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Function |
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| 1. |
Functional Ingredients |
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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. |
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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. |
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Enzymes synthesized and excreted
outside the cells by Bacillus subtilis after exponential
multiplication. |
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| 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). |
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(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. |
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| 2. |
Mode of Action |
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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. |
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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. |
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| 3. |
Scientific Data
on NKCP Functionality |
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| 1) |
Functionality
in animals |
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| a) |
Thrombolytic
action of NKCP in rat thrombosis models |
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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. |
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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. |
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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. |
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| b) |
In vivo effect of oral
NKCP in experimental thrombolysis models
Laboratory of Physiology, Faculty of Nutrition,
Kobe Gakuin University |
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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. |
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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). |
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| 2) |
Activity in
humans |
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| a) |
Relation between
NKCP ingestion and activity in humans
Daiwa Pharmaceutical Report |
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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. |
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Doses of 2 tablets or more (250
mg in terms of NKCP) daily for a minimum of 4
days showed stable activity. |
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| b) |
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 |
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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. |
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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. |
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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. |
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Changes in subjective
symptoms due to NKCP intake |
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| Symptom |
Conditions |
Before intake |
At 1 month |
At 2 months |
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| 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. |
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| 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 |
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| 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. |
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Figures represent the numbers
of patients |
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Significant difference by Multiple
Range Test: N.S. Not Significant, p<0.05 Significant
difference (5% of the danger rate) |
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Changes in fibrinolysis/coagulation
parameters due to NKCP intake (n=23) |
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| 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 |
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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. |
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| c) |
Anticoagulative/fibrinolytic
effect of new natto extract
The 26th meeting of the Japanese
Society of Biorheology |
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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. |
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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. |
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| d) |
Effect of the dried filtrate
of natto bacilli culture "NKCP" on
blood fluidity
Journal of the Japanese Society
of Hemorhelogy Vol. 5 (1), 2002 |
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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. |
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