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 cQuímicadProgramas 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 la TA a los niveles normales en las ratas hipertensas tratadas con L-NAME. Además, EacSh pudo prevenir el aumento de TA inducido por el L-NAME. Al final del tratamiento no se observó ninguna diferencia en el peso del cuerpo y órganos vitales entre los grupos controles y tratados. Estudios hematológico y bioquímicos no revelaron diferencias entre los grupos controles y tratados, excepto los perfiles de AST y ALT, lo que puede haber sido por la presencia de L-NAME. Estos resultados sugieren que la administración oral de EacSh puede previnir el aumento de TA en ratas con hipertensión inducida por L-NAME, lo que puede estar mediado parcialmente por la presencia del nitrite y nitrato en lo EacSh. 

Palabras chaves: NG-nitroarginine methyl éster (L-NAME); hipertensión; Óxido nítrico;  Stephanolepis hispidus extracto acuoso. 


            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 Northeastern Brazil animal-based medicines have, for long, been used. Popular medicines have been elaborated from parts of the animal body, from products of their corporal secretions and from excrements (4). Despite their importance, studies on the pharmacological properties and therapeutic use of animals and their body parts have been neglected when compared to plants.

            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 Rio Grande, Southern of Brazil, the fish Balistes capriscus and Stephanolepis hispidus (Tetraodontiformes), commonly named triggerfish or plane head filefish, has been used by fishermen in the treatment of respiratory disorders, such as asthma and bronchitis, with a water infusion of dried and powdered skin (9). In a recent study we have reported that the aqueous extract Balistes capriscus displays a vasorelaxing effect mediated by endothelium-dependent (NO/EDRF) and endothelium-independent factors, and neurally induced by nonadrenergic and noncholinergic nerves (9).   

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


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, Brazil) tap water were available ad libitum.  

            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 (St Louis, MO, USA).

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), Rio Grande - Brazil. The animals were frozen at 18°C until used for the assays.

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, Brazil).

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 University Hospital (Rio Grande, RS, Brazil).

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.                                                                             


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, š 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, š 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.  



Treatment Group  (weight g)





AqeSh/l-NAME (n=8)

























seminal vesicles








 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.  



Treatment Group



L-NAME/AqeSh (n8)



Hemoglobin (g/dL)




MCHC (g/dL)




Platelets (103/mm3)




Haematocrit (%)




Total WBC (103/mm3)




Monocytes (%)




Lymphocytes (%)




Granulocytes  (%)








 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.




Treatment Group







Urea (mmol/l)




Bilirubin total (mg/100ml)




AST (U/l)




ALT (U/l)
























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.  .


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