한수지 51(2), , 2018 Original Article Korean J Fish Aquat Sci 51(2), ,2018 혈관내피세포에서 TNF-α 로유도되는혈관염증에대한쏙 (Upogebia major) 효소가수분해물의억제효과 김소연 양지

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1 한수지 51(2), , 18 Original Article Korean J Fish Aquat Sci 51(2), ,18 혈관내피세포에서 TNF-α 로유도되는혈관염증에대한쏙 (Upogebia major) 효소가수분해물의억제효과 김소연 양지은 송재희 1 맹상현 이지현 윤나영 국립수산과학원식품위생가공과, 1 서해수산연구소갯벌연구센터 Inhibition Effect of Enzymatic Hydrolysate from Japanese Mud Shrimp Upogebia major on TNF-α-induced Vascular Inflammation in Human Umbilical Vein Endothelial Cells (HUVECs) So-Yeon Kim, Ji-Eun Yang, Jae-Hee Song 1, Sang-Hyun Maeng, Ji-Hyun Lee and Na-Young Yoon Food Safety and Processing Research Division, National Institute of Fisheries Science, Busan 483, Korea 1 Tidal Flat Research Center, National Institute of Fisheries Science, Gunsan 514, Korea Arteriosclerosis is the major cause of coronary artery and cerebrovascular disease, which are leading causes of death. Pro-inflammatory cytokines induce injury to vascular endothelial cells by increasing cell adhesion molecules, leading to vascular inflammation, a major risk factor for the development of arteriosclerosis. In the current study, we investigated the inhibitory effect of enzymatic hydrolysate from Japanese mud shrimp Upogebia major on the inflammation of tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). We first evaluated the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of eight U. major enzymatic hydrolysates: alcalase, papain, α-chymotrypsin (α-chy), trypsin, pepsin, neutrase, protamex and flavourzyme. Of these, α-chy exhibited potent antioxidant and ACE inhibitory activities. The α-chy hydrolysate was fractionated by two ultrafiltration membranes of 3 and 1 kda. The α-chy hydrolysate of U. major and its molecular weight cut-off fractions resulted in a significant reduction in NO production and a decrease in cell adhesion molecules [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] and pro-inflammatory cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)] in TNF-α-stimulated HUVECs. These results suggest that enzymatic hydrolysate from U. major can be used in the control and prevention of vascular inflammation and arteriosclerosis. Key words: Upogebia major, Enzymatic hydrolysate, Atherosclerosis, HUVEC, TNF-α 서론,,,,, (Lee et al., 12a). (Packard and Libby, 8).,,,,,,,, (Lee et al., 12b; Cho et al., 16).. (Upogebia major), Korean J Fish Aquat Sci 51(2) , April 18 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 6 February 18; Revised 27 February 18; Accepted 2 March 18 Corresponding author: Tel: Fax: address: Copyright 18 The Korean Society of Fisheries and Aquatic Science 127 pissn: , eissn:

2 128 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영,,,.,,,, (Hong, 13).,, carotenoids,,,, (Pan et al., ; Rosa and Barracco, 1; Sindhu and Sherief, 11; Oh and Jung, 15).,., human umbilical vein endothelial cells (HUVECs) tumor necrosis factor- (TNF- ), NO, [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)]. 재료및방법 재료 (U. major) kg, - C. Alcalase 2.4L (Alc), papain (Pa), -chymotrypsin ( -Chy), trypsin (Try), pepsin (Pep), serine, o-phthaldialdehyde (OPA) Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Neutrase (Neu), Protamex (Pro), Flavourzyme (Fla) Novo Co. (Novozyme Laboratories, Copenhagen, Denmark). 2, 2 -azino-bis [3-ethylbenzothiazoline-6-sulfonicacid] (ABTS + ), potassium persulfate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium phosphate, potassium ferricyanide [K 3 Fe(CN) 6 ], trichloroacetic acid (TCA), L-ascorbic acid, hippuryl-l-histidyl-lleucine (HHL), sodium borate, angiotensin I-converting enzyme (ACE), hydrochloric acid, pyridine, benzene sulfonyl chloride (BSC), captopril, tumor necrosis factor- (TNF- ), Griess reagent Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), iron (III) chloride (FeCl 3 ) Junsei Chemical Co. (Tokyo, Japan), cell lysis buffer Cell signaling technology, Inc (Danvers, MA, USA), nitrite standard Promega (Madison, WI, USA). human umbilical vein endothelial cells (HUVECs) Endothelial Growth Medium-2 (EGM TM -2 Medium) Lonza (Walkersville, MD, USA), penicillin-streptomycin solution Cellgro (Herndon, VA, USA), trypsin ethylenediaminetetraacetic acid (Trypsin-EDTA) Gibco (Grand Island, NY, USA), Cell Proliferation Reagent water-soluble tetrazolium salt (WST-1) Roche (Mannheim, Germany) ELISA kit R&D systems, Inc. (Minneapolis, MN, USA). 쏙효소가수분해 1 g 8 (Alc, Pa, -Chy, Try, Pep, Neu, Pro, Fla), 1% g/ g ( ). 8 Table 1 8., 1. 4,45 g. -. Table 1.The conditions of enzymatic hydrolysis and degree of hydrolysis of the enzymatic hydrolysates of Japanese mud shrimp Upogebia major Enzyme Buffer ph Temperature ( ) DH 1 (%) Alcalase (Alc).1 M Na ±.4 α-chymotrypsin (α-chy).1 M Na ±.2 Flavourzyme (Fla).1 M Na ±.3 Neutrase (Neu).1 M Na ±.8 Papain (Pa).1 M Na ±.5 Pepsin (Pep).1 M Glycine-HCl ±.6 Protamax (Pro).1 M Na ±.9 Trypsin (Try).1 M Na ±.4 DH 1, degree of hydrolysis. Enzymatic hydrolysates were obtained from 8 hour under the optical conditions.

3 쏙효소가수분해물의혈관염증억제효과 129 가수분해도 (Degree of hydrolysis, DH) Adler-Nissen (1979), o-phthaldialdehyde (OPA) free amino groups (Adler-Nissen, 1986). DH = h / h tot h tot protein equivalent peptide bonds, h. 가수분해물분획 8 1 ml, 3 kda 1 kda ultrafiltration membranes (Amicon Ultrafilter devices; Millipore, Billerica, MA, USA) 9 g (<3 kda, 3-1 kda, >1 kda).. ABTS + 라디칼소거활성 ABTS + Roberta et al. (1999). 7.4 mm ABTS 2.6 mm potassium persulfate buffer 1:1 4. ABTS nm L ABTS + 19 L nm. L-ascorbic acid, ABTS + 5% IC 5 (mg/ ml). DPPH 라디칼소거활성 DPPH Blois (1958)..2 mm DPPH 1 L L, 37 3, 5 nm. L-ascorbic acid, DPPH 5% IC 5 (mg/ml). Reducing power 측정 Reducing power Oyaizu (1986). L.2 M sodium phosphate buffer (ph 6.6) 1% K 3 Fe(CN) 6 L, 5. 1% TCA L, 2, g 1. L.1% FeCl 3 L, 7 nm. L-ascorbic acid, 5% EC 5 (mg/ml). ACE 억제효과측정 ACE Li et al. (5). L 5 L 5 mm HHL,.1M sodium borate buffer (ph 8.3) U ACE 1 L M HCl L sodium borate buffer (3 L), pyridine ( L), BSC ( Table 2. The antioxidant and ACE inhibitory activities of eight enzymatic hydrolysates of Japanese mud shrimp Upogebia major (mg/ml) ABTS + (IC 51 ) DPPH (IC 5 ) Reducing power (EC 52 ) ACE (IC 5 ) Alcalase (Alc).9±. c,3 3.23±.22 c 3.1±.18 b,c.7±.6 c α-chymotrypsin (α-chy).9±. c 2.87±.18 d 1.77±.13 d.8±.5 c Flavourzyme (Fla).9±. c 4.28±.21 b 2.81±.19 c.17±.8 a Neutrase (Neu).8±. c 5.61±.69 a 3.38±.42 b.18±. a Papain (Pa).13±. b 3.56±.61 c 2.±.9 c.11±.28 b Pepsin (Pep).18±. a 4.72±.28 b 5.93±.66 a.17±.5 a Protamax (Pro).9±. c 4.59±.17 b 2.64±.32 c.2±.5 d Trypsin (Try).12±. b 3.68±.38 c 3.62±.22 b.11±.8 b A L-Ascorbic acid (μg/ml) 5.61±.9 d 4.46±.27 e 4.71±. e B Captopril (ng/ml).25±.5 e 1 IC 5 (5% inhibition concentration) values of ABTS + and DPPH radical scavenging, and ACE inhibitory activities were expressed as a mean ±SD. 2 The reducing power was expressed as an EC 5 (concentration of the.5 absorbance) value. 3 Mean within the same row with different superscripts are significantly different by Duncan s multiple range test (P<.5). A L-ascorbic acid was used as a positive control of ABTS + and DPPH radicals scavenging and reducing power. B Captopril was used as a positive control of ACE inhibitory activity. ABTS +, 2, 2 -azinobis [3-ethylbenzothiazoline-6-sulfonicacid]; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ACE, angiotensin I-converting enzyme.

4 13 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영 Cell viability (%) 1 1 Nitric oxide (%) Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Fig. 1. The cell viability of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions in HUVEC. HUVEC, human umbilical vein endothelial cell. L) 37 3, 41 nm. captopril, ACE ACE 5% IC 5 (mg/ml). 세포배양 HUVEC 2% fetal bovine serum (FBS) vascular endothelial growth factor (VEGF) Endothelial Growth Medium-2 (EGM TM -2 Medium) 37, 5% CO 2. 세포독성측정 HUVEC WST-1 cell viability. 96 well plate HU- VEC 5 1³ cells/well,, well WST-1 1 L 2, ELISA reader (US/MQX ; Bio-Tek Instruments Inc., Winooski, VT, USA) 45 nm. Nitric oxide (NO) 생성억제능측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml)., lysis buffer L cell, 14, rpm 1. L Griess reagent L, 5 nm. Nitrite standard NO. Fig. 2. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the production of nitric oxide (NO) in TNF-αstimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α-nontreated group. TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell. 세포부착인자 (Cell adhesion molecules, CAMs) 생성량측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml). ELISA kit (R&D systems, Inc., Minneapolis, MN, USA) VCAM-1, ICAM- 1, E-selectin ELISA reader (US/MQX ; Bio- Tek Instruments Inc., Winooski, VT, USA) 45 nm. 사이토카인 (Cytokines) 생성량측정 HUVEC 6 well plates 24, 2 TNF- ( ng/ml). ELISA kit (R&D systems, Inc., Minneapolis, MN, USA) IL-6, IL- 8, MCP-1 ELISA reader (US/MQX ; Bio-Tek Instruments Inc., Winooski, VT, USA) 45 nm. 통계 (SD), SAS (SAS Institute, Cary, NC, USA), Duncan multiple-comparison test Student t-test P<.5.

5 쏙효소가수분해물의혈관염증억제효과 131 결과및고찰 효소가수분해및가수분해도 8, (Table 1). -Chy ( %)>Fla ( %)>Pep ( %)>Pro ( %)>Neu ( %) >Pa (65.9.5%)>Alc ( %) >Try ( %). 8 -Chy. 항산화및 ACE 억제활성 DNA,,, (Nardin, 1). cytokine, growth factor,,, apoptosis (Kunsch and Medford, 1999).. angiotensin-ii, superoxide anion, VCAM-1, IL-6, MCP-1. ACE angiotensin-i angiotensin-ii,. ACE (Parish and Miller, 1992; Paul et al., 6; Takahashi et al., 11). ACE 8 (ABTS + DPPH, reducing power) ACE (Table 2). ABTS + IC mg/ml, Alc, -Chy, Fla, Neu, Pro. DPPH 8 -Chy IC mg/ml, Reducing power -Chy EC mg/ml. ACE Alc, -Chy, VCAM-1 (%) ICAM-1 (%) E-selectin (%) Fig. 3. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the expression of adhesion molecules (VCAM- 1, ICAM-1 and E-selectin) in TNF-α-stimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α-nontreated group. VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; E- selectin, endothelial-selectin; TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell. Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda

6 132 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영 Pro. 8 ACE -Chy. HUVEC 세포에대한독성 HUVEC -Chy (Fig. 1). -Chy ( mg/ml), -Chy.125- %,. NO 생성억제효과 NO (Korhonen et al., 5). TNF- HUVEC -Chy NO (Fig. 2). -Chy HUVEC TNF- NO. 세포부착인자 (Cell adhesion molecules, CAMs) 의발현에미치는영향,, (Peter et al., 1997; Koo et al., 14). (VCAM-1, ICAM-1, E-selectin) (IL-6, IL-8, MCP-1), (Bae et al., 8). TNF- HUVEC -Chy (Fig. 3). TNF- VCAM-1 -Chy,,., -Chy 3, <3 kda, 3-1 kda, >1 kda ( mg/ ml) VCAM-1, >1 kda. HUVEC ICAM-1 -Chy, IL-6 (%) IL-8 (%) MCP-1 (%) 1 Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Blank Control α-chy <3 kda 3-1 kda >1 kda Fig. 4. The inhibition effect of the α-chymotrypsin hydrolysates of Japanese mud shrimp Upogebia major and its molecular weight cut-off fractions on the expression of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in TNF-α-stimulated HUVEC. All data are presented as means±sd of three independent experiments. P<.5 compared with TNF-α treatment only. P<.1, compared with the TNF-α -nontreated group. IL-6, interleukin-6; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α; HUVEC, human umbilical vein endothelial cell.

7 쏙효소가수분해물의혈관염증억제효과 kda >1 kda mg/ml ICAM-1, <3 kda. HUVEC TNF- E-selectin -Chy mg/ml. 3-1 kda >1 kda mg/ml E-selectin, <3 kda., >1 kda. -Chy, >1 kda. 염증성사이토카인 (Pro-inflammatory cytokines) 의발현에미치는영향 IL-6, IL-8, MCP-1. IL-6, IL-8 chemoattractant, MCP-1 chemoattractant (Yang et al., 4; Sprague and Khalil, 9). HUVEC TNF-, -Chy (Fig. 4). TNF- HUVEC IL-6 -Chy IL mg/ml IL-6. HUVEC TNF- IL-8 -Chy 1 mg/ml. <3 kda.25-1 mg/ml, 3-1 kda >1 kda mg/ml IL kda IL-8,. TNF- MCP-1 -Chy mg/ml mg/ml, >1 kda MCP-1. -Chy HUVEC TNF- NO, -Chy., -Chy >1 kda,,. 사사 (R1). References Adler-Nissen J Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27, org/1.121/jf226a42. Adler-Nissen J Enzymic hydrolysis of food proteins. Elsevier Applied Science Publishers. Barking, Essex, U.K., Bae JH, Park JS, Hong GR, Shin DG, Kim YJ and Shim BS. 8. Correlation between inflammatory markers and the progression of atherosclerosis in the patients with coronary artery and disease. Korean J Medicine 74, Blois MS Antioxidant determinations by the use of a stable free radical. Nature 181, org/1.138/ a. Cho YM, Song HS, Jang SA, Park DW, Shin YS, Jeong YJ and Kang SC. 16. Suppression of VCAM-1 expression in human aortic smooth muscle cells treated with ethanol extracts of Cynanchum wilfordii radix, Arctium lappa L., and Dioscorea opposita. Korean J Plant Res 29, dx.doi.org/1.7732/kjpr Hong JS. 13. Biology of the mud shrimp Upogebia major (de Haan, 1841), with particular reference to pest management for shrimp control in manila clam bed in the West Coast of Korea. Ocean Polar Res 35, org/1.4217/opr Koo HJ, Park HJ, Byeon HE, Kwak JH, Um SH, Kwon ST, Rhee DK and Pyo SN. 14. Chimese yam extracts containing β-sitosterol and ethyl linoleate protect against ath-

8 134 김소연ㆍ양지은ㆍ송재희ㆍ맹상현ㆍ이지현ㆍ윤나영 erosclerosis in apolipoprotein e-deficient mice and inhibit muscular expression of VCAM-1 in vitro. J Food Sci 79, H719-H Korhonen R, Lahti A, Kankaanranta H and Moilanen E. 5. Nitric oxide production and signaling in inflammation. Curr Drug Targets Inflamm Allergy 4, org/1.2174/ Kunsch C and Medford RM Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 85, Lee AS, Kim JS, Lee YJ and Kang DG. 12a. Anti- TNF-α activity of Portulaca oleracea in vascular endothelial cells. Int J Mol Sci 13, Lee SM, Lee YJ, Kim YC, Kim JS, Kang DG and Lee HS. 12b. Vascular protective role of vitexicarpin isolated from Vitex rotundifolia in human umbilical vein endothelial cells. Inflammation 35, Li GH, Liu H, Shi YH and Le GW. 5. Direct spectrophotemetric measurement of angiotensin I-converting enzyme inhibitory activity for screening bioactive peptides. J Pharm Biomed Anal 37, jpba Nardin ED. 1. The role of inflammatory and immunological mediators in periodontitis and cardiovascular disease. Ann periodontal 6, Oh GW and Jung WK. 15. Biomedical materials for regenerating bone tissue utilizing marine invertebrate. Korean J Fish Aquat Sci 48, KFAS Oyaizu M Studies on product of browning reaction prepared from glucoseamine. Jpn J Nutr 44, dx.doi.org/1.5264/eiyogakuzashi Packard RR and Libby P. 8. Inflammation in atherosclerosis: from vascular biology to biomarker discovery and risk prediction. Clin Chem 54, clinchem Pan J, Kurosky A, Xu B, Chopra AK, Coppenhaver DH, Singh IP and Baron S.. Broad antiviral activity in tissues of crustaceans. Antiviral Res 48, org/1.116/s ()117-. Parish RC and Miller LJ Adverse effects of angiotensin converting enzyme (ACE) inhibitors. Drug Saf 7, Paul M, Mehr AP and Kreutz R. 6. Physiology of local rennin-angiotensin systems. Physiol Rev 86, dx.doi.org/1.1152/physrev Peter K, Nawroth P, Conradt C, Nordt T, Weiss T, Boehme M, Wunsch A, Allenberg J, Kubler W and Bode C Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intracellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin. Arterioscler Thromb Vasc Biol 17, Roberta R, Nicoletta P, Anna P, Ananth P, Min Y and Catherine RE Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26, (98) Rosa RD and Barracco MA. 1. Antimicrobial peptides in crustaceans. Invertebrate Surviv J 7, Sindhu S and Sherief PM. 11. Extraction, characterization, antioxidant and anti-inflammatory properties of carotenoids from the shell waste of Arabian red shrimp Aristeus alcocki, Ramadan Open Conf Proc J 2, org/1.2174/ Sprague AH and Khalil RA. 9. Inflammatory cytokines in vascular dysfunction and vascular disease. Biochem Pharmacol 78, Takahashi H, Yoshika M, Komiyama Y and Nishimura M. 11. The central mechanism underlying hypertension: a review of the roles of sodium ions, epithelial sodium channels, the rennin-angiotensin-aldosterone system, oxidative stress and endogenous digitalis in the brain. Hypertens Res 34, Yang YH, Lai HJ, Huang CM, Wang LC, Lin YT and Chiang BL. 4. Sera from children with active Henoch-Schonlein purpura can enhance the production of interleukin 8 by human umbilical venous endothelial cells. Ann Rheum Dis 63,

한수지 51(2), , 2018 Original Article Korean J Fish Aquat Sci 51(2), ,2018 쏙 (Upogebia major) 효소가수분해물의면역증강효과 이지현 양지은 송재희 1 맹상현 김소연 윤나영 * 국립수

한수지 51(2), , 2018 Original Article Korean J Fish Aquat Sci 51(2), ,2018 쏙 (Upogebia major) 효소가수분해물의면역증강효과 이지현 양지은 송재희 1 맹상현 김소연 윤나영 * 국립수 한수지 51(2), 135141, 18 Original Article Korean J Fish Aquat Sci 51(2),135141,18 쏙 (Upogebia major) 효소가수분해물의면역증강효과 이지현 양지은 송재희 1 맹상현 김소연 윤나영 국립수산과학원식품위생가공과, 1 서해수산연구소갯벌연구센터 Immunopotentiating Activities