lingonberry juice lowers blood pressure of spontaneously hypertensive rats (shr)
TRANSCRIPT
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 4 3 2 – 1 4 4 0
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Lingonberry juice lowers blood pressureof spontaneously hypertensive rats (SHR)
1756-4646/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jff.2013.05.012
* Corresponding author. Tel.: +358 191 25348; mobile: +358 415013771; fax: +358 191 25364.E-mail address: [email protected] (A.S. Kivimaki).
Anne S. Kivimaki*, Aino Siltari, Pauliina I. Ehlers, Riitta Korpela, Heikki Vapaatalo
Institute of Biomedicine, Pharmacology, University of Helsinki, P.O. Box 63, Helsinki 00014, Finland
A R T I C L E I N F O A B S T R A C T
Article history:
Received 8 February 2013
Received in revised form
22 May 2013
Accepted 30 May 2013
Available online 22 June 2013
Keywords:
Alkaline phosphatase
Blood pressure
Endothelial function
Lingonberry
Polyphenols
SHR
Lingonberries (Vaccinium vitis-idaea) are rich in polyphenols, such as proanthocyanidins,
anthocyanins, flavonols and flavanols. Polyphenol-rich foods affect vascular health. We
previously described improved endothelium-dependent vascular function as well as anti-
inflammatory and anti-atherothrombotic effects in spontaneously hypertensive rats
(SHR) fed with lingonberry juice. In the present study, we investigated the effects of lingon-
berry juice dose on blood pressure, vascular function and vascular inflammation in SHR in
an 8 weeks’ study. Diluted (1:5) cold-compressed lingonberry juice was given as drinking
fluid ad libitum to 5 week old SHR for 8 weeks. Control group (SHR) and normotensive refer-
ence group (Wistar-Kyoto) received tap water. Systolic blood pressure (SBP) was monitored
weekly. Function of mesenteric artery rings was assessed in standard organ-bath chambers
after 8 weeks. Biochemical and clinical chemistry variables were measured from plasma
and urine. Lingonberry juice lowered SBP of SHR (p = 0.007). Endothelium-dependent vascu-
lar relaxation was not improved. Lingonberry treatment slightly affected plasma inflamma-
tory markers (reduction of NOx and sICAM-1) and clinical chemistry variables (decreased
alkaline phosphatase and increased chloride and calcium levels). In conclusion, Lingon-
berry juice at small concentrations lowers blood pressure in a long-term treatment in SHR.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Increased blood pressure is a world-wide health problem (Lo-
pez, Mathers, Ezzati, Jamison, & Murray, 2001). Cardiovascular
risk factors, such as hypertension, arterial stiffness, hyper-
cholesterolemia, insulin resistance and metabolic syndrome
often coexist leading to increased cardiovascular morbidity
and health problems (Krousel-Wood, Muntner, He, & Whel-
ton, 2004). The main treatment for hypertension in addition
to lifestyle changes is antihypertensive drugs, e.g. diuretics,
beta-blockers, angiotensin-converting enzyme (ACE) inhibi-
tors, angiotensin type 1 receptor antagonists and calcium
antagonists (Mancia et al., 2007). Polyphenols have been
suggested to have protective effects against cardiovascular
diseases (Stoclet et al., 2004). An inverse correlation between
the consumption of berries, fruits and vegetables and cardio-
vascular risk factors has been found (Rissanen et al., 2003).
Consumption of polyphenol-rich cocoa has been shown to
lower blood pressure and improve endothelial function (Kylli
et al., 2011; Stoclet et al., 2004). Lingonberry is a berry from
Nordic countries rich in polyphenols and vitamins C and E
(Finnish Food Composition Database; Sudano et al., 2012).
Polyphenols are secondary metabolites in plants, and differ-
ent groups of polyphenols are suggested to be mainly respon-
sible for the health effects of fruits and vegetables. Especially
dietary flavanols inhibit development of atherosclerosis in
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 4 3 2 – 1 4 4 0 1433
animal models. Flavanols also improve endothelial function,
reduce blood pressure and inhibit platelet reactivity (Grassi
et al., 2009; Heiss, Keen, & Kelm, 2010).
In our previous study lingonberry juice enhanced endothe-
lium-dependent vascular relaxation in spontaneously hyper-
tensive rats (SHR) (Kivimaki, Ehlers, Turpeinen, Vapaatalo, &
Korpela, 2011). It is suggested that blood pressure – lowering
effect of polyphenol-rich foods is partly due to inhibition of
angiotensin-converting enzyme (ACE), which is a key regula-
tor of blood pressure (Actis-Goretta, Ottaviani, & Fraga, 2006;
Persson, Persson, Hagg, & Andersson, 2011). Vascular endo-
thelium has an important role in the regulation of blood pres-
sure and vascular function by controlling vascular tone.
Endothelium maintains the balance between vasoconstric-
tion and vasodilatation synthesizing and releasing various
relaxing factors, like nitric oxide (NO), cyclo-oxygenase
(COX) – derived prostanoids and endothelium-derived hyper-
polarizing factor (EDHF) (Feletou & Vanhoutte, 2006; Vapaat-
alo & Mervaala, 2001). In large arteries, NO is the most
important vasorelaxing factor (Shimokawa & Tsutsui, 2010;
Shimokawa et al., 1996).
Polyphenols are also suggested to act as antioxidants due
to aromatic structure and functional groups (Grassi et al.,
2009; Michalska et al., 2010). Accordingly, 6 weeks‘ interven-
tion with wild blueberry juice reduced oxidative stress in hu-
mans (Riso et al., 2012) and lingonberry extract
supplementation for 6 weeks decreased the total oxidant sta-
tus and affected positively antioxidant defence enzymes in
red blood cells and liver in rats (Mane, Loonis, Juhel, Dufour,
& Malien-Aubert, 2011).
Anti-inflammatory actions of polyphenols have also been
described. In in vitro studies, many flavonoids affect favour-
ably several inflammatory markers, like NO, cytokines and
adhesion molecules (Conzalez et al., 2011). In our previous
study, lingonberry and cranberry juices given at high concen-
trations in drinking fluid inhibited mRNA expressions of
cyclooxygenase 2 (COX2), monocyte chemoattractant protein
1 (MCP1) and P-selectin in SHR (Kivimaki et al., 2012).
The aim of the present study was to investigate whether
long-term consumption of lingonberry juice, in smaller doses
(less phenolic compounds) than in our previous study, is able
to lower blood pressure and improve endothelial function of
SHR.
2. Materials and methods
2.1. Experimental protocol
The protocol was approved by National Animal Experimenta-
tion Committee of Finland according to EC Directive 86/609/
ECC and Finnish Experimental Animal Act 62/2006. Sixteen
five weeks old (weight approx. 220 g) male spontaneously
hypertensive rats (SHR) and eight age-matched male
Wistar-Kyoto (WKY) rats were purchased from Charles River
Laboratories (Sulzfeld, Germany). The rats were housed in a
standard experimental animal laboratory, randomized into
groups according to the weight and systolic blood pressure
(SBP), and placed four in a cage. One SHR group received
cold-compressed diluted lingonberry juice. Tap water (+1%
sucrose) was given to the SHR control group. WKY rats were
used as a normotensive control e.g. to follow possible effects
of aging on the variables measured. The lingonberry puree
was purchased from Bandedosa PLC (Ilmajoki, Finland),
cold-compressed to lingonberry extract and diluted to one
part of lingonberry extract and four parts of water. Sucrose
was added (1% w/w) in the juice because rats did not drink
unsweetened juice. Same amount of sucrose was added to
the tap water of control and reference groups. The consump-
tion of juice and water was recorded daily during the 8 weeks’
treatment. The rats had free access to standard rat pellet
(2018 Teklad Global 18% Protein Rodent Diet; Harlan Laborato-
ries, Madison, WI, USA) and consumption of the feed was re-
corded weekly.
2.2. Metabolic caging
Rats were housed individually in metabolic cages for 24 h
after 7 weeks of treatment. They received same feed and
drinking fluid as during the experiment ad libitum. Urine of
the rats was collected during 24 h and stored at �80 �C until
analysis.
2.3. Systolic blood pressure and heart rate
Systolic blood pressure (SBP) and heart rate were measured
with non-invasive blood pressure measurement system
(tail-cuff method) CODA (Kent Scientific Corporation, Torring-
ton, CT, USA) once a week by the same researcher at the same
daytime (before noon). The rats were pre-warmed for 10–
15 min at 32 �C to intensify the pulsation of the tail artery.
Measurements were done in three parts, which all included
eight measurements. SBP and heart rate were calculated as
the mean of the measurements accepted by the CODA
program.
2.4. Collection of samples
After 8 weeks’ treatment (SHR) or 10 weeks without treatment
(WKY) (age difference because of practical reasons), the rats
were rendered unconscious with CO2/O2 (30/70%, AGA, Riihi-
maki, Finland) and decapitated. Blood was collected with or
without ethylenediamine tetraacetic acid (EDTA) as an antico-
agulant, and centrifuged 3000·g for 10 min at +4 �C. Aliquots
of the plasma or serum were stored in �80 �C until analysis.
Superior mesenteric arteries were excised and placed in ice-
cold oxygenated Krebs buffer for vascular reactivity studies.
The hearts, left kidneys, left ventricles and adrenal glands
were excised and weighted.
2.5. Vascular reactivity studies
Mesenteric arteries were carefully cleaned from adherent
connective tissue, 4 mm section from proximal end of mesen-
teric-aorta junction was cut off and following 4 mm section
was used for vascular reactivity measurements. The artery
rings were placed in oxygenated (O2/CO2 95/5%, AGA) ice-cold
Krebs buffer (pH 7.4–7.6 composition in mM: NaCl 119.0,
NaHCO3 25.0, glucose 11.1, KCl 4.7, CaCl2 1.6, KH2PO4 1.2,
MgSO4 1.2). The rings were placed between stainless steel
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hooks (diameter 0.1 mm) and mounted in 10 ml organ bath
chambers in oxygenated Krebs buffer (composition as above,
37 �C). The rings were at the beginning equilibrated for an
hour with the resting tension of 1.5 g. The force of contraction
was measured with isometric force–displacement transducer
using a computerized system (EMKA Technologies, Paris,
France).
After the equilibration period, the rings were exposed to
60 mM KCl to study the contraction response. Then, endothe-
lium-dependent and -independent relaxations were tested
using acetylcholine (ACh, 1 nM–10 lM) or sodium nitroprus-
side (SNP, 1 nM–1 lM) cumulatively after phenylephrine (PE,
1 lM) contraction. The roles of cyclo-oxygenase (COX) and ni-
tric oxide synthase (NOS) were studied incubating arteries for
15 min with a non-selective COX inhibitor diclofenac (3 lM) or
a non-selective NOS inhibitor L-NG-Nitroarginine methyl es-
ter (L-NAME, 100 lM) before PE contraction and cumulative
ACh (1 nM–10 lM). The rings were equilibrated for 20–30 min
between different experiments and washed three times with
Krebs buffer. The concentrations reported are the final con-
centrations in the organ chamber. All compounds were pur-
chased from Sigma–Aldrich (St. Louis, MO, USA).
2.6. Ultra performance liquid chromatography – UPLC
UPLC-method was used to detect concentrations of phenolic
compounds in the diluted lingonberry juice. Measurements
were done at the Department of Food and Environmental Sci-
ences, University of Helsinki (Hellstrom, Sinkkonen, Karonen,
& Mattila, 2007; Kahkonen, Hopia, & Heinonen, 2001; Lamu-
ela-Raventos & Waterhouse, 1995). Before chromatography,
phenolic compounds were eluted with 70% acetone. Flavo-
noids and other phenolic compounds were assayed in their
natural form or in their glycosides or other conjugates.
Hydroxybenzoic acids were quantified with gallic acid,
hydroxycinnamic acids with chlorogenic acid, flavan-3-ols
and procyanidins with catechin, flavonols with rutin and
anthocyanins with cyaniding-3-glucoside, respectively. All
samples were measured in three replicates. Series-diode-
detector (UV/Vis) and fluorometer were used in the detection.
2.7. Biochemical variables
Total NO (NOx), high-sensitive CRP (hsCRP), interleukin 10
(IL-10), 6-keto prostaglandin F1a (6-keto PGF1a) and aldoste-
rone were measured from plasma with commercial kits
according to the manufacturers’ instructions (Parameter total
NO/Nitrite/Nitrate Immunoassay, R&D Systems, Minneapolis,
MN, USA, Rat high-sensitive CRP ELISA, Kamiya Biomedical
Company, Seattle, WA, USA; Rat IL-10, R&D Systems, 6-keto-
prostaglandin F1a, Cayman Chemicals, Ann Arbor, MI, USA).
Intracellular adhesion molecule 1 (ICAM-1) was determined
from serum samples according to the manufacturers’ instruc-
tions (ICAM-1/CD 54 Quantikine ELISA Kit, R&D Systems).
From urine samples, creatinine, albumin, cyclic GMP and 8-
isoprostane were determined (Parameter Creatinine Assay,
R&D Systems; Rat Albumin, Alpha diagnostic, San Antonia,
TX, USA; Cyclic GMP EIA kit, Cayman Chemicals; 8-isopros-
tane EIA kit). Serum and kidney extract ACE activity were
measured using the method of Santos et al. (Santos et al.,
1985).
2.8. Clinical chemistry
Plasma electrolytes (K, Na, Cl, Ca), alanine aminotransferase
(ALAT), alkaline phosphatase (ALP), aspartate transaminase
(AST), creatinine (Crea), albumin (Alb), urea, creatine kinase
(CKNAC), total bilirubin (TBil), inorganic phosphate (Pi), glu-
cose (Glu), high-density lipoprotein (HDL), low-density lipo-
protein (LDL), triglycerides (TG) and total cholesterol (TC)
were measured using routine clinical chemistry (ADVIA 1650
Chemistry System, Siemens Healthcare Diagnostics Inc.,
Deerfield, IL, USA).
2.9. Statistical analyses
The main comparison was made between lingonberry-treated
and non-treated (control) SHRs. All data are presented as
mean ± SEM. Values of normotensive Wistar-Kyoto rats were
used as a reference group representing healthy rats to find
out normal concentrations of physical, biochemical and
chemical markers. Statistical analysis was performed using
IBM SPSS Statistics 20 software. General linear model for re-
peated measurements was used to evaluate blood pressure,
heart rate and vascular reactivity data. General linear model
for univariate analysis were used to compare groups in clini-
cal chemistry measurements. Differences were considered
significant when p < 0.05. Figures were made with GraphPad
Prism� software.
3. Results
3.1. Phenolic content of the lingonberry juice and dailyintake
Phenolic content is presented in Table 1. The most abundant
phenolic compound of the lingonberry juice was procyani-
dins. Total phenolic concentration of the diluted lingonberry
juice was 34.1 ± 0.2 mg/100 g and total daily intake was
12.7 mg/rat.
3.2. Drinking fluid and food consumption
The consumption of drinking fluid and food did not show sig-
nificant differences between the groups (Table 2).
3.3. Body and organ weights of the rats
The average body weights at the end of the experiment in the
SHR lingonberry group, control group and normotensive refer-
ence group were 315.7 ± 8.7 g, 319.3 ± 7.4 g and 406.0 ± 11.5 g,
respectively. WKY rats were 2 weeks older than SHR. There
were no significant differences in the organ weights related
to the body weight between the two SHR groups. Obviously
due to higher body weight, the proportioned kidney and heart
weights of the WKY rats were slightly lower than those of the
SHR (data not shown). Also the long lasting high blood
Table 1 – Phenolic content of the lingonberry juice (Mean ± SEM, n = 3).
Phenolic compound Content in juice mg/100 g Daily intake mg/rat
Hydroxybenzoic acids 0.1 ± 0.0 0.04
Hydroxycinnamic acids 2.5 ± 0.1 0.9
Anthocyanins 5.9 ± 0.1 2.2
Flavonols 3.8 ± 0.1 1.4
Flavan-3-ols 3.0 ± 0.1 1.1
Procyanidins 18.7 ± 0.6 7.0
Total 34.1 ± 0.2 12.7
Table 2 – The average drinking fluid and food consumption during the study. (Mean ± SEM, n = 8).
Group Drink (ml/rat) Food (g/rat)
Lingonberry SHR 37.2 ± 2.7 18.1 ± 0.9
Control SHR 46.0 ± 3.4 17.6 ± 0.2
Control WKY 47.7 ± 4.4 20.2 ± 0.6
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pressure of the SHR could explain the hypertrophy of these
organs.
3.4. Blood pressure and heart rate
Blood pressure and heart rate values during the 8 weeks’
treatment are presented in Fig. 1A and B. Systolic blood pres-
sure of the lingonberry group was significantly lower during
the study when compared to the SHR control group
(p = 0.007). The average blood pressures after 8 weeks of treat-
ment of the lingonberry, control and normotensive reference
Fig. 1 – (A) Effect of lingonberry treatment on systolic blood
pressure (SBP) of the SHR during 8 weeks’ treatment,
p = 0.007 (SHR lingonberry vs. SHR control) (General linear
model for repeated measurements). (B) Effect of lingonberry
treatment on heart rate (HR) of the SHR during 8 weeks’
treatment (Mean ± SEM, n = 8).
groups were 172.1 ± 3.6 mmHg, 193.0 ± 5.3 mmHg and
119.0 ± 6.3 mmHg, respectively. Average heart rate at the end
of the study seemed to be lower in the lingonberry
group (467.9 ± 91 beats per minute), than in the control group
(593.8 ± 60) (p = 0.28) and in the normotensive reference
group (606.4 ± 42).
3.5. Vascular reactivity studies
ACh-induced mesenteric artery relaxation was impaired in
both SHR groups vs. the normotensive reference group
(Fig. 2A). The lingonberry treatment tended to improve the
relaxation when compared to the SHR control group
(p = 0.160). Interestingly, cyclooxygenase (COX) inhibition
with diclofenac improved the ACh-induced relaxation in both
SHR groups similarly (Fig. 2B). However, inhibition of COX did
not influence the endothelium-dependent relaxation of nor-
motensive reference group. Nitric oxide synthase (NOS) inhi-
bition by L-NAME attenuated the relaxation equally in both
SHR groups (Fig. 2C). Inhibition of NOS reduced only slightly
the ACh relaxation of the normotensive reference rats (max-
imal relaxation about 70%). Thus, in SHR, ACh-relaxation is
mediated almost only via NO. Diclofenac and L-NAME to-
gether abolished the relaxation of SHR totally, but a slight dif-
ference between the lingonberry and the control group rats
(p = 0.066) (Fig. 2D) was seen. The maximal relaxation of the
mesenteric artery rings from normotensive reference rats still
reached the level of 50% which suggests the role of EDHF in
the control of vascular tone of the WKY animals.
Endothelium-independent relaxation with sodium nitro-
prusside (SNP) was over 80% in both SHR groups and reached
100% in the normotensive reference group (Fig. 2E). The ling-
onberry group had slightly higher sensitivity to the SNP than
the SHR control group (p = 0.380).
3.6. Biochemical variables
Biochemical variables are presented in Fig. 3A–F. The total
NOx and aldosterone were higher in the SHR groups than in
Fig. 2 – Vascular responses of mesenteric artery rings after 8 weeks’ treatment with lingonberry juice (Mean ± SEM, n = 6–8).
(A) ACh-induced relaxation was impaired in both SHR groups. Lingonberry slightly improved the relaxation (p = 0.160). (B–D)
The SHR relaxation is mediated mainly by NO, but in WKY the relaxation is also due to endothelium-dependent
hyperpolarizing factor (EDHF). (E) Lingonberry group showed slightly higher sensitivity to the SNP than the SHR controls.
1436 J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 4 3 2 – 1 4 4 0
the WKY group. There were no significant differences
between SHR groups in NOx (p = 0.160), hs CRP (p = 0.703),
aldosterone (p = 0.240), ACE (p = 0.519), sICAM-1 (p = 0.471) or
6-keto PGF 1a (p = 0.270) concentrations. The level of IL-10
was under detection limit (data not shown).
There were no significant differences in biochemical
markers (albumin (kidney function), 8-isoprostane (oxidative
stress) and cGMP (nitric oxide production) measured from ur-
ine between the SHR groups (p = 0.793, p = 0.200 and p = 0.813,
respectively). (Fig. 3G–I).
3.7. Clinical chemistry
Clinical chemistry data are presented in Table 3. Values of the
normotensive reference rats followed the clinical chemistry
reference values of Wistar rats presented by Boehm
et al.(Boehm et al., 2007). Plasma Cl, Ca and alkaline phospha-
tase (ALP) concentrations were significantly different between
the SHR groups (p = 0.03, p = 0.002 and p = 0.009, respectively).
Lingonberry increased the concentration of Cl and Ca and
decreased the concentration of ALP. Liver function markers,
alanine transaminase (ALT), ALP and aspartate transaminase
(AST) levels were noteworthy lower in the normotensive con-
trol group than in the SHRs. ALP and AST concentrations of
the lingonberry group were slightly lower vs. the SHR con-
trols. Interestingly, blood lipid values, HDL, LDL, Trig and Chol
were lower in the SHR groups than in the normotensive WKY
group. However, all the values were in the normal range.
(Boehm et al., 2007).
4. Discussion
We demonstrated that low concentration (1:5) of cold-com-
pressed lingonberry juice lowered systolic blood pressure of
spontaneously hypertensive rats in a long-term treatment
and tended to improve endothelium-dependent vascular
relaxation. As far as we know, this was the first study to inves-
tigate the effects of a low concentration of lingonberry juice
on blood pressure and vascular function of hypertensive rats.
Normotensive WKY rat groups were included in the study as a
Fig. 3 – (A–F) Concentrations of biochemical variables in plasma of the rats after 8 weeks’ treatment with lingonberry
(Mean ± SEM, n = 6–8). There were no significant differences. (G–I) Concentrations of biochemical variables of urine of the rats
related to creatinine concentration. (Mean ± SEM, n = 6–8). p not significant.
Table 3 – Clinical chemistry data of plasma samples of the rats after 8 weeks’ treatment with lingonberry. (Mean ± SEM,n = 6–8).
Lingonberry SHR Control SHR Normotensive WKY p (lingonberry vs. control)
K (lmol/l) 6.6 ± 0.2 7.1 ± 0.3 6.3 ± 0.2 0.13
Na (lmol/l) 145.4 ± 1.2 145.6 ± 0.3 147.9 ± 0.4 0.868
Cl (lmol/l) 105.3 ± 0.4 103.9 ± 0.4 105.0 ± 0.5 0.03
Ca (lmol/l) 3.0 ± 0.1 2.8 ± 0.0 2.8 ± 0.1 0.002
Pi (mmol/l) 2.5 ± 0.1 2.5 ± 0.1 2.5 ± 0.1 0.557
Glu (mmol/l) 8.7 ± 0.1 9.1 ± 0.2 9.5 ± 0.2 0.22
Chol (mmol/l) 2.4 ± 0.1 2.2 ± 0.1 3.0 ± 0.1 0.167
HDL (mmol/l) 0.6 ± 0.0 0.6 ± 0.0 0.8 ± 0.0 0.871
LDL (mmol/l) 0.18 ± 0.0 0.22 ± 0.0 0.27 ± 0.0 0.278
Trig (mmol/l) 1.3 ± 0.1 1.1 ± 0.1 2.0 ± 0.2 0.133
Alb (g/l) 41.4 ± 0.5 41.7 ± 0.4 38.5 ± 0.3 0.484
ALT (U/l) 66.6 ± 2.9 65.9 ± 2.8 56.9 ± 3.0 0.871
ALP (U/l) 342.2 ± 17.0 401.1 ± 10.3 299.3 ± 10.5 0.009
AST (U/l) 146.9 ± 6.1 154.4 ± 7.3 100.3 ± 5.2 0.489
CK (U/l) 4120 ± 424 4285 ± 386 4211 ± 623 0.786
Crea (lmol/l) 47.8 ± 0.8 49.3 ± 0.8 41.4 ± 1.1 0.224
Urea (mmol/l) 7.2 ± 0.3 7.3 ± 0.2 7.3 ± 0.1 0.621
Tbil (lmol/l) 0.7 ± 0.1 0.6 ± 0.1 0.2 ± 0.0 0.321
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reference group to detect possible changes in blood pressure,
vascular function and biochemical markers related to
increasing age.
In our previous study (Kivimaki et al., 2011), lingonberry
juice at higher concentrations normalized endothelial func-
tion, without affecting the development of hypertension in
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the SHR. These two experiments differed from dilution and
thus the dose of the lingonberry juice, and the berries were
picked up from different places. In the present study, the
phenolic concentration of the lingonberry juice remained
about half of that in the previous study (Kivimaki et al.,
2011). Proportions of phenolic compounds were different,
but procyanidins and anthocyanins were most dominant
in both juices. Ratio of procyanidins to the total phenolic
content was about 55% in the present study and 35% in
the previous study. Possibly the most remarkable deviation
between these two studies were the levels of blood pressure
in the beginning of the experiments. Thus in the
present study, we tested the antihypertensive effect of the
juice on already hypertensive animals whereas the inhibi-
tion of the development of hypertension was tested in the
previous study.
Renin-angiotensin system (RAS) is one of the major regu-
lators of blood pressure and vascular tone locally and system-
ically. Angiotensin-converting enzyme (ACE) is an important
factor controlling vascular tone by producing extremely po-
tent vasoconstrictor angiotensin II (Fyhrquist & Saijonmaa,
2008). Particularly, procyanidins, which are flavanol oligo-
mers, are demonstrated to inhibit ACE activity (Actis-Goretta,
Ottaviani, & Fraga, 2003; Actis-Goretta, Ottaviani, Keen, &
Fraga, 2003). In the present study, there were no differences
in circulating ACE activity between the groups. Accordingly,
plasma aldosterone levels did not differ. Thus, the inhibition
of ACE seems not to be the main mechanism behind the blood
pressure-lowering effect of the lingonberry juice. If the results
of the present and previous study are compared, (Kivimaki
et al., 2012) it can be concluded that the antihypertensive ef-
fect of lingonberry juice cannot be explained by improved
vascular relaxation. In some cases high volumes of the drink-
ing fluid can cause hypotensive effects (Quinones, Miguel,
Muquerza, & Aleixandre, 2011). In this study the amount of
drinking fluids were approximately same in all groups, thus
the amount of drinking fluid do not explain the antihyperten-
sive effect of lingonberry juice. Interestingly, there are studies
demonstrating that high quantity of polyphenols could be
pro-oxidative and higher doses of polyphenols may not de-
crease blood pressure anymore (Duarte et al., 2001; Lambert
& Elias, 2010).
In hypertension, increased levels of plasma oxidative
agents, superoxide, hydrogen peroxide and reduced levels of
an antioxidant vitamin C have been found (Hamilton et al.,
2004). Increase of reactive oxygen species (ROS) are related
to cardiovascular diseases (Schnackenberg, Welch, & Wilcox,
1998). Because lingonberry is very rich in antioxidative vita-
mins C and E (Finnish Food Composition Database), it may
have antioxidative actions. Also the structure of polyphenols
supports this assumption (Michalska et al., 2010). In the pres-
ent study, measurement of 8-isoprostane from urine was per-
formed to find out possible oxidative stress in the rats.
However, there were no differences in 8-isoprostane concen-
trations between the hypertensive animal groups. Even so, we
could speculate that the decrease in blood pressure in lingon-
berry group could be partly related to antioxidative properties
of polyphenols and vitamins C and E. Supporting this
speculation, increased endothelial production of superoxide
has been established in SHR (Landmesser et al., 2007).
Respectively, ROS production has been reported to decrease
by antihypertensive drugs (Antoniades et al., 2010).
Low-grade inflammation is present in cardiovascular dis-
eases, such as hypertension and endothelial dysfunction (Lib-
by, 2001). Dysfunctional endothelium releases inflammatory
molecules causing also platelet activation and adhesion to
the vascular wall (Hazewindus, Haenen, Weseler, & Bast,
2012; Libby, 2008). Association between formation of reactive
oxygen species (ROS) and low-grade inflammation is
established. Lipid peroxidation caused by ROS initiates
inflammatory actions which then again produce ROS.
Pro-inflammatory transcription factors, like NF-jB, are acti-
vated by ROS (Hamalainen, Nieminen, Vuorela, Heinonen, &
Moilanen, 2007), which can be inhibited by some flavonoids
(Schini-Kerth, Etienne-Selloum, Chataigneau, & Auger, 2011).
In our previous study (Kivimaki et al., 2012), we found that
lingonberry and cranberry juices have anti-inflammatory
and anti-atherothrombotic actions in SHR, but in the present
study we could not verify those findings with the methods
used. NO has several roles in vascular function and inflam-
mation. It is an 2.important vasodilator, an inhibitor of mono-
cyte chemoattractant protein (MCP1), adhesion molecules
and other pro-inflammatory and pro-atherothrombotic medi-
ators and it also affects in entire inflammatory process
(Feletou & Vanhoutte, 2006; Guzik, Korbut, & Adamek-Guzik,
2003; Schmitt & Dirsch, 2009; Shimokawa et al., 1996). NO pro-
duction has been shown to be influenced by various polyphe-
nols (Tonelli et al., 2009) and some flavonoids inhibit NO
production of macrophages (Schini-Kerth, Etienne-Selloum,
Chataigneau, & Auger, 2011). Here we measured higher total
plasma concentration of NOx in SHR groups than in the
WKY rats, which is in line with the data of proinflammatory
actions of NO. Lingonberry treatment tended to decrease
the level of NOx and parallel to that its main second messen-
ger, cyclic GMP. Lingonberry treatment also tended to de-
crease the concentrations of two other inflammation
markers sICAM-1 and 6-keto PGF 1a. We suggest that these
small though not significant changes towards favourable
direction in anti-inflammatory markers can at least partly ex-
plain the blood pressure-lowering effect of the lingonberry
juice.
We made also routine clinical chemistry of the plasma
samples. Markers of liver function, ALT, ALS, ALP, and Tbil
were increased, but not to pathological levels, in the SHR
group compared to normotensive reference group. An inter-
esting finding was that lingonberry juice significantly lowered
the plasma concentration of alkaline phosphatase. ALP is also
an inhibitor of vascular calcification and the concentration is
highest in bone, liver and kidneys. Epidemiologically a posi-
tive correlation between high levels of ALP and increased risk
of all cause death and kidney failure has been found. (Tonelli
et al., 2009).
Accordingly, plasma calcium concentration was higher in
the lingonberry group than in the other groups. Dietary cal-
cium has been shown to have positive effect on blood pres-
sure both in experimental and epidemiological studies
(McCarron, 1989; Sallinen et al., 1996). The exact role of quite
high concentrations of calcium in lingonberry juice (Kivimaki
et al., 2011) remains to be clarified.
J O U R N A L O F F U N C T I O N A L F O O D S 5 ( 2 0 1 3 ) 1 4 3 2 – 1 4 4 0 1439
In conclusion, the present study showed that lingonberry
juice as drinking fluid in quite low concentrations lowers
systolic blood pressure in a long-term treatment in SHR.
There seems not to be only a single mechanism explaining
the slight antihypertensive effect. A tendency to a decrease
in some inflammatory markers and increase in total plasma
calcium might at least partly explain our findings which seem
not to be associated with inhibition of renin-angiotensin sys-
tem. Endothelial dysfunction was not improved by diluted
lingonberry juice like it was improved with more concen-
trated juice in our previous study.
Acknowledgements
Orion Corporation, Orion Pharma Espoo, Finland is greatly
acknowledged for analyzing clinical chemistry markers of
plasma samples. We want to thank Professor Marina Heinon-
en for analyzing the phenolic compound of the lingonberry
juice. We are grateful to Piet Finckenberg for the scientific
assistance during the study. Sari Laakkonen and Paivi Lein-
ikka are warmly acknowledged for the assistance with animal
care.
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