Antioxidant and myocardial preservation activities of natural phytochemicals from mung bean (Vigna radiata L.) seeds

Article abstract of DOI:10.1021/acs.jafc.6b01538

Mung bean (Vigna radiata L.) seeds (MBS) contain abundant nutrients with biological activities. This study was aimed to isolate key bioactive components from MBS with antioxidant and myocardial preservation activities. A new flavonoid C-glycoside, isovitexin-6″-O-α-l-glucoside, and 14 known compounds were obtained. Their structures were identified by extensive 1D and 2D NMR and FT-ICR-MS spectroscopic analyses. The antioxidant activities of these compounds were evaluated. Compounds 1-5 and 7-10 displayed 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS^?+) scavenging activity, but only 5 and 7 exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH?) scavenging activity. The myocardial preservation effect of 2, 3, and MBS were investigated by measuring the serum levels of LDH, CK, and AST as well as the tissue level of MDA and SOD. The results demonstrated that 2, 3, and MBS had a significant protective effect against ISO-induced myocardial ischemia. MBS can be regarded as a potential new source of antioxidants and myocardial preservation agents.

Full text of DOI:10.1021/acs.jafc.6b01538

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Article
Antioxidant and myocardial preservation activities of natural
phytochemicals from Mung Bean (Vigna radiate L.) seeds
Yan Bai, Jiawei Chang, Yan Xu, Dan Cheng, Hongxin Liu, Yunli Zhao, and Zhiguo Yu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01538 • Publication Date (Web): 17 May 2016
Downloaded from http://pubs.acs.org on May 17, 2016
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Antioxidant and myocardial preservation activities of
natural phytochemicals from Mung Bean (Vigna
radiata L.) seeds
†
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Yan Bai*, Jiawei Chang*, Yan Xu*, Dan Cheng*, Hongxin Liu , Yunli Zhao*, Zhiguo
Yu*
*Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang
Pharmaceutical University, Shenyang 110016, China
†Dalian Weida Pharmacy Co., Limited, 12ꢀ7 Zhuhai Street, Xigang District, Dalian
116011, China
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ABSTRACT
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Mung bean (Vigna radiata L.) seeds (MBS) contain abundant nutrients with
biological activities. This study was aimed to isolate key bioactive components from
MBS with antioxidant and myocardial preservation activities. A new flavonoid Cꢀ
glycoside, isovitexinꢀ6''ꢀOꢀαꢀLꢀglucoside, and fourteen known compounds were
obtained. Their structures were identified by extensive 1D and 2D NMR and FTꢀICRꢀ
MS spectroscopic analyses. The antioxidant activities of these compounds were
•+
evaluated. 1-5, 7ꢀ10 displayed ABTS scavenging activity, but only 5 and 7 exhibited
•
DPPH scavenging activity. The myocardial preservation effect of 2, 3 and MBS were
investigated by measuring the serum levels of LDH, CK and AST as well as the tissue
level of MDA and SOD. The results demonstrated that 2, 3 and MBS had a significant
protective effect against ISOꢀinduced myocardial ischemia. MBS can be regarded as a
potential new source of antioxidants and myocardial preservation agents.
KEYWORDS: mung bean seeds; vitexin; isovitexin; myocardial preservation;
antioxidant; functional foods.
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INTRODUCTION
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Reactive oxygen species (ROS) are free radicals found naturally in the human body,
which are well recognised for playing a dual role as both deleterious and beneficial
1
species. Beneficial effects of ROS occur at low/moderate concentrations. When
environmental conditions or stress result in excession of ROS, damage to cellular
2
systems and DNA can occur. The damage caused by ROS is linked to several
diseases, such as cancer, rheumatoid arthritis, diabetes, neurological disorders, aging,
1
and cardiovascular diseases. Among these, ROSꢀinduced oxidative stress plays a role
in various cardiovascular diseases, such as atherosclerosis, congestive heart failure,
3
hypertension, cardiomyopathies, cardiac hypertrophy, and ischemic heart disease.
Ischemic heart disease secondary to acute myocardial infarction is a severe health
4
problem in the world, and is a primary cause of morbidity and mortality. The intake
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of foods rich in antioxidants can result in a lower risk of cardiovascular disease. In
recent years, there has been increasing interest in identifying new sources of natural
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antioxidants and other healthꢀpromoting compounds. Many natural bioactive
constituents, including flavones from vegetables, fruits, or grains, have been used
7
experimentally as effective protection against ROS or myocardial ischemic injury.
Mung bean, the seeds of Vigna radiata L. (MBS), are popular food legumes in
China, India, Korea, Japan, and other parts of Southeast Asian countries. It is well
known for its detoxification activities and is used to refresh mentality, alleviate heat
8
stroke, and reduce swelling in the summer. The seeds and sprouts of mung beans are
widely used as a fresh salad vegetable or common food in India, Bangladesh,
Southeast Asia, and western countries. In China, they not only have been widely
consumed as a food, in the form of soup, cake, or ice cream, but also used as a
cosmetic, such as mung bean mask. As a food, MBS contain abundant nutrients with
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biological activities, including proteins, phenolic acids, fatty acid, and minerals. In
recent years, the sprouts of mung beans have more bioactive components, including
10, 11
flavonoids, phenolic acids, organic acids, amino acids, carbohydrates, and lipids.
Interest in MBS as a functional food ingredient is increasing for its physiological
1
2
13
14
functionalities, such as antiinflammatory,
antibacterial,
antitumor,
1
5
16
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18
hypolipidemic,
antioxidant activity.
extract focus on two active components, vitexin and isovitexin.
antidiabetic,
detoxication,
hepatoprotective effect,
and
19, 20
However, until now, reports of antioxidant activity in MBS
20
Besides these
findings, there is no literature published on a systematic phytochemical study of MBS.
Accordingly, the active ingredients in the management of myocardial health from
MBS are not yet clear.
This contribution investigates the phytochemical profiles of MBS and studies
isolated key bioactive components from MBS with antioxidant and myocardial
preservation activities firstly. These results suggest that bioactive components from
MBS are new sources of natural active compounds that can be administered to
prevent myocardial ischemic.
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MATERIALS AND METHODS
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Experimental animals. Healthy male SpragueꢀDawley rats weighing 200ꢀ220 g, 7ꢀ8
weeks old, obtained from the Central Animal House of Shenyang Pharmaceutical
University (Shenyang, China) were used in this study. They were housed (three
rats/cage) in polypropylene cages (47 × 34 × 20 cm) lined with husk, renewed every
24 h under a 12:12 h light and dark cycle at around 22 °C. The rats had free access to
standard laboratory water and food. Animal experiments were carried out according to
the Guidelines for Animal Experimentation of Shenyang Pharmaceutical University
and approved by the Animal Ethics Committee of this institution.
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Chemicals and Reagents. All of the solvents (analytical grade) for the extraction
were purchased from Qingdao Haiyang Chemical Co., Ltd (Qingdao, China).
Methanol and glacial acetic acid (chromatography grade) were purchased from
Concord Chemical Reagents Co. (Tianjin, China). The water used during HPLC
analysis and for sample preparation was obtained from Wahaha Group Co., Ltd.
(Hangzhou, China). DPPH (2, 2ꢀdiphenylꢀ1ꢀpicrylhydrazyl, purity ≥ 98.0%), 2, 2′ꢀ
azinoꢀbis (3ꢀethylbenzothiazolineꢀ6ꢀsulphonic acid) (ABTS), lactate dehydrogenase
(LDH), creatine kinase (CK), aspartate aminotransferase (AST), methane dicarboxylic
aldehyde (MDA), and superoxide dismutase (SOD) test kits were obtained from the
Jiancheng Institute of Biotechnology (Nanjing, China).
1
13
Instrumentation. H NMR (400 MHz and 600 MHz), C NMR (100 MHz and 150
MHz) and 2D NMR were acquired on BrukerꢀARXꢀ400 and BrukerꢀAVꢀ600 NMR
spectrometers with TMS as an internal standard. FTꢀICRꢀMS spectra were obtained
on a Bruker Solarix 7.0T FTꢀICRꢀMS system. Semipreparative HPLC separations
were carried out on a LCꢀ10AT instrument with an SPDꢀ10A VP UV/vis detector
using YMCꢀPackꢀODSꢀA column (250 × 10 mm, 5 ꢁm) at 210 nm. The UPLCꢀ
MS/MS analysis was carried out on an Acquity UPLC IꢀClass System (Waters Corp.,
Milford, MA, USA) combined with a Xevo TQꢀS mass spectrometer (Waters Corp.,
Milford, MA, USA). The compositions of samples were analyzed by a Thermo
Hypersil GOLD C18 column (2.1 × 50 mm, 1.9 ꢁm). GC was performed in an Agilent
7890A gas chromatograph system (Agilent technologies, Inc., Santa Clara, CA, USA)
coupled to a flame ionization detector (FID) by use of a HPꢀ5 capillary column (30 m
× 0.32 mm × 0.25 ꢁM). Antioxidant and myocardial preservation activities were
measured by a microplate reader (imark, BIOꢀRAD, USA). Column chromatography
was performed using silica gel (100ꢀ200 and 200ꢀ300 mesh; Qingdao Ocean
1
1
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Chemical Co. Ltd., Qingdao, China), C18 reversedꢀphase silica gel (50 µm; YMC Co.
Ltd., Kyoto, Japan), and Sephadex LHꢀ20 (GE Healthcare, Uppsala, Sweden). TLC
plates were precoated with silica gel GF254 (Qingdao Ocean Chemical Co. Ltd.,
Qingdao, China).
Plant Material. Mung bean (Vigna radiata L.) seeds in Jilin Province of China were
purchased from Carrefour supermarket in Shenyang, Liaoning, China in March, 2014.
A voucher specimen (No. 20140801) has been deposited in the Herbarium of
Shenyang Pharmaceutical University, Liaoning, China.
Extraction and Isolation. MBS (50 kg) were extracted three times with 95% EtOH
(3 × 8 L) under reflux (2 h each). After concentrating the extract solutions under
reduced pressure at 45 °C, the crude residue was suspended in water (1.5 L), which
was successively partitioned with petroleum ether (3 × 1.5 L), dichloromethane (3 ×
1.5 L), and nꢀbutanol (3 × 1.5 L) to obtain petroleum ether, dichloromethane, and nꢀ
butanol fractions. The nꢀbutanol fraction (63.2 g) was chromatographed over a silica
gel column (80 × 650 mm, 386 g) by use of a gradient system of increasing polarity
2 2
with CH Cl ꢀMeOH (from 100:0 to 1:1, v/v) to afford five fractions (AꢀE), and
compounds 2 (8.2 g), 6 (108 mg), 9 (1.6 g), 13 (28 mg), and 15 (159 mg). The
fraction A (3.1 g) was subjected to a Sephadex LHꢀ20 (25 × 1300 mm) eluting with
MeOH to afford two fractions: frs. A to A . Fraction A was purified by preparative
1
2
1
TLC, eluting with PEꢀAcetoneꢀHAc (400:50:1, v/v/v) to provide compounds 10 (37
mg) and 11 (14 mg). Fr. A was passed over an ODS column (50 × 500 mm, 60 g)
eluting with MeOHꢀH O (from 1:4 to 1:1, v/v) to afford three fractions: frs. A2a to A2c
Fractions A2a to A2c were purified by preparative TLC, eluting with CH Cl ꢀMeOH
2
2
.
2
2
(30:1, v/v) to obtain 7 (19 mg), 12 (6 mg), and 14 (29 mg), respectively. The fraction
D (7.5 g) was subjected to an ODS column (50 × 500 mm, 60 g) eluting with MeOHꢀ
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H
2
O (from 1:9 to 1:1, v/v) to obtain fraction D
1
and to yield 3 (4.2 g). Fraction D
1
was
purified by preparative TLC, eluting with CH
2
Cl ꢀMeOHꢀHAc (250:50:1, v/v/v) to
2
give 5 (7 mg). The fraction E (6.8 g) was subjected to a Sephadex LHꢀ20 (25 × 1300
mm) with MeOH to afford two fractions: frs. E to E . Fraction E was purified by
1
2
1
preparative TLC, eluting with CH Cl ꢀMeOHꢀHAc (150:50:1, v/v/v) to give 1 (253
2
2
mg), and 4 (14 mg). Fraction E
45:55) to afford 8 (85 mg, t 19.2 min).
Isovitexinꢀ6''ꢀOꢀαꢀDꢀglucoside (1): yellow oil; UV (MeOH)
2 2
was separated by preparative HPLC (MeOH/H O,
R
λmax (log ε) 334
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(0.59) and 271 (0.51); IR (KBr)
ν
max 3409, 2924, 1653, 1510, 1355, 1020, 835, and
ꢀ1
+
780 cm . FTꢀICRꢀMS m/z: 593.151194 [MꢀH] (C27
H
29
O15, calculated for
1
13
593.150365). For H and C NMR, see Table 1.
Acid Hydrolysis of Compound 1. Compound 1 (2 mg) was hydrolyzed with 2 M
HCl (2 mL) at 90°C for 3 h. After cooling, the mixture was extracted with EtOAc (3 ×
3.0 mL). The EtOAc layer and the aqueous layer were then evaporated under vacuum
to give residues. The solutions were examined by TLC and compared with authentic
samples. Flavone was detected in the EtOAc layer solution and glucose was detected
in the aqueous layer solution. The remaining sugar residue was dissolved in
anhydrous pyridine (2 mL) and mixed with Lꢀcysteine methyl ester hydrochloride (2
mg). The mixture was kept at 60 °C for 3 h, and 0.5 mL of trimethylsilyimidazole
(TMSI) was added. The reactant was then cooled to ambient temperature and
2
partitioned between nꢀhexane and H O (2 mL each). The nꢀhexane fraction was
analysed by GC under the following conditions: column (HPꢀ5, 30 m × 0.32 mm ×
0.25 ꢁm, Agilent); detection (FID); detector temperature (280 °C); injection
temperature (270 °C); injection volume (1 mL); column temperature (120 to 280 °C at
the rate of 8 °C/min); carrier gas (N
2
, 1.4 mL/min). The peaks of authentic samples of
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Dꢀglucose and Lꢀglucose after treatment in the same manner were detected at 20.25
min and 21.48 min, respectively.
UPLC-MS/MS Analysis. The mass spectrometry was operated in positive scan mode.
The mobile phase was composed of methanolꢀ0.1% formic acid in water (28:72, v/ v).
The total running time was 5 min with a flow rate of 0.3 mL/min. The main working
parameters were set as follows: cone voltage, 30 V; the desolvation temperature, 350
°C; ion source temperature, 150 °C; gas flow (N ), 700 L/h.
2
Assay for ABTS Radical Scavenging Activity. The ABTS 2, 2′ꢀazinobis (3ꢀ
ethylbenzothiazolineꢀ6ꢀsulphonic acid) diammonium salt assay was carried out
2
1
according to the method of Cai et al. with slight modifications. The ABTS radical
cation solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium
persulphate. The mixture was incubated in darkness at room temperature for 12 h
before use, and the radical was stable in this form for more than 2 days when stored in
the dark at room temperature. The ABTS cation radical solution was then diluted with
water to obtain an absorbance of 0.70 ± 0.02 at 734 nm. One hundred microlitres of
test sample with various concentrations were added to 3.0 mL of diluted
•+·
ABTS solution and mixed vigorously, incubated in darkness for 6 min at room
temperature and the absorbance was measured at 734 nm. LꢀAscorbic acid was used
as the positive control. The negative control used 100 ꢁL ethanol instead of the
sample. The test was carried out in triplicate and the results were mean values. The
•+
•+
ABTS scavenging effect was calculated as follows: ABTS scavenging effect (%) =
[1 ꢀ (SꢀS )/(CꢀC )] × 100%, where S, S , C, and C are the absorbances of the sample,
b
b
b
b
blank sample, negative control, and blank control, respectively.
Assay for DPPH Radical Scavenging Activity. The DPPH free radicals scavenging
22
activity of fifteen compounds was determined by the method mentioned by Hatano
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with slight modifications. One hundred microlitres of different concentrations of the
1
ethanol solution of the sample (the negative control used 100 ꢀL ethanol instead of
1
1
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1
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1
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1
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2
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the sample) was added to 96ꢀwells, followed by 100 ꢀL of 0.15 mM DPPH. The
mixtures were incubated for 30 min in darkness at room temperature. The reduction of
the DPPH radical was determined by measuring the absorbance at 517 nm with a
microplate reader (imark, BIOꢀRAD, USA). LꢀAscorbic acid was used as the positive
control. The test was carried out in triplicate and the results were mean values. The
percentage of DPPH free radical scavenging activity was calculated using the same
formula as for the ABTS assay.
Myocardial Preservation Experimental Design. The animals were grouped into five
groups of six rats each, including a normal control, a isoproterenol (ISO) model
control, a vitexin group (VG), an isovitexin group (IVG), and a mung bean seeds
extract group (MBSG). Rats in the VG and the IVG were given an oral dose of vitexin
and isovitexin saline solution at dosages of 6 mg/kg body weight. Rats in the MBSG
were given an oral dose of 750 mg/kg (6.75 mg for 2 and 11.25 mg for 3) saline
solution. Rats in the normal control and ISO model control groups were given equal
volumes saline (5 mL/kg). Then, all of the rats, except for the normal control group
were injected subcutaneously with ISO (6 mg/kg body weight) at an interval of 24 h
23
for 2 days to induce myocardial infarction (MI) (on 6th and 7th day). All treatments
were performed once daily for 7 consecutive days. Two hours after the second dose of
ISO injection (on 7th day), all of the rats were anesthetized with diethyl ether. Blood
was collected in dry tubes without anticoagulant. The blood samples were centrifuged
at 10000 × g for 10 min at 4 °C to obtain the serum. The serum samples were stored at
ꢀ80 °C until required for the assay of LDH, CK, and AST. All of the rats were
sacrificed by cervical decapitation. Heart tissues were excised immediately and rinsed
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in iceꢀchilled saline. The homogenate was centrifuged at 10000 × g for 10 min and the
supernatant was collected for determination of MDA content and SOD activity. A
portion of the heart was retained for histopathologic observation.
Biochemical Analyses. LDH, CK, AST, MDA, and SOD in heart tissues were
measured by the detection kits according to the manufacturer’s instructions (Nanjing
Jiancheng Institute of Biotechnology, Nanjing, China).
Histopathological Examination. Portions of the heart tissues were fixed in 10%
formalin. After the proper dehydration, the tissues were embedded in paraffin wax.
Sections (5 ꢁm) were prepared and stained with hematoxylin and eosin.
Statistical Analysis. The compound content of MBS and antioxidant activities were
repeated in triplicate and data were analyzed by an ANOVA test with SPSS 17.0
(Statistical Program for Social Sciences, SPSS Inc., Chicago, IL, USA). All statistical
comparisons were made by means of a oneꢀway ANOVA test followed by Dunett’s tꢀ
test. All of the results were expressed as mean ± standard deviation (SD). P values <
0.05 were considered significant.
225 RESULTS AND DISCUSSION
+
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Compound 1 was obtained as a yellow oil; FTꢀICRꢀMS m/z: 593.151194 [MꢀH]
15, calculated for 593.150365). Its UV spectrum showed absorbances at λmax
271 and 334 nm, and the IR spectrum showed absorption bands for hydroxyl (3409
29
(C27H O
ꢀ1
ꢀ1
1
cm ) and carbonyl (1653 cm ) groups. Its HꢀNMR (600 MHz, DMSOꢀd ) spectrum
6
showed signals for two phenolic hydroxyl δ
signals at δ 6.75 (1H, s) and 6.48 (1H, s), and an AA′BB′ system at δ
= 8.6 Hz) and 6.92 (2H, d, J = 8.6 Hz) of ring B, which indicated it was a flavone,
with apigenin as the aglycone. The proton signals at δ 4.57 (1H, d, J = 9.8 Hz) and
4.72 (1H, d, J = 3.5 Hz) were assigned to a βꢀglucopyranosyl and αꢀglucopyranosyl,
H
13.52 and 10.35, two aromatic protons
H
H
7.90 (2H, d, J
H
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respectively. The C NMR spectrum of 1 (Table 1) also suggested the presence of βꢀ
2
glucopyranosyl and αꢀglucopyranosyl residues. The chemical shift value of Cꢀ1′′ (δc
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73.3 ppm) suggested that 1 was a Cꢀglycoside. In addition, the (1→6) glycosidic bond
of the outside glucose to the inner glucose was characterized by the HMBC
correlations of δ 3.60 and 3.70 (Hꢀ6′′) to δ 98.6 (Cꢀ1′′′). The linkage position of the
glucose chain to flavone nucleus was determined at Cꢀ6 from the HMBC correlations
of δ 4.57 (Hꢀ1′′) to δ 160.6 (Cꢀ5), 108.8 (Cꢀ6) and 163.4 (Cꢀ7) (Fig. 2). After acid
hydrolysis, isovitexin was detected in the EtOAc solution (Isovitexin was a flavonoid
Cꢀglycoside, which was hard to be hydrolyzed under acid condition). The Lꢀ
configuration of the glucopyranosyl moiety was determined by acid hydrolysis
followed by derivatization and GC analysis. The structure of compound 1 was
therefore characterized as a new flavone Cꢀglycoside and named isovitexinꢀ6''ꢀOꢀαꢀLꢀ
glucoside (Fig. 1).
24
Fourteen known compounds (Fig. 1) were identified as vitexin (2), isovitexin
25
26
27
28
29
(3), dulcinoside (4), (2R, 3R)ꢀtaxifolin (5), scopolin (6), pꢀcoumaric acid (7),
3
0
31
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Lꢀtryptophan (8), Dꢀ3ꢀOꢀmethyl inositol (9), muconic acid (10), benzoic acid
33
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(11), 2Hꢀ1, 3ꢀthiazineꢀ2, 4 (3H)ꢀdione (12), nicotinic acid (13), nicotinamide
36
37
(14), and methylꢀαꢀDꢀglucoside (15).
Quantitation is the key issue for quality control of functional foods. In this study,
content analysis was conducted to determine the content of 1ꢀ4, 8, and 13 in 95%
EtOH extract by UPLCꢀMS/MS analysis. Product ion scan spectra and representative
multipleꢀreaction monitoring (MRM) chromatograms of the six analytes are shown in
Fig. 3. The contents of the six analytes exhibited remarkable differences. Among the
analytes, the content of 3 with 5.32 mg/g was the highest. The second one is 2 with
2.97 mg/g. In the remaining analytes, 8 showed a content of 2.29 mg/g, followed by 1
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277
278
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280
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283
284
with 1.70 mg/g, 13 with 0.38 mg/g, while 4 showed the lowest content with 0.13 mg/g
(Table 2). Given these results, 2 and 3 were the major secondary metabolite
components in MBS.
•+
•
In the present study, the ABTS scavenging assay and DPPH scavenging assay
were performed to study the antioxidant properties of MBS extract and isolated
•+
compounds. As shown in Table 3, 1ꢀ5, 7-10 demonstrated ABTS scavenging activity
with EC50 values ranging from 2.21 to 71.12 ꢁM. Significantly, 3ꢀ5, 7, 8, and 10
•+
exhibited significant ABTS scavenging activity with EC50 values less than 10 ꢁM,
which were more potent than the positive control, Lꢀascorbic acid (EC = 11.06 ꢁM).
50
However, most of isolates (EC50 > 100 ꢁM) were found to be less active than the
•
positive control Lꢀascorbic acid (EC50 = 21.55 ꢁM) in the DPPH scavenging assay
except for 5 (EC50 = 17.32 ꢁM) and 7 (EC50 = 19.33 ꢁM). This finding is in
accordance with the knowledge that the compounds with more phenolic hydroxyl
38
groups show stronger radical scavenging activity. Significant differences were
•
•+
observed between DPPH and ABTS scavenging assays. The antioxidant capacity
•+
•
detected by ABTS scavenging assay was markedly higher than by DPPH
2
9
scavenging assay for fruits, vegetables, and beverages, which is in agreement with
our results.
7
Vitexin (2) has a reported myocardial preservation effect. Thus, the question
arises, whether isovitexin (3) and extract from MBS could have a similar effect. LDH,
CK, and AST are major metabolic enzymes in the heart and can be released into the
serum during cardiac damage. As shown in Fig. 4, serum LDH, CK, and AST levels
increased quickly in the model control group (P < 0.01), indicating that the ISOꢀ
induced myocardial ischemia model in rats was successfully established. Vitexin,
isovitexin, or MBS extract preconditioning showed a significant protective effect on
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the ISOꢀinduced myocardial ischemia by markedly preventing any elevation in the
serum levels of LDH, CK, and AST (P < 0.01). However, both VG and IVG showed a
better protective effect than MBSG (P < 0.05). The MDA levels and SOD activities in
heart tissues are shown in Fig. 4. Compared with the levels in the normal control
group, there was a significant increase in the MDA level and a marked reduction in
SOD activity in the ISOꢀinduced myocardial ischemia model group (P < 0.01). The
administration of vitexin, isovitexin or MBS extract resulted in a significant decrease
in the level of MDA and a marked increase in the level of SOD, compared with the
ISOꢀinduced myocardial ischemia model group (P < 0.01). The changed trend of
MDA and SOD is in accordance with the serum LDH, CK, and AST levels.
Obvious morphological changes were observed in the heart cells of the
myocardial ischemia rats. As shown in Fig. 5, in the hearts of the normal rats, cells
had clear borders with intact cytoplasm and prominent nuclei. In addition, no
inflammatory cell infiltrates were observed (Fig. 5A). In contrast, changes in the
features of the myocardium appeared after injection of ISO. Cardiac interstitial edema
and infiltration of inflammatory cells were observed in the ISOꢀinduced myocardial
ischemia model group (Fig. 5B). After administration of vitexin or isovitexin, cardiac
interstitial edema diminished and less infiltration of inflammatory cells was observed
(Fig. 5C and D). Rats fed with MBS extract exhibited still less cardiac interstitial
edema and infiltration of inflammatory cells, but the histopathologic changes were
markedly improved compared with ISOꢀinduced myocardial ischemia model group
(Fig. 5E). These data suggest a myocardial preservation effect of MBS and vitexin as
well as isovitexin appear to be the major compounds responsible for the effect.
Administertion of either vitexin or isovitexin alone showed much better effects on
myocardial preservation than treatment with MBS extract. Isovitexinꢀ6''ꢀOꢀαꢀLꢀ
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334
glucoside (1) would be hydrolyzed in vivo to produce the isovitexin (3). Thus, 1 may has
effect on myocardial preservation as well, which should be further studied.
The correlation between antioxidant and cardioproctetive activities of flavone Cꢀ
glycosides has been reported. Antioxidant activity of orientin and isoorientin (two
flavone Cꢀglycosides) appears to reduce the LꢀNAME induced damage in rats to make
3
9
Lagenaria siceraria fruit possess antihypertensive and cardioprotective activity.
Therefore, the antioxidant property of vitexin and isovitexin may contribute to the
myocardial preservation effect. Further studies should be carried out to assess the link
between antioxidant activity and myocardial preservation action of vitexin and
isovitexin.
In conclusion, we report here the structures of 15 compounds isolated from MBS,
as well as their in vitro antioxidant activities for the first time. 1ꢀ5, 7ꢀ10 displayed
•+
•
ABTS scavenging activity, and only 5 and 7 exhibited DPPH scavenging activity.
By UPLCꢀMS/MS analysis, 2 and 3 were the major secondary metabolite components
in the MBS extract. In the investigation of the myocardial preservation effect, 2, 3,
and MBS extract showed significantly decreased the activities of LDH, CK, and AST
of the myocardial ischemic, MDA contents decreased and the activity of SOD
increased in the hearts of ISOꢀinduced myocardial ischemic rats. In addition,
histopathologic changes induced by ISO were markedly improved by 2, 3, and MBS
extract. 2 and 3 are the major constituents responsible for the myocardial preservation
effect in MBS. This finding is in accordance with the knowledge that many natural
bioactive constituents have protection effect against myocardial ischemic injury. The
results obtained in our study provide a potential justification for the use of the seeds
from Vigna radiata L. industrial byproducts as a valuable source of raw material for
new antioxidant and myocardial preservation agents.
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357
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
UV, IR, NMR, and FTꢀICRꢀMS data of compound 1. (PDF)
AUTHOR INFORMATION
Corresponding Author
*(Z.ꢀG.Y.) Mail: School of Pharmacy, Shenyang Pharmaceutical University, 103
Wenhua Road, Shenyang Liaoning 110016, China. Phone: +86ꢀ24ꢀ23986295.
Eꢀmail: zhiguoꢀyu@163.com.
Funding Sources
This research was supported by the National Undergraduate Training Programs for
Innovation and Entrepreneurship (201410163026).
Notes
The authors declare that there are no conflicts of interest.
ABBREVIATIONS USED
NMR, nuclear magnetic resonance spectrometry; HPLC, highperformance liquid
chromatography; HSQC, heteronuclear singleꢀquantum coherence; HMBC,
heteronuclear multiplebond correlation; FTꢀICRꢀMS, fourier transform ion cyclotron
resonance mass spectrometry; UV, ultraviolet spectrometry; IR, infared absorption
+
spectrum; DPPH, 2, 2ꢀDiphenylꢀ1ꢀpicrylhydrazyl; ABTS , 2, 2'ꢀazinobisꢀ(3ꢀ
ethylbenzothiazolineꢀ6ꢀsulfonate) radical cation.
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Figure captions
Figure 1. Chemical structures of the isolated compounds 1ꢀ15 from mung bean seeds.
Figure 2. Key HMBC correlations of compound 1.
Figure 3. Product ion scan spectra and representative multipleꢀreaction monitoring
(MRM) chromatograms of six analytes.
Figure 4. Effects of vitexin, isovitexin and extract from MBS on serum and tissue
biochemical paramters in rats with ISOꢀinduced myocardial ischemia. Normal: normal
control group; Model: ISO model control group; VG: vitexin group; IVG: isovitexin group;
MBSG: mung bean seeds extract group. Values were presented as mean ± standard deviation
(
n = 6). ** P < 0.01 vs. normal control group; # P < 0.05 vs. model control group; ## P < 0.01
vs. model control group by oneꢀway ANOVA.
Figure 5. HEꢀstained sections of heart tissues from the different experimental groups.
(
A) normal control group, (B) ISO model control group, (C) VG (6 mg/kg), (D) IVG
6 mg/kg), (E) MBSG (750 mg/kg).
(
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1
13
6
Table 1 H and C NMR data of compound 1 (in DMSOꢀd )
position δC
163.4
δH
position δC
δH
2
3
4
5
6
7
8
9
1
1
2
3
4
1
2′′
70.1 4.02 m
79.0 3.20 m
70.0 3.06 m
79.6 3.32 m
102.7
181.7
160.6
108.8
163.4
93.8
6.75 s
3′′
4′′
5′′
6′′
66.8 3.60 m, 3.70 m
98.6 4.72 d (3.5)
72.0 3.15 m
73.1 3.39 m
70.2 3.30 m
72.5 3.37 m
60.6 3.39 m, 3.51 m
13.52 s
1′′′
2′′′
3′′′
4′′′
5′′′
6.48 s
156.3
103.1
121.1
128.4
116.0
161.2
73.3
0
′
′, 6′
′, 5′
′
7.90 d (8.6) 6′′′
6.92 d (8.6) 5ꢀOH
7ꢀOH
10.35 br s
′′
4.57 d (9.8)
1
NMR spectroscopic data were recorded at 600 MHz ( H NMR) and
1
3
1
50 MHz ( C NMR)
a
Table 2 Calibration and quantitation of six analytes (n = 3)
Correlation 2
coefficient (r )
b
No. Analytes
Regression equation
X ranges (ꢀg/mL) Contents (mg/g)
isovitexinꢀ6''ꢀOꢀαꢀ
Lꢀglucoside
1
2
3
4
8
Y = 68294Xꢀ2422
0.9998
0.1397ꢀ5.5897
0.1884ꢀ7.5370
0.4373ꢀ17.4904
0.0103ꢀ0.4130
0.2447ꢀ9.7893
0.0031ꢀ1.2333
1.70 ± 0.22
2.97 ± 2.00
5.32 ± 0.27
0.13 ± 0.08
2.29 ± 0.25
0.38 ± 0.09
vitexin
Y = 1E+06X+2E+06
Y = 2E+06X+8E+06
Y = 30760X+29378
Y = 3E+06X+4E+07
0.9991
0.9985
0.9993
0.9985
isovitexin
dulcinoside
Lꢀtryptophan
nicotinic acid
1
3
Y = 136549X+434939 0.9997
a
n = number of samples; data represent the mean ± SD of triplicate tests for each sample.
Milligram per 1 g of 95% EtOH extract.
b
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Table 3 Antioxidant activities of MBS extract and compounds 1-15
a
a
compound
ABTS (EC50, ꢁM) DPPH (EC50, ꢁM)
1
2
3
4
5
6
7
8
9
12.20 ± 2.66
38.29 ± 3.86
4.09 ± 0.37
3.70 ± 0.12
2.21 ± 0.15
>100
>100
>100
>100
>100
17.32± 2.01
>100
3.02 ± 0.18
4.17 ± 0.62
71.12 ± 4.28
9.08 ± 0.07
>100
19.33 ± 1.99
>100
>100
10
11
12
13
14
15
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
MBS extract
0.13 ± 0.26 mg/mL 3.00 ± 0.23 mg/mL
11.06 ± 0.62 21.55 ± 1.61
LꢀAscorbic acid
a
EC50 values correspond to the sample concentration
achieving 50% of activity
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OH
6
'''
'''
5
R
1
OH
HO
3'
6'
'''
O
OH
4
4' OH
5'
1
'''
2'
1
HO
R2
O
O
HO
HO
1
2''' OH
8
3
'''
O
HO
7
6
9
O
2
HO
O
O
'
6
4
''
''
5''
O
1''
3
OH
10
OH
HO
HO
5
4
2'' OH
2
3
R1 = Glc R2 = H
R1 = H R2 = Glc
3''
OH
O
OH
1
4 R
1
= H
R
2
= Rha(1 6)Glc
5
COOH
NH2
OHOH
O
O
O
COOH
OH
OH
HO
HO
O
O
O
HO
OH
HO
N
H
OH
7
8
9
6
COOH
O
COR
HO
COOH
O
NH
HO
OCH
3
HO
HOOC
N
HO
S
O
1
3 R = OH
10
11
12
14 R = NH2
15
Figure 1
OH
O
OH
HO
HO
OH
HO
O
O
O
O
HO
HO
OH
OH
HMBC, H to C
Figure 2
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Figure 3
2
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2
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A
B
C
D
E
Figure 5
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Graphic Abstract
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Supporting Information
Antioxidant and myocardial preservation activities of
natural phytochemicals from Mung Bean (Vigna
radiata L.) seeds
†
Yan Bai*, Jiawei Chang*, Yan Xu*, Dan Cheng*, Hongxin Liu , Yunli Zhao*, Zhiguo
Yu*
*
Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang
Pharmaceutical University, Shenyang 110016, China
†
Dalian Weida Pharmacy Co., Limited, 12ꢀ7 Zhuhai Street, Xigang District, Dalian
116011, China
29
ACS Paragon Plus Environment