Evaluation of the safety of long-term oral administratio ofnasturtium officinalein healthy rats

Shahnaz Shekarforoush＊, Morteza Nabizadeh, Reza Soleimani, Hamideh Eftekhari

Department of Physiology, Arsanjan Branch, Islamic Azad University, Fars, Iran

Evaluation of the safety of long-term oral administratio ofnasturtium officinalein healthy rats

Shahnaz Shekarforoush＊, Morteza Nabizadeh, Reza Soleimani, Hamideh Eftekhari

Department of Physiology, Arsanjan Branch, Islamic Azad University, Fars, Iran

Nasturtium officinaleis a leafy vegetable of the Brassicaceae family used as a home remedy or a medicinal plant due to anticancer, antioxidant, and hepatoprotective properties. The purpose of this study was to evaluate the effects of the water extract ofN. officinaleadministered daily for 60 days at dose levels of 500 and 1000 mg/kg body weight in male Wistar rats. The effects on haematological and serum biochemical parameters were measured as indices of organ toxicity. The behaviour of rats was assessed in two animal models of anxiety, the open field test and the elevated plus-maze. After sixty-day intake ofN. officinalewater extract by gavage, the rats showed no significant changes in behavioural outcomes. The extract did not affect the biochemical profile related to serum lipid metabolism (TG, total cholesterol, HDL, and LDL) and markers of liver and kidney function (AST, ALT, total protein, albumin, and creatinine). However, a significant reduction in alkaline phosphatase (ALP) and BUN levels occurred in the treated groups. The extract did not affect WBC, RBC, Hb, HCT MCH, MCHC, MCV levels. It also produced no alteration in the body weight gain and relative weight of the organs. This study confirms that water extract ofN. officinaleadministered up to the dose of 1000 mg/kg for long term is not likely to produce toxic effects on some organs or haematological and biochemical indices in rats.

Nasturtium officinale; blood cell counts; behaviour

1 Introduction

Nasturtium officinale(N. officinale), a member of Brassicaceae family, is consumed as a vegetable in salads and used as a home remedy or a medicinal plant by the people of south eastern region of Iran. Its common name is “Watercress”. It contains high levels of vitamins especiallyvitamin C, lipid- and aqueous-soluble antioxidants in particular tocopherols and carotenoids [1], high amount of calcium [2] and anti-cancer chemicals isothiocyanates [3]. Isothiocyanates by inhibition of cytochrome P450 enzymes induce apoptosis and cell cycle arrest in several cancer cell lines [4]. In addition,N. officinalehas high hypolipidaemic [5], hepatoprotective [6] and anti-inflammatory properties [7]. Previous studies have demonstrated that watercress supplementation effectively protects against exercise-induced oxidative stress [8] and against lymphocyte DNA damage associated with human colon cancer cells, H2O2exposure [3], and smoking [1]. It was reported that consumption ofwatercress can reduce cancer risk in humans via a decrease in DNA damage along with an elevation in plasma antioxidant concentrations ofβ-carotene and lutein [1].N. officinaleenhances antioxidant enzyme activities, suppresses lipid peroxidation and increases glutathione level in rat liver tissue [5]. It has been pointed to the fact that antioxidant activity of most plants is due to their phenolic compounds [9]. The total phenolic content ofN. officinaleextract is 820 µg/g in terms of galic acid equivalent [10]. High contents of phenols are the cause of its high radical scavenging activity [9]. Moreover, xanthophyll, an effective scavenger of singlet oxygen, was elevated following watercress supplementation [8]. The results of a study indicated that water and ethanol extracts of watercress may prevent the formation of toxic carbonyl compounds such as MDAin vitro[11].N. officinalehas the most potent activity against some drug-resistant variants of Mycobacterium tuberculosis [12] and can stimulate bone formation via the proliferation and differentiation of osteoblast-like MG-63 cells [13].

The use of herbal extracts as a form of complementary or alternative medicine is fairly common and may be based on clinical experience. Because the scientific studies to support their safety for continuing therapeutic application are limited, there is need for a perfect scientific evaluation to predict the safety and effects of long term exposure to these plants. Taking into account thatN. officinaleis a good source of key nutrients associated with beneficial effects on human health, the aim of the present study was to investigate the subchronic toxicity ofN. officinaleon rats.

2 Materials and Methods

2.1 Preparation of plant exract

Whole parts (stems, leaves and flowers) ofN. officinalewere collected from natural populations from Shir stream near Sepidan city in the north of Fars province in Iran. Samples were identified by a botanist (Z. Taheri) and authenticated by the Herbarium of Kharazmi University, Tehran, Iran.

After washing and cleaning of the waste materials, the plant parts were dried at room temperature and milled into fine powder. Dried powder (100 g) was mixed in 600 ml of distilled water for 24 hours. The mixture was filtered and then the filtrate was concentrated with a rotary evaporator, and 39 g of the extract was obtained. The dried water extract was stored in airtight containers and refrigerated until use.

2.2 Animals

Rats weighing about 150-180 g obtained from the animal house of Shiraz University were used for the study. Animals were acclimated to the housing conditions for one week prior to the commencement of the study. They were also maintained under standard conditions of temperature (20-22 °C) and humidity (40%-60%) with a 12-h light-dark cycle and fed on standard pellets with free access to water. The body weight was measured for all rats once at the beginning of the study and once on the day of sacrifice. All experiments were approved by Animal Ethics Committee of Islamic Azad University.

2.3 Experimental design

The animals were randomly divided into three groups of 8 animals each:

Group 1 served as normal control and received distilled water 1 ml/kg, groups 2 and 3 received 500 (N500) and 1000 (N1000) mg/kg body weight of water extract ofN. officinaleby gavage for 60 days. The selection of the dose range was based on a previous study on the antigenotoxic activity of the water juice ofN. officinaleagainst damage induced by cyclophosphamide in rats [14]. Previousstudy on acute toxicity ofN. officinaleshowed an oral LD50 of ＞ 5 g/kg. Moreover, the dose of 500 mg/kg presented high anti-inflammatory activities [7]. Rats were monitored daily for general appearance and mortality during the experimental period.

Two behavioural tests were performed, i.e. open-field and plus-maze tests. The time interval between the two tests was 24 hours. Twentyfour hours after the behavioural tests, the animals were exsaguinated under ether anaesthesia. Blood samples drawn from the heart were subjected to haematological and biochemical analysis. After that, animals were quickly sacrificed and dissected.

2.4 Open field test

The open field test is a commonly used model for assessment of emotional behaviour in rats [15]. It consists of a square arena (80 × 80 cm2) with 40 cm walls made of glass and the box floor is painted black. On the 58th day of administration, each rat was placed in the central part of the arena, and the total distance travelled, time spent in the inner and outer zone, and mean speed were measured for 5 min. Rats' behaviour was continuously videotaped by a video camera placed above the arena. After each test, the box was cleaned with 70% ethanol to remove animal odors.

An increase of duration in the inner zone without modification of total locomotion was considered as an anxiolytic-like effect while the contrary, that is a decrease in these variables, is associated with anxiogenic effects [16].

2.5 Elevated plus-maze test

The elevated plus-maze test measures anxietylike behaviour in rodents [17]. The apparatus was elevated 100 cm off the floor, made of opaque plexiglas and consisted of two open arms (no walls)and two closed arms (20 cm high walls) extending from a central square platform (10 cm × 10 cm). Anxiety reduction was indicated with increase of time spent in the open arms or with the number of entries into the open arms while a decrease in these variables was considered as an anxiety-like behaviour [18].

On the 59th day of administration, each rat was placed on the central platform for 5 minutes and its behaviour was videotaped with a video camera above the maze. The time spent in the open and closed arms, the number of entries into the arms, the total distance travelled, and the mean speed were analyzed. The analysis was performed using Ethovision 7.1.

2.6 Pathological examination

All animals were firstly subjected to a thorough macroscopic examination of external surface of organs. Then different organs (heart, liver, kidneys, and testes) were quickly excised and weighted individually. The relative organ weight of each rat was calculated as follows: absolute organ weight (g)×100/ body weight (g).

2.7 Hematological and biochemical analysis

The hematological tests were performed on blood samples with ethylene diamine tetraacetic acid (EDTA) by using an automated blood analyzer (Sysmex kx21, Japan): haemoglobin (Hb), hematocrit (HCT), red blood cell (RBC) count, white blood cell (WBC) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

For biochemical parameters, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TG), total protein, albumin, alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine, and urea were determined by colorimetric methods. All biochemical determinations were performed using commercial kits (BioSystems, Spain) with Prestige 24i (Japan).

2.8 Statistical analysis

Data are expressed as the mean±standard error of mean (SEM). The analyses were performed through SPSS for Windows (Version 16.0. Chicago, SPSS Inc.). One-way analysis of variance (ANOVA) followed by a Tucky test for multiple comparisons was used. Differences were considered significant atP ＜0.05.

3 Results

3.1 Open field test

Fig. 1 shows that after the long-term administration of 500 and 1000 mg/kg ofN. officinale, there were no statistical differences among the groups in total distance travelled, mean speed, time spent in the inner zone, and distance travelled in the inner zone.

Fig. 1 Open field test performed after sixty days of oral administration of 500 and 1000 mg/kg ofNasturtium officinale. Total distance travelled (A), mean speed (B), time spent in the inner zone (C), and the distance travelled in the inner zone of the apparatus (D). Data are presented as the mean±SEM (n= 8). No significant differences were observed among groups.

3.2 Elevated plus-maze test

No differences among groups were shown in Fig. 2 in mean speed, total distance travelled, time spent in the closed arms and in the open arms, the frequency of entries into the closed arms and into the open arms. These results confirmed the data obtained in the open field test in which administration of 500 and 1000 mg/kg ofN. officinaledid not cause anxiety-like behaviour.

Fig. 2 Elevated plus-maze test performed after sixty days of oral administration of 500 and 1000 mg/kg ofNasturtium officinale. Mean speed (A), total distance travelled (B), time spent in closed arms (C), time spent in the open arms (D), the frequency of entries into the closed arms (E), and the frequency of entries into the open arms (F). Data are presented as mean±SEM (n =8). No significant differences were observed among the groups.

3.3 Oral toxicity study

After 60 days of treatment, no remarkable changes in general appearance or death were observed in all animals. All rats appeared healthy during the experiment. Visceral examinations of the control and treated rats revealed no visible lesions at the end of the study. There were no statistical difference between groups in final mean body weight and in the relative weights of the kidney, heart, liver, and testes among treated and control groups (Table 1).

Table 1 Effects ofN. officinaleextract on body weight (g) and the relative weight of visceral organs (g)×100/ body weight (g) after a 60 days study in rats

3.4 Hematological and biochemical parameters

Values of hematological parameters like red blood cells (RBC) count, haemoglobin (Hb), hematocrit (Hct), MCV, MCH, MCHC, and white blood cells (WBC) count were not significantly different between groups (Table 2). A marked but not significant decrease in the level of TG was observed in the N1000 group. Furthermore, there were significant reductions in the level of ALP and BUN in N1000 compared to the control group. However, there were no significant differences among groups in the rest of biochemical parameters analysed including: AST, ALT, albumin, total protein, LDH, cholesterol, LDL, HDL, and creatinine compared to the control group (Table 3).

Table 2 Hematological profiles of rats following oral administration ofN. officinalefor 60 days

Table 3 Serum biochemical evalutions of rats following oral administration ofN. officinalefor 60 days

4 Discussion

N. officinaleis a green leafy vegetable that widely used as a folk medicine for treatment of some disease such as diabetes and bronchitis [19]. Previous studies demonstrated that water extract ofN. officinalehas anti-inflammatory, antiulcerogenic, and antioxidant properties [10] and contains, per gram weight, one of the highest concentrations of glucosinolates and carotenoids of anyvegetable [20]. Glucosinolates during the chewing process are converted to isothiocyanates which has anticarcinogenic properties and reduces DNA damage in both animal models and humans [21].N. officinaleat the daily dose of 500 and 1000 mg/kg were chosen for investigating their effects on baseline aspects of rat physiology, considering its antigenotoxic activities described previously [14].

The present study demonstrated that oral administration of water extract ofN. officinalefor 60 days had no adverse effects on hematologic, metabolic, and behavioural outcomes in normal male rat. Additionally, the macroscopic appearance of all the tissues was normal. In accordance with this, no significant changes occurred in the final body weight, organ/body weight, and mortality rate among groups.

The open field and elevated plus maze are very popular rodent behavioral tests for research [16]. In the open field test, long term administration of N. officinal did not affect total distance travelled, mean speed, time and distance travelled in the inner zone. Similarly, in the elevated plus maze, no significant difference was observed in total distance travelled, mean speed, time spent, and the number of entries in open and closed arms in the control and treated rats. These results indicate thatN. officinaledid not cause alterations in anxiety-like behaviours.

We searched for signs of undesirable effects induced by long-term administration of N. officinal through precise hematological, metabolic, and tissue assessments. In clinical diagnosis, the measurement of serum enzymes and metabolic parameters is a valuable tool. Markers of liver and kidney function (ALT, AST, total proteins, albumin, creatinine) and biochemical profile related to serum lipid metabolism (TG, total cholesterol, HDL, and LDL) were not changed by the intake ofN. officinaleextract. The levels of ALP and BUN significantly decreased in the N1000 group compared to the control group; however, none of the values were lower than the normal range for rats [22]. In addition, under macroscopic evaluation, there was no sign of alterations in kidney and liver byN. officinaleextract. These results are in accordance to the findings of Fares (2009) regarding the intake of water extract ofN. officinale(100, 250 and 500 mg/kg body weight) via drinking water for one month. Their findings showed that the extract did not cause any significant changes in the serum total cholesterol, HDL, TG and glucose levels. They observed no hepatotoxic effects over the study period [10]. In this study, there were no significant changes in haematological parameters in treated rats compared to the control rats. These results confirm the lack of structural or functional abnormalities and support the safety nature of the plant extract. However, more examinations on these tissues through histologic, histochemical and immunohistochemical investigations are required in this issue.

Antioxidant activity and hepatoprotective effect ofN. officinalehas been proven in previous studies [1, 6]. Large amounts of flavonoids in theN. officinaleextract might be responsible for suppressing lipid peroxidation in the liver of rats fed a high-fat diet [5]. Fogarty et al demonstrated that consumption of rawN. officinale(85 g) 2 h before an exhausting exercise test attenuates exercise-induced DNA damage and lipid peroxidation, probably due to the higher amounts of antioxidants [8]. The 1000 mg of water and ethanol extracts ofN. officinalecontain 88.60 ± 2.41 and 74.18 ± 1.72 μg pyrocatechol equivalent of phenolic compounds which are very important because of ability in scavenging of radicals and inhibiting lipid peroxidation [11]. Systemic usage ofN. officinaleat doses of 250, 500 and 750 mg/kg significantly reduces tissue edema induced by carrageenan, that is, N. officinale possesses anti-inflammatory activity [7].

Physiological features as well as values forphysiological variables of rodents are remarkably similar to humans and can be used in evaluation of both the effectiveness and toxicity of the agents and pharmacological strategies [23].

This study provides evidence that sixty days administration ofN. officinalehas a level of safety. Due to its beneficial effects and a pharmacological level of safety,N. officinalemay be offered as a safe herbal supplement. Further investigations should be completed to obtain safety and effectiveness data.

5 Conclusions

This study indicated for the first time that 60 days of oral administration of water extract ofN. officinaledid not affect specific aspects of male rat physiology. In fact, normal locomotor ability, lipid homeostasis, blood cells counts, as well as markers of normal functionality of liver and kidney were observed in treated rats suggesting a level of pharmacological safety. Since no major alterations were found on behavioral, hematological, and biochemical parameters, the results of this study indicated that long term oral administration ofN. officinalewas safe up to 1000 mg/kg and had no systemic side effects in male Wistar rats.

Acknowledgements

The present paper was extracted from Msc thesis written by Nabizadeh and Soleimani, financially supported by Arsanjan Branch, Islamic Azad University. We gratefully acknowledge Dr. Rafiee and Mr. Taheri for their cooperation to identify the plant and the assistance of Mrs Esmaeelzadeh to perform behavioural tests in this research.

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* Author to whom correspondence should be addressed. Address: Arsanjan Branch, Islamic Azad University, Fars, Iran. Tel: +98-9173376859; Email: shek@iaua.ac.ir or sh.shekar@yahoo.com

Received: 2014-07-21 Accepted: 2016-09-21