Biotransformation of phenolic tetrahydroprotoberberines in plant cell cultures followed by LC-NMR, LC-MS, and LC-CD

Article abstract of DOI:10.1021/np900440d

A metabolic pathway of 2,3,10,11-oxygenated tetrahydroprotoberberines having the OH group on ring D was demonstrated. Metabolism of ¹³C- or D₂-labeled precursors was studied in cell cultures of Macleaya, Corydalis, and Nandina species. The structures of alkaloid metabolites obtained from feeding experiments were determined by application of combined LC-NMR, LC-MS/MS, and LC-CD techniques. (S)-Tetrahydropseudoprotoberberine (5) was stereospecifically O-methylated to the 5-isomer (12) in cell cultures of three plant species. This 5-isomer was further N-methylated to the (5)-α-N-methyl salt (15), which was oxidized to produce the pseudoprotopine-type alkaloid (10) in cell cultures of Macleaya and Corydalis species. These transformations were the same as those of 2,3,9,10-oxygenated protoberberines. The tetrahydropseudoprotoberberines (5, 6, and 12) were dehydrogenated to pseudoprotoberberines (13, 16, and 14), respectively. Both the R- and 5-enantiomers of 5 were dehydrogenated in Macleaya cordata different from the case of 2,3,9,10-oxygenated protoberberines. Precursor 7, with OH groups at C-10 and C-11was O-methylated at C-10 in M. cordata and C. ochotensis var. raddeana, which was distinct from O-methylation in N. domestica, in which 7 was O-methylated at both C-11 and C-10. Stereoselective O-demethylation [(S)-S to (5)-18] occurred in N. domestica.

Full text of DOI:10.1021/np900440d

J. Nat. Prod. 2010, 73, 115–122
115
Biotransformation of Phenolic Tetrahydroprotoberberines in Plant Cell Cultures Followed by
LC-NMR, LC-MS, and LC-CD
Kinuko Iwasa,*,† Wenhua Cui,† Teturo Takahashi,† Yumi Nishiyama,† Miyoko Kamigauchi,† Junko Koyama,†
Atsuko Takeuchi,† Masataka Moriyasu,† and Kazuyoshi Takeda‡
Kobe Pharmaceutical UniVersity, 4-19-1 Motoyamakita, Higashinada-ku, Kobe-shi 658-8558, Japan, and Yokohama College of Pharmacy,
601 Matanocyo, Hodogayaku, Yokohama-shi 245-0066, Japan
ReceiVed July 27, 2009
A metabolic pathway of 2,3,10,11-oxygenated tetrahydroprotoberberines having the OH group on ring D was
demonstrated. Metabolism of 13C- or D2-labeled precursors was studied in cell cultures of Macleaya, Corydalis, and
Nandina species. The structures of alkaloid metabolites obtained from feeding experiments were determined by application
of combined LC-NMR, LC-MS/MS, and LC-CD techniques. (S)-Tetrahydropseudoprotoberberine (5) was stereospe-
cifically O-methylated to the S-isomer (12) in cell cultures of three plant species. This S-isomer was further N-methylated
to the (S)-R-N-methyl salt (15), which was oxidized to produce the pseudoprotopine-type alkaloid (10) in cell cultures
of Macleaya and Corydalis species. These transformations were the same as those of 2,3,9,10-oxygenated protoberberines.
The tetrahydropseudoprotoberberines (5, 6, and 12) were dehydrogenated to pseudoprotoberberines (13, 16, and 14),
respectively. Both the R- and S-enantiomers of 5 were dehydrogenated in Macleaya cordata different from the case of
2,3,9,10-oxygenated protoberberines. Precursor 7, with OH groups at C-10 and C-11, was O-methylated at C-10 in M.
cordata and C. ochotensis var. raddeana, which was distinct from O-methylation in N. domestica, in which 7 was
O-methylated at both C-11 and C-10. Stereoselective O-demethylation [(S)-5 to (S)-18] occurred in N. domestica.
We previously investigated the biosynthesis of isoquinoline
alkaloids in Papaveraceae and Fumariaceae plants and in their cell
cultures, and also evaluated the metabolites for various types of
biological activity.1–5 Numerous compounds with antimicrobial,
antimalarial, anti-HIV, and anticancer activities were revealed.2–5
(2,3,10,11-oxygenated tetrahydroprotoberberines), together with 4
(a 2,3,9,10-oxygenated tetrahydroprotoberberine as a comparison),
was studied in cell cultures of Corydalis ochotensis var. raddeana
(Fumariaceae), Macleaya cordata (Papaveraceae), and Nandina
domestica Thumb. (Berberidaceae). Without prior isolation, the
structures of the metabolites were determined by LC-NMR, LC-
MS, and LC-CD analyses.
Although protoberberine alkaloids differ in the number and
position of oxygen functions on the aromatic rings A and D, two
oxygenation patterns most frequently encountered were at carbons
2,3,9,10 and 2,3,10,11. The former group occurs most commonly,
while the latter has been labeled “pseudoprotoberberine” and is not
as widespread.6 Some 2,3,10,11-oxygenated alkaloids display higher
activity in biological assays (e.g., antimalarial activity) than the
corresponding 2,3,9,10-substituted analogues.3 The biosynthetic
conversion of the 2,3,9,10-oxygenated protoberberines to protopines,
benzophenanthridines, rhoeadines, benzindanoazepines, and spiroben-
zylisoquinolines has been demonstrated.1,7 Earlier we found that
2,3,10,11-oxygenated protoberberines (tetrahydropseudocoptisine
and tetrahydropseudopalmatine) underwent bioconversion into
pseudoprotopine-type alkaloids via their R-N-methyl salts.8 Phenolic
pseudoprotoberberine-type alkaloids with an OH group on aromatic
rings A or D occur in some plants,6,9 including Corydalis species.
Except for our studies,10–12 the metabolic pathway leading to the
formation of these alkaloids has not been investigated. Results from
prior feeding experiments of 1-benzyltetrahydroisoquinolines with
one (e.g., 1 and 2) or two OH groups (e.g., 3) at C-3′, C-4′, C-6,
or C-7 have demonstrated that the presence of an OH group at
C-3′ is essential for the biotransformation of 1-benzyltetrahydroiso-
quinolines into 2,3,9,10- and 2,3,10,11-oxygenated tetrahydro-
protoberberines.10–12 Tetrahydropalmatrubine (4)13 is derived from
norlaudanine (1, C-3′ OH) and its N-methyl derivative. Corytenchine
(5)14 is formed from 1 and its N-methyl derivative and also from
the N-methyl derivative of 3 (C-3′ and -4′ OH). However,
10-demethylxylopinine (6)15 and spinosine (7)16 are not produced
from compounds 2 (C-4′ OH) and 3 (C-3′ and -4′ OH), respectively.
In our current investigation, the metabolism of 5, 6, and 7
Results and Discussion
Synthesis. [8-13C]-Tetrahydroprotoberberines [(()-[8-13C]-4-7]
and (()-[8-D2]-7 were prepared by the Mannich reaction of 13C-
or D2-labeled 37% formaldehyde with 1-benzyltetrahydroisoquino-
lines (1, 2, and 3) (Supporting Information, Scheme S1), which
were prepared from 3,4-dimethoxyphenylethylamine and appropri-
ate 3,4-dioxygenated phenylacetic acids, according to reported
procedures.17,18 Structures of the synthetic compounds ([8-D2]-7,
1
[8-13C]-4-7) were confirmed by analysis of their MS, HRMS, H
NMR, and NOESY spectra.
Feeding Experiments. Callus tissues of Macleaya cordata,
Corydalis ochotensis var. raddeana, and Nandina domestica were
incubated at 25 °C (N. domestica 27 °C) on an agar medium
containing the substrate for four weeks (Table 1). Following
incubation, media and cells were separated and extracted according
to the procedure described previously.11 The ether and CHCl3-
soluble alkaloid fractions (E-1, E-2 and C-1, C-2) (Supporting
Information, Figure S1) were subjected to LC-NMR, LC-MS, and
LC-CD.
Corydalis and Macleaya Species. The metabolism of [8-13C]-
tetrahydroprotoberberines [(()-[8-13C]-4-6] and [(()-8-D2]-spi-
nosine [(()-[8-D2]-7] (Scheme 1), which possess OH groups on
the aromatic ring D, were examined in cultured cells of C.
ochotensis var. raddeana and M. cordata (Table 1). Initially,
* To whom correspondence should be addressed. E-mail: k-iwasa@
kobepharma-u.ac.jp. Tel: 081-78-453-0031. Fax: 081-78-435-2080.
† Kobe Pharmaceutical University.
‡ Yokohama College of Pharmacy.
10.1021/np900440d  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/19/2010

116 Journal of Natural Products, 2010, Vol. 73, No. 2
Iwasa et al.
Table 1. Administration of Tetrahydroprotoberberines (4-7) to
Cultured Cells of Macleaya cordata, Corydalis ochotensis var.
raddeana, and Nandina domesticaa
Peak f2 displayed a [M]+ ion at m/z 353 and product ion at m/z
337. The 1H NMR spectrum of peak f2 showed four O-CH3 groups
(δ 3.86, 3.91, 3.97, and 4.04), four aromatic proton singlets (δ 7.01,
7.46, 7.53, and 8.47), and two aromatic proton doublets at δ 7.56
(1H, d, J ) 2.5 Hz, 13C-H long-range coupling) and 9.14 (1H, d,
J ) 188 Hz, 13C-H coupling). These data indicated that the
compound represented by peak f2 was [8-13C]-pseudopalmatine ([8-
13C]-14) (Scheme 1). This structure was confirmed by comparison
of the 1H NMR data with that of authentic unlabeled sample8
(Supporting Information, Figure S3).
no.
callusb
substrate (mg)
[8-13C]-4
wt of dry cells (g)
1
2
3
4
5
6
7
8
A
A
A
A
B
A
A
A
B
A
B
C
C
C
C
C
5
4
7
10
10
5
2.7
2.5
4.9
2.0
1.9
2.5
2.3
3.4
5.0
1.3
1.3
2.0
2.0
1.9
2.2
1.7
(S)-[8-13C]-4
(R)-[8-13C]-4
[8-13C]-5
[8-13C]-5
(S)-[8-13C]-5
(R)-[8-13C]-5
[8-13C]-6
[8-13C]-6
[8-D2]-7
5
The metabolites obtained from feeding (()-[8-13C]-5 to C.
ochotensis var. raddeana (Table 1, no. 5) were found to be 10, 11,
12, and 13. As shown in Scheme 1, 5 was likely converted to 11
and 12, and the latter further converted to 10. Compound 11 could
be produced by oxidation at C-13a after formation of the methyl-
endioxy group from the OH and OCH3 groups on ring D, a
metabolic pathway comparable to the conversion of 4 to 9 and then
to 8. Compounds 5 and 12 could also be dehydrogenated to give
13 and 14, respectively (Scheme 1).
10
10
10
10
10
10
10
10
10
9
10
11
12
13
14
15
16
[8-D2]-7
[8-13C]-4
[8-13C]-5
[8-13C]-6
[8-13C]-7
[8-D2]-7
a MS medium: 400 mL; incubation period: 4 weeks. b A: Macleaya
cordata; B: Corydalis ochotensis var. raddeana; C: Nandina domestica.
In a similar fashion, the metabolic path of (()-[8-13C]-10-
demethylxylopinine [(()-[8-13C]-6] was examined in M. cordata
and C. ochotensis var. raddeana. Precursor [8-13C]-6 was admin-
istered to cultured cells of M. cordata (Table 1, no. 8). The resulting
fraction E-2 showed LC peaks a3-h3 (Figure 2), which produced
[M + H]+ or [M]+ ions at m/z 387, 371, 369, 343, 339, 353, 357,
and 355, respectively. The compounds associated with peaks a3,
d3, f3, and g3 were identified as [8-13C]-10, -6, -14, and -12 (Scheme
1), respectively. The amount of [8-13C]-12 was smaller than that
obtained from feeding of [(()-[8-13C]-5]. Thus, 5 is a better
precursor of 12 than of 6 in M. cordata. LC peak b3 showed a [M
+ H]+ ion at m/z 371 and a product ion at m/z 206, corresponding
to [8-13C]-cis-N-methyltetrahydropseudopalmatinium salt ([8-13C]-
15) (Scheme 1). The structure of 15 was confirmed by comparison
of its LC-MS/MS data with that of authentic 15 ([M]+ ion at m/z
370 and a product ion at m/z 206).8 Peak e3 displayed a [M]+ ion
at m/z 339 and a product ion at m/z 323 in the LC-MS/MS. The 1H
NMR spectrum (A, Supporting Information, Figure S4) of the
metabolite showed three OCH3 groups (δ 3.86, 3.91, and 4.04),
four aromatic singlets (δ 7.01, 7.44, 7.52, and 8.43), an aromatic
doublet (δ 9.06, J ) 143 Hz, 13C-H coupling), and two methylene
multiplets (δ 3.14 and 4.65). The structure of the metabolite was
deduced to be 13C-labeled 10-demethyldehydroxylopinine ([8-13C]-
16) (Scheme 1). Peak h3 showed a [M + H]+ ion at m/z 355 and
precursor [(()-8-13C]-tetrahydropalmatrubine [(()-[8-13C]-4] was
administered to cells of M. cordata (Table 1, no. 1). Fraction E-1
showed four LC peaks, a1-d1 (I, Figure 1). In LC-APCI-MS, peaks
a1-d1 showed [M + H]+ ions at m/z 371, 354, 343, and 341,
respectively. From LC-NMR and LC-MS data, peaks b1 and c1 were
attributed to protopine (a component of M. cordata) and precursor
4, respectively. Peak a1 showed product ions at m/z 222, 205, and
204 and was identified as [8-13C]-cryptopine ([8-13C]-8) (Scheme
1) by comparison of its LC-NMR and LC-MS data with those of
an unlabeled authentic sample.10 Peak d1 showed a product ion at
m/z 192 in LC-MS/MS and corresponded to [8-13C]-tetrahy-
droepiberberine ([8-13C]-9) (Scheme 1) by comparison of the LC-
MS with that of an unlabeled authentic sample.11 Thus, the feeding
of (()-[8-13C]-4 to M. cordata resulted in two metabolites, [8-13C]-
8 and -9. As shown in Scheme 1, 4 was converted to 9 (the
methylenedioxy group of 9 being formed from the 9-hydroxy and
10-methoxy groups on ring D of 4), which was further converted
to 8. This metabolic pathway has been demonstrated previously.1,7
Next, [(()-8-13C]-corytenchine ((()-[8-13C]-5) was administered
to cultured cells of M. cordata (Table 1, no. 4). Peaks a2-f2 (II,
Figure 1) in fractions E-1 and C-2 showed [M + H]+ or [M]+ ions
at m/z 387, 371, 343, 357, 339, and 353, respectively, in the LC-
MS. Peaks a2 and b2 also showed product ions at m/z 222, 205,
and 204 and were identified as [8-13C]-pseudomuramine ([8-13C]-
10) and [8-13C]-pseudocryptopine ([8-13C]-11) (Scheme 1) by
comparison of their LC-MS data with those of protopine-type
alkaloids.10 From LC-MS/MS data, peak c2 was deduced to be
precursor [8-13C]-5. Peak d2 displayed a [M + H]+ ion at m/z 357
1
a product ion at m/z 339. The H NMR spectrum of peak h3 (B,
Supporting Information, Figure S4) showed four aromatic singlets
(δ 6.92, 7.13, 7.18, and 7.36), an aromatic doublet (δ 7.58, J )
3.5 Hz, 13C-H long-range coupling), three OCH3 groups (δ 3.83,
3.87, and 3.93), and two methylene multiplets (δ 2.90 and 3.24).
The structure of the metabolite associated with peak h3 was
determined to be 13C-labeled 8-oxo-10-demethyldehydroxylopinine
([8-13C]-17) (Scheme 1).
1
and product ion at m/z 192. The H NMR spectrum of peak d2
showed four O-CH3 groups at δ 3.76, 3.78 (6H), and 3.79 and four
aromatic protons at δ 6.78 (1H, d, J ) 3.0 Hz, 13C-H long-range
coupling), 6.82 (2H, s), and 6.86 (1H, s). These data indicated that
peak d2 was [8-13C]-tetrahydropseudopalmatine ([8-13C]-12) (Scheme
The metabolites obtained from feeding [8-13C]-6 to C. ochotensis
(Table 1, no. 9) were deduced to be [8-13C]-10, -12, -14, -16, and
-17, by analysis of 1H NMR and LC-MS/MS data. These metabo-
lites were identical to those obtained from the feeding experiment
of [8-13C]-6 to M. cordata.
In our proposed biosynthetic pathway, 6 was O-methylated to give
12, which was converted via its N-methyl derivative (15) into 10. In
addition, 12 can be dehydrogenated to 14. Precursor 6 could also be
dehydrogenated to give 16, which was oxidized to the 8-oxo derivative
17 (Scheme 1). Although 11 was obtained from 5, it was not obtained
from 6. This result suggested that a methylenedioxy group may not
be formed from an OH at C-10 and an OCH3 at C-11.
1
1). This structure was confirmed by comparison of the H NMR
data with that of authentic unlabeled sample8 measured under the
same conditions (Supporting Information, Figure S2). The signal
pattern for H-8 in the 1H NMR spectrum of [8-13C]-12 was different
from that of unlabeled 12, because of the 13C-H coupling. Peak e2
showed a [M]+ ion at m/z 339 and product ion at m/z 323 in the
LC-MS/MS. The 1H NMR spectrum of peak e2 showed three O-CH3
groups at δ 3.84 and 3.89 (6H), aromatic proton singlets at δ 6.95,
6.97, 7.47, and 8.05, and aromatic proton doublets at δ 7.30 (1H,
d, J ) 2.5 Hz, 13C-H long-range coupling) and 8.74 (1H, d, J )
181 Hz, 13C-H coupling). These data indicated that peak e2 was
[8-13C]-dehydrocorytenchine ([8-13C]-13) (Scheme 1). This structure
In the next stage of our study, the metabolism of [(()-8-D2]-
spinosine ([(()-[8-D2]-7]), which has two OH groups on the
aromatic D ring, was examined in M. cordata and C. ochotensis
(Table 1, no. 10 and 11, respectively). From the LC-MS/MS data,
1
was confirmed by comparison of the H NMR data with that of
authentic unlabeled sample11 (Supporting Information, Figure S3).

Biotransformation of Phenolic Tetrahydroprotoberberines
Journal of Natural Products, 2010, Vol. 73, No. 2 117
Scheme 1. Metabolism of (()-[8-13C]-Tetrahydropalmatrubine, -Corytenchine, -10-Demethylxylopinine, and -Spinosine,
(()-[8-D2]-Spinosine [(()-[8-13C]-4, -5, -6, -7, and (()-[8-D2]-7] in M. cordata (M.c.), C. ochotensis var. raddeana (C.o.), and
Nandina domestica (N.d.)
[8-D2]-5 ([M + H]+ ion at m/z 344 and product ion at m/z 192)
and [8-D2]-12 ([M + H]+ ion at m/z 358 and product ion at m/z
192) were identified as the metabolites in M. cordata. The only
metabolite from C. ochotensis was identified as [8-D2]-5 from LC-
MS/MS data. Compound 7 was O-methylated selectively at C-10
to give 5, which in turn was O-methylated on ring D to afford 12
(Scheme 1).
Chiral purities of the tetrahydroprotoberberine metabolites were
determined by online LC-CD and LC-MS/MS analyses. Enantio-
meric separations were achieved for (()-corytenchine (pseudotype
protoberberine) and (()-tetrahydropalmatrubine using a Chiralcel
OJ-RH column with 0.1 M NH4OAc-MeCN (0.05% TFA) as
eluent. The relative amounts were determined by the ratio of the
peak areas in the MS and CD chromatograms. Both compounds
showed a positive Cotton effect for the faster eluting peak and
negative Cotton effect for the slower peak in the LC-CD spectra at
236 nm (Figure 3). The absolute configurations at C-13a for the
enantiomers were previously established as R and S for the faster
and slower peaks, respectively.10
fied the metabolic pathways of these compounds as shown in
Scheme 1. The metabolic pathways for tetrahydropseudoprotober-
berines (5-7) with one or two OH group on ring D were demon-
strated for the first time, except for conversion of 12 into 14.8
Metabolism of (R/S)-[8-13C]-Tetrahydroprotoberberines
([8-13C]-4 and -5) in Macleaya cordata. Because the metabolism
of tetrahydroprotoberberines likely involves stereospecific biocon-
versions, the metabolism of (R/S)-[8-13C]-tetrahydropalmatrubine
and -corytenchine [(R/S)-[8-13C]-4 and -5)] was examined in
Macleaya cordata. These precursors, (R/S)-[8-13C]-4 and -5, were
resolved by preparative HPLC on a chiral column (OJ-RH).
Fraction E-1 (I, Figure 4) obtained from the feeding (Table 1,
no. 2) of (S)-[8-13C]-4 to M. cordata showed three peaks, a4-c4, in
the LC (I, Figure 4). In the LC-APCI-MS, peaks a4-c4 showed
[M + H]+ ions at m/z 371, 354, and 341, respectively, and product
ions at m/z 204 and 190, 188, and 189 and 192, respectively. From
LC-NMR and LC-MS data, peaks a4, b4, and c4 were attributed to
[8-13C]-8, protopine (a component of M. cordata), and [8-13C]-9,
respectively (Scheme 2). Metabolite [8-13C]-9 showed a negative
Cotton effect indicating the S-configuration at C-13a. Metabolites
[8-13C]-8 and -9 were not detected from the feeding (Table 1, no.
3) of (R)-[8-13C]-4. Thus, (S)-4 was likely to be converted to (S)-
9, which was further converted to 8 as shown in Scheme 2.
(S)-[8-13C]-Corytenchine [(S)-[8-13C]-5] was also administered
to cultured cells of M. cordata (Table 1, no. 6). The resulting
fractions E-1 and C-1 showed peaks a5-d5 in the LC (II, Figure
4), with [M + H]+ or [M]+ ion at m/z 343, 353, 357, and 339,
respectively, and product ions at m/z 204 and 192, 337, 192, and
323, respectively. The compounds associated with peaks a5, b5, c5,
and d5 were identified as precursor ([8-13C]-5), [8-13C]-14, -12, and
-13 (Scheme 2), respectively, by analysis of 1H NMR and LC-MS/
MS data. Metabolite [8-13C]-12 displayed a negative Cotton effect
that corresponds to the S-configuration at C-13a. Compound [8-13C]-
13 was produced in the feeding (Table 1, no. 7) of (R)-[8-13C]-5;
however, [8-13C]-12 and -14 were not detected.
The CD spectrum of the [8-13C]-tetrahydropalmatrubine me-
tabolite, 9, showed a negative Cotton effect that matched that of
the S-enantiomer. Thus, (S)-4 was bioconverted via (S)-9 to 8.
Metabolite 12 obtained from (()-[8-13C]-5 and (()-[8-13C]-6 was
determined to be the S-enantiomer by LC-CD analysis. The
metabolite of (()-[8-13C]-6, 15, showed a negative Cotton effect
in its CD spectra; thus, it is the S-enantiomer. Corytenchine (5)
and 12, metabolites of (()-[8-D2]-7, displayed negative Cotton
effects in the LC/CD. Both 5 and 12 were S-enantiomers.
Metabolites 5 and 12 showed protonated molecular ions [M +
H]+ at m/z 344 and 358 ([8-D2]-5 and -12), respectively, as well as
[M + H]+ - 1 ions at m/z 343 and 357 ([8-D]-5 and -12),
respectively, in M. cordata. The ratios of 344:343 and 358:357 were
ca. 3:1. The [M + H]+ - 1 ion was also observed in metabolite 5
in C. ochotensis. The ratio of 344:343 was ca. 95:5. Pseudopro-
toberberines [8-D]-13 and [8-D]-14 formed from [8-D2]-5 and -12,
respectively, may be stereoselectively reduced to (S)-[8-D]-5 and
-12.
Thus, (S)-4 was transformed to (S)-9, and the R-isomer did not
serve as a substrate. The enzyme responsible for formation of the
methylenedioxy bridge was highly specific for the S-configurated
The results obtained from feeding experiments with [8-13C]-
tetrahydroprotoberberines [(()-[8-13C]-4-6] and (()-[8-D2]-7 clari-

118 Journal of Natural Products, 2010, Vol. 73, No. 2
Iwasa et al.
Figure 1. LC data of the alkaloid fractions obtained from the feedings of (()-[8-13C]-tetrahydropalmatrubine ([8-13C]-4) (I) and (()-[8-
13C]-corytenchine ([8-13C]-5) (II) to M. cordata. Eluent: A, 0.1 M NH4OAc (0.05% TFA); B, MeCN (0.05% TFA). Gradient A/B: initial
80:20, 25 min 55:45, 26 min 0:100. Flow rate: 1 mL/min. UV detection: 280 nm.
stereoselectivity. The pathway shown in Scheme 2 was demon-
strated for the first time. (S)-Tetrahydroprotoberberine oxidase
discovered in Berberis wilsoniae suspension cultures exhibits strict
specificity for the S-enantiomer of tetrahydroprotoberberines.19 (S)-
Canadine was stereospecifically dehydrogenated to berberine by
(S)-canadine oxidase in Coptis japonica cell cultures.20 These results
obtained with 2,3,9,10-oxygenated tetrahydroprotoberberines differ
from those with 2,3,10,11-oxygenated tetrahydroprotoberberines in
M. cordata.
Nandina domestica. Trace amounts of the O-methylated
metabolite (tetrahydropalmatine) and its dehydro derivative (pal-
matine) were obtained from the feeding (Table 1, no. 12) of (()-
[8-13C]-4. The enzyme responsible for the formation of the
methylenedioxy bridge may be inactive in N. domestica, in contrast
to M. cordata (Scheme 1).
The precursor [(()-8-13C]-corytenchine ([(()-[8-13C]-5]) was
administered to cultured cells of N. domestica (Table 1, no. 13).
Figure 2. LC data of the alkaloid fraction (E-2) obtained from the
The resulting fractions E-1 and C-1 showed peaks a6-g6 (Figure
feeding of (()-[8-13C]-10-demethylxylopinine ([8-13C]-6) to M.
5). The protonated molecular ions ([M + H]+ or [M]+ ion) and
cordata. Eluent: A, 0.1 M NH4OAc (0.05% TFA); B, MeCN (0.05%
product ions are included in Figure 5. Peak a6 displayed a [M +
TFA). Gradient A/B: initial 80:20, 25 min 55:45. Flow rate: 1 mL/
H]+ion at m/z 329, 14 mass units less than the precursor, and a
min. UV detection: 280 nm.
1
product ion at m/z 178. The H NMR spectrum of peak a6 (A,
Supporting Information, Figure S5) showed OCH3 groups at δ 3.78
and 3.80 and aromatic signals at δ 6.72 (1H, s), 6.74 (1H, s), 6.76
(1H, d, J ) 4.0 Hz, 13C-H long-range coupling), and 6.79 (1H, s).
These data suggested that the compound represented by peak a6
isomer. (S)-Corytenchine was converted to (S)-12 by a highly
stereoselective O-methylation at C-11, while the R-isomer did not
serve as a substrate. Both (R)- and (S)-5 were converted to 13 by
dehydrogenation of the C ring. This enzyme did not show any

Biotransformation of Phenolic Tetrahydroprotoberberines
Journal of Natural Products, 2010, Vol. 73, No. 2 119
Figure 3. UV and CD chromatograms from the LC-CD of (()-tetrahydropalmatrubine (4), (()-corytenchine (5), (()-10-demethylxylopinine
(6), (()-[8-D2]-spinosine ([8-D2]-7), and (()-tetrahydropseudopalmatine (12). Column: Chiralcel OJ-RH (4.6 × 150 mm). Eluent: A, 0.1
M NH4OAc (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
mL/min. UV detection: 236 nm.
unlabeled samples (18 and 19). The compound associated with peak
b6 was identified as [8-13C]-5 by analysis of 1H NMR and LC-MS/
MS data. Peak c6 showed a [M + H]+ ion at m/z 357, 14 mass
units more than the precursor, and a product ion at m/z 192. The
1H NMR spectrum of peak c6 displayed four OCH3 groups and
four aromatic protons including a proton with 13C-H long-range
coupling. These data indicated that peak c6 was [8-13C]-12. This
1
structure was confirmed by comparison of the H NMR data with
that of an authentic unlabeled sample. Metabolites, [8-13C]-18 and
-12 displayed negative CD bands (I, Supporting Information, Figure
S6), corresponding to the S-configuration at C-13a. Peaks d6 and
e6 (Figure 5) in the CHCl3-soluble alkaloid fraction (fraction C-1)
showed [M + H]+ or [M]+ and product ions at m/z 339 and 323
and at m/z 353 and 337, respectively, in the LC-MS/MS. These
data indicated that the compounds represented by peaks d6 and e6
were [8-13C]-13 and -14, respectively. Peaks f6 and g6 were
identified as berberine and palmatine, which are components of
the callus21 (Scheme 1).
Thus, as shown in Scheme 1, (S)-5 was converted to (S)-12 by
O-methylation at C-11, which was then dehydrogenated to 14. In
addition, 5 was converted to 13 by the dehydrogenation of the C
ring. This metabolism is the same as that in Corydalis and Macleaya
species. However, in N. domestica, (S)-5 was also demethylated at
C-3 to give (S)-18, and this metabolism was not observed in
Corydalis and Macleaya species.
In a similar fashion, the metabolism of (()-[8-13C]-10-demeth-
ylxylopinine ([8-13C]-6) was examined in N. domestica (Table 1,
no 14). Two metabolites showing [M + H]+ ions at m/z 357 and
329 and product ions at m/z 192 and 178, respectively, were detected
in the LC-MS/MS of the Et2O-soluble alkaloid fraction (fraction
E-1). The former was deduced to be [8-13C]-12 by comparison with
an authentic sample using LC-MS. The latter was suggested to be
the 13C-labeled metabolite obtained from O-demethylation of ring
A [329 (343-14), 178 (192-14)]. The amounts of both metabolites
were less than those obtained in the feeding of (()-[8-13C]-5.
Therefore, 5 is a better precursor than 6 for 12 in N. domestica.
This result was identical to that of M. cordata.
Figure 4. LC data of the alkaloid fractions obtained from the
feedings of (S)-[8-13C]-tetrahydropalmatrubine ([8-13C]-4) (I) and
(S)-[8-13C]-corytenchine ([8-13C]-5) (II) to M. cordata. Eluent: A,
0.1 M NH4OAc (0.05% TFA); B. MeCN (0.05% TFA). Gradient
A/B: initial 80:20, 25 min 55:45, 26 min 0:100. Flow rate: 1 mL/
min. UV detection: 280 nm.
was [8-13C]-isocoreximine or -coreximine ([8-13C]-18 or -19)
(Scheme 1). The structure was confirmed to be [8-13C]-18 by
comparison of the 1H NMR data (A, Supporting Information, Figure
S5) those (B and C, Supporting Information, Figure S5) of authentic
In the next stage of our study, the metabolism of [(()-8-13C]-
spinosine ([(()-[8-13C]-7]) was examined in N. domestica (Table

120 Journal of Natural Products, 2010, Vol. 73, No. 2
Iwasa et al.
Scheme 2. Metabolism of (S)- and/or (R)-[8-13C]-Tetrahydropalmatrubine and -Corytenchine (4 and 5) in M. cordata
1, no. 15). By comparison of the LC-MS/MS with authentic
samples, (S)-[8-13C]-12 ([M + H]+ion at m/z 357 and product ion
at m/z 192), (S)-[8-13C]-5, and (S)-[8-13C]-6 ([M + H]+ ion at m/z
343 and product ion at m/z 192) were identified as the metabolites.
As shown in Scheme 1, three (S)-tetrahydroprotoberberines (5,
6, and 12) were obtained from 7. (S)-Spinosine (7) was O-
methylated at C-10 and C-11 to give (S)-5 and (S)-6, respectively.
This result was different from that in Corydalis and Macleaya
species, where the metabolite 5, O-methylated at C-10, was
obtained. Both (S)-5 and -6 could be further O-methylated to give
(S)-12.
[(()-8-D2]-Spinosine ([(()-[8-D2]-7]) was administered to N.
domestica (Table 1, no. 16) in order to examine whether a redox
process shown in M. cordata was present in N. domestica. [8-D2]-
Tetrahydropseudopalmatine ([8-D2]-12) ([M + H]+ ion at m/z 358
and product ion at m/z 192), [8-D2]-5, and [8-D2]-6 ([M + H]+ion
at m/z 344 and product ion at m/z 192) were identified as the
metabolites. It was demonstrated that these alkaloids are S-
configured at C-13a by LC-MS/MS and LC-CD analyses (II,
Supporting Information, Figure S6). Protonated molecular ions at
m/z 343 or 342 corresponding to [8-D]-5 and -6 or 5 and 6 and
those at m/z 357 or 356 corresponding to [8-D]-12 and 12 were
not detected. Thus, a redox process was not present in N. domestica
as opposed to Corydalis species.
The metabolic pathway was clarified by the results obtained from
feeding experiments of (()-[8-13C]-4-7 and (()-[8-D2]-7 as shown
in Scheme 1. Metabolism in N. domestica (Berberidaceae) differed
from that in M. cordata (Papaveraceae) and C. ochotensis var.
raddeana (Fumariaceae), which are closely related families in some
pathways. Therefore it can be concluded that in cell cultures of
Macleaya, Corydalis, and Nandina species S-configured 2,3,10,11-
oxygenated tetrahydropseudoprotoberberine (e.g., 5) was O-me-
thylated stereospecifically to afford the S-isomer (e.g., 12), which
was N-methylated to the (S)-R-N-methyl salt (e.g., 15). The (S)-
R-N-methyl salt was oxidized to produce the pseudoprotopine-type
alkaloid (e.g., 10). These transformations were similar to those of
Figure 5. LC and LC-MS/MS data of the alkaloid fractions (E-1 and C-1) obtained from the feeding of (()-[8-13C]-corytenchine ([8-13C]-
5) to Nandina domestica.

Biotransformation of Phenolic Tetrahydroprotoberberines
Journal of Natural Products, 2010, Vol. 73, No. 2 121
in Me2CO (30 mL)-concd HCl (0.5 mL). The solvent was evaporated
and the residue was crystallized from EtOAc-Me2CO (2:1) to give
the hydrochloride of 110 (550 mg, yield 27%, mp 213-214 °C). This
product (250 mg) in H2O (25 mL) was adjusted to pH 5 with dilute
HCl, and aqueous 37% H13CHO (100 mg) was added. After standing
overnight at rt, the resulting crystals were filtered to give (()-[8-13C]-
5 (62 mg, mp 171 °C).
2,3,9,10-oxygenated protoberberines. The tetrahydropseudoproto-
berberines (5, 6, and 12) were dehydrogenated to the pseudopro-
toberberines (13, 16, and 14), respectively. Both the R- and
S-enantiomers of 2,3,10,11-oxygenated protoberberine (5) were
dehydrogenated in M. cordata, which differed from 2,3,9,10-
oxygenated protoberberines. The tetrahydropseudoprotoberberine
7, with OH groups at C-10 and C-11, was O-methylated at C-10 in
M. cordata and C. ochotensis, as distinct from O-methylation in
N. domestica, in which 7 was O-methylated at C-11 as well as
C-10. Stereoselective O-demethylation was also observed in N.
domestica. O-Demethylation [from (S)-5 to (S)-18] occurred in the
metabolic pathway after formation of the tetrahydroprotoberberines
in N. domestica, which differed from that in M. cordata and C.
ochotensis. In conclusion, a metabolic pathway of 2,3,10,11-
oxygenated tetrahydroprotoberberines having one or two OH groups
on ring D was demonstrated for the first time by application of
LC-MS, LC-NMR, and LC-CD techniques.
Crystals of (()-[8-13C]-5/(()-[8-13C]-4, 1:1 (71 mg), which precipi-
tated in the filtrate were collected. The mother liquid [(()-[8-13C]-5/
(()-[8-13C]-4], 3:1, was separated by PHPLC. The eluents were purified
by HPLC [H2O (0.05% TFA)/MeOH (0.05% TFA)] to give (()-[8-
13C]-5 (40 mg) and (()-[8-13C]-4 (15 mg) as trifluoroacetates. (()-[8-
13C]-5: 1H NMR data were identical with those of the unlabeled
compound10 except for H-8 (δ 4.45, d, J ) 143.5 Hz, 13C-H coupling)
and H-9 (δ 6.78, d, J ) 5.5 Hz, 13C-H long-range coupling). SIMS
m/z 343 [M + H]+ (100), 192 (53), HR-SIMS m/z 343.1749 (calcd for
C1913CH24NO4: 343.1738). [(()-[8-13C]-4: 1H NMR data were identical
to those of the unlabeled compound,10 except for H-8 (δ 4.46, d, J )
143 Hz, 13C-H coupling). SIMS m/z 343 [M + H]+ (100), 192 (27),
HR-SIMS m/z 343.1753 (calcd for C1913CH24NO4: 343.1738).
Experimental Section
Preparation of (()-[8-13C]-10-Demethylxylopinine [(()-[8-13C]-6].
A mixture of 3,4-dimethoxyphenethylamine (1.06 g) and homovanillic
acid (1.04 g) was heated in an oil-bath at 180-190 °C for 30 min. The
hydrochloride of 2 was prepared as described above (for 1) from the
O-protected 1-benzyltetrahydroisoquinoline (818 mg). A solution of
its hydrochloride (110 mg) in H2O (11 mL) was adjusted to pH 3 with
dilute HCl, and aqueous 37% H13CHO (20 mg) was added. After
standing overnight at rt, the resulting white powder was filtered to give
a mixture of (()-[8-13C]-6 and 2 (85:15), which was separated and
purified by PHPLC to give (()-[8-13C]-6 (82 mg) and 2 (16.7 mg) as
trifluoroacetates. [(()-[8-13C]-6: 1H NMR (500 MHz, CD3OD) δ 6.95
(1H, s, H-1), 6.87 (1H, s, H-12), 6.83 (1H, s, H-4), 6.66 (1H, d, J )
5.0 Hz, H-9, 13C-H long-range coupling), 4.64 (1H, brd, J ) 10 Hz),
4.42 (2H, d, J ) 143 Hz, H-8, 13C-H coupling), 3.87 (6H, s, 2- and
11-OMe), 3.84 (3H, s, 3-OMe), 3.77 and 3.03 (each 1H, m, H-13),
3.74 and 3.42 (each 1H, m, H-6), 3.24 and 3.04 (each 1H, m, H-5);
13C NMR (125 MHz, CD3OD) δ 26.90 (C-5), 34.40 (C-13), 51.44 (C-
6, br), 56.48, 56.51, 56.78 (C-2, 3-, and 11-OMe], 56.63 (13C-8), 61.48
(C-13a, br), 110.07 (C-1), 112.57 (C-12, d, J ) 3.25 Hz), 112.85 (C-
4), 113.45 (C-9, d, J ) 6.0 Hz), 121.70 (C-12a), 123.47 (C-8a), 124.89
(C-4a), 125.85 (C-1a), 147.29 (C-10, d J ) 5.63 Hz), 149.47 (C-11),
150.15 (C-2), 150.65 (C-3); SIMS m/z 343 [M + H]+ (100), 192 (45);
HR-SIMS m/z 343.1751 (calcd for C1913CH24NO4, 343.1738).
General Experimental Procedures. Conventional 1H NMR and
NOESY spectra were obtained on a Varian VXR-500S spectrometer
(1H: 499.99 MHz) in CD3OD. Mass spectra were determined on a
Hitachi M-4100 instrument at 75 eV. Secondary ion mass spectra
(SIMS) were measured using glycerol as matrix. HPLC and PHPLC
analyses were performed using a Hitachi M-6200 intelligent pump (1
mL/ min) or Hitachi M-6250 intelligent pump (6 mL/min), respectively,
with a Hitachi L-4000 UV detector (280 nm). Cosmosil 5C18-AR
reversed-phase columns (4.6 i.d. × 150 mm and 20 i.d. × 250 mm)
were used for HPLC and PHPLC, respectively. Analyses with a Hitachi
HPLC system were made using a solvent system, (A) 0.1 M NH4OAc
(0.05% TFA)/(B) MeOH (0.05% TFA) under the following gradient
conditions: A/B, initial (80:20), 25 min (55:45), 26 min (0:100) for
LC with a Hitachi HPLC system or initial (80:20), 10 min (60:40), 20
min (60:40), 30 min (0:100) for LC-MS and LC-CD or initial (80:20),
6 min (0:100) for PHPLC (flow rate: 60 mL/min, detection: 280 nm).
PHPLC purifications were performed using a solvent system, (A) H2O
(0.05% TFA)/(B) MeOH (0.05% TFA) under the following gradient
conditions: A/B 50:50 to 0:100, 30 min. The chiral analytical separation
was carried out on a chiral OJ-RH column (4.6 i.d. × 150 mm, Daicel
Chemical Ltd.) at rt for LC/MS and 40 °C for LC-CD. The flow rate
was 0.5 mL/min (detection: LC-MS 280 nm, LC-CD 236 nm).
Materials. The calli of M. cordata, C. ochotensis var. raddeana,
and N. domestica were derived in 1974, 1981, and 2003 from 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 4 or 5 weeks on the same fresh medium at 25 °C in
the dark. 3,4-Dimethoxyphenylethylamine, 3-hydroxy-4-methoxyphe-
nylacetic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid,
DCDO (99 atom % D), CD3I (99.5 atom % D), and H13CHO (99 atom
Preparation of (()-[8-13C]-Spinosine [(()-[8-13C]-7]. A mixture
of 3,4-dimethoxyphenethylamine (1.4 g) and 3,4-dihydroxyphenylacetic
acid (1.45 g) was heated in an oil-bath at 180-190 °C for 30 min. The
product was dissolved in anhydrous EtOH (30 mL), and anhydrous
K2CO3 (4.0 g) and benzyl chloride (4.0 g) were added. The mixture
was refluxed for 2.5 h and filtered using Celite 545. After cooling, the
resulting crystals were collected to give the di-O-benzyl derivative (2.49
g, yield 63%, mp 125-126 °C). This product (3.88 g) was dissolved
in anhydrous C6H5CH3 (100 mL), and C6H6 (50 mL) and POCl3 (8
mL) were added. The mixture was refluxed for 30 min, solvent removed,
and the residue washed with Et2O to give the 3,4-dihydroisoquinoline,
which was dissolved in Me2CO-MeOH and dilute HCl (0.5 mL).
Solvent was evaporated, and the residue was crystallized from Me2CO
to give the hydrochloride (2.5 g). The hydrochloride (12.4 g) was
dissolved in MeOH (70 mL), and NaBH4 (2 g) was added in small
portions with stirring at rt. The mixture was then refluxed for 25 min,
and H2O (100 mL) was added and concentrated. The mixture was
extracted with CHCl3; then the extract was dried and evaporated. The
residue was dissolved in EtOH, and concentrated HCl (10 mL) was
added and refluxed for 2 h. Solvent was removed and the residue
crystallized from Me2CO to give the hydrochloride of 3 (1.1 g). A
solution of this hydrochloride (250 mg) in H2O (30 mL) was adjusted
to pH 3 with dilute HCl, and aqueous 37% H13CHO (100 mg) was
added. After standing overnight at rt, the resulting white powder was
%
13C) were obtained from commercial suppliers. (S)-Coreximine and
(S)-isocoreximine were obtained from Monathotaxis fornicata (An-
nonaceae).
LC-APCI-MS, LC-NMR, and LC-CD Methods. LC-APCI-MS
(/MS), LC-NMR, and LC-CD were measured using procedures
described previously.11
Preparation of (()-[8-13C]-Corytenchine [(()-[8-13C]-5] and (()-
[8-13C]-Tetrahydropalmatrubine [(()-[8-13C]-4]. A mixture of 3,4-
dimethoxyphenethylamine (1.0 g) and 3-hydroxy-4-methoxypheny-
lacetic acid (1.0 g) was heated in an oil-bath at 180-190 °C for 1 h.
Crystallization of the product from CHCl3-C6H6 afforded the amide.
To a solution of the phenolic amide and triethylamine (1 mL) in
anhydrous CHCl3 (50 mL) was added dropwise ethyl chloroformate (2
mL) in C6H6 (10 mL) with stirring at 5-10 °C. Stirring was continued
for 30 min at rt, solvent was removed, and the residue was dissolved
in CHCl3. The CHCl3 layer was washed with NaHCO3 solution, dried
over Na2SO4, and evaporated to give the O-protected amide. A mixture
of the amide, POCl3 (2 mL), and dry benzene (20 mL) was refluxed
for 2.5 h. The solvent was removed, the residue was washed with Et2O
and dissolved in MeOH (70 mL), and NaBH4 (700 mg) was then added
in small portions with stirring at rt. The mixture was refluxed for 4.5 h,
and H2O (30 mL) was added and concentrated. The mixture was
extracted with CHCl3, the CHCl3 extract dried, and the residue dissolved
1
filtered to give (()-[8-13C]-7 (128 mg). (()-[8-13C]-7: H NMR (500
MHz, CD3OD) δ 6.94 (1H, s, H-1), 6.82 (1H, s, H-4), 6.72 (1H, s,
H-12), 6.62 (1H, d, J ) 5.0 Hz, H-9, 13C-H long-range coupling),
4.64 (1H, d, J ) 10 Hz), 4.40 (2H, d, J ) 143 Hz, H-8, 13C-H
coupling), 3.86 (3H, s, 2-OMe), 3.84 (3H, s, 3-OMe), 3.74 and 3.43
(each 1H, m, H-6), 3.69 and 2.98 (each 1H, m, H-13), 3.24 and 3.04
(each 1H, m, H-5); 13C NMR (125 MHz, CD3OD) δ 26.86 (C-5), 34.20
(C-13), 51.40 (C-6, br), 56.51 (C-2 and 3-OMe], 56.76 (13C-8), 61.53

122 Journal of Natural Products, 2010, Vol. 73, No. 2
Iwasa et al.
(C-13a, br), 110.05 (C-1), 112.82 (C-4), 113.49 (C-9, d, J ) 5.0 Hz),
116.10 (C-12, d, J ) 2.75 Hz), 119.70 (C-12a), 123.47 (C-8a), 124.82
(C-4a), 125.84 (C-1a), 146.24 (C-10, d J ) 5.63 Hz), 147.02 (C-11),
150.14 (C-2), 150.62 (C-3); SIMS m/z 329 [M + H]+ (100), 192 (15);
HR-SIMS m/z 329.1598 (calcd for C1813CH22NO4, 329.1582).
Preparation of (()-[8-D2]-Spinosine [(()-[8-D2]-7]. A solution of
the hydrochloride of 3 (200 mg) in D2O (20 mL) was adjusted to pH
3 with dilute 20% DCl (11 µL), and aqueous 37% DCDO (100 mg)
was added. After standing overnight at rt, the resulting white powder
was filtered to give (()-[8-D2]-7 (133 mg, mp 212-222 °C). (()-[8-
D2]-7: 1H NMR data were identical to those of the unlabeled compound,
except for disappearance of H-8. SIMS m/z 330 [M + H]+ (100), 192
(45); HR-SIMS m/z 330.1668 (calcd for C19H20D2NO4, 330.1673).
Resolutions of (R)-and (S)-[8-13C]-Corytenchine [(R)- and (S)-
[8-13C]-5] and (R)- and (S)-[8-13C]-Tetrahydropalmatrubine [(R)-
and (S)-[8-13C]-4]. Chiral separation of (()-[8-13C]-5 (50 mg) was
performed using a chiral OJ-RH reversed-phase column (6.0 i.d. ×
150 mm), a Jasco 880-PU intelligent pump, and a Jasco 875-UV
detector. Solvents (A) 0.1 M NH4OAc (0.05% TFA) and (B) MeCN
(0.05% TFA) were used for nonlinear gradient: initial 20% of B, 10
min 40% of B, and 20 min 40% of B; 6 mL/min (detection: 280 nm).
Eluents obtained from the peaks corresponding to (R)-5 (tR ) 14.5 min)
and (S)-5 (tR ) 15.1 min) were evaporated, and the residues were further
purified by HPLC [H2O (0.05% TFA)-MeOH (0.05% TFA)] to give
(R)-5 (5.5 mg) and (S)-5 (2.0 mg) as trifluoroacetates.
Supporting Information Available: Scheme 1S and Figures S1-S6.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
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Chiral separation of (()-[8-13C]-4 (30 mg) was undertaken in the
same manner. Eluents obtained from the peaks corresponding to (R)-4
(tR ) 19.3 min) and (S)-4 (tR ) 22.5 min) were evaporated and the
residue further purified by HPLC [H2O (0.05% TFA)-MeOH
(0.05%TFA)] to give (R)-4 (7.9 mg) and (S)-4 (12 mg) as trifluoro-
acetates. Optical purity of each enantiomer was demonstrated by LC-
CD.10
Feeding Experiments. Substrates were dissolved in H2O (2-4 mL)
and introduced through a sterile bacterial filter into 100 mL conical
flasks to which 40 mL of autoclaved MS medium (as that employed in
the subculture) was added. Calli (ca. 4-5 g) were transferred to each
conical flask and incubated at 25 °C (C. ochotensis and M. cordata)
and 27 °C (N. domestica) in the dark for four weeks. After incubation,
calli and medium were freeze-dried. Water was separated, and callus
and medium were extracted with hot MeOH. The H2O and MeOH
extracts were concentrated and, after acidification, were washed with
Et2O, made basic with NH4OH, and extracted with Et2O and then CHCl3
to give fractions E-1 and C-1 (callus) and fractions E-2 and C-2
(medium), which were subjected to LC-NMR, LC-MS, and LC-CD
measurements.
NP900440D