Food Chemistry 126 (2011) 1909–1915

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Food Chemistry

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Analytical Methods

Screening assay of angiotensin-converting enzyme inhibitory activity fromcomplex natural colourants and foods using high-throughput LC-MS/MS

Koichi Inoue a,⇑, Marie Kitade a, Tomoaki Hino a,b, Hisao Oka a,b

a Department of Physical and Analytical Chemistry, School of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya 463-8521, Japanb Graduate School of Human Ecology, Human Ecology Major, Kinjo Gakuin University, Japan

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

Article history:Received 19 August 2010Accepted 2 December 2010Available online 22 December 2010

Keywords:Angiotensin-converting enzymeInhibitorsLiquid chromatography mass spectrometryStable isotope dilution

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.12.019

Abbreviations: ACE, angiotensin-converting enzymLC, liquid chromatography; SID, stable isotope dilutionMS/MS, tandem mass spectrometry; MRM, multiphippuric acid; HHL, hippuryl-L-histidyl-L-leucine; RAS⇑ Corresponding author. Tel./fax: +81 52 798 0982.

E-mail address: kinoue@kinjo-u.ac.jp (K. Inoue).

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACEinhibitors derived from natural components may be of therapeutic value in preventive medicine. In thisstudy, we report a novel screening assay of ACE inhibitors from complex natural colourants and foodsthat employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, andstable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target samplewas subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuricacid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identifythe effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. Themajor HA and HA-d5 fragment ions at m/z 180 ? 105 and 185 ? 110 in the multiple reaction monitoring(MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS methoddescribed here is a rapid, selective, sensitive, and highly reproducible method for the determination ofHA in various samples. Based on the assay developed, all samples such as natural colourants, infant for-mula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee couldbe accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assayhas no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The renin-angiotensin system (RAS) is a critical regulator ofblood pressure and fluid homeostasis. Angiotensin II, the primarybioactive peptide of the RAS, is generated from angiotensin I byangiotensin-converting enzyme (ACE). ACE plays an important rolein the RAS in maintaining blood pressure levels. Elevated ACEactivity leads to an increased concentration of angiotensin II andhypertension. Thus, ACE inhibition is considered to be a targetfor antihypertensive agents and an important therapeutic ap-proach in the management of blood pressure levels.

Hypertension affects up to 30% of the adult population inmost countries. On an estimate, 7.6 million premature deaths,92 million deaths, and disability-adjusted life years can be attrib-uted to high blood pressure (Lawes, Vander Hoorn, & Rodgers,

ll rights reserved.

e; SPE, solid phase extraction;; ESI, electrospray ionisation;

le reaction monitoring; HA,, Renin-angiotensin system.

2008). Untreated hypertension can lead to a stroke, coronaryheart disease, and kidney dysfunction. In recent years, nutraceu-ticals and functional foods have attracted considerable interest aspotential alternative therapies for the treatment of hypertension,especially for pre-hypertensive patients. Chen et al. suggest thatblood pressure-lowering nutraceuticals and functional foods playan important role in the treatment of hypertension and reductionin the risk of cardiovascular diseases (Chen et al., 2009). Martinet al. indicated that a significant opportunity exists for studyingthe effects and mechanisms of action of soy isoflavones onhypertension in both animals and humans (Martin, Song, Mark,& Eyster, 2008a). The blood pressure-lowering properties of thesenutraceuticals and functional foods are most likely mediated bythe inhibition of ACE activity (Guang & Phillips, 2009). A numberof animal-derived peptides, including peptides from milk, egg,and muscle proteins, have exhibited in vitro ACE inhibition activ-ity (Balti, Nedjar-Arroume, Adjé, Guillochon, & Nasri, 2010; Hiro-ta et al., 2007; Majumder & Wu, 2009; Miguel et al., 2007). Someof these have displayed significant antihypertensive activity inrat and human studies (FitzGerald, Murray, & Walsh, 2004;Murray & FitzGerald, 2007). For the discovery of new targetedcompounds from natural products, an extensive research has

1910 K. Inoue et al. / Food Chemistry 126 (2011) 1909–1915

been carried out to detect ACE inhibitory substances such as pep-tides, proteins, and chemicals from food and natural sources.

Accordingly, a convenient, reliable, simple, and sensitive meth-od is needed to evaluate the ACE activity of effective compoundsfrom various materials. ACE activity is usually determinedin vitro by monitoring the transformation of a substrate to a prod-uct, as catalysed by ACE. There are spectrophotometric methods forthe determination of ACE inhibitory peptides (Hernández-Ledesma, Martín-Alvarez, & Pueyo, 2003; Li, Liu, Shi, & Le, 2005).Usually, a target sample and Hippuryl-His-Leu (HHL) solution isincubated with ACE, stopped by the addition of HCl, and extractedof hippuric acid (HA) using ethyl acetate. Then, an aliquot of eachethyl acetate layer is evaporated, redissolved in pure water, andthe absorbance determined at 228 nm (Cushman & Cheung,1971; Ibarra-Rubio, Pena, & Pedraza-Chaverri, 1989). However,the sensitivity, reliability, and resolution of spectrophotometric as-says are not sufficient for the direct evaluation of the ACE activityof high-matrix samples such as food and natural products. Recentstudies have reported that liquid chromatography (LC) withultraviolet (UV) and mass spectrometric (MS) detections were usedfor screening the ACE activity (Geng, He, Yang, & Wang, 2010;Lahoguea, Réhela, Taupina, Harasa, & Allaume, 2010; Siemerinket al., 2010; Xiao, Luo, Chen, & Yao, 2006). These chromatographicapproaches provide an accurate method for screening the ACEactivity and inhibition in various materials. Moreover, MS detec-tion is useful and powerful in the selective determination of a sub-strate to the product, as catalysed by ACE. A few LC-MS methodsattempted to quantify the ACE activity and inhibition (van Elswijket al., 2003). Unfortunately, these reported methods could not beapplied to evaluate high-matrix and complex samples from naturalfoods and products. For the investigation of blood pressure-lowering nutraceuticals and functional foods, a useful screening as-say of ACE activity and inhibition would be needed for high-matrixand complex samples.

In this study, we describe a novel, sensitive, and reliablemethod for the detection and quantification of ACE inhibitionactivity in high-matrix natural colourants and food samples thatemploys solid phase extraction (SPE), high-throughput LC separa-tion, and stable isotope dilution electrospray tandem massspectrometry (SID-ESI-MS/MS) in multiple reaction monitoring(MRM) mode. This method is applied to determine a productionof HA from HHL by ACE activity and allows a completeassessment of systemic and enzymatic changes. To the best ofour knowledge, the LC/SID-ESI-MS/MS with SPE is the mostcomprehensive and rapid screening method with high sensitivityin a single analysis. This approach is applicable to the monitor-ing of a variety of targeted ACE inhibitors in various foodmatrixes.

2. Experimental

2.1. Reagents

N-Hippuryl-His-Leu hydrate (HHL), ACE (25 unit, 5.35 units/mgsolid) and glycerol (for molecular biology, purity: 99%) were ob-tained from Sigma–Aldrich (St. Louis, MO). Hippuric acid (HA, pur-ity: 98.0%), captopril (purity: 96.0%), sodium dihydrogenphosphatedehydrate, potassium dihydrogenphosphate, formic acid, methanol(for HPLC analysis) and acetonitrile (for HPLC analysis) wereobtained from Wako Chemical Co. (Osaka, Japan). N-Benzoyl-d5-glycine (HA-d5, purity: 99.4%) was obtained from CDN IsotopesCo. (Quebec, Canada). Natural colourants were obtained fromSan-Ei Gen F.F.I. Co. (Osaka, Japan) and Kanto Chemical Co. (Tokyo,Japan). Purified water was obtained using a Milli-Q Simplicity� UVsystem (Millipore, Bedford, MA, USA).

2.2. Equipment

HPLC was performed using a LC-20AD pump, SPD-20AV detec-tor, CTO-20AC column oven with injector, and C-R8A recorder sys-tem (Shimadzu Co., Kyoto, Japan). HPLC separation was performedusing a TSK-GEL ODS 100 V column (2.0 � 150 mm, 3 lm: TosohCo., Tokyo, Japan). LC/MS/MS analyses were performed using aWaters Alliance 2695/Micromass Quattro Premier system with anelectrospray ionisation (ESI) source (Waters, Milford, MA, USA).LC column was a TSK-GEL ODS-140HTP (2.1 � 50 mm, 2.3 lm: Tos-oh Co., Tokyo, Japan).

2.3. HPLC assay

The mobile phase consisted of 0.1% aqueous formic acid (Sol-vent A) and 0.1% formic acid in acetonitrile (Solvent B). The HPLCstep-wise gradient was as follows: 15% Solvent B at 0 min, 15% Sol-vent B at 15 min, 98% Solvent B at 15.1 min, and 98% Solvent B at20 min with a flow rate of 0.2 mL/min. The elution of HA was mon-itored by absorbance at 225 or 280 nm.

2.4. LC/ESI-MS/MS assay

The mobile phase consisted of 0.1% aqueous formic acid(Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). TheLC step-wise gradient was as follows: 25% Solvent B at 0 min,25% Solvent B at 3.5 min, 95% Solvent B at 3.6 min, 95% Solvent Bat 6.0 min, and 25% Solvent B at 6.1 min with a flow rate of0.2 mL/min. The injection volume was 1.0 lL. The column temper-ature was 40 �C. The ESI source conditions in the positive ionisationmode were as follows: capillary voltage 2.5 kV, extractor voltage4 V, RF lens voltage 0 V, source temperature of 110 �C, and desolv-ation temperature of 380 �C. The cone and desolvation gas flowswere 50 L/h and 900 L/h, respectively, and were obtained using anitrogen source. We used argon as the collision gas and regulatedit at 0.35 mL/h, setting the multipliers to 650 V. Cone voltage andcollision energies of HA were investigated using infusion analysisat 17 V and 15 eV, respectively.

2.5. Preparation of ACE, HHL and sample solutions

ACE was dissolved in its original vial with water and glycerol(50/50, V/V) to yield a 0.25 unit/mL solution (Hernández-Ledesmaet al., 2003). HHL was dissolved with phosphate buffer (pH 7.4) toyield 1 and 0.1 mM concentration. These solutions were stored at�20 �C till use. Natural colourants and captopril were dissolvedin water or ethanol to yield 5000 lg/mL concentration. For theevaluation of this system, the captopril solution was prepared bydissolving the appropriate amount of standard in pure water.

2.6. Preparation of food and supplement samples

Infant formula (n = 6), soy paste (Japanese miso) (n = 5), ketch-up (n = 1), mayonnaise (n = 1), wheat flour (n = 3), orange juice(n = 1), supplement drink (n = 1), tea (n = 3, barley, black, andgreen), and coffee (n = 1) were obtained from a local store inNagoya, Japan.

For preparation of the infant formula, 20 mg samples wereweighed in tubes. Then, 1.0 mL water was added, and the samplevortex mixed for 1 min. Centrifugal ultrafiltration was used withAmicon Ultra-4 (Ultracel-3 K, regenerated cellulose 3000 M.W.for volumes <4 mL, Milliore Co., Ltd., Billerica, MA, USA). 0.5 mLsample solution was eluted through centrifugal ultrafiltration car-tridges by centrifugation at 3500 rpm for 15 min and evaluated byACE inhibition assay.

K. Inoue et al. / Food Chemistry 126 (2011) 1909–1915 1911

For preparation of solid samples, 10 mg samples were weighedin tubes. Then, 1.0 mL water was added, and the sample vortexmixed for 1 min. 0.5 mL sample solution was eluted through cen-trifugal ultrafiltration cartridges by centrifugation at 3500 rpmfor 15 min and evaluated by ACE inhibition assay.

For preparation of liquid samples, 0.5 mL sample solution waseluted through centrifugal ultrafiltration cartridges by centrifuga-tion at 3500 rpm for 15 min and evaluated by ACE inhibition assay.

Table 1Recovery tests of HA in the developed ACE assays by solid phase extraction(Method 2).

Concentration of HA(lg/mL)

Assay Calibration Recovery ± SD (%),n = 3

3.0 HPLC Exterminal 99.3 ± 3.11.0 LC/SID-ESI-

MS/MSInternal 100.4 ± 0.5

0.5 LC/SID-ESI-MS/MS

Internal 100.1 ± 0.9

0.1 LC/SID-ESI-MS/MS

Internal 100.1 ± 0.7

Rel

ease

d H

A le

vels (A)

2.7. Assays for the evaluation of ACE activity

The assays used to measure ACE activity were adopted fromglobal standards (Hernández-Ledesma et al., 2003) (Method 1)and our developed methods (Method 2).

Method 1: 150 lL reaction solution (1 mM HHL in phosphatebuffer, pH 7.4) was initiated by 30 lL ACE (0.25 unit/mL) solutionand 30 lL blank solvent (pure water or ethanol), and incubatedfor 150 min at 37 �C. The reaction was terminated by the additionof 250 lL HCl (1 mol/L), and then 1.5 mL ethyl acetate was addedand shaken vigorously for 2 min at room temperature and centri-fuged for 5 min at 3000 rpm. One mL of the upper layer was placedin tube and evaporated. Water (1 mL) was added to dissolve theresidue prior to measurement of absorbance at 225 or 280 nm. Inaddition, this solution was evaluated by HPLC assay.

Method 2: 150 lL reaction solution (1 mM HHL in phosphatebuffer, pH 7.4) was initiated by 30 lL ACE (0.25 unit/mL) solutionand 30 lL blank solvent (pure water or ethanol), and incubatedfor 150 min at 37 �C. In case of LC with LC/SID-ESI-MS/MS assay,HA-d5 solution (10 lg/mL, 50 lL) was added as an internal stan-dard. Reactions were terminated by removing the substrates usingSPE. An OASIS-HLB (1 mL, 30 mg, Waters Co., Milford, MA, USA)cartridge was used. Before extracting the reacted solutions, theSPE cartridge was conditioned by eluting 1.0 mL methanol fol-lowed by 1.0 mL 0.1% aqueous formic acid. After this reaction,the sample solution was diluted with 1.0 mL 0.1% aqueous formicacid and eluted through an SPE cartridge. The cartridge was thenwashed with 1.0 mL 0.1% aqueous formic acid. Methanol (1 mL)was added at a low flow rate to elute the analytes that were re-tained in the cartridges. This solution was then evaporated.Water/methanol (50/50, V/V) (1 mL) was added to dissolve the res-idue prior to measurement of HPLC or LC/SID-ESI-MS/MS assays.

Abu

ndan

ce (2

80 n

m)

Retention time (min)0 5

HA (6.9 min)

10

Abu

ndan

ce (2

80 n

m)

Retention time (min)0 5 10

(B) (C)

0 50 100 150 200 250

Reaction time (min)

HA (7.0 min)

Fig. 1. Reaction time and HPLC chromatograms of HA for ACE-induced hydrolysis ofHHL in phosphate buffer (pH 7.4). (A) Time course of HA concentration in ACE-induced hydrolysis of HHL in phosphate buffer (pH 7.4) at 37 �C. (B) HPLCchromatogram of HA from hydrolysis of HHL by ACE without any test sample(Control: water). (C) HPLC chromatogram of HA from hydrolysis of HHL by ACE withcaptopril (5.0 ng/mL).

2.8. Screening assay for the inhibition of ACE activity

To evaluate the ACE inhibition, the above-described reactionwas performed at 37 �C with 1 mM HHL, ACE (0.25 unit/mL) solu-tion, and a sample solution in phosphate buffer of pH 7.4 for150 min. The reaction was terminated by SPE and measured usingLC/SID-ESI-MS/MS. In this experiment, the test substances werecaptopril, natural colourants, foods, and supplement samples. Acontrol sample, prepared without test substances in water or eth-anol, was used to determine the background ACE-inhibition level.This activity was expressed as the inhibition value, and was calcu-lated using the following formula:

ACE inhibition activity of test substances (%) = [(HA concentra-tion levels without test substances) � (HA concentration levelswith test substances)/(HA concentration levels without testsubstances)] � 100.

All analyses were performed thrice and their results are pre-sented as the mean ± standard deviation (SD). If the test substancedisplayed inhibition activity toward ACE, these values would beincreased significantly (up to 100%). On the other hand, if the sub-stance did not display inhibition activity, these values would onlybe altered slightly by the test substances.

3. Results and discussion

3.1. HPLC assay of ACE inhibition

It is difficult to determine the ACE inhibition of complex mate-rials, components, substances and colourants derived from foodand natural sources using spectrometric methods. Therefore, weevaluated the natural colourants for the inhibition of ACE activityusing Method 1. In this experiment, test substances of red colou-rants (red cabbage, grape skin, elderberry, and red radish) couldnot be evaluated using a spectrometric method (225 and280 nm). Moreover, the extraction of HA using ethyl acetate wasshown to absorb at 228 nm and accounted for 12% of the totalabsorbance (Matsui, Matsufuji, & Osajima, 1992; Wu, Aluko, &Muir, 2002). Spectrometric methods without chromatographicseparation have the advantages of being simple, rapid, and easytechniques. However, spectrometric methods without separationare considered unsuitable for the universal, useful, and reliable as-says of ACE inhibitors of various food and natural sources. Thus, we

1912 K. Inoue et al. / Food Chemistry 126 (2011) 1909–1915

adopted HPLC analysis of HA for the assay of ACE inhibition in thesubsequent study.

Usually, the ACE reaction is terminated by HCl, and then HA re-leased is extracted by ethyl acetate for the assay of ACE inhibitors(Cushman & Cheung, 1971). Many assays are modifications of thispreparation. Thus, we evaluated the recovery of HA based on Meth-od 1 with the HPLC method. Our results showed a recovery value of

Table 2Evaluation of ACE inhibitions of natural colourants by the developed assays.

Colourants ACE inhibition’s activity ± SD (%), n = 3

HPLC assay LC/SID-ESI-MS/MS assay

Annatto extract x 3.9 ± 4.0Beefsteak plant x 43.5 ± 2.0Beet red 0.1 ± 4.5 0.1 ± 5.5Carthamus yellow x 62.6 ± 3.4Cochineal extract x 14.4 ± 5.4Elderberry x 20.9 ± 1.9Gardenia yellow 13.3 ± 3.0 11.4 ± 4.7Grape skin x 29.9 ± 3.3Marigold 0.8 ± 1.6 3.2 ± 2.3Monascus 54.3 ± 2.1 58.2 ± 1.9Monascus yellow 0.1 ± 10.5 0.1 ± 3.7Papria x 0.1 ± 3.6Persimmon x 32.6 ± 1.7Purple cone x 49.4 ± 2.5Purple potato x 20.2 ± 2.7Red cabbage x 25.9 ± 0.5Red radish x 39.3 ± 4.1Sepia pigment 61.0 ± 6.7 63.3 ± 0.4Turmeric 30.9 ± 1.4 36.1 ± 0.2

Retention time (min)

0 5 10

HA

0 5 10

0 5 10 0 5 10

HA

HA?

(A) (B)

(C) (D)

Abu

nd

ance

(280

nm

)

HA?

Fig. 2. HPLC chromatograms of HA for the ACE inhibition assay of naturalcolourants. (A) Evaluation of gardenia yellow (ACE inhibition activity: 13.3 ±3.0%). (B) Evaluation of Monascus (ACE inhibition activity: 54.3 ± 2.1%). (C)Evaluation of red cabbage. (D) Evaluation of red radish.

23.1 ± 5.7% (n = 3, detection level: 3.0 lg/mL). The recovery andreproducibility of HA are highly important factors for the evalua-tion of ACE activity and hydrolysed reaction. The hydrolysed HAfrom HHL was confirmed by various detectors, but these sampleswere not sufficiently large for terminal reactions after storage inacidic solutions. Moreover, the use of various test substancesand/or materials has an adverse effect on HPLC chromatograms(leading to noise peaks) in the UV and MS/MS detectors. Thus,we used SPE to eliminate the matrix and other chemicals insteadof adding acidic solutions for stable stopping procedure, and negat-ing the effect of UV absorbance and MS/MS ionisation. We deviseda useful, convenient, and reliable SPE preparation method to termi-nate the ACE reaction and to extract the released HA for the assayof ACE inhibitors. In studies of SPE optimisation, the Bond Elut-C18

(100 mg, 1 mL, VARIAN) and OASIS-HLB (30 mg, 1 mL, Waters)were investigated with water, acetic acid, and formic acid solu-tions. In this study, good recovery (99.3 ± 3.1%, n = 3) using OASISHLB was achieved using 0.1% aqueous formic acid (Table 1). ThisSPE preparation has several advantages over other methods thatterminate the ACE reaction and extract the released HA for the as-say of ACE inhibition. SPE avoids extraction with ethyl acetate, ter-minal reaction with HCl, and troublesome work-up procedures.Modified preparation with SPE can provide the high-throughputmethod with a 96-well plate and automatic SPE system. Thus, thisdeveloped SPE preparation of HA released has the possibility ofbeing a universal and standard method for the assay of ACE inhibi-tion. Based on this method of HA released from HHL, reaction timewas investigated for ACE activity. The HA concentration over timeis shown in Fig. 1A. In this experiment, the reaction time is150 min; the point at which ACE activity becomes saturated.

m/z100 200

%

0

100109.8

81.8

100 200

%

0

100 104.8

76.8

m/z

(A) (B)m/z 179.9 m/z 184.8

NH

OH

O

O

77

105

NH

OH

O

O

D

DD

D

D82 110

Time1.0 2.0 3.0

%%

184.8 109.71.20e5

1.06

179.9 104.71.21e5

1.07HA

HA-d5

Time1.0 2.0 3.0

%%

184.8 109.71.42e51.08

179.9 104.72.09e51.10

HA

HA-d5(C) (D)

Fig. 3. LC/ESI-MS/MS spectra and chromatograms of HA and HA-d5 for the ACEinhibition assay of natural colourants. (A) MS/MS spectrum and fragment pattern ofHA from m/z 180. (B) MS/MS spectrum and fragment pattern of HA-d5 from m/z 185.(C) MRM chromatograms of HA and HA-d5 for evaluation of red cabbage (ACEinhibition activity: 25.9 ± 0.5%). (D) MRM chromatograms of HA and HA-d5 forevaluation of red radish (ACE inhibition activity: 39.3 ± 4.1%).

K. Inoue et al. / Food Chemistry 126 (2011) 1909–1915 1913

The results of these experiments indicate that the HPLC assay canbe utilised to monitor ACE-induced hydrolysis of HHL in phosphatebuffer (pH 7.4) for 150 min. The HPLC chromatogram of blank(water) with HHL and ACE is shown in Fig. 1B. Also, the HPLC chro-matogram of captopril (5.0 ng/mL) with HHL and ACE is shown inFig. 1C.

For the investigation of ACE activity of functional foods, a testsubstance was evaluated using peptides derived from a multitudeof plant and animal proteins, such as milk, soy, and fish, represent-ing sources of health-enhancing substances (De Leo, Panarese,Gallerani, & Ceci, 2009). It is easy to evaluate colourless and polarpeptides from foods using HPLC and/or spectrometric assays. In thisstudy, we intended to develop a useful assay that is applicable tothe monitoring of a variety of targeted ACE inhibitors in variousmatrices. Thus, the HPLC assay was evaluated using various naturalcolourants as test substances. ACE inhibition activity of natural col-ourants is shown in Table 2. The HPLC chromatograms of HA for theACE inhibition assay are shown in Fig. 2. The evaluations of garde-nia yellow (Fig. 2A) and Monascus (Fig. 2B) colourants were foundto be 13.3 ± 3.0% and 54.3 ± 2.1%, respectively. However, the redcabbage (Fig. 2C) and red radish (Fig. 2D) colourants could not beevaluated by HPLC assay. In fact, in Table 2 and 12 colourants werenot evaluated because of the noise and background peaks that weredetected by HPLC assay. Thus, HPLC assay has limitations for theevaluation of ACE inhibition activity of complex samples.

3.2. LC/ESI-MS/MS assay of ACE inhibition

Xiao et al. and Geng et al. reported a technique for the assay ofACE inhibition based on MS detection of HA (Geng, He, Yang, &

Inhibition activity of ACE (%)

(n=3)

Inhibition activity of ACE (%)

(n=3)

(A)

(C)

Fig. 4. ACE inhibition activity of various samples using LC/SID-ESI-MS/MS method

Wang, 2010; Xiao, Luo, Chen, & Yao, 2006). They suggest that theaccuracy and precision of the LC-MS method are almost the sameas for the conventional HPLC method with UV detection. AnMS-based assay could be used to identify potential analytes forthe quantification of HA based on the reaction of HHL and ACE.In addition, an LC/MS assay may be utilised in the high-throughputscreening of the inhibitors of ACE. However, LC/MS assay to screeninhibitors of ACE activity has not been validated for the determina-tion of ACE inhibitors from complex substances such as foods,natural product, and colourants. While using an MS detector, aproblem occurs in ion suppression and enhancement in thedetermination of analytes in high-matrix samples. Moreover, ahigh-throughput screening assay may be utilised with theLC/ESI-MS/MS method. Here, we adopt the development of anLC/ESI-MS/MS assay with stable isotope HA to screen for inhibitionof ACE activity. Our approach overcomes the limitations of previousHPLC and LC/MS assays, and allows high-throughput measures ofthe specific ACE inhibition activity in various samples.

Full-scan mass spectrum was evaluated to determine the rela-tive intensities of ions in a given mass range, using positive ESImode. The mass spectra of HA and HA-d5 showed signals at m/z179.9 and m/z 184.8 [M + H]+, respectively. From the MS/MS spec-tra shown in Fig. 3A and B, we observe that the m/z peaks at 104.7and 109.7 from Phe-CO+ have a higher intensity than other ionswith collision energy of 15 eV. The major HA and HA-d5 fragmentions at m/z 180 ? 105 and 185 ? 110 in the MRM mode can deter-mine lower quantities of 0.05 ng/mL standard solution with S/N = 3than other methods. The SID-ESI-MS/MS method described herecan be a rapid, selective, sensitive, and highly reproducible methodfor the determination of HA in various samples based on

Inhibition activity of ACE (%)

(n=3)

(B)

. (A) Infant formula samples. (B) Solid food samples. (C) Liquid food samples.

1914 K. Inoue et al. / Food Chemistry 126 (2011) 1909–1915

high-throughput LC separation. The retention time is only 1.1 minusing a TSK-GEL ODS-140HTP column. Recovery is almost 100% forvarious concentrations (Table 1). The basic underlying advantageof this optimised method that utilises a very simple SPE procedureand a short chromatographic run time is that it may be particularlysuitable for routine screening assay.

This high-throughput LC/SID-ESI-MS/MS assay was evaluatedusing various natural colourants for test substances. ACE inhibitionactivity of natural colourants is shown in Table 2. The MRM chro-matograms of HA for the ACE inhibition assay of red cabbage andred radish are shown in Fig. 3C and D. In this study, all colourantscould be evaluated by this assay. Thus, high-throughput LC/SID-ESI-MS/MS assay has no limitations for the evaluation of inhibitionactivity of various natural samples such as colour, high-matrix,complex and processed foods.

3.3. Application of developed method for the determination of ACEinhibition

Various foods, supplements, and artifacts were evaluated forthe substantive experiment of the universal, useful, and rapid as-say of ACE inhibition. The results are shown in Fig. 4. Based onthe developed assay, all samples could be accurately measuredfor ACE inhibition in various matrices. A number of food-derivedpeptides have been shown to have in vitro ACE inhibition activity(Guang & Phillips, 2009). Further research into the bioavailabilityand targeting of the RAS of these food-derived peptides may leadto the development of more effective drugs and foods as novelalternatives to current treatment options (Hong et al., 2008).However, it is possible that the various extracted contents,including polyphenols from fruits such as pomegranate juicesand bilberry have the same effect as ACE in vitro assay (Basu &Penugonda, 2008; Persson, Persson, & Andersson, 2009). Thus,available alternatives of ACE inhibitors may be investigated withnot only peptides, but also other compounds in various foods.This signifies that we must develop a useful assay for ACE inhi-bition to support the discovery of new drugs and functionalfoods. In this study, we developed a screening assay of ACEinhibition using a high-throughput LC/SID-ESI-MS/MS method.The ACE inhibitions of hypoallergenic infant formula (inhi-bition activity from 78.1 ± 1.1% to 82.5 ± 0.3%) (Martin, Wellner,Ossowski, & Henle, 2008b) and soy paste (inhibition activityfrom 39.6 ± 2.2% to 61.8 ± 0.2%) (Inoue et al. 2009) weredemonstrated (Fig. 4A and B), and novel effects of orange juice(inhibition activity 99.2 ± 0.1%) were also detected (Fig. 4C) usingthis method.

4. Conclusions

To screen for novel therapeutic strategies for the treatment ofblood pressure and prevention of hypertension, various methodshave been utilised to evaluate ACE inhibition. Almost all previouslydeveloped analytical assays are able to detect peptides and stan-dards directly in simple matrices. As of today, not a single analyt-ical method has targeted specific components of various matrixfood samples. In the present study, we focused on the rapid, selec-tive, sensitive, highly reproducible, useful, and universal evaluationof ACE inhibition in various foods, and developed an assay toscreen inhibitors of ACE using high-throughput LC/SID-ESI-MS/MS. This novel screening assay of potential ACE inhibition allowsinvestigation of the interesting effects of various foods, naturalextraction, and biological samples. Thus, the use of this newlydeveloped assay may allow us to determine new drugs and

functional foods that are effective against ACE in the RAS as newtreatment options for hypertension.

References

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