Dipeptide (Tyr-Ile) Acting as an Inhibitor of Angiotensin-I-Converting Enzyme (ACE) from the Hydrolysate of Jellyfish Nemopilema nomurai

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  • ABSTRACT

    The jellyfish Nemopilema nomurai was hydrolyzed with papain and a novel dipeptide purified via ultrafiltration, gel filtration chro-matography with Sephadex LH-20, and reverse phase chromatography using C18 and C12 columns. The IR, 1H NMR, 13C NMR, and MS spectrometer analyses showed that the dipeptide comprised tyrosine-isoleucine (Tyr-Ile). The IC50 and Ki values were 6.56 ± 1.12 and 3.10 ± 0.28 μM, respectively, indicating competitive inhibition of angiotensin-Ι-converting enzyme (ACE). As a novel ACE-inhibitory active peptide, Tyr-Ile may have potential for use in antihypertensive therapy.


  • KEYWORD

    ACE , Jellyfish , Nemopilema nomurai , Papain , Tyrosine-isoleucine

  • Introduction

    Functional foods may contain bioactive peptides since they exert a physiological effect in the body. These peptides are released from dietary protein by enzymatic hydrolysis or dur-ing processing. Bioactive peptides that regulate blood pres-sure are believed to be inhibitors of angiotensin-Ι-converting enzyme (ACE). Those peptides are, in general, inactive within the sequence of the parent protein, but can be released dur-ing gastrointestinal digestion or food processing (Shahidi and Zhong, 2008). Recently, many ACE-inhibitory peptides have been isolated and characterized from various protein hydroly-sates such as cheese (Smacchi and Gobbetti, 1998), milk pro-tein (Gobbetti et al., 2000), egg white (Miguel et al., 2007), plant protein (Dziuba et al., 1999; Wu et al., 2008), meat (Jang and Lee, 2005), and marine resources (Je et al., 2005; He et al., 2006). Various ACE-inhibitory peptides have also been isolated from fish protein, e.g., sardines (Matsui et al., 1993), tuna (Kohama et al., 1988), and cod (Kim et al., 2000). Pro-tein hydrolysates are usually produced by gastrointestinal en-zymes (e.g., pepsin or pancreatin) or microbial enzymes (e.g., Protamex; Novozymes, Bagsvaerd, Denmark) under varying conditions.

    ACE (EC 3.4.15.1) is a circulating enzyme that partici-pates in the rennin-angiotensin system and plays an important physiological role in regulating blood pressure. ACE acts as an exopeptidase that cleaves dipeptides from the C-terminus of various oligopeptides. It converts the inactive form of de-capeptide (angiotensin Ι) to a potent octapeptide vasoconstric-tor (angiotensin II), and inactivates the catalytic function of bradykinin, which has a depressor action (Ondetii et al., 1977; FitzGerald et al., 2004).

    Nemopilema nomurai is a giant jellyfish species that has been blooming offshore of Korea, China, and Japan in the last several years. Jellyfish have traditionally been consumed to treat asthma and hypertension in East Asian countries. Despite this, knowledge of the active compound(s) is lacking. Thus, the aim of this study was to determine the functional prop-erties of the active compounds of N. nomurai and assess its possible application in antihypertensive therapy. We hydro-lyzed N. nomurai jellyfish with papain, and then purified and elucidated the structure and the inhibition pattern of an ACE-inhibitory peptide.

    Materials and Methods

      >  Materials

    A giant jellyfish, N. nomurai, was caught south of Jeju Is-land, Korea. Only the umbrella (mesogloea) was collected, washed with deionized water, and then stored at -23℃ until required. ACE from rabbit lung, the substrate hipuryl-L-histi-dyl-L-leucine (Hip-His-Leu), trifluoroacetic acid (TFA), aceto-nitrile (CH3CN), and papain were purchased from Sigma-Al-drich Chemical Co. (St. Louis, MO, USA). Sephadex LH-20 (Amersham Pharmacia Biotech, Tokyo, Japan) used for gel filtration chromatography, and C18 and C12 columns used for reverse-phase HPLC were obtained from Phenomenex Inc. (Torrance, CA, USA). Other reagents were of reagent grade and used without further purification.

      >  Enzymatic hydrolysis

    Papain hydrolysis of the jellyfish was conducted under the following conditions. First, jellyfish samples were lyophilized to remove excess water. Enzymatic hydrolysis (100 mL) was carried out for 4 h with 10% (w/v) lyophilized jellyfish at 60℃ and pH 6.0. The ratio of enzyme to reaction volume was varied from 0 to 2% (w/v). Each enzyme reaction was stopped by heat treatment at 90℃ for 15 min. The resultant slurry was centrifuged at 3,000 g for 10 min, and the supernatant was ly-ophilized and then used for analysis. The degree of hydrolysis was defined as the proportion (%) of α-amino nitrogen with respect to the total-N in the samples (Taylor, 1957).

    Degree of hydrolysis (%) = (h/htot) × 100,

    where htot is the total-N of lyophilized samples, and h is the quantity of α-amino nitrogen.

      >  Determination of ACE-inhibitory activity

    ACE-inhibitory activity was determined using the modified method of Cushman and Cheung (1971). The standard reac-tion mixture contained 5 mM Hip-His-Leu as a substrate, 0.3 M NaCl, and 5 mU ACE in 50 mM sodium borate buffer (pH 8.3). A sample (50 μL) was added to enzyme solution (50 μL) and then mixed with 8.3 mM Hip-His-Leu (150 μL) contain-ing 0.5 M NaCl to obtain the same concentration as the stan-dard reaction mixture. After incubation at 37℃ for 30 min, the reaction was stopped by the addition of 1.0 N HCl (250 μL). The resulting hippuric acid was extracted by addition of 1.5 mL ethyl acetate. After centrifugation (800 g, 15 min), 1 mL of the upper layer was transferred to a new glass tube and evapo-rated at room temperature for 2 h in a vacuum. The extracted hippuric acid was dissolved in 3.0 mL distilled water, and the absorbance at 228 nm was measured using a spectrophotom-eter (model U-3210; Hitachi Co., Tokyo, Japan).

      >  Fractionation of jellyfish hydrolysates

    The papain hydrolysate showing the greatest inhibitory ac-tivity was selected and the resultant hydrolysate was fraction-ated through an Amicon Millipore membrane (1 kDa cutoffs; Amicon, Beverly, MA, USA). The resultant fraction (FII) was lyophilized and then stored at -82℃ until required.

      >  Purification of ACE-inhibitory peptide

    The active fraction (FII) was further purified by Sephadex LH-20 (25 × 250 mm) gel filtration chromatography, eluting with 30% methanol at a flow rate of 0.5 mL/min, and reverse-phase HPLC eluting with a linear gradient of MeOH-H2O (A eluent; H2O:MeOH:TFA = 90:10:0.1 (v/v/v), B eluent; MeOH:H2O: TFA = 90:10:0.1 (v/v/v)) at a flow rate of 2 mL/min (C18 5 μM ODS 3100A, 10 × 250 mm, UV detection at 214 nm; Phenomenex). To obtain pure peptide, a Jupiter Pro-teo C12 column (90 A, 10 ㎛, 21.2 × 150 mm; Phenomenex) was used at a flow rate of 2 mL/min with a linear CH3CN-H2O gradient (A eluent; H2O:CH3CN:TFA = 95:5:0.1 (v/v/v), B eluent; CH3CN:H2O: TFA = 55:45:0.08 (v/v/v).

      >  Structure of the purified peptide

    The IR spectra were recorded on a Fourier transform (FT)-IR model IFS-88 spectrometer (Bruker, Billerica, MA, USA). NMR spectra were recorded on a JEOL JNM ECP-400 (400 MHz for 1H, 100 MHz for 13C) spectrometer (JEOL Ltd., Tokyo, Japan). Chemical shift (δ) values were expressed in ppm and were referenced to the residual solvent signals with resonances at δH/δC, 7.26/77.0 (CDCl3). To calculate molecu-lar weights, purified samples were processed on an 1100 LC/MSD spectrometer (Agilent Technologies, Santa Clara, CA, USA) with direct injection onto an electrospray interface in the positive or negative mode. The structure of the purified peptide was conformed using Chemdraw 11 (CambridgeSoft, Cambridge, MA, USA).

      >  IC50 values and kinetic analysis

    The IC50 value of the purified peptide was determined by a standard method, and was defined as the concentration of inhibitor required to inhibit 50% of ACE activity. For a Line-weaver-Burk plot, 0-4 μg/mL of purified peptide was added to the reaction solution as an inhibitor, and the inhibition pattern and Ki were estimated using GraFit 5.0 (GraFit Software, Sur-rey, UK).

    Results and Discussion

      >  Papain digestion of jellyfish

    The hydrolysis rates of jellyfish mesogloea ranged from 2 to 17%, depending on the papain concentration used. Ex-amination of the ACE-inhibitory activity of each hydrolysate revealed that the activity increased significantly by as much as 62% after 4 h digestion, although negligible activity was detected in intact jellyfish. The greatest inhibitory activity was obtained with an enzyme concentration of 0.2% (w/v), and it then leveled off even in a prolonged reaction (Fig. 1). Pa-pain can digest most protein substrates and exhibits a broad substrate specificity, cleaving the peptide bonds of basic amino acids, leucine, or glycine. Because of this broad sub-strate specificity, many peptides and amino acids have been produced, leading to higher ACE-inhibitory activity. Lee et al. (2001) and Kim et al. (1996) reported that no relationship existed between the degree of hydrolysis of laver and fresh-water fish and ACE-inhibitory activity. In the case of laver hydrolysate, both neutrase and papain achieved a similar de-gree of hydrolysis (50.7% and 51.3%, respectively) and ACE-inhibitory activity (34.3% and 34.9%, respectively), whereas the hydrolysis rate and ACE-inhibitory activity of jellyfish differed markedly. This indicates that different substrates will show clear differences in reaction pattern.

      >  Fractionation and characterization of papain hydrolysate

    The papain hydrolysate was fractionated by ultrafiltration (1 kDa MWCO) and the activity determined. The resulting fraction (FII, <1 kDa) possessed 61.1% of the total activity, whereas the retained fraction (FI, >1 kDa) possessed 35.0% (Fig. 2). These findings suggested that the most active com-pounds were present in the FII fraction and that this activity was newly exposed by papain treatment because no recogniz-able inhibitory activity was detected before papain hydrolysis. Molecular weights (Mw) of fraction FII were less than 1 kDa, since a 1 kDa MWCO membrane was used.

    To inhibit ACE in vivo, peptides must reach the blood intact. Many barriers in the body might limit or enhance the activity of the peptides in vivo. When proteins or peptides are ingested, the first barriers are the stomach and small intestine, where proteins and peptides are broken down by enzymes such as pepsin, trypsin, and α-chymotrypsin. The resulting oligopep-tides and free amino acids are absorbed into the blood. During absorption, peptides are further hydrolyzed by brush border peptidases and peptidases in enterocytes (Vermeirssen et al., 2004). Byun and Kim (2001) observed a correlation between

    the molecular weight of hydrolysate and the specificity of the ACE-inhibitory activity, and revealed that activities increased with decreasing molecular weight. However, we were unable to detect a correlation between molecular wieght and ACE-inhibitory activity.

      >  Purification of the ACE-inhibitory peptide

    To purify the ACE-inhibitory peptide, fraction FII was sub-jected to a Sephadex LH-20 column, and two major fractions were obtained: FII-A and FII-B (Fig. 3). The ACE-inhibitory activities of FII-A and FII-B represented 54.01% and 55.08%, respectively. FII-B was further purified by HPLC using a re-verse-phase column (ODS C18). Inhibitory activity was detect-ed in a wide range of eluted fractions (FII-B(I)-FII-B(VII)), indicating that many inhibitory peptides were exposed by papain treatment. For further purification, FII-B(IV), which

    exhibited the highest ACE-inhibitory activity, was applied to another reverse-phase column (ODS C12), and a pure active peptide, FII-B(IV)-VII, was obtained (Fig. 4). The purifica-tion yield and IC50 value of FII-B(IV)-VII were 0.08 ± 0.02% and 1.93 ± 0.33 μg/mL, respectively (Table 1). Based on the molecular weight (294.16 Da) determined by an Agilent 1100 LC/MSD spectrometer, the IC50 value was 6.56 ± 1.12 μM.

    Lee et al. (2005) purified ACE-inhibitory peptides from goat’s milk casein hydrolysates of pepsin, and showed their sequences to be Ala-Tyr-Phe-Tye and Pro-Tyr-Tyr. Gao et al. (2010) also produced ACE-inhibitory peptides from papain-digested cottonseed hydrolysate, and calculated the IC50 val-ues to range from 0.159 to 0.792 mg/mL, even though they did not elucidate the structure.

    In this work, we hydrolyzed jellyfish with papain and puri-fied an ACE-inhibitory peptide (FII-B(IV)-VII) from the hy-drolysate. These findings led us to postulate that many proteo-lytic enzymes, including papain, may be applied to produce ACE-inhibitory peptides of varying molecular weight from several jellyfish species.

      >  Structure of the purified peptide

    To elucidate the structure of FII-B(IV)-VII, several tech-niques were employed. The IR spectrum showed an NH/OH vibrational frequency at 3,398 cm-1 and peptide bond amine peak (CN) at 1,203 cm-1, whereas the carbonyl and broad peaks of the cyclic ring appeared at 1,672 and 1,468 cm-1, re-spectively. These IR data indicated that the peptide contains a phenolic ring.

    The 1H, 13C NMR, and DEPT data allowed assignment of seven methine, two methylene, and two methyl groups. The remaining quaternary centers consisted of a carbonyl (172.4 ppm), hydroxyl (155.2 ppm), and double-bonded (163.3 ppm) oxygenated carbon signals. The 1H NMR spectrum showed that the peaks at δ 1.14, 1.32, 2.95, and 3.08 ppm were related to the methylene protons of the ester group (Table 2). These data convinced us that FII-B(IV)-VII contains tyrosine and isoleucine. The methine protons of isoleucine appeared at δ 1.87 and 3.73 ppm, and those of tyrosine appeared at δ 3.97 ppm. The methine protons attached to the cyclic side chain appeared at δ 6.71 and 7.01 ppm as a doublet. The 13C NMR spectrum showed two carbonyl peaks at δ 172.4 (C=O ester) and δ 163.3 ppm (C=O amide), δ 116.1-155.2 ppm (benzilic carbons), δ 35.1, 58.3, and 62.6 ppm (methine carbons), δ 11.1 and 14.4 ppm (methyl carbons), which correlated with the structure of tyrosine-isoleucine (Tyr-Ile) (Table 2). These data strongly suggest that the purified peptide, FII-B(IV)-VII, is a Tyr-Ile dipeptide, which is a novel dipeptide reported for the first time in this work. The molecular weight and isoelec-tric point (pI) of Tyr-Ile were calculated to be 294.35 Da and 5.9, respectively, using the ChemBioDraw 11 program, cor-responding to the LC/MS data (294.16 Da).

    Kawasaki et al. (2000) reported that a dipeptide (Val-Tyr)

    purified from sardine muscle hydrolysate had a significant an-tihypertensive effect on mildly hypertensive subjects via ACE inhibition. Erdmann et al. (2006) reported that sardine muscle hydrolysate contained a Met-Tyr dipeptide that showed ACE-inhibitory activity and was capable of diminishing free radical formation in human endothelial cells.

      >  IC50 value and kinetic analysis

    The ACE inhibition pattern of the purified peptide was in-vestigated by applying a Lineweaver-Burk plot. Tyr-Ile acts as a competitive inhibitor of ACE, suggesting that Tyr-Ile from N. nomurai competes with the substrate at the active site of ACE. The IC50 and Ki values of Tyr-Ile were 6.56 ± 1.12 μM (1.93 ± 0.33 μg/mL) and 3.10 ± 0.28 μM, respectively (Fig. 5). The inhibition pattern of Tyr-Ile was similar to those of fibrinogen pentapeptides, casein fragments, porcine plasma tripeptides, and tuna muscle octapeptide (Sugiyama et al., 1991; Astawan et al., 1995). Cheung et al. (1980) reported that tryptophan, tyrosine, proline, or phenylalanine at the carboxy-terminal and branched-chain aliphatic amino acids at the amino-terminal are suitable for competitive inhibition of ACE. Fujita and Yoshikawa (1999) reported that LKPNM is a prodrug type of ACE-inhibitory peptide because LKPNM was hydrolyzed by ACE to produce LKP, which exhibited an ACE-inhibitory activity eightfold higher than LKPNM. After oral administration in SHR, the antihypertensive effect of LKPNM was maximal after 6 h and that of LKP at 4 h.

    Some antihypertensive drugs are known to produce side ef-fects, such as an abnormal elevation of blood pressure. How-ever, jellyfish is a favorite seafood in Southeast and East Asian nations. Thus, jellyfish may be a useful functional food for maintenance of blood pressure within the normal range. Our results also suggest that an ACE inhibitor derived from N. no-murai can be utilized in the development of physiologically functional foods.

  • 1. Astawan M, Wahyuni M, Yasuhara T, Yamada K, Tadokoro T, Maekawa A 1995 Effects of angiotensin-I converting enzyme in-hibitory substances derived from Indonesian dried-salted fish on blood pressure of rats. [Biosci Biotechnol Biochem] Vol.59 P.425-429 google doi
  • 2. Byun HG, Kim SK 2001 Purification and characterization of an-giotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin. [Process Biochem] Vol.36 P.1155-1162 google doi
  • 3. Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW 1980 Binding of peptide substrates and inhibitors of angiotensin con-verting enzyme. Importance of the COOH-terminal dipeptide se-quence. [J Biol Chem] Vol.255 P.401-407 google
  • 4. Cushman DW, Cheung HS 1971 Spectrophotometeric assay and properties of angiotensin-converting enzyme of rabbit lung. [Bio-chem Pharmacol] Vol.20 P.1637-1648 google doi
  • 5. Dziuba J, Minkiewicz P, Nalecz D 1999 Biologically active pep-tides from plant and animal proteins. [Pol J Food Nutr Sci] Vol.8 P.3-16 google
  • 6. Erdmann K, Grosser N, Schipporeit K, SchrOder H 2006 The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. [J Nutr] Vol.136 P.2148-2152 google
  • 7. FitzGerald RJ, Murray BA, Walsh DJ 2004 The emerging role of dairy protein and bioactive peptides in nutrition and health: hypo-tensive peptides from milk proteins. [J Nutr] Vol.134 P.980S-988S google
  • 8. Fujita H, Yoshikawa M 1999 LKPNM: a prodrug-type ACE-in-hibitory peptide derived from fish protein. [Immunopharmcology] Vol.44 P.123-127 google doi
  • 9. Gao D, Chang T, Li H, Cao Y 2010 Angiotensin I-converting en-zyme inhibitor derived from cottonseed protein hydrolysate. [Afr J Biotechnol] Vol.9 P.8977-8983 google
  • 10. Gobbetti M, Ferranti P, Smacchi E, Goffredi F, Addeo F 2000 Pro-duction of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. Bul-garicus SS1 and Lactococcus lactis subsp. [Cremoris FT4. Appl Environ Microbiol] Vol.66 P.3898-3904 google doi
  • 11. He H, Chen X, Sun C, Zhang Y, Gao P 2006 Preparation and functional evaluation of oligopeptide-enriched hydrolysates from shrimp (Acetes chinesis) treated with crude protease from Bacillus sp. SM98011. [Bioresour Technol] Vol.97 P.385-390 google doi
  • 12. Jang A, Lee M 2005 Purification and identification of angioten-sin converting enzyme inhibitory peptides from beef hydrolysates. [Meat Sci] Vol.69 P.653-661 google doi
  • 13. Je JY, Park JY, Jung WK, Park PJ, Kim SK 2005 Isolation of angio-tensin I converting enzyme (ACE) inhibitor from fermented oyster sauce Crassostrea gigas. [Food Chem] Vol.90 P.809-814 google doi
  • 14. Kawasaki T, Seki E, Osajima K, Yoshida M, Asada K, Matsui T, Osajima Y 2000 Antihypertensive effect of valyl-tyrosine a short chain peptide derived from sardine muscle hydrolyzate on mild hypertensive subjects. [J Hum Hypertens] Vol.14 P.519-523 google doi
  • 15. Kim SK, Choi YR, Park PJ, Choi JH, Moon SH 2000 Screening of biofunctional peptides from cod processing wastes. [J Korean Soc Agric Chem Biotechnol] Vol.43 P.225-227 google
  • 16. Kim TJ, Yoon HD, Lee DS, Jang YS, Suh SB, Yeum DM 1996 Angiotensin I converting enzyme inhibitory activity of hot-water extract and enzymatic hydrolysate of fresh water fish. [J Korean Soc Food Sci Nutr] Vol.25 P.871-877 google
  • 17. Kohama Y, Matsumoto S, Oka H, Teramoto T, Okabe M, Mimura T 1988 Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. [Biochem Biophys Res Commun] Vol.155 P.332-337 google doi
  • 18. Lee HD, Kim KS, Do JR 2001 Separation and purification of angio-tensin-I converting enzyme inhibitory peptides from laver hydroly-sate. [J Korean Fish Sci] Vol.34 P.164-172 google
  • 19. Lee KJ, Kim SB, Ryu JS, Shin HS, Lim JW 2005 Separation and purification of angiotensin converting enzyme inhibitory peptides derived from goat’s milk casein hydrolysates. [Asian-Aust J Anim Sci] Vol.18 P.741-746 google
  • 20. Matsui T, Matsufuji H, Seki E, Osajima K, Nakashima M, Osajima Y 1993 Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle. [Biosci Biotechnol Biochem] Vol.57 P.922-925 google doi
  • 21. Miguel M, Alonso MJ, Salaices M, Aleixandre A, Lopez-Fandi?o R 2007 Antihypertensive ACE-inhibitory and vasodilator properties of egg white hydrolysates: effect of a simulated intestinal diges-tion. [Food Chem] Vol.104 P.163-168 google doi
  • 22. Ondetii MA, Rubin B, Cushman DW 1977 Design of specific in-ibitors of angiotensin-converting enzyme: a new class of orally active antihypertensive agents. [Science] Vol.196 P.441-444 google doi
  • 23. Shahidi F, Zhong Y 2008 Bioactive peptides. [J AOAC Int] Vol.91 P.914-931 google
  • 24. Smacchi E, Gobbetti M 1998 Peptides from several Italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria Pseudo-monas fluorescens ATCC 948 and to the angiotensin I-converting enzyme: design synthesis and utilization. [Enzyme Microb Tech-nol] Vol.22 P.687-694 google doi
  • 25. Sugiyama K, Takada K, Egawa M, Yamamoto I, Onzuka H, Oba K 1991 Hypotensive effect of fish protein hydrolysate. [Nippon Nogeikagaku Kaishi] Vol.65 P.35-43 google doi
  • 26. Taylor WH 1957 Formol titration: an evaluation of its various modifi-cations. [Analyst] Vol.82 P.488-498 google doi
  • 27. Vermeirssen V, Van Camp J, Verstraete W 2004 Bioavailability of angiotensin I converting enzyme inhibitory peptides. [Br J Nutr] Vol.92 P.357-366 google doi
  • 28. Wu H, He AL, Che XL, Sun CY, Zhang YZ, Zhou BC 2008 Purifi-cation and identification of novel angiotensin-I-converting enzyme inhibitory peptides from shark meat hydrolysate. [Process Biochem] Vol.43 P.457-461 google doi
  • [Fig. 1.] Degree of hydrolysis and angiotensin-Ι-converting enzyme (ACE)-inhibitory activity of jellyfish hydrolysate. Papain was used to hydrolyze Nemopilema nomurai and the inhibitory activity of the resulting hydrolysate was determined by the method mentioned in Materials and Methods. ■ hydrolysis (%); -●- ACE inhibition (%).
    Degree of hydrolysis and angiotensin-Ι-converting enzyme (ACE)-inhibitory activity of jellyfish hydrolysate. Papain was used to hydrolyze Nemopilema nomurai and the inhibitory activity of the resulting hydrolysate was determined by the method mentioned in Materials and Methods. ■ hydrolysis (%); -●- ACE inhibition (%).
  • [Fig. 2.] Angiotensin-Ι-converting enzyme (ACE)-inhibitory activity of the fractionated jellyfish hydrolysate. The resultant hydrolysate was fractionated using amicon millipore membrane filters (1kDa cut offs) then the inhibitory activity was determined by the standard method. PH papain hydrolysate before fractionation; FI >1 kDa; FII <1 kDa.
    Angiotensin-Ι-converting enzyme (ACE)-inhibitory activity of the fractionated jellyfish hydrolysate. The resultant hydrolysate was fractionated using amicon millipore membrane filters (1kDa cut offs) then the inhibitory activity was determined by the standard method. PH papain hydrolysate before fractionation; FI >1 kDa; FII <1 kDa.
  • [Fig. 3.] Gel filtration chromatography of the fraction FII on Sephadex LH-20. Separation was performed with 30% aqueous methanol solution at a flow rate of 0.5 mL/min and collected with fraction volume of 5 mL. The fraction corresponding to FII-B was collected and determined the inhibitory activities.-●- Absorbance at 214 nm;■ ACE inhibition (%).
    Gel filtration chromatography of the fraction FII on Sephadex LH-20. Separation was performed with 30% aqueous methanol solution at a flow rate of 0.5 mL/min and collected with fraction volume of 5 mL. The fraction corresponding to FII-B was collected and determined the inhibitory activities.-●- Absorbance at 214 nm;■ ACE inhibition (%).
  • [Fig. 4.] Separation chromatogram of the FII-B(IV) by using C12 reverse-phase column. The resultant active fraction (FII-B(IV)) after C18 column chromatography was loaded on a Jupiter Proteo C12 column (90A 10 ㎛ 21.2 × 150 mm) and eluted with a linear gradient of CH3CN-H2O [A eluent; H2O:CH3CN:TFA = 95:5:0.1 (v/v/v) B eluent; CH3CN:H2O:TFA = 55:45:0.08(v/v/v)] at a flow rate of 2 mL/min. The inhibitory activity of each elute from I to X was determined and the resultant active fraction FII-B(IV)-VII was obtained.
    Separation chromatogram of the FII-B(IV) by using C12 reverse-phase column. The resultant active fraction (FII-B(IV)) after C18 column chromatography was loaded on a Jupiter Proteo C12 column (90A 10 ㎛ 21.2 × 150 mm) and eluted with a linear gradient of CH3CN-H2O [A eluent; H2O:CH3CN:TFA = 95:5:0.1 (v/v/v) B eluent; CH3CN:H2O:TFA = 55:45:0.08(v/v/v)] at a flow rate of 2 mL/min. The inhibitory activity of each elute from I to X was determined and the resultant active fraction FII-B(IV)-VII was obtained.
  • [Table 1.] ACE-inhibitory activity and purification yield of each step
    ACE-inhibitory activity and purification yield of each step
  • [Table 2.] 1H and 13C NMR chemical shifts for the FII-B(IV)-VII
    1H and 13C NMR chemical shifts for the FII-B(IV)-VII
  • [Fig. 5.] Lineweaver-Burk plot of FII-B(IV)-VII on ACE reaction. A series of FII-B(IV)-VII (0 0.5 1.0 2.0 4.0 μg/mL) was included in the reaction solution and the Ki was calculated by using the Grafit 5.0 program. HHL hipuryl-L-histidyl-L-leucine; ○ 0 μg/mL; ● 0.5 μg/mL; □ 1.0 μg/mL; ■ 2.0 μg/mL; Δ 4.0 μg/mL.
    Lineweaver-Burk plot of FII-B(IV)-VII on ACE reaction. A series of FII-B(IV)-VII (0 0.5 1.0 2.0 4.0 μg/mL) was included in the reaction solution and the Ki was calculated by using the Grafit 5.0 program. HHL hipuryl-L-histidyl-L-leucine; ○ 0 μg/mL; ● 0.5 μg/mL; □ 1.0 μg/mL; ■ 2.0 μg/mL; Δ 4.0 μg/mL.