Antioxidant activity of the new black vinegar “IZUMI”

Abstract

Objectives

“IZUMI” is a new kind of vinegar resulting from an improvement in the manufacturing method of Kurosu, a traditional vinegar product made from unpolished rice. We evaluated the antioxidant activity of this new Kurosu by means of measuring the level of diacron-reactive oxygen metabolites (d-ROM), the biological antioxidant potential (BAP), as well as RBC deformability using the microchannel array flow method.

Participants

Ten healthy, untrained female volunteers participated in this study. Measurements: All subjects drank 50 ml of “IZUMI” on a daily basis, and blood samples were collected pre-“IZUMI” (I), after one month “IZUMI” consumption (II), and after two months “IZUMI” consumption (III). The subjects continuously wore a lifecorder during a 7-day period and the nutritional intake was measured before the initial blood sample collection.

Results

There were no significant changes in weight, BMI, fat mass, or fat-free mass. There were no significant differences in daily energy consumption, physical activity and nutritional intake. Peripheral blood variables did not change significantly. The drinking of “IZUMI” increased serum BAP level gradually, and after 30 days it was significantly higher as compared to the pre-drinking level. The serum level of d-ROM and blood filtration time (BFT) decreased by drinking “IZUMI”; with d-ROM significantly lower than the pre-drinking level after 30 days and BFT significantly decreased after 60 days (all P<0.05).

Conclusions

These results suggest that “IZUMI”, a Kurosu containing a higher level of amino acids, increases antioxidant activity and reduces oxidative stress and blood filtration time in female subjects.

Introduction

One of the Japanese traditional rice vinegars, Kurosu is an unpolished black rice vinegar produced by a traditional static fermentation process. As Kurosu is produced from unpolished rice, it is characterized by higher levels of amino acids and organic acids as compared to other vinegars (1). An extract of Kurosu was recently shown to suppress lipid peroxidation more effectively than extracts of other vinegars, because the former shows higher antioxidative activity in a radical-scavenging system (2, 3). Kurosu also exhibited anti-tumor activity in a mouse skin model of carcinogenesis, and it had a suppressive effect on the growth of a variety of lines of cultured tumor cells (2, 4). However, as far as we know, there are no studies on the antioxidative activity of Kurosu in human beings. Recently, the diacron-reactive oxygen metabolites (d-ROM) level and the biological antioxidant potential (BAP) have been used to evaluate the oxidative condition, and their significance as clinical markers has been reported in various fields (5, 6, 7, 8, 9, 10, 11, 12, 13). The d-ROM level represents the total level of peroxidized metabolites, and BAP reflects serum antioxidant capacity. “IZUMI” is a novel vinegar with increased amino acid content resulting from an improvement in the manufacturing method of Kurosu, a traditional vinegar product from unpolished rice. The purpose of this study was to assess the effects of “IZUMI”, during a 60-day period, on the oxidative condition of women by way of measuring the d-ROM and BAP levels. It is well known that reactive oxygen species induce membrane damage in RBC and reduce RBC deformability (14, 15). We also measured RBC deformability using the microchannel array flow method (16, 17).

Subjects and Methods

Ten healthy untrained female volunteers (age 54 ± 6 years, height 154.0 ± 8.0 cm, weight 61.3 ± 4.7 kg, BMI 25.9 ± 1.9) participated in the study. All subjects were non-smokers and were not taking any routine medication or vitamin supplements. None of them participated regularly in sport or exercise training.

RBC deformability measurement

RBC deformability has been assessed by a variety of methods. We measured RBC deformability using a microchannel array flow analyzer system (McFAN HR300, Mc Lab., Tokyo, Japan), which is a negative-pressure filtration method (16, 17). A syringe containing 100 μL blood sample was connected to the apparatus with a disposable tip (Bloody 7). Bloody 7 consisted of a single-crystal silicon chip with 7854 microchannels (groove size, 5 μm wide, 4.5 μm deep, and 30 μm long) simulating the size of a capillary blood vessel. A negative water pressure difference of 20 cm in height was applied at room temperature and the time for 100 μL heparinized blood to pass through the microchannel arrays of the chip was measured to estimate deformability. The passage time measurement of the 100-μL blood sample was automatically calibrated with the passage time of saline adjusted to 12 sec. The passage time through the microchannels of the silicon chip was measured for every 10 μL of the 100-μL sample by monitoring the volume in the syringe. RBC deformability was assessed by the passage time generated by the flow of 100-μL sample (BFT).

Body composition

Body composition was measured with a body composition analyzer (Inbody720; Biospace, Seoul, Republic of Korea) using the biophysical impedance analysis method. Inbody720 uses 8-point tactile electrodes and multifrequency (1, 5, 50, 250, 500 and 1000 kHz) analysis to measure body weight, fat mass, and fat free mass (18).

Nutrition intake

Nutritional intake was measured with a food frequency questionnaire (FFQ) PC program (Wellness WinQ, Okayama, Japan) that included 11 food groups with 74 food items. For each item on the food list, subjects were asked to estimate the frequency of consumption based on specified frequency categories that indicated the number of times the food is usually consumed per month, as well as the calculated protein, fat, and carbohydrate energy rations. It is well known that the FFQ has high reproducibility and reasonably good validity (19), is useful in assessing the usual intake of nutrients, foods and food groups, and is consistent throughout the Japanese population (20).

Daily physical activity

Daily physical activity was measured with a computerized accelerometer (Lifecorder EX; Suzuken, Nagoya, Japan) and associated software (Lifelyzer 02 Pro; Suzuken). Steps and accelerations were recorded at 4-sec intervals, and acceleration data were categorized into 11 physical activity levels (PA) (level 0, 0.5 and 1 to 9). The accelerometer reported the time spent in each physical activity level per day. A strong correlation was shown between the activity levels detected by the accelerometer and the metabolic equivalents (METs).

PA 0 represents the quiet resting state and PA 0.5 subtle movements (movements with less than 1.3 METs). PA 1 to 9 are for walking and exercise. PA 1 is for gentle walking (about 1.3 METs) and 9 is for running (about 9.1 METs). Movements of PA 1 to 9 are counted as step numbers, but the subtle movements of grades PA 0 and 0.5 are not recorded as steps. The daily physical activity related to energy consumption, calorimetry with this device, is calculated by the computed METs data, body height and weight, and subtracting the basal metabolic consumption (21).

Peripheral blood counts

The peripheral blood counts were measured using a cell counter (Celltac E, Nihon Koden Co., Tokyo, Japan).

Measurement of d-ROM level and BAP

Values for d-ROM and BAP were ascertained using the Free Radical Analytical System 4 (FRAS4; H&D srl, Parma, Italy). The d-ROM level is proportional to the serum hydroperoxide concentration. Hydroperoxides are products of the peroxidation of proteins, peptides, amino acids, lipids, and fatty acids. Measurement of the d-ROM level is based on the ability of transition metals to catalyze in the presence of peroxides, the formation of free radicals, which are trapped by an alchilamine. The alchilamine reacts to form a colored radical that is detectable at 505 nm. The results are expressed in conventional units called U.CARR (Carratelli units) with 1 U.CARR corresponding to 0.08 mg/100 ml H₂O₂ (5, 6).

The BAP test is a photometric test that measures the plasma biological antioxidant potential as the capacity of the plasma sample to reduce iron from the ferric (Fe³⁺) to the ferrous form (Fe²⁺).

Briefly, a 10-μL plasma sample is added to a solution of ferric chloride and thiocyanate derivate, and the intensity of any resulting decolourization is proportional to the ability of plasma to reduce ferric ions. The BAP test provides a global measurement of many antioxidants, including uric acid, ascorbic acid, proteins, -tocopherol and bilirubin (9, 12). The results of the BAP test are expressed in 1 mol/L of reduced iron.

For the d-ROM and BAP assays, the intraday coefficients of variation were 3.2% and 6.5% (n=5), respectively, and the interday coefficients of variation were 6.2% and 5.0% (n=5), respectively.

Experimental Procedure

All subjects drank 50 ml “IZUMI” every day, and blood samples were collected pre-“IZUMI” (I), after one month “IZUMI” consumption (II), and after two months “IZUMI” consumption (III). Subjects continuously wore a Lifecoder (except while sleeping or bathing) during a 7-day period and nutritional intake was measured with a food frequency questionnaire (FFQ) PC program prior to the initial blood sample collection. All subjects were asked to maintain the same physical activity and diet during the study period. The subjects' physical activity and food intake during the study period were measured.

The protocol was approved by the Ethics Committee of the National Institute of Fitness and Sports in Kanoya, and all subjects gave informed written consent. The study was in accordance with the Declaration of Helsinki.

Statistical Analysis

Values are presented as mean ± standard deviation (SD). Data were analyzed by repeated measurements ANOVA non-parametric (Friedman) tests, P < 0.05 was considered to be statistically significant. Differences among groups were analyzed by the Wilcoxon signed-rank test, with the Bonferroni correction, and P < 0.016 was considered to be statistically significant.

Results

Table 1 shows changes in characteristics pre-“IZUMI” (I), after one month “IZUMI” consumption (II), after two months “IZUMI” consumption (III). There were no significant changes in weight, BMI, fat mass, or fat-free mass throughout the study period.

Table 1.

Subjects' physical characteristics throughout the experimental period

	I	II	III	P
Weight (kg)	61.3±4.7	61.2±4.5	61.0±4.5	0.3012
BMI	25.9±1.9	25.9±1.9	25.8±1.9	0.3012
Fat mass (kg)	22.3±3.6	22.8±3.6	22.6±4.0	0.5836
Fat Free Mass (kg)	39.0±4.0	38.5±4.1	38.4±3.6	0.1317

I: Pre-“IZUMI”, II: One month “IZUMI” consumption, III: Two months “IZUMI” consumption. Values are presented as mean ± standard deviation. Data were analyzed by repeated measurements ANOVA non-parametric (Friedman) tests, p<0.05 was considered to be statistically significant

Daily energy consumption was 1860 ± 220 kcal/day (I), 1808 ± 144 kcal/day (II), and 1815 ± 194 kcal/day (III). The daily number of steps was 10544 ± 5719 (I), 9521 ± 4826 (II), and 9586 ± 5509 (III). There were no significant differences in daily energy consumption and physical activity throughout the study period.

There were also no significant differences in nutritional intake throughout the study (Table 2).

Table 2.

Effects of “IZUMI” consumption on dietary energy and nutrient intake

	I	II	III	P
Energy (kcal/d)	2181+730	2129+713	2074+506	0.4966
Protein (g/d)	87.5+40.9	85.1+34.6	76.6+26.3	0.4966
Fat (g/d)	68.3+25.5	62.2+18.8	58.4+13.3	0.2725
Carbohydrate (g/d)	296.9+89.3	299.4+108.8	304.7+79.2	0.7408
Vitamin C (mg/d)	108+42	128+62	131+61	0.7408

I: Pre-“IZUMI”, II: One month “IZUMI” consumption, III: Two months “IZUMI” consumption. Values are presented as mean ± standard deviation. Data were analyzed by repeated measurements ANOVA non-parametric (Friedman) tests. No statistically significant differences were noted between groups for any of the measured variables (P > 0.05).

Table 3 shows the counts of peripheral blood variables of all subjects throughout this study. None of the variable counts changed significantly. The drinking of “IZUMI” increased serum BAP level gradually, and after 30 days it was significantly higher than the pre-drinking level. The serum level of d-ROM and BFT decreased by drinking “IZUMI”; d-ROM was significantly lower than the pre-drinking level after 30 days, and BFT was significantly decreased after 60 days (Table 4).

Table 3.

Effects of “IZUMI” consumption on blood cell parameters

	I	II	III	P
WBC (μ1)	5296+1128	4761+951	5058+1089	0.2231
RBC (x104/μ1)	444+27	449+31	453+37	0.0724
Hb (g/dl)	13.0+0.8	13.1+1.0	13.2+1.1	0.0807
Hct (%)	40.5+2.3	40.9+2.7	41.7+3.0	0.0889
PLT (x104/μ1)	25.6+7.0	25.0+7.3	26.4+6.2	0.6873

I: Pre-“IZUMI”, II: One month “IZUMI” consumption, III: Two months “IZUMI” consumption. Values are presented as mean ± standard deviation. Data were analyzed by repeated measurements ANOVA non-parametric (Friedman) tests, P < 0.05 was considered to be statistically significant. WBC: white blood cell; RBC: red blood cell; Hb: hemoglobin; Hct: hematocrit; PLT: platelet cell.

Table 4.

Effects of “IZUMI” consumption on oxidative stress and total blood filtration time

	I	II	III	P
BAP (μmol/L	2371+169∗	2809+207∗∗	3073+98∗∗∗	0.0001
d-ROM (U.CARR)	338+48∗	284+37	248+31∗∗∗	0.0006
BFT (sec)	52.2+14.1	46.7+7.5	41.4+3.4∗∗∗	0.0165

I: Pre-“IZUMI”, II: One month “IZUMI” consumption, III: Two months “IZUMI” consumption. Values are presented as mean ± standard deviation. Data were analyzed by repeated measurements ANOVA non-parametric (Friedman) tests, P < 0.05 was considered to be statistically significant. Differences among groups were analyzed by the Wilcoxon signed-rank test, with the Bonferroni correction. ∗P < 0.016 for I vs. II; ∗∗P < 0.016 for II vs. III; ∗∗∗P < 0.016 for I vs. III

Discussion

Recently, Nishidai et al. (2) investigated the in vitro antioxidative activities of various kinds of vinegar in a linoleic acid autoxidation system with the thiobarbituric acid (TBA) method, as well as the dose-dependent radical scavenging activity of the vinegar samples by using a representative colorimetric system with a stable DPPH radical. They reported that Kurosu completely inhibited linoleic acid autoxidation and this activity, comparable to -tocopherol, was significantly higher than that of any other vinegar sample (P < 0.01). Also, the radical scavenging activity of Kurosu was enhanced with increasing concentration and again exhibited the highest radical scavenging activity among the vinegar samples tested. They also demonstrated that Kurosu showed anti-inflammatory and anti-tumor promoting effects in mouse skin.

The level of thiobarbituric acid reactive substances (TBARS) is a well-known biomarker for the overall oxidative damage to cellular constituents, including membrane lipids. They examined the inhibitory effects on TPA (12-O-tetradecanoylphorbol-13-acetate)-induced lipid peroxidation as quantified by the amount of TBARS and the myeloperoxidase (MPO) activity (2). Kurosu reduced both TBARS formation and MPO activity in mouse skin. MPO is a lysosomal enzyme that catalyzes the formation of hypochlorous acid from the substrate H₂O₂. Hypochlorous acid is highly toxic, mutagenic (22), and also activates carcinogens (23). The observation that Kurosu potentially inhibited both MPO activity and H2O2 generation strongly suggests that Kurosu may effectively suppress the generation of hypochlorous acid in an inflammatory region, thus inhibiting oxidative damage.

They also evaluated the in vivo anti-tumor promoting activity of Kurosu by a two-stage carcinogenesis experiment in mouse skin. The anti-tumor promoting activity was determined by evaluating both tumor incidence and the number of tumors per mouse. The average number of tumors per mouse was markedly reduced in the group treated with Kurosu.

Therefore, leukocyte-derived ROS production is believed to be involved as a common mechanism for inflammation-related carcinogenesis, including skin cancer (24). Based on these results, Nishidai et al. suggest that Kurosu strongly inhibits lipid peroxidation both in vitro and in vivo in mouse skin. The significant suppression of TPA-induced oxidative stress and tumor promotion by Kurosu may be mediated by scavenging of the free radicals produced by leukocytes and by interfering with leukocyte infiltration into the region of inflammation.

Shimoji et al. (3) investigated the modifying effects of administering an ethyl acetate extract of Kurosu (EK) in drinking water on the development of azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats and reported that EK administration significantly inhibited the incidence and multiplicity of colon adenocarcinoma (P < 0.05) compared with those in the AOM alone group. These findings suggest that EK may also be effective in inhibiting colon carcinogenesis.

EK strongly inhibited both in vitro and in vivo lipid peroxidation in mouse skin. However, there have been no reports on the antioxidative activity of Kurosu in humans.

There are studies that show some clinical benefits after the intake of antioxidants (25), and others with the opposite results (26, 27). Two possibilities are suggested for this discrepancy. The first possibility is that antioxidants could act as prooxidants (28). This may happen in the case of high supplement dosages or because of unpredictable clinical/biochemical conditions (29). The second possibility is a problem concerning the bioavailability of antioxidants taken as supplements, which may not be sufficiently absorbed to generate any biological effects. Bioavailability issues are especially problematic if no reliable assessment methods are available. Therefore, the effect of their intake on oxidative stress is not predictable. Recently, a new test for the evaluation of the derivatives of reactive oxygen metabolites in serum, the d-ROM test, has become available (5, 6, 7, 8). On the basis of this method, Cornelli et al. (6) tested three different formulas in 14 apparently healthy volunteers (11 men and 3 women), and a 15% deviation of levels was chosen as the cut-off value for a significant change in oxidative stress. They reported that one subject increased oxidative stress to a level that reached the cut-off value. Based on these results, they concluded that the event is at least uncommon, antioxidant may act as proxidants in some subjects. Additionally, the d-ROM test was confirmed to be a reliable tool for the determination of oxidative stress.

In our results, the value of U.CARR decreased in the serum of all subjects after treatment with “IZUMI” for 60 days (Figure 1). This means that “IZUMI” may not act as prooxidant. Furthermore, in eight cases out of ten, this value decreased more than 15% compared to pre-treatment levels.

Figure 1.

U.CARR values in serum samples before and after 60 days of “IZUMI” consumption. P value was 0.0051 by the Wilcoxon signed-rank test. Solid lines represent cases that showed > 15% decrease, broken lines represent cases that showed < 15% decrease

In general, antioxidant enzymes and antioxidant substances are mobilized when reactive oxidative stress (ROS) is increased, thereby ameliorating ROS, thus maintaining the balance between ROS and antioxidant force. Oxidative stress is common in several physiological and pathological conditions, and a diminished value in the d-ROM test may indicate both a decrease in the production of damaging free radicals and/or an increase in antioxidant defenses.

The BAP test is a photometric test that measures plasma biological antioxidant potential as the capacity of the plasma sample to reduce iron from the ferric (Fe³⁺) to the ferrous form (Fe²⁺). In this study, drinking “IZUMI” increased serum BAP level gradually, and after 30 days it was significantly higher than the pre-drinking level. The rate of increase was 17.4 - 48.3%. This elevation in the BAP may play an important role in the mechanism responsible for decreasing the level of serum U.CARR after drinking “IZUMI”.

Some interfering factors must be considered when the d-ROM and BAP tests are used, because the use of medical drugs, administration of supplements, as well as a state of fatigue after physical exercise have been shown to influence their levels (5, 12). We asked all subjects to maintain the same physical activity and diet during the study period, and the physical activity and food intake of all subjects during the study period were measured. There were no significant differences in the nutritional intake and physical activity throughout the study.

Blood filtration time is determined by various factors, such as hematocrit, WBC, and osmolality, but of these factors RBC deformability is the most important (30). It is well known that human red blood cells (RBC) are susceptible to oxidative damage, with both structural and functional properties altered as a consequence of oxidant attack. Such oxidant-related alterations may lead to changes in RBC deformability (14, 15).

Recently, Miyawaki et al. (31) investigated the effects of Astaxanthin (AX) on human blood rheology, measuring whole blood transit time by using a microchannel array flow analyzer (MC-FAN apparatus). After AX administration (6 mg/day for 10 days), the whole blood transit time decreased from 52.8 ± 4.9 s to 47.6 ± 4.2 s (P < 0.01). AX, the main carotenoid pigment in aquatic animals, has great antioxidant activity and protects against lipid peroxidation in vitro and in vivo (32). Based on such reports, they suggested that the improvement in blood rheology from AX could be due to an antioxidant effect on the erythrocyte membrane. Our data on RBC deformability is confirmed by their data.

Furthermore, HOCl produced in WBC is highly toxic and also decreases RBC deformability markedly (30, 33). In this study, there were no significant changes in peripheral blood counts, and the shortening of BFT is likely the cause of a reduced ROS.

Conclusion

“IZUMI”, a new Kurosu containing higher levels of amino acids, increased anti-oxidant activity (BAP test) and reduced ROS (d-ROM test) in female subjects. Furthermore, blood filtration time decreased significantly due to a reduction in ROS.

Acknowledgements: The authors would like to thank all of the subjects who participated in this study. We also thank Maeda K. for medical assistance.

Financial disclosure: None of the authors had any financial interest or support for this paper.