1
Supplementary File
2
3
Article title:
4
Characterization of bioactive agents in five types of marketed sprouts and comparison of their
5
antihypertensive, antihyperlipidemic, and antidiabetic effects in fructose-loaded SHRs
6
Journal name:
7
Journal of Food Science and Technology
8
Author names:
9
Kozo Nakamura, Masahiro Koyama, Ryuya Ishida, Takashi Kitahara, Takero Nakajima, Toshifumi
10
Aoyama
11
Corresponding author name:
12
Kozo Nakamura
13
Affiliation and e-mail address of the corresponding author:
14
Faculty of Agriculture, Shinshu University. E-mail: knakamu@shinshu-u.ac.jp
15
1
16
17
The analyses of the functional ingredients in powdered sprouts (PSs)
-Aminobutyric acid (GABA), CoenzymeQ10 (CoQ10), and rutin in the PSs were measured at
18
the Collaborated Research Center for Food Functions at Faculty of Agriculture in Shinshu University
19
(CREFAS); myo-Inositol-1,2,3,4,5,6-hexakisphosphate (IP6) was determined at the Research Center
20
for Human and Environmental Science at Shinshu University.
21
GABA: One gram of each PS was extracted with hot water (20 mL) for 1 h in a reflux apparatus
22
followed by filtration. The precipitate was extracted once more. The combined extract was
23
evaporated to prepare analytical samples. The GABA in each PS extract was dissolved at 2.0 mg/mL
24
in 0.1 M HCl. Capillary electrophoresis was performed with a 75 μm i.d. fused silica column (100
25
cm length, Beckman Coulter Inc., California, USA) at 25°C with electrophoretic solvent (61.0 μM
26
CaCl2, 50.5 μM MgSO4, 13.4 μM KCl, 7.25 mM NaCl) on a P/ACE™ MDQ capillary
27
electrophoresis analysis system (Beckman Coulter Inc.). Pressure injection (20 psi × 10 sec) was
28
used to inject the samples. The applied voltage was 30 kV, and compounds were detected by UV
29
monitoring at 190 nm. GABA was identified by retention time relative to a standard. A calibration
30
curve was obtained over a range of 0.025–0.1 mg/mL GABA in 0.1 M HCl with a correlation
31
coefficient of 0.9992. Linearity was described by the equation y = 3.1740 × 10-5 x – 0.002 (y:
32
concentration of GABA, x: area of GABA peak on HPLC chromatogram).
2
33
CoQ10: One gram of each PS was added to 0.5 M NaCl aqueous solution (5.0 mL) followed by
34
ethanol (5.0 mL) and n-hexane (25 mL), and CoQ10 was extracted three times in an incubator with
35
shaking at 250 rpm for 20 min at 25°C. The combined extract was collected by centrifugation (450 g,
36
5 min, Eppendorf Centrifuge 5810 R; Eppendorf AG, Hamburg, Germany) and lyophilized for HPLC.
37
One milligram of lyophilized extract was dissolved in 1.0 mL of the eluent described below.
38
Chromatography was performed on a LC2010CHT HPLC system (Shimadzu, Co., Kyoto, Japan).
39
Elution was performed isocratically with methanol, ethanol, 2-propanol, and 1.0 M ammonium
40
acetate buffer at pH 4.4 (53:24:21:2 v/v/v/v) at a flow rate of 0.7 mL/min, 35°C through COSMOSIL
41
5C18-AR-300 (4.6 × 150 mm, Nacalai Tesque, Kyoto, Japan). Chromatograms were acquired at 275
42
nm and the injection volume was 5.0 μL. A calibration curve was obtained over the range 12.5–50.0
43
μg/mL with a correlation coefficient of 0.9997. Linearity was described by the equation y = 7.69 ×
44
10-5 x − 0.384 (y: concentration of CoQ10, x: area of CoQ10 peak on HPLC chromatogram).
45
Rutin: Rutin was extracted using 50% aqueous methanol and analytical samples were prepared
46
using procedures similar to those for CoQ10. One milligram lyophilized extract was dissolved in 1.0
47
mL of 10% acetonitrile in water with 0.1% formic acid. Separations were performed using a
48
LC2010CHT HPLC system (Shimadzu, Co.) with a CHEMCOBOND 5-ODS-W reversed-phase
49
column (150 mm × 4.6 mm i.d., Chemco Scientific Co., Ltd., Osaka, Japan). Gradient elution for
3
50
rutin was performed with a mobile phase of acetonitrile with 0.1% formic acid (Solvent B) and 0.1%
51
formic acid in distilled water (Solvent A): 0–10 min, 0–5% Solvent B; 10–15 min, 5–5% Solvent B;
52
15–20 min, 5–10% Solvent B; 20–40 min, 10–20% Solvent B; and 40–50 min, 20–100% Solvent B.
53
Chromatography was performed at 40°C with a flow rate of 0.8 mL/min, injection volume of 10 μL,
54
and chromatograms were acquired at 280 nm. A calibration curve was obtained in the concentration
55
range of 0.125–1.25 μg/mL with a correlation coefficient of 0.9999. Linearity was described by the
56
equation y = 4.0335 × 10-4 x + 0.023 (y: concentration of rutin, x: area of rutin peak on HPLC
57
chromatogram).
58
IP6: Six grams of each PS were extracted three times with 30 mL of 0.75 M HCl in a shaking
59
incubator at 250 rpm for 2 h at 25°C. After removal of the residue by centrifugation at 3600 g for 30
60
min at 5°C (Centrifuge 5810 R), the combined supernatant was added to 0.5 M EDTA at ratio of 5:2
61
(supernatant:0.5 M EDTA, v/v) and pH was adjusted to 6.0 using 2.0 M NaOH. The mixture was
62
lyophilized, dispersed in 40 mL water, and filtered (0.45 μm pore size). The filtrate was adjusted to
63
pH 12.6 using solid NaOH and lyophilized again for NMR. 31P NMR analysis was performed on a
64
solution of 200 mg sample in D2O. 1H decoupled 31P NMR spectra were recorded at 25°C on a
65
JNM-LA400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 161.7 MHz and the number of
66
scans was set to 512. To quantify 31P in the PS samples, 2-aminoethylphosphonic acid (10 mM) was
4
67
used as an internal standard.
68
69
The analyses of the nutritional components in PSs
70
The nutritional contents (water, protein, lipid, carbohydrate, minerals, dietary fiber, and
71
vitamins) and total energy of each PS were analyzed by Food Research Laboratories, Japan,
72
according to the Analytical Manual of Standard Tables of Food Composition in Japan.
73
Water content was determined by the vacuum oven-drying method. Total ash content was
74
determined by the direct ashing method. Total fat content was estimated by an acid hydrolysis
75
method. Protein content was estimated by multiplying the total nitrogen content by a factor of 6.25,
76
where total nitrogen contents were determined by the Kjeldahl method. Total dietary fiber was
77
determined using the enzymatic−gravimetric method based on the weight of ethanol precipitate after
78
enzymatic digestion. Total carbohydrate content was estimated by subtracting the percentages of
79
water, protein, fat, ash, and dietary fiber from 100%. Total energy was calculated according to the
80
following equation:
81
Energy (kcal) = 4 × protein (g) + 9 × lipid (g) + 4 × carbohydrate (g) + 2 × dietary fiber (g).
82
Vitamin B1 (Thiamin) and B2 (riboflavin) were fluorimetrically determined. Total vitamin C
83
(ascorbic acid) was determined by the hydrazine method of HPLC.
5