G. latifolia leaves are used to treat loss of appetite, cough and stomach ache (Okafor, 1980) and it has been found very efficacious as an antidiarrhoea (Sofowora, 1982). The leaves are also used as spices in the soup of nursing mothers, where they are believed to serve as stimulants for rapid contraction of the womb and the return of menstrual cycle (Okafor, 1987). The use of the plant leaves in the management of diabetes mellitus, and high blood pressure is on the increase in Nigeria (Gamaniel and Akah, 1996). In another study, the sugar and lipid lowering effect of the plant was studied, and it was found out that prolonged consumption of the plant could possibly help in the management of diabetes and atherosclerosis (Obasi and Okoro 1997). The plant also has been found to reduce hepatic phospholipids and this could adversely affect the health of the consumers (Obasi and Njoku, 2000). Industrially, the plant has been found as a suitable substitute for hops in beer production (Okafor and Aniche, 1983).

Antioxidants are substances produced either endogenously or exogenously, and they are capable of reducing free radicals produced during oxidation reactions in biological systems. Antioxidants terminate the chain reactions by reducing free radical intermediates and many compounds with antioxidant activity are readily oxidizable (Larson, 1995). The antioxidant activity of several transition metals involves the donation of a single electron to a free radical species (Cadenas, 1995). Most antioxidants contain groups such as the thiols and polyphenols which are used in the reduction of these free radicals.

Antioxidants that reduce the rate of chain propagation are known as preventive antioxidants (Batham and Mayes, 2006). The preventive antioxidants are also known as enzymatic antioxidants and include the superoxide dismutase, glutathione peroxide, and catalase. These antioxidants counteract the hazardous reaction initiated by oxygen metabolites, the reactive oxygen species (ROS) (Rosa et al., 2003).

In the search for possible antioxidant compounds, plant phytochemicals have been extensively used and studied. These compounds are non-nutritive plant chemicals that contain proteive, disease preventing compounds (Ijeh et al., 1996).

More than 900 phytochemicals have been identified and characterized (Ajali, 2004). The groups that are of interest include the alkaloids, steroids, flavonoids, tannins, resins, saponins, glycosides, and terpenes. In this study, to compliment the increasing research interest in Gongronema latifolia, we investigated further the phytochemicals and antioxidant activities of the ethanolic extract of the leaves of Gongronema latifolia as most of the disease conditions claimed to be cured by this plant could trigger free radical reactions.

MATERIALS AND METHODS:

MATERIALS:

Plant Material:

The leaves and stem of Gongronema latifolia were used for this study. The leaves were bought from the Nsukka market and were identified by Mr. Njokuocha of the Department of Botany, University of Nigeria, Nsukka. The specimen was stored in the herbarium of the Department of Botany, University of Nigeria Nsukka.

Animals:

Six (6) Adult albino rats were used for the lipid peroxidation scavenging tests. Twenty one (21) adult albino mice were used for the acute toxicity study. All the animals used were bought from the Animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka. The rats were fed with pellets and water ad libitum.

Preparation of Plant Material:

The freshly collected leaves of Gongronema latifolia were chopped, dried and milled to coarse powder using the hammer mill.

Extraction of Plant Material:

A weighed quantity, 50g of the powdered plant was extracted with 250ml of absolute ethanol to obtain the extract. The extract was used for the lipid peroxidation scavenging tests.

Phytochemical Analysis of Gongronema latifolia:

The phytochemical analysis of the plant was carried out on both fresh and dried samples according to the method of Harborne (1973) to identify the active constituents of Gongronema latifolia.

Acute Toxicity Study (LD₅₀) of the Extract:

The acute toxicity study (LD₅₀) of the extract was determined using the method of Lorke (1983).

Preparation of Extract of Gongronema latifolia for Nitric Oxide/Lipid Peroxidation-Induced Scavenging Test:

A quantity, 1g of the ethanol extract of Gongronema latifolia. was suspended in 100ml of 20% tween 80 in standard phosphate buffer (0.025M, pH 7.4) to give the stock solution containing 10,000µg/ml of the extract. The stock solution was then used for the nitric oxide/lipid peroxidation-induced scavenging tests.

Nitric Oxide Scavenging Activity:

Nitric oxide scavenging assay was performed by Sreejayan (1997)

Carbon Tetrachloride-Induced Lipid Peroxidation Scavenging Test:

Carbon tetrachloride induced lipid peroxidation scavenging test was performed using the method of Comporti (1989).

Ferrous Sulphate-Induced Lipid Peroxidation Scavenging Test:

The method of Okhawa et al (1979) was used with minor modification by Tripathi and Sharma (1998).

Statistical Analysis:

The data in the results were expressed as mean ± SD and test of statistical significance was carried out using one way ANOVA. The statistical package used was statistical package for social sciences (SPSS), version 18.

RESULTS:

Percentage Yield of Extract:

Table 1: Percentage yield of the ethanol extract of the leaves stems of Gongronema latifolia

EXTRACT (g)	Percentage (%)
50.0	17.94

The result of the percentage yield from the table 1 is found to be 17.94%.

Phytochemical Analysis:

Table 2: Phytochemical analysis of both the fresh and dried leaves of Gongronema latifolia.

	Fresh Sample	Dried Sample
1. Alkaloid	++	+++
2. Flavonoids	+	++
3. Glycosides	ND	ND
4. Reducing Sugars	ND	ND
5. Saponin	++	++
6. Protein	+	++
7. Carbohydrates	++	++
8. Fats and Oils	++	+
9. Tannins	+	+
10. Steroids	+	++
11. Terpenoids	+	+
12. Test for Resins	+	+

KEY: ND Not Detected

+ Present in little amount; ++ Moderately present; +++ present in large amount

From the result of the phytochemical studies as shown in Table 2, alkaloids, Flavonoids, Saponins, Resins, Proteins, carbohydrate, fat and oils, terpenoids, tannins and steroids were present in the leaves and stems of both fresh and dried Gongronema latifolia.

Acute Toxicity Studies of Gongronema latifolia:

Table 3: Shows the result of the acute toxicity studies (LD₅₀) of the ethanol extract of Gongronema latifolia.

Dose (mg/kg)

after weight body

No. of Animals before

administration

No. of deaths

administration

250

500

750

1000

2000

3000

Control

LIPID PEROXIDATION-INDUCED SCAVENGING TESTS:

Effects of nitric oxide scavenging activity of the extract of Gongronema latifolia:

The extract of Gongronema latifolia showed significant free radical scavenging activity on nitric oxide (NO)-induced release of free radicals. Different concentrations (100µg/ml, 200µg/ml, 400µg/ml and 800µg/ml) exhibited different percentage of inhibition. Ascorbic acid was used as reference standard.

Table 4: shows the percentage inhibitions of different concentrations of the extract of Gongronema latifolia in the nitric oxide scavenging activity test.

	Concentration (µg/ml)	Absorbance 546nm (mean ± S.E)	Inhibition (%)
Ethanol extract of Apium graveolens	100 200 400 800	0.391 ± 0.032 0.387 ± 0.004 0.358 ± 0.002 0.221 ± 0.010	24.95 25.72 31.29 57.58
Ascorbic acid	100 200 400 800	0.426 ± 0.003 0.421 ± 0.001 0.403 ± 0.006 0.333 ± 0.005	18.23 19.19 22.65 36.66
Control		0.521 ± 0.035

From the table, the absorbance of the extract decreased with increasing concentration, which corresponds with an increase in the inhibition of the nitric oxide produced.

Effect of the Extract on Carbon tetrachloride-Induced Lipid Peroxidation in Rat Liver Homogenate:

Lipid peroxidation induced by carbon tetrachloride was inhibited by the extract of Gongronema latifolia at all tested doses (100µg/ml, 200µg/ml, 400µg/ml and 800µg/ml). The percentage inhibition of peroxide formation was increased in a dose-dependent manner. Also, the standard, vitamin C (ascorbic acid), showed significant reduction in lipid peroxidation formation.

Table 5: Inhibition of the extract on carbon tetrachloride induced lipid peroxidation test.

	Concentration (µg/ml)	Absorbance 543nm (mean ± S.E)	Inhibition (%)
Ethanol extract of Murraya koenigii	100 200 400 800	1.331 ± 0.014 1.078 ± 0.047 0.787 ± 0.039 0.704 ± 0.044	21.52 36.44 53.60 58.51
Ascorbic acid	100 200 400 800	0.402 ± 0.0055 0.348 ± 0.0033 0.267 ± 0.0069 0.187 ± 0.042	76.30 79.48 84.26 88.95
Control		1.696 ± 0.0126

From Table 5, the concentration of the extract increased as the absorbance decresed, and there was a corresponding increase in the inhibition to the carbon tetrachloride-induced lipid peroxidation.

Table 6: Inhibition of the extract on ferrous sulphate induced lipid peroxidation test.

	Concentration (µg/ml)	Absorbance 535nm (mean ± S.E)	Inhibition (%)
Ethanol extract of Apium graveolens	100 200 400 800	0.223 ± 0.033 0.211 ± 0.044 0.179 ± 0.008 0.149 ± 0.025	53.54 56.04 62.71 68.96
Ascorbic acid	100 200 400 800	0.324 ± 0.027 0.236 ± 0.021 0.200 ± 0.041 0.147 ± 0.005	32.50 50.83 58.33 69.38
Control		0.480 ± 0.069

From the table, as the concentration of the extract increased as the absorbance decreased and there was a corresponding increase in the inhibition to the ferrous sulphate-induced lipid peroxidation.

Effect of the Extract on Ferrous Sulphate-Induced Lipid Peroxidation in Rat Liver Homogenate:

The extract showed significant reduction in the lipid peroxidation formation against ferrous sulphate-induced lipid peroxidation in a dose-dependent manner. Ascorbic acid, the standard antioxidant used also showed significant reduction in the lipid peroxidation.

DISCUSSION:

This study was carried out to determine the antioxidant activities of the leaves of Gongronema latifolia. From the studies, the acute toxicity test of the ethanol extract on mice was found to be 2450mg/kg body weight, which indicates that the plant is safe for human consumption.

The investigation on the phytochemical constituents of Gongronema latifolia revealed the presence of alkaloids, tannins, sterols, flavonoids, fats and oils, carbohydrate, resins and protein. The content of the phytochemicals in the dried plants were higher than those found in the fresh plants and this could possibly be attributed to the water loss during the drying process, thus making the phytochemicals to be possibly concentrated. This result was consistent with earlier studies (Obasi and Okoro, 1997; Gamaniel and Akah, 1996; Kupchan, 1971).

In the various thiobarbituric acid (TBA) assays carried out, the ethanol extract of Gongronema latifolia showed potency in the scavenging of nitric oxide. As shown in Table 4, 100µg/ml of the extract gave an inhibition of 24.95% in the formation of nitric acid and 800µg/ml of the extract gave 57.58% inhibition. This shows that the plant has antioxidant properties. From the study also, as the concentration of the extract increased, the inhibition as recorded by the formation of nitric acid equally increased. This trend was also found using ascorbic acid, a known potent antioxidant drug.

The plant extract also gave a similar inhibitory activity with carbon tetrachloride and ferrous sulphate as shown in Table 5 and 6. The extract of the plant contains very high concentration of alkaloids and flavonoids as shown in Table 2. The alkaloids and flavonoids are known antioxidants and may have contributed to the inhibitory activities of the plant against formation of the free radicals. This plant could serve an important role against the peroxidation of membrane lipids, alteration of redox balance, enzyme inactivation and DNA damage. The several implications are that this plant could possibly alleviate the incidence of oxidative stress in biological systems and may justify its use as a medicinal plant. This again supplements its role in nutrition, especially as a rich source of mineral element and fibre.

The antioxidant activity of this plant have also sshown that the leaves have a promising role in the treatment of several physiological ailments as reported by Gamaniel and Akah (1996) and could serve as a possible neutraceuticals.

REFERENCES:

1. Agbo, C.U. and Obi, I.U.(2007). Studies on Seed Germination of Gongronema latifolia Benth. Bio-res. 5(1): 175-178.

2. Ajali, U. (2004). Chemistry of Bio-compounds. Ryce Kerex publishers. Enugu, Pp. 81-161.

3. Batham, K.M. and Mayes, P.A. (2006). Lipids of physiological significance. In “Harper’s Illustrated Biochemistry”, 27^th ed. Murray, R.K., Granner, D.K. and Rodwell, V.W. (eds). McGraw-Hill Companies, Inc. Connecticut. Pp. 128-129.

4. Cadenas, E. (1995). Mechanisms of Oxygen Activation and Reactive Oxygen Species identification. In “Oxidative Stress and Antioxidant Defense in Biology”. Ahmad, sS (ed) Chapman and Hall, New York. Pp. 25

5. Comporti, M. (1989). Three Models of Free Radical Induced Cell Injury. Chem. Biol. 72: 1-56.

6. Gamaniel, K.S. and Akah, P.A. (1996). Analysis of the gastrointestinal relaxing effect of the stem extract of Gongronema latifolia. phytomedicine. 2(4): 293-620.

7. Harborne, J.B. (1973). Phytochemicals Methods. A guide to modern Technology of Plant Analysis, 2^nd ed. Chapman and Hall, New York, Pp. 88-125.

8. Ijeh, I.I., Nwaugu, V.O. and Obidoa, O. (1996). Comparative Studies on the Nutritive, Phytochemical and Antimicrobial properties of two varieties of Vermonia amygdalina plant product research comm. 1: 63-68.

9. Kupchan, S.M. (1971). Drugs from natural products- plant sources in drug discovery, science and development. American Chemical Society. Washington D.C. p. 86.

10. Larson, A.R. (1995). Antioxidant Mechanisms of Secondary Natural products. In “oxidative Stress and Anitioxidant Defense in Biology”. Ahmad, S. (ed). Champman and Hall, New York. Pp. 215.

11. Lorke, D. (1983). Determination of Acute Toxicity. Arch. Toxicol. 53: 275.

12. Obasi, S.C. and Njoku, O.U. (2000). Reduction of rats serum hepatic phospholipids levels by aqueous extract of G. latifolia. W. African J. Pharm. Drug Research. 16(1,2): 52-57.

13. Obasi, S.C. and Okoro, O. (1997). Hyperglycemic effects of Gongronema latifolium (Benth) in albino rats. Environ. Toxicology. 1(1): 22-25.

14. Okafor, C.N. (1987). Identification and Conservation of Plants Used in Tradition Medicine. A paper presented at the international workshop on evaluation of traditional medicine. March 11^th – 14^th in the University of Nigeria.

15. Okafor, J.C. (1980). The Nigerian Rain Forest Ecosystem. O.U.U. Okali (ed). Ibadan University Press, pp. 262-300.

16. Okafor, L. and Aniche, G.N. (1983). West African hop substitute for sorghum larger. J. Brew. Distill. Inter. 12: 20-22.

17. Okhawa, H., Ohishi, N. and Yagi, K. (1979). Assay for lipid peroxide in Animal Tissues by Thiobarbituric acid reactions. Anal. Biochem.95(5): 351-358.

18. Osemeobo, G.I. and Ujor, G. (1991). Tha non-wood forest products in Nigeria. Report of the EC-FAO partnership program (1998-2000), Nigeria, Federal Department of Forest.

19. Rosa, E.V.C., Valgas, C., Souza-sierra, M.M., Cirrea, A.X.R. and Radetski, C.M. (2003). Biomass growth, micronucleus induction and Antioxidant stress enzyme responses in Vicia faba exposed to Cadium in solution. Environ. Toxicol. And Chem. 22(3(: 645-649.

20. Singh, N.P. (1988). Flora of Eastern Karnataka, Vol. 1. Mittal publication, Delhi, Pp. 141-142.

21. Sofowora, A. (1982). Medicinal Plants and Traditional Medicine in Africa (2^nd edu.). Spectrum Books Publishers Ltd. Ibadan. Pp. 140-145.

22. Sreejayan, N.M.R. (1997). Nitric Oxide Scavenging by Curcuminoids. J. Pharm. Pharmacol. 49(1): 105-107.

23. Tripathi. and Sharma, M. (1998). Comparism of the antioxidant action of the alcohol extract of Rubia codifolia with rubiadin. India J. Biochem. Biophsy. 35(5): 313-316.

Received on 12.05.2011

Modified on 02.06.2011

Accepted on 05.07.2011

Research J. Science and Tech. 3(4): July-August. 2011: 204-207