236 J ournal of Natural Products, 1999, Vol. 62, No. 2
Ye et al.
Exp er im en ta l Section
between H-1′′ (δ 6.21) of the inner glucose and C-28 (δ
176.5), as well as between H-1′′′ (δ 4.96) of the central
glucose and C-6′′ (δ 69.2) of the inner glucose. Moreover,
the HMBC spectrum revealed a correlation cross-peak
between H-1 (δ 5.84) of the terminal rhamnose and C-4′′′
(δ 78.3) of the central glucose. All available evidence
suggested that 3 was a new saponin whose structure could
be deduced as 3-O-â-D-glucopyranosyl bayogenin 28-O-R-
L-rhamnopyranosyl(1f4)-â-D-glucopyranosyl(1f6)-â-D-glu-
copyranosyl ester.
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter using a
sodium lamp operating at 589 nm in MeOH. 1H NMR (600,
500, or 400 MHz), 13C NMR(150, 125 or 100 MHz), and 2D
NMR spectra were determined on Varian Unity INOVA-600,
-500 and J EOL J NM-EX 400 spectrometers. Fast-atom
bombardment mass spectra (FABMS) were recorded in NBA
matrix in the positive ion mode on a Finnigan MAT TSQ 7000
spectrometer. Column chromatography was carried out with
ODS (10-40 µm, Merck). TLC was conducted on silica gel 60
F254 and RP-18 F254 S plates (Merck).
P la n t Ma ter ia l. The roots of P. patens var. multifida
(Pritz.) S. H. Li et Y. H. Huang were collected at Chifun, the
Inner Mongolia Autonomous Region of China in May 1994,
and authenticated by Dr. Xian-Min Cui. A voucher specimen
has been deposited in the Herbarium of China Pharmaceutical
University.
Extr a ction a n d Isola tion . Dried roots of P. patens var.
multifida (800 g) were extracted with MeOH (1500 mL × 3, 2
h each). The extracts were combined, evaporated, dissolved
in H2O and defatted with CHCl3 (H2O-CHCl3, 1:1). The
defatted extract was partitioned with n-BuOH and the n-
BuOH layer was evaporated to dryness. The dark brown
residue was redissolved in a small amount of MeOH and
subjected to column chromatography on D101 resin (ca. 1000
g) using EtOH-H2O as eluant. Fractions were collected and
checked by TLC (CHCl3-MeOH-H2O, 6.5:3.5:1, lower layer).
Fractions 8-11 (2.2 g) containing a mixture of saponins 3-5
were separated using a reversed-phase C18 column (ca. 100 g,
10-40 µm) eluted with MeOH-H2O (30:70-45:55) to yield
pure compounds 5 (105 mg), 4 (60 mg), and 3 (200 mg).
Fractions 20-24 (1.6 g) containing a mixture of 1, 2 and two
known triterpene glycosides were separated by column chro-
matography eluted with MeOH-H2O (40:60 f 60:40) to yield
pure compounds 1 (85 mg), 2 (50 mg), bayogenin 3-O-â-D-
glucopyranoside (6, 40 mg) and hederagenin 3-O-â-D-gluco-
pyranosyl(1f2)-R-L-arabinopyranoside (7, 70 mg).
Acid Hyd r olysis of 1-5. Solutions of compounds (10 mg
each) in 5% HCl-EtOH (5 mL) were heated in a boiling water
bath for 8 h. The reaction mixture was diluted with H2O and
neutralized with Ag2CO3. The neutralized solution was ex-
tracted with EtOAc. The EtOAc layer was evaporated and
chromatographed on Sephadex LH-20 using MeOH as eluant
to yield the aglycon. The aglycon was analyzed by mmp, IR,
and NMR and compared with an authentic sample. The H2O
layers were analyzed by HPTLC (n-BuOH-HOAc-H2O,
4:1:1) to reveal the presence of glucose, galactose, rhamnose,
or arabinose.
The FABMS of 4 showed a [M + Na]+ ion at m/z 1273,
consistent with a molecular formula of C60H98O27. Com-
parison of the NMR spectral data of 4 with those of 1-3
showed a difference in the aglycon portion. The most
1
significant feature of the H NMR spectrum of 4 was the
presence of seven methyl singlets in addition to a doublet
due to the rhamnose residue. The aglycon of 4 was then
18
identified as oleanolic acid
and confirmed by acidic
1
hydrolysis. The H and 13C NMR spectra (Tables 1-3) of 4
revealed the presence of five sugar residues. The carbon
signals at δ 88.5 (C-3) and at δ 176.1 (C-28) further
suggested that both positions were glycosylated. Careful
analysis of the NMR data revealed that the compound had
the same glycosidic chains at C-3 and C-28 positions as in
1 and 3, respectively. The HMBC and ROESY experiments
also supported the above observation. Thus, the HMBC
spectrum of 4 exhibited correlation signals between H-1
(δ 4.99) of galactose and C-3 (δ 88.5) of the aglycon, as well
as between H-1 (δ 5.23) of glucose and C-2 (δ 81.4) of
galactose. Moreover, correlations were also demonstrated
between H-1′′ (δ 6.20) of the inner glucose and C-28 (δ
176.1), between H-1′′′ (δ 4.96) of the central glucose and
C-6′′ (δ 68.7) of the inner glucose, as well as between H-1
(δ 5.82) of the rhamnose and C-4′′′ (δ 77.6) of the central
glucose. Further information was derived from the results
of an ROESY experiment. The ROESY spectrum revealed
correlation peaks between H-1 of glucose (δ 5.23) and H-2
of galactose (δ 4.54), as well as between H-1 of rhamnose
(δ 5.82) and H-4′′′ of the central glucose (δ 4.37). All the
available evidence led to the conclusion that 4 had the
structure of 3-O-â-D-glucopyranosyl(1f2)-â-D-galactopyra-
nosyl oleanolic acid 28-O-R-L-rhamnopyranosyl(1f4)-â-D-
glucopyranosyl(1f 6)-â-D-glucopyranosyl ester.
The FABMS of 5 displayed a quasimolecular ion [M +
Na]+ at m/z 1451, consistent with a molecular formula of
C66H108O33. The 13C and DEPT NMR spectra of 5 contained
66 signals, of which 36 belonged to the saccharide portion
and 30 to the triterpene aglycon. Acid hydrolysis of 5
afforded hederagenin, glucose, galactose, and rhamnose.
Further comparison of 13C NMR data for the saccharide
portions indicated that 5 had the same glycosidic chains
at C-3 and C-28 positions as 2 and 4, respectively. Upon
alkaline hydrolysis with 5 N NH4OH, 5 yielded a prosa-
pogenin which was identical with 2 by direct comparison
of NMR data. The oligosaccharide structure was confirmed
by 2D NMR. Thus, in the HMBC spectrum of 5, correlation
cross-peaks between H-1 (δ 5.23) of glucose and C-2 (δ 82.1)
of galactose, between H-1′ (δ 4.95) of glucose and C-6 (δ
69.1) of galactose, between H-1′′′ (δ 4.96) of the central
glucose and C-6′′ (δ 69.0) of the inner glucose, as well as
between H-1 (δ 5.82) of rhamnose and C-4′′′ (δ 78.1) of the
central glucose were displayed. Hence, the structure of 5
was established as 3-O-[â-D-glucopyranosyl(1f2)][â-D-glu-
copyranosyl(1f6)]-â-D-galactopyranosyl hederagenin 28-
O-R-L-rhamnopyranosyl(1f4)-â-D-glucopyranosyl(1f6)-â-
D-glucopyranosyl ester.
Alk a lin e Hyd r olysis of 1-5. Each compound (30 mg) was
refluxed with 5 N NH4OH in 50% EtOH (10 mL) for 15 h. After
cooling, the reaction mixture was neutralized with 2N HCl and
extracted with n-BuOH. The n-BuOH layer was evaporated
to dryness to give a residue, which was chromatographed on
a C18 column (10-40 µm) eluted with MeOH-H2O to yield the
prosapogenin. The prosapogenins were identified by NMR
spectra and/or by direct comparison with authentic samples.
D
Com p ou n d 1: Amorphous powder; mp 239-241 °C; [R]20
+20.8° (c 0.19, MeOH); IR νmax (KBr) 3400 (OH), 1730 (COOR),
1640 (CdC) cm-1; 1H NMR (500 MHz, pyridine-d5) δ 5.42 (1H,
brs, H-12), 4.35 (1H, overlapped, H-23a), 4.13 (1H, overlapped,
H-3), 3.73 (1H, overlapped, H-23b), 3.15 (1H, brd, J ) 9.8 Hz,
H-18), 1.17 (3H, s, Me), 1.09 (3H, s, Me), 1.06 (3H, s, Me), 0.90
1
(3H, s, Me), 0.86 (3H, s, Me), 0.85 (3H, s, Me); H NMR data
of the saccharide residues, see Table 3; 13C NMR (125 MHz,
pyridine-d5), see Tables 1 and 2; FABMS m/z 981 [M + Na]+.
D
Com p ou n d 2: Amorphous powder; mp 208-212 °C; [R]20
+21.2° (c 0.15, MeOH); IR νmax (KBr) 3410 (OH), 1697 (COOH),
1630 (CdC) cm-1; 1H NMR (400 MHz, pyridine-d5) δ 5.44 (1H,
brs, H-12), 4.36 (1H, overlapped, H-23a), 4.15 (1H, overlapped,
H-3), 3.74 (1H, overlapped, H-23b), 3.26 (1H, brd, J ) 9.7 Hz,
H-18), 1.20 (3H, s, Me), 1.06 (3H, s, Me), 0.98 (3H, s, Me), 0.92
1
(3H, s, Me), 0.92 (3H, s, Me), 0.90 (3H, s, Me); H NMR data