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Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks
Comparative Biochemistry and Physiology Part C 128 Ž2001. 27᎐33
Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks Hiroshi Yoshii a,1, Norihide Tachi a,U , Riichiro Ohbab, Osamu Sakamuraa , Hidemaro Takeyamaa , Toru Itani a a
Department of Hygiene and Occupational Health, Nagoya City Uni¨ ersity Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan b Department of Applied Microbial Technology, Faculty of Engineering, Sojo Uni¨ ersity, 4-22-1, Ikeda, Kumamoto 860-0082, Japan Received 31 March 2000; received in revised form 8 September 2000; accepted 21 September 2000
Abstract Oligopeptides of 1 KDa or less were obtained by hydrolysis of chicken egg yolks with a crude enzyme, and by dialysis with a semipermeable membrane filter. Since the extracted peptides had an inhibitory action on the activity of angiotensin I-converting enzyme ŽACE. in vitro, they were orally administered at 20, 100 and 500 mgrkg body weight to spontaneously hypertensive rats ŽSHR. for 12 weeks to analyze the physiological role on cardiovascular functions. The administered oligopeptides suppressed the development of hypertension at all dosages. After 12 weeks at 500 mgrkg body weight, the values for systolic, mean, and diastolic blood pressure were approximately 10% less in SHRs administered than controls. Furthermore, serum ACE activity of the peptide-administered groups was significantly lower than that of the control group in a dose-related manner. Our results imply that oligopeptides extracted from hen’s egg yolks could potentially suppress the development of hypertension in SHR, and this effect might be induced by the inhibition of ACE activity. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Angiotensin I-converting enzyme ŽACE.; Antihypertensive effect; Hypertension; Systolic blood pressure; Oligopeptides; Physiological functional food; Egg yolk; Spontaneously hypertensive rat ŽSHR.
1. Introduction More than 20 million people suffer from hypertension in Japan alone, and 90% or more of them are patients with essential hypertension ŽMuratani and Fukiyama, 1992.. Essential hypertension has been considered a disease induced by various U
Corresponding author. Tel.: q81-52-853-8171; fax: q8152-859-1228. 1 Present address: Fine Chemical Laboratory Co. Ltd., 5-7-8 Shimoshinjo, Higashiyodogawa-ku, Osaka 533-0021, Japan
factors relating with life-style and heredity ŽMuratani and Fukiyama, 1992.. Blood pressure should be controlled in these hypertensive patients, because high blood pressure is a wellknown risk factor for cerebro- and cardiovascular disorders ŽLevy et al., 1991.. At the same time, it is necessary to properly regulate the blood pressure, since the sudden and excessive declines in blood pressure have also been documented to increase the risks in diseases other than hypertension ŽCruickshank et al., 1987; Furberg et al., 1995; Psaty et al., 1995; Pahor et al., 1996.. There
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 1 7 2 - 1
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H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
may be some methods to moderately control the blood pressure in hypertensive patients. Of these, a nutritional approach is suspected to be relatively easy and effective to control the blood pressure without any excessive decrease, and particularly, physiologically functional foods may be candidates in the nutritional approach. Physiological functions of food constituents have been elucidated, and it is important to use such food constituents for the maintenance and promotion of health ŽKawakami et al., 1995.. For instance, some peptides have recently been reported to play an important role in controlling the development of hypertension by regulating the renin᎐angiotensin system ŽShiono and Sokabe, 1976; Markov et al., 1990; Haws et al., 1994; Ashida et al., 1996.. Also, several studies have shown an antihypertensive effect of angiotensin I-converting enzyme ŽACE. inhibitory peptides derived from soy sauce, fish sauce, tuna, Japanese sake, fermented soybeans and others ŽMaruyama et al., 1985; Kohama et al., 1988; Maruyama et al., 1989; Kinoshita et al., 1993; Matsumura et al., 1993; Saito et al., 1994a,b; Kawakami et al., 1995; Matsufuji et al., 1995; Okamoto et al., 1995a,b,c.. It has been assumed from these findings that such peptides could be utilized to control the blood pressure especially among people with high risk of essential hypertension. In this study, we separated ACE inhibitory oligopeptides with a commercial crude enzyme from chicken eggs, and aimed to show the inhibitory influence of the oligopeptides on the development of hypertension in SHR. Through the experiments, we tried to evaluate the availability of the oligopeptides as a physiologically functional food to control blood pressure.
2. Methods 2.1. Preparation of oligopeptides Chicken egg yolks were hydrolyzed with a commercial crude enzyme, according to the method of Ohba et al. Ž1993, 1994a,b, 1995.. Namely, 25% yolk solution diluted with distilled water, 0.05 M lactic acid-sodium lactate buffer ŽpH 2.5., and Newlase F solution Žderived from the genus Rhizopus, with an activity equivalent to 18 IUrl of protease and 24 IUrl of lipase, Amano Pharmaceutical Co. Ltd., Tokyo, Japan. were
mixed at a ratio of 4:5:1. The solution was incubated for 5 h at 50⬚C while rotating at 60 rev.rmin. The reaction was terminated by inactivating the enzyme at 100⬚C in a water bath for 5 min. Then, the crude solution was filtrated by using a 0.45 m-membrane filter with a nominal cut-off of 1 kDa. The filtrate was powdered after lyophilization to obtain granular oligopeptides. Using this powder, the effects on the cardiovascular functions were evaluated in vivo. We also treated the filtrate with commercial proteases to know whether the digestion with gastrointestinal enzymes, when administered into the alimentary canal, would alter the ACE inhibitory action of acquired oligopeptides. The filtrate was treated at 37⬚C with either pepsin Žprotease activity: 14 IUrl, Nacalai-Tesque Inc., Tokyo, Japan., trypsin Žprotease activity: 24 IUrl, Sigma Chemical Co., St. Louis, MO., chymotrypsin Žprotease activity: 26 IUrl, Sigma Chemical Co.., or all of these for 5 h. Following the incubation, the enzymes were inactivated at 100⬚C for 5 min, and the solution was filtered again using a 0.45-m membrane filter. The filtrate was freeze-dried. 2.2. Measurement of ACE inhibitory acti¨ ity The 50% inhibitory concentration of ACE activity ŽIC 50 . was assayed in the obtained oligopeptides and other peptides with the treatment by endogenous enzymes, according to the modified method of Cushman et al. ŽCushman and Cheung, 1971; Lieberman, 1975.. 2.3. Effects of oligopeptides on the cardio¨ ascular functions in SHR. Forty male SHR ŽSHRrIzm, supplied from Disease Model Cooperative Research Association, Kyoto, Japan. were housed individually in polycarbonate cages at 23 " 0.5⬚C with a humidity of 55 " 5%, and 12 h lightr 12 h dark Žlights on 0800 h.. This study was carried out according to the guidelines for the animal experimentation of Nagoya City University Medical School. Animals were assigned at 5 weeks of age to one of the following groups; a control group orally administered only water at 5 mlrkg body weightrday, and three groups of animals administered the oligopeptides at 20, 100 or 500 mgrkg b.w.rday. The oligopeptides were dissolved in water to adjust the concentration at 4, 20 and 100
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27 ᭚133
mgrml for the groups of 20, 100 and 500 mgrkg b.w. administrations, respectively. The injection volume was maintained at 5 mlrkg b.w. in all groups. The administration was performed every day for 12 weeks. Systolic and mean blood pressure was measured once a week by a tail cuff method using BP-98A ŽSoftron, Tokyo, Japan.. Briefly, a tail was put through a rubber cuff in animals held in a plastic tube at 35⬚C, and the cuff was inflated with air. The pressure level at which first pulse appeared after blood flow had been interrupted with the inflated cuff, was designated systolic blood pressure, and the level of maximum amplitude of the pulse wave during measurement was considered the mean blood pressure. Diastolic blood pressure was automatically calculated by the equipment. The measurement was started 1 h or later after the daily administration. Preliminary results revealed that blood pressure was not influenced when the measurement was performed later than 1 h after the oral injection of the oligopeptides or water Ždata not shown.. The difference between the weights of food given on a certain day and what remained 7 days later was designated the weekly food intake. The water intake was measured in the same way as food intake. On the 87th day of the experiment, blood was collected from the vena cava in SHR under pentobarbital anesthesia. After the blood sampling, animals were killed by heart puncture to weigh heart, liver, kidneys and lungs. Collected blood was clotted to obtain the serum. ACE activity was measured according to the method of Kasahara and Ashihara Ž1981. and Yotsumoto Ž1987.. Blood pressure, and food and water intakes were analyzed by two-way analysis of variance ŽANOVA, time = treatment ., and by StudentNewman-Keuls post hoc test. Student-NewmanKeuls test was also employed to show a difference in the IC 50 value, serum ACE activity and organ weight between groups. A statistical significance was considered at P - 0.05.
3. Results 3.1. ACE inhibitory action in ¨ itro The oligopeptides obtained with a crude enzyme exerted an ACE inhibitory action, and the
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Table 1 ACE inhibitory activities of the oligopeptides alone or with additional treatment of some proteases a IC50 Žmgrml. b
O Pc Td Ce PTCf
1.22" 0.08 1.33" 0.15 1.15" 0.16 1.33" 0.19 1.23" 0.08
a
Each value represents mean "S.D. of 5 samples. There is no significant difference between groups. b O: the oligopeptides powder without additional protease treatment. c P: pepsin treatment powder. d T: trypsin treatment powder. e C: chymotrypsin treatment powder. f PTC: Powder treated with all three enzymes.
50% ACE inhibitory concentration ŽIC 50 . was 1.22 mgrml ŽTable 1.. There was no significant difference in IC 50 between the oligopeptides and the other peptides treated individually or with mixture of pepsin, trypsin, and chymotrypsin. 3.2. Influences of the oligopeptides on physiological ¨ ariables in SHR
Systolic, mean and diastolic blood pressures ŽSBP, MBP and DBP, respectively. increased in all groups with time ŽFigs. 1᎐3.. All blood pressures were lower from the 3rd weeks of administration in the oligopeptide-injected groups than in the control. At the 12th week, the blood pressure values were 4᎐8% less in the groups administered the oligopeptides than the control. Particularly, two-way ANOVA revealed a statistical significance in SBP in time, treatment and their interaction Žtime; F s 273.9, d.f.s 12, P - 0.001, treatment; F s 8.597, d.f.s 3, P - 0.001, and time = treatment; F s 1784, d.f.s 36, Ps 0.004.. There were significant differences in SBP between the group administered oligopeptides at the highest dose and the control from the 3rd to 12th weeks except at the 9th and 10th weeks. ANOVA failed to show a statistical significance in the interaction between time and treatment in both MBP Ž F s 1.320, df s 36, Ps 0.107. and DBP Ž F s 1.003, d.f.s 36, Ps 0.468., although there were significant differences in time and treatment ŽMBP; F s 257.90, d.f.s 12, P - 0.001 in time, and F s 7.544, d.f.s 3, P - 0.001 in treatment, and DBP; F s 18592, d.f.s 12, P - 0.001 in time, and F s
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
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Fig. 1. Changes in systolic blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Each point represents a mean " SEM of 10 animals. Two-way ANOVA revealed a statistical significance in time, treatment, and their interaction Ž P - 0.005, see text...U Significantly different Ž P 0.05. from the control, detected by post hoc analysis.
6.537, d.f.s 3, P - 0.001 in treatment .. No significant difference was shown in body weight ŽFig. 4., weekly food or water intake ŽFigs. 5 and 6., or organ weights ŽTable 3. between groups. The serum ACE activity value was significantly 6᎐13% lower in the oligopeptide groups than that of the control ŽTable 2. in a dose-dependent manner.
4. Discussion The oligopeptides obtained by hydrolyzing
Fig. 2. Changes in mean Ž" SEM. blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Two-way ANOVA revealed a statistical significance in time, treatment Ž P - 0.001., although not in their interaction.
Fig. 3. Changes in diastolic blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Two-way ANOVA revealed a statistical significance in time, and treatment Ž P - 0.001., although not in their interaction. Table 2 Serum ACE activity in SHR administered oligopeptides for 12 weeks a Dose
Serum ACE activity ŽIUrl.
Control 20 mgrkg 100 mgrkg 500 mgrkg
42.2" 2.15 U 39.8" 2.12 U 38.8" 2.12 U 36.6" 3.09
a U
Each value represents mean "S.D. of 10 animals. Significantly different from the control at P - 0.05.
chicken egg yolks had an inhibitory effect on ACE activity in vitro in the present study. The IC 50 value of the peptides was 1.22 mgrml. The IC 50 was not changed even after the oligopeptides had been incubated with other gastrointestinal enzymes such as pepsin, trypsin and chymotrypsin. We therefore conclude that the
Fig. 4. Changes in mean Ž" SEM. body weight in SHRs administered the oligopeptides for 12 weeks.
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27 ᭚133
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Table 3 Percent organ weight on the 87th day of the experiment in SHRs administered the oligopeptidesa Dose
Heart Ž%.
Liver Ž%.
Kidneys Ž%.
Control 20 mgrkg 100 mgrkg 500 mgrkg
0.419" 0.030 0.409" 0.022 0.412" 0.026 0.413" 0.010
3.35" 0.313 3.46" 0.337 3.36" 0.289 3.29" 0.152
0.843" 0.032 0.838" 0.033 0.831" 0.019 0.830" 0.029
a Percent weight to body weight. Each value represents percent weight to body weight mean "S.D. of 10 animals.
Fig. 5. Changes in weekly food intake in SHRs administered the oligopeptides for 12 weeks.
oligopeptides were not further hydrolyzed by major digestive proteases. In excess of 90% of the powder was peptides and approximately 5% was carbohydrates. One of the authors ŽR.O.. is proceeding to purify the powder, and it has become clear that the substances inhibiting the ACE activity in vitro are oligopeptides Ždata not shown.. The IC 50 value of the oligopeptides was similar to the ACE inhibiting activity of other crude peptides reported in earlier studies Ž1.18᎐1.86 mgrml, Ohba, 1996.. We expect that additional purification will result in stronger ACE inhibiting peptides, because the peptides used in the present study were extracted very crudely. The level of SBP, MBP and DBP was generally lower from the 3rd week of the experiment in animals administered the oligopeptides than in the control. The increase in blood pressures was reduced in a dose-dependent manner from the 3rd to 6th weeks of the experiment. These results
Fig. 6. Changes in weekly water intake in SHRs administered the oligopeptides for 12 weeks.
suggest that the oligopeptides play a role in suppressing or delaying the development of increases in blood pressures. Since the value of serum ACE activity was significantly decreased in the oligopeptide-treated groups, compared with the control group, the peptides appear not to be digested in the alimentary canal, as expected from the in vitro study mentioned above. ACE is an enzyme in the renin᎐angiotensin system, and converts an inactive peptide, angiotensin I to angiotensin II which has a hypertensive function ŽMurakami, 1993.. ACE also inactivates a vasodilator, bradykinin ŽMurakami, 1993.. Inhibition of ACE activity therefore results in decrease of blood pressure. Based on the concept, the egg yolk oligopeptides exerted an antihypertensive action possibly by inhibiting the ACE activity. However, a constant dose-dependent relationship was not observed throughout the entire period of the experiment in this study. This may partly be due to an inadequate inhibition of tissue ACE activities, especially aortic ACE activity, because it has been indicated that aortic ACE activity was more important in SHRs to increase blood pressure than that in serum, and that the inhibition of aortic ACE activity reduced the blood pressure ŽOkunishi et al., 1991.. Also, many systems other than renin᎐angiotensin system contribute to control the blood pressure, such as autonomic nervous system, endothelin system, and nitric oxide system. It is possible that compensatory activation of these systems could reduce the difference between the high and low dosage groups in this study. We have not measured the activity in the aorta in the present study, nor did we observe other systems. Thus, the precise mechanism remains unclear, and further analysis is needed. Neither weekly food nor water intake was dif-
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H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
ferent among groups. Body weight was also similar in all groups. These findings are likely to imply that the oligopeptides administered to animals had no effect on the gross metabolic conditions in this study. The SBP value was higher in the oligopeptide groups than that in normotensive rats reported in the earlier studies Žapprox. 180 mmHg at 17 weeks of age in the group at the highest dose in this study, and 130 mmHg in normotensive WKY rats, Okamoto et al., 1988.. The finding indicates that the administered oligopeptides had a relatively moderate action on suppressing the blood pressure increase. This characteristic of the peptides, together with little effect on the general physical condition, may be potentially useful as a physiologically functional food, because excessive reduction of blood pressure has been documented to induce myocardial infarction ŽPsaty et al., 1995., and aggravation of survival rate ŽCruickshank et al., 1987; Furberg et al., 1995.. In conclusion, we have shown that oligopeptides obtained by hydrolyzing chicken egg yolks inhibit an ACE action in vitro as well as in vivo, and prevent the development of hypertension in SHRs. These results suggest that the increase in blood pressure could be partially inhibited by taking the peptides derived from common foods. Finally, we expect that such oligopeptides could be used as a physiologically functional food to control blood pressure in patients with essential hypertension, although further investigations should be undertaken to determine the amino acid sequence, mechanisms of action, and potential side effects. References Ashida, T., Yoshimi, H., Kawano, Y., Matsuoka, H., Omae, T., 1996. Effects of dietary salt and driven Ca pump and NarCa exchange in aortic rings of Dahl rats. Clin. Exp. Hypertens. 18, 851᎐871. Cruickshank, J., Thorp, J.M., Zacharias, F.J., 1987. Benefits and potential harm of lowering high blood pressure. Lancet 1, 581᎐584. Cushman, D.W., Cheung, H.S., 1971. Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637᎐1648. Furberg, C.D., Psaty, B.M., Meyer, J.V., 1995. Nifedipine; dose-related increase in patients with coronary heart disease. Circulation 62, 1326᎐1331.
Haws, R.M., Shaul, P.W., Arant, B.S., Atiyeh, B.A., Seikaly, M.G., 1994. Glomerular losartan ŽDup753.sensitive angiotensin II receptor density is increased in young spontaneously hypertensive rats. Pediatr. Res. 36, 671᎐676. Kasahara, Y., Ashihara, Y., 1981. Colorimetry of angiotensin I-converting enzyme activity in serum. Clin. Chem. 27, 1922᎐1925. Kawakami, A., Kayahara, H., Tadasa, K., 1995. Taste evaluations of angiotensin I-converting enzyme inhibitors, Leu-Lys-Tyr analogues. Biosci. Biotech. Biochem. 59, 709᎐710. Kinoshita, E., Yamakashi, J., Kikuchi, M., 1993. Purification and identification of an angiotensin I-converting enzyme inhibitor from soy sauce. Biosci. Biotech. Biochem. 57, 1107᎐1110. Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M., Mimura, T., 1988. Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochem. Biophys. Res. Commun. 155, 332᎐337. Levy, D., Garrison, R.J., Savage, D.D., Kannel, W.B., Castelli, W.P., 1991. Prognostic implication of echocardiographically determined left ventricular mass in the Framingham Study. N. Engl. J. Med. 322, 1561᎐1566. Lieberman, J., 1975. Elevation of serum angiotensin converting enzyme ŽACE. level in sarcoidosis. Am. J. Med. 59, 356᎐372. Markov, M., Zhelyazkov, D., Markova, M., 1990. Influence of isoteoline on the angiotensin-converting enzyme activity. Med. Arch. 27, 45᎐49. Maruyama, S., Miyoshi, S., Kaneko, T., Tanaka, H., 1989. Angiotensin I-converting enzyme inhibitory activities of synthetic peptides related to the tandem repeated sequence of a maize endosperm protein. Agric. Biol. Chem. 53, 1077᎐1081. Maruyama, S., Nakagomi, K., Tomizuka, N., Suzuki, H., 1985. Angiotensin I-converting enzyme inhibitory derived from an enzymatic hydrolysate of casein. II. Isolation and bradykinin-potentiating activity on the uterus and the ileum of rats. Agric. Biol. Chem. 49, 1405᎐1409. Matsufuji, H., Matsui, T., Ohshige, S., Kawasaki, T., Osajima, K., Osajima, Y., 1995. Antihypertensive effects of angiotensin fragments in SHR. Biosci. Biotech. Biochem. 59, 1398᎐1401. Matsumura, N., Fujii, M., Takeda, Y., Sugita, K., Shimizu, T., 1993. Angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels autolysate. Biosci. Biotech. Biochem. 57, 695᎐697. Murakami, K., 1993. Biochemical and molecular biological studies on renin᎐angiotensin system. Seikagaku 65, 260᎐276 Žin Japanese..
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Muratani, H., Fukiyama, K., 1992. Pathogenesis of essential hypertension᎐a perspective. Nippon-Rinsho 50, 250᎐256 Žin Japanese.. Ohba, R., 1996. Physiologically functional foods with a special view to new hen’s egg products. Denpun-toShokuhin 21, 11᎐18 Žin Japanese.. Ohba, R., Ide, S., Yoshida, A., Nagata, Z., Ueda, S., 1995. Effects of mixed enzyme preparations of proteins for separating egg yolk oil from a fresh yolk suspension. Biosci. Biotech. Biochem. 59, 949᎐951. Ohba, R., Nakashima, Y., Ueda, S., 1994a. Solubilization of proteins for clarification of egg yolk powder suspensions and separation of lipids using enzymes. J. Ferment. Bioeng. 78, 197᎐199. Ohba, R., Nakashima, Y., Ueda, S., 1994b. Separation and formation of egg yolk oil by solubilizing the lipoproteins of spray-dried egg yolk into polypeptides. Biosci. Biotech. Biochem. 58, 2159᎐2163. Ohba, R., Teramoto, Y., Ueda, S., 1993. Clarification of spray-dried egg yolk suspensions and solubilization of proteins from lipoproteins. J. Food Sci. 58, 307᎐309. Okamoto, A., Hanagata, H., Kawamura, Y., Yanagida, F., 1995a. Anti-hypertensive substances in fermented soybean, Natto. Plant Foods Hum. Nutr. 47, 39᎐47. Okamoto, A., Hanagata, H., Matsumoto, E., Kawamura, Y., Yanagida, F., 1995b. Angiotensin I-converting enzyme inhibitory activities of various fermented foods. Biosci. Biotech. Biochem. 59, 1147᎐1149. Okamoto, A., Matsumoto, E., Iwashita, A., Yusuhara, T., Kawamura, Y., Koizumi, Y., Yanagida, F., 1995c.
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Angiotensin I-converting enzyme inhibitory action of fish sauce. Food Sci. Technol. 1, 101᎐106. Okamoto, K., Suzuki, A., Sunano, S., 1988. Alterations of sympathetic transmission and vascular muscle reactivities in spontaneous hypertension. Prog. Hypertens. 1, 47᎐63. Okunishi, H., Kawamoto, T., Kurobe, Y., Oka, Y., Ishii, K., Tanaka, T., Miyazaki, M., 1991. Pathogenetic role of vascular angiotensin converting enzyme in the spontaneously hypertensive rat. Clin. Exp. Pharmacol. Physiol. 18, 649᎐659. Pahor, M., Guralink, J.M., Furberg, C.D., Carbonin, P., Havilik, R.J., 1996. Risk of gastrointestinal hemorrhage with calcium antagonists in hypertensive persons over 67 years old. Lancet 347, 1061᎐1065. Psaty, B.M., Hechbert, S.R., Koepsell, T.D et al., 1995. The risk of myocardial infarction associated with antihypertensive drug therapy. JAMA 274, 620᎐625. Saito, Y., Wanezaki, K., Kawato, A., Imayasu, S., 1994a. Structure and activity of angiotensin I converting enzyme inhibitory peptides from Sake and Sake lees. Biosci. Biotech. Biochem. 48, 1767᎐1771. Saito, Y., Wanezaki, K., Kawato, A., Imayasu, S., 1994b. Antihypertensive effects of peptide in Sake and its by-products on spontaneously hypertensive rats. Biosci. Biotech. Biochem. 58, 812᎐816. Shiono, K., Sokabe, H., 1976. Renin᎐angiotensin system in spontaneously hypertensive rats. Am. J. Physiol. 231, 1295᎐1297. Yotsumoto, H., 1987. Angiotensin converting enzyme. Med. Practice 4, 217᎐219 Žin Japanese..
Antihypertensive effect of ACE inhibitory oligopeptides from chicken egg yolks Hiroshi Yoshii a,1, Norihide Tachi a,U , Riichiro Ohbab, Osamu Sakamuraa , Hidemaro Takeyamaa , Toru Itani a a
Department of Hygiene and Occupational Health, Nagoya City Uni¨ ersity Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan b Department of Applied Microbial Technology, Faculty of Engineering, Sojo Uni¨ ersity, 4-22-1, Ikeda, Kumamoto 860-0082, Japan Received 31 March 2000; received in revised form 8 September 2000; accepted 21 September 2000
Abstract Oligopeptides of 1 KDa or less were obtained by hydrolysis of chicken egg yolks with a crude enzyme, and by dialysis with a semipermeable membrane filter. Since the extracted peptides had an inhibitory action on the activity of angiotensin I-converting enzyme ŽACE. in vitro, they were orally administered at 20, 100 and 500 mgrkg body weight to spontaneously hypertensive rats ŽSHR. for 12 weeks to analyze the physiological role on cardiovascular functions. The administered oligopeptides suppressed the development of hypertension at all dosages. After 12 weeks at 500 mgrkg body weight, the values for systolic, mean, and diastolic blood pressure were approximately 10% less in SHRs administered than controls. Furthermore, serum ACE activity of the peptide-administered groups was significantly lower than that of the control group in a dose-related manner. Our results imply that oligopeptides extracted from hen’s egg yolks could potentially suppress the development of hypertension in SHR, and this effect might be induced by the inhibition of ACE activity. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Angiotensin I-converting enzyme ŽACE.; Antihypertensive effect; Hypertension; Systolic blood pressure; Oligopeptides; Physiological functional food; Egg yolk; Spontaneously hypertensive rat ŽSHR.
1. Introduction More than 20 million people suffer from hypertension in Japan alone, and 90% or more of them are patients with essential hypertension ŽMuratani and Fukiyama, 1992.. Essential hypertension has been considered a disease induced by various U
Corresponding author. Tel.: q81-52-853-8171; fax: q8152-859-1228. 1 Present address: Fine Chemical Laboratory Co. Ltd., 5-7-8 Shimoshinjo, Higashiyodogawa-ku, Osaka 533-0021, Japan
factors relating with life-style and heredity ŽMuratani and Fukiyama, 1992.. Blood pressure should be controlled in these hypertensive patients, because high blood pressure is a wellknown risk factor for cerebro- and cardiovascular disorders ŽLevy et al., 1991.. At the same time, it is necessary to properly regulate the blood pressure, since the sudden and excessive declines in blood pressure have also been documented to increase the risks in diseases other than hypertension ŽCruickshank et al., 1987; Furberg et al., 1995; Psaty et al., 1995; Pahor et al., 1996.. There
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 1 7 2 - 1
28
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
may be some methods to moderately control the blood pressure in hypertensive patients. Of these, a nutritional approach is suspected to be relatively easy and effective to control the blood pressure without any excessive decrease, and particularly, physiologically functional foods may be candidates in the nutritional approach. Physiological functions of food constituents have been elucidated, and it is important to use such food constituents for the maintenance and promotion of health ŽKawakami et al., 1995.. For instance, some peptides have recently been reported to play an important role in controlling the development of hypertension by regulating the renin᎐angiotensin system ŽShiono and Sokabe, 1976; Markov et al., 1990; Haws et al., 1994; Ashida et al., 1996.. Also, several studies have shown an antihypertensive effect of angiotensin I-converting enzyme ŽACE. inhibitory peptides derived from soy sauce, fish sauce, tuna, Japanese sake, fermented soybeans and others ŽMaruyama et al., 1985; Kohama et al., 1988; Maruyama et al., 1989; Kinoshita et al., 1993; Matsumura et al., 1993; Saito et al., 1994a,b; Kawakami et al., 1995; Matsufuji et al., 1995; Okamoto et al., 1995a,b,c.. It has been assumed from these findings that such peptides could be utilized to control the blood pressure especially among people with high risk of essential hypertension. In this study, we separated ACE inhibitory oligopeptides with a commercial crude enzyme from chicken eggs, and aimed to show the inhibitory influence of the oligopeptides on the development of hypertension in SHR. Through the experiments, we tried to evaluate the availability of the oligopeptides as a physiologically functional food to control blood pressure.
2. Methods 2.1. Preparation of oligopeptides Chicken egg yolks were hydrolyzed with a commercial crude enzyme, according to the method of Ohba et al. Ž1993, 1994a,b, 1995.. Namely, 25% yolk solution diluted with distilled water, 0.05 M lactic acid-sodium lactate buffer ŽpH 2.5., and Newlase F solution Žderived from the genus Rhizopus, with an activity equivalent to 18 IUrl of protease and 24 IUrl of lipase, Amano Pharmaceutical Co. Ltd., Tokyo, Japan. were
mixed at a ratio of 4:5:1. The solution was incubated for 5 h at 50⬚C while rotating at 60 rev.rmin. The reaction was terminated by inactivating the enzyme at 100⬚C in a water bath for 5 min. Then, the crude solution was filtrated by using a 0.45 m-membrane filter with a nominal cut-off of 1 kDa. The filtrate was powdered after lyophilization to obtain granular oligopeptides. Using this powder, the effects on the cardiovascular functions were evaluated in vivo. We also treated the filtrate with commercial proteases to know whether the digestion with gastrointestinal enzymes, when administered into the alimentary canal, would alter the ACE inhibitory action of acquired oligopeptides. The filtrate was treated at 37⬚C with either pepsin Žprotease activity: 14 IUrl, Nacalai-Tesque Inc., Tokyo, Japan., trypsin Žprotease activity: 24 IUrl, Sigma Chemical Co., St. Louis, MO., chymotrypsin Žprotease activity: 26 IUrl, Sigma Chemical Co.., or all of these for 5 h. Following the incubation, the enzymes were inactivated at 100⬚C for 5 min, and the solution was filtered again using a 0.45-m membrane filter. The filtrate was freeze-dried. 2.2. Measurement of ACE inhibitory acti¨ ity The 50% inhibitory concentration of ACE activity ŽIC 50 . was assayed in the obtained oligopeptides and other peptides with the treatment by endogenous enzymes, according to the modified method of Cushman et al. ŽCushman and Cheung, 1971; Lieberman, 1975.. 2.3. Effects of oligopeptides on the cardio¨ ascular functions in SHR. Forty male SHR ŽSHRrIzm, supplied from Disease Model Cooperative Research Association, Kyoto, Japan. were housed individually in polycarbonate cages at 23 " 0.5⬚C with a humidity of 55 " 5%, and 12 h lightr 12 h dark Žlights on 0800 h.. This study was carried out according to the guidelines for the animal experimentation of Nagoya City University Medical School. Animals were assigned at 5 weeks of age to one of the following groups; a control group orally administered only water at 5 mlrkg body weightrday, and three groups of animals administered the oligopeptides at 20, 100 or 500 mgrkg b.w.rday. The oligopeptides were dissolved in water to adjust the concentration at 4, 20 and 100
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27 ᭚133
mgrml for the groups of 20, 100 and 500 mgrkg b.w. administrations, respectively. The injection volume was maintained at 5 mlrkg b.w. in all groups. The administration was performed every day for 12 weeks. Systolic and mean blood pressure was measured once a week by a tail cuff method using BP-98A ŽSoftron, Tokyo, Japan.. Briefly, a tail was put through a rubber cuff in animals held in a plastic tube at 35⬚C, and the cuff was inflated with air. The pressure level at which first pulse appeared after blood flow had been interrupted with the inflated cuff, was designated systolic blood pressure, and the level of maximum amplitude of the pulse wave during measurement was considered the mean blood pressure. Diastolic blood pressure was automatically calculated by the equipment. The measurement was started 1 h or later after the daily administration. Preliminary results revealed that blood pressure was not influenced when the measurement was performed later than 1 h after the oral injection of the oligopeptides or water Ždata not shown.. The difference between the weights of food given on a certain day and what remained 7 days later was designated the weekly food intake. The water intake was measured in the same way as food intake. On the 87th day of the experiment, blood was collected from the vena cava in SHR under pentobarbital anesthesia. After the blood sampling, animals were killed by heart puncture to weigh heart, liver, kidneys and lungs. Collected blood was clotted to obtain the serum. ACE activity was measured according to the method of Kasahara and Ashihara Ž1981. and Yotsumoto Ž1987.. Blood pressure, and food and water intakes were analyzed by two-way analysis of variance ŽANOVA, time = treatment ., and by StudentNewman-Keuls post hoc test. Student-NewmanKeuls test was also employed to show a difference in the IC 50 value, serum ACE activity and organ weight between groups. A statistical significance was considered at P - 0.05.
3. Results 3.1. ACE inhibitory action in ¨ itro The oligopeptides obtained with a crude enzyme exerted an ACE inhibitory action, and the
29
Table 1 ACE inhibitory activities of the oligopeptides alone or with additional treatment of some proteases a IC50 Žmgrml. b
O Pc Td Ce PTCf
1.22" 0.08 1.33" 0.15 1.15" 0.16 1.33" 0.19 1.23" 0.08
a
Each value represents mean "S.D. of 5 samples. There is no significant difference between groups. b O: the oligopeptides powder without additional protease treatment. c P: pepsin treatment powder. d T: trypsin treatment powder. e C: chymotrypsin treatment powder. f PTC: Powder treated with all three enzymes.
50% ACE inhibitory concentration ŽIC 50 . was 1.22 mgrml ŽTable 1.. There was no significant difference in IC 50 between the oligopeptides and the other peptides treated individually or with mixture of pepsin, trypsin, and chymotrypsin. 3.2. Influences of the oligopeptides on physiological ¨ ariables in SHR
Systolic, mean and diastolic blood pressures ŽSBP, MBP and DBP, respectively. increased in all groups with time ŽFigs. 1᎐3.. All blood pressures were lower from the 3rd weeks of administration in the oligopeptide-injected groups than in the control. At the 12th week, the blood pressure values were 4᎐8% less in the groups administered the oligopeptides than the control. Particularly, two-way ANOVA revealed a statistical significance in SBP in time, treatment and their interaction Žtime; F s 273.9, d.f.s 12, P - 0.001, treatment; F s 8.597, d.f.s 3, P - 0.001, and time = treatment; F s 1784, d.f.s 36, Ps 0.004.. There were significant differences in SBP between the group administered oligopeptides at the highest dose and the control from the 3rd to 12th weeks except at the 9th and 10th weeks. ANOVA failed to show a statistical significance in the interaction between time and treatment in both MBP Ž F s 1.320, df s 36, Ps 0.107. and DBP Ž F s 1.003, d.f.s 36, Ps 0.468., although there were significant differences in time and treatment ŽMBP; F s 257.90, d.f.s 12, P - 0.001 in time, and F s 7.544, d.f.s 3, P - 0.001 in treatment, and DBP; F s 18592, d.f.s 12, P - 0.001 in time, and F s
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
30
Fig. 1. Changes in systolic blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Each point represents a mean " SEM of 10 animals. Two-way ANOVA revealed a statistical significance in time, treatment, and their interaction Ž P - 0.005, see text...U Significantly different Ž P 0.05. from the control, detected by post hoc analysis.
6.537, d.f.s 3, P - 0.001 in treatment .. No significant difference was shown in body weight ŽFig. 4., weekly food or water intake ŽFigs. 5 and 6., or organ weights ŽTable 3. between groups. The serum ACE activity value was significantly 6᎐13% lower in the oligopeptide groups than that of the control ŽTable 2. in a dose-dependent manner.
4. Discussion The oligopeptides obtained by hydrolyzing
Fig. 2. Changes in mean Ž" SEM. blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Two-way ANOVA revealed a statistical significance in time, treatment Ž P - 0.001., although not in their interaction.
Fig. 3. Changes in diastolic blood pressure levels in SHRs administered the oligopeptides for 12 weeks. Two-way ANOVA revealed a statistical significance in time, and treatment Ž P - 0.001., although not in their interaction. Table 2 Serum ACE activity in SHR administered oligopeptides for 12 weeks a Dose
Serum ACE activity ŽIUrl.
Control 20 mgrkg 100 mgrkg 500 mgrkg
42.2" 2.15 U 39.8" 2.12 U 38.8" 2.12 U 36.6" 3.09
a U
Each value represents mean "S.D. of 10 animals. Significantly different from the control at P - 0.05.
chicken egg yolks had an inhibitory effect on ACE activity in vitro in the present study. The IC 50 value of the peptides was 1.22 mgrml. The IC 50 was not changed even after the oligopeptides had been incubated with other gastrointestinal enzymes such as pepsin, trypsin and chymotrypsin. We therefore conclude that the
Fig. 4. Changes in mean Ž" SEM. body weight in SHRs administered the oligopeptides for 12 weeks.
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27 ᭚133
31
Table 3 Percent organ weight on the 87th day of the experiment in SHRs administered the oligopeptidesa Dose
Heart Ž%.
Liver Ž%.
Kidneys Ž%.
Control 20 mgrkg 100 mgrkg 500 mgrkg
0.419" 0.030 0.409" 0.022 0.412" 0.026 0.413" 0.010
3.35" 0.313 3.46" 0.337 3.36" 0.289 3.29" 0.152
0.843" 0.032 0.838" 0.033 0.831" 0.019 0.830" 0.029
a Percent weight to body weight. Each value represents percent weight to body weight mean "S.D. of 10 animals.
Fig. 5. Changes in weekly food intake in SHRs administered the oligopeptides for 12 weeks.
oligopeptides were not further hydrolyzed by major digestive proteases. In excess of 90% of the powder was peptides and approximately 5% was carbohydrates. One of the authors ŽR.O.. is proceeding to purify the powder, and it has become clear that the substances inhibiting the ACE activity in vitro are oligopeptides Ždata not shown.. The IC 50 value of the oligopeptides was similar to the ACE inhibiting activity of other crude peptides reported in earlier studies Ž1.18᎐1.86 mgrml, Ohba, 1996.. We expect that additional purification will result in stronger ACE inhibiting peptides, because the peptides used in the present study were extracted very crudely. The level of SBP, MBP and DBP was generally lower from the 3rd week of the experiment in animals administered the oligopeptides than in the control. The increase in blood pressures was reduced in a dose-dependent manner from the 3rd to 6th weeks of the experiment. These results
Fig. 6. Changes in weekly water intake in SHRs administered the oligopeptides for 12 weeks.
suggest that the oligopeptides play a role in suppressing or delaying the development of increases in blood pressures. Since the value of serum ACE activity was significantly decreased in the oligopeptide-treated groups, compared with the control group, the peptides appear not to be digested in the alimentary canal, as expected from the in vitro study mentioned above. ACE is an enzyme in the renin᎐angiotensin system, and converts an inactive peptide, angiotensin I to angiotensin II which has a hypertensive function ŽMurakami, 1993.. ACE also inactivates a vasodilator, bradykinin ŽMurakami, 1993.. Inhibition of ACE activity therefore results in decrease of blood pressure. Based on the concept, the egg yolk oligopeptides exerted an antihypertensive action possibly by inhibiting the ACE activity. However, a constant dose-dependent relationship was not observed throughout the entire period of the experiment in this study. This may partly be due to an inadequate inhibition of tissue ACE activities, especially aortic ACE activity, because it has been indicated that aortic ACE activity was more important in SHRs to increase blood pressure than that in serum, and that the inhibition of aortic ACE activity reduced the blood pressure ŽOkunishi et al., 1991.. Also, many systems other than renin᎐angiotensin system contribute to control the blood pressure, such as autonomic nervous system, endothelin system, and nitric oxide system. It is possible that compensatory activation of these systems could reduce the difference between the high and low dosage groups in this study. We have not measured the activity in the aorta in the present study, nor did we observe other systems. Thus, the precise mechanism remains unclear, and further analysis is needed. Neither weekly food nor water intake was dif-
32
H. Yoshii et al. r Comparati¨ e Biochemistry and Physiology Part C 128 (2001) 27᎐33
ferent among groups. Body weight was also similar in all groups. These findings are likely to imply that the oligopeptides administered to animals had no effect on the gross metabolic conditions in this study. The SBP value was higher in the oligopeptide groups than that in normotensive rats reported in the earlier studies Žapprox. 180 mmHg at 17 weeks of age in the group at the highest dose in this study, and 130 mmHg in normotensive WKY rats, Okamoto et al., 1988.. The finding indicates that the administered oligopeptides had a relatively moderate action on suppressing the blood pressure increase. This characteristic of the peptides, together with little effect on the general physical condition, may be potentially useful as a physiologically functional food, because excessive reduction of blood pressure has been documented to induce myocardial infarction ŽPsaty et al., 1995., and aggravation of survival rate ŽCruickshank et al., 1987; Furberg et al., 1995.. In conclusion, we have shown that oligopeptides obtained by hydrolyzing chicken egg yolks inhibit an ACE action in vitro as well as in vivo, and prevent the development of hypertension in SHRs. These results suggest that the increase in blood pressure could be partially inhibited by taking the peptides derived from common foods. Finally, we expect that such oligopeptides could be used as a physiologically functional food to control blood pressure in patients with essential hypertension, although further investigations should be undertaken to determine the amino acid sequence, mechanisms of action, and potential side effects. References Ashida, T., Yoshimi, H., Kawano, Y., Matsuoka, H., Omae, T., 1996. Effects of dietary salt and driven Ca pump and NarCa exchange in aortic rings of Dahl rats. Clin. Exp. Hypertens. 18, 851᎐871. Cruickshank, J., Thorp, J.M., Zacharias, F.J., 1987. Benefits and potential harm of lowering high blood pressure. Lancet 1, 581᎐584. Cushman, D.W., Cheung, H.S., 1971. Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637᎐1648. Furberg, C.D., Psaty, B.M., Meyer, J.V., 1995. Nifedipine; dose-related increase in patients with coronary heart disease. Circulation 62, 1326᎐1331.
Haws, R.M., Shaul, P.W., Arant, B.S., Atiyeh, B.A., Seikaly, M.G., 1994. Glomerular losartan ŽDup753.sensitive angiotensin II receptor density is increased in young spontaneously hypertensive rats. Pediatr. Res. 36, 671᎐676. Kasahara, Y., Ashihara, Y., 1981. Colorimetry of angiotensin I-converting enzyme activity in serum. Clin. Chem. 27, 1922᎐1925. Kawakami, A., Kayahara, H., Tadasa, K., 1995. Taste evaluations of angiotensin I-converting enzyme inhibitors, Leu-Lys-Tyr analogues. Biosci. Biotech. Biochem. 59, 709᎐710. Kinoshita, E., Yamakashi, J., Kikuchi, M., 1993. Purification and identification of an angiotensin I-converting enzyme inhibitor from soy sauce. Biosci. Biotech. Biochem. 57, 1107᎐1110. Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M., Mimura, T., 1988. Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochem. Biophys. Res. Commun. 155, 332᎐337. Levy, D., Garrison, R.J., Savage, D.D., Kannel, W.B., Castelli, W.P., 1991. Prognostic implication of echocardiographically determined left ventricular mass in the Framingham Study. N. Engl. J. Med. 322, 1561᎐1566. Lieberman, J., 1975. Elevation of serum angiotensin converting enzyme ŽACE. level in sarcoidosis. Am. J. Med. 59, 356᎐372. Markov, M., Zhelyazkov, D., Markova, M., 1990. Influence of isoteoline on the angiotensin-converting enzyme activity. Med. Arch. 27, 45᎐49. Maruyama, S., Miyoshi, S., Kaneko, T., Tanaka, H., 1989. Angiotensin I-converting enzyme inhibitory activities of synthetic peptides related to the tandem repeated sequence of a maize endosperm protein. Agric. Biol. Chem. 53, 1077᎐1081. Maruyama, S., Nakagomi, K., Tomizuka, N., Suzuki, H., 1985. Angiotensin I-converting enzyme inhibitory derived from an enzymatic hydrolysate of casein. II. Isolation and bradykinin-potentiating activity on the uterus and the ileum of rats. Agric. Biol. Chem. 49, 1405᎐1409. Matsufuji, H., Matsui, T., Ohshige, S., Kawasaki, T., Osajima, K., Osajima, Y., 1995. Antihypertensive effects of angiotensin fragments in SHR. Biosci. Biotech. Biochem. 59, 1398᎐1401. Matsumura, N., Fujii, M., Takeda, Y., Sugita, K., Shimizu, T., 1993. Angiotensin I-converting enzyme inhibitory peptides derived from bonito bowels autolysate. Biosci. Biotech. Biochem. 57, 695᎐697. Murakami, K., 1993. Biochemical and molecular biological studies on renin᎐angiotensin system. Seikagaku 65, 260᎐276 Žin Japanese..
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Muratani, H., Fukiyama, K., 1992. Pathogenesis of essential hypertension᎐a perspective. Nippon-Rinsho 50, 250᎐256 Žin Japanese.. Ohba, R., 1996. Physiologically functional foods with a special view to new hen’s egg products. Denpun-toShokuhin 21, 11᎐18 Žin Japanese.. Ohba, R., Ide, S., Yoshida, A., Nagata, Z., Ueda, S., 1995. Effects of mixed enzyme preparations of proteins for separating egg yolk oil from a fresh yolk suspension. Biosci. Biotech. Biochem. 59, 949᎐951. Ohba, R., Nakashima, Y., Ueda, S., 1994a. Solubilization of proteins for clarification of egg yolk powder suspensions and separation of lipids using enzymes. J. Ferment. Bioeng. 78, 197᎐199. Ohba, R., Nakashima, Y., Ueda, S., 1994b. Separation and formation of egg yolk oil by solubilizing the lipoproteins of spray-dried egg yolk into polypeptides. Biosci. Biotech. Biochem. 58, 2159᎐2163. Ohba, R., Teramoto, Y., Ueda, S., 1993. Clarification of spray-dried egg yolk suspensions and solubilization of proteins from lipoproteins. J. Food Sci. 58, 307᎐309. Okamoto, A., Hanagata, H., Kawamura, Y., Yanagida, F., 1995a. Anti-hypertensive substances in fermented soybean, Natto. Plant Foods Hum. Nutr. 47, 39᎐47. Okamoto, A., Hanagata, H., Matsumoto, E., Kawamura, Y., Yanagida, F., 1995b. Angiotensin I-converting enzyme inhibitory activities of various fermented foods. Biosci. Biotech. Biochem. 59, 1147᎐1149. Okamoto, A., Matsumoto, E., Iwashita, A., Yusuhara, T., Kawamura, Y., Koizumi, Y., Yanagida, F., 1995c.
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Angiotensin I-converting enzyme inhibitory action of fish sauce. Food Sci. Technol. 1, 101᎐106. Okamoto, K., Suzuki, A., Sunano, S., 1988. Alterations of sympathetic transmission and vascular muscle reactivities in spontaneous hypertension. Prog. Hypertens. 1, 47᎐63. Okunishi, H., Kawamoto, T., Kurobe, Y., Oka, Y., Ishii, K., Tanaka, T., Miyazaki, M., 1991. Pathogenetic role of vascular angiotensin converting enzyme in the spontaneously hypertensive rat. Clin. Exp. Pharmacol. Physiol. 18, 649᎐659. Pahor, M., Guralink, J.M., Furberg, C.D., Carbonin, P., Havilik, R.J., 1996. Risk of gastrointestinal hemorrhage with calcium antagonists in hypertensive persons over 67 years old. Lancet 347, 1061᎐1065. Psaty, B.M., Hechbert, S.R., Koepsell, T.D et al., 1995. The risk of myocardial infarction associated with antihypertensive drug therapy. JAMA 274, 620᎐625. Saito, Y., Wanezaki, K., Kawato, A., Imayasu, S., 1994a. Structure and activity of angiotensin I converting enzyme inhibitory peptides from Sake and Sake lees. Biosci. Biotech. Biochem. 48, 1767᎐1771. Saito, Y., Wanezaki, K., Kawato, A., Imayasu, S., 1994b. Antihypertensive effects of peptide in Sake and its by-products on spontaneously hypertensive rats. Biosci. Biotech. Biochem. 58, 812᎐816. Shiono, K., Sokabe, H., 1976. Renin᎐angiotensin system in spontaneously hypertensive rats. Am. J. Physiol. 231, 1295᎐1297. Yotsumoto, H., 1987. Angiotensin converting enzyme. Med. Practice 4, 217᎐219 Žin Japanese..