Tigloylshikonin, a new minor shikonin derivative, from the roots and the commercial root extract of Lithospermum erythrorhizon

Article abstract of DOI:10.1248/cpb.59.117

Tigloylshikonin, a new shikonin derivative esterified with tiglic acid ((E)-2-methylbut-2-enoic acid), was isolated as a minor pigment from a food colorant "Shikon color," a commercial root extract from Lithospermum erythrorhizon SIEBOLD et ZUCCARINI. The structure of tigloylshikonin was elucidated using ¹H, ¹³C, the difference nuclear Overhauser effect (NOE), and 2D NMR techniques. Its stereochemistry was determined by chiral-phase HPLC analysis. Tigloylshikonin was also found in the roots of L. erythrorhizon, which indicated that this new shikonin derivative is a typical component of naphthoquinone pigments in the roots of L. erythrorhizon.

Full text of DOI:10.1248/cpb.59.117

January 2011
Note
Chem. Pharm. Bull. 59(1) 117—119 (2011)
117
Tigloylshikonin, a New Minor Shikonin Derivative, from the Roots and the
Commercial Root Extract of Lithospermum erythrorhizon
Yusai ITO,* Kenichi ONOBORI, Takeshi YAMAZAKI, and Yoko KAWAMURA
Division of Food Additives, National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501,
Japan. Received July 15, 2010; accepted November 2, 2010; published online November 5, 2010
Tigloylshikonin, a new shikonin derivative esterified with tiglic acid ((E)-2-methylbut-2-enoic acid), was
isolated as a minor pigment from a food colorant “Shikon color,” a commercial root extract from Lithospermum
1
13
erythrorhizon SIEBOLD et ZUCCARINI. The structure of tigloylshikonin was elucidated using H, C, the difference
nuclear Overhauser effect (NOE), and 2D NMR techniques. Its stereochemistry was determined by chiral-phase
HPLC analysis. Tigloylshikonin was also found in the roots of L. erythrorhizon, which indicated that this new
shikonin derivative is a typical component of naphthoquinone pigments in the roots of L. erythrorhizon.
Key words Lithospermum erythrorhizon; shikonin; tiglic acid; angelic acid; Shikon color
The red pigments from the roots of Lithospermum ery-
throrhizon SIEBOLD et ZUCCARINI (Boraginaceae) have been
used in Asia since ancient times as dyestuffs for fabrics, cos-
metics, and food and as crude drugs for anti-inflammatory
1)
ointment. The pigments were naphthoquinone natural prod-
2)
ucts, shikonin (1) and its ester derivatives. In many previous
studies, various shikonin derivatives, such as those of b-hy-
3
)
4)
droxyisovalerylshikonin (2), acetylshikonin (3), propionyl-
5)
6)
shikonin (4), isobutyrylshikonin (5), b,b-dimethylacryl-
oylshikonin (6), isovalerylshikonin (7), and a-methylbu-
tyrylshikonin (8), were reported from the roots of L. ery- Fig. 1. The Structures of Shikonin (1) and Its Ester Derivatives (2—10)
throrhizon (Fig. 1). Shikonin derivatives have several biologi-
6)
7)
7)
8)
9)
cal activities such as anti-inflammatory, antiviral, antibac-
7,10)
9)
11)
terial,
antifungal, and antitumor activities, and recent
studies showed that the level of these activities were affected
12)
by the structures of the derivatives. Therefore, the evalua-
tion of various natural and synthetic shikonin analogues as
1
,8,13—15)
new drug candidates has been eagerly reported.
In our continuous study to secure safe for the natural food
additives, the commercial root extract of L. erythrorhizon
used as food colorant (Shikon color) was analyzed using liq-
uid chromatography-electrospray ionization mass spectrome-
try (LC/ESI-MS). In addition to shikonin (1) and its ester
derivatives (2—8), we found a new shikonin derivative,
tigloylshikonin (9), as a minor pigment. Here we report for
the first time the isolation and structural elucidation of 9 with
mass spectrometry and NMR techniques.
Results and Discussion
The commercial root extract of L. erythrorhizon, “Shikon
color,” was diluted with MeOH and subjected to reversed-
Fig. 2. The Reversed-Phase HPLC Chromatogram Detected at 515 nm of
the Commercial Root Extract from L. erythrorhizon
phase LC/ESI-MS analysis. The chromatogram detected at elucidated as a mixture.
15 nm indicated some pigment peaks (Fig. 2), and the posi- A minor shikonin derivative (9) was found between the
5
tive-mode ESI-MS analysis of each peak showed ion peaks at peaks of isobutyrylshikonin (5) and b,b-dimethylacryloyl-
ꢀ
m/z 311, 411, 353, 367, 381, 393, and 395 as [MꢀNa] , cor- shikonin (6) on the chromatogram (Fig. 2). The positive ESI-
responding to shikonin (1), b-hydroxyisovalerylshikonin (2), MS spectrum of 9 showed the ion peak at m/z 393 as
ꢀ
acetylshikonin (3), propionylshikonin (4), isobutyrylshikonin [MꢀNa] , which was the same as that of 6. Therefore, it was
(
(
5), b,b-dimethylacryloylshikonin (6), and isovalerylshikonin presumed that 9 was a structural isomer of 6 and a derivative
7) with a-methylbutyrylshikonin (8), respectively. Except esterified with tiglic acid ((E)-2-methylbut-2-enoic acid; cis
for 7 and 8, each compound was purified with preparative form) or angelic acid ((Z)-2-methylbut-2-enoic acid; trans
HPLC and its structure was identified with NMR experi- form) as a substitution of b,b-dimethylacrylic acid of 6. The
1
ments. Because 7 and 8 could not be clearly separated on
H-NMR spectrum of 9 recorded in CDCl showed the set of
3
HPLC, both compounds were purified and their structures resonances about shikonin (1) as well as other derivatives. In
∗
To whom correspondence should be addressed. e-mail: yuito@nihs.go.jp
© 2011 Pharmaceutical Society of Japan

1
18
Vol. 59, No. 1
21)
Echium italicum collected in Macedonia. The root extract
of L. erythrorhizon has been traditionally used as dye and
crude drag in Japan, and the pigment constitution in it has
been eagerly studied ever since Japanese scientists first puri-
fied shikonin (1) as its acetate (3) from the roots of L. ery-
22)
throrhizon. Therefore, the discovery of a new shikonin de-
rivative was surprising and suggested the presence of uniden-
tified minor pigments in the roots of this species.
Experimental
1
1
Instruments The NMR spectra were recorded on an ECA-500 spec-
trometer (JEOL, Tokyo, Japan). LC/ESI-MS analysis was performed on a
Waters system (Waters, Milford, MA, U.S.A.) equipped with an Alliance
Fig. 3. Characteristic Correlations Observed in H– H COSY (Bold
Lines) and H– C HMBC (Arrows) of 9
1
13
2695 (LC), a 2996 photodiode array (PDA) detector, and a Quattro micro
addition, a doublet methyl signal at dH 1.83 ppm (H-4ꢁ), a (quadrupole ESI-MS). High-resolution mass spectrometry analysis was per-
formed on LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA,
U.S.A.). HPLC purification was performed on a Shimadzu LC-10ADvp
singlet methyl signal at d 1.86 ppm (H-5ꢁ), and a multiplet
H
1
H signal at d 6.97 ppm (H-3ꢁ) were observed. The
H
(Shimadzu, Kyoto, Japan) equipped with a SPD-M10Avp PDA detector. The
1 1
shikonin moiety in 9 was confirmed by the H– H correlation
IR spectrum was recorded on a Fourier transform (FT)/IR-4100 (JASCO,
1
13
spectroscopy (COSY) and H– C heteronuclear multiple Tokyo Japan). The UV spectra were obtained in MeOH on a V-650 spec-
1
1
trophotometer (JASCO, Tokyo, Japan).
bond coherence (HMBC) correlations (Fig. 3). The H– H
Standard Samples and Materials The shikonin standard sample (99%)
and the mixture of optical isomers (shikonin : alkanninꢂabout 6 : 1) were
purchased from Wako Pure Chemical Industries (Osaka, Japan). The com-
mercial root extract of L. erythrorhizon, or Shikon color, was obtained from
COSY experiment of 9 indicated the spin couplings between
1
13
H-3ꢁ and H-4ꢁ and the H– C HMBC experiment of 9 re-
vealed the structure of a 2,3-dimethylacrylic acid moiety by
the correlations from the H-5ꢁ signal to the d 167.1 ppm (C- a food company in Japan. The commercial extract was prepared by the root
C
extraction of L. erythrorhizon with ethanol, following evaporation. A crude
1
ꢁ), d 128.3 (C-2ꢁ) and d 138.6 (C-3ꢁ) signals as well as by
the correlation from the H-3ꢁ and H-4ꢁ signals to the C-2ꢁ
signal (Fig. 3). The geochemistry of the acid was determined
C
C
drug “Ko-shikon” (the chipped roots of L. erythrorhizon) was purchased
from a crude-drug company in Tokyo, which had collected in China.
LC/ESI-MS Analysis The commercial root extract (500 mg) was di-
in the cis form (tiglate) by in the difference nuclear Over- luted with MeOH (10 ml) and filtrated with a polyvinylidene difluoride
hauser effect (NOE) experiments between H-3ꢁ and H-5ꢁ and (PVDF) membrane syringe filter (pore sizeꢂ0.45 mm, Whatman Interna-
tional, Piscataway, NJ, U.S.A.). The crude drug “Ko-shikon” (50 g) was ex-
tracted with EtOH (500 ml) and the extract was filtrated. Each aliquot (10 ml)
of both samples was injected to the LC/ESI-MS system. A COSMOSIL
between H-4ꢁ and H-5ꢁ. The chemical shift at H-3ꢁ of 9 (d
H
6
.97 ppm) also supported this interpretation, because previ-
ous studies reported the chemical shift at H-3 of angelate was
5
C -MS-II column (4.6 mm i.d.ꢃ150 mm, Nacalai Tesque, Kyoto, Japan)
18
16)
17)
6
.08 ppm, whereas that of tiglate was 6.95 ppm. Finally, was employed for separation at 40 °C. As the mobile phase, H O (solvent A)
2
the chiral HPLC analysis of the saponificates of 9 revealed and MeOH (solvent B) were used. The gradient condition was linear from
0 to 100% of solvent B in solvent A for 25 min, followed by an isocratic
condition of 100% (solvent B) continuously for 10 min. The flow rate was
.5 ml/min. UV absorption was recorded at 515 nm on PDA. The retention
times of 1, 2, 3, 4, 5, 6, 7 with 8 and 9 were 13.07, 16.02, 17.96, 20.56,
5
that the naphthoquinone structure of 9 was composed of
18)
96.5% of 1 and 3.5% of alkannin, an enantiomer of 1. Re-
0
cent studies showed that naphthoquinone pigments in the
roots of Boraginaceae plants are a mixture of optical isomers, 22.73, 23.97, 24.62 and 23.57 min, respectively. The ESI-MS was operated
in positive ion mode of SCAN analysis. The source temperature was 120 °C
and cone nitrogen gas flow was 50 l/h. Desolvation nitrogen gas flow was
1
and alkannin, and that the ratio of isomers varies with the
1)
species of plant. In the case of L. erythrorhizon, it is known
that the shikonin type is a major isomer. Thus, the structure
of 9 was established as tigloylshikonin.
5
4
00 l/h and desolvation temperature was 400 °C. The capillary voltage was
.25 kV and the cone voltage was 30 V.
The Purification of Shikonin Derivatives (1—9) The commercial ex-
To confirm that tigloylshikonin (9) was not an artifact in tract (2.0 g) was diluted with MeOH (20 ml) and purified with a reversed-
phase HPLC to afford 1 (8.5 mg), 2 (24.7 mg), 3 (34.2 mg), 4 (3.7 mg), 5
commercial products, the roots of L. erythrorhizon as a crude
traditional drug, designated as “Ko-shikon,” were extracted
with ethanol and the extract was subjected to LC/ESI-MS
(22.3 mg), 6 (14.3 mg), 7 with 8 (19.8 mg) and 9 (3.5 mg). A COSMOSIL
5
C -MS-II column (20 mm i.d.ꢃ250 mm) was employed for separation at
18
4
0 °C. As an eluent, H O containing 0.05% (v/v) trifluoroacetic acid (TFA)
2
analysis. The obtained profile of shikonin derivatives in the (solvent A) and acetonitrile containing 0.05% (v/v) TFA (solvent B) were
extract was the same as that in the food colorant, and the used. The gradient condition was linear from 70 to 100% of solvent B in sol-
vent A for 10 min, and then an isocratic condition of 100% (solvent B) was
continuous for 5 min. The flow rate was 9.0 ml/min. UV absorption was
recorded at 515 nm on PDA. Compounds (1—9) were manually collected.
The red fraction containing shikonin derivatives were evaporated to remove
presence of 9 was also observed. The purification of 9 from
the extract was carried out in the same manner, and the struc-
1
tural identification of 9 was achieved by H-NMR analysis.
Thus, the results demonstrated that 9 was a typical shikonin volatile and then diluted with H O to settle the red pigments, which were
2
derivative in the roots of L. erythrorhizon.
In the past, angeloylshikonin (10), a geometrical isomer of
tigloylshikonin (9), was reported as a major pigment from the
extracted with EtOAc. The red EtOAc fraction was dried with anhydrous
Na SO and concentrated to dryness in vacuo. The obtained shikonin deriva-
2
4
tives were stored at ꢄ20 °C.
Shikonin (1) Red purple solid. ESI-MS (positive mode) m/z: 311
19)
roots of Alkanna hirsutissima collected in northern Iraq.
ꢀ 1
[
MꢀNa] , H-NMR (in CDCl ) d: 1.65 (3H, s, H-6ꢅ), 1.76 (3H, s, H-5ꢅ),
3
As the optical isomer of 10, angeloylalkannin was also 2.37 (1H, m, H-2ꢅ), 2.63 (1H, m, H-2ꢅ), 4.91 (1H, m, H-1ꢅ), 5.20 (1H, m, H-
3
ꢅ), 7.04 (1H, s, H-3), 7.18 (1H, d, Jꢂ9.7 Hz, H-6 or H-7), 7.22 (1H, d, ꢂ
reported from the roots of Alkanna tinctoria, a common
20)
9.7 Hz, H-6 or H-7), 12.5 (1H, s, OH-5), 12.5 (1H, s, OH-8).
source of alkannin derivatives. However, there are no re-
ports on the isolation and structural elucidation of 9 from any
Boraginaceae plants, although HPLC analysis recently sug-
b-Hydroxyisovalerylshikonin (2) Red purple solid. ESI-MS (positive
ꢀ
1
mode) m/z: 411 [MꢀNa] , H-NMR (in CDCl ) d: 1.31 (3H, s, H-4ꢁ), 1.33
3
(3H, s, H-5ꢁ), 1.59 (3H, s, H-6ꢅ), 1.69 (3H, s, H-5ꢅ), 2.52 (1H, m, H-2ꢅ),
gested the presence of 9 in addition to 10 in the roots of 2.61 (1H, m, H-2ꢅ), 2.61 (2H, s, H-2ꢁ), 5.11 (1H, m, H-3ꢅ), 6.10 (1H, dd,

January 2011
119
Jꢂ6.9, 4.6 Hz, H-1ꢅ), 7.03 (1H, s, H-3), 7.18 (1H, s, H-6), 7.18 (1H, s, H-7),
ring for 12 h at room temperature, 1 M HCl (3 ml) was added for neutraliza-
12.4 (1H, s, OH-5), 12.6 (1H, s, OH-8).
tion and extracted with CHCl (10 ml), which was dried and evaporated in
3
Acetylshikonin (3) Red purple solid. ESI-MS (positive mode) m/z: 353 vacuo. The residue was dissolved in MeOH (1 ml) and subjected to reversed-
ꢀ
1
[
2
MꢀNa] , H-NMR (in CDCl ) d: 1.57 (3H, s, H-6ꢅ), 1.68 (3H, s, H-5ꢅ), phase LC/ESI-MS analysis as described above to check the achievement of
3
.13 (3H, s, H-2ꢁ), 2.46 (1H, m, H-2ꢅ), 2.60 (1H, m, H-2ꢅ), 5.11 (1H, t, saponification of 9. Subsequently, the saponificate of 9 was subjected to chi-
Jꢂ7.5 Hz, H-3ꢅ), 6.01 (1H, dd, Jꢂ6.9, 5.2 Hz, H-1ꢅ), 6.98 (1H, s, H-3), 7.18 ral-phase HPLC analysis to determine the ratio of shikonin to alkannin on
(1H, s, H-6), 7.18 (1H, s, H-7), 12.4 (1H, s, OH-5), 12.4 (1H, s, OH-8).
the peak areas of both compounds. For the chiral column, a Chiralcel-OJ-H
Propionylshikonin (4) Red purple solid. ESI-MS (positive mode) m/z: column (4.6 mm i.d.ꢃ250 mm, Daicel Chemical Industry, Osaka, Japan) was
ꢀ
1
3
5
67 [MꢀNa] , H-NMR (in CDCl ) d: 1.57 (3H, s, H-6ꢅ), 1.68 (3H, s, H- used at 40 °C. For the mobile phase, n-hexane/2-propanol/acetic acid
3
ꢅ), 2.40 (1H, d, Jꢂ7.7 Hz, H-2ꢁ), 2.41 (1H, d, Jꢂ7.7 Hz, H-2ꢁ), 2.48 (1H, (95 : 5 : 0.3, v/v) was used. The flow rate was 1.0 ml/min. The UV absorption
m, H-2ꢅ), 2.59 (1H, m, H-2ꢅ), 5.11 (1H, t, Jꢂ9.4 Hz, H-3ꢅ), 6.02 (1H, dd, was recorded at 515 nm on PDA. The retention times of shikonin and alkan-
Jꢂ6.3, 4.6 Hz, H-1ꢅ), 6.97 (1H, s, H-3), 7.18 (1H, s, H-6), 7.18 (1H, s, H-7),
nin were 12.3 min and 14.7 min, respectively.
1
2.42 (1H, s, OH-5), 12.4 (1H, s, OH-8).
Isobutyrylshikonin (5) Red purple solid. ESI-MS (positive mode) m/z: References
ꢀ
1
3
81 [MꢀNa] , H-NMR (in CDCl ) d: 1.20 (3H, d, Jꢂ6.8 Hz, H-3ꢁ), 1.20
1) Papageorgiou V. P., Assimopoulou A. N., Couladouros E. A., Hep-
worth D., Nicolau K. C., Angew. Chem. Int. Ed., 38, 271—300 (1999).
2) Brockmann H., Justus Liebigs Ann. Chem., 521, 1—47 (1936).
3) Morimoto I., Hirata Y., Tetrahedron Lett., 31, 3677—3680 (1966).
4) Hisamichi S., Yoshizaki F., Shoyakugaku Zasshi, 36, 154—159 (1982).
5) Cho M. H., Paik Y, S., Hahn T. R., Arch. Pharm. Res., 22, 414—416
(1999).
3
(
3H, d, Jꢂ6.8 Hz, H-4ꢁ), 1.58 (3H, s, H-6ꢅ), 1.68 (3H, s, H-5ꢅ), 2.48 (1H, m,
H-2ꢅ), 2.60 (1H, m, H-2ꢅ), 2.61 (1H, m, H-2ꢁ), 5.11 (1H, t, Jꢂ7.4 Hz, H-3ꢅ),
.01 (1H, dd, Jꢂ7.4, 4.6 Hz, H-1ꢅ), 6.97 (1H, s, H-3), 7.18 (1H, s, H-6), 7.18
6
(1H, s, H-7), 12.43 (1H, s, OH-5), 12.43 (1H, s, OH-8).
b,b-Dimethylacryloylshikonin (6) Red purple solid. ESI-MS (positive
ꢀ
1
mode) m/z: 393 [MꢀNa] , H-NMR (in CDCl ) d: 1.57 (3H, s, H-6ꢅ), 1.68
3
(3H, s, H-5ꢅ), 1.93 (3H, s, H-5ꢁ), 2.15 (3H, s, H-4ꢁ), 2.48 (1H, m, H-2ꢅ),
6) Morimoto I., Kishi T., Ikegami S., Hirata Y., Tetrahedron Lett., 52
4737—4739 (1965).
7) Kyogoku K., Terayama H., Tachi Y., Suzuki T., Komatsu M., Shoyaku-
gaku Zasshi, 27, 24—30 (1973).
2
6
.60 (1H, m, H-2ꢅ), 5.14 (1H, dd, Jꢂ6.9, 5.7 Hz, H-3ꢅ), 5.78, (1H, s, H-2ꢁ),
.00 (1H, dd, Jꢂ6.9, 5.8 Hz, H-1ꢅ), 6.97 (1H, s, H-3), 7.18 (1H, s, H-6), 7.18
(1H, s, H-7), 12.43 (1H, s, OH-5), 12.43 (1H, s, OH-8).
Isovalerylshikonin (7) Red purple solid. ESI-MS (positive mode) m/z:
8) Papageorgiou V. P., Assimopoulou A. N., Ballis A. C., Curr. Med.
Chem., 15, 3248—3267 (2008).
9) Li C., Fukushi Y., Kawabada J., Tahara S., Mizutani J., Uyeda I., J.
Pesticide Sci., 23, 54—57 (1998).
ꢀ
1
3
(
95 [MꢀNa] , H-NMR (in CDCl ) d: 0.96 (3H, d, Jꢂ2.3 Hz, H-4ꢁ), 0.97
3
3H, d, Jꢂ2.3 Hz, H-5ꢁ), 2.12 (1H, m, H-3ꢁ), 2.26 (2H, m, H-2ꢁ), 1.59 (3H,
s, H-6ꢅ), 1.68 (3H, s, H-5ꢅ), 2.45 (1H, m, H-2ꢅ), 2.59 (1H, m, H-2ꢅ), 5.12
(
1H, t, Jꢂ6.3 Hz, H-3ꢅ), 6.03 (1H, m, H-1ꢅ), 6.98 (1H, s, H-3), 7.18 (1H, s, 10) Tanaka Y., Odani T., Kanaya T., Shoyakugaku Zasshi, 28, 173—178
H-6), 7.18 (1H, s, H-7), 12.42 (1H, s, OH-5), 12.42 (1H, s, OH-8).
(1974).
a-Methylbutyrylshikonin (8) Red purple solid. ESI-MS (positive
11) Sankawa U., Ebizuka Y., Miyazaki T., Isomura Y., Otsuka H., Shibata
S., Inomata M., Fukuoka F., Chem. Pharm. Bull., 25, 2392—2395
(1977).
ꢀ
1
mode) m/z: 395 [MꢀNa] , H-NMR (in CDCl ) d: 0.92 (3H, t, Jꢂ7.5 Hz,
3
H-4ꢁ), 1.16 (3H, d, Jꢂ6.9 Hz, H-5ꢁ), 1.52 (1H, m, H-3ꢁ), 1.58 (3H, s, H-6ꢅ),
1
2
6
1
.68 (3H, s, H-5ꢅ), 1.70 (1H, m, H-3ꢁ), 2.45 (1H, m, H-2ꢅ), 2.46 (1H, m, H- 12) Hashimoto S., Xu M., Masuda Y., Aiuchi T., Nakajo S., Cao J.,
ꢁ), 2.59 (1H, m, H-2ꢅ), 5.12 (1H, t, Jꢂ6.3 Hz, H-3ꢅ), 6.03 (1H, m, H-1ꢅ),
.98 (1H, s, H-3), 7.18 (1H, s, H-6), 7.18 (1H, s, H-7), 12.42 (1H, s, OH-5),
Miyakosi M., Ida Y., Nakaya K., J. Biochem., 125, 17—23 (1999).
13) Ahn B. Z., Baik K. U., Kweon G. R., Lim K., Hwang B. D., J. Med.
Chem., 38, 1044—1047 (1995).
2.42 (1H, s, OH-8).
Tigloylshikonin (9) Red purple solid. IR (KBr) n
ꢄ1
cm : 1715, 1610, 14) Xuan Y., Hu X., Cancer Lett., 274, 233—242 (2009).
455, 760. ESI-MS (positive mode) m/z: 393 [MꢀNa] , HR-ESI-MS (posi- 15) Wang W., Dai M., Zhu C., Zhang J., Lin L., Ding J., Duan W., Bioorg.
max
ꢀ
1
ꢀ
tive mode) m/z: 371.14968 [MꢀH] (Calcd for C H O , 371.14946).
Med. Chem. Lett., 19, 735—737 (2009).
2
1
23
6
UV–Vis. l
(MeOH) nm (log e): 560 (3.6), 515.5 (3.8), 485 (3.6), 275.5 16) Noda N., Kogetsu H., Kawasaki T., Miyahara K., Phytochemistry, 31,
max
1
(
3.9). H-NMR (CDCl ) d: 1.57 (3H, s, H-6ꢅ), 1.68 (3H, s, H-5ꢅ), 1.83 (3H,
2761—2766 (1992).
3
d, Jꢂ8.0 Hz, H-4ꢁ), 1.86 (3H, s, H-5ꢁ), 2.53 (1H, m, H-2ꢅ), 2.64 (1H, m, H-
ꢅ), 5.14 (1H, t, Jꢂ8.0 Hz, H-3ꢅ), 6.04 (1H, dd, Jꢂ6.3, 5.1 Hz, H-1ꢅ), 6.95
1H, s, H-3), 6.97 (1H, m, H-3ꢁ) 7.18 (1H, s, H-6), 7.18 (1H, s, H-7), 12.39
17) Alvarez-Garcia R., Torres-Valencea J. M., Roman L. U., Hernandez J.
D., Cerda-Gracia-Rojas C. M., Joseph-Nathan P., Phytochemistry, 66,
639—642 (2005).
2
(
(
(
1
6
1
3
1H, s, OH-5), 12.43 (1H, s, OH-8), C-NMR (CDCl ) d: 12.2 (C-4ꢁ), 14.6 18) Ikeda Y., Ihida N., Fukuya C., Yokoyama K., Tabata M., Fukui H.,
3
C-5ꢁ), 18.1 (C-5ꢅ), 25.9 (C-6ꢅ), 33.0 (C-2ꢅ), 69.6 (C-1ꢅ), 111.7 (C-10),
11.9 (C-9), 117.9 (C-3ꢅ), 128.3 (C-2ꢁ), 131.5 (C-3), 132.6 (C-7), 132.8 (C-
), 136.1 (C-4ꢅ), 138.6 (C-3ꢁ), 149.0 (C-2), 166.5 (C-5), 166.8 (C-8), 167.1
C-1ꢁ), 177.4 (C-1), 178.9 (C-4).
Honda G., Chem. Pharm. Bull., 39, 2351—2352 (1991).
19) Afzal M., Tofeeq M., J. Chem. Soc. Perkin Trans. 1, 14, 1334 (1975).
20) Papageorgiou V. P., Digenis G. A., Planta Med., 39, 81—84 (1980).
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1216, 3156—3162 (2009).
(
Chiral Analysis of the Saponificates of 9 Chiral analysis was based on
18)
the manner reported previously. Briefly, the purified 9 (0.5 mg) was dis- 22) Majima R., Kuroda C., Acta Phytochim., 1, 43—65 (1922).
solved in MeOH (1 ml), and to this 1 M NaOH (3 ml) was added. After stir-