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The Beneficial Effects On Blood Pressure Dyslipidemia and Oxidative Stress of Sambucus Nigra Extract Associated With Renin Inhibitors
The Beneficial Effects On Blood Pressure Dyslipidemia and Oxidative Stress of Sambucus Nigra Extract Associated With Renin Inhibitors
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
RESEARCH ARTICLE
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.
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.
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.
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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