Bioactive saponins and glycosides part 29. Acylated oleanane-type triterpene saponins: Theasaponins A6, A7, and B 5 from the seeds of Camellia sinensis

Article abstract of DOI:10.1002/hlca.200790240

Three new acylated oleanane-type triterpene saponins, theasaponins A ₆ (1), A₇ (2), and B₅ (3), were isolated from the saponin fraction of the seeds of the Japanese tea plant Camellia sinensis together with the known constituent foliatheasaponin III (4). The structures of the glycosides 1-3 were elucidated on the basis of spectroscopic, chemical, and physico-chemical evidence.

Full text of DOI:10.1002/hlca.200790240

2
342
Helvetica Chimica Acta – Vol. 90 (2007)
Bioactive Saponins and Glycosides
Part 291)
Acylated Oleanane-Type Triterpene Saponins: Theasaponins A , A , and B from the
6
7
5
Seeds of Camellia sinensis
a
b
a
a
c
c
a
by Toshio Morikawa ) ), Hisashi Matsuda ), Ning Li ) ), Xian Li ), and Masayuki Yoshikawa* )
a) Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
(
phone: þ 81-75-595-4633; fax: þ 81-75-595-4768; e-mail: myoshika@mb.kyoto-phu.ac.jp)
b) Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-osaka,
Osaka 577-8502, Japan
c) Research Department of Natural Medicine, Shenyang Pharmaceutical University, Shenyang 110016,
P. R. China
Three new acylated oleanane-type triterpene saponins, theasaponins A (1), A (2), and B (3), were
6
7
5
isolated from the saponin fraction of the seeds of the Japanese tea plant Camellia sinensis together with
the known constituent foliatheasaponin III (4). The structures of the glycosides 1 – 3 were elucidated on
the basis of spectroscopic, chemical, and physico-chemical evidence.
Introduction. – As part of our research on saponin constituents from the seeds of
the Japanese tea plant, Camellia sinensis (L.) O. Kuntze (C. sinensis L. var. sinensis),
Theaceae, we have reported over the years the isolation and structure elucidation of
theasaponins A – A , B , C , E – E , F – F , H , G , and G [2 – 6], as well as those of
1
5
1
1
1
13
1
3
1
1
2
four flavanol oligoglycosides, theaflavanosides I – IV [7]. Among the isolates,
theasaponins A , E , E , and E , as well as assamsaponins A, C, and D, were found to
2
1
2
5
show protective effects on EtOH-induced gastric lesions in rats [3][4], hepatopro-
tective activity [7], and anti-hyperlipidemic activity [8].
Our continuing search now led to the isolation of three new acylated oleanane-type
triterpene saponins, theasaponins A (1), A (2), and B (3), which were obtained from
6
7
5
the seeds of C. sinensis, together with a known saponin, foliatheasaponin III (4) [1],
which was isolated for the first time from this plant. Herein, we report the structure
elucidation of the new compounds.
Results and Discussion. – The saponin fraction of the MeOH extract of tea seeds,
cultivated in Shizuoka Prefecture, Japan, as described previously [3], was purified by
HPLC to afford compounds 1, 2, 3, and 4 in yields of 60, 100, 60, and 330 ppm,
respectively.
Compound 1, obtained as colorless fine crystals from CHCl /MeOH (m.p. 226.1 –
3
2
3
2
27.48) exhibited a positive optical rotation ([a] ¼ þ8.4 (MeOH)). The IR spectrum
D
1)
For Part 28, see [1].
ꢀ
2007 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta – Vol. 90 (2007)
2343
À1
of 1 showed absorption bands at 1716 and 1651 cm ascribable to COOH and olefin
À1
moieties, and broad bands at 3431 and 1084 cm , suggestive of an oligoglycoside
structure. Positive- and negative-ion FAB-MS analysis of 1 showed the quasimolecular
þ
À
ions at m/z 1285 ([M þ Na] ) and 1261 ([M À H] ), respectively, and positive HR-
FAB-MS analysis revealed the molecular formula of 1 as C H O .
60
94 28
Alkaline hydrolysis of 1 with 10% aqueous KOH in H O/1,4-dioxane 1:1 gave
2
desacyl-theasaponin A (1a) [5], together with two organic acids, acetic acid (AcOH)
and angelic acid (¼(Z)-2-methylbut-2-enoic acid; AngOH), which were identified by
HPLC analysis of the corresponding 4-nitrobenzyl derivatives [3 – 5]. Previously, we
reported that hydrolysis of 1a with 5% aqueous H SO /1,4-dioxane 1:1 gave d-
2
4
glucuronic acid (d-GlcA), d-galactose (d-Gal), l-arabinose (l-Ara), and d-glucose (d-
Glc), as identified by GLC analysis of their trimethylsilyl thiazolidine derivatives [5].
1
13
The H- and C-NMR spectra of 1 (Tables 1 and 2, resp.), which were assigned by
various NMR experiments (including DEPT, DQF, HMQC, and HMBC) showed
signals assignable to six Me singlets at d(H) 0.78, 0.85, 1.04, 1.12, 1.29, and 1.42 (Me(26),
Me(25), Me(24), Me(29), Me(30), and Me(27)); two oxygenated CH groups at d(H)
2
3
.65, 3.92 (2d, J ¼ 10.7 Hz each, CH (28)), and at 3.74 – 3.80and 4.35 – 4.42 (2 m,
2
CH (23)); four oxygenated CH groups at d(H) 4.11 – 4.17 (m, HÀC(3)), 4.78 and 5.97
2
(
2d, J ¼ 9.8 Hz each, HÀC(22), HÀC(21)), and 5.92 (br. s, HÀC(16)); an olefinic H-
atom at d(H) 5.34 (br. s, HÀC(12)); and four glycosyl moieties at d(H) 5.08 (d, J ¼
.9 Hz, HÀC(1’) of GlcA), 5.14 (d, J ¼ 7.4 Hz, HÀC(1’’’’) of Glc), 5.79 (d, J ¼
.6 Hz, HÀC(1’’) of Gal), and 5.84 (d, J ¼ 5.8 Hz, HÀC(1’’’) of Ara), together with
an acetyl (Ac) group at d(H) 2.51 (s) and an angeloyl (Ang) group at d(H) 1.94 (s, Me),
6
7
2
.02 (d, J ¼ 7.3 Hz, Me), and 5.91 (qd-like, ¼CH).
The positions of the AcO and AngO groups in 1 were clarified on the basis of an
1
13
HMBC experiment, which showed H ! C long-range correlations between the

2
344
Helvetica Chimica Acta – Vol. 90 (2007)
Table 1. 1H-NMR Data of 1 – 3. Recorded at 500 MHz and 408C in (D )pyridine; d in ppm, J in Hz.
5
Arbitrary atom numbering; for abbreviations, see text.
Atom
1
2
3
HÀC(3)
4.11 – 4.17 (m)
5.34 (br. s)
5.92 (br. s)
2.95 (dd-like)
5.97 (d, J ¼ 9.8)
4.78 (d, J ¼ 9.8)
3.74 – 3.80( m),
4.12 – 4.16 (m)
5.38 (br. s)
5.60(br. s)
3.01 (m)
5.87 (d, J ¼ 10.4)
6.15 (d, J ¼ 10.4)
3.75 – 3.79 (m),
4.36 – 4.41 (m)
–
1.0 s)
0.86 (s)
0.77 (s)
1.41 (s)
3.28 (dd, J ¼ 7.4, 12.5)
5.37 (br. s)
4.87 (br. s)
2.93 (dd-like)
6.49 (d, J ¼ 9.8)
4.81 (d, J ¼ 9.8)
–
HÀC(12)
HÀC(16)
HÀC(18)
HÀC(21)
HÀC(22)
CH (23)
2
4
–
.35 – 4.42 (m)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
1.29 (s)
4 ( 1.13 (s)
1.04 (s)
0.85 (s)
0.78 (s)
1.42 (s)
0.81 (s)
0.86 (s)
1.85 (s)
CH (28)
3.65 (d, J ¼ 10.7),
3.46 (d, J ¼ 10.4),
3.59 (d, J ¼ 10.4)
1.0 s)
3.69 (d, J ¼ 10.4),
2
3
.92 (d, J ¼ 10.7)
3.96 (d, J ¼ 10.4)
Me(29)
1.12 (s)
6 ( 1.14 (s)
Me(30)
1.29 (s)
1.27 (s)
1.33 (s)
HÀC(1’) of GlcA
HÀC(1’’) of Gal
HÀC(1’’’) of Ara
HÀC(1’’’’) of Glc
HÀC(1’’’’) of Xyl
5.08 (d, J ¼ 6.9)
5.79 (d, J ¼ 7.6)
5.84 (d, J ¼ 5.8)
5.14 (d, J ¼ 7.4)
–
5.08 (d, J ¼ 6.9)
5.79 (d, J ¼ 7.9)
5.84 (d, J ¼ 5.8)
5.14 (d, J ¼ 6.4)
–
4.96 (d, J ¼ 7.4)
5.78 (d, J ¼ 7.3)
5.81 (d, J ¼ 5.8)
–
5.04 (d, J ¼ 7.6)
1
6-AcO
2.51 (s)
2.51 (s)
–
HÀC(3) of Ang
Me(4) of Ang
Me(5) of Ang
5.91 (qd-like)
2.02 (d, J ¼ 7.3)
1.94 (s)
5.99 (qd-like)
2.06 (d, J ¼ 7.3)
1.98 (s)
5.90( qd-like)
2.06 (d, J ¼ 7.0)
1.99 (s)
2
2-AcO
–
2.04 (s)
–
following pairs: HÀC(16) and d(C) 170.0 (C¼O of Ac), HÀC(21) and d(C) 168.4
(
(
C(1) of Ang), HÀC(1’) of GlcA and d(C) 82.7 (C(3)), HÀC(1’’) of Gal and d(C) 78.8
C(2’) of GlcA), HÀC(1’’’) of Ara and d(C) 84.7 (C(3’) of GlcA), and HÀC(1’’’’) of
13
Glc and d(C) 81.2 (C(2’’’) of Ara. Furthermore, comparison of the C-NMR data of 1
with those of 1a revealed acylation shifts at the 16- and 21-positions of the desacyl-
theasaponin A moiety [1a: d(H) 5.00 (br. s, HÀC(16)), 4.77 (d, J ¼ 9.8 Hz, HÀC(21));
d(C) 67.8 (C(16)), 78.7 (C(21))] [5].
On the basis of the above evidence, the structure of theasaponin A (1) was
6
determined as 16-O-acetyl-21-O-angeloyltheasapogenol A 3-O-b-d-galactopyranosyl-
(
1 ! 2)-[b-d-glucopyranosyl-(1 ! 2)-a-l-arabinopyranosyl-(1 ! 3)]-b-d-glucurono-
2
pyranoside ).
Compound 2, obtained as colorless fine crystals from CHCl /MeOH (m.p. 224.8 –
2
3
2
4
25.78), exhibited a positive optical rotation ([a] ¼ þ7.4 (MeOH)). The IR spectrum
D
À1
of 2 showed absorption bands at 3453, 1725, 1645, and 1076 cm , ascribable to OH,
2)
For systematic names, see Exper. Part.

Helvetica Chimica Acta – Vol. 90 (2007)
2345
Table 2. 13C-NMR Data of 1 – 3. Recorded at 125 MHz and 408C in (D )pyridine; d in ppm. Arbitrary
5
atom numbering; for abbreviations, see text.
Position
1
2
3
Position
1
2
3
1
2
3
4
5
6
7
8
9
38.7
25.6
82.7
43.5
48.048.055.8
18.018.018.4
32.7
40.0
47.046.9
36.6
23.8
124.5
141.9
41.4
30.9
71.6
47.6
39.8
47.3
38.7
25.6
82.7
43.5
38.8
26.6
89.5
39.6
1
2
33.1
40.0
22-AcO:
1
2
b-d-GlcA:
’
’
3’
4’
’
’
b-d-Gal:
1’’
’’
3’’
4’’
5’’
6’’
a-l-Ara:
1’’’
’’’
3’’’
4’’’
170.4
21.0
104.3
78.8
84.7
71.5
77.3
171.9
104.3
78.7
84.8
71.4
77.3
171.9
105.7
79.0
83.9
71.1
77.3
172.2
32.7
40.0
46.9
36.7
23.8
5
1
1
1
1
1
1
1
1
1
1
036.6
6
23.8
122.7
1
2
3
4
5
6
7
8
9
125.1
141.0143.5
41.1
30.9
71.4
46.9
39.5
47.1
2
78.3
73.3
64.6
13.6
16.1
16.8
27.027.3
63.7
29.4
19.7
103.2
73.8
75.1
69.9
76.6
61.9
103.5
73.8
75.1
69.9
76.6
61.9
103.5
73.3
75.1
70.1
76.4
61.9
2
41.8
34.4
67.8
47.8
40.4
46.9
101.7
81.2
72.3
67.5
64.7
101.8
81.1
72.3
67.5
64.7
101.7
82.1
73.8
68.3
65.9
2
3 05 3. 96 .0
36.1
2
2
2
2
2
2
2
2
2
3
1
1
2
2
1
2
3
4
5
1
2
3
4
5
6
7
8
80.5
70.8
64.7
13.6
16.2
16.8
27.1
64.7
30.0
20.2
81.6
73.0
28.05
16.8
15.8
16.8
’’’
b-d-Glc:
1’’’’
2’’’’
’’’’
4’’’’
5’’’’
6’’’’
b-d-Xyl:
1’’’’
’’’’
105.9
75.8
78.4
71.6
78.4
62.6
105.9
75.9
78.5
71.5
78.5
62.6
3
65.9
29.9
20.4
9
0
6-AcO:
170.0
22.3
169.8
22.02
106.8
75.7
78.3
70.8
67.5
1-AngO:
3’’’’
4’’’’
5’’’’
168.4
129.2
136.8
16.016.015.9
21.1
167.8
128.4
138.1
168.6
129.5
136.0
20.9
21.0
COOH, olefin, and ether functions. The molecular formula C H O was determined
6
þ
2
96 29
À
by positive- and negative-ion FAB-MS (m/z 1327 ([M þ Na] ) and 1303 ([M À H] ),
resp.), and by HR-FAB-MS (positive-ion mode). The fragmentation patterns in the
negative-ion FAB mass spectrum of 2 indicated the loss of a terminal mono-hexose
À
(
1
m/z 1141 ([M À C H O ] ) and a disaccharide part (pentose and hexose units; m/z
6
11
À
5
009 ([M À C H O ] ).
11
19
9

2
346
Helvetica Chimica Acta – Vol. 90 (2007)
Treatment of 2 with 10% aqueous KOH in H O/1,4-dioxane 1:1 liberated 1a and
2
both AcOH and AngOH, as identified by HPLC analysis of their 4-nitrobenzyl
derivatives.
1
13
The H- and C-NMR spectra of 2 (Tables 1 and 2, resp.) revealed two AcO groups
at d(H) 2.04, 2.51 (2s) and an AngO group at d(H) 1.98 (s, Me), 2.0 6 (d , J ¼ 7.3 Hz,
Me), and 5.99 (qd-like, ¼CH). Their positions were clarified by an HMBC experiment.
Thus, long-range correlations were observed between HÀC(16) and d(C) 169.8 (C¼O
of Ac), between HÀC(21) and d(C) 167.8 (C(1) of Ang), and between HÀC(22) and
13
d(C) 170.4 (C¼O of Ac). Furthermore, comparison of the C-NMR data of 2 with
those of 1 revealed an acetylation shift near the 22-position of the aglycone.
Consequently, the structure of theasaponin A (2) was determined as 16,22-di-O-
7
acetyl-21-O-angeloyltheasapogenol A 3-O-b-d-galactopyranosyl-(1 ! 2)-[b-d-gluco-
pyranosyl-(1 ! 2)-a-l-arabinopyranosyl-(1 ! 3)]-b-d-glucuronopyranoside.
Compound 3, obtained as colorless fine crystals from CHCl /MeOH (m.p. 233.1 –
3
2
3
2
33.98), exhibited a positive optical rotation ([a] ¼ þ4.0(MeOH)). Analysis of the
D
positive- and negative-ion FAB mass spectra of 3 showed quasimolecular ion peaks at
þ
À
m/z 1197 ([M þ Na] ) and 1173 ([M À H] ), respectively. HR-FAB-MS Analysis
(
positive-ion mode) revealed the molecular formula to be C H O .
57
90 25
Alkaline hydrolysis of 3 with 10% aqueous KOH in H O/1,4-dioxane 1:1 liberated
2
desacyl-assamsaponin E (3a) [9] and AngOH, which was identified by HPLC analysis
of its 4-nitrobenzyl derivative. Previously, we reported that hydrolysis of 3a with 5%
aqueous H SO /1,4-dioxane 1:1 gave d-GlcA, d-Gal, l-Ara, and d-xylose (d-Xyl),
2
4
which were identified by GLC analysis of their trimethylsilyl thiazolidine derivatives
9].
The H- and C-NMR Spectra of 3 (Tables 1 and 2, resp.) indicated the presence of
a theasapogenol-B (barringtogenol-C) moiety, with seven Me singlets at d(H) 0.81,
.86, 1.13, 1.14, 1.29, 1.33, and 1.85 (Me(25), Me(26), Me(24), Me(29), Me(23),
[
1
13
0
Me(30), and Me(27)), a CH moiety and four oxygenated CH groups [d(H) 3.28 (dd,
2
J ¼ 7.4, 12.5 Hz, HÀC(3)); 3.69, 3.96 (2d, J ¼ 10.4 Hz each, CH (28)); 4.81, 6.49 (2d,
2
J ¼ 9.8 Hz, HÀC(22), HÀC(21)); 4.87 (br. s, HÀC(16))], an olefinic H-atom at d(H)
5
.37 (br. s, HÀC(12)), and four glycosyl moieties at d(H) 4.96 (d, J ¼ 7.4 Hz, HÀC(1’)
of GlcA), 5.04 (d, J ¼ 7.6 Hz, HÀC(1’’’’) of Xyl), 5.78 (d, J ¼ 7.3 Hz, HÀC(1’’) of Gal),
and 5.81 (d, J ¼ 5.8 Hz, HÀC(1’’’) of Ara), together with an Ang group at d(H) 1.99 (s,
Me), 2.06 (d, J ¼ 7.0Hz, Me), and 5.90( qd-like, ¼CH). In the HMBC spectrum of 3, a
1
13
long-range H ! C correlation was observed between HÀC(21) and d(C) 168.6 (C(1)
of Ang).
From the above data, the structure of theasaponin B (3) was determined as 21-O-
5
angeloyltheasapogenol B 3-O-b-d-galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 !
2
)-a-l-arabinopyranosyl-(1 ! 3)]-b-d-glucuronopyranoside.
Experimental Part
General. Column chromatography (CC): silica gel BW-200 (150– 300 mesh; Fuji Silysia Chemical,
Ltd., Japan). HPLC: Shimadzu RID-6A refractive-index and SPD-10A UV/VIS detectors, LC-6AD
pump, CTO-10A oven, and Chromatopac C-R6A column. M.p: Yanagimoto MP-500D micro-melting-
point apparatus; uncorrected. Optical rotations: Horiba SEPA-300 digital polarimeter (l ¼ 5 cm). IR

Helvetica Chimica Acta – Vol. 90 (2007)
2347
Spectra: Shimadzu FT-IR-8100 spectrometer; in cm . 1H- and 13C-NMR Spectra: Jeol JNM-LA500
À1
spectrometer; at 500/125 MHz, resp., d in ppm rel. to Me Si, J in Hz. FAB- and HR-FAB-MS: Jeol JMS-
4
SX 102A mass spectrometer; in m/z.
Plant material. The seeds of Camellia sinensis were cultivated in 2004 at Shizuoka Prefecture, Japan,
as described previously [2].
Extraction and Isolation. Compounds 1 – 4 were isolated as described below from three previously
reported [2] fractions, Fr. 5 (2.20g), Fr. 6 (0.96 g), and Fr. 8 (0.97 g), originally obtained from the saponin
fraction (eluted with MeOH) of the seeds of C. sinensis (1.0kg).
Fr. 5 (2.20g) was separated by HPLC [ YMC-Pack ODS-A, 250 ꢀ 20mm i.d.; MeCN/1% aq. AcOH
4
0:60; 9.0ml/min] to afford the following twelve subfractions: Fr. 5.1 [13 mg (50ppm), theasaponin A ;
4
tR 15.5 min], Fr. 5.2 [53 mg (210ppm), theasaponin A ; t 21.9 min], Fr. 5.3 [23 mg (90ppm),
1
R
theasaponin F
37 mg), Fr. 5.6 (164 mg), Fr. 5.7 (100 mg), Fr. 5.8 (328 mg), Fr. 5.9 (148 mg (59 ppm), theasaponin
A ; t 42.2 min]; Fr. 5.10 (200 mg), Fr. 5.11 (645 mg), and Fr. 5.12 (85 mg). Then, Fr. 5.8 (328 mg) was
1 R 6 R
; t 25.3 min], Fr. 5.4 [14 mg (60ppm), theasaponin A (1); t 27.0min], Fr. 5.5
(
3
R
subjected to HPLC [Develosil C30-UG-5, 250 ꢀ 20mm i.d.; MeCN/MeOH/1% aq. AcOH 35 :16 :49;
.0ml/min] to afford theasaponin A [2; 26 mg (100 ppm); t 48.2 min], together with camelliasaponin C
1
9
7
R
[
[
10mg (40ppm); tR 41.9 min], theasaponin C1 [77 mg (310ppm); tR 44.7 min], and theosaponin F2
54 mg (210ppm); tR 46.5 min].
Fr. 6 (960mg) was subjected to HPLC [ YMC-pack ODS-A, 250 ꢀ 20mm i.d.; MeCN/1% aq. AcOH
4
2
3 :57; 9.0ml/min] to give the following nine fractions: Fr. 6.1 [16 mg (60ppm), theasaponin B 5 (3); t
R
0.7 min], Fr. 6.2 [56 mg (220ppm), assamsaponin I; tR 25.1 min], Fr. 6.3 [323 mg (1300 ppm),
assamsaponin C; tR 27.3 min], Fr. 6.4 (65 mg), Fr. 6.5 [39 mg (160ppm), floratheasaponin A; tR
9.3 min], Fr. 6.6 (15 mg), Fr. 6.7 (75 mg), Fr. 6.8 (20mg), and Fr. 6.9 [126 mg (500 ppm), theasaponin
2
E ; t 37.1 min].
5
R
Fr. 8 (0.97 g) was subjected to HPLC [YMC-Pack ODS-A, 250 ꢀ 20mm i.d., MeCN/1% aq. AcOH
4
3 :57; 9.0ml/min] to afford five fractions: Fr. 8.1 [323 mg (1300 ppm), theasaponin A ; t 28.0min],
2 R
Fr. 8.2 [136 mg (540ppm), theasaponin F 3; tR 31.8 min], Fr. 8.3 (46 mg), Fr. 8.4 (84 mg), and Fr. 8.5
[
82 mg (330ppm), foliatheasaponin III ( 4); tR 39.3 min].
Theasaponin A6 (¼(3b,16a,21b,22a)-16-Acetoxy-22,23,28-trihydroxy-21-{[(2Z)-2-methylbut-2-
enoyl]oxy}olean-12-en-3-yl b-d-Galactopyranosyl-(1! 2)-[b-d-glucopyranosyl-(1! 2)-a-l-arabinopyr-
anosyl-(1 ! 3)]-b-d-glucopyranosiduronic Acid; 1). Colorless, fine crystals. M.p. 226.1 – 227.48 (CHCl /
3
2
3
1
13
MeOH). [a] ¼ þ8.4 (c ¼ 0.55, MeOH). IR (KBr): 3431, 1716, 1651, 1084. H- and C-NMR: see
D
þ
À
Tables 1 and 2, resp. FAB-MS (pos.): 1285 ([M þ Na] ). FAB-MS (neg.): 1261 ([M À H] ), 1099 ([M À
À
À
þ
þ
C H O ] ), 967 ([M À C H O ] ). HR-FAB-MS (pos.): 1285.5839 ([M þ Na] , C H NaO ; calc.
6
11
5
11 19
9
60 94
28
1
285.5829).
Theasaponin A7 (¼(3b,16a,21b,22a)-16,22-Diacetoxy-23,28-dihydroxy-21-{[(2Z)-2-methylbut-2-
enoyl]oxy}olean-12-en-3-yl b-d-Galactopyranosyl-(1! 2)-[b-d-glucopyranosyl-(1! 2)-a-l-arabinopyr-
anosyl-(1 ! 3)]-b-d-glucopyranosiduronic Acid; 2). Colorless, fine crystals. M.p. 224.8 – 225.78 (CHCl /
3
2
4
1
13
MeOH). [a] ¼ þ7.4 (c ¼ 1.01, MeOH). IR (KBr): 3453, 1725, 1645, 1076. H- and C-NMR: see
D
þ
À
Tables 1 and 2, resp. FAB-MS (pos.): 1327 ([M þ Na] ). FAB-MS (neg.): 1303 ([M À H] ), 1141 ([M À
À
À
þ
þ
29
C H O ] ), 1009 ([M À C H O ] ). HR-FAB-MS (pos.): 1327.5929 ([M þ Na] , C H NaO ; calc.
6
11
5
11 19
9
62 96
1
327.5935).
Theasaponin B
olean-12-en-3-yl b-d-Galactopyranosyl-(1 ! 2)-[b-d-xylopyranosyl-(1 ! 2)-a-l-arabinopyranosyl-(1 !
)]-b-d-glucopyranosiduronic Acid; 3). Colorless, fine crystals. M.p. 233.1 – 233.98 (CHCl /MeOH).
5
(¼(3b,16a,21b,22a)-16,22,28-Trihydroxy-21-{[(2Z)-2-methylbut-2-enoyl]oxy}-
3
[
3
2
3
1
13
a] ¼ þ4.0( c ¼ 0.88, MeOH). IR (KBr): 3453, 1716, 1645, 1084. H- and C-NMR: see Tables 1 and 2,
D
þ
À
À
5
resp. FAB-MS (pos.): 1197 ([M þ Na] ). FAB-MS (neg.): 1173 ([M À H] ), 1011 ([M À C H O ] ).
6
11
þ
þ
25
HR-FAB-MS (pos.): 1197.5657 ([M þ Na] , C H NaO ; calc. 1197.5669).
57
90
Alkaline Hydrolysis of 1 – 3. A soln. of the appropriate theasaponin (10mg) in 50% aq. 1,4-dioxane
1 ml) was treated with 10% aq. KOH (1 ml), and the mixture was stirred at 378 for 1 h. An aliquot
0.1 ml) of the reaction mixture was concentrated under reduced pressure, and the resulting residue was
(
(
dissolved in 1,2-dichloroethane (2 ml). This soln. was treated with ꢁpara-nitrobenzyl-N,N’-diisopropyl-
isoureaꢂ (10mg) and stirred at 80 8 for 1 h. The reaction mixture was then subjected to HPLC analysis

2
348
Helvetica Chimica Acta – Vol. 90 (2007)
[
YMC-Pack ODS-A, 250 ꢀ 4.6 mm i.d.; MeOH/H O 70:30; 0.9 ml/min, UV detection at 254 nm], and
2
the para-nitrobenzyl esters of AcOH (t 6.3 min) from 1 and 2, as well as angelic acid (t 16.0min) from
1
R
R
þ
– 3 were detected. The rest of each reaction mixture was neutralized over Dowex HCR W2 resin (H
form), which was then removed by filtration. The filtrate was concentrated under reduced pressure, and
the resulting product was subjected to CC (2 g SiO ; CHCl /MeOH/H O 6 :4 :1) to give desacyl-
2
3
2
theasaponin A [5] (1a; 6 mg each from 1 and 2) or desacyl-assamsaponin E [9] (3a; 6 mg, from 3).
M. Y. and H. M. were supported by the 21st COE Program, Academic Frontier Project, and a Grant-
in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of
Japan (MEXT). T. M. was supported by the High-tech Research Center Project (2007) and a Grant-in Aid
for Scientific Research from MEXT.
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2
[
[
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[
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Received July 26, 2007