Isolation and characterisation of a novel angiotensin i-converting enzyme (ace) inhibitory peptide from the algae protein waste

Food Chemistry 115 (2009) 279–284 Contents lists available at Isolation and characterisation of a novel angiotensin I-converting enzyme(ACE) inhibitory peptide from the algae protein waste I.-Chuan Sheih a, Tony J. Fang a,1, Tung-Kung Wu b,* a Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang, Taichung 40227, Taiwan, ROCb Department of Biological Science and Technology, National Chiao Tung University,75 Po-Ai Street, Hsin-Chu 30068, Taiwan, ROC A hendeca-peptide with angiotensin I-converting enzyme (ACE) inhibitory activity was isolated from the Received 19 August 2008 pepsin hydrolysate of algae protein waste, a mass-produced industrial by-product of an algae essence Received in revised form 3 November 2008 from microalgae, Chlorella vulgaris. Edman degradation revealed its amino acid sequence to be Val-Glu- Accepted 4 December 2008 Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe. Inhibitory kinetics revealed a non-competitive binding modewith IC50 value against ACE of 29.6 lM, suggesting a potent amount of ACE inhibitory activity comparedwith other peptides from the microalgae protein hydrolysates which have a reported range between 11.4 and 315.3 lM. In addition, the purified hendeca-peptide completely retained its ACE inhibitory activity at a pH range of 2–10, temperatures of 40–100 °C, as well as after treatments in vitro by a gastrointestinal enzyme, thus indicating its heat- and pH-stability. The combination of the biochemical properties of this Angiotensin I-converting enzyme isolated hendeca-peptide and a cheap algae protein resource make an attractive alternative for producinga high value product for blood pressure regulation as well as water and fluid balance.
Ó 2008 Elsevier Ltd. All rights reserved.
great attention, due to their potential beneficial effects related tohypertension.
Hypertension is identified as a cardiovascular risk factor, and is A large variety of algae protein resources exists in the ocean, but often called a ‘‘silent killer" because persons with hypertension very few papers report the functional peptides from algae protein are often asymptomatic for years. This disease currently affects 15–20% of all adults (The renin- Among the known species of algae, Chlorella vulga- angiotensin system (RAS) plays an important role in the regulation ris has been the most popular edible microalgae with no side ef- of an organism's water, electrolytes and blood ( fects. Algae essence is an industrial product derived from water the angiotensin I-converting enzyme (ACE) participates in regulat- extracts of microalgae, and high molecular weight algae protein ing blood pressure. ACE inhibitors such as enalapril and captopril waste is a by-product of production. More than 100 tons of algae are used as antihypertensive drugs protein wastes are harvested every year in Taiwan, and it is all However, since synthetic ACE inhibitors remade into low economical-value animal feed. However, this by- cause a number of undesirable side effects such as cough, lost of product might become an important protein source for the selection taste, renal impairment, and angioneurotic oedema ( of novel ACE inhibitory peptides by enzymatic hydrolysis. This is a ), there has been a trend towards the development comparatively cheap protein source in contrast to most ACE inhib- of a natural ACE inhibitors. In recent years, peptides have been itory peptides originating from costly animal proteins and plant shown to possess many physiological functions, including im- proteins. In this study, we screened an ACE inhibitory peptide from mune-modulation antioxi- algae protein waste digested with commercial enzymes. We also dation (), antihypertension ( investigated the ACEs inhibitory potency, inhibition mechanism, and antimicrobial and stability against temperature, pH, and gastric proteases of the activities Among the different groups purified peptide from algae protein waste in vitro.
of bioactive peptides, the ACE inhibitory peptides have received 2. Materials and methods * Corresponding author. Tel.: +886 3 5712121/56917; fax: +886 3 5725700.
E-mail addresses: (T.J. Fang), Algae protein waste was dried and kept at 20 °C prior to use.
1 Tel.: +886 4 22861505; fax: +886 4 22876211.
Hippuryl-L-histidyl-L-leucine (HHL), ACE obtained from human 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.12.019 I.-C. Sheih et al. / Food Chemistry 115 (2009) 279–284 skin, hippuryl acid, and pancreatin from porcine were purchased with antihypertensive activity in the elutent were pooled for fur- from Sigma Chemical Co. (St. Louis, MO, USA). Flavourzyme Type ther experiments.
A and alcalase were purchased from Novo Nordisk A/S (Copenha-gen, Denmark) and papain was obtained from Amano (Nagoya, 2.4.4. Reverse-phase high-performance (RP-HPLC) chromatography Japan). Pepsin was obtained from Nacalai Tesque (Kyoto, Japan).
The lyophilised fraction was further purified on an Inertsil ODS- Sephacryl S-100 HR, Q-sepharose Fast Flow, and the Sephasil 3 C18 semi-prep column (10  250 mm). The column was eluted peptide C8 column were purchased from Pharmacia Biotech. Co.
with a linear gradient of acetonitrile (25–40% in 30 min) containing (Uppsala, Sweden).
0.1% TFA at 2 ml/min. The active fraction was re-chromatographiedon a Sephasil peptide C8 column (4.6  250 mm) at a flow rate of 2.2. Preparation of enzymatic hydrolysate Algae protein waste (10%, w/v) was digested with commercial 2.5. Determination of amino acid sequence proteases at the concentration of 0.2% (w/v) for 15 h at an appro-priate pH and temperature for each enzyme reaction, using the The amino acid sequence of the purified peptide was deter- reaction conditions suggested by the manufacturer. At the end of mined by an automated Edman degradation with an Applied Bio- the reaction, the digestion was heated in a boiling water bath for systems Procise 494 protein sequencer (Foster City, CA, USA) 10 min in order to inactivate the enzyme. The commercial enzymesused in this study included pepsin, flavourzyme, alcalase, and pa- 2.6. Stability of ACE inhibitory peptide pain. The protein yield was defined as the ratio of total protein inthe respective enzymatic hydrolysate over the total protein in The purified peptide solutions were incubated at different tem- the algae protein waste without enzyme hydrolysis.
peratures (40, 60, 80, and 100 °C) for 1 h, and then assayed forresidual ACE inhibitory activity. The peptide solutions were alsoincubated at 37 °C, and pH values of 2, 4, 6, 8 and 10, for 1 h. Sta- 2.3. Measurement of ACE inhibitory activity bility against gastrointestinal protease was also assayed in vitro. 1%(w/w) of ACE inhibitory peptide solution in 0.1 M KCl–HCl (pH 2.0) The ACE inhibitory activity was measured according to the buffer with pepsin was incubated for 3 h in a water bath at 37 °C, method of with some modifications.
then neutralised to pH 7.8 before heating to boiling for 10 min. The A mixture (190 ll) containing 100 mM sodium borate buffer (pH remaining suspension was further digested by a 1% (w/w) porcine 8.3), 1.68 mU ACE enzyme, and an appropriate mount of peptide pancreatin for 4 h at 37 °C. The sample was boiled for 10 min fol- solution was pre-incubated for 5 min at 37 °C. The reaction was ini- lowed by centrifugation (10,000 g, 10 min) and then assayed for tiated by adding 15 ll of HHL at a final concentration of 3.94 mM, residual ACE inhibitory activity and terminated by adding 190 ll of 1 M HCl after 1 h of incubation.
Five microlitres of the solution were injected directly onto an Inert- 2.7. Determination of the inhibition pattern on ACE sil (octadecylsilane) ODS-3 C18 column (4.6  250 mm) (). The mobile phase was 0.1% TFA in 50% Various substrate (HHL) concentrations were co-incubated with methanol with 0.8 ml/min and monitored at 228 nm to evaluate purified peptides and the ACE solution, and each reaction mixture the degree of inhibition of ACE activity by the bioactive peptides.
was assayed as described in Section . Standard hippuric acid The IC50 value was defined as the concentration of inhibitor re- solution was injected as a reference. The K quired to inhibit 50% of the ACE activity.
m and Vmax values for the reaction at different concentrations of purified peptides weredetermined according to Lineweaver–Burk plots.
2.4. Purification of ACE inhibitory peptides from algae protein waste 2.8. Statistical analysis 2.4.1. Ammonium sulfate fractionation Ammonium sulfate was added to a concentration of 20% satura- Results were presented as means of experiments done in tripli- tion in the supernatant from the pepsin hydrolysates; and precip- cate ± standard deviation. The Student's t-test was used to deter- itated protein was removed by centrifugation (10,000 g, 20 min).
mine the level of significance. A p value of less than 0.05 was The ammonium sulfate concentration was continually raised to taken as significant.
80% saturation in the permeate, stepwise. Each fraction was as-sessed for ACE inhibitory activity. A strong ACE inhibitory activityfraction was collected and lyophilised for the next step.
3. Results and discussion 3.1. Preparation of ACE inhibitory peptides from algae protein waste 2.4.2. Gel filtration chromatography The 40–80% precipitate was dissolved in distilled water and the Many ACE inhibitory peptides have been discovered from enzy- solution was fractionated using a Sephacryl S-100 high HR column matic hydrolysates of different food proteins, but so far, there has (u2.6  70 cm), pre-equilibrated with distilled water. The column been no research focused on cheaper algae protein waste which was eluted with the same buffer, and 6 ml fractions were collected consists of over 50% protein content. In this study, the algae protein at a flow rate of 1.5 ml/min. Fractions showing ACE inhibitory waste hydrolysates were prepared by means of hydrolysis with activity were pooled and lyophilised.
commercial proteases including pepsin, papain, alcalase, and fla-vourzyme. The hydrolysis was necessary in order to release ACE 2.4.3. Ion exchange chromatography inhibitory peptides from the inactive forms of intact algae protein A strong ACE inhibitory activity fraction subsequently was waste. The results indicated the specificity of the enzymes in the loaded onto a Q-sepharose Fast Flow column (u2.6  40 cm), generation of the ACE inhibitory peptides, as shown in . The which was pre-equilibrated with 20 mM Tris–HCl buffer solution pepsin treatment released specific peptides from the inactive algae (pH 7.8), then eluted with a linear gradient of NaCl (0.0–1.0 M) protein, with the highest ACE inhibitory activity and protein yield in the same buffer at a flow rate of 1.5 ml/min. Bioactive peptides among the hydrolysates (p < 0.05). Other reports indicate that pep- I.-C. Sheih et al. / Food Chemistry 115 (2009) 279–284 ACE inhibition activity (%) Flavouzyme Alcalase ACE inhibition activity (%) Protein yield (%) Flavouzyme Alcalase Fig. 1. (A) ACE inhibitory activity and (B) protein yield of algae protein waste ACE inhibition activity (%) hydrolysed by various enzymes, respectively. The protein yield was defined as the ratio of total protein in the respective enzymatic hydrolysate over the total protein observed for the control. The control was algae protein waste without enzymehydrolysis. The values were represented as the mean of the triplicate ± SD.
Fig. 2. (A) The ACE inhibitory activity of ammonium sulfate fractionation in the *Significant difference from control at p < 0.05.
pepsin hydrolysate. (B) The resultant ACE inhibitory activity of fractions (desig-nated as A and B) from 40% to 80% ammonium sulfate fraction on a Sephacryl S-100HR column. The values were represented as the mean of the triplicate ± SD.
sin was capable of producing ACE inhibitory peptides from algaeprotein ().
3.2. Purification of ACE inhibitory peptide from pepsin hydrolysate of The peptides present in pepsin hydrolysates from the algae pro- tein were fractionated with ammonium sulfate, and then separated into four fractions. The 40–60% and 60–80% fraction exhibited higher ACE inhibitory activity than other fractions These two fractions were combined, precipitated, and re-dissolved in a Absorbance at 215 nm small volume of distilled water, and subsequently purified using column chromatographic methods. Size exclusion chromatographyof the ammonium sulfate fraction on a Sephacryl S-100 high HR column resulted in two fractions (designated as A and B). Fraction B was found to possess higher ACE inhibitory activity (b), so itwas further subjected to a Q-sepharose Fast Flow column with a linear gradient of NaCl (0.0–1.0 M) (The bound peptides(B2 fraction), which were eluted at 0.35–0.45 M NaCl concentra- tion, had no ACE inhibitory activity, but the non-adsorption frac- tion (B1 fraction) expressed strong ACE inhibitory activity. TheB1 fraction was pooled, lyophilised, and further separated by RP- HPLC on an Inertsil ODS-3 C18 reverse-phase semi-prep column (10  250 mm). Fraction B1a showed the most potent ACEinhibitory activity, and was reloaded on a Sephasil peptide C8 re- ACE inhibition activity (%) verse-phase analytical column (4.6  250 mm) to attain a purifiedpeptide (P fraction) (The purified peptide was shown to in- Fig. 3. Elution profile and of active fraction B on a Q-sepharose Fast Flow column hibit ACE in a dose-dependent manner with an IC50 value 29.6 lM and its ACE inhibitory activity. The separation was performed at a flow rate of ). There have been few studies on ACE inhibitory peptides 90 ml/h with a linear gradient of NaCl (0–1.0 M) in 20 mM Tris–HCl buffer, pH 7.8.
from algae protein hydrolysates. re- The fractions were designated B1–B2, and activity was determined as the downpanel. The values were represented as the mean of triplicate ± SD.
ported IC50 values of Ala-Ile-Tyr-Lys, Tyr-Lys-Tyr-Tyr, Lys-Phe-Tyr-Gly, and Tyr-Asn-Lys-Leu from the peptic digest of wakame,Undaria pinnatifida, were 213, 64.2, 90.5 and 21 lM, respectively.
Ala-Glu-Leu and Val-Val-Pro-Pro-Ala from the peptic digest of The IC50 values of Ile-Val-Val-Glu, Ala-Phe-Leu, Phe-Ala-Leu, microalgae, C. vulgaris were 315.3, 63.8, 26.3, 57.1 and 79.5 lM, I.-C. Sheih et al. / Food Chemistry 115 (2009) 279–284 to validate the ACE inhibitory activity of the purified peptide, a synthetic hendeca-peptide with the same sequence was synthes- ised and tested. The synthetic peptide exhibited the same ACE inhibitory activity as the purified peptide from algae protein hydrolysate (data not shown). The result suggests that the purifiedpeptide actually possesses ACE inhibitory activity.
The hendeca-peptide sequence was next subjected to secondary structure prediction to elucidate possible structure-activity corre- lations ). The results showed thatthe hendeca-peptide contains 18.2% extended and 81.8% coiled Absorbance at 215 nm secondary structure. previously reported that the function of a bioactive peptide was dependenton its amino acid composition; however, the activity of these ami- no acid residues was also limited by the structure of the polypep- Retention time (min) tide (). Therefore, the extended and coiled structure in this peptide might contribute to ACE inhibitory activ- ity. In parallel, other structure-activity correlation studies haveindicated that ACE binding was strongly influenced by the C-termi- nal tripeptide sequence of the substrate, and the tripeptide could interact with the subsites S1, S0 and S0 of ACE ( ). The ACE preferred substrates containing branched amino acid residues at the N-terminal position, and hydrophobic aminoacid residues (aromatic or branched-side chains) at the C-terminal Absorbance at 215 nm ). The hydrophilic amino acid residues in the peptide Retention time (min) sequence could also affect inhibitory activity by disrupting the ac-cess of the peptide to the active site of ACE. The hydrophilic–hydro- Fig. 4. (A) Reverse-phase HPLC pattern of active fraction B1 on a ODS C18 reverse - phobic partitioning in the sequence was also a critical factor in the phase column (10  250 mm) and the separation was carried out with a linear inhibitory activity (). The peptide with comparative gradient from 25% to 40% acetonitrile in 0.1% TFA for 30 min at a flow rate of 2 ml/min. (B) The reverse-phase HPLC pattern of active fraction B1a on a Sephasil peptide low IC50 value had a high content of branched and aromatic amino C8 column (4.6  250 mm) at a flow rate of 1.0 ml/min. The P fraction represented acids such as Pro, Glu, Val, Phe, and Tyr in its peptide sequence.
the purified peptide.
Thus, it was very likely to have a higher antihypertensive potentialPerhaps the abundance of theabove-mentioned amino acids in the purified peptide might ac-count for the exhibited potency of its ACE inhibitory activity.
3.4. Stability of the purified peptide The processing stability of the purified peptide after various pH and temperature treatments was a prerequisite in preparing foodswith ‘‘functional peptides". To investigate the pH and heat-stability of the purified peptide, the peptide was subjected to incubation atpH 2–10 and temperature 40–100 °C for 1 h and measured for residual activity (data not shown). The results showed that the ACE inhibition activity (%) 21.61 µM 43.23 µM purified hendeca-peptide completely retained its ACE inhibitory Fig. 5. The ACE inhibitory activity of various concentrations of the purified peptide activity (p > 0.05), indicating that the purified peptide was both derived from algae protein waste. The values were represented as the mean of the pH and heat-stable.
triplicate ± SD.
Gastrointestinal enzyme incubation in vitro provided an easy process to imitate the fate of these peptides under oral adminis-tration. Some ACE inhibitory substances failed to show the hypo- respectively. The IC50 values of Ile-Ala-Glu, Ile-Ala-Pro-Gly and Val- tensive activity after oral administration in vivo, due to the Ala-Phe from peptic digest of the microalgae, Spirulina platensis, possible hydrolysis of these peptides by ACE or gastrointestinal were 34.7, 11.4 and 35.8 lM, respectively ( ). Therefore, the peptide purified from algae protein waste To evaluate the stability of the purified peptide under gastroin- in this study had potent ACE inhibitory activity, when compared testinal enzymes digestion, the purified peptide was first incu- to the results with the aforementioned peptides which ranged bated with various gastrointestinal enzymes, including pepsin from 11.4 to 315.3 lM.
and pancreatin, then subjected to ACE inhibitory activity assaysand HPLC profile comparisons. The results showed that no appar- 3.3. Determination of amino acid sequence ent change was observed after in vitro incubation with gastroin-testinal enzymes (p > 0.05), suggesting that there is resistance Most of the reported peptides exhibiting ACE inhibitory activity of the purified peptide to digestion in the gastrointestinal tract, contained 5–13 amino acids (The and that the active sequence of the peptide would not be de- purified peptide described herein was subjected to Edman degra- stroyed by these enzymes. The low susceptibility of the purified dation experiments for amino acid sequence determination. The peptide to hydrolysis by gastric proteases was similar to that of determined sequence was obtained as Val-Glu-Cys-Tyr-Gly-Pro- shorter oligopeptides, as shown by Wu and Ding for small pep- Asn-Arg-Pro-Gln-Phe, with a molecular mass of 1309 Da. In order I.-C. Sheih et al. / Food Chemistry 115 (2009) 279–284 didate in future industrial production of functional peptides forblood level regulation in hypertensive patients.
We thank National Chiao Tung University, MOE ATU Program and National Science Council, ROC, Project No. NSC-96-2313-B-005-008-MY3 for financially supporting this research. We are also grateful to the staffs of TC3 Proteomics, Technology Commons, Col- lege of Life Science, NTU for help with protein sequencing.
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FOKUSTHEMA 2: Pharmakologisches Enhancement in der Sexualität von Männern und Frauen Hintergründe, Fakten und Methoden zur natürlichen Luststeigerung (Bild:, Abdruck nicht gestattet) Potenzmittel– harmloses Vergnügen oder Gefahr? Das pharmakologische Enhancement ist längst in der täglichen Praxis angekommen. Die 1998

Endod Dent Traumatol 2000; 16: 1–8 Copyright C Munksgaard 2000 Printed in Denmark . All rights reserved ISSN 0109-2502 Macrophages in periapical lesions Metzger Z. Macrophages in periapical lesions. Endod Dent Z. Metzger Traumatol 2000; 16: 1–8. C Munksgaard, 2000. Department of Oral Biology, Goldschleger Schoolof Dental Medicine, Tel Aviv University, Tel Aviv,Israel and Department of Endodontics, University