한수지 47(2), , 2014 Original Article Kor J Fish Aquat Sci 47(2), ,2014 별불가사리 (Asterina pectinifera) 조직별초산추출물의생리활성탐색 고혜진, 조미정 1, 김군도 1, 박남규

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1 한수지 47(2), , 2014 Original Article Kor J Fish Aquat Sci 47(2), ,2014 별불가사리 (Asterina pectinifera) 조직별초산추출물의생리활성탐색 고혜진, 조미정 1, 김군도 1, 박남규 * 부경대학교생물공학과, 1 부경대학교미생물학과 Biological Activities of Acidic Extracts of the Starfish Asterina pectinifera Hye-Jin Go, Mi Jeong Jo 1, Gun-Do Kim 1 and Nam Gyu Park* Department of Biotechnology, Pukyong National University, Busan , Korea 1 Department of Microbiology, Pukyong National University, Busan , Korea The present study was performed to examine the contraction and relaxation responses of the smooth muscles, and search for antimicrobial and antioxidant activities in the tissues, of the starfish Asterina pectinifera. Frozen samples were extracted with distilled water containing 1% acetic acid. Extracts from all tissues showed potent antimicrobial activity against Escherichia coli D31. Relatively high levels of antimicrobial activity were also detected in the body extracts. Liver, tube feet, and body extracts caused contraction responses in the dorsal retractor muscles (DRM) of the starfish. In contrast, all tissues examined exhibited contractile activity in the esophagus of squid Todarodes pacificus. In addition, liver and gonad extracts caused contraction responses upon application to the intestine of the puffer fish Takifugu pardalis. Relaxation effects on the DRM of starfish were identified in most of the extracts, while no relaxant activity was detected in body extracts. Extracts from all tissues examined also exhibited antioxidant activities. The results of this study suggest that starfish are a potential source of novel bioactive compounds. Key words: Starfish, Asterina pectinifera, Antimicrobial activity, Contraction and relaxation responses, Antioxidant activity 서론,,, (Donia and Hamann, 2003)., (Hancock and Diamond, 2000). (innate immunity) (adaptive immunity).,. (Matsunaga et al, 1985), (Charlet et al., 1996; Mitta et al., 1999; Mitta et al., 2000), (Miyata et al., 1989), (Ovchinnikova et al., 2004), (Iijima et al., 2003), (Destoumieux et al., 1997), (Li et al., 2008), (Ovchinnikova et al., 2006). eledosin (Erspamer and Anastasi, 1962) (neuropeptides) (Krieger, 1986). (Elliott and Barchas, 1979; Krieger, 1983),,, (Zadina et al., 1986). Kor J Fish Aquat Sci 47(2) , April 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 14 February 2014; Revised 20 March 2014; Accepted 27 March 2014 *Corresponding author: Tel: Fax: address: ngpark@pknu.ac.kr Copyright 2014 The Korean Society of Fisheries and Aquatic Science 122 pissn: , eissn:

2 별불가사리추출물의생리활성 123 (Chauvet et al., 1994), (Irwin and Wong, 1995), (Iwakiri et al., 1990), (Jensen and Colon, 1992)., (Ames et al., 1993; Rice-Evans and Miller, 1996)., tert-butylhydroxytoluene (BHT), tert-butylhydroxyanisol (BHA) -tocopherol, vitamin C, carotenoids, flavonoids, (Kang et al., 2003; Kim et al., 1997; Koshino et al., 1996; Park et al., 1991). (Sampath Kumar NS et al., 2012), (Fan et al., 2012), mycosporin-like amino acids (MAAs) (Dunlap and Yamamoto, 1995).,. (Asterias amurensis) (Kwon et al., 2007) (Chae et al., 2007).. (Asterina pectinifera),. 시료 재료및방법 (Asterina pectinifera, 6-7 cm) 10 (body), (liver), (gonad), (muscle), (gut) (tubefeet). -70 일반시약 Tryptic soy broth (TSB), yeast extract, tryptone (Pancreatic digest of casein), agarose (Low EEO), acetylcholine chloride, carbachol 1,1-diphenyl-2-picrylhydrazyl (DPPH) Sigma (St. Louis, USA). 추출물의조제 (Asterina pectinifera) 1% 1:3 (V/V), ,000 g., 2. 생리활성탐색 항균활성 ultra radial diffusion assay (URDA) (Lehrer et al., 1991). North Carolina State University Edward J. Noga E. coli D31. 5 L 37, clear zone clear zone diameter (mm). 별불가사리 dorsal retractor muscle (DRM) 에서의수축및이완활성측정 eye spot 5 DRM mess 20 mm., isometric transducer (NEC- Sanei, Tokyo, Japan) resting tension 1.0 g 90., DRM tension 1.0 g acetylcholine (ACh) 10-4 M. 1/10. ACh 100%, ACh., tension 1.0 g 10-4 M ACh, M ACh, M Ach 100% %. 55 mm Mg 2+, ASW : MgCl 2.6H 2 O 55 mm, NaCl 445 mm, KCl 10 mm, CaCl 2.2H 2 O 10 mm, Glucose 10 mm, Tris-HCl 10 mm (ph 7.8). 오징어소화관에서의수축활성 7 cm, 1.0 cm. buffer, buffer : NaCl 466 mm, MgCl 2 54 mm, CaCl 2 11 mm, KCl 10 mm, NaHCO 3 3 mm, Na-HEPES 10 mm. 2 ml physiography system (NECSanei, Tokyo, Japan) isometric

124 고혜진ㆍ조미정ㆍ김군도ㆍ박남규 transducer 1.5 g, 15 60. 10-5 M carbachol (Carb) 15 3. 1/10 physiography. 졸복장관평활근에대한수축활성, 1 cm 2.0-3.0 cm., 1.0 cm. buffer, buffer : NaCl 165 mm, KCl 3 mm, MgSO 4 7H 2 O 1.

3 124 고혜진ㆍ조미정ㆍ김군도ㆍ박남규 transducer 1.5 g, M carbachol (Carb) /10 physiography. 졸복장관평활근에대한수축활성, 1 cm cm., 1.0 cm. buffer, buffer : NaCl 165 mm, KCl 3 mm, MgSO 4 7H 2 O 1.25 mm, NaHCO 3 2 mm, CaCl 2.2H 2 O 1.5 mm, Glucose 10 mm (ph 7.4). 2 ml physiography system isometric transducer 1.5 g, M ACh /10. physiography M ACh 100%, ACh. 항산화활성의측정 DPPH methanol M, 3 mg 0.01% 1 ml, 1/50. 1 ml DPPH solution UV cuvette 520 nm. (control) 1 ml DPPH 4 ml methanol. DPPH radical (%). DPPH radical (%) = [(O.D. of Control - O.D. of Sample)/O.D. of Control] 100 통계처리 3, GraphPad Prism 5.0 for Window ANOVA test, P<0.05 Dunnett s multiple comparison test. 결과및고찰 Fig. 1. Antimicrobial activity of various tissues' extracts of the starfish Asterina pectinifera against E. coli D31. 별불가사리추출물들의항균활성 E. coli D31 (Fig. 1) mm, 18 2 mm, 15 1 mm, 14 1 mm, 13 2 mm, 19 3 mm (positive control: Piscidine ) (Seo et al., 2005; Seo et al, 2013)..,.,. (Edward et al., 2004).. (Christina et al., 2001; Martinage et al., 1983),,

4 별불가사리추출물의생리활성 125 A. The DRM of Starfish, Asterina pectinifera 2 min 1.0 g Muscle Gut Liver Body Tube feet B. The esophagus squid, Todarodes pacificus 2 min 1.0 g Muscle Gut Liver Tube feet Body Gonad C. The intestine of panther puffer, Takifugu pardalis 1.0 g Gonad Liver 2 min Fig. 2. The contractile activity of various tissues' extracts of the starfish Asterina pectinifera on the smooth muscle of other organisms. A; DRM of the starfish Asterina pectinifera, B; Esophagus of the squid Todarodes pacificus, C; Intestine of the panther puffer T akifugu pardalis. The extracts were applied at the time indicated by arrows.... 별불가사리 DRM, 오징어식도및졸복의장관평활근에대한수축활성 DRM, 10-4 M ACh 100%, ACh. Table 1 DRM.,, DRM.,, 100%, 80%.,.. Fig. 2B Table M Carb 100%, Carb. (164.3%) (103.6%), (71.4%) Table 1. The contractile activity of various tissues' extracts of the starfish Asterina pectinifera on the smooth muscle of the other organisms (n=3) Sample DRM (A. pectinifera) Contractile activity (%) Esophagus (T. pacificus) Intestine (T. pardalis) Muscle ±4 N.T. Gut ±1 N.T. Gonad N.T ±3 52.3±2 Liver 77.8± ±2 36.4±1 Tube Feet 22.2±3 14.3±5 N.T. Body 100±1 42.1±2 N.T. 1 N.T.; not tested.

5 126 고혜진ㆍ조미정ㆍ김군도ㆍ박남규 Muscle Gut Liver Tube feet Gonad Body Ach 10-4 M Ach 10-4 M Ach 10-4 M Ach 10-4 M Ach 10-4 M Ach 10-4 M Fig. 3. The relaxant activity of various tissues' extracts of starfish Asterina pectinifera on the starfish Asterina pectinifera DRM. The extracts were applied at the time indicated by arrows...,, (phasic contraction) (Fig. 2A, Table 1)., (amplitude) (tone), (Fig. 2C). DRM Fig. 3 Table 2. Table %, 92.1%, 76.8%, 78.9%, 62.5%.,, ACh (Fig. 3)., (Stichopus japonicus) longitudinal muscle body wall, 2 (Iwakoshi et al., 1995), (Holothuria glaberrima) (Diaz-Miranda et al., 1995). Asterias rubens Asterias forbesi radial nerve cords FMRFamide peptide S1 S2 (Elphick et al., 1991), cardiac stomach (Elphick and Melarange, 2001). Table 2. Relaxant activity of various tissues' extracts on the starfish Asterina pectinifera DRM (n=3) Sample Relaxant activity (%) Muscle 41.4±8 Gut 92.1±3 Gonad 76.8±2 Liver 78.9±4 Tube Feet 62.5±5 Body 0 Table 3. In vitro DPPH radical scavenging activities of various tissues extracts of the starfish Asterina pectinifera (n=3).. 항산화활성 Sample DPPH DPPH radical. Vitamin C 0.8, 4, 20, g/ml, DPPH radical 13, 19, 60, 88 90% (data not shown). Table 3 DPPH radical %, 100 1%, %, %,, 100 1%., 27.2%., (Asterias amurensis) (Kwon MC et al., 2007), (Kim IH et al., 2001).,. 사사 2007 ( ) (No. KRF F00042) References Radical Scavenging Effect (RSE, %) Muscle 27.2±5 Gut 95.8±2 Gonad 100.0±1 Liver 97.3±2 Tube Feet 67.3±3 Body 100.0±1 Ames BN, Shigenaga MK and Hagen TM Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl

6 별불가사리추출물의생리활성 127 Acad Sci USA 90, Charlet M, Chernysh S, Philippe H, Hetru C, Hoffmann JA and Bulet P Innate immunity. Isolation of several, cysteine-rich antimicrobial peptides from the blood of a mollusk, Mytilus edulis. J Biol Chem 271, Chauvet J, Rouille Y, Chauveau C, Chauvet MT and Acher R Special asvatocin and phasvatocin, two new oxytocinlike peptides in the spotted dogfish (Scyliorhinus caniculus). Proc Natl Acad Sci USA 91, Chea SY, Kim MJ, Kim DS, Park JE, Jo SK and Yee ST Effect of Asterina pectinifera extracts on the activation of immune cells. J Korean Soc Food Sci Nutr 36, Destoumieux D, Bulet P, Loew D, Van DA, Rodriguez J and Bachere E Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus Vannamei (Decapoda). J Biol Chem 272, Diaz-Miranda L, Blanco RE and Garcia-Arraras JE Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic study. J Comp Neurol 352, Donia M and Hamann MT Marine natural products and their potential applications as anti-infective agents. Lancet Infect Dis 3, Dunlap WC and Yamamoto Y Small-molecule antioxidants in marine organims: antioxidant activity of mycosporine-glycine. Comp Biochem Physiol B 112, Edward ER, Richard SF and Robert DB Invertebrate Zoology 7th edition. Thomson Learning, CA, U.S.A., Elliott GR and Barchas JD Neuroregulators: neurotransmitters and neuromodulators. Behav Brain Sci 2, Elphick MR and Melarange R Neural control of muscle relaxation in echinoderms. J Exp Biol 204, Elphick MR, Price DA, Lee TD and Thorndyke MC The SALMFamides: a new family of neuropeptides in the starfish Asterias rubens. J Exp Biol 198, Erspamer V and Anastasi A Structure and pharmacological actions of eledoisin, the active endecapeptide of the posterior salivary glands of Eledone. Experientia 18, Fan J, He J, Zhuang Y and Sun L Purification and Identification of Antioxidant Peptides from Enzymatic Hydrolysates of Tilapia (Oreochromis niloticus) Frame Protein. Molecules 17, Hancock REW and Diamond G The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol 8, Iijima N, Tanimoto N, Emoto Y, Morita Y, Uematsu K, Murakami T and Nakai T Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major. Eur J Biochem 270, Irwin DM and Wong J Trout and chicken proglucagon: alternative splicing generates mrna transcripts encoding glucagon-like peptide. Mol Endocrinol 9, Iwakiri M, Sugiyama A, Ikeda T, Muneoka Y and Kubota I A novel oxytocin-like peptide isolated from the neural complexes of tunicate, Styele plicata. Zool Sci 7, Iwakoshi E, Ohtani M, Takahashi T, Muneoka Y, Ikeda T, Fujita T, Minakata H and Nomoto K Comparative aspects of structure and action of bioactive peptides isolated from the sea cucumber Stichopus japonicus. In: peptide Chemistry. Ohno M. ed. Protein Research Foundation, Osaka, Japan, Jensen J and Colon JM Substance-P-related and neurokinin-a related peptides from the brain of the cod and trout. Eur J Biochem 206, Kang DG, Yun CK and Lee HS Screening and comparison of antioxidant activity of solvent extracts of herbal medicines used in Korea. J Ethnopharm 87, Kim WG, Kim JP, Koshino H, Shin-Ya K, Seto H and Yoo ID Benzastatin E, F, and G: new indoline alkaloids with neronal cell protectin activity form Streptomyces nitrosporesus. Tetrahedron 53, Koshino H, Lee IK, Kim JP, Kim WG, Uzawa J and Yoo ID Agrocybenine, novel class alkaloid from the Korean mushroom Agrocybe cylindracea. Tetrahedron Letters 37, Krieger DT Brain peptides: what, where, and why? Science 222, Krieger DT An overview of neuropeptides. In: Neuropeptides in Neurologic and Psychiatric Disease. Martin JB and Barchas JD eds. Raven Press, New York, U.S.A., Kwon MC, Kim CH, Kim HS, Hwang BY and Lee HY Anti-wrinkle Activity of Low Molecular Weight Peptides Derived from the Collagen Isolated from Asterias amurensis. Korean J Food Sci Technol 3, Lehrer RI, Rosenman M, Harwig SSL, Jackson R and Eisenhaur P Ultrasensitive assay for endogenous antimicrobial polypeptides. J Immunol Methods 137, Li C, Haug T, Styrvold OB, Jørgensen TØ and Stensvåg K Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis. Dev Comp Immunol 32, dci Matsunaga S, Fusetani N and Konosu S Bioactive marine metabolites, Ⅳ. Isolation and the amino acid composition of discodermin A, an antimicrobial peptide, from the marine sponge Discodermia kiiensis. J Nat Prod 48, Martinage A, Belaiche D, Dupressoik T and Sautiere P Primary Structure of Histone H2A from Gonads of the Starfish Asterias rubens. Eur J Biochem 130, Mitta G, Hubert F, Dyrynda EA, Boudry P and Roch P

7 128 고혜진ㆍ조미정ㆍ김군도ㆍ박남규 Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis. Dev Comp Immunol 24, Mitta G, Hubert F, Noel T and Roch P Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis. Eur J Biochem 265, Miyata T, Tokunaga F, Yoneya T, Yoshikawa K, Iwanaga S, Niwa M, Takao T and Shimonishi Y Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity. J Biochem 106, Ovchinnikova TV, Aleshina GM, Balandin SV, Krasnosdembskaya AD, Markelov ML, Frolova EI, Leonova YF, Tagaev AA, Krasnodembsky EG and Kokryakov VN Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina. FEBS Lett 577, org/ /j.febslet Ovchinnikova TV, Balandin SV, Aleshina GM, Tagaev AA, Leonova YF, Krasnodembsky ED, Men shenin AV, Kokryakov VN Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita with structural features of defensins and channel-blocking toxins. Biochem Biophys Res Commun 348, Park CH, Erika V, Valore, Waring AJ and Ganz T Hepcidin, a Urinary Antimicrobial Peptide Synthesized in the Liver. J Biol Chem 276, Park JH, Kang KC, Baek SB, Lee YH and Rhee KS Separation of antioxidant compounds from edible marine algae. Korean J Food Sci Technol 23, Rice-Evans CA and Miller NJ Antioxidant activities of flavonoids as bioactive compounds of food. Biochem Soc Trans 24, Sampath Kumar NS, Nazeer RA and Jaiganesh R Purification and identification of antioxidant peptides from the skin protein hydrolysate of two marine fishes, horse mackerel ( Magalaspis cordyla) and croaker (Otolithes ruber). Amino Acids 42, s Seo JK, Crawford JM, Stone KL and Noga EJ Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica. Biochem Biophys Res Comm 338, Seo JK, Lee MJ, Go HJ, Kim GD, Jeong HD, Nam BH and Park NG Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas. Mol Immunol 53, molimm Zadina JE, Banks WA and Kastin AJ Central nervous system effects of peptides, : a cross-listing of peptides and their central actions from the first six years of the journal peptides. Peptides 7,

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untitled 3. 농업환경연구과 과제구분 기본연구 수행시기 전반기 연구과제 및 세부과제 수행 기간 소 속 책임자 농가에 적합한 부식성곤충 대량 사육기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 1) 부식성 곤충 먹이 제조 기술 개발 12~ 13 농업환경연구과 곤충팀 이영혜 색인용어 부식성곤충, 장수풍뎅이, 계통, 먹이제조 ABSTRACT In first check,