Antihypertensive drugs: a perspective on the value of

improved blood pressure control in the USA

Aims We estimated what the improved treatment of high blood pressure

with anithypertensive drug therapy has contributed to U.S. public health, in
terms of reductions in excess premature deaths from cardiovascular disease,
myocardial infarctions, and strokes
.
Methods and Results Using national survey data to estimate blood
pressures in the absence of antihypertensive therapy and comparing them
with blood pressures actually observed in 1999-00, we inferred the impact of
antihypertensive therapy on blood pressure. Using risk equations from the
Framingham Heart Study, we estimated the impact of lowered blood
pressures on the risk and number of myocardial infarctions and strokes
(2002) and deaths (2001). Assigning a monetary value from the literature to
the improvement in overall life expectancy related to antihypertensive
therapy, we compared it to average spending on antihypertensives.

Conclusion Antihypertensive therapy has had a major impact on U.S. public

health. In the absence of antihypertensives, 1999–00 blood pressures for
adults age 40 + would have been 10 to 13 percent higher, and 86000 excess
premature deaths from cardiovascular disease (2001), and 833,000 hospital
discharges for stroke and myocardial infarction (2002) would have occurred.
Treatment has generated a benefit-to-cost ratio of at least 6:1, but much
more can be achieved.
Hydralazine as antihypertensive therapy in obesity-related
hypertension

Abstract

OBJECTIVE: The objectives were two-fold: (1) determine whether the use of
hydralazine as antihypertensive therapy during obesity development exacerbated
obesity-related cardioacceleration and hormonal abnormalities; (2) determine
whether the absence of hypertension in obesity attenuated obesity-related
abnormalities in hemodynamics, cardiac hypertrophy, and hormonal profile.

DESIGN: Female New Zealand White rabbits were divided into lean control (n=12),
lean hydralazine-treated (n=9), obese control (n=11), and obese hydralazine-
treated (n=8) groups. Pretreatment mean blood pressure (BP) and heart rate
(HR) were determined using telemetry. Pretreatment BP was maintained during
12 weeks of obesity development using hydralazine.

MEASUREMENTS: Chronically measured BP and HR; plasma/blood volume; wet and

dry ventricular weights; body fat/water; and hormonal profile (plasma renin
activity, aldosterone, cortisol, atrial natriuretic peptide, adrenaline, and
noradrenaline).

RESULTS: Hydralazine treatment in obese animals attenuated obesity-related

renin–angiotensin system (RAS) activation. In contrast, RAS was activated in lean
hydralazine, as indicated by increased plasma aldosterone. The absence of
hypertension in obese hydralazine did not result in attenuation of
cardioacceleration, cardiac hypertrophy, or intravascular volumes.

CONCLUSIONS: Hydralazine treatment in obese rabbits did not exacerbate obesity-

related cardiovascular and hormonal alterations. Cardioacceleration and cardiac
hypertrophy persisted in obese hydralazine despite BP control, suggesting
hypertension-independent effects of obesity on these variables. Hydralazine's
effects on RAS activation differed in lean and obese rabbits, suggesting that the
systemic effects of hydralazine as a control therapy in evaluation of
antihypertensive medications may differ depending on the underlying pathology.

Keywords:

hydralazine, renin–angiotensin system, rabbit model of obesity, hypertension, cardiac hypertrophy

Topof page
Introduction

Determining the independent influence of obesity on cardiac, cardiovascular, renal, and

hormonal abnormalities is complicated by the concomitant occurrence of hypertension.
Approaches to resolving this issue include statistical methods1,2,3or cross-sectional studies
that recruit separate groups of obese-normotensive and obese-hypertensive
subjects.4 These approaches engender two related problems. First, they assume that the
negative sequelae of hypertension occur in a dichotomous manner and that the independent
influence of obesity therefore is manifest when blood pressure (BP) is <140/80 mmHg.
Secondly, they ignore potential interindividual differences in baseline BP. It has been
hypothesized that obese individuals with BP <140/80 mmHg are actually 'hypertensive'
relative to what their BP would be if they were lean.5 This observation is supported by data
which show a positive linear relationship between BP and body mass index even in the
normotensive range,6 and also by data demonstrating that obese-normotensive individuals
almost always reduce BP when they lose weight.7,8

In contrast, it is possible to study hypertension-independent effects of obesity on

cardiovascular function with an animal model of diet-induced obesity,9 by utilizing a
longitudinal analysis wherein a pre-obese BP is determined and subsequently maintained
during obesity development. However, the choice of antihypertensive treatment is
problematic. In evaluating various antihypertensive medications, animal studies most often
use hydralazine as the control drug because of its pure vasodilatory action and lack of effect
on other body systems.10,11,12,13,14,15,16In comparison, antihypertensive treatments such as
angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers have
significant effects on cardiac and cardiovascular structure and/or function independent of
their BP-lowering effects.11,17,18

Despite its advantages, the antihypertensive efficacy of hydralazine is thought to be blunted

by undesirable baroreceptor-mediated side effects such as reflex tachycardia, renin release,
fluid retention, and activation of the sympathetic nervous system, a phenomenon known as
pseudotolerance.19 Yet, occurrence of this reflex response is seldom documented in animal
models. In most cases, data on hormonal reflex responses are not
reported,10,13,14,15,16,20,21,22 while the heart rate (HR) response has been reported as either
increased20 or unchanged.21,22,15,16 Use of hydralazine as antihypertensive therapy in an
obesity-hypertension model is further complicated by the fact that potential side effects are
similar to physiological changes often seen in obesity (e.g., tachycardia and activation of the
renin–angiotensin system (RAS)).9,23,24

Therefore, the purposes of this study were two-fold. First, using the rabbit model, we
sought to determine whether the use of hydralazine as an antihypertensive therapy would
exacerbate cardiovascular and hormonal alterations already present in obesity. If
cardiovascular and hormonal effects of hydralazine were neutral, we would be able to
determine whether the absence of hypertension would attenuate obesity-related
abnormalities in hemodynamics, cardiac hypertrophy, and hormonal profile.

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Methods

Animals
Experimental protocols were approved by the Institutional Animal Care and Use Committee
of the University of North Texas Health Science Center. All animal care and use programs
were carried out according to the Guide for the Care and Use of Laboratory Animals (NIH
Publication 86-23, revised 1985) and the regulations of the Animal Welfare Act. Female New
Zealand White rabbits were purchased when they were approximately 15–17 weeks old,
weighing 3.25–3.75 kg (Myrtle's Rabbitry, Thompson Station, TN, USA). They were housed
individually in a humidity- and temperature-controlled room with a 12-h light cycle, and fed
100–120 g per day of standard rabbit chow (Prolab® Breeder Rabbit chow, Purina Mills, St
Louis, MO, USA). After a 1-week acclimation period, they were randomly divided into four
groups: lean control (n=12), lean hydralazine-treated (n=9), obese control (n=11), and
obese hydralazine-treated (n=8). Lean and obese controls served as benchmarks for normal
and abnormal structure/function, respectively. In the obese hydralazine-treated group,
obesity-related increases in BP were blocked in order to produce an obese-normotensive
group. The lean groups ate a maintenance diet,25 while obese groups were given a high fat
diet,ad libitum, consisting of standard rabbit chow with 10% added fat. The excess fat in the
diet was 2/3 corn oil and 1/3 lard. Experiments were performed after rabbits had been on
their respective diets for 12 weeks.

Telemetry monitoring
In order to monitor daily HR and BP, telemetry transmitters (TA11PA-D70, Data Sciences
International, Minneapolis, MN, USA) were aseptically implanted in the iliac artery under
isoflurane anesthesia, prior to beginning the dietary protocol. Data were collected for a 4 s
interval every 3 min. The daily averages of BP and HR were calculated from data collected
between 1100 and 0700 h. From the daily averages, a weekly average was calculated. After
13–16 day postsurgical recovery, data were collected for 1 week to establish each animal's
control value of BP.

Hydralazine treatment
Starting in week 2 of the dietary protocol, lean and obese hydralazine-treated groups
received 6 and 10–14 mg/kg/day, respectively, of hydralazine (s.c.) dissolved in saline. The
dosages were chosen to maintain the control value of mean BP.

Acute experiment

Hormone analyses

After 12 weeks, fasting arterial blood samples were taken for measurement of plasma
protein, hematocrit, plasma renin activity (PRA), aldosterone (ALDO), cortisol, atrial
natriuretic peptide (ANP), adrenaline, and noradrenaline. Samples were placed in
appropriate chilled blood collection tubes and centrifuged at 1200 g for 15 min at 4°C.
Plasma was stored at -90°C until assay. Hematocrit was measured in duplicate using
microhematocrit tubes. Plasma protein was measured in duplicate by refractometry. Plasma
catecholamines were extracted with alumina, separated by HPLC, and quantified
amperometrically in an electrochemical detector. Peptide and steroid hormones were
determined in double–antibody and charcoal-based radioimmunoassay systems using both
commercial and locally developed reagents. Where applicable, radioligands were iodinated
with chloramine–T and purified by ion exchange/size exclusion chromatography.

Blood and plasma volume

Blood and plasma volumes were measured using the Evan's blue dye procedure. First, a
control arterial blood sample (3 ml) was taken. This was followed by a venous injection of
Evan's blue dye (5 mg/kg); 2 ml arterial blood samples were then taken after 10, 20, and
40 min. The optical density of plasma samples was determined and dye concentration at
time zero was extrapolated from a plot of time vs plasma dye concentration. Dye
concentration at time zero, total amount of dye injected, and hematocrit were used to
determine plasma and blood volumes.

Cardiac hypertrophy
After sacrifice, the heart was excised, atria cut away, and right and left ventricles weighed
separately. Ventricles were subsequently freeze-dried to a constant weight and weighed
again to the nearest 0.1 mg.

Body composition analysis

To determine fat and water composition, 2–3 g samples of whole body homogenate were
assayed as described previously.9 Triplicate samples from each animal were assayed and
the average value used in all analyses.

Statistical analyses
To determine whether hydralazine treatment exacerbated obesity-related abnormalities, we
used an unpaired t-test comparing obese control and obese hydralazine-treated groups.
When the effects of hydralazine were neutral, this analysis was used to test whether the
absence of hypertension during obesity development attenuated obesity-related
abnormalities. Unpaired t-tests between lean control and lean hydralazine-treated groups
were used to test for the independent effects of hydralazine. Our initial analysis indicated
that there were differences in body weight between control and respective hydralazine-
treated groups. Therefore, analyses were repeated using subsets of animals that did not
differ significantly in body weight. Feed consumption data were analyzed using repeated-
measures analysis of variance.
Topof page

Results

Animal characteristics
Group characteristics are listed in Table 1. Mean daily BP measured during week 12 was
significantly lower in obese hydralazine-treated compared with obese controls (P 0.05).
However, obese controls had greater body weight as well as greater body fat percentage
and fat weight compared with obese hydralazine-treated (P 0.05). Lean controls had
greater body fat percentage and fat weight compared with lean hydralazine-treated, despite
significantly lower body weight (P 0.05).
Table 1. Group characteristics

Next table|Figure and tables index

Lean Lean Obese Obese

control hydralazine control hydralazine

Body weight
3.71 0.03 3.80 0.04* 5.05 0.12* 4.65 0.13
(kg)

Mean BP
62.5 3.9 64.1 4.5 75.1 4.5* 62.9 2.3
(mmHg)
 Lean Lean Obese Obese
control hydralazine control hydralazine

HR (beats/min) 180 6§ 160 7 217 5 212 6

Body fat (%) 13.3 1.5* 7.9 1.1 30.0 1.9* 20.5 1.6

Fat weight (g) 0.49 0.05* 0.30 0.04 1.53 0.12* 0.96 0.10

Dry RV weight
0.50 0.05 0.43 0.03 0.63 0.04 0.56 0.05
(g)

Dry LV weight
0.98 0.07 0.90 0.05 1.27 0.05 1.27 0.06
(g)

Plasma volume
130 6 142 6 143 7 143 10
(ml)

Blood volume
193 8 213 10 223 10 218 17
(ml)

Hematocrit (%) 35.5 0.7 35.7 0.7 38.6 0.8§ 36.6 0.5

Protein (mg/dl) 5.0 0.1 5.0 0.1 5.2 0.1* 4.9 0.1

Values are mean s.e.

BP=average daily blood pressure measured during week 12; HR=average daily heart rate
measured during week 12; RV=right ventricle; LV=left ventricle.
*
P 0.05, greater than the respective body weight group.
§
p 0.10, greater than the respective body weight group.

Despite differences in body weight and body composition between control and hydralazine-
treated groups, dry right and left ventricular weights did not differ significantly in either lean
or obese animals (Table 1). Similarly, intravascular volumes did not differ between control
and hydralazine-treated animals. However, plasma protein concentration was significantly
higher (P 0.05, Table 1), while hematocrit tended to be higher (P 0.10, Table 1) in obese
controls compared with obese hydralazine-treated.

Lower body weight in obese hydralazine-treated after 12 weeks was associated with lower
overall feed consumption (11.8 0.4 vs 13.5 0.4 kg, respectively, P 0.05). Feed
consumption during week 1 of the protocol was similar in obese controls and obese
hydralazine-treated (170 6 vs 169 9 g, respectively), but dropped in obese hydralazine-
treated beginning in week 2 when hydralazine treatment began (137 5 vs 164 10 g,
respectively). This may be related to nausea, a potential side effect of hydralazine.19

Hormone concentrations
PRA did not differ significantly between lean groups, but ALDO was significantly higher in
lean hydralazine-treated compared with lean controls (P 0.05, Figure 1a, b). In contrast,
hydralazine treatment in obese rabbits resulted in lower PRA (P 0.10, Figure 1a) and ALDO
(P 0.05, Figure 1b) concentrations. There were no significant differences between controls
and hydralazine-treated animals in plasma concentrations of cortisol, ANP, noradrenaline, or
adrenaline (Table 2).

Table 2. Hormone concentrations

Next table|Previous table|Figure and tables index

Lean Lean Obese Obese

control hydralazine control hydralazine

ALDO/PRA 201 56 261 46 99 14 69 14

Cortisol (nM) 26.2 6.9 35.3 8.8 57.2 11.7 36.9 8.6

ANP (pM) 7.8 2.3a 3.0 1.2b 2.0 0.7c 1.1 0.5d

Noradrenaline
4.2 0.8 3.4 0.8 3.1 0.5 2.6 0.8
(pmol/ml)

Adrenaline
0.7 0.2 0.6 0.2 0.5 0.3 0.4 0.1
(pmol/ml)

Values are mean s.e. PRA=plasma renin activity; ALDO=aldosterone; ANP=atrial

natriuretic peptide.

a
n=8;

b
n=6;

c
n=8;

d
n=6.

Animal characteristics (groups matched for body weight)

Since control and hydralazine-treated groups differed in body weight, it was possible that
group differences in other variables were due to differing body weight and not to
hydralazine treatment. In order to test the effect of differing body weights, the heaviest
animal from lean hydralazine-treated and the four heaviest animals from obese controls
were eliminated from analyses so that control and respective hydralazine-treated groups no
longer differed significantly in body weight. Results of these analyses are shown in Table 3.
BP in obese hydralazine-treated was 10% lower than in obese controls, but this difference
was not significant. We believe that this outcome was due to the reduced sample size since
the percentage difference is similar to the significant differences that we have previously
noted between lean and obese rabbits.9,26,27,28 Further, acutely measured BP was significantly
lower in obese hydralazine-treated compared with obese controls (84.7 1.8 vs 90.2
1.5 mmHg, respectively, P 0.05)

Table 3. Group characteristics (groups matched for body weight)

Next table|Previous table|Figure and tables index

Lean Lean Obese Obese

control hydralazine control hydralazine

Body weight
3.71 0.03 3.78 0.04 4.86 0.14 4.65 0.13
(kg)

Mean BP
62.5 3.9 63.7 5.4 69.7 3.9 62.9 2.3
(mmHg)

HR (beats/min) 180 6 163 8 216 6 212 6

Body fat (%) 13.3 1.5* 7.8 1.2 26.5 1.8* 20.5 1.6

Fat weight (g) 0.49 0.05* 0.29 0.05 1.30 0.11* 0.96 0.10

Dry RV weight
0.50 0.05 0.43 0.03 0.68 0.04 0.56 0.05
(g)

Dry LV weight
0.98 0.07 0.90 0.06 1.30 0.07 1.27 0.06
(g)

Plasma volume
130 6 142 7 140 9 143 10
(ml)

Blood volume
193 8 212 12 220 13 218 17
(ml)

Hematocrit (%) 35.5 0.7 35.4 0.8 39.2 1.0* 36.6 0.5

Protein (mg/dl) 5.0 0.1 5.0 0.1 5.3 0.1* 4.9 0.1
 Values are mean s.e. BP=average daily blood pressure measured during week 12;
HR=average daily heart rate measured during week 12; RV=right ventricle; LV=left
ventricle;

*
P 0.05, greater than the respective weight-matched group.

Despite equivalent body weight, body fat weight and percentage were still significantly
higher in obese controls compared with obese hydralazine-treated (P 0.05), while body
water percentage was lower (50.4 1.4 vs 54.8 1.1%, respectively, P 0.05). In lean rabbits,
percent body fat and fat weight were lower in lean hydralazine-treated compared with lean
controls (P 0.05). Body water percentage tended to be higher in lean hydralazine-treated
compared with lean controls (65.9 0.7 vs 63.0 1.2%, respectively, P=0.06). There were no
significant differences between control and respective hydralazine-treated groups in the dry
right and left ventricular weights or intravascular volumes (Table 3). However, hematocrit
and plasma protein concentrations were significantly higher in obese controls compared with
obese hydralazine-treated (P 0.05).

Hormone concentrations (groups matched for body weight)

Using control and hydralazine-treated groups that did not differ significantly in weight, we
re-analyzed plasma hormones (Table 4). In lean rabbits, hydralazine treatment elevated
ALDO (P 0.05, Table 4). In contrast, ALDO was 57% lower (P 0.05) and ALDO/PRA 40%
lower (P=0.06) in obese hydralazine-treated compared with obese controls. There were no
significant differences between controls and respective hydralazine-treated animals in
plasma concentrations of cortisol, ANP, noradrenaline, or adrenaline (data not show

TABLE 4
FROM:
Hydralazine as antihypertensive therapy in obesity-related hypertension
J F Carroll, J W King and J S Cohen
BACK TO ARTICLE

Table 4. Renin–angiotensin system in groups matched for body weight

Previous table|Figure and tables index

Lean Lean Obese Obese

control hydralazine control hydralazine

PRA (ng
2.17 0.60 2.00 0.48 5.88 1.16 3.84 0.63
AI/ml/h)

ALDO
249 62 474 60* 609 91* 259 56
(pmol/l)

ALDO/PRA 201 56 258 53 115 17§ 69 14

Values are mean s.e. PRA=plasma renin activity; AI=angiotensin I; ALDO=aldosterone.

*
P 0.05, greater than the respective weight-matched group;

§
P=0.06, greater than the respective weight-matched group.

Topof page

Discussion

We demonstrated that the arterial vasodilator hydralazine is effective in controlling BP

during development of obesity in the rabbit model, and that the pseudotolerance
phenomenon associated with vasodilation-induced, baroreceptor-mediated sympathetic
activation19 is either absent or differently expressed in lean and obese animals. In obese
rabbits, hydralazine treatment controlled BP, attenuated activation of RAS, and did not
exacerbate obesity-related cardioacceleration. In contrast, RAS was activated in
hydralazine-treated lean rabbits, as evidenced by increased plasma ALDO. Finally, these
data indicate that control of hypertension in obesity did not attenuate obesity-related effects
on cardioacceleration, cardiac hypertrophy, and intravascular volumes.

Hydralazine is commonly used in animal studies as the control treatment in the evaluation
of other antihypertensive therapies.10,11,12,13,14,15,16 However, because hydralazine induces
direct relaxation of vascular smooth muscle in resistance vessels, it is thought to provoke
baroreceptor-mediated sympathetic activation. As a result, the potential side effects include
increased HR and cardiac output, and RAS activation.19 In humans, these side effects are
alleviated by combining hydralazine with -blockers to blunt cardiac sequelae, and diuretics
to control fluid retention and plasma volume expansion. In contrast, hydralazine is
commonly used as monotherapy in animal studies, but its side effects are often not
reported. As some of the potential side effects of hydralazine (eg, tachycardia, RAS
activation, and plasma volume expansion) are already exhibited in obesity,9,23,24 we
documented whether the use of hydralazine as antihypertensive therapy exacerbated these
effects.

Interestingly, hydralazine treatment resulted in different manifestations in lean and obese

animals. In lean animals, RAS was activated, as evidenced by increased ALDO. However,
PRA was not altered, demonstrating that measuring single components of RAS may give an
incomplete picture of physiological function. In contrast, RAS activation was attenuated in
obese hydralazine-treated, with PRA and ALDO lowered by 35 and 57%, respectively. In
light of RAS activation seen in lean hydralazine-treated, this reduction may be particularly
significant. The cause of RAS attenuation in obese hydralazine-treated is unknown, but may
be related to improved renal blood flow as a result of arterial vasodilation. RAS attenuation
is not likely due to inhibition by ANP29,30 since ANP was not increased in obese hydralazine-
treated. These data suggest that effects of hydralazine as a control treatment may be
altered by underlying pathology or phenotype. Further, despite RAS attenuation in obese
hydralazine-treated, a comparison of obese hydralazine-treated and lean controls
demonstrated that PRA tended to be higher (P=0.09) and ALDO/PRA lower (P=0.08) in
obese hydralazine-treated, suggesting that obesity still exerted effects on RAS independent
of hypertension.

Cardiac hypertrophy in obesity may be related to preload- and afterload-induced ventricular

remodeling,31 prolonged neurohumoral activation of RAS,32,33,34 and/or alterations in other
hormones such as ANP35 or insulin.31 Despite reduced BP and attenuated activation of RAS in
obese hydralazine-treated, cardiac hypertrophy persisted, suggesting that preload played a
more important role in ventricular hypertrophy in this model than hypertension-induced
afterload or RAS. Further, our data are in agreement with findings that hydralazine does not
have an independent effect on cardiac hypertrophy.12,13,14,16,36 Hydralazine-treated groups did
not have significantly different wet or dry ventricular weights compared with their respective
controls. While we did not measure chamber volumes, the neutral effect of hydralazine on
intravascular volumes suggested that preload-initiated obesity-related cardiac chamber
remodeling37 would be unaltered by hydralazine. The lack of difference in wet ventricular
weights suggested that, despite greater overall body water in hydralazine-treated groups,
cardiac tissue was not subject to significant edema; this is reinforced by similar dry
ventricular weights. However, hypertrophy as measured by direct measurement of
ventricular weight or by echocardiography may be an incomplete measure of hydralazine's
effect. One investigation demonstrated that hydralazine treatment in stroke-prone
spontaneously hypertensive rats reduced BP and the left ventricular interstitial collagen
volume fraction without concomitant changes in ventricular weight.38While our data would
suggest a hypertension-independent effect of obesity on cardiac hypertrophy, further
analyses are necessary to determine whether BP control in obesity induced alterations in
cardiac composition.

While there were no differences between control and hydralazine-treated animals in

intravascular volumes, body water was greater and body fat lower in hydralazine-treated
animals. This may be related to the purported hydralazine-related side effect of
edema.19 Since lean groups were maintained at equivalent body weights, the gradual
increase in body water occurred at the expense of body fat. Obese animals were fed ad
lib so that the increasing body water along with the increasing body weight obscured the
attenuation in body fat accretion. These data demonstrate the importance of body
composition analysis in addition to body weight measurement in assessing the effects of
obesity-related interventions. However, the mechanism responsible for increased body
water in lean and obese hydralazine-treated groups is unclear, since both ALDO and
ALDO/PRA changed in opposite directions in these groups. Further studies of renal function
and sympathetic nervous system influences on the kidney are needed to clarify these
questions.

We did not find that hydralazine treatment altered sympathetic activation in either lean or
obese animals, as indicated by unchanged plasma adrenaline and noradrenaline. While it is
unlikely that a single measurement of plasma catecholamines adequately reflects the
chronic state of the sympathetic nervous system, a lack of sympathetic activation was also
suggested by unaltered HR responses in hydralazine-treated animals compared with
respective controls. Nevertheless, we cannot rule out sympathetically mediated effects in
individual organs/tissues.

An emerging area of study in obesity is that of adipose tissue as an endocrine organ. All
components of RAS are evident in adipocytes, but their contribution to hypertension and
metabolic disease is still uncertain.39,40 Further, different levels of expression of RAS
components are evident in subcutaneous vs visceral adipose tissue.41 Other peptide
hormones secreted by adipose tissue include leptin, adipsin, plasminogen activator inhibitor-
1 (PAI-1), adiponectin, and resistin, and their regulation is altered when fat mass is
markedly altered.42 Therefore, we cannot exclude the possibility that reduced fat mass in
obese hydralazine-treated contributed to altered plasma RAS components or to reduced BP.
However, because we did not see similar directional changes in lean hydralazine-treated, we
hypothesize that reduced adipocyte hormone secretion was not responsible for attenuated
RAS in obese hydralazine-treated.

From these data, we conclude that hydralazine treatment did not exacerbate cardiovascular
and hormonal alterations in obesity, and in fact resulted in an unexpected reduction in
baroreceptor-mediated RAS activation. Further, cardioacceleration and cardiac hypertrophy
persisted in obese hydralazine-treated despite BP control, suggesting hypertension-
independent effects of obesity on these variables. It is evident that documentation of
systemic effects of hydralazine as a control therapy in evaluation of antihypertensive
medications is often lacking, but may provide crucial information regarding integrated
physiological function, which may differ depending on the underlying pathology. Finally,
further studies are clearly warranted to determine the differential effects of hydralazine in
lean and obese animals on cardiac composition, renal function, sympathetic nervous
system/renal function relationships, and adipocyte biology.

Topof page

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Antioxidant activities and fatty acid composition of wild

grown myrtle (Myrtus communis L.) fruits

Abstract

The fruits of eight myrtles, Myrtus communis L. accessions from the Mediterranean region of
Turkey were evaluated for their antioxidant activities and fatty acid contents. The antioxidant
activities of the fruit extracts were determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH)
and β-carotene-linoleic acid assays. The fatty acid contents of fruits were determined by using
gas chromatography. The methanol extracts of fruits exhibited a high level of free radical
scavenging activity. There was a wide range (74.51-91.65%) of antioxidant activity among the
accessions in the β-carotene-linoleic acid assay. The amount of total phenolics (TP) was
determined to be between 44.41-74.44 µg Gallic acid equivalent (GAE)/mg, on a dry weight
basis. Oleic acid was the dominant fatty acid (67.07%), followed by palmitic (10.24%), and
stearic acid (8.19%), respectively. These results suggest the future utilization of myrtle fruit
extracts as food additives or in chemoprevention studies.

Keywords: Antioxidant activity, diphenylpicrylhydrazyl, Mediterranean, myrtle

Introduction

Myrtaceae (Myrtus communis L.) is an evergreen shrub belonging to the family of Myrtaceae
growing spontaneously throughout the Mediterranean area. It is a typical annual shrub of the
Mediterranean countries including Turkey, Greece, Italy, Algeria, Tunisia, and Morocco. In
Turkey, myrtle plants are found within the natural pine forests and riversides in the
Mediterranean region, particularly in the Taurus Mountains, 500 to 600 m above sea level. [1]
People living in Mediterranean region have consumed myrtle fruits, called as 'hambeles', 'mersin'
or 'murt' in Turkish, as food, and used them for some medicinal purposes. [2] In folk medicine, a
decoction of leaves and fruits or infusion of myrtle are used for stomachic, hypoglycemic, cough
and oral diseases, antimicrobic, for constipation, appetizing, antihemorrhagic and externally for
wound healing. [3],[4] The oils of the fruits are used both in the flavor and fragrance industries. [2],[5]
The fruits are very astringent and are used as a condiment, as a substitute for pepper, and are
considered a rich source of tannins. [6] The plant contains many biologically active compounds
such as fibers, sugars, and antioxidants. [2],[7]

Recent studies have focused on the healthy functions of aromatic and medicinal plants, which
have antioxidant, antimicrobial, and mutagen properties. [8] Dietary intake of antioxidant
compounds is important for health. [9] Although there are some synthetic antioxidants such as
butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which are commonly
used in processed foods, it has been reported that these compounds have some side effects. [10]

Another healthy function of aromatic and medicinal plants is their essential fatty acid
composition, which humans cannot synthesize, and must be obtained through diet. Essential fatty
acids are necessary for the formation of healthy cell membranes and the proper development and
functioning of the brain and nervous system. [11]

There is little information with regard to the antioxidant activity and fatty acid content of myrtle
accessions from different regions of the world. Thus far there have been few attempts to study
the fatty acid content in the fruits of myrtle, in Turkey. [12] Moreover, little information has been
published with regard to the antioxidant activity of myrtle. In these studies, generally cultivated
plants were used. The fatty acid content and antioxidant activities of wild growing myrtle fruits
have not been reported in detail. In the present work, we report the investigation on bioactive
compounds, such as, fatty acids, TP, and antioxidant activities of myrtle, to exploit its potential
as a food and natural preservative.

Materials and Methods

Chemicals

All the chemicals used in this study were purchased from Sigma (USA). The chemicals were of
analytical grade.

Plant materials

The fruits of wild growing myrtle were harvested manually from eight accessions found in
Hatay, situated in the Mediterranean region of Turkey. The fruit colors of the accessions were
white except 31-04, which was dark blue. Identification of plants was carried out by Dr. Meryem
Sengul (Department of Botany, Faculty of Science, Ataturk University). A voucher specimen has
been kept in Horticulture Department of the Ataturk University for future reference. The fruits
were packed in a portable refrigerator during transportation to the laboratory (2-3 hours). The
air-dried and powdered plant materials (100 g) were extracted in a soxhlet with methanol at 60°C
for six hours. The extract was then filtered and concentrated in vacuo at 45°C. Finally, the
extracts were kept in the dark at 4°C until tested.

The fatty acid composition was analyzed by Gas Chromatograph (GC) Clarus 500 with an
autosampler (Perkin Elmer, USA) equipped with a flame ionization detector and a fused silica
capillary SGE column (30 m x 0.32 mm, ID x 0.25 µm, BP20 0.25 UM, USA). The oven
temperature was 140°C, held for five minutes, raised to 200°C at a rate of 4°C/minute and to
220°C at a rate of 1°C/minute, while the injector and the detector temperature were set at 220°C
and 280°C, respectively. The sample size was 1 µl and the carrier gas was controlled at 16 ps.
The split ratio was 1:100. Fatty acids were identified by comparing the retention times of the
Fatty Acid Methyl Ester (FAME) with the standard component FAME mixture (Supelco).
Triplicate GC analyses were performed and the results were expressed in GC area percent as a
mean value and ± standard deviation.

The hydrogen atom or electron donation ability of the corresponding extracts and some pure
compounds was measured from the bleaching of the purple-colored methanol solution of
diphenylpicrylhydrazyl (DPPH). This spectrophotometric assay uses stable radical DPPH as a
reagent (Sigma-Aldrich). [13] Various concentrations of extracts, of 100 µl, in methanol, were
added to 5 ml of 0.004% methanol solution of DPPH. After a 30-minute incubation period at
room temperature the absorbance was read against a blank, at 517 nm. Inhibition-free radical
DPPH in percent (I%) was calculated as follows:

I% = (A blank 2 A sample/ A blank ) 100

where A blank is the absorbance of the control reaction (containing all reagents except the test
compound), and A sample is the absorbance of the test compound. The extract concentration
providing 50% inhibition (IC50) was calculated from the graph-plotted inhibition percentage
against extract concentration. Tests were carried out in triplicate and á-tocopherol was used as a
positive control. In this assay, the antioxidant capacity was determined by measuring the
inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising
from linoleic acid oxidation. [14] A stock solution of β-carotene/linoleic acid (Sigma-Aldrich) was
prepared as follows. First, 0.5 mg of β-carotene was dissolved in 1 ml of chloroform (HPLC
grade), and then 25 µl of linoleic acid and 200 mg of Tween 40 (Merck) were added. The
chloroform was subsequently evaporated using a vacuum evaporator. Next, 100 ml of distilled
water saturated with oxygen (30 minutes at 100 ml/minute) was added with vigorous shaking.
Aliquots (2.5 ml) of this reaction mixture were transferred to test tubes, and 350 µl portions of
the extracts (2 g/l in methanol) were added before incubating for 48 hours at room temperature.
The same procedure was repeated with á-tocopherol at the same concentration and a blank
containing only 350 µl of ethanol. After the incubation period the absorbance of the mixtures
was measured at 490 nm. The antioxidant capacities of the samples were compared with those of
á-tocopherol and the blank.

The TP content of myrtle fruits was evaluated by employing the literature methods involving the
Folin-Ciocalteu reagent and Gallic acid as a standard. [15] The extract solution (0.1 ml) containing
1000 µg extract was taken in a volumetric flask, and 46 ml of distilled water and 1 ml of Folin-
Ciocalteu reagent were added and flask was shaken thoroughly. After three minutes, 2% Na 2 CO
3 was added to 3 ml of the solution and the mixture was allowed to stand for two hours with

intermittent shaking. Absorbance was measured at 760 nm. The same procedure was repeated for
all standard gallic acid solutions (0-1000 mg/0.1 ml) and a standard curve was obtained.

The experiment was a completely randomized design with four replications. The data were
subjected to analysis of variance (ANOVA) and the means were separated by the Duncan
multiple range test at P < 0.05 significant level.

Results and Discussion

Fatty acid analysis has shown that the myrtle fruits studied contained 14 fatty acids [Table 1].
Oleic acid was found to be the dominant fatty acid (67.07%) followed by palmitic acid (10.24%)
and stearic acid (8.19%), respectively [Table 1]. The total peak areas of the mentioned fatty acids
were between 94.74-98.87% in accessions and 73.68% of these were unsaturated and 19.97%
were saturated, respectively [Table 1]. The results of the present study also indicated that the
fruits of myrtle are rich sources of essential fatty acids (18:2). 31-04 had a dark fruit color and
several fatty acids that were not present in the other white-fruited accessions. It is possible that
there is an association between fruit color and fatty acid composition.

It was previously reported that the main fatty acids in the fruits of myrtle were oleic and palmitic
acids, [12] which supported our findings. However, Cakir [12] also reported the presence of myristic
and lauric acids in myrtle fruits. In our study we did not detect these fatty acids.

The free radical-scavenging activities of the methanol extracts, measured as a decolorizing

activity following the trapping of the unpaired electron of DPPH, are shown in [Table 2]. The
free radical scavenging activities were found between 2.34 (31-01) and 8.24 µg/ml (31-07). The
highest free radical scavenging activity was determined in 31-01 (2.34 µg/ml). The IC50 values
of the other accessions were also close to this accession and there were no statistical differences
between the accessions [Table 2]. This result indicated that the extracts of myrtle fruit were
extremely potent radical scavengers as also electron donors. Therefore, the extract could also
react with free radicals, converting them to more stable products and terminating the radical
chain reaction. This might also be important in protecting cellular DNA, lipids, and proteins
from free radical damage. The results also indicated that the aqueous extracts showed
considerable antioxidant activity, as measured by their capacity to scavenge the stable-free
radical DPPH. As for as our literature survey, we could reach only one report that showed that
the IC50 values of the methanol extract of myrtle fruit, sampled from Tunisia, was 6.5 µg/ml, [7]
which supported our results.

In the β-carotene/linoleic acid assay, there were statistical differences between the extracts of
accessions and also accessions and á-tocopherol [Table 2]. The inhibition ratio was 96.83% in á-
tocopherol followed by 31-01 (91.65%), 31-06 (89.80%), and 31-03 (88.64%), respectively. This
result suggested that in the β-carotene/linoleic acid assay, oxidation of linoleic acid was
effectively inhibited by the myrtle fruit extract. Therefore, it can be concluded that myrtle fruits
have a strong antioxidant capacity and can be important both in the human diet and for food
safety, when mixed with other foods. In the previous studies conducted on different cultivars of
myrtle, the antioxidant activity was found to be between 76.7 and 99.0% in aqueous, methanol,
and á-tocopherol extracts, respectively, which was very close to our results.

The TP of accessions was between 44.41 and 88.56 µg GAE/mg dry weight equivalents of
phenolic compounds in the extract. There were statistical differences among accessions in terms
of TP [ P < 0.05, [Table 2]]. The antioxidative effectiveness in natural sources was reported to be
mostly due to TP. [16] The results for TP in fruit extracts clearly outline the rich phenolic sources.
The variability among accessions could be due to the plants used, environmental factors, and
collection period. Phenolic compounds are the antioxidants that contribute to the high
antioxidant capacity observed in edible plants. [17]

The chemical composition of myrtle leaf extracts have been studied previously and the results
have shown and determined that flavonoids, coumarins, and tannins are the major compounds. [7]
Flavonoids are the most probable candidates among the compounds, known to be present in the
methanol extracts, which prevent oxidative lesions. [18]

Conclusion

As a conclusion from these results, it can be suggested that the consumption of myrtle can
probably offer some dietary benefits, as they contain antioxidant constituents, which can protect
against lipid peroxidation and can scavenge free radicals. This report further suggests that myrtle
may have beneficial chemopreventive effects in addition to providing potential new sources of
natural antioxidants as well as genes, and also for use as food and for medicinal purposes.
However, further studies to fractionate the active extracts, to identify the active compounds, and
to determine the exact mechanism of action, are required. Moreover, the sampling date and
phenotype factors should be considered in future efforts, to improve and domesticate myrtle as a
major agricultural crop.

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