Toxicity Evaluation of Cucurbita maxima. Seed Extract in Mice

Abstract

Pumpkin seeds of Cucurbita maxima. Duch. (Cucurbitaceae) are frequently used for stomach pain, as an anti-inflammatory and antipyretic, and for the treatment of worms. This study was carried out to determine the possible acute and subacute toxicity effect of the hydroalcohol extract from seeds of C. maxima. after oral administration to mice and to identify the target organs of its possible toxic effects. The results showed that its average lethal dose (DL₅₀) is higher than 5000 mg/kg and subacute treatment shows increases in body weight. The levels of serum alkaline phosphatase, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and the hematological parameters did not show any alterations. The results suggest that the hydroalcoholic extract from C. maxima. seeds, at a dose of 5000 mg/kg, presents a considerable safety margin, being devoid of acute toxicity.

Keywords:

• Acute toxicity
• Cucurbita maxima
• subacute toxicity

Introduction

Cucurbita. sp. is a vegetable belonging to the family Curcubitaceae and has been used as a popular vegetable in cooking since antiquity. Pumpkin seeds are also commonly used as a traditional medicine in several countries for the treatment of ascariasis (Srivastava & Singh, 1967; Giove Nakazawa, 1996), cestodiasis (Chung & Ko, 1976; Yang et al., 1994; Mohamoud & El Alfy, 2003), and schistosomiasis (Chou & Ming, 1960). Pharmacological studies have also demonstrated antitumor activities (Xia et al., 2003).

In Brazil, Cucurbita maxima. Duch., is popularly known as pumpkin-moranga. or jerimum., where it is cultivated annually (Correa, 1926). It is yellowish-white in color and is commonly used in folk medicine to treat stomach pain and as an anti-inflammatory and antiparasitic (Vanaclocha & Cañigueral, 2003). It is also used as a traditional intestinal antiparasitic in rural and urban areas (Srivastava & Singh, 1967). Previous pharmacological studies have demonstrated that this plant presents strong antimalarial activity (Amorim et al., 1991) and anthelmintic activity (Amorim & Borba, 1993; Diaz Obregon et al., 2004).

To the best of our knowledge, only one report exists in the literature concerning the possible toxic effect of C. maxima. (Queiroz-Neto et al., 1994). This study aimed to evaluate the toxicity of the hydroalcoholic extract from C. maxima. in order to define the limits of safety regarding the medicinal use of its seeds.

Materials and Methods

Plant extract

Pumpkin-moranga. (Cucurbita maxima. Duch.) were authenticated by MSc. Rene Artur Ferreira (Curso de Farmácia, Univali, Itajaí, SC, Brazil). A voucher specimen was deposited in the Barbosa Rodrigues Herbarium (BRH, Itajaí, SC, Brazil) under number VC Filho 060. A pumpkin seed hydroalcoholic extract was prepared by triturating air-dried seeds and macerating with ethanol 50% at room temperature for 15 days. The solvent was then removed under reduced pressure.

Animals

Female and male Swiss mice, 25–35 g, from the animal laboratory of Univali, were kept in a temperature-controlled environment (25 ± 2°C) with a 12-h light-dark cycle. Food and water were freely available for the acute toxicity experiment. The same occurred for the subacute toxicity experiment, with food and water being recorded every 2 days.

Acute toxicity

The mice were randomly divided into four experimental groups and a control group, with 10 animals in each group, and kept in propylene boxes. The control group received saline, and each of the experimental groups received hydroalcoholic extract at doses of 100, 500, 1000, and 5000 mg/kg, administered through an orogastric tube. All the animals were kept under constant observation for 3 h, and for 24 h after administering the extract, to observe any changes in general behavior or other physiological activities. At the end of the experiment, each animal was sacrificed by cervical displacement.

Subacute toxicity

The mice were divided at random into an experimental group and a control group, with 10 animals in each. The control group received saline, and the experimental group received hydroalcoholic extract (1000 mg/kg), administered orally through an orogastric tube for 30 days. The animals were weighed every 2 days. At the end of the experimental period, blood was collected by orbital sinus venipuncture, under ether anesthesia, for biochemical and hematological analysis. After the blood collection, the animals were sacrificed by cervical displacement, and the target organs (liver, heart, spleen, left kidney, and left lung) were removed for macroscopic analysis.

The biochemical parameters evaluated included assays for serum alkaline phosphatase (Alk. Phosph.), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and urea using commercial kits (LABTEST, Lagoa Santa, MG, Brazil). The hematology parameters were determined for the control and 1000 mg/kg group and included a red blood cell count, hematocrit and leukocyte count.

Statistical methods

The results are presented as mean ± SD, and the statistical significance between the groups was analyzed by means of analysis of variance, followed by Dunnett's multiple comparison test. P values of less than 0.05 were considered as indicative of significance.

Results and Discussion

Oral administration of hydroalcohol extract from C. maxima. seeds in mice, at doses from 100 to 5000 mg/kg,did not produce any significant changes in behavior, breathing, cutaneous effects, sensory nervous system responses, or gastrointestinal effects. During the 24-h experimental period, no deaths occurred in any of the groups. These results indicate that DL₅₀ of hydroalcoholic extract of C. maxima. is higher than 5000 mg/kg and presented a considerable safety margin, being apparently devoid of acute toxicity for male and female mice.

The results show that the body weight of the animals treated with the 1000 mg/kg dose during the experimental 30-day period presented significant changes compared with the control group (Table 1). These results can be justified by the chemical composition and favorable nutritional value of the pumpkin seed (Vanaclocha & Cañigueral, 2003; Achu et al., 2005). Zdunczyk and co-workers (1999) have reported that the nutritional value of pumpkin seed has a higher protein efficiency ratio (PER) value than the soybean meal diet.

Table 1 Variation of the mean weight of the mice with treatment (oral route) for 30 days, with Cucurbita maxima. seed extract at a dose of 1000 mg/kg

Time (day)	Control	1000 mg/kg
1	30.75 ± 3.15	28.01 ± 2.68
5	31.72 ± 3.22	35.62 ± 4.50
10	33.02 ± 3.75	39.00 ± 5.17
15	35.61 ± 4.10	41.62 ± 5.33
20	35.70 ± 4.57	43.42 ± 4.43
25	37.50 ± 4.50	45.52 ± 4.73
30	39.37 ± 4.72	48.01 ± 3.74

Mean values of 10 animals ± SD. p < 0.05.

The macroscopic analysis of the target organs of the treated animals (liver, heart, lung, spleen, and left kidney) did not show any significant changes (color, texture) when compared with the control group (Table 2).

Table 2 Effect of Cucurbita maxima. seed extract on body and organ weight

Parameter	Control	1000 mg/kg
Body (g)	39.37 ± 4.72	48.01 ± 3.74*
Liver (mg)	1812.50 ± 106.25	2550.00 ± 168.75**
Heart (mg)	185.93 ± 4.69	214.06 ± 7.81*
Left lung (mg)	215.62 ± 6.25	243.75 ± 12.50*
Spleen (mg)	145.83 ± 15.83	175.00 ± 16.66*
Left kidney (mg)	208.33 ± 8.33	307.50 ± 14.16**

Mean values of 10 animals ± SD. *p < 0.05; **p < 0.01 vs. control group (Dunnett's test). Treatment for 30 days. The control group received saline.

The hematology parameters, red blood cell, hematocrit, leukocyte, and differential of cells, did not show any considerable alterations in the animals that received the hydroalcohol extract compared with the control group. These results are summarized in Table 3, and suggest that the hydroalcoholic extract from C. maxima. seeds, at a dose of 1000 mg/kg, does not interfere in the level of production/inhibition of red blood cells.

Table 3 Blood profile after treatment with the Cucurbita maxima. seed hydroalcoholic extract

Parameter	Control	1000 mg/kg
Hemoglobin (mg/dl)	13.67 ± 0.44	13.82 ± 0.23
Red blood cell (mm^3)	8.53 ± 0.43	8.08 ± 0.20
Hematocrit (%)	42.88 ± 1.17	44.83 ± 0.29
Leukocyte (10^6/ml)	8.43 ± 0.68	6.76 ± 0.69

Mean values of 10 animals ± SD. p < 0.05 vs. control group (Dunnett's test).

Table 4 shows the results of the biochemical assays with the serum, for mice treated, or not treated, with a hydroalcoholic extract from C. maxima. seeds. The serum evaluation of the enzymes Alk. Phosph., AST, and ALT is an important reference for the evaluation of hepatotoxicity, and these results do not demonstrate any significant alteration. However, we stress that the creatinine and urea levels (Table 4) presented abnormal alterations when compared with the control group, which can be related to the protection of the catabolism or high protein diet, therefore the urinary flow of the mice was considered normal.

Table 4 Effect of Cucurbita maxima. seed extract on biochemical parameters

Parameter	Control	1000 mg/kg
Alkaline phosphatase (U/l)	204.83 ± 15.63	224.37 ± 24.00
ALT (U/l)	32.54 ± 3.05	46.06 ± 5.09
AST (U/l)	111.86 ± 5.09	103.72 ± 10.17
Creatinine (mg/dl)	0.216 ± 0.03	0.283 ± 0.03
Urea (mg/dl)	40.00 ± 3.75	56.25 ± 2.50

Mean values of 10 animals ± SD. p < 0.05 vs. control group (Dunnett's test).

Our results showed that hydroalcoholic extract from C. maxima. seeds was devoid of acute and subacute toxicity at 5000 mg/kg and 1000 mg/kg, respectively, for mice. These results reinforce the findings for this pumpkin against rats and swine (Queiroz Neto et al., 1994).