Leonticins A-C three octasaccharide saponins from Leontice kiangnanensis

Article abstract of DOI:10.1021/np960185g

Three octasaccharide saponins, leonticins A, B, and C (1-3), were isolated from the tubers of Leontice kiangnanensis. Their structures were elucidated by a combination of chemical degradation and spectral methods including negative FABMS and NMR measurements as 3-O-β-D-glucopyranosyl(1→ 2)-α-L-arabinopyranosylhederagenin 28-O-α-L-rhamnopyranosyl(1→4)-β-D- glucopyranosyl(1→6)-β-D-glucopyranosyl(1→4)-α-L-rhamnopyranosyl(1→4)- β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (1), 3-O-[β-D- xylopyranosyl(1→3)-β-D-galactopyranosyl(1→4)-β-D- glucopyranosyl(1→3)][β-D-glucopyranosyl(1→2)]-α-L- arabinopyranosyloleanolic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D- glucopyranosyl(1→6)-β-D-glucopyranoside (2), and 3-O-[β-D- xylopyranosyl(1→3)-β-D-galactopyranosyl(1→4)-β-D- glucopyranosyl(1→3)][β-D-glucopyranosyl(1→2)-α-L- arabinopyranosylechinocystic acid 28-O-α-L-rhamnopyranosyl(1→4)-β-D- glucopyranosyl(1→6)-β-D-glucopyranoside (3), respectively. The complete assignments of the proton and carbon resonances for 1-3 were achieved based on extensive 2D NMR analysis (DQF-COSY, TOCSY, ROESY, HSQC, and HMBC).

Full text of DOI:10.1021/np960185g

722
J . Nat. Prod. 1996, 59, 722-728
Leon ticin s A-C, Th r ee Octa sa cch a r id e Sa p on in s fr om Leon tice kia n gn a n en sis†
Min Chen,‡ Wei Wei Wu,§ and Otto Sticher*
Department of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich, Winterthurerstrasse 190,
CH-8057 Zu¨rich, Switzerland
Daniel Nanz
Department of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland
Received J anuary 17, 1996X
Three octasaccharide saponins, leonticins A, B, and C (1-3), were isolated from the tubers of
Leontice kiangnanensis. Their structures were elucidated by a combination of chemical
degradation and spectral methods including negative FABMS and NMR measurements as 3-O-
â-D-glucopyranosyl(1f2)-R-L-arabinopyranosylhederagenin 28-O-R-L-rhamnopyranosyl(1f4)-
â-D-glucopyranosyl(1f6)-â-D-glucopyranosyl(1f4)-R-L-rhamnopyranosyl(1f4)-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranoside (1), 3-O-[â-D-xylopyranosyl(1f3)-â-D-galactopyranosyl(1f4)-â-D-
glucopyranosyl(1f3)][â-D-glucopyranosyl(1f2)]-R-L-arabinopyranosyloleanolic acid 28-O-R-L-
rhamnopyranosyl(1f4)-â-D-glucopyranosyl(1f6)-â-D-glucopyranoside (2), and 3-O-[â-D-xylo-
pyranosyl(1f3)-â-D-galactopyranosyl(1f4)-â-D-glucopyranosyl(1f3)][â-D-glucopyranosyl(1f2)]-
R-L-arabinopyranosylechinocystic acid 28-O-R-L-rhamnopyranosyl(1f4)-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranoside (3), respectively. The complete assignments of the proton and carbon
resonances for 1-3 were achieved based on extensive 2D NMR analysis (DQF-COSY, TOCSY,
ROESY, HSQC, and HMBC).
Leontice kiangnanensis P.L. Chiu, identified as a new
species of the genus Leontice (Berberidaceae) in 1987,1
is one of the four species of this genus distributed in
China. Its tubers have been used as a folk medicine in
southeastern China for the treatment of rheumatism
and hemorrhages associated with gastric ulcers. As
part of our screening program aimed at the discovery
of new bioactive agents from plant sources, the 70%
EtOH extract of this crude drug exhibited cytotoxic
activity against P-388 lymphocytic leukemia in vitro and
anti-inflammatory effects on carrageenin-induced swell-
ing of the ankle in rats and xylene-induced inflamma-
tion of the ear in mice. Bioassay-guided fractionation
showed that the n-BuOH extract was responsible for the
anti-inflammatory activity, while the most prominent
cytotoxic fraction (ED50 ) 1.21 µg/mL) was located in
an aqueous effluent from a styrene polymer gel purifica-
tion of the aqueous phase after solvent partition.
Chemical investigation of this cytotoxic aqueous fraction
resulted in the isolation of several saponins.2 This
paper reports the isolation and structure elucidation of
three octasaccharide saponins obtained in our continu-
ing efforts to reveal the chemical constituents from this
saponin-rich fraction.
the negative-ion FABMS corresponding to a molecular
formula of C76H124O41. Analysis of the 1H- and 13C-NMR
spectra indicated that leonticin C is a C-3 and C-28
bisdesmosidic octasaccharide saponin with echinocystic
acid as the aglycon. Acid hydrolysis of 3 afforded
arabinose, xylose, rhamnose, galactose, and glucose as
the sugar components on TLC by comparison with
authentic samples. Alkaline hydrolysis gave the pen-
tasaccharide prosapogenin (3a ), along with rhamnose
and glucose identified in the same manner as above.
The negative-ion FABMS spectra of 3 and 3a provided
the diagnostic fragment ions generated from glycosidic
cleavage along the sugar chain so that the composition
and the sequence of monosaccharides in the molecule
of 3 were deduced from the characteristic fragmentation
pattern. In addition, the weak ions at m/ z 1089 [M -
132 - H]- and 1059 [M - 162 - H]- in 3a (Figure 1),
corresponding to the separate loss of either a pentose
or a hexose residue, suggested that the pentasaccharide
unit at C-3 of 3 was branched.
1
The H-NMR spectrum of 3 was typical of a saponin.
The sugar proton resonances, except for the relatively
well-resolved eight anomeric proton resonances, par-
tially or completely overlapped between 3.4 and 4.4
ppm. The signals arising from the aglycon moiety were
in good agreement with that of echinocystic acid from
the observation of seven methyl singlets, one of which
was shifted downfield to δ 1.81 (CH3-27) due to 1,3-
diaxial interaction in the presence of the axially oriented
16R-OH and the characteristic broad singlet (H-16eq) at
δ 5.25.3 Identification of the spin-systems of individual
monosaccharides and the complete assignment of proton
resonances were achieved through the TOCSY experi-
ment (tmix ) 106 ms) complemented by a DQF-COSY
spectrum. The expanded sugar proton region of the
TOCSY spectrum of 3 identified most of the spin-
systems associated with the eight individual monosac-
charides. Spin-systems with consistently large vicinal
Resu lts a n d Discu ssion
Leonticin C (3) was obtained as a colorless amorphous
powder, mp 226-228 °C; [R]20D -13.49° (c 0.43, MeOH).
Compound 3 exhibited a [M - H]- ion at m/ z 1691 in
* To whom correspondence should be addressed. Phone: ++ 41 1
257 6050. FAX: 41 1 262 5223. E-mail: sticher@phyto.pharma.ethz.ch.
† Presented at the 210th ACS National Meeting, Chicago, IL, Aug
20-24, 1995; see Book of Abstracts Part 1, AGFD, Poster 172.
‡ Current address: Shanghai Institute of Pharmaceutical Industry
(SIPI), 1320 Bejing Xi Lu, Shanghai 200040, People’s Republic of
China.
§ Department of New Drug Research, SIPI, Shanghai, 200040,
People’s Republic of China.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0163-3864(96)00185-1 CCC: $12.00
© 1996 American Chemical Society and American Society of Pharmacognosy

Leonticins A-C
J ournal of Natural Products, 1996, Vol. 59, No. 8 723
F igu r e 1. FABMS (negative) fragments of 3a .
Ta ble 1. 1H-NMR Chemical Shifts and HMBC Correlations for the Aglycon Moieties of 1-3 and 1a -3a a,b
proton
1
2
3
1a
2a
3a
HMBC
3
12
16
18
23
4.16c
5.44 br s
3.25 dd (12, 4)
5.45 br s
3.26 dd (12, 4)
5.60 br s
5.25 brs
3.50 dd (14, 3)
1.24 s
4.18c
5.47 br s
3.24 dd (12, 4)
5.47 br s
3.26 dd (12, 4)
5.61 br s
5.23 br s
3.68c
C: 1, 2, 4, 5, Ara C1
C: 9, 11, 14, 18
C: 14, 15, 17, 18, 22
C: 14, 16, 17, 20
C: 3, 4, 5, 24
3.21 dd (13, 4)
3.69 d (11)
4.13c
0.95 s
0.98 s
1.00 s
1.26 s
0.96 s
0.96 s
3.22 dd (13, 3)
1.26 s
3.28 dd (14, 3)
3.71 d (11)
4.28 d (12)
1.05 s
0.95 s
1.04 s
1.24 s
0.95 s
1.02 s
3.32 dd (14, 4)
1.27 s
1.25 s
24
25
26
27
29
30
1.09 s
0.87 s
0.97 s
1.32 s
0.99 s
0.99 s
1.09 s
0.90 s
0.99 s
1.81 s
1.05 s
1.12 s
1.10 s
0.85 s
1.00 s
1.31 s
0.98 s
1.03 s
1.09 s
0.87 s
1.02 s
1.83 s
1.07 s
1.20 s
C: 3, 4, 5, 23
C: 1, 5, 9, 10
C: 7, 8, 9, 14
C: 8, 13, 14, 15
C: 19, 20, 21, 30
C: 19, 20, 21, 29
a
b
Recorded at 600.13 MHz in pyridine-d5/MeOH-d4. J values in Hz in parentheses. c Overlapped signals.
coupling constants (3J ) 7-9 Hz) in xylose and glucose
displayed a magnetization transfer from the anomeric
protons (δ 6.24, 5.51, 5.27, 5.23, and 4.97) up to H-5 or
H-6A and H-6B. In contrast, magnetization transfer is
inefficient in arabinose, galactose, and rhamnose (δ 4.78,
5.13, and 5.83 ppm) ring systems because of the small
coupling 3J 4,5 < 1.5 Hz4 for the former two and the weak
coupling 3J 2,3 in the latter, which block the transfer from
H-4 to H-5 and from H-2 to H-3, respectively. This
J -network information allowed us to discern the indi-
vidual sugar residue types. However, an unambiguous
assignment of the sugar proton resonances to specific
protons in a given residue could not be determined at
this stage. Therefore, the DQF-COSY experiment was
utilized to trace each magnetization transfer between
the vicinal coupled protons starting from each well
resolved anomeric proton (H-1). Using this approach,
the assignment of the sugar proton signals of 3 were
established as shown in Table 3.
With the proton resonances assigned, the sugar
sequence and the linkage sites of 3 and 3a were
examined by cross-relaxation experiments. Inter-
residual cross peaks in the ROESY spectrum of 3a (tmix
) 300 ms) showed the following proton pairs to be close
in space (Figure 2): ara H-1 (δ 4.76) and aglycon H-3 (δ
3.26), glc′ H-1 (δ 5.50) and ara H-2 (δ 4.72), glc H-1 (δ
5.26) and ara H-3 (δ 4.27), gal H-1 (δ 5.13) and glc H-4
(δ 4.27), and xyl H-1 (δ 5.23) and gal H-3 (δ 4.19). The
three correlations arising from the arabinose residue
clearly indicated it to be (a) the inner pentose residue
bonded to the aglycon and (b) the branched center for
the unit of five monosaccharides at C-3. The combina-
tion of ROE data and the sequence information obtained
from FABMS (negative) spectrum of 3a enabled us to
establish the partial structure of 3. Analogously, the
sequence and linkage sites of the remaining trisaccha-
ride unit linked at C-28 were solved by using a similar
approach. In addition to the information of the connec-
tivities, the intraresidual ROE correlation, particularly
the 1,3-diaxial dipolar interaction in the xylose and
glucose residues, allowed the verification of the proton
assignment based on J -coupling information. Further-
more, ROE data and the diagnostic coupling constants
of the anomeric protons of 3 permitted us to establish
their relative stereochemistry as R-arabinopyranose,
R-rhamnopyranose, â-xylopyranose, â-galactopyranose,
and â-glucopyranose, as would be expected for most
naturally occurring carbohydrates.
The 13C-NMR chemical shifts of 3, which were readily
assigned by a heteronuclear single-quantum correlation
(HSQC) spectrum, were found to agree well with these
results. It is worthy of note that, compared with the
routinely used HMQC experiment, the HSQC spectrum
exhibited better resolution in the F1 dimension,5,6 which
was particularly useful to distinguish signals in exten-
1
sively overlapped regions in both the 13C- and H-NMR
spectra. The glycosidation shifts obtained from the data
analysis fully supported the linkage site assignment
based on the ROESY spectrum (Table 4).7,8 Final
confirmation of the connectivities and assignments for
both aglycon and sugar moieties was obtained by
exhaustive analysis of an HMBC spectrum of 3. The
2
3
observed J H,C and J H,C long-range correlations arising
from the separately resolved H-12, H-16, H-18, and
methyl protons in the aglycon are summarized in Table
1. Similarly, the detection of all possible two- and three-

724 J ournal of Natural Products, 1996, Vol. 59, No. 8
Chen et al.
Ta ble 2. 13C-NMR Chemical Shifts for the Aglycon Moieties of 1-3 and 1a -3a a
carbon
1
2
3
1a
2a
3a
DEPTb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
39.23
26.34
82.74
43.88
48.19
18.67
32.99
40.35
48.58
37.36
24.31
123.28
144.59
42.61
28.73
23.80
47.52
42.15
46.73
31.19
34.46
33.16
65.03
13.72
16.56
17.94
26.43
177.02
33.47
24.07
39.37
27.01
90.11
40.18
56.47
19.02
32.99
40.37
48.58
37.50
24.28
123.32
144.56
42.61
28.74
23.84
47.55
42.18
46.79
31.23
34.50
33.58
28.38
17.04
16.00
17.89
26.43
177.03
33.50
24.09
39.46
27.04
90.18
40.19
56.55
19.04
33.91
40.54
47.69
37.54
24.30
123.13
144.80
42.50
36.35
74.67
49.63
41.71
47.69
31.22
36.30
32.47
28.36
17.05
16.13
17.96
27.53
176.44
33.52
25.05
39.26
26.49
82.71
44.05
48.45
18.75
33.41
40.28
48.65
37.46
24.39
123.05
145.31
42.68
28.87
24.22
47.18
42.51
46.95
31.48
34.74
33.78
65.41
13.99
16.60
17.99
26.67
180.66
33.78
24.31
39.29
27.18
89.53
40.25
56.42
19.04
33.84
40.28
48.57
37.55
24.34
123.03
145.33
42.69
28.87
24.26
47.22
42.54
47.03
31.52
34.79
33.74
28.58
17.29
16.02
17.95
26.71
180.74
33.84
24.34
39.47
27.27
89.60
40.32
56.58
19.13
34.11
40.49
47.81
37.62
24.42
122.78
145.83
42.72
36.71
75.44
49.02
42.18
47.94
31.61
36.83
33.18
28.57
17.33
16.19
18.13
27.85
181.18
33.99
25.58
t
t
d
s
d
t
t
s
d
s
t
s
s
s
t
t, d
s
d
t
s
t
t
t, q
q
q
q
q
s
q
q
a
b
Recorded at 150.91 MHz in pyridine-d5/MeOH-d4. Multiplicity by DEPT.
Ta ble 3. 1H-NMR Chemical Shifts for the Sugar Moieties of Compounds 2, 3, 2a , and 3a a,b
proton
2
3
2a
3a
proton
2
3
C-3
C-28
Ara 1
2
3
4
5A
5B
4.78 d (6.6)
4.72 t (7, 8)
4.34c
4.54 br s
3.72 d (11)
4.26c
4.78 d (6.8)
4.71 t (7, 8)
4.32c
4.53 br s
3.72 d (11)
4.26c
4.75 d (7.0)
4.72 t (7, 8)
4.28c
4.45 br s
3.67 d (10)
4.16c
4.76 d (6.9)
4.72 t (7, 8)
4.27c
Glc′′ 1
2
3
4
5
6A
6.25 d (8.2)
4.10c
6.24 d (8.2)
4.04c
4.21c
4.17c
4.46 br s
4.24 t (8)
4.16c
3.68c
4.10c
4.09c
4.18c
4.34c
4.32c
6B
4.70 d (10)
4.68 d (12)
Glc′ (1f2) Ara
Glc′′′ (1f6) Glc′′
1
2
3
4
5
6A
6B
5.53 d (8.2)
3.97c
5.51 d (7.8)
3.95 t (8, 9)
4.13c
5.52 d (7.9)
4.03 t (8)
4.21c
5.50 d (7.7)
4.02 t (8)
4.18c
1
2
3
4
5
6A
6B
4.99 d (7.9)
3.91 t (8, 9)
4.13c
4.97 d (7.9)
3.91 t (8, 9)
4.11c
4.15c
4.05 t (9)
3.77 m
4.02c
4.13c
4.14c
4.33c
4.32c
3.76 m
3.72 m
4.29 dd (11, 3)
4.37 dd (11, 3)
3.72 m
3.73c
3.71c
4.24c
4.22c
4.28c
4.12c
4.12c
4.40 br d (12)
4.40 br d (10)
4.38 dd (12, 3)
4.27c
4.27c
Glc (1f3) Ara
Rha (1f4) Glc′′′
1
2
3
4
5
6A
6B
5.27 d (7.8)
4.00 t (8)
4.20c
5.27 d (7.9)
4.00c
5.26 d (7.8)
3.98 t (8)
4.22 t (9)
4.27 t (9)
3.87 m
5.26 d (8.0)
3.98 t (8, 9)
4.22 t (9)
4.27 t (9)
3.89 m
1
2
3
4
5
6
5.85 br s
4.62 br s
4.48c
4.26 t (8)
4.85 m
5.83 br s
4.61 br s
4.47c
4.19c
4.27c
4.26c
4.24c
3.92c
3.91c
4.85 m
1.71 d (6)
4.46c
4.47c
4.41 dd (11, 3)
4.48 dd (12, 4)
4.42 dd (11, 3)
4.48 dd (11, 3)
1.71 d (6)
4.52c
4.47c
Gal (1f4) Glc
1
2
3
5.13 d (7.7)
4.59 t (8, 9)
4.20c
5.13 d (7.9)
4.58 t (8, 9)
4.20c
5.11 d (7.6)
4.66 t (8)
4.18c
5.13 d (7.7)
4.66 t (8)
4.19c
4
5
6A
6B
4.68 br s
4.68 br s
4.67 br s
4.67 br s
4.19c
4.19c
4.17c
4.16c
4.33c
4.32c
4.43 dd (12, 3)
4.33c
4.46 d (12)
4.33 dd (12, 4)
4.44c
4.44c
Xyl (1f3) Gal
1
2
3
4
5A
5B
5.24 d (7.5)
3.97 t (8)
4.10c
5.23 d (7.5)
3.97 t (8, 9)
4.10c
5.23 d (7.6)
3.99 t (8, 9)
4.15c
5.23 d (7.7)
3.99 t (8, 9)
4.15c
4.13c
4.12c
4.18c
4.17c
3.70 d (11)
3.70 d (10)
3.67 d (11)
3.69c
4.32c
4.31c
4.30c
4.30c
a
b
Recorded at 600.13 MHz in pyridine-d5/MeOH-d4. J values (in Hz) in parentheses. c Indicates overlapped signals.

Leonticins A-C
J ournal of Natural Products, 1996, Vol. 59, No. 8 725
F igu r e 2. Key HMBC and ROE correlations for establishing the sugar linkage sites of compounds of 2 and 3.
Ta ble 4. 13C-NMR Chemical Shifts for the Sugar Moieties of Compounds 2, 3, 2a , and 3a a
carbon
2
3
2a
3a
carbon
2
3
C-3
C-28
Ara 1
106.19
77.64
84.21
69.67
66.81
106.18
77.61
84.24
69.66
66.81
106.10
77.97
83.87
69.49
66.67
106.12
77.92
83.93
69.50
66.71
Glc′′ 1
2
3
4
5
6
96.32
74.40
79.06
71.33
78.62
69.78
96.42
74.43
79.03
71.29
78.62
69.81
2
3
4
5
Glc′ (1f2) Ara
Glc′′′ (1f6) Glc′′
1
2
3
4
5
6
104.69
76.69
79.06
72.87
78.46
63.85
104.65
76.66
79.03
72.86
78.44
63.85
104.85
76.79
79.15
72.88
78.09
63.76
104.82
76.77
79.14
72.88
78.14
63.78
1
2
3
4
5
6
105.23
75.84
77.09
79.22
77.64
61.95
105.27
75.76
77.06
79.27
77.61
61.95
Glc (1f3) Ara
Rha (1f4) Glc′′′
1
2
3
4
5
6
105.35
75.31
76.88
81.96
77.08
62.29
105.35
75.30
76.86
81.89
77.05
62.25
105.26
75.21
76.91
82.28
77.07
62.31
105.28
75.28
76.89
82.20
77.07
62.27
1
2
3
4
5
6
103.48
73.04
73.09
74.40
71.03
18.95
103.48
72.97
73.01
74.37
71.03
18.92
Gal (1f4) Glc
1
2
3
4
5
6
105.73
71.98
84.83
70.12
77.57
62.56
105.70
71.96
84.81
70.11
77.54
62.56
105.89
71.89
84.89
70.08
77.66
62.48
105.85
71.89
84.88
70.08
77.65
62.49
Xyl (1f3) Gal
1
2
3
4
5
107.47
75.88
78.46
71.52
67.71
107.43
75.83
78.44
71.50
67.69
107.66
75.95
78.81
71.59
67.79
107.60
75.94
78.78
71.59
67.78
a
Recorded at 150.91 MHz in pyridine-d5/MeOH-d4.
bond inter- and intra-residual correlations of the ano-
meric protons confirmed unambiguously the glycosidic
linkages (Figure 2) and the assignments for proton and
carbon resonances. Thus, the chemical structure of
leonticin C was established as 3-O-[â-D-xylopyranosyl-
(1f3)-â-D-galactopyranosyl(1f4)-â-D-glucopyranosyl-
(1f3)][â-D-glucopyranosyl(1f2)]-R-L-arabinopyranosyl-
echinocystic acid 28-O-R-L-rhamnopyranosyl(1f4)-â-D-
glucopyranosyl(1f6)-â-D-glucopyranoside (3).
molecular weight of 1206 [m/ z 1205, (M - H)-]. Full
spectroscopic characterization indicated that 2 was
similar to 3, except that 2 has oleanolic acid as the
aglycon. The complete assignments of the proton and
carbon resonances of 2 were based on the heteronuclear
correlation experiments (HSQC and HMBC) of 2 and
homonuclear correlations (DQF-COSY and ROESY
spectra) of 2a . Consequently, the determination of the
connectivities and the relative stereochemistry of the
sugar moieties in 2 were achieved by the methods
described above. The chemical structure of leonticin B
was thus deduced as 3-O-[â-D-xylopyranosyl(1f3)-â-D-
Leonticin B (2) was found to have a molecular formula
of C76H124O40 [m/ z 1675 (M - H)-]. Alkaline hydrolysis
gave the pentasaccharide prosapogenin (2a ), with a

726 J ournal of Natural Products, 1996, Vol. 59, No. 8
Chen et al.
F igu r e 3. Key HMBC and ROE correlations for establishing the sugar linkage sites of compound 1.
galactopyranosyl(1f4)-â-D-glucopyranosyl(1f3)][â-D-
glucopyranosyl(1f2)]-R-L-arabinopyranosyloleanolic acid
28-O-R-L-rhamnopyranosyl(1f4)-â-D-glucopyranosyl-
(1f6)-â-D-glucopyranoside (2).
Leonticin A (1) showed a molecular ion at m/ z 1705
[(M - H)-, C77H126O41] along with the characteristic
fragments for a successive loss of monosaccharide
residues in the negative-ion FABMS spectrum. Acid
hydrolysis of 1 afforded hederagenin, arabinose, rham-
nose, and glucose. Alkaline hydrolysis yielded the
disaccharide glycoside cauloside C9 as prosapogenin
(1a ), indicating that a hexasaccharide unit is attached
at C-28 of 1.
although only six anomeric protons could be seen in the
1D proton spectrum. The HSQC spectrum disclosed
that the anomeric protons of two glucose and two
rhamnose residues resonate both at δ 4.99 (2H, d, J )
7.7 Hz) and δ 5.85 (2H, br s), respectively. The overlap
1
of both H- and 13C-NMR signals due to the repetition
of similar structural moieties prevented the precise
characterization of the monosaccharide ring systems
from the homonuclear J -coupling correlations as de-
scribed above. The unambiguous assignments of the
proton and carbon resonances and the connectivities of
1 were established by a careful study of the HMBC
spectrum. The anomeric configuration of each sugar
component was determined from the magnitude of the
coupling constant of the anomeric proton with the aid
of ROEs detected in the ROESY spectrum (Figure 3).
Thus, the structure of leonticin A was elucidated as 3-O-
â-D-glucopyranosyl(1f2)-R-L-arabinopyranosylheder-
agenin 28-O-R-L-rhamnopyranosyl(1f4)-â-D-glucopy-
ranosyl(1f6)-â-D-glucopyranosyl(1f4)-R-L-rhamnopy-
ranosyl(1f4)-â-D-glucopyranosyl(1f6)-â-D-glucopyrano-
side (1).
Leonticins A-C (1-3) and the prosapogenins (1a -
3a ) were tested for cytotoxic activity against KB cells.10
None of them was found to be active at the concentration
of 4 µg/mL.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rota-
tions were determined on a Perkin-Elmer 241 polarim-
eter. FABMS were recorded in a glycerol matrix in the
negative-ion mode on a VG ZAB2-Seq spectrometer and
EIMS on a VG Tribrid spectrometer. All NMR experi-
ments were performed on a Bruker AMX-600 spectrom-
eter in pyridine-d5 with two drops of MeOH-d4 at 300
K, and chemical shifts were referenced to TMS. HPLC
was performed on a Waters 590 programmable pump
with a Knauer differential refractometer. Vacuum
liquid chromatography was carried out with Si gel 60
(0.040-0.063 mm, Merck). TLC was conducted on Si
gel 60 F254 (Merck) and RP-18 F254 S (Merck).
Analysis of the sugar region of the 1H-NMR and
DEPT spectra of 1 indicated the presence of one arabi-
nose, two rhamnose, and five glucose residues in the
molecule, which were readily recognized by the char-
acteristic two methyl doublets at 1.71 and 1.83 ppm (rha
CH3-6) in the proton spectrum and the multiplicities of
the carbon signals (60-70 ppm) in the DEPT spectrum,

Leonticins A-C
J ournal of Natural Products, 1996, Vol. 59, No. 8 727
Ta ble 5. NMR Chemical Shifts for the Sugar Moieties of Compounds 1 and 1a a,b
1 (δH)
1a (δH)
1 (δC)
1a (δC)
1 (δH)
1 (δC)
C-3
Rha (1f4) Glc′′
Ara 1
2
3
4
5A
5B
5.14 d (6.0)
4.56 t (6, 7)
4.31c
5.17 d (6.0)
4.58 t (6, 7)
4.28c
4.33 br s
3.75 d (11)
4.24c
104.49
81.33
74.01
68.82
65.44
104.51
81.92
74.18
68.81
65.41
1
2
3
4
5
6
5.85 br s
4.61 br s
4.62 m
4.43 t (9)
4.95 m
103.38
72.63
73.03
84.84
69.22
19.09
4.37c
3.71 d (10)
4.23c
1.83 d (6)
Glc (1f2) Ara
Glc′′′ (1f4) Rha
1
2
3
4
5
6A
6B
5.21 d (7.5)
4.05 t (8)
4.14c
5.18 d (7.4)
4.08 t (8)
4.19 t (8)
4.22 t (8)
3.82 m
106.22
76.62
78.60
71.91
78.88
63.07
106.54
76.79
78.75
71.90
78.82
63.04
1
2
3
4
5
6A
6B
5.29 d (7.9)
3.99 t (8)
4.11c
106.67
76.61
78.89
71.77
77.86
70.61
4.15c
4.13c
3.83c
3.91c
4.34c
4.36 dd (12, 4)
4.46 dd (11, 3)
4.29c
4.47c
4.67 d (12)
C-28
Glc′′′′ (1f6) Glc′′′
Glc′ 1
2
3
4
5
6A
6B
6.26 d (8.0)
4.10c
96.31
74.40
79.06
71.30
78.57
69.81
1
2
3
4
5
6A
6B
4.99 d (7.7)
3.93 t (9)
4.13c
105.71
75.85
77.02
78.49
77.65
61.90
4.21c
4.26c
4.36c
4.11c
3.70 m
4.34c
4.08c
4.69 d (12)
4.27c
Glc′′ (1f6) Glc′
Rha′ (1f4) Glc′′′′
1
2
3
4
5
6A
6B
4.99 d (7.7)
3.92 t (8, 9)
4.13c
105.32
76.05
77.05
79.16
77.71
62.03
1
2
3
4
5
6
5.85 br s
4.61 br s
4.47c
102.83
73.03
73.03
74.40
71.02
18.96
4.33c
4.25c
3.70c
4.88 m
1.71 d (6)
4.08c
4.27c
a
b
Recorded at 600.13 MHz and 150.91 MHz in pyridine-d5/MeOH-d4. J values (in Hz) in parentheses. c Indicates overlapped signals.
P la n t Ma ter ia l. The tubers of L. kiangnanensis
were collected in Anhui Province of the People’s Repub-
lic of China in May 1992. A voucher specimen (No. N
88006) has been deposited in the herbarium of Shanghai
Institute of Pharmaceutical Industry, Shanghai, Peo-
ple’s Republic of China.
Extr a ction a n d Isola tion . The dried and pulver-
ized tubers (1 kg) were extracted with 70% EtOH. The
concentrated aqueous extract was subjected to solvent
partition and gel chromatography as previously de-
scribed.2 The H2O effluent obtained from the porous
polymer gel column was rechromatographed on the
same column. Fractions eluting with 30% aqueous
EtOH were combined for further fractionation by vacuum
liquid chromatography on Si gel 60 using CHCl3-
MeOH-H2O (5:5:1) as eluent. Final purification was
performed with HPLC on Spherisorb C18 column [5 µm,
16 × 250 mm, MeOH-H2O (6:4), flow rate 5 mL/min]
to afford 1 (56 mg), 2 (63 mg), and 3 (31 mg).
Acid Hyd r olysis of 1-3. A 5-mg quantity of each
sample was refluxed with 2 M HCl in MeOH (2 ml) for
4 h. The reaction solution was evaporated under
reduced pressure, and the hydrolysate was extracted
with ether. The ether extract was evaporated to afford
the aglycon, which was identified by a combination of
EIMS measurement11 and comparison of its 13C-NMR
data with published values.12,13 The H2O layer was
neutralized with alkali solution and concentrated at
reduced pressure. The residues were compared with
standard sugars on Si gel TLC [CHCl3-MeOH-H2O
(30:12:4), 9 mL of lower layer + 1 mL of HOAc], which
showed the sugars to be arabinose, rhamnose, and
glucose for compound 1 and arabinose, xylose, rham-
nose, galactose, and glucose for 2 and 3.
h, and after the usual workup the prosapogenins 1a ,
2a , and 3a were obtained.
Leon ticin A (1): white amorphous powder; mp 219-
1
221 °C; [R]20 -16.31° (c 1.03, MeOH); H NMR (pyri-
D
dine-d5/MeOH-d4, 600.13 MHz), see Tables 1 and 5; 13
C
NMR (pyridine-d5/MeOH-d4, 150.91 MHz), see Tables
2 and 5; negative-ion FABMS m/ z [M - H]- 1705, [M
- rha - H]- 1559, [M - glc - H]- 1543, [M - rha - glc
- H]- 1397, [M - rha - 2glc - H]- 1235, [M - 2rha -
2glc - H]- 1089, [M - 2rha - 3glc - H]- 927, [M -
2rha - 4glc - H]- 765, [M - 2rha - 5glc - H]- 603,
[M - 2rha - 5glc - ara - H]- 471.
P r osa p ogen in 1a : white amorphous powder; mp
253-255 °C; 1H NMR (pyridine-d5/MeOH-d4, 600.13
MHz), see Tables 1 and 5; 13C NMR (pyridine-d5/MeOH-
d4, 150.91 MHz), see Tables 2 and 5; negative-ion
FABMS m/ z [M - H]- 765, [M - glc - H]- 603, [M -
glc - ara - H]- 471.
Hed er a gen in : white needles; mp 304-305 °C; EIMS
m/ z [M]+ 472 (2), 426 (3), 248 (100), 203 (62).
Leon ticin B (2): white amorphous powder; mp 244-
246 °C; [R]20D -4.06° (c 0.96, MeOH); 1H NMR (pyridine-
d5/MeOH-d4, 600.13 MHz), see Tables 1 and 3; 13C NMR
(pyridine-d5/MeOH-d4, 150.91 MHz), see Tables 2 and
4; negative-ion FABMS m/ z [M - H]- 1675, [M - xyl
- H]- 1543, [M - xyl - glc - H]- 1381, [M - xyl - gal
- glc - H]- 1219, [M - rha - 2glc]- 1205, [M - rha -
2glc - xyl - H]- 1073, [M - rha - 3glc - H]- 1043, [M
- rha - 3glc - xyl - H]- 911, [M - rha - 3glc - gal -
xyl - H]- 749, [M - rha - 4glc - xyl - gal - H]- 587,
[M - rha - 4glc - xyl - gal - ara - H]- 455.
P r osa p ogen in 2a : white amorphous powder; mp
1
264-266 °C; [R]20 +22.30° (c 1.00, MeOH); H NMR
D
(pyridine-d5/MeOH-d4, 600.13 MHz), see Tables 1 and
3; 13C NMR (pyridine-d5/MeOH-d4, 150.91 MHz), see
Tables 2 and 4; negative-ion FABMS m/ z [M - H]-
Alk a lin e Hyd r olysis of 1-3. Compounds 1-3 were
each hydrolyzed with 0.5 M KOH (4 mL) at 80 °C for 2

728 J ournal of Natural Products, 1996, Vol. 59, No. 8
Chen et al.
1205, [M - xyl - H]- 1073, [M - glc - H]- 1043, [M -
xyl - glc - H]- 911, [M - xyl - gal - glc - H]- 749,
[M - xyl - gal - 2glc - H]- 587, [M - xyl - gal - 2glc
- ara - H]- 455.
Olea n olic a cid : white amorphous powder; mp 290-
292 °C; EIMS m/ z [M]+ 456 (1), 248 (100), 203 (46), 189
(24).
Amrein, Department of Chemistry, ETH, for recording
the mass spectra; Dr. E. Zass, Department of Organic
Chemistry, ETH, for carrying out literature searches;
D.Q. Wang, Department of Botany, Anhui Traditional
Chinese Medicine School, People’s Republic of China,
for collection of the plant material; and X.H. Chen,
Department of Pharmacology, Shanghai Institute of
Pharmaceutical Industry, for primary screening of the
biological activities, and B. Frei, Department of Phar-
macy, ETH, for the evaluation of cytotoxic activity
against KB cells.
Leon ticin C (3): white amorphous powder; mp 226-
1
228 °C; [R]20 -13.49° (c 0.43, MeOH); H NMR (pyri-
D
dine-d5/MeOH-d4, 600.13 MHz), see Tables 1 and 3; 13
C
NMR (pyridine-d5/MeOH-d4, 150.91 MHz), see Tables
2 and 4; negative-ion FABMS m/ z [M - H]- 1691, [M
- xyl - H]- 1559, [M - xyl - glc - H]- 1397, [M - xyl
- gal - glc - H]- 1235, [M - rha - 2glc]- 1221, [M -
rha - 2glc - xyl - H]- 1089, [M - rha - 3glc - H]-
1059, [M - rha - 3glc - xyl - H]- 927, [M - rha -
3glc - gal - xyl - H]- 765, [M - rha - 4glc - xyl -
gal - ara - H]- 471.
Refer en ces a n d Notes
(1) Wang, L. Z.; Yang, J .; Wang, D. Q. Auhui Zhong Yi Xue Yuan
Xue Bao 1987, 6(3), 48-49.
(2) Chen, M.; Wu, W. W.; Yu, Y.; Shen, Q. Y. Abstracts of UNESCO
Regional Seminar on Chemistry of Medicinal Plants; Chendu:
People’s Republic of China, 1992; p 253.
(3) Kitagawa, I.; Yoshikawa, M.; Yosioka, I. Tetrahedron Lett. 1974,
469-472.
(4) Homans, S. W. Prog. Nucl. Magn. Reson. Spectrosc. 1990, 22,
55-81.
P r osa p ogen in 3a : white amorphous powder; mp
1
249-250 °C; [R]20 +10.81° (c 0.74, MeOH); H NMR
D
(5) Bax, A.; Ikura, M.; Kay, L. E.; Torchia, D. A.; Tschudin, R. J .
Magn. Reson. 1990, 86, 304-318.
(pyridine-d5/MeOH-d4, 600.13 MHz), see Tables 1 and
3; 13C NMR (pyridine-d5/MeOH-d4, 150.91 MHz), see
Tables 2 and 4; negative-ion FABMS m/ z [M - H]-
1221, [M - xyl - H]- 1089, [M - glc - H]- 1059, [M -
xyl - glc - H]- 927, [M - xyl - gal - glc - H]- 765,
[M - xyl - gal - 2glc - H]- 603, [M - xyl - gal - 2glc
- ara - H]- 471.
(6) Norwood, T. J .; Boyd, J .; Heritage, J . E.; Soffe, N.; Campbell, I.
D. J . Magn. Reson. 1990, 87, 488-501.
(7) Yamasaki, K.; Kohda, H.; Kobayashi, T.; Kasai, R.; Tanaka, O.
Tetrahedron Lett. 1976, 1005-1008.
(8) Kasai, R.; Suzuo, M.; Asakawa, J .; Tanaka, O. Tetrahedron Lett.
1977, 175-178.
(9) Strigina, L. I.; Chetyrina, N. S.; Elyakov, G. B. Chem. Nat. Comp.
1970, 6, 569-572.
(10) Swanson, S. M.; Pezzuto, J . M. In Drug Bioscreening: Drug
Evaluation Techniques in Pharmacology; Thompson, E. B., Ed.;
VCH Publishers: New York, 1990; pp 273-297.
(11) Budzikiewicz, B.; Wilson, J . M.; Djerassi, C. J . Am. Chem. Soc.
1963, 85, 3688-3699.
Ech in ocystic a cid : white amorphous powder; mp
297-299 °C; EIMS m/ z [M]+ 471 (1), 428 (2), 410 (42),
395 (100), 187 (21).
(12) Tori, K.; Seo, S.; Shimaoka, A.; Tomita, Y. Tetrahedron Lett.
1974, 4227-4230.
(13) Warashina, T.; Miyase, T.; Ueno, A. Chem. Pharm. Bull. 1991,
39, 388-396.
Ack n ow led gm en t. We thank N. Walch and D.
Rentsch, Department of Organic Chemistry, University
of Zurich, for performing all NMR experiments on the
Bruker AMX-600; R. Ha¨fliger, O. Greter, and Dr. W.
NP960185G