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Potential Lipid-Lowering Mechanisms of Biochanin A Zhaohui Xue, Qian Zhang, Wancong Yu, Haichao Wen, Xiaonan Hou, Dan Li, and Xiaohong Kou J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

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Journal of Agricultural and Food Chemistry

Potential Lipid-Lowering Mechanisms of Biochanin A Short title: Biochanin A Lipid-Lowering Mechanisms Zhaohui Xue1#, Qian Zhang1#, Wancong Yu2, Haichao Wen1, Xiaonan Hou1, Dan Li1, Xiaohong Kou1* 1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China 2. Medical Plant Lab, Tianjin Research Center of Agricultural Biotechnology, Tianjin 3000381, China

*Corresponding author. Department of Food Science, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. Tel./fax: +86 022 83727262. E-mail address: [email protected] #

These authors make equal contributions to this manuscript. 1

ACS Paragon Plus Environment


1

Abstract

2

Extensive studies have demonstrated that biochanin A (BCA) has a significant

3

hypolipidemic effect. However, its mechanism of action is not clear. In this context,

4

the effect of BCA on a high-fat diet (HFD)-induced hyperlipidemia in mice was

5

determined. The results showed that treatment with a medium dose of biochanin A

6

(BM) significantly decreased low density lipoprotein cholesterol (LDL-C) 85% (from

7

1.196 ± 0.183 to 0.181 ± 0.0778 mM) and total cholesterol (TC) 39% (from 5.983 ±

8

0.128 to 3.649 ± 0.374 mM) levels, increased lipoprotein lipase (LPL) 96% (from

9

1.421 ± 0.0982 to 2.784 ± 0.177 U/mg protein) and hepatic triglyceride lipase (HTGL)

10

78% (from 1.614 ± 0.0848 to 2.870 ± 0.0977 U/mg protein) activities, significantly

11

improved fecal lipid levels and lowered the epididymal fat index in hyperlipidemic

12

mice compared with the HFD control mice (p < 0.05). In vitro, the high antioxidant

13

capacity of BCA was determined by the FRAP assay, ABTS+. scavenging methods

14

and an ROS assay. In RAW 264.7 macrophages, a dose of 10 µm BCA significantly

15

increased the cholesterol efflux by 18.7% compared with the control cells. Moreover,

16

molecular docking of BCA on cholesterol ester transfer protein (CETP) (Asn24 and

17

Thr27 at the N-terminal; Ala274 and Phe270 at the C-terminal) gave new insights into

18

the role of BCA in preventing cholesterol ester transport.

19

Key words: biochanin A, hypolipidemic, antioxidant, cholesterol efflux, CETP

20

21

2


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1. Introduction

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According to extensive research1-3 and Pharmacopoeia records,4 a diet that

24

includes chickpeas (Cicer arietinum L.) has beneficial effects on dyslipidemia

25

because chickpeas play a role in lowering plasma lipid levels. One of the compounds

26

(5,7-dihydroxy-4'-methoxy isoflavone) found in chickpeas5 and other foods with

27

estrogenic properties may play an important role in regulating lipid metabolism.

28

Bhathena6 and others7 investigated the effects of soy isoflavones on atherosclerosis,

29

obesity and diabetes. The results showed that dietary phytoestrogens, such as

30

genistein (a precursor of biochanin A), have protective effects against a variety of

31

disorders, including hyperlipidemia. Biochanin A possesses excellent hypolipidemic

32

properties.8 However, the mechanism of how biochanin A regulates lipid metabolism

33

is unclear.

34

Lipid metabolism is a complicated process in which exogenous lipid is digested

35

and absorbed in the gut, transported into the blood circulation from the lymphatic

36

system by lipoproteins, and then stored in fat tissues after conversion by the liver

37

(Figure 7). Numerous epidemiological studies have already established a link between

38

hyperlipidemia and the incidence of several diseases, such as atherosclerosis (AS),

39

cardiovascular diseases (CAD),9,10 hypertension and diabetes mellitus.11 In particular,

40

hyperlipidemia is a common dyslipidemia characterized by increased blood levels of

41

total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol

42

(LDL-C) and decreased levels of high-density lipoprotein cholesterol (HDL-C). High

43

TC levels are the leading cause of hypercholesterolemia. Plasma LDL cholesterol 3



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contributes to the formation of atherosclerotic lesions, and HDL particles promote

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cholesterol uptake in the liver thus accelerating lipid metabolism and the fecal

46

excretion of cholesterol.12 Therefore, according to the guidelines for the detection and

47

treatment of high cholesterol, low-density lipoprotein cholesterol is the focus when

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lipid metabolism is in disorder. As a hydrophobic glycoprotein circulating in the plasma, cholesterol ester

49 50

transfer protein (CETP) is thought to facilitate the exchange of cholesterol esters (CEs)

51

and

52

(apoB)-containing particles [i.e., very low-density lipoprotein (VLDL) and LDL].13,14

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The net effect of CETP is that it enables the reduction of HDL-C levels and the

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increase of LDL-C levels (Figure 7). Thus, CETP inhibition is expected to be a

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potential therapy for raising plasma HDL cholesterol levels.15,16

triacylglycerols

and

transfers

between

HDL

and

apolipoprotein

B

56

Cholesterol efflux (Figure 7) can effectively prevent macrophages from

57

transforming into foam cells, thereby preventing the subsequent development of

58

atherosclerosis. Additionally, cholesterol efflux from the macrophage to HDL is the

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initial key step of reverse cholesterol transport and is associated with atheroprotection

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in animal studies. Therefore, as an important protective mechanism against lipid loads

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in the atherosclerotic plaque, cholesterol efflux from macrophage-derived foam cells

62

should be increased.17

63

A high-fat diet (HFD) promotes oxidative stress, which in turn aggravates the

64

attack by reactive oxygen species (ROS) and the formation of lipid peroxides (LPO)

65

(Figure 7).18 Oxidative stress levels present in cells or tissues have been implicated in 4


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the pathogenesis of hypertension, atherosclerosis, and cancer.18 Moreover, many

67

studies have shown that several active components in plants have protective effects

68

against HFD-induced oxidative stress.7,19

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Biochanin A is likely to play a role in many events involved in lipid metabolism

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including affecting lipid levels, lipid metabolic rates, cholesterol ester transport and

71

cholesterol efflux. The objective of this study was to gain insight into the role that

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biochanin A plays in hyperlipidemia, particularly atherosclerosis, and to better

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understand the mechanisms of its cholesterol-lowering effect.

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2. Materials and Methods

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2.1 Chemicals

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2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonate)

(ABTS),

77

1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Solarbio (Beijing,

78

China). MTT was from Amresco (Beijing, China), DMSO from Jiangtianhuagong

79

(Tianjin, China), and FBS from MD Genics Incorporation (Beijing, China). HDL and

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ox-LDL were provided by Peking Union-Biology Co., Ltd (Beijing, China),

81

25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol

82

obtained from Shanghai Pumai Biotechnology Co., Ltd. (Shanghai, China).,

83

Simvastatin from TCI (Shanghai) Development Co., Ltd. Reactive oxygen species

84

(ROS), TC, TG, HDL-C, LDL-C, SOD, MDA, HTGL and LPL assay kits were

85

obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA

86

(>98% purity) was purchased from Nantong Feiyu (Jiangsu, China). The other

5


was


87

reagents were of analytical grade.

88

2.2 Hyperlipidemia mouse model and experimental design20,21

89

Six-week-old Kunming male mice (certificate number: SCXK (Jing) 2014-0004)

90

weighing 18-22 g were purchased from Beijing HFK Bio-science Co., Ltd and were

91

allowed to adapt to the animal room conditions (a temperature of 22-25°C, relative

92

humidity of 55-75%, and a 12 h light/dark cycle) for three days. Mouse body weights

93

were monitored weekly during the feeding process.

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The mice were randomly allocated to six groups (11 mice per group): a normal

95

control (NC) group fed with the standard diet; a positive control group fed with a

96

high-fat diet for three weeks and treated with 0.4 mL of simvastatin (10 mg/(kg·d)) in

97

the fourth week; a low-dose biochanin A (BL) group (high-fat diet + 30 mg/(kg·d)

98

BCA); a medium-dose biochanin A (BM) group (high-fat diet + 60 mg/(kg·d) BCA);

99

a high-dose biochanin A (BH) group (high-fat diet + 120 mg/(kg·d) BCA) and a

100

high-fat diet control group (HFD). All groups were administered a 0.5% sodium

101

carboxymethyl cellulose (CMC-Na) solution. The high-fat diet contained 78.8% of

102

the standard diet, 1% cholesterol, 10% egg yolk powder, 10% lard and 0.2% bile.

103

After 4 weeks of experimental treatment and ether anesthesia, blood was gathered

104

from the eyeball of each 12-h-fasted mouse. The liver was removed and weighed,

105

frozen in liquid nitrogen, and stored at -70 ℃ for further analysis. The viscera weight

106

and viscera index were measured and calculated. The morphological changes in the

107

liver tissue were observed using hematoxylin and eosin (HE) staining and an optical

108

microscope. Three days before the end of the animal experiment, mice feces were 6


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collected and stored at -20 ℃; these samples were subsequently used in Soxhlet

110

extractions to determine the fecal fat content of each sample.

111

Animal experimental operations followed the principles outlined in the Guide for

112

the Care and Use of Laboratory Animals issued by the Animal Care and Use

113

Committee of the National Veterinary Research and Quarantine Service (NVRQS).

114

2.3 Determination of the lipid content and levels in serum and liver

115

tissues20

116

Mouse blood samples were centrifuged for 10 min at 1500 g and 4 ℃ before the

117

serum levels of TC, TG, HDL-C and LDL-C were measured using enzymatic kits.

118

Liver homogenates (1:9, w/v) were prepared in cold saline and the lipids extracted to

119

determine TC and TG. Total protein content was determined using a Coomassie

120

brilliant blue assay.

121

2.4 Determination of malondialdehyde (MDA) levels and superoxide

122

dismutase (SOD), hepatic triglyceride lipase (HTGL) and lipoprotein

123

lipase (LPL) activities

124

The supernatants from the liver homogenates were used to measure SOD, MDA,

125

HTGL and LPL activities. SOD and MDA in serum and liver tissues, HTGL and LPL

126

activities in liver tissue were determined with related kits. Serum was obtained by

127

centrifuging the blood.

128

2.5 Cholesterol efflux assay22

129

RAW 264.7 macrophages were prepared by culturing in RPMI 1640 medium. 7



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The cells were seeded in 96-well plates at a density of 2×105/well for 6 h. After

131

removal of RPMI 1640, serum-free medium containing 25-NBD cholesterol was

132

added in. After 24 h, the medium was removed and the cells were washed twice with

133

cold PBS. The positive control and sample groups were treated for 6 h with

134

serum-free medium containing 10 µM simvastatin and 0.1 to 20 µM BCA. A

135

microplate reader was used for determining the 25-NBD cholesterol content (N) in the

136

media and cells at an emission wavelength of 485 nm and an absorption wavelength

137

of 535 nm (three replicates). The cholesterol efflux rate was calculated as

138

100×NMedium/(NCell+NMedium) %.

139

2.6 Testing BCA antioxidant activity and the ROS assay

140

The antioxidant activity of biochanin A was evaluated using the ferric reducing

141

antioxidant

power

(FRAP)

assay

and

142

(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+.) scavenging method. A previously

143

described method by Gao and others was followed for the FRAP assay.20,23 One

144

milliliter of BCA with various concentrations was mixed with 2 mL of a 0.2 M

145

phosphate buffer (pH 6.6) and 2 mL of a potassium ferricyanide solution (1%). After

146

reacting for 20 min at 50 ℃, 2 mL of a trichloroacetic acid solution (10%) was added.

147

Then, 2 mL of reaction solution was combined with 2 mL of distilled water and 0.4

148

mL of a ferric chloride solution (0.1%). After incubation in the dark for 30 min, the

149

absorbance of the solution at 700 nm was measured. The ABTS+. scavenging capacity

150

was determined according to previously published methods by Ozgen and others.19,24

151

Briefly, 50 µL of BCA solution was added to 3 mL of ABTS+. solution, the mixture 8


the

2,2'-azinobis

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152

was incubated in the dark for 10 min and the absorbance of the mixture at 734 nm was

153

measured.

154

The reactive oxygen species (ROS) assay in MCF-7 cells was evaluated using

155

DCFH-DA, a cell permeable non-fluorescent molecular probe that is oxidized by ROS

156

to the fluorescent compound 2',7'-dichlorofluorescein (DCF). MCF-7 cells were

157

seeded into 6-well plates at 106 cells/well and treated for 48 h with 0.25 to 2.0 mM

158

BCA. The cells were washed twice with PBS and then incubated with 1 µL of

159

DCFH-DA at 37 ℃ for 30 min in the dark. A flow cytometer (BD FACS Calibur, BD

160

Bioscience, San Jose, CA) was used to determine fluorescence intensity.

161

2.7 Molecular docking of BCA in CETP

162

The atomic type and coordinate data of BCA (PDB ID: 2QYO) and the protein

163

structure of CETP (PDB ID: 2OBD) were obtained from the RCSB Protein Data Bank

164

(PDB, http://www.rcsb.org) and saved in a .pbd format. Autodock 4.2 software was

165

used to perform all the docking runs. The ligands and receptors file was read by

166

abstract data type (ADT) and a PDBQT file was generated. The CETP protein

167

structure was fixed through hydrogenation, correction with amino acid residues of the

168

C-terminal and N-terminal domain and removal of excess water molecules and

169

heteroatoms to keep the protein molecules and the original ligand. CETP was fully

170

rigid and BCA was semi-flexible. PDBQT files of the ligand and receptor were read

171

in and the number of grid points, box center, and grid spacing were regulated in the

172

Grid Box and saved as a .gpf file. The docking coordinates of the N-terminal domain

173

of CETP were x = 20.00, y = 18.00, ƶ = 18.00 and the box size was 60×60×60 Å. The 9



174

docking coordinates of the C-terminal domain of CETP were x = 15.00, y = 0.00, ƶ =

175

48.00 and the box size was 60×60×60 Å. After the Dock Parameter File (DPF) was

176

generated, the receptor and ligand reading, selection of the docking number, RMS,

177

and Lamarckian genetic algorithm (LGA) were performed and saved as a .dpf file.

178

AutoGrid and AutoDock were run and a .glg and .dlg file were generated. The docked

179

results were ranked and clustered based on the root-mean square deviation (RMSD) of

180

the binding free energy. The conformation with the lowest docked energy was chosen

181

from the most populated clusters and further analyzed. Molecular simulation was

182

performed using a Dell workstation computer, Intel Xeon X5675 CPU, Ubuntu10.04

183

OS.

184

2.8 Statistical analyses

185

Statistics and analysis of experimental data were done using SPSS V.19.0

186

statistical software and GraphPad Prism 5; the tests used were a one-factor ANOVA

187

and the Duncan multiple comparisons test. The mean values ± standard error of the

188

mean (SEM) were calculated. Statistical differences were significant at 95% (p < 0.05)

189

and remarkably significant at the 99% level (p < 0.01).

190

3. Results

191

3.1 Animal experiments

192

3.1.1 Serum lipid levels

193

In the third week, the TC, TG and LDL-C serum levels of mice on the HFD were

194

increased by 22.8%, 37.3% and 9.76%, respectively, compared with the NC group. 10


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The HDL-C level decreased 9.1%, which indicated that this was a successful mouse

196

model of hyperlipidemia. Serum LDL-C levels in the BL, BM, and BH groups were

197

significantly lower than that in the HFD group, demonstrating the important role of

198

BCA in decreasing serum LDL-C. Although no significant differences in the serum

199

HDL-C levels were observed for the NC, BM, BH and HFD groups of mice, there

200

were dose-effect relationships between BCA and HDL-C. Compared with the HFD

201

treatment, BH treatment led to a 5% higher level of HDL-C. Moreover, BCA

202

treatment significantly decreased TC levels compared with the HFD (p < 0.05, Figure

203

1A).

204

3.1.2 Changes in HTGL and LPL activity

205

HTGL activity was lowest in the HFD group and subsequently increased with an

206

increase in BCA content. BCA treatment restored HTGL activity to normal and

207

maintained nearly the same level of activity as that measured in the NC group. LPL

208

activity was the lowest in the HFD group and was more than twice that of the NC and

209

HFD groups in the BCA-treatment groups (p < 0.05) (Figure 1B).

210

3.1.3 Comparison of liver histology

211

Histopathological observations exhibited marked pathological changes among

212

different treatments, i.e., NC mouse livers were characterized by a dull-red, clear-cut

213

margin and cut without a greasy feeling; HFD mouse livers were characterized by a

214

cream-yellow color, hard texture, greasy sections and degeneration of local lesions;

215

the sample groups (BL, BM, BH) and simvastatin positive control (PC) group were 11



216

characterized by dull-red livers with a slight yellow color, clear-cut margins and

217

slightly greasy sections (Figure 3A).

218

A 100× magnification was used to examine the HE stained liver sections to

219

evaluate pathological changes in the livers. The liver tissues in the NC and PC groups

220

presented clear structures, mild edema and degeneration of partial hepatocytes,

221

punctate or small focal necrosis of liver cells, mild dilatation and congestion of the

222

interlobular veins and central veins, and chronic inflammatory cell infiltration of the

223

stroma. HFD and BL liver histology sections showed extensive and moderate edema

224

and degeneration of hepatocytes, punctate or small focal necrosis of liver cells,

225

steatosis, mild dilatation and congestion of the interlobular veins and central veins,

226

and chronic inflammatory cell infiltration of the stroma. Liver histology sections from

227

the BM and BH groups had clear structures, mild to moderate edema, degeneration of

228

hepatocytes and punctate or small focal necrosis of liver cells. It was clear that a

229

high-fat diet initiated dysfunctional lipid metabolism in mice. The results showed that

230

hepatic lesions from mice fed BCA were lighter than the lesions of those fed an HFD,

231

indicating that BCA could reverse the liver lesions caused by a high-fat diet (Figure

232

3B).

233

TC and TG levels in the liver tissues of HFD-treated mice were higher than those

234

of mice in the other groups, supporting the hypothesis of the physiological role of

235

BCA in decreasing TC and TG levels. Simvastatin returned TG to normal levels and

236

maintained nearly the same level as that of the mice in the NC group. Nevertheless,

237

high-dose BCA and middle-dose BCA treatment significantly reduced the TG level (p 12


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< 0.05) (Figure 3C).

239

3.1.5 Determination of fecal fat and epididymal fat indexes

240

Fecal lipid excretion in all the BCA-treated groups at the end of the fourth week

241

of treatment was more than that at the second week of treatment. Moreover, fecal lipid

242

content increased with increasing BCA concentration. More importantly, the fecal fat

243

excretion in all the BCA treatment groups was lower than that of the HFD group at

244

both the second and fourth weeks. These results once again prove the role of BCA in

245

promoting lipid metabolism and fat excretion. The weight ratio (weight of fat/body

246

weight) of the epididymis is also shown in Figure 1C. The epididymal fat of PC and

247

BM mice was 20% less than that of HFD mice, indicating that BCA and simvastatin

248

accelerated lipid metabolism and reduced lipid accumulation (p < 0.05) (Figure 1C).

249

3.2 Effect of BCA on cholesterol efflux

250

Cholesterol efflux had a dose-response relationship with BCA. The 10 and 20

251

µM doses of BCA had the same effect as simvastatin on cholesterol efflux in

252

RAW264.7 macrophages; the treatments significantly improved the cholesterol efflux

253

capacity of macrophages. Both the 10 and 20 µM BCA treatments significantly

254

promoted cholesterol efflux compared with the control. Cholesterol efflux in the

255

group treated with 20 µM BCA was 17.91% higher than that in the group treated with

256

0.1 µM BCA (p < 0.05) (Figure 2).

257

3.3 Antioxidation of BCA

258

The dose-dependent reducing capacity of BCA is shown in Figure 4A. The 13



259

reducing capacity of BCA was strongest at a concentration of 8.0 mM (with an

260

absorbance value of 0.301). From 0.1 to 3.0 mM BCA, the ABTS free radical

261

scavenging ability of BCA significantly increased (Figure 4B). The free

262

radical-scavenging rate of ABTS+. reached 99.2% at a concentration of 4.0 mM BCA

263

(p < 0.05).

264

Increased serum and liver SOD activity was observed in the BCA-treated groups

265

(BL, BM, BH); the BH group had 89.19 U/mg protein in the liver and 137.56 U/ml in

266

the serum, which were more than the HFD group (p < 0.05, p < 0.01) (Figure 4D).

267

Serum and liver MDA levels in the HFD group (particularly the liver levels)

268

were higher than those of the other groups. MDA levels obviously declined after BCA

269

and simvastatin treatment. The MDA level in the BM group decreased by 4.01 U/mg

270

protein in the liver and 1.89 U/ml in the serum compared with the HFD group (p

Potential Lipid-Lowering Mechanisms of Biochanin A - ACS Publications

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