Triterpene glycosides from Pulsatilla chinensis

Article abstract of DOI:10.1023/A:1021381308598

Four triterpene glycosides were isolated from the roots of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae). Two new glycosides, chinensiosides A (1a) and B (2), were identified as 3-O-[α-L-rhamnopyranosyl-(1→ 2)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1→4) -β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-3β, 23-dihydroxylup-20(29)-en-28-oic acid and 3-O-{α -L-rhamnopyranosyl-(1→-2)-[β-D-glucopyranosyl-(1→4)] -α-L-arabinopyranosyl}-28-O-[α-L-rhamnopyranosyl-(1→4) -β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-3β, 23-dihydroxylup-20(29)-en-28-oic acid. The other two glycosides were identified as previously known hederasaponin C (3) from Hedera helix and glycoside III (4) from Pulsatilla cernua.

Full text of DOI:10.1023/A:1021381308598

Russian Chemical Bulletin, International Edition, Vol. 51, No. 10, pp. 1945—1950, October, 2002
1945
Triterpene glycosides from Pulsatilla chinensis
ꢀ
L. I. Glebko , N. P. Krasovskaj, L. I. Strigina, K. P. Ulanova, V. A. Denisenko, and P. S. Dmitrenok
Pacific Institute of Bioorganic Chemistry, Russian Academy of Sciences,
159 prosp. 100ꢀletiya Vladivostoka, 690022, Vladivostok, Russian Federation,
Fax: +7(423 2) 314 050. Eꢀmail: piboc@stl.ru
Four triterpene glycosides were isolated from the roots of Pulsatilla chinensis (Bunge) Regel
(Ranunculaceae). Two new glycosides, chinensiosides A (1a) and B (2), were identified as
3ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ(1→2)ꢀαꢀLꢀarabinopyranosyl]ꢀ28ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ
(1→4)ꢀβꢀDꢀglucopyranosylꢀ(1→6)ꢀβꢀDꢀglucopyranosyl]ꢀ3β,23ꢀdihydroxylupꢀ20(29)ꢀenꢀ28ꢀoic
acid and 3ꢀOꢀ{αꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ[βꢀDꢀglucopyranosylꢀ(1→4)]ꢀαꢀLꢀarabinopyranoꢀ
syl}ꢀ28ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ(1→4)ꢀβꢀDꢀglucopyranosylꢀ(1→6)ꢀβꢀDꢀglucopyranosyl]ꢀ
3β,23ꢀdihydroxylupꢀ20(29)ꢀenꢀ28ꢀoic acid. The other two glycosides were identified as previꢀ
ously known hederasaponin C (3) from Hedera helix and glycoside III (4) from Pulsatilla
cernua.
Key words: Pulsatilla chinensis, Ranunculaceae, triterpene glycosides, glycosides of
1
23ꢀhydroxybetulinic acid, chinensiosides A and B, H and 13C NMR spectra, MALDIꢀTOF
mass spectra.
Chinese anemone Pulsatilla chinensis (Bunge) Regel
(Ranunculaceae) is one of the most wellꢀknown means of
alternative Oriental medicine. The roots of this plant are
used to treat amebic dysentery, malaria, vaginal trichomoꢀ
niasis, and bacterial infections. During previous studies of
P. chinensis, a new lupanic triterpenoid, viz., pulsatillic
acid, 23ꢀhydroxybetulinic acid, and three new lupaneꢀ
type triterpene glycosides, viz., pulsatillosides А, B, and C,
were isolated from plants that grow in China,1,2 and two
new and several known oleananeꢀtype glycosides and two
known lignanes were isolated from plants that grow in
Japan.3 The glycoside fraction of the methanol extract
from the roots of P. chinensis exhibits a cytotoxic activity
against the HLꢀ60ꢀcells of human leukemia.3
HPLC using the RPꢀ18 reversed phase, aqueous MeCN
as the eluent, and refractometric detection. As a result, a
new glycoside called chinensioside A (1a) and known
hederasaponin C 7 (3) were isolated from fraction A and a
new glycoside called chinensioside B (2) and previously
known8 glycoside III (4) were isolated from fraction B.
A brief communication on the structures of these comꢀ
pounds has been published.9
To continue the studies of triterpene metabolites of
the Pulsatilla Mill. plants growing in the Russian Far
East,4—6 we studied the triterpene composition of the
roots of P. chinensis.
Results and Discussion
Usual techniques including ethanol extraction and
chromatographic separation on silica gel columns using
gradient elution with CHCl3—EtOH and CHCl3—MeOH
solvent mixtures resulted in the isolation of two glycoside
fractions А and B, which were first considered to be indiꢀ
vidual compounds. Examination of the 13C NMR spectra
showed that each fraction is a mixture of two glycosides
that are difficult to separate. The ultimate isolation and
purification of individual compounds were performed by
R1 = αꢀLꢀRhapꢀ(1→2)ꢀαꢀLꢀArap→ (1a,b, 3),
R
R
R
2 = αꢀLꢀRhapꢀ(1→4)ꢀβꢀDꢀGlcpꢀ(1→6)ꢀβꢀDꢀGlcp→ (1a, 3),
2 = H (1b);
1 = αꢀLꢀRhapꢀ(1→2)ꢀ[βꢀDꢀGlcpꢀ(1→4)]ꢀαꢀLꢀArap→,
R2 = αꢀLꢀRhapꢀ(1→4)ꢀβꢀDꢀGlcpꢀ(1→6)ꢀβꢀDꢀGlcp→ (2, 4)
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1792—1797, October, 2002.
1066ꢀ5285/02/5110ꢀ1945 $27.00 © 2002 Plenum Publishing Corporation

1946
Russ.Chem.Bull., Int.Ed., Vol. 51, No. 10, October, 2002
Glebko et al.
Compound 1a is a white amorphous powder whose IR
spectrum exhibits absorption bands due to an exo methylꢀ
ene group (1643, 881 cm–1), an ester carbonyl group
(1732 cm–1), and hydroxy groups (3419 cm–1). Total acid
hydrolysis of compound 1a yielded 23ꢀhydroxybetulinic
acid as the aglycon; this product was identified by comꢀ
paring the IR, 1H NMR, and 13C NMR spectra with the
corresponding data for an authentic sample.1 Using a carꢀ
bohydrate analyzer, the aqueous fraction of the hydrolyꢀ
sate produced from 1a was found to contain arabiꢀ
nose, rhamnose, and glucose in 1 : 2 : 2 ratio. The
MALDIꢀTOF(+) mass spectrum (detection of positive
ions) of compound 1a exhibts a pseudoꢀmolecularꢀion
peak [M + Na]+ with m/z 1243.6071, which is in good
agreement with the molecular formula C59H96O26 + Na
(calculated, 1243.6083). In addition, this mass spectrum
contains a clear peak with m/z 493 corresponding to the
[23ꢀhydroxybetulinic acid + Na – 2 H]+ ion.
The 1H NMR spectrum of the aglycon moiety of comꢀ
pound 1a exhibits singlets at δH 0.89, 0.96, 1.06, 1.16, and
1.69, due to the protons of five tertiary methyl groups,
and at δH 4.67 (C(29)HB) and 4.84 (C(29)HA) for two
olefinic protons of the exo mehylene group and also a
multiplet at δH 3.39 for the methine proton (C(29)H).
The chemical shifts of the olefinic protons, the methine
proton, and the (C(30)H3) protons completely coincide
with the corresponding values in the spectra of known
lupaneꢀtype glycosides.1,2
published data,10 this signal corresponds to the olefinic
C(29) atom.
The chemical shift of the signal of the aglycon C(3)
atom in the 13C NMR spectrum of 1a is shifted downfield
(+7.25 ppm) relative to a similar signal in the spectrum of
C(3)ꢀunsubstituted 23ꢀhydroxybetulinic acid. The subꢀ
stantial βꢀ (–2.23 ppm) and γꢀeffects (–3.24 ppm) of
glycosylation are also observed on the aglycon C(2) and
C(23) atoms; these effects are comparable in magnitude
with and have the same size as those in the spectrum of
pulsatilloside A.1 The signal of the C(28) atom in this
spectrum is shifted upfield (–4.39 ppm) with respect to
the corresponding signal for the free acid. A similar effect
of glycosylation of a carboxy group has been found previꢀ
ously for pulsatilloside C.2 The presence of a carbohydrate
chain at C(28) is confirmed additionally by the presence
of an absorption band for the ester group (1732 cm–1) in
the IR spectrum of 1a. On the basis of these results, we
concluded that compound 1a is the 3,28ꢀOꢀbis(glycoside)
of 23ꢀhydroxybetulinic acid.
The 1H NMR spectrum of the carbohydrate fragment
of compound 1a exhibits doublets for five anomeric proꢀ
tons with δH 4.98, 5.13, 5.88, 6.28, and 6.37. In addition,
in the high field, proton signals occur at δH 1.64 and 1.72.
Undoubtedly, these belong to the secondary methyl groups
of two rhamnose residues (Table 1). Correspondingly, the
13C NMR spectrum of 1a contains signals for the five
anomeric C atoms at δC 94.97, 101.31, 102.40, 103.73,
and 104.72 ppm and a combined signal at δC 18.15 corꢀ
responding to two C atoms of the secondary methyl
groups in the two rhamnose residues (Table 2); this
confirms the presence of five monosaccharide resiꢀ
dues in 1a.
The 13C NMR spectrum of 1a confirmed the presence
of five C atoms of the tertiary methyl groups (δC 13.32,
14.58, 16.12, 16.61, and 19.12), two C atoms of the exo
methylene group (δC 109.70 and 150.50), the methineꢀ
group C atom (δC 49.55), and the ester carbonyl group C
atom (δC 174.60). In the 13C NMR spectrum of 1 reꢀ
corded without proton decoupling (gated decoupling, GD
spectrum), the C signal at δC 109.70 is a triplet with the
spinꢀspin coupling constant JC,H = 151 Hz; according to
By analogy with other triterpene glycosides of the plant
origin, one can suggest that the arabinose and rhamnose
residues in 1a belong to the Lꢀseries, while glucose resiꢀ
dues correspond to the Dꢀseries.
1
Table 1. H NMR data for anomeric and methyl protons of the carbohydrate fragments of comꢀ
pounds 1a,b and 2 (in pyridineꢀd5)
Proton
Monosaccharide
residue
δ
(J/Hz)
H
1a
1b
2
at the aglycon C(3) atom
5.13 (d, J = 6.2)
6.28 (d, J = 1.2)
1.64 (d, J = 6.2)
—
С(1)Н
С(1)Н
C(6)H3
С(1)Н
Arap
5.12 (d, J = 6.2)
6.25 (d, J = 1.2)
1.64 (d, J = 6.2)
—
5.00 (d, J = 6.2)
6.27 (d, J = 1.5)
1.65 (d, J = 6.3)
5.13 (d, J = 7.8)
Rhap (1→2)
Rhap (1→2)
Glcp (1→4)
at the aglycon C(28) atom
6.37 (d, J = 8.1)
4.98 (d, J = 7.8)
5.88 (d, J = 1.0)
1.72 (d, J = 6.1)
С(1)Н
С(1)Н
С(1)Н
C(6)H3
Glcp (intern.)
Glcp (1→6)
Rhap (1→4)
Rhap (1→4)
—
—
—
—
6.36 (d, J = 8.1)
4.95 (d, J = 7.5)
5.87 (d, J = 1.5)
1.71 (d, J = 6.3)

Pulsatilla chinensis triterpene glycosides
Russ.Chem.Bull., Int.Ed., Vol. 51, No. 10, October, 2002
1947
Table 2. 13C NMR data for carbohydrate parts (δ and
direct spinꢀspin coupling constants (JC(1),H(1)/Hz) for
compounds 1 and 2 (in pyridineꢀd5)
protons of five tertiary methyl groups and signals at δH 4.93
(C(29)HB) and 4.84 (C(29)HA) for two protons of the exo
methylene group and contain a multiplet at δH 3.55 for
the methine proton of the C(19)H group.
The carbohydrate part of the 1H NMR spectrum of 1b
was found to contain doublets for the anomeric protons of
the arabinopyranose residues (δH 5.12) and rhamnoꢀ
pyranose (δH 6.25) and a signal for the protons of the
secondary methyl group of the rhamnopyranosyl residues
at δH 1.64 (see Table 1). The data obtained for progenin
1b proved that the initial glycoside contains a disacchaꢀ
ride fragment consisting of the arabinopyranose and
rhamnopyranose residues.
The 13C NMR spectrum confirmed that compound 1b
contains five C atoms of tertiary methyl groups (δC 13.53,
14.64, 16.19, 16.68, and 19.19), the C(29) and C(20)
atoms of the exo methylene group (δC 109.73 and 151.02),
the C(19) atom of the methine group (δC 49.50), and the
C(28) atom of the free carboxy group (δC 178.62). In the
GD spectrum of 1b, the signal at δC 109.73 for C(29) is a
triplet with the spinꢀspin coupling constant JC,H = 157 Hz.
The signal of the aglycon C(3) atom in the 13C NMR
spectrum of 1b is shifted downfield (+7.32 ppm) relaꢀ
tive to the corresponding signal of C(3)ꢀunsubstituted
23ꢀhydroxybetulinic acid. Upfield βꢀshifts were noted for
the C(2) (–2.15 ppm) and C(4) (–0.37 ppm) atoms and
γꢀshifts were found for C(5) (–1.28 ppm) and C(23)
(–2.67 ppm) atoms. These data indicate that the disacꢀ
charide fragment in 1b is localized at the aglycon C(3)
atom. Comparison of the carbohydrate region of the
13C NMR spectrum of 1b with the published data14 for
hederasaponin C showed them to be fully identical.
In the GD spectrum of 1b, the direct spinꢀspin couꢀ
pling constants JC(1),H(1) are 172 Hz for the rhamnoꢀ
pyranosyl residue and 163 Hz for the arabinopyranosyl
residue, which proves unambiguously the axial and equaꢀ
torial positions of the oxygen atoms, respectively.
C
Atom C
1a
2
δ
J
δ
J
C
C
Arap
С(1)
С(2)
С(3)
С(4)
103.73
75.63
73.92
68.67
63.73
160.1
171.1
103.90
76.12
74.26
79.66
63.69
160.3
С(5)
Rhap (1→2)
С(1)
101.31
71.97
72.19
73.80
69.41
18.15
101.36
71.89
72.21
73.85
69.36
18.25
172.6
159.3
164.9
159.3
168.6
С(2)
С(3)
С(4)
С(5)
C(6)H3
Glcp (1→4)
C(1)
106.24
75.16
78.23
71.09
78.40
62.31
C(2)
C(3)
C(4)
C(5)
C(6)
Glcp (intern.)
С(1)
94.97
73.80
78.35
70.68
77.71
69.22
163.7
159.1
168.3
94.99
73.75
78.40
70.76
77.69
69.26
С(2)
С(3)
С(4)
С(5)
С(6)
Glcp (1→6)
С(1)
104.72
74.94
76.18
78.19
76.79
61.11
104.77
74.96
76.22
78.23
76.83
61.11
С(2)
С(3)
С(4)
С(5)
С(6)
The downfield shift of the signal for C(2) of the
arabinopyranosyl residue (+3.44 ppm) relative to the corꢀ
responding signal of methyl αꢀLꢀarabinopyranoside15 conꢀ
firms the presence of the 1→2 bond between the rhamnoꢀ
pyranosyl and arabinopyranosyl residues.
It follows from the obtained data that progenin 1b is
3ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ(1→2)ꢀαꢀLꢀarabinopyranoꢀ
syl]ꢀ3β,23ꢀdihydroxylupꢀ20(29)ꢀenꢀ28ꢀoic acid.
Rhap (1→4)
С(1)
102.40
72.19
72.41
73.63
70.01
18.15
102.44
72.15
72.45
73.67
70.04
18.15
С(2)
С(3)
С(4)
С(5)
C(6)H3
The direct spinꢀspin coupling constants JC(1),H(1) of
the doublet signals of the anomeric C atoms of the
monosaccharide residues in the GD spectrum of 1a (see
Table 2) attest unambiguously, according to published
data,11—13 to the αꢀconfiguration of the anomeric centers
of arabinopyranose and rhamnopyranoses and to the
βꢀconfiguration of the anomeric centers of glucopyranoses.
Harsh alkaline hydrolysis of 1a afforded progenin 1b,
The remaining three signals of anomeric protons and
1
anomeric C atoms in the H and 13C NMR spectra of
glycoside 1a, which belong to the αꢀLꢀrhamnopyranose
residue and two βꢀDꢀglucopyranose residues, point to the
presence of an oligosaccharide chain at the aglycon C(28)
atom. Comparison of the spectral data for this chain with
the data for the αꢀLꢀRhapꢀ(1→4)ꢀβꢀDꢀGlcpꢀ(1→6)ꢀβꢀ
DꢀGlcp fragment in pulsatilloside C 2 demonstrated that
they are fully identical. The MALDIꢀTOF(+) mass specꢀ
trum of 1a exhibits clear peaks with m/z 965 and 773,
1
whose H NMR spectrum exhibits (in the aglycon part)
signals at δH 0.88, 1.02, 1.06, 1.07, and 1.77 due to the

1948
Russ.Chem.Bull., Int.Ed., Vol. 51, No. 10, October, 2002
Glebko et al.
corresponding to the [M + Na – Rha – Ara]+ and
[M + Na – Rha – 2 Glc]+ ions. On the basis of the results
obtained, glycoside 1a, called chinensioside A, was idenꢀ
tified as 3ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ(1→2)ꢀαꢀLꢀarabinoꢀ
pyranosyl]ꢀ28ꢀOꢀ[αꢀLꢀrhamnopyranosylꢀ(1→4)ꢀβꢀDꢀ
glucopyranosylꢀ(1→6)ꢀβꢀDꢀglucopyranosyl]ꢀ3β,23ꢀdiꢀ
hydroxylupꢀ20(29)ꢀenꢀ28ꢀoic acid.
residues were attributed to the Lꢀseries and the glucose
residues were ascribed to the Dꢀseries.
The MALDIꢀTOF(+) mass spectrum of compound 2,
like the mass spectrum of 1a, exhibits a peak with m/z 935
for the [M + Na – Rha – 2 Glc]+ ion, resulting from
elimination of the trisaccharide fragment. In addition,
this spectrum contains peaks with m/z 1259 and 1244 for
the [M + Na – Rha]+ and [M + Na – Glc]+ fragments,
resulting from elimination of rhamnose or glucose resiꢀ
dues. Based on examination of the results, the structure of
compound 2, called chinensioside B, was identified as
3ꢀOꢀ{αꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ[βꢀDꢀglucopyranosylꢀ
(1→4)]ꢀαꢀLꢀarabinopyranosyl}ꢀ28ꢀOꢀ[αꢀLꢀrhamnopyraꢀ
nosylꢀ(1→4)ꢀβꢀDꢀglucopyranosylꢀ(1→6)ꢀβꢀDꢀglucopyraꢀ
nosyl]ꢀ3β,23ꢀdihydroxylupꢀ20(29)ꢀenꢀ28ꢀoic acid.
The MALDIꢀTOF(+) mass spectrum of compound 3
exhibits a peak for the pseudoꢀmolecular ion [M + Na]+
with m/z 1243.6097, which corresponds to the molecular
formula C59H96O26 + Na (calculated, 1243.6083). Comꢀ
parison of the spectroscopic data for glycosides 3 and 1a
revealed differences in the triterpenoid parts of these molꢀ
ecules and full identity of the carbohydrate parts. The 1H
and 13C NMR spectra of compound 3 are in good agreeꢀ
ment with published data7,14 for hederasaponin C from
the Hedera helix plant. Thus, glycoside 3 was identified as
hederasaponin C.
Compound 2 was prepared as a white amorphous powꢀ
der. The IR spectrum of compound 2 exhibits the same
absorption bands as the IR spectrum of glycoside 1a.
The MALDIꢀTOF(+) spectrum of compound 2 conꢀ
tains a pseudoꢀmolecularꢀion peak [M + Na]+ with
m/z 1405.6643, which is consistent with the molecular
formula C65H106O31 + Na (calculated, 1405.6612). The
peak with m/z 493 occurring in this spectrum implies the
presence of the [23ꢀhydroxybetulinic acid + Na – 2 H]+
ion in 2. Comparison of the 1H and 13C NMR spectra of
compounds 2 and 1a revealed the full identity of their
aglycon fragments. These results lead to the conclusion
that the aglycon in compound 2, as in glycoside 1a, is
C(3)ꢀ and C(28)ꢀglycosylated 23ꢀhydroxybetulinic acid.
1
The H NMR spectrum of glycoside 2 contain six
doublets for anomeric protons at δH 4.95, 5.00, 5.13, 5.87,
6.27, and 6.36 and signals for the protons of the secondary
methyl groups of two rhamnose residues with δH 1.65 and
1.71 (see Table 1). Correspondingly, the 13C NMR specꢀ
trum of 2 contains signals for six anomeric C atoms and
signals for two C atoms of the secondary methyl groups of
two rhamnose residues (see Table 2). Comparative exꢀ
amination of the 1H and 13C NMR spectra of compounds
2 and 1a showed that these compounds contain an identiꢀ
cal trisaccharide fragment at the aglycon C(28) atom and
that 2, unlike 1a, contains an additional monosaccharide
residue (glucose). In the 13C NMR spectrum of comꢀ
pound 2, the signal for the arabinose C(4) atom occurs in
a lower field than the corresponding arabinose signal in
the spectrum of 1a. This means that the additional
monosaccharide residue is attached to the arabinose
C(4) atom.
The fact that the proton chemical shifts and the correꢀ
sponding spinꢀspin coupling constants as well as the carꢀ
bon chemical shifts in the NMR spectra of 2 coinꢀ
cide almost completely with analogous data for the
αꢀLꢀRhapꢀ(1→2)ꢀβꢀDꢀGlcpꢀ(1→4)ꢀαꢀLꢀArap fragment in
pulsatille saponin D 16 confirmed the structure of the
branched trisaccharide chain at the aglycon C(3) atom of
compound 2.
The MALDIꢀTOF(+) mass spectrum of compound 4
contains a pseudoꢀmolecularꢀion peak, [M + Na]+ with
m/z 1405.6637, consistent with the formula C65H106O31
+
Na (calculated, 1405.6612). Analysis of the spectroscopic
data of glycosides 4 and 2 showed that the compounds
being compared have identical carbohydrate chains and
differ only in the structure. The 1H and 13C NMR spectra
of compound 4 are in good agreement with published
data8 for glycoside III, isolated previously from P. cernua.
As a result, compound 4 was identified as glycoside III.
Since the previous publication8 reports only partly the
spectroscopic data for glycoside III, we considered it perꢀ
tinent to report the NMR spectra of compound 4 in the
Experimental.
Thus, compounds 1a,b and 2 can be considered to be
new glycosides of 23ꢀhydroxybetulinic acid. Compounds
1a, 2—4, which were not found previously in P. chinensis,
were found to be the major glycosides in the roots of
this plant.
Experimental
The direct spinꢀspin coupling constants JC(1),H(1) for
the doublets of the anomeric C atoms of monosaccharide
residues in the GD spectrum of 2 (see Table 2) are in good
agreement with the analogous data reported in the literaꢀ
ture11—13 and point to the αꢀconfiguration of the anomeric
centers of arabinopyranose and rhamnopyranose and to
the βꢀconfiguration of the anomeric centers of glucopyraꢀ
nose. By analogy with 1a, the arabinose and rhamnose
1
H and 13C NMR spectra were recorded on Bruker WMꢀ250,
Bruker DPXꢀ300, and Bruker DRXꢀ500 spectrometers for soluꢀ
tions of compounds in pyridineꢀd5 with Me4Si as the internal
standard; the chemical shifts are given in the δꢀscale (ppm) and
spinꢀspin coupling constants are in Hertz. IR spectra (КBr)
were recorded on a Bruker 240 spectrometer; MALDIꢀTOF(+)
mass spectra were run on a Bruker BiflexꢀIII mass spectrometer

Pulsatilla chinensis triterpene glycosides
Russ.Chem.Bull., Int.Ed., Vol. 51, No. 10, October, 2002
1949
(Germany, N2 laser, 337 nm); analyses were done with 1ꢀµL
samples of MeOH solutions (1 mg mL–1) (with 2,5ꢀdihydroxyꢀ
benzoic acid as the matrix). The optical rotation was measured
on a Perkin—Elmer 141 polarimeter. HPLC was performed usꢀ
ing a GCPꢀ100 chromatograph (Czechia), a column with
Separon SGX (150×3.3 mm, 7 µm), and a refractometric detecꢀ
tor. Monosaccharides were analyzed on a Biotronikꢀ5000 carꢀ
bohydrate analyzer (Germany).
Column chromatography was carried out using silica gel
KSK (100—200 mesh, Russia) and silica gel L (40—100 µm,
Chemapol, Czechia) as the sorbents. TLC was performed on
glass plates (9×6 cm) with a layer of KSK silica gel fixed by a
silicic acid sol.
Plant specimens were collected during the blossoming peꢀ
riod (May and June) in the vicinity of the Chernyatino village,
Oktyabr´sk region, Primorski Krai (Russia) and identified using
references (No. 63997) kept in the herbarium of the Pacific
Institute of Bioorganic Chemistry of the FarꢀEastern Branch of
the RAS.
Isolation of compounds 1a, 2—4. Crushed airꢀdried roots
(weight 525 g) were treated with CHCl3 (1 L × 3) and exhausꢀ
tively extracted with 80% EtOH at ∼20 °C. The combined ethaꢀ
nol extract was concentrated in vacuo to remove ethanol, 1 L of
water was added, and the mixture was repeatedly extracted with
nꢀbutanol. The combined butanol extract was concentrated in
vacuo at a temperature not higher than 50 °C, and the residue
(76 g) was dissolved in 100 mL of warm MeOH and poured in
1 L of acetone. The resulting precipitate was washed on a filter
with cold acetone and dried at ∼20 °C to give 47 g of a mixture of
triterpenoid compounds. A part of the mixture (35 g) was passed
through a column with silica gel with the CHCl3—EtOH solvent
system as the eluent (7 : 1→2 : 2). With increasing polarity of the
eluent, two fractions were isolated, I (10.4 g) and II (6.0 g).
Fraction I (5.0 g) was passed additionally through a similar
column with the CHCl3—MeOH solvent system (5 : 1→3 : 1) as
the eluent to give the glycoside fraction А consisting of comꢀ
pounds 1a and 3 (2.87 g, TLC, BuOH—EtOH—25% NH4OH
(5 : 2 : 3), one spot with Rf 0.35). Fraction II (2.5 g) was passed
through a column with silica gel using the CHCl3—MeOH solꢀ
vent system (4 : 1→2 : 1) as the eluent to give glycoside
fraction B consisting of compounds 2 and 4 (0.93 g, TLC,
BuOH—EtOH—25% NH4OH (5 : 2 :3), one spot with Rf 0.20).
The overall fractions А (210 mg) and B (190 mg) were separated
and purified by HPLC on an RPꢀ18 column using MeCN—H2O
solvent systems (27.5 : 72.5 and 30 : 70) to give glycosides 1a
(57 mg), 2 (19 mg), 3 (75 mg), and 4 (45 mg).
30.59 (C(15)), 31.96 (C(16)), 56.69 (C(17)), 47.54 (C(18)),
49.55 (C(19)), 150.50 (C(20)), 29.88 (C(21)), 36.73 (C(22)),
64.84 (C(23)), 13.32 (C(24)), 16.61 (C(25)), 16.12 (C(26)),
14.58 (C(27)), 174.60 (C(28)), 109.70 (C(29)), 19.12 (C(30));
the spectrum of the carbohydrate part is given in Table 2.
Acid hydrolysis of glycoside 1a. Glycoside 1a (20 mg) in 3 mL
of MeOH was treated with 2 M HCl (10 mL) for 2 h at 100 °C.
The hydrolysate was diluted with water and extracted with
AcOEt, and the extract was washed with water, dried, and conꢀ
centrated. The residue was crystallized from MeOH to give 6 mg
of a compound. By comparing the IR and NMR spectra with
published data,1 the product was identified as 23ꢀhydroxyꢀ
betulinic acid. Using a carbohydrate analyzer, arabinose, rhamꢀ
nose, and glucose in 1 : 2 : 2 ratio were detected in the aqueous
fraction.
Alkaline hydrolysis of glycoside 1a. Glycoside 1a (10 mg) was
hydrolyzed with a 5% solution of KOH (10 mL) at ∼20 °C for
24 h. The hydrolysate was neutralized by the KUꢀ2 cation exꢀ
changer in the H+ form to pH 5, the resin was separated on a
filter, the solution was extracted with nꢀbutanol, the extract was
concentrated to dryness, and the residue was chromatographed
on a column with silica gel using the CHCl3—MeOH solvent
system (40 : 5→40 : 35) to give progenin 1b (3.7 g).
1
Progenin 1b. H NMR, aglycon part: 0.88, 1.02, 1.06, 1.07,
1.77 (all s, each 3 H, C(24)H3, C(25)H3, C(26)H3, C(27)H3,
C(30)H3); 3.55 (m, 1 H, C(19)H), 4.93 and 4.75 (both br.s,
each 1 H, C(29)HB, C(29)HA); the spectrum of the carbohydrate
part is presented in Table 1. 13C NMR, aglycon part: 39.01 (C(1)),
25.85 (C(2)), 80.93 C((3)), 42.60 (C(4)), 47.67 (C(5)), 17.93
(C(6)), 34.19 (C(7)), 40.84 (C(8)), 50.73 (C(9)), 36.79 (C(10)),
21.01 (C(11)), 26.18 (C(12)), 38.35 (C(13)), 43.40 (C(14)), 30.96
(C(15)), 32.69 (C(16)), 56.37 (C(17)), 47.54 (C(18)), 49.50
(C(19)), 151.02 (C(20)), 30.47 (C(21)), 37.32 (C(22)), 65.41
(C(23)), 13.53 (C(24)), 16.68 (C(25)), 16.19 (C(26)), 14.64
(C(27)), 178.62 (C(28)), 109.73 (C(29)), 19.19 (C(30)); carboꢀ
hydrate part: Ara: 104.14 (C(1)), 75.64 (C(2)), 74.48 C((3)),
69.10 (C(4)), 63.71 (C(5)); Rha: 101.48 (C(1)), 72.16 (C(2)),
72.32 C((3)), 73.92 (C(4)), 69.50 (C(5)), 18.33 (C(6)).
Chinensioside B (2), amorphous; C65H106O31; [α]D –9.0
(c 0.40, MeOH). IR (KBr), ν/cm–1: 3418 and 1053 (OH), 2932
(CH), 1745 (C(28)=O), 1693, 1646 and 883 (C(20)=C(29)H2),
1455, 1381. MALDIꢀTOF(+) MS, m/z (Irel (%)): 1405.6
[M + Na]+ (100), 1259 [M + Na – Rha]+ (8.7), 1244
[M + Na – Glc]+ (4.7), 1097 [M + Na – Rha – Glc]+ (6), 935
[M + Na – Rha – 2 Glc]+ (43), 493 [23ꢀhydroxybetulinic
1
acid + Na – 2 H]+ (57). H NMR, aglycon part: 0.88, 0.97,
Chinensioside A (1a), amorphous; C59H96O26; [α]D –17.0
(c 0.44, MeOH); IR (KBr), ν/cm–1: 3419 and 1056 (OH), 2938
(CH), 1732 (C(28)=O), 1643 and 881 (C(20)=C(29)H2), 1452,
1386. MALDIꢀTOF(+) MS, m/z (Irel (%)): 1243.6 [M + Na]+
(100), 1097 [M + Na – Rha]+ (10), 965 [M + Na – Rha – Ara]+
(11), 773 [M + Na – Rha – 2 Glc]+ (7), 493 [23ꢀhydroxyꢀ
1.05, 1.16, 1.70 (all s, each 3 H, C(24)H3, C(25)H3, C(26)H3,
C(27)H3, C(30)H3); 3.40 (m, 1 H, C(19)H); 4.69 and 4.85
(both br.s, each 1 H, C(29)HB, C(29)HA); the spectrum of the
carbohydrate part is presented in Table 1. 13C NMR, aglycon
part: 39.02 (C(1)), 25.81 (C(2)), 80.88 C((3)), 42.54 (C(4)),
47.60 (C(5)), 17.86 (C(6)), 34.05 (C(7)), 40.91 (C(8)), 50.74
(C(9)), 36.61 (C(10)), 20.92 (C(11)), 26.12 (C(12)), 38.12
(C(13)), 43.31 (C(14)), 30.62 (C(15)), 31.98 (C(16)), 56.72
(C(17)), 47.54 (C(18)), 49.59 (C(19)), 150.56 (C(20)), 29.92
(C(21)), 36.75 (C(22)), 64.73 (C(23)), 13.41 (C(24)), 16.65
(C(25)), 16.14 (C(26)), 14.60 (C(27)), 174.65 (C(28)), 109.71
(C(29)), 19.15 (C(30)); the spectrum of the carbohydrate part is
presented in Table 2.
1
betulininc acid + Na – 2 H]+ (14). H NMR spectrum of an
aliquot portion: 0.89, 0.96, 1.06, 1.16, and 1.69 (all s, each 3 H,
C(24)H3, C(25)H3, C(26)H3, C(27)H3, C(30)H3); 3.39 (m, 1 H,
C(19)H); 4.67 and 4.68 (both br.s, each 1 H, C(29)HB,
C(29)HA); the spectrum of the carbohydrate part is given in
Table 1. 13C NMR spectrum of the aglycon part: 39.06 (C(1)),
25.77 (C(2)), 80.86 C((3)), 42.50 (C(4)), 47.60 (C(5)), 17.85
(C(6)), 34.01 (C(7)), 40.88 (C(8)), 50.70 (C(9)), 36.59 (C(10)),
20.90 (C(11)), 26.01 (C(12)), 38.09 (C(13)), 43.28 (C(14)),
Glycoside 3, amorphous; C59H96O26; [α]D –15.1 (c 0.49,
MeOH). IR (KBr), ν/cm–1: 3420 (OH), 1732 (C(28)=O), 1642

1950
Russ.Chem.Bull., Int.Ed., Vol. 51, No. 10, October, 2002
Glebko et al.
(C(12)H=C(13)). MALDIꢀTOF(+) MS, m/z (Irel (%)): 1243.6
[M + Na]+ (100), 1097 [M + Na – Rha]+ (10), 965 [M + Na –
Rha – Ara]+ (6), 773 [M + Na – Rha – 2 Glc]+ (10), 493
[hederagenin + Na – 2 H]+ (47).
2. W. Ye, A. He, S. Zhao, and C. T. Che, J. Nat. Prod., 1998,
61, 658.
3. Y. Mimaki, M. Kuroda, T. Asano, and Y. Sashida, J. Nat.
Prod., 1999, 62, 1279.
Glycoside 4, amorphous; C65H106O31; [α]D –12.9 (c 0.41,
MeOH). IR (KBr), ν/cmꢀ1: 3418 (OH), 1745 (C(28)=O), 1642
(C(12)H=C(13)). MALDIꢀTOF(+) MS, m/z (Irel (% )): 1405.6
[M + Na]+ (100), 1259 [M + Na – Rha]+ (17), 1244 [M + Na –
Glc]+ (47), 935 [M + Na – Rha – 2 Glc]+ (30), 493 [hederageꢀ
nin + Na – 2 H]+ (60). 1H NMR, aglycon part: 0.86, 0.89, 0.99,
1.11, 1.13, 1.18 (all s, each 3 H, C(25)H3, C(29)H3, C(24)H3,
C(26)H3, C(27)H3, C(30)H3); 3.17 (m, 1 H, C(18)H); 5.41
(br.s, 1 H, C(12)H=C(13)); the spectrum of the carbohydrate
fragment at the aglycon C(3) atom: Ara: 4.99 (d, 1 H, C(1)H,
J = 6.5 Hz); Rha: 6.28 (s, 1 H, C(1)H); 1.67 (d, 3 H, C(6)H3,
J = 6.1 Hz); Glc: 5.13 (d, 1 H, C(1)H, J = 7.9 Hz); the specꢀ
trum of the carbohydrate fragment at the aglycon C(28) atom:
Rha: 5.87 (d, 1 H, C(1)H, J = 1.1 Hz); 1.71 (d, 3 H, C(6)H3,
J = 6.2 Hz); Glc: 5.00 (d, 1 H, C(1)H, J = 7.7 Hz), Glc: 6.25 (d,
1 H, C(1)H, J = 8.1 Hz). 13C NMR, aglycon part: 38.14 (C(1)),
25.96 (C(2)), 80.81 (C(3)), 43.23 (C(4)), 47.57 (C(5)), 17.92
(C(6)), 32.58 (C(7)), 39.68 (C(8)), 47.94 (C(9)), 36.64 (C(10)),
23.58 (C(11)), 122.63 (C(12)), 143.80 C((13)), 41.92 (C(14)),
28.06 (C(15)), 23.14 (C(16)), 46.80 (C(17)), 41.43 (C(18)),
45.98 (C(19)), 30.45 (C(20)), 33.77 (C(21)), 32.33 (C(22)),
64.80 (C(23)), 13.64 (C(24)), 15.91 (C(25)), 17.32 (C(26)),
25.76 (C(27)), 176.20 (C(28)), 32.79 (C(29)), 23.44 (C(30));
the spectrum of the carbohydrate fragment at the aglycon C(3)
atom: Ara: 103.94 (C(1)), 76.12 (C(2)), 74.31 (C(3)), 79.73
(C(4)), 63.76 (C(5)); Rha: 101.38 (C(1)), 71.91 (C(2)), 72.23
(C(3)),73.86 (C(4)), 69.38 (C(5)), 18.27 (C(6)); Glc: 106.28
(C(1)), 75.17 (C(2)), 78.24 (C(3)), 71.09 (C(4)), 78.41 (C(5)),
62.32 (C(6)); the spectrum of the carbohydrate fragment at the
aglycon C(28) atom: Glc: 95.37 (C(1)), 73.64 (C(2)), 78.41
(C(3)), 70.75 (C(4)), 77.74 (C(5)), 69.07 (C(6)); Glc: 105.54
(C(1)), 75.02 (C(2)), 76.28 (C(3)), 78.24 (C(4)), 76.84 (C(5)),
61.15 (C(6)); Rha: 102.48 (C(1)), 72.18 (C(2)), 72.46 (C(3)),
73.70 (C(4)), 70.04 (C(5)), 18.16 (C(6)).
4. S. A. Zinova, K. P. Ulanova, Zh. I. Ul´kina, and L. I.
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(Syktyvkar, September 25—30, 2000), Abstrs., Syktyvkar,
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Received February 26, 2001;
in revised form May 17, 2002