Antihypertensive Effect of a Combination of Uracil and Glycerol

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Antihypertensive Effect of a Combination of Uracil and Glycerol Derived from Lactobacillus plantarum Strain TWK10-Fermented Soy Milk Yi-Yen Liu,†,∥ Shih-Yu Zeng,†,∥ Yann-Lii Leu,§ and Tsung-Yu Tsai*,† †

Department of Food Science, Fu Jen Catholic University, New Taipei City, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan City, Taiwan

§

S Supporting Information *

ABSTRACT: We previously demonstrated that angiotensin-converting enzyme (ACE) could be inhibited by soy milk that had been fermented with the Lactobacillus plantarum strain TWK10, suggesting great potential for the development of antihypertensive products. In this work, the bioactive ACE inhibitors in TWK10-fermented soy milk water extracts were isolated, and a combination of uracil and glycerol (CUG) was identified as one of the ACE inhibitors. We then examined the physiological effects of CUG treatment in short-term and long-term studies using spontaneously hypertensive rats (SHRs) as an experimental model. The results revealed that the fermented soy milk extracts and CUG decreased blood pressure by 11.97 ± 3.71 to 19.54 ± 9.54 mmHg, 8 h after oral administration, and exhibited antihypertensive effects in SHRs in a long-term study. In addition, CUG was shown to decrease blood pressure by suppressing either the renin activity or the ACE activity and, thus, decreasing the downstream vasoconstricting peptide angiotensin II and the hormone aldosterone. CUG also promoted nitric oxide production, resulting in vasodilation and further improvement to hypertension. This important finding suggests that TWK10-fermented soy milk and its functional ingredients, uracil and glycerol, exhibit antihypertensive effects via multiple pathways and provide a healthier and more natural antihypertensive functional food. KEYWORDS: angiotensin-converting enzyme, combination of uracil and glycerol, hypertension, spontaneous hypertensive rats, TWK10-fermented soy milk

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INTRODUCTION Soy milk is a processed soybean product that is common in Taiwan. It is an inexpensive source of nutrition that is particularly favored by those who do not drink animal milk such as vegans and individuals with lactose intolerance or milk allergies. Previous studies have shown that soy milk that is fermented with probiotics inhibits the growth of potential pathogens,1 inhibits melanin production,2,3 reduces serum cholesterol,4 exerts an antiobesity effect,5 and modulates the immune system.6 Probiotic-fermented soy milk also contains certain proteins and aglycone isoflavones, which have been shown to have antitumor activity, improve the symptoms of menopause, and reduce the risk of atherosclerosis.7 Hypertension has particular relevance to modern medicine as it is a key symptom of metabolic syndromes, themselves important risk factors for the development of cardiovascular disease, which can in turn lead to myocardial infarction or stroke.8 Studies have suggested that angiotensin-converting enzyme (ACE) is a multifunctional enzyme associated with the blood pressure (BP)-regulating renin−angiotensin system (RAS); specifically, it catalyzes both the production of the vasoconstricting peptide angiotensin II (AngII) and the inactivation of the vasodilator bradykinin.9 Several previous studies have suggested that lactic acid bacteria (LAB)-fermented milk or soy milk with high bioavailability can improve hypertension in humans.10,11 ACE inhibitory peptides are released from precursor soybean proteins by the action of microbial proteases during the fermentation processes.11,12 We previously demonstrated that a water extract of soy milk that has been fermented © XXXX American Chemical Society

with the Taiwanese fermented cabbage-derived Lactobacillus plantarum strain TWK10 effectively inhibited ACE activity.13 In this work, we sought to identify the bioactive ingredients in the TWK10-fermented soy milk water extract that inhibited ACE activity. In addition, the physiological effects of these active compounds were evaluated in short- and long-term studies using spontaneously hypertensive rats (SHRs), and the inhibitory mechanism of co-administration of these active compounds was revealed.

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MATERIALS AND METHODS

Materials. Lactobacilli de Man, Rogosa, and Sharpe (MRS) broth and Bacto agar were purchased from Becton, Dickinson and Co. (Franklin Lakes, NJ, USA). Nongenetically modified soybean (Glycine max L. Merrill BB50) was obtained from ChuanGui Bio-Organic Co. (Taoyuan, Taiwan). The bacterial strain L. plantarum TWK10 was isolated from Taiwanese fermented cabbage and stored at −80 °C in Lactobacilli MRS with 20% glycerol.3 Purified ACE from rabbit lung, captopril (CAP), γ-aminobutyric acid (GABA), hippuric acid, uracil, glycerol, hippuryl-L-histidyl-L-leucine (HHL), N-[3-(2-furyl)acryloyl]L-phenylalanylglycyl-glycine (FAPGG), and DMSO were purchased from Sigma (St. Louis, MO, USA). HPLC grade acetonitrile and acetic acid were obtained from Merck KGaA (Darmstadt, Germany). Isolation and Identification the ACE-Inhibitory Ingredients in TWK10-Fermented Soy Milk Water Extract. Water extracts of TWK10-fermented soy milk were prepared according to a previously Received: April 1, 2015 Revised: August 4, 2015 Accepted: August 5, 2015

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DOI: 10.1021/acs.jafc.5b01649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry published procedure.13 Preparative silica gel (SiO2) chromatography was used to fractionate TWK10-fermented soy milk water extract. Sequential elution using ethyl acetate (EA)/methanol (MeOH) (9:1), EA/MeOH/H2O (7:3:0.5), EA/MeOH/H2O (7:3:2), EA/MeOH/ H2O (7:3:4), and isopropanol/H2O (7:3) resulted in five fractions (F1−F5) being obtained. Fractions F2 and F4 were further separated using a RP-HPLC system (Thermo, Waltham, MA, USA) coupled to a variable UV absorbance detector (Thermo) operated at 254 nm. The chromatogram was recorded using an electronic integrator (Thermo). Chromatographic separation was performed using a C18 RP analytical HPLC column [YMC-Pack ODS-AQ (25 μm, 12 nm) 250 × 10 mm i.d.] (YMC Co., Ltd., Kyoto, Japan). The composition of the mobile phase during the gradient elution was increased linearly from 0 min (100% water and 0% acetonitrile) to 30 min (85% water and 15% acetonitrile). The flow rate was held constant at 2 mL/min. Five isolates were collected from each fraction, named F2-1−F2-5 and F4-1−F4-5, and the inhibitory effects of these subfractions on ACE activity were investigated. Moreover, F2-2 and F2-3 were further isolated using TLC [solvent EA/MeOH/H2O (8:3:1)], and detected at a UV absorbance of 254 nm. Four subfractions were obtained from F2-2 and F2-3, which were named F2-2A, F2-2B, F2-3A, and F2-3B, respectively. The inhibitory effects of these four subfractions on ACE activity were then determined. Finally, the ACE inhibitor, F2-2A, was identified. Time-of-flight mass spectrometry TOF MS data were obtained using a Microflex MALDI-TOF MS (Bruker Daltonics, Billerica, MA, USA). NMR spectra were obtained using Varian NMR spectrometers (Varian Gemini 500 MHz; Varian Inc., Palo Alto, CA, USA) using D2O as solvent. Measurement of ACE Inhibition. ACE inhibition was measured according to the method described by Cushman and Cheung with some modifications.14 Aliquots (50 μL) of buffered substrate solution (12.5 mM HHL in 100 mM borate buffer solution containing 300 mM NaCl; pH 8.3) were mixed with samples (50 μL) at each tested concentration, and then pre-incubated at 37 °C for 5 min. ACE (25 μL; 25 mU/mL) was added to the reaction, which was incubated at 37 °C for 20 min. The enzymatic reaction was stopped by adding 0.5 N HCl (1 mL). The hippuric acid that was released because of ACE activity was extracted with ethyl acetate (1 mL), which was then dried. The sediment was dissolved in deionized water (1 mL), and the absorbance at 228 nm was measured. The inhibitory activity was calculated using the following equation: ACE inhibitory activity (%) = 100 × [(A − B) − (C − D)]/(A − B), where A is the absorbance of a solution containing ACE but no sample, B is the absorbance of a solution containing ACE that had been previously inactivated by the addition of HCl but no sample, C is the absorbance of a solution containing both ACE and the sample, and D is the absorbance of a solution containing HCl-inactivated ACE and the sample. Lineweaver−Burk Plots. The kinetics parameters were evaluated by Lineweaver−Burk’s method. Briefly, 160 μL of FAPGG (dissolved in 50 mM Tris-HCl buffer containing 0.3 M NaCl, pH 7.5) was mixed with 20 μL of ACE (final activity of 20 mU) and 20 μL of CUG. The rate of decrease in absorbance at 345 nm was recorded for 10 min at room temperature. Distilled water was used instead of CUG solution in the blank experiment. The kinetics of ACE inhibition was studied with 0.0625, 0.125, 0.25, and 0.5 mM FAPGG. In Vivo Assay of Antihypertensive Effect over Short- and Long-Term Intake. A total of 42 SHRs (13 weeks old, 260−300 g) and 6 Wistar Kyoto (WKY) rats (13 weeks old, 260−300 g) were used in this experiment and were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The animals were maintained at a temperature of 21 ± 2 °C and a relative humidity of 55 ± 10% with 12 h light/dark cycles in the Fu Jen Laboratory Animal Center (Taipei, Taiwan). Rats were fed tap water and a standard diet (5001-Laboratory Rodent Diet; LabDiet, St. Louis, MO, USA). Each sample was dissolved in distilled water (1 mL) and was administered orally by gastric intubation between 12 and 1 p.m. Distilled water was used as a negative control, and the known antihypertensive agents CAP (50 mg/kg) and GABA (0.76 mg/kg) were used as positive controls.15 The dose was based on 400 mL/day/ person (weight was 65 kg and height of 170 cm), and human doses

were converted to animal equivalent doses using the Boyd formula.16 The dose of TWK10-fermented soy milk water extract powder was 0.51 g/kg. The dose of combined uracil and glycerol (CUG) was based on the content of TWK10-fermented soy milk water extracts. The 1:1, 1:2, and 1:3 ratios of uracil/glycerol (1.24/1.24 mg/kg, 1.24/2.48 mg/kg, and 1.24/3.72 mg/kg, respectively) were dissolved in distilled water and administrated orally, and their antihypertensive effects in SHRs were evaluated 8 h after treatment. Moreover, low- and high-doses of soy milk extracts (0.51 and 2.55 g/kg, respectively) and low (uracil, 1.24 mg/kg; glycerol, 3.72 mg/kg) and high (uracil, 6.20 mg/kg; glycerol, 18.60 mg/kg) doses of CUG were administered. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded using the tail-cuff method before administration and at 4, 8, and 24 h postadministration using a BP-2000 Blood Pressure Analysis System (Visitech System, Inc., Apex, NC, USA). Before measurements, the rats were kept at 37 °C for 40 min to detect pulsations in the tail artery. Five readings were taken, and the mean of all measurements was calculated. To minimize stress-induced variations in BP, the same individual made all measurements in the same peaceful environment. A training period of 2 weeks was established before the actual trial time, during which time the rats became accustomed to the procedure, to guarantee the reliability of the measurements. After the short-term tests, the SHRs continued to receive low and high doses of CUG for 8 weeks, and SBP and DBP were recorded biweekly. Twenty-four hours before sacrifice, all food was removed. Animals were anesthetized by carbon dioxide inhalation and sacrificed. Blood was collected by cardiac puncture, and serum samples were obtained by drawing the blood into a serum separated tube, allowing it to clot, and centrifuging it for 10 min at 3000g to separate serum. In addition, plasma was obtained by collecting blood in heparinized syringes containing 5% heparin and 2% sodium citrate and centrifuging at 3000g for 15 min. The aorta, liver, lung, and kidney tissues were cleaved and divided into two parts; one part was homogenized (2 g in 10 mL of PBS) using a FastPrep System (MP Biomedicals, Santa Ana, CA, USA), and the other was immediately fixed in 10% neutral buffered formalin for further histological analysis. The experiment was reviewed and approved by the Animal Care and Research Ethics Committee of the Fu Jen Catholic University (IACUC approval A10105). Biochemical and Histological Assessment. The activity of ACE in liver, lung, and kidney tissues was determined as previously described (see Measurement of ACE Inhibition). The serum level of bradykinin in SHRs was determined using a bradykinin enzyme immunoassay kit (Uscn Life Science Inc., Houston, TX, USA). The levels of aldosterone and renin activity in plasma were evaluated using a Wallac 2470 WIZARD2 gamma counter (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA), and AngII levels were measured using an AngII EIA kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). The level of nitrite (NO2−) and nitrate (NO3−) anions in plasma was measured using a nitric oxide EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA). The level of lipid peroxide in serum was determined by measuring the thiobarbituric acid reactive substance (TBARS) concentration as an index of lipid peroxidation, according to the Yagi method.17 In addition, the activity of SOD was measured using an SD125 kit supplied by Randox Laboratories Ltd. (Crumlin, UK). The aorta samples from each of the rats were divided into two parts. One part was for evaluating the vascular elastin distribution and nitric oxide synthase (eNOS) protein expression. The other part was used to determine the protein expression of calmodulin (CaM). To prepare them for optical microscopy, the aorta samples were embedded in paraffin, and sections were stained with hematoxylin−eosin. Verhoff ’s stain was used to evaluate the expression and distribution of vascular elastin. eNOS protein expression was quantified with an immunohistochemical assay according to the method of Zhao et al. and examined using an Nikon TS-100 microscope (Tokyo, Japan). Motic Images 2000 software (Xiamen, China) was used for examination of hypertensive tissues changes. In addition, CaM protein levels were B

DOI: 10.1021/acs.jafc.5b01649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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investigated using Western blotting. The aorta samples (0.2 g) were homogenized with Nonidet P-40/SDS lysis buffer (200 μL; 1% Nonidet P-40, 0.01% SDS, 0.1 M Tris-HCl at pH 7.2, 100 mM phenylmethanesulfonyl fluoride, and 1 mg/mL of aprotinin) using a FastPrep System (MP Biomedicals). The lysate was centrifuged at 11000g for 20 min at 4 °C, and supernatants were collected. Each gel lane was loaded with total protein from the cell extracts (40 μg). Proteins were resolved by SDS-PAGE using a 7.5% acrylamide gel and then transferred to a nitrocellulose membrane. Blocking was performed in Tris-buffered saline containing 2% nonfat milk powder. Membranes were incubated overnight at 4 °C, in blocking solution containing anti-CaM antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) (1:500) and mouse antiβ-actin monoclonal antibody (Chemicon International, Inc., Temecula, CA, USA) (1:5000). Blots were then incubated in blocking buffer containing the corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at 25 °C. Protein bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL, USA), and a UVP image analysis system (UVP, Upland, CA, USA). Analysis was performed using Gel-Pro Analyzer 4 (Media Cybernetics, Inc., Rockville, MD, USA). Statistical Analysis. All values represent means and standard deviations of three repeats. Data were then compared using Duncan’s multiple-range method using SPSS statistical analysis software.

CAP uracil, 10 μg/mL glycerol, 30 μg/mL 1:1 ratio of uracil/glycerol (10 μg/mL/10 μg/mL) 1:2 ratio of uracil/glycerol (10 μg/mL/20 μg/mL) 1:3 ratio of uracil/glycerol (10 μg/mL/30 μg/mL)

RESULTS AND DISCUSSION

ACE-Inhibitory Ingredients in TWK10-Fermented Soy Milk Extract. The half-maximal inhibitory concentration (IC50) value of water extract from TWK10-fermented soy milk for ACE inhibition was 5.61 mg/mL. After extensive fractionation of the TWK10-fermented soy milk water extract, the IC50 values of F2, F3, and F4 were 43.44, 100.00, and 36.91 μg/mL, respectively. Fractions F2 and F4 had the higher inhibitory effect on ACE and were further separated using a RP-HPLC system. In addition, subfractions F2-3 and F4-3, derived from F2 and F4, respectively, had the same retention time (13 min) during the separation using the RP-HPLC system. Moreover, the IC50 values of F2-2, F2-3, and F4-3 were 7.26, 9.19, and 9.35 μg/mL, respectively. According to our findings, F2-3 and F4-3 had similar retention times and IC50 values; there is an empirical basis to support the fact that these two isolates were hypothesized as the same one. Furthermore, F2-2A and F2-3B had better ACE inhibitory effect than others, and the IC50 values of F2-2A and F2-3B were 1.35 and 2.37 μg/mL, respectively, after extensive fractionation of F2-2 and F2-3. A component fraction, F2-2A, was isolated as a white amorphous solid and identified as one of the inhibitors having ACE inhibitory activity (68.94 ± 7.47 and 49.3 ± 4.7% at 10 and 1 μg/mL, respectively). The following spectral data allowed the fraction to be identified as a mixture of uracil and glycerol (Supplemental Figure 1): TOF MS, m/z 113.0498 [M + H]+ for uracil and 115.0516 [M + Na]+ for glycerol (Supplemental Figure 2); 1H NMR (D2O, 500 MHz) δ 3.54 (2H, dd, J = 6.5 and 11.5, H-7 and 9), 3.63 (2H, dd, J = 6.5 and 12.0, H-7 and 9), 3.76 (H, tt, H-8), 5.78 (H, d, J = 8.0, H-5), and 7.51 (H, d, J = 8.0, H-6) (Supplemental Figure 3); 13C NMR (D2O, 125 MHz) δ 63.46 (C-7 and 9), 79.04 (C-8), 101.38 (C-5), 144.33 (C-6), 153.99 (C-2), and 168.35 (C-4) (Supplemental Figure 4). Sugar nucleotides act as substrates during the biosynthesis of polysaccharides in higher plants. Uridine diphosphoglucuronic acid donates D-glucuronosyl units for the synthesis of several types of structural polysaccharides. It is also the precursor of other nucleotide sugars, including uridine diphosphate galacturonic acid, uridine diphosphate xylose, uridine diphosphate L-arabinose, and uridine diphosphate D-apiose, which are incorporated into pectin and hemicelluloses in plants.18 Previous

Table 1. Inhibitory Effect of the Uracil and Glycerol on ACEa group

Article

ACE inhibitory activity (%) 74.21 ± 0.73 A 24.31 ± 8.45 B ND 72.92 ± 3.14 A 72.58 ± 2.88 A 70.94 ± 3.83 A

Data are presented as the mean ± SD (N = 3). Values with different letters in the same column are significantly different by Duncan’s multiple-range test (p < 0.05). ND, not detected; CAP, captopril at a concentration of 3.4 mM.

a

Figure 1. Lineweaver−Burk plots of ACE inhibition by different concentrations of combination of uracil and glycerol at various substrate concentrations (0.0625−0.5 mM). V = initial rate of reaction [ΔA345 (nm)/min]. C

DOI: 10.1021/acs.jafc.5b01649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry studies suggested that uracil formation is a consequence of the enzymatic hydrolysis of uridine by microorganisms.19 In addition, Hidalgo et al. reported that the presence of uracil in tomato products is an index of LAB contamination, suggesting that uracil is a metabolic product that correlates with the growth of LAB in the presence of uridine.20 In the present study, lipids were present in unfermented soy milk (1.4%, w/w), and lipase activity was observed (data not shown). Taken together, these data suggest that the functional ingredients, uracil and glycerol, were present TWK10-fermented soy milk. In addition, Schanker and Tocco demonstrated that uracil is absorbed across the rat intestinal epithelium via an active transport mechanism.21 Moreover, the principal transport mechanism for glycerol in the rat small intestine is passive (diffusive) transport via the paracellular route and an Na+-dependent carrier-mediated transport system. Therefore, these two functional ingredients are theoretically both absorbed in the rat small intestine. Inhibitory Effects of Uracil and Glycerol on ACE Activity in Vitro. The uracil content in TW10-fermented soy milk water extracts was analyzed using HPLC according to a method described by Deporte et al.22 and was found to be 2.44 ± 0.07 mg/g in dried powder. In addition, the glycerol content of soy milk extract was 7.31 ± 0.58 mg/g as shown using enzymatic kits (GY105, Randox Laboratories Ltd., Crumlin, UK). The ratio of uracil and glycerol was nearly 1:3. The ACE inhibitory effects of different ratios of pure uracil and glycerol were then evaluated. As shown in Table 1, there were no significant differences between each ratio of uracil/ glycerol (p > 0.05) and the positive control. Each substance alone had little or no effect on ACE activity. Glycerol is a water-soluble trivalent alcohol normally present in human tissues as an integral part of both fats and triglycerides. It is readily metabolized, primarily in the liver, and systemic hyperosmolarity may be reduced and dehydration limited. MacDonald and Uden (1982)23 indicated that intravenous glycerol and mannitol were equally effective in lowering acute elevations of intracranial pressure. Previous studies employing glycerol as a major osmotherapy agent entailed pharmacologically inert substances that increase the osmotic pressure of plasma, promoting movement of water from interstitial spaces to vascular spaces.24 Osmotic agents include mannitol, urea, sorbitol, glycerol, and hypertonic saline, and they have primarily been applied in the treatment of cerebral infarction, experimental ischemia, traumatic intracranial hypertension, or edema.25 In the current study, uracil and glycerol had low or no ACEI activity, respectively, and the inhibitory effect rose with an increase in the ratio of uracil/glycerol (from 1:1 to 1:3), indicating that the presence of glycerol increased the inhibitory effects of ACE by increasing the level of uracil to bind to ACE. The mode of ACE inhibition by CUG was investigated by kinetic studies using Lineweaver−Burk plots. As shown in Figure 1, the double-reciprocal plots of ACE-catalyzed reactions in the absence and presence of a combination of uracil and glycerol indicate mostly uncompetitive inhibition of ACE activity. The result suggests that the combination of uracil and glycerol exhibited activities by binding to only the enzyme− substrate complex. The combination of uracil and glycerol as an uncompetitive inhibitor can bind to the enzyme−substrate complex to prevent catalysis. The Km value for ACE activity in the absence of the combination of uracil and glycerol was 0.08 mM FAPGG, and Vmax was 0.005. The presence of the

Figure 2. Effect of the single oral administration of TWK10-fermented soy milk water extract and combined uracil and glycerol treatment on (A) systolic and (B) diastolic blood pressure in spontaneously hypertensive rats. Control, administration of H2O; captopril, administration of captopril at a dose of 50 mg/kg; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg; uracil-glycerol-1×, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively, 0.76 mg/kg bw; uracil-glycerol-5×, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively, 0.76 mg/kg bw; TWK10-1×, administration of soy milk extracts at a dose of 0.51 g/kg bw; TWK10-5×, administration of soy milk extracts at a dose of 2.55 g/kg bw. Data are presented as means ± SD (n = 6). Values with different letters were significantly different according to Duncan’s multiplerange test (p < 0.05).

combination of uracil and glycerol resulted in concentrationdependent decreases in both Vmax and Km values. In the uncompetitive inhibition system, the inhibitor bound only to the enzyme−substrate complex and decreased the maximum enzyme activity, taking longer for the substrate or product to leave the active site. Most ACE inhibitory peptides obtained from the hydrolysis of pea (Pisum sativum var. Bajka) globulins,26 rapeseed protein digest,27 chickpea,28 or wakame (Undaria pinnatifida)29 belong to uncompetitive inhibitors. In addition, nonpeptide compounds such as usnic acid from lichen species Usnea complanata under submerged fermentation or polyphenols from tea (Camellia sinensis) also showed an uncompetitive type of ACE inhibition.30,31 According to our D

DOI: 10.1021/acs.jafc.5b01649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 2. Effect of Chronic Oral Administration of the Mixture of Uracil and Glycerol on Systolic Blood Pressure in SHRa SBP (mmHg) group N C GABA LDG HDG

0 weeks 157.18 185.54 185.30 185.47 184.93

± ± ± ± ±

4.38 3.62 4.21 2.47 2.54

2 weeks a Aa Aa Aa Aa

156.05 199.84 185.73 185.60 186.97

± ± ± ± ±

5.20 4.05 3.45 4.62 4.96

4 weeks a Ab Ba Ba Ba

153.46 204.03 187.44 192.09 187.13

± ± ± ± ±

8.33 2.65 4.41 2.22 5.31

6 weeks a Ac Cab Bb Ca

150.79 206.89 189.15 193.97 188.28

± ± ± ± ±

5.45 2.27 3.64 1.38 3.95

8 weeks a Ac Cab Bbc Ca

151.25 207.12 190.72 195.82 188.67

± ± ± ± ±

8.53 3.11 3.12 2.01 3.31

a Ac Cb Bc Ca

Data are presented as means ± SD (N = 6). Values with different upper and lower case letters in columns and rows, respectively, are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively; HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

a

Table 3. Effect of Chronic Oral Administration of the Mixture of Uracil and Glycerol on Diastolic Blood Pressure in SHRa DBP (mmHg) group N C GABA LDG HDG

0 weeks 133.92 165.85 165.89 166.25 168.48

± ± ± ± ±

9.13 6.01 6.79 3.00 5.30

2 weeks a Aa Aa Aa Aa

139.67 181.52 169.13 168.24 168.14

± ± ± ± ±

7.32 6.10 3.26 6.37 4.79

4 weeks a Ab Ba Ba Ba

131.79 184.06 166.71 173.50 170.15

± ± ± ± ±

6.15 4.15 8.19 2.16 2.12

6 weeks a Abc Ca Bb BCa

133.64 185.94 167.26 174.75 171.06

± ± ± ± ±

5.37 3.89 4.28 2.60 5.43

8 weeks a Abc Ca Bb BCa

133.36 189.02 167.86 177.45 168.14

± ± ± ± ±

5.16 3.98 5.99 2.45 5.27

a Ac Ca Bb Ca

Data are presented as means ± SD (N = 6). Values with different upper and lower letters in columns and rows, respectively, are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively; HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

a

findings, based on kinetic studies using Lineweaver−Burk plots, the CUG demonstrated mostly uncompetitive inhibition of ACE activity, and we can hypothesize that a conformational change in ACE occurs upon binding to the substrate, which presents a binding site for CUG. As a result, inhibition occurs because the substrate−enzyme−inhibitor complex cannot form the final product in vitro. Antihypertensive Effects of Soy Milk Extract and CUG on SHRs in Short-Term Study. The antihypertensive effects of TWK10-fermented soy milk water extracts and CUG were investigated in SHRs. The initial SBP and DBP were measured as reference values before the oral administration of samples. As shown in Figure 2, there was no significant difference in the SBP and DBP of the control group during the 24 h after oral administration of the samples (p > 0.05). In the CAP group, SBP and DBP were suppressed by 20.11 ± 8.35 and 20.20 ± 7.84 mmHg, respectively, after 4 h, and by 35.31 ± 11.52 and 46.91 ± 13.92 mmHg after 8 h (p < 0.05). GABA was used as an additional positive control and suppressed SBP by 17.90 ± 5.75 and 19.96 ± 6.25 mmHg (p < 0.05) after 4 and 8 h, respectively. The uracil content in the low dose of soy milk extract was 1.24 mg/kg, and this was used to determine the low dose of uracil in CUG. Doses of 1:1, 1:2, and 1:3 ratios of uracil/glycerol (1.24/1.24 mg/kg, 1.24/2.48 mg/kg, and 1.24/3.72 mg/kg, respectively) were administered orally to evaluate their antihypertensive effect 8 h after treatment of SHRs. The SBP and DBP of rats was suppressed most effectively by the 1:3 uracil/glycerol, which induced reductions of 12.18 ± 3.13 and 10.47 ± 3.07 mmHg, respectively (p < 0.05). This ratio was used in subsequent experiments. As shown in Figure 2, there was no significant difference between the SBP and DBP in the negative control group rats and those that received the low dose of soy milk extract (0.51 g/kg) 4 h after treatment (p > 0.05). However, 8 h after

treatment, the extract decreased SBP and DBP significantly by 19.54 ± 9.54 and 28.19 ± 7.14 mmHg, respectively (p < 0.05 versus negative control). A high dose of soy milk extract (2.55 g/kg) significantly suppressed DBP 4 h after treatment and SBP after 4 and 8 h, compared with the negative control group (p < 0.05). Moreover, the SBP of rats treated with low and high doses of CUG was decreased by 8.83 ± 4.18 and 15.42 ± 4.21 mmHg, respectively, 4 h after oral administration (p < 0.05), compared with the control group. It was also reduced 8 h after oral administration (p < 0.05), compared with the low and high doses of soy milk extract. The low and high doses CUG suppressed SBP significantly by 11.97 ± 3.71 and 16.89 ± 2.59 mmHg and DBP by 13.01 ± 4.77 and 18.03 ± 3.74 mmHg, respectively, compared with the control group. The initial DBP and SBP values were restored to the initial levels after 24 h. These data show that CUG was more effective than soy milk extract 4 h after oral administration. However, the TWK10-fermented soy milk water extract suppressed DBP more potently than CUG after 8 h, suggesting that there might be additional functional ingredients in the other fractionated extract. Antihypertensive Effects of CUG on SHR in LongTerm Study. Tables 2 and 3 show the changes in SBP and DBP during 8 weeks of CUG administration. There was no significant difference in the SBP and DBP of the all groups before oral administration of the samples (p > 0.05). During the 8 week period, SBP and DBP of the control group increased. The low and high doses of CUG significantly suppressed SBP and DBP after 4 and 8 weeks of administration (p < 0.05) compared to control. In the low-dose CUG group, SBP and DBP both increased significantly between the baseline and after 4 weeks of administration and between 4 and 8 weeks (p < 0.05). However, in the high-dose CUG group neither SBP nor DBP had significant changes over the 8 weeks (p > 0.05). In summary, although long-term oral administration of a E

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Figure 3. Effect of the combination of uracil and glycerol on microscopic examination of aorta elastin thin section in spontaneously hypertensive rats: (A) normal; (B) control; (C) GABA treated; (D) low dose; (E) high dose (×100 magnification).

Table 4. Effect of Mixture of Uracil and Glycerol on Microscopic Examination of Aorta Elastin Band Number in SHRa group N C GABA LDG HDG

Table 5. Effect of the Mixture of Uracil and Glycerol on ACE Activity in Organs of SHRsa ACE activity (U/mL)

aortic elastin bands (number) 6.50 10.67 8.00 8.67 7.50

± ± ± ± ±

0.55 1.03 0.63 0.52 0.55

group

D A BC B C

N C GABA LDG HDG

Data are presented as means ± SD (N = 6). The elastin bands were counted from three different sections/specimens. Values with different letters are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively; HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

a

kidney 12.01 21.54 18.40 14.08 7.95

± ± ± ± ±

2.90 4.82 4.87 2.27 2.96

lung BC A A B C

24.74 37.00 31.57 18.29 13.20

± ± ± ± ±

4.76 3.54 3.27 2.60 2.98

liver C A B D E

26.67 29.70 23.60 26.86 18.28

± ± ± ± ±

4.19 5.99 3.42 6.79 3.24

AB A BC AB C

Data are presented as means ± SD (N = 6). Values with different letters are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively; HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

a

vessels.33 In this study, elastin bands in arteries were counted (Table 4), and significantly fewer were found in the two CUGtreated groups (p < 0.05). In summary, the elastin fibers in the aorta of CUG-treated SHRs were significantly straighter than those of controls, affecting the elasticity of vessels and aiding in the improvement of hypertension. Regulatory Effects of Long-Term Administration of CUG on the RAS in SHRs. Figure 4A shows the aldosterone levels in plasma of SHRs after 8 weeks of administration of CUG and indicates that administration of low and high doses of CUG caused significant decreases in aldosterone level, compared with the control group (p < 0.05). The results also show that AngII was significantly suppressed by both low and high doses of CUG, compared to the control group (p < 0.05) (Figure 4B). Figure 4C shows that only a high dose of CUG significantly increased the concentration of bradykinin after long-term administration. These results indicate that suppression of aldosterone levels may lead to the inactivation of the upstream catalytic enzyme. Moreover, Table 5 shows the effects of 8 weeks of CUG administration on ACE activity in the kidney, lung, and liver of SHRs. CUG was found to dose-dependently

low-dose CUG suppresses the increase in SBP and DBP in SHRs, a high dose of CUG is required to prevent an increase in BP entirely. In addition, the difference in effectiveness between low and high doses of CUG after 8 weeks of administration reached significant levels (p < 0.05), indicating a positive correlation between the dose of CUG and its efficacy. Effect of Long-Term CUG Treatment on Elastin Arrangement in Arcuate Arteries of SHRs. Elastin is a protein that maintains vascular elasticity and gives blood vessels the capacity to stretch and contract. Previous studies have indicated that hypertension causes excessive pressure within blood vessels, leading to an altered distribution of elastic fibers.32 Figure 3 shows that elastin in untreated SHRs was the most disorganized. After 8 weeks of treatment with higher doses of CUG, fibers were more ordered, indicating a positive correlation between CUG dose and elastin arrangement. This result indicated that CUG can improve the elasticity and tensile strength of blood vessels. In addition, there have been reports that spontaneous hypertension increases levels of elastin and causes inelastic fibrous masses to accumulate within blood F

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Figure 4. Effect of the combination of uracil and glycerol on (A) angiotensin II, (B) aldosterone, (C) bradykinin, and (D) rennin activity in SHR. Data are presented as means ± SD (n = 6). Bars with different letters are significantly different by Duncan’s multiple-range test (p < 0.05).

Table 6. Effect of the Mixture of Uracil and Glycerol on Superoxide Dismutase and Malondialdehyde Concentration in Serum of SHRa group

SOD (U/mL)

N C GABA LDG HDG

2.28 1.80 3.04 2.38 2.43

± ± ± ± ±

0.32 0.12 0.30 0.22 0.18

B A C B B

Table 7. Effect of the Mixture of Uracil and Glycerol on Immunohistochemical of eNOS in the Sections of Aorta and Nitric Oxide Concentration in Plasma in SHRsa

MDA (μM) 10.13 17.00 10.38 10.00 10.75

± ± ± ± ±

1.73 4.14 2.45 1.69 2.71

group B A B B B

N C GABA LDG HDG

eNOS density (%) 4.50 3.87 3.39 6.75 18.23

± ± ± ± ±

2.84 1.76 2.59 2.91 4.89

A A A A B

nitrate + nitrite (μM) 16.73 16.23 16.58 15.50 22.45

± ± ± ± ±

1.12 3.09 4.06 2.39 5.89

A A A A B

a

Data are presented as means ± SD (N = 6). Values with different letters are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively, HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

a

Data are presented as means ± SD (N = 6). Values with different letters are significantly different by Duncan’s multiple-range test (p < 0.05). N, normal control group; C, hypertension control group; GABA, administration of γ-aminobutyric acid at a dose of 0.76 mg/kg bw; LDG, administration of combined uracil and glycerol at doses of 1.24 and 3.72 mg/kg bw, respectively; HDG, administration of combined uracil and glycerol at doses of 6.20 and 18.60 mg/kg bw, respectively.

suppress ACE activity in the liver, kidneys, and lungs, and the results were significant (p < 0.05). In addition, GABA decreased ACE activity in the lung and liver because GABA suppresses BP

through inhibition of sympathetic nerve activation and reduction of binding with α1-receptors on vascular smooth muscle cells that connect with the RAS.34,35 G

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These downstream factors were regulated by suppressing renin activity and reducing ACE activity. Effects of Long-Term Administration of CUG on Antioxidant Capacity and Nitric Oxide (NO) Production in SHRs. Free radicals bind with NO, which then is then oxidized into peroxynitrate, reducing NO levels and affecting vasodialation. Additionally, the accumulation of products of lipid overoxidationsuch as malondialdehyde (MDA)in blood vessels causes atherosclerosis, resulting in an increase in BP. The effect of CUG administration on SOD activity and MDA was determined. Table 6 shows that the administration of low and high doses of CUG and GABA significantly increased SOD activity and decreased MDA levels compared to the control group (p < 0.05). Table 7 shows the effect of CUG administration on plasma NO level and eNOS expression in SHRs. Plasma NO levels following a high dose of CUG increased 1.38 times, compared with the control group (p < 0.05); however, low-dose CUG administration made no difference (p > 0.05). Immunostaining showed that there was no difference in eNOS expression between low-dose CUG-treated and GABA-treated groups, compared to the control group (p > 0.05). However, the highdose CUG administration significantly increased eNOS expression within arcuate arteries, 4.71 times greater than the control group (p < 0.05). This result corresponds with the NO level in the treated SHRs. Figure 5 shows that long-term administration of low and high doses of CUG increased CaM protein expression by 1.36 and 2.19 times, respectively, compared to the control group (p < 0.05). It can mediate eNOS activity by binding calcium ions to further produce NO.37 In summary, administration of low and high doses of CUG can increase the SOD activity and decrease the MDA level to prevent NO bound with free radicals. In addition, eNOS and CaM expressions were also improved to increase the NO

Figure 4D shows that only the high dose of CUG caused a significant decrease in renin activity in SHRs, compared to the control group (p < 0.05). Previous studies indicate that blockade of the RAS leads to a feedback increase in renin secretion and synthesis.36 The inhibitory effect of CUG on renin activity was determined in vitro in this study using a Renin Inhibitor Screening Kit (K799-100, BioVision, Inc., Milpitas, CA, USA). It was found that renin activity was significantly inhibited by 52.94 ± 6.79, 29.41 ± 3.23, 20.59 ± 3.19, 17.65 ± 4.24, and 5.88 ± 1.23% at concentrations of 1000, 100, 50, 10, and 1 μg/mL, respectively. In summary, CUG can decrease BP through AngII and the hormone aldosterone, downstream products of RAS.

Figure 5. Expression of calmodulin by administration of combination of uracil and glycerol. Data are presented as means ± SD (n = 3). Bars with different letters are significantly different by Duncan’s multiplerange test (p < 0.05).

Figure 6. Mechanism of antihypertensive effect of combined uracil and glycerol treatment. H

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production. These factors can promote vasodilation, resulting in the improvement of hypertension (Figure 6). BP is controlled by a number of different biochemical pathways. NO levels can be increased indirectly by enhancing the activity of endothelial eNOS. Upon entering smooth muscle cells, NO can activate guanylyl cyclase, increasing the accumulation of cyclic guanosine monophosphate and promoting smooth muscle relaxation.38−40 Along with NO, ACE is an important factor that exerts a cardiovascular protective effect and regulates BP.41 Oxidative stress has been implicated as a causal factor in diseases such as hypertension and atherosclerosis.42,43 Martinez-Villaluenga reported that Enterococcus faecium-fermented soy milk inhibited ACE, had antioxidant activity, and had an antihypertensive effect.10 The regulation of BP involves the RAS, which involves ACE; therefore, inhibiting ACE is a key target for regulating BP. Specifically, ACE inhibitors can lower BP by reducing the production of AngII and inhibiting the degradation of bradykinin.44 This study indicated that SBP and DBP were suppressed following treatment with the CUG. A previous study indicated that the administration of LAB or related fermentation products suppressed SBP and DBP by 4.4−16 and 3.4−11 mmHg, respectively,45−47 which was similar to the effect exerted by CUG in this study. In this work, we confirmed that the mechanism by which CUG reduces BP is by inhibiting ACE activity and suppressing the downstream vasoactive peptide AngII and the hormone aldosterone. Additionally, in the case of ACE inhibitors, blockade of the RAS leads to a feedback increase in renin activity,36 and angiotensin I (AngI) can be observed; however, the renin activity of SHRs administered with CUG was suppressed. Moreover, the results revealed that renin activity was inhibited by CUG in vitro. It has been suggested that the CUG derivative derived from TWK10-fermented soy milk can be effective in lowering BP as either renin or ACE inhibitors and is an important novel finding as a functional foods. In conclusion, we identified that a combination of uracil and glycerol was one of the ACE inhibitory functional ingredients in TWK10-fermented soy milk and have demonstrated that it works as a renin inhibitor, an ACE inhibitor, and a promotor of NO production and have shown that it is as effective as an existing drug at lowering BP in vivo for the first time. We are currently in the process of confirming another ACE inhibitor, F2-3B. Compared with peptides or other organic compounds described previously, CUG has significant BP-lowering effects, suggesting that they could be novel and potent natural ingredients in antihypertensive functional foods.48−50

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Financial support (MOST 102-2221-E-030-006 and MOST 103-2221-E-030-013) from the Ministry of Science and Technology of Taiwan is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We express our gratitude to James Chang, Chuan Gui BioOrganic Co., who kindly provided soybean (Glycine max (L.) Merrill BB50).

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ABBREVIATIONS USED ACE, angiotensin-converting enzyme; AngI, angiotensin I; BP, blood pressure; CaM, calmodulin; CAP, captopril; CUG, combined uracil and glycerol; DBP, diastolic blood pressure; EA, ethyl acetate; EIA, enzyme immunoassay; eNOS, endothelial nitric oxide synthase; GABA, γ-aminobutyric acid; HHL, hippuryl-L-histidyl-L-leucine; LAB, lactic acid bacteria; MDA, malondialdehyde; MeOH, methanol; MRS, de Man, Rogosa, and Sharpe; NO, nitric oxide; RAS, renin−angiotensin system; SBP, systolic blood pressure; SHR, spontaneous hypertensive rat; TBARS, thiobarbituric acid reactive substance; WKY, Wistar Kyoto

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs/jafc/5b01649. Supplemental figures 1−4 (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(T.-Y. Tsai) Phone: +886-2-2905-2539. E-mail: tytsai@ mail.fju.edu.tw. Mail: No. 510 Zhongzheng Road, Xinzhuang District, 24205 New Taipei City, Taiwan. Author Contributions ∥

Y.-Y. Liu and S.-Y. Zeng made equal contributions to this paper. I

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DOI: 10.1021/acs.jafc.5b01649 J. Agric. Food Chem. XXXX, XXX, XXX−XXX