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Scientific Research on Nattokinase
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Scientific Research on Nattokinase

NATTOKINASE

Nattô is a traditional Japanese food made from fermented soybeans. It has a very strong smell and a sticky consistency, but is very rich in protein. To make Nattô, the soybeans are fermented with the beneficial bacteria Bacillus natto (it is sometimes listed as Bacillus subtilis natto).1 During fermentation, the Bacilli produce enzymatic proteases, the most important of which is nattokinase.2,3

Like other proteases, nattokinase has potent anti-inflammatory activity as well as several other beneficial properties. However, probably the most important aspect of nattokinase is its fibrinolytic activity. To understand fibrinolytic activity, first one must understand the basic process of coagulation or clotting of blood. In damaged tissue, the broken blood vessel releases a compound called thromboplastin. At the same time platelets adhere to the broken edges of the vessel and disintegrate, releasing platelet factor 3. Both of these react with protein factors and calcium ions to form prothrombin activator. Once the prothrombin activator is formed, the process from prothrombin to a clot follows.11


Of course, clotting forms an important function in tissue repair. However, a clot in the blood stream (a thrombus) can be very dangerous, even fatal.

In vitro and in vivo studies have consistently demonstrated the potent fibrinolytic effect of Nattokinase.4 This enzyme is a serine protease type enzyme which appears to reduce the formation of dangerous clots and inhibit arterial thickening, both by direct fibrinolysis of clots and inhibition of the plasma protein plasminogen activator inhibitor.5 Research with nattokinase demonstrates that it may help avoid or reduce the likelihood of deep vein thrombosis, cardiac infarction, pulmonary emboli and stroke. It appears to accomplish this via its finbrinolytic, anti-inflammatory and modulating effect on blood pressure. Studies on hypertension demonstrate an average drop of 10.9% in Systolic Blood Pressure and a 9.7 percent drop in Diastolic Blood Pressure.6,7,21

Blood clots (thrombi) form when strands of fibrin accumulate in the circulatory system. These clots can cause blockage of blood flow. If blood flow is blocked, the oxygen supply to that tissue is cut off and it eventually dies. In the heart, this can result in myocardial infarction (heart attack). In the brain, it can result in strokes or mini-strokes. Deep vein thrombosis can result in pulmonary emboli. All these events can be life-threatening. An in vitro study not only demonstrated the powerful fibrinolytic activity of nattokinase, but also showed the significant reduction in the aggregation of red blood cells and overall lowered whole blood viscosity. The net results are vascular conditions that are less likely to produce blood clots. The authors suggest that nattokinase possesses very real potential as a therapeutic agent in cardiovascular health.8

As a result of scientific research, nattokinase is available as a dietary supplement for human use. Because nattokinase is considered a systemic enzyme, it is important to demonstrate its absorption from the GI tract. An in vivo study of the duodenal absorption of nattokinase in rats showed the intact absorption of the enzyme. This was based upon the subsequent degradation of plasma fibrinogen which clearly demonstrated transport of nattokinase across the intestinal tract. The action of nattokinase on the cleavage of fibrinogen in the plasma was remarkably prolonged being present in plasma samples drawn 3 to 5 hours after administration of the enzyme.9

The process of forming a clot is complex and involves several enzymes. However, the body mainly produces one central enzyme for dissolving a clot, plasmin. It happens that the properties of nattokinase are very similar to plasmin. Nattokinase is particularly effective because it enhances the body's natural ability to fight blood clots in several different ways. It dissolves fibrin directly and appears to enhance the body's natural production of both plasmin and other clot-dissolving enzymes like urokinase. An in vivo study was undertaken to demonstrate the thrombolytic activity of nattokinase, plasmin and elastase on an induced clot in the common carotid artery of laboratory rats. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo in this model.10

A study conducted with natto on 12 healthy adults (6 men and 6 women, between the ages of 21 and 55) sought to demonstrate fibrinolytic activity. The volunteers were given 200 grams of natto (the food) before breakfast, then their fibrinolytic activity was tested over time. The results indicate natto generates an increased ability to dissolve blood clots. As a control, researchers later fed the same amount of boiled soybeans to the same volunteers and tracked their fibrinolytic activity. The tests showed no significant change.2

The accumulation of fibrin in blood vessels significantly increases the likelihood of thrombosis formation resulting in a cardiovascular event. For thrombolytic therapy, microbial fibrinolytic enzymes are now much more accepted. The physiochemical properties of this enzyme is becoming well characterized and its effectiveness in thrombolysis in vivo has been further identified.12,13

A fascinating study was conducted to measure the effect of nattokinase in the prevention of deep vein thrombosis and superficial vein thrombosis on extended flights of 7-8 hours on high-risk individuals. The nattokinase had no thromboses. The placebo group had 5 deep vein thromboses and 2 superficial vein thromboses or 7.6% of 94 individuals. After the flight, the degree of edema was increased by 12% in the placebo group and decreased and the nattokinase group decreased by 15%. The authors concluded that nattokinase was effective in reducing thrombotic events and in controlling edema in high-risk subjects on long flights.14

In a study following 238 high-risk patients for stroke, the authors demonstrated a significant relationship between increased common carotid artery intima-media thickness (CCA-IMT) and the occurrence of myocardial infarction and stroke. For each increment of 0.1 mm in CCA-IMT the probability of experiencing recurrent stroke increased by 18.0%.15

Anatomy of Arterial Wall

A study with rat femoral artery investigating the effect of dietary supplementation with natto extracts on the intimal thickening of arteries was conducted. It was shown that dietary natto extract supplementation suppressed intimal thickening (0.06 +/- 0.01; P < 0.05) compared with the control group. These findings suggest that natto extracts, because of their thrombolytic activity, suppress intimal thickening after vascular injury as a result of the inhibition of thrombi formation.16,18

In another study by the same group, they demonstrated that nattokinase (NK) inactivates plasminogen activator inhibitor type 1, which in turn potentiates fibrinolytic activity. Further, they investigated the effect on neointima formation and on thrombolysis at the site of endothelial injury. They concluded that dietary natto-extracts supplementation suppress intimal thickening produced by endothelial injury in rat femoral artery. These effects are at least partially attributable to NK, which showed enhanced thrombolysis near the vessel wall.17,18,19

At least 20 or more proteins form amyloid fibrils, which are causative agents in Alzheimer's disease, prion disease, and systematic amyloidosis. Improving amyloid clearance is a major target of the therapy for these diseases. Degradation of amyloids may help prevent or at least alleviate these diseases. In this study, the authors demonstrated the amyloid-degrading ability of nattokinase. They also determined that this ability is shared by proteinase K and subtilisin, but not by trypsin or plasmin.20

While more research is needed, it seems clear that nattokinase offers significant benefits to those who are at risk for a cardiovascular event. Indeed, nattokinase may reduce the risk of deep vein thrombosis, myocardial infarction, pulmonary emboli and stroke. Further, its effect to reduce the thickness of the intima-media of arterial walls may result in reduced blood pressure as well as other risk factors like atherosclerosis. The demonstrated reduction of the aggregation of red blood cells and lowered whole blood viscosity leads one to conclude that nattokinase helps provide vascular conditions that are less likely to produce blood clots. Additional research is needed in the area of amyloid degradation. Nattokinase or related compounds may offer hope for the prevention and treatment of amyloid-related diseases.

Nattokinase has anticoagulant properties. It is recommended that healthcare professionals carefully review patient profiles before using nattokinase. This is especially true if patients are taking aspirin, coumadin or other medicines that increase clotting time.
SCIENTIFIC ABSTRACTS PERTAINING TO
NATTOKINASE AND FIBRINOLYTIC RESEARCH


The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1. J Biol Chem. 2001 Jul 6;276(27):24690-6. Epub 2001 Apr 26. Department of Physiology, Hamamatsu University School of Medicine, 3600, Handa-cho, Hamamatsu, 431-3192, Japan. Urano T, Ihara H, Umemura K, Suzuki Y, Oike M, Akita S, Tsukamoto Y, Suzuki I, Takada A.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis

Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase
Acta Haematol. 1990;84(3):139-43. Department of Physiology, Miyazaki Medical College, Japan.Sumi H, Hamada H, Nakanishi K, Hiratani H.

The existence of a potent fibrinolytic enzyme (nattokinase, NK) in the traditional fermented food called 'natto', was reported by us previously. It was confirmed that oral administration of NK (or natto) 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 mass produced.

A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet Experientia. 1987 Oct 15;43(10):1110-1. Department of Physiology, Miyazaki Medical College, Japan. Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H.

A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.

Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo Appl Microbiol Biotechnol. 2005 Nov;69(2):126-32. Epub 2005 Nov 12. College of Life Sciences, Sichuan University, Chengdu, PR China. Peng Y, Yang X, Zhang Y.

Accumulation of fibrin in the blood vessels usually results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. For thrombolytic therapy, microbial fibrinolytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesirable side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus from traditional fermented foods. The physiochemical properties of these enzymes have been characterized, and their effectiveness in thrombolysis in vivo has been further identified. Therefore, microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases.

Purification and Characterization of a Strong Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto, a Popular Soybean Fermented Food. Japan Biochemical and Biophysical Research Communications, Vol. 197, Issue 3, 30 December 1993, pp. 1340-1347 Fujita M., Nomura K., Hong K., Ito Y., Asada A. and Nishimuro S.Jcr Pharmaceut Co Ltd, Biotechnol Res Labs, 3 2 61 Takatsukadai, Nishi Ku, Kobe 65122, Japan

A strong fibrinolytic enzyme (nattokinase) was purified from the vegetable cheese natto. Nattokinase was extracted from natto with saline and isolated by sequential use of hydrophobic chromatography on Butyl-Toyopearl, ion-exchange chromatography on CM-Toyopearl, and gel-filtration on Sephadex G-50. The isolated protein gave a single sharp band on SDS-PAGE either before or after reduction. The sequence, as determined by automated Edman degradation of the uncleaved molecule and its enzymatically derived peptide, consisted of a total 275 amino acid residues (M.W=27,728) and exhibited a high homology with the subtilisins. The purified nattokinase digested not only fibrin but also several synthtic substrates. Among the synthetic substrates, the most sensitive substrate was Suc-Ala-Ala-Pro-Phe-pNA for subtilisin. PMSF inhibited both the fibrinolytic activity and the amidolytic activity. The results indicate that nattokinase is a subtilisin-like serine protease.

A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet Experientia 1987, Oct 15; 43(10):1110-1. H. Sumi, H. Hamada*, H. Tsushima, H. Mihara and H. Muraki** Department of Physiology, Miyazaki Medical College, Miyazaki 889-16 (Japan), *Department of Fundamental Natural Science, Okayama University' of Science, Okayama 700 (Japan), and **Department of Fermentation Technology, Faculty of Engineering, Yamanashi University, Kofu 400 (Japan), 23 June 1986

A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1 -hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.

Enhancement of the Fibrinolytic Activity in Plasma by Oral Administration of Nattokinase Acta Haematol 1990; 84:139-143 Hiroyuki Sumia, Hiroki Hamadab, Koichiro Nakanishic, Hajime Hiratanic
aDepartment of Physiology, Miyazaki Medical College, Miyazaki, Japan;
bDepartment of Biochemistry, Oklahoma State University, Okla., USA;
cBiochemistry Research Laboratories, JCR Pharmaceuticals, Kobe, Japan

The existence of a potent fibrinolytic enzyme (nattokinase, NK) in the traditional fermented food called 'natto', was reported by us previously. It was confirmed that oral administration of NK (or natto) 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 mass produced.

Determination and Properties of the Fibrinolysis Accelerating Substance (FAS) in Japanese Fermented Soybean "Natto" Nippon Nogeikagaku Kaishi. 74 (11) 1259-1264 (2000) Sumi Hiroyuki, Sasaki Tomohiro, Yatagai Chieko, Kozaki Yasutaka Kurashiki Univ. Sci. and the Arts, Coll. Sci. and Industrial Technol., JPN

Using trypsin-fibrin plate method, strong activity of the fibrinolysis accelerating substance (FAS) was proved in water extract of Japanese fermented soybean "natto". In 7 commercial natto preparations, high concentrations of 74,100.+-.54,500 units (equivalent to .MU.g surfactin)/100 g wet weight (Mean.+-.SD) were detected. FAS strongly accelerated not only the activities of direct fibrin degradating enzymes such as plasmin, trypsin, and earthworm protease, but also that of the plasminogen activator (urokinase) and pro-urokinase activator (nattokinase). It was easily separated as dark yellow powder from natto extract by two-step procedures of acid-precipitation (pH 2.0) and ethanol treatment (soluble fraction). The final yield and purity from 1 kg natto were about 817,000 units (dry weight 4.6 g) and 177.6 units/mg, respectively.

Effects of nattokinase, a pro-fibrinolytic enzyme, on red blood cell aggregation and whole blood viscosity Clin Hemorheol Microcirc 2006; 35(1-2):139-42. Pais E, Alexy T, Holsworth RE, Meiselman HJ

The vegetable cheese-like food, natto, is extremely popular in Japan with a history extending back over 1000 years. A fibrinolytic enzyme, termed nattokinase, can be extracted from natto; the enzyme is a subtilisin-like serine protease composed of 275 amino acid residues and has a molecular weight of 27.7 kDa. In vitro and in vivo studies have consistently demonstrated the potent pro-fibrinolytic effect of the enzyme. However, no studies to date have evaluated the effects of nattokinase on various hemorheological parameters and thus we have begun to assess the effects of the enzyme on RBC aggregation and blood viscosity. Blood samples were incubated with nattokinase (final activities of 0, 15.6, 31.3, 62.5 and 125 units/ml) for 30 minutes at 37 degrees C. RBC aggregation was measured using a Myrenne MA-1 aggregometer and blood viscosity assessed over 1-1000 s(-1) with a computer controlled scanning capillary rheometer (Rheolog). Our in vitro results showed a significant, dose-dependent decrease of RBC aggregation and low-shear viscosity, with these beneficial effects evident at concentrations similar to those achieved in previous in vivo animal trials. Our preliminary data thus indicate positive in vitro hemorheological effects of nattokinase, and suggest its potential value as a therapeutic agent and the need for additional studies and clinical trials.

Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo Applied Microbiology and Biotechnology Vol. 69, No. 2, November, 2005, pp-126-132
Yong Peng1, 2, Xiaojuan Yang1, 3 and Yizheng Zhang1
1. College of Life Sciences, Sichuan University, Chengdu, 610064, PR China
2. Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH, USA
3. Program of Molecular, Cellular and Developmental Biology, Ohio State University, Columbus, OH, USA

Accumulation of fibrin in the blood vessels usually results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. For thrombolytic therapy, microbial fibrinolytic enzymes have now attracted much more attention than typical thrombolytic agents because of the expensive prices and the undesirable side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus from traditional fermented foods. The physiochemical properties of these enzymes have been characterized, and their effectiveness in thrombolysis in vivo has been further identified. Therefore, microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases.

Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects
Nutrient Research, Volume 29, Issue 3, Pages 145-220 (March 2009) Chien-Hsun Hsia, Ming-Ching Shen, Jen-Shiou Lin, Yao-Ke Wen, Kai-Lin Hwang, Thau-Ming Cham, Nae-Cherng Yang

Nattokinase, a serine proteinase from Bacillus subtilis, is considered to be one of the most active functional ingredients found in natto. In this study, we hypothesized that nattokinase could reduce certain factors of blood clotting and lipids that are associated with an increase risk for cardiovascular disease (CVD). Thus, an open-label, self-controlled clinical trial was conducted on subjects of the following groups: healthy volunteers (Healthy Group), patients with cardiovascular risk factors (Cardiovascular Group), and patients undergoing dialysis (Dialysis Group). All subjects ingested 2 capsules of nattokinase (2000 fibrinolysis units per capsule) daily orally for 2 months. The laboratory measurements were performed on the screening visit and, subsequently, regularly after the initiation of the study. The intent-to-treat analysis was performed on all 45 enrolled subjects. By use of mixed model analysis, a significant time effect, but not group effect, was observed in the change from baseline of fibrinogen (P = .003), factor VII (P < .001), and factor VIII (P < .001), suggesting that the plasma levels of the 3 coagulation factors continuously declined during intake; also, the extents of decrease were similar between groups. After 2 months of administration, fibrinogen, factor VII, and factor VIII decreased 9%, 14%, and 17%, respectively, for the Healthy Group; 7%, 13%, and 19%, respectively, for the Cardiovascular Group; and 10%, 7%, and 19%, respectively, for the Dialysis Group, whereas blood lipids were unaffected by nattokinase. No significant changes of uric acid or notable adverse events were observed in any of the subjects. In summary, this study showed that oral administration of nattokinase could be considered as a CVD nutraceutical by decreasing plasma levels of fibrinogen, factor VII, and factor VIII.