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研究生: 胡喬伊
Cesarea Hulda Joel
論文名稱: 自長莖葡萄蕨藻篩選製備具血管收縮素轉化酶抑制活性之胜肽
Screening of Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from Caulerpa lentillifera
指導教授: 徐睿良
Jue-Liang Hsu
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
畢業學年度: 106
語文別: 英文
論文頁數: 62
中文關鍵詞: ACE抑制胜肽生物活性指引分群海葡萄抗高血壓作用
外文關鍵詞: ACE inhibitory peptide, antihypertensive effect, sea grapes, bioassay-guided fractionation
DOI URL: http://doi.org/10.6346/THE.NPUST.BST.005.2018.D01
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  • 具血管收縮素轉化梅(Angiotensin Converting Enzyme,ACE)抑制活性之胜肽,由於其具備抗高血壓之潛力及消費者對於合成藥物安全性之疑慮,使其成為現今食品工業中極具吸引力之研究主題。本研究之目的為利用不同蛋白酶水解長莖葡萄蕨藻 Caulerpa lentillifera,俗稱:海葡萄),並由其水解物中篩選並製備具ACE抑制活性之胜肽。藉由不同酵素水解16小時獲得其水解物,並透過3kDa超過濾膜過濾,進而得到其短鏈胜肽。結果顯示,經由嗜熱菌蛋白酶水解之水解物,對ACE具有最高的抑制活性,其IC50值為11.86±0.98μg/ mL。將水解物利用逆相高效能液相層析儀進行生物活性指引分群,結果顯示,分液9具有最高的ACE抑制活性。將分液9利用液相層析-串聯式質譜儀進行胜肽鑑定分析,並透過從頭定序法(de novo)計算得到二段胜肽,序列分別為Phe-Asp-Gly-Ile-Pro (FP-5) 和Ala-Ile-Asp-Pro-Val-Arg-Ala (AA-7),其IC50分別為58.89±0.68μM和65.76±0.92μM,且酵素動力學結果顯示,FP-5和AA-7皆為競爭型抑制劑。此研究首度報導自長莖葡萄蕨藻之水解物中篩選出具有抑制ACE之活性肽,將有益於高血壓之預防且具做為功能性食品開發之潛力。

    Peptides with Angiotensin Converting Enzyme (ACE, EC 3.4.15.1) inhibitory activity have received considerable interest from the food industry due to their potential as antihypertensive and the consumer’s concern over the safety of the synthetic counterparts. The objectives of this study were to isolate ACE inhibitory peptide from Caulerpa lentillifera (known commonly as sea grape) protein hydrolysate using different proteases. Short-chain peptides were obtained after 16 hours of hydrolysis by various enzymes and followed by ultrafiltration using 3 kDa molecular weight cut off (MWCO) membrane. The results indicated that thermolysin hydrolysate had the highest ACE inhibitory activity with the IC50 value of 11.86±0.98μg/mL. Bioassay-guided fractionation was performed using reversed-phase high performance liquid chromatography (RP-HPLC). The fraction 9 showed the highest ACE inhibitory activity. Peptides in fraction 9 were sequenced by liquid chromatography tandem mass Spectrometry (LC-MS/MS). LC-MS/MS analysis of fraction 9 gave the peptides sequence as Phe-Asp-Gly-Ile-Pro (FP-5) and Ala-Ile-Asp-Pro-Val-Arg-Ala (AA-7) identified using de novo method. Furthermore, their IC50 values were determined as 58.89±0.68μM and 65.76±0.92μM, respectively. The kinetic study revealed that FP-5 and AA-7 are competitive inhibitors. Therefore, we can conclude that it is the first report about ACE inhibitory peptides derived from Caulerpa lentillifera and it may be beneficial for preventing hypertension and functional food development.

    TABLE OF CONTENT
    COVER .............................................................................................................. I
    CHINESE ABSTRACT ................................................................................... II
    ABSTRACT .................................................................................................... III
    ACKNOWLEDGEMENTS ............................................................................ V
    TABLE OF CONTENTS ............................................................................. VII
    LIST OF FIGURES ......................................................................................... IX
    LIST OF TABLES .......................................................................................... X
    I. INTRODUCTION ......................................................................................... 1
    1.1 Background .............................................................................................. 1
    1.2 Future Impact ........................................................................................... 3
    II. LITERATURE RIVIEW .............................................................................. 4
    2.1 Hypertension ............................................................................................ 4
    2.2 Cause of Hypertension ............................................................................. 5
    2.3 Angiotensin Converting Enzyme (ACE) ................................................. 6
    2.4 ACE Inhibition Mechanism ..................................................................... 8
    2.5 Caulerpa lentillifera ............................................................................... 10
    2.6 Bioactive Peptide ................................................................................... 11
    2.7 Enzymatic Hydrolysis ............................................................................ 12
    2.8 Ultrafiltration.......................................................................................... 14
    2.9 LC-MS/MS Analysis.............................................................................. 14
    2.10 Peptide Synthesis ................................................................................. 15
    2.11 Molecular Docking .............................................................................. 16
    III. MATERIALS AND METHODS .............................................................. 18
    3.1 Experimental Design .............................................................................. 18
    3.2 Materials ................................................................................................. 19
    3.3 Instruments ............................................................................................. 19
    3.4 Experimental Protocol ............................................................................ 20
    3.4.1 Sample Preparation ...................................................................... 20
    3.4.2 Extraction of Caulerpa lentillifera Protein .................................. 20
    3.4.3 Protein Purification ...................................................................... 20
    3.4.4 Enzymatic Digestion .................................................................... 21
    3.4.5 Protein Content Determination .................................................... 21
    3.4.6 Desalting ....................................................................................... 22
    3.4.7 Fractionation Using RP-HPLC .................................................... 22 3.4.8 ACE Inhibitory Assay and Determination of IC50 ........................ 22
    3.4.9 Identification Peptide Sequence Using LC-MS/MS .................... 23 3.4.10 De Novo Sequencing .................................................................... 24 3.4.11 Peptide Synthesis .......................................................................... 24
    3.4.12 Peptide Stability towards ACE ..................................................... 25
    3.4.13 Molecular Docking Simulation .................................................... 25
    3.4.14 Inhibition Pattern of ACE Inhibitory Peptide .............................. 26
    3.4.15 Statistical Analysis ........................................................................ 26
    IV RESULTS .................................................................................................. 27
    4.1 ACE Inhibitory Assay of Caulerpa lentillifera Protein Hydrolysate .... 27
    4.2 IC50 of Thermolysin Hydrolysate ........................................................... 28
    4.3 Fractionation .......................................................................................... 28
    4.4 Peptide Sequence Identification ............................................................. 30
    4.5 Ion Fragments Confirmation .................................................................. 31
    4.6 IC50 Determination Using Synthetic Peptide ......................................... 34
    4.7 Lineweaver-Burk Plot ............................................................................ 34
    4.8 Molecular Docking Simulation .............................................................. 36
    4.9 Stability of ACE Inhibitory Peptides under ACE Preicubation............. 37
    V. DISCUSSION ............................................................................................ 39
    VI. CONCLUSION ......................................................................................... 45
    REFERENCES ................................................................................................ 46
    INFORMATION OF AUTHOR ..................................................................... 52

    Ahluwali M, Bangalore S. Management of hypertension in 2017 targets and therapies. Current Opinion in Cardiology 2017; 32(4):413–421.
    Ambigailapan, Priyatharini, Abdulrahaman S, Al-Khalifa, Fereidoon S. Antioxidant and angiotensin-I converting enzyme (ACE) inhibitory activities of date seed protein hydrolysates prepared using alcalase, flavourzyme and thermolysin. Journal of Functional Foods 2015; 18:1125-1137.
    American Heart Association. Guideline: Understanding and Managing High Blood Pressure 2014. http://www.heart.org. Accessed at October 2017.
    Anderson C, Arnolda L, Cowley D. Guideline for the diagnosis and management of hypertension in adults. National Heart Foundation of Australia 2016; ABN 98 008 419 761.
    Andrews PR, Carson JM, Caselli A, Spark MJ, Woods R. Conformational analysis and active site modeling of angiotensin converting enzyme inhibitors. Journal Medical Chemistry 1985; 28:393-399.
    Arora PK, Chauhan A. ACE inhibitors: a comprehensive review. International Journal of Pharmaceutical Sciences and Research 2013; 4(2):532-549.
    Beldent V, Michaud A, Wei L, Chauvet MT, Corvol P. Proteolytic release of human angiotensin-I converting enzyme: localization of the cleavage site. The Journal of Biological Chemistry 1993; 268:428–434.
    Bhuyan BJ, Mugesh G. Synthesis characterization and antioxidant activity of angiotensin converting enzyme inhibitors. Biomolecules Chemistry 2011; 9:1356-1365.
    Burt VL, Cutler JA, Higgins M. Trends in the prevalence, awareness, treatment, and control of hypertension in the adult US population: data from the health examination surveys, 1960 to 1991. Hypertension 1995; 26:60.
    Byun, Hee G, Kim SK. Purification and characterization of Angiotensin-I converting enzyme (ACE) inhibitory peptides from alaska pollack
    (Theragra chalcogramma) skin. Process Biochemistry 2001; 36(12):1155-1162. Capon RJ, Ghisalberti EL, Jefferies PR. Metabolites of the green-algae, Caulerpa Species. Phytochemistry 1983; 22(6):1465-1467.
    Cha SH, Lee KW, Jeon YJ. Screening of extracts from red algae in Jeju for potentials marine angiotensin-I converting enzyme (ACE) inhibitory activity. Algae 2006; 21:343–348.
    De Dios AM. Angiotensin-converting enzyme inhibitors in the treatment of hypertension, Rev. Argent. Cardiol 2011; 79(2):103–105.
    De Wet H, Ramulondi M, Ngcobo ZN. The use of indigenous medicine for the treatment of hypertension by a rural community in Northern Maputaland, South Africa. South Africa Journal of Botany 2016; 103:78-88.
    Diego A, Vital L, Mojica L, de Mejía EG, Mendoza S, Loarca-Piña G. Biological potential of protein hydrolysates and peptides from common bean (Phaseolus vulgaris). Food Research International 2015; 76:39-50.
    Esther CR, Jr. Marine EM, Bernstein KE. The role of angiotensin-converting enzyme in blood pressure control, renal function, and male fertility. Trends in Endocrinology and Metabolism 1997; 8(5):181-186.
    Fakim GA. Medicinal plants: traditions of yesterday and drugs of tomorrow. Molecular Aspects of Medicine 2006; 27:1–93.
    Fujita H, Yoshikawa M. LKPNM: a prodrug-type ACE inhibitory peptide derived from fish protein. Immunopharmacology 1999; 44:123–127.
    Huang ZG. Marine species and their distributions in China seas. Beijing China Ocean Press 1994; 224.
    Ikeda K, Kitamura A, Machida H, Watanabe M, Negishi H, Hiraoka J. Effect of Undaria pinnatifida (WAKAME) on the development of cerebrovascular disease in stroke-prone spontaneously hypertensive rats. Clinical and Experimental Pharmacology and Physiology 2003; 30:44–48.
    Iwaniak A, Minkiewicz P, Darewicz M. Food-originating ACE inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction. Comprehensive Reviews in Food Science and Food Safety 2014; 13:114–134. Katzung BG, Masters SB, Trevor AJ. Basic and clinical pharmacology. 12th ed. New York: McGraw-Hill; 2012.
    Kamath V, Niketh S, Chandrashekar A, Rajini PS. Chymotryptic hydrolysates of α-kafirin, the storage protein of sorghum (Sorghum bicolor) exhibited angiotensin converting enzyme inhibitory activity. Food Chemistry 2007; 100: 306–311.
    Kong X, Zhou H, Qian H. Enzymatic preparation and functional properties of wheat gluten hydrolysates. Food Chemistry 2007; 101:615–620.
    Lee SH, Qian ZJ, Kim SK. A novel angiotensin-I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chemistry 2010; 118: 96–102.
    Lewington S, Clarke R, Qizilbash N, Peto R, Collins R. Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 2002; 360:1903–1913.
    Li GH, Le GW, Shi YH, Shrestha S. Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition Research 2004; 24:469–486. Li GH, Qu MR, Wan JZ, You JM. Antihypertensive effect of rice protein hydrolysate with in vitro angiotensin-I converting enzyme inhibitory activity in spontaneously hypertensive rats. Asia Pacific Journal of Clinical Nutrition 2007; 16:275-280.
    Maeda R, Ida T, Ihara H, Sakamoto T. Induction of apoptosis in MCF-7 cells by β-1,3-xylooligosaccharides prepared from Caulerpa lentillifera. Bioscience Biotechnology Biochemistry 2012; 76:1032-1034.
    Manilal A, Sujith S, Kiran GS, Selvin J, Shakir C, Gandhimathi R. Bio-potentials of seaweeds collected from Southwest Coast of India. Journal of Marine Science and Technology 2009; 17:67–73.
    Mary A, Mary V. Lorella A, Matias, JR. Rediscovery of naturally occurring seagrape Caulerpa lentillifera from the gulf of mannar and its mariculture. Current Science 2009; 97:1418–20. Matanjun P, Mohamed S, Mustapha NM, Muhammad K. Nutrient content of tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera, and Sargassum polycystum. Journal of Applied Phycology 2009; 21(1):75-80.
    Millioni R, Franchin C, Pivato M, Tessari P, Arrigoni, G. Sample loading influences studies comparing isoelectric focusing vs. strong cation exchange peptide fractionation. Journal of Chromatography A 2013; 1307:207–208.
    Morihara K, Tswzuki H. Thermolysin: kinetic study with oligopeptides. European Journal of Biochemistry 1970; 15:374-380.
    Natesh R, Schwager SL, Sturrock ED, Acharya KR. Crystal structure of the human angiotensin-converting enzyme lisinopril complex. Nature 2003; 421(6922):551-554.
    NHLBI (National High Blood Pressure Education Program). Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. Bethesda MD, National Heart, Lung, and Blood Institute 2003. http://www.nhlbi.nih.gov/guidelines/ hypertension/. Accessed at October 2017. Ni H, Li L, Liu G, Hu SQ. Inhibition mechanism and model of an angiotensin-I converting enzyme (ACE) inhibitory hexapeptide from yeast (Saccharomyces cerevisiae). Plose one 2012; 7(5): e37077
    Ogihara T, Mikami H, Katahira K, Otsuka, A. Comparative study of the effects of three angiotensin converting enzyme inhibitors on the cough reflex. American Journal of Hypertension 1991; 4(2):46–51.
    Ondetti MA, Rubin B, Cushman DW. Design of specific inhibitors of angiotensin-I converting enzyme: new class of orally active antyhypersentive agents. Science 1977; 196(4288):441-444.
    Pank M, Kirret O, Paberit N, Aaviksaar A. Hydrophobic interaction in thermolysin specificity. FEBS Letters 1982; 142: 297-300.
    Pickering G. High blood pressure. London: Churchill; 1968.
    Pihlanto-Leppälä A. Bioactive peptides derived from bovine whey proteins: opioid and ace-inhibitory peptides. Trends in Food Science and Technology 2000; 11:347–356.
    Priyanto AD, Doerksen RJ, Chang CI, Sung WC, Widjanarko SB, Kusnadi J, Lin YC, Wang TC, Hsu JL. Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. Journal of Proteomics 2015; 128:424-435.
    Pujiastuti YP, Shih YH, Chen WL, Sukoso, Hsu JL. Screening of angiotensin-I converting enzyme inhibitory peptides derived from soft-shelled turtle yolk using two orthogonal bioassay-guided fractionations. Journal of Fucntional Foods 2017; 28:36-47. Rosendorff C, Lackland DT, Allison M, Aronow WS, Black HR, Blumenthal RS, Cannon CP, de Lemos JA, Elliott WJ, Findeiss L, Gersh BJ, Gore JM, Levy D, Long JB, O'Connor CM, O'Gara PT, Ogedegbe O, Oparil S, White WB, American Heart Association, American College of Cardiology, American Society of Hypertension. Treatment of hypertension in patients with coronary artery disease: a scientific statement from the American Heart Association, American College of Cardiology, and American Society of Hypertension. Journal of the American Society of Hypertension 2015; 9(6):453-98. Sato M, Hosokawa T, Yamaguchi T, Nakano T, Muramoto K, Kahara T, Funayama K, Kobayashi A. Angiotensin-I converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. Journal of Agriculture Food Chemistry 2002; 50:6245-6252.
    Singh BP, Vij S, Hati S. Functional significance of bioactive peptides derived from soybean. Peptides 2014; 54:171-179.
    Skeggs LT, Kahn JR, Shumway NP. The purification of hypertensin-II. The Journal of Experimental Medicine 1956; 103:301-307.
    Suetsuna K, Nakano T. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). Journal of Nutritional Biochemistry 2000; 11:450-454.
    Sunantha K, Rawdkuen S. Purification and characterization of ACE inhibitory peptide from aquatic resources: review Sunantha Ketnawa and Saroat Radwuken program of food technology, school of agro-industry, Mae Fah Luang University, to whom correspondence shou. International Journal of Plant, Animal, and Environment Science 2013; 3(1):220-233.
    Wei L, Alhenc-Gelas F, Corvol P, Cluuser E. The two homologous domains of human angiotensin-I converting enzyme interact differently with competitive inhibitors. The Journal of Biological Chemistry 1992; 267:13398-13405.
    Wijesekara I, Kim SK. Angiotensin-I converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical Industry. Marine Drugs 2010; 8:1080–1093.
    Yokoyama K, Chiba H, Yoshikawa M. Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Bioscience Biotechnology Biochemistry 1992; 56(10):1541-1545.

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