Phytochemical Screening and Acute- and Organ- Toxicity Evaluation of Telfairia occidentalis Root Aqueous Extract on Normal Wister Rats

Ogbonnaya, E. Anthony*, Monago, C. Comfortand Belonwu, D. Chuka

Department of Biochemistry, University of Port Harcourt, Choba, Rivers State, Nigeria

ABSTRACT:

The phytochemical compositions of both aqueous and ethanolic extracts, and acute and organ toxicities of Telfairia occidentalis Root Aqueous Extract (TAE) were investigated in this study. Thirty five (35) healthy wister albino rats (99-140g) were separated into two groups of fifteen (15) and twenty (20) animals and used for acute- and organ- toxicity testing respectively. For acute toxicity 15 animals of both sexes were divided into 3 groups of 5 animals each and dosed 10, 100, and 1000mg/kg body weight (bw) of TAE respectively. For organ toxicity testing, 20 female animals were placed into 4 groups of 5 animals each. The first three groups were dosed as in the acute toxicity test, while the fourth group received equal dose of normal saline. Animals were sacrificed after 24 hours of administration of TAE. Phytochemical assay results show the presence of flavonoids, steroids, terpenes, tannins, saponins and carbohydrates in both aqueous and ethanolic extracts. The presence of plant steroids was more pronounced compared to other phytochemicals and alkaloids and tannins were absent or present in undetectable level. The TAE was lethal at the administered doses (10mg/kg bw, 100mg/kg bw and 1000mg/kg bw) to only male rats, although at the highest dose (1000mg/kg bw) the surviving animals were unconscious but regained consciousness minutes later. The biochemical assays show significant increase in the activities and levels of SGOT, SGPT, Creatinine, Urea, and Total protein, an indication of liver and renal insufficiency. Thus this study shows that at a dose as low as 10mg/kg bw, Telfairia occidentalis root aqueous extract (TAE) could exert hepatotoxic and nephrotoxic effects on rats, and also indicates that these toxicities may be dose- and sex-dependent.

KEYWORDS: Telfairia occidentalis, toxicity, hepatotoxicity, nephrotoxicity

INTRODUCTION:

Telfairia occidentalis hook F (Curcurbitaceae) is a tropical vine grown in West Africa as a leaf vegetable and for its edible seeds known variously as fluted pumpkin, fluted gourd, and ugu and iroko (Nigeria, West Africa). It is a dioecious, perennial and drought tolerant food crop. It is widely cultivated in Eastern Nigeria and the young shoots and leaves of the female plant form the main vegetable ingredients of the Nigerian Edikangikon, a popular delicacy of the Calabar area of South-South Nigeria (Akoroda, 1990).

]It is usually grown trellised or free range in some cultures where it is allowed to climb any vertical object on its route. The fruit is large, up to 13kg, but unedible. The seed is up to 5cm in diameter, dark red and rich in fat and protein (Okoli and Mgbeoku, 1983).

The seeds of fluted pumpkin are high in carbohydrate, fat and phosphorus. They also contain Vitamin A (Christian et al., 2007). The fatty acid of pumpkin seed oil is of high molecular weight and its iodine value indicates high degree of unsaturation compared to palm oil (Agatemor, 2006). The leaf laminae were shown to have abundant calcium crystals (Okoli and McEven, 1986).

The pure leaf extract of pumpkin of its mixture with egg is taken to treat anemia (Okoli and Mgbeoku, 1983). The leaf infusion is given as blood tonic to help convalescents, and can also help in treatment of malaria and loss of appetite (Obute and Adubor, 2007).

Oboh (2005) demonstrated a dose- dependent hepatoprotective effect of Telfairia occidentalis leaf extracts. He suggested that aqueous extract of the leaf could be more effective than the ethanolic extract due to higher antioxidant activity of the aqueous extract. The ethanolic fruit extract has also been shown to have a dose-dependent hypercholesterolemic, hyperproteinemic, hypertri glyceridemic and hyper-conjugated bilirubinemic effects on rats, suggesting that the fruit may not be safe for consumption (Olorunfemi et al., 2006).

Eseyin et al. (2006), investigating the effect of the root ethanolic extract of the plant on glucose level of normoglycemic rats observed that, unlike the leaf extract, the root extract of Telfairia occcidentalis did not possess hypoglycemic activity. Their work also could not confirm the claim of toxicity of the root. The co-administration of the leaf extract of Telfairia occidentalis before or simultaneously with chloroquine affected the pharmacokinetics of the drug (Eseyin et al., 2007).

Telfairia occidentalis contains considerable amount of antinutrients (Ajabade et al., 2006). It was noted that the roots of Telfairia occidentalis were potent poisons, while the seed contain agglutinin, which accounts for one-third of total extractable Telfairia occidentalis protein (Togun et al., 1994). Hart et al. (2005) estimated the concentration of trace metals – lead, iron, copper and zinc in crops harvested in oil prospecting locations, and showed that Telfairia occidentalis leaves had the highest uptake. The plant can also thrive in organic nutrient-enhanced, crude oil polluted soil containing water soluble fractions of the crude oil (Wegwu and onyeike, 2005). The aim of this study is to investigate the phytochemical composition of Telfairia occidentalis root and to ascertain the acute and organ toxicities of its aqueous extracts in rats.

MATERIALS AND METHODS:

Chemicals and Reagents

All chemicals used in this study are of analytical grade, and products of M&B (chloroform) and BDH England (ethanol). The reagents are commercial kits obtained from QCA (creatinine kit) and Randox, UK (GOT, GPT, Urea and protein kits).

Plant Sample

Telfairia occidentalis (Fluted pumpkin) root was obtained from a farmland in Warri, Delta State, South-South Nigeria and promptly identified in the Herbarium of Plant Science and Biotechnology, University of Port Harcourt. The sample was chopped into bits and air-dried for 2 weeks before being reduced to powder using a mechanical grinder.

Extraction

Powdered pumpkin root (350g) was macerated in 1500ml distilled water for 24 hours. Filtration was achieved with a sieve cloth and then Vacuo-filtration on filter paper. Filtrate was concentrated to small bulk using rotary evaporator and reduced to constant dry weight in an electric oven at 25^oC. Part of the extract was used for Phytochemical screening and the other for acute toxicity testing. Also, a weight of 100g of ground plant material was extracted by maceration in 300ml 80% ethanol for 24 hours. Extract was filtered under pressure using Whatman filter paper. Filtrate was evaporated to dryness in a rotary evaporator and reduced to constant dry weight in an electric oven at 25^oC. This extract was used for phytochemical screening only.

Animals

Thirty five (35) healthy albino rats (99-140g) were obtained from the Animal house of Faculty of veterinary Medicine, University of Nigeria, Nsukka. Animals were acclimatized for 2 weeks under standard laboratory conditions with unlimited access to water and standard rat chow.

Phytochemical Screening

The following tests were carried out on both the aqueous and ethanolic extracts of the sample to screen for the presence of some phytochemicals: Carbohydrates, was done using both Fehling and Molish tests. Alkaloids, flavonoids, steroids, terpenes, tannins, saponins, were assayed using Wagner reagent method, lead acetate test, acetic anhydride test, Liberman- Burchard test, ferric chloride test and the frothing test respectively.

Animal Treatment

(A) Acute Toxicity Test

Fifteen Wister albino rats were used for this test. Animals were placed into 3 groups of 5 animals each and administered orally, 10mg/kg b.w, 100mg/kg b.w and 1000mg/kg b.w respectively. Animals were observed for 24 hours for physical and behavioral changes and mortality.

(B) Organ Toxicity Testing

Twenty female rats were used for this test. Animals were placed into 4 groups of 5 animals each. Four (4) groups were dosed as in acute toxicity test, while the last group received normal saline, 0.002ml/ kg body weight. Animals were sacrificed after 24 hours under mild chloroform anesthesia. Blood was collected by cardiac puncture, allowed to stand for 15 minutes and centrifuged to obtain the serum. Serum was used for biochemical analysis.

Biochemical Analysis

Estimation of Serum Glutamate-Oxaloacetate Transaminase (SGOT) and Serum Glutamate-Pyruvate Transaminase (SGPT) were assayed by colorimetric method of Reitman and Frankel, 1957. Estimation of serum urea and creatinine Levels were done using Urease-Berthelot method as described by Weatherburn (1967) and modified Jaffe method as described by Blass et al. (1974) respectively. Total protein was estimated by biuret method as described by Tietz (1995).

Statistical Analysis

The data obtained from assay were analyzed statistically using student’s t- test and Analysis of Variance (ANOVA). Post-hoc comparisons were made using the Bonferonni’s test. A P< 0.05 was considered statistically significant.

RESULTS:

Table 1: Phytochemical Screening Results

Assay	Aqueous Extract	Ethanolic Extract

Carbohydrates	+	+
Alkaloids	-	-
Flavonoids	+	+
Steroids	++	++
Terpenes	+	+
Tannins	-	-
Saponins	+	+

Keys:

++ = Present in higher concentration

+ = Present

- = Absent or present in undetectable amount

Table 2: Acute Toxicity Test Results

Group n=5	Treatment	Mortality Ratio	Sex of Dead animals
I	10mg/kg bw extract	3/5	Male
II	100mg/kg bw extract	5/5	Male
III	1000mg/kg bw extract	2/5	Male
IV	Normal Saline	0/5	-

DISCUSSION:

Phytochemical assay results (Table 1) show the presence of flavonoids, steroids, terpenes, tannins, saponins and carbohydrates in both the ethanolic and aqueous extracts. The presence of plant steroids was more pronounced compared to other phytochemicals. Alkaloids and tannins were absent or present in undetectable level. The acute toxicity test (Table 2) shows a mortality rate range of between 40 and100percent. The extract was lethal at the administered doses (10mg/kg bw, 100mg/kg bw and 1000mg/kg bw) to only male rats, although at the highest dose (1000mg/kg bw) the surviving animals were unconscious but regained consciousness minutes later. This is an indication of sex-dependent toxicity of Telfairia occidentalis Root Aqueous Extract (TAE). The relationship between sex of organisms, on one hand and the ability of the organism to metabolize/ eliminate some xenobiotics has remained enigmatic. Certain substances are known to have pharmacological effect on organisms of one sex, but not on others of same species, but different sex. Substances such as Oxamniquine, Acetaminophen, 2,4-dichlorophenylacetic acid (2,4-D), and Benzene has been shown by various workers to exhibit sex dependent toxicity (Malcolm et al., 1983; Tarloff et al., 1996; Griffin et al., 1997; and Bauer et al., 2003). One form of cytochrome P450, CYP3A2, has been implicated in the sex-dependent metabolism of one of the substances (Kaneko et al., 2001). The liver function assays (Table 3) show a significant increase in the activities of the transaminases (SGOT and SGPT) for the extract treated groups compared to control. This shows that the extract may be hepatotoxic at the doses administered. The groups administered the highest dose (Group III) show increased activity of the enzymes compared to groups I and II. This is an indication that the toxicity of the extract may be dose dependent. The Renal function assay results (Table 3) show changes in the levels of creatinine and urea in the serum. This is an indication of a possible nephrotoxicity of TAE. While the extract causes increase in the levels of urea in the treated animals (Groups I, II, and III), compared to normal (Group IV), it lead to reduction in the level of creatinine in the same animal groups (I, II, and III), a trend that cannot be explained. The serum Total protein assay result (Table 3) shows a significant increase in the level of total protein, compared to control. This increase may result from the combined liver and renal conditions induced by TAE administration. No dose dependent effect was observed here. This study, therefore indicates that Telfairia occidentalis root aqueous extract (TAE) may be both hepatotoxic and nephrotoxic and these toxicities may be both dose- and sex-dependent. Further study to isolate the active component of the extract and investigate its effect at lower doses is underway.

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Received on 08.06.2010

Accepted on 14.08.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(5): Sept.-Oct. 2010, 417-420

Group N= 5	Treatment	SGOT Mean ± SD (U/L)	SGPT Mean ± SD (U/L)	Creatinine Mean ± SD (mg/dl)	Urea Mean ± SD (mg/dl)	Total Protein Mean ± SD (g/dl)
I	10mg/kg bw extract	25.12 ± 4.29^ab	47.17 ± 41.45^ab	1.88 ± 0.04^a	83.06± 3.17^ab	7.42 ± 0.48^a
II	100mg/kg bw extract	25.88 ± 2.06^ab	55.70 ± 0.59^ab	1.84 ± 0.00^a	81.59± 0.20^ab	7.45 ± 1.07^a
III	1000mg/kg bw extract	17.20 ± 6.65^a	84.20 ± 4.38^a	1.60 ± 0.13^a	91.16 ± 6.56^a	7.51 ± 1.96^a
IV	Normal Saline	13.01 ± 0.75	30.00 ± 3.54	5.83 ± 0.33	29.95 ± 4.10	5.20 ± 0.29