Anticoagulating Effects of Water-Extractive Components from Heshiko and Narezushi, Japanese Fermented Aquatic Products

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1 J Nutr Sci Vitaminol, 61, 84 89, 2015 Anticoagulating Effects of Water-Extractive Components from Heshiko and Narezushi, Japanese Fermented Aquatic Products Koji ITO Department of Marine Bioscience, Fukui Prefectural University, Gakuencho, Obama, Fukui , Japan (Received March 5, 2014) Summary Antithrombotic effects of water-extractive components (extracts) from heshiko and narezushi, traditional fermented aquatic products in Japan, were investigated in terms of blood coagulation and fibrinolysis by means of animal experiments. Blood clot formation was suppressed by administration of heshiko and narezushi extracts to both groups of Wistar rats fed artificial and high-fat experimental diets. Prothrombin time and activated partial thromboplastin time of plasma prepared from the rats, as indices of blood coagulation, were not prolonged by administration of heshiko or narezushi extracts. In contrast, the activities of plasmin and tissue plasminogen activator (tpa), key enzymes in the fibrinolytic system, were significantly increased by administration of heshiko and narezushi extracts. These results suggest that the anticoagulating effects of heshiko and narezushi extracts are associated with the promotion of the fibrinolytic system via enhancement of plasmin and tpa activities. Key Words antithrombotic effect, fermented mackerel, fibrinolysis, plasmin, tpa Thrombosis is closely associated with circulatory diseases including myocardial and cerebral infarction. In maintaining blood homeostasis, thrombus formation is regulated by two systems: the coagulation system and the fibrinolytic system. The former is largely regulated by the balance among platelet aggregation, fibrin formation and coagulation inhibitors, e.g., antithrombin III. The latter is mainly controlled by the balance between the levels of plasminogen activator (PA) and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). A blood clot adhering to the wall of a blood vessel is called a thrombus and is formed when the activity of the coagulation system exceeds that of the fibrinolytic one (1). Antithrombotic effects as physiological functions of some foodstuffs, especially certain vegetables, have been reported (2 5). Furthermore, polyphenols from wine (6, 7), catechins from tea (8), mushroom (Shiitake and Hiratake) extracts (9), and sesaminol from sesame (10) are known as food components having antithrombotic effects. With respect to fibrinolysis, fibrinolytic enzymes have been found in natto (11, 12), siokara (13) and earthworms (Lumbricus rubellus) (14), and enhancement of fibrinolysis was reported in natto (12, 15 17), wine (7) and mushrooms (9). In spite of numerous reports on antithrombotic effects from plant material as mentioned above, little information is available on those of animal products. Hypertension and hyperlipidemia are reported to be factors contributing to thrombosis. Skurk et al. (18) suggested that angiotensin II, which is an important substance for increasing blood pressure and is produced by angiotensin-i converting enzyme (ACE), increased PAI-1 kitou@fpu.ac.jp levels in plasma. Furthermore, ACE inhibitors including cinarapril have the effects of suppressing thrombus formation (19) and reducing PAI-1 levels in plasma (20). Hyperlipidemia also increased the concentration of PAI-1 in plasma (21, 22). In addition, as is already known, the increase of blood triacylglycerol concentration has a close relationship to the activity of the factors in the blood coagulation system, resulting in the acceleration of the blood coagulation (23 25). We previously reported that extracts from heshiko and narezushi, traditional fermented aquatic products produced in the Wakasa Bay area of Japan, had hypotensive and hypocholesterolemic effects (26 29) in animal experiments. Heshiko and narezushi were prepared by the aging of salted mackerel with rice bran or cooked rice, respectively, for several months at ambient temperature. The physiological functions of heshiko and narezushi were attributable to the large amount of water-extractive components including free amino acids, peptides and organic acids produced during aging (28 31). Inhibition of ACE activity and reduction of plasma triacylglycerol levels by the extracts from heshiko and narezushi suggests their potential to prevent thrombosis. The objective of this study was to elucidate if the extracts from heshiko and narezushi had the antithrombotic effects in terms of coagulation and fibrinolysis by means of animal experiments. MATERIALS AND METHODS Preparation of extracts from heshiko and narezushi. Extracts from heshiko and narezushi experimentally manufactured in the laboratory were prepared according to the method reported previously (30, 31). About 84

2 Anticoagulating Effects of Japanese Fermented Mackerel Products 85 Table 1. Composition of high-fat experimental diet. Components Composition (%) Sucrose 21.3 Cornstarch 40 Gluten 10 Casein 10 Corn oil 10 Free cholesterol 0.5 Vitamin mixture 1 1 Mineral mixture Sodium chloride 0.7 Choline chloride 0.2 Water 3 1 AIN-93 (Oriental Yeast Co., Ltd., Tokyo) was used as the vitamin mixture. 2 AIN-93G (Oriental Yeast Co., Ltd., Tokyo) was used as the mineral mixture. 10 g of dorsal ordinary muscle from mackerel of heshiko or narezushi was homogenized with 100 ml of boiled distilled water. After heating at 100 C for 5 min, the homogenate was cooled to room temperature, and then centrifuged at 10,000 g for 20 min at 2 C. The first supernatant was decanted and 50 ml of distilled water was added to the precipitate to obtain a second supernatant in the same manner. The two supernatants were combined, filtered with filter paper (No. 2; Toyo, Tokyo, Japan) and the filtrate was diluted to 200 ml with distilled water to prepare each extract. Peptide contents in the extracts were determined according to the method of Lowry et al. (32) using bovine -globulin as the standard. Animal experiments. Seven-week-old male Wistar S/T (Japan SLC, Inc., Hamamatsu, Japan) were divided into eight groups of six individuals each, and housed for 1 wk before the start of experiments under the following conditions: 23 2 C, 55 5% relative humidity, 12:12 h light:dark cycle, with ad libitum access to artificial diet (CE-2; CLEA Japan, Inc., Osaka, Japan) and sterilized tap water. The artificial diet for four groups and the high-fat experimental diet for the other groups were then provided for 30 d. The composition of the high-fat experimental diet is shown in Table 1. As the high-fat experimental diet contained 10% of triacylglycerol (corn oil) and 0.5% of free cholesterol, its fat energy ratio was 22.5 kcal% and was higher than that of the artificial diet (12.0 kcal%). The extracts or the control were administered successively (for 30 d) to rats by using stainless sonde, and the dose (10 mg/kg) and the volume (10 ml/kg) for administration were set as described previously (26). The concentration of NaCl in extracts or control was adjusted to 1.0%, which was equivalent to that of heshiko extract. The care and treatment of animals conformed to the internal guidelines on animal experiments of Fukui Prefectural University. Measurement of blood clotting time. The rats were sacrificed under diethyl ether anesthesia, and blood was drawn from the aorta. An aliquot of blood (2 ml) was immediately poured into a plastic cup to coagulate spontaneously, and blood viscosity was monitored for 6 min using a tuning fork vibration viscometer (SV-1A; A&D, Tokyo, Japan). Measurement of the activity of blood coagulation factors. To prevent blood coagulation, 0.2 ml of 3.2% sodium citrate solution was immediately added to 1.8 ml of blood and stirred well in a plastic tube. The plasma was then separated by centrifugation at 735 g for 10 min at 5 C. As indices of blood coagulation factors in plasma, both prothrombin time (PT) and activated partial thromboplastin time (APTT) were automatically measured by using an automated blood coagulation analyzer (CA-50; Sysmex, Kobe, Japan). Measurement of plasmin activity. Plasmin activity in plasma was measured using a chromogenic substrate (S-2251, H-D-valyl-L-leucyl-L-lysine-p-nitroaniline dihydrochloride; Sekisui Medical, Tokyo, Japan). A 400 L aliquot of S-2251 solution (1.5 mg/ml) was added to 12 L of plasma and reacted at 37 C for 50 s. The reaction was terminated by adding 1 ml of 2% citric acid. The activity was estimated from the difference in absorbance at 405 nm and 505 nm and expressed as a relative value to that of the standard (normal human plasma; Daiichi, Sekisui Medical). Measurement of plasminogen activity. Plasminogen activity in plasma was measured using a commercial kit (TestzymS PLG; Sekisui Medical) according to the protocol provided by the manufacturer. The activity was estimated as a relative value to that of the standard (normal human plasma; Daiichi, Sekisui Medical). The measurement of tissue plasminogen activator activity (tpa). As plasma plasminogen activator (PA), the activity of tpa in plasma was measured using a commercial kit (Rat tpa ELISA kit, Innovative Research Inc., Novi, MI) according to the protocol provided by the manufacturer. Statistics. Data are expressed as the mean standard deviations of six individuals. The differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. Different superscripts in each data column of tables indicate significant differences (p 0.05). RESULTS AND DISCUSSION The effect of heshiko and narezushi extracts on blood clotting time A blood clot is formed by the activation of the blood coagulation system upon bleeding. As formation of artificial blood clots with spontaneous blood coagulation decreases vibration detected by the viscometer sensor, increases in viscometer readings are used to monitor the blood clot formation. Figures 1 and 2 demonstrate the progress of blood clot formation of rats fed the artificial or high-fat experimental diet, respectively. As shown in Fig. 1, the blood viscosity of the control group fed the artificial diet gradually increased from 10 mpa S to 44 mpa S in 4.5 min, thereafter greatly rose and reached 147 mpa S at 6 min. The changes in blood viscosity of the raw mackerel extract group

3 86 ITO K Fig. 1. Effect of administration of heshiko and narezushi extracts on successive changes in blood viscosity of Wistar rats fed an artificial diet. The blood samples drawn from Wistar rats fed an artificial diet with or without administration of heshiko and narezushi extracts for 30 d were subjected to monitoring of the successive changes in viscosity. An underline in the figure indicates significant differences between the control and heshiko/narezushi extract at each time point (p 0.05). Fig. 2. Effect of administration of heshiko and narezushi extracts on successive changes in blood viscosity of Wistar rats fed a high-fat experimental diet. The blood samples drawn from Wistar rats fed a high-fat experimental diet with or without administration of heshiko and narezushi extracts for 30 d were subjected to monitoring of the successive changes in viscosity. An underline in the figure indicates significant differences between the control and heshiko/narezushi extract at each time point (p 0.05). were similar to that of the control group throughout the observation. On the other hand, a similar trend was observed in the increase of blood viscosity of the heshiko and narezushi extract groups to that of the control group in 4 min, but the final viscosity remained considerably lower than that of the control group. In the test for the rats fed the high-fat experimental diet as shown in Fig. 2, the blood viscosity of the control group rapidly increased and exceeded 147 mpa S in 2 min (Fig. 2), which was similar to maximum value of the control group fed the artificial diet at 6 min (Fig. 1). The blood viscosity of the control group reached 400 mpa S at 6 min, indicating that high-fat experimental diet administration promoted blood clot formation. Hanzawa et al. (23, 25) reported that the activities of factor II and V and platelets involved in blood coagulation increased under the hyperlipidemia. It is, therefore, presumed that the remarkable increase in the blood viscosity of rats fed the high-fat experimental diet is caused by activation of the factors related to blood coagulation. The blood viscosity of the group administered raw mackerel extract remained lower than that in the control group, though it increased greatly from 1.5 min and reached 350 mpa S at 6 min. The blood viscosity of the groups administered heshiko and narezushi extract mildly increased compared to that of above two groups in 3.5 min and the final values of viscosity were less than 250 mpa S. These results indicated that the administration of heshiko and narezushi extracts suppressed blood clot formation in the rats fed either an artificial or high-fat experimental diet. The administration of raw mackerel extract somewhat suppressed blood clot formation, but the suppressive effect was very small. Presumably, the components produced and accumulated in the mackerel meat during the processing of heshiko and narezushi were involved in the suppression of blood clot formation. However, it remains unclear how the extracts affect blood coagulation activity and fibrinolytic activity. Next, to elucidate the preventive effects of heshiko and narezushi extracts against thrombosis, the activities of blood coagulation factors in the plasma from the rats fed the artificial diet or high-fat experimental diet were investigated. The effect of heshiko and narezushi extracts on the coagulation system Table 2 presents the PT and APTT values, as indices of coagulation system activity, of the plasma from the rats fed the artificial and high-fat experimental diet with or without administration of heshiko and narezushi extracts for 30 d. The PT value reflects the activities of coagulation factors II, V, VII and X, while the APTT value represents those of VIII, IX and XI. These coagulation factors act on the fibrin formation process. The PT and APTT values of the plasma from the rats prior to feeding with the artificial diet with or without the administration of heshiko and narezushi extracts were s and s, respectively. The 30 d feeding with the artificial diet barely changed PT and APTT values for the control, heshiko extract and narezushi extract groups. Similarly, the PT and APTT values of all groups after the 30 d feeding with the high-fat experi-

4 Anticoagulating Effects of Japanese Fermented Mackerel Products 87 Table 2. Effect of administration of heshiko and narezushi extracts on plasma PT and APTT values. Before After administration for 30 d Artificial diet Control Heshiko extract Narezushi extract PT value (s) a a a a APTT value (s) a a a a High-fat experimental diet Control Heshiko extract Narezushi extract PT value (s) a a a a APTT value (s) a a a a Different superscripts in each column indicate a significant difference (p 0.05). Table 3. Effect of administration of heshiko and narezushi extracts on the activity of fibrinolytic system factors. Before After administration for 30 d Artificial diet Control Heshiko extract Narezushi extract Plasmin (%) ac a b bc Plasminogen (%) a a a a tpa 2 (ng/ml) a a b b High-fat experimental diet Control Heshiko extract Narezushi extract Plasmin (%) a b c ac Plasminogen (%) a a a a tpa 2 (ng/ml) a b c c 1 Data were expressed as percentage relative to normal human plasma. 2 tpa, tissue plasminogen activator. Different superscripts in each column indicate a significant difference (p 0.05). mental diet were at almost the same level as that before administration irrespective of the administration of heshiko and narezushi extracts. Moreover, there were no differences in the PT or APTT values between the rats fed the artificial diet and high-fat experimental diet in any group, though the blood clot formation progressed rapidly in the rats fed the high-fat experimental diet (Fig. 2). The reason is unclear at present and further research should be conducted on this point. Wojewódzka-Zelezniakowicz et al. (19) reported that ACE inhibitors, such as perindopril and cilazapril, reduced fibrin formation and prolonged PT and APTT values. They hypothesized that ACE inhibitors suppressed the expression of coagulation system factors via the effect on the vessel endothelium, not as a result of ACE inhibition. The formation of fibrin from fibrinogen by activation of coagulation factors is the most important event in the blood coagulation system (33). However, the prolongation of both PT and APTT values was not observed in the heshiko or narezushi extract groups, suggesting the administration of heshiko and narezushi extracts to the rats yielded no impact on the blood coagulation factors. Blood clots diminish due to retraction and fibrinolysis as time goes by (34). Platelet aggregation activates thrombin (coagulation factor II) and is also closely related to clot retraction (35). However, no changes in PT values including thrombin activity were observed by administration of heshiko or narezushi extracts (Table 2). In addition, since these extracts were prepared using hot water, the proteins in the extracts from heshiko and narezushi were likely to lose enzyme-like activity for direct degradation of fibrin fiber. Therefore, the enhancement of fibrinolytic activity might be a cause for the suppression in blood clot formation by the administration of heshiko and narezushi extracts. Next, the effects of the extracts on fibrinolytic activity of the plasma were investigated. The effect of heshiko and narezushi extracts on fibrinolytic system factors Table 3 shows fibrinolytic enzyme activities in rats fed the artificial or high-fat experimental diet with or without administration of heshiko and narezushi extracts. When the rats were fed the artificial diet, the plasmin activities of the control groups barely changed with administration of 1.0% NaCl for 30 d. In contrast, the plasmin activity of the heshiko extract group significantly increased with the administration for 30 d and

5 88 ITO K an increasing tendency in the activity of the narezushi extract group was also observed. Furthermore, the activity of plasminogen, the precursor of plasmin, slightly decreased in the control group with administration of 1.0% NaCl for 30 d, but not significantly. A decreasing tendency in plasminogen activity was observed in the heshiko and narezushi extract groups, though the decreases were not significant. On the other hand, when the rats were fed the highfat experimental diet, the plasmin activities of all groups decreased after the administration for 30 d. The decrement of the control group was largest among them, while the decrease of plasmin activity was significantly suppressed by the administration of heshiko and narezushi extracts. However, no differences in the plasminogen activity were observed in any of the groups fed the high-fat experimental diet regardless of the administration of heshiko and narezushi extract. These results suggested that the administration of heshiko and narezushi extracts promoted the activation of plasmin. The activities of tpa, one of plasminogen activators, of the control, heshiko and narezushi extract groups after the 30-d feeding with the artificial diet were ng/ml, ng/ml and ng/ml, respectively. The tpa activities of the heshiko and narezushi extract groups were significantly higher than that before administration ( ng/ml). The tpa activity of the control group fed the high-fat experimental diet was largely decreased to ng/ml from ng/ml after the administration for 30 d, while the activities of the heshiko and narezushi extract groups were significantly increased by the administration (over 0.13 ng/ml). No significant changes in the activity of urokinase, another PA in plasma, were observed in any group irrespective of the diet (data not shown). Plasmin is the most important fibrinolytic enzyme in fibrin degradation, and its activation requires a plasminogen activator such as urokinase or tpa (33). The significant decreases of plasmin and tpa activity after feeding with the high-fat experimental diet were possibly related to the remarkable progress in the blood clot formation as shown in Fig. 2. The enhancement of plasmin activity via an increase of tpa activity by the administration of the heshiko and narezushi extracts was thought to contribute to the suppression of blood clot formation. That is, the acceleration of tpa activity by the heshiko and narezushi extracts was suggested to be the key function of their antithrombotic effects. However, plasmin-like and tpa-like activities were not observed in the extracts from heshiko or narezushi as mentioned above, and it was unlikely that their extracts directly affected plasminogen as tpa did. The tpa is also a fibrinolytic enzyme secreted from the endothelium of vessels into the blood (36). PAI-1 is the inhibitor of tpa and is secreted from platelets, the endothelium and fat cells (33). There are two possibilities as the reason for the increase of tpa activity by the administration of the extracts from heshiko and narezushi. One is the effect on the endothelium of vessels after absorption in the intestine to accelerate the secretion of tpa, and the other is the inhibition of the PAI-1 activity. The concentration of PAI-1 in plasma increases with hyperlipidemia (21). Fogari and Zoppi (20) reported that ACE inhibitors suppressed PAI-1 levels in plasma, and Skurk et al. (18) showed the increase of PAI-1 levels in plasma from angiotensin II, the product of ACE. We already reported that the administration of the heshiko and narezushi extracts decreased systolic blood pressure and ACE activity in the aorta and plasma in the spontaneously hypertensive rat (26, 27). Furthermore, the increases in plasma cholesterol and triacylglycerol levels in the rats fed the high-fat experimental diet were also suppressed by the administration of heshiko and narezushi extracts (28, 29). In the rats administered heshiko and narezushi extracts, the PAI-1 activity of plasma decreased despite the difference in diet (data not shown). These results indicate that the anticoagulating effect of the extracts from heshiko and narezushi is closely related to the acceleration of fibrinolysis via the increase of tpa activity and the decrease of PAI-1 activity. Antihypertensive and hypocholesterolemic effects of the extracts from heshiko and narezushi were likely to be attributable waterextractive components including peptides produced during processing (26 29). Therefore, the water-extractive components including peptides produced by protein hydrolysis during processing also may enhance tpa activity through the inhibition of PAI-1 activity directly or through hypotensive and hypolipidemic effects. However, the component(s) in the extracts responsible for the anticoagulating effect remains unclear. The change of PAI-1 activity by the administration of heshiko and narezushi extracts and the possible component(s) will be reported in the next paper. In conclusion, it was revealed that heshiko and narezushi extracts prolonged the blood clotting time as an anticoagulating effect and that the effect was associated with the enhancement of plasmin activity in the fibrinolytic system via increased tpa activity. The results of this study provide valuable information to elucidate the physiological function of the fermented aquatic products, heshiko and narezushi, on the blood circulatory system. 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