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Elucidating the improvement in vascular endothelial function of Sakurajima Daikon and its mechanism of action: a comparative study with Raphanus sativus Rei Kuroda, Kimiko Kazumura, Miki Ushikata, Yuji Minami, and Katsuko Kajiya J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01750 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Journal of Agricultural and Food Chemistry

Elucidating the improvement in vascular endothelial function of Sakurajima Daikon and its mechanism of action: a comparative study with Raphanus sativus

Rei Kuroda1, Kimiko Kazumura2, Miki Ushikata3, Yuji Minami3, Katsuko Kajiya3* 1

Major in Biochemical Science & Technology, Graduate School of Agriculture, Kagoshima

University, Kagoshima, Japan 2

Central Research Laboratory, Hamamatsu Photonics K. K., Japan

3

Department of Food Science & Biotechnology, Faculty of Agriculture, Kagoshima University,

Kagoshima, Japan

*Corresponding author E-mail: [email protected] Phone: +81-99-285-8631

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ACS Paragon Plus Environment


1

Abstract

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Vascular diseases such as myocardial or cerebral infarction are the leading causes of death. Some

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vascular diseases occur due to a decrease in vascular endothelial function. The innermost layer of the

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vasculature is formed by vascular endothelial cells (VECs) that are critical for nitric oxide (NO)

5

synthesis. In our search for active constituents in farm products with the potential for improving the

6

vascular system, we examined the effect of Raphanus sativus cv. Sakurajima Daikon on NO

7

production in VECs. In this study, we found that the underlying mechanism for stimulating NO

8

production by Sakurajima Daikon extract involves endothelial NO synthase (eNOS) activation by the

9

phosphorylation of Ser1177 and the dephosphorylation of Thr495, which is triggered by elevated

10

concentrations of cytoplasmic Ca2+, resulting from the activation of Ca2+ channels in VECs. We

11

observed that trigonelline, an active constituent of Sakurajima Daikon, improves the NO production

12

in VEC cultures.

13 14

Keywords: Raphanus sativus, endothelial function, nitric oxide, simultaneous monitoring system

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INTRODUCTION

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Cerebrovascular diseases, such as stroke, and heart diseases, such as angina pectoris and myocardial

18

infarction, account for nearly 25% of deaths in Japan1 and the world2. Since brain and heart functions

19

remain normal until just prior to attacks, these diseases are not caused by organ dysfunction, but by

20

an impairment of blood vessel networks in those organs. The economic burden of vascular diseases

21

on patients and society is indicated by the fact that these patients endure not only long-term

22

treatments and treatment sequelae, but also are confined to bed for extended periods. Hence, there is

23

a need to improve vascular function and prevent vascular diseases.

24

Blood vessels consist of three layers, the outermost tunica adventitia, the tunica media, and the

25

innermost tunica intima containing vascular endothelial cells (VECs). Nitric oxide (NO) is released

26

from VECs to protect blood vessels by regulating their contraction and relaxation and by preventing

27

thrombus formation caused by the attachment of white blood cells and other blood components to the

28

vascular endothelium. However, if VECs are damaged by oxidative stress caused by reactive oxygen

29

species or oxidized low-density lipoprotein (LDL), the production of NO is suppressed, increasing

30

the risk of cardiovascular diseases. Thus, improving the NO production by VECs is critical for

31

protecting blood vessels.

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The Kagoshima Prefecture in Japan is famous for the largest radish cultivar, Raphanus sativus cv.

34

Sakurajima Daikon (Sakurajima Daikon; Figure 1A), which was certified as the world’s biggest

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35

radish by the Guinness Book of Records3. The radishes regularly weigh about 4 to 5 kg, but large

36

ones weigh around 30 kg, with a girth of approximately 110 cm. Although the common radish

37

reportedly possesses antioxidant, antihypertensive, and anti-thrombogenic activities4-6, there are no

38

studies that directly compare the potential health benefits, like improvement of blood vessel function,

39

of Sakurajima Daikon with the benefits of a common variety such as Raphanus sativus var.

40

Longipinnatus (Aokubi Daikon). Here, we used Aokubi Daikon as a reference in our study about the

41

effect of Sakurajima Daikon on the NO production in human coronary artery endothelial cells and

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porcine aortic endothelial cells and in our analysis of the underlying mechanism of this effect that

43

potentially applies to blood vessels.

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MATERIALS AND METHODS

46

Materials

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Human and porcine VECs were purchased from KURABOU INDUSTRIES LTD. (Osaka, Japan)

48

and Cosmo Bio Co. Ltd. (Tokyo, Japan), respectively. Experiments were performed using both

49

human and porcine VECs but the figures were prepared using the data of the porcine VECs

50

experiments that included a high number of replicates (n=8). [1,2-a]pyrazine-3-one hydrochloride

51

(MCLA), diaminofluorescein-2 diacetate (DAF-2 DA ), and Fluo4-acetoxy methyl ester (Fluo4-AM)

52

obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), GORYO Chemical Inc.(Hokkaido,

53

Japan), and DOJINDO Laboratories (Kumamoto, Japan). Trigonelline and γ-aminobutyric acid

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(GABA) were purchased from FUJIFILM Wako Chemical Corporation (Osaka, Japan). Western blot

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reagents obtained from Bio-Rad Laboratories. Inc. (CA, USA). The primary antibodies (β-Actin,

56

1/5000; eNOS, 1/1000; P-eNOS (Ser1177), 1/1000; P-eNOS (Thr495), 1/1000) and the secondary

57

antibody (anti-rabbit IgG, HRP-linked antibody, 1/10,000) were purchased from Cell Signaling

58

Technology (MA, USA).

59 60

Sample preparation

61

Sakurajima Daikon cultivated in Kagoshima City, Japan, was used in this study. Aokubi Daikon was

62

obtained from the Kagoshima Prefectural Institute for Agricultural Development, Japan, and used as

63

reference material. The crops were harvested in January of 2017. Uto et al. investigated the effects of

64

different parts of Angelica acutiloba Kitagawa, such as the aerial parts and the root, on NO

65

production and isolated constituents with anti-inflammatory activity from its aerial parts7. In this

66

study, after removing all the inedible material from the radish, the edible parts like root, peel, and

67

leaves were separated and cut into small pieces, processed using a homogenizer, and lyophilized to

68

generate powdered raw material. One milliliter of methanol/H2O/acetic acid solvent (95.0/9.5/0.5,

69

v/v/v) was added to 25 mg raw material and mixed in a vortex followed by 5 min of ultrasonic

70

treatment. The sample was centrifuged twice at 1,600×g for 10 min at 4 °C; the supernatant was

71

collected and concentrated by drying. The dry sample material was weighed and, prior to

72

experiments, dissolved in an appropriate solvent.

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73 74

NO quantification using a modified Griess method and a fluorescence method

75

Because NO has a short half-life and is rapidly oxidized to NO2- and NO3- in aqueous solution, its

76

concentration is indirectly determined via NO2-. Nitrate reductase-mediated reduction of NO3- is used

77

to ensure that the NO2- concentration represents the original NO level of a sample. Typically, NO2- is

78

measured using the Griess method8. A fluorescence method9 using 2,3-diaminonaphthalene (DAN) is

79

a newer NO2- assay with higher sensitivity than the Griess method. Because NO2- reacts with DAN

80

under acidic conditions to form a fluorescent adduct, naphthalenetriazole, we quantified the product

81

by measuring its fluorescence intensity with a microplate reader (TECAN). VECs of the normal

82

human coronary artery and normal porcine aorta were adjusted to 5.0 × 104 cells/mL and cultured in

83

96-well plates until 80% confluency was reached. Then, incubation continued overnight (12 h) in

84

medium either with or without the radish extract supplement. Culture supernatants were collected,

85

cleared from cells, and reduced by a 30-min incubation at 37 °C with nitrate reductase and respective

86

enzyme cofactors (iron, molybdenum, and cytochrome), followed by a 15-min incubation with DAN.

87

The assay was terminated by measuring the fluorescence intensity (λex=360 nm, λem=450 nm). The

88

amount of NO per sample was calculated by transforming raw data, using a calibration curve

89

prepared with NaNO3, and expressing the result as a relative value derived from a comparison with a

90

control value of 1. The t-test was applied for statistical analysis.

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A simultaneous monitoring system using fluorescence and chemiluminescence for real-time

93

measurement of the NO production, the cytoplasmic Ca2+ concentration, and the production of

94

superoxide anion radical O2-

95

A simultaneous monitoring system, CFL-C2000 (Hamamatsu Photonics K.K., Shizuoka, Japan),

96

employing fluorescence and chemiluminescence was used. This device continuously collects

97

chemiluminescence data, while fluorescence emission is measured only if excitation light is on.

98

Therefore, fluorescence and chemiluminescence can be simultaneously measured by quick repeating

99

the on/off sequence of the excitation light10. In this study, we measured NO production and

100

cytoplasmic Ca2+ concentration based on fluorescence data, along with the production of superoxide

101

anion radicals by chemiluminescence.

102 103

Cells were seeded in T-25 flasks and cultured while regularly changing the medium until 80 to 90%

104

confluency was reached. Cells were washed once with HEPES buffer before 5 mL of fresh, phenol

105

red-free medium was added. In a dark environment, 50 µL of DAF-2 DA (final concentration, 50

106

µmol/L) was added for measuring NO, or 33 µL (final concentration, 3 µmol/L) of Fluo4-AM was

107

added for measuring cytoplasmic Ca2+ concentration. Cell culture samples were incubated for 1 h at

108

37 °C with 5% CO2 atmosphere. Then, cells were harvested and suspended in a 1 mM CaCl2 solution,

109

adjusted with HEPES buffer, at a concentration of 1.0 × 105 cells/mL. To measure superoxide anion

110

radicals by chemiluminescence, 2 mL cell suspension was dispensed in a cuvette and 500 µM MCLA

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111

was added to start a 7-min preincubation at 37 °C. The cuvette was placed into the CFL-C2000 prior

112

to initiating measurements for 4 h. After 10 min, 100 µL of 2.0 mg/mL plant material in sterilized

113

water was added to each sample at a final concentration of 100 µg/mL. The control sample was

114

supplemented with 100 µL of sterilized water.

115 116

Measuring the activation level of vascular endothelial nitric oxide synthase by western blotting

117

VEC test cultures were adjusted to a concentration of 1×106 to 1×107 cells/mL, using 1 mM

118

CaCl2-containing medium prepared with various supplements in a volume of 5 mL per culture.

119

L-arginine cultures were supplemented with 500 µM L-arginine hydrochloride whereas endothelial

120

NO synthase (eNOS) inhibitor cultures were prepared with 500 µM NG-nitro-L-arginine methyl ester

121

hydrochloride (L-NAME) and 500 µM L-arginine hydrochloride. The radish extract cultures were

122

prepared using either 1 mg/mL of the Sakurajima Daikon aqueous extract or 1 mg/mL of the Aokubi

123

Daikon aqueous extracts at a final concentration of 100 µg/mL. The incubation was performed for 4

124

h at 37 °C in a 5% CO2 atmosphere. Then, cells were recovered and lysed in HEPES buffer, yielding

125

a precipitate that was subjected to electrophoresis and transferred to a polyvinylidene difluoride

126

membrane. After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline-Tween 20

127

(TBST), primary antibodies were added for overnight incubation at 4 °C. Incubation with the

128

secondary antibody (anti-rabbit IgG, HRP-linked antibody, 1/10,000) was done for 1 h at room

129

temperature (15–20 °C). Chemiluminescence on ClarityTM Western ECL Substrate was detected

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using the ChemiDocTMXRS+ System and Image Lab Software (BIO-RAD). A β-actin preparation

131

was used as a loading control. The expression levels of total eNOS and active, phosphorylated eNOS

132

were expressed as relative values using the L-arginine-treated VEC culture sample as a standardized

133

reference with the value 1. The t-test was applied for statistical analyses.

134 135

Identification of active constituents in Sakurajima Daikon extracts

136

A preparation containing 200 µg/mL of Sakurajima Daikon aqueous extract was subjected to an

137

analysis by an information-dependent acquisition system, which can efficiently provide numerical

138

measurements of desired ions in real time using LC-MS/MS (LC system; Shimadzu, 3200QTRAP;

139

SCIEX). Data were collected using the Analyst® software (version1.5.1), and results were analyzed

140

using the databases Mass Bank and METLIN. After performing an initial survey scan using the

141

enhanced mass scan (EMS) system, an improved resolution was employed to correct mass errors and

142

check isotopic distributions. Next, the product ion scan was combined with the enhanced product ion

143

scan for obtaining fragment information. HPLC analysis was performed using a TSKgel ODS-100Z

144

(150 nm × 4.6 mm ID, 5 µm; TOSOH) column, a distilled water/acetonitrile mobile phase (60/40,

145

v/v), a 0.4-mL/min flow rate, a 5-µL injection volume, a UV wavelength of 210 nm, and a

146

temperature range of 20–25 °C (ambient). MS analyses were performed on a 3200 QTRAP system

147

employing the ESI+ ionizing method and EMS combined with the enhanced power scan, using High

148

Collision Gas; the value of the Curtain Gas was 20.00, the ionspray voltage was 5.5 kV, the

9



149

temperature was maintained at 500 °C, the values of the ion source gases 1 and 2 were 40 and 50 psi,

150

respectively, and the value of the collision energy was 30 eV. Standard compound preparations with

151

known properties and the Sakurajima Daikon aqueous extract preparation were subjected to the

152

analysis. Retention times and UV spectra of the HPLC runs were searched for compounds with a hit

153

in the LC-MS/MS database, using standard compounds as reference values.

154 155

Measuring NO with fluorescence microscopy

156

Cell suspensions containing 5.0 × 104 cells/mL were cultured at 37 °C in a 5% CO2 atmosphere until

157

80–90% confluency was reached; 100-µL aliquots of 10 µM DAF-2 DA solution were added to the

158

cultures and incubated for 1 h in the dark. After removing the DAF-2 DA solution, 100 µL of sample

159

containing test material at concentrations between 1 ng/mL and 1 mg/mL or reference compounds in

160

phenol red-free medium was added to each test cell sample, whereas 100 µL phenol red-free medium

161

was added to each blank control cell sample. Incubation was conducted for 2 h in the dark, and

162

fluorescence measurements were performed using a fluorescence microscope (KEYENCE). Using

163

the hybrid cell counter of the BZ-X Analyzer, cell numbers were determined according to their

164

brightness.

165 166

RESULTS AND DISCUSSION

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NO production

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The VECs maintain vascular endothelial function by producing NO. Here, we investigated if

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aqueous extracts of Sakurajima Daikon root, leaves, and peel contain components that can promote

170

NO production in VECs, and, thus, could potentially improve vascular endothelial function. The NO

171

levels in test cultures were monitored using a fluorescence assay with DAN. Prior to the VEC culture

172

experiment, we obtained a linear calibration curve for the assay, which had a correlation coefficient

173

(R2) of 0.9905. Importantly, we found that at concentrations above 10 µg/mL, the aqueous root

174

extract of Sakurajima Daikon caused a concentration-dependent of NO2- and NO3- levels in porcine

175

VECs (Figure 1B). The data also showed that the effects of the leave and peel extracts on the NO

176

production were similar to the effects of the root extract (Figure 1C).

177 178

Real-time measurement of the concentrations of NO, cytoplasmic Ca2+, and superoxide anion

179

radical using a simultaneous monitoring system for fluorescence and chemiluminescence

180

Based on the finding that Sakurajima Daikon promotes the production of NO in VECs, we proceeded

181

to examine the underlying mechanism. We hypothesized that Sakurajima Daikon might stimulate a

182

cellular function that activates vascular eNOS by Ca2+-calmodulin binding, induced by elevated

183

cytoplasmic Ca2+ concentrations. In addition, we examined the effect of the Sakurajima Daikon root

184

extract on the production of superoxide anion radicals that are known to damage VECs and decrease

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NO production. We used a simultaneous monitoring system that measures fluorescence and

186

chemiluminescence. In this experiment, real-time measurements were simultaneously performed

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187

using a fluorescent reagent, which after incorporation into cells directly detects intracellular NO and

188

Ca2+. In addition, a chemiluminescent reagent was used for detecting the superoxide anion radicals

189

released from the cell.

190 191

The experiment showed that NO production (Figure 2A) and cytoplasmic Ca2+ concentration (Figure

192

2B) increased in a time-dependent manner in the presence of Sakurajima Daikon root extract. We

193

also found that Sakurajima Daikon root extract generated a stronger response in VECs than the root

194

extract of Aokubi Daikon or the control. Furthermore, none of the extracts affected the production of

195

superoxide anion radicals in VECs (Figure 2C). These results suggest that the root of Sakurajima

196

Daikon can activate eNOS by triggering the activity of the calcium channels in the cell membrane or

197

by utilizing the calcium storage inside the cells for increasing NO production. Hence, the Sakurajima

198

Daikon root may be an effective stimulant for improving vascular function.

199 200

Measurement of the activation of eNOS by western blotting

201

As a primary signal generator, eNOS is crucial for the synthesis of NO from L-arginine and oxygen

202

within VECs. The enzyme has a molecular weight of 140 kDa and resides in caveolae, invaginated

203

cell membrane structures that are abundantly present in VECs. In the absence of any stimulation, the

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activity of eNOS is controlled by binding to caveolin11. However, a concentration increase of

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cytoplasmic Ca2+ causes a higher level of Ca2+-bound calmodulin, which replaces the caveolin bound

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to eNOS, and binds to the enzyme instead. This replacement liberates eNOS from the caveolae and

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makes it accessible for activation. Two modifications, the phosphorylation of Ser1177 and the

208

dephosphorylation of Thr495, are involved in eNOS activation, and measuring the phosphorylation

209

status in response to the treatment with Sakurajima Daikon root extract would help to elucidate the

210

underlying mechanism12.

211 212

We found that the phosphorylation of eNOS at Ser1177 was significantly more stimulated in porcine

213

VECs by the Sakurajima Daikon root extract than by the L-arginine preparation (Figure 3A, B).

214

However,

215

1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid, sequestration

216

phosphorylation of Ser1177 (data not shown). We also observed that eNOS dephosphorylation at

217

Thr495 tended to be more promoted by the Sakurajima Daikon root extract than by the other three

218

preparations (Figure 3A, C). However, dephosphorylation at Thr495 is less critical for eNOS activity.

219

Furthermore, none of the preparations affected the cellular eNOS protein levels (Figure 3A, D).

220

These results suggested that the Sakurajima Daikon root extract activated eNOS by phosphorylation

221

of Ser1177 and dephosphorylation of Thr495, whereas the extract had no effect on the cellular

222

protein level of eNOS.

in

the

presence

of

a

chelating

of Ca2+ prevented

223 224

Identification of the active constituents in Sakurajima Daikon aqueous extract

13


agent,


225

To identify the Sakurajima Daikon constituents that improve NO production in VECs, all detectable

226

extract compounds were examined by LC-ESI-MS/MS and quantified by HPLC. The total ion

227

chromatography (TIC) showed that the highest ion intensity was obtained at a retention time of 3.47

228

min, followed by the second highest at 3.83 min (Figure 4A). The mass spectrum at TIC 3.47 min

229

showed a strong peak at m/z = 104.0 [M+H+], which was confirmed by MS/MS. In the database, the

230

highest score was obtained for GABA, although lower-scoring hits were also obtained for

231

aminobutyric acid isomers and dimethylglycine. GABA, an amino acid, is widely distributed in

232

animals and plants. In mammalians, it primarily acts as a neurotransmitter in the suppressive system.

233

It is also reported to have blood pressure reducing effects13. When we examined whether the

234

Sakurajima Daikon extract contained GABA14, no HPLC peak was detected at 7.75 min, the

235

retention time of the GABA standard. Therefore, we confirmed that Sakurajima Daikon does not

236

contain GABA. The mass spectrum at the second highest ion intensity region at TIC 3.83 min

237

showed an m/z = 138.1 [M+H+], which was confirmed by a detailed MS/MS (Figure 4B, C). A search

238

of the database identified trigonelline (Figure 4D) as the highest score, whereas another hit was

239

obtained with N-methylnicotinamide. Trigonelline is a betaine-type molecule with two charged

240

groups in one molecule. The compound is found in coffee and some agricultural and marine products.

241

It is decomposed to a niacin analog by heat. Trigonelline has been reported to reduce brain aging and

242

Alzheimer-type dementias, and it has inhibitory effects on the invasion of cancer cells15-16. A

243

trigonelline standard produced two peaks at the retention times of 3.4 min and 3.65 min, which we

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were unable to resolve (Figure 4E)17. It was possible that the peaks corresponded to structural

245

isomers or stereoisomers, thus, we treated them as a single peak. Interestingly, the Sakurajima

246

Daikon extract also showed two peaks at the same retention times, indicating that the extract

247

contained trigonelline (Figure 4E). Therefore, using a linear calibration curve with a correlation

248

coefficient of 0.999992, we performed quantitative analysis and found that 1 mg of concentrated and

249

dried root extract of Sakurajima Daikon contained approximately 9 µg of trigonelline.

250 251

Measurement of NO production using fluorescence microscopy

252

Fluorescence imaging combined with microscopy is a dependable method for obtaining

253

molecule-specific spatial information such as its cellular localization. The intracellular site of NO

254

production was examined by fluorescence microscopy using a fluorescent probe that detects

255

intracellular NO, which was expressed as a relative value that depends on the number of fluorescent

256

cells. Figure 5A shows microscopic images processed for the fluorescence detection of NO

257

production in porcine VECs induced by Sakurajima or Aokubi Daikon using fluorescence

258

microscope. The number of fluorescent cells was determined by merging a fluorescent image with

259

the corresponding non-fluorescent micrograph (Figure 5B and 5C). The cells supplemented with

260

Sakurajima Daikon aqueous extract were associated with higher NO values per visual field than the

261

cells of the blank control and the cells supplemented with Aokubi Daikon extract (Figure 5).

262

Furthermore, trigonelline increased the production of NO (Figure 6A and 6B), which was confirmed

15



263

for an extended concentration range from 10 ng/mL to 100 µg/mL (data not shown). In Figure 6C,

264

both preparations activated P-eNOS(Ser1177) but 9 µg trigonelline standard was a stronger stimulant

265

than 1 mg Sakurajima Daikon contained approximately 9 µg of trigonelline. However, in

266

experiments measuring the concentrations of NO and cytoplasmic Ca2+, no significant differences

267

were observed between the trigonelline standard and the Sakurajima Daikon preparation (data not

268

shown). Thus, our results indicated that trigonelline improved the production of NO in porcine VECs,

269

suggesting that it is the active constituent in Sakurajima Daikon aqueous extracts. In contrast, GABA

270

did not increase the production of NO.

271 272

The results of this study suggested that the underlying mechanism for stimulating NO production by

273

Sakurajima Daikon extract involves eNOS activation by the phosphorylation of Ser1177 and the

274

dephosphorylation of Thr495, which is triggered by elevated concentrations of cytoplasmic Ca2+,

275

resulting from the activation of Ca2+ channels in VECs (Figure 7). It is reported that elevated

276

cytoplasmic Ca2+ concentrations induce a mechanism that activates vascular eNOS by Ca2+–

277

calmodulin binding18. Here, we confirmed that trigonelline, which is thought to be an active

278

constituent of Sakurajima Daikon, improves the NO production in VEC culture. It has been reported

279

that trigonelline has growth-promoting functions in radish seedlings19 and that trigonelline isolated

280

from pumpkins improved hypertension and diabetes in a mouse model20. Furthermore, trigonelline is

281

enzymatically or non-enzymatically (by thermal decomposition) converted to a niacin analog.

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Studies show that niacin analogs act as constituents of coenzymes like NAD and NADP, which are

283

involved in redox reactions19. Importantly, we determined that the NO-production stimulant in

284

extracts of Sakurajima Daikon, the world’s biggest radish, is trigonelline. Furthermore, trigonelline is

285

thought to act as an agonist for receptors including the muscarinic receptor, which stimulates

286

receptor-activated Ca2+ channels21. Because these receptors are associated with phosphatidylinositol

287

responses, Ca2+ release from intracellular storages, instead of Ca2+ influx from outside the cell, is

288

possible and needs to be investigated in the future. A report suggests that the Ca2+ channels in VECs

289

are either transient receptor potential (TRP) C4 or TRPV4 channels, belonging to the TRP channel

290

family22. Examining the interaction between trigonelline and these Ca2+ channels might provide

291

clues for a better understanding of the underlying mechanism. Because NADPH is involved in the

292

activation of eNOS, the niacin analog obtained by the thermal decomposition of trigonelline might

293

have contributed to the increase in NO production, instead of intact trigonelline.

294 295

Among

296

endothelial-dependent hyperpolarizing factor, NO is the strongest. However, NO cannot be made

297

available as a drug. Therefore, the identification of NO-stimulating constituents in farm products that

298

can be consumed with regular meals would contribute to the prevention of vascular diseases.

the

endothelium-derived

relaxing

factors

NO,

299 300

ABBREVIATIONS USED

17


prostaglandin

I2

(PGI2),

and


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301

BSA,

302

2,3-diaminonaphthalene; EMS, enhanced mass scan; eNOS, endothelial NO Synthase; GABA,

303

γ-aminobutyric acid; L-NAME, NG-nitro-L-arginine methyl ester hydrochloride; LDL, low density

304

lipoprotein; MCLA, [1,2-a]pyrazine-3-one hydrochloride; NO, nitric oxide; TIC, total ion

305

chromatography; VECs, vascular endothelial cells

bovine

serum

albumin;

DAF-2

DA,

diaminofluorescein-2

diacetate;

DAN,

306 307

ACKNOWLEDGMENT

308

The authors would like to thank Dr. Fumio Yagi for advice with the experiments. This work was

309

supported in part by JSPS KAKENHI [grant number 17K07795], Sapporo Bioscience Foundation,

310

The Foundation for Dietary Scientific Research, and Public Foundation Yonemori-seishinikuseikai.

311

The funding agencies had no role in study design, data collection and analysis, decision to publish or

312

preparation of the manuscript.

313 314

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Figure captions

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Figure 1. (A) Photo of Sakurajima Daikon: leaves, root, and peel of Sakurajima Daikon were used

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for extract preparation. Photo credit: Mr. Hiromi Fukidome (B) The level of NO production in

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porcine VECs was measured in the presence of Sakurajima Daikon root extract (*P

Elucidating the Improvement in Vascular Endothelial Function from

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