Indonesian medicinal plants. XXIV. Stereochemical structure of perseitol x K+ complex isolated from the leaves of Scurrula fusca (Loranthaceae).

Article abstract of DOI:10.1248/cpb.50.489

A complex of perseitol (D-glycero-D-galacto-heptitol) and K+ ions in a molar ratio of 20:1 was isolated from the leaves of Scurrula fusca (Loranthaceae), which has been traditionally used for the treatment of cancer in Sulawesi Island, Indonesia. The ster

Full text of DOI:10.1248/cpb.50.489

April 2002
Chem. Pharm. Bull. 50(4) 489—492 (2002)
489
Indonesian Medicinal Plants. XXIV.1) Stereochemical Structure of
Perseitol·K؉ Complex Isolated from the Leaves of Scurrula fusca
(Loranthaceae)
Takashi ISHIZU, Hendig WINARNO, Etsuji TSUJINO, Tetsuo MORITA, and Hirotaka SHIBUYA*
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Sanzo, 1 Gakuen-cho, Fukuyama, Hiroshima
729–0292, Japan. Received November 7, 2001; accepted January 8, 2002
A complex of perseitol (D-glycero-D-galacto-heptitol) and K؉ ions in a molar ratio of 20 : 1 was isolated from
the leaves of Scurrula fusca (Loranthaceae), which has been traditionally used for the treatment of cancer in Su-
lawesi Island, Indonesia. The stereochemical structure of the complex in H2O solution has been elucidated by use
of several kinds of NMR techniques. Furthermore, it has been found that the complex exhibits a potent in-
hibitory effect on [3H]-leucine incorporation for protein synthesis in Ehrlich ascites tumor cells in mice.
Key words Scurrula fusca; perseitol; potassium ion; complex; Indonesian medicinal plant; Loranthaceae
The parasite plant Scurrula fusca (BL.) G. DON. (Loran- breadth of perseitol (1a) at 1.67ϫ10Ϫ1 M solution in H2O by
thaceae), whose host-plant is Ficus riedelii MIQ. (Moraceae), adding regular amounts of KSCN as a potassium source to
is locally called benalu alus, and the decoction of its leaves adjust the molar ratio of 1a to the Kϩ ion from 26 : 1 to 10 : 1
has been traditionally used to cure cancer in Sulawesi Island, (Fig. 2). The proton signals of 1a gradually broadened with
Indonesia.
increases in the amount of Kϩ ions, from 26 : 1 to 20 : 1, and
After cooling, from the methanol extract of the dried the signal breadth was widest at a molar ratio of 20 : 1. These
leaves were obtained amorphous precipitates, which were pu- findings indicated that the proton motions of perseitol (1a)
rified by HPLC on a gel partition resin to afford a complex were restricted due to complex formation with the Kϩ ions,
(1) of perseitol and Kϩ ions in 1.0% yield. This paper deals and that 20 molecules of 1a formed a complex (1) with one
with the structural elucidation of the complex (1)2) and its bi-
ological activity.
The complex (1), an amorphous powder, shows physico-
chemical properties of a melting point at 174—177 °C and
specific rotation, [a]D Ϫ2.0° (H2O). Acetylation of the com-
plex (1) with pyridine and acetic anhydride furnished an ac-
etate (2), which was identified as perseitol heptaacetate3) by
comparison of physicochemical properties including its spe-
cific rotation [a]D Ϫ14.8° (MeOH) with those in the litera-
ture.3) Therefore, it has been found that the organic part of 1
is perseitol (D-glycero-D-galacto-heptitol).
Fluorescence X-ray analysis indicated that the complex (1)
contained Kϩ ions and did not have other metal ions. Atomic
absorption analysis showed that the complex (1) consisted of
perseitol (1a) and Kϩ ions in a molar ratio of 20 : 1.
In order to clarify the stereochemical structure of the com-
plex (1) in H2O solution, detailed NMR analysis of 1a and 1
was performed. The conformation of perseitol (1a) in H2O
solution was found to be a “zigzag” with a planar carbon
chain by using a Kurplus type equation concerning heptitol
between the coupling constants and dihedral angles.4—6) Fur-
thermore, no significant change in chemical shift or signal
1
breadth in the H- and 13C-NMR spectra of 1a was observed
at 1.67ϫ10Ϫ2—1.67ϫ100 M in H2O, which indicates that self-
assembly did not occur between those concentration ranges.
1
Next, we investigated changes in the H-NMR signal
RϭH Peresitol (D-glycero-D-galacto-heptitol) (1a)
RϭAc Peresitol heptaacetate (2)
1
Fig. 2. Changes in H-NMR Signals of Perseitol (1a) (10.61 mg, 1.67ϫ
10Ϫ1 M) by Adding KSCN
Fig. 1
To whom correspondence should be addressed. e-mail: shibuya@fupharm.fukuyama-u.ac.jp
© 2002 Pharmaceutical Society of Japan

490
Vol. 50, No. 4
mole of the Kϩ ion in H2O solution state. Thereafter, the
widening proton signals of 1a became sharper again, from
20 : 1 to 10 : 1, due to the untying of the complex (1).
seven hydroxyl protons was similarly observed as in the case
of a molar ratio of 20 : 1 (KSCN/perseitolϭ0.05) (Fig. 5).
As a result, it may be presumed that 20 molecules of per-
seitol (1a), while maintaining the “zigzag” conformation, as-
sociate with the surrounding Kϩ ions by the above-men-
tioned interaction (Fig. 5) to form the complex (1), which has
a spherical stereochemical structure in H2O solution. Fur-
thermore, the supposition that the spherical stereochemical
structure was composed of one layer of perseitol (1a) (Fig. 3)
1
The H-NMR spectrum of the complex (1) gave a similar
signal pattern to that of perseitol (1a), except for the signal
breadth, which indicates that the planar “zigzag” conforma-
tion of 1a is also maintained in the complex (1).
Figure 3 shows plots for the chemical shifts of methine
and methylene proton signals of perseitol (1a) against a
molar ratio of 1a and Kϩ ions. The result indicated that every
proton signal at a molar ratio of 20 : 1 shifted upfield most
prominently. Therefore, the stoichiometry in complex (1) of
perseitol (1a) with the Kϩ ion was confirmed to be 20 : 1.
Furthermore, changes in chemical shifts of the seven hy-
droxyl proton signals of 1.67ϫ10Ϫ1 M solution of perseitol
(1a) in DMSO-d6 were observed by adding regular amounts
of KSCN to adjust the molar ratio of Kϩ ion to 1a from 0 : 1
to 1 : 1 (Fig. 4). The 3-hydroxyl proton signal shifted down-
field most prominently, before the 2-, 4-, and 5-hydroxyl pro-
ton signals shifted. The downfield shifts of 1-, 6-, and 7-hy-
droxyl proton signals were not as prominent as those of other
hydroxyl proton signals. These facts indicated that the 3-hy-
droxyl group of perseitol (1a) most strongly attracted the Kϩ
ions in forming complex (1), while the 2-, 4-, and 5-hydroxyl
groups attracted these ions more strongly than the 1-, 6-, and
7-hydroxyl groups. The tendency of a downfield shift of the
1
was accounted for by the one-signal pattern in the H-NMR
spectrum of the complex (1).
The aqueous solution containing persitol (1a) and Kϩ ions
in a molar ratio of 20 : 1, in fact, gave complex (1) as an
amorphous powder, with physicochemical properties includ-
ing melting point and specific rotation which were identical
with those of complex (1) isolated from the dried leaves of
Scurrula fusca. However, the molecular weight of complex
(1) was not detected by several kinds of MS techniques, i.e.
EI, FAB, ESI, and TOF.
We also investigated the interaction between perseitol (1a)
and other metal ions, Naϩ from NaSCN, Mg2ϩ from MgCO3,
Ca2ϩ from Ca(SCN)2, and Agϩ from Ag2CO3, by the same
1H-NMR analysis. However, no combination afforded a simi-
lar complex in H2O solution. It was therefore considered that
a cation size of Kϩ ion might be the essential factor in the
formation of complex (1).
It should be noted that complex (1) exhibits an inhibitory
effect on protein synthesis in Ehrlich ascites tumor cells in
1
Fig. 3. Shifts in H-NMR Signals of Methine and Methylene Protons of
Perseitol (1a) (10.61 mg, 1.67ϫ10Ϫ1 M) by Adding KSCN
Fig. 5
1ab-H (᭛), 2-H (᭿), 3-H (᭺), 4-H (᭹), 5-H (᭞), 6-H (ϫ), 7a-H (ᮀ), 7b-H (᭝).
Fig. 6. Changes in Incorporation Rates of [3H]-Leucine into Ehrlich As-
cites Tumor Cells by Complex (1) and Perseitol (1a)
1
Fig. 4. Shifts in H-NMR Signals of Hydroxyl Protons of Perseitol (1a)
(10.61 mg, 1.67ϫ10Ϫ1 M) by Adding KSCN
The molecular weight of complex (1) was calculated to be 212 (1a).

April 2002
491
perseitol and Kϩ ions (20 : 1) (1, 2.5 g, 1.0% from the dried leaves). The
complex of perseitol and Kϩ ions (20 : 1) (1): an amorphous powder, mp
174—177 °C, [a]D Ϫ2.0° (cϭ3.0, in H2O at 25 °C). IR (KBr) cmϪ1: 3284.
1H-NMR (H2O) all protons of 1 appeared as broadening signal. d: 3.64 (1H,
3-H), 3.65 (1H, 7-Ha), 3.68 (2H, 1-Ha, 1-Hb), 3.75 (1H, 6-H), 3.79 (1H, 5-
H), 3.85 (1H, 7-Hb), 3.89 (1H, 4-H), 3.97 (1H, 2-H). 13C-NMR (D2O) dC:
66.2 (1-C), 66.2 (7-C), 71.2 (4-C), 72.1 (5-C), 72.2 (3-C), 73.2 (2-C), 73.9
1
(6-C). H-NMR (D2O) all protons of 1 appeared as broadening signal. d:
3.66 (1H, 3-H), 3.67 (1H, 7-Ha), 3.68—3.69 (2H, 1-Ha, 1-Hb), 3.77 (1H, 6-
H), 3.81 (1H, 5-H), 3.87 (1H, 7-Hb), 3.91 (1H, 4-H), 3.98 (1H, 2-H). 13C-
NMR (D2O) dC: 66.0 (1-C), 66.0 (7-C), 71.0 (4-C), 71.9 (5-C), 72.0 (3-C),
73.0 (2-C), 73.7 (6-C). FAB-MS m/z: 213 [highest peak, (perseitolϩH)ϩ].
Anal. Calcd for C140H320O140K·5H2O: C, 38.45; H, 7.61. Found: C, 38.23;
H, 7.53.
NMR Spectrum of Perseitol (1a): 1H-NMR (H2O) d: 3.64 (1H, dd,
3,4ϭ9.5 Hz, 3-H), 3.65 (1H, dd, J6,7aϭ5.8 Hz, J7a,7bϭ11.9 Hz, 7-Ha), 3.68
J
(2H, m, 1-Ha, 1-Hb), 3.75 (1H, ddd, J6,7bϭ2.7 Hz, 6-H), 3.79 (1H, dd,
5,6ϭ8.8 Hz, 5-H), 3.85 (1H, dd, 7-Hb), 3.90 (1H, dd, J4,5ϭ1.0 Hz, 4-H), 3.97
Fig. 7. Changes in Incorporation Rates of [3H]-Leucine into Ehrlich As-
cites Tumor Cells by the Various Mole Ratios of Perseitol (1a) and Kϩ Ions
(at 10Ϫ4 M)
J
(1H, dd, J2,3ϭ1.2 Hz, 2-H). 1H-NMR (DMSO-d6) d: 3.37 (1H, m, 7-Ha),
3.40 (2H, m, 1-Ha, 1-Hb), 3.45 (1H, m, 3-H), 3.45 (1H, m, 6-H), 3.57 (1H,
m, 5-H), 3.61 (1H, m, 7-Hb), 3.68 (1H, m, 4-H), 3.71 (1H, m, 2-H), 3.95
(1H, d, Jϭ7.6 Hz, 3-OH), 4.02 (1H, d, Jϭ7.9 Hz, 5-OH), 4.06 (1H, d,
Jϭ7.3 Hz, 4-OH), 4.12 (1H, d, Jϭ5.5 Hz, 2-OH); 4.34 (1H, dd, Jϭ5.5,
5.8 Hz, 7-OH), 4.40 (1H, d, Jϭ6.7 Hz, 6-OH), 4.43 (1H, dd, Jϭ5.5, 5.8 Hz,
1-OH). 13C-NMR (DMSO-d6) dC: 63.1 (7-C), 63.8 (1-C), 68.4 (5-C), 69.0
mice. Figure 6 shows plots for [3H]-leucine incorporation
rates into the Ehrlich ascites tumor cells7) by administrating
complex (1) and Kϩ ion free perseitol (1a).
1
(4-C), 69.5 (5-C), 70.0 (6-C), 71.5 (2-C). The H- and 13C-NMR spectra in
Furthermore, [3H]-leucine incorporation rates into the
Ehrlich ascites tumor cells in the presence of aqueous solu-
tions containing the various mole ratios of perseitol (1a) and
Kϩ ions were also investigated (Fig. 7). The solution of the
molar ratio 20 : 1 most strongly inhibited the protein synthe-
sis, with 40.3% inhibition at 10Ϫ4 M. However, complex (1)
and perseitol (1a) did not show cytotoxicity against L1210
cells.
D2O were identical with those in the literature.6)
Preparation of the Complex (1) from Perseitol (1a) and K؉ Ions
(20 : 1) A solution of perseitol (1a, 10.6 mg, 0.05 mmol) and KSCN (0.24
mg, 0.0025 mmol) in H2O (300 ml), when widening proton signals were ob-
served in the 1H-NMR spectrum, was lyophilized to give an amorphous
powder. The powder was purified by HPLC [column, TSK-GEL G-3000 PW
XL (TOSOH); elution, purified water] to afford the complex (1) of perseitol
and Kϩ ions (20 : 1) (1, 2.2 mg, 1.0%), which was identified with the com-
plex isolated from the leaves of Scurrula fusca by comparison of physico-
chemical data, including the specific rotation.
It was therefore concluded that complex (1) of perseitol
and Kϩ ions in a molar ratio 20 : 1 might be one of the bio-
logically active substances in the leaves of Scurrula fusca
(Loranthaceae).
The Interaction between Perseitol (1a) and Na؉, Mg2؉ Ca2؉, and Ag؉
Ions To the solution of perseitol (1a, 10.6 mg, 0.05 mmol) in H2O (300 ml)
was added regular amounts of NaSCN, MgCO3, Ca(SCN)2, and Ag2CO3, ad-
justing the molar ratio of 1a to the Naϩ, Mg2ϩ, Ca2ϩ and Agϩ ions from
1
26 : 1 to 10 : 1, respectively. In every case, the breadth of the H-NMR sig-
Experimental
nals assignable to 1a was not changed, indicating no formation of a complex
of perseitol (1a) and the metal cations in H2O solution.
1H- and 13C-NMR spectra were taken on a JEOL JMN-LA 500 (500 MHz
1
1
for H and 125 MHz for 13C) spectrometer. H and 13C chemical shifts (d)
are quoted in ppm relative to sodium 2, 2-dimethyl-2-silapentane-5-sulfate
(DSS, dϭ0) in H2O and tetramethylsilane (TMS, dϭ0) in DMSO-d6 as an
internal standard, and coupling constants are given in Hz. The measuring
temperature was 25.0 °C. The following abbreviations were used: singlet (s),
doublet (d), double doublet (dd), double double doublet (ddd), triplet (t),
quartet (q), multiplet (m), and broad signal (br).
Acetylation of the Complex (1) A solution of complex (1, 143 mg) in
pyridine (0.5 ml) was treated with Ac2O (1.0 ml) and left at room tempera-
ture for 2 h. The solvent was removed in vacuo to give a residue, which was
purified by column chromatography on silica gel and eluted with n-hexane-
ethyl acetate (3 : 2) to give perseitol heptaacetate (2, 150 mg, 44%). Perseitol
heptaacetate (2): colorless needles from diethyl ether, mp 119—120 °C, [a]D
Ϫ14.8° (cϭ1.2, in MeOH at 25 °C). IR (KBr) cmϪ1: 1749. 13C-NMR
(CDCl3) dC: 20.5, 20.6, 20.7, 20.8 (totally 7C, –OCOCH3ϫ7), 61.8, 62.1 (1-
C, 7-C), 66.5, 67.1, 67.5, 67.6, 67.9 (4-, 3-, 5-, 2-, 6-C), 169.5, 169.8, 170.1,
170.3, 170.4 (totally 7C, –OCOCH3ϫ7). FAB-MS m/z: 507 (MϩH)ϩ. High-
resolution FAB-MS m/z: Calcd for C21H31O14: 507.1713. Found: 507.1711
(MϩH)ϩ. The 1H-NMR spectrum was identical with that in the literature.3)
Determination of [3H]-Leucine Incorporation Rate Samples at the in-
dicated concentrations were dissolved into the suspension of [3H]-leucine
(18.5 kBq/dish) in Ehrlich ascites tumor cells in mice which had been incu-
bated in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 5.0 mM glu-
cose and 2.0% albumin for 30 min. The incubated Ehrlich ascites tumor cells
were washed twice with ice-cold 50 mM Tris–HCl buffer (pH 7.4) and ho-
mogenized in 0.5 ml of the same buffer for 10 s by sonication. The ho-
mogenate was spotted on a strip of filter paper (1.5ϫ1.5 cm, Whatmann,
3MM) and treated by the TCA-precipitate method to obtain the
radioactivity.8,9)
HPLC was carried out with a Shimadzu LC-10AD. Melting points were
measured on a Yanagimoto micro hot-stage melting point apparatus without
correction. Optical rotations were measured with a JASCO DIP-360 auto-
matic polarimeter. FAB-MS was obtained with a JEOL JMS-DX 300L spec-
trometer using glycerol as a matrix. Fluorescence X-ray was obtained with a
Rigaku-Kevex energy-dispersive X-ray spectrometer (ultra-trace system).
Atomic absorption was recorded on a Shimadzu atomic absorption flame
emission spectrophotometer AA-670.
Plant Material Scurrula fusca (Loranthaceae) was collected in the Poso
area of Sulawesi Island, Indonesia, in August, 1992, and was identified in the
Herbarium Bogoriense, Research Centre for Biology-LIPI, Indonesia.
Sample Tube for NMR Measurements A double sample tube system
for measurements in H2O was used. Inside: an NMR sample tube (f 3 mm)
contained D2O (200 ml) for a field-frequency lock, and DSS (0.1 mg) was
used as the internal reference. Outside: an NMR sample tube (f 5 mm) con-
tained H2O (300 ml) as the solvent, plus sample (0.05 mmol).
Isolation of the Complex (1) The dried leaves (250 g) of Scurrula fusca
(Loranthaceae) were extracted three times with methanol (each 500 ml)
under reflux for 3 h. The combined extract solution was filtered, and the fil-
trate was cooled to room temperature to give precipitates (12.5 g). The pre-
cipitates were collected and stirred in purified water for 24 h. The resulting
insolubility was removed by filtration and the filtrate was lyophilized to give
an amorphous powder, which was purified by HPLC [column, TSK-GEL G-
3000 PW XL (TOSOH); elution, purified water] to afford the complex (1) of
Acknowledgements This work was partially supported by Grants-in-
Aid for International Scientific Research (No. 02041054 and No. 04041069)
from the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
References
1) Part XXIII: Ohashi K., Bohgaki T., Matsubara T., Shibuya H., Chem.
Pharm. Bull., 48, 433—435 (2000).

492
2) Preliminary communication: Ishizu T., Tsujino E., Winarno H., Ohashi
Vol. 50, No. 4
6) Lewis D., J. Chem. Soc. Perkin Trans II, 1986, 467—470.
K., Shibuya H., Tetrahedron Lett., 42, 6887—6889 (2001).
3) Moore R. E., Barchi J. J., Jr., Bartolini G., J. Org. Chem., 50, 374—
379 (1985).
4) Karplus M., J. Chem. Phys., 30, 11—15 (1959).
5) Haasnoot C. A. G., Leeuw F. A. A. M. DE., Altona C., Tetrahedron,
36, 2783—2792 (1980).
7) Morita T., Sakata K., Kanagawa A., Ueki H., Biol. Pharm. Bull., 17,
577—580 (1994).
8) Ashyby P., Bennett D. P., Spencer I. M., Robinson D. S., Biochem. J.,
176, 865—872 (1978).
9) Sera M., Tanaka K., Morita T., Ueki H., Arch. Biochem. Biophys., 279,
291—297 (1990).