Inhibition of angiotensin-converting enzyme by aqueous extract of tomato

ORIGINAL CONTRIBUTION

Inhibition of angiotensin-converting enzyme by aqueous extractof tomato

Dipankar Biswas • Md. Main Uddin •

Lili L. Dizdarevic • Aud Jørgensen •

Asim K. Duttaroy

Received: 9 November 2013 / Accepted: 18 February 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract

Purpose To investigate the presence of anti-angiotensin

converting enzyme (ACE) factors in aqueous extract of

tomato.

Methods The bio-guided fractionation of the aqueous

extract of tomato produced a sugar-free, heat-stable frac-

tion with molecular mass \1,000 Da from tomatoes. The

sugar-free tomato extract (TE) was tested for its anti-ACE

activity using human plasma and rabbit lung pure ACE. In

addition, its effect on human platelet aggregation induced

by ADP, collagen or arachidonic acid was determined. The

mechanism of platelet inhibitory action of TE was inves-

tigated by measuring platelet factor 4 (PF4) release and

cAMP synthesis by platelets.

Results Typically, 100 g tomatoes produced 72.2 ±

4.7 mg of TE. This extract inhibited both platelet aggre-

gation and plasma ACE activity in a dose-dependent

manner. It inhibited platelet aggregation in response to

ADP, collagen or arachidonic acid, and inhibitory action

was mediated in part by reducing platelet PF4 release and

by stimulating cAMP synthesis. The IC50 value of TE for

ADP-induced platelet aggregation was 0.4 ± 0.02 mg/ml,

whereas the IC50 value for ACE enzyme inhibition

was 1.40 ± 0.04 mg/ml. Both the TE and commercially

available sugar-free TE, Fruitflow�-2 had similar amount

of catechin, and also had equal inhibitory potencies against

platelet aggregation and plasma ACE activity.

Conclusion Together these data indicate that aqueous

extract of tomatoes contain anti-ACE factors in addition to

previously described anti-platelet factors.

Keywords Platelet � Tomatoes � Tomato extract �Angiotensin converting enzyme (ACE) � Platelet

aggregation � cAMP � Platelet factor 4 (PF4)

Introduction

Cardiovascular disease (CVD) is a leading cause of mor-

bidity and mortality, and, therefore prevention of CVD is a

public health priority [1, 2]. High blood pressure, hyperac-

tivity of platelets and hyperlipidemia are recognized risk

factors for CVD [3–5]. Untreated hypertension can lead to

CVD, stroke, hypertensive retinopathy, gout, kidney dys-

function, disability, and even death [6]. Treatment of mod-

erate to severe hypertension is a life-long commitment and

requires drug therapy in combination with changes in life-

style and food habits. Essential hypertension can be treated

with one of several types of medications, including diuret-

ics, b-adrenoreceptor blockers, inhibitors of angiotensin

converting enzyme (ACE), calcium channel blockers,

a-adrenoreceptor antagonists, vasodilators, and centrally

acting agents.

The renin–angiotensin system is a powerful mecha-

nism for controlling blood pressure [7, 8]. In hypertensive

patients with elevated plasma rennin–angiotensin activity,

a fivefold increased incidence of myocardial infarction

was demonstrated [5]. ACE (EC 3.4.15.1, dipeptidyl

carboxypeptidase) is a glycoprotein peptidyldipeptide

Dipankar Biswas and Md. Main Uddin have contributed equally to

this work.

D. Biswas � Md. M. Uddin � L. L. Dizdarevic � A. Jørgensen �A. K. Duttaroy (&)

Department of Nutrition, Faculty of Medicine, Institute of Basic

Medical Sciences, University of Oslo, PO Box 1046,

Blindern, 0316 Oslo, Norway

e-mail: [email protected]

123

Eur J Nutr

DOI 10.1007/s00394-014-0676-1

hydrolase that cleaves histidyl leucine dipeptide from

angiotensin I forming the potent vasoconstrictor angio-

tensin II. Studies demonstrated that ACE inhibitors

(ACEIs) significantly reduced the morbidity and mortality

in patients with myocardial infarction, and the incidence

of ischemic events in patients with CVD, even in the

absence of their blood pressure lowering effects [3, 9,

10]. The therapeutic administration of certain ACEIs has

also been associated with positive health effects beyond

the regulation of blood pressure [11]. Mechanisms of

action of ACEIs in the cardiovascular system are not well

understood, but it has been postulated that in addition to

antioxidant properties, these may have multiple effects

such as lowering of blood pressure, anti-proliferative

effect, and anti-platelet effects [7, 12–15]. The main

ACEIs in foods are peptides, flavonoids, and polyphenols.

Flavonoid-rich plant extracts have been demonstrated as

natural competitive ACEIs where the ACE activity is

identified as a critical factor in regulating high blood

pressure [16]. These compounds are known to be inhib-

itors of cyclic nucleotide phosphodiesterase and TxA2

synthesis, the two main determining factors in human

blood platelet activation/aggregation processes. Conse-

quently, it is possible that consumption of these bioactive

components might reduce more than one CVD risk fac-

tor, such as platelet hyperactivity and hypertension

[17, 18].

In recent years, there has been considerable interest in

the potential for using natural food components as func-

tional foods to treat hypertension, especially for people

with borderline to mild high blood pressure that does not

warrant the prescription of anti-hypertensive drugs [19].

The polyphenols inhibit and down-regulate expression of

ACE and renin [19]. Polyphenols have also been associated

with the formation of endothelial nitric oxide leading to

vasodilation and lowering of blood pressure [20]. These

purified polyphenols also inhibit ACE activity in vitro [21].

Lycopene-rich tomato extract (TE) was shown to lower

blood pressure in human volunteers, and this effect was

thought to be associated with its high antioxidant content

[22–24]. Tomatoes contain several polyphenols [22, 25],

but there is no information available as to whether tomato

has any anti-ACE activity. We and others reported the

cardio-protective potentials of TE both in vitro and in vivo

[22–28].

We earlier showed that constituents of aqueous

extract of tomatoes have a range of anti-platelet activi-

ties and are bioavailable [25, 27, 29]. Here we describe,

for the first time, that in addition to the platelet inhib-

itors, similarly prepared aqueous extract of tomatoes

contained ACEIs. The presence of these diverse cardio-

protective factors makes tomato a true cardiovascular

fruit.

Materials and methods

Materials

Tomatoes and other fruits were obtained from a grocery

shop in Oslo. Fruitflow�-2 (sugar-free tomato aqueous

extract) was kindly provided by DSM Nutritional Products,

Basel, Switzerland. Collagen and arachidonic acid were

obtained from Chrono-Log (Havertown, USA), while

ADP was obtained from Helena, USA. A Bond Elut ENV

cartridge was obtained from Agilent, USA. Angiotensin

Converting Enzyme Assay REA kit was from BUHL-

MANN laboratories AG, Switzerland. cAMP assay kit and

IBMX were obtained from Cayman, USA. Platelet factor 4

(PF4) human ELISA kit was obtained from Abcam, UK.

ACE of rabbit lung (EC 3.4.15.1), prostaglandin E1 and

captopril were obtained from the Sigma chemical com-

pany. A Lipidex-1000 column was obtained from the

Packard, USA. All other reagents used were of analytical

grade quality.

Preparation of sugar-free tomato aqueous extract (TE)

To prepare the 100 % fruit juice, the tomatoes were

homogenized with Brown Turbo Mixer for 20–30 s at

highest speed; tomato juice (TJ) was initially prepared after

centrifugation at 9,0009g at 4 �C for 15 min. For further

fractionation of the aqueous extract of tomato, the whole

homogenate as prepared above was boiled at 90 �C for

20 min, and the homogenate was then centrifuged at

22,0009g for 15 min at 4 �C, as described before [29]. The

supernatant was then freeze-dried overnight. The dried

extract was dissolved in double distilled water and sub-

jected to ultrafiltration with molecular weight cut-off of

1,000 kDa using MicrosepTM Centrifugal Devices (Pall

Corporation, USA). The ultrafiltrate was freeze-dried and

reconstituted in water, and pH was adjusted to 7.4 for

further studies. The TE as prepared above contained more

than 50 % water-soluble sugars. Solid phase extraction

column chromatography was used for the removal of

sugars using a Bond Elut ENV cartridge (Agilent) [25].

This cartridge was conditioned with 2 9 4 ml 100 %

methanol and then equilibrated with 2 9 4 ml distilled

water. Typically, 1 g freeze-dried extract (as prepared

above) was dissolved in 4 ml MilliQ water, loaded onto the

cartridge, and water-soluble components were eluted 3–4

times with 4 ml of MilliQ water. The cartridge was then

dried out completely before elution of the non-sugar

components with 3 9 2 ml 100 % methanol under slow

(drop wise) flow rates. The eluted fractions were evapo-

rated to dryness under N2 at 45 �C and then dissolved in

500 ll MilliQ water. Both the water-eluted and methanol-

eluted fractions were then used for their inhibitory

Eur J Nutr

123

activities against platelet aggregation and serum ACE

activity. The freeze-dried material from each of these steps

was used for the presence of anti-platelet and ACE inhib-

itory factors.

In some cases, sugar-free extracts of other fruits were

also prepared following the same procedure as described

above.

Determination of total phenolic contents in the tomato

juice or extract

The concentration of phenolics in sugar-free TE or Fruit-

flow�-2 (FF2) was determined using spectrophotometric

method [30]. A methanolic solution of the extract at a

concentration of 1 mg/ml was used in the analysis. The

reaction mixture was prepared by mixing 0.5 ml of meth-

anolic solution of extract (1 mg/ml), 2.5 ml of 10 % Folin–

Ciocalteu’s reagent dissolved in water and 2.5 ml 7.5 %

NaHCO3. A blank was simultaneously prepared, contain-

ing 0.5 ml methanol, 2.5 ml 10 % Folin–Ciocalteu’s

reagent dissolved in water and 2.5 ml of 7.5 % of

NaHCO3. Samples were prepared in triplicate for each

analysis, and the mean value of absorbance was obtained.

The same procedure was repeated for the standard solution

of catechin, and the calibration line was constructed. Total

polyphenol content was calculated from absorption values

and a linear regression equation using catechin as standard.

Results were shown as mg or lg CE (catechin equivalent).

Effect of TE and Fruitflow�-2 (FF2) on serum ACE

The effect of TE and FF2 on the serum ACE activity was

measured using the ACE assay REA Direct kit, as described

before [31]. Serum was prepared after human blood was left to

clot completely at room temperature. Serum was then sepa-

rated by centrifugation within 2 h of collection. Typically, the

serum (100 ll) was incubated with different concentrations of

TE or FF2 for 15 min at 37 �C. After the incubation, the ACE

activity of the serum was measured. ACE inhibitory activity

(%) = (A - B)/(A - C) 9 100 where A represents cpm in

the presence of ACE and sample, B is absorbance of control,

and C is absorbance of the reaction blank.

IC50 values (the amounts in mg/ml required to produce

50 % ACE inhibition) were calculated using GraphPad

Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA).

Nonlinear regression was performed on the dose–response

data and a sigmoidal curve with variable slope was fitted to

each of the data sets. The equation used for the sigmoidal

curve with variable slope was

Y ¼ bottomþ top� bottomð Þ= 1þ 10 logIC50�Xð ÞHillslopeÞ� �

where bottom is the Y value at the bottom plateau; top is

the Y value at the top plateau; log IC50 is the X value of

response halfway between top and bottom. Hill slope is the

Hill coefficient or slope factor (controls slope or curve).

For curve fitting, only the mean value of each data point,

without weighting, was considered.

The minimum inhibitory concentration and the maximal

inhibitory concentration were calculated from the x values

at the intercept between the slope and the bottom plateau

and top plateau, respectively. The IC50 was calculated from

the x value of the response halfway between top and bot-

tom plateau.

Effects of TE and FF2 on rabbit lung ACE

In order to investigate whether the observed inhibitory effect

of TEs in serum was mediated through direct interaction with

enzyme, we investigated the effect of TE or FF2 on the

activity of rabbit lung ACE. Rabbit lung ACE (6.6 mU) was

incubated with increasing concentrations of sugar-free

extracts for 30 min at 37 �C. After the incubation, residual

ACE activity was then measured as described elsewhere.

Effects of TE and FF2 on human blood platelet

aggregation

Anti-platelet activity of juice or extract prepared from

fruits at different steps of the preparation was investigated.

The pH of all samples was adjusted to 7.4 with KOH

solution prior to testing their effect on platelet aggregation.

This study was approved by the local ethical committee,

Oslo university hospital. Venous blood was collected from

volunteers who had not taken any medications for at least

14 days before donation. Blood (20–30 ml) was collected.

Blood coagulation was prevented by mixing the blood

samples with acid citrate buffer (135 mM), pH 7.4 in the

ratio of nine parts by volume of blood with one part by

volume of buffer. Platelet-rich plasma (PRP) was prepared

from the samples by centrifuging the blood at 1809g for

15 min at room temperature. The pH-adjusted juice or

extract prepared at each level of purification of TE or FF2

was then incubated with 0.225 ml of PRP at 37 �C for

15 min after which the effect of the fruit extract on ADP-

induced platelet aggregation was monitored with the

addition of either ADP (1–5 lM), collagen (1–5 lg/ml) or

arachidonic acid (500 lg/ml). Controls were run in parallel

using 10–30 ll of 0.9 % NaCl, pH 7.4, instead of the fruit

extract. Platelet aggregation in PRP was monitored using

Aggram, Helena, USA, at a constant stirring speed of

1,000 rpm at 37 �C [29]. The measurement of the extent of

ADP-induced platelet aggregation in PRP was carried out

at each time point. The maximal aggregation (100 %) was

Eur J Nutr

123

defined as the maximum change in light transmission

observed over 15 min without extracts.

Inhibition of platelet aggregation was expressed as the

decrease in the area under the curve compared with the

control. In some cases, PGE1 and aspirin were used as

controls. Each sample was measured in triplicate. The IC50

values (the concentration necessary to reduce the induced

platelet aggregation by 50 % with respect to control) were

obtained from concentration-effect curves.

Cyclic AMP assay

Cyclic AMP was determined in PRP as previously

described [32]. PRP aliquots (700 ll) were incubated with

different concentrations of TE or FF2 for 12 min in the

absence and presence of 100 lM IBMX (a phosphodies-

terase inhibitor). In some cases, PGE1 (10 lM) was used

in order to compare its stimulation of cAMP with those of

TE or FF2 in PRP. After 12 min of incubation, ethanol

was added to the plasma at a ratio of 2:1 and vortexed for

10 s. The mixture was kept at 4 �C for 15 min. Subse-

quently, the samples were centrifuged at 1,5009g for

15 min at 4 �C, and the resultant supernatant was dried

under N2 at 55 �C. The dried material was then recon-

stituted in assay buffer, and cAMP was measured using

the cAMP assay kit.

Inhibition of platelet factor 4 (PF4) release by sugar-

free tomato extract and FF2 in platelet-rich plasma

In order to measure the inhibitory effect of TE or FF2 on

PF4 release, PRP (130 ll) was incubated in the presence

of these extracts at different concentrations and followed

by 3 lM ADP treatment, as described [33]. After 5 min

of incubation, PRP was centrifuged to prepare platelet-

poor plasma, and PF4 was the measured using the PF4

assay kit.

Statistics

Results are presented as the mean ± SD. Results were

analyzed by the Student’s t test. Other statistical analyses

were performed using ANOVA where appropriate, values

were considered to be significantly different when

p \ 0.05.

Results

Sugar-free aqueous extract of tomatoes

Table 1 summarizes the effects of TJ (100 % fruit juice

after adjusting the pH to 7.4) on plasma ACE activity and

platelet aggregation. TJ inhibited the platelet aggregation

and ACE activity in a dose-dependent manner. TJ at 30 ll

(1.32 lg CE/ml PRP) inhibited 79 ± 12 % platelet

aggregation and 40.3 ± 3.6 % of plasma ACE activity.

Bioactive compounds of the juice were fractionated fol-

lowing the methods as outlined in Fig. 1 to investigate

which compounds/fraction of the juice has effects on ACE

activity or platelet aggregation. Delipidation followed by

ultrafiltration of TE indicated that the active factors in

tomatoes were water soluble, heat stable, and molecular

Table 1 Effects of 100 % freshly prepared tomato juice on platelet

aggregation and serum ACE activity

Inhibition of platelet

aggregation (%)

Inhibition of

ACE activity

(%)

Control 0 0

Tomato juice (10 ll)

(0.44 lg CE/ml PRP)

62 ± 9* 30.2 ± 1.3*

Tomato juice (20 ll)

(0.88 lg mg CE/ml PRP)

77 ± 11* 35.2 ± 2.3*

Tomato juice (30 ll)

(1.32 lg CE/ml PRP)

79 ± 12* 40.3 ± 3.6*

Results are expressed as mean ± SD. Each experiment was carried

out in triplicate (n = 6). The ACE activity was measured in serum as

described in section ‘‘Methods’’

* p \ 0.05 statistically significant difference from controls (incubated

in the absence of extract)

Fig. 1 Outline of tomato extract preparation. Tomatoes were homog-

enized and the juice was boiled at 90 �C for 15 min and centrifuged at

22,0009g for 15 min. The supernatant was then delipidated using

Lipidex-1000 column followed by filtration using a filter with

molecular cutoff \1,000 Da. The filtrated fraction was de-sugarized

using a hydrophobic column as described in section ‘‘Methods’’

Eur J Nutr

123

mass was \1,000 Da (data not shown). Soluble sugars

present in fruit extract were removed using SPE column

chromatography, as described by us before [25]. Typically,

100 g of tomatoes produced about 72 mg of TE. Soluble

sugars had no effects on ACE activity or platelet aggre-

gation (data not shown). Catechin contents of TE and FF2

were 0.019 ± 0.002 and 0.020 ± 0.001 lg/mg, respec-

tively (p [ 0.05).

Effects of TE and FF2 on human serum ACE activity

Both TE and FF2 inhibited serum ACE activity in a dose-

dependent manner (Table 2). The IC50 values of TE and

FF2 for ACE inhibition in serum were 1.95 ± 0.34 mg/ml

(0.037 lg CE/ml) and 1.91 ± 0.24 mg/ml (0.038 lg CE/

ml), respectively, whereas for captopril the value was

0.56 ± 0.08 lg/ml. FF2 also inhibited rabbit lung ACE

activity in a dose-dependent manner (Fig. 2). The IC50

values of FF2 and TE for ACE were 1.45 ± 0.21 mg/ml

(0.029 lg CE/ml) and 1.40 ± 0.04 mg/ml (0.027 lg CE/

ml), respectively. Incubation of ACE with 3 mg/ml of FF2

for 30 min at 37 �C completely abolished the ACE activity

(data not shown). Orange or banana extract prepared under

similar conditions did not show any inhibition of ACE

activity (data not shown).

Inhibition of platelet aggregation by TE and FF2

Both TE and FF2 inhibited platelet aggregation induced by

different aggregating agents, ADP, collagen, and arachi-

donic acid with equal potency (data not shown). Figure 3

shows the inhibition of platelet aggregation in response to

ADP by different concentrations of FF2 and TE. The

concentration of TE or FF2 required to inhibit platelet

aggregation by 50 % (IC50) in PRP induced by ADP was

determined. The IC50 value for ADP-induced platelet

aggregation was 0.40 ± 0.02 mg/ml.

Effects of TE and FF2 on PF4 release

To examine the effect of TE and FF2 on the extracellular

release of granule contents, we measured PF4 (a constitu-

ent of a granules) in the supernatants from ADP-stimulated

platelets in the presence and absence of TE or FF2. The

level of PF4 was determined in PRP in duplicates (n = 2).

The presence of increasing amounts of TE or FF2 inhibited

ADP-induced PF4 release in a dose-dependent manner.

The release of PF4 was inhibited by 50 and 72 % by 1.0

and 1.5 mg/ml of FF2 and TE, respectively, compared with

the control (p \ 0.05).

Effect of TE and FF2 on cAMP synthesis

cAMP levels in PRP were determined after treating these

cells with different concentrations of TE and FF2. The

presence of 0.6 mg/ml of TE and FF2 increased cAMP by

twofold compared with the basal levels (p \ 0.05). PGE1 at

10 lM increased cAMP level by fourfold compared with

the basal levels (p \ 0.05).

Discussion

This paper reports that TE exhibits an ability to inhibit both

ACE activity and platelet aggregation. The water soluble,

Table 2 Effects of FF2 and sugar-free TE on serum ACE activity

Amounts (lg) Inhibition of serum ACE activity (mean % ± SEM)

FF2 TE

65 20.4 ± 5.2 18.37 ± 1.99

130 36.2 ± 5.1 35.21 ± 4.83

195 47.0 ± 6.5 45.1 ± 1.73

260 56.1 ± 5.0 54.1 ± 3.41

325 62.0 ± 8.5 61.93 ± 1.16

Results are expressed as mean ± SD. The ACE activity was mea-

sured in serum as described in section ‘‘Methods’’. Each experiment

was carried out in triplicate (n = 6)

* p \ 0.05, statistically significant from controls (incubated in the

absence of extracts)

Fig. 2 Inhibitory effect of FF2 on ACE activity. ACE was incubated

with different concentrations of FF2 for 30 min at 37 �C. The residual

activity of ACE was then measured as described in section

‘‘Methods’’ p \ 0.01; statistically significant difference from controls

(incubated in the absence of extracts). For details, see section

‘‘Methods’’. Each experiment was done in triplicate (n = 6)

Eur J Nutr

123

colorless components in TE have molecular weight

\1,000 Da. This TE was prepared following the method

used for preparation of anti-platelet components from

tomatoes [25, 29]. Effects of anti-platelet components of

aqueous TE were demonstrated both in vitro and ex vivo

[25, 27]. This TE was commercially branded as Fruitflow�

and later developed in two variant forms named Fruitflow�

and its sugar-free derivative Fruitflow�-2 (FF2).

Now, our new data demonstrate that TE or FF2, in

addition to anti-platelet factors, contained anti-ACE factors.

This is not however surprising as the similar procedures are

used in order to isolate the sugar-free aqueous extract of

tomatoes (TE or FF2). In fact TE and FF2 had similar

catechin contents, and equal inhibitory potencies against

platelet aggregation and plasma ACE. At present, the

compounds responsible for anti-ACE effect are not known.

The FF2 was standardized on the total quantity of 37 bio-

active components [25]. FF2 contains a range of nucleo-

sides and derivatives, sensory components, and several

phenolic acids and some glycosidic derivatives such as

chlorogenic acid. In addition, several flavonoids, including

rutin, quercetin, kaempferol, and luteolin are also shown to

be present. The anti-ACE activity has largely been ascribed

to the presence of flavonoids [34]. A number of extracts and

compounds obtained from plants have been shown to con-

tain ACEIs [35, 36]. Different studies have revealed the

important role that flavonoid structure plays in its biological

function; the position and number of substituents in the

flavonoid basic structure significantly affect the anti-pro-

liferative, cytotoxic, antioxidant, and anti-enzymatic

activities of such molecules [37]. Flavonoids can be dif-

ferentiated into several subfamilies according to their

degree of unsaturation and the degree of oxidation of the

oxygenated heterocycle and can be characterized as flava-

nones, flavones, flavonols, isoflavones, flavanols (essen-

tially, flavan-3-ols) and anthocyanidins, all of which are

most relevant for the human diet [38]. Recent data provided

flavonoid structure–activity relationships and established

the structural features needed for the ACEI activity of

flavonoids [37]. Inhibition of ACE has been considered to

be one of the effective therapeutic approaches for the

treatment of CVDs. In recent years, it has been recognized

that many dietary constituents may contribute to human

cardiovascular health. There has been an increased focus on

identifying these natural components of foods, describing

their physiological activities and mechanisms of actions.

A

B

Fig. 3 Dose-dependent-inhibition of platelet aggregation by TE and

FF2. PRP was prepared as described in section ‘‘Methods’’. PRP (final

volume, 0.225 ml) was then incubated with different amounts of

sugar-free TE and FF2 for 15 min at 37 �C before ADP-induced

aggregation was initiated. Aggregation was followed at 37 �C with

stirring. A representative inhibition profile of TE (a) and FF2 (b) on

ADP-induced aggregation of platelets is shown. For details see

section ‘‘Methods’’

Eur J Nutr

123

Grain, vegetables, fruits, milk, cheese, meat, chicken, egg,

fish, soybean, tea, wine, mushrooms, and lactic acid bac-

teria are various food sources with potential antihyperten-

sive effects. ACEIs have more consistent data of mortality

benefit in the setting of prior myocardial infarction, coro-

nary artery disease or heart failure. The flavonoids as

inhibitors of ACE in vivo may be useful as nutritional

supplements or in pharmaceutical formulations to obtain a

sufficient dose/response efficacy. As part of our search for a

therapeutic approach to the treatment of high blood pres-

sure, we have been conducting in vitro screening for the

ACE inhibitory effects of various extracts exhibited dis-

tinctive ACE inhibitory activity including from kiwifruits

[31]. Bioassay-directed further purification of different

extracts using various chromatographic methods afforded

flavonoids with anti-ACE activity.

Modulation of blood pressure by the TE thereby pro-

vides a prophylactic and therapeutic benefit in preventing

the pathological processes that lead to CVD. Accordingly,

the TE described, taken orally, may be safely and effec-

tively used in CVD prophylaxis. In particular, composi-

tions of the sugar-free TEs provided herein are effective as

cardio-protective agents especially for people with obesity,

insulin resistance, and sedentary life styles. At present our

data do not allow us to make any recommendation as to

how many tomatoes or how much TE or juice should be

consumed in order to have a reasonable blood pressure

lowering effect in moderately hypertensive patients.

However, studies demonstrated that that consumption of

200 g of tomatoes (equivalent to two tomatoes) per day for

8 weeks significantly lowered both diastolic and systolic

blood pressure and platelet aggregation response [26, 27].

This new functionality (anti-ACE activity) of the sugar-

free TE makes it as versatile cardio-protective agent.

However, further studies are required in order to establish

the bioavailability and efficacy of the anti-ACE activity

along with the anti-platelet activity of the TE.

Acknowledgments This work was supported in part by the Throne

Holst Foundation, Norway.

Conflict of interest The authors have declared no conflict of

interest.

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