Steroidal alkaloids from the bulbs of Fritillaria unibracteata

Article abstract of DOI:10.1080/10286020.2011.619980

Two new steroidal alkaloids peimisine-3-O-β-D-glucopyranoside (1) and puqiedinone- 3-O-β-D-glucopyranoside (3), together with three known compounds peimisine (2), puqiedinone (4), and puqiedine (5), were isolated and characterized from the bulbs of Fritillaria unibracteata. Their structures were fully elucidated by spectroscopic and chemical methods. Compound 1 showed moderate protection effect on neurotoxicity of PC12 cell lines induced by rotenone.

Full text of DOI:10.1080/10286020.2011.619980

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Steroidal alkaloids from the bulbs of
Fritillaria unibracteata
Qing-Jian Zhang a , Zhong-Fei Zheng a & De-Quan Yu a
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College , Beijing,
100050, China
Published online: 25 Nov 2011.
To cite this article: Qing-Jian Zhang , Zhong-Fei Zheng & De-Quan Yu (2011) Steroidal alkaloids from
the bulbs of Fritillaria unibracteata , Journal of Asian Natural Products Research, 13:12, 1098-1103,
DOI: 10.1080/10286020.2011.619980
To link to this article: http://dx.doi.org/10.1080/10286020.2011.619980
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Journal of Asian Natural Products Research
Vol. 13, No. 12, December 2011, 1098–1103
Steroidal alkaloids from the bulbs of Fritillaria unibracteata
Qing-Jian Zhang, Zhong-Fei Zheng and De-Quan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
(Received 13 June 2011; final version received 31 August 2011)
Two new steroidal alkaloids peimisine-3-O-b-D-glucopyranoside (1) and puqiedinone-
3-O-b-D-glucopyranoside (3), together with three known compounds peimisine (2),
puqiedinone (4), and puqiedine (5), were isolated and characterized from the bulbs of
Fritillaria unibracteata. Their structures were fully elucidated by spectroscopic and
chemical methods. Compound 1 showed moderate protection effect on neurotoxicity of
PC12 cell lines induced by rotenone.
Keywords: Fritillaria unibracteata; steroidal alkaloid; peimisine-3-O-b-D-glucopyr-
anoside; puqiedinone-3-O-b-D-glucopyranoside
1. Introduction
these alkaloids and their protective
effects of PC12 cells.
Fritillaria unibracteata belongs to the
genus Fritillaria of the family Liliaceae,
whose bulbs, named ‘Chuan Bei Mu’, have
been used as an antitussive, antiasthmatic,
and expectorant agents in traditional
Chinese medicine for hundreds of years
and included in the Chinese Pharmaco-
poeias [1]. Previous investigation on the
pharmacology and phytochemistry of the
title plant suggested that the steroid
alkaloids were responsible for the anti-
tussive effects of these herbs [2]. In our
phytochemical investigation on bioactive
constituents in the bulbs of F. unibrac-
teata, two new steroidal alkaloids peimi-
sine-3-O-b-D-glucopyranoside (1) and
puqiedinone-3-O-b-D-glucopyranoside
(3), together with three known compounds
peimisine (2), puqiedinone (4), and puqie-
dine (5), were isolated and characterized
(Figure 1). This paper describes the
isolation and structural elucidation of
2. Results and discussion
Compound 1 was isolated as a white
20
powder with ½aꢀD 233.3 (c ¼ 0.19,
MeOH), showing a positive Dragendorff
reaction. The HR-ESI-MS exhibited a
quasi-molecular ion at m/z 590.3701
[M þ H]þ, corresponding to the molecular
formula C33H51NO8, with nine degrees of
unsaturation. The ESI-MS2 exhibited a
fragment ion at m/z 428.4 [M þ H-162]þ,
indicating the presence of a hexose
moiety in compound 1. The IR spectrum
showed absorption bands of hydroxyl
(3393 cm21), carbonyl (1704 cm21), the
CvC double bond (1649 cm21), and the
typical absorptions of a tetrahydrofuran
ring at 1121, 986, and 929 cm21 [3,4].
1
In the H NMR spectrum, the signals
due to two tertiary methyl groups at d 0.51
(3H, s, H-19) and 1.69 (3H, s, H-18), two
secondary methyl groups at d 0.82 (3H, d,
*Corresponding author. Email: dqyu@imm.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.619980
http://www.tandfonline.com

Journal of Asian Natural Products Research
1099
27
25
26
H
N
H
H
21
26
N
N
18
18
22
20
22
H
H
H
H
13
17
13
12
20
11
12
11
25
27
19
10
19
10
17
23
O
9 H
H
9 H
21
H
H
14
14
H
H
H
H
H
H
RO
RO
HO
3
3
6
6
4
4
H
H
H
O
OH
O
1 R = b-D-glc
2 R = H
3 R = b-D-glc
4 R = H
5
Figure 1. The structures of compounds 1–5.
J ¼ 6.3 Hz, H-27) and 1.09 (3H, d,
J ¼ 6.9 Hz, H-21), and two oxygenated
methine groups at d 3.93–4.08 (over-
lapped, H-3) and 3.40 (m, H-23) were
observed. The 13C NMR spectrum
(Table 1) revealed the presence of one
carbonyl group at d 209.7 (C-6); a double
bond moiety at d 128.9 (C-12) and 140.7
(C-13); three oxygenated carbons at d 76.9
(C-3), 85.1 (C-17), and 75.6 (C-23); two
nitrogenated carbons at d 66.9 (C-22) and
54.9 (C-26); and four methyls at d 13.4
(C-18), 12.3 (C-19), 11.2 (C-21), and 19.0
Table 1. The 13C NMR spectral data of
compounds 1–5.
No 1a
2b
3a
3b
4b
5b
1
2
3
4
5
6
7
8
9
39.4 39.0 36.8 37.0 37.0 39.1
31.9 31.6 28.8 28.7 30.5 31.3
76.9 70.7 76.9 75.6 70.9 71.9
37.1 37.0 27.0 26.4 30.2 34.8
56.3 56.6 56.6 56.6 56.8 48.3
209.7 210.6 210.0 211.6 211.2 72.8
45.9 45.7 45.9 45.9 46.0 38.5
46.5 46.0 40.2 40.2 40.2 34.7
54.4 54.4 56.5 56.5 56.8 57.6
10 38.4 38.4 38.4 38.6 38.4 35.4
11 27.0 28.5 30.0 29.7 30.1 29.3
12 128.9 128.4 40.2 41.0 41.2 40.2
13 140.7 141.0 40.7 41.0 41.0 44.6
14 48.7 48.5 44.3 44.2 44.4 44.0
15 24.7 24.1 25.0 25.2 25.3 24.7
16 29.2 29.9 24.4 24.4 24.5 25.5
17 85.1 85.0 45.9 46.1 46.4 46.5
18 13.4 13.1 63.0 64.3 64.7 61.5
19 12.3 12.4 12.6 12.9 12.8 15.1
20 40.2 39.5 45.9 44.2 44.8 41.3
21 11.2 10.6 14.7 14.8 14.8 14.9
22 66.9 66.1 68.3 68.5 68.4 68.4
23 75.6 75.5 33.0 33.2 33.4 30.2
24 28.7 30.3 29.2 28.6 28.8 33.5
25 31.3 31.6 30.0 30.4 30.8 30.8
26 54.9 54.5 63.0 60.9 61.2 64.7
27 19.0 18.8 19.3 19.5 19.6 19.7
1
(C-27). In the H and 13C NMR spectra,
the signals of a hexose moiety at dH 5.05
(1H, d, J ¼ 7.8 Hz, H-10), 3.93–4.08 (2H,
m, H-20,50), 4.21–4.32 (2H, m, H-30,40),
4.38–4.44 (1H, m, H-6b), and 4.62 (1H, br
d, J ¼ 11.2 Hz, H-6a), and at dC 102.2
(C-10), 75.4 (C-20), 78.6 (C-30), 71.8 (C-40),
78.6 (C-50), and 63.0 (C-60) were assign-
able to a b-glucopyranosyl moiety [5].
The spectral data of aglycon moiety
mentioned above were almost identical to
those of peimisine (2), except for the
obvious downfield shift of C-3 from dC
70.7 to dC 76.9 [3], suggesting the
structure of 1 was peimisine glucoside,
and the glucosidated site at C-3. In the
HMBC spectrum (Figure 2), the long-
range correlations of H-10/C-3 was
observed, which indicated that the glucose
was connected to C-3. After compound 1
was hydrolyzed with b-glucosidase [5],
10 102.2
102.2 101.0
75.4 73.5
78.6 78.0
71.8 70.0
78.6 76.4
63.0 61.9
20
30
40
50
60
75.4
78.6
71.8
78.6
63.0
a C5D5N.
b CDCl3.

1100
Q.-J. Zhang et al.
H
H
N
N
H
H
H
H
H
O
H
H
O
H
H
O
HO
HO
O
O
O
O
H
H
OH
OH
OH
OH
OH
OH
1
3
Figure 2. Key HMBC correlations of compounds 1 and 3.
both of peimisine and D-glucose were
detected. This further supported the above
result. Thus, the structure of 1 was
elucidated as peimisine-3-O-b-D-gluco-
pyranoside (Figure 1).
(C-21), and 19.3 (C-27), one carbonyl
group at d 210.0 (C-6), besides the six
carbon signals at dC 102.2, 75.4, 78.6,
71.8, 78.6, and 63.0 assignable to the b-
glucopyranosyl moiety. To compare the
carbon signals of aglycon with those of
known compounds, the 13C NMR spec-
trum of 3 was tested in the commonly used
solvent CDCl3, in which the signals of
aglycon were similar to those of puqiedi-
none (4), except for the obvious downfield
shift of C-3 from dC 70.9 to dC 75.6 [6].
The spectral data suggested that compound
3 was puqiedinone glucoside and the
glycosidated site at C-3. In the HMBC
spectrum, the long-range correlations of
H-10/C-3 and H-3/C-10 confirmed that the
glucose located at C-3 (Figure 2). After the
hydrolysis of compound 3 in the same way
as 1, D-glucose and puqiedinone were
detected. Thus, the structure of compound
3 was elucidated as puqiedinone-3-O-b-D-
glucopyranoside (Figure 1).
Compound 3 was obtained as a white
20
powder with ½aꢀD 241.6 (c ¼ 0.08,
MeOH) and showed a positive Dragendorff
reaction. The molecular formula
C33H53NO7 was assigned on the basis of
the quasi-molecular ion peak at m/z
576.3907 [M þ H]þ in its HR-ESI-MS.
The ion at m/z 414 [M 2 162 þ H]þ
indicated the presence of a hexose moiety.
The IR spectrum of 3 displayed the
absorption bands of hydroxyl (3398 cm21),
carbonyl (1704 cm21), and trans-quinolizi-
dine (2750 cm21) moieties [6].
The 1H NMR (500 MHz, C5D5N)
spectrum disclosed three methyl signals
at d 0.69 (3H, br s, H-19), 0.78 (6H, br d,
J ¼ 5.5 Hz, H-21, 27). An oxy-substituted
methine at d 3.94 (1H, m, H-3) with
W1/2 . 25 Hz indicated a b-configuration
of RO-3 [7]. The signals of a b-
glucopyranosyl moiety at d 5.02 (1H, d,
J ¼ 7.5 Hz, H-10), 4.02–4.10 (1H, m,
H-20), 4.20–4.30 (2H, m, H-30,40), 3.95–
4.01 (1H, m, H-50), 4.60 (1H, d,
J ¼ 12.0 Hz, H-60), and 4.40 (1H, dd,
J ¼ 6.0, 12.0 Hz, H-60) were observed. 13C
NMR (125 MHz, C5D5N) spectrum
(Table 1) disclosed the presence of three
methyl groups at dC 12.6 (C-19), 14.7
Compounds 2, 4, and 5 were deduced
to be peimisine (2) [3], puqiedinone (4)
[6], and puqiedine (5) [8] by comparison of
their spectral data with those reported in
the literature.
The effect of compounds 1–4 on
neurotoxicity of PC12 cell lines induced
by rotenone was evaluated (Table 2).
Compound 1 showed moderate protection
effect with cell viability rate 46.3 ^ 0.6%
( p , 0.01) at 10 mM.

Journal of Asian Natural Products Research
1101
Institute of Medicine Plant Development,
Chinese Academy of Medical Sciences and
Peking Union Medical College. A voucher
specimen (ID-S-2371) has been deposited
at the Herbarium of Institute of Materia
Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College.
Table 2. The effect of compounds 1–4 on
neurotoxicity of PC12 cell lines induced by
rotenone (means ^ SD, n ¼ 3).
Cell survival rate (%)
Control
Model
100.0 ^ 5.4
42.3 ^ 1.6###
1
2
3
4
46.3 ^ 0.6
40.8 ^ 2.6
45.0 ^ 2.7
39.9 ^ 1.8
**
3.3 Extraction and isolation
The dried plant materials (4.9 kg) were
powdered and refluxed with MeOH for
three times under reflux to give 230 g of an
extract, which was dissolved in 1% HCl.
After filtered, the acidic solution was
extracted with petroleum ether. Then, it
was basified with ammonia to pH . 11 and
extracted with chloroform to give the crude
total alkaloid 1.45 g, which was further
fractionated by column chromatography on
silica gel with petroleum ether–acetone–
Et3N (200:20:1 to 40:20:1) to yield 4
(15 mg) and seven fractions. Fraction 5
(27 mg) and fraction 6 (57 mg) were
subjected to a silica gel H column (f
1.5 £ 20 cm) and eluted with petroleum
ether–acetone–Et3N (30:10:1) to provide
compounds 5 (2 mg) and 2 (20 mg),
respectively. Fraction 7 (1.17 g) was iso-
lated with HPLC to yield 1 (10 mg, Rt
27.3 min) and 3 (6 mg, Rt 53.7 min). The
HPLC was carried out with YMC column
(C-18, 20 £ 250 mm), mobile phase aceto-
nitrile-0.2% Et3N/H2O (acetonitrile
increased from 10% to 100% in 60 min,
5 ml/min), and the Alltech 2000 ELSD
detector (3.1 l/min velocity of air flow and
1108C of the drift tube).
Note: ###p , 0.001 vs. control;
model.
p , 0.01 vs.
**
3. Experimental
3.1 General experimental procedures
Optical rotations were obtained on a
Perkin-Elmer 241 polarimeter (Perkin-
Elmer, Waltham, MA, USA). IR spectra
were recorded on a Nicolet IMPACT-400
spectrophotometer (Thermo Electron,
Madison, WI, USA) as KBr disks. Mass
spectra were obtained on the AutoSpec
Ultima-TOF instruments (Micromass,
Manchester, UK). NMR spectra were
recorded with Mercury-300 and INOVA-
500 spectrophotometer (Varian, Palo
Alto, CA, USA). Silica gel (200–300
mesh) for column chromatography and
silica gel GF254 for TLC were obtained
from Qingdao Marine Chemical Factory
(Qingdao, China). Size-exclusion chroma-
tography was carried out using Sephadex
LH-20 (Pharmacia, Uppsala, Sweden).
HPLC were carried out on a Lumtech KT-
501 chromatograph (Knauer, Berlin, Ger-
man) with YMC ODS-A C-18 column
(20 £ 250 mm, YMC, Kyoto, Japan) and an
Alltech 2000 ELSD detector (Alltech,
Deerfield, IL, USA). GC was conducted
on an Agilent 7890A instrument (Agilent,
Waldbronn, Germany).
3.3.1 Peimisine-3-O-b-D-glucoside (1)
20
White powder, ½aꢀD 233.3 (c ¼ 0.19,
MeOH), positive Dragendorff reaction. IR
(FT-IR microscope transmission) nmax
3393, 2925, 2856, 1704, 1649, 1121, 1077,
:
3.2 Plant material
1
The bulbs of F. unibracteata were collected
in July 2007 from the Ruo-Er-Gai County,
Sichuan Province, China, which were
identified by Prof. Shi-Lin Chen, the
1035, 986, 929 cm21. H NMR (300MHz,
C5D5N): d 0.51 (3H, s, H-19), 0.82 (3H, d,
J ¼ 6.3 Hz, H-27), 1.09 (3H, d, J ¼ 6.9 Hz,
H-21), 1.69 (3H, s, H-18), 3.93–4.08

1102
Q.-J. Zhang et al.
(overlapped, H-3), 3.40 (m, H-24) 5.05(1H,
d, J ¼ 7.8 Hz, H-10), 3.93–4.08 (2H, m, H-
20,50), 4.21–4.32 (2H, m, H-30,40), 4.38–
4.44 (1H, m, H-6b), 4.62 (1H, br d,
J ¼ 11.2 Hz, H-6a). 13C NMR spectral
data (125MHz, C5D5N), see Table 1. ESI-
MS: m/z 612.5 [M þ Na]þ, 590.5
[M þ H]þ. ESI-MS2: m/z 428.4 [M þ H-
162]þ. HR-ESI-MS: m/z 590.3701
[M þ H]þ (calcd for C33H52NO8,
590.3687).
ester hydrochloride (2 mg) was added and
stirred at 608C for 3 h. After the mixture
evaporated in vacuo to dryness, 0.2 ml of N-
trimethylsilylimidazole was added and
kept at 608C for another 3 h. The reaction
mixture was partitioned between n-hexane
and H2O, and the n-hexane extract analyzed
by GC under the following conditions:
capillary column, HP-5 (30 m £ 0.25 mm,
with a 0.25 mm film, Dikma, Lake Forest,
CA, USA); detection, Flame ionization
detector; detector temperature, 2808C;
injection temperature, 2508C; initial tem-
perature 1608C, then raised to 280 at
58C/min, final temperature maintained for
10 min; carrier, N2 gas. From the hydro-
lysate of 1, D-glucose was confirmed by
comparison of the retention time of its
derivative with that of authentic sugar
derivatized in a similar way, which showed
the retention time of 19.1 min. Compound 3
was hydrolyzed and detected in the same
method as compound 1, and puqiedinone
and D-glucose were identified in the MeOH
and water fractions, respectively.
3.3.2 Puqiedinone-3-O-b-D-
glucopyranoside (3)
20
White powder, ½aꢀD 241.6 (c ¼ 0.08,
MeOH), positive Dragendorff reaction. IR
(FT-IR microscope transmission) nmax
3398, 2915, 2749, 1704, 1455, 1373, 1078,
:
1
1034cm21. H NMR (500MHz, C5D5N):
d 0.69 (3H, br s, H-19), 0.78 (6H, br d,
J ¼ 5.5 Hz, H-21 and H-27), 5.02 (1H, d,
J ¼ 7.5 Hz, H-10), 4.02–4.10 (1H, m, H-20),
4.20–4.30 (2H, m, H-30,40), 3.95–4.01 (1H,
m, H-50), 4.60 (1H, d, J ¼ 12.0Hz, H-60),
4.40 (1H, dd, J ¼ 6.0, 12.0Hz, H-60), 3.94
(1H, m, W1/2 . 25Hz, H-3). 13C NMR
(125MHz) spectral data, see Table 1. ESI-
MS: m/z 598.4 [M þ Na]þ, 576.5
[M þ H]þ, 414.3 [M-162 þ H]þ. HR-ESI-
MS: m/z 576.3907 [M þ H]þ (calcd for
C33H54NO7, 576.3895).
3.4 PC12 cells protective assay
PC12 cells at a density of 5 £ 103 cells per
well in 96-well plates were cultured in
Dulbecco’s modified eagle medium media
supplemented with 5% fetal bovine serum
and 5% horse serum, and L-glutamine
(2 mM). Cultures were maintained at 378C
in 5% CO2 in a humidified incubator. On
the second day after plating, compounds at
concentrations of 10 and 4 mM rotenone
were added to the cells. After incubation
for another 48 h, 10 ml of the 5 mg/ml 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide was added and main-
tained for 4 h. Absorbance was measured
at 570 nm using an Ultramark microplate
reader (Molecular Devices, Sunnyvale,
CA, USA). Cell viability was evaluated.
3.3.3 The hydrolysis of 1 and 3 with b-
glucosidase
Compound 1 (2 mg) was dissolved in
100 ml DMSO, and 0.5 mg b-glucosidase
(from almonds, TCI Corp., Tokyo, Japan)
was added. The mixture was diluted to 1 ml,
and hydrolyzed at 408C for 10 h. The
mixture was separated through the ODS
solid phase extraction eluted with water and
MeOH, respectively. Peimisine was
detected in the MeOH-eluted fraction by
TLC analysis. The water-eluted fraction
was evaporated under vacuum, and the
residue was dissolved in anhydrous pyr-
idine (1 ml), to which L-cysteine methyl
Acknowledgements
This research program was financially sup-
ported by National Science and Technology

Journal of Asian Natural Products Research
1103
Project of China (No. 2009ZX09311-004), and
the Key Laboratory of Bioactive Substances
and Resources Utilization of Chinese Herbal
Medicine, Peking Union Medical College,
Ministry of Education. Our gratitude is
extended to Prof. Jin-Feng Hu at the Institute
of Materia Medica, Chinese Academy of
Medical Sciences & Peking Union Medical
College by bioassays.
[2] T.Z. Wang, L.L. Du, and S. Wang, West
China J. Pharm. Sci. 16, 200 (2001).
[3] F.P. Wang, R. Zhang, and X.Y. Tang, Acta
Pharm. Sin. 27, 273 (1992).
[4] J.Z. Wu, Chin. Tradit. Herb. Drugs 15, 38
(1984).
[5] Z.Z. Qian and T. Nohara, Phytochemistry
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[6] G. Lin, Y.P. Ho, P. Li, and X.G. Li, J. Nat.
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