INTRODUCTION

Free radicals such as Reactive Oxygen Species (ROS) in living organisms are known to cause damage to all the cell targets (proteins, lipids, DNA) implicating in the processes of aging as well as in wide range of degenerative diseases (Duan et al., 2006). A study shows that antioxidant substances with scavenge free radicals play an important role in the prevention of free radical-induced diseases. Since the natural antioxidant mechanism in humane body under some circumstances can be inefficient, a dietary intake of antioxidant compounds become an alternative. Recent investigations have shown that the antioxidant properties of plants could be correlated with oxidative stress defense and different human diseases (Malencic et al., 2000; Sanchez-Moreno et al., 1999; Stajner et al., 1998).

Eupatorium odoratum L. (synonym: Chromolaena odorata), a perennial shrub belonging to the Astereaceae family, invasive weed native to South and Central America. It has been introduced into the tropical regions of Asia, Africa and other parts of the world. Although, it is considered as one of the world’s worst weeds, it has been used as folk medicine for the treatment of cough, malaria, diarrhea and skin infection (Iwu, 1993). The juice of aerial parts of this plant is used as a local antiseptic agent (Taylor et al., 1996). Many researchers reported that its leaf exhibited antimicrobial (Iwu and Chiori, 1984), antifungal (Ngono Ngane et al., 2006), anti-inflammatory, antipyretic and antispasmodic (Taiwo et al., 2000), wound healing (Phan et al., 2001), platelet-activating factor receptor binding inhibitory (Ling et al., 2007) and haemostatic activity (Triratana et al., 1991). Even though there are reports on the antioxidant activity of leaf of E. odoratum, different methods and or different solvents in extraction were used (Alisi and Onyeze, 2008; Rao et al., 2010; Chomnawang et al., 2007; Akinmoladun et al., 2007). Therefore, the present study has been designed to evaluate the total antioxidant and free radical scavenging activities of the ethanol extracts of leaf, stem and root and flower parts of Eupatorium odoratum L. with a view to contributing to the search for beneficial uses of this plant.

MATERIALS AND METHODS

Chemicals: Chemicals and reagents, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene, linoleic acid were purchased from Sigma Chemical Co. (MO, USA). All other chemicals and solvents used in this study were of the reagent grade.

Plant materials: Root, leaf, flower and stem of E. odoratum were collected on July 2004 from TINPIPLAY, KAVRE district, Nepal. Plant was authenticated in the Central Department of Botany, Tribhuvan University (TU) and its voucher herbarium specimen (19-TUCH) was deposited at Tribhuvan University Central Herbarium, Central Department of Botany, TU, Nepal.

Root was washed with water and dried in sun for three days and finally dried in shade. Stem was dried in sunlight while leaf was dried in sunshade. These materials were ground to powder. Flower was shredded into coarse powder.

Preparation of the extracts: Powdered root, stem and leaf (50 g each) of E. odoratum were separately extracted with ethanol (400 mL) in a Soxhlet extractor for 7 h. On the other hand, the coarsely powdered flower parts (50 g) were first defatted with hexane. Thus defatted flowers (14 g) were extracted with ethanol (150 mL). Each extract liquid obtained from above plant materials was evaporated completely on Rotavapor in vacuo to obtain crude extract.

MEASUREMENT OF ANTIOXIDANT ACTIVITY

β-Carotene bleaching assay: Total antioxidant activity of ethanolic extracts of different parts of plant and standard (BHA) was measured according to literature (Bruni et al., 2004). The Relative Antioxidant Activity (RAA) of extracts was calculated with the following formula:

Where:

DPPH free radical scavenging assay: Effect of extracts on DPPH radical and standard (BHA) was measured by slightly modified method of Brand-Williams et al. (1995). The plant extract (0.5 g) was dissolved in methanol to make 10 mL stock solution. The volumes of 0.1, 0.2, 0.4 and 1 mL of stock solution were pipette out separately into 10 mL volumetric flasks and diluted further with methanol up to the mark to make four sample solutions corresponding to 0.5, 1, 2 and 5 mg mL^-1 concentrations, respectively. Positive controls (BHA) of similar concentrations were also prepared by same method.

The test was performed in 4 mL reaction mixtures containing 3.9 mL methanolic DPPH solution (6x10^-5 M) and 0.1 mL of sample solution of various concentrations (0.5, 1, 2 and 5 mg mL^-1 corresponding to 50, 100, 200 and 500 μg extract or BHA) or 0.1 mL of methanol (as blank). Three replicates were made for each concentration of each extract and BHA. After 30 min of incubation in the dark at room temperature, absorbance of the reaction mixtures was measured at 517 nm using MeOH as reference. The blank reading was measured every time just before the experiment of each concentration of extract or BHA starts. The inhibitory effect of DPPH by the sample was calculated according to the formula (Yen and Duh, 1994):

Where:

Inhibition concentration (IC₅₀) is the concentration (μg mL^-1) of antioxidant required to inhibit 50% of the DPPH free radicals. The IC₅₀ was calculated by using linear regression equation at four different concentrations of sample.

Statistical analysis: The RAA value was expressed as mean of three experiments with standard deviation. Percentage inhibition was expressed as mean of three experiments with standard error mean. Paired-sample Student’s t-test was performed for the significant differences between sample and positive control, BHA using SPSS 11.5 software. The antioxidant activity of extract was considered not significantly different from BHA when p>0.01. The antioxidant activity was significantly different from BHA if p<0.01.