Effects of aqueous extract
from Stephanolepis hispidus
on blood pressure in the normal and in L-NAME-induced hypertensive rats
Efeito do extrato aquoso da pele do peixe Stephanolepis hispidus sobre a pressão sanguínea de ratos normotenso e hioertensos inducidos por NG-nitro-L-arginine metilo ester (L-NAME).
Efecto del extracto acuoso del piel del pez Stephanolepis hispidus en el normotenso y NG-nitro-L-arginine metilo ester (L-NAME) hipertensión-inducido ratos
Ana Luiza Muccillo-Baischadef, Daniela Brum da Silvaa, Andressa de Andradea, Daniela Carrazzonia, Marta Regina Cezar Vazbde, Gianni Goulart Perazaa, Eliana Badiale Furlongcg, Maria Cristina Flores Soaresade .
Fundação Universidade do Rio Grande, Rio Grande, RS, Brazil, aDepartamentos de Ciências Fisiológicas, bEnfermagem e cQuímica, dProgramas de Pós-Graduação em Enfermagem, eCiências da Saúde, fCiências Fisiológicas e gEngenharia e Ciências de Alimentos, iversidade Federal do Rio Grande.
Abstract . The aim of the study was to
investigating the antihypertensive effect of the aqueous extract from skin fish
Stephanolepis hispidus
(AqeSh) in normotensive
and NG-nitro-L-arginine methyl ester
(L-NAME)-induced hypertension. The treatment of rats with L-NAME (60 mg/l,
drinking water) caused a sustained increased in rat blood pressure (BP). AqeSh restored rat BP
to normal levels in the L-NAME-treated hypertensive rats. Moreover, AqeSh was able to prevent the increase of BP induced
by L-NAME. At the end of the treatment no difference in body weight and vital
organs were noted between the control and treated groups. Hematological and
biochemical studies revealed no differences in treated animal except AST and
ALT profile, may due to the presence of L-NAME treatment. These results suggest
that oral administration of AqeSh prevent the
increase of BP in L-NAME-induced hypertension that may be mediated partially by
nitrite and nitrate present in the skin.
Key words: NG-nitroarginine methyl ester (L-NAME); hypertension; Nitric
oxide; Stephanolepis hispidus
water extract.
Resumo . O objetivo deste estudo foi o de investigar o efeito antihipertensivo do extrato aquoso da pele do peixe Stephanolepis hispidus (AqeSh) em ratos normotensos e hipertensos induzidos pelo tratamento com NG-nitro-L-arginina metil ester (L-NAME). O tratamento dos animais de experimentação com L-NAME (60 mg/l, água de beber) provocou um aumento sustentado na pressão arterial. O tratamento com AqeSh restaurou a pressão sanguínea para os níveis normais nos animais hipertensos. Além disso, o AqeSh preveniu o aumento da pressão sanguínea induzida pelo L-NAME. Ao final dos tratamentos não foram observadas diferenças significativas nos pesos corporal e dos órgãos vitais entre o grupo controle e os tratados. A avaliação hematológica e bioquímica não revelou diferenças entre os grupos exceto para os valores de AST e ALT no grupo hipertenso, provavelmente devido à presença do tratamento com L-NAME. Estes resultados sugerem que a administração oral do AqeSh previne o aumento da pressão sanguínea induzida pelo L-NAME. Este efeito pode ser mediado parcialmente por nitrito e nitrato presentes na pele do peixe.
Palavras-chave: NG-nitroarginine methyl éster (L-NAME); Hipertensão; Óxido nítrico; Stephanolepis hispidus extrato aquoso.
Résumen. Eso estudio ha tenido como objetivo investigar el efecto
antihipertensivo del extracto acuoso del piel del pez Stephanolepis
hispidus en el normotenso
y NG-nitro-L-arginine metilo ester (L-NAME) hipertensión-inducido ratos. El tratamiento
de ratas con el L-NAME (60 mg/l) causó un persistente
aumento en la tensión arterial (TA). El extracto acuoso de Stephanolepis
hispidus (EacSh)
restauró
Palabras chaves: NG-nitroarginine methyl éster (L-NAME); hipertensión; Óxido nítrico; Stephanolepis hispidus extracto acuoso.
Introduction
Natural products have, for centuries, served as a major source of drugs.
Medicinal plants are known to be an important source of new substances with
potential therapeutics effects (1). In the past 30-40 years, marine plants and
animals have been the focus of a worldwide effort to define natural products
with potential therapeutic use (2).
Animals, and tissues derived from different organs,
have constituted part of the inventory of medicinal substances used by various
cultures since ancient times (3). The history of healing with animals is
revised by Lev (3), and historical surveys have located and identified 286
medicinal substances, of which, 81.8% are from plants, 5.2% from minerals, 3.5%
from other materials, and 9.5% of animal origin.
In
Popular plants and other remedies are widely used in the treatment of a variety
of disorders. However, only few of them have been clinically controlled or
chemically and biologically studied to identify their active constituents.
Despite the complexity of natural products, investigation on their
efficacy is feasible and desirable, particularly vis-à-vis their popularity.
In the International Conference of Primary Health Care (5) all present
government representative showed national policies strategies for the
development medicinal plants. In developing countries, the practice of traditional
medicine has been stimulated by the World Health Organization (WHO). The
poverty and lack of access to modern medicine leads 65 to 80% of the world’s
population in these countries to depend essentially on plants for primary
health care. The limited access to formal and adequate health services, leads
people to seek help from traditional healers, who provide such alternative
health care service (6).
Sutherland
(7) identifies herbs that consumers commonly select for the treatment of
hypertension, and make some nursing care consideration for their use.
Nurses working in the Brazilian Health System need to communicate with
patients, families and other health-care professional, and the relationship
between communication and popular culture may help to obtain information
regarding popular practices (8). Traditional medicine occupies a central place
among rural or peripheral communities of developing countries in the provision
of health care due to the absence of an efficient primary health care system.
In the city of
The WHO has proposed new and more aggressive
international guidelines for the treatment of hypertension (10). Many studies
have been performed to discover antihypertensive properties if substances from
natural sources. Among them Fritillaria (11);
Brazilian propolis (12); vegetable drinks with
peptides derived from sardine protein (13).
The aim of the present experimental study was to
evaluate the effects of skin aqueous extract from Stephanolepis
hispidus (AqeSh)
on arterial blood pressure (indirect non-invasive blood pressure study
techniques, tail cuff) in normal and L-NAME hypertensive rats. Therefore we
also investigated the effects of the extract on biochemical and hematological
parameters, body and organs weight.
Material and methods
Animals
The experimental animals consisting of a total of 26 male Wistar rats. They were randomly divided into three groups of rats namely control
(n=10), L-NAME/AqeSh (n=8) and AqeSh/ L-NAME (n=8). The animals were obtained of
the Animal House of Fundação Universidade
Federal do Rio Grande (FURG). They were housed in groups of five, in plexiglass cage (42x25x15cm), in climatized
rooms (21o C; 55% humidity) on a 12 hlight/dark
cycle, with lightoff at 07:00. The rats were 5-6
months old on arrival. Foods in pellets (Virtu R-Lab,
Housing conditions and experimentations were in accordance with the Brazilian
regulations on the care and use of animals for scientific purposes, from the
Guide to the Care and Use of Laboratory Animals (14). The animals were
accustomed to our housing conditions for at least four weeks before being
used.
Ethical procedures
The animal
procedures were in strict accordance with the Colégio
Brasileiro de Experimentação
Animal for the Guide to The Care and Use of Laboratory Animals (14).
Animal treatments
The rats were divided
into three groups: a control group (n=10) with free access to tap water for 4
weeks; a second group (n=8) was intragastrically
supplemented with 23 mg/ml per day of AqeSh
for four weeks. After the second week L-NAME (60 mg/l) was
co-administered with the drinking water; a third group (n=8) was allowed to
drink water containing L-NAME (60 mg/l) for four weeks. After the second
week AqeSh (23mg/ml) was co-administered with
the drinking water. L-NAME was dissolved in the drinking water at a concentration
of 60 mg/l, resulting in a daily intake of approximately 6 mg/kg. Body weight,
and food and water (or L-NAME solution) intake were recorded. L-NAME was
purchased from the Sigma Chemical Company (
Fish identification and collection
The fish, Stephanolepis hispidus,
were identified by Dr. Ricardo Robaldo from
Department of Physiological Sciences of Fundação Universidade Federal do Rio Grande (FURG),
Preparation
of extract
Fish skin was
toasted at a temperature of 150º C during 45 min. Coarsely powdered
dried skin (20g) was then extracted with 200 ml of bi-distilled water with the
aid of a Soxhlet extractor during 1 hour. The
final concentration was 23mg/ml (AND-AD 4714,
Mean arterial pressure measuring
The systolic blood-pressure (SBP) was measured weekly in awake rats by tail
cuff plethysmography using a Letica
LE 5007 system (Letica, Barcelona, Spain), after
warming the animals at 37o C for 10 min. Animals were trained for at
least 3 weeks to stay under a piece of cloth until the arterial pressure was
steadily recorded with minimal restraint and stress. The first measurement of
arterial pressure was discarded, and the mean of ten subsequent determinations
taken as SBP level.
Body, vital organ and viscera
weights
The average pre,
during and post-treatment body weights of the chronically treated animals were
compared with the control group. Immediately after death the animals underwent laparotomy and the positions, shapes and colors of internal
organs, namely liver, kidney, lung, heart, spleen, testis, seminal vesicle and epididymis, were visually observed for any signs of gross
lesion. These organs were collected and weighed.
Biochemical and hematological
studies
Trunk blood was
collected by decapitation. The serum was separed and aspartate aminotransferase (AST),
alanine amino trasferase
(ALT), urea, total bilirubin, cholesterol,
triglycerides and HDL were measured. These parameters were determined using an
automatic analyzer (Vitalab Selectra
2) at the clinical analysis laboratory from the
Blood was
collected in separate tubes containing EDTA (1.5mg) and hematological parameters,
including hemoglobin content, total white blood cell count (WBC), differential
count of lymphocytes (%),monocytes (%) and
granulocytes (%), haematocrit (%), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and
platelet counts, were determined by automated analysis (ABX Diagnostics micros
60, Brazil).
Statistical analysis
All data are
represented as mean ± S.E.M. Descriptive statistical analysis was carried out
and the difference between the groups results were determined using one way
analysis of variance (ANOVA) followed by Tukey’s post
hoc test and considered statistically significant when P <
0.05.
Results
Effects on systolic blood pressure (SBP)
Oral administration of L-NAME (60 mg/l) during 2 weeks
increased SBP from 142.0±8.5 to 165.1±9.8 mmHg. After this period
co-administration of AqeSh during 2 weeks
dropped the SBP to 123.0±8.6 mmHg (Fig. 1).
AqeSh alone had no effect on SBP, and the values were
128.7±6.5 and 131.3±2.6 mmHg before and after the administration period
respectively. Co-administration of L-NAME failed to increase the SBP, and a
progressive drop in SBP continued up to 4 weeks, after which it was 128.7±4.3
mmHg, similar to control group. In the control group, the SBP values were
between 140.1±8.2 and 119.3±5.4 mmHg during the experimental period (Fig. 1).
Fig. 1. Effects of AqeSh (23 mg/ml) on rat
systolic blood pressure. *P<0.05, compared with the value
before L-NAME intake in the same group.
*AqeSh: Aqueous skins extract of Stephanolepis
hispidus. Control, ¯ L-NAME/AqeSh, AqeSh/l-NAME
Body and organ weights
Water intake and food consumption of all treated
animals were similar to the control group. Weight gain in the treated groups
was comparable to that of the control one (Fig. 2).
Fig. 2. Effects of AqeSh (23 mg/ml) on rat body. *P<0.05,
compared with the value before L-NAME intake in the same group.
*AqeSh: Aqueous skins extract of Stephanolepis
hispidus. Control, ¯ L-NAME/AqeSh, AqeSh/l-NAME
There were significant differences in body weights
over the experimental period in all groups.
The administration of AqeSh
at dose of 23 mg/ml did not produce any significant organ weight change (Table
1)
Table 1. Effect of Stephanolepis
hispidus aqueous extract in lieu of
drinking water for 12 weeks on organ weight of rats.
|
Organ |
Treatment Group (weight g) |
||
|
Control (n=10) |
L-NAME/AqeSh (n=8) |
AqeSh/l-NAME (n=8) |
|
|
liver |
8.3±0.30 |
7.7±0.22 |
7.8±0.30 |
|
kidney |
1.9±0.04 |
2.0±0.20 |
1.9±0.10 |
|
lung |
1.4±0.04 |
1.5±0.05 |
1.4±0.10 |
|
heart |
0.9±0.03 |
1.0±0.05 |
1.0±0.10 |
|
spleen |
0.7±0.02 |
0.8±0.03 |
0.7±0.04 |
|
testis |
3.5±0.10 |
3.4±0.10 |
3.6±0.10 |
|
seminal vesicles |
0.9±0.10 |
0.9±0.00 |
0.9±0.10 |
|
epididymis |
1.3±0.10 |
1.5±0.10 |
1.5±0.10 |
Values
given are absolute wet weight of the organ (g) expressed as means ± SEM (n=10).
No significant differences were observed in any parameters between the three
groups. #AqeSh: Aqueous skins
extract of Stephanolepis hispidus. For details see text.
Blood biochemistry and hematological studies
There were no significant changes in the number of
white blood cell (WBC), lymphocytes (%),monocytes
(%),granulocytes (%), hematocrit (%), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration
(MCHC) and platelet in treated animals (Table 2).
Table 2. . Effect of Stephanolepis
hispidus aqueous extract in lieu of
drinking water for 12 weeks on hematological parameters of rats.
|
Parameter |
Treatment Group |
||
|
Control (n10) |
L-NAME/AqeSh (n8) |
AqeSh/l-NAME (n10) |
|
|
Hemoglobin (g/dL) |
14,73±0,22 |
15,06±0,26 |
15,23±0,29 |
|
MCHC (g/dL) |
38.4±0.4 |
71.8±3.4 |
80.1±1.6 |
|
Platelets (103/mm3) |
696,6±56,33 |
757±93,31 |
626,5±42,20 |
|
Haematocrit (%) |
38,41±0,79 |
40,9±0,53 |
40,44±1,07 |
|
Total WBC (103/mm3) |
4.2±0.4 |
6.6±2.4 |
3.7±0.5 |
|
Monocytes (%) |
15.9±2.3 |
14.8±0.8 |
14.2±1.1 |
|
Lymphocytes (%) |
75.1±3.0 |
71.8±3.4 |
80.1±1.6 |
|
Granulocytes (%) |
8.9±1.9 |
13.3±3.8 |
5.6±0.7 |
|
|
|
|
|
Values are expressed as
means ± SEM (n=10). No significant differences were observed in any parameters
between the three groups. #AqeSh:
Aqueous skins extract of Stephanolepis hispidus. For details see text.
No significant alteration in biochemical parameters
urea, total bilirubin, cholesterol, triglycerides and
HDL were detected (Table 3). Nevertheless, the level of aspartate
aminotransferase (AST) and alanine
amino trasferase (ALT) were significantly altered in
both treated groups.
Table 3. . Effect of Stephanolepis
hispidus aqueous extract in lieu of
drinking water for 12 weeks on some plasma biochemical parameters of rats.
|
Parameter |
Treatment Group |
||
|
Control (n=10) |
L-NAME/AseSh* (n=8) |
AseSh*/l-NAME (n=8) |
|
|
Urea (mmol/l) |
32.2±1.6 |
29.8±0.9 |
35.5±2.3 |
|
Bilirubin total (mg/100ml) |
0.14±0.0 |
0.2±0.0 |
0.5±0.2 |
|
AST (U/l) |
204.2±15.7 |
132.8±8.3* |
214.3±19.4* |
|
ALT (U/l) |
37.7±2.9 |
40.6±3.3* |
45.7±3.8* |
|
Triglycerides |
105.2±9.6 |
61.3±4.4 |
50.6±15.7 |
|
Cholesterol |
65.9±2.9 |
82.8±9.7 |
50.9±13.1 |
|
HDL |
27.2±1.4 |
27.7±2.8 |
31.7±1.8 |
Values are
expressed as means ± SEM (n=10). #AqeSh:
Aqueous skins extract of Stephanolepis hispidus. *P<0.05, compared with the control
group. For details see text. .
Discussion
The obtained results clearly demonstrate that in
L-NAME-induced hypertensive rats the oral administration of AqeSh
attenuated the increase of SBP restoring it to normal levels.
The vasorelaxant effect of
NO could be competitively inhibited by several L-arginine
analogues such L-NAME (15). The decrease levels of NO may be important
pathological factor in the development and sustenance of hypertension. The
chronic treatment with L-NAME increased the blood pressure and decreased plasma
NO and aortic tissue NO levels (11). Animals treated chronically with NOS
inhibitors develop hypertension, with a resetting of the pressure-natriuresis relation toward higher blood-pressure.
A nitrate-derived NO formation pathway is a possible
mechanism for the hypotensive effect of vegetable and
fruit-rich diet (16). Diet constitutes an important source of human and animal
exposure to nitrite and nitrate. Vegetables as the principal source of dietary
nitrate but small amounts any also be present in fish and dairy products (17).
Nitrite is known as a vasodilator in vitro (18). The orally ingested
nitrite can be an alternative to L-arginine as a
source of NO in vivo and L-NAME-treated rats lowered the systolic blood pressure
(19).
A recent study has reported that nitrite and nitrate
(20) are the components of the aqueous extract from the skin of Balistes capriscus
and Menthicirrus litoralis.
Furthermore, the aqueous extract Balistes capriscus skin induced the relaxation of mesenteric
arterial bed by endothelium-dependent mechanism mediated by EDHF or NO, and neurally induced vasorelaxation
associated with NO from nonadrenergic noncholinergic nerves (9). Accordingly, AqeSh
may also have an antihypertensive effect via stimulation of NO system.
The change in body weight has been used as an
indicator of adverse effects of drugs or illness and the ability of an animal
to gain, or maintain, weight may be considered a sensitive indicator of health
((21). There was no change in animal behavior, and the body weight was not
significantly different in the treated groups as compared to the control.
At the end of the experiments the average weights of
the vital organs and conditions of the viscera were similar to the control
group. The weight of testes, caudal epididymis and
seminal vesicles were normal and also comparable to the control group.
Blood is an important index of physiological and
pathological status in man and animals, and the parameters usually evaluated
are hemoglobin, packed cell volume, white blood cell count and platelets
count. The normal range of these parameters can be altered by the
ingestion of some toxic products, like plants or other natural products (22).
There were no significant alterations in the hematological parameters of rats
treated with either L-NAME or L-NAME and AqeSh
when compared to control values.
Kidney eliminates waste products of metabolism from
the body. In renal failure, waste products accumulate particularly nitrogenous
substances like non-protein nitrogen, urea and uric acid. It has been reported
that the non-protein nitrogen values is elevated when blood urea is also raised
(23). In the present study, no significant alteration in urea level in both
treated groups could be observed. However, both treatments alter blood AST and
ALT levels.
The levels of transaminases,
ALT and AST, are good indicators of liver function. The chronic
inhibition of NO synthesis by the oral administration of L-NAME has been
related to arterial hypertension, vascular smooth muscle contraction, renal
dysfunction, and increased blood pressures (24, 25). Elevated transaminases levels in L-NAME-treated rats may be perhaps
interpreted as a toxic effect on liver, since their increase is a common
finding in liver disorders. The inhibition of NOS by L-NAME resulted in a
progressive injury observed in liver and lung. NOS inhibition aggravated liver
injury as alanine aminotransferase
increased by 61% compared with rats subjected to reperfusion injury (26).
The use of popular remedies by clients with mild to
moderate hypertension is of greater concern to the medical community than their
use by those simply desiring health maintenance or the treatment of mild
illnesses such as the common cold. Nursing action in regard to
alternative therapies involves direct questioning of the client regarding the
use and dosages of such components, and client education should incorporate
information regarding their toxicity (7).
From the above investigation, it can be concluded that
the AqeSh did not significantly alter
hematological parameters and kidney function. However, treated groups exhibited
significant alteration of AST and ALT profile, what may be due to the presence
of L-NAME.
The inactivation of NO system was reversed, along with
SBP normalization in the L-NAME induced hypertensive rats, by co-administration
of AqeSh. This study thus provides evidences
that the skin of Stephanolepis hispidus has hypotensive
effects in L-NAME hypertensive rats. This effect may be, at least partially,
mediated by nitrite and nitrate. Further studies are in progress in our
laboratory to support the idea that fish skin may be an effective dietary
supplement for the improvement of blood pressure control.
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Received: Feb 23,
2007
Revised Apr 9th, 2007
Accept Jun 23rd, 2007