A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Process Biochemistry 47 (2012) 2005–2011

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A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats Seok-Chun Ko a , Nalae Kang a , Eun-A. Kim a , Min Cheol Kang a , Seung-Hong Lee a , Sung-Myung Kang a , Joon-Baek Lee b , Byong-Tae Jeon c , Se-Kwon Kim d , Sun-Joo Park d , Pyo-Jam Park e , Won-Kyo Jung f , Daekyung Kim g,∗,1 , You-Jin Jeon a,∗∗,1 a

Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea Department of Earth Marine Sciences, Jeju National University, Jeju 690-756, Republic of Korea c Korean Nokyong Research Center, College of Medicine Konkuk University, Chungju 380-701, Republic of Korea d Department of Chemistry, Pukyoung National University, Busan 608-737, Republic of Korea e Department of Biotechnology, College of Medicine Konkuk University, Chungju 380-701, Republic of Korea f Department of Marine Life Science, Marine Life Research center, Chosun University, Gwangju 501-759, Republic of Korea g Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 24 March 2012 Received in revised form 5 July 2012 Accepted 10 July 2012 Available online 17 July 2012 Keywords: Marine Chlorella ellipsoidea Angiotensin I-converting enzyme (ACE) Peptide Antihypertensive effect

a b s t r a c t Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, ␣-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC50 value: 128.4 ␮M), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and ␣-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Angiotensin I-converting enzyme (peptidyl carboxy peptidase, EC 3.4.15.1, ACE) belongs to the class of zinc proteases that requires zinc and chloride for activation [1]. ACE plays an important role in the regulation of blood pressure by virtue of the renin–angiotensin system (RAS) and kallikrein kinnin system (KKS) [1–3]. In KKS, ACE inactivates the vasodilator bradykinin, while in the RAS, ACE acts as an exopeptidase that cleaves His–Leu from the C-terminal of decapeptide angiotensin I, and produces the potent vasoconstrictor octapeptide angiotensin II [4]. Therefore,

∗ Corresponding author. Tel.: +82 64 800 4930; fax: +82 64 805 7800. ∗∗ Corresponding author. Tel.: +82 64 754 3475; fax: +82 64 756 3493. E-mail addresses: [email protected] (D. Kim), [email protected], [email protected] (Y.-J. Jeon). 1 You-Jin Jeon and Daekyung Kim contributed equally to this study. 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.07.015

inhibition of ACE activity is considered to be a pivotal therapeutic approach for controlling hypertension. ACE inhibitors such as captopril and enalapril have been used to down regulate blood pressure in hypertensive subjects in both in vivo and clinical studies [5–7]. However, the subjects presented certain side effects such as headache, insomnia, and fever. Therefore, the development of ACE inhibitors from natural products has become a major area of research. Potent bioactive peptides can be induced from enzymatic hydrolysis of various proteins and may act as potential physiological modulators in metabolism processes during the intestinal digestion of food [3]. Bioactive peptides are liberated depending on their structure, composition, and amino acid sequence [8]. These peptides exhibit various bioactivities such as antioxidative [9], antimicrobial [10] and antihypertensive effects [11]. Recently, many ACE inhibitory peptides were reported as natural alternative bioactive peptides that are safer than synthetic ACE inhibitors. Various ACE inhibitory peptides have been isolated from food proteins,

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such as tuna back bone [5], sheep milk yogurt [12], egg whites [13] canola meal [14], yak milk casein [15] and oyster [16]. Microalgae are rich in proteins, carbohydrates, minerals, and diverse functional pigments [17]. Among the microalgae, Chlorella spp., a type of single-celled green algae, has been shown to possess nutrients and bioactive substances [18,19]. Chlorella spp. has various functional properties such as antioxidant [20], antidiabetes [21], anti-inflammatory, and immunomodulatory effects [22]. Among Chlorella spp., the bioactivities of Chlorella ellipsoidea used in this study have been rarely reported. In addition, the ACE inhibitory effect of C. ellipsoidea has not yet been reported. Although the protein content of C. ellipsoidea is more than 45%, it is usually used as animal feed after the production of algae essence [17,19]. Therefore, appropriate processing of the C. ellipsoidea protein is further required to better obtain all of its potential bioactivities. The objective of this study was to isolate ACE inhibitory peptide from C. ellipsoidea protein hydrolysate and identify the purified peptides with regards to ACE inhibitory activity. Furthermore, we have also investigated the antihypertensive action by oral administration in SHRs.

The molecular weight and amino acid sequence of the purified peptide from C. ellipsoidea protein was determined using a quardrupole time-of-flight mass spectrometer (Q-TOF MS; Micromass, Altrincham, UK) coupled with electrospray ionization (ESI) source. The purified peptide dissolved in methanol/water (1:1, v/v) was infused into the ESI source and the molecular weight was determined by doubly charged (M+2H)2+ state analysis in the mass spectrum. Following the molecular weight determination, the peptide was automatically selected for fragmentation and sequence information was obtained by tandem MS analysis.

2. Materials and methods

2.7. Stability of the peptide during in vitro digestion by gastrointestinal enzymes

2.1. Materials

The 1% (w/w) pepsin solution was prepared in a 20 mM glycine–HCl buffer adjust to pH 2.0, while the 1% (w/w) trypsin and ␣-chymotrypsin solution in 50 mM sodium phosphate buffer was adjusted to pH 8.0. The peptide was dissolved at 0.5 mg/ml in the pepsin, trypsin and ␣-chymotrypsin solution and reacted at 37 ◦ C for 4 h. Reactions were terminated by boiling at 100 ◦ C for 15 min. Then, these solutions were centrifuged at 10,000 g for 25 min. The ACE inhibitory activity (IC50 values) was then measured. The stability of supernatant was analyzed using by LC–MS.

Marine C. ellipsoidea, was obtained from Marine Bio Process Co., Korea, and lyophilized at −70 ◦ C using a freeze dryer. Lyophilized C. ellipsoidea powder was stored at −80 ◦ C until use. Protamex, Kojizyme 500 MG, Neutrase 0.8 L, Flavourzyme 500 MG, Alcalase 2.4 L FG were purchased from Novo Co. (Novozyme Nordisk, Bagsvaerd) and trypsin, ␣-chymotrypsin, pepsin, papain, N-hippuryl-HisLeu tetrahydrate (HHL), angiotensin I converting enzyme (lung acetone powder from rabbit) and Sephadex G-25 were obtained from Sigma chemical Co. (St. Louis, MO, USA). The other chemicals and reagents used were of analytical grade. 2.2. Preparation of C. ellipsoidea protein hydrolysate (CEPH) For the production of ACE inhibitory peptide from C. ellipsoidea, enzymatic hydrolysis was performed using various enzymes (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, ␣-chymotrypsin, pepsin and papain) at their optimal conditions. At enzyme/substrate ratio of 1/100 (w/w), substrate and enzyme were mixed in a 100 ml flask with buffer, temperature and pH control devices. As soon as the enzymatic reaction completed, the hydrolysate was boiled for 10 min at 100 ◦ C to inactivate the enzyme. The hydrolysate was clarified by centrifugation (1800 × g, for 20 min at 4 ◦ C) to remove the residue. The resultant CEPH was fractionated through ultra-filtration (UF) membranes with a range of molecular weight (MW) cut-off of 5 and 10 kDa, respectively. Fractionates were designed as follows: CEPH-I with distribution of below 5 kDa, CEPH-II with distribution of 5–10 kDa and CEPH-III with distribution of above 10 kDa. All recovered CEPH fractions were lyophilized and stored at −70 ◦ C until used.

extracted with 500 ␮l of ethyl acetate. Then a 200 ␮l aliquot of the extract was removed and evaporated in a dry-oven at 80 ◦ C. The residue was dissolved in 1 ml distilled water and its UV absorbance was measured at 228 nm. The IC50 value was defined as the concentration of inhibitor required to inhibit 50% of ACE inhibitory activity. 2.5. Determination of molecular weight and amino acid sequence

2.6. Determination of ACE inhibition pattern To clarify the inhibitory mechanism of purified peptide on ACE. Different concentrations of purified peptide were added to each reaction mixture. The enzyme activities were measured with different concentrations of substrate (HHL). ACE inhibitory pattern in presence of the inhibitor was obtained with Lineweaver–Burk plot.

2.8. Animals and measurement of systolic blood pressure Spontaneously hypertensive rats (SHRs, 10-week-old male, specific pathogenfree, 250–300 g body weight) with tail systolic blood pressure (SBP) over 180 mmHg were obtained from SLC Inc. (Shizuoka, Japan). SHRs were housed individually in steel cages in room kept at 24 ± 1 ◦ C with 12 h light/dark cycle and fed a standard laboratory diet. Tap water was freely available. The peptide was dissolved in saline at a dose of 10 mg/kg body weight and injected orally using a metal gastric sonde in SHRs. The lowering efficacy of the peptide on systolic blood pressure (SBP) was compared with that of captopril. Control rats were administrated with the same volume of saline solution. Following oral administration, SBP was measured by tail-cuff method with a CODATM blood pressure monitor (Kent Scientific Corp., Torrington, USA) after warming up SHRs in warming platform maintained at 37 ◦ C for 15 min. 2.9. Statistical analysis All data were expressed as mean ± standard deviation of three determinations. The significance of the differences of SBPs between control group and administration group was analyzed using Student’s t-test.

2.3. Purification of ACE inhibitory peptide The most active fraction by ultradiltered hydrolysate was again filtered and applied to a column (2.5 cm × 75 cm) packed with Sephadex G-25 resin which was previously equilibrated with distilled water. The flow rate was 2 ml/min, and elution peaks were monitored at 280 nm. The fractions were collected at 5 min intervals with a fraction collector, and fractions showing ACE inhibitory activity were pooled and lyophilized. The fraction with the highest ACE inhibitory activity was dissolved in distilled water, and separated by reversed-phase high performance liquid chromatography (RP-HPLC) on a J sphere ODS-H80 column (C18 , 4 ␮m, 4.6 mm × 250 mm, YMC, Kyoto, Japan). For RP-HPLC analysis, mobile phases used in the gradient elution consisted of eluent A consisting of 0.1% trifluoroacetic acid (TFA) in distilled water (v/v); and eluent B of 0.1% trifluoroacetic acid (TFA) in acetonitrile. The separation was performed with a linear gradient from 0 to 50% eluent B at a flow rate of 1.0 ml/min. The UV absorbance of the eluent was monitored at 280 nm. Finally, the fraction with the ACE inhibitory activity was collected and lyophilized; this was followed by identification of the amino acid sequence. 2.4. Measurement of ACE inhibitory activity The ACE inhibitory activity assay was performed according to the methods of Cushman and Cheung [23] with slight modification. For each assay, 50 ␮l of the hydrolysate solution with 50 ␮l of ACE solution (25 mU/ml) was pre-incubated at 37 ◦ C for 30 min, and then incubated with 100 ␮l of substrate (25 mM hippuryl-HisLeu in 50 mM sodium borate buffer containing 500 mM NaCl at pH 8.3) at 37 ◦ C for 60 min. The reaction stopped by adding 250 ␮l of 1 N HCl. Hippuric acid was

3. Results and discussion 3.1. Preparation of C. ellipsoidea protein hydrolysates (CEPH) and their ACE inhibitory activity To produce ACE inhibitory peptides, C. ellipsoidea protein was separately hydrolyzed using various commercial digestive enzymes. In the ACE inhibitory activity assay (Table 1), the highest IC50 value was exhibited by Alcalase-proteolytic hydrolysate at 1.47 mg/ml. The occurrence of ACE inhibitory peptides in protein hydrolysates has already been reported from marine bioresources such as rotifer, S. plicata and S. clava [8,25,26]. Compared to what has been observed in previous reports, the ACE inhibitory activities of enzymatic hydrolysates from C. ellipsoidea exhibited moderate IC50 values. Many previous reports have found that Alcalase is capable of producing bioactive peptides when it is incorporated into hydrolyzing food proteins [8,27,28]. Moreover, Alcalase produces shorter peptide sequences as well as terminal amino acid sequences responsible for various bioactivities [8,29].

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Table 1 Optimum conditions and time of enzymatic hydrolysis for various enzymes. Enzyme

pH

Temp. (◦ C)

Time (h)

Kojizyme Flavourzyme Neutrase Alcalase Protamex Pepsin Trypsin ␣-Chymotrypsin Papain

6.0 7.0 6.0 8.0 6.0 2.0 8.0 8.0 6.0

40 50 50 50 40 37 37 37 37

12 12 12 12 12 12 12 12 12

Table 2 ACE inhibitory activity of enzymatic hydrolysates from C. ellipsoidea. Enzyme

IC50 value (mg/ml)a

Kojizyme Flavourzyme Neutrase Alcalase Protamex Pepsin Trypsin ␣-Chymotrypsin Papain

1.94 1.92 1.60 1.47 1.78 2.35 2.40 2.52 2.50

± ± ± ± ± ± ± ± ±

0.03 0.02 0.03 0.04 0.02 0.03 0.04 0.04 0.03

The values of IC50 were determined by at triplicate individual experiments. a The concentration of an inhibitor required to inhibit 50% of the ACE activity.

The molecular weight of hydrolyzed protein is an important factor in producing protein hydrolysates that are desirable as functional material [30]. One of the methods for fractionating various molecular weights of hydrolysate is the use of UF membranes with different molecular weight cut-offs [31]. In this study, Alcalase-proteolytic hydrolysate was fractionated with an UF system into three individual fractions with three molecular weight (MW) groups of CEPH-I (MW < 5 kDa), CEPH-II (MW = 5–10 kDa), and CEPH-III (MW > 10 kDa), using UF membranes (MW cut-off of 5 and 10 kDa). The three groups were investigated for ACE inhibitory activity. Among all of the MW groups, CEPH-I evidenced the strongest ACE inhibitory activity and had an IC50 value of 0.89 mg/ml (Table 2). It has been previously reported that yellow sole frame hydrolysate was fractionated into three fractions (>10, 5–10 and <5 kDa) by UF according to molecular weights and the <5 kDa fraction exhibited strongest ACE inhibitory activity and had an IC50 value of 0.883 mg/ml and similar with our results [11]. The low molecular weight fraction had more potent ACE inhibitory activity than that of the high molecular weight fraction [11]. Therefore, we selected CEPH-I for purification as an ACE inhibitory peptide (Table 3).

Table 3 ACE inhibitory activity of molecular weight fractions from Alcalase-proteolytic hydrolysate of C. ellipsoidea. IC50 value (mg/ml)a

Fraction b

Unfractionated >10 kDac 5–10 kDa <5 kDa

1.47 1.42 1.38 0.89

± ± ± ±

0.04 0.04 0.03 0.04

The values of IC50 were determined by at triplicate individual experiments. a The concentration of an inhibitor required to inhibit 50% of the ACE activity. b Alcalase-proteolytic hydrolysate. c UF membrane (molecular weight cut-off of 5 and 10 kDa) was used.

Fig. 1. Sephadex G-25 gel filtration chromatogram of <5 kDa fraction of Alcalaseproteolytic hydrolysate from C. ellipsoidea. (A) Separation was performed with 2 ml/min and collected at a fraction volume (10 ml). The fractions isolated by Sephadex G-25. Gel column were separated into five fractions (F1–F5). (B) ACE inhibitory activity of each fraction.

3.2. Purification of ACE inhibitory peptide CEPH-I was fractionated on a Sephadex G-25 column using size exclusion chromatography. As shown in Fig. 1(A), there were five major absorbance peaks at 280 nm and five fractions (F1–F5) associated with the peaks were pooled and lyophilized for ACE inhibitory activity. Among the fractions, F4 exhibited the strongest ACE inhibitory activity and had an IC50 value of 0.301 mg/ml (Fig. 2(B)). The lyophilized active F4 fraction was further separated by RP-HPLC on an ODS column with a linear gradient of acetonitrile (0–50% for 50 min) containing 0.1% TFA. The elution profiles of the peaks are shown in Fig. 2(A). The peaks were separated into three fractions (F4-I–F4-III) and each fraction was pooled and lyophilized for ACE inhibitory activity. The F4-I fraction showed the most potent ACE inhibitory activity, with an IC50 value of 0.06 mg/ml (Fig. 2(B)). Typical result obtained during the purification steps are summarized in Table 4. The ACE inhibitory peptide was purified 24.5-fold from the enzymatic hydrolysate using a three-step purification procedure, with a 0.08% yield. After consecutive chromatography, we finally obtained the purified peptide from C. ellipsoidea protein and its amino acid sequence was determined by Q-TOF ESI mass spectroscopy.

Table 4 Purification of ACE inhibitory peptide of Alcalase-proteolytic hydrolysate from C. ellipsoidea. Purification step

IC50 value (mg/ml)a

Alcalase hydrolysate Ultrafiltration (<5 kDa) Gel filtration chromatography RP-HPLC

1.47 0.89 0.30 0.06

± ± ± ±

0.04 0.04 0.03 0.01

Yield (%)

Purification fold

100 60.85 2.56 0.08

1 1.65 4.90 24.5

The values of IC50 were determined by at triplicate individual experiments. a The concentration of an inhibitor required to inhibit 50% of the ACE activity.

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Fig. 2. RP-HPLC chromatogram of the potent ACE inhibitory activity fraction F4 isolated from Sephadex G-25. (A) Separation into sub-fractions (F4-I to F4-III) was performed with linear gradient of acetonitrile from 0% to 50% at a flow rate of 1.0 ml/min and a J sphere ODS-H80 (4 ␮m, 4.6 mm × 250 mm) column. The elution was monitored at 280 nm. (B) ACE inhibitory activity of each fraction.

3.3. Amino acid sequence and inhibition pattern of purified ACE inhibitory peptide

Table 5 Comparison with ACE inhibitory activity by purified and synthetic peptide. Peptide

It was previously reported that several peptides were derived from the peptic hydrolysate of Chlorella vulgaris, such as Ile–Val–Val–Glu (IC50 value = 315.3 ␮M) [32]. In this study, an amino acid sequence was identified using MS/MS and proved to be a decapeptide (Fig. 3), Val–Glu–Gly–Tyr (MW: 467.2 Da), and exhibited a higher activity (IC50 value = 128.4 ␮M) compared to C. vulgaris. In order to validate the ACE inhibitory activity of the purified peptide, a synthetic peptide with the same sequence was synthesized and tested. The synthetic peptide exhibited the same

VEGY a

IC50 value (␮M) a Purified peptide

Synthetic peptide

128.4

128.6

The concentration of an inhibitor required to inhibit 50% of the ACE activity.

ACE inhibitory activity as the purified peptide from marine C. ellipsoidea protein hydrolysate (Table 5). Bioactive peptides usually contain 3–20 amino acid residues and low MW peptides are

Fig. 3. Identification of molecular mass and amino acid sequence of the purified peptide from C. ellipsoidea. MS/MS experiments were performed on a Q-TOF tandem mass spectrometer (Micromass Co., Manchester, UK) equipped with a nano-ESI source. Sequencing of purified peptide was sequenced by using the PepSeq de nove sequencing algorithm.

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peptide had competitive inhibition pattern [8,38]. Captopril has been reported to show competitive inhibition competition with substrates for binding to active ACE sites [39]. In this study, the ACE inhibitory peptide from C. ellipsoidea protein hydrolysate contained a hydrophobic amino acid at the N-terminal, as well as Trp at the C-terminal tripeptide sequence, which may contribute to ACE inhibitory activity. 3.4. Stability of the peptide against gastrointestinal enzymes

Fig. 4. The ACE inhibition pattern of purified peptide was estimated using Lineweaver–Burk plots. 1/V [␮M/min] and 1/S [mM] represents the mutual of reaction velocity and substrate, respectively.

more potent as bioactive peptides than high MW peptides [5]. Structure–activity relationships among different peptide inhibitors of ACE indicate that binding to ACE is strongly affected by the C-terminal tripeptide sequence of the substrate, and it is proposed that peptides, which include hydrophobic amino acids at these positions, are potent inhibitors [8]. Moreover, Cushman and Cheung [23] reported that Trp, Tyr, Pro, and Phe at the C-terminal, and branched-chain aliphatic amino acids at the N-terminal, were suitable for a peptide binding to ACE, as a competitive inhibitor. According to previous reports on ACE inhibitory peptides, hydrophobic amino acids in the N-terminal region of the active peptide play pivotal roles in binding to the ACE active site, and Val and Leu residues are the amino acids most frequently observed in other ACE inhibitory peptides [33–36]. Recently, Wu et al. [37] proposed models for ACE inhibitory peptides through computational analysis. According to their proposal, hydrophobic amino acid residues at the N-terminus, positively charged amino acids at the middle, and aromatic amino acids at the C-terminus of the peptides evidence high ACE inhibition. The ACE inhibition pattern of the purified peptide from C. ellipsoidea was investigated using Lineweaver–Burk plots, and found to be competitive (Fig. 4). This result was consistent with the previous studies of ACE inhibitory peptide in which the purified

To evaluate the stability of the peptide, the synthetic peptide was incubated under simulated gastrointestinal conditions using pepsin, trypsin and ␣-chymotrypsin. The chromatogram of the synthetic peptide was not affected by simulated gastrointestinal incubation with pepsin, trypsin and ␣-chymotrypsin as compared to the synthetic peptide, and showing no impact of gastrointestinal enzymes on ACE inhibitory activity of VEGY. (Fig. 5(A)–(D)). Our results suggest that VEGY is stable against gastrointestinal enzymes of pepsin, trypsin and ␣-chymotrypsin. Based inhibition mechanism, ACE inhibitory peptides can be classified into three groups [40,41]. The inhibitor-type peptides, i.e., true inhibitors, which activity is not changed after treatment by ACE or gastrointestinal enzymes. The substrate type peptides exhibit an elevation of activity by preincubation with ACE. The prodrug-type peptides were converted to their true inhibitors with higher activity by ACE or gastrointestinal enzymes. The VEGY in this study is true inhibitor because the ACE inhibitory activity of the peptide is not affected by pepsin, trypsin and ␣-chymotrypsin, and the Lineweaver–Burk plot indicates that VEGY competes with HHL for the binding sites of ACE. 3.5. Antihypertensive effect of purified peptide on SHRs Antihypertensive effect of the purified peptide was evaluated by measuring the change of systolic blood pressure (SBP) at 2, 4, 6 and 8 h after oral administration of 10 mg/kg of body weight. Captopril was used as a positive control, and the control group was injected with the same volume of saline. As shown in Fig. 5, the SBP in quiescent state of SHR was 189.5 ± 1.4 mmHg. After oral administration of the peptide and captopril, SBP was clearly decreased and activities were maintained for 6 h. The maximal decrements in SBP of the peptide and captopril treatment groups were 22.8 and 35.4 mmHg at 4 h, respectively. Lee et al. [5] reported that the maximal decrement in SBP of ACE inhibitory peptide from tuna frame protein was 21 mmHg at 6 h, and similar with our result. The small peptides (di- or tripeptide) are easily absorbed in their intact forms

Fig. 5. Chromatograms for VEGY after treatments by gastrointestinal enzymes. The VEGY (A) was treated by pepsin (B), trypsin (C) and ␣-chymotrypsin (D).

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Fig. 6. Change of SBP after oral administration of purified peptide in SHR. Captopril was used as positive control. Single oral administration was performed with a dose of 10 mg/kg body weight, and SBP was measured 0, 2, 4, 6 and 8 h after oral administration of purified peptide. The significance of the difference from control at *p < 0.01.

in the intestine [5]. Also, the purified peptide has relatively low MW compared to other reports [5,8,11], the results clearly showed that the purified peptide exert a substantial effect on reduction of SBP in SHR (Fig. 6). 4. Conclusion In this study, we evaluated the ACE inhibitory activity of a purified peptide from enzymatic hydrolysate of C. ellipsoidea. Using consecutive chromatographic methods, the peptide was determined to exhibit potent ACE inhibitory activity with an IC50 value of 128.4 ␮M. The ACE inhibitory pattern of the purified peptide from C. ellipsoidea was shown by Lineweaver–Burk plots to be a competitive inhibition pattern, and stable against the gastrointestinal enzymes. Antihypertensive effect in SHR also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results of this study suggest that this ACE inhibitory peptide from C. ellipsoidea hydrolysate could be used as a possible food supplement or in pharmaceutical applications. Acknowledgment This research was supported by a grant (T32607) from the Korea Basic Science Institute (KBSI) to D. Kim. References [1] Alper AB, Calhoun DA, Oparil S. Hypertension. In: Encyclopedia of life sciences. Nature Publishing Group; 2001. pp. 1–8. [2] Unger T. The role of rennin–angiotensin system in the development of cardiovascular disease. Am J Cardiol 2002;89:3–9. [3] Je JY, Park JY, Jung WK, Park PJ, Kim SK. Isolation of angiotensin I converting enzyme (ACE) inhibitor from fermented oyster sauce, Crassostrea gigas. Food Chem 2005;90:809–14. [4] Segura-Campos MR, Chel-Guerrero LA, Betancur-Ancona DA. Purification of angiotensin I-converting enzyme inhibitory peptides from a cowpea (Vigna unguiculata) enzymatic hydrolysate. Process Biochem 2011;46: 864–72. [5] 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 Chem 2010;118:96–102. [6] Hata Y, Yamamoto M, Ohni M, Nakajima K, Nakajima Y, Takano T. A placebocontrolled study of the effect of sour milk on blood pressure in hypertensive subjects. Am J Clin Nutr 1996;64:767–71. [7] Maria MC, Rosalia C, Maria JM, Mercedes R, Isidra R. Novel casein-derived peptides with antihypertensive activity. Int Dairy J 2009;19:566–73.

[8] Lee JK, Hong S, Jeon JK, Kim SK, Byun HG. Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, brachionus rotundiformis. Bioresour Technol 2009;100:5255–9. [9] Liu R, Wang M, Duan J, Guo J, Tang Y. Purification and identification of three novel antioxidant peptides from Cornu Bubali (Water buffalo horn). Peptides 2010;31:786–93. [10] Kim JB, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. J Pept Res 2001;58:349–56. [11] Jung WK, Mends E, Je JY, Park PJ, Son BW, Kim HC, et al. Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chem 2006;94:26–32. [12] Papadimitriou GC, Vafopoulou MA, Silva VS, Gomes A, Malcata XF, Alichanidis E. Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity. Food Chem 2007;105:647–56. [13] Miguel M, Alonso JM, Salaices M, Aleixandre A, Lopez FR. Antihypertensive, ACE-inhibitory and vasodilator properties of an egg white hydrolysate: effect of a simulated intestinal digestion. Food Chem 2007;104: 163–8. [14] Wu J, Aluko RE, Muir AD. Purification of angiotensin I-converting enzymeinhibitory peptides from the enzymatic hydrolysate of defatted canola meal. Food Chem 2008;111:942–50. [15] Maoa X, Ni J, Sun W, Hao P, Fan L. Value-added utilization of yak milk casein for the production of angiotensin-I-converting enzyme inhibitory peptides. Food Chem 2007;103:1282–7. [16] Wang J, Hu J, Cui J, Bai X, Du Y, Miyaguchi Y. Purification and identification of ACE inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysates in spontaneously hypertensive rats. Food Chem 2008;111:302–8. [17] Lee SH, Chang DW, Lee BJ, Jeon YJ. Antioxidant activity of solubilized Tetraselmis suecica and Chlorella ellipsoidea by enzymatic digests. J Food Sci Nutr 2009;14:21–8. [18] Chen CL, Liou SF, Chen SJ, Shih MF. Protective effects of chlorella-derived on UVB-induced production of MMP-1 and degradation of procollagen genes in human skin fibroblast. Regul Toxicol Pharm 2011;60:112–9. [19] Jeon SJ, Lee JH, Song KB. Isolation of calcium-binding peptide from chlorella protein hydrolysates. J Food Sci Nutr 2010;15:282–6. [20] Lee SH, Kang HJ, Lee HJ, Kang MH, Park YK. Six-week supplementation with Chlorella has favorable impact on antioxidant status in Korean male smokers. Nutrition 2010;26:175–83. [21] Rodriguez-Lopez M, Lopez-Quijada C. Plasma glucose and plasma insulin in normal and alloxanized rats treated with Chlorella. Life Sci 1971;10: 57–68. [22] Guzma S, Gato A, Lamela M, Freire-Garabal M, Calleja JM. Anti-inflammatory and immunomodulatory activities of polysaccharide from Chlorella stigmatophora and Phaeodactylum tricornutum. Phytother Res 2003;17: 665–70. [23] Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 1971;20:1637–48. [25] Ko SC, Kang MC, Lee JK, Byun HG, Kim SK, Lee SC, et al. Effect of angiotensin I-converting enzyme (ACE) inhibitory peptide purified from enzymatic hydrolysates of Styela plicata. Eur Food Res Technol 2011;233: 915–22. [26] Ko SC, Lee JK, Byun HG, Lee SC, Jeon YJ. Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue. Process Biochem 2012;47:34–40. [27] Li GH, Wan JZ, Le GW, Shi YH. Novel angiotensin I-converting enzyme inhibitory peptides isolated from alcalase hydrolysate of mung bean protein. J Pept Sci 2006;12:509–14. [28] Wijesekara I, Qian ZJ, Ryu BM, Ngo DH, Kim SK. Purification and identification of antihypertensive peptides from seaweed pipefish (Syngnathus schlegeli) muscle protein hydrolsyate. Food Res Int 2011;44:703–7. [29] Saito Y, Wanezaki K, Kawato A, Imayasu S. Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees. Biosci Biotechnol Biochem 1994;58:1767–71. [30] Byun HG, Kim SK. Purification and chracterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalogramma) skin. Process Biochem 2001;36:1155–62. [31] Jeon YJ, Byun HG, Kim SK. Improvement of functional properties of cod frame protein hydrolysate using ultrafiltration membranes. Process Biochem 2000;35:471–8. [32] Suetsuna K, Chen JR. Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulina platensis. Mar Biotechnol 2001;3:305–9. [33] Rho SJ, Lee JS, Chung YI, Kim YW, Lee HG. Purification and identification of an antiotensin I-converting enzyme inhibitory peptide form fermented soybean extract. Process Biochem 2009;44:490–3. [34] Clare DA, Swaisgood HE. Bioactive milk peptides: a prospectus. J Dairy Sci 2000;83:1187–95. [35] Astawan M, Wahyuni M, Yasuhara T, Yamada K, Tadokoro T, Maekawa A. Effects of angiotensin I-converting enzyme inhibitory substances derived from Indonesian dried-salted fish on blood pressure of rats. Biosci Biotechnol Biochem 1995;59:425–9.

S.-C. Ko et al. / Process Biochemistry 47 (2012) 2005–2011 [36] Wu J, Ding X. Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats. J Agric Food Chem 2006;54:732–8. [37] Wu J, Aluko RE, Nakai S. Structural requirements of angiotensin I-converting enzyme inhibitory peptides: quantitative structure–activity relationship study of di- and tripeptides. J Agric Food Chem 2006;54:732–8. [38] Chen J, Wanga Y, Zhong Q, Wua Y, Xiaa W. Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein. Peptides 2012;33:52–8.

2011

[39] Tsai JS, Lin TC, Chen JL, Pan BS. The inhibitory effects of fresh water clam (corbicula fluminea, Muller) muscle protein hydrolysates on antiotensin I converting enzyme. Process Biochem 2006;4:2276–81. [40] Zhao Y, Li B, Dong S, Liu ZL, Zhao Z, Wang J, et al. A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate. Peptides 2009;30: 1028–33. [41] Chen J, Wang Y, Zhong Q, Wu Y, Xia W. Purification and characterization of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein. Peptides 2012;33:52–8.