Bioactive isoquinoline alkaloids from Corydalis saxicola

Article abstract of DOI:10.1055/s-0031-1280126

Twelve isoquinoline alkaloids including two new nitro-containing tetrahydroprotoberberines, (-)-2,9-dihydroxyl-3,11-dimethoxy-1,10- dinitrotetrahydroprotoberberine (1) and (+)-4-nitroisoapocavidine (2), were isolated from the whole plant of Corydalis saxicola Bunting. The structures of the new compounds were established by spectroscopic analysis and chemical evidence. The inhibitory activity of these isolates against cholinesterase and canine parvovirus were evaluated. Compounds 1 and 1a, (+)-1-nitroapocavidine (5), berberine (8), palmatine (9), dehydrocavidine (10), and sanguinarine (11) showed potent inhibitory activity against acetylcholinesterase with IC ₅₀ values of less than 10M, while only compound 1 possessed weak activity against canine parvovirus. Structure-activity studies demonstrated that the nitro substituents at ring A in the tetrahydroprotoberberines led to an increase in the anti-acetylcholinesterase activity.

Full text of DOI:10.1055/s-0031-1280126

Original Papers 65
Bioactive Isoquinoline Alkaloids
from Corydalis saxicola
Authors
Qiao-Qin Huang1,2, Jun-Long Bi3, Qian-Yun Sun4, Fu-Mei Yang4, Yue-Hu Wang1, Gui-Hua Tang1, Fu-Wei Zhao1,
Huan Wang1, Jin-Jin Xu1, Edward J. Kennelly5, Chun-Lin Long1,5, Ge-Fen Yin3
Affiliations
The affiliations are listed at the end of the article
Key words
Abstract
berberine (8), palmatine (9), dehydrocavidine
(10), and sanguinarine (11) showed potent inhib-
itory activity against acetylcholinesterase with
IC50 values of less than 10 µM, while only com-
pound 1 possessed weak activity against canine
parvovirus. Structure-activity studies demon-
strated that the nitro substituents at ring A in the
tetrahydroprotoberberines led to an increase in
the anti-acetylcholinesterase activity.
"
l Corydalis saxicola Bunting
!
"
l Fumariaceae
Twelve isoquinoline alkaloids including two new
nitro-containing tetrahydroprotoberberines, (−)-
2,9-dihydroxyl-3,11-dimethoxy-1,10-dinitrote-
trahydroprotoberberine (1) and (+)-4-nitroiso-
apocavidine (2), were isolated from the whole
plant of Corydalis saxicola Bunting. The structures
of the new compounds were established by spec-
troscopic analysis and chemical evidence. The in-
hibitory activity of these isolates against cholines-
terase and canine parvovirus were evaluated.
Compounds 1 and 1a, (+)-1-nitroapocavidine (5),
"
l isoquinoline alkaloids
"
l acetylcholinesterase
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
!
Materials and Methods
!
Corydalis saxicola Bunting (Fumariaceae) is a pe-
rennial herb distributed mainly in southwest Chi-
na [1]. As a traditional Chinese folk medicine, it is
used for the treatment of hepatitis, diarrhea,
stomachaches, and bleeding hemorrhoids [2].
The plant is rich in isoquinoline alkaloids with
General experimental procedures
received
revised
accepted
June 3, 2011
July 2, 2011
July 5, 2011
Melting points were determined using an X-4
melting point apparatus (Yingyu Yuhua Appara-
tus Factory) and were not corrected. Optical rota-
tions were determined on a Horiba SEPA-300 po-
larimeter. UV spectra were recorded on a Shimad-
zu double-beam 210A spectrometer. CD spectra
were measured in a Chirascan circular dichorism
spectrometer. IR spectra were recorded on a Bio-
Rad FTS-135 infrared spectrophotometer with
KBr disks. 1D and 2D NMR spectra were recorded
on Bruker AM-400 and DRX-500 spectrometers
with TMS as an internal standard. ESIMS and
HRESI analyses were carried out on an API Qstar
Pulsar 1 instrument. EIMS and HREIMS were car-
ried out on a Waters Autospec Premier P776 mass
spectrometer. Column chromatography was per-
formed over silica gel G (80–100, 200–300, and
300–400 mesh), Sephadex LH-20 (40–70 µm;
Amersham Pharmacia Biotech AB), HPLC (Agilent
1200, chromatography column: Zorbax SB‑C18, ϕ
9.4 × 250 mm, 5 µm), C-18 silica gel (40–75 µm;
Fuji Silysia Chemical Ltd.), and MCI gel CHP 20P
(polystyrene type, 75–150 µm; Mitsubishi Chem-
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1280126
Published online August 19,
2011
Planta Med 2012; 78: 65–70
© Georg Thieme Verlag KG
Stuttgart · New York ·
ISSN 0032‑0943
activities of anti-hepatitis
B virus and DNA
topoisomerase I inhibition [3–7]. In our con-
tinuing study to find bioactive alkaloids from
medicinal plants [8–11], the chemical constitu-
ents of C. saxicola were investigated, which led
to the isolation of 12 isoquinoline alkaloids in-
cluding two new nitrotetrahydroprotoberber-
ines: 2,9-dihydroxy-3,11-dimethoxy-1,10-dini-
trotetrahydroprotoberberine (1) and 4-nitroiso-
apocavidine (2). Isoquinoline alkaloids from
Corydalis were known for their antivirus and
Correspondence
Prof. Dr. Chun-Lin Long
Kunming Institute of Botany
Chinese Academy of Sciences
132# Lanhei Road, Heilongtan
Kunming 650201
P.R. China
anti-acetylcholinesterase
activities
[6,12].
Phone: + 861068930381
Fax: + 861068930381
long@mail.kib.ac.cn
Therefore, all of these isolates were evaluated for
the activity against canine parvovirus (CPV), ace-
tylcholinesterase (AChE), and butyrylcholinester-
ase (BuChE). The structural elucidation of the
new compounds and bioassay results are re-
ported.
Correspondence
Dr. Ge-Fen Yin
Yunnan Agricultural University
Kunming 650201
P.R. China
yingefen383@sohu.com
Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

66 Original Papers
ical Corporation). TLC was conducted on precoated silica gel
plates GF 254 (Qingdao).
C143 (60 mg) was purified by preparative TCL (CHCl3-MeOH,
1:1) to gain 3 (27 mg) and 6 (20 mg). The purity of compounds
1–12 was greater than 95% as determined by TLC and NMR.
(−)-2,9-Dihydroxy-3,11-dimethoxy-1,10-dinitrotetrahydroproto-
berberine (1): yellow amorphous powder (MeOH); [α]1D4 − 586.3(c
0.17, MeOH); UV (MeOH) λmax (log ε) 342 (3.82), 274 (3.73) nm;
CD Δε (c 0.0020, MeOH) − 0.60 (240), − 1.52 (207); IR (KBr) νmax
Plant material
The whole plants of C. saxicola were collected from Jingxi County,
Guangxi Zhuang Autonomous Region in June 2007. The plant was
identified by Professor Chun-Lin Long (Kunming Institute of Bot-
any, Chinese Academy of Sciences), and a voucher specimen
(No. JX0701) was deposited at the Key Laboratory of Economic
Plants and Biotechnology, Kunming Institute of Botany.
13
3432, 1629, 1517, 1327 cm−1
;
1H and C NMR, see l Table 1;
"
ESIMS m/z 418 [M + H]+; HRESIMS m/z 418.1251 [M + H]+ (calcd.
for C19H20N3O8, 418.1250).
Preparation of 1a: K2CO3 (10.0 mg), Me2CO (10 mL), and Me2SO4
(0.5 mL) were added to a solution of 1 (8.0 mg). The mixture was
stirred for 12 h at 50°C. Then, 25% NH3 ·H2O (5 mL) was added
and evaporated in vacuo. The residue was partitioned between
H2O (10 mL) and CHCl3 (3 × 10 mL). The CHCl3 layer was concen-
trated and purified by Sephadex LH-20 column chromatography
(MeOH) to afford 1a (7.9 mg): pink amorphous powder (MeOH);
Extraction and isolation
The whole plants of C. saxicola (1.5 kg) were extracted three
times with MeOH (3 × 5 L) under reflux. The solvent was evapo-
rated under reduced pressure to give a residue (379 g). The ex-
tract was separated by silica gel column chromatography
(80 × 10 cm, No. 1007388, 80–100 mesh; Qingdao), eluted with
CHCl3–MeOH (30:1, 20:1, 10:1, 5:1, 2:1, and 1:1). Fractions
were combined based on TLC analysis, and 6 fractions (A–F) were
obtained. Fraction B (38 g) was subjected to C-18 reversed-phase
(6 × 50 cm, LiChroprep RP-18; Merck) eluted with MeOH–H2O
(gradient from 1:20 to 95% MeOH) to afford 7 fractions (B1–B7).
Fraction B3 (5.4 g) was separated on silica gel CC (50 × 4 cm,
No. 0803136, 200–300 mesh; Qingdao) eluted with CHCl3-MeOH
(20:1 to 0:1) to afford 6 fractions (B31–B36). The fraction B36
(468 mg) was applied to Sephadex LH-20 (200 cm, Sephadex LH-
20) with MeOH and further purified by column chromatography
(40 × 2 cm, No. 0100349, 300–400 mesh; Qingdao) eluted with
CHCl3: MeOH (5:1) to gain 1 (12.0 mg) and 10 (110 mg). The frac-
tion B33 (676 mg) was purified on silica gel CC (40 × 2 cm,
No. 0100349, 300–400 mesh; Qingdao) eluted with petroleum
ether-Me2CO (10:1 to 0:1) to afford 5 fractions (B331–B335), then
the fraction B333 (50 mg) was purified by preparative TCL (petro-
leum ether-Me2CO, 2:1) to gain 11 (5.0 mg) and 12 (6.0 mg).
The fraction B2 (400 mg) was separated by Sephadex LH-20
[α]2D3 − 112.1 (c 0.19, MeOH); CD (c 0.0020, MeOH) Δε·–0.60
13
(240), − 1.52 (207); 1H and C NMR, see l Table 1; ESIMS m/z
"
460 [M]+.
(+)-4-Nitroisoapocavidine (2): yellow powder (CHCl3); m.p. 290–
293°C; [α]2D5 + 82.7 (c 0.20, CHCl3); UV (CHCl3) λmax (log ε) 424
(3.05), 343 (3.49), 279 (3.78), 240 (3.93) nm; CD Δε (c 0.0021,
MeOH) + 0.23 (207); IR (KBr) νmax 3430, 1628, 1534, 1510, 1467,
1358, 1292 cm−1; 1H and 13C NMR, see l Table 1; ESIMS m/z 385
"
[M + H]+; EIMS m/z 384 [M]+ (45), 369 (8), 354 (7), 223 (5), 162
(100); HREIMS m/z 384.1322 [M]+ (calcd. for C20H20N2O6,
384.1321).
General procedure for preparation of hydrochlorides
A drop of concentrated hydrochloric acid was added to a solution
of the alkaloids (1.0 mg) in MeOH (0.5 mL). The mixture was
evaporated under reduced pressure to yield the hydrochlorides
of compounds 1–7.
(200 cm, Sephadex LH-20) with MeOH to get 5 fractions (B21
–
Bioassays
B25). The fraction B23 (110 mg) was subjected to column chroma-
tography (40 × 2 cm, No. 0100349, 300–400 mesh; Qingdao)
eluted with CHCl3-MeOH-diethylamine (40:2:1) to get 3 frac-
tions (B231–B234), and the fraction B232 (58 mg) was chromato-
graphed on a Sephadex LH-20 column (200 cm, Sephadex LH-
20) to obtain 8 (15 mg) and 9 (17 mg).
The fraction C (4.0 g), which was subjected to column chromatog-
raphy over MCI gel CHP 20P (80 × 10 cm, polystyrene type, 75–
150 µm) was eluted with MeOH‑H2O (4:1 to 100% MeOH) to ob-
tain 3 fractions (C1–C3). The fraction C1 (3.2 g) was flashed on C18
silica gel (4 × 50 cm, LiChroprep RP-18; Merck) with MeOH‑H2O
(gradient from 1:20 to 95% MeOH) to get 6 fractions (C11–C16).
The fraction C15 (500 mg) was separated on silica gel CC
(40 × 2 cm, No. 0100349, 300–400 mesh; Qingdao) eluted with
Microplate assay for AChE and BuChE: The AChE inhibitory activ-
ities of samples were measured quantitatively by the improved
Ellmanʼs method with tacrine (Sigma, purity 98%) as the positive
control (IC50 = 0.19 µM) [13,14]. Inhibitory activity of compounds
against BuChE was performed by the improved Ellmanʼs method
using tetraisopropylpyrophosphoramide (Sigma, purity 98%) as
the positive control (IC50 = 1.35 µM) [13,15].
Cell culture and cytotoxicity assays: The embryonic feline kidney
F81 cell line (Cell Bank of the Chinese Academy of Sciences,
Shanghai) was used for growing canine parvovirus, which was
isolated by the authors in Yunnan Agricultural University. The
cells were grown in monolayer in a 5% carbon dioxide and 95%
atmosphere at 37°C, with minimal essential medium (MEM; Hy-
Clone) containing 10% fetal bovine serum (HyClone). Cytotoxicity
assays were performed by the WST-8 method [16,17], using Cell
Counting Kit-8 (CCK-8; Dojindo) according to the supplier recom-
mendations. Briefly, cells were incubated in a 96-well microcul-
ture plate (Corning) in the absence or presence of twofold serial
dilutions of compounds 1–12 and ribavirin (Sigma). After 4 days
of culture, 10 µL of CCK-8 solution was added, and the cells were
incubated for 1 hour. The number of surviving cells was mea-
sured with a Bio-Tek ELx 800.
CHCl3-ethyl acetate (1:0 to 0:1) to obtain 5 fractions (C151
–
C
155). The fraction C152 (106 mg) was purified by preparative TCL
(petroleum ether-ethyl acetate, 2:1) to obtain a mixture and 5
(31 mg). The mixture was then submitted to Sephadex LH-20
(200 cm, Sephadex LH-20) with MeOH and preparative TCL (pe-
troleum ether-ethyl acetate, 2:1) to gain 2 (11 mg). The fraction
C151 (86 mg) was subjected to preparative TCL (petroleum ether-
ethyl acetate, 2:1) to gain 7 (50 mg).
The fraction C14 (400 mg) was then submitted to Sephadex LH-20
(200 cm, Sephadex LH-20) with MeOH to obtain 4 fractions
(C141–C144); then the fraction C142 (53 mg) was purified by pre-
parative TCL (CHCl3-MeOH, 6:1) to get 4 (15 mg). The fraction
ELISA microplate reader could show the detection wavelength of
450 nm (L1) and the reference wavelength of 650 nm (L2). The
50% cytotoxic concentration (CC50) was obtained by nonlinear
Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

Original Papers 67
Table 1 1HNMR data for compounds 1, 1a, and 2 (δ in ppm, J in Hz).
Position
1
1a
2
a
b
c
d
e
f
δH
δC
δH
δC
δH
δC
1
140.0 (qC)
139.7 (qC)
149.0 (qC)
114.4 (CH)
127.2 (qC)
29.8 (CH2)
144.9 (qC)
140.7 (qC)
154.8 (qC)
116.9 (CH)
127.8 (qC)
23.9 (CH2)
6.77 (1H, s)
110.0 (CH)
148.8 (qC)
142.0 (qC)
137.5 (qC)
119.1 (qC)
22.3 (CH2)
2
3
4
6.93 (1H, s)
7.45 (1H, s)
3.60 (2H, m)
4.10 (2H, m)
4a
5
3.01 (1H, m)
3.89 (1H, m)
2.85 (1H, m)
3.95 (1H, m)
3.14 (1H, m)
2.88 (1H, ddd, 16.3, 5.3, 5.3)
3.13 (1H, m)
6
8
48.2 (CH2)
53.8 (CH2)
51.0 (CH2)
61.0 (CH2)
51.2 (CH2)
2.69 (1H, ddd, 11.4, 7.5, 5.3)
4.16 (1H, d, 16.5)
5.42 (1H, d, 16.4)
5.15 (1H, d, 16.4)
4.90 (1H, d, 15.5)
4.02 (1H, d, 15.5)
52.0 (CH2)
3.78 (1H, d, 16.5)
8a
9
122.9 (qC)
150.3 (qC)
140.4 (qC)
146.2 (qC)
108.1 (CH)
126.2 (qC)
32.2 (CH2)
119.8 (qC)
150.9 (qC)
143.2 (qC)
151.6 (qC)
111.1 (CH)
122.9 (qC)
33.1 (CH2)
108.4 (qC)
143.7 (qC)
146.4 (qC)
109.0 (CH)
121.6 (CH)
130.2 (qC)
36.1 (CH)
10
11
12
12a
13
6.76 (1H, d, 8.0)
6.70 (1H, d, 8.0)
7.67 (1H, s)
7.90 (1H, s)
3.19 (1H, dd, 18.0, 3.6)
2.98 (1H, dd, 18.0, 11.4)
3.90 (1H, dd, 11.4, 3.6)
4.00 (1H, m)
3.64 (1H, m)
4.66 (1H, br s)
3.89 (3H, s)
3.56 (1H, m)
14
55.4 (CH)
5.01 (1H, dd, 11.3, 5.6)
61.3 (CH)
116.0 (qC)
62.5 (CH3)
57.2 (CH3)
50.9 (CH3)
61.7 (CH3)
56.9 (CH3)
63.6 (CH)
120.9 (qC)
56.8 (CH3)
14a
122.3 (qC)
2-OMe
3.94 (3H, s)
4.01 (3H, s)
3.52 (3H, s)
4.04 (3H, s)
4.02 (3H, s)
3-OMe 3.92 (3H, s)
7-NMe
56.8 (CH3)g
9-OMe
11-OMe 3.93 (3H, s)
13-Me
57.0 (CH3)g
1.28 (3H, d, 6.8)
5.97 (1H, d, 1.5)
5.93 (1H, d, 1.5)
17.9 (CH3)
OCH2O
102.1 (CH2)
a Measured in CD3OD-DMSO-d6 (6:1) at 500 MHz. b In CD3OD-DMSO-d6 (6:1) at 100 MHz. c In acetone-d6 at 500 MHz. d In acetone-d6 at 100 MHz. e In CDCl3-CD3OD (6:1) at
400 MHz. f In CDCl3-CD3OD (6:1) at 100 MHz. g Data under the same entry are interchangeable
regression analysis of logistic curves (the value of L1 – L2 to differ-
ent concentrations of compounds).
stituted phenyl rings [δH 7.67 (1H, s, H-12), 6.93 (1H, s, H-4); δC
150.3 (qC), 149.0 (qC), 146.2 (qC), 140.4 (qC), 140.0 (qC), 139.7
(qC), 127.2 (qC), 126.2 (qC), 122.9 (qC), 122.3 (qC), 114.4 (CH),
Protection assay for CPV-infected F81 cells: By the determination
of cell viability, the ability of the compounds to protect CPV-in-
fected F81 cells from death was evaluated [18,19] using the
WST-8 method as described above. Tissue culture medium infec-
tive dose (TCID50) of 300 viral particles with twofold serial dilu-
tions of the compounds were added to each test well, and the
plates were reincubated for 4 days to allow development of a cy-
topathologic effect (CPE) if any. The number of surviving cells was
measured. The 50% effective concentration (EC50) and therapeu-
tic index (TI) were calculated. Ribavirin (Sigma, purity 99.5%) was
1
108.1 (CH)]. The H NMR spectrum also showed signals for two
aryl methoxy groups, three aliphatic protons (H2-13 and H-14)
as an AMX system, and two aliphatic protons of the same methy-
lene (H2-8) as an AX system, together indicating that 1 may be a
tetrahydroprotoberberine [20].
1
1
"
Using the H– H COSY and HMBC results (l Fig. 2), the carbon
skeleton of tetrahydroprotoberberine 1 was established. The RO-
ESY spectrum showed the correlations of H-4/3-OMe and H-12/
11-OMe, helping to establish that the two methoxy groups were
located at C-3 and C-11, respectively.
"
used as a positive control (EC50 = 121.72 µM; TI = 10.5) (l Fig. 1).
The remaining four substituent groups in the two benzene rings
were deduced as two hydroxy groups and two nitro groups ac-
cording to the molecular formula of 1. In order to determine their
locations, a derivative (1a) of 1 was prepared.
Supporting information
The optical rotation values and CD data of compounds 1–7 as well
as 1D NMR and 2D NMR spectra of compounds 1, 1a, and 2 are
available as Supporting Information.
The ESI‑MS afforded the positive ion of 1a at m/z 460 [M]+, which
implied that three more O- or N-methyl groups were linked to 1
1
"
through the reaction. The H‑NMR spectrum (l Table 1) of 1a in-
dicated four O-methyl groups (δH 3.94, 4.01, 4.02, and 4.04) and
one N-methyl group (δH 3.52). The 3-OMe and 11-OMe groups
Results and Discussion
!
"
The molecular formula C19H19N3O8 of 1 was determined from the
HRESIMS ([M + H]+ 418.1251, calcd. 418.1250). The IR spectrum
of 1 indicated the presence of hydroxy (3432 cm−1), aromatic
(1629 cm−1), and nitro (1327 and 1517 cm−1) functional groups.
were located on 1a by the ROESY correlations (l Fig. 2) of H-4/
3-OMe and H-12/11-OMe, respectively, while 2-OMe and 9-
OMe groups were determined by the HMBC correlations
"
(l Fig. 2) from H-4 and 2-OMe to C-2, and H2-8 and 9-OMe to C-
"
NMR data of 1 (l Table 1) showed the presence of two pentasub-
9, respectively. Thus, the remaining two nitro groups must be
Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

68 Original Papers
Fig. 1 Structures of compounds 1–7.
Fig. 2 Key 2D NMR correlations of 1, 1a, and 2.
linked to C-1 and C-10, respectively. Therefore, the planar struc-
ture of 1 was elucidated as 2,9-dihydroxy-3,11-dimethoxy-1,10-
dinitrotetrahydroprotoberberine.
pound 2 is very similar to the two known alkaloids except for
the substitution pattern in ring A. The presence of ROESY correla-
"
tions (l Fig. 2) between H-1 and 2-OMe implied that the nitro
The absolute configuration of 1 was determined as 14S according
to the strong negative Cotton effect at 207 nm (Δε·–1.52) in the
CD spectrum of 1 hydrochloride [21].
group should be located at C-4 in 2 rather than at C-1 in 5. In the
13-methyl tetrahydroprotoberberines with a cis configuration
between H-13 and H-14, the carbon chemical shift of 13-Me is
about 18 ppm rather than 22 ppm in those with a trans configu-
ration [22,24,25]. Therefore, the relative configuration of 2 was
confirmed as 4-nitroisoapocavidine. The absolute configuration
of 2 was deduced as 13S, 14R by comparison of its CD spectrum
with that of known (+)-cavidine (6) (see Supporting Information)
[26,27].
Ringdahl et al. have studied the CD spectra of a series of 14S-tet-
rahydroprotoberberines and suggested that the intense 206–
210 nm negative Cotton effects correlate directly with the abso-
lute configuration of 14S [21]. However, 14R-tetrahydroprotober-
Compound 2 was obtained as yellow powder, which has the mo-
lecular formula of C20H20N2O6 according to its HREIMS at m/z
384.1322 [M]+ (calcd. 384.1321). The fragments with m/z 223
(5%) and 162 (100%) in the EIMS suggested the substitution pat-
tern of the 13-methyl-9,10-methylenedioxy moiety at rings C
and D [3]. The 1H‑NMR spectrum displayed one O-methyl group
at δ 3.89 (s), one methyl group at δ 1.28 (d), and one methylene-
dioxy group at δ 5.97 (d) and 5.93 (d), respectively. Comparison of
NMR data of compound 2 with those of known (+)-1-nitroapoca-
vidine (5) [3] and isoapocavidine [22,23] revealed that com-
Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

Original Papers 69
Conflict of Interest
!
Table 2 AChE inhibitory activity of alkaloids from Corydalis saxicola.
Compound
IC50 (µM)
There are no conflicts of interest among the authors.
1
8.77 0.20
3.34 0.69
53.30 0.50
43.20 1.20
64.40 1.60
1.70 0.31
1a
Affiliations
1
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of
2
Botany, Chinese Academy of Sciences, Kunming, Peopleʼs Republic of China
College of Landscape and Horticulture, Yunnan Agricultural University,
3
2
4
Kunming, Peopleʼs Republic of China
College of Animal Science and Technology, Yunnan Agricultural University,
5
3
6
> 200
Kunming, Peopleʼs Republic of China
Key Laboratory of Chemistry for Natural Products, Guizhou Province and
Chinese Academy of Sciences, Guiyang, Peopleʼs Republic of China
College of Life and Environmental Sciences, Minzu University of China,
4
7
14.50 0.20
1.87 0.48
2.20 0.46
9.92 0.23
1.93 0.01
58.70 1.34
0.19 0.02
8
5
9
Beijing, Peopleʼs Republic of China
10
11
References
12
1 Flora of Guangxi. Nanning: Guangxi Science and Technology Press;
1993: 410
Tacrine
2 Zhonghua Bencao, Vol. 3. Shanghai: Shanghai Science and Technology
Press; 1999: 638–640
3 Li HL, Zhang WD, Zhang W, Zhan C, Liu RH. A new nitro alkaloid from
Corydalis saxicola bunting. Chin Chem Lett 2005; 16: 367–368
4 Wu YR, Ma YB, Zhao YX, Yao SY, Zhou J, Zhou Y, Chen JJ. Two new quater-
nary alkaloids and anti-hepatitis B virus active constituents from
Corydalis saxicol. Planta Med 2007; 73: 787–791
5 Li HL, Zhang WD, Han T, Zhang C, Liu RH, Chen HS. Tetrahydroprotober-
berine alkaloids from Corydalis saxicola. Chem Nat Comp 2007; 43:
173–175
berines were not reported in the literature. We found that 14R
compounds (2 and 5–7) showed positive Cotton effects at 206–
210 nm, whose intensity was changed according to the configu-
ration of C-13. In the case of the cis configuration between H-13
and H-14 (2, 5, and 6), the Cotton effects are much weaker than
that in the trans one (7) (see Supporting Information).
To the best of our knowledge, compounds 1 and 2 are among the
very few examples of the natural tetrahydroprotoberberine alka-
loids with a nitro group.
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The known alkaloids (−)-tetrahydropalmatine (3) [21,28],
(−)-corynoxidine (4) [29], (+)-1-nitroapocavidine (5) [3], (+)-cav-
idine (6) [26,27], (+)-thalictrifoline (7) [26,27,30], berberine (8)
[31,32], palmatine (9) [33], dehydrocavidine (10) [34], sanguina-
rine (11) [31], and 8-acetonyldihydrochelerythrine (12) [35]
were determined by comparison of their spectral data with those
in the literature.
The inhibitory activity of all compounds against AChE, BuChE,
and canine parvovirus were evaluated. The results showed that
compounds 1 and 1a, (+)-1-nitroapocavidine (5), berberine (8),
palmatine (9), dehydrocavidine (10), and sanguinarine (11)
owned potent inhibitory activity against acetylcholinesterase
"
with IC50 values less than 10 µM (l Table 2). Structure-activity
relationships indicated that (i) all of tested berberines (8–10)
were active (IC50 < 10 µM). Other berberines such as jatrorrhizine,
dehydrocorydaline, pseudocolumbamine, coptisine, and pseudo-
palmatine possessed similar potency [20,36]. However, phenolic
hydroxy groups (dehydrocorydalmine and dehydrocoryten-
chine) could reduce the activity [20]; (ii) nitro substitutions at
ring A, especially at C-1, in the tetrahedroprotoberberines (2, 5,
and 6) could increase the activity.
All of these compounds were inactive against BuChE (IC50
> 200 µM), while compound 1 showed weak protection activity
of CPV-infected F81 cells from death (EC50 = 182.06 µM; SI = 2.2).
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A colorimetric assay for viral agents that produce cytopathic effects.
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21 Ringdahl B, Chan RPK, Craig JC, Manske RHF. Proterberine alkaloids: chi-
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Acknowledgements
!
This work was funded by the National Natural Science Founda-
tion of China (Grant Nos. 20972166 and 31070288), Yunnan Pro-
vincial Program for Excellent Scientists (No. 2009CI125), National
Basic Research Program of China (973 Program) (No.
2009CB526512), and Ministry of Education of China through its
985 and 111 projects (MUC 98506-01000101 and B08044).
Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

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Huang Q-Q et al. Bioactive Isoquinoline Alkaloids… Planta Med 2012; 78: 65–70

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