New Phenolic Compounds from the Roots of Lentil (Lens?culinaris)

Article abstract of DOI:10.1002/hlca.201600117

While lentil (Lens?culinaris) seeds are phytochemically well characterized, very little is known about secondary metabolites from lentil roots. Our research on lentil roots led to isolation of five phenolic compounds and five group B soyasaponins. Their structures were established using NMR spectroscopy and mass spectrometry. Four phenolics have not been hitherto described in the literature: 4-O-β-d-glucopyranosyl-2-methoxybenzoic acid, (αS)-4,4′-di-O-β-d-glucopyranosyl-α,2′-dihydroxydihydrochalcone, (αS)-4′-O-β-d-glucopyranosyl-α,2′,4-trihydroxydihydrochalcone, and keto-2-hydroxyglycitein. The DPPH^?radical-scavenging activity of the purified phenolic compounds was additionally evaluated.

Full text of DOI:10.1002/hlca.201600117

674
Helv. Chim. Acta 2016, 99, 674 – 680
FULL PAPER
New Phenolic Compounds from the Roots of Lentil (Lens culinaris)
a
a
b
a
_
by Jerzy Zuchowski* ), Łukasz Pecio ), Emilia Reszczy nꢀ ska ), and Anna Stochmal )
a
)
Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation - State Research Institute, ul.
Czartoryskich 8, PL-24-100 Puławy (phone: +48814786887; e-mail: jzuchowski@iung.pulawy.pl)
b
)
Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, pl. Marii Curie-Skłodowskiej 1,
PL-20-031 Lublin
While lentil (Lens culinaris) seeds are phytochemically well characterized, very little is known about secondary
metabolites from lentil roots. Our research on lentil roots led to isolation of five phenolic compounds and five group B
soyasaponins. Their structures were established using NMR spectroscopy and mass spectrometry. Four phenolics have not
0
been hitherto described in the literature: 4-O-b-D-glucopyranosyl-2-methoxybenzoic acid, (aS)-4,4 -di-O-b-D-glucopyranosyl-
0
0
0
a,2 -dihydroxydihydrochalcone, (aS)-4 -O-b-D-glucopyranosyl-a,2 ,4-trihydroxydihydrochalcone, and keto-2-hydroxyglycitein.
The DPPH radical-scavenging activity of the purified phenolic compounds was additionally evaluated.
∙
Keywords: Natural products, Phenolic compounds, Glycosides, a-Hydroxydihydrochalcones, Lentil.
phenolic compounds and several saponins. It seems that the
roots are practically devoid of quercetin and kaempferol
Many plant species belonging to the family Fabaceae are acylated glycosides, which are present in significant
Introduction
a rich source of diverse phenolic compounds and triter-
pene saponins [1 – 3]. While secondary metabolites of
soybean, pea, common bean, or numerous species of
amounts in leaves and stems of this plant [11]. Instead,
derivatives of phenolic acids, dihydrochalcones, and numer-
ous undefined compounds were detected. The application
Medicago, Trifolium, and Lupinus have been broadly of a three-step purification procedure, including C18 vac-
investigated, many other legumes are not so well charac-
terized. Lentil (Lens culinaris MEDIK.) is one of the oldest
cultivated plants, originating from the Middle East.
Nowadays, it is an important crop (the global production
of 4.885 million tons in 2014), most extensively cultivated
in southern and western Asia, North America, and Aus-
tralia [4][5]. Aside from major nutrients, lentil seeds con-
tain different types of secondary metabolites, and their
phytochemical composition is well described. They con-
tain different types of phenolic compounds (phenolic
uum liquid chromatography (VLC), Sephadex LH-20 col-
umn, and semipreparative RP-HPLC, enabled us to isolate
5 phenolic constituents of the extract (Fig.) Soyasaponin
0
Bb, soyasaponin Bc, soyasaponin Bb , soyasaponin Be, and
soyasaponin bg were additionally purified from the F5 VLC
fraction. Structures of the isolated compounds were deter-
mined by NMR and ESI-MS analyses.
ESI-MS analyses of compounds 1 and 2, performed in
the negative ionization mode, gave deprotonated mole-
cules at m/z 329. These precursor ions fragmented to pro-
acids, lignans, resveratrol, catechins, flavonoids), soyas- duce ions at m/z 167 [(M ꢀ H) ꢀ 162]ꢀ after the loss of
ꢀ
aponins, as well as tocopherols, phytosterols, and carote-
noids [6 – 10]. However, there is still very little precise ing the additional loss of CO . The fragmentation pat-
hexose, and at m/z 123 [(M ꢀ H) – 162 ꢀ 44] , indicat-
2
information about phytochemicals present in other parts
of this plant.
Recently, we have reported the occurrence of numer-
ous flavonol glycosides in the aerial parts of lentil plants
[11]. The aim of the current study was to isolate and iden-
tify secondary metabolites from their roots.
terns, UV spectra (similar to that of vanillic acid), and
short retention times of 1 and 2 suggested that they might
be hexose derivatives of methoxy-hydroxybenzoic acids.
This deduction was confirmed by NMR analyses of these
1
3
compounds. The C-NMR spectrum of 1 showed 14 sig-
1
3
nals, sorted by C and distortionless enhancement by
polarization transfer (DEPT) experiments into one Me,
one CH , eight CH, and four quaternary C-atoms (see
2
Results and Discussion
Table 1). The latter were attributed to the presence of a
free COOH group at d(C) 166.4 (further supported by
the loss of 44 mass units in ESI-MS/MS spectrum), two
UHPLC/MS analysis of 80% MeOH crude extract of len-
til roots showed the presence of a variety of putative
©
2016 Wiley-VHCA AG, Z u€ rich
DOI: 10.1002/hlca.201600117

Helv. Chim. Acta 2016, 99, 674 – 680
675
Figure. Structures of compounds 1 – 5.
1
1
Table 1. The H- and C-NMR data ( C, 125 MHz; H, 500 MHz)
13
13
1
of the H and COSY spectra of 1 exhibited the presence
of one set of ABX-type aromatic H-atoms at d(H) 7.67 (1
H, d, J = 8.7, H–C(6)), 6.63 (1 H, dd, J = 8.7, 2.2, H–C
a
for 1 ) in (D
6 3
)DMSO (0.08% CF COOH) at 25 °C; d in ppm, J in
Hz.
(5)) and 6.73 (1 H, d, J = 2.2, H–C(3)). Additionally, a
Position
d(H)
d(C)
Me group at d(H)/d(C) 3.80 (3 H, s)/55.6 was determined
to be attached to the C(2) of the aromatic moiety based
on the correlation between the MeO and C(2) (d(C)
C=O
166.4
113.6
160.3
100.7
161.5
107.4
132.8
1
2
3
4
5
6
1
60.3) as observed in the HMBC spectrum. It was further
6.73 (1 H, d, J = 2.2)
supported by NOE effect in ROESY spectrum between
Me group (d(H) 3.80) and H–C(3) (d(H) 6.73), as well as
correlations observed in the HMBC spectrum between H-
atom H–C(3) and C-atoms C(2) and C(4) (d(C) 161.5).
6.63 (1 H, dd, J = 8.7, 2.2)
7.67 (1 H, d, J = 8.7)
4
1
2
3
4
5
6
-O-Glc
0
4.97 (1 H, d, J = 7.4)
3.25 (1 H, dd, J = 8.9, 7.4)
3.29 (1 H, t, J = 8.7)
3.14 (1 H, t, J = 9.0)
3.38 (1 H, ddd, J = 9.4, 6.1, 1.5)
3.70 (1 H, dd, J = 11.6, 1.5)
99.9
73.1
76.6
69.8
77.2
60.7
1
The H-NMR data for the aglycone are consistent with
0
0
0
0
0
those of the rarely occurring 2-methoxy-4-hydroxybenzoic
acid [12]. The b-glucopyranosyl unit was positioned at
C(4), which is in agreement with correlations observed
0
between the anomeric H-atom H–C(1 ) (d(H) 4.97) and
3.44 (1 H, dd, J = 11.7, 6.3)
C(4) in the HMBC spectrum. Acid hydrolysis of com-
pound 1 and determination of absolute configuration of
the released monosaccharide by UHPLC/MS demon-
strated the presence of D-glucose. Therefore, the
compound 1 was identified as a new compound and
named as 4-O-b-D-glucopyranosyl-2-methoxybenzoic acid
(4-O-b-D-glucopyranosyl pluchoic acid, Fig.)
OCH
3
3.80 (3 H, s)
55.6
a
1
Assignments were done by HSQC, H2BC, HMBC, and H, H-
1
)
COSY experiments.
O-bearing aromatic C-atoms at d(C) 161.5 and 160.3, and
non-O-bearing aromatic C-atom at d(C) 113.6. Further-
more, one anomeric C-atom at d(C) 99.9, four O-bearing
CH C-atoms at d(C) 73.1, 76.6, 69.8, and 77.2, and one O-
The compound 2 was identified, on the basis of its
NMR spectra, as 2-O-b-D-glucopyranosyl-5-methoxyben-
bearing CH2 C-atom at d(C) 60.7 were assigned to the zoic acid, previously isolated from flowers of Bom-
1
sugar moiety. The H-NMR of 1 contained anomeric H-
atom signal at d(H) 4.97 (1 H, d, J = 7.4), which sug-
gested b-configuration of the sugar moiety. On the basis
of 1D-TOCSY, HSQC, and H2BC spectra, the sugar unit
bax ceiba tree [13].
Compounds 3 and 4 had practically the same UV
spectra (k
274 and 317 nm), their MS analyses gave
max
ꢀ
[M ꢀ H] ions signals at m/z 597 and m/z 435, respec-
was identified as b-glucopyranoside. The aromatic region tively. The CID of the ion m/z 597 generated fragments
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2016 Wiley-VHCA AG, Z u€ rich
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676
Helv. Chim. Acta 2016, 99, 674 – 680
ꢀ
0 0
273 J = 8.6, H–C(3,5)), in accordance with AA BB system.
at
m/z
435
[(M ꢀ H) – 162]
and
m/z
ꢀ
[
(M ꢀ H) – 162 – 162] , indicating the loss of two hexose The other set corresponded to trisubstituted Ph group at
0
moieties. The deprotonated compound 4 [at m/z 435] d(H) 6.58 (1 H, d, J = 2.4, H–C(3 )), 6.61 (1 H, dd,
0
0
fragmented to give a m/z 273 ion, formed after the loss of
hexose. These data suggested that 3 and 4 could be di-
and monohexosides of the same aglycone, respectively.
This hypothesis was confirmed by further experiments.
Acid hydrolysis of these compounds and determination of
J = 9.0, 2.4, H–C(5 )), and 7.97 (1 H, d, J = 9.0, H–C(6 )),
in accordance with ABX type aromatic H-atoms. Due to
deshielding effect of the CO group and formation of a
0
strong intramolecular H-bond, OH group located at C(2 )
appeared at d(H) 12.15 (OH, s), indicating the presence
0
0
absolute configuration of released monosaccharides of a,4,2 ,4 -tetrahydroxydihydrochalcone aglycone [14].
demonstrated the presence of D-glucose. The UHPLC/MS
analysis of nonpolar products of the hydrolysis showed
that 3 and 4 contained a chromatographically identical dance with the literature [15][16]. The carbohydrate
The aglycone has been isolated from some other legume
1
13
plants, and our H- and C-NMR data are in good accor-
1
aglycone. The precise structure of both compounds was
1
elucidated by NMR spectroscopy. The H-NMR spectrum
region of H-NMR spectrum showed the presence of the
oxymethine H-atoms in the range d(H) 3.10 – 3.75. More-
of 3 contained CH2 H-atom signals appearing at d(H) over, two anomeric H-atom signals at ((H) 4.99 (1 H, d,
00 000
.99 (1 H, dd, J = 13.9, 4.3, H –C(b)) and 2.73 (1 H, dd, J = 7.5, H–C(1 )) and 4.80 (1 H, d, J = 7.5, H–C(1 ))
a
2
J = 13.9, 8.4, H –C(b)) and an oxymethine H-atom res-
were also observed, indicating the presence of two sugar
b
onating at d(H) 5.07 (1 H, dd, J = 8.4, 4.4, H–C(a)). The units. Based on the values of coupling constants
1
aromatic region of the H and COSY spectra of 3 exhib- (J > 7 Hz), and the analysis of 1D-TOCSY, HSQC, and
ited the presence of two sets of aromatic H-atoms (see
Table 2). One set corresponded to a disubstituted aro-
matic ring with two ortho-coupling H-atoms and appeared
H2BC data, the two sugar units were elucidated as b-glu-
copyranosides. The C-NMR and DEPT spectra of 3
1
3
indicated 27 C-atoms, including three CH , 18 tertiary,
2
at d(H) 7.15 (2 H, d, J = 8.6, H–C(2,6)) and 6.93 (2 H, d, and six quaternary C-atoms. The latter were attributed to
1
Table 2. The H- and C-NMR data ( H, 500 MHz; C, 125 MHz) for 3 and 4 ) in (D
13
1
13
a
6
)DMSO (0.08% CF
3
COOH) at 25 °C, d in ppm, J in
Hz.
3
4
Position
d(H)
d(C)
d(H)
d(C)
C=O
a
ba
bb
1
204.0
73.9
39.4
204.4
5.07 (1 H, dd, J = 8.4, 4.4)
2.99 (1 H, dd, J = 13.9, 4.3)
2.73 (1 H, dd, J = 13.9, 8.4)
5.04 (1 H, dd, J = 8.2, 4.7)
2.92 (1 H, dd, J = 13.9, 4.6)
2.68 (1 H, dd, J = 13.9, 8.2)
73.9
39.5
131.3
130.2
115.9
156.0
113.3
163.5
103.6
163.3
108.3
132.7
128.0
130.3
114.8
155.7
113.3
163.5
103.5
163.3
108.3
132.7
2
3
4
1
2
3
4
5
6
4
1
2
3
4
5
6
,6
7.15 (2 H, d, J = 8.6)
6.93 (2 H, d, J = 8.6)
7.02 (2 H, d, J = 8.5)
6.63 (2 H, d, J = 8.5)
,5
0
0
0
0
0
0
6.58 (1 H, d, J = 2.4)
6.57 (1 H, d, J = 2.4)
6.61 (1 H, dd, J = 9.0, 2.4)
7.97 (1 H, d, J = 9.0)
6.59 (1 H, dd, J = 8.9, 2.4)
7.93 (1 H, d, J = 9.0)
-O-Glc
0
0
0
0
0
0
0
0
0
0
0
0
4.99 (1 H, d, J = 7.5)
3.24 (1 H, dd, J = 9.0, 7.5)
3.29 (1 H, t, J = 8.9)
3.17 (1 H, dd, J = 9.5, 8.3)
3.38 (1 H, ddd, J = 8.5, 5.9, 2.2)
3.68 (1 H, dd, J = 11.9, 2.5),
99.6
73.1
77.0
69.5
77.1
60.5
4.99 (1 H, d, J = 7.5)
3.23 (1 H, dd, J = 9.0, 7.6)
3.28 (1 H, t, J = 8.8)
3.17 (1 H, dd, J = 9.6, 8.3)
3.38 (1 H, ddd, J = 9.5, 5.6, 2.0)
3.69 (1 H, dd, J = 11.6, 1.6),
3.47 (1 H, dd, J = 11.9, 5.6)
99.6
73.1
76.4
69.5
77.1
60.5
3
.47 (1 H, dd, J = 11.9, 4.4)
0
0
0
0
0
0
0
4
1
2
3
4
5
6
-O-Glc
00
4.80 (1 H, d, J = 7.5)
3.21 (1 H, dd, J = 9.0, 7.5)
3.25 (1 H, t, J = 8.5)
3.15 (1 H, dd, J = 9.5, 8.3)
3.29 (1 H, ddd, J = 9.1, 6.0, 2.3)
3.68 (1 H, dd, J = 11.9, 2.9),
100.5
73.2
76.6
69.7
76.4
60.7
00
00
00
00
00
3
.45 (1 H, dd, J = 11.9, 5.0)
a
1
Assignments were done by HSQC, H2BC, HMBC, and H, H-COSY experiments.
1
)
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Helv. Chim. Acta 2016, 99, 674 – 680
677
the presence of a CO group at d(C) 204.0, three O-bear-
ing aromatic C-atoms at d(C) 163.5, 163.3 and, 156.0, and
163.3 and 155.7, and two non-O-bearing aromatic
C-atom at d(C) 113.3 and 128.0. Furthermore, one
two non-O-bearing aromatic C-atoms at d(C) 113.3 and anomeric C-atom at d(C) 99.6, four O-bearing CH
31.3. Furthermore, two anomeric C-atoms at d(C) 99.6 C-atoms at d(C) 73.1, 76.4, 69.5, 77.1, and one O-bear-
and 100.5, eight O-bearing CH C-atoms at d(C) 73.1, 77.0, ing CH C-atom at d(C) 60.5 were assigned to the sugar
1
2
6
9.5, 77.1, 73.2, 76.6, 69.7, and 76.4, and two O-bearing
moiety. The b-glucopyranosyl unit was positioned at C
0
CH C-atoms at d(C) 60.5 and 60.7 were assigned to the
(4 ), which is in agreement with correlations observed
0
2
0
sugar moieties according to HSQC-TOCSY spectra. One
0
between the anomeric H-atom H–C(1 ) (d(H) 4.99) and
0
b-glucopyranosyl unit was positioned at C(4 ), which is in C(4 ) (d(C) 163.3) in the HMBC spectrum, and NOE
0
0
agreement with correlations observed between the effect in ROESY spectrum between H–C(1 ) and both
0
0
0
0
0
anomeric H-atom H–C(1 ) (d(H) 4.99) and C(4 ) (d(C)
63.3) in the HMBC spectrum, and NOE effect in
H–C(3 ) (d(H) 6.57) and H–C(5 ) (d(H) 6.59). The abso-
lute configuration was determined as (aS) by CD spec-
tral analysis. Similarly to 3, the compound 4 presented
one negative Cotton effect at 245 nm and two positive
1
0
0
0
ROESY spectrum between H–C(1 ) and both H–C(3 ) (d
0
(
H) 6.58) and H–C(5 ) (d(H) 6.61). The other b-glucopyr-
anosyl unit was positioned at C(4), which is in agreement Cotton effects (305 nm and 340 nm). Therefore, the
0
with correlations observed between the anomeric H-atom
0
compound 4 was identified as (aS)-4 -O-b-D-glucopyrano-
H–C(1 ) (d(H) 4.80) and C(4) (d(C) 156.0) in the HMBC syl-a,2 ,4-trihydroxydihydrochalcone (Fig.)
00
0
spectrum, and NOE effect in ROESY spectrum between
The MS analysis of the compound 5 revealed it did
not contain any carbohydrate group, and it gave a depro-
0
00
H–C(1 ) and H-atoms H–C(3,5) (d(H) 6.93). The abso-
lute configuration of asymmetric C(a) of 3 was deter-
mined as aS by comparison of its CD spectrum
1
tonated molecule at m/z 299. The C-NMR spectrum of
3
1
5 showed 16 signals, sorted by C and DEPT experi-
3
(
sequential negative and positive Cotton effects at 245 nm ments into one Me, and seven tertiary and eight quater-
0
0
and 319 nm, resp.) with that of (aR)-a,2 -dihydroxy-4,4 - nary C-atoms (Table 3). The latter were attributed to the
dimethoxydihydrochalcone (positive Cotton effect at presence of two CO groups at d(C) 176.9 and 167.2, four
2
from Pterocarpus angolensis [17]. Therefore, the com-
46 nm and negative Cotton effect at 310 nm), isolated
O-bearing aromatic C-atoms at d(C) 148.8, 146.7, 152.5,
and 155.9, and two non-O-bearing aromatic C-atom at d
(C) 114.3 and 143.4. Furthermore, one CH C-atom,which
showed upfield shifted resonance at d(C) 88.1, indicated
that compound 5 exhibited structural change in ring C,
compared to glycitein [18]. This was in agreement with
0
pound 3 was identified as (aS)-4,4 -di-O-b-D-glucopyrano-
0
syl-a,2 -dihydroxydihydrochalcone (Fig.)
The spectroscopic analysis of compound 4 indicated
that its structure is closely related to that of compound
1
3. The H-NMR spectrum of 4, contained CH2 H-atom correlations observed between H-atom H–C(3) (d(H)
signals appearing at d(H) 2.92 (1 H, dd, J = 13.9, 4.6, 5.04, s) and both C-atom C(2) (d(C) 167.2) and C(4) (d
H –C(b)) and 2.68 (1 H, dd, J = 13.9, 8.2, H –C(b)) and (C) 176.9). It was further supported by NOE effect visible
a
b
an oxymethine H-atom resonating at d(H) 5.04 (1 H, in ROESY spectrum between H-atom H–C(3) and
1
0
0
1
dd, J = 8.2, 4.7, H–C(a)). The aromatic region of the H H–C(2 /6 ) (d(H) 7.16). The aromatic region of H and
and COSY spectra of 4, similarly to 3, exhibited the COSY spectra of 5 exhibited the presence of two sets of
presence of two sets of aromatic H-atoms (see Table 2).
One set corresponded to a di-substituted aromatic ring
with two ortho-coupling H-atoms and appeared at d(H)
1
13
1
13
Table 3. The H- and C-NMR data ( H, 500 MHz; C, 125 MHz)
a
for 5 ) in (D
6
)DMSO (0.08% CF
3
COOH) at 25 °C, d in ppm, J in
7
.02 (2 H, d, J = 8.5, H–C(2,6)) and 6.63 (2 H, d,
Hz.
0
0
J = 8.5, H–C(3,5)), in accordance with AA BB system.
The other set corresponded to tri-substituted Ph group
at d(H) 6.57 (1 H, d, J = 2.4, H–C(3 )), 6.59 (1 H, dd,
J = 8.9, 2.4, H–C(5 )) and 7.93 (1 H, d, J = 9.0, H–
C(6 )), in accordance with ABX type aromatic H-atoms.
1
The carbohydrate region of H-NMR spectrum showed
Position
d(H)
d(C)
0
2
3
4
5
6
7
8
9
167.2
88.1
0
5.04 (1 H, s)
7.31 (1 H, s)
0
176.9
104.8
146.7
148.8
102.8
152.5
114.3
143.4
121.8
116.4
155.9
55.8
the presence of the oxymethine H-atoms in the range d
(
H) 3.10-3.75. Moreover, an anomeric H-atom signal at
6.92 (1 H, s)
0
0
d(H) 4.99 (1 H, d, J = 7.5, H–C(1 )) was observed, indi-
cating the presence of one sugar unit. Based on the val-
ues of coupling constants (J > 7 Hz), and the analysis of
1
1
2
3
4
0
0
0
0
0
,6
7.16 (1 H, d, J = 8.9)
6.86 (1 H, d, J = 8.9)
1
D-TOCSY, HSQC, and H2BC data, the sugar unit was
1
0
0
,5
3
elucidated as b-glucopyranoside. The
DEPT spectra of 4 indicated 21 C-atoms, including two
C-NMR and
OCH3
3.85 (3 H, s)
CH , 13 tertiary, and six quaternary C-atoms. The latter
2
a
1
Assignments were done by HSQC, H2BC, HMBC, and H, H-
1
)
COSY experiments.
were attributed to the presence of a CO group at d(C)
2
04.4, three O-bearing aromatic C-atoms at d(C) 163.5,
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2016 Wiley-VHCA AG, Z u€ rich
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678
Helv. Chim. Acta 2016, 99, 674 – 680
aromatic H-atoms, characteristic for the glycitein agly-
cone. One set corresponded to a disubstituted aromatic
ring with two ortho-coupling H-atoms and appeared at d
DMSO or d(H) 7.58 and d(C) 135.91 for (D )pyridine
(both calibrated to TMS at 0.00 ppm). UV-VIS Spectra
were determined using a Thermo Evolution 260 Bio spec-
5
0 0
(
H) 7.16 (2 H, d, J = 8.9, H–C(2 ,6 )) and 6.86 (2 H, d, trophotometer (Thermo Fisher Scientific, Waltham, MA,
0
0
0
0
J = 8.9, H–C(3 ,5 )), in accordance with AA BB system. USA). IR Spectra were obtained with a Thermo Nicolet
The other two singlet H-atom peaks at d(H) 7.31 (1 H, s) iS 50 FT-IR spectrometer (Thermo Fisher Scientific, Wal-
and d(H) 6.92 (1 H, s), is characteristic for tetrasubsti-
tham, MA, USA), equipped with a Pike GladiATR acces-
tuted benzene ring. Therefore, the compound 5 was iden- sory (Pike Technology, Madison, WI, USA). Optical
tified as keto-2-hydroxyglycitein (Fig.), first time isolated rotations were measured using an automatic digital
from natural sources [19].
The ability of the purified phenolic compounds to
scavenge DPPH radicals was assessed using a rapid TLC-
polarimeter (P-2000, Jasco, Tokyo, Japan). A Chirascan-
plus spectrometer (Applied Photophysics Limited,
Leatherhead, Surrey, UK) was used in ECD experiments,
scans (190 – 400 nm) were performed in a 10 mm path
∙
DPPH test. The investigated substances were generally
∙
poor DPPH scavengers. Compounds 1, 2, and 3 showed length quartz cuvette.
no detectable antiradical activity, while activities of com-
pounds 4 and 5 were 0.267 (ꢁ 0.044) and 0.313 (ꢁ 0.062)
Plant Material
of that of rutin, respectively. Since compounds 1 and 2
have no free OH group in their aglycone parts, the lack
of antioxidant properties is not surprising. Similarly, the
Seeds of lentil (Lens culinaris MEDIK.) cv. Tina were pro-
vided by the Department of Agrotechnology and Crop
0
0
aglycone of compounds 3 and 4, a,2 ,4,4 -tetrahydroxydi- Management, University of Warmia and Mazury, Olsztyn,
hydrochalcone, is a poor DPPH scavenger, so the low Poland. Lentil plants were grown in the experimental field
∙
antiradical activity of its glucoside derivatives should also
be expected [20].
of the Institute of Soil Science and Plant Cultivation in
Puławy, Poland, and harvested during the flowering per-
iod. The collected lentil roots were lyophilized in
This work was financed from a grant of the National CHRIST GAMMA 2 – 16 LSC laboratory freeze-dryer.
Science Centre, Poland (No. 2011/01/B/NZ9/04679).
Extraction and Fractionation of Root Extract
Freeze-dried lentil roots were milled into a powder and
defatted with CHCl . The defatted material (42.3 g) was
Experimental Part
3
three times extracted with boiling 80% MeOH (500 ml)
under reflux. The obtained soln. was filtered, concentrated
General Details
UHPLC/MS analyses of the root extract and its fractions
in a rotary evaporator, and freeze-dried to yield 5.2 g of
were carried out using an ACQUITY UPLC System chro- the crude extract. The extract was fractionated by VLC
matograph (Waters, Milford, MA, USA), equipped with a
PDA detector and coupled with a triple quadrupole mass
on
a C18 column (10 9 5.5 cm; Lichroprep RP-18
40 – 63 lm, Merck), the bound compounds were eluted
spectrometer (ACQUITY TQD, Waters). Samples were with increasing concentrations of MeOH in H O (20%,
2
chromatographed on an ACQUITY BEH C18 40%, 60%, 80%, and 98%; 500 ml portions). The result-
(
100 9 2.1 mm, 1.7 lm; Waters) column; details of the ing fractions (F1 – F5) were concentrated under reduced
separation conditions and the MS settings were described
previously [11]. Exact mass measurements were per-
pressure and freeze-dried, to obtain 0.365 g of F1 (20%
MeOH), 0.287 g of F2 (40% MeOH), 0.306 g of F3 (60%
formed using a SYNAPT G2-S HDMS system (Waters, MeOH), 0.426 g of F4 (80% MeOH), and 0.333 g of F5
Milford, MA, USA). Fragmentation analyses were carried
out by direct infusion electrospray mass spectrometry,
using an ACQUITY TQD mass spectrometer (Waters,
Milford, MA, USA). NMR analyses were performed
using an Avance III HD 500 MHz spectrometer (Bruker
(98% MeOH).
Isolation of Phenolic Compounds
Preparations containing compounds eluted with 20 – 60%
were further purified by Sephadex LH-20 chromatogra-
BioSpin, Rheinstetten, Germany), in (D )DMSO contain-
6
ing 0.08% CF COOH (phenolic compounds) or (D )pyri- phy. The fraction F1 was separated on a 2.2 9 85 cm col-
3
5
dine (saponins), at 25 °C. Typical pulse sequences and
parameters were applied to obtain 1D- H, 1D-selective and 80% MeOH were used for fractions F2 and F3. The
umn, using MeOH as an eluent; a 3.4 9 37 cm column
1
TOCSY and ROESY, gCOSY, TOCSY (mixing flow rate was 2 ml/min, and 7 ml of fractions were col-
1
3
time = 120 ms), ROESY (mixing time = 250 ms), 1D- C lected. The composition of the chromatographic fractions
and DEPT-135, gHSQC, gHSQC-TOCSY (mixing was monitored by TLC on SiO plates (80:15:15:5 MeCN/
2
time = 80 ms), gH2BC, and gHMBC spectra. The chemi-
cal shift referencing was performed using the internal sol-
CHCl /H O/HCOOH). Chromatographic spots were visu-
3 2
alized under UV, or plates were sprayed with a H SO /
2 4
vent resonances at d(H) 2.50 and d(C) 39.51 for (D6) MeOH mixture (1:5), and heated at 130 °C.
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Helv. Chim. Acta 2016, 99, 674 – 680
679
D
Chromatographic fractions containing the same dominant
compounds were combined, evaporated, dissolved in H O
solid. ½aꢃ = ꢀ33.2 (c = 0.10, MeOH). UV (c = 0.07 mM,
2
0
MeOH): 274 (4.1), 317 (3.7). ECD (c = 0.17 mM, MeOH):
or mixtures of H O and BuOH, and freeze-dried (F1 – 204 (–0.167), 234 (0.253), 245 (–0.295), 319 (0.705). IR
2
t
2
groups 1 – 4; F2 – groups 5 – 9; F3 – groups 10 – 15). (ATR): 3360, 3193, 2922, 2851, 1658, 1632, 1469, 1412,
1
The obtained preparations were subsequently separated 1136, 1015. H- and
13
C-NMR: Table 2. ESI-MS/MS
ꢀ
ꢀ
by reversed-phase semipreparative HPLC (analytical to
(TQ): 597 ([M ꢀ H] ), 435 ([M – H – 162] ), 273
ꢀ
semiprep. HPLC system, equipped with a ELSD detector; ([M ꢀ H – 162 – 162] ). HR-ESI-MS (Q-TOF): 621.1791
+
þ
15
Gilson Inc., Middleton, WI, USA) on a C18 column ([M + Na] , C H NaO ; calc. 621.1795).
2
7
34
0
0
(Kromasil 100-5-C18, 250 9 10 mm, 5 lm). Preparations
(aS)-4 -O-b-D-Glucopyranosyl-a,2 ,4-trihydroxydihydrochal-
cone (= 3-Hydroxy-4-[(2S)-2-hydroxy-3-(4-hydroxyphenyl)-
propanoyl]-phenyl b-D-Glucopyranoside; 4). It was eluted
were separated isocratically, using aq. MeCN solns. of
different concentrations (from 6% to 28% MeCN),
containing 0.2% FA. The mobile phase flow was 6 ml/
with 14% MeCN from the Kromasil C18 semiprep. HPLC
D
min, the column temp. was maintained at 30 °C. The column. White solid. ½aꢃ = ꢀ13.0 (c = 0.18, MeOH). UV
2
0
group 2 yielded compound 3 (7.7 mg), group 3 yielded
compounds 1 (2.2 mg) and 2 (0.8 mg), group 8 and group
(c = 0.09 mM, MeOH): 274 (4.2), 317 (3.8). ECD (c
0.17 mM, MeOH): 204 (ꢀ0.587), 236 (0.344), 245 (ꢀ0.336),
340 (0.711). IR (ATR): 3363, 3205, 2922, 2852, 1633, 1509,
1
2 yielded compound 4 (3.2 mg) and compound 5
0.4 mg), resp.
1
13
(
1240, 1070, 1018. H- and C-NMR: Table 2. ESI-MS/MS
ꢀ
(
TQ):
ꢀ
435
([M ꢀ H] ),
273
([M – H –
+
162] ). HR-ESI-MS (Q-TOF): 459.1261 ([M + Na] ,
C H NaO ; calc. 459.1267).
Determination of the Absolute Configuration of
Sugars
þ
2
1
24
10
7
-Hydroxy-3-(4-hydroxyphenyl)-6-methoxy-2H-chromene-
Samples of new compounds (0.25 mg) were subjected to
acid hydrolysis (1 ml of 2M HCl, 100 °C, 2 h). After cool-
ing, hydrolysates were thrice extracted with 1 ml portions
of AcOEt, to remove aglycones. Monosaccharide-contain-
2,4(3H)-dione (5). It was eluted with 28% MeCN from
the Kromasil C18 semiprep. HPLC column. Yellow-
D
brown solid. ½aꢃ = 19.0 (c = 0.02, MeOH). UV
2
0
(c = 0.13 mM, MeOH): 280 (3.8), 310 (3.8). ECD
(c = 0.33 mM, MeOH): 241 (0.490). IR (ATR): 3237,
2962, 1681, 1627, 1575, 1510, 1475, 1392, 1267, 1227, 1151.
ing H O phases were neutralized with Amberlite IRA-400
2
ꢀ
(
OH form) and dried. The absolute configuration of sug-
1
13
ars was determined according to the procedure of Tanaka
et al. [21]; a modified sample analysis method (UPLC-
ESI-MS/MS) was applied, as described in [22].
H- and
([M ꢀ H] ). HR-ESI-MS (Q-TOF): 301.0705 ([M + H] ,
C-NMR: Table 3. ESI-MS/MS (TQ): 299
+
ꢀ
þ
C H O ; calc. 301.0707).
1
6
13
6
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[
ꢂ
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ꢀ
1
67 ([M – H – 162] ), 123 ([M – H – 162 – 44] ). HR-
+
þ
ESI-MS (Q-TOF): 353.0847 ([M + Na] , C H NaO ;
1
4
18
9
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1
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© 2016 Wiley-VHCA AG, Z u€ rich