New triterpenoid saponins with strong α-glucosidase inhibitory activity from the roots of Gypsophila oldhamiana

Article abstract of DOI:10.1016/j.bmc.2007.12.053

Seven new triterpenoid saponins (1-7), have been isolated and elucidated from the roots of Gypsophila oldhamiana together with five known triterpenoid saponins (8-12). These saponins which could be classified into three series: 3-O-monoglucosides (1, 8, 9), 28-O-monoglucosides (2-4, 12) and 3, 28-O-bidesmosides (5-7, 10, 11), have been evaluated for their α-glucosidase inhibition activity. As a result, the preliminary structure-activity relationships were discussed based on the position of sugar linkage attached to the aglycone, and 28-O-monoglucosides 2-4 and 12 showed significant inhibitory activities on α-glucosidase.

Full text of DOI:10.1016/j.bmc.2007.12.053

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2912–2920
New triterpenoid saponins with strong a-glucosidase
inhibitory activity from the roots of Gypsophila oldhamiana
Jian-Guang Luo, Li Ma and Ling-Yi Kong*
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009,
People’s Republic of China
Received 3 November 2007; revised 26 December 2007; accepted 27 December 2007
Available online 1 January 2008
Abstract—Seven new triterpenoid saponins (1–7), have been isolated and elucidated from the roots of Gypsophila oldhamiana
together with five known triterpenoid saponins (8–12). These saponins which could be classified into three series: 3-O-monogluco-
sides (1, 8, 9), 28-O-monoglucosides (2–4, 12) and 3, 28-O-bidesmosides (5–7, 10, 11), have been evaluated for their a-glucosidase
inhibition activity. As a result, the preliminary structure–activity relationships were discussed based on the position of sugar linkage
attached to the aglycone, and 28-O-monoglucosides 2–4 and 12 showed significant inhibitory activities on a-glucosidase.
ꢀ
2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Gypsophila oldhamiana (Miq.) (Caryophyllaceae) is a
small perennial herb widely distributed in the north re-
gions of China, its roots have been used as a substitute
for the traditional Chinese medicine Yin-Chai-Hu (roots
of stellria dichotoma var. Lanceolata Bge) to treat fever,
consumptive disease, and infantile malnutrition syn-
drome. Its roots are also used as a remedy for diabetes
The 70% EtOH extract of the roots of G. oldhamiana
was partitioned by water and n-BuOH. The n-BuOH-
soluble fraction, subjected to chromatographic purifica-
tion over a silica gel column and repeated RP-C18
column, followed by HPLC purification, afforded seven
new triterpenoid saponins (1–7) and five known sapo-
2
b,4
nins (8–12) (Scheme 1).
1
in folk herb medicines of China. In previous chemical
study, saponins, sterols, and a cyclic peptide have been
Compound 1 was obtained as an amorphous powder.
The HRESIMS showed a pseudo molecular ion peak
2
reported from G. oldhamiana. Our continuing research
ꢀ
on anti-diabetes activity constituents from the n-BuOH-
soluble extracts of its roots resulted in the isolation of
seven new triterpenoid saponins (1–7), together with five
known triterpenoid saponins (8–12). In order to evalu-
ate their anti-diabetes effects, all isolated saponins (1–
at m/z 985.4643 [MꢀH] (calcd 985.4649) corresponding
to molecular formula of C H O . The IR spectrum
4
8
74 21
ꢀ1
showed absorption bands at 3427 cm (–OH),
1722 cm (CO), and 1077 cm (C–O–C). Acid hydro-
ꢀ
1
ꢀ1
lysis of 1 with 2 M HCl afforded an aglycone identified
5
1
can retard the uptake of dietary carbohydrates and thus
2) have been screened as a-glucosidase inhibitors that
as quillaic acid (1a) by TLC comparison with an
authentic sample, which was confirmed on the basis of
3
1
13
suppress postprandial hyperglycemia. In this paper, we
the H, C NMR (Table 1), HMQC, and HMBC spec-
tra of 1. The sugars obtained from the saponin hydrol-
ysates were identified as D-galactose and D-glucuronic
acid (2:1) based on GC–MS analysis of their chiral
derivatives. The assignments of the carbon signals of
the sugar components (Table 2) were determined from
TOCSY, HSQC, and HMBC spectra. The three sugar
report the structure elucidation of these seven new sap-
onins as well as the strong a-glucosidase inhibitory
activities of 28-O-monoglucosides 2–4 and 12.
anomeric carbons were detected at d 103.8, 104.2,
C
1
3
and 105.0 in C NMR spectrum, attached to protons
at dH 4.84 (d, J = 7.5 Hz), 5.47 (d, J = 7.6 Hz), and
5.20 (d, J = 7.6 Hz), respectively, in the HSQC experi-
Keywords: Gypsophila oldhamiana; Caryophyllaceae; Triterpenoid sap-
onins; a-Glucosidase inhibitors.
Corresponding author. Tel.: +86 25 8539 1232; fax: +86 25 8530
1528; e-mail: cpu_lykong@126.com
*
1
3
ment. In addition, the C NMR data of compound 1
0
968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.053

J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
2913
O
COOH
C
OH
R2
OH Me
HO
GlcA
HOOC
HO
R1O
O
O
H
O
O
Fuc
O
O
HO
CHO
OH
R
Ara'
O
Rha
OH
O
Xyl
OH
O
O
Me
HO
O
O
Ara
HO
HO
O
O
1
R1=Gal R2=OH
R1=Xyl R2=OH
HO
HO
OH
OH
OH
OH
Gal
OH
8
9
R1=Xyl R2=H
2 R= OH
1
2 R=CHO
O
C
Glc-II
OH
OH
OH
HO
Me
O
O
O
HO
O
OH
O
O
Fuc
O
HO
OH
OH
OH
O
O
O
OH
HO
HO
OH Ara'
OH
O
Rha
O
Xyl
O
O
O
O
Me
O
Ara HO
HO
Glc-I
O
OH
OH
HO
O
O
CHO
OH HO
HO
α-D-Gal
OH
OH
Glc-III
OH
3
4
O
R1
R2
R3
R4
C
OH
R1 Me
5
6
H
H
H
H
H
O
O
O
Fuc HO
GlcA
HOOC
HO
R2O
OH OH
O
H
Xyl-
Ara
O
CHO
HO
7
H
H
Xyl- (6-O-Ac)-Glc-
H
H
O
Me
O
O
O
R4O
Rha
O
10
Xyl-
Xyl-
Glc-
H
R3O OH
OH
Xyl
HO
OH
1
1
OH
Ara-(1→4)-Ara
Gal
Scheme 1. Structures of triterpenoid saponins 1–12.
indicated that the sugar residue was attached to C-3 of
aglycone (d 84.5), which was confirmed by cross-peak
in the HMBC spectrum between dH 4.84 (H-1 of glucu-
carbons (d 122.5 and 144.6) indicated that the aglycone
C
was of an olean-12-ene skeleton. Detailed analysis of the
TOCSY, HMQC, HMBC, and ROESY data indicated
that the aglycone in 2 possessed the same partial struc-
ture in the C, D, and E-rings as that of quillaic acid.
Additionally, a salient downshift (+20.0) at C-4 (dC
76.0) and the absence of CHO indicated that a hydroxyl
was located at C-4 to replace the CHO group of quillaic
acid. The b-configuration of methyl group (24-Me) was
evident from the ROESY spectrum which showed signif-
icant correlations through space interaction between 24-
Me (dH 1.38) and 25-Me (dH 0.92). The above informa-
tion suggested that the aglycone in 2 was segetalic acid,
which was first isolated as an aglycone of a triterpenoid
saponin from Vaccaria segetalis (Neck) Garcke (Caryo-
C
ronic acid) and d 84.5 (C-3 of the aglycone). The link-
C
ages of the two other monosaccharides to glucuronic
acid were established from the following HMBC corre-
lations: H-1 (dH 5.47) of D-galactose with C-3 (dC
8
7.8) of D-glucuronic acid and H-1 (d 5.20) of another
H
D-galactose with C-2 (d 78.3) of D-glucuronic acid. The
C
3
JH1, H2 coupling constants (7.5–8.0 Hz) suggested b-
anomeric configurations for three monosaccharide moi-
eties. On the basis of these results, the structure of 1 was
determined to be quillaic acid 3-O-b-D-galactopyrano-
syl-(1 ! 2)-[b-D-galactopyranosyl-(1 ! 3)]-b-D-glucurono-
pyranoside.
6
phyllaceae). Acid hydrolysis of 2 furnished a rear-
ranged aglycone and identified as 3-keto, 16a-hydroxy,
24-noroleanolic acid (2a), and the monosaccharide
Compounds 2 and 3, respectively, displayed quasi-
molecular ion peaks [MꢀH] at m/z 1161.5688 and
ꢀ
6
1143.5560 in the negative HRESIMS, which was consis-
tent with the molecular formula of C H O and
components were identified as D-fucose, L-rhamnose,
D-xylose, and L-arabinose (1:1:1:2) based on GC–MS
analysis of their chiral derivatives. The presence of five
5
6
90 25
1
C H O , respectively. H NMR and C NMR spec-
13
5
6
88 24
1
tra indicated that compounds 2 and 3 had the same su-
gar moieties (Table 2) but differed in the aglycone parts
sugar moieties was further evidenced by the H and
C NMR spectra which displayed five anomeric pro-
1
3
(
Table 1). The aglycone part of 2 showed six methyl
proton singlets (dH 0.92, 0.96, 1.00, 1.17, 1.38, and
.70) and a triplet-like vinyl proton (dH 5.63). The
characteristic t-like signal coupled with the two olefinic
tons at d 6.00 (d, J = 8.7 Hz), 6.51 (br s), 5.02 (d,
J = 7.0 Hz), 5.12 (d, J = 7.0 Hz), and 4.98 (d,
J = 7.3 Hz), and carbons at dC 94.7, 101.1, 106.6,
105.7, and 106.8, respectively. On the basis of the
1

2914
J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
1
Table 1. C NMR data for aglycone moieties of compounds 1–7
3
bon (d 216.5) besides the characteristic information of
the olean-12-ene skeleton including five angular methyl
groups (dH 0.86, 0.88, 0.95, 1.18, and 1.38), and a trip-
C
a
1
a
2
b
3
a
4
a
5
a
6
a
7
Carbon
1
2
3
4
5
6
7
8
9
38.0
23.7
84.5
55.1
48.6
20.4
32.8
40.1
48.8
36.2
25.2
38.8
28.6
41.5
38.3
38.6
27.1
71.7
56.3
47.8
21.1
32.5
40.2
48.0
36.2
23.8
38.1
25.1
83.6
55.0
48.6
20.7
32.6
40.2
47.8
36.2
23.3
37.9
25.2
84.6
54.9
48.6
20.6
32.4
40.0
47.6
36.0
23.0
38.1
25.4
84.4
55.1
48.9
21.0
32.7
40.2
47.8
36.3
23.4
let-like vinyl proton (d 5.34) coupled with the two ole-
H
finic carbons (d 123.4 and 144.9). Detailed analysis of
79.7 216.5
C
the HMQC, HMBC, and ROESY data indicated that
the aglycone in 3 was a rearranged product of segelic acid
and identified as 3-keto, 16a-hydroxy, 24-noroleanolic
76.0
56.3
18.0
32.9
40.2
47.2
38.0
24.0
45.9
55.3
25.0
31.9
40.6
45.8
37.8
23.4
6
acid (2a). The a-configuration of methyl group (23-
Me) was evident from the ROESY spectrum which
showed significant correlation through space interaction
between 23-Me (d 0.99) and H-5 (d 1.09, a-H), but no
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
correlation between 23-Me with 25-Me (d 1.18, b-Me).
H
122.1 122.5 123.4 122.7 122.4 122.2 122.4
145.2 144.6 144.9 144.1 144.1 143.9 144.1
Based upon the above information, the structure of 3 was
elucidated to be 3-keto, 16a-hydroxy, 24-noroleanolic
acid 28-O-a-L-arabinopyranosyl-(1 ! 4)-a-L-arabinopyr-
anosyl-(1 ! 3)-b-D-xylopyranosyl-(1 ! 4)-a-L-rhamnopyr-
anosyl-(1 ! 2)-b-D-fucopyranosyl ester.
42.1
36.3
74.6
47.0
41.4
47.2
31.0
36.1
32.8
209.9
11.0
15.7
17.4
27.2
42.3
36.5
74.0
49.3
41.7
47.4
30.8
36.0
32.1
42.8
36.5
74.6
50.1
42.4
48.0
31.3
36.5
33.2
42.2
28.3
23.2
47.0
41.7
46.2
30.7
34.0
32.4
42.2
28.4
23.7
46.6
42.0
46.3
30.7
33.9
32.3
42.1
28.4
23.5
46.9
41.9
46.1
30.6
33.8
32.2
42.2
28.2
23.7
47.0
42.0
46.4
30.7
33.9
32.4
Compound 4 was isolated as an amorphous powder,
with the molecular formula C H O , as determined
from data of the negative-ion HRESIMS (m/z
5
4
86 24
ꢀ
1
gypsogenin (4a) as the aglycone, and D-glucose and D-
117.5406 [MꢀH] ). On acid hydrolysis, 4 afforded
—
12.2 207.3 209.6 210.4 210.2
6
17.6
15.4
17.5
27.2
—
9.7
15.8
17.4
26.1
11.0
15.7
17.4
25.9
11.0
15.6
17.2
25.7
11.2
15.8
17.4
26.0
galactose in the ratio of 3:1 as component sugars. The
H and C NMR chemical shift assignments of sugar
moieties (Table 2) were accomplished by a combination
13.7
1
13
17.8
27.1
179.9 176.0 177.3 176.2 176.6 176.5 176.5
of TOCSY, HMQC, and HMBC experiments. The b-
anomeric configurations for the glucose were determined
coupling constants (7.8–8.2 Hz), while
33.3
24.7
33.2
24.5
33.3
24.8
33.1
23.7
33.1
23.7
33.0
23.5
33.1
23.8
3
from their J
H1,H2
a
Measured in pyridine-d
OD.
5
.
the a-anomeric configuration for the galactose was deter-
b
3
mined from its small J
Measured in CD
3
coupling constant (3.5 Hz)
H1,H2
and a direct comparison of the NMR data with those
9
of the literature data. The tetrasaccharide moiety at-
tached to C-28 was established by the following HMBC
combined analysis of TOCSY, HMQC, HMBC, and
ROESY spectra, all proton and carbon signals of these
sugars were assigned (Table 2). The b-anomeric configu-
correlations: H-1 of galactose (d 5.46) with C-6 of glu-
H
cose III (d 68.4), H-1 of glucose III (d 4.90) with C-6 of
C
H
rations for the D-fucose and D-xylose units, the a-ano-
glucose I (d 69.3), H-1 of glucose II (d 5.24) with C-3
of glucose I (d 88.5), H-1 of glucose glucose I (d 6.19)
C H
C H
4
meric configurations for two L-arabinose ( C
1
configuration) units were determined from their
J
with C-28 of the aglycone (d 176.2). Thus, the structure
C
3
13
coupling constants (7–8 Hz) and C NMR
of 4 was determined to be gypsogenin 28-O-a-D-galacto-
pyranosyl-(1 ! 6)-b-D-glucopyranosyl-(1 ! 6)-[b-D-glu-
copyranosyl-(1 ! 3)]-b-D-glucopyranosyl ester.
H1,H2
7
data. And the a-anomeric configuration of L-rhamnose
8
was judged by its C (dC 68.3). The HMBC spectrum
5
elucidated the sugar sequence as a-L-arabinopyrano-
syl-(1 ! 4)-a-L-arabinopyranosyl-(1 ! 3)-b-D-xylopyr-
anosyl-(1 ! 4)-a-L-rhamnopyranosyl-(1 ! 2)-b-D-fuco-
pyranosyl as supported by the cross-peaks of H-1 (dH
Compound 5 was obtained as an amorphous powder. It
revealed an [MꢀH] ion peak at m/z 1231 in the nega-
ꢀ
tive ESIMS. The molecular formula of C H O was
59
92 27
6
.51) of rhamnose with C-2 (dC 73.9) of fucose, and
confirmed by HRESIMS. On acid hydrolysis, 5 afforded
6
gypsogenin (4a) as the aglycone, and D-xylose, D-gal-
H-1 (d 5.02) of xylose with C-4 (d 84.8) of rhamnose,
H
C
H-1 (dH 5.12) of inner arabinose with C-3 (d 87.6) of
xylose, and H-1 (dH 4.98) of terminal arabinose with
actose, D-fucose, D-glucoronic acid, and L-rhamnose in
the ratio of 1:1:1:1:1 as component sugars. The chemical
shifts of dC 83.6 (C-3) and 176.6 (C-28) revealed that
compound 5 was a bisdesmosidic glycoside. The ano-
meric proton signals at dH 6.41 (s), 6.00 (d,
J = 8.2 Hz), 5.21 (d, J = 7.5 Hz), 5.00 (d, J = 7.2 Hz),
and 4.90 (d, J = 7.5 Hz) gave correlations with anomeric
carbon signals at dc 101.3, 94.7, 106.3, 107.7, and 103.5
C
C-4 (d 78.5) of inner arabinose (Fig. 1). The same con-
C
clusion with regard to the sugar sequence was also
drawn from the ROESY experiments (Fig. 1). The
pentasaccharide moiety was attached to C-28 of segetalic
acid by the observation of HMBC correlation between
C-28 (d 176.0) of aglycone and H-1 (dH 6.00) of D-fu-
C
1
13
cose. Thus, the structure of 2 was determined as segetalic
acid 28-O-a-L-arabinopyranosyl-(1 ! 4)-a-L-arabinopyr-
anosyl-(1 ! 3)-b-D-xylopyranosyl-(1 ! 4)-a-L-rhamnopyr-
anosyl-(1 ! 2)- b-D-fucopyranosyl ester. The aglycone
part of 3, unlike segelic acid, showed a doublet methyl
in HMQC spectrum, respectively. The H and C NMR
chemical shift assignments (Table 3) were accomplished
by a combination of TOCSY, HMQC, and HMBC
experiments. The linkage of the sugar units at C-3 of
the aglycone was established from the following HMBC
group (d 0.99, d, J = 6.5 Hz) and a ketone carbonyl car-
H
correlations: H-1 of galactose (d 5.21) with C-2 of glu-
H

3
-O-b-D
-galactopyranosyl-(1 ! 2)-b-D
-glucuronopyr-
J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
2915
1
3
Table 2. C and H NMR data for sugar moieties of 1–4
1
1
2
3
4
d
C
d
H
d
C
d
H
d
C
d
H
d
C
d
H
3-O–
GlcA
28-O–
28-O–
GlcI
Fuc
1
1
103.7 4.84 (d, 7.5)
78.3 4.26 (dd, 9.2, 7.5)
87.8 4.21 (t, 9.2)
71.6 4.48 (t, 9.2)
77.2 4.47 (d, 9.3)
171.7
94.7 6.00 (d, 8.7)
95.2 5.32 (d, 8.2)
74.5 3.82 (dd, 8.5, 8.2)
76.6 3.69 (m)
1
2
3
4
5
6
95.0 6.19 (d, 8.2)
72.8 4.12 (m)
2
2
73.9 4.67 (dd, 9.2, 8.7)
76.8 4.15 (dd, 9.2, 3.2)
73.2 3.90 (d, 3.2)
3
3
88.5 4.22 (t, 8.2)
69.1 4.22 (t, 8.2)
77.6 4.04 (m)
4
4
73.6 3.57 (m)
72.7 3.68 (br d, 6.4)
16.5 1.23 (d, 6.4)
5
5
72.6 3.89 (br d, 6.5)
16.9 1.46 (d, 6.5)
6
6
69.3 4.25 (d,11.0, 4.0)
4.62 (br d, 11.0)
Gal
1
Rha
1
104.4 5.47 (d, 7.6)
101.1 6.51 (s)
101.2 5.40 (d, 1.5)
71.9 3.84 (br s)
GlcII
2
73.7 4.47 (dd, 9.7, 7.6)
75.6 4.12 (dd, 9.7, 3.2)
70.1 4.55 (br d, 3.3)
76.6 3.92(dd, 7.7, 4.0)
61.8 4.52 (dd, 11.3, 4.0)
2
71.9 4.78 (br d, 3.0)
72.4 4.66 (dd, 9.4, 3.0)
84.8 4.30 (t, 9.4)
1
2
3
4
5
6
105.8 5.24 (d, 7.8)
75.4 4.00 (t, 7.8)
78.3 4.11 (m)
3
3
72.3 3.94 (br d, 8.5)
84.6 3.54 (t, 8.5)
68.7 3.80 (dd, 8.5, 6.2)
18.3 1.33 (d, 6.2)
4
4
5
5
68.1 4.42 (dd, 9.4, 6.4)
18.4 1.58 (d, 6.4)
71.7 4.13 (m)
78.5 3.97 (m)
6
6
4.41(dd,11.3, 7.7)
Xyl
1
62.5 4.22 (br d, 11.8, 4.2)
4.50 (br d, 11.8)
Gal
1
106.6 5.02 (d, 7.1)
75.4 3.95 (dd, 9.0)
87.6 3.94 (m)
106.7 4.55 (d, 7.8)
75.2 3.41 (dd, 10.7, 7.8) GlcIII
105.2 5.20 (d, 7.6)
73.0 4.47 (dd, 9.8, 7.6)
77.3 4.10 (dd, 9.8, 3.4)
70.2 4.45 (br d, 3.4)
75.4 4.10 (dd, 7.8,4.1)
62.0 4.46 (m)
2
2
3
87.2 3.50 (t, 10.7)
69.7 3.57 (m)
1
2
3
4
5
6
105.4 4.90 (d, 7.8)
74.9 3.95 (t, 8.0)
78.2 4.14 (m)
3
4
68.9 3.94 (m)
66.9 4.08(dd, 11.5, 5.1)
3.41 (d, 5.1)
4
5
66.9 3.94 (dd, 12.0, 5.3)
3.24 (d, 5.3)
5
71.7 4.06 (m)
76.2 3.90 (m)
6
Ara
1
4.30 (dd, 11.9, 4.1)
105.7 5.12 (d, 7.0)
73.2 4.42 (dd, 8.7, 7.0)
73.4 4.16 (dd, 8.7, 3.1)
78.5 4.25 (m)
105.3 4.49 (d, 7.0)
73.0 3.70 (dd, 8.5, 7.0)
74.3 3.84 (br d, 8.5)
79.4 3.90 (m)
68.4 4.28 (dd, 11.5, 4.4)
4.45 (br d, 11.5)
2
3
Gal
1
4
100.6 5.46 (d, 3.5)
70.6 4.63 (dd, 9.6, 3.5)
71.7 4.51 (dd, 9.6, 3.2)
71.1 4.55 (m)
5
66.2 4.44 (dd, 11.0, 5.0)
3
66.8 4.20 (dd, 11.5, 5.2)
3.61 (d, 5.3)
2
.47 (d, 5.0)
3
Ara
1
4
106.8 4.98 (d, 7.3)
72.9 4.43 (dd, 9.6, 7.3)
74.5 4.06 (dd, 9.6, 3.2)
69.6 4.33 (m)
106.9 4.38 (d, 7.2)
72.8 3.86 (dd, 9.5, 7.2)
74.1 3.80 (br d, 9.5)
69.8 3.80 (m)
5
72.5 4.61 (m)
62.7 4.39 (2H, m)
2
6
3
4
5
67.2 3.68 (dd, 11.1, 5.0)
4
67.2 3.54 (dd, 11.0, 5.1)
3.88 (d, 5.1)
.17 (d, 5.0)
anosyl gypsogenin 28-O-b-D-xylopyranosyl-(1 ! 4)-a-L-
O
rhamnopyranosyl-(1 ! 2)-b-D-fucopyranoside.
C
OH
OH
H3C
H
Compound 6 had a molecular formula of C H107O35 as
6
established by HRESIMS. Acid hydrolysis of 6 afforded
6
gypsogenin (4a) and monosaccharides D-fucose, L-
9
O
O
O
Fuc
HO
HO
HO
H
Rha
O
H
rhamnose, D-xylose, D-galactose, L-arabinose, and D-
glucuronic acid (1:1:2:1:1:1). The chemical shifts of dC
84.6 (C-3) and 176.5 (C-28) revealed that compound 6
was a bisdesmosidic glycoside. The anomeric proton sig-
nals at dH 6.44 (s), 6.01 (d, J = 8.2 Hz), 5.55 (d,
J = 7.7 Hz), 5.34 (d, J = 7.7 Hz), 5.19 (d, J = 7.1 Hz),
H3C
O
O
OH
HO
O
Ara
HO
HO
OH
O
O
O
H
H
OH
H
H
OH H
HO
OH
HMBC
ROESY
H
Figure 1. Selected HMBC and ROESY correlations for 2.
4
.95 (d, J = 7.6 Hz), and 4.86 (d, J = 7.5 Hz) gave corre-
lations with anomeric carbon signals at dc 101.2, 94.7,
03.9, 104.9, 105.4, 107.0, and 104.0 in HMQC spec-
1
1
13
curonic acid (d 82.2), H-1 of glucuronic acid (d 4.90)
trum, respectively. The H and C NMR chemical shift
assignments (Table 3) were accomplished by a combina-
tion of TOCSY, HMQC, and HMBC experiments. The
linkage of the sugar units at C-3 of the aglycone was
established from the following HMBC correlations: H-
1 of galactose (dH 5.55) with C-2 of glucuronic acid
(d 78.2), H-1 of xylose (d 5.34) with C-3 of glucuronic
C
H
with C-3 of the aglycone (d 83.6). Similarly, the sugar
C
chain at C-28 was established from the following
HMBC correlations: H-1 of rhamnose (dH 6.41) with
C-2 of fucose (dC 74.7), H-1 of xylose (dH 5.00) with
C-4 of rhamnose (dC 85.5), and H-1 of fucose (dH
6.00) with C-28 of the aglycone (dC 176.6). From the
above evidences, the structure of 5 was elucidated as
C
H
acid (d 78.2), and H-1 of glucuronic acid (d 4.86) with
C
H

2916
J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
1
Table 3. C and H NMR data for sugar moieties of 5–7
3
1
5
6
7
d
C
d
H
d
C
d
H
d
C
d
H
3-O-Sugars
GlcA
1
103.5
82.2
77.6
70.8
77.2
172.3
4.90 (d, 7.5)
4.23 (dd, 9.2, 7.5)
4.28 (t, 9.2)
104.0
78.2
85.8
71.2
77.2
171.7
4.86 (d, 7.5)
4.30 (dd, 9.1, 7.5)
4.21 (t, 9.1)
104.0
78.6
85.9
70.8
76.3
170.9
4.89 (d, 7.5)
4.32 (t, 7.5)
2
3
4.22 (dd, 9.5, 7.5)
4.48 (t, 9.5)
4.48 (d, 9.5)
4
4.22 (t, 9.5)
4.51 (d, 9.7)
4.43 (t, 9.3)
4.47 (d, 9.3)
5
6
Gal
1
106.3
72.5
74.0
70.2
74.8
62.1
5.21 (d, 7.5)
103.9
73.5
75.4
70.0
76.4
61.3
5.55 (d, 7.7)
104.2
73.7
75.2
70.2
76.7
61.7
5.50 (d, 7.6)
4.45 (t, 8.0)
4.13 (dd, 8.2, 3.0)
4.52 (d, 3.0)
4.00 (m)
2
4.53 (dd, 9.5, 2.7)
4.11 (dd, 8.5, 3.3)
4.56 (d, 3.3)
4.44 (dd, 9.6, 7.7)
4.13 (dd, 9.6, 3.2)
4.55 (d, 3.2)
3
4
5
3.96 (m)
4.52 (2H, m)
3.96 (m)
4.50 (m), 4.38 (m)
6
4.40 (2H, m)
Xyl
1
104.9
75.2
78.5
70.7
67.2
5.34 (d, 7.7)
104.9
75.3
78.6
70.8
67.3
5.26 (d, 7.7)
3.90 (m)
2
3.92 (dd, 7.7, 8.3)
4.08 (t, 8.3)
4.07 (m)
3
4.12 (t, 8.4)
4.09 (m)
4
5
3.64 (dd, 12.0, 10.2)
4
3.63 (dd, 11.4, 10.0)
4.20 (br d, 11.4, 5.1)
.22 (dd, 12.0, 5.4)
28-O-Sugars
Fuc
1
94.7
74.7
76.7
73.2
72.2
16.9
6.00 (d, 8.2)
4.67 (t, 8.2)
4.15 (dd, 8.2, 4.0)
3.93 (d, 4.0)
3.88 (m)
94.7
73.8
76.7
73.0
72.2
16.8
6.01 (d, 8.2)
4.64 (t, 8.2)
4.13 (dd, 8.4, 2.7)
3.90 (d, 3.0)
3.84 (m)
94.8
74.1
75.1
73.1
72.4
16.9
5.99 (d, 8.2)
4.63 (t, 8.2)
2
3
4.15 (dd, 8.2, 3.3)
3.96 (d, 3.3)
4
5
3.90 (dd, 6.1, 3.3)
1.48 (d, 6.1)
6
1.46 (d, 6.3)
1.45 (d, 6.3)
Rha
1
101.3
71.8
72.9
85.5
68.2
18.5
6.41 (s)
4.82 (br s)
101.2
71.5
72.5
85.6
67.8
18.4
6.44 (s)
4.82 (br s)
101.3
71.3
82.7
78.8
68.2
18.9
6.30 (s)
5.00 (br s)
2
3
4.67 (br d, 8.5)
4.30 (t, 8.5)
4.47 (dd, 8.5, 6.2)
1.69 (d, 6.2)
4.59 (dd, 8.8, 2.3)
4.25 (t, 8.8)
4.65 (br d, 8.3)
4.48 (t, 8.3)
4.47 (dd, 8.3, 5.5)
1.67 (d, 5.5)
4
5
4.38 (dd, 8.8, 6.1)
1.65 (d, 6.1)
6
Xyl
1
107.7
76.3
78.7
68.7
66.8
5.00 (d, 7.2)
4.04 (m)
107.0
75.2
86.4
68.7
66.8
4.95 (d, 7.6)
3.92 (t, 8.9)
3.95 (m)
105.3
75.9
79.5
71.2
67.1
5.34 (d, 7.8)
2
3.89 (dd, 10.7, 7.8)
4.01 (t, 10.7)
4.14 (m)
3
4.02 (m)
4.10 (dd, 10.2, 8.5)
4.25 (dd, 11.3, 5.3)
4
4.01 (dd, 9.9, 8.6,)
3.45 (t, 11.1)
4.14 (dd, 11.1, 5.4)
5
3.33 (t, 11.0)
4.13 (dd, 11.0, 5.4)
3
.50 (t, 11.2)
Ara
Glc
1
2
3
4
5
6
105.4
72.6
74.4
69.1
67.1
5.19 (d, 7.1)
105.1
75.1
78.7
71.7
78.2
64.4
5.23 (d, 7.7)
3.90 (t, 7.8)
4.10 (t, 8.0)
3.98 (m)
4.51 (dd, 8.6, 7.1)
4.15 (dd, 8.5, 3.0)
4.27 (m)
3.73 (dd, 11.5, 5.3)
4.30 (d, 5.3)
3.90 (m)
4.65 (br d, 11.0)
4
.90 (br d, 11.0)
COCH
COCH
3
170.9
20.9
3
2.12 (s)
C-3 of the aglycone (d 84.6). Similarly, the sugar chain
at C-28 was established from the following HMBC cor-
opyranosyl gypsogenin 28-O-a-L-arabinopyranosyl-
(1 ! 3)-b-D-xylopyranosyl-(1 ! 4)-a-L-rhamnopyrano-
syl-(1 ! 2)-b-D-fucopyranoside.
C
relations: H-1 of rhamnose (d 6.44) with C-2 of fucose
H
(
(
(
d 73.8), H-1 of xylose (d 4.95) with C-4 of rhamnose
C H
d 85.6), H-1 of arabinose (d 5.19) with C-3 of xylose
Compound 7 displayed quasi-molecular ion peak
[MꢀH] at m/z 1567.6743 in the negative HRESIMS, in
C
H
ꢀ
dC 86.4), and H-1 of fucose (dH 6.01) with C-28 of the
aglycone (dC 176.5). From the above evidences, the
structure of 6 was elucidated as 3-O-b-D-galactopyrano-
syl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 3)]-b-D-glucuron-
accordance with empirical molecular formula of
C H O . Its negative-ion ESIMS showed a quasi-
7
2
112 37
ꢀ
molecular ion peak at m/z 1567 [MꢀH] , 42 mass units

J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
2917
higher than that of known compound 10 (m/z 1643
4a
Table 4. a-Glucosidase inhibition of compounds 1–12 and their
aglycones
ꢀ
1
13
[
MꢀH] ). The H and C NMR data (Table 3) of 7 as-
signed from TOCSY, HMQC, and HMBC experiments
were similar to those of known compound 10, except for
the appearance of an additional acetyl group. The down-
Compound a-Glucosidase
inhibition constant
Compound a-Glucosidase
a
inhibition
(IC50) [lM]
b
(IC50) [lM]
b
field signals of Glc-6 at d 4.90, 4.65 gave two cross-peaks
H
1
2
3
4
5
6
7
8
Not active
9
10
Not active
Not active
Not active
78.5 ± 7.1
59.7 ± 3.2
98.2 ± 1.9
85.2 ± 1.2
b
b
with the downfield signal of C-6 at d 64.4 in the HMQC
23.1 ± 1.8
65.5 ± 4.5
15.2 ± 1.8
C
spectrum, respectively, and revealed the location of the
acetyl group at this position, which was confirmed by cor-
relations between two proton signals [dH 4.90 (br d,
J = 11.0 Hz), 4.65 (br d, J = 11. 0 Hz)] of Glc-6 and car-
bonyl carbon (dH 170.9) of acetyl group in the HMBC
spectrum, respectively. On the basis of the above results,
compound 7 was determined as 3-O-b-D-galactopyrano-
syl-(1 ! 2)-[b-D-xylopyranosyl-(1 ! 3)]-b-D-glucurono-
11
12
b
Not active
1a
2a
b
Not active
b
Not active
4a
Acarbose
b
c
Not active
388.0 ± 9.6
a
IC50 is defined as the concentration that resulted in a 50% a-gluco-
sidase inhibition and the results are means ± standard deviation of
three independent replicates.
pyranosyl
gypsogenin
28-O-(6-O-acetyl)-b-D-gluco-
b
c
No inhibition at 400 lM concentration.
Positive control substance.
pyranosyl-(1 ! 3)-[b-D-xylopyranosyl-(1 ! 4)]-a-L-rham-
nopyranosyl-(1 ! 2)-b-D-fucopyranoside.
Compounds 2 and 3 are newly described triterpenoid
saponins bearing identical sugar chain. As described
above, acid hydrolysis of 2 furnished a rearranged agly-
cone and identified as 3-keto, 16a-hydroxy, 24-norole-
anolic acid (2a), which is the aglycone of 3. It
suggested that 3 originates from 2 by a sequence of
dehydrated and rearranged reactions in the plant. The
fact that 2, 3, and 12 with the identical sugar sequences
coexist in the same plant leads us to suggest a reasonably
plausible biosynthetic pathway for 2 and 3 originated
from 12 (Scheme 2).
against a-glucosidase. As shown in Table 4, compounds
–4, 12, 1a, 2a, and 4a exhibited more potent a-glucosi-
dase inhibitory activities than that of acarbose, a widely
2
used clinically useful drug, used as a positive control
(IC50 = 388 ± 9.6 lM). While the other saponins (1, 5–
1
1) were found to be inactive in this test (no inhibition
1
2
at 400 lM concentration). To our knowledge, 2–4
and 12 are the first examples of the triterpenoid saponins
with the a-glucosidase inhibitory activities. Interest-
ingly, compound 4 is a triterpenoid saponin with a-ano-
meric monosaccharide (a-galactose), and a similar
a-anomeric structural unit exists in acarbose (a-glucose).
It showed the stronger inhibitory activity with
IC50 = 15.2 ± 1.8 lM, compared with the other saponins
Since some triterpenes have been reported to exert anti-
1
0
diabetes activities or potent a-glucosidase inhibitory
1
1,12
activities,
the isolated saponins (1–12), with their
2
, 3, and 12.
aglycones quillaic acid (1a), 3-keto, 16a-hydroxy, 24-
noroleanolic acid (2a) and gypsogenin (4a) as the refer-
ences, have been evaluated for the inhibitory activities
As described above, the isolated saponins (1–12) from
G. oldhamiana, based on the position of sugar linkage at-
[O]
COO Sugars
OH
-CO2
COO Sugars
OH
COO Sugars
OH
HO
HO
HO
COOH
CHO
12
OH-
COO Sugars
OH
-H2O
Rearrangement
COO Sugars
OH
COO Sugars
OH
HO
O
HO
OH
3
2
Scheme 2. Hypothetical biogenetic pathway for 2 and 3 originated from 12.

2918
J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
tached to the aglycone, could be classified into three ser-
ies: 3-O-monoglucosides (1, 8, 9), 28-O-monoglucosides
rated by HPLC (CH CN–H O, 47:53, UV detection at
3 2
210 nm), and yielded pure 2 (10 mg, t = 7.7 min), 12
R
(
2–4, 12), and 3, 28-O-bidesmosides (5–7, 10, 11). On the
(30 mg, t = 12.2 min), and 3 (7 mg, t = 28.3 min),
R
R
basis of our results, the preliminary structure–activity
relationships based on the position of sugar linkage at-
tached to the aglycone may be suggested. In particular,
compared with the aglycones (1a, 2a, and 4a), only the
respectively. Fraction 4 was subjected to HPLC
(CH CN-0.05% TFA in H O, 45:55) to afford 4 (10 mg,
3
2
t = 3.5 min), 1 (24 mg, t = 6.5 min), 8 (18 mg, t =
R
R
R
7.8 min), and 9 (15 mg, t = 8.4 min). Fraction 5 was
R
2
8-O-monoglucosidic glycosides (2–4, 12) still showed
passed through a Sephadex LH-20 column (MeOH as elu-
ent) to obtain 6 (20 mg) and a mixture. This mixture was
further separated by HPLC (CH CN-0.05% TFA in H O,
significant a-glucosidase inhibitory activities. The gyl-
cosylation of aglycone at 3-OH (1, 5–11) led to loss of
a-glucosidase inhibitory activity, which was probably
due to the presence of a free polar group (OH or
C@O) at C-3 of aglycone and it may interact with the
enzyme binding sites through intermolecular interac-
tions. Of course, more saponins need to be tested to con-
firm this result.
3
2
48:52) to afford 5 (15 mg, t = 11.6 min), 7 (25 mg,
R
t = 16.2 min), 10 (30 mg, t = 8.9 min), and 11 (25 mg,
R
R
tR = 6.1 min), respectively.
2
5
ꢂ
3.3.1. Compound 1. White powder; ½aꢁ þ 17:8 (c 0.22;
D
KBr
max
ꢀ1
.
MeOH); IRm : 3427, 2945, 1722, 1680, 1077 cm
H NMR (500 MHz, pyridine-d ) d 0.78, 0.94, 1.04,
1
5
1.16, 1.43, 1.79 (3H each, s, Me-25, 26, 29, 30, 24, 27),
2.79 (1H, t, J = 13.5 Hz, 19-H), 3.57 (1H, dd, J = 3.0,
3. Experimental
1
5.20 (1H, br s, 16-H), 5.57 (1H, t-like, 12-H), 9.90
3.5 Hz, 18-H), 4.04 (1H, dd, J = 3.0, 13.5 Hz, 3-H),
3
.1. General
1
3
1H, s, 23-H); C NMR data of the aglycone, see Table
(
1. H and C NMR data of glycosidic part, see Table 2.
1
13
Optical rotations were measured with a JASCO P-1020
polarimeter. IR (KBr-disks) spectra were recorded by
Nicolet Impact-410 spectrometer. 1D and 2D NMR
spectra were recorded at 300 K on Bruker, ACF-500
NMR instrument ( H: 500 MHz, C: 125 MHz), with
TMS as internal standard. Mass spectra were obtained
on a MS Agilent 1100 Series LC/MSD Trap mass spec-
trometer (ESIMS) and a Micro Q-TOF MS (HRE-
SIMS), respectively. TLC was performed on pre-
coated silica gel 60 F254 (Qingdao Haiyang Chemical
Co. Ltd) and detection was achieved by 10% H SO -
+
ESIMS m/z: 1009 [M+Na] ; HRESIMS m/z: 985.4643
ꢀ
[MꢀH] (calcd for C H O , 985.4649).
4
8
73 21
1
13
25
D
ꢂ
3.3.2. Compound 2. White powder; ½aꢁ ꢀ 6:0 (c 0.03;
KBr
max
ꢀ1
.
MeOH); IRm : 3428, 2931, 1731, 1632, 1062 cm
H NMR (500 MHz, pyridine-d ) d 0.92, 0.96, 1.00,
1
5
1.17, 1.38, 1.70 (3H each, s, Me-25, 29, 30, 26, 24, 27),
2.79 (1H, t, J = 13.2 Hz, 19-H), 3.44 (1H, dd, J = 3.8,
14.3 Hz, 18-H), 3.84 (1H, m, 3-H), 5.17 (1H, br s, 16-
1
3
H), 5.63 (1H, t, J = 3.3 Hz, 12-H); C NMR data of
the aglycone, see Table 1. H and C NMR data of gly-
2
4
1
13
EtOH for saponins, aniline–phthalate reagents for sug-
ars. Silica gel H (Qingdao Haiyang Chemical Co. Ltd),
Sephadex LH-20 (Pharmacia), and RP-C18 (40–63 lm,
FuJi) were used for column chromatography. Prepara-
tive HPLC was carried out using Agilent 1100 Series
with Shim-park RP-C18 column (200 · 20 mm i.d.) and
ꢀ
cosidic part, see Table 2. ESIMS m/z: 1161 [MꢀH] ,
ꢀ
HRESIMS: m/z 1161.5688 [MꢀH]
(calcd for
C H O , 1161.5688).
6
5
89 25
2
D
5
ꢂ
3.3.3. Compound 3. White powder; ½aꢁ ꢀ 4:8 (c 0.05;
KBr
max
ꢀ1
.
1
100 Series Multiple Wavelength detector.
MeOH); IRm : 3410–3499, 2931, 1724, 1060 cm
H NMR (500 MHz, CD OD) d 0.86, 0.88, 0.95, 1.18,
1
3
3
.2. Plant material
1.38 (3H each, s, Me-26, 29, 30, 25, 27), 0.99 (3H, d,
J = 6.5 Hz, 23-Me), 2.30 (1H, t, J = 13.3 Hz, 19-H),
2.96 (1H, dd, J = 4.0, 14.3 Hz, 18-H), 4.51 (1H, br s,
The roots of G. oldhmiana were collected in suburbs of
Jinan, Shandong Province, China, in October 2004
and identified by Prof. Yun-Yao Li of the Department
of Pharmacognosy, Shandong University. A voucher
specimen (No. 041022) was deposited in the Department
of Natural Medicinal Chemistry, China Pharmaceutical
University.
1
3
16-H), 5.34 (1H, t, J = 3.3 Hz, 12-H); C NMR data
of the aglycone, see Table 1. H and C NMR data
of glycosidic part, see Table 2. ESIMS m/z: 1143
1
13
ꢀ
ꢀ
+
[MꢀH] , 1167 [M+Na] ; HRESIMS m/z: 1143.5560
[MꢀH] (calcd for C H O , 1161.5592).
5
6
87 24
2
5
ꢂ
3
.3.4. Compound 4. White powder; ½aꢁ þ 50:0 (c 0.03;
D
KBr
max
ꢀ1
.
3
.3. Extraction and isolation
MeOH); IRm : 3439, 2932, 1730, 1634, 1066 cm
H NMR (500 MHz, pyridine-d ) d 0.85, 0.86, 0.93,
1
5
The air-dried roots (30 kg) of G. oldhmiana were extracted
with aq. 70% EtOH (40 l · 2 h · 3) under reflux. After re-
moval of the solvent in vacuum, the residue was sus-
1.04, 1.20, 1.34 (3H each, s, Me-30, 29, 25, 26, 27, 24),
3.14 (1H, dd, J = 4.1, 14.0 Hz, 18-H), 4.04 (1H, m, 3-
1
3
H), 5.40 (1H, t-like, 12-H), 9.60 (1H, s, 23-H);
NMR data of the aglycone, see Table 1. H and
C
C
1
13
pended in H O and partitioned between n-BuOH and
2
water. The n-BuOH-soluble portion (724 g) was fraction-
ated by a silica gel column (100–200 mesh), which was
eluted with CHCl /MeOH/H O (100:10:0 ! 100:100:1)
NMR data of glycosidic part, see Table 2. ESIMS m/z:
+
ꢀ
1141 [M + Na] , HRESIMS m/z: 1117.5406 [MꢀH]
(calcd for C H O , 1117.5436).
54 85 24
3
2
to give five fractions (fractions 1–5), each fraction was fur-
ther subjected to repeated RP-C18 column with MeOH/
2
D
5
ꢂ
3.3.5. Compound 5. White powder; ½aꢁ þ 5:1 (c 0.04;
KBr
max
ꢀ1
.
H O 4:6 ! 8:2 as eluents. Fraction 3 was further sepa-
MeOH); IRm : 3421, 2941, 1727, 1649, 1048 cm
2

J.-G. Luo et al. / Bioorg. Med. Chem. 16 (2008) 2912–2920
2919
1
H NMR (500 MHz, pyridine-d ) d 0.79, 0.86, 0.87,
actose (tR 7.538 min), and D-glucose (tR 8.032 min)
derivatives prepared in the same way, respectively.
5
1
3
5
.03, 1.24, 1.40 (3H each, s, Me-25, 29, 30, 26, 27, 24),
.10 (1H, br d, J = 14.0 Hz, 18-H), 4.02 (1H, m, 3-H),
.38 (1H, t-like, 12-H), 9.90 (1H, s, 23-H); C NMR
1
3
3.4.1. 3-Keto, 16a-hydroxy, 24-noroleanolic acid (2a). An
1
13
25
ꢂ
KBr
max
ESIMS m/z: 457
data of the aglycone, see Table 1. H and C NMR data
of glycosidic part, see Table 3. ESIMS m/z: 1231
[MꢀH] ; HRESIMS m/z: 1231.5715 [MꢀH] (calcd
for C H O , 1231.5753).
amorphous solid, ½aꢁ þ 30:5 (c 0.10; CHCl ); IRm
D
3434, 1778, 1706, 1457 cm
[M + H] . H NMR (500 MHz, CDCl ) d 0.84, 0.85,
3
ꢀ
1
;
ꢀ
ꢀ
+
1
3
0.91, 1.12, 1.14 (3H each, s, Me-30, 26, 29, 25, 27-
H), 1.00 (3H, d, J = 6.5 Hz, 23-H) 2.54 (1H, t,
J = 14.0 Hz, H-19), 3.23 (1H, dd, J = 3.3, 14.0 Hz,
5
9
91 27
2
D
5
ꢂ
3
.3.6. Compound 6. White powder; ½aꢁ þ 4:8 (c 0.05;
KBr
max
ꢀ1
.
MeOH); IRm : 3429, 2929, 1731, 1633, 1074 cm
18-H), 5.12 (1H, br s, H-16), 5.34 (1H, t-like, 12-H);
1
13
H NMR (500 MHz, pyridine-d ) d 0.73, 0.82, 0.84,
C NMR (125 MHz, CDCl ) d 40.2 (C-1), 37.4 (C-
5
3
1
3
.01, 1.22, 1.40 (3H each, s, Me-25, 30, 29, 26, 27, 24),
.05 (1H, br d, J = 14.0 Hz, 18-H), 4.03 (1H, m, 3-H),
2), 213.6 (C-3), 44.8 (C-4), 53.7 (C-5), 24.0 (C-6),
31.7 (C-7), 39.2 (C-8), 45.4 (C-9), 36.7 (C-10), 22.1
(C-11), 122. 6 (C-12), 143.9 (C-13), 41.9 (C-14), 36.0
(C-15), 74.1 (C-16), 48.6 (C-17), 41.3 (C-18), 46.9
(C-19), 30.7 (C-20), 35.9 (C-21), 32.4 (C-22), 11.7
(C-23), 13.2 (C-25), 17.1 (C-26), 26.8 (C-27), 180.6
(C-28), 33.1 (C-29), 23.6 (C-30).
1
.36 (1H, t-like, 12-H), 9.91 (1H, s, 23-H); C NMR
3
5
data of the aglycone, see Table 1. H and C NMR data
of glycosidic part, see Table 3. ESIMS m/z: 1495
[MꢀH] , HRESIMS m/z: 1495.6571 [MꢀH] (calcd
for C H O , 1495.6598).
1
13
ꢀ
ꢀ
6
9
107 35
2
5
ꢂ
3
.3.7. Compound 7. White powder; ½aꢁ ꢀ 1:5 (c 0.20;
D
3440, 1734, 1041 cm
3.5. Enzyme inhibition assay
KBr
max
ꢀ1
1
MeOH); IRm
.
H NMR
(
500 MHz, pyridine-d ) d 0.86, 0.87, 0.89, 1.10, 1.21,
5
The a-glucosidase inhibition assay was performed
according to the slightly modified method of Pierre
et al. a-Glucosidase (E.C.3.2.1.20) was purchased
1.52 (3H each, s, Me-25, 29, 30, 26, 27, 24), 3.09 (1H,
br d, J = 14.0 Hz, 18-H), 4.05 (1H, m, 3-H), 5.36 (1H,
13
1
3
t-like, 12-H), 9.93 (1H, s, 23-H); C NMR data of the
from Sigma company (No. G-5003, Lot. 081k7415).
The inhibition was measured spectro-photometrically
at pH 6.8 and at 37 ꢁC for 10 min, using 0.01 M p-nitro-
phenyl a-D-glucopyranoside (PNPG) as a substrate and
1 U/mL of enzyme, in 0.067 M KH PO –Na HPO buf-
1 13
aglycone, see Table 1. H and C NMR data of glyco-
ꢀ
sidic part, see Table 3. ESIMS m/z: 1567 [MꢀH] ,
HRESIMS m/z: 1567.6743 [MꢀH]ꢀ (calcd for
C H O , 1567.6809).
111 37
7
2
2
4
2
4
fer. Acarbose were used as positive controls. The incre-
ment in absorption at 410 nm due to the hydrolysis of
PNPG by a-glucosidase was monitored continuously
with an auto multi-functional microplate reader (BIO-
RAD680).
3
.4. Acid hydrolysis and sterochemistry of sugars of 1–7
Each compound (5 mg) was heated in 2 M HCl (5 mL)
at 90 ꢁC for 4 h. The reaction mixture was extracted
with EtOAc (3 · 5 mL). The EtOAc extract was purified
by chromatography on Sephadex LH-20 (2.0 · 100 cm).
Comparing TLC with authentic samples, the aglycone
Acknowledgment
of 1 was determined to be quillaic acid (1a, R : 0.25,
f
CHCl –MeOH, 20:1), while those of 4–7 were gypsoge-
3
This research work was supported by the National Nat-
ural Science Foundation of China for Outstanding
Young Scientists (No. 30525032).
nin (4a, R : 0.43, CHCl –MeOH, 20:1). In addition,
f
3
3
tained from 2 and 3. The H O layer was concentrated
-keto, 16a-hydroxy, 24-noroleanolic acid (2a) was ob-
2
under reduced pressure to dryness, to give a residue of
the sugar fraction. The residue was dissolved in pyridine
References and notes
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