New minor spirostane glycosides from Ypsilandra thibetica

Article abstract of DOI:10.1002/hlca.201100368

A further phytochemical investigation on the whole plants of Ypsilandra thibetica yielded three new spirostane glycosides, named ypsilandrosides M-O (1-3). Their structures were established as (3β,11α,25R)-3,11- dihydroxyspirost-5-en-12-one 3-{O-α-L-rhanmopyranosyl-(1→4)-O-L- rhanmopyranosyl-(1→4)-O-[α-L-rhanmopyranosyl-(1→2)] -β-D-glucopyranoside} (1), (3β,7β,25R)-spirost-5-ene-3,7-diol 3-{O-α-L-rhanmopyranosyl-(1→4)-O-α-L-rhanmopyranosyl-(1→4) -O-[α-L-rhanmopyranosyl-(1→2)]-β-D-glucopyranoside} (2), and (3β,7α,25R)-spirost-5-ene-3,7,17-triol 3-{O-α-L- rhanmopyranosyl-(1→4)-O-α-L-rhanmopyranosyl-(1→4) -O-[α-L-rhanmopyranosyl-(1→2)]-β-D-glucopyranoside} (3) by means of a combination of MS, 1D- and 2D-NMR spectroscopic methods, and chemical degradation. Among them, compound 3 is the first pennogenin (=(3β,25R)- spirost-5-ene-3,17-diol) saponin whose aglycone contains an OH group at C(7). Compounds 1-3 were evaluated for the inhibition of the growth of five tumor cell lines, but all of them proved to be inactive. Copyright

Full text of DOI:10.1002/hlca.201100368

Helvetica Chimica Acta – Vol. 95 (2012)
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New Minor Spirostane Glycosides from Ypsilandra thibetica
by Xu-Dong Zhanga)b), Chang-Xiang Chena), Jun-Yun Yanga), Wei Ni a), and Hai-Yang Liu*a)
a) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China
(phone: þ 86-871-5223246; fax: þ 86-871-5223245; e-mail: haiyangliu@mail.kib.ac.cn)
b) College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,
Qingdao 266042, P. R. China
A further phytochemical investigation on the whole plants of Ypsilandra thibetica yielded three new
spirostane glycosides, named ypsilandrosides M – O (1 – 3). Their structures were established as
(3b,11a,25R)-3,11-dihydroxyspirost-5-en-12-one 3-{O-a-l-rhanmopyranosyl-(1 ! 4)-O-l-rhanmopyra-
nosyl-(1 ! 4)-O-[a-l-rhanmopyranosyl-(1 ! 2)]-b-d-glucopyranoside} (1), (3b,7b,25R)-spirost-5-ene-
3,7-diol 3-{O-a-l-rhanmopyranosyl-(1 ! 4)-O-a-l-rhanmopyranosyl-(1 ! 4)-O-[a-l-rhanmopyranosyl-
(1 ! 2)]-b-d-glucopyranoside} (2), and (3b,7a,25R)-spirost-5-ene-3,7,17-triol 3-{O-a-l-rhanmopyrano-
syl-(1! 4)-O-a-l-rhanmopyranosyl-(1! 4)-O-[a-l-rhanmopyranosyl-(1! 2)]-b-d-glucopyranoside} (3)
by means of a combination of MS, 1D- and 2D-NMR spectroscopic methods, and chemical degradation.
Among them, compound 3 is the first pennogenin (¼(3b,25R)-spirost-5-ene-3,17-diol) saponin whose
aglycone contains an OH group at C(7). Compounds 1 – 3 were evaluated for the inhibition of the growth
of five tumor cell lines, but all of them proved to be inactive.
Introduction. – Steroidal saponins, which are almost exclusively present in the
monocotyledonous angiosperms, have attracted a growing interest owing to the range
of their biological actions including antidiabetic, antitumor, antitussive, and anti-
dementia activity and as platelet aggregation inhibitors [1]. Ypsilandra thibetica
Franch. is a perennial herb of the family Liliaceae and grows in southwestern China
[2]. The whole plant has been used as hemostatic agent in Chinese folk medicine [3]. In
our recent study, we found that this species is a rich source of steroidal saponins. Two
sapogenin, 22 spirostanol saponins, and two C(22) steroidal lactone glycosides were
isolated from the title plant [4 – 7]. In continuation of our investigations on the
chemistry of this species, we obtained three new minor spirostane glycosides,
ypsilandrosides M – O (1 – 3; Fig. 1). Herein, we report the isolation, structural
elucidation, and cytotoxic evaluation of the new spirostane glycosides.
Results and Discussion. – Compound 1, obtained as a colorless amorphous powder,
gave a quasimolecular-ion peak at m/z 1043.5057 ([M ꢀ H]ꢀ) in its HR-ESI-MS.
Combined with 13C-NMR spectroscopic data (Table), its molecular formula was
determined as C51H80O22. The IR spectrum showed absorption for OH groups at
3441 cmꢀ1, a C¼O group at 1710 cmꢀ1, and an olefin moiety at 1638 cmꢀ1. The 1H-NMR
spectrum of 1 (Table) showed signals due to two tertiary Me groups at d(H) 1.11 and
1.36 (2s), two secondary Me groups at d(H) 0.68 (d, J ¼ 5.5 Hz) and 1.29 (d, J ¼ 6.9 Hz),
one olefinic H-atom at d(H) 5.33 (d, J ¼ 3.9 Hz), as well as two H-atom signals
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 95 (2012)
Fig. 1. Compounds 1 – 3, isolated from Ypsilandra thibetica
attributable to an O-bearing CH2(26) at d(H) 3.45 (t, J ¼ 10.5 Hz) and 3.54 – 3.57 (m).
The 13C-NMR and DEPT spectrum (Table) showed a total of 27 C-atoms arising from
the aglycone moiety. Furthermore, a dioxygenated quaternary C-atom at d(C) 109.4
and olefinic C-atoms at d(C) 141.5 and 121.3 suggested that 1 possessed a
hydroxyspirost-5-ene skeleton. Comparison of the 1H- and 13C-NMR spectra indicated
that 1 differed from ypsilandroside J (¼(3b,7a,25R)-3-[(O-6-deoxy-a-l-mannopyra-
nosyl-(1 ! 4)-O-6-deoxy-a-l-mannopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyrano-
syl-(1 ! 2)]-b-d-glucopyranosyl)oxy]-7-hydroxyspirost-5-en-12-one) [6] by the pres-
ence of a CH2 (d(C) 32.0; d(H) 1.48 – 1.50 (m) and 1.80 – 1.83 (m)) instead of an O-
bearing CH moiety (d(C) 64.3; d(H) 4.06 (m)) comprising C(7) in the latter, which was
confirmed by the 1H,1H correlations HꢀC(6) (d(H) 5.33)/CH2(7) in the 1H,1H-COSY
plot of 1. The ROESY correlations HꢀC(11) (d(H) 4.72)/Me(18) (d(H) 1.11)
confirmed that OHꢀC(11) was a-oriented. Other ROESY correlations suggested that
compound 1 has the same ring junctions as those of ypsilandroside J. The stronger
intensity of the band at 899 compared with that at 919 cmꢀ1 in its IR spectrum suggested
1
the (R) absolute configuration at C(25) [8]. The anomeric region in the H- and
13C-NMR spectra of 1 showed signals for four anomeric H-atoms at d(H) 4.97 (d, J ¼
7.0 Hz), 5.85 (br. s), 6.29 (br. s), and 6.41 (br. s) with their corresponding anomeric C-
atoms at d(C) 100.4, 102.3, 103.3, and 102.2, respectively (Table). Acid hydrolysis of 1
produced d-glucose and l-rhamnose (¼6-deoxy-l-mannose) as sugar residues, which
were determined by GC analysis of their corresponding trimethylsilated l-cysteine
1
adducts. The H-NMR coupling constant (3J(1,2) ꢁ 7 Hz) for the anomeric H-atom
revealed that the glucose unit has a b-configuration, while the three rhamnose units
have the a-configuration according to the chemical-shift values of C(3’’) (d(C) 72.9
(d)), C(5’’) (d(C) 69.6 (d)), C(3’’’) (d(C) 73.3 (d)), C(5’’’) (d(C) 68.4 (d)), C(3’’’’) (d(C)
72.9 (d)), and C(5’’’’) (d(C) 70.4 (d)), compared with the corresponding C-atoms of
methyl a- and b-rhamnopyranoside [9][10]. In the HMBC spectrum of 1, the
correlations HꢀC(1’) (d(H) 4.97)/C(3) (d(C) 77.9), HꢀC(1’’) (d(H) 6.41)/C(2’) (d(C)
78.2), HꢀC(1’’’) (d(H) 5.85)/C(4’) (d(C) 78.0), and HꢀC(1’’’’) (d(H) 6.29)/C(4’’’) (d(C)
80.5) revealed an inner glucopyranosyl unit linked at C(3) of the aglycone, a terminal
rhamnopyranosyl unit at C(2’) of the glucopyranosyl unit, an inner rhamnopyranosyl

Helvetica Chimica Acta – Vol. 95 (2012)
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Table. 1H- and 13C-NMR Data (C5D5N) of Compounds 1 – 3. d in ppm, J in Hz.
Position
CH2(1)
1a)
2a)
3b)
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
1.23 – 1.25,
39.1 (t)
0.96 – 0.99,
37.1 (t)
0.93 (dd,
38.0 (t)
1.87 – 1.88 (2m)
1.72 – 1.74 (2m)
J ¼ 4.0, 14.2),
1.69 – 1.71 (m)
1.84 – 1.87,
CH2(2)
1.92 – 1.95,
29.2 (t)
1.83 – 1.85,
30.3 (t)
30.0 (t)
2.04 – 2.06 (2m)
3.86 – 3.88 (m)
2.77 (t, J ¼ 11.8),
2.81 – 2.83 (m)
2.07 – 2.09 (2m)
77.9 (d) 3.92 – 3.95 (m)
2.00 – 2.03 (2m)
77.9 (d) 3.75 – 3.77 (m)
HꢀC(3)
77.9 (d)
39.0 (t)
CH2(4)
39.4 (t)
2.75 (t, J ¼ 12.3),
38.6 (t)
2.69 – 2.71 (m),
2.82 (dd ,
2.84 – 2.87 (m)
J ¼ 4.7, 12.2)
C(5)
141.5 (s)
121.3 (d) 5.68 (br. s)
32.0 (t) 4.00 – 4.03 (m)
141.5 (s)
143.9 (s)
126.1 (d)
64.7 (d)
HꢀC(6)
CH2(7) or
HꢀC(7)
HꢀC(8)
HꢀC(9)
C(10)
5.33 (d, J ¼ 3.9)
1.48 – 1.50 (m),
1.80 – 1.83 (m)
1.44 – 1.46 (m)
1.38 – 1.40 (m)
128.8 (d) 5.81 (d, J ¼ 4.8)
72.5 (d) 4.03 – 4.05 (m)
31.7 (d) 1.81 – 1.83 (m)
60.4 (d) 1.08 – 1.11 (m)
39.7 (s)
40.8 (d) 1.66 – 1.68 (m)
48.7 (d) 1.65 – 1.67 (m)
37.3 (s)
38.7 (d)
42.6 (d)
37.3 (s)
20.9 (t)
HꢀC(11) or 4.72 (d, J ¼ 9.9)
73.9 (d) 1.45 – 1.47 (m)
21.3 (t)
40.0 (t)
41.0 (s)
1.63 – 1.65 (m)
CH2(11)
C(12) or
CH2(12)
C(13)
213.4 (s) 1.12 – 1.15,
1.70 – 1.72 (2m)
54.0 (s)
56.0 (d) 1.34 – 1.36 (m)
31.8 (t)
1.67 – 1.69 (m)
32.1 (t)
45.0 (s)
46.3 (d)
32.1 (t)
HꢀC(14)
1.41 – 1.43 (m)
1.77 – 1.80,
56.4 (d) 3.03 – 3.05 (m)
CH2(15)
2.03 – 2.05,
35.3 (t)
2.12 – 2.15,
2.06 – 2.09 (2m)
4.42 – 4.44 (m)
HꢀC(17) or 2.85 – 2.87 (m)
2.83 – 2.85 (2m)
79.9 (d) 4.63 – 4.66 (m)
54.2 (d) 1.80 – 1.82 (m)
2.72 – 2.74 (2m)
81.7 (d) 4.55 – 4.58 (m)
62.6 (d)
HꢀC(16)
90.4 (d)
90.3 (s)
C(17)
Me(18)
Me(19)
HꢀC(20)
1.11 (s)
1.36 (s)
1.85 – 1.88 (m)
15.7 (q) 0.86 (s)
19.1 (q) 1.00 (s)
42.5 (d) 1.97 (dd,
J ¼ 7.0, 13.5)
16.5 (q) 1.03 (s)
19.0 (q) 1.08 (s)
42.1 (d) 2.21 – 2.24 (m)
17.2 (q)
19.6 (q)
45.0 (d)
Me(21)
C(22)
CH2(23)
1.29 (d, J ¼ 6.9)
13.9 (q) 1.15 (d, J ¼ 7.0)
109.4 (s)
15.2 (q) 1.25 (d, J ¼ 7.1)
109.3 (s)
9.7 (q)
109.9 (s)
32.1 (t)
1.59 – 1.61,
31.8 (t)
1.62 – 1.65,
31.9 (t)
1.67 – 1.69 (m, 2 H)
1.62 – 1.65 (2m)
1.52 – 1.54 (m)
1.57 – 1.60 (m)
3.45 (t, J ¼ 10.5),
3.54 – 3.57 (m)
0.68 (d, J ¼ 5.5)
1.65 – 1.68 (2m)
1.53 – 1.55 (m)
30.5 (d) 1.57 – 1.59 (m)
CH2(24)
HꢀC(25)
CH2(26)
30.2 (t)
29.3 (t)
30.7 (d) 1.72 – 1.75 (m)
66.9 (t)
1.57 – 1.59 (m)
28.9 (t)
30.5 (d)
66.7 (t)
67.0 (t)
3.49 (t, J ¼ 10.5),
3.44 – 3.46,
3.47 – 3.49 (2m)
3.55 – 3.57 (m)
Me(27)
Glc:
17.3 (q) 0.67 (d, J ¼ 5.2)
17.4 (q) 0.64 (d, J ¼ 5.8)
17.3 (q)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
CH2(6’)
4.97 (d, J ¼ 7.0)
4.20 – 4.22 (m)
4.22 – 4.25 (m)
4.40 – 4.43 (m)
3.52 – 3.55 (m)
4.02 (d, J ¼ 11.1),
4.15 (d, J ¼ 11.6)
100.4 (d) 4.95 (d, J ¼ 7.5)
78.2 (d) 4.19 – 4.22 (m)
77.8 (d) 4.22 – 4.25 (m)
78.0 (d) 4.40 – 4.42 (m)
77.0 (d) 3.58 – 3.60 (m)
100.4 (d) 4.87 (d, J ¼ 7.7)
78.1 (d) 4.18 – 4.21 (m)
77.6 (d) 4.21 – 4.23 (m)
77.7 (d) 4.40 – 4.43 (m)
77.1 (d) 3.60 – 3.62 (m)
100.4 (d)
78.1 (d)
77.8 (d)
78.1 (d)
77.0 (d)
61.4 (t)
61.2 (t)
4.05 (dd,
61.3 (t)
4.05 – 4.07 (m),
4.19 (d, J ¼ 13.0)
J ¼ 3.0, 13.0),
4.21 – 4.23 (m)

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Helvetica Chimica Acta – Vol. 95 (2012)
2a)
Table (cont.)
1a)
3b)
d(H)
d(C)
d(H)
d(C)
d(H)
d(C)
Rha:
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
HꢀC(5’’)
Me(6’’)
6.41 (br. s)
102.2 (d)
72.5 (d)
72.9 (d)
74.2 (d)
69.6 (d)
18.7 (q)
6.39 (br. s)
102.2 (d)
72.5 (d)
73.0 (d)
74.2 (d)
69.6 (d)
18.7 (q)
6.41 (br. s)
102.2 (d)
72.5 (d)
72.9 (d)
74.2 (d)
69.6 (d)
18.7 (q)
4.86 – 4. 88 (m)
4.62 – 4.64 (m)
4.35 – 4.37 (m)
4.96 – 4.98 (m)
1.59 (d, J ¼ 5.8)
4.85 – 4.87 (m)
4.62 – 4.65 (m)
4.37 – 4.39 (m)
4.95 – 4.97 (m)
1.59 (d, J ¼ 6.3)
4.86 – 4.88 (m)
4.66 – 4.69 (m)
4.38 – 4.40 (m)
4.95 – 4.98 (m)
1.59 (d, J ¼ 6.0)
Rha:
HꢀC(1’’’)
HꢀC(2’’’)
HꢀC(3’’’)
HꢀC(4’’’)
HꢀC(5’’’)
Me(6’’’)
Rha:
5.85 (br. s)
102.3 (d)
72.9 (d)
73.3 (d)
80.5 (d)
68.4 (d)
18.9 (q)
5.84 (br. s)
102.3 (d)
72.9 (d)
73.4 (d)
80.5 (d)
68.4 (d)
18.9 (q)
5.84 (br. s)
102.3 (d)
72.9 (d)
73.3 (d)
80.5 (d)
68.4 (d)
18.9 (q)
4.53 – 4.55 (m)
4.55 – 4.58 (m)
4.42 – 4.44 (m)
4.94 – 4.96 (m)
1.59 (d, J ¼ 5.8)
4.52 – 4.54 (m)
4.56 – 4.58 (m)
4.44 – 4.47 (m)
4.93 – 4.95 (m)
1.59 (d, J ¼ 6.3)
4.52 – 4.54 (m)
4.57 – 4.59 (m)
4.45 – 4.48 (m)
4.93 – 4.95 (m)
1.59 (d, J ¼ 6.0)
HꢀC(1’’’’)
HꢀC(2’’’’)
HꢀC(3’’’’)
HꢀC(4’’’’)
HꢀC(5’’’’)
Me(6’’’’)
6.29 (br. s)
103.3 (d)
72.7 (d)
72.9 (d)
74.1 (d)
70.4 (d)
18.5 (q)
6.29 (br. s)
103.4 (d)
72.7 (d)
73.0 (d)
74.1 (d)
70.5 (d)
18.5 (q)
6.29 (br. s)
103.3(d)
72.7 (d)
72.9 (d)
74.1 (d)
70.4 (d)
18.5 (q)
4.91 – 4.93 (m)
4.54 – 4.56 (m)
4.28 – 4.30 (m)
4.95 – 4.97 (m)
1.77 (d, J ¼ 6.2)
4.90 – 4.93 (m)
4.55 – 4.57 (m)
4.31 – 4.33 (m)
4.96 – 4.98 (m)
1.75 (d, J ¼ 6.2)
4.92 – 4.94 (m)
4.55 – 4.57 (m)
4.32 – 4.35 (m)
4.97 – 4.99 (m)
1.76 (d, J ¼ 6.1)
a) Recorded at 400 MHz. b) Recorded at 500 MHz.
unit at C(4’) of the inner glucopyranosyl unit, and another terminal rhamnopyranosyl
unit at C(4’’’) of the inner rhamnopyranosyl unit. Therefore, the structure of 1 was
established as (3b,11a,25R)-3,11-dihydroxyspirost-5-en-12-one 3-{O-a-l-rhanmopy-
ranosyl-(1 ! 4)-O-a-l-rhanmopyranosyl-(1 ! 4)-O-[a-l-rhanmopyranosyl-(1 ! 2)]-b-
d-glucopyranoside}, and named ypsilandroside M.
Compound 2 was obtained as a colorless amorphous powder. Its molecular formula
C51H82O21 was determined by the HR-ESI-MS (m/z 1029.5274 ([M ꢀ H]ꢀ) and
required eleven degrees of unsaturation. The 1H-NMR spectrum of 2 (Table) showed
signals for four steroid Me groups (d(H) 0.67 (d, J ¼ 5.2 Hz), 0.86 (s), 1.00 (s), and 1.15
(d, J ¼ 7.0 Hz)), as well as signals for four anomeric H-atoms (d(H) 4.95 (d, J ¼ 7.5 Hz ) ,
5.84 (br. s), 6.29 (br. s), and 6.39 (br. s)). Comparison of the NMR spectra of the
aglycone moiety of 2 with those of diosgenin (¼(3b,25R)-spirost-5-en-3-ol) revealed
that the d(H) and d(C) for the rings of A and C – F of 2 were in good agreement with
those of diosgenin, but indicated the lack of a CH2 group and the presence of an O-
bearing CH group (d(C) 72.5 (d)) in ring B [11]. This suggested that that the CH2 group
at C(7) was oxygenated to an O-bearing CH group in 2, which was corroborated by the
1H,1H correlations of the olefinic H-atom at d(H) 5.68 (br. s, HꢀC(6)) with the proton
linked to an O-bearing C-atom (d(C) 72.5 (d)) at d(H) 4.00 – 4.03 (m, HꢀC(7)). The
location of OHꢀC(7) also could be confirmed by the HMBCs HꢀC(7)/C(5), C(6),
C(8), C(9), and C(14). The b-orientation of OHꢀC(7) was determined by the ROESY

Helvetica Chimica Acta – Vol. 95 (2012)
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correlations of HꢀC(7)/Heq – C(4), HꢀC(9), and HꢀC(14) (Fig. 2), which was
confirmed by the chemical shift of C(7) (d(C) 72.5), while the signal for C(7) would
appear at d(C) ca. 64.6 for the 7a-isomer [12 – 15]. Hence, the aglycone of 2 was
identified as (3b,7b,25R)-spirost-5-ene-3,7-diol. The 13C-NMR signals arising from the
tetraasaccharide moiety composed of one b-d-glucopyranosyl and three a-l-rhamno-
pyranosyl units were in good agreement with those of 1. On the basis of the above
evidence, the structure of 2 was established as (3b,7b,25R)-spirost-5-ene-3,7-diol 3-{O-
a-l-rhanmopyranosyl-(1 ! 4)-O-a-l-rhanmopyranosyl-(1 ! 4)-O-[a-l-rhanmopyrano-
syl-(1 ! 2)]-b-d-glucopyranoside}, and named ypsilandroside N.
Fig. 2. Key ROESY correlations for the aglycones of compounds 2 and 3
Compound 3 was isolated as colorless amorphous powder. The negative-ion HR-
ESI-MS of 3 displayed a peak at m/z 1045.5199 ([M ꢀ H]ꢀ), corresponding to the
molecular formula C51H82O22, which differs from that of 2 by one additional O-atom.
Its 13C-NMR spectrum showed 51 resonance lines which included 5 quaternary C-
atoms, 29 CH groups, 10 CH2 groups, and 7 Me groups. Among them, 27 signals were
assigned to the aglycone part and 24 to four monosaccharide units. The above data
revealed that 3 should be a spirostane glycoside similar to 2. A major difference
between the two molecules was the appearance of an O-bearing quaternary C-atom
(d(C) 90.3) in compound 3. The downfield shifts of C(13) (Dd þ 4.0), C(16) (Dd þ 8.7),
and C(20) (Dd þ 2.9) with respect to 2 and the HMBCs C(17) (d(C) 90.3)/CH2(12)
(d(H) 1.67 – 1.69 (m)), HꢀC(14) (d(H) 3.03 – 3.05 (m)), CH2(15) (d(H) 2.12 – 2.15 and
2.72 – 2.74 (2m)), HꢀC(16) (d(H) 4.55 – 4.58 (m)), Me(18) (d(H) 1.03 (s)), HꢀC(20)
(d(H) 2.21 – 2.24 (m)), and Me(21) (d(H) 1.25 (d, J ¼ 7.1 Hz)) confirmed that 3 had an
OH group at C(17). A second major difference was the upfield shift of C(7) (d(C) 64.7)
compared to 2 (Dd ꢀ 7.8). This indicated the a-orientation of OHꢀC(7) which could be
further confirmed by the ROESY correlations HꢀC(7)/HꢀC(8), HaxꢀC(15), and
HeqꢀC(15) (Fig. 2). Thus, the structure of 3 was identified as (3b,7a,25R)-spirost-5-
ene-3,7,17-triol 3-{O-a-l-rhanmopyranosyl-(1 ! 4)-O-a-l-rhanmopyranosyl-(1 ! 4)-
O-[a-l-rhanmopyranosyl-(1 ! 2)]-b-d-glucopyranoside}, and named ypsilandroside O.
Ypsilandroside N (2) is a rare spirostane saponin, whose aglycone contains a 7b-OH
group. Only one spirostane sapogenin and three spirostane saponins, which contained
7b-OH group, were previously reported. They were isolated from Paris pollyphylla
Smith var. yunnanensis (Liliaceae) [13], Dioscorea septemloba (Dioscoreaceae) [15],
and Urginea sanguinea (Hyacynthaceae) [16]. Ypsilandroside O (3) is the first report
of a 7-hydroxylated pennogenin (¼(3b,25R)-spirost-5-ene-3,17-diol) saponin. Since
steroidal saponins are reported to possess cytotoxic activity against various cancer cell
lines [1], the cytotoxic activities of compounds 1 – 3 were evaluated against five tumor

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Helvetica Chimica Acta – Vol. 95 (2012)
cell lines (MCF-7, SW480, A-549, HL-60, and SMMC-7721). Cisplatin and taxol were
used as the positive control. The results showed that none of the tested compounds had
any discernible cytotoxic activity against these cell lines (IC50 > 40 mm).
This work was financially supported by the National Natural Science Funding of China (No.
31170333), the Natural Science Foundation of Yunnan Province (No. 2009CC019), the West Light
Foundation of the Chinese Academy of Sciences (No. 2908025712W1), and a fund (No. 540806321211) of
the State Key Laboratory of Phytochemistry and Plant Resources in West China, and the Germplasm
Bank of Wild Species and CAS Innovation Program of the Kunming Institute of Botany.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2, 200 – 300 mesh; Qingdao Marine Chemical
Inc., P. R. China), SiO2 H (10 – 40 mm; Qingdao Marine Chemical Inc.), or Lichroprep Rp-18 (43 – 63 mm;
Merck). TLC: visualization by heating SiO2 plates sprayed with 10% H2SO4 in EtOH. GC: Shimadzu-
GC-2010 instrument; H2 flame ionization detector. Semi-prep. HPLC: Agilent-1100 apparatus; Zorbax-
SB-C-18 column (9.4 mm ꢂ 25 cm, 5 mm; Agilent); tR in min. Optical rotations: Horiba-SEAP-300
polarimeter. IR Spectra: Bio-Rad-FTS-135 spectrometer; KBr pellets; ˜n in cmꢀ1 1H- and 13C-NMR
.
Spectra: Bruker-AM-400 (400 and 100 MHz) and -DRX-500 (500 and 125 MHz) instruments; d in ppm
rel. to Me4Si as internal standard, J in Hz. FAB-MS: VG-Auto-Spec-3000 mass spectrometer; in m/z. ESI-
MS and HR-ESI-MS: API-QSTAR-TOF spectrometer; in m/z.
Plant Material. The whole plants of Y. thibetica were collected in November 2006 from Luding
County, Sichuan Province, P. R. China, and identified by Prof. Xin-Qi Chen, Institute of Botany, Chinese
Academy of Sciences, Beijing. A voucher specimen (No. HY0002) was deposited with the State Key
Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany.
Extraction and Isolation. The powdered air-died plants of Y. thibetica (10 kg) were exhaustively
extracted three times with 70% EtOH (3 ꢂ 50 l) under reflux. After evaporation, the resulting residue
was passed through a YWD-3F macroporous resin column eluted with EtOH/H2O 0 :1, 4 :6, 7:3, and 1:0.
The 70% EtOH fraction (70 g) was subjected to CC (SiO2, CHCl3/MeOH/H2O 10 :1:0 ! 7:3 :0.5 (v/v)):
Fractions 1 – 4. Fr. 3 (10.5 g) was subjected to MPLC (Rp-18, MeOH/H2O 6 :4 ! 8 :2): Fr. 3.1 – 3.3.
Subfractions of interest were purified by semi-prep. HPLC (MeOH/H2O 65 :35; flow rate 3 ml/min): 1
(9 mg; tR 12.4), 2 (6 mg; tR 37.0), and 3 (7 mg; tR 9.6).
Ypsilandroside M (¼(3b,11a,25R)-3-[(O-6-Deoxy-a-l-mannopyranosyl-(1 ! 4)-O-6-deoxy-a-l-
mannopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyranosyl-(1 ! 2)]-b-d-glucopyranosyl)oxy]-11-hy-
droxyspirost-5-en-12-one; 1): Colorless amorphous powder. [a]1D8 ¼ ꢀ113.5 (c ¼ 0.1, MeOH). IR (KBr):
3441, 2932, 2875, 1710, 1638, 1456, 1383, 1051, 981, 919, 899, 866 (intensity: 899 > 919). 1H- and 13C-NMR:
Table. ESI-MS (neg.): 1043 ([M ꢀ H]ꢀ). HR-ESI-MS: 1043.5057 ([M ꢀ H]ꢀ, C51H79O2ꢀ2 ; calc.
1043.5063).
Ypsilandroside N (¼(3b,7b,25R)-7-Hydroxyspirost-5-en-3-yl O-6-Deoxy-a-l-mannopyranosyl-(1 !
4)-O-6-deoxy-a-l-mannopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyranosyl-(1 ! 2)]-b-d-glucopyrano-
side; 2): Colorless amorphous powder. [a]2D5 ¼ ꢀ79.5 (c ¼ 0.15, CHCl3/MeOH 1:1). IR (KBr): 3423, 2935,
2874, 1648, 1457, 1385, 1051, 981, 919, 910, 898, 865 (intensity: 898 > 910). 1H- and 13C-NMR: Table. FAB-
MS (neg.): 1029 ([M ꢀ H]ꢀ), 883 ([M ꢀ H ꢀ C6H10O4]ꢀ), 737 ([M ꢀ H ꢀ 2C6H10O4]ꢀ). HR-ESI-MS:
1029.5274 ([M ꢀ H]ꢀ, C51H81O2ꢀ1 ; calc. 1029.5270).
Ypsilandroside O (¼(3b,7a,25R)-7,17-Dihydroxyspirost-5-en-3-yl O-6-Deoxy-a-l-mannopyranosyl-
(1 ! 4)-O-6-deoxy-a-l-mannopyranosyl-(1 ! 4)-O-[6-deoxy-a-l-mannopyranosyl-(1 ! 2)]-b-d-gluco-
pyranoside; 3): Colorless amorphous powder. [a]2D5 ¼ ꢀ103.3 (c ¼ 0.30, CHCl3/MeOH 1:1). IR (KBr):
3417, 2932, 2876, 1664, 1455, 1384, 1053, 980, 954, 913, 896, 867 (intensity: 896 > 913). 1H- and 13C-NMR:
Table. FAB-MS (neg.): 1046 (Mꢀ), 900 ([M ꢀ C6H10O4]ꢀ), 754 ([M ꢀ 2C6H10O4]ꢀ). HR-ESI-MS:
1045.5199 ([M ꢀ H]ꢀ, C51H81O2ꢀ2 ; calc. 1045.5219).
Acid Hydrolysis and GC Analysis. Each compound 1 – 3 (2 mg) was refluxed in 4m CF3COOH/
dioxane 1:1 ((v/v), 2 ml) on a water bath for 4 h. After cooling, the mixture was extracted with CHCl3

Helvetica Chimica Acta – Vol. 95 (2012)
1093
(3 ꢂ 5 ml). The aq. layer was concentrated with MeOH until neutral. The dried residue was dissolved in
1 ml anh. pyridine and treated with l-cysteine methyl ester hydrochloride (1.5 mg) under stirring at 608
for 1 h. Then 1-(trimethylsilyl)-1H-imidazole (1.0 ml) was added and the mixture kept at 608 for 30 min.
The supernatant (4 ml) was analyzed by GC (H2 flame ionization detector; 30QC2/AC-5 quartz capillary
column (30 m ꢂ 0.32 mm); column temp. 180 – 2808, heating rate 38/min; carrier gas N2 (1 ml/min);
injector temp. 2508; split ratio 1:50). The configurations of d-glucose and l-rhamnose (¼6-deoxy-l-
mannose) for compounds 1 – 3 were determined by comparison of the retentions times of the
corresponding derivatives with those of standard d-glucose and l-rhamnose, giving a single peak at 19.01
and 15.43 min, resp.
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Received September 19, 2011