300 J ournal of Natural Products, 1998, Vol. 61, No. 2
Notes
genol B 3-O-â-D-glucopyranosyl(1f2)-â-D-glucopyrano-
syl(1f2)-6′-O-methyl-â-D-glucuronopyranoside.
Lathyrus saponin methyl ester (3) was obtained as
an amorphous white powder: mp 273-279 °C; [R]20
D
+24.6° (c 0.53, MeOH); IR (KBr) νmax 3414, 2949, 1744,
Exp er im en ta l Section
1638, 1074 cm-1 1H NMR (pyridine-d5, 500 MHz) δ
;
Gen er al Exper im en tal P r ocedu r es. Melting points
were measured on a Mitamura-Riken apparatus and are
uncorrected. The optical rotations were determined on
a Rudolph Autopol III automatic polarimeter. The IR
spectra were obtained on a J ASCO FT/IR-5300 spec-
trometer. The FAB mass spectrum was obtained in a
3-nitrobenzyl alcohol matrix in a positive-ion mode on
a VG-VSEQ spectrometer. The NMR spectra were
measured in pyridine-d5 on a Bruker AMX-500 instru-
ment, and the chemical shifts were referenced to TMS.
GC analysis was performed with a Hewlett-Packard
5890 Series II gas chromatograph equipped with an H2
flame ionization detector. The column was an HP-5
capillary column (30 m × 0.32 mm × 0.25 µm). Condi-
tions: column temperature, 200 °C for TMS ethers of
thiazolidine derivatives and 150 °C for TMS ethers of
methyl glycopyranosides; injector and detector temper-
ature, 290 °C; He flow rate, 30 mL/min. TLC was
performed on silica gel 60F254 (Merck) and cellulose
plates (Art No. 5716, Merck).
0.73, 0.95, 0.98, 1.21, 1.24, 1.28, 1.37 (21H, all s, 7 ×
Me), 2.39 (1H, brd, J ) 12.8 Hz, H-18), 3.37 (1H, t-like,
J ) 10.5 Hz, H-24), 3.43 (1H, dd, J ) 4.4, 7.7 Hz, H-3),
3.65 (1H, brdt, J ) 9.5 Hz, H-5′′′), 3.74 (3H, s, OMe),
3.74 (1H, m, H-22), 3.97 (1H, m, H-5′′), 4.57 (1H, d, J )
10 Hz, H-5′), 4.67 (1H, t, J ) 9.5 Hz, H-3′), 5.05 (1H, d,
J ) 7.7 Hz, H-1′), 5.22 (1H, d, J ) 7.6 Hz, H-1′′′), 5.30
(1H, brs, H-12), 5.58 (1H, J ) 7.8 Hz, H-1′′); 13C NMR
(pyridine-d5, 125.8 MHz) δ 90.7 (C-3), 122.4 (C-12), 144.9
(C-13), 75.6 (C-22), 63.4 (C-24), 104.7 (C-1′), 82.2 (C-2′),
77.2 (C-3′), 72.1 (C-4′), 77.2 (C-5′), 170.3 (C-6′), 52.2
(OMe), 103.5 (C-1′′), 85.9 (C-2′′), 77.6 (C-3′′), 71.5 (C-
4′′), 79.5 (C-5′′), 62.8 (C-6′′), 106.8 (C-1′′′), 77.0 (C-2′′′),
77.6 (C-3′′′), 69.4 (C-4′′′), 78.1 (C-5′′′), 61.2 (C-6′′′);
positive FAB MS, see text.
Acid Hyd r olysis of 3. Saponin 3 (8 mg) was
refluxed with 5% HCl in 60% aqueous dioxane (10 mL)
for 2 h. The resulting solution was evaporated under
reduced pressure, and the hydrolysate was extracted
with ether. The ether extract was evaporated to yield
soyasapogenol B, which was identified by direct com-
parison with an authentic sample.8 The H2O layer was
neutralized with Ag2CO3 and filtered, and the filtrate
was concentrated under reduced pressure. The residue
was compared with standard sugars by cellulose TLC
[pyridine-EtOAc-HOAc-H2O (36:36:7:21)], which in-
dicated the sugars to be glucuronic acid and glucose.
P la n t Ma ter ia l. The whole plant of L. japonicus was
collected in May 1996, Dukjeok island in Kyungki
Province, Korea, and authenticated by one of us (K.B.).
A voucher specimen (CNU 1146) was deposited in the
Department of Pharmacognosy, Chungnam National
University.
Extr a ction a n d Isola tion . Fresh whole plant (5 kg)
of L. japonicus was dried, and the dry plant material
(1.3 kg) was extracted three times with 80% MeOH at
room temperature. The MeOH extract was evaporated
to dryness, and the dry residue was partitioned in
succession between H2O and n-hexane, CHCl3, and then
n-BuOH, affording 23.6, 11.4, and 85 g of the respective
extracts. A portion of the BuOH fraction (37 g) was
passed through a Sephadex LH-20 column eluting with
80% MeOH to give subfractions rich in flavonoids (14.2
g) and saponins (21 g). The subfraction rich in fla-
vonoids was subjected to SiO2 column chromatography.
Elution with EtOAc saturated with H2O with increasing
amounts of MeOH and then MeOH gave astragalin (7
mg) and an MeOH-soluble portion. The MeOH-soluble
portion was purified by a column with Sephadex LH-
20 eluted with 50% MeOH to afford isoquercitrin (15
mg) and a mixture of compounds, which was further
purified with Toyopearl HW 40F eluted with H2O, 30%
EtOH, and then MeOH to yield kaempferol 3-O-rutino-
side (5 mg) and rutin (6 mg). The subfraction rich in
saponins was methylated with CH2N2 to yield methy-
lated saponin fraction. A portion of the methylated
saponin fraction (5.7 g) was subjected column chroma-
tography on SiO2 eluted with EtOAc saturated with H2O
with increasing amounts of MeOH to give compound 1
(7 mg) and a mixture of compounds 2 and 3 (100 mg).
The mixture of 2 and 3 was repeatedly chromatographed
on LiChroprep RP-18 with 80% MeOH as eluent to yield
saponin 2 (50 mg) and then 3 (30 mg). The known
P a r tia l Acid Hyd r olysis of Sa p on in 3. Saponin 3
(7 mg) was refluxed with HCl (one drop) in MeOH (3.6
mL) for 10 min, and on usual workup two prosapogenins
(soyasapogenol B 3-O-â-D-glucuronopyranoside methyl
ester and 1) were identified by TLC [CHCl3-MeOH (6:
1)] with authentic samples.7 To identify sugar compo-
nents, the reaction mixture was evaporated under a N2
stream and dried in vacuo. The residue was dissolved
in pyridine (0.05 mL), and then the solution was
trimethylsilylated with TMS-HT (0.1 mL) at 60 °C for
30 min. After the addition of n-hexane and water, the
n-hexane layer was removed and analyzed by GC. The
retention times (tR) of the peaks were 26.7 and 29.4 min
for methyl glucopyranoside.
Refer en ces a n d Notes
(1) Perry, L. M. Medicinal Plants of East and Southeast Asia:
Attributed Properties and Uses; The MIT Press: Cambridge,
1980; p 229.
(2) Bell, E. A. In Comparative Phytochemistry; Swain, T., Ed.;
Academic Press: London, 1966; Chapter 12, pp 195-209.
(3) Hatanaka, S.-I.; Kaneko, S. Phytochemistry 1977, 16, 1041-
1042.
(4) Hatanaka, S.-I.; Kaneko, S. Phytochemistry 1978, 17, 2027.
(5) Kang, S. S.; Kim, J . S.; Kim, Y. H.; Choi, J . S. J . Nat. Prod.
1997, 62, 1060-1062.
(6) Kang, S. S.; Cordell, G. A.; Soejarto, D. D.; Fong, H. H. S. J .
Nat. Prod. 1985, 48, 155-156.
(7) Kang, S. S.; Lee, Y. S.; Lee, E. B. Arch. Pharm. Res. 1988, 11,
197-202.
(8) Kang, S. S.; Lim, C.-H.; Lee, S. Y. Arch. Pharm. Res. 1987, 10,
9-13.
1
compounds were identified by TLC and H and 13C NMR
(9) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35,
501-506.
data as well as by direct comparison with authentic
samples.6,7
NP970449E