한수지 49(4), 445-453, 16 Originl Artile Koren J Fish Aqut Si 49(4),445-453,16 빅벨리해마 (Hippompus dominlis) 유래펩신가수분해물의 In vitro 와 In vivo 에서의항산화효능 김현수 신병옥 김서영 Lei Wng 이원우 김윤택 1 노섬 1 조문제 2 전유진 * 제주대학교해양의생명과학부, 1 한국해수관상어센터, 2 제주대학교의학전문대학원 Antioxidnt Ativity of Pepsin Hydrolyste Derived from Edile Hippompus dominlis in vitro nd in Zerfish Models Hyun-Soo Kim, Byeung-Ok Shin, Seo-Young Kim, Lei Wng, WonWoo Lee, Yoon Tek Kim 1, Sum Rho 1, Moonje Cho 2 nd You-Jin Jeon* Deprtment of Mrine Life Siene, Jeju Ntionl University, Jeju 63243, Kore 1 Center of Ornmentl Reefs nd Aquriums, Jeju 63354, Kore 2 Deprtment of Mediine, Medil Shool, Jeju Ntionl University, Jeju 63243, Kore Sehorse Hippompus dominlis mrine teleost fish, hs long een used s one of the essentil mterils in trditionl Chinese mediine. However, the uses of sehorse hve een limited due to its high ost, despite its enefiil iologil tivities. Sehorse hs not een widely explored for its iofuntionl properties nd tive omponents. In the present study, the enzymti hydrolystes of sehorse were prepred y using two digestive enzymes (trypsin nd pepsin) nd five food grde enzymes (neutrse, protmex, llse, kojizyme, nd flvourzyme). The enzymti hydrolystes indited higher hydrolysis yields thn its wter extrt. Among them, the distilled wter-pepsin hydrolyste (DP) whih ws otined y distilled wter extrtion followed y pepsin hydrolysis, showed the highest yield nd protein ontent s well s the highest lkyl rdil svenging tivity. Also, it provided protetive effets ginst oxidtive stress indued y AAPH in vero ell nd zerfish. Further frtiontion sed on the moleulr weight ws rried out to identify it s tive omponents, nd < 5 kd (less thn 5 kd) moleulr weight frtion ws onfirmed to hve the highest ntioxidnt tivity. In onlusion, this study suggests tht DP of sehorse hs ntioxidnt properties, nd might e novel nd useful mteril from the mrine origin for helthy funtionl foods nd osmetis. Key words: Sehorse, Hippompus dominlis, Enzymti hydrolysis, Antioxidnt tivity, Zerfish 서론 (Syngnthiformes), (Syngnthide) 52 215 (Hippompus) 32, 7% - (Myers, 1979; Vri, 1982; Lourie et l., 1999). (Hippompus orontus), (Hippompus histrix), (Hippompus kud), (Hippompus mohnikei) (Hippompus trimultus) 5 (Kim nd Lee, 1995).,, (Vinent, 1996; Choi et l., 6). (Zhu, 5), (She et l., 1995; Zhng et l., 1994), (Xu nd Xu, 1997), (Ryu et l., 1; Ryu et l., 1), http://dx.doi.org/1.5657/kfas.16.445 Koren J Fish Aqut Si 49(4) 445-453, August 16 This is n Open Aess rtile distriuted under the terms of the Cretive Commons Attriution Non-Commeril Liens (http://retiveommons.org/lienses/y-n/3./) whih permits unrestrited non-ommeril use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. Reeived 3 June 16; Aepted 11 July 16 *Corresponding uthor: Tel: +82. 64. 754. 3475 Fx: +82. 64. 756. 3493 E-mil ddress: youjin@jejunu..kr Copyright 16 The Koren Soiety of Fisheries nd Aquti Siene 445 pissn:374-8111, eissn:2287-8815
446 김현수ㆍ신병옥ㆍ김서영ㆍ Lei Wng ㆍ이원우ㆍ김윤택ㆍ노섬ㆍ조문제ㆍ전유진 (Chen et l., 11; Qin et l., 8), (Xu et l., 3), (Himy et l., 12) in vitro. (Hippompus dominlis) (Syngnthifromes), (Syngnthide).,, (Woods, 7),. (Hippompus kud Bleeker), (Hippompus guttultus), Short-snouted sehorse (Hippompus hippompus). 2 (trypsin, pepsin) 5 (neutrse, protmex, llse, kojizyme, flvourzyme),,,,,,,, (Bekmn et l., 199; Cho et l., 12; Heo et l., 5; Ahn et l., 7; Prk et l., 4). 실험시약및재료 재료및방법 (CCORA) m (Fig. 1),. Fig. 1. The photo of different sizes of sehorses Hippompus dominlis. 2 (trypsin, pepsin) Sigm Chemil Co. (USA) 5 (neutrse, protmex, llse, kojizyme, flvourzyme) Novozyme Co. (Denmrk). 2,2-zois-(2- midinopropne) dihydrohloride (AAPH), -(4-Pyridyl-1- oxide)-n-tert-utylnitrone (4-POBN), 3-(4,5-dimethylthizol- 2-yl)-2,5-diphenyltetrzolium romide (MTT), 2,7-dihlorofluoresein diette (DCF-DA), sodium dodeyl sulfte (SDS), dimethyl sulfoxide (DMSO), ovine serum lumin (BSA) Sigm-Aldrih (St. Louis, MO, USA). Piere BCA protein ssy kit Thermo Sientifi (Rokford, IL, USA) RPMI 16 fetl ovine serum (FBS) Gio/BRL (Cnd).. 해마의일반성분분석 AOAC (Helrih, 199). 15 24, 55 3. Kjeldhl, Soxhlet. 해마의단백질효소가수분해물의제조 Ko et l. (13), 7 (distilled wter, DW). 1:1, ph 24. DW 24 pepsin trypsin 12 DW-pepsin (DP) DW-trypsin (DT). 1 1 ph 7.. pore size 7 m 7. 단백질효소가수분해물의수율및단백질함량측정 1. 7 Piere BCA protein ssy kit, BSA.
빅벨리해마효소가수분해물의항산화효능 447 Alkyl rdil 소거능측정 lkyl rdil Kng et l. (13)., µl, DW µl, mm AAPH µl, mm 4-POBN µl 37 3 pillry tue ; Eletron spin resonne (JES-FA ESR, JEOL, Tokyo, Jpn) signl. DW. Power: 1 mw, Amplitude: 1, Modultion width:.2 mt, Sweep width: 1 mt, Sweep time: 3 s, Time onstnt :.3 s 세포배양 Vero ell 1% FBS, 1% ntiioti RPMI 16. Cell ulture flsk 37, 5% CO 2 2-3. Vero ells 에서의독성평가 Ko et l. (12) MTT ssy. Vero ells (1 1 5 ells/ml) 96 wellplte 19 µl, 37, 5% CO 2 24. 37 24 2 mg/ml MTT 5 µl. 37 3 dimethylsufoxide (DMSO) 12 ELISA reder 5 nm. Vero ells 에서의 ROS 생성측정 ROS Ko et l. (12) AAPH ROS. 1 1 5 vero ells 96 wellplte 18 µl, 37 24. 37 3 4 mm AAPH 1 µl, 37 3.5% DCF-DA 1 µl 37 1 ELISA reder. Vero ells 에서 AAPH 에의한산화적손상에대한보호효과측정 AAPH Ko et l. (12) AAPH. 1 1 5 vero ells 96 wellplte 18 µl seeding, 37 24. 37 1 4 mm AAPH 1 µl. 37 24 2 mg/ml MTT 5 µl. 37 3, 37 3 DMSO 12 ELISA reder 5 nm.. Zerfish emryo 에서의 AAPH 산화보호효과측정 Zerfish (Dnio rerio) (Kim et l., 14). 7-9 hpf (hours post-fertiliztion) zerfish emryo 12 well pltes 15 emryo medium 1.8 ml (25, 5, 1, µg/ml) 1 µl. 1 15 mm AAPH 1 µl.. 통계분석. SPSS (SPSS In. Ver. 12.) Onewy ANOVA-test Turkey s multiple rnge test P<.5. 해마의일반성분 결과및고찰 Tle 1,,,,. 58.47%. Tle 1. Chemil ompositions of sehorse Hippompus dominlis (%, w/w) Composition Moisture Ash Crude protein Crude lipid Hippompus Adominlis 5.18 33.75 58.47 2.6
448 김현수ㆍ신병옥ㆍ김서영ㆍ Lei Wng ㆍ이원우ㆍ김윤택ㆍ노섬ㆍ조문제ㆍ전유진 해마의단백질효소가수분해물들의수율및단백질함량, DW (Tsi et l., 6). DW 24 1 g 374.5 3.54 mg, 7,. DP 786.67 15.28 mg, DT 5. 25.17 mg (Tle 2). 5. DP neutrse llse, 783.33. mg, 78. 1. mg (Tle 2). 1 g DW 24 93.38 3.4 mg, DW 24 2 (Tle 2). DP 221.21 7.2 mg, 5 (Tle 2). DW DP DT, 5. 해마단백질효소가수분해물들의 Alkyl rdil 소거능 Alkyl rdil. IC 5 (mg/ml) DP.15. mg/ml, DT.15. mg/ml, Neutrse.18.16 mg/ml, DP DT lkyl rdil (Tle 2).. DP,, lkyl rdil DP. DP 의세포독성및 AAPH 산화보호효과 MTT ssy MTT (suinte dehydrogense) (formzn), (Yng nd Boo, 13). DP Fig. 2A. 5 g/ml, 1 g/ml, g/ml, 4 mm AAPH. DP AAPH (Fig. 2B), AAPH AAPH ROS, AAPH Tle 2. Yields nd lkyl rdil svenging tivity of enzymti hydrolystes prepred from sehorse Hippompus dominlis y seven proteinses Yields (mount tined from 1 g of rw smple) Rdil svenging tivity (IC 5, mg/ml) Hydolystes ontents (mg) Protein ontents (mg) Alkyl DW 374.5±3.54 93.38±3.4.38±.6 Trypsin 41.5±2.12 127.67±2.79 <.25 Pepsin 445.±1.41 144.21±3.29.24±.6 DT 1 5.±25.17 147.5±7.14.15±. DP 2 786.76±15.28 221.21±7.2.15±. Neutrse 783.33±. 222.19±5.67.18±.16 Protmex 67.±. 156.12±6.21 <.25 Allse 78.±1. 179.±3..73±.9 Kojizyme 673.33±7. 228.39±3.15.25±. Flvourzyme 626.67±3. 251.±1.56.7±.3 1 Distilled wter-trypsin hydrolyste (DT). 2 Distilled wter-pepsin hydrolyste (DP).
빅벨리해마효소가수분해물의항산화효능 449 ROS (Fig. 2C). DP AAPH ROS. AAPH ron rdil, ron rdil ron rdil peroxy rdil, peroxy rdil,, (Niki, 199). DP 및분자량별분획물의 SDS PAGE 를이용한분석및각분자량분획물의 Alkyl rdil 소거활성 DP. DP 5 kd 1 kd 1 kd, 5-1 kd, 5 kd, Fig. 3A SDS-PAGE., 5 kd lkyl rdil IC 5.15.1 mg/ml (Fig. 3B). DP lkyl rdil DP. (Ko et l., 12, Je et l., 5, Ren et l., 8, Zho et l., 9),. DP 분자량별분획물의세포독성및 AAPH 에의해산화된 Vero ells 의보호효과 DP Fig. 4A. MTT ssy 1 kd, 5-1 kd, 5 kd 5 g/ml, 1 g/ml, Vero ells. 4 mm AAPH 5 g/ml, 1 g/ml Vero ells, Vero ells., 5 kd Frtion 1 g/ml 77.17 2.12% (Fig. 4B). 4 mm AAPH 5 g/ml, 1 g/ml, Vero ells Totl ROS, 5 kd 1 g/ml Totl ROS 61.1.49%. 5 kd Totl ROS Cell viility (%) 1 1 8 1 1 (A) (B) (C) Cell viility (%) d 1 8 ROS prodution (%) 1 8 d CON 5 1 CON 5 1 CON 5 1 DP (µg/ml) 4 mm AAPH + DP (µg/ml) 4 mm AAPH + DP (µg/ml) Fig. 2. (A) The ytotoxi effet of distilled wter-pepsin hydrolyste (DP) on viility in vero ells. Cells were treted with DP t the indited onentrtions (5, 1 nd µg/ml). (B) Protetive effet of DP on AAPH-indued oxidtive dmge in norml ells. The viilities of ells on AAPH tretment ws ssessed y MTT ssy. (C) Inhiitory effet of DP on the intrellulr ROS generted in AAPHindue oxidtive ells. Experiments were performed in triplite, nd the dt re expressed s men ± SD. - P<.5 indites signifintly different in eh groups.
45 김현수ㆍ신병옥ㆍ김서영ㆍ Lei Wng ㆍ이원우ㆍ김윤택ㆍ노섬ㆍ조문제ㆍ전유진 (A) 75 kd 63 kd 48 kd 35 kd 25 kd 17 kd 11 kd Mrker DP 1 kd 1-5 kd 5 kd Alkyl rdil svenging tivity (%) 1 9 8 7 5 3 1 (B) IC 5 vlues (mg/ml).125 mg/ml.25 mg/ml 1 kd 1-5 kd 5 kd 1 kd 1-5 kd 5 kd.24±..21±..15±.1 Fig. 3. (A) SDS-PAGE ptterns of moleulr weight frtions from DP. (B) Alkyl rdil svenging tivities of the moleulr weight frtions prepred from DP. Alkyl rdil svenging tivities of the three smples (.125 mg/ml,.25 mg/ml) were mesured y ESR spetrometry. - P<.5 indites signifintly different in eh groups. 1 1 (A) 5 μg/ml 1 μg/ml 1 1 (B) d 5 μg/ml 1 μg/ml 1 1 (C) d 5 μg/ml 1 μg/ml Cell viility (%) 8 Cell viility (%) 8 ROS prodution (%) 8 CON 1 kd 1-5 kd 5 kd CON 1 kd 1-5 kd 5 kd 4 mm AAPH CON 1 kd 1-5 kd 5 kd 4 mm AAPH Fig. 4. (A) The ytotoxi effets of the moleulr weight frtions from DP on viility in vero ells. Cells were treted with the moleulr weight frtions from pepsin digestive hydrolyste t the indited onentrtions (5, 1 µg/ml). After 24 h inution, ell viilities ws ssessed y MTT ssy. (B) Protetive effets of the moleulr weight frtions from DP on AAPH-indued oxidtive dmge in norml ells. The viilities of ells with AAPH tretment ws ssessed y MTT ssy. (C) The intrellulr ROS ws deteted y DCFH-DA ssy. Grph showed the intrellulr ROS svenging tivity. Experiments were performed in triplite, nd the dt re expressed s men±sd. -d P<.5 indites signifintly different in eh groups. (Fig. 4C). ESR lkyl rdil, DP in vivo 5 kd zerfish emryo. DP 5 kd 이하분획물의 AAPH 에의해산화된 Zerfish emryo 의보호효과 Zerfish., emryo,, zerfish
빅벨리해마효소가수분해물의항산화효능 451 in vivo (Kng et l., 15; Ko et l., 14). 7% epioly zerfish emryo 15 mm AAPH zerfish emryo, 3 DP 5, g/ml, 24, 48, 72. g/ml 5% 5 kd AAPH. in vivo zerfish emryo DP AAPH., lkyl. DP Vero Cells ROS, AAPH. DP, lkyl, 5 kd, vero ells ROS AAPH DP Survivl rte of zerfish (%) 1 1 8 CON 25 5 1 15 mm AAPH + DP 5 kd frtions (μg/ml) 3 h 24 h 48 h 72h Elpsed times (h) Fig. 5. Survivl rtes fter tretment with AAPH or o-tretment with 5 kd moleulr frtion from DP. The emryos were treted with 15 mm AAPH with or without 5 kd moleulr frtion from DP t the different onentrtions (25, 5, 1, µg/ml).., 5 kd AAPH zerfish emryo., DP,. 사사 15 " " Referenes Ahn GN, Kim KN, Ch SH, Song CB, Lee J, Heo MS, Yeo IK, Lee NH, Jee YH, Kim JS, Heu MS nd Jeon YJ. 7. Antioxidnt tivities of phlorotnnins purified from Ekloni v on free rdil svenging using ESR nd H 2 O 2 - medited DNA dmge. Eur Food Res Tehnol 226, 71-79. http://dx.doi.org/1.17/s217-6-51-y. Bekmn JS, Bekmn TW, Chen J, Mrshell PA nd Freemn BA. 199. Apprent hydroxyl rdil prodution y peroxynitrile implitions for endothelil injury from nitri oxide nd superoxide. PNAS 87, 16-1624. http://dx.doi. org/1.173/pns.87.4.16. Choi YU, Rho S, Jung MM, Lee YD nd Noh GA. 6. Prturition nd Erly Growth of Crowned Sehorse, Hippompus orontus in Kore. N Am J Aquult 19, 19-118. Chen CB, Yun XH, Chen Z nd Yi MH. 11. Study on extrtion nd ntioxidnt tivities of liquefied protein of threespot hippompus. Chinese Tropil Mediine 11, 329-331. Cho CH, Lee BH, Kim HY, Kim YC nd Kim DO. 12. Effet of feedstuffs on minerl omposition, ntioxidnt pity, nd protetion of neuronl PC-12 ells of deer tlers. KSBB J 27, 243-25. http://dx.doi.org/1.7841/ksj.12.4.243. Helrih K. 199. Offiil Methods of Anlysis of AOAC Intl. 15th ed.; Assoition of Offiil Anlysis Chemists, Wshington, DC, USA. Chpter 11. 1-31. Himy SWA, Ryu BM, Qin ZJ nd Kim SK. 12. Peonol from Hippompus kud Bleeler suppressed the neuro-inflmmtory responses in vitro vi NF-kB nd MAPK signling pthwys. Toxiol In Vitro 26, 878-887. http://dx.doi. org/1.116/j.tiv.12.4.22. Heo SJ, Prk EJ, Lee KW nd Jeon YJ. 5. Antioxidnt tivities of enzymti extrts from rown seweeds. Bioresoure Tehnol 96, 1613-1623. http://dx.doi.org/1.116/j. ioteh.4.7.13. Je JY, Prk PJ nd Kim SK. 5. Antioxidnt tivity of peptide isolted from Alsk Pollk (Thergr hlogrmm) frme protein hydrolyste. Food Res Int 38, 45-5. http://dx.doi.org/1.116/j.foodres.4.7.3.
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from Hippompus. Chinese Phrmeutil Affirs 19, 23-24. Zhng ZH, Xu GJ, Xu LS nd Wng Q. 1994. Inhiitory effets of Hippompus SPP. extrts on L-glutmi id indued C influx in rts neurons. Chinese J Mr Drugs 52, 6-9. Zho Y, Li B, Dong S, Liu Z, Zho X, Wng J nd Zeng M. 9. A novel ACE inhiitory peptide isolted from Audin molpdioide hydrolyste. Peptides 3, 128-133. http://dx.doi.org/1.116/j.peptides.9.3.2. 빅벨리해마효소가수분해물의항산화효능 453