Nattokinase

5/21/2018 Nattokinase

1/6

686

Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012, Pages 686-691 doi:10.6227/jfda.2012200317

Nattokinase Reduces Brain Infarction, Fibrinogen and

Activated Partial Thromboplastin Time against Cerebral

Ischemia-Reperfusion InjuryJIUNN-MIN WANG1,4, HSUAN-YING CHEN2, SHIU-MIN CHENG3, SSU-HUA CHEN3,

LIN-LAN YANG3AND FU-CHOU CHENG3*

1.Department of Medical Laboratory, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.2.Stem Cell Center, Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.

3.Department of Psychology, Asia University, Taichung, Taiwan, R.O.C.4.Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan, R.O.C.

(Received: August 9, 2011; Accepted: January 19, 2012)

ABSTRACT

Natto is a traditional Japanese food prepared from fermented soybeans. nattokinase (NK) is considered one of the most active func-

tional ingredients in natto. The present study evaluated the neuroprotective effects of NK in gerbils subjected to focal cerebral ischemia.

The gerbils were separated into four groups: control group, ischemic group and two NK groups (4 and 40 mg/day i.g. for 7 days). All gerbils

were fed standard chow with or without NK prior to cerebral ischemia. Focal cerebral ischemia was induced by the occlusion of the right

common carotid artery and middle cerebral artery for 60 min, followed by reperfusion. Infarct volumes were determined by 2,3,5-triphen-

yltetrazolium chloride staining. Anti-thrombotic effects of NK, such as fibrinogen,prothrombin time and activated partial thromboplastin

time, were also determined. Our data showed that NK significantly reduced infarct volumes by 54-68% (p< 0.01), compared to the isch-

emic group. Prothrombin time was not significantly different in all groups. However, low dosage NK (4 mg/day) reversed ischemic-induced

increase in fibrinogen concentrations and activated partial thromboplastin time. In summary, the data indicated that oral intake of NK for

7 days may alter fibrinolytic activity and provide neuroprotective effects against focal cerebral ischemia in gerbils.

Key words: cerebral ischemia, gerbil, nattokinase, 2,3,5-triphenyltetrazolium chloride

INTRODUCTION

Extracts of fermented soybeans (Natto), a traditionalJapanese diet, promote brinolytic activity in the circulation

in a similar manner to oral urokinase(1-2). A major component

from natto is nattokinase (NK). NK is a single polypeptide

chain composed of 275 amino acid residues (molecular

weight of 27,724 Da) and exhibits a high homology with

subtilisins(1,3-4). In recent studies(2,5), natto and NK were used

as traditional medicine for heart and vascular diseases, and

as anti-beriberi agents. After the oral administration of NK,

lysis of thrombi in induced-thrombosis dogs was observed

by angiograpy(2). NK also demonstrated strong brinolytic

activity in the digestion of brin and plasmin substrates(5).

Previous studies concluded that NK is one of the most

potent brinolytic enzymes in traditional fermented soybean

food. In general, the brinolytic activity of NK was measured

by clot-lysis assay and compared with that of plasmin(2-3,5).

NK had about 4 times the activity of plasmin. Previousstudies demonstrated that oral administration of natto and

NK not only hydrolyzed thrombiin vivo, but also converted

plasminogen to plasmin(2,5). In addition, NK can improve

brinolysis for long periods of time and this is related to the

tissue plasminogen activator (t-PA) acting as an endogenous

plasminogen activator(1).

Over decades, various brinolytic agents such as uroki-

nase and t-PA have been developed for thrombolytic therapy

in routine clinics. However, the biological activity of these

substances is short-lived in the bodys circulation. Sumi and

his colleagues claimed that NK has higher brinolytic activity

than others; it could be retained in the blood for more than 3 h

upon oral administration(2,5) . These results suggest that NK

may be a potent natural agent for oral thrombolytic therapy.

Cerebral ischemia results from limited availability of* Author for correspondence. Tel: +886-4-23592525 ext. 4018;

Fax: +886-4-23592705; E-mail: [email protected]


2/6

Journal of Food and Drug Analysis, Vol. 20, No. 3, 2012

687

oxygen and glucose supply. It impairs oxidative metabolism

in severely ischemic regions of the affected brain tissues

and leads to rapid decrease in ATP and other energy-related

metabolites(6-8).Various ATP-driven membrane-bound pumps

or reuptake processes that work to maintain the homeostasisof important metabolites or ions become impaired in brain

tissues. Moderate to severe neuronal damage might occur

following ischemic events. Several models of focal ischemia

have been developed to study the pathology and treatment

of ischemic stroke in different animal species. In one widely

used technique, cauterization or clipping of the middle cere-

bral artery in mice or gerbils through a subtemporal craniec-

tomy is generally accepted as a valid experimental model of

cerebral ischemia(9-12).

In the present study, the effects of NK against cerebral

ischemia were investigated in a gerbil stroke model. The

roles of thrombolytics in the pathogenesis of cerebral isch-

emic reperfusion injury have been studied extensively(10-11).

Although brinolytic activity after the oral administration of

NK had been reported in vascular diseases, there remains a

need to explore its protective effects on the central nervous

system. In addition, plasma brinolytic activity after the oral

intake of NK for 7 days was also investigated in gerbils.

MATERIALS AND METHODS

I.NK and Animals

NK with a molecular mass of 27.7 kDa was isolated frombacteria Bacillus Subtilis natto and puried by gel ltra-

tion chromatography. NK was identied by high resolution

mass spectrometry (20,000 FU/g, Synmax, Taipei, Taiwan).

The brinolysis unit (FU) was determined by the method of

Fujita(3), which employed a standard brin plate. In order to

determine the neuroprotective effects of NK as an anti-isch-

emic agent in vivo, a focal cerebral ischemic gerbil model was

adopted. Adult male gerbils (n = 42, 60-75 g) were obtained

from the Laboratory Animal Center of Taichung Veterans

General Hospital. All animals were housed at a temperature

of 25C in a light-controlled room (12 : 12 h light-dark

cycle) with standard rat chow and water ad libitum. Animalcare and experimental procedures were approved by the

Institutional Animal Care and Use Commettee (IACUC) of

TCVGH (#La-97477). The gerbils were randomly separated

into two experiments. One experiment (n = 18) was evalu-

ated based on 2,3,5-triphenyltetrazolium chloride (TTC)

staining and the other experiment (n = 24) was subjected to

blood sampling for bioassays. All gerbils were divided into

four groups: group 1 (control group with sham-operated) was

only pretreated with saline, group 2 (ischemia group) was

pretreated with saline and subjected to cerebral ischemia,

group 3 (LNKI group) was pretreated with low dosage NK (4

mg/day for 7 days; i.g.) and subjected to cerebral ischemia,

group 4 (HNKI group) was pretreated with high dosage NK

(40 mg/day for 7 days; i.g.) and subjected to cerebral isch-

emia, according to experimental protocol.

II.Stroke Model in Gerbils

Each gerbil was anesthetized with chlorohydrate (400 mg/

kg, i.p.) and its body temperature was maintained at 37C

with a heating pad (CMA/150). A midline neck incision wasmade and the right carotid artery was exposed and separated

from the vago-sympathetic trunk. The right carotid artery was

loosely encircled with a 4-0 suture for later occlusion. The

gerbils head was placed in a stereotaxic frame (David Kopf,

CA, USA) with the nose bar positioned 4.0 mm below the

horizontal line. Following a midline incision, the skull was

partially removed to expose the right middle cerebral artery

(MCA). The MCA was loosely encircled with an 8-0 suture

for later occlusion. A focal cerebral ischemia was induced by

occlusion of the right common carotid artery (CCA) and MCA

for 60 min, followed by reperfusion(12). Twenty-three hours

after cerebral ischemia, each gerbil was anesthetized and

perfused transcardially with 2% isotonic heparinized saline.

III.2,3,5-Triphenyltetrazolium Chloride (TTC) Staining

The brain was then removed and sliced into ve

2-mm-thick coronal sections for TTC staining as described by

Bederson et al.(13). The brain slices were immersed in a 2%

solution of TTC in normal saline for 20 min. They were then

washed and immersed in 10% phosphate-buffered formalin

in the dark and refrigerated until photographed. Infarct size

was determined on projected tracings normal and infarct area

of the same TTC stained section by computer image analysis.

In general, TTC staining effectively detected areas of infarc-tion 24 h after the onset of cerebral ischemia.

IV.Measurement of Coagulation Function

After feeding either with or without NK for 7 days, all

animals received cuts 4 mm from the tail end under general

anesthesia with isourane. The tail was soaked in saline

1 cm from the cutting point at 37C. Blood samples were

obtained into heparinized microtubes. After collection,

bleeding can be stopped by gently compressing the wound for

a few seconds. Plasma samples were separated, frozen and

stored at -80C until assays were performed. For all clottingtests, Dade reagents, Innovin and Actin FSL and Fibrinogen

(Siemens Healthcare Products GMbH, Marburg, Germany)

were used and assayed according to our medical laboratory

standard instructions. Prothrombin time (PT) determination

was performed in 50-L plasma samples and 100 L of

Dade Innovin (containing thromboplastin, calcium, buffers

and stablizers). The coagulation cascade was activated by

incubating plasma with the optimal thromboplastin and

calcium. The clotting time was then measured. Activated

partial thromboplastin time (APTT) was assessed using the

Dade Actin FSL reagent containing phospholipid. Dade Actin

FSL (100 L) was added to the plasma samples (50 L). The

mixture was incubated at 37C for 120 s, and 50 L of 0.02 M

CaCl2was added to activate the reaction. Clot formation was

the end point of the reactions and the results were reported in


3/6


688

of a unilateral MCA and CCA is known to result in ischemic

infarcts of various sizes and degrees. In rats, there is a need

to occlude both the right and left CCA and one of MCA to

produce predictable infarctions. However, the gerbil brain

lacks the connection between the carotid and vertebrobasilar

circulation, resulting in an incomplete circle of Willis. There-

fore, the occlusion of unilateral CCA and MCA in gerbils

was proposed in the present study. The results of this study

demonstrated that a consistent infarction volume (15.3

1.2%) can be achieved 23 h after the occlusion of unilateral

CCA and MCA in gerbils. Our results are less severe but

consistent with previous studies that utilized MCA occlusion

or endovascular sutures(9-11). The experimental models of

cerebral ischemia developed by Baskaya et al.(9), Yamamoto

et al.(10)and Yoshimine et al.(11) were successful because of

seconds. Fibrinogen (FIB) can be converted from a soluble

protein to an insoluble polymer by the action of thrombin

resulting in the formation of a brin clot. Bovine thrombin

(25 L, 100 IU/mL, Dade Fibrinogen reagent) was added to

the plasma samples (50 L) to induce an insoluble polymer

for the brinogen measurement at 660 nm(14).

V.Statistical Analysis

All data are expressed as the meanSEM. The statis-

tical signicance of differences between two groups was

determined by Students t-test. A p value less than 0.05 is

considered signicant.

RESULTS AND DISCUSSION

I.Effect of NK on Cerebral Ischemia

All animals had infarction in the cortex and some severecases in the caudate-putamen. Typical infarct sizes (as visual-

ized in white by TTC staining) in brain slices for each group

were shown in Figure 1. Mean total infarct sizes (expressed

as volumes and percentages of the right hemisphere) in the

ischemia, LNKI and HNKI groups were 79.4 6.8 mm3(15.3

1.2%), 36.6 7.9 mm3(7.5 1.6%) and 25.2 10.4 mm3

(5.0 2.0%), respectively, as shown in Figure 2. Pretreat-

ments with 4 mg and 40 mg of NK for seven days signi-

cantly reduced infarct sizes by 53.9 and 68.3% (p< 0.01),

respectively, when compared with that of the ischemia group.

In general, the occlusion of MCA and CCA does not

always result in a predictable infarction volume in animal

studies. Collateral pathways in different species may redis-

tribute blood ow sufciently to prevent infarction in partic-

ular regions. In rats, cats and dogs, a simultaneous occlusion

Figure 2. Effects of NK on the infarct volume in gerbils. Infarct

volumes were determined by TTC staining in gerbils subjected to 1 h

MCAO+CCAO, followed by reperfusion for 23 h. Pretreatments with

NK (4 mg/day for LNKI and 40 mg/day for HNKI; n = 6 for both)

were given for seven days prior to the MCA+CCA-occlusion. Data

are expressed as mean + SEM. **p< 0.01 for infarct volumes vs. the

control group (n = 6) as determined by Students t-test.

**

**

0

10

20

30

40

50

60

70

80

90

ischemia

Infarct(mm

3)

LNKI HNKI

ischemia

LNKI

HNKI

Figure 1. Typical image of 2-mm thick brain slices stains with TTC after 23 h of 1 h MCAO+CCAO in gerbils. Pretreatments with saline, low and

high dosage of NK (4 and 40 mg/day; i.g.) were given for seven days prior to the MCAO+CCAO.

ischemia 4 mg NK 40 mg NK Control 4 mg NK 40 mg NK


4/6


689

ischemic insults, as well as dosages (4 mg and 40 mg/day) of

NK therapy. Further investigation will be needed to explore

the mechanisms of PT during cerebral ischemia.

Thrombolytic therapy is one of the important advances

in the management of acute ischemic stroke and is evaluatedin several randomized trials(15). NK was indicated as a potent

natural agent for oral brinolytic therapy. In the present

study, APTT and FIB levels were increased by 17% and 16%,

respectively. Both APTT and FIB levels were signicantly

decreased in the LNKI group than those of the ischemia group

(Figure 3B & C). In the low dosage NK group, APTT and

FIB levels were reversed and even down to approximately

40% of the ischemic gerbils. APTT and FIB levels might be

indicating that a hypercoagulable state existed in cerebral

ischemic gerbils.

Fibrinogen (FIB) is a marker of coagulation and inam-

mation, and also inuences brinolysis. NK is now widely

used as a health-promoting food for reducing the risk of throm-

bosis due to its brinolytic activity(1-5,16). Increased plasma

FIB levels may be associated with cardiovascular diseases(17)

and NK decreased plasma levels of FIB in a clinical trial(18).

Hsia et al. observed a reduction of FIB after long-term

administration of NK(18). The low dosage of NK (4 mg/day)

reversed the ischemia-induced increase in FIB concentration;

however, oral intake of high dosage NK (40 mg/day) did not.

In general, aspirin, a potent anticoagulant agent, is used for

the prevention of heart attacks, stroke and atherothrombotic

diseases. It exerts anti-platelet action by inhibiting cyclooxy-

genase (COX), thus reducing the synthesis of thrombogenic

thromboxane A2(TXA2) in platelets. However, it also inhibitsCOX in vascular endothelial cells and reduces the production

incomplete anastomosis in gerbils. The advantages of the

present model include simple craniectomy procedures and

consistent infarct volume.

II.Effect of NK on Coagulation Function

There was no difference in the PT values among all groups

as shown in Figure 3A. The APTT value was signicantly

higher in the ischemia (127% of the basal) and HNKI (128%

of the basal) groups than that of the control group (Figure

3B). The FIB concentration was signicantly increased in the

ischemia (173% of the basal) and HNKI (164% of the basal)

groups and was signicantly decreased in the LNKI (72% of

the basal) group when compared to that in the control group

(Figure 3C).

The PT and APTT were used to reect the extrinsic and

intrinsic coagulation pathways, respectively. Particularly,

PT is a sensitive monitoring test for patients with hereditary,

acquired coagulation disorders and using oral anti-coagulant

therapy. A recent study discovered that natto extract (16 mg/

day for 7 days) had anti-thrombotic effects that included

increasing 17% PT and decreasing 16% FIB values with direct

administration to rats subjected to sciatic nerve injury(14).

However, it had only a little effect on APTT in rats(14). In

sciatic nerve injury, thrombi formation after endothelial injury

was correlated with intimal thickening and it was found that

combination of a t-PA and an anti-platelet agent inhibited

mural thrombi formation after endothelial injury. Our recent

experimental data showed that the PT values had no signi-

cant change after cerebral ischemia. These differences in PTmight depend on different animal models and various cerebral

(A)

0

1

2

3

4

5

6

7

8

9

10

control ischemia LNKI HNKI



Prothro

mbinTime(sec)

controlLNKI

ischemiaHNKI

(B)

(C)

0

100

200

300

400

500

600

Fibrinogen(mg/dl)

controlLNKI

ischemiaHNKI

0

10

20

30

40

50

60

70

80

90

100

ActivatedP

artialThromboplastin

Time(sec)

control ischemiaLNKI HNKI

Figure 3. Effects of saline and NK on (A) PT, (B) APTT, and (C) FIB

in gerbils subjected to 1 h MCAO+CCAO, followed by reperfusion for

23 h. Data were expressed as mean + SEM. Control (n = 6): pretreat-

ment with saline for seven days; Ischemia (n = 6): 1 h MCAO+CCAO,

followed by reperfusion for 23 h; LNKI (n = 6): pretreatment with NK

(4 mg/day) for seven days and 1 h MCAO+CCAO, followed by reper-

fusion for 23 h; HNKI (n = 6) : pretreatment with NK (40 mg/day) forseven days and 1 h MCAO+CCAO, followed by reperfusion for 23 h.

*p< 0.01 compared with control group; #p< 0.01 compared with isch-

emia group; p< 0.01 compared with LNKI group.


5/6


690

of prostaglandin I2 (PGI2), which has anti-thrombotic effect.

Low-dose (40 mg/day) aspirin is able to reduce the forma-

tion of TXA2 in platelets, producing an inhibitory effect on

platelet aggregation, without the inhibition of PGI2 synthesis.

However, higher doses of aspirin are required to attain furtherinhibition(19). In this study, the effects of low or high dosages

of NK on FIB levels may be mediated by a different pathway

similar to that of aspirin. The reason why high dosage NK did

not reverse FIB levels in cerebral ischemic model remained

unclear.

Although the neuroprotective mechanism of NK is

unclear, the in vivocerebral ischemic model suggested that

a reduction of neuronal injury might involve thrombosis,

excessive stimulation of free radical formation, glutamate

receptors, and inammation following focal cerebral isch-

emia. The brain produces free radicals during cerebral isch-

emia at a sufcient rate to overcome endogenous antioxidant

defenses(20). Many studies demonstrated that natto fractions

have inhibitory effect on LDL oxidation as a result of their

radical scavenging activity. Iwai et al. found that natto frac-

tions might help to prevent arteriosclerosis, as they appear

to reduce lipid peroxidation and enhance antioxidation in

vivo(21). This may, in part, involve free radical scavenging

abilities and be benecial to neuroprotective effects of NK in

cerebral ischemic models.

CONCLUSIONS

NK provides neuroprotective effects against focalcerebral ischemia in a gerbil model. Reduced infarct volumes

were signicant in both low and high dosage NK groups

when compared to that of the control group. PT was not

signicantly different in all groups. However, low dosage NK

(4 mg/day) reversed the ischemic-induced increase in FIB

concentrations and APTT. The role of NK in the neuropro-

tection against cerebral ischemia remains unclear, but may

involve the alternation of brinolytic activity in the central

nervous system. Further investigations should be carried out

to examine the roles of NK and exact mechanisms which are

responsible for the neuroprotective effects.

ACKNOWLEDGMENTS

This study was supported by grants from Taichung

Veterans General Hospital (TCVGH-997302B).

REFERENCES

1. Fujita, M., Hong, K., Ito, Y., Fujii, R., Kariya, K. and

Nishimuro, S. 1995. Thrombolytic effect of nattokinase

on a chemically induced thrombosis model in rat. Biol.

Pharm. Bull. 18: 1387-1391.

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

1990. Enhancement of the fibrinolytic activity in plasma

by oral administration of nattokinase. Acta Haematol. 84:

139-143.

3. Fujita, M., Ito, Y., Hong, K, and Nishimuro, S. 1995.

Characterization of Nattokinase-degraded products from

human fibrinogen or cross-linked fibrin. Fibrinolysis 9:157.

4. Yanagisawa, Y., Chatake, T. and Chiba-Kamoshida, K.

et al. 2010. Purification, crystallization and prelimi-

nary X-ray diffraction experiment of nattokinase from

Bacillus subtilis natto. Acta Crystallogr. Sect. F. Struct.

Biol. Cryst. Commun. 66: 1670-1673.

5. Sumi, H., Hamada, H., Tsushima, H., Mihara, H. and

Muraki, H. 1987. A novel fibrinolytic enzyme (natto-

kinase) in the vegetable cheese Natto; a typical and

popular soybean food in the Japanese diet. Experientia

43: 1110-1111.

6. Ito, U., Spatz, M., Walker, J. T. Jr. and Klatzo, I. 1975.

Experimental cerebral ischemia in mongolian gerbils. I.

Light microscopic observations. Acta Neuropathol. 32:

209-223.

7. Lipton, S. A. and Rosenberg, P. A. 1994. Excitatory

amino acids as a final common pathway for neurologic

disorders. N. Engl. J. Med. 330: 613-622.

8. Meyer, F. B., Sundt, T. M. Jr., Yanagihara, T. and

Anderson, R. E. 1987. Focal cerebral ischemia: patho-

physiologic mechanisms and rationale for future avenues

of treatment. Mayo Clin. Proc. 62: 35-55.

9. Baskaya, M. K., Dogan, A. and Dempsey, R. J. 1999.

Application of endovascular suture occlusion of middle

cerebral artery in gerbils to obtain consistent infarction.Neurol. Res. 21: 574-578.

10. Yamamoto, K., Akai, F., Yoshimine, T. and Yanagihara,

T. 1987. Immunohistochemical investigation of cerebral

ischemia after middle cerebral artery occlusion in gerbils.

J. Neurosurg. 67: 414-420.

11. Yoshimine, T. and Yanagihara, T. 1983. Regional cere-

bral ischemia by occlusion of the posterior communi-

cating artery and the middle cerebral artery in gerbils. J.

Neurosurg. 58: 362-367.

12. Chung, S.Y., Cheng, F. C., Lee, M. S., Lin, J. Y., Lin, M.

C. and Wang, M. F. 2006. Ginkgo biloba Leaf Extract

(EGb761) Combined with Neuroprotective AgentsReduces the Infarct Volumes of Gerbil Ischemic Brain.

Am. J. Chin. Med. 34: 803-817.

13. Bederson, J. B., Pitts, L. H., Germano, S. M., Nishimura,

M. C., Davis, R. L. and Bartkowski, H. M. 1986. Evalu-

ation of 2,3,5-triphenyltetrazolium chloride as a stain

for detection and quantification of experimental cerebral

infarction in rats. Stroke 17: 1304-1308.

14. Pan, H. C., Cheng, F. C. and Chen, C. J. et al. 2009.

Dietary supplement with fermented soybeans, natto,

improved the neurobehavioral deficits after sciatic nerve

injury in rats. Neurol. Res. 31: 441-452.

15. Abboud, H. and Amarenco, P. 2004. Thrombolysis and

acute cerebral infarction. Med. Sci. (Paris) 20: 1104-1108.

16. Koenig, W. 2003. Fibrin (ogen) in cardiovascular disease:

an update. Thromb. Haemost. 89: 601-609.


6/6


691

17. Hsia, C. H., Shen, M. C., Lin, J. S., Wen, Y. K., Hwang,

K. L., Cham, T. M. and Yang, N. C. 2009. Nattokinase

decreases plasma levels of fibrinogen, factor VII, and

factor VIII in human subjects. Nutr. Res. 29: 190-196.

18. Omura, K., Hitosugi, M., Zhu, X., Ikeda, M., Maeda, H.and Tokudome, S. 2005. A newly derived protein from

Bacillus subtilis natto with both antithrombotic and fibri-

nolytic effects. J. Pharmacol. Sci. 99: 247-251.

19. Tohgi, H., Konno, S., Tamura, K., Kimura, B. and

Kawano, K. 1992. Effects of low-to-high doses of aspirin

on platelet aggregability and metabolites of thromboxane

A2 and prostacyclin. Stroke 23: 1400-1403.

20. Wang, M. J., Huang, H. M., Chen, H. L., Kuo, J. S. and

Jeng, K. C. 2001. Dehydroepiandrosterone inhibits lipo-

polysaccharide-induced nitric oxide production in BV-2

microglia. J. Neurochem. 77: 830-838.

21. Iwai, K., Nakaya, N., Kawasaki, Y. and Matsue, H. 2002.Antioxidative functions of natto, a kind of fermented

soybeans: effect on LDL oxidation and lipid metabo-

lism in cholesterol-fed rats. J. Agric. Food Chem. 50:

3597-3601.

Documents

Nattokinase