Crotalionosides A - C, three new megastigmane glucosides, two new pterocarpan glucosides and a chalcone C-glucoside from the whole plants of Crotalaria zanzibarica

Article abstract of DOI:10.1248/cpb.58.1026

From a 1-BuOH-soluble fraction of the MeOH extract of the leaves of Crotalaria zanzibarica collected in the Okinawa Islands, three new megastigmane glucosides, named crotalionosides A - C, two new pterocarpan glucosides and a chalcone C-glucoside were iso

Full text of DOI:10.1248/cpb.58.1026

1026
Regular Article
Chem. Pharm. Bull. 58(8) 1026—1032 (2010)
Vol. 58, No. 8
Crotalionosides A—C, Three New Megastigmane Glucosides, Two New
Pterocarpan Glucosides and a Chalcone C-Glucoside from the Whole
Plants of Crotalaria zanzibarica
Junko SHITAMOTO,a Katsuyoshi MATSUNAMI,a Hideaki OTSUKA,*,a Takakazu SHINZATO,b and
c
Yoshio TAKEDA
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-
b
ku, Hiroshima 734–8553, Japan: Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1
Sembaru, Nakagami-gun, Okinawa 903–0213, Japan: and c Faculty of Pharmacy, Yasuda Women’s University; 6–13–1
Yasuhigashi, Asaminami-ku, Hiroshima 731–0153, Japan.
Received March 3, 2010; accepted April 30, 2010; published online May 13, 2010
From a 1-BuOH-soluble fraction of the MeOH extract of the leaves of Crotalaria zanzibarica collected in the
Okinawa Islands, three new megastigmane glucosides, named crotalionosides A—C, two new pterocarpan gluco-
sides and a chalcone C-glucoside were isolated together with two known flavonoid glycosides and one known
megastigmane glucoside. The structures of the new compounds were elucidated by a combination of spectro-
scopic analyses. The absolute configurations of allenic megastigmane glucosides were determined by application
of the modified Mosher’s method. Those of the allenic moieties were determined by interpretation of the circular
dichroism (CD) spectra of the reduction products derived from citrosides A and B. The aglycones of pterocarpan
glucosides were found to be melilotocarpan B and the absolute structure of the chalcone C-glucoside was deter-
mined by comparison of the CD spectral behavior with the reported values.
Key words Crotalaria zanzibarica; Leguminosae; megastigmane glucoside; crotalionoside; pterocarpan glucoside; chalcone C-
glucoside
The Crotalaria genus comprises about 350 species, which (1—3), pterocarpane glucosides (4, 5), and chalcone C-glu-
are mainly found in tropical areas. C. zanzibarica BENTHAM coside (6) were elucidated on the basis of spectroscopic evi-
(Leguminosae)1) (syn. C. usaramoensis BAKER f.)2) is a dence, and those of the known compounds (7—9) were iden-
perennial shrubby herb and was introduced to the Okinawa tified by comparison of spectroscopic data with those re-
Islands from Eastern Africa as a green manure crop. These ported in the literature.
days, this plant is widely found in the Okinawa Islands. It
Crotalionoside A (1), [a]D21 Ϫ18.9°, was isolated as an
grows up to about 1.5 m in height and bears yellow flowers amorphous powder and its elemental composition was deter-
all year. Some pyrrolidine alkaloids, usaramine,3) and us- mined to be C19H32O9 by high resolution (HR)-electrospray
aramoensine,4) have been isolated from C. usaramoensis ionization (ESI)-mass spectrometry (MS). The IR spectrum
(synonym of C. zanzibarica). A related pyrrolidine alkaloid, indicated the presence of hydroxyl (3367 cmϪ1) and allenic
1
integerrimine, has also been isolated from C. incana,4) and groups (1959 cmϪ1). The prominent H-NMR resonances
monocrotaline and trichodesmine from C. sessiliflora.5) From were assigned to three singlet methyls, one doublet methyl,
a 1-BuOH-soluble fraction, new megastigmane glucosides, one set of methylene protons [dH 1.38 (dd, Jϭ12, 12 Hz) and
named crotalionosides A—C (1—3), two pterocarpane glu- 1.83 (dd, Jϭ12, 5 Hz)], three oxymethine protons, one
cosides (4, 5), and a chalcone C-glucoside (6) were isolated, olefinic proton (dH 5.35), and an anomeric proton. In the 13C-
together with three known compounds, 3-hydroxy-5,6- NMR spectrum, resonances for four methyls, one methylene,
epoxy-b-ionol 9-O-b-D-glucopyranoside (7),6) kaempferol 3- three oxymethines, two quaternary carbons and three olefinic
O-robinoside (8),7) and robinin (9).8) This paper deals with carbons attributable to an allenic unit along with six signals
structural elucidation of the new compounds and stereo- assignable to a glucopyranose moiety were observed (Table
chemical discussion of allene-possessing megastigmane 1). The above evidence indicates that crotalionoside A (1) is
aglycones.
a derivative of megastigmane glucoside with an allenic unit
and three hydoxyl groups, as shown in Fig. 1. Two proton
Results and Discussion
sequences, –CH2–CHO(H)–CHO(H)– and ϭCH–CHO(H)–
Air-dried whole plants of C. zanzibarica were extracted CH3, were observed in the H–1H correlation spectroscopy
with MeOH three times and the concentrated MeOH extract (COSY) spectrum, and the protons involed were correlated
was partitioned with solvents of increasing polarity. The 1- with the carbon signals in the heteronuclear single quantum
BuOH-soluble fraction was separated by means of various coherence (HSQC) experiment. The heteronuclear multiple
chromatographic procedures including column chromatogra- bond connectivity (HMBC) correlations from methyl protons
phy (CC) on a highly porous synthetic resin (Diaion HP-20), on two gem-dimethyl groups with a methylene carbon placed
and then normal silica gel and reversed-phase octadecyl sil- the methylene carbon at the 2-position, and since the
ica gel (ODS) CC, droplet counter-current chromatography anomeric proton (dH 4.62) showed a cross peak with one of
(DCCC), and high-performance liquid chromatography the oxymethine carbons and the oxymethine proton (dH 3.26)
(HPLC), which afforded nine compounds (1—9). The details on the oxymethine carbon (dC 91.7) showed a cross peak
and yields are given under Experimental. The structures of with the adjacent oxymethine proton (dH 4.22) (Fig. 2), the
the new megastigmane glucosides, crotalionosides A—C glucosidic linkage was placed on the hydroxyl group at the
1
∗ To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp.
© 2010 Pharmaceutical Society of Japan

August 2010
1027
Table 1. 13C-NMR Spectroscopic Data for Compounds 1, 2 and 3
(CD3OD, 100 MHz)
C
1
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
35.5
47.6
69.5
91.7
74.9
117.4
201.0
100.5
67.2
23.6
29.4
32.9
27.1
106.1
76.2
78.3
71.6
78.2
62.8
35.6
47.7
69.5
91.7
74.9
117.5
200.9
100.5
67.2
23.6
29.4
32.7
27.2
106.1
76.2
78.3
71.6
78.1
62.8
44.5
49.4
76.8
48.7
82.3
92.8
126.7
134.3
78.1
21.4
25.9
32.8
31.6
102.9
75.5
78.2
71.5
78.0
62.8
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
Fig. 3. Results of Modified Mosher’s Method (DdS–dR
)
nuclear Overhauser effect spectroscopy (NOESY) spectrum
revealed that these protons were on the same side. Enzymatic
hydrolysis of crotalionoside A (1) gave an aglycone (1a) and
glucose, which was found to be of the D-series from its posi-
tive optical rotation sign, and thus the mode of the glucosidic
linkage was determined to be b from the coupling constant
of the anomeric proton (Jϭ8 Hz). To the aglycone (1a), the
modified Mosher’s method was applied.9) (R)- and (S)-a-
Methoxy-a-trifluoromethylphenyacetic acid (MTPA) deriva-
tives (1b, 1c) were prepared from the aglycone (1a) using 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC) and N,N-dimethyl-4-aminopyridine (4-DMAP), which
afforded 3,9-diesters. As a result, the absolute configurations
at the 3- and 9-positions were determined to 3S and 9S, re-
spectively (Fig. 3, 1b, 1c). In turn, those at the 4- and 5-posi-
tions were R and S, respectively. The remaining stereochemi-
cal issue of the axis chirality in the allene part will be dis-
cussed later.
Crotalionoside B (2), [a]D27 Ϫ12.1°, was isolated as an
amorphous powder and its elemental compsotion was found
to be the same as that of crotalionoside A (1). The physico-
chemical properties of crotalionoside B (2) were almost iden-
tical to those of 1 (Table 1), except for the optical rotation
value. Since crotalionosides A and B (1, 2) exhibited differ-
ent retention times in the same HPLC run, they were unam-
biguously different compounds. As there was a probable chi-
Fig. 1. Structures
ral probe, b- -glucopyranose, at the hydroxyl group of the
D
ring moiety, the stereochemistry of 2 must be the same as
that of 1, although the result of the modified Mosher’s
method, applied to its aglycone, did not show a regular distri-
bution of signs. To clarify the stereochemsitry of the side
chain, the aglycone (2a) of crotalionoside B (2), obtained by
enzymatic hydrolysis, was analyzed by the modified Mosher’s
method, as shown in Fig. 3 (2b, 2c). The absolute configura-
Fig. 2. HMBC Correlations of 1
4-position. Thus, the planar structure of crotalionoside A (1) tion at the 9-position was revealed to be R, which was oppo-
was elucidated to be megastima-6,7-diene-3,4,5,9-tetrol 4-O- site to that of crotalionoside A (1).
glucopyranoside (Fig. 1). The proton–proton coupling con-
Compounds structurally related to crotalionosides A and
stant (Jϭ9 Hz) of H-3 and H-4 indicated that these protons B (1, 2), citrosides A (10) and B (11), were first isolated
were in axial positions, and the significant correlation be- from Citrus unshiu, and they are epimers as to the allene
tween H-4 and H3-13 observed in the phase-sensitive (PS)- axis, 7R and 7S, respectively.10) Citrosides A (10) and B (11),

1028
Vol. 58, No. 8
Fig. 5. HMBC Correlations of 3
Fig. 6. Results of Modified Mosher’s Method (DdS–dR
)
mined to be C19H32O8. The 13C-NMR spectrum indicated
the presence of 19 resonances, including six assignable to
glucopyranose, and four methyl, two methylene, one trans
double bond, two oxymethine, two oxygenated quarternary
carbon and one quarternary carbon signals. The 1H–1H
COSY spectrum exhibited two proton sequences, that is,
Fig. 4. Partial 1H-NMR Chemical Shifts for 10, 10b, 10c, 11, 11b and 11c
used in this experiment, were independently obtained from A: –C(2)H2–C(3)HO(H)–C(4)H2– and B: –C(7)HϭC(8)H–
Elaeocarpus japonicus SIEBOLD et ZUCCARINI11) and Sambucus CH(9)O(H)–C(10)H3. In the HMBC spectrum, geminal
chinensis LINDLEY,12) respectively. Citrosides A (10) and B methyl proton signals showed long-range correlation with a
(11) were reduced with NaBH4 in the presence of CeCl3 to methylene carbon, A: C(2) and an oxygen-bearing quater-
give the corresponding alcohols (10a, 11a).13) In the circular nary carbon (dC 92.8), to which two olefinic protons (dH
dichroism (CD) spectra, an positive Cotton effect at 225 nm 5.78, 5.80) also correlated (Fig. 5). From the degree of unsat-
was observed for both alcohols, and the absorption position uration, calculated from the elemental composition, other
and sign of the Cotton effect were also the same as those of than the six-membered ring, crotalionoside C (3) was ex-
crotalionosides A (1) and B (2). Thus, CD spectral analyses pected to possess one more cyclic system, whose presence
were not effective for determining the absolute configuration was substantiated by the HMBC correlation cross peak be-
of the allene axis. The alcohols (10a, 11a) comprised R and S tween H-3 (dH 4.33) and C-6 (dC 92.8). The position of the
mixtures as to the 9-position and they were separated by sugar linkage was revealed to be at the hydroxyl group on the
HPLC, which yielded pure compounds 10b and 10c, and 11b side chain by the HMBC spectrum and the mode of the link-
and 11c, of which the absolute configurations at the 9-posi- age was determined to be b from the coupling constant of the
1
tion remain uncertain. Figure 4 shows the H-NMR spectral anomeric proton (dH 4.36, Jϭ8 Hz), together with the results
data for 10b and 10c, and 11b and 11c, the chemical shifts of chirality analysis of glucose. From the above evidence, the
for H-8, 9 and 10 for 7R compounds being essentially the structure of crotalionoside C (3) was assigned as megastag-
same for 10b and 10c, and those for 7S compounds being man-7-en-3,6-epoxy-5,9-diol 9-O-b-D-glucopyranoside. En-
different from those for 7R compounds, but also essentially zymatic hydrolysis of 3 gave an aglycone, crotalionol C (3a),
the same for 11b and 11c. These different chemical shifts and application of the modified Mosher’s method to 3a estab-
solely depended upon the configurations at the 7-postition, lished the absolute configuration at the 9-position to be R
that is, not upon those at the 9-positions. However, since de- (Fig. 6). However, the ring region lacks a clue for determing
termination of axis configurations of citrosides A (10) and B the absolute configuration. The PS-NOESY correlation be-
(11) still has some ambiguity,10,14) the absolute configurations tween H3-13 (dH 1.19) and H-4eq (dH 1.95) revealed that the
at the 7-positions of crotalionosides A (1) and B (2) were epoxy ring and H3-13 were on the same side, namely the ring
concluded to be the same as that of citroside A and tenta- has a 3S*,5R*,6R* relative configuration, as shown in Fig. 1.
tively assigned as 7R. Therefore, the structures of crotaliono-
sides A (1) and B (2) were elucidated to be (3S,4R,5S,7R, powder and its elemental composition was determined to be
9S)- and (3S,4R,5S,7R,9R)-megastigma-6,7-diene-3,4,5,9- C22H24O10 by HR-ESI-MS. The IR spectrum indicated that
Compound 4, [a]D Ϫ65.6°, was isolated as an amorphous
tetrol 4-O-b-D-glucopyranosides, respectively, as shown in compound
4 possessed aromatic rings (1615, 1505,
Fig. 1.
1470 cmϪ1) with a glycosidic moiety (3388 cmϪ1), the pres-
Crotalionoside C (3), [a]D25 ϩ15.8°, was isolated as an ence of the aromatic rings being also supported by the UV
amorphous powder and its elemental composition was deter- absorption at 282 nm. In the 1H-NMR spectrum, four

August 2010
1029
glucose was of the D-series. Thus, the structure of compound
5 was elucidated to be melilotocarpan 4Ј-O-b-D-glucopyra-
noside.
Compound 6, [a]D ϩ61.5°, was isolated as an amorphous
powder and its elemental composition was determined to be
1
C21H24O10. The H- and 13C-NMR spectroscopic data were
essentially the same as those for coatline A, which was iso-
lated from Elysenhardtia polystachya by Beltrami et al.16)
Although Beltrami et al. did not disscuss the stereochemistry
of coatline A, compound 6 was expected to be a stereochemi-
cal isomer of coatline A ([a]D Ϫ45.2), since their optical ro-
tation signs and values were opposite to each other. Thus,
they are diastereomers, however, their NMR spectra were
identical due to the remoteness of the chiral centers, such
as the sugar moiety and a-carbon. Augustyn et al. examined
the CD spectra of enantioselectively synthesized a-hydroxy-
Fig. 7. H–H COSY and HMBC Correlations of 4
aliphatic proton signals, two aromatic proton signals coupled dihydrochalcones.17) A similar compound, (aR)-a,2Ј4Ј-trihy-
in an AB doublet system [dH 6.78 (d, Jϭ9 Hz) and 7.24 (d, droxy-4-methoxydihydrochalcone, (12) exhibited negative
Jϭ9 Hz)], three aromatic proton signals coupled in an ABX Cotton effect at 313 nm ([q] Ϫ12000) and a positive one at
system [dH 6.24 (d, Jϭ2 Hz), 6.33 (dd, Jϭ8, 2 Hz), and 7.09 245 nm ([q] ϩ4000), which were opposite to those of com-
(d, Jϭ8 Hz)], one methoxy signal and an anomeric proton pound 6. Thus, compound 6 has the a-S configuration and
signal [dH 4.90 (d, Jϭ8 Hz)] were observed. The 13C-NMR coatline A a-R. This conclusion is in a good accord with that
spectrum exhibited 22 resonances, including six signals as- made by Alvarez and Delgado on determination of the ab-
signable to glucopyranose, 12 aromatic signals, and one solute configuration of related a-hydroxydihydrochalcone.18)
methoxyl, one oxymethylene, one oxymethine and one me-
Experimental
thine signal. A sequence of oxymethylene, methine and
General Experimental Procedures Mp was measured with a Yanagi-
moto micromelting point apparatus and is uncorrected. Optical rotations and
oxymethine protons was observed in the 1H–1H COSY spec-
CD spectra were measured on JASCO P-1030 and JASCO J-720 polarime-
ters, respectively. IR and UV spectra were measured on Horiba FT-710 and
trum (Fig. 7). One of the aromatic rings was substituted with
three oxygen atoms, and these substituted carbon atoms were
correlated with methoxy, anomeric and oxymethylene pro-
tons in the HMBC spectrum. On irradiation of methoxyl pro-
tons, one (dH 6.78) of the aromatic protons was signifcantly
enhanced in the difference NOE experiment and this proton
showed cross peaks with dC 135.4 (C-8) and 116.6 (C-10).
The HMBC correlation from the oxymethine proton to C-10
and other correlations shown in Fig. 7 revealed the structure
of ring A. While the other trisubstituted aromatic ring pos-
sessed two oxygen atoms, and HMBC correlation cross
peaks between oxymethylene protons and the quartenary car-
bon, dC 119.2, methine proton and oxygen bearing quarte-
nary carbon, dC 160.0, together with other correlations sug-
1
JASCO V-520 UV/Vis spectrophotometers, respectively. H- and 13C-NMR
spectra were taken on a JEOL JNM a-400 spectrometer at 400 MHz and
100 MHz, respectively, with tetramethylsilane as an internal standard. Posi-
tive-ion HR-MS were performed with an Applied Biosystem QSTAR XL
system ESI-time-of-flight (TOF)-MS.
A highly-porous synthetic resin (Diaion HP-20) was purchased from
Mitsubishi Kagaku (Tokyo, Japan). Silica gel CC and reversed-phase
[octadecyl silica gel (ODS)] open CC were performed on silica gel 60
(Merck, Darmstadt, Germany) and Cosmosil 75C18-OPN (Nacalai Tesque,
Kyoto, Japan) [Fϭ50 mm, Lϭ25 cm, linear gradient: MeOH–H2O (1 : 9,
1 l)→(1 : 1, 1 l), fractions of 10 g being collected], respectively. Droplet
counter-current chromatography (DCCC) (Tokyo Rikakikai, Tokyo, Japan)
was equipped with 500 glass columns (Fϭ2 mm, Lϭ40 cm), and the lower
and upper layers of a solvent mixture of CHCl3–MeOH–H2O–n-PrOH
(9 : 12 : 8 : 2) were used for the stationary and mobile phases, respectively.
gested the structure of around B ring was as shown in Fig. 1. Five-gram fractions were collected and numbered according to their order of
elution with the mobile phase. HPLC was performed on an ODS column
The above evidence substantiated that compound 4 was a 8-
(Inertsil; GL Science, Tokyo, Japan; Fϭ6 mm, Lϭ25 cm), and the eluate
was monitored with a UV detector at 254 nm and a refractive index monitor.
Emulsin was purchased from Tokyo Chemical Industries Co., Ltd.
O-glucoside of pterocarpan, melilotocarpan B, isolated from
Melilotus alba.15) On enzymatic hydrolysis of 4, meliloto-
carpan B (4a) was obtained. The optical rotation value and
sign of 4a showed good accordance with those reported in
the literature.15) Thus, compound 4 was concluded to have
the 3R,4R configuration. The negative Cotton effect at
234 nm observed in the CD spectrum also supported the con-
figuration. Sugar analysis, using a chiral detector, revealed
that the glucose was of the D-series and the coupling con-
stant (Jϭ8 Hz) of the anomeric proton established the mode
of sugar linkage was b.
Compound 5, [a]D Ϫ94.1°, was isolated as an amorphous
powder and its elemental composition was found to be the
same as that of compound 4. Other spectroscopic data were
similar to those for compound 4. In the HMBC spectrum, the
anomeric proton (dH 4.84) showed a cross peak with dC
160.4, to which was also correlated the aromatic proton, H-5Ј
(Tokyo, Japan), and crude hesperidinase was a gift from Tokyo Tanabe Phar-
maceutical Co., Ltd. (Tokyo, Japan). (R)- and (S)-a-MTPAs were the prod-
ucts of Wako Pure Chemical Industry Co., Ltd. (Tokyo, Japan).
Plant Material Whole plants of C. zanzibarica BENTHAM (Legumi-
nosae) were collected in Kunigami-son, Kunigami-gun, Okinawa, Japan, in
July 2003, and a voucher specimen was deposited in the Herbarium of Phar-
maceutical Sciences, Graduate School of Biomedical Sciences, Hiroshima
University (03-CZ-Okinawa-0630).
Extraction and Isolation Dried aerial parts of C. zanzibarica (18.9 kg)
were extracted three times with MeOH (45 l) at 25 °C for one week and then
concentrated to 6 l in vacuo. The extract was washed with n-hexane (6 l,
167 g) and then the MeOH layer was concentrated to a gummy mass. The
latter was suspended in water (6 l) and then extracted with EtOAc (6 l) to
give 260 g of an EtOAc-soluble fraction. The aqueous layer was extracted
with 1-BuOH (6 l) to give a 1-BuOH-soluble fraction (228 g), and the re-
maining water-layer was concentrated to furnish 1.21 kg of a water-soluble
fraction.
A portion (96.8 g) of the 1-BuOH-soluble fraction was subjected to a Di-
[dH 6.64 (dd, Jϭ8, 2 Hz)]. Sugar analysis also revealed that aion HP-20 column (Fϭ60 mm, Lϭ50 cm) using H2O–MeOH (4 : 1, 4 l),

1030
Vol. 58, No. 8
Table 2. 13C-NMR Spectroscopic Data for Pterocarpans (4, 5) and Chal-
cone (6) (CD3OD, 100 MHz)
(2 : 3, 4 l), (3 : 2, 4 l), and (1 : 4, 4 l), and MeOH (4 l), 1 l fractions were being
collected. The residue (15.4 g in fractions 3—6) of the 20—40% MeOH
eluent was subjected to silica gel (500 g) CC (Fϭ50 mm, Lϭ50 cm), with
elution with CHCl3–MeOH [(9 : 1, 2 l), (4 : 1, 2 l), and (3 : 1, 2 l)],
CHCl3–MeOH–H2O (35 : 15 : 2, 2 l), and MeOH (2 l), 200 ml fractions being
collected. Combined fractions 18—23 (1.02 g) of the 20% MeOH eluate
were purified by ODS CC to give 88.4 mg of 7 in fractions 118—124. Com-
bined fractions 24—30 (735 mg) of the 20—30% MeOH eluate were sepa-
rated by ODS CC to give two fractions (59.1 mg in fractions 122—133 and
32.6 mg in fractions 134—144). Purification of the former by repeated
HPLC [MeOH–H2O (7 : 13) and then (11 : 29)] afforded 0.5 mg of 2. The
latter was purified by HPLC [MeOH–H2O (2 : 3)] to give 3.7 mg of 6 and
2.7 mg of 1 from the peaks at 6 min and 10 min, respectively. An aliquot
(2.5 g) of combined fractions 31—40 (4.02 g) was subjected ODS CC and
the residue (89.1 mg) in 139—161 was purified by DCCC. The residue
(8.8 mg) in fractions 37—45 was finally purified by HPLC [MeOH–H2O
(2 : 3)], 0.5 mg of 2 and 1.8 mg of 1 being yielded from the peaks at 7.5 min
and 9 min, respectively.
The residue (12.7 g) in fractions 7—10, obtained from the 40% MeOH
eluate on Diaion HP-20 CC, was separated by silica gel (600 g) CC
(Fϭ58 mm, Lϭ49 cm), with elution with CHCl3–MeOH [(9 : 1, 3 l), (4 : 1,
3 l), and (3 : 1, 3 l)], CHCl3–MeOH–H2O [(35 : 15 : 2, 3 l) and (65 : 35 : 4, 3 l),
and MeOH (3 l), 300 ml fractions being collected. Combined fractions 9—
18 (2.23 g) were purified by ODS CC to give a residue (150 mg in fractions
142—152) and 65.8 mg of 4 in fractions 157—161. The residue was then
purified by DCCC to give a residue (17.1 mg in fractions 80—88) and
40.6 mg of 5 in fractions 108—115. The residue was finally purified by
HPLC (MeOH–H2O, 37 : 63) to give 5.3 mg of 3 from the peak at 16 min.
Combined fractions 19—26 (2.20 g) were purified by ODS CC to give a
residue (384 mg in fractions 75—84) and 68.7 mg of 9. The residue was
separated by DCCC to give 28.8 mg of 6 in fractions 24—27. On partial
evaporation of combined fractions 32—46 (5.70 g), 236 mg of 8 was ob-
tained as a yellow precipitate.
C
4
5
6
1
2
3
4
5
6
7
8
9
10
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
1Љ
129.3
131.6
116.2
157.2
116.2
131.6
68.2
41.0
79.8
67.9
41.2
80.2
127.8
107.7
154.6
135.4
150.7
116.6
119.2
160.0
98.8
162.0
108.9
126.1
105.1
75.8
122.1
107.1
150.0
135.8
145.6
115.7
122.6
161.9
100.5
160.4
110.4
126.0
102.6
75.0
112.2
165.4
112.9
165.6
109.7
133.2
75.6
2Љ
72.9
3Љ
78.4
78.2
80.2
4Љ
71.5
71.5
71.8
5Љ
6Љ
77.9
62.7
78.0
62.6
82.7
62.8
–OCH3
a
57.1
57.8
74.4
42.2
205.7
b
ϾCϭO
Crotalionoside A (1): Amorphous powder; [a]D21 Ϫ18.9° (cϭ0.19,
MeOH): IR nmax (film) cmϪ1: 3367, 2926, 1959, 1649, 1454, 1371, 1076,
1029; UV lmax (MeOH) nm (log e): 208 (3.32); 1H-NMR (CD3OD,
400 MHz) d: 5.35 (1H, d, Jϭ6 Hz, H-8), 4.62 (1H, d, Jϭ8 Hz, H-1Ј), 4.27
(1H, qd, Jϭ6, 6 Hz, H-9), 4.22 (1H, ddd, Jϭ12, 9, 5 Hz, H-3), 3.85 (1H, dd,
Jϭ12, 2 Hz, H-6Јa), 3.69 (1H, dd, Jϭ12, 5 Hz, H-6Јb), 3.39 (1H, m, H-5Ј),
3.36 (1H, dd, Jϭ9, 9 Hz, H-3Ј), 3.33 (1H, dd, Jϭ10, 9 Hz, H-4Ј), 3.29 (1H,
dd, Jϭ9, 8 Hz, H-2Ј), 3.27 (1H, dd, Jϭ10, 9Hz, H-3Ј), 3.26 (1H, d, Jϭ9 Hz,
H-4), 1.83 (1H, dd, Jϭ12, 5 Hz, H-2a), 1.45 (3H, s, H3-13), 1.38 (1H, dd,
Jϭ12, 12 Hz, H-2b), 1.31 (3H, s, H3-11), 1.26 (3H, d, Jϭ6 Hz, H3-10), 1.06
(3H, s, H3-12); 13C-NMR (CD3OD, 100 MHz): Table 1; CD De (nm): ϩ2.46
(224) (cϭ3.01ϫ10Ϫ5 M); HR-ESI-MS (positive-ion mode) m/z: 427.1935
[MϩNa]ϩ (Calcd for C19H32O9Na: 427.1938).
(CD3OD, 100 MHz) d: 7.24 (1H, d, Jϭ9 Hz, H-5), 7.09 (1H, d, Jϭ8 Hz, H-
6Ј), 6.78 (1H, d, Jϭ9 Hz, H-6), 6.33 (1H, dd, Jϭ8, 2 Hz, H-5Ј), 6.24 (1H, d,
Jϭ2 Hz, H-3Ј), 5.51 (1H, d, Jϭ7 Hz, H-4), 4.90 (1H, d, Jϭ8 Hz, H-1Љ), 4.31
(1H, dd, Jϭ10, 3 Hz, H-2a), 3.87 (3H, s, –OCH3), 3.76 (1H, dd, Jϭ12, 3 Hz,
H-6Љa), 3.65 (1H, dd, Jϭ12, 5 Hz, H-6Љb), 3.61 (1H, dd, Jϭ10, 10 Hz, H-2b),
3.58 (1H, ddd, Jϭ10, 7, 3 Hz, H-3), 3.47 (1H, dd, Jϭ9, 8 Hz, H-2Љ), 3.42
(1H, dd, Jϭ10, 9 Hz, H-4Љ), 3.40 (1H, dd, Jϭ10, 9 Hz, H-3Љ), 3.19 (1H, m,
H-5Љ); 13C-NMR (CD3OD, 100 MHz): Table 2; CD De (nm): ϩ4.90 (285),
Ϫ13.1 (234), Ϫ17.2 (213) (cϭ4.29ϫ10Ϫ5 M); HR-ESI-MS (positive-ion
mode) m/z: 471.1264 [MϩNa]ϩ (Calcd for C22H24O10Na: 471.1261).
Compound 5: Amorphous powder; [a]D25 Ϫ94.1° (cϭ0.19, MeOH); IR
nmax (film) cmϪ1: 3360, 2937, 1620, 1491, 1269, 1100, 1070, 707; UV lmax
(MeOH) nm (log e): 280 (3.61), 233sh (3.92), 213 (4.25); 1H-NMR
(CD3OD, 100 MHz) d: 7.18 (1H, d, Jϭ8 Hz, H-6Ј), 6.95 (1H, d, Jϭ9 Hz, H-
5), 6.69 (1H, d, Jϭ9 Hz, H-6), 6.64 (1H, dd, Jϭ8, 2 Hz, H-5Ј), 6.57 (1H, d,
Jϭ2 Hz, H-3Ј), 5.53 (1H, d, Jϭ7 Hz, H-4), 4.84 (1H, d, Jϭ8 Hz, H-1Љ), 4.30
(1H, dd, Jϭ10, 3 Hz, H-2a), 3.84 (3H, s, –OCH3), 3.87 (1H, dd, Jϭ12, 2 Hz,
H-6Љa), 3.68 (1H, dd, Jϭ12, 5 Hz, H-6Љb), 3.61 (1H, dd, Jϭ10, 10 Hz, H-2b),
3.58 (1H, dd, Jϭ10, 7 Hz, H-3), 3.44 (1H, dd, Jϭ9, 8 Hz, H-2Љ), 3.39 (1H,
dd, Jϭ9, 10 Hz, H-4Љ), 3.45 (1H, dd, Jϭ9, 9 Hz, H-3Љ), 3.40 (1H, m, H-5Љ);
13C-NMR (CD3OD, 100 MHz): Table 2; CD De (nm): ϩ2.96 (285), Ϫ13.9
(233), Ϫ11.1 (212) (cϭ4.17ϫ10Ϫ5 M); HR-ESI-MS (positive-ion mode) m/z:
471.1254 [MϩNa]ϩ (Calcd for C22H24O10Na: 471.1261).
Crotalionoside B (2): Amorphous powder; [a]D27 Ϫ12.1° (cϭ0.03,
MeOH): IR nmax (film) cmϪ1: 3363, 2925, 1959, 1453, 1370, 1074, 1034;
1
UV lmax (MeOH) nm (log e): 209 (3.35); H-NMR (CD3OD, 400 MHz) d:
5.34 (1H, d, Jϭ6 Hz, H-8), 4.62 (1H, d, Jϭ8 Hz, H-1Ј), 4.28 (1H, qd, Jϭ6,
6 Hz, H-9), 4.21 (1H, ddd, Jϭ12, 9, 5 Hz, H-3), 3.84 (1H, dd, Jϭ12, 2 Hz,
H-6Јa), 3.69 (1H, dd, Jϭ12, 5 Hz, H-6Јb), 3.39 (1H, m, H-5Ј), 3.33 (1H, dd,
Jϭ10, 9 Hz, H-4Ј), 3.29 (1H, dd, Jϭ9, 8 Hz, H-2Ј), 3.27 (1H, dd, Jϭ10,
9 Hz, H-3Ј), 3.26 (1H, d, Jϭ9 Hz, H-4), 1.83 (1H, dd, Jϭ12, 5 Hz, H-2a),
1.44 (3H, s, H3-13), 1.38 (1H, dd, Jϭ12, 12 Hz, H-2b), 1.31 (3H, s, H3-11),
1.26 (3H, d, Jϭ6 Hz, H3-10), 1.07 (3H, s, H3-12); 13C-NMR (CD3OD,
100 MHz): Table 1; CD De (nm): ϩ3.55 (225) (cϭ2.98ϫ10-5 M); HR-ESI-
MS (positive-ion mode) m/z: 427.1936 [MϩNa]ϩ (Calcd for C19H32O9Na:
427.1938).
Compound 6: Amorphous powder; [a]D21 ϩ61.5° (cϭ0.27, MeOH); IR
nmax (film) cmϪ1: 3367, 2928, 16180, 1514, 1444, 1396, 1249, 1077, 1028,
711; UV lmax (MeOH) nm (log e): 319sh (3.72), 283 (3.97), 239sh (3.86),
220 (4.17); 1H-NMR (CD3OD, 100 MHz) d: 7.71 (1H, d, Jϭ9 Hz, H-6Ј),
7.01 (2H, d, Jϭ8 Hz, H-2, 6), 6.67 (2H, d, Jϭ8 Hz, H-3, 5), 6.41 (1H, d,
Jϭ9 Hz, H-5Ј), 5.17 (1H, dd, Jϭ7, 5 Hz, H-a), 4.94 (1H, d, Jϭ10 Hz, H-1Љ),
4.05 (1H, dd, Jϭ10, 10 Hz, H-2Љ), 3.86 (1H, dd, Jϭ12, 2 Hz, H-6Љa), 3.74
(1H, dd, Jϭ12, 5 Hz, H-6Љb), 3.49 (1H, dd, Jϭ10, 10 Hz, H-4Љ), 3.48 (1H,
dd, Jϭ10, 10 Hz, H-3Љ), 3.42 (1H, m, H-5Љ), 3.04 (1H, dd, Jϭ14, 5 Hz, H-
ba), 2.86 (1H, dd, Jϭ14, 7 Hz, H-bb); 13C-NMR (CD3OD, 100 MHz): Table
2; CD De (nm): ϩ32.0 (325), ϩ17.9 (293), Ϫ5.80 (254), [q] nm ϩ105600
(325), ϩ59070 (293), Ϫ19140 (254) (cϭ3.00ϫ10Ϫ5 M); HR-ESI-MS (posi-
tive-ion mode) m/z: 459.1236 [MϩNa]ϩ (Calcd for C21H24O10Na:
459.1261).
Crotalionoside C (3): Amorphous powder; [a]D25 ϩ15.8° (cϭ0.31,
MeOH); IR nmax (film) cmϪ1: 3395, 2970, 1368, 1305, 1243, 1074, 1036;
1H-NMR (CD3OD, 400 MHz) d: 5.80 (1H, d, Jϭ16 Hz, H-7), 5.78 (1H, dd,
Jϭ16, 5 Hz, H-8), 4.39 (1H, qd, Jϭ6, 5 Hz, H-9), 4.36 (1H, d, Jϭ8 Hz, H-
1Ј), 4.33 (1H, br dd, Jϭ6, 6 Hz, H-3), 3.81 (1H, dd, Jϭ12, 2 Hz, H-6Јa), 3.66
(1H, dd, Jϭ12, 5 Hz, H-6Јb), 3.39 (1H, dd, Jϭ9, 9 Hz, H-3Ј), 3.35 (1H, m,
H-5Ј), 3.32 (1H, dd, Jϭ9, 9 Hz, H-4Ј), 3.18 (1H, dd, Jϭ9, 8 Hz, H-2Ј), 1.95
(1H, ddd, Jϭ12, 6, 2 Hz, H-4a), 1.77 (1H, ddd, Jϭ12, 6, 2 Hz, H-2a), 1.64
(1H, dd, Jϭ12, 6 Hz, H-4b), 1.59 (1H, d, Jϭ12 Hz, H-2b), 1.40 (3H, s, H3-
11), 1.30 (3H, d, Jϭ6 Hz, H3-10), 1.19 (3H, s, H3-13), 0.58 (3H, s, H3-12);
13C-NMR (CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion mode)
m/z: 411.1993 [MϩNa]ϩ (Calcd for C19H32O8Na: 411.1989).
Compound 4: Amorphous powder; [a]D25 Ϫ65.6° (cϭ0.16, MeOH); IR
Enzymatic Hydrolysis of Crotalionoside A (1) Crotalionoside A (1)
(4.2 mg) in 2 ml of H2O was hydrolyzed with emulsin (5.2 mg) and crude
hesperidinase (5.0 mg) for 24 h at 37 °C. The reaction mixture was evapo-
n
max (film) cmϪ1: 3388, 2897, 1615, 1505, 1470, 1288, 1099, 1027, 800; UV
lmax (MeOH) nm (log e): 282 (3.56), 230sh (3.90), 212 (4.21); 1H-NMR

August 2010
1031
rated to dryness, and then the methanolic solution was adsorbed on silica gel
and subjected to silica gel CC (20 g, Fϭ22 mm, Lϭ11 cm) with CHCl3
(100 ml) and CHCl3–MeOH (19 : 1, 100 ml, 9 : 1, 100 ml, 17 : 3, 100 ml, and
7 : 3, 300 ml), 12 ml fractions being collected. Crotalionol A (1a) (1.8 mg,
72%) and D-glucose (1.6 mg, 86%) were recovered in fractions 26—31 and
36—42, respectively. Crotalionol A (1a): an amorphous powder, [a]D27
1.06 (3H, s, H3-12), H-2b could not be assigned, due to overlapping signals;
HR-ESI-TOF-MS (positive-ion mode) m/z: 697.2213 [MϩNa]ϩ (Calcd for
C33H36O8F6Na, 697.2206). Crotalionol B 3,9-di-O-(S)-MTPA ester (2c): an
amorphous powder, 1H-NMR (CDCl3, 400 MHz) d: 7.46—7.62 (4H, m, aro-
matic protons), 7.33—7.43 (6H, m, aromatic protons), 5.56 (1H, qd, Jϭ6,
6 Hz, H-9), 5.43 (1H, d, Jϭ6 Hz, H-8), 5.43 (1H, ddd, Jϭ12, 9, 5 Hz, H-3),
Ϫ34.2° (cϭ0.12, MeOH); 1H-NMR (CD3OD, 400 MHz) d: 5.35 (1H, d, 3.61 (3H, q, Jϭ1 Hz, –OCH3), 3.56 (3H, q, Jϭ1 Hz, –OCH3), 3.16 (1H, d,
Jϭ6 Hz, H-8), 4.27 (1H, qd, Jϭ6, 6 Hz, H-9), 3.95 (1H, ddd, Jϭ12, 9, 5 Hz, Jϭ9 Hz, H-4), 1.88 (1H, dd, Jϭ12, 5 Hz, H-2a), 1.42 (3H, s, H3-11), 1.42
H-3), 2.99 (1H, d, Jϭ9 Hz, H-4), 1.82 (1H, dd, Jϭ12, 5 Hz, H-2a), 1.36 (3H,
s, H3-13), 1.34 (1H, dd, Jϭ12, 12 Hz, H-2b), 1.30 (3H, s, H3-11), 1.25 (3H,
(3H, d, Jϭ6 Hz, H3-10), 1.36 (3H, s, H3-13), 1.02 (3H, s, H3-12), H-2b could
not be assigned, due to overlapping signals; HR-ESI-TOF-MS (positive-ion
d, Jϭ6 Hz, H3-10), 1.06 (3H, s, H3-12); 13C-NMR (CD3OD, 100 MHz) d: mode) m/z: 697.2204 [MϩNa]ϩ (Calcd for C33H36O8F6Na, 697.2206).
200.8 (C-7), 117.6 (C-6), 100.3 (C-8), 82.2 (C-4), 74.7 (C-5), 69.2 (C-3),
67.2 (C-9), 47.6 (C-2), 35.6 (C-1), 32.9 (C-12), 29.4 (C-11), 26.7 (C-13),
NaBH4 Reduction of Citrosides A (10) and B (11) to the Correspond-
ing Alcohols (10a, 10b) To a solution of citroside A (10) (10 mg) in 0.5 ml
23.5 (C-10); HR-ESI-TOF-MS (positive-ion mode) m/z: 265.1409 of MeOH was added 10 mg of CeCl3·7H2O and then 2 mg of NaBH4, the re-
[MϩNa]ϩ (Calcd for C13H22O4Na, 265.1410). D-Glucose: [a]D27 ϩ35.2° action mixture being stirred for 5 min at 25 °C. Excess NaBH4 was quenched
(cϭ0.11, H2O, 24 h after being dissolved in the solvent).
by the addition of 100 ml of (CH3)2CO and then the reaction mixture was
Preparation of Crotalionol A (R)- and (S)-MTPA 3,9-Diesters (1b, 1c) evaporated and purified by preparative TLC (developed with CHCl3–
from 1a A solution of 1a (1.1 mg) in 1 ml of dehydrated CH2Cl2 was re- MeOH–H2O, 15 : 6 : 1, and then eluted with CHCl3–MeOH, 1 : 1) to afford
acted with (R)-MTPA (66 mg) in the presence of EDC (17 mg) and 4-DMAP 1.7 mg of 10a. Citroside B (11) (1.5 mg) was similarly reduced to give
(12 mg), the mixture being occasionally stirred at 25 °C for 6 h. After the ad-
dition of 1 ml of CH2Cl2, the solution was washed with H2O (1 ml), 5% HCl
(1 ml), NaHCO3-saturated H2O, and then brine (1 ml), successively. The or-
0.5 mg of 11a. 10a: CD De (nm): ϩ2.32 (226) (cϭ4.90ϫ10Ϫ5 M, MeOH).
11a: CD De (nm): ϩ1.29 (225) (cϭ5.15ϫ10Ϫ5 M, MeOH).
HPLC Separation of 10a and 11a Compound 10a was separated by
ganic layer was dried over Na2SO4 and then evaporated under reduced pres- HPLC (MeOH–H2O, 2 : 3) to give 0.4 mg of 10b and 0.2 mg of 10c from the
sure. The residue was purified by preparative TLC [silica gel (0.25 mm peaks at 11 min and 12 min, respectively. Compound 11a was similarly sepa-
thickness), being applied for 18 cm, development with CHCl3–(CH3)2CO
(19 : 1) for 9 cm, and then eluted with CHCl3–MeOH (9 : 1)] to furnish an
ester, 1b (0.5 mg, 29%). Through a similar procedure, 1c (1.0 mg, 45%) was (1H, d, Jϭ6 Hz, H-8), 4.50 (1H, d, Jϭ8 Hz, H-1Ј), 4.28 (2H, overlapped, H-
prepared from 1a (0.8 mg) using (S)-MTPA (46 mg), EDC (17 mg), and 4- 3, 9), 3.79 (1H, dd, Jϭ12, 3 Hz, H-6Јa), 3.64 (1H, dd, Jϭ12, 5 Hz, H-6Јb),
rated by HPLC to give 50 mg of 11b and 50 mg of 11c from the peaks at
1
20 min and 23 min, respectively. 10b: H-NMR (CD3OD, 400 MHz) d: 5.33
DMAP (12 mg). Crotalionol A 3,9-di-O-(R)-MTPA ester (1b) of 1a: an
3.18—3.36 (3H, m, H-3Ј, 4Ј, 5Ј), 3.12 (1H, dd, Jϭ9, 8 Hz, H-2Ј), 2.41 (1H,
amorphous powder; 1H-NMR (CDCl3, 400 MHz) d: 7.46—7.59 (4H, m, aro- ddd, Jϭ13, 4, 2 Hz, H-4a), 1.84 (1H, ddd, Jϭ13, 4, 2 Hz, H-2a), 1.41 (3H, s,
matic protons), 7.34—7.43 (6H, m, aromatic protons), 5.55 (1H, qd, Jϭ6,
6 Hz, H-9), 5.46 (1H, ddd, Jϭ12, 9, 5 Hz, H-3), 5.41 (1H, dd, Jϭ6, 1 Hz, H-
8), 3.59 (3H, s, –OCH3), 3.54 (3H, s, –OCH3), 3.29 (1H, d, Jϭ9 Hz, H-4),
1.96 (1H, dd, Jϭ12, 5 Hz, H-2a), 1.45 (1H, dd, Jϭ12, 12 Hz, H-2b), 1.42
(3H, d, Jϭ6 Hz, H3-10), 1.37 (3H, s, H3-11), 1.34 (3H, s, H3-13), 0.96 (3H,
H3-13), 1.29 (3H, s, H3-12), 1.26 (3H, d, Jϭ6 Hz, H3-10), 1.09 (3H, s, H3-
11), H-2b and 4b could not be assigned, due to overlapping signals; CD De
(nm): ϩ1.20 (226) (cϭ5.15ϫ10Ϫ5 M, MeOH); HR-ESI-TOF-MS (positive-
ion mode) m/z: 411.1985 [MϩNa]ϩ (Calcd for C19H32O8Na, 411.1989). 10c:
1H-NMR (CD3OD, 400 MHz) d: 5.32 (1H, d, Jϭ6 Hz, H-8), 4.50 (1H, d,
s, H3-12); HR-ESI-TOF-MS (positive-ion mode) m/z: 697.2209 [MϩNa]ϩ Jϭ8 Hz, H-1Ј), 4.27 (2H, overlapped, H-3, 9), 3.79 (1H, dd, Jϭ12, 3 Hz, H-
(Calcd for C33H36O8F6Na, 697.2206). Crotalionol A 3,9-di-O-(S)-MTPA 6Јa), 3.64 (1H, dd, Jϭ12, 5 Hz, H-6Јb), 3.18—3.36 (3H, m, H-3Ј, 4Ј, 5Ј),
ester (1c) of 1a: an amorphous powder, 1H-NMR (CDCl3, 400 MHz) d:
7.48—7.67 (4H, m, aromatic protons), 7.34—7.42 (6H, m, aromatic pro-
tons), 5.53 (1H, qd, Jϭ6, 6 Hz, H-9), 5.48 (1H, d, Jϭ6 Hz, H-8), 5.47 (1H,
3.12 (1H, dd, Jϭ9, 8 Hz, H-2Ј), 2.41 (1H, ddd, Jϭ13, 4, 2 Hz, H-4a), 1.84
(1H, Jϭ13, 4, 2 Hz, H-2a), 1.42 (3H, s, H3-13), 1.29 (3H, s, H3-12), 1.26
(3H, d, Jϭ6 Hz, H3-10), 1.08 (3H, s, H3-11), H-2b and 4b could not be as-
ddd, Jϭ12, 9, 5 Hz, H-3), 3.60 (3H, q, Jϭ1 Hz, –OCH3), 3.51 (3H, q, signed, due to overlapping signals; CD De (nm): ϩ1.65 (226) (cϭ5.15ϫ
Jϭ1 Hz, –OCH3), 3.34 (1H, d, Jϭ9 Hz, H-4), 1.90 (1H, dd, Jϭ12, 5 Hz, H-
10Ϫ5 M, MeOH); HR-ESI-TOF-MS (positive-ion mode) m/z: 411.1983
2a), 1.41 (1H, dd, Jϭ12, 12 Hz, H-2b), 1.38 (3H, s, H3-11), 1.36 (3H, d, [MϩNa]ϩ (Calcd for C19H32O8Na, 411.1989). 11b: 1H-NMR (CD3OD,
Jϭ6 Hz, H3-10), 1.36 (3H, s, H3-13), 1.01 (3H, s, H3-12); HR-ESI-TOF-MS 400 MHz) d: 5.43 (1H, d, Jϭ6 Hz, H-8), 4.53 (1H, d, Jϭ 8Hz, H-1Ј), 4.51
(positive-ion mode) m/z: 697.2204 [MϩNa]ϩ (Calcd for C33H36O8F6Na,
697.2206).
Enzymatic Hydrolysis of Crotalionoside B (2) Crotalionoside B (2)
(1H, overlapped with HDO signal, H-9), 4.30 (1H, m, H-3), 3.80 (1H, dd,
Jϭ12, 2 Hz, H-6Јa), 3.65 (1H, dd, Jϭ12, 5 Hz,. H-6Јb), 3.12—3.36 (4H, m,
H-2Ј, 3Ј, 4Ј, 5Ј), 2.41 (1H, m, H-4a), 1.38 (1H, m, H-2a), 1.38 (3H, s, H3-
(0.9 mg) was similarly hydrolyzed with emulsin (1.5 mg) and crude hes- 13), 1.33 (3H, d, Jϭ6 Hz, H3-10), 1.30 (3H, s, H3-12), 1.08 (3H, s, H3-11),
peridinase (1.5 mg). The reaction mixture was evaporated to dryness, and H-2b and 4b could not be assigned, due to overlapping signals; HR-
then the methanolic solution was adsorbed on silica gel and subjected to sil- ESI-TOF-MS (positive-ion mode) m/z: 411.1991 [MϩNa]ϩ (Calcd for
1
ica gel CC (7 g, Fϭ10 mm, Lϭ17 cm) with CHCl3 (50 ml) and CHCl3– C19H32O8Na, 411.1989). 11c: H-NMR (CD3OD, 400 MHz) d: 5.40 (1H, d,
MeOH (19 : 1, 50 ml, 9 : 1, 50 ml, 17 : 3, 50 ml, and 7 : 3, 150 ml), 5 ml frac-
tions being collected. Crotalionol B (2a) (0.4 mg, 74%) and D-glucose
Jϭ6 Hz, H-8), 4.53 (1H, d, Jϭ8 Hz, H-1Ј), 4.51 (1H, overlapped with HDO
signal, H-9), 4.30 (1H, m, H-3), 3.80 (1H, dd, Jϭ12, 2 Hz, H-6Јa), 3.65 (1H,
(0.2 mg, 50%) were recovered in fractions 28—34 and 45—53, respectively. dd, Jϭ12, 5 Hz, H-6Јb), 3.12—3.36 (4H, m, H-2Ј, 3Ј, 4Ј, 5Ј), 2.41 (1H, m,
Crotalionol B (2a): amorphous powder; [a]D25 ϩ3.77° (cϭ0.038, MeOH);
H-4a), 1.83 (1H, m, H-2a), 1.40 (3H, s, H3-13), 1.31 (3H, d, Jϭ6 Hz, H3-10),
1H-NMR (CD3OD, 400 MHz) d: 5.35 (1H, d, Jϭ6 Hz, H-8), 4.28 (1H, qd, 1.29 (3H, s, H3-12), 1.06 (3H, s, H3-11), H-2b and 4b could not be assigned,
Jϭ6, 6 Hz, H-9), 3.94 (1H, ddd, Jϭ12, 9 5 Hz, H-3), 3.00 (1H, d, Jϭ9 Hz, due to overlapping signals; HR-ESI-TOF-MS (positive-ion mode) m/z:
H-4), 1.82 (1H, dd, Jϭ12, 5 Hz, H-2a), 1.36 (3H, s, H3-13), 1.35 (1H, dd, 411.1984 [MϩNa]ϩ (Calcd for C19H32O8Na, 411.1989).
Jϭ12, 12 Hz, H-2b), 1.30 (3H, s, H3-11), 1.25 (3H, d, Jϭ6 Hz, H3-10), 1.06
(3H, s, H3-12); 13C-NMR (CD3OD, 100 MHz) d: 200.7 (C-7), 117.6 (C-6),
100.4 (C-8), 82.2 (C-4), 74.7 (C-5), 69.2 (C-3), 67.2 (C-9), 48.0 (C-2), 35.7
(C-1), 32.7 (C-12), 29.4 (C-11), 26.8 (C-13), 23.5 (C-10); HR-ESI-TOF-MS
Enzymatic Hydrolysis of Crotalionoside C (3) Crotalionoside C (3)
(4.5 mg) was hydrolyzed with emulsin (4.5 mg) and crude hesperidinase
(4.0 mg) at 37 °C for 24 h. The reaction mixture was evaporated to dryness,
and then the methanolic solution was adsorbed on silica gel and subjected to
(positive-ion mode) m/z: 265.1408 [MϩNa]ϩ (Calcd for C13H22O4Na, silica gel CC (25 g, Fϭ22 mm, Lϭ13 cm) with CHCl3 (100 ml) and
265.1410).
CHCl3–MeOH (19 : 1, 100 ml, 9 : 1, 100 ml, 17 : 3, 100 ml, and 7 : 3, 300 ml),
Preparation of Crotalionol B (R)- and (S)-MTPA 3,9-Diesters (2b, 2c) 5 ml fractions being collected. Crotalionol C (3a) (2.2 mg, 86%) and glucose
from 2a From 2a (0.2 mg each), 2b (0.2 mg, 36%) and 2c (0.3 mg, 54%)
(1.4 mg, 67%) were recovered in fractions 21—24 and fractions 49—55,
were prepared with the respective amounts of (R)- and (S)-MTPA (51, repectively. Crotalionol C (3a): Amorphous powder; [a]D24 Ϫ23.1° (cϭ0.15,
1
41 mg), EDC (13, 15 mg), and 4-DMAP (10, 18 mg). Crotalionol B 3,9-di-
O-(R)-MTPA ester (2b): an amorphous powder, 1H-NMR (CDCl3, 400 MHz)
d: 7.49—7.58 (4H, m, aromatic protons), 7.36—7.43 (6H, m, aromatic pro-
tons), 5.58 (1H, qd, Jϭ6, 6 Hz, H-9), 5.52 (1H, d, Jϭ6 Hz, H-8), 5.46 (1H,
ddd, Jϭ12, 9, 5 Hz, H-3), 3.58 (3H, q, Jϭ1 Hz, –OCH3), 3.51 (3H, q,
Jϭ1 Hz, –OCH3), 3.28 (1H, d, Jϭ9 Hz, H-4), 2.00 (1H, dd, Jϭ12, 5 Hz, H-
2a), 1.39 (3H, s, H3-11), 1.36 (3H, d, Jϭ6 Hz, H3-10), 1.32 (3H, s, H3-13),
MeOH); H-NMR (CD3OD, 400 MHz) d: 5.74 (1H, d, Jϭ16 Hz, H-7), 5.70
(1H, dd, Jϭ16, 5 Hz, H-8), 4.32 (1H, br dd, Jϭ6, 6 Hz, H-3), 4.29 (1H, qd,
Jϭ6, 5 Hz, H-9), 1.95 (1H, ddd, Jϭ12, 6, 2 Hz, H-4a), 1.77 (1H, ddd, Jϭ12,
6, 2 Hz, H-2a), 1.65 (1H, d, Jϭ12 Hz, H-4b), 1.59 (1H, d, Jϭ12 Hz, H-2b),
1.40 (3H, s, H3-11), 1.24 (3H, d, Jϭ6 Hz, H3-10), 1.17 (3H, s, H3-13), 0.86
(3H, s, H3-12); 13C-NMR (CD3OD, 100 MHz) d: 136.2 (C-8), 124.6 (C-7),
92.7 (C-6), 82.2 (C-5), 76.7 (C-3), 69.3 (C-9), 49.4 (C-2), 48.7 (C-4), 44.5

1032
Vol. 58, No. 8
(C-1), 32.7 (C-12), 31.4 (C-13), 25.9 (C-11), 24.0 (C-10); HR-ESI-TOF-MS
(positive-ion mode) m/z: 249.1463 [MϩNa]ϩ (Calcd for C13H22O3Na,
249.1461).
9.5 min exhibiting positive rotation. The peaks were also identified with au-
thetic D-glucose.
Known Compounds Isolated 3-Hydroxy-5,6-epoxy-b-ionol 9-O-b-D-
Preparation of Crotalionol C (R)- and (S)-MTPA 9-Esters (3b, 3c) glucopyranoside (7): [a]D19 Ϫ50.7° (cϭ0.88, MeOH).6) Kaempferol 3-O-
from 3a From 3a (1.1 mg each), 3b (1.3 mg, 60%) and 3c (1.2 mg, 56%)
robinoside (8): [a]D25 Ϫ27.1°(cϭ0.75, MeOH).7) Robinin (9): [a]D25 Ϫ113.4°
were prepared with the respective amounts of (R)- and (S)-MTPA (26, (cϭ0.47, pyridine).8)
21 mg), EDC (15, 17 mg), and 4-DMAP (10, 10 mg). Crotalionol C 9-O-(R)-
MTPA ester (3b): amorphous powder; 1H-NMR (CDCl3, 400 MHz) d:
7.51—7.54 (2H, m, aromatic protons), 7.37—7.39 (3H, m, aromatic pro-
tons), 5.88 (1H, d, Jϭ16 Hz, H-7), 5.75 (1H, dd, Jϭ16, 7 Hz, H-8), 5.61
(1H, dq, Jϭ7, 6 Hz, H-9), 4.36 (1H, br dd, Jϭ6, 6 Hz, H-3), 3.53 (3H, q,
Jϭ1 Hz, –OCH3), 2.00 (1H, ddd, Jϭ12, 6, 2 Hz, H-4a), 1.81 (1H, ddd,
Jϭ12, 6, 2 Hz, H-2a), 1.65 (1H, d, Jϭ12 Hz, H-4b), 1.59 (1H, d, Jϭ12 Hz,
Acknowledgements The authors are grateful for access to the supercon-
ducting NMR instrument (JEOL a-400) at the Analytical Center of Molecu-
lar Medicine and an Applied Biosystem QSTAR XL system ESI (Nano
Spray)-MS at the Analysis Center of Life Science of the Graduate School of
Biomedical Sciences, Hiroshima University. This work was supported in
part by a Grant-in-Aid from each of the Ministry of Education, Science,
H-2b), 1.39 (3H, s, H3-11), 1.38 (3H, d, Jϭ6 Hz, H3-10), 1.16 (3H, s, H3- Sports, Culture and Technology of Japan and the Japan Society for the Pro-
13), 0.85 (3H, s, H3-12); HR-ESI-TOF-MS (positive-ion mode) m/z: motion of Science. Thanks are also due to the Research Foundation for Phar-
465.1840 [MϩNa]ϩ (Calcd for C23H29O5F3Na, 465.1859). Crotalionol C 9-
O-(S)-MTPA esters (3c): amorphous powder; H-NMR (CDCl3, 400 MHz)
maceutical Sciences and the Takeda Science Foundation for their financial
support.
1
d: 7.51—7.54 (2H, m, aromatic protons), 7.37—7.39 (3H, m, aromatic pro-
tons), 5.77 (1H, d, Jϭ16 Hz, H-7), 5.68 (1H, dd, Jϭ16, 7 Hz, H-8), 5.62
(1H, dq, Jϭ7, 6 Hz, H-9), 4.34 (1H, br dd, Jϭ6, 6 Hz, H-3), 3.55 (3H, q,
Jϭ1 Hz, –OCH3), 1.99 (1H, ddd, Jϭ12, 6, 2 Hz, H-4a), 1.79 (1H, ddd,
Jϭ12, 6, 2 Hz, H-2a), 1.64 (1H, d, Jϭ12 Hz, H-4b), 1.58 (1H, d, Jϭ12 Hz,
H-2b), 1.43 (3H, d, Jϭ6 Hz, H3-10), 1.36 (3H, s, H3-11), 1.14 (3H, s, H3-
13), 0.82 (3H, s, H3-12); HR-ESI-TOF-MS (positive-ion mode) m/z:
465.1838 [MϩNa]ϩ (Calcd for C23H29O5F3Na, 465.1859).
Enzymatic Hydrolysis of 4 Compound 4 (21.5 mg) in 2 ml of H2O was
incubated with 9.4 mg of b-glucosidase at 37 °C for 24 h. The dried reaction
mixture was subjected to silica gel (38 g) CC with a solvent system of
CHCl3 (200 ml) and CHCl3–MeOH [(19 : 1, 200 ml) and (9 : 1, 200 ml)],
12 ml fractions being collected. An aglycone, melilotocarpan B (4a)
(7.6 mg) was obtained in fractions 25—30, which was recrystallized from
CHCl3–MeOH to give 3.2 mg (23%) of colorless needles. Melilotocarpan B
(4a): colorless needles (CHCl3–MeOH), mp 111—113 °C, [a]D Ϫ149.5°
(cϭ0.21, dioxane); 1H-NMR (acetone-d6, 400 MHz) d: 7.13 (1H, d, Jϭ8 Hz,
H-6Ј), 6.95 (1H, d, Jϭ8 Hz, H-5), 6.71 (1H, d, Jϭ8 Hz, H-6Ј), 6.37 (1H, dd,
Jϭ8, 2 Hz, H-5Ј), 6.28 (1H, d, Jϭ2 Hz, H-3Ј), 5.53 (1H, d, Jϭ6 Hz, H-4),
4.31 (1H, dd, Jϭ10, 3 Hz, H-2b), 3.84 (3H, s, –OCH3), 3.60 (2H, m, H-2a,
3); 13C-NMR (acetone-d6, 100 MHz) d: 161.8 (C-2Ј), 159.7 (C-4Ј), 149.0
(C-7), 145.2 (C-9), 136.1 (C-8), 125.9 (C-6Ј), 121.5 (C-5), 119.1 (C-1Ј),
115.6 (C-10), 108.4 (C-5Ј), 106.9 (C-6), 98.6 (C-3Ј), 79.4 (C-4), 67.6 (C-2),
56.7 (–OCH3), 40.6 (C-3); HR-ESI-TOF-MS (positive-ion mode) m/z:
309.0734 [MϩNa]ϩ (Calcd for C16H14O5Na, 309.0733).
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Sugar Analyses Glucoses derived on enzymatic hydrolyses of crotal-
ionosides B (2) and C (3) were dissolved in 100 ml of H2O, and an aliquot
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