56(5) 12.fm

Size: px

Start display at page:

Download "56(5) 12.fm"

태곤 봉
5 years ago
Views:

1 약학회지제 56 권제 5 호 309~313 (2012) Yakhak Hoeji Vol. 56, No. 5 종설 Isoflavone-Free 검은콩펩타이드의항고혈압및 ACE 활성억제효과 안창원 신동석 박수현 홍순선 * 강주희 ** 박창신 **,# ( 주 ) 농심 R&BD 센터, * 인하대학교의과대학의학전문대학원특성화교실임상시험센터, ** 약리학교실의약물독성연구소 (Received September 7, 2012; October 26, 2012; Accepted October 30, 2012) Antihypertensive and ACE Inhibitory Effects of Novel Isoflavone-free Black Soy Peptide Mixture Chang-Won Ahn, Dong-Seok Shin, Soo-Hyun Park, Soon-Sun Hong*, Ju-Hee Kang** and Chang-Shin Park**,# R&BD Center, NongShim Co. Ltd., Seoul , Korea *Department of Biomedical Sciences and Clinical Research Center, Inha University, **Department of Pharmacology and Medicinal Toxicology Research Center, Center for Advanced Medical Education, Inha University College of Medicne by BK-21 Project, Inha University, Incheon Korea Abstract Antiobesity, hypotriglyceridemic and antihypertensive activities of isoflavone-free peptide mixture (black soybean peptide, BSP) were reported in our previous experiments. In the present study, angiotensin converting enzyme inhibitory (ACEi) activity was decreased in the aorta tissues of spontaneously hypertensive rats (SHRs) treated with BSP (1% in drink water) for 4 weeks, but not in serum. BSP administration significantly decreased ACE activity by 17.5% (from 33.2±4.5 to 27.4±1.96 munit/mg, p=0.0013) in aorta tissue hydrolysate. BSP treatment also decreased significantly mean blood pressure (BP) (from 213.0±16.96 to 184.0±6.53 mmhg, p<0.0001) as expected. These results indicate that BSP has antihypertensive activity as well as ACEi activity. Keywords isoflavone-free peptide mixture, ACE, SHR, antihypertension 혈압조절기작의 Renin-Angiotensin계에서 angiotensin converting enzyme(ace, EC ) 은혈관수축을유도하고, aldosterone 물질분비를촉진하여혈압상승을촉진하는효소이다. 1) 또한 ACE는혈압강하작용을가지는 bradykinin과같은물질을분해하여불활성화시키는한편, 불활성상태의 angiotensin- I을절단하여혈압상승작용을하는 angiotensin-ii로활성화시킴으로써혈압상승을유도시키는것으로알려져있다. 따라서, ACE 활성억제효과를나타내는믈질은 captopril과같은항고혈압제재로사용되고있다. 2) 식품유래 ACE 억제활성을나타내는성분은화학적으로합성한성분에비하여안전성측면에서장점을가진다는중요한의미를가진다. Maruyama 등 3) 이우유유래펩타이드의 ACE 저해활성을보고한이후, 다양한종류의단백질분해물과펩타이드 # 본논문에관한문의는저자에게로 ( 전화 ) ( 팩스 ) ( ) parkshin@inha.ac.kr 에서 ACE 활성억제효과가보고되었다. 특히, Sekiya 등 4) 은우유의트립신분해물의섭취시인체내 ACE 저해활성을확인하였고, 정어리펩타이드, 5) 발효유유래펩타이드, 6) 밀단백펩타이드, 7) 간장유래펩타이드, 8) 된장유래펩타이드 9) 등다수의펩타이드에서 ACE 활성억제효과가보고되었고일부는상품화되기도하였다. 콩은동양에서된장, 간장, 청국장과같은발효식품이나두부, 두유와같은다양한콩가공식품의형태로섭취되고있으며, 많은연구에서이들은만성질환을예방하는데도움을주는것으로알려져왔다. 10) 특히, 나토, 된장, 간장, 미소와같은콩발효식품으로부터다양한종류의 ACE 저해펩타이드가보고되어콩단백유래펩타이드의혈압조절가능성을시사하였다. 8-14) 최근, 검은콩 (Rhynchosia volubilis) 유래펩타이드의다양한기능성이보고되었다. ( 주 ) 농심에서분리및정제하여개발한 BSP 는 15) 체중조절및혈당조절효과가확인되었으며, 작용기전으로 AMPK(AMP-activated protein kinase) 활성촉진과 ER stress/ 인슐린저항성의개선효과기전이각각제시되었다. 16,17) 또한, 309

2 310 안창원 신동석 박수현 홍순선 강주희 박창신 BSP의항고혈압활성을 SHR 동물모델에서가능한 hemeoxygenase-1(ho-1) 작용기전제시와함께확인하였다. 18) 본연구에서는우리의이전결과에서확인한 BSP의항고혈압활성의새로운기전으로써펩타이드의 ACE 활성억제효과를복부대동맥조직에서입증하고, SHR 동물모델에서혈압강하효과와함께제시하였다. 결과통계분석본실험에서는대조군 (n=10) 과실험군 (n=10) 의평균혈압과 ACE 활성도측정치에서두군의차이와통계적유의성을위하여 Unpaired t test 방법을사용하였다. P 값이 0.05 이하 (95% confidence Interval) 인경우를두군간유의한차이로평가하였으며모든값을평균 ± 표준편차로제시하였다. 재료및실험방법 실험결과및고찰 실험재료검정콩 (Rhynchosia volubilis) 펩타이드는 ( 주 ) 농심 ( 서울, 한국 ) 에서분리정제하여개발한 isoflavone-free peptide mixture(bsp: 50.7% 조단백량, 4.6% 수분량 ) 10) 를동물실험에사용하였다. 본실험에서조직및혈청내 ACE저해활성측정을위하여사용한 ACE(8.2 unit/g) 및기질 (HHL, hippuryl-l-histidyl-l-leucine) 은 Sigma-Aldrich(St. Louis, MO) 에서구입하여사용하였다. 혈압의변화 SHR 모델을이용한동물실험에서는 BSP 또는물을섭취한실험군과대조군을분리하여꼬리에서평균혈압을측정하였다. Fig. 1에서보는바와같이, 1% BSP를 4주간처리한후측정된실험군의평균혈압은 184.0±6.53 mmhg로대조군에서측정된평균혈압 213.0±16.96 mmhg보다유의하게감소하였다 (P< ). 이는 BSP의강력한혈압강하효과를의미한다. 동물모델실험동물모델실험과정은인하대학교의과대학 Animal Ethics Committee에서승인되었고, 미국국립보건원 (NIH, National Institutes of Health) 과 Korean Academy of Medical Science에의해제시된동물실험에대한가이드에따라수행하였다. 14주된수컷 SHR(n=20, 310±10 g) 을아산생명연구센터 ( 서울, 한국 ) 에서구입하여 1주일간안정기간을두고실험을진행하였다. 동물모델 SHR을검정콩펩타이드 1% 수용액섭취군 ( 실험군, n=10) 과일반물을복용하게한대조군 (n=10) 으로나누어 4주간자유롭게섭취할수있도록두었다. BSP 또는물의섭취를마친 19주된동물들의혈압은 tail-cuff 방법 19) 으로측정하였으며, 실험이종료된후안지오텐신전환효소 (angiotensin converting enzyme, ACE) 저해활성을측정하기위한복부대동맥 (abdominal aorta) 조직과혈청을확보하였다. ACE 활성의변화 BSP를처리한실험군의복부대동맥 (abdominal aorta) 에서측정된 ACE 활성정도는 27.4±1.96 munit/mg로대조군배대동맥조직에서측정된 33.2±4.5 munit/mg보다유의하게감소하였다 (Fig. 2, P=0.0013). 이는 BSP의 ACE 활성억제효과를의미한다. 그러나실험군과대조군조직내 ACE 활성도는차이는있는반면, Serum에서는두군간유의한차이가없었다 (Fig. 3). 최근에우리는 ( 주 ) 농심에서개발한 BSP의 in vivo 뿐아니라 in vitro 효과에서도다양한생리활성효과와가능한작용기전들을보고한바있다 ) 특히, 즉, 신등 (2012) 은 SHR 동물모델과인체정상혈관내피세포인 HUVECs을재료로 BSP의항고혈압효과와가능한작용기전으로 heme-oxygenase-1(ho-1) 의유도발현결과를제시하였다. 18) 항산화효소군중하나인 HO-1 ACE 억제활성측정 ACE 억제활성측정은 Cushman과 Cheung의방법에따랐다. 20) 간략하게기술하면, 복부대동맥조직및혈청을이용하여 sodium borate 완충액을첨가하여현탁시료를만들고, 효소를추출하였으며원심분리하여상등액을 ACE 조효소로사용하였다. HHL 기질, ACE 조효소및시료 (20~30 mg/ml) 또는증류수를동일용량비율로혼합하여반응시킨다. HCl을이용하여시료반응을정지시키고 ethyl acetate를첨가하고원심분리하여상등액을확보한다. 확보된시료들은가열하여건조시키고, 증류수로다시용해시킨후 228 nm에서흡광도를측정하였다. 이때 rabbit lung 유래 ACE(Sigma) 로측정한표준곡선을이용하여흡광도를 ACE 활성으로환산하였다. Fig. 1 In vivo effects of BSP on the mean blood pressure (BP) in SHR model (n=10 each group, ***P<0.0001). J. Pharm. Soc. Korea

3 항고혈압및 ACE 억제효과 311 Fig. 2 In vivo effects of BSP on the ACE activity in aorta tissues of SHR model (n=10 each group, **P<0.01). Fig. 3 In vivo effects of BSP on the ACE activity in serum of SHR model (n=10 each group, P>0.05). 의혈관내유도발현은 carbon monoxide(co)-cgmp의생성촉진뿐아니라기관손상과관련된세포신호전달기전들에서세포및기관보호활성이잘알려져있다 ) 더욱이 HO-1의혈압조절의주요활성기전은우리의 BSP의연구결과와이전의여러연구결과들에서제시되고있다 ) 본연구결과에서확인된 BSP의 ACE 활성억제효과와 HO-1 의유도발현기전은모두가능한혈압조절기전으로제시될뿐아니라입증되고있으며, 특히 ACE 억제활성은 captopril과같은 ACE 억제제약제와같이항고혈압기전의가장주요경로임을알수있다 ) Fig. 1과 2에서보는바와같이, 4주동안 ACE 활성에대한 1% 의 BSP 활성억제효과는 17.5% 로다소낮게측정되지만유의성있는차이의결과로 BSP의항고혈압활성에대한새로운기전을제시하는것이다. 더욱이복부대동맥조직에서는 ACE 활성차이를확인할수있었으나, 혈청내활성의차이는없었던결과도 BSP의혈관조직내 ACE 활성억제효과를반영하고있다. 특히, 혈청내 ACE 활성도는기대한것보다높게측정되었는데, ACE 활성정도에서차이가없었던것은조직간 활성도의차이를의미하는것보다현재실험조건에서의반응정도의차이를반영하는것일수있다. 이와같은결과는 Miguel 등에의해보고된흥미로운결과처럼가수분해된계란흰자의섭취로나타난 ACE 활성에미치는 local 효과로 in vivo 실험시조직간뚜렷한차이를보였고, 특히혈장내에서는처리시간에따라서그활성의차이가뚜렷했다. 여기서또한 local ACE 활성의차이뿐아니라그반응정도에서도조직또는기관에서차이가있음을제시하였다. 33) 우리는본연구의실험군에서확인된 BSP의 ACE 활성억제효과는혈관조직의반응민감도 (sensitivity) 가혈청또는혈장에비하여높음을알수있었고, 이는현재실험조건에서는혈청내차이가없었던 ACE 활성도정도가투여정도뿐아니라투여시간에따라서그효과가나타날수있음도제시할수있다. 우리가섭취하는식품들의분해산물에서생성되는다양한펩타이드및아미노산들은상기에서기술한 HO-1 유도발현과 ACE 활성억제효과의촉진물질로보고되었다. 31,32,34) Met-Tyr, Metphe, captopril 등은 ACE 활성억제효과를나타내고 HO-1의발현도유도하지만서로독립된작용경로로확인되고있다. 즉, 이와같은생리활성펩타이드들은두지경로모두를촉진시키면서항고혈압활성을더강력하게나타내는것이다. Fig. 1에서보는것처럼강력한 BSP의항고혈압효과는 17.5% 정도의 ACE 활성억제만으로설명하기에는부족하지만, 이전 HO-1의연구결과와함께제시된 AMPK-NO-cGMP 활성경로를종합하면 BSP 의항고혈압효과는다양한기전으로서로독립적또는연관된신호전달경로로작용할것으로예측된다. 펩타이드류의 ACE 억제활성은다양한종류가보고되었으며, 콩단백질유래 His-His-Leu 은동물실험에서도그효과가확인된바있다. 9) BSP는분자량이 10 kda 이하의작은크기의펩타이드성분으로되어있고특히, hepta-펩타이드 (IPPGVPY) 의높은농도는 ACE 활성억제효과뿐아니라 HO-1 그리고또다른생리활성능의가능성이충분한것으로판단된다. 또한 BSP 원료로사용한검정콩펩타이드에는 His-His-Lue을포함한새로운펩타이드도있어 ACE 활성을억제하는효과뿐아니라 HO-1과다른경로들의활성기전을통하여본실험에서처럼 SHR에서의혈압감소효과를나타낸것으로추정된다. 결론검정콩펩타이드 BSP(1%) 의 4주간처리는 SHR 고혈압동물모델에서항고혈압활성을나타냈으며, HO-1 유도발현뿐아니라 18) ACE 활성억제효과의새로운기전이확인되어, BSP의혈압강하효과는최소한 HO-1과혈관조직내 ACE 억제활성을포함하는다양한관련기전들이복합적으로작용하는것으로판단된다. Vol. 56, No. 5, 2012

4 312 안창원 신동석 박수현 홍순선 강주희 박창신 감사의말씀 본연구는 ( 주 ) 농심의연구지원으로수행되었으며이에감사드립니다. 참고문헌 1) Saxena, P. R. : Interaction between the renin-angiotensinaldosterone and sympathetic nervous systems. J. Cardiovasc. Pharm. 19, 6 (1992). 2) Kim, J. E., Hwang, K. and Lee, S. P. : ACE inhibitory and hydrolytic enzyme activities in textured vegetable protein in relation to the solid state fermentation period using Bacillus subtilis HA. Food Sci. Biotechnol. 19, 487 (2010). 3) Maruyama, S. and Suzuki, H. : A peptide inhibitor of angiotensin I-converting enzyme in a triptic hydrolysate of casein. Agric. Biol. Chem., 46, 1393 (1982). 4) Sekiya, S., Kobayashi, Y., Kita, E., Imamura, Y. and Toyama. S. : Antihypertensive effects of tryptic hydrolysate of casein on mormotensive and hypertensive volunteers. J. Nutr. Food Sci. 45, 513 (1992). 5) Seiki, E., Osajima, K., Matsui, T. and Osajima, Y. : Separation and purification of angiotensin-i converting enzyme inhibitory peptides from heated sardine meat by treatment with alkaline protease. 일본식품공업학회지 40, 783 (1993). 6) Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S. and Takano, T. : Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. J. Dairy Sci. 78, 777 (1995). 7) Motoi, H. and Kodama, T. : Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides from wheat gliadin hydrolysate. Nahrung 47, 354 (2003). 8) Oka, S. and Nagata, K. : Isolation and characterization of neutral peptides in soy sauce. Agri. Biol. Chem. Tokyo 38, 1185 (1974). 9) Shin, Z. I., Yu, R. N., Park, S. A., Chung, D. K., Ahn, C. W., Nam, H. S., Kim, K. S. and Lee, H. J. : His-His-Leu, an angiotensin I converting enzyme inhibitory peptide deribed from korean soybean paste, Exerts antihypertensive activity in vivo. J. Agric. Food Chem. 49, 3004 (2001). 10) Song, H. N. and Jung, K. S. : Quality characteristics and physiological activities of fermented soybean by lactic acid bacteria. Korean J. Food Sci. Technol. 38, 475 (2006). 11) Okamoto, A. Hanagata, H. Kawamura, Y. and Yanagida, F. : Antihypertensive substances in fermented soybean, natto. Plant Foods Hum. Nutr. 47, 39 (1995). 12) Shin, Z. I., Nam, H. S., Lee, H. J. and Moon, T. H. : Fractionation of angiotensin converting enzyme (ACE) inhibitory peptides from soybean paste. Korean J. Food Sci. Technol. 27, 230 (1995). 13) Teranaka, T., Ezawa, M., Matsuyama, J., Ebine, H. and Kiyosawa, I. : Inhibitory effects of extracts from rice-koji miso, barley-kji miso, and soybean-koji miso on activity of angiotensin I converting enzyme. Nippon Nogeik. Kaishi. 69, 1163 (1995). 14) Rho, S. J., Lee, J. S., Chung, Y. I., Kim, Y. W. and Lee, H. G. : Purification and identification of an angiotensin I converting enzyme inhibitory peptide from fermented soybean extract. Process Biochem. 44, 490 (2009). 15) Rho, S. J., Park, S., Ahn, C. W., Shin, J. K. and Lee, H. G. : Diabetic and hypocholeseroaemic action of black soy peptide in dietary obesic rats. J. Sci. Food Agric. 87, 908 (2007). 16) Jang, E. H., Moon, J. S., Ko, J. H., Ahn, C. W., Lee, H. H., Shin, J. K., Park, C. S. and Kang, J. H. : Novel black soy peptides with antiobesity effects: activation of leptin-like signaling and AMP-activated protein kinase. Int. J. Obes. 32, 1161 (2008). 17) Jang, E. H., Ko, J. H., Ahn, C. W., Lee, H. H., Shin, J. K., Chang, S. J., Park, C, S. and Kang, J. H. : In vivo and in vitro application of black soybean peptides in the amelioration of endoplasmic reticulum stress and improvement of insulin resistance. Life Sci. 86, 267 (2010). 18) Shin, M. K., Kwon, Y, H., Ahn, C. W., Shin, D. S., Park, S. H., Choi, B. H., Hong, S. S., Kang, J. H. and Park, C. S. : Antihypertensive effects of novel isoflavone-free black soy peptide mixture as HO-1 inducer. Yakhak Hoeji 56, 191 (2012). 19) Buag, R. D. : Validation in awake rats of a tail-cuff method for measuring systolic pressure. J. Appl. Physiol. 34, 279 (1973). 20) Cushman, D. W. and Cheung, H. S. : Spectrophotometric assay and properties of the angiotensin converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637 (1971). 21) Brune, B. and Ullrich, V. : Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol. Pharmacol. 32, 497 (1987). 22) Johnson, R. A., Colombari, E., Colombari, D. S., Lavesa, M., Talman, W. T. and Nasjletti, A. : Role of endogenous carbon monoxide in central regulation of arterial pressure. Hypertension. 30, 962 (1997). 23) Ryter, S. W. and Choi, A. M. : Heme oxygenase-1: molecular mechanisms of gene expression in oxygen-related stress. Antioxid. Redox. Signal. 4, 625 (2002). 24) Morita, T. : Heme oxygenase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 25, 1786 (2005). 25) Abraham, N. G. and Kappas, A. : Heme oxygenase and the cardiovascular-renal system. Free. Radic. Biol. Med. 39, 1 (2005). 26) Kim, H. P., Ryter, S. W. and Choi, A. M. : CO as a cellular signaling molecule. Annu. Rev. Pharmacol. Toxicol. 46, 411 (2006). J. Pharm. Soc. Korea

5 항고혈압및 ACE 억제효과 ) Ndisang, J. F., Zhao, W. and Wang, R. : Selective regulation of blood pressure by heme oxygenase-1 in hypertension. Hypertension. 40, 315 (2002). 28) Ndisang, J. F., Tabien, H. E. and Wang, R. : Carbon monoxide and hypertension. J. Hypertens. 22, 1057 (2004). 29) Elmarakby, A. A., Faulkner, J., Posey, S. P. and Sullivan, J. C. : Induction of hemeoxygenase-1 attenuates the hypertension and renal inflammation in spontaneously hypertensive rats. Pharmacol. Res. 62, 400 (2010). 30) Jadhav, A. and Ndisang J. F. : Interaction of the heme oxygenase system with the renin-angiotensin system in DOCA-salt hypertensive rats. The FASEB J. 21, (2007). 31) Matsufuji, H., Matsui, T., Seki, E., Osajima, K., Nakashima, M. and Osajima, Y. : Angiotensin I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived from sardine muscle. Biosci. Biotechnol. Biochem. 56, 2244 (1994). 32) Vercruysse, L., Van Camp, J. and Smagghe, G. : ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein: a review. J. Agric. Food Chem. 53, 8106 (2005). 33) Miguel, M., Manso, M. A., Martin-Alvarez, P. J. and Alexixandre, A. : Angiotensin-converting enzyme activity in plasma and tissues of spontaneously hypertensive rats after the short- and long-term intake of hydrolysed egg white. Mol. Nutr. Food Res. 51, 555 (2007). 34) Erdmann, K., Grosser, N., Schipporeit, K. and Schroder, H. : The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. J. Nutr. 136, 2148 (2006). Vol. 56, No. 5, 2012

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract