Omics Analyses of Gut Microbiota in a Circadian Rhythm Disorder

Jul 22, 2019 - In addition, Kyoto Encyclopedia of Genes and Genomes pathways of ATP-binding cassette transporters, ... of molecular weight and number ...
2 downloads 0 Views 944KB Size
Subscriber access provided by BUFFALO STATE

Bioactive Constituents, Metabolites, and Functions

Omics analyses of gut microbiota in a circadian rhythm disorder mouse model fed with oolong tea polyphenols Tongtong Guo, Dan Song, Chi-Tang Ho, Xin Zhang, Chundan Zhang, Jinxuan Cao, and Zufang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03000 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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 31

Journal of Agricultural and Food Chemistry

Omics Analyses of Gut Microbiota in a Circadian Rhythm Disorder Mouse Model Fed with Oolong Tea Polyphenols Tongtong Guo†, Dan Song†, Chi-Tang Ho‡, Xin Zhang†, § ,*1, Chundan Zhang§, Jinxuan Cao†, Zufang Wu† †Department

of Food Science and Engineering, Ningbo University, Ningbo 315211,

P.R. China ‡Department

of Food Science, Rutgers University, New Brunswick, New Jersey

08901, United States §Key

Laboratory of Animal Protein Deep Processing Technology of Zhejiang

Province, Ningbo University, Ningbo 315211, P.R. China



Corresponding author: Xin Zhang; E-mail address: [email protected] 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

ABSTRACT: Microbiome has been revealed as a key element involved in

2

maintaining the circadian rhythms. Oolong tea polyphenols (OTP) has been shown to

3

have a potential prebiotic activity. Therefore, this study focused on the regulation

4

mechanisms of OTP on host circadian rhythms. After 8 weeks of OTP administration,

5

a large expansion in the relative abundance of Bacteroidetes with a decrease in

6

Firmicutes was observed, which reflected the positive modulatory effect of OTP on

7

gut flora. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways

8

of ATP-binding cassette (ABC) transporters, two-component system and the

9

biosynthesis of amino acids enriched the most differentially expressed genes (DEGs)

10

after OTP treatment. Of the differentially expressed proteins (DEPs) identified, most

11

were related to metabolism, genetic information processing and environmental

12

information processing. It underscores the ability of OTP to regulate circadian rhythm

13

by enhancing beneficial intestinal microbiota and affecting metabolic pathways,

14

contributing to the improvement of host micro-ecology.

15

KEYWORDS: oolong tea polyphenols, circadian clock, modulatory effect, intestinal

16

microbiota

2

ACS Paragon Plus Environment

Page 2 of 31

Page 3 of 31

Journal of Agricultural and Food Chemistry

17

Introduction

18

Tea is widely consumed around the world. Oolong tea is a unique semi-fermented tea

19

containing various ingredients, and the putative effective compounds is attributed to

20

polyphenols.1 Oolong tea polyphenols (OTP) have been shown to have a potential

21

prebiotic activity.2 The main phenolic compounds in OTP include (-)-epigallocatechin

22

gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and

23

(-)-epicatechin (EC).3 Furthermore, as the O-methylated form of EGCG,

24

(-)-epigallocatechin 3-O-(3-O-methyl)gallate (EGCG3″Me) shows a satisfied effect

25

on host intestinal micro-ecology by modulating gut microbiota.4

26

The rotation of the earth around its axis generates a cycle of light and darkness.

27

Circadian rhythm is an endogenous oscillation of physiology synchronizes many

28

biological processes with changes in environmental factors.5 Therefore, almost all

29

living organisms have evolved circadian clock to adapt and respond to the physical

30

environment.6 The mammalian circadian rhythm is generated by a central clock that

31

receives direct light input from the retina and synchronizes other central and

32

peripheral tissues phases.7 In a peripheral clock system, the clock in the liver is

33

critical because it affects many physiological processes.8 Therefore, natural products

34

have been noted as potential functional components with circadian modulatory

35

activity. In recent years, the relationship between circadian rhythm and intestinal flora

36

has aroused widespread concern.9

37

In the gut, the microbiota has been revealed as a critical element affecting host

38

health,10 and the disturbance of microbiota composition has been demonstrated to be 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

39

related to the development of certain metabolic syndromes.11 Circadian clocks and

40

metabolism are inseparable, both central and liver circadian clocks coordinate

41

metabolic events in response to the awakening-sleep cycle.12 The gut microbiome is

42

important in maintaining the host's circadian rhythm. Despite the existence of light

43

and dark signals, sterile mice exhibited impaired circadian clock gene expression,

44

whereas the intestinal microbiota of the normal mice showed different diurnal

45

variation depending on dietary composition.13 Dietary tea polyphenols have been

46

reported to ameliorate memory impairment via a circadian clock-related mechanism.14

47

In addition, plant polyphenols could effective regulate the expression and rhythm of

48

circadian clock genes as well as the internal environment.15 Our previous studies have

49

also shown that tea polyphenols are beneficial for the stability of gut flora, especially

50

in environmentally induced microbial imbalances.16,17

51

Prebiotics not only modulate gut flora, but also improve the integrity of intestinal

52

tight junctions.18 Tea catechins have been showed prebiotic activity and generated

53

short-chain fatty acids (SCFAs) in our previous study.19 In addition, we have

54

identified KEGG pathways enriched the most DEGs after OTP intervention.16 At

55

present, the research on the regulation of tea catechins on intestinal flora is an

56

emerging field, and there is still a lack of systematic understanding. Tea catechins can

57

affect the circadian rhythm of peripheral clock systems, but the molecular regulation

58

mechanisms are still unclear. Therefore, investigating the inter-relationship between

59

intestinal flora and host circadian rhythm is a breakthrough in promoting the theory of

60

circadian rhythm regulation. 4

ACS Paragon Plus Environment

Page 4 of 31

Page 5 of 31

Journal of Agricultural and Food Chemistry

61

Materials and Methods

62

Chemicals. Polyamide resin was purchased from Ocean Chemical Co., Ltd.

63

(Qingdao, China). Standards of tea catechin monomers were prepared according to

64

the method we reported.19 All other chemicals were of analytical grade.

65

Preparation of OTP. Oolong tea was obtained from a local tea plantation in

66

Ningbo. The OTP preparation was performed according to our reported method.16

67

Briefly, the tea was extracted with hot water at a ratio of 1:16 (m: v) at 96 °C for 40

68

min, and purified by a polyamide column. The elutions were analyzed by HPLC,16

69

and the desired fractions containing OTP were concentrated and lyophilized.

70

Animals and experimental design. We initially colonized young adult (6 weeks

71

old) male C57BL/6J mice by a microbial community present in freshly stool samples

72

from 5 healthy volunteers (3 females and 2 males, 25-30 years old). Fresh human

73

stool samples were diluted in 10 mL of PBS under anaerobic conditions, and 0.2 mL

74

of the vortexed stool suspension was introduced by gavage into each sterile recipient.

75

The mice were acclimated to the environment for 7 days in constant darkness, and

76

then randomly divided into 3 groups: a 12 h light-dark cycle group (control), constant

77

darkness group (CD) and constant darkness with OTP group (CD-OTP) fed with 0.1%

78

(w/w) OTP. The body weight, food and water consumption of each animal was

79

recorded weekly, and faecal samples were collected from the CD-OTP group after 0

80

(OTP-0), 2 (OTP-2), 4 (OTP-4) and 8 weeks (OTP-8). After 8 weeks, the laparotomy

81

was performed under pentobarbital anesthesia, and liver and epididymal fat were

82

isolated and immediately weighed. 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

83

Analysis of intestinal microbiota. DNA was extracted using E.Z.N.A. ®Stool

84

DNA Kit by the manufacturer's instructions.16 Prior to sequencing, the above 16S

85

rDNA V3-V4 region of each sample was amplified with a set of primers targeting the

86

16S rRNA gene region. Sequences were analyzed using QIIME (Quantitative Insights

87

Into Microbial Ecology) (version 1.2.8) software package.20 High quality reads were

88

clustered into operational taxonomic units (OTUs) using CD-HIT software (version

89

4.6.1). The OTUs reached 97% nucleotide similarity level were used for richness

90

(Chao1), alpha diversity (Shannon and Simpson).21 Phylogenetic beta diversity

91

measures unweighted UniFrac distance metrics analysis and principal coordinate

92

analysis (PCoA) were performed using OTUs.

93

Construction of a gut metagenome reference. Firstly, we performed taxonomic

94

assignments and functional annotations using the Non-Redundant (NR), KEGG, and

95

Gene Ontology (GO) databases. 16 In addition to setting the E value was set to 10-5, all

96

genes were searched against integrated microbial genomes (IMG, version 3.4) using

97

default parameters. The taxonomic association of a gene was determined by the

98

lowest common ancestor of all taxonomically annotated results.

99

Protein extraction, digestion and LC-MS/MS measurements. Faecal samples

100

were suspended with PBS and rotated on a Thermo shaker (MSC-100) overnight at

101

4 °C. The precipitates were collected by centrifugation and re-suspended in 90%

102

pre-cooled acetone. The extract was centrifuged, then the supernatant was transferred

103

to a new tube and the protein was precipitated by methanol chloroform.

104

Protein samples were digested according to previously reported method.22 The 6

ACS Paragon Plus Environment

Page 6 of 31

Page 7 of 31

Journal of Agricultural and Food Chemistry

105

peptides were dried under vacuum, re-suspended in 2% acetonitrile and 0.1% TFA

106

and desalted with Sep-Pak C18 (Waters, WAT023590). Peptides were analyzed on a

107

Q Exactive mass spectrometer coupled with Easy-nLC 1200. The peptides were

108

loaded onto a C18-reversed phase column and eluted with a gradient of 5-80%

109

acetonitrile + 0.1% formic acid at a flow rate of 300 nL/min.

110

Taxonomic analysis of peptides and identification of proteins. The MS/MS

111

spectra were searched according to the bacterial database from the Uniprot database

112

(http://www.uniprot.org/) and the decoy database.23 Protein quantification was

113

performed in Peaks Studio 8 software based on MS1 peak intensity, and functional

114

analysis

115

(http://www.genome.jp/kegg/pathway.html).

of

identified

proteins

were

assigned

to

the

KEGG

database

116

Statistical analysis. Data obtained were analyzed by SAS and expressed as mean

117

± standard deviation (SD). Comparisons between groups were performed using

118

one-way analysis of variance (ANOVA) and Duncan’s multiple-comparison test. All

119

results were considered statistically significant at P < 0.05.

120

Results

121

The tea catechin contents in oolong tea. The HPLC chromatogram of oolong tea

122

infusion in Fig. S1 shows that peaks 1-13 are gallic acid, (-)-gallocatechin (GC),

123

theobromine,

124

(-)-gallocatechin-3-gallate (GCG), EGCG3″Me, ECG, and (-)-catechin gallate (CG).

125

It indicated that the EGCG content is the highest (Table S1), making it the main tea

126

catechin, while the content of EGCG3″Me is high.

EGC,

(-)-catechin

(C),

theophylline,

7

ACS Paragon Plus Environment

EGCG,

caffeine,

EC,

Journal of Agricultural and Food Chemistry

127

The effect of OTP on body and organ weight of the mouse model. The increase

128

in body weight after 2 weeks was significantly higher (P < 0.05) in the CD group than

129

in the CD-OTP group (Fig. 1), which showed the direct weight-reducing activity of

130

OTP. In Fig. S2, the values for the liver and epididymal weight in the CD group were

131

significantly higher than those of the control (P < 0.05), while the weight gain of the

132

CD-OTP group was significantly reduced (P < 0.05). The results of food (Fig. S3A)

133

and water consumption (Fig. S3B) showed no significant difference (P > 0.05) in all

134

groups.

135

The effect of OTP on bacterial diversity of the mouse model. The estimator of

136

Chao1 richness for total bacterial community diversity was increased during OTP

137

treatment (Table S2). The functional role of OTP in improving species richness and

138

diversity was further supported by a significantly higher Shannon and lower Simpson

139

indexes (P < 0.05). A Venn diagram showed that there were only 243 of the total

140

richness of 1,029 OTUs were shared among all samples, demonstrating that >23% of

141

the OTUs observed after OTP treatment were the same as in the initial treatment.

142

In each group, Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria were

143

the most abundant phyla (Fig. 2). After 8 weeks of OTP treatment, a dramatically

144

decrease in the relative abundance of Firmicutes with an increase of Bacteroidetes

145

was observed. The corresponding Firmicutes/Bacteroidetes (F/B) ratio was decreased

146

from 0.80 (OTP-0) to 0.41 (OTP-8), indicating that Firmicutes was largely inhibited

147

by OTP. In the control group, the F/B ratio was also decreased. However, in the CD

148

group the ratio showed an opposite trend, which suggested the circadian rhythm 8

ACS Paragon Plus Environment

Page 8 of 31

Page 9 of 31

Journal of Agricultural and Food Chemistry

149

disorder may lead to imbalance of intestinal flora, and the result was similar to the

150

consequences of obesity-induced gut dysbiosis.

151

At the family level (Fig. 3A), it can be seen that both Prevotellaceae and

152

Bacteroidaceae showed significant increasing trends (P < 0.05) during the treatment

153

of OTP. Prevotellaceae was the predominant bacterium, and the relative abundance of

154

which increased from 0.34 ± 0.02 (OTP-0) to 0.44 ± 0.02 at the 8th week. For

155

Bacteroidaceae, it showed a similar trend, while Eubacteriaceae, Ruminococcaceae

156

and Lachnospiraceae showed a decreasing trend after OTP intervention. Prevotella

157

and Bacteroides accounted for the majority at the genus level (Fig. 3B), whose

158

relative abundances were increased after OTP treatment, while Faecalibacterium,

159

Mitsuokella and Ruminococcus were decreased. In addition, as shown in Fig. S4, the

160

faecal flora in the OTP-fed group at 0, 2, 4 and 8-week were separated clearly by

161

PCoA.

162

The effect of OTP on the gut microbiome of humanized mouse. The imputed

163

relative abundances of KEGG pathways in OTP-0 and OTP-8 were used to predict

164

metabolic function changes within the microbiomes (Fig. 4). The KEGG pathways

165

indicated that membrane transport, carbohydrate metabolism and signal transduction

166

accounted for the top three. At the same time, the largest statistical differences

167

between OTP-0 and OTP-8 were cell motility, membrane transport and environmental

168

adaptation. After 8 weeks, the OTP-associated DEGs were enriched in various KEGG

169

pathways involved in ABC transporters, two-component system, and biosynthesis of

170

amino acids (Fig. S5). 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

171

GO analysis of DEGs between OTP-0 and OTP-8 indicated that most genes in

172

biological processes including regulation of transcription, DNA-templated, transport,

173

translation, metabolic process and carbohydrate metabolic process; cellular

174

components including cytosol, plasma membrane, cytoplasm, integral component of

175

membrane and membrane; and molecular functions including transcription factor

176

activity, sequence-specific DNA binding, ATP binding, structural constituent of

177

ribosome, protein binding and catalytic activity (Fig. 5).

178

Metaproteomic analysis was applied to study the proteins of the intestinal

179

microbiota of the CD group fed with OTP. A total of 312,676 MS/MS spectra were

180

generated from the faecal samples, and 65,535 peptides could be identified. The

181

distribution of molecular weight (A), number (B) of peptides and sequence coverages

182

(C) by label-free proteomics were shown in Fig. S6A-C. Of the differentially

183

expressed proteins (DEPs) between OTP-0 and OTP-8 identified, most were related to

184

metabolism, genetic information processing and environmental information

185

processing (Fig. 6). After OTP intervention, DEPs enriched in the metabolism

186

domains included carbohydrate metabolism, global and overview maps and amino

187

acid metabolism; genetic information processing including translation, folding,

188

sorting degradation and transcription; environmental information processing including

189

membrane transport and signal transduction.

190

Discussion

191

The human gut is considered to be the main site for the interaction between OTP and

192

intestinal flora.24 Meanwhile, evidence has showed an inextricable link between gut 10

ACS Paragon Plus Environment

Page 10 of 31

Page 11 of 31

Journal of Agricultural and Food Chemistry

193

microbiota and metabolic syndromes. Previous studies underscored the ability of

194

microbial-derived metabolites to alter circadian rhythms as well as the metabolic

195

function of the host.14

196

Sleep defects and circadian rhythm disruption are closely related to metabolic

197

disorders, which may lead to obesity by regulating feeding time and amount of food

198

intake.25 Studies have shown that tea polyphenols could modulate the relatively

199

shallow daily oscillations of circadian clock gene expression in the liver caused by

200

continuous darkness.15 People with persistent circadian rhythm disorders, such as

201

insomnia and night shift workers, have higher incidence of hyperlipidemia,

202

atherosclerosis, hypertension and other diseases.26 The occurrence of these chronic

203

diseases was closely related to the human intestinal micro-ecological imbalance, and

204

showed circadian rhythm oscillation similar to the intestinal flora.27 A common

205

characteristic of these chronic diseases is that they appear to be accelerated by the

206

inflammatory

207

characteristic of circadian clock,29 which maintaining biological homeostasis through

208

a number of specialized metabolic and signaling pathways.30 It has been demonstrated

209

that environmental circadian rhythm disruption can lead to disorders of the gut flora,

210

especially when under dietary stress.31

process.28

Most

intestinal

microbial

communities

showed

a

211

Tea catechins exhibited proliferative effects on certain beneficial bacteria in our

212

previous study; in addition, it promoted the production of total SCFAs.19 The

213

production of SCFAs is the result of intestinal microbiota metabolism, which plays a

214

vital role in regulating host metabolism and cell proliferation.25 In this study, an 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

215

increase in the relative abundances of genera known to produce acetate and butyrate

216

were observed in mice supplemented with OTP. Different strains of gut microbiota

217

exhibited varying degrees of growth sensitivity to the metabolites of polyphenols.32

218

The changes of microbiota populations after circadian rhythm disruption have been

219

characterized by the expansion of pro-inflammatory bacteria and a decrease in

220

putative anti-inflammatory, SCFAs-producing ones.33

221

In the present study, circadian disorder leads to symptoms similar to obesity. After

222

8 weeks of treatment, the body weight and epididymal fat were significantly increased

223

(P < 0.05) in the CD group, while a decrease in the relative abundance of Firmicutes

224

was observed in the circadian rhythm disorder group with OTP intervention. In

225

addition, OTP increased the relative abundance of Bifidobacteria dramatically after 8

226

weeks, which may enhance intestinal barrier function, stimulate the host immune

227

system and regulate lipid metabolism, and considered to be important bacterial group

228

associated with host health.34 An increased ratio of F/B has been observed in obese

229

people.35 However, the generation of circadian rhythm disorder involves more

230

complex issues, which cannot only be simply explained by an imbalance in the F/B

231

proportion.

232

Although the relationships between intestinal flora and metabolic syndromes

233

remain unclear, high-throughput sequencing offers the opportunity to explore

234

taxonomy and genes through less biased and more comprehensive measurements.36

235

The mechanism by which tea polyphenols regulate the flora in the gut includes the

236

interactions with the basic development and metabolic aspects of bacteria, as well as 12

ACS Paragon Plus Environment

Page 12 of 31

Page 13 of 31

Journal of Agricultural and Food Chemistry

237

interference with cell membrane function and bacterial energy metabolism.37 The

238

effect of tea polyphenols on the gut microbiota depends on the structure of both the

239

polyphenols and the microbial strain.38,39 Tea polyphenols can form hydrogen bonds

240

with stacking interactions of nucleobases, which may explain their inhibition on

241

bacterial DNA and RNA synthesis.40 In our study, enriched GO terms suggesting that

242

OTP alleviated the negative consequences of circadian rhythm disorder by affecting

243

cellular components. Moreover, tea polyphenols can influence bacteria through a

244

variety of cellular targets. For instance, plant polyphenols can form complex with

245

proteins via hydrogen bonding, covalent bonding and hydrophobic interactions.41

246

KEGG analysis of DEGs showed the most enriched metabolic pathways between

247

OTP-0 and OTP-8 in the present study. The ABC transporters are widely distributed,

248

and involved in detoxification and transport processes.42 In addition, the results

249

revealed by metaproteomic indicated that after 8 weeks, most DEPs were enriched in

250

the metabolic domains, and that may be due to that the gut microbiota can regulate

251

energy harvest of the host.43 To be more specific, intestinal microflora affects the

252

host's energy metabolism system by regulating the efficiency of energy extraction

253

from diet and the way of using or storing this harvested energy.

254

EGCG has been reported to relieve diet-induced metabolic syndromes related to

255

the circadian clock,44 and dietary tea polyphenols can ameliorate memory impairment

256

through a circadian clock-related mechanism.14 However, due to the limited

257

bioavailability of tea polyphenols in vivo, the majority ingested are metabolized into

258

various derivatives by intestinal microbiota. The activity of tea polyphenols depends 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

259

to a large extent on their conversion in the intestine. It has been indicated that the

260

disruption of circadian clock by changing the light/dark circadian cycle induced the

261

changes in the intestinal microbiota, including composition and circadian rhythm

262

oscillation.45 Recent studies have also demonstrated the ability of microbial metabolic

263

derivatives to regulate central and liver circadian rhythm as well as host metabolic

264

function, which implies prebiotics may alleviate circadian rhythm misalignment.46

265

Furthermore, this study will better assess the feasibility of tea polyphenols' microbial

266

metabolites in the treatment of circadian rhythm disruption and the related metabolic

267

syndromes.

268

In conclusion, OTP ameliorated circadian rhythm disorder induced gut dysbiosis,

269

and showed activities to maintain micro-ecology balance. For the DEPs identified by

270

metaproteomic analysis, most were related to metabolism, genetic and environmental

271

information processing. Finally, our results suggested that OTP has prebiotic activity

272

for ameliorate metabolic syndrome associated with circadian rhythm disorders.

273

Acknowledgment

274

We thank LC-Bio Technology for the bio-information analysis.

275

Supporting Information description

276

Figure S1. Representative elution profiles of tea catechins, gallic acid, caffeine,

277

theobromine and theophylline from oolong tea (1, gallic acid; 2, (-)-gallocatechin

278

(GC); 3, theobromine; 4, EGC; 5, (-)-catechin (C); 6, theophylline; 7, EGCG; 8,

279

caffeine; 9, EC; 10, (-)-gallocatechin-3-gallate (GCG); 11, EGCG3″Me, 12, ECG; 13,

280

(-)-catechin gallate (CG)). 14

ACS Paragon Plus Environment

Page 14 of 31

Page 15 of 31

Journal of Agricultural and Food Chemistry

281

Figure S2. Effect of OTP on liver weight (A) and epididymal fat weight (B) of the

282

circadian rhythm disorder mouse model. Different letters indicate significant

283

differences (P < 0.05) among different groups.

284

Figure S3. Effect of OTP on water intake (A) and food intake (B) of circadian rhythm

285

disorder mouse model.

286

Figure S4. Principal coordinate analysis (PCoA) plot of the faecal microbiota based

287

on the unweighted UniFrac metric.

288

Figure S5. KEGG analysis of differentially expressed genes (DEGs) between OTP-0

289

and OTP-8.

290

Figure S6. The distribution of molecular weight (A), number (B) of peptides and

291

sequence coverages (C) by label-free proteomics.

292

Table S1. Contents of tea catechins, gallic acid, caffeine, theobromine and

293

theophylline in oolong tea.

294

Table S2. The influence of OTP on the biodiversity of faecal microbiota in the

295

circadian rhythm disorder mouse model.

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 31

296

References

297

(1) Zhang, X.; Wu, Z. F.; Weng. P. F. Antioxidant and hepatoprotective effect of

298

(-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3''Me) from Chinese

299

oolong tea. J. Agric. Food Chem. 2014, 62, 10046-10054.

300

(2) Cheng, M.; Zhang, X.; Miao, Y. J.; Cao, J. X.; Wu, Z. F.; Weng, P. F. The

301

modulatory effect of (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3''Me)

302

on intestinal microbiota of high fat diet-induced obesity mice model. Food Res.

303

Int. 2017, 92, 9-16.

304

(3) Zhang, X.; Wu, Z. F.; Weng, P. F.; Yang, Y. Analysis of tea catechins in vegetable

305

oils by high performance liquid chromatography combined with liquid-liquid

306

extraction. Int. J. Food Sci. Technol. 2015, 50, 885-891.

307

(4) Zhang, X.; Chen, Y. H.; Zhu, J. Y.; Zhang, M.; Ho, C. T.; Huang, Q. R.; Cao, J. X.

308

Metagenomics analysis of gut microbiota in a high fat diet-induced obesity

309

mouse

310

(EGCG3″Me). Mol. Nutr. Food Res. 2018, 62, e1800274.

model

fed

with

(-)-epigallocatechin

3-O-(3-O-methyl)

gallate

311

(5) Mi, Y.; Qi, G.; Gao, Y.; Li, R.; Wang, Y.; Li, X.; Huang, S.; Liu, X.

312

(-)-Epigallocatechin-3-gallate ameliorates insulin resistance and mitochondrial

313

dysfunction in HepG2 cells: involvement of Bmal1. Mol. Nutr. Food Res. 2017,

314

61, 1700440.

315

(6) He, B.; Nohara, K.; Park, N.; Park, Y. S.; Guillory, B.; Zhao, Z.; Garcia, J. M.;

316

Koike, N.; Lee, C. C.; Takahashi, J. S.; Yoo, S. H.; Chen, Z. The small molecule

16

ACS Paragon Plus Environment

Page 17 of 31

Journal of Agricultural and Food Chemistry

317

nobiletin targets the molecular oscillator to enhance circadian rhythms and

318

protect against metabolic syndrome. Cell Metab. 2016, 23, 610-621.

319

(7) Wang, Y.; Kuang, Z.; Yu, X.; Ruhn, K. A.; Kubo, M.; Hooper, L. V. The

320

intestinal microbiota regulates body composition through NFIL3 and the

321

circadian clock. Science 2017, 357, 912-916.

322

(8) Thaiss, C. A.; Zeevi, D.; Levy, M.; Zilberman-Schapira, G.; Suez, J.; Tengeler, A.

323

C.; Abramson, L.; Katz, M. N.; Korem, T.; Zmora, N.; Kuperman, Y.; Biton, I.;

324

Gilad, S.; Harmelin, A.; Shapiro, H.; Halpern, Z.; Segal, E.; Elinav, E.

325

Transkingdom control of microbiota diurnsal oscillations promotes metabolic

326

homeostasis. Cell 2014, 159, 514-529.

327

(9)

Marchesi, J. R.; Adams, D. H.; Fava, F.; Hermes, G. D.; Hirschfield, G. M.;

328

Hold, G.; Quraishi, M. N.; Kinross, J.; Smidt, H.; Tuohy, K. M.; Thomas, L. V.;

329

Zoetendal, E. G.; Hart, A. The gut microbiota and host health: a new clinical

330

frontier. Gut 2016, 65, 330-339.

331

(10) David, L. A.; Maurice, C.; Carmody, R. N.; Gootenberg, D. B.; Button, J. E.;

332

Wolfe, B. E.; Ling, A. V.; Devlin, A. S.; Varma, Y.; Fischbach, M. A.;

333

Biddinger, S. B.; Dutton, R. J.; Turnbaugh, P. J. Diet rapidly and reproducibly

334

alters the human gut microbiome. Nature 2013, 505, 559-563.

335

(11) Turnbaugh, P. J.; Ridaura, V. K.; Faith, J. J.; Rey, F. E.; Knight, R.; Gordon, J. I.

336

The effect of diet on the human gut microbiome: a metagenomic analysis in

337

humanized gnotobiotic mice. Sci. Transl. Med. 2010, 1, 6799-6806.

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

338 339

(12) Huang, W.; Ramsey, K.M.; Marcheva, B.; Bass, J. Circadian rhythms, sleep, and metabolism. J. Clin. Invest. 2011, 121, 2133-2141.

340

(13) Leone, V.; Gibbons, S. M.; Martinez, K.; Hutchison, A. L.; Huang, E. Y.; Cham,

341

C. M.; Pierre, J. F.; Heneghan, A. F.; Nadimpalli, A.; Hubert, N.; Zale, E.;

342

Wang, Y.; Huang, Y.; Theriault, B.; Dinner, A. R.; Musch, M. W.; Kudsk, K. A.;

343

Prendergast, B. J.; Gilbert, J. A.; Chang, E. B. Effects of diurnal variation of gut

344

microbes and high fat feeding on host circadian clock function and metabolism.

345

Cell Host Microbe 2015, 17, 681-689.

346

(14) Qi, G.; Mi, Y.; Liu, Z.; Fan, R.; Qiao, Q.; Sun, Y.; Ren, B.; Liu, X. Dietary tea

347

polyphenols ameliorate metabolic syndrome and memory impairment via

348

circadian clock related mechanisms. J. Funct. Foods 2017, 34, 168-180.

349

(15) Liu, F.; Zhang, X.; Zhao, B.; Tan, X.; Wang, L.; Liu, X. Role of food

350

phytochemicals in the modulation of circadian clocks. J. Agric. Food Chem.

351

2019, doi: 10.1021/acs.jafc.9b02263.

352

(16) Cheng, M.; Zhang, X.; Zhu, J. Y.; Cheng, L.; Cao, J. X.; Wu, Z. F.; Weng, P. F.;

353

Zheng, X. J. A metagenomics approach to the intestinal microbiome structure

354

and function in high fat diet-induced obesity mice fed with oolong tea

355

polyphenols. Food Funct. 2018, 9, 1079-1087.

356

(17) Guo, X. J.; Cheng, M.; Zhang, X.; Cao, J. X.; Wu, Z. F.; Weng, P. F. Green tea

357

polyphenols reduces obesity in high fat-diet induced mice by modulating

358

intestinal microbiota composition. Int. J. Food Sci. Technol. 2017, 52,

359

1723-1730. 18

ACS Paragon Plus Environment

Page 18 of 31

Page 19 of 31

Journal of Agricultural and Food Chemistry

360

(18) Chang, C. J.; Lin, C. S.; Lu, C. C.; Martel, J.; Ko, Y. F.; Ojcius, D. M.; Tseng, S.

361

F.; Wu, T. R.; Chen, Y. Y.; Young, J. D.; Lai, H. C. Ganoderma lucidum reduces

362

obesity in mice by modulating the composition of the gut microbiota. Nat.

363

Commun. 2015, 6, 7489.

364

(19) Zhang, X.; Zhu, X. L.; Sun, Y. K.; Hu, B.; Sun, Y.; Jabbar, S.; Zeng, X. X.

365

Fermentation in vitro of EGCG, GCG and EGCG3″Me isolated from Oolong tea

366

by human intestinal microbiota. Food Res. Int. 2013, 54, 1589-1595.

367

(20) Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.;

368

Costello, E. K.; Fierer, N.; Peña, A. G.; Goodrich, J. K.; Gordon, J. I.; Huttley,

369

G. A.; Kelley, S. T.; Knights, D.; Koenig, J. E.; Ley, R. E.; Lozupone, C. A.;

370

McDonald, D.; Muegge, B. D.; Pirrung, M.; Reeder, J.; Sevinsky, J. R.;

371

Turnbaugh, P. J.; Walters, W. A.; Widmann, J.; Yatsunenko, T.; Zaneveld, J.;

372

Knight, R. QIIME allows analysis of high-throughput community sequencing

373

data. Nat. Methods 2010, 7, 335-336.

374

(21) Schloss, P. D.; Westcott, S. L.; Ryabin, T.; Hall, J. R.; Hartmann, M.; Hollister,

375

E. B.; Lesniewski, R. A.; Oakley, B. B.; Parks, D. H.; Robinson, C. J.; Sahl,

376

J.W.; Stres, B.; Thallinger, G. G.; van Horn, D. J.; Weber, C. F. Introducing

377

mothur: Open-source, platform-independent, community-supported software for

378

describing and comparing microbial communities. Appl. Environ. Microbiol.

379

2009, 75, 7537-7541.

380

(22) Yin, X.; Zhang, Y.; Liu, X.; Chen, C.; Lu, H.; Shen, H.; Yang, P. Systematic

381

comparison between SDS-PAGE/RPLC and high-/low-pH RPLC coupled 19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

382

tandem mass spectrometry strategies in a whole proteome analysis. Analyst 2015,

383

140, 1314-1322.

384

(23) Zhang, J.; Xin, L.; Shan, B.; Chen, W.; Xie, M.; Yuen, D.; Zhang, W.; Zhang, Z.;

385

Lajoie, G. A.; Ma, B. PEAKS DB: de novo sequencing assisted database search

386

for sensitive and accurate peptide identification. Mol. Cell. Proteomics 2012, 11,

387

1-8.

388

(24) Cheng, M.; Zhang, X.; Guo, X. J.; Wu, Z. F.; Weng, P. F. The interaction effect

389

and mechanism between tea polyphenols and intestinal microbiota: role in

390

human health. J. Food Biochem. 2017, 41, e12415.

391

(25) Kawabe, Y.; Nakamura, Y.; Kikuchi, S.; Murakami, Y.; Tanaka, T.;

392

Takebayashi, T.; Okayama, A.; Miura, K.; Okamura, T.; Ueshima, H.

393

Relationship between shift work and clustering of the metabolic syndrome

394

diagnostic components. J. Atheroscler. Thromb. 2014, 21, 703-711.

395 396 397 398

(26) Bass, J.; Takahashi, J. S. Circadian integration of metabolism and energetics. Science 2010, 330, 1349-1354. (27) Huang, W.; Ramsey, K. M.; Marcheva, B.; Bass, J. Circadian rhythms, sleep, and metabolism. J. Clin. Invest. 2011, 121, 2133-2141.

399

(28) Clemente, J. C.; Ursell, L. K.; Parfrey, L. W.; Knight, R. The impact of the gut

400

microbiota on human health: an integrative view. Cell 2012, 148, 1258-1270.

401

(29) Bellet, M. M.; Deriu, E.; Liu, J. Z.; Blaschitz, C.; Zeller, M.; Edwards, R. A.;

402

Sahar, S.; Dandekar, S.; Baldi, P.; George, M. D.; Raffatellu, M.; Sassone-Corsi,

403

P. Circadian clock regulates the host response to Salmonella. Proc. Natl. Acad. 20

ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31

404

Journal of Agricultural and Food Chemistry

Sci. U S A. 2013, 110, 9897-9902.

405

(30) Sharon, G.; Garg, N.; Debelius, J.; Knight, R.; Dorrestein, P. C.; Mazmanian, S.

406

K. Specialized metabolites from the microbiome in health and disease. Cell

407

Metab. 2014, 20, 719-730.

408

(31) Voigt, R. M.; Summa, K. C.; Forsyth, C. B, Green, S. J.; Engen, P.; Naqib, A.;

409

Vitaterna, M. H.; Turek, F. W.; Keshavarzian, A. The circadian clock mutation

410

promotes intestinal dysbiosis. Alcohol. Clin. Exp. Res. 2016, 40, 335-347.

411

(32) Stoupi, S.; Williamson, G.; Drynan, J. W.; Barron, D.; Clifford, M. N. A

412

comparison of the in vitro biotransformation of (-)-epicatechin and procyanidin

413

B2 by human faecal microbiota. Mol. Nutr. Food Res. 2010, 54, 747-759.

414

(33) Voigt, R. M.; Forsyth, C. B.; Green, S. J.; Mutlu, E.; Engen. P.; Vitaterna, M. H.;

415

Turek, F. W.; Keshavarzian, A. Circadian disorganization alters intestinal

416

microbiota. PLoS One 2014, 9, e97500.

417 418

(34) Gibson, G. R. Prebiotics as gut microflora management tools. J. Clin. Gastroenterol. 2008, 2, S75-S79.

419

(35) Turnbaugh, P. J.; Ley, R. E.; Mahowald, M. A.; Magrini, V.; Mardis, E. R.;

420

Gordon, J. I. An obesity-associated gut microbiome with increased capacity for

421

energy harvest. Nature 2006, 444, 1027-1031.

422

(36) Zoetendal, E.; Rajilic-Stojanovic, M. V. W. High-throughput diversity and

423

functionality analysis of the gastrointestinal tract microbiota. Gut 2008, 57,

424

1605-1615.

425

(37) Barbieri, R.; Coppo, E.; Marchese, A.; Daglia, M.; Sobarzo-Sanchez, E.; Nabavi, 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

426

S. F.; Nabavi, S. M. Phytochemicals for human disease: An update on

427

plant-derived compounds antibacterial activity. Microbiol. Res. 2017, 196,

428

44-68.

429 430

(38) Hervert-Hernández, D.; Goñi, I. Dietary polyphenols and human gut microbiota: a review. Food Rev. Int. 2011, 27, 154-169.

431

(39) Sirk, T. W.; Friedman, M.; Brown, E. F. Molecular binding of black tea

432

theaflavins to biological membranes: Relationship to bioactivities. J. Agric. Food

433

Chem. 2011, 59, 3780-3787.

434 435

(40) Williamson, G.; Clifford, M. N. Colonic metabolites of berry polyphenols: the missing link to biological activity? Br. J. Nutr. 2010, 104, S48-S66.

436

(41) Cheng, L.; Chen, Y.; Zhang, X.; Zheng, X.; Cao, J.; Wu, Z.; Qin, W.; Cheng, K.

437

A metagenomic analysis of the modulatory effect of Cyclocarya paliurus

438

flavonoids on the intestinal microbiome in a high fat diet-induced obesity mouse

439

model. J. Sci. Food Agr. 2019, 99, 3967-3975.

440

(42) Aszalos A. Role of ATP-binding cassette (ABC) transporters in interactions

441

between natural products and drugs. Curr. Drug Metab. 2008, 9, 1010-1018.

442

(43) Zheng, H.; Chen, M.; Li, Y.; Wang, Y.; Wei, L.; Liao, Z.; Wang, M.; Ma, F.;

443

Liao, Q.; Xie, Z. Modulation of gut microbiome composition and function in

444

experimental colitis treated with sulfasalazine. Front. Microbiol. 2017, 8, 1703.

445

(44) Mi, Y.; Qi, G.; Fan, R.; Ji, X.; Liu, Z.; Liu, X. EGCG ameliorates diet-induced

446

metabolic syndrome associating with the circadian clock. BBA - Mol. Basis. Dis.

447

2017, 3, 1575-1589. 22

ACS Paragon Plus Environment

Page 22 of 31

Page 23 of 31

Journal of Agricultural and Food Chemistry

448

(45) Deaver, J. A.; Eum, S. Y.; Michal, T. Circadian disruption changes gut

449

microbiome taxa and functional gene composition. Front. Microbiol. 2018, 9,

450

737.

451

(46) Tahara, Y.; Yamazaki, M.; Sukigara, H.; Motohashi, H.; Sasaki, H.; Miyakawa,

452

H.; Haraguchi, A.; Ikeda, Y.; Fukuda, S.; Shibata, S. Gut microbiota-derived

453

short chain fatty acids induce circadian clock entrainment in mouse peripheral

454

tissue. Sci. Rep. 2018, 8, 1395.

455

Funding

456

This work was sponsored by Zhejiang Provincial Natural Science Foundation of

457

China (LY19C200006), the Key Research and Development Project of Zhejiang

458

Province (2017C02039 & 2018C02047) and K.C. Wong Magna Fund in Ningbo

459

University.

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

460

Figure captions

461

Figure 1. Effect of OTP on the body weight of the circadian rhythm disorder mouse

462

model (n=8). *indicates significantly differences (P < 0.05) between CD and CD-OTP

463

group.

464

Figure 2. The relative abundance of the top phylum from samples (n=3).

465

Figure 3. Relative abundance analyses at the family (A) and genus (B) levels from

466

the faecal microbiota of the circadian rhythm disorder mouse model.

467

Figure 4. Imputed metagenomic differences between OTP-0 and OTP-8 in KEGG

468

pathway maps. The relative abundance of metabolic pathways encoded in each

469

imputed sample metagenome was analysed using STAMP.

470

Figure 5. GO analysis of differentially expressed genes (DEGs) between OTP-0 and

471

OTP-8.

472

Figure 6. KEGG classification of differentially expressed proteins (DEPs) in the

473

faecal microbiota of the circadian rhythm disorder mouse model between OTP-0 and

474

OTP-8 by metaproteomic analyses.

24

ACS Paragon Plus Environment

Page 24 of 31

Page 25 of 31

Journal of Agricultural and Food Chemistry

Figure 1

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 2

26

ACS Paragon Plus Environment

Page 26 of 31

Page 27 of 31

Journal of Agricultural and Food Chemistry

Figure 3

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 4

28

ACS Paragon Plus Environment

Page 28 of 31

Page 29 of 31

Journal of Agricultural and Food Chemistry

Figure 5

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 6

30

ACS Paragon Plus Environment

Page 30 of 31

Page 31 of 31

Journal of Agricultural and Food Chemistry

Graphic for table of contents

31

ACS Paragon Plus Environment