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Elucidating the Improvement in Vascular Endothelial Function from Sakurajima Daikon and Its Mechanism of Action: A Comparative Study with Raphanus sativus Rei Kuroda,† Kimiko Kazumura,‡ Miki Ushikata,§ Yuji Minami,§ and Katsuko Kajiya*,§ †

Major in Biochemical Science & Technology, Graduate School of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu 434-8601, Japan § Department of Food Science & Biotechnology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan J. Agric. Food Chem. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 08/18/18. For personal use only.

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ABSTRACT: Vascular diseases, such as myocardial and cerebral infarctions, are the leading causes of death. Some vascular diseases occur as the result of decreases in vascular endothelial function. The innermost layer of the vasculature is formed by vascular endothelial cells (VECs), which are critical for nitric oxide (NO) synthesis. In our search for active constituents in farm products with the potential for improving the vascular system, we examined the effect of Raphanus sativus cv. Sakurajima Daikon on NO production in VECs. In this study, we found that the underlying mechanism for stimulating NO production by Sakurajima Daikon extract involves endothelial-NO-synthase (eNOS) activation by the phosphorylation of Ser1177 and the dephosphorylation of Thr495, which are triggered by elevated concentrations of cytoplasmic Ca2+ resulting from the activation of Ca2+ channels in VECs. We observed that trigonelline, an active constituent of Sakurajima Daikon, improves NO production in VEC cultures. KEYWORDS: Raphanus sativus, endothelial function, nitric oxide, simultaneous-monitoring system

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INTRODUCTION Cerebrovascular diseases, such as stroke, and heart diseases, such as angina pectoris and myocardial infarction, account for nearly 25% of deaths in Japan1 and the world.2 Because brain and heart functions remain normal until just prior to attacks, these diseases are not caused by organ dysfunction but by an impairment of the blood-vessel networks in these organs. The economic burden of vascular diseases on patients and society is indicated by the fact that these patients not only endure longterm treatments and treatment sequelae but also are confined to bed for extended periods. Hence, there is a need to improve vascular function and prevent vascular diseases. Blood vessels consist of three layers: the outermost tunica adventitia; the tunica media; and the innermost tunica intima, which contains the vascular endothelial cells (VECs). Nitric oxide (NO) is released from VECs to protect blood vessels by regulating their contraction and relaxation and by preventing thrombus formation caused by the attachment of white blood cells and other blood components to the vascular endothelium. However, if VECs are damaged by oxidative stress caused by reactive oxygen species or oxidized low-density lipoproteins (LDLs), the production of NO is suppressed, increasing the risk of cardiovascular diseases. Thus, improving NO production by VECs is critical for protecting blood vessels. Kagoshima Prefecture in Japan is famous for the largest radish cultivar, Raphanus sativus cv. Sakurajima Daikon (Figure 1A), which was certified as the world’s biggest radish by the Guinness Book of Records.3 The radishes regularly weigh about 4 to 5 kg, but large ones weigh around 30 kg, with a girth of approximately 110 cm. Although the common radish reportedly possesses antioxidant, antihypertensive, and antithrombogenic activities,4−6 there are no studies that directly © XXXX American Chemical Society

compare the potential health benefits, like the improvement of blood-vessel function, of Sakurajima Daikon with the benefits of common varieties, such as Raphanus sativus var. longipinnatus (Aokubi Daikon). Here, we used Aokubi Daikon as a reference in our study about the effect of Sakurajima Daikon on NO production in human coronary-artery endothelial cells and porcine aortic endothelial cells and in our analysis of the underlying mechanism of this effect that potentially applies to blood vessels.

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MATERIALS AND METHODS

Materials. Human and porcine VECs were purchased from Kurabou Industries Ltd. (Osaka, Japan) and Cosmo Bio Company Ltd. (Tokyo, Japan), respectively. Experiments were performed using both human and porcine VECs, but the figures were prepared using the data from the porcine-VEC experiments, which included a high number of replicates (n = 8). [1,2-a]Pyrazine-3-one hydrochloride (MCLA), diaminofluorescein-2 diacetate (DAF-2 DA), and fluo4acetoxy methyl ester (Fluo4-AM) were obtained from Tokyo Chemical Industry Company Ltd. (Tokyo, Japan), Goryo Chemical Inc. (Hokkaido, Japan), and Dojindo Laboratories (Kumamoto, Japan). Trigonelline and γ-aminobutyric acid (GABA) were purchased from Fujifilm Wako Chemical Corporation (Osaka, Japan). Western-blot reagents were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). The primary antibodies (β-actin, 1/ 5000; eNOS, 1/1000; P-eNOS(Ser1177), 1/1000; and P-eNOS(Thr495), 1/1000) and the secondary antibody (anti-rabbit-IgG HRP-linked antibody, 1/10 000) were purchased from Cell Signaling Technology (Danvers, MA). Received: Revised: Accepted: Published: A

April 7, 2018 July 21, 2018 July 23, 2018 July 23, 2018 DOI: 10.1021/acs.jafc.8b01750 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

harvested in January of 2017. Uto et al. investigated the effects of different parts of Angelica acutiloba Kitagawa, such as the aerial parts and the root, on NO production and isolated constituents with antiinflammatory activity from its aerial parts.7 In this study, after removing all the inedible material from the radish, the edible parts (i.e., the roots, peels, and leaves) were separated, cut into small pieces, processed using a homogenizer, and lyophilized to generate powdered raw material. One milliliter of methanol/H2O/acetic acid solvent (95.0/9.5/0.5, v/v/v) was added to 25 mg of raw material and mixed in a vortexer; this was followed by 5 min of ultrasonic treatment. The sample was centrifuged twice at 1600g for 10 min at 4 °C; the supernatant was collected and concentrated by drying. The dry sample material was weighed and dissolved in the appropriate solvents prior to the experiments. NO Quantification Using a Modified Griess Method and a Fluorescence Method. Because NO has a short half-life and is rapidly oxidized to NO2− and NO3− in aqueous solution, its concentration is indirectly determined via that of NO2−. Nitratereductase-mediated reduction of NO3− is used to ensure that the NO2− concentration represents the original NO level of a sample. Typically, NO2− is measured using the Griess method.8 A fluorescence method9 using 2,3-diaminonaphthalene (DAN) is a newer NO2− assay with higher sensitivity than that of the Griess method. Because NO2− reacts with DAN under acidic conditions to form a fluorescent adduct, naphthalenetriazole, we quantified the product by measuring its fluorescence intensity with a microplate reader (Tecan, Männedorf, Switzerland). VECs of the normal human coronary artery and normal porcine aorta were adjusted to 5.0 × 104 cells/mL and cultured in 96-well plates until 80% confluency was reached. Then, incubation continued overnight (12 h) in medium either with or without the radish-extract supplement. Culture supernatants were collected, cleared from cells, and reduced by a 30 min incubation at 37 °C with nitrate reductase and the respective enzyme cofactors (iron, molybdenum, and cytochrome); this was followed by a 15 min incubation with DAN. The assay was terminated by measuring the fluorescence intensity (λex = 360 nm, λem = 450 nm). The amount of NO per sample was calculated by transforming the raw data using a calibration curve prepared with NaNO3 and expressing the result as a relative value derived from a comparison with a control value of 1. The t-test was applied for statistical analysis. Simultaneous-Monitoring System Using Fluorescence and Chemiluminescence for Real-Time Measurement of NO Production, Cytoplasmic-Ca2+ Concentration, and Production of the Superoxide Anion Radical O2−•. A simultaneousmonitoring system, CFL-C2000 (Hamamatsu Photonics K.K., Shizuoka, Japan), employing fluorescence and chemiluminescence was used. This device continuously collects chemiluminescence data, whereas the fluorescence emission is measured only if the excitation light is on. Therefore, fluorescence and chemiluminescence can be simultaneously measured by quickly repeating the on−off sequence of the excitation light.10 In this study, we measured NO production and cytoplasmic-Ca2+ concentration on the basis of the fluorescence data, and we measured the production of superoxide anion radicals by chemiluminescence. We seeded the cells in T-25 flasks and cultured them while regularly changing the medium until 80 to 90% confluency was reached. Cells were washed once with HEPES buffer before 5 mL of fresh, phenol-red-free medium was added. In a dark environment, 50 μL of DAF-2 DA (final concentration of 50 μmol/L) was added for measuring NO, or 33 μL (final concentration of 3 μmol/L) of Fluo4AM was added for measuring the cytoplasmic-Ca2+ concentration. Cell-culture samples were incubated for 1 h at 37 °C with a 5% CO2 atmosphere. Then, cells were harvested and suspended in a 1 mM CaCl2 solution, adjusted with HEPES buffer, at a concentration of 1.0 × 105 cells/mL. To measure superoxide anion radicals by chemiluminescence, 2 mL of the cell suspension was dispensed in a cuvette and 0.5 μM MCLA was added to start a 7 min preincubation at 37 °C. The cuvette was placed into the CFL-C2000 prior to initiating the measurements for 4 h. After 10 min, 100 μL of 2.0 mg/ mL plant material in sterilized water was added to each sample to a

Figure 1. (A) Photo of Sakurajima Daikon. The leaves, roots, and peels of Sakurajima Daikon were used for extract preparation. Photo credit: Hiromi Fukidome. (B) Level of NO production in porcine VECs measured in the presence of Sakurajima Daikon root extract (*P < 0.01 vs control). (C) Level of NO production in porcine VECs measured in the presence of extracts derived from different parts of Sakurajima Daikon. Columns are marked as follows: shaded column, root; dotted column, peel; and checkered column, leaves (*P < 0.01 vs root-extract control, +P < 0.01 vs peel-extract control, #P < 0.01 vs leaf-extract control). Similar results were obtained using human VECs (data not shown). Sample Preparation. Sakurajima Daikon cultivated in Kagoshima City, Japan, was used in this study. Aokubi Daikon was obtained from the Kagoshima Prefectural Institute for Agricultural Development, Japan, and was used as the reference material. The crops were B

DOI: 10.1021/acs.jafc.8b01750 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry final concentration of 100 μg/mL. The control sample was supplemented with 100 μL of sterilized water. Measuring the Activation Level of Vascular Endothelial Nitric Oxide Synthase by Western Blotting. VEC test cultures were adjusted to a concentration of 1 × 106 to 1 × 107 cells/mL, using medium containing 1 mM CaCl2 and prepared with various supplements in a volume of 5 mL per culture. L-Arginine cultures were supplemented with 500 μM L-arginine hydrochloride, whereas endothelial-NO-synthase (eNOS) inhibitor cultures were prepared with 500 μM NG-nitro-L-arginine methyl ester hydrochloride (LNAME) and 500 μM L-arginine hydrochloride. The radish-extract cultures were prepared using either 1 mg/mL Sakurajima Daikon aqueous extract or 1 mg/mL Aokubi Daikon aqueous extract at a final concentration of 100 μg/mL. The incubation was performed for 4 h at 37 °C in a 5% CO2 atmosphere. Then, cells were recovered and lysed in HEPES buffer, yielding a precipitate that was subjected to electrophoresis and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline−Tween 20 (TBST), primary antibodies were added for an overnight incubation at 4 °C. Incubation with the secondary antibody (anti-rabbit-IgG HRP-linked antibody, 1/10 000) was done for 1 h at room temperature (15−20 °C). Chemiluminescence on a Clarity Western ECL Substrate was detected using the ChemiDocTMXRS+ System and Image Lab Software (Bio-Rad, Hercules, CA). A β-actin preparation was used as a loading control. The expression levels of total eNOS and active phosphorylated eNOS were expressed as relative values using the L-arginine-treated-VECculture sample as a standardized reference with a value of 1. The t-test was applied for statistical analysis. Identification of Active Constituents in Sakurajima Daikon Extracts. A preparation containing 200 μg/mL Sakurajima Daikon aqueous extract was subjected to analysis with an informationdependent acquisition system, which could efficiently provide numerical measurements of desired ions in real-time using LC-MS/ MS (LC system, Shimadzu, Kyoto, Japan; 3200QTRAP, SCIEX, Framingham, MA). Data were collected using the Analyst software (version 1.5.1), and results were analyzed using the databases Mass Bank and METLIN. After performing an initial survey scan using the enhanced-mass-scan (EMS) system, an improved resolution was employed to correct mass errors and check isotopic distributions. Next, the product-ion scan was combined with the enhanced production scan for obtaining fragment information. HPLC analysis was performed using a TSKgel ODS-100Z column (150 nm × 4.6 mm i.d., 5 μm; Tosoh, Tokyo, Japan), a distilled water/acetonitrile mobile phase (60/40, v/v), a 0.4 mL/min flow rate, a 5 μL injection volume, a UV wavelength of 210 nm, and a temperature range of 20−25 °C (ambient). MS analyses were performed on a 3200 QTRAP system employing the ESI+ ionizing method and EMS combined with the enhanced power scan, using high-collision gas. The value of the curtain gas was 20.00; the ionspray voltage was 5.5 kV; the temperature was maintained at 500 °C; the values of ion-source gases 1 and 2 were 40 and 50 psi, respectively; and the value of the collision energy was 30 eV. Standard-compound preparations with known properties and the Sakurajima Daikon aqueous-extract preparation were subjected to the analysis. Retention times and UV spectra of the HPLC runs were searched for compounds with a hit in the LC-MS/MS database, using standard compounds as reference values. Measuring NO with Fluorescence Microscopy. Cell suspensions containing 5.0 × 104 cells/mL were cultured at 37 °C in a 5% CO2 atmosphere until 80−90% confluency was reached; 100 μL aliquots of 10 μM DAF-2 DA solution were added to the cultures and incubated for 1 h in the dark. After removing the DAF-2 DA solution, 100 μL of sample containing the test material at concentrations between 1 ng/mL and 1 mg/mL or reference compounds in phenolred-free medium was added to each test-cell sample, whereas 100 μL of phenol-red-free medium was added to each blank control-cell sample. Incubation was conducted for 2 h in the dark, and fluorescence measurements were performed using a fluorescence microscope (Keyence, Osaka, Japan). Using the hybrid cell counter of

the BZ-X Analyzer, cell numbers were determined according to their brightness.

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RESULTS AND DISCUSSION NO Production. VECs maintain vascular endothelial function by producing NO. Here, we investigated if aqueous extracts of Sakurajima Daikon roots, leaves, and peels contain components that can promote NO production in VECs and thus potentially improve vascular endothelial function. The NO levels in test cultures were monitored using a fluorescence assay with DAN. Prior to the VEC-culture experiment, we obtained a linear calibration curve for the assay, which had a correlation coefficient (R2) of 0.9905. Importantly, we found that at concentrations above 10 μg/mL, the aqueous root extract of Sakurajima Daikon caused concentration-dependent increases in NO2− and NO3− levels in porcine VECs (Figure 1B). The data also showed that the effects of the leaf and peel extracts on NO production were similar to the effects of the root extract (Figure 1C). Real-Time Measurements of the Concentrations of NO, Cytoplasmic Ca2+, and the Superoxide Anion Radical Using a Simultaneous-Monitoring System for Fluorescence and Chemiluminescence. On the basis of the finding that Sakurajima Daikon promotes the production of NO in VECs, we proceeded to examine the underlying mechanism. We hypothesized that Sakurajima Daikon might stimulate a cellular function that activates vascular eNOS by Ca2+−calmodulin binding induced by elevated cytoplasmicCa2+ concentrations. In addition, we examined the effect of the Sakurajima Daikon root extract on the production of superoxide anion radicals, which are known to damage VECs and decrease NO production. We used a simultaneousmonitoring system that measures fluorescence and chemiluminescence. In this experiment, real-time measurements were simultaneously performed using a fluorescent reagent, which, after incorporation into cells, directly detected intracellular NO and Ca2+. In addition, a chemiluminescent reagent was used for detecting the superoxide anion radicals released from the cell. The experiment showed that NO production (Figure 2A) and cytoplasmic-Ca2+ concentration (Figure 2B) increased in a time-dependent manner in the presence of Sakurajima Daikon root extract. We also found that Sakurajima Daikon root extract generated a stronger response in VECs than the root extract of Aokubi Daikon or the control. Furthermore, none of the extracts affected the production of superoxide anion radicals in VECs (Figure 2C). These results suggest that the root of Sakurajima Daikon can activate eNOS by triggering the activity of the calcium channels in the cell membrane or by utilizing the calcium storage inside the cells for increasing NO production. Hence, Sakurajima Daikon root may be an effective stimulant for improving vascular function. Measurement of the Activation of eNOS by Western Blotting. As a primary signal generator, eNOS is crucial for the synthesis of NO from L-arginine and oxygen within VECs. The enzyme has a molecular weight of 140 kDa and resides in caveolae, invaginated-cell-membrane structures that are abundantly present in VECs. In the absence of any stimulation, the activity of eNOS is controlled by binding to caveolin.11 However, increases in cytoplasmic-Ca2+ concentrations result in higher levels of Ca2+−bound calmodulin, which replaces the caveolin bound to eNOS and binds to the enzyme instead. This replacement liberates eNOS from the caveolae and makes it accessible for activation. Two modifications, the phosphorC

DOI: 10.1021/acs.jafc.8b01750 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. eNOS-activation status was determined for porcine VECs treated with L-arginine, L-NAME, Sakurajima Daikon, or Aokubi Daikon preparations. (A) Evaluation of the eNOS-phosphorylation level using Western blotting. (B) Relative levels of Ser1177 phosphorylation in eNOS derived by quantitative analysis of Western-blot images (P < 0.05 vs L-arginine). (C) Relative levels of Thr495 phosphorylation in eNOS derived by quantitative analysis of Western-blot images. (D) Relative cellular amounts of eNOS derived by quantitative analysis of Western-blot images. Similar results were obtained using human VECs (data not shown).

Figure 2. Level of NO production (A), concentration of cytoplasmic Ca2+ (B), and level of superoxide-anion-radical production measured in extract-treated porcine VECs using a simultaneous-monitoring system. The solid line represents Sakurajima Daikon root extract, the dotted line represents Aokubi Daikon root extract, and the dashed line represents the control. Similar results were obtained using human VECs (data not shown).

phosphorylation of Ser1177 (data not shown). We also observed that eNOS dephosphorylation at Thr495 tended to be promoted more by the Sakurajima Daikon root extract than by the three other preparations (Figure 3A,C). However, dephosphorylation at Thr495 is less critical for eNOS activity. Furthermore, none of the preparations affected cellular eNOS protein levels (Figure 3A,D). These results suggested that the Sakurajima Daikon root extract activated eNOS by phosphorylation of Ser1177 and dephosphorylation of Thr495, whereas the extract had no effect on the cellular protein level of eNOS. Identification of the Active Constituents in the Sakurajima Daikon Aqueous Extract. To identify the Sakurajima Daikon constituents that improve NO production in VECs, all detectable extract compounds were examined by LC-ESI-MS/MS and quantified by HPLC. Total-ion chroma-

ylation of Ser1177 and the dephosphorylation of Thr495, are involved in eNOS activation, and measuring the phosphorylation status in response to treatment with Sakurajima Daikon root extract would help to elucidate the underlying mechanism.12 We found that the phosphorylation of eNOS at Ser1177 in porcine VECs was significantly more stimulated by the Sakurajima Daikon root extract than by the L-arginine preparation (Figure 3A,B). However, in the presence of a chelating agent, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid, the sequestration of Ca 2+ prevented D

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Figure 4. (A) Total-ion-current chromatogram (TIC) of Sakurajima Daikon extract. (B) Mass spectrum and (C) MS/MS spectrum of TIC 3.83. (D) Structural formula of trigonelline. (E) Trigonelline chromatogram of the standard reagent (blue) and Sakurajima Daikon extract (red).

MS (Figure 4B,C). A search of the database identified trigonelline (Figure 4D) as the highest score, whereas another hit was obtained with N-methylnicotinamide. Trigonelline is a betaine-type molecule with two charged groups in one molecule. The compound is found in coffee and some agricultural and marine products. It is decomposed to a niacin analogue by heat. Trigonelline has been reported to reduce brain aging and Alzheimer-type dementias, and it has inhibitory effects on the invasion of cancer cells.15,16 A trigonelline standard produced two peaks at retention times of 3.4 and 3.65 min, which we were unable to resolve (Figure 4E).17 It was possible that the peaks corresponded to structural isomers or stereoisomers; thus, we treated them as a single peak. Interestingly, the Sakurajima Daikon extract also showed two peaks at the same retention times, indicating that the extract contained trigonelline (Figure 4E). Therefore, using a linear calibration curve with a correlation coefficient of

tography (TIC) showed that the highest ion intensity was obtained at a retention time of 3.47 min, and the secondhighest was at 3.83 min (Figure 4A). The mass spectrum at TIC 3.47 min showed a strong peak at m/z 104.0 [M + H]+, which was confirmed by MS/MS. In the database, the highest score was obtained for GABA, although lower-scoring hits were also obtained for aminobutyric acid isomers and dimethylglycine. GABA, an amino acid, is widely distributed in animals and plants. In mammalians, it primarily acts as a neurotransmitter in the suppressive system. It is also reported to have blood-pressure-reducing effects.13 When we examined whether the Sakurajima Daikon extract contained GABA,14 no HPLC peak was detected at 7.75 min, the retention time of the GABA standard. Therefore, we confirmed that Sakurajima Daikon does not contain GABA. The mass spectrum at the second-highest-ion-intensity region at TIC 3.83 min showed an m/z 138.1 [M + H]+, which was confirmed by detailed MS/ E

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Daikon aqueous extracts were associated with higher NO values per visual field compared with those of the cells of the blank control and the cells supplemented with Aokubi Daikon extract (Figure 5). Furthermore, trigonelline increased the production of NO (Figure 6A,B), which was confirmed for an

0.999 992, we performed quantitative analysis and found that 1 mg of concentrated and dried root extract of Sakurajima Daikon contained approximately 9 μg of trigonelline. Measurement of NO Production Using Fluorescence Microscopy. Fluorescence imaging combined with microscopy is a dependable method for obtaining molecule-specific spatial information such as its cellular localization. The intracellular site of NO production was examined by fluorescence microscopy using a fluorescent probe that detects intracellular NO, which was expressed as a relative value that depended on the number of fluorescent cells. Figure 5A shows microscopic images processed for the fluorescence detection of NO production in porcine VECs induced by Sakurajima or Aokubi Daikon using a fluorescence microscope. The number of fluorescent cells was determined by merging the fluorescent image with the corresponding nonfluorescent micrograph (Figure 5B,C). The cells supplemented with Sakurajima

Figure 6. (A) NO production induced by GABA or trigonelline detected in porcine VECs using fluorescence microscopy. (B) Number of fluorescent cells expressed as luminance per visual field and derived from the corresponding images (shown in panel A): white column, control; dotted column, Sakurajima Daikon; black column, GABA; and striped column, trigonelline. (C) Relative level of Ser1177 phosphorylation in eNOS derived by quantitative analysis of Western-blot images. Porcine VECs were treated with L-arginine, 1 mg/mL Sakurajima Daikon, or 9 μg/mL trigonelline (P < 0.05 vs Larginine). Similar results were obtained using human VECs (data not shown).

extended concentration range from 10 ng/mL to 100 μg/mL (data not shown). In Figure 6C, both preparations activated PeNOS(Ser1177), but 9 μg of the trigonelline standard was a stronger stimulant than 1 mg of Sakurajima Daikon extract containing approximately 9 μg of trigonelline. However, in experiments measuring the concentrations of NO and cytoplasmic Ca2+, no significant differences were observed between the trigonelline standard and the Sakurajima Daikon preparation (data not shown). Thus, our results indicated that trigonelline improved the production of NO in porcine VECs, suggesting that it is the active constituent in Sakurajima

Figure 5. (A) NO production stimulated by Sakurajima Daikon extract or Aokubi Daikon extract detected in porcine VECs using fluorescence microscopy. (B) Number of fluorescent cells expressed as luminance per visual field and derived from the corresponding images (shown in panel A): white column, control; dotted column, Sakurajima Daikon; and black column, Aokubi Daikon. (C) Number of NO-producing VECs per visual field. Similar results were obtained using human VECs (data not shown). F

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Figure 7. Mechanism of NO synthesis and vasorelaxation.

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Daikon aqueous extracts. In contrast, GABA did not increase the production of NO. The results of this study suggest that the underlying mechanism for stimulating NO production by Sakurajima Daikon extract involves eNOS activation by the phosphorylation of Ser1177 and the dephosphorylation of Thr495, which is triggered by elevated concentrations of cytoplasmic Ca2+ resulting from the activation of Ca2+ channels in VECs (Figure 7). It is reported that elevated cytoplasmic-Ca2+ concentrations induce a mechanism that activates vascular eNOS by Ca2+−calmodulin binding.18 Here, we confirmed that trigonelline, which is thought to be an active constituent of Sakurajima Daikon, improves NO production in VEC culture. It has been reported that trigonelline has growth-promoting functions in radish seedlings19 and that trigonelline isolated from pumpkins improved hypertension and diabetes in a mouse model.20 Furthermore, trigonelline is enzymatically or nonenzymatically (by thermal decomposition) converted to a niacin analogue. Studies show that niacin analogues act as constituents of coenzymes like NAD and NADP, which are involved in redox reactions.19 Importantly, we determined that the NO-production stimulant in extracts of Sakurajima Daikon, the world’s biggest radish, is trigonelline. Furthermore, trigonelline is thought to act as an agonist for receptors, including the muscarinic receptor, which stimulates receptoractivated Ca2+ channels.21 Because these receptors are associated with phosphatidylinositol responses, Ca2+ release from intracellular storage, instead of Ca2+ influx from outside the cell, is possible and needs to be investigated in the future. A report suggests that the Ca2+ channels in VECs are either transient-receptor-potential (TRP) C4 or TRPV4 channels, belonging to the TRP channel family.22 Examining the interaction between trigonelline and these Ca2+ channels might provide clues for a better understanding of the underlying mechanism. Because NADPH is involved in the activation of eNOS, the niacin analogue obtained by the thermal decomposition of trigonelline might have contributed to the increase in NO production, instead of intact trigonelline. Among the endothelium-derived relaxing factors (i.e., NO, prostaglandin I2 (PGI2), and endothelial-dependent hyperpolarizing factor), NO is the strongest. However, NO cannot be made available as a drug. Therefore, the identification of NO-stimulating constituents in farm products that can be consumed with regular meals would contribute to the prevention of vascular diseases.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +81-99285-8631. ORCID

Katsuko Kajiya: 0000-0001-9043-5515 Funding

This work was supported in part by JSPS KAKENHI (grant number 17K07795), the Sapporo Bioscience Foundation, The Foundation for Dietary Scientific Research, and Public Foundation Yonemori-seishinikuseikai. The funding agencies had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Fumio Yagi for advice with the experiments.

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ABBREVIATIONS USED BSA, bovine serum albumin; DAF-2 DA, diaminofluorescein-2 diacetate; DAN, 2,3-diaminonaphthalene; EMS, enhanced mass scan; eNOS, endothelial NO synthase; GABA, γaminobutyric acid; L-NAME, NG-nitro-L-arginine methyl ester hydrochloride; LDL, low-density lipoprotein; MCLA, [1,2a]pyrazine-3-one hydrochloride; NO, nitric oxide; TIC, totalion chromatography; VECs, vascular endothelial cells

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REFERENCES

(1) Statistics & Other Data; Ministry of Health, Labour and Welfare: Tokyo, Japan, 2016. Available at http://www.mhlw.go.jp/english/ database/index.html. (2) World Health Organization. The top 10 causes of death. http:// www.who.int/mediacentre/factsheets/fs310/en/ (accessed March 12, 2018). (3) Guinness World Records. Heaviest radish. http://www. guinnessworldrecords.com/world-records/heaviest-radish (accessed March 12, 2018). (4) Hanlon, P. R.; Barnes, D. M. Phytochemical composition and biological activity of 8 varieties of radish (Raphanus sativus L.) sprouts and mature taproots. J. Food Sci. 2011, 76 (1), C185−C192. (5) Chung, D.-H.; Kim, S.-H.; Myung, N.; Cho, K. J.; Chang, M. J. The antihypertensive effect of ethyl acetate extract of radish leaves in G

DOI: 10.1021/acs.jafc.8b01750 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry spontaneously hypertensive rats. Nutr. Res. Pract. 2012, 6 (4), 308− 314. (6) Ku, S.-K.; Bae, J.-S. Antithrombotic activities of sulforaphane via inhibiting platelet aggregation and FIIa/FXa. Arch. Pharmacal Res. 2014, 37, 1454−1463. (7) Uto, T.; Tung, N. H.; Taniyama, R.; Miyanowaki, T.; Morinaga, O.; Shoyama, Y. Anti-inflammatory activity of constituents isolated from aerial part of Angelica acutiloba Kitagawa. Phytother. Res. 2015, 29, 1956−1963. (8) Tsikas, D. Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: Appraisal of the Griess reaction in the L-arginine/nitric oxide area of research. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2007, 851, 51−70. (9) Hu, T. M.; Chiu, S. J.; Hsu, Y. M. Nitroxidative chemistry interferes with fluorescent probe chemistry: implications for nitric oxide detection using 2,3-diaminonaphthalene. Biochem. Biophys. Res. Commun. 2014, 451, 196−201. (10) Kazumura, K.; Sato, Y.; Satozono, H.; Koike, T.; Tsuchiya, H.; Hiramatsu, M.; Katsumata, M.; Okazaki, S. Simultaneous monitoring of superoxides and intracellular calcium ions in neutrophils by chemiluminescence and fluorescence: Evaluation of action mechanisms of bioactive compounds in foods. J. Pharm. Biomed. Anal. 2013, 84, 90−96. (11) Omura, M.; Kobayashi, S.; Mizukami, Y.; Mogami, K.; Todoroki-Ikeda, N.; Miyake, T.; Matsuzaki, M. Eicosapentanoic acid (EPA) induces Ca2+-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation. FEBS Lett. 2001, 487, 361−366. (12) Zhang, X.-H.; Yokoo, H.; Nishioka, H.; Fujii, H.; Matsuda, N.; Hayashi, T.; Hattori, Y. Beneficial effect of the oligomerized polyphenol oligonol on high glucose-induced changes in eNOS phosphorylation and dephosphorylation in endothelial cells. Br. J. Pharmacol. 2010, 159, 928−938. (13) Ma, P.; Li, T.; Ji, F.; Wang, H.; Pang, J. Effect of GABA on blood pressure and blood dynamics of anesthetic rats. Int. J. Clin. Exp. Med. 2015, 8 (8), 14296−14302. (14) Nacalai Tesque Research. COSMOSIL Application data. https://www.nacalai.co.jp/global/ (accessed March 12, 2018). (15) Tohda, C.; Kuboyama, T.; Komatsu, K. Search for natural products related to regeneration of the neuronal network. Neurosignals 2005, 14, 34−45. (16) Hirakawa, N.; Okauchi, R.; Miura, Y.; Yagasaki, K. Anti-invasive activity of niacin and trigonelline against cancer cells. Biosci., Biotechnol., Biochem. 2005, 69 (3), 653−658. (17) Shailajan, S.; Menon, S.; Singh, A.; Mhatre, M.; Sayed, N. A validated RP-HPLC method for quantitation of trigonelline from herbal formulations containing Trigonella foenum-graecum (L.) seeds. Pharm. Methods 2011, 2 (3), 157−160. (18) Fleming, I.; Busse, R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am. J. Physiol. Regul. Integr. Comp. Physiol 2003, 284 (1), R1−R12. (19) Taguchi, H. New physiological functions of niacin-related compounds on plants, isolation of nicotinic acid-N-glucoside and biosynthesis of homarine. Vitamins 1997, 71 (1), 1−15. (20) Yoshinari, O.; Sato, H.; Igarashi, K. Anti-diabetic effects of pumpkin its components, trigonelline and nicotinic acid, on GotoKakizaki rats. Biosci., Biotechnol., Biochem. 2009, 73 (5), 1033−1041. (21) Tse, S. Y. H. Cholinomimetic compound distinct from caffeine contained in coffee. II: Muscarinic actions. J. Pharm. Sci. 1992, 81 (5), 449−452. (22) Muraki, K. Analyses of Ca-related ion channel currents and their involvement in Ca mobilization in smooth muscle and endothelial cells. Nippon Yakurigaku Zasshi 2003, 121, 143−151.

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DOI: 10.1021/acs.jafc.8b01750 J. Agric. Food Chem. XXXX, XXX, XXX−XXX