MRC Letter

Received: 29 July 2011

Revised: 31 December 2011

Accepted: 5 January 2012

Published online in Wiley Online Library: 10 February 2012

(wileyonlinelibrary.com) DOI 10.1002/mrc.3797

Structure elucidation and complete NMR

spectral assignments of glucosylated saponins
of cantalasaponin I
Li-ping Kang,a,b Yong-ze Wang,a,b Bing Feng,a Hong-zhi Huang,b
Wen-bin Zhou,a Yang Zhao,a Cheng-qi Xiong,a Da-wei Tan,a
Xin-bo Songb and Bai-ping Maa*
Five new glucosylated steroidal glycosides, cantalasaponin I-B1 (1), I-B2 (2), I-B3 (3), I-B4 (4) and I-B5 (5), were isolated and
puried from the transformed product of the cantalasaponin I by using Toruzyme 3.0 l as biocatalyst. Their structures were
elucidated on the basis of high-resolution electrospray ionization mass spectrometry, one-dimensional (1H and 13C NMR)
and two-dimensional [COSY, heteronuclear single-quantum correlation (HSQC), HMBC and HSQC-TOCSY] NMR spectral analyses and chemical evidence. Copyright 2012 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: NMR; 1H NMR; 13C NMR; 2D NMR; steroidal saponins; glucosylation; cantalasaponin I

Introduction
Toruzyme 3.0 L, a kind of cyclodextrin glucanotransferase (CGTase),
is an enzyme, which is capable of catalyzing intramolecular and
intermolecular transglycosylations, as well as hydrolyzation of starch
and cyclodextrin.[1,2] CGTase has been used in the glucosylation of
natural products, such as hesperidin, hydroquinone and rutin.[3]
Cantalasaponin I, a spirostanoside from Agave sisalanta Perrine,
was reported to be able to inhibit the J TC226 cancer cells.[4,5] To
synthesize steroidal saponins with novel sugar chains for further
pharmacological research and to improve their poor solubility,
cantalasaponin I was employed as substrates to be glucosylated
by Toruzyme 3.0 L, and ve new glucosylated products were
isolated and identied. This paper reports the purication and identication of ve glucosylated products of cantalasaponin I (Fig. 1).

Result and Discussion

Magn. Reson. Chem. 2012, 50, 7983

* Correspondence to: Bai-ping Ma, Department of Biotechnology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China. E-mail:
mabaiping@sina.com

These authors contributed equally to this study.

a Beijing Institute of Radiation Medicine, Beijing, China

b Tianjin University of Traditional Chinese Medicine, Tianjin, China

Copyright 2012 John Wiley & Sons, Ltd.

79

Compound 1 was isolated as a white amorphous powder, showing

positive reaction to the LiebermannBurchard reagent and
negative reaction to the Ehrlich reagent. The molecular formula of
1 was determined as C51H84O25 on the basis of its high-resolution
electrospray ionization mass spectrometry (HRESIMS) at m/z
1119.5203 (calcd. 1119.5199 [M(C51H84O25)Na]+). Fast atom
bombardment mass spectrometry (FABMS) showed the main ions
at m/z 1119.0 [M+Na]+, 1097.0 [M+H]+, 1079.0 [M+H18]+, 917.2
[M+H18162]+, 755.3 [M+H18162
2]+, 593.3 [M+H18
162
3]+ and 431.4 [M+H18162
4]+, implied that 1 had four
hexose units. Comparing the 1H and 13C NMR data with cantalasaponin I,[5] the aglycone of 1 was identied as (23S, 25R)-5a- spirostane3b, 6a, 23-triol. The observation of four anomeric proton signals at d
5.08 (H-Glc1-1, d, J = 7.8 Hz), 5.78 (H-Glc2-1, d, J = 3.6 Hz), 5.91 (H-Glc31, d, J = 3.6 Hz) and 4.86 (H-Glc4-1, d, J = 7.8 Hz) in the 1H NMR

spectrum and four anomeric carbon signals at d 101.3, 103.0, 103.3

and 106.3 in the 13C NMR spectrum, and related in the heteronuclear
single-quantum correlation (HSQC) spectrum, also suggested that
this compound possesses four sugar moieties. The b-congurations
for two glucoses were judged by their relative large vicinal (3JHH)
coupling constants (7 Hz), and the a-congurations for the other
two glucoses were identied by their relative small vicinal (3JHH)
coupling constants (4 Hz).[68] The structure elucidation of the sugar
portion was achieved by COSY, HSQC, HSQC-TOCSY and HMBC
experiments. The HSQC-TOCSY experiment for the isolated anomeric
proton (d 5.08, 5.78, 5.91 and 4.86) signals at the uncrowned region
of the spectrum revealed four spin systems of the monosaccharide
units. For example, starting from the anomeric proton signal at d
5.08 (H-Glc1-1), correlations for H-1/C-1, H-1/C-2, H-1/C-3, H-1/C-4
and H-1/C-5 were observed, but correlation for H-1/C-6 in this glucose unit could not be obtained. Starting from proton signal at d
3.57 (H-Glc1-5), correlations for H-5/C-4, H-5/C-5 and H-5/C-6 were
observed (Fig. 2). Analysis of the HSQC and COSY experiments
allowed the sequential assignments of all proton and carbon
resonances within each sugar residue, starting from the well-isolated
proton signals (Table 1). The sequence of sugar chains and their
linkage sites to the aglycone moiety were deduced from the HMBC
spectrum, in which long-range correlations were observed from d
5.08 (H-Glc1-1) to d 76.8 (C-3), d 5.78 (H-Glc2-1) to d 81.9 (C-Glc1-4),

L. Kang et al.

Figure 1. Structure of compounds 15.

80

d 5.91 (H-Glc3-1) to d 81.9 (C-Glc1-4), and d 4.86 (H-Glc4-1) to d

80.0 (C-6). Thus, the structure of 1 was identied as (23S, 25R)-5aspirost-3b, 6a, 23-triol-3-O-{a-D-glucopyranosyl-(1!4)-a-D-glucopyranosyl-(1!4)-b-D-glucopyranoside} 6-O-b-D-glucopyranoside,
named cantalasaponin I-B1.
Compound 2 was isolated as a white amorphous powder. The
molecular formula of 2 was determined to be C45H74O20 on the
basis of its HRESIMS at m/z 957.4650 (calcd. 957.4666 [M+Na]+).
The main fragment ions at m/z 917.2 [M+H18]+, 755.2 [M+H
18162]+, 593.3 [M+H 18162
2]+, 431.3 [M+H18162
3]+
and 413.3 [M+H18
2162
3]+ in FABMS implied that 2 had

wileyonlinelibrary.com/journal/mrc

three hexose units. Comparison of the NMR spectra with 1

(Table 1) indicated that 2 has the same skeleton but different
number of sugar units in the molecule. The 1H NMR spectrum
displayed three anomeric proton signals at d 5.02 (1H, d,
J = 7.2 Hz), 4.85 (1H, d, J = 7.8 Hz) and 5.88 (1H, d, J = 3.6 Hz), which
were attributed to the H-1 of two b-glucoses and one a-glucose.
In HSQC-TOCSY spectrum, starting from the anomeric proton
signal at d 5.88, correlations for H-1/C-1, H-1/C-2, H-1/C-3 and
H-1/C-4 were observed, and starting from proton signal at d
4.16, correlations for H-4/C-4, H-4/C-5 and H-4/C-6 were
observed. In the HMBC spectrum, the anomeric proton signals
at d 5.02 (H-Glc1-1), 5.88 (H-Glc2-1) and 4.85 (H-Glc3-1) showed
long-range correlations with the carbon signals at d 77.0 (C-3),
81.4 (C-Glc1-4) and 80.0 (C-6), respectively. On the basis of
these data, 2 was identied as (23S, 25R)-5a-spirost-3b, 6a,
23-triol-3-O-{a-D-glucopyranosyl-(1!4)-b-D-glucopyranoside}
6-O-b-D-glucopyranoside, named cantalasaponin I-B2.
Compound 3 was isolated as a white amorphous powder.
HRESIMS spectrum of 3 showed ion at m/z 1095.5238 (calcd.
1095.5223 [C51H83O25]), indicating the molecular formula also
was C51H84O25. FABMS showed similar ions to compound 1.
Comparing the 1H and 13C NMR (Table 1) and HMBC data with
1, compound 3 has the same skeleton and substituent groups,
the structure difference existed in the sugar sequence and their
linkage site. Anomeric region in the 1H and 13C NMR spectra of
3 showed signals for four anomeric proton signals at d 5.03 (1H,
d, J = 7.8 Hz), 5.89 (1H, d, J = 4.2 Hz), 4.77 (1H, d, J = 7.8 Hz) and
5.92 (1H, d, J = 3.6 Hz) with their corresponding anomeric carbon
signals at d 101.4, 103.3, 106.1 and 13.1, respectively. In the HMBC
spectrum, the proton signals at d 5.03 (H-Glc1-1), 5.89 (H-Glc2-1),
4.77 (H-Glc3-1) and 5.92 (H-Glc4-1) showed long-range correlations with carbon signals at d 76.9 (C-3), 81.5 (C-Glc1-4), 80.0
(C-6) and 81.1 (C-Glc3-4), respectively. Thus, 3 was identied
as (23S,25R)-5a-spirost-3b, 6a, 23-triol-3-O-{a-D-glucopyranosyl(1!4)-b-D-glucopyranoside} 6-O-{a-D- glucopyranosyl-(1!4)-bD-glucopyranoside}, named cantalasaponin I-B3.
Compound 4 was isolated as a white amorphous powder. 4
was identied as an isomer of 1 and 3 by its HRESIMS at m/z
1095.5267 (calcd. 1095.5223 [C51H83O25]). Comparison of the
MS and 1H and 13C NMR of 4 with those of 1 and 3 revealed that
they have the same aglycone and their structural difference in
the sugar sequence and their linkage site. The 1H NMR spectrum
displayed four anomeric proton signals at d 5.10 (1H, d, J = 7.8 Hz),
4.84 (1H, d, J = 7.8 Hz), 5.82 (1H, d, J = 3.6 Hz) and 5.90 (1H, d,
J = 4.2 Hz) which were attributed to the H-1 of two b-glucoses
and two a-glucoses, respectively. The HMBC spectrum of 4
showed the long-range correlations between d 5.10 (H-Glc1-1)
and d 76.9 (C-3), d 4.84 (H-Glc2-1) and d 80.0 (C-6), d 5.82
(H-Glc3-1) and d 81.6 (C-Glc2-4), and d 5.90 (H-Glc4-1) and d 81.7
(C-Glc3-4), indicated the sugar sequence and their linkage
sites to the aglycone moiety. So, 4 was identied as
(23S, 25R)-spirost-5a-3b, 6a, 23-triol-3-O-b-D-glucopyranoside
6-O-{a-D-glucopyranosyl-(1!4)-a-D-glucopyranosyl-(1!4)-b-Dglucopyranoside}, named cantalasaponin I-B4.
Compound 5 was isolated as a white amorphous powder. The
molecular formula of 5 was determined as C45H74O20 on the basis
of its HRESIMS at m/z 957.4650 (calcd. 957.4666 [M+Na]+).
Comparing the MS and 1H and 13C NMR with 2, the structural
difference of 5 could be deduced in the sugar sequence. The
1
H NMR spectrum displayed three anomeric proton signals at d
5.10 (1H, d, J = 7.8 Hz), 4.77 (1H, d, J = 7.8 Hz) and 5.92 (1H, d,
J = 3.8 Hz), which were attributed to the H-1 of two b-glucoses

Copyright 2012 John Wiley & Sons, Ltd.

Magn. Reson. Chem. 2012, 50, 7983

NMR study on glucosylated saponins of cantalasaponin I

Figure 2. Heteronuclear single-quantum correlation-TOCSY spectrum of compound 1.

Table 1. The 1H (600 MHz) and 13C NMR (150 MHz) data of compounds 15 (pyridine-d5)
No.

1
dC

dH J (Hz)

37.5

29.8

3
4

76.8
28.4

5
6
7

50.9
80.0
41.3

0.80 (ddd,
3.3, 10.3, 13.6)
1.49 (ma)
1.62 (m)
1.96 (ob)
3.94 (m)
1.41 (m)
3.39 (br d, 11.8)
1.23 (br t, 11.6)
3.55 (m)
1.16 (o)
2.57 (br d, 12.4)

8
9

33.9
53.8

10
11

36.7
21.2

12

40.3

13
14
15

41.5
56.3
32.0

16
17
18

81.6
62.5
16.9

1.50 (m)
0.53 (ddd,
3.0, 8.5, 11.5)

1.14 (m)
1.34 (br d, 12.0)
1.04 (dt, 4.8, 11.9)
1.67 (br d, 11.9)

1.03 (o)
1.43 (m)
1.98 (o)
4.54 (m)
1.84 (dd, 7.4, 8.3)
0.95 (s)

2
dC
37.5

29.8
77.0
28.5
50.9
80.0
41.3

33.9
53.8
36.7
21.2
40.3
41.5
56.3
32.0

81.6
62.5
16.9

3
dH J (Hz)

0.81(ddd,
3.6, 10.2, 13.8)
1.48 (br d, 13.8)
1.61 (m)
1.99 (m)
3.93 (m)
1.40 (m)
3.37 (br d, 13.2)
1.23 (br t, 13.2)
3.51 (m)
1.13 (m)
2.58 (dt, 12.6, 4.2)
1.51 (m)
0.52 (ddd,
3.6, 7.8, 11.4)

1.11 (m)
1.35 (br d, 13.8)
1.04 (dt, 5.1, 12.0)
1.67 (br d, 12.0)

1.03 (o)
1.39 (m)
1.97 (o)
4.52 (o)
1.84 (dd, 7.8, 8.4)
0.94 (s)

dC
37.5

29.7
76.9
28.4
50.8
80.0
41.3

33.9
53.8
36.7
21.2
40.3
41.4
56.3
32.0

81.6
62.5
16.9

4
dH J (Hz)

0.79 (ddd,
3.6, 9.6, 13.2)
1.47 (br d, 13.2)
1.60 (m)
1.97 (m)
3.93 (m)
1.38 (m)
3.33 (br d, 12.6)
1.21 (br t, 12.6)
3.53 (m)
1.11 (m)
2.48 (dt, 12.6,
4.2)
1.47 (o)
0.51 (ddd,
4.2, 7.8, 12.0)

1.12 (m)
1.34 (br d, 13.2)
1.04 (m)
1.67 (br d, 12.0)

1.02 (m)
1.40 (m)
1.91 (ddd,
6.6, 8.4, 12.0)
4.54 (m)
1.84 (dd, 7.8, 7.8)
0.95 (s)

dC
37.5

29.8
76.9
28.5
50.9
80.0
41.3

dH J (Hz)
0.79 (ddd,
3.6, 9.6, 13.2)
1.47 (br d, 13.2)
1.62 (m)
2.00 ( m)
3.94 (m)
1.41 (m)
3.35 (br d, 11.5)
1.22 (br t, 12.6)
3.53 (m)
1.13 (m)
2.52 (dt, 12.6, 4.2)

dC
37.5

29.8
76.9
28.5
50.8
80.0
41.3

33.9
53.8

1.46 (o)
0.51 (br t, 8.7 )

33.9
53.8

36.7
21.2

1.10 (m)
1.34 (m)
1.04 (m)
1.66 (br d, 12.0)

1.03 (m)
1.42 (m)
1.94 (ddd, 5.5,
6.4, 12.0)
4.52 (o)
1.85 (dd, 7.2, 8.5)
0.94 (s)

36.7
21.2

40.3
41.4
56.3
32.0

81.6
62.5
16.9

40.3
41.5
56.3
32.0

81.6
62.5
16.9

dH J (Hz)
0.78 (ddd,
3.2, 10.3, 13.4)
1.48 m
1.64 (m)
2.00 (m)
3.94 (m)
1.39 (m)
3.34 (br d, 12.1)
1.21 (br t, 12.6)
3.51 (m)
1.13 (m)
2.50 (dt, 12.6, 4.2)
1.48 (m)
0.51(ddd, 3.6,
11.4, 15.0)

1.10 (m)
1.34 (br d, 13.3)
1.04 (m)
1.67 (br d, 11.6)

1.05 (m)
1.40 (m)
1.92 (ddd, 5.5,
6.8, 12.2)
4.52 (m)
1.84 (dd, 7.2, 8.6)
0.95 (s)

81

Magn. Reson. Chem. 2012, 50, 7983

Copyright 2012 John Wiley & Sons, Ltd.

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L. Kang et al.

Table 1. (Continued)
No.

1
dC

dH J (Hz)

dC

dH J (Hz)

dC

dH J (Hz)

dC

13.3
35.8
14.7
111.7
67.5
38.8

13.3
35.8
14.7
111.7
67.4
38.8

0.61 (s)
3.00 (dq, 6.6, 7.2)
1.17 (d, 7.2)

3.83 (dd, 4.5, 11.1)

1.75 (dd, 11.1, 12.0)

13.3
35.8
14.7
111.7
67.5
38.8

31.8
66.0

31.8
66.0

2.09 (m)
1.79 (m)
3.46 (dd, 10.8, 11.4)

31.8
66.0

16.9

0.60 (s)
3.00 (dq, 6.6, 7.2)
1.17 (d, 7.2)

3.83 (m)
1.76 (dd, 11.6,
12.0)
2.09 (m)
1.80 (m)
3.46 (dd, 10.8,
11.4)
3.52 (m)
0.74 (d, 6.3)

16.9

3.53 (br d, 11.4)

0.74 (d, 6.0)

16.9

dH J (Hz)

dC

dH J (Hz)

0.60 (s)
3.00 (dq, 6.6, 7.0)
1.17 (d, 7.0)

3.83 (o)
1.75 (dd, 11.4,
12.7)
2.09 (m)
1.79 (m)
3.46 (dd, 10.2,
10.8)
3.53 (m)
0.74 (d, 6.3)

13.3
35.8
14.7
111.7
67.5
38.8

0.60 (s)
3.00 (dq, 6.6, 7.2)
1.17 (d, 6.6)

3.83 (o)
1.76 (dd, 11.4,
11.7)
2.09 (br d, 11.7)
1.80 (m)
3.46 (dd, 10.5,
11.1)
3.53 (m)
0.74 (d, 6.0)

19
20
21
22
23
24

13.3
35.8
14.7
111.7
67.5
38.8

25
26

31.8
66.0

0.61 (s)
3.00 (dq, 6.6, 6.9)
1.17 (d, 6.9)

3.83 (m)
1.75 (dd, 11.6,
12.0)
2.09 (m)
1.80 (m)
3.44 (dd, 9.3, 11.4)

27
Glu1
1
2
3
4
5
6

16.9

3.53 (m)
0.73 (d, 6.2)

101.3
74.9
77.8
81.9
76.6
61.7

5.08 (d, 7.8)

4.01 (o)
4.33 (m)
4.30 (m)
3.57 (br d, 7.2 )
4.27 (m)
4.30 (m)

101.5
74.9
77.8
81.4
76.5
61.8

5.02 (d, 7.2)

4.00 (o)
4.33 (o)
4.36 (o)
3.56 (m)
4.28 (br d 11.4)
4.36 (o)

101.4
74.9
77.9
81.5
76.6
61.8

5.03 (d, 7.8)

4.01 (dd, 7.8, 8.4)
4.34 (o)
4.35 (o)
3.58 (m)
4.31 (m)
4.38 (dd, 3.6, 12.0)

101.6
75.5
78.7
71.8
78.2
62.7

5.10 (d, 7.8)

4.04 (dd 7.8, 8.4)
4.26 (m)
4.24 (m)
3.84 (o)
4.33 (m)
4.51 (m)

101.6
75.5
78.6
71.8
78.2
62.7

5.10 (d, 7.8)

4.04 (dd, 7.8, 8.4)
4.26 (o)
4.24 (o)
3.83 (o)
4.32 (o)
4.43 (br d, 11.2)

Glu2
1
2
3
4
5
6

103.0
74.0
75.1
81.7
73.5
61.9

5.78 (d, 3.6)

4.10 (m)
4.67 (dd 9.0, 9.6)
4.25 (m)
4.38 (m)
4.39 (m)
4.45 (br d, 10.8)

103.2
74.6
75.5
71.9
75.3
62.8

5.88 (d, 3.6)

4.16 (br d 9.0)
4.57 (dd 9.0, 9.6)
4.17 (o)
4.53 (o)
4.33 (o)
4.52 (o)

103.3
74.5
75.5
71.9
75.3
62.8

5.89 (d, 3.6)

4.15 (o)
4.53 (o)
4.16 (o)
4.57 (m)
4.33 (o)
4.53 (o)

106.3
75.2
77.6
81.6
76.6
62.2

4.84 (d, 7.8)

3.99 (dd, 7.8, 9.0)
4.28 (o)
4.29 (o)
3.85 (o)
4.43 (o)
4.42 (o)

106.1
75.2
77.7
81.2
76.5
62.2

4.77 (d, 7.8)

3.99 (dd, 7.8, 8.7)
4.28 (dd, 8.7, 9.8)
4.35 (m)
3.77 (br d, 9.5)
4.44 (br d, 11.3)
4.51 (o)

103.3
74.5
75.5
71.9
75.3
62.7

5.91 (d, 3.6)

4.19 (m)
4.58 (dd, 9.1, 9.4)
4.17 (m)
4.56 (m)
4.32 (m)
4.52 (o)

106.3
75.7
78.0
71.9
78.5
63.2

4.85 (d, 7.8)

4.01 (o)
4.22 (o)
4.23 (o)
3.95 (m)
4.39 (m)
4.52 (o)

106.1
75.2
77.7
81.1
76.6
62.2

4.77 (d, 7.8)

3.99 (dd 7.8, 9.0)
4.27 (dd 9.0, 9.6)
4.33 (o)
3.77 (br d, 9.0)
4.44 (br d, 11.6)
4.50 (dd, 3.6, 11.6)

102.9
74.1
75.1
81.7
73.6
62.0

5.82 (d, 3.6)

4.11 (m)
4.68 (dd, 9.3, 9.5)
4.23 (o)
4.43 (o)
4.38 (m)
4.47 (br d, 12.0)

103.1
74.6
75.6
72.0
75.4
62.8

5.92 (d, 3.8)

4.17 (dd, 3.8, 9.2)
4.59 (dd, 9.2, 9.0)
4.16 (o)
4.58 (o)
4.33 (o)
4.56 (o)

106.3
75.7
78.0
71.8
78.5
63.2

4.86 (d, 7.8)

3.99 (m)
4.21 (o)
4.23 (o)
3.94 (m)
4.38 (m)
4.52 (o)

103.1
74.6
75.6
72.0
75.4
62.8

5.92 (d, 3.6)

4.16 (o)
4.59 (m)
4.16 (o)
4.57 (m)
4.33 (o)
4.56 (o)

103.2
74.6
75.5
71.9
75.3
62.5

5.90 (d, 4.2)

4.18 (o)
4.57 (dd, 7.0, 9.3)
4.17 (o)
4.53 (m)
4.33 (o)
4.43 (o)

31.8
66.0

16.9

Glu3

Glu4
1
2
3
4
5
6

m: multiplet signals.
o: overlapped with other signals.

82

and one a-glucose, respectively. In the HMBC spectrum, the

anomeric proton signals at d 5.10 (H-Glc1-1), 4.77 (H-Glc2-1) and
5.92 (H-Glc3-1) showed long-range correlations with carbon
signals at d 76.9 (C-3), 80.0 (C-6) and 81.2 (C-Glc2-4), respectively.
Thus, 5 was identied as (23S, 25R)-5a-spirost-3b, 6a, 23-triol-3-Ob-D-glucopyranoside 6-O-{a-D-glucopyranosyl-(1!4)-b-D-glucopyranoside}, named cantalasaponin I-B5.

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Experimental
General
HPLC was performed using Waters 2695 Alliance Separations
Module; Empower Pro. Detector: Alltech ELSD 2000, temperature:
100  C, gas ow: 2.4 L/min; Column: ODS (5 mm, 250
4.6 mm,
YMC, Japan); FABMS was measured on a Micromass Zabspec,

Copyright 2012 John Wiley & Sons, Ltd.

Magn. Reson. Chem. 2012, 50, 7983

NMR study on glucosylated saponins of cantalasaponin I

and HRESIMS was recorded on 9.4 T Q-FT-MS Apex Qe (Bruker
Co., Germany). TLC was performed on precoated kieselgel GF254
plate (0.20.25 mm, 200
100 mm, Qingdao Haiyang Chemical
Group Co., Qingdao, Peoples Republic of China) using CHCl3MeOH-H2O, and detection was achieved by spraying Ehrlich
and 10% H2SO4/EtOH solution reagent followed by heating. The
enzyme, Toruryme 3.0 L, was obtained from Novozymes, China.

Biotransformation of cantalasaponin I
Cantalasaponin I (900 mg) and dextrin (4500 mg) were dissolved
in 900 ml of 70% methanolwater, 4.5 ml of Toruzyme solution
were added to the substrate solution and allowed to react at
50  1  C for 24 h. The reaction mixtures were boiled to stop
the reaction.
Purication of converted products

NMR spectra
NMR spectra were recorded on Varian UNITYINOVA 600 spectrometer (599.8 MHz for 1H NMR and 150.8 MHz for 13C NMR) instrument in pyridine-d5 (1H, d 8.71, 7.55, 7.19 ppm; 13C, d 149.9,
135.5, 123.5 ppm) at 300 k. The chemical shifts were given in d
(ppm) with tetramethylsilane as an internal standard. All
compounds were carried out with the following parameters: 1H
NMR spectrum: spectral width 12 000 Hz, acquisition time 2.67 s,
relaxation delay 3.0 s and digital resolution 0.18 Hz/pt. 13C NMR
spectrum: spectral width 40 000 Hz, acquisition time 0.80 s, relaxation delay 1.0 s and digital resolution 0.61 Hz/pt. COSY spectrum:
spectral width 6000 Hz, acquisition time 0.17 s, FT size
2048
2048, relaxation delay 1.5 s. HSQC spectrum: spectral
width of the proton dimension 5800 Hz, spectral width of the carbon dimension 28 000 Hz, relaxation delay 1.0 s, data points
1024
128 complex points, data matrix for FT was 2048
1024
complex point, before FT, the data points of F1 were linear
predicted to 256. HMBC spectrum: spectral width of the proton
dimension 5800 Hz, spectral width of the carbon dimension
34 000 Hz, relaxation delay 1.0 s, data points for proton and
carbon were set as 1024
256 complex points, data for carbon
were linear predicted to 512 before FT, FT data matrix was
2048
1024 complex points. HSQC-TOCSY spectrum: spectral
width of the proton dimension 5800 Hz, spectral width of the
carbon dimension 28 000 Hz, data points for proton and carbon
were set as 1024
256 complex points, data points of F1 linear
predicated to 512, and mixing time was set at 80 ms.

The reaction mixtures were extracted three times with n-butanol,

and the n-butanol layer was concentrated in a vacuum to give
the crude extract. The crude extract was further separated with
prep. TLC of silica gel H and developed with CHCl3-CH3OH-H2O
(60: 36: 10, lower phase) to give four bands. The samples were
recovered with methanol and concentrated under reduced
pressure to give four fractions (Fr. 14). Fr. 2 was separated on
RP-C18 by HPLC, eluted with CH3CN-H2O (27: 73) to give
compounds 2 (32 mg) and 5 (18 mg); Fr. 3 was separated on a
RP-C18 by HPLC, eluted with CH3CN-H2O (26: 74) to give
compounds 1 (10 mg), 3 (9 mg) and 4 (9 mg).
Cantalasaponin IB1 (1), White amorphous powder; HR ESIMS:
m/z 1119.5203 (calcd for 1119.5199 [M(C51H84O25)Na]+); 1H and
13
C NMR data; Table 1.
Cantalasaponin IB2 (2), White amorphous powder, HR ESIMS:
m/z 957.4650 (calcd for 957.4666 [M(C45H74O20)Na]+); 1H and 13C
NMR data; Table 1.
Cantalasaponin IB3 (3), White amorphous powder, HR ESIMS:
m/z 1095.5238 (calcd for 1095.5223 [C51H83O25]); 1H and 13C NMR
data; Table 1.
Cantalasaponin IB4 (4), White amorphous powder, HR ESIMS:
m/z 1095.5267 (calcd for 1095.5223 [C51H83O25]); 1H and 13C NMR
data; Table 1.
Cantalasaponin IB5 (5), White amorphous powder, HR ESIMS:
m/z 957.4650 (calcd for 957.4666 [M(C45H74O20)Na]+); 1H and 13C
NMR data; Table 1.
Acknowledgements

Plant material
The material was collected from Hainan province, China, in August 2005 and was identied as Agave sisalanta by Prof. Jianmei Huang. A voucher specimen (No. 040123) was deposited in
the Herbarium of Beijing Institute of Radiation Medicine, Beijing.

This work was supported by the Major Program of Municipal

Natural Science Foundation of Beijing (7090001) and the National
Natural Science Foundation of China (30973632). MS and NMR
spectra were recorded at the National Center of Biomedical
Analysis.

References
Extraction and isolation
Fresh leaves of Agave sisalana Perrine were extracted three times
with 50% ethanolH2O. The extract was concentrated under
reduced pressure, chromatographed on macroporous resin
AB-8 and eluted with gradient acetoneH2O (10%, 45% and 80%)
to give three fractions (Fr. A-C). Fr. B was further chromatographed
on silica gel and eluted with solvent CHCl3-CH3OH-H2O (70:25:5,
v/v/v, lower phase) to give the crude cantalasaponin I, which was
recrystallized to give cantalasaponin I[5] (purity 99%, 1 g).

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