Ipomotaosides A-D, resin glycosides from the aerial parts of ipomoea batatas and their inhibitory activity against COX-1 and COX-2

Article abstract of DOI:10.1021/np100283t

Four new resin glycosides, namely, ipomotaosides A-D (1-4), were isolated from the dried aerial parts of Ipomoea batatas. The structures of 1-4 were elucidated by analysis of their spectroscopic data and by chemical derivatization and were tested for their anti-inflammatory activity against cyclooxygenase (COX)-1 and -2.

Full text of DOI:10.1021/np100283t

J. Nat. Prod. 2010, 73, 1763–1766
1763
Ipomotaosides A-D, Resin Glycosides from the Aerial Parts of Ipomoea batatas and Their
Inhibitory Activity against COX-1 and COX-2
Kazuko Yoshikawa,* Chiho Yagi, Hiroshi Hama, Masami Tanaka, Shigenobu Arihara, and Toshihiro Hashimoto
Faculty of Pharmaceutical Sciences, Tokushima Bunri UniVersity, Yamashiro-Cho, Tokushima 770-8514, Japan
ReceiVed April 27, 2010
Four new resin glycosides, namely, ipomotaosides A-D (1-4), were isolated from the dried aerial parts of Ipomoea
batatas. The structures of 1-4 were elucidated by analysis of their spectroscopic data and by chemical derivatization
and were tested for their anti-inflammatory activity against cyclooxygenase (COX)-1 and -2.
The rhizomes of Ipomoea batatas (L.) Lam. (Convolvulaceae)
are used for food and as a folk medicine, and the plant has been
cultivated as a health food in western Japan.2 In mainland China,
the roots and leaves have been used in folkloric medicine for
anemia, diabetes, hemorrhage, hypertension, and leukemia.3 The
genus Ipomoea was shown to be a rich source of resin glycosides,
which have been reported from the roots of I. batatas.4-10 However,
the aerial parts of this species are disposed of without being used
in Japan. In the course of a study on the development of new
medicinal resources, a chemical study was initiated of the aerial
parts of I. batatas. The ethyl acetate extract was fractionated and
subjected to column chromatography and subsequent preparative
HPLC to afford four new resin glycosides, designated as ipomo-
taosides A (1), B (2), C (3), and D (4). The structures of 1-4 were
elucidated by various NMR techniques, including COSY, HMQC,
HMBC, DEPT, and ROESY experiments and by chemical degrada-
tion. The anti-inflammatory activity of 1-4 against COX-1 and
COX-2 was examined.
Results and Discussion
A mixture of 1-4 was treated with 5% KOH to afford the organic
acid components, n-decanoic acid (deca), n-dodecanoic acid (dodca),
and trans-cinnamic acid (cin), from the diethyl ether layer. The
n-BuOH layer furnished a glycosidic acid (5), which showed a quasi
molecular ion peak at m/z 879 [M + Na]+ in its HRFABMS,
indicative of a molecular formula of C40H72O19. On mild acidic
hydrolysis, 5 furnished (11S)-jalapinolic acid (Jla) (6),11 in addition
to D-fucose and L-rhamnose, in a ratio of 1:3, as sugar components.
NMR experiments (COSY, HMQC, HMBC, and ROESY) of 5
allowed the full assignments of the sugar units (Tables 1 and 2).
Compound 5 was identified as operculinic acid C, obtained
previously from Ipomoea operculata.11
Ipomotaoside A (1) gave a [M + Na]+ peak at m/z 1327.7911
in its HRFABMS, corresponding to a molecular formula of
C71H116O21, requiring 14 unsaturation equivalents. The IR spectrum
of 1 showed absorptions due to hydroxy (3445 cm-1), carbonyl
(1715 cm-1), and aromatic (1635 cm-1) functions. The negative
FABMS of 1 exhibited fragment ion peaks at m/z 1149 [M -154
(deca) - H]-, 1019 [1149 - 130 (cin)]-, 837 [1019 - 182
(dodeca)]-, 545, 417, and 271, suggesting that this compound
includes one unit each of n-decanoic acid, trans-cinnamic acid,
n-dodecanoic acid, and 5. The diagnostic fragment ion peaks at
m/z 545 [417 + 128 (146 - H2O)]-, 417 [271 + 146]-, and 271
[Jla - H]- indicated that a macrocyclic disaccharide ester formed
between the second sugar (Rha′) and the carbonyl group of
jalapinolic acid (6).12(Moreover, these diagnostic fragment ion peaks
were observed in the FABMS of each of 2-4, to be described
below.) The 1H NMR spectrum of 1 exhibited three triplet methyls
and many methylene signals assignable to the organic acids (Org),
a trans-cinnamoyl group as confirmed by the distinctive trans-
coupled olefinic protons at δ 6.61 (d, J ) 16.0 Hz) and 7.88 (d, J
) 16.0 Hz) and aromatic protons at δ 7.36-7.44 (m, 5H), four
acylated protons at δ 5.84 (t, J ) 9.9 Hz), 5.85 (dd, J ) 9.3, 3.0
Hz), 5.94 (dd, J ) 3.2, 1.6 Hz), and 5.95 (dd, J ) 2.2, 1.5 Hz),
and four anomeric protons at δ 4.77 (d, J ) 7.4 Hz), 5.50 (d, J )
1.5 Hz), 5.70 (d, J ) 1.6 Hz), and 6.23 (d, J ) 1.6 Hz) (Table 1).
The chemical shifts and coupling constants of the signals due to
the sugar moieties were compatible with a ꢀ-linkage for the
fucopyranosyl in 4C1 conformation and R-linkages for all the
rhamnopyranosyl units in 1C4 conformation. The 13C NMR spectrum
* To whom correspondence should be addressed. Tel: +81-88-602-8439.
Fax: +81-88-655-3051. E-mail: yosikawa@ph.bunri-u.ac.jp.
10.1021/np100283t  2010 American Chemical Society
Published on Web 10/20/2010

1764 Journal of Natural Products, 2010, Vol. 73, No. 11
Yoshikawa et al.
Table 1. 1H NMR Spectroscopic Data of Compounds 1-5 (in pyridine-d5, 600 MHz)
position
1
2
3
4
5
aglycone
11
Cin-2
3
5,9
3.87 (m)
3.84 (m)
3.88 (m)
3.92 (m)
3.89 (m)
6.61 (d, 16.0)
7.88 (d, 16.0)
7.36 (m)
6.45 (d, 15.9)
7.75 (d, 15.9)
7.28 (m)
6.43 (d, 15.9)
7.72 (d, 15.9)
7.29 (m)
6.56 (d, 15.9)
7.85 (d, 15.9)
7.34 (m)
6,8
7
7.44 (m)
7.36 (m)
7.33 (m)
7.28 (m)
7.31 (m)
7.29 (m)
7.40 (m)
7.34 (m)
Me
Me
0.82 (t, 7.3)
0.86 (t, 7.1)
0.82 (t, 7.3)
0.84 (t, 7.3)
0.83 (t, 7.3)
0.86 (t, 7.3)
0.83 (t, 7.3)
0.86 (t, 7.3)
0.83 (t, 7.3)
Me
Fuc-1
0.88 (t, 7.1)
4.77 (d, 7.4)
0.86 (t, 7.3)
4.73 (d, 7.9)
1.03 (t, 7.3)
4.79 (d, 6.8)
1.03 (t, 7.1)
4.80 (d, 7.7)
4.73 (d, 7.9)
2
3
4
4.19 (dd, 9.5,7.4)
4.11 (dd, 9.5,3.4)
4.02 (d, 3.4)
4.16 (dd, 9.5, 7.9)
4.05 (dd, 9.5, 3.4)
3.98 (d, 3.4)
4.54 (dd, 9.5, 6.8)
4.19 (dd, 9.5, 3.3)
3.93 (d, 3.3)
4.56 (dd, 9.1, 7.7)
4.20 (dd, 9.1, 3.0)
3.93 (d, 3.0)
4.44 (dd, 9.4, 7.9)
4.11 (dd, 9.4, 3.4)
3.92 (d, 3.4)
5
6
3.80 (q, 6.3)
1.50 (d, 6.3)
3.75 (q, 6.4)
1.53 (d, 6.4)
3.82 (q, 6.8)
1.52 (d, 6.8)
3.83 (q, 6.3)
1.52 (d, 6.3)
3.75 (q, 6.4)
1.50 (d, 6.4)
Rha′-1
5.50 (d, 1.5)
5.50 (d, 1.2)
6.38 (d, 1.7)
6.40 (d, 1.4)
6.19 (d, 1.2)
2
5.95 (dd, 2.2, 1.5)
5.06 (dd, 9.6, 2.2)
4.26 (dd, 9.6, 9.3)
4.50 (dq, 9.3, 6.0)
1.67 (d, 6.0)
5.94 (dd, 3.3, 1.2)
5.01 (dd, 9.6, 3.3)
4.23 (dd, 9.8, 9.6)
4.49 (dq, 9.8, 5.8)
1.70 (d, 5.8)
5.29 (dd, 3.3, 1.7)
5.61 (dd, 9.4, 3.3)
4.67 (dd, 9.7, 9.4)
5.02 (dd, 9.7, 5.8)
1.59 (d, 5.8)
5.27 (dd, 2.2, 1.4)
5.70 (dd, 9.6, 2.2)
4.73 (t, 9.6)
5.10 (dd, 9.6, 6.2)
1.60 (d, 6.2)
4.74 (dd, 3.3, 1.2)
4.52 (dd, 9.6, 3.3)
4.39 (dd, 9.8, 9.6)
4.30 (dd, 9.8, 5.9)
1.54 (d, 5.9)
3
4
5
6
Rha′′-1
6.23 (d, 1.6)
6.05 (d, 1.6)
5.63 (d, 1.7)
5.92 (d, 1.4)
6.21 (d, 1.6)
2
4.99 (dd, 3.0, 1.6)
5.85 (dd, 9.3, 3.0)
4.62 (dd, 9.6, 9.3)
4.46 (dq, 9.6, 6.2)
1.67 (d, 6.2)
6.07 (dd, 3.2, 1.6)
4.75 (dd, 9.0, 3.2)
4.25 (dd, 9.4, 9.0)
4.46 (dq, 9.4, 6.0)
1.64 (d, 6.0)
5.83 (dd, 3.3, 1.7)
4.64 (dd, 9.0, 3.3)
4.27 (dd, 9.4, 9.0)
4.38 (dq, 9.4, 6.0)
1.65 (d, 6.0)
4.77 (dd, 3.0, 1.4)
5.74 (dd, 9.3, 3.0)
4.57 (dd, 9.6, 9.3)
4.42 (dq, 9.6, 6.0)
1.64 (d, 6.0)
4.62 (dd, 3.2, 1.6)
4.58 (dd, 9.0, 3.2)
4.29 (dd, 9.5, 9.0)
4.31 (dq, 9.5, 6.0)
1.55 (d, 6.0)
3
4
5
6
Rha′′′-1
5.70 (d, 1.6)
6.20 (brd, 1.1)
6.23 (dd, 3.3, 1.1)
4.80 (dd, 9.7, 3.3)
5.87 (dd, 9.9, 9.7)
4.53 (dq, 9.9, 5.8)
1.58 (d, 5.8)
6.13 (d, 1.1)
5.59 (d, 1.4)
6.27 (d, 1.1)
2
3
4
5
6
5.94 (dd, 3.2, 1.6)
4.74 (dd, 9.9, 3.2)
5.84 (t, 9.9)
4.43 (dq, 9.9, 6.3)
1.50 (d, 6.3)
6.21 (dd, 3.3, 1.1)
4.75 (dd, 9.6, 3.3)
5.83 (dd, 9.8, 9.6)
4.48 (dq, 9.8, 6.2)
1.55 (d, 6.2)
5.85 (dd, 3.3, 1.4)
4.63 (dd, 9.5, 3.3)
5.80 (dd, 9.9, 9.5)
4.36 (dq, 9.9, 6.3)
1.52 (d, 6.3)
4.79 (dd, 3.3, 1.1)
4.43 (dd, 9.6, 3.3)
4.25 (dd, 9.9, 9.6)
4.82 (dq, 9.9, 6.0)
1.57 (d, 6.0)
Table 2. 13C NMR Spectroscopic Data of Compounds 1-5
(in pyridine-d5, 150 MHz)
(Table 2). Detailed assignments of the proton and carbon signals
of 1 were obtained using a group of NMR experiments (COSY,
1
HMQC, HMBC, and ROESY). In a comparison of the H NMR
carbon
1
2
3
4
5
spectrum with that of 5, the signals of H-2 (δ 5.95) of Rha′, H-2
(δ 5.85) of Rha′′, and H-2 (δ 5.94) and H-4 (δ 5.84) of Rha′′′
were shifted by +1.21, +1.27, +1.15, and +1.59 ppm, respectively.
The HMBC spectrum of 1 permitted the unambiguous assignments
of the esterified positions of the oligosaccharide core to be made
from the correlations between the carbonyl ester group and the
pyranose ring proton. The HMBC spectrum showed correlations
between H-2 of Rha′ (δ 5.95)/C-1 of Jla (δ 173.2), H-3 of Rha′′ (δ
5.85)/C-1 of Org (δ 173.7), H-2 of Rha′′′ (δ 5.94)/the signal at δ
166.4 correlated with those of the olefinic protons due to the
cinnamoyl, and H-4 of Rha′′′ (δ 5.84)/C-1 of Org (δ 173.8),
respectively. Therefore, these observations suggested that the
carboxyl group of jalapinolic acid combined with C-2 of Rha′ to
form a macrocyclic ring, with the cinmamoyl moiety attached at
C-2 of Rha′′′ and the dodecanoyl and decanoyl moieties located at
C-3 of Rha′′ and/or C-4 of Rha′′′. In order to clarify the location
of the ester linkage of the two organic acids, 1 was acylated to
give a pentaacetate (1a). The positive FABMS of 1a exhibited,
besides a [M + Na]+ ion peak at m/z 1538, a diagnostic fragment
ion peak at m/z 501 [HRFABMS m/z 501.2901 (calcd for C29H41O7,
501.2853)] assignable to a dodeca unit attached at C-4 of Rha′′′,
but no fragment ion peak at m/z 473, which would be expected if
a deca unit had been located at this position. Accordingly, the
structure of 1 was concluded to be (S)-jalapinolic acid 11-O-(2-
O-trans-cinnamoyl)-[(4-O-n-dodecanoyl)]-R-L-rhamnopyranosyl-
(1f4)-O-(3-O-n-decanoyl)-R-L-rhamnopyranosyl-(1f4)-O-R-L-
rhamnopyranosyl-(1f2)-O-ꢀ-D-fucopyranoside, intramolecular 1,2′′-
ester.
11
Cin-1
2
3
4
5,9
6,8
7
CdO
CdO
CdO
Me
Me
Me
Fuc-1
2
82.3
166.7
118.5
145.5
134.6
129.2
128.6
130.7
173.2
173.7
173.8
14.2
14.3
14.3
104.4
80.2
73.4
73.0
70.9
17.4
98.8
73.9
70.3
81.0
68.8
19.4
103.4
70.0
75.6
79.1
68.6
18.8
100.5
74.2
68.0
82.5
166.4
118.7
145.3
134.8
129.2
128.6
130.7
173.1
173.2
173.6
14.6
14.6
14.6
104.5
80.2
73.7
73.1
70.8
17.7
98.7
73.9
70.3
81.4
69.0
19.7
100.4
74.2
71.1
80.4
68.4
19.2
100.5
74.2
68.3
79.3
166.3
118.6
145.2
134.7
129.1
128.5
130.6
173.2
173.6
174.8
14.5
14.6
14.9
101.6
73.2
76.8
73.6
71.3
17.2
100.4
69.6
78.2
78.1
67.7
19.1
79.8
166.5
118.5
145.5
134.6
129.2
128.6
130.7
173.1
173.6
174.4
14.3
14.3
14.6
101.7
73.0
76.9
73.6
71.2
17.8
100.3
69.7
79.0
76.1
67.4
19.4
78.2
176.1
14.3
101.4
75.1
76.6
73.4
71.2
17.4
101.5
72.7
73.2
80.6
67.2
19.4
102.9
73.1
73.4
79.6
68.4
18.9
102.8
72.4
72.8
73.9
70.2
18.4
3
4
5
6
Rha′-1
2
3
4
5
6
Rha′′-1
2
3
4
5
6
100.3
74.1
70.6
80.5
68.3
18.7
100.4
73.9
68.2
75.2
102.4
70.3
75.2
79.1
69.0
18.5
100.6
74.0
67.8
74.9
Rha′′-1
2
3
4
5
6
Ipomotaoside B (2) exhibited a molecular formula of C73H120O21
([M + Na]+, m/z 1355.8227), differing from 1 by an additional
C2H4 unit. Also, the negative FABMS of 2 exhibited fragment ion
peaks at m/z 1149 [M - 182 (dodeca) - H]-, 1019 [1149 - 130
(cin)]-, 837 [1019 - 182 (dodeca)]-, 545, 417, and 271, suggesting
that 2 consists of one unit of trans-cinnamic acid, two units of
74.9
68.3
17.9
75.4
68.2
18.4
68.1
18.0
68.2
17.3
showed four ester carbonyl carbons at δ 173.8, 173.7, 173.2, and
166.4 and four anomeric carbons at δ 98.8, 100.5, 103.4, and 104.4

Resin Glycosides from Ipomoea batatas
Journal of Natural Products, 2010, Vol. 73, No. 11 1765
Table 3. IC50 Valuesa (µM) of Compounds 1-4
n-dodecanoic acid, and 5, with the jalapinolic acid unit esterified
with the first rhamnose (Rha′). The sites of the four ester linkages
in 2 were defined by use of the HMBC spectrum, and correlations
were exhibited between H-2 of Rha′ (δ 5.94)/C-1 of Jla (δ 173.1),
H-2 of Rha′′ (δ 6.07)/C-1 of dodeca (δ 173.2), H-2 of Rha′′′ (δ
6.23)/C-1 of cin (δ 166.4), and H-4 of Rha′′′ (δ 5.87)/C-1 of dodeca
(δ 173.6), respectively. Hence, the structure of ipomotaoside B (2)
was concluded to be (S)-jalapinolic acid 11-O-(2-O-trans-cin-
namoyl)-[(4-O-n-dodecanoyl)]-R-L-rhamnopyranosyl-(1f4)-O-(2-
O-n-dodecanoyl)-R-L-rhamnopyranosyl-(1f4)-O-R-L-rhamnopyr-
anosyl-(1f2)-O-ꢀ-D-fucopyranoside, intramolecular 1,2′′-ester.
Ipomotaoside C (3) gave the same molecular formula, C71H116O21
([M + Na]+, m/z 1327.7927), as 1. The negative FABMS of 3
exhibited the same fragment peaks at m/z 1149 [M - 154 (deca)
- H]-, 1019 [1149 - 130 (cin)]-, and 837 [1019 - 182 (dodeca)]-,
in addition to the same diagnostic fragment ion peaks at m/z 545,
417, and 271, suggesting it to be an isomer of 1. In the HMBC
spectrum of 3, a macrocyclic ring was formed between jalapinolic
acid and C-3 of Rha′ at δ 5.61 (dd, J ) 9.4, 3.3 Hz), as shown by
correlation between H-3 of Rha′/C-1 of Jla (δ 174.8), along with
correlations with H-2 of Rha′′ (δ 5.83)/C-1 of Org (δ 173.2), H-2
of Rha′′′ (δ 6.21)/C-1 of cin (δ 166.4), and H-4 of Rha′′′ (δ 5.83)/
C-1 of Org (δ 173.6) similar to 2. Using a similar strategy to that
for 1, acetylation of 3 gave a pentaacetate (3a), and its FABMS
showed a diagnostic fragment ion at m/z 501 [HRFABMS m/z
501.2873 (C29H41O7)] along with a [M + Na]+ ion peak at m/z
1538, but no fragment ion peak at m/z 473. Consequently, the
structure of ipomotaoside C (3) was concluded to be (S)-jalapinolic
acid 11-O-(2-O-trans-cinnamoyl)-[(4-O-n-dodecanoyl)]-R-L-rham-
nopyranosyl-(1f4)-O-(2-O-n-decanoyl)-R-L-rhamnopyranosyl-(1f4)-
O-R-L-rhamnopyranosyl-(1f2)-O-ꢀ-D-fucopyranoside, intramolec-
ular 1,3′′-ester.
Ipomotaoside D (4) gave the same molecular formula of
C71H116O21 and fragment ion peaks at m/z 1173 [M - 130 (cin) -
H]-, 1019 [1173 - 154 (deca)]-, 837 [1019 - 182 (dodeca)]-,
545, 417, and 271 similar to those of 1 and 3 in the FABMS. The
pentaacetate (4a) showed the same diagnostic fragment ion peak
at m/z 501 [HRFABMS m/z 501.2859 (C29H41O7)], but no peak at
m/z 473. These MS data and the 1H NMR spectra of 3 and 4 differed
only in the position of the decanoyl residue. The HMBC spectrum
of 4 gave the correlations of H-3 of Rha′ (δ 5.70)/C-1 of Jla (δ
174.4), H-3 of Rha′′ (δ 5.74)/C-1 of deca (δ 173.1), H-2 of Rha′′′
(δ 5.85)/C-1 of cin (δ 166.4), and H-4 of Rha′′′(δ 5.80)/C-1 of
deca (δ 173.6). These findings clarified that the deca at C-2 in 3 is
transposed to C-3 in 4. Thus, the structure of ipomotaoside D (4)
was concluded to be (S)-jalapinolic acid 11-O-(2-O-trans-cin-
namoyl)-[(4-O-n-dodecanoyl)]-R-L-rhamnopyranosyl-(1f4)-O-(3-
O-n-decanoyl)-R-L-rhamnopyranosyl-(1f4)-O-R-L-rhamnopyranosyl-
(1f2)-O-ꢀ-D-fucopyranoside, intramolecular 1,3′′-ester.
The resin glycosides are known to exhibit several biological
effects such as cytotoxicity to cancer cells and antimicrobial,
antifungal,13,14 antituberculosis,15 and antidepressant activities.16
However, anti-inflammatory activity had not been reported. In one
of the inflammatory processes, two distinct isoforms of the
cyclooxygenase enzymes, COX-1 and COX-2, convert arachidonic
acid to prostaglandins. The inducible COX-2 enzyme is associated
with inflammatory conditions, whereas extensively expressed
COX-1 enzyme is responsible for the cytoprotective effects of
prostaglandins.17 The enzymes COX-1 and COX-2 have been used
extensively as tools for studying the anti-inflammatory effects of
natural compounds.18-22 Each of the isolates from I. batatas was
evaluated for its inhibitory activity against these enzymes, using a
protocol according to Futaki et al.23 Aspirin was used as positive
control, and the results are listed in Table 3. Compound 1 showed
an equivalent potency against both COX-1 and COX-2 to aspirin,
whereas weak or no inhibitory activity was observed for compounds
2-4. The degree to which these resin glycosides inhibited inflam-
compound
COX-1
COX-2
1
2
3
4
9.3
14.5
147.0
NIb
132.0
NIb
NIb
4.5
NIb
aspirin
13.9
a IC50 based on triplicate five-point titration. b NI: no inhibition.
matory mediators might depend on the size of the macrocyclic
structure in each molecule. Our results suggest that a mixture of
resin glycosides contained in the aerial parts of I. batatas is capable
of inhibiting COX-1 and -2 enzymes. Therefore, their consumption
may contribute to reduced inflammation.
Experimental Section
General Experimental Procedures. Optical rotations were taken
on a JASCO DIP-1000 polarimeter. UV and IR spectra were recorded
on a Shimazu UV-1650PC and a JASCO FT/IR-410 spectrophotometer,
respectively. NMR spectra were measured on a Varian UNITY 600
spectrometer in C5D5N using TMS as internal standard. NMR experi-
ments included COSY, DEPT, HMQC, HMBC, and ROESY. Coupling
constants (J values) are given in Hz. MS were measured on a JEOL
JMS-HX 100 mass spectrometer. Silica gel column chromatography
was performed on Kieselgel 60 (230-400 mesh). HPLC was performed
on a JASCO PU-1580 HPLC system equipped with a JASCO UV-970
detector.
Plant Material. Ipomoea batatas was collected at Nakagawa,
Tokushima Prefecture, Japan, in October 2002. The plant was identified
by one of the authors (S.A.), and a voucher specimen (TB 5427) was
deposited in the Herbarium of the Faculty of Pharmaceutical Sciences,
Tokushima Bunri University, Tokushima, Japan.
Extraction and Isolation. Dried aerial parts (2.0 kg) of I. batatas
were extracted with EtOAc at room temperature for six weeks. The
EtOAc extract (50 g) was subjected to silica gel column chromatog-
raphy, eluted with hexane-EtOAc (10:1 f 1:30) for elution to afford
fractions 1-7. Fraction 5 (7.0 g) was passaged over a silica gel column,
with hexane-EtOAc (3:7-0:10) for elution, to afford fractions 5-1-4.
Fraction 5-2 was subjected to silica gel column chromatography, eluted
with hexane-EtOAc (3:7-0:10), and was purified finally by preparative
HPLC (ODS, 88% MeOH), to afford ipomotaosides A (1, 35.0 mg), B
(2, 90.0 mg), and C (3, 75.0 mg). Fraction 5-3 was purified by
preparative HPLC (ODS, 80-100% MeOH) to afford ipomotaosides
A (1, 521.5 mg), B (2, 20.0 mg), and D (4, 195.0 mg).
Ipomotaoside A (1): white powder; mp 100-102 °C; [R]25D -27.9
(c 1.5, MeOH); UV (MeOH) λmax (log ε) 230 (3.98), 237 (4.03), 244
1
(3.88) nm; FT-IR (dry film) νmax 3445, 1715, 1635, 1050 cm-1; H
and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 1327.7911
(calcd for C71H116O21Na, 1327.7907).
Ipomotaoside B (2): white powder; mp 112-114 °C; [R]25 -8.9
D
(c 1.7, MeOH); UV (MeOH) λmax (log ε) 232 (4.06), 238 (4.24), 246
1
(3.87) nm; FT-IR (film) νmax 3450, 1740, 1640, 1075 cm-1; H and
13C NMR data, see Tables 1 and 2; HRFABMS m/z 1355.8227 (calcd
for C73H120O21Na, 1355.8220).
Ipomotaoside C (3): white powder; mp 105-107 °C; [R]25D -37.4
(c 3.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.98), 237 (4.03), 244
1
(3.88) nm; FT-IR (film) νmax 3440, 1730, 1640, 1030 cm-1; H and
13C NMR data, see Tables 1 and 2; HRFABMS m/z 1327.7927 (calcd
for C71H116O21Na, 1327.7907).
Ipomotaoside D (4): white powder; mp 106-108 °C; [R]25D -18.3
(c 3.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.98), 237 (4.03), 244
1
(3.88) nm; FT-IR (dry film) νmax 3445, 1740, 1640, 1050 cm-1; H
and 13C NMR data, see Tables 1 and 2; HRFABMS m/z 1327.7962
(calcd for C71H116O21Na, 1327.7907).
Alkaline Hydrolysis of Compounds 1-4. A mixture of 1-4 (70.0
mg) in MeOH (2 mL) was treated with 5% KOH (2 mL), and the
mixture was heated at 90 °C for 2 h. The reaction mixture was adjusted
to pH 4.0 with 5% HCl and extracted with diethyl ether to give a
mixture of organic acids. A solution of the organic acids (8 mg) and
O-p-nitrobenzyl-N,N′-diisopropylisourea (30 mg) in CH2Cl2 (3 mL) was
heated at 80 °C for 2 h. The reaction mixture was evaporated under
reduced pressure to afford their p-nitrobenzyl esters. The p-nitrobenzyl
esters were compared by HPLC (C30, 90% MeOH, 1 mL/min, 273 nm)

1766 Journal of Natural Products, 2010, Vol. 73, No. 11
Yoshikawa et al.
with authentic samples. The organic acid portion gave the following
peaks, tR: trans-cinnamic acid, 5.2 min [HREIMS m/z 283.0852 (calcd
for C16H13O4N, 283.0845)]; n-decanoic acid, 11.6 min [HREIMS m/z
307.1785 (calcd for C17H25O4N, 307.1784)]; n-dodecanoic acid, 21.0
min [HREIMS m/z 335.2102 (calcd for C19H29O4N, 335.2097)]. The
n-BuOH layer was subjected to silica gel column chromatography,
eluting with CH2C12-MeOH-H2O (8:3:1), to afford operculinic acid
Acknowledgment. We are grateful to Ms. A. Yoshikawa for
gathering the plants and are indebted to Ms. I. Okamoto for measure-
ments of mass spectra.
Supporting Information Available: 1D and 2D NMR spectra of
compounds 1-4. This material is available free of charge via the
Internet at http://pubs.acs.org.
C (5, 37 mg).11 Compound 5: amorphous solid; [R]25 -65.3 (c 1.7,
D
MeOH); FT-IR (dry film) νmax 3410 (OH), 1700 (CdO) cm-1; 1H and
13C NMR (C5D5N, 600 MHz), see Tables 1 and 2; FABMS m/z 837
[M - H]-.
References and Notes
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Acid Hydrolysis of Compound 5. A solution of 5 (30 mg) in 5%
H2SO4-dioxane was heated at 100 °C for 1 h. The reaction mixture
was diluted with H2O and then was adjusted to pH 4.0 with 5% HCl
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The H2O layer was neutralized with Amberlite IRA-35 and evapo-
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of each sugar was determined by using RI detection (Shimadzu RID-
10A) and chiral detection (Shodex OR-1) by HPLC (Shodex RSpak
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HRFABMS m/z 1537.8424 (calcd for C81H126O26Na, 1537.8435) and
m/z 501.2901 (calcd for C29H41O7, 501.2853). 3a: (+)-FABMS m/z 1538
(18), 501 (26), 131 (100); HRFABMS m/z 1537.8448 (calcd for
C81H126O26Na, 1537.8435) and m/z 501.2873 (calcd for C29H41O7,
501.2853). 4a: (+)-FABMS m/z 1538 (18), 501 (39), 131 (100);
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m/z 501.2859 (calcd for C29H41O7, 501.2853).
COX-1- and COX-2-Catalyzed Prostaglandin Biosynthesis Assay
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were suspended in 0.1 M of Tris-HCl buffer (pH 7.5) containing
hematin (1 mM) and phenol (2 mM), as cofactors. The reaction medium
was preincubated with sample for 2 min at 37 °C, and 51.4 µM [1-14C]
arachidonic acid (Sigma, St. Louis, MO) was added. The reaction
mixture was incubated for 2 min at 37 °C. To terminate the reaction
and extract PGE2, 400 µL of n-hexane-EtOAc (2:1) was added to the
reaction mixture, and the preparation was centrifuged at 2000 rpm for
1 min. The organic solvent phase was discarded. The extraction
procedure was repeated twice; then 50 µL of EtOH was added to the
aqueous phase and the preparation centrifuged at 2000 rpm for 1 min.
The amount of PGE2 was measured by radioimmunoassay using a liquid
scintillation counter. COX-1 (EC1.14.99.1, isolated from ram seminal
vesicles, Cayman Chemical Company, Ann Arbor, MI) and COX-2
(isolated from sheep placenta, purity 70%, Cayman Chemical Company)
were used.
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NP100283T