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Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

The beneficial effects on blood pressure,


dyslipidemia and oxidative stress of Sambucus
nigra extract associated with renin inhibitors

Manuela Ciocoiu, Magda Badescu, Oana Badulescu & Laurentiu Badescu

To cite this article: Manuela Ciocoiu, Magda Badescu, Oana Badulescu & Laurentiu Badescu
(2016) The beneficial effects on blood pressure, dyslipidemia and oxidative stress of Sambucus
nigra extract associated with renin inhibitors, Pharmaceutical Biology, 54:12, 3063-3067, DOI:
10.1080/13880209.2016.1207088

To link to this article: https://doi.org/10.1080/13880209.2016.1207088

Published online: 14 Jul 2016.

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PHARMACEUTICAL BIOLOGY, 2016
VOL. 54, NO. 12, 3063–3067
http://dx.doi.org/10.1080/13880209.2016.1207088

RESEARCH ARTICLE

The beneficial effects on blood pressure, dyslipidemia and oxidative stress of


Sambucus nigra extract associated with renin inhibitors
Manuela Ciocoiua, Magda Badescua, Oana Badulescua and Laurentiu Badescub
a
Department of Pathophysiology, Faculty of Medicine, University of Medicine and Pharmacy ‘Grigore T. Popa’ Iasi, Romania; bDepartment of
Cellular and Molecular Biology, University of Medicine and Pharmacy ‘Grigore T. Popa’, Iasi, Romania

ABSTRACT ARTICLE HISTORY


Context: The health effects of Sambucus nigra L. (Caprifoliaceae) could be due to polyphenols whose Received 21 December 2015
modes of action differ from the traditional one proposed for exogenous antioxidants. Accepted 24 June 2016
Objective: The study emphasizes the effects of the association between the renin inhibitor and the poly- Revised 16 June 2016
phenolic extract on biochemical parameters and systolic (TAS) and diastolic (TAD) blood pressure within an Published online 14 July 2016
L NAME-induced experimental model of arterial hypertension (AHT).
KEYWORDS
Materials and methods: The polyphenols are extracted with ethanol from isolated and purified vegetable Blood pressure; aliskiren;
material represented by the mature fruit of the S. nigra with a dosage of 0.046 g/kg body weight (PS), polyphenols; oxidative stress
every 2 days, for 8 weeks. The dose represents 1/20 of LD50. The Wistar white rat blood pressure values
were recorded using a CODATM system, which uses a non-invasive blood pressure measuring method.
Results and discussion: The total antioxidant capacity levels were significantly decreased (p < 0.001) in
AHT group as compared to the rats in the AHT þ PS group. A combination of a renin inhibitor (Aliskiren)
and polyphenolic extract generated a superior antioxidant effect compared to administering the two separ-
ately. Both TAS and TAD in rats with drug-induced hypertension were reduced by polyphenolic extract.
The homogeneous values of TAS record a significant decrease (p < 0.001) of the average values in
AHT þ PS group or AHT þ Aliskiren group.
Conclusion: The combination of two different classes of substances, namely, renin inhibitors and natural
polyphenol extracts, reduces arterial pressure and also might reduce the side effects of the major classes
of antihypertensive agents and improve the quality of live.

Introduction hyperoside) (up to 3.0%), triterpenes (about 1%), sterols (about


1%), phenolic acids (about 3%) and essential oil (up to 0.15%)
Polyphenols (flavonoids, lignans, stilbenes, etc.) are the most
(Ghosh & Scheepens 2009; Cunha et al. 2016).
diverse group of phytochemicals distributed in vegetables, fruits,
The renin–angiotensin–aldosterone system (RAAS) plays a key
olive oil and wine, and exhibit wide range of protective roles such
role in the regulation of blood pressure and volume homeostasis.
as hypolipidemic, antioxidative, antiproliferative and anti-inflam-
The renin inhibitors (Aliskiren) could be an alternative of the
matory effects to reduce the onset of disease progression (Liu
angiotensin-converting enzyme inhibitors (ACEI) or the angioten-
2003; George et al. 2009).
sin receptor blockers (ARB), in treating AHT and it could also
Among the great family of polyphenols, flavonoids play an
accomplish a multi-protection of the organs (heart, kidney, brain)
important role. Bioflavonoids are biologically active compounds
(Gradman et al. 2005). Since ACEI and ARB increase the level of
that, once inside the human body, determine a positive biologic
plasma renin, Aliskiren combination can be beneficial because it
response (Mennen et al. 2004). Bioflavonoids are recommended
as supplement in preventing and treating those deficiencies that reduces plasma renin activity (Allikmets 2007).
The purpose of the study was to emphasize the effects of the
involve capillary fragility and permeability (Yao et al. 2004).
Several epidemiological studies (Dauchet et al. 2005; Bayard association between the renin inhibitor and the S. nigra extract on
et al. 2007) suggest that regular consumption of foods and bever- biochemical parameters and heart rate, systolic and diastolic blood
ages rich in polyphenols is associated with a reduction in the risk pressure.
of a range of pathological conditions, such as arterial hypertension
(AHT), coronary heart disease, stroke, diabetes mellitus and can- Materials and methods
cer. Co-active compounds identified in the fresh berries of
Sambucus nigra L. (Caprifoliaceae) (Elderberry) known as Ripe berries of S. nigra were shade-dried at room temperature for
European elder include proteins and several vitamins (e.g., vita- 1 week. Dried berries (100 g) were chopped into small pieces and
mins C, folic acid and pantothenic acid) (Sidor & Gramza- extracted with 3  700 mL ethanol using a magnetic stirrer (FALC
Michałowska 2015). This fruit contains many primary metabolites, F30ST), each time for 3 h. The combined extracts were taken to
as sugars and organic acids, and secondary metabolites, as flavo- dryness by evaporation under reduced pressure (BUCHI € R-210
noids (kaempferol, astragalin, quercetin, rutin, isoquercetin, € €
rotavapor, BUCHI V-850 vacuum controller, BUCHI V-700

CONTACT Magda Badescu magda.badescu@gmail.com Department of Pathophysiology, University of Medicine and Pharmacy ‘Grigore T. Popa’, 16, Universitatii
Street, 700115 Iasi, Romania
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
3064 M. CIOCOIU ET AL.

vacuum pump). Total phenolics quantification was performed by out by means of Siedel’s modified colorimetric method. The
Folin-Ciocalteu method. The absorbance was measured at 765 nm exploration of the lipid profile included the measurement by pho-
after 2 h of incubation at room temperature. A calibration curve tocolorimetry, in the serum obtained after separation, of high-
was plotted using gallic acid as standard. The amount of total phen- density lipoproteins (HDLc) (Lopes-Virella 1977), for all the ani-
olic content was expressed as gallic acid equivalents (GAE)/100 g mals included in the experiment.
extract. The absorbencies of all the solutions were determined by The rat blood pressure values were recorded using a CODATM
using a V-550 Able Jasco UV-VIS spectrophotometer. The result is system purchased from Kent Scientific Corporation (Torrington,
the mean of triplicates ± standard deviation. The total content of CT), which uses a non-invasive blood pressure measuring
polyphenols was 7.63 ± 0.12 mg GAE/100 g from S. nigra extract. method. It records the blood volume–pressure by a band attached
The experiment used active therapeutic doses, well-determined to the tail, homologated by Bland-Altman testing designed to
fractions of LD50 on an experimental model of AHT. Fractions of reveal conformity with an invasive method (radiotelemetry),
LD50 are doses representing 1/5, 1/10, 1/20, 1/40 of LD50. The dose which enjoys proven accuracy, yet it is difficult to use in our
representing 1/20 of LD50 was chosen, as it is the smallest dose that study. The method is also recommended by the AHA in its blood
determined the pharmacodynamic effect that is being researched, pressure measuring guide for laboratory animals. The actual
without producing significant toxic effects. S-Sambunigrin contains experiment consists of performing at least six blood pressure
hydrogen cyanide (HCN), which is toxic (3 to 17 mg HCN/100 g measurements in each laboratory animal, data collection by means
fresh weight in leaf and 3 mg HCN/100 g of fruit) (www.ema.eur- of the CODATM software and subsequent data processing.
opa.eu, 2013). Elderberry extracts used in our experiment are gen-
erally without side effects when taken in the suggested dosages.
Statistical data interpretation
The research was performed on Wistar white rats, with an
average weight of 250–280 g, which were divided into 6 groups of All the data are shown as mean value ± standard error of the
12, namely: W Group – control, normal animals, that did not mean (SEM). In order to assess the normal distribution of the
receive natural polyphenols; PS Group – animals that were groups, Shapiro–Wilk test was performed. Statistical data inter-
administered polyphenols under the form of solution, from the pretation considered the corresponding differences for a given sig-
extract obtained from the S. nigra fruit, with a dosage of 0.046 g/ nificance threshold: p > 0.05, statistically insignificant; p < 0.05,
kg body, p.o. (by tube feeding), at every 2 days, for 8 weeks. The statistically significant; p < 0.01, strong statistical significance;
dry polyphenol extract was diluted in DMSO, 100 mL poly- p < 0.001, very strong statistical significance.
phenolic solution containing 840 mg natural polyphenols, 95 mL
distilled water and 5 mL DMSO; AHT Group – animals which
were administered N(G)-nitro-L-arginine-methyl ester (L-NAME) Results
40 mg/kg body/day, i.p., at every 2 days, for 8 weeks; AHT þ PS The TAC levels were significantly decreased (p < 0.001) in AHT
Group – animals which were administered polyphenols in the group (1.31 ± 0.16 mmol/L) as compared to the rats in the W
dosage mentioned p.o. at every 2 days, concomitantly with L- (1.55 ± 0.29 mmol/L) and AHT þ PS (1.53 ± 0.27 mmol/L) groups.
NAME, for 8 weeks; AHT þ Alisk Group – animals with AHT As expected, severe oxidative stress disturbs the antioxidant bal-
induced by L-NAME, that were given Aliskiren 30 mg/kg body/ ance by generating reactive species and decreasing the TAC in the
day, administered s.c. for 8 weeks; AHT þ Alisk þ PS Group – ani- extracellular space. There are similar TAC values in group W and
mals with AHT that were given Aliskiren and polyphenols in the group PS (1.58 ± 0.31 mmol/L) and the differences between the
above-mentioned dosage, for 8 weeks. AHT and AHT þ PS groups are statistically significant (p < 0.01).
The in vivo experiments, on experimental animal sample, were The highest average values of HDLc can be found in W group,
carried out in specially equipped premises, where conditions of very significantly higher (p < 0.001) compared to the AHT group.
habitat in accordance with standard norms were created. Between PS group has average values of HDLc significantly higher
experiments, the rats were kept in individual cages and were given (p < 0.001) than those recorded in the AHT, AHT þ PS and
food and water. A 12-h light/dark cycles was employed. AHT þ Alisk groups. The lowest average values are noticed for
The experiments on murine model complied with the ethical the rats in AHT group (Figure 1). Supplementing the diet with S.
norms in experimental research, yet also with the terms enforced nigra determines average values of HDLc close to those recorded
by the Iasi ‘Grigore T. Popa’ University of Medicine and in the W group. Significant correlations of the individual values
Pharmacy Commission of Bioethics. At the same time, the EU of HDLc with blood pressure have been met only in the groups:
norms concerning lab rats were taken into consideration (the AHT þ Alisk þ PS with systolic blood pressure values (TAS) a
Directive of the European Council Committee 86/609/EEC). weak indirect correlation (r ¼ 0.11) (Figure 2);
For the extracellular response the total antioxidant capacity AHT þ Alisk þ PS with diastolic blood pressure values (TAD) a
(TAC) was determined by using a RANDOX kit for manual use moderate indirect correlation (r ¼ 0.50).
by Randox Laboratories Ltd. The major advantage of this test is The lowest average values of the ceruloplasmin have been
to measure the antioxidant capacity of all antioxidants in a bio- recorded in the AHT group. The highest average values of the
logical sample and not just the antioxidant capacity of a single ceruloplasmin have been recorded in the W group and in the
compound. The method is based on formation of the groups treated with S. nigra. Administration of S. nigra led to
ABTS•þ cation [2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic similar average values to those found in the W group, which sug-
acid)] and its scavenging by antioxidant sample constituents (e.g. gests it is the most efficient administration.
serum or food) measured by spectrophotometry (decay of green/ The highest average values of the uric acid have been recorded
blue chromophore absorbance is inversely associated with antioxi- in AHT group, significantly higher than in the other groups. The
dant sample content and the control antioxidant is Trolox, a lowest average values of the uric acid have been recorded in the
hydrophilic vitamin E analogue). groups treated with S. nigra and in the W group, significantly
Ceruloplasmin determination was performed on an EOS lower than those registered in the other researched groups. The
BRAVO automatic analyser. Uric acid determination was carried statistical processing confirms the homogeneity of the uric acid
PHARMACEUTICAL BIOLOGY 3065

40

35

30

mg/dL
25

20

15
W PS AHT AHT+PS AHT+Alisk AHT+Alisk+ PS
Study groups
Figure 1. Average values of HDLc on study groups.

AHT + PS Group AHT+ Alisk + PS Group


175 175
y = −0.25x + 148.25
R2 = 0.0126
150 150
y = −0.5607x + 165.36
TAS/TAD

TAS/TAD

R2 = 0.1047
125 125
y = −0.9583x + 126.79
R2 = 0.2524
100 100
y = −0.8218x + 122.25
R2 = 0.3244
75 75
10 20 30 40 10 20 30 40
HDLc (mg/dL) HDLc (mg/dL)
Figure 2. Correlation of HDLc with TAS in AHT þ PS and AHT þ Alisk þ PS groups. Group AHT þ Alisk þ PS shows a weak, indirect correlation between HDLc with TAS
and a moderate indirect correlation between HDLc with TAD. TAS: systolic blood pressure; TAD: diastolic blood pressure.

average values in the groups treated with S. nigra no matter of 450

the association, or not, with AHT.


The highest average values of heart rate were recorded in the 425
AHT group, significantly higher than in other investigated groups.
At the significant limit of 95%, the average values of the heart 400
rate in AHT, AHT þ PS, AHT þ Alisk groups were significantly
higher compared to those recorded in W and PS group
Heart rate

375
(Figure 3). AHT þ Alisk þ PS group has average values that are
significantly lower than the AHT group. To be noted that in
groups with AHT the association with Aliskiren and/or polyphe- 350

nols reduces the heart rate. In the PS group, the average values of
heart rate are comparable to those recorded in the W group. 325
The highest average values of TAS can be found in the AHT
group, presenting strong differences, statistically significant com-
300
pared to other investigated groups. Depending on the investigated W PS AHT AHT+PS AHT+Alisk AHT+Alisk+PS
group, the homogeneous values of TAS record a significant Study groups
decrease (p < 0.001) of the average values in AHT þ PS group or Figure 3. Average values of the heart rate on study groups.
AHT þ Alisk group (Figure 4). The highest TAD can be found in
the AHT group, significantly higher (p < 0.001) than other analysed neurological pathologies (Ferrari & Torres 2003). Various epi-
groups. The PS and AHT þ Alisk þ PS groups has average values of demiological studies have shown an inverse association between
TAD comparable to those recorded in the W group (Figure 5). the consumption of polyphenols or polyphenol-rich foods and
the risk of cardiovascular diseases (Bell & Gochenaur 2006;
Pascual-Teresa & Sanchez-Ballesta 2008). In this sense, research-
Discussion
ers had materially proved that following consumption of diets
The role of antioxidant nutrients in fighting against oxidative stress rich in fruits and vegetables there was an increase in serum
is well-established in several diseases including cardiovascular and TAC (Cao et al. 1998).
3066 M. CIOCOIU ET AL.

180 Phytochemicals like curcumin, resveratrol, epigallocatechin-3-


gallate and isothiocyanates share common properties and play an
170
important role to activate the phase II detoxifying and antioxidant
enzymes like heme oxygenase-1 (HO-1), glutathione-peroxidase
and glutathione-S-transferase by targeting the common transcrip-
160
tion factor Nrf2 [nuclear factor (erythroid derived 2) related fac-
tor] (Liby et al. 2007; Tosetti et al. 2009).
TAS, mmHg

150 The redox-sensitive transcription factor Nrf2 plays an import-


ant role in regulating induction of phase II detoxifying or antioxi-
dant enzymes (HO-1, SOD, etc.), which result in cellular defense
140
against oxidative stress and exert cytoprotective mechanism. A
cytoskeleton binding protein called Kelch-like erythroid CNC
130 homologue (ECH)- associated protein 1 (Keap1) binds to Nrf2
regulating its translocation to the nucleus or its activation (Lee-
Hilz et al. 2006). Following nuclear translocation, Nrf2 binds not
120
W PS AHT AHT+PS AHT+Alisk AHT+Alisk+PS
only to the specific consensus cis-element called antioxidative
responsive element (ARE) or electrophile response element (Na &
Study groups
Surh 2008) present in the promoter region of genes encoding
Figure 4. Average values of systolic arterial pressure (mmHg) on study groups.
many antioxidant enzymes, but also to other trans-acting factors
such as small Maf-F/G/K as well as the coactivators of ARE
140
including cAMP response element binding protein (CREB-binding
protein or CBP), p300 that can coordinately regulate the ARE-
driven antioxidant gene transcription (Dhakshinamoorthy &
Jaiswal 2000; Shen et al. 2004).
120
Endothelial dysfunction determines changes in cellular redox
characteristics. Starting from these premises and from previous
studies that have discussed the potential antioxidant extract of S.
nigra is proved that incorporation of anthocyanins from these
TAD, mmHg

100
fruits into endothelial cells causes protective effect against oxida-
tive stress (Skoczynska et al. 2007).
Association within the inhibitor of renin hypertensive group
and separate polyphenols reveal an improvement in mean systolic
80 values of hypertension compared with values obtained in the
group that received only Aliskiren. Similar data were obtained for
diastolic component of hypertension.
The key question is whether this polyphenolic extract can be
60 used as both preventive and acute therapeutic agents in the treat-
W PS AHT AHT+PS AHT+Alisk AHT+Alisk+PS ment of cardiovascular disease. In a preventive approach, S. nigra
Study groups may lower blood pressure and increased HDLc. Acutely, polyphe-
Figure 5. Average values of diastolic arterial pressure (mmHg) on study groups. nols inhibit oxidative stress. Due to the pleiotropic properties of
the polyphenols and their potential synergies of action on vascular
endothelium, they appear to be good candidates for both the pre-
In this experimental model, all groups were put on the same
vention and treatment of cardiovascular diseases.
diet. The reduction in antioxidant mechanisms can be neutralized
The use of the direct renin inhibitor in the therapy combined
through natural polyphenol compounds of S. nigra, which will
with polyphenolic extract of S. nigra ensures an increased protec-
maintain TAC to levels capable of neutralizing ROS effects. The
tion and improves the results in comparison with the mono-ther-
TAC evaluation, used with other oxidative stress and antioxidant
apy. The combined inhibition of RAAS may allow the use of
defense biomarkers, constitutes the first step in search for a
smaller doses of each component in order to obtain a more effi-
healthy body status.
cient and more durable suppression of RAAS, and probably less
The beneficial antiradicalar effect of ceruloplasmin is suggested
adverse effects.
by the fact that all groups present higher average values of cerulo-
Future studies are required to understand on the mechanisms
plasmin compared to the hypertensive group. Through stimulat-
of the in vivo effects of polyphenolic extract. Additional aspects
ing activity of ceruloplasmin, the polyphenols effect is favourable,
about bioactivity, absorption and stability of dietary polyphenols
proving its additional antioxidant role. Moreover, low ceruloplas-
min values were associated with low HDLc values and increased with respect to modulation arterial pressure by renin inhibitors
heart rate. are also important areas of further research.
The ceruloplasmin and uric acid were determined as non-spe-
cific oxidative stress markers. Uric acid acts as a scavenger of free Conclusions
radicals due to covalent bonds that can be set with singlet oxygen.
On animal testing, hyperuricemia predisposes to hypertension Both systolic and diastolic arterial pressure in rats with drug-
through various mechanisms such as endothelial dysfunction, induced hypertension were reduced by polyphenolic extract from
inflammation and vascular changes in the renal microcirculation, S. nigra. When polyphenols are associated with renin inhibitor,
activation of the RAAS system (Manach et al. 2004; Schroeter this occurrence is further enhanced. The polyphenolic extract has
et al. 2010). both an antioxidant and hypolipemiant effect. In order to form
PHARMACEUTICAL BIOLOGY 3067

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diseases, an understanding of the basic molecular mechanisms by 2005. Aliskiren, a novel orally effective renin inhibitor, provides dose-
dependent antihypertensive efficacy and placebo-like tolerability in hyper-
prophylactic agents is required.
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Lee-Hilz YY, Boerboom AM, Westphal AH, van Berkel WJH, Aarts JMM,
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The authors declare that they have no competing interests. functional agents for the prevention and treatment of cancer. Nat Rev
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