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A dietary wolfberry extract modifies oxidative stress by controlling the expression of inflammatory mRNAs in overweight and hypercholesterolemic subjects: a randomized, double-blind, placebo-controlled trial You Jin Lee, Youngsook Ahn, Oran Kwon, Mee Youn Lee, Choong Hwan Lee, Sungyoung Lee, Taesung Park, Sung Won Kwon, and Ji Yeon Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04701 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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Journal of Agricultural and Food Chemistry

A dietary wolfberry extract modifies oxidative stress by controlling the expression of inflammatory mRNAs in overweight and hypercholesterolemic subjects: a randomized, double-blind, placebocontrolled trial You Jin Lee†, Youngsook Ahn†, Oran Kwon†, Mee Youn Lee‡, Choong Hwan Lee‡, Sungyoung Lee§, Taesung Park§, ⊥, Sung Won Kwon*# and Ji Yeon Kim*△

†Department

of Nutritional Science and Food Management, Ewha Womans University, Seoul

120-750, Republic of Korea ‡Department

of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of

Korea §Interdisciplinary

Program in Bioinformatics, Seoul National University, Seoul 151-747,

Republic of Korea ⊥Department

#

of Statistics, Seoul National University, Seoul 151-747, Republic of Korea

Department of Surgery, CHA Bundang Medical Center, CHA University, Seongnam,

Gyeonggi-do 463-712, Republic of Korea △Department

of Food Science and Technology, Seoul National University of Science and

Technology, Seoul 139-743, Republic of Korea

*

Correspondence: 1

ACS Paragon Plus Environment


Professor Sung Won Kwon, [email protected], Fax: +82-31-780-5250 Professor Ji Yeon Kim, [email protected], Fax: +82-2-976-6460

2


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1

Abstract

2 3

In the present study, we evaluated the anti-oxidative and anti-inflammatory effects of an

4

aqueous extract of wolfberry fruit (WBE) in mild hypercholesterolemic and overweight

5

subjects. This study was a double-blind randomized trial of two parallel groups of free-living

6

subjects (n=53). The participants consumed the contents of an 80-mL pouch containing 13.5

7

g WBE or placebo after one meal per day over an 8-week period. Following 8 weeks of WBE

8

supplementation, we observed a slight but significant decrease in erythrocyte superoxide

9

dismutase activity and an increase in catalase activity. Furthermore, to assess endogenous

10

DNA damage in lymphocytes, the alkaline comet assay was performed, showing that the

11

percentage of DNA in the tail was significantly decreased by 8-week WBE intake.

12

Additionally, the proportion of significantly deregulated mRNAs related to oxidative or

13

inflammatory stress was considerably higher in the WBE intake group. The present data

14

indicate that WBE intake has anti-oxidative and anti-inflammatory effects in overweight and

15

hypercholesterolemic subjects by modulating mRNA expression.

16 17

Keywords: DNA damage; inflammation; mRNA array; postprandial inflammatory response;

18

wolfberry fruit extract

3



19 20

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Introduction Wolfberry is a well-known traditional herbal medicine in East Asia, as well as a health

21

food product in Western countries.1,

22

including polysaccharides, which are often referred to as Lycium barbarum polysaccharides,

23

as well as carotenoids, flavonoids, β-sitosterol, minerals, and vitamins.3-5 There are several

24

human intervention studies regarding the plasma anti-oxidant levels and immunomodulatory

25

effects of wolfberry.6-8 However, although there is a relatively large amount of literature on

26

this subject, the mechanisms activated following wolfberry consumption are not fully

27

understood; hence, there is a need for hypothesis-generating research to identify the

28

unexplored

29

immunomodulatory activities of a commercial product marketed as GoChi (water extract of L.

30

barbarum) have been confirmed.1 Wolfberry is commercially available as two closely related

31

species, L. barbarum and L. chinense.1 GoChi products sold outside Asia exclusively contain

32

L. barbarum berries; on the contrary, L. chinense is used widely in Korea.1, 9 Although L.

33

chinense has traditionally been shown to have similar pharmacological properties to L.

34

barbarum, there is little scientific evidence regarding the properties of L. chinense.

mechanisms

of

the

2

Wolfberry contains many bioactive compounds,

effects

of

wolfberry.

The

anti-oxidative

and

35

Many chronic metabolic diseases, such as hypercholesterolemia, obesity, metabolic

36

syndrome, and type 2 diabetes, are significantly associated with chronic low-grade

37

inflammation and oxidative stress. However, due to the general lack of sensitive and specific

38

biomarkers for these states, it is challenging to determine the degree to which inflammation

39

and oxidative markers are changed by food components or nutrients.10 Recently,

40

nutrigenomic technologies have prompted analyses of various aspects of the oxidative and

41

inflammatory responses that are modulated by nutritional intervention.11

42

The present human intervention study was aimed at assessing the anti-oxidative and anti-

43

inflammatory activity of a standardized wolfberry (L. chinense) fruit aqueous extract (WBE). 4


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The study subjects were mild hypercholesterolemic and overweight subjects who were in a

45

low-grade oxidative and inflammatory stressed state. There were reports that overweight

46

subjects having metabolic dysfunction showed higher concentrations of circulating cytokines

47

and chemokines in overweight subjects compared to normal weight individuals.10 Therefore,

48

in order to measuring anti-oxidative and anti-inflammatory effects of WBE, overweight

49

having hypercholesterolemia subjects were included. The effects of WBE were characterized

50

by measuring established, classic oxidative stress markers, such as anti-oxidant enzyme

51

activities, lipid oxidative markers and deoxyribonucleic acid (DNA) oxidative markers, in

52

combination with profiling targeted oxidative and inflammatory gene expression arrays and

53

postprandial inflammatory proteomic responses.

54 55

Materials and Methods

56

Participants

57

Healthy overweight and mild hypercholesterolemic subjects who were at least 30 y of

58

age, had body mass index (BMI) values (in kg/m2) of 23 to 30, and low-density lipoprotein

59

(LDL)-cholesterol concentrations of 130 to 165 mg/dL were recruited by local advertising.

60

One hundred nineteen subjects were recruited from the CHA Bundang Medical Center

61

(Bundang, Gyeonggi-do, Korea). The study protocol was approved by the Institutional

62

Review Boards of CHA Bundang Medical Center (No. BD2012-149D) and registered at the

63

International Clinical Trials Registry Platform of the WHO (KCT0000729).

64 65

Study design

66

This study was an 8-week, double-blind randomized trial of two parallel groups of free-

67

living subjects. After the initial screening and the subsequent 2-week run-in phase, 53 eligible

68

subjects were randomized into either the WBE or placebo group. The randomization code 5



69

was prepared using random, computer-generated numbers by a staff member who was not

70

involved in running the trial. The principal investigator was provided with a sealed envelope

71

for each subject that was only to be opened after all of the data were entered into the

72

computer or in response to a medical emergency. Allocation concealment was maintained

73

successfully, as no sealed envelope was opened voluntarily or accidently during the study.

74

The participants were advised to maintain their usual lifestyle, level of activity and diet but to

75

avoid consuming anti-oxidant-rich foods during the run-in and intervention periods. The

76

subjects were instructed to ingest one pouch of test sample or placebo (80 mL/pouch) after a

77

meal for 8 weeks. Each test pouch contained 13.5 g of WBE, other colorants and water. To

78

monitor the subjects’ nutrient and dietary compliances, 3-day dietary records were collected

79

during the intervention period. The subjects’ nutrient and energy intakes were calculated

80

using a nutritional analysis program (Can-Pro 3.0 software, Korean Nutrition Society, Seoul,

81

Korea).

82 83

Test drinks

84

WBE was provided by the Rural Development Administration (Jeonju, Korea). Briefly, dried

85

wolfberry fruits were purchased from Cheongyang (Chungcheongnam-do, Korea) and

86

extracted in water at 95°C for 3 h. The placebo beverage contained brown sucrose, dextrin,

87

anhydrous glucose, magnesium chloride, caramel powder, sodium citrate, and citric acid in

88

place of WBE. Placebo and WBE were matched to have the same taste, appearance and

89

flavor using stevioside. Then, the test products were packed in invisible pouches and stored at

90

4°C before use.

91 92 93

Chemical analysis of WBE The extracted sample was completely dried and then stored at -20 °C until the analyses 6


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were conducted. The chemical characteristics of the wolfberry sample were analyzed using

95

ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-tandem mass

96

spectrometry (UHPLC-LTQ-ESI-IT-MS). Three hundred milligrams of the dried sample

97

powder was dissolved in 30 mL of water by brief vortexing and was allowed to partition with

98

ethyl acetate (1:1). Then, the ethyl acetate fraction was dried using a speed-vacuum apparatus.

99

For UPLC-Q-TOF-MS analysis, dried extracts were re-dissolved in methanol and

100

syringe-filtered (0.2 µm PTFE filter, Chromdisc, Daegu, Korea). The UPLC was performed

101

using a Waters Micromass Q-TOF Premier with a UPLC Acquity system (Waters, Milford,

102

MA, USA) equipped with a binary solvent delivery system, an autosampler, and an UV

103

detector. The UPLC system was set up with an Acquity UPLC BEH C18 column [100 mm ×

104

2.1 mm, 1.7 µm particle size; (Waters.)]. The mobile phase consisted of water (A) and

105

acetonitrile (B) with 0.1% formic acid (v/v). The mobile phase gradient system was as

106

follows: 5% B for 1 min, increased to 100% B for 9 min, held at 100% for 1 min, decreased

107

to 5% B over 2 min, and finally held at 5% B for 1 min. Five microliters of a sample extract

108

solution was injected, and the flow rate was maintained at 0.3 mL/min. The waters Q-TOF

109

Primer (Micromass MS Technologies, Manchester, U.K.) was operated in quadrupole mode

110

for the MS data, and the TOF data were collected in the range of m/z 100−1000 in both

111

negative (−) and positive (+) ion modes. The operating parameters were as follows: capillary

112

voltage, 2.8 kV; and cone voltage, ≤40 V; desolvation gas flow, 700 L/h at 200 °C; ion

113

source temperature, 100 °C; The V mode of the mass spectrometer was used, and data were

114

collected in the centroid mode with a scan accumulation time of 0.2 s.

115

A method for tandem mass analysis using UHPLC-LTQ-IT-MS/MS analysis, The LTQ

116

XL linear ion trap mass spectrometer consists of an electrospray interface (Thermo Fisher

117

Scientific, Inc., Waltham, MA, USA) coupled to a Dionex UltiMate 3000 RS Pump, RS 7



118

Autosampler, RS Column Compartment, and RS Diode Array Detector (Dionex Corporation,

119

Sunnyvale, CA, USA). Ten microliters of sample was injected and separated in a Thermo

120

Scientific Syncronis C18 UHPLC column (100 mm × 2.1 mm i.d.; 1.7 µm particle size). The

121

mobile phase composition is same as that described previously. The solvent gradient was

122

increased from 10% to 100% of solvent B over 15 min, maintained for 3 min, and re-

123

equilibrated to the initial condition for 4 min. The column temperature was maintained at 35

124

°C during the measurements. The photodiode array was set to 200–600 nm for detection and

125

managed by a three-dimensional (3D) field. Ion trap analysis was performed in full-scan ion

126

mode within a range of 100–1000 m/z. The operating parameters were set as follows: source

127

voltage, ±5 kV; capillary voltage, 39 V; and capillary temperature, 275 °C. After the full-scan

128

MS analysis, tandem MS analyses were performed using scan-type turbo data-dependent

129

scanning (DDS) under the same conditions used for MS scanning. Metabolites were

130

positively identified using standard compounds. When standard compounds were not

131

available, a tentative identification was made on the basis of MS spectra using the Combined

132

Chemical Dictionary version 7.2 (Chapman & Hall/CRC), references, and an in-house library.

133

Among the chemicals contained in WBE, betaine was used as an index material. For the

134

quantitative analysis, HPLC was performed using a isocratic mobile phase system (Waters

135

e2695, Waters Co., MA, USA) equipped with an evaporative light scattering detector (Waters

136

2424, Waters Co., MA, USA) and a Capcell Pak C18 UG 120 column (4.6×250 mm, 5 µm,

137

Shiseido Co., Tokyo, Japan). The mobile phase was 5% acetonitrile in water (0.32%

138

perfluoropentanoic acid) at a flow rate of 0.5 mL/min. The injection volume was 20 µL. The

139

level of betaine was quantified using standard solutions of 0.00625, 0.012, 0.025, 0.05, 0.1

140

and 0.2 mg/mL betaine.

141 142

Biochemical analysis 8


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After the run-in and the 8-week intervention periods were complete, fasting blood

144

samples were collected from the subjects’ veins into serum separator tubes (BD Biosciences,

145

San Jose, CA, USA). The erythrocyte superoxide dismutase (SOD) and catalase (CAT)

146

activities were determined using the methods described by Miquel J et al. and Johnsson et

147

al.,12, 13 respectively. The erythrocyte glutathione peroxidase (GPx; Cayman, Ann Abor, USA)

148

and plasma oxidized LDL (oxLDL; Mercodia, Uppsala, Sweden) levels were measured using

149

kits. All procedures were performed in accordance with the manufacturer’s instructions. The

150

plasma malondialdehyde (MDA) levels were measured with a high-performance liquid

151

chromatograph (HPLC; Shiseido Co., Ltd., Tokyo, Japan) connected to a fluorescence

152

detector using a reverse-phase column (C18, 4.6 mm inner diameter × 150 mm; Shiseido

153

Co., Ltd., Tokyo, Japan). The endogenous DNA damage in lymphocytes was analyzed using

154

the alkaline comet assay (single-cell gel electrophoresis), according to the method described

155

by Olive et al.14 Hematological and biochemical variables were measured using routine

156

clinical techniques to ensure the subjects’ safety.

157 158

mRNA array analysis

159

Fasting blood was collected after the run-in and the 8-week intervention periods in PAX

160

gene™ blood RNA tubes (Qiagen, Valencia, CA, USA), and RNA was isolated from these

161

blood samples using the PAX gene™ blood RNA kit (Qiagen, Valencia, CA, USA),

162

according to the manufacturer’s instructions. The total RNA concentrations and the 260/280

163

nm ratio were evaluated using a spectrophotometer (Biospec-nano; Shimadzu Corp., Tokyo,

164

Japan). Only samples with a 260/280 nm ratio between 1.7 and 2.1 were processed further.

165

cDNA was generated using a high-quality RNA and a cDNA kit (Bioneer, Daejeon, Korea).

166

PCR was performed with a Step-One-Plus RT-PCR system (Applied Biosystems, Foster City, 9



167

CA, USA) in a 96-well plate using a final volume of 30 µL of the following components: 0.6

168

µL of ROX dye, 1 µL of template, 15 µL of 2X GreenStar qPCR master mix, and 13.4 µL of

169

nanofiltered water (Bioneer, Daejeon, Korea) per well. Amplifications were performed using

170

a 10-min template pre-denaturation step at 95ºC, followed by 40 cycles of 95ºC for 5 sec,

171

58ºC for 25 sec and 72ºC for 30 sec. A total of 88 genes were classified into the following

172

categories and are shown in Supplementary Table 1: inflammatory mediators and signaling

173

molecules (47 genes), plaque formation and coagulation (3 genes), anti-oxidant (14 genes),

174

blood cell differentiation (2 genes) and lipid/lipoprotein metabolism (22 genes). The relative

175

amounts of these mRNAs were normalized to the quantity of beta-2 microglobulin (B2M),

176

and the relative amounts of all RNAs were calculated using the comparative CT method.

177 178

Statistical analysis

179

Sample size was calculated to detect differences in erythrocyte SOD activity between the

180

treated groups. Using a 2-sided 0.05 significance level, the study had greater than 80% power,

181

with up to 20% dropout. All data collection processes and subsequent statistical analysis were

182

performed with the SAS program package version 9.3 (SAS Institute, Cary, NC, USA). The

183

data were presented as the means and standard errors (SE). The variables were tested for a

184

normal distribution using the Shapiro-Wilk W-test, and log transformation was performed on

185

the skewed variables. Differences in the baseline characteristics, blood profiles, nutritional

186

assessment, and biochemical parameters in the different groups were determined with

187

Student’s t-test. Differences in the anti-oxidant variables from the baseline values following

188

the 8-week intervention were compared in the WBE-treated group and placebo group using

189

an analysis of covariance (ANCOVA). Stepwise selection was applied to choose the

190

covariates. For each comparison, P-values less than 0.05 were considered statistically

191

significant. 10


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Results and Discussion

193 194

Chemical profile of WBE

195

The chemical profile is presented in Figure 1A and Table 1. Using UV spectrum and

196

MS/MS pattern, major chemicals contained WBE was tentatively identified. Identified

197

compounds were betaine, lyciumide A, lyciumoside V or IV, lyciumoside III, lyciumoside I

198

and lyciumoside X. Among these constituents, only betaine was determined using standard

199

compound, while others were confirmed based on previous reports owing to lack of standard

200

materials. The major flavonoids in L. barbarum that were investigated included quercetin,

201

caffeoylquinic acid, caffeic acid and vanillic acid.15-18 L. barbarum also contains carotenoids

202

such as zeaxanthin, β-cryptoxanthin and β-carotene.19 However, because WBE was prepared

203

using water extraction from L. chinense, chemical profiles were seemed to be different from

204

previous reports.15-19 Lyciumide A and lyciumosides were reported to be found in Lycium

205

species.,1, 20 however, their physiological activities or relation to the beneficial effects of

206

Lycium species were not reported, yet. Besides differences in species and varieties,

207

phytochemical content of berries depends on geological origin.16 Among these compounds,

208

betaine is the most well-known index material for quality control of L. chinense;21 therefore,

209

the quantity of betaine in WBE was analyzed using standard compounds and determined to

210

be 11.63 ± 0.27 mg/g (Figure 1B).

211 212

Compliance

213

The flow chart of the study is shown in Figure 2. One hundred nineteen participants were

214

screened, and fifty-three eligible subjects were enrolled. No serious adverse events were

215

reported during the study. All participants reported that they adhered to the dietary guideline,

216

which was confirmed by their consistent nutrient intake levels (data not shown). The subjects 11



217

maintained their habitual lifestyle, which was supported by their unchanged BMI, blood

218

pressure, fasting blood glucose, cholesterol and liver metabolizing enzyme levels, such as

219

GOT and GPT (Table 2).

220 221

WBE treatment induced changes in anti-oxidant capacity

222

After the 8-week WBE supplementation, we observed significant changes in the SOD

223

and CAT activities (Figure 3A). SOD activity was significantly reduced in the WBE intake

224

group (P = 0.0209), while CAT activity was slightly but significantly increased in the WBE

225

group (P = 0.0003). The GPx, plasma MDA and oxLDL levels did not change as a result of

226

WBE intake (Figure 3A and B). On the contrary, based on the comet assay, DNA damage

227

(based on the percent of DNA in the tail) was significantly decreased following the 8-week

228

WBE supplementation (P = 0.0003, Figure 3C). In a previous study, the effects of the

229

commercial wolfberry juice GoChi were investigated in young, healthy adults, and the results

230

showed that various oxidative biomarkers, including SOD and GSH-Px activities and MDA

231

levels, were beneficially changed.7 The chemical composition of L. chinense fruits has been

232

reported to be similar to that of L. barbarum.22 According to Korean Pharmacopoeia, L.

233

chinensis and L. barbarum are regarded as same Lycium Fructus.23 Although, these two

234

species have been regarded as similar medicinal plant, the contents of betaine contained in

235

fruits of those species were slightly different.21 Additionally, rutin and chlorogenic acid were

236

reported to be contained more in leaves of L. chinense than that of L. barbarum.24 However,

237

there have been fewer investigations on L. chinense fruit compared to L. barbarum fruit, and

238

there have been no clinical trials of L. chinense. The focus of previous reports regarding the

239

anti-oxidative properties of L. chinense has been on hepatoprotection using in vitro cell lines

240

or animal models.9, 25 Therefore, the present human intervention study was the first trial to

241

examine the effects of L. chinense fruits. In the present study, we confirmed that 8-week 12


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WBE (standardized with betaine) supplementation significantly diminished DNA damage in

243

overweight and mild hypercholesterolemic participants. SOD activity in erythrocytes was

244

slightly but significantly decreased, but CAT activity was increased. Endogenous defense

245

systems against oxidative stress are partly composed of SOD and CAT, which convert

246

superoxide radical anions to H2O2 and continuously reduce H2O2 to water.26, 27 Excessive

247

ROS production can lead to lipid, protein and DNA damage. Endogenous antioxidant enzyme

248

activity can be stimulated indirectly by modulation of ROS signaling pathways; however, in

249

some cases, reduced SOD or CAT activity was found in subjects with elevated oxidative

250

stress, such as those at risk for CVD or non-insulin-dependent diabetes mellitus patients.28-30

251

According to several published reports, the consumption of berries, such as blueberry and

252

bilberry, has a protective effect on DNA base oxidation or antioxidant capacity without

253

affecting erythrocyte SOD activity.31,

254

response to WBE consumption were inconsistent regarding SOD and CAT activities, the

255

differences were extremely small, and DNA damage (comet assay) decreased upon WBE

256

consumption. Therefore, we concluded that the daily consumption of 13.5 g of WBE may

257

help reduce oxidative stress.

32

Therefore, although the significant changes in

258 259

Changes in the mRNA levels of anti-oxidant and inflammatory genes

260

Emerging data have linked ROS generation to inflammatory markers. However,

261

according to several previously published reports, the relief of oxidative stress by the

262

consumption of plant flavonoids or polyphenols has not been proven to reduce inflammatory

263

protein levels.31, 33, 34 Thus, it is worthwhile to explore the mRNA expression levels of genes

264

related to the inflammatory response after WBE consumption to elucidate the molecular

265

mechanisms of WBE in subjects with low-grade inflammation. Considering the complex

266

molecular networks involved in oxidative and inflammatory stress, a huge number of mRNAs 13



267

should be explored. High-throughput array techniques represent a potentially efficient

268

strategy, and among the various array formats, PCR arrays are the most reliable tools for

269

analyzing the expression of a focused panel of genes.35, 36 Therefore, to evaluate whether

270

WBE supplementation may modulate oxidative or inflammatory stress at the mRNA level,

271

we measured the expression profiles of targeted genes involved in the regulation of oxidative

272

or inflammatory responses (88 genes, see Supplementary Table 1S). The altered genes, which

273

exhibited a mean percentage change in response to WBE supplementation, are shown in

274

Table 3 and Figure 4. Thirty-three of these genes showed the highest levels (fold change) and

275

statistically significant differences between the placebo and WBE intake groups (P < 0.05).

276

WBE intervention affected Toll-like receptor 4 (TLR4) and signal transducer and activator of

277

transcription 2 (STAT2). Consequently, WBE intake decreased the expression of translation

278

initiation factor IF-1 (INFA1), TNF, IL-6, tumor necrosis factor receptor superfamily

279

member 1B (TNFRSF1B) and CC chemokine ligand 22 (CCL22), among others. Moreover,

280

in addition to the TLR signaling cascade, WBE also affected platelet endothelial aggregation

281

receptor 1, which is related to plaque formation and coagulation; antioxidant-related genes,

282

such as SOD2, heme oxygenase 1 (HMOX1), cytochrome b-245 beta chain (CYBB),

283

cytochrome b-245 alpha chain (CYBA), neutrophil cytosolic factor 1 (NCF1), thioredoxin

284

reductase 1 (TXNRD1) and advanced glycosylation end product-specific receptor (AGER);

285

and genes related to lipid/lipoprotein metabolism, such as low density lipoprotein receptor

286

(LDLR) and apolipoprotein E (APOE). In agreement with the change in antioxidant capacity,

287

SOD expression levels in whole-cell extracts were down-regulated. Based on these data, we

288

postulated that WBE might have a role as a ROS scavenger, which could explain the decrease

289

in SOD expression and capacity, although CAT activity slightly increased. ROS can damage

290

a large number of molecules, such as lipids, proteins and DNA, and these damaged molecules

291

can stimulate inflammation at the cellular and molecular levels. According to a recent study, 14


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the elevated DNA damage in patients with type 2 diabetes was related to inflammation,

293

according to microarray data, which is in agreement with the findings of the present study.37

294

There is no consensus as to which biomarkers represent low-grade inflammation.10 Thus, a

295

range of blood cell markers, antioxidant capacity, and proinflammatory proteins are

296

frequently measured.38-40 Although we observed no change in oxidative biochemical markers

297

such as MDA and oxLDL, there was a clear indication that WBE influenced inflammatory

298

responses and DNA damage. However, the extent to which these changes are related to health

299

should be elucidated further in future studies.

300

Food components used as supplements must be well characterized in terms of their

301

metabolism, the cellular processes they influence and their associations with specific health

302

outcomes. The lack of WBE chemical compositions or metabolomics data is the major

303

limitation of the present study. Nonetheless, the present data indicate that WBE intake exerts

304

anti-oxidative and anti-inflammatory effects in overweight and hypercholesterolemic subjects

305

by modulating mRNA expression levels. Although the extent to which these changes are

306

related to health remains to be elucidated, the results of our study show that mRNA array

307

technology and techniques to measure perturbations in homeostasis may represent effective

308

strategies for detecting metabolic changes in the human body.

309 310

Abbreviations

311

CAT, catalase; DNA, deoxyribonucleic acid; GPx, glutathione peroxidase; MDA,

312

malondialdehyde; mRNA, messenger RNA; oxLDL, oxidized LDL; SOD, superoxide

313

dismutase.

314 315

Acknowledgements 15



316

We are grateful for the contributions of the participants in this study. Funding was provided

317

by the “Cooperative Research Program for Agriculture Science & Technology Development

318

(No. PJ01169502)”, Rural Development Administration, Republic of Korea, and the Bio-

319

Synergy Research Project (No.2012M3A9C4048761) of the Ministry of Science, ICT and

320

Future Planning through the National Research Foundation.

321

16


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References

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1. Potterat, O., Goji (Lycium barbarum and L. chinense): Phytochemistry, pharmacology and

324

safety in the perspective of traditional uses and recent popularity. Planta Med. 2010, 76, 7-

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2. Lin, C. C.; Chuang, S. C.; Lin, J. M.; Yang, J. J., Evaluation of the antiinflammatory

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hepatoprotective and antioxidant activities of Lycium chinense from Taiwan.

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Phytomedicine : international journal of phytotherapy and phytopharmacology 1997, 4,

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22


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457


Figure captions

458 459

Figure 1. The UPLC/Q-TOF-MS base peak chromatograms in a positive ionization mode of

460

the WBE (A) and HPLC chromatogram of betaine in WBE (B). The numbers of the

461

compounds in parentheses of (A) are the same as those referred to in Table 1.

462 463

Figure 2. Flow diagram of participant selection and analysis.

464 465

Figure 3. The antioxidant biomarkers during the 8-week intervention period. Data are

466

presented as the LS mean ± SE. Statistical significance was analyzed using ANCOVA with

467

stepwise selection of covariates. Bonferroni’s multiple comparison test was performed as the

468

post hoc analysis (*P < 0.05 and **P < 0.01).

469 470

Figure 4. Heat map of the targeted mRNA array data.

471

23



Page 24 of 30

Table 1. Chemical characteristics of WBE analyzed using UPLC-Q-TOF-MS UPLC-Q-TOF-MS NO. Tentative metabolites 1)

UHPLC-LTQ-IT-MS/MS

RT M. W.

-

[M-H]

+

[M+H]

Error 2)

[M+H]+ MSn

(mDa)

MS/MS pattern

3) UV λmax Identified

M. F

(min) 118.0868 C5H11NO2

-0.7

1

Betaine

0.87

117

116.0624

2

Lyciumide A

4.79

313

312.1242 314.1439 C18H19NO4 -0.5

314> 296, 193

220

Ref [17]

3

Lyciumoside V or IV

5.85

776

775.4121 799.4249 C38H64O16

-

-

Ref [21][41]

4

Lyciumoside III

6.03

648

647.3616 671.3737

* 647>485

209, 381 Ref [21], [42]

5

Lyciumoside I

6.19

630

629.3566 653.364

0.9

630>467>305

213, 282 Ref [42][43]

6

Lyciumoside X

6.33

486

485.3096 509.3104 C26H46O8

0.7

509>487,343

218

0.5

C32H56O13 1.4 C32H54O12

118 > 59

(nm) 226, 268 STD

RT, retention time; M.W., molecular weight; M.F. Ref., Reference; *, positive mode; 1) Metabolites were tentatively identified by matching molecular weight and formula, UV λmax, MSn and references. 2) Differences between observed mass and calculated mass. mDa stands for error in milliDaltons. 3) Identification: STD, Ref, references.

24


Ref [43]

Page 25 of 30


Table 2. Pre- and post-intervention characteristics of subjects assigned to each test group Placebo

WBE

P-value2)

51.59 ± 1.461)

49.85 ± 1.40

0.393

3 / 24

9 / 17

0.961

Body mass index (kg/m2)

25.37 ± 0.43

25.53 ± 0.41

0.775

Fasting blood glucose (mg/dL)

103.15 ± 2.43

104.27 ± 1.19

0.651

Fasting insulin (IU/dL)

6.89 ± 0.67

6.32 ± 0.49

0.877

HOMA-IR

1.82 ± 0.21

1.62 ± 0.14

0.953

Total cholesterol (mg/dL)

224.22 ± 3.67

225.00 ± 4.37

0.932

HDL-cholesterol (mg/dL)

54.06 ± 2.29

53.92 ± 1.92

0.928

LDL-cholesterol (mg/dL)

141.44 ± 3.63

139.54 ± 4.19

0.712

Triglyceride (mg/dL)

118.00 ± 7.27

148.58 ± 16.43

0.129

ALT

18.63 ± 2.46

19.00 ± 1.50

0.539

AST

18.11 ± 0.82

19.23 ± 1.21

0.537

GGT

18.63 ± 5.43

24.46 ± 3.30

0.031

Body mass index (kg/m2)

25.37 ± 0.43

25.53 ± 0.41

0.775

Fasting blood glucose (mg/dL)

105.52 ± 2.02

106.96 ± 2.36

0.687

Fasting insulin (IU/dL)

7.25 ± 0.98

6.56 ± 0.57

0.934

HOMA-IR

1.95 ± 0.33

1.73 ± 0.15

0.999

Total cholesterol (mg/dL)

221.22 ± 4.80

232.20 ± 24.90

0.113

HDL-cholesterol (mg/dL)

53.29 ± 2.39

54.87 ± 1.64

0.437

LDL-cholesterol (mg/dL)

139.74 ± 4.91

146.54 ± 4.60

0.304

Triglyceride (mg/dL)

122.93 ± 9.25

134.85 ± 14.15

0.737

ALT

22.48 ± 3.65

19.54 ± 1.91

0.512

AST

21.70 ± 3.05

19.50 ± 1.53

0.591

GGT

19.00 ± 2.98

26.15 ± 3.63

0.084

Variables Age (y) Gender (male/female) Pre-intervention

Post-intervention

1) Data are presented as the mean ± SE. 2) Differences between groups were evaluated using Student's t-test.



Page 26 of 30

Table 3. Significantly changed mRNA expression levels compared to baseline values (arbitrary units)1) Biological function

Gene

Placebo

WBE

P-value2)

TLR4 1.15 ± 0.09 0.69 ± 0.20 0.015 TNF 1.63 ± 0.28 0.74 ± 0.12 0.007 IL6 1.15 ± 0.09 0.62 ± 0.10 0.005 PTGDR 1.46 ± 0.24 0.90 ± 0.24 0.039 IFNA1 0.56 ± 0.15 0.25 ± 0.04 0.024 CCL22 1.07 ± 0.24 0.38 ± 0.04 0.003 SELL 1.19 ± 0.14 0.76 ± 0.18 0.025 ITGAL 2.12 ± 0.39 0.81 ± 0.13 0.005 GRB2 1.05 ± 0.09 0.75 ± 0.15 0.044 STAT3 1.15 ± 0.10 0.57 ± 0.13 0.005 BCL6 1.17 ± 0.10 0.60 ± 0.15 0.002 TNFRSF1B 1.23 ± 0.20 0.63 ± 0.10 0.010 CRLF1 1.29 ± 0.31 0.63 ± 0.09 0.048 AKT1 2.05 ± 0.29 0.86 ± 0.12 0.001 BCL2 1.75 ± 0.29 0.92 ± 0.15 0.025 PEAR1 1.98 ± 0.45 0.62 ± 0.15 0.009 Plaque formation and coagulation SOD2 1.18 ± 0.11 0.63 ± 0.14 0.003 Antioxidant HMOX1 2.00 ± 0.41 0.74 ± 0.19 0.004 CYBB 1.20 ± 0.22 0.65 ± 0.10 0.028 CYBA 1.19 ± 0.19 0.75 ± 0.14 0.035 NCF1 1.28 ± 0.17 0.66 ± 0.08 0.002 TXNRD1 1.27 ± 0.14 0.72 ± 0.12 0.007 AGER 1.39 ± 0.14 0.72 ± 0.13 0.003 CCL21 1.38 ± 0.28 0.48 ± 0.07 0.002 Blood cell differentiation GK 0.95 ± 0.14 0.53 ± 0.11 0.013 Lipid/lipoprotein metabolism LPAR2 1.92 ± 0.39 0.70 ± 0.14 0.008 LDLR 1.17 ± 0.20 0.60 ± 0.10 0.018 SLC27A1 1.12 ± 0.16 0.59 ± 0.07 0.004 SCD 0.86 ± 0.21 0.32 ± 0.06 0.018 ACACB 1.49 ± 0.27 0.89 ± 0.15 0.044 DGKQ 0.86 ± 0.12 0.49 ± 0.07 0.029 MOGAT3 1.31 ± 0.30 0.56 ± 0.11 0.041 APOE 1.70 ± 0.48 0.65 ± 0.09 0.030 1) Changes are presented relative to baseline values. Data are presented as the mean ± SE. Inflammatory signaling molecules

2) Differences between groups after the 8-week intervention were evaluated using Student’s t-test (*P < 0.05)


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Figure graphics




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TOC 338x190mm (96 x 96 DPI)


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Dietary Wolfberry Extract Modifies Oxidative Stress ... - ACS Publications

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