Maintaining vascular function is important to human health, because vascular dysfunction could be a risk factor of cardiovascular diseases (CVD) [1
]. Oxidative stress is a major trigger of vascular dysfunction. It is widely accepted that oxidative stress is closely related to the aging process [2
], and decline in vascular function is an inevitable result of aging. Recently, many researchers are focusing on the effect of dietary intake of polyphenols on the prevention of CVD in vivo and in vitro [3
]. Nitric oxide (NO) is another indicator for regulation of vascular function. It contributes to the relaxation of blood vessels and eventually leads to improvement in vascular stiffness [5
]. Oxidative stress and aging quenches the production of NO and hampers the NO-mediated responses that eventually lead to vascular dysfunction [6
]. Black soybean is a nutrient-rich food that contains abundant polyphenols in its seed coat and grain and is widely eaten in Eastern Asian countries. It contains anthocyanins and flavan-3-ols including epicatechin, procyanidin B2, procyanidin C1, and cinnamtannin A2 in its seed coat, in contrast to yellow soybean [7
]. In addition, isoflavones are major polyphenols in the grain of black soybean, as they are in the yellow one. Previous studies reported that polyphenols in the black soybean had various beneficial physiological functions, such as antioxidant in HepG2 cells [9
], anti-obesity, and anti-diabetic activities in C57BL/6 mice [10
Since antioxidant activity of black soybean polyphenols is expected to exert prevention of CVD, many researchers addressed this issue. However, amelioration and prevention effects of black soybean on CVD were mainly reported in animal experiments [11
]. Recently, we reported that an intake of roasted black soybean at 30 g/day for 4 and 8 weeks improves vascular function and vascular age through increasing NO production and reducing oxidative stress in healthy women by an open-label trial [15
]. Moreover, we found that black soybean seed coat extract increased the NO production accompanied by Glucagon-like peputide-1 (GLP-1) secretion from the intestine of rats [16
]. Another research group demonstrated that consumption of black soybean (35% of experiment diet) for 10 weeks inhibited oxidative stress by increasing antioxidant activity and improving lipid profiles in ovariectomized rats, resulting in the improvement of risk factors associated with CVD [11
]. It was also demonstrated that an oral administration of black soybean extract at 50 and 100 mg/kg body weight for 14 days reduced the risk of CVD by improving blood circulation through inhibiting platelet aggregation and thrombus formation in rats [12
]. From these reports, it can be expected that black soybean improves vascular function and prevents CVD.
However, there is not enough evidence for the improvement of vascular function in humans after consumption of black soybean. As mentioned above, we reported that consumption of roasted black soybean improves vascular function in healthy women, but there are two remaining issues. One is that previous participants were only woman, and another is the design of study. In the previous study, all participants were aware of the treatment because of the open-label trial without a placebo meal. To elucidate the exact effects of black soybean on the improvement of vascular function, in this study, we performed a randomized, placebo-controlled, crossover trial in both healthy men and women using a test cookie containing black soybean powder and a placebo cookie. In this trial, the ingestion amounts of black soybean were reduced from 30 g to 20 g, and the period was shortened from 8 weeks to 4 weeks; we also investigated vascular function, production of nitric oxide, oxidative stress, and polyphenol concentrations in the plasma and the urine.
2. Materials and Methods
2.1. Participants Eligibility
This study was approved and conducted by the Institutional Review Board of Fujicco Co., Ltd. (randomized controlled trials registration number: #5801) in accordance with the Declaration of Helsinki. To perform the study, informed consent was obtained from all participants. Participants were excluded if they met any of the following exclusion criteria: (1) having a clinical history of severe gastrointestinal disease, liver disease, kidney disease, or heart disease; (2) currently undergoing treatment for metabolic syndrome or its associated diseases; (3) using medication for blood flow and/or pressure, such as Warfarin and Captopril; and (4) having a physical condition considered inappropriate for this study by a physician. The main inclusion criterion was that the vascular age of the participant was higher than their chronological age.
2.2. Design of Human Study
This study was a randomized, placebo-controlled, crossover trial. The required sample size assumed 34 participants (17 per group) with a power of 80%, a two-tailed α level of 0.05, and a 50% treatment effect by G*power free software [17
]. From this assumption, 23 participants (12 males and 11 females) aged from 30 to 60 years were enrolled for this study. Participants were randomly allocated to receive daily about 40 g/day of cookie containing either 20 g of roasted black soybeans powder or 20 g of flour as a placebo for 4 weeks, with a washout period of 4 weeks between the treatments. In the previous study [15
], participants consumed 30 g of roasted black soybean (whole grain) as the test meal for 4 and 8 weeks, and consumption of black soybean markedly improved the vascular function after 4 weeks. Therefore, in the present study, we reduced the intake amount of black soybean to 20 g and decided on a test period of 4 weeks. Since polyphenols are recognized as xenobiotics, they are easily excreted into urine and feces by 24 h [18
]. In addition, many crossover trials employ a 4 weeks washout period [19
]. Thus, we also decided on the 4 weeks washout period in the present study. At the beginning and the end of each treatment, anthropometrics, accelerated plethysmogram (APG), and blood pressure and assessments of health-related quality of life (HRQOL) were measured; blood and urine samples were also collected from participants under the fasting condition before breakfast.
2.3. Study Diets
Test cookies were produced by Fujicco Co., Ltd. (Kobe, Japan) and Bakery Miki. The composition of the cookies is shown in Table 1
A, and their nutrients composition was calculated and is shown in Table 1
B. The used black soybean cultivar was “Hikariguro”. Indigestible dextrin was used to form the cookie (Pine Fiber W, Matsutani Chemical Industry Co., Ltd., Itami, Japan). Nutritional composition of test cookies was calculated using the Standard Tables of Food Composition in JAPAN, 2015. Polyphenol contents of each test cookie were also measured using the same methods as described in the previous report [15
]. In brief, a cookie was pulverized in a mortar, and the powdered cookie was washed by hexane to remove fat. After removing the hexane layer, the residue was extracted with 70% acetone containing 0.5% acetic acid. The acetone extract was provided for the measurement of polyphenol contents by a high-performance liquid chromatography (HPLC). The detailed method is written in the later section (Section 2.8
, Extraction and Quantification of Polyphenols.) For the antioxidant activity, the 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH) radical absorbance capacity of black soybean and placebo cookies was measured [9
2.4. Measurements of Body Composition
Body composition, body weight, body mass index (BMI), body fat percentage, visceral fat percentage, biological age, basal metabolic rate, estimated bone mass, and muscle mass were measured by a Bioelectrical Impedance Analysis using a body composition meter (BC-610-PB, TANITA. Co., Ltd. Tokyo, Japan).
2.5. Measurements of Vascular Function
Participants took a rest of about 10 min, and then vascular function and blood pressure were measured. Vascular function was evaluated by acceleration plethysmogram (APG) using a Pulse Analyzer®
device (Pulse Analyzer Plus View®
, YKC Corporation, Tokyo, Japan) as described in the previous report [15
]. Briefly, APG was measured with the device attached to the left middle finger of each participant. APG is the second derivative wave of the photoplethysmogram, which consisted of a
waves, namely, early systolic positive wave, early systolic negative wave, late systolic re-increasing wave, and late systolic re-decreasing wave, respectively. Their magnitudes and the height ratios of b/a, c/a, and d/a were measured. Vascular age was calculated from the second derivative of the photoplethysmogram aging index, which is [(b-c-d-e)/a] wave ratio [20
]. Vascular waveform, waveform score, and peripheral vascular health were calculated from a wave pattern, and the ratios of these 4 waves were calculated using software for the Pulse Analyzer®
. Systolic, diastolic, and central blood pressure were measured in the right upper arm using an automated sphygmomanometer (HEM-9000AI, OMRON Corporation, Kyoto, Japan).
2.6. Measurements of Biomarkers in Blood and Urine
Plasma and urine were used for measurement of NO2/NO3, 8-hydroxy-2′-deoxyguanosine (8-OHdG), hexanoyl-lysine (HEL), and myeloperoxidase (MPO) by corresponding commercial kit [NO: NO2/NO3 Assay Kit-C II (DOJINDO LABORATORIES, Kumamoto, Japan); 8-OHdG: New 8-OHdG Check ELISA (Japan Institute for the Control of Aging, NIKKEN SEIL Co., Ltd. (JaICA) Shizuoka, Japan); HEL: HEL ELISA kits (JaICA); and MPO: Human serum MPO and urine MPO ELISA kits (JaICA)]. Urinary NO2/NO3 level was corrected by creatinine equivalent, which was measured by using Creatinine (urinary) Colorimetric Assay Kit (Cayman CHEMICAL, Ann Arbor, MI, USA).
Analyses of hematologic biochemical markers including creatinine, total protein, blood urea nitrogen, glucose, lactate dehydrogenase, alkaline phosphatase, γ- glutamyltranspeptidase, aspartate aminotransferase, alanine aminotransferase, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, Na+, Cl−, K+, white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean hemoglobin concentration, and platelet were measured by LSI Medience Co. (Tokyo, Japan).
2.7. Extraction and Quantification of Polyphenols
Extraction and quantification of polyphenols were performed by the same method as described in our previous report [15
]. An aliquot of 10 mL urine was concentrated to 2 mL using a centrifugal separator in vacuo. Concentrated urine or 500 μL of plasma were mixed with 2% (w/v) ascorbic acid (200 μL for urine and 50 μL for plasma) to prevent oxidation during extraction and were transferred to polypropylene centrifuge tubes (15 mL, BD Biosciences, San Jose, CA, USA), which were siliconized by Sigmacote®
(Sigma-Aldrich, St. Louis, MO, USA) before use. These plasma and urine samples were hydrolyzed with 500 U of β-glucuronidase from Escherichia coli
(type IX-A, Sigma-Aldrich) and 10 U of sulfatase from Abalone entrails
(type VIII, Sigma-Aldrich) for deconjugation according to our previous reports [15
]. A solid phase extraction method was used to extract polyphenols from the mixture: C18 Sep-Pak cartridge (50 mg resin, Waters Co., Milford, MA, USA) was conditioned with 5 mL of methanol and 5 mL of ultrapure water. Plasma and urine samples were centrifuged at 3000× g
for 15 min to remove precipitated protein and applied to the cartridge. After the cartridge was washed with 5 mL of 10% methanol, polyphenols were eluted with 2 mL of 95% (v/v) methanol and evaporated to dryness using the centrifugal separator. Obtained extracts were used for analysis of polyphenols by HPLC.
HPLC was performed using a system equipped with a DGU-20A 3R degas unit, LC-20AD XR binary pump, SIL-20AC XR auto sampler, RF-20A XS fluorescence detector, SPD-M20A diode array detector, CTO-20AC column oven, and CBM-20A communications bus module connected to an LC work station (Shimadzu Corporation, Kyoto, Japan). The analytical column was a Cadenza CL-C18 column (φ 250 mm × 4.6 mm, 3 μm, Imtakt, Kyoto, Japan) protected by a guard column (Cadenza CL-C18, φ 5 mm × 2 mm, 3 μm, Imtak). Quantification of cyanidin-3-O
-glucoside (C3G), flavan-3-ols, and isoflavones was separately carried out under the same analytical conditions with corresponding authentic compound as described in our previous report [15
2.8. Assessments of HRQOL
HRQOL was assessed using the Japanese version of the 36-item Short-Form Health Survey (SF-36) questionnaire developed by iHope International Co., Ltd. (Kyoto, Japan) [24
]. SF-36 was used as a profile-type evaluation of HRQOL. In this study, the Japanese version 1.20 was used; the reliability and the validity of the Japanese version of SF-36 have been confirmed in normal subjects [25
]. SF-36 can comprehensively evaluate physical and mental functions and can also assess social activities. In this study, eight subscales of SF-36 (physical functioning, role physical, bodily pain, general health, vitality, social functioning, role emotional, and mental health) were used to investigate health-related quality of life.
2.9. Statistical Analysis
Data are expressed as the means ± standard deviation. Statistical analysis was performed with a Welch’s t test using JMP statistical software version 11.2.0 (SAS Institute, Cary, NC, USA). The level of significance was set as p < 0.05.
Recently, we reported that intake of black soybean improved vascular function in healthy women by an open-labeled trial. This study expanded the human trial from the previous study to confirm the intake of black soybean improved vascular function. In the present study, we conducted and performed the randomized, single blind, placebo controlled, crossover trial for 4 weeks by reducing the intake amount of black soybean from 30 g to 20 g. We confirmed that the improvement of vascular function, including lowered vascular age and decreased blood pressure (Table 4
and Table S2
), was accompanied by increased concentration of polyphenols derived from black soybean (Figure 1
and Figure 2
and Tables S4 and S5
). Obtained results in the current study support our previous one. Moreover, smaller intake amount (20 g of black soybean/day) is enough to improve the vascular function regardless of gender. Therefore, daily intake of black soybean is possible to improve vascular stiffness and to reduce the risk of CVD.
Oxidative stress is closely related to the underlying mechanism of vascular dysfunction. In this study, we used three biomarkers, 8-OHdG, HEL, and MPO. These biomarkers reflect the different aspects of the oxidative stress: 8-OHdG is a marker for oxidative DNA damage [28
]; HEL is one of the lipid peroxidation markers [29
]; and MPO is another biomarker for lipid peroxidation and inflammation [30
]. Of these, 8-OHdG level specifically decreased in plasma and urine (Table 5
and Table S3
), suggesting that the amelioration of oxidative DNA damage would contribute to the improvement of vascular function, because oxidative DNA damage is a trigger of the development of CVD [31
]. Our previous report demonstrated that black soybean seed coat polyphenols prevented 2,2′-azobis(2-methylpropionamide) dihydrochloride (AAPH)-induced formation of 8-OHdG in HepG2 cells [9
]. It was also reported that drinking red wine suppressed 8-OHdG formation in humans with a Mediterranean diet and an Occidental diet for 3 months [32
]. These results indicate that polyphenols would contribute to the improvement of vascular function. In our previous human trial, we found 8-OHdG and HEL levels significantly decreased in the plasma [15
]. In the current study, we did not observe the decreasing HEL level. This discrepancy may be due to the difference of the intake amount of black soybean. Taken together, these results show black soybean polyphenols have a potential to decrease oxidative stress markers, and 8-OHdG is the most sensitive biomarker for improvement of vascular function through suppressing oxidative stress.
In this study, we measured 11 black soybean polyphenols and one intestinal bacterial metabolite of daidzein, namely, (S
)-equol, in the plasma and the urine by HPLC and found epicatechin, daidzein, and genistein in the plasma (Figure 1
and Table S4
) and C3G, procyaniding B2, daidzin, genistin, glycitin, daidzein, genistein, and glycitein in the urine (Figure 2
and Table S5
). These results are reasonable, because it is known that bioavailability of epicatechin and isoflavones is relatively higher than that of C3G and procyanidins [33
], indicating that epicatechin and isoflavones in black soybean are considerable active compounds for the improvement of vascular function though possessing their antioxidant activity. It is known that soy isoflavones improves vascular functions [35
]. Interestingly, certain amounts of isoflavones were detected as their aglycone forms in plasma and urine in this study. Usually, isoflavones are contained in soybean as the glycoside forms. This result coincides with those in our previous human trial [15
]. Another study also demonstrated the same metabolic conversion after the intake of soybean [37
]. Thus, isoflavone glycosides underwent hydrolysis in the intestines and formed aglycones and contributed to the improvement of vascular function. It is reported that (S
)-equol affects vascular function [38
]. In this study, only aglycine form of (S
)-equol slightly increased with statistical significance in the plasma after consumption of black soybean cookie (Figure 1
and Table S4
). It is therefore suggested that contribution of (S
)-equol to the improvement of vascular function might be limited. Although active compound was unclear in this study, the results from our previous study demonstrated that monomer to tetramer procyanidins from black soybean reduced 8-OHdG and oxidative stress levels to the same degree [9
]. It was therefore suggested that these polyphenols coordinately acted for the improvement of vascular function. Further study is needed to clarify the active compound in the future.
NO is known to regulate vascular function through the relaxation of blood vessels and eventually leads to improvement in vascular stiffness [5
]. Oxidative stress decreases the production of NO and its functions, resulting in induction of the vascular dysfunction [6
]. In this study, NO2
concentration in plasma and urine significantly increased in the black soybean group compared to that in the placebo group. In our previous open-label trial, consumption of black soybean significantly increased the urinary NO2
level after 4 and 8 weeks. Recently, we found that flavan-3ols in black soybean promoted NO production through activation of endothelial nitric oxide synthase [16
]. Isoflavones and equol were also reported to promote NO production [35
]. These results indicated that consumption of black soybean improved vascular function and blood pressure through increasing NO concentration in addition to antioxidant activity of polyphenols in humans.
Components other than polyphenols in the black soybean may also contribute to the observed effects. It was reported that soy protein, accounting for approximately 36% of dry soybeans by weight, was reported to reduce the risk of CVD through regulating the lipid profile, including lowering total cholesterol, low-density lipoprotein, and triglycerides without affecting high-density lipoprotein [41
]. Dietary fiber also reportedly caused a reduction in the blood pressure and improved endothelial function [42
]. Actually, the nutrient content of soybean cookie was quite different from that of the placebo cookie in the present study. These nutrients and polyphenols might coordinately play a role in the improvement of vascular function. Therefore, black soybean is an attractive functional food for prevention and/or improvement of vascular function. Daily intake of black soybean may contribute to maintaining human health.