Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin

Subscriber access provided by - Access paid by the | UCSB Libraries

Bioactive Constituents, Metabolites, and Functions

Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin, and 5-Demethyltangeretin from Citrus Peel in Saccharomyces cerevisiae Meiyan Wang, Dan Meng, Peng Zhang, Xiangxing Wang, Gang Du, Charles Brennan, Shiming Li, Chi-Tang Ho, and Hui Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00509 • Publication Date (Web): 11 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin,

2

and 5-Demethyltangeretin from Citrus Peel in Saccharomyces cerevisiae

3

Meiyan Wang,†,⊥ Dan Meng,†,⊥ Peng Zhang,†,‡ XiangxingWang,† Gang Du,† Charles

4

Brennan,†,§ Shiming Li,*,‡,ǁ Chi-Tang Hoǁ and Hui Zhao*,†

5

†

6

Tianjin University of Commerce, Tianjin, China.

7

‡

8

Chemical Engineering, Huanggang Normal University, Hubei, China.

9

§

Tianjin Key Laboratory of Food and Biotechnology, School of Biotechnology and Food Science,

Hubei Key Laboratory for Processing & Application of Catalytic Materials, College of Chemistry &

Centre for Food Research and Innovation, Department of Wine, Food and Molecular Bioscience,

10

Lincoln University, Lincoln 7647, New Zealand.

11

ǁ

12

Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA.

⊥

Equal contribution authors.

13

*Correspondence should be addressed to:

14

Hui Zhao, Ph.D.

15

School of Biotechnology and Food Science, Tianjin University of Commerce

16

No. 409 Guangrong Rd., Building IV 408

17

Tianjin, Tianjin 300134, China

18

E-mail: [email protected] (H. Zhao)

19

Shiming Li, Ph.D.

20

Department of Food Science, Rutgers University

21

New Brunswick, NJ 08901, USA

22

E-mail: [email protected] (S. Li) 1

ACS Paragon Plus Environment


23

ABSTRACT: Aging and oxidative-related events are closely associated with the

24

oxidative damages induced by excess reactive oxygen species (ROS). The

25

phytochemicals nobiletin (NBT) and tangeretin (TAN), and their 5-demethylated

26

derivatives 5-demethylnobiletin (5-DN) and 5-demethyltangeretin (5-DT) are the

27

representative polymethoxyflavones (PMFs) compounds found in aged citrus peel.

28

Although the health benefits from PMFs due to their antioxidant activities have been

29

well documented, a systematic assessment regarding the antioxidation process of

30

PMFs is still lack of attention. Here, we investigated the effects of the four PMFs

31

subjected to oxidative stress including hydrogen peroxide, carbon tetrachloride, and

32

cadmium sulfate using an emerging model organism Saccharomyces cerevisiae. As

33

expected, all of the four PMFs exhibited improved cellular tolerance with decreasing

34

lipid peroxidation and ROS. Furthermore, by using the mutant strains deficient in

35

catalase, superoxide dismutase, or glutathione synthase, NBT, 5-DN, and TAN appear

36

to contribute to the increased tolerance by activating cytosolic catalase under CCl4,

37

while the antioxidant protection conferred by 5-DT against H2O2and CdSO4 seems to

38

require cytosolic catalase and glutathione respectively. However, the involvement of

39

Ctt1 and Sod1 is achieved neither by decreasing lipid peroxidation nor by scavenging

40

intracellular ROS according to our results. In addition, a comparison of antioxidant

41

capability of the four PMFs was conducted in this study. In general, this research tries

42

to explore the antioxidant mechanism of PMFs in Saccharomyces cerevisiae, hoping

43

to provide an example for developing more efficacious dietary antioxidants to battle

44

against oxidative- or age-related illness. 2


Page 2 of 34

Page 3 of 34


45

KEYWORDS: PMFs, antioxidant, lipid peroxidation, ROS, Sacharomyces

46

cerevisiae

3



Page 4 of 34

47

INTRODUCTION

48

Reactive oxygen species (ROS), including superoxide radical, hydroxyl radical and

49

hydrogen peroxide, are usually formed as normal by-products of cellular metabolism.

50

As key signalling molecules, ROS play significant roles in the complex signalling

51

network from bacteria to mammalian cells.1,2 However, excess ROS arising from

52

exogenous environment as well as endogenous oxidative metabolism may lead to

53

oxidative stress and further result in protein and lipid peroxidation and DNA

54

damage.3-5 A free radical theory was proposed to elucidate aging and age-related

55

disorders by Dr. Harman as early as 1956.6,7 Since then, numerous researchers have

56

confirmed that cellular and organismal decline induced by oxidative damage are

57

positively correlated with the increase of ROS level.8 Therefore, minimizing

58

macromolecular damage caused by ROS might be an effective strategy not only for

59

slowing aging but also for combating age-related diseases such as cancer, Alzheimer

60

and cardiovascular disease.

61

Given the important role of ROS plays in oxidative damages and age-related

62

maladies, attempts in exploring novel antioxidant molecules to scavenge ROS have

63

proliferated.9-12 Of note, aged tangerine peels, namely “chenpi”, which are rich in

64

polyphenols, flavonoids and carotenoids, were reported as a representative

65

antioxidant.13,14 Glycosides, including hesperidin and naringin, are a class of

66

polymethoxyflavones (PMFs) existing in the peels of citrus genus. Another class of

67

PMFs in orange peels is O-methylated aglycones of flavones, represented by nobiletin

68

(5,6,7,8,3’,4’-hexamethyoxflavone,

NBT) 4


and

tangeretin

Page 5 of 34


69

(5,6,7,8,4’-pentamethoxyflavone,TAN), which have been documented to exhibit

70

various

71

anti-carcinogenic17, anti-diabetic properties18, as well as antioxidant effects.19,20 PMFs

72

are capable of entering into the membrane interface and trapping aqueous free radicals

73

to form stable phenoxy radicals, and therefore prevent the initiation of lipid

74

peroxidation.21 As a typical structure of PMFs, the phenyl benzopyrone group is

75

considered as a key determinant of antioxidant activity of PMFs.22,23

bioactivities,

such

anti-atherogenic15,

as

anti-inflammatory16,

76

Although many studies have been performed to characterize the antioxidant

77

properties of PMFs, the molecular mechanisms of how they function in organism

78

remain unclear. Saccharomyces cerevisiae can be effectively controlled to make

79

modifications such as gene mutation or disruption, which facilitates the identification

80

of target genes related to oxidative stress or response pathways.24 Furthermore,

81

because of the behaviour of similar antioxidants,25 S. cerevisiae is considered as a

82

powerful eukaryotic model to study the molecular mechanisms associated with

83

oxidative stress resistance. In this work, four natural citrus PMFs, NBT and TAN and

84

their

85

(5-hydroxy-6,7,8,3’,4’-pentamethoxyflavone,

86

(5-hydroxy-6,7,8,4’-tetramethoxyflavone, 5-DT), were selected to explore the

87

mechanism by which these four citrus PMFs protect S. cerevisiae cells from oxidative

88

damage imposed by hydrogen peroxide (H2O2), carbon tetrachloride (CCl4) or

89

cadmium. In addition, we investigate whether 5-methyl substitute was involved in the

90

antioxidant action of PMFs by comparing the bioactivities of NBT and TAN with

corresponding

demethylated

counterparts 5-DN)

5


and

5-demethylnobiletin 5-demethyltangeretin


Page 6 of 34

91

their 5-demethylated counterparts, respectively. Overall, this research provided novel

92

insights into the molecular mechanisms relevant to antioxidant protection

93

MATERIALS AND METHODS

of PMFs.

94

Chemicals and reagents. PMFs including NBT, TAN, 5-DN and 5-DT (Figure 1)

95

were prepared as our previous report.26 All other chemicals were purchased from

96

Sigma-Aldrich (St. Louis, MO, USA).

97

S. cerevisiae strains, culture media, and growth conditions. Wild-type (WT)

98

strain BY4741 (Matα his3∆1 leu2∆0 met15∆0 ura3∆0)27 was obtained from

99

EUROSCARF (European S. cerevisiae Archive for Functional Analysis, Institute of

100

Molecular Biosciences Johann Wolfgang Goethe-University Frankfurt, Germany). Its

101

isogenic mutants ctt1∆, sod1∆ and gsh1∆, harboring the gene CTT1, SOD1 or GSH1

102

respectively, interrupted by gene KanMX4, were generously gifted from Prof. Pereira

103

(Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de

104

Janeiro). Stocks of yeast strains were maintained on solid 2% YPD (1% yeast extract,

105

2% glucose, 2% peptone and 2% agar, plus 0.02% geneticine for mutant strains).

106

Yeast cells were grown on liquid 2% YPD medium in an orbital shaker at 28 °C/160

107

rpm. Growth of yeast cells was monitored by optical density at 600 nm (OD600).

108

Oxidative stress conditions. Yeast cells (50 mg) at the first exponential phase

109

growing on 2% YPD were exposed to oxidants (10 mM CCl4, 2 mM H2O2, or 2.5 mM

110

CdSO4) at 28 °C/160 rpm for 1 h, to build an oxidative stress damage yeast model.28,29

111

Cytotoxicity assay of PMFs. Yeast cells at the mid-long phase (OD600 = 1.0) were

112

10-fold diluted to a density of 1.0 ×106 cells/mL with fresh medium containing or not 6


Page 7 of 34


113

PMFs (25 µg/mL, 50 µg/mL or 100 µg/mL), and incubated at 28 °C/160 rpm for 2 h.

114

Then, the diluted yeast cells were spotted adjacently on YPD agar plates. After 72 h of

115

incubation at 28 °C, the number of the colonies was counted and calculated.

116

Cytotoxicity of PMFs in this work was expressed as viability based on the literature.30

117

Tolerance analysis. Tolerance analysis was conducted in a similar procedure as for

118

cytotoxicity assay with small modifications. After pre-treatment with, or without,

119

PMFs at 28 °C/160 rpm for 2 h, yeast cells were exposed to oxidative stress condition

120

for 1 h. The tolerance of cells was calculated and expressed as survival rates in a same

121

method with above cytotoxicity test.

122

Detection of lipid peroxidation. The level of lipid peroxidation was measured in

123

yeast cells exposed to oxidative stress with 2.5 µL of 50 µg/mL PMFs pre-treated or

124

not. Cells cooled on ice were harvested by centrifugation for 2 min at 4 °C/6000 rpm,

125

and washed three times with distilled Millipore purified water. The pellets were

126

resuspended in 500 µL of 0.1 g/mL trichloroacetic (TCA) in the absence of glass

127

beads, and then cells lysis were performed by using vortex with 6 cycles of 20 s

128

agitation. Extracts were subsequently cooled on ice for 20 s and then centrifuged at

129

6000 rpm for 2 min. The supernatant was mixed with 100 µL of 0.1 M EDTA and 600

130

µL of 0.01 g/mL thiobarbituric acid (TBA). The mixture was incubated in a boiling

131

water bath for 20 min and then cooled, the absorbance was detected at 532 nm.31 The

132

results were shown as a ratio between absorbance of stressed, pre-adapted or not with

133

PMFs, and non-stressed cells.

134

Determination

of

intracellular

oxidation.

The

7


oxidant-sensitive

probe


135

2′7′-dichlorofluorescein diacetate (H2DCF-DA) was utilized to evaluate the level of

136

intracellular oxidation.32 A fresh 5 mM stock solution of H2DCF-DA was added to the

137

culture diluted to a concentration of 10 µM and incubated with shaking for 15 min at

138

28 °C to allow uptake of the probe. Subsequently, half of the culture was exposed

139

directly to oxidative stress, while the other part was treated with 2.5 µL of 50 µg/mL

140

PMFs for 2 h and, thereafter, exposed to oxidative stress. Cell extracts were prepared

141

as previous report presented by Pereira et al.33 The fluorescence was measured on a

142

Photo Technology International (PTI) spectrofluorimeter at an excitation/emission

143

wavelength of 504 nm/524 nm. The fluorescence in cells that had not been exposed to

144

oxidative stress was used as a control. The results were exhibited as a ratio between

145

H2DCF fluorescence of exposed to stressors, pre-adapted with PMFs or not, and

146

non-stressed yeast cells.

147

Statistical analysis. The experiments were conducted in triplicate and results were

148

expressed as mean value ± standard deviation (SD). Statistical Package for Social

149

Science 12.0 (SPSS 12.0) for Windows was employed to analyze data in this work.

150

The mean difference was determined by Tukey’s multiple comparison test at p < 0.05.

151

To evaluate the level of lipid peroxidation and intracellular oxidation, we compared

152

the homogeneity between stressed and non-stressed cells of each strain at p < 0.05.

153

The statistically different results were represented by different letters.

154

RESULTS AND DISCUSSION

155

Cytotoxicity assay of four PMFs. To ensure the elimination of direct toxic effects

156

on yeast cells, we investigated if the treatment of NBT, 5-DN, TAN, or 5-DT would 8


Page 8 of 34

Page 9 of 34


157

kill S. cerevisiae cells. The WT cells were directly exposed to four PMFs for 2 h, and

158

then cell survival rate was measured. The results (Figure 2) showed that cells

159

continued to reach 100% tolerance after treatment with four PMFs under the

160

concentrations of 25 µg/mL, 50 µg/mL and 100 µg/mL respectively, which indicated

161

that four PMFs in the range of 25-100 µg/mL were all nontoxic for the WT strain

162

BY4741 and can be used to study the antioxidant mechanisms of PMFs.

163

Tolerance analysis of S. cerevisiae cells pre-treated with PMFs against

164

oxidative damage. The antioxidant property of NBT, 5-DN, TAN, and 5-DT were

165

assessed by exposing S. cerevisiae cells, pre-adapted or not with 2.5 µL of 50 µg/mL

166

PMFs, to 10 mM CCl4, 2 mM H2O2, or 2.5 mM CdSO4. Compared with CCl4 and

167

CdSO4, 2 mM H2O2 was much more toxic for all the strains (Figure 3). This result

168

was predictable since H2O2 produces the most virulent and highly reactive hydroxyl

169

radical against the organisms. The viabilities of the WT strain imposed by all stresses

170

were significantly improved when pre-treated with PMFs, which demonstrated that

171

NBT, 5-DN, TAN, and 5-DT reliably protected WT yeast cells from oxidative

172

damage. Previous research suggested that an antioxidant might be associated with the

173

protection mechanism if a mutant strain deficient in a specific antioxidant system

174

could not acquire tolerance after the adaptive treatment.28 Therefore, the mutant

175

strains sod1∆, ctt1∆, and gsh1∆ were employed to determine whether superoxide

176

dismutase, catalase, or glutathione is connected with the mechanism of acquisition of

177

tolerance.

178

Under oxidative stress induced by 10 mM CCl4, the viabilities of S. cerevisiae cells 9



179

previously adapted with PMFs or not were depicted in Figure 3A. 5-DT was observed

180

to increase the tolerance of all mutant strains to CCl4, signifying that Ctt1, Sod1 and

181

GSH are all not necessary for this adaptive treatment. By contrast, the protection

182

achieved by NBT, 5-DN, or TAN against CCl4 appeared to need the cytosolic catalase,

183

since the survival of ctt1∆ strain was not improved when treated with three of the

184

PMFs. It is noteworthy that 5-DT conferred the ctt1∆ strain the strongest ability to

185

defend CCl4 stress by increasing the viability from 52.7% to 89.2%, which revealed

186

that the treatment with 5-DT reduced the damage of CCl4 to ctt1∆ strain. Interestingly,

187

we found that the mutant strain deficient in Ctt1 acquired a higher tolerance to CCl4

188

stress than the WT strain. This could be correlated with super expression of other

189

antioxidant systems as a compensation of a deficiency in one antioxidant system.34,35

190

A similar phenomenon was observed in ctt1∆ strain stressed with 2.5 mM CdSO4

191

(Figure 3C), which could be explained as well.

192

The survival rates of yeast cells stressed with 2 mM H2O2 or 2.5 mM CdSO4 are

193

displayed in Figure 3B and 3C, respectively. After pre-adaption with 5-DT, neither

194

ctt1∆ strain exposed to H2O2 nor gsh1∆ strain stressed with CdSO4 was found to

195

improve cell viability. This result suggested that cytosolic catalase and glutathione

196

may be involved in the antioxidant protection afforded by 5-DT under H2O2 and

197

CdSO4, respectively. On the other hand, all Ctt1, Sod1 and GSH seemed to be

198

nonessential to the protection induced by NBT, 5-DN, or TAN against both H2O2 and

199

CdSO4, as the survivals of all tested strains were apparently enhanced in statistics

200

when pre-treating with above three PMFs. 10


Page 10 of 34

Page 11 of 34


201

In addition, we compared the antioxidant activities of four PMFs according to

202

Figure 3. NBT was found to perform similarly with TAN in protecting yeast cells

203

from all oxidant stresses. However, the increasing tolerances provided by NBT and

204

5-DN (or TAN and 5-DT), were unpredictable and changed with the strains and the

205

stresses designed in the experiment.

206

Detection of lipid peroxidation levels. Extensive studies have revealed that a

207

critical target of free radical attack is the membrane proteins, leading to lipid

208

peroxidation, cell leakage and death.36,37 In theory, all the stressors including CCl4,

209

H2O2, or CdSO4 have the abilities of attacking against the cell membrane and lead to

210

lipid peroxidation. Lipid peroxidation was assessed to determine whether the four

211

PMFs were responsible for their antioxidant protective actions by reducing the levels

212

of membrane peroxidation. As expected, all of the stresses increased the levels of

213

lipid peroxidation of WT strain (Figure 4). H2O2 produced the most aggressive stress

214

to the cell membranes by tripling the lipid peroxidation levels (Figure 4B), which is in

215

agreement with the lower survival rates depicted in Fig. 2. In contrast, CCl4 (Figure

216

4A) and CdSO4 (Figure 4C) generated a modest increase in lipid peroxidation by

217

giving an enhancement of 130% and 50%, respectively. Research shows that PMFs

218

can be preferentially combined into membrane lipid bilayers and served as a hydrogen

219

donor, trapping free radicals, and further inhibiting the formation of lipid radicals.38,39

220

Consistent with this, all lipid peroxidation damages were alleviated by four PMFs,

221

which implied that protection achieved by the four PMFs against membrane oxidation

222

seems to contribute to improved cell viabilities. The greatest reduction of lipid 11



223

oxidation levels was reached during CCl4 exposure, especially pre-treating cells with

224

5-DN, nearly offsetting the increase of lipid oxidation under CCl4 (Figure 4A).

225

Besides, the lipid peroxidation could be completely suppressed by four PMFs in

226

Cd2+-induced cells (Figure 4C). Consistent with the observation, none of the mutant

227

strains related to redox genes showed the main response to the PMFs treatment. This

228

might hint that the PMFs′ solution of lipid peroxidation induced by the stressor

229

occurred mainly at the cell membrane although the further evidence needs to be

230

investigated.

231

Furthermore, considering the vastly different roles of Ctt1, Sod1 and GSH played

232

in the mechanism of acquisition of increased tolerance to environmental stresses, both

233

ctt1∆ strain and sod1∆ strain were taken as the representatives to study the

234

involvement of catalase and superoxide dismutase in lipid peroxidation. However, the

235

behaviour of both mutant strains was similar to that of the WT strain. In both mutants,

236

the four PMFs could well relieve the membrane peroxidation caused by all stresses,

237

although the ctt1∆ strain had not acquired tolerance under CCl4 with NBT, 5-DN or

238

TAN adaptation or under H2O2 with 5-DT treatment. These results signify that neither

239

Ctt1 nor Sod1 is correlated to the protection against lipid oxidation conferred by four

240

PMFs.

241

Interestingly, cells adapted with NBT (or TAN) was found to exhibited a relatively

242

higher lipid peroxide levels than treated with its 5-demethylated counterparts 5-DN

243

(or 5-DT), indicating that citrus PMFs with 5-demethylated structure may be

12


Page 12 of 34

Page 13 of 34


244

beneficial to cope with lipid peroxidation, and providing a new clue to identify and

245

synthesize the antioxidants.

246

Analysis of intracellular oxidation. The consequent accumulations of ROS result

247

in oxidative damages and cell death, which was reported to be associated with

248

oxidative- or various age-related disorders.40 Many of the bioactivities reported to be

249

present in the PMFs have been achieved by scavenging radicals.41,42 The level of

250

intracellular oxidation was evaluated by using the fluorescent probe H2DCF-DA, in

251

order to investigate whether reduction of intracellular ROS contribute to the increase

252

in tolerance achieved by four PMFs. H2DCF-DA is widely utilized to measure the

253

intracellular ROS level as, once inside the cell, it is susceptible to ROS attack and

254

then yields amore fluorescent compound.43 The level of ROS was not detected to rise

255

when exposing cells to 2.5 mM CdSO4 for 1 h, but CdSO4 did induce lipid

256

peroxidation in this study, which implies that the toxicity of Cd2+ is related to the

257

destruction of the membrane. Thus, the stresses used in ROS analysis, summarized in

258

Figure 5, were only CCl4 and H2O2.

259

It is obvious that the ROS levels dramatically increased after exposure of WT strain

260

to H2O2 or CCl4 (Figure 5). Especially imposed by H2O2, ROS in WT cells enhances

261

almost 15-folds (Figure 5B), which can be linked to the higher sensitivity exhibited by

262

H2O2-stressed cells (Figure 3B). However, the high ROS levels induced by both

263

stresses are all decreased after treatment with four PMFs, and the alleviation is much

264

more pronounced under CCl4. Among of which, the level of ROS formed in response

265

to CCl4 in WT strain was almost 4-fold lower after 5-DN adaption, demonstrating that 13



266

5-DN is well capable of resisting free radical damage (Figure 5A).

267

The ROS levels in ctt1∆ and sod1∆ strains were concurrently detected and the

268

results are presented in Figure 5. Akin to what occurred in the tests of lipid

269

peroxidation, all four PMFs alleviated the intracellular oxidation of both stressed

270

mutants, which indicates that neither Ctt1 nor Sod1 was involved in scavenging

271

intracellular ROS conferred by four PMFs under CCl4 or H2O2. After treatment with

272

the four different PMFs, the increased ROS caused by CCl4 or H2O2 was down to a

273

similar level in all strains, which reveals that NBT, 5-DN, TAN, and 5-DT share

274

roughly the same abilities in scavenging intracellular ROS. In addition, we found that

275

the increase of ROS level induced by H2O2 in mutant deficient in Ctt1 or Sod1 was

276

lower than in WT strain (Figure 5B). This is no surprise since a deficiency in one

277

antioxidant system might be overcome by compensation of the remaining defence

278

system. 34,35

279

The correlation research indicated that oxidative damages resulted from an

280

overload of ROS were involved in aging as well as in the onset and evolution of over

281

a hundred diseases.28 Accordingly, biomolecules become less prone to oxidation

282

under a lower level of ROS. Previous research has revealed that restraining DNA

283

oxidation appeared to contribute to a reduction in cancer, while the prevention of

284

low-density protein oxidation was beneficial to protect against Alzheimer.44,45 In this

285

study, four PMFs alleviated oxidative damages by reducing intracellular oxidation as

286

well as lipid peroxidation, which could explain why the survival rates of yeast cells

287

were improved when preadapted with these PMFs. 14


Page 14 of 34

Page 15 of 34


288

According to our results, all the four PMFs investigated here can improve the

289

tolerance of yeast cells to oxidative conditions, and the significant antioxidant

290

function may be related to their capacities to diminish the level not only of lipid

291

peroxidation but also of intracellular ROS. More importantly, because signalling

292

pathways involved in the oxidative stress response are highly conserved in yeast and

293

human46,47, S. cerevisiae has been well developed for high-throughput screening of

294

chemical inhibitors of human proteins48,49. Hence, the yeast model utilized here

295

provides reliable insight for assessing the antioxidant activity of dietary natural

296

products including PMFs.

297

Previous

structure–activity

relationship

studies

suggested

that

298

demethylation-modified analogues of the methoxy group at the C-5 position on the A

299

ring of NBT and TAN have more profound bioactivities including inhibiting cancer

300

and inflammation50,51. Indeed, compounds 5-DN and 5-DT were also found stronger

301

ability to reduce the lipid peroxidation damage than their corresponding parent

302

counterparts NBT and TAN. On the other hand, a previous report showed that the

303

increased tolerance conferred by different natural antioxidants may need various free

304

radical scavenging enzymes28. As expected, our results indicate that Ctt1 may be

305

involved in the increased tolerance afforded by NBT, 5-DN, or TAN under CCl4,

306

while Ctt1 and GSH appeared to be respectively associated with antioxidant

307

protection of 5-DT against CCl4 and CdSO4.

308

Collectively, our current study suggested a fundamental structure–activity

309

relationship concerning antioxidant activity of the four natural PMFs in S. cerevisiae 15



310

model. We hope our data will provide enlightenment regarding PMFs for the coming

311

studies in animal model and human wellness. More importantly, the findings

312

indicated that NBT, 5-DN, TAN and 5-DT afforded outstanding antioxidant capacity

313

and free radical scavenging potential, and might be promising candidates as dietary

314

natural products in the treatment of oxidative- or age-related diseases.

315

CONFLICT OF INTEREST

316

The authors declare no conflicts of interest.

317

ACKNOWLEGEMENTS

318

We appreciated Prof. Pereira (Laboratório de Investigação de Fatores de Estresse

319

(LIFE), Departamento de Bioquímica, Instituto de Química, Universidade Federal do

320

Rio de Janeiro, Rio de Janeiro, RJ, Brazil 21941-909) for his generous gifts of the

321

mutant strains which are key to our current research. This study was supported by

322

National Natural Science Foundation of China (Grant No. 31571832, 31701172),

323

Tianjin Innovative Research Team Grant (TD-12-5049), Tianjin Natural Science

324

Grant (16JCQNJC14600), Talent grant of Tianjin University of Commerce (R160124,

325

R170106).

326

REFERENCES

327

(1) Mittler, R.; Vanderauwera, S.; Suzuki, N.; Miller, G.; Tognetti, V. B.; Vandepoele, K.; Gollery, M.;

328

Shulaev, V.; Van Breusegem, F. ROS signaling: the new wave? Trends Plant Sci. 2011, 16 (6), 300-309.

329

(2) Apel, K.; Hirt, H. Reactive Oxygen Species: Metabolism, Oxidative Stress, and Signal

330

Transduction. Annu. Rev. Plant Biol. 2004, 55, 373-399.

331

(3) Foyer, C. H.; Noctor, G. Redox regulation in photosynthetic organisms: signaling, acclimation, 16


Page 16 of 34

Page 17 of 34


332

and practical implications. Antioxid. Redox Sign. 2009, 11 (4), 861-905.

333

(4) Møller, I. M.; Jensen, P. E.; Hansson, A. Oxidative modifications to cellular components in plants.

334

Annu. Rev. Plant Biol. 2007, 58 (1), 459-481.

335

(5) Mittler, R.; Vanderauwera, S.; Gollery, M.; Van, B. F. Reactive oxygen gene network of plants.

336

Trends Plant Sci. 2004, 9 (10), 490-498.

337

(6) Harman, D. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 1956, 11

338

(3), 298-300.

339

(7) Harman, D. Free radical theory of aging: The “free radical” diseases. Age 1984, 7 (4), 111-131.

340

(8) Haenold, R.; Wassef, D. M.; Heinemann, S. H.; Hoshi, T. Oxidative damage, aging and anti-aging

341

strategies. Age 2005, 27 (3), 183-199.

342

(9) Zhang, Q.; Li, N.; Zhou, G.; Lu, X.; Xu, Z.; Li, Z. In vivo antioxidant activity of polysaccharide

343

fraction from Porphyra haitanesis (Rhodephyta) in aging mice. Pharmacol. Res. 2003, 48 (2), 151-155.

344

(10) Kalaycıoğlu, Z.; Erim, F. B. Total phenolic contents, antioxidant activities, and bioactive

345

ingredients of juices from pomegranate cultivars worldwide. Food Chem. 2017, 221, 496-507.

346

(11) Leutner, S.; Eckert, A.; Müller, W. E. ROS generation, lipid peroxidation and antioxidant enzyme

347

activities in the aging brain. J. Neural Transm. 2001, 108 (8-9), 955.

348

(12) Jin, S. L.; Yin, Y. G. In vivo antioxidant activity of total flavonoids from indocalamus leaves in

349

aging mice caused by D-galactose. Food Chem. Toxicol. 2012, 50 (10), 3814.

350

(13) Zia-ur, R. Citrus peel extract – A natural source of antioxidant. Food Chem. 2006, 99 (3),

351

450-454.

352

(14) Bocco, A.; Cuvelier, M. E.; Hubert Richard, A.; Berset, C. Antioxidant Activity and Phenolic

353

Composition of Citrus Peel and Seed Extracts. J. Agric. Food Chem. 2015, 46 (6), 2123-2129. 17



354

(15) Kurowska, E. M.; Manthey, J. A. Hypolipidemic effects and absorption of citrus

355

polymethoxylated flavones in hamsters with diet-induced hypercholesterolemia. J. Agric. Food Chem.

356

2004, 52 (10), 2879-2886.

357

(16) Gosslau, A.; Chen, K. Y.; Ho, C. T.; Li, S. Anti-inflammatory effects of characterized orange peel

358

extracts enriched with bioactive polymethoxyflavones. Food Sci. Hum. Wel. 2014, 3 (1), 26-35.

359

(17) Lai, C. S.; Li, S.; Miyauchi, Y.; Suzawa, M.; Ho, C. T.; Pan, M. H. Potent anti-cancer effects of

360

citrus peel flavonoids in human prostate xenograft tumors. Food Funct. 2013, 4 (6), 944.

361

(18) Guo, J.; Tao, H.; Cao, Y.; Ho, C. T.; Jin, S.; Huang, Q. Prevention of Obesity and Type 2 Diabetes

362

with Aged Citrus Peel (Chenpi) Extract. J. Agric. Food Chem. 2016, 64 (10), 2053-2061.

363

(19) Fu, M.; Xu, Y.; Chen, Y.; Wu, J.; Yu, Y.; Zou, B.; An, K.; Xiao, G. Evaluation of bioactive

364

flavonoids and antioxidant activity in Pericarpium Citri Reticulatae (Citrus reticulata 'Chachi') during

365

storage. Food Chem. 2017, 230, 649-656.

366

(20) Li, S.; Pan, M. H.; Lo, C. Y.; Tan, D.; Wang, Y.; Shahidi, F.; Ho, C. T. Chemistry and health

367

effects of polymethoxyflavones and hydroxylated polymethoxyflavones. J. Funct. Foods 2009, 1 (1),

368

2-12.

369

(21) Soobrattee, M. A.; Neergheen, V. S.; Luximon-Ramma, A.; Aruoma, O. I.; Bahorun, T. Phenolics

370

as potential antioxidant therapeutic agents: mechanism and actions. Mutat. Res. 2005, 579 (2), 200-213.

371

(22) Acker, S. A. V.; Berg, D. J. V. D.; Tromp, M. N. J. L.; Griffioen, D. H.; Bennekom, W. P. V.; Vijgh,

372

W. J. V. D.; Bast, A. Structural aspect of antioxidant activity of flavonoids. Free Radic. Res. 1996, 20

373

(3), 331-342.

374

(23) Puiggros, F.; Llópiz, N.; Ardévol, A.; Bladé, C.; Arola, L.; Salvadó, M. J. Grape seed procyanidins

375

prevent oxidative injury by modulating the expression of antioxidant enzyme systems. J. Agric. Food 18


Page 18 of 34

Page 19 of 34


376

Chem. 2005, 53 (15), 6080-6086.

377

(24) Kim, J. H.; Campbell, B. C.; Yu, J.; Mahoney, N.; Chan, K. L.; Molyneux, R. J.; Bhatnagar, D.;

378

Cleveland, T. E. Examination of fungal stress response genes using Saccharomyces cerevisiae as a

379

model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link. Appl.

380

Microbiol. Biotechnol. 2005, 67 (6), 807-815.

381

(25) Mager, W. H.; Winderickx, J. Yeast as a model for medical and medicinal research. Trends

382

Pharmacol. Sci. 2005, 26 (5), 265-273.

383

(26) Li, S.; Lo, C. Y.; Ho, C. T. Hydroxylated polymethoxyflavones and methylated flavonoids in

384

sweet orange (Citrus sinensis) peel. J. Agric. Food Chem. 2006, 54 (12), 4176-4185.

385

(27) Brachmann, C. B.; Davies, A.; Cost, G. J.; Caputo, E.; Li, J.; Hieter, P.; Boeke, J. D. Designer

386

deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for

387

PCR-mediated gene disruption and other applications. Yeast 1998, 14 (2), 115-132.

388

(28) Dani, C.; Bonatto, D.; Salvador, M.; Pereira, M. D.; Henriques, J. A.; Eleutherio, E. Antioxidant

389

protection of resveratrol and catechin in Saccharomyces cerevisiae. J. Agric. Food Chem. 2008, 56 (11),

390

4268-4272.

391

(29) de Sá, R. A.; de Castro, F. A.; Eleutherio, E. C.; de Souza, R. M.; Da, S. J.; Pereira, M. D.

392

Brazilian propolis protects Saccharomyces cerevisiae cells against oxidative stress. Braz. J. Microbiol.

393

2013, 44 (3), 993-1000.

394

(30) Silva, C. G.; Herdeiro, R. S.; Mathias, C. J.; Panek, A. D.; Silveira, C. S.; Rodrigues, V. P.; Rennó,

395

M. N.; Falcão, D. Q.; Cerqueira, D. M.; Minto, A. B. M. Evaluation of antioxidant activity of Brazilian

396

plants. Pharmacol. Res. 2005, 52 (3), 229-233.

397

(31) Steels, E. L.; Learmonth, R. P.; Watson, K. Stress tolerance and membrane lipid unsaturation in 19



398

Saccharomyces cerevisiae grown aerobically or anaerobically. Microbiology 1994, 140 ( Pt 3) (Pt 3),

399

569-576.

400

(32) Pereira, M. D.; Herdeiro, R. S.; Fernandes, P. N.; Eleutherio, E. C.; Panek, A. D. Targets of

401

oxidative stress in yeast sod mutants. Biochim. Biophys. Acta 2003, 1620 (1-3), 245-251.

402

(33) Pereira, M. D.; Eleutherio, E. C.; Panek, A. D. Acquisition of tolerance against oxidative damage

403

in Saccharomyces cerevisiae. BMC Microbiol. 2001, 1 (1), 11.

404

(34) Fernandes, P. N.; Mannarino, S. C.; Silva, C. G.; Pereira, M. D.; Panek, A. D.; Eleutherio, E. C. A.

405

Oxidative stress response in eukaryotes: effect of glutathione, superoxide dismutase and catalase on

406

adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae. Redox Rep. 2007, 12 (5),

407

236-244.

408

(35) França, M. B.; Panek, A. D.; Eleutherio, E. C. The role of cytoplasmic catalase in dehydration

409

tolerance of Saccharomyces cerevisiae. Cell Stress Chaperon. 2005, 10 (3), 167-170.

410

(36) Richards, D. M.; Dean, R. T.; Jessup, W. Membrane proteins are critical targets in free radical

411

mediated cytolysis. BBA - Biomembrane 1988, 946 (2), 281-288.

412

(37) Atiba, A. S.; Abbiyesuku, F. M.; Niranatiba, T. A.; Oparinde, D. P.; Ajose, O. A.; Akindele, R. A.

413

Free Radical Attack on Membrane Lipid and Antioxidant Vitamins in the Course of Pre-Eclamptic

414

Pregnancy. Ethiop. J. Health. Sci. 2014, 24 (1), 35-42.

415

(38) Riceevans, C. A.; Miller, N. J.; Paganga, G. Structure-antioxidant activity relationships of

416

flavonoids and phenolic acids. Free Radic. Biol. Med. 1996, 20 (7), 933-956.

417

(39) Cotelle, N. Role of flavonoids in oxidative stress. Curr. Top. Med. Chem. 2001, 1 (6), 569-590.

418

(40) Esposito, E.; Rotilio, D.; Matteo, V. D.; Giulio, C. D.; Cacchio, M.; Algeri, S. A review of specific

419

dietary antioxidants and the effects on biochemical mechanisms related to neurodegenerative processes. 20


Page 20 of 34

Page 21 of 34


420

Neurobiol. Aging 2002, 23 (5), 719-710.

421

(41) Loziene, K.; Venskutonis, P. R.; Sipailiene, A.; Labokas, J. Radical scavenging and antibacterial

422

properties of the extracts from different Thymus pulegioides L. chemotypes. Food Chem. 2007, 103 (2),

423

546-559.

424

(42) Zaugg, J.; Potterat, O.; Plescher, A.; Honermeier, B.; Hamburger, M. Quantitative analysis of

425

anti-inflammatory and radical scavenging triterpenoid esters in evening primrose seeds. J. Agric. Food

426

Chem. 2006, 54 (18), 6623-6628.

427

(43) Rota, C.; Fann, Y. C.; Mason, R. P. Phenoxyl free radical formation during the oxidation of the

428

fluorescent dye 2',7'-dichlorofluorescein by horseradish peroxidase. Possible consequences for

429

oxidative stress measurements. J. Biol. Chem. 1999, 274 (40), 28161-28168.

430

(44) Aldred, S.; Bennett, S. P. Increased low-density lipoprotein oxidation, but not total plasma protein

431

oxidation, in Alzheimer's disease. Clin. Biochem. 2010, 43 (3), 267-271.

432

(45) Cao, S.; Zhu, X.; Zhang, C.; Qian, H.; Schuttler, H. B.; Gong, J. P.; Xu, Y. Competition between

433

DNA methylation, nucleotide synthesis and anti-oxidation in cancer versus normal tissues. Cancer Res.

434

2017, 77 (15), 4285-4195.

435

(46) Kachroo, A. H.; Laurent, J. M.; Yellman, C. M.; Meyer, A. G.; Wilke, C. O.; Marcotte, E. M.

436

Evolution. Systematic humanization of yeast genes reveals conserved functions and genetic modularity.

437

Science 2015, 348 (6237), 921-925.

438

(47) Sherman, M. Y.; Muchowski, P. J. Making yeast tremble: yeast models as tools to study

439

neurodegenerative disorders. Neuromolecular Med. 2003, 4 (1-2), 133-146.

440

(48) Bovee, T. F.; Helsdingen, R. J.; Koks, P. D.; Kuiper, H. A.; Hoogenboom, R. L.; Keijer, J.

441

Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein. 21



442

Gene 2004, 325, 187-200.

443

(49) Tucker, C. L.; Fields, S. A yeast sensor of ligand binding. Nat. Biotechnol. 2001, 19 (11),

444

1042-1046.

445

(50) Zheng, J.; Fang, X.; Cao, Y.; Xiao, H.; He, L. Monitoring the chemical production of

446

citrus-derived bioactive 5-demethylnobiletin using surface-enhanced Raman spectroscopy. J. Agric.

447

Food Chem. 2013, 61 (34), 8079-8083.

448

(51) Qiu, P.; Dong, P.; Guan, H.; Li, S.; Ho, C. T.; Pan, M. H.; McClements, D. J.; Xiao, H. Inhibitory

449

effects of 5-hydroxy polymethoxyflavones on colon cancer cells. Mol. Nutr. Food Res. 2010, 54 Suppl

450

2, S244-252.

451 452

22


Page 22 of 34

Page 23 of 34


453

FIGURE CAPTIONS

454

Figure 1. Structures of NBT, 5-DN, TAN, and 5-DT.

455

Figure 2. Survival rate of S. cerevisiae cells directly exposed to increasing NBT,

456

5-DN, TAN, or 5-DT concentrations. The PMFs density used were 25 µg/mL, 50

457

µg/mL and 100 µg/mL, respectively. Data represent means ± SD of three independent

458

experiments.

459

Figure 3. Effect of 10 mM CCl4 (A), 2 mM H2O2 (B), or 2.5 mM CdSO4 (C) on the

460

viability in S. cerevisiae cells (wild type and mutants strains ctt1∆, sod1∆, and gsh1∆)

461

and the anti-oxidative effect of NBT, 5-DN, TAN, or 5-DT pre-treatment. The

462

concentration of PMFs used was 50 µg/mL. Data represent means ± SD of three

463

independent experiments. The statistically different results were represented by

464

different letters in each oxidative stress group, p

Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin

Recommend Documents