Three new E-secoursane triterpenoid saponins from the leaves of Ilex dunniana

Article abstract of DOI:10.1002/hlca.201000385

Three new triterpenoid saponins with an 18,19-secours-13(18)-ene skeleton, dunnianaolactones A-C (1-3, resp.), together with nine known compounds i.e., the ursane-type triterpene saponin 4, the two benzofuran lignans 5 and 6, five flavonoid glycosides, and 4-hydroxybenzoic acid, were isolated from the leaves of Ilex dunniana Levl. (Aquifoliaceae). Their structures were elucidated by means of spectroscopic and chemical methods. The configuration of dunnianaolactone A (1) was further confirmed by X-ray crystal-structure analysis. Copyright

Full text of DOI:10.1002/hlca.201000385

Helvetica Chimica Acta – Vol. 94 (2011)
2207
Three New E-Secoursane Triterpenoid Saponins from the Leaves of Ilex
dunniana
by Yue Zhang, Lu-Jun Li, Peng Zhang*, Hui-Fang Pi, Han-Li Ruan, and Ji-Zhou Wu
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of
Pharmacy, Huazhong University of Science and Technology, No. 13 Hangkong Rd., Wuhan 430030,
P. R. China (e-mail: zpeng023@gmail.com)
Three new triterpenoid saponins with an 18,19-secours-13(18)-ene skeleton, dunnianaolactones A –
C (1 – 3, resp.), together with nine known compounds i.e., the ursane-type triterpene saponin 4, the two
benzofuran lignans 5 and 6, five flavonoid glycosides, and 4-hydroxybenzoic acid, were isolated from the
leaves of Ilex dunniana Levl. (Aquifoliaceae). Their structures were elucidated by means of
spectroscopic and chemical methods. The configuration of dunnianaolactone A (1) was further
confirmed by X-ray crystal-structure analysis.
Introduction. – Species of the genus Ilex, from the family Aquifoliaceae, have a
wide range of pharmacological activities and are generally used to treat cardiovascular
disease, fever, cough, and inflammation [1]. Ilex dunniana Levl., an evergreen tree, is
distributed in the west of Hubei Province, southwest of Sichuan Province, east of
Yunnan Province, and northeast of Guizhou Province in P. R. China [1]. It is locally
used as a beverage for the treatment of sore throats and arthritis, and the prevention of
wound infections through external application in the northwest regions of Hubei
Province. However, there are no reports on the chemical composition of this plant.
In the present study, three new 18,19-secoursane glycosides, namely dunnianao-
lactones A – C (1 – 3), were isolated from the leaves of I. dunniana, together with nine
known compounds, including one ursane-type triterpenoid saponin (see 4), two
benzofuran lignans (see 5 and 6), five flavonoid glycosides, and 4-hydroxybenzoic acid
(
Fig. 1). The structures of the new compounds were elucidated by means of
spectroscopic and chemical methods. To confirm the configuration of the new 18,19-
secourane glycosides, an X-ray diffraction analysis of 1 was also carried out.
Results and Discussion. – The leaves of I. dunniana were extracted with 90% EtOH.
The EtOH extract was partitioned with petroleum ether, AcOEt, and BuOH. Three
new compounds 1 – 3 and nine known compounds were isolated from the BuOH-
soluble fraction.
Compound 1 was obtained as colorless, acicular crystals. The HR-ESI-MS
(
negative-ion mode) suggested that the molecular formula was C H O . The
C-NMR (including DEPT) spectrum (Table 1) showed 47 C-atom signals, with 30
47 76 18
1
3
attributed to the aglycone part, including one C¼O (d(C) 181.5), one trisubstituted
C¼C (d(C) 150.2 and 117.2), and seven Me groups (d(C) 8.1, 17.2, 17.4, 18.2, 18.9, 22.4,
1
and 28.6). The H-NMR spectrum of 1 (Table 1) displayed signals corresponding to five
ꢀ
2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

2
208
Helvetica Chimica Acta – Vol. 94 (2011)
Table 1. 1H- and 13C-NMR Data (400 and 100 MHz, resp., CD OD) of 1 – 3 ). d in ppm, J in Hz.
a
3
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
CH (1)
1.78 – 1.80 (m),
40.5 1.82 – 1.85 (m),
1.10 – 1.15 (overlap)
29.8 1.79 – 1.82 (m),
1.63 – 1.64 (overlap)
90.1 3.17 – 3.24 (m)
40.5
57.5 0.82 (d, J ¼ 11.4)
19.3 1.47 – 1.64 (m)
35.6 1.53 – 1.57 (overlap)
42.4
40.2 1.74 – 1.78 (m),
1.07 – 1.17 (overlap)
29.8 1.78 – 1.84 (m),
1.58 – 1.63 (overlap)
90.6 3.19 – 3.25 (m)
40.5
57.3 0.83 (d, J ¼ 11.2)
19.3 1.41 – 1.61 (m)
35.8 1.48 – 1.55 (overlap)
42.4
40.5
2
1
.05 – 1.13 (overlap)
1.80 – 1.82 (m),
.63 – 1.64 (overlap)
CH (2)
29.8
2
1
HꢀC(3)
C(4)
3.14 – 3.21 (m)
90.2
40.5
57.6
19.3
35.8
42.4
50.7
38.4
HꢀC(5)
0.84 (d, J ¼ 11.4)
1.41 – 1.54 (m)
1.51 – 1.54 (overlap)
CH (6)
2
CH (7)
2
C(8)
HꢀC(9)
1.52 – 1.58 (overlap)
50.6 1.55 – 1.59 (overlap)
38.4
50.6 1.53 – 1.57 (overlap)
38.4
C(10)
CH (11)
1.90 (dd, J ¼ 12.2, 11.2), 32.9 1.93 (dd, J ¼ 12.2, 11.5), 33.2 1.90 (dd, J ¼ 12.2, 11.5), 33.2
2
1.30 – 1.40 (m)
1.34 – 1.39 (m)
70.3 4.25 – 4.30 (m)
150.2
1.29 – 1.39 (m)
70.4 4.18 – 4.27 (m)
150.2
HꢀC(12)
C(13)
C(14)
4.18 – 4.28 (m)
70.4
150.2
44.2
44.2
44.2
CH (15)
1.87 – 1.92 ,
27.5 1.87 – 1.92 (m),
1.73 – 1.80 (m)
27.4 1.98 – 2.05,
1.68 – 1.79 (2m)
28.6 2.66 (td, J ¼ 13.7, 3.0),
1.16 – 1.22 (overlap)
45.7
27.5
2
1
.73 – 1.80 (2m)
2.66 (td, J ¼ 12.9, 2.5),
.17 – 1.21 (overlap)
CH (16)
28.6 2.69 (td, J ¼ 13.7, 2.9),
28.6
2
1
1.21 – 1.25 (m)
C(17)
45.6
117.2 5.90 (s)
45.6
117.2
79.8
45.3
76.0
43.0
HꢀC(18)
HꢀC(19)
HꢀC(20)
HꢀC(21)
5.86 (s)
4.85 – 4.96 (m)
1.70 – 1.80 (m)
117.2 5.85 (s)
79.8 4.91 – 4.96 (m)
45.3 1.74 – 1.79 (m)
76.0 4.24 (dd, J ¼ 6.5, 2.1)
43.0 2.21 (dd, J ¼ 12.5, 5.1),
1.88 – 1.93 (m)
79.8 4.85 – 4.95 (m)
45.3 1.70 – 1.77 (m)
76.0 4.20 (dd, J ¼ 6.4, 2.2)
43.0 2.17 (dd, J ¼ 12.5, 5.2),
1.83 – 1.92 (m)
4.22 (dd, J ¼ 6.4, 2.2)
CH (22)
2.18 (dd, J ¼ 12.5, 5.2),
2
1.85 – 1.94 (m)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
C(28)
1.06 (s)
0.88 (s)
0.96 (s)
0.99 (s)
1.19 (s)
28.6 1.12 (s)
17.2 0.91 (s)
17.4 1.00 (s)
18.9 1.03 (s)
22.4 1.23 (s)
181.5
28.6 1.08 (s)
17.0 0.88 (s)
17.2 0.96 (s)
18.9 0.99 (s)
22.4 1.18 (s)
181.6
28.6
17.3
17.4
18.9
22.4
181.5
18.2
8.1
Me(29)
Me(30)
1.25 (d, J ¼ 6.4)
0.94 (d, J ¼ 7.0)
18.2 1.29 (d, J ¼ 6.5)
18.2 1.25 (d, J ¼ 5.8)
8.1 0.98 (d, J ¼ 7.0)
8.1 0.94 (d, J ¼ 7.0)
3
-Xyl or 3-Glc
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
4.41 (d, J ¼ 6.6)
106.4 4.34 (d, J ¼ 7.4)
79.0 3.18 – 3.26 (overlap)
78.8 3.37 – 3.44 (m)
107.6 4.44 (d, J ¼ 7.4)
75.6 3.43 – 3.50 (m)
78.2 3.40 – 3.50 (m)
71.4 3.23 – 3.32 (m)
66.8 3.20 – 3.27 (m)
105.8
79.7
79.1
72.2
77.8
3.39 – 3.47 (m)
3.39 – 3.47 (m)
3.37 (dt, J ¼ 6.5, 2.2)
3.88 (dd, J ¼ 11.4, 5.0),
3.22 (dd, J ¼ 12.2, 2.7)
71.7 3.49 – 3.55 (m)
CH
2
(5’)
66.6 3.86 (dd, J ¼ 11.8,2.4),
or HꢀC(5’)
3.17 (dd, J ¼ 11.8,5.3)
CH (6’)
3.87 (dd, J ¼ 11.8, 1.9),
63.0
2
3
.70 (dd, J ¼ 11.9, 5.2)

Helvetica Chimica Acta – Vol. 94 (2011)
2209
Table 1 (cont.)
1
2
3
d(H)
d(C) d(H)
d(C) d(H)
d(C)
2’-Rha
HꢀC(1’’) 5.35 (d, J ¼ 1.5)
HꢀC(2’’) 3.76 (dd, J ¼ 9.6, 3.4)
HꢀC(3’’) 3.97 – 3.99 (m)
HꢀC(4’’) 3.36 – 3.41 (m)
HꢀC(5’’) 3.96 – 3.98 (m)
102.1
72.3
72.2
74.1
70.2
18.3
5.39 (d, J ¼ 1.5)
102.0
72.3
72.2
74.1
70.1
18.3
3.77 (dd, J ¼ 9.6, 3.4)
3.97 (dd, J ¼ 3.3, 1.7)
3.40 (dd, J ¼ 9.6, 3.4)
4.00 (d, J ¼ 6.0)
Me(6’’)
9-Glc
1.24 (d, J ¼ 3.1)
1.25 (d, J ¼ 3.1)
1
HꢀC(1’’’) 4.31 (d, J ¼ 7.8)
HꢀC(2’’’) 3.15 – 3.22 (m)
HꢀC(3’’’) 3.32 – 3.38 (m)
HꢀC(4’’’) 3.31 – 3.39 (m)
HꢀC(5’’’) 3.23 – 3.29 (m)
103.4 4.35 (d, J ¼ 7.8)
75.2 3.18 – 3.22 (overlap)
78.4 3.37 – 3.42 (m)
71.8 3.38 – 3.40 (m)
77.8 3.28 – 3.36 (m)
103.4 4.30 (d, J ¼ 7.8)
103.4
75.2
78.4
71.8
77.8
62.8
75.3 3.14 – 3.19 (m)
78.4 3.33 – 3.38 (m)
71.8 3.31 – 3.37 (m)
77.9 3.23 – 3.27 (m)
62.9 3.83 (dd, J ¼ 11.9, 2.2),
3.70 (dd, J ¼ 11.9, 5.2)
CH (6’’’)
3.83 (dd, J ¼ 11.9, 2.3),
.71 (dd, J ¼ 11.9, 5.3)
62.8 3.82 (dd, J ¼ 11.9, 2.3),
2
3
3.75 (dd, J ¼ 11.9, 5.3)
a) Assignments based on HSQC, HMBC, H, H-COSY, and NOESY experiments.
1
1
Fig. 1. Compounds isolated from I. dunniana
tertiary Me groups (d(H) 0.88, 0.96, 0.99, 1.06, and 1.19), two secondary Me groups
d(H) 0.94, 1.25) and one olefinic H-atom signal (d(H) 5.86). These features suggested
that 1 was an ursolic acid type triterpenoid derived from (3b)-3-hydroxyurs-12-en-28-
(

2
210
Helvetica Chimica Acta – Vol. 94 (2011)
oic acid. However, the presence of only three non-O-bearing CH (d(C) 45.3, 50.6, and
7.5) suggested a secoursane structure [2]. Two diagnostic olefinic C-atom signals at
5
d(C) 117.2 and 150.2, the characteristic resonances of HꢀC(12) (d(H) 4.18-4.28 (m);
d(C) 70.3) correlated with the olefinic C(13) (d(C) 150.2) and CH(18) (d(H) 5.86 (s,
13
1
H); d(C) 117.2) according to the 1 H, C-HMBC data, and the Me(30) (d(H) 0.94 (d,
J ¼ 7 Hz); d(C) 8.1) vicinal to an OR group, was characteristic of a 18,19-secoursane
derivative, which possesses a C(13)¼C(18) bond and a b-hydroxy group at C(12) or
C(21) [3][4].
The nature of the sugar units of 1 was first determined by the comparison of their
C-NMR with the corresponding monosaccharides (d-glucose, l-rhamnose, and d-
13
xylose), and was confirmed by HPLC analysis after acid hydrolysis. Acid hydrolysis of
compounds 1 – 3 afforded the same triterpene derivative 1a which lost one H O
2
compared with the aglycone of 1 – 3 to form a 11,13(18)-diene moiety (d(C) 129.3,
129.2, 145.5, and 121.1). The structure of 1a was established as rel-(3b,19R,20S,21R)-
3,19-dihydroxy-18,19-secoursane-11,13(18)-diene-28,21-lactone [5] (for NMR data, see
Table 2).
Table 2. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; CDCl ) of Compound 1a. d in ppm, J in Hz.
3
d(H)
d(C)
d(H)
d(C)
CH (1)
1.03 (dd, J ¼ 13.4, 4.3),
38.3
CH (16)
C(17)
2.44 (td, J ¼ 13.5, 3.5)
28.6
45.0
121.1
67.6
44.6
78.0
42.5
2
2
1.83 – 1.86 (m)
CH (2)
1.59 – 1.69 (m)
3.22 (dd, J ¼ 11.2, 5.0)
25.1
79.2
39.1
55.1
18.3
HꢀC(18)
HꢀC(19)
HꢀC(20)
HꢀC(21)
5.36 (s)
2
HꢀC(3)
4.14 (qd, J ¼ 6.4, 2.2)
1.64 – 1.67 (overlap)
4.52 (dt, J ¼ 10.1, 5.4)
2.19 (dd, J ¼ 13.5, 3.5),
1.80 – 1.87 (m)
0.97 (s)
0.76 (s)
0.80 (s)
0.88 (s)
0.95 (s)
C(4)
HꢀC(5)
0.75 – 0.77 (m)
1.39 – 1.44 (m),
CH (6)
CH (22)
2
2
1.59 – 1.61 (m)
CH (7)
1.30 – 1.40 (overlap)
32.2
41.0
54.6
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
C(28)
28.1
15.3
16.6
18.1
20.3
178.8
20.6
8.3
2
C(8)
HꢀC(9)
1.93 (d, J ¼ 3.0)
C(10)
41.1
HꢀC(11)
HꢀC(12)
C(13)
5.89 (dd, J ¼ 10.1, 3.0)
5.66 (d, J ¼ 10.1)
129.3
129.2
145.5
37.0
Me(29)
Me(30)
1.19 (d, J ¼ 6.5)
0.84 (d, J ¼ 7.0)
C(14)
CH (15)
1.55 – 1.65 (m)
27.3
2
In the HMBC spectrum of 1 (Fig. 2), the anomeric H-atom of the Glc unit (d(H)
.31 (d, J ¼ 7.8 Hz, HꢀC(1’’’)) was long-range coupled with C(21) (d(C) 76.0) and C(19)
4
(d(C) 79.8). The anomeric H-atom of the Rha unit (d(H) 5.35 (d, J ¼ 1.5 Hz, HꢀC(1’’)))
was long-range coupled with C(2’) of the Xyl unit (d(C) 79.0), and the anomeric H-
atom of the Xyl unit (d(H) 4.41 (d, J ¼ 6.6 Hz, HꢀC(1’))) was long-range coupled with
C(3) (d(C) 90.1). These features established that Xyl was linked at C(3) of the aglycon,
Rha was linked at C(2’) of Xyl, and Glc was linked at C(19) of the aglycon. The relative
configuration of 1 was determined by NOESY analysis (Fig. 3) and comparison with
documented data [5][6]. Definitive confirmation of the structure of 1 was obtained
from X-ray analysis of a single crystal grown in AcOEt/MeOH/H O. Since the absolute
2

Helvetica Chimica Acta – Vol. 94 (2011)
2211
configuration of the sugar residues had been established by HPLC after acid hydrolysis,
the absolute configuration of the aglycone of 1 could be deduced from the X-ray data
(
Fig. 4). Thus compound 1 was finally identified as (3b,12b,19R,20S,21R)-19-(b-d-
glucopyranosyloxy)-12-hydroxy-3-{[O-a-l-rhamnopyranosyl-(1 ! 2)-b-d-xylopyrano-
syl]oxy}-18,19-secours-13(18)-ene-28,21-lactone, and named dunnianaolactone A.
Fig. 2. Key HMBC features of compound 1
Fig. 3. Key NOESY correlations of compound 1
Fig. 4. ORTEP Drawing of compound 1. Partially arbitary atom numbering.
Compound 2 was obtained as white, minute acicular crystals. Comparison of the
NMR data of 1 and 2 in CD OD (Table 1) indicated that 2 had the same aglycone part
3
as 1. HR-ESI-MS (negative-ion mode) analysis suggested that the molecular formula
was C H O . Acidic hydrolysis of 2 gave d-xylose and d-glucose.
41
68 15

2
212
Helvetica Chimica Acta – Vol. 94 (2011)
The position of their attachment was deduced from the HMBC spectrum and
further confirmed by comparison with the data reported for alatoside A (same 3-O-
substituent) and alatosides B and C (similar 19-O-substituent) [5]. Thus compound 2
was established as (3b,12b,19R,20S,21R)-19-(b-d-glucopyranosyloxy)-12-hydroxy-3-
(
b-d-xylopyranosyloxy)-18,19-secours-13(18)-ene-28,21-lactone, and named dunnia-
naolactone B.
Compound 3 was obtained as white, amorphous solid, which had the same aglycone
part as 1 and 2, according to the NMR data (Table 1). HR-ESI-MS analysis suggested
that the molecular formula of 3 was C H O . On acid hydrolysis, 3 furnished d-
48
78 19
glucose and l-rhamnose in the ratio 2 :1.
The position of attachment and the interglycosidic linkage were established from
the HMBC data and were corroborated by comparison with the data reported for
alatoside C (same 3-O-substituent and 19-O-substituent) [5]. Compound 3 was thus
established as (3b,12b,19R,20S,21R)-19-(b-d-glucopyranosyloxy)-12-hydroxy-3-{[O-a-
l-rhamnopyranosyl-(1 ! 2)-b-d-glucopyranosyl]oxy}-18,19-secours-13(18)-ene-28,21-
lactone, and named dunnianaolactone C.
Nine known compounds were also isolated from this plant, i.e., an ursane-type
triterpenoid saponin, rotundioic acid 28-b-d-glucopyranosyl ester (¼ ilexoside XXX)
(4) [7] and two benzofuran lignans, 4-[erythro-2,3-dihydro-3-(hydroxymethyl)-5-(3-
hydroxypropyl)-7-methoxybenzofuran-2-yl]-2,6-dimethoxyphenyl b-d-glucopyrano-
side (5) [8] and (7S,8R)-dihydrodehydrodiconiferyl alcohol 9-(b-d-glucopyranoside)
(
¼[(2S,3R)-2,3-dyhidro-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxyprophyl)-7-me-
thoxybenzofuran-3-yl]methyl b-d-glucopyranoside; 6) [9] (Fig. 1) besides five flavo-
noid glycosides, namely quercetin (¼2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-
bensopygran-4-one) [10], quercetin 3-(b-d-glucopyranoside) [11], quercetin 3-[a-l-
rhamnopyranosyl-(1 ! 6)-b-d-glucopyranoside] (rutin) [12], kaempferol 3-(a-l-rham-
nopyranosyl-(1 ! 6)-b-d-glucopyranoside [13] and kaempferol 3-[a-l-rhamnopyrano-
syl-(1 ! 6)-b-d-galactopyranoside] [13] (kaempferol ¼ 3,5,7-trihydroxy-2-(4-hydroxy-
phenyl)-4H-1-benzopyran-4-one), and 4-hydroxybenzoic acid [14].
Discussion. – To date, 19 E-secoursane-type triterpenoids have been reported, and
twelve of them are from the genus Ilex, including I. crenata, I. cornuta, and I. aculeolata
[
15 – 17]. The most similar isolates are alatosides A – C from Sesamum alatumThonn.,
which possess the same carbon skelekon as dunnianaolactones A – C [5]. Interestingly,
alatosides A – C possess an OHꢀC(18) group and a C(12)¼C(13) bond, whereas the
situation is reversed in dunnianaolactones A – C, with an OHꢀC(12) group and a
C(13)¼(18) bond.
Experimental Part
General. Column chromatography (CC): silica gel H (SiO ; 200– 300 mesh; Qingdao Marine
2
Chemical Industry), reversed-phase SiO2 CG161 (75 mm; Rohm and Hass), Sephadex LH-20 (20 –
1
50 mm, Amersham), macroreticular resin HPD-100 (60 – 16 mesh; Bonchem Chemical Industry). Anal.
HPLC: Hitachi HPLC system with UV detector L-2400 and pump L-2130; lmax (log e) in nm. M.p.: Tech
X-5 microscope melting point apparatus (Beijing Instrument Co., Ltd.); uncorrected. ORD: Jasco-P-
ꢀ
1
1
010 spectropolarimeter. IR Spectra: Bruker-Vertex-70 spectrophotometer; n in cm . 1D- and 2D-NMR
1
13
Spectra: Bruker-AM-400 spectrometer; at 400 ( H) and 100 MHz ( C) in CD OD or CDCl ; d in ppm
3
3

Helvetica Chimica Acta – Vol. 94 (2011)
2213
rel. to Me Si as internal standard, J in Hz. MS: Bruker Microtof-Q-II-10238 mass spectrometer with
4
Bruker Compass Datanalysis 4.0 software; in m/z.
Plant Material. Ilex dunniana Levl. was collected in October 2007 in Enshi, Hubei Province, China.
The plant was identified and authenticated by Prof. Yingming Wang at the Wuhan Botanical Garden,
Chinese Academy of Sciences. A voucher specimen (ID 20071003) was deposited with the Pharmacy
School, Huazhong University of Science and Technology, Wuhan, P. R. China.
Extraction and Isolation. Dried I. dunniana leaves were coarsely powdered. Exactly 10 kg of the
dried, powdered plant material was extracted 5 ꢁ with 95% EtOH (ca. 20 l each time), at r.t., for 1 d each
time. The EtOH soln. was concentrated resulting in 390 g (3.9%) of extract. The residue was suspended
in H O (1.5 l) and partitioned successively with petroleum ether (5 ꢁ 2.0 l), AcOEt (5 ꢁ 2.0 l), and
2
BuOH (5 ꢁ 2.0 l). A total of 55.3 g of the BuOH-soluble residue was subjected to CC (HPD-100, 20, 50,
and 90% EtOH). The 50% EtOH fraction (10.9 g) was then subjected to CC (SiO , gradient CHCl /
2
3
MeOH/H O): Frs. A – G. Fr. B was subjected to CC (SiO , CHCl /MeOH 100 :1 ! 1:1): quercetin
2
2
3
(
80 mg) and quercetin 3-(b-d-glucopyranoside(44 mg). Fr. C was subjected to CC (SiO , AcOEt/MeOH/
2
H O 80 :20 :1): 1 (1.1 g) and 3 (90 mg). Fr. D was subjected to CC (SiO , AcOEt/MeOH/H O 90 :10 :1):
2
2
2
Fr. D1. Fr. D1 was then subjected to CC (CG161, MeOH/H O 6 :1): 2 (6 mg) and 4 (8 mg), which were
2
purified by CC (Sephadex LH-20, MeOH). Fr. E was subjected to CC (CG161, MeOH/H O 6 :1): 5
2
(
11 mg) and 6 (23 mg). Fr. F was separated by CC (Sephadex LH-20, MeOH): Frs. F1 and F 2. Fr. F1 was
subjected to CC (SiO , AcOEt/MeOH/H O 50 :50 :1): to give quercetin 3-[a-l-rhamnopyranosyl-(1 !
2
2
6
)-b-d-glucopyranoside] (20 mg), kaempferol 3-[a-l-rhamnopyranosyl-(1 ! 6)-b-d-glucopyranoside]
(
4
6 mg), and kaempferol 3-[a-l-rhamnopyranosyl-(1 ! 6)-b-d-glucopyranoside] (9 mg). From Fr. F 2,
-hydroxybenzoic acid (6 mg) was separated and purified by CC (Sephadex LH-20, MeOH).
Dunnianaolactone A (¼(3b,12b,19R,20S,21R)-3b-{[2-O-(6-Deoxy-a-l-mannopyranosyl) b-d-xylo-
pyranosyl]oxy}-19-(b-d-glucopyranosyl)oxy)-12,21-dihydroxy-18,19-secours-13(18)-en-28-oic Acid g-
2
8
Lactone; 1): Colorless, acicular crystals (AcOEt/MeOH/H O). M.p. 285 – 2868. [a] ¼ ꢀ102.1 (c ¼ 1.0,
2
D
1
13
MeOH). UV (MeOH): 238 (1.8). IR (KBr): 3550 – 3250, 2935, 1767, 1649, 1459, 1382. H- and C-NMR:
ꢀ
ꢀ
Table 1. HR-ESI-MS: 963.4722 ([M þ Cl] , C H O Cl ; calc. 963.4720).
47
76 18
Dunnianaolactone B (¼(3b,12b,19R,20S,21R)-19-(b-d-Glucopyranosyloxy)-12,21-dihydroxy-3-(b-
d-xylopyranosyloxy]-18,19-secours-13(18)-en-28-oic Acid g-Lactone; 2): Colorless, minute acicular
2
8
crystals (AcOEt/MeOH/H O). M.p. 196 – 2008. [a]
D
¼ ꢀ83.6 (c ¼ 0.17, MeOH). UV (MeOH): 236
2
1 13
(
2.4). IR (KBr): 3550 – 3250, 2940, 1751, 1650, 1460, 1383. H- and C-NMR: Table 1. HR-ESI-MS:
ꢀ
ꢀ
8
17.4153 ([M þ Cl] , C H O Cl ; calc. 817.4141).
41
66 14
Dunnianaolactone C (¼(3b,12b,19R,20S,21R)-3-{[2-O-(6-Deoxy-a-l-mannopyranosyl)-b-d-gluco-
pyranosyl]oxy}-19-(b-d-glucopyranosyloxy)-12,21-dihydroxy-18,19-secours-13(18)-en-28-oic Acid g-
2
8
Lactone; 3): White solid. M.p. 316 – 3198. [a] ¼ ꢀ95.5 (c ¼ 0.99, MeOH). UV (MeOH): 238 (1.9). IR
D
1
13
(
(
KBr): 3550 – 3250, 2945, 1769, 1631, 1459, 1377. H- and C-NMR: Table 1. HR-ESI-MS: 957.5037
[M ꢀ H] , C H O19 ; calc. 957.5059).
ꢀ
ꢀ
48
77
Sugar Identification [18][19]. Each compound 1 – 3 (2.0 mg) was heated seperately at 808 in 2m HCl
2 ml) for 4 h. After cooling, the mixture was extracted with CHCl (3 ꢁ 2 ml). The CHCl extract was
(
3
3
washed to give 1a as an amorphous powder (see Table 2). The H O-soluble fraction was concentrated
2
and the residue dissolved in pyridine (1 ml) and treated with l-cysteine methyl ester (10 mg) for 1.5 h at
6
08. Then, O-tolyl isothiocyanate (20 ml) was added and the mixture heated at 608 for 1.5 h. The mixture
was analyzed by anal. HPLC (Thermo ODS Hypersil Dim (5 mm, 4.6 ꢁ 250 mm): at 358, MeCN/50 mm
H PO in H O 3 :7 for 40 min and washing of the column with 90% MeCN; detection at 250 ml). d-
3
4
2
Glucose (tR 16.76 min), l-rhamnose (tR 26.65 min), d-xylose (tR 18.34 min), and l-arabinose (tR
7.67 min), identified by comparison with authentic samples.
X-Ray Crystal-Structure Analysis of Dunnianaolactone A (1) ). Crystal data: C H O · 4(H O)O;
1
1
47
77 18
2
M 1018.15; crystal size 0.10 ꢁ 0.10 ꢁ 0.12 mm, monoclinic, space group C2 (No. 5); a ¼ 35.23900 ꢂ, b ¼
r
3
ꢀ3
6
F
.58900 ꢂ, c ¼ 24.74800 ꢂ, a ¼ 908, b ¼ 109.86008, g ¼ 908; V¼ 5404.48 ꢂ ; Z ¼ 4; D ¼ 1.251 gcm
;
calc
ꢀ
1
¼ 2204; m(MoK ) ¼ 0.099 mm . Data collection was performed with a Smart Apex CCD and
000
a
1)
CCDC-796161 contains the supplementary crystallographic data for this article. These data can be
obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif.

2
214
Helvetica Chimica Acta – Vol. 94 (2011)
graphite-monochromated radiation (l 0.71073 ꢂ), 6565 unique reflections were collected to qmax ¼ 25.08,
in which 4401 reflections were observed (I > 2.0 s(I)). The structure was solved by direct methods
(
SHELXTL) and refined by full-matrix least squares on I. In the structure refinements, non-H-atoms
were refined anisotropically. H-Atoms bonded to C-atoms were placed on the geometrically ideal
positions by the ꢃride onꢄ method. H-Atoms bound to O-atom were located by the difference Fourier
method and were included in the calculation of structure factors with isotropic temp. factors. The final
indices were R ¼ 0.0659, R ¼ 0.1791, and S ¼ 0.96.
w
REFERENCES
[
1] Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, ꢃFlora
Reipublicae Popularis Sinicaeꢄ, Science Press, Beijing, 1999, Vol. 45, Fasc. 2, p. 3. and 166.
2] R.-T. Li, J.-T. Li, J.-k. Wang, Q.-B. Han, Z.-Y. Zhu, H.-D. Sun, Helv. Chim. Acta 2005, 88, 252.
3] Y.-B. Zhou, J.-H. Wang, X.-M. Li, X.-C. Fu, Z. Yan, Y. Cong, X. Li, Helv. Chim. Acta 2008, 91, 1244.
4] M.-A. Ouyang, X. Zhang, D.-P. Li, Nat. Prod. Lett. 2002, 16, 137.
5] O. Potterat, K. Hostettmann, H. Stoeckli-Evans, M. Saadou, Helv. Chim. Acta 1992, 75, 833.
6] T. Kakuno, K. Yoshikawa, S. Arihara, M. Takei, K. Endo, Tetrahedron 1991, 47, 7219.
7] K. Amimoto, K. Yoshikaw, S. Arihara, Chem. Pharm. Bull. 1992, 40, 3138.
[
[
[
[
[
[
[
8] K. Takara, D. Matsui, K. Wada, T. Ichiba, I. Chinen, Y. Nakasone, Biosci. Biotechnol. Biochem. 2003,
6
7, 376.
9] M.-R. Kim, H.-I. Moon, J. H. Chung, Y. H. Moon, K.-S. Hahm, E.-R. Woo, Chem. Pharm. Bull. 2004,
2, 1466.
[
5
[
10] D. Q. Yu, J. S. Yang, ꢃ Handbook of Analytical Chemistryꢄ, Chemical Industry Press, Beijing, 1999,
Vol. 7, p. 820.
[
[
[
[
[
[
[
11] J. X. Wang, X. J. You, Z. C. Zhu, X. M. Chen, Tianran Chanwu Yanjiu Yu Kaifa 1990, 2, 63.
12] Y. C. Wu, Z. T. Zhang, B. Q. Zhen, Q. G. Liu, J. Northwest Sci-Tech Univ. Agric. Forest. 2004, 32, 99.
13] W.-C. Hou, R.-D. Lin, T.-H. Lee, Y.-H. Huang, F.-L. Hsu, M.-H. Lee, J. Sci. Food Agric. 2005, 85, 615.
14] C. OꢄConnor, D. McLennan, D. Calvert, A. Mitha, Aust. J. Chem. 1987, 40, 677.
15] T. Kakuno, K. Yoshikawa, S. Arihara, Tetrahedron Lett. 1991, 32, 3535.
16] T. Wu, L. Yan, Q.-J. Tang, Z.-T. Wang, Food Chem. 2008, 111, 78.
17] N. Zeng, Y. Shen, L.-Z. Li, W.-H. Jiao, P.-Y. Gao, S.-J. Song, W.-S. Chen, H.-W. Lin, J. Nat. Prod.
2011, 74, 732.
[
[
18] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, I. Kouno, Chem. Pharm. Bull. 2007, 55, 899.
19] T. Murata, T. Miyase, T. Warashina, F. Yoshizaki, J. Nat. Prod. 2009, 72, 1049.
Received October 19, 2010