1. BACKGROUND
Heart disease and cerebrovascular disease (strokes) are respectively the first and the third leading causes of all deaths in developed parts of the world, with heart disease accounting for as much as 30% of all deaths and cerebrovascular disease accounting for nearly 10%. And these figures are only the tip of the iceberg in regards to the devastation of these diseases: for every one who actually dies from them, there are several others suffering from their ill effects — a stroke or a heart attack, for example, can leave a person bedridden. More mild symptoms might include neck stiffness, chronic headache, cold hands and feet, breathlessness, chest pains, poor mental function and dizziness.
Any focus on increasing length and quality of life must look, therefore, at methods to maintain a healthy vascular system and blood flow. This is fundamental to good health and was the reason why Daiwa Pharmaceutical Co. Ltd. started research into the health properties of a traditional Japanese food called natto which was supposed to be so beneficial to health and the circulation. What they discovered was that there is a good good bacteria in this traditional fermented food, B. subtilis natto, which contains enzymes that help the body to maintain optimum blood flow and viscosity by breaking down a substance in the blood called PAI-1 that interferes with the body's natural processes. PAI-1 concentrations increase as we get older which is why our blood gets more sticky as we age and why we are increasingly at risk from vascular diseases.
The problem with natto is is that it is a very unpleasant food to eat (it has an "acquired" taste/smell and a slimy texture); it contains high amounts of vitamin K2 which can interfere with anticoagulants such as Warfarin; the active enzyme content of natto can vary enormously from brand to brand (see below) and it is not easy to get hold of in Western countries. Daiwa Pharaceutical Co. Ltd., therefore, decided to extract the active ingredient (in the same way that good bacteria is extracted from yogart), remove the vitmain K2 in the process and to produce a standardized small tablet that would allow people to enjoy the benefits of natto without any of the drawbacks associated with it. They have called this "natto in a tablet" NKCP.
Comparison of good bacteria activity in 89 different natto brands found in Japan showing the variation in peptidase activity. Daiwa Pharmaceutical therefore decided to produced a standardized "natto in a tablet" equivalent.
2. DAIWA PHARMACEUTICAL CO. LTD.
Daiwa Pharmaceutical Co. is a new breed of pharmaceutical company focused entirely on the development of natural food solutions. Already renowned for producing what is now the leading serious immune system supplement, BioBran MGN-3, a food extract made entirely from rice bran, Daiwa Pharmaceutical have now developed the highest quality and most active Natto extract (sometimes called nattokinase, although this is just one of its components) — NKCP. What sets Daiwa Pharmaceutical apart from many other developers of innovative foods and food supplements is its dedication to high quality research and production. Daiwa is not afraid to developing natural (and therefore not patentable) products, something which most pharmaceutical companies would be reluctant to do.
3. MANUFACTURING PROCESS
NKCP is manufactured by culturing B. subtilis natto -- the good bacteria isolated from the natto food -- in a liquid medium under conditions suitable for the production of the active protein. The cell bodies, insoluble matter and low-molecular weight substances (which include the characterisitic odour molecules) are then removed. The peptide concentration is then standardized and the NKCP is powdered and then pressed into tablets. Below is the manufacturing flow chart for NKCP.
1. FUNCTIONAL INGREDIENTS
The functional ingredient in the food natto and the NKCP natto extract responsible for reducing the risk of thrombosis is a protein (sometimes called Nattokinase) produced by natto bacillus (B. subtilis natto). This is one of a number of proteins with differing molecular weights contained in natto. Its activity is confirmed through its ability to hydrolyze synthetic substrates for plasmin and fibrin. Improved thrombolytic activity has been demonstrated in a study on dogs given natto or partially purified extract of natto and in a study on humans taking natto.
NKCP has stable peptidase activity and its major active ingredient is a 34,000-45,000-dalton protein produced by the natto bacillus, belonging to the bacillopeptidase F family. The activity is stable at pH 6.0 ~ pH 9.0 under 60ºC and the optimum is pH 9.0. Its activity is measured by determining the ability to hydrolyze synthetic substrate for plasmin (S-2251) and its quantitative determination is carried out by measuring the amount of antigen reacting with the antibody specific for the protein, using ELISA.
Enzymes synthesized and excreted outside the cells by Bacillus subtilis after exponential multiplication.
| Types | Gene | Properties |
| Subtilisin (alkaline) protease | apr | Molecular weight by SDS-PAGE: 20 kDa (1), 28 kDa (2) Nattokinase. Its casein decomposing activity and direct fibrinolytic activity have been confirmed. The ability to decompose and inactivate plasminogen activator inhibitor type 1 (PAI-1) has also been reported (2). |
| Neutral protease | npr | Major exoproteinase as with apr. |
| Extracellular protease | epr | Molecular weight by SDS-PAGE: 40-34 kDa |
| Metallo protease | mpr | Molecular weight by SDS-PAGE: 28 kDa (3) |
| Bacillopeptidase F | bpr | Molecular weight by SDS-PAGE: 47 kDa (3), 48 kDa (4). Secreted as a 92 kDa protein and converted into 80 kDa and 48 kDa proteins (4). Has high esterase activity as well as proteinase activity (5). |
(1) Sumi, et al., Experientia, Vol. 43, 1110-1111, 1987.
(2) The Journal of Biological Chemistry, Vol. 276, pp. 24690-24696, 2001.
Mol Gen Genet 1990 May; 221 (3): 486-90
(3) Journal of Bacteriology, Vol. 172, pp. 1019-1023, 1990.
(4) The Journal of Biological Chemistry, Vol. 265, pp. 6845-6850, 1990.
(5) Journal of Bacteriology, Vol. 172, pp. 1470-1477, 1990.
2. MODE OF ACTION
NKCP has a thrombolytic effect in vitro and in vivo by ingestion and the effect is milder compared to the plasminogen activator (t-PA). NKCP has been confirmed to decompose and inactivate plasminogen activator inhibitor (PAI-1) in cell culture systems and it also reduces blood viscosity. Ingestion of NKCP reduces the plasminogen activator inhibitor (PAI-1) and helps the plasminogen activator (t-PA) to work efficiently. t-PA activates plasmin and reduces blood fibrin. Thus, NKCP facilitates the activation of fibrinolysis cascade reactions by reducing PAI-1 and helps maintain the balance between coagulation and fibrinolysis. Consequently, NKCP ingestion is unlikely to dissolve fibrin excessively.
Because of decreased PAI-1, NKCP fully activates the fibrinolytic cascade process and helps in maintaining the balance between coagulation and fibrinolysis of the blood. In other words, NKCP ingestion is unlikely to cause unexpected fibrinolysis. NKCP also reduces blood viscosity in vitro and in vivo. Although the association between this action and increased fibrinolytic activity is unknown, reduced blood viscosity is likely to contribute to maintaining sound blood conditions since it prevents stagnant blood flow.
3. SCIENTIFIC DATA ON NKCP FUNCTIONALITY
3.1 Functionality in animals
3.1.1 Thrombolytic action of NKCP in rat thrombosis models
A comparative study was conducted by the Material Research Center to evaluate the thrombolytic action of NKCP by the in situ loop method using thrombosis models.
Thrombosis was produced in rats through platelet adhesion and agglutination, induced by injuries in the endothelial cells of the abdominal descending aorta. Six hours after the induction of thrombosis, the activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as indicators for blood clotting activity.
The APTT values were 33.5 ± 2.4 sec, 52.0 ± 4.5 sec and 63.3 ± 2.9 sec for the control, NKCP 100 mg/kg group and NKCP 250 mg/kg group, respectively, indicating a significant prolongation for NKCP. The PT values were 16.7?0.5 sec, 20.6 ± 0.9 sec and 21.3 ± 1.7 sec for the control, NKCP 100 mg/kg group and NKCP 250 mg/kg group, respectively, indicating a similar result for APTT. Since NKCP showed a significant prolongation of the coagulation time, as indicated above, its possible role in reducing thrombosis was suggested.
3.1.2 In vivo effect of oral NKCP in experimental thrombolysis models
Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University
The thrombolytic activity of NKCP after 14 weeks ingestion of feed containing 0.2% NKCP was evaluated in rat experimental thrombolysis models for arterial thrombosis mainly consisting of platelets using mesenteric microvessels.
The NKCP group showed an obvious increase in endogenous thrombolytic activity in a dose-dependent manner compared to the control group. The activity was equivalent to 0.2 mg/kg of tissue-type plasminogen activator (t-PA).
3.2 Activity in humans
3.2.1 Relation between NKCP ingestion and activity in humans
Daiwa Pharmaceutical Report
The effectiveness and safety of NKCP in a dose range between 1 and 8 tablets daily (125-1,000 mg on the NKCP basis) was evaluated in 40 healthy adults. The subjects ingested NKCP for a period from 1 day to 3 weeks. Various tests were performed to determine the optimum intake recommendations using the euglobulin lysis time (ELT) as the main indicator.
Doses of 2 tablets or more (250 mg in terms of NKCP) daily for a minimum of 4 days showed stable activity.
3.2.2 Effect of the natto bacillus culture filtrate "NKCP" on the coagulative/fibrinolytic system
Published as "Fibrinolytic and anti-thrombotic effect of the protein from Bacillus subtilis (natto) by the oral administration" - Japanese Society of Biorheology
The effect on the coagulation/fibrinolysis system of NKCP at a dose of 2 tablets daily (250 mg on NKCP basis) for 2 weeks was evaluated in 28 adults including patients with metabolic disease carrying the risk of thrombosis.
NKCP decreased the ELT by 10.1% without affecting the tissue-type plasminogen activator (t-PA) or the activated partial thromboplastin time (APTT), and this suggests that NKCP enhances fibrinolysis without intermediation by plasmin.
The effect of chronic use of NKCP at the dose of 2 tablets daily (250 mg on the NKCP basis) was evaluated in 23 subjects for 2 months. The ELT showed a significant decrease at 1 and 2 months and t-PA showed a significant increase at 2 months. Improvement in neck stiffness was observed at 1 and 2 months.
Changes in subjective symptoms due to NKCP intake
| Symptom | Conditions | Before intake | At 1 month | At 2 months |
| Headache | Severe | 1 | 1 | 1 |
| Moderate | 7 | 3 | 4 |
| No symptom (including mild neck stiffness) | 15 | 16 | 16 |
| Remarkable improvement | – | 3 | 2 |
| Shirley-Williams multiple test | – | N.S. | N.S. |
| Neck stiffness | Severe | 5 | 1 | 1 |
| Moderate | 10 | 9 | 10 |
| No symptom (including mild neck stiffness) | 8 | 9 | 11 |
| Remarkable improvement | – | 4 | 1 |
| Shirley-Williams multiple test | – | P<0.05 | P<0.05 |
| Dizziness | Severe | 0 | 0 | 0 |
| Moderate | 6 | 4 | 4 |
| No symptom (including mild neck stiffness) | 17 | 18 | 18 |
| Remarkable improvement | – | 1 | 1 |
| Shirley-Williams multiple test | – | N.S. | N.S. |
Figures represent the numbers of patients
Significant difference by Multiple Range Test: N.S. Not Significant, p<0.05 Significant difference (5% of the danger rate)
Changes in fibrinolysis/coagulation parameters due to NKCP intake (n=23)
| Parameters | Normal values | Before intake | At 1 month | At 2 months |
| ELT(1) | 6-12 hrs. | 9.0 ± 1.3 | 8.1 ± 1.5** | 8.0 ± 1.5** |
| t-PA(2) | ≤ 10ng/mL | 5.4 ± 2.6 | 5.8 ± 2.8 | 6.4 ± 2.2* |
| FDP(3) | ≤ 4μg/mL | 3.0 ± 0.7 | 2.0 ± 0.6* | 3.0 ± 0.7 |
Figure represents ± Standard deviation. According to the significant difference by Duncan's Multiple Range Test, * shows the significant difference in 5 % of danger rate, and ** shows the significant difference in 1 % of danger rate.
1) Upper limit of measurement (ULM) is 12 hrs.
2) Lower limit of measurement (LLM) is 1.5 ng/mL.
3) LLM is 2 μg/mL.
3.1.3 Anticoagulative/fibrinolytic effect of new natto extract
The 26th meeting of the Japanese Society of Biorheology
Natto culture filtrate without B. subtilis natto cells or vitamin K (hereafter abbreviated as NKCP) was subjected to gel filtration using a carrier such as Toyopearl HW-40F to extract NKCP with a molecular weight of 45 KDa. NKCP was shown to hydrolyze S-2251, a synthetic substrate specific to plasmin at 10 IU/mg/min. A saline solution of NKCP was added to human blood immediately after collection to measure laboratory parameters related to coagulation/fibrinolysis. As a result, NKCP treatment decreased both concentrations of thrombin-antithrombin III compound (TAT) and fibrin monomer (FM), showing that it has an anticoagulant action like heparin. Moreover, the concentration of D-dimmer (D-d) was high and that of fibrinogen (Fbn) was low, showing that it has a fibrinolytic effect like alteplase. Different from alteplase, however, the fibrinolytic effect was accompanied by no increase in the concentration of α2 plasmin inhibitor-plasmin compound (PIC), suggesting the fibrinolytic effect is independent of plasmin. As the concentration of NKCP was higher, the effect became larger.
These results demonstrated that the new extract with a molecular weight of 45 KDa from natto culture fluid has an anticoagulant effect and a plasmin-independent fibrinolytic effect on human blood.
3.1.4 Effect of the dried filtrate of natto bacilli culture "NKCP" on blood fluidity
Journal of the Japanese Society of Hemorhelogy Vol. 5 (1), 2002
NKCP was given orally to 13 healthy adults for a week to measure the euglobulin lysis time (ELT) as the indicator for fibrinolysis activity. Doses of 2-4 tablets (equivalent to 250-500 mg of NKCP) daily increased fibrinolysis activity. One healthy adult volunteer, who took 8 tablets (equivalent to 1,000 mg of NKCP) of NKCP daily continuously after meals, showed a remarkably shortened whole blood passage time as determined with a micro channel array flow analyzer (MC-FAN) on Day 7 or later.
