Isolation and structural determination of triterpenoid glycosides from the aerial parts of alsike clover (Trifolium hybridum L.)

Article abstract of DOI:10.1021/jf305541e

Five azukisapogenol glycosides (1-5) have been isolated from the aerial parts of alsike clover (Trifolium hybridum L.), and their structures were elucidated by combined spectroscopic, spectrometric (1D and 2D NMR; HRESIMS, ESI-MS/MS), and chemical methods. Three of them are new compounds and were identified as 3-O-[-α-l-arabinopyranosyl(1→2)]-β-d- glucuronopyranosyl azukisapogenol (1), 3-O-[-β-d- glucuronopyranosyl(1→2)-β-d-glucuronopyranosyl]-29-O-β-d- glucopyranosyl azukisapogenol (2), and 3-O-[-α-l- arabinopyranosyl(1→2)-β-d-glucuronopyranosyl]-29-O-β-d- glucopyranosyl azukisapogenol (3). The remaining two (4, 5) are known compounds but have not been previously described as saponins constituents of the genus Trifolium. Also, azukisapogenol is reported here as a triterpenoid aglycone for the first time in this genus. Finally, the main chemotaxonomic features that may be recognized as specific of Trifolium species were discussed.

Full text of DOI:10.1021/jf305541e

Article
pubs.acs.org/JAFC
Isolation and Structural Determination of Triterpenoid Glycosides
from the Aerial Parts of Alsike Clover (Trifolium hybridum L.)
,
†
†
‡
‡
†
Andy J. Per
́
ez,* Mariusz Kowalczyk, Ana M. Simonet, Francisco A. Macías, Wiesław Oleszek,
†
and Anna Stochmal
†Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100,
Puławy, Poland
‡
Grupo de Alelopatía, Departamento de Química Organ
1510 Puerto Real, Cadiz, Spain
́
́
ica, Facultad de Ciencias, Universidad de Cad
́
iz, C/Repub
́
lica Saharaui, s/n,
1
*
S Supporting Information
ABSTRACT: Five azukisapogenol glycosides (1−5) have been isolated from the aerial parts of alsike clover (Trifolium hybridum
L.), and their structures were elucidated by combined spectroscopic, spectrometric (1D and 2D NMR; HRESIMS, ESI−MS/
MS), and chemical methods. Three of them are new compounds and were identified as 3-O-[-α-L-arabinopyranosyl(1→2)]-β-D-
glucuronopyranosyl azukisapogenol (1), 3-O-[-β-D-glucuronopyranosyl(1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyrano-
syl azukisapogenol (2), and 3-O-[-α-L-arabinopyranosyl(1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl azukisapoge-
nol (3). The remaining two (4, 5) are known compounds but have not been previously described as saponins constituents of the
genus Trifolium. Also, azukisapogenol is reported here as a triterpenoid aglycone for the first time in this genus. Finally, the main
chemotaxonomic features that may be recognized as specific of Trifolium species were discussed.
KEYWORDS: alsike clover, Trifolium hybridum L., triterpenoid saponins, azukisapogenol, structural elucidation, HRESIMS,
ESI−MS/MS, NMR
INTRODUCTION
The qualitative studies of saponins in the seeds of T.
10
■
hybridum and 56 other species of Trifolium confirmed the
presence of glycosides of soyasapogenol B in all tested species,
as well as 22-O-glucosylated or 22-O-diglucosylated derivatives
of soyasaponin I in some of them. Phytochemical analysis of the
aerial parts of a number of species of Trifolium proved the
presence of phenolic acids, clovamides, and flavonoids in T.
Alsike clover (Trifolium hybridum L., Fabaceae) is a perennial
plant, which has been widely cultivated as forage, cover crops,
and for erosion control. It is distributed worldwide, especially in
Europe, North and South America, Northwest and South of
1
Africa, and Western Asia.
Apart from its potential interest as forage crop, it has been
also of interest for its content of saponins. This group of
secondary metabolites is well-known to possess a broad
spectrum of biological properties, such as molluscicidal, anti2-
inflammatory, antifungal, hemolytic, cytotoxic, and antitumor.
Saponins have also been used as food and feed additives,
providing benefits to human and animal health, and perform-
ance. The beneficial effects of saponins, such as the defaunation
11
hybridum. However, the occurrence of saponins in the aerial
parts of this plant has not been investigated so far.
Therefore, the aim of the current investigation was to isolate
and elucidate the chemical structures of the saponins in aerial
parts of T. hybridum, and additionally to contribute to the
chemotaxonomic knowledge in the genus Trifolium.
3
MATERIALS AND METHODS
of microorganisms in ruminants, have been shown. In
■
addition, several human feeding studies suggest that the
consumption of soybean and other legumes, which contain
soyasaponins, has hypocholesterolemic activity and may be
beneficial in diabetes control.
As indicated by several studies on the species of the genus
Plant Material. Seeds of authenticated material of T. hybridum
were provided by Genebank, Zentralinstitute fu
̈
r Pflanzen-genetik and
Kulturpflanzenforschung (Gatersleben, Germany). Seeds were planted
(1 m × 1 m plots) in an experimental field of the Institute of Soil
Science and Plant Cultivation in Puławy, Poland. The plants were
harvested at the beginning of flowering, lyophilized, finely powdered,
and used for the successive extraction.
4
5
6,7
8
Trifolium, such as T. incarnatum, T. repens, T. alexandrinum,
9
and T. resupinatum, the saponins in these plants are the
mixture of triterpenic pentacyclic glycosides being most often
derivatives of soyasapogenol A, B, C, and E. These have as a
common feature the hydroxymethyl group at C-24 and a
double bond between C-12 and C-13. The sugars portion
consists of one or three units, where a β-D-glucuronic acid
moiety is linked at the C-3 position of the aglycone. The
bidesmosidic saponins that have been also identified usually
had a diglucoside linkage at C-22.
Extraction and Purification. The dried and finely powdered T.
hybridum aerial parts (leaves, stems, and flowers, 300 g) were defatted
with CHCl in a Soxhlet apparatus (fats made up 3.8% of dried
3
material). Defatted material (288 g) was then extracted with 70%
Received: December 28, 2012
Revised: February 25, 2013
Accepted: February 26, 2013
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/jf305541e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
MeOH with reflux for 2 h. The solvent was removed under reduced
pressure, and the residue was resuspended in water. The solution was
applied onto a 10 cm × 6 cm, 40−63 μm LiChroprep RP-18 glass
column (Merck, Darmstadt, Germany), previously conditioned with
water. The column was washed first with water and then with 40%
MeOH to remove sugars and phenolics. Total saponins were eluted
with 80% MeOH and dried under vacuum; 11.7 g (4.1%, of defatted
material) of crude saponins was obtained.
fragmentation amplitude of 1.0 V, threshold set at 100, and ion charge
control on, with maximum acquiring time set at 250 ms.
NMR Measurements. All experiments were performed on a
1
15
31
Varian INOVA-600 spectrometer equipped with 5 mm H { N− P}
1
PFG high-field inverse detection z-gradient probe. H (599.78 MHz)
and 13C (150.83 MHz) NMR spectra were recorded in pyridine-d or
5
pyridine-d /D O (9:1) at 25 °C. Chemical shifts are given on the δ
5
2
scale and were referenced to residual pyridine, δ 8.70, 7.55, 7.18 and
H
Fractionation and Separation. Crude saponins powder (2 g)
δC 149.84, 135.50, 123.48. Varian pulse sequences using gradient were
applied, and all 2D spectra, except for HMBC, were recorded in the
phase-sensitive mode. The 1D TOCSY and 1D ROESY spectra were
recorded using a PFG selective excitation. For 1D TOCSY, the
MLEV17, mixing time was set with an acquisition array of 15, 30, 55,
was suspended in 30% MeOH and loaded onto a 40 cm × 3 cm, 40−
63 μm LiChroprep RP-18 column (Millipore Corp., Bedford, MA).
The column was washed with a 30−100% step gradient (steps of 10%)
of MeOH in water. Ten milliliter fractions were collected and checked
by TLC on Si 60 F254 silica gel (Merck, Warsaw, Poland), developed
with ethyl acetate/acetic acid/water (7:2:2). Chromatograms were
sprayed with Liebermann−Burchard reagent and heated at 130 °C.
Fractions showing similar profiles were combined. Column
chromatography gave 22 fractions, from F6 to F22, each comprising
two to four major saponins.
7
0, 100, 150, 200 ms and for 1D ROESY, a 200 ms spin lock mixing
1
time. On the basis of 1D and 2D NMR analysis, assignments of H and
13
C signals were obtained.
Acid Hydrolysis of Saponins. Compounds 1−5 (each 2 mg)
were treated with 1 N HCl (1 mL) at 80 °C for 3 h. After cooling, the
solvent was eliminated with a stream of N , dry residue was suspended
2
Single saponins were separated from each fraction by Si 60 silica gel
in water, and aglycones were extracted with ethyl acetate (3 × 2 mL).
(
Merck) chromatography on a 20 cm × 1 cm column, 15−25 μm,
The aqueous layer, containing sugars, was neutralized with Amberlite
12
LiChroprep eluted with ethyl acetate/acetic acid/water (9:2:2). In
−
IRA-400 (OH form). For each samples, both the organic and the
this way, five (1−5) pure compounds were isolated (Figure 1).
aqueous solution were dried under N and stored for the subsequent
2
analyses.
Identification of Aglycones. All saponins yielded the same
aglycone, which was identified as azukisapogenol by comparison of its
1
13
physical data ([α] , melting point, H NMR, C NMR, and MS) with
reported values.
D
1
3
Determination of Absolute Configurations of Sugars. To
determine the absolute configuration of monosaccharide constituents
14
of isolated compounds 1−5, the method of Tanaka et al. was used
with slight modifications. Sugars of each sample were dissolved in
pyridine (0.5 mL) containing L-cysteine methyl ester hydrochloride (2
mg) and heated at 60 °C for 1 h; o-tolyl isothiocyanate (2 μL) was
then added, and the mixture was heated at 60 °C for 1 h. Each reaction
mixture was directly analyzed by reversed-phase HPLC using a model
6
16 pump, a 996 photodiode array detector, and a 717-plus
autosampler (Waters, Milford, MA). The column used was a 250
mm × 4 mm i.d., 5 μm, Eurospher 100 C18 (Knauer, Berlin,
Germany); mobile phase MeCN−H O (25:75, v/v) containing 50
2
mM H PO ; detection UV (250 nm); flow rate 0.8 mL/min; column
3
4
temperature 35 °C. The HPLC column was washed with MeOH after
each injection.
The derivatives of monosaccharides L-arabinose, D-glucuronic acid
in 1, D-glucuronic acid, D-glucose in 2, L-arabinose, D-glucose, D-
glucuronic acid in 3, D-glucuronic acid in 4, and D-glucose, D-
glucuronic acid in 5 were identified by comparison of their retention
times with those of authentic samples (Sigma-Aldrich, Steinheim,
Figure 1. Structures of saponins 1−5 isolated from T. hybridum aerial
parts.
Germany) treated in the same way as described above (R : D-glucose
14.57 min, L-glucose 13.33 min, D-arabinose 17.55 min, L-arabinose
t
1
6.63 min, D-glucuronic acid 14.48 min, L-glucuronic acid 13.95 min).
Melting points were determined using a Buchi (Uster, Switzerland)
̈
apparatus. Optical rotations were measured on a Perkin-Elmer 241
polarimeter.
Mass Spectra. Exact masses of the new saponins were measured
by an UPLC-QTOF ESI (Waters Synapt G2, Manchester, UK) high
resolution mass spectrometer (HRMS). The samples were dissolved
with methanol and analyzed. Mass spectra were recorded in positive
ion mode with the range of m/z 100−2000, mass resolution of 20 000,
and acceleration voltage of 0.7 kV.
2
0
Chemical Data of Saponins 1−5. Saponin 1: [α]
+11.0
D
(
MeOH, c 0.1). HRESIMS, m/z 803.4201 [M + Na]+ calcd for
C H O Na, 803.4194. ESI−MS (negative ion mode), m/z (relative
41 64 14
−
intensity) 779.4 (100%) [M − H] , which fragmented in the MS/MS
−
giving 629 (100%) [(M − H) − 150(Ara + H O)] , 471 (29%) [(M
The fragmentation patterns of the saponins were obtained using a
Thermo LCQ Advantage Max ion-trap mass spectrometer. The
2
−
1
13
−
H) − 150(Ara + H O) − 158(GlcA−H O)] . H and C NMR
2
2
samples were dissolved in MeOH/H O (8:2) at the concentration of
spectra are reported in Tables 1 and 2.
Saponin 2: [α]20
−6 (MeOH/H
1009.4611 [M + Na] calcd for C48H O21Na, 1009.4620. ESI−MS
74
2
100 ppm and injected by direct infusion at a flow rate of 5 μL/min
D
2
O, 1:1, c 0.3). HRESIMS, m/z
+
with a syringe pump. The spectrometer was operated in the negative
electrospray mode with the following parameters: spray voltage 3.9 kV,
capillary voltage −47 V, tube lens offset −60 V, capillary temperature
(negative ion mode), m/z (relative intensity) 985.5 (100%) [M −
−
H] , which fragmented in the MS/MS giving 823.3 (100%) [(M − H)
−
−
2
40 °C. Nitrogen sheath and auxiliary gas flows were of 65 and 10
− 162(Glc)] , 647.4 (2%) [(M − H) − 162(Glc) − 176(GlcA)] ,
−
arbitrary units, respectively. Full scan spectra were acquired over the
range of m/z 150−2000. Automated MS/MS was performed by
isolating the molecular ion using an isolation width of m/z 1.0,
471.5 (2%) [(M − H) − 162(Glc) − 176(GlcA) − 176(GlcA)] ,
−
1
13
351.1 (54%) [(GlcA(1→2)GlcA − H O) − H] . H and C NMR
2
spectra are reported in Tables 1 and 2.
B
dx.doi.org/10.1021/jf305541e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
a
Table 1. NMR Spectroscopic Data (600 MHz) for the Aglycones of Compounds 1−3
b
b
c
1
2
3
carbon
1
δC, type
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
38.6, CH2
1.30 m
.74 m
2.23 m
.91 dddd (13.5, 12.9, 12.1, 3.8)
38.8
1.36 m
37.5
1.25 m
0.68 m
2.33 m
1.76 m
0
0.82 m
2
26.7, CH2
26.6
2.23 m
25.3
1
1.95 m
3
4
5
6
90.4, CH
44.0, C
3.36 dd (12.1, 4.5)
90.0
44.0
56.2
19.0
3.47 dd (11.5, 4.5)
89.3
42.6
55.0
17.3
3.33 dd (11.9, 4.1)
56.0, CH
18.6, CH2
0.76 m
1.53 m
0.78 dd (6.9, 2.2)
1.61 m
0.62 m
1.35 m
1.00 m
1.17 m
1.00 m
1
.28 m
1.35 m
.20 m
1.41 m
7
33.0, CH2
33.1
1.33 m
31.8
1
1.17 m
8
9
40.0, C
39.9
47.6
38.7
46.4
47.6, CH
36.5, C
1.49 m
1.42 m
1.24 m
1
1
1
1
1
1
0
1
2
3
4
5
36.5
35.2
24.0, CH2
122.9, CH
144.5, C
41.8, C
1.75 m (2H)
5.22 t (3.7)
23.9
1.71 m (2H)
5.12 t (3.8)
22.8
1.54 m (2H)
4.98 t (3.8)
123.0
144.2
41.7
121.9
143.1
40.6
26.4, CH2
1.71 m
26.3
1.64 m
0.84 m
25.1
1.48 m
0.69 m
1.78 m
0.60 m
0
.92 m
2.13 m
.84 brd (12.6)
1
6
27.2, CH2
27.0
1.94 ddd (13.6, 13.3, 4.5)
0.72 m
25.8
0
1
1
1
7
8
9
32.7, C
32.6
46.2
40.6
31.4
45.0
39.3
46.4, CH
41.5, CH2
2.11 m
2.01 dd (13.5, 4.3)
2.43 dd (13.8, 13.5)
1.61 m
1.87 dd (13.6, 2.9)
2.26 dd (13.6, 13.8)
1.44 m
2.54 dd (13.7, 13.7)
1
.68 m
2
2
0
1
42.8, C
43.1
29.5
42.0
28.2
29.9, CH2
2.25 m
.70 m
1.53 m
.36 m
1.59 m
1.22 m
1.43 m
1.24 m
1.41 s
1.89 m
1.45 m
1.27 m
1.11 m
1.15 s
1
2
2
36.5, CH2
36.0
34.8
1
2
2
3
4
22.5, CH3
63.3, CH2
1.26 s
22.8
63.3
21.4
62.1
4.29 d (11.5)
4.35 d (11.6)
3.63 d (11.6)
0.76 s
4.10 d (11.7)
3.21 d (11.7)
0.51 s
3
.36 d (11.5)
2
2
2
2
2
3
5
6
7
8
9
0
15.6, CH3
16.8, CH3
26.0, CH3
28.3, CH3
181.2, C
0.71 s
0.88 s
1.22 s
0.91 s
15.5
16.7
25.9
28.2
177.7
19.5
14.4
15.6
24.8
27.0
177.5
18.3
0.84 s
0.66 s
1.08 s
0.93 s
0.84 s
0.70 s
19.9, CH3
1.46 s
1.36 s
1.24 s
a
1
1
b
The assignments were based on H− H COSY, 2D-TOCSY, HSQC, HSQC-TOCSY, and HMBC experiments. Acquired in pyridine-d5.
c
Acquired in pyridine-d /D O (9:1).
5
2
Saponin 3: [α]20 +26 (MeOH/H O, 1:1, c 0.1). HRESIMS, m/z
D
2
RESULTS AND DISCUSSION
■
9
65.4709 [M + Na]+ calcd for C H O Na, 965.4722. ESI−MS
47
74 19
Structure Elucidation. Crude saponins were fractionated
(
negative ion mode), m/z (relative intensity) 941.4 (100%) [M −
by reverse-phase (RP-18) column chromatography in a
gradient of water/methanol to yield a number of fractions,
which then were further purified on a silica gel column using
acidified solvent to suppress ion formation on the column. As a
result, the saponins 1−5 were obtained in pure forms (Figure
), out of which 1−3 are reported for the first time in this
study. To elucidate the chemical structures of the new
compounds, combined NMR, HRESIMS, and ESI−MS/MS
techniques were employed. The assignments of all C and H
signals were performed on the basis of 2D NMR experiments
−
H] , which fragmented in the MS/MS giving 779.4 (100%) [(M − H)
−
−
162(Glc)] , 629.3 (3%) [(M − H) − 162(Glc) − 150(Ara +
−
H O)] , 471.3 (1%) [(M − H) − 162(Glc) − 150(Ara + H O) −
2
2
−
1
13
1
58(GlcA − H O)] . H and C NMR spectra are reported in Tables
2
1
1
and 2.
Saponin 4: ESI−MS (negative ion mode), m/z (relative intensity)
−
6
47.3 (100%) [M(C H O ) − H] , which fragmented in the MS/
36
56 10
13
1
−
MS giving 471.4 (100%) [(M − H) − 176(GlcA)] .
Saponin 5: ESI−MS (negative ion mode), m/z (relative intensity)
(
HSQC, HMBC, DQF-COSY, TOCSY, ROESY, HSQC-
−
8
09.3 (100%) [M(C H O ) − H] , which fragmented in the MS/
42
66 15
TOCSY) and selective excitation methods (1D-ROESY and
1D-TOCSY acquired “in array”) and are reported in Tables 1
and 2. The exact molecular weights were obtained by
−
MS giving 647.4 (100%) [(M − H) − 162(Glc)] , 471 (3%) [(M −
−
H) − 162(Glc) − 176(GlcA)] .
C
dx.doi.org/10.1021/jf305541e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Table 2. NMR Spectroscopic Data (600 MHz) for the Sugar
Units of Compounds 1−3
which correlate in the HSQC experiment with the carbon
signals at δ 15.6, 16.8, 28.3, 26.0, 22.5, and 19.9, respectively.
An additional feature was the signal at δ 5.22 (t, J = 3.7 Hz)
a
b
b
c
1
2
3
12
typical of H-12 of the Δ -oleanene skeleton, which was
confirmed by the presence in the 13C NMR spectrum of the
olefinic signals at δ144.5 and 122.9 attributable to C-13 and C-
δH (J in
Hz)
δH (J in
Hz)
carbon
β-D-GlcA
105.2 4.91 d
δ
C
δ
C
δ
H
(J in Hz)
δ
C
β-D-GlcA
β-D-GlcA
1
2. The occurrence of a carboxylic group was suggested for the
1′
2′
3′
4′
5′
6′
104.7 5.02 d (7.2)
103.2 4.68 d (8.0)
13
signal at δ 181.2 in the C NMR spectrum, which was assigned
as C-29 due to the HMBC correlation throughout three bonds
with the methyl group at δ 1.46 (C-30). Its equatorial
orientation was also shown by 2D ROESY spectrum, in
which a correlation between the signal at δ 2.11 (H-18) and the
C-30 methyl group was clearly observed (Figure 2). A signal
(7.7)
78.6 4.40 dd
81.1 4.37 dd (7.2,
9.0)
79.0 4.03 dd
(8.0, 8.0)
(7.7, 9.0)
78.0 4.34 dd
78.0 4.39 dd (9.0,
8.2)
76.7 4.17 dd
(8.0, 9.1)
(9.0, 9.0)
73.5 4.54 dd
73.2 4.54 dd (8.2,
9.7)
71.9 4.14 dd
(9.1, 9.7)
(9.0, 9.6)
77.6 4.58 d
77.6 4.61 d (9.7)
76.7 4.18 d (9.7)
(9.6)
172.7
α-L-Ara
104.8 5.48 d
7.5)
73.6 4.43 dd
172.3
171.9
β-D-GlcA
α-L-Ara
1
2
3
″
″
″
105.1 5.74 d (7.2)
103.5 5.10 d (7.7)
(
75.7 4.27 dd (7.2,
9.2)
71.7 4.21 dd
(7.7, 9.5)
(7.5, 9.4)
74.9 4.04 dd
77.8 4.28 dd (9.2,
4.9)
73.2 3.87 dd
(9.5, 3.5)
(9.4, 3.4)
4
5
″
″
70.3 4.13 m
67.5 4.23 d
73.2 4.55 m
77.7 4.55 m
68.7 4.03 m
66.1 4.12 d
(12.3)
(
12.3)
.66 d
12.3)
3
3.50 d
(12.3)
(
6
″
170.0
β-D-Glc
β-D-Glc
Figure 2. Main correlations observed in 2D ROESY spectrum for
aglycone moiety of 1, azukisapogenol.
1
2
″′
″′
96.0 6.37 d (8.1)
74.3 4.19 dd (8.1,
94.5 6.06 d (8.2)
72.6 4.00 dd
(8.2, 8.7)
9
.0)
78.7 4.27 dd (9.0,
.9)
71.0 4.34 dd (8.9,
.7)
79.5 4.04 ddd (9.7,
.5, 2.6)
62.1 4.45 d (12.0,
.6)
.37 dd (12.0,
3
4
5
6
″′
″′
″′
″′
76.7 4.13 dd
(8.7, 9.2)
1
present in H NMR at δ 3.36 and its correlation in HSQC
8
experiment with the carbon resonance at δ 90.4 suggested the
presence of a secondary alcoholic group. The location of this
hydroxyl group was determined to be at 3β (equatorial), by
means of analysis of the value of coupling constants of H-3 (dd,
69.5 4.03 dd
(9.2, 9.5)
9
77.7 3.87 m
4
3
3
60.6 4.23 d
(12.3)
J = 12.1, 4.5 Hz), which indicate both J (axial−axial) and J
2
(
axial−equatorial) coupling with H-2 (δ 1.91) and H-2 (δ
.23), respectively. At the same time, the typical 24-
ax
eq
4
4.08 dd
(12.3,
2
4
.5)
1
5
.1)
hydroxymethyl group of the aglycone was deduced by the H
NMR signals at δ 3.36 (d, J = 11.5 Hz) and δ 4.29 (d, J = 11.5
Hz), which showed a cross peak in HSQC experiment with the
carbon resonance signal at δ 63.3, as well as in HMBC
spectrum with carbon signals at δ 22.5 (C-23, methyl group)
and δ 90.4 (C-3), being consistent with the data reported in the
a
1
1
The assignments were based on H− H COSY, 1D-TOCSY, 1D-
ROESY, HSQC, HSQC-TOCSY, and HMBC experiments. Acquired
in pyridine-d . Acquired in pyridine-d /D O (9:1).
b
c
5
5
2
HRESIMS and the fragmentation ions by means of ESI−MS/
MS, which were also used to establish sugar chain in the
molecules. The absolute configurations of the sugar residues of
each compound were determined after hydrolysis using the
6,8,15
literature.
The NOEs observed in 2D ROESY spectrum
(Figure 2) were used to establish the three-dimensional
disposition of the rest of substituents in the aglycone, as well
as the relative configurations of chiral centers. Therefore, the
structure of aglycone of 1 was elucidated as (20R,4S)-3β,24-
dihydroxy-12-en-olean-29-oic acid, named azukisapogenol, and
previously identified as a constituent of saponins in Vigna
17
14
method of Tanaka et al. with slight modifications, and the
aglycones moieties were confirmed by comparison of their
physical data with those already reported in the literature.
Compound 1 (6 mg) was isolated as an amorphous white
solid. The HRESI mass spectrum supported a molecular
formula of C H O . The ESI−MS showed a molecular ion
13
16
angulariz, Oxytropis glabra, and Oxytropis myriophylla.
However, it is reported here for the first time in the genus
Trifolium. This result was confirmed after hydrolysis of 1, which
41
64 14
−
peak at m/z 779 [M − H] , and the MS/MS spectrum of this
ion showed the fragment ion peaks at m/z 629 [(M − H) −
yielded an aglycone identified as azukisapogenol by [α] ,
D
−
1
13
1
50] , corresponding to the loss of a pentose unit plus water,
melting point, H NMR, C NMR, and MS data as compared
−
13,16,17
and m/z 471 [(M − H) − 150 − 158] , due to further loss of a
hexosyluronic acid unit less water. The triterpenoid glycosidic
structure for this compound was also confirmed by NMR
spectra (Tables 1 and 2). The H NMR spectrum (in pyridine-
d5) showed for the aglycone moiety (Table 1) signals of six
tertiary methyl groups at δ 0.71, 0.88, 0.91, 1.22, 1.26, and 1.46,
to those reported in the literature.
In addition, H and 13C NMR spectra of 1 showed the
presence of two anomeric protons at δ 4.91, 5.48 and carbons
at δ 105.2, 104.8 (Table 2). The individual sugar units were
identified by a combination of DQF-COSY, 1D TOCSY, and
1D ROESY experiments. 1D TOCSY and 1D ROESY spectra
1
1
D
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Journal of Agricultural and Food Chemistry
Article
were acquired from selective excitation of each anomeric
proton and were in particular very helpful. Selective TOCSY
experiment for signal at δ 4.91 revealed a typical spin system of
β-glucuronic acid (Figure 3A); while for the signal at δ 5.48
Compound 2 (16 mg) was isolated as an amorphous white
solid; the molecular formula was determined as C H O . Its
4
8
74 21
−
ESI−MS showed a molecular ion peak at m/z 985 [M − H] .
Other fragmentation ion peaks were observed at m/z 823 [(M
H) − 162] , 647 [(M − H) − 162 − 176] , and 471 [(M −
H) − 162 − 176 − 176] , corresponding to the successive loss
of one hexose residue and two hexosyluronic acid moieties,
respectively. ESI−MS/MS also showed a peak at m/z 351,
which corresponded to two hexosyluronic acid units obtained
by the breaking of glycosidic bond with the aglycone and losing
of a molecule of water. Assignments of all NMR signals of the
aglycone portion of 2 were performed from extensive 2D NMR
−
−
−
−
1
13
analysis. The H and C NMR chemical shifts (in pyridine-d )
5
of the aglycone moiety of 2 (Table 1) were similar to those of
1
, indicating also the presence of azukisapogenol. Only slight
differences were detected between them, mainly in the carbon
resonance signal of carboxylic group at C-29 (δ 177.7), which
was found to be 3.5 ppm shifted to higher field than that of 1,
1
due to a glycosidic esterification. The H NMR spectrum of 2
showed signals for three anomeric protons at δ 5.02, 5.74, and
6
.37, which had correlations in the HSQC spectrum with the
anomeric carbon signals at δ 104.7, 105.1, and 96.0,
respectively. The nature of sugar units was determined using
the same procedure as described above for 1. In this way, the
signals at δ 5.02 and δ 5.74 were identified as β-glucuronic acid
and δ 6.37 as β-glucose. The connections of sugars to the
aglycone were suggested by HMBC experiment, where long-
range correlations between the anomeric signal at δ 5.02 (H-
1GlcA′) and signal at δ 90.0 (C-3), as well as between signal at δ
Figure 3. 1D TOCSY experiments acquired “in array”. Shown
subspectra corresponding to 15, 30, 55, and 200 ms, obtained by
irradiation of anomeric protons of 1 at (A) δ 4.91 (β-glucuronic acid)
and (B) δ 5.48 (α-arabinose).
6
.37 (H-1 ) and signal at δ 177.7 (C-29), were observed, and
Glc
confirmed the presence of a bidesmosidic saponin. The chain
GlcA″(1→2)GlcA′ was also shown by a cross peak between the
signal at δ 5.74 (H-1 ″) and δ 81.1 (C-2 ) observed in
HMBC experiment, and confirmed by a ROESY correlation
between δ 5.74 (H-1GlcA″) and δ 4.37 (H-2GlcA′). The acid
hydrolysis of 2 and subsequent HPLC analysis of sugar units
confirmed the presence of D-glucose and D-glucuronic acid; the
peaks at 14.69 and 14.45 min were coincident with the
(
Figure 3B), it allowed one to observe correlations for H-1″/H-
GlcA
GlcA′
2
″ and H-2″/H-3″, and a relatively small coupling constant of
H-3″/H-4″. This evidence together with the 1D ROESY
correlations of H-1″/H-3″ and H-1″/H-5″ indicated the
presence of α-arabinose as a sugar unit. Analysis of the
HSQC and HSQC-TOCSY experiments allowed the assign-
1
3
ment of the signals in C NMR spectrum corresponding to
both sugar units. The sequence of sugars chain and connection
with the aglycone then were determined by the employment of
the HMBC experiment. Thus, the anomeric signal at δ 4.91 (H-
1GlcA) showed a long-range correlation with the signal at δ 90.4
derivative of D-glucose and D-glucuronic acid (R of D-glucose,
t
1
4.57; R of D-glucuronic acid, 14.48), respectively. This
t
compound was elucidated as 3-O-[-β-D-glucuronopyranosyl-
(
1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl azu-
kisapogenol and represents a new bidesmosidic natural saponin.
Compound 3 (9 mg) was isolated as an amorphous white
solid; the molecular formula was determined as C H O . Its
(
C-3), indicating that the glucuronic acid is directly linked to
the triterpenic aglycone at C-3, which was confirmed by the
4
7
74 19
observed correlation in 1D ROESY between δ 4.91 (H-1GlcA
and δ 3.36 (H-3). The chain Ara(1→2)GlcA was also shown by
HMBC experiment, where a clear cross peak between the signal
at δ 5.48 (H-1 ) and δ 78.6 (C-2GlcA) was observed, and
confirmed by 1D ROESY correlation between δ 5.48 (H-1Ara)
and δ 4.40 (H-2GlcA). To verify the nature of the sugar units and
to determine their absolute configurations, the sugars obtained
after acid hydrolysis of 1 were converted into the thiazolizine
derivatives to arylthiocarbamate using L-cysteine methyl ester
and o-tolylisothiocyanate. The reaction mixture then was
analyzed by C18 HPLC, and the retention times (R ) were
compared to values obtained for derivatives of each D- or L-
authentic sugars. The peaks at 14.45 and 16.59 min coincided
with derivatives of D-glucuronic acid and L-arabinose (R of D-
)
−
ESI−MS displayed a molecular ion peak at m/z 941 [M − H] .
The MS/MS of this ion showed a pattern similar to that in 1,
with the fragmentation ion peaks at m/z 779 [(M − H) −
Ara
−
−
1
1
62] , 629 [(M − H) − 162 − 150] , and 471 [(M − H) −
−
62 − 150 − 158] , corresponding to the successive loss of an
hexose, a pentose plus water, and an hexosyluronic acid less
water, respectively. The H NMR spectrum of 3 (in pyridine-
1
d /D O, 9:1) showed for the aglycone moiety (Table 1) signals
5
2
of six tertiary methyl groups at δ 0.51, 0.66, 0.70, 0.93, 1.15, and
.24, which correlate in the HSQC experiment with the carbon
signals at δ 14.4, 15.6, 27.0, 24.8, 21.4, and 18.3, respectively.
1
t
12
The Δ -oleanane skeleton was suggested due to the presence
1
of an olefinic signal in H NMR at δ 4.98 (t, J = 3.8 Hz), as well
t
13
glucuronic acid, 14.48; R of L-arabinose, 16.63), respectively.
as the C NMR signals at δ 143.1 and 121.9, assigned as C-13
and C-12, respectively. After a comprehensive analysis of 2D
NMR experiments, the presence of a carboxylic group at C-29,
a hydroxyl group at C-3, and 24-hydroxymethyl group was also
t
Finally, the structure of this saponin was elucidated as 3-O-[α-L-
arabinopyranosyl(1→2)]-β-D-glucuronopyranosyl azukisapoge-
nol, which represents a new identified natural compound.
E
dx.doi.org/10.1021/jf305541e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
−9
5
established. Therefore, it suggested that the aglycone of 3 was
the same as that in 1 and 2.
saponins previously reported from other Trifolium species.
So it could be recognized as a chemotaxonomic feature in the
Trifolium genus. However, this evidence is not conclusive,
because an exhaustive phytochemical research in all organs of
the plant has not been carried out in the present work. In
addition, α-L-arabinose and β-D-glucuronic acid were eventually
found linked 1→2 to β-D-glucuronic acid. Bidesmosidic
saponins are characterized by the presence of a β-D-glucose
linked at C-29, as a glycosidic esterification.
On the basis of our results in the investigation of T.
hybridum, we are studying the saponins composition in other
species of this genus for a better understanding of their
metabolism and biological roles in these plants.
1
13
The H and C NMR spectra of 3 showed also the presence
of three anomeric protons at δ 4.68, 5.10, and 6.06, and carbons
at δ 103.2, 103.5, and 94.5 (Table 2), which were identified in
the same way as in 1 and 2 as β-glucuronic acid, α-arabinose,
and β-glucose, respectively. Their absolute configurations were
determined as D-glucuronic acid, L-arabinose, and D-glucose. By
means of HMBC experiment, their connectivity was estab-
lished. The anomeric signal at δ 4.68 (H-1GlcA) showed a long-
range correlation with the signal at δ 89.3 (C-3), while a cross
peak between the signal at δ 6.06 (H-1 ) and the signal at δ
Glc
177.5 (C-29) indicated, as in 2, that it is a bidesmosidic
saponin. The Ara(1→2)GlcA linkage was also established by
HMBC experiment, where a clear cross peak between the signal
ASSOCIATED CONTENT
■
at δ 5.10 (H-1 ) and δ 79.0 (C-2GlcA) was observed, and
Ara
*
S
Supporting Information
confirmed by a ROESY correlation between δ 5.10 (H-1Ara)
and δ 4.03 (H-2GlcA). Consequently, this compound was
elucidated as 3-O-[-α-L-arabinopyranosyl(1→2)-β-D-glucurono-
pyranosyl]-29-O-β-D-glucopyranosyl azukisapogenol and repre-
sents a new bidesmosidic natural saponin.
1
13
NMR spectra ( H and C NMR, HSQC, HMBC, ROESY, and
DQF-COSY) for the new compounds (1−3), including
TOCSY spectra for compounds 2 and 3. ESI−MS/MS spectra
of compounds 1−3. This material is available free of charge via
the Internet at http://pubs.acs.org.
For compound 4, 7 mg, MW 648, its ESI−MS/MS indicated
the presence of only one hexosyluronic acid unit, which was
identified after hydrolysis as β-D-glucuronic acid. It had
spectroscopic characteristics identical to those of 3-O-β-D-
glucuronopyranosyl azukisapogenol (myrioside B), which was
AUTHOR INFORMATION
■
*
Corresponding Author
Phone: +48 8188 63421, ext 206. Fax: +48 8188 63421, ext
295. E-mail: andy.perezdear@alum.uca.es.
17
previously reported from O. myriophylla.
For compound 5, 3 mg, MW 810, its ESI−MS/MS showed
the loss of hexose unit (β-D-glucose) and hexosyluronic acid
unit (β-D-glucuronic acid). Spectroscopic characteristics of this
saponin were consistent with those of 3-O-[-β-D-glucuronopyr-
anosyl]-29-O-β-D-glucopyranosyl azukisapogenol (myrioside
Funding
The work was supported by the seventh Framework Program
of European Community, PROFICIENCY (Contract No.
245751), and by statutory activity of the Institute of Soil
Science and Plant Cultivation − State Research Institute (1.06,
2012−2014).
17
C), which was previously described in O. myriophylla.
In the present work, three new triterpenoid saponins 3-O-
-α-L-arabinopyranosyl(1→2)]-β-D-glucuronopyranosyl azuki-
Notes
[
The authors declare no competing financial interest.
sapogenol, 3-O-[-β-D-glucuronopyranosyl(1→2)-β-D-glucuro-
nopyranosyl]-29-O-β-D-glucopyranosyl azukisapogenol, and 3-
O-[-α-L-arabinopyranosyl(1→2)-β-D-glucuronopyranosyl]-29-
O-β-D-glucopyranosyl azukisapogenol, from the aerial parts of
T. hybridum, have been isolated and their structures elucidated.
Additionally, two known triterpenoid saponins, myrioside B
and C, were also isolated. An interesting feature of those
saponins is the identification of azukisapogenol as a new
aglycone for saponins of Trifolium species. The most common
aglycones isolated so far within this genus have been
soyasapogenol A, B, and E, and their glycosylated deriva-
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dx.doi.org/10.1021/jf305541e | J. Agric. Food Chem. XXXX, XXX, XXX−XXX