Hepatotoxicity of the Methanol Extract of Carica papaya. (Paw-Paw) Seeds in Wistar Rats

Abstract

Activity of Carica papaya. Linn. (commonly known as paw-paw.) seed extract in liver physiology of albino (Wistar) rats was studied. The MeOH extract of C. papaya. seeds were Soxhlet extracted. Tolerated doses of C. papaya. were estimated in acute toxicity studies and administered orally, single or repeated doses, for 30 days to adult male rats weighing between 190 and 200 g, which were divided into four groups of five rats per group. Group 1 received 10 mg/kg; group 2 received 50 mg/kg; group 3 received 200 mg/kg; group 4 received normal saline (1 ml/rat) as control. Twenty-four hours after treatments, the animals of all groups were sacrificed and blood samples collected by heart puncture into centrifugal tubes. The blood samples were allowed to coagulate before centrifuged at 400 rpm at 4°C for 15 min to separate the serum for enzyme assays. A portion of liver was cut off and fixed in 10% normal saline. The result showed that C. papaya. seed extract treatments caused elevation of rat serum levels of acid phosphatase (ACP), alkaline phosphatase (ALP), and aspartate amino transferase (AST). Also revealed was mild to severe metaplasia of hepatocytes in a dose-related manner as well as proliferation of Kupfer cells and hepatic cells cirrhosis. These biochemical and pathological changes indicated liver cell damage and malfunction. These results, therefore, suggest that seeds of C. papaya. should be used in herbal medicine with care to avoid toxicity.

Keywords:

• Carica papaya
• hepatotoxicity
• liver sections
• Wistar rats

Introduction

Carica papaya. Linn. (Caricaceae) is a fruit tree commonly cultivated in some parts of southern Nigeria. It is popularly called paw-paw tree. and its fruits are called paw-paw.. The ripe paw-paw fruits are used for preparing Nigerian salads, sweets, or taken alone with a meal to enhance digestion. The unripe fruit extract is used for the treatment of hypertension in Nigerian herbal medicine. The seeds have no nutritive value, but in herbal medical practice, they have been used to treat a variety of ailments such as typhoid fever and malaria fever. Recently, our preliminary investigation into the activity of the seeds extract of C. papaya. on male fertility revealed antifertility properties (Udoh & Kehinde, 1999).

The phytochemical analysis of the ripe fruit paw-paw indicated the presence of vitamins A and C (Georges & Pandelai, 1949). The bark of the tree contains latex, which is the commercial source of an enzyme used in the production of digestive medicine, canned meat, leather tanning, and so forth (Georges & Pandelai, 1949).

The roots of C. papaya. have been claimed by herbalists to possess antimicrobial properties against microorganisms like gonorrhea, and so forth. In West Africa, the plant is mainly used as diuretic (root and leaves), antihelmintic (leaves and seeds), and to treat bilious conditions (fruit) (Dalziel, 1937). Ripe and unripe fruits (epicarp, endocarp, and seeds) showed very significant antibacterial activity with Staphylococcus aureus., Bacillus cereus., Escherichia coli., Pseudomonas aeruginosa., and Shigella flexneri. (Georges & Pandelai 1949). Literature searches revealed that few scientific investigations have been carried out on the pharmacodynamic properties of C. papaya.. However, Nigerian traditional medical practitioners use the seed extract for treatment of various types of ailments without considering adverse side effects.

Based on the above facts, it became necessary to investigate the action of C. papaya. on the liver function of Wistar rats.

Materials and Methods

Animals

Adult male albino Wistar rats weighing between 190 and 200 g were bought from the Animal House Unit, Department of Biological Sciences, University of Calabar, Nigeria. The animals were housed at controlled temperature (28±2°C) with a 12-h dark-light cycle and free access to rat pellets (Agro Feeds Ltd, Calabar, Nigeria) and water. The animals were allowed an acclimatization period of 7 days before treatments.

Extraction

Ripe fruits of Carica papaya. were bought from Watt Market, Calabar, Nigeria, between the months of October and December 1999. They were identified by Frank Apejoye of the Department of Botany Herbarium, University of Calabar (voucher specimen no. 73). The fruits were cut open to remove the seeds and were washed clean to remove debris. The clean seeds were sun-dried for 2 days and then oven-dried at 40°C for 1 week to ensure complete dryness. The dried seeds were ground into power with the help of an electric blender (Inoulmex, France).

The powdered sample (100 g) of C. papaya. seeds was wrapped in a thimble and placed in a 500 cm³ Soxhlet extractor (M&G Scientific Co., England). The sample was Soxhlet extracted following standard analytical laboratory method at 60°C in absolute ethanol (450 ml) for 72 h. The extract was evaporated to a paste form at 40°C for 8 h to produce 11% yield of raw sample of C. papaya.. The powder plant-drug was stored for use in the experiment in a refrigerator.

Extract concentration

A gram weight of C. papaya. extract was dissolved in 10 ml of normal saline to give a stock concentration of 100 mg/ml. The doses of the C. papaya. extract administered were calculated based on kilogram body weight of rat.

Treatment

Forty adult male Wistar rats weighing between 190 and 200 g were divided into four groups (n = 10). Group 1 received a single dose of 5 ml of normal saline (0.85% NaCl) orally as control; groups 2, 3, and 4 were treated with a single dose of extract of C. papaya. (10, 50, and 200 mg kg⁻¹ day⁻¹), respectively. Another set of 40 male rats weighing between 190 and 200 g were grouped into four groups (n = 10) so that group 1 received 5 ml of normal saline (0.85 NaCl) orally, daily for 3 days, and groups 2, 3, and 4 received 1, 50, and 200 mg kg⁻¹ day⁻¹ of C. papaya. extract daily for 3 days, respectively. After 7 days post-treatment, the animals of all the groups were anesthetized with chloroform (Sigma, St. Louis, Mo, USA) to allow easy collection of cardiac blood sample for biochemical assay and the animals later sacrificed, and the liver tissues isolated were fixed in 10% formal-saline.

Biochemical assays

Animals treated with either normal saline as control or extract of C. papaya. (10, 50, and 200 mg kg⁻¹ day⁻¹) in single dose or repeated doses for 3 days were anesthetized with chloroform, and blood samples were collected through heart puncture. The blood samples were centrifuged at 4000 rpm at 4°C for 15 min to separate the serum. The method for measurement of enzyme activity of acid phosphatase (ACP), alkaline phosphatase (ALP), and aspartate amino transferase (AST) described by Reitman and Frankel (1957) were used. Enzyme activity was measured colorimetrically at 546 nm using the standard method described by Reitman and Frankel (1957) and Oyedapo and Araba (2001).

Histopathology

A part of each liver was cut off and prepared for light microscopy by paraffin embedding, 6-µm sectioning, and staining with hematoxylin and eosin (H&E). The tissue specimens were histologically processed for morphological investigations and viewed and photographed using a light microscope (Olympus, Japan) and camera (Verorex Company, USA).

Phytochemical analysis

The ethanol extract of C. papaya. seeds was phytochemically screened and found to contain glycosides and polyphenol, and some traces of alkaloid, saponins, flavonoids, reducing compounds, phlobatanins, and hydroxymethyl anthaquinons (Table 1).

Table 1.. The photochemical screening of Carica papaya. seed extract.

Serial no.	Chemical constituent	Alcoholic extract
1	Akaloid	+
2	Glycosides	+ +
3	Saponins	+
4	Flavonoids	+
5	Reducing compounds	+
6	Polyphenol	+ +
7	Phlobatanins	+
8	Hydroxymethyl-anthraquinones	+

+, present; + +, present in excess.

Statistical analysis

Results were expressed as means ±SEM based on at least five experiments. Significance was determined by using Student's t.-test for paired data.

Results

Biochemical assays

Acid phosphatase (ACP)

Oral administration of the methanol extract of C. papaya. to rats in doses of 10, 50, and 200 mg/kg reduced the concentration of serum ACP with respect to control (Table 2). In contrast, rats treated with the C. papaya. (50 and 200 mg/kg) caused elevated concentration of ACP in a dose-related manner (Table 2). Decreased or increased concentration of the serum ACP was significantly different (p < 0.05) compared with the control.

Table 2.. Effect of Carica papaya. seeds extract on alkaline phosphatase (ALP), acid phosphatase (ACP), and aspartate amino transferase (AST) serum levels of rats.

Group	Experiment	Dosage mg kg^−1 day^−1	ACP IU/L	ALP IU/L	AST IU/L
A	Control	1.0 ml of 0.85% NaCl	5.0±0.2	140±0.4	49±0.1
B	Carica papaya.	200	7.4±0.2^d.	250±0.3^a.	95.7±0.3
C	Carica papaya.	50	6.7±0.2^e.	223±0.4^b.	85±0.2**
D	Carica papaya.	10	4.6±0.1^f.	205±0.07^c.	74.0±0.4***

Results are means±SEM of 5 experiments.

^a.p < 0.01;

^b.p < 0.01;

^c.p < 0.05;

^d.p < 0.05;

^e.p < 0.05;

^f.p < 0.05.

*p < 0.01;

**p < 0.05;

***p < 0.01; versus control by Student's t.-test.

Alkaline phosphatase (ALP)

The methanolic extract of C. papaya. (10, 50, and 200 mg/kg) administered to rats orally raised serum concentration of ALP in a dose-related manner (Table 2). The increase in the concentration of the serum ALP was significantly different compared to control (p < 0.01).

Aspartate amino transferase (AST)

Serum samples of rats treated with the MeOH extract of C. papaya. (10, 50, and 200 mg/kg) exhibited increased concentrations of the enzyme AST. The elevation of the enzyme concentration was dose-related and significantly different compared with the control (p < 0.05) (Table 2).

Histopathology

The histology of the liver sections of rats treated with normal saline (control experiments) showed normal cell morphology (Fig. 1a). Comparatively, the histology of the sections of livers of rats treated with C. papaya. seed extract (10 mg/kg) showed a mild metaplasia of the hepatocytes without liver cell damage (Fig. 1d). However, rats treated with C. papaya., 50 and 200 mg/kg, revealed severe metaplasia of binucleated cells, proliferation of Kupfer cells, and hepatic cell cirrhosis that was significant and more pronounced in the rats treated with 200 mg/kg (Figs. 1b and c).

Figure 1 Photomicrographs of sections of liver morphology: (a) Control; (b) C. papaya. (200 mg kg⁻¹ day⁻¹) treated rat liver (arrows “A” show binucleated cell metaplasia and “B” Kupffer cells proliferation); (c) C. papaya. (50 mg kg⁻¹ day⁻¹) treated rat liver [arrows “A” and “B” indicated as for part (b)]; (d) C. papaya. (10 mg kg⁻¹ day⁻¹) treated liver morphology (arrows show metaplasia of the hepatocytes) (H & E, × 400)

Discussion

C. papaya. has been employed in Nigerian traditional medicine for the treatment of a variety of ailments. Liver is a known major metabolic organ in which most substances foreign to the body are biologically transformed or metabolized into nontoxic or more toxic compounds than the original substance (Bowman & Rand, 1980; Jie, 1995, Loch et al., 1995). This investigation showed that the administration of a methanol seed extract of C. papaya. to rats elevated serum levels of ACP and AST in a dose-related fashion.

Elevated serum levels of ALP and AST signified liver cell cirrhosis and malfunction (Busik et al., 1993; Grgurevich et al., 1995; Oyedapo, 1996; Oyedapo & Araba, 2001). It was reported that an extract of the seed of Garcinia kola. (bitter kola) given to rats caused liver cell damage (Braide, 1990). Similarly, administration of C. papaya. seed extract to rodents resulted in elevation of liver transaminases and could cause liver cell lesion in the same manner as reported for Garcinia kola. seed extract (Braide, 1990; Cummings & Metcalf, 1995). This observation, therefore, indicated that the use of the seed extract of C. papaya. in herbal medicine practice without proper scientific evaluation of its activity is dangerous to health. Therefore, proper dose regimens should be designed to avoid toxicity. The photomicrographs of the liver sections of the rats treated with a high dose of C. papaya. (200 mg/kg) confirmed this fact. However, the extent of liver cell damage administered at low doses of C. papaya. (10 and 50 mg/kg) was not significant. Therefore, the seeds of C. papaya. should be used in herbal medicine with caution.