1. Introduction

The destructive nature of reactive oxygen species and their diverse pahophysiological effects on vital organs are becoming of great interest to toxicologists. Toxicity of many xenobiotics is associated with the production of free radicals, which have been implicated in the pathophysiology of many diseases. There are extensive evidence for oxidative stress as an important mechanism of degeneration in brain, kidney and liver disorders ^[1]. The human body system are equipped with natural antioxidants that protect against oxidative damages and also repair damaged molecules ^[2]. However, this natural antioxidant mechanism can be inefficient and hence dietary intake of antioxidant compounds is important.

Oxidative stress results when there is an increased production of free radicals and diminished levels of antioxidant system resulting in cell damage leading to the pathogenesis of a variety of human diseases including chronic and permanent damage to tissues and organs ^[3]. Free radicals are highly reactive oxygen centered species causing lipid oxidation and enormous biological damage. They attack proteins, DNA, polyunsaturated fatty acids and almost any biological molecules in living systems and induce functional anomalies and pathological changes ^[4].

Recent reports indicated an inverse relationship between dietary intake of antioxidant rich foods and incidence of human diseases ^[5]. Many natural antioxidants including tocopherol, vitamin C, carotenoid, and flavonoids have been found from various kinds of plants, such as cereals, vegetables, fruits and herbs ^[6].

Ageratum conyzoides (Yoruba: imi esu) is a common annual herbaceous weed with long history of traditional medicinal use in many countries especially in the tropical regions ^[7]. It is an erect, herbaceous annual plant used in folk remedies as a purgative, treatment of ulcers, skin diseases, fever and as a wound dressing ^[8]. The plant is also utilized in the treatment of mental and infectious diseases. Ageratum contains many bioactive compounds including flavonoids, alkaloids, comarins, essential oils, benzofurans, terpenoids and tannins. A research conducted in India reported that extract from Ageratum leaves protected mice from radiation damage and ulcers. Both cellular protection actions were attributed in part to the antioxidant effects of Ageratum ^[9].

Zanthoxylum zanthoxyloides (common name: Fagara-English, Orin ata-Yoruba) which belongs to the family Rutaceae is use as chewing stick in most African countries. The plant have been used as components of antiseptic, anti-parasitic and analgesic preparations for managing small pox, syphilis and related disease conditions ^[10]. Traditional healers throughout Nigeria have used different parts of Zanthoxylum for the treatment of a wide range of disorders, including toothache, urinary and venereal diseases and rheumatism ^[11]. Metabolites isolated from Zanthoxylum stem bark have shown anticonvulsant, antisickling, anaesthetic, antibacterial, anti-hypertensive and anti-inflammatory properties ^[12]. The extract contain flavonoids, chelerythrine, berberine and phenol ^[13]. This study intend to evaluate and compare the antioxidative potential of Ageratum conyzoides leaves and Zanthoxylum zanthoxyloides stem bark in rats tissues so as to justify their ethnobotanical value for the treatment of degenerative diseases.

2. Materials and Methods

Chemical kits; Total protein, superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPX), catalase (CAT) and malondialdehyde (MDA) were obtained from Nanjing Jiancheng Biological Engineering Institute, China. All other chemicals used were of analytical grade and obtained from Sigma Chemical Company, England.

Zanthoxylum zanthoxyloides stem bark and Ageratum conyziodes leaves were obtained from their natural source in Osogbo, Nigeria and properly identified by a botanist in the Department of Biological Sciences, Osun State University Osogbo, Nigeria. The plants were cut into small pieces and then air-dried at room temperature to constant weight after which they were pulverized with a mechanical grinder. They were then stored in a polythene bag until use. Powdered samples were soaked in 5 volumes 80% methanol for 3 days and then filtered using a Whatman No.1 filter paper (125mm). Crude extract was obtained by evaporating the methanol in a rotatory evaporator (SM-8A Surgifield Medical England) at a temperature of 80°C.

Thirty male albino rats weighing between 120-140g were used for the study. They were obtained and raised at the Central Animal House, Osun State University, Nigeria. The animals were housed in well ventilated wooden cages and cleaned twice daily under optimum laboratory conditions (12 hours light, 12 hours darkness, temperature range 25-28°C and relative humidity). They were acclimatized for 2 weeks before drug administration. The animals were given rat pellets and water ad libitum and sorted into 6 groups A, B, C, D, E and F. Group A served as the control and received distilled water. Oxidative stress was induced in the tissues by oral administration of 5mg/kg bw cadmium chloride to Groups B-F. Group C and D were treated with 200 and 500 mg/kg bw of Zanthoxylum zanthoxyloides extract respectively while Group E and F were treated with 200 and 500 mg/kg bw of Ageratum conyzoides extract respectively. Group B were left untreated. The drug and extracts were prepared with distilled water and administered to the rats orally by the aid of canular for a period of 21 days.

Rats were sacrificed by cervical dislocation. They were quickly dissected and the brain, kidney and liver removed. The excised organs were rinsed in ice-cold 1.15 % KCl and homogenized in 4 volume of ice-cold 0.1M Tris-KCL buffer (pH 7.4). The homogenates were centrifuged at 12,500 rpm for 15 minutes at 4^oC in an Eppendorf refrigerated centrifuge. The supernatant (post mitochondria fractions-PMF) obtained were refrigerated and subsequently used for antioxidant analysis.

Catalase activity was determined by the method of Johansson and Borg ^[14] based on its peroxidatic activity. The method of Kakkar et al. ^[15] was employed in the determination of superoxide dismutase activity. Glutathione–S-transferase activity was measured using the method of Habig et al. ^[16] based on its ability to form conjugate with 1, 2-dichloro 4-nitrobenzene (CDNB). The procedure described by Tappel ^[17] was used to estimate gluthathione peroxidase activity. Thiobarbituric acid reactive substances (TBARS) was assayed in the tissues as described by Ohkawa et al. ^[18] by measuring lipid peroxide product of the reaction between malondialdehyde and thiobarbituric acid. Protein concentration was estimated using the biuret method ^[19].

Data analysis was done using SPSS computer software version 12.0 and values were presented as mean ± SD. Statistical comparisons were done using one-way ANOVA. The level of significance was set at p <0.05.

3. Results

Levels of antioxidants in the brain, kidney and liver of experimental rats is shown in Table 1- Table 4 while TBARS concentration is shown in Table 5. The liver has the highest contents antioxidants followed by the kidney. It can be observed that rats administered cadmium chloride only had significantly reduced antioxidant levels and elevation of TBARS compared with the control group. Treatment of rats with graded doses of Zanthoxylum zanthoxyloides stem bark and Ageratum conyzoides leaves significantly reversed the insult caused by cadmium toxicity as it normalized the antioxidants and TBARS levels close to that obtained in the control.

Table 1. Catalase (CAT) activity (U/mg protein) in the tissues of experimental rats

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Table 2. Superoxide dismutase (SOD) activity (U/mg protein) in the tissues of experimental rats

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Table 3. Glutathione-S-transferase (GST) activity (U/mg protein) in the tissues of experimental rats

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Table 4. Glutathione peroxidase (GPX) activity (U/mg protein) in the tissues of experimental rats

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Table 5. TBARS Concentration (nmol MDA/mg protein) in the tissues of experimental rats

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4. Discussion

In the present investigation, we observed that cadmium chloride induced oxidative stress in the brain, kidney and liver of the rats. This was evident from the significant elevation of TBARS and depletion of antioxidant capacity in the tissues of the rats. The liver is the most affected followed by the kidney. The liver is the largest organ in the body and is often the target organ for chemically induced injuries ^[20]. Therefore it must show the highest level of lipid peroxidation. Cadmium is a popular toxic agent found in tobacco, cereals, vegetables and fishes. It can accumulates in the kidneys, liver and other tissues where it stimulates lipid peroxidation and induce toxicity ^[21]. Production of free radicals in excess can cause tissue injury, however, tissue injury can itself cause free radical generation ^[22]. Free radicals production is a naturally occurring process which can be protected by a variety of enzymatic and nonenzymatic mechanisms ^[23]. Some of these mechanisms are impaired after long-term consumption of prooxidative drugs and may therefore contribute to damage to the brain, kidney and liver ^[24].

Results obtained in this study demonstrate that Zanthoxylum zanthoxyloides stem bark and Ageratum conyzoides posses excellent antioxidant activity. The two extracts significantly inhibited the formation of TBARS in a dose-dependent manner and boosted enzymatic and non-enzymatic antioxidants capacity in the brain, kidney and liver of the rats. The observed antioxidant properties of the plants might be due to the presence of polyphenolic compounds such as flavonoid and proanthocyanidin as obtained in their phytochemical analysis in previous studies ^[25]. Polyphenolic compounds have been proven to possess significant antioxidant activities and are responsible for many pharmacological properties observed in plants ^[26]. The antioxidant activities of polyphenolic compounds complement and sometimes synergize the antioxidant activities of vitamin C, vitamin E and carotenoids making them an important nutritional component in the body’s defenses against free radical damage ^[27].

Exogenous antioxidant compounds (including those from plant sources) could attenuate oxidative damage of a tissue indirectly or directly by various mechanisms, including the prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity, and radical scavenging ^[28]. The antioxidant activity of Zanthoxylum zanthoxyloides stem bark extracts was found to be better than Ageratum conyzoides leaf extract which could be due to the presence of different compounds in the extracts ^[29].

5. Conclusion

This study clearly demonstrate that methanolic extracts of Zanthoxylum zanthoxyloides stem bark and Ageratum conyzoides leaves contain some potent antioxidants which can function as free radical scavengers thereby ameliorating drug induced oxidative damage in rats tissues. The plants act possibly by augmenting endogenous antioxidant defense mechanisms thereby protecting tissues against damage from free radicals released from cadmium metabolism. The observed antioxidant activity of the extracts may be a contributing factor to their suitability for the prevention and treatment of wide range of disorders.

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