dertaken for the isolation of active constituents from the crude Therefore, it could be concluded that the cytotoxic effect of the
saponin fraction of the extract. By the serial chromatography of root extract of P. grandiflorum on cultured human tumor cell
the saponin fraction, eight triterpenoid saponins (1 ± 8) were lines might be attributed predominantly to the saponin fraction
isolated and their chemical structures were established by com- (1.0 ± 2.0% of the extract), particularly to the components, platy-
parison of their spectral data with published ones. Among the codin D2 (6), deapioplatycodin D (7), and platycodin D (8).
isolates, compound 1 (Fig.1) was found to be a new component
which had not been reported previously.
Materials and Methods
Compound 1, an amorphous powder, [a]D20: ±22 (c, 0.1 in EtOH),
possessed the molecular formula C64H104O34 as determined by Extraction and isolation: The species P. grandiflorum cultivated
MALDI-TOF-MS (m/z = 1439: [M + Na]+; Voyager; PE Biosys- for three years in Kangwon Province, Korea was harvested in Sep-
tems USA). The 1H-NMR and 13C-NMR spectral data of 1 indicat- tember 2000. The roots were cut into slices and dried. For future
ed that it had the sapogenin, 2b,3b,16a,23,24-pentahydroxy- reference, a voucher specimen (herbarium No. KM-00 024) has
olean-12-en-28-oic acid and oligosaccharide moieties at C-3 been preserved at the Herbarium of Kookmin University, Insti-
and C-28.
tute of Forest Science, Korea.
Particularly, the 13C-NMR spectrum of 1 is quite imposable with The dried roots (1.0 kg) were soaked in methanol at room tem-
that of platycoside E (2; Fig.1) except for some typical signals perature for 7 days. Concentration of the solvent gave 220 g of a
(d = 111.7, 81.0 and 65.9) due to the apiose moiety of 2. These re- brown syrupy MeOH extract which was suspended in 2.2 L of
sults suggested that l be a congener of 2 (platycoside E), which H2O and poured into a Diaion HP-20 column (é = 5.0100 cm),
has been found recently in this species [7]. By the scrutiny of which was stabilized with H2O. The column was eluted with ad-
the spectral data of 1 with those of 2, the chemical structure of ditional 10 L of 20% MeOH. The eluate was combined and con-
1 was established to be 3-O-b-D-glucopyranosyl-(1®6)-b-D-glu- centrated under reduced pressure to give 190 g of syrupy residue
copyranosyl-(1®6)-b-D-glucopyranosyl-2b,3b,16a,23,24-pentahy- (Fr. A). The Diaion HP-20 column was further washed with addi-
droxyolean-12-ene-28-oic acid 28-O-b-D-xylopyranosyl-(1®4)- tional 10 L of 85% MeOH. The eluate was concentrated under re-
a-L-rhamnopyranosyl-(1®2)-a-L-arabinopyranoside, which bis duced pressure to give 24 g of brown powder (Fr. B). The column
the deapio analogue of platycoside E (2). Compound 2 was was finally washed out with MeOH. The washings were concen-
found to be converted slowly to 1 by mild acid hydrolysis in trated under reduced pressure to give 0.8 g of oily residue (Fr. C)
0.1 N HCl.
[10]. The crude MeOH extract of the root and each fraction ob-
tained from the MeOH extract by the Diaion HP-20 column chro-
All of the isolated saponins were examined for the cytotoxicity matography (Fr. A ± Fr. C) were evaluated for cytotoxic effects on
against five cultured human tumor cell lines [8], the currently the cultured human tumor cell lines. Among the tested, only the
used cell lines in the National Cancer Institute (USA) for their in Fr. B exhibited a moderate cytotoxicity upon each of the tested
vitro anti-cancer drug screening program, i.e., A549 (non-small cell lines. Therefore, the Fr. B was purified by repeated prepara-
cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (cen- tive HPLC (Futecs NS-3000i system equipped with GROM-SIL
tral nerve system) and HCT-15 (colon), in vitro [9]. When the ex- 120 ODS-4 HE column; 250 cm20 mm) with 26% acetonitrile
amined tumor cells were exposed continuously to each of com- in 20 mM KH2PO4 as eluent, which led to the isolation of eight
pounds 1 ± 8 for 48 hours, their proliferation was significantly triterpenoidal saponins (1 ± 8), i.e., 22 mg of 1 (tR = 12.8 min,
decreased in a dose-dependent manner. The ED50 values of each GROM-SIL 120 ODS-5, ST column; 250 cm5 mm, flow rate 0.7
component (1 ± 8) on the proliferation of the five human tumor mL/min), 150 mg of 2 (tR = 14.4 min), 15 mg of 3 (tR = 24.3
567
cells are summarized in Table 1.
min), 28 mg of 4 (tR = 27.9 min), 14 mg of 5 (tR = 37.3 min),
20 mg of 6 (tR = 65.3 min), 24 mg of 7 (tR = 74.7 min) and
42 mg of 8 (tR = 77.4 min). The structure of the new saponin 1
was determined on the basis of spectral analysis and chemical
evidences. The detailed chemical shifts of 1 in the 13C-NMR (in
pyridine-d5) spectrum are as follows (the data in the parenthesis
are those for the corresponding signals of 2 [10]); C1:45.3 (45.3),
C2:68.7 (68.8), C3:88.8 (88.8), C4:48.2 (48.2), C5:47.6 (47.6),
C6:19.4 (19.5), C7:33.6 (33.5), C8:40.5 (40.6), C9:45.0 (45.0),
C10:38.0 (38.0), C11:24.1 (24.1), C12:123.1 (123.3), C13:144.4
(144.7), C14:42.5 (42.5), C15:36.1 (36.2), C16:74.0 (73.9),
C17:49.7 (49.7), C18:41.6 (41.7), C19:47.2 (47.2), C20:31.0
(31.0), C21:36.1 (36.1), C22:32.2 (32.2), C23:63.5 (63.7),
C24:67.3 (67.3), C25:19.2 (19.2), C26:17.7 (17.7), C27:27.1
(27.1), C28:176.0 (176.1), C29:33.4 (33.3), C30:24.8 (24.8), iG1
(inner glucose): 106.1 (106.1), iG2:74.9 (74.9), iG3:78.4 (78.5),
iG4:72.4 (72.3), iG5:76.5 (76.6), iG6:70.2 (70.7), cG1 (central
glucose): 105.0 (105.0), cG2:75.4 (75.4), cG3:78.5 (78.4),
cG4:71.1 (71.3), cG5:77.2 (77.2), cG6:70.2 (70.2), tG1 (terminal
glucose): 105.7 (105.6), tG2:75.2 (75.2), tG3:78.7 (78.7),
Fig. 1 Structures of saponins 1 and 2.
Letter¼ Planta Med 2005; 71: 566±568