Brazilian natural medicines. III1 structures of triterpene oligoglycosides and lipase inhibitors from mate, leaves of ilex paraguariensis

Article abstract of DOI:10.1248/cpb.57.257

The methanolic extract from the leaves of Ilex paraguariensis (Aquifoliaceae) was found to show an inhibitory activity on porcine pancreatic lipase. From the methanolic extract, three new triterpene oligoglycosides, mateglycosides A, B, and C, were isolat

Full text of DOI:10.1248/cpb.57.257

March 2009
Chem. Pharm. Bull. 57(3) 257—261 (2009)
257
1)
Brazilian Natural Medicines. III.
Structures of Triterpene
Oligoglycosides and Lipase Inhibitors from Mate, Leaves of Ilex
paraguariensis
Sachiko SUGIMOTO, Seikou NAKAMURA, Sachiyo YAMAMOTO, Chihiro YAMASHITA, Yoshimi ODA,
Hisashi MATSUDA, and Masayuki YOSHIKAWA*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received November 11, 2008; accepted December 15, 2008; published online January 5, 2009
The methanolic extract from the leaves of Ilex paraguariensis (Aquifoliaceae) was found to show an in-
hibitory activity on porcine pancreatic lipase. From the methanolic extract, three new triterpene oligoglycosides,
mateglycosides A, B, and C, were isolated together with 18 known compounds. The chemical structures of new
oligoglycosides were elucidated on the basis of chemical and physicochemical evidence. Several constituents
showed inhibitory activities on pancreatic lipase.
Key words mateglycoside; Ilex paraguariensis; lipase inhibitor; mate tea; Brazilian natural medicine; triterpene oligoglycoside
An Aquifolium plant, Ilex (I.) paraguariensis ST. HIL., is cosides A (1), B (2), and C (3) together with 18 known con-
distributed in South American countries: Brazil, Argentina, stituents. This paper deals with the structure elucidation of
Paraguay, etc., and is locally called as mate, erva-mate, or new saponins (1—3) as well as the inhibitory effects of the
yerba-mate. The leaves of this plant are commonly consumed principal constituents on pancreatic lipase.
in South American countries for the preparation of several
Isolation from the Leaves of I. paraguariensis The
types of beverages, such as mate tea or Jesuit’s tea. The methanolic extract (33.3% from the dried leaves) from the
leaves of I. paragueriensis are also known to have the thera- leaves of Ilex paraguariensis cultivated in Brazil, which
peutic effects on arthritis, slow digestions, liver diseases, showed the inhibitory effect on porcine pancreatic lipase,
headache, rheumatism, and obesity in Brazilian traditional was partitioned into an EtOAc and water (1 : 1, v/v) mixture
medicine. As chemical constituents of I. paraguariensis, var- to furnish an EtOAc-soluble fraction (11.2%) and an aqueous
2)
ious triterpene saponins together with flavonoids, caf- phase. The aqueous phase was further extracted with 1-
feoylquinic acids, and xanthines were isolated from the aerial BuOH to give 1-BuOH (16.7%)- and H O (6.4%)-soluble
2
parts. However, the biological activities of the saponin con- fractions. As shown in Table 1, the EtOAc- and 1-BuOH-sol-
stituents have not yet characterized.
uble fractions were found to exhibit the inhibitory effects on
Previously, we have reported the isolation and structure pancreatic lipase, whereas the H O-soluble fraction showed
2
elucidation of flavonoids, pseudoalkaloids, and steroidal weak activity. The active fractions were subjected to normal-
saponins with antidiabetic activities from several Brazilian and reversed-phase silica gel column chromatographies and
1
,3—5)
natural medicines
and also, we have examined the bio- finally HPLC to provide mateglycosides A (1, 0.20%), B (2,
logical activities of triterpene saponins from natural medi- 0.02%), C (3, 0.001%), and D (13, 0.10%), which was reported
6
—11)
12—21)
cines
and medicinal foodstuffs.
As a continuing as an acidic hydrolysis product of oleanolic acid tetraglycoside
22)
23)
study, the methanolic extract from the leaves of I. paraguar- from Aralia elata, together with ursolic acid (4, 1.02%),
iensis was found to exhibit an inhibitory effect against 3b-hydroxyurs-11-en-13b(28)-olide
porcine pancreatic lipase. From the methanolic extract, we saponins 1 (5, 0.09%), 2 (6, 0.01%), 3 (7, 0.034%),
2
4)
(16, 0.039%), mate-
25)
26)
26)
26)
27)
28)
have isolated three new triterpene saponins named mategly-
4
(8, 0.034%), 5 (9, 0.009%), J3 (10, 0.06%), 3-O-b-
Chart 1. New Saponins (1—3) from the Leaves of Ilex paraguariensis
∗
To whom correspondence should be addressed. e-mail: myoshika@mb.kyoto-phu.ac.jp
© 2009 Pharmaceutical Society of Japan

2
58
Vol. 57, No. 3
Table 1. Inhibitory Effects of the MeOH Extract and EtOAc-, 1-BuOH-,
and H O-Soluble Fractions from the Leaves of I. paraguariensis against
2
Porcine Pancreatic Lipase Activity
Inhibition (%)
Conc. (mg/ml)
0
50 100 200 400 800 1000
MeOH ext.
EtOAc-soluble fraction
0.0 28
0.0 42
0.0 31
0.0 21
30
55
37
32
33
69
39
43
35
80
94
48
81
89
99
53
95
88
99
58
1-BuOH-soluble fraction
H O-soluble fraction
2
(
Table 2) spectra of 1, which were assigned by various NMR
38)
experiments, indicated the presence of the following func-
tions: an oleanolic acid part {seven methyls [d 0.87, 0.88,
0
.90, 1.09, 1.13, 1.21, 1.26 (all s, 25, 29, 30, 26, 24, 23, 27-
H )], a methine bearing an oxygen function [d 3.30 (dd, Jꢂ
3
3.5, 11.0 Hz, 3-H)], and an olefin [d 5.42 (m, 12-H)]}, an a-
L-arabinopyranosyl moiety [d 4.86 (d, Jꢂ7.2 Hz, 1ꢃ-H)],
three b-D-glucopyranosyl moieties [d 5.05 (d, Jꢂ7.6 Hz, 1ꢄꢅ-
H), 5.11 (d, Jꢂ7.6 Hz, 1ꢅ-H), and 6.27 (d, Jꢂ8.2 Hz, 1ꢄꢄ-H)],
and an a-L-rhamnopyranosyl moiety [d 6.15 (br s, 1ꢄ-H)]. In
the heteronuclear multiple bond connectivity (HMBC) exper-
iment of 1, long-range correlations were observed between
the following protons and carbons: the 1ꢃ-proton and 3-car-
bon, the 1ꢄ-proton and 2ꢃ-carbon, the 1ꢅ-proton and 3ꢃ-car-
bon, the 1ꢄꢄ-proton and 28-carbon, and the 1ꢄꢅ-proton and
6
ꢄꢄ-carbon. On the basis of those findings, the structure of
mateglycoside A was determined to be 3-O-a-L-rhamnopyra-
nosyl(1—2)[b-D-glucopyranosyl(1ꢅ-3)]-a-L-arabinopyra-
nosyloleanolic acid 28-O-b-D-glucopyranosyl(1—6)-b-D-
glucopyranoside (1).
Chart 2. Known Constituents from the Leaves of Ilex paraguariensis
Mateglycoside B (2) was also obtained as a white powder
2
5
D-glucopyranosyl(1—3)-a-L-2-O-acetylarabinopyranosyl- with positive optical rotation ([a] ꢁ18.7° in MeOH). The
D
29)
urolic acid 28-O-b-D-glucopyranoside (11, 0.047%), nudi- IR spectrum of 2 showed absorption bands at 3600, 1716,
3
0)
ꢀ1
caucin C (14, 0.12%), 3-O-a-L-rhamnopyranosyl(1—2)-a- 1650, and 1070 cm due to hydroxyl, carbonyl, and olefin
L-arabinopyranosyloleanolic acid 28-O-b-D-glucopyrnosyl(1— functions. In the positive- and negative-ion FAB-MS spectra
3
1)
6
)-b-D-glucopyranoside
(15, 0.29%), (R)-linalyl 6-O- of 2, pseudomolecular ion peaks were observed at m/z 951
32)
ꢁ ꢀ
a-L-arabinopyranosyl-b-D-glucopyranoside (18, 0.090%), (MꢁNa) and m/z 927 (MꢀH) , respectively and high-reso-
3
3)
34)
rutin
0
(19, 0.512%), kaempferol-3-O-rutinoside
(20, lution FAB-MS analysis of the pseudomolecular ion peak
35)
ꢁ
.178%), 5-O-caffeoyl-D-quinic acid (21, 0.046%), caf- (MꢁNa) revealed the molecular formula of 2 to be
feine (0.55%), and theobromine (0.15%).
The Structures of Mateglycosides A (1), B (2), and C 1,4-dioxane provided 23-hydroxyursolic acid (17) together
3) Mateglycoside A (1) was isolated as a white powder with L-arabinose and D-glucose, which were identified by
3
6)
37)
C H O . Acid hydrolysis of 2 with 5% aqueous H SO -
47 76 18 2 4
39)
(
2
5
1,3,6)
1
with negative optical rotation ([a] ꢀ5.6° in MeOH). The HPLC analysis using an optical rotation detector.
The H-
D
1
3
38)
IR spectrum of 1 showed absorption bands at 1719 and (pyridine-d ) and C-NMR (Table 2) spectra of 2 showed
5
ꢀ
1
1
650 cm ascribable to carbonyl and olefin functions and signals assignable to a 23-hydroxyursolic acid part, an a-L-
ꢀ1
broad bands at 3600 and 1075 cm suggestive of an oligo- arabinopyranosyl moiety [d 4.98 (d, Jꢂ7.2 Hz, 1ꢃ-H)], and
glycoside structure. In the positive-ion FAB-MS spectrum of two b-D-glucopyranosyl moieties [d 5.34 (d, Jꢂ7.6 Hz, 1ꢄ-
1
, a quasimolecular ion peak was observed at m/z 1243 (Mꢁ H), 6.29 (d, Jꢂ7.9 Hz, 1ꢅ-H)]. The HMBC experiment on 2
ꢁ
Na) , whereas its negative-ion FAB-MS spectrum showed a showed long-range correlations between the 1ꢃ-proton and 3-
ꢀ
quasimolecular ion peak at m/z 1219 (MꢀH) together with carbon, between the 1ꢄ-proton and 3ꢃ-carbon, and between
fragment ion peak at m/z 895 (MꢀC H O ) , which was the 1ꢅ-proton and 28-carbon. Consequently, the structure of
ꢀ
1
2
21 10
derived by cleavage of the ester glycoside linkage. The mo- mateglycoside B was clarified to be 3-O-b-D-glucopyra-
lecular formula of 1 was determined to be C H O by nosyl(1—3)-a-L-arabinopyranosyl 23-hydroxyurolic acid 28-
5
9
96 26
high-resolution positive-ion FAB-MS analysis of the quasi- O-b-D-glucopyranoside (2).
ꢁ
molecular ion peak (MꢁNa) . Acid hydrolysis of 1 with 5%
Mateglycoside C (3), which was isolated as a white pow-
2
9
aqueous H SO in 1,4-dioxane (1 : 1, v/v) liberated oleanolic der with positive optical rotation ([a] ꢁ47.3° in MeOH),
acid (12) together with L-arabinose, D-glucose, and L-rham- showed absorption bands due to hydroxyl, carbonyl, and
2
4
D
nose, which were identified by HPLC analysis using an opti- olefin functions in the IR spectrum. The molecular formula,
1
,3,6)
1
13
cal rotation detector.
The H- (pyridine-d ) and C-NMR C H O , of 3 was determined from the positive-ion FAB-
5 41 66 13

March 2009
259
13
Table 2.
C-
C-NMR (125, 150 MHz) Data of Mateglycosides A (1), B (2) and C (3) (Measured in Pyridine-d5)
1
2
3
C-
1
2
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
39.0
26.6
88.2
39.6
56.0
18.6
32.5
39.9
48.0
37.0
23.4
123.0
144.1
42.1
28.3
23.7
47.0
41.7
46.2
30.7
33.1
33.1
28.1
17.0
15.7
17.5
26.0
176.5
23.6
33.1
39.3
26.3
81.8
43.5
47.5
19.2
33.2
39.1
48.1
36.8
24.7
126.7
138.4
42.5
28.7
24.7
48.4
53.3
40.2
39.0
30.8
36.8
64.2
13.7
16.4
17.7
23.7
176.2
17.4
21.3
39.1
26.2
81.9
43.5
47.6
18.2
33.2
40.2
48.1
36.9
24.7
126.2
138.4
42.5
28.5
24.7
48.4
53.3
39.1
39.4
30.8
36.8
64.5
13.7
16.4
17.4
23.8
176.2
17.8
21.3
3-O-Ara
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
2ꢃ-O-Rha
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
3ꢃ-O-Glc
1ꢅ
2ꢅ
3ꢅ
4ꢅ
5ꢅ
6ꢅ
28-O-Glc
1ꢄꢄ
2ꢄꢄ
3ꢄꢄ
4ꢄꢄ
5ꢄꢄ
6ꢄꢄ
6ꢄꢄ-O-Glc
1ꢄꢅ
2ꢄꢅ
104.6
74.7
82.0
68.2
64.8
106.6
72.2
84.2
69.3
67.1
106.7
73.2
74.8
69.7
67.0
101.9
72.3
72.5
73.9
70.0
18.6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
104.7
74.9
78.3
71.4
78.5
62.5
106.4
75.7
78.4
71.6
78.4
62.7
95.6
75.1
78.2
70.8
77.9
69.3
95.7
74.1
78.9
71.2
78.8
62.3
95.7
74.1
79.2
71.2
78.9
62.3
105.3
75.1
78.4
71.3
78.7
62.5
3
4
5
6
ꢄꢅ
ꢄꢅ
ꢄꢅ
ꢄꢅ
ꢁ
MS [m/z 789 (MꢁNa) ] and by high-resolution MS meas- Table 3. Inhibitory Effects of Principal Constituents from I. paraguarien-
sis against Porcine Pancreatic Lipase Activity
urement. The acid hydrolysis of 3 liberated 17, L-arabinose,
1,3,6)
1
13
and D-glucose.
The H- (pyridine-d ) and C-NMR
5
Inhibition (%)
38)
(
Table 2) spectra of 3 showed signals assignable to a 23-
hydroxylursolic acid part, an a-L-arabinopyranosyl moiety [d
.99 (d, Jꢂ7.2 Hz, 1ꢃ-H)], and a b-D-glucopyranosyl moiety
d 6.29 (d, Jꢂ8.1 Hz, 1ꢄ-H)]. In the HMBC experiment on 3,
Conc. (mM)
0
50
100
200
400
800
4
[
1
4
5
7
9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
ꢀ2.7
13
47
2.1
5.2
36
8.0
39
ꢀ7.8
10
94
ꢀ2.3
0.5
78
77
83
24
12
ꢀ2.5
16
95
3.9
ꢀ0.9
94
94
102
27
23
39
15
22
46
17
long-range correlations were observed between the 1ꢃ-proton
and the 3-carbon and between the 1ꢄ-proton and 28-carbon.
This evidence led us to formulate the structure of mateglyco-
side C (3) as shown.
96
96
15
33
ꢀ3.1
96
ꢀ7.8
96
1
1
1
1
2
4
5
8
9
0
97
98
In the course of characterization studies on antiobesitic
constituents from medicinal foodstuffs, we have reported the
isolation and structure elucidation of several saponins and
diterpenes with the inhibitory effect on pancreatic lipase
101
36
99
39
15
10
37
38
49
21
39
53
47
4
0)
41)
from Salvia officinalis,
Camellia sinensis,
Aesculus
8)
42,43)
turbinata, and Sapindus rarak.
Pancreatic lipase is well
known to play an important role in lipid digestion. Since the antiobesitic activity of mate tea.
mate tea is used for the treatment of obesity and also, the
methanolic extract and its EtOAc- and 1-BuOH-soluble frac-
tions from the leaves of I. paraguariensis showed an in-
Experimental
The following instruments were used to obtain physical data: specific ro-
tations, Horiba SEPA-300 digital polarimeter (lꢂ5 cm); IR spectra, Shi-
hibitory activity on porcine pancreatic lipase, we examined madzu FTIR-8100 spectrometer; FAB-MS and high-resolution MS, JEOL
1
JMS-SX 102A mass spectrometer; H-NMR spectra, JEOL EX-270
the inhibitory effects of the principal constituents from the
EtOAc- and 1-BuOH-soluble fractions. As shown in Table 3,
several triterpene saponins (5, 14, 15) and monoterpene oli-
(
270 MHz), JNM-LA500 (500 MHz), and JEOL ECA-600K (600 MHz)
1
3
spectrometers; C-NMR spectra, JEOL EX-270 (68 MHz) JNM-LA500
125 MHz), and JEOL ECA-600K (150 MHz) spectrometers with tetra-
(
goglycosides (18) were found to exhibit the potent inhibitory methylsilane as an internal standard; and HPLC detector, Shimadzu RID-6A
activities and these compounds seems to be responsible for refractive index and SPD-10Avp UV-VIS detectors. HPLC column, YMC-

2
60
Vol. 57, No. 3
Pack ODS-A (250ꢆ4.6 mm i.d.) and (250ꢆ20 mm i.d.) columns were used [600 g, H O : MeOH (10 : 90)→MeOH] to give six fractions [fr. 4-1 (12.3 g),
2
for analytical and preparative purposes, respectively.
4-2 (1.1 g), 4-3 (477 mg), 4-4 (663 mg), 4-5 (1.2 g), 4-6 (3.5 g)]. Fraction 4-2
The following experimental conditions were used for chromatography: or- (500 mg) was purified by HPLC [H O : MeOH (65 : 35)] to give mateglyco-
2
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji side C (3, 11.3 mg). Fraction 4-5 (250 mg) was purified by HPLC [H O :
2
Silysia Chemical, Ltd., 150—350 mesh); reverse-phase silica gel column MeOH (65 : 35)] to furnish 5 (21.6 mg). The known compounds were identi-
1
13
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., fied by comparison of their physical data ([a] , IR, MS, and H- and C-
D
22—35)
1
0
00—200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck, NMR) with reported values.
The obtained caffeine and theobromine
.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (re- were identified by comparison of the MS, H- and C-NMR data with those
1
13
verse phase); reversed-phase HPTLC, precoated TLC plates with Silica gel of commercial products.
2
5
RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spraying
with 1% Ce(SO ) –10% aqueous H SO followed by heating.
Mateglycoside A (1): A white powder; [a]D ꢀ5.6 (cꢂ0.64, MeOH);
high-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa) :
ꢁ
4
2
2
4
59 96 26
ꢀ1
Plant Material The leaves of I. paraguariensis, which were cultivated 1243.6089. Found: 1243.6094; IR (KBr): n 3600, 1719, 1650, 1075 cm
;
max
1
in Brazil, were purchased from Mitsuboshi Pharmaceutical Co., Ltd., Nara,
Japan. The botanical identification was undertaken by research members of (3H each, all s, H -25, 29, 30, 26, 23, 24, 27), 3.30 (1H, dd, Jꢂ3.5, 11.0 Hz,
H-NMR (600 MHz, pyridine-d ) d 0.87, 0.88, 0.90, 1.09, 1.13, 1.21, 1.26
5
3
Mitsuboshi Pharmaceutical Co., Ltd. A voucher specimen of this plant is on
file in our laboratory.
H-3), 4.86 (1H, d, Jꢂ7.2 Hz, H-1ꢃ), 5.05 (1H, d, Jꢂ7.6 Hz, H-1ꢄꢅ), 5.11
(1H, d, Jꢂ7.6 Hz, H-1ꢅ), 5.42 (m, H-12), 6.15 (1H, br s, H-1ꢄ), 6.27 (1H, d,
1
3
Isolation of Mateglycosides A (1), B (2), C (3), and D (4) The dried Jꢂ8.2 Hz, H-1ꢄꢄ); C-NMR: given in Table 2; positive-ion FAB-MS: m/z
ꢁ
ꢀ
leaves (3.0 kg) of I. paraguariensis were finely cut and extracted three times 1243 (MꢁNa) ; negative-ion FAB-MS: m/z 1219 (MꢀH) , m/z 895
ꢀ
ꢀ
ꢀ
with methanol under reflux for 3 h. Evaporation of the solvent under reduced (MꢀC H O ) , m/z 733 (MꢀC H O ) , m/z 587 (MꢀC H O ) , m/z
1
2
21 10
18 31 15
24 41 19
ꢀ
pressure provided a methanolic extract (1.0 kg). The methanolic extract 455 (MꢀC H O ) .
2
9
49 23
2
5
(
500 g) was partitioned into an EtOAc–H O (1 : 1, v/v) mixture to furnish an
Mateglycoside B (2): A white powder; [a]D ꢁ18.7 (cꢂ0.13, MeOH);
EtOAc-soluble fraction (168.0 g) and aqueous phase, which was extracted high-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa) :
2
ꢁ
4
7
76 18
ꢀ1
with 1-BuOH to give 1-BuOH- (251 g) and H O- (96.5 g) soluble fractions.
951.4933. Found: 951.4938; IR (KBr): n
3600, 1716, 1650, 1070 cm ;
2
max
1
The 1-BuOH-soluble fraction (150 g) was subjected to normal-phase sil-
H-NMR (600 MHz, pyridine-d ) d 0.89. 0.94 (both 3H, both d, Jꢂ5.9 Hz,
5
ica gel column chromatography [BW-200 (Fuji Silysia Co., Ltd., 3.0 kg), H -30, 29), 0.95, 0.98, 1.15, 1.18 (3H each, all s, H -23, 25, 27, 26), 3.72
3
3
CHCl : MeOH : H O (10 : 3 : 1→7 : 3 : 1 lower phase→6 : 4 : 1, v/v/v)→MeOH]
(1H, d, Jꢂ10.5 Hz, H-24), 4.26 (1H, m, H-24), 4.28 (1H, m, H-3), 4.98 (1H,
to give six fractions [fr.1 (14.7 g), 2 (26.9 g), 3 (30.8 g), 4 (41.3 g), 5 (34.5 g), d, Jꢂ7.2 Hz, H-1ꢃ), 5.34 (1H, d, Jꢂ7.6 Hz, H-1ꢄ), 6.29 (1H, d, Jꢂ7.9 Hz, H-
3
2
1
3
ꢁ
6
(5.5 g)]. Fraction 1 (14.0 g) was separated by reversed-phase silica gel 1ꢅ); C-NMR: given in Table 2; positive-ion FAB-MS: m/z 951 (MꢁNa) ;
ꢀ ꢀ
6
column chromatography [420 g, H O–MeOH (90 : 10→70 : 30→50 : 50→ negative-ion FAB-MS: m/z 927 (MꢀH) , m/z 765 (MꢀC H O ) , m/z 603
2
11
5
ꢀ
ꢀ
3
0 : 70, v/v)→MeOH] to give theobromine (1.30 g), and caffeine (4.80 g). (MꢀC H O ) , m/z 471 (MꢀC H O ) .
1
2
21 10
17 29 14
2
9
Fraction 2 (25.0 g) was subjected to reversed-phase silica gel column chro-
Mateglycoside C (3): A white powder; [a]D ꢁ47.3 (cꢂ0.63, MeOH);
ꢁ
matography [750 g, H O : MeOH (90 : 10→70 : 30→50 : 50→30 : 70→20 : high-resolution positive-ion FAB-MS: Calcd for C H O Na (MꢁNa) :
2
41 66 13
ꢀ1
1
8
(
0)→MeOH] to furnish seven fractions [fr. 2-1 (9.4 g), 2-2 (157 mg), 2-3 789.4412. Found: 789.4407; IR (KBr): nmax 3507, 1653, 1072 cm
; H-
1.2 g), 2-4 (9.0 g), 2-5 (1.7 g), 2-6 (1.5 g), 2-7 (238 mg)]. Fraction 2-2 (157
NMR (500 MHz, pyridine-d ) d 0.89. 0.93 (both 3H, both d, Jꢂ6.2 Hz, H -
5
3
mg) was purified by HPLC [H O : MeOH (40 : 60)] to give (R)-linalyl 6-O- 30, 29), 0.94, 0.99, 1.14, 1.19 (3H each, all s, H -23, 25, 27, 26), 3.73, 4.30
2
3
arabinopyranosyl-b-D-glucopyranoside (18, 74.8 mg). Fraction 2-4 (500 mg)
(both 1H, both d, Jꢂ10.7 Hz, H -24), 4.26 (1H, m, H-3), 4.99 (1H, d, Jꢂ
2
1
3
was purified by HPLC [H O : MeOH (30 : 70)] to furnish matesaponin 1 (5,
7.2 Hz, H-1ꢃ), 6.29 (1H, d, Jꢂ8.1 Hz, H-1ꢄ); C-NMR: given in Table 2;
ꢁ
2
3
1.4 mg). Fraction 2-7 (238 mg) was separated by HPLC [1% aqueous acetic positive-ion FAB-MS: m/z 789 (MꢁNa) .
acid : MeOH : MeCN (49 : 16 : 35)] to provide matesaponins J3 (10, 17.4 mg)
and mateglycoside D (13, 30.3 mg), and 3-O-b-D-glucopyranosyl(1—3)-a-
L-2-O-acetyl-arabinopyranosyl ursolic acid 28-O-b-D-glucopyranoside (11,
Acid Hydrolysis of Mateglycosides A (1), B (2), and C (3) A solution
of 1 (33.1 mg), 2 (17.0 mg), and 3 (3.5 mg) in 5% aqueous H SO -1,4-diox-
ane (1 : 1, 1.0 ml) was heated under reflux for 2 h, respectively. After cool-
2
4
1
3.6 mg). Fraction 3 (30.0 g) was separated by reversed-phase silica gel col- ing, the reaction mixture was poured into ice-water and neutralized with
ꢀ
umn chromatography [900 g, H O : MeOH (90 : 10→70 : 30→50 : 50→30 : Amberlite IRA-400 (OH form), and the resin was removed by filtration.
2
7
0→20 : 80)→MeOH] to give rutin (19, 2.2 g), kaempferol 3-O-rutinoside
Then, the filtrate was extracted with EtOAc to give an EtOAc-soluble frac-
(
(
(
20, 1.5 g), and five fractions [fr. 3-1 (4.3 g), 3-2 (2.4 g), 3-3 (6.3 g), 3-4 tion and aqueous phase. The EtOAc-soluble fraction was purified by normal-
11.0 g), 3-5 (2.2 g)]. Fraction 3-3 (3.0 g) was subjected HPLC [H O : MeOH phase silica gel column chromatography [1.0 g, CHCl →CHCl : MeOH
2
3
3
30 : 70)] to give matesaponin 2 (6, 6.3 mg), nudicaucin C (14, 252 mg), and
(20 : 1)] to give oleanolic acid (12, 13.0 mg) from 1 and 23-hydroxyursolic
acid (17, 4.9 mg and 1 mg) from 2 and 3. Those triterpenes (12, 17) were
3
-O-a-L-rhamnopyranosyl(1—2)-a-L-arabinopyranosyl oleanolic acid 28-
1
13
O-b-D-glucopyranosyl(1—6)-b-D-glucopyranoside (15, 430 mg). Fraction 4
identified by comparison of their H- and C-NMR and MS data with those
40.0 g) was separated by reversed-phase silica gel column chromatography of authentic samples.
(
[
1.2 kg, H O : MeOH (90 : 10→70 : 30→50 : 50→30 : 70→20 : 80)→MeOH]
The aqueous layer was subjected to HPLC analysis under the following
2
to give 19 (2.2 g) and six fractions [fr. 4-1 (26.4 g), 4-2 (1.0 g), 4-3 (2.7 g), 4-
conditions: HPLC column, Kaseisorb LC NH –60–5, 4.6 mm i.d.ꢆ250 mm
2
4
(2.9 g), 4-5 (0.79 g), 4-6 (3.9 g)]. Fraction 4-2 (500 mg) was separated by (Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Shodex
HPLC [H O : MeOH (40 : 60)] to give matesaponin 3 (7, 44.8 mg). Fraction OR-2 (Showa Denko Co., Ltd., Tokyo, Japan)]; mobile phase, MeCN–H O
2
2
4
-3 (2.4 g) was purified by HPLC [H O : MeOH (30 : 70)] followed by HPLC (80 : 20, v/v); flow rate 0.80 ml/min; column temperature, room temperature.
2
[
H O : MeCN (70 : 30)] to give mateglycoside A (1, 513 mg) and mate-
Identification of L-arabinose (from 1, 2, and 3) D-glucose (from 1, 2, and 3),
and L-rhamnose (from 1), which were confirmed by comparison of its reten-
2
saponin 5 (9, 72.4 mg). Fraction 4-4 (1.5 g) was purified by HPLC
[
(
[
H O : MeCN (70 : 30)] to furnish 1 (457 mg) and 9 (97.0 mg). Fraction 4-5 tion time and optical rotation with that of an authentic sample; t : 8.3 min
2
R
0.79 g) was purified by HPLC [H O : MeOH (30 : 70)] followed by HPLC
(L-arabinose, positive optical rotation), 9.5 min (D-glucose, positive optical
2
H O : MeCN (70 : 30)] to give 1 (20.8 mg), matesaponin 4 (8, 29.7 mg), and
rotation), 6.5 min (L-rhamnose, negative optical rotation).
2
1
1 (65.7 mg). Fraction 5 (33.0 g) was subjected by reversed-phase silica gel
Effect on Pancreatic Lipase Activity A suspension of triolein (80 mg),
column chromatography [1.2 kg, H O : MeOH (90 : 10→70 : 30→50 : 50→ phosphatidylcholine (10 mg), and sodium taurocholate (5 mg) in 9 ml of 0.1
2
3
0 : 70)→MeOH] followed by HPLC [H O : MeOH (90 : 10)] to give 5-O-
M Tris–HCl buffer (pHꢂ7.0) containing 0.1 M NaCl was sonicated for 10
min. This sonicated substrate suspension (0.1 ml) in a test tube was pre-incu-
2
caffeoyl-D-quinic acid (21, 20.8 mg).
The EtOAc-soluble fraction (100 g) was subjected to normal-phase silica bated with 5 ml of test sample in DMSO and 95 ml of the tris–HCl buffer for
gel column chromatography [3.0 kg, hexane : EtOAc (5 : 1→1 : 1)→EtOAc→ 3 min at 37 °C. An aliquot of porcine pancreatic lipase (250 mg/ml, type II,
CHCl →CHCl : MeOH (5 : 1→1 : 1)→MeOH] to provide four fractions [fr. Sigma Chemical Co.) (50 ml) or the tris–HCl buffer (50 ml) as a blank test
3
3
1
(21.1 g), 2 (13.3 g), 3 (29.9 g), 4 (20.4 g)]. Fraction 3 (13.3 g) was sepa- was then added to start the reaction. After 30 min of incubation, the test tube
rated by reversed-phase silica gel column chromatography [390 g, H O : was immediately immersed in boiling water for 2 min to stop the reaction,
2
MeOH (10 : 90)→MeOH] to give ursolic acid (4, 900 mg) and five fractions then cooled with water. Free fatty acid concentration was determined by a
[fr. 3-1 (599 mg), 3-2 (10.2 g), 3-3 (312 mg), 3-4 (151 mg), 3-5 (993 mg)].
commercial kit (NEFA C-test Wako, Wako Pure Chemical Industries, Ltd.).
Fraction 3-2 (400 mg) was purified by HPLC [H O : MeOH (10 : 90)] to give
2
3
4
-b-hydroxyurs-11-en-13b(28)-olide (17, 13.1 mg) and 4 (308 mg). Fraction
Acknowledgments This research was supported by the 21st COE Pro-
(20.3 g) was subjected to reversed-phase silica gel column chromatography gram, Academic Frontier Project, and a Grant-in Aid for Scientific Research

March 2009
261
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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