Implication on significance of the dietary compatibility: Based on the

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Implication on significance of the dietary compatibility: Based on the antioxidant and anti-inflammatory interactions with different ratios of hydrophilic and lipophilic antioxidants among four daily agricultural crops Yao Pan, Hongyan Li, Shi-Lian Zheng, Bing Zhang, and zeyuan deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01690 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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Journal of Agricultural and Food Chemistry

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Implication on significance of the dietary compatibility:

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Based on the antioxidant and anti-inflammatory

3

interactions with different ratios of hydrophilic and

4

lipophilic antioxidants among four daily agricultural crops

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Yao Pan1, Hongyan Li1*, Shilian Zheng1, Bing Zhang1, Ze-yuan Deng1,2**

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1

State Key Laboratory of Food Science and Technology, University of Nanchang, Nanchang 330047, Jiangxi, China

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2

Institute for Advanced Study, University of Nanchang, Nanchang 330031, Jiangxi, China

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*Corresponding author. Tel.: +86 791 88314447-8226; fax: +86 791 88304402 E-mail address: [email protected] ** Corresponding author. Tel.: +86 791 88304402; fax: +86 791 88304402 E-mail address: [email protected] 1

ACS Paragon Plus Environment


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Abstract

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The hydrophilic extracts of eggplant peel (HEEP) and purple sweet potato

14

(HEPP), lipophilic extracts of tomato (LET) and carrot (LEC) were mixed in different

15

ratios to assess the significance on the compatibility of aliments, based on their

16

antioxidant and anti-inflammatory interactions in H9c2 cells. The results indicated

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that groups of some combinational extracts (HEPP-HEEP: F1/10, LEC-HEEP: F3/10,

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LEC-HEPP: F3/10) showed stronger synergistic antioxidant and anti-inflammatory

19

effects than individual groups. For example, the GPx activity of LEC-HEEP (F3/10)

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group (86.71 ± 1.88) was higher than that in HEEP (79.97 ± 1.68) and LEC (77.31 ±

21

1.85) groups. The level of ROS was 30.37 ± 0.25 in LEC-HEEP (F3/10) group while

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the levels were 34.34 ± 0.36 and 46.23 ± 0.51 in HEEP group and LEC group,

23

respectively. And the level of MDAwas 1.82 ± 0.24 in the LEC-HEEP (F3/10) group

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while the levels were 2.48 ± 0.13 and 3.01 ± 0.24 in HEEP group and LEC group,

25

respectively. The expressions of inflammatory mediators (IL-1β, IL-6, IL-8) and cell

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adhesion molecules (VCAM-1, ICAM-1) showed the similar tendency. However,

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some groups (LET-LEC: F5/10, LET-LEC: F9/10, LET-HEPP: F7/10) showed

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antagonistic effects based on these indicators. The principal component analysis

29

showed that samples could be defined by two principal components. PC1: the main

30

phenolic acids and flavonoids. PC2: carotenoids. Moreover, phenolics and

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anthoyanins were in the majority in synergistic groups, and carotenoids were in the

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majority in antagonistic groups. These results indicated that there exist synergistic or

33

antagonistic interactions of aliments on anti-oxidation and anti-inflammation, which 2


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implied the significance of food compatibility.

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Key words: antioxidant, anti-inflammatory, interaction, compatibility, vegetable extracts

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3



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1 Introduction

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The oxygen consumption leads to the generation of a series of reactive oxygen

41

species (ROS) such as superoxide anion radicals (O•2), hydroxyl radicals (OH•) and

42

hydrogen peroxide (H2O2)1. ROS also plays a significant role in transduction cascades

43

and pathways2. Epidemiological studies have shown that high consumption of fruits

44

and vegetables has health benefits in the prevention of chronic diseases3. In fact,

45

antioxidant phytochemicals (carotenoids, anthocyanins and phenolics) in vegetables

46

and fruits can delay or preventthe lipid oxidation, inhibit the initiation or propagation

47

of

48

phytochemicals are also involved in scavenging free radicals4. Other studies showed

49

that inflammatory responses can be reduced by the increased expression of

50

antioxidant genes5,6.

oxidizing

chain

reactions,

reduce

inflammatory

response.

Antioxidant

51

Different phytochemical combinations showed various antioxidant interactions

52

including synergistic, additive, and antagonistic effects7. It was shown that the

53

antioxidant interactions of food mixture maybe not only depended on the chemical

54

reaction since sometimes there was no change of the main component in the mixture

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before and after combination7. At present, three points were provided by some

56

researchers8. One of them was that the regeneration of antioxidants may be the main

57

reason of the antioxidant interaction9. Moreover, the generation of new material can

58

further influence the antioxidant interaction. For example, rutin could scavenge free

59

radicals which results in a rutin-phenoxyl-radical, and the phenoxylradicals may be

60

recycled by carotenoids10. α-Tocopheroxylradical, which is derived from α-tocopherol 4


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after reaction with a free radical, can act as pro-oxidant. Rutin may act as a

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supplement to ascorbic acid sparing α-tocopherol and/or regenerating the

63

α-tocopheroxyl radical at the surface, thus preventing α-tocopheroxyl from promoting

64

oxidation10. On the other hand, it was revealed that protein kinase-related signaling

65

pathways including phosphoinositide 3-kinase and protein kinase C.Their activities

66

were modulated by phytochemicals. And it was also suggested that these relevant

67

protein can affect the absorption and bioavailability of phytochemicals in cells11 and

68

antioxidant interaction.

69

The antioxidant interaction was a hot topic at recent years. However, most of

70

researches were carried out by chemical standards, and focused on the antioxidant

71

interaction of same polar extracts (hydrophilic or lipophilic). In fact, there are both

72

hydrophilic and lipophilic antioxidant components in dietary foods. Whereas,

73

available information regarding the antioxidant interactions between the mixture of

74

lipophilic and hydrophilic extracts in daily vegetables are still limited10. Moreover,

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most of the researches reported the in vitro antioxidant interaction on ratio of 1:1, less

76

was studied on other ratios or in vivo antioxidant interactions.

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Four daily agricultural crops such as eggplant (which has a dark purple skin and

78

contains anthocyanins and phenolics), purple sweet potatoes (which constitute

79

polyphenols and anthocyanins)12, carrot and tomatoes (which are good sources of

80

carotenoids such as β-carotene and lycopene) have shown remarkable antioxidant and

81

anti-inflammation activities13. Phenolic compounds and anthocyanins are hydrophilic

82

antioxidant which protect against oxidative stress and inflammation12. Carotenoids are 5



83

lipophilic antioxidants which can protect biological systems from oxidative stress and

84

modulate the enzymatic antioxidant pathways14. Carotenoids can also ward off heart

85

disease and several types of cancers such as lung, prostate, cervical, digestive tract

86

and breast cancers15.Not only these agricultural crops are daily foods, but also are

87

important source of hydrophilic and lipophilic antioxidants for human health.

88

Therefore, they were selected to explore the effect of hydrophilic-lipophilic

89

antioxidants ratio (different vegetable extracts) on antioxidant and anti-inflammatory

90

interactions. And we utilized the combination index (CI) method, which is based on

91

the physico-chemical principle of the median-effect equation (MEE) of the

92

mass-action law and has been most widely used16in drug combinations17, to analyze

93

the influences of various hydrophilic-lipophilic antioxidants ratio.

94 95

2 Materials and methods

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2.1 Chemicals and reagents

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β-Carotene,

gallic

acid,

p-hydroxybenzoic

acid,

epigallocatechingallate,

98

chlorogenic acid, catechin, epigallocatechin, protocatechuate, p-coumaric acid, ferulic

99

acid, isoferulic acid, caffeic acid, rutin, myricetin, quercetin, kaempferol and DPPH

100

were obtained from Aladdin (Fengxian, shanghai, China). Lycopene, delphinidin,

101

petunidin, peonidin, gallic acid were gained from ChengShiKangPu Institute of

102

Chemical Technology (Beijing, China). ABTS was purchased from Beyotime (Jiangsu,

103

China). Solvents including methanol, acetonitrile, methyltert-butylether (MTBE) were

104

purchased from Anpel (Shanghai, China). 6


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2.2 Plant materials

107

Tomato, carrot, purple sweet potato, and eggplant were purchased from local

108

supermarket. The mature vegetables (2.5 kg/each kind) were selected, and the edges

109

were removed. The vegetables were washed with tap water. Carrot, tomato and purple

110

sweet potato (with peels) were cut into small pieces (every piece was 0.5 cm length,

111

0.5 cm wide and 0.5 cm thick). Eggplant peels (purple part only) were scraped off and

112

cut into small pieces (every pieces was 1 cm length, 1 cm wide and10 mm thick ), and

113

ground into homogenate with a commercial triturator (JJ-2B, JintanRonghua

114

Instrument Manufacture Co. Ltd, Jiangsu, China). The homogenate was freeze-dried

115

(FD-1, Beijing Detianyou Technology and Development Co., Ltd., Beijing, China)

116

and ground into fine powders, and then stored at -80°C until analysis.

117 118

2.3 Phytochemical extractions

119

The carotenoids of tomato and carrot were extracted according to the method18

120

with some modifications. Briefly, 0.5 g dried vegetable powder was transferred into

121

50 mL tube containing 10 mL of acetone for tomatoes and carrots, and the mixture

122

was carried out in a water bath at 4°C for 12 h. The mixture was centrifuged at 4200

123

rpm for 3 min (TDL-5-A, Anke, Shanghai, China). After the supernatant was

124

separated, the residue was re-extracted twice. The supernatant (water phase) was

125

gathered, the lipophilic extracts were evaporated to dryness with nitrogen.

126

Phenolics including anthocyanins of purple sweet potato and eggplant peel were 7



127

extracted according to the previous methods18 with minor modifications. Briefly, 0.5 g

128

dried vegetable powder was transferred into 50 mL tube and were topped up to 10 mL

129

with 0.1% HCl (v/v) in 80% methanol. The mixture was carried out in a water bath at

130

4°C for 12 h and then was centrifuged at 4200 rpm for 3 min (TDL-5-A, Anke,

131

Shanghai, China). After the supernatant was separated, the residue was re-extracted

132

twice. And the hydrophilic extracts were freeze-dried.

133

All procedures were conducted in dark to avoid oxidation and three separate

134

samples were extracted. The extractions were then filtered through a 0.2-µm PTFE

135

syringe filter (AA-56316, Troody), for the further analysis

136 137

2.4 The identification and quantification of carotenoids

138

The Agilent 1290 UPLC system contained a binary pump Bin Pump SL, a

139

degasser, a TCC SL column oven, a thermostatedHiP-ALS autosampler and a DAD

140

detector was used (Agilent Technologies, Santa Clara, CA, USA). The identification

141

of carotenoids followed the method as described in our previous report19 with minor

142

modifications. The mobile phase consisted of methanol (A) and acetonitrile (B).

143

Isocratic elution with 65% A and 35% B was used. The flow rate was 0.3 mL/min and

144

the column temperature was controlled at 30°C. All standards and samples were

145

dissolved in acetone and the sample injection volume was 3 µL. In order to generate a

146

specific calibration plot for the calibration and quantification, β-carotene standard (1.0

147

mg) and lycopene standard (1.0 mg) were accurately weighted and dissolved in 1 mL

148

acetone. The stock solution was diluted to obtain corresponding solutions (0.1, 0.2, 8


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0.4, 0.6, 0.8 mg/mL).

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2.5 The identification and quantification of phenolics

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The instrument is the same as the preceding article and the method as described

153

in previous report20 with little modification. For phenolics, the mobile phase consisted

154

of 0.1% formic acid in de-ionized water (A) and acetonitrile (B). The gradientdient

155

program was as follows: 0-10 min, 90-80% A; 10-15 min, 80-60% A; 15-18 min,

156

60-90% A. A 6 min post-run procedure was set to re-equilibrate the column. The flow

157

rate was 0.1 mL/min and the column temperature was controlled at 25°C. The UV-vis

158

absorbance data of the peaks was acquired over a range from 230 to 600 nm. All

159

standards and samples were dissolved in methanol and the sample injection volume

160

was 3 µL. In order to generate a specific calibration plot for the calibration and

161

quantification, each standard (1.0 mg) was accurately weighted and dissolved in 1 mL

162

methanol. The stock solution was diluted to obtain corresponding solutions (10, 25,

163

50, 100, 150 and 200 µg/mL).

164 165

2.7 The identification of anthocyanins

166

The HPLC-QTOF-MS consisted of an Agilent system G6430 (Waldbronn,

167

Germany) equipped with ODS C18 column (2.1×100 mm, 1.8 µm) and a detector

168

(series 1100-DAD G1315B) was used. As the mobile phase, (A) 0.2% formic

169

acid-water and (B) 0.2% formic acid-acetonitrile were used. The gradient dient

170

program was as follows: 0-15 min 7%-13% B, 15-18 min 13%-13% B, 18-25 min 9



171

13%-16% B, 25-30 min 16%-16% B, 30-40 min 16-20% B, 40-41 min 20%-7% B.

172

The flow rate was 0.3 mL/min and the column temperature was controlled at 35°C.

173

The UV-vis absorbance data of the peaks was acquired in range of 280-520 nm.

174

ESI-MSn experiments were performed using the following conditions: positive ion

175

and alternating mode, detection range of m/z was 100-1700, capillary voltage was 4

176

KV, gas flow temperature is 350°C, nebulizer (N2) was 60 psi, dry gas was 11 L/min.

177 178

2.8 The total phenolic, flavonoid, anthocyanin and carotenoid contents

179

The total phenolic contents (TPCs) of dried vegetable powder were carried out

180

by Folin-Ciocalteu method7 using gallic acid as a standard. The TPC was expressed as

181

milligram of gallic acid equivalent (GAE) in gram of dry weight (DW). The total

182

flavonoid contents (TFCs) of dried vegetable powder were determined according to

183

previously reported methods21. TFC was expressed as mg catechin equivalents per g

184

dry weight extract (mg CAE/g DW) using the catechin calibration curve. The total

185

anthocyanin contents (TACs) of purple potato and eggplant were measured using pH

186

differential method18. Total carotenoid contents (TCCs) were measured using pervious

187

method22. All samples were tested in triplicate.

188 189

2.9 Cell culture and viability assays

190

H9c2 cells (2-1, Zhichenhui BiotechnologyCo., Shanghai, China) were plated in

191

Dulbecco’s modified Eagle’s medium (DMEM) (01-051-1ACS, Biological Industries,

192

Shanghai, China) supplemented with heat inactivated 10% fetal bovine serum (FBS) 10


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(04-001-1A/B, Biological Industries, Shanghai, China), 100 U/mL penicillin, 100

194

mg/mL streptomycin (Solarbio Co., Beijing, China) and cultured in a humidified

195

incubator at 37°C, 5% CO2. Cells were plated in the suitable plates for 24 h before the

196

appropriate treatments for the different assays. Cell viability was assayed with trypan

197

blue within 1 h of cell isolation. Only preparations with cell viability greater than 95%

198

were used for subsequent experiments. The hydrophilic extracts were dried

199

underlyophilization,

200

(Zhongshanjinqiao Biotechnology Co., Beijing, China) as the hydrophilic stock

201

solution (200 mg/mL). The lipophilic extracts were dried under nitrogen, and

202

re-dissolved in a small volume of dimethylsulfoxide (DMSO) (Damao Co. Ltd.,

203

Tianjing, China) as the lipophilic stock solution (200 mg/mL). Both these stock

204

solutions were diluted in cell culture medium as different ratios.

and

re-dissolved

in

phosphate

buffer

solution

(PBS)

205 206

2.10 Determination of MTT assay by combination index (CI).

207

The MTT assay were determined according to our previous study23 with some

208

modifications.

H2O2-induced

cytotoxicity

was

209

5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cells were

210

seeded in 96-well microstate plates, after the treatments with H2O2 (0, 50, 100, 150,

211

200, 300 µmol/L) for 1 h, respectively, and 0.5 mg/mL MTT (Sigma Co., USA) was

212

added to each well and incubated for 4 h to form formazan crystals. Then, the medium

213

was gently removed, and the crystals were dissolved in 100 mL of DMSO. The

214

formed formazan crystals were quantified and measured at 490 nm in microplate 11


measured

using

3-(4,


215

kinetic reader (Thermo Scientific varioskan flash, Vantaa, Finland). The data was

216

expressed as a percentage of viability compared to untreated control cells. The

217

appropriate concentration for H2O2 (cell relative viability was under 50%) was

218

selected.

219

Five different fractions (1/10, 3/10, 5/10, 7/10, and 9/10) of each vegetable

220

extracts were designed to mix with different fractions of another one (9/10, 7/10, 5/10,

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3/10 and1/10). Cells were divided into 36 groups: control group, H2O2 group, four

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individual groups (HEEP, HEPP, LEC, LET), HEEP-HEPP+H2O2 groups (with five

223

different fractions), LEC-HEEP+H2O2 groups (with five different fractions),

224

HEEP-LET+H2O2 groups (with five different fractions), LEC-HEPP+H2O2 (with five

225

different fractions), LET-HEPP+H2O2 (with five different fractions), LEC-LET+H2O2

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(with five different fractions). After incubation of combination extracts in different

227

concentration (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL) for 12 h and treatment with

228

H2O2 (200 µmol/L, the optimal concentration from the results of preliminary

229

experiment) for 1 h, the cell viability was determined by MTT assay. Based on

230

different concentration from different fractions, the CI value can be generated

231

automatically by CompuSyn17. According to the CI values, 6 groups (3 synergistic

232

and 3 antagonistic groups) were chosen for the further experiments.

233 234

2.11 Measurement of reactive oxygen species (ROS) production

235

A fluorescent probe, dichlorodihydrofluoresceindiacetate (DCF-DA), and

236

flowcytometry were used to measure the intracellular ROS formation. Briefly, cells 12


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were seeded into 6-well plates for 24 h and treated with extracts for 12 h, induced by

238

H2O2 for 30 min, followed by incubation with 5 mM DCF-DA in medium at 37°C for

239

10 min in the dark. After centrifugation at 1000 rpm for 5 min, the supernatants were

240

removed and the pellets were resuspended in 1% Triton X-100. Fluorescence was

241

measured by flow cytometry. Flow cytometry was performed using a FACS calibur

242

(BD biosciences) system with cell quest software. The percentages of cells in different

243

phases of the cell cycle within the GFP-positive population were determined using the

244

software program ModFit.

245

2.12 The enzymatic activities of SOD, GSH, GPx, GST, CAT and the level of

246 247

MDA

248

Total cell lysates were prepared in RIPA lysis buffer containing protease

249

inhibitors (Beyotime Biotech, Shanghai, China). Total protein concentration was

250

determined by the BCA protein assay (Beyotime Biotech, Shanghai, China).

251

Superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx),

252

glutathione transferase (GST), catalase (CAT) enzymatic activities and the level of

253

malondialdehyde (MDA) were determined using colorimetric kits (Beyotime Biotech,

254

Shanghai, China) according to the manufacturer’s protocols.

255 256 257 258

2.13 Western blot analysis of the inflammatory mediators (IL-1β, IL-6, IL-8, TNF-α) and cell adhesion molecules (VCAM-1, ICAM-1) H9c2 cells were incubated with combination groups for 12 h in 6-well culture 13



259

plates, respectively. Then, cells were induced by H2O2 for 1 h. Immediately following

260

the incubation, the cells were washed three times with ice-cold PBS and the total

261

protein was extracted by 50 µL cell lysis buffer (1 mL RIPA + 10 µL PMSF). After

262

incubation for 5 min at -4°C, the sample was centrifuged at 12000 rpm for 15 min and

263

the supernatant was separated and stored at -80°C. Proteins (about 80 µg) were

264

separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF)

265

membranes (Roche Diagnostics GmbH, Mannheim, Germany). After blocking in 5%

266

fat-free milk in Tris-buffered saline-Tween-20 (TBST) for 2 h at room temperature,

267

the PVDF membranes were incubated with primary antibodies (Anti-IL-6 antibody

268

ab9324, Anti-VCAM 1 antibody [EPR5047] ab134047, Anti-IL1 beta antibody

269

ab9787, Anti-ICAM 1 antibody [EPR19938] ab206398, Anti-TNF alpha antibody

270

ab6671, Anti-IL-8 antibody ab7747, all of these antibodies were punched from Abcam,

271

Cambridge, MA) overnight at 4°C. The membranes were washed with TBST and then

272

incubated with horseradish peroxidase-conjugated anti-rabbit (L3012, Signalway

273

Antibody, Nanjing, China) or anti-mouse (L3032, Signalway Antibody) secondary

274

antibodies in TBST (1:1000) for 2 h at room temperature, and then visualized by a

275

super enhanced chemiluminescence detection reagent (Beyotime Institute of

276

Biotechnology, Haimen, China). The signals were detected using Image Station

277

4000R (Kodak, Rochester, NY, USA). Quantification of results was performed using

278

Quantity One. Each experiment was repeated at least three times.

279 280

2.14 Statistical analysis 14


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The data are expressed as mean ± standard error of the mean (SEM) from at least

282

three independent assays, each one in duplicate. Statistical differences were analyzed

283

by one- or two-way ANOVA followed by multiple comparisons performed with post

284

hoc Tukey’s test using SPSS version 18.0 (SPSS Inc., Chicago, IL). Differences were

285

considered as statistically significant if P< 0.05.

286 287

3 Results

288

3.1 Phytochemical contents and antioxidant activities

289

The

main

anthocyanins

of

hydrophilic

extracts

were

290

cyanidin-3-sophoroside-5-glucoside,

peonidin-3-sophoroside-5-glucoside,

291

delphinidin-3-glucoside,

292

pelargonidin-3-lutinoside-5-glucoside,

293

acid-sophoroside-5-glucoside (Table 1, Figure 1). It showed that the anthocyanins of

294

purple sweet potato and eggplant peel frequently occurred as glycoside. The main

295

phenolic compounds in purple sweet potato and eggplant peel weregallic

296

acid,p-hydroxybenzoic

297

p-coumaric acid. (Table 2, Figure 2). Moreover, the TPCs of HEEPand HEPP were

298

18.28 ± 0.73 and 6.17 ± 0.51 mg GAE/g DW, the TFCs of HEEP and HEPP were 7.89

299

± 0.68 and 2.96 ± 0.49 mg G3G/100g DW, and the TACs of HEEP and HEPP were

300

1.96 and 0.33 mg G3G/g DW, respectively (Table 3A).

petunidin-3-double-mulberry-cloth-glucoside, and

acid,chlorogenic

cyanidin-3-p-hydroxybenzoic

acid,protocatechuate,quercetin

and

301

On the other hand, the main carotenoids of LEC were β-carotene (735.02 ± 19.01

302

µg/g DW) and lycopene (132.03 ± 13.88 µg/g DW), and its TCCs was 937.01 ± 21.13 15



303

µg/g DW. Lycopene (538.26 ± 12.31 µg/g DW) and β-carotene (254.13 ± 10.97 µg/g

304

DW) were also the main carotenoids of LET and its TCC was 851.12 ± 17.67 µg/g

305

DW (Figure 3,Table 3B).

306 307 308

3.2 The interaction effects of different fractions among different vegetable extracts on the relative cell viability

309

After treated with 200 µmol/L H2O2 for 1 h, the relative cell viability was below

310

50%. Hence, the treated concentration (200 µmol/L) was chosen to set up the damage

311

cell model with H2O2. Compared with the control group, cell viability was under 95%

312

when the concentrations of HEEP and HEPP were above 1.5 mg/mL and the

313

concentrations of LEC and LET were above 2.0 mg/mL. Therefore, in order to ensure

314

the cell viability after the addition of extracts, appropriate concentration (1 mg/mL) of

315

these four extracts was chosen for the further study.

316

The combination indexes of relative cell viability at different ratios of four

317

vegetable extractions were showed in Table 4. Three groups [HEPP-HEEP: F1/10

318

(S1), LEC-HEEP: F3/10 (S2), LEC-HEPP: F3/10 (S3)] showed lower CI values

319

(0.825±0.01, 0.701±0.014, 0.797±0.097, respectively) which indicated synergistic

320

effects (CI＜1) .However, there were three groups[LET-LEC: F5/10 (A1), LET-LEC:

321

F9/10 (A2), LET-HEPP: F3/10 (A3)]had higher CI values (1.465±0.045, 1.264±0.091,

322

1.354±0.023, respectively) which showed antagonistic effects (CI＞1).Therefore,

323

these 6 groups(S1, S2, S3, A1, A2, A3) were chosen for the further experiments.

324

Different combinational groups with various ratios presented diverse antioxidant 16


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interactions in this study on account of the fact that people usually ingest the mixture

326

of phytochemicals in their daily food, included lipophilic and hydrophilic

327

phytochemicals. Apparently, from the MTT results of 30 kinds of different

328

combinational groups, six groups (S1, S2, S3, A1, A2, A3) showed more significant

329

effects on H2O2-induced H9c2 cells than the individual groups.

330

3.3 The combination groups inhibited the generation of intracellular ROS

331

The intracellular ROS generation is a marker of oxidative stress. Cells induced

332

by H2O2 exhibited a significant increase of intracellular ROS generation (75.07 ± 0.31,

333

Figure 4) as compared to the control group (36.64 ± 0.22). When the cells were

334

pretreated with the individual extracts (HEPP, HEEP, LET, LEC), there was a

335

significant decrease in intracellular ROS generation compared to H2O2-induced group

336

(Figure 4). For example, the intracellular ROS generation of LEC group (46.23 ± 0.51)

337

was inferior to the H2O2-induced group (75.07 ± 0.31).

338

However, when pretreated with the combination of extracts, some groups

339

showed the synergistically inhibited effects on the intracellular ROS generation

340

(Figure 4). For instance, the ROS generation in LEC-HEEP (F3/10) was 30.37 ± 0.25,

341

which was lower than those in HEEP (34.34 ± 0.36) and LEC groups (46.23 ± 0.51).

342

In the same way, groups of HEPP-HEEP (F1/10) and LEC-HEPP (F3/10) showed the

343

synergistic effect. However, there are also some groups (LET-LEC: F5/10, LET-LEC:

344

F9/10, LET-HEPP: F3/10) showed antioxidant antagonistic effects (Figure 5). For

345

instance, the ROS generation in LET-LEC (F9/10) was 52.35 ± 0.38, which was

346

higher than those in LEC and LET groups (34.34 ± 0.36 and 34.27 ± 0.43, 17



347 348 349

respectively). 3.4 Effects of different ratios among different extracts on GPx, SOD, CAT activities, the level of GSH and MDA in H2O2-induced H9c2 cells

350

The groups pretreated with different extracts could effectively prevent the GSH

351

depletion in H9c2 cells exposed to H2O2 (200 µmol/L) for 1 h (Figure 5). It was noted

352

that the GSH levels of individual group, such as HEEP group (84.55 ± 1.79) could be

353

recovered compared with the H2O2-induced groups (70.47 ± 1.46). In addition, three

354

combination groups (S1, S2, S3) showed synergistic effects on recovery of GSH

355

depletion (Figure 6). The activity of GSH on HEPP-HEEP (F1/10) group (94.01 ±

356

1.87) was significant higher than those of HEPP and HEEP groups (78.83 ± 1.27 and

357

84.55 ± 1.79, respectively). However, another three groups (A1, A2, A3) showed

358

antagonistic effect on recovery of GSH depletion (Figure 5). The value of GSH on

359

LET-LEC(F5/10) group (69.37 ± 1.49) was significant decreased compared the

360

individual LET and LEC groups (75.97 ± 1.86 and 72.09 ± 1.99, respectively).

361

In the present study, the groups treated with H2O2 resulted in the significant

362

decrease on activity of GPx (% of control group) in H9c2 cells (P0.05).However, the SOD activities (197.34 ± 1.62

375

U/mg prot) in S2 group were significantly higher than HEEP (153.11 ± 4.87 U/mg

376

prot) and LEC groups and (156.23 ± 5.85 U/mg prot).

377

Similarly, the activity of CAT on the S2 group (7.23 ± 0.84 U/mg prot) showed

378

synergistic effect compared with the individual groups (HEEP: 5.01 ± 0.91, HEPP:

379

4.92 ± 0.75, LEC: 4.85 ± 0.63). However, the activity of CAT showed no significant

380

difference among A1, A2, A3 groups and the individual groups (LEC, LET,

381

HEPP).On the other hand, the expression of MDA (Figure 5) in the H2O2-induced

382

group (3.48 ± 0.16 nmol/mL) was significantly higher than other groups. Meanwhile,

383

the expression of MDA in the S2 group (LEC-HEEP F3/10, 2.01 ± 0.21 nmol/mL)

384

was significantly lower than those in HEEP and LEC groups (2.37 ± 0.19 and 3.01 ±

385

0.25 nmol/mL), which showed synergistic effect (P

Implication on significance of the dietary compatibility: Based on the

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