Protective Effects of Japanese Black Vinegar “Kurozu” and Its Sediment “Kurozu Moromimatsu” on Dextran Sulfate Sodium-induced Experimental Colitis

Kurozu is a traditional Japanese black vinegar that is used in the preparation of foods. It is manufactured, mainly in Kagoshima prefecture in Japan, by fermentation of unpolished rice with lactobacillus and Koji bacillus in earthenware jars for more than one year, during which time it gradually becomes black. The supernatant is known as Kurozu, and the solid sediment, which is rich in organic materials, minerals, amino acids and so on, is known as Kurozu Moromimatsu (Kurozu-M). Many products containing Kurozu and Kurozu-M are available in Japan as health foods or supplements.


Introduction
Kurozu is a traditional Japanese black vinegar that is used in the preparation of foods.It is manufactured, mainly in Kagoshima prefecture in Japan, by fermentation of unpolished rice with lactobacillus and Koji bacillus in earthenware jars for more than one year, during which time it gradually becomes black.The supernatant is known as Kurozu, and the solid sediment, which is rich in organic materials, minerals, amino acids and so on, is known as Kurozu Moromimatsu ( Kurozu-M).Many products containing Kurozu and Kurozu-M are available in Japan as health foods or supplements.
As reported by Murooka et al. (Murooka Y & Yamashita M, 2008), Kurozu has ameliorating effects on hyperlipemia and hypertension, as well as anti-cancer activity against colon cancer in vitro and in vivo (Nanda K et al., 2004;Shimoji Y et al., 2003;Shimoji Y et al., 2004).Further, we reported that Kurozu-M treatment reduced the activity of gelatinases (metalloproteinase-2, -9) in tumor tissues, inhibited the growth of human colon cancer cells, LoVo, in an animal model (Fukuyama N et al., 2007), and inhibited the growth of hepatocellular carcinoma in a diethylnitrosamine-induced animal model (Shizuma T et al., 2011).Kurozu has also been reported to have free radical-scavenging activity (Murooka Y & Yamashita M, 2008).Since active oxygen species or radicals are related to inflammation and tissue injury, Kurozu and Kurozu-M may be potential functional foods with preventive or therapeutic effects against inflammatory diseases.
Ulcerative colitis (UC) is an obstinate inflammatory bowel disease (IBD).The causes of UC are not well-established, but multiple genetic factors (van Lierop et al., 2009), immune responses of the colon (Hong SK et al., 2010) intestinal flora, and inflammatory cytokines (Polińska B et al., 2009;Ghosh N et al., 2010) have been suggested to be involved.Moreover, oxidative stress is thought to influence the severity of UC (Beckman JS et al., 1990;Babbs CF, 1992;Grudziński IP & Frankiewicz-Jóźko A, 2001;Hong SK et al., 2010).Enhanced release of reactive oxygen species (ROS), such as superoxide and hydroxyl radical, and reactive nitrogen species (RNS), such as peroxynitrite generated from nitric oxide (NO), is associated with aggravation of both clinical UC and dextran sulfate sodium (DSS)-induced colitis in an animal model (Elson CO et al., 1995).

Experimental procedures
After initial DSS administration, changes of body weight and bloody stool frequency were monitored every 2 days for 12 days in all mice.Then, the mice were sacrificed and the proximal colon was resected.Microscopic examination (hematoxylin-eosin (H.E.) staining) was performed in all groups, and myeloperoxidase (MPO) staining, as a marker of leukocyte activation, was performed for all groups except the acetic acid group.Moreover, histological findings in the colon were evaluated and scored in the 4 groups according to the reported grading system (Tomita T et al., 2008), as follows.Mucosal damage: 0, normal; 1, 3-10 intraepithelial cells (IEL)/high power field (HPF) and focal damage; 2, >10 IEL/HPF and rare crypt abscesses; 3, >10 IEL/HPF, multiple crypt abscesses and erosion/ulceration.Submucosal damage: 0, normal or widely scattered leukocytes; 1, focal aggregates of leukocytes; 2, diffuse leukocyte infiltration with expansion of submucosa; 3, diffuse leukocyte infiltration.Muscularis damage: 0, normal or widely scattered leukocytes; 1, widely scattered leukocyte aggregates between muscle layers; 2, leukocyte infiltration with focal effacement of the muscularis; 3, extensive leukocyte infiltration with transmural effacement of the muscularis.
Moreover, enzyme-linked immunosorbent assay (ELISA) of serum tumor necrosis factor (TNF)-α and interleukin (IL)-2 as pro-inflammatory cytokines was carried out in the control and Kurozu groups.ELISA measurement of nitrotyrosine levels of resected colonic tissues at 12 days after initial DSS administration, as a marker of oxidative or nitration stress, was performed in all groups except the acetic acid group.Moreover, urinary excretion/day (during 11-12 days after initial DSS administration) of nitrite and nitrate (NOx) as a parameter of the bioavailability of NO was measured by means of the Griess method (Griess reagent kit; Invitrogen Japan K.K, Tokyo, Japan) in the (DSS-induced) control group, the Kurozu group, and the group given standard CE-2 diet without administration of DSS (each group: n=10).

Statistical analysis
The significance of differences of body weight, serum cytokines (TNF-α and IL-2), histological scores, and nitrotyrosine and NOx levels among the groups was examined by one-way analysis of variance (ANOVA) and Tukey's multiple comparison post-hoc test.The significance of differences in bloody stool frequency was examined by contingency table analysis.The criterion of significance was p<0.05.
Body weight after DSS administration is given as a percentage of basal body weight before DSS administration, taken as 100%.Levels of serum cytokines (TNF-α and IL-2) and histological scores, nitrotyrosine in colonic tissues, and NOx in urine, are presented as mean and standard deviation (SD).The frequency of mice with bloody stool after DSS administration is given as a percentage of the number of animals in each group.

Change of body weight
There were no significant differences of diet or water intake among the four groups throughout the 12 days (data not shown).
The Kurozu group showed a significantly reduced body weight loss in the period of 6-12 days after initial DSS administration compared with the control group (p<0.001) and in the period of 8-12 days after initial DSS administration compared with the acetic acid group (p<0.001).The Kurozu-M group showed a significantly reduced body weight loss in the period of 6-8 days after initial DSS administration compared with the control group (p<0.05) and at 8 and 12 days after initial DSS administration compared with the acetic acid group (p<0.05).The acetic acid group showed a significantly reduced body weight loss only at 6 days after initial DSS administration compared with the control group (p<0.01).The results are summarized in Table 1.

Frequency of bloody stool
The appearance of bloody stool was noted in all mice of the control and acetic acid groups in the period of 4-12 days after initial DSS administration.In contrast, bloody stool was rarely noted in the Kurozu group: the frequency was 0% (0/10) during 2-8 days after initial DSS administration and only 20% (2/10) at 12 days after initial DSS administration.Similarly, in the Kurozu-M group, bloody stool was not noted during 2-8 days after initial DSS administration and the frequency was only 30% (3/10) at 12 days (Table 2 The bloody stool frequency in mice after initial DSS administration is given as a percentage of the number of animals in each group.(Shizuma T et al., 2011) Table 2. Frequencies of bloody stool after initial administration of DSS

Histology
H.E. staining revealed epithelial abrasions, cryptal disturbance, hemorrhage and inflammatory cell invasion, and thickening of layers in the mucosa and submucosa of the colon in the control group, but these changes were minimal in the Kurozu and Kurozu-M groups.There was no marked difference between the control group and the acetic acid group (Fig. 1a~d).Moreover, the Kurozu and the Kurozu-M groups showed fewer MPOpositive cells than the control group (Fig. 2a~c).
Histological scores (Tomita T et al., 2008) were as follows: control group, 5.88±0.33;Kurozu group, 0.50±0.50;Kurozu-M group, 0.88±0.33;acetic acid group, 4.38±0.48.In the Kurozu group and Kurozu-M groups, the scores were significantly (p<0.001)reduced compared with the control and acetic acid groups.Moreover, there was no significant difference between the control group and acetic acid group.

Levels of cytokines
The levels of TNF-α (pg/ml) were 17.2±3.25 in the control group with administration of DSS and 15.9±5.77 in the Kurozu group (normal range: 0.6~2.8).Levels of IL-2 (U/ml) were 1.23±0.37 in the control group with administration of DSS and 1.13±0.39 in the Kurozu group (normal range: below 0.8).There were no significant differences in the levels of either of the cytokines between the two groups.

Level of nitrotyrosine in colonic tissue
The Kurozu group showed a significantly reduced nitrotyrosine level (53.1±7.1 ng/g protein) in resected colonic tissue compared with the control group (86.9±11.7 ng/g protein, p<0.001).However, the difference from the control was not significant in the Kurozu-M group (74.2±15.1 ng/g protein) (Fig. 3).
(ng/g protein) Kurozu treatment significantly (p<0.001)reduced the nitrotyrosine level in resected colonic tissue, in comparison to the control group.On the other hand, the difference from the control was not significant in the Kurozu-M group.

Level of NOx in urine
NOx levels in urine (μM) in the (DSS-induced) control group (807±172) and the Kurozu group (723±196) were significantly increased compared with the group that did not receive DSS administration (517±87).However, there were no significant difference between the (DSS-induced) control group and the Kurozu group (Fig. 4).

Discussion
Our results indicate that Kurozu and Kurozu-M exert protective effects against DSSinduced colitis in mice.Kurozu and Kurozu-M both reduced the bloody stool frequency and attenuated inflammatory changes of colon tissue, as well as inhibiting body weight loss.
Further, Kurozu and Kurozu-M decreased the number of MPO-positive cells.Since MPO is a marker of activation of leukocytes (Schindhelm RK., 2009), both Kurozu and Kurozu-M treatments appear to have anti-inflammatory effects in our model.Moreover, Kurozu, but not Kurozu-M, significantly reduced the nitrotyrosine level in colonic tissues in comparison with the control, although there was no significant difference in NOx level between the control and Kurozu groups.Nitrotyrosine is produced via at least two pathways, reaction of superoxide and NO, and reaction of nitrite and MPO (Beckman JS et al., 1990;Radi R, 2009).Therefore, Kurozu may suppress either of these pathways, or both.On the other hand, NOx is a good index of generation of NO by NO synthase (NOS) (Akuta T et al., 2006;Yang GY et al., 2009).Since no significant reduction of NOx level by Kurozu treatment was found in this study, suppression of NO and nitrite formation can be ruled out as a mechanism of reduction of nitrotyrosine generation.Therefore, Kurozu may reduce nitrotyrosine formation by suppressing the generation of superoxide or the activity of MPO.
In inflammatory states, including UC, generation of superoxide and NO is generally accelerated (Cross RK & Wilson KT, 2003).Superoxide itself is cytotoxic and is associated with tissue damage in many diseases.Moreover, superoxide and NO rapidly react with each other to form highly reactive peroxynitrite, leading to severe tissue damage via nitration of protein tyrosine residues to form nitrotyrosine, which is consequently considered to be a marker of oxidative and nitration stress.Therefore, peroxynitrite may have played a key role in the induction of colitis in this study.
Regarding the MPO pathway, MPO is mainly released from activated neutrophils.Since it is possible that Kurozu may block MPO release from neutrophils in the colonic tissues, we can not rule out the possibility that Kurozu suppresses the reaction of MPO and nitrite, thereby leading to a reduction of nitrotyrosine formation.However, Kurozu-M treatment did not reduce the generation of nitrotyrosine significantly, although infiltration of MPO-positive cells was remarkably suppressed.Moreover, the cytotoxity of MPO itself is unclear; our previous study demonstrated that MPO was protective against tissue injury in MPO knockout mice, although in that case, we examined brain tissue (Takizawa S et al., 2002).However, at present, we have no evidence that the reduction of nitrotyrosine level in the Kurozu group involves suppression of MPO.Rather, our findings indicate that the mechanism predominantly mediating the anti-colitis effect of Kurozu is suppression of superoxide production, leading to decreased generation of peroxynitrite, which is strongly cytotoxic, or other ROS and RNS, although at this stage we can not rule out the possibility that suppression of MPO also contributes.
Overall, our results indicate that Kurozu and Kurozu-M both have an anti-colitis effect in the DSS-induced mouse colitis model, though Kurozu is more potent.This, in turn, suggests that the active materials are predominantly soluble compounds, because Kurozu-M is the precipitate formed during the manufacture of Kurozu.
Kurozu contains acetic acid, free amino acids, peptides, minerals, water-soluble vitamins, organic compounds including bacterial fermentation products, lipids and saccharides.Its main component is acetic acid, but we found here that acetic acid did not affect body weight loss, bloody stool frequency, or pathological findings in DSS-induced mice, compared with the control.Further, acetic acid is not present in Kurozu-M.Therefore, acetic acid is not one of the major protective components against colitis.
Amino acids seem to be good candidates for the anti-colitis agents in Kurozu and Kurozu-M.For example, glutamine has a protective effect on gut function, and has been suggested to have an anti-colitis effect in an animal model (Ameho CK et al., 1997).Other amino acids or their metabolites or degradation products, including arginine, asparagine, cysteine, serine (Faure M et al., 2006), methionine, and tryptophan, are known to have anti-oxidative effects (Wu G, 2009).Moreover, cysteine (Kim CJ et al., 2009), serine and tryptophan (Kim CJ et al., 2010) were found to have protective effects in animal models of colitis.Therefore, free amino acids or peptides may be among the active agents present in Kurozu and Kurozu-M.
Other organic materials, including lactic acid and products of lactobacillus or Koji bacillus fermentation, are also candidate protective agents.Kurozu is fermented with lactobacillus or Koji bacillus for several years in earthenware jars, and lactobacillus is well known as a beneficial component of human intestinal flora.Moreover, clinically significant anti-colitis effects of administration of probiotics (Sartor RB, 2004) or synbiotics (Kanauchi O et al., www.intechopen.comProtective Effects of Japanese Black Vinegar "Kurozu" and Its Sediment "Kurozu Moromimatsu" on Dextran Sulfate Sodium-induced Experimental Colitis 173 2009) have been reported in patients with mild UC.Further, anti-oxidative effects and protective effects against animal colitis were reported for Koji bacillus (Fukuda Y et al., 2006;Lee IH et al.,2008).Therefore, many components of Kurozu and Kurozu-M, including amino acids and oligopeptides, as well as other organic materials, may contribute to the anti-colitis effects observed here.
At present, UC is generally treated with anti-inflammatory agents, such as 5-aminosalicylic acid (5-ASA) and prednisolone (Rogler G, 2009).However, administration of these drugs is sometimes accompanied with severe side effects.Therefore, a dietary therapy would be desirable, particularly from the viewpoints of safety and long-term effectiveness.
In order to further examine the mechanism of action of Kurozu, additional study will be needed to evaluate the quantitative changes of pro-inflammatory cytokines in resected colonic tissues, for example, measurement of mRNA expression levels in tissues of the DSSinduced control and Kurozu groups, because there was no difference in the serum levels of pro-inflammatory cytokines.It could not be determined in this study whether Kurozu has a direct anti-oxidant effect, because the absorption, distribution, metabolism and excretion characteristics of Kurozu and its constituents in the mouse remain to be determined.This question might be addressed by means of in vitro experiments on colonic cell lines or rectal application of Kurozu in an animal model.
For the practical use of Kurozu in the management of colitis patients, it will be necessary to identify optimum dosages and administration schedules.Many products containing Kurozu are available in Japan as health foods or supplements, and in this study, the amounts of Kurozu used were based on volumes typically ingested by humans, adjusted for body weight.The next step will be a clinical trial to examine the anti-colitis effects of Kurozu as an oral supplement in patients with mild ulcerative colitis, focusing initially on typical dose and administration schedules recommended for existing health foods and supplements.

Conclusion
Our results indicate that Kurozu exerts a protective effect against DSS-induced colitis in mice, and one of the mechanisms involved may be an anti-oxidative or anti-nitration stress activity.

References
Akuta, T.,; Zaki, MH.,; Yoshitake, J., ;Okamoto, T. & Akaike, T. (2006).Nitrative stress through formation of 8-nitroguanosine: insights into microbial pathogenesis.Nitric Oxide, vol.14:101-108 . staining of resected colon revealed abrasions of epithelium, cryptal disturbance, and inflammatory cell infitration in mucosa and submucosal areas of colon in the control group.Kurozu and Kurozu-M treatment remarkably attenuated these changes in comparison to the control group.The acetic acid group showed no marked attenuation of colitis compared with the control group (a, the control group; b, the Kurozu group; c, the Kurozu-M group; d, the acetic acid group).

Fig. 4 .
Fig. 4. NOx levels among the three groups This work was supported by grants in 2009 Tokai University, School of Medicine Research Aid, 2009 and 2010 Grant-in-Aid for Scientific Research in Japan Society for the Promotion of Science (No. 21659295 and No.22659106) and 2009 Grant-in-Aid for Japanese Society for Parenteral and Enteral Nutrition.

Table 1 .
). Changes of body weight after initial adminsitration of DSS c p<0.05; *days after initial administration of DSS Body weight after DSS administration is given as a percentage of the basal body weight before DSS administration, taken as 100% (mean SD).(ShizumaTet al., 2011)*p<0.01;† days after initial administration of DSS Protective Effects of Japanese Black Vinegar "Kurozu" and Its Sediment "Kurozu Moromimatsu" on Dextran Sulfate Sodium-induced Experimental Colitis 167 Protective Effects of Japanese Black Vinegar "Kurozu" and Its Sediment "Kurozu Moromimatsu" on Dextran Sulfate Sodium-induced Experimental Colitis 169 www.intechopen.com(a) Control group www.intechopen.comColitis 168 (b) Kurozu group www.intechopen.com (c) Kurozu-M group MPO-positive cells are indicated by θ and ι (a, control group; b, Kurozu group; c, Kurozu-M group).The Kurozu and the Kurozu-M groups showed fewer MPO-positive cells than the control group.