Flavonoids from Malus hupehensis and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells

Article abstract of DOI:10.1016/j.phytochem.2012.11.020

Three biflavonoid glycosides along with 12 known flavonoids, were isolated from leaves of Malus hupehensis. The complete structures of two of the compounds were established from analysis of MS, NMR spectroscopic and CD data, as well as DFT CD calculations they were determined to be atropisomeric along a central biaryl axis. The antioxidant activities and protective effects of the compounds against doxorubicin-induced cardiomyopathy in H9c2 cells were also investigated. Amongst all of the isolated compounds, quercetin was the most active radical scavenger with EC₅₀ values of 3.2 μM and 17.8 μM by the DPPH and ABTS⁺ methods, respectively. The results indicated that three of the flavanoids also had a strong protective influence against doxorubicin-induced cell death with EC₅₀ values of 8.3, 5.2 and 7.6 μM, respectively.

Full text of DOI:10.1016/j.phytochem.2012.11.020

Phytochemistry 87 (2013) 119–125
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Flavonoids from Malus hupehensis and their cardioprotective effects against
doxorubicin-induced toxicity in H9c2 cells
⇑
Shu-Qi Wang, Xiao-Feng Zhu, Xiao-Ning Wang, Tao Shen, Feng Xiang, Hong-Xiang Lou
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, Shandong University, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2012
Received in revised form 6 September 2012
Available online 28 December 2012
Three biflavonoid glycosides along with 12 known flavonoids, were isolated from leaves of Malus
hupehensis. The complete structures of two of the compounds were established from analysis of MS,
NMR spectroscopic and CD data, as well as DFT CD calculations they were determined to be atropisomeric
along a central biaryl axis. The antioxidant activities and protective effects of the compounds against
doxorubicin-induced cardiomyopathy in H9c2 cells were also investigated. Amongst all of the isolated
Keywords:
Malus hupehensis
Rosaceae
Biflavonoid glycosides
Circular dichroism
DFT CD calculations
Cardioprotection
compounds, quercetin was the most active radical scavenger with EC50 values of 3.2 lM and 17.8 lM
by the DPPH and ABTSÅ+ methods, respectively. The results indicated that three of the flavanoids also
had a strong protective influence against doxorubicin-induced cell death with EC50 values of 8.3, 5.2
and 7.6 lM, respectively.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
be effective in protecting against doxorubicin (Dox)-induced car-
diotoxicity (Goodman and Hochstein, 1977; Gille and Nohl, 1997;
Minotti et al. (2004)). Previous studies also showed that flavonoids
protect against both cardiomyopathy and cardiomyocyte apoptosis
induced by Dox (Han et al., 2008; Wang et al., 2010a,b). Therefore,
an objective of the present study was to determine whether treat-
ment with the isolated flavonoids protects against Dox-induced
cardiotoxicity in H9c2 cardiomyocytes. The isolation, structural
elucidation and biological activities of 15 isolated compounds is
reported.
Malus hupehensis (Pamp.) Rehder, a medicinal herb belonging to
the Rosaceae family, is widely distributed throughout southern
China. The young leaves of this plant, known as ‘Tea crabapple’,
are traditionally manufactured in the form of tea bags. Various
medicinal uses of M. hupehensis, such as the treatment of inflam-
mation, hypoxia, fatigue, and hypoglycaemia, have been reported
in traditional medicine (Jie et al., 2006). Phytochemical investiga-
tion of M. hupehensis indicated that its leaves are a rich source of
phloridzin, which can significantly lower plasma glucose levels
(Boccia et al., 1999; Bradford and Allen, 2007; Janssen et al.,
2003; Wang et al., 2010a,b). Extracts from the Malus plant species
also exhibited antiproliferative (Cetkovic et al., 2011), antihyper-
glycaemic (Barbosa et al., 2010), hepatoprotective (Yang et al.,
2010) and antimicrobial effects (Tian et al., 2010).These effects
have been attributed in part to the presence of polyphenols and
their related antioxidant capacity.
A bioassay-guided fractionation technique was used to separate
components from crude M. hupehensis extract which displayed sig-
nificant antioxidant properties in both DPPHÅ and ABTSÅ+ radical
scavenging assays, as part of our investigations into antioxidants
from traditional Chinese medicines (Du et al., 2007; Du and Lou,
2008; Han et al., 2008). Three biflavonoid glycosides (1–3, Fig. 1)
and 12 known flavonoids were isolated from the leaves of
M. hupehensis. Natural phenolic compounds have been found to
2. Results and discussion
EtOH extracts (95%) of dried leaves of M. hupehensis were con-
centrated, dissolved in distilled water and successively partitioned
into CH2Cl2, n-BuOH, and H2O fractions. The n-butanol extract was
subjected to repeated separation by silica gel column chromatogra-
phy, as well as by Sephadex LH-20 and semi-preparative HPLC, to
obtain three previously unreported compounds, 1–3, along with
12 known compounds viz. phloridzin (4) (Lu and Foo, 1998), acace-
tin (5) (Fazilatun et al., 2004), phloretin (6) (Zhang et al., 2008),
chrysin (7) (Shang et al., 2009), avicularin (8) (Sirat et al., 2010),
quercetin (9) (Li et al., 2010), 5,7-dihydroxychromone-7-O-b-
glucoside (10) (Shirataki et al., 1988), 3-hydroxyphloridzin (11)
(El Naggar et al., 1980), quercetin-3-O-b- -glucopyranoside (12)
(Gluchoff-Fiasson et al., 1997), luteolin-5-O-b- -glucopyranoside
(13) (Dias et al., 1998; Harborne, 1967), phloretin-20,40-di-O-b-
glucopyranoside (14) (Bognar et al., 1969) and epipinoresinol-4-
b- -glucoside (15) (Chiba et al., 1980), respectively. The known
D-
D
D
D-
⇑
Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382019.
E-mail address: louhongxiang@sdu.edu.cn (H.-X. Lou).
D
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.11.020

120
S.-Q. Wang et al. / Phytochemistry 87 (2013) 119–125
Fig. 1. Structures of compounds 1–3.
compounds were identified by comparing their spectroscopic data
to literature values.
13C NMR spectroscopic data (Table 1) suggested that 2 was an iso-
mer of 1. Further analysis of the 2D NMR spectroscopic data
(1H–1H COSY, HMQC, and HMBC spectra) indicated that both com-
pounds 1 and 2 had the same molecular structure. Nonetheless, the
CD spectra of the two dimers were different.
Compound 1 was obtained as a pale yellow powder with a
molecular formula of C42H46O21 as determined by HRESIMS. The
IR spectrum exhibited a broad OH absorption band at 3378 cmꢀ1
and a carbonyl band at 1628 cmꢀ1. The 1H and 13C NMR data
(Table 1) suggested the presence of six sp3 methylenes, 10 sp3
methines, nine sp2 methines and 17 sp2 quaternary carbons.
Duplicate signals characteristic of dihydrochalcone units were
observed in the 1H NMR spectrum. The resonances for H-7 and
H-8 were located at dH 2.44 (2H, m, H-7) and 3.25 (2H, m, H-8)
for unit I and 2.79 (2H, m, H-7), 3.32 (2H, m, H-8) for unit II. The
aromatic A-ring protons resonated at dH 6.64 (1H, s, H-2, I-A) and
6.33 (1H, s, H-6, I-A) for unit I and 7.05 (2H, d, J = 8.4 Hz, H-2 and
H-6, II-A), 6.65 (2H, d, J = 8.4 Hz, H-3 and H-5, II-A) for unit II.
The A2B2 spin system indicated the presence of a 4-hydroxy substi-
tuted A-ring in unit II. Three aromatic protons remained: dH 6.11
(1H, d, J = 1.2 Hz, H-30, I-B), 5.91 (1H, d, J = 1.2 Hz, H-50, I-B), and
6.27 (1H, s, H-50, II-B).
Of all the structural elements, only the central biaryl axis
could be responsible for the striking chiroptical difference (Xu
et al., 2010). The absolute configurations of 1 and 2 were as-
signed by comparing their experimental CD spectra (Hirasawa
et al., 2010). Previous studies have reported that atropisomeric
cupressuflavone 40-O-b-
D-glucopyranosides possess similar CD
spectroscopical properties to 40,4000,7,700-tetra-O-methylcupressuf-
lavone (Inatomi et al., 2005). Glucosyl moieties have little effect
on the long-wavelength region of the ECD spectra because
atropisomeric biflavonoids exhibit both intense positive and
negative Cotton signals at 300–400 nm (Harada et al., 1992;
Inatomi et al., 2005; Li et al., 1997). To reduce the computa-
tional cost, compounds 1 and 2 were modelled as two simpler
atropisomers (denoted as 10 and 20) that contained the essential
biflavonoid structural features (Fig. 3).
In addition, the anomeric protons at dH 4.90 (1H, d, J = 7.2 Hz,
H-100, unit I) and 4.92 (1H, d, J = 7.2 Hz, H-100, unit II) borne by the
carbon atoms at dC 101.4 (C-100, unit I) and 101.3 (C-100, unit II),
respectively, indicated the presence of two glycosidic moieties in
the molecule. Glucosyl moieties were deduced by analysis of the
13C NMR data (Lu and Foo, 1997; Wang et al., 2009). The presence
The calculated CD spectra of the isomers produced similar CD
patterns to those of both 1 and 2 as shown in Fig. 4. By this means,
the absolute configurations of 1 and 2 were unambiguously eluci-
dated as P and M, respectively (Fig. 1).
Compound 3, a pale, yellow, amorphous solid, gave an HRESIMS
of two
2007).
D-glucose was confirmed via sugar analysis (Tanaka et al.,
[M+Na]+ peak at m/z 905.2133, corresponding molecular formula
C
42H42O21 which indicates 22 unsaturated sites. The 13C NMR spec-
The 13C NMR resonances of the biflavonoids were similar to
troscopic data suggested the presence of four sp3 methylenes, 10
sp3 methines, 11 sp2 methines, and 17 sp2 quaternary carbons
(Table 1).
those of phloridzin (Lu and Foo, 1997) and phloretin-40-O-b-
D-glu-
coside (Wang et al., 2009) (Table 1). The COSY, HSQC and HMBC
experiments confirmed the presence of these two flavanone units
(Fig. 2). The (I-5, II-30) connection between the two flavanone units
was indicated by the significant correlation between the proton
signal at dH 6.33 (H-6, I-A) and the carbon resonance at dC 110.4
(C-30, II-B) in the HMBC spectrum. The linkage at C-5 (I-A) in the
phloridzin unit was confirmed by a long-range correlation between
H-6 (I-A) at dH 6.33 and the carbons at dC 26.7 (C-7, I-A) and 11.5
(C-2, I-A) in the HMBC spectrum. Compound 1 was thus estab-
lished as phloridzin-(I-5,II-30)-phloretin-40-O-b-glucoside (Fig. 1).
Compound 2 possessed the molecular formula C42H46O21 as
determined by HRESIMS. The molecular weight and both 1H and
The 1H NMR spectrum possessed two distinct flavonoid reso-
nances, which indicate a biflavonoid. Protons typical of a dihydr-
ochalcone moiety were observed at dH 2.88 (2H, t, J = 7.2 Hz, H-7,
unit I) and 3.47 (2H, m, H-8, unit I). The 4-hydroxyphenyl signals
were observed at dH 7.25 (2H, d, J = 8.4 Hz, H-2 and H-6, I-A) and
6.86 (2H, d, J = 8.4 Hz, H-3 and H-5, I-A). Four protons were ob-
served for the tetrasubstituted aromatic rings at dH 6.14 (1H, d,
J = 2.4 Hz, H-30, I-B), 5.93 (1H, d, J = 2.4 Hz, H-50, I-B), 6.31 (1H, d,
J = 1.8 Hz, H-6, II-A), and 6.39 (1H, d, J = 1.8 Hz, H-8, II-A). Three
other aromatic protons remained: dH 6.71 (1H, s, H-3, II-C), 6.37
(1H, s, H-30, II-B), and 7.70 (1H, s, H-60, II-B). Two anomeric protons

S.-Q. Wang et al. / Phytochemistry 87 (2013) 119–125
121
Table 1
1H and 13C NMR spectroscopic data for compounds 1, 2 and 3.
Position
1
2
3
dH
dC
dH
dC
dH
dC
Unit I
1
2
3
4
5
6
7
8
131.8
115.5
144.7
142.7
123.8
118.9
26.7
131.8
115.5
144.7
142.7
123.8
118.9
26.7
136.2
129.9
117.3
155.8
117.3
129.9
28.9
6.64 (1H, br s)
6.64 (1H, br s)
7.25 (1H, d, 8.4)
6.86 (1H, d, 8.4)
6.86 (1H, d, 8.4)
7.25 (1H, d, 8.4)
2.88 (2H, t, 7.2)
3.47 (2H, m)
6.33 (1H, br s)
2.44 (2H, m)
3.25 (2H, m)
6.33 (1H, br s)
2.44 (2H, m)
3.25 (2H, m)
44.3
44.3
44.4
9
205.5
105.6
161.3
94.7
164.8
97.3
165.6
101.4
73.6
77.7
69.8
77.3
61.0
205.5
105.6
161.3
94.7
164.8
97.3
165.6
101.4
73.6
77.7
69.8
77.3
61.0
204.4
105.1
160.9
94.4
165.3
96.8
164.5
100.9
73.0
77.2
69.3
76.7
60.5
10
20
30
40
50
60
100
200
300
400
500
600
6.11 (1H, d, 1.2)
5.91 (1H, d, 1.2)
6.11 (1H, d, 1.2)
5.91 (1H, d, 1.2)
6.14 (1H, d, 2.4)
5.93 (1H, d, 2.4)
4.90 (1H, d, 7.2)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.35 (1H, m)
4.90 (1H, d, 7.2)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.35 (1H, m)
4.93 (1H, d, 7.2)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.35 (1H, m)
3.70 (1H, m)
3.70 (1H, m)
4.67 (1H, m)
Unit II
1
2
3
4
5
6
7
132.1
129.7
115.4
155.7
115.4
129.7
29.5
132.1
129.7
115.4
155.7
115.4
129.7
29.5
7.05 (1H, d, 8.4)
6.65 (1H, d, 8.4)
7.05 (1H, d, 8.4)
6.65 (1H, d, 8.4)
149.0
103.1
178.4
157.0
97.6
6.71 (1H, s)
6.65 (1H, d, 8.4)
7.05 (1H, d, 8.4)
2.79 (2H, m)
6.65 (1H, d, 8.4)
7.05 (1H, d, 8.4)
2.79 (2H, m)
6.31 (1H, d, 1.8)
6.39 (1H, d, 1.8)
167.4
92.3
8
3.32 (2H, m)
45.6
3.32 (2H, m)
45.6
9
205.3
205.3
157.0
103.6
114.3
145.9
107.1
146.2
142.3
116.4
99.6
73.2
77.2
69.3
76.7
10
10
20
30
40
50
60
100
200
300
400
500
600
104.8
164.0
110.4
160.0
93.9
162.7
101.3
73.4
77.6
69.4
77.1
60.5
104.8
164.0
110.4
160.0
93.9
162.7
101.3
73.4
77.6
69.4
77.1
60.5
6.37 (1H, s)
6.27 (1H, s)
6.27 (1H, s)
7.70 (1H, s)
4.92 (1H, d, 7.2)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.35 (1H, m)
4.98 (1H, d, 7.2)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.32–3.51 (1H, m)
3.35 (1H, m)
5.07 (1H, d, 7.2)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.17–3.52 (1H, m)
3.35 (1H, m)
60.5
3.70 (1H, m)
3.70 (1H, m)
3.69 (1H, m)
Recorded in DMSO-d6; all chemical shift assignments were based on both 1D and 2D NMR techniques. 1H and 13C NMR spectra were recorded at 600 and 150 MHz,
respectively.
Fig. 2. Selected correlations from the HMBC (?) and COSY (–) NMR spectra of
compounds 1 and 2.
Fig. 3. Structures of compounds 10 and 20.
at dH 4.93 (1H, d, J = 7.2 Hz, H-100, unit I) and 5.07 (1H, d, J = 7.2 Hz,
H-100, unit II) were attributed to the glycosidic moieties.
Harborne, 1967). C-20 in II-B should be involved in the interflavo-
noid ether linkage with C-4 in I-B, as the C-20 resonance was shifted
downfield by 27.6 ppm from the corresponding carbon resonance
The 13C NMR data of 3 closely resembled those of phloridzin
(Lu and Foo, 1998) and luteolin-5-O-glucoside (Dias et al., 1998;

122
S.-Q. Wang et al. / Phytochemistry 87 (2013) 119–125
effects against doxorubicin-induced cell death with EC50 values
of 8.3, 5.2 and 7.6 lM, respectively (Table 2).
1 (expt.)
1' (calcd.)
2 (expt.)
2' (calcd.)
20
0
3. Conclusions
Δ
ε
As part of our investigations of antioxidants from traditional
Chinese medicine, three biflavonoid glycosides (1–3) along with
12 known flavonoids, were isolated from the leaves of M. hupehen-
sis. Compound 9 with the highest radical scavenging activity by
both DPPH and ABTSÅ+ radical methods, can be considered as the
major antioxidant constituent in this plant. Compounds 8, 9, and
12 had a strong protective effect against doxorubicin-induced cell
death in cell line H9c2. Thereby, traditional Chinese medicine is a
valuable resource for seeking potential agents for protecting
cardiomyocytes against doxorubicin-induced cell death.
20
expt.
calcd.
0.5
A
240 260 280 300 320 340 360 380 400
4. Experimental
Fig. 4. Experimental and simulated CD/UV spectra of compounds 1 and 2. The
dotted lines denote the simulated Boltzmann-averaged CD/UV spectra.
4.1. General experimental procedures
Optical rotations were measured using a GYROMAT-HP polarim-
eter. UV spectra were obtained with an Agilent 8453E UV–Visible
spectroscopy system. CD spectra were obtained on a Chirascan
spectropolarimeter. IR spectra were recorded on a Thermo Nicolet
NEXUS 470 FT-IR spectrometer using KBr discs. NMR spectra were
measured on a Bruker Avance DRX-600 spectrometer operating at
600 (1H) and 150 (13C) MHz with TMS as internal standard. HRE-
SIMS was conducted on a LTQ-Orbitrap XL. Semipreparative HPLC
was performed on an Agilent 1100 liquid chromatograph with a
of luteolin-5-O-glucoside. This linkage was confirmed by the
NOESY spectrum of 3, which possessed two strong correlations
between H-30 (II-B) and H-3 (I-A) and H-3 (II-C) and H-3 (I-A).
Therefore, structure 3 was determined to be phloridzin-(I-4, O,
II-20)-luteolin-5-O-glucoside (Fig. 1).
Many studies have shown that the major molecular mechanism
involved in doxorubicin-induced cardiotoxicity is the generation of
reactive oxygen species (ROS) by the catalytic quinone moiety in
doxorubicin. Therefore, removing excess ROS or suppressing their
generation using antioxidants may be effective in preventing Dox
cardiotoxicity (Bagchi et al., 2003). In the present study, 15 flavo-
noids were tested in vitro for antioxidative activity using the stan-
dard DPPH and ABTSÅ+ radical-scavenging assays. The EC50 values
were compared (Table 2), and the results showed that 1, 2 and 9
exhibited good free radical scavenging activity, stronger than the
positive control, ascorbic acid. The compounds were also evaluated
for their cytoprotective effects against Dox-induced myocardial
toxicity in H9c2 cells using the MTT colorimetric method. The
results indicated that 8, 9 and 12 in particular had protective
ZORBAX SB-C18 column (9.4 mm ꢁ 250 mm, 5
lm). HPLC was
performed on an Agilent 1200 liquid chromatograph with a
Phenomenex C18 column (4.6 mm ꢁ 250 mm). Silica gel (200–300
mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and
Sephadex LH-20 (25–100 lm; Pharmacia Biotek, Denmark) were
used for column chromatography (CC). TLC was performed using
high-performance TLC plates precoated with silica gel GF254
(Qingdao Haiyang Chemical Co., Ltd.). Spots were detected using
UV light or by spraying with H2SO4–EtOH (1:9) and then heating.
4.2. Plant material
Leaves of M. hupehensis were collected in the suburbs of Linyi
City, Shandong Province, PR China, in July 2008 and identified by
Prof. Lan Xiang of Shandong University. A voucher specimen (No.
HBHT0807) was deposited in the Herbarium of the School of Phar-
maceutical Sciences, Shandong University.
Table 2
DPPHÅ and ABTSÅ+ radical scavenging and cytoprotective effects of compounds 1–15.a
Compound
DPPHÅ EC50
M)b
ABTSÅ+ EC50
M)c
Cytoprotection EC50
(l
M)d
(l
(
l
1
2
3
4
5
6
7
8
3.1
2.8
8.6
31.5
40.6
21.5
53.3
6.5
27.3
24.9
17.26
49.6
29.1
15.0
61.5
23.4
17.8
>160
46.1
14.2
23.6
86.2
>160
42.1
>80
>80
11.3
>80
41.2
>80
>80
8.3
4.3. Extraction and isolation
Dried leaves of M. hupehensis (2.5 kg) were extracted three
times (2 h, 2 h, 1 h) with EtOH–H2O (3 ꢁ 3 L 95:5, v/v) under con-
ditions of reflux. The combined EtOH extracts were concentrated in
vacuo to yield the crude material (890 g). The latter was then sus-
pended in H2O (1 L) and successively partitioned into CH2Cl2
(3 ꢁ 1 L), n-BuOH (3 ꢁ 1 L) and H2O fractions. After filtering and
concentrating under reduced pressure, the n-BuOH extract
(210 g) was subjected to silica gel (200–300 mesh) CC and eluted
with a gradient of CH2Cl2–MeOH (100/1; 20/1; 10/1; 5/1; 2/1)
and finally MeOH. The collected fractions were combined into 13
major fractions (MH1–MH13) based on their TLC behaviour. Sub-
fraction MH2 (5 g) was separated via silica gel CC using a gradient
system of increasing polarity of CH2Cl–MeOH (50:1–10:1, v/v) to
yield three fractions (MH2A–MH2C). MH2B was purified on Sepha-
dex LH-20 eluted with CHCl3–MeOH (1:1) to yield chrysin (7,
350 mg) and chromone (10, 210 mg). Sub-fraction MH3 (2.3 g)
9
10
11
12
13
14
15
Ascorbic
3.2
5.2
>160
16.1
9.7
27.5
72.33
158.3
7.2
>80
>80
7.6
13.5
>80
>80
>80
acide
a
b
c
Each experiment was independently performed at least three times.
Effective concentration required to scavenge 50% of the DPPHÅ free radical.
Effective concentration required to scavenge 50% of the ABTSÅ+ cation radical.
Effective concentration required to protect the cell viability at 50%.
Ascorbic acid was used as the positive control.
d
e

S.-Q. Wang et al. / Phytochemistry 87 (2013) 119–125
123
was subjected to Sephadex LH-20 CC using CH2Cl2–MeOH (1:1) as
eluent to yield four fractions (MH3A–MH3D). MH3A was further
separated by HPLC on a semi-preparative ZORBAX SB-C18 column
and stirring at 60 °C for another 1.5 h. Separation by HPLC on a
C-18 column eluted with H2O (containing 0.2% TFA)–CH3CN
(68:32) provided the derivative S-1.
(9.4 mm ꢁ 250 mm, 5
l
m), using MeOH–H2O (70:30) to afford
Compound 1 was hydrolysed with HCl and extracted with
CH2Cl2. The aqueous layer was passed through a Sephadex LH-20
column using EtOH as eluent, and the eluate was concentrated.
The residue was dissolved in pyridine (0.4 mL) and stirred with
30,5,50,7-tetrahydroxyflavanone (5, 30 mg). MH3C was fractionated
in a similar way to afford phloretin (6, 45 mg). Sub-fraction MH4
(24 g) was subjected to silica gel CC, using a CH2Cl2–MeOH
(40:1–1:1) gradient system, to give sub-fractions MH4A through
MH4E based on TLC analysis. MH4B was subjected to a Sephadex
LH-20 column eluted with EtOH to afford quercetin (9, 720 mg).
Further separation of sub-fraction MH4D (12 g) by repeated silica
gel CC with CH2Cl2–MeOH (9:1) afforded phloridzin (4, 5 g). Sub-
fraction MH5 (1.5 g) was subjected to a Sephadex LH-20 column
using EtOH as eluent to yield two major sub-fractions, MH5A and
MH5B. MH5A was further purified by ODS CC eluted with a gradient
MeOH–H2O system (from 50:50 to 90:10) to yield avicularine (8,
52 mg). MH5B was further separated by HPLC on a Phenomenex
C18 column using MeOH–H2O (75:25) as the mobile phase to pro-
vide 3-hydroxyphloridzin (11, 60 mg). MH8 (3.5 g) was applied to a
Sephadex LH-20 column using EtOH as eluent to yield sub-frac-
tions MH8A to MH8B by TLC analysis. Separation of sub-fraction
MH8B (1.1 g) followed similar procedures to sub-fraction MH5B
and yielded P-phloridzin-(I-5, II-30)-phloretin-40-O-glucoside (1,
26 mg), M-phloridzin-(I-5, II-30)-phloretin-40-O-glucoside (2,
L-cysteine methyl ester (10 mg) for 1.5 h at 60 °C before adding
o-tolyl isothiocyanate (40 L) to the mixture and heating at 60 °C
l
for 1.5 h. The reaction mixture was analysed by HPLC using
250 nm detection. Analytical HPLC was performed on a Phenome-
nex C18 column (4.6 ꢁ 250 mm) at 25 °C using a gradient of CH3CN:
0.2%TFA in H2O: 0–23 min (32:68), 23–30 min (32:68 to 70:30),
and 30–60 min (70:30) as the mobile phase. Peaks were detected
using an Agilent DAD detector.
sugar moieties in 1, as the product had the same retention times
as S-1 (tR 11.9 min). Two -glucose moieties were identified in 2
D-Glucoses were identified as the
D
and 3 by the same method.
4.8. Calculation of CD spectra
All calculations were performed using the GAUSSIAN 03 soft-
ware package under a DFT framework (Frisch et al., 2004). The
semiempirical AM1 method and DFT approach (B3LYP/6-31G⁄)
were chosen to determine the potential energy surface (PES). At
the B3LYP/6-31G⁄ level, the ground-state geometries were fully
optimised without any symmetry restrictions, and frequency cal-
culations were conducted to confirm these minima and calculate
the free energy at 298.15 K. The electronic excitation energies
and rotational strengths in both the gas phase and methanol were
calculated using TDDFT at the same level for both the velocity and
length formalism for the first 90 states. The rotatory strengths
were summed and energetically weighted according to Boltzmann
statistics. The CD curves were then simulated using the Gaussian
function (Stephens and Harada, 2010):
35 mg), and quercetin-3-O-b-D-glucopyranoside (12, 40 mg),
respectively. MH8C (600 mg) was purified by HPLC on a semi-pre-
parative ZORBAX SB-C18 column using CH3OH–H2O (60:40) as the
mobile phase to afford luteolin-5-O-b-D-glucopyranoside (13,
50 mg) and epipinoresinol (15, 45 mg). MH10 (550 mg) was further
fractioned by HPLC on a Phenomenex C18 column using MeOH–H2O
(47:53) as the mobile phase to yield phloridzin-(I-4,O,II-20)-
luteolin-5-O-b-
-glucopyranoside (14, 18 mg).
D
-glucoside (3, 15 mg) and phloretin-20,40-di-O-b-
D
4.4. P-phloridzin-(I-5, II-30)-phloretin-4-O-glucoside (1)
X
D
EiÞ=r 2
ꢂ
Amorphous light yellow powder; ½a D25
ꢂ
ꢀ17.6 (c 2.87, MeOH); UV
1
1
r p
EiRieꢀ½ðEꢀ
ð1Þ
pffiffiffi
D
e
ðEÞ ¼
ꢁ
D
2:296 ꢁ 10ꢀ39
(MeOH) kmax (loge) 229 (4.05), 287 (3.16) nm; CD (MeOH) kmax
i
(D
e
) 315 (3.3), 287 (ꢀ1.5) nm; IR (KBr) mmax 3378, 2932, 2861,
where
r
is half the band width at a height of 1/e, and
the excitation energies and rotatory strengths for transition i,
respectively. Values of = 0.4 eV and Ri = Rvel were used here.
D
Ei and Ri are
1628, 1608, 1515, 1455, 1373 cmꢀ1; For 1H and 13C NMR spectro-
scopic data, see Table 1; HRESIMS m/z 887.2641 [M+H]+ (calcd
for C42H47O21, 887.2610).
r
4.9. 1,1-Diphenyl-2-picrylhydrazil (DPPH) free radical scavenging
activity
4.5. M-phloridzin-(I-5, II-30)-phloretin-4-O-glucoside (2)
Amorphous light yellow powder; ½a D25
ꢀ12.1 (c 1.58, MeOH); UV
ꢂ
The DPPH radical scavenging activities of compounds 1–15
were measured as described previously (Huang et al., 2003). The
bleaching rate of a stable free radical, DPPHÅ, was monitored at a
characteristic wavelength in the presence of the sample. In its rad-
ical form, DPPHÅ absorbs at 515 nm; however, its absorption de-
creases upon reduction by an antioxidant or radical species.
Briefly, a 1 mM solution of DPPHÅ in EtOH was prepared and 1 ml
was added to 5 mL solutions of the studied compounds in EtOH
at different concentrations. After 30 min, the absorbance at
515 nm was measured. The capability to scavenge the DPPHÅ radi-
cal was calculated using the following equation:
(MeOH) kmax (loge) 229 (3.73), 287 (2.96) nm; CD (MeOH) kmax
(D
e
) 315 (ꢀ4.1), 287 (3.6) nm; IR (KBr) mmax 3363, 2934, 2892,
1624, 1607, 1515, 1463, 1373 cmꢀ1; For 1H and 13C NMR spectro-
scopic data, see Table 1; HRESIMS m/z 887.2647 [M+H]+ (calcd
for C42H47O21, 887.2610).
4.6. Phloridzin-(I-4, O, II-2)-luteolin-5-O-glucoside (3)
Amorphous light yellow powder; ½a D25
ꢀ11.6 (c 2.38, MeOH); UV
ꢂ
(MeOH) kmax (loge) 224 (4.03), 257 (3.66) nm; CD (MeOH) kmax
(D
e
) 201 (ꢀ5.3) nm; IR (KBr) mmax 3356, 2928, 1720, 1608,
1468 cmꢀ1; For 1H and 13C NMR spectroscopic data, see Table 1;
HRESIMS m/z 905.2133 [M+Na]+ (calcd for C42H42O21Na,
905.2116).
DPPHÅ scavenging effectð%Þ ¼ ð1 ꢀ ASample=AControlÞ ꢁ 100
where ASample is the absorbance in the presence of the compounds,
and AControl is the absorbance without those compounds.
4.7. Sugar identification
4.10. ABTS radical cation decolourisation assay
D
-Glucose (72 mg) and
(90 mg) were dissolved in pyridine (3 mL) and stirred at 60 °C for
1.5 h before adding o-tolyl isothiocyanate (360 L) to the mixture
L-cysteine methyl ester hydrochloride
ABTSÅ+ radical scavenging activity was determined using a total
antioxidant capacity assay kit for the ABTS method according to
l

124
S.-Q. Wang et al. / Phytochemistry 87 (2013) 119–125
Fazilatun, N., Nornisah, M., Zhari, I., 2004. Superoxide radical scavenging properties
of extracts and flavonoids isolated from the leaves of Blumea balsamifera.
Pharm. Biol. 42, 404–408.
the manufacturer’s instructions (Beyotime Institute of Biotechnol-
ogy Institute) (Wang et al., 2010a,b). The scavenging capability for
the ABTSÅ+ radical was calculated using the following equation:
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R.,
Montgomery Jr., J.A., Vreven, T., Kudin, K.N., Burant, J.C., Millam, J.M., Iyengar,
S.S., Tomasi, J., Barone, V., Mennucci, B., Cossi, M., Scalmani, G., Rega, N.,
Petersson, G.A., Nakatsuji, H., Hada, M., Ehara, M., Toyota, K., Fukuda, R.,
Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Klene, M., Li,
X., Knox, J.E., Hratchian, H.P., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J.,
Gomperts, R., Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C.,
Ochterski, J.W., Ayala, P.Y., Morokuma, K., Voth, G.A., Salvador, P., Dannenberg,
J.J., Zakrzewski, V.G., Dapprich, S., Daniels, A.D., Strain, M.C., Farkas, O., Malick,
D.K., Rabuck, A.D., Raghavachari, K., Foresman, J.B., Ortiz, J.V., Cui, Q., Baboul,
A.G., Clifford, S., Cioslowski, J., Stefanov, B.B., Liu, G., Liashenko, A., Piskorz, P.,
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ABTSÅþ scavenging effectð%Þ ¼ ð1 ꢀ ASample=AControlÞ ꢁ 100
where AControl is the initial ABTSÅ+ absorbance, and ASample is the
absorbance of the remaining ABTSÅ+ in the presence of the studied
compounds.
4.11. Cell viability measurements
Rat cardiac H9c2 cells (ATCC Rockville, MD) were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% foetal bovine serum, 100 U/ml of penicillin, 100 lg/ml of
streptomycin under 5% CO2 at 37 °C. These cells were fed every
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were then cotreated with doxorubicin (20
l
M in DMEM) for 24 h. The pretreated cells
M in DMEM) and the
l
test compounds for another 24 h. After incubation, the cells were
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Acknowledgements
Financial support from the Natural Science Foundation of China
(No. 30925038), Science and Technology Development Project of
Shandong Province (No. 2011GSF11909) and Independent Innova-
tion Foundation of Shandong University (IIFSDU) is gratefully
acknowledged.
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