terrestribisamide. The stopped-flow H NMR
spectrum of peak c3 displayed olefinic signals for both E and Z
double bonds (Figure 13). Therefore, peak c3 was identified as (E,Z)-
terrestribisamide.
protons
(E,E)-terrestribisamide
(Z,Z)-terrestribisamide
2-H
5-H
6-H
7-H
7.12
7.08
6.82a
7.04a
7.35b
6.41b
3.23
6.75c
6.83c
6.63d
5.83d
3.07
Similarly, in the LC-MS/MS of Callus Fr. C, peaks a4, b4, and
d4 (Figure 14) showed the same protonated molecular ion at m/z
441 and product ion at m/z 177. Peaks a4, b4, and d4 were identified
as (Z,Z)-, (E,Z)-, and (E,E)-terrestribisamide, respectively. Peaks
c4, e4, and f4 in Figure 14 displayed molecular ions at m/z 338,
352, and 336 and product ions at m/z 322, 336, and 320,
respectively, in the LC-MS/MS. The stopped-flow 1H NMR
spectrum of peak c4 showed four aromatic proton singlets at δ 9.57,
8.61, 7.55, and 6.88, two aromatic proton doublets (J ) 9.0 Hz) at
δ 8.02 and 7.94, and three methoxy groups at δ 4.05, 4.03, and
3.93 (Figure 15, A). The compound associated with peak c4 was
determined to be jatrorrhizine by comparison of LC-NMR data with
those of an authentic sample. The LC-NMR spectrum of peak e4
exhibited four aromatic proton singlets at δ 9.60, 8.64, 7.55, and
7.03, two aromatic proton doublets (J ) 9.5 Hz) at δ 8.03 and
7.95, and four methoxy groups at δ 4.06, 4.03, 3.92, and 3.87
(Figure 15, B). The compound related to peak e4 was confirmed to
be palmatine by comparison of LC-NMR data with those of an
authentic sample. The compound corresponding to peak f4 was
8-H
10-H
11-H
3-OCH3
1.53
3.80
1.28
3.73
a d J ) 8.0 Hz. b d J ) 16.0 Hz. c d J ) 7.5 Hz. d d J ) 12.0 Hz.
The compounds associated with peaks b3-d3 had the same
protonated molecular ion (m/z 441) and, thus, were isomers of
terrestribisamide with different double-bond configurations. The
compound corresponding to peak d3 had identical data to those
1
described above for (E,E)-terrestribisamide. The stopped-flow H
NMR spectrum of peak b3 showed an aromatic proton singlet at δ
7.08, two aromatic proton doublets (J ) 7.5 Hz) at δ 6.83 and
6.75, two olefinic proton doublets (J ) 12.0 Hz) at δ 6.63 and
5.83, a methoxy group at δ 3.73, and two methylene proton singlets
at δ 3.07 and 1.28 (Figure 12). The coupling constant (12 Hz) of
the olefinic protons indicated that the configuration of both double
bonds was Z. Thus, the compound responsible for peak b3 was
Figure 8. 13C NMR spectrum of (E,E)-terrestribisamide.

Alkaloids in Crude Extracts of Nandina domestica
Journal of Natural Products, 2008, Vol. 71, No. 8 1381
Table 2. 13C NMR HMBC Data of (E,E)-Terrestribisamide
C-1
C-2
C-3
C-4
C-5
C-6
C
CH
C
C
CH
CH
128.25
111.53
149.27
149.82
116.46
123.15
C-7
C-8
C-9
C-10
C-11
C(3)-OCH3
CH
CH
CdO
CH2
CH2
CH3
142.02
118.73
169.23
40.14
27.92
56.36
Scheme 1
measured on a Varian VXR-500 spectrometer (125 MHz). Mass spectra
were determined on a Hitachi M 80 instrument at 75 eV.
Materials. In 2003, calli of N. domestica were derived from the
stems of wild plants grown in Kobe (Japan) on Murashige and Skoog’s
medium containing 2,4-dichlorophenoxyacetic acid (1 mg/L), kinetin
(0.1 mg/L), yeast extract (0.1%), and agar (1%). The callus tissues were
subcultured every four or five weeks on fresh medium at 25 °C in the
dark.
Extraction of N. domestica. Ground parts of N. domestica (217 g)
were cut into pieces, extracted several times with MeOH under reflux,
and concentrated after addition of H2O. Methanol extracts were
extracted with 2% HCl several times. The extracts were washed with
Et2O, made basic with NH4OH, and extracted with Et2O and then CHCl3
to give Plant Frs. E (11.0 mg) and C (10.2 mg), which were subjected
to LC-NMR, LC-MS, and LC-CD measurements.
identical with berberine in LC-NMR (Figure 15, C), also recognized
in Plant Fr. C.
Accordingly, jatrorrhizine, palmatine, berberine, (R)-carnegine,
and (E,E)-, (E,Z)-, and (Z,Z)-terrestribisamide have been identified
in the callus of N. domestica. (R)-Carnegine and (E,E)-, (E,Z)-,
and (Z,Z)-terrestribisamide were characterized herein for the first
time. Identification of unstable (E,Z)- and (Z,Z)-terrestribisamide
formed by trans-cis isomerization of (E,E)-terrestribisamide
indicates the usefulness of LC-NMR for structural analysis.
Experimental Section
General Experimental Procedures. Conventional 1H NMR and
NOESY spectra were obtained on a Varian VXR-500 spectrometer (500
MHz) in CD3OD as solvent. 13C NMR and DEPT spectra were
Figure 9. LC data of the alkaloid fraction (Plant Fr. E) obtained from N. domestica. Column: Cosmosil 5C18-AR-II (4.6 × 150 mm).
Eluent: A: 0.1 M NH4OAc/D2O (0.05% TFA); B: MeCN (0.05% TFA). Gradient A/B: initial 80/20, 10 min 60/40, 20 min 60/40, 30 min
0/100. Flow rate: 1.0 mL/min. UV detector: 280 nm.

1382 Journal of Natural Products, 2008, Vol. 71, No. 8
Iwasa et al.
Figure 10. Stopped-flow LC-1H NMR spectrum of peak a3 in LC of the alkaloid fraction (Callus Fr. E) obtained from N. domestica and
1
1
carnegine. (A) H NMR spectrum of peak a3 (carnegine). (B) H NMR spectrum of standard carnegine.
Figure 11. LC-CD data of the alkaloid fraction (Callus Fr. E) obtained from N. domestica and (R) or (S)-carnegine. Column: Chiralcel
OD-RH (4.6 × 150 mm). Eluent: A: 0.1 M NH4OAc (0.05% TFA); B: MeCN (0.05% TFA). Gradient A/B: initial 100/0, 5 min 95/5, 15
min 75/25, 25 min 75/25, 35 min 0/100. Flow rate: 0.5 mL/min. Temp: 40 °C. UV detector: 236 nm.
Extraction of Callus of N. domestica. After being subcultured for
5-6 weeks, calli (441 g) and medium were freeze-dried. Water was
separated, and callus and medium were extracted several times with
hot MeOH. H2O and MeOH extracts were concentrated. The extracts
were washed with Et2O after acidification, made basic with NH4OH,
and extracted with Et2O and then CHCl3 to give Callus Frs. E (15.2
mg) and C (20.6 mg), which were subjected to LC-NMR, LC-MS, and
LC-CD measurements.
A/B initial 80/20, 75/25 (5 min), 75/25 (15 min), 0/100 (30); 6 mL/
min; 280 nm].
Optical Resolution of (()-Carnegine. A solution of the free base
obtained from (()-carnegine hydrochloride (4.12 g)29 in H2O (10 mL)
was added to a solution of L-(+)-tartaric acid (2 g) in H2O (5 mL).
The mixture was left in the refrigerator overnight. The resulting white
crystals were filtered and washed with cold water to provide the salt
[(+)-carnegine·(+)-tartaric acid] (2.72 g, R/S: 70/30). This salt (910
mg) was dissolved in H2O (5 mL) and left in the refrigerator for 3
days. The resulting crystals were filtered, and the collected salt (216
mg, R/S: 93/7) was dissolved in H2O, basified with aqueous NH3, and
Peak d4 (E,E-terrestribisamide) in LC of Callus Fr. C (Figure 14)
was separated by preparative HPLC [Cosmosil 5 C18-AR (20 × 250
mm), (A) 0.1 M NH4OAc (0.05% TFA) and (B) CH3CN (0.05% TFA):

Alkaloids in Crude Extracts of Nandina domestica
Journal of Natural Products, 2008, Vol. 71, No. 8 1383
Figure 12. Stopped-flow LC-1H NMR spectrum of peak b3 in LC of the alkaloid fraction (Callus Fr. E) obtained from N. domestica.
Figure 13. Stopped-flow LC-1H NMR spectrum of peak c3 in LC of the alkaloid fraction (Callus Fr. E) obtained from N. domestica.
*Signals attributed to (E,E)-terrestribisamide. **Signals attributed to (Z,Z)-terrestribisamide. ***Signals attributed to (E,E)-terrestribisamide
and (Z,Z)-terrestribisamide.
extracted with Et2O. The ether extract was dried and evaporated to
give (+)-carnegine as an oil (118 mg). Similarly, a solution of the free
base obtained from (()-carnegine hydrochloride (7.50 g) in H2O (10
mL) was added to a solution of D-(-)-tartaric acid (2.7 g) in H2O (14
mL). The mixture was left in the refrigerator for 4 days. The resulting
white crystals were filtered and washed with cold H2O to give the salt
[(-)-carnegine·(-)-tartaric acid] (3.34 g, R/S: 40/60). This salt was
dissolved in H2O (5 mL) and left in the refrigerator for 2 days. The
resulting crystals were filtered, and the resulting salt (1.01 g, R/S: 4/96)
was converted to the free base [(-)-carnegine].
LC-APCI-MS Method. LC-APCI-MS (/MS) was measured on an
Applied Biosystems API 3000 triple quadrupole mass spectrometer (MS/
MS) with a heated nebulizer interface as described in a previous paper.30
LC-CD Method. LC-CD analyses were carried out on a chiral phase
column at 236 nm. Chromatographic separations were performed using
a Jasco PU-2080Plus intelligent pump with a column oven (Jasco 860-
CO), Jasco Browin NT, HSS-2000 data processor, and Jasco CD-
2095Plus CD chiral detector (Hg-Xe lamp), simultaneously monitoring
the CD and UV signals at one specific wavelength (range 220-420
nm).
LC-NMR Method. LC-NMR data were acquired using a Varian
UNITY-INOVA-500 spectrometer (1H: 499.83 MHz) equipped with a
1
60 µL triple-resonance microflow NMR probe. 1D H NMR spectra
were obtained in stopped-flow mode as described in the previous
paper.30
LC-1H NMR: isoboldine δ 2.74 (1H, t, J ) 13.5 Hz, H-7), 2.94
(1H, m, H-4), 2.99 (3H, s, NCH3), 3.18 (1H, m, H-7), 3.22 (1H, m,
H-4), 3.36 (1H, m, H-5), 3.65 (1H, m, H-5), 3.82 (3H, s, OCH3-10),
3.85 (3H, s, OCH3-2), 4.12 (1H, m, H-6a), 6.77 (1H, s, H-3), 6.83
(1H, s, H-8), 8.02 (1H, s, H-11); sinoacutine δ 1.95 (overlap with H2O,

1384 Journal of Natural Products, 2008, Vol. 71, No. 8
Iwasa et al.
Figure 14. LC data of the alkaloid fraction (Callus Fr. C) obtain from N. domestica. Column: Cosmosil 5C18-AR-II (4.6 × 150 mm).
Gradient: A: 0.1 M NH4OAc/D2O (0.05% TFA), B: MeCN (0.05% TFA).A/B: initial 80/20, 5 min 70/30, 15 min 70/30, 25 min 0/100.Flow
rate: 1.0 mL/min.UV detector: 280 nm.
Figure 15. Stopped-flow LC-1H NMR spectra of protoberberine-type alkaloids in the alkaloid fraction (Callus Fr. C) obtained from N.
domestica. (A) 1H NMR spectrum of peak c4 (jatrorrhizine). (B) 1H NMR spectrum of peak e4 (palmatine). (C) 1H NMR spectrum of peak
f4 (berberine).
H-15), 2.49 (1H, m, H-15), 2.70 (3H, s, NCH3), 2.81 (1H, m, H-16),
3.07 (1H, m, H-16), 3.29 (1H, dd, J ) 19.0, 5.5 Hz, H-10), 3.42 (1H,
d, J ) 19.0 Hz, H-10), 3.67 (3H, s, OCH3-6), 3.77 (3H, s, OCH3-3),
4.26 (1H, d, J ) 5.5 Hz, H-9), 6.47 (1H, s, H-8), 6.72 (1H, d, J ) 8.0
Hz, H-1), 6.90 (1H, d, J ) 8.0 Hz, H-2), 7.78 (1H, s, H-5); domesticine
δ 2.73 (1H, t, J ) 14.0 Hz, H-4), 2.91 (1H, m, H-7), 2.97 (3H, s,
NCH3), 3.16 (1H, m, H-7), 3.20 (1H, m, H-4), 3.33 (1H, m, H-5), 3.62
(1H, m, H-5), 3.83 (3H, s, OCH3), 4.09 (1H, s, H-6a), 5.94 (2H, s,
OCH2O), 6.76 (1H, s, H-3), 6.86 (1H, s, H-8), 7.87 (1H, s, H-11);
nantenine δ 2.66 (1H, t, J ) 14.0 Hz, H-4), 2.87 (3H, s, NCH3), 2.89
(1H, m, H-7), 3.13 (1H, m, H-7), 3.16 (1H, m, H-4), 3.18 (1H, m,
H-5), 3.50 (1H, br s, H-5), 3.58 (3H, s, OCH3-1), 3.81 (3H, s, OCH3-
2), 5.96 (2H, s, OCH2O), 6.81 (1H, s, H-3), 6.86 (1H, s, H-8), 7.77
(1H, s, H-11); menispermine δ 2.83 (1H, t, J ) 13.0 Hz, H-7), 2.92
(3H, s, NCH3), 3.06 (1H, m, H-4), 3.25 (1H, m, H-7), 3.30 (3H, s,
NCH3), 3.34 (1H, m, H-4), 3.59 (1H, m, H-5), 3.62 (3H, s, OCH3-1),
3.71 (1H, m, H-5), 3.83 (3H, s, OCH3-10), 3.88 (3H, s, OCH3-2), 4.27
(1H, d, J ) 13.0 Hz, H-6a), 7.01 (1H, d, J ) 8.5 Hz, H-8), 7.02 (1H,
s, H-3), 7.04 (1H, d, J ) 8.5 Hz, H-9); O-methylpallidine δ 1.95
(overlap with H2O, H-15), 2.15 (1H, m, H-15), 2.74 (3H, s, NCH3),

Alkaloids in Crude Extracts of Nandina domestica
Journal of Natural Products, 2008, Vol. 71, No. 8 1385
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2.88 (1H, m, H-16), 3.06 (1H, m, H-16), 3.33 (1H, dd, J ) 19.0, 5.5
Hz, H-10), 3.45 (1H, d, J ) 19.0 Hz, H-10), 3.75 (3H, s, OCH3-6),
3.76 (3H, s, OCH3-2), 3.80 (3H, s, OCH3-3), 4.31 (1H, d, J ) 5.5 Hz,
H-9), 6.50 (1H, s, H-8), 6.83 (1H, s, H-1), 6.93 (1H, s, H-5). 7.07 (1H,
s, H-4).
HPLC Parameters for LC-NMR, LC-MS, and LC-CD. Chro-
matographic preparations were performed using a Cosmosil 5 C18-AR
(4.6 i.d. × 150 mm) reversed-phase column. The mobile phases [(A)
0.1 M NH4OAc (0.05% TFA, D2O for LC-NMR) and (B) CH3CN
(0.05% TFA) or CH3OH (0.05% TFA)] were used by nonlinear gradient
elution. Chiral analytical separation was carried out on a chiral OJ-RH
column (4.6 i.d. × 150 mm, Daicel Chemical Ltd.) at 40 °C for LC-
CD.
References and Notes
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