Food Chemistry 128 (2011) 968–973

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Food Chemistry

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Antihypertensive effect of corn peptides, produced by a continuous productionin enzymatic membrane reactor, in spontaneously hypertensive rats

Wen-Hao Huang a, Jie Sun b, Hui He a,⇑, Hua-Wei Dong a, Jiang-Tao Li a

a College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Chinab General Mills, 9000 Plymouth Ave N JFB Center, Minneapolis, MN 55414, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 November 2010Received in revised form 20 March 2011Accepted 31 March 2011Available online 7 April 2011

Keywords:Corn peptide (CP)ACE-inhibitory peptidesUltrafiltrationEnzymatic membrane reactorsSpontaneously hypertensive rat (SHR)Antihypertension

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.03.127

⇑ Corresponding author. Tel.: +86 27 87280346; faxE-mail address: hehui@mail.hzau.edu.cn (H. He).

The aim of this study was to produce corn peptides (CP) with potent angiotensin converting enzyme(ACE)-inhibitory activity and to investigate antihypertensive effects in spontaneously hypertensive rats(SHRs). Results showed that the hydrolysate yield and quality were stable during continuous CP produc-tion using an enzymatic membrane reactor (EMR). The hydrolysate was then filtered through a 3 kDa cut-off membrane and the IC50 value of permeate was decreased fourfold (IC50 = 0.29 mg protein/ml). Gastric-intubation CP (Mw < 3 kDa) at a dose of 100 mg/kg bw revealed the best antihypertensive effects in bothacute and long-term animal studies using SHRs’ models, the reductions in maximal systolic blood pres-sure (SBP) were 26.57 mm Hg and 34.45 mm Hg, respectively. Long-term antihypertensive effect of CPadministration at a dose of 100 mg/kg bw was comparable to Captopril at 2 mg/kg bw dose. The CP sig-nificantly inhibited the ACE activities in SHRs’ lungs and testes (p < 0.01 � p < 0.05) suggesting that thesewere CP’s target sites.

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1. Introduction

In recent years, hypertension, which carries with it a high risk ofcerebrovascular, cardiac, and renal complications, has become themost common yet serious worldwide chronic health problem.Treatment, however, has been effective in reducing the risk ofthe disease (Collins et al., 1990). Lifestyle modifications and diettherapy are two of the most important tools in the preventionand treatment of hypertension (Hermansen, 2000).

The most common mechanism underlying a blood pressure-lowering effect seems to be inhibition of the angiotensin-I-convert-ing enzyme (ACE); hence ACE inhibitors are frequently used intherapy to reduce the morbidity and mortality of patients withhypertension and other related diseases. Synthetical ACE inhibitorssuch as Captopril, Enalapril, and Lisinopril are effective for decreas-ing blood pressure. However, some undesirable side effects havebeen reported, including coughing, dizziness, headache, abnormaltaste (metallic or salty taste), and kidney and liver problems. Re-cently, an increasing number of studies have suggested that somefood proteins have functions other than energetic and nutritionalones; several peptides with potent ACE-inhibitory activity havebeen isolated from such proteins (Miguel & Aleixandre, 2006;Murray & FitzGerald, 2007). The in vivo effects of antihypertensiveagents are usually tested using a spontaneously hypertensive rat

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(SHR) model, which has shown to be a comparable model for hu-man essential hypertension (FitzGerald, Murray, & Walsh, 2004).

Commercial production of ACE-inhibitory peptides has beenlimited by the lack of suitable large-scale technologies. Membraneseparation techniques have been used for the enrichment of pep-tides with a specific molecular weight (Mw) range (Korhonen &Pihlanto, 2003). Ultrafiltration is routinely employed to enrich bio-active peptides from protein hydrolysates. Enzymatic hydrolysiscan be performed through conventional batch hydrolysis or contin-uous hydrolysis using ultrafiltration membranes. Compared withconventional batch hydrolysis, enzymatic membrane reactors(EMR) for continuous production of specific peptides have beenwidely applied for total conversion of food proteins to hydrolysateswith improved nutritional and/or functional properties (Martin-Orue, Henry, & Bouhallab, 1999; Perea & Ugalde, 1996). EMR hasbeen shown to improve the efficiency of enzyme-catalysed biocon-version, and to increase product yields, and they can be easilyscaled up for industrial purposes.

Corn gluten meal (CGM), a by-product of the starch industrywith abundant protein, is mainly comprised of zein (�40%) andglutelin (�40%). Although presently mainly used as forage, CGMmay become a good source in the preparation of ACE-inhibitorypeptides due to its high proportions of hydrophobic amino acidand praline (Kim, Whang, Kim, Koh, & Suh, 2004). For example, ahexapeptide (Pro-Ser-Gly-Gln-Tyr-Tyr) and a dipeptide (Ala-Tyr)were isolated from the CGM hydrolysate and the IC50 value of theseACE-inhibitors was 0.1 mM and 14.2 lM, respectively (Suh,

W.-H. Huang et al. / Food Chemistry 128 (2011) 968–973 969

Whang, & Lee, 1999; Yang, Tao, Liu, & Liu, 2007). Miyoshi et al.(1991) separated several ACE-inhibitory peptides from a-zeinhydrolysate. One of them, a tripeptide (Leu-Arg-Pro), was synthe-sized and exerted potent ACE-inhibitory activity in SHRs.

The objectives of this study were (1) to investigate the use ofultrafiltration membrane reactors for corn protein hydrolysate pro-duction with ACE inhibitory activity; (2) to evaluate operation sta-bility and hydrolysate product quality measured by peptideconcentration, ACE inhibitory activity, and IC50 values during pro-cessing; and (3) to evaluate the in vivo antihypertension effects ofpurified peptides using an SHR model.

2. Materials and methods

2.1. Materials

Corn gluten meal (CGM) was purchased from Zheng Da Com-pany (Wuhan, China), which is a by-product of starch industrycontaining abundant protein. It is mainly comprised of zein(�40%) and glutelin (�40%). Alcalase 2.4 L, Commercial enzymes,was purchased from NOVO Industri A/S (Novo Nordisk BiochemInc., Tianjin, China). The hydrolysis effects of corn protein usingAlcalase and Neutrase were compared in our previous work (Sui,He, Wang, He, & Hu, 2006). Due to corn protein’s higher solubilityin alkaline condition and higher hydrolysis efficiency, Alcalase waschosen in the study. ACE (from rabbit lung), substrate peptide ofACE hippuryl-histidyl-leucine (HHL) and 2-deoxy-D-ribose werepurchased from Sigma Chemical Co. (St. Louis, MO, USA). Poly-meric-Cellulose spiral-wound membranes, including molecularweight cut-off (MwCO) at 1 kDa, 3 kDa and 5 kDa, were purchasedfrom Millipore Inc. (Billerica, Massachusetts, USA).

2.2. Preparation of concentrated corn protein

The concentrated corn protein was prepared from CGM as wedescribed previously (Guo, Sun, He, Yu, & Du, 2009). Its crude pro-teins content was 92.55%, as determined by Kjeldahl method. Theconversion factor from nitrogen to protein is 6.24.

2.3. Continuous preparation of corn protein hydrolysate by anenzymatic membrane reactor

To increase the degree of hydrolysis, 4% concentrated corn pro-tein suspensions (w/v) were heated at 90–100 �C for 30 min (Guoet al., 2009). The temperature of suspensions was then droppedto 45 �C and the pH value was adjusted to 8.0 (the optimum pHfor Alcalase activity), and Alcalase was then added to the suspen-sion in the reaction vessel at a 1.5% (w/w) ratio. The reaction vesselwas then incubated at 45 �C, equipped with spiral-wound ultrafil-tration modules containing 5 kDa nominal MwCO polymeric-cellu-lose membranes and a peristaltic pump. The mixture of substrateand the enzyme in the reaction vessel was recirculated from theprimary side of the membrane module pumped by a peristalticpump. The secondary side was under slight vacuum condition cre-ated by an ejector jet pump controlled by a by-pass valve. Perme-ate was collected in special traps, namely the enzymaticmembrane reactor. Inlet pressure and flow velocity were properlyadjusted. The reaction mixture was pumped through the ultrafil-tration membrane where the larger-Mw hydrolysate would recycleto the reaction vessel. The permeation with the lower Mw fraction,which was small enough to penetrate the membrane, was collectedfor further tests. The level of reaction mixture in the vessel wasmaintained at a constant level by the addition of corn protein pre-treated as described earlier.

2.4. The effect of treatment by different MwCO ultrafiltrationmembranes on ACE inhibitory activity of corn peptides (CP)

Hydrolysates (Mw < 5 kDa) produced in the EMR were then sub-jected to ultrafiltration using membranes with 1 kDa, 3 kDa MwCO,respectively. Each permeate fraction was collected and lyophilised.The two fractions and original hydrolysates were tested for ACE-inhibitory activity and the Mw < 3 kDa fraction was used in the fol-lowing animal test.

2.5. Assay for ACE inhibitory activity

ACE inhibitory activity was analysed by spectrophotometryusing hippuryl-histidyl-leucine (HHL) as substrate, according tothe method of Cushman and Cheung (1971) method with minormodifications. Testing solutions (100 ll) were mixed with 100 llof 0.1 M borate buffer (pH 8.3) containing 5 mM HHL and 0.3 MNaCl and 150 ll of ACE (2 mU). The mixture was incubated at37 �C for 30 min. The reaction was then stopped by the additionof 250 ll of 1 M HCl. Formed hippuric acid was extracted withethyl acetate (1500 ll) and, after removal of ethyl acetate(500 ll) by heat evaporation, the hippuric acid was redissolvedin 1 M NaCl (3 ml) and measured spectrophotometrically at228 nm. The IC50 value was defined as the concentration of ACEinhibitor namely protein hydrolysate needed to reduce 50% ofACE activity, and determined by regression analysis of ACE inhibi-tory activity (%) versus protein concentration. The IC50 value wasexpressed as mg protein/ml. The ACE inhibitory activity (%) was ex-pressed as follow:

ACE inhibitory activityð%Þ ¼ ðA� SÞ%ðA� CÞð2� 1Þ

wherein A is the absorbance at 228 nm without protein hydro-lysate; S is the absorbance with protein hydrolysate; and C is theabsorbance without protein hydrolysate and ACE.

2.6. Deoxyribose assay for hydroxyl radical

Deoxyribose assay was used to evaluate the hydroxyl radicalscavenging ability of CP as we described previously (Sun, He, &Xie, 2004).

2.7. Treatment of animals

Spontaneously hypertensive rats (SHRs) (8-weeks-old, males,SPF, 180–200 g body weight) with tail systolic blood pressure(SBP) over 180 mm Hg were purchased from Vital River Experi-ment Animal Inc., Beijing, China. All animals were cared for inaccordance with the standards for laboratory animals establishedby the People’s Republic of China (GB14925-2001) and Animalhandling followed the Declaration of Helsinki and the GuidingPrinciples in the Care and Use of Animals. The rats were allowedto acclimate a week prior to the experiments. They were housedindividually in steel cages in a room kept at 25 �C with a relativehumidity of 50% and a 12 h light–dark cycle, and fed a standardlaboratory diet. Tap water was freely available. SBP was measuredby tail-cuff method with a tail measurement device (ALC–NIBP sys-tem, Shanghai Alcott Biotech Co., Ltd., Shanghai, China).

After one week of acclimation, 40 SHRs were randomly dividedinto five groups, namely the SHR model group, the Captopril con-trol group, and CP groups (including low dose group (25 mg/kg bw), middle dose group (50 mg/kg�bw), and high dose group(100 mg/kg bw)). CP with Mw < 3 kDa were dissolved with distilledwater at different concentrations and intragastric administrated toSHRs via gavage tube at multiple doses of 25, 50, 100 mg/kg bw. CPtest dosages were selected by preliminary tests. Captopril, a knownACE inhibitor, was administrated at a dose of 2 mg/kg bw as a

970 W.-H. Huang et al. / Food Chemistry 128 (2011) 968–973

positive control. Model control SHRs were administrated with sal-ine water. All volumes of oral administration were at a dose of5 ml/kg bw. The SBP was measured in rats by the tail-cuff methodboth before administration and 1, 2, 3, 4, 5 and 7 h post-administration.

For our long-term experiments, 40 SHRs were randomly dividedinto five groups, similar to those mentioned in the previous acuteoral administration experiments. Eight Wistar-Kyoto rats (8-weeks-old, males, SPF, 180–200 g body weight) were then ran-domly chosen as the normal control group. The CP (Mw < 3 kDa)were dissolved with distilled water at different concentrationsand intragastric administrated in SHRs at multiple doses of 25,50, 100 mg/kg bw. Positive control rats were administrated withCaptopril at a dose of 2 mg/kg; SHRs in the model control groupwere administrated with saline water; and rats in the normal con-trol group were administrated CP (Mw < 3 kDa) at dose of 100 mg/kg bw. All volumes of oral administration were at 5 ml/kg bw. Alltreatments were administrated once a day between 9:00 am and12:00 noon for 30 days. The SBP was measured on days 0, 6, 12,18, 24, 30.

2.8. Determination of biochemical parameters in tissues

Thirty days after oral sample administrations, blood sampleswere collected and serums were isolated for further tests. All ani-mals were then sacrificed. Lung, heart, kidney, and testes were ex-cised and ACE extracts prepared by the method described byYasunori, Osamu, and Toshiaki (1996). Tissue ACE inhibitory activ-ities were assayed by the method described in Section 2.2.

2.9. Statistical analysis

Quantitative data were expressed as mean ± SD and all statisti-cal comparisons were made by means of one-way ANOVA test fol-lowed by Tukey’s test. Differences were considered statisticallysignificant when p < 0.05.

3. Results and discussion

3.1. Continuous preparation of corn peptides in enzymatic membranereactor

Fig. 1 shows that the quality and yield of hydrolysates(Mw < 5 kDa) were fairly stable during the continuous 5 h prepara-tion process in the EMR. From the first to the second hour, the pep-tide concentration and their ACE-inhibitory activities changed onlyslightly in the permeate fraction; or the peptides level of permeatedecreased slightly whilst their ACE-inhibitory activities increasedwith time. It may be reasoned that at the beginning of the reactionthe permeate fraction contained a high level of Mw peptide, whichhas lower ACE-inhibitory activities, compared to a lower level of

30

40

50

60

70

80

90

100

1 2 3 4 5time(h)

ACE

inhi

bitio

n (%

)

2

2.5

3

3.5

4

4.5

5

pept

ide

conc

entra

tion(

mg/

ml)

Fig. 1. Peptides concentrations (�) and ACE-inhibitory activities (j) at differenthydrolysis time. Results are mean values of at least three replicates. Standarddeviation did not exceed 3% of the recorded values.

Mw peptides. After the initial 2 h reactions, the permeate peptideconcentration reached a constant level of 4 mg/ml, and ACE-inhib-itory activities were at approximately 84.00% from 2 h to 5 h. ACE-inhibitory activity of the permeation reached 89.82% at 5 h. Utilis-ing the enzymatic ultrafiltration membrane reactor, the continu-ous production method improved both the yield and productquality compared to the batch process. The desired corn peptideswere continuously produced and collected by a constant feed ofcorn protein and enzymes. The new process not only avoidedunnecessary further hydrolysis of needed corn peptides but alsoused protease more efficiently.

3.2. ACE-inhibitory activities and �OH scavenging activities of cornpeptides isolated through different ultrafiltration membranes

3.2.1. ACE-inhibitory activities of corn peptides with differentmolecular weight

Using different MwCO ultrafiltration membranes, fractions ofCP were isolated and each contained a range of molecular-weightpeptides. The fraction with Mw < 1 kDa exhibited a much lowerIC50 value (IC50 = 0.44 mg protein/ml) than that of the Mw < 5 kDafraction (IC50 = 1.27 mg protein/ml) (Table 1), whilst theMw < 3 kDa fraction exhibited the lowest IC50 value (IC50 = 0.29 mgpeptides/ml) amongst all fractions, with the IC50 value decreasingfourfold of the Mw < 5 kDa fraction.

Based on their action mechanisms, ACE inhibitors can be gener-ally divided into two categories: (1) those that compete with avail-able ACE substrate to react with ACE; and (2) those that combinewith the ACE bioactive region to inhibit its enzymatic activity.ACE substrate competitors are normally composed of more thanfour amino acids whilst ACE active site inhibitors are composedof two or three amino acids. The ACE-inhibitory peptides derivedfrom protein are mostly composed of 2–15 amino acids. Themolecular size of the ACE-inhibitory peptide plays an importantrole in its inhibitory activity (Fujita, Yoshikaw, & LKPNM, 1999).Membrane separation techniques provide the most up-to-datetechnology for peptide enrichment with a specific molecularweight range (Korhonen & Pihlanto, 2003). Hyun and Shin (2000)studied activities of ACE-inhibitory peptides derived from Alcalasehydrolysate of bovine serum albumin and trypsin hydrolysate ofcasein. The peptides were isolated using ultrafiltration membraneswith 10 kDa, 3 kDa and 1 kDa MwCO respectively, and the authorsfound that the ACE-inhibitory activities increased with a decreaseof peptide’s MwCO range. However, Mullally, Meisel, and FitzGer-ald (1997) found that the ACE-inhibitory activities of whey proteinhydrolysates increased by ultrafiltration purification, but the ACE-inhibitory activity of peptide fraction with Mw < 1 kDa was lowerthan those with Mw < 3 kDa. Lee and Song (2003) also found a sim-ilar result when they processed bovine serum albumin hydrolysateusing ultrafiltration membrane.

Our study suggested similar findings to Mullally’s and Lee’sobservations (Lee & Song, 2003; Mullally et al., 1997). The peptidefraction with Mw < 3 kDa showed the highest ACE-inhibitory activ-ity, followed by the Mw < 1 kDa fraction and then the Mw < 5 kDafraction. Although the corn peptides with ACE-inhibitory activity

Table 1IC50 value of ACE-inhibitory activities and OH scavenging activity of the corn peptideswith different molecular weight.

Sample IC50(mg/ml)

ACE-inhibitory activity OH scavenging activity

Mw < 1 kDa permeate 0.44 2.32Mw < 3 kDa permeate 0.29 1.99Mw < 5 kDa permeate 1.27 2.08

Time(h)

Sy

sto

lic

blo

od

pre

ssu

re (

mm

Hg

)

****

**

**

**

****

****

**

**

*

** **** **

****

**

Fig. 2. Changes of systolic blood pressure (SBP) of spontaneously hypertensive rats(SHRs) in test groups with acute oral administration where model control: 25 mg/kg of saline water (j); Captopril: 2 mg/kg�bw (�); corn peptide groups: 25 mg/kg�bw (N), 50 mg/kg�bw (h) and 100 mg/kg�bw (s), Data are expressed asmean ± SD (n = 8). ⁄Significantly different (p < 0.05) versus the model control.⁄⁄Significantly different (p < 0.01) versus the model control.

W.-H. Huang et al. / Food Chemistry 128 (2011) 968–973 971

reported all were Mw < 1 kDa (Suh et al., 1999; Yang et al., 2007;Yano, Suzuki, & Funatsu, 1996), our results demonstrated thatthe CP fraction with Mw < 3 kDa had the strongest ACE-inhibitoryactivity, suggesting that the CP fraction with Mw between 1 kDaand 3 kDa might be synergist.

Peptides could be absorbed in an intact form through the inter-stitial space into the intestinal lymphatic system. The ability ofcompounds to enter the intestinal lymphatic system was affectedby their permeability via the capillary of the portal circulationand lipid solubility (Deak & Csaky, 1984). Molecular size and struc-tural properties, such as hydrophobicity, affected the major trans-port route of peptides (Shimizu, Tsunogai, & Arai, 1997). Researchfindings indicated that peptides with 2–6 amino acids were mosteasily absorbed in comparison with protein and free amino acids(Grimble, 1994). Roberts, Burney, Black, and Zaloga (1999) have re-ported that small (di- and tripeptides) and large (10–51 aminoacids) peptides could cross the intestinal barrier intact and exhib-ited their biological functions at the tissue level. It implied that theCP fraction with Mw < 3 kDa could be absorbed in an intact formand exhibited biological functions at the tissue level.

3.2.2. �OH scavenging activities of corn peptides with differentmolecular weight

Table 1 indicated that IC50 value of �OH scavenging activity ofthe CP fraction with Mw < 3 kDa was the lowest amongst all CPfractions, which was however similar to that of other fractionswith statistical difference. The Mw < 3 kDa fraction not only exhib-ited the highest ACE-inhibitory activity, but also the highest � scav-enging activity. Its ACE-inhibitory activity decreased fourfold(Table 1) in comparison with the Mw < 5 kDa fraction. Neverthe-less, their � scavenging activities were similar.

Vercruysse, Smagghe, Beckers, and Camp (2009) have reportedthat insect protein hydrolysates, exerting both ACE-inhibitoryand antioxidant activity, might be incorporated as multifunctionalingredient into functional foods. Miguel, Alonso, Salaices, Aleixan-dre, and Lópze-Fandiño (2007) have investigated the hydrolysateof egg white (HEW). They found the combination of ACE-inhibitoryactivity and vascular relaxation properties, in addition to antioxi-dant effects, could provide superior cardiovascular protection,due to an additive effect, suggesting that HEW or HEW < 3 kDacould be useful in the prevention and/or treatment of hypertensionand other associated disorders. The correlation between ACE-inhibitory activity and antioxidant activity about CP is intricate,which will be studied in further experiments.

3.3. Antihypertensive activity of corn peptides in vivo

In our acute oral administration experiments (Fig. 2), therewere little SBP changes observed in the SHR model control group.After oral administration of Captopril and CP (Mw < 3 kDa) sampleswith several dosages, significant SBP reductions were found at 1 h(p < 0.01), compared to the SHR model control group. The maxi-

Table 2Changes of systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs) in tes

Group/dose SBP/mm Hg

0d 6d 12d

SHR model control 188.21 ± 2.17 186.69 ± 5.08 186Captopril/2 mg/kg�bw 189.68 ± 4.84 157.42 ± 4.85** 153CP/100 mg/kg�bw 189.86 ± 3.34 158.70 ± 2.02** 157CP/50 mg/kg�bw 189.80 ± 4.21 165.98 ± 4.74** 163CP/25 mg/kg�bw 186.01 ± 2.49 166.57 ± 3.13** 166Normal control/100 mg/kg�bw 119.97 ± 5.63 113.46 ± 5.42 111

Data are expressed as mean ± SD (n = 8)** Significant differences (p < 0.01) versus model control.

mum SBP reduction levels of these treatment groups were, indescending order: Captopril group (38.42 mm Hg); CP high dosegroup (26.57 mm Hg); CP middle dose group (19.57 mm Hg); andCP low dose group (17.91 mm Hg). The antihypertensive effectsof these treatments were maintained for 5 h. The SBP began to re-store 3 h after treatment and returned to initial levels 7 h afteradministration. The best antihypertensive effect was observed inthe positive control group (Captopril) in the first three hours. At4 h, however, a high dose of CP at 100 mg/kg bw treatment showedan even longer lasting antihypertensive effect than a dose of 2 mg/kg bw Captopril. Overall, our results clearly showed that CP treat-ment could depress SBP rapidly in SHRs.

In our long-term experiments (Table 2), the SBP of all treatmentgroups was significantly lowered (p < 0.01) as compared to the SHRmodel control group. At any given time, Captopril (2 mg/kg bw)showed the strongest SBP reduction effects amongst all treat-ments. After 30 days of treatment, the maximal SBP reductions inall groups were, in descending order: Captopril group(37.28 mm Hg), CP high dose group (34.45 mm Hg), CP middledose group (30.95 mm Hg) and CP low dose group (27.49 mm Hg).SBP reductions of more than 30 mm Hg were observed in both theCP middle dose and high dose groups. The antihypertensive effectimproved with a CP dosage increase. The SBP reduction of the Cap-topril and CP high dose groups exceeded 30 mm Hg on the sixthday. Interestingly, the SBP of Wistar-Kyoto rats showed no signifi-cant changes during long-term oral treatment with high dose CP.These results indicated that CP not only had good long-term anti-hypertensive effects, but also had no side effects for normal rats.

Many ACE inhibitory peptides have recently been found fromenzyme-digested food protein. Miguel et al. (2007) reported

t groups over time after long-term administration.

18d 24d 30d

.72 ± 2.24 182.68 ± 3.9 191.51 ± 8.93 186.15 ± 7.27

.03 ± 2.83** 154.86 ± 2.70** 150.29 ± 11.87** 152.4 ± 1.77**

.84 ± 2.91** 157.15 ± 3.25** 156.91 ± 11.09** 155.41 ± 3.48**

.12 ± 3.28** 163.25 ± 4.43** 160.59 ± 7.82** 158.85 ± 3.21**

.04 ± 3.78** 165.62 ± 5.83** 166.61 ± 4.49** 158.52 ± 5.53**

.04 ± 4.95 117.84 ± 9.85 120.97 ± 10.97 114.79 ± 5.01

Table 3ACE activity in tissues in SHR after oral administration of corn peptide at 30 days (mU/ mg prot, n = 8, x ± SD).

Group/dose Serum Kidney Heart Lung Testes

Model control 20.75 ± 6.64 2.95 ± 0.76 67.02 ± 20.45 388.83 ± 44.97 264.75 ± 49.98Captopril/2 mg/kg�bw 22.85 ± 5.67 2.02 ± 0.50* 68.64 ± 20.27 293.73 ± 30.48** 204.69 ± 45.29*

CP/100 mg/kg�bw 24.62 ± 4.26 3.04 ± 1.03 64.36 ± 8.62 283.78 ± 61.51** 204.90 ± 20.57*

CP/50 mg/kg�bw 21.54 ± 6.05 3.14 ± 0.92 69.80 ± 5.08 316.61 ± 43.54** 235.27 ± 53.46CP/25 mg/kg�bw 21.05 ± 8.64 3.00 ± 0.97 68.69 ± 14.10 383.96 ± 40.90 246.69 ± 27.01

* Significant differences (p < 0.05) versus model control.** Significant differences (p < 0.01) versus model control.

972 W.-H. Huang et al. / Food Chemistry 128 (2011) 968–973

significant SBP decrease (�20 mm Hg) of SHR 2-4 h after oraladministration of Mw < 3 kDa egg white hydrolysate fraction(100 mg/kg bw). A hexapeptide (Pro-Ser-Gly-Gln-Tyr-Tyr) was iso-lated from CGM, which may antagonise a rat’s pressor response toangiotensin-I. Maximum blood pressure reduction of 6 mm Hg wasobserved 15 min after intravenous administration of hexapeptide,at a dose of 30 mg/kg body weight of the rat (Suh et al., 1999). Yanget al. (2007) found a dipeptide (Ala-Tyr), which was also isolatedfrom CGM, and exhibited a maximum SBP reduction of 9.5 mm Hg2 h after oral administration of Ala-Tyr at doses of 50 mg/kg bw.Miyoshi et al. (1991) isolated a tripeptide (Leu-Arg-Pro) from zein.At a dose of 30 mg/kg of body weight, the intravenous administra-tion of this tripeptide reduced a rat’s SBP by 15 mm Hg in 2 min;however, the SBP level returned to the initial level five min aftertripeptide administration. Base on acute oral administration testresults of these studies, it seemed that although antihypertensiveeffects were observed soon after intravenous administration of asingle peptide, the effect duration was short and the antihyperten-sive effect seemed inferior to that of mixed peptides. In our study,CP, a mixture of peptides, was continuously prepared in an EMR,and then subjected to ultrafiltration using 3 kDa MwCO mem-brane. The mixture of peptides (CP) not only showed potent ACE-inhibitory activity in vitro, but also decreased SBP by 16.9 mm Hgin SHRs 1 h after acute oral administration (25 mg/ml). In addition,the significant antihypertensive effects lasted for 5 h. This sug-gested that the CP antihypertensive effect was better than the hex-apeptide, tripeptide and dipeptide effects mentioned above.Moreover, we observed a long-term CP antihypertensive actionin vivo; CP at a dose of 100 and 50 mg/kg bw reduced the SBP morethan 30 mm Hg.

Further more, corn gluten meal (CGM) is an abundant byprod-uct of the starch industry. Hence, CP derived from CGM is not onlymore abundant and costs less than a single peptide, it also demon-strated more potent antihypertensive effects through synergistaction.

3.4. Effects of CP treatment on ACE in tissues of SHRs in a long-termexperiment

To establish the CP dose–response relationship and its inhibi-tory effects in various tissues, the Mw < 3 kDa CP fraction was cho-sen for our long-term animal experiment. Compared to the modelcontrol group (Table 3), ACE activity in kidney tissue was reducedby 31.4% in Captopril-administrated SHRs. This change was signif-icant (p < 0.05). Lung ACE activities were also significantly de-creased (p < 0.01) by Captopril (2 mg/kg bw) and in all CP groupsexcept the low dose group (25 mg/kg bw). Moreover, CP treatmentat a dose of 100 mg/kg bw exhibited more effective lung ACE inhi-bition than was caused by 2 mg/kg bw Captopril. Compared to themodel control group (Table 3), the ACE activities in testes tissuedecreased 22.7% and 22.6%, respectively, in both the Captopriland high dose CP group (p < 0.05). The ACE-inhibitory activity ofCP at a dose of 100 mg/kg bw was similar to that of Captopril(2 mg/kg bw); however, the serum and heart ACE activities

amongst all groups were not significantly different from the modelcontrol group.

It is well known that ACE exists in the vascular endothelium ofvarious organs. In order to exert an antihypertensive effect afteroral ingestion, bioactive peptides must be absorbed from the intes-tine intact and be resistant to degradation by plasma peptidases toreach the target sites (Ondetti & Cushman, 1982). Researchersthink that the prolonged antihypertensive action of ACE inhibitorsmight be related to persistent inhibition of ACE activity of tissues,such as vascular wall and kidneys. The antihypertensive activitiesof Captopril, an ACE-inhibitor, are known to be related to the inhi-bition of ACE activities in such organs as the lung, kidney, aorta,and brain (Unger, Ganten, Lang, & Schölkens, 1985). Masuda,Nakamura, and Takano (1996) found that the aorta ACE activityin SHRs was significantly decreased compared to the model controlgroup by administration of acidophilus milk that contained twokinds of tripeptide, Val-Pro-Pro and Ile-Pro-Pro. The antihyperten-sive effect of oligopeptides of Mw < 1 kDa obtained by hydrolysis ofchicken egg yolks was due to its inhibiting ability on serum ACEactivity (Yoshii et al., 2001). The ACE inhibitors with differentenzymology and biological characteristics had different sites of ac-tion and expressed different lower blood-pressure effects (Cottonet al., 2002). Miguel, Contreras, Recio, and Aleixandre (2009) indi-cated that ACE inhibitors with different molecular structures haddifferent metabolism pathways and tissue distribution. These char-acteristics determined an ACE inhibitory effect in tissue and thusexhibited different antihypertensive effects. In this study, we mea-sured the ACE activities in various organs and serum as a first stepin clarifying the CP antihypertensive-activity mechanism. Wefound that the lung and testes were the target sites of CP, and thatCP showed antihypertensive effects through inhibiting the ACEactivity in both the lung and testes.

4. Conclusions

The hydrolysate’s quality and yield were well maintained dur-ing continuous production by the EMR. The IC50 value of ACE-inhibitory activities of CP fraction with Mw < 3 kDa decreased four-fold than that of CP fraction with Mw < 5 kDa. The IC50 value of �OHradical scavenging activity of the CP fraction with Mw < 3 kDa wasthe lowest amongst all CP fractions, which was however similar tothat of other fractions with statistical difference. In the acute oraladministration experiment, CP administered at a dose of 100 mg/kg bw showed the best antihypertensive effect in CP treatmentgroups. Compared with the SHR model control group, the maximalSBP reduction of 26.57 mm Hg occurred 2 h after administering CPat dose of 100 mg/kg bw, and the significant reduction of SBP couldpersist for 5 h longer than Captopril at a dose of 2 mg/kg bw. In ourlong-term experiment, exceeded 30 mm Hg reduction of SBP wasobserved on the sixth day by administering CP at a dose of100 mg/kg bw. After 30 successive days of administering CP, themaximal SBP reduction was 34.45 mm Hg; and the SBP reductionof more than 30 mm Hg was also observed in the CP middle dose(50 mg/kg bw) group on day 30. The lung and testes ACE activities

W.-H. Huang et al. / Food Chemistry 128 (2011) 968–973 973

were significantly decreased by CP (100 mg/kg bw) administration(p < 0.05), which demonstrated that these organs were the targetsites that CP exerted its most antihypertensive effects.

Acknowledgements

We gratefully thank the National ‘‘863’’ Program of China(Grant No. 2008AA10Z314) and the National Natural Science Foun-dation of China (Grant No. 30972043) for the financial support onthis research.

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