Antioxidant activity of Gloriosa superba in albino rats

Pradeep Kumar Samal

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh.

ABSTRACT:

The objective of this study was to investigate the antioxidant activity of Gloriosa superba leaves against carbon tetracholoride (CCl4) induced hepatotoxic model. The plant materials were dried in shade, powdered with help of hand grinder and extracted with alcohol. Preliminary phytochemical test shows the presence of Alkaloids, Carbohydrates, Tannins, Amino acids and Flavanoids. The alcoholic extract of Gloriosa superba (AEGS) showed a significant dose dependent (250 mg and 500 mg/kg p.o.) antioxidant activity. The degree of antioxidant activity was measured by using serum biochemical parameters like hepatic antioxidant enzymes like SOD, CAT, GPX and LPO. AEGS at a dose level of 250mg/kg and 500mg/kg produce significant (P<0.001) antioxidant activity by decreasing the level Malondialdehyde, while it significantly increased the levels of glutathione peroxidase (GPX) , superoxide dismutase (SOD) and Catalase activity (CAT) in a dose dependent manner. The effect of AEGS was comparable to that of standard drug Silymarin.

KEYWORDS: Gloriosa superba, SOD, GPX, CAT, MDA

INTRODUCTION:

Free radicals, from both endogenous and exogenous sources are implicated in the etiologic of several degenerative diseases such as coronary artery diseases, stroke, rheumatoid arthritis, diabetes and cancer.¹ High consumption of fruits and vegetables is associated with low risk for these diseases, which is attributed to the antioxidant vitamins and other phytochemical.^2-4 There is a great deal of interest in edible plants that contain antioxidants and health promoting phytochemicals, in view of their health implications.

Liver is often abused by environmental toxins, poor eating habits, alcohol and over the counter drug use, that damage and weaken the liver leading to important public health problems like hepatitis, cirrhosis and alcoholic liver diseases.⁵ The conventional drugs used in the treatment of liver diseases viz., corticoasteroids, antiviral and immunosuppressant agents are sometimes inadequate and may lead to serious adverse effects. In India, numerous medicinal plants and their formulations are used for liver disorders in traditional systems of medicine. It is cardinal to treat liver disorders since it directly affects the biochemistry of the cell through participating events like oxidative stress, redox change, etc.⁶ which induces lipid peroxidation. Peroxidation of membrane phospholipids not only alters the lipid mileu and structural as well as functional integrity of cell membranes, but also affects the activities of various membrane-bound enzymes including total ATPase, Mg2+ATPase, Ca2+ATPase and Na+K+ATPase.^7-8 In view of lack of synthetic agents for the treatment of hepatic disorder, there is a growing focus to evaluate traditional herbal medicines for hepatoprotective activity.⁹ Therefore; there is a need to develop satisfactory hepatoprotective drugs.

Gloriosa superba (Colchicacea) is a large, evergreen shurb growing up to 1.5 – 4 m tall. Leaves are 10-20 cm long oblanceolate to obovate, entire, tip narrow with short stalks. Its bark is used in Arthritis, Rheumatism where as its leaf is used in Fabrifuge generally healing, stomach troubles.

2. MATERIALS AND METHODS: -

2.1 Plant Materials:

The leaves of Gloriosa superba (Colchicacea) were collected from Thakur Chedilal Barristor Agriculture College and Research Centre, Bilaspur, India, in the month of Oct 2012, and air dried at room temperature after wash with tape water. The Plant identification was done by Dr. H. B. Singh Chief Scientist Head of the Raw Materials Herbarium and Museum, NISCAIR, New Delhi (NISCAIR/RHMD/Consult/-2012-13/2164/170).

2.2 Drugs and Chemicals:

Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like ethanol (CDH, Mumbai), alcohol (CDH, Mumbai) anesthetic ether (CDH, Mumbai) and CCl₄ (Ranbaxy, Delhi) Thiobarbituric acid, Trichloro acetic acid, H₂O₂, Epinephrine, EDTA, Tric Hcl buffer, GSH, NADP and other phytochemical reagents were obtained from Institute.

2.3 Animals:

Each experiment had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of SLT Institute of Pharmaceutical Sciences, Bilaspur (Chhattisgarh) was taken prior to the experiments (Reference No. 25/IAEC/Pharmacy/2013 dated 15/06/13). All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

2.4 Preparation of plant Extracts:

About 300 g of the leaves powder of Gloriosa superba was extracted with 1.2 L of alcohol using Soxhlet apparatus for 72 hrs at 40-50°C. The extract was concentrated to ¼ of its original volume by distillation as it was adapted to recover the solvent, which could be used again for extraction.¹⁰

2.5 Acute toxicity study (AOT):

Acute oral toxicity study was performed according to the procedure OECD guideline no. 425.¹¹ AOT was performed on Swiss albino mice and the animal were kept fasting for overnight providing water ad libitum, after which the alcoholic extract of Gloriosa superba (AEGS) was administered orally 5000 mg/kg and observed the mortality of animals.

2.6 Preliminary Phytochemical Analysis:

The extracts obtained were subjected to various chemical tests to detect the chemical constituents present in extracts of Gloriosa superba .^12-15

2.7 Assessment of liver function:

The alcoholic extract of Gloriosa superba was evaluated for their antioxidant activity by using CCl₄ induced acute model. Male Wistar rats, weighing (150-200) were divided into 5 groups consisting of 6 animals in each group. Group 1 received distilled water (6 ml/kg, p.o.) for 10 days. Group 2 were treated with vehicle (0.5% of Tween 40, 1 ml/kg, p.o.) for 10 days. Group 3 received silymarin (50 mg/kg, p.o.) for 10 days. Group 4, 5 pretreated with alcoholic extract of Gloriosa superba in the dose of 250 mg and 500 mg/kg body weight respectively for 10 days. Food was withdrawn 16 hrs before administration to enhance the acute liver toxicity. Group 2, 3, 4 and 5 were treated with CCl₄ was administered (2 ml/kg, s.c.) diluted in olive oil (1:1) was administered on 10^th day after 1 hrs of extracts treatment. After the treatment animals were sacrificed by cervical decapitation under excessive anaesthesia on the 11^th day. Immediately after sacrifice, the liver was dissected out, washed in the ice-cold saline, and the homogenate was prepared in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant liquid was used for the assay of marker enzymes, namely Glutathione peroxidise by Paglia, D.E., Valentine, W.N., (1967), superoxide dismutase by Saggu et al., (1989)., Misra and Fridovich, (1972), catalase by Chance and Maehly., (1955) and MDA by Esterbauer and Cheeseman (1990).

2.8 Statistical analysis:

The experimental results were expressed as the Mean ± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way ANOVA followed by Tukey Kramer’s post hoc test. P value of < 0.05 was considered as statistically significant.

3. RESULTS:

Preliminary phytochemical studies with extract revealed the phytoconstituents like cardiac glycoside, carbohydrates, phytosterols, saponins, phenolics and tannins. Different doses of alcoholic extract of Gloriosa superba leaves (AEGS) was screened in albino mice for their acute oral toxicity. No mortality was recorded till 5000 mg/kg body weight. Hence the extract was found to be safe up to the dose levels of 5000 mg/kg. So 1/10^th and 1/20^th of these dose i.e. 500 and 250 mg/kg body weight of AEGS for oral dose was select as therapeutic dose for pharmacological activity screening.

The effects of AEGS on rat liver Glutathione peroxidase (GP_X), Malondialdehyde (MDA) and enzyme antioxidants namely SOD and CAT levels are shown in Fig.1-4. A significant decrease in the level of glutathione and enzyme antioxidants (SOD and CAT) and increase in MDA were noted after single administration of CCl₄. Upon administration of AEGS, the activities of glutathione and enzymic antioxidants were significantly (P<0.05) reversed to near normal. The effects of AEGS were comparable with that of standard reference drug silymarine.

4. DISCUSSION:

In biological systems, lipid peroxidation (oxidative degradation of polyunsaturated fatty acid in the cell membranes) generates a number of degradation products, such as malondialdehyde (MDA), and is found to be an important cause of cell membrane destruction and cell damage .¹⁶ CCl₄ is commonly used to induce hepatoxicity in animal models. Metabolic processes convert CCl₄ in to the trichloromethyl radical (CCl₃^-) which interacts with O₂ to yield the highly reactive trichloromethylperoxy radical (CCl₃O₂^-). Both radicals are capable of binding to protein and lipid or abstracting a hydrogen atom from unsaturated lipid, which induced lipid peroxidation and leads to changes in the endoplasmic reticulam, reduction in protein synthesis and elevation of serum transaminase enzyme levels.^17-18

Lipid peroxidation as measured by MDA formation increased in the liver tissue of rats treated with CCl₄.^19-20 MDA a secondary product of lipid peroxidation is a major reactive aldehyde, levels can lead to peroxidation of biological membrane. A major defense mechanism involves the antioxidant enzymes, GSH, CAT, and SOD.²¹ Decrease in SOD activity is a sensitive index of hepatocellular damage. SOD scanvenges the superoxide anion to form hydrogen peroxide, thus diminishing toxic effects caused the free radical. CAT is an enzymatic antioxidant widely distributed in all animals’ tissue, highest concentrations are found in erythrocytes and liver cell. CAT decomposed H₂O₂ and protect the tissue from highly reactive hydroxyl radicals.²²

CCl₄- treated rats showed significant decreases in GP_X, CAT, and SOD level compare with control. In contrast GSH, CAT, and SOD levels were significantly increased in AEGS treated groups, suggesting enhanced antioxidant properties.²³

The qualitative analysis of Gloriosa superba leaves extract indicated the presence of flavonoids, polyphenols, is natural antioxidants but have also been reported to significantly increase SOD, Glutathion and catalase and decrease MDA level.

5. ACKNOWLEDGEMENTS:

The authors wish to thank Prof. J.S. Dangi, Head of the Institute for facilities for technical assistance.

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Received on 20.10.2013

Modified on 25.11.2013

Accepted on 03.12.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(6): November –December 2013, 288-292