Roasted Barley Extract Affects Blood Flow in the Rat Tail and

Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

Roasted barley extract affects blood flow in the rat tail and increases cutaneous blood flow and skin temperature in humans Hiroshi Ashigai, Yoshimasa Taniguchi, Yasuko Matsukura, Emiko Ikeshima, Keiko Nakashima, Mai Mizutani, and Hiroaki Yajima J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04930 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 44

Journal of Agricultural and Food Chemistry

Title Roasted barley extract affects blood flow in the rat tail and increases cutaneous blood flow and skin temperature in humans

1

Hiroshi Ashigai,†* Yoshimasa Taniguchi,‡ Yasuko Matsukura,‡ Emiko Ikeshima,† Keiko

2

Nakashima,† Mai Mizutani,† and Hiroaki Yajima§

†

Research Laboratories for Health Science & Food Technologies, Kirin Co. Ltd. 1-17-5,

Namamugi, Tsurumi-ku, Yokohama 230-8628, Japan ‡ Research

Laboratories for Key Technologies, Kirin Co. Ltd. 1-13-5 Fukuura, Kanazawa-ku,

Yokohama 236-0004, Japan § Research

& Development Planning Department, Research & Development Division, Kirin

Co., Ltd. 4-10-2 Nakano, Nakano-ku 164-0001, Japan

*

Corresponding author: Hiroshi Ashigai

Tel: +81-90-1930-9950, Fax: +81-45-500-8311, E-mail:[email protected]

1

ACS Paragon Plus Environment


3

Abstract

4

Roasted barley extract (RBE, “Mugicha”) is a traditional Japanese beverage reported to

5

improve blood viscosity and affect food functionality. RBE is suggested to contain

6

2,5-diketopiperazines, which are the functional component with neuroprotective and

7

immunostimulatory effects that are produced in food through roasting. In this study, we

8

investigated the effects of RBE on blood circulation, both clinically and in rats. At first, we

9

confirmed five 2,5-diketopiperazine derivatives in RBE by LC-MS analysis. Secondary, we

10

revealed that RBE affects blood flow in the rat tail, and compared the efficacy on rat tail

11

blood flow among five 2,5-diketopiperazine in RBE. Especially, cyclo (D-Phe-L-Pro) was

12

the most effective in increasing blood flow in the rat tail. We also researched the mechanism

13

of cyclo (D-Phe-L-Pro) with rat aorta study. As results, we confirmed that cyclo

14

(D-Phe-L-Pro) has effect on vasodilatation through the release of nitric oxide in the vascular

15

endothelium. Finally, we also confirmed that RBE affects cutaneous blood flow and increases

16

skin temperature in humans.

17 18

Keywords

2


Page 2 of 44

Page 3 of 44


19

Cyclo (D-Phe-L-Pro); roasted barley extract; vasodilatation; nitric oxide; clinical study

20 21 22

Introduction

23

The traditional Japanese beverage “Mugicha” is a roasted barley extract (RBE) and is

24

suggested to contain the cyclic peptide, 2,5-diketopiperazine, which is a bitter compound

25

found in food ingredients, in particular roasted food ingredients1-3. For example, roasted

26

coffee beans, cocoa beans, and beef contain various 2,5-diketopiperazines4–6.

27

2,5-Diketopiperazine is usually produced during protein decomposition in food via roasting.

28

It has a lactam structure and is formed from the intramolecular cyclization of dipeptides7.

29

Previously, 2,5-diketopiperazine was reported to be a feedback signal molecule of hormone

30

degradation8. Furthermore, cyclo (His-Pro) is reported to be a degradation signal molecule

31

for thyrotropin releasing hormone. Cyclo (His-Pro) has neuroprotective9, anti-tumor10,

32

anti-inflammatory11, anti-obesity12, and immunomodulatory13 effects. Moreover, cyclo

33

(Pro-Phe) has been reported to have a beneficial effect in Alzheimer’s disease14. There have

34

been no reports on whether 2,5-diketopiperazines can affect on blood flow or circulation.

3



35

Blood flow has two important roles in the human body: delivery of bioactive compounds,

36

such as oxygen, carbon dioxide, and various hormones15, around the body, and maintaining

37

homeostasis by regulating osmotic pressure and body temperature16. Blood also serves to

38

remove CO2 and other waste products from the body.

39

In some pathological situations involving blood circulation such as hypertension and

40

arteriosclerosis, vasoconstriction occurs, and blood flow is lower than that in the normal

41

situation17. Furthermore, many people with nervous conditions develop vasoconstriction.

42

In this study, we investigated whether RBE has effect on blood flow in the rat tail and

43

investigated the underlying mechanism in an ex vivo study. Additionally, we compared the

44

efficacies of five 2,5-diketopiperazine compounds derived from RBE. Furthermore, we

45

conducted a randomized, double-blind, placebo-controlled, parallel-group comparison study

46

to investigate the effects of RBE on blood flow in the skin. In addition, the effects of RBE on

47

the temperature of skin under cold water stress were evaluated.

48 49

Materials and Methods

50

Preparation of RBE

4


Page 4 of 44

Page 5 of 44


51

Roasted barley was purchased from MC FOODS Ltd. (Tokyo, Japan). “Mugicha” was

52

prepared by adding 1 L of boiling water to 100 g of roasted barley for 30 min. Unroasted

53

barley extract (URBE) was similarly prepared with unroasted barley.

54 55

Chemicals

56

Cyclo (L-Pro-L-Phe) was purchased from Bachem AG (Bubendorf, Switzerland).

57

2,5-Diketopiperazine derivatives were purchased from Peptide Institute, Inc. (Osaka, Japan).

58

Norepinephrine (NE) was purchased from Wako Pure Chemical Industries (Osaka, Japan).

59

NG-nitro-L-arginine methyl ester (L-NAME) was purchased from Dojindo Laboratories

60

(Kumamoto, Japan).

61 62

Measurement of 2,5-diketopiperazine levels

63

Aliquots (1 µL) of the extracts were analyzed by liquid chromatography-mass spectrometry

64

(LC-MS) using a Develosil column (C30-UG-3; 2.0 × 250 mm; Phenomenex, Torrance, CA,

65

USA) coupled to a 4000 QTRAP LC-MS system (AB Sciex, Tokyo, Japan) in selected-ion

66

monitoring (SIM) mode. The mobile phase consisted of formic acid (0.1% in water) and

5



67

acetonitrile, and was set at a flow rate of 0.2 mL/min. The analysis was started with 10%

68

acetonitrile for 10 min. Next, the acetonitrile content was increased to 30% within 30 min,

69

increased to 100% within 10 min, and then held at 100% for 10 min. The mass spectrometer

70

was carried out with the positive electrospray ionization mode. Scan type was a quadrupole

71

mass spectrometer with a capillary voltage of 3kV. The desolvation temperature was 650°C.

72

Collision gas was argon gas. 2,5-Diketopiperazines were detected by measuring the

73

corresponding mass traces in SIM mode by setting [M+1]+. Cyclo (L-Pro-D-Phe) and cyclo

74

(L-Pro-L-Phe) were detected by 245 (m/z). Cyclo (L-Pro-D-Leu) and cyclo (L-Pro-L-Leu)

75

were detected by 211 (m/z). Cyclo (L-Pro-L-Pro) was detected by 195 (m/z).

76 77

Animals

78

Specific pathogen-free male Wistar rats were purchased from Charles River Laboratories

79

(Yokohama, Japan) and used in this study. The rats were housed under a 12/12-h light/dark

80

cycle in a thermoregulated room maintained at a temperature of 24 ± 2.0°C and a relative

81

humidity of 55 ± 10%. Food (CE-2; Clea, Tokyo, Japan) and water were provided ad libitum.

82

Blood flow measurement was performed at the age of 9–10 weeks, and weigh or 300-350

6


Page 6 of 44

Page 7 of 44


83

g/rat. The animals were treated in accordance with Kirin Pharmaceutical’s ethical guidelines

84

for animal care, handling, and termination.

85 86

Measurement of blood flow in the tails of the rats

87

On the day of the experiment, the animals were not allowed access to food for 16 h prior to

88

the induction of anesthesia. The rats were anesthetized with 1 g/kg urethane by

89

intraperitoneal injection, after which they were cannulated intratracheally. A laser Doppler

90

blood flow meter (ALF-21; Advance Co. Ltd., Tokyo, Japan) was fixed to the tail of each

91

animal for the measurement of blood flow. Analog signals were converted to digital signals

92

using an A/D converter (Power-Lab; AD Instruments, Dunedin, New Zealand). Before the

93

rats were administered any sample, they were stabilized for 15 min to minimize signal

94

fluctuation. After stabilization, water (3.3 ml water/kg) or 2,5-diketopiperazine sample (30

95

µg 2,5-diketopiperazine/3.3 ml water/kg) was injected by intragastric adiministration into the

96

rats, after which blood flow measurement was started20. Data have been expressed as mean ±

97

standard error.

98

7



99

Measurement of vasodilatation of rat aorta rings

100

The rats were adapted to the study environment for at least one week prior to the experiment.

101

Anesthesia was induced with diethyl ether by inhalation, after which the animals were

102

exsanguinated by abdominal aorta dissection. The thoracic aorta was removed and cut into

103

rings (2 mm in length). The rings were set in a 5-mL organ bath filled with Krebs buffer

104

solution (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM NaH2PO4,

105

25.0 mM NaHCO3, and 10 mM glucose; pH 7.35) that was continuously bubbled with 95%

106

O2/5% CO2. Tension was measured with an isometric force transducer (micro easy Magnus

107

UC-2TD; Iwashiya Kishimoto Ika Sangyo, Kyoto, Japan). The rings were equilibrated for 10

108

min before the experiment was started. After equilibration, vessels were contracted by

109

treatment with 0.1 µM NE19,02.

110

Vasodilatation ratio was calculated as follows: We evaluated 1 µM acetyl choline

111

vasodilatation tension as 100% vasodilatation tension19,20. We standardized vasodilatation

112

ratio with 1 µM acetyl choline. After acetyl choline evaluation, we reset the a 5-mL organ

113

bath filled with Krebs buffer solution. Then, we added test chemical (2,5-diketopiperazine

114

derivative) in organ bath, evaluating vasodilatation tension and calculating vasodilation

8


Page 8 of 44

Page 9 of 44


115

tension.

116

For evaluating that vasodilatation was depended on endothelium, we did the vasodilatation

117

experiment with endothelium denuded aorta rings (denuded vessel) 21.

118

For evaluating that vasodilatation was depended on NO synthesis pathway, we did the same

119

vasodilatation experiment with 100 µM L-NAME, which is an inhibitor of NO synthetase22.

120 121

Measurement of NO concentration in rat aorta

122

We evaluated total amount of NO2 and NO3 for NO substitution, because NO is oxidized to

123

NO2 or NO3 immediately. The rats were anesthetized with diethyl ether by inhalation and

124

then exsanguinated by abdominal aorta dissection. The thoracic aorta was removed, cut into

125

rings (2 mm in length), and incubated in the presence of 100 µM cyclo (D-Phe-L-Pro), 1 mM

126

(D-Phe-L-Pro), or 100 µM acetylcholine for 15 min. After incubation, the rings were

127

homogenized in 0.1 mM HCl using a multi-bead shocker (Yasui Kikai Corporation, Osaka,

128

Japan). The homogenates were centrifuged at 15,000 rpm for 10 min, after which the amount

129

of NO2 and NO3 was measured using NO2/NO3 Assay kit (Dojindo Laboratories,, Kumamoto,

130

Japan) according to manufactures’ instruction.

131 132

Ethical considerations for the clinical study

133

This clinical study was performed in accordance with the ethical standards stated in the 9



134

Helsinki Declaration (1964) (revised version) and the ethical guidelines for epidemiological

135

research of the Ministry of Education, Culture, Science, and Technology (Japan), and the

136

Ministry of Health, Labour, and Welfare (Japan). The study protocol (no. 22674) was

137

approved by “The Board of Evaluation for Research Studies involving Human Subjects.”

138

(Osaka, Japan). The clinical trial was performed from September to November 2010.

139 140

Participants

141

All the subjects (n = 48) were healthy Japanese women. They provided informed consent

142

complying with the Helsinki Declaration. Healthy non-smoking women aged 20–40 years

143

and who felt cold sensations were enrolled in the study. The inclusion criteria were as

144

follows: females who were aged 20 to 40 years old, feel cold sensation as subjective

145

symptom, not smoking provided informed consent complying with the Helsinki

146

Declaration. The exclusion criteria were as follows: using oral medications that affect blood

147

circulation, excessive alcohol-drinking behavior, unstable menstrual cycle, undergoing some

148

form of treatment, possible onset of allergic symptoms, history of a serious disease (e.g.,

149

heart disease, diabetes, kidney disease, liver disease), severe anemia, possibility of pregnancy,

10


Page 10 of 44

Page 11 of 44


150

pregnancy, lactation, judged as unsuitable for the study following analysis of a filled

151

questionnaire, planned participation in another clinical study, and judged as unsuitable for the

152

study by the investigator for any other reason.

153 154

Beverages used in the clinical study

155

RBE was prepared as previously indicated. The beverage was diluted with 4000 g water and

156

each subject was administered an aliquot of 250 g. A suitable food color was used to prepare

157

the placebo beverage so that it did not differ in appearance or taste from the test beverage.

158

Other measures were taken to ensure that the two beverages had a similar taste. Before the

159

experiment, the site investigator checked that they were not distinguishable in appearance or

160

taste. Table 1 shows macronutrients of RBE beverage and placebo beverage. The RBE

161

beverage contained 258 µg cyclo (D-Phe-L-Pro)/250 mL bottle.

162 163

Clinical study design

164

The study was a randomized, double-blind, placebo-controlled, parallel-group comparison

165

study. The site investigator enrolled the participants in the study. The participants were

11



166

divided into two groups, RBE or placebo, through stratified randomization by the assigning

167

controller, who kept the assignment list in a sealed container until the trial was completed.

168

Participants, investigators, and other persons concerned with the study, excluding the

169

assigning controller, were blinded to the study. The participants had to drink RBE or the

170

placebo beverage.

171

During the test period, participants were forbidden to consume any pharmaceutical product

172

containing caffeine or vitamin K. Furthermore, on the day before the experiment, participants

173

were forbidden to drink excessively, exercise vigorously, or consume any beverage/food

174

containing an ingredient that has an effect on blood flow. Smoking was forbidden on the

175

experiment day.

176 177

Participants

178

Forty-eight individuals were evaluated to participate in the study. All of them were found to

179

be eligible and were enrolled in the study. No participant dropped out of the study. Each

180

subject underwent a full analysis as a safety evaluation procedure. Supplemental information

181

1 shows the background data of the subjects.

12


Page 12 of 44

Page 13 of 44


182 183

Measurement of skin temperature and blood flow in the skin

184

All subjects were restricted to wear light clothes such as cotton short-sleeved T-shirt on the

185

experiment day. In addition, they had to wear the same clothes on alternate days during the

186

study. The test room conditions were as follows: temperature, 25.5 ± 0.5°C; humidity, 52.5 ±

187

7.5%; and air flow, < 0.1 m/sec. Subjects were allowed to acclimatize to the test room

188

conditions for 45 min before the experiment was started. Subject drank the test beverage 250

189

mL/bottle before cold stress (-10 min). Next, the subjects were asked to put their left hand in

190

cold water (15°C) for 1 min. Cold water set by thermos-stated water bath. Subjects remained

191

seated and did not move during this period. The second finger of the left hand was used as the

192

skin site in the measurements. Blood flow and temperature at this site were measured at

193

intervals of 5, 10, 15, 20, and 30 min. A laser Doppler blood perfusion imager (Periscan PIM

194

3 system; Perimed AB, Datavägen, Sweden) was used for blood flow measurement. Skin

195

temperature was estimated by infrared thermography (H2640; Nippon Avionics Company,

196

Tokyo, Japan). Fig. 1 is a schematic representation of the present study.

197

13



198

Statistical analysis

199

Results have been expressed as mean ± standard deviation of the mean. Ekuseru-Toukei 2010

200

(Social Survey Research Information Co. Ltd., Tokyo, Japan) was used for the statistical

201

analysis. Data obtained from the animal experiments were analyzed by Dunnett’s test or

202

unpaired t-test. Significant differences in data between RBE- and placebo-treated groups in

203

the clinical study were estimated using unpaired t-test. Statistical significance was considered

204

at p < 0.05.

205 206

Results

207

Measurement of the amounts of 2,5-diketopiperazine derivatives in RBE

208

The LC-MS analysis showed that RBE contained five 2,5-diketopiperazine derivatives (Table

209

2). Cyclo (D-Phe-L-Pro) was 2.71 ppm, it was the largest amount 2,5-diketopiperazine in

210

RBE. URBE contained almost no 2,5-diketopiperazine (Table 2).

211 212

RBE increased blood flow in the rat tails

213

The effect of RBE on cutaneous blood flow was examined in the animal study. Fig. 2 shows

14


Page 14 of 44

Page 15 of 44


214

the changes in blood flow at the proximal site of the dorsal tail skin after treatment with RBE.

215

Treatment with water (control) led to a decline in blood flow of the rat tail. Rat tails’ blood

216

flow of RBE administration group was higher than control group in 10, 20, 30 min (Fig. 2).

217

We confirmed that RBE administration didn’t lower rat tail blood flow rather than water

218

administration.

219 220

Comparing the efficacies of 2,5-diketopiperazine derivatives on blood flow in the rat tail

221

Fig. 3 shows the changes in blood flow at the proximal site of the dorsal tail skin after

222

treatment with 2,5-diketopiperazines in the rats. Cyclo (D-Phe-L-Pro) caused the most

223

effective increase in blood flow in the tails of the animals (Fig. 3). Additionally, it was

224

observed that cyclo (D-Phe-L-Pro) increased blood flow in a concentration-dependent

225

manner (Fig. 4).

226 227

Vasodilatation experiment with rat aorta rings

228

Cyclo (D-Phe-L-Pro) induced vasodilatation in NE-treated rat aorta rings (Fig. 5). However,

229

this effect was suppressed by the removal of the endothelium (Fig. 5). L-NAME inhibited

15



230

cyclo (D-Phe-L-Pro) vasodilatation (Fig. 5). These results show that Cyclo (D-Phe-L-Pro)

231

has effect on vasodilatation depending on endothelium and related to NO synthetase.

232 233

NO measurement in rat aorta rings

234

Cyclo (D-Phe-L-Pro) raised total NO2 and NO3 production in concentration depending

235

manner (Fig. 6). Acetyl choline (Ach) was used as positive control, Ach also raised total NO2

236

and NO3 production (Fig. 6). This result shows that cyclo (D-Phe-L-Pro) induces NO

237

production in rat aorta (blood vessel).

238 239

Effects of RBE on blood flow in the human skin

240

Blood flow in the skin was higher in the RBE-treated subjects (p < 0.05 at 10, 15, 20, and 30

241

min) than it was in the placebo-treated subjects (Fig. 7(a)). Additionally, RBE induced

242

significantly higher changes in blood flow at 15, 20, and 30 min (Fig. 7(b)). We confirmed

243

that RBE administraion causes rapidly elevation of skin blood frow rather than placebo.

244 245

Effects of RBE on human skin temperature

16


Page 16 of 44

Page 17 of 44


246

Skin temperature was higher in the RBE-treated group (p < 0.05 at 5, 10, 15, 20, and 30 min)

247

than it was in the placebo-treated group (Fig. 8(a)). Furthermore, RBE induced a higher

248

change in skin temperature from 0 min than the placebo did (Fig. 8(b)). We confirmed that

249

RBE administraion causes rapidly elevation of skin temperature rather than placebo.

250 251

Safety endpoints

252

No adverse events occurred during the study.

253 254

Discussion

255

Blood flow is mainly regulated by endothelium-derived relaxing factors, such as nitric oxide

256

(NO)23 and prostacyclin24, and the autonomic nervous system. NO plays important roles in

257

vasodilatation and the control of physiological conditions23. It activates guanylate cyclase in

258

smooth muscles and causes vessel relaxation24. Cutaneous arterial sympathetic nerve activity

259

(CASNA), one of the functions of the autonomic nervous system, is involved in the control of

260

blood flow in the skin18.

261

The results revealed that RBE contains five 2,5-diketopiperazine derivatives, whereas URBE

17



262

barely contains any 2,5-diketopiperazines. Roasting food ingredient is one of the major

263

synthetic methods to generate 2,5-diketopiperazines25. In the present study, it was assumed

264

that the 2,5-diketopiperazine derivatives in RBE were generated during roasting of the barley.

265

The results also showed that RBE increases blood flow in the rat tail (Fig. 2). In the present

266

study, cyclo (D-Phe-L-Pro) and cyclo (L-Pro-D-Leu) significantly increased blood flow in

267

the tails of the rats when their effects were compared to those of the control treatment (Fig. 3).

268

It was noted that cyclo (D-Phe-L-Pro) showed the highest efficacy among the five

269

2,5-diketopiperazines detected in RBE (Fig. 3). Moreover, it was the most abundant

270

2,5-diketopiperazine in RBE (Table 1). Therefore, we believe that cyclo (D-Phe-L-Pro) is the

271

key chemical responsible for the effect of RBE on blood circulation. We also found that

272

cyclo (D-Phe-L-Pro) increased blood flow in a concentration-dependent manner (Fig. 4)

273

Blood flow in the tails of the control rats slightly declined after the treatment was

274

administered. In the present study, we induced anesthesia with urethane, which activates the

275

sympathetic nervous system by causing the release of adrenaline26. Adrenalin induced by

276

urethane caused vasoconstriction and a decline in peripheral blood flow27.

277

Furthermore, the results indicated that cyclo (D-Phe-L-Pro) has an effect on vasodilatation

18


Page 18 of 44

Page 19 of 44


278

(Fig. 5). We also observed that the vasodilatory effect of cyclo (D-Phe-L-Pro) was

279

diminished

280

cyclo-(D-Phe-L-Pro)-induced vasodilatation is dependent on the vascular endothelium (Fig.

281

5). There are two mechanisms that underlie vasodilatation in the endothelium: the NO-cyclic

282

guanosine monophosphate (cGMP) pathway23 and the prostanoid-cyclic adenosine

283

monophosphate pathway24. We found that cyclo-(D-Phe-L-Pro)-induced vasodilatation

284

occurs via the NO-cGMP pathway. This is because L-NAME, which is an inhibitor of

285

endothelial NO synthase (eNOS), inhibited cyclo-(D-Phe-L-Pro)-induced vasodilatation (Fig.

286

5). We also confirmed that cyclo-(D-Phe-L-Pro) caused NO production in rat aorta (Fig. 6).

287

NO activates guanylate cyclase in vascular smooth muscle cells, which results in an increase

288

in cGMP levels. Consequently, Ca2+ ions are released from vascular smooth muscle cells, and

289

this result in vasodilatation. The results of the study show that cyclo-(D-Phe-L-Pro)-induced

290

vasodilatation occurs via NO production resulting from eNOS activation in the endothelium.

291

Blood flow is controlled by the autonomic nervous system18. In the present study, we focused

292

on vasodilatation mechanisms that occur through effects on the endothelium. In future studies,

293

we hope to investigate whether cyclo (D-Phe-L-Pro) has an effect on the autonomic nervous

in

denuded

aorta

rings

(Fig.

5).

Therefore,

19


it

appears

that


294

system.

295

In previous study, 2,5-diketopiperazine derivatives were reported to be metabolized like

296

dipeptide, Mizuma reported that cyclo (Gly-Phe) or cyclo (Ser-Tyr) are imported via peptide

297

transporter: PEPT1, and metabolized to amino acids by peptidase28-31. 2,5-diketopiperazine

298

derivatives are immediatly introduced in circulation after orally administration25. We assume

299

that cyclo-(D-Phe-L-Pro) is also absorbed through PEPT1 and absorbed in circulation

300

immediately.

301

In the present study, we were unable to clarify what the receptor for cyclo (D-Phe-L-Pro) is.

302

In a previous study, 2,5-diketopiperazine derivatives were shown to have an effect on

303

transient receptor potential melastatin 8 (TRPM8) channel32, which is involved in

304

vasodilatation33. Therefore, we assumed that cyclo (D-Phe-L-Pro) is also a TRPM8 channel

305

agonist. Menthol is a known TRPM8 channel agonist33. L-NAME inhibits TRPM8 channel

306

activity of TRPM833. These results support our hypothesis that cyclo (D-Phe-L-Pro) and

307

menthol have a similar mechanism of action.

308

Generally, dysfunctions in blood flow circulation cause a cold sensation34. We observed that

309

RBE induced higher changes in blood flow in the skin than the placebo did (Fig. 7). Similar

20


Page 20 of 44

Page 21 of 44


310

results were obtained in the animal study (Fig. 2).

311

We also observed that changes in skin temperature were higher in the RBE-treated group than

312

they were in the placebo-treated group (Fig. 8). These results strongly indicate that RBE has

313

an effect on the control of skin temperature in humans. The changes in skin temperature were

314

similar to the changes in blood flow in the skin (Fig. 8). Regarding the control of body

315

temperature, it is reported that blood flow has a thermal effect in homeostasis in humans35.

316

However, we assumed that a rapid increase in skin temperature was caused by a thermal

317

transfer from the core of the body to peripheral skin tissues though blood circulation.

318

In previous study, there were no reports that RBE has effect on blood circulation. We had

319

firstly demonstrated that the RBE has effect on blood circulation with animal and clinical

320

study. Moreover, we revealed that cyclo-(D-Phe-L-Pro) in RBE also has effect on blood flow

321

circulation and vasodilatation via NO production in endothelium.

322

Blood flow circulation is related to hypertension36, arteriosclerosis37, and cold sensation38.

323

Although RBE might be beneficial in improving the above conditions, blood flow is also

324

regulated by the autonomic nervous systems34. RBE may have effects on the autonomic

325

nervous system. In a previous study, cold water stress activated CASNA and induced

21



326

vasoconstriction39. In the present study, we confirmed that RBE suppressed CASNA (data not

327

shown). However, it was assumed that RBE has an effect on blood flow regulation through

328

CASNA.

329

One limitation of this study is that only Japanese subjects were enrolled. We hope that

330

clinical studies on RBE will be conducted in other countries.

331 332

Abbreviations used: RBE, Roasted barley extract; L-NAME, NG-Nitro-L-arginine methyl

333

ester hydrochloride; NE, Norepinephrine; CASNA, Cutaneous arterial sympathetic nerve

334

activity; URBE, Unroasted barley extract; NO, nitric oxide; eNOS, endothelial NO synthase;

335

TRPM8, transient receptor potential melastatin 8.

336 337

Acknowledgment

338

We thank Kyoko Kato for the helpful technical support.

339 340

Conflict of interest

341

HA, YT, YM, EI, MM, and HY are employees of Kirin Co. Ltd., the study sponsor.

22


Page 22 of 44

Page 23 of 44


342 343 344

References

345

1.

346 347

bioactive natural products. Chem. Rev. 2012, 112, 3641–3716. 2.

348 349

Borthwick, A.D. 2,5-Diketopiperazines: synthesis, reactions, medicinal chemistry, and

Borthwick, A.D.; Da Costa, N.C. 2,5-diketopiperazines in food and beverages: Taste and bioactivity. Crit. Rev. Food Sci. Nutr. 2017, 57, 718–742.

3.

Ryan, L.A.; Dal Bello, F.; Arendt, E.K.; Koehler, P. Detection and quantitation of

350

2,5-diketopiperazines in wheat sourdough and bread. J. Agric. Food Chem. 2009, 57,

351

9563–9568.

352

4.

353 354

Ginz, M.; Engelhardt, U.H. Identification of proline-based diketopiperazines in roasted coffee. J. Agric. Food Chem. 2000, 48, 3528–3532.

5.

Stark, T.; Hofmann, T. Structures, sensory activity, and dose/response functions of

355

2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao). J. Agric. Food Chem.

356

2005, 53, 7222–7231.

23



357

6.

Chen, M.Z.; Dewis, M.L.; Kraut, K.; Merritt, D.; Reiber, L.; Trinnaman, L.; Da Costa,

358

N.C. 2, 5-diketopiperazines (cyclic dipeptides) in beef: identification, synthesis, and

359

sensory evaluation. J. Food Sci. 2009, 74, C100–C105.

360

7.

Wyatt, P.G.; Allen, M.J.; Borthwick, A.D.; Davies, D.E.; Exall, A.M.; Hatley, R.J.; Irving,

361

W.R.; Livermore, D.G.; Miller, N.D.; Nerozzi, F.; Sollis, S.L.; Szardenings, A.K.

362

2,5-Diketopiperazines as potent and selective oxytocin antagonists 1: Identification,

363

stereochemistry and initial SAR. Bioorg. Med. Chem. Lett. 2005, 15, 2579–2582.

364

8.

Kanzaki, H.; Yanagisawa, S.; Nitoda, T. Enzymatic synthesis of dehydro cyclo(His–Phe)s,

365

analogs of the potent cell cycle inhibitor, dehydrophenylahistin, and their inhibitory

366

activities toward cell division. Biosci. Biotechnol. Biochem. 2004, 68, 2341–2345.

367 368 369

9.

Cornacchia, C.; Cacciatore, I.; Baldassarre, L.; Mollica, A.; Feliciani, F.; Pinnen, F. 2,5-diketopiperazines as neuroprotective agents. Mini Rev. Med. Chem. 2012, 12, 2–12.

10. Mollica, A.; Costante, R.; Fiorito, S.; Genovese, S.; Stefanucci, A.; Mathieu, V.; Kiss, R.;

370

Epifano, F. Synthesis and anti-cancer activity of naturally occurring

371

2,5-diketopiperazines. Fitoterapia 2014, 98, 91–97.

24


Page 24 of 44

Page 25 of 44

372


11. Nishanth Kumar, S.; Dileep, C.; Mohandas, C.; Nambisan, B.; Ca, J.

373

Cyclo(D-Tyr-D-Phe): a new antibacterial, anticancer, and antioxidant cyclic dipeptide

374

from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode. J.

375

Pept. Sci. 2014, 20, 173–185.

376

12. Song, M.K.; Rosenthal, M.J.; Song, A.M.; Uyemura, K.; Yang, H.; Ament, M.E.;

377

Yamaguchi, D.T.; Cornford, E.M. Body weight reduction in rats by oral treatment with

378

zinc plus cyclo-(His-Pro). Br. J. Pharmacol. 2009, 158, 442–450.

379

13. Kim, K.; Kim, N.J.; Kim, S.Y.; Kim, I.H.; Kim, K.S.; Lee, G.R. Cyclo(Phe-Pro)

380

produced by the human pathogen Vibrio vulnificus inhibits host innate immune responses

381

through the NF-κB pathway. Infect. Immun. 2015, 83, 1150–1161.

382

14. Song, M.K.; Bischoff, D.S.; Song, A.M.; Uyemura, K.; Yamaguchi, D.T. Metabolic

383

relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus

384

zinc treatment. BBA Clin. 2016, 7, 41–54.

385 386

15. de Aguilar-Nascimento, J.E. The role of macronutrients in gastrointestinal blood flow. Curr. Opin. Clin. Nutr. Metab. Care 2005, 8, 552–556.

25



387

16. Kadekaro, M.; Summy-Long, J.Y. Centrally produced nitric oxide and the regulation of

388

body fluid and blood pressure homeostases. Clin. Exp. Pharmacol. Physiol. 2000, 27,

389

450–459.

390 391 392

17. González-Alonso, J. Human thermoregulation and the cardiovascular system. Exp. Physiol. 2012, 97, 340–346. 18. Horii, Y.; Tanida, M.; Shen, J.; Fujisaki, Y.; Fuyuki, R.; Hashimoto, K.; Niijima, A.;

393

Nakashima, T.; Nagai, K. Skin application of urea-containing cream affected cutaneous

394

arterial sympathetic nerve activity, blood flow, and water evaporation. Skin Res. Technol.

395

2011, 17, 75–81.

396 397

19. Zhao, Y.; Vanhoutte, P.M.; Leung, S.W. Vascular nitric oxide: Beyond eNOS. J. Pharmacol. Sci. 2015, 129, 83–94.

398

20. Lindauer, U.; Megow, D.; Matsuda, H.; Dirnagl, U. Nitric oxide: a modulator, but not a

399

mediator, of neurovascular coupling in rat somatosensory cortex. Am. J. Physiol. 1999,

400

277, H799–H811.

26


Page 26 of 44

Page 27 of 44

401


21. Yanaga, A.; Goto, H.; Nakagawa, T.; Hikiami, H.; Shibahara, N.; Shimada, Y.

402

Cinnamaldehyde induces endothelium-dependent and -independent vasorelaxant action

403

on isolated rat aorta. Biol. Pharm. Bull. 2006, 29, 2415–2418.

404

22. Sasaki, Y.; Suzuki, M.; Matsumoto, T.; Hosokawa, T.; Kobayashi, T.; Kamata, K.;

405

Nagumo, S. Vasorelaxant activity of Sappan Lignum constituents and extracts on rat

406

aorta and mesenteric artery. Biol. Pharm. Bull. 2010, 33, 1555–1560.

407

23. Patel, R.P.; Hogg, N.; Kim-Shapiro, D.B. The potential role of the red blood cell in

408

nitrite-dependent regulation of blood flow. Cardiovasc. Res. 2011, 89, 507–515.

409

24. Hristovska, A.M.; Rasmussen, L.E.; Hansen, P.B.; Nielsen, S.S.; Nüsing, R.M.;

410

Narumiya, S.; Vanhoutte, P.; Skøtt, O.; Jensen, B.L. Prostaglandin E2 induces vascular

411

relaxation by E-prostanoid 4 receptor-mediated activation of endothelial nitric oxide

412

synthase. Hypertension 2007, 50, 525–530.

413

25. Taga Y, Kusubata M, Ogawa-Goto K, Hattori S. Identification of Collagen-Derived

414

Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability:

415

Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating. J

416

Agric Food Chem. 2017, 65, 9514-9521.

27



417

26. Himori, N., Ishimori, T. Different responses to beta-adrenoceptor blocking drugs of the

418

blood pressure and heart rate in the urethane-anesthetized dog and rat. Jpn. J. Pharmacol.

419

1988, 47, 71–80.

420

27. Horii, Y., Fujisaki, Y., Fuyuki, R., Nagai, K. L-Carnosine's dose-dependent effects on

421

muscle sympathetic nerves and blood flow. Neurosci. Lett. 2015, 591, 144–148.

422 423 424

28. Mizuma T, Masubuchi S, Awazu S. Intestinal absorption of stable cyclic dipeptides by the oligopeptide transporter in rat. J Pharm Pharmacol. 1998, 50, 167-172. 29. Mizuma T, Masubuchi S, Awazu S. Intestinal absorption of stable cyclic

425

glycylphenylalanine: comparison with the linear form. J Pharm Pharmacol. 1997, 49,

426

1067-1071.

427

30. Brandsch M, Knütter I, Thunecke F, Hartrodt B, Born I, Börner V, Hirche F, Fischer G,

428

Neubert K. Decisive structural determinants for the interaction of proline derivatives

429

with the intestinal H+/peptide symporter. Eur J Biochem. 1999, 266, 502-508.

430

31. Tateoka R, Abe H, Miyauchi S, Shuto S, Matsuda A, Kobayashi M, Miyazaki K, Kamo

431

N. Significance of substrate hydrophobicity for recognition by an oligopeptide

432

transporter (PEPT1). Bioconjug Chem. 2001, 12, 485-492.

28


Page 28 of 44

Page 29 of 44

433


32. De Petrocellis, L.; Arroyo, F.J.; Orlando, P.; Schiano Moriello, A.; Vitale, R.M.; Amodeo,

434

P.; Sánchez, A.; Roncero, C.; Bianchini, G.; Martín, M.A.; López-Alvarado, P.;

435

Menéndez, J.C. Tetrahydroisoquinoline-derived urea and 2,5-diketopiperazine

436

derivatives as selective antagonists of the transient receptor potential melastatin 8

437

(TRPM8) channel receptor and antiprostate cancer agents. J. Med. Chem. 2016, 59,

438

5661–5683.

439

33. Johnson, C.D.; Melanaphy, D.; Purse, A.; Stokesberry, S.A.; Dickson, P.; Zholos, A.V.

440

Transient receptor potential melastatin 8 channel involvement in the regulation of

441

vascular tone. Am. J. Physiol. Heart Circ. Physiol. 2009, 296, H1868–H1877.

442 443 444 445 446 447

34. Lahera, V.; Navarro-Cid, J.; Cachofeiro, V.; García-Estañ, J.; Ruilope, L.M. Nitric oxide, the kidney, and hypertension. Am. J. Hypertens. 1997, 10, 129–140. 35. González-Alonso, J. Human thermoregulation and the cardiovascular system. Exp. Physiol. 2012, 97, 340–346. 36. Prior, B.M.; Lloyd, P.G.; Ren, J.; Li, Z.; Yang, H.T.; Laughlin, M.H.; Terjung, R.L. Arteriogenesis: role of nitric oxide. Endothelium 2003, 10, 207–216.

29



448

37. Galea, E.; Golanov, E.V.; Feinstein, D.L.; Kobylarz, K.A.; Glickstein, S.B.; Reis, D.J.

449

Cerebellar stimulation reduces inducible nitric oxide synthase expression and protects

450

brain from ischemia. Am. J. Physiol. 1998, 274, H2035–H2045.

451 452

38. Liu, C.; Yavar, Z.; Sun, Q. Cardiovascular response to thermoregulatory challenges. Am. J. Physiol. Heart Circ. Physiol. 2015, 309, H1793–H1812.

453

39. Takumi, H.; Fujishima, N.; Shiraishi, K.; Mori, Y.; Ariyama, A.; Kometani, T.;

454

40. Hashimoto, S.; Nadamoto, T. Effects of alpha-glucosylhesperidin on the peripheral body

455

temperature and autonomic nervous system. Biosci. Biotechnol. Biochem. 2010, 74, 707–

456

715.

457

30


Page 30 of 44

Page 31 of 44


458

Fig. 1 Schematic representation of intervention.

459

Subject was acclimatized with test room condition for 45 min. Before intake of test beverage,

460

we measured subject skin blood flow and skin temperature. After drinking test beverage,

461

subject put their hand into cold water (15ºC) for 1min. Then subject had to sit on the chair

462

and keep their body position same (not moving), and we measured their skin blood flow and

463

skin temperature every 5 min for 30 min.

464 465

Fig. 2 Rat tail’s blood flow change on administrating roasted barley extract (RBE).

466

Control(●), RBE (▲).

467

flow of 0min. The differences between control and RBE administration were statistically

468

significant by student t-test (p

Roasted Barley Extract Affects Blood Flow in the Rat Tail and

Recommend Documents