Nattokinase Research

Suzuki Y, Kondo K, Matsumoto Y,Zhao BQ, Otsuguro K,Maeda T, Tsukamoto Y, Urano T,

Umemura K.

Life Sci. 2003 Jul 25;73(10):1289-98

Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu City, Shizuoka 431-3192, Japan. yapplel@hama-med.ac.jp

We have previously demonstrated that Nattō-extracts containing Nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with Nattō-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary Nattō-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary Nattō-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall.

PMID: 12850244 [PubMed - indexed for MEDLINE]

Nutrition. 2003 Mar;19(3):261-4. 

Dietary supplementation with fermented soybeans suppresses intimal thickening. 

Suzuki Y, Kondo K, Ichise H, Tsukamoto Y, Urano T, Umemura K. 

Department of Pharmacology, Hamamatsu University School of Medicine, Shizuoka , Japan . yapplel@hama-med.ac.jp 

Although soy foods have been consumed for more than 1000 y, it is only in the past 20 y that they have made inroads into Western diets. We investigated the effect of dietary supplementation with Nattō extracts produced from fermented soybeans on intimal thickening of arteries after vessel endothelial denudation. Nattō extracts include Nattokinase, a potent fibrinolytic enzyme having four times greater fibrinolytic activity than plasmin. Intimal thickening was induced in the femoral arteries by intravenous infusion of rose bengal followed by focal irradiation with a transluminal green light. Dietary Nattō extract supplementation was started 3 wk before endothelial injury and continued for another 3 wk after. In ex vivo studies, euglobulin clot lysis times were measured 3 wk after the initial supplementation. Neointima formation and thickening were also initiated successfully. The intima media ratio 3 wk after endothelial injury was 0.15 +/- 0.03 in the control group. Dietary Nattō extract supplementation suppressed intimal thickening (0.06 +/- 0.01; P < 0.05) compared with the control group. Nattō extracts shortened euglobulin clot lysis time, suggesting that their thrombolytic activities were enhanced. These findings suggest that Nattō extracts, because of their thrombolytic activity, suppress intimal thickening after vascular injury as a result of the inhibition of mural thrombi formation. 

PMID: 12620531 [PubMed - indexed for MEDLINE]

Life Sci. 2003 Jul 25;73(10):1289-98.

Dietary supplementation of fermented soybean, Nattō, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. 

Suzuki Y, Kondo K, Matsumoto Y, Zhao BQ, Otsuguro K, Maeda T, Tsukamoto Y, Urano T, Umemura K. 

Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu City , Shizuoka 431-3192, Japan . yapplel@hama-med.ac.jp 

We have previously demonstrated that Nattō-extracts containing Nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with Nattō-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary Nattō-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary Nattō-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall. 

PMID: 12850244 [PubMed - indexed for MEDLINE]

Biol Pharm Bull. 1995 Oct;18(10):1387-91.

Thrombolytic effect of Nattokinase on a chemically induced thrombosis model in rat. 

Fujita M, Hong K, Ito Y, Fujii R, Kariya K, Nishimuro S. 

Biotechnology Research Laboratories, JCR Pharmaceuticals Co., Ltd., Kobe , Japan . 

Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of Nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase ( UK ; 50000 IU/kg, i.v.) or tissue plasminogen activator (tPA; 13300 IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of Nattokinase using this model and compared it with fibrino(geno)lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with Nattokinase, plasmin and elastase were 62.0 +/- 5.3%, 15.8 +/- 0.7% and 0%, respectively. The results indicate that the thrombolytic activity of Nattokinase is stronger than that of plasmin or elastase in vivo. 

PMID: 8593442 [PubMed - indexed for MEDLINE]

Life Sci. 2003 Jul 25;73(10):1289-98.

Enhancement of the fibrinolytic activity in plasma by oral administration of Nattokinase. 

Sumi H, Hamada H, Nakanishi K, Hiratani H. 

Department of Physiology, Miyazaki Medical College , Japan . 

The existence of a potent fibrinolytic enzyme (Nattokinase, NK) in the traditional fermented food called 'Nattō', was reported by us previously. It was confirmed that oral administration of NK (or Nattō) produced a mild and frequent enhancement of the fibrinolytic activity in the plasma, as indicated by the fibrinolytic parameters, and the production of tissue plasminogen activator. NK capsules were also administered orally to dogs with experimentally induced thrombosis, and lysis of the thrombi was observed by angiography. The results obtained suggest that NK represents a possible drug for use not only in the treatment of embolism but also in the prevention of the disease, since NK has a proven safety and can be massproduced. 

PMID: 2123064 [PubMed - indexed for MEDLINE]

J Nutr Sci Vitaminol ( Tokyo ). 2004 Apr;50(2):114-20.

Promotion of bone formation by fermented soybean (Nattō) intake in premenopausal women. 

Katsuyama H, Ideguchi S, Fukunaga M, Fukunaga T, Saijoh K, Sunami S. 

Department of Public Health, Kawasaki Medical School , Kurashiki 701-0192, Japan . katsu@med.kawasaki-m.ac.jp 

A therapeutic agent of vitamin K2 is approved for the treatment of osteoporosis in Japan . However, little is known about the efficacy of dietary intake of vitamin K2 for bone health. We compared the effects of various levels of fermented soybeans (Nattō) intake, which contains plenty of vitamin K2, on bone stiffness and bone turnover markers in healthy premenopausal women. Seventy-three healthy premenopausal women were randomly divided into four groups matched for age and parity categories. Nattō was supplied as follows: Group 1 (no intake), Group 2 (once per month), Group 3 (once per week) and Group 4 (three times per week). Subjects took Nattō at a lunch for 1 y, and the stiffness index by quantitative ultrasound and bone turnover markers were assessed at baseline, 6 mo and 1 y. There was no statistical difference in the stiffness index during the 1 y observation. However, bone specific alkaline phosphatase (BAP) in Group 4 was higher than that in Group 3 at 1 y and undercarboxylated osteocalcin (Glu) in Group 4 was significantly lower than those in Groups 1, 2 and 3 at 6 mo. Logistic regression analysis showed that the risk of reduction of bone formation markers declined to 0.07 in Group 4 based on that in Group 1. In premenopausal women who had to keep the stiffness index as high as possible before menopause, Nattō intake may have contributed to the promotion of bone formation. 

Publication Types:

Clinical Trial

Randomized Controlled Trial 

PMID: 15242015 [PubMed - indexed for MEDLINE]

J Nutr Sci Vitaminol ( Tokyo ). 2002 Jun;48(3):207-15.

Usual dietary intake of fermented soybeans (Nattō) is associated with bone mineral density in premenopausal women.

Katsuyama H, Ideguchi S, Fukunaga M, Saijoh K, Sunami S.

Department of Public Health, Kawasaki Medical School , Kurashiki , Japan . katsu@med.kawasaki-m.ac.jp 

Fermented soybeans (Nattō), a traditional Japanese food, contain more than 100 times as much vitamin K2 as various cheeses and are considered to promote gamma-carboxylation. Thus it is conceivable that Nattō may play a preventive role in the development of osteoporosis. In this study, the relationships between the bone stiffness index measured by ultrasound, bone turnover markers, and lifestyle factors, including Nattō intake, were examined in relation to vitamin D receptor (VDR) polymorphism. Among 117 premenopausal volunteers, approximately 75% were bb homozygotes, 20% were Bb heterozygotes, and only 5% were BB homozygotes. The B allele group and the bb group were subdivided according to Nattō intake. In a monovariate analysis, no significant differences in indices for dietary intake, including Ca and vitamin D intake, were observed. The stiffness index in the B allele group, however, was slightly lower than in the bb groups when there was no Nattō intake. There were no significant differences in serum ALP and Gla-osteocalcin, bone formation markers, or NTx and Ca in urine, bone resorption markers. A logistic regression test, including the interactional effect of Nattō intake and VDR RFLP, indicated that the B allele group was a risk factor of bone mineral loss and that Nattō was effective in maintaining bone stiffness in this group. Although the present study was cross sectional and requires longitudinal investigation, Nattō may improve the bone health of people who have a low affinity receptor for vitamin D. 

PMID: 12350079 [PubMed - indexed for MEDLINE]

Nutrition. 2001 Apr;17(4):315-21.

Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk. 

Kaneki M, Hedges SJ, Hosoi T, Fujiwara S, Lyons A, Crean SJ, Ishida N, Nakagawa M, Takechi M, Sano Y, Mizuno Y, Hoshino S, Miyao M, Inoue S, Horiki K, Shiraki M, Ouchi Y, Orimo H. 

Department of Geriatric Medicine, University of Tokyo Graduate School of Medicine, Tokyo , Japan . 

Increasing evidence indicates a significant role for vitamin K in bone metabolism and osteoporosis. In this study, we found a large geographic difference in serum vitamin K2 (menaquinone-7; MK-7) levels in postmenopausal women. Serum MK-7 concentrations were 5.26 +/- 6.13 ng/mL (mean +/- SD) in Japanese women in Tokyo, 1.22 +/- 1.85 in Japanese women in Hiroshima, and 0.37 +/- 0.20 in British women. We investigated the effect of Japanese fermented soybean food, Nattō, on serum vitamin K levels. Nattō contains a large amount of MK-7 and is eaten frequently in eastern ( Tokyo ) but seldom in western ( Hiroshima ) Japan . Serum concentrations of MK-7 were significantly higher in frequent Nattō eaters, and Nattō intake resulted in a marked, sustained increase in serum MK-7 concentration. We analyzed the relation between the regional difference in Nattō intake and fracture incidence. A statistically significant inverse correlation was found between incidence of hip fractures in women and Nattō consumption in each prefecture throughout Japan . These findings indicate that the large geographic difference in MK-7 levels may be ascribed, at least in part, to Nattō intake and suggest the possibility that higher MK-7 level resulting from Nattō consumption may contribute to the relatively lower fracture risk in Japanese women. 

PMID: 11369171 [PubMed - indexed for MEDLINE] 

Prolonged intake of fermented soybean (Nattō) diets containing vitamin K2 (menaquinone-7) prevents bone loss in ovariectomized rats.

Yamaguchi M, Kakuda H, Gao YH, Tsukamoto Y.

Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka , Japan .

The effect of the prolonged intake of dietary vitamin K2 (menaquinone-7, MK-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing the fermented soybean (Nattō; including 9.4 micrograms MK-7/100 g diet) without or with supplemental MK-7 (containing 14.1 or 18.8 micrograms of MK-7 as total per 100 g diet) for 150 days. Feeding produced a significant elevation of MK-7 concentration in the serum of OVX rats. In this case, the femoral MK-4 content was significantly increased, but MK-7 was not detected in the femoral tissues, indicating degradation of MK-7. Serum gamma-carboxylated osteocalcin concentration was significantly decreased by OVX. This decrease was significantly prevented by the feeding of the Nattō diets with supplemental MK-7 (18.8 micrograms/100 g diets). OVX caused a significant decrease in femoral dry weight, femoral calcium content, and mineral density. These decreases were significantly prevented by feeding with diets containing Nattō with MK-7 (total, 18.8 micrograms/100 g diets). This study demonstrates that the prolonged intake of Nattō dietary including MK-7 has a preventive effect on bone loss induced by OVX. Dietary MK-7 may be useful in the prevention of osteoporosis.

PMID: 10701161 [PubMed - indexed for MEDLINE]

Effect of vitamin K2 (menaquinone-7) in fermented soybean (Nattō) on bone loss in ovariectomized rats. 

Yamaguchi M, Taguchi H, Gao YH, Igarashi A, Tsukamoto Y. 

Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka , Japan . 

The effect of dietary vitamin K2 (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12mg/100g diet) or menaquinone-7 (MK-7; 18.1mg/100g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (Nattō; including 9.4 microg MK-7/100g diet) without or with added MK-7 (37.6 microg/100g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing Nattō with MK-7 added (37.6 microg/100g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7. 

PMID: 10084398 [PubMed - indexed for MEDLINE] 

J Nutr. 2004 Nov;134(11):3100-5.

Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study.

Geleijnse JM, Vermeer C, Grobbee DE, Schurgers LJ, Knapen MH, van der Meer IM, Hofman A, Witteman JC. 

Department of Epidemiology & Biostatistics, Erasmus Medical Center Rotterdam , Rotterdam , The Netherlands . 

Vitamin K-dependent proteins, including matrix Gla-protein, have been shown to inhibit vascular calcification. Activation of these proteins via carboxylation depends on the availability of vitamin K. We examined whether dietary intake of phylloquinone (vitamin K-1) and menaquinone (vitamin K-2) were related to aortic calcification and coronary heart disease (CHD) in the population-based Rotterdam Study. The analysis included 4807 subjects with dietary data and no history of myocardial infarction at baseline (1990-1993) who were followed until January 1, 2000 . The risk of incident CHD, all-cause mortality, and aortic atherosclerosis was studied in tertiles of energy-adjusted vitamin K intake after adjustment for age, gender, BMI, smoking, diabetes, education, and dietary factors. The relative risk (RR) of CHD mortality was reduced in the mid and upper tertiles of dietary menaquinone compared to the lower tertile [RR = 0.73 (95% CI: 0.45, 1.17) and 0.43 (0.24, 0.77), respectively]. Intake of menaquinone was also inversely related to all-cause mortality [RR = 0.91 (0.75, 1.09) and 0.74 (0.59, 0.92), respectively] and severe aortic calcification [odds ratio of 0.71 (0.50, 1.00) and 0.48 (0.32, 0.71), respectively]. Phylloquinone intake was not related to any of the outcomes. These findings suggest that an adequate intake of menaquinone could be important for CHD prevention. 

PMID: 15514282 [PubMed - indexed for MEDLINE]