NATTOKINASE SLIDES

Japan Bio Science Laboratory- 1 -

Nattokinase NSK-SD®

The pioneer of Nattokinase

Established in 1974


Osaka Head Office

Kyushu

Factory

Beppu Onsen

Kyushu

Factory

Kyushu Factory (Oita pref.)

Yufuin Onsen

Tokyo Office

Kyoto

Factory

http://www.mapion.co.jp/imap/admi/japan.map

http://www.mapion.co.jp/imap/admi/japan.map

http://www.mapion.co.jp/imap/admi/admi44.map

http://www.mapion.co.jp/imap/admi/admi44.map


• Japanese traditional fermented food

• Has been eaten for more than 1000 years

• Prepared by fermenting boiled soy beans in the presence of Bacillus natto

What is Natto?


Production Flow of Natto

Fermentation

Inoculation of Natto bacteria

Boil the soybean

Soak the soybean in water


Active Ingredients of Natto

Nattokinase

(by Bacillus natto)

Vitamin K2

Polyamine

Polyglutamic AcidVitamin E

Protein

Lecithin

Isoflavone

Calcium

Active Ingredients contained in the sticky part produced by BacillusNatto

Active Ingredients from Soybeans

(From Soybeans)

Vitamin B


What is Nattokinase?

• Found in Natto in 1980

• Serine protease

• Composed of 275 amino

acids

• Molecular weight is 27,728

• Specifically degrades fibrin

(thrombi): dissolving blood

clots without inhibiting

wound healing

• Activity is indicated as

FU （Fibrin Degradation Unit）


• 1907: Dr. S. Sawamura Natto bacteria is product of Protease

• 1925: Dr. K. Oshima Characterization report of Protease (Protease degrades fibrin and gelatin)

• 1953: Dr. S. Miyake et al Crystalization of Protease

• 1956: Dr. S. Miyake et al First amino acid composition defined

• 1987: Dr. H. Sumi et al Named “Nattokinase”

• 1989: Dr. M. Fujita et al Identification of amino acid sequence

• 1992: Dr. E. Ichishima et al Identification of DNA sequence

• 1995: Dr. M. Fujita et al Transport of nattokinase across rat intestinal tract.

Study History



History of nattokinase for supplement

1998: The first Nattokinase supplement in the world was launched

by JBSL (NSK-FD)

2001: Nattokinase supplement without the odor was launched

by JBSL (NSK-SD)

2003: Japan NattoKinase Association (JNKA) was established

JNKA mark was set up

2003: The standard of specification of fermented soybean extract

was established through Japan Health and

Nutrition Food Association (JHFA mark)

2006: JBSL obtained the process patent (JP3834048)

2006: JBSL obtained the substance & process patent (JP3881494)



JBSL’s Nattokinase Raw Material: NSK-SD

• Nattokinase Activity: >20,000FU/g

• No VK2 contained (<0.1ppm)

• No smell of natto

• Recommended daily intake:

2,000FU (100mg of NSK-SD)

• Product application:

Capsules, tablets, powdery supplement etc.


1. Fibrinolytic Effect

2. Reduction of Hypertension Kim, J. et al. Effects of NSK-SD (nattokinase) on blood pressure: A randomized controlled trial. ISSN 0916-9636. Vol. 31, No. 8, August 2008, Hypertension Research.

3. Decrease of Blood Viscosity Pais, E., Alexy, T., Holsworth, R.E., and Meislman, H.J., 2006. Effects of nattokinase, a pro-fibrinolytic enzyme, on red blood cell aggregation and whole blood viscosity. Clin. Hemorheol. Microcirc. 35: 139-142.

4. Antiplatelet aggregating activity Takaoka, S., Murata, Y., Moriyama, H. and Hoshi, K. The 123rd Annual Meeting of the Pharmaceutical Society of Japan, Tokyo, Japan. Nagasaki, Antiplatelet effects of nattokinase (NSK-SD), p.134.

5. Effect on blood flow improvement Iuchi, S., Dokai, H. and Shionoya, K. Improvement of blood flow increase by ingestion of NSK II. FOOD Style 21, Vol. 10, No. 8, 78-90 (2006).

6. Reduction of Plasminogen Activator Inhibitor type 1 (PAI-1)Urano, T., Ihara, H., Suzuki, Y., Oike, M., Akita, S., Tsukamoto. Y., Suzuki, I., and Takada, A., 2001. The pro-fibrinolytic enzyme Subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor. J. Bio. Chem. 276: 24690-24696.

Functions of Nattokinase


Plasmin

Attack

Increase

Nattokinase

Fibrin

Degradation

Products

（FDP)Degradation

➊AttackFibrin

Thrombus

Fibrinogen

Thrombin

Tissue

Plasminogen

Activator

（t-PA)

➋Activation(in vitro)➌Degradation

Plasminogen

Activator

Inhibitor

Type1(PAI-1)

Urokinase

Pro-Urokinase

Plasminogen

Direct & Indirect Effects of NK on the Fibrinolytic System

Mechanism of Nattokinase


Degradation of Artificial Thrombus by Nattokinase

As time goes by, nattokinase degrades the artificial thrombus and it becomes clear liquid.

Mechanism of Nattokinase

0 hour 1 hour 3 hours


Safety of NSK-SD

• Single Oral Toxicity Study

Max. Dose : 1,000 mg/kg・day

Result : No Influence

• 13-Weeks Oral Toxicity StudyMax. Dose : 1,000 mg/kg・day

Result : No Influence

• Reverse-Mutation Assay (Ames Test)Result : Negative

• Chromosomal Aberration TestResult : Negative

• Pathogenicity Study of Nattokinase Producing BacteriaResult : No Influence

Safety Tests: Animal & Microorganism


Safety of NSK-SD

1) An Oral Safety study of NK containing food, NSK II Randomized, Double-blinded Study

31 participants (22=NSKII 3 capsule/day, 9=placebo)

4 wks + 2-wk observation

Result : Safe

2) Clinical Study of Effect and Safety of NK Administration on CV

Patients with Warfarin Double-blinded, Placebo Controlled Study

60 participants with cardiovascular disease (taking warfarin)

Low dose: 15=1,700FU/ day, 15=placebo

High dose: 15=3,400FU/ day, 15=placebo

26 wks

Result : Safe

Safety Tests: Human Clinical Studies


3) An Open Clinical Pilot Study Study to Evaluate the Safety &

Efficacy of NK as an Add-on Oral Fibrinolytic Agent Tolow

Molecular weight Heparin & Anti-platelet in Acute Ischeamic

Stroke

Prospective Multicenter Proof-of-concent Open Pilot Study

12 participants in hospital after a stroke (taking heparin & antiplatelet drugs)

6,000FU/ day for 7 days + 90-day monitoring

Result : Safe

Safety of NSK-SD


Natto vs. Nattokinase

NK Amount

Calorie

Odor

Purines

Vitamin K2

VSNatto

NattokinaseSupplement

Unstable(Varies among products too)

Approx. 100kcal/50g

Has unique odor

Contained

Contained

Stable

A few kcal/g

Odorless

Not contained

Not contained


Preservation Stability of Nattokinase

0102030405060708090

100

0 2 4 6 8 10 12 14

Day

Rel

ativ

e ac

tivi

ty （％）

Preservation stability of commercially Natto （preserved at 5℃）

Preservation stability of NSK-SD (preserved at RT)

0

20

40

60

80

100

120

0 5 10 15 20 25 30 35 40 45

Time (months)

Res

idua

l act

ivit

y (%

)


Comparison of NK activities of commercially available natto per pack

FU


The growth of nattokinase market in Japan

JBSL occupies more than 70% of

nattokinase market in Japan.


Areas JBSL is directly exporting NSK-SD • USA• EU• China• Korea• Taiwan• Vietnam• Indonesia• Malaysia• Singapore

(As of Dec 2012)

Occupies about 80% of the world market

= Approx. US$300M as the end products

NSK-SD in Other Countries


NSK-SD in Korea Latest News!


Korea Food & Drug Administration （KFDA）

Health Functional Food, Certification of Functional Raw Material

Raw material: Fermented soybean extract powder

Indicator component: Fibrin degrading enzyme activity

1) Characteristics: White to milk-white color powder

2) Fibrin degrading enzyme activity (FU/g) : 18,000~28,0003) Lead (mg/kg): <1.04) Total arsenic (mg/kg): <1.05) Cadmium (mg/kg): <0.56) Total mercury (mg/kg): <0.57) Total coliforms: Negative8) Total aflatoxin (㎍/kg): <10.0

Functions claimed: Effective for the hypertensive

(Physiological activation function 2nd grade)

Daily intake: 100mg as fermented soybean extract powder

Test Method: Assay Method of Nattokinase (Fibrin

degradation method)

The key points of Health Functional Food in Korea


NSK-SD in USA

• Raw Material & Bulk Softgels

→ Brand/Contract Manufacturer- No.1 direct marketer of doctor-formulated nutritional product, etc

• Started in 2003

• Channel- Clinics (Doctors)

- Direct Marketing (Internet, Mail Order)

- Retail Shops (DS, Health Food)


More than 50 products are on the U.S. market

NSK-SD in USAPicture taken in a supplement shop


Case: Direct Marketing

• Premium Brand by Clinical Doctor- Formulated & Recommended by Doctor

- Dr’s Expert Knowledge & Experience

→ ↑Customer’s trust on the product

• Clinical Data → Doctors- Doctors highly value clinical studies/data

- Communication with doctors

NSK-SD in USA


123456789

10111213141516171819202122232425

26 272829303132333435363738394041424344454647484950

Coenzyme Q10CollagenNatokinaseSoy IsoflavonesSoy Peptide DHAGinkgo extractTurmeric extractSoy lecithinInstant Green JuiceCatechinSoy saponinMaca LycopeneGlucosamineCeramideHyaluronic acidHeme ironChondroitinChinese blackberry extractChitosanTaurineSesaminGaba(γ-aminobutyric acid)Citric acid

BCAA（Branched chain amino acid）RutinCapsaicinβ-caroteneSqualene （The ocean shark extract）Indigestible dextrin（Dietary fiber）Gymnema EPA(Eicosapentaenoic acid)CCP（Casein calcium peptide）GarciniaFucoidanLactoferrinFlavangenolSardine peptideFructo-oligosaccharideMelilotPlant sterolL-carnitineAnhydrideⅢCreatineCPP（Casein phosphopeptide）β-glucanWheat albuminDiacylglycerolGlobin protein resolutions

24.214.113.613.412.012.011.311.311.311.110.29.79.49.08.88.78.18.17.67.67.67.67.17.16.9

6.56.46.26.26.25.85.85.55.35.35.14.94.44.44.44.44.13.93.93.73.73.53.23.03.8

Ranking of most expected functional nutrients (%)

Note: The figure shows the percentage of people answered they wish to buy products with the nutritional component.

(Ref: Nikkei new product review Vol.78)

Survey


63.4

59.0

55.4

53.9

48.9

46.3

45.6

40.7

39.6

34.5

32.2

31.1

29.0

28.7

24.9

0 10 20 30 40 50 60 70％Make blood flow smooth and

improve the blood circulation

Heal the eyestrain

Enhance the brain work such as memorization

Abstain the obesity

Heal the stiff shoulders

Abstain and control stress

Activate the skin cells

Abstain the activities of negative Active Oxygen

Providing the good sleep

Providing hair with nutrition

Enhance the foot muscle’s abilities

Improve the food and hand’s bone abilities

Enhance the duration and muscle abilities for sports

Improving the good circulation

Abstain the side effects of drinking alcohol

Functional nutrients expected to be developed in the future(Multiple answer up to 3)Survey


What is thrombus?

A scab made with hemocyte and platelet tangled with

fibrin to repair injured vessel.

Injury

Platelet Aggregation

Coagulation

Reparation

Fibrinolysis

Thrombus will not be removed if

fibrinolytic activity is decreased

due to various reasons.

Process: Injury to Repair


What is thrombosis?

Arteriosclerosis make the brain vessel narrow and thrombus can get stuck there. This will make the blood flow stop all of sudden.

Arteriosclerosis Thrombus

Cholesterol precipitated and

accumulated in a blood

vessel

How cerebral

infarction occurs


Approx. 100,000km in total

= 2.5 x earth

circumference!!

The length of vessels in our body

is…

In this length of vessel, thrombus

are produced every time vessels get

injured in our daily life.

Blood Vessels of Whole Body


Others17%

Cerebrovasucular Diseases

14%

HeartDiseases

15%

Cancer31%

ChronicObstructive

Lung Diseases1%

Lung Diseases2%

Renal Failure2%

Senile Decay2%

Suic ide3%

Accidents4%

Pneumonia9%

28.8%Ref. 2002 Vital Statistics

The rate of the cause of death

Thrombosis in data


Dementia10%

Heart Diseases5%

Fracture&Fall13%

Colds&Pneumonia3%

Decay15%

Others10%

Rheumatic&Arthritis6%

Patients with cerebrovascular diseases (e.g. cerebral Infarction, hemorrhage) will need nursing and rehabilitation. This brings a big effect on the society and now is a social problem.

Causes of Bedridden: Age Over 65

Thrombosis in data

Cerebrovascular Diseases

38%

Comprehensive Survey of Living Condition 2004


・Genetic diabetes・Obesity・Smoking・Lack of exercise・Stress・Excess intake of alcohol

・Excess intake of salt・Aging etc.

Obesity

High-bloodPressure

Hyperlipidaemia

Diabetes

Lifestyle diseasesCauses of lifestyle disease

Cardiac infarction, cerebral Infarction, anginacerebral hemorrhage, cerebral embolismrenal sclerosis, renal insufficiencyfundal hemorrhage

Metabolic

Syndrome

How thrombosis occurs?

Onset of complications


Nattokinase and Prevention of Thrombosis

To prevent thrombosis…

Prevent arteriosclerosis by reconsidering

your lifestyle habit

Proper diet, proper amount of exercise

Don’t forget thrombus are produced inside

our body all the time for vessel recoveryPrevention: Intake of Nattokinase

TOO LATE TO TAKE ACTIONS

AFTER THROMBOSIS HIT YOU!


It is widely known that the crisis rate of thrombosis is higher in a

cold season. HOWEVER the mortality rate of thrombosis

DRAMATICALLY INCREASES OVER 32 deg C.

The graphs above show the results of the study on the relationship between the

temperature and thrombosis. A significant increase in the mortality rate was seen at 32 deg

C. especially in cerebral infarction (1.66 times more compared to 31 deg C.). (Source：Pan WH et al., Lancet 345: 353-5, 1995)

Appendix: Thrombosis vs. TemperatureM

ort

alit

y ra

te

(per 100,0

00 p

pl)

Temperature (Ave.)

1.66 times higher

Increase by 3%

Cardiac Infarction Cerebral Infarction

Age: Over 65

Age: 25-44 Age: 45-64

NATTOKINASE SLIDES

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