Secondary metabolites of Astragalus danicus Retz. and A. inopinatus Boriss

Article abstract of DOI:10.1023/A:1011302309943

The chemical composition of the above-ground parts of Astragalus danicus and A. inopinatus collected in the Baikal region (Eastern Siberia) was studied for the first time. From A. danicus, pentacyclic triterpene saponins were isolated and identified, viz., 3-O-(β-glucuronopyranosyl)-3β,22β,24-trihydroxyolean-12-ene, 3-O-[O-(β-D-xylopyranosyl)-(1→2)-(β-glucuronopyranosyl)]-3β, 22β,24-trihydroxyolean-12-ene, 3-O-[O-(β-D-glucopyranosyl)-(1→2)-(β-glucuronopyranosyl)]-3β ,22β,24-trihydroxyolean-12-ene, 3-O-[O-(α-L-rhamnopyranosyl)-(1→2)-O-(β-D-xylopyranosyl) -(1→2)-(β-glucuronopyranosyl)]-3β,22β,24-trihydroxyolean-12-ene, 3-O-[O-(α-L-rhamnopyranosyl)-(1→2)-O-(β-D-glucopyranosyl)-(l-2)-(β-glucuronopyranosyl)]-3β,22β,24-trihydroxyolean-12-ene, 3-O-methyl-D-chiro-inositol, and linolenic acid. In A. inopinatus, the same saponins were identified as well as tricosan-1-ol and tetracosan-1-ol, 5,7,4′-trihydroxyflavon (apigenin), and atetracyclic triterpenoid, 20(R),24(S)-epoxycyclolanost-9(11)-ene-3β,6α, 16β, 25-tetrol (cycloastragenol). All reported compounds from the both genus of Astragalus were isolated for the first time. Methanolic extracts of A. danicus and A. inopinatus exhibited low inhibitory activity with respect to the growth of HeLa cells. The chloroform fraction of A. danicus showed a strong antimicrobial activity against Staphylococcus aureus and a strong cytotoxic activity against HeLa cells.

Full text of DOI:10.1023/A:1011302309943

Russian Chemical Bulletin, International Edition, Vol. 50, No. 6, pp. 1107—1112, June, 2001
1107
Secondary metabolites of Astragalus danicus Retz. and A. inopinatus Boriss.
A. S. Gromova,a V. I. Lutsky,a J. G. Cannon,b D. Li,b and N. L. Owenb
aA. E. Favorsky Irkutsk Institute of Chemistry,
Siberian Branch of the Russian Academy of Sciences,
1 ul. Favorskogo, 664033 Irkutsk, Russian Federation.
Fax: +7 (395 2) 35 6046. E-mail: admin@irioch.irk.ru
bDepartment of Chemistry and Biochemistry, Brigham Young University,
Provo, Utah 84602, US.
Fax: (801) 378 5474
The chemical composition of the above-ground parts of Astragalus danicus and A. inopinatus
collected in the Baikal region (Eastern Siberia) was studied for the first time. From A. danicus,
pentacyclic triterpene saponins were isolated and identified, viz., 3-O-(β-glucuronopyranosyl)-
3β,22β,24-trihydroxyolean-12-ene, 3-O-[O-(β-D-xylopyranosyl)-(1→2)-(β-glucuronopyra-
nosyl)]-3β,22β,24-trihydroxyolean-12-ene, 3-O-[O-(β-D-glucopyranosyl)-(1→2)-(β-glu-
curonopyranosyl)]-3β,22β,24-trihydroxyolean-12-ene, 3-O-[O-(α-L-rhamnopyranosyl)-
(1→2)-O-(β-D-xylopyranosyl)-(1→2)-(β-glucuronopyranosyl)]-3β,22β,24-trihydroxyolean-12-
ene, 3-O-[O-(α-L-rhamnopyranosyl)-(1→2)-O-(β-D-glucopyranosyl)-(1→2)-(β-glucurono-
pyranosyl)]-3β,22β,24-trihydroxyolean-12-ene, 3-O-methyl-D-chiro-inositol, and linolenic acid.
In A. inopinatus, the same saponins were identified as well as tricosan-1-ol and tetracosan-1-ol,
5,7,4´-trihydroxyflavon (apigenin), and a tetracyclic triterpenoid, 20(R),24(S)-epoxycyclolanost-
9(11)-ene-3β,6α,16β,25-tetrol (cycloastragenol). All reported compounds from the both
genus of Astragalus were isolated for the first time. Methanolic extracts of A. danicus and A.
inopinatus exhibited low inhibitory activity with respect to the growth of HeLa cells. The
chloroform fraction of A. danicus showed a strong antimicrobial activity against Staphylococ-
cus aureus and a strong cytotoxic activity against HeLa cells.
Key words: Astragalus, saponins, cycloastragenol, O-methylinositol, apigenin, higher
alcohols, linolenic acid, biological activity.
The genus Astragalus (Fabaceae family) comprises
about 2000 species; of these, about 80 species produce
saponins. Biological activities of many saponins have
been established. The chemical composition of Astraga-
lus inopinatus and triterpene substances A. danicus has
not been studied previously. A. danicus is used in alter-
native and oriental medicine as a tonic and in treatment
of gynaecological disorders and hypertension;1 no pub-
lished data on the use of A. inopinatus have been found.
tetracoosan-1-ol (9), a flavon (apigenin) (10), and a
tetracyclic triterpenoid, viz., cycloastragenol (11) were
obtained from a similar fraction of A. inopinatus. These
compounds were identified by chemical and spectral
methods, namely, 1D and 2D NMR spectroscopy
(DEPT, COSY, HETCOR, HMBC), high-resolution
mass spectrometry (HR MS), and EI MS, FAB MS
(positive and negative ions), and GC/MS.
The compounds were isolated by column chroma-
tography on silica gel; in addition, reversed-phase
preparative HPLC was used to purify compounds
2—5 and 7.
Results and Discussion
Compound 1 has the formula C36H58O9 (HR FAB
MS). Acid hydrolysis of monoside 1 yielded glucuronic
acid (GlcA) (identified by TLC with an authentic
sample*) and a genin C30H50O3 (HR FAB MS). The 1H
and 13C NMR spectra of glycoside 1 confirm the iden-
tification of GlcA and attest to the pyranose form of its
oxide ring and to the β-configuration of the glycosidic
bond (δH 4.91, d, H(1), J = 7.8 Hz; δC 105.9, C(1)). In
terms of its physicochemical parameters and 1H and
We studied the chemical composition of the above-
ground parts of the species under consideration col-
lected in the blooming period in the Baikal region,
Eastern Siberia. Methanolic extracts of air-dry finely
ground plant material were treated successively with
chloroform and n-butanol. Five pentacyclic triterpene
glycosides were isolated from the butanolic extract of
À. danicus, namely, one monoside (1), two biosides (2
and 3), and two triosides (4 and 5). From the butanolic
extract of A. inopinatus, glycosides 2—5 were isolated.
O-Methylcyclitol ((+)-pinitol) (6) and linolenic acid
(7) were found in the chloroform extract of À. danicus.
Two saturated alcohols, viz., tricosan-1-ol (8) and
* The absolute configuration (D- or L-) of the glucuronic acid
residues in compounds 1—5 has not been determined; this
monosaccharide is depicted in the D-form for convenience.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1059—1063, June, 2001.
1066-5285/01/5006-1107 $25.00 © 2001 Plenum Publishing Corporation

1108
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 6, June, 2001
Gromova et al.
Table 1. 13C NMR spectral data of triterpene fragments of
compounds 1—5, 11, and 12 (δ, 1 in C5D5N; 2—5 and 11 in
CD3OD; 12 in CDCl3)
12
22
COOH
OH
O
C atom 12
1
2
3
4
5
11
1
OH
1
2
3
4
5
6
7
8
38.4
27.7
80.9
42.8
55.9
18.4
33.1
39.7
47.7
36.7
23.7
39.1
26.8
89.4
44.7
57.0
19.2
33.7
40.4
48.2
36.9
24.5
39.4
26.9
91.1
44.7
56.9
19.3
33.8
40.4
48.2
37.0
24.4
39.1
26.8
91.5
44.2
56.7
19.0
33.7
40.4
48.2
36.9
24.4
40.0
27.0
92.3
45.0
57.7
19.6
34.5
40.9
47.6
37.6
25.0
40.0
27.0
92.3
44.7
57.6
19.6
34.5
40.9
47.6
37.6
25.0
33.6
31.2
79.5
42.8
54.6
69.9
34.2
46.0
22.2
31.0
26.8
32.5
47.0
47.2
47.0
74.7
59.2
22.0
29.0
88.5
28.6
35.6
26.9
82.8
72.6
26.8
27.1
27.8
16.0
20.7
HO
3
OH
RO
S1
24 CH2OH
R = S1 (1), S2 (2), S3 (3), S4 (4), S5 (5), H (12)
COOH
COOH
O
OH
O
9
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
COOH
COOH
HO
CH2OH
O
HO
O
122.5 122.9 122.8 122.8 138.9 138.8
143.5 145.2 145.2 145.2 145.3 135.3
OH
OH
O
O
O
O
HO
CH2OH
42.1
25.9
28.2
37.4
44.7
46.2
30.5
41.5
76.6
22.3
64.5
16.1
16.9
25.4
28.2
32.8
20.0
42.8
26.8
29.1
38.4
45.7
47.2
31.3
42.7
75.9
23.7
63.5
16.0
17.5
26.2
29.1
33.7
21.6
42.8
26.9
29.1
38.4
45.8
47.2
31.2
42.7
76.0
23.2
63.3
16.0
17.5
26.1
29.1
33.7
21.5
42.8
26.8
29.1
38.4
45.8
47.2
31.2
42.7
76.0
23.0
63.7
16.0
17.4
26.1
29.0
33.6
21.5
43.5
27.1
30.1
38.7
46.9
47.6
31.5
42.4
77.1
23.2
64.1
16.3
17.7
25.5
29.2
32.7
20.5
43.5
23.2
30.1
38.7
46.9
47.6
31.5
42.4
77.1
23.3
64.4
16.5
17.7
25.5
29.2
32.7
20.5
HO
OH
OH
O
O
O
O
OH
OH
OH
OH
OH
OH
OH
O
O
O
O
OH
Me
Me
OH
OH
OH OH
S4
OH OH
S5
S2
S3
HO
OH
COOH
1
3
HO
OMe
HO
OH
6
Note. The signals presented in Tables 1 and 2 were assigned
based on 2D NMR spectroscopy (DEPT, COSY, HETCOR,
ROESY, HMBC).
Me(CH2)21CH2OH
Me(CH2)22CH2OH
9
8
The attachment of GlcA in glycoside 1 to the O(3)
atom of genin 12 is indicated by the downfield shift of
the signal of C(3) in the 13C NMR spectrum of monoside
1 by 11.6 ppm with respect to that in the spectrum of
genin 12 (Table 1) and by the presence of a cross peak
between H(1) of the GlcA residue (δH 4.91) and C(3) of
the genin (δC 89.4) in the HÌÂC NMR spectrum of
the monoside. Thus, compound 1 is 3-O-β-glucu-
ronide of soyasapogenin Â. Previously,2 a deriva-
tive of this glycoside at the carboxy group has been
isolated.
HO
O
2
4
´
OH
7
5
OH
O
OH
10
24
O
20
21
7
Biosides 2 and 3 have the molecular formulas
C41H66O13 and C42O68O14 (HR FAB MS) and charac-
teristic ions with m/z 633 [(Ì – H) – 132] , 457
19
OH
16
–
–
–
3
[(Ì – H) – 132 – 176)] and 633 [(Ì – H) – 162] ,
–
HO
6
457 [(Ì – H) – 162 – 176)] , respectively, which
points to the presence of a pentose and a uronic acid in
compound 2 and a hexose and a uronic acid in com-
pound 3. Partial acid hydrolysis of biosides yielded one
progenin identical with monoside 1. According to 1H
and 13C NMR spectroscopy (Table 2), the pentose was
OH
11
13C
NMR
spectroscopy,
3β,22β,24-trihydroxyolean-12-ene; it was identified as
soyasapogenin  (12).2,3
the
genin
is

Components of Astragalus danicus and A. inopinatus
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 6, June, 2001
1109
Table 2. 13C NMR spectrl data of the carbohydrate parts of
glycosides 1—5 (δ, 1 in C5D5N; 2—5 in CD3OD)
Thus, biosides
2
and
3
were identified as
3-O-[O-(β-D-xylopyranosyl)-(1→2)-(β-glucurono-
pyranosyl)]-3β,22β,24-trihydroxyolean-12-ene and
3-O-[O-(β-D-glucopyranosyl)-(1→2)-(β-glucurono-
pyranosyl)]-3β,22β,24-trihydroxyolean-12-ene, respec-
tively. Previously,6 a bioside 2 derivative has been iso-
lated and characterized; bioside 3 has been isolated7
from Galega officinalis.
The molecular formulas of triosides 4 and 5 are
C47H76O17 and C48H78O18 (HR FAB MS). The nega-
tive-ion FAB mass spectra contain fragment ions with
Carbohydrate
residue,
1
2
3
4
5
C atom
GlcA
1
2
3
4
5
105.9
75.4
78.5
73.9
77.0
175.3
105.2
81.2
78.5
73.5
78.2
176.0
105.2
82.4
78.5
73.5
78.2
176.0
105.6
78.8
76.9
74.2
78.0
176.0
105.5
78.8
76.9
74.1
78.0
176.0
–
m/z 765 [(Ì – H) – 146] , 633 [(Ì – H) – 146 –
6
–
–
132] and 457 [(Ì – H) – 146 – 132 – 176)]
for compound 4 and fragment ions with m/z 795
Glc
1
2
3
4
104.5
75.8
78.2
70.5
78.3
62.0
102.4
79.5
79.0
71.2
79.3
61.9
–
–
[(Ì – H) – 146] , 633 [(Ì – H) – 146 – 162] and
–
457 [(Ì – H) – 146 – 162 – 176)] for compound 5.
These data indicate that trioside 4 contains a deoxy-
hexose, a pentose, and a uronic acid, and trioside 5
contains a deoxyhexose, a hexose, and a uronic acid. In
the products of partial acid hydrolysis of both glyco-
sides, one progenin (1H and 13C NMR, negative ion
FAB MS) identical with monoside 1 was found. In turn,
biosides 2 and 3 are progenins of triosides 4 and 5; this
follows from comparison of the 1H and 13C NMR
spectra of the biosides and triosides, respectively (see
Table 2). According to the data from 1H and 13C NMR
spectroscopy, the deoxyhexose in both triosides was
identified as rhamnopyranose. In establishing the con-
figuration of the rhamnosidic bond, the chemical shifts
of C(3) and C(5) (δC 72.5 and 69.4 for α-configuration
and δC 75.4 and 73.5 for β-configuration) rather than
the chemical shift of the anomeric C atom are the
characteristic values in the 13C NMR spectrum.8 In
compounds 4 and 5, the chemical shifts of C(3) are 72.4
ppm and those of C(5) are 69.8 ppm (see Table 2);
hence, rhamnose has the α-configuration of the glyco-
sidic center in both cases. The binding of this residue to
Xyl in glycoside 4 and to Glc in glycoside 5 at the O(2)
atoms is judged by the downfield shifts of signals for the
C(2) atoms, equal to 3.5 ppm and 3.7 ppm in triosides 4
and 5, respectively, relative to the positions of these
signals in the spectra of biosides 2 and 3 (see Table 2).
The same is indicated by the cross peaks between the
H(1) atoms of rhamnose (δH 5.19 in 4 and δH 5.22 in 5)
and the C(2) atoms of Xyl (δC 79.3) and Glc (δC 79.5)
in the HMBC NMR spectra of compounds 4 and 5,
respectively.
5
6
Xyl
1
2
3
4
105.1
75.8
77.1
71.0
67.1
103.2
79.3
78.6
70.5
66.8
5
Rha
1
2
3
4
102.3
72.4
72.4
74.4
69.8
18.4
102.1
72.4
72.4
74.4
69.8
18.4
5
6
identified as xylose, and the hexose was identified as
glucose having the pyranose forms of the oxide rings
and the β-configurations of anomeric centers (δH 4.50,
J = 8.0 Hz, H(1), δC 105.0 C(1) Xyl; δH 4.12, J = 7.5 Hz,
H(1), δC 103.2, C(1) Glc). The attachment of the Xyl
residue in compound 2 and the attachment of Glc in 3
to the O(2) atom of the glucuronic acid residues is
indicated by the downfield shift of the signals of C(2) of
the GlcA residues in the 13C NMR spectra of these
biosides with respect to that in monoside 1 (see Table 2).
The above-considered results were confirmed by analy-
sis of the 1H—13C HÌÂC NMR spectra of biosides 2
and 3. These spectra contain cross peaks between the
H(1) atoms of the Xyl residue (δH 4.64) in compound 2
(and H(1) of the Glc residue (δH 4.78) in 3) and the
C(2) atoms of the GlcA residues (δC 81.2 and 82.4 in 2
and 3, respectively). Since the terminal xylose and
glucose residues were fouind to have β-configurations,
we assigned them to the D-series on the basis of Klyne´s
calculations of molecular rotation.4 For compound 2:
[M]D(2) – [M]D(1) = –26.0 (cf. Ref. 5: [M]D (methyl
β-D-xylopyranoside) –108.1, [M]D (methyl α-D-xylo-
pyranoside +251.2). For compound 3: [M]D(3) –
[M]D(1) = –96.9 (cf. Ref. 5: [M]D (methyl β-D-gluco-
pyranoside) –66.3, [M]D (methyl α-D-glucopyrano-
side) +308.3).
The assignment of terminal α-rhamnopyranosides in
compounds 4 and 5 to the L-series was performed by the
Klyne method.4 For compound 4: [M]D(4) – [M]D(2) =
–247.3; for compound 5: [M]D(5) – [M]D(3) = –272.4
(cf. Ref. 5: [M]D (methyl α-L-rhamnopyranoside) –111.3,
[M]D (methyl β-L-rhamnopyranoside) +169.8).
Thus, triosides
4 and 5 were identified as
3-O-[O-(α-L-rhamnopyranosyl)-(1→2)-O-(β-D-xylo-
pyranosyl)-(1→2)-(β-glucuronîpyranosyl)]-3β,22β,24-
trihydroxyolean-12-ene and 3-O-[O-(α-L-rham-
nopyranosyl)-(1→2)-O-(β-D-glucopyranosyl)-

1110
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 6, June, 2001
Gromova et al.
Dynamax SD-200 chromatograph (C(18); 5 µm; a 4.6½250 mm
analytical column, and a 21.4½250 mm preparative column).
Low-pressure column chromatography was carried out using
silica gel 200—400 mesh (Fisher); GPC was carried out on
Sephadex G-25 and ion exchange chromatography was done on
(1→2)-(β-glucuronîpyranosyl)]-3β,22β,24-trihydroxy-
olean-12-ene, respectively. Compound 4 was first iso-
lated from A. membranacea,6 and compound 5 has been
isolated from Medicàgo hisðida.9
O-Methylinositol10 (6) and linolenic acid11 (7) were
isolated from the chloroform extract of À. danicus by
flash chromatography on silica gel. O-Methylinositol
(6) was identified as 3-O-methyl-D-chiro-inositol
((+)-pinitol), acid 7 was identified using the data from
HR MS, 1H and 13C NMR spectra, and GC/MS of the
methyl ester.
From a similar fraction from A. inopinatus, two
saturated alcohols 8 and 9, flavon (10), and tetracyclic
triterpenoid (11) were isolated by chromatography on
silica gel. The alcohols were identified by GC/MS and
HR MS in the form of O-acetates as tricosan-1-ol (8)
and tetracosan-1-ol (9).12 The compounds 10, 11 were
identified as apigenin13 and cycloastragenol,14 using
physicochemical constants and NMR, MS spectra, re-
spectively. Triterpenoid 11 was isolated for the first time
from the products of acid hydrolysis of saponins from
À. måmbranacåus Bunge roots.6
Aqueous and methanolic extracts of both types of
astragal and the chloroform and n-butanolic fractions of
the methanolic extracts of A. danicus were assayed in
vitro for cytotoxic (HeLa cells), fungistatic (Candida
albicans), and antimicrobial (Staphylococcus aureus and
Pseudomonas aeruginosa) activities. The aqueous ex-
tracts were found to be inactive in all of the assays listed
above. The methanolic extracts of A. danicus and
A. inopinatus inhibit the growth of the HeLa cells by
30% (here and below, the concentration used is
1000 µg mL–1); the chloroform fraction of the methanolic
extract of A. danicus inhibits it by 95%. In addition, the
same fraction suppresses the growth of Gram-positive
bacteria Staphylococcus aureus by 82%.
–
Amberlite IRÀ-68 (OH ) and Amberlite IRC-50 (H+) ion
exchange resins; TLC was performed on Whatman Al SilG/UV
silica gel. The substances on chromatograms were visualized by
spraying the plates with a 0.5% solution of vanillin in 50%
H3ÐO4. For column and thin layer chromatography, the fol-
lowing solvent systems were used (in systems (1) and (2), the
proportion of the polar solvent increased from 0 to 15% (v/v)):
(1) hexane—acetone; (2) chloroform—methanol; chloro-
form—methanol—water: (3) 70 : 15 : 1; (4) 70 : 23 : 1;
(5) 70 : 23 : 4; (6) 80 : 36 : 7.
Plant material. The above-ground parts of the plants
Astragalus danicus Retz. and A. inopinatus Boriss. were gathered
during the blooming period (July 1998, Buryatia, Tunkinskii
region). The herbarium specimens are deposited with the M. G.
Popov Academic Herbarium of the Siberian Institute of Physi-
ology and Biochemistry of Plants of the Siberian Branch of the
RAS (Irkutsk).
Extraction and fractionation of the extract. Air-dry finely
ground raw material of A. danicus and A. inopinatus (0.5 kg
each) was extracted successively with H2O and ÌåOH (3½3 L).
The methanolic extract was concentrated to 1/4 initial volume
and treated successively with chloroform (3½250 mL) and
n-butanol (5½300 mL).
A. danicus. The chloroform extract (2.1 g) was subjected to
flash chromatography on silica gel in system (1). The fraction
containing component 6 was eluted with 5% acetone in hexane,
and the fraction containing component 7, with 15% acetone in
hexane. Re-chromatography on silica gel in system (2) (5%)
gave individual compound 6.
Preparative HPLC (MeCN—H2O, 45 : 55 → 55 : 45 (v/v))
gave linolenic acid11 (7) identified from the data from NMR
spectroscopy and GC/MS of the corresponding methyl ester
carried out after treatment with the MethPrep I reagent (Alltech).
The butanolic extract (3.5 g) was chromatographed on silica
gel using systems (3)—(6) for elution. The use of system (4)
gave a fraction containing monoside 1. This fraction was
chromatographed once again on silica gel in systems (3) and (4)
and then successively purified on Sephadex G-25 and the
Amberlite IRC-50 cation exchange resin using gradient elution
with the H2O—ÌåOH system. This gave compound 1 (10 mg).
Further washing of the column with system (5) yielded a
fraction containing a mixture of biosides 2 and 3 (∼250 mg) and
elution with system (6) afforded a mixture of triosides 4 and 5
(∼320 mg). Compounds 2—5 were isolated by preparative HPLC
in the MeCN—H2O system (45 : 55 (v/v)).
Thus, eleven compounds were isolated and identi-
fied from A. danicus and A. inopinatus. Compounds 1
and 2 have not been isolated previously in the native
state; their physicochemical parameters and spectral
characteristics were determined for the first time. None
of the compounds described have been isolated previ-
ously from both types of astragal. It was found that the
chloroform fraction of A. danicus exhibits high cytotoxic
and antimicrobial activities.
3-O-Methyl-D-chiro-inositol (6), m.p. 186—187 °C
23
(MeOH), [α]D +57 (c 1.09; H2O) (cf. Ref. 10: m.p. 188 °C;
23
[α]D +59). IR, ν/cm–1: 3409 (OH), 1070, 1040 (C—O—).
Experimental
Found, m/z: 195.0862 [M
+
H]+. C7H15O6. Calculated:
[Ì + 1]+ 195.0869. 1H NMR (CD3OD), δ: 3.84 (m, 2 H,
H(1), H(6)); 3.64 (dd, 1 H, H(2), J = 2.4, 9.7 Hz); 3.59 (dd,
1 H, H(5), J = 2.4, 9.7 Hz); 3.48 (t, 1 H, H(4), J = 9.7 Hz);
3.18 (t, 1 H, H(3), J = 9.7 Hz).
Melting points were determined on a Bristoline stage;
optical rotations were measured on a Perkin—Elmer 241 instru-
ment. IR spectra were recorded on a Mattson Polaris spectrom-
eter in pellets with ÊÂr and the NMR spectra were run at
–
Linolenic acid (7). Found, m/z: 277.2245 [M – H] .
–
C18H29O2. Calculated: [Ì – 1] 277.2251. 1H NMR (CDCl3),
ambient temperature on
a Varian VXR-500 spectrometer
(500 ÌHz for 1H; 125 ÌHz for 13C). The FAB and HR mass
spectra were recorded on a JEOL SX102A instrument with
double focusing (glycerol or thioglycerol as the matrix, xenon
as the ionizing gas); GC/MS and EI MS were run on the same
instrument. HPLC analysis was carried out on a Rainin/Varian
δ: 5.36 (m, 6 H, CH=CH); 2.80; 2.35; 2.05; 1.62 (all m,
each 2 H, CH2); 1.32 (m, 12 H, CH2); 0.96 (t, 3 H, Me).
13C NMR (CDCl3), δ: 180.5; 131.9; 130.2; 128.3; 128.2; 127.7;
127.1; 34.0; 29.5; 29.1; 29.0; 28.9; 27.2; 25.6; 25.5; 24.6;
20.5; 14.5. Methyl linolenate: C19H32O2. MS (EI, 70 eV),

Components of Astragalus danicus and A. inopinatus
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 6, June, 2001
1111
m/z (Irel (%)) : 292 [Ì]+ (13), 261 [Ì – 31]+ (10), 121 (27),
95 (65), 79 (100), 55 (45).
Acid hydrolysis. Monoside 1 (3 mg) (or 7 mg of a mixture
of biosides 2 and 3, or 9 mg of a mixture of triosides 4 and 5) in
5 mL of a 6% H2SO4—dioxane mixture (1 : 1) was heated for
5 h at 100 °C. The reaction mixture was neutralized with the
3-O-(β-Glucuronîpyranosyl)-3β,22β,24-trihydroxyolean-
12-ene (1). White plates, m.p. 242—244 °C (MeOH),
23
–
[α]D +25.7 (c 0.5; ÌåOH). IR, ν/cm–1: 3400 (OH), 1600 br
Amberlite IRÀ-68 (OH ) anion exchange resin, the solution
–
(COO ), 1068, 1040 (C—O—). Negative-ion FAB MS, m/z:
was concentrated, and the residue was dissolved in water and
extracted with 3½7 mL of CHC13. TLC of the aqueous layer in
solvent systems (6) and (7) allowed identification (by compari-
son with authentic samples) of GlcA in monoside 1; Xyl (or
Glc) and GlcA in biosides 2 and 3; and Xyl (or Glc), Rha, and
GlcA in triosides 4 and 5.
–
–
633 [M – H] , 457 [(Ì – H) – 176)] . Found, m/z: 633.3990
[M – H] . C36H57O9. Calculated: [Ì – 1] 633.4003. 1H NMR
(C5D5N), δ: 5.40 (s, 1 H, H(12)); 4.40 and 3.65 (both d,
each 1 H, CH2OH, J = 10.7 Hz); 3.75 (m, 1 H, H(22)); 3.65
(m, 1 H, H(3)); 4.91 (d, 1 H, H(1) GlcA, J = 7.8 Hz); 4.31 (m,
1 H, H(5) GlcA); 4.26 (m, 1 H, H(4) GlcA); 4.13 (m, 1 H,
H(3) GlcA); 3.95 (m, 1 H, H(2) GlcA). The 13C NMR spectral
data are listed in Tables 1 and 2.
–
–
Partial acid hydrolysis. A mixture of saponins 2 and 3
(50 mg) or triosides 4 and 5 (57 mg) in 7 mL of a 3%
H2SO4—dioxane mixture (1 : 1) was heated on a water bath for
4 h at 80 °C. The reaction mixture was neutralized with the
3-O-[O-(β-D-Xylopyranosyl)-(1→2)-(β-glucuronîpyrano-
syl)]-3β,22β,24-trihydroxyolean-12-ene (2). White plates, m.p.
–
Amberlite IRÀ-68 (OH ) anion exchange resin, the solution
23
220—222 °C (MeOH), [α]D
+17.9 (c 0.6; ÌåOH). IR,
was concentrated, and the residue was dissolved in water and
extracted with ÂunOH ( 3½7 mL). The extract was concen-
trated and the residue was chromatographed on a column with
SiO2. Elution with system (2) (1—5% ÌåOH in CHCl3) gave
fractions (1) and (2) containing genin 12 and monoside 1,
respectively. Re-chromatography of fraction (1) in system (2)
(3% ÌåOH in CHCl3) yielded 12 mg of genin 12; flash
chromatography of fraction 2 in system (3) afforded 11 mg of
monoside 1.
ν/cm–1: 3410 (OH), 1600 br (COO ), 1070, 1045 (C—O—).
Negative-ion FAB MS, m/z: 765 [Ì – H] , 633 [(Ì – H) –
–
–
–
–
132] and 457 [(Ì – H) – 132 – 176)] . Found, m/z:
–
–
765.4420 [M – H] . C41H65O13. Calculated: [Ì – 1] 765.4425.
1H NMR (the signals for the anomeric protons of carbo-
hydrates, CD3OD), δ: 4.50 (d, 1 H, H(1) GlcA, J = 7.1 Hz);
4.64 (d, 1 H, H(1) Xyl, J = 8.0 Hz). The 13C NMR spectral
data are listed in Tables 1 and 2.
3-O-[O-(β-D-Glucopyranosyl)-(1→2)-(β-glucuronîpyra-
nosyl)]-3β,22β,24-trihydroxyolean-12-ene (3). White plates,
3β,22β,24-Trihydroxyolean-12-ene (12), m.p. 258—259 °C
23
(EtOH), [α]D
+90.6 (c 0.7; CHCl3) (cf. Ref. 2: m.p.
23
30.5
m.p. 283—285 °C (MeOH), [α]D + 8.3 (c 0.5; ÌåOH) (cf.
258—260 °C; [α]D
+92.4 (CHCl3)). MS (EI, 70 eV),
Ref. 7: m.p. 285—287 oC, for C42H68O14•2H2O: [α]D25 + 27.5
m/z (Irel (%)): 458 [Ì]+ (7), 440 [̗H2O]+ (5); C30 H50O3.
1H NMR (CDCl3), δ: 4.25, 3.35 (both d, each 1 H, CH2OH,
J = 11.0 Hz); 3.43 (m, H(3)); 3.43 (m, H(2)); 1.25; 1.12; 1.04;
0.95; 0.91; 0.90; 0.87 (all s, 7 Me groups, each 3 H).3 The
13C NMR spectral data are listed in Table 1.
–
(c 0.2; ÌåOH)). IR, ν/cm–1: 3410 (OH), 1600 br (COO ),
1072, 1068, 1035 (C—O—). Negative-ion FAB MS, m/z:
–
–
795 [(Ì – H)] , 633 [(Ì – H) – 162] and 457 [(Ì – H) –
–
–
162 – 176)] . Found, m/z : 795.4542 [M – H] . C42H67O14
.
Calculated: [Ì – 1] 795.4531. 1H NMR (the signals for
the anomeric protons of carbohydrates, CD3OD), δ: 4.50 (d,
1 H, H(1) GlcA, J = 7.1 Hz); 4.78 (d, 1 H, H(1) Glc,
À. inopinatus. From the butanolic extract (3.2 g), saponins
2—5 were isolated under conditions described for a similar
fraction of A. danicus. The chloroform extract of A. inopinatus
(1.8 g) was subjected to flash chromatography on silica gel.
Elution with system (1) (3% Me2CO in C6H14) gave a fraction
containing alcohols 8 and 9. The mixture was acetylated
(Ac2O/Py, 50 °C) and the products were analyzed by GC/MS
and HR MS; tricosan-1-ol 8 was identified as the correspond-
ing acetate (Found, m/z 382.5020; C25H50O2; Calculated:
382.5026) and tetracosan-1-ol 9 was identified as the acetate
(Found, m/z: 396.5219; C26H52O2; Calculated: 396.5227). Fur-
ther elution of the column with system (1) (15% Me2CO in
C6H14) gave a fraction containing a mixture of components 10
and 11. Flash chromatography of this fraction on silica gel in
system (2) (3% and 5% MeOH in CHCl3) yielded 17 mg of
flavon 10 and 20 mg triterpenoid 11.
5,7,4´-Trihydroxyflavon (apigenin) (10), m.p. 343—344 °C
(EtOH) (cf. Ref. 13: m.p. 344 °C). IR, ν/cm–1: 3426 (OH),
1655 (C=O), 1611 (C=C), 1489 (arom.), 830 (C—H). C15H10O5.
MS (EI, 70 eV), m/z (Irel (%)): 270 [Ì]+ (7), 214 [M – 56]+
(17). 1H NMR (CD3OD), δ: 8.04 (d, 2 H, H(2´), H(6´),
J = 8.9 Hz); 7.20 (d, 2 H, H(3´), H(5´), J = 8.9 Hz); 6.85 (s,
1 H, H(3)); 6.80 (d, 1 H, H(8), J = 2.4 Hz); 6.73 (d, 1 H,
H(6), J = 2.4 Hz).
–
J
= 7.5 Hz). The 13C NMR spectral data are listed in
Tables 1 and 2.
3-O-[O-(α-L-Rhamnopyranosyl)-(1→2)-O-(β-D-xylopyra-
nosyl)-(1→2)-(β-glucuronîpyranosyl)]-3β,22β,24-trihydroxy-
olean-12-ene (4). White needles, m.p. 223—225 °C (MeOH),
23
[α]D –11.2 (c 1.2; ÌåOH) (cf. Ref. 6: m.p. 223—224 °C,
[α]D –12.1 (c 1.0; ÌåOH)). IR, ν/cm–1: 3420 (OH), 1600 br
23
–
(COO ), 1070, 1045 (C—O—). Negative-ion FAB MS, m/z:
–
–
911 [M – H] , 765 [(Ì – H) – 146] , 633 [(Ì – H) – 146 –
–
–
132] and 457 [(Ì – H) – 146 – 132 – 176] Found, m/z:
–
–
911.4992 [M – H] . C47H75O17. Calculated: [Ì – 1] 911.5004.
1H NMR (the signals for the anomeric protons of carbo-
hydrates, CD3OD), δ: 5.19 (d, 1 H, H(1) Rha, J = 1.5 Hz);
4.78 (d, 1 H, H(1) Xyl, J = 8.0 Hz); 4.40 (d, 1 H, H(1) GlcA,
J
=
7.0 Hz). The 13C NMR spectral data are listed in
Tables 1 and 2.
3-O-[O-(α-L-Rhamnopyranosyl)-(1→2)-O-(β-D-glucopyra-
nosyl)-(1→2)-(β-glucuronîpyranosyl)]-3β,22β,24-trihydroxy-
olean-12-ene (5). White needles, m.p. 244—246 °C (MeOH),
23
[α]D –21.9 (c 0.6; ÌåOH) (cf. Ref. 9: m.p. 245—247 °C,
–
[α]D –22.5 (c 0.42; ÌåOH)). IR: 3420 (OH), 1600 (br, COO ),
1070, 1045 (C—O—). Negative-ion FAB MS, m/z:
20(R),24(S)-Epoxycyclolanost-9(11)-ene-3β,6α,16β,25-
tetrol (cycloastragenol) (11), m.p. 239—240 °C (EtOH),
–
–
941[M – H] , 795 [(Ì – H) – 146] , 633 [((Ì – H) – 146 –
–
–
23
162)] and 457 [(Ì – H) – 146 – 162 – 176)] . Found, m/z:
[α]D +46.6 (c 0.8; ÌåOH) (cf. Ref. 14: m.p. 239—241 °C;
–
–
23
941.5112 [M – H] . C48H77O18. Calculated: [Ì – 1] 941.5110.
1H NMR (the signals of anomeric protons of carbohydrates,
CD3OD), δ: 5.22 (d, 1 H, H(1) Rha, J = 1.5 Hz); 4.92 (d, 1 H,
H(1) Glc, J = 7.5 Hz); 4.42 (d, 1 H, H(1) GlcA, J = 7.0 Hz).
The 13C NMR spectral data are listed in Tables 1 and 2.
[α]D +50.6). IR, ν/cm–1: 3450—3250 (OH), 3045 (>CH2 of
the cyclopropane ring). MS (EI, 15 and 70 eV), m/z (Irel (%)):
490 [Ì]+ (2), 472 [Ì – H2O]+ (18), 143 [C18H15O2]+ (100).
–
Found, m/z: 489.3569 [M – H] . C30H49O5. Calculated:
–
[M – H] 489.3571. 1H NMR (CD3OD), δ: 4.60 (dd, 1 H,

1112
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 6, June, 2001
Gromova et al.
H(16), J = 6.8, 7.8 Hz); 3.78 (t, 1 H, H(24), J = 6.4 Hz); 3.45
(td, 1 H, H(6), J = 2.9, 10.9 Hz); 3.21 (dd, 1 H, H(3), J = 4.4,
10.3 Hz); 0.54, 0.48 (both d, each 1 H, 2 H(19), J = 4.9 Hz).
The 13C NMR spectral data are listed in Table 1.
3. R. L. Baxter, K. R. Price, and G. R. Fenwick, J. Natural
Products, 1990, 53, 298.
4. W. Klyne, Biochem. J., 1950, 47, xli.
5. Slovar' organicheskikh soedinenii [Dictionary of Organic Com-
pounds], Izd-vo inostr. lit., Moscow, 1949, 2, 116; 3, 599,
936 (Russ. Transl.).
6. I. Kitagawa, H. K. Wang, and M. Yoshikawa, Chem. Pharm.
Bull., 1983, 31, 716.
7. T. Fukunaga, K. Nishiya, and K. Takeya, Chem. Pharm.
Bull., 1987, 35, 1610.
The cytotoxic activity studied by K. Metcalf (Labo-
ratory of Biochemistry, Brigham Young University) and
the bacteriostatic activity was assayed by J. Donaldson
and L. Ford (Department of Botany, Brigham Young
University).
8. S. Seo, J. Tomita, K. Tori, and Y. Yashimura, J. Am. Chem.
Soc., 1978, 100, 3331.
9. S. B. Mahato, Phytochemistry, 1991, 30, 3389.
10. Beilst., Å III 6, 6927.
The authors are grateful to B. Jackson (Brigham
Young University) for recording mass spectra, to S. Wood
(Brigham Young University) for fruitful discussion, and
to D. M. Grant (University of Utah, Salt Lake City,
UT, US) for support in performing this work.
11. Beilst., E III 2, 1508.
12. N. Ina and N. Iida, Chem. Pharm. Bull., 1986, 34, 726.
13. W. Karrer, Konstitution und Vorkommen der organi-
schen Pflanzenstoffe, Birkhauser-Verlag, Basel—Stuttgart,
1958, 1449.
14. M. I. Isaev, M. B. Gorovits, and N. K. Abubakirov, Khim.
Prirod. Soedinenii, 1989, 156 [Chem. Nat. Compd., 1989
(Engl. Transl.)].
References
1. Rastitel'nye resursy SSSR [Plant Resources of the USSR],
Ed. P. D. Sokolov, Nauka, Moscow, 1987, 3, 112 (in
Russian).
2. I. Kitagawa, M. Yoshikawa, and I. Yosioka, Chem. Pharm.
Bull., 1976, 24, 121.
Received July 12, 2000;
in revised form March 16, 2001