Aromatic glucosides from the seeds of Prunus davidiana

Article abstract of DOI:10.1021/np4000922

Chemical investigation of the seeds of Prunus davidiana afforded seven new aromatic glucosides, i.e., the prupersins A-E (1-5) and compounds 6 and 7, as well as 11 known compounds. The structures of 1-7 were elucidated by spectroscopic data analysis and chemical evidence, and configurations were determined by hydrolysis experiments (1, 2, and 5) or electronic circular dichroism (6). Compounds 1-6 exhibited antioxidant activity aganist Fe ²⁺-cysteine-induced rat liver microsomal lipid peroxidation, with malondialdehyde inhibitory rates of 50-67% and 53-57% at concentrations of 10^-5 and 10^-6 mol/L, respectively.

Full text of DOI:10.1021/np4000922

Article
pubs.acs.org/jnp
Aromatic Glucosides from the Seeds of Prunus davidiana
Xiao-Yan Chen, Hong-Qing Wang, Ting Zhang, Chao Liu, Jie Kang, Ruo-Yun Chen,* and De-Quan Yu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Chemical investigation of the seeds of Prunus davidiana
afforded seven new aromatic glucosides, i.e., the prupersins A−E (1−5)
and compounds 6 and 7, as well as 11 known compounds. The
structures of 1−7 were elucidated by spectroscopic data analysis and
chemical evidence, and configurations were determined by hydrolysis
experiments (1, 2, and 5) or electronic circular dichroism (6).
Compounds 1−6 exhibited antioxidant activity aganist Fe2+-cysteine-
induced rat liver microsomal lipid peroxidation, with malondialdehyde
inhibitory rates of 50−67% and 53−57% at concentrations of 10−5 and
10−6 mol/L, respectively.
he seeds of Prunus davidiana (Carr.) Franch or Prunus
persica (L.) Batsch, persicae semen, have been used in
hydroxyphenyl)glycerol, and β-glucopyranosyl moieties. In
the HMBC spectrum, the correlations of H-2″, H-6″, H-3″
(weak), and H-5″ (weak)/C-7″ confirmed the presence of a
benzoyl moiety, and the correlations of H-2, H-6/C-7; H-7/C-
1, C-2, C-8, and C-9; H-8/C-9 and C-7 (weak); and H-9/C-7;
H-9b/C-8 confirmed the presence of a phenylglycerol moiety.
In addition, the correlations of H-6′/C-7″; H-9/C-1′; and H-
1′/C-9 indicated the benzoyl group was attached to C-6′ of the
glucopyranosyl moiety, and C-1′ of the glucopyranosyl unit was
connected to C-9 of the phenylglycerol moiety.
T
traditional Chinese medicine to treat cardiovascular, cerebro-
vascular, and gynecological diseases.1 Previous phytochemical
studies on persicae semen have led to the isolation and
identification of cyanogenic glycosides, glycerides, sterols, and
emulsins.2 As part of our program to study traditional Chinese
medicine, an ethanolic extract of the seeds of P. davidiana
(Carr.) Franch was investigated. This led to the identification of
18 aromatic glucosides: the new prupersins A−E (1−5); two
synthetic compounds, 6 and 7, that have not yet been found in
nature; and 11 known analogues. Herein, we report the
isolation and structural elucidation of these new glucosides. In
addition, we report the results of antioxidant activity assays
against Fe2+-cysteine-induced rat liver microsomal lipid
peroxidation.
Alkaline hydrolysis of 1 (Scheme 1) gave benzoic acid, glucose,
and 1-(4-hydroxyphenyl)glycerol (1c). The presence of
benzoic acid and glucose was confirmed by comparison with
authentic samples. The D-configuration of glucose was
determined by optical rotation (Experimental Section). A β-
anomeric configuration for the glucosyl unit was assigned via its
3
1
large J1,2 coupling constant (7.5 Hz). The H NMR spectrum
of 1-(4-hydroxyphenyl)glycerol (1c) was in good agreement
with that of threo-1-(4-hydroxyphenyl)glycerol,3 and the
RESULTS AND DISCUSSION
■
Prupersin A (1) was obtained as an amorphous powder, [α]20
D
−25.3 (c 2.35, MeOH). The IR spectrum of 1 showed
absorption bands at 3339, 1717, 1614, 1600, and 1516 cm−1,
indicating the presence of hydroxy, ester carbonyl, and aromatic
functional groups. The molecular formula, C22H26O10, was
determined from positive-ion HRESIMS (473.1428 [M + Na]+,
calcd 473.1418) and also supported by the NMR spectroscopic
positive [α]D value {[α]20 +18.0 (c 0.06, EtOH)} indicated
D
that the absolute configuration was 7S, 8S.4 On the basis of the
above data, prupersin A was characterized as 1 (Figure 1).
Prupersin B (2) was obtained as a colorless gum, [α]20D −82.0
(c 0.37, MeOH). The molecular formula was determined to be
C21H22O9 on the basis of positive-ion HRESIMS (441.1158 [M
+ Na]+, calcd 441.1156), corresponding to 11 indices of
hydrogen deficiency. Similar to 1, the 1D NMR data (Tables 1
and 2) indicated the presence of a benzoyl group and a β-
glucopyranosyl moiety. HMBC correlations of H-6′a and H-6′b
with C-7″ confirmed that the connection between the benzoyl
1
data. The H NMR spectrum (Table 1) revealed the presence
of two oxymethine hydrogens at δH 4.35 (d, J = 4.5 Hz, H-7)
and 3.65 (m, H-8), one oxymethylene at δH 3.82 (dd, J = 10.0,
2.5 Hz, H-9a) and 3.37 (overlapped, H-9b), nine aromatic
protons at δH 7.97 (dd, J = 8.0, 1.0 Hz, H-2″, 6″), 7.68 (br dd, J
= 8.0, 8.0 Hz, H-4″), 7.55 (dd, J = 8.0, 8.0 Hz, H-3″, 5″), 7.07
(d, J = 8.0 Hz, H-2, 6), and 6.62 (d, J = 8.0 Hz, H-3, 5), and a
glucopyranosyl unit. The 13C NMR spectrum (Table 2)
displayed characteristic signals for benzoyl, 1-(4-
Received: January 31, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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a
1
Table 1. H NMR Spectroscopic Data of Compounds 1−5
position
1
2
3
4
5
b
b
b
b
b
2
3
4
5
6
7
7.07, d (8.0)
6.62, d (8.0)
7.36, d (6.0)
7.36, d (7.5)
7.98, dd (7.5, 2.0)
7.52, dd (7.5, 7.5)
7.67, dd (7.5, 7.5)
7.52, dd (7.5, 7.5)
7.98, dd (7.5, 2.0)
7.57,
7.45,
7.45,
7.45,
7.57,
m
m
m
m
m
b
7.20−7.27,
7.20−7.27,
7.20−7.27,
m
m
m
7.31, dd (7.5, 7.5)
7.26, dd (7.5,7.5)
7.31, dd (7.5, 7.5)
7.36, d (7.5)
b
b
6.62, d (8.0)
7.07, d (8.0)
4.35, d (4.5)
7.36, d (6.0)
5.21, s
a 4.78, d (12.5)
b 4.53, d (12.5)
5.98, s
8
3.65, m
9a
3.82, dd (10.0, 2.5)
b
9b
3.37
1′
2′
3′
4′
4.22, d (7.5)
3.99, d (7.5)
4.19, d (7.8)
5.57, dd (6, 2.0)
4.21, d (7.5)
b
b
b
b
3.03, dd (7.5, 7.5)
3.20−3.44,
m
m
m
m
2.97−3.08,
m
3.30,
3.30,
3.18,
3.52,
m
m
m
m
3.08,
3.08,
3.08,
m
m
m
b
b
b
b
b
b
b
3.22
3.20−3.44,
3.20−3.44,
3.20−3.44,
3.11, ddd (8.5, 8.5, 5.0)
b
b
b
3.21
2.97−308, m
b
b
b
b
5′
3.48
3.31,
m
3.2−3.4,
m
6′a
4.55, dd (11.5, 1.5)
4.27, dd (11.5, 6)
4.64, d (11.0)
4.00, d (11.4)
3.6, dd (11.4, 7.5)
5.14, d (5.0)
3.97, br d (11.5)
4.00, br d (12.0)
6′b
2′-OH
3′-OH
4′-OH
1″
2″
3″
4″
5″
6″a
6″b
2″-OH
3″-OH
4″-OH
2‴
3‴
4‴
5‴
6‴
4.41, dd (11.0, 6.5)
3.63, dd (11.5, 6.0)
3.62, dd (12.0, 7.5)
5.02, d (5.0)
5.02, d (5.0)
4.38, d (7.5)
4.29, d (8)
4.50, d (7.5)
b
b
7.97, dd (8.0, 1.0)
7.55, dd (8.0, 8.0)
7.68, br dd (8.0, 8.0)
7.55, dd (8.0, 8.0)
7.97, dd (8.0, 1.0)
8.08, d (7.5)
2.97−3.08,
3.20, m
m
3.01, dd (6.0, 6.0)
3.08,
m
b
b
7.52, dd (7.5, 7.5)
7.64, dd (7.5, 7.5)
7.52, dd (7.5, 7.5)
8.08, d, (7.5)
3.20,
3.18,
3.42,
m
m
m
3.2−3.4,
3.2−3.4,
3.53, m
m
m
b
b
b
3.23, m
3.46, dd (6.0, 6.0)
a 4.56, d (11.5)
b 4.29, dd (11.5, 6)
5.09, d (5.5)
4.51, dd (12.0, 2.0)
4.23, dd (12.0, 6.5)
4.60, dd (11.5, 2.0)
4.35, dd (11.5, 6.0)
5.10, d (5.5)
5.27, d (5.5)
7.98, dd (7.5, 1.0)
7.53, dd (7.5, 7.5)
7.66, dd (7.5, 7.5)
7.53, dd (7.5, 7.5)
7.98, dd (7.5, 1.0)
7.95, dd (7.5, 2.0)
7.52, dd (7.5, 7.5)
7.65, dd (7.5, 7.5)
7.52, dd (7.5, 7.5)
7.95, dd (7.5, 2.0)
9.11, d (2.0)
8.30, ddd (8.0, 2.0, 2.0)
b
7.59,
m
8.83, dd (8.0, 2.0)
a
1H NMR data (δ) were measured at 500 MHz in DMSO-d6 for 1, 3, 4, 5 and in methanol-d4 for 2. Coupling constants (J) in Hz are given in
parentheses. The assignments were based on HSQC and HMBC experiments. Signal overlapped.
b
and β-glucopyranosyl moieties was identical to that of 1. In
addition, the H NMR data revealed the presence of one
NMR data (Tables 1 and 2) indicated that, as with compounds
1
1 and 2, there was a 6-benzoylglucosyl moiety present in 3,
which was verified by correlations from H-2‴, H-6‴, H-6″a,
oxymethine proton at δH 5.21 (s, H-7) and the protons of a
monosubstituted aromatic ring at δH 7.36 (d, J = 6.0 Hz, 2H)
and 7.20−7.27 (m, 3H), which in combination with the 13C
NMR data suggests the presence of a mandelic acid moiety.
The position of the 6-benzoylglucosyl group was determined to
be at C-7 of the mandelic acid moiety through the connectivity
of C-1′−O−C-7, based on the HMBC correlations of H-1′/C-
7. Hydrolysis of 2 yielded benzoic acid, β-D-glucopyranose with
1
and H-6″b to C-7‴ in the HMBC spectrum. The H NMR
spectrum showed signals attributable to a monosubstituted
aromatic ring at δH 7.36 (d, J = 7.5 Hz, H-2, 6), 7.31 (dd, J =
7.5, 7.5 Hz, H-3, 5), and 7.26 (dd, J = 7.5, 7.5 Hz, H-4) and an
oxymethylene group at δH 4.78 (d, J = 12.5, H-7a) and 4.53 (d,
J = 12.5, H-7b), revealing the presence of a benzyl group. Two
doublets attributed to anomeric protons (δH 4.19 and 4.38),
together with the coupling patterns of oxymethylene and
oxymethine protons resonating between δH 3.00 and 4.56, as
well as six hydroxy signals, indicated the presence of two β-
glucopyranosyl units. This was confirmed by acidic hydrolysis
of 3, which produced β-D-glucopyranose as the sole sugar, as
identified by TLC comparison and the optical rotation value. In
the HMBC spectrum, correlations of H-6′a, H-6′b/C-1″; H-
1″/C-6′; H-2, H-6, and H-1′/C-7; and H-7a and H-7b/C-1′
showed the connections of C-1″−O−C-6′ and C-1′−O−C-7.
Therefore, prupersin C was deduced to have structure 3
(Figure 1).
[α]20D +27.4 (c 0.06, H2O), and (−)-mandelic acid with [α]20
D
−95.3 (c 0.19, H2O). The(2R absolute configuration was
confirmed by the negative [α]D value of mandelic acid.5
Therefore, prupersin B was determined to have structure 2
(Figure 1).
Prupersin C (3) was obtained as an amorphous powder, [α]20
D
−29.8 (c 0.24, MeOH). The molecular formula was determined
to be C26H32O12 from analysis of HRESIMS data (m/z
559.1796 [M + Na]+, calcd 559.1786), corresponding to 11
indices of hydrogen deficiency. The IR spectrum indicated the
presence of hydroxy (3362 cm−1), ester carbonyl (1716 cm−1),
and aromatic (1601, 1496 cm−1) functionalities. Analysis of 1D
B
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a
Table 2. 13C NMR Spectroscopic Data of Compounds 1−5
position
1
2
3
4
5
1
133.3 C
137.7, C
138.0, C
129.7, C
133.8, C
2
128.0 CH
114.3 CH
156.1 C
129.7, CH
129.6, CH
129.8, C
127.7, CH
128.1, CH
127.3, CH
128.1, CH
127.7, CH
69.6, CH2
129.5, CH
128.7, CH
133.7, CH
128.7, CH
129.5,CH
164.6, C
127.3, CH
129.0, CH
129.6, CH
129.0, CH
127.3, CH
66.7, CH
118.8, C
3
4
5
114.3 CH
128.0 CH
73.4 CH
73.8 CH
71.4 CH2
103.7 CH
73.6 CH
76.1 CH
70.1 CH
73.6 CH
64.3 CH
129.8 CH
129.2 CH
128.8 CH
133.4 CH
128.8 CH
129.2 CH2
165.7 C
129.6, CH
129.7, CH
79.4, CH
6
7
8
9
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
7″
1‴
2‴
3‴
4‴
5‴
6‴
7‴
99.7, CH
74.9, CH
77.3, CH
72.2, CH
75.9, CH
65.4, CH
131.5, C
102.0, CH
73.4, CH
76.6, CH
70.1, CH
75.8, CH
68.6, CH2
103.5, CH
73.5, CH
76.5, CH
70.0, CH
73.6, CH
64.2, CH2
94.9, CH
72.4, CH
76.1, CH
69.4, CH
76.5, CH
68.0, CH2
102.9, CH
73.4, CH
76.4. CH
70.0, CH
73.7, CH
64.2, CH2
101.5, CH
73.1, CH
76.5, CH
70.0, CH
76.5, CH
68.5, CH2
103.8, CH
73.5, CH
76.1, CH
70.0, CH
73.7, CH
64.6, CH2
130.8, CH
129.8, CH
134.6, CH
129.8, CH
130.8, CH
167.9, C
129.7, C
129.1, C
129.2, CH
128.7, CH
133.3, CH
128.7, CH
129.2, CH
165.7, C
129.2, CH
128.7, CH
133.2, CH
128.7, CH
129.2, CH
165.6, C
150.0, CH
125.7, C
136.9, CH
124.0, CH
153.7, CH
164.6, C
a
13C NMR data (δ) were measured in DMSO-d6 at 100 MHz for 1 and 5 and at 125 MHz for 3 and 4 and in methanol-d4 at 100 MHz for 2. The
assignments were based on HSQC and HMBC experiments.
Scheme 1. Hydrolysis of 1
Prupersin D (4) was obtained as an amorphous powder, [α]20
Therefore, prupersin D was elucidated to have structure 4
(Figure 1).
D
−24.3 (c 0.21, MeOH). The molecular formula was determined
to be C26H30O13 from analysis of HRESIMS data (m/z
573.1594 [M + Na]+, calcd 573.1579), corresponding to 12
indices of hydrogen deficiency.
Prupersin E (5) was obtained as an amorphous powder that
crystallized from MeOH, [α]20 −53.6 (c 0.02, MeOH). The
D
molecular formula was determined to be C26H30N2O12 from
analysis of HRESIMS data (m/z 585.1709 [M + Na]+, calcd
585.1731), corresponding to 12 indices of hydrogen deficiency.
The IR spectrum indicated the presence of ester carbonyl
(1726 cm−1) and aromatic (1592, 1494 cm−1) functionalities.
The NMR spectrum resembled that of the known compound
amygdalin except for signals attributed to a 3-substituted
pyridine moiety at δH 9.11 (d, J = 2.0 Hz, H-2″), 8.83 (dd, J =
8.0, 2.0 Hz, H-6‴), 8.30 (ddd, J = 8.0, 2.0, 2.0 Hz, H-4‴), and
7.59 (m, H-5‴) and one carbonyl carbon at δc 164.6. These
data indicated 5 as an amygdalin derivative, which was further
The UV, IR, and NMR spectra of 4 were similar to those of 3.
1
Comparison of the H and 13C NMR data of 4 (Tables 1 and
2) with those of 3 shows a carbonyl signal (δC 164.6) instead of
the oxymethylene signal (δC 69.6) in 3, and the signal of
anomeric carbon 1′ shifts from δC 102.0 to 94.9, indicating the
presence of a benzoyl group in 4 rather than the benzyl group
in 3.6 This assignment was in agreement with HMBC
correlations between H-1′, H-2, and H-6 and C-7. Acidic
hydrolysis of 4 produced β-D-glucopyranose as the sole sugar.
C
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confirmed by hydrolysis (Experimental Section). HMBC
correlations between H-6″a, H-6″b, H-2‴, and H-4‴ and the
carbonyl carbon indicated the connectivity of C-3‴−C-7‴ and
C-7‴−O−C-6″. Therefore, the structure of prupersin E was
determined as 5 (Figure 1).
In addition to the five new compounds (1−5), two
compounds that have not previously been found in nature,
ethyl amygdalinate (6)7,8 and 4-hydroxymethyl-2-methoxy-
phenyl 6-O-benzoyl-β-D-glucopyranoside (7),9 were isolated
from the seeds of P. davidiana. Full NMR data are presented
here for the first time (Table 3). Compound 6 was obtained as
an amorphous, white powder, [α]20 −100.2 (c 0.09, MeOH).
D
The molecular formula was determined to be C22H32O13 from
HRESIMS data (m/z 527.1706 [M + Na]+), corresponding to
1
seven indices of hydrogen deficiency. The H and 13C NMR
spectra for 6 corresponded to those for amygdalin, except for
the presence of an ester in place of the cyano moiety. The
NMR signals of the ester moiety and the HMBC correlations of
H-7 and H-9/C-8 (Table 3) indicated the presence of an ethyl
mandelate group. A 7R absolute configuration can be
1
unambiguously assigned on the basis of the positive Lb CEs
in the ECD spectrum.10 Acid hydrolysis of 6 yielded D-glucose.
A β-anomeric configuration for the glucosyl unit was assigned
3
from the large J1, 2 coupling constant (7.6 Hz). Thus, 6 was
determined to be ethyl amygdalinate, which has been
previously synthesized by Campbell and Haworth.7
Compound 7 was obtained as an amorphous powder. The
molecular formula was determined to be C21H24O9, based on
HRESIMS data (m/z 443.1323 [M + Na]+, calcd 443.1313),
Figure 1. Structures of compounds 1−7 and amygdalin.
a
Table 3. NMR Spectroscopic Data of Compounds 6 and 7
6
7
position
δC
δH (J in Hz)
HMBC
4, 7
δC
δH (J in Hz)
HMBC
1
136.5, C
147.0, C
2
129.0, CH
129.7, CH
130.0, CH
129.7, CH
129.0, CH
78.9, CH
172.9, C
7.48−7.51, m
7.32−7.38, m
7.32−7.38, m
7.32−7.38, m
7.48−7.51, m
5.42, s
150.9, C
3
1
112.6, CH
137.8, C
6.89, d (1.8)
1, 2, 5, 7
4
2, 6
5
1
120.5, CH
118.1, CH
65.0, CH2
6.57, dd (8.1, 1.8)
6.97, d (8.1)
4.4, s
1, 3, 7
1, 2, 4
3, 5
6
4, 7
7
1, 2, 6, 8, 1′
8
b
9
62.7, CH2
14.3, CH3
101.3, CH
74.8, CH
78.0, CH
71.4, CH
77.2, CH
69.6, CH2
4.07−4.21, m
8, 10
9
10
1′
2′
3′
4′
5′
6′a
6′b
1″
2″
3″
4″
5″
6″a
6″b
7″
OMe
1.05, t (6.9)
b
4.07−4.21, m
102.8, CH
74.9, CH
77.8, CH
72.0, CH
75.6, CH
65.3, CH2
4.82, d (7.0)
1
b
3.20−3.40
3.20−3.40
3.20−3.40
3.20−3.40
3.32−3.48, m
3.32−3.48, m
3.32−3.48, m
b
b
3.67, ddd (7.5, 7.5, 2.1)
b
4.07−4.21, m
5′, 1″
6′, 2″
4.60, dd (12, 2.1)
7″, 5′
3.76, dd (11.6, 5.6)
4.32, dd (12, 7.5)
104.8, CH
75.2, CH
78.0, CH
71.6, CH
77.5, CH
62.7, CH2
4.39 d, (7.6)
131.3, C
b
3.20−3.40, m
3.20−3.40, m
3.20−3.40, m
3.20−3.40, m
130.7, CH
129.6, CH
134.3, CH
129.6, CH
130.7, CH
7.92, d (7.2)
4″, 6″, 7″
1″, 5″, 4″
2″, 6″
1″, 3″, 4″
2″, 4″, 7″
b
7.40, dd (7.2, 7.2)
7.53, dd (7.2, 7.2)
7.40, dd (7.2, 7.2)
7.92, d (7.2)
b
b
3.85, dd, (12.0, 2.0)
4″
3.65, dd, (12.0,3.9)
167.8, C
56.7
3.75, s
2
a
1H NMR data (δ) were measured in methanol-d4 at 400 MHz for 6 and in methanol-d4 at 500 MHz for 7. Coupling constants (J) in Hz are given in
b
parentheses. The assignments were based on HSQC and HMBC experiments. Signal overlapped.
D
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Table 4. Antioxidant Activity of Compounds 1−7
compound
concentration (M/L)
inhibitiory rates of MDA (%)
compound
concentration (M/L)
inhibitiory rates of MDA (%)
1
10−4
10−5
10−6
10−4
10−5
10−6
10−4
10−5
10−6
10−4
10−5
10−6
88
64
57
67
60
53
55
50
59
56
66
56
5
10−4
10−5
10−6
10−4
10−5
10−6
10−4
10−5
10−6
88
67
56
51
51
56
51
42
41
2
3
4
6
7
Plant Material. The seeds of Prunus davidiana (Carr.) Franch.
were collected at Yuncheng, Shanxi Province, in China, in July 2010
and identified by Professor Lin Ma from the Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College. A voucher specimen (Annonaceae No. S-2443) was
deposited in the Herbarium of the Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical
College.
corresponding to 10 indices of hydrogen deficiency. Analysis of
1D NMR data indicated the presence of a 6-benzoylglucosyl
moiety in 7, as for compounds 1 and 2, which was verified by
correlations from H-2″, H-6″, H-6′a, and H-6′b to C-7″ in the
HMBC spectrum of 7. The presence of a 1,2,4-trisubstituted
aromatic ring, a methoxy group, and an oxymethylene group
was also determined from the 1H NMR spectrum. According to
the analysis of 2D NMR data (Table 3), the methoxy group is
located at C-2 and the oxymethylene group at C-4. Thus, 7 was
determined to be 4-hydroxymethyl-2-methoxyphenyl 6-O-
benzoyl-β-D-glucopyranoside.
Extraction and Isolation. Air-dried and powdered seeds of P.
davidiana (17 kg) were exhaustively extracted with 95% aqueous
EtOH (3 × 75 L) at reflux. The combined extracts were concentrated
under reduced pressure to dryness. The residue was suspended in H2O
and partitioned with petroleum ether, EtOAc, and n-BuOH,
successively. The n-BuOH-soluble residue (500 g) was crystallized in
95% aqueous EtOH to remove the major constituent amygdalin. The
filtrate was concentrated under reduced pressure to dryness. The
residue (350 g) was subjected to column chromatography on silica gel
and eluted with a gradient of CHCl3−MeOH (10:1; 5:1; 3:1; 1:1;
100% MeOH). Fraction B (19 g) was subjected to silica gel column
chromatography (CHCl3−MeOH, 7:1; 5:1; 1:1; 100% MeOH) to
afford two subfractions. Fraction B-a (15.5 g) was further separated by
MPLC (ODS, 50 μm, YMC), eluted with 15, 20, 25, 35, and 100%
MeOH−H2O, to afford 40 subfractions. Fraction B-a-25 (42 mg) was
purified by preparative HPLC using 14% MeCN−H2O (8 mL/min) as
the mobile phase to yield compound 1 (14 mg, tR 60.3 min). Fraction
B-a-37 (155 mg) was subjected to preparative HPLC using 30%
MeCN−H2O (8 mL/min) to give compound 3 (29 mg, tR 56.3 min).
Fraction B-a-34 (185 mg) was purified by preparative HPLC using
28% MeCN−H2O (8 mL/min) to give compound 4 (34 mg, tR 42.3
min). Fraction B-a-27 (121 mg) was crystallized in MeOH to afford
compound 5 (19 mg). Fraction B-a-2 was separated on Sephadex LH-
20 eluted with H2O to afford three subfractions. Fraction B-b (4 g)
was separated on Sephadex LH-20, eluted with MeOH−H2O, 1:1, to
afford 7 (5 mg). Fraction C (60 g) was subjected to silica gel column
chromatography (CHCl3−MeOH, 5:1; 2:1; 1:1; 100% MeOH) to give
two subfractions. Fraction C-b (10 g) was further separated by MPLC
(ODS, 50 μm, YMC), eluted with 20, 50, 70, and 100% MeOH−H2O,
to afford 25 subfractions. Fraction C-b-20 (50 mg) was subjected to
preparative HPLC using 60% MeOH−H2O (8 mL/min) to give
compound 2 (10 mg, tR 45.3 min). Fraction B-a-34 (300 mg) was
purified by preparative HPLC using 12% MeCN−H2O (8 mL/min) to
give compound 6 (200 mg, tR 40.2 min).
The known compounds were identified as amygdalin,11 1-O-
vanilloyl-β-D-glucose,12 vanilloloside,13 androsin,14 2-β-D-gluco-
pyranosyloxy-2-phenylacetic acid amide,15 benzyl-β-D-glucopyr-
anoside,16 benzyl-β-D-glucopyranosyl(1−6)-β-D-glucopyrano-
side,17 amygdalinic acid,18 mandelic acid β-D-glucopyranoside,19
and a mixture of prunasin and sambunigrin,19 by NMR analysis
and comparison with literature data.
The antioxidant activity of compounds 1−7 was assessed
from the amount of malondialdehyde (MDA) that was
produced during microsomal lipid peroxidation induced by
Fe2+-cysteine. As shown in Table 4, compounds 1−6 showed
antioxidant activities with inhibitory rates of >50% at
concentrations of 10 μM. The compounds were also bioassayed
for cytotoxicity against five human tumor cell lines, HCT-8
(human ileocecal adenocarcinoma cell line), Bel-7402 (human
hepatoma cell line), BGC-823 (human gastric cancer cell line),
A549 (human lung epithelial cell line), and A2780 (human
ovarian cancer cell line). However, all the compounds were
inactive against these cell lines (showing IC50 value of >10
μM).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Jasco P-2000 polarimeter, and UV spectra with a
Jasco V-650 spectrophotometer. ECD spectra were measured on a
Jasco J-815 spectrometer. IR spectra were recorded on a Nicolet 5700
spectrometer by an FT-IR microscope transmission method. NMR
measurements were performed using VNS-600, Inova-500, Bruker
AV500-III, Mp-400, and Mercury-300 spectrometers. HRESIMS was
performed using an Agilent 1100 series LC/MSD ion trap mass
spectrometer. HPLC was performed on a Lumtech instrument
equipped with an Alltech 500 ELSD detector, using a YMC-Pack
ODS-A column (250 × 20 mm, 5 μm). Silica gel (200−300 mesh,
Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-
20 (GE), and ODS (50 μm, YMC, Japan) were used for column
chromatography. TLC was carried out with GF254 plates (Qingdao
Marine Chemical Factory). Spots were visualized by spraying with 10%
H2SO4 in EtOH followed by heating.
Prupersin A (1): amorphous, white powder; [α]20 −25.3 (c 2.35,
D
MeOH); UV(MeOH): λmax (log ε) 205 (3.77) nm; IR νmax 3339,
2890, 2257, 2128, 1717, 1614, 1600, 1516, 1452, 1317, 1278, 1172,
1
1051, 1025, 827, 764, 717, 834, 577 cm−1; H NMR (DMSO-d6, 500
MHz) and 13C NMR (DMSO-d6, 100 MHz) see Tables 1 and 2;
positive-ion ESIMS m/z 473.0 [M + Na]+; positive-ion HRESIMS m/
z 473.1428 [M + Na]+ (calcd for C22H26O10Na, 473.1418).
Prupersin B (2): colorless gum; [α]20 −82.0 (c 0.37, MeOH); UV
D
(MeOH): λmax (log ε) 194 (4.80) nm; IR νmax 3372, 3066, 2894, 1719,
1603, 1492, 1452, 1411, 1317, 1278, 1196, 1071, 1025, 968, 801, 748,
E
dx.doi.org/10.1021/np4000922 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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1
714, 629, 579, 517 cm−1; H NMR (methanol-d4, 500 MHz) and 13C
evaporated in vacuo. The residue was extracted with MeOH and
purified by preparative HPLC using 20% MeOH−H2O (5 mL/min) as
the mobile phase to yield amygdalin (1 mg, tR 40 min), as determined
by comparison with an authentic sample with analaytical HPLC (20%
MeOH−H2O, tR 10.5 min).
NMR (methanol-d4, 125 MHz) see Tables 1 and 2; negative-ion
ESIMS m/z 417 [M − H]−; positive-ion HRESIMS m/z 441.1158 [M
+ Na]+ (calcd for C21H22O9Na, 441.1156).
Prupersin C (3): amorphous, white powder; [α]20 −29.8 (c 0.24,
D
Acid Hydrolysis of Compounds 3−7. Compounds 3 (5 mg), 4 (5
mg), 5 (5 mg), 6 (5 mg), and 7 (5 mg) were individually refluxed in
6% HCl (5.0 mL) at 80 °C for 2 h. Each reaction mixture was
extracted with CHCl3 (3 × 6 mL), and the H2O phase was dried using
a N2 stream. The residues were separately subjected to column
chromatography over silica gel with EtOAc−MeOH−H2O (7:5:1) as
MeOH); UV (MeOH) λmax (log ε) 193 (4.99) nm; IR νmax 3362,
2883, 2256, 2128, 1716, 1601, 1496, 1453, 1363, 1316, 1277, 1165,
1048, 1025, 825, 715 cm−1; 1H NMR (DMSO-d6, 500 MHz) and 13
C
NMR (DMSO-d6, 125 MHz) see Tables 1 and 2; positive-ion ESIMS
m/z 559 [M + Na]+; positive-ion HRESIMS m/z 559.1796 [M + Na]+
(calcd for C26H32O12Na, 559.1786).
eluent to yield (+)-D-glucose (2.10 mg) from 3, [α]20 +44.3 (c 0.14,
Prupersin D (4): amorphous, white powder; [α]20 −24.3 (c 0.21,
D
D
H2O); (+)-D-glucose (2.60 mg) from 4, [α]20 +52.5 (c 0.17, H2O);
MeOH); UV (MeOH) λmax (logε) 193 (5.06) nm; IR νmax 3445, 3073,
D
(+)-D-glucose (2.00 mg) from 5, [α]20 +25.5 (c 0.01, H2O); (+)-D-
2920, 2879, 1727, 1602, 1585, 1493, 1453, 1421, 1352, 1318, 1274,
D
glucose (3.20 mg) from 6, [α]20 +51.5 (c 0.16, H2O); and (+)-D-
1180, 1079, 1026, 995, 909, 853, 808,713, 686, 565 cm−1; H NMR
1
D
glucose (1.60 mg) from 7, [α]20 +22.5 (c 0.01, H2O). The solvent
(DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz) see
Tables 1 and 2; positive-ion ESIMS m/z 573 [M + Na]+; positive-ion
HRESIMS m/z 573.1594 [M + Na]+ (calcd for C26H30O13Na,
573.1579).
D
system EtOAc−MeOH−H2O (7:5:1) was used for TLC identification.
Antioxidant Assay. The antioxidant activity of compounds 1−7 was
assayed in vitro by measuring the inhibiton of MDA production using
Fe2+/cysteine-induced rat liver microsomal lipid peroxidation. MDA
was detected using the thiobarbituric acid (TBA) method. Briefly,
different concentrations of compound or vehicle (10 μL), 1 mg/mL of
microsomal protein (100 μL), and 0.2 mM cysteine (10 μL) in 0.1 M
PBS (0.82 mL) were incubated for 15 min at 37 °C, 0.5 mM FeSO4
(50 μL) was added, and the solution was mixed and incubated for a
further 15 min at 37 °C again. An equal volume of 20% trichloroacetic
acid was added to terminate the reaction, and the mixture was
centrifuged for 10 min at 3000 rpm. The supernatant (1 mL) was
reacted with 0.67% TBA (1 mL) for 10 min at 100 °C. After cooling,
the amount of MDA was quantified by determining the absorbance at
532 nm, and then the inhibition rate was calculated, from which the
inhibition rate (IR) was calculated as IR (%) = 100% − At/(Ap − Ac) ×
100, where Ap, At, and Ac refer to the absorbance of Fe2+-cysteine, the
test compound, and control, respectively.20
Prupersin E (5): colorless needles (MeOH); mp 148−151 °C;
[α]20 −53.6 (c 0.02, MeOH); IR νmax 3371, 2964, 2935, 2887, 1726,
D
1661, 1592, 1495, 1457, 1422, 1371, 1336, 1295, 1266, 1231, 1197,
1161, 1145, 1124, 1079, 1046, 1018, 1000, 970, 895, 880, 851, 836,
761, 743, 703, 679, 610, 533 cm−1; H NMR (DMSO-d6, 500 MHz)
1
and 13C NMR (DMSO-d6, 100 MHz) see Tables 1 and 2; positive-ion
ESIMS m/z 585 [M + Na]+; positive-ion HRESIMS m/z 585.1709 [M
+ Na]+ (calcd for C26H30N2O12Na, 585.1691).
Ethyl amygdalinate (6): amorphous, white powder; [α]20D −100.2
(c 0.09, MeOH); UV (MeOH) λmax (log ε) 192 (5.04) nm; ECD
(MeOH) 261 (Δε +8.56 × 10−3) nm; IR νmax 3564, 3536, 3292, 2955,
2917, 2871, 1740, 1611, 1499, 1452, 1418, 1370, 1322, 1301, 1270,
1238, 1211, 1165, 1134, 1109, 1080, 1035, 949, 894, 731,614 cm−1; 1H
NMR (methanol-d4, 300 MHz) and 13C NMR (methanol-d4, 100
MHz) see Table 3; positive-ion ESIMS m/z 527 [M + Na]+; positive-
ion HRESIMS m/z 527.1706 [M + Na]+.
4-Hydroxymethyl-2-methoxyphenyl 6-O-benzoyl-β-D-glucopyra-
noside (7): amorphous, white powder; 1H NMR (D2O, 500 MHz) and
13C NMR (D2O, 125 MHz) see Table 3; positive-ion ESIMS m/z 443
ASSOCIATED CONTENT
* Supporting Information
MS, NMR, and ECD spectra for compounds 1−7, threo-1-(4-
hydroxyphenyl)glycerol, and mandelic acid. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
[M + Na]+; positive-ion HRESIMS m/z 443.1323 [M + Na]+ (calcd
for C21H24O9Na, 443.1313).
Alkaline and Enzymatic Hydrolysis of Compounds 1, 2, and 5.
Compound 1 (5.5 mg) was dissolved in a 0.01 M solution of NaOH in
H2O−MeOH (2 mL, v/v, 1:1) at rt for 4 h. The reaction mixture was
neutralized with dilute HCl, and the MeOH was removed under
vacuum. The remaining aqueous solution was extracted with n-BuOH.
The n-BuOH layer was evaporated in vacuo and purified by preparative
HPLC using 14% MeCN−H2O (5 mL/min) as the mobile phase to
yield compound 1-(4-hydroxyphenyl)glycerol 3-O-β-D-glucopyrano-
side (2.5 mg, tR 20 min) and benzoic acid (1.2 mg, tR 35 min). Benzoic
acid was identified by comparison with an authentic sample using
analaytical HPLC (14% MeCN−H2O, tR 6.5 min). 1-(4-
Hydroxyphenyl)glycerol 3-O-β-D-glucopyranoside was hydrolyzed
with 15 mg of β-glucosidase (BCBF2889 V, RS-Sigma) in 1.5 mL of
H2O at 37 °C for 10 h. The reaction mixture was extracted with n-
BuOH. The n-BuOH layer was evaporated in vacuo and subjected to
preparative HPLC using 3% MeOH−H2O (5 mL/min) to give the
aglycone (0.5 mg, tR 25 min) as a colorless gum: [α]20D +18.0 (c 0.06,
EtOH). The 1H NMR spectrum was in agreement with that of threo-1-
(4-hydroxyphenyl)propane-1,2,3-triol.3 The aqueous layer was evapo-
rated and subjected to column chromatography over silica gel with
EtOAc−MeOH−H2O (7:5:1) as eluent to yield (+)-D-glucose (0.91
mg), [α]20D +30.3 (c 0.09, H2O), as confirmed by comparison with an
authentic sample (EtOAc−MeOH−H2O (7:5:1), Rf 0.59).
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-10-83161622. Fax: +86-10-63017757. E-mail: rych@
imm.ac.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Department of Medicinal Analysis, Institute of
Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, for the measurements of IR,
NMR, and HRESIMS spectra.
REFERENCES
■
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Journal of Natural Products
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