Three new steroidal saponins from Fritillaria pallidiflora

Article abstract of DOI:10.1080/10286020.2011.619979

Three new steroidal saponins, pallidiflosides A (1), B (2), and C (3), have been isolated from the dry bulbs of Fritillaria pallidiflora Schrenk. Their structures were elucidated as 26-O-β-D-glucopyranosyl-(25R)-furost-5,20(22) -dien-3β,26-diol-3-O-β-D-xylopyranosyl(1 → 4)-[α-L- rhamnopyranosyl(1 → 2)]-β-D-glucopyranoside (1); 26-O-β-D- glucopyranosyl-3β,26-dihydroxyl-20,22-seco-25(R)-furost-5-en-20, 22-dione-3-O-α-L-rhamnopyranosyl(1 → 2)- β-D-glucopyranoside (2); and (25R)-spirost-5-ene-3β,17a-diol-3-O-β-D-glucopyranosyl(1 → 4)- β-D-galactopyranoside (3) by spectroscopic techniques and chemical means.

Full text of DOI:10.1080/10286020.2011.619979

This article was downloaded by: [Florida State University]
On: 23 December 2014, At: 06:48
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Three new steroidal saponins from
Fritillaria pallidiflora
Shuo Shen a b , Chao-Jun Chen a , Ren Bu a , Lu Ga a , Guo-Yu Li c
Yong Tan c , Xian Li b & Jin-Hui Wang a b
,
a School of Pharmacy, Inner Monglia Medical College , Hohhot,
010110, China
b School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University , Shenyang, 110016, China
c School of Pharmacy, Shihezi University , Shihezi, 832002, China
Published online: 19 Oct 2011.
To cite this article: Shuo Shen , Chao-Jun Chen , Ren Bu , Lu Ga , Guo-Yu Li , Yong Tan , Xian Li
& Jin-Hui Wang (2011) Three new steroidal saponins from Fritillaria pallidiflora , Journal of Asian
Natural Products Research, 13:11, 1014-1022, DOI: 10.1080/10286020.2011.619979
To link to this article: http://dx.doi.org/10.1080/10286020.2011.619979
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research
Vol. 13, No. 11, October 2011, 1014–1022
Three new steroidal saponins from Fritillaria pallidiflora
Shuo Shenab, Chao-Jun Chena, Ren Bua, Lu Gaa, Guo-Yu Lic, Yong Tanc, Xian Lib and
Jin-Hui Wangab*
b
aSchool of Pharmacy, Inner Monglia Medical College, Hohhot 010110, China; School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016,
China; cSchool of Pharmacy, Shihezi University, Shihezi 832002, China
(Received 27 May 2011; final version received 31 August 2011)
Three new steroidal saponins, pallidiflosides A (1), B (2), and C (3), have been
isolated from the dry bulbs of Fritillaria pallidiflora Schrenk. Their structures were
elucidated as 26-O-b-D-glucopyranosyl-(25R)-furost-5,20(22)-dien-3b,26-diol-3-O-b-
D-xylopyranosyl(1 ! 4)-[a-L-rhamnopyranosyl(1 ! 2)]-b-D-glucopyranoside (1);
26-O-b-D-glucopyranosyl-3b,26-dihydroxyl-20,22-seco-25(R)-furost-5-en-20,22-
dione-3-O-a-L-rhamnopyranosyl(1 ! 2)-b-D-glucopyranoside (2); and (25R)-spirost-
5-ene-3b,17a-diol-3-O-b-D-glucopyranosyl(1 ! 4)-b-D-galactopyranoside (3) by
spectroscopic techniques and chemical means.
Keywords: Fritillaria pallidiflora; steroidal saponins; NMR
1. Introduction
Herein, we report their isolation and
structural elucidation.
Fritillaria pallidiflora Schrenk is one of
the well-known traditional Chinese medi-
cines. It belongs to the Fritillaria genus of
Liliaceae family and is mainly distributed
in Xinjiang Province of China. The dry
bulbs of F. pallidiflora have been used as
antitussive, antiasthmatic, and expectorant
agents for hundreds of years [1]. The
alkaloids and saponins are the main
effective ingredients in the Fritillaria
genus of Liliaceae family [2]. However,
a literature survey concerning the second-
ary metabolites of F. pallidiflora showed
that no systematic chemical work on the
saponin constituents of the plant had been
carried out. Therefore, a phytochemical
investigation on the saponin constituents
of the dry bulbs of F. pallidiflora was
carried out. As a result, three new steroidal
saponins, pallidiflosides A (1), B (2), and
C (3) (see Figure 1), were obtained.
2. Results and discussion
Compound 1 was obtained as a white
20
amorphous solid with ½aꢀD -56.1 (MeOH, c
0.052) and gave positive results with
Liebermann–Burchard and Ehrlich reagent
tests. The IR spectrum showed absorption
bands for hydroxyl (3364 cm21) and a
glycosidic linkage (1000–1100cm21). Its
molecular formula C50H80O21 was deduced
from HR-ESI-MS at m/z 1015.5105
[M 2 H]2. The ESI-MS in positive-ion
mode showed ion peaks at m/z 1039
[M þ Na]þ, 907 [(M þ Naþ)-132]þ, 893
[(M þ Naþ)-146]þ, 877 [(M þ Naþ)-
162]þ, 761 [(M þ Naþ)-132-146]þ, and
599 [(M þ Naþ)-132-146-162]þ. The 1H
NMR spectrum of 1 showed three methyl
singlets at d 0.74, 1.04, and 1.66; one methyl
doublet at d 1.03 (d, J ¼ 6.5 Hz); one
*Corresponding author. Email: wjh.1972@yahoo.com.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.619979
http://www.tandfonline.com

Journal of Asian Natural Products Research
1015
R2O
21
18
26
25
22
O
20
23
24
27
19
3
5
R1O
6
1
R1 = S1, R2 = S4
R2O
26
25
21
20
22
O
O
23
27
H
17
O
19
16
H
H
3
5
R1O
6
2
R1 = S2, R2 = S4
25
26
O
20 22
27
18
23
24
17
16
O
19
5
OH
3
RO
6
3
R = S3
OH
OH
O
O
HO
HO
1'''
O
HO
O
O
HO
HO
1'
H
H
OH
O
S2
=
H
S1
=
1'
H
O
HO
H
1''
O
HO
HO
1''
OH
HO
OH
OH
OH
O
HO
HO
1''
OH
O
S3
=
S4
=
O
OH
HO
HO
H
O
HO
OH
1'
H
OH
H
Figure 1. The structures of compounds 1–3.

1016
S. Shen et al.
olefinicprotonatd5.30(d, J ¼ 4.0 Hz); four
anomeric proton signals at d 4.96 (d,
J ¼ 7.2 Hz), 6.33 (s), 5.06 (d, J ¼ 7.0 Hz),
and 4.89 (d, J ¼ 7.8 Hz). The 13C NMR
spectrum of 1 revealed characteristic signals
for two pairs of olefinic carbons at d 140.8
and 121.9 and d 103.6 and 152.4; four
anomeric carbons of sugars at d 100.0,
102.0, 105.9, and 105.0. The above
evidences indicated that 1 was a furostanol
saponin with four sugar units. Comparison
of the NMR spectral data of 1 (Table 1) with
the literature data [3] allowed the identifi-
cation of the aglycone as the previously
reported furost-5,20(22)-dien-3b,26-diol.
The geminal proton resonances of H2-26
appeared at d 3.64 (Ha-26) and 3.94(Hb-26),
which showed the Dab of dHa-dHb as
0.30ppm (,0.48ppm), indicating that the
configurationofC-25was R[4]. TheHMBC
spectrum (Figure 2) of 1 showed corre-
lations between the proton signal at d 1.66
(H-21) and the carbon signals at d 64.5
(C-17), 103.6 (C-20), and 152.4 (C-22);
between the proton signal at d 1.04 (H-19)
and the carbon signals at d 140.8 (C-5), 50.3
(C-9), and 37.1(C-10). Theabove evidences
convinced the presence of two double bonds
at C-5 (6) and C-20 (22). Acid hydrolysis
of 1 afforded L-rhamnose, D-xylose, and
D-glucose in a ratio of 1:1:2 on the basis
of gas chromatography (GC) analysis. The
b-anomeric configurations for both glucose
and xylose were judged from their coupling
Table 1. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectral data of the aglycone moieties of
compounds 1 and 2 in pyridine-d5.
1
2
No.
dC
dH
dC
dH
1
2
3
4
5
6
7
8
37.6
30.2
78.4
39.0
140.8
121.9
32.4
31.5
50.3
37.1
21.3
39.7
43.4
54.9
34.5
84.5
64.5
14.1
19.5
103.6
11.9
152.4
23.7
31.5
33.5
75.0
17.4
0.98 m, 1.77 m
1.94 o, 2.16 m
3.89 m
37.5
30.1
78.0
39.0
141.0
121.6
31.9
31.0
50.4
37.1
20.7
38.2
42.3
54.1
35.5
74.2
66.7
13.8
19.5
205.6
30.5
173.3
32.3
29.0
33.5
74.8
17.0
0.92 m, 1.71 o
1.85 o, 2.11o
3.89 m
2.78 m, 2.85 m
2.75 d (12.6), 2.83 m
5.30 d (4.0)
1.52 o, 1.88 m
1.52 o
5.30 d (4.5)
1.85 o
1.52 m
9
0.92 m
0.88 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.45 m, 1.75 m
1.16 m, 1.79 m
1.37 m, 1.47 m
1.07 m, 2.15 o
0.88 m
0.82 m
1.31 td (3.9, 12.8), 2.43 o
5.64 m
2.47 d (7.8)
1.21 s
1.05 s
1.50 o, 2.13 td (7.8, 13.6)
4.79 d (7.8)
2.46 d (9.9)
0.74 s
1.04 s
1.66 s
2.12 s
2.23 m
1.49 m, 1.85 m
1.97 o
3.64 dd (9.5, 6.0), 3.94 m
1.03 d (6.5)
2.42 o
1.53 m, 1.94 m
1.83 o
3.50 dd (6.0, 9.3), 3.89 m
0.90 d (6.5)
Note: Overlapped signals are indicated by ‘o.’

Journal of Asian Natural Products Research
1017
OH
O
HO
HO
1''''
O
OH
21
H
27
O
19
OH
3
O
O
HO
HO
1'''
H
O
HO
O
1'
H
OH
O
O
HO
H
1''
OH
HO
1
OH
O
HO
21
1'''
HO
O
OH
H
O
O
27
H
O
19
H
OH
H
3
O
HO
O
HO
HO
1'
H
O
O
H
HO
1''
OH
2
21
O
27
O
OH
19
OH
O
1''
HO
OH
O
HO
OH
H
3
O
HO
O
1'
OH
H
3
Figure 2. Key HMBC correlations of compounds 1–3.

1018
S. Shen et al.
Table 2. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectral data of the sugar moieties of
compounds 1 and 2 in pyridine-d5.
1
2
No.
dC
dH
dC
dH
(3-O)-b-D-Glc
20
100.0
77.5
77.4
81.5
76.3
61.8
102.0
72.6
72.9
74.2
69.6
18.7
105.9
75.0
78.7
70.8
67.4
105.0
75.3
78.6
71.7
78.4
62.9
4.96 d (7.2)
100.4
79.7
78.4
71.7
77.9
62.5
102.1
72.6
72.9
74.0
69.5
18.7
5.03 d (7.8)
4.26 dd (7.8, 9.0)
4.21 dd (9.0, 9.0)
4.14 o
4.23 dd (7.2, 9.1)
4.23 m
4.19 m
3.87 m
4.36 m, 4.49 d (12.5)
6.33 s
4.82 d (3.5)
4.64 dd (3.5, 9.4)
4.42 m
30
40
50
3.88 o
60
4.29 m, 4.51 d (10.2)
6.38 s
4.81 brs
4.62 dd (3.3, 9.3)
4.35 o
4.97 o
(Glc2)-a-L-Rha
200
300
400
500
4.95 o
600
1.81 d (6.1)
5.06 d (7.0)
4.03 dd (7.0, 9.0)
4.10 m
1.77 d (6.1)
(Glc4)-b-D-Xyl
2000
3000
4000
4.15 m
5000
3.69 d (10.6), 4.29 d (10.6)
4.89 d (7.8)
4.03 m
4.26 m
4.25 m
(26-O)-b-D-Glc
105.0
75.2
78.6
71.5
78.6
62.9
4.78 d (7.5)
4.03 dd (7.5, 9.0)
4.24 m
4.20 m
3.92 m
4.32 m, 4.52 d (11.4)
20000
30000
40000
50000
60000
3.95 d (9.0)
4.43 m, 4.54 d (11.5)
Note: Overlapped signals are indicated by ‘o.’
constants(3JH1,H2 . 7.0)[5]. Thea-anome-
ric configuration for the rhamnose was
determined by its C-500 data (d 69.6) [6]. The
HMBC spectrum (Figure 2) showed corre-
lations between the anomeric proton signal
H-10 at d 4.96 and the carbon signal C-3 at d
78.4, between H-100 at d 6.33 and C-20 at
d 77.5, between H-1000 at d 5.06 and C-40 at d
81.5, and between H-10000 at d 4.89 and
C-26 at d 75.0. According to the accumu-
lated evidence above, the structure of 1
was characterized as 26-O-b-D-glucopyra-
nosyl-(25R)-furost-5,20(22)-dien-3b,26-
diol-3-O-b-D-xylopyranosyl(1 ! 4)-[a-L-
rhamnopyranosyl(1 ! 2)]-b-D-glucopyra-
noside, named as pallidifloside A (1).
absorption bands for hydroxyl (3363 cm21),
carbonyl of ester (1745 cm21), and carbonyl
of ketone (1713 cm21). Its molecular
formula C45H72O19 was deduced from the
HR-ESI-MS at m/z 915.4598 [M 2 H]2,
1
and this was further supported by the H
NMR, 13C NMR, and DEPT spectra (in
pyridine-d5; see Tables 1 and 2). The ESI-
MS in positive-ion mode showed the ions at
m/z 939 [M þ Na]þ, 793 [(M þ Naþ)-
146]þ, 777 [(M þ Naþ)-162]þ, and 631
[(M þ Na)þ-146-162]þ. The 1H NMR
spectrum of 2 showed three methyl singlets
at d 1.21, 1.05, and 2.12; one methyl doublet
at d 0.90 (d, J ¼ 6.5 Hz); one olefinic proton
at d 5.30(d, J ¼ 4.5 Hz); and three anomeric
protons at d 5.03 (d, J ¼ 7.8 Hz), 6.38 (s),
and 4.78 (d, J ¼ 7.5 Hz). These data,
combined with the analysis of its 13C
NMR spectrum (four methyl groups at d
13.8, 19.5, 30.5, and 17.0, trisubstituted
Compound 2 was obtained as a white
20
amorphous solid with ½aꢀD -59.2 (MeOH, c
0.052) and it gave positive results with
Liebermann–Burchard and positive Ehrlich
reagent tests. The IR spectrum showed

Journal of Asian Natural Products Research
1019
double bonds at d 141.0 and 121.6, an ester
carbonyl at d 173.3, a ketone carbonyl at
d 205.6, and three anomeric carbon signals
atd100.4,102.1,and105.0), indicatedthat2
was a furostanol saponin with three sugar
units. Comparison of the NMR spectral data
of 2 (Table 1) with the literature data [3]
allowed the identification of the aglycone as
the previously reported (3b,25R)-20,22-
seco-25-furost-5-en-20,22-dione-3,26-diol.
The HMBC correlations between the proton
at d 1.05 (H-19) and the carbons at d 141.0
(C-5), 50.4 (C-9), and 37.1 (C-10), between
theprotonatd2.12(H-21)andthecarbonsat
d 66.7 (C-17) and 205.6 (C-20), and no
HMBC correlation between the proton at d
2.12 (s, H-21) and the carbon at d 173.3
(C-22) were observed, which convinced the
presence of one double bond at C-5 (6) and
theEringof2wasopenedbetweenC-20and
C-22. In the NOESY spectrum of 2, the
proton at d 0.82 (m, H-14) showed the
correlations with the protons at d 5.64 (m,
H-16) and d 2.47 (d, J ¼ 7.8 Hz, H-17),
suggesting H-16 and H-17 were a-oriented.
The HMBC and NOESY correlations
mentioned above supported the conclusion,
i.e. the aglycone of 2 was (3b, 25R)-20,22-
seco-25-furost-5-en-20,22-dione-3,26-diol.
Acid hydrolysis of 2 afforded L-rhamnose
and D-glucose in a ratio of 1:2 on the basis of
GC analysis. The b-anomeric configur-
ations for two glucoses were judged from
their coupling constants (3JH1,H2 . 7.0) [5].
The a-anomeric configuration for the
rhamnose was determined by its chemical
shift of C-500 at d 69.5 [6]. The HMBC
spectrum (Figure 2) showed correlations
between H-100 at d 5.03 and C-3 at d 78.0,
between H-100 at d 6.38 and C-20 at d 79.7,
and between H-1000 at d 4.78 and C-26 at d
74.8. According to the above evidence, the
structure of 2 was determined as 26-O-b-D-
glucopyranosyl-3b,26-dihydroxyl-20,22-
seco-25(R)-furost-5-en-20,22-dione-3-O-
a-L-rhamnopyranosyl(1 ! 2)-b-D-gluco-
pyranoside, named as pallidifloside B (2).
Compound 3 was obtained as a white
c 0.051) and gave results positive in
Liebermann–Burchard and negative in
Ehrlich reagent tests. The IR spectrum
showed absorption bands for hydroxyl
(3421 cm21) and a glycosidic linkage
(1000–1100 cm21). Its molecular for-
mula, C39H62O14, was deduced from the
HR-ESI-MS at m/z 753.4061 [M 2 H]2.
The ESI-MS in positive-ion mode
showed the ions at m/z 777 [M þ Na]þ,
615 [(M þ Naþ)-162]þ, and 453
[(M þ Naþ)-162-162]þ. The 1H NMR
spectrum of 3 showed two methyl
singlets at d 0.94 and 0.92; two methyl
doublets at d 1.25 (d, J ¼ 6.9 Hz) and
0.69 (d, J ¼ 5.1 Hz); one olefinic proton
at d 5.30 (d, J ¼ 4.5 Hz); two anomeric
proton signals at d 4.90 (d, J ¼ 7.5 Hz)
and 5.33 (d, J ¼ 8.1 Hz). The 13C NMR
spectrum of 3 revealed characteristic
signals for one quaternary carbon signal
at d 109.5 and two olefinic carbon signals
at d 140.6 and 121.4 as well as two
anomeric carbons of sugars at d 102.6
and 106.8. The above evidences indi-
cated that 3 was a spirostanol saponin
1
with two sugar units. Comparing the H
and 13C NMR spectral data of 3 (Table 3)
with the literature data [7] allowed the
identification of the aglycone as the
previously reported spirost-5-ene-
3b,17a-diol and the C-25R configuration
was determined by the inspection of the
13C NMR spectral data of C-27 (d 17.0),
C-26 (d 66.7), C-25 (d 30.0), and C-24 (d
28.5) [6]. Acid hydrolysis of 3 afforded
D-galactose and D-glucose in a ratio of
1:1 on the basis of GC analysis. The b-
anomeric configurations for the glucose
and galactose were judged from their
coupling constants (3JH1,H2 . 7.0) [5].
The HMBC spectrum (Figure 2) showed
the correlation between H-10 at d 4.90
and C-3 at d 77.7 and between H-100 at
d 5.33 and C-40 at d 79.7. Thus, the
structure of
3 was elucidated as
(25R)-spirost-5-ene-3b,17a-diol-3-O-b-
D-glucopyranosyl(1 ! 4)-b-D-galacto-
pyranoside, named as pallidifloside C (3).
20
amorphous solid with ½aꢀD -47.2 (MeOH,

1020
S. Shen et al.
Table 3. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectral data of compound 3 in pyridine-d5.
3
3
No.
dC
dH
No.
dC
dH
1
2
3
4
5
6
7
8
37.2
30.1
77.7
38.9
140.6
121.4
32.0
31.8
49.9
36.7
20.7
32.2
44.9
53.0
32.9
89.7
89.8
16.9
19.1
44.5
9.5
0.97 m, 1.72 m
1.57 m, 2.07 m
3.82 m
(3-O)-b-D-Gal
20
102.6
73.2
75.1
79.7
75.8
60.7
106.8
74.9
78.5
72.0
78.2
62.8
4.90 d (7.5)
4.43 dd (7.5, 9.6)
4.09 o
4.72 m
4.10 o
4.23 o, 4.67 m
5.33 d (8.1)
4.19 dd (8.1, 8.4)
4.32 m
4.12 o
3.92 m
4.22 m, 4.62 d (11.5)
30
2.65 d (12.4), 2.71 m
40
50
5.30 d (4.5)
1.47 m, 1.90 m
1.55 m
60
(Gal4)-b-D-Glc
200
300
400
500
600
9
0.91 o
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1.50 o
1.89 m, 2.21 m
2.05 m
1.51 m, 2.19 m
4.49 t (7.5)
0.94 s
0.92 s
2.29 d (6.9)
1.25 d (6.9)
109.5
32.4
28.5
30.0
66.7
17.0
1.52 o
1.58 m
1.33 m
3.52 o, 3.95 m
0.69 d (5.1)
Note: Overlapped signals are indicated by ‘o.’
3. Experimental
with an H2 flame ionization detector
and a DB-5 quartz capillary column
(30 m £ 0.25 mm £ 0.25 um). Chroma-
tography was performed on silica gel
(200–300 mesh; Qingdao Marine Chemi-
cal Group, Co., Qingdao, China) and
preparative HPLC [Shimadzu-LC-8A
pump, Shimadzu-SPD-20A UV spectro-
photometric detector at 203 nm, Shimadzu
Shim-pack PRC-ODS (10 mm, 20 £
250 mm; flow rate 12.0 ml/min)].
3.1 General experimental procedures
Optical rotations were determined on a
Perkin-Elmer 241 MC polarimeter. UV
spectra were obtained on a Shimadzu UV-
2201 spectrophotometer. IR spectra were
recorded on a Shimadzu IRPrestige-21
spectrophotometer. The NMR data were
recorded on a Bruker AV-600 spectrometer
(600 MHz) and a Varian INOVA-300
1
spectrophotometer (300 MHz for H and
75 MHz for 13C) in C5D5N. The ESI-MS
data were obtained on a Finnigan LCQ
mass spectrometer. The HR-ESI-MS data
were obtained on a Waters LCT Premier
XE time-of-flying mass spectrometer. GC
analysis was performed on a Shimadzu
GC-2010 gas chromatograph equipped
3.2 Plant material
The dry bulbs of F. pallidiflora Schrenk
(2.0 kg) were collected in Yining, Xinjiang
Province of China in June 2008, and
identified by Dr Yong Tan of Shihezi
University. A voucher specimen (no.

Journal of Asian Natural Products Research
1021
2008090158) has been deposited in
Research Department of Natural
Medicine, Shenyang Pharmaceutical
University.
[(M þ Na)-132-146-162]þ; HR-ESI-MS:
m/z 1015.5105 [M 2 H]2 (calculated for
C50H79O21, 1015.5114).
3.3.2 Pallidifloside B (2)
3.3 Extraction and isolation
A white amorphous solid, positive Lie-
bermann–Burchard and positive Ehrlich
The dry bulbs of F. pallidiflora (2.0 kg)
were extracted with 70% EtOH (3 £
20 liters) for 3 h at 958C. The extracts
were combined and concentrated under
reduced pressure to give the brown residue
(98 g). The residue was suspended in water
(2 liters) and then extracted with petroleum
ether (2 liters), CHCl3 (2 liters), EtOAc
(2 liters), and n-BuOH (2 liters), respect-
ively. The n-BuOH layer was concentrated
under reduced pressure to give the brown
extract (26.0), which was chromato-
graphed on silica gel eluting with gradient
CHCl3/MeOH (100:1–100:100) to yield
10 fractions 1–10. Fraction 4 (1.2 g) was
further purified by preparative HPLC
(column: 20 £ 250 mm, RP-18, 10 mm;
flow rate: 12.0 ml/min) with MeOH–H2O
(75:25) as a mobile phase to afford
compound 3 (18.2 mg; retention time
102.2 min). Fraction 6 (0.7 g) was further
purified by preparative HPLC (column:
20 £ 250 mm, RP-18, 10 mm; flow rate:
12.0 ml/min) with MeOH–H2O (60:40) as
a mobile phase to afford compound 1
(35.4 mg; retention time 42.2 min) and
compound 2 (39.4 mg; retention time
13.6 min)
20
reagent tests; ½aꢀD -59.2 (MeOH, c 0.052);
UV (MeOH) lmax (nm): 203.5, 238.5; IR
(KBr) nmax (cm21): 3363, 1745, 1713,
1
1652, 1074, 1040, 949, 912, and 840; H
and 13C NMR spectral data are shown in
Tables 1 and 2; ESI-MS: m/z 939
[M þ Na]þ, 793 [(M þ Na)-146]þ, 777
[(M þ Na)-162]þ, 631 [(M þ Na)-146-
162]þ; HR-ESI-MS: m/z 915.4598
[M 2 H]2 (calculated for C45H71O19,
915.4590).
3.3.3 Pallidifloside C (3)
A white amorphous solid, positive Lie-
bermann–Burchard and negative Ehrlich
20
reagent tests; ½aꢀD -47.2 (MeOH, c 0.051);
UV (MeOH) lmax (nm): 203.5; IR (KBr)
nmax (cm21): 3421, 2927, 1458, 1377,
1245, 1159, 1077, 918, and 893; 1H
and 13C NMR spectral data are shown in
Table 3; ESI-MS: m/z 777 [M þ Na]þ,
615 [(M þ Na)-162]þ, and 453 [(M þ
Na)-162-162]þ. HR-ESI-MS: m/z 753.
4061 [M 2 H]2 (calculated for C39H61
O14, 753.4061).
3.4 Acid hydrolysis of 1, 2, and 3
3.3.1 Pallidifloside A (1)
Compound 1 (2.0 mg) was hydrolyzed
with 2 M HCl (5 ml), heated for 2 h at 958C
and extracted with CHCl3 (3 £ 5 ml). The
aqueous layer was concentrated to dryness
under a stream of nitrogen. The aqueous
residue was redissolved in anhydrous
pyridine (2 ml) and L-cysteine methyl
ester hydrochloride (2.5 mg) was added
to the solution of pyridine. The mixture
was heated at 608C for 2 h and acetic
anhydride (2 ml) was added, followed by
heating at 908C for 0.5 h. The solution was
A white amorphous solid, positive Lie-
bermann–Burchard and Ehrlich reagent
20
tests; ½aꢀD -56.1 (MeOH, c 0.052); UV
(MeOH) lmax (nm): 204.0; IR (KBR) nmax
(cm21): 3364, 2923, 1654, 1465, 1457,
1378, 1161, 1074, 1043, 913, and 839; 1H
and 13C NMR spectral data are shown in
Tables 1 and 2; ESI-MS: m/z 1039
[M þ Na]þ, 907 [(M þ Na)-132]þ, 893
[(M þ Na)-146]þ, 877 [(M þ Na)-162]þ,
761 [(M þ Na)-132-146]þ, and 599

1022
S. Shen et al.
concentrated to dryness under a stream of
nitrogen. The reaction product was sub-
jected to GC analysis in Shimadzu GC-
2010 (DB-5, 30 m £ 0.25 mm, 0.25 mm)
and FID detection. Column temperature:
1208C, carrier gas: N2 (30 ml/min), split
ratio: 10:1, injection temperature: 2608C,
detector temperature: 2608C, and injection
volume: 1 ul are the conditions for GC
analysis. The absolute configurations of
the monosaccharides of 1 were confirmed
to be L-rhamnose (tR ¼ 18.8 min), D-
glucose (tR ¼ 21.2 min), and D-xylose
(tR ¼ 16.9 min) by comparison of the
retention time of monosaccharide deriva-
tives with those of standard sugars (the
standard sugars were subjected to the same
reaction): L-rhamnose (tR ¼ 18.6 min), D-
glucose (tR ¼ 21.4 min), and D-xylose
(tR ¼ 16.8 min).
ides of 3 were confirmed to be D-galactose
(tR ¼ 21.9 min) and D-glucose (tR ¼
21.3 min).
Acknowledgment
This work was financially supported by
National Key Technologies R&D Program of
China during the 11th Five-Year Plan Period
(2009ZX09501-011) and National Major
Special Science and Technology Project
(2010ZX09401-304-104A).
References
[1] D.M. Xu, N.X. Huang, S.Q. Wang,
X.G. Wen, and X.Y. Wu, Acta Bot. Sin.
32, 789 (1990).
[2] H.L. Yuan, Chin. Tradit. Herb. Drugs 33,
858 (2002).
[3] A. Yokosuka, Y. Mimaki, and Y. Sashida,
J. Nat. Prod. 65, 1293 (2002).
[4] P.K. Agrawal, Steroids 70, 715 (2005).
[5] I. Kostova and D. Dinchev, Phytochem.
Rev. 4, 111 (2005).
[6] P.K. Agrawal, D.C. Jain, P.K. Gupta, and
R.S. Thakur, Phytochemistry 24, 2479
(1985).
[7] M. Ono, C. Takamura, F. Sugita,
C. Masuoka, H. Yoshimitsu, T. Ikeda,
and T. Nohara, Chem. Pharm. Bull. 55, 551
(2007).
Compound 2 (2.1 mg) was subjected to
acid hydrolysis as described for 1 and the
absolute configurations of monosacchar-
ides of 2 were confirmed to be L-rhamnose
(tR ¼ 18.7 min) and D-glucose (tR ¼
21.5 min).
Compound 3 (2.0 mg) was subjected to
acid hydrolysis as described for 1 and the
absolute configurations of monosacchar-