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Food and Beverage Chemistry/Biochemistry

Daily dietary antioxidant interactions are due to not only the quantity but also the ratios of hydrophilic and lipophilic phytochemicals Yao Pan, zeyuan deng, Shi-Lian Zheng, Xuan Chen, Bing Zhang, and Hongyan Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03412 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Journal of Agricultural and Food Chemistry

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Daily dietary antioxidant interactions are due to not only the

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quantity but also the ratios of hydrophilic and lipophilic

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phytochemicals Yao Pan1, Ze-yuan Deng1,2, Shi-lian Zheng1, Xuan Chen1, Bing Zhang1,

4

Hongyan Li1*

5 6

1

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

7 8

2

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

9 10

*Corresponding

author. Tel.: +86 791 88314447-8226; fax: +86 791 88304402

E-mail address: [email protected] 1


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Abstract

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The hydrophilic extracts of mulberry (HEM) and blueberry (HEB), lipophilic

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extracts of mango (LEM) and watermelon (LEW) were mixed in different ratios to

14

assess the antioxidant interactions by chemical (DPPH and ABTS assay) and H9c2

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cell-based models. There were both synergistic and antagonistic antioxidant

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interactions among daily fruits. Some groups with combinational extracts showed

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stronger synergistic antioxidant effects than the individual groups and others

18

(HEM-LEW: F1/10, LEW-LEM: F5/10, HEB-LEM: F3/10) showed antagonistic

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effects than the individual groups based on the indicators (the values of DPPH, ABTS,

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MTT; the expression of SOD, GSH-Px, CAT, MDA; the release of LDH and the

21

quantification of CAA). The principal component analysis (PCA) showed that

22

samples could be defined by two principal components: PC1, the main phenolic acids

23

and anthocyanins; PC2, carotenoids. From our results, primarily, carotenoids were in

24

the majority on antagonistic groups, and phenolics and anthocyanins were in the

25

majority on synergistic groups. However, the combinational groups containing only

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hydrophilic compounds did not always show synergistic effects. Therefore, the

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compatibility of diets implies to balance the ratios of hydrophilic and lipophlic

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compounds in our daily food. In addition, the expression of enzymes (SOD, GSH-Px,

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CAT) may not be sensitive to the changes of antioxidant activity caused by the

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combinations with different ratios of hydrophilic compounds and lipophilc

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compounds. And the different structures of lipophilic compounds (β-carotene and

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lycopene) could influence the antagonistic effects.

33 2



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Key words: synergistic effects, antagonistic effects, compatibility, fruit extracts

3


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1 Introduction

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Accumulation of excessive free radicals exceeding cellular antioxidant capacity

37

results in oxidative stress1. This process can damage macromolecules (DNA, lipids,

38

and proteins), organelles (membranes and mitochondria), and whole tissues in human

39

body2. Furthermore, oxidative stress is implicated in the exacerbation of

40

cardiovascular, and other chronic diseases, both through direct molecular damage and

41

secondary activation of stress-associated signaling pathways2,3. Diets rich in fruits are

42

recommended by many health organizations and epidemiological studies. Based on

43

the Dietary Guidelines for Americans, the standards require schools to offer salad bars

44

with greater quantities of fruits for school-age children4. Another study also indicated

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that fruit salad is a super diet for health5. Other studies have shown that some

46

compounds from fruits can decrease the incidence of several chronic diseases,

47

including coronary heart disease, stroke, diabetes mellitus and some cancers6. Among

48

the bioactive compounds in fruits, phytochemicals (carotenoids, anthocyanins and

49

phenolics) are good antioxidants and can effectively scavenge the excessive free

50

radicals in body7.

51

Previous research indicated that phytochemicals showed various antioxidant

52

interactions including synergistic, additive, and antagonistic effects when they were

53

combined mutually8. The reported mechanisms of antioxidant interactions can be

54

summarized as three points: First of all, the main reason of the antioxidant interaction

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is due to the regeneration of antioxidants9. It means that one of the antioxidants can

56

scavenge free radicals and then be recycled by another antioxidant in the 4



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combination10. The second point is that different phytochemicals play antioxidant role

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through different manners11, because there were different antioxidant pathways to

59

inhibit the radical such as hydrogen atom transfer (HAT) and single electron transferin

60

the external chemical models7,11. Thirdly, it was revealed that various proteins

61

kinase-related signaling pathways were modulated by different phytochemicals. And

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the bioavailability of phytochemicals in cells and antioxidant interaction can be

63

affected by these proteins12.

64

In fact, more and more researches were focused on antioxidant interactions in

65

recent years. For example, Anwesa13 and others evaluated the possible synergistic

66

interactions on antioxidant efficacy of essential oils of some selected spices and herbs.

67

Marwa14 and others evaluated the functional constituents, antioxidant and

68

anti-inflammatory activities of Malaysian Ganoderma lucidum aqueous extract and

69

Egyptian

70

lipopolysaccharide-stimulated white blood cells. Wang15 and others indentified the

71

antioxidant compounds and synergistic antioxidant effects between Huang-qi and

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Sheng-ma. However, most of researches were still pay attention to the interactions of

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same polar extracts (hydrophilic or lipophilic). In fact, we might not intake a sort of

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single fruit in our daily diet and antioxidant interactions maybe affected by both of

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hydrophilic and lipophilic antioxidant phytochemicals in fruits.

Chlorella

vulgaris

ethanolic

extract

in

vitro

model

of

76

Among dietary phytochemicals in fruits, carotenoids and phenolics represent the

77

most abundant lipid and water soluble antioxidants, respectively. Several studies have

78

proven their pharmacological potential as inflammatory, anti-diabetic, antioxidant 5


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effects

. Fruit salad is a dish consisting of various kinds of fruit (including

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berries, mango, watermelon), and is a common form of diet for human5. Hence, four

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daily fruits such as mulberry and blueberry (which constitute phenolics and

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anthocyanins)20, mango and watermelon (which are good sources of carotenoids such

83

as β-carotene and lycopene)16,21 have been chosen to explore the rules of antioxidant

84

interactions in phytochemical combinations with different ratios and different

85

compounds (hydrophilic and lipophilic extracts). These four fruits can be consumed

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together in fruit salad in our daily life. Both of the in vitro chemical based models

87

(DPPH, ABTS) and cell-based models (MTT, ROS, LDH, the cellular antioxidant

88

activity, antioxidant enzymes) were used to assess their antioxidant interactions based

89

on combination index (CI)16,21. Moreover, in order to explore the relationships

90

between different ratios (hydrophilic and lipophilic parts) of extracts and their

91

antioxidant interactions, principal component analysis (PCA) was used to analyze the

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main compounds of different combinations.

93

2 Materials and Methods

94

2.1 Chemicals and Reagents

95

β-Carotene, chlorogenic acid, catechin, caffeic acid, rutin, quercetin, and DPPH

96

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

97

petunidin, peonidin, cyanidin, malvdin were gained from ChengShiKangPu Institute

98

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

99

(Jiangsu, China). Solvents including methanol, acetonitrile, were purchased from

100

Anpel (Shanghai, China). 6



101

2.2 Fruit Materials

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Mulberry, blueberry, mango, and watermelon were purchased from a local

103

supermarket. Fruits were washed with tap water. Mulberry, blueberry (with peels),

104

mango (without peels and stones) and watermelon (without peels and nuts) were cut

105

into small pieces (every piece was 0.5 cm long, 0.5 cm wide, and 0.5 cm thick). Both

106

of these fresh fruits were ground into homogenate with a commercial triturator (JJ-2B,

107

JintanRonghua Instrument Manufacture Co. Ltd., Jiangsu, China). The homogenates

108

were freeze-dried (FD-1, Beijing Detianyou Technology and Development Co., Ltd.,

109

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

110

2.3 Phytochemical Extractions

111

The carotenoids of mango and watermelon were extracted according to the

112

previously published method7 with some modifications. Briefly, 1 g dried fruit powder

113

was transferred into 100 mL tube containing 20 mL of acetone, and the mixture was

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

115

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

116

residue was re-extracted twice. The supernatant was gathered, and the lipophilic

117

extracts were evaporated to dryness with nitrogen.

118

Phenolics including anthocyanins of mulberry and blueberry were extracted

119

according to previous methods7 with minor modifications. Briefly, 1 g dried fruit

120

powder was transferred into 100 mL tube and topped up to 20 mL with 0.1% HCl (v/v)

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in 80% methanol. The mixture was carried out in a water bath at 4°C for 12 h and then

122

was centrifuged at 4200 rpm for 3 min (TDL-5-A, Anke, Shanghai, China). After the 7


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supernatant was separated, the residue was re-extracted twice. And the supernatant

124

was collected and freeze-dried as hydrophilic extracts.

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

126

samples were extracted. The extractions were then filtered through a 0.22-µm PTFE

127

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

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2.4 Identification and Quantification of Carotenoids

129

The Agilent 1290 UPLC system was used (Agilent Technologies, Santa Clara,

130

CA, USA), which contained a binary pump Bin Pump SL, a degasser, a TCC SL

131

column oven, a thermostated HiP-ALS auto sampler, and a DAD detector. The

132

identification of carotenoids followed the method described in our previous

133

report22with minor modifications. The mobile phase consisted of methanol (A) and

134

acetonitrile (B). Isocratic elution with 65% A and 35% B was used. The flow rate was

135

0.3 mL/min, and the column temperature was controlled at 30°C. All standards and

136

samples were dissolved in acetone, and the sample injection volume was 3 µL. In

137

order to generate a specific calibration plot for the calibration and quantification,

138

β-carotene standard (1.0 mg) and lycopene standard (1.0 mg) were accurately

139

weighted and dissolved in 1 mL acetone. The stock solution was diluted to obtain the

140

corresponding solutions (0.1, 0.2, 0.4, 0.6, 0.8 mg/mL).

141

2.5 Identification and Quantification of Phenolics and Anthocyanins

142

The UPLC-QTOF-MS2 consisted of an LC30 system (Shimadzu, Japan)

143

equipped with Shim-pack GIST C18 column (2.7×75 mm, 2µm) and a detector (series

144

1100-DAD G1315B) was used. The mobile phase consisted of 0.1% formic acid in 8



145

deionized water (A) and acetonitrile (B). The gradient program was as follows: 0-10

146

min, 97-90% A; 10-15 min, 90-88% A; 15-20 min, 88-80% A; 20-35 min, 80-70%;

147

35-36 min, 70-97%. A 2 min postrun procedure was set to re-equilibrate the column.

148

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

149

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

150

volume was 3 µL. The UV-vis absorbance data of the peaks were acquired in range of

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280-520 nm. ESI-MSn experiments were performed using the following conditions:

152

positive ion and alternating mode, detection range of m/z was 100-1700, capillary

153

voltage was 4 KV, gas flow temperature is 350°C, nebulizer (N2) was 60 psi, dry gas

154

was 11 L/min. In order to generate a specific calibration plot for the calibration and

155

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

156

methanol. The stock solution was diluted to obtain corresponding solutions (10, 25, 50,

157

100, 150, and 200 µg/mL).

158

2.6 Hydrolysis and Identification of Anthocyanins

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Twenty milliliter crude extract and 5 mL of 6M HCl were mixed in a 40 mL tube

160

tightly sealed with a screw cap, flushed with nitrogen, and then incubated in a shaking

161

water bath at 90°C for 2 h to hydrolyze the anthocyanins23. The samples were allowed

162

to cool down, and then centrifuged at 2000 rpm for 5 min. The supernatant was filtered

163

through a 0.22-μm PTFE membrane filter and subjected to UPLC analysis.

164

Stock solutions of the standards were prepared separately by dissolving 10 mg of

165

each compound in 5mL DMSO and then topped up to 100 mL in a volumetric flask

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with methanol (final concentration 100 μg/mL). All anthocyanidins in the samples 9


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were quantified with external standards by using respective standard curves generated

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from serial dilutions of 10, 20, 40, 60, 80, and 100 μg/mL.

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2.7 Total phenolic, Flavonoid, Anthocyanin and Carotenoid Contents

170

The total phenolic contents (TPCs) of dried fruit powder using gallic acid as a

171

standard were carried out by Folin-Ciocalteu method8. The TPC is expressed as

172

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

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

174

previously reported method24. TFC was expressed as milligram catechin equivalents

175

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

176

The total anthocyanin contents (TACs) of mulberry and blueberry were measured

177

using the pH differential method7. The Total carotenoid contents (TCCs) were

178

measured using previously used method25. All samples were tested in triplicate.

179

2.8 Antioxidant Assays

180

The combination index (CI)，based on the physico-chemical principle of the

181

median-effect equation (MEE) of the mass-action law and had been most widely

182

used26 in drug combinations27，was used to analyze the interactions.

183

DPPH radical scavenging capacity: The antioxidant activity of the fruit extracts

184

was measured spectrophotometrically7 with slight modification. Briefly, 20 µL fruit

185

extracts and control solution were mixed with 100 µL methanolic DPPH solution

186

(0.26 mM, dissolved in methanol) in 96-well plate and incubated in the dark at 25°C

187

for 30 min. Then the absorbance was measured at 517 nm in microplate kinetic reader

188

(Thermo Scientific varioskan flash, Vantaa, Finland). The DPPH radical scavenging 10



189

activity was expressed as the value of combination index (CI25, CI50, CI75, which

190

means the CI value at the scavenging capacity of 25%, 50%, 75%, respectively). The

191

CI value was calculated by Compusyn software. It can also be calculated by an

192

equation: CI=(D)1/(DX)1+(D)2/(DX)2. In this equation, D1 and D2 mean the dose of two

193

extracts when they achieved the effect alone, respectively. And the (DX)1, (DX)2 mean

194

the dose of two extracts when they achieved same effect after combination,

195

respectively. When the CI＞1, means the antagonistic effects were shown. When CI＜

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1, means the synergistic effects were shown. When the CI=1, means the additive

197

effects were shown. All procedures were operated in dim light and all samples were

198

done in triplicate.

199

ABTS radical scavenging capacity: ABTS radical scavenging capacity was

200

determined using the method of Rice-Evans group7. Briefly, the ABTS solution was

201

prepared by ABTS radical solution and oxidant solution and let stand for 12 to 16 h at

202

room temperature (20-25°C) in the dark. The ABTS solution was adjusted with 90 to

203

100 mL 80% ethanol to obtain an absorbance of 0.7±0.05 at 734 nm before usage. The

204

fruit extracts/control solutions (10 µL) were mixed with 200 µL fresh ABTS solution

205

and let stand for 6 min at room temperature (20-25°C) before the absorbance was

206

measured at 734 nm in the aforementioned microplate kinetic reader (Thermo

207

Scientific varioskan flash). The radical scavenging capacity was expressed as CI value.

208

All samples were conducted in triplicate.

209

2.9 Cell Culture and Viability Assays

210

H9c2 cells (Zhichenhui Biotechnology Co., Shanghai, China) were plated in 11


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Dulbecco’s modified Eagle’s medium (DMEM, 01-0511 ACS, Biological Industries,

212

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

213

Biological Industries, Shanghai, China), 100 U/mL penicillin, 100 mg/mL

214

streptomycin (Solarbio Co., Beijing, China) and cultured in a humidified incubator at

215

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

216

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

217

1 h of cell isolation. Only preparations with cell viability greater than 95% were used

218

for subsequent experiments. The hydrophilic extracts were dried under lyophilization,

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and redissolved in PBS as the hydrophilic stock solution (200 mg/mL). The lipophilic

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extracts were dried under nitrogen, and redissolved in a small volume of dimethyl

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sulfoxide (DMSO, Damao Co. Ltd., Tianjing, China) as the lipophilic stock solution

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(200 mg/mL). Both these stock solutions were diluted in cell culture medium as

223

different ratios.

224

2.10 Determination of MTT Assay by Combination Index (CI)

225

H2O2-induced cytotoxicity was measured using 3-(4, 5-dimethylthiazol-2-yl)-2,

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5-diphenyl tetrazolium bromide (MTT) assay. Cells were seeded in 96-well microstate

227

plates for 1×104 per well, after treated with H2O2 (0, 50, 100, 150, 200, 300 µmol/L)

228

for 1 h, respectively, and 0.5 mg/mL MTT (Sigma Co., USA) was added to each well

229

and incubated for 4 h to form formazan crystals. Then, the medium was gently

230

removed, and the crystals were dissolved in 100 mL of DMSO. The formed formazan

231

crystals were quantified and measured at 490 nm in a microplate kinetic reader

232

(Thermo Scientific varioskan flash, Vantaa, Finland). The data is expressed as a 12



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percentage of viability compared to the untreated control cells. The appropriate

234

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

235

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

236

were designed to mix with different fractions of another one (9/10, 7/10, 5/10, 3/10

237

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

238

individual groups (HEM, HEB, LEM, LEW), HEM-HEB+H2O2 groups (with five

239

different fractions: 1/10, 3/10, 5/10, 7/10, 9/10 of HEM mixed with 9/10, 7/10, 5/10,

240

3/10, 1/10 of HEB), HEM-LEM+H2O2 groups (with five different fractions),

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HEM-LEW+H2O2 groups (with five different fractions), HEB-LEM+H2O2 (with five

242

different

243

LEM-LEW+H2O2 (with five different fractions). After incubated by combinational

244

extracts in different concentrations (0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/mL) for 12 h

245

and treatment with H2O2 (200 µmol/L, the optimal concentration from the results of

246

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

247

Based on different concentration from different fractions, the CI value can be

248

generated automatically by CompuSyn17. According to the CI values, six groups

249

(three synergistic and three antagonistic groups) were chosen for the further

250

experiments.

251

2.11 Measurement of Reactive Oxygen Species (ROS) Production

fractions),

HEB-LEW+H2O2

(with

five

different

fractions),

252

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

253

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

254

seeded into 6-well plates at 5×105 per well for 24 h and treated with extracts for 12 h, 13


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induced by H2O2 for 30 min, followed by incubation with 5 mM DCF-DA in medium

256

at 37°C for 10 min in the dark. After centrifugation at 1000 rpm for 5 min, the

257

supernatants were removed, and the pellets were resuspended in 1% Triton X-100.

258

The fluorescence was measured by flow cytometry. Flow cytometry was performed

259

using a FACS Calibur (BD biosciences) system with cell quest software. The

260

percentages of cells in different phases of the cell cycle within the GFP-positive

261

population were determined using the software program ModFit.

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2.12 Enzymes activities of SOD, GSH-Px, CAT, the Expression of MDA and Release

263

of LDH

264

Total cell lysates were prepared in RIPA lysis buffer containing protease

265

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

266

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

267

Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT)

268

enzymatic activities, and the level of malondialdehyde (MDA), lactate dehydrogenase

269

(LDH) were determined using colorimetric kits (Beyotime Biotech, Shanghai, China)

270

according to the manufacturer’s protocols.

271

2.13 The Cellular Antioxidant Activity (CAA)

272

The CAA assay was referred to the methods from Kelly and others28 with a little

273

modification. H9c2 cells were seeded at a density of 6 × 104 per well on a 96-well

274

microplate in 100 µL of growth medium. The outside wells of the plate were not used

275

as there was much more variation from them than from the inner wells. Twenty-four

276

hours after seeding, the growth medium was removed and the wells were washed with 14



277

PBS. Triplicate wells were treated for 1 h with 100 µu of pure fruit extracts. And then,

278

wells were washed with 100 µL of PBS. Next, 25 µM DCFH-DA were dissolved in

279

treatment medium with H2O2 (200 µmol/L). And the 96-well microplate was placed

280

into a Fluoroskan Ascent FL plate-reader (Thermo Lab systems, Franklin, MA) at

281

37 °C. Emission at 538 nm was measured with excitation at 485 nm every 5 min for 1

282

h.

283

2.14 Statistical Analysis

284

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

285

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

286

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

287

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

288

considered as statistically significant if P< 0.05.

289

3. Results

290

3.1 The Profiles and Contents of Phytochemicals

291

The TPCs of HEM and HEB were 45.15±5.12 and 32.17±6.51 mg GAE/g DW,

292

the TFCs of HEM and HEB were 18.10±2.68 and 11.06±3.49 mg G3G/100g DW, and

293

the TACs of HEM and HEB were 21.65±0.18 and 15.33±0.03 mg G3G/g DW,

294

respectively (Table 1).

295

According to the UPLC-MS-MS analysis, the main anthocyanins of hydrophilic

296

mulberry and blueberry extracts were identified to cyanidin 3-O-glucoside (peak 1, 4,

297

2*, fragment ions at m/z 285[M-Glc]-, 447 [M-H]-), peonidin 3-O-glucoside (peak

298

4*,fragment ions at m/z 301[M-Glc]-, 463 [M-H]-), petunidin 3-O-glucoside (peak 1*, 15


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fragment ions at m/z 316[M-Glc]-, 478 [M-H]-), delphindin 3-O-glucoside (peak 2, 5,

300

3*, fragment ions at m/z 303[M-Glc]-, 465 [M-H]-), malvidin3-O-glucoside (peak 5*,

301

fragment ions at m/z 331[M-Glc]-, 493 [M-H]-)29-30. The main phenolics of

302

hydrophilic mulberry and blueberry extracts were characterized as chlorogenic acid

303

(peak 3, 6*, m/z 353 [M-H]-), caffeic acid (peak 6, 7*, m/z 179 [M-H]-), rutin (peak 7,

304

8*, m/z 609 [M-H]-), quercetin (peak 8*, m/z 301 [M-H]-), and catechin (peak 9*, m/z

305

289 [M-H]-, Figure S1, Figure S2, Tables 2A and 2B).

306

Moreover, from the profiles of hydrolysis, the anthocyandins of HEM were

307

mainly cyanidin and delphinidin, while petunidin, cyanidin, delphinidin, peonidin,

308

and malvidin were found in HEB (Figure S4, Table 3). On the other hand, the main

309

carotenoids of LEM were β-carotene (764.87±97 µg/mg Extracts) and lycopene

310

(35.67±2.64 µg/mg Extracts), and its TCC was 835.01±31.21 µg/g DW. Lycopene

311

(694.23±5.86 µg/mg Extracts) and β-carotene (127.04±3.67 µg/mg Extracts) were

312

also the main carotenoids of LET and the TCC was 856.48 ± 12.56 µg/g DW (Figure

313

S3, Table 1, Table 3).

314

3.2 Interaction Effects of Different Fractions among Different Fruits Extracts on the in

315

vitro Chemical Based Models

316

The combination index of DPPH and ABTS values (CI75, CI50, CI25) among

317

different fruit mixtures are presented in Tables 4A and 4B. In the ABTS assay, some

318

groups (HEM-LEM: F9/10, HEM-LEW: F9/10, HEB-HEM: F5/10, HEB-LEM: F9/10,

319

HEB-LEW: F9/10) showed lower CI values than other groups. For example, the

320

HEM-LEM (F9/10) group indicated synergistic effects (CI＜1), and the CI values 16



321

were 0.66±0.12 (CI25), 0.82±0.09 (CI50), 0.93±0.05 (CI75), respectively. However,

322

some groups (HEM-HEB: F7/10, HEB-LEM: F3/10 and F5/10, LEM-LEW: F1/10,

323

F3/10, F5/10 and F7/10) showed much higher CI values than other groups. For

324

example, the LEM-LEW (F5/10) group indicated antagonistic effect (CI＞1), and the

325

CI values were 2.14±0.21 (CI25), 2.71±0.13 (CI50), 3.48±0.24 (CI75), respectively.

326

Similarly, in the DPPH assay, some groups (HEM-LEM: F9/10, HEM-HEB:

327

F5/10, HEM-LEW: F9/10, HEB-LEM: F7/10 and F9/10, HEB-LEW: F9/10) showed

328

lower CI values than other groups. For instance, the HEB-LEM (F9/10) group

329

indicated synergistic effects (CI＜1), the CI values were 0.84±0.07 (CI25), 0.89±0.03

330

(CI50), 0.93±0.05 (CI75), respectively. Other groups (HEM-HEB: F7/10, HEB-LEM:

331

F1/10 and F3/10, LEM-LEW: F1/10, F5/10 and F9/10) showed high CI values. For

332

instance, the LEM-LEW (F5/10) group indicated antagonistic effect (CI＞1), and the

333

CI values were 2.24±0.15 (CI25), 2.94±0.08 (CI50), 3.47±0.12 (CI75), respectively.

334

Interestingly, we found that different fractions of fruit combination showed

335

different antioxidant interactions. In the ABTS assay, the CI values of F5/10 group

336

(HEM and HEB) were 0.83±0.09 (CI25), 0.93±0.06 (CI50), 0.99±0.07 (CI75),

337

respectively. However, the values of F7/10 group were 1.28±0.22 (CI25), 1.71±0.19

338

(CI50), 2.00±0.05 (CI75), respectively. It means an increase or decrease in

339

concentrations of one composition in the binary mixtures was found to affect the

340

antioxidant interactions In addition, our previous study7 also found similar

341

phenomenon. In order to further explore the relationship between interaction and

342

different ratios of composition (esp. the hydrophilic and lipophilic contents), the in 17


Page 18 of 52

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343

vitro H2O2-induced H9c2 cell-based models were established.

344

3.3 Interaction Effects of Different Fractions among Different Fruits Extracts on the

345

Relative Cell Viability

346

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

347

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

348

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

349

when the concentrations of HEM and HEB were above 0.9 mg/mL, and the

350

concentrations of LEM and LEW were above 1.0 mg/mL. Therefore, in order to

351

ensure the cell viability after the addition of extracts, the appropriate concentration

352

(0.8 mg/mL) of these four extracts was chosen for the further study.

353

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

354

are shown in Table 5. Six groups (HEM-LEM: F9/10, HEM-LEW: F9/10, HEM-HEB:

355

F5/10, HEB-LEM: F7/10 and F9/10, HEB-LEW: F9/10) showed low CI values

356

(0.63±0.04, 0.94±0.04, 0.67±0.08, 0.71±0.12, respectively), which indicated

357

synergistic effects (CI＜1). While another six groups (HEM-LEW: F1/10, HEB-LEM:

358

F1/10 and F3/10, LEM-LEW: F1/10, F5/10, F9/10) with high CI values (1.94±0.13,

359

1.12±0.08, 1.36±0.12, 1.16±0.07, 2.24±0.17, 1.29±0.09, respectively) showed

360

antagonistic effects (CI＞1).

361

Coincidentally, some groups in the results or MTT showed the same interactions

362

with results of ABTS and DPPH assays and had more significant interactions

363

compared to other combinational groups (Table 4, Table 5). Hence, six groups

364

[HEM-LEW: F9/10 (S1), HEB-HEM: F5/10 (S2), HEB-LEM: F9/10 (S3), 18



365

HEM-LEW: F1/10 (A1), LEW-LEM F5/10 (A2), HEB-LEM: F3/10 (A3)] with

366

significant interactional effects were chosen for the further experiments.

367

3.4 Combination Groups Inhibited the Generation of Intracellular ROS and the

368

Quantification of CAA

369

Intracellular ROS generation is a marker of oxidative stress2. Cells induced by

370

H2O2 exhibited a significant increase of intracellular ROS generation (80.49±0.64,

371

Figure 5) as compared to the control group (23.17±0.29). When the cells were

372

pretreated with the individual extracts (HEM, HEB, LEM, LEW), there was a

373

significant decrease of intracellular ROS generation compared to the H2O2-induced

374

group (Figure 1). For example, the intracellular ROS generation of HEM group

375

(69.30±0.39) was inferior to the H2O2-induced group (80.49±0.64).

376

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

377

showed the synergistical inhibitions on the intracellular ROS generation (Figure 1).

378

For instance, intracelluar ROS generation in S1 group was 48.36±3.28, which was

379

lower than those in HEM (M2=69.30±0.39) and LEW (M2=46.23±0.51) groups

380

(M2=69.76±3.66). Similarly, S2 and S3 showed the synergistic effect. However, some

381

groups showed antagonistic effects with no significant difference (A2, A3, Figure 1).

382

For instance, the ROS generation in A2 group was 72.56±2.48, while the ROS

383

generation in LEW and LEM groups was 72.56±2.48, 72.51±1.93, respectively.

384

In addition, we made a quantification of CAA to detect the changes of ROS in

385

cells within 1 h (Figure 2). The results showed that A1, A2, A3 groups showed

386

significant differences with the individual groups. On the other hand, S1, S2, S3 19


Page 20 of 52

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387

groups showed synergistic antioxidant effects compared to the individual groups.

388

Previous studies indicated that phytochemicals could enhance the expression of

389

various antioxidant defensive enzymes such as catalase (CAT), glutathione peroxidase

390

(GSH-Px), and superoxide (SOD)31 to eliminate ROS. Hence, some antioxidant

391

enzymes (GSH-Px, SOD, CAT) in H2O2-induced H9c2 cells were measured to fully

392

explore the antioxidant interactions.

393

3.5 Effects of Different Ratios among Different Extracts on GSH-Px, SOD, CAT,

394

Activities in H2O2-Induced H9c2 cells

395

For example, the activity of SOD in cells induced by H2O2 was 52.78±1.72 U/mL,

396

which was significant lower than those of other groups which incubated by the

397

individual extracts (P< 0.05, Figure 3).

398

All of S1, S2 and S3 groups showed higher expression of GSH-Px and SOD than

399

the individual groups (Figure 3), which means that these groups showed significant

400

synergistic effects. For examples, the activity of SOD (100.98±2.31 U/mL) in S1

401

group was significantly higher than those in HEM (86.37±4.23 U/mL) and LEW

402

(71.24±3.34 U/mL) groups. The activity of SOD (98.23±1.89 U/mL) in S2 group was

403

significantly higher than HEM (86.37±4.23 U/mL) and HEB (72.31±3.74 U/mL)

404

groups. The S3 group showed the similar trends.

405

On the other hand, A1 and A3 groups showed lower expression of SOD than the

406

individual groups, and A2 group was the only group expressed significant antagonistic

407

effect. The activity of SOD in A2 group (65.32±2.56 U/mL) was significant lower

408

than those of LEM (70.11±2.88 U/mL) and LEW (71.24±3.34 U/mL) groups. 20



409

Meanwhile, the expression of GSH-Px showed the similar trend as SOD.

410

In addition, S1, S2, S3 groups showed lower expression of CAT than the

411

individual groups. And A1, A2, A3 groups showed higher expression than the

412

individual groups (P>0.05). However, all of these groups showed no significant

413

differences in the expression of CAT.

414

3.6 The Levels of MDA and LDH in H2O2-Induced H9c2 Cells

415

From the expression of MDA, all of S1, S2, and S3 groups showed significant

416

lower expression of MDA than the individual groups. For instance, the expression of

417

MDA in S1 group (2.56±0.15 µmol/L) was significantly lower than those in HEM

418

(2.98±0.21 µmol/L) and LEW (3.37±0.22 µmol/L) groups. And all of A1, A2, A3

419

groups showed higher expression of MDA than the individual groups. For example,

420

the expression of MDA in A2 group (3.67±0.18 µmol/L) was significantly lower than

421

those of HEM (3.23±0.15 µmol/L) and LEW (3.37±0.22 µmol/L) groups. In addition,

422

the quantification of LDH showed the same trends. For example, the level of LDH in

423

S2 group was 27.94±0.64, which was significant lower than the individual groups

424

(HEM: 34.31±1.30, HEB: 35.32±0.59). The level of A1 group was 42.42±2.14, which

425

was significant higher than the individual groups (HEM: 34.31±1.30, LEW:

426

32.87±0.35).

427

4. Discussion

428

Primarily, it seems that some interactions existed in these four daily fruit

429

extracts evaluated by these indicators (the results of DPPH, ABTS; the expression of

430

antioxidant enzymes, MDA, ROS; the release of LDH and the quantification of CAA). 21


Page 22 of 52

Page 23 of 52


431

And they appeared some special rules between the different ratios of extracts

432

(hydrophilic and lipophilic compounds) and antioxidant interactions in evidence.

433

4.1 Antioxidant Interactions were Influenced by the Ratios of Hydrophilic and

434

Lipophilic Components in Combinations

435

From the results, in the combinational groups which contained both hydrophilic

436

and lipophilc compounds, when the hydrophilic components were in the majority, the

437

combination groups would tend to show synergistic effects. And when the lipophilic

438

components were in the majority, the combination groups would tend to show

439

antagonistic effects. Principal component analysis (PCA) was used to analyze the

440

main compounds of extracts and antioxidant interactions according to results of UPLC

441

and MTT, while the results of MTT were agree with the chemical based indicators.

442

From the results, those components of combinational groups could be divided into

443

two contributors. The first contained most of the phenolics and flavonoids

444

(contribution rate was 76.45%), and the second contained carotenoids and several

445

phenolics (contribution rate was 12.76%). The total contribution rate was 89.21% of

446

two-dimensional diagram (Figure 4). The synergism groups were in the right of

447

Figure 4B (the hydrophilic components were dominant) while the antagonism groups

448

were in the left of Figure 4B (the lipophilic components were dominant). For instance,

449

the S1 group (HEM-LEW F9/10) and the A1 group (HEM-LEW F1/10) showed

450

different interaction effects while they were combined by same extracts with different

451

ratios. In the S1 group, the HEM (hydrophilic compounds) was in the majority while

452

the LEW (lipophilic compounds) was in the majority in the A1 group. 22



453

Moreover, from our results, some groups which only contained hydrophilic

454

compounds still showed antagonistic effects compared individual groups. For

455

example, the groups (HEM-HEB, F1/10; HEM- HEB, F7/10; HEM-HEB F9/10)

456

showed antagonistic effects with the CI value were 1.24±0.05, 1.38±0.15, 1.35±0.09,

457

respectively, and other groups showed no significant synergistic effects (HEM-HEB

458

F5/10, CI=0.94±0.03, Table 5), although their main compounds were hydrophilic

459

extracts (HEM and HEB). Actually, the hydrophilic compounds may have competitive

460

inhibition of absorption and their absorption may affected by their different

461

structures32. That is to say, the combinational groups may not always show synergistic

462

effects when they only contained hydrophilic compounds, even the hydrophilic

463

compounds were in the majority. Therefore, a single intake of large amounts of

464

hydrophilic compounds without lipophilic ones may not improve the antioxidant

465

capacity in our daily diet.

466

4.2 The Expression of Antioxidant Enzymes May Not Sensitive to the Changes of

467

Antioxidant Activity Caused by Interactions

468

In our results, groups (S1, S2, S3, A1, A2, A3) showed more significant

469

interaction than other groups both in chemical based models and the results of MTT.

470

However, these six groups showed no significant antagonistic effects on the

471

expression of enzymes (SOD, GSH-Px, CAT), although the change of expression on

472

enzymes caused by interactions had the same trend compared with other indicators.

473

Our previous study33 had also found the similar phenomenon.

474

In the first place, the expression of antioxidant enzymes may be not sensitive to 23


Page 24 of 52

Page 25 of 52


475

all the change of antioxidant activity caused by interactions. New findings34 revealed

476

that the antioxidant activity of some flavonoids was not limited to their

477

radical-scavenging activity, but also the antioxidant defense system included

478

antioxidant enzymes (CAT, SOD, GPx). From our results, hydrophilic compounds

479

(polyphenols, flavonoids and anthocyanins) were in the majority of synergistic groups

480

and these groups showed significant synergistic effects on expression of antioxidant

481

enzymes (SOD, GPx). For example, the S1 groups (HEM-LEW F9/10), whose

482

hydrophilic extracts were in the majority, showed significant higher expression of

483

SOD (100.98±2.31 U/mL) and GSH-Px (8.32±1.56 U/mg protein) than the individual

484

groups (HEM and LEW: SOD were 86.37±4.23 U/mL and 71.24±3.34 U/mL,

485

GSH-Px were 6.38±1.24 U/mg protein and 4.97±1.07 U/mg protein, respectively,

486

Figure 3). While other studies35 indicated that carotenoids likely took over the SOD

487

activity, acting firstly in the detoxification chain of superoxide radicals, resulting in its

488

reduced activity, whereas they promoted the CAT activity, acting secondly in the

489

detoxification chain. And carotenoids may chemically interact with superoxide

490

radicals and release hydrogen peroxides by yet unknown redox reaction, which were

491

then detoxified by CAT activity. From our results, lipophilic compounds (carotenoids)

492

were in the majority of antagonistic groups and these groups showed significant

493

antagonistic effects on the results of chemical models and most of indicators on

494

cell-based models, except the expression antioxidant enzymes (SOD, GSH-Px). For

495

example, the A1 group (HEM-LEW F1/10), which had lipophilic extracts in the

496

majority, showed no significant expression of SOD (70.21±1.97 U/mL) and GSH-Px 24



497

(4.76±1.71 U/mg protein) compared with the individual groups (HEM and LEW),

498

whose SOD were 86.37±4.23 U/mL and 71.24±3.34 U/mL, while GSH-Px were

499

6.38±1.24 U/mg protein and 4.97±1.07 U/mg protein, respectively (Figure 3).

500

Hence, when the hydrophilic extracts were in the majority, a small amount of

501

lipophilic extracts may also have less effect on expressions of antioxidant enzymes,

502

which made the interaction (reflected by the expression of enzymes) easier detected.

503

However, when the lipophilic extracts were in the majority, large amounts of

504

carotenoids may affect the expression of antioxidant enzymes and make interaction

505

(reflected by the expression of enzymes) not obvious even though carotenoids can

506

scavenge free radicals. Maybe the expression of antioxidant enzymes showed weaker

507

sensitivity to the antioxidant interactions when lipophilic extracts were in the

508

majority.

509

In addition, from the results of MDA, groups which contained more lipophilic

510

compounds showed significant antagonistic effects (Figure 3). Studies indicated that

511

carotenoids could react with oxygen to produce a carotenoid peroxyl radical, which

512

was capable of acting as a prooxidant and caused the lipid peroxidation, that might be

513

why the greater carotenoid concentrations increased lipid oxidation36 (which can be

514

reflected by the level of MDA). Hence, the level of MDA maybe more sensitive to

515

those groups contained more lipophilic compounds and showed more antagonistic

516

effects compared withthe individual groups. Hence, maybe it is necessary to judge the

517

antioxidant interaction by comprehensive indicators.

518

4.3 Antioxidant Interactions were affected by Different Structures of Carotenoids 25


Page 26 of 52

Page 27 of 52


519

From our results, some groups which contained more LEW showed higher

520

antagonistic effects than groups contained more LEM. For example, on the ABTS

521

models, the CI25, CI50, CI75 on group (HEB-LEM F1/10) were 1.11±0.09,

522

1.13±0.12, 1.24±0.11, respectively (Table 4A). While the CI25, CI50, CI75 on group

523

(HEB-LEW F1/10) were 1.61±0.07, 1.87±0.11, 1.28±0.09, respectively (Table 4A).

524

Although both of these two groups had the same hydrophilic compounds (HEB) in the

525

same ratios, group contained more LEW still have higher CI value which means more

526

significant antagonistic effects than group contained more LEM. Similarly, the same

527

trend was also found in DPPH models. For example, the CI25, CI50, CI75 on group

528

(HEB-LEM F1/10) were 1.32±0.09, 1.47±0.07, 1.58±0.08, respectively (Table 4B).

529

While the CI25, CI50, CI75 on group (HEB-LEW F1/10) were 1.34±0.05, 1.53±0.21,

530

1.63±0.09, respectively (Table 4B).

531

In reality, this phenomenon was also found on cell-based models. For instance,

532

the CI value of the group (HEM-LEM, F1/10) was 1.13±0.05, which was much lower

533

than the group (HEM-LEW, F1/10, CI value was 1.94±0.13), although both of these

534

two groups had the same ratios of hydrophilic compounds (HEM, Table 5).

535

Interestingly, the CI value of the group (HEB-LEM, F1/10) was 1.12±0.08, which was

536

much lower than the group (HEB-LEW, F1/10, CI value was 1.44±0.20), although

537

both of these two groups had the same ratios of hydrophilic compounds (HEB, Table

538

5).

539

From the UPLC analysis (Figure S3, Table 3), β-carotene was in the majority of

540

LEM (764.87±3.97 µg/mg Extracts), while lycopene was in the majority of LEW 26



541

(694.23±5.86 µg/mg Extracts). And the combinational groups contained LEW

542

(lycopene was in the majority) showed more significant antagonistic effects than the

543

combinational groups contained LEM (β-carotene was in the majority).

544

On the one hand, these two kinds of carotenoids had different structures.

545

β-Carotene is a member of the carotenes, which are terpenoids (isoprenoids) and

546

synthesized biochemically from eight isoprene units and thus having 40 carbons. It is

547

distinguished by having beta-rings at both ends of the molecule37. Lycopene is a

548

symmetrical tetraterpene assembled from eight isoprene unit consists entirely of

549

carbon and hydrogen37. And in its natural, its molecule is long and straight,

550

constrained by its system of eleven conjugated double bonds37. Lycopene has longer

551

hydrocarbon chains and more double bond of carbon compared to β-carotene. And in

552

lycopene, all the C=C bonds are coplanar, allowing that the unpaired electrons formed

553

at C-4 to be readily delocalized38. This may be the reason why they showed different

554

antioxidant interactions on chemical models. On the other hand, the different

555

structures of them may influence their different antioxidant on scavenging ROS in

556

cells. Actually, carotenoids are chain-breaking antioxidants and can scavenge ROO

557

by three main mechanisms: electron transfer, allylic hydrogen abstraction and radical

558

addition to the conjugated double bonds system39. Previous study38 indicated that the

559

opening of the β-ionone ring and the chromophore extension (number of conjugated

560

double bonds) of carotenoids seemed to be the factors with the biggest impact on the

561

ROO·(a kind of ROS which formed by hydroperoxides decomposition) scavenging

562

capacity. And other study40 found that β-carotene also exhibited high efficiency in 27


Page 28 of 52

Page 29 of 52


563

preventing the induced-oxidative damage in human erythrocytes than lycopene.

564

Hence, we can possibly indicate that the combinational groups (hydrophilic

565

compounds mixed lycopene) would show more significant antagonistic effects than

566

other groups (hydrophilic compounds mixed β-carotene) because their different

567

structures.

568

4.4 Antioxidant Interactions Provide an Implication for Dietary Fruits

569

Overall, our results still indicated the relationship between different ratios of

570

compounds (lipophilic and hydrophilic) and antioxidant interactions. In recent years,

571

the compatibility (a theory of drug interactions) of traditional Chinese medicine (TCM,

572

esp. many materials from food）has been accepted and becoming popular in Western

573

medicine41. In this theory, antagonism (reducing curative effects of other drugs) and

574

rejection (increasing toxicity of each other) will present if drugs were combined

575

incorrectly41,42. Although, a large number of studies indicated that compound

576

prescriptions often achieve better effects than the single ones in medicines43,41. This

577

study probably put forward the interaction effects (compatibility) on daily fruits in

578

dietary guidance and implication. Hence, we can possibly speculate that certain

579

proportions of fractions among daily fruits are better than the single food for health.

580

This is similar to the compatibility of TCM theory. In addition, although the

581

combinational groups with hydrophilic compounds in the majority showed more

582

synergistic effects than the individual groups, maybe the antioxidant capacity can be

583

enhanced by intake hydrophilic with lipophilic compounds according to proper ratios

584

rather than intake only hydrophilic foods in our daily diet. It is necessary to balance 28



585

the ratios of hydrophilic and lipophlic compounds in our daily food.

586 587 588 589

Acknowledgments This project was funded by the National Natural Science Foundations of China (Grant No.: 31760432 and 31301433).

29


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and Anti-Inflammatory Properties of the Citrus Flavonoids Hesperidin and

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Enzymatic Antioxidant Defences. Dev. Comp. Immunol. 2015, 49 (2),

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Glutathione Induced by ROS in Human Erythrocytes. Life Sci. 2014, 99 (1-2),

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The Rediscovery of Ancient Chinese Herbal Formulas. Phyther. Res. 2004, 18

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Zhou, M.; Hong, Y.; Lin, X.; Shen, L.; Feng, Y. Recent Pharmaceutical

741

Evidence on the Compatibility Rationality of Traditional Chinese Medicine. J.

742

Ethnopharmacol. 2017, 206 (June), 363–375.

743 36



744

37


Page 38 of 52

Page 39 of 52


745

Figure 1. Generation of intracellular ROS in H9c2 cells. M2 means the percentage of ROS level

746

and in cells. HEM-HEB: hydrophilic extracts of mulberry and hydrophilic extracts of blueberry;

747

LEM-LEW: lipophilic extracts of mango and lipophilic extracts of watermelon. F1/10 (HEM-HEB)

748

refers to the quantity of mulberry (1/10) to blueberry (9/10) in the binary mixture. S1: HEM-LEW

749

F9/10, S2: HEM-HEB F5/10, S3: HEB-LEM F9/10, A1: HEM-LEW F1/10, A2: LEM-LEW:

750

F5/10, A3: HEB-LEM: F3/10. Values with different letters in the same column showed significant

751

difference between the H2O2-induced group and extracts-induced groups. (P < 0.05)

752 753 754

38



755

756 757

Figure 2. The peroxyl radical-induced oxidation of DCFH to DCF in H9c2 cells and the inhibition

758

of oxidation by extracts. CAA units can be calculated by this equation: CAA unit = 100-(∫SA⁄ ∫CA)

759

×100. HEM-HEB, hydrophilic extracts of mulberry and hydrophilic extracts of blueberry;

760

LEM-LEW, lipophilic extracts of mango and lipophilic extracts of watermelon. F1/10 (HEM-HEB)

761

refers to the quantity of mulberry (1/10) to blueberry (9/10) in the binary mixture. S1: HEM-LEW

762

F9/10, S2: HEM-HEB F5/10, S3: HEB-LEM F9/10, A1: HEM-LEW F1/10, A2: LEM-LEW:

763

F5/10, A3: HEB-LEM: F3/10. Values with different letters in the same column showed significant 39


Page 40 of 52

Page 41 of 52


764

difference between the H2O2-induced group and extracts-induced groups. (P < 0.05).

765

40



766

767

768 769

Figure 3. Effects of fruit extracts on SOD, GSH-Px, CAT activities and the level of MDA and

770

LDH induced by H2O2in H9c2 cells. HEM-HEB: hydrophilic extracts of mulberry and hydrophilic

771

extracts of blueberry; LEM-LEW: lipophilic extracts of mango and lipophilic extracts of

772

watermelon. F1/10 (HEM-HEB) refers to the quantity of mulberry (1/10) to blueberry (9/10) in

773

the binary mixture. S1: HEM-LEW F9/10, S2: HEM-HEB F5/10, S3: HEB-LEM F9/10, A1:

774

HEM-LEW F1/10, A2: LEM-LEW, F5/10, A3: HEB-LEM: F3/10. Values with different letters in

775

the same column showed significantly difference between the H2O2-induced group and 41


Page 42 of 52

Page 43 of 52


776

extracts-induced groups. (P < 0.05)

777

42



778 779

Figure 4A. The PCA analysis of compounds in combination groups. The compound in the same

780

circle means these compounds may have the similar effects on the combination groups. The

781

greencircle represents the hydrophilic compounds, and the red circle represents the lipophilic

782

compounds.

43


Page 44 of 52

Page 45 of 52


783 784

Figure 4B. PCA analysis of combination groups. F means the different fractions. Three synergistic

785

groups (S1: HEM-LEW F9/10, S2: HEB-HEM F5/10, S3: HEB-LEM F9/10) were represented by filled

786

triangle in different colors. Three antagonistic groups (A1: HEM-LEW F1/10, A2: LEW-LEM F5/10,

787

A3: HEB-LEM F3/10) were represented by solid circular in different colors. The HEM, HEB means

788

the hydrophilic extracts of mulberry and blueberry, respectively. The LEW, LEM mean the lipophilic

789

extracts of watermelon and mango, respectively. F1/10 (HEM-HEB) refers to the quantity of mulberry

790

(1/10) to blueberry (9/10) in the binary mixture.

44



791

Page 46 of 52

Table 1. Main Antioxidant Components and Antioxidant Activities in Fruit Extracts. extracts

TPCb

TFC

TAC

TCC

(mg GAE/g

(mg G3G/100g DW)

(mg /100g

(µg/g DW)

DW)

DW)

HEMa

45.15 ± 5.12

18.10 ± 2.68

21.65± 0.18

-

HEB

32.17 ± 6.51

11.06 ± 3.49

15.33± 0.03

-

LEM

0.26 ± 0.03

-

835.01 ± 31.21

LEW

0.35 ± 0.04

-

856.48 ± 12.56

792

a

793

LEM mean the lipophilic extracts of watermelon and mango, respectively.

794

b

The HEM, HEB means the hydrophilic extracts of mulberry and blueberry, respectively. The LEW,

TPC, total phenolic acid; TFC, total flavonoid acid; TAC, total anthocyanins; TCC, total carotenoids

795

45


Page 47 of 52


796

Table 2A. Hydrophilic Contents of Mulberry. peak

UPLC RT

absorption

[M-H]-(m/z)

MS2(m/z)

tentative identification

(min) wavelength 1,4

11.40

280, 520

477

285

Cyanidin 3-O-glucoside

2,5

13.47

280, 520

465

303

Delphin 3-O-glucoside

3

7.95

280

353

179, 191

Chlorogenic acid

6

14.02

280

179

Caffeic acid

7

14.52

280

609

Rutin

8

21.97

280

301

Quercetin

797 798

Table 2B. Hydrophilic Contents of Bluelberry. peak

UPLC RT

absorption

[M-H]-(m/z)

MS2(m/z)

tentative identification

(min) wavelength 1*

7.241

520

478

316

Petunidin 3-O-glucoside

2*

11.23

520

447

301

Cyanidin 3-O-glucoside

3*

12.90

520

465

303

Delphin 3-O-glucoside

4*

14.08

520

463

301

Peonidin 3-O-glucoside

5*

15.13

520

493

331

Malvidin-3 O-glucoside

6*

8.023

280

353

179, 191

Chlorogenic acid

7*

13.90

280

179

Caffeic acid

8*

14.32

280

609

Rutin

9*

11.59

280

289

245, 179

799

46


Catechin


800

Page 48 of 52

Table 3. Main Contents in the Combination Groups. group number

compound

concentration (µg/mg Extracts)

S1 HEM-LEWa F9/10

S2 HEM-HEB F5/10

S3 HEB-LEM F9/10

cyanidin

287.09

delphinidin

160.34

chlorogenic acid

48.57

caffeic acid

46.27

rutin

91.69

quercetin

18.51

β-carotene

12.70

lycopene

69.42

cyanidin

166.10

delphinidin

147.63

petunidin

29.55

peonidin

144.52

malvidin

55.35

chlorogenic acid

53.27

caffeicacid

102.82

rutin

59.43

quercetin

10.29

catechin

13.32

cyanidin

11.88

delphinidin

105.39

petunidin

53.19

peonidin

260.13

malvidin

99.63

chlorogenic acid

47.30

caffeicacid

138.81

rutin

15.28

47


Page 49 of 52


A1

A2

A3

HEM-LEW F1/10

LEM-LEW F5/10

HEB-LEM F3/10

catechin

23.98

β-carotene

76.49

lycopene

3.57

cyanidin

31.90

delphinidin

17.82

chlorogenic acid

5.40

caffeic acid

5.14

rutin

10.19

quercetin

2.06

β-carotene

114.34

lycopene

624.81

β-carotene

445.96

lycopene

364.95

cyanidin

3.96

delphinidin

35.13

petunidin

17.73

peonidin

86.71

malvidin

33.21

chlorogenic acid

15.77

caffeic acid

46.27

rutin

5.09

catechin

7.99

β-carotene

535.41

lycopene

24.97


801

a

802


803

b

804

mixture.

F1/10 (HEM-HEB) refers to the quantity of mulberry (1/10) to blueberry (9/10) in the binary

48



805 806

Page 50 of 52

Table 4A. Combination Index (CI) and Synergistic or Antagonistic Effects of Fruit Extracts on ABTS Models. Fruit extractsa

Fractionsb F1/10

HEM-LEM

HEM-HEB

HEM-LEW

HEB-LEM

HEB-LEW

LEM-LEW

CI25c 1.00±0.12

CI50 ad a

CI75 b

1.71±0.09b

a

1.56±0.08

2.26±0.12a

1.29±0.11

F3/10

1.11±0.13

F5/10

1.07±0.07a

1.47±0.05ab

2.04±0.10a

F7/10

0.87±0.07b

0.95±0.10c

1.02±0.06c

F9/10

0.66±0.12c

0.82±0.99d

0.93±0.05cd

F1/10

1.14±0.21ab

1.25±0.08b

1.38±0.12b

F3/10

0.91±0.14

bc

c

0.96±0.09

1.22±0.11b

F5/10

0.83±0.09c

0.93±0.06c

0.99±0.07c

F7/10

1.28±0.22a

1.71±0.19a

2.00±0.05a

F9/10

1.35±0.14a

1.28±0.11b

1.23±0.08b

F1/10

1.87±0.05

a

b

1.53±0.12

1.24±0.08c

F3/10

1.77±0.09ab

1.28±0.10c

0.92±0.12d

F5/10

1.61±0.23b

1.80±0.09a

2.02±0.21a

F7/10

1.03±0.08c

1.37±0.09bc

1.84±0.11b

F9/10

0.65±0.13d

0.82±0.12d

0.93±0.08d

F1/10

1.11±0.09

b

b

1.13±0.12

1.24±0.11c

F3/10

1.24±0.13a

1.71±0.09a

2.50±0.21a

F5/10

1.25±0.07a

1.80±0.13a

2.64±0.09a

F7/10

0.80±0.05c

1.14±0.09b

1.66±0.10b

F9/10

0.82±0.05

c

c

0.89±0.10

0.99±0.05d

F1/10

1.61±0.07a

1.87±0.11a

1.28±0.09b

F3/10

0.92±0.08b

1.14±0.12b

2.55±0.09a

F5/10

0.93±0.09b

1.15±0.10b

2.55±0.21a

F7/10

0.61±0.08d

0.82±0.09c

0.94±0.17c

F9/10

0.74±0.13

c

c

0.86±0.09

0.95±0.05c

F1/10

1.12±0.13c

1.97±0.22c

2.06±0.13c

F3/10

2.14±0.09ab

2.80±0.12b

3.68±0.14b

F5/10

2.14±0.21ab

2.71±0.13b

3.48±0.24b

F7/10

2.74±0.13a

3.32±0.09a

6.44±0.23a

F9/10

c

c

3.40±0.22b

1.09±0.08

1.91±0.12


807 808

a

809 810 811 812 813 814

b

815 816 817

Values with different letters (a, b, c) in the same column showed significantly difference with each other (P< 0.05)


F1/10 (HEM-HEB) refers to the quantity of mulberry (1/10) to blueberry (9/10) in the binary mixture.

c

The CI value (CI25, CI50, CI75) was calculated by Compusyn software (CI25, CI50, CI75, which

means the CI value at the inhibition of 25%, 50%, 75%, respectively). When the CI＞1, means the antagonistic effects. When CI＜1, means the synergistic effects. When the CI=1, means the additive effects. d

49


Page 51 of 52


818 819

Table 4B. Combination Index (CI) and Synergistic or Antagonistic Effects of Fruit Extracts on DPPH Models. Fruit extractsa

Fractionsb F1/10

HEM-LEM

HEM-HEB

HEM-LEW

HEB-LEM

HEB-LEW

LEM-LEW

CI25c 1.23±0.11

CI50 ab bc

CI75 b

1.64±0.13b

a

1.58±0.12

2.03±0.22a

1.21±0.09

F3/10

1.12±0.09

F5/10

1.03±0.08b

1.25±0.05b

1.47±0.11b

F7/10

1.36±0.06a

1.23±0.09b

0.98±0.10c

F9/10

0.63±0.13d

0.85±0.09b

0.89±0.11c

F1/10

1.24±0.21b

1.37±0.06b

1.45±0.08b

F3/10

1.12±0.04

c

c

1.15±0.09

1.20±0.13c

F5/10

0.74±0.09d

0.82±0.12d

0.94±0.14d

F7/10

1.38±0.20a

1.67±0.21a

1.90±0.09a

F9/10

1.35±0.11a

1.25±0.09bc

1.20±0.05c

F1/10

1.94±0.11

a

a

1.91±0.04

2.21±0.09a

F3/10

1.68±0.20b

1.83±0.11ab

1.97±0.09b

F5/10

1.23±0.04c

1.51±0.05b

1.85±0.11bc

F7/10

0.96±0.12cd

0.98±0.05c

1.04±0.07c

F9/10

0.67±0.08d

0.74±0.11d

0.88±0.09d

F1/10

1.32±0.09

a

ab

1.58±0.08ab

F3/10

1.26±0.11ab

1.56±0.09a

1.69±0.12a

F5/10

1.04±0.05b

1.17±0.13b

1.20±0.14b

F7/10

0.71±0.09d

0.84±0.12c

0.96±0.11c

F9/10

0.84±0.07

c

c

0.89±0.03

0.93±0.05c

F1/10

1.34±0.05a

1.53±0.21a

1.63±0.09a

F3/10

0.93±0.09b

1.14±0.11bc

1.43±0.10b

F5/10

0.98±0.07b

1.23±0.13b

1.37±0.08bc

F7/10

0.62±0.12d

1.00±0.20bc

1.31±0.13bc

F9/10

0.82±0.08

c

c

0.93±0.06

0.95±0.08c

F1/10

1.16±0.08c

1.95±0.13b

2.08±0.12bc

F3/10

1.18±0.09c

0.95±0.15d

0.87±0.09de

F5/10

2.24±0.15a

2.94±0.08a

3.47±0.12a

F7/10

1.04±0.06c

1.34±0.07c

1.47±0.06c

F9/10

b

b

2.46±0.12b

1.29±0.07

1.47±0.07

1.93±0.08


820 821

a

822 823 824 825 826 827

b

828 829 830




c



50



831

Page 52 of 52

Table 5. Combination Index (CI) and Synergistic or Antagonistic Effects of Fruit Extracts. Fruit extractsa

Fractionsb F1/10

HEM-LEM

HEM-HEB

F3/10

c

1.36±0.12a

＞1

F9/10

0.63±0.04de

＜1

F1/10

1.24±0.05

b

＞1

1.12±0.11

c

＞1

F5/10

0.94±0.03

cd

＜1

F7/10

1.38±0.15a

＞1

F9/10

1.35±0.09

a

＞1

1.94±0.13

a

＞1

F3/10

=1

F3/10

1.68±0.21

b

＞1

F5/10

1.23±0.07c

＞1

F7/10

0.96±0.06d

＜1

F9/10

e

＜1

b

＞1

a

＞1

0.67±0.08 1.12±0.08

F3/10

1.36±0.12

F5/10

1.04±0.11b

F7/10

d

＜1

0.84±0.14

c

＜1

F1/10

1.44±0.20

a

＞1

F3/10

0.93±0.11b

＜1

F5/10

b

=1

de

＜1

c

＜1

F1/10

1.16±0.07

bc

＞1

F3/10

1.18±0.04bc

＞1

F5/10

2.24±0.17

a

＞1

1.04±0.08

c

=1

b

＞1

F7/10 F9/10 LEM-LEW

＞1

1.12±0.10

1.03±0.08

F9/10 HEB-LEW

＞1

b

F7/10

F1/10 HEB-LEM

1.13±0.05

Synergistic or antagonistic effect bd

F5/10

F1/10 HEM-LEW

CIc

F7/10 F9/10

0.71±0.12

0.98±0.06 0.62±0.09

0.82±0.13

1.29±0.09

=1

832 833 834 835 836 837 838 839

a

840 841



LEM mean the lipophilic extracts of watermelon and mango, respectively. b


c



51


Daily Dietary Antioxidant Interactions Are Due to ... - ACS Publications

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